key: cord-258881-74aijckl authors: wang, maomao; luo, limin; bu, haiji; xia, hu title: case report: one case of coronavirus desease 2019(covid-19) in patient co-nfected by hiv with a low cd4+ t cell count date: 2020-04-23 journal: int j infect dis doi: 10.1016/j.ijid.2020.04.060 sha: doc_id: 258881 cord_uid: 74aijckl abstract the ongoing outbreak of covid-19 that began in wuhan, china has become an emergency of international concern when thousands of peolple were infected around the world.we report a case infected by sars-cov-2 and hiv simultaneously,which showed a longer course of disease and slower generation of specific antibody. this case highlights the coinfection of sars-cov-2 and hiv may impaire the immune system worse. since december 2019, an outbreak of coronavirus disease, officially named by the world health organization as covid-19, appeared in pneumonia and respiratory illness. lymphopenia has been considered as a poor prognostic factor for severe acute respiratory syndrome(sars) [1] as well as in covid-19 [2] . here, we report clinical findings in a patient confirmed with covid-19, who was also co-infected by human immunodeficiency virus (hiv) . here we report a patient infected by sars-cov-2 , who had a relatively long course of disease with unstable state. then eight markers of infectious diseases was checked and the result showed that abtibodies to hiv and syphilis were positive .then the patient was transferred to specialty hospital for further treatment on march 8. in the specialty hospital, the cd4 cell count was 34/ul, cd8 cell count was 737/ul and cd4/cd8 was 0.05. the dectection of cryptococcus antigen in serum was negative and the patient was then given anti-hiv treatment . on february 11, 2020, a 37-year-old man was presented to wuhan huo shen shan hoapital, with a history of fever, dry cough and chest pain since january 10, 2020. the chest ct of this patient on covid-19 is caused by a novel type of coronavirus sars-cov-2. people are generally susceptible to sars-cov-2 infection, especially the elderly patients and those with underlying diseases [2] . the median time from onset of symptoms to first hospital admission was 7 days, to shortness of breath was 8 days, to ards was 9days, to mechanical ventilation was 10.5 days, and to icu admission was 10.5 days. [3] the patient described here were admitted to our hospital because of fever which lasting nearly one month and typical changes of viral pneumonia in lung ct imaging. the prominent complaint was dyspnea, the study of qin showed, the total number of b cells, t cells, and nk cells significantly decreased in patients with covid-19, and more evident in the severe cases compared to the non-severe group. the author suggested that sars-cov-2 might damage lymphocytes, especially t lymphocytes, and the immune system was impaired during the period of disease [2] . corticosteroids may delay viral clearance [3] . in this case, corticosteroid therapy was used( methylprednisone 200mg totally) accompanied with arbidol for anti-virus therapy.the body temperature turned normal. we also used tocilizumab one time to fight inflammation storm, which did not show the reduction of il-6 in serum. in conclusion, we report the clinical features of a patient infected by sars-cov-2 and hiv. the case appeared to be a long course of disease for more than 2 months. and until the later period of the course the igm in serum could be detected, which may due to the destroy of the immune response by the two viruses cooperatively. the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic:an analysis of all 1755 patients dysregulation of immune response in patients with covid-19 in wuhan we declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. key: cord-031289-uxoz0xhk authors: coccolini, federico; tartaglia, dario; puglisi, adolfo; giordano, cesira; pistello, mauro; lodato, marianna; chiarugi, massimo title: sars-cov-2 is present in peritoneal fluid in covid-19 patients date: 2020-05-18 journal: ann surg doi: 10.1097/sla.0000000000004030 sha: doc_id: 31289 cord_uid: uxoz0xhk background: the excretion pathomechanisms of sars-cov-2 are actually unknown. no certain data exist about viral load in the different body compartments and fluids during the different disease phases. material and methods: specific real-time reverse transcriptase–polymerase chain reaction targeting 3 sars-cov-e genes were used to detect the presence of the virus. results: sars-cov-2 was detected in peritoneal fluid at a higher concentration than in respiratory tract. conclusion: detection of sars-cov-2 in peritoneal fluid has never been reported. the present article represents the very first positive result describing the presence of the virus in peritoneal fluid during an emergency surgical procedure in a covid-19 sick patient. this article thus represents a warning for increasing the level of awareness and protection for surgeon especially in emergency surgical setting. (ann surg 2020;272:e240-e242) article a ctual pandemia posed several safety issues especially for those categories not directly involved in airway management. in fact, thousands of health care workers have been infected and died amid the ongoing coronavirus outbreak. actually, they must be among the best protected people. they face long hours, changing protocols, potential medical supply shortages, and pose at risk their own personal health and that of their loved ones. 1 ultraspecialistic branches of health system providing a unique service that cannot be performed by other medical disciplines should be even more secured. critically ill and injured patients, in fact, will continue to need emergent care. 1 the lack of precise data about viral load in the different body compartments and fluids forced health care workers to work in a situation of uncertainty and unsafety. the excretion pathomechanisms of sars-cov-2 are actually mostly unknown. sars-cov-2 rna has been found in blood and feces of covid-19 patients. 2, 3 the presence in peritoneal fluid has never been demonstrated. the present article is the very first one showing that sars-cov-2 is present in peritoneal fluid. the patient in whom the virus was detected was a 78 years' old man who came to the hospital from his house for abdominal pain associated to alteration of the alvus. at the admission, associated to the signs and symptoms of intestinal occlusion, he presented fever, cough, and mild respiratory symptoms with o 2 saturation of 92% maintained with an o 2 therapy at 2 lt/min with nasal cannula. no information about his infectious state did exist. his medical history was positive for arterial hypertension, type ii diabetes insulindependent, atrial fibrillation, mild chronic renal insufficiency, asymptomatic abdominal aortic aneurysm (maximum diameter 5 cm), and previous open appendectomy (20 years ago). thoracoabdominal computed tomography scan showed bilateral pneumonia and intestinal occlusion due to a small bowel volvulus with no signs of gut ischemia. the respiratory nasal swab was positive for sars-cov-2. he was admitted with a diagnosis of intestinal mechanical obstruction due to small bowel volvulus associated to sars-cov-2 pneumonia. he was operated and at the laparotomy free reactive clear fluid was found. neither perforation nor bowel ischemia was present. the volvulus was due to an omental band attached to the right iliac fossa. two swabs were obtained from peritoneal fluid and sent for sars-cov-2 detection. adhesiolysis was performed without intestinal resection. the subsequent abdominal cavity exploration showed the whole bowel vital and viable; neither colonic diverticula nor other evident pathological findings were found. after the intervention the patient was sent awake to the covid medical ward. his respiratory condition after the intervention remained stable. 98% o 2 saturation was maintained with a venturi mask with fio 2 of 35%, gradually diminished up to a complete independency from o 2 therapy. the postoperative period was uneventful, and the patient was discharged at home in postoperative day 10. two respiratory nasal swabs collected 24 hours apart and performed before discharge were negative. the real-time reverse transcriptase-polymerase chain reaction used to detect the sars-cov-2 rna genome in peritoneal fluid and nasal swabs detects 3 targets, namely rna-dependent rna polymerase, nucleoprotein (n), and envelope (e). the assay was performed according to the who guidelines 4 and corman et al' protocols 5 (fig. 1) . this method amplifies the number of copies of 3 targets at levels detectable by the instrument. although qualitative, the method allows to infer the amount of the viral rna genome based on the threshold at which the amplified signal becomes detectable. the nasal swab and the respiratory fluid were collected 1 day apart. interestingly, the nasal swab contained less sars-cov-2 rna virus compared to the viral fluid that scored positive in 2 targets out of 3. furthermore, the peritoneal fluid remained positive and at levels comparable to the nasal swab when retested 10-fold diluted. this indicates that the viral load in the peritoneal fluid was higher compared to the upper respiratory material and suggests that the surgical operation was indeed a procedure at risk of infection. viral isolation, which would have provided stronger evidence of infectivity, could not be performed. this new result poses an important warning for the safety of the operating staff and requests an immediate update of the rules to protect surgical teams. all surgical procedure in fact may potentially provoke aerosolization of the virus and the infection of the personnel. either laparoscopic or open surgical procedures may result in gas/ vapor forming maneuver. electrocautering, advanced coagulation, and cutting devices produce gas and vapor that aerosolize the peritoneal fluid and consequently the virus. previous studies covid-19 the show down for mass casualty preparedness and management: the cassandra syndrome covid 19 pandemic and gynaecological laparoscopic surgery: knowns and unknowns. facts views vis obgyn detection of sars-cov-2 in different types of clinical specimens detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr detectable serum sars-cov-2 viral load (rnaaemia) is closely correlated with drastically elevated interleukin 6 (il-6) level in critically ill covid-19 patients surgery in covid-19 patients: operative indications copyright © 2020 wolters kluwer health, inc. all rights reserved. demonstrated activated corynebacterium, human papillomavirus, hepatitis b virus, and human immunodeficiency virus in surgical smoke. 2 data from the literature showed as no defined direct relation exists between viremia and the severity of clinical picture. patient conditions seem to be influenced more by the host response to the infection that can be approximately calculated using hematic level of interleukin-6. 6 however, in presence of mild to moderate symptoms is less likely to detect a positive viremia than in critically ill patients. 6 if we hypothesize the same mechanism for the other body fluids, the greater the viremia, the higher the risks. as no information exist about the virus passage to peritoneal cavity and fluids, present data may suggest that potentially all people even those with mild to moderate respiratory symptoms by sars-cov-2 could present viral load in peritoneal fluid, thus increasing the exposure and contagion risks for the entire surgical staff.peritoneal fluid contamination with blood of feces may interfere with the virus detection. in present case, no contamination with faces or blood was present. the skin was adequately prepared with 2 preps with alcoholic solution lasting at least 2 minutes so the potential viral contamination from skin was significantly reduced.due to the lack of convincing data, scarce definitive instructions exist to prevent the potential contagion deriving from peritoneal fluid to the surgical staff. few protocols have been recently published to direct and help doctors and surgeons in their daily practice. 7 this present article represents a warning for increasing the level of awareness and protection for surgical staff especially in emergency surgery situations even in absence of intestinal perforation or ischemia. sars-cov-2, in fact is present in peritoneal fluids and it potentially aerosolizes to the environment. key: cord-255552-k1retwa4 authors: gassen, nils c.; papies, jan; bajaj, thomas; dethloff, frederik; emanuel, jackson; weckmann, katja; heinz, daniel e.; heinemann, nicolas; lennarz, martina; richter, anja; niemeyer, daniela; corman, victor m.; giavalisco, patrick; drosten, christian; müller, marcel a. title: analysis of sars-cov-2-controlled autophagy reveals spermidine, mk-2206, and niclosamide as putative antiviral therapeutics date: 2020-04-15 journal: biorxiv doi: 10.1101/2020.04.15.997254 sha: doc_id: 255552 cord_uid: k1retwa4 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) poses an acute threat to public health and the world economy, especially because no approved specific drugs or vaccines are available. pharmacological modulation of metabolism-dependent cellular pathways such as autophagy reduced propagation of highly pathogenic middle east respiratory syndrome (mers)-cov. here we show that sars-cov-2 infection limits autophagy by interfering with multiple metabolic pathways and that compound-driven interventions aimed at autophagy induction reduce sars-cov-2 propagation in vitro. in-depth analyses of autophagy signaling and metabolomics indicate that sars-cov-2 reduces glycolysis and protein translation by limiting activation of amp-protein activated kinase (ampk) and mammalian target of rapamycin complex 1 (mtorc1). infection also downregulates autophagy-inducing spermidine, and facilitates akt1/skp2-dependent degradation of autophagy-initiating beclin-1 (becn1). targeting of these pathways by exogenous administration of spermidine, akt inhibitor mk-2206, and the beclin-1 stabilizing, antihelminthic drug niclosamide inhibited sars-cov-2 propagation by 85, 88, and >99%, respectively. in sum, sars-cov-2 infection causally diminishes autophagy. a clinically approved and well-tolerated autophagy-inducing compound shows potential for evaluation as a treatment against sars-cov-2. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) poses an acute threat to public health 22 and the world economy, especially because no approved specific drugs or vaccines are available. compound-based targeting of cellular proteins that are essential for the virus life cycle has led to the 50 discovery of broadly reactive drugs against a range of covs (3-6). as virus propagation strongly 51 depends on energy and catabolic substrates of host cells, drug target identification should consider 52 the metabolism of infected cells (3). autophagy, a highly conserved cytosolic degradation process of 53 long-lived proteins, lipids, and organelles in eukaryotic cells, is tightly controlled by metabolism (7, 8). during autophagy, intracellular macromolecules are recycled by incorporation into lc3b-lipidated 55 autophagosomes (ap) and degradation into their monomers, such as fatty and amino acids, after 56 fusion with low ph lysosomes (9). in the case of highly pathogenic middle east respiratory syndrome 57 (mers)-cov, we recently showed that autophagy is limited by a virus-induced akt1-dependent 58 activation of the e3-ligase s-phase kinase-associated protein 2 (skp2), which targets the key autophagy 59 initiating protein beclin-1 (becn1) for proteasomal degradation (10). congruently, inhibition of skp2 propagation up to 50% (figure 4a, lower right, figure s3d,e) . akt1 blocks mtorc1 inhibitor tsc2 (30) 172 and further supports the suggestion that up-regulation of mtorc1 components has antiviral effects. as akt1 inhibition results in becn1 up-regulation and autophagy induction (10, 31), sars-cov-2 174 growth inhibition was expected. direct blocking of the negative becn1 regulator spk2 by previously 175 described inhibitors smip004, smip004-7, valinomycin, and niclosamide (10) showed sars-cov-2 176 growth inhibition from 50 (smip004, smip004-7) to over 99% in case of valinomycin and niclosamide 177 (figure 4a, lower panel, figure s3d,e) . we further confirmed that the dominant intervention of 178 niclosamide during sars-cov-2 infection acts on autophagy induction, as adding bafa1 after 179 niclosamide treatment showed an enhancing effect on the lipidation of lc3b as reflected by 180 comparable lc3b-ii/i ratios between mock-and sars-cov-2-infected cells (figure 4b) . however, we 181 cannot exclude that the activity of niclosamide as a hydrogen ionophore has additional inhibitory 182 functions, e.g. by blocking endosomal acidification (32), which is important for sars-cov-2 entry (6). twenty-four hours later, cells were fixed and analyzed by fluorescence microscopy. vesicles with both green and red fluorescence (aps) and with red fluorescence only (autolysosomes, al) were counted. in all panels error bars denote standard error of mean derived from n = 3 biologically independent experiments. tp < 0.1, *p < 0.05, ***p < 0.001 (two-way anova in a,b, one-way anova in c,d, t-test in e. abbreviations: lc3b, microtubule-associated protein 1a/1b light chain 3b; mrfp, monomeric red fluorescent protein; egfp, enhanced green fluorescent protein. clinical characteristics of coronavirus disease 2019 in china. n engl 216 antiviral potential of erk/mapk and pi3k/akt/mtor signaling 218 modulation for middle east respiratory syndrome coronavirus infection as identified 219 by temporal kinome analysis the sars-coronavirus-host interactome: identification of cyclophilins 221 as target for pan-coronavirus inhibitors targeting membrane-bound viral rna synthesis reveals potent 223 inhibition of diverse coronaviruses including the middle east respiratory syndrome 224 virus sars-cov-2 cell entry depends on ace2 and tmprss2 and is 226 blocked by a clinically proven protease inhibitor historical landmarks of autophagy research metabolomics profiling reveals differential adaptation of major 229 energy metabolism pathways associated with autophagy upon oxygen and glucose 230 reduction the reversible modification regulates the membrane-binding state of 232 apg8/aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway skp2 attenuates autophagy through beclin1-ubiquitination and its 235 inhibition reduces mers-coronavirus infection guidelines for the use and interpretation of assays for monitoring 237 autophagy dissection of the autophagosome maturation process 239 by a novel reporter protein, tandem fluorescent-tagged lc3 ampk: regulation of metabolic dynamics in the context 242 of autophagy ampk and mtor in cellular energy homeostasis and drug 244 targets ampk--sensing energy while talking to other signaling pathways sars-coronavirus replication is supported by a reticulovesicular 250 network of modified endoplasmic reticulum autophagy during viral infection -a double-edged 252 sword how mitochondria produce reactive oxygen species spatially distinct pools of torc1 balance protein homeostasis induction of autophagy by spermidine promotes longevity polyamines and eif5a hypusination modulate mitochondrial 260 respiration and macrophage activation polyamines control eif5a hypusination, tfeb translation, and 262 autophagy to reverse b cell senescence inhibitors of polyamine metabolism: review article inhibition of polyamine biosynthesis is a broad-spectrum strategy 266 against rna viruses inhibition of lipolysis and lipogenesis in isolated rat adipocytes with 268 aicar, a cell-permeable activator of amp-activated protein kinase aicar inhibits nfkappab dna binding 271 independently of ampk to attenuate lps-triggered inflammatory responses in human 272 macrophages network-based drug repurposing for novel coronavirus 2019-274 ncov/sars-cov-2 phase ii trial of akt inhibitor mk-2206 in patients with advanced breast 276 cancer who have tumors with pik3ca or akt mutations, and/or pten loss/pten 277 mutation tsc2 is phosphorylated and inhibited by akt and 279 suppresses mtor signalling akt-mediated regulation of autophagy and tumorigenesis through 281 beclin 1 phosphorylation niclosamide is a proton carrier and targets acidic endosomes with 283 broad antiviral effects the effect of spermidine on memory performance in older adults at 285 risk for dementia: a randomized controlled trial safety and tolerability of spermidine supplementation in mice and 290 older adults with subjective cognitive decline results of an abbreviated phase-ii study with the akt inhibitor mk-292 2206 in patients with niclosamide as a treatment for hymenolepis diminuta and dipylidium 294 caninum infection in man key: cord-273451-xnce010o authors: salisbury-afshar, elizabeth m.; rich, josiah d.; adashi, eli y. title: vulnerable populations: weathering the pandemic storm date: 2020-04-22 journal: am j prev med doi: 10.1016/j.amepre.2020.04.002 sha: doc_id: 273451 cord_uid: xnce010o nan in the fog of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic, vulnerable populations will be overlooked. marginalized in the best of times, the millions of people experiencing homelessness, struggling with substance use disorder, or incarcerated must not be left out. as in prior epidemics, these populations will pay the greatest price. viewed through the lens of the universal declaration of human rights and related treaties, all humans deserve medical care, necessary social services, and security of livelihood in situations out of one's control. 1 a similar conclusion will be arrived at on pragmatic public health grounds if the sars-cov-2 epidemic curve is to be -flattened.‖ social distancing may either be physically infeasible or else unlikely to be meticulously adhered to. every group represents a potential reservoir of infection that could infect others. yet, even with awareness that all individuals deserve access to services, and that supporting marginalized populations will slow the spread of sars-cov-2, resource limitations will demand difficult allocation determinations. the governmental, medical, and public health responses will dictate whether existing health inequities are worsened during the pandemic. containing the pandemic requires a vigorous attempt to redress the needs of the voiceless. failure to do so is not an option. the most important public health intervention to reduce the spread of sars-cov-2 is social distancing through self-isolation and cancellation of group gatherings. however, social distancing is not possible for homeless or incarcerated people. few if any alternatives exist for these populations and expedient planning and alternative solutions are needed. homeless shelters are minimally staffed and overcrowded with large fluxes of people and limited bathroom access-ideal for viral transmission. amid this pandemic, shelters are reducing staffing and clientele while experiencing increasing demand for services. consistent with guidance from the centers for disease control and prevention, symptom screening protocols are being implemented to identify individuals with fever or respiratory illness, 2 and although facilities strive to isolate symptomatic individuals, they are often unable to do so owing to space limitations. the long incubation period of sars-cov-2 and the heterogeneous clinical presentation may render symptom screening to be a limited tool, especially given that individuals may be asymptomatic with sars-cov-2. preliminary reports from post-exposure testing at a homeless shelter in boston found a 36% case rate of sars-cov-2 from their cohort of 408 clients who received the polymerase chain reaction test, but only a small percentage had cough (7.5%), shortness of breath (1.4%), or fever (0.7%). 3 many individuals experiencing homelessness with sars-cov-2 will not meet hospitalization criteria and will be discharged into the general population. inadequate social services will result in infected people spending more time in public places, thereby contributing to the spread of the virus. the sars-cov-2 response must prioritize access to testing in congregate settings and ensure access to shelters that can follow isolation and quarantine recommendations. local coordination of shelter services will be required to ensure that both asymptomatic individuals without covid-19 and those who are exposed or diagnosed with covid-19 have safe places to quarantine and isolate. in most localities, this will require identification of new facilities; examples of such facilities could include hotels, dormitories, and closed public buildings. higher baseline prevalence in many communities, and ongoing staff movement into and out of the facility makes it unlikely the virus will stay out. this will lead to added strain on local healthcare facilities when they have limited capacity to handle additional patients. the centers for disease control and prevention currently recommends avoiding cohorting those at higher risk for severe illness from covid-19 with others. if it is unavoidable, then accommodations should be made to prevent transmission to the higher-risk individuals. 6 at the time of this report, at least one correctional facility (j clarke, ri doc, personal communication, 2020) has separated individuals at risk for severe disease from others as much as possible, yet the efficacy of this practice is still unclear. in addition to the measures taken inside correctional facilities, authorities must move quickly to reassess security and public health risks. release of individuals who do not pose an immediate threat to public safety while reducing arrests and delaying sentencing is prudent. individuals who are being held in jails because of their inability to afford bail, or for minor infractions or violations, should be released with support to ensure safe shelter. similarly, those eligible for parole can and should be released. already, it has been observed that sars-cov-2 outbreaks in correctional settings spread quickly, with reports from cook county jail in illinois going from two positive cases to more than 350 confirmed positive cases in slightly more than 2 weeks. 7 the u.s., which has the highest worldwide incarceration rate, and a disproportionally greater minority and disadvantaged population, must act swiftly to protect the health and safety of incarcerated individuals and the community at large. this pandemic comes to the u.s. in the midst of the opioid epidemic. experiences from disasters like hurricanes katrina and sandy have taught that illicit drug market disruptions place people who use drugs at risk for supply discontinuity, paraphernalia shortage, and social disconnection. 8, 9 this will happen now. furthermore, shutdowns will disrupt the ability to generate income to support their use. the result will be decreased use, loss of tolerance, and risk for overdose. sharing or reusing scarce syringes will increase infectious diseases. individuals who use opioids may be more interested in initiating medication for opioid use disorder. however, the treatments with the strongest evidence-methadone and buprenorphine-are not readily accessible in many parts of the country. 10 one million individuals with opioid use disorder did not have access to medication for opioid use disorder in 2012. 11 in response to sars-cov-2, the substance abuse and mental health services administration has developed emergency regulations to support medication for opioid use disorder via telehealth, 12 and temporarily waived the requirement for in-person physical exam to be able to initiate buprenorphine. to increase access further, the drug addiction treatment act of 2000 waiver should be suspended to allow any prescriber with a drug enforcement administration license to prescribe buprenorphine. at a minimum, patient limits should be removed that would allow existing buprenorphine prescribers to take on more new patients. these often forgotten populations-people incarcerated, homeless, or using drugs-are likely to experience higher risk of exposure to sars-cov-2 because of their social circumstances. they also have high rates of medical comorbidities compared with the general population, thereby putting them at risk for more severe disease or death once exposed. if existing surge capacity models hold true, 13 hospitals will reach bed capacity quickly and triaging to prioritize resources for those at least risk for death may ensue, similar to what is happening in italy. vulnerable populations will likely experience further marginalization in this pandemic. for example, if there is one remaining ventilator and several patients in need, will the individual who is experiencing homelessness and using illicit drugs be the one selected? stigma against people who use drugs 14, 15 and other biases of care teams will become more pronounced in stressed and underresourced hospital settings, thereby impacting allocation of resources and exacerbating existing health inequities. central to the sars-cov-2 response must be a focus on forgotten populations. planning should incorporate dedicated efforts, funding, and policies/guidelines specific to individuals who experience homelessness, are incarcerated, or are coping with substance use disorders both because these populations deserve care and services, and because not doing so poses great risk to the broader community. although decades of inattention and underfunding for these systems will not be remedied in the midst of a national emergency, the responsibility to support these populations is now more critical than ever. careful consideration of and assistance for these vulnerable communities will be required to achieve a comprehensive and effective public health response. universal declaration of human rights. www.un.org/en/universal-declarationhuman-rights interim guidance for homeless service providers to plan and respond to coronavirus disease covid-19 outbreak at a large homeless shelter in boston: implications for universal testing epidemiology of covid-19 among people experiencing homelessness: early evidence from boston influenza control can be achieved in a custodial setting: pandemic (h1n1) 2009 and 2011 in an australian prison covid-19) in correctional and detention facilities chicago's jail is top us hot spot as virus spreads behind bars drug market reconstitution after hurricane katrina: lessons for local drug abuse control initiatives immediate impact of hurricane sandy on people who inject drugs in new york city characteristics of us counties with high opioid overdose mortality and low capacity to deliver medications for opioid use disorder national and state treatment need and capacity for opioid agonist medication-assisted treatment provision of methadone and buprenorphine for the treatment of opioid use disorder in the covid-19 emergency ihme covid-19 health service utilization forecasting team. forecasting covid-19 impact on hospital bed-days, icu-days, ventilator-days and deaths by us state in the next 4 months stigma among health professionals towards patients with substance use disorders and its consequences for healthcare delivery: systematic review stigma and the toll of addiction only people who have made substantive contributions to the study are included as authors and all authors have provided consent for their names to be used. the research presented in this paper is that of the authors and does not reflect the official policy of the nih.dr. rich is supported, in part, by grant p20gm125507 from the nih. all authors contributed to the writing and editing of this current issues manuscript.drs. salisbury-afshar and rich report no financial disclosures. dr. eli adashi serves as co-chair of the safety advisory board of ohana biosciences, inc. key: cord-262266-m0fjt483 authors: peddu, vikas; shean, ryan c; xie, hong; shrestha, lasata; perchetti, garrett a; minot, samuel s; roychoudhury, pavitra; huang, meei-li; nalla, arun; reddy, shriya b; phung, quynh; reinhardt, adam; jerome, keith r; greninger, alexander l title: metagenomic analysis reveals clinical sars-cov-2 infection and bacterial or viral superinfection and colonization date: 2020-05-07 journal: clin chem doi: 10.1093/clinchem/hvaa106 sha: doc_id: 262266 cord_uid: m0fjt483 background: more than two months separated the initial description of sars-cov-2 and discovery of its widespread dissemination in the united states. despite this lengthy interval, implementation of specific quantitative reverse transcription (qrt)-pcr-based sars-cov-2 tests in the us has been slow, and testing is still not widely available. metagenomic sequencing offers the promise of unbiased detection of emerging pathogens, without requiring prior knowledge of the identity of the responsible agent or its genomic sequence. methods: to evaluate metagenomic approaches in the context of the current sars-cov-2 epidemic, laboratory-confirmed positive and negative samples from seattle, washington were evaluated by metagenomic sequencing, with comparison to a 2019 reference genomic database created before the emergence of sars-cov-2. results: within 36 hours our results showed clear identification of a novel human betacoronavirus, closely related to known betacoronaviruses of bats, in laboratory-proven cases of sars-cov-2. a subset of samples also showed superinfection or colonization with human parainfluenza virus 3 or moraxella species, highlighting the need to test directly for sars-cov-2 as opposed to ruling out an infection using a viral respiratory panel. samples negative for sars-cov-2 by rt-pcr were also negative by metagenomic analysis, and positive for rhinovirus a and c. unlike targeted sars-cov-2 qrt-pcr testing, metagenomic analysis of these sars-cov-2 negative samples identified candidate etiological agents for the patients’ respiratory symptoms. conclusion: taken together, these results demonstrate the value of metagenomic analysis in the monitoring and response to this and future viral pandemics. on january 20, 2020, less than one month after the initial reports of a series of viral pneumonia cases in wuhan, china, the first case of infection with the novel sars-cov-2 was confirmed in the united states (1) . rapid person-to-person transmission has resulted in 614,482 total cases and 27,085 deaths within the united states as of april 15, 2020 (2) . epidemiological analyses have shown increased mortality risk in elderly patients above 65 years age, especially with underlying comorbidities (3) (4) . reported clinical complications that develop include sepsis in 59% of cases and acute respiratory distress syndrome in 17-29% of cases, often progressing to require mechanical ventilation (5) (6) (7) . for rapidly emerging infectious diseases, metagenomic next-generation sequencing (mngs) offers an opportunity to both recover whole viral genomes for epidemiological purposes and to agnostically determine co-infections that may be associated with increased morbidity and mortality in emerging infectious diseases (8) . here, we evaluated the performance of metagenomic sequencing on eight samples sent for sars-cov-2 diagnostic testing. mngs was performed in under 36 hours from sample collection to analysis, and the results were confirmed using validated qrt-pcr based methods. eight nasopharyngeal swabs in viral transport medium were sent to the university of washington clinical virology laboratory for diagnostic or confirmatory testing. qrt-pcr was performed using a modified protocol of the world health eight unique patient samples consisting of six positive and two negative cases of suspected sars-cov-2 were sequenced using rna extracted for a qrt-pcr diagnostic assay. in parallel we created mngs sequencing libraries using a previously published protocol using ds-cdna synthesis, followed by nextera xt tagmentation and 20 cycles of pcr amplification (10) . these libraries were sequenced on an illumina miseq using a 1x185 run with the miseq reagent kit v3 (150-cycle). reads per million (rpm) calculations and inter-sample comparisons were performed using the rpm_summary.r script (11). output from the pipeline was visualized using the pavian metagenomics data explorer and interpreted by a bioinformatician as well as two board-certified pathologists, who were blinded to clinical information on the samples prior to interpretation ( table 1) . reads that neither aligned to hg38 nor the nt database were re-trimmed using trimmomatic (13) . mitochondrial sequences were depleted prior to assembly reads by alignment to the human mitochondrial genome (mn540528.1) using bowtie2 with default options (15) . aligned reads were removed using samtools (17) , and then converted back to fastq format using samtools fastq. bbtools bbfakereads.sh was used to split the single end sequence into a pseudo-paired end sequence for assembly with metaspades (14, 18) . all available sars-cov-2 sequences from the global initiative on sharing all influenza data were downloaded on 3/18/2020, consisting of 806 unique samples. sequences with more than 5% n content were manually removed. genome alignment was done using mafft with the default settings. phylogenetic trees were built using raxml using the gtrcati model with 1000 bootstrap replicates. any taxa with an rpm < 10 were filtered out in order to exclude misclassified reads, possible water contaminants, and nasal flora. independent blinded analysis by both a bioinformatician and board-certified pathologist arrived at concordant interpretation of the results described. despite our reference database not containing any sars-cov-2 genomes, the six samples that were positive for sars-cov-2 by qrt-pcr had reads classified to table 2) . we were able to similarly detect and assemble sars-cov-2 with a c t of 29.5. as expected, our approach scaled with c t (r 2 = 0.80) (table 2, figure 2 ). sample wa6-uw3 showed substantial evidence of hpiv3 infection with an rpm of 4002 consisting of 4,027 unique reads. reads from this sample aligning to hpiv3 were also successfully de novo assembled using the geneious 9.1.8 assembler (19) . from this assembly we were able to reproduce the full hipv3 genome with a mean depth of coverage of 66.4. the two sars-cov-2 negative samples, sc5683 and sc5698, contained reads classifying to rhinovirus species a and c respectively. sc5683 contained reads classifying to both rhinovirus a71 (rpm = 1,592), as well as human rhinovirus spp. (rpm = 19,061). sc5698 contained reads classifying only to rhinovirus c3 (rpm = 454) ( figure 3 ). common skin flora cutibacterium acnes were present to some degree in nearly out of the eight total samples, the six with sars-cov-2 detected by metagenomic sequencing had c t s of below 30 for both the e and rdrp genes by qrt-pcr for sars-cov-2. in contrast, the two samples negative for sars-cov-2 by metagenomic sequencing, sc5683 and sc5698, had no amplification of either gene ( table 2 ). the exo internal control was successfully amplified in all tested samples. phylogenetic analysis revealed that the six sars-cov-2 sequences found cluster within two clades representing the washington state and european outbreaks. wa3-uw1, a traveler from korea to washington state, was the only sequence to cluster in the european clade. all genomes were over 99.5% identical by nucleotide relative to the reference strain (nc_045512.1). wa3-uw1 contained 3 amino acid mutations in the using mngs, we were able to successfully detect sars-cov-2 in six out of six positive samples, which were also confirmed by qrt-pcr. in addition, we were able to recover nearly full sars-cov-2 genomes from taxonomically unassigned reads (20) . the total time required for this testing was approximately 36 hours from receiving the sample to taxonomical assessment. such rapid turnaround could prove invaluable in the future when presented with an unknown infectious agent. the six sars-cov-2 sequences we present here represent two distinct clades from the pandemic: one european and one from washington state. sample wa3-uw1, the only sample to cluster within the european clade, was derived from a traveler from south korea. this sample diverged early within the clade and seems to be the terminal isolate within the united states. all other samples clustered with others from washington state and are representative of the larger washington state outbreak. a consequence of our reference database having been built from the 2019 genbank nt database is that it does not contain any sars-cov-2 sequences. despite this, reads with sequence homology to sars-cov were able to classify sars-cov-2 reads to the taxa "severe acute respiratory syndrome-related coronavirus" (national center for biotechnology information taxid 694009) . this was confirmed with assembly of unassigned reads, from which we were able to retrieve nearly the whole sars-cov-2 genome for all positive samples. we also demonstrate that with as few as 5 million reads we can de-novo assemble a full sars-cov-2 genome from a sample with a c t as high as 29.5 ( figure 2 ). of note, the number of sars-cov-2 reads is driven not only by viral load, but also the number of reads going to bacterial or human sequence (online supplemental table 1 ). additionally, reference genome length is not taken into account in this implementation of our pipeline. as a result, a bias is introduced as rpm will scale with the abundance of the organism, as well as increase linearly with the length of its genome. in addition to being positive for sars-cov-2, samples wa6-uw3, wa8-uw5, and wa9-uw6 also showed positive metagenomic results for m. catarrhalis, a gramnegative diplococcus that colonizes the nares of up to 75% of children, but only 0-4% of adults (21) (22) . the rpm of m. catarrhalis from wa8-uw5 and wa9-uw6 were 100x higher than that of wa6-uw3. these results further illustrate the ability of mngs to detect bacterial infections and/or colonizations on a patient-by-patient basis. first case of 2019 novel coronavirus in the united states an interactive web-based dashboard to track covid-19 in real time characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention cov-2: virus dynamics and host response clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a singlecentered, retrospective, observational study epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan metagenomic analysis identified co-infection with human rhinovirus c and bocavirus 1 in an adult suffering from severe pneumonia comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children rapid metagenomic next-generation sequencing during an investigation of hospital-acquired human parainfluenza virus 3 infections pavian: interactive analysis of metagenomics data for microbiome studies and pathogen identification trimmomatic: a flexible trimmer for illumina sequence data fast gapped-read alignment with bowtie 2 faster and more accurate sequence alignment with snap the sequence alignment/map format and samtools metaspades: a new versatile metagenomic assembler geneious | bioinformatics software for sequence data analysis a metagenomic analysis of pandemic influenza a (2009 h1n1) infection in patients from north america moraxella catarrhalis: from emerging to established pathogen co-infection with sars-cov-2 and influenza a virus in patient with pneumonia, china. emerg infect dis the authors declare they have no competing interests. key: cord-265322-3854ddb9 authors: vavougios, george d. title: a data-driven hypothesis on the epigenetic dysregulation of host metabolism by sars coronaviral infection: potential implications for the sars-cov-2 modus operandi date: 2020-04-23 journal: med hypotheses doi: 10.1016/j.mehy.2020.109759 sha: doc_id: 265322 cord_uid: 3854ddb9 covid-19, the disease caused by the novel sars-cov-2, a betacoronavirus structurally similar to sars-cov. based on both structural and syndromic similarities with sars-cov, a hypothesis is formed on sars-cov-2 potential to affect the host’s metabolism as part of its lifecycle. this hypothesis is evaluated by (a) exploratory analysis of sars-cov / human transcriptomic interaction data and gene set enrichment analysis (b) a confirmatory, focused review of the literature based on the findings by (a). a string viruses (available search for human – sars-cov (ncbi taxonomy id: 9606 vs. ncbi taxonomy id: 694009) genomic interactions reveals ten human proteins, interacting with sars-cov: sgta, fgl2, specc1, stat3, phb, bcl2l1, ppp1ca, cav1, jun, xpo1. gene set enrichment analyses (gsea) with string on this network revealed their role as a putative protein – protein interaction network (ppi; enrichment p-value=0.0296) mediating, viral parasitism, interleukin as well as insulin signaling, diabetes and triglyceride catabolism. in the literature, sars-cov has been known to cause de novo diabetes by ace2-dependent uptake on pancreatic isle cells, and furthermore dysregulate lipid autophagy in favor of the viral lifecycle. conversely, currently there are only non-causative, observational evidence of worse outcomes for covid-19 patients with comorbid diabetes or hyperglycemia. no study has reported on the lipid profiles of covid-19 patients; however, lipid-targeting molecules have been proposed as agents against sars-cov-2. future studies, reporting on lipid and glucose metabolism of covid-19 patients could help elucidate the disease’s seculae and aid drug design. . covid-19 is caused by the novel sars-cov-2, a betacoronavirus structurally similar (approximately 79%) to sars-cov (2) . in a recent study by hoffmann et al. (3) , the similarities between the sars viruses extend to ace2 dependent host cell entry. at present, sars-cov-2 has shown significant similarities with sars-cov, both in clinical characteristics and exploited host intracellular functions (5) . due to these commonalities, sars-cov remains an attractive substitute for the yet to be determined specifics of the sars-cov-2 / human protein interaction (6) and its consequences. among the first studies to report clinical data on covid19 was a recent publication by yang and colleagues (6) . their study provided the foundation for a hypothesis put forth by fang and colleagues indicating that diabetic and hypertensive patients exposed to ace2 inhibitors may be at an increased risk of more severe covid-19 (7) . meta-analyses on sars cohorts have indicated that both a history of diabetes and hyperglycemia were independent factors of worse outcomes including more severe respiratory symptoms and death, regardless of medication (9) . in another study, sars-cov was shown to cause diabetes by ace2-dependent infection of pancreatic isle cells (10) . interestingly, the significantly enriched "camp signaling" pathway is an indirect link between diabetes and ace2 signaling, based on experimental evidence associating camp levels and ace2 activity in diabetic patients (11) (false discovery rate (fdr) <0.05); table 1 . following entry to host cells, lipid metabolism is a subsequent important target of single strand rna viruses, critical for the formation of the viral envelope in subsequent lifecycles (12) . autophagy mediated triglyceride and lipid droplet catabolism is one such mechanism, as identified in denv infection (13) . hijacking the host cells' lipid metabolism has been shown to be a critical step in establishing hcov-22e and mers -coronavirus latency (14) . in sars-cov patients, alterations in lipid metabolism have been detected as far as 12 years after the initial infection (15) . evidence on covid-19's interplay with diabetes is only recently emerging, and can currently only be considered within the context of epidemiological studies, determining diabetes mellitus (dm) as frequent comorbidity. furthermore, dm has recently been characterized as a determinant of more severe respiratory syndrome, along with other comorbidities (16) . aside from dm, novel hyperglycemia was recently associated with an increased with worse outcomes in covid-19 patients, however this association was not independent of other predictors in the multivariate model (17) . epidemiological associations between covid-19 and lipid metabolism are currently not possible, since even large scale cohorts do not report on relevant measurements (18) . on the experimental level, lipid metabolism on the cellular level has been proposed as a treatment target for covid-19; specifically, both interactions between sars-cov's spike protein with lipid rich membrane compartments, as well as the epigenetic modulations in lipid metabolism were considered as the end-point targets for the development of small molecules, aiming to prevent sars-cov-2 infection (19) . barring actual proteomics sars-cov-2, sars-cov based in silico analyses of the sars-cov -human interaction partially support the hypothesis of fang and colleagues, insofar as to warrant further scrutiny on covid19 patient's metabolic states and concomitant medication. while the approach presented here is inherently limited due to setting its basis on the sars-cov proteomic interactions, it nevertheless presents in silico and literature evidence supporting sars-cov-2 potential to affect human metabolism. furthermore, as genes affected by sars-cov infection are significantly enriched for other infections, they may represent a common interface, targeted by viruses. future studies should determine sars-cov-2 interaction and effect on the human transcriptome, further identifying drug targets using pharmacogenomic enrichment analyses. the author confirms that there are no known conflicts of interest associated with this manuscript and there has been no financial support for this work that could have influenced its outcome. sars-cov-2 and covid-19: the most important research questions genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor differences and similarities between severe acute respiratory syndrome (sars)-coronavirus (cov) and sars-cov-2. would a rose by another name smell as sweet? receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection string v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars binding of sars coronavirus to its receptor damages islets and causes acute diabetes activation of the ace2/angiotensin-(1-7)/mas receptor axis enhances the reparative function of dysfunctional diabetic endothelial progenitors host lipids in positive-strand rna virus genome replication dengue virus-induced autophagy regulates lipid metabolism modulation of lipid droplet metabolism-a potential target for therapeutic intervention in infections altered lipid metabolism in recovered sars patients twelve years after risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease epidemiological, clinical characteristics of cases of sars-cov-2 infection with abnormal imaging findings prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis natural small molecules as inhibitors of coronavirus lipid-dependent attachment to host cells: a possible strategy for reducing sars-cov-2 infectivity? the author confirms that there are no known conflicts of interest associated with this manuscript and there has been no financial support for this work that could have influenced its outcome. george vavougios md, phd 20. 21. key: cord-272603-nbosceoz authors: lin, qiuyuan; wen, donghua; wu, jing; liu, liling; wu, wenjuan; fang, xueen; kong, jilie title: microfluidic immunoassays for sensitive and simultaneous detection of igg/igm/antigen of sars-cov-2 within 15 min date: 2020-07-02 journal: anal chem doi: 10.1021/acs.analchem.0c01635 sha: doc_id: 272603 cord_uid: nbosceoz [image: see text] the outbreak of sars-cov-2 is posing serious global public health problems. facing the emergence of this pandemic, we established a portable microfluidic immunoassay system for easy-to-use, sensitive, rapid (<15 min), multiple, and on-site detection of igg/igm/antigen of sars-cov-2 simultaneously. this integrated method was successfully applied for detecting sars-cov-2 igm and igg antibodies in clinical human serum as well as sars-cov-2 antigen in pharyngeal swabs from 26 patients with covid-19 infection and 28 uninfected people. the assay demonstrated high sensitivity and specificity, which is promising for the diagnosis and monitoring as well as control of sars-cov-2 worldwide. t he ongoing outbreak of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), identified as the causative agent of the corona virus disease 2019 (covid19) , rapidly spreads to cause a global pandemic and poses a huge challenge for global public health. 1−3 sars-cov-2 can spread rapidly through direct human-to-human transmission resulting in high infectivity. 4 the indefinite latency and nonspecific symptoms of sars-cov-2 infection makes the pandemic situation more serious. 5−8 considering the seriously increasing number of infected cases and widening geographical spread of sars-cov-2, and when in absence of effective antiviral therapeutics and vaccines for covid-19, there is an urgent need for easy-to-use, high-throughput, timely, accessible, and on-site methods for rapidly and sensitively detecting sars-cov-2 infection at an early stage for responses against the ongoing coronavirus outbreak and prevent and control the pandemic. 9−11 reverse transcription-polymerase chain reaction (rt-pcr) is the primary method for the diagnosis of sars-cov-2. 4,12−14 however, rt-pcr requires time-consuming and laborintensive rna preparation, a reverse transcription step, and professional operation, which decreases detection sensitivity and is difficult to achieve on-site detection. computed tomography (ct) imaging is an essential tool for fast diagnosis of sars-cov-2. 15, 16 while the specialized equipment of ct fails to meet a large scale of requirement, it may not provide the benefit for point-of-care diagnosis of covid-19. various enzyme-linked immunosorbent assay (elisa)-based methods have been developed for sars-cov-2 diagnosis. 17 for example, the extensively utilized colloidal gold-immunochromatographic assays 18 and lateral flow immunochromatographic assays (lfas) 19, 20 offer an immunoassay method for detecting covid-19, which show the advantages of simplicity, costeffectiveness, fast, and point-of-care testing. still, there are remaining limitations such as limited sensitivity and incapability of quantitative detection. microfluidics-based diagnostic systems have been extensively developed and applied in various fields. 21−23 microfluidic technologies are able to integrate sample preparation, reaction, and detection steps into a miniaturized chip. microfluidicsbased platform offers many advantages: (1) it enables rapid, laboratory-quality, sensitive detection at the point of need; (2) portability, high throughput, multiplex, and automatic; (3) it significantly saves the volume of reagents and reduces the testing price. 24 the determination of specific antibodies (such as immunoglobulin g/m, igg/m) and antigen is an easy, fast, reliable, and accessible strategy for the diagnosis of sars-cov-2 as well as efficient and large-scale screening of suspected cases at point-of-care settings. 25, 26 the detection of igg and igm in serum or whole blood has been demonstrated to be a reliable method for diagnosing covid-19 with high specificity and sensitivity. 18, 27 additionally, detecting sars-cov-2 antigen protein in nasopharyngeal swab samples has exhibited outstanding advantages in clinical testing. 26 to meet the challenge of the large epidemic, we describe the development of a point-of-care microfluidic platform integrating a homemade fluorescence detection analyzer ( figure 1a ), sars-cov-2 diagnostic microchips ( figure 1b) , and multiple immunoassays ( figure 1c ) for detecting three biomarkers (igg, igm, and antigen). the microchip fluorescence detector ( figure 1a ) measuring 28 cm × 22 cm × 14 cm and weighing 3.8 kg integrates centrifugation, fluorescence detection, and result display function, which is portable for use in the field. this proposed platform allowed analysis of three samples or biomarkers on the fluorescence detector simultaneously. the simple and low-cost microchip (length × width × height, 55 mm × 35 mm × 5.2 mm) ( figure 1b and figure s1 ) was designed and fabricated by assembling top and bottom plates (made of polycarbonate) that sandwich the middle layer containing the sample analysis channel (made of double-sided adhesive tape). the microchip is composed of a sample loading chamber, a waste reservoir, and a fluorescence immunoassay fluid channel comprising a capture region and test region. figure 1c describes the lab-on-a-chip fluorescence immunoassay for detecting three biomarkers of sars-cov-2. the combination of multiple biomarker detection offers outstanding performance such as improving the sensitivity and accuracy for sars-cov-2 diagnosis. the preparation of the immunoassay microchip for detecting igg, igm, and antigen of sars-cov-2 was achieved by matrix nanospotting, which is listed in the supporting information. the cost of a ready-touse immunoassay microchip is only about 5 yuan (0.71 dollar). when 10 μl of specimen (blood, serum, plasma, pharyngeal swabs, alveolar lavage fluid, or fecal suspension) is added into the loading chamber of the microchip followed by the addition of 70 μl of sample dilution buffer, the biomarker of sars-cov-2 (igg/igm/antigen) can specifically bind to the fluorescent microsphere (fms) labeled capture antibody. due to the large nanoscale of fms, the antigen−antibody complexes are easily migrated with the flow under the capillary effect. then the "antigen−antibody complexes" are immobilized on the fluorescence test region through a second "antigen−antibody" affinity interaction with sars-cov-2 antigen or detection antibody. if the specimen does not include sars-cov-2 biomarkers, no fms-labeled complexes bind on the test region. after 10 min, immunoassay chips are placed in the portable fluorescence analyzer followed by 10 s centrifugation to remove the residual liquid in the channel into the waste chamber. finally, the fluorescence detection results are read and obtained from the analyzer. the whole assay only takes less than 15 min. this robust microfluidic immunoassay system can provide a useful tool for sars-cov-2 diagnosis in public health laboratories as well as for timely screening potentially infected patients to monitor and prevent the epidemic owning to its capability of easy, fast, cost-effective, and point-of-care detection. moreover, this multiple detection system contributes to enhancing the accuracy and sensitivity of the detection. this sample-to-answer microfluidic immunoassay platform for multiple biomarkers detection was initially determined by clinical samples from confirmed covid-19 patients and healthy samples. all clinical specimens were collected and tested in shanghai east hospital (affiliated east hospital of tongji university, shanghai, china). figure 2a and 2b, respectively, show the fluorescence immunoassay detection results of igg in serum samples from 2 patients and 10 healthy people. we can observe that there is a significant difference between the infected patients and healthy people by comparing their final fluorescence value obtained from peak fluorescence intensity minus background fluorescence intensity, which was defined as the t value, and the t value could be automatically read from the analyzer ( figure 2c , p < 0.0001, using the mann−whitney no parametric test). figure 2d −f illustrates the similar detection results of igm in serum samples from 9 patients and 7 healthy people. the antigen testing results from nasopharyngeal swabs also exhibited an obvious distinction between 10 patients and 9 healthy people ( figure 2g−i) . these results proved the feasibility of the developed method. the cutoff values for igg, igm, and antigen detection were determined as 200 (t value), 200, and 100, respectively, according to these results. it was also found that the fluorescence values of serum samples from covid-19 patients ( figure 2b ,e) were significantly higher than the detection results of pharyngeal swab samples ( figure 2h ), which is probably because of the different levels of these biomarkers' concentrations between serum samples and pharyngeal swab "+" represents positive, 200−500; "++" represents medium positive, 500−1500; "+++" represents strong positive, >1500; "−" represents negative, <200). samples. these results indicated that it was more easy and sensitive to diagnose sars-cov-2 infection via serum samples than pharyngeal swab samples. the performance of the microfluidic diagnosis system was evaluated for monitoring the progression of sars-cov-2 infection by igg and igm testing. the testing results are summarized in table 1 results; only one false negative result of patient 2 was found for the igm test, which is probably because the igm level had not significantly increased on the 2nd day of infection. these results demonstrated that this immunoassay platform had a high sensitivity and specificity for the diagnosis of covid-19, which provides a rapid approach to adequately meet the urgent need for the ongoing global outbreak management of sars-cov-2. the proposed method was further evaluated by dividing the sars-cov-2 infection into three stages according to the patient's infection time. as shown in figure 3a , the t value for igg was increasing when the patients' infection time was changed from stage 1 (infected 1−7 days) to stage 3 (infected over 14 days). such a growth trend was also observed when detecting igm in serum from covid-19 patients ( figure 3b ). these findings further proved that the microchip immunoassay could not only accurately identify between sars-cov-2infected and uninfected cases but also roughly discriminate the severity of the patient's infection development with high sensitivity and reliability. the proposed microfluidic immunoassay exhibits the comparable superiority with the traditional colloidal gold-immunochromatographic assay method for sars-cov-2 detection, 18 such as easy-to-use, low cost, and point-of-care detection. moreover, our method offers the ability of sensitive, multiple, and quantitative detection based on fluorescence intensity (t value), which shows promising applications for covid-19 diagnosis. our method has been approved by the center for medical device evaluation (cmde) in china and obtained european ce certification. in conclusion, this study successfully developed a multiple detection tool for diagnosis of sars-cov-2, which integrated diagnostic microchips, a homemade portable fluorescence detector, and a microfluidic immunoassay approach. the multiple-testing platform was demonstrated to be easy-to-use, rapid, portable, and highly sensitive for point-of-care detection of sars-cov-2 within 15 min. the novel integrated diagnostic tool holds great promise for applications in sars-cov-2 pandemic monitoring and control. meanwhile, this sample-to-answer system is expected to be readily applicable for quantitative and sensitive detection of biomarkers of many diseases. the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.0c01635. figure s1 , scheme of microchip for fluorescence immunoassay; experimental section including materials, conjugation of fluorescent microspheres with capture antibodies, preparation of the immunoassay microchip, and immunoassays on a microchip (pdf) corresponding authors china; department of chemistry and institutes of biomedical sciences china; department of chemistry and institutes of biomedical sciences are first co-authors. all authors have given the national key r & d program of china (grant 2017yfa0205100), the national natural science foundation of china (grant 21974028), and the natural science foundation of shanghai (grants 19441903900, 12zr1401700, and 17jc1400100) for financial support key: cord-260048-yis26g81 authors: mcnamara, ryan p.; caro-vegas, carolina; landis, justin t.; moorad, razia; pluta, linda j.; eason, anthony b.; thompson, cecilia; bailey, aubrey; villamor, femi cleola s.; lange, philip t.; wong, jason p.; seltzer, tischan; seltzer, jedediah; zhou, yijun; vahrson, wolfgang; juarez, angelica; meyo, james o.; calabre, tiphaine; broussard, grant; rivera-soto, ricardo; chappell, danielle l.; baric, ralph s.; damania, blossom; miller, melissa b.; dittmer, dirk p. title: high-density amplicon sequencing identifies community spread and ongoing evolution of sars-cov-2 in the southern united states date: 2020-10-20 journal: cell rep doi: 10.1016/j.celrep.2020.108352 sha: doc_id: 260048 cord_uid: yis26g81 sars-cov-2 is constantly evolving. prior studies focused on high case-density locations, such as the u.s. northern and western metropolitan areas. this study demonstrates continued sars-cov-2 evolution in a suburban southern u.s. region by high-density amplicon sequencing of symptomatic cases. 57% of strains carried the spike d614g variant, which was associated with higher genome copy numbers and its prevalence expanded with time. four strains carried a deletion in a predicted stem loop of the 3’ untranslated region. the data are consistent with community spread within local populations and the larger continental u.s. the data instill confidence in current testing sensitivity and validate “testing by sequencing” as an option to uncover cases, particularly non-standard covid-19 clinical presentations. this study contributes to the understanding of covid-19 through an extensive set of genomes from a non-urban setting and informs vaccine design by defining d614g as a dominant and emergent sars-cov-2 isolate in the u.s. cov and mers-cov (lu, roujian et al., 2020) . residues at the receptor-binding site have evolved for 84 better association with ace2 compared to sars-cov (wan, yushun et al., 2020; wrapp et al., 2020) 85 and can be attributed to these molecular features: five of the residues critical for binding to ace2 are 86 different in sars-cov-2 as compared to sars-cov (wan, yushun et al., 2020; wrapp et al., 2020) 87 and a functional polybasic cleavage site (rrar) is present at the s1/s2 boundary of the sars-cov initial analyses of human sars-cov-2 genomes established three major variant types worldwide 98 (forster et al., 2020) . clade b was derived from a by a synonymous t8782c mutation in orf1ab; 99 and a nonsynonymous c28144t mutation that changes a leucine to serine in orf8 (ceraolo and to provide finer granularity about biological changes during sars-cov-2 transmission, we southeastern u.s. from the start of the u.s. epidemic on march 3, 2020, until past the peak of the first 115 major wave of infections. the first case in north carolina (nc) was reported on march 26, 2020. the 116 samples cover the period when community spread in nc was established, and when the state-wide 117 stay at home order was issued (march 30 -may 8, 2020). sars-cov-2 testing remains limited in many countries due to a shortage of personal 119 protective equipment, testing kits, and diagnostic capacity. the centers for disease control (cdc) 120 guidelines during the time of sampling prioritized patients with specific clinical symptoms (fever, 121 cough, and shortness of breath) and curtailed testing to only a subset of all probable cases. individuals not fitting the clinical criteria for testing, as well as asymptomatic individuals, were distribution of 10x coverage for all samples is presented in figure 1a . as expected, more mapped 151 reads yielded higher coverage. of the 33 negative controls, none had >10 2 total reads aligned. of the 152 positive samples, greater than 5*10 3 total mapped reads were needed to obtain 1x coverage of the 153 whole genome, a minimum of 3.1x10 4 reads were needed to obtain >90% coverage at 10x. the 154 number of reads aligned varied depending on the viral load, as determined by real-time qpcr using 155 cdc primer n1, but not total rna, as determined using rnase p, of the samples ( figure 1b) . in this 156 assay, any cp <35 for sars-cov-2 qpcr yielded reliable coverage, which increased linearly with 157 viral load. at a cp ≥35 most positive samples still yielded reads that mapped to the target genome 158 and thus allowed detection of sars-cov-2 sequences; however, the results were less consistent, 159 and coverage was more variable. as expected, total rna (measured by rnase p) was not 160 associated with sequencing coverage and varied considerably across samples, even though each 161 sample used the same amount of virus transport medium (vtm). the coverage level distribution is shown in figure 1c a 50-fold range of input rna; it was higher than rna seq, except in the terminal regions that were not 185 covered by pcr amplicons. in some cases, as little as five microliters of vtm from a single swab had 186 sufficient virus to obtain a full-length viral genome sequence at 1000x. this data is consistent with the 187 astonishingly high reported genome copy numbers of sars-cov-2 in some cases 188 and demonstrates the principal suitability of "testing by sequencing" as a diagnostic option for sarscov-2 and other rapidly evolving viruses. the average quality score per read is set to a minimal average phred score of 20 isolates, supported by multiple, independent junction-spanning reads (figure 3 a, b) . junctions were 271 mapped to single nucleotide resolution directly from individual reads. to confirm our deep-sequencing 272 results, we performed 3' utr site-specific amplification and sanger-based sequencing (figure 3 e g). the variant 3' end does not destroy overall folding but introduces a shorter stable hairpin ( figure 274 3 c, d). how this mutation affects viral fitness remains to be established. in sum, this study generated exhaustive snv information representing the introduction and 276 spread of sars-cov-2 across a suburban low-density area in the southern u.s. all samples were 277 from symptomatic cases and the majority of genomes clustered with variants that predominate the 278 outbreak in the u.s., rather than europe or china. this supports the notion that the majority of u.s. there seems to be partial overlap between the bulged stem-loop and the pseudoknot, suggesting that 330 these two structures are mutually exclusive and may serve as a switch to regulate the ratio of full gotaq promega master mix (#m712c), 2.5µl primers (0.5µm), and brought to volume with nuclease-551 free water. the pcr was performed with an initial denaturation step a 95°c for 2 minutes, the pcr 552 cycled at 95°c for 30 seconds, 56°c for 1 minute, and 73°c for 1 minute for 40 cycles, followed by a 553 final extension at 73°c for 10 minutes and a 4°c hold. the annealing temperature was derived from 554 the primer pair melting temperatures. primer pcr products were visualized on a 1.5% agarose gels 555 and pcr bands were gel purified using the qiagen gel purification kit (qiagen inc.). purified dna 556 was eluted in 30µl nuclease-free water and pcr products were cloned using topo-ta for snv were called using the clc bio algorithm (qiagen inc.) for human genome snv calling. the threshold for reporting was set at >90% frequency and a minimum coverage of 10-fold with 574 balanced forward and reverse reads for all snv. targeted regions were determined via thermo sars-cov-2 designed bed file and 576 sequences with 1x coverage across more than 99% of the 237 sars-cov-2 amplicons were 577 considered complete sequences. any sequences with 1x coverage between 5% and 99% were 578 considered partial genomes. partial genomes are included in the variant calling analysis but were not 579 submitted to genbank or gisaid. all consensus sequences derived from this study were manually curated to revert poly all positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, 598 missing data, and ambiguous bases were allowed at any position (partial deletion option). there were coronavirus susceptibility to the antiviral remdesivir (gs-5734) is 617 mediated by the viral polymerase and the proofreading exoribonuclease the proximal origin of 619 sars-cov-2 presymptomatic sars-cov-2 infections and transmission in 622 a skilled nursing facility sars-cov-2 phylogenetic analysis sars-cov-2 viral spike g614 mutation exhibits higher case 627 fatality rate covid-19 in critically ill patients in the seattle 630 region -case series amino acid variation analysis of surface spike glycoprotein at 614 in sars-cov-2 strains genomic variance of the 2019-ncov coronavirus epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in 638 wuhan, china: a descriptive study molecular evolution of the sars coronavirus during the course of the 640 sars epidemic in china detection of 2019 novel coronavirus (2019-ncov) by real-643 time rt-pcr the species 645 severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-646 cov-2 could the d614 g substitution in the 648 sars-cov-2 spike (s) protein be associated with higher covid-19 mortality? coast-to-coast spread of sars-cov-2 during the early 652 epidemic in the united states phylogenetic network analysis of sars-654 cov-2 genomes genomic epidemiology of hcov-19 characterization of the rna 658 components of a putative molecular switch in the 3' untranslated region of the murine coronavirus 659 genome a live, 661 impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of 662 lethal disease evaluation of a 664 recombination-resistant coronavirus as a broadly applicable clinical characteristics of coronavirus disease 2019 in china nextstrain: real-time tracking of pathogen evolution temporal dynamics in viral shedding and transmissibility of covid-19 sars-cov-2 transmission from 677 presymptomatic meeting attendee faster quantitative real-time pcr protocols may 679 lose sensitivity and show increased variability sars-cov-2 cell entry depends on ace2 and tmprss2 682 and is blocked by a clinically proven protease inhibitor evolutionary and structural analyses of sars-685 cov-2 d614g spike protein mutation now documented worldwide accurate sampling and 687 deep sequencing of the hiv-1 protease gene using a primer id phylogenetic analysis 690 and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and 691 proteolytically sensitive activation loop mafft multiple sequence alignment software version 7: 693 improvements in performance and usability infection and rapid transmission of sars-cov-2 in ferrets tracking changes in sars-cov-2 spike: evidence that 699 d614g increases infectivity of the covid-19 virus cov-2 variants collected in russia during the covid-19 outbreak mega x: molecular evolutionary 704 genetics analysis across computing platforms functional assessment of cell entry and receptor usage 706 for sars-cov-2 and other lineage b betacoronaviruses early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia. 709 the 711 impact of mutations in sars-cov-2 spike on viral infectivity and antigenicity efficiency clustering for low-density 714 microarrays and its application to qpcr antibody responses to sars-cov-2 in patients with covid-19 genomic epidemiology of sars-cov-2 in guangdong province genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins 723 and receptor binding genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins 726 and receptor binding us cdc real-time reverse transcription pcr panel for detection of severe 729 acute respiratory syndrome coronavirus 2 standardization of sequencing coverage depth in ngs: recommendation for 732 detection of clonal and subclonal mutations in cancer diagnostics the neighbor-joining method: a new method for reconstructing 734 phylogenetic trees burden of respiratory viral infection in persons with 737 human immunodeficiency virus. influenza other respir viruses structural basis of receptor recognition by sars-cov-2 gisaid: global initiative on sharing all influenza data -from vision to 743 reality whole genome and phylogenetic analysis of two sars-746 additional clues on multiple introductions 747 and further circulation in europe prospects for inferring very large phylogenies by using the 749 neighbor-joining method coronavirus disease 2019 in children -united states rapid reconstruction of sars-cov-2 using a synthetic 754 genomics platform aerosol and surface stability of sars-cov-757 2 as compared with sars-cov-1 emergence of genomic diversity and recurrent mutations an outbreak of severe kawasaki cov-2 epidemic: an observational cohort study antigenicity of the sars-cov-2 spike glycoprotein receptor recognition by the novel 769 coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus receptor recognition by the novel 772 coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus a phylogenetically conserved hairpin-type 3' 775 untranslated region pseudoknot functions in coronavirus rna replication virological assessment of hospitalized patients with covid-778 2019 cryo-em structure of the 2019-ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china factors 786 associated with prolonged viral rna shedding in patients with covid-19 characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral 790 shedding genetic cluster analysis of sars-cov-2 and the 792 identification of those responsible for the major outbreaks in various countries quantitative detection and viral load analysis of sars-cov-2 in infected patients genomic characterization and phylogenetic analysis of sars-799 cov-2 in italy viral and 801 host factors related to the clinical outcome of covid-19 clinical 804 course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a 805 retrospective cohort study a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china sars-cov-2 viral load in upper respiratory specimens of infected patients genetic interactions between an 815 essential 3' cis-acting rna pseudoknot, replicase gene products, and the extreme 3' end of the mouse 816 coronavirus genome key: cord-254636-3lr008th authors: shishir, tushar ahmed; naser, iftekhar bin; faruque, shah m. title: in silico comparative genomics of sars-cov-2 to determine the source and diversity of the pathogen in bangladesh date: 2020-08-16 journal: biorxiv doi: 10.1101/2020.07.20.212563 sha: doc_id: 254636 cord_uid: 3lr008th the covid19 pandemic caused by sars-cov-2 virus has severely affected most countries of the world including bangladesh. we conducted comparative analysis of publicly available whole-genome sequences of 64 sars-cov-2 isolates in bangladesh and 371 isolates from another 27 countries to predict possible transmission routes of covid19 to bangladesh and genomic variations among the viruses. phylogenetic analysis indicated that the pathogen was imported in bangladesh from multiple countries. the viruses found in the southern district of chattogram were closely related to strains from saudi arabia whereas those in dhaka were similar to that of united kingdom and france. the 64 sars-cov-2 sequences from bangladesh belonged to three clusters. compared to the ancestral sars-cov-2 sequence reported from china, the isolates in bangladesh had a total of 180 mutations in the coding region of the genome, and 110 of these were missense. among these, 99 missense mutations (90%) were predicted to destabilize protein structures. remarkably, a mutation that leads to an i300f change in the nsp2 protein and a mutation leading to d614g change in the spike protein were prevalent in sars-cov-2 genomic sequences, and might have influenced the epidemiological properties of the virus in bangladesh. the pandemic of coronavirus disease referred to as covid-19 pandemic, which originated in wuhan, china in december 2019 is ongoing and has now spread to 213 countries and territories. as of july 2020, the pandemic has caused about 16 million cases and over half a million death. this novel virus of the coronaviridae family and betacoronavirus genus (1, 2) designated as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is the causative agent of covid-19. previously two other coronaviruses, namely sars-cov and mers-cov have demonstrated high pathogenicity and caused epidemic with mortality rate ~10% and ~34% respectively affecting more than 25 countries each time (3) (4) (5) . however, sars-cov-2 has proven to be highly infectious and caused pandemic spread to over 213 countries and territories. besides its devastating impact in north america and europe, the disease is now rapidly spreading in south america including brazil, and in south asian countries particularly india, pakistan and bangladesh (6) . the virus was first detected in bangladesh in march 2020 (7) . although infections remained low until the end of march it began to rise steeply in april. by the end of june, new cases in bangladesh grew to nearly 150,000 and the rate of detection of cases compared to the total number of samples tested increased to about 22% which was highest in asia (6) . sars-cov-2 is a positive-sense single-stranded rna virus with a genome size of nearly 30kb. the 5' end of the genome codes for a polyprotein which is further cleaved to viral nonstructural proteins whereas the 3' end encodes for structural proteins of the virus including surface glycoproteins spike (s), membrane protein (m), envelop protein (e) and nucleocapsid protein (n) (8) . like other rna viruses, sars-cov-2 is also inherently prone to mutations due to high recombination frequency resulting in genomic diversity (9) (10) (11) . due to the rapid evolution of the virus, development of vaccines and therapies may be challenging. to monitor the emergence of diversity, it is important to conduct comparative genomics of viruses isolated over time and in various geographical locations. comparative analysis of genome sequences of various isolates of sars-cov-2 would allow to identify and characterize the variable and conserved regions of the genome and this knowledge may be useful for developing effective vaccines, as well as in molecular epidemiological tracking. thousands of sars-cov-2 virus genomes have been sequenced and submitted in public databases for further study. this include 66 sars-cov-2 genomic sequences submitted from bangladesh in the global initiative on sharing all influenza data (gisaid) database, till 11th june 2020 (12) . we conducted comparative analysis of publicly available genome sequences of sars-cov-2 from 27 countries to predict the origin of viruses in bangladesh by studying a time-4 resolved phylogenetic relationship. later, we analyzed the variants present in different isolates of bangladesh to understand the pattern of mutations in relation to the ancestral wuhan strain, find unique mutations, and possible effect of these mutations on the stability of encoded proteins, and selection pressure on genes. a total of 435 whole genome sequences of sars-cov-2 including 64 sequences isolated in bangladesh (detail information in s1 table) , and that of 5 isolates of each month between january to may, 2020 isolated in 27 different countries with high frequency of infection were included in this analysis. source and number of sequence are presented in table 1 (detail information are provided in s2 table) . however, since only number of sequences were reported from different african countries, we included all sequences from the african countries and categorized collectively as african sequence (12) . reference sequence included in various analysis was the sequence of the ancestral strain from wuhan, china (nc_045512.2) (13). selected sequences were annotated by viral annotation pipeline and identification (vapid) tool (14) , and multiple sequence alignment was carried out using mafft algorithm (15) . maximum likelihood phylogenetic tree was constructed with iq-tree (16), the generated tree was reconstructed based on time-calibration by treetime (17) , and visualized on itol server (18) . for analysis of mutations, sequence were mapped with minimap2 (19) , and variants were detected using samtools (20) and snp-sites (21) . a haplotype network was generated based on mutations in genome using popart (22) . sequences were then put into different clades based on specific mutations proposed in gisaid (23) and further classified as d614g type (24, 25) . subsequently, another phylogenetic tree and haplotype network containing only sars-cov-2 sequences from bangladeshi was constructed and categorized using the same tools, and additionally one step further clustered with treecluster (26) . the direction of selection in sequences from bangladesh was calculated by the slac algorithm (27) in the datamonkey server (28) . finally, the effects of the mutations on protein stability were predicted using deepddg (29). a total of 435 genomic sequences of sars-cov-2 reported from various countries (table 1) which included 64 sequences from bangladesh and the sequence of the ancestral sars-cov-2 isolated in wuhan, china were analyzed in the time-resolved phylogenetic tree. sequences from bangladesh belonged to three different clusters of which one cluster carried 43 of the 64 sequences, and shared the same node with sequence from germany while they had a common ancestry with isolates from the united kingdom (fig 1) . the remaining two clusters of sars-cov-2 sequences contained 4 and 5 sequences respectively from bangladesh, and they shared the same node with sequence of sars-cov-2 reported from india, and also shared a common ancestry with isolates from saudi arabia. besides, 12 lone sequences that did not belong to any of these clusters were found to have similarity with sequences from europe including united kingdom, germany, france, italy, and russia. one of these sequences was closely related to sequence reported from the usa. subsequently, all sars-cov-2 sequences from representative countries were clustered based on some specific mutations sustained, into 7 different clades as mentioned by gisaid. in this analysis, the sequences from bangladesh were found to be distributed in all clades except v (figs 1 and 2) . classification. in order to understand the evolutionary relationship and possible transmission dynamics of sars-cov-2 in bangladesh at a higher resolution, another time-resolved phylogenetic tree carrying only sequences of the pathogen isolated in various regions of bangladesh was generated using the sequence of the first sars-cov-2 reported from yuhan, china as a reference. of the three clusters produced in this analysis, cluster-1 included mostly isolates from chattogram and one isolate from dhaka, cluster-2 included isolates from dhaka, narayanganj and chattogram districts, whereas cluster-3 included isolates from chattogram only. as mentioned above, the isolates from bangladesh were found to be distributed in all 7 gisaid clades based on specific mutations, except in clade v (fig 2) . most isolates of dhaka and narayanganj (47 of 52) belonged to the gr clade, whereas those of chattogram belonged to five different gsid clades (g, gh, gr, o, and s). the major international airport in bangladesh is situated in the capital city dhaka, whereas the major seaport is located in chattogram. based on the phylogenetic analysis, all isolates of dhaka were the descendant of sars-cov-2 found in european countries, more specifically france and the united kingdom. on the other hand, most isolates of chattogram were found closely related to saudi arabian isolates. moreover, considering the gsid clades, the presence of s clade was absent among dhaka whereas most isolates of chattogram was found to belong to the s clade. clearly these two genomic variants of sars-cov-2 were initially imported by travelers from different countries, and the two variants initially spread in the two areas. that the isolates of narayanganj and two isolates of dhaka are closely related, indicates that the sars-cov-2 strain imported initially through international traveler to dhaka later spread to narayanganj, which is a densely populated city with river ports and large business centres. the sars-cov-2 sequences were also categorized according to d614g type mutation (fig 1) . this particular subtype with a non-silent (aspartate to glycine) mutation at 614th position of the spike protein is presumed to have rapidly outcompeted other preexisting subtypes, including the ancestral strain. the d614g mutation generates an additional serine protease (elastase) cleavage site near the s1-s2 junction of the spike protein (25) . all but one sequence from dhaka and narayanganj were found to be of g type which carries glycine at position 614 whereas sequences of chattogram carry sequences of both types (fig 2) . in addition, the first sequence from bangladesh carried g614 type of surface glycoprotein, which indicate that this dominant variant was present since the first isolation of sars-cov-2 in bangladesh and the mutant virus might have been imported to the country from europe, and the presence of the mutation might have facilitated viral transmission. relationships among dna sequences within a population are often studied by constructing and visualizing a haplotype network. we constructed a haplotype network by the median joining algorithm and found that 338 of 434 sars-cov-2 sequences from representative countries were alike, therefore formed a large haplo group (fig 3a) . however, there were presences of a significant number of unique lineages too consisting of a single or multiple sars-cov-2 sequences (fig 3a) . this network demonstrated the closeness of the sequences and their pattern of mutation beyond the geographical boundary. several sars-cov-2 isolates appeared to have sustained certain common mutations along with certain unique mutations. although a large proportion of sequences from bangladesh belonged to the common cluster (fig 3a) , there was a significant number of unique nodes as well due to mutations overtime subsequent to being carried into bangladesh (fig 3a) . therefore analysis of the sequences from bangladesh provided further insight of their mutation patterns. the haplotype network revealed that viruses isolated in bangladesh had certain unique mutations in them, and as a result they belonged to different haplo groups and no significant cig group (fig. 3a) . most of the isolates sustained a significant number of mutations compared with each other. in addition it further confirms that most isolates from chattogram ( fig 3b) were not directly related to those isolated in dhaka or narayanganj. we detected the presence of 209 point mutations in 64 sars-cov-2 isolates from bangladesh when compared to the reference sequence from yuhan, china. in addition, 19 isolates were found to have lost significant portions of their genome, and as a result lost sequences for some non-structural proteins such as orf7 and orf8 while other deletions were upstream or downstream gene variants (s3 table) . among the point mutations, 29 mutations were in the non-coding region of the genome and 180 were in coding regions. ten of the 29 non-coding mutations were in upstream non-coding region and rest was in downstream non-coding region of the genome. seventy mutations in the coding region were synonymous and 110 mutations predicted substituted amino acids. among twelve predicted orfs, orf1ab which comprises approximately 67% of the genome encoding 16 nonstructural proteins had more than 60 percent of the total mutations while gene e encoding envelope protein and orf7b were conserved and did not carry any mutation. though orf1 harbored the highest number of mutations, mutation density was highest in orf10 considering orf lengths. details and distribution of the mutations are presented in table 2 and full analysis report is placed in s3 table. in sequences from bangladesh, 241c>t and 3037c>t changes were the two most abundant mutations found in 58 out of 64 isolates, and often found simultaneously (table 3) . position 241 is located in the non-coding region whereas the mutation in position 3037 was synonymous. on the other hand, 57 sequences were found to harbor 14408c>t and 23403a>g mutations which altered amino acid pro>leu and asp>gly respectively, and these two mutations were found to be present simultaneously as well. in addition, other cofig 4) . orf6 was predicted to have dn/ds value of 17.8 due to the presence of higher number of missense than synonymous mutations. this finding indicates that orf6 is rapidly evolving and is highly divergent. the orf6 protein is an accessory protein whose function is yet to be fully elucidated (30) . the orf7a and nucleocapsid phosphoprotein had dn/ds values 1.08 and 1.55 respectively which confer their strong evolution to cope up with challenges under positive selection pressure. orf10 is predicted to be conversed with dn/ds value 0 while envelope protein and orf7b did not harbor any mutation and was conserved. on the other hand, orf3a and surface glycoprotein might approach toward positive selection pressure and evolve but rests of the proteins were under negative selection pressure. table 4) . none of the mutations in structural proteins were predicted to increase stability. all mutations were synonymous and found this gene conserved in summary, mutation analysis revealed point mutations as well as deletion of base pairs. deletions of the base pairs were associated with missing non-structural proteins and predictably affected certain viral properties since orf7a protein is the growth factor of the coronavirus family, induce apoptosis, and promotes viral encapsulation (31) (32) (33) while orf8 is associated with viral adaptation by playing role in host-virus interaction (34, 35) . furthermore, we have found that some genes are under positive selection pressure indicating that the virus is fast-evolving presumably to evade host cell's innate immunity; which should be taken into special consideration prior to vaccine development or other treatment strategies. finally, a missense mutation at 1163a>t changing the amino acid isoleucine to phenylalanine in nsp2 protein was found uniquely among 44 isolates in bangladesh. nsp2 is a methyltransferase like domain that interacts with phb and phb2, and modulates the host cell survival strategy by affecting cellular differentiation, mitochondrial biogenesis, and cell cycle progression to escape from innate immunity (35, 36) . this unique and high-frequency mutation might be a further interest of study, considering death rate against the infection rate in bangladesh. virus taxonomy: the database of the international committee on taxonomy of viruses (ictv) epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china sars and mers: recent insights into emerging coronaviruses sars and other coronaviruses as causes of pneumonia webmeter. coronavirus age, sex, demographics (covid-19) -worldometer. 2020. available from: www.worldometers.info covid-19 outbreak situations in bangladesh: an empirical analysis origin and evolution of pathogenic coronaviruses coronaviruses: an rna proofreading machine regulates replication fidelity and diversity two 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marrow stromal antigen 2 virion tethering through a novel mechanism of glycosylation interference the orf8 protein of sars-cov-2 mediates immune evasion through potently downregulating mhc-i. biorxiv the proteins of severe acute respiratory syndrome coronavirus-2 (sars cov-2 or n-cov19), the cause of covid-19 covid-2019: the role of the nsp2 and nsp3 in its pathogenesis key: cord-258914-g6pv8zz9 authors: proud, pamela c.; tsitoura, daphne; watson, robert j.; chua, brendon y; aram, marilyn j.; bewley, kevin r.; cavell, breeze e.; cobb, rebecca; dowall, stuart; fotheringham, susan a.; ho, catherine m. k.; lucas, vanessa; ngabo, didier; rayner, emma; ryan, kathryn a.; slack, gillian s.; thomas, stephen; wand, nadina i.; yeates, paul; demaison, christophe; jackson, david c.; bartlett, nathan w.; mercuri, francesca; carroll, miles w. title: prophylactic intranasal administration of a tlr2 agonist reduces upper respiratory tract viral shedding in a sars-cov-2 challenge ferret model date: 2020-09-25 journal: biorxiv doi: 10.1101/2020.09.25.309914 sha: doc_id: 258914 cord_uid: g6pv8zz9 respiratory viruses such as coronaviruses represent major ongoing global threats, causing epidemics and pandemics with huge economic burden. rapid spread of virus through populations poses an enormous challenge for outbreak control. like all respiratory viruses, the most recent novel human coronavirus sars-cov-2, initiates infection in the upper respiratory tract (urt). infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. a therapy that restricts initial replication in the urt has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. we show that prophylactic intra-nasal administration of the tlr2/6 agonist inna-051 in a sars-cov-2 ferret infection model effectively reduces levels of viral rna in the nose and throat. the results of our study support clinical development of a therapy based on prophylactic tlr2/6 innate immune activation in the urt to reduce sars-cov-2 transmission and provide protection against covid-19. as with other respiratory covs, sars-cov-2 primarily spreads via the airborne route, 86 with respiratory droplets expelled by infected individuals 6 . virus can be transmitted 87 from symptomatic, as well as pre-or asymptomatic individuals 7,8 , with asymptomatic 88 individuals being able to shed virus, and therefore being capable to transmit the disease, 89 for longer than those with symptoms 9 . as with other respiratory viruses such as influenza, 90 recent evidence suggests that, the epithelium of the upper respiratory tract (urt) is the 91 initial site of sars-cov-2 infection 10, 11 . this is consistent with the abundant nasal 92 epithelial cell expression of the sars-cov-2 receptor, angiotensin-converting enzyme 2 93 (ace2) and its decreasing expression throughout the lower respiratory tract 11 . 94 a topical treatment of the urt that boosts anti-viral immunity and restricts viral replication 95 is a promising method to promote viral clearance, reduce viral shedding and 96 transmission. the tlrs are key microbe-recognition receptors with a crucial role in 97 activation of host defence and protection from infections and therefore attractive drug 98 targets against infectious diseases [12] [13] [14] to determine whether tlr2/6 agonists are also active against sars-cov-2, we used in life samples were taken at days 1, 3, 5, 7, 10 and 12, with scheduled culls at days 137 3 (n=6) and end of study days 12-14 (n=18) (fig 1a) . comparison test, significant (>10 fold) reduction in nasal viral rna was observed at 5 186 dpc (p=0.0057) and highly significant (p<0.0001), greater than 10-fold reduction in 187 throat viral rna was apparent from 5 to 7 dpc following inna-051 i.n. treatment 188 ( figure s1 ). group 2 (20ug/ml) appears to be the most optimal dosing in this study to assess sars-cov-2 detected beyond the urt, lung tissue samples were 196 collected, on scheduled cull day 3 (6/24 animals) and days 12-14 (18/24 animals) dpc 197 and analysed for viral rna levels. on day 3 dpc, two culled ferrets from the control 198 vehicle group had detectable viral rna levels (7.42x10 4 and 2.86x10 4 copies/ml) (fig 199 2c ). there was one ferret in group 1 showing detectable, but below the quantifiable access to food and water was ad libitum and environmental enrichment was provided. ferrets were anaesthetised with ketamine/xylazine (17.9 mg/kg and 3.6 mg/kg 401 bodyweight) and exsanguination was effected via cardiac puncture, followed by 402 injection of an anaesthetic overdose (sodium pentabarbitone dolelethal, vetquinol uk 403 ltd, 140 mg/kg). a necropsy was performed immediately after confirmation of death. the left lung was dissected and used for subsequent virology procedures. x 10 6 pfu/ml sars-cov-2. nasal wash and throat swabs were collected at days 1, 3, 586 5, 7, 10 & 12 post challenge (p.c.) for all 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that provides rapid immune-system-mediated protection and subsequent long-term adaptive generation 523 of adaptive immune responses following influenza virus challenge is not 524 compromised by pre-treatment with the tlr-2 agonist pam2cys dose-dependent response to infection with sars-cov-2 in 527 the ferret model: evidence of protection to re-challenge. biorxiv ferret models of viral pathogenesis mapping influenza transmission in the ferret model 532 to transmission in humans receptor recognition by 534 the novel coronavirus from wuhan: an analysis based on decade structural studies of sars coronavirus pathology of experimental sars coronavirus 538 infection in cats and ferrets susceptibility of ferrets, cats, dogs, and other domesticated animals 541 to sars-coronavirus 2 are most highly expressed in nasal goblet and ciliated cells within human 545 airways the role of tlr2 in 547 tlr-dependent human mucosal epithelial cell 550 responses to microbial pathogens the current state of h5n1 vaccines and the use of 553 the ferret model for influenza therapeutic and prophylactic development antiviral efficacies of fda-approved drugs against sars-557 sars-cov-2 vaccines: should we focus on mucosal immunity? expert opinion 560 on biological therapy chadox1 ncov-19 vaccination prevents sars-cov-562 2 pneumonia in rhesus macaques. biorxiv tlr1-and tlr6-independent recognition of 565 bacterial lipopeptides transloading of tumor antigen-derived peptides into antigen-568 presenting cells the synthetic bacterial lipopeptide pam3csk4 modulates 571 respiratory syncytial virus infection independent of tlr activation isolation and rapid sharing of the 2019 novel coronavirus cov-2) from the first patient diagnosed with covid-19 in australia. the medical 575 journal of australia metagenomic nanopore sequencing of influenza virus 577 direct from clinical respiratory samples viral rna was quantified by rt-qpcr. 600 (a) nasal wash (b) throat swab (c) lung tissue. geometric mean +/-standard 601 deviation are displayed on the graphs. dashed horizontal lines denote the lower limit 602 of quantification (lloq) and lower limit of detection (llod) dunnett's multiple comparisons test are displayed above the error bars (*) key: cord-024133-zv0ysi8m authors: saxena, shailendra k.; kumar, swatantra; maurya, vimal k.; sharma, raman; dandu, himanshu r.; bhatt, madan l. b. title: current insight into the novel coronavirus disease 2019 (covid-19) date: 2020-04-30 journal: coronavirus disease 2019 (covid-19) doi: 10.1007/978-981-15-4814-7_1 sha: doc_id: 24133 cord_uid: zv0ysi8m sars-cov-2 is a novel strain of coronavirus that has not been previously identified in humans. it has been declared a pandemic and has infected at least 1,844,683 individuals and caused 117,021 deaths as of 14th april 2020. transmission among humans occurs via close contact with an infected individual that produces respiratory droplets. patients have been shown to undergo acute respiratory distress syndrome, which is defined as cytokine storm. the diagnosis relies on detection of nucleic acid, igg/igm antibodies, and a chest radiograph of the suspected individuals. the genome of sars-cov-2 is similar to other coronaviruses that comprise of ten open reading frames (orfs). sars-cov-2 spike protein exhibits higher affinity to ace2 receptor as compared with sars-cov. repurposing drugs like favipiravir, remdesivir, chloroquine, and tmprss2 protease inhibitors have been shown to be effective for the treatment of covid-19. personal protective measures should be followed to prevent sars-cov-2 infection. in addition, a clinical trial of sars-cov-2 vaccine, mrna-1273, has been started. this chapter provides a glimpse of advancements made in the area of sars-cov-2 infection by proving recent clinical and research trials in the field. on 11 march 2020, the world health organization (who) declared severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as a pandemic that causes novel coronavirus disease 2019 (covid-19) (world health organization 2020a). by 14 april 2020, around 1,844,683 confirmed cases with 117,021 deaths were reported from at least 213 countries, areas, or territories (world health organization 2020b). sars-cov-2 is a novel strain of coronavirus that has not been previously identified in humans. phylogenetic analysis suggests that sars-cov-2 might have emerged from the zoonotic cycle and rapidly spread by human to human transmission (chan et al. 2020a ). however, the exact source of sars-cov-2 has not been identified yet. transmission among humans occurs via close contact with an infected individual that produces respiratory droplets while coughing or sneezing within a range of about 6 ft (ghinai et al. 2020) . infected individuals have been reported with common clinical symptoms involving fever, nonproductive cough, myalgia, shortness of breath, as well as normal or decreased leukocyte counts ( fig. 1.1) ). in addition, severe cases of infection cause pneumonia, severe acute respiratory syndrome, kidney failure, and death xiong et al. 2020) . even with the implementation of strong travel restrictions, a large number of individuals exposed to sars-cov-2 have been traveling internationally without being detected, leading to spread of the virus worldwide (chinazzi et al. 2020) . however, extensive measures have been implemented by outstanding public health action to reduce person-to-person transmission of sars-cov-2 ( fig. 1.2 ). in addition, the scientific fraternity worldwide has been continuously working on covid-19 from the beginning by publishing the genome and developing highly specific diagnostic tools for the detection of sars-cov-2 infection. 1.2 sars-cov-2 genome and pathogenesis sars-cov-2 is a single-stranded rna virus of~30 kb genome size, which belongs to the genus coronavirus and family coronaviridae. the genome of sars-cov-2 is similar to other coronaviruses that comprise of ten open reading frames (orfs). the first orfs (orf1a/b), about two-thirds of viral rna, are translated into two large polyproteins pp1a and pp1ab, which processed into non-structural proteins (nsp1-nsp16) (chan et al. 2020b ). the size of each sars-cov-2 virion is about 70-90 nm (kim et al. 2020) . the genome of sars-cov-2 encodes for four structural proteins similar to other coronaviruses. these proteins are s (spike), e (envelope), m (membrane), and n (nucleocapsid) protein which are required to make complete virus particle. s protein is responsible for the attachment and entry of sars-cov-2 to the host target cell receptor, probably angiotensin-converting enzyme 2 (ace2) mainly expressed on alveolar epithelial type ii (aecii) cells, including extrapulmonary tissues such as heart, kidney, endothelium, and intestine ). sars-cov-2 has been shown to exhibit novel glycosylation sites in the spike glycoprotein of 2019-ncov, suggesting that the virus may utilize different glycosylation sites to interact with its receptors (kumar et al. 2020) . studies have demonstrated that sars-cov-2 spike protein has higher affinity to the ace2 receptor as compared with sars (walls et al. 2020 ). upon entry into the host target cells, the viral antigens get presented via antigenpresenting cells (apcs) to virus-specific cytotoxic t lymphocytes (ctl). so far, studies have not been conducted that reveal the peptide presentation. however, ctl epitopes of sars-cov-2 have been predicted by several studies, which may be used for understanding the pathogenesis and development of peptide-based vaccines (kumar et al. 2020; walls et al. 2020) . studies have been conducted in sars-cov-2 infected patients showing the activation and reduction in cd4 + and cd8 + t cell counts (li et al. 2020a ). in addition, sars-cov-2 patients have been found to present with acute respiratory distress syndrome (ards) (zumla et al. 2020 ). ards is a cytokine storm syndrome (css) which is a lethal uncontrollable inflammatory response resulting from the release of large pro-inflammatory cytokines (il-1β, ifn-α, ifn-γ, il-12, il-6, il-18, tnf-α, il-33, tgfβ, etc.) and chemokines (ccl3, ccl2, cxcl8, ccl5, cxcl9, cxcl10, etc.) by immune cells (li et al. 2020a ). suspected patients get diagnosed for sars-cov-2 infection by collecting various specimens, including nasopharyngeal or oropharyngeal swabs, nasopharyngeal or oropharyngeal aspirates or washes, bronchoalveolar lavage, sputum, tracheal aspirates, and blood. specimens can be stored at 4 c for up to 72 h after sample collection and may be stored at à70 c for longer periods of time (centre for disease control and prevention 2020a). diagnosis tests such as nucleic acid test, elisa, ct scan, and blood cultures are being implemented for the detection of sars-cov-2 infection. commonly used nucleic acid tests are rt-qpcr and highthroughput sequencing, where rt-qpcr is the effective and straightforward method for detection of pathogenic viruses in respiratory secretions and blood. specific primers and probes against orf1ab and n gene regions have been recommended to use for the detection of sars-cov-2 (wang et al. 2020a ). in addition, immunological detection of igm and igg antibodies are being performed to diagnose the covid-19 patients (li et al. 2020b ). patients reporting respiratory discomfort were evaluated using ct scan ). there is no specific treatment available for sars-cov-2 and the current treatment relies on supportive care of the infected patients (centre for disease control and prevention 2020b). however, some evidences suggest the use of repurposing drugs as the current choice of therapy. remdesivir, a nucleoside analogue-based drug that is currently under clinical trial for treating ebola virus infection, has been shown to block sars-cov-2 infection in vitro (wang et al. 2020b ). in addition, favipiravir, a type of rna-dependent rna polymerase inhibitor that has been designed to treat influenza virus infection, has been found to exhibit antiviral activity against sars-cov-2 . use of chloroquine, especially hydroxychloroquine, has been found to be effective against sars-cov-2 in vitro, which interferes with the glycosylation of cellular receptors . apart from attachment inhibitors, tmprss2 protease inhibitors have also been found to block sars-cov-2 infection in lung cells (hoffmann et al. 2020 ). the current preventive strategies of sars-cov-2 infection relies on personal protective measures such as covering of nose/mouth when coughing or sneezing, use of ffp3 or n95 mask, use of tissues to contain respiratory secretions and dispose of these in nearest waste receptacle, and hand hygiene after contact with contaminated objects/materials or respiratory secretion ( fig. 1. 3) (centre for disease control and prevention 2020c). healthcare professionals are at the highest risk of getting sars-cov-2 infection from infected patients and therefore extreme precaution needs to be taken while handling covid-19 patients. international travelers presenting any symptoms of sars-cov-2 should be isolated and quarantined to prevent further infections (gostic et al. 2020) . apart from these personal protective measures, development of effective vaccine is the ultimate way of controlling sars-cov-2 infection. using bioinformatics approaches, novel cytotoxic t lymphocyte (ctl) generated from spike glycoproteins may be used to develop effective vaccine for sars-cov-2. the first sars-cov-2 vaccine, namely, mrna-1273, is under clinical trial and involves 45 volunteers who have received two intramuscular injections at an interval of 28 days (u.s. national library of medicine 2020). sars-cov-2 has been declared a pandemic that causes covid-19. infected individuals have been reported with common flu-like symptoms and cytokine storm syndrome in severe cases. diagnosis of covid-19 relies on the detection of nucleic acid tests by rt-qpcr. current treatment relies on the symptomatic relief of the patients. however, several repurposing drugs like favipiravir, remdesivir, chloroquine, and tmprss2 protease inhibitors have been shown to be effective. personal protective measures should be followed to prevent sars-cov-2 infection. in addition, a clinical trial of sars-cov-2 vaccine, mrna-1273, has been started. fig. 1.3 steps needed to be taken by covid-19 patients in order to prevent the spread of sars-cov-2 infection the emergence of any infectious viral disease is difficult to anticipate, even though use of spatial epidemiology and mathematical modeling may predict the occurrence of emerging or re-emerging diseases like covid-19. rna recombination, mutation, and reassortment as well as other factors including globalization, expanding human population, deforestation, and altered ecosystems are the most convergent forces for the emergence of viral infectious diseases. sars-cov-2 infection is of global public health and economic importance and therefore needs collective government and societal response. considering the escalating number of cases worldwide, the who has declared sars-cov-2 as a pandemic. the global shutdown of trade and travel may result in a reduction in the transmission rate of sars-cov-2. personal protective measures should be implemented to reduce the risk of sars-cov-2. in order to prevent the spread of sars-cov-2 infection, several major steps should be taken, for instance strengthening surveillance and conducting awareness programs. in addition, extensive applied research should be funded and executed to understand the molecular mechanism and to develop effective prevention and control strategies for covid-19. interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (covid-19). centre for disease control and prevention centre for disease control and prevention (2020b) interim clinical guidance for management of patients with confirmed coronavirus disease (covid-19). centre for disease control and prevention centre for disease control and prevention (2020c) interim infection prevention and control recommendations for patients with suspected or confirmed coronavirus disease 2019 (covid-19) in healthcare settings. centre for disease control and prevention a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak. science. pii: eaba9757 discovering drugs to treat coronavirus disease 2019 (covid-19) illinois covid-19 investigation team (2020) first known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the usa estimated effectiveness of symptom and risk screening to prevent the spread of covid-19 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor identification of coronavirus isolated from a patient in korea with covid-19 structural, glycosylation and antigenic variation between 2019 novel coronavirus (2019-ncov) and sars coronavirus (sars-cov) molecular immune pathogenesis and diagnosis of covid-19 ye f (2020b) development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis safety and immunogenicity study of 2019-ncov vaccine (mrna-1273) to prevent sars-cov-2 infection veesler d (2020) structure, function, and antigenicity of the sars-cov-2 spike glycoprotein combination of rt-qpcr testing and clinical features for diagnosis of covid-19 facilitates management of sars-cov-2 outbreak remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro world health organization (2020a) coronavirus disease 2019 (covid-19) situation report-51. world health organization world health organization (2020b) coronavirus disease 2019 (covid-19) situation report-85. world health organization clinical and high-resolution ct features of the covid-19 infection: comparison of the initial and follow-up changes structural basis for the recognition of the sars-cov-2 by full-length human ace2. science. pii: eabb2762 in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) clinical characteristics of 140 patients infected with sars-cov-2 in wuhan a comparative study on the clinical features of covid-19 pneumonia to other pneumonias ct features of coronavirus disease 2019 (covid-19) pneumonia in 62 patients in wuhan, china reducing mortality from 2019-ncov: host-directed therapies should be an option acknowledgments we are grateful to the vice chancellor, king george's medical university (kgmu), lucknow, india, for the encouragement of this work. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. key: cord-268476-3lxsh1zz authors: skoog, hunter; withrow, kirk; jeyarajan, harishanker; greene, benjamin; batra, hitesh; cox, daniel; pierce, albert; grayson, jessica w.; carroll, william r. title: tracheotomy in the sars‐cov‐2 pandemic date: 2020-04-29 journal: head neck doi: 10.1002/hed.26214 sha: doc_id: 268476 cord_uid: 3lxsh1zz the severe acute respiratory syndrome (sars)‐cov‐2 pandemic continues to produce a large number of patients with chronic respiratory failure and ventilator dependence. as such, surgeons will be called upon to perform tracheotomy for a subset of these chronically intubated patients. as seen during the sars and the sars‐cov‐2 outbreaks, aerosol‐generating procedures (agp) have been associated with higher rates of infection of medical personnel and potential acceleration of viral dissemination throughout the medical center. therefore, a thoughtful approach to tracheotomy (and other agps) is imperative and maintaining traditional management norms may be unsuitable or even potentially harmful. we sought to review the existing evidence informing best practices and then develop straightforward guidelines for tracheotomy during the sars‐cov‐2 pandemic. this communication is the product of those efforts and is based on national and international experience with the current sars‐cov‐2 pandemic and the sars epidemic of 2002/2003. the severe acute respiratory syndrome (sars)-cov-2 pandemic continues to produce a large number of patients with chronic respiratory failure and ventilator dependence. surgeons will be called upon to perform tracheotomy for a subset of these chronically intubated patients. as seen during the sars and the sars-cov-2 outbreaks, aerosol-generating procedures (agp) have been associated with higher rates of infection of medical personnel and potential acceleration of viral dissemination throughout the medical center. therefore, a thoughtful approach to tracheotomy (and other agps) is imperative and maintaining traditional management norms may be unsuitable or even potentially harmful. in this backdrop, we sought to review the existing evidence informing best practices and then develop straightforward guidelines for tracheotomy during the sars-cov-2 pandemic. this communication is the product of those efforts and is based on national and international experience with the current sars-cov-2 pandemic and the sars epidemic of 2002/2003. our priorities in establishing these guidelines included: optimal patient care, protection of medical personnel, minimizing further spread of the virus and preservation of important resources (icu beds, ventilators, and ppe). these recommendations represent a consensus of stakeholders from our medical center including otolaryngologists, trauma surgeons, interventional pulmonologists, anesthesiologists, and critical care providers. due to the paucity of data regarding the current sars-cov-2 epidemic, the literature from the sars epidemic of in canada, 43% of cases occurred in health care workers as a result of agps. 1,2 one instance involved a difficult intubation of a patient who was under investigation for sars. this intubation was linked to the infection of seven hospital staff all of whom had been wearing full ppe including n95 masks during patient contact. 2 there are multiple reports 3,4 of safely performing tracheotomy on patients with sars without infecting health care workers. standard processes included strict infection control measures, seamless and regulated surgical intervention, and if possible delayed (>30 days from diagnosis) tracheotomy in patients who had been sars positive. infection control measures (in addition to standard airborne and contact precautions) taken during these procedures included: double gowning, papr suits, double gloving, a changing room after the procedure (ante room), performing the procedure in a negative pressure room. the surgical personnel were the most experienced available to minimize operating/exposure time. patients were completely paralyzed to minimize air movement and coughing and thus viral dissemination via aerosolization. prior to the procedure, a trial run was completed to ensure maximum efficiency of the procedure. just prior to airway entry, the patients were pre-oxygenated, ventilation was held, and the cuff on the endotracheal tube was dropped to minimize air movement over the respiratory mucosa. while the patient was apneic, the tracheotomy incision was performed. open suctioning of the trachea was avoided. instead, a closed suctioning system with a viral filter was used. 3,4 during the current sars-cov-2 pandemic, reports from multiple countries suggest that otolaryngologists and support staff are at a unique risk of sars-cov-2 exposure. an early case in china with devastating results involved endoscopic pituitary surgery in a patient with flu-like symptoms. 5 contact tracing confirmed that 14 health care workers became infected with sars-cov-2 from this one procedure. in wuhan, otolaryngologists and ophthalmologists were among the most commonly infected health care workers. n95 masks did not appear to completely prevent infection of health care workers. papr suits were more effective. 5 in iran, over 20 otolaryngologists have been hospitalized after testing positive for sars-cov-2 with at least two deaths, including a chief resident. 5 two ent physicians required ventilator support in the united kingdom, with at least one ultimately succumbing to the infection. to date, over 60 physicians have died in italy as a result of sars-cov-2 infection. 6 in spain, over 12 000 health care workers have tested positive for the disease (14.4% of total reported cases). 7 in the united states, ohio and minnesota have reported 16%-28% of their covid positive cases involve health care workers. 7 the american academy of otolaryngology: head and neck surgery has recommended a multidisciplinary approach in determining indications for tracheotomy in patients with sars-cov-2. in addition to the necessity of donning appropriate ppe, they recommend not performing tracheotomy until 2-3 weeks postintubation and repeat sars-cov-2 testing is negative. the procedures should be performed under a closed circuit with a minimal number of care providers and duration of procedure. 8 emergent tracheostomy secondary to upper airway distortion or obstruction precluding endotracheal intubation has not been reported in patients with sars-cov-2. early experience suggests that patients with sars-cov-2 produce relatively little mucus and secretions in relation to other causes of respiratory failure. for these reasons, tracheotomy appears less critical for pulmonary toilet for patients with sars-cov-2. 9 5 | recommendations for tracheotomy during sars-cov-2 pandemic as the number of infected patients requiring intubation and ventilator support climbs, a growing population of patients would normally qualify for tracheotomy due to failed extubation or prolonged ventilatory needs. considering the clear evidence from both sars and sars-cov-2 infection, 9 it is imperative that this patient population be managed appropriately to minimize infectious risk to the health care team. currently, there are no published reports on tracheotomy in patients with covid-19. we felt there were numerous reasons to develop consensus guidelines including: • an anticipated high volume in our center (third largest public hospital in the united states). • known risk to intraoperative and postoperative medical staff. • anticipated shortages of resources (icu beds, ventilators, ppe) which could be impacted by decisions around tracheotomy. • high potential for conflict between surgeons and critical care staff over an emotionally charged issue. to address these concerns, we assembled a task force of stakeholders in our medical center involved with tracheotomy including otolaryngology, trauma surgery, critical care medicine, and anesthesiology. the group reviewed existing literature and met virtually to draft recommendations. ideas were refined with final recommendations approved by all task force members as summarized below. the availability of highly predictive testing for sars-cov-2 has greatly simplified the decision process in our medical center; improving the safety of medical staff, preserving resources like ppe and freeing up resources like ventilators and icu space. severe respiratory failure should be exceedingly rare (figure 1 ). in patients with prolonged intubation, elective tracheotomy can be delayed well beyond the usual 14-day timeline. despite concern for airway stenosis due to prolonged intubation, recent data suggests early tracheotomy may be less crucial and the safety issues around the pandemic outweigh the risks of late airway stenosis. 10 the sars-cov-2 pandemic is rapidly evolving with continuous updates in treatment and guidelines. at the time of writing, there were over 492 000 cases in the united states with almost 19 000 deaths. 12 the management algorithm recommended by our multidisciplinary team draws on experiences with the 2002-2003 sars epidemic and the current experience of surgeons, anesthesiologists, intensivists, and other health care workers with sars-cov-2. the goals of these recommendations are to provide optimal patient care, protection of medical personnel, minimizing further spread of the virus, and preservation of important resources (icu beds, ventilators, and ppe). the chronology of the 2002-2003 sars mini pandemic cluster of severe acute respiratory syndrome cases among protected health care workers-toronto tracheostomy in a patient with severe acute respiratory syndrome safe tracheostomy for patients with severe acute respiratory syndrome precautions for endoscopic transnasal skull base surgery during the covid-19 pandemic more than 60 doctors in italy have died in covid-19 pandemic how many medical workers have contracted covid-19? aao position statement: tracheotomy recommendations during the covid-19 pandemic covid-19 and the otolaryngologist-preliminary evidencebased review laryngotracheal stenosis in early vs late tracheostomy: a systematic review suspension laryngoscopy-assisted percutaneous dilatational tracheostomy in high-risk patients tracheotomy in the sars-cov-2 pandemic we thank michel thomas for his assistance in the graphic design of our flowchart.orcid hunter skoog https://orcid.org/0000-0001-6510-9064 key: cord-032751-pmclolvh authors: head, katharine j.; kasting, monica l.; sturm, lynne a.; hartsock, jane a.; zimet, gregory d. title: a national survey assessing sars-cov-2 vaccination intentions: implications for future public health communication efforts date: 2020-09-23 journal: sci commun doi: 10.1177/1075547020960463 sha: doc_id: 32751 cord_uid: pmclolvh with sars-cov-2 vaccines under development, research is needed to assess intention to vaccinate. we conducted a survey (n = 3,159) with u.s. adults in may 2020 assessing sars-cov-2 vaccine intentions, intentions with a provider recommendation, and sociodemographic and psychosocial variables. participants had high sars-cov-2 vaccine intentions (m = 5.23/7-point scale), which increased significantly with a provider recommendation (m = 5.47). hierarchical linear regression showed that less education and working in health care were associated with lower intent, and liberal political views, altruism, and covid-19-related health beliefs were associated with higher intent. this work can inform interventions to increase vaccine uptake, ultimately reducing covid-19-related morbidity and mortality. the covid-19 (coronavirus disease 2019) pandemic, caused by the sars-cov-2 (severe acute respiratory syndrome coronavirus 2) virus, emerged in late 2019 with u.s. cases presently at 5.9 million, and >180,000 attributable deaths (centers for disease control and prevention [cdc], 2020b) . with no available vaccine, public health agencies like the centers for disease control have advised the public on specific behaviors to limit transmission (e.g., "social distancing," wearing a face mask, etc.; cdc, 2020a). beyond individual behaviors, local and state governments across the country enacted various "stay-at-home" orders and closed nonessential businesses during parts of march, april, and may (lee et al., 2020) . despite these measures, covid-19 has caused a serious disease burden to the u.s. health care system. consensus among medical experts is that until a vaccine is available and we reach high-vaccine coverage, nonpharmaceutical interventions will only be able to curb the spread of the virus (corey et al., 2020) . several sars-cov-2 vaccines are in development and might be available by early 2021, though availability will depend on successful clinical trials demonstrating efficacy and safety (lurie et al., 2020) . public health and medical practitioners must prepare to promote acceptance of these vaccines. vaccine hesitancy, which describes a range of stances toward vaccination, from deep skepticism about vaccine efficacy and safety to more mild concerns, has been identified by the world health organization as a major global health threat and is particularly prevalent in the united states (macdonald, 2015; quinn et al., 2019; world health organization, 2019) . because scholars have argued that vaccine hesitancy is driven by contextspecific factors including time and place as well as individual factors such as beliefs about threat of disease (brewer et al., 2007; dubé et al., 2015; larson et al., 2014) , it is important to understand perceptions related to sars-cov-2 vaccination and to assess what factors may contribute to higher or lower intentions to vaccinate. previous research with other vaccine-preventable diseases show that there are identifiable factors that may influence vaccination intentions and acceptance. for example, certain sociodemographic factors have played a role in adult vaccination acceptance, such as socioeconomic status, age, race and ethnicity, and geographic location (abbas et al., 2018; almario et al., 2016; galarce et al., 2011) . since vaccination relies on the principle of "herd immunity," prosocial motives for behaviors that benefit others, such as general altruism, prosociality, and sympathy, can play a role in some vaccination decision making (li et al., 2016; vietri et al., 2012) . additionally, theoretical models like the health belief model have long recognized that variables like perceived severity and susceptibility to a disease may predict behavioral intentions, which in turn, predict behavior (brewer et al., 2007; bish et al., 2011; champion & skinner, 2008; gerend & shepherd, 2012; yang, 2015) . the extended parallel process model further posits that health promotion and message design must consider the balance between addressing issues of severity and susceptibility in a way that promotes message acceptance, rather than provoking too much or not enough fear and thus causing people to reject the message (prati et al., 2012; quick et al., 2018; vorpahl & yang, 2018; witte, 1992) . vaccine communication and promotion work has long relied on theoretical models like these not only for guiding formative work with target populations (cameron et al., 2009; chen et al., 2011) but also to develop and test behavioral interventions (gerend & shepherd, 2012; gore & bracken, 2005) . finally, research demonstrates that a provider recommendation remains an important predictor of vaccination behavior in the united states reiter et al., 2013) . more important, strong provider recommendations are needed to maximize the effect on patient vaccination decisions (gilkey et al., 2016; lu et al., 2018) . given the novel nature of covid-19, research is needed to assess the public's intentions to get the sars-cov-2 vaccine, when it becomes available, as well as what factors may be associated with higher or lower intent. to ensure high vaccination coverage, public health campaigns must be carefully designed based on evidence about target populations and may even need to employ targeted communication strategies based on sociodemographic and psychosocial variables dubé et al., 2015 , kriss et al., 2017 minor et al., 2010; stockwell et al., 2012) . otherwise, we risk disseminating counterproductive messaging that may reinforce hesitancy in those already hesitant (bloom et al., 2014) . therefore, a national survey of adults in the united states was used to address the following research questions: research question 1: what are the sars-cov-2 vaccine behavioral intentions of adults in the u.s.? research question 2: what are the sars-cov-2 vaccine behavioral intentions of adults in the u.s. when a health care provider recommends the vaccine? research question 3: what factors are associated with sars-cov-2 vaccine behavioral intentions of adults in the united states? the data for this study come from a survey assessing knowledge, beliefs, and behaviors related to the covid-19 pandemic. data were collected between may 4 and may 11, 2020 through an online survey. participant recruitment was facilitated by dynata, a market research firm that maintains panels of 62 million volunteer survey respondents throughout 100 countries. panelists receive monetary incentives tailored to both the time and effort required for participation and regional preferences. email invitations were sent to members of dynata's u.s. panel who met eligibility criteria of being 18 years or older and able to read english. the study was approved by the university's institutional review board as exempt and not requiring written informed consent. a total of 4,042 participants opened the survey and 351 (8.6%) chose not to continue after reading the informed consent welcome page. we excluded anyone who did not answer the intention outcome measures for the current study. importantly, because vaccine intent and/or need may be different for people who were previously infected with sars-cov-2 and perceived threat variables (discussed below) are usually only measured for future threats, only participants who answered "no" to the question "do you believe that you've had covid-19" are included in the current study (n = 3,159). in addition to demographic information, the study team collected data on participants' vaccine behavioral intentions, sociocultural beliefs, experiences with covid-19, and health beliefs regarding personal risk and threat of covid-19. detailed information on variables measured at the categorical level as well as their response options can be found in table 1 ; variables measured at the continuous level are described below. vaccine behavioral intentions. two items, adapted from previous vaccine work, assessed participants' likelihood to receive a sars-cov-2 vaccine (gerend & shepherd, 2012) . based on pretesting of our survey instruments, it was determined that using the term "covid-19 vaccine" in the survey was more appropriate for lay audiences, since sars-cov-2 is less frequently used in lay communication. these two vaccine intent items included "how likely is it that you'll get a covid-19 vaccine, if it becomes available?" (individual intent) and "if your healthcare provider strongly recommended a covid-19 vaccine in the next year, how likely is it that you'd get vaccinated?" (provider rec intent). both items were assessed using a 7-point likert-type scale (1 = very unlikely to 7 = very likely). because these two items were highly correlated with high reliability (cronbach's α = .91), the two behavioral intention items were averaged into a single overall intent measure (overall vaccine intent). altruism. we assessed participants' altruism using an 18-item scale adapted from rushton et al. (1981) . participants responded to each item on a 5-point likert-type scale where 1 = never to 5 = very often. we conducted a principal components exploratory factor analysis, which extracted two factors. we labeled the first factor, which consisted of five items (cronbach's α = .83), high commitment altruism (i.e., behaviors that require a relatively high level of personal involvement; e.g., "i have helped push a stranger's car out of the snow or mud."). we labeled the second factor, which consisted of four items (cronbach's α = .81), low commitment altruism (i.e., behaviors that require a relatively low level of personal involvement; e.g., "i have given money to charity."). covid-related worry. a three item scale adapted from liau et al. (1998) and fan et al. (2018) was used to measure participants' personal worry about covid-19 ("i am scared about getting infected with covid-19," "the possibility of getting infected in the future with covid-19 concerns me," and "i don't really worry about getting infected with covid-19"). participants responded to each item on a 5-point likert-type scale where 1 = strongly disagree to 5 = strongly agree. the last item was reverse coded, and then the three items were summed and averaged to derive a single covid-related worry score (cronbach's α = .82). perceived severity of covid. a four-item scale adapted from cahyanto et al.'s (2016) work on ebola was used to measure participants' perceptions of the severity of covid-19 (e.g., "i am afraid that i may die if i contract covid-19."). participants responded to each item on a 5-point likert-type scale where 1 = strongly disagree to 5 = strongly agree. the items were summed and averaged to derive a single perceived severity of covid score (cronbach's α = .706). likelihood of infection. personal susceptibility was measured with a single item: "how likely do you believe it is that you will get infected with covid-19?" participants responded on a 5-point likert-type scale where 1 = not at all to 5 = extremely. threat to physical health. perceived threat to physical health was measured with a single item: "if you got infected with covid-19, how threatening would it be to your physical health?" participants responded on a 5-point likert-type scale where 1 = not at all to 5 = extremely. first, the sample was described using frequency distributions or means and standard deviations, as appropriate. we then examined our two vaccination intent variables (individual intent and provider rec intent) and examined if the participant changed their likelihood of receiving a sars-cov-2 vaccine when they were told a provider recommended it. we then examined bivariate associations between the overall vaccine intent score and each of the potential predictor variables using linear regression. any variable that was significant at p < .01 in bivariate linear regression was included in subsequent analyses. we used .01, rather than .05 as the cutoff because, with our large sample size, a cutoff level of .05 might identify trivial relationships. we then conducted a three-step hierarchical multiple linear regression analysis. in the first step, we included demographic characteristics, in the second step we added in health care characteristics, and in the third step we included health belief characteristics. this approach was used to determine if health beliefs influenced likelihood of receiving a sars-cov-2 vaccine, above and beyond demographic and health care characteristics. the final analytic sample included 3,159 participants who reported no previous covid-19 diagnosis. mean age was 46.9 years (sd = 16.8) and the majority of participants were female (n = 1,657; 52.8%) and non-hispanic white (n = 2,039; 65.1%). for a complete inventory of sample descriptive statistics, see table 1 . when asked how likely they were to get the sars-cov-2 vaccine, the mean score was 5.24 (sd = 2.0). this average intention increased to a mean score of 5.48 (sd = 1.93) when they were asked the likelihood of receiving the vaccine if their health care provider strongly recommended it. for a categorical breakdown of responses to each of the intent variables, see table 2 . the mean increase from individual intent to provider recommendation intent was significant, t = −12.343 (p < .0001). when examining change in intent from individual intent to intent due to provider recommendation, the majority of the sample (n = 2,144; 67.9%) did not change their response to the likelihood of receiving the vaccine. however, almost one quarter of the sample (n = 730; 23.1%) became more likely to receive the vaccine if a provider recommended it and a smaller percentage (n = 285; 9.0%) became less likely to receive the vaccine if a provider recommended it; see figure 1 . in bivariate analyses with overall intent score (individual intent and provider recommendation intent combined; m = 5.36, sd = 1.88), variables that had associations at p > .01 included region (p = .207), knowing someone who has had covid-19 (p = .028), and sex (p = .013). these variables were not included in subsequent analyses. see table 1 for all bivariate analyses. multivariable regression analyses can be found in table 3 . the first step of the hierarchical multiple regression including only demographic variables that had an adjusted r 2 value of .136. when personal health care variables were added in step 2, the adjusted r 2 value increased to .220. finally, in the third step of the hierarchical multiple regression, the adjusted r 2 increased to .318 when the health belief variables were included. in step 3 of the hierarchical regression model, with all variables included, less education was associated with lower intent to receive a sars-cov-2 vaccine. likewise, being currently employed in health care was also negatively associated with intent to receive a vaccine as compared with those who were never employed in the health care system (β = −0.36; 95% ci [−0.56, −0.15]). participants who self-identified as liberal reported the highest intent to receive a sars-cov-2 vaccine (β = 0.27; 95% ci [0.11, 0.43]), followed by moderates, and then conservatives. the health belief variables that were significant in the full regression model were all positively associated with intent to receive a sars-cov-2 vaccine. specifically, as low-commitment altruism increased, likelihood of receiving a sars-cov-2 vaccine increased (β = 0.19; 95% ci [0.11, 0.28]). furthermore, as perceived threat to physical health increased, likelihood of receiving a sars-cov-2 vaccine increased (β = 0.11; 95% ci [0.04, 0.18]). those who believed covid-19 was a major problem in their community had higher likelihood of receiving a sars-cov-2 vaccine compared with those who did not (β = 0.21; 95% ci [0.08, 0.35]). worry was most strongly associated with sars-cov-2 vaccine intent; as worry increased, intent likewise increased (β = 0.43; 95% ci [0.36, 0.51]). this article aimed to examine u.s. respondents' intentions to receive the sars-cov-2 vaccine when it becomes available, and investigate factors associated with those intentions. overall, participants in this study reported step 1: demographic variables step 2: including health care variables step 3 ref. ref. ref. ref. ref. ref. ref. ref. step 1: demographic variables step 2: including health care variables step 3 ref. ref. ref. ref. work in health care currently employed in health care ref. ref. ref. ref. household income (2019) less than $25,000 ref. ref. ref. ref. ref. ref. ref. ref. health care characteristics received a flu vaccine, 12 months yes ref. ref. ref. (continued) step 1: demographic variables step 2: including health care variables step 3 ref. step 1 r 2 = .143 (adjusted = .136); step 2 r 2 = .227 (adjusted = .220); step 3 ref. = reference group. boldface type indicates statistical significance (p<0.05). high intentions to receive a sars-cov-2 vaccine, which were even higher with a strong provider recommendation. several sociodemographic and health belief variables were also associated with higher and lower sars-cov-2 vaccine intentions. below, we discuss the implications of these findings and suggest areas for future work, including research and practical application. importantly, participants reported relatively high individual intent to receive a sars-cov-2 vaccine. on a 7-point scale, participants in this study reported an average of 5.23. while not quite a ceiling effect, we believe this suggests strong support for a vaccine, more so because no vaccine has been fully tested and made available to the public. our findings are consistent with other recent work examining perceptions of the sars-cov-2 vaccine, also showing highvaccine intentions in the united states (reiter et al., 2020; thigpen & funk, 2020) . interestingly, this level of intention to receive the sars-cov-2 vaccine is markedly higher than what is seen for actual u.s. adult vaccination behaviors for influenza. the cdc reports that 2018-2019 flu vaccination coverage among adults ≥18 years was only 45.3% (cdc, 2019). related, research shows that the relationship between intention and actual behavior, while usually significantly positive, is not always a perfect correlation and that different predictors (e.g., perceived susceptibility, doctor recommendation) may differently predict intentions versus actual behavior (juraskova et al., 2011; krawczyk et al., 2012; schwenk & möser, 2009; webb & sheeran, 2006) . therefore, while participants in this study expressed high sars-cov-2 vaccine intentions, these findings should be interpreted cautiously. actual uptake of a future vaccine will likely depend on many factors, including the status of the covid-19 pandemic at the time of vaccine debut. of note for communication scholars, these findings suggest that social normative messaging could capitalize on the high level of vaccine intention. social norms campaigns use descriptive norms (i.e., descriptive statistics) to correct or reinforce the frequency with which others are performing a behavior, with the assumption that individuals seek to conform to the pressures of societal norms (i.e., subjective norms; burchell et al., 2013) . while most social norms campaigns target audiences who may be overestimating the frequency of an unhealthy behavior (e.g., binge drinking; campo et al., 2004) , the same normative principles have been found to significantly predict hpv vaccination intentions and uptake among young women (de visser et al., 2011) . for example, social norms messages can address sars-cov-2 vaccine hesitancy by highlighting the high intentions to vaccinate expressed by the majority of people in one's social network. this approach will require communication scientists to engage in formative research to develop and test messages with different audiences, especially given the differences in intention across subgroups of population found in this study. participants in this study also were significantly more likely to receive the vaccine if their health care provider strongly recommended it. this finding is consistent with previous work showing a doctor's recommendation is a significant predictor of vaccination behavior (gorman et al., 2012; rahman et al., 2015; sturm et al., 2017) , including when newer vaccines, such as the 2009 h1n1 influenza vaccine, are being considered (coe et al., 2012) . a key limitation of this study is that the single-item measure only asked participants about intentions if their provider strongly recommended the vaccine; no information was gathered about what information they may want about the sars-cov-2 vaccine from their provider. providers are the most trusted source of health information for patients (jackson et al., 2019) , including information about vaccines (eller et al., 2019) , which may be important once a sars-cov-2 vaccine becomes widely available. vaccine promotion campaigns may need to emphasize the importance of talking with a health care provider about the vaccine, including asking for information to address any concerns or questions. at the same time, health care providers may need support and training such as that already offered through the cdc (cdc, 2016; cdc, 2018) to be most effective in recommending a sars-cov-2 vaccine. specific sociodemographic and health belief variables were associated with intentions to vaccinate, and are worthy of consideration for future work, especially for communication interventions seeking to promote a sars-cov-2 vaccine. demographics. participants with less education expressed a lower intention to receive a sars-cov-2 vaccine. education is often associated with health literacy (kutner et al., 2006; paasche-orlow et al., 2005) , suggesting the critical importance of educating the public on the role of vaccines in reducing covid-19 prevalence through herd immunity. these efforts may need to be done in conjunction with messages about how herd immunity works, as previous work has shown that limited understanding can undermine vaccination intentions and behavior (sobo, 2016) . the effective deployment of "flatten the curve"-a phrase previously not commonly used among lay audiences when discussing a disease outbreak-via social media is an example of effectively educating the public about complex health terms in accessible ways (boboltz, 2020) . interestingly, participants who were employed in health care indicated a lower vaccine intention. this was contrary to what was expected. previous work has shown that some health care providers express vaccine hesitancy and low-vaccine acceptance themselves (collange et al., 2016; verger et al., 2015) . additionally, our question only queried whether the individual worked in health care and did not distinguish positions entailing direct patient care or type of training. given that many health care-related positions are nonclinical (e.g., janitorial, receptionist), some participants who answered this question may have limited understanding about the role of vaccines in preventing infectious diseases. we believe further work is needed to clarify this finding. participants' self-reported political views were associated with vaccine intent, with liberals expressing the strongest sars-cov-2 vaccine intentions, followed by moderates, and then conservatives. the united states has a complex and often partisan political environment, which may be compounded by mass media news consumption and "echo chambers" within social media platforms (bakshy et al., 2015; iyengar & hahn, 2009 ). one group espousing significantly lower intentions than other groups represents a potential challenge for high vaccine community coverage; however, these media trends may also represent an arena for targeted messaging going forward. we make an especially strong call for future work on this issue and implore other health and science communication researchers and practitioners to devote particular attention to targeted work on political ideology as we inch closer to an available sars-cov-2 vaccine. finally, we found that as individuals' level of low commitment altruism increased, so too did their likelihood of receiving a sars-cov-2 vaccine. importantly, we all must remember that vaccines provide both a personal benefit and public health benefit. research on the relationship between concepts like altruism and vaccination is an area that has received increasing, but still inadequate, attention in the vaccine literature (korn et al., 2020; li et al., 2016; quadri-sheriff et al., 2012; vietri et al., 2012) . going forward, research examining individual's concern for the "other" as a potential motivating factor for sars-cov-2 vaccination, as well as a potential message design strategy, is an important focus. intentions. consistent with frameworks like the health belief model and the extended parallel process model, individuals who expressed fear-measured in this study as higher worry, perceived threat to physical health, and perceived covid-19 to be a major problem in their community-were more likely to intend to get the sars-cov-2 vaccine when it becomes available. the data for this study were collected in early may 2020, when many states in the united states were still in "lock down" mode and covid-19 rates and hospitalizations were high but steady. if covid-19 rates and hospitalizations are high when the vaccine debuts, these perceived threat variables may continue to be positively associated with intention. however, if infection rates drop or individuals become numb to the threat posed by the disease, these variables may not be as strongly associated with intentions. it will be important, therefore, to do both longitudinal and cross-sectional surveys over time to monitor changes in public attitudes and perceptions about covid-19 disease and a sars-cov-2 vaccine as well as examine the potential association of other social and behavioral determinants of health such as access and cost issues. in the meantime, communication scientists can capitalize on these findings by exploring messaging strategies that address individuals' fears about covid-19. a limitation of this study is that we used a national but not a population representative sample. participants were members of an opt-in panel and may not reflect all u.s. adults. furthermore, the cross-sectional survey design precludes determination of causal direction in the relationships identified and necessarily represents a snapshot in time, rather than the evolving landscape of the public's knowledge and attitudes about covid-19. as previously noted, intent can be an imperfect predictor of subsequent behavior. finally, two measurement limitations worth mentioning include a mismatch in the wording of our intention measures (i.e., the provider intention measure specified a timeline of "in the next year" while the individual intention item did not) and excluding participants who believed they had a previous sars-cov-2 infection from the health belief items (e.g., perceived severity, worry, likelihood of infection, threat). this study examined sars-cov-2 vaccine intentions and factors associated with these intentions. in addition to high intentions to receive the vaccine, provider recommendation increased intentions and will likely be an important factor in achieving the level of vaccination needed for herd immunity. several sociodemographic and health belief variables were associated with demographics, perceptions, and socioeconomic factors affecting influenza vaccination among adults in the united states persistent racial and ethnic 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perspectives effect of a text messaging intervention on influenza vaccination in an urban, low-income pediatric and adolescent population: a randomized controlled trial pediatrician-parent conversations about human papillomavirus vaccination: an analysis of audio recordings most americans expect a covid-19 vaccine within a year; 72% say they would get vaccinated vaccine hesitancy among general practitioners and its determinants during controversies: a national cross-sectional survey in france vaccinating to help ourselves and others who is to blame? framing hpv to influence vaccination intentions among college students does changing behavioral intentions engender behavior change? a meta-analysis of the experimental evidence putting the fear back into fear appeals: the extended parallel process model ten threats to global health in 2019 predicting young adults' intentions to get the h1n1 vaccine: an integrated model key: cord-275454-an8xvow3 authors: clark, andrew e; lee, francesca m title: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) screening with specimen pools: time to swim, or too deep for comfort? date: 2020-09-28 journal: clin infect dis doi: 10.1093/cid/ciaa1145 sha: doc_id: 275454 cord_uid: an8xvow3 nan the outbreak and global spread of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) continues to challenge the way physicians, laboratories, and public health officials diagnose and track cases. testing was initially relegated to reference laboratories and academic medical centers with the expertise to rapidly design and validate laboratorydeveloped diagnostic assays to detect a novel virus. these laboratories quickly became overburdened, resulting in turnaround times beyond what is clinically actionable. the availability of commercial sars-cov-2 diagnostics decentralized coronavirus disease 2019 (covid-19) testing algorithms, resulting in increased testing capacity on a national level. however, despite the herculean efforts of physicians, public health officials, and clinical laboratory personnel, significant challenges and case backlogs remain. as we learn more about covid-19 transmission, countries worldwide have tried to curtail disease spread via containment efforts, including quarantines, closure of municipal spaces, and compulsory social distancing. due to limited availability, testing initially focused on the identification of symptomatic individuals. as parts of the world report decreased caseloads, the focus has shifted to include screening of asymptomatic individuals in community settings in order to prevent future outbreaks. similar to routine diagnostics, screening tests need to be both analytically and clinically sensitive and easy to perform. screening tests also carry an inherent requirement to be reasonably inexpensive and able to identify large numbers of affected yet asymptomatic patients. the optimal specimen type, diagnostic technology, sampling format, and screening strategy for sars-cov-2 remain unknown; this uncertainty is compounded by unstable testing supply chains. we read with interest the study appearing in this issue by li et al, who utilized a pooled sample strategy and a point-of-care (poc) reverse transcriptase-polymerase chain reaction (rt-pcr) assay for screening asymptomatic airline passengers arriving from areas of high sars-cov-2 prevalence. the study demonstrates sample pooling as a mechanism for both cost reduction and resource conservation, using a commercial molecular platform with a rapid turnaround time and strong analytical sensitivity. while we view the results of this work as an overwhelmingly positive step forward, it is important to consider the context in which this strategy was deployed and the impact of the location and population on the results. sample pooling is a method of increasing the throughput of diagnostic assays. in this approach, small volumes of samples from multiple patients are combined into a single test, resulting in substantial reagent savings. if the pooled sample returns a negative result, all patients with specimens comprising that pool are considered not to be infected. a positive result requires that the pool be deconvoluted, or split, into its representative parts, and each component sample is tested individually to identify the infected patient(s). sample pooling was evaluated for tracking sars-cov-2 in a community setting early in the us outbreak [1] , and has been used historically as a public health tool for the detection of other viral agents, including human immunodeficiency virus. at the time of this writing, 2 reference laboratories in the united states (quest diagnostics and labcorp) have received emergency use authorizations from the us food and drug administration to use pooled specimens for sars-cov-2 detection [2] . in this work, pooling was performed in a 10:1 ratio, meaning 10 patient specimens were combined and tested using a single sars-cov-2 assay. the number of sars-cov-2 specimens per pool varies in the limited number of investigations reported to date [1, [3] [4] [5] . while the potential financial and reagent savings are obvious, careful and rigorous investigation is necessary to assure the pooling of specimens does not impact the analytical sensitivity of the assay [3, 4] . this is particularly important in a screening test used for asymptomatic individuals, who may not have high levels of detectable viral material present in their clinical specimens. indeed, in this work, rt-pcr cycle threshold (ct) values of nasopharyngeal swabs from the 2 positive, asymptomatic patients were 40. 6 and 41.6 when tested alone, and 43.8 and 43.9 when tested in the context of a 10:1 pool. these values are at the extreme end of the analytical sensitivity of this assay, highlighting the need for careful experimental design. li et al pooled specimens from patients traveling from high-risk areas in mainland china to hainan island as part of a sars-cov-2 disembarkation screening process at sanya airport. a particularly remarkable aspect of this work is that all testing (including positive pool deconvolution) was performed on site at the airport. most investigations of sars-cov-2 sample pooling utilize laboratory-developed assays that must be performed in approved biosafety level 2 settings, require specialized instrumentation, require trained technical staff, and are not portable. by taking advantage of the ease of operation of the cepheid platform, sample analyses were able to be performed in real time, allowing for rapid intervention. this approach may be feasible for an island featuring limited points of entry and a more isolated population. it is, however, challenging to extrapolate this study to an effective screening protocol for use in schools, workplaces, and sporting events in geographical areas with more porous borders. additionally, for sample pooling to be an effective strategy, the test population must have a low prevalence rate. the total number of covid-19 cases in hainan at the time of writing was 170. this number is significantly lower than the daily number of cases reported by some us cities, highlighting the utility of this approach in a low-prevalence setting. locales with higher sars-cov-2 prevalences will require more frequent deconvolution of positive patient pools, thus chipping away at or negating the benefits of a pooling approach. there are several practical points to resolve before a test-based screening policy can be deployed. beyond the obvious financial concerns, a major issue is the testing turnaround time. to date, most of the available "rapid" poc platforms repeatedly demonstrate lower levels of analytical sensitivity. in the united states, this means the molecular testing would need to be performed in a clinical laboratory improvement amendments (clia)certified laboratory, which could again lead to the overburdening of clinical laboratories; a notable exception is the cepheid platform for sars-cov-2, which is clia-waived and has good analytical sensitivity, hence its use in this study. moreover, the simple reality of specimen collection, transportation, testing, and reporting further prolongs turnaround times in settings without access to an on-site laboratory (ie, schools, business, etc.). this might imply that patients need to be tested the day before results are to be reviewed; in other words, get swabbed on sunday to go to work on monday. this leads to a second and possibly more important question: what does a negative or positive result mean? at our institution, we are aware of patients who underwent preprocedure sars-cov2 screening utilizing the same assay deployed in this work, only to be diagnosed with active, symptomatic covid-19 within 5 days of testing. as knowledge about covid-19 increases, it appears that the predictive value of a negative test in an asymptomatic person depends on the timing of sample collection in the context of the patient's disease course [6] . therefore, if an institution utilizes a weekly screening protocol, and screened individuals are still in their incubation period, it is possible they could test negative on monday, become infectious by wednesday, but not show symptoms until friday. if people misinterpret negative test results as justification to relax their nonpharmacologic interventions (mask wearing), especially at traditionally social times like lunch and coffee breaks, the magnitude of disease spread could be significant. conversely, in the absence of robust data concerning viral shedding, infectivity, and correlation to rt-pcr ct values, testing of asymptomatic patients using an assay with high analytical sensitivity begs the question as to whether such patients are truly contagious. of the 2 positive patients detected in this work, both exhibited immunoglobin g seropositivity and had nasopharyngeal samples with elevated ct values; 1 had an exposure history spanning up to 6 months before testing. it is not possible to know whether these patients are truly infectious without additional studies. thus, until we learn more about the biology and transmission kinetics of sars-cov-2, it is difficult to ascertain the clinical significance of such results. it might be reasonable to deploy a test-based screening strategy in a setting with controlled points of entry, a rapid turnaround time, effective means of contacting those who are tested, a low disease prevalence, and high compliance with masking. as these prerequisites are unlikely to be met in the contiguous united states, we either need to reconsider the utility of test-based screening or find faster, cheaper, and more sensitive poc tests. until this happens, pooled testing is an option to reduce costs and speed results. potential conflicts of interest. the authors: no reported conflicts of interest. both authors have sample pooling as a strategy to detect community transmission of sars-cov-2 united states food and drug administration pooling of samples for testing for sars-cov-2 in asymptomatic people assessment of specimen pooling to conserve sars cov-2 testing resources coronavirus (covid-19) update: fda issues first emergency authorization for sample pooling in diagnostic testing • cid 2020:xx (xx xxxx) • editorial commentary variation in false-negative rate of reverse transcriptase polymerase chain reactionbased sars-cov-2 tests by time since exposure editorial commentary • cid 2020:xx (xx xxxx) • 3 submitted the icmje form for disclosure of potential conflicts of interest. key: cord-158628-71n1tgrw authors: russo, giulia; pennisi, marzio; viceconti, marco; pappalardo, francesco title: in silico trial to test covid-19 candidate vaccines: a case study with uiss platform date: 2020-05-05 journal: nan doi: nan sha: doc_id: 158628 cord_uid: 71n1tgrw sars-cov-2 is a severe respiratory infection that infects humans. its outburst entitled it as a pandemic emergence. to get a grip on this, outbreak specific preventive and therapeutic interventions are urgently needed. it must be said that, until now, there are no existing vaccines for coronaviruses. to promptly and rapidly respond to pandemic events, the application of in silico trials can be used for designing and testing medicines against sars-cov-2 and speed-up the vaccine discovery pipeline, predicting any therapeutic failure and minimizing undesired effects. here, we present an in silico platform that showed to be in very good agreement with the latest literature in predicting sarscov-2 dynamics and related immune system host response. moreover, it has been used to predict the outcome of one of the latest suggested approach to design an effective vaccine, based on monoclonal antibody. uiss is then potentially ready to be used as an in silico trial platform to predict the outcome of vaccination strategy against sars-cov-2. s the epicenter of coronavirus disease 2019 (covid19) and emerging severe acute respiratory syndrome (sars) caused by novel coronavirus (2019-ncov) spread is making its way across the world, global healthcare finds itself facing tremendous challenges. according to the world health organization (who) situation report (91st), updated on 20 april 2020, there have been globally 72846 confirmed cases of 2019-ncov and 5296 cases of death caused by the virus itself [1] . 2019-ncov (also referred to as sars-cov-2 or hcov-19) [2] , is the seventh coronavirus known to infect humans along with sars-cov, mers-cov, hku1, nl63, oc43 and 229e [3] . while these last four coronaviruses are associated with mild symptoms, sars-cov, mers-cov and sars-cov-2 can cause severe acute respiratory syndrome [4] , especially in elderlies, of which men, and those individuals with comorbidities and immunocompromised conditions [5] ). although it is similar to sars-cov, sars-cov-2 has an improved ability for pathogenicity [6] . in particular, latest evidences during the ongoing pandemic reveal that patients affected by sars-cov-2 can progress their clinical picture from fever, cough, ageusia and anosmia, sore throat, breathlessness, fatigue, or malaise to pneumonia, acute respiratory distress syndrome (ards) and multi organ dysfunction illness [7] . significantly, in most critically ill patients, sars-cov-2 infection is also associated with a severe clinical inflammatory picture based on a serious cytokine storm that is mainly characterized by elevated plasma concentrations of interleukins 6 (il-6) [8] . in this scenario, it seems that il-6 owns an important driving role on the cytokine storm, leading to lung damage and reduced survival [9] . to get a grip on this outbreak and flatten the curve of infection, a specific therapeutic intervention to prevent the severity of the disease is urgently needed to reduce morbidity and mortality because, until now, there are no existing vaccines for coronaviruses. the ideal profile for a targeted sars-cov-2 vaccine must address the need of vaccinating human population, with particular regard of those individuals classified as at high risk, comprising, for example, frontline healthcare workers, individuals over the age of 60 and those that show debilitating chronic diseases. recently, specific findings about the genome sequencing of sars-cov-2 in different countries where cases of infection were registered, revealed its relative intrinsic genomic variability, its virus dynamics and the related host response mechanisms, unveiling interesting knowledge useful for the formulation of innovative strategies for preventive vaccination. specifically, sars-cov-2 sequencing along with its relative intrinsic genomic variability [10] , the presence of minority variants generated during sars-cov-2 replication [11] , the involved cellular factors that favors sars-cov-2 cell entry [12] , the timing in which viral load peaks (during the first week of illness), its gradual decline (over the second week) and the increasing of both igg and igm antibodies (around day 10 after symptom onset) represent some of the relevant insights so far delineated and considered by research community about sars-cov-2 virus [13] . even though these findings are having several practice consequences and suggest valuable conclusions, sars-cov-2 a dynamics has not been yet fully understood. information about which parts of sars-cov-2 sequence are recognized by the human immune system is still limited and scarcely available. such knowledge would be of immediate relevance and great help for the design of new vaccines, facilitating the evaluation of potential immunogenic candidates, as well as monitoring the virus mutation events that would be transmitted through the human population. currently, there are at least 42 vaccine candidates around the world under development and evaluation at different stages against covid-19 [14] , also accordingly from what reported by who through its continuously undergoing landscapes documents concerning the covid-19 candidate vaccines. these promising vaccine candidates deal with several vaccine technologies based on recombinant protein subunits [15] , nucleic acids [16] , non-replicating and replicating viral vectors [17] , [18] , protein constructs [19] , virus-like particles [20] , liveattenuated virus strains [21] , inactivated virus [14] , or human monoclonal antibodies (mabs) [22] . today, challenges of continuing development of solutions for covid-19 pandemic are a mandatory need. as never before, the application of modeling and simulation can actively design better vaccine prototypes, support decision making, decrease experimental costs and time, and eventually improve success rates of the trials. to this aim, in silico trials (ists) for design and testing medicines [23] [25] can accelerate and speed-up the vaccine discovery pipeline, predicting any therapeutic failure and minimizing undesired effects. beyond traditional modeling techniques or applications, agent-based models (abms) represent a paradigm that can cover the entire spectrum of the vaccine development process [26] , especially for the quantification and prediction of the humoral and cellular response of a specific candidate vaccine as well as its efficacy [27] . the simulation platform we use from fifteen years, named universal immune system simulator (uiss), is based on agentbased methodology, which is able to brilliantly simulate each single entity of the immune system (and consequently its dynamics), along with the significant immune responses induced by a specific pathogen or stimulus. recently, uiss provided different success stories in immunology field as it is most widely reported in the literature [28] [31] . we chose to analyze, within the wide landscape of potential candidate vaccines against sars-cov-2, a specific crossneutralizing antibody that wang et al. [32] suggest to be promising in targeting and binding a communal conserved epitope of sars-cov-2 and sars-cov on the spike receptor binding domain [33] , through an independent mechanism of receptor binding inhibition. as a case study, here we report a first application of uiss in silico platform to provide predictions of the efficacy of a potential therapy against covid-19 based on a mab strategy intervention like the one proposed by wang et al. agent-based models (abms) belong to the class of mechanistic models, a family of models that, differently from data-driven models, uses a description of the underlying mechanisms of a given phenomenon to reproduce it. such a description is usually based on different observational data, previous knowledge and/or hypotheses, and is usually aggregated and rationalized into a conceptual map (i.e., a flow chart and/or a schematic disease model) that reassumes the cascade of events of the phenomenon under investigation. the conceptual map is then translated into mathematical/computational terms and then executed by computers to observe, in silico, the evolution of the phenomenon over time. besides abms, other modeling techniques based on the mechanistic approach can be used. among these, we recall, for example, ordinary and partial differential equations [34] [36] and petri nets [37] , [38] . as the name suggests, agent-based models are based on the paradigm of 'agents', autonomous entities that behave individually according to established rules. such entities can be heterogeneous in nature, and are usually represented on a simulation space where they are free to move, interact eachother and change their internal state as a consequence of interactions. from a computer science perspective, agents can be seen as stochastic finite-state machines, capable of assuming a limited number of discrete states. using abms, the global evolution of the phenomena is observed by taking into account the sum of the individual behaviors of all agents, and sometimes unexpected "emergent" behaviors may be observed. abms have been successfully applied in many research fields, from social sciences to ecology, from epidemiology to biology. in the field of immunology, we developed the universal immune system simulator (uiss), an agent-based framework that has been extended through the last decades to simulate the behavior of the immune system response when challenged against many diseases. in uiss agents are used to describe cells and molecules of the immune system, as well as external actors that can destabilize (i.e., pathogens such as viruses and bacteria) or restore (i.e. prophylactic and therapeutic treatments) the normal health of the host. one of the main features of uiss is its ability to mimic the adaptive immune response mechanisms. mammals have in fact developed an advanced immune system machinery capable to specifically recognize pathogens in order to better react against them. this advanced response is based on the ability to exactly recognize foreign proteins (i.e., epitopes) on pathogens surface by means of receptors, through a key-to-lock mechanism. while an explicit implementation would be both unfeasible and partially inaccurate from a computational point of view, in uiss we mimic such a process through the use of binary strings. binary strings are used for both representing epitopes and immune system cells' receptors, and the probability that an immune system cell recognizes a pathogen is proportional to the hamming distance (the number of mismatching bits) between the two strings involved into the interaction. although this abstraction may seem binding, millions of interactions can be simulated quickly on modern computers, making easier the reproduction of many features of the immune system such as memory, specificity, tolerance and homeostasis. for example, this abstraction demonstrated able to allow the selection of the best adjuvant among a series of candidates for an influenza vaccine when properly coupled with results coming from existing binding prediction tools [28] . this suggests how such an abstraction is able to capture the complexity of the problem. besides of receptors, uiss implements many other immune system mechanisms, as thymus selection, haematopoiesis, cell maturation, hayflick limit, aging, immunological memory, antibody hyper-mutation, bystander effect, cell anergy, antigen processing and presentation. up to now, uiss in silico platform has been successfully applied to the design and verification of novel treatments for many diseases in both preclinical and clinical environments, including pathologies such as mammary carcinoma [39] and derived lung metastases [40] , melanoma [41] , atherosclerosis [42] , multiple sclerosis [31] and influenza [28] . more recently, uiss has been used as the centerpiece of the strituvad h2020 project with the aim to create an in silico trial for tuberculosis. in this context, observations from virtual patients will be coupled with results from a real clinical trial to obtain an in silico augmented clinical trial, with greater accuracy and more statistical power [30] . the sars-cov-2 disease model has been implemented in uiss computational framework starting by identifying a question of interest. the question of interest describes the specific question, decision or concern that is being addressed with a computational model. in other words, the question of interest lays out the engineering question that is to be answered (at least in part) through a model. the next step is to define the context of use (cou), which provides a detailed and complete explanation of how the computational model output will be used to answer the question of interest. in this specific study, the question of interest is how potential prophylactic or therapeutic vaccines could cure covid-19, building or stimulating an effective immune response against sars-cov-2 virus. uiss must then represent and reproduce the fundamental sars-cov-2 -immune system competition and dynamics. to this end, we first selected all the players that have a role in the viral infection both at cellular and molecular scale and then we categorized all the interactions among entities that play a relevant role in this biological scenario. finally compartment assumptions have to be done to let the entities move and interact each other. in our case, we considered the lung compartment that models the main organ target of the virus and the generic lymph node that allows immune system entities to be activated and selected. figure 1 gives a detailed sketch on the main compartments, entities and interactions. sars-cov-2 first entry is located in the upper respiratory tract. then it proceeds to bronchial and finally to lungs in which it reaches its main cellular target i.e., the epithelial lung cells (lep) [43] . the virus is eventually captured by dendritic cells (dc) and macrophages (m). dc are the main antigen processing cells of the immune system [44] that are able to present the peptides antigen complexed in both major histocompatibility class i and class ii (mhc-i and mhc-ii, respectively). if a dc encounters the native virus form, it can be able to process it and present its peptides complexed with mhc-ii to cd4 t cells for further actions. dc, upon virus activation, release interferon type a and b (ifn-a and ifn-b) and interleukin-12 (il-12) that are important cytokines in fighting intracellular pathogens. also, m are able to capture the native form of the sars-cov-2 and, if properly activated by pro-inflammatory cytokines, be able to internally destroy it. after their successful activation, macrophages release a proinflammatory cytokine that is tumor necrosis factor alpha (tnf-alpha). a fraction of sars-cov-2 viruses reach lep and through the envelope spike glycoprotein binds to their cellular receptor, ace2. doing that, the viral rna genome starts to be released into the cytoplasm and is translated into two polyproteins and structural proteins, after which the viral genome begins to replicate inside the cell [45] . following the flux of the conceptual disease model represented in figure 1 , after a certain amount of time (that we tuned with available data, as described in the next sections), new copies of the virus are released from the infected lep that eventually dies. new released copies of functional sars-cov-2 infect new cells, spreading further the infection in the lungs. when a cell is infected by a virus, it can be susceptible of different destinies. one of them is the shutting down of mhc-i expression to avoid peripheral blood compartment is seen as connecting duct, not explicitly represented. the starting point is the sars-cov-2 droplets entrance in the upper respiratory tract (not shown). then, all the main infection dynamics is described. the immune system cascade is shown as it was implemented, based on the latest research results published in specialized literature. for each entity, the localization (i.e., the biological compartment in which the entities are present) and the status (i.e., the differentiation states that an entity can own) are defined. the results of the immune system mounting process is the killing of the infected lung epithelial cells by the cytotoxic t lymphocyte and the local release of both chemokine factors and cytokines. at the humoral level, specific igm (first) and igg (after) directed against sars-cov-2 virus are released by plasma b cells. regulatory system is also involved in the process. if the immune system machinery works correctly, regulatory arm shutdowns excessive cytokines storm, avoiding the severe prognosis of covid-19. immune system recognition from specific cd8 t cells. in this case, a population of innate immunity cells, natural killer cells (nk) may identify them and proceed to kill them through specific actions. the other one is represented by a different mhc-i presentation on the cell surface, as the virus has modified the normal behavior of cell to let the host to make functioning virus copies. in this circumstance, (that we supposed to happen during sars-cov-2 infection) cell mhc-i presentation is different from the normal case. dc are able (through a mechanism known as "nibbling" process [46] ) to cross present the antigen complexed with mhc-i proteins to let adaptive immune response to recognize and kill virus infected cells. activated and antigen presenting cells (both dc and/or m) migrate into the proximal lymph nodes to present their content to adaptive immune cells i.e., t cells and b cells. also, a portion of viruses could eventually migrate to the lymph nodes. here, b cells can be activated by virus if specific immunoglobulin receptor in b cell surface binds to it. in this context, b cell is activated, and it immediately releases immunoglobulins of m class (igm) that are the first antibody response that can be measured. further, apc cells activate cd4 t cells (helper t cells, th) that under the influence of specific cytokines released before, differentiate into helper t cell type 1 (th1). th1 migrate under chemokines gradient to the site of infection. there, they release interferon gamma (ifn-g) that makes macrophages able to destroy captured viral particles and allow them to release il-12 that promotes immune system activation against the virus. th1 cells allow the differentiation and the iso-switching b cells into immunoglobulins class g (igg) producing plasma cells. igg are specific antibodies that bind against virus receptors, eventually inhibiting its capacity to infect cells. mhc-i/peptides dc presenting cells are also able to activate cd8 cytotoxic t cells (tc) to destroy sars-cov-2 infected cells and then eliminate the reservoir of infection. eventually, tc migrate into the site of infection and recognize and kill infected lep. tc killed infected lep release chemokines and interleukin 1 and 6 (il-1 and il-6). il-1 is the main cytokine that induces several systemic effects in the host, for example fever. il-6 is a proinflammatory cytokine that can change the severity of covid-19 disease as reported in very recent literature [47] . our disease model takes good account of the cytokines storm in the prognosis of the severity of the disease. entities (both cellular and molecular) move and diffuse in a simulation space represented as a l x l lattice (l is set depending on the dimension of the compartment one intends to reproduce), with periodic boundary conditions. there is no correlation between entities residing on different sites at a fixed time as the interactions among cells and molecules take place within a lattice-site in a single time step. all entities are allowed to move with a uniform probability between neighboring lattices in the grid and with an equal diffusion coefficient (brownian motion). scientific knowledge about sars-cov-2 is still not complete and research contributions appear every day. apart from this, we used all the available literature data to compare the dynamics predicted by the uiss platform with all findings we were able to fetch. the first task we accomplished with success was the evaluation of the replication kinetic of sars-cov-2. to this end, we set a first use case simulation considering a digital patient in which a virus challenge dose of 0.1 multiplicity of infection (moi) was administered at day 0. inoculation. il-6 dynamics and its related plasma levels (fg/âµl) are also shown in the inner panel (purple line). in the right one, the dynamics of cpe on the lung infected cells is measured: they started at day 3.5 and peak around day 5. after 21 days, the simulated digital patient almost recovers from the infection. one can notice how uiss is capable to simulate, accordingly to the recent literature, the early viral clearance by day 10 post-onset in mild cases. in addition, it is wort to note that virus persists after day 10, until day 15, and its complete clearance is around day 19. in the inner panel (purple line), il-6 dynamics and its related plasma levels (fg/âµl) are shown. il-6 dynamics shows a much more prominent peak of values. this is in very good agreement with latest literature data, as explained within the manuscript. in the right panel, the dynamics of cpe on the lung infected cells is measured: in this case, cpe are much more severe and the recover from infection is clearly delayed. uiss is capable to simulate, accordingly to the recent literature, how the severe cases tend to have a higher viral load both at the beginning and later on. simulation space was 5 cubic millimeters of lung tissue, 5 cubic millimeters of lymph tissue and 5 microliters of peripheral blood. figure 2 (right panel) shows that peak viral titers are reached by 48 h post-inoculation. we also plotted il-6 dynamics: as reported in [9] the levels of il-6 could be provide a prognosis on the severity of the infection. we also measured cytopathic effects (cpe) on the lung compartment. cpe are defined as changes occurred in the infected cell that eventually lead its lysis or inability to reproduce. figure 2 (left panel) highlights the dynamics of cpe: they started at day 3.5 and peak around day 5. after 21 days, the simulated digital patient almost recovers from the infections. these findings are in good agreement with actual literature [13] , [48] . in a recent work, liu et al [49] reported that mild cases were found to have an early viral clearance, with 90% of these patients repeatedly testing negative by day 10 post-onset. at the same time, they found that all severe cases still tested positive at or beyond day 10 post-onset. moreover, severe cases tended to have a higher viral load both at the beginning and later. in contrast, mild cases had early viral clearance 10 days post onset. uiss was also able to reproduce this scenario. as one can see, figure 2 is in very good agreement for viral clearance. we also were able to reproduce severe conditions acting on the immune system aging parameters obtaining results showed in figure 3 . in this case, figure 3 (right panel) shows virus presence after day 10, until day 15, and its complete clearance about day 19. moreover, cpe are much more severe and the recover from infection was clearly delayed (left panel). il-6 dynamics shows a much more prominent peak of values. this is in very good agreement with latest literature data, as explained before. to validate the main immune system response of mild-tomoderate covid-19 patients, we used the results available in [50] . in this work, the authors report the kinetics of immune responses in terms of activated cd4 + t cells, cd8 + t cells, igm and igg antibodies, detected in blood before symptomatic recovery. as one can see from figure 4 , the kinetics of activated th1 cells (panel a), activated cd8 t cells (panel b) and the igm and igg (panel c) predicted by the simulator are in good agreement with their findings. uiss is an immune system simulation platform that was designed to be applied to several and different scenarios, especially to carry on in silico trials to predict the efficacy of a specific prophylactic or therapeutic vaccine against a particular disease. in silico trials aim to strongly reduce the time to develop new effective therapeutics: this is particularly crucial in situation like the one we are facing with. as soon as a disease model incorporated into uiss is tuned and validated against available data, it can be used as an in silico lab to test new vaccines. in the previous section, we demonstrated that uiss-sars-cov-2 is able to reproduce and predict the main aspects of the viral infection. as a working example, here we show how the platform can be immediately used to predict the efficacy of a human monoclonal antibody that neutralizes sars-cov-2 developed by wang et al. [32] . in this work the authors suppose that the developed antibody (named 47d11) neutralizes sars-cov-2 through a yet unknown mechanism that is different from receptor binding interference. hence, in implementing the mechanism of action of 47d11 into uiss computational framework we used the alternative mechanisms of coronavirus neutralization by receptor-binding domain (rbd) targeting antibodies that have been reported, including spike inactivation through antibody-induced destabilization of its prefusion structure, which wang et al. indicated also applicable for 47d11. we then modeled the receptor interaction to trigger irreversible conformational changes in coronavirus spike proteins enabling membrane fusion, as described in [51] . the validation in silico trial consists in simulating the in vitro experiment conducted by wang et al. where they showed that the monoclonal antibody was effective in contrasting sars-cov-2 to infect the target cells. to mimic the in vitro system that is an isolated system, we disabled both the lung and the lymph node compartments and we turned off all the immune system interactions in uiss. we then injected virus particles in the peripheral blood compartment of the simulator along with different concentrations of mab. only lep-sars-cov-2 interaction has been allowed to happen. figure 5 shows the obtained results. as one can envisage from the figure, we reproduced with great accuracy the in vitro results reported by wang et al. in particular, the computational framework was able to correctly predict the efficacy of 47d11 mab simulating its mechanism of action that induces the entering of the virus inside lep. the most effective concentration is 10 ng/ml. this step makes the simulation platform ready for usage as a in silico trial to predict now the effects of a mab therapy. for this purpose, we designed two kind of in silico experiments. the first one dealt with mab vaccination strategy used to prevent the onset of infection. the second one involved mab as interventional drug to treat already infected hosts. figure 6 clearly demonstrates that if a patient enters in contact with the virus just after 7 days post vaccination, he is fully protected from the infection: as one can see, sars-cov-2 actively infected values of lep are low. this is also true if a potential subject is being infected after two weeks (panel b), one month (panel c) or three months (panel d) after vaccination. things change for the other two cases i.e., panel e (subject infected after 6 months) or panel f (subject infected after one year). in this circumstance, the computational framework predicts that the mab vaccination is practically ineffective in protecting the onset of the disease. a second injection of the mab is suggested around month 4 to extend the protection of the host for one year. the second experiment we designed is to use our in silico trial platform to predict the efficacy of a mab-based vaccine in therapeutic settings. we envisaged both mild-moderate case (the same digital patient type shown in figure 2 ) and severe case (the same digital patient shown in figure 3 ). for both cases, we administered the mab vaccine one day and two days after the onset of infection. figure 7 shows different behavior for both mild-moderate case (panel a, mab injected one day after the onset of infection, and panel b, mab injected two days after the onset of infection) and severe case (panels c, mab injected one day after the onset of infection, and panel d, mab injected two days after the onset of infection). as figure 7 depicts, mab vaccine is effective in preventing or strongly limiting the cpe. if the mab vaccine is injected after two days, it is not able to protect the lpe of the host to be infected by the virus and consequently from covid-19 pathology. in this paper, we present an in silico platform that was demonstrated able to reproduce the main dynamics of sars-cov-2 virus and the elicited host immune response against it. the disease model was implemented inside uiss fig. 7 . in silico trial of 47d11 to predict therapeutic efficacy. different behaviors for both mild-moderate case (panels a and b) and severe case (panels c and d) are shown. in panel a, mab is injected one day after the onset of infection. in panel b, mab is injected two days after the onset of infection. in panel c, mab is injected one day after the onset of infection. in panel d, mab is injected two days after the onset of infection. blue lines depict actively infected lep while red lines represent lep to show the cpe. as one can notice, mab vaccine is effective in preventing or strongly limiting the cpe only by two days of the infection. computational framework, an in silico trial platform that has been applied to several biological scenario. uiss shows that the simulated sars-cov-2 dynamics is in very good agreement with the one described in the latest literature; also, the immune system response predicted by uiss against the virus mirrored the one observed in state of the art research data. this validation step entitled uiss-sars-cov-2 to be used as a in silico lab to test the efficacy of potential vaccines for covid-19, knowing a priori their mechanism of action. hence, we set an in silico trial to test a recent vaccination strategy based on the employment of monoclonal antibodies directed against a specific target protein of the virus. the simulator is in good agreement in predicting the in vitro experiment outcome performed by the inventors of 47d11 mab. finally, we designed two experimental settings to predict the efficacy of mab vaccination when used in both preventive and therapeutic cases. we predicted that mab is an effective therapy when used as a preventive vaccine (granting up one year protection when injected with a two times schedule). moreover, we envisaged that 47d11 mab if effective only if administered in a very stringent time-frame if employed as a therapeutic strategy. novel 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with 2019 novel coronavirus in wuhan, china severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, united states viral dynamics in mild and severe cases of covid-19 breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 unexpected receptor functional mimicry elucidates activation of coronavirus fusion the authors would like to thank all the researchers that are involved in finding potentially effective vaccines candidates against sars-cov-2 virus. we will be more than happy to collaborate with any of them to support the process. key: cord-256217-fnjer0e0 authors: neri, piergiorgio; pichi, francesco title: covid-19 and the eye immunity: lesson learned from the past and possible new therapeutic insights date: 2020-04-20 journal: int ophthalmol doi: 10.1007/s10792-020-01389-2 sha: doc_id: 256217 cord_uid: fnjer0e0 nan corona virus represents nowadays the hot topic in the scientific world due to the outbreak of a novel serotype formerly named coronavirus disease (covid)-19 and now identified as severe acute respiratory syndrome (sars)-cov-2 [1] . the disease has been firstly reported in wuhan, yubei district of the republic of china, when in december 2019 its outbreak affected around 80,000 people and provoked about 3000 deaths due to pulmonary complications [1] . the virus rapidly spread all over the world and was recognized as a pandemic by the world health organization (who) on march 11, 2020 [2] . in 4 months, the disease was recognized in about 1,521,252 people and made 92,798 victims mainly in elderly and compromised patients. on an official report released on april 10, 2020, sars-cov-2 carried a mortality rate around 6.1% compared to influenza that is supposed be around 1% [3] . all countries are encountering massive and dramatic problems in terms of prevention and sustainability of treatments for this disease which does not have a universally agreed therapeutic protocol. current management of covid-19 is mainly supportive, even though the medical literature has reported several rescue strategies which might minimize the mortality rate in those who have severe pulmonary complications. hydroxychloroquine and azithromycin combination seems to be a valid possible therapeutic protocol [4] and is currently under urgent investigation. unfortunately, the real challenge is represented by those cases where the severe form of sars-cov-2 interstitial pneumonia occurs, with acute respiratory distress syndrome (ards) that might lead to fatal events [5] . the urgent need to find a treatment for such lifethreatening condition has led to off-label approaches based on the possible hypercytokinemia with multiorgan failure [6] . an experimental protocol using an anti-interleukin (il)-6 receptor monoclonal antibody named tocilizumab (actemra, roche pharma (schweiz) ltd, b2084b21) has given hope of a possible effective therapy. tocilizumab was used as a rescue treatment in a series of patients affected by severe interstitial pneumonia by zhang et al. [7] which observed an evident improvement in both systemic symptoms and the specific pulmonary impairment within a few days. these interesting results were followed by a series of other publications speculating on how the inhibition of immune checkpoints might have exerted a possible good control of the disease. on the other hand, the severe contingent situation created by the outbreak of sars-cov-2 infection does not allow to have good models to rely on. in the medical literature, sars coronavirus is associated with the occurrence of hemophagocytic lymphohistiocytosis (shlh) which represents a lifethreatening hyperinflammatory syndrome leading to a rapid multiorgan deterioration due to severe hypercytokinemia [6] . at this stage, can the ocular immune system represent a valid model upon which to rely in order to hypothesize a possible therapeutic strategy? in 1990, robbins et al. reported on a possible animal model where murine coronavirus induced an acute and long-lasting disease of the retina [8] . this model was identified as experimental coronavirus retinopathy (ecor) [8] . few months later, the same group of researchers reported that murine coronavirus had a specific retinotropism when introduced by several direct routes [9] . the researchers found that the viral insult leads to a progressive impairment of all the structures of the retinal tissue, including photoreceptors and retinal pigment epithelium (rpe). murine coronavirus showed an evident tropism to the neural tissue of the retina which was independent of route of inoculation, such as onto the cornea, the anterior chamber (ac), intravitreally, intracerebrally, or intranasally even. interestingly, viral antigens were located primarily in the inner nuclear layer, photoreceptors, muller cells, and rpe. on the other hand, at day 10, ganglion cell layer was the only and still infected, indicating a spontaneous clearance of the virus from the other retinal tissues. this trend in affecting the nervous system might be the case of sars-cov-2 as well, since a recent report described guillan-barrè syndrome as the first manifestation of the disease [10] . interestingly, infection induces fibrosis of rpe cells as well as moderate retinal atrophy in the long term, which suggests a certain role played by fibroblasts in attempting a repair of the damaged tissue. although ecor was used to study retinal degeneration specifically, it might represent a possible experimental model for interesting speculations on how to approach severe sars-cov-2 pulmonary complications. going through the medical literature, very little information is available on the possible role played by the immune system in lung involvement, which shows moderate multinucleated giant cells, minimal lymphocytes, eosinophils, cd4-positive t cells, and neutrophils [11] . lungs blood vessels of alveolar septum are congested, edematous, and widened, with modest infiltration of monocytes and lymphocytes. hyaline thrombi are also found in a minority of microvessels, as well as local hemorrhage in lung tissue, organization of exudates in some alveolar cavities, and pulmonary interstitial fibrosis. on the other hand, all those findings were taken postmortem and there is lack of data on different phases of the disease. looking at the ecor model, it gives the impression that coronavirus creates two different phases: the first is represented by the primary infection which induces the triggering of the immune system, while the second phase is likely to be an autoimmune disease where the role of the severe postviral inflammation represents a severe occurrence worth of prompt intervention. coronavirus animal model suggests that such hypothesis might be justified [9] : as we previously said, coronavirus was detected into the ganglion cell layer only after 10 days, even though in 26% of the experimental animals a severe and massive inflammation of most ocular structures was observed. the authors speculated that gliosis might have been present in affected retinas as the result of breakdown of the blood-ocular barrier which can allow coronavirus-bearing macrophages to access the eye. regarding the specific vascular involvement, massive inflammation leads to damage to the walls of veins, evidenced by loss of continuity and erythrocytes extravasation into the surrounding tissue. similarly, the lung polythrombosis observed in sars-cov-2 cases seems to be a result of the interstitial inflammation where hyaline thrombi of the microvessels might be induced by the perivasculitis affecting the vessels walls embedded into the affected tissue [11] . at this stage, the therapeutic strategy is still opened for discussion and might offer a series of fascinating hypotheses for a rescue treatment in severe sars-cov-2 pneumonia. albeit it is true that anti-il-6 receptor monoclonal antibody has given promising results for the control of severe sars-cov-2 pneumonia, it is interesting to notice that retinal degeneration in ecor is associated with an evident increase in tnf-alpha, as well as soluble tnfr2, inducing an anomaly of tnf-alpha signaling [12] . the clinical picture offered by sars-cov-2 pneumonia seems to present a series of similarities to so-called interstitial pneumonia with autoimmune features (ipaf) recently described by the european respiratory society and the american thoracic society [13] . in ipaf, the immune reaction plays a primary role in its pathogenesis. however, there is no general agreement on any recommendations; therefore, even the diagnosis and management are left to the individual caregiver [13] . while the use of anti-tnf-alpha appears quite risky due to the possible occurrence of fulminant tuberculosis or might play a very limited role, other new generation immunosuppressives, such as intravenous immunoglobulin, selective il-1 cytokine blockade, and jak inhibition [6] , were proposed. however, it is unclear why the scientific world is still strongly reluctant to promptly use steroids to target the second phase of the disease, while this is not the case for monoclonals. similarly to ebola virus disease (evd), where steroids were successfully used even though empirically, the number of circulating t lymphocytes is dramatically reduced in severe cases, and elevated cytokine levels in the serum are observed [14] . under that light, steroids might appear a potent weapon against fatal immune system activation. since world health organization provided no guideline on corticosteroids use for the management of sars-cov-2, they can be only used empirically with no indication on timing, duration, and dosage. in addition, early use of steroids seems to exert a more than promising control of inflammatory response in other severe viral diseases [14] . the same issue was addressed several times for more common diseases like herpes virus encephalitis [15] , which is universally recognized as a postviral autoimmune response. herpetic postviral central nervous system inflammation has corticosteroids as the first line drug in the therapeutic armamentarium [16] and might be considered a possible model for a prompt response in treating earlier life-threatening forms of sars-cov-2. on the basis of the current knowledge, corticosteroids might be a potent therapeutic weapon for the second phase of the disease, when the immune system targets dramatically multiple organs and specifically the lungs, while it has to be kept in mind that their use can be risky when the activation of inflammatory processes remains within the normal range. however, the risk/benefit ratio of corticosteroids for severe form of sars-cov-2 disease should be clarified by the health authorities [17] , as well as the weight of cost-effectiveness compared to other therapies like monoclonals. moreover, the sars-cov-2 pneumonia animal model described in 2010 by smits et al. [18] presents many similarities with ecor reported almost 2 decades earlier, in terms of viral clearance and timing of immune response. considering the course of the disease, it seems the supposed postcoronavirus immune response happens within 10 to 14 days after the onset of the disease in both models. consequently, that might be the appropriate timing to treat, while the viral insult might be over already as per the animal model. at that time, a prompt intervention with no delay either by monoclonals or by steroids may rescue the patient and avoid the local immune-driven irreversible tissue damage. it might have been appropriate to test these hypotheses by an appropriate trial, but nowadays time does not play in favor and there is the urgent need of a prompt and brave action. can the coronavirus disease 2019 (covid-19) affect the eyes? a review of coronaviruses and ocular implications in humans and animals who (2020) who director-general's opening remarks at the media briefing on covid-19-11 who (2020) coronavirus disease 2019 (covid-19) situation report-81 chloroquine and hydroxychloroquine as available weapons to fight covid-19 world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) hlh across speciality collaboration, uk covid-19: consider cytokine storm syndromes and immunosuppression the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality retinopathy following intravitreal injection of mice with mhv strain jhm ocular tropisms of murine coronavirus (strain jhm) after inoculation by various routes guillain-barré syndrome associated with sars-cov-2 infection: causality or coincidence? a pathological report of three covid-19 cases by minimally invasive autopsies retinal degeneration in experimental coronavirus retinopathy (ecor) is associated with increased tnf-alpha, soluble tnfr2 and altered tnf-alpha signaling ers/ ats task force on undifferentiated forms of ctd-ild''. an official european respiratory society/american thoracic society research statement: interstitial pneumonia with autoimmune features do corticosteroids have a role in treating ebola virus disease? sci china on the use of corticosteroids for 2019-ncov pneumonia herpes simplex virus encephalitis update diagnosis and treatment of viral encephalitis exacerbated innate host response to sars-cov in aged non-human primates key: cord-252232-vgq6gjpx authors: hou, yuxuan; peng, cheng; yu, meng; li, yan; han, zhenggang; li, fang; wang, lin-fa; shi, zhengli title: angiotensin-converting enzyme 2 (ace2) proteins of different bat species confer variable susceptibility to sars-cov entry date: 2010-06-22 journal: arch virol doi: 10.1007/s00705-010-0729-6 sha: doc_id: 252232 cord_uid: vgq6gjpx the discovery of sars-like coronavirus in bats suggests that bats could be the natural reservoir of sars-cov. however, previous studies indicated the angiotensin-converting enzyme 2 (ace2) protein, a known sars-cov receptor, from a horseshoe bat was unable to act as a functional receptor for sars-cov. here, we extended our previous study to ace2 molecules from seven additional bat species and tested their interactions with human sars-cov spike protein using both hiv-based pseudotype and live sars-cov infection assays. the results show that ace2s of myotis daubentoni and rhinolophus sinicus support viral entry mediated by the sars-cov s protein, albeit with different efficiency in comparison to that of the human ace2. further, the alteration of several key residues either decreased or enhanced bat ace2 receptor efficiency, as predicted from a structural modeling study of the different bat ace2 molecules. these data suggest that m. daubentoni and r. sinicus are likely to be susceptible to sars-cov and may be candidates as the natural host of the sars-cov progenitor viruses. furthermore, our current study also demonstrates that the genetic diversity of ace2 among bats is greater than that observed among known sars-cov susceptible mammals, highlighting the possibility that there are many more uncharacterized bat species that can act as a reservoir of sars-cov or its progenitor viruses. this calls for continuation and expansion of field surveillance studies among different bat populations to eventually identify the true natural reservoir of sars-cov. electronic supplementary material: the online version of this article (doi:10.1007/s00705-010-0729-6) contains supplementary material, which is available to authorized users. severe acute respiratory syndrome coronavirus (sars-cov) is the aetiological agent responsible for the sars outbreaks during 2002-2003, which had a huge global impact on public health, travel and the world economy [4, 11] . the host range of sars-cov is largely determined by the specific and high-affinity interactions between a defined receptor-binding domain (rbd) on the sars-cov spike protein and its host receptor, angiontensin-converting enzyme 2 (ace2) [6, 7, 9] . it has been hypothesized that sars-cov was harbored in its natural reservoir, bats, and was transmitted directly or indirectly from bats to palm civets and then to humans [10] . however, although the genetically related sars-like coronavirus (sl-cov) has been identified in horseshoe bats of the genus rhinolophus [5, 8, 12, 18] , its spike protein was not able to use the human ace2 (hace2) protein as a receptor [13] . close examination of the crystal structure of human sars-cov rbd complexed with hace2 suggests that truncations in the receptor-binding motif (rbm) region of sl-cov spike protein abolish its hace2-binding ability [7, 10] , and hence the sl-cov found recently in horseshoe bats is unlikely to be the direct ancestor of human sars-cov. also, it has been shown that the human sars-cov spike protein and its closely related civet sars-cov spike protein were not able to use a horseshoe bat (r. pearsoni) ace2 as a receptor [13] , highlighting a critical missing link in the bat-to-civet/human transmission chain of sars-cov. there are at least three plausible scenarios to explain the origin of sars-cov. first, some unknown intermediate hosts were responsible for the adaptation and transmission of sars-cov from bats to civets or humans. this is the most popular theory of sars-cov transmission at the present time [10] . second, there is an sl-cov with a very close relationship to the outbreak sars-cov strains in a non-bat animal host that is capable of direct transmission from reservoir host to human or civet. third, ace2 from yet to be identified bat species may function as an efficient receptor, and these bats could be the direct reservoir of human or civet sars-cov. unraveling which scenario is most likely to have occurred during the 2002-2003 sars epidemic is critical for our understanding of the dynamics of the outbreak and will play a key role in helping us to prevent future outbreaks. to this end, we have extended our studies to include ace2 molecules from different bat species and examined their interaction with the human sars-cov spike protein. our results show that there is great genetic diversity among bat ace2 molecules, especially at the key residues known to be important for interacting with the viral spike protein, and that ace2s of myotis daubentoni and rhinolophus sinicus from hubei province can support viral entry. hela cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (gibco, usa). rabbit polyclonal antibodies against ace2 of r. pearsoni (rpace2) were generated using r. pearsoni ace2 protein expressed in escherichia coli at the wuhan institute of virology following standard procedures. bats were sampled from their natural habitats in hubei, guangxi, guizhou, henan and yunnan provinces in china as described previously [8] . bat identification was initially determined in the field by morphology and later confirmed in the laboratory by sequencing the mitochondrial cytochrome b gene from samples of blood cells or rectal tissue as described previously [1] . total rna was extracted from bat rectal tissue using trizol reagent (invitrogen, usa) and treating with rnase-free dnase i at 37°c for 30 min. first-strand cdna was synthesized from total rna by reverse transcription with random hexamers. full-length bat ace2 fragments were amplified using the forward primer baf2 (5 0 -cttgg taccatgtcaggctcttyctgg-3 0 ) and the reverse primer bar2 (5 0 -ccgctcgagctaaaab[g/t/c]ga v[g/a/c]gtctgaacatcatc-3 0 ). the pcr mixture (25 ll) contained 0.5 ll cdna, 1.5 mm mgcl 2 and 0.2 lm of each primer, and the fragments were amplified using the following parameters: 95°c for 5 min, 35 cycles of 94°c for 30 s, 55°c for 45 s and 68°c for 3 min, with a final elongation step at 68°c for 10 min. all bat ace2s were cloned into pcdna3.1 with kpni and xhoi, and this was verified by sequencing. for samples in which full-length ace2 amplification was unsuccessful, the n-terminal region (1-1,170 bp) was amplified using the forward primer baf2 and the reverse primer rmr (5 0 -ttagctccatttcttagcaggtag g-3 0 ). chimeric ace2 was constructed by combining the n-terminal region of bat ace2 with the c-terminal portion of human ace2 at the unique bamhi site (1,070-1,075 bp). the chimera was subsequently cloned into pcdna3.1 with kpni and xhoi and sequenced as above. construction of bat ace2 mutants ace2 from m. daubentoni was chosen to generate a series of ace2 mutants using a quikchange ii site-directed mutagenesis kit (stratagene, usa). the altered amino acid codon for each mutant is indicated as follows: i27t, n31k, k35e, and h41y. mutants were confirmed by sequencing. all bat ace2s were submitted to genbank (ef569964, gq999931-gq999938). sequence alignment was performed using clustalx version 1.83 [15] and corrected manually. a phylogenetic tree based on amino acid (aa) sequences was constructed using the neighbor-joining (nj) method in mega version 4.1. [14] . lysates of hela cells expressing human ace2 or bat ace2 were separated on a 4-10% sds-page gel, followed by transfer to a polyvinylidene difluoride (pvdf) membrane using a semi-dry protein transfer apparatus (bio-rad, usa). the membrane was probed with a rabbit polyclonal antibody against the bat ace2 protein (1:200) at room temperature for 1 h, followed by incubation with alkaline-phosphatase-conjugated goat anti-rabbit igg (1:1,000) (chemicon, australia). the probed proteins were visualized using nbt and bcip color development (promega, usa). an hiv-1-luciferase pseudotype virus carrying the sars-cov bj01 s protein, hiv/bj01-s, was prepared as described previously [13] . hela cells were seeded onto 96-well plates for 18 h and then transfected with 0.2 lg recombinant plasmid containing bat or human ace2 using 0.5 ll lipofectamine 2000 (invitrogen, usa) according to the manufacturer's protocol. at 24 h post-transfection, 30 ll medium containing hiv/bj01-s was added to each well. at 2-3 h postinfection, unadsorbed viruses were removed, and fresh medium was added. the infection was monitored by measuring luciferase activity, expressed from the reporter gene carried by the pseudovirus, using a luciferase assay kit (promega, usa). cells were lysed at 48 h postinfection by adding 20 ll lysis buffer provided with the kit, and 10 ll of the resulting lysates were tested for luciferase activity by the addition of 20 ll luciferase substrate in a turner designs td-20/20 luminometer. each infection experiment was conducted in triplicate, and all experiments were repeated three times. live sars-cov infection was carried out under bsl4 conditions at the australian animal health laboratory (aahl) as described previously [16, 17] . briefly, 48 h after transfection, the time at which expression of the ace2 receptor on the hela cell surface is optimal, 2 9 10 6 tcid 50 of virus was added to the cells for infection. the cells were fixed 24 h later by treatment with 100% methanol for 10 min and washed five times with pbst. the primary antibody, chicken anti-sars-cov s (produced against the recombinant s protein expressed in e. coli at aahl), at a 1:500 dilution in 1% bsa/pbs, was added and incubated with the cells for 1 h at room temperature. an fitc anti-chicken conjugate (chemicon, australia) at 1:1,000 in 1% bsa/pbs was added after washing the cells five times and incubated with the cells for 1 h. infection was monitored by immunofluorescent microscopic analysis. cloning and expression of ace2 genes from different bat species ace2 genes from seven bat species were amplified and cloned (fig. 1, sfig. 1) . full-length genes were obtained from rhinolophus ferrumequinum from hubei province (rf-hb), r. macrotis from hubei province (rm-hb), r. pearsoni from guangxi (rp-gx), r. pusillus from hubei province (rpu-hb), r. sinicus from guangxi province (rs-gx) and r. sinicus from hubei province (rs-hb). for the following bat species, amplification of the full-length coding region was not successful, and instead,the n-terminal region was cloned in frame with the c-terminal region of the human ace2 gene to form a chimeric fulllength ace molecule: r. pearsoni from guizhou province (rp-gz), myotis daubentonii bat from yunnan province (md-yn) and hipposideros pratti bat from henan province (hp-hn) . the full-length sequences of bat ace2 are identical in size to that of hace2 (805 aa in total). sequence comparison showed that bat ace2s are closely related to ace2s of other mammals and have an aa sequence identity of 80-82% to human and civet ace2. the aa identity of ace2 from different bat families ranges from 78 to 84%, and within the genus rhinolophus, the sequence identity increases to 89-98%. the major sequence variation among bat ace2s is located in the n-terminal region, which has been identified in structural studies as the sars-cov-binding region [6, 7] . a phylogenetic tree was constructed based on the sequences of bat ace2 (sfig. 2) using the mega package [14] . all ace2 genes were cloned into a eukaryotic expression vector and used to transfect hela cells. western blot analysis showed that all ace2s were expressed efficiently and at very similar levels and were recognized by a rabbit anti-bat ace2 antibody with an apparent molecular weight of approximately 100-130 kda (fig. 2c) . to examine the susceptibility of different bat ace2 molecules to sars-cov entry, the hiv/bj01-s pseudovirus system was used to infect hela cells transiently expressing bat ace2 or human ace2 genes. among the bat ace2s, only mdace2 (mdace2) and rs-hb ace2 demonstrated significant pseudovirus infection, as deduced from the significantly higher level of luciferase activity in comparison to background activity in the negative control (fig. 2a) . although such assays are not to be viewed as an absolute quantification of receptor activity, it is nevertheless worth noting that mdace2-mediated infection seemed to be more efficient than with rs-hb ace2. in the same context, it is clear that the bat ace2s were less efficient overall than the human ace2 in this particular assay system. the biological significance of this observation remains to be determined. the functionality of mdace2 and rs-hb ace2 as sars-cov entry receptors was further confirmed by infection with live virus. as shown in fig. 2b , both bat ace2 proteins could clearly support sars-cov infection. no attempt was made to quantify infection efficiency in this study due to difficulties encountered in conducting experiments under bsl4 conditions. homologous structural modeling of human sars-cov rbd complexed with mdace2 supports mdace2 as a receptor for human sars-cov s protein. the crystal structure of human sars-cov rbd complexed with hace2 shows that two salt bridges at the sars-cov-hace2 interface, between hace2 lys31 and glu35 and between hace2 lys353 and hace2 glu38, are both buried in a hydrophobic environment and contribute critically to the sars-cov-hace2 interactions (fig. 3a , c) [7] . disturbance of the formation of either of these salt bridges weakens sars-cov-hace2 binding. the lys31-glu35 salt bridge at the sars-cov-hace2 interface becomes an asn31-lys35 hydrogen bond at the sars-cov-md-ynace2 interface (fig. 3b) , which possibly weakens virus-receptor binding but still is largely compatible with the virus-receptor interface. thr27 on hace2 supports the lys31-gu35 salt bridge through hydrophobic interactions with tyr475 (fig. 3a) ; ile27 on mdace2 supports the asn31-lys35 hydrogen bond more efficiently than thr27 through tighter hydrophobic interactions with tyr475 (fig. 3b) . moreover, tyr41 on hace2 supports the lys353-glu38 salt bridge (fig. 3c) ; his41 on mdace2 supports the same salt bridge less efficiently than tyr41 (fig. 3d) . overall, mdace2 is an efficient receptor for sars-cov, despite the fact that its receptor activity is lower than that of hace2. compared with mdace2, rs-hb ace2 contains glu31 and glu35, which are not compatible with each other due to their same negative charges, which disfavor . the alignment was generated using clustalx v1.83. in black are single, fully conserved residues. in gray are strongly conserved residues. in light gray are weakly conserved residues. asterisks indicate residues that interact directly with the receptor-binding domain of the sars-cov s protein virus-receptor binding. however, rs-hb ace2 also contains thr27 and tyr41, both of which support sars-cov entry by contributing favorably to the hydrophobic interactions at the virus-receptor interface. thus, rs-hb is a low-efficiency receptor for sars-cov. all of the other bat ace2 molecules contain combinations of the aforementioned key residues that are completely incompatible with virus-receptor interactions. more specifically, they either contain same-charged residues at the 31 and 35 positions, which repel each other, or contain his41 and lys27, which disfavor sars-cov binding (fig. 1) . in particular, lys27 on some of these bat ace2 molecules is incompatible with certain hydrophobic residues, such as leu443 and phe460, on sars-cov rbd (fig. 3a, b) . therefore, these bat ace2 molecules are not receptors for sars-cov. to confirm the above homologous structural analysis, we carried out site-directed mutagenesis on mdace2. our results show that mutations e31k, k35e, and i27t all dramatically decrease the receptor activity of mdace2, whereas mutation h41y greatly increases its receptor activity (fig. 2a) . therefore, our mutagenesis data further confirmed that key residues in ace2 determine the receptor activity of mdace2. our finding that m. daubentoni and r. sinicus could support sars-cov infection has important implications in relation to the origin of sars-cov. since all lines of investigation have indicated that ace2-binding affinity is among the important determinants for sars-cov host range, our data would suggest that m. daubentonii and r. sinicus have the potential to serve as the direct reservoirs for human sars-cov or its highly related civet sars-cov. to further investigate the potential of m. daubentonii and r. sinicus as reservoirs for sars-cov, more efforts will have to be directed toward widening the surveillance of bats in these families and in different geographical locations. another important finding of our current study is the great genetic diversity of bat ace2 proteins, which is in contrast to the genetically homogenous hace2 [10] . sequence variations of bat ace2, especially in positions that are critical to sars-cov binding, such as residues 27, 31, 35, and 41, suggest that, in addition to the md-yn and rs-hb ace2s, there may be many other bats with an ace2 protein that makes them susceptible to sars-cov entry. this again highlights the need for more field surveillance and molecular characterization of different bat ace2 proteins until the true reservoir host of sars-cov is identified and its spillover mechanisms and transmission pathways are fully characterized. interactions. a critical salt bridge between hace2 lys31 and glu35 and the hydrophobic residues surrounding it, based on the experimentally determined crystal structure of sars-cov rbd complexed with hace2 (pdb 2ajf). b homologous structural modeling of the hydrogen bond between mdace2 asn31 and lys35. the modeling was done in the program o [3] . c critical salt bridge between hace2 lys353 and glu38 and the hydrophobic residues surrounding it, based on the structure of sars-cov rbd complexed with hace2. d homologous structural modeling of the salt bridge between mdaace2 lys353 and sars-cov glu38 and the hydrophobic residues surrounding it. structural illustrations were prepared using the program povscript [2] evolutionary relationships between bat coronaviruses and their hosts povscript?: a program for model and data visualization using persistence of vision raytracing improved methods for building protein models in electron density maps and the location of errors in these models a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats structure of sars coronavirus spike receptor-binding domain complexed with receptor structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections bats are natural reservoirs of sars-like coronaviruses receptor and viral determinants of sars-coronavirus adaptation to human ace2 animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions coronavirus as a possible cause of severe acute respiratory syndrome full-length genome sequences of two sars-like coronaviruses in horseshoe bats and genetic variation analysis difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin mega4: molecular evolutionary genetics analysis (mega) software version 4.0 the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools determination and application of immunodominant regions of sars coronavirus spike and nucleocapsid proteins recognized by sera from different animal species intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans bats as reservoir of sars-cov progenitor virus 1569 key: cord-272010-kc0gi3cj authors: anand, sai priya; chen, yaozong; prévost, jérémie; gasser, romain; beaudoin-bussières, guillaume; abrams, cameron f.; pazgier, marzena; finzi, andrés title: interaction of human ace2 to membrane-bound sars-cov-1 and sars-cov-2 s glycoproteins date: 2020-09-29 journal: viruses doi: 10.3390/v12101104 sha: doc_id: 272010 cord_uid: kc0gi3cj severe acute respiratory syndrome virus 2 (sars-cov-2) is responsible for the current global coronavirus disease 2019 (covid-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. the viral entry of sars-cov-2 depends on an interaction between the receptor-binding domain of its trimeric spike glycoprotein and the human angiotensin-converting enzyme 2 (ace2) receptor. a better understanding of the spike/ace2 interaction is still required to design anti-sars-cov-2 therapeutics. here, we investigated the degree of cooperativity of ace2 within both the sars-cov-2 and the closely related sars-cov-1 membrane-bound s glycoproteins. we show that there exist differential inter-protomer conformational transitions between both spike trimers. interestingly, the sars-cov-2 spike exhibits a positive cooperativity for monomeric soluble ace2 binding when compared to the sars-cov-1 spike, which might have more structural restraints. our findings can be of importance in the development of therapeutics that block the spike/ace2 interaction. severe acute respiratory syndrome virus 2 (sars-cov-2) is the cause of the current and rapidly evolving coronavirus disease 2019 (covid-19) pandemic. one potential therapeutic target receiving significant attention is the interaction between the sars-cov-2 spike (s) glycoprotein and its receptor, human angiotensin-converting enzyme 2 (ace2). the s glycoprotein is a heavily glycosylated type i membrane protein present as a trimer on mature virions and consists of three s1/s2 heterodimers [1, 2] . the s1 subunit contains a receptor-binding domain (rbd) that specifically binds to ace2 and can undergo hinge-like movements to switch between an "up" position for receptor binding and "down" position for potential immune evasion [1] [2] [3] [4] . the s glycoprotein can only bind to ace2 with the rbd in the "up" state and this results in the dissociation of the trimer [5, 6] . ace2, the primary receptor for the plasmids expressing the different human coronavirus spikes (sars-cov-2 and sars-cov-1) were previously reported [7] . the d614g mutation was introduced using the quikchange ii xl site-directed mutagenesis protocol (agilent technologies, santa clara, ca, usa). the presence of the desired mutations was determined by automated dna sequencing. the plasmid encoding for soluble human ace2 (residues 1-615) fused with an 8xhistag was reported elsewhere [2] . to generate the recombinant ace2-fc fusion plasmid, dna encoding ace2 (1-615) was linked to fc segment of human igg1 and the whole fusion fragment was cloned into pacp-tag(m)-2 vector using nhei/noti as restriction sites. the vesicular stomatitis virus g (vsv-g)-encoding plasmid (psvcmv-in-vsv-g) was previously described [21] . 293t human embryonic kidney cells (obtained from atcc, manassas, va, usa) were maintained at 37 • c under 5% co 2 in dulbecco's modified eagle's medium (dmem) (wisent, st. bruno, qc, canada) containing 5% fetal bovine serum (vwr) and 100 µg/ml of penicillin-streptomycin (wisent). the generation and maintenance of 293t-ace2 cell line were previously reported [22] . freestyle 293f cells (invitrogen, rockford, il, usa) were grown in freestyle 293f medium (invitrogen) to a density of 1 × 10 6 cells/ml at 37 • c with 8% co 2 with regular agitation (150 rpm). cells were transfected with a plasmid coding for soluble ace2 or ace2-fc using expifectamine 293 transfection reagent, as directed by the manufacturer (invitrogen). one week later, cells were pelleted and discarded. supernatants were filtered using a 0.22 µm filter (thermo fisher scientific, waltham, ma, usa). the recombinant sace2 protein was purified by nickel affinity columns (invitrogen) and ace2-fc was purified using protein a affinity column (cytiva, marlborough, ma, usa), as directed by the manufacturers. the protein preparations were dialyzed against phosphate-buffered saline (pbs) and stored in aliquots at −80 • c until further use. to assess purity, recombinant proteins were loaded on sds-page gels and stained with coomassie blue. using the standard calcium phosphate method, 10 µg of spike expressor and 2 µg of a green fluorescent protein (gfp) expressor (pires-gfp) were transfected into 2 × 10 6 293t cells. to determine the hill coefficients, cells were preincubated with increasing concentrations of soluble ace2 (0 to 11,500 nm), ace2-fc (0 to 500 nm), or the monoclonal antibody cr3022 (0 to 270 nm) 48 h post-transfection. sace2 binding was detected using a polyclonal goat anti-ace2 (rnd systems). alexafluor-647-conjugated goat anti-human igg (h+l) ab (invitrogen) and alexafluor-647-conjugated donkey anti-goat igg (h+l) ab (invitrogen) were used as secondary antibodies. the percentage of transfected cells (gfp+ cells) was determined by gating the living cell population based on viability dye staining (aqua vivid, invitrogen). samples were acquired on an lsrii cytometer (bd biosciences, mississauga, on, canada) and data analysis was performed using flowjo vx.0.7 (tree star, ashland, or, usa). hill coefficient analyses were done using graphpad prism version 8.0.1 (graphpad, san diego, ca, usa). target cells were infected with single-round luciferase-expressing lentiviral particles. briefly, 293t cells were transfected by the calcium phosphate method with the lentiviral vector pnl4.3 r-e-luc (nih aids reagent program) and a plasmid encoding for sars-cov-2 spike (wt or d614g), sars-cov-1 spike, or vsv-g at a ratio of 5:4. two days after transfection, the cell supernatants were harvested. each virus preparation was frozen and stored in aliquots at −80 • c until use. 293t-ace2 target cells were seeded at a density of 1 × 10 4 cells/well in 96-well luminometer-compatible tissue culture plates (perkin elmer, waltham, ma, usa) 24 h before infection. luciferase-expressing recombinant viruses in a final volume of 100 µl were incubated with increasing concentrations of soluble ace2 (0 to 11,500 nm), ace2-fc (0 to 500 nm), or the monoclonal antibody cr3022 (0 to 270 nm) for 1 h at 37 • c and were then added to the target cells for an additional 4 hours followed by incubation for 48 h at 37 • c; the medium was then removed from each well, and the cells were lysed by the addition of 30 µl of passive lysis buffer (promega, madison, wi, usa) followed by three freeze-thaw cycles. an lb 941 tristar luminometer (berthold technologies, bad wildbad, germany) was used to measure the luciferase activity of each well after the addition of 100 µl of luciferin buffer (15 mm mgso4, 15 mm kpo4 [ph 7.8], 1 mm atp, and 1 mm dithiothreitol) and 50 µl of 1 mm d-luciferin potassium salt (prolume, pinetop, az, usa). the neutralization half-maximal inhibitory concentration (ic 50 ) represents the ligand concentration required to inhibit 50% of the infection of 293t-ace2 cells by recombinant lentiviral viruses bearing the indicated surface glycoproteins. ic 50 values were determined using a normalized non-linear regression using graphpad prism software. to better understand the interactions between membrane-bound sars-cov-1 and sars-cov-2 s glycoproteins with their receptor, human ace2, we sought to determine the cooperativity of ace2 within the respective trimers. to assess this, we calculated the hill coefficient, which is the steepness of a concentration-response curve and reflects the degree of cooperativity between a ligand and its receptor [23, 24] . briefly, hek293t cells were transfected with plasmids expressing the full-length sars-cov-1 s and sars-cov-2 s glycoproteins. we also tested the sars-cov-2 s d614g mutant that is associated with higher infectivity and is now the strain circulating worldwide [25] [26] [27] . transfected cells were incubated with increasing concentrations of sace2 and bound sace2 was revealed with an anti-ace2 antibody. these results were used to calculate the hill coefficient as indicated in material and methods. both the sars-cov-2 s and its d614g counterpart demonstrated a positive cooperativity of sace2 binding (hill coefficient > 1) ( figure 1a) , thus, indicating that the d614g mutation does not overly affect s conformation, at least regarding sace2 interaction, in line with recent findings [26, 27] . interestingly, sars-cov-1 s presented negative cooperativity (hill coefficient < 1), suggesting that the interaction of sace2 with one sars-cov-1 s protomer reduces the efficiency with which additional sace2 molecules can engage adjacent s protomers. to evaluate if the differential hill coefficients observed between sars-cov-2 and sars-cov-1 was conserved among different rbd ligands, we tested two additional rbd-binding ligands: ace2-fc, a molecule presenting two ace2 domains (residues 1-615) fused to a fc fragment, and the cr3022 monoclonal antibody, which is specific to the sars-cov-1 rbd and has been shown to cross-react strongly with the rbd of sars-cov-2 and does not compete with the binding of ace2 [28, 29] . interestingly, no differential cooperativity between sars-cov-2 and sars-cov-1 was observed for ace2-fc, suggesting that the enhanced avidity provided by ace2-fc, which allows for multiple spike protomers to bind, is able to overcome potential structural restraints present in the sars-cov-1 s ( figure 1b ). of note, we observed negative cooperativity of cr3022 for all tested s glycoproteins ( figure 1c ), in line with previous findings showing that the binding of cr3022 leads to the destruction of the prefusion sars-cov-2 s trimer [30] . thus, it is possible that the binding of cr3022 to one rbd protomer distorts the trimer structure, preventing additional cr3022 molecules from binding. next, we tested the abilities of sace2 and ace2-fc to neutralize sars-cov-1 and sars-cov-2 spike-bearing pseudovirions. we observed a higher neutralization potency of sace2 against sars-cov-2 s (wt or d614g) when compared to sars-cov-1 (table 1) . however, whether the negative cooperativity observed for the sars-cov-1 s/sace2 interaction could explain the ~5-fold lower neutralization potency of monomeric ace2 when compared to sars-cov-2 (ic50 = 245 nm for sars-cov-2 s; 1359 nm for sars-cov-1 s) ( figure 2a ; table 1 ) remains to be determined. in agreement with previous reports [18] , ace2-fc neutralized both sars-cov-1 s and sars-cov-2 s with a higher efficiency compared to monomeric sace2 ( figure 2b ; table 1 ), further supporting previous observations that ligand multimerization enhances potency by providing higher avidity [18, 20, 31, 32] . interestingly, we observed that sace2 ic50 was only reached upon its half-maximal binding to the respective s proteins, suggesting a model where the occupancy of at least two or more protomers of the sars-cov-1 s or sars-cov-2 s by sace2 is needed for virus neutralization ( figure 2d,e) . next, we tested the abilities of sace2 and ace2-fc to neutralize sars-cov-1 and sars-cov-2 spike-bearing pseudovirions. we observed a higher neutralization potency of sace2 against sars-cov-2 s (wt or d614g) when compared to sars-cov-1 (table 1) . however, whether the negative cooperativity observed for the sars-cov-1 s/sace2 interaction could explain the~5-fold lower neutralization potency of monomeric ace2 when compared to sars-cov-2 (ic 50 = 245 nm for sars-cov-2 s; 1359 nm for sars-cov-1 s) (figure 2a ; table 1 ) remains to be determined. in agreement with previous reports [18] , ace2-fc neutralized both sars-cov-1 s and sars-cov-2 s with a higher efficiency compared to monomeric sace2 ( figure 2b ; table 1 ), further supporting previous observations that ligand multimerization enhances potency by providing higher avidity [18, 20, 31, 32] . interestingly, we observed that sace2 ic 50 was only reached upon its half-maximal binding to the respective s proteins, suggesting a model where the occupancy of at least two or more protomers of the sars-cov-1 s or sars-cov-2 s by sace2 is needed for virus neutralization ( figure 2d ,e). cryo-em data collected on free (receptor-unbound) sars-cov-2 s indicate that it assumes three distinct states. preferential are the 3-rbd-down state (41%) and the 1-rbd-up state (45%) that exists at a near 1:1 ratio, whereas less preferential is the 2-rbd-up conformation (approximately 10%) [1, 16, 33] . this indicates that there is a relatively low barrier for the open-closed transition in its energy landscape. consistent with this more easy-to-open propensity of the sars-cov-2 spike, zhou et al. have identified 15% one-ace2-bound, 43% two-ace2-bound, and 38% three ace2-bound sars-cov-2 spike by single-particle cryo-em when mixing spike and ace2 in a 1:3 molar ratio [15] . the higher percentage of the two/three-ace2 bound state over the single ace2-bound state corroborates the positive cooperativity seen between monomeric receptor and sars-cov-2 trimer. interestingly, in all ace2-bound spike trimers, rbds not bound to ace2 are in the "down" conformation, suggesting that the down-to-up rearrangement of the spike trimer is the rate-limiting step in receptor binding. in contrast, available data reveal differences in the conformational dynamics of the sars-cov-1 spike which seems to have less propensity to engage multiple ace2 monomers. with a ratio of 1:3 (spike:ace2), as described by zhou et al. for the sars-cov-2 spike, the prevailing complex fraction observed for sars-cov-1 was the one ace2 monomer bound spike [34] . interestingly, the cryo-em structure of this complex revealed three distinct conformations of spike in which the loaded rbd adopted three different tilted orientations and the other two rbds remained in the down state [34] . this is different from the multiple ace2 loading of the sars-cov-2 spike which displays one essentially identical rbd up conformation. based on the structural dynamic data described above we propose a model as shown in figure 3 . according to our model, the sars-cov-1 spike has more structural restraints from the ntd, sd1, sd2 and s2 domains and, therefore, needs to overcome a higher energy barrier to open. as a result, the sars-cov-1 spike has a smaller population in the open conformation in the absence of ace2, which is in line with the unavailability of structures of multi-ace2 bound sars-cov-1 spikes and our finding that monomeric sace2 binding suppresses the opening of the other two rbds. interestingly, these differences can be detected only if monomeric ace2 is used to form the complex but not with ace2-fc. there is still a question of debate if ace2 monomer or dimer is involved in the entry process of both sars-cov-1 and sars-cov-2. if the latter, the differences we observe for monomeric ace2 resulting from the intrinsic structural features of the two coronavirus spikes which leads to different energy landscapes could be mitigated during the process of viral entry in vivo. however, whether these differences are important for antibody-mediated neutralization remains to be determined. altogether, our results show differential inter-protomer conformational transitions between sars-cov-2 and sars-cov-1 s glycoproteins upon sace2 binding. a better understanding of conformational differences between the s glycoproteins between these two beta-coronaviruses might prove useful for the development of new therapeutics and/or vaccine design. we assume conformational landscapes for both species of spikes (sars-cov-1: black; sars-cov-2: red) that permit equilibration among four distinct states: all-receptor-binding-domains (rbds) -down ("0"), one-rbd-up ("1"), two-rbds-up ("2"), and three-rbds-up ("3"). we hypothesize that the sars-cov-2 spike energetic barrier for transitioning from all-rbds-down to one-rbd-up ( * ) is lower for sars-cov-2 than for sars-cov-1, while both experience the same barrier for the reverse transition, making the one-rbd-up state more stable for sars-cov-2 than for sars-cov-1, as illustrated by the relative energy differences ∆ . however, sars-cov-1 spike likely needs to overcome substantially higher energy barriers to transit from the one-rbd-up state to the two-rbdsup state ( * ), as compared with sars-cov-2 spike. this underlying conformational selection mechanism results in a larger population of sars-cov-2 spike in two or three rbds up state and has higher chance to engage multiple ace2, which eventually spurs the complete open and dissociation of s1 from s2 and induces membrane fusion. cartoon representation of closed, one-rbd-up, two-rbds-up states of spike were generated from deposited structures in protein data bank (6zwv, 6vsb, 6 × 2b, respectively). the three-rbds-up model was generated by c3 symmetry superposition of three one-rbd-up protomer in 6vsb. we assume conformational landscapes for both species of spikes (sars-cov-1: black; sars-cov-2: red) that permit equilibration among four distinct states: all-receptor-binding-domains (rbds) -down ("0"), one-rbd-up ("1"), two-rbds-up ("2"), and three-rbds-up ("3"). we hypothesize that the sars-cov-2 spike energetic barrier for transitioning from all-rbds-down to one-rbd-up (e * 01 ) is lower for sars-cov-2 than for sars-cov-1, while both experience the same barrier for the reverse transition, making the one-rbd-up state more stable for sars-cov-2 than for sars-cov-1, as illustrated by the relative energy differences ∆e 01 . however, sars-cov-1 spike likely needs to overcome substantially higher energy barriers to transit from the one-rbd-up state to the two-rbds-up state (e * 12 ), as compared with sars-cov-2 spike. this underlying conformational selection mechanism results in a larger population of sars-cov-2 spike in two or three rbds up state and has higher chance to engage multiple ace2, which eventually spurs the complete open and dissociation of s1 from s2 and induces membrane fusion. cartoon representation of closed, one-rbd-up, two-rbds-up states of spike were generated from deposited structures in protein data bank (6zwv, 6vsb, 6 × 2b, respectively). the three-rbds-up model was generated by c3 symmetry superposition of three one-rbd-up protomer in 6vsb. author contributions: s.p.a., j.p., and a.f. conceived the study; s.p.a., j.p., and a.f. designed experimental approaches; s.p.a. and r.g. performed the experiments; s.p.a., y.c., j.p., c.f.a., m.p., and a.f. analyzed and interpreted the experiments; g.b.-b., y.c., and m.p. contributed unique reagents; s.p.a., y.c., m.p., and a.f. wrote the paper. every author has read, edited, and approved the final manuscript. all authors have read and agreed to the published version of the manuscript. funding: this work was supported by "ministère de l'économie et de l'innovation du québec, programme de soutien aux organismes de recherche et d'innovation", by the fondation du chum, by the canada's covid-19 immunity task force (citf), in collaboration with the canadian institutes of health research (cihr) and a cihr foundation grant #352417 to a.f. this work was also supported by the national institute of health grants r01 ai116274 to mp and r01 ai129769 to m.p. and a.f. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors declare no competing interests. a.f. is the recipient of a canada research chair on retroviral entry #rchs0235 950-232424. s.p.a., j.p., and g.b.b. are supported by cihr fellowships. r.g. is supported by a mitacs accélération postdoctoral fellowship. structure, function, and antigenicity of the sars-cov-2 spike glycoprotein cryo-em structure of the 2019-ncov spike in the prefusion conformation cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding distinct conformational states of sars-cov-2 spike protein structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus angiotensin-converting enzyme 2 is an essential regulator of heart function inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin furin cleavage site is key to sars-cov-2 pathogenesis structural basis of receptor recognition by sars-cov-2 a ph-dependent switch mediates conformational masking of sars-cov-2 spike structures and distributions of sars-cov-2 spike proteins on intact virions characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine trimeric sars-cov-2 spike interacts with dimeric ace2 with limited intra-spike avidity sars-cov-2 and three related coronaviruses utilize multiple ace2 orthologs and are potently blocked by an improved ace2-ig neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig the membrane-proximal intracytoplasmic tyrosine residue of hiv-1 envelope glycoprotein is critical for basolateral targeting of viral budding in mdck cells cross-sectional evaluation of humoral responses against sars-cov-2 spike the hill equation revisited: uses and misuses cooperativity in binding processes: new insights from phenomenological modeling tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus decline of humoral responses against sars-cov-2 spike in convalescent individuals the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants neutralization of sars-cov-2 by destruction of the prefusion spike the sequence of human ace2 is suboptimal for binding the s spike protein of sars coronavirus 2 engineering human ace2 to optimize binding to the spike protein of sars coronavirus 2 controlling the sars-cov-2 spike glycoprotein conformation cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 the authors thank m. gordon joyce (u.s. mhrp) for the monoclonal antibody cr3022. the authors declare no conflict of interest. the views expressed in this presentation are those of the authors and do not reflect the official policy or position of the uniformed services university, us army, the department of defense, or the us government.viruses 2020, 12, 1104 key: cord-262958-tmp6yxlv authors: pinto, dora; park, young-jun; beltramello, martina; walls, alexandra c.; tortorici, m. alejandra; bianchi, siro; jaconi, stefano; culap, katja; zatta, fabrizia; de marco, anna; peter, alessia; guarino, barbara; spreafico, roberto; cameroni, elisabetta; case, james brett; chen, rita e.; havenar-daughton, colin; snell, gyorgy; telenti, amalio; virgin, herbert w.; lanzavecchia, antonio; diamond, michael s.; fink, katja; veesler, david; corti, davide title: structural and functional analysis of a potent sarbecovirus neutralizing antibody date: 2020-04-09 journal: biorxiv doi: 10.1101/2020.04.07.023903 sha: doc_id: 262958 cord_uid: tmp6yxlv sars-cov-2 is a newly emerged coronavirus responsible for the current covid-19 pandemic that has resulted in more than one million infections and 73,000 deaths1,2. vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. the sars-cov-2 spike (s) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. here we describe multiple monoclonal antibodies targeting sars-cov-2 s identified from memory b cells of a sars survivor infected in 2003. one antibody, named s309, potently neutralizes sars-cov-2 and sars-cov pseudoviruses as well as authentic sars-cov-2 by engaging the s receptor-binding domain. using cryo-electron microscopy and binding assays, we show that s309 recognizes a glycan-containing epitope that is conserved within the sarbecovirus subgenus, without competing with receptor attachment. antibody cocktails including s309 along with other antibodies identified here further enhanced sars-cov-2 neutralization and may limit the emergence of neutralization-escape mutants. these results pave the way for using s309 and s309-containing antibody cocktails for prophylaxis in individuals at high risk of exposure or as a post-exposure therapy to limit or treat severe disease. sars-cov-2 is a newly emerged coronavirus responsible for the current covid-23 19 pandemic that has resulted in more than one million infections and 73,000 24 deaths 1,2 . vaccine and therapeutic discovery efforts are paramount to curb the 25 pandemic spread of this zoonotic virus. the sars-cov-2 spike (s) glycoprotein 26 promotes entry into host cells and is the main target of neutralizing antibodies. 27 here we describe multiple monoclonal antibodies targeting sars-cov-2 s 28 identified from memory b cells of a sars survivor infected in 2003. one 29 antibody, named s309, potently neutralizes sars-cov-2 and sars-cov 30 pseudoviruses as well as authentic sars-cov-2 by engaging the s receptor-31 binding domain. using cryo-electron microscopy and binding assays, we show 32 that s309 recognizes a glycan-containing epitope that is conserved within the 33 sarbecovirus subgenus, without competing with receptor attachment. antibody 34 cocktails including s309 along with other antibodies identified here further 35 enhanced sars-cov-2 neutralization and may limit the emergence of 36 neutralization-escape mutants. these results pave the way for using s309 and 37 s309-containing antibody cocktails for prophylaxis in individuals at high risk of 38 exposure or as a post-exposure therapy to limit or treat severe disease. coronavirus entry into host cells is mediated by the transmembrane spike (s) 41 glycoprotein that forms homotrimers protruding from the viral surface 3 . the s 42 glycoprotein comprises two functional subunits: s1 (divided into a, b, c and d domains) 43 that is responsible for binding to host cell receptors and s2 that promotes fusion of the 44 viral and cellular membranes 4,5 . both sars-cov-2 and sars-cov belong to the 45 sarbecovirus subgenus and their s glycoproteins share 80% amino acid sequence 46 identity 6 . sars-cov-2 s is closely related to the bat sars-related cov (sarsr-cov) 47 ratg13 with which it shares 97.2% amino acid sequence identity 1 . we and others 48 recently demonstrated that human angiotensin converting enzyme 2 (hace2) is a 49 functional receptor for sars-cov-2, as is the case for sars-cov 1,6-8 . the ranging between 1.4 and 6,100 ng/ml, and 0.8 and 254 ng/ml, respectively (fig. 1a-94 b). mabs were further evaluated for binding to the sars-cov-2 and sars-cov s b 95 domains as well as to the prefusion-stabilized oc43 s 28 , mers-cov s 29,30 , sars-96 cov s 30 and sars-cov-2 s 6 ectodomain trimers. none of the mabs studied bound to 97 prefusion oc43 s or mers-cov s ectodomain trimers, indicating a lack of cross-98 reactivity outside the sarbecovirus subgenus (extended data fig.1) . mabs s303, 99 s304, s309 and s315 recognized the sars-cov-2 and sars-cov rbds. in 100 particular, s309 bound with nanomolar affinity to both s b domains, as determined by 101 biolayer interferometry (fig. 1c-d, extended data fig. 2) . unexpectedly, s306 and 102 s310 stained cells expressing sars-cov-2 s at higher levels than those expressing 103 sars-cov s, yet it did not interact with sars-cov-2 or sars-cov s ectodomain 104 trimers and rbd constructs by elisa. these results suggest that they may recognize 105 post-fusion sars-cov-2 s, which was recently proposed to be abundant on the 106 surface of authentic sars-cov-2 viruses 31 (fig. 1a-b and extended data fig.3) . 107 the s 3-fold molecular axis. cdrh3 and cdrl2 sandwich the sars-cov-2 s glycan 142 at position n343 through contacts with the core fucose moiety (in agreement with the 143 detection of sars-cov-2 n343 core-fucosylated peptides by mass-spectrometry 34 ) 144 and to a lesser extent with the core n-acetyl-glucosamine (fig. 2d) . these latter 145 interactions bury an average surface of ~170 å 2 and stabilize the n343 oligosaccharide 146 which is resolved to a much larger extent than in the apo sars-cov-2 s structures 6,9 . 147 the structural data explain the s309 cross-reactivity between sars-cov-2 and 148 sars-cov as 19 out of 24 residues of the epitope are strictly conserved ( fig. 2f and 149 extended data fig. 6a to further investigate the mechanism of s309-mediated neutralization, we 175 compared side-by-side transduction of sars-cov-2-mlv in the presence of either 176 s309 fab or s309 igg. both experiments yielded comparable ic50 values (3.8 and 3.5 177 nm, respectively), indicating similar potencies for igg and fab (fig. 3d) . however, the 178 s309 igg reached 100% neutralization, whereas the s309 fab plateaued at ~80% 179 neutralization (fig. 3d) . this result indicates that one or more igg-specific bivalent 180 mechanisms, such as s trimer cross-linking, steric hindrance or aggregation of 181 virions 37 , may contribute to the ability to fully neutralize pseudovirions. to gain more insight into the epitopes recognized by our panel of mabs, we 205 used structural information, escape mutants analysis 23,27,30 , and biolayer 206 inteferometry-based epitope binning to map the antigenic sites present on the sars-207 cov and sars-cov-2 s b domains ( fig. 4a and extended data fig.10 ). this analysis 208 identified at least four antigenic sites within the s b domain of sars-cov targeted by 209 our panel of mabs. the receptor-binding motif, which is targeted by s230, s227 and 210 s110, is termed site i. sites ii and iii are defined by s315 and s124, respectively, and 211 the two sites were bridged by mab s304. site iv is defined by s309, s109, and s303 212 mabs. given the lower number of mabs cross-reacting with sars-cov-2, we were 213 able to identify sites iv targeted by s309 and s303, and site ii-iii targeted by s304 and 214 s315 (fig. 4b) . movie frame alignment, estimation of the microscope contrast-transfer function 439 parameters, particle picking and extraction were carried out using warp 49 . particle 440 images were extracted with a box size of 800 binned to 400 yielding a pixel size of 1.05 441 å. for each data set two rounds of reference-free 2d classification were performed 442 using cryosparc 50 to select well-defined particle images. subsequently, two rounds 443 of 3d classification with 50 iterations each (angular sampling 7.5˚ for 25 iterations and 444 1.8˚ with local search for 25 iterations), using our previously reported closed sars-445 cov-2 s structure 6 as initial model, were carried out using relion 51 without imposing 446 symmetry to separate distinct sars-cov-2 s conformations. 3d refinements were 447 carried out using non-uniform refinement along with per-particle defocus refinement in 448 cryosparc 50 . particle images were subjected to bayesian polishing 52 before 449 performing another round of non-uniform refinement in cryosparc 50 followed by per-450 particle defocus refinement and again non-uniform refinement. supplemented with 30% glycerol. the dataset was collected at als beamline 5.0.2 472 and processed to 3.3 å resolution in space group p41212 using mosflm 64 and 473 aimless 65 . the structure of fab s309 was solved by molecular replacement using 474 phaser 66 and homology models as search models. the coordinates were improved 475 and completed using coot 55 and refined with refmac5 67 . crystallographic data 476 collection and refinement statistics are shown in table 3 . s309 mab. a-b , ribbon diagrams of s309 and ace2 bound to sars-cov-2 s b . this composite model was generated using the sars-cov-2 s/s309 cryoem structure reported here and a crystal structure of sars-cov-2 s bound to ace2 16 . c, competition of s309 or s230 mabs with ace2 to bind to sars-cov s b (left panel) and sars-cov-2 s b (right panel). ace2 was immobilized at the surface of biosensors before incubation with s b domain alone or s b precomplexed with mabs. the vertical dashed line indicates the start of the association of mab-complexed or free s b to solid-phase ace2. d, neutralization of sars-cov-mlv by s309 igg1 or s309 fab, plotted in nm (means ±sd is shown, one out of two experiments is shown). e, mab-mediated adcc using primary nk effector cells and sars-cov-2 s-expressing expicho as target cells. bar graph shows the average area under the curve (auc) for the responses of 3-4 donors genotyped for their fcgriiia (mean±sd, from two independent experiments). f, activation of high affinity (v158) or low affinity (f158) fcgriiia was measured using jurkat reporter cells and sars-cov-2 sexpressing expicho as target cells (one experiment, one or two measurements per mab). g, mab-mediated adcp using cell trace violet-labelled pbmcs as phagocytic cells and pkf67labelled sars-cov-2 s-expressing expicho as target cells. bar graph shows the average area under the curve (auc) for the responses of four donor (mean±sd, from two independent experiments). h, activation of fcgriia measured using jurkat reporter cells and sars-cov-2 s-expressing expicho as target cells (one experiment, one or two measurements per mab). fig. 9) . b, competition of mab pairs for binding to the sars-cov-2 s b domain . c-d, neutralization of sars-cov-2-mlv by s309 combined with an equimolar amount of s304 or s315 mabs. for mab cocktails the concentration on the x axis is that of the individual mabs. table 1 : characteristics of the antibodies described in this study. vh and vl % identity refers to v gene identity compared to germline (imgt). isolation and characterization of a bat 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function of antibodies elicited 632 by zika virus infection enhanced antibody half-life improves in vivo activity potent binding of 2019 novel coronavirus spike protein by a sars 637 coronavirus-specific human monoclonal antibody automated molecular microscopy: the new leginon system addressing preferred specimen orientation in single-particle 642 cryo-em through tilting real-time cryo-electron microscopy data 645 preprocessing with warp cryosparc: 648 algorithms for rapid unsupervised cryo-em structure determination new tools for automated high-resolution cryo-em structure 651 determination in relion-3 a bayesian approach to beam-654 induced motion correction in cryo-em single-particle analysis prevention of overfitting in cryo-em structure 657 determination visualizing density maps with ucsf 659 chimera features and development 662 of coot tools for macromolecular model building and refinement into 665 electron cryo-microscopy reconstructions automated structure refinement of macromolecular 668 assemblies from cryo-em maps using rosetta automatically fixing errors in glycoprotein structures with 672 molprobity: all-atom structure validation for macromolecular 674 crystallography emringer: side chain-directed model and map validation for 677 3d cryo-electron microscopy macromolecular structure determination using x-rays, 681 neutrons and electrons: recent developments in phenix privateer: software for the conformational validation of 684 carbohydrate structures ucsf chimerax: meeting modern challenges in 687 visualization and analysis 689 imosflm: a new graphical interface for diffraction-image processing with 690 mosflm how good are my data and what is the 693 resolution? phaser crystallographic software refmac5 for the refinement of macromolecular crystal 698 structures identifying sars-cov-2 related coronaviruses in malayan 701 pangolins key: cord-268468-036i1082 authors: asif, muhammad; ajmal, muhammad; ashraf, ghazala; muhammad, nadeem; aziz, ayesha; iftikhar, tayyaba; wang, junlei; liu, hongfang title: the role of biosensors in covid-19 outbreak date: 2020-09-18 journal: curr opin electrochem doi: 10.1016/j.coelec.2020.08.011 sha: doc_id: 268468 cord_uid: 036i1082 herein, we have summarized and argued about biomarkers and indicators used for the detection of sars-cov-2. antibody detection methods are not considered suitable to screen individuals at early stages and asymptomatic cases. the diagnosis of covid-19 using biomarkers and indicators at point of care level is much crucial. therefore, it is urgently needed to develop rapid and sensitive detection methods which can target antigens. we have critically elaborated key role of biosensors to cope the outbreak situation. in this review, the importance of biosensors including electrochemical, surface enhanced raman scattering, field-effect transistor and surface plasmon resonance biosensors in the detection of sars-cov-2 has been underscored. finally, we have outlined pros and cons of diagnostic approaches as-well-as future directions. the coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was declared as pandemic on 13 march 2020. it has not only come to be the leading cause of mortalities around the globe but has also become the unexpected socioeconomic burden [1] . since the covid-19 outbreak reported in early december 2019 in wuhan, millions of people have been infected, thousands of them died and even the economy of many countries has halted [2] . the transmission of virus may occur through breathing, aerosols particles and direct touching of abiotic surfaces. there are also evidences of virus transmission through fecal since the sars-cov-2 has been found in feces samples. the transfer of virus through asymptomatic patients has also been observed in many cases [3, 4] . in this regards; world health organization (who) has urged the scientific community to carry out huge amount of diagnostic tests to curb the spread of virus since testing is an important tool to understand the epidemiology of the outbreak. furthermore, fast diagnostic testing is very crucial in making prompt decisions to treat and isolate the infected patients which can ultimately slow down the transmission of infectious disease. the testing platforms together with the risk management and the healthcare system are vital responses in all outbreaks. in this outbreak, three different types of diagnosis tests are being used including (i) chest ct scan along with clinical indications, (ii) rna detection using rt-pcr assay and (iii) lateral flow assays, full automatic chemiluminescence method, enzyme-linked immunosorbent assay (elisa) for the determination of antibodies [5] . nevertheless, there are some drawbacks related to ct scan method such as the use of ct scan diagnosis is limited to big hospitals, rural hospitals do not have the facility of ct scan, well-trained radiologists are required to analyze the images of ct scan and ct scan method cannot distinguish whether the infection is caused by sars-cov-2 or any other virus. on contrary, rt-pcr is a time consuming assay that may take four hour to execute one test and possesses great possibility of false-negative results. the patients with initial false-negative test results can transmit the virus to healthy individuals while preventing the proper control of infection. in the meantime, antibody response appears at about 10 th day after the onset of symptoms so all the assays that can target antibodies cannot be reliable in case of early diagnosis and identification of asymptomatic individuals. the false-positive results are also most likely owing to the interference caused by other proteins that are present in serological samples. the more precise and targeted detection of the virus can be carried out using biosensor based approaches. the technology that exists behind the testing is biosensing platforms that apply the strategy of bio-recognition elements or binding target molecules in a particular way for the detection of biological analytes [6, 7] . this type of binding acts as transducers which create measurable signals either directly through impedance measurements, surface plasmon resonance, or labeling the molecules such as enzymes/optical compounds [8, 9] . in this review, we have summarized the biosensor based technologies which are able to detect sars-cov-2 effectively. there are several biomarkers and indicators that can be used for the detection of sars-cov-2. single-stranded rna is very crucial biomarker which is used to detect sars-cov-2. generally, conserved or fully expressed genes are the desired targets for rt-pcr assays [5, 10] . there are some special primers that can specifically target these genes with high sensitivity in detecting sars-cov-2 while rule out the detection of other similar types of viruses including mers, (oc43 and 229e) and influenza [11] . three different kinds of novel rt-pcr assays were developed that targeted n and s genes of sars-cov-2 and also compared with each other [12] . the excellent sensitivity was achieved while using nasopharyngeal samples and no crossreactivity with other coronaviruses was observed. the diagnosis of covid-19 may also be carried out by using the structural proteins such as s, m, e and n proteins ( figure 1a ) as antigens. expectedly, sars-cov-2 has 28 different types of proteins [13] . it has been reported in several studies that m and e proteins are very crucial in assembling the virus structure [14] . the s protein is of much importance that combines with host j o u r n a l p r e -p r o o f cells and receptor-binding domain of s protein interacts with ace2 receptors. it is more likely that s and n proteins could be imperative antigen biomarkers which might be employed for the detection of sars-cov-2 because the same proteins have also been used previously in various methods for the detection of sars-cov [15, 16] . recently, jiang et al. fabricated the proteome microarray using 18 out of 28 proteins and employed to monitor antibody responses. the patients in recovering stage show full antibodies response to proteome particularly to protein n, s1 but not s2 [17] . moreover, detection technique based on viral proteins has also been investigated using lateral-flow assays for the diagnosis of covid-19. the diagnosis of covid-19 can also be carried out by detecting specific antibodies to sars[20] . till this end, the diagnosis of covid-19 using antibodies detection assays is not reliable. the antibody detection can be helpful for the patients at recovering phase as well as much crucial for the design of vaccine since it exact level correlates with virus neutralization titre [18]. there are some other biomarkers as well that can be monitored for the diagnosis of covid-19 including blood and urine samples, infection index, hemagglutination level, blood gas index, and cytokine levels. table 1 a biosensor is defined as an analytical tool consisting of a transducer portion and a biological element. besides the clinically employed approaches for the diagnostics purposes in hospitals, various biosensor based technologies are being developed and some have already been established for the diagnosis of covid-19 pneumonia. figure 1b shows the schematic illustration of currently used diagnostic techniques and possible biosensing platforms for covid-19, (i) covid-19 patient, (ii) sampling ways, iii) biomarkers and indicators, (iv) diagnostic methods, (v) promising biosensors. biosensors being capable for continuous monitoring of biomarkers would be potential candidates for diagnosing covid-19 patients with mild to critical conditions and evaluating the success rate of anti-inflammation therapies [22] . though the nucleic acid testing and antibody detection using rt-pcr and elisa respectively, have been well-developed but these approaches still suffer from some practical limitations. therefore, biosensors are the ideal alternative tools which show rapid response, high accuracy, the surface plasmonic resonance (spr) biosensors are now essential tools and have obtained the key role in characterizing and quantifying bio-analytical targets both in life science and pharmaceutical research. these biosensors are label-free, highly sensitive and can be applied to different types of clinically interested target analytes. the spr biosensors have also been used for the detection of antibody of sars-cov using a protein which was created by genetically fusing gold binding polypeptides to a sars coronaviral surface antigen [27] . recently, masson's research group has reported the use of human serum sample without dilution for the detection of nucleocapsid antibodies which are specific against the sars-cov-2 employing spr biosensing technology [28] . the peptide monolayer was successfully coated on spr biosensor and further functionalized with virus nucleocapsid protein which was finally able to detect sars-cov-2 antibodies at nanomolar level. the portable spr instrument was used to carry out the bioassay. the working mechanism is that when the sensor is exposed to sars-cov-2, the immune system gives response by expressing antibodies at levels which can be considering the availability of current diagnostic approaches, field-effect transistor (fet)-based biosensing platforms have many promising benefits such as capability to be very sensitive and to detect small volume of target analyte instantaneously. these biosensors have potential use in clinical analysis, point-of-care tests, and on-site diagnostics [30] . graphene with the hexagonal carbon atoms exposed on its surface, being electronically conductive, having high charge mobility and specific surface area, has proved to be ultrasensitive in sensing systems owing to its capability to detect nearby variations on their surface and to provide an ideal sensing platform. therefore, graphene-based fet biosensors are very important to carry out the immunological diagnosis with high sensitivity. in this regard, seo and co-workers have successfully fabricated a device based on fet technology for the detection of sars-cov-2 in clinical specimens as shown in figure 2b [31]. the graphene sheets of the fet were conjugated with specific antibodies against sars-cov-2 spike protein in order to construct the biosensor. the sensing aptitude of the biosensor was evaluated employing antigen protein, self-cultured virus, and nasopharyngeal swab samples taken from people infected with covid-19 pneumonia. the fet biosensor was able to detect sarscov-2 spike protein 1 fg/ml in phosphate-buffersaline and 100 fg/ml clinical transport medium. additionally, fet biosensor performed very well in detection of sarcov-2 in self-cultured medium and nasopharyngeal swab samples with detection limits of 1.6 × 10 1 plaque-forming units/ml (pfu/ml) and 2.42 × 10 2 copies/ml. interestingly, the fabricated biosensing device showed no any quantifiable cross-reactivity with mers-cov antigen. the [39] . the low possible concentration detected with this biosensor was 1×10 7 copies/ml for these three viruses. the sers spectra enabled the discrimination of viruses. j o u r n a l p r e -p r o o f the previous treatment of sars-cov-1 and mers patients with corticosteroids gave disparate results that is why who has discouraged the usage of corticosteroids for covid-19 treatment. however some reports show good results if they are administered at cautious doses [47, 48] . kinetic profile of cytokine like il-6 can convey important data which further can guide the onset of corticosteroids therapy and regulate the doses to lower the inflammation while keeping side effects at minimum level [49] . the severe covid-19 infected people have high level of lymphopenia and a proinflammatory cytokine storm compared with mild infection persons. table 3 shows some other anti-inflammatory therapies that can take advantages from biosensorbased guide for administration of hydroxychloroquine, [50] immunoglobulins, azithromycin [51] and convalescent plasma treatments [52] . other drugs such as tocilizumab [53] or anakinra [54] have also anti-inflammatory effects for covid-19. the mechanism of these treatments is to halt the particular pro-inflammatory signaling pathways. these drugs can be administrated using some analogous techniques "companion diagnostics" which are used for cancer care. the exact monitoring of specific pro-inflammatory factors would guide how to administrate these drugs. for instance, the mechanism of tocilizumab is to bind with receptor il-6 and hinder the interaction with membrane binding that consequently stops the stimulation of downstream janus kinase accountable for signal cascading [55] . these blockers of il6-mediated inflammatory response including tocilizumab and sarilumab must be directed by measuring il-6 [56, 57] . it is well-documented that antibodies are crucial in the treatment of unwanted cytokine excrete conditions in immune anti-cancer treatments. moreover, measuring serial il-6 shows that after appropriately administrating tocilizumab there is small upsurge in il-6 following the decrease in time [56] . the aforementioned studies show that the improvement of inflammatory infections can be monitored by executing kinetic measurements of biomarkers. pandemic. the wearable biosensors are able to monitor the patients continuously, a much-desired feature of biosensors. the authors declare no conflict of interest. dear editor, professor zbigniew stojek, it gives us a lot of pleasure to submit the enclosed manuscript entitled "the role of biosensors in covid-19 outbreak: a topical mini review" to current opinion in electrochemistry. it is hereby made sure that the manuscript's approval and consent have been taken from all authors and no author claims any conflict of interest. it is also made sure that the research work we are going to submit is not under any review or publication process in any of the journals. therefore, we are hopeful that this work can turn out as an effective contribution to covid-19 diagnostics. we wish that it could be published in current opinion in electrochemistry. your efforts in reviewing the manuscript are greatly appreciated. thanking you in anticipation. sincerely yours, papers of particular interest, published within the period of review, have been highlighted as: * of special interest * * of outstanding interest covid-19 navigating the uncharted trends and innovations in biosensors for covid-19 mass testing evidence of sars-cov-2 infection in returning travelers from a familial cluster of infection associated with the 2019 novel coronavirus indicating possible person-to-person transmission during the incubation period molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia metal oxide intercalated layered double hydroxide nanosphere: with enhanced electrocatalyic activity towards h 2 o 2 for 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covid-19: results of an open-label non-randomized clinical trial effectiveness of convalescent plasma therapy in severe covid-19 patients the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality continuous intravenous anakinra infusion to calm the cytokine storm in macrophage activation syndrome can we use interleukin-6 (il-6) blockade for coronavirus disease 2019 (covid-19)-induced cytokine release syndrome (crs)? tocilizumab treatment in covid----19: a single center experience why tocilizumab could be an effective treatment for severe covid-19? key: cord-262276-5nue46dm authors: roussel, yanis; giraud-gatineau, audrey; jimeno, marie-thérèse; rolain, jean-marc; zandotti, christine; colson, philippe; raoult, didier title: sars-cov-2: fear versus data date: 2020-03-19 journal: int j antimicrob agents doi: 10.1016/j.ijantimicag.2020.105947 sha: doc_id: 262276 cord_uid: 5nue46dm sars-cov-2, the novel coronavirus from china, is spreading around the world, causing a huge reaction despite its current low incidence outside china and the far east. four common coronaviruses are in current circulation and cause millions of cases worldwide. this article compares the incidence and mortality rates of these four common coronaviruses with those of sars-cov-2 in organisation for economic co-operation and development countries. it is concluded that the problem of sars-cov-2 is probably being overestimated, as 2.6 million people die of respiratory infections each year compared with less than 4000 deaths for sars-cov-2 at the time of writing. coronaviridae represent a very important family of animal and human viruses [1 , 2] that are in permanent circulation. four common human coronaviruses (hku1, nl63, oc43 and e229) cause 10-20% of respiratory infections worldwide and are present in all continents [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] ( table 1 ) . mortality is poorly assessed, but it is clear that there are chronic carriers as well as asymptomatic carriers. studies have shown that there are as many asymptomatic carriers as symptomatic patients [3 , 9] . three epidemic episodes of emerging coronaviruses have been reported. the first, severe acute respiratory syndrome (sars) coronavirus, had very little impact on global morbidity and mortality, with more than 80 0 0 recognized cases and 774 deaths [15 , 16] . the second, middle east respiratory syndrome (mers)-coronavirus, remained localized in saudi arabia, with a small epidemic of mainly nosocomial infections in south korea [17] . mers-coronavirus, like sars-coronavirus, highlighted the major danger of nosocomial transmission to healthcare personnel, the health of whom is essential in these epidemics [18] . finally, sars-cov-2, the novel coronavirus that appeared in december 2019, has expanded and has now affected more than 90 0 0 0 people worldwide [2 , 19 , 20] . at the time of writing, a significant number of cases had occurred in the far east. it is incontestably contagious, as a quasi-experimental study on the diamond princess cruise ship showed that confinement of infected patients with uninfected patients resulted in rapid infection of the uninfected patients, leading to 700 additional cases on board [21] . however, coronaviruses in common circulation remain predominant because of their global distribution and their non-negligible mortality [14 , 22] . the aim of this study was to share the experience of a reference laboratory representing approximately 1% of serious and diagnosed respiratory infections, particularly seasonal, in france. this will allow the evaluation of the relative mortality of different human coronaviruses presented in hospitals in marseille compared with that of sars-cov-2. assistance publique-hôpitaux de marseille (ap-hm) covers all public hospitals in marseille, including four university hospitals: la timone hospital, conception hospital, north hospital and south hospital; this corresponds to 3400 beds and 125 000 admissions each year [23] . the ihu méditerranée infection diagnostic laboratory tests all samples from ap-hm in which respiratory viruses are suspected. molecular biology is used for diagnosis. the results are monitored by a weekly automated surveillance system in the diagnostic laboratory, coupled with a laboratory informa[10] guangzou, china 13 048 244 (2.25%) zeng [11] guangzou, china 11 399 489 (4.3%) killerby [14] usa 18 806 2.2% kiyuka [13] kenya 5573 10.1% owusu [9] ghana 593 cases 13.7% dube [30] south africa 620 controls 214 (tuberculosis) 10 .5% 8% sipulwa [12] kenya 417 8.4% subramoney [31] south africa 860 4.8% nunes [32] south africa 1026 15% le-viet [3] vietnam 378 4 (1.05%) tion system (nexlabs) [23] . sars-cov-2 epidemiological data were obtained through an online platform gathering data from public agencies [24] . statistical analyses were performed using biostatgv software. in 2016, there were 594 0 0 0 deaths in france; 59.2% of these deaths occurred in a care establishment [25] . in the same year, ap-hm reported 2854 deaths. as such, it can be estimated that approximately 0.8% of deaths in care establishments in france occurred in ap-hm hospitals. this estimate provides an approximation of the number of people affected by a pathogen in france according to the number of people who died each year at ap-hm hospitals. from 1 january 2013 to 31 december 2019, 21 662 samples were tested by the ihu méditerranée infection diagnostic laboratory. among these, 770 samples were positive for coronavirus, with eight deaths (mortality rate 1%). among identified coronaviruses, 63 were identified as hku1 (one death, mortality rate 1.6%), 74 were identified as nl63 (two deaths, mortality rate 2.7%), 92 were identified as e229 (one death, mortality rate 1.1%) and 160 were identified as oc43 (four deaths, mortality rate 2.5%). three hundred and eighty-one coronaviruses, diagnosed before 2017, were not assigned to any of these four strains ( table 2 ) . systematic testing (molecular biology) for sars-cov-2 was performed from 1 january 2020 to 2 march 2020. in total, 7059 samples from patients presenting with infectious symptoms were tested by the ihu méditerranée infection diagnostic laboratory. among them, 543 samples were positive for coronaviruses, with two deaths (mortality rate 0.36%): 277 samples were hku1, 146 samples were nl63, 77 samples were oc43 and 43 samples were 229e. no cases of sars-cov-2 were identified among these samples. of the two deaths, one patient had oc43 (mortality rate 1.3%) and one patient had hku1 (mortality rate 0.36%). there were no deaths from nl63 or e229 during this period. over the same period, ihu méditerranée infection was the regional centre for detection of the novel coronavirus sars-cov-2. at the time of writing, 596 analyses have been performed on suspected cases since the emergence of the novel pathogen, from which four cases of sars-cov-2 have been identified. in addition, 337 french nationals returning from hubei province have been tested twice, and all were negative for sars-cov-2. by 2 march 2020, a total of 90 307 patients have tested positive for sars-cov-2 worldwide, with 3086 deaths (mortality rate 3.4%). among the organisation for economic co-operation and development (oecd) countries, 7476 patients have tested positive for sars-cov-2, with 96 deaths (mortality rate 1.3%) ( table 3 ). in france, 191 people have tested positive for sars-cov-2, with three deaths (mortality rate 1.6%). this study compared the mortality rate of sars-cov-2 in oecd countries (1.3%) with the mortality rate of common coronaviruses identified in ap-hm patients (0.8%) from 1 january 2013 to 2 march 2020. chi-squared test was performed, and the p -value was 0.11 (not significant). this study found that the mortality rate of common coronavirus infections is 0.8% in france. in comparison, the mortality rate of sars-cov-2 in european or american developed countries of a comparable economic level is 1.3% ( table 3 ). if the extrapolation of deaths in ap-hm hospitals is correct, in metropolitan france, this would represent 543/0.8 * 100 = 67 875 cases of patients hospitalized with a respiratory infection with common coronaviruses in 2 months, which is almost as many cases as for sars-cov-2 worldwide. in fact, mortality from respiratory infections is extremely dependent on the quality of care and access to care, and [27] . there is little chance that the emergence of sars-cov-2 could change this statistic significantly. fear could have a larger impact than the virus itself; a case of suicide motivated by the fear of sars-cov-2 has been reported in india [28] . in addition, coronaviruses that have rarely been tested systematically around the world may persist in the pharynx of asymptomatic people, representing a potential source of population immunity [29] . furthermore, it should be noted that systematic studies of other coronaviruses (but not yet for sars-cov-2) have found that the percentage of asymptomatic carriers is equal to or even higher than the percentage of symptomatic patients. the same data for sars-cov-2 may soon be available, which will further reduce the relative risk associated with this specific pathology. funding: this work was supported by the french government under the 'investments for the future' programme managed by the national agency for research, méditerranée-infection 10-iahu-03. ethical approval: not applicable. testing of repatriates was approved by the ethical board of the committee for the protection of persons (cpp ile de france vi, 6 february 2020). from sars coronavirus to novel animal and human coronaviruses characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention [published online ahead of print prospective case-control analysis of the aetiologies of acute undifferentiated fever in vietnam severe respiratory illness outbreak associated with human coronavirus nl63 in a long-term care facility intensive care admission for coronavirus oc43 respiratory tract infections human coronavirus-hku1 infection among adults in cleveland human coronavirus and severe acute respiratory infection in southern brazil coronavirus hku1 and other coronavirus infections in hong kong human coronaviruses associated with upper respiratory tract infections in three rural areas of ghana epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of hcov-oc43 during 2010-2015 in guangzhou epidemiology and clinical characteristics of human coronaviruses oc43, 229e, nl63, and hku1: a study of hospitalized children with acute respiratory tract infection in guangzhou, china molecular characterization of human coronaviruses and their circulation dynamics in kenya human coronavirus nl63 molecular epidemiology and evolutionary patterns in rural coastal kenya human coronavirus circulation in the united states the severe acute respiratory syndrome sars and mers: recent insights into emerging coronaviruses isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission world health organization. coronavirus disease (covid-19) outbreak, geneva: who novel coronavirus pneumonia emergency response epidemiology team the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china covid-19 outbreak on the diamond princess cruise ship: estimating the epidemic potential and effectiveness of public health countermeasures outbreaks of human coronavirus in a pediatric and neonatal intensive care unit real-time microbiology laboratory surveillance system to detect abnormal events and emerging infections institut national de la statistique et des études économiques. 594 0 0 0 personnes décédées en france en 2016, pour un quart d'entre elles à leur domicile the nasopharyngeal microbiota in patients with viral respiratory tract infections is enriched in bacterial pathogens collaborators global, regional, and national age-sex-specific mortality and life expectancy fear of covid 2019: first suicidal case in india! coronavirus infections: epidemiological, clinical and immunological features and hypotheses. cell stress, in press [published online ahead of print respiratory microbes present in the nasopharynx of children hospitalised with suspected pulmonary tuberculosis in cape town, south africa human bocavirus, coronavirus, and polyomavirus detected among patients hospitalised with severe acute respiratory illness in south africa clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in hiv-infected and hivuninfected south african children key: cord-274122-n9jnu2ah authors: mielech, anna m.; kilianski, andy; baez-santos, yahira m.; mesecar, andrew d.; baker, susan c. title: mers-cov papain-like protease has deisgylating and deubiquitinating activities date: 2014-02-01 journal: virology doi: 10.1016/j.virol.2013.11.040 sha: doc_id: 274122 cord_uid: n9jnu2ah coronaviruses encode papain-like proteases (plpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (dub)/deisgylating activity, which is hypothesized to modify the innate immune response to infection. here, we investigate the predicted dub activity of the plpro domain of the recently described middle east respiratory syndrome coronavirus (mers-cov). we found that expression of mers-cov plpro reduces the levels of ubiquitinated and isgylated host cell proteins; consistent with multifunctional plpro activity. further, we compared the ability of mers-cov plpro and severe acute respiratory syndrome coronavirus (sars-cov) plpro to block innate immune signaling of proinflammatory cytokines. we show that expression of sars-cov and mers-cov plpros blocks upregulation of cytokines ccl5, ifn-β and cxcl10 in stimulated cells. overall these results indicate that the plpro domains of mers-cov and sars-cov have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis. middle east respiratory syndrome coronavirus (mers-cov) is a recently described coronavirus with high mortality. as of november 18, 2013, there have been 157 confirmed cases and 66 deaths (http://www.who.int/csr/don/2013_11_18/en/index.html). mers disease is characterized primarily by respiratory symptoms but several patients also developed renal failure drosten et al., 2013) . in most cases reported thus far, immunosuppression or other types of medical disorders have been associated with more severe disease (assiri et al., 2013) . the sequence of the rna genome of mers-cov is most similar to bat coronaviruses hku4 and hku5 ; however, the origin of mers-cov is not known. a recent report showed that dromedary camels have high levels of neutralizing serum antibodies against mers-cov, suggesting a possible zoonotic source (reusken et al., 2013) . in addition, analysis of fecal samples from bats identified an egyptian tomb bat as a potential source of infection (memish et al., 2013) , but more work is needed to identify the animal reservoir(s) for mers-cov. limited humanto-human transmission of mers-cov has been reported, which considering the high mortality, raises a concern that the virus has a potential to become a threat to public health (assiri et al., 2013; guery et al., 2013) similar to severe acute respiratory syndrome coronavirus (sars-cov). the sars-cov pandemic from 2002-2003 was controlled by public health measures of identification and isolation of infected, symptomatic individuals and their contacts which broke the chain of human-to-human transmission. a sars-cov-like virus is endemic in chinese horseshoe bats, but changes in the sequence of the spike glycoprotein are required for this virus to efficiently infect humans (lau et al., 2005; rockx et al., 2007) . for mers-cov, it is unclear if the virus can jump directly from bats to humans, if there are any mutations in the viral genome that facilitate infection or disease in humans, and if there are both symptomatic and asymptomatic cases, which would make any potential epidemic more difficult to control by public health measures alone. our goal was to apply the knowledge gained from the study of sars-cov to identify and characterize the mers-cov papain-like protease domain as an innate immune antagonist and as a potential target for therapeutics. mers-cov, similar to other coronaviruses, is a positive-strand rna virus that upon entry into cells is translated to produce a viral replicase polyprotein. the replicase polyprotein is processed by viral proteases to generate a membrane-associated viral replication complex (snijder et al., 2006; perlman and netland, 2009 ). sequence analysis of mers-cov indicates that the canonical papain-like protease (plpro) and 3c-like proteinase (3clpro) are likely responsible for processing the polyprotein to generate 16 nonstructural proteins that assemble to form the replication complex. the majority of coronavirus papain-like proteases (plps), including sars-cov plpro, have been shown thus far to act as deubiquitinases and interferon antagonists (clementz et al., 2010; frieman et al., 2009; ratia et al., 2006; zheng et al., 2008; xing et al., 2013; barretto et al., 2005; sulea et al., 2005; chen et al., 2007) . the ubiquitin pathway is important for regulating a number of innate immune pathways and the ability of a viral protein to cleave ubiquitin from host cell proteins can contribute to virus pathogenesis. in addition to ubiquitination, modification of cellular proteins with interferon-stimulated gene 15 (isg15) is known to have a broad-spectrum antiviral activity. isg15 is ubiquitin-like protein that can be conjugated to cellular targets via a mechanism called isgylation, regulating innate immune responses. coronavirus plps are known to have the ability to remove isg15 conjugates from cellular substrates (clementz et al., 2010; lindner et al., 2005 lindner et al., , 2007 . in this study, we demonstrate the deisgylating and deubiquitinating (dub) activities of the papain-like protease from mers-cov, and provide new information on the potential role of coronavirus protease/dubs to inhibit the innate immune response. mers-cov plpro is encoded within nonstructural protein 3 (nsp3) of the replicase polyprotein (fig. 1a) . to gain insight into the potential of mers-cov plpro to recognize and cleave ubiquitin and isg15 from proteins, we used the high-resolution x-ray structure of sars-cov plpro in apo-enzyme form (pdb:2fe8, chain c) to generate a homology model of mers-cov plpro. we threaded the mers-cov primary amino acid sequence into the sars-cov structure and then energy minimized the structure. the homology model displays several conserved structural features between mers-cov and sars-cov plpro; including the ubiquitinlike domain (ubl), a catalytic triad consisting of c1594-h1761-d1776 and the ubiquitin-binding domain at the zinc finger. to model ubiquitin (ub) into the zinc finger and palm domains of mers-cov plpro, we used the x-ray structure and associated electron density of sars-cov plpro in complex with ub aldehyde (ubal) (pdb:4mm3) for refinement and energy minimization of the model in complex with ub. the resulting mers-cov-ubal model displays a nearly ideal fit of the ub moiety within the palm and the zinc finger regions of the enzyme with the c-terminal extension of ubiquitin oriented properly towards the mers-cov substrate subsites and catalytic triad (fig. 1b) . from this model, we hypothesize that the plpro domain from mers-cov, like sars-cov is a multifunctional enzyme with protease, deubiquitinating and likely deisgylating activity. we recently described expression and protease activity of mers-cov plpro in cell culture (kilianski et al., 2013) . to determine the deisgylating activity of mers-cov plpro, we transfected hek293t cells with c-myc-isg15 plasmid, isg15 conjugation machinery, and increasing amounts of plasmids expressing mers-cov plpro wild-type and catalytic mutant c1594a (plproca). cysteine 1594 is predicted to be the active site cysteine nucleophile that attacks the substrate peptide bond and mutation to alanine should significantly reduce or abolish enzymatic activity (fig. 1 ). in addition, we transfected cells with plasmids expressing sars-cov plpro wild-type or catalytic mutant (c1651a). we harvested cell lysates at 20 h posttransfection to evaluate the presence of isgylated proteins. we found that both mers-cov and sars-cov plpro can deconjugate isg15 from multiple cellular substrates in a dose-dependent manner. in contrast, plpro catalytic mutants did not deconjugate isg15, indicating that catalytic activity of plpro is required for its deisgylating activity ( fig. 2a) . thus, mers-cov plpro like sars-cov plpro (lindner et al., 2007) has deisgylating activity. to assess the dub activity of mers-cov plpro, we transfected hek293t cells with plasmid expressing flag-ub and increasing amounts of wild-type plpro or plproca. we determined that plpro can deubiquitinate multiple cellular substrates, and that plpro catalytic activity is required for dub activity (fig. 2b ). this dub activity is also observed with expression of sars-cov plpro, consistent with previous reports (frieman et al., 2009; ratia et al., 2006; barretto et al., 2005; lindner et al., 2005 lindner et al., , 2007 . in these experiments, we noticed the difference in the expression levels of sars-cov and mers-cov plpros in transfected cells, which may be due to differences in codon optimization in the mers-cov plpro construct. further in vitro studies using purified enzymes are needed to determine the relative kinetics of sars-cov and mers-cov plpro dub and deisgylating activities. taken together, our data indicate that mers-cov plpro is a potent deisgylating enzyme that also exhibits dub activity and that both activities require cysteine 1594 for catalysis, likely in the context of the predicted catalytic triad (fig. 1b) . coronavirus plps have been shown to block interferon β (ifnβ) induction in transfected cells (clementz et al., 2010; frieman et al., 2009; devaraj et al., 2007) . in addition, the deubiquitinase function of an arterivirus papain-like protease has been shown to have a role in interferon antagonism during virus infection (van kasteren et al., 2013) . therefore, we assessed the ability of mers-cov plpro to antagonize interferon production. first, we addressed if mers-cov plpro can inhibit mda5 induced ifnβ reporter, since mda5 has been implicated in recognition of coronaviruses during virus infection (zust et al., 2011) . we transfected hek293t cells with plasmids expressing ifn-β-luciferase, renilla luciferase, pef-bos-mda5 (rothenfusser et al., 2005) and increasing amounts of wild-type plpro or plproca. at 16 h posttransfection we assessed luciferase reporter activity. we determined that mers-cov plpro can potently inhibit mda5 mediated induction of ifnβ in a dose-dependent manner and that catalytic activity of mers-cov plpro is required for ifnβ antagonism (fig. 3a ). using overexpression of an active form of rig-i, we determined that mers-cov plpro can also inhibit n-rig-i induced ifnβ reporter. similarly to the experiment with mda5 stimulation, the catalytic activity of mers-cov plpro is necessary for ifnβ antagonism upon n-rig-i stimulation (fig. 3b) . upon recognition of viral rna by pattern recognition receptors (prrs) such as mda5 or rig-i the signal is transmitted downstream via mitochondrial antiviral signaling protein (mavs). thus, we tested if plpro is able to inhibit mavs induced ifnβ reporter. to stimulate the ifnβ reporter, we overexpressed pef-bos-mavs (rothenfusser et al., 2005) in hek293t cells, co-expressed reporters, and either the wild-type plpro or plproca. we found that plpro, but not plproca inhibits mavs induced ifnβ reporter (fig. 3c ). finally, we tested the ability of mers-cov plpro to inhibit nf-κb reporter activity as observed with sars-cov plpro. we transfected cells with plasmids expressing nf-κb luciferase, renilla luciferase, and mers-cov wild-type plpro or plproca, treated cells with tnfα to activate the nf-κb pathway, and harvested cell lysates at 4 h post-treatment to assess luciferase activity. we determined that wild-type plpro can reduce induction of nf-κb reporter in a dose-dependent manner and that the catalytic cysteine residue is required for this activity (fig. 3d) . taken together these results indicate that mers-cov plpro is an interferon antagonist and that catalytic activity is required for the antagonism. in addition, plpro can reduce tnfα-mediated induction of nf-κb reporter activity and catalytic activity is also required. to further investigate the role of coronavirus plpros in inhibiting innate immune responses we tested the effect of mers-cov plpro on the expression of endogenous cytokines. first, using the human innate and adaptive immune responses pcr array (sabiosciences) we determined that in hek293t cells ccl5 (rantes), ifnβ, and cxcl10 (ip-10) mrna levels are upregulated more than 20-fold upon mda5 stimulation (data not shown) and therefore selected these genes for further analysis. to determine the effect mers-cov plpro and sars-cov plpro on cytokine expression, we performed qrt-pcr to measure mrna encoding ccl5, ifnβ, and cxcl10 levels in the presence of cov plpros. hek293t cells were transfected with pef-bos-mda5, and wild-type or catalytic mutants of mers-cov or sars-cov plpros. at 18 h posttransfection the total rna was extracted and qrt-pcr was performed. we found that both mers-cov and sars-cov plpro can potently inhibit (over 3-fold reduction) expression of ccl5 upon mda5 stimulation and that catalytic activity is required for this inhibition (fig. 4a ). in agreement with the results from luciferase reporter assays, we observed that expression of ifnβ in mda5 stimulated cells is inhibited in the presence of wild-type mers-cov plpro and sars-cov plpro (fig. 4b ). cxcl10 mrna levels were also significantly reduced (po0.0005) when wild-type, but not catalytic mutant versions of mers-cov plpro and sars-cov plpro were expressed (fig. 4c) . to our knowledge, this is the first report showing that both mers-cov plpro and sars-cov plpro can reduce induction of endogenous proinflammatory cytokines in cells, and that the mechanism requires catalytic activity. viruses must "do more with less" because of the compact nature of their genomes. one example of this is the multifunctional plp domain encoded in all members of the order nidovirales. nidoviruses, including those in the coronavirus and arterivirus families, encode one or more plp domain. these plps are critical for proteolytic processing of the viral replicase polyprotein. in addition to protease activity, many of these plps have also been shown to act as viral deubiquitinating enzymes (dubs), able to deconjugate ubiquitin and isg15 from cellular substrates. coronavirus dub activity was first proposed by molecular modeling of the sars-cov plpro domain which predicted that the protease may be multifunctional . indeed, analysis of the dub activity of purified cov plps and the x-ray crystal structure of sars-cov plpro fully support the initial prediction of viral dub activity (ratia et al., 2006; barretto et al., 2005; lindner et al., 2005; ratia et al., 2008) . analysis of plps from coronaviruses and arteriviruses have revealed conserved dub activity; although the enzymes in the coronavirus family fall into the ubiquitin specific protease (usp) family whereas the arterivirus plps are in the ovarian tumor (otu) domain family of enzymes. the identification of a newly emerged coronavirus mers-cov provides an opportunity to evaluate plpro enzymatic activity and develop new hypotheses about how this protease/dub may contribute to viral pathogenesis. our modeling of the mers-cov plpro domain onto the structure of sars-cov led to the prediction of viral dub/deisgylating activity. although the enzymes are only $ 30% identical to sars-cov plpro at the amino acid level, we show that deisgylating, dub and interferon antagonism activities are conserved. importantly, we show that coronavirus plpro activity can modulate the innate immune response. coronaviruses have been shown to modulate immune responses upon infection, however the mechanisms involved in the regulation are not yet clear (totura and baric, 2012) . cytokine and chemokine responses to sars-cov in non-lymphatic cells and in infected patients results in low levels of several cytokines, including ccl5 and ifnβ (wong et al., 2004; spiegel and weber, 2006) . in addition, cxcl10, ccl5, and ifnβ among others, are not induced in cloned bronchial epithelial cell line and human alveolar type ii cells infected with sars-cov early post infection (yoshikawa et al., 2010; qian et al., 2013) . the innate immune response to mers-cov is also intriguing. microarray analysis of mers-cov infection of calu-3 cells results in distinct immune response compared to sars-cov infection. expression of multiple genes involved in activation of adaptive immune responses, such as mhc class i and ii, are downregulated in mers-cov infected cells (josset et al., 2013) . the ability of sars-cov and mers-cov to modulate early immune responses is likely due to multiple proteins encoded within virus genomes that may act as interferon antagonists. previous reports showed that several coronavirus proteins can block the activation of innate immune responses, particularly production and induction of ifnβ response (reviewed in (totura and baric, 2012) ). structural proteins, such as sars-cov nucleocapsid (n) and membrane protein (m), in addition to being critical elements of the viral particles, have been shown to block the ifn response. several accessory proteins (sars-cov orf3b, orf6, and mouse hepatitis virus ns2) are known to act as antagonists of innate immunity (kopecky-bromberg et al., 2007; zhao et al., 2012) . indeed, mers-cov accessory protein 4a has been reported to block induction of ifn (niemeyer et al., 2013) . in addition, nonstructural proteins including nsp1, nsp7, nsp15 have been implicated as ifn antagonists (frieman et al., 2009; kamitani et al., 2009) . importantly, plps encoded within nsp3 have been shown to block ifnβ induction. sars-cov plpro and hcov-nl63 plp2 are interferon antagonists and catalytic activity is important for plpro antagonism (clementz et al., 2010; frieman et al., 2009; devaraj et al., 2007) . sars-cov plpro is the only plp in the sars-cov genome and it is both a deisgylating and a deubiquitinating enzyme (ratia et al., 2006; lindner et al., 2005; lindner et al., 2007) . on the other hand, hcov-nl63 encodes two papain-like proteases, plp1 and plp2, in the genome but only plp2, which has 22% homology to sars-cov plpro, is an ifn antagonist with deisgylating and dub activities (clementz et al., 2010) . hcov-nl63 plp1 is devoid of these activities . in addition, mouse hepatitis virus plp2 and porcine epidemic diarrhea virus plp have dub activity and act as ifn antagonists (zheng et al., 2008; xing et al., 2013) . plps from arteriviruses are also known to block ifn responses. the n terminal region of nsp2 encodes the papain-like protease in porcine reproductive and respiratory syndrome virus (prrsv). this plp has been characterized as an otu with deubiquitinating and deisgylating ability (frias-staheli et al., 2007) . in addition, sun et al. (2010) , showed that prrsv plp domain can block sendai virus induced ifnβ, and can also inhibit nf-κb by preventing iκbα degradation by its deubiquitination. a more recent report showed that prrsv plp has also deisgylating activity which suggests multiple roles of prrsv papain-like protease in antagonism of innate immunity (sun et al., 2012) . nsp2 of another member of the arteriviridae, equine arteritis virus (eav), has deubiquitinating and deisgylating activities as well (frias-staheli et al., 2007) . the deubiquitinating ability of eav plp can block rig-i induced ifn by inhibiting ubiquitination of rig-i, which is required for its activation . co-crystal structure of eav plp with ubiquitin reveled potential interaction sites between those molecules, and mutagenesis studies showed that plp dub activity is required for inhibition of innate immunity in infected cells (van kasteren et al., 2013) . specific deubiquitinating and deisgylating activities have been shown for crimean-congo hemorrhagic fever virus (cchfv) which is a highly pathogenic, negative-strand rna virus belonging to the family bunyaviridae. the l protease of cchfv contains otu domain with the ability to cleave isg15 modification. l protease can remove isg15-meidated immune protection in type i ifn receptor knock-out mice and make them highly susceptible to sindbis virus infection (frias-staheli et al., 2007) . overall, the results from multiple laboratories studying a variety of coronavirus, arterivirus, and bunyavirus proteases indicate that deubiquitinating and deisgylating activity of viral proteases have an important role for inhibition of innate immune responses and possibly virus pathogenesis. here, we characterized the papain-like protease from mers-cov revealing the deisgylating and deubiquitinating activities, and that it can act as an interferon antagonist. further, we showed for the first time that sars-cov plpro and mers-cov plpro can block induction of several endogenous proinflammatory cytokines. our data suggest that antagonism of innate immune responses mediated by mers-cov and sars-cov plpros is not limited to ifnβ, but may affect expression of many cellular cytokines. our results suggest that plpro might contribute to the modulation of innate immune responses upon sars-cov and mers-cov infection, however, the exact mechanism and the role of coronavirus plps and their associated dub and deisgylating activities in these processes remains to be determined. the crystal structure sars-cov plpro (pdb:2fe8) was used as the template structure to generate a homology model of mers-cov plpro using the automated web-based homology modeling server 3d-jigsaw (bimolecular modeling laboratory, cancer research uk, england). ccp4 program suite 6.2.0 and coot version 0.6.2 were used for final refinement, energy minimization and modeling of ub into the zinc finger and palm regions of mers-cov plpro by using the electron density of sars-cov plpro in complex with ubiquitin-aldehyde (pdb:4mm3). hek293t cells were cultured in dulbecco's modified eagle medium (dmem) with 10% fetal calf serum (fcs) and 2% glutamine. transfections were performed with 70% confluent hek293t cells in cell bind plates (corning) using transit-lt1 reagent (mirus) according to manufacturer's protocol. the mers-cov plpro (pcdna-mers-plpro) expression plasmid and generation of catalytic mutant were described previously (kilianski et al., 2013) . pcdna-sars-plpro wild-type and catalytic mutant expression plasmids were described elsewhere (barretto et al., 2005) . for the luciferase assay experiments we used ifnβ-luc deisgylating activity assay hek293t cells in 12-well plates were transfected with 10, 25, 50, 100 ng of pcdna-mers-plpro wild-type or catalytic mutant, and 250 ng pisg15-myc, 125 ng pubch8, 125 ng pube1l, and 125 ng pherc5. at 20 h post-transfection, cells were lysed with lysis buffer (20 mm tris (ph 7.5), 150 mm nacl, 1 mm egta, 1 mm edta, 1% triton x-100, 2.5 mm na pyrophosphate, 1 mm betaglycerophosphate, 1 mm na ortho-vanadate, 1 mg/ml leupeptin). proteins were separated by sds-page, and transferred to pvdf membrane using a semi-dry transfer apparatus (biorad). following transfer, the membrane was blocked using 5% dried skim milk in tbst buffer (0.9% nacl, 10 mm tris-hcl, ph ¼7.5, 0.1% tween 20) overnight at 4 1c. the membrane was incubated with mouse anti-myc antibody (mbl) at the dilution of 1:2500. the membrane was washed 3 times for 15 min in tbst buffer. following the membrane was incubated with secondary goat-anti-mouse-hrp antibody at the dilution 1:5000 (amersham). then the membrane was washed 3 times for 15 min in tbst buffer. the detection was performed using western lighting chemiluminescence reagent plus (perkinelmer) and visualized using fluorocheme imager (protein simple). to verify expression of the plpro the membrane was probed with mouse anti-v5 antibody (invitrogen) at the dilution 1:5000. mouse anti-calnexin antibody (cell signal) at the dilution 1:2000 was used to determine loading standard. to assess dub activity, hek293t cells in 12-well plates were transfected with 400 ng pcdna3.1-flag-ub and 0.25, 0.5, or 1 mg pcdna-mers-plpro wild-type or catalytic mutant. at 18 h posttransfection, cells were lysed with 100 ml of lysis buffer. proteins were separated by sds-page and transferred to pvdf membrane as described above. membrane probing was performed using mouse anti-flag m2 antibody (sigma) at the dilution of 1:2000. hek293t cells in 24-well plates were transfected with 50 ng renilla-luciferase, 100 ng ifn-β-luc, and 25, 50, and 100 ng pcdna-mers-plpro wild-type or catalytic mutant expression plasmids. as a stimulation 150 ng pef-bos mda5, or 50 ng pef-bos mavs, or 50 ng n-rig-i per well was transfected. empty pcdna3.1-v5/his-b vector plasmid was used to standardize the total amount of dna used for transfection. at 16 h post-transfection cells were lysed using 1x passive lysis buffer (promega). alternatively, the cells were transfected with 50 ng pgl4 32 [luc2 nf-κb-re hyrgo], 100 ng ifnβ-luc and pcdna-mers-plprov5 wild-type or catalytic mutant for 12 h and then treated with 10 ng/ml tnfα (roche) for 4 h, and lysed. for all experiments firefly and renilla luciferase were measured using dual luciferase reporter assay system (promega) and luminometer (veritas). results were normalized to renilla luciferase expression control. experiments were performed in triplicate. remaining lysates were incubated with lysis buffer a (0.9% nacl, 10 mm tris-hcl, ph 7.5, 0.1% tween-20) and analyzed by sds-page as described above. hek293t cells in 12-well plates were transfected with 300 ng pef-bos mda5 and 200 ng pcdna-mers-plpro wild-type or catalytic mutant expression plasmids, or 200 ng pcdna-sars-plpro wild-type or catalytic mutant expression plasmids. empty vector plasmid pcdna3.1-v5/his-b vector was used to standardize the total amount of dna in each sample. the cells were lysed 18 h posttransfection with buffer rlt (qiagen) and rna was extracted using rneasy mini (qiagen). reverse transcription was performed using 1 mg of total rna and the rt 2 first strand kit (qiagen) according to manufacturer's protocol. 1 ml of cdna was used to set up qrt-pcr reaction according to the manufacturer's protocol using single primer assay for ifnβ, cxcl10, and ccl5 (sabiosciences). c t values were normalized to housekeeping gene (rpl13). hospital outbreak of middle east respiratory syndrome coronavirus the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity deubiquitinating and interferon antagonism activities of coronavirus papainlike proteases proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl63 clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection regulation of irf-3-dependent innate immunity by the papain-like protease 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coronavirus ns2 protein is required for virus replication and liver pathology we thank dr. xufang deng for helpful discussions and review of the manuscript. this work was supported by the nih grant r01 ai085089 (to scb and adm). amm was supported by arthur j. schmitt dissertation fellowship from loyola university chicago. ak was supported by the nih training grant in experimental immunology (nih t32 ai512795). key: cord-104500-m0kfom0x authors: kyriakopoulos, anthony m.; papaefthymiou, apostolis; georgilas, nikolaos; doulberis, michael; kountouras, jannis title: the potential role of super spread events in sars-cov-2 pandemic; a narrative review date: 2020-09-21 journal: arch acad emerg med doi: nan sha: doc_id: 104500 cord_uid: m0kfom0x coronaviruses, members of coronaviridae family, cause extensive epidemics of vast diseases like severe acute respiratory syndrome (sars) and coronavirus disease-19 (covid-19) in animals and humans. super spread events (sses) potentiate early outbreak of the disease and its constant spread in later stages. viral recombination events within species and across hosts lead to natural selection based on advanced infectivity and resistance. in this review, the importance of containment of sses was investigated with emphasis on stopping covid-19 spread and its socio-economic consequences. a comprehensive search was conducted among literature available in multiple electronic sources to find articles that addressed the “potential role of sses on severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic” and were published before 20(th) of august 2020. overall, ninety-eight articles were found eligible and reviewed. specific screening strategies within potential super spreading host groups can also help to efficiently manage severe acute respiratory syndrome coronavirus 2 (sars-cov-2) epidemics, in contrast to the partially effective general restriction measures. the effect of sses on previous sars epidemics has been documented in detail. however, the respective potential impact of sses on sars-cov-2 outbreak is composed and presented in the current review, thereby implying the warranted effort required for effective sse preventive strategies, which may lead to overt global community health benefits. this is crucial for sars-cov-2 pandemic containment as the vaccine(s) development process will take considerable time to safely establish its potential usefulness for future clinical usage. severe acute respiratory syndrome (sars) has periodically emerged as epidemics and its natural history could be utilized as a "compass" to comprehend and manage the current pandemic of sars-cov-2. sars-cov-2 the etiologic agent of the novel coronavirus disease 2019 (covid19) , be-although the prediction and subsequently the prevention of sses seems to be complicated, the virus, host, environmental, and mass behaviors determine relative approaches to prevent and control sses; core community health programs can inhibit and decrease the incidence and the effect of sses (9) . nevertheless, horizontal austerity measures, such as recommending or compelling individuals to self-isolate at home, which might cause serious social and psychological burden, and quarantine, also leading to loss of income due to social distancing, are associated with negative psychological and religious effects, which can be long lasting (10) , thereby leading to serious instability of the global society. prolonged social isolation and loneliness are associated with increased mortality (11) . currently, limited piece of information exists regarding the effect of sses on coronavirus epidemics. the aim of this narrative review is to mainly focus on the potential impact of sses on large outbreaks of coronavirus. the development of an emergency sars-cov-2 vaccine has its potential usefulness and/or limitations and may result in severe health outcomes, which prompts better screening for sses in order to control coronavirus pandemics. to avoid, in most respects, literature selection bias (12) , multiple electronic sources: medline/pubmed, scifinder, science direct and goggle scholar as well as researchgate and general (google) were investigated via queries with a nonrestricted time frame reaching the 20th of august 2020. initial investigation of sses and sars, sses and mers, and sses and covid-19, gave narrative results from pubmed. the selected literature, which is included in the study, is presented in table 1. same items were also searched in all other mentioned sources. the scope of the study was not only to investigate the transmission of sars-cov-2 due to sses, its comparison with sars-cov-1 and mers-cov, but also to assess the general global impact due to sses by covid-19. therefore, further literature investigation was performed using the same electronic sources. further investigation was made on: a) the prevention of sses by coronaviruses causing sar-1, mers and covid-19, b) the socio-economic relation of sars-cov-1, mers and covid-19 due to sses, c) the austerity caused by sses of covid-19, and d) the relation of sses containment to future vaccination programs. for further investigation, the following items were searched: "sars, mers and covid-19 epidemic prevention", "sars mers and covid-19 infectivity and pathogenicity", "coronavirus sse prevention", sse coronavirus crisis and socio-economics", "holy cup reli-gion and transmission of pathogens and sses", and "coronavirus immunity and vaccination". studies providing an adequate determination of an sse related to sars, mers and covid-19 were primarily screened and selected by two reviewers (authors) blinded to one another. the results were thereafter cross-matched and duplicates were removed. based on this primary search, the socio-economic impact of coronavirus, produced by sses, was extrapolated by two other reviewers (authors). following this initial selection stage, further screening was performed by all reviewers, using the previously described search items to identify parameters determining the global impact of covid-19 due to sses. identified parameters included the global impact of immunity and vaccination, the holy cup and religion transmission, and the austerity caused by covid-19 and other coronavirus epidemics due to restrictions applied. all search results were cross-matched to remove duplicates and thereafter, exclusion and inclusion criteria were applied. after removing the duplicates, review was conducted on titles and abstracts. also, a decision was made to remove "news press opinions". computational model methodologies producing contradictory results, studies with wrong interpretation of sses, and studies with non-clear-cut results were also removed. studies using the interpretation "a super spreading individual, known as the index case, produces a cluster of sars, mers, and covid-19 secondary infections" were included. a second exclusion criterion was applied. in this stage, peer reviewed literature of recent dates, studies assessing sars, mers, and covid-19 epidemiology measures, studies on covid-19 restriction measures producing social and economic austerity, articles discussing the perspective for future vaccination and population immunity, and finally genetic studies on coronaviruses causing sars, mers, and covid-19. by following the described methodology, on medline/pubmed: a) 23 articles were found on sars and mers and sse, and b) 11 articles were found on covid-19 and sses. out of: a) 13 of the 23 articles on sars and mers and sse, and b) 7 out of the 11 articles on covid-19 and sse were deemed relevant hits. after applying the exclusion criteria, 12 articles from the first category, and 4 from the second category were included in the study. suitable articles found by searching, which were selected and reviewed for each part, are illustrated in figure 1 . further investigation in all other electronic sources described, using the same method(21) original research description of sse importance in covid-19 epidemic ! severe acute respiratory syndrome; ∧ middle east respiratory syndrome; &coronavirus disease-19; *super spread events; **when clearly indicated in article, the type of study is also mentioned. ology, increased the number of the included literature to a) 17 and b) 14, for their respective categories of search. studies included from pubmed in these categories of searches are briefly described and listed in table 1. further, assessing the general global impact of sses related to covid-19, using all the mentioned sources, via the same methodology, led to the inclusion of a) 10 articles related to genetic analysis of sars-cov-1 and mers-cov and sars-cov-2, b) 5 articles related to super spread events, c) 2 articles related to austerity, d) 18 articles related to infectivity and pathogenicity of sars, mers and covid-19, e) 17 articles related to prevention of sses concerning human coronaviruses, f) 9 articles related to socio-economic impact, and g) 9 articles related to immunity and future vaccination. table 2 illustrates the initial numbers of hits using all search items in all sources, and the final number of articles reviewed in each category. the involvement of sses in sars extensive outbreaks (1, 4, 5, (13) (14) (15) (16) (17) , necessitates urgent elucidation as global tranquility is disturbed by covid-19 pandemic. epidemiological research has proposed that the outbreak was related to a seafood market in wuhan (hubei, china), underlining the ongoing risk of viral transmission from animals to induce severe diseases in humans. metagenomic rna sequencing of bronchoalveolar lavage fluid from a patient with pneumonia identified a novel rna virus strain from the coronaviridae family (called sars-cov-2); and phylogenetic analysis (by introducing the widely used in silico protein screening) (18) (19) (20) (21) of the complete viral genome (29,903 nucleotides) disclosed that the virus was most closely connected (89.1% nucleotide similarity) with a group of sars-like coronaviruses (genus betacoronavirus, subgenus sarbecovirus) formerly isolated from bats in china (18) (19) (20) (21) (22) . (1, 5, 13, 15) , that closely related sars-like viral genes were traceable in chinese bat populations. authors claimed that these viruses were capable of infecting humans, by selective adaptations or adjustments, and thereby, causing a new epidemic (23) . enhancement of virulence is also attributed to these adaptations due to acquisition of spike protein via adaptive mutations (24) . continuous viral random mutations are possible through intermediate host transmission, until a deadly virus develops, as illustrated in figure 2 . recent evidence revealed that recombination within intermediate hosts has contributed to development of sars-cov-2 (1, 24). asian outdoor markets could constitute the ideal places for continuous viral mutation exchanges (25) . as presented in table 3 , the best way to circumvent continuous virus production is targeted surveillance; to at least stop the overspreading by sses (2, 3, 22, 26) . this has also been proposed by menachery et al. (23) . sars-cov-2 is accountable for the unprecedented covid-19 pandemic (27) , and the interplaying mechanisms involved in the pathophysiology of covid-19 include sars-cov-2 virulence, host immune response, and complex inflammatory reactions (28) . emerging data, also, imply that the reser-voirs of sars-cov-1 infection may be similar to covid-19 (1, 4, 5, 13, 29) , as remarkable similarities exist between sars and swine acute diarrhea syndrome (sads) in topographical, temporal, environmental and etiological backgrounds. however, the increasing coronavirus variety and spread in bats were recognized as a potential target to diminish future epidemics that might impend livestock, community health, and financial progress (30) . probably, identification of animal and insect vectors that transmit the disease, identification and control of alternative routes of transmission like fecal-oral route, and identification of super spreader patient groups could help minimize the epidemiological extent compared to the one observed for sars-cov-2 infection worldwide. lessons from sars epidemic taught us that the key to control is minimizing the time from the diagnosis of infection to prompt hospital isolation and diminishing the probability table 3 : key clinical and laboratory screening functions to appropriately forecast, prevent, and confront sars! coronavirus 2, and future coronavirus epidemic waves to estimate the probability of a major outbreak, use simulations of stochastic compartmental epidemic models. use of diagnostic tests to detect asymptomatic susceptibility and pre-symptomatic infectivity estimation of super spread events of current and previous coronavirus epidemics introduction of individual reproductive number. integrated and computational analysis of the influence of individual variation by binomial distribution and use of branching process analysis of disease data. genetic characterization of inpatient viral isolates to identify intermediate animal hosts facilitating the infection next generation sequencing of samples and cultured viral isolates to obtain full sequence and phylogenetic analysis application. environmental detection and continuous sewage monitoring rt-qpcr# screening on sewage systems, vectorsùĺ and potential air transmission. autopsies and detection of serology conversion of potential vectors. heptad repeat region screening for positive selection computer simulation models to detect positive selection events e.g. codeml branch-site test coupled with bayes empirical bayes procedure, and mixed effects model of evolution. receptor recognition analysis of ace-ii+ to identify origin of crossspecies and human to human transmissions coronaviruses genetic sequencing and phylogenetic analysis of ace-ii to provide origin and efficiency of cross-species and human to human transmission and identification of intermediate hosts. !severe acute respiratory syndrome; #reverse transcriptase quantitative polymerase chain reaction; +angiotensin-converting enzyme-ii. fecal -oral frequent contact with wild animal reservoirs (including domestic animals) and birds** airborne and fecal -oral construction area workers air particles sewage system workers*** fecal -oral *in both community and hospital environments. **including slaughter houses, pet shops, animal and bird collectors and breeders, cow, and pig farmers. ***including workers coming in contact with environment contamination. !human immunodeficiency virus, #methicillin resistant staphylococcus aureus. of another sse (5). the typically recognized 20-80 rule or the so-called "pareto rule", states that 20% of efforts lead to 80% of results (31) . more specifically, this comprises a principally convenient state when tackling infectious diseases and is applied to investigate infection transmission, and initially among cattle farms. in this regard, woolhouse et al. (17) reported that targeted actions concerning disease control and prevention in 20% of the farms that mainly supplied the basic reproduction number (ro) decreased spread by 80% (32) . focusing on the covid-19 virus, ro is a sign of virus transmissibility, denoting the average figure of novel infections caused by an infectious individual in a totally naive population. for r0 > 1, the number of infected people tends to increase, whereas for r0 < 1, transmission is likely to stop; ro represents a chief model in the epidemics, signifying the risk of an infectious mediator with regard to epidemic spread (33) . recent data indicate that the estimated mean ro for covid-19 is almost 3.28, with a median of 2.79 and the interquartile range (iqr) of 1.16, which is substantially higher than who's estimation of 1.95. however, due to biased methodology, ro for covid-19 is expected to be about 2-3, which is approxi-mately consistent with the who estimate (33) . sses appear to be a main limitation of the ro concept. ro, when calculated as a mean or median value, does not include the heterogeneity of transmission between infected individuals (4); two infective agents with equal r0 estimates might have noticeably diverse patterns of transmission. moreover, the goal of a health care system is to achieve ro <1, which is probably only phenomenally feasible in certain conditions without scheduled prevention, recognition, and response to sses (9) . naturally, epidemics follow the aforementioned 20 / 80 rule (17) . specifically, in human population, due to heterogeneous exposure to infectious agent, the 20% core population may transmit the disease, widely. for sars, the rate might have been even lower than 20% (4). the increased infectious potential of a small population subgroup seems to be related to immunodeficiency, such as in hemodialysis, cancer, immunosuppressive therapies (4, 5, 15, 34) . additionally, facilitation of disease spread and transmission due to vector exposure has been investigated in relation to cockroaches (35) . possible mechanical transportation by rats and cat (13, 36) and air transmission (37) in sars-cov-1 have also been studied. other animals capable of being sars-cov-2 carriers (excluding mice and rats), like pigs, ferrets, cats, and nonhuman primates have recently been introduced (3), and contamination of sewage with sars-cov-2, has probably preceded covid-19 outbreak in france (29) . all these agents may contribute to a minimum of 80% of the total transmission potential (17), maybe even more (4, 5) . table 4 displays possible super spreader groups; thus, indicating screening targets to prevent sses. sars epidemic taught us that control programs were inefficient in controlling the epidemic within a population, and failed to identify and provide a targeted infection diagnosis in groups causing potential sses (5, 17) . on the other hand, sars-cov-2 having the ability to cause a pandemic rather than an epidemic, resulted in an increased number of cases and deaths; albeit having a lower mortality rate than sars coronavirus (2) . sses during covid-19 may involve not only one city, but also a whole country or many countries, requiring investigation of their effects on a national or international level (2, 38, 39). preventing and decreasing covid-19-related sses necessitates the decryption of the mechanism through which sars-cov-2 spreads through super spreader individuals, for example within healthcare facilities (7, 9) . healthcare facilities are essential for prevention and control of sses (9) . sse prevention may enable us to even overcome initial low covid-19 virus infectiveness. the capability of the virus to produce sses troubles the epidemiological attempts to restrict viral spread only by isolating individuals at high risk and performing obsolete isolation at home for the general population as carried out in countries such as greece (5) . during the sars epidemic in china (beijing) and singapore, the vast majority of infected individuals were barely infective and only 6% of the population was highly infectious, in contrast to many published sars models (4, 5) . other ways of potential coronavirus transmission between hosts may provide explanations for enormous outbreaks (16) . it should not be disregarded that coronaviruses cause both respiratory and intestinal infections and share common evolutionary roots with hepatitis viruses (40, 41) . passing the cross-species barrier and genetic adaptation within hosts may promote virulence of coronaviruses in humans (14) . this, prompts to specifically identify potential super spreader groups within populations through targeted diagnosis. some of these groups are listed in table 2 . for this purpose, a usual infection must be distinguished from a super spread infection (4, 5) . during sars epidemic, the coronavirus infectiousness mostly occurred in the late stages of infection (5, 17) , whereas in covid-19, viruses are transmitted even in pre-symptomatic stages (42) . as with influenza a virus subtype h1n1 transmission (43) , accurate diagnosis of covid-19 in potentially asymptomatic super spreaders may help contain the magnitude of large outbreaks (44) . in the case of diamond princess cruise ship, an earlyassessed r0 of 14.8 (âl'ĺ4 times higher than the r0 in the epicenter of the outbreak in wuhan, china) was decreased to an assessed effective ro of 1.78 following on-board isolation and quarantine processes (45) . similarly, in china (wuhan) the application of non-pharmaceutical interventions in the society, including a cordon sanitaire of the town; interruption of community transport, school, and most employment; and termination of all community events decreased the ro from 3.86 to 0.32 over a 5-week period (46) . nevertheless, these strategies could not be maintained. emerging research evidence (29) regarding sewage contamination that preceded paris covid-19 epidemic is pointing to the reports of 2003 from the health department of hong kong (35, 36) , the noble work by ng (13) , and urge for extensive environmental monitoring (29, 37) to prevent future covid-19 relapses. however, the flow of genetic variation may be even more complex as illustrated in figure 2 . therefore, advanced clinical and laboratory monitoring is required to prevent sses and thereafter, new coronavirus epidemics. assembly of key functions and screening techniques of reference centers is presented in table 3 . newer therapeutic agents and protocol applications are promising (47) , although probably carrying the possibility of resistance state (48) . first, these also require specific diagnostic and surveillance strategies to overcome any unknown adverse epidemiology consequences (48) . inhibiting wild meat markets and related consumption of wild meat by creating vivid campaigns could be a critical for interrupting the introduction of coronaviruses crossing from animals to the human population, as was the case for sars (1, 4, 5) and middle east respiratory syndrome (mers) (49) epidemics, and probably now for covid-19 pandemic (1-3) . furthermore, the food production process requires radical reconsideration, concerning the industrial environment of current food production and serious violations of natural ecosystems (50) . current industrial procedures for preparing food increasingly favor conditions where viral evolution produces new mutations and increased rates of mutations (25) , thus raising the probability of new and more infectious viral strains. in sars and mers epidemics, the role of sses in vigorously distributing the epidemics has been substantially proven (51) (52) (53) (54) (55) . the new covid-19 epidemiology evidence also adequately highlights the important role of sses in homeland of china (21, 56, 57) , although surprising evidence from neighboring countries show the unlikely role of sse in the spread of the disease (58). the coronaviridae family is characterized by a positive-sense single-stranded rna genome. mouse hepatitis virus is a representative member of the family (41) . additionally, human hepatitis e virus also has a positive-sense single stranded rna genome and shares a common evolution pathway with coronaviruses (40) . hepatitis-related incidents were described for sars (59) . the genetic recombination of these viruses within arbitrary intermediate hosts produced contagious strains that are extremely pathogenic to humans (40, 60) . in this respect, the relation of sars-cov genetic sequences isolated from human, civets, and bats permitted us to find the reason for such a dangerous epidemic, which affected people on a worldwide scale in 2003 (61) . moreover, the unpredictable epidemic of mers-cov posed a serious risk to the health of communities worldwide. these underscored the necessity for further research of the virus epidemiology and pathophysiology to develop successful therapeutic and preventive medications against mers-cov infection (62) . while sars-cov-2 is genetically and structurally connected with mers-cov, it has its own exclusive structures which are responsible for its quick spread throughout the world (60). specifically, variations in coronavirus pathogenicity within different species (63) make the understanding of sars epidemics even more unclear through their capability to overcome the barrier for cross species transmission, which also alters their infectivity status (14, 64) . as a result, boosting the pathogenic behavior of coronavirus strains, within species (65) , and across species barriers (49) , which is a reflection of their positive adaptation to rapid recombination events (49) . the recent mers epidemic revealed the tendency of the strain to genetically adapt and produce greater outbreaks (49) as occurred in sars epidemic in 2003 (66) . however, mainly for socioeconomic reasons, alarm signals were ignored until recently (67) . a new phylogenetic analysis technique employed on clustered covid-19 strains displayed a geographic variation preference in infectivity and pathogenesis (39) . this is probably due to predominating strain's tendency to cause an sse as an outcome of a multi-factorial epidemic process presented in figure 2 (23, 24) . marked sses for covid-19 have already been fully characterized and warrant urgent investigation (23, 24) . as presented in tables 3 and 4 , each way of transmission should be investigated. heterogeneity of epidemic characteristics across nations (39) implies that in this way we may minimize coronavirus transmission. therefore, salvation of national economic catastrophes will also be achieved in this way (66) . thus, the whole biomedical science machinery needs to perform targeted diagnosis of sses and share the obtained experience. subsequently, central authorities will no longer need excessive non-specific contact measures, which will in turn normalize both societal and economic activities. on the other hand, improper understanding of how covid-19 spreads resulted in societal imbalance due to arbitrary restriction of social and religious life including holy communion cup. it has been consecutively demonstrated by expert research that the holy cup (chalice) and the holy cloth are not sources or pathways, for potential spreading of infectious diseases including human immunodeficiency virus (hiv) (68), hepatitis b virus (hbv) (69) as well as other communicable pathogens (70) . specifically, a review (69), considered other 129 relative studies. in this review, the possibilities that the shared communion cup can act as a vehicle for indirect transmission of human immunodeficiency virus, since it was detected in the saliva of infected individuals, was investigated. it was emphasized that although for bacterial contamination, the alcoholic content of the wine, the material that the cup is made of, or the practice of partially rotating the cup, cannot stop the occasional transmission of microbes, the microbial transmission was considerably reduced by the intervening use of a cloth to swab the lip of the cup between communicants. notably, it was emphasized that transmission means not an obligatory inoculation or infection. furthermore, it was also emphasized that out of the epidemiology of microbes transmitted via saliva, particularly for the transmission of the herpes viruses, the indirect transmission is rare, and indeed transmission is highly possible by other means than by the saliva. it was also emphasized that neither hepatitis b virus nor human immunodeficiency virus infection can be transmitted by saliva, rendering their indi-rect transmission also less likely by inorganic objects. finally, the study concluded that no episode of disease transmission has ever been reported as a result of the shared communion cup use, and that there was not any scientific evidence that the communion cup practice should be abandoned due to the possible risk of spreading of any infection (71, 72) . likewise, kingston et al. (68) , by considering 44 relative papers, also concluded that there is no evidence that the holy communion cup spreads infections. moreover, more recent estimations also demonstrated that no infections have ever been observed as a result of religious rituals including christian common communion chalice practice (70) ; whereas, data of previous studies implied that saliva could play a role in hbv transmission, are likely to be trivial (69) . similarly, recent evidence indicate that, although hbv dna and hcv rna can be discovered in the saliva of infected patients, they seem unlikely to transmit infection (72) . it should be noted that, as in the case of coronavirus (73, 74) , hbv also exists in many body fluids including saliva, nasopharyngeal fluid or tears by measures of qualitative and pcr methods (75) . the detection of hbv dna in saliva motivated our study group to investigate the potential viral transmission through the holy communion cup. two successive retrospective studies were conducted to investigate the role of holy communion as an independent risk factor of hbv dispersion. the first preliminary study included patients from our registry of those with chronic hepatitis b under entecavir (jannis kountouras-personal communication) treatment (76) , and in the next step, the relative registry of another department of the same hospital was incorporated. other parameters studied, the substantial independent categorical variable to evaluate our hypothesis was the patients' occupation, thereby introducing two sub-groups; priests and non-priests. this classification was performed based on a standard active and perpetual exhibition (at least once weekly) of priests to many people's saliva, as a part of the grounded process of the holy communion cup. the control group comprised of the aggregate of orthodox priests in greece (10,338) and the rest general population (10, 680, 866) at that timeframe. approval of the institutional ethics committee was obtained and all predispositions of the helsinki declaration were fulfilled. the reservoir database did not include any personified information (name, id number, etc.) and thus no informed consent was required. pearson's chi-squared test with 1 degree of freedom was performed to evaluate whether there was a statistically significant difference between the frequencies of hbv infection in case and control groups and statistical significance was set at p <0.05. the first single-centre registry included 71 patients and one (1.4%) of them was a priest. chronic hepatitis b was significantly more frequent among non-priests compared to priests (x2 (1, n=71)=12.65, p <0.05). the extended sample (n=429) included the registry of another department and an aggregate of four (0.93%) priests were diagnosed with chronic hepatitis b. likewise, the chi-square test revealed that non-priest subjects were more likely to suffer from chronic hepatitis from hbv infection compared to priests (x2 (1, n=429) = 31, p <0.001). in conclusion, both of our analyses indicated a lower prevalence of hbv chronic hepatitis among priests when compared to other occupations. currently, vaccines for covid-19 are in pre-clinical development, and no final clinical phase has been ended due the recent emergence of the disorder. many global entities have stated their plans to produce a vaccine for covid-19. according to the who, 41 candidate vaccines are being produced for covid-19 as of march 13, 2020 (77) . importantly, for production of highly effective and safe covid-19 vaccines, features such as the possibility of the induction of antigen-dependent enhancement (ade) and additional severe opposing effects previously detected with sars and mers should be considered. ade is a phenomenon that occurs when non-neutralizing antibodies against proteins of a virus increase, also increasing virus infectivity (78) . in this regard, coronaviruses can escape the immunity provided by inactivated or recombinant protein vaccines via fast evolution (79) . the problem with live attenuated vaccines is that the coronavirus can recover its virulence via serial passages in cell culture or in vivo (80) . moreover, vaccination in animals and humans could facilitate, rather than inhibit, the pathogenesis of the targeted viruses. this can be the consequence of an ade phenomenon. this underlines a mechanism by which specific antibodies facilitate infection with the targeted virus, or cell-based augmentation, a process resulting in an allergic inflammatory response induced by immunopathology (81, 82) . many experimental sars-cov-1 vaccines have been formulated from whole inactivated viruses, due to their advantage of large-scale production, multiple epitope presentation and high conformation stability (83) . one such vaccine uses viruses from ay71a217 strain of sars-cov-1, which are double inactivated using formalin and uv irradiation, the socalled double-inactivated virus (div) vaccine (84) . although div had initially been demonstrated to induce neutralizing antibodies and to protect against sars-cov-1 viral replication, both in tissue culture and in young mice, it soon became apparent that older mice suffered from vaccine-induced immune pathologies, including failure to contain viral replication, augmented clinical disease and associated symptoms, and increased inflammatory response and eosinophilic influx (84, 85) . in this respect, there is an overlap between the immunopathologic responses connected with coronavirus disease and vaccination, and the role of t helper (th) 17 cells in immune augmentation and eosinophilic lung immunopathology; host th17 polarized inflammatory reactions portray an important role in the pathophysiology of covid-19 pneumonia and edema (86, 87) . eosinophilic pathology, indicating increased pathogenesis and disease severity in the elderly, has been attributed to the nucleocapsid (n) protein, despite the incorporation of multiple sars-cov-1 antigens in the div (82, 84) . this is on grounds that the n protein is a strong modulator of innate immunity, also acting as an interferon antagonist, and therefore, it has the capability to induce inflammation with subsequent immune pathology in situations of heterologous viral challenge or in immune senescence, where patients fail to mount effective immune responses against the disease (84, 88) . the route of transmission is important to be established for sars cov-2. as seen with other important infectious diseases of a) air borne transmission such as tuberculosis (89), b) orofecal transmission such as hev (90) and c) blood transmission such as hbv and hepatitis d virus (91) , even if efficient vaccination is established, understanding of sses is still important. recent research data on the immune receptors used by coronaviruses, which reflect their ability to propagate in the human population, imply that complex immune reactions are responsible for a cell to cell transmission. in addition to ace-ii receptor, as is the case with sars-cov, mers-cov (92) and possibly for sars-cov-2 (92, 93), viruses use complex receptor recognition systems common to immunopathology damage mechanisms in coronavirusinfected individuals, which clearly define the clinical outcome (94) . therefore, application of vaccines that may interfere with antibody-mediated infection by coronaviruses (95) without true epidemiologic containment of coronaviruses, to restrict genetic adaptation events and inevitably producing an sse, may be a miscellaneous attempt. however, synergy of sse prevention measures with proper vaccination can provide a robust attempt for disease containment. this study aimed to perform a literature review. although effort was made to decrease the risk of bias of results via double-blind screening of literature and employment of multiple electronic search engines, bias cannot be eliminated due to incomplete retrieval of identified research and biased estimations of included literature conclusions and methods used. outcome of the study may also contain biased estimations originating from wrong interpretation of super spreading individuals in literature reviewed for sars, mers, and covid-19 outbreaks. although the importance of sses in covid-19 was recognized by this study, more data from future accumulated epidemiology studies are needed to justify these findings. taken all together, management of sses is mandatory to yield efficient control over sars-cov-2. this is achievable through early diagnosis of pre/asymptomatic infected individuals within potential super spreading groups. prevention of outbreaks is more essential, especially due to the lack of efficient vaccination and therapeutic protocols, which necessitates efficient monitoring, as sars-cov-2 virus follows complex infectious patterns. the sars-cov-2 epidemiological models that do not take sses into consideration seem to lead to confusing results with high uncertainty. sars-cov-2 causes prolonged "pandemics" through complex adaptation routes. currently, in addition to the high technology utilized for diagnosis, clinical observation is indispensable to deeply comprehend sses and prohibit further outspread of covid-19. reference laboratories with efficient and accredited molecular and serological diagnosis must be inter-linked between countries. all these parameters could contribute to avoiding a second blind unjustified response that characterized the first covid-19 pandemic spread. understanding the epidemiology of covid-19 through sses could be preventive for future epidemics. a systematic meta-analysis research methodology, when covid-19 epidemiology data accumulate further, would be advisable to confirm the conclusions of this study. this study did not involve the participation of any humans or animals as it was based only on literature research. ak inspired the conception and drafted the initial manuscript. jk revised substantially the manuscript, intellectual content and provided disclosed data for 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super-spreading events of sars in a hospital setting: who, when, and why super-spreaders in infectious diseases all authors agree to publish this manuscript. all data used for this manuscript are available upon request all authors declare that they have no competing interests. no funding or grant was received for this study. we thank our families for providing moral assistance to accomplish this study. key: cord-262045-r2iqpmmc authors: smits, saskia l.; raj, v. stalin; pas, suzan d.; reusken, chantal b.e.m.; mohran, khaled; farag, elmoubasher a.b.a.; al-romaihi, hamad e.; alhajri, mohd m.; haagmans, bart l.; koopmans, marion p. title: reliable typing of mers-cov variants with a small genome fragment date: 2014-12-15 journal: j clin virol doi: 10.1016/j.jcv.2014.12.006 sha: doc_id: 262045 cord_uid: r2iqpmmc background: middle east respiratory syndrome coronavirus (mers-cov) is an emerging pathogen that causes lower respiratory tract infection in humans. camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. human-to-human transmission occurs sporadically. the wide geographic distribution of mers-cov among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a mers-cov variant with efficient human-to-human transmission capabilities. phylogenetic analysis of mers-cov has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples. objective: to develop a simple, reliable mers-cov variant typing assay to facilitate monitoring of mers-cov diversity in animals and humans. study design: phylogenetic analysis of presently known full-length mers-cov genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable mers-cov variant typing. rt-pcr assays targeting these regions were designed and optimized. results: a reverse-transcription pcr assay for mers-cov targeting a 615 bp spike fragment provides a phylogenetic clustering of mers-cov variants comparable to that of full-length genomes. the detection limit corresponds to a cycle treshold value of ∼35 with standard upe real time pcr assays on rna isolated from mers-cov emc. nasal swabs from rt-pcr positive camels (ct values 12.9–32.2) yielded reliable sequence information in 14 samples. conclusions: we developed a simple, reliable mers-cov variant typing assay which is crucial in monitoring mers-cov circulation in real time with relatively little investment on location. middle east respiratory syndrome coronavirus (mers-cov; family coronaviridae) may cause severe lower respiratory tract infection in humans [1, 2] . camels are considered the likely animal source for zoonotic infection; sporadic human-to-human transmission does occur, but is considered to be inefficient based on currently available data [3] [4] [5] [6] [7] [8] [9] [10] . until recently, new mers-cov infections in humans were reported at a steady low rate reaching ∼200 confirmed human cases early 2014. in march and april 2014, however, a surge of mers-cov infections occurred mainly in hospitals around jeddah, kingdom of saudi arabia (ksa) and united arab emirates (uae). this increased case load was in part attributed to an increase in community cases, but mostly to transmission within hospitals, with no evidence for evolution of a mers-cov variant with more efficient human-to-human transmission capabilities (who;http://www.who.int/csr/disease/coronavirus infections/ mers cov update 09 may 2014.pdf?ua=1). the ongoing occurrence of new cases, however, and the finding that mers-cov is endemic in dromedary camels in a wide geographic region [3, 10, 11] , stresses the need for surveillance of strain diversity, to help unravel the epidemiology of this newly identified pathogen, and to provide a reference for studies into mers-cov evolution. all currently sequenced human and camel mers-cov genomes share >99% nucleotide identity across the ∼30 kb genome. phylogenetic analysis has occurred mainly by analysing full-length genomes or multiple concatenated genome fragments, to provide reliable phylogenetic information [5, [12] [13] [14] . however, full length genome sequencing capacity is not widely available, and requires relatively high viral load, leading to limited success when trying to sequence animal or human samples [5] . an accurate typing of mers-cov variants, preferably with a simple assay encompassing a short region of the mers-cov genome, is crucial in monitoring the mers-cov outbreak in real time with relatively little investments on location. in this study, we describe the development of a mers-cov variant typing assay, which can be used in monitoring mers-cov circulation, especially when more information on virus type is required rapidly from a large number of viruses from animals/humans. full or near full-length mers-cov genomes encompassing nucleotides 215-29770 (numbering corresponding to mers-cov emc genome jx869059) were aligned with mafft version 7 (http://mafft.cbrc.jp/alignment/server/) ( table 1) . to remove the redundancy from the dataset, fastgroupii analysis (http://fastgroup.sdsu.edu/fg tools.htm) was performed grouping all currently available viral genomes based on nucleotide composition, resulting in 15 groups (table 1) . one viral genome from each group was taken as representative in subsequent analyses. a summary of nucleotide positions that vary across the genomes was created using bioedit v7.2.0 [15] and the number of nucleotide positions with variations was plotted over 1000 nucleotide windows. phyml trees were generated using seaview 4 software with the approximate likelihood ratio test based on a shimodaira-hasegawa-like procedure which used general time reversible as substitution model. nearest neighbor interchange, subtree pruning, and regrafting-based tree search algorithms were used to estimate tree topologies, as described previously [5] . total nucleic acids were isolated using an automated mag-napure 96 extraction with the total nucleic acid isolation kit (roche, mannheim, germany) as described previously [13] . the detection limit of the assay was determined on rna isolated from 10x dilutions of cell culture derived mers-cov emc 2012 (jx869059), as described above. virus stocks were prepared as described previously [16] . rna was isolated from serial 10-fold dilutions of mers-cov emc 2012 [13] . serial 10-fold dilutions of this rna were amplified in parallel with the mers-cov variant typing assay described above and the upe and n gene real time pcr assays [17, 18] . the sensitivity of the mers-cov variant typing assay was expressed as cycle threshold value based on the upe real time pcr assay. on may 13 and 15, 2014, the first two mers-cov infected patients in the netherlands who became infected upon travel to saudi arabia were reported to who; throat swabs from these patients were available [19] . in february and april 2014, nasal swabs were taken from dromedary camels of different age and sex from a slaughterhouse in doha, qatar [13] , which were available for this study. to identify genome regions for reliable phylogenetic analysis comparable to that of full-length genome, (near) full-length mers-cov genomes (table 1) were aligned. viral genomes that were 99.9% identical were grouped and one viral genome from each group was taken as representative in the analysis (table 1 ) and a phyml tree was generated (fig. 1a) . was plotted over 1000 nt windows (fig. 1b) . four genome fragments, two located in orf1a, one in s and one in orf4b, showed a relatively high number of snps (fig. 1b) and for this reason already had been used as concatenated genome fragment for phylogenetic analysis [12] . however, phylogenetic trees created for these four fragments separately did not accurately reflect the phylogenetic positions of the currently known full-length mers-cov genomes (data not shown). in a subsequent analysis all identified snps were inspected visually and regions containing snps with phylogenetic information regarding the previously identified four clusters of viruses were identified (fig. 1a) . the s2 domain of the spike protein contains a number of these mutations (fig. 1b) . a fragment of 615 bp, containing three of these snps, provided a phylogenetic tree similar to the one obtained upon full-genome analysis regarding the previously identified four mers-cov clusters (fig. 2) , whereas other mers-cov genome regions did not provide similar results. as observed for the full-length genomes, human, and camel mers-cov genomes shared >99% nucleotide identity across the 615 bp s2 domain fragment. mers-cov genomes that were released recently and not taken along in the variation analysis were typed using the 615 bp s2 domain and clustered similar to their phylogenetic positions as upon full genome analysis ( fig. 2 and table 1 ), thereby validating the assay. a sequencing rt-pcr targeting the identified genome fragment was developed and optimized using rna isolated from cell culturederived mers-cov. in limiting dilution experiments, the mers-cov variant typing assay amplified the 615 bp fragment down to a cycle treshold value of ∼35 as determined by diagnostic upe real time pcr assays [17, 18] . the mers-cov variant typing assay was used to type rt-pcr positive nose swabs from the first two human dutch mers-cov cases in 2014. the results showed grouping consistent with previous findings based on long sequence fragments [19] (fig. 2 ). in addition, the mers-cov variant typing assay was performed on camel samples from a slaughterhouse in qatar [13] and sequences for 14 mers-cov positive animals with cycle threshold values ranging from 12.9 to 32.2 as determined by upe real time rt-pcr [17, 18] were obtained (fig. 2) . five different camel mers-cov variants in clusters b1 and b2 were detected, without the need for full genome sequencing. phylogenetic analysis of representative mers-cov full-length genomes indicated that four regions in the mers-cov genome exist with a substantially higher nucleotide variation across genomes. however, phylogenetic analysis of these genome regions sepa-rately did not provide reliable phylogenetic information, in contrast to an analysis of the concatenated fragments [5, 12, 19] . subsequent analyses revealed a region in the open reading frame that encodes the spike protein with a number of positions in which nucleotide variation occurs between mers-cov variants with a strong phylogenetic signal regarding previously identified clusters of viruses based on full-length mers-cov genomes. the here[19] and from camels from a slaughterhouse in doha, qatar [13] . the observed detection limit of a cycle treshold value of ∼35 allows variant typing in clinical samples obtained from humans and animals with relatively low viral loads. it enables inclusion of samples in phylogenetic analysis that would not have been included when only full length genomes would have been accepted. this mers-cov variant typing assay, targeting a part of the mers-cov spike gene, is a relatively simple rt-pcr sequencing assay that could be performed more widely as initial screening assay in laboratories with basic sequencing capacity. it provides accurate crude mers-cov type information, applicable in monitoring viral variants in real time. new variants identified through this initial screening could then be sent to a reference laboratory for further characterization. the continued occurrence of transmission between humans in health care and family settings is an ongoing concern as stated by the world health organization (http://www.who.int/csr/disease/coronavirus infections/mers cov update 27 march 2014.pdf?ua=1), although the outbreaks appear to be self-limiting or extinguishable with rigorous implementation of appropriate infection control guidelines at present. however, as the primary route of transmission to humans is uninterrupted, human-to-human transmissions will continue to occur. the data obtained from the mers-cov variant typing assay would aid in informing the most effective international preparedness and response, allowing ad hoc risk assessment and implementation of containment strategies if necessary. state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia human infection with mers coronavirus after exposure to infected camels antibodies against mers coronavirus in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands this work was funded by zonmw top project 91213058z. this does not alter our adherence to all the policies on sharing data and materials. all authors contributed to gathering and analysis of the information. saskia smits, bart haagmans, and marion koopmans drafted and revised the manuscript based on all authors contributions. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2014.12.006. key: cord-263481-w5ytp1q7 authors: lokman, syed mohammad; rasheduzzaman, m.d.; salauddin, asma; barua, rocktim; tanzina, afsana yeasmin; rumi, meheadi hasan; hossain, m.d. imran; siddiki, a.m.a.m. zonaed; mannan, adnan; hasan, m.d. mahbub title: exploring the genomic and proteomic variations of sars-cov-2 spike glycoprotein: a computational biology approach date: 2020-06-02 journal: infect genet evol doi: 10.1016/j.meegid.2020.104389 sha: doc_id: 263481 cord_uid: w5ytp1q7 the newly identified sars-cov-2 has now been reported from around 185 countries with more than a million confirmed human cases including more than 120,000 deaths. the genomes of sars-cov-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins need to be explored to understand the biology of the virus. spike glycoprotein is one of the major targets to be explored because of its role during the entry of coronaviruses into host cells. we analyzed 320 whole-genome sequences and 320 spike protein sequences of sars-cov-2 using multiple sequence alignment. in this study, 483 unique variations have been identified among the genomes of sars-cov-2 including 25 nonsynonymous mutations and one deletion in the spike (s) protein. among the 26 variations detected, 12 variations were located at the n-terminal domain and 6 variations at the receptor-binding domain (rbd) which might alter the interaction of s protein with the host receptor angiotensin converting enzyme-2 (ace2). besides, 22 amino acid insertions were identified in the spike protein of sars-cov-2 in comparison with that of sars-cov. phylogenetic analyses of spike protein revealed that bat coronavirus have a close evolutionary relationship with circulating sars-cov-2. the genetic variation analysis data presented in this study can help a better understanding of sars-cov-2 pathogenesis. based on results reported herein, potential inhibitors against s protein can be designed by considering these variations and their impact on protein structure. wuhan, hubei province of china in december 2019. the death toll rose to more than 68,000 among 1,250,000 confirmed cases around the globe (until april 4, 2020) [1] . the virus causing covid-19 is named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). based on the phylogenetic studies, the sars-cov-2 is categorized as a member of the genus betacoronavirus, the same lineage that includes sars coronavirus (sars-cov) [2] that caused sars (severe acute respiratory syndrome) in china during 2002 [3] . recent studies showed that sars-cov-2 has a close relationship with bat sars-like covs [4, 5] [7] ]. interestingly, s glycoprotein is characterized as the critical determinant for viral entry into host cells which consists of two functional subunits namely s1 and s2. the s1 subunit recognizes and binds to the host receptor through the receptor-binding domain (rbd) whereas s2 is responsible for fusion with the host cell membrane [ [8] , [9] , [10] ]. mers-cov uses dipeptidyl peptidase-4 (dpp4) as entry receptor [11] whereas sars-cov and sars-cov-2 utilize ace-2 (angiotensin converting enzyme-2) [12] , abundantly available in lung alveolar epithelial cells and enterocytes, suggesting s glycoprotein as a potential drug target to halt the entry of sars-with remarkable properties like glutamine-rich 42 aa long exclusive molecular signature (dsqqtvgqqdgsednqtttiqtivevqpqlemeltpvvqtie) in position 983-1024 of polyprotein 1ab (pp1ab) [16] , diversified receptor-binding domain (rbd), unique furin cleavage site (prrar↓sv) at s1/s2 boundary in s glycoprotein which could play roles in viral pathogenesis, diagnosis and treatment [17] . to date, few genomic variations of sars-cov-2 are reported [ [18] , [19] ]. there is growing evidence that spike protein, a 1273 amino acid long glycoprotein having multiple domains, possibly plays a major role in sars-cov-2 pathogenesis. viral entry to the host cell is initiated by the receptor-binding domain (rbd) of s1 head. upon receptor-binding, proteolytic cleavage occurs at s1/s2 cleavage site and two heptad repeats (hr) of s2 stalk form a six-helix bundle structure triggering the release of the fusion peptide. as it comes into close proximity to the transmembrane anchor (tm), the tm domain facilitates membrane destabilization required for fusion between virus-host membranes [ [20] , [21] ]. insights into the sequence variations of s glycoprotein among available genomes are key to understanding the biology of sars-cov-2 infection, developing antiviral treatments and vaccines. in this study, we have analyzed 320 genomic sequences of sars-cov-2 to identify mutations between the available genomes followed by the amino acid variations in the glycoprotein s to foresee their impact on the viral entry to host cell from structural biology viewpoint. analysis. the ncbi reference sequence of sars-cov-2 s glycoprotein, accession number yp_009724390 was used as the canonical sequence for the analyses of spike protein variants. variant analyses of sars-cov-2 genomes were performed in the genome detective coronavirus typing tool version 1.13 which is specially designed for this virus the dataset was then aligned with muscle [24] . entropy (h(x)) plot of nucleotide variations in sars-cov-2 genome was constructed using bioedit [25] . mega x (version 10.1.7) was used to construct the msas and the phylogenetic tree using pairwise alignment and neighborjoining methods in clustalw [26, 27] . tree structure was validated by running the analysis on 1000 bootstraps [28] replications dataset and the evolutionary distances were calculated using the poisson correction method [29] . variant sequences of sars-cov-2 were modeled in swiss-model [30] using the cryo-em spike protein structure of sars-cov-2 (pdb id 6vsb; [8] ) as a template. the overall quality of models was assessed in rampage server [31] by generating ramachandran plots (supplementary table 1 ). pymol and biovia discovery studio were used for structure visualization and superpose [32, 33] . j o u r n a l p r e -p r o o f 3. results multiple sequence alignment of the available 320 genomes of sars-cov-2 were performed and 483 variations were found throughout the 29,903 bp long sars-cov-2 genome with in total 115 variations in utr region, 130 synonymous variations that cause no amino acid alteration, 228 non-synonymous variations causing change in amino acid residue, 16 indels, and 2 variations in non-coding region (supplementary table 2 ). among the 483 variations, 40 variations (14 synonymous, 25 non-synonymous mutations and one deletion) were observed in the region of orf s that encodes s glycoprotein which is responsible for viral fusion and entry into the host cell [34] . notable that, most of the sars-cov-2 genome sequences were deposited from the usa (250) and china (50) (supplementary fig. 1 ). positional variability of the sars-cov-2 genome was calculated from the msa of 320 sars-cov-2 whole genomes as a measure of entropy value (h(x)) [35] . excluding 5′ and 3′ utr, ten hotspot of hypervariable position were identified, of which seven were located at orf1ab (1059c>t, 3037c>t, 8782c>t, 14408c>t, 17747c>t, 17858a>g, 18060c>t) and one at orf s (23403a>g), orf3a (25563g>t), and orf8 (28144t>c) respectively. the variability at position 8782 and 28144 were found to be the highest among the other hotspots ( fig. 1 ). the phylogenetic analysis of a total of 66 sequences (26 unique sars-cov-2 and 40 different coronavirus s glycoprotein sequences) was performed. the evolutionary distances showed that all the sars-cov-2 spike proteins cluster in the same node of the phylogenetic tree confirming the sequences are similar to refseq yp_009724390 (fig. 2) . bat coronaviruses has a close evolutionary relationship as different strains were found in the nearest outgroups and clades (bat coronavirus bm48-31, bat hp-beta coronavirus, bat coronavirus hku9) conferring that j o u r n a l p r e -p r o o f journal pre-proof coronavirus has vast geographical spread and bat is the most prevalent host (fig. 2) . in other clades, the clusters were speculated through different hosts which may describe the evolutionary changes of surface glycoprotein due to cross species transmission. viral hosts reported from different spots at different times is indicative of possible recombination. the s glycoprotein sequences of sars-cov-2 were retrieved from the ncbi virus variation resource repository and aligned using clustalw. the position of sars-cov-2 spike protein domains was measured by aligning with the sars-cov spike protein (fig. 3 ) [36, 37] . from the sequence identity matrix, 26 unique variants among unique sars-cov-2 spike glycoprotein sequences were identified to have 25 substitutions and a deletion ( fig. 4a and supplementary table 3 ). 215 sequences were found identical with sars-cov-2 s protein reference sequence (yp_009724390) while 64 sequences were identical with the same variation of d614g (supplementary table 4 respectively due to substitution of amino acid that differs in charge. the remaining 15 variants were mutated with the amino acids that are similar in charge (fig. 4 a) . the sars-cov-2 spike protein variants were superposed with the cryo-electron microscopic structure of sars-cov-2 spike protein [8] . fig. 3) . the s2 subunit of spike protein, especially the heptad repeat region 2, fusion peptide domain, transmembrane domain, and cytoplasmic tail, were found to be highly conserved in the sars-cov and the sars-cov-2 variants while the s1 subunit was more diverse, specifically the n-terminal domain (ntd) and receptor-binding domain (rbd). the spatial distribution of s protein sequences having different variation over time reveals that most of the variants (17 out of 240 s glycoprotein sequences) were reported from the us j o u r n a l p r e -p r o o f journal pre-proof followed by 3 out of 2 sequences (including y145 deletion) and 2 out of 50 sequences from india and china, respectively (fig. 5) . only one variant was found out of only one available sequence in the repository from sweden, australia, south korea and peru. interestingly, all sequences are unique among countries from the sequence reported except d614g, which was found in the us and peru (fig. 5) . moreover, we have also analyzed sequences from brazil, italy, nepal, pakistan, spain, taiwan and vietnam but there is no variation in the s glycoprotein sequence was found when compared to refseq yp_009724390. covid 19 is one of the most contagious pandemics the world has ever had with 1,250,000 confirmed cases to date (april 4, 2020) and the cases have increased as high as 5 times in less than a month [1] . phylogenetic analysis showed that the sars-cov-2 is a unique coronavirus presumably related to bat coronavirus (bm48-31, hp-betacoronavirus). during this study, we [38] , [39] , [40] ]. likewise, a number of studies targeting sars-cov-2 spike protein have been undertaken for the therapeutic measures [41] , but the unique structural and functional details of sars-cov-2 spike protein are still under scrutiny. we also found a variant (r408i) at receptor binding domain (rbd) that mutated from positively charged arginine residue to neutral and smaller sized isoleucine residue (fig. 4 i) . this change might alter the interaction of viral rbd with the host receptor because the r408 residue of sars-cov-2 is known to interact with the ace2 receptor for viral entry [42] . similarly, alterations of rbd (g476s, v483a, h519q, and a520s) also could affect the interaction of sars-cov-2 spike protein with other molecules j o u r n a l p r e -p r o o f which require further investigations. qia98583 and qis30615 variants were found to have an alteration of alanine to valine (a930v), and aspartic acid to tyrosine (d936y) respectively in the alpha helix of the hr1 domain. previous reports have indicated that hr1 domain plays a significant role in viral fusion and entry by forming helical bundles with hr2, and mutations including alanine substitution by valine (a1168v) in hr1 region are predominantly responsible for conferring resistance to mouse hepatitis coronaviruses against hr2 derived peptide entry inhibitors [43] . this study hypothesizes the mutation (a930v) found in that of sars-cov-2 might also have a role in the emergence of drug-resistance virus strains. also, the mutation (d1168h) found in the heptad repeat 2 (hr) sars-cov-2 could play a vital role in viral pathogenesis. moreover, we found that 20 variants including one deletion out of 26 were located within s1 especially within ntd and rbd region of glycoprotein s (fig. 4a) which region is responsible for the preliminary interaction with the host cell receptor ace2. this indicates that the ntd and rbd are very prone to mutations. however, the ntd and rbd portions harbour potential epitopes that might serve as potential peptide vaccine candidates against sars-cov-2 as reported in different studies [44] [45] [46] . the reason behind choosing the sequences from s protein domain ntd and rbd is they are situated in the outer surface of the virus that could be more accessible for the immune system (fig. 3c ). so the variations reported herein within the outer domains of s glycoprotein could help to design effective epitope-based vaccines or antivirals. the sars-cov-2 s protein contains additional furin protease cleavage site, prrars, in s1/s2 domain which is conserved among all 320 sequences as revealed during this study ( supplementary fig. 3 ). this unique signature is thought to make the sars-cov-2 more virulent than sars-cov and regarded as novel features of the viral pathogenesis [9] . according to previous reports the more the host cell protease can process the coronavirus s can accelerate viral tropism accordingly in influenza virus [[9] , [47] , [48] , [49] ]. apart from that, this could also promote viruses to escape antiviral therapies targeting transmembrane protease j o u r n a l p r e -p r o o f tmprss2 (clinicaltrials.gov, nct04321096) which is well reported protease to cleave at s1/s2 of s glycoprotein [50] . comparative analyses between sars-cov and sars-cov-2 spike glycoprotein showed 77% similarity between them where the most diverse region was coronavirus disease (covid-2019) situation reports severe acute respiratory syndrome-related coronavirus--the species and its viruses, a statement of the coronavirus study group lim, others, a novel coronavirus associated with severe acute respiratory syndrome bats are natural reservoirs of sars-like coronaviruses, science (80-. ) huang, others, a pneumonia outbreak associated with a new coronavirus of probable bat origin pei, others, a new coronavirus associated with human respiratory disease in china genome composition and divergence of the novel coronavirus (2019-ncov) originating in china cryo-em structure of the 2019-ncov spike in the prefusion conformation, science (80-. ) structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structure analysis of the receptor binding of 2019-ncov fouchier, others, dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc greenough, others, angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses a. nitsche, others, sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor an exclusive 42 amino acid signature in pp1ab protein provides insights into the evolutive history of the 2019 novel human-pathogenic coronavirus (sars-cov2) the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade genomic variance of the 2019-ncov coronavirus genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors muscle: multiple sequence alignment with improved accuracy and speed bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt mega x: molecular evolutionary genetics analysis across computing platforms the neighbor-joining method: a new method for reconstructing phylogenetic trees bootstrap confidence levels for phylogenetic trees evolutionary divergence and convergence in proteins swiss-model: homology modelling of protein structures and complexes structure validation by calpha geometry: phi, psi and cbeta deviation pymol: an open-source molecular graphics tool receptor recognition mechanisms of coronaviruses: a decade of structural studies a parvovirus b19 synthetic genome: sequence features and functional competence cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein role of changes in sars-cov-2 in the interaction with the human ace2 receptor: an in silico analysis coronavirus escape from heptad repeat 2 (hr2)-derived peptide entry inhibition as a result of mutations in the hr1 domain of the spike fusion protein development of epitope-based peptide vaccine against novel coronavirus 2019 (sars-cov-2): immunoinformatics approach in silico identification of novel b cell and t cell epitopes of wuhan coronavirus (2019-ncov) for effective multi epitope-based peptide vaccine production epitope-based chimeric peptide vaccine design against s, m and e proteins of sars-cov-2 etiologic agent of global pandemic covid-19: an in silico approach host cell proteases controlling virus pathogenicity role of hemagglutinin cleavage for the pathogenicity of influenza virus host cell proteases: critical determinants of coronavirus tropism and pathogenesis coronaviruses: an overview of their replication and pathogenesis receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins laude, others, aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev positional organization of major structural protein-encoding genes in orange color (s = spike protein, e = envelope protein, m = membrane protein, n = nucleocapsid protein) and accessory protein orfs in blue colors. b. variability within 320 sars-cov-2 genomic sequences represented by entropy (h(x)) value across genomic location key: cord-124012-5zxkd2jy authors: schwab, patrick; schutte, august dumont; dietz, benedikt; bauer, stefan title: predcovid-19: a systematic study of clinical predictive models for coronavirus disease 2019 date: 2020-05-17 journal: nan doi: nan sha: doc_id: 124012 cord_uid: 5zxkd2jy coronavirus disease 2019 (covid-19) is a rapidly emerging respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). due to the rapid human-to-human transmission of sars-cov-2, many healthcare systems are at risk of exceeding their healthcare capacities, in particular in terms of sars-cov-2 tests, hospital and intensive care unit (icu) beds and mechanical ventilators. predictive algorithms could potentially ease the strain on healthcare systems by identifying those who are most likely to receive a positive sars-cov-2 test, be hospitalised or admitted to the icu. here, we study clinical predictive models that estimate, using machine learning and based on routinely collected clinical data, which patients are likely to receive a positive sars-cov-2 test, require hospitalisation or intensive care. to evaluate the predictive performance of our models, we perform a retrospective evaluation on clinical and blood analysis data from a cohort of 5644 patients. our experimental results indicate that our predictive models identify (i) patients that test positive for sars-cov-2 a priori at a sensitivity of 75% (95% ci: 67%, 81%) and a specificity of 49% (95% ci: 46%, 51%), (ii) sars-cov-2 positive patients that require hospitalisation with 0.92 auc (95% ci: 0.81, 0.98), and (iii) sars-cov-2 positive patients that require critical care with 0.98 auc (95% ci: 0.95, 1.00). in addition, we determine which clinical features are predictive to what degree for each of the aforementioned clinical tasks. our results indicate that predictive models trained on routinely collected clinical data could be used to predict clinical pathways for covid-19, and therefore help inform care and prioritise resources. c oronavirus disease 2019 (covid19) was first discovered in december 2019 in china, and has since rapidly spread to over 200 countries [1] . the covid-19 pandemic challenges healthcare systems worldwide as a high peak capacity for testing and hospitalisation is necessary to diagnose and treat affected patients, particularly if the spread of sars-cov-2 is not mitigated. to avoid exceeding the available healthcare capacities, many countries have adopted social distancing policies, imposed travel restrictions, and postponed non-essential care and surgeries in order to reduce peak demand on their healthcare systems [2] , [3] , [4] . we study the use of predictive models (light purple) to estimate whether patients are likely (i) to be sars-cov-2 positive, and whether sars-cov-2 positive patients are likely (ii) to be admitted to the hospital and (iii) to require critical care based on clinical, demographic and blood analysis data. accurate clinical predictive models stratify patients according to individual risk, and, in this manner, help prioritise healthcare resources, such as testing, hospital and critical care capacity. the adoption of clinical predictive models that accurately predict who is likely to require testing, hospitalisation and intensive care from routinely collected clinical data could potentially further reduce peak demand by ensuring resources are prioritised to those individuals with the highest risk ( figure 1 ). for example, a clinical predictive model that accurately identifies patients that are likely to test positive for sars-cov-2 a priori could help prioritise limited sars-cov-2 testing capacity. however, developing accurate clinical prediction models for sars-cov-2 is difficult as relationships between clinical data, hospitalisation, and intensive care unit (icu) admission have not yet been established conclusively due to the recent emergence of sars-cov-2. in this systematic study, we develop and evaluate clinical predictive models that use routinely collected clinical data to identify (i) patients that are likely to receive a positive sars-cov-2 test, (ii) sars-cov-2 positive patients that are likely to require hospitalisation, and (iii) sars-cov-2 positive patients that are likely to require intensive care. using the developed predictive models, we additionally determine which clinical features are most predictive for each of the aforementioned clinical tasks. our results indicate that predictive models could be used to predict clinical pathways for covid-19 patients. such predictive models may be of significant utility for healthcare systems as preserving healthcare capacity has been linked to successfully combating sars-cov-2 [5] , [6] . this work contains the following contributions: • we develop and systematically study predictive models for estimating the likelihoods of (i) a positive sars-cov-2 test in patients presenting at hospitals, (ii) hospital admission in sars-cov-2 positive patients, and (iii) critical care admission in sars-cov-2 positive patients. • we validate the performance of the developed clinical predictive models in a retrospective evaluation using realworld data from a cohort of 5644 patients. • we determine and quantify the predictive power of routinely-collected clinical, demographic, and blood analysis data for the aforementioned clinical prediction tasks. a substantial body of work is dedicated to the study, validation and implementation of predictive models for clinical tasks. clinical predictive models have, for example, been used to predict risk of septic shock [7] , [8] , risk of heart failure [9] , readmission following heart failure [10] , [11] , [12] , false alarms in critical care [13] , risk scores [14] , outcomes [15] and mortality in pneumonia [16] , [17] , and mortality risk in critical care [18] , [19] , [20] . predicting clinical outcomes for individual patients is difficult because a large number of confounding factors may influence patient outcomes, and collecting and accounting for these factors in an unbiased way remains an open challenge in clinical practice [21] . systematic studies, such as the one presented in this work, enable medical practitioners to better understand, assess and potentially overcome these issues by systematically evaluating generalisation ability, expected predictive performance, and influential predictors of various clinical predictive models. beyond the need for systematic evaluation, missingness [22] , [23] , [24] , [25] , noise [26] , [27] , multivariate input data [13] , [28] , [29] , [30] , and the need for interpretability [31] , [32] , [33] , [34] have been highlighted as particularly important considerations in healthcare settings. in this work, we build on recent methodological advances to develop and systematically study clinical predictive models that may aid in prioritising healthcare resources [35] for covid-19, and thereby help prevent a potential overextension of healthcare system capacity. several clinical predictive models have recently been proposed for covid-19, for example, for predicting potential covid-19 diagnoses using data from emergency care admission exams [36] and chest imaging data [37] , [38] , [39] , [40] , [41] , [42] , for predicting covid-19 related mortality from clinical risk factors [43] , [44] , and for predicting which patients will develop acute respiratory distress syndrome (ards) from patients' clinical characteristics [45] . [46] presented a review of epidemiology and clinical features associated with covid-19, and [47] a critical review that assessed limitations and risk of bias in diagnostic and prognostic models for covid-19. in addition, [48] performed a cohort study for clinical and laboratory predictors of covid-19 related inhospital mortality that identified baseline neutrophil count, age fig. 2 : the presented multistage machine-learning pipeline consists of preprocessing (light purple) the input data x, developing multiple candidate models using the given dataset (orange), selecting the best candidate model for evaluation (blue), and evaluating the selected best model's outputsŷ. and several other clinical features as top predictors of mortality. beyond prediction, [49] have argued for the responsible use of data in tackling the challenges posed by sars-cov-2. owing to the recent emergence of sars-cov-2, there currently exists, to the best of our knowledge, no prior systematic study on clinical predictive models that predict likelihood of a positive sars-cov-2 test, hospital and intensive care unit admission from clinical, demographic and blood analysis data that accounts for the missingness that is characteristic for the clinical setting. we additionally assess the influence of various clinical, demographic, and blood analysis measurements on the predictions of the developed clinical predictive models. 1) problem setting: in the given setting, we are given 106 routine clinical, laboratory and demographic measurements, or features, x i ∈ x for presenting patients. features may be discrete or continuous, and some features may be missing as not all tests are necessarily performed on all patients. the clinical predictive tasks consist of utilising the routine clinical features x i to predict, for a newly presenting patient, (i) the likelihoodŷ sars-cov-2 of receiving a positive sars-cov-2 test result, (ii) the likelihoodŷ admission of requiring hospital admission, and the (iii) likelihoodŷ icu of requiring intensive care. in addition, we are given a development dataset consisting of n patients, their corresponding observed routine clinical features x i , sars-cov-2 test results y sars-cov-2 ∈ {0, 1}, hospital admissions y admission ∈ {0, 1}, and icu admissions y icu ∈ {0, 1}, where 1 indicates the presence of an outcome. using this development dataset, our goal is to derive clinical predictive modelsf sars-cov-2 ,f admission andf icu for the respective before-mentioned tasks in order to inform care and help prioritise scarce healthcare resources. 2) methodology: to derive the clinical predictive modelŝ f sars-cov-2 ,f admission andf icu from the given development dataset, we set up a systematic model development, validation, and evaluation pipeline (fig. 2) . to evaluate the generalisation ability of the developed clinical predictive models and to rule out overfitting to patients in the evaluation cohort, the development data is initially split into independent and stratified training, validation, and test folds without any patient overlap. concretely, the multistage pipeline consists of (i) preprocessing, (ii) model development, (iii) model selection, and (iv) model evaluation stages. for preprocessing and model development, only the training fold is used, and only the validation and test folds of the development data are used for model selection and model evaluation, respectively. we outline the pipeline stages in detail in the following paragraphs. 3) preprocessing: in the preprocessing stage, we first drop all input features that are missing for more than 99.8% of all training set patients to ensure we have a minimal amount of data for each feature. this removes a total of 9 features from the original 106 routine clinical, laboratory and demographic features. we then transform all discrete features for each patient into their one-hot encoded representation with one out of p indicator variables set to 1 to indicate the discrete value for this patient, and all others set to 0 with p being the number of unique values for the discrete feature. we defined those features as discrete that have fewer than 6 unique values across all patients in the training fold. for discrete features, missing features were counted as a separate category in the one hot representation. next, we standardised all continuous features to have zero mean and unit standard deviation across the training fold data. lastly, we performed multiple imputation by chained equations (mice) to impute all missing values of every continuous feature from the respective other features in an iterative fashion [50] . we additionally added a missing indicator that indicates 1 if the feature was imputed by mice and 0 if it was originally present in order to preserve missingness information in the data after imputation. after the preprocessing stage, continuous input features are standardised and fully imputed, and discrete input features are one-hot encoded. all preprocessing operations are derived only from the training fold, and naïvely applied without adjustment to validation and test folds in order to avoid information leakage. 4) model development: in the model development stage, we train candidate clinical predictive modelsf sars-cov-2 ,f admission andf icu using supervised learning on the training fold of the preprocessed data. to derive the models from the preprocessed training fold data, we optimise various types of predictive models, and perform a hyperparameter search with m runs for each of them. the model development process yields m candidate models with different hyperparameter choices and predictive performances for each model category. 5) model selection: in order to select the best model amongst the set of candidate models, we evaluate their predictive performance against the held-out validation fold that had not been used for model development. we choose the top candidate model by ranking all models by their evaluated predictive performance. the model selection stage using the independent validation fold enables us to optimise hyperparameters without utilising test fold data. 6) model evaluation: in the model evaluation stage, we evaluate the selected best clinical predictive model against the held-out test fold that had not been used neither for training nor model selection in order to estimate the expected generalisation error of the models on previously unseen data. using this approach, every selected best model from the model selection stage is evaluated exactly once against the test fold. using the presented standardised model development, selection and evaluation pipeline, we compare various types of clinical predictive models in the same test setting, with exactly the same amount of hyperparameter optimisation and input features against the same test fold. this process enables us to systematically study the expected generalisation ability, predictive performance and influential features of clinical predictive models for predicting sars-cov-2 test results, hospital admission for sars-cov-2 positive patients, and icu admission for sars-cov-2 positive patients. we conducted retrospective experiments to evaluate the predictive performance of a number of clinical predictive models on each of the presented clinical prediction tasks using the standardised development, validation and evaluation pipeline. concretely, our experiments aimed to answer the following questions: 1 what is the expected predictive performance of the various clinical predictive models in predicting (i) sars-cov-2 test results for presenting patients, (ii) hospital admission for sars-cov-2 positive patients, and (iii) icu admission for sars-cov-2 positive patients? 2 which clinical, demographic and blood analysis features were most important for the respective best encountered predictive models for each clinical prediction task? the following subsections describe the conducted experimental evaluation in detail. we used anonymised data from a cohort of 5644 patients seen at the hospital israelita albert einstein in são paulo, brazil in the early months of 2020 1 . over the data collection time frame, the rate of sars-cov-2 positive patients at the hospital was around 10% of which around 6.5% and 2.5% required hospitalisation and critical care, respectively (table i) . notably, younger patients were underrepresented in the sars-cov-2 positive group relative to the general patient population which may have been caused by the reportedly more severe disease progression in older patients [51] . information on patient sex was not included in our dataset. we randomly split the entire available patient cohort into training (50%), validation (20%) and test folds (30%) within strata of patient age, sars-cov-2 test result, hospital admission status, and icu admission status. after stratification, the three folds were approximately balanced across the stratification dimensions. using the presented systematic evaluation methodology, we trained five different model types: logistic regression (lr), neural network (nn), random forest (rf), support vector machine (svm), and gradient boosting (xgb) [52] . the nn was a multi-layer perceptron (mlp) consisting of l hidden layers with n hidden units each followed by a non-linear activation function (relu [53] , selu [54] , or elu [55] ) and batch normalisation [56] , and was trained using the adam optimiser [57] for up to 300 epochs with an early stopping patience of 12 epochs on the validation set loss. we followed an unbiased, systematic approach to hyperparameter selection and optimisation. for each type of clinical predictive model, we performed a maximum of 30 hyperparameter optimisation runs with hyperparameters chosen from predefined ranges (table ii) . the performance of each hyperparameter optimisation run was evaluated against the validation cohort. after computing the validation set performance, we selected the best candidate predictive model across the 30 hyperparameter optimisation runs by area under the receiver operator curve for further evaluation against the test set. d. metrics 1) predictive performance: to assess the predictive performance of each of the developed clinical predictive models, we evaluated their performance in terms of area under the receiver operator curve (auc), area under the precision recall curve (aupr), sensitivity, specificity, and specificity at greater than 95% sensitivity (spec.@95%sens.) on the held-out test set cohorts for each task (table i) . after model development and hyperparameter optimisation, we evaluated each model type exactly once against the test set to calculate the final performance metrics. operating thresholds for each model were the operating points on the receiver operator characteristic curve closest to the top left coordinate as calculated for the validation cohort. we chose a variety of complementary evaluation metrics in order to give a comprehensive picture of the expected performance of each clinical predictive model on the evaluated tasks. for each of the performance metrics, we additionally computed 95% confidence intervals (cis) using bootstrap resampling with 100 bootstrap samples on the test set cohort in order to quantify the uncertainty of our analysis results. we also assessed whether differences between clinical predictive models were statistically significant at significance level α = 0.05 using pairwise t-tests with the respective best models for each task as measured by auc. 2) importance of test types: to quantify the importance of specific clinical, demographic and blood analysis features on each of the predicted outcomes, we utilised causal explanation (cxplain) models [34] . cxplain provides standardised relative feature importance attributions for any predictive model by computing the marginal contribution of each input feature towards the predictive performance of a model [58] , and is therefore particularly well-suited for assessing feature importance in our diverse set of models. we used the test fold's ground truth labels to compute the exact marginal contribution of each input feature without any estimation uncertainty. in terms of predictive performance (table iii) , we found that the overall best identified models by auc were xgb for predicting sars-cov-2 test results, rf for predicting hospital admissions for sars-cov-2 positive patients, and svm for predicting icu admission for sars-cov-2 positive patients . notably, we found that predicting positive sars-cov-2 results from routinely collected clinical measurements was a considerably more difficult task for clinical predictive models than predicting hospitalisation and icu admission. nonetheless, the best encountered clinical predictive model for predicting sars-cov-2 test results (xgb) achieved a respectable sensitivity of 75% (95% ci: 67%, 81%) and specificity of 49% (95% ci: 46%, 51%). after fixing the operating threshold of the model to meet a sensitivity level of at least 95% (spec.@95% sens.), the best xgb model for predicting sars-cov-2 test results would achieve a specificity of 23% (95% ci: 7%, 32%). we additionally found that the differences in predictive performance between the best xgb model for predicting sars-cov-2 test results and the other predictive models was significant at a pre-specified significance level of α = 0.05 (ttest) for all but the aupr metric, where nn achieved a significantly better aupr of 0.22 and the difference to svm was not significant at the pre-specified significance level. on the task of predicting hospital admissions for sars-cov-2 positive patients, the best encountered rf model achieved a sensitivity of 55% (95% ci: 19%, 85%), a high specificity of 96% (95% ci: 92%, 98%), and a specificity at a fixed sensitivity of at least 95% (spec.@95% sens.) of 34% (95% ci: 29%, 97%). owing to the lower sample size due to the smaller cohort of sars-cov-2 positive patients, the performance results for predicting hospital admission generally had wider uncertainty bounds but were nonetheless significantly better for rf than the other predictive models at the pre-specified significance level of α = 0.05 (t-test) for most performance metrics with the exception of auc where xgb achieved an auc of 0.91 and aupr where lr achieved an aupr of 0.44. on the task of predicting icu admission for sars-cov-2 positive patients, svm had a sensitivity of 80% (95% ci: 36%, 100%), a specificity of 96% (95% ci: 92%, 98%), and a specificity at a fixed sensitivity of at least 95% (spec.@95% sens.) of 95% (95% ci: 91%, 100%). due to the small percentage of around 3% of sars-cov-2 positive patients that were admitted to the icu (table i) , uncertainty bounds were wider than for the models predicting hospital admissions, and the results of the best encountered svm were found to be not significantly better than lr and rf in terms of auc, lr and nn in terms of sensitivity, and nn in terms of spec.@95% sens. at the pre-specified significance level of α = 0.05 (t-test). in terms of feature importance, we found that importance scores were distributed highly unequally, relatively uniform and highly uniform for the best models encountered for predicting sars-cov-2 test results, for predicting hospital admissions for sars-cov-2 positive patients, and for predicting icu admission, respectively (figure 4 ). most notably, we found that 71.7% of the importance for the best xgb model for predicting sars-cov-2 test results was assigned to the missing indicator corresponding to the arterial lactic acid measurement, i.e. much of the marginal predictive performance gain of the xgb model was attributed to whether or not the arterial lactic acid test had been ordered. beyond arterial lactic acid being missing, age, leukocyte count, platelet count, and creatinine were implied to be associated with a positive sars-cov-2 test result by the best encountered predictive model, which further substantiates recent independent reports of those factors being potentially associated with sars-cov-2 [59] , [60] , [61] , [62] , [48] . similarly to the best encountered xgb model for predicting sars-cov-2 test results, the top encountered predictive models for hospital admission and icu admission for sars-cov-2 positive patients assigned a considerable degree of importance to missingness patterns associated with a number of measurements. a possible explanation for missingness appearing as a top predictor across the different tasks is that decisions whether or not to order a certain test to be performed for a given patient were influenced by patient characteristics that were not captured in the set of clinical measurements that were available to the predictive models. a controlled setting with standardised testing guidelines would be required to determine which confounding factors are behind the predictive power of the missingness patterns that have been implied to be associated with covid-19 by the predictive models. beyond missingness patterns, top predictors for predicting hospital admission were lactate dehydrogenase [63] , gammaglutamyltransferase, which through abnormal liver function has been reported to be implicated in covid-19 severity [64] , and hco 3 [65] . for predicting icu admission in sars-cov-2 positive patients, pco 2 and ph [48] were top predictors. blood ph, and in particular respiratory alkalosis, has been reported to be associated with severe covid-19 [66] . we presented a systematic study of predictive models that predict sars-cov-2 test results, hospital admission for sars-cov-2 positive patients, and icu admission for sars-cov-2 positive patients using routinely collected clinical measurements. models that predict sars-cov-2 test results could help prioritise scarce testing capacity by identifying those individuals that are more likely to receive a positive result. similarly, predictive models that predict which sars-cov-2 positive patients would be most likely to require hospital and critical care beds could help better utilise existing hospital capacity by prioritising those patients that have the highest risk of deterioration. facilitating the efficient utilisation of scarce healthcare resources is particularly important in dealing with sars-cov-2 as its rapid transmission significantly increases demand for healthcare services worldwide. the main limitation of the presented study is that its experimental evaluation was based on data collected from a single study site, and its results may therefore not generalise to settings with significantly different patient populations, admission criteria, patterns of missingness, and testing guidelines. in addition, we did not have access to mortality data for the analysed cohort, and we were therefore not able to correlate our predicted individual risk scores with patient mortality, which is another related prediction task that may be of clinical importance. future studies should include a broader set of clinical measurements and outcomes, cohorts from multiple distinct geographical sites and under varying patterns of missingness in order to determine the robustness of the clinical predictive models to these confounding factors. finally, we believe that the inclusion of data from other modalities, such as genomic profiling and medical imaging, and data on co-morbidities, symptoms and treatment histories could potentially further improve predictive performance of clinical predictive models across the presented prediction tasks. we presented a systematic study in which we developed and evaluated clinical predictive models for covid-19 that estimate (i) the likelihood of a positive sars-cov-2 test in patients presenting at hospitals, (ii) the likelihood of hospital admission and (iii) intensive care unit admission in sars-cov-2 positive patients. we evaluated our developed clinical predictive models in a retrospective evaluation using a cohort of 5644 hospital patients seen in são paulo, brazil. in addition, we determined the clinical, demographic and blood analysis measurements that were most important for accurately predicting sars-cov-2 status, hospital admissions, and icu admissions. our experimental results indicate that clinical predictive models may in the future potentially be used to inform care and help prioritise scarce healthcare resources by assigning personalised risk scores for individual patients using routinely collected clinical, demographic and blood analysis data. furthermore, our findings on the importance of routine clinical measurements towards predicting clinical pathways for patients increase our understanding of the interrelations of individual risk profiles and outcomes in sars-cov-2. based on our study's results, we conclude that healthcare systems should explore the use of predictive models that assess individual covid-19 risk in order to improve healthcare resource prioritisation and 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factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical features of covid-19-related liver damage respiratory support in novel coronavirus disease (covid-19) patients, with a focus on resource-limited settings covid-19 in iran, a comprehensive investigation from exposure to treatment outcomes key: cord-268388-kkhuzf3p authors: sharif-yakan, ahmad; kanj, souha s. title: emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives date: 2014-12-04 journal: plos pathog doi: 10.1371/journal.ppat.1004457 sha: doc_id: 268388 cord_uid: kkhuzf3p nan two years have passed since the initial description of the middle east respiratory syndrome coronavirus (mers-cov), yet the epidemic is far from being controlled. the high case fatality rate, the recent steep increase in reported cases, and the potential to cause a global pandemic during the upcoming hajj season are serious concerns. although a wealth of information about the pathophysiology, proposed animal reservoir, and intermediate host has been revealed, many questions remain unanswered. we herein review mers-cov, covering its proposed origins, route of transmission, treatment options, and future perspectives. first reported in 2012 [1] , middle east respiratory syndrome coronavirus (mers-cov) is a novel coronavirus and the first lineage 2c betacoronavirus known to infect humans [2] . with a case fatality rate of 35%, an urgent response is needed to prevent a global pandemic [3] . prior to 2003, coronaviruses were not considered serious human pathogens since they only caused mild upper respiratory tract infections (urtis) [4] . the first zoonotic introduction of a coronavirus into the human population occurred with the severe acute respiratory syndrome coronavirus (sars-cov) in 2002. sars-cov caused a global pandemic, with 8,400 recorded cases and 800 deaths [5] . mers-cov marks the second known zoonotic introduction of a highly pathogenic coronavirus, probably originating from bats. three lines of evidence currently support this theory: (1) the very close phylogenetic similarity with the bat betacoronaviruses: btcov-hku4 and btcov-hku5 [6] ; (2) closely related coronavirus sequences have been recovered from bats in africa, asia, the americas, and eurasia; and (3) mers-cov uses the evolutionary conserved dipeptidyl peptidase-4 (dpp4) protein in pipistrellus pipistrellus bats for cell entry [7] . since human-bat contact is limited, camels have been implicated as probable intermediate hosts. mers-cov appears to have been circulating in dromedary camels for over 20 years [8] . mers-cov uses the dpp4 (cd26) receptor to gain entry and effectively replicate in camel cell lines [9] . neutralizing antibodies for mers-cov have been detected in dromedary camels from oman, canary islands, egypt, jordan, united arab emirates, and saudi arabia [10, 11] . the exact mode of transmission from camels to humans remains to be confirmed [12] . camel milk was investigated as a possible route of transmission, given the common practice of consuming camel milk in the arabian peninsula. the first reported case of mers-cov in yemen occurred in a yemeni pilot who consumed raw camel milk [13] , and reusken et al. reported the finding of mers-cov in camel milk in qatar [14] . however, respiratory transmission is currently considered as the most likely route of transmission [15] . mers-cov has been detected by reverse transcription pcr (rt-pcr) from the nasal swabs of three camels in qatar and was linked to two confirmed human cases with high similarity upon sequencing, suggesting a possible respiratory mode of transmission [16] . several clusters of mers-cov cases have been reported, mainly among household members and health care workers (hcws), suggesting that transmission is through close contact. the largest cluster reported so far has been in 23 hcws in a hospital in al hasa, kingdom of saudi arabia (ksa), while the largest family cluster has been in three infected brothers from riyadh, ksa [17, 18] . the basic reproductive rate for mers-cov has still not been determined with certainty [11] . using two transmission scenarios, breban et al. reported an r 0 of 0.60 and 0.69 [18] . cauchemez et al. reported a similar r 0 at 0.63, but warned that in the absence of infection control measures, r 0 may range from 0.8-1.3 and could lead to a self-sustaining transmission [19] . propensity for the mers-cov to replicate in the lower respiratory tract may account for the observed limited transmission [20] . the united states centers for disease control and prevention (cdc) recommends standard contact and airborne precautions with the use of an n-95 mask when caring for an infected patient [21] . the cdc defines a laboratory-confirmed case of mers-cov as a patient with a positive pcr from a respiratory sample, and a probable case as a patient who had close contact with a confirmed case but inconclusive laboratory evidence [22] . the incubation period for the presentation of mers-cov symptoms is 2-14 days and it remains unknown whether patients are infectious during the incubation period [23] . the average age of presentation is 50 years, with a male predominance [24] . clinically, mers-cov causes symptoms of upper and lower rtis [23] . the severity of symptoms varies widely. most asymptomatic cases have been discovered through screening after contact with a known case [2] . presenting signs and symptoms may include highgrade fever, non-productive cough, dyspnea, headache, myalgia, nausea, vomiting, and diarrhea that may precede the respiratory symptoms [25, 26] . renal failure has been frequently reported, yet no conclusive evidence of a direct viral invasion of renal tissues exists [11, 27, 28] . notably, most patients who developed complications had coexisting medical co-morbidities [11] . laboratory findings on admission may include leukopenia, lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase levels [25] . mers-cov can also cause severe pneumonia with acute respiratory distress syndrome (ards), requiring mechanical ventilation and intensive care admission [24] . to date, there is still a lack of surgical and pathological information from patients infected with mers-cov, which hampers full understanding of the pathogenesis. lastly, coinfection with other respiratory viruses and with community-acquired bacteria has been also reported in mers-cov patients [11, 29, 30] . as of june 26, 2014, who officially reported 707 affected patients in 21 countries in three continents. two-hundred and fifty-two patients have died of mers-cov, setting the case fatality rate at 35% [3] . the cases so far have been acquired either directly through a probable zoonotic source, or as a result of human-human transmission via close contact. retrospective analysis tracked the first outbreak to a hospital in the city of al-zarqa in jordan in april 2012 [31] . an unexplained observation has been the seasonal variation in reported numbers, with a peak between april and june of each year. the number of cases reported during april 2014 alone was alarming, because it was greater than the cumulative number of cases reported since the outbreak began [32] . the recent increase in the number of infected patients may arguably be attributed to better case detection and active surveillance programs. yet other factors may have contributed to the observed surge, including suboptimal infection control practices in affected hospitals in saudi arabia, as documented in a recent report of the who mission to jeddah [33] . another explanation for the seasonal variation may be that it coincides with camel birthing season, and younger camels seem to be more often infected than their older counterparts [34] . the distribution of the total reported cases by country is as follows: 85.8% in the ksa, 8.1% in the united arab emirates, 1.7% in jordan, and 1% in qatar [32] . cases have also been reported in kuwait, yemen, oman, iran, lebanon, tunisia, algeria, bangladesh, malaysia, france, italy, germany, the netherlands, united kingdom, greece, italy, and the united states [32] . furthermore, seropositive camels for mers-cov were detected in egypt, kenya, nigeria, tunisia, and ethiopia, suggesting that there may be mers-cov cases that are undetected in africa [8, 35, 36] . in 2012, 3,161,573 muslims from 188 countries gathered in mecca to perform the annual hajj, the largest gathering of muslims in the world. the identification of mers-cov in saudi arabia has generated international concern of a global pandemic. as a response, the saudi government requested that elderly and chronically ill muslims avoid hajj in 2013 and restricted the number of pilgrims to 2,061,573. consequently, no cases were reported during the pilgrimage of that year [37] . nasopharyngeal specimens collected from 5,235 pilgrims revealed no cases of mers-cov nasal carriage [38] . a prospective cohort study of 129 french pilgrims did not reveal any mers-cov cases [39] . nevertheless, the potential for spreading of mers-cov during the 2014 hajj season (october 2-6) remains possible, especially since documented transmission occurred this year in patients from iran and malaysia after their return from umrah in mecca. it is worth noting that the two most frequently visited cities during the hajj, mecca and medina, have so far reported 32 and 35 cases respectively [32] . mers-cov binds to the dpp4 (cd26) surface receptor using the spike (s) surface protein with subsequent cell entry [40] . the exact mechanism of entry after receptor binding is still unknown. the s surface protein is composed of a core subdomain that shares similarity with that of sars-cov and a receptor binding subdomain (rbsd) that exhibits significant variation from the sars-cov rbsd. the development of vaccines targeting the rbsd of mers-cov is currently under investigation because they are thought to be safer and more effective than vaccines based on inactivated virus, dna, or viral vectors [40] . another potential therapeutic approach is the inhibition of the papain-like and/or 3c-like protease of mers-cov [41] . to date, no effective therapy or prophylaxis for mers-cov exists. supportive therapy remains the cornerstone of management. current treatment is based on previous experience with the sars-cov, in-vitro studies, and case series. various agents have been tried, including those that block virus entry, inhibit viral replication, or interfere with host immune response [42] . the international severe acute respiratory and emerging infection consortium (isaric) suggested therapeutic options for treatment of mers-cov infection with various agents alongside continuous evaluation of efficacy, and in the setting of clinical trials [43] . based on experience with sars-cov, the use of convalescent plasma, hyper-immune globulin, or human monoclonal antibodies that contain neutralizing antibodies may be efficacious and is recommended as first-line treatment when available [43] . ribavirin and interferon alpha-2b both showed promising results, especially when used in combination, both in vitro and in animal studies using rhesus macaques monkeys [44] . however, these positive results did not translate clinically in an observational study in five patients, all of whom succumbed to the infection [45, 46] . repurposing of currently available agents may be an efficient approach. dyall et al. screened various agents with potential therapeutic efficacy [46] . cyclosporin a, mycophenolic acid, interferon-beta, homoharringtonine, cycloheximide, anisomycin, and emetine dihydrochloride hydrate were found to have the most potent in vitro activity against mers-cov. despite the progress in our understanding of mers-cov, many questions remain unanswered. the definitive origin, exact mechanism of transmission, and the reason behind seasonal variability are still unclear. although most cases have been described in countries of the arabian peninsula, the increasing travel to the region and the hajj season in ksa pose a threat of a potential global pandemic. extensive efforts are required to speed up the development of an effective therapy and vaccine. repurposing of currently available pharmaceutical agents is highly desirable for a more rapid drug development. meanwhile, hcws who encounter patients with respiratory symptoms who have lived or traveled to areas with mers-cov should have a low threshold to consider a diagnosis of mers-cov, with testing and immediate implementation of proper infection control practices to prevent further spread. finally, given the important role that camels may play in transmission of the virus, the common practices in the arabian peninsula of herding and consuming unpasteurized camels' milk should be discouraged until conclusive evidence is obtained that such practices do not contribute to infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers: emergence of a novel human coronavirus middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of 26 identification of a novel coronavirus in patients with severe acute respiratory syndrome consensus document on the epidemiology of severe acute respiratory syndrome (sars) genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc antibodies against mers coronavirus in dromedary camels replicative capacity of mers coronavirus in livestock cell lines middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study the emergence of the middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? global alert and response: middle east respiratory syndrome coronavirus (mers-cov) -update middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels, qatar middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation hospital outbreak of middle east respiratory syndrome coronavirus interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques interim infection control and prevention recommendations for hospitalized patients with mers-cov mers case definition mers clinical update from the idsa center for disease control and prevention cdc epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission family cluster of middle east respiratory syndrome coronavirus infections a patient with severe respiratory failure caused by novel human coronavirus a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection epidemiological findings from a retrospective investigation who concludes mers-cov mission in saudi arabia mers-cov enigma deepens as reported cases surge geographic distribution of mers coronavirus among dromedary camels mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of 20 prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj lack of mers coronavirus but prevalence of influenza virus in french pilgrims after 2013 hajj the receptor binding domain of mers-cov: the dawn of vaccine and treatment development proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease what are our pharmacotherapeutic options for mers-cov? international severe acute respiratory & emerging infection consortium (isaric)-clinical decision making tool for treatment of mers-cov v.1.1 an animal model of mers produced by infection of rhesus macaques with mers coronavirus ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study repurposing of clinically developed drugs for treatment of middle east respiratory coronavirus infection the authors would like to acknowledge sima l. sharara for editing the manuscript. key: cord-278362-pwi48i20 authors: khan, abbas; ali, syed shujait; khan, muhammad tahir; saleem, shoaib; ali, arif; suleman, muhammad; babar, zainib; shafiq, athar; khan, mazhar; wei, dong-qing title: combined drug repurposing and virtual screening strategies with molecular dynamics simulation identified potent inhibitors for sars-cov-2 main protease (3clpro) date: 2020-06-18 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1779128 sha: doc_id: 278362 cord_uid: pwi48i20 the current coronavirus (sars-cov-2) pandemic and phenomenal spread to every nook and cranny of the world has raised major apprehensions about the modern public health care system. so far as a result of this epidemic, 4,434,653 confirmed cases and 302,169 deaths are reported. the growing infection rate and death toll demand the use of all possible approaches to design novel drugs and vaccines to curb this disease. in this study, we combined drugs repurposing and virtual drug screening strategies to target 3clpro, which has an essential role in viral maturation and replication. a total of 31 fda approved anti-hiv drugs, and traditional chinese medicines (tcm) database were screened to find potential inhibitors. as a result, saquinavir, and five drugs (tcm5280805, tcm5280445, tcm5280343, tcm5280863, and tcm5458190) from the tcm database were found as promising hits. furthermore, results from molecular dynamics simulation and total binding free energy revealed that saquinavir and tcm5280805 target the catalytic dyad (his41 and cys145) and possess stable dynamics behavior. thus, we suggest that these compounds should be tested experimentally against the sars-cov-2 as saquinavir has been reported to inhibit hiv protease experimentally. considering the intensity of coronavirus dissemination, the present research is in line with the idea of discovering the latest inhibitors against the coronavirus essential pathways to accelerate the drug development cycle. communicated by ramaswamy h. sarma. the coronaviruses (covs) are placed within four genera, alphacoronavirus, beta-coronavirus, gamma-coronavirus and deltacoronavirus (a, b, c, and d) of subfamily orthocoronavirinae. however, the members of beta-coronavirus such as severe acute respiratory syndrome coronavirus (sars-cov1 and sars-cov2) and the middle east respiratory syndrome coronavirus (mers-cov) have been involved in pneumonia epidemics in the 21st century. sars-cov1, mers-cov and sars-cov2 outbreaks caused by zoonotic viruses (lau et al., 2005; reusken et al., 2013); with the case fatality ratio of 10% (cheng et al., 2007; lee et al., 2003; zaki et al., 2012) , 35% (de groot et al., 2013; zaki et al., 2012) ; and 5%, respectively. the recent outbreak and phenomenal spread of sars-cov-2 to every nook and cranny of the world (3.66 million infections and 309774 deaths) compelled world health organization (who) to declare it pandemic on march 20, 2020. the virus causing covid-19 was named 2019-ncov (2019 novel coronavirus) on january 12, 2020 by who zhu et al., 2020) , however the international virus classification commission (ictv) on february 11, 2020, named this virus as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this threat of global concern to humanity, mainly due to the unavailability of proper treatment pushed the investigators to discover and design therapeutic drugs and vaccines to combat the coronavirus. infection symptoms include fever, dyspnea, shortness of breath, and cough, whereas severe cases lead to kidney failure and death (rothan & byrareddy, 2020) . the sars-cov-2 is composed of spike (s) protein, membrane (m) protein, envelope (e) protein, and nucleocapsid (n) protein. in anti covs therapies, the main focus is to boost the human immune system or to block the binding of spike protein with the receptor proteins. therefore, the treatments based on targeting coronavirus rely on blocking of virus binding to receptors, inhibition of virus replication, prevention of the synthesis of viral rna and inhibition of the virus self-assembly process (bosch et al., 2003; khan et al., 2018; omrani et al., 2014) . to combat the latest covs threats, the researchers are following multiple approaches for novel drug development (zumla et al., 2016) and testing the efficacy of existing drugs. in one approach, experiments were performed to test the existing broad-spectrum antiviral drugs (ribavirin, interferons, and cyclophilin inhibitors) against sars-cov-2 (chan et al., 2013; zumla et al., 2016) . the use of these antiviral drugs is approved against viral infections, and their dosages, efficacy, metabolic characteristic, and side effects are well known. however, there is a possibility that this "broad-spectrum" treatment against covs would not be effective. in the second approach, molecular databases were subjected to highthroughput screening for potential drug molecules to combat infection caused by a coronavirus (de wilde et al., 2014; dyall et al., 2014) . this strategy was proved successful in the discovery of lopinavir/ritonavir for the treatment of hiv. some researchers are using the genomic and pathological information of covs for the development of a new specific drug from scratch. theoretically, this is an effective approach; however, it is very time consuming and might cost several years or even more than ten years (khan et al., 2018; omrani et al., 2014) . structural bioinformatics based approaches are the fastest way for finding the potential molecules from the marketed drugs or bioactive compounds for effective treatment of sars-cov-2. main-protease or 3clpro inhibition is a promising target to control the recent sars-cov-2 infection due to its essential role in viral maturation and replication (ul qamar et al., 2020) . 3clpro has three important domains i-iii, which correspond to positions 8-101, 102-184, and 201-303, respectively. there is a connecting loop that corresponds to position 185-200, which connects domains ii and iii. the structure of 3clpro has an important catalytic dyad consist of his41 and cys145. in this study, the protein of sars-cov-2 (3clpro, also named 3-chymotrypsin-like protease) was subjected to drug repurposing and virtual screening for potent drug identification followed by molecular dynamics simulation and binding free energy calculation. our findings revealed that saquinavir, which is an hiv protease inhibitor reported experimentally (kim et al., 1998) and tcm5280805, are promising hits, which need in vitro validation for antiviral effects. we hope this study will provide useful information for the clinical treatment of novel coronavirus associated pneumonia. (b) the electrostatic potential of 3clpro and displays the important active site residues in the sticks, including the two essential residues his41 and cys145 (catalytic dyad). protein databank (http://www.rcsb.org/) (rose et al., 2016) was used for retrieval of 3clpro (6lu7) crystal structure. using the protein preparation implemented in schr€ odinger software (schr€ odinger, llc, new york, ny), the structure was prepared and optimized. the opls_2005 force field was used for protein-energy minimization. for ligands preparation such as assigning appropriate ionization, stereochemistry, ring conformations, and tautomer (release, 2017; schrodinger, 2011) , a ligprep module was used. apbs tool (lerner & carlson, 2006) implemented in pymol was used for electrostatic potential calculation. drug repositioning or repurposing approach is used to speed up the drug development cycle by finding a new therapeutic application for a marketed drug that has been licensed for a particular use (sleigh & barton, 2010) . this approach was fruitful in the case of sildenafil for leprosy, erectile dysfunction, and pulmonary hypertension, and multiple myeloma thalidomide (hernandez et al., 2017) . literature mining was carried out to collect anti-hiv drugs for screening against 3clpro (sars-cov-2). multiple drugs were retrieved from drugbank database. a total of 31 drugs were shortlisted for screening against the 3clpro (sars-cov-2). schr€ odinger binding site was used for finding the binding site of proteins using the default parameters, and the generated maps show the binding cavity. the identified binding sites have the descriptions regarding hydrogen bonding, a degree of exposure and enclosure, size, linking site points, tightness, hydrophobic and hydrophilic nature. the grid with dimensions 12 å â 12 å â 12 å was generated. the final active site grid identified was based on the experimentally reported residues by a recent crystallographic study and the maps generated by schr€ odinger maestro. three steps of virtual screening (htvs, sp, and xp) were used to screen the anti-hiv and tcm compounds databases. furthermore, the bioactivity of these compounds was predicted by using molinspiration cheminformatics tool. molinspiration is an efficient tool that has been used by several studies ($4500) to predict bioactivity results. top hits from anti-hiv drugs and tcm database were subjected to molecular dynamics simulation using the amber18 package (case et al., 2005) . the antechamber was used to generate the drugs topologies.tip3p water model was to solvate the system, and na þ counter ions were used to neutralizing the system. two steps energy minimization of the system followed by heating and equilibration was performed. particle mesh ewald (pme) algorithm was applied to calculate the long-range electrostatic interactions (price & brooks iii, 2004) . for van der waals interactions, a 1.4 nm cutoff values were set and also for short-range columbic, respectively. a total of 100 ns md simulation was performed with a time step of 2 fs. the behavior of the ligand-protein complex and stability were analyzed. post-simulation analysis such as rmsd, rmsf, rog and hydrogen bonds occupancy were performed using cpptraj and ptraj (roe & cheatham iii, 2013) . the script mmpbsa.py was used to calculate the free binding energy for all the protein-ligand complexes (chen et al., 2016; hou et al., 2012; miller iii et al., 2012; sun et al., 2014) ; considering 500 snapshots from md trajectories using the following equation: in this equation, dg bind represents total free binding energy, while others show the free energy of complex, the protein, and the ligand. specific energy term contributes to the whole free energy was calculated by the equation: g bond , g ele and g vdw specify interactions among bonded, electrostatic, and van der waals states. in contrast, g pol and g npol represent the polar and non-polar interaction to the table 1 . a list of anti-hiv drugs used for docking against the proteinase enzyme of coronavirus (sars-cov-2). targeted virus(es) free energy presumed through precise gb (generalized born). this free energy calculation method is widely used by different studies to understand the binding energy of different ligands wang et al., 2019) . 3clpro structure retrieval and preparation the crystallographic structure (306 amino acids long) of the 3clpro (sars-cov-2) was retrieved from the rcsb database using accession id: 6lu7. the structure of 3clpro was analyzed for missing residues and refinement. the structure was subjected to energy minimization and structure preparation using maestro. the structure of 3clpro has three domains, as given in figure 1 (a). these domains i-iii correspond to position 8-101, 102-184, and 201-303, respectively. while there is a connecting loop corresponds to position 185-200, which connects domains ii and iii. all these domains are coloured differently. the ligand in the active site (yellow colour (sphere)) has also been shown in figure 1 (a). the electrostatic potential of the 3clpro shows that the active site region possesses a strong electronegative potential, while weak electronegative potential around the active site can also be observed. however, the weak electropositive and neutral potential is distributed too. the active site grid (x¼ à10.71, y ¼ 12.41, z ¼ 68.83) was generated, and important residues were identified. important active site residues his41, met49, tyr54, phe140, leu141, asn142, cys145, his163, met165, glu166, leu167, phe185, asn187, and gln192 were identified and shown in figure 1 (b). the binding cleft is at the same place as in the previously reported sars with the two critical residues his41 and cys145 forms a catalytic dyad. in the current study, the repurposing of anti-hiv drugs against the sars-cov-2 main protease was carried out using structure-based screening methods. anti-hiv drugs such as lopinavir-ritonavir are reported to be active against the sars-cov-2. clinical trials of these drugs on different groups of patients showed significant improvement. thus, anti-hiv drugs possess the potential to work against srs-cov-2 targets. therefore, immediate testing of anti-hiv drugs may lead to a discovery of potent inhibitor against the sars-cov-2. a list of 31 anti-hiv drugs ( these drugs were prepared for molecular docking against the proteinase enzymes of coronavirus (sars-cov-2). the docking scores were ranged from à9.09 kcal/mol to à4.16 kcal/mol. all the conformations were analyzed for the best interactions with the key residues. initial criteria were set to shortlist the drugs based on binding affinity and interactions with the dyad residues (his41 and cys145). the docking score à9.09 kcal/mol was reported for the hiv protease inhibitor drug saquinavir (figure 2 ). this drug has been reported to inhibit the hiv proteinase enzymes too. in this case, (sars-cov-2), the saquinavir formed six hydrogen bonds, including two with the central dyad residues (his41 and cya145). previous reports on different sars, such as the octa-peptide, also stressed the blocking of these two residues. other interactions include four hydrogen bonds with glu166, gln189, met49, and gly143, while some pi-alkyl interactions, including one with cys145 and other residues, are also formed. thus, we speculate that the clinical testing of figure 4 . rmsds of all the systems including 3clpro-squanavir (red), 3clpro-5280805 (green), 3clpro-5280445 (blue), 3clpro-5280343 (cyan), 3clpro-5280863 (magenta) and 3clpro-5458190 (navy). the x-axis shows the time in nanoseconds while the y-axis shows the rmsd in å. saquinavir should be done at the earliest. it is also reported by different scientists that hiv drugs could be potentially used against the recent coronavirus. virtual screening of tcm database against the 3clpro (sars-cov-2) furthermore, we also performed virtual screening of the traditional chinese medicines database (tcm). a total of 37,800 compounds were screened after excluding the drugs violating the lipinski rules of five. important residues in the active site (his41 and cys145) were targeted. the top five compounds based on the docking scores were selected. the best docking score, à11.183 kcal/mol, was reported for the compound (5280805). more than ten bonds, including hydrogen bonds, van der waals, electrostatics, and pi-alkyl interactions, were formed. among the seven hydrogen bonds, two hydrogen bonds with the residue his41 were also formed, which is a key residue in the active site dyad. among the other phe140, leu141, asn142, his164, glu166, and thr190 also formed hydrogen bonds with the ligand (5280805). on the other hand, compound 5280863, with the docking score à7.688 kcal/mol formed a strong hydrogen bond with the key residue his41. the compound 5280863 also formed five hydrogen bonds with the other active site residues (glu166, arg188, gln189, and thr190). the other three compounds (5280445, 5280343, and 5458190) were also analyzed for the interaction with the key residues. it can be seen from figure 3 that his41 is mainly involved in the interaction with the ligands. table 2 is showing the 2d structures of the top five compounds with their docking scores. cross validation of our predicted compounds by comparing with the experimentally tested compounds showed that the saquinavir and tcm5280805 possess a similar scaffold with myricetrin and scutellarin which are flavonoids and reported to have strong inhibitory effects by targeting 3clpro. on the other hand, the four compounds (tcm5280343, tcm5280445, tcm5280863 and tcm5458190) from tcm database shares structural similarity with scutellarein, dihydromyricetin, quercetagetin, myricetin, 5,6-dihydroxyflavone, 6,7-dihydroxyflavone, chrysin, herbacetin and baicalein which are flavonoids and testes to have strong inhibitory effects in an experimental condition by targeting 3clpro (chen & du, 2020; dai et al., 2020; yang et al., 2020) . their structural analyses showed that these compounds mainly form bonds with phe140 and glu166 while our compounds specifically target the catalytic dyad with supplementary interactions with the other active site residues. thus, we speculate that our compounds could also show the same potential activity against the 3clpro in the experimental assay because our compounds are also plants derived extracts and belongs to a group of flavonoids. furthermore, results obtained from the server shows that all these shortlisted compounds are active against the protease target. from the scores it can be seen that saquinavir with score 0.40 possesses strong inhibitory effects against the proteases while the others the reported score for tcm5280805 (à0.7), tcm5280863 (à0.27), tcm5280445 (à0.22), tcm5280343 (à0.25) while tcm5458190 possess (à0.12) bioactivity score against the protease target. thus, these results strongly suggest that the shortlisted compounds could efficiently inhibit the 3clpro in the experimental setup and could be tested for clinical trials. figure 5 . rmsfs of all the systems including 3clpro-squanavir (red), 3clpro-5280805 (green), 3clpro-5280445 (blue), 3clpro-5280343 (cyan), 3clpro-5280863 (magenta) and 3clpro-5458190 (navy). the x-axis shows the time in nanoseconds while the y-axis shows the rmsf in å. the right panel shows the secondary structure of 3clpro. to understand the conformational and dynamics features of the selected hits against the 3clpro of sars-cov-2molecular dynamics (md) simulation is an imperative method to explore the behavior of each system in real-time. dynamics features of the six selected systems (saquinavir, 528085, 5280863, 5280445, 5280343 , and 5458190 were calculated after 100 ns simulation. for stability rmsd, while to evaluate the flexibility at residue level rmsf of the complexes were calculated to comprehend the overall stability and flexibility of the system. the stability of all the six systems was calculated by using root mean square deviation (rmsd). we tested the stability of the docked drugs in the active pocket and its effects on the stability of the whole system. the results suggest that all the six systems were stable with acceptable deviation (as shown in figure 4 ). the average rmsd for all the systems was found to be between 1 and 4å. saquinavir-3clpro exhibited an average rmsd of 2.5 å soon after reaching 35 ns; the system attained stability remains uniform for the rest of the time. it can be seen that the complex 3clpro-5280805 remained stable, but after 60 ns, the system showed an acceptable deviation for a little time. after that, the system gained stability and entered the production stage. for a complex 3clpro-5280445, the system showed a slight deviation between 40 and 45ns but remained stable during the course of the simulation. on the other hand, 3clpro-5280343 and 3clpro-5280863 reported divergence between 40 and 60ns, but the system soon gained stability. similarly, in the case of 3clpro-5458190, the examination of rmsd of the ca backbone has an average rmsd of 2.3 å. the system shows a deviation in figure 6 . rogs (radius of gyration) of all the systems including 3clpro-squanavir (red), 3clpro-5280805 (green), 3clpro-5280445 (blue), 3clpro-5280343 (cyan), 3clpro-5280863 (magenta) and 3clpro-5458190 (navy). the rmsd for the first 15 ns while remained uniformed for the rest of the simulation time. thus, these results revealed the stable internal motions and negligible fluctuations during the course of the simulation. the differences in the rmsd of each system is due to the binding and unbinding of the ligand at different time intervals. also, the systems with more stable behaviour show that the ligand remained intact, and the system entered the production phase soon when compared to the others. aside, the binding of small molecules affects the system differently as the ligand-binding orientation changes over the simulation time. the rmsf depicts the flexibility at residues level. as shown in figure 5 , the pattern of flexibility is almost similar and was recorded around 1-4å in all secondary components except loop regions with slightly fluctuated residues. however, active site residues seem stable in the course of the simulation, which is due to the ligand recognition by the ligandbinding regions. it can be seen that 3clpro-squanavir and 3clpro-5458190 showed an average rmsf of 2.5 å with significant fluctuations in the regions 40-45, 90-110, 150-165, and 245-260 . a similar pattern of fluctuation was also observed for systems including 3clpro-5280805, 3clpro-5280445, and 3clpro-5280863. however, the average rmsf remained below the 2 å. only the system 3clpro-5280343 wed higher fluctuation than the rest, but the residual flexibility pattern was similar. the average rmsf for all the five systems was around 1-4å, as clearly seen in figure 5 (left panel). these findings here show that the target protein is stabilized by binding of all five chosen drugs docked against it. thus the binding of ligand has significantly affected the residue fluctuation, which is due to the internal residues disturbed by the binding of different ligands, and this both correlated and non-correlated motions are affected. the secondary structure components are also given in figure 5 (right panel). the radius of gyration (rog) conveys the information of stable and unstable folding protein while interacting with ligands. less compactness (more unfolded) shows higher rog values, whereas low rog values indicate strong compactness and higher structural stiffness (more folded). thus, rog was determined to assess the system's compactness over time. as shown in figure 6 , the simulated complexes show gyration scores between 21 and 24å. in the case of 3clpro-squanavir, the gyration score for the initial (2000) frames was reported to be 22.50 å. however, a little fluctuation between (2000-3000) frames was observed, but soon after 3500, the values become lower and uniform. in the case of 3clpro-5280805, 3clpro-5280445, 3clpro-5280863, and 3clpro-5458190 showed an average gyration of 22-22.50 å. similarly, 3clpro-5280445 also showed similar fluctuation, but in the last few frames, the values fluctuated a little higher, which is assumed to be in an acceptable range. the binding and unbinding of ligands greatly affected the overall compactness of the complexes. as given in figure 7 , in 3clpro-squanavir, 3clpro-5280805, and 3clpro-5280343 complexes, the hydrogen bond with his41 was reported in 87%, 65% and 39% of the trajectories. hydrogen bond with cys145 residue was reported in 65% trajectories only in the 3clpro-squanavircomplex. furthermore, leu141 and glu166 were found in almost all complexes. however, another important residue gln189 was found in 76% of the 3clpro-squanavir trajectories, 36% in 3clpro-5280805, while 21% in 3clpro-tcm5280445 trajectories. other active site residues were not detected in a significant population of the md trajectories. binding free energy mm/gbsa, a popular approach, was used to estimate the binding free energy of all the six systems. the binding free energy determines the binding affinity between ligands and 3clpro. each energy term, including van der waals energy, electrostatic energy, polar solvation energy, solvent accessible surface area energy, and total binding free energy of all the systems are given in table 3 . it can be seen that the 3clpro-saquinavir system possesses the highest total binding energy of à74.4061 kcal/mol. thus it confirms that saquinavir, an anti-hiv drug that has been reported to inhibit hiv protease experimentally, possesses strong inhibitory effects against the 3clpro of sars-cov-2. recently published results also shortlisted saquinavir as a potential candidate, but they only performed 20 ns simulation (khan et al., 2020) , which could not reflect better results than what we revealed by adding 5-folds more simulation time. furthermore, the top five hits from tcm virtual screening results were also subjected to the free binding energy as reported by the virtual screening results tcm5280805 was reported with the best docking score and interaction. herein, tcm5280805 also possesses the best total binding energy,29.486 kcal/mol, against the 3clpro. also, the other hits from including tcm5280445, tcm5280343, tcm5280863, and tcm5458190 possess total binding energy of à16.342 kcal/ mol, à17.184 kcal/mol, à10.315 kcal/mol and à15.695 kcal/ mol. while the other energy terms such as van der waals energy, electrostatic energy, polar solvation energy, solventaccessible surface area are given in table 3 , thus these results strongly suggest that saquinavir and tcm5280805 should be tested experimentally against the sars-cov-2 at earliest. to date, a total of 4,434,653 confirmed cases and 302,169 deaths are reported from the sars-cov-2 epidemic, which indicates that the current viral treatment is not satisfactory, and more diverse studies are needed for the incorporation of various natural and fda approved compounds for the effective treatment of sars-cov-2. further, our existing capacity for treating zoonotic coronavirus infections is still in the trial, and the available resources seem very limited to fight this life-threatening hazard. although extensive research efforts have been initiated during 2003 and 2012 outbreaks of sars and mers-cov, respectively, however, no effective drugs so far have been synthesized, nor the matter may be taken seriously to prevent the future pandemics of zoonotic coronavirus. drug repositioning is a 'universal strategy' for neglected diseases due to reduced number of clinical trial steps required could reduce the time and cost of the medicine to reach the market, existing pharmaceutical supply chains could facilitate the 'formulation and distribution' of the drug, the known possibility of combining with other drugs could enable more efficient treatment, repositioning could encourage the development of 'new modes of action for old drugs and new types of medicines, eliminating 'activation obstacles' from early stages of study could allow the project to progress rapidly towards disease-oriented development (pushpakom et al., 2019) . one of the primary reasons why no prototype coronavirus inhibitor has progressed to the (early) preclinical stage so far is due to the transient nature of this epidemic. like the sars virus 17 years ago, the new sars-cov-2, and the new evolving coronaviruses that may continue to pose a threat to global public health in the future. hence, discovering broad-spectrum inhibitors that can reduce the symptoms of human coronavirus infection remains a formidable subject of study. considering the time-consuming process of designing and documenting antiviral medications, proven therapies for certain diseases may be the only fastest therapeutic choice for emerging infectious diseases. the prescription has ample experience and dosage for most of those medications that have been formulated, and their health and adme properties are well known. in this study, based on the results of bioinformatics analysis, we targeted 3clpro from sars-cov-2 using drugs repurposing (anti-hiv drugs) virtual drugs screening (tcm) approaches to shortlist the most potent compounds for the possible treatment. the results of the entire article stressed the potential inhibitory role of saquinavir and tcm5280805, which are based on computer-based virtual drug screening. we have not conducted further in vivo and in vitro antiviral experiments yet, because we want to share our results with scientists in anti-sars-cov-2 research as soon as possible. this study will help to repurpose drug design, perform in vivo and in vitro evaluations for candidate drugs obtained in this study, and prepare for clinical trial applications. 3c-like protease (3clpro), also called the main protease, is an attractive target for the treatment of sars-cov-2 because of its role in the viral replication cycle. the current study focused on structure based approaches repurposed anti-hiv drugs and screened traditional chinese medicines databases against the active site residues of 3clpro. initial analysis such as molecular docking scores and interactions with important residues shortlisted one drug (saquinavir) from anti-hiv drugs while five compounds from tcm database.these compounds were then subjected to molecular dynamics simulation and post-simulation analysis. among the six complexes subjected to molecular dynamics simulation saquinavir, tcm5280805 showed the highest hydrogen bond occupancy. the rest were also found to have good activity against the 3clpro. in addition, the bioactivity of these compounds were predicted which reported saquinavir as strong potential inhibitor followed by the others.binding free energy calculations of all the complexes suggested that these compounds significantly interacting and binds with the receptor. thus, here from all the analyses, we suggest that saquinavir and tcm5280805 should be tested experimentally for possible treatment of sars-cov-2. ak, zb, mk, and ssa conceptualized the study and did the analysis. aa, ms, ss wrote the manuscript. ak, ss and ssa revised the manuscript and improved the write-up. dqw is an academic supervisor. he supervised the study. the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the amber biomolecular simulation programs broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus assessing the performance of the mm/pbsa and mm/gbsa methods. 6. capability to predict protein-protein binding free energies and rerank binding poses generated by protein-protein docking potential natural compounds for preventing sars-cov-2 (2019-ncov) infection. preprints emerging coronaviruses: genome structure, replication, and pathogenesis severe acute respiratory syndrome coronavirus 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structure of mpro from covid-19 virus and discovery of its inhibitors. biorxiv deeplearning-based target screening and similarity search for the predicted inhibitors of the pathways in parkinson's disease allosteric ligands for the pharmacologically important flavivirus target (ns5) from zinc database based on pharmacophoric points, free energy calculations and dynamics correlation identification of chymotrypsin-like protease inhibitors of sars-cov-2 via integrated computational approach saquinavir, an hiv protease inhibitor, is transported by p-glycoprotein severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats a major outbreak of severe acute respiratory syndrome in hong kong apbs plugin for pymol mmpbsa.py: an efficient program for end-state free energy calculations ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study a modified tip3p water potential for simulation with ewald summation drug repurposing: progress, challenges and recommendations maestro. schr€ odinger, llc middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. the lancet infectious diseases ptraj and cpptraj: software for processing and analysis of molecular dynamics trajectory data the rcsb protein data bank: integrative view of protein, gene and 3d structural information the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak schrodinger software suite (p. 670). schr€ odinger assessing the performance of mm/pbsa and mm/gbsa methods. 4. accuracies of mm/pbsa and mm/gbsa methodologies evaluated by various simulation protocols using pdbbind data set structural basis of sars-cov-2 3clpro and anti-covid-19 drug discovery from medicinal plants the systematic modeling studies and free energy calculations of the phenazine compounds as anti-tuberculosis agents drugbank 5.0: a major update to the drugbank database traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus from patients with pneumonia in china coronaviruses -drug discovery and therapeutic options the authors declare no conflict of interest. key: cord-034354-4xu97je3 authors: wang, hongye; wu, xian; zhang, xiaomei; hou, xin; liang, te; wang, dan; teng, fei; dai, jiayu; duan, hu; guo, shubin; li, yongzhe; yu, xiaobo title: sars-cov-2 proteome microarray for mapping covid-19 antibody interactions at amino acid resolution date: 2020-10-21 journal: acs cent sci doi: 10.1021/acscentsci.0c00742 sha: doc_id: 34354 cord_uid: 4xu97je3 [image: see text] comprehensive profiling of humoral antibody response to severe acute respiratory syndrome (sars) coronavirus-2 (cov-2) proteins is essential in understanding the host immunity and in developing diagnostic tests and vaccines. to address this concern, we developed a sars-cov-2 proteome peptide microarray to analyze antibody interactions at the amino acid resolution. with the array, we demonstrate the feasibility of employing sars-cov-1 antibodies to detect the sars-cov-2 nucleocapsid phosphoprotein. the first landscape of b-cell epitopes for sars-cov-2 igm and igg antibodies in the serum of 10 coronavirus disease of 2019 (covid-19) patients with early infection is also constructed. with array data and structural analysis, a peptide epitope for neutralizing antibodies within the sars-cov-2 spike receptor-binding domain’s interaction interface with the angiotensin-converting enzyme 2 receptor was predicted. all the results demonstrate the utility of our microarray as a platform to determine the changes of antibody responses in covid-19 patients and animal models as well as to identify potential targets for diagnosis and treatment. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has since proven to be highly contagious, with the median incubation period of 4 d. 1−3 infection of sars-cov-2, called covid-19, results in a range of symptoms, ranging from a mild cough to pneumonia. it is estimated that 17.9% of patients might be asymptomatic, 4 which may lead to two or even three transmissions per infected individual. 3, 5, 6 particular subsets of the population are extremely vulnerable to covid-19, including the elderly, those with underlying conditions, and immunocompromised individuals. on the evening of january 30, 2020, the world health organization listed the novel coronavirus outbreak as a public health emergency of international concern. 7 the novel coronavirus had spread worldwide by august 11, 2020, 8 with 20 014 574 confirmed cases and 734 755 deaths in 188 countries. 9 the high transmission rates of sars-cov-2, limited diagnostic tests, and no antiviral treatment options pose huge challenges for the control and treatment of sars-cov-2-infected patients. 10, 11 sars-cov-2 is 82% similar to the original sars virus attributed to the outbreak in 2003. 12 generally, a sars-cov-2 virus has a polyprotein (the open reading frame 1a and 1b, orf1ab), four structural proteins (envelope, e; membrane, m; nucleocapsid phosphoprotein, n; spike, s), and five accessary proteins (orf3a, orf6, orf7a, orf8, orf10). 13 the largest polyprotein encoded by orf1ab can be proteolytically cleaved into 16 putative nonstructural proteins (nsps), which might be involved in viral rna replication and transcription. 12 the e and m proteins are important in the viral assembly of a coronavirus. the n protein forms complexes with genomic rna and is important to enhance the efficiency of viral transcription and assembly. 14 the s protein is on the surface of the viral particle, enabling the infection of host cells by binding to the host cell receptor, angiotensin-converting enzyme 2 (ace2), via the s-protein's receptor binding domain (rbd) within the s-protein's subunit 1. 15, 16 the accessory proteins may have functions in signaling inhibition, apoptosis induction, and cell cycle arrest. 13 the identification of b-cell and t-cell epitopes for sars-cov-2 proteins is essential in developing effective diagnostic tests and vaccines, especially for structural n and s proteins. these epitopes have thus far been predicted either by bioinformatics or measured using t-cell based assays. 17−20 however, proteome-wide analysis of the humoral antibody response to sars-cov-2 proteins using an immunoproteomics platform has not been performed to date. here, we use a peptide-based sars-cov-2 peptide microarray to analyze antibody interactions in high throughput at the amino acid resolution. to produce the sars-cov-2 proteome microarray ( figure 1a ), we first extracted the reference sequences of 10 proteins encoded by the sars-cov-2 coronavirus genome from the national center for biotechnology information (ncbi) database (accession no. mn908947.3). using these reference sequences, we prepared a peptide library containing 966 peptides representing sars-cov-2 proteins, in which each peptide was 15 amino acids long with a 5 amino acid overlap. all peptides were labeled with a c-terminal biotin group and printed onto a three-dimensional (3d) modified microscope slide using biotin−streptavidin chemistry, 21 such that the dynamic range of serum antibody detection using sars-cov-2 proteome microarray. the lod was calculated using the signal of the buffer control plus two standard deviations. (c) reproducibility of serum antibody detection using the sars-cov-2 proteome microarray. (d) epitope binding of the anti-sars-cov-1 n protein antibody using the sars-cov-2 proteome microarray. the specific antibody binding to the target epitope is selected with a z-score higher than 3 as a threshold. the false-colored rainbow color from blue to red corresponds to the z-score from low to high, respectively. figure 2 . landscape of humoral igm antibody response to sars-cov-2 orf1ab proteome. the x-axis represents the sequence of amino acids of sars-cov-2 nonstructural proteins (nsps) from the n-terminal to c-terminal. the y-axis represents the serum samples from covid-19 patients. the false-colored rainbow color from blue to red corresponds to the signals of antibody binding from low to high, respectively. peptides were immobilized on the slide via their c-terminus. full-length sars-cov-2 n protein, full-length e, and five s truncated proteins were also printed (supporting information table 1 ). using serum spiked with anti-sars antibodies, we next determined the optimal lengths of time to block the array, incubate with serum samples, and incubate with the detection antibody. optimal signal-to-noise ratios were obtained with blocking for 1 min, serum incubation for 30 min, and detection antibody incubation for 30 min (supporting information, figures 1−3) . serum screening using the sars-cov-2 proteome microarray can be performed in 1.5 h while keeping a good dynamic range (∼2 orders of magnitude) and lowest limit of detection (lod) (94 pg/ml) (figure 1b ). this represents a significant decrease in time compared to the standard ∼18 h using protein microarrays. 22 the intra-and figure 3 . landscape of the humoral igg antibody response to the sars-cov-2 orf1ab proteome. the x-axis represents the sequence of amino acids of the sars-cov-2 nonstructural proteins (nsps) from the n-terminal to c-terminal. the y-axis represents the serum samples from the covid-19 patients. the false-colored rainbow color from blue to red corresponds to the signals of antibody binding from low to high, respectively. inter-array r correlations were 0.9992 and 0.9978, respectively, demonstrating that the sars-cov-2 proteome microarray has a high reproducibility ( figure 1c ). epitope mapping of sars-cov-1 antibodies for sars-cov-2 n protein detection. since the sars-cov-1 and sars-cov-2 genomes are highly similar, we tested rabbit monoclonal and polyclonal anti-sars-cov-1 n protein antibodies on the sars-cov-2 proteome microarray ( figure 1d and supporting information, figure 4 ). the monoclonal ab displayed high specificity to two epitopes (rrgpe and paadl) on the sars-cov-2 n protein with a z-score higher than 3. 23 minor cross-reactivity was observed on the epitope (svllf) of the e protein. the polyclonal antibody bound to 11 epitopes (e1-e11) on the n protein with cross reactivity to six epitopes on m, s, orf8, and orf1ab proteins. the crossreactive epitopes on m, s, orf8, and orf1ab proteins are different than those present in the n-protein (figure 1d) , and the results were validated using full-length n-and s-proteins (supporting information, figure 5 ). , and orf10), respectively. the x-axis represents the sequence of amino acids of sars-cov-2 proteins from the n-terminal to c-terminal. the y-axis represents the serum samples from covid-19 patients. the false-colored rainbow color from blue to red corresponds to the signals of antibody binding from low to high, respectively. antibodies in the serum of covid-19 patients. using the sars-cov-2 proteome microarray, we screened igm and igg antibodies in the serum of 10 covid-19 patients who were in the early stage of infection (days of symptoms onset, 3.0 ± 5.92) (supporting information, table s2 ) to construct a landscape of humoral responses to the sars-cov-2 proteome (figure 2 ). sixty-one (61) igg and igm antibody epitopes were identified in seven sars-cov-2 proteins (m, n, s, orf1ab, orf3a, orf7a, and orf8) with a z-score higher than 3 in at least one covid-19 patient (table 1) . 23 the orf1ab has the maximal number of igm and igg epitopes (n = 32). these epitopes were distributed on the proteins of nsp1−4, nsp6, nsp8−10, and nsp12−16 (figures 2 and 3) . additional binding epitopes were identified on s (n = 8), n (n = 8), m (n = 5), orf3a (n = 4), orf7a (n = 3), and orf8 (n = 1) proteins ( figure 4 and table 1 ). notably, four immunodominant epitopes with antibodies in more than 80% of the covid-19 patients were present in the n (residue 206−210, sparm), s (residue 816−820, sfied), and orf3a (residue 136−140, knpll; residue 176−180, spise) proteins. however, antibodies to e, orf6, and orf10 were not detected ( figure 4) . furthermore, with overlapping peptides representing the full-length s protein, human igm and human igg antibodies were found to target three and six epitopes, respectively ( figure 4 and table 1 ). likewise for the n-protein, igm antibodies targeted two epitopes, and igg antibodies bound to eight epitopes (figure 4 and table 1 ). structural analysis shows that all epitope peptides within the rna binding domain loop of the n protein are easily accessible to antibodies (figure 5a ). six epitopes were identified in the s protein, with three epitopes located at the surface and three epitopes located inside the protein (figure 5b) . to help understand the translational potential of these peptide epitopes in covid-19 diagnosis, we compared the expression of serum antibodies targeting these immunogenic epitopes in 10 covid-19 patients with 10 control patients with nucleic acid testing negative (supporting information, table 1 ). these control patients were suspected to have covid-19 due to displaying similar symptoms but were confirmed to not have covid-19 via polymerase chain reaction (pcr) testing. statistical analyses identified one igg epitope and five igm epitopes (mann−whitney u-test, p < 0.01) (figure 6a) . one igg and igm epitope (816-sfied820) was located on the s protein, and one igm epitope (206-sparm-210) was located on the n protein; both of these proteins have been utilized as biomarkers in covid-19 diagnosis. in addition, we identified three potential new epitope biomarkers from orf3a (136-knpll-140 and 176-tspis-180) and nsp2 (296-fmgri-300), which should be validated in a different cohort in the future ( table 1) . identification of an epitope for potential neutralizing antibodies in the serum of covid-19 patients. there is a subdomain (residue 438−498) within the sars-cov-2 s-protein's rbd that directly engages the ace2 receptor, which makes it a potential target for developing neutralizing antibodies. 24 however, the identification of neutralizing antibodies to competitively inhibit the binding of the sars-cov-2 virus to the host ace2 receptor has proved challenging. in this work, we analyzed the immunological response to seven peptide sequences within the rbd subdomain (residue 438−498). some igm antibodies from patients "p45" and "p52" and igg antibodies from patients "p10", "p15", "p33", "p45", and "p52" bind to the same epitope (residues 456−460, frksn) (figure 6b,c) . structural analysis of the rbd-ace2 complex demonstrates that the epitope located within the rbd loop engages with the ace2 receptor 25 (figure 6d ), thus supporting our data. interestingly, this epitope (residues 456−460, frksn) was validated from a neutralizing antibody (b38) isolated from a convalescent patient. 26 with a mouse model, the antibody blocked the binding of s-rbd to ace2 and reduced virus titers in infected lung. the results provide evidence for the existence of a linear epitope for neutralizing antibodies in covid-19 patients. comprehensive profiling of the humoral antibody response to sars-cov-2 proteins is essential to understand the host immunity and identify the targets for covid-19 diagnostics and treatment. in this work, we created an sars-cov-2 proteome microarray with good reproducibility and sensitivity (figure 1a −c) that enables the high-throughput scanning of serum antibodies with sars-cov-2 proteins within 1.5 h. by epitope mapping a set of monoclonal and polyclonal antibodies previously prepared to target sars-cov-1 proteins, we demonstrate that the antibodies can also be used to detect sars-cov-2 proteins (figure 1d and supporting information figure s5 ). 27 sars-cov-1 antibodies could provide a quick alternative for developing an immunoassay to detect sars-cov-2 antigens. furthermore, we constructed the first landscape of b-cell epitopes of serum igm and igg antibodies, representing the comprehensive antibody response of covid-19 patients to sars-cov-2 infection (figures 2−4) . in addition, we experimentally validated four b-cell epitopes previously predicted by bioinformatics, 17, 19 including two epitopes on the s protein (residues 806−820, lpdpskpskrsfied; residues 456−460, frksn), one epitope on the n protein (residues 166−170, tlpkg), and one epitope on the m protein (residues 6−10, gtitv). igg and igm serum antibodies to one and five epitopes, respectively, were differentially expressed between 10 covid-19 patients and 10 control patients with similar symptoms but negative for sars-cov-2. these epitopes should be validated in future studies ( figure 6a ). as part of the humoral response of the adaptive immune system, neutralizing antibodies is critical in viral clearance and saving the lives of covid-19 patients. 16, 26 in this work, we identified a peptide epitope (residue 456−460, frksn) located at the interface of the sars-cov-2 s-rbd-ace2 receptor interaction in the serum of five mild covid-19 patients (p10, p15, p33, p45, p52) (figure 6b ). this epitope may serve as an antigen to stimulate neutralizing antibodies to the rbd-ace2 interaction and increase cd4+/cd8+ t-cell responses. 17, 28 the number of days after symptom onset ranged from 1 to 20 d (p10, 20 d; p15, 1 d; p33, 3 d; p45, 5 d; p52, 2 d). the result is consistent with previous reports and indicates humoral antibodies in early sars-cov-2 infection may confer protection. 29, 30 the results might also explain why most infected people can recover without medical intervention. there are several limitations to this study. first, the chemically synthesized peptides on the microarray do not have conformational epitopes. to address this issue, we included full-length n, s, and e proteins on our microarrays as a comparison. second, the peptides do not have posttranslational modifications, yet the sars-cov-2 s protein is glycosylated in vivo. 15 however, specific glycosylation on peptides is challenging and thus was not considered in this study. 31 third, 80 000 genomic sequences of sars-cov-2 have been submitted to the global initiative on sharing all influenza data (gisaid) database (https://www.gisaid.org/) since the preparation of our proteome microarray. these new strains could be included in the next version of the sars-cov-2 proteome microarrays. fourth, we specifically studied the binding of igg and igm antibodies in serum to the peptide array; however, the influence of total immunoglobulins on binding was not explored. total immunoglobulin profiling and the analysis of purified antibodies should be investigated in the future. altogether, we demonstrate that our sars-cov-2 peptidebased microarray can serve as a platform to determine the changes of humoral antibody response in covid-19 patients and animal models. the array can also help identify potential targets for covid-19 diagnosis and treatment. scientists who wish to acquire these arrays to help fight this covid-19 pandemic are encouraged to contact us. preparation of sars-cov-2 proteome microarray. all biotin-labeled peptides were obtained from china peptides and guoping pharmaceutical company. all sars-cov-2 e, n, and s proteins were obtained from sino biological, inc. among them, the sars-cov-2 e protein (catalogue no. dra33) was expressed in escherichia coli. the n protein (catalogue no. 40588-v08b) was expressed in insect cells. three s proteins (s1+s2 ecd: catalogue no. 40589-v08b1, s2 ecd: catalogue no. 40590-v08b, s1 subunit: catalogue no. 40591-v08b1) were expressed in insect cells, and two s proteins (s1 subunit: catalogue no. 40591-v08h, rbd: catalogue no. 40592-v05h) were expressed in human hek293 cells. these peptides and proteins were printed onto a 3d modified slide surface (capital biochip corp) in parallel and in duplicate using an arrayjet microarrayer. phosphate buffered saline (pbs), bovine serum albumin (bsa, 100 μg/ml) (sigma-aldrich), and hemagglutinin (ha) peptides (500 μg/ml) (china peptides) were used as negative controls. biotinylated bsa (100 μg/ml), human igg and igm (10 μg/ml), and polio peptides (500 μg/ml) (china peptides) were used as positive controls. the peptide microarrays were stored at −20°c until ready to use. no unexpected or unusually high safety hazards were encountered in this work. characterization of anti-sars antibody using sars-cov-2 proteome microarray. the peptide microarrays were assembled in an incubation tray and blocked with 5% (w/v) milk in 1x pbs with 0.2% (v/v) tween-20 (pbst) for 1 min at room temperature. after it was washed with pbst three times, the array was incubated with a rabbit anti-sars-cov-1 n-protein monoclonal or a rabbit anti-sars-cov-1 n-protein polyclonal antibody (1 μg/ml) (catalogue nos. 40143-r001 and 40143-t62, respectively; sino biological) for 30 min at room temperature. after it was washed again, the array was incubated with an alexa fluor 555 labeled goat antirabbit igg (h+l), cross-adsorbed, secondary antibody (jackson immu-noresearch) for 30 min. the arrays were washed, dissembled from the tray, and dried with centrifugation for 2 min at 2000 rpm. the resulting array was scanned with a genepix 4300a microarray scanner (molecular devices). the median fluorescent signal intensity of each spot was extracted using genepix pro7 software (molecular devices). the median background signal was subtracted from the median spot signal intensity. detection of serum antibody using sars-cov-2 proteome microarray. prior to the antibody detection, the peptide microarrays were assembled in an incubation tray and blocked with 5% (w/ v) milk in 1x pbs with 0.2% (v/v) tween-20 (pbst) for 1 min at room temperature. after it was washed with pbst three times, the array was incubated with 1:300 diluted serum for 30 min at room temperature. after it was washed again, the array was then incubated for 30 min with a mixture containing cy3 affinipure donkey antihuman igg(h+l) and alexa fluor 647 affinipure goat antihuman igm fc5 μantibody (jackson immunoresearch) (2 μg/ml). finally, the array was washed with pbst and water, dissembled from the tray, and dried with centrifugation for 2 min at 2000 rpm. the array was scanned with a genepix 4300a microarray scanner (molecular devices) at 10 μm resolution using a laser at 532 nm with 100% power/pmt gain 800 for igg and 635 nm with 100% power/pmt gain 900 for igm. the median fluorescent signal intensity with background subtraction was extracted using genepix pro7 software (molecular devices). data analysis. the raw fluorescence signal intensity was the median signal intensity subtracted by the median background intensity of each spot, and then averaged across duplicate spots. the resulting signals were normalized with a z-score, which is shown below. 23 z score (intensity mean intensity )/sd p p 1 p n p1 pn where p is any peptide or protein on the microarray, and p1··· pn represents the aggregate measure of all of the peptides or proteins. the heatmap of antibody response to the peptides was visualized using the multiexperiment viewer software version 4.9 (dana-farber cancer institute). 32 statistical analyses were performed using the graphpad prism software version 6.0 (graphpad software, inc.) with the mann− whitney u-test (p-value < 0.01). clinical features of patients infected with 2019 novel coronavirus in early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of coronavirus disease 2019 in china estimating the asymptomatic proportion of 2019 novel coronavirus onboard the princess cruises ship china coronavirus: mild but infectious cases may make it hard to control outbreak, report warns importation and human-to-human transmission of a novel coronavirus in vietnam a novel coronavirus outbreak of global health concern first case of 2019 novel coronavirus in the united states an interactive web-based dashboard to track covid-19 in real time outbreak of a novel coronavirus a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster genomic characterization of the 2019 novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan sars coronavirus accessory proteins structural basis of rna recognition by the sars-cov-2 nucleocapsid phosphoprotein structural and functional basis of sars-cov-2 entry by using human ace2 immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov preliminary identification of potential vaccine targets for the covid-19 sars-cov-2) based on sars-cov immunological studies a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals in-depth serum proteomics reveals biomarkers of psoriasis severity and response to traditional chinese medicine analysis of microarray data using z score transformation a human monoclonal antibody blocking sars-cov-2 infection structure of dimeric full-length human ace2 in complex with b0at1 a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its receptor ace2 a roadmap to generate renewable protein binders to the human proteome neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications antibody responses to sars-cov-2 in patients with covid-19 glycosylation of biosimilars: recent advances in analytical characterization and clinical implications mev+r: using mev as a graphical user interface for bioconductor applications in microarray analysis key: cord-264968-ctx39vhi authors: woo, patrick cy; lau, susanna kp; tsoi, hoi-wah; chan, kwok-hung; wong, beatrice hl; che, xiao-yan; tam, victoria kp; tam, sidney cf; cheng, vincent cc; hung, ivan fn; wong, samson sy; zheng, bo-jian; guan, yi; yuen, kwok-yung title: relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia date: 2004-03-13 journal: lancet doi: 10.1016/s0140-6736(04)15729-2 sha: doc_id: 264968 cord_uid: ctx39vhi background: although the genome of severe acute respiratory syndrome coronavirus (sars-cov) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. methods: we cloned and purified the nucleocapsid protein and spike polypeptide of sars-cov and examined their immunogenicity with serum from patients with sars-cov pneumonia. an elisa based on recombinant nucleocapsid protein for igg detection was tested with serum from 149 healthy blood donors who donated 3 years previously and with serum positive for antibodies against sars-cov (by indirect immunofluorescence assay) from 106 patients with sars-cov pneumonia. the seroprevalence of sars-cov was studied with the elisa in healthy blood donors who donated during the sars outbreak in hong kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. all positive samples were confirmed by two separate western-blot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide). findings: western-blot analysis showed that the nucleocapsid protein and spike polypeptide of sars-cov are highly immunogenic. the specificity of the igg antibody test (elisa with positive samples confirmed by the two western-blot assays) was 100%, and the sensitivity was 94·3%. three of 400 healthy blood donors who donated during the sars outbreak and one of 131 non-pneumonic paediatric inpatients were positive for igg antibodies, confirmed by the two western-blot assays (total, 0·48% of our study population). interpretation: our findings support the existence of subclinical or non-pneumonic sars-cov infections. such infections are more common than sars-cov pneumonia in our locality. background although the genome of severe acute respiratory syndrome coronavirus (sars-cov) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. methods we cloned and purified the nucleocapsid protein and spike polypeptide of sars-cov and examined their immunogenicity with serum from patients with sars-cov pneumonia. an elisa based on recombinant nucleocapsid protein for igg detection was tested with serum from 149 healthy blood donors who donated 3 years previously and with serum positive for antibodies against sars-cov (by indirect immunofluorescence assay) from 106 patients with sars-cov pneumonia. the seroprevalence of sars-cov was studied with the elisa in healthy blood donors who donated during the sars outbreak in hong kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. all positive samples were confirmed by two separate westernblot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide). findings western-blot analysis showed that the nucleocapsid protein and spike polypeptide of sars-cov are highly immunogenic. the specificity of the igg antibody test (elisa with positive samples confirmed by the two western-blot assays) was 100%, and the sensitivity was 94·3%. three of 400 healthy blood donors who donated during the sars outbreak and one of 131 non-pneumonic paediatric inpatients were positive for igg antibodies, confirmed by the two western-blot assays (total, 0·48% of our study population). severe acute respiratory syndrome (sars) has now affected 30 countries in five continents, with more than 8400 cases and more than 910 deaths. a novel virus, the sars coronavirus (sars-cov), is known to be the aetiological agent. [1] [2] [3] [4] [5] [6] [7] [8] [9] the viral genome has been completely sequenced. 10, 11 we have also reported the isolation of viruses resembling sars-cov from himalayan palm civets found in a live animal market in the guangdong province of china; this finding implied that animals could be a reservoir of the virus. 12 detection of sars-cov from a non-pneumonic case in singapore (http://www.who.int/csr/don/2003_09_16/en/) suggested that non-pneumonic and subclinical sars-cov infections are possible. however, extensive seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking. at present, the most widely used methods for detection of antibodies against sars-cov are indirect immunofluorescence assay and elisa with cell-culture extract. 1, 5 however, antibody detection by these methods is difficult to standardise and has not been compared with recombinant-antigen-based antibody-detection tests. a recent seroprevalence study, which used the indirect immunofluorescence assay for antibody detection, did not detect sars-cov antibodies in any of 674 health-care workers from a hospital in which a sars outbreak had occurred. 13 an approach of elisa-based antibody-detection tests using recombinant antigens with positive results confirmed by western-blot assays that use two different antigenic proteins offers higher sensitivity, specificity, and reproducibility than indirect immunofluorescence assay and elisa with cell-culture extract and is easier to standardise. [14] [15] [16] [17] [18] [19] in this study, we used a sensitive and specific elisa based on the highly immunogenic nucleocapsid protein of sars-cov and confirmed positive results by western-blot assays with recombinant nucleocapsid protein and recombinant spike polypeptide (another highly immunogenic protein) of sars-cov to examine the seroprevalence of non-pneumonic sars-cov in the general population, non-pneumonic patients in hospital, and health-care workers during the sars epidemic. pellet was resuspended in 10 l dnase-free, rnase-free double-distilled water and was used as the template for rt-pcr. cloning and purification of (his) 6 -tagged recombinant proteins to produce a fusion plasmid of the nucleocapsid protein of the sars-cov for protein purification, primers lpw723 and lpw726 (panel) were used to amplify the gene encoding this protein by rt-pcr. the sequence coding for aminoacid residues 1-422 of the nucleocapsid protein was amplified and cloned into the bamhi and ecori sites of expression vector pet-28b(+) (novagen, madison, wi, usa) in frame and downstream of the series of six histidine residues. the (his) 6 -tagged recombinant nucleocapsid protein was expressed and purified by means of the nickel-loaded hitrap chelating system (amersham pharmacia, piscataway, nj, usa) according to the manufacturer's instructions. roughly 3 mg purified protein was routinely obtained from 1 l escherichia coli carrying the fusion plasmid. for the spike protein of the sars-cov, primers lpw742 and lpw931 (panel) were used to amplify the gene encoding aminoacid residues 14-667 by rt-pcr. this portion of the spike protein was used because the complete protein could not be expressed in e coli. the pcr product was cloned into the bamhi and kpni sites of vector pqe-31 (qiagen, hilden, germany). the resulting clone was digested by psti, and the fragment that contained the gene encoding aminoacid residues 250-667 of the spike protein was cloned into expression vector pqe-30 (qiagen, hilden, germany) in frame and downstream of the series of six histidine residues. expression and purification of the (his) 6 -tagged recombinant spike polypeptide were done as described for the nucleocapsid protein. western-blot analysis was done according to our published protocols, with slight modifications. [20] [21] [22] briefly, 200 ng purified (his) 6 -tagged recombinant nucleocapsid protein or (his) 6 -tagged recombinant spike polypeptide was loaded into each well of a sodium dodecyl sulphate 10% polyacrylamide gel, then electroblotted onto a nitrocellulose membrane (bio-rad, hercules, ca, usa). the blot was cut into strips, and the strips were incubated separately with 1 in 1000 dilution of three serum samples, obtained from three patients with sars-cov pneumonia, positive for antibodies against sars-cov detected by our indirect immunofluorescence assay. 1 antigen-antibody interaction was detected with an ecl fluorescence system (amersham life science, buckinghamshire, uk). serum samples from three healthy blood donors were used as controls. assessment of recombinant nucleocapsid protein elisa serum samples from 149 healthy blood donors who donated blood 3 years previously (aged 18 years or older) and 106 patients with pneumonia positive for antibodies against sars-cov detected by our indirect immunofluorescence assay 1 were used for the assessment of the elisa-based igg antibody test. the test was modified from our previous publications. 15, 17 briefly, each well of a nunc immunoplate (roskilde, denmark) was coated with 20 ng purified (his) 6 -tagged recombinant nucleocapsid protein for 12 h, then blocked in phosphate-buffered saline with 2% bovine serum albumin. 100 l diluted human serum (1 in 40) was added to each well of the protein-coated plates, and they were incubated at 37°c for 2 h. after five washes with washing buffer, 100 l horseradish-peroxidase-conjugated goat antibody to human igg (1 in 4000 dilution; zymed laboratories inc, south san francisco, ca, usa) was added to each well, and the plates were incubated at 37°c for 1 h. after a further five washes with washing buffer, 100 l diluted 3,3ј,5,5ј-tetramethylbenzidine (zymed laboratories) was added to each well and incubated at room temperature for 15 min. 100 l 0·3 mol/l sulphuric acid was added, and the absorbance at 450 nm of each well was measured. each sample was tested in duplicate, and the mean absorbance for each sample was calculated. the presence of specific antibodies in positive samples was confirmed by retesting of the sample by the elisa based on recombinant nucleocapsid protein and western-blot assays with recombinant nucleocapsid protein and recombinant spike polypeptide separately. using the protocol described above, we tested for the presence of igg antibodies against the nucleocapsid protein in serum samples from 400 healthy blood donors (aged 18 years or older) who donated blood in march-may, 2003; 131 non-pneumonic paediatric inpatients (aged less than 18 years) and 264 nonpneumonic adult inpatients (aged 18 years or older) admitted to queen mary hospital in may, 2003; and 33 symptom-free health-care workers. the presence of specific antibodies in all positive samples was confirmed by two separate western-blot assays, one with recombinant nucleocapsid protein and the other with recombinant spike polypeptide as the antigen. the study sponsors had no role in the study design; collection, analysis, or interpretation of data; or the writing of the report. the purified (his) 6 -tagged recombinant nucleocapsid protein and spike polypeptide were separated on denaturing polyacrylamide gels followed by western-blot analysis with serum from three patients with pneumonia positive for antibodies against the sars-cov. prominent immunoreactive protein bands of about 50 kda were visible on the western blots that used either antigen (figure 1). these sizes were consistent with the expected size of 49·6 kda for the full-length (his) 6 -tagged nucleocapsid protein and 48·6 kda for the (his) 6 -tagged spike polypeptide. an elisa-based sars-cov antibody test was developed for the detection of specific antibodies against the (his) 6 -tagged recombinant nucleocapsid protein. box titration was carried out with different dilutions of (his) 6tagged recombinant nucleocapsid protein coating antigen and pooled serum from three patients with pneumonia positive for antibody against the sars-cov. the results identified 20 ng purified (his) 6 -tagged recombinant nucleocapsid protein per elisa well as the ideal amount for plate coating for igg detection (data not shown). to establish the baseline for the tests, serum samples from 149 healthy blood donors who donated blood 3 years previously were tested in the elisa. for these samples, the mean optical density at 450 nm for igg detection was 0·127 (sd 0·068). an absorbance value of 0·263 was selected as the cut-off value (the mean value for the healthy controls plus 2 sd; figure 2) . seven of the samples from 149 healthy blood donors had values of more than 0·263 in the igg elisa (figure 2), but none of them had the specific antibody when tested by the nucleocapsid protein or the spike polypeptide westernblot assay. the specificity of the igg antibody test (elisa confirmed by western-blot assays) was 100%. the mean value for the samples obtained from the 106 patients with sars-cov pneumonia, positive for igg antibodies against the sars-cov by our indirect immunofluorescence assay, was 1·153 (sd 0·702). 100 samples had optical density of more than 0·263 in the igg elisa (figure 2). all 100 were confirmed to have the specific antibody by both the nucleocapsid protein and the spike polypeptide western-blot assays. the sensitivity of the igg antibody test, with the immunofluorescence assay as the gold standard, was therefore 94·3%. 16 (4·0%) of the 400 healthy blood donors who donated blood in march-may, 2003, eight (6·1%) of the 131 non-pneumonic paediatric patients, eight (3·0%) of the 264 non-pneumonic adult patients, and one (3%) of the 33 symptom-free health-care workers who had cared for the patients with sars-cov pneumonia were positive for igg antibodies by elisa ( figure 2) . however, only three (0·8%) healthy blood donors who donated blood in march-may, 2003, and one (0·8%) non-pneumonic paediatric patient were confirmed to have specific sars-cov antibodies by both the nucleocapsid protein and spike polypeptide western-blot assays. up to the end of may, 1728 patients from a population of about seven million in hong kong (0·025%) had developed sars-cov pneumonia, compared with a rate of non-pneumonic sars-cov infections in our study population of about 0·48% (p<0·001 by poisson exact test of equality). previous studies in animal coronaviruses, such as infectious bronchitis virus, have shown that the nucleocapsid protein and spike protein are highly immunogenic, are abundantly expressed during infection, and can be used for serodiagnosis of animal coronavirus infections. [23] [24] [25] in this study, detection of igg antibodies to both proteins was highly sensitive and specific for sars-cov infections. six (5·7%) serum samples that were seropositive by the immunofluorescence assay were negative by the elisa. the reason for this discrepancy may be that the nucleocapsid protein did not elicit antibody response in this minority group of patients. conversely, of five patients with sars-cov pneumonia who were seronegative by the immunofluorescence assay but rt-pcr positive for sars-cov, two were seropositive by our elisa, with clearly positive opticaldensity values of 0·874 and 1·228 and confirmation by western-blot assay (unpublished). furthermore, in four of 20 patients with sars coronavirus pneumonia who had serial serum samples, igg was detected earlier by the elisa than by the immunofluorescence test (unpublished). in another study that used elisa based on cell-culture extract, five of 15 patients with sars-cov pneumonia were positive according to that elisa but negative by indirect immunofluorescence during the time when the elisa titres were low. 5 this finding accords with the results of a previous study on human coronavirus 229e, which showed that three of 51 serum samples were positive by recombinant-nucleocapsid-protein-based western blot but negative by immunofluorescence, and six of 51 serum samples were positive by immunofluorescence but negative by recombinant-nucleocapsid-protein-based western blot. 26 all these findings show that our elisa may be able to detect additional cases that the immunofluorescence assay has missed. with this potentially more sensitive elisa for igg antibody detection, we assessed the seroprevalence of non-pneumonic sars-cov infections in both the general population and our hospital population; all positive serum samples detected by elisa were confirmed by two separate western-blot assays, with two immunologically unrelated antigens. the spike polypeptide was used in addition to the nucleocapsid protein for western-blot confirmation to eliminate the possibility of cross-reactivity between antibodies against the nucleocapsid proteins of other human coronaviruses and that of sars-cov. however, the aminoacid identities between the nucleocapsid protein of sars-cov and those of the human coronaviruses oc43 and 229e are only 32·7% and 21·3%, respectively, and there were no crossreactions between 13 pairs of oc43 and 14 pairs of 229e human coronavirus serum samples and the sars-cov. 5 in fact, of the 33 individuals who were igg positive by elisa, 26 (79%) were positive by the nucleocapsidprotein-based western-blot assay, but only four were positive by both the nucleocapsid protein and the spike polypeptide western-blot assays. this apparent high rate of false-positive non-pneumonic cases, as detected by the recombinant nucleocapsid protein elisa, is due to the use of a single antigen for screening asymptomatic or nonpneumonic infections. it is well known that the positive predictive values of serological tests are much affected when the prevalence of the infection is low, especially in clinically incompatible cases. this is the reason why an immunologically unrelated antigen, the spike protein, has to be used for confirmation of the cases detected as "positive" by the nucleocapsid-protein-based assays. cross-reactivity with human coronavirus oc43 or other sars-cov-like viruses remains an important issue for future studies on sars-cov serology. three of the four individuals with non-pneumonic sars-cov infections were healthy blood donors, and one was a paediatric inpatient. this patient was a 19-month-old girl who was admitted to hospital in may, 2003, because of fever for 2 days. she was also noted to have had a cough for the previous 2 weeks and repeated vomiting before admission. there had been no breathing difficulty or diarrhoea. the child had no history of direct contact with patients with sars-cov pneumonia. however, a colleague in her mother's workplace had recently been diagnosed as having sars-cov pneumonia. physical examination of the girl revealed only shotty (palpable but too small to be measured) cervical lymph nodes and congested throat. her chest radiograph was normal. apart from mild lymphopenia (lymphocyte count 0·63ϫ10 9 /l), her blood results were normal. nasopharyngeal aspirate was negative for influenza viruses, parainfluenza viruses, adenovirus, and respiratory syncytial virus antigens. 27 she was given antipyretic treatment and was discharged the next day. subsequent antibody testing showed that her serum was positive for both igg and igm antibodies against the nucleocapsid protein as well as igg antibodies against the spike polypeptide of sars-cov. the difference between the rate of non-pneumonic sars-cov infections in our study population and the rate of sars in hong kong suggests that non-pneumonic infections are more common than sars-cov pneumonia and may explain cases of sars-cov pneumonia in patients who had no obvious contact with other patients with sars. contributors p c y woo and k y yuen are coprincipal investigators, jointly wrote the report, and coordinated and supervised the study. s k p lau supervised the analysis of all serological data and corrected the report. h w tsoi cloned and purified the nucleocapsid protein and did the serological assays. k h chan supervised the immunofluorescence assay. b h l wong and v k p tam did the serological assays and analysed the data. x y che cloned and purified the spike polypeptide. s c f tam coordinated the collection of clinical specimens. v c c cheng and i f n hung managed the patients' database. s s y wong, b j zheng, and y guan corrected the report. none declared. coronavirus as a possible cause of severe acute respiratory syndrome development of a standard treatment protocol for severe acute respiratory syndrome prospective study of the clinical progression and viral load of sars-associated coronavirus pneumonia in a community outbreak lung 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for serodiagnosis of aspergillosis detection of specific antibodies to an antigenic cell wall galactomannoprotein for serodiagnosis of aspergillus fumigatus aspergillosis enzyme-linked immunosorbent assay for detecting antibodies to borne disease virusspecific proteins an indirect elisa for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen cloning and characterization of male in burkholderia pseudomallei groel encodes a highly antigenic protein in burkholderia pseudomallei aflmp1 encodes an antigenic cell wall protein in aspergillus flavus localization of linear b-cell epitopes on infectious bronchitis virus nucleocapsid protein recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus an elisa for antibodies against infectious bronchitis virus using an s1 spike polypeptide detection of human coronavirus 229e-specific antibodies using recombinant fusion proteins cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients this work is partly supported by a research grant council grant (hku 7532/03m), the sars research fund, and the university sars donation fund, the university of hong kong. we thank members of the clinical microbiology laboratory, queen mary hospital, and members of the department of medicine, united christian hospital, hong kong for their assistance. key: cord-262328-q7mt0xve authors: wajnberg, ania; mansour, mayce; leven, emily; bouvier, nicole m; patel, gopi; firpo-betancourt, adolfo; mendu, rao; jhang, jeffrey; arinsburg, suzanne; gitman, melissa; houldsworth, jane; sordillo, emilia; paniz-mondolfi, alberto; baine, ian; simon, viviana; aberg, judith; krammer, florian; reich, david; cordon-cardo, carlos title: humoral response and pcr positivity in patients with covid-19 in the new york city region, usa: an observational study date: 2020-09-25 journal: lancet microbe doi: 10.1016/s2666-5247(20)30120-8 sha: doc_id: 262328 cord_uid: q7mt0xve background: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused a global pandemic. the proportion of infected individuals who seroconvert is still an open question. in addition, it has been shown in some individuals that viral genome can be detected up to 3 months after symptom resolution. we investigated both seroconversion and pcr positivity in a large cohort of convalescent serum donors in the new york city (ny, usa) region. methods: in this observational study, we ran an outreach programme in the new york city area. we recruited participants via the redcap (vanderbilt university, nashville, tn, usa) online survey response. individuals with confirmed or suspected sars-cov-2 infection were screened via pcr for presence of viral genome and via elisa for presence of anti-sars-cov-2 spike antibodies. one-way anova and fisher's exact test were used to measure the association of age, gender, symptom duration, and days from symptom onset and resolution with positive antibody results. findings: between march 26 and april 10, 2020, we measured sars-cov-2 antibody titres in 1343 people. of the 624 participants with confirmed sars-cov-2 infection who had serologies done after 4 weeks, all but three seroconverted to the sars-cov-2 spike protein, whereas 269 (37%) of 719 participants with suspected sars-cov-2 infection seroconverted. pcr positivity was detected up to 28 days from symptom resolution. interpretation: most patients with confirmed covid-19 seroconvert, potentially providing immunity to reinfection. we also report that in a large proportion of individuals, viral genome can be detected via pcr in the upper respiratory tract for weeks after symptom resolution, but it is unclear whether this signal represents infectious virus. analysis of our large cohort suggests that most patients with mild covid-19 seroconvert 4 weeks after illness, and raises questions about the use of pcr to clear positive individuals. funding: none. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which causes covid-19, has rapidly spread around the world, leading to unprecedented strain on health-care systems and economies and causing more than 405 000 infections and 25 000 deaths in new york state (new york state, department of health, nysdoh covid-19 tracker) as of july 20, 2020. substantial disruptions to daily life have been enacted to flatten the epidemic curve. to avoid spread of sars-cov-2 and to help standardise definitions of clearance, it is important to understand the duration of sars-cov-2 nucleic acids within the nasopharynx and the time course to the mounting of an antibody response to this new viral pathogen. current us centers for disease control and prevention (cdc) guidelines suggest that people with confirmed or suspected sars-cov-2 infection should remain in isolation for at least 10 days from symptom onset and return to work if they have been asymptomatic for at least 72 h. however, so far, there are limited data that help to define the time to viral clearance from illness onset and cessation of symptoms. a previous case study suggested that sars-cov-2 can exhibit ongoing viral shedding for a median of 2·5 days (range 1·0-8·0) after complete symptom resolution, but it remains unclear whether this viral shedding poses a risk for forward transmission. 1 a small case sample of four patients admitted to hospital were sars-cov-2 positive on repeat rt-pcr testing 5-13 days after discharge. 2 other studies have found viral shedding for up to 3 months after symptoms resolve. 3 from work with the 2003 sars-cov, it is not clear whether detection of viral genome of this duration indicates prolonged infectivity or the presence of non-viable virus. 4 a clearer understanding of the duration of viral shedding is crucial for preventing transmission by infected individuals, particularly as they begin to feel well enough to resume normal activities. an understanding of the time to pcr clearance might also help to guide isolation durations and return to work clearance, as well as clarify the usefulness of negative pcr testing as part of defining disease clearance. there are limited data worldwide on the development of antibodies against sars-cov-2, particularly the formation of igg. there is concern regarding efficacy of antibody testing for diagnosis of sars-cov-2, and little is known about long-lasting immunity. one study measured neutralising antibodies in 175 patients admitted to hospital and found that 64% had high antibody titres, 30% had weak antibody response, and 6% had undetectable titres. 5 studying the plasma from previously infected individuals might improve our understanding of the timing and strength of different populations' antibody response to this novel illness, delineate the duration of antibody presence, and identify cases of possible reinfection. additionally, individuals with high antibody titres might become donors for convalescent plasma treatment for patients who are critically ill as part of ongoing studies of this therapeutic option. [6] [7] [8] we present a large dataset of serum antibody testing completed at mount sinai hospital (new york, ny, usa) in people who have fully recovered after mild illness from sars-cov-2 infection. we aimed to describe the time to sars-cov-2 pcr clearance from the nasopharynx, the rates of igg development, and time to serum igg development from onset and resolution of symptoms in participants with previously confirmed or suspected sars-cov-2 infection. in this observational study, we ran an outreach programme in the new york city (ny, usa) area, including parts of connecticut and new jersey, to identify people who had recovered from sars-cov-2 infection for nasopharyngeal pcr (cobas 6800; roche diagnostics, indianapolis, in, usa) and serum igg titre measurement (elisa; icahn school of medicine at mount sinai, new york, ny, usa). 9, 10 we recruited participants via the redcap (vanderbilt university, nashville, tn, usa) online survey response, which was advertised on our hospital website, and subsequently shared by several news organisations and public officials in new york. redcap respondents were deemed to be eligible if they had previously tested positive evidence before this study we searched pubmed, medrxiv, and biorxiv for research articles and preprints published in english between jan 1 and may 1, 2020, using the terms "sars cov 2", "covid 19", "elisa antibody", and "pcr". we did not find reports of elisa antibody assays as large as this one from areas with major covid-19 hotspots, and found mixed and growing reports of igg response to and pcr positivity for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) over time. we report on a large cohort of patients who recovered from mild covid-19, and our findings show that the majority of patients with pcr-confirmed sars-cov-2 infection have an igg response. more than a third of patients without pcr-confirmed sars-cov-2 had positive igg, and a significant minority of patients had positive pcr results despite full resolution of symptoms for more than 2 weeks. our findings suggest that igg antibodies develop over a period of 7-50 days from symptom onset and 5-49 from symptom resolution, suggesting that the optimal timeframe for widespread antibody testing is at least 3-4 weeks after symptom onset and at least 2 weeks after symptom resolution. we also present the common finding of persistent pcr positivity, which raises issues regarding use of pcr testing for clearance of disease. both of these findings have policy implications as the pandemic continues to spread around the world. symptom duration 9 (6-12) 9 (6-12) 7 (4-10) 10 (4-15) 0·030 data are mean (sd; range), n (%), or median (iqr). na=not applicable. for sars-cov-2 or if they were symptomatic with suspected sars-cov-2 and in a high-risk group. symptomatic individuals deemed to be at high risk were those who either lived with someone with a positive sars-cov-2 pcr test, had been told by a physician that they had symptoms consistent with sars-cov-2, or were healthcare workers. we only included participants who selfreported suspicious symptoms after feb 1, 2020, as sars-cov-2 is believed to have begun to spread in new york city from this time. additionally, only participants who were asymptomatic at the time of survey were contacted. respondents self-reported date of symptom onset, date of positive sars-cov-2 test (if applicable), and last date of symptoms. duration of symptoms was calculated from these self-reported dates. this study was reviewed and approved by our institutional review board. during the first 2 weeks of the survey, we tested for sars-cov-2 in the nasopharynx by pcr as well as igg antibody in the serum of every individual, whereas in the third week the testing was limited to sars-cov-2 antibodies only. the rationale for this change was that more data are available on lack of infectiousness with symptom resolution more than 14 days before testing. during week 1, participants were brought in 10 days after they had a confirmed or suspected diagnosis and had been asymptomatic for at least 3 days. in week 2, as we identified more potential donors and learned more about our antibody assay, we extended our timeline to 14 days after symptom onset, with at least 3 days asymptomatic. in week 3, we included participants 21 days or more after symptom onset, who had been completely asymptomatic for at least 14 days. sars-cov-2 pcr was considered to be positive if detected on nasopharyngeal swab. 11 a close agreement has been shown between the cobas 6800 used in this study and two other widely implemented sars-cov-2 pcr tests: cepheid genexpert (cepheid, sunnyvale, ca, usa) and hologic panther fusion (hologic, marlborough, ma, usa). [12] [13] [14] we measured serum igg antibody titres using a serological elisa developed at icahn school of medicine at mount sinai and described on march 18, 2020; this serum test has a sensitivity of 92% and a specificity of more than 99% as per our us food and drug administration (fda) emergency use authorisation. 15 increasing titres in the mount sinai elisa assay have been shown to correlate with viral neutralisation. 9, 16 serum igg titres were considered to be strongly positive if they were detected at titres of 1:320 or higher (highest dilutions were 1:320, 1:960, and 1:2880), and considered to be weakly positive if detected at titres of 1:80 and 1:160. as per the fda emergency use authorisation, detected means that a sample was above the optical density cutoff of 0·15 in the receptor binding domain screening elisa and had a titre of at least 1:80 in the spike confirmatory titration. negative was defined as titres below 1:80 (and is shown in figures as 1:40 for representation purposes). all interested partici pants with antibody titres of more than 1:320 and negative sars-cov-2 pcr swabs were screened by the new york blood center using standard criteria for plasma donation and included as donors in our convalescent plasma study if eligible. participants with weakly positive antibody titres were invited to return for repeat serum titre testing at least 7 days after their initial antibody test. participants with positive pcr swabs and antibodies were asked to return for pcr testing at least 3 days after initial pcr test so they could be referred for plasma donation once the virus had fully cleared. given the frequent serial detection of sars-cov-2 by nasopharyngeal pcr using 3-day incre ments, on april 3, 2020, we began to ask participants to return at least 10 days after the last positive pcr for retesting. one-way anova and fisher's exact test were used to measure the association of age, gender, symptom duration, and days from symptom onset and resolution with positive antibody results. there was no funding source for this study. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. between march 26 and april 10, 2020, we measured sars-cov-2 antibody titres in 1343 people. the mean age of the participants was 40·35 years (sd 12·18) with 256 (19%) participants aged 17-29 years, 968 (72%) aged 30-59 years, and 119 (9%) aged 60 years or older. 706 (53%) participants were male and 624 (46%) had confirmed sars-cov-2 diagnosis by previous pcr testing. the median number of days between symptom onset to serum antibody test was 24 (iqr 21-28), the median number of days between symptom resolution to antibody test was 15 (12) (13) (14) (15) (16) (17) (18) (19) (20) , and median duration of symptoms was 9 days (6-13). of the 1343 participants, 18 (1%) were referred to our redcap after an emergency room visit or admission to hospital in the mount sinai hospital system; the other 1325 (99%) were self-referred individuals who had mild to moderate symptoms. in the 584 participants for whom both nasopharyngeal pcr testing and serum antibody testing was available, sars-cov-2 rna was detected in 249 (43%) at a median of 20 days (iqr 18-23) from symptom onset and 12 days (9-14) from symptom resolution. 624 participants had confirmed sars-cov-2 disease by pcr before coming for testing; 606 (97%) were by selfreport and 18 (3%) were documented in our electronic medical record. if self-reported, participants provided the date of testing. in this subgroup, the mean age was 39·14 years (sd 12·09) and 368 (59%) participants were male (table 1) . at first test, 511 (82%) were strongly antibody positive at a titre of 1:320 or more, 42 (7%) were weakly positive, and 71 (11%) were negative (figure 1a). we asked 113 (18%) of 624 participants with an initial negative or weakly positive antibody response to return for a second test 10 or more days later. at the time of the first test, 217 (35%) were still pcr positive (range 5-22 days from symptom resolution; figure 2 ). median duration of symptoms in this group was 9 days (iqr 6-13). age was not associated with a strong antibody response (p=0·17), whereas male gender was associated with a stronger response (p=0·0015). longer duration between symptom onset and antibody test was associated with a higher titre antibody test (median 23 days [iqr 20-27] for positive titre vs 20 days [18] [19] [20] [21] [22] for weakly positive, p<0·0001; table 1). symptom duration was also associated with higher antibody titres (median 9 days [iqr 6-12] for positive titre vs 7 days [4] [5] [6] [7] [8] [9] [10] for weakly positive, p=0·030; table 1). this finding can also be clearly observed in figure 1a , which plots titres against days after symptom onset. in the subgroup of 719 participants with suspected disease who did not have confirmed sars-cov-2 infection, mean age was 41·39 years (sd 12·16) and 338 (47%) were male (table 2) . 250 (35%) were strongly antibody positive, 19 (3%) were weakly positive, and 436 (62%) were negative at the first test. antibodies were measured a median of 32 days (iqr 28-38) from symptom onset and 23 days (18-29) from date of symptom resolution. in this group, younger age was individual might be shown if tested more than once on different days. only results for individuals for whom a date of symptom resolution was available are shown. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 of the 113 participants with pcr-confirmed sars-cov-2 infection and weakly positive or negative titres on their first serum antibody test, 64 have returned for follow-up antibody titres as of may 1, 2020. of these participants, 57 (89%) displayed increased titres between the two tests, a median of 13 days (iqr 11-17) later (figure 1b). four (6%) participants remained weakly positive, and three (5%) remained negative. the three that remained negative all self-reported positive pcr testing (none was documented in our electronic medical record; table 3). although all 1343 survey participants self-reported complete resolution of symptoms before testing, 249 (19%) tested positive for naso pharyngeal sars-cov-2 rna. the maximum time of positive nasopharyngeal pcr testing was 43 days from symptom onset and 34 days from symptom resolution. for the 182 individuals who returned for repeat nasopharyngeal swabbing at least 3 days after a previous positive test, 112 (62%) were negative on the repeat test, a median of 10 days (iqr 7-12) after the first test. 70 (38%) remained positive and were rescheduled for another nasopharyngeal pcr 7-10 days later (table 4 ). our survey provides a large cross-sectional representation of sars-cov-2 rna and antibodies found in participants recruited after recovery from sars-cov-2 during the early weeks of the outbreak in the new york city region. an understanding of the duration of potential infectiousness and the time to igg antibody response is crucial to the containment of sars-cov-2 and the plans for widespread antibody testing over the coming months. some countries, states, and organisations might even be con sidering antibody testing before letting individuals return to work. in contrast to some of the previous literature on formation of antibodies, more than 99% of the patients who self-reported or had laboratory-documented sars-cov-2 infection developed igg antibodies using our assay. additionally, our findings suggest that igg antibodies develop over a period of 7-50 days from symptom onset and 5-49 from symptom resolution, with a median of 24 days from symptom onset to higher antibody titres, and a median of 15 days from symptom resolution to higher antibody titres. these results suggest that the optimal timeframe for widespread antibody testing is at least 3-4 weeks after symptom onset and at least 2 weeks after symptom resolution. 28% of patients with a positive antibody response were tested within 2 weeks of symptom resolution, 67% within 3 weeks of resolution, and 94% within 4 weeks of resolution. in our survey, we did not find evidence for a decrease in igg antibody titre levels on repeat sampling. although we do not yet know what, if any, immunity is conferred by igg or the duration of the igg response, at this time it seems likely that igg against sars-cov-2 might confer some level of immunity based on what is known about viral immunity to other pathogens. in previous studies of patients with sars-cov and mers-cov infection, igg peaked within months of primary infection and waned over time. [17] [18] [19] similar observations have been made with human coronaviruses, whereby immunity can confer at least limited protection. 19 to study the duration of igg antibody response to sars-cov-2, we plan to follow our cohort for the next 6 months to track titre levels. among participants who did not have a previous pcr test but who were deemed to be high risk-ie, had symptoms consistent with sars-cov-2 infection and were told by a health-care provider that they had presumed infection, lived with someone with confirmed infection, or were health-care workers themselves-37% had igg antibodies against sars-cov-2. this finding suggests that a majority of participants suspected of having covid-19 actually were not infected with sars-cov-2; however, it might also include a false negative rate of our assay (which has a 92% sensitivity) or insufficient time for participants to mount an igg antibody response. although some patients with negative antibodies might have been falsely negative, the negative predictive value for the mount sinai elisa is 99·6%, 20 and symptoms in the many patients who tested negative in this study are likely to be explained by alternative diagnoses. furthermore, in the group of pcr-positive individuals, more than 99% of them were captured due to the testing algorithm that recalled any of them who were negative or had low titres. this indicates an even higher sensitivity for the elisa antibody test, further underscoring the likelihood that many in the non-pcr-confirmed group did not have sars-cov-2. this high lights the importance of expanded pcr testing to improve diagnosis of this disease even in minimally symptomatic individuals. the 19% of participants who remained pcr positive despite self-reporting full resolution of symptoms bring to light important considerations regarding the possible duration of viral transmission, and the limited usefulness of pcr testing to ensure clearance. this positive pcr finding could represent shedding of nonviable virus, non-infectious genome fragments or viruses engulfed by immune cells, asymptomatic carriers of sars-cov-2, or ongoing infection despite full resolution of symptoms. detection of viral genome even months after resolution of infection has been shown for viruses such as measles virus. 21 so far, studies have indicated that this is not live virus, 22 and further studies are warranted to determine whether nasopharyngeal pcr positivity is related to transmission and, if so, for how long. this should have substantial implications in terms of guidance of when individuals who have recovered from sars-cov-2 infection should end selfisolation; the current cdc recommendation is at least 10 days after symptom onset with at least 72 h without fever off of antipyretics. if pcr positivity is a result of identifying non-infectious genome or non-viable virus, it might be necessary to avoid use of pcr as a definition of clearance of sars-cov-2. there are limitations to our evaluation. most participants had mild disease, and thus these data might not reflect pcr or antibody findings in a moderately or severely ill population. participants were recruited based on selfreferral in the context of a convalescent plasma donation programme; although individuals were not monetarily reimbursed for their testing, they received antibody results before widespread availability of antibody testing. addition ally, all individuals self-reported their pcr dates and symptom timing, which might have led to recall bias in terms of dates of symptom onset, resolution, and duration, and might have led us to miss asymptomatic carriers who did not inquire about testing. additionally, given recruitment via an english-language survey and our use of a single collection site, our sample findings might not be generalisable to a more diverse patient population. given online recruitment, our sample probably also included individuals younger than those most affected with symptomatic covid-19. furthermore, we did not collect rigorous data regarding symptom severity, which could potentially be related to the timeline and strength of igg antibody response to sars-cov-2. future studies are planned to help us to understand the magnitude and duration of the igg response in patients who have recovered from sars-cov-2 infection, and what antibody titre might be necessary to protect individuals from reinfection. we also hope to better understand which, if any, patients do not mount an igg immune response. finally, the clinical significance of prolonged positive sars-cov-2 nasopharyngeal pcr in the absence of symptoms requires further clarification. duration of nasopharyngeal sars-cov-2 pcr detection and time to mount igg antibody response have important implications for the spread of this virus and risk for reinfection among individuals. in our sample, we found that 19% of people continue to have nasopharyngeal pcr positivity 2 or more weeks after symptom resolution, and that it takes 3 or more weeks to mount an igg antibody response believed to be potentially protective against future infection. reassuringly, we found that almost all participants with confirmed sars-cov-2 infection in our study mounted an igg immune response to this disease. taken together, these findings will be pivotal in understanding disease activity of sars-cov-2 moving forward. mount sinai has licensed serological assays to commercial entities and has filed for patent protection for serological assays. time kinetics of viral clearance and resolution of symptoms in novel coronavirus infection positive rt-pcr test results in patients recovered from covid-19 profile of rt-pcr for sars-cov-2: a preliminary study from 56 covid-19 patients duration of rt-pcr positivity in severe acute respiratory syndrome neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications effectiveness of convalescent plasma therapy in severe covid-19 patients deployment of convalescent plasma for the prevention and treatment of covid-19 treatment of 5 critically ill patients with covid-19 with convalescent plasma a serological assay to detect sars-cov-2 seroconversion in humans sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup comparison of sars-cov-2 detection from nasopharyngeal swab samples by the roche cobas 6800 sars-cov-2 test and a laboratory-developed realtime rt-pcr test detection of sars-cov-2 by use of the cepheid xpert xpress sars-cov-2 and roche cobas sars-cov-2 assays test agreement between roche cobas 6800 and cepheid genexpert xpress sars-cov-2 assays at high cycle threshold ranges comparison of two highthroughput reverse transcription-polymerase chain reaction systems for the detection of severe acute respiratory syndrome coronavirus 2 accelerated emergency use authorization (eua) summary covid-19 elisa igg antibody test sars-cov-2 infection induces robust, neutralizing antibody responses that are stable for at least three months disappearance of antibodies to sars-associated coronavirus after recovery middle east respiratory syndrome coronavirus infection dynamics and antibody responses among clinically diverse patients, saudi arabia a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease eua authorized serology test performance prolonged persistence of measles virus rna is characteristic of primary infection dynamics us centers for disease control and prevention. duration of isolation and precautions for adults with covid-19 work on sars-cov-2 immunity in the krammer laboratory (icahn school of medicine at mount sinai, new york, ny, usa) is partly supported by the us national institute of allergy and infectious diseases centers of excellence for influenza research and surveillance contract hhsn272201400008c (fk), collaborative influenza vaccine innovation centers contract 75n93019c00051 (fk), and the generous support of the jpb foundation, the open philanthropy project (number 2020-215611), and other philanthropic donations.we thank all the patients who generously came in for testing and plasma donation. we thank kim stone and kim muellers (icahn school of medicine at mount sinai) for their work on data analysis. we also thank the medical students who helped to facilitate this project. key: cord-274506-fzcuu4ma authors: jo, seri; kim, hyojin; kim, suwon; shin, dong hae; kim, mi‐sun title: characteristics of flavonoids as potent mers‐cov 3c‐like protease inhibitors date: 2019-09-12 journal: chem biol drug des doi: 10.1111/cbdd.13604 sha: doc_id: 274506 cord_uid: fzcuu4ma middle east respiratory syndrome‐coronavirus (mers‐cov) is a zoonotic virus transmitted between animals and human beings. it causes mers with high mortality rate. however, no vaccine or specific treatment is currently available. since antiviral activity of some flavonoids is known, we applied a flavonoid library to probe inhibitory compounds against mers‐cov 3c‐like protease (3clpro). herbacetin, isobavachalcone, quercetin 3‐β‐d‐glucoside and helichrysetin were found to block the enzymatic activity of mers‐cov 3clpro. the binding of the four flavonoids was also confirmed independently using a tryptophan‐based fluorescence method. the systematic comparison of the binding affinity of flavonoids made it possible to infer their scaffolds and functional groups required to bind with mers‐cov 3clpro. an induced‐fit docking analysis revealed that s1 and s2 sites play a role in interaction with flavonoids. the experimental and computational study showed that flavonol and chalcone are favourite scaffolds to bind with the catalytic site of mers‐cov 3clpro. it was also deduced that some flavonoid derivatives with hydrophobic or carbohydrate attached to their core structures have a good inhibitory effect. therefore, we suggest that flavonoids with these characteristics can be used as templates to develop potent mers‐cov 3clpro inhibitors. coronaviruses (covs) are a positive sense, single-stranded rna virus coated with viral particles (fehr & perlman, 2015) . together with arteriviridae and roniviridae, covs belong to the coronaviridae family in the order nidovirales. these covs can infect a wide variety of hosts, including avian, swine and humans. human coronaviruses (hcovs) represent a major group of covs associated with various respiratory diseases from common cold to serious pneumonia and bronchitis (mesel-lemoine et al., 2012) . today, hcovs are recognized as one of the most rapidly evolving viruses originated from their characteristic high genomic nucleotide substitution rates and recombination. in human, their infection is known to cause approximately one-third of common cold. severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) covs are highly pathogenic and caused a fatal first major outbreak in 2003 and 2012 , respectively (de wit, doremalen, falzarano, & munster, 2016 . sars-and mers-covs genomes contain two open reading frames orf1a and orf1b translated to two respective viral polyproteins pp1a and pp1ab by host ribosomes. orf1a encodes two cysteine proteases, a papain-like protease (plpro) and a 3c-like protease (3clpro). while plpro cuts the first three cleavage sites of its polyprotein, 3clpro is responsible for cleavage of the remaining eleven locations resulting in release of a total of 16 non-structural proteins (nsp) in both sars-and mers-covs. the homodimeric form of 3clpro is active in the presence of substrates. the crystal structure of 3clpro showed that each monomer is composed of three structural domains: domains i and ii form a chymotrypsinlike architecture with catalytic cysteine and are connected to a third c-terminal domain via a long loop (neddle, lountos, & waugh, 2015) . in the proteolytic site, all 3clpros prefer glutamine at p1 position and leucine, basic residues, small hydrophobic residues at p2, p3 and p4 positions, respectively (chuck, chow, wan, & wong, 2011) . at p1′ and p2′ positions, small residues are required. since the autocleavage process is essential for viral propagation, 3clpro is a good drug target for anti-coronaviral infection. in this study, we used a proteolytic method to probe mers-cov 3clpro inhibitory compounds with a synthetic peptide labelled with the edans-dabcyl fret (fluorescence resonance energy transfer) pair (liu et al., 2005) . since emission wavelengths of edans are widely overlapped with absorbance wavelengths of dabcyl, the energy emitted from edans will be quenched by dabcyl in a close proximity (10-100 å). therefore, an increment of fluorescence can be a sign to judge whether a substrate is cleaved or not in this design. hence from the fluorescence intensity change, the proteolytic activity of protease could be detected. with a synthetic peptide with the fret pair, a flavonoid library was screened to search mers-cov 3clpro inhibitory compounds. recent studies showed that some flavonoids have antiviral activity in some viruses (frabasile et al., 2017; jucá et al.,2018; kiat et al., 2006; yang, lin, zhou, liu, & zhu, 2017; zakaryan, arabyan, oo, & zandi, 2017) . therefore, we assayed flavonoids and tried to induce their structural property crucial to bind with mers-cov 3clpro. the coding sequence of mers-cov nsp5, a 3c-like protease (ncbi ref. seq. nc_019843.3) was synthesized chemically by bioneer and cloned into a bacteriophage t7-based expression vector. the plasmid dna was transformed into e. coli bl21 (de3) for protein expression. e. coli bl21 (de3) cells were grown on luria-bertani (lb) agar plates containing 150 μg/ml ampicillin. several colonies were picked and grown in capped test-tubes with 10 ml lb broth containing 150 μg/ml ampicillin. a cell stock composed of 0.85 ml culture and 0.15 ml glycerol was prepared and frozen at 193 k for use in a large culture. the frozen cell stock was grown in 5 ml lb medium and diluted into 2,000 ml fresh lb medium. the culture was incubated at 310 k with shaking until an od 600 of 0.6-0.8 was reached. at this point, expression of mers-cov 3clpro was induced using isopropyl-β-d-1-thiogalactopyranoside (iptg) at a final concentration of 1 mm. the culture was further grown at 310 k for 3 hr in a shaking incubator. cells were harvested by centrifugation at 7,650 g (6,500 rev min −1 ) for 10 min in a high-speed refrigerated centrifuge at 277 k. the cultured cell paste was resuspended in 25 ml of a buffer consisting of 50 mm tris-hcl ph 8.0, 100 mm nacl, 10 mm imidazole, 1 mm phenylmethylsulfonyl fluoride (pmsf) and 10 μg/ml dnase i. the cell suspension was disrupted using an ultrasonic cell disruptor (digital sonifier 450; branson). cell debris was pelleted by centrifugation at 24,900 g (15,000 rev min −1 ) for 30 min in a highspeed refrigerated ultra-centrifuge at 277 k. the protein was purified by affinity chromatography using a 5 ml hi-trap q column (ge healthcare) followed by a 5 ml hi-trap blue column (ge healthcare). the custom-synthesized fluorogenic substrate, dabcyl-ktsavlqsgfrkme-edans (anygen), was used as a substrate for the proteolytic assay using mers-cov 3clpro (kuo, chi, hsu, & liang, 2004) . this substrate contains the nsp4/nsp5 cleavage sequence, gvlq↓sg (wua et al., 2015) , and works as a generic peptide substrate for many coronavirus including mers-cov 3clpro. the peptide was dissolved in distilled water and incubated with each protease. a spectramax i3x multi-mode microplate reader (molecular devices) was used to measure spectral-based fluorescence. the proteolytic activity was determined at 310 k by following the increase in fluorescence (λ excitation = 340 nm, λ emission = 490 nm, bandwidths = 9, 15 nm, respectively) of edans upon peptide hydrolysis as a function of time. assays were conducted in black, 96-well plates (nunc) in 350 μl assay buffers containing protease and substrate as follows: for the mers-cov 3clpro assay, 1.84 μl of 0.19 mm protease containing 20 mm tris ph 8.0 was incubated with 8.75 μl of 0.1 mm substrate at 310 k for 2 hr before measuring relative fluorescence unit (rfu). before the assay, the emission spectra of 40 flavonoids were surveyed after illuminating at 340 nm to avoid the overlapping with the emission spectrum of edans. every compound was suitable to be tested. the final concentration of the protease, peptide and chemical used at the assay was 1, 2.5 and 20 μm each. at first, mers-cov 3clpro and chemical were mixed and preincubated at room temperature for 1 hr. the reaction was initiated by the addition of the substrate, and each well was incubated at 310 k for 2 hr. after 2 hr, we measured the fluorescence of the mixture on the black 96-well plate using the end-point mode of spectramax i3x where the excitation wavelength was fixed to 340 nm and the emission wavelength was set to 490 nm using 9, 15 nm bandwidth, respectively. all reactions were carried out in triplicate. among the first forty flavonoids (table s1 ), four of them were picked up to further assay at a concentration range of 2 μm-320 μm. ic50 value which is the value causing 50% inhibition of catalytic activity of mers-cov 3clpro was calculated by non-linear regression analysis using graphpad prism 7.03 (graphpad software). the proteolytic assay using mers-cov 3clpro in the presence of triton x-100 has been performed to differentiate artificial inhibitory activity of chemicals through non-specific binding with proteases by forming aggregate or complexation. the concentration used in this study was 0.01%. to confirm the feasibility of the assay method independently, the fluorescence spectra from tryptophans of mers-cov 3clpro with candidate inhibitors were investigated (lin, lan, guan, sheng, & zhang, 2009 ). the fluorescence measurements were recorded with a spectramax i3x multi-mode microplate reader (molecular devices) at excitation and emission wavelengths of 295 nm and 300-500 nm, respectively. the optimal excitation and emission wavelengths were determined by softmax pro. five tryptophans of mers-cov 3clpro showed a strong fluorescence emission with a peak at 340 nm at the excitation wavelength of 295 nm. in contrast, the flavonoids were almost non-fluorescent under the same experiment condition. each 40 μm chemical was incubated with 1 μm mers-cov 3clpro for 1 hr, and the fluorescence intensity of mers-cov 3clpro was measured. all the docking and scoring calculations were performed using the schrödinger suite of software (maestro, version 11.5.011). the compounds were extracted from the pubchem database in sdf format and were combined in one file. the file was then imported into maestro and prepared for docking using ligprep. the atomic co-ordinates of the crystal structure of mers-cov 3clpro (5wkj) were retrieved from the protein data bank and prepared by removing all solvent and adding hydrogens and minimal minimization in the presence of bound ligand using protein preparation wizard. ionizer was used to generate ionized state of all compounds at target ph 7.0 ± 2.0. this prepared low-energy conformers of the ligand were taken as the input for docking analysis. grids for molecular docking were built using receptor grid generation. compounds were docked using ligand docking mode with postdocking minimization including 5 poses per ligand. the docked poses were then refined using an xp (extra precision) option with the threshold value for rejecting minimized pose of 0.5 kcal/mol. the energies were calculated using the opls3 force field. the induced-fit docking protocol (sherman, day, jacobson, friesner, & farid, 2006) was run from the graphical user interface accessible within maestro 11.5.011. receptor sampling and refinement were performed on residues within 5.0 å of each ligand for each of the 20 ligand-protein complexes. with prime (jacobson et al., 2004) , a side-chain sampling and prediction module, the side-chains, as well as the backbone of the target protein, were energy minimized. a total of 20 induced-fit receptor conformations were generated for each of the eight test ligands. re-docking the test ligands into their respective 20 structures within 30.0 kcal/mol of their lowest energy structure. finally, the ligand poses were scored using a combination of prime and glidescore scoring functions . the amount of cell harvested for purification of mers-cov 3clpro was 3.01 g per 2,000 ml of e. coli culture. the protein was purified by ion chromatography using a 5 ml hi-trap q column (ge healthcare). the column was equilibrated with a buffer consisting of 20 mm bis-tris ph 7.0, and the pooled fractions were loaded. the column was eluted using a linear nacl gradient to 1.0 m nacl, and the protein was eluted at 0.16 m nacl. the pooled fractions were loaded on a 5 ml hi-trap blue column (ge healthcare) equilibrated with a buffer consisting of 10 mm sodium phosphate ph 7.0. the target protein was detected in unbound fractions. sds-page showed one band around 33(33,737.77da) kda, corresponding to the molecular weight of mers-cov 3clpro. the protein was concentrated to 31.14 mg/ml for protease assays in a buffer consisting of 10 mm sodium phosphate ph 7.0. the purified mers-cov 3clpro was different from the previous method (kumar et al., 2016) where the native form was obtained after removing of a his 6 -tag wih factor-xa. in our experiment, the enzyme activity was 10-fold decreased when the his 6 -tag protein was used (data not shown). that is due to the location of the his 6 -tag connected to the n-terminus of mers-cov 3clpro. the published crystal structure showed that the active site pocket might be easily hindered by the flexible n-terminal his 6 -tag. a flavonoid library consisting of ten different scaffolds was also built (figure 1 ). it contains three isoflavones, one isoflavane, six flavones, nine flavonols, four flavanols, five flavanones, one flavanonol, one prenylflavonoid, eight chalcones and two unclassified flavonoids (table s1 ). we applied the library to assay mers-cov 3clpro. since flavonoids are known to aggregate through a complexation and thus non-specifically inhibit various proteases, the assay in the presence of triton x-100 was performed. before assay, we tested the influence of triton x-100 on the catalytic activity of mers-cov 3clpro. as shown in the figure s1 , only a slight increment of the catalytic activity was observed even up to 0.1% triton x-100. therefore, we performed the experiment at the concentration of 0.01% triton x-100 where no significant interference was detected. using forty flavonoids, an inhibitory effect of each compound at 20 μm was tested. among them, herbacetin (3,4′,5,7,8-pentahydroxyflavone), isobavachalcone (2′,4,4′-trihydroxy-3′-(3-methyl-2-butenyl)chalcone), quercetin 3-β-d-glucoside (3,3′,4′,5,7-pentahydroxyflavone 3-β-d-glucoside) and helichrysetin (4,2′,4′-trihydroxy-6′methoxychalcone) were found to have prominent inhibitory activity (figure 2 ). the four compounds showed the severely reduced fluorescent intensity and thus represented their mers-cov 3clpro inhibitory activity. the experimental data were plotted as log inhibitor concentration versus per cent fluorescence inhibition (figure 2) . ic50 values obtained from the dose-dependent inhibitory curves of herbacetin, isobavachalcone, quercetin 3-β-d-glucoside and helichrysetin are 40.59, 35.85, 37.03 and 67.04 μm, respectively. in order to confirm the inhibitory activity of the flavonoids independently, a general tryptophan-based assay method was employed. tryptophan was well-known to emit its own fluorescence. therefore, if tryptophan is positioned adequately in proteins, the change in fluorescence intensity can reflect the binding state of chemicals and be used to judge interaction between proteins and chemicals. mers-cov 3clpro contains five tryptophan residues and thus displays a high fluorescence peak at 340 nm at the tryptophan excitation wavelength of 295 nm. we monitored the change in the fluorescence intensities of mers-cov 3clpro depending on the presence or absence of four chemicals. since each compound in the flavonoid library was almost non-fluorescent under the experiment condition, a change in fluorescence intensity reflects interactions between mers-cov 3clpro and chemicals. only the four inhibitory compounds obviously reduced the fluorescence intensity of mers-cov 3clpro (figure 3) . the decreased emission intensity confirmed the complex formation between mers-cov 3clpro and inhibitory compounds. in order to deduce the binding modes of the inhibitory flavonoids, an in-depth theoretical investigation through an induced-fit docking study was carried out. the interactions between mers-cov 3clpro and the inhibitory flavonoids were analysed to predict their binding affinities. top ranked structures (according to the glide scores) from the induced-fit docking results for herbacetin (−10.246), isobavachalcone (−9.364), quercetin 3-β-d-glucoside (−9.751) and helichrysetin (−9.953) were selected and hypothesized to be biological complexes. in order to compare binding affinities to their closest homologues ( figure s2 figure s3 . it is very obvious that the s1 site and hydrophobic s2 site of mers-cov 3clpro play a key role in predicted complexes. flavonoids are natural compounds with multiple pharmacological characteristics such as antioxidant, anti-inflammatory, analgesic, anti-carcinogenic, antibacterial infection, antifungal and antiviral properties (frabasile et al., 2017) . naringenin has therapeutic effects on various neurological, cardiovascular, gastrointestinal, rheumatological, metabolic and malignant disorders (rani et al., 2016) . it also represents antiviral function (zakaryan et al., 2017) . fisetin is a commercially available nutraceutical with anti-inflammatory, antioxidant, anti-tumorigenic, anti-invasive, anti-angiogenic, anti-diabetic, neuroprotective and cardioprotective effects (pal, pearlman, & afaq, 2016) . interestingly, fisetin has an anti-noroviral activity by inducing cytokines (seo & choi, 2017) . although some of flavonoids show an antiviral effect, a molecular mechanism of their antiviral activity was rarely known. in this study, we assayed the inhibitory activity of various flavonoids against mers-cov 3clpro. for the trial, a flavonoid library composed of nine different scaffolds plus one undefined group was constructed. they are classified based on a c6-c3-c6 skeleton and differ in the overall hydroxylation patterns and in the saturation of the heteroatomic ring c together with the position of the attached aromatic ring b (at the positions c-2 or c-3 of ring c) (jucá et al., 2018) . the flavonoid library was tested, and thus, a systematic analysis was possible. among the ten groups, isoflavones, isoflavane, flavanols and flavanones did not show any inhibitory activity over mers-cov 3clpro. the other groups contains some flavonoids displaying moderate inhibitory activity. however, four flavonoids, herbacetin, isobavachalcone, quercetin 3-β-d-glucoside and helichrysetin exhibited prominent inhibitory activity. the immediate inference of the primary scaffold required for binding with mers-cov 3clpro was as follows: first, the orientation of the aromatic ring b at the position c-2 of ring c is essential as shown in isoflavones and isoflavane. second, the saturated heteroatomic ring c is not preferred as shown in flavanols and flavanones. third, chalcone and flavonol scaffolds show a promising binding property. the more detailed structural comparison also provided valuable structural information required for the binding affinity of each flavonoid. helichrysetin is a chalcone derivative (van puyvelde et al., 1989) . therefore, we compared its inhibitory activity with a chalcone, cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) which is the structurally identical homologue except one hydroxyl group at the 4-position of the benzyl moiety of chalcone. the inhibitory activity of cardamonin at the concentration of 40 μm was lower than that of helichrysetin ( figure 4 ). it implies that the 4-hydroxyl group of helichrysetin is functionally important to bind with mers-cov 3clpro. the structural comparison of cardamonin with isobavachalcone also indicated the modification effect of acetophenone ring of chalcone: the hydrophobic modification at the 3′-position of the acetophenone ring moiety of isobavachalcone improves its binding affinity to mers-cov 3clpro. intriguingly, the docking study shows that the 4-hydroxyl group of helichrysetin forms a hydrogen bond with the hydroxyl group of tyr54 of mers-cov 3clpro. since tyr54 is located deep inside of the hydrophobic s2 site, helichrysetin is inserted into deeper than cardamonin ( figure s3a ). as a result, helichrysetin seems to have a better affinity and thus represents a better inhibitory activity. in the flavonoid library, there are quite similar homologues of herbacetin, isobavachalcone and quercetin 3-β-d-glucoside. they are kaempferol (3,4′,5,7-tetrahydroxyflavone), 2,2′,4′-trihydroxychalcone and quercitrin (3,3′,4′,5,7-pentahydroxyflavone-3-l-rhamnoside), respectively ( figure s2 ). their mers-cov 3clpro inhibitory activity is lower than their corresponding compounds at 40 μm. herbacetin is a kaempferol derivative where one more hydroxide is attached at 8-position of kaempferol. the docking study indicates kaempferol derivatives occupy the s1 and s2 sites of mers-cov 3clpro ( figure s3b ). the hydroxyl group at 7-position looks important to bind at the s1 binding site. a f i g u r e 4 comparison of inhibitory activity between homologue flavonoids. the two homologue flavonoids are compared side by side. each of two bars represents the effect of two homologue inhibitory compounds at 40 μm against mers-cov 3clpro compared to the control. each bar is expressed as the mean ± standard error of the mean (n = 3). rfu = relative fluorescence units bit better inhibitory activity of herbacetin indicates the hydroxide at 8-position is favourable for its binding affinity to mers-cov 3clpro. in the predicted complex, the hydroxyl group at 8-position of herbacetin induces a hydrogen bond with his41 at the s1 site. the improved activity of isobavachalcone compared with 2,2′,4′-trihydroxychalcone again points out the importance of its hydrophobic substituent. coincided with the experimental result, the prenyl moiety of isobavachalcone makes hydrophobic interactions with met25 and leu49 at the hydrophobic s2 site ( figure s3c) . quercetin 3-β-d-glucoside is a homologue of quercitrin where rhamnoside is substituted for glucoside. the better inhibitory activity of quercetin 3-β-d-glucoside means that hydroxymethyl is preferred to hydroxide in this position of the glucoside to interact with mers-cov 3clpro. the docking study represents that the hydroxymethyl group of the glucoside moiety makes a hydrogen bond with glu169 and thus contributes its tighter binding to the s1 site than the rhamnoside moiety of quercitrin ( figure s3d ). helichrysetin and isobavachalcone inhibiting the activity of mers-cov 3clpro are chalcone derivatives. chalcone has immunomodulation, antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, anticancer and anti-diabetic activities (yadav, prasad, sung, & aggarwal, 2011) . recently, the non-competitive inhibition of cardamonin against the dengue virus protease has been reported (kiat et al., 2006) . as discussed above, cardamonin is a strong homologue of helichrysetin. the analysis of the crystal structures of three viral proteases, mers-cov 3clpro, dengue virus ns2b/ns3 protease and norovirus 3clpro were performed, and their active sites were compared ( figure s4 ) (erbel et al., 2006; nakamura et al., 2005) . despite their sequential divergence, their overall architecture resembles each other. furthermore, the spatial positions of their catalytic residues were also highly conserved. the observation implies that chalcone and flavonol can be used as templates to develop a potential antiviral agent by targeting these viral proteases. in this study, we showed that the antiviral effects of some flavonoids may be directly from their inhibition of main viral proteases and thus nullify a process of virus peptides. therefore, one of antiviral mechanisms of flavonoids may be originated from their direct binding capability to viral proteases. consequently, flavonoid derivatives can be used as a template to design not only for broad-spectrum but also for virus-specific antiviral agents. a further study is going on to develop better inhibitory lead compounds derived from this study. we built a flavonoid library to systematically search mers-cov 3clpro inhibitory compounds with a fret method. we found four flavonoids effectively reducing the proteolytic activity of mers-cov 3clpro. the binding of the flavonoids was independently proven by a tryptophanbased fluorescence method. the assay in the presence of triton x-100 also eliminated the chance of the false-positive result from the aggregation of flavonoids. the analysis of the four compounds with their homologs using an induced-fit docking study provided an insight of flavonoid scaffolds required to bind with mers-cov 3clpro. the prominent activity of helichrysetin and isobavachalcone indicates that the flexibility of the chalcone scaffold is good to bind with mers-cov 3clpro. in contrast, the comparable activity of flavones with a rigid γ-pyrone ring indicates that their spatial rearrangement together with various functional groups may be an alternative strategy to interact with mers-cov 3clpro. therefore, this study suggests that an antiviral effect of some flavonoids is directly from their structural characteristics binding to viral proteases. profiling of substrate specificities of 3c-like proteases from group 1, 2a, 2b, and 3 coronaviruses sars and mers: recent insights into emerging coronaviruses structural basis for the activation of flaviviral ns3 proteases from dengue and west nile virus coronaviruses: an overview of their replication and pathogenesis the citrus flavanone naringenin impairs dengue virus replication in human cells extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes a hierarchical approach to allatom protein loop prediction flavonoids: biological activities and therapeutic potential inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, boesenbergia rotunda (l.), towards dengue-2 virus ns3 protease identification, synthesis and evaluation of sars-cov and mers-cov 3c-like protease inhibitors characterization of sars main protease and inhibitor assay using a fluorogenic substrate spectroscopic investigation of interaction between mangiferin and bovine serum albumin screening of drugs by fret analysis identifies inhibitors of sars-cov 3cl protease a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes a norovirus protease structure provides insights into active and substrate binding site integrity structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity fisetin and its role in chronic diseases pharmacological properties and therapeutic potential of naringenin: a citrus flavonoid of pharmaceutical promise inhibitory mechanism of five natural flavonoids against murine norovirus novel procedure for modeling ligand/receptor induced fit effects isolation of flavonoids and a chalcone from helichrysum odoratissimum and synthesis of helichrysetin prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus the role of chalcones in suppression of nf-κb-mediated inflammation and cancer activity of compounds from taxillus sutchuenensis as inhibitors of hcv ns3 serine protease flavonoids: promising natural compounds against viral infections the authors declare no conflict of interest. the data that support the findings of this study are available from the corresponding author upon reasonable request. https://orcid.org/0000-0002-2205-1453mi-sun kim https://orcid.org/0000-0002-4092-4203 key: cord-025119-201ac32t authors: salman, saad; shah, fahad h; idrees, jawaria; idrees, fariha; velagala, shreya; ali, johar; khan, abid a title: virtual screening of immunomodulatory medicinal compounds as promising anti-sars-cov-2 inhibitors date: 2020-05-21 journal: nan doi: 10.2217/fvl-2020-0079 sha: doc_id: 25119 cord_uid: 201ac32t aim: severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a pernicious viral disease, causes acute respiratory distress responsible for mortality and morbidity worldwide. to screen different immunomodulatory medicinal compounds to unravel their interaction with sars-cov-2 viral proteins. materials & methods: a library of immunomodulatory medicinal compounds with antiviral capability were analyzed against sars proteases, spike protein and nonstructural proteins (nsp-9, 15) using autodock vina. results: out of more than 300 medicinal compounds, only six compounds: arzanol, ferulic acid, genistein, resveratrol, rosmanol and thymohydroquinone showed significant interaction with the sars viral proteins by forming hydrogen bonds with the active site residues with low binding energy. further admet (absorption, distribution, metabolism, excretion and toxicity) analysis showed good pharmacokinetic properties and low acute toxicity of these compounds. conclusion: the current study provides convincing evidence that these medicinal compounds exert antiviral activity against the sars-cov-2 virus and could be further exploited for the treatment of this disease. severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a novel pathogen from the class of coronaviruses, is taking over the world at an unprecedented rate. it is one of the most debilitating and deadly viral respiratory diseases, and has raised many concerns among healthcare professionals and the general public. the dilemma is the severity profile, enhanced surged capacity of diagnostic and laboratory tools, the spectrum of illness and that there is no specific therapeutic intervention, while the lack of sufficient understanding of viral pathogenesis makes it even more enigmatic [1, 2] . recent studies are currently underway to decipher the molecular mechanism adopted by these viruses to understand the nature of the disease and to identify potential therapeutic targets for vaccine and drug development. coronaviruses, by taxonomical hierarchy, belong to order nidovirales and family coronaviridae. the family is further genetically classified into four different genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronvirus. the first two genera (alpha and beta-) of coronaviridae cause diseases in mammals, whereas the other two affect birds. both sars-cov-1 (middle east respiratory syndrome) and sars-cov-2 (novel coronavirus19) belong to genus betacoronavirus that are highly contagious viruses and notorious for causing a multitude of diseases with neurological, respiratory, enteric and hepatic manifestations [3] [4] [5] . they are assumed to be transmitted from bats to different organisms, which ultimately lead to transmission to humans. sars-cov-2, previously known as novel coronaviruses , are enveloped positive-sense singlestranded rna viruses with a genome size of 27-30 kb and are equipped with 5 -cap structure and 3 poly-a tails that act as typical messenger rna upon transduction [6, 7] . the genomic organization of sars-cov-2 is comprised of 14 annotated open-reading frames (orfs) translating 27 proteins. at the 5 -terminus, lies orf1a and orf1b, which encode for polyproteins that after proteolytic processing produce 16 nonstructural proteins (nsp), involved in direct rna replication and transcription. the structural region is congregated adjacent to the 3 -end and encodes for four structural proteins (spike, envelope, membrane and nucleocapsid) and other accessory proteins through ribosomal frameshifting. the function of sars spike glycoprotein is to recognize angiotensin converting enzyme-2 receptor on human epithelial cells to attach and transduce its viral rna, to commence their proliferation [8] , whereas membrane and envelope proteins are responsible for viral assembly and packaging. nucleocapsid protein encapsulates the viral genetic material inside the virion [9] . among nsps, nsp-5 protease [10] and peptidase [11] facilitate polyprotein cleavage, nsp-9 replicase [12] encourages replication of viral rna and nsp-15 endoribonuclease, [13] (a subunit of sars-cov-2 rna-dependent rna polymerase) catalyzes viral rna replication. the role of other nsps is still elusive. all these protein culprits together cause serious respiratory distress sometimes leading to death. inhibiting some of these viral proteins might impede viral replication and could be considered invaluable therapeutic targets for this disease. to recognize potential inhibitors of these viral proteins, initial screening of various pharmacological agents is essential. among these agents, medicinal compounds are promising drug candidates for sars-cov-2, as they are known for their antiviral and immunomodulatory activity since medieval times and some of them are well studied [14] [15] [16] . other abilities of these compounds include immune response amelioration, rejuvenation of damaged tissues [16, 17] and reduction of viral load [15, 18] . here, we analyzed different medicinal compounds using a virtual screening method to obtain promising inhibitors for these viral proteins that could be further utilized for sars-cov-2 treatment. in this study, essential sars-cov-2 proteins facilitating viral-host interaction, polyprotein processing and vital replication proteins were selected from the rcsb protein databank. these proteins are sars coronavirus peptidase (2gtb) [11] , sars-cov-2 protease (6lu7) [10] , spike glycoprotein (6vsb) [8] , nsp-9 replicase protein (6w4b) [12] and nsp-15 endoribonuclease (6vww) [13] . modrefiner was used for structure refinement and energy minimization of these protein receptors [19] . the refined protein structures were used to predict their active site residues using metapocket 2.0 [20] and subsequently exploited for docking analysis. medicinal compounds, known for antiviral and the immunomodulatory activity [14] [15] [16] 18] , were selected as ligands and prepared with the prodrg server [21] prior to docking. the entire docking study was facilitated by autodock vina equipped with raccoon2 (a virtual screening plugin), and the ligands were focused at the predicted active site of the viral protein receptors to perform site-specific docking to procure potential inhibitors. the successful ligands obtained from screening analysis were further investigated for admet (absorption, distribution, metabolism, excretion and toxicity) properties facilitated by gusar [22] and swissadme database [23] . molecular docking is a computer-assisted drug design and development method utilized to predict the interaction and binding mode of different compounds with a target protein. these compounds (ligands) when focused at the active site of a particular protein (receptor) establish different types of chemical bonds with their active residues. these chemical bonds include hydrogen, vanderwaal, salt bridges, π-π interaction, π-sigma bond, π-sulfur and many other hydrophobic interactions. among these bonds, hydrogen bonds play a critical role in protein-ligand interaction and lower the binding energy in order to stabilize the docked complex [24] . it is already established that when a protein active site is blocked by a chemical substrate (ligand) this abrogates its functional enzymatic activity. this approach was adopted to find novel inhibitors for sars-cov-2 by inhibiting critical proteins responsible for virus attachment and replication. more than 300 medicinal compounds with immunomodulatory and antiviral activity were added to the raccoon2 plugin of autodock vina to perform virtual screening to obtain promising inhibitors for sars-cov-2 proteins. both target proteins and compounds were prepared for docking studies by subjecting them for energy minimization and structure refinement. molecular docking was performed by exposing these compounds to the predicted active site residues of these viral proteins. the results obtained from this analysis were filtered based on compounds interaction with the active site residues, low binding energy and hydrogen and hydrophobic interaction. it was observed that, among these medicinal compounds, only six compounds showed promising interaction with the active site of sars proteins as summarized in table 1 and molecularly represented in figure 1 and supplementary figures 1, 3 , 5 and 7, respectively. the majority of these compounds exploited oxygen (o-, o=) and hydroxyl groups (-oh) to form hydrogen bonds with the sars proteins whereas carbon rings participated in establishing hydrophobic interactions. some compounds showed similar binding affinity for the active residues of these viral proteins to establish hydrogen bonds, such as in the case of peptidases (2gtb), threonine-111 and asparagine-151 were favored by arzanol, ferulic acid and thymohydroquinone while threonine-199 by genistein, and resveratrol as shown in figure 2 . mentary figure 8 ). the binding energy observed for these compounds was approximately 5-7.0 kcal/mol and they were further subjected for ligand conformational analysis, which was predicted by the ligrmsd database [25] . the prime purpose of root-mean-square deviation (rmsd) analysis is to evaluate experimentally solved ligand structure against predicted docked ligand conformations. the predicted value of the rmsd for these compounds was approximately 0.9-1.5å, which is considered successful and stable. a value beyond 2å indicates instability and aberrancy in ligand conformation and docking parameters. the principal aim of acute toxicity prediction is to determine unwanted side effects of a compound after single or multiple exposures to an organism by a known administration route (subcutaneous, oral, inhalation, intravenous or intraperitoneal). the successful compounds were further used to evaluate their acute toxicity using gusar. gusar analyses compounds based on the quantitative neighborhoods of atoms descriptors and prediction of activity spectra for substances algorithm and correlate the obtained results with symyx mdl toxicity database and further classify them on the organisation for economic co-operation and development (oecd) chemical classification manual [22] . it has been observed that these compounds elicit toxicity when the compound dose surpasses more than 500,000 mg/kg in case of the oral route, more than 7000 mg/kg for intravenous route and more than 20,000 mg/kg for subcutaneous and intraperitoneal route database as shown in table 2 . the results of the oecd chemical classification of these compounds revealed that arzanol, genistein, rosmanol and thymohydroquinone are class 4 chemicals, whereas ferulic acid and resveratrol are class 5 chemicals. to ascertain the behavior of these compounds inside an organism in terms of absorption, distribution, metabolism, and excretion (adme), it is imperative to discover their pharmacokinetic properties prior to animal and clinical studies. for this reason, the swissadme database [23] was exploited to elucidate the pharmacokinetics and drug likeness of these compounds. the lipophilicity of arzanol had a logp o/w value of 3.42 that indicates high sublingual absorption, whereas for other compounds the value remained in between 1.3 and 2.8 suggesting high oral, intestinal and central nervous system absorption. all these compounds possess high gastrointestinal absorption and watersoluble capability and ferulic acid, resveratrol and thymohydroquinone are permeable to the blood-brain barrier. out of six compounds, two of them; ferulic acid and resveratrol are cyp1a2 inhibitors, which increases the drug half-life of these compounds and also avert serious drug interactions. the drug-likeness criteria are qualified by all these compounds with no violations and possess an appreciable bioavailability score. the results are summarized in table 3 . with a growing number of infected cases with thousands of mortalities worldwide, it is imperative to discover potential therapies in order to curb this devastating situation. who approved chloroquine on an emergency basis and hydroxychloroquine based on promising in vitro and clinical results [26] . but in hindsight, these drugs are highly toxic, capable of jeopardizing patient's health by disrupting essential heart and neurological functions [27, 28] . on the other hand, hundreds of different anti-hiv protease inhibitors have been reported that are capable of terminating sars-cov-2 viral replication and rna extension. however, these viruses possess proof-reading and other coping mechanisms to attenuate the interaction, thus making these antiprotease drugs inefficient as evident 10 .2217/fvl-2020-0079 future virol. (epub ahead of print) future science group from recent clinical trials [9, 28, 29] . to address these problems, we utilized a novel approach by gathering medicinal compounds with antiviral and immunomodulatory activity to check their inhibitory interaction against critical sars-cov-2 proteins. the viral proteins include structural proteins such as spike glycoprotein that allow these viral particles to attach themselves to ace-2 receptor of the host cell, whereas nonstructural proteins (nsp-9, nsp-15) facilitate viral replication and proteases modulate the production of different proteins through proteolytic cleavage of sars-cov-2 polyproteins. this study aimed to obtain novel drug candidates that have the capability to interact with the active site of all of these viral proteins and should possess efficient pharmacokinetic profile with low toxicity to ensure safety during administration. both proteins and ligands were prepared with modrefiner and prodrg server to eradicate any bad contacts, unwanted potential energy and structural anomaly that might result in false interaction. these structurally refined molecules were used and added to raccoon2 virtual screening plugin of autodock vina to perform virtual screening of these ligands and to filter out possible drug candidates establishing hydrogen bonds, other than vanderwaal interaction, with the active site residues of these viral protein receptors. only six medicinal compounds: arzanol, ferulic acid, genistein, resveratrol, rosmanol and thymohydroquinone formed hydrogen bonds with these viral proteins. these compounds were further analyzed for ligand rmsd to evaluate their interaction stability through ligrmsd [25] . the predicted value of rmsd of these compounds was in between 0.9-1.5å, which is considered adequate and stable [30] . determining the acute toxicity and pharmacokinetic properties of these compounds was facilitated by gusar software [22] and swissadme [23] . the toxicity profile of these compounds is relatively low and require high doses to elicit toxic response. the majority of the compounds are class 4 chemicals that have mild toxic effects (piloerection and diarrhea), whereas ferulic acid and resveratrol are class 5 chemicals with low toxic effects [31] . hence, the dosage of these compounds must be calibrated to exploit its full benefits and avert adverse effects. the pharmacokinetic attributes are in favor of these compounds to be exploited as promising drug candidate for sars-cov-2 treatment. apart from their antiviral activity, these medicinal compounds rejuvenate immunological response, and prevent the onset of cytokine storm that is classical hallmark of this disease. medicinal compounds combined with standard antiviral medications, synergistically ameliorate the inhibitory action on antiviral proteins [32] , reduce toxic effects [33] promote tissue repair and alleviate patients' symptoms [16] . incorporating these compounds with temporary approved anti-sars drugs as adjuvants might develop and sustain good immunological response against this lethal infection. another probable action of these compounds is to encourage phagocytotic functions, regulate the proliferation of macrophages and neutrophils and expedite the development of adaptive immunity by promoting t-cells cytokine production, natural killer cells activity and dendritic cells stimulation, which in reality takes 4-7 days for activation [14, 16] . such intervention might be able to build immunity and ameliorate the deplorable condition of sars-cov-2-affected individuals. virtual screening of different immunomodulatory compounds successfully filtered promising drug candidates for the treatment of sars-cov-2. these compounds have low toxicity, efficient pharmacokinetic profile and an excellent binding affinity for sars-cov-2 proteins. they may be further subjected to in vitro and in vivo experimentations to determine their therapeutic efficacy to be further exploited as lead compounds for clinical trials. this study elucidated the chemical affinity and mechanism of these medicinal compounds against sars-cov-2 protein targets. the results obtained from this in silico study could be used as a vehicle by other researchers and clinicians looking for promising anti-sars therapies. • docking interaction of immunomodulatory medicinal compounds library filtered six promising medicinal compounds against severe acute respiratory syndrome coronavirus-2 (sars-cov-2) viral proteins. • these six compounds, arzanol, ferulic acid, genistein, resveratrol, rosmanol and thymohydroquinone, possess strong binding affinity for sars-cov-2 proteins by forming hydrogen bonds within active site residues of these proteins. • these compounds have low acute toxicity profile, excellent pharmacokinetic properties and show nominal deviation from the viral protein backbone, as evident from their root-mean-square deviation values. • this study suggests that these compounds are promising anti-sars-cov-2 inhibitors and can also serve as immunomodulatory agents to enhance immunogenicity. • the findings of this study might serve as a guide for clinicians, pharmaceutical experts and other researchers to further validate these insights in vitro and in large clinical settings to confirm their therapeutic efficacy in this disease. to view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/sup pl/10.2217/fvl-2020-0079 all the authors equally contributed for this study. financial & competing interests disclosure pathological findings of covid-19 associated with acute respiratory distress syndrome characteristics of covid-19 infection in beijing covid-19: gastrointestinal manifestations and potential fecal-oral transmission the neuroinvasive potential of sars-cov2 may be at least partially responsible for the respiratory failure of covid-19 patients liver injury in covid-19: management and challenges genome composition and divergence 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review of the 2019 novel coronavirus (covid-19) new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? prolonged neuropsychiatric effects following management of chloroquine intoxication with psychotropic polypharmacy a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 is it reliable to take the molecular docking top scoring position as the best solution without considering available structural data? biometrical evaluation of the performance of the revised oecd test guideline 402 for assessing acute dermal toxicity medicinal plants used by traditional medicine practitioners to boost the immune system in people living with hiv/aids in uganda synergy and antagonism in natural product extracts: when 1+ 1 does not equal 2 the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. key: cord-265128-i0d4lxko authors: gurung, arun bahadur; ali, mohammad ajmal; lee, joongku; farah, mohammad abul; al-anazi, khalid mashay title: unravelling lead antiviral phytochemicals for the inhibition of sars-cov-2 m(pro) enzyme through in silico approach date: 2020-05-22 journal: life sci doi: 10.1016/j.lfs.2020.117831 sha: doc_id: 265128 cord_uid: i0d4lxko a new sars coronavirus (sars-cov-2) belonging to the genus betacoronavirus has caused a pandemic known as covid-19. among coronaviruses, the main protease (m(pro)) is an essential drug target which, along with papain-like proteases catalyzes the processing of polyproteins translated from viral rna and recognizes specific cleavage sites. there are no human proteases with similar cleavage specificity and therefore, inhibitors are highly likely to be nontoxic. therefore, targeting the sars-cov-2 m(pro) enzyme with small molecules can block viral replication. the present study is aimed at the identification of promising lead molecules for sars-cov-2 m(pro) enzyme through virtual screening of antiviral compounds from plants. the binding affinity of selected small drug-like molecules to sars-cov-2 m(pro), sars-cov m(pro) and mers-cov m(pro) were studied using molecular docking. bonducellpin d was identified as the best lead molecule which shows higher binding affinity (−9.28 kcal/mol) as compared to the control (−8.24 kcal/mol). the molecular binding was stabilized through four hydrogen bonds with glu166 and thr190 as well as hydrophobic interactions via eight residues. the sars-cov-2 m(pro) shows identities of 96.08% and 50.65% to that of sars-cov m(pro) and mers-cov m(pro) respectively at the sequence level. at the structural level, the root mean square deviation (rmsd) between sars-cov-2 m(pro) and sars-cov m(pro) was found to be 0.517 å and 0.817 å between sars-cov-2 m(pro) and mers-cov m(pro). bonducellpin d exhibited broad-spectrum inhibition potential against sars-cov m(pro) and mers-cov m(pro) and therefore is a promising drug candidate, which needs further validations through in vitro and in vivo studies. j o u r n a l p r e -p r o o f coronaviruses (covs) are positive-sense rna enveloped viruses which derive their name from the crown-like spikes on their surface and they belong to coronaviridae family. they are classified into four main subgroups-alpha, beta, gamma, and delta depending on their genomic structure [1] . alpha-and beta coronaviruses cause respiratory infections in humans and gastroenteritis in other mammals [2, 3] . likewise, the middle east respiratory syndrome coronavirus (mers-cov) caused a disastrous pandemic in 2012 leading to 37% mortality [1] . all coronaviruses infecting humans usually known to have intermediate hosts such as bats or rodents [4] . previous outbreaks of sars-cov and mers-cov involved civet cats and dromedary camels for their direct transmission to humans [1] . a new coronavirus caused an outbreak of the pulmonary disease in wuhan (the capital of hubei province in china) in december 2019 and has since spread across different parts of the world [5, 6] . since its rna genome shows about 82% identity to that of the sars coronavirus (sars-cov), the new virus has been termed as sars-cov-2 [7] . however, both these viruses belong to the same clade of the genus betacoronavirus [5, 6] . the sars-cov-2 caused a disease known as covid-19. at the initial outbreak, cases were linked to the huanan seafood and animal market in wuhan but active human-to-human transmission caused exponential growth in the number of reported cases. the world health organization (who) confirmed the outbreak a pandemic on march 11, 2020. there have been more than 170,000 cumulative cases worldwide accounting for approximately 3.7% case-fatality rate as of march 15, 2020 [8] . due to the close similarity to sars-cov, the biochemical interactions and the pathogenesis of sars-cov-2 are highly likely to be similar [1] . the virus entry into the host cell is mainly mediated through the binding of the sars spike (s) protein to the angiotensinconverting enzyme 2 (ace-2) receptor on the cell surface [9] . among coronaviruses, the main protease (m pro , also called 3cl pro ) has emerged as the best-described drug target [10] . the j o u r n a l p r e -p r o o f 4 polyproteins that are translated from the viral rna are processed by this enzyme together with the papain-like protease(s) [11] . the m pro recognizes and acts remarkably on eleven cleavage sites typically leu-gln↓(ser,ala,gly) on the large polyprotein 1ab (replicase 1ab) of approximately 790 kda. blocking the activity of this enzyme would help in inhibiting viral replication. there are no reported human proteases with a similar cleavage specificity and therefore, inhibitors against this enzyme are less probable to be toxic [8] . the three dimensional x-ray crystal structure of this enzyme in complex with α-ketoamide inhibitor 13b (o6k) was recently solved by zhang et al. (2020) (pdb id: 6y2f) which offers an opportunity for structure-based drug design against the enzyme target. understanding the relevance of the steady rise in the number of infected and death cases in recent time from covid-19 and lack of effective therapeutic interventions such as drugs and vaccines, computer-aided drug design is an important strategy to be sought after. this rational based drug design will reduce the cost and time incurred in the drug discovery process. structure-based drug design primarily relies on molecular docking to identify lead molecules against the target proteins from chemical libraries [12, 13] . compared to the synthetic inhibitors plant based-drugs have less toxicity and much safer to use. the natural products such as traditional medicines and plant-derived compounds (phytochemicals) are the rich sources of promising antiviral drugs [14] . around 44% of the approved antiviral drugs between 1981 and 2006 were derived from natural products [15] . the plant extracts have been extensively used and screened for drug molecules to evaluate theirs in vitro antiviral activities. few examples of medicinal plants with proven antiviral activities include phyllanthus amarus schum. and thonn which blocks human immunodeficiency virus (hiv) replication both in vitro and in vivo [16] ; azadirachta indica juss. (neem) shows in vitro and in vivo inhibition properties against dengue virus type-2 (denv-2) [17] ; geranium sanguineum l. significantly inhibits the replication of herpes simplex virus type-1 and 2 (hsv-1 and hsv-2) in vitro [18] ; acacia nilotica l. possesses activity against hepatitis c virus (hcv) in vitro etc [19] . in the present study, we have screened small drug-like molecules from a dataset of phytochemicals possessing antiviral activities using drug-like filters and toxicity studies. the the information about a set of thirty-eight phytochemicals from medicinal plants with antiviral activities was retrieved through literature search [14] . the summary of the selected phytochemicals (class, plant source and antiviral activity) is provided in suppl. table 1 the phytochemicals were screened based on physicochemical properties obeying lipinski's rule of five (rof) filters [23] and further tested for in silico toxicity studies such as mutagenicity, tumorigenicity, reproductive effects and irritancy. the physicochemical properties of the phytochemicals were determined using datawarrior program version 4.6.1 (sander et al., 2015) . table 1 . the sequence percentage identity of the sars-cov-2 m pro to sars-cov m pro and mers-cov m pro was determined using a standard protein basic local alignment search (blastp) tool (https://blast.ncbi.nlm.nih.gov/blast.cgi?page=proteins). multiple sequence alignment of the three sequences were performed using clustal w algorithm [26] . pairwise structural clustering of sars-cov-2 m pro , sars-cov m pro and mers-cov m pro was analyzed using ucsf chimera tool [27] . to check the suitability of molecular docking parameters and algorithm to reproduce the native binding poses, a redocking experiment was performed using the co-crystal compound. where ∆g is the binding energy in kcal/mol, r is the universal gas constant (1.987 calk −1 mol −1 ) and t is the temperature (298.15 k) a stable complex is formed between a protein and ligand which exhibits more negative free energy of binding and low k i indicates high potency of an inhibitor [29, 30] . the hydrogen bonds and hydrophobic interactions between the compounds and the target enzymes were studied using a total of 38 bioactive phytochemicals possessing antiviral activities were selected for the study. these compounds were chosen based on the previous reports of their potent antiviral effects against various pathogenic viruses such as adenovirus, influenza virus, respiratory syncytial virus, human cytomegalovirus, herpes simplex virus, poliovirus, varicella-zoster virus etc. (suppl. table 1 ). the compound set consists of different classes of phytochemicals including active flavonoids (n=14), active organic acids (n=5), active alkaloids (n=5), active essential oils (n=3), active stilbenes (n=6) and other phytoconstituents (n=5). the three-dimensional structures the compounds were modelled and optimized. a list of these phytochemicals is enumerated in table 2 . these optimized structures were used further for virtual screening and molecular docking studies. from a total of 38 phytochemicals, 10 compounds (four active flavonoids, two active alkaloids, two active essential oils and two other phytoconstituents) were found to be orally bioactive with j o u r n a l p r e -p r o o f 8 respect to rof criterion (molecular weight (mw) ≤500, clogp (partition coefficient between noctanol and water) ≤5, number of hydrogen bond donors (hbd) ≤5 and number of hydrogen bond acceptors (hba) ≤10 [23] ) without any significant toxicity issues such as being nonmutagenic, non-tumourigenic, non-irritant and no adverse effects on reproductive health (table 3 ). these drug-like compounds were further taken for molecular docking studies. the drugattrition rate in preclinical and clinical trials is quite high due to the poor pharmacokinetic studies and therefore initial screening of these drug-like molecules can increase the chances of passing through the clinics. his163, his164, met165, pro168, asp187, gln189, thr190 and gln192) ( figure 3d ). interestingly, the residues his41 and cys145 which form catalytic dyad residues are also found interacting with the inhibitor. thus all the three lead molecules have better binding affinity to sars-cov-2 m pro compared to the standard inhibitor. (n=17) ( figure 4d ). it also shows good binding to mers-cov m pro which involves seven hydrogen bonds with cys145, ser147, cys148, gln167 and glu169 and hydrophobic interactions via residues met25, thr26, leu27, his41, phe143, leu144, gly146, his166, met168, leu170, ala171, gln192, val193, his194 and gln195 (n=15) ( figure 5d ). the binding energies and inhibition constants of the phytochemicals with the sars-cov-2 m pro enzyme were compared with that of a set of twelve fda approved antiviral drugs-a) viral sars-cov-2 and coronavirus disease 2019: what we know so far, pathogens origin and evolution of pathogenic coronaviruses fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin bat coronaviruses in china others, a pneumonia outbreak associated with a new coronavirus of probable bat origin others, a new coronavirus associated with human respiratory disease in china severe acute respiratory syndrome-related coronavirus--the species and its viruses, a statement of the coronavirus study group crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus--induced lung injury coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design molecular docking: a powerful approach for structure-based drug discovery molecular docking in modern drug discovery: principles and recent applications antiviral properties of phytochemicals natural products as sources of new drugs over the last 25 years concerted inhibitory activities of phyllanthus amarus on hiv replication in vitro and ex vivo inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type-2 replication antiherpes virus activity of extracts from the medicinal plant geranium sanguineum l antiviral activity of acacia nilotica against hepatitis c virus in liver infected cells merck molecular force field. i. basis, form, scope, parameterization, and performance of mmff94 exploring the physicochemical profile and the binding patterns of selected novel anticancer himalayan plant derived active compounds with macromolecular targets pubchem substance and compound databases lead-and drug-like compounds: the rule-of-five revolution datawarrior: an open-source program for chemistry aware data visualization and analysis molecular docking of the anticancer bioactive compound proceraside with macromolecules involved in the cell cycle and dna replication clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice ucsf chimera--a visualization system for exploratory research and analysis autodock4 and autodocktools4: automated docking with selective receptor flexibility insights into protein-ligand interactions: mechanisms, models, and methods molecular docking studies of lonchocarpus cyanescens triterpenoids as inhibitors for malaria ligplot+: multiple ligand-protein interaction diagrams for drug discovery structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design in silico study of fucoxanthin as a tumor cytotoxic agent molecular structures and antiviral activities of naturally occurring and modified cassane furanoditerpenoids and friedelane triterpenoids from caesalpinia minax antibacterial, antifungal, and antiviral activities of some flavonoids list of abbreviations ace-2 :angiotensin converting enzyme 2 blast: basic local alignment search clogp: partition coefficient between n-octanol and water hba: hydrogen bond acceptor hbd: hydrogen bond donor mers-cov: middle east respiratory syndrome coronavirus mpro: main protease mw: molecular weight pdb: protein data bank rmsd: root mean square deviation rof: rule of five sars-cov: severe acute respiratory syndrome coronavirus ligplot analysis for top three lead compounds along with the control against sars 83 å) 91 å) key: cord-019048-29wzpwvr authors: franks, teri j.; galvin, jeffrey r. title: coronavirus date: 2013-08-26 journal: viruses and the lung doi: 10.1007/978-3-642-40605-8_13 sha: doc_id: 19048 cord_uid: 29wzpwvr name of virus: coronavirus there are no synonyms. the term coronavirus is typically used in conjunction with a species designation, for example, miniopterus bat coronavirus-hku8 or human coronavirus-229e, or severe acute respiratory syndrome-related coronavirus. sars-cov is the most aggressive human coronavirus known to date, and its epidemiology is quite different from that of the fi ve non-sars human coronaviruses. between november 2002 and july 2003, sars-cov emerged, swept around the globe via routes of international air travel, and caused 8,098 sars cases in 26 countries with 774 deaths. this strained the healthcare system in the countries with infections and led to travel restrictions and signifi cant effects on the global economy (who 2004a ). on july 5, 2003, the who declared the chain of person-to-person transmission of sars-cov in the epidemic broken (who 2003c ) . since july 2003, sars infection has been documented on several occasions. three incidents were attributed to breaches in laboratory biosafety. the fourth incident involved four communityacquired cases attributed to animal or environmental exposure in three and an undetermined source of infection in the fourth (liang et al. 2004 ) . in contrast, most of the non-sars human coronaviruses have been in continuous circulation globally since their initial isolation. they emerge in winter and spring and demonstrate periodicity with epidemics occurring at 2 to 3 year intervals. they primarily cause upper respiratory tract infections that are more common in children than in adults, and they account for an estimated 15 % of adult colds and up to 35 % of upper respiratory tract infections during peak viral activity. less commonly, they are associated with lower respiratory tract disease in infants, immunocompromised patients, and the elderly (gerna et al. 2006 ; principi et al. 2010 ; van der hoek 2007 ) . in late 2012, hcov-emc was isolated in a 60-year-old male who presented with acute pneumonia, subsequently developed renal failure, and had a fatal outcome (zaki et al. 2012 ) . from discovery to mid-september 2013, hcov-emc, renamed mers-cov, (de groot 2013 ) caused 132 laboratory-confi rmed cases of severe acute pneumonia including 58 deaths. (who 2013 ) . sars-cov is an animal virus that crossed the species barrier when environmental change increased chances for the virus to enter humans and enable human-to-human transmission (antia et al. 2003 ) . supporting this, research has identifi ed a sars-cov-like virus in himalayan masked palm civets, raccoon dogs, and chinese ferret badgers sold in live-animal markets for human consumption in southern china, as well as, in humans working in the same markets indicating a route of interspecies transmission. horseshoe bats have been identifi ed as a natural reservoir of sars-cov-like viruses (guan et al. 2003 ; li et al. 2005 ; song et al. 2005 ) . sars-cov is highly contagious, and spread occurs primarily by close person-to-person contact via droplet transmission or fomite. virus is shed in respiratory secretions, feces, and urine. at room temperature it retains its infectivity for 4 days in diarrheal stool samples, for up to 6 days when dried, and for more than 7 days in respiratory specimens. the virus is readily inactivated by commonly used disinfectants (lai et al. 2005 ; rabenau et al. 2005 ). coronaviruses are the largest of all rna viruses and have a positive-sense, single-stranded rna genome of 30-32 kilobases. virions are enveloped and spherical with widely spaced clubshaped surface projections that give the virus its unique coronal fringe by negative-staining electron microscopy ( fig. 13.1 ). cryo-electron microscopy reveals an outer envelope diameter of 85 ± 5 nm with 20 nm club-shaped surface projections, an exceptionally thick, 7.8 ± 0.7 nm envelope and a loosely wound, helical nucleocapsid separated from the envelope by a 4 nm gap. certain structural proteins are common to all coronaviruses: the spike glycoprotein s, an envelope glycoprotein that mediates receptor-binding and membrane fusion; the envelope spanning glycoprotein m, which contributes to the thickness of the envelop; the envelope protein e, which has been identifi ed as a virulence factor sars-cov ; and the nucleocapsid protein n, with its function in genome encapsidation, rna synthesis and translation, and as a type i interferon antagonist ( fig. 13 .2 ). additional accessory proteins vary by species; for sars-cov, structural proteins 3a, 6, and 7 and nonstructural proteins nsp2-5 and nsp9 have been described (goldsmith et al. 2004 ; king et al. 2011 ; neuman et al. 2006 ). sars-cov is internalized through binding of the spike glycoprotein to the host cell surface receptor angiotensin-converting enzyme 2 (ace2) (wang et al. 2004 ). binding initiates conformational change in the spike that mediates fusion of the viral and host cell membranes and release of the nucleocapsid into the target cell allowing for disassembly and replication of the genome. spike-mediated cell-to-cell fusion can occur and promotes syncytium formation and viral spread (cheng et al. 2007 ) . once internalized, the specifi c mechanism by which the human immune system responds to sars-cov is not well understood, and a particular area of controversy is the role of interferon (ifn). cameron and colleagues measured plasma levels of ifn during the natural history of sars in 40 patients. they found high ifn-alpha, ifngamma, and ifn-stimulated chemokine levels, and robust antiviral ifn-stimulated gene (isg) expression was present early in the course of illness. patients entered a crisis phase starting at approximately day 8, and most patients resolved ifn responses at crisis and expressed adaptive immune genes as they recovered. in contrast, patients with poor outcomes demonstrated deviated isg and immunoglobulin gene expression levels, persistent chemokine levels, and defi cient anti-sars spike antibody production, suggesting a malfunction in the switch from innate to adaptive immunity (cameron et al. 2007 ). the mean incubation period for sars is 5 days with a range of 2-10 days. the clinical course of sars follows a typical pattern that parallels viral load. the fi rst week of illness is an infl uenza-like prodrome with fever, malaise, myalgia, headache, and rigors that coincide with increasing viral load. a decreasing viral load accompanies the second week of illness that is characterized by dry cough, dyspnea, and hypoxemia. up to 70 % of patients develop large volume watery diarrhea. clinical deterioration with rapidly progressive respiratory distress occurs in severe cases with approximately 20 % requiring intensive care. progression to respiratory failure is the most common cause of death. transmission occurs primarily in the second week (hui and chan 2010 ) . chest radiographic and ct changes occur 3-4 days after onset of illness in most patients despite the lack of respiratory signs. initial unilateral peripheral areas of ground glass and consolidation progress to multiple bilateral areas involving more than 80 % of lung characteristic of diffuse alveolar damage. in patients who survive the acute episode, traction bronchiectasis heralds the development of fi brosis and honeycomb lung (fig. 13. 3 ) (chang et al. 2005 ). sars-cov affects multiple organs but the major pathology is in the lungs. diffuse alveolar damage (dad) is the primary histologic fi nding, and the phase of dad varies based on duration of illness. cases of short duration, 10 days or less, demonstrate acute-phase dad characterized by hyaline membranes lining alveolar walls, interstitial and airspace edema, mild chronic interstitial infl ammation, and vascular congestion (fig. 13.4 ) . bronchiolar injury is evidenced by luminal collections of fi brin associated with loss of cilia, denudation of bronchiolar epithelium, and deposition of fi brin on exposed basement membranes. cases of more than 10 days duration exhibit organizing-phase dad characterized by interstitial and airspace fi broblast proliferation accompanied by repair including type ii pneumocyte hyperplasia and airway-centered squamous metaplasia. hyperplastic type ii cells show marked cytologic change, including cytomegaly, nucleomegaly, clearing of nuclear chromatin, and prominent nucleoli. alveolar spaces contain a combination anteroposterior portable chest radiograph ( center ) acquired 3 days later demonstrates consolidation of all fi ve lobes with the patient intubated. anteroposterior chest radiograph ( right ) acquired 3 months later demonstrates reticular opacities in the lung periphery. a chest ct acquired at the same time confi rms the presence of fi brosis with traction bronchiectasis and reticular opacities of macrophages and desquamated pneumocytes including multinucleated forms of both. acute bronchopneumonia is a common feature in organizing-phase dad, and fi brin thrombi may also be present. intranuclear and intracytoplasmic inclusions have been variably reported, but sars lacks a unique tissue response and cytopathic effect, making diagnosis by light microscopy alone difficult. after several weeks there can be progression of the organizing phase to the fi brotic phase, with extensive restructuring of the lung parenchyma and development of honeycomb lung (franks et al. 2003 ). there are no clinical or laboratory fi ndings that reliably diagnose sars-cov infection early or rapidly enough to inform management decisions that must be made soon after a patient enters the healthcare system in order to contain potential infection. the centers for disease control and prevention (cdc) recommends that the diagnosis of sars-cov infection and initiation of isolation and stringent infection control measures should be based on risk of exposure. in the absence of person-to-person transmission of sars-cov anywhere in the world, the diagnosis of sars-cov infection should be considered only in patients who require hospitalization for radiologically confi rmed pneumonia and who have an epidemiologic history that raises suspicion of sars-cov infection. suspicion is heightened when the patient, within 10 days of onset of illness, has a history of recent travel to mainland china, hong kong, or taiwan, or close contact with ill persons with a history of travel to these areas, or is employed in an occupation at risk for sars-cov, or is part of a cluster of cases of atypical pneumonia without an alternative diagnosis. laboratory testing for sars-cov is available, including antibody detection by enzyme immunoassay (eia) and reverse transcription polymerase chain reaction (rt-pcr). however, the positive predictive value of a diagnostic test is very low in the absence of personto-person transmission worldwide, and the cdc recommends testing be performed judiciously and in consultation with local or state health departments (cdc 2005 ). initial signs and symptoms of sars are nonspecifi c and common, which generates a wide differential diagnosis of respiratory pathogens including infl uenza virus, parainfl uenza fig. 13.4 acute-phase dad in sars patient. acute-phase dad is characterized by eosinophilic hyaline membranes plastered against alveolar walls, interstitial and airspace edema, and mild chronic interstitial infl ammation (100×, hematoxylin-eosin stain) viruses, respiratory syncytial virus, haemophilus infl uenza, mycoplasma pneumonia, chlamydia species, legionella species, coxiella burnetii, and other human coronaviruses (who 2004b ). at the time of this writing in october 2013, the world is in an interepidemic period for sars. the greatest risk of recurrence is from emergence or introduction of sars-cov from laboratories and emergence of sars-cov-like viruses from wildlife or other animal reservoirs. if sars recurs, early detection of infected individuals is essential to contain local spread of infection and prevent international dissemination. primary responsibility for risk assessment and management of sars is with national health authorities, for example, the cdc in the united states. however, in its role coordinating global and regional surveillance, the who has revised its guidelines for global surveillance and reporting of sars and has provided a framework of activities at national and international levels for risk assessment of sars (who 2004b , c ). many drugs were empirically tried during the epidemic, but no treatment has been shown to consistently improve the outcome of sars patients, and supportive medical care remains the primary therapy. the case fatality ratio for sars ranges from 0 % to more than 50 % depending on age group, with an overall estimate of 11 %. case fatality is estimated to be less than 1 % for people aged 24 years and younger, 6 % for 25-44 years, 15 % for 45-64 years, and over 50 % for people aged 65 years and older (who 2003b ). efforts to develop a sars vaccine have been ongoing since the epidemic of 2002-2003, and signifi cant advances have been made in our understanding of sars-cov. notably, the domains of the s glycoprotein that allow for viral infection have been identifi ed, ace2 has been determined to be a surface receptor for binding the s glycoprotein, and the regions of interaction between the s glycoprotein and ace2 have been mapped. all of these present targets for vaccine development. however, much of the immunology and pathogenesis of sars is incompletely understood. of particular concern is the potential for a sars vaccine to trigger immunopathogenic mechanisms which could lead to more severe disease in vaccines, as has been observed with some veterinary coronavirus vaccines. additionally, coronaviruses are notorious for their frequent mutations which further complicate development of a suitable vaccine. currently there are no licensed vaccines for use in sars (niaid 2012 ). of the six human coronaviruses recognized to date, sars-cov is the most aggressive. four of the non-sars coronaviruses mainly cause upper respiratory tract infections that are more common in children than in adults. the fi fth non-sars coronavirus, mers-cov, reportedly causes severe acute pneumonia similar to sars-cov. sars-cov has been identifi ed as the etiologic agent for sars, which caused 8,098 infections in 26 countries with 774 deaths during the 2002-2003 epidemic. initial signs and symptoms of sars are nonspecifi c and common, thereby generating a wide differential diagnosis of more commonly occurring lower respiratory tract pathogens. sars primarily targets the lungs and produces a viral pneumonia with a high mortality rate. dad is the histopathologic hallmark of sars and the phase of dad varies with the duration of illness: acutephase dad is seen in illnesses of 10 days or less, whereas organizing-phase dad is associated with illnesses greater than 10 days in duration. sars has no vaccine and no treatment. currently, the world is in an interepidemic period for sars. resurgence of sars remains a distinct possibility, as the circumstances that allowed a sars-cov-like virus to cross the species barrier from animals to humans in the live-animal markets of southern china still exist. all countries must be 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mabillard, holly; sayer, john a. title: electrolyte disturbances in sars-cov-2 infection date: 2020-07-22 journal: f1000res doi: 10.12688/f1000research.24441.2 sha: doc_id: 269289 cord_uid: 6uog10j4 the global pandemic secondary to the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is leading to unprecedented global morbidity and mortality. with a bewildering array of complications, renal involvement in various forms is common, including serum electrolyte derangements. hypokalaemia secondary to sars-cov-2 was common in a reported chinese cohort. here we review the emerging evidence on hypokalaemia and sars-cov-2 infection, the potential pathophysiological mechanisms based on early clinical and histopathological data and important clinical implications. mechanisms of hypokalaemia are multifactorial and so the electrolyte disturbance can be difficult to avoid. we provide further support to the theory of renin-angiotensin-aldosterone (ras) activation, discuss the strengths and weaknesses of implicating ras involvement and highlight the importance of calculating the transtubular potassium gradient to identify those at risk of hypokalaemia and its complications. the global pandemic secondary to the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is leading to unprecedented global morbidity and mortality. common symptoms include fever (43% of patients), cough (50%) and dyspnoea (29%) but other features such as myalgia (36%), diarrhoea (19%), anosmia and hypogeusia (10%) are also common 1 . the most frequent serious manifestation of infection is pneumonia with 15% of patients developing serious manifestations such as hypoxia, dyspnoea, extensive pulmonary involvement and acute respiratory distress syndrome (ards) 2-5 . a recent meta-analysis including 50,466 patients with sars-cov-2 infection described an ards incidence of 14.8% 6 . anecdotal data and clinical observation from patients with the world health organization named disease coronavirus disease 2019 (covid-19) has highlighted a bewildering array of presentations and pathologies in those infected with sar-cov-2 many of which, it should be stressed, are rare. these include additional respiratory complications (pulmonary fibrosis -reported in 21% of those hospitalised with sars-cov-2 9 months post-discharge in one study 3 ) 7 , cardiovascular complications (acute cardiac injury (7% 8 ), cardiomyopathy (1/3 patients 9 ), cardiac tamponade, heart failure, dysrhythmias (17% 8 ) and venous thromboembolic events (20% 10 )) 11 , neurological complications (myopathy, acute stroke (5.7% of those with severe infection 12 ), guillain-barre syndrome (0.4% hospitalised patients 11 ) and encephalopathy) 13 , acute liver and/or pancreatic injury (29% and 17% respectively in one cohort) 14 , cytokine storm syndrome, septic shock, dic, diarrhoea, kawasaki-like disease 14 and renal complications (acute tubular injury, rhabdomyolysis, proteinuria, secondary focal segmental glomerulosclerosis and possible renin-angiotensinaldosterone system activation) 15 . a few small studies have demonstrated that sars-cov-2 can affect the kidneys directly but it is unclear if this is a key mechanism in acute kidney injury seen in patients with sars-cov-2 infection. involvement of the renin-angiotensin-aldosterone (ras) pathway has been speculated as a mechanism to lead to more severe covid-19 disease and patients with severe disease were more likely to have hypertension, chronic kidney disease (ckd), diabetes mellitus and cardiovascular disease than those with milder disease. this is, however not confirmed and is preliminary data that may be the subject of bias with disregard of age as a potential confounding factor 16 . here we aim to review the evidence, potential clinical implications and possible mechanisms of electrolyte disturbances and kidney injury in patients with sars-cov-2 infection. firstly, it is important to highlight how transmission of the virus occurs in humans. the sars-cov-2 spike protein enters host cells via the angiotensin-converting enzyme 2 (ace2) receptor on the surface of pulmonary type 2 alveolar cells 16,17 . ace2 is crucial to counter-regulate ras and shares approximately 60% homology with ace. ras pathway components are co-expressed in most organs and tissues in the body and communicate via both paracrine and autocrine signalling. ace2 converts angiotensin-ii into angiotensin-(1-7), which acts on the mas receptor expressed on a variety of tissue cell lineages relevant to cardiovascular disease in addition to type 2 alveolar epithelial cells. it lowers blood pressure through vasodilation and promotion of renal sodium and water excretion and it also attenuates inflammation by producing nitric oxide 18 . meanwhile, ace converts angiotensin-i to angiotensin-ii which has directly opposing effects to signalling mediated by ace2. angiotensin-ii acts at the type 1 angiotensin receptor (at1r) to increase blood pressure by the induction of vasoconstriction and renal sodium and water reabsorption. this also creates oxidative stress which promotes inflammation and fibrosis. the balance between these two opposing pathways determines potential tissue injury, predominantly in the heart and kidneys 16, 17 . not only has the ace2 receptor found to be used for host cell entry by the sars-cov virus, but the affinity for sars-cov-2 is 10-20-fold higher 18 . during host cell entry, proteolytic cleavage of ace2 by transmembrane serine protease 2 (tmprss2) occurs which is thought to suppress ace2 expression. this in theory would lead to a reduction in angiotensin-(1-7) generation and angiotensin-ii levels would increase resulting in oxidative-stress mediated tissue damage and hypertension 16, 17 . this mechanism resulting in lung parenchymal injury has already been demonstrated in mice injected with sars-cov virus 19 . involvement of the ras pathway has therefore been speculated as a mechanism to lead to more severe covid-19 disease and patients with severe disease were more likely to have hypertension, chronic kidney disease (ckd), diabetes mellitus and cardiovascular disease than those with milder disease. this is, however not confirmed and is preliminary data that may be the subject of bias with disregard of age as a potential confounding factor 17 . hypokalaemia is known to cause muscle weakness, paraesthesia, thirst, muscle cramps and weakness, but hypokalaemia is an important complication of any disease due to it being a potentially life-threatening condition 20 . in fact, the incidence of ventricular fibrillation has been shown to be fivefold higher in patients with hypokalaemia compared to those with hyperkalaemia 21 . furthermore, inadequate management of hypokalaemia has been identified in 24% of hospitalised patients 22 , reiterating that we need to be even more vigilant with the management of this life-threatening condition during a pandemic where resources are even more scarce. what is the evidence for hypokalaemia in sars-cov-2 infection so far? the release of a pre-print retrospective chinese study initially sparked interest into hypokalaemia as a potentially prevalent biochemical disturbance in sars-cov-2 infection 23 . this was released around a similar time to concerns around ras inhibitors and sars-cov-2 where speculation that ras inhibitors may increase risk of sars-cov-2 infection and its severity due to the knowledge that ace2 is the viral binding site for host cell entry. it has been stressed by multiple organisations globally that ras inhibitors should be continued unless otherwise medially indicated as the risks of stopping these important drugs have numerous serious complications to the patient. this study claimed that hypokalaemia was prevalent amongst sars-cov-2 infected patients, affecting 108 of 175 patients (62%) that had a serum potassium of <3.5 mmol/l and only 10 patients had a serum k >4.0 mmol/l which is the value required for those with myocardial dysfunction. of the patients, 22% had severe hypokalaemia (serum potassium <3.0 mmol/l). in total, 11% of all patients and 28% of those with severe hypokalaemia displayed metabolic alkalosis (ph >7.45) compared to 4% with normokalaemia suggesting the possibility of ras activation. the study suggested that the hypokalaemia was not due to gastrointestinal (gi) loss of potassium as only a small proportion of patients had gi symptoms, there was no significant difference between serum potassium in those with or without diarrhoea and significant urinary potassium wasting was shown. a total of 20 patients had a spot urine potassium/ creatinine ratio and there were significantly higher urinary potassium/creatinine ratios in those with hypokalaemia compared with normokalaemia. the study reported that the degree of hypokalaemia correlated with severity of sars-cov-2 symptoms and they suggested that hypokalaemia can be difficult to correct as seen in two patients because the renal potassium wasting persists until clinical recovery from the virus. this study is relatively small, limited to a chinese population and did not disclose medications or diuretic use which could be a significant confounding factor here. as diuretics (especially "loop" diuretics) are used to improve oxygenation in patients with ards, some of these patients may well have been prescribed them. it was postulated that hypokalaemia in these patients is due to the effects of increased angiotensin-2 resulting from the proteolytic cleavage of ace2 from virion invasion of the host although this conclusion cannot be made without measuring components of the ras system in these patients. further studies including declaration of medications/diuretic use and measurement of components of the ras pathway are necessary to make conclusions about aetiology of hypokalaemia in sars-cov-2 infection although hypokalaemia was clearly a clinical risk in these patients that should be communicated to the medical community. a study performed in hong kong during the 2003 sars-cov epidemic compared symptoms and laboratory findings in sars-cov rt-pcr swab-positive patients to negative patients 24 . hypokalaemia was present in 41.3% of rt-pcr positive patients and was reported as a 'common laboratory finding'. it was also suggested in this paper that the hypokalaemia was due to the increased effects of angiotensin-2 but, again, the ras components were not measured in patients. in contrast, the largest sars-cov-2 case series so far (1099 patients included) did not demonstrate any major difference in serum potassium between those with mild and those with severe disease 2 . serum potassium was mostly reported as normal in this cohort. sars-cov was found in the urine in one patient but urine was not routinely tested for the virus in this cohort. a small chinese, retrospective cohort of 12 patients who were hospitalised with varying degrees of severity of sars-cov-2 infection were studied to comprehensively assess renal function, risk factors for, and incidence of early renal injury. 50% of patients had hypokalaemia and 50% also had hyponatraemia 25 . however, this study did not acknowledge if any of the patients were taking diuretics or other medications that could alter serum potassium or sodium concentration. the theory of ras activation causing hypokalaemia is popular, however it is recognised that hyperaldosteronism does not always cause hypokalaemia. this is explained by the 'renal potassium switch' mechanism ( figure 1 ), which involves the paradoxical actions of aldosterone on potassium; for example, aldosterone increases sodium reabsorption in states of low circulating volume without significantly altering potassium balance, but it also increases serum potassium excretion in high potassium states without affecting sodium balance. this paradoxical action all depends on the sodium delivery to the aldosterone-sensitive distal nephron (collecting duct) which is generated by the sodium-chloride co-symporter (ncc) in the early distal convoluted tubule. activation of this 'switch' is by phosphorylation of ncc by the wnk/spak/osr1 pathway 26, 27 and is done by aldosterone in response to low or high serum potassium i.e. ncc is maximally phosphorylated when serum potassium is <2.5 mmol/l and not at all phosphorylated when serum potassium is >5.2 mmol/l 28 . in states of decreased circulating volume, enac activity is increased due to the effects of excess aldosterone (which generates a sufficient intracellular electrochemical gradient to cause active transport of potassium from blood via na + k + -atpase which would be passively excreted into the tubular lumen by romk and bk channels in the principal cell). whether this generates an effect on potassium or not depends on the integrated activity of the entire distal nephron. in a state of low circulating volume, ncc in the early dct is 'switched on' which results in increased reabsorption of sodium and chloride in the early dct and, along with sodium reabsorption in the pct, a net increase of sodium reabsorption from the kidney occurs. as a result, sodium delivery to the aldosterone sensitive distal nephron (collecting duct) is decreased which offsets the effects of enac and potassium excretion is therefore unchanged. this results in a state of hyperaldosteronism without hypokalaemia 28 . it should be noted that, although the renal potassium 'switch' mechanism is a popular theory, it has never been proven although there are case reports of patients with normokalaemic primary hyperaldosteronism that develop hypokalaemia with a thiazide diuretic 29 . in states of hyperaldosteronism and volume depletion the renal potassium switch is 'active'. angiotensin-ii (ang-ii) stimulates kir4.1/5.1 to hyperpolarise the basolateral membrane potential and lowers intracellular chloride. this action activates wnk1 (with-no-lysine-kinase-1) and wnk4 (with-no-lysine-kinase-4) by phosphorylation which subsequently phosphorylates sps1-related proline / alanine-rich kinase (spak) and oxidative stress-responsive kinase (osr1) which, again, phosphorylates the sodium chloride co-symporter (ncc). ncc then avidly reabsorbs sodium (and chloride) back into the body in the early part of the distal convoluted tubule (dct1). this results in reduced tubular sodium delivery to the collecting duct. subsequently there is less sodium for the epithelial sodium channel (enac) to reabsorb and its action is offset. enac usually reabsorbs sodium and, via an electrochemical gradient made by sodium-potassium-atpase, potassium is normally excreted into the tubular lumen by romk (renal outer medulla potassium channel) and bk (big potassium/maxi-k channel) and eliminated from the body. when enac is less active, as a consequence of less tubular sodium delivery, less potassium is excreted. this results in a state of hyperaldosteronism with a normal serum potassium. in states of hyperaldosteronism and normal cardiovascular volume (in response to high serum potassium) the switch is 'inactive'. ncc is not phosphorylated which results in more distal sodium delivery to the collecting duct and subsequent enac activation resulting in excretion of potassium into the tubular lumen and a state of hypokalaemia 26,27 . arrows represent the direction of movement of electrolytes. red arrows reflect a reduction in movement and green arrows represent an increase in movement. clc-kb, chloride voltagegated channel kb; kir4.1/5.1, inwardly rectifying potassium channel 4.1; at1r, type 1 angiotensin receptor. other electrolyte disturbances various case reports describe the onset of syndrome of inappropriate anti-diuretic hormone secretion (siadh) in some patients with sars-cov-2 infection 30 . although siadh is frequently associated with atypical pneumonia and pneumonia is a frequent complication of sars-cov-2 infection, the literature does describe this phenomenon in the absence of respiratory symptoms, fever and any alternative explanation for siadh 31 . it is therefore prudent to consider testing for sars-cov-2 infection in cases of siadh without a clear aetiology. additional studies are required to ascertain incidence and pathogenesis of siadh in sars-cov-2 infection. one retrospective cohort study of 42 laboratory-confirmed covid-19 patients without a history of chronic kidney disease have shown a high incidence of novel acquired incomplete renal fanconi syndrome, often preceding acute kidney injury and/or resolving during the clinical recovery phase of the infection 32 . 75% of patients admitted to hospital for mild, moderate or severe respiratory failure due to sars-cov-2 infection had acute incomplete renal fanconi syndrome. proteinuria was always associated and often with severe renal phosphate leak (in addition to frequent hypophosphataemia, hyperuricosuria and glycosuria). renal associated hypokalaemia and renal tubular acidosis was not analysed in this study due to many patients requiring potassium supplementation in addition to the effects of mechanical ventilation, gfr alterations and other factors. the authors suggest that the development of incomplete renal fanconi syndrome could be used as a predictor of acute kidney injury (and a prognostic marker) as 88% of patients with severe stage 2 and 3 kdigo acute kidney injury experienced proximal tubule injury prior to acute kidney injury onset. none of these patients had significant haemodynamic instability or had high mean sofa scores to suggest an alternative pre-renal aetiology for their acute kidney injury. a quarter of the patients were given lopinavir/ ritonavir but this is rarely associated with acute kidney injury and not associated with renal fanconi syndrome. there are two studies so far (one in pre-print form) to look at renal histopathology on post-mortem findings in sars-cov-2 patients who died of the disease (9 of the 26 had pre-morbid clinical kidney injury signs) 33,34 . the first demonstrated prominent proximal tubule injury (observed in all 26 patients) with frank necrosis observed in some specimens and three patients had high creatinine phosphokinase and pigmented casts on light microscopy, probably representing rhabdomyolysis. there was occasional distal tubule and collecting duct swelling with some interstitial oedema and virus particles were seen in seven of the specimens on electron microscopy in proximal tubule epithelial cells and the podocyte, both of which are the only sites of renal ace2 expression 33 . the second post-mortem case series in (currently in pre-print) examined six post-mortem renal histopathology samples of patients who died with sars-cov-2 infection 34 . all specimens had histopathological evidence of acute tubular necrosis, two patients had severe lymphocytic infiltration of the tubulointerstitium and sars-cov-2 nucleocapsid antigens were observed in renal tubule cells. transmission electron microscopy was used in samples from two different patients, both of which demonstrated the presence of virions and virus-like particles in renal cells and these cells were markedly swollen with mitochondrial, lysosomal and endoplasmic reticulum expansion. these finding suggest that sars-cov-2 directly infects kidneys and specifically infects and damages kidney tubules. furthermore, strong cd68 + macrophage presence and c5b-9 deposition seen in the tubulointerstitium of all samples (yet absent in the rest of the kidney tissue and very little in the glomeruli and capillaries in two patients) suggest that further tubular damage occurs as a consequence of macrophage recruitment and c5b-9 activation and deposition. it would be logical to suggest that histopathological evidence of damage to tubular cells would result, clinically, as a tubulopathy, of which we know urine electrolyte wasting is a recognised complication and these case series are histopathological evidence to support this suggestion. finally, we know that tmprss2 primes sars-cov-2 to gain cellular entry and tmprss2 is only detectable (in low levels) at the s3 section of the proximal convoluted tubule in the kidney 35 . it is therefore assumed that this is one way in which the kidney is 'infected' by the virus and reiterates the probability of direct virus-induced tubular damage. various case series have demonstrated the virus in urine 36,37 and it is unclear as to how it enters the tubular lumen and whether this is via the apical membrane of tubule cells. however, one study did look at the presence of sars-cov-2 in bodily fluids over time and did not detect the virus in urine in any of the nine patients studied 38 . a couple of case reports have demonstrated that the virus may affect the kidney in other ways which may be an alternative explanation for the entry of virus into the urine 39,40 . collapsing focal segmental glomerulosclerosis (fsgs) has been described twice in the literature so far in patients of african or african american origin and both of these patients demonstrated severe tubular injury. it was noted that severe acute tubular necrosis (atn) was observed in the absence of haemodynamic compromise and severe pulmonary involvement suggesting that the tubulopathy was not ischaemic and more likely to be because of either direct viral toxicity or cytokine-mediated damage. various clinical and histopathological studies have demonstrated evidence of hypokalaemia, hyponatraemia, siadh, incomplete fanconi syndrome and tubulopathy in patients with sars-cov-2 infection. the data from china and evidence from the sars-cov case series provide good clinical justification to monitor potassium levels in patients infected with sars-cov-2. we do need to see this clinical finding replicated in multi-centres to be able to make definitive conclusions about this clinical sequela. although hyperkalaemia can occur for many reasons in patients with sars-cov-2 infection, the incidence of ventricular fibrillation has been shown to be five-fold higher in patients with hypokalaemia compared to those with hyperkalaemia 21 . based on evidence of significant urine potassium wasting which can be prolonged, we suggest checking the transtubular potassium gradient (ttkg) in patients with sars-cov-2 infection. the ttkg adjusts the urinary potassium for the concentrating effects that occur in the collecting duct where water is removed from urine. this would help identify those at risk of severe or prolonged hypokalaemia and prompt preemptive cautious electrolyte monitoring and replacement which should help reduce the risk of hypokalaemic complications which can be fatal. the ttkg is easily measured and only requires measurements of serum and urine osmolality and potassium (urine potassium/serum potassium)/(urine osmolality/ serum osmolality). a high ttkg in the setting of hypokalaemia suggests that potassium wasting is of renal origin. this, for example, may be due to hyperaldosteronism, pseudohyperaldosteronism or a potassium wasting tubulopathy. the validity of this measurement depends on three assumptions: (1) few solutes are reabsorbed in the medullary collecting duct, (2) potassium is neither secreted or reabsorbed in the medullary collecting duct and (3) the osmolality of fluid in the terminal collecting duct is iso-osmolar to plasma. as water is reabsorbed in the medullary collecting duct, urine potassium concentration is not an accurate index evaluating distal potassium secretion because the effect of water is not taken into account. the urine to plasma osmolality ratio adjusts for the degree of medullary water reabsorption which increases urine potassium concentration as more water is reabsorbed. therefore, ttkg is relatively accurate providing the urine is not dilute, that the osmolality of urine is greater or equal to the osmolality of plasma (because vasopressin is required for optimal potassium excretion in the distal nephron) and that urine sodium concentration is >25mmol/l so that distal sodium delivery is not limiting 41 . the main premise underlying the ttkg is the absence of significant solute transport in the collecting duct so that any change in urinary potassium concentration only occurs due to medullary water reabsorption and the ttkg does not overestimate the gradient for collecting duct potassium secretion. however, we now know that some urea is reabsorbed in the late cortical collecting duct and this urea recycling aids the tubular secretion of potassium so it should be noted that the ttkg should not be used as a diagnostic tool in hyperkalaemia aetiology as it is not reliable 42 . in situations of dilute urine and high urine flow rate, the ttkg also underestimates potassium secretory capacity in the hyperkalaemic patient 43 . however, we believe that the utility of the ttkg is still of value in hypokalaemic patients to differentiate between renal and extra-renal potassium wasting providing the aforementioned criteria necessitating its accuracy is adhered to. measuring components of the renin-angiotensin-aldosterone pathway would help us to identify if there really is involvement of the sars-cov-2 virus; however, measuring these components in the acute setting has its limitations. the ras system is heavily influenced by a multitude of factors. many of the patients with sars-cov-2 infection are critically unwell and often present to hospital in states of low cardiovascular volume. it has been recommended that patients with ards also be kept in a relative state of volume depletion as to not worsen pulmonary interstitial oedema and thus oxygenation. both of these factors would typically result in ras activation and could be very misleading when determining virus-related ras pathway involvement. some studies have shown that low urine ph (causing acidic urine) can be a predictor of ras activation 44 so future studies determining ras system involvement of sars-cov-2 infection should, as well as ttkg, also consider urine ph as an early marker of ras system activation. potassium levels <3.0 mmol/l can be arrhythmogenic and specifically can cause qtc interval prolongation, torsade de pointes, ventricular fibrillation and sudden cardiac death 45 . this is particularly relevant for a couple of reasons. first, many of the drugs currently undergoing clinical trials in sars-cov-2 patients prolong the qtc interval (hydroxychloroquine 46 , azithromycin 46 , favipiravir, lopinavir/ritonavir and fingolimod: nct04280588) and some drugs also risk hypokalaemia which may worsen pre-existing electrolyte disturbance (e.g. methylprednisolone: nct04273321, nct04263402; thalidomide: nct04273529, nct04273581; and bevacizumab: nct04275414). second, cardiac involvement in sars-cov-2 is high (44.4% of infected patients admitted to icu experienced an arrhythmia). induction of arrhythmias can be due to multi-factorial aetiologies of cardiac injury in sars-cov-2 patients such as hypoxia-mediated, direct tissue damage, cytokine-storm syndrome, worsening coronary perfusion and the direct effects of medications 47 . both of these factors really emphasise the importance of maintaining normokalaemia in these patients to reduce morbidity and mortality. in addition to the arrhythmogenic effects of both the sars-cov-2 cardiac sequelae and various clinical trial drugs, many patients are being given diuretics to improve the oxygenation in ards, which also risks hypokalaemic complications. loop diuretics can induce hypokalaemia by blocking the na + -k + -2clcotransporter (nkcc2) in the thick ascending limb of the loop of henle which results in failure of sodium, potassium and chloride to be reabsorbed into the concentrated medullary interstitium (this transporter normally reabsorbs about 25% of the sodium load). this enhances distal tubular concentration of sodium, reduced hypertonicity of the surrounding interstitium and less water reabsorption in the collecting duct. distal sodium delivery increases potassium loss via the na+/k+-atpase pumps at the apical membrane of the principal cell of the collecting duct as there is more sodium to be exchanged with potassium for excretion. thiazide diuretics can cause hypokalaemia by the same principle of enhanced distal sodium delivery as they block the sodium-chloride co-symporter (ncc) in the distal convoluted tubule. furthermore, loop diuretics also block nkcc2 at the macula densa which (along with the ras-activation response to initial volume reduction) induces renin secretion, making ras system measurement inaccurate if patients are on diuretics. if diuretics are to be used, it would be wise to consider potassium-sparing agents in hypokalaemic patients to reduce the cardiac complications of worsening hypokalaemia that can occur with those that are not potassium sparing. it would be logical that with clear histopathological data suggesting tubular injury, hypokalaemia is to be expected in patients infected with sars-cov-2 48 . tubular injury seems to be multifactorial in these patients; atn from sirs/cytokine storm syndrome, peritubular congestion, virion invasion, drug toxicity and pigment-cast nephropathy from rhabdomyolysis and therefore difficult to avoid. the data on outcomes in cardiac arrest for sars-cov-2 patients is unfortunately poor. chinese data suggests that in a series of 136 patients who underwent resuscitation efforts, return of spontaneous circulation was only achieved in 18 (13.2%) and only four patients were alive at 30 days 49 . the strong association of hypokalaemia with arrhythmia and sudden cardiac death reiterates the importance of vigilance for detection and treatment of this electrolyte disturbance during and after admission given such poor outcomes when spontaneous circulation is lost. to truly know the extent of involvement of the ras pathway as a potential cause of hypokalaemia in sars-cov-2 patients, further studies require measurement of all components of the ras pathway. until we know this, we cannot make definite conclusions about the mechanisms of hypokalaemia in these patients outside of the histopathological evidence of tubulopathy published, of which multiple aetiological factors are likely. no data are associated with this article. "hypokalaemia is known to cause muscle weakness, paraesthesia, thirst, muscle cramps and weakness, but hypokalaemia is an important complication of any disease due to it being a potentially life-threatening condition". please rephrase -hypokalaemia is important, but at the same time rare (does not occur with 'any disease'). â�� "furthermore, inadequate management of hypokalaemia has been identified in 24% of hospitalised patients 22 , reiterating that we need to be even more vigilant with the management of this life-threatening condition during a pandemic where resources are even more scarce". please reference, or clarify. management of hypokalaemia is not usually resource-intensive, and i have not seen evidence that potassium replacement is difficult to come by in the pandemic. â�� "this study claimed that hypokalaemia was prevalent amongst sars-cov-2 infected patients, affecting 108 of 175 patients (62%) that had a serum potassium of 4.0 mmol/l which is the value required for those with myocardial dysfunction." hypokalaemia affected 62% of patients with a k less than 3.5? or hypokalaemia as defined by k<3.5 affected 62% of the cohort. â�� "only 10 patients had a serum k >4.0 mmol/l which is the value required for those with myocardial dysfunction". please clarify what is meant by this phrase. â�� "principal cell" does not warrant capitalisation. â�� "although hyperkalaemia can occur for many reasons in patients with sars-cov-2 infection, the incidence of ventricular fibrillation has been shown to be five-fold higher in patients with hypokalaemia compared to those with hyperkalaemia [21] ." this data is from the setting of acute myocardial infarction, not sars-cov-2, and therefore may not be applicable. please add a qualifying statement. â�� you recommend measuring the transtubular potassium gradient. does this significantly alter management in these patients, outside of a research context? â�� "it has been recommended that patients with ards also be kept in a relative state of volume depletion as to not worsen pulmonary interstitial oedema and thus oxygenation." this advice has since been dropped in the context of sars-cov-2 -please make this clear. coronavirus disease 2019 case surveillance united states covid-19 and the cardiovascular system its occurrence in types 1 and 2 rta despite sustained correction of systemic acidosis pubmed abstract | publisher full text | free full text in-hospital cardiac arrest outcomes among patients with covid-19 pneumonia in wuhan, china. resuscitation fetal programming and the angiotensin-(17) axis: a review of the experimental and clinical data pubmed abstract | publisher full text | free full text renal ace2 expression in human kidney disease some aspects of the writing were difficult to follow please broaden abstract to include electrolyte disturbances in addition to hypokalaemia, reflecting the article as a whole. in the first paragraph, there is quite a lot of text dealing with the many complications of covid infection, which is a bit irrelevant.â�� "â�¦proximal tubule epithelial cells and the podocyte, both of which are the only sites of renal ace2 expression" this is not true; see the human protein atlas or for example 1 .â�� "a few small studies have demonstrated that sars-cov-2 can affect the kidneys directly, but it is unclear if this is a key mechanism in acute kidney injury seen in patients with sars-cov-2 infection"? reference. it would be useful to include a comment on the prevalence of acute kidney injury in sars-cov-2 patients. reviewer expertise: renal tubular physiology.we confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.reviewer report 20 august 2020 https://doi.org/10.5256/f1000research.27922.r67707 â© 2020 welling p. this is an open access peer review report distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. johns hopkins university, school of medicine, baltimore, md, usa i have read the revised review. it is much improved. i would accept without further revision. competing interests: no competing interests were disclosed. johns hopkins university, school of medicine, baltimore, md, usa as pointed out by this review, the evidence for hypokalemia in covid-19 is weak, promulgated by a single unpublished article that was not corroborated by larger series studies. my chief concern about the review, is that it amplifies the myth. furthermore, the frequent use of loop diuretics in ards, which cause hypokalemia, makes a causal connection between the development of hypokalemia and covid-19 precarious.at the very least, the title should be changed to minimize the connection between hypokalemia and covid-19.paragraph 1 is somewhat misleading. although covid-19 does have an array of different sequela, acute respiratory distress syndrome (ards) seems to be the most common serve form of the disease. the first paragraph should provide an incidence of this and the other sequela. reference to large studies from china, europe and north american should be cited.paragraph 2 the statement "sars-cov-2 may affect the kidneys directly and indirectly by both renal and renin-angiotensin-aldosterone system (ras) involvement," has no factual basis. there are a few small studies that demonstrate that sars-cov-2 can infect the kidney but it is presently unclear if this is a key mechanism of aki in covid19.paragraph 3 provide a reference to mas receptor on type 2 alveolar epithelial cells as stated. paragraph 3 last sentence. this is evidence for sars-cov, not sars-cov2provide a reference to "hyperaldosteronism does not always cause hypokalaemia." although "switch" activation has been a popular explanation for this, it has never been proven. perhaps, the authors could cite clinical studies, demonstrating the development of severe hypokalemia in normokalemic pa patients following thiazide administration as evidence.the limitations of ttkg should be articulated. are all factual statements correct and adequately supported by citations? partly are arguments sufficiently supported by evidence from the published literature? partly competing interests: no competing interests were disclosed.reviewer expertise: i am an expert on molecular underpinnings of potassium balance and electrolyte disorders. i coined the "potassium switch"i confirm that i have read this submission and believe that i have an appropriate level of expertise to state that i do not consider it to be of an acceptable scientific standard, for reasons outlined above.the benefits of publishing with f1000research:your article is published within days, with no editorial bias â�¢ you can publish traditional articles, null/negative results, case reports, data notes and more â�¢ the peer review process is transparent and collaborative â�¢ your article is indexed in pubmed after passing peer review â�¢ dedicated customer support at every stage â�¢ for pre-submission enquiries, contact research@f1000.com key: cord-256020-wrui3i2l authors: fadaka, adewale oluwaseun; sibuyi, nicole remaliah samantha; adewale, olusola bolaji; bakare, olalekan olanrewaju; akanbi, musa oyebowale; klein, ashwil; madiehe, abram madimabe; meyer, mervin title: understanding the epidemiology, pathophysiology, diagnosis and management of sars-cov-2 date: 2020-08-26 journal: j int med res doi: 10.1177/0300060520949077 sha: doc_id: 256020 cord_uid: wrui3i2l the emergence of coronavirus disease 2019 (covid-19) in december 2019 has resulted in over 20 million cases and 741,808 deaths globally, affecting more than 200 countries. covid-19 was declared a pandemic on 11 march 2020 by the world health organization. the disease is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2). there is limited information on covid-19, and treatment has so far focused on supportive care and use of repurposed drugs. covid-19 can be transmitted via person-to-person contact through droplet spread. some of the recommended precautionary measures to reduce the rate of disease spread include social distancing, good hygiene practices, and avoidance of crowded areas. these measures are effective because the droplets are heavy and can only travel approximately 1 meter in the air, settling quickly on fixed surfaces. promising strategies to combat sars-cov-2 include discovery of therapeutic targets/drugs and vaccines. in this review, we summarize the epidemiology, pathophysiology, and diagnosis of covid-19. we also address the mechanisms of action of approved repurposed drugs for therapeutic management of the disease. covid-19 has become one of the most dangerous pandemics in recent history. the pandemic has claimed more than 740,000 lives, with more than 20 million reported cases since the original outbreak. the disease is caused by sars-cov-2, a zoonotic pathogen that acquired mutations as it crossed the species barrier from bat to pangolin enabling it to infect humans. 1 sars-cov-2 was confirmed as a novel coronavirus using molecular methods and initially named 2019 novel coronavirus (2019-ncov). 2 the disease caused by this virus was later renamed covid-19 by the world health organization (who). 3 sars-cov-2 is highly infectious and has spread to more than 200 countries in all continents. hence, the virus was declared a global threat (pandemic) by the who. sars-cov-2 has a single-stranded positive sense rna genome (þssrna) approximately 30 kb long. the sars-cov-2 genome is organized similarly to those of sars-cov-1 and middle east respiratory syndrome (mers)-cov. 4 using phylogenetic analysis, the sars-cov-2 genome was demonstrated to share 96% sequence similarity with a bat cov genome. sars-cov-2 belongs to the b-coronavirus genus that includes sars-cov-1 and mers-cov. 5 the clinical symptoms of covid-19 include fever, cough, and pneumonia, which makes the disease enormously dangerous with a high case fatality rate. 6, 7 in contrast to other b-coronaviruses, many sars-cov-2 deaths have resulted from multiple organ dysfunction syndrome (msof) rather than respiratory failure. 8 this could be attributed to the widespread distribution of angiotensin converting enzyme-2 (ace-2), the primary receptor for sars coronaviruses, in multiple organs. 9,10 ace-2 is expressed as a cellsurface molecule in the respiratory tract (epithelium, arterial and venous endothelium), the small intestinal epithelium, and arterial smooth muscle cells. 11 sars-cov-2 is morphologically spherical and is characterized by presence of a spike protein, lower pathogenicity and higher transmissibility between humans. 12 some of the primary measures taken to reduce the number of infections and prevent community transmission are to avoid crowds and gatherings and to practice good hygiene. interestingly, countries with the highest reported prevalence and mortality such as the united states, spain, italy, the united kingdom, russia, germany, brazil, france, turkey, iran and china, are more concerned with flattening the curve through early case detection, isolation, and development of therapeutic drugs and vaccines. because of the novel nature of this disease, there is limited information regarding risk factors for severe outcomes. specific factors such as the serial interval, viral lifespan, incubation period, pathogenic mechanisms, clinical features and optimal disease management remain unclear. therefore, this review aimed to summarize the epidemiology and pathophysiology of sars-cov-2 as well as the use of repurposed food and drug administration (fda) approved drugs for therapy. the entry of this virus into host cells and possible downstream complications deserve closer attention. covs are members of the subfamily othocoronavirinae of the family coronaviridae. the subfamily consist of four genera: alpha, beta, gamma and delta covs. 13 both alpha-and beta-covs can infect mammals, including humans, while the gamma-and delta-covs only infect birds. 14 about seven covs have been isolated from humans ( figure 1 ). these include two alpha-covs, human coronavirus 229e (hcov-229e) and human coronavirus nl63 (hcov-nl63), and five beta-covs: human coronavirus oc43 (hcov-oc43), human coronavirus hku1 (hcov-hku1), sars-cov-1, mers-cov, and sars-cov-2. sars-cov-1, mers-cov, and sars-cov-2 are pathogenic and cause severe infections in humans following contact with the respective intermediate hosts (bats) (figure 2 ). however, hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1 do not appear to cause severe infections in humans. 14 covs are enveloped viruses with þssrna genomes. they have the largest genomes (approximately 26-33 kb) among rna viruses. all covs possess nonsegmented genomes with similar organization. 15 generally, about two-thirds of the genome consists of two large and overlapping open reading frames (orf1a and orf1b), which are translated into polyproteins pp1a and pp1ab and subsequently processed to yield 16 non-structural proteins (nsp1 to nsp16). 16 the remaining one-third of the genome consists of orfs encoding structural proteins including the spike (s) glycoprotein embedded in the envelope and the envelope (e), matrix (m), and nucleoproteins (n). 17 there are short untranslated regions at both the 3 0 and 5 0 ends. the s protein plays a role in receptor-binding and entry of virus into host cells and is thus considered a major therapeutic target. the m and e proteins are important for viral assembly, while the n protein is necessary for synthesis of rna. 18 overview of the sars-cov family sars-covs belongs to a global family of viruses causing respiratory disease and influenza-like symptoms such as fever, muscle pain, sore throat, headache, and cough. 19 figure 3 highlights the onset and progression of sars-cov-1, mers-cov, and sars-cov-2 infection. the first case of sars-cov-1, reported in china, resulted in an outbreak that caused hundreds of deaths and thousands of infected cases in the early 2000s. 20 a pneumonia-like syndrome (mers) was first discovered in saudi arabia and then spread to several countries, where it incurred a mortality rate of about 3% to 6%. marra et al (21) observed that the mers mortality rate increased with age and was as high as 43% to 55% in people older than 60 years. in december 2019, sars-cov-2, caused an outbreak in china and then spread worldwide. the resulting disease (covid-19) is a serious threat mostly in people with compromised immune systems or underlying conditions such as lung disease, diabetes mellitus, and human immunodeficiency virus infection. 22 sars-cov-1. sars-cov-1 causes a viral respiratory disease and belongs to lineage b of the beta-covs. 11 of uncooked meat, milk or urine, as shown in figure 2 . human-to-human infection also occurred through nosocomial transmission. 11 symptoms of human sars-cov-1 infections include headache, fever and respiratory complications such as cough, dyspnea, and pneumonia. 23 the sars-cov-1 genome is 29,727 nucleotides in length. the 5 0 end of the genome contains orf1a and orf1b. the polyproteins encoded by these orf are auto-catalytically processed to yield a number of viral proteases as well as the rna-dependent rna polymerase (rdrp). the remainder of the genome encodes the viral structural proteins (s, e, m and n) as well as several accessory proteins. 18 the receptor for sars-covs is ace-2, a surface molecule found on cells of the respiratory tract, the small intestinal epithelium, and smooth muscle. in the respiratory tract, ace-2 is expressed on epithelial cells of the alveoli, bronchi, trachea, and bronchial serous glands, as well as on alveolar monocytes and macrophages. the ace-2 enzyme plays an important role in protection against lung failure. 11 mers-cov. mers-cov causes a viral respiratory disease and belongs to lineage c of the beta-covs. 11 the first cases of human infection by mers-cov were reported in saudi arabia in 2012. cases were subsequently reported in other countries including qatar, egypt, and the republic of korea following contact with infected camels (figure 3b ). cases of mers-cov were identified in about 27 countries between 2012 and 2018. 18 mers-cov rna in camels showed more than 99% genomic sequence similarity to human mers-cov. 23 bats are the natural hosts of mers-cov (figure 2) , and a high prevalence of mers-cov infections in dromedary camels (intermediate hosts) was confirmed in the middle east, spain, and africa. mers-cov infections were transmitted to humans following contact with camels infected with the virus. 23 the mers-cov genome is about 30,119 nucleotides in length and has a 5 0 terminal cap structure and a poly (a) tail at the 3 0 end. the genome encodes 16 non-structural proteins (nsp1-nsp16) at the 5 0 end, four structural proteins (s, e, m, and n) at the 3 0 end, 24 and five accessory proteins in orf3, orf4a, orf4b, orf5, and orf8. 18 risk factors for severe mers-cov include age and the presence of underlying conditions such as diabetes, obesity, hypertension, chronic renal disease, and lung diseases. 25 the receptor for mers-cov is dipeptidyl peptidase 4 (dpp4 or cd26). dpp4 is a multifunctional cell surface protein and is expressed on the epithelial cells of the respiratory tract, liver, kidney, small intestine, and prostate, as well as on activated white blood cells. dpp4 also plays a vital role in activation of t cells and providing co-stimulatory signals for immune responses of t cells. 11, 18 consequently, mers-cov causes acute pneumonia and renal dysfunction with associated clinical symptoms such as cough, chest pain, sore throat, fever, diarrhea, vomiting, and abdominal pain. mers-cov can infect human dendritic cells and macrophages in vitro, thereby contributing to dysregulation of the immune system. 18 sars-cov-2. sars-cov-2, a newly discovered coronavirus, has a þssrna genome and a spherical morphology when observed under the electron microscope. 26,27 sars-cov-2 encodes a richly glycosylated spike protein responsible for binding to the ace-2 receptor. 28 the virion's shape and the ability of spike proteins to form a crownlike structure gave coronaviruses their name. 29, 30 the sars-cov-2 genetic material is surrounded by a lipid-bilayer envelope. other structural components include the nucleocapsids, membrane, envelope, and hemagglutinin. unlike the envelope, the membrane exists in higher quantities within the virions. 31 among other functions, the envelope serves to release viral particles from the host cells. 32 the nucleocapsid assist in rna packaging during virion assembly. 33, 34 hemagglutinin enhances the entry and pathogenesis of coronaviruses. 33 some of the characteristics of sars-cov-2 are summarized in table 1 . many of these features are shared with sars-cov-1. more information on the epidemiology and general characteristics of sars-cov-1 can be found later in the review. other important human covs include alpha-covs (hcov-229e and hcov-nl63) and two beta-covs (hcov-oc43 and hcov-hku1). the receptor for hcov-229e is human aminopeptidase n (cd13), a cell-surface metalloprotease present on cells of the kidney and lung, as well as on epithelial and intestinal cells. the receptor for hcov-nl63 is also ace-2. the receptor for hcov-oc43 is 9-o-acetylated sialic acid, while no receptor has been identified for hcov-hku1. 42, 43 generally, the most common diagnostic tools for human covs are molecular detection techniques such as reverse transcription-polymerase chain reaction (rt-pcr) using rna extracted from respiratory tract samples as template. other methods include serological tests and viral cultures. 11 although several agents against covs, including antibodies, antiviral peptides, and cell or viral protease inhibitors, have been shown to be effective both in vitro and/or in vivo, clinical trial outcomes have not been reported. 11 therefore, clinical treatments for covs are still lacking. nevertheless, supportive and symptomatic therapy are used for at present, 215 countries and territories are affected by the sars-cov-2 outbreak, including the united states, italy, germany, south africa, and nigeria. the distribution of sars-cov-2 confirmed cases, including mortality and recovery, is shown in figure 4 . the united states, china, and some european countries have high case numbers and mortality rates. recovery rates are increasing worldwide with higher numbers reported in china. the cfr is defined as the percentage of deaths recorded among confirmed cases. as of 14 april 2020, the number of deaths among confirmed cases was estimated at 126,066 to 1,992,189, resulting in a cfr of 6.33%. this differs from the cfr of 3.70% calculated on 15 march 2020. however, several factors can prevent the accurate determination of the cfr. we compared the global cfr to the african cfr. the highest cfr was observed in italy, followed by spain and the netherlands (figure 5a ). in africa, countries such as egypt, algeria, burkina faso, and morocco had cfrs above 5% (figure 5b) . a major challenge in the accurate calculation of the cfr is the denominator (the number of confirmed cases). asymptomatic cases of covid-19, patients with mild symptoms, or individuals who are misdiagnosed may be left out of the denominator, leading to underestimation or overestimation of the cfr. 45 high cfrs reflect limited access to health care for the most vulnerable patients and limitations in health-care systems, including limited capacity of surveillance systems to trigger a timely response (who, 2020). to date, over 20 million cases of covid-19 have been reported globally. it is important to estimate the reproductive number for this virus to enable accurate predictions. two primary factors, the reproductive ratio (r o ) and the serial interval (s i ), are essential to estimate the rate of transmission of this disease. serial interval (s i ). to understand the case turnover and transmissibility of covid-19, the serial interval (s i ) from the onset of illness in a primary case to the onset of illness in a secondary case is important. 49 recent studies estimated the average s i for covid-19 as 3.77 (2.23-4.82) days. 49-51 a shorter s i makes covid-19 harder to contain and more likely to rapidly transmit within populations ( figure 6 ). taking the r 0 and s i of covid-19 into consideration, it can be inferred that approximately 4 days are required for an infected person to transmit covid-19. it is highly likely that the individual can infect approximately two or three other persons, making the spread of covid-19 extremely rapid and dangerous ( figure 7a ). the extent of spread is totally dependent on the r o value (figure 7b ). if r 0 < 1, each existing infection causes less than one new infection. in this case, the disease will decline and eventually die out. if r o ¼ 1, each existing infection causes one new infection. the disease will remain stable in the population, but will not result in epidemic spread. if r 0 > 1, each existing infection causes more than one new infection. the disease will spread between individuals and eventually lead to an outbreak. covid-19 was regarded as an outbreak (30 january 2020) with a r 0 > 1 (figure 7b) . importantly, the r 0 value of a disease only applies when everyone in a population is completely susceptible. this means that no one has been vaccinated or had the disease previously, and there is no way to control the spread of the disease using interventions such as drugs or vaccines. there are currently two known modes of covid-19 transmission: the fecal-oral route and respiratory droplets. 6, [52] [53] [54] [55] [56] droplets have potential to come into contact and infect a healthy person within 3 to 6 feet (1 meter). droplets that stick to surfaces can survive for more than 24 hours, remaining infectious. the virus can remain airborne for about 3 hours, long enough to permit transmission. upon infection with sars-cov-2, the virus infects type ii pneumocytes of the alveoli. 57 these pneumocytes are responsible for surfactant production. surfactant decreases the surface tension within alveoli and reduces the collapsing pressure. the spike protein of the virus binds to ace-2 on the pneumocytes (figure 8 ) permitting virion entry into the host cell. 58 the virus hijacks the host cell's machinery (ribosomes) to enable translation of its þ ssrna genome into different protein molecules. the virus can also use its rdrp to produce additional copies of its þ ssrna genome (figure 9 ). the translated polyproteins are further processed into different individual components within the host cell. these processes give rise to multiple virions, which are then released upon pneumocyte damage. in response to this process, type ii pneumocytes releases specific inflammatory mediators that instruct macrophages to secrete interleukins 1 and 6 (il-1 and il-6) and tumor necrosis factor-alpha. these cytokines cause the endothelial cells lining blood vessels to dilate, leading to increased capillary permeability. in response, fluids accumulate in the alveoli leading to edema. 59 as surface tension increases, the collapsing pressure of the alveoli increases. a decrease in gas exchange is also observed through this process, which in turn leads to hypoxia and difficulty breathing (dyspnea). this can progress to a critical condition such as acute respiratory distress syndrome (ards). inflammatory mediators further stimulate neutrophils, which release reactive oxygen species and proteases. this process damages the alveoli (both type 1 and 2), leading to consolidation and alveolar collapse. high levels of il-1 and il-6 travel through the blood to the central nervous system, instructing the hypothalamus to release prostaglandins and causing fever. severe lung inflammation leads to systemic inflammatory respiratory syndrome. progression can lead to increased capillary permeability. the overall blood volume decreases, and through a series of processes involving hypotension and decreased perfusion of multiple different organs, multiple system organ failure (msof) can occur. during msof, elevated levels of blood urea, nitrogen, and creatinine accrue in the kidney. the liver also releases specific inflammatory and acute phase response biomolecules (aspartate transaminase, alanine transaminase, bilirubin, c-reactive protein [crp], fibrinogen and il-6) that can serve as biomarkers for patients with covid-19. there is continued research by multiple groups into the mechanism of transmission of sars-cov-2 through the eyes. investigations were necessary because health care providers, including a perking university physician, may have contracted the virus while not wearing eye protection when treating patients. 61 some researchers have asserted that avoidance of touching the eyes, nose or mouth with unwashed or unsterilized hands can reduce covid-19 transmission. the mucous membranes that line various cavities in the body are generally most susceptible to viral transmission. 62 ocular symptoms such as viral conjunctivitis can result from sars-cov2 upper respiratory tract infections. 63 this was confirmed in 9 of 1,099 patients in china. 64 other research also found that 1 of 30 patients hospitalized with covid-19 were diagnosed with conjunctivitis. 65 in a study conducted by the american optometric association, covid-19 was linked to ocular signs and symptoms including photophobia, irritation, conjunctival infection and ocular discharge. 66 thus, the superficial blood vessels of the conjunctiva are an alleged route of exposure and transmission of sars-cov-2. the clinical manifestations of sars-cov-2 are uncertain and change frequently. some infections are asymptomatic. symptoms can include respiratory distress syndrome, pneumonia of different levels of severity, 67 and sometimes death. according to the who, the most common symptoms of covid-19 are fever, fatigue, dry continuous cough, and shortness of breath. 68, 69 some patients may have a runny nose, sore throat, nasal congestion, aches and pains, and diarrhea. 70 some patients report a loss of sense of smell and taste. 71 in some cases, symptoms are mild and similar to those of the common cold; in such patients, recovery can occur without any treatment. 72 the least commonly observed symptoms include nausea or vomiting, coughing up blood or bloody mucus, and viral conjunctivitis causing red eyes, watery discharge from the eyes, swollen eyelids and light sensitivity. 3 occasional upper respiratory and gastrointestinal symptoms, accompanied by changes vital signs such as increased respiration (heart rate) and low blood pressure may also be observed, especially in the elderly and among individuals suffering from heart disease, chronic respiratory conditions and diabetes. 73 additionally, patients critically ill with covid-19 may present with increased venous thromboembolism including thrombocytopenia, elevated d-dimer, prolonged prothrombin time and disseminated intravascular coagulation (dic). these coagulation abnormalities are associated with a systemic inflammatory response and an imbalance between pro-coagulant and anticoagulants homeostasis mechanisms. and increase the risk of mortality. 74 some of these clinical features are also observed in cases of dic observed in septic patients. these features are very distinct in covid-19 patients as their levels are higher than the standards for sepsis. 75, 76 diagnosis, prognosis, treatment, and management of sars-cov-2 sars-cov-2 causes various complications ranging from fever, dry cough, and pneumonia to decreased organ perfusion leading to msof. early symptoms are similar to those of influenza, and the first step to differentiate covid-19 from flu and pneumonia is a nasopharyngeal swab test (viral testing for influenza a/b). 77 quantitative polymerase chain reaction-based (qpcr) methods are the major diagnostic tests for sars-cov-2 using nasal swab, aspirate, sputum, or blood as samples. these method have some limitations as they are time consuming and have variable sensitivity (30%-80%). [78] [79] [80] another diagnostic method is the newly approved nucleic acid test, which is carried out based on the principle of fluorescence pcr. 81 the main goal of sars-cov-2 diagnosis is to accurately detect the virus and to minimize further transmissions by timely isolation and treatment of infected patients. other tests (not specific for sars-cov-2) used in conjunction with the methods above are based on clinical manifestations. these include blood tests such as complete blood count, comprehensive metabolic panels, basic metabolic panels, and assessment of liver/kidney markers and procalcitonin levels (for bacterial infections). inflammatory markers may also be assessed including crp, erythrocyte sedimentation rate, il-6, lactate dehydrogenase, d-dimer, ferritin, troponin and creatine kinase-mb. imaging investigations are typically computed tomography scans: in covid-19 patients these often show glass opacities, areas of consolidation, and paving patterns in cases of severe and progressive disease. ground glass opacities can also be observed on chest x-ray. finally, ultrasound can show b-lines, pleural line thickening, and lung consolidation. air bronchograms can also be used for assessment. these tests are non-specific but helpful to determine patients' health status. covid-19 patients with severe ards could potentially present with pneumonia. pneumonia may be severe, leading to ards and msof. it is therefore imperative to mechanically ventilate the lungs to avoid ards and msof. although the risk factors for covid-19 remain unclear, some risk factors put patients at a significantly higher risk of mortality, especially in individuals with underlying conditions. 6, 35, 40, 82 some medical disorders are correlated with higher risk of mortality in covid-19 patients. these include, cardiovascular diseases mortality risk 10.6%), 41 lung disease (mortality risk 7.3%), type 1 and 2 diabetes (mortality risk 6.3%), immunosuppression (e.g., cancer patients; mortality risk 5.6%), [83] [84] [85] [86] and age ( figure 10 ). 87 covid-19 mortality rates increase with advancing age, and are especially high in those aged >60 years. elevated inflammatory markers in response to tissue damage (elevated levels of d-dimer, ferritin, creatine kinase-mb, and troponin) have been associated with higher mortality rates. the first line of treatment for patients presented with the symptoms of covid-19 (fever, dry cough, and shortness of breath) is self-quarantine for at least 14 days. cases are monitored for progression of symptoms such as increased fever (>40 c), significant difficulty breathing or shortness of breath, mouth breaks, constant coughing, and dehydration. if there is no significant improvement in symptoms, it is advisable to consult a clinician for confirmation of the diagnosis and to avoid further spread of the virus. the main treatment currently available is supportive care. although there is limited information on covid-19, it has been linked to ards. for patients with high fevers that could potentially lead to dehydration, intravenous fluids such as normal saline or lactated ringer's fluid can be administered sparingly to prevent lung overload. to reduce fever, antipyretic drugs such as paracetamol or acetaminophen can be administered. drugs such as remdesivir, chloroquine, ritonavir, tocilizumab, corticosteroids have been repurposed for the treatment of covid-19 (figure 11 ), although their clinical effectiveness has not yet been confirmed. [88] [89] [90] [91] [92] [93] unfortunately, the use of chloroquine and derivatives such as hydroxychloroquine, alone or in combination with other drugs, resulted in cardiac toxicity. investigations of these drugs were recently suspended by the who. 94 mechanism of action of selected repurposed drugs for treatment of covid-19. the elucidation of potential targets could lead to covid-19specific treatments (figure 11 ). approaches to anti-sars-cov-2 drug development include (a) inhibition of sars-cov-2 entry, (b) disruption of sars-cov-2 þ ssrna synthesis after entry, (c) inflammatory response suppression, and (d) disruption of sars-cov-2 translation. an fda-approved immunosuppressive and anti-malarial parasite drug (chloroquine) can inhibit the entry of covid-19 into the endosome after binding to ace-2 receptors, thereby preventing the release of þ ssrna for translation. 89, 93, 95, 96 studies also showed that hydroxychloroquine, an analog of chloroquine, was more potent and could be used in place of chloroquine. 97 remdesivir, a novel antiviral drug and nucleotide analog used to treat ebola virus infection, is currently under clinical development. the mechanism of the drug is reported to be at the post viral entry stage. 89 its mode of action is to inhibit the rdrp, preventing synthesis of the viral þ ssrna (figure 11 (2)). this drug is currently in phase 2 clinical trials. the protein inhibitor ritonavir has also been proposed for the treatment of covid-19. this drug interferes with the protease enzymes (proteinases) by inhibiting the conversion of polyproteins into the mature components (spike proteins, nucleocapsids) needed by the virus for multiplication (figure 11 (3)). another immunosuppressive drug, tocilizumab, has also been repurposed for covid-19 because of its ability to block il-6 and inhibit inflammatory responses. corticosteroids can also decrease inflammation by inhibiting phospholipases such as phospholipase a 2 , and thus suppress the excessive production of prostaglandins (figure 11 (4) ). sars-cov-2 appears unfamiliar to the human innate immune system. coupled with its emergence and its spread globally via human-to-human transmission, the development of vaccines for prevention is no longer a debate but a necessity. several vaccine platforms are being developed and some have entered clinical trials. however, approval by regulatory agencies such as the fda and manufacturing may take 12 to 18 months. 98 studies of the antiviral activity of host-directed drugs and compounds have identified two classes of molecules (protein biogenesis inhibitors and ligands of the sigma1 and sigma2 receptors) as effective inhibitors of viral infectivity. molecules that target the sigma1 and sigma2 receptors perturb the virus through different mechanisms from translation inhibitors, potentially including modulation of cellular stress responses. 99 the ligands haloperidol, pb28, pd-144418, and hydroxychloroquine are currently undergoing clinical trials in covid-19 patients. 100 these molecules exert their antiviral effects during viral replication by inhibiting nucleoprotein expression after viral entry has occurred. the lack of selectivity of chloroquine and hydroxychloroquine, including off-target effects on the human ether-a`-go-go-related gene (herg) and other molecules, may be related to the adverse cardiac reactions that have limited their use. 101 a recent study by gordon et al identified 332 highconfidence sars-cov-2-human protein interactions connected to multiple biological processes, including protein trafficking, translation, transcription and ubiquitination regulation. 102 the study identified 69 ligands, including fda approved drugs, compounds in clinical trials, and preclinical compounds that might theoretically have therapeutic effects as host-directed interventions against covid-19. to date, no known antiviral drugs nor vaccines against sars-cov-2 with proven clinical efficacy have been identified. part of the reason for the absence of such agents is limited information regarding the molecular details of the infection. to develop therapeutic interventions against covid-19, it is crucial to understand how the virus interactions with the host during infection. ace-2, a potential target for covid-19 treatment. ace-2 has been identified as the functional receptor for sars-cov-2. it is an outer membrane enzyme expressed in vascular endothelial cells, the renal tubular epithelium, and leydig's cells of the testes, lungs, heart, kidney, and gastrointestinal tract. 59, 103 it is a type-ii transmembrane metallocarboxypeptidase with its enzymatically active domain exposed at the cell surface. 104 ace-2 is a key player in the renin-angiotensin system (ras) and a target for treatment of hypertension. 105 ace-2 catalyzes the cleavage of angiotensin ii, a vasoconstrictor, into angiotensin 1-7, a vasodilator, thereby lowering blood pressure by negatively regulating ras. 106 ace-2 is a promising drug target for treating cov infection as well as other cardiovascular diseases. 5 ace-2 confers a protective effect against lung injury induced by viruses by increasing the production of angiotensin 1-7. the virus presumably causes lung damage by reducing ace-2 levels on cells through the process of degradation and internalization. 107, 108 because studies have shown that ace inhibitors and angiotensin ii receptor blockers could be used to up-regulate the expression and activity of ace-2 in hypertensive patients, similar strategies might reduce the severity of covid-19. 109, 110 recent studies have shown that the expression of human ace-2 is associated with sars-cov infection and that genetic variations of this receptor may contribute to susceptibility and/or resistance against infection. 111 for instance, a single-cell rna sequencing analysis of ace-2 revealed that type ii alveolar cells had higher expression of ace-2 in eight individual lung tissues obtained from normal donors. ace-2 expression was higher in the two male samples compared with the six female samples. additionally, ace-2 expression was higher in the only asian male in the study compared with caucasian and black americans, 59 which might explain why the four german cases showed mild clinical symptoms with no severe illness. 112 this implies that variation in ace-2 expression in covid-19 patients is likely to affect susceptibility, symptoms and intervention outcomes following sars-cov-2 infection. the degree of spread of covid-19 is currently about 5.3% and could potentially increase if precautionary measures are not considered. global prevention of the spread of covid-19 is therefore a crucial and urgent goal. to prevent further spread of this disease, detection and isolation of individuals with covid-19 is of the utmost priority. examples of measures to curb spread include self-quarantine, isolation of infected individuals, social distancing, good personal hygiene (frequent hand washing with soap and water/alcohol-based sanitizers and avoiding touching the eyes, nose and the mouth), and use of personal protective equipment. certain classes of compounds, called surfactants, are contained in soap and have the ability to neutralize microbes such as sars cov-2. 113 this is because soap can assemble into bubblelike structures called micelles that trap viral matter and other biomaterials. 114 surfactants in soap lather have their hydrophilic parts pointing outwards and interacting with solvent and their hydrophobic heads pointing inwards. this opens the coronavirus outer membrane and encapsulates viral molecules within micelles, thus making insoluble viral molecules easily soluble in water and effectively removing them from hands, surfaces or other areas after about 20 s. 115 therefore surfactants in soap can disrupt and sequester viruses and other contaminants while sanitizer and disinfectants are designed to kill sars-cov-2. 116 the outbreak of sars-cov-2 has become a global threat. however, information regarding this virus remains limited. the available information is inconsistent and there are constant data updates, which may contribute to variation between study results. for more consistent and accurate results, well-annotated data from clinical patients and subclinical subjects in normal populations could help to better understand the pandemic. the information provided in this review is based on data provided by the center for systems science and engineering (csse) at johns hopkins university during specific date ranges. key insights into the prevalence, pathophysiology, diagnosis, and potential treatment of sars-cov-2 are herein summarized. research efforts are being intensified to address the current challenges in the quest for adequate treatments, diagnostics, and vaccines. clinical studies into the genetic variation of receptors such as ace-2 in tissues and across populations will remain an active area of research until relevant targets and therapies are found. while the development of adequate treatments and vaccines for covid-19 is underway, it is advisable that good hygiene practices including washing of hands and social distancing should be practiced, and government guidance/guidelines should be followed. this will help to reduce the spread of the disease. we hope that the insights gained from this review will enable researchers to help patients develop accommodating lifestyles and improve the efficiency of health care practitioners. data are freely available at the following sources. centers for disease control and prevention (https://www.cdc.gov/). center for systems science and engineering (csse) at johns hopkins university (https://coronavirus.jhu. edu/map.html). worldometers (https://www. worldometers.info/coronavirus/?). zoonotic origins of human coronaviruses detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 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as tentative sars-cov-2 therapeutics structural variations in human ace2 may influence its binding with sars-cov-2 spike protein comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations cationic gemini-surfactants based on waste cooking oil as new 'green' inhibitors for n80-steel corrosion in sulphuric acid: a combined empirical and theoretical approaches the effect of surfactant concentration, salinity, temperature, and ph on surfactant adsorption for chemical enhanced oil recovery: a review the micelle thermodynamics and mixed properties of sulfobetaine-type zwitterionic gemini surfactant with nonionic and anionic surfactants managing neonates with respiratory failure due to sars-cov-2-authors' reply the authors declare that there is no conflict of interest. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. adewale oluwaseun fadaka https://orcid. org/0000-0002-3952-2098 nicole remaliah samantha sibuyi https:// orcid.org/0000-0001-7175-5388 ashwil klein https://orcid.org/0000-0002-5606-886x key: cord-264267-weat0qs6 authors: kleine-weber, hannah; schroeder, simon; krüger, nadine; prokscha, alexander; naim, hassan y.; müller, marcel a.; drosten, christian; pöhlmann, stefan; hoffmann, markus title: polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of middle east respiratory syndrome coronavirus date: 2020-01-21 journal: emerg microbes infect doi: 10.1080/22221751.2020.1713705 sha: doc_id: 264267 cord_uid: weat0qs6 middle east respiratory syndrome (mers) coronavirus (mers-cov) causes a severe respiratory disease in humans. the mers-cov spike (s) glycoprotein mediates viral entry into target cells. for this, mers-cov s engages the host cell protein dipeptidyl peptidase 4 (dpp4, cd26) and the interface between mers-cov s and dpp4 has been resolved on the atomic level. here, we asked whether naturally-occurring polymorphisms in dpp4, that alter amino acid residues required for mers-cov s binding, influence cellular entry of mers-cov. by screening of public databases, we identified fourteen such polymorphisms. introduction of the respective mutations into dpp4 revealed that all except one (δ346-348) were compatible with robust dpp4 expression. four polymorphisms (k267e, k267n, a291p and δ346-348) strongly reduced binding of mers-cov s to dpp4 and s protein-driven host cell entry, as determined using soluble s protein and s protein bearing rhabdoviral vectors, respectively. two polymorphisms (k267e and a291p) were analyzed in the context of authentic mers-cov and were found to attenuate viral replication. collectively, we identified naturally-occurring polymorphisms in dpp4 that negatively impact cellular entry of mers-cov and might thus modulate mers development in infected patients. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus with a single-stranded rna genome of positive polarity. it belongs to the coronaviridae family (genus betacoronavirus), which is part of the order nidovirales. mers-cov was isolated in 2012 from the sputum of a 60 year old man suffering from acute pneumonia and renal failure in saudi arabia [1] . since its discovery, mers-cov has caused 2,442 human infections of which 842 (34.5%) had a fatal outcome (as of may, 2019) [2] . dromedary camels are reservoir hosts of mers-cov and display only common cold-like symptoms upon infection but constitute the main source of human infections. transmission to humans occurs via close contact to animals or contaminated animal products [3] [4] [5] [6] . human-to-human transmissions seem limited and were mainly observed in health care settings, leading to mers outbreaks in hospitals [7] [8] [9] [10] [11] [12] . finally, differences in the tissue specific expression of the cellular receptor for mers-cov, dpp4, were recently suggested to account for the differences in mers-cov transmission and disease induction in camels and humans, respectively [13, 14] . in order to infect a host (cell) and replicate, mers-cov has to deliver its genome into the cellular cytoplasm for gene translation and genome replication. this process is facilitated by the viral spike (s) glycoprotein, a type-i transmembrane protein embedded in the viral envelope. for host cell entry, the surface unit, s1, of mers-cov s binds to the cellular type-ii transmembrane protein dipeptidyl peptidase 4 (dpp4, cd26) [15] . the structure of the interface between dpp4 and mers-cov-s was resolved on the atomic level and fifteen residues in dpp4 were found to make direct contact with residues in the viral s protein [16] . upon dpp4 engagement, mers-cov s undergoes proteolytic activation through the cellular serine protease tmprss2 or the endosomal cysteine protease cathepsin l [17] [18] [19] , which allows the transmembrane unit, s2, of mers-cov s to fuse the viral membrane with cellular membranes. dpp4 is a prolyl oligopeptidase that is expressed in various tissues [20] and involved in multiple biological processes including t-cell activation [21] , control of the activity of growth factors, chemokines and bioactive peptides [22] [23] [24] , and regulation of the glucose metabolism [25] . mature dpp4 is embedded in the plasma membrane as a homodimer and each monomer consists of an n-terminal cytoplasmic domain, followed by a transmembrane domain and a large ectodomain, which can be further subdivided into a short stalk domain, a glycosylation-rich and a cysteine-rich region as well as the c-terminal catalytic domain (α/ β-hydrolase domain) [26] . polymorphisms in the dpp4 gene were implicated in several diseases and conditions, including diabetes [27, 28] and myocardial infarction [29] but their potential impact on mers-cov infection has not been analyzed. we asked whether naturally-occurring amino acid polymorphisms in dpp4 residues making contact with mers-cov s have an impact on mers-cov entry. we identified fourteen polymorphisms by screening public databases and introduced the respective mutations into a dpp4 expression plasmid. we identified four mutations that reduced mers-cov s binding to dpp4 and mers-cov s-driven host cell entry without affecting dpp4 expression at the cell surface. 293t cells were transfected with expression vectors for wt or mutant dpp4, or empty expression vector (negative control). at 16 h post transfection, the culture medium was replaced and the cells were further incubated for additional 32 h. then, the cells were washed with pbs and mixed with 2x sds-sample buffer (0.03 m tris-hcl, 10% glycerol, 2% sds, 0.2% bromophenol blue, 1 mm edta). cell lysis was achieved by incubating the samples for 10 min at room temperature followed by incubation at 96°c for an additional 10 min. the samples were further loaded on polyacrylamide gels and sds-page (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed. next, the proteins were transferred onto nitrocellulose membranes (hartenstein gmbh) by immunoblotting. the membranes were further blocked by incubation in pbs-t (pbs containing 0.5% tween 20 and 5% skim milk powder) for 30 min at room temperature. afterwards, the membranes were incubated overnight at 4°c with undiluted supernatant of a hybridoma cell line secreting anti-cmyc antibody 9e10 (for dpp4 detection) or pbs-t containing anti-ß-actin (actb) antibody (mouse, 1:1,000, sigma aldrich). following three washing intervals with pbs-t, the membranes were further incubated with pbs-t containing horseradish peroxidaseconjugated anti-mouse antibody (goat, 1:5,000, dianova) for 1 h at room temperature before an in house-prepared enhanced chemiluminescent solution (0.1 m tris-hcl [ph 8.6], 250 µg/ml luminol, 1 mg/ ml para-hydroxycoumaric acid, 0.3% h 2 o 2 ) was added and signals were recorded using the chemocam imaging system and the chemostar professional software (intas science imaging instruments gmbh). in order to quantify the signal intensity of the protein bands, the program imagej (fiji distribution) [30] was used. to account for differences in the total protein content of the samples and variations, we normalized the dpp4 signals against the respective signals of the loading control (actb). 293t (human kidney cells, dsmz no. acc 635), bhk-21 (hamster kidney cells, dsmz no. acc 61) and vero 76 (african green monkey kidney cells, kindly provided by andrea maisner, philipps-university marburg) were cultivated in dulbecco's modified eagle medium (pan-biotech) while caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in minimum essential medium (thermofisher scientific). the media were supplemented with 10% fetal bovine serum (biochrom), 100 u/ml of penicillin and 0.1 mg/ml of streptomycin (pan-biotech). all cell lines were incubated at 37°c and 5% co2 in a humidified atmosphere. for subcultivation and seeding, cells were washed with phosphate-buffered saline (pbs) and detached by incubation with trypsin/ edta solution (pan-biotech) (bhk-21, vero 76 and caco-2) or by resuspending the cells in culture medium (293t). transfection of 293t and bhk-21 cells was carried out by calcium-phosphate precipitation or with the help of icafectin-441 (in-cell-art) or fugene hd (promega). all dpp4 mutants were generated based on a pcdna3.1/zeo(+)-based expression vector in which the coding sequence for human dpp4 (genbank: xm_005246371.3) containing an c-terminal cmyc epitope was inserted into via bamhi/ecori restriction sites. the following aa (amino acid) substitutions were introduced via overlap-extension pcr: k267e, k267n, q286k, t288i, t288s, a289v, a291p, a291v, r317k, y322h, i346t, i346v and k392n . in addition, a deletion mutant was generated that lacks aa residues 346-348 (δ346-348). information on dpp4 polymorphisms was retrieved from the ensembl database (https://www.ensembl.org/index.html) [31] and the single nucleotide polymorphism database (dbsnp) of the national center for biotechnology information (ncbi) (https://www.ncbi.nlm.nih.gov/snp) [32] , and is based on data provided by the gnomad database (genome aggregation database, https://gnomad. broadinstitute.org/), topmed program (trans-omics for precision medicine, https://www.nhlbiwgs.org/), exac consortium (exome aggregation consortium, http://exac.broadinstitute.org/) [33] and the 1000g project (1,000 genomes project, http://www. internationalgenome.org/) [34] (for detailed information see supplementary table 1) . we further utilized pcaggs-based expression vectors for vesicular stomatitis virus (vsv) glycoprotein (g), mers-cov s wildtype (wt) and mers-cov s (d510g) (the latter two either untagged or equipped with a c-terminal v5 epitope) that have been described elsewhere [35] [36] [37] . in addition, a previously described expression vector for angiotensin converting enzyme 2 was employed [38] . similar to the strategy used for the generation of dpp4 mutants, we employed the overlap-extension pcr technique to introduce a single mutation into the mers-cov s open reading frame, thus generating untagged and v5-tagged mers-cov s (d539n). soluble s comprising the s1 subdomain of mers-cov s (aa residues: 1-747) fused to a human igg fc tag was generated by inserting the pcr-amplified s1 sequences into the pcg1fc vector [39] (kindly provided by georg herrler, university of veterinary medicine hannover) making use of the bamhi/sali restriction sites. in addition, we generated an expression vector for the enhanced green fluorescent protein (egfp) by inserting the egfp coding sequence, which was pcr-amplified from the pegfp-c1 vector (clontech), into the pcaggs plasmid using the ecori/xhoi restriction sites. all pcr-amplified sequences were subjected to automated sequence analysis (microsynth seqlab) to verify their integrity. sequences of primers used for cloning of the different constructs are available upon request. analysis of dpp4 surface expression by immunofluorescence analysis bhk-21 cells were grown on coverslips and transfected with the different dpp4 constructs or empty expression vector using icafectin-441 (in-cell-art) at 24 h post seeding according to the manufacturer's instructions. after changing the culture medium at 4 h post transfection, the cells were incubated for additional 20 h. then, the culture medium was aspirated and the cells were washed with pbs, before they were fixed by incubated with pbs containing 4% paraformaldehyde (pbs/pfa) for 15 min at room temperature. subsequently, the cells were washed with 0.1 m glycine/ pbs solution followed by a washing step with pbs. next, the coverslips were incubated with anti-dpp4 antibody (mouse, diluted 1:200 in pbs containing 1% bovine serum albumin [pbs/bsa], abcam) for 1 h at 4°c. for this, the coverslip was put on a drop (20 µl) of antibody solution that was added on a sheet of parafilm inside a humidity chamber (a glass dish in which the parafilm was placed on wet paper tissue). thereafter, the cells were washed 3x with pbs before incubation with alexafluor568-conjugated antimouse antibody (goat, 1:1000, diluted in pbs/bsa, thermofisher scientific) for 30 min at 4°c was performed. subsequently, the cells were washed 3x with pbs. finally, the cells were incubated with dapi (4',6-diamidino-2-phenylindole, carl roth) and mounted in prolong gold antifade mountant (ther-mofisher scientific) before they were analyzed using a zeiss lsm800 (zeiss) confocal laser scanning microscope and the zen imaging software (zeiss). analysis of dpp4 surface expression by flow cytometry bhk-21 cells were transfected with expression vectors for wt or mutant dpp4, or empty expression vector (negative control). at 16 h post transfection, the culture medium was replaced and the cells were further incubated for additional 32 h. then, the cells were washed with pbs, resuspended in pbs/ bsa and pelleted by centrifugation (600 x g, 5 min, 4°c). after aspiration of the supernatant, the cells were resuspended in pbs/bsa containing anti-dpp4 antibody (mouse, diluted 1:100, abcam) and incubated for 1 h at 4°c. next, the cells were pelleted, washed with pbs/bsa, pelleted again, resuspended in pbs/bsa containing alexafluor488-conjugated anti-mouse antibody (donkey, diluted 1:500, thermo-fisher scientific) and incubated for 1 h at 4°c. subsequently, the cells were washed (as described above) and resuspended in pbs/pfa for 2 h at 4°c for fixation. finally, the cells were washed (as described above) and resuspended in pbs/bsa for flow cytometric analysis using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of dpp4 surface expression, the mean fluorescence intensity (mfi) value of the negative control was subtracted from all samples. for normalization of dpp4 surface expression, values obtained for cells expressing dpp4 wt were set as 100% and the relative surface expression of the respective dpp4 mutants was calculated accordingly. in order to generate soluble mers-cov s for binding studies, 293t cells were transfected with an expression vector for the s1 subunit of mers-cov s fused to the fc fragment of human immunoglobulin g (solmers-s1-fc). at 24 h post transfection, the culture medium was exchanged and the cells were further incubated for 24 h before culture supernatants were harvested and freed from cellular debris by centrifugation (4,700 x g, 10 min, 4°c). the clarified supernatants were loaded on vivaspin protein concentrator columns with a molecular weight cut-off of 30 kda (sartorius) and centrifuged at 4,700 x g at 4°c until the sample was 10-fold concentrated. for the binding studies with solmers-s1-fc, a similar protocol was followed as described for the analysis of dpp4 surface expression with the exceptions that sol-mers-s1-fc was used instead of the primary antibody (1:10 dilution in pbs/bsa) and that an alexafluor488conjugated anti-human antibody (goat, 1:500 dilution in pbs/bsa, thermofisher scientific) was employed as the secondary antibody. bhk-21 cells transfected with expression vectors for wt or mutant dpp4, ace2 or empty expression vector (both negative controls) were analyzed by flow cytometry for solmers-s1-fc binding using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of solmers-s1-fc binding, the mfi value obtained for cells transfected with empty expression vector was subtracted from all samples. further, binding of solmers-s1-fc to cells expressing dpp4 wt was set as 100% and the relative binding efficiencies to cells expressing the respective dpp4 mutants or ace2 were calculated accordingly. 293t cells (grown in 6-well plates) were cotransfected with expression plasmids coding for solmers-s1-fc and wt or mutant dpp4. cells transfected with empty expression vector instead of dpp4 or sol-mers-s1-fc (or both) served as controls. at 16 ,400 x g at 4°c, before 400 µl of the supernatant were mixed with 50 µl of protein a-sepharose (1 g protein a-sepharose [sigma-aldrich] in 4 ml pbs) while the residual 100 µl of the cell lysate were mixed with 100 µl 2x sds-sample buffer and incubated for 15 min at 96°c (these samples were later analyzed to confirm comparable total protein levels [via detection of actb] as well as comparable dpp4 and solmers-s1-fc levels before the co-immunoprecipitation [co-ip] step.). following incubation of the lysate/protein a-sepharose mixtures for 2 h at 4°c in an overhead shaker, the samples were centrifuged for 5 min at 16,400 x g at 4°c to pellet the protein a-sepharose/solmers-s1-fc/dpp4-complexes. after aspiration of the supernatant, 500 µl of np40 lysis buffer (without protease inhibitors) were added and the cells were mixed by vortexing, before being centrifuged again. this washing routine was repeated three times, before finally 50 µl of 2x sdssample buffer were added to the pelleted complexes and the samples were further incubated for 15 min at 96°c. thereafter, the samples were subjected to sds-page and western blot analysis (see above). detection of dpp4 (lysate and co-ip samples) and actb (lysate samples) was carried out as described above. solmers-s1-fc was detected (lysate and co-ip samples) by incubation with a peroxidase-conjugated anti-human antibody (goat, 1:5,000, dianova). signal intensities of the protein bands were quantified as described above. further, signals obtained for dpp4 were normalized against the respective signals for solmers-s1-fc in order to account for variations in transfection efficiency and sample processing. for the binding studies with soluble dpp4, a similar protocol was followed as described for the analysis of binding of solmers-s1-fc with the exceptions that a soluble dpp4 fused to the fc region of human igg (soldpp4-fc, acro biosystems) was used instead of solmers-s1-fc (1:200 dilution in pbs/bsa) and that an alexafluor488-conjugated anti-human antibody (goat, 1:500 dilution in pbs/bsa, thermofisher scientific) was employed as the secondary antibody. 293t cells transfected with expression vectors for wt or mutant (d510g and d539n) mers-cov s, or empty expression vector (negative control) were analyzed by flow cytometry for soldpp4-fc binding using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of soldpp4-fc binding, the mfi value obtained for cells transfected with empty expression vector was subtracted from all samples. further, binding of soldpp4-fc to cells expressing mers-cov s wt was set as 100% and the relative binding efficiencies to cells expressing the respective mers-cov s mutants were calculated accordingly. we employed a previously described protocol for the generation of vsv pseudotype particles (vsvpp) that is based on a replication-deficient vsv vector that lacks the genetic information for vsv-g but instead contains the genetic information for egfp and firefly luciferase (fluc) as reporters of transduction efficiency (vsv*δg-fluc, kindly provided by gert zimmer, institute of virology and immunology, mittelhäusern/switzerland) [37, 40] . in brief, 293t cells transfected with expression vectors for mers-cov s, vsv-g (positive control) or empty expression vector (negative control) were inoculated with vsv*δg-fluc for 1 h before being washed with pbs and further incubated for 16 h with culture medium that was supplemented with anti-vsv-g antibody (i1, mouse hybridoma supernatant from crl-2700; atcc) (except for cells expressing vsv-g). the produced vsvpp were inoculated onto bhk-21 cells expressing wt or mutant dpp4, or no dpp4 (empty expression vector, negative control) and incubated for 16-18 h before fluc activity in cell lysates was quantified as an indicator for transduction efficiency using the beetle-juice kit (pjk) and a plate luminometer (hidex) [41] . bhk-21 cells were transfected with expression vectors for wildtype or mutant dpp4 (k267e or a291p), or empty expression vector (negative control) using fugene hd (promega) according to the manufacturer's instructions. at 24 h posttransfection, the cells were infected with mers-cov (human betacoronavirus 2c emc/2012, mers-cov emc-2012, genbank accession number: jx869059) at a multiplicity of infection of 0.01 for 1 h. thereafter, the inoculum was removed and the cells were washed 3x with pbs before fresh medium was added and the first sample (time point 0 h postinfection) was taken. the cells were further incubated and additional samples were taken at 24 and 48 h postinfection. viral titers in the culture supernatant were analyzed by quantitative reversetranscriptase pcr, using the upe assay according to a published protocol [42] . in brief, viral rna was isolated from cell culture supernatant using the nucleospin rna virus kit (macherey-nagel), reversetranscribed into cdna using the superscript iii one step rt-pcr system (thermofisher scientific) and analyzed on a lightcycler 480 qpcr cycler platform (roche) with primers and conditions as specified for the upe assay [42] . in vitro-transcribed standard samples containing defined amounts of mers-cov fragments (10, 100, 1,000 and 10,000 copies) were included for absolute quantification as genome equivalents (ge). the dpp4 protein structure (4pv7) [43] and the structure of the complex formed by the mers-cov s receptor binding domain bound to dpp4 (4l72) [16] were retrieved from the research collaboratory for structural bioinformatics protein database (rscb pdb, https://www.rcsb.org/). structure visualization and colorization was performed using the yasara software (http://www.yasara.org/index.html) [44] and ucsf chimera version 1.14 (developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco) [45] . one-way or two-way analysis of variance (anova) with dunnett's posttest was used to test for statistical significance. only p values of 0.05 or lower were con identification of polymorphisms in dpp4 that alter amino acid residues which make contact with mers-cov s the binding interface between mers-cov s and the cellular receptor dpp4 was resolved by wang and colleagues using crystallography, revealing the interacting amino acid residues for each binding partner [16] : fourteen residues of mers-cov s (y499, n501, k502, l506, d510, r511, e513, d537 g538, d539, y540, r542, w553 and v555) interact with a total of fifteen residues in dpp4 (k267, f269, q286, t288, a289, a291, l294, i295, h298, r317, y322, r336, q344, i346 and k392) [16] , which are distributed over the glycosylation-rich domain and the cysteinerich domain ( figure 1a-c) . in order to identify polymorphic residues in dpp4 that contact mers-cov s, we screened public databases that provide information on polymorphic amino acid residues based on data derived from different bio projects (i.e. gnomad, topmed, exac, 1000g; more information is given in the materials and methods section). by this method we found that nine out of the fifteen dpp4 residues interacting with mers-cov s are polymorphic (k267, q286, t288, a289, a291, r317, y322, i346 and k392) ( figure 1c ). while five of these residues can be replaced by only a single different amino acid residue (q286[k], a289 . circles with sticks represent glycosylation sites, while small numbers indicate the amino acid residues. triangles below the domains highlight the positions of amino acid residues that directly interact with mers-cov s (grey triangles mark residues for which no polymorphism has been reported, while red triangles indicate polymorphic residues). (b) side (left) and top (right) view of homodimeric dpp4 (the dotted line indicates the border between the two monomers and the cellular plasma membrane is schematically depicted below the side view model of dpp4). the protein model was constructed on the published crystal structure (4pv7) deposited in rscb pdb and the binding interface with mers-cov s has been highlighted (green). (c) close-up on the dpp4 residues that directly interact with mers-cov s and for which no polymorphic (yellow) or polymorphic (red) residues have been reported. in addition, the specific residues in dpp4 (regular letters and numbers), including the respective polymorphic residues (letters in brackets), and the corresponding interacting residues in mers-cov s (italicized letters and numbers) are indicated. (d) frequency of polymorphic dpp4 residues in the human population. public databases (see supplementary table 1 and the materials and methods section for detailed information) were screened for the frequency of the polymorphic residues under study (y-axis). error bars indicate standard error of the mean (sem) and refer to polymorphic residues found in more than one database. table 1 ). finally, the frequency of these polymorphisms in the human population is low, ranging roughly from 1:19,000 (a289v) to 1:245,000 (t288i) ( figure 1d and supplementary table 1 ). we next introduced the polymorphisms in a dpp4 expression plasmid. western blot analysis and signal quantification revealed that all resulting dpp4 variants were robustly expressed and total expression levels were comparable (figure 2a-b) . as dpp4 needs to be transported to the plasma membrane to be engaged by mers-cov s for host cell entry, we next investigated whether the presence of the polymorphic dpp4 residues has an impact on dpp4 cell surface localization. for this, we performed flow cytometry and confocal laser scanning microscopy, using transfected bhk-21 cells and an antibody targeting the dpp4 ectodomain. we found that all dpp4 variants but one, a deletion variant lacking amino acid residues 346-348 (δ346-348), displayed comparable cell surface expression levels ( figure 3a-b) . we next investigated whether polymorphic dpp4 residues impact mers-cov host cell entry. for this, we made use of vesicular stomatitis virus (vsv) pseudotypes (vsvpp) bearing mers-cov s or vsv g, which does not bind to dpp4 and served as negative control [46] . as expected, vsvpp harboring vsv g were able to efficiently transduce bhk-21 target cells irrespective of dpp4 expression. in contrast, transduction of bhk-21 cells mediated by mers-cov s critically depended on ectopic expression of human dpp4, in accordance with published findings [47] ( figure 4) . notably, four dpp4 polymorphisms -k267e, k267n, a291p and δ346-348 -severely reduced mers-cov s-driven transduction compared to dpp4 wt (figure 4 ). in order to analyze whether the reduction in mers-cov s-driven host cell entry would translate into attenuated mers-cov replication, we next investigated two dpp4 polymorphisms (k267e and a291p) in the context of infection with authentic mers-cov. when followed over a period of two days post infection it was observed that mers-cov replication in bhk-21 cells expressing human dpp4 was significantly reduced when dpp4 contained either k267e or a291p ( figure 5 ). after the identification of dpp4 polymorphisms that reduce s-driven cellular entry of rhabdoviral vectors as well as mers-cov replication, we next sought to investigate whether the attenuating phenotype was due to reduced binding of mers-cov s to dpp4. for this, we used soluble mers-cov s, produced by fusing the s1 subunit, which contains the dpp4 binding domain, to the fc portion of human immunoglobulin g (solmers-s1-fc). co-immunoprecipitation analysis demonstrated that dpp4 variants harboring polymorphisms k267e, k267n or a291p, which were not compatible with efficient mers-cov s-driven host cell entry, displayed significantly reduced ability to interact with mers-cov s as indicated by weaker dpp4 signals upon protein a-sepharosemediated pull-down of dpp4/solmers-s1-fc (as compared to dpp4 wt, figure 6a-b) . notably, dpp4 variant δ346-348 could be as efficiently coimmunoprecipitated as dpp4 wt, indicating that its inefficient receptor function was solely due to its defect in proper surface transport. the findings obtained by co-ip analysis were confirmed by flow cytometry. it was revealed that polymorphisms that reduced mers-cov s-driven host cell entry (k267e, k267n, a291p and δ346-348) and spread of authentic mers-cov (k267e and a291p) also reduced mers-cov s binding to cells expressing dpp4 on the cell surface ( figure 6c ). in addition, polymorphism a289v, which decreased mers-cov s-driven transduction to a lesser extent than the aforementioned polymorphisms (figure 4) , also reduced mers-cov s binding to dpp4. thus, dpp4 polymorphisms k267e, k267n and a291p reduce mers-cov s-driven host cell entry and mers-cov infection by diminishing mers-cov s binding to dpp4. host cell entry of mers-cov critically depends on the interaction between the viral s protein and the cellular receptor dpp4. a link between obesity or underlying diseases like diabetes mellitus, which both can affect dpp4 expression levels [48] , and the risk of fatal outcome of mers-cov infection has been made [49] . moreover, alanine scanning mutagenesis identified dpp4 residues critical for mers-cov entry, including k267, l294, i295, r317 and r336 [50, 51] . however, the impact of natural-occurring variations on host cell entry of mers-cov has not been addressed so far. we identified dpp4 polymorphisms that reduce s protein-driven host cell entry and replication of authentic mers-cov by lowering the binding efficiency of mers-cov s to dpp4, suggesting that the dpp4 phenotype may impact the course of mers-cov infection. western blot analysis, flow cytometry and confocal laser scanning microscopy revealed that none of the polymorphisms studied, except deletion of amino acids 346-348, had a significant impact on total or cell surface expression of dpp4, at least in the context of dpp4 transfected cells. four polymorphisms located at three different sites in dpp4 (k267e, k267n, a291p and δ346-348) severely reduced s protein-driven host cell entry. as dpp4 δ346-348 was shown to be incompatible with robust cell surface transport but able to interact with mers-cov s in co-ip analysis, we conclude that the reduction in entry efficiency is solely due to insufficient dpp4 surface levels. in contrast, reduction of host cell entry by k267e, k267n and a291p could not be explained by reduced dpp4 expression and these polymorphisms were thus further investigated. mers-cov infection of bhk-21 cells transfected to express dpp4 wt and variants k267e or a291p revealed that k267e or a291p were not compatible with robust mers-cov replication. finally, co-ip analyses and binding studies with soluble mers-cov s showed that these dpp4 polymorphisms reduced s protein binding to dpp4. when looking at the crystal structure of the complex consisting of the mers-cov s receptor binding domain bound to dpp4, these observations do not come as a surprise. dpp4 residue k267 has been reported to contact mers-cov s residues g538 and d539, including a salt bridge interaction with d539 [16] . the exchange of k267 to either glutamate (e) . whole cell lysates (wcl) were prepared and analyzed for dpp4 expression by sds-page under non-reducing conditions and wb using a primary antibody targeting the c-terminal cmyc epitope and a peroxidase-conjugated secondary antibody. further, expression of beta-actin (actb) was analyzed as a loading control. shown are the expression data from a representative experiment. numbers at the left indicate the molecular weight in kilodalton (kda). (b) quantification of total dpp4 expression in wcl. after normalization of dpp4 band intensities with that of the corresponding actb bands. dpp4 wt expression was set as 100% and the relative expression of mutant dpp4 was calculated accordingly. presented are the combined data of three independent experiments with error bars indicating the sem. no statistical significance for differences in total expression between wt and mutant dpp4 was observed by one-way analysis of variance with dunnett's posttest (p > 0.05, not significant [ns]). . surface expressed dpp4 was stained by subsequent incubation of the non-permeabilized cells with a primary antibody that targets the dpp4 ectodomain and an alexafluor488-conjugated secondary antibody. fluorescent signals representing surface-expressed dpp4 were analyzed by flow cytometry and the mean fluorescence intensity (mfi) values for each sample were calculated. for normalization, the mfi value of the negative control was subtracted from all samples. further, surface expression of dpp4 wt was set as 100% and the relative surface expression of the dpp4 mutants was calculated accordingly. shown are the combined data of three experiments with error bars indicating the sem. statistical significance for differences in surface expression between wt and mutant dpp4 was tested by one-way analysis of variance with dunnett's posttest (p > 0.05, not significant; p ≤ 0.05, *). (b) dpp4 surface expression was further analyzed by immunofluorescence analysis. for this, dpp4 wt or dpp4 mutants were expressed in bhk-21 cells grown on coverslips (cells transfected with empty expression vector served as negative control). after fixation of the cells, surface expressed dpp4 was stained by subsequent incubation of non-permeabilized cells with a primary antibody that targets the dpp4 ectodomain and an alexafluor568-conjugated secondary antibody. in addition, cellular nuclei were stained with dapi. finally, images were taken using a confocal laser scanning microscope at a magnification of 80x. or asparagine (n) likely abolishes/decreases the interaction with mers-s due to the different biochemical properties of k267 (positively charged, basic) versus e267 (negatively charged, acidic) and n267 (not charged, acidic) (supplementary figure 1) . for dpp4 residue a291, which has been reported to contact the mers-cov s residue e513, no information on the type of interaction is available [16] . here, we speculate that the bulky and distorted side chain of proline (in comparison to the small side chain of alanine) abolishes/decreases interaction with mers-cov s residue e513 (supplementary figure 1) . in contrast to that, valine contains a small side chain and also has identical biochemical properties as alanine and thus might be efficiently contacted by e513 of mers-cov s, which is why we did not observe any impact of polymorphisms a291v on mers-cov s-driven entry and mers-cov s mers-cov s binding/interaction (supplementary figure 1 ). the observation that certain polymorphisms in dpp4 reduced mers-cov s binding and viral entry triggered the question whether residues in mers-cov s that are in direct contact with the respective dpp4 residues are also polymorphic. indeed, we obtained initial evidence to support such a concept. thus, we found that residue 539 in mers-cov s which contacts dpp4 residue 267 is polymorphic, with certain mers-cov variants harboring an asparagine instead of an . reduced mers-cov s-driven host cell entry is caused by inefficient s protein binding to dpp4 harboring polymorphic amino acid residues. in order to investigate whether reduced mers-cov s-driven host cell entry and mers-cov replication is due to inefficient mers-cov s binding to dpp4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-ip) as well as binding experiments with a soluble s protein comprising the s1 subunit of mers-cov s fused to the fc region of human igg. (a) 293t cells were cotransfected with expression plasmids coding for soluble, fc-tagged mers-cov s1 (solmers-s1-fc) and the indicated dpp4 variant containing a c-terminal cmyc-tag. cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solmers-s1-fc or dpp4 served as controls. at 48 h posttransduction, cells were lysed and incubated with protein a sepharose. next, samples were subjected to sds-page and western blot analysis. dpp4 levels were detected via antibodies specific for the cmyc-tag, whereas solmers-s1-fc was detected using a peroxidase-coupled anti-human antibody. similar results were obtained in three individual experiments. analysis of whole cell lysates (wcl) for expression of solmers-s1-fc, dpp4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solmers-s1-fc and dpp4. (b) for quantification of mers-cov s/dpp4 interaction we first normalized the dpp4 signals against the respective solmers-s1-fc signals. then, mers-cov s/dpp4 interaction was set as 100% for wildtype (wt) dpp4 and the relative interaction efficiency for each dpp4 mutant was calculated accordingly. presented are the mean data from three independent experiments. error bars indicate the sem. statistical significance of differences in mers-cov s/dpp4 interaction between wt and mutant dpp4 was analyzed by one-way analysis of variance with dunnett's posttest (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (c) soluble mers-cov s1-fc was incubated with bhk-21 cells expressing wildtype (wt) or mutant dpp4, or cells transfected with empty expression vector or an ace2-expression plasmid (controls). to detect bound s protein, the cells were subsequently incubated with an alexafluor488-conjugated anti-human antibody directed against the fc-tag. fluorescent signals representing bound solmers-s1-fc were analyzed by flow cytometry and mfi values for each sample were calculated. for normalization, the mfi value of the negative control (empty expression vector) was subtracted from all samples. further, binding of sol-mers-s1-fc to cells expressing dpp4 wt was set as 100% and the relative binding to cells expressing the dpp4 mutants was calculated accordingly. shown are the combined data of five independent experiments with error bars indicating the sem. statistical significance of differences in solmers-s1-fc binding to cells expressing wt or mutant dpp4 was analyzed by one-way analysis of variance with dunnett's posttest (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). aspartate residue at this position. d539n reduced entry into cells expressing relatively low amounts of dpp4 but had no effect on entry into cells expressing high amounts of dpp4 (supplementary figure 2) . moreover, and more interestingly, d539n slightly rescued mers-cov s-driven entry from the negative effect exerted by dpp4 polymorphism k267n (supplementary figure 2) . similarly, residue 510 in mers-cov s, which is known to interact with dpp4 residues 317 and 322, was found to be polymorphic, and previous studies demonstrated that polymorphism d510g reduced dpp4 binding but also increased resistance to neutralizing antibodies [37] . notably, d510g slightly increased entry via dpp4 harboring polymorphism y322h and allowed mers-cov s to use dpp4 with polymorphism r317k with the same efficiency as wt dpp4. it should be stated that none of these effects was statistically significant and that dpp4 and mers-cov s polymorphisms occur with low frequency. although it is unlikely that the dpp4 polymorphisms have emerged as a result of evolutionary pressure from mers-cov infections, our results suggest that certain existing dpp4 polymorphism(s) might foster the emergence of mers-cov variants with altered biological properties. the polymorphisms studied here occur with relatively low frequencies of one per ∼19,000 (a289v) to ∼245,000 (t288i) individuals. however, detailed information on the geographic distribution or incidence in certain ethnical groups is largely missing. thus, dpp4 polymorphisms could contribute to the perplexing absence of mers cases in africa, where the virus circulates in camels [52] [53] [54] [55] [56] [57] . however, recent evidence suggests that sequence variations between african and arabian mers-cov might be a factor [53, 57] . more importantly, it remains to be analyzed how frequent dpp4 polymorphisms that affect s protein binding occur in the middle east and 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respiratory syndrome coronavirus disease from saudi arabia: a descriptive study identification of residues on human receptor dpp4 critical for mers-cov binding and entry dipeptidyl peptidase-4 levels are increased and partially related to body fat distribution in patients with familial partial lipodystrophy type 2 middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses from camels in africa exhibit region-dependent genetic diversity antibodies against mers coronavirus in dromedary camels mers coronavirus neutralizing antibodies in camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus in dromedaries in ethiopia is antigenically different from the middle east isolate emc we are grateful to thank g. herrler, a. maisner and g. zimmer for providing plasmids and reagents. we further thank a.-s. moldenhauer for excellent technical support. no potential conflict of interest was reported by the authors. this work was supported, including the efforts of stefan pöhlmann and christian drosten, by the bundesministerium für bildung und forschung [grant numbers 01ki1723d and 01ki1723a], network project rapid (risikobewertung bei präpandemischen respiratorischen infektionserkrankungen). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord-256146-d599uera authors: kuiken, thijs; fouchier, ron am; schutten, martin; rimmelzwaan, guus f; van amerongen, geert; van riel, debby; laman, jon d; de jong, ton; van doornum, gerard; lim, wilina; ling, ai ee; chan, paul ks; tam, john s; zambon, maria c; gopal, robin; drosten, christian; van der werf, sylvie; escriou, nicolas; manuguerra, jean-claude; stöhr, klaus; peiris, j s malik; osterhaus, albert dme title: newly discovered coronavirus as the primary cause of severe acute respiratory syndrome date: 2003-07-26 journal: lancet doi: 10.1016/s0140-6736(03)13967-0 sha: doc_id: 256146 cord_uid: d599uera background: the worldwide outbreak of severe acute respiratory syndrome (sars) is associated with a newly discovered coronavirus, sars-associated coronavirus (sarscov). we did clinical and experimental studies to assess the role of this virus in the cause of sars. methods: we tested clinical and postmortem samples from 436 sars patients in six countries for infection with sarscov, human metapneumovirus, and other respiratory pathogens. we infected four cynomolgus macaques (macaca fascicularis) with sars-cov in an attempt to replicate sars and did necropsies on day 6 after infection. findings: sars-cov infection was diagnosed in 329 (75%) of 436 patients fitting the case definition of sars; human metapneumovirus was diagnosed in 41 (12%) of 335, and other respiratory pathogens were diagnosed only sporadically. sars-cov was, therefore, the most likely causal agent of sars. the four sars-cov-infected macaques excreted sars-cov from nose, mouth, and pharynx from 2 days after infection. three of four macaques developed diffuse alveolar damage, similar to that in sars patients, and characterised by epithelial necrosis, serosanguineous exudate, formation of hyaline membranes, type 2 pneumocyte hyperplasia, and the presence of syncytia. sars-cov was detected in pneumonic areas by virus isolation and rt-pcr, and was localised to alveolar epithelial cells and syncytia by immunohistochemistry and transmission electron microscopy. interpretation: replication in sars-cov-infected macaques of pneumonia similar to that in human beings with sars, combined with the high prevalence of sars-cov infection in sars patients, fulfill the criteria required to prove that sars-cov is the primary cause of sars. published online july 22, 2003 http://image.thelancet.com/extras/03art6318web.pdf severe acute respiratory syndrome (sars) is an emergent disease that was first reported in guangdong province, people's republic of china, in november, 2002, from where it spread to other asian countries, north america, and europe. [1] [2] [3] [4] [5] by july 3, 2003, this epidemic had resulted globally in 8439 reported cases, of which 812 were fatal. 6 pulmonary lesions in sars patients have been diagnosed as diffuse alveolar damage. histological changes include desquamation of pneumocytes, formation of hyaline membranes, flooding of alveolar lumina with oedema fluid mixed with inflammatory cells, and the presence of enlarged pneumocytes and syncytia. alveolar walls are thickened by mild mononuclear infiltrate, and in later stages air spaces contain fibromyxoid-organising exudate. 2, 4, 5, 7, 8 coronaviruslike particles have been detected by transmission electron microscopy in cells from a lung biopsy sample 2 and a bronchoalveolar lavage sample, 7 and in pneumocytes from a postmortem lung sample. 8 until now, immunohistochemical detection of sars-associated coronavirus (sars-cov) in sars-associated lesions has not succeeded. 7 identification of the causal agent of sars is essential to make an accurate case definition, to diagnose the disease accurately, and to develop appropriate preventive and curative treatment. several agents have been proposed in the course of investigation of sars. during initial investigations in china, mycoplasma pneumoniae and chlamydia sp were suggested as possible causes. 9, 10 subsequent studies ruled out these agents and other known viral and bacterial pathogens, 3,4 except for human metapneumovirus. 5 a previoulsy unknown coronavirus was identified in patients with sars. 2, 7, 11 it was suggested that this coronavirus, alone or in combination with human metapneumovirus or other causal agents, might be the primary cause of sars. we investigated the causal role of the newly discovered sars-cov by analysis of the results of investigations done by the who network of laboratories, showing that sars-cov was the only agent seen consistently in patients with probable sars. we also investigated whether respiratory lesions of sars could be replicated in experimentally infected cynomolgus macaques (macaca fascicularis). replication was based on histopathology, as in a preliminary report, 12 and on localisation of sars-cov to the typical pulmonary lesions by immunohistochemistry and transmission electron microscopy. newly discovered coronavirus as the primary cause of severe acute respiratory syndrome all patients in this study fitted the who case definition for sars (table 1). 1 some of the data on various proportions of patients in cohorts 1, 2 2, 13 3, 8 [5] [6] [7] [8] 7 and 9, 11, and 12 11 have been published previously. antibody to sars-cov was detected in paired acute and convalescent sera by use of an indirect immunofluorescence assay 2 for patients in all cohorts. in addition, an enzyme-linked immunosorbent assay 7 was used with minor modifications in cohorts 8 and 10. antibody to human metapneumovirus was detected in paired acute and convalescent sera by use of an immunofluorescence assay on human-metapneumovirusinfected fetal rhesus monkey kidney (frhk-4) cells 14 for 32 patients in cohort 1, and for all patients who were human-metapneumovirus culture positive in cohorts 3 and 4. iga-specific and igg-specific eia were used for the remaining five patients in cohort 1, and for all patients in cohort 8. an iga-specific eia was used for patients in cohort 5. these tests are under development for commercial distribution (meddens diagnostics, vorden, netherlands). the clinical samples from which rna extracts were obtained varied among cohorts and included swabs from nose, pharynx, or conjunctiva, aspirates from nasopharynx or trachea, bronchoalveolar lavage fluid, faeces, urine, sputum, and blood. samples included postmortem lung tissue in cohort 3, and from various organs in cohort 8. the rt-pcr for sars-cov on clinical samples was done according to peiris and colleagues' method 2 for cohorts 1-3, and according to that by drosten and colleagues 11 for cohorts 9, 11, and 12. comparable tests that used primer pair cor-1/cor-2 were used for cohorts [5] [6] [7] 15 primer pairs cor-p-f3/cor-p-r1 and 2bp/4bm for cohort 10, 15, 16 and primer pairs cor-1/cor-2 and sar1s/sar1 as for cohort 8. 15 virus isolation procedures for sars-cov were done by inoculation of samples on to vero cells for cohorts 4-7, and on to hela, human embryonic lung, llc, madin-darby canine kidney cells, and vero cell lines for cohort 8. presence of virus was confirmed by immunofluorescence, rt-pcr, or both. the rt-pcr for human metapneumovirus on clinical samples was done according to peiris and colleagues' method 2 for cohorts 1 and 3, with use of primer pair l6 (5ј-catgcccactataaaaggtcag-3ј) and l7 (5ј-caccccagtctttcttgaaa-3ј) for cohorts 5-7, according to the method of peret and colleagues, 17 with conventional and real-time pcr for cohort 8, with use of a family-specific primer pair according to drosten and colleagues 11 for cohort 9, for cohort 10 with use of n and f gene-based primers, and by nested pcr with use of the outer primer pair p9 (5ј-gatcaacatatct tcagtccagac-3ј) and p10 (5ј-aaaagcatgatc cgatatgaaccc-3ј) and l6 and l7 as inner primer pair for cohorts 11 and 12. in addition, for cohort 8 samples were inoculated onto hela, human embryonic lung, llc, madin-darby canine kidney, and vero cell lines for 28 days at 33ºc. for cohort 4, nasopharyngeal aspirate samples were inoculated onto llc-mk2 rhesus monkey kidney cell and human laryngeal carcinoma cell (hep-2) monolayers and incubated at 37ºc for 12-14 days (hep-2 cells) or 21 days (llc-mk2 cells) in a roller tube culture system. these cell lines were selected based on the results of an initial assessment. 18 irrespective of the presence of cytopathic effect, cell culture supernatants were testd for human metapneumovirus by nested rt-pcr with use of the outer primer pair (5ј-agctgttccattgg cagca-3ј) and (5ј-atgctgttcrccytcaac ttt-3ј; r=a or g, y=c or t) and the inner primer pair (5ј-gagtagggatcatcaagca-3ј) and (5ј-gct tagctgrtatacagtgtt-3ј). all pcr products were confirmed by nucleotide sequencing. in addition, all cell cultures positive for human metapneumovirus rtpcr were passaged on to another llc-mk2 rhesus monkey kidney cell culture tube and incubated for 21 days. randomly selected supernatants of cell cultures with cyopathic effect were examined by electron microscopy for the presence of virus particles. we made the virus stock used to inoculate cynomolgus macaques from the fourth passage of a sars-cov isolate, obtained from patient 5688, who died of sars, and inoculated it on to vero 118 cells cultured in iscove's modified dulbeco's medium (bio whitaker, walkersville, md, usa). after centrifugation at 270 g for 5 min, 1 ml samples were made from the supernatant and from the pelleted cells, which were resuspended in 5 ml medium. the titre of this virus stock was 1ϫ10 6 median tissue culture infectious dose (tcid 50 ) per ml. all cell cultures were done under biosafety level 3 conditions. four adult cynomolgus macaques, two males and two females, were placed in negatively pressurised glove boxes. they were infected with 1ϫ10 6 tcid 50 of sars-cov suspended in 5 ml phosphate-buffered saline. 4 ml was applied intratracheally, 0·5 ml intranasally, and 0·25 ml on each conjunctiva. we checked the macaques daily for clinical signs. just before infection and at days 2, 4, and 6 after infection, we anaesthetised the macaques with ketamine and collected 10 ml blood from inguinal veins, and took nasal, oral, pharyngeal, and rectal swabs, which were placed in 1 ml virus transport medium. 19 from macaques 3 and 4, we also collected sputum samples by use of swabs under the tongue and sponges placed in the buccal cavity during anaesthesia. we extracted fluid from the sponges by centrifugation. the macaques were euthanised 6 days after infection by exsanguination under ketamine anesthesia. we did necropsies of the macaques according to a standard protocol. for histological examination we collected the following tissues: adrenal gland, aorta, axillary lymph node, brachial biceps muscle, brain stem, caecum, cerebellum, cerebrum, colon, duodenum, eye, tissues for light-microscope examination were fixed in 10% neutral-buffered formalin, embedded in paraffin, and 4 m sections were stained with haematoxylin and eosin. selected lung sections were stained with monoclonal antibody ae1/ae3 (neomarkers, fremont, ca, usa) for the identification of epithelial cells, and monoclonal antibody cd68 (dako, glostrup, denmark) for the identification of macrophages according to standard immunohistochemical procedures. we developed an immunohistochemical method for detecting sars-cov antigen. duplicate sections of all tissue samples were stained with an avidin-biotin complex peroxidase method. paraffin was removed from sections, which were rehydrated and pretreated with protease (sigma, st louis, mo, usa) for 10 min at 37ºc. endogenous peroxidase was revealed with 4-chloro-1-naphthol (sigma). endogenous biotin was blocked with an avidin-biotin blocking kit (vector laboratories, burlingame, ca, usa). slides were briefly washed with 0·05% phosphate-buffered saline tween 20 (fluka, buchs, switzerland) and incubated with biotinylated purified human igg from a convalescent sars patient, negative control biotinylated purified human igg, or the dilution buffer (phosphate-buffered saline containing 1% bovine serum albumin) for 1 h at room temperature. after washing, sections were incubated with avidin-biotin complex-horseradish peroxidase (dako) for 1 h at room temperature. horseradish peroxidase activity was revealed by incubating slides in 3-amino-9-ethylcarbazole (sigma) solution for 10 min, resulting in a bright red precipitate. sections were counterstained with haematoxylin. tissue sections from cynomolgus macaques that had not been infected with sars-cov were included as negative controls. we included formalin-fixed, paraffin-embedded sars-cov-infected or uninfected vero 118 cells in each staining as positive and negative controls, respectively. for negative contrast electron microscopy, samples of tissue culture supernatants were centrifuged at 17 000 g at 4ºc, after which the pellet was resuspended in phosphatebuffered saline and stained with phosphotungstic acid. samples of lung from macaque 3 were fixed for transmission electron microscopy in 4% formaldehyde and 1% glutar(di)aldehyde after brief fixation in 10% neutral-buffered formalin, and post-fixed in 1% osmium tetroxide. after embedding in epoxy resin, thin sections were prepared, stained with 6% saturated uranyl acetate and lead citrate, and examined with a philips morgagni 268d electron microscope. samples from lung, duodenum, jejunum, kidney, liver, and spleen were collected post mortem for virus isolation and rt-pcr. additionally, we collected the following samples for rt-pcr only: cerebellum, cerebrum, ileum, intestinal contents from the colon, mesenteric lymph node, nasal septum, pancreas, skin, spleen, stomach, tonsil, trachea, tracheobronchial lymph node, urinary bladder, and urine. from macaques 3 and 4, samples of intestinal contents from the jejunum, ileum, and caecum were also collected for rt-pcr. intestinal contents were pretreated with stool transport and recovery buffer (roche diagnostics, mannheim, germany) before further processing. tissue samples were homogenised in 4 ml transport medium 19 with polytron pt2100 tissue grinders (kinematica, littau-lucerne, switzerland). after lowspeed centrifugation, the homogenates were frozen at -70ºc until inoculation on vero 118 cell cultures in logarithmic dilutions. the infectious virus titres were expressed as tcid 50 /g of tissue. the identity of the isolated virus was confirmed as sars-cov by rt-pcr of supernatant. we developed an rt-pcr with primers and probe specific for the nucleoprotein gene of sars-cov because the nature of the coronavirus replication cycle 20 suggested that such an assay may be more sensitive than rt-pcrs based on the polymerase gene, which are currently in use for diagnostics. nucleic acid isolation was done on the magnapure lc automated nucleic acid isolation system (roche diagnostics). swabs, faeces, and serum were processed with the magnapure lc total nucleic acid serum plasma blood isolation kit. postmortem tissue samples were processed with the magnapure lc rna isolation kit i on the magnapure lc station using the external lysis protocol. sars-cov rna was detected on the abi prism 7700, with use of the ez rtth rna amplification kit (applied biosystems, foster city, ca, usa). primers sarsnp fpr1 (5ј-caaacattggccg caaatt-3ј), sarsnp rpr1 (5ј-caatgcgtgaca ttccaaaga-3ј), and probe sarsnp prb1 (5ј-cacaatttgctccaagtgcctctgca-3ј) (eurogentec) specific for the nucleoprotein (np) gene of sars-cov were used for amplification. amplification parameters were 2 min at 50ºc, 30 min at 60ºc, 5 min at 95ºc, and 45 cycles of 30 s at 95ºc, and 1 min at 62ºc. we compared the sensitivity of the sars-cov np rtpcr with a sars-cov polymerase rtpcr that used essentially the same methods with primers and probe specific for the sars-cov polymerase (sarstm fpr1 . serial dilutions of the sars-cov virus stock and sars-cov-infected vero cells from patient 5688 were made and tested with the np and polymerase-specific rt-pcrs. samples from the respiratory tract (nasal swabs, pharyngeal swabs, postmortem trachea, and lung samples) were also monitored for influenza a and b virus, respiratory syncytial virus a and b, rhinovirus, coronavirus (oc43 and 229e), and human metapneumovirus with use of essentially the same rt-pcr methods but with specific primers. we detected antibody to sars-cov in macaque sera by use of an indirect immunofluorescence assay. sars-cov-infected vero 118 cells that had developed cytopathic effect were used to coat microscope slides. after incubation of the serum for 30 min at 37ºc, slides were washed with phosphate-buffered saline and incubated with antihuman igg, iga, and igm, conjugated with fluorescein thiocyanate (dako). after washing and drying, slides were examined with a fluorescence microscope (zeiss axioscope, oberkochen, germany). we the sponsors of the study had no role in the study design, data collection, data analysis, data interpretation, or in the writing of the report. overall, 75% of patients who had suspected sars according to the who case definition were diagnosed as being infected with probable sars-cov, whereas 12% were diagnosed as having human metapneumovirus infection (table 2). the high proportion of patients diagnosed with human metapneumovirus infection could be attributed mainly to cohort 4, in which 36% of patients were positive for this infection. without this cohort, the overall proportion of human metapneumovirus diagnoses decreased to 4%. alternative diagnoses in patients who tested negative for sars-cov infection were influenza (five), m pneumoniae infection (one), and legionella sp infection (one). three macaques (1, 2, and 4) became lethargic from days 2-3 after infection onwards. macaques 1 and 2 developed a temporary skin rash at day 4 after infection. macaque 2 had respiratory distress, consisting of an increased respiratory rate and dyspnoa, from day 4 after infection onwards. macaques 2-4 had multiple foci of pulmonary consolidation in both lungs. the consolidated lung tissue was grey-red, firm, level, and less buoyant than normal. the tracheobronchial lymph nodes and spleen in these macaques were about twice the normal size. the other organs in these three macaques, as well as the respiratory tract and other organs of macaque 1 were normal on microscopic inspection. the main lesion in the consolidated pulmonary tissue of macaques 2-4 involved the alveoli and bronchioles, and consisted of areas with acute or more advanced phases of diffuse alveolar damage. in such areas the lumina of alveoli and bronchioles were variably filled with proteinrich oedema fluid, fibrin, erythrocytes, and cellular debris, a moderate number of alveolar macrophages, and fewer neutrophils and lymphocytes (figure 1). the cytoplasm of some of these macrophages contained erythrocytes or pools of oedema fluid. there was extensive loss of epithelium from alveolar and bronchiolar walls. in areas with more advanced diffuse alveolar damage, the alveolar walls were moderately thickened and lined by cuboidal epithelial cells (type 2 pneumocyte hyperplasia), and the alveolar lumina contained mainly alveolar macrophages (figure 1). regeneration of epithelium was seen in some bronchioles, visible as one irregular layer of squamous to high cuboidal epithelial cells with hyperchromatic nuclei. in some areas, the alveolar walls were lined by deep eosinophilic hyaline membranes (figure 1). there were occasional multinucleated giant cells (syncytia) in bronchioles and alveoli, either attached to the wall or free in the lumen (figure 1). these syncytia had up to about 30 peripheral nuclei, abundant hyaline eosinophilic cytoplasm, and, based on positive cd68 staining and negative pan-keratin staining, originated from marcophages. enlarged type 2 pneumocytes with large vacuolated nuclei, prominent nucleoli and abundant vesicular cytoplasm were frequently found attached to the alveolar walls (figure 2). the epithelial origin of these cells was confirmed by keratin expression (figure 2). by contrast, alveolar macrophages had smaller nuclei, less prominent nucleoli, and were generally loose in the alveolar lumina (figure 2). the identity of these cells as macrophages was confirmed by cd68 staining (figure 2). alveolar and bronchiolar walls were thickened by oedema fluid, mononuclear cells, and neutrophils. there were aggregates of lymphocytes around small pulmonary vessels. moderate numbers of lymphocytes and macrophages were present in the lamina propria and submucosa of the bronchial walls, and a few neutrophils in the bronchial epithelium. changes in other tissues of macaques 2-4 were diffuse lymphoid hyperplasia and sinus histiocytosis of the tracheobronchial lymph nodes. macaques 2 and 3 also had diffuse intrafollicular hyalinosis of the spleen. there were minimum multifocal inflammatory lesions in the pulmonary tissue of macaque 1, consisting of increased numbers of alveolar macrophages (about ten per alveolus) and occasional syncytia in alveoli and bronchioles. with use of a biotinylated igg fraction from a sars patient, sars-cov expression was detected in a few to moderate numbers of alveolar epithelial cells (type 2 pneumocytes; figure 3 ) and rare intrabronchiolar and intra-alveolar syncytia ( ultrastructurally, coronavirus-like particles measuring about 70 nm in diameter with typical internal nucleocapsid-like structure and club-shaped surface projections were found in enlarged alveolar epithelial cells (type 2 pneumocytes) of inflamed lung tissue from macaque 3. the particles were localised within smoothwalled vesicles, often closely associated with the golgi apparatus ( figure 4) . the particles in inflamed lung tissue were similar in size and structure to coronavirus particles in vero 118 cells infected with sars-cov ( figure 4) . the np-specific rt-pcr was about 100-fold more sensitive than the polymerase-gene-based rt-pcr on the virus stock and the infected cell dilution series. the np rt-pcr also was more sensitive based on detection of viral rna in samples obtained from the sars-covinfected macaques (data not shown). the macaques shed sars-cov from sputum, nose, and pharynx from 2 days after infection onwards (table 3) . sars-cov was isolated from the nasal and throat swabs from macaque 2 at 2 days after infection, from the throat swabs of macaque 4 at days 2, 4, and 6 after infection, and from sputum samples of macaque 4 at days 2 and 6 after infection. the virus titre in these samples was not measured. several other clinical samples were positive only by rt-pcr (table 3) . by negative-contrast electron microscopy, coronavirus particles were seen in cell cultures obtained from nasal swabs (figure 4) and closely resembled those in the virus stock used to infect the macaques ( figure 4) . virus was not detected in rectal swabs. sars-cov was isolated from the lung (1ϫ10 5 tcid 50 /g tissue) and kidney (1ϫ10 3 tcid 50 /g tissue) of macaque 2, and from the lung (1ϫ10 4 tcid 50 /g tissue) of macaque 4. no virus was isolated from the postmortem samples of macaques 1 or 3. several other tissues were positive only by rt-pcr (table 4) . no macaque had detectable antibody to sars-cov by day 6 after infection. virological examinations of nasal and pharyngeal swabs, and tracheal and lung samples from all four macaques by rt-pcr for influenza a and b virus, respiratory syncytial virus a and b, rhinovirus, coronavirus (oc43 and 229e) and human metapneumovirus were negative. no relevant pathogens were identified on bacteriological culture of lung and blood samples. the lung homogenates tested negative for chlamydia sp and c pneumoniae by pcr. according to koch's postulates, as modified by rivers for virus diseases, 23 six criteria need to be fulfilled for a particular micro-organism to be the causal agent of a disease. laboratory investigations of clinical and postmortem samples from sars patients, as presented here and in earlier studies 2,5,7,11 already fulfilled the first three criteria-isolation of the virus from diseased hosts, cultivation in experimental hosts or host cells, and proof of filterability (to exclude larger pathogens). the results of our studies on sars-cov-infected macaques fulfill the remaining postulates-production of a comparable disease in the original host or a related species, and reisolation of the virus. detection of a specific immune response to the virus after experimental infection was already reported. 12 together, these findings prove that sars-cov is the primary cause of sars. the primary role of sars-cov in the cause of sars is suggested by the cumulative studies at who laboratories, in which most sars patients were diagnosed as having sars-cov infection, frequently in the absence of other respiratory pathogens. the most common co-infection in sars patients was with human metapneumovirus. the sars patients in cohort 4 co-infected with human metapneumovirus were mostly health-care workers from the same ward and who shared resting areas. the circulation of this infection among them during the sars outbreak probably explains the frequency of the infection in cohort 4. similarly, four sars patients from canada were infected with sars-cov and human metapneumovirus. 5 the clinical symptoms of human metapneumovirus infection vary from mild upper-respiratory-tract disease to severe bronchiolitis and pneumonia. 24 the possible role of human metapneumovirus infection in exacerbating sars remains to be assessed. alternative diagnoses, such as influenza, were occasionally made among patients fitting the case definition of sars but testing negative for sars-cov infection. on the basis of the current case definition, therefore, disease from sars-cov infection overlaps with respiratory illnesses of other causes. alternative diagnoses are most likely in geographical areas where sars is not endemic. the pulmonary lesions in sars-cov-infected macaques are comparable to those in sars patients, 2, 4, 5, 7, 8 and to those in other respiratory coronavirus infections, such as sialodacryoadenitis virus infection in rats, 25 n d 3 ----4 results from rt-pcr or virus isolation. nd=not done. syncytia were found commonly in bronchioles, and less frequently in alveolar ducts and alveoli, of sars-covinfected macaques. syncytia also were prominent in the alveoli of sars patients. 3, 7, 8 based on expression of cd68 and pan-keratin, the syncytia were of histiocytic origin in macaques (this study), and of histiocytic or epithelial origin in sars patients. 8 the formation of syncytia in these macaques may have been induced by sars-cov infection, because some syncytia were positive for sars-cov by immunohistochemistry, and the spike protein of coronavirus induces cell to cell fusion. 20 pneumocytes showing cytomegaly, enlarged nuclei, and prominent nucleoli were common both in sars-cov-infected macaques (this study) and in sars patients. 2, 8 such enlargement and cytologic atypia of hyperplastic type 2 pneumocytes occurs commonly in organising diffuse alveolar damage, and is non-specific. 27 the development of a specific immunohistochemical test to identify sars-cov antigen in histological samples allowed us to assess the cell tropism of sars-cov infection in macaques. expression of sars-cov in the lung was restricted to pneumonic areas and localised to the cytoplasm of type 2 pneumocytes and syncytia. the infection of type 2 pneumocytes by coronavirus was confirmed by transmission electron microscopy. these findings correspond to the detection of coronavirus-like particles in pneumocytes of a postmortem lung sample of a sars patient, 8 and with the tropism of respiratory coronaviruses in pigs and rats for respiratory epithelium, and occasionally alveolar macrophages. 25, 26 on the basis of histological changes, sars-cov infection in the macaques primarily affected the epithelium of alveoli and bronchioles. at the time of euthanasia, 6 days after infection, most pneumonic areas showed early to advanced type 2 pneumocyte hyperplasia, indicating repair of alveolar walls after loss of type 1 pneumocytes. the temporal sequence of the histological changes corresponds with that of experimental infection with porcine respiratory coronavirus in pigs, in which acute changes (loss of epithelium, presence of macrophages and fibrin in airway lumina) were seen at days 2-6 after infection, and more advanced changes (type 2 pneumocyte hyperplasia, interstitial mononuclear cell infiltration) were seen from days 7-11 after infection. 26 because diffuse alveolar damage from different causes follows a common pathway, 27,28 more chronic changes in these macaques probably would have included organisation of the intra-alveolar exudate, resulting in alveolar fibrosis and bronchiolitis obliterans, as seen in sars patients who died later in the course of disease. [3] [4] [5] the development of fibrosis is dependent on the deposition of fibrin in the alveoli rather than on the continued presence of virus infection. onset of fibrosis is a critical feature of chronic diffuse alveolar damage, because it leads to loss of alveolar function and is irreversible. 27 in respiratory coronavirus infections in pigs and rats, viral infection of respiratory epithelium is maximum at days 3-4 after infection, and is no longer measurable by days 6-9. 25, 29 the rapid disappearance of infected cells after initial infection might explain why sars-cov was not found in type 1 pneumocytes, and was only occasionally found in type 2 pneumocytes in these macaques. it also might decrease the chance of detecting sars-cov by immunohistochemistry in postmortem lung tissue of sars patients who die after a protracted course of disease. the lymphoid depletion of splenic follicles in experimentally infected macaques corresponds to that seen in a sars patient 8 and in pigs infection with porcine respiratory coronavirus. 29 based on these findings, together with the leucopenia observed in sars patients, [2] [3] [4] [5] we speculate that sars-cov infection suppresses immunity and may predispose infected hosts to secondary infections, such as in measles virus infection. 30 virological examination of clinical and postmortem samples of experimentally infected macaques indicates that the respiratory tract was the most important source of virus, as is probably the case in human beings. 13 unlike in sars patients, 13 sars-cov was not detected in urine or faeces of these macaques, although faeces did test positive in a previous experiment. 12 this finding may be partly explained by the early cut-off point of the experiment (6 days after infection), because sars-cov rna was detected in the faeces of sars patients in the late convalescent phase. 11 the sporadic detection by rt-pcr of sars-cov in the urinary bladder, stomach, duodenum, cerebrum, and spleen in infected macaques in the absence of evidence of viral replication-based on virus culture or immunohistochemistry-suggests overspill from other tissues, for example via blood. the isolation of sars-cov from the kidney of one macaque suggests viral replication at that site, but this theory could not be confirmed by immunohistochemistry. the rt-pcr based on nucleoprotein primers proved to be about 100-fold more sensitive than the existing rt-pcr, based on polymerase primers. presumably, this difference is due to the gradient in the transcription of coronavirus rna, with high concentrations of nucleoprotein rna and low concentrations of polymerase rna. 20 collectively, these results of laboratory studies of sars patients and experimental infections of macaques prove that the newly discovered sars-cov is the primary causal agent of sars. based on histopathological and immunohistochemical analysis of postmortem tissues of these macaques, sars-cov infection primarily affects epithelium of the lower respiratory tract, with potentially severe consequences for respiratory function. none declared. t kuiken and r a m fouchier participated in the joint planning and coordination of the study. t kuiken wrote the report and supervised and interpreted the pathology, immunohistochemistry, and electron microscopy components of the experimental infections. r a m fouchier and m schutten developed, supervised, and assessed the rt-pcr for sars-cov. g f rimmelzwaan and g van amerongen planned and carried out the infection experiments. d van riel and j d laman were involved in the design, execution, and assessment of the immunohistochemistry test to detect sars-cov in tissues. t de jong did the electron microscopy and detected sars-cov in pneumocytes. g van doornum supervised the virological analyses of samples from the experimental infections. k stöhr participated as secretary of the who laboratory network on sars diagnosis and played a substantial part in the initiation of the study. a d m e osterhaus was the principal investigator and was responsible for the overall planning and coordination of the study. all other researchers were involved in the development, application, assessment of diagnostic tests on samples from sars patients, or a combination of these. berend niemeyer, georgina aron, and judith guldemeester for virological assistance; rob van herwijnen, european veterinary laboratory, woerden, for purification and biotinylation of primary antisera for immunohistochemistry; frank van der panne for assistance with preparation of figures; a m burguière for contributions to analysis of the data; s azebi, c batejat, g coralie, b crescenzo-chaigne, f fichenick, s gerbaud, v lorin, c rousseaux, and m tardy-panit for technical assistance; n tordo for the design of primers p9 and p10 and for helpful discussions; and f freymuth for providing free the human severe acute respiratory syndrome (sars) coronavirus as a possible cause of severe acute respiratory syndrome a major 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from 11 coronaviruses and development of a consensus polymerase chain reaction assay characterization of human metapneumoviruses isolated from patients in north america detection of human metapneumovirus from patients with severe acute respiratory syndrome: a methodological evaluation detection of influenza a viruses from different species by pcr amplification of conserved sequences in the matrix gene coronaviridae: the viruses and their replication detection of chlamydia trachomatis in clinical specimens by the polymerase chain reaction rapid and standardised detection of chlamydia pneumoniae using lightcycler real-time fluorescence pcr a dictionary of virology a newly discovered human metapneumovirus isolated from young children with respiratory tract disease experimental infection of adult axenic rats with parker's rat coronavirus pathogenicity of experimental infection with 'pneumotropic' porcine coronavirus common pathways and patterns of injury the respiratory system pathogenicity of porcine respiratory coronavirus isolated in québec measles virus and immunomodulation: molecular bases and perspectives we thank, at the erasmus medical centre, rotterdam, the staff of the histology and immunohistochemistry laboratories of the department of pathology for technical assistance; alewijn ott and arjen van vliet, diagnostic unit, department of medical microbiology and infectious diseases, for bacteriological examination of macaque tissues; and alex van belkum and liesbeth van der zwaan, department of medical microbiology and infectious diseases, for examination of macaque lung homogenates for chlamydia. we thank theo bestebroer, metapneumovirus. we thank the french medical team, the medical staff of the hanoi french hospital, and the medical teams at the hospitals in tourcoing, besançon, strasbourg, bordeaux, montpellier, hôpital d'instruction des armées, brest, rennes, chu bichat claude bernard, paris, and chu pitié-salpétrière, paris for providing the samples. we thank the staff of the department of pathology, virology section, singapore general hospital, who did the bulk of the investigations there; and the staff of the defence medical research institute, who did some of the stool pcr tests. key: cord-033551-eojpkxz9 authors: shekh, shamasoddin; reddy, k. kasi amarnath; gowd, konkallu hanumae title: in silico allicin induced s-thioallylation of sars-cov-2 main protease date: 2020-09-16 journal: nan doi: 10.1080/17415993.2020.1817457 sha: doc_id: 33551 cord_uid: eojpkxz9 coronavirus disease 2019 (covid-19) is an ongoing pandemic caused due to new coronavirus infection with (3)716075 deaths across the world as reported by the world health organization (who). sars-cov-2 main protease (m(pro)) plays a vital role in the replication of coronavirus and thus an attractive target for the screening of inhibitors for the therapy of covid-19. the preclinical drugs ebselen and px-12 are potent inhibitors of sars-cov-2 m(pro) and covalently modifies the active site cys-145 residue of m(pro) through selenosulfide/disulfide. in the current report, using virtual screening methods, reactive sulfur species allicin is subjecting for covalent docking at the active site of sars-cov-2 m(pro) using px-12 as a benchmark reference compound. the results indicate that allicin induces dual s-thioallylation of cys-145 and cys-85/ cys-156 residues of sars-cov-2 m(pro). using density functional theory (dft), gibbs free energy change (dg) is calculated for the putative reactions between n-acetylcysteine amide thiol and allicin/allyl sulfenic acid. the overall reaction is exergonic and allyl disulfide of cys-145 residue of m(pro) is involved in a sulfur mediated hydrogen bond. the results indicate that allicin causes dual s-thioallylation of sars-cov-2 m(pro) which may be of interest for treatment and attenuation of ongoing coronavirus infection. coronavirus infection is a global emergency with 19,187,943 confirmed cases and 716,075 deaths across the world as on 8th august 2020 reported by world health organization (who) [1] . the disease caused due to sars-cov-2 virus which spreads rapidly through aerosols of cough and sneezes or secretions of bodily fluids of infected persons [2] [3] [4] . the uncontrolled spreading of the corona infection is a great threat for mankind which demands immediate development of therapies to combat the disease. it is a great challenge to treat the deadly infection due to a lack of specific drugs, viable vaccines to immunize against the virus, and unproven history of using ayurvedic medicine to control the disease [5, 6] . in the independent reports, zhang et al., 7 and jin et al., 8 have reported the crystal structure of the sars-cov-2 main protease that plays a crucial role in replication and proliferation of the coronavirus [7, 8] . sars-cov-2 main protease (m pro ) is involved in proteolytic processing of translated polyproteins of the coronavirus genome into the functional polypeptides which eventually assemble to form new coronavirus [7, 8] . the active site of sars-cov-2 m pro contains catalytic dyad motif involving his-41 and cys-145 residues which facilitates the hydrolysis of peptide bonds at > 11 sites of the viral genome polyprotein [7, 8] . the recognition site for cleavage of the peptide bond by sars-cov-2 m pro is the c-terminus of glutamine of sequence -leu-gln-ser/ala/glyin the polyprotein replicase lab of coronavirus [7] . inhibition of the function of sars-cov-2 m pro has a direct impact on the formation of functional polypeptides which are critical in the assembly of new viruses thereby attenuating the replication and proliferation of coronavirus. interestingly, proteases with similar specificity for cleavage of peptide bond are not evident in humans [7] which is an added advantage in choosing the sars-cov-2 m pro as a target for the screening of drugs to combat covid19 . attempts have been made through a structure-based drug design approach to identify the inhibitors for sars-cov-2 m pro to attenuate coronavirus infection [8] . the approved drugs and natural products have been virtually screened against sars-cov-2 m pro to assess their ability to attenuate coronavirus replication and treatment of covid-19 disease [9] [10] [11] . the natural product extract of garlic allium sativum has a long-documented history in the human civilization as food spices, traditional medicine, antibacterial/antiviral and antioxidant agent and also for the treatment of common cold and infection [12] . allicin is the heart of garlic extract which was isolated and characterized by cavallito and bailey in 1944 and accounts for the large section of pharmacological activity of garlic extract [13, 14] . allicin is a thiosulfinate containing organosulfur species produced by the allium sativum as part of a defense mechanism to protect garlic plants against pathogens and predators [12, 15] . allicin is most abundant in garlic and formed through condensation of two molecules of allyl sulfenic acid in an enzymatic reaction during tissue damage of raw garlic or wetting of dried/pulverized garlic powder [16] . allicin is an oxidizing agent and potentially reacts with cellular protein thiols and glutathione leading to the formation of s-allyl-mercapto-proteins/glutathione [17] . the multi-faceted role of allicin as antiviral agent, antimicrobial agent, modulator of the immune system, lowering risks of cardiovascular diseases may be useful in combating the on-going covid-19 pandemic [12, 15, 18] . the oxidizing nature of allicin through s-thioallylation may be of special interest due to the presence of cysteine thiol at the active site of sars-cov-2 m pro [7, 8] . jin et.al., 2020 have reported tethering of active site cys-145 residue of sars-cov-2 m pro with inhibitors ebselen and px-12 through selenosulfide/disulfide. in the current report, virtual screening methods were used to assess the ability of allicin to covalently modify cysteine residues of sars-cov-2 m pro . the report indicates the allicin as a covalent inhibitor of sars-cov-2 m pro and may be useful in attenuating the coronavirus infection. crystal structures of sars-cov-2 m pro were retrieved from the protein databank (pdb). the pdb code of sars-cov-2 m pro used in the studies: apo form is 6y2e and inhibitor bound form are 6lu7, 5rfv, 5rfw. three-dimensional structures of sars-cov-2 m pro were processed using the maestro version 12.0.012 platform of schrödinger software. the alignment of structures of m pro and measurement of the distance between the atoms was achieved using option quick align and measurement of distance, respectively. the residue around 3 a°distance to the cysteine in the structure was identified using maestro version 12.0.012. the relative surface accessibility of the cysteine residues in the 3d-structure of m pro was determined using the software get-access (http://curie.utmb.edu/getarea.html) [19] . the pka of cysteine thiols of m pro were calculated using the protein preparation wizard of glide, maestro version 12.0.012 platform of schrödinger software. the graph is plotted using origin-pro software. the virtual screening of allicin as an inhibitor of sars-cov-2 m pro was performed using glide, maestro version 12.0.012 platform of schrödinger software. screening of allicin was conducted against the four pdb co-crystal structures of sars-cov-2 m pro . the crystal structure of sars-cov-2 m pro was processed using the protein preparation wizard. hydrogen atoms were added, sample water orientations were achieved using propka at ph 7, and waters with less than three hydrogen bonds to non-waters were removed from the m pro . the restrained minimization of m pro -ligand complex was achieved using opls3e force field. the ligand allicin was processed using ligprep of schrödinger software with opls3e force field. docking simulations were achieved using the default option of the glide docking process of schrödinger software. receptor grid was generated by choosing the centroid of the workspace ligand. allicin docking was performed using standard precision (sp) mode with flexible ligand sampling by adding epik state penalties for the docking score. using the custom covalent reaction type provided by the schrödinger, covalent docking was performed using pose prediction docking mode of schrödinger software. the covalent docking affinity score was used to prioritize the binding site of the allyl sulfenic acid. gibbs free energy change ( g) for putative reactions between n-acetylcysteine amide and allicin were calculated by density functional theory (dft) on the maestro materials science 3.4.012 platform of schrödinger software. molecules were optimized using b3lyp-d3 on the jaguar platform (version: 10.2, schrödinger release 2019-2) with a 6-31g** basis set and polarization function on all atoms. accuracy level was set to ultrafine and maximum iteration steps of 200 with the switch to analytical integrals near convergence. gibbs free energy change ( g) for the reaction was calculated using the pre-optimized molecules on jaguar reaction (release 2019-2) platform. the accuracy level was set to ultrafine with default convergence criteria and the solvent model was none. gibbs free energies are stated in (kcal/mol) assuming standard condition (t = 298.15 k and p = 1.0 bar). in independent studies, zhang et.al., 2020 and jin et.al., 2020 have reported crystal structures of sars-cov-2 m pro (or) covid-19 virus m pro . structure of sars-cov-2 m pro is a homodimer with active site projecting outside the dimer interface (figure-s1). thus, the monomer structure is used for virtual screening of inhibitor against the sars-cov-2 m pro [8, 10, 11] . figure 1a shows the structure of sars-cov-2 m pro with free cysteine thiols and active site dyad residues. [7] with the cysteine free thiols and the active site dyad motif. cysteine thiols and catalytic residues are highlighted through the ball representation of schrödinger software. n-and c-terminus of the protein is also indicated. (b) allicin derived from garlic (allium sativum) and optimized structure of allicin with homo and lumo orbitals by dft (density functional theory). the 2d-structure of allicin is also indicated. interestingly, both the reports have documented the covalent binding of the inhibitors, both peptidomimetic and sulfur/selenium contain small molecules, at the catalytic cys-145 residue of sars-cov-2 m pro . specific interest is ebselen and px-12 which covalently bind to cys-145 residue of sars-cov-2 m pro through selenosulfide and disulfide bond, respectively. virtual screening methods have revealed various natural products as inhibitors of sars-cov-2 m pro including bioactive sulfur compounds derived from garlic essential oils [15] . allicin is of special interest due to the antiviral activity and the reactive nature of the thiosulfinate group [12] . figure 1b shows allicin natural products derived from garlic (allium sativum) and its optimized structure with homo and lumo orbitals by density functional theory (dft). homo and lumo orbitals indicate the nucleophilic attack at the thiosulfinate group of allicin. the current report has evaluated allicin as a covalent inhibitor of sars-cov using the custom-made covalent reaction type provided by schrödinger for reactions-1 and reaction-2 (scheme-s1), covalent docking was performed between allicin/px-12 and active site of sars-cov-2 m pro . figure 2b shows the formation of cysteine allyl disulfide at the cys-145 residue of sars-cov-2 m pro after covalent docking with allicin. similar results are also observed with px-12 (figure-s5) . these observations support that allicin covalently modifies the cys-145 residue of sars-cov-2 m pro through the formation of a disulfide bond. the by-product of the reaction between cys-145 thiol and the allicin is an allyl sulfenic acid which is a reactive sulfur species that can potentially react with thiols to form corresponding allyl disulfide. sars-cov-2 m pro contains a total of twelve cysteine residues around the binding site of allicin; cys-16, cys-22, cys-38, cys-44, cys-85, cys-117, cys-128, cys-156, cys-160, cys-265, and cys-300. interestingly, all these cysteine residues are present in the reduced state of the thiol form. figure 3 shows the relative accessible surface area of cysteine residues around the binding site of allicin of sars-cov-2 m pro . except for cys-85, cys-156, and cys-300, the remaining cysteine residues are buried in the protein (rasa is < 5 %) and may not be accessible for the reaction with allyl sulfenic acid. the cys-85 residue is accessible to external agents and independent of binding of the inhibitor to the m pro . the cys-156 residue is accessible to external agents and the extent of accessibility is affected due to the binding of the inhibitor to the m pro . figure-s6 shows the residues in the vicinity of cys-156 in unbound and bound form of sars-cov-2 m pro . particularly, in the n3 bound form of sars-cov-2 m pro , lys-100 and tyr-154 residues move over cys-156 retarding its accessibility to the external agent. the cys-300 residue is exposed in the apo form and buried in the inhibitor bound form of sars-cov-2 m pro . the flanking residues to the c-terminus of cys-300 are unstructured in nature and projects in opposite orientation between the apo and inhibitor bound form of sars-cov-2 m pro (figure-s7 ). such moments are affecting the solvent accessibility of cys-300 residue. figure-s8 shows pka of thiols of cysteine residues in the unbound and ligand-bound form of sars-cov-2 m pro . the pka of cys-85, cys-145, and cys-156 are (11.6-11.8), 10.0, and (8.9-9.5), respectively. the pka of thiol of cys-145 and cys-156 are comparable indicating similar reactivity towards s-thioallylation. in the case of cys-156, relatively higher ph conditions may be required for cysteine thiol to exist in thiolate form. based on the above information, allyl sulfenic acid was covalently docked at cys-85 and cys-156 residue of sars-cov-2 m pro . using a custom-made covalent reaction type provided by schrödinger for the reactions-3 (scheme-s1), covalent docking was performed between allyl sulfenic acid and solvent accessible cys-85/cys-156 thiol of sars-cov-2 m pro . figure 4b and c show the formation of cysteine allyl disulfide at cys-85 and cys-156 residue of sars-cov-2 m pro after covalent docking with allyl sulfenic acid. table-s2 provides a summary of covalent docking of allicin/px-12/allyl sulfenic acid at cysteine thiols of four different co-crystal structures of sars-cov-2 m pro . covalent docking affinity score and retention of accessibility in inhibitor bound form supports the formation of cysteine allyl disulfide at cys-85 over cys-156 residue ( table-s2 to support the observed covalent docking results between allicin and cysteine thiols of sars-cov-2 m pro , the energetics of reactions between cysteine thiol with allicin was figure2b is 2.04 a°. as stated in schrödinger's software, noncovalent docking is 'docking ligands to a protein target using pre-generated receptor grids. includes settings for ligand sampling, pose filtering and post-processing; constrains to the receptor, a ligand core, or on ligand torsions; and using similarity and dissimilarity screening'. covalent docking is 'the reactive functional group on the ligand and reactive residues in the receptor are identified, and the bond is formed between the correct atoms on each'. figure 5a shows the possible reactions of cysteine thiols with allicin and their reactive intermediates. figure-s9 shows the optimized structures of reactants and products of putative reaction by dft. the initial reaction of cysteine thiol with allicin results in the formation of products cysteine allyl disulfide and allyl thioaldehyde. the gibbs free energy change for the initial reaction of cysteine thiol with allicin is + 4.79 kcal/mol indicating the endergonic and non-spontaneous nature of the reaction. these features suggest that additional energy is required to facilitate the occurrence of the reaction in the form of input energy or through the stabilization of the products. in the context of present studies, the later may be possible at the binding site of the allicin in sars-cov-2 m pro . figure 5b shows the sulfur mediated hydrogen bonding by the sulfur of allyl disulfide formed after covalent docking of allicin at the active site of sars-cov-2 m pro . the sulfur mediated hydrogen bonds are well documented in peptides and proteins [21, 22] . sulfur centered hbonds (schbs) are increasingly evident in proteins; n−h···s h-bonds in thioredoxins, o−h···s h-bonds are found in catalase and s−h···o h-bonds in globular proteins [19] . the role of sulfur mediated hydrogen bonding is becoming very significant in molecular assemblies, structural biology, and functional materials [19] . schbs are now considered as worthy participants in hydrogen bonding with distinct features compared to the conventional x−h···y (x, y = o, n) hydrogen bonds [23] . the sulfur of allyl disulfide is involved in bifurcated hydrogen bond; hydrogen bond distance between gly-143 nh−s-allyl is 2.43 a°with the angle of 147.8°and cys-145 nh−s-allyl is 2.33 a°with the angle of 153.4°. in addition to the hydrogen bond, hydrophobic and van der waals interaction also contributes to stabilizing the newly formed product of cysteine allyl disulfide. it is worth noting that in the absence of such stabilization of product or input energy, the initial reaction may not be feasible between cysteine thiol and allicin. rather, all the exposed cysteine residues in the 3d-structure of proteins may not react with allicin to yield the product of cysteine allyl disulfide. newly formed product of allyl thioaldehyde may undergo tautomerization to allyl sulfenic acid which is an exergonic reaction with g of −19.59 kcal/mol. net reaction between cysteine thiol and allicin to yield cysteine allyl disulfide and allyl sulfenic acid has g of − 14.80 kcal/mol indicating preference for the forward reaction. the allyl sulfenic acid is a reactive sulfur species and may participate in the reaction with nearby cysteine thiol to yield another molecule of cysteine allyl disulfide and water. this consecutive reaction is also exergonic with g of −22.39 kcal/mol. the reaction of multiple thiol-containing m pro with a single molecule of allicin may result in the formation of two cysteine allyl disulfide with the elimination of water. these calculations support the formation of cys-145 allyl disulfide during the covalent docking of allicin at the active site figure 6 shows steps involved in the dual modification of cysteine residues of sars-cov-2 m pro as cysteine allyl disulfide by the allicin. the coronavirus genome encodes for 29 proteins (one may not get expressed): four structural proteins (spike, envelop, membrane, nucleocapsid) and 25 non-structural proteins [24, 25] . the spike protein/s-protein binds to cell surface angiotensin-converting enzyme 2 (ace2) receptors of the host cells for entry into the host cells [26] . the nonstructural proteins are mainly involved in the virus assembly, replication, and modulation of the host system and initially expressed as two long polyprotein pp1a and pp1ab. these polyproteins were further protested into 16 smaller proteins with the help of sars-cov-2 m pro and papain-like protease. interestingly, sars-cov-2 m pro performs 11 of these cleavages, thus, it is an attractive target for the drug discovery against covid-19 [7, 8] . the other non-structural proteins involved in the life-cycle of coronavirus is rna-dependent rna polymerase (rdrp), helicase and nsp3 protein that associated with immunomodulation of host system to protect the virus. the structural spike glycoprotein and non-structural sars-cov-2 m pro , papain-like protease, rdrp, helicase, and nsp3 are an important target for anti-covid drugs [27] . the spike protein contains an exposed cys-136 residue and twelve disulfides (pdb id:6vxx) and further, cys-136 residue is the distance from the binding surface with ace2. interestingly, the active site of sars-cov-2 m pro and papain-like protease contains cysteine residue. the catalytic motif of m pro consists of dyad residues (his-41 and cys-145) [7, 8] and papain-like protease consists of triad residue (cys-111, his-272, and asp-287) [28] . the covalent modification of active site cysteine residues in these proteases results in functional loss which eventually terminates the formation of non-structural proteins and replication of coronavirus. using in silico approach, the present studies have evaluated the allicin as a covalent inhibitor of sars-cov-2 m pro . the reactive nature of allicin and its reaction by-products with cysteine thiols results in the dual s-thioallylation of sars-cov-2 m pro . thus, allicin may attenuate the replication of coronavirus and may be useful in attenuating the covid-19. synergistic effects of allicin induced s-thioallylation of coronavirus proteins may play a role in attenuating the replication and propagation of coronavirus. the active site of sars-cov-2 main protease that cleaves eleven sites of long polyprotein to release functional proteins required for assembly/replication of coronavirus contains cysteine free thiol. allicin natural product derived from allium sativum has proven medicinal properties including antiviral and antimicrobial activity and lowering risks of cardiovascular diseases contains reactive thiosulfinate which can cause protein s-thioallyllation. in the current report, using in silico non-covalent and covalent docking screening methods, allicin is evaluated as a covalent inhibitor of sars-cov-2 m pro . allicin causes dual sthioallylation of cys-145 and solvent-exposed cys-85/cys-156 residue of sars-cov-2 m pro thereby acts as a potent inhibitor of sars-cov-2 m pro . the multi-faceted roles of allicin as a booster of immune system [29] and covalent inhibitor of coronavirus protease may be useful in treating covid-19 infection. coronavirus disease 2019 (covid-19) situation report -201 data as received by who from national authorities by 10:00 cest a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019 (covid-19) unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors structure of mpro from sars-cov-2 and discovery of its inhibitors research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases rapid identification of potential inhibitors of sars-cov-2 main protease by deep docking of 1.3 billion compounds structural basis of sars-cov-2 3clpro and anti-covid-19 drug discovery from medicinal plants allicin: chemistry and biological properties allicin, the antibacterial principle of allium sativum. i. isolation, physical properties and antibacterial action allicin, the antibacterial principle of allium sativum. ii. determination of the chemical structure investigation into sars-cov-2 resistance of compounds in garlic essential oil allicin bioavailability and bioequivalence from garlic supplements and sarlic foods allicin induces thiol stress in bacteria through sallylmercapto modification of protein cysteines the antioxidant properties of garlic compounds: alyl cysteine, alliin, allicin, and allyl disulfide exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules the human allicin-proteome: s-thioallylation of proteins by the garlic defence substance allicin and its biological effects hydrogen bonds involving sulfur: new insights from ab initio calculations and gas phase laser spectroscopy critical assessment of the strength of hydrogen bonds between the sulfur atom of methionine/cysteine and backbone amides in proteins the prodigious hydrogen bonds with sulfur and selenium in molecular assemblies, structural biology, and functional materials genome composition and divergence of the novel coronavirus (2019-ncov) originating in china the architecture of sars-cov-2 transcriptome structural basis of receptor recognition by sars-cov-2 therapeutic options for the 2019 novel coronavirus (2019-ncov) activity profiling and structures of inhibitor-bound sars-cov-2-plpro protease provides a framework for anti-covid-19 drug design immunomodulation and anti-inflammatory effects of garlic compounds we acknowledge dr. pritesh bhat and dr. sudharsan pandiyan from schrödinger for guiding in executing the current research work. mr. shamasoddin shekh is the recipient of a ph.d. fellowship from the directorate of minorities from the government of karnataka. this research work is funded by the dst-serb-ecr research grant. we also acknowledge dr. harish holla, department of chemistry, cuk. no potential conflict of interest was reported by the author(s). this research work is funded by department of science and technology. key: cord-275946-ofd2ipvs authors: cheng, matthew p.; yansouni, cedric p.; basta, nicole e.; desjardins, michaël; kanjilal, sanjat; paquette, katryn; caya, chelsea; semret, makeda; quach, caroline; libman, michael; mazzola, laura; sacks, jilian a.; dittrich, sabine; papenburg, jesse title: serodiagnostics for severe acute respiratory syndrome–related coronavirus-2: a narrative review date: 2020-06-04 journal: ann intern med doi: 10.7326/m20-2854 sha: doc_id: 275946 cord_uid: ofd2ipvs accurate serologic tests to detect host antibodies to severe acute respiratory syndrome–related coronavirus-2 (sars-cov-2) will be critical for the public health response to the coronavirus disease 2019 pandemic. many use cases are envisaged, including complementing molecular methods for diagnosis of active disease and estimating immunity for individuals. at the population level, carefully designed seroepidemiologic studies will aid in the characterization of transmission dynamics and refinement of disease burden estimates and will provide insight into the kinetics of humoral immunity. yet, despite an explosion in the number and availability of serologic assays to test for antibodies against sars-cov-2, most have undergone minimal external validation to date. this hinders assay selection and implementation, as well as interpretation of study results. in addition, critical knowledge gaps remain regarding serologic correlates of protection from infection or disease, and the degree to which these assays cross-react with antibodies against related coronaviruses. this article discusses key use cases for sars-cov-2 antibody detection tests and their application to serologic studies, reviews currently available assays, highlights key areas of ongoing research, and proposes potential strategies for test implementation. s ince the initial identification of severe acute respiratory syndrome-related coronavirus-2 (sars-cov-2) as the etiologic agent of coronavirus disease 2019 (covid19) , there have been over 5.3 million confirmed cases and around 340 000 deaths reported worldwide, according to the world health organization (who) (1) . however, given the prevalence of asymptomatic or minimally symptomatic individuals (2, 3) , the imperfect sensitivity of molecular assays performed at a single time point (4) , and limited molecular testing capacity in several parts of the world, the true number of infections probably exceeds the who's estimate by several fold. in addition to scaling up molecular testing for diagnosis of active disease, several countries have incorporated serologic surveillance studies to their covid-19 pandemic response. these studies can help elucidate disease transmission dynamics and improve disease burden estimates by identifying persons who were previously infected, even if pauci-or asymptomatic (5); assess transmission within and between subgroups in the population; and provide insight into the kinetics of humoral immunity after infection (6, 7) . serologic testing may also serve as an adjunct to molecular methods for covid-19 diagnosis in certain clinical scenarios (8) . despite a rapid increase in the number and availability of serologic assays to test for antibodies against sars-cov-2 (9), most have undergone minimal or no external validation or have poorly described validation panels, which hinders assay selection and interpretation of results. in addition, interpretation of serologic assays is limited at present because of critical knowledge gaps. for example, no definite serologic correlates of protection from infection or disease have been identified in humans, and the degree to which these assays cross-react with antibodies against related coronaviruses is poorly described. we discuss key use cases for sars-cov-2 antibody detection tests and their application to serologic studies. we review currently available assays, highlight key areas of ongoing research, and propose potential strategies for test implementation. we searched the medline ovid database for articles on sars-cov-2 serologic assays (the appendix, available at annals.org, shows the search strategy). additional studies were identified by hand-searching references of selected articles, consulting international experts, and searching covid-19 and sars-cov-2 preprints on medrxiv and biorxiv. this search was last updated on 20 may 2020. innate responses. of note, infection prevalence in the population being tested must always be considered. in patients with clinical features of covid-19, a highly specific test, such as sars-cov-2 polymerase chain reaction (pcr), has a high positive predictive value for true infection. conversely, if testing asymptomatic individuals when the true seroprevalence of a population is only 5%, an assay with a specificity of 95% would produce a false-positive rate of 50%. low specificity is particularly problematic in cases where incorrectly identifying an individual as immune could place them at significant risk-for instance, if they were to enter settings with high risk for exposure without appropriate personal protective equipment. the sensitivity of a serologic assay can be established by testing sera from patients who have been identified as infected on the basis of a reference standard. however, a single estimate of sensitivity to de-scribe test performance can be difficult to interpret when samples are collected at different time points since infection. sensitivity estimates will vary according to time since infection in the validation cohort. early (<7 days since symptom onset) and mid-stage (8 to 14 days) pcr-confirmed cases of covid-19 will have lower rates of seroconversion than in the later stage (>14 days); thus, antibody tests will have lower sensitivity to detect infection in earlier phases. likewise, antibody responses may be more easily detectable in severe cases (hospitalized patients) than in mild or asymptomatic infections (11) . establishing the analytic specificity of sars-cov-2 seroassays presents a challenge because of potential for cross-reactivity with antibodies to related coronaviruses (11, 12) . to address this, test reactivity thresholds used to define a positive result can be adjusted to optimize the tradeoff between sensitivity and specificity (13). with higher thresholds, sensitivity decreases as cases with low serum antibody levels are categorized as negative, but specificity improves as low amounts of nonspecific antibody are no longer considered positive. physicochemical assay variables can also be modified so that less specific antibodies, with less "avidity" for the antigen, are excluded. this also improves specificity at some expense to sensitivity. tests that target igm, which by its nature can be nonspecific, will probably have increased risk for false-positive results. validation of the clinical specificity of a serologic assay requires sera from different types of sources. in the case of covid-19, sera collected before the end of 2019 are presumed to be seronegative for sars-cov-2 (14). the samples chosen should be representative of the population of interest. in addition, individuals known to have been infected with various common pathogens, including other human coronaviruses, but who could not have been infected with sars-cov-2, should be evaluated to demonstrate the absence of cross-reactivity. finally, patients with illnesses known to stimulate high levels of polyclonal antibodies, such as epstein-barr virus infection, malaria, or conditions associated with production of rheumatoid factor, can be evaluated for cross-reactivity (15) (16) (17) . without these validations, assay specificity will be difficult to establish. once a particular assay is shown to have high sensitivity and high specificity, this assay can serve as a surrogate "gold standard" for the validation of other assays, as well as a standard for quantitative assays. to date, most published sars-cov-2 serologic assay validations have classified patient sera according to sars-cov-2 pcr results (18) . polymerase chain reaction assay is an imperfect comparator for sars-cov-2 diagnosis because of variable analytic performance across assays (19) , and because pcr sensitivity depends on sample type, quality of sampling, and timing relative to illness onset (4, 20) . this can lead to unpredictable directions of bias for seroassay accuracy estimolecular testing on respiratory specimens, the current gold standard for diagnosis of sars-cov-2 infection, is hampered by imperfect sensitivity and limited testing capacity. antibody testing has potential to aid in particular diagnostic scenarios, such as in rt-pcr negative patients who present later during disease course. antibody testing should not be used as the sole basis for diagnosis of acute covid-19. appropriately designed seroepidemiologic studies will play an essential part in the public health response to the covid-19 pandemic by characterizing transmission dynamics, refining disease burden estimates, and providing insight into the kinetics of humoral immunity to sars-cov-2. validation of novel antibody detection tests for sars-cov-2 must pay careful attention to the choice of source populations and reference standards, and to possible cross-reactivity with antibodies to other human coronavirus infections. plaque reduction neutralization assays are currently the reference standard for determination of host antibodies capable of inhibiting viral replication, but must be performed in a biosafety level 3 laboratory. urgent research is needed to determine the serologic correlates of immunity against sars-cov-2. mates. there is an urgent need for validation studies to provide more detail on pcr comparators and on study populations, especially regarding disease severity and timing in the illness course. furthermore, to enable a better understanding of the diagnostic accuracy of various sars-cov-2 serologic tests, the development of reference panels, including seroconversion panels, by using well-characterized sera is necessary. coronavirus spike (s) and nucleocapsid (n) envelope proteins are highly immunogenic and constitute important antigenic targets for the development of serologic assays (11, 21) . as with sars-cov-1, the s protein of sars-cov-2 binds to the cell surface angiotensin-converting enzyme 2 (ace2) receptor (21) (22) (23) . host neutralizing antibodies (nabs) appear to be predominantly directed at the s protein (24) . the n protein plays crucial roles in viral replication and assembly, is highly conserved, and induces antibodies sooner than s during infection (6, 25, 26) . commercial sars-cov-2 serologic assay development has focused on enzyme immunoassays, such as laboratory-based enzyme-linked immunosorbent assays (elisas) and rapid lateral flow assays (lfas). more complex serum neutralization assays are important as a reference standard and to assess immunity. only a subset of antibodies raised against a specific antigen have the property of neutralizing viral replication. neutralization assays, such as plaque reduction and microneutralization methods, provide essential data for the validation of candidate diagnostic tests and to define correlates of protective immunity. the primary drawback of functional assays of sars-cov-2 neutralization is that they can only be performed by experienced staff in a biosafety level (bsl) 3 laboratory owing to the need to culture live virus, which increases complexity and cost. thus, efforts to circumvent these obstacles have converged on finding surrogates of traditional neutralization titers. live pseudotyped viruses have been developed that incorporate the s protein of sars-cov-2, can be cultivated in bsl-2 conditions, and express a reporter enzyme when infecting cells through binding to the ace2 receptor, thereby allowing for automated quantification (27) . such reporter virus sys the figure shows a decision tree for interpreting antibody test results by symptomatology (symptomatic, postsymptomatic, asymptomatic or subclinical) and whether the patient is a suspected case. it is presumed herein that antibody tests with the highest possible sensitivity and specificity are used, and that the symptomatology is occurring early in the pandemic, when seroprevalence is low and before the availability of a vaccine. for sars-cov-2, the accuracy of antibody test results and the appropriate test interpretation both depend on clinical context. in some situations, the clinical context does not enable a single interpretation of the antibody test result. for example, a positive antibody test in a low-risk population could be the result of prior infection, or it could be a false-positive result. similarly, a negative antibody test in a high-risk population cannot a priori differentiate among preseroconversion, undetectable seroconversion, a false-negative result, or the absence of infection. sars-cov-2 = severe acute respiratory syndrome-related coronavirus-2. * the relationship between positive antibody results and protective immunity will vary among assays and must be validated individually. † includes high exposure, high risk, hot spots, and contact tracing. serodiagnostics for sars-cov-2: a narrative review tems would offer substantial advantages in terms of speed, cost, and scalability while providing a quasifunctional assessment of the host neutralizing antibody response (18) . other groups are striving to create surrogates of neutralization that bypass the need for viral culture through the use of blocking elisa formats (28) . for high-throughput and inexpensive (after initial capital outlay) screening in clinical laboratories, relevant antigenic targets can be purified or synthesized, and 1 or more can be incorporated into an elisa test platform. specific antibody-antigen reactivity is detected by using enzyme conjugates that produce color changes or other detector labels that can be objectively measured (29) . the elisas detect antibodies directed at the chosen antigen without regard for their ability to elicit viral neutralization. thus, interpretation of immune status from elisa results requires rigorous characterization of the assay with respect to a reference standard. for the moment, this work has not been done for sars-cov-2. furthermore, universal standards for reporting are lacking (some assays produce semiquantitative results, others are qualitative), and assays have variable test detection limits and reproducibility and use different analytes (igg, igm, iga) or combinations thereof, with unclear effect on performance (30) . it is thus not surprising that estimates of elisa test sensitivity and specificity vary widely across assays and even within assays evaluated by different investigators ( table 2 ) (31-47). the lfas leverage the same capture agents as an elisa in a lateral flow strip format (48) . the lateral flow format enables a simple and fast time to result (10 to 30 minutes), but with tradeoffs in detection that is severalfold less sensitive than their elisa counterpart, a higher cost per test, and lower throughput (49) . for lfas, follow-up confirmatory testing is typically recommended. most provide qualitative, visual results subjectively interpreted by the operator. the use of a small instrument reader can increase test sensitivity and may permit quantitative and more reproducible results (50, 51). to enable community-based and home testing, lfas should be paired with minimally invasive samples, such as finger-prick or oral fluid or swabs, and minimal sample processing. these tests are ideal for near-patient testing and low infrastructure settings, such as the lower levels of the public health system in low-and middle-income countries (52), where they have been used to effectively screen and triage cases of epidemic and nonepidemic diseases. particularly where resources are constrained, inexpensive lfas may be useful to expand diagnostic test capacity. many sars-cov-2 lfa antibody tests are available; however, the performance of these tests is still under evaluation, and their value needs to be carefully weighed depending on the use case. a recent large study found heterogeneous and inconsistent results among 10 lfas and identified signal interpretation as a major obstacle (41). population-based seroepidemiologic studies are an important source of evidence about sars-cov-2 transmission dynamics and will be critical for informing interventions to mitigate the effects of the covid-19 pandemic (53) . whereas reports of clinical cases identify persons with acute disease, seroepidemiologic studies identify those who were infected previously, including those who experienced mild disease or subclinical infections and thus may not be subject to biases due to health care-seeking behavior and limitations on eligibility for testing during acute disease. these assessments of seroprevalence overall and in specific groups can be used to estimate important characteristics of the pandemic (54 -56) . serologic surveillance studies can also assess the accumulation of persons with antibody responses over time to estimate incidence of sars-cov-2 infection (57, 58) and can track age-and jurisdiction-specific disease susceptibility and identify at-risk populations (59) . utilizing standard protocols for the design and implementation of serologic studies (60) and making protocols publicly available can improve scientific rigor and ensure comparability across studies undertaken in different populations. of note, the who unity studies aim to combine worldwide seroepidemiologic study data (61) . cross-sectional serologic surveillance studies are a key first step toward determining the proportion of a population that has been infected with sars-cov-2. when estimating age-specific seroprevalence is the primary aim, the gold-standard study design is the conduct of appropriately powered, cross-sectional, age-stratified, population-representative, randomly sampled, serologic studies in each population of interest. this study design, when implemented appropriately, ensures that the estimates obtained are representative of the population of interest and minimizes the potential that the results may have common sources of bias (62) . in addition, many variations of this design are also valuable for estimating agespecific seroprevalence, especially when statistical methods are used that can account for alternative design elements and sources of uncertainty (63) . layering seroprevalence surveys onto other existing observational or interventional studies or utilizing residual sera from blood donors or from routine lab tests can increase feasibility and timeliness of estimating seroprevalence at some risk to generalizability. to determine sars-cov-2 seroincidence, or the proportion of the population seroconverting over a certain time frame, longitudinal studies can be conducted among cohorts of individuals who are at high risk for exposure (such as health care workers) or among those for whom little is known about the risk for infection (such as children). furthermore, longitudinal serologic surveillance can be implemented to provide insight in situations where prevention and control measures are for instance, household-or workplace-based serologic studies can aid in the determination of secondary attack rates, especially when the proportion of asymptomatic infections may be high. in addition, well-designed seroepidemiologic studies are critical for informing mathematical models and forecasting tools to guide prevention and control strategies. a critical aspect in the interpretation of serologic tests is an understanding of the dynamic nature of the humoral response to sars-cov-2 infection. a few studies have defined the kinetics of antibody formation in patients with disease ranging from mildly symptomatic to critically ill. these studies have consistently shown that most patients seroconvert by 2 weeks after the onset of symptoms, and almost all patients have detectable antibodies by day 28 (6, 7, 20, 64, 65) . antibodies can be detected as early as 1 day after illness onset, with peak igm and iga titers occurring in the ensuing 7 to 14 days and waning thereafter. the igg response appears to peak simultaneously in some cases, or slightly later in others (66) , and plateaus between 15 and 21 days (6). in some cases, igg titer declines significantly within weeks (67) . some patients appear to have weak or undetectable seroconversion (44, 66) . illness severity probably affects antibody responses. critically ill patients had a delayed but more robust formation of igm and igg in one study (7) . anti-sars-cov-2 responses in subclinical infections have yet to be characterized. finally, the suitability of alternative specimen types to serum, such as saliva or dried blood spots (68, 69), must be established for sars-cov-2 serodiagnostics. correlates of protection are empirically derived, specific immune markers associated with protection against infection or disease (53) . seropositivity is often a useful correlate for clinical immunity, though cell-mediated immunity is known to be essential and antibody production is not the sole mechanistic contribution to protection (70) . the relationship between seropositivity and immune protection has not yet been established for coronaviruses. a recent report on 175 patients who recovered from covid-19 showed that nab titers were moderately correlated with antibodies binding to s protein domains (24) . surprisingly, 30% of patients developed only low titers of nabs after recovery, with younger patients (15 to 39 years of age) having significantly lower anti-sars-cov-2 and nab titers. this suggest that innate and adaptive cellular immunity are also likely to play a significant role in viral clearance and immunity to coronaviruses (71) . little is known regarding seropositivity and risk for reinfection to coronaviruses. in a challenge study with hcov-229e, healthy volunteers who had lower specific igg titers at baseline were more likely to develop clinically overt infection (72) . after the challenge, specific igg and nab peaked at 3 weeks and fell considerably at 12 weeks. one year later, 6 out of 9 previously infected participants became infected after a rechallenge, though they were asymptomatic and the duration of viral shedding was shorter than during the first challenge-suggesting at least partial protection induced by the first infection. of note, the immune response dynamics after sars-cov-1 and middle east respiratory syndrome-coronavirus (mers-cov) infection differ substantially from what was seen with hcov-229e challenge. values for igg and nab peaked 4 months after sars-cov-1 and decreased after 16 months. after mers-cov infection, 86% of patients had detectable igg and nabs for at least 34 months (11). evaluations of sars-cov-2 serologic assays must account for potential cross-reactivity with other coronaviruses, including the 4 endemic human coronaviruses: hku1, oc43, nl63, and 229e. a systematic review of antibody-mediated immunity to coronaviruses found that studies of serologic responses to human coronavirus n proteins suggest cross-reactivity within human alphacoronaviruses (229e and nl63) and human betacoronaviruses (oc43 and hku1), but not between human alpha-and betacoronaviruses (11) . the available evidence suggests that natural infections with endemic coronaviruses produce little cross-reactivity to emerging coronaviruses sars-cov-1 and mers-cov. regarding sars-cov-2 elisa using s1 protein epitopes, several pilot studies report positive results with sera from patients with sars-cov-1, and a lack of significant cross-reactivity when using sera from small numbers of patients seropositive for the endemic human coronaviruses (18, 44) . data regarding sars-cov-2 elisa based on the n protein are more limited. the specificity of 2 elisa and 10 lateral flow assays has also been assessed against 108 pre-covid-19 sera from u.s. patients collected in july 2018, and ranged from 84.3 to 100% (41). finally, in keeping with these results, a surrogate assay of sars-cov-2 viral neutralization tests was found to be highly specific among sera positive for endemic human coronaviruses antibodies but showed some degree of cross reactivity with sars-cov-1 positive sera (28) . thus, cross reactivity of sars-cov-2 serologic assays may be a concern in areas where sars-cov-1 and mers-cov circulated widely. overall, serologic tests based on s protein appear to distinguish between emerging and endemic coronaviruses. assays based on the n protein can serve as a marker of recent infection but might be expected to cross react more with endemic coronaviruses. convalescent plasma therapy, as a means of providing "passive" immunity to susceptible individuals and as early therapy after infection, has been used for many viral infections (73) . this approach was used in a small number of patients with sars-cov-1 and mers-cov and has shown promise in a few case series of sars-cov-2 infection (74 -77) . use of covid-19 convalescent plasma has been approved in several jurisdictions under the category of an emergency investigational new drug (78) . as a general principle, the efficacy of plasma therapy is a function of several factors, including timing of plasma donation (plasma obtained a few weeks after recovery during convalescence is considered more immunogenic, with higher titers of polyclonal neutralizing antibodies), dosage, and timing of administration in relation to onset of disease in the recipient. for covid-19, identifying "optimal" donors will prove to be an additional challenge, given the heterogeneity in antibody titers during convalescence and the lack of an established correlation between specific antibody titers and clinical efficacy (79) . as an example, in the treatment of influenza, plasma with high nab titers collected from a nonconvalescent general population did not show efficacy (80, 81) , suggesting that donor selection should not be based solely on serologic titers. eventually, antibody derived from vaccinated donors may deserve further study. serologic tests are essential to better understand the determinants of sars-cov-2 immunity and to guide vaccine development. for sars-cov-1 and mers-cov, the s protein was shown to be the most important antigen leading to production of nabs and inhibition of viral entry into the host cells (82) . since then, s protein has been the major target for vaccine candidates. previous experience using sars-cov-1 subunit vaccine based on the full-length s protein showed potent nab responses and protective immunity in animal models. however, some of these vaccines were also associated with a harmful immune enhance-review serodiagnostics for sars-cov-2: a narrative review ment, as seen in vaccine candidates for dengue or respiratory syncytial virus, leading to a potentially more severe disease in vaccinated individuals (83) . antibodydependent enhancement has also been seen among sars-cov-1-infected macaques injected with antispike igg (84) . for sars-cov-1 and mers-cov, the receptor-binding domain (rbd) of the s protein was shown to be the major immunodominant region. subunit vaccines targeting rbd specifically elicited high nab titers but were not associated with immune enhancement (82, 85) . in sars-cov-2-infected patients, among the binding antibodies to the different regions of the s protein (s1, s2, rbd), rbd-specific igg correlated best with nabs, suggesting that rbd is be a promising target for sars-cov-2 vaccine candidates (24) . however, because rbd is the most variable region of the genome (86) , there is still a theoretical risk for immunologic "escape," as well as immune enhancement development (87) . the n protein, a more conserved region of the genome, has been of interest for sars-cov-1 and mers-cov vaccine candidates and was thought to be at lower risk for immune enhancement; however, it was not shown to elicit nabs (82) . the role of the n protein in sars-cov-2 immune response is still unknown. in conclusion, the covid-19 pandemic has revealed several gaps in our diagnostic arsenal and is highlighting the essential role of serodiagnostics as part of our public health response. with the use of carefully validated assays, appropriately designed serologic studies will help characterize transmission dynamics and refine disease burden estimates. urgent scientific research is needed to link specific serologic variables with immunity against 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kurdgelashvili, george; williams, riley j title: long-term hydroxychloroquine use in patients with rheumatic conditions and development of sars-cov-2 infection: a retrospective cohort study date: 2020-09-21 journal: lancet rheumatol doi: 10.1016/s2665-9913(20)30305-2 sha: doc_id: 257399 cord_uid: p6of5fno background: hydroxychloroquine is one of several agents being evaluated in the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. we aimed to examine whether patients with rheumatological conditions receiving chronic hydroxychloroquine therapy are at less risk of developing sars-cov-2 infection than those not receiving hydroxychloroquine. methods: this retrospective cohort study included de-identified information of all veterans in the us veterans health administration clinical administrative database aged 18 years or older with rheumatoid arthritis, systemic lupus erythematosus, or associated rheumatological conditions (based on international classification of diseases, 10th edition, diagnostic codes) who were alive on march 1, 2020. a propensity score was calculated for each patient, and each patient who was receiving hydroxychloroquine was matched to two patients who were not receiving hydroxychloroquine (controls). the primary endpoint was the proportion of patients with pcr-confirmed sars-cov-2 infection among those receiving chronic hydroxychloroquine versus the propensity-matched patients not receiving chronic hydroxychloroquine between march 1 and june 30, 2020. secondary outcomes were hospital admission associated with sars-cov-2 infection; intensive care requirement associated with sars-cov-2 infection; mortality associated with sars-cov-2 infection; and overall rates of any hospital admission and mortality (ie, all cause). multivariate logistic regression analysis was done to determine independent variables for the development of active sars-cov-2 infection. findings: between march 1 and june 30, 2020, 10 703 patients receiving hydroxychloroquine and 21 406 patients not receiving hydroxychloroquine were included in the primary analysis. the incidence of active sars-cov-2 infections during the study period did not differ between patients receiving hydroxychloroquine and patients not receiving hydroxychloroquine (31 [0·3%] of 10 703 vs 78 [0·4%] of 21 406; odds ratio 0·79, 95% ci 0·52–1·20, p=0·27). there were no significant differences in secondary outcomes between the two groups in patients who developed active sars-cov-2 infection. for all patients in the study, overall mortality was lower in the hydroxychloroquine group than in the group of patients who did not receive hydroxychloroquine (odds ratio 0·70, 95% ci 0·55–0·89, p=0·0031). in multivariate logistic regression analysis, receipt of hydroxychloroquine was not associated with the development of active sars-cov-2 infection (odds ratio 0·79, 95% ci 0·51–1·42). interpretation: hydroxychloroquine was not associated with a preventive effect against sars-cov-2 infection in a large group of patients with rheumatological conditions. funding: none. the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in wuhan (hubei province, china) in late 2019 sparked a global pandemic that, towards the end of august, 2020, had resulted in close to 25 million known cases with almost 800 000 attributed deaths. [1] [2] [3] the pandemic reached the usa in january, 2020, and by the end of august more than 180 000 deaths had been recorded among 5·8 million cases. 4 as the research community launched several efforts to develop vaccine candidates, various potential pharmacotherapy options began to be explored on the basis of previous in-vitro and in-vivo studies of existing coronaviruses and on the few early in-vitro studies on sars-cov-2. 5, 6 in addition to antiviral agents, some atypical drugs, such as the antimalarial drugs chloroquine and hydroxychloroquine, demonstrated in-vitro activity. in the usa, chloroquine was not on the market, but hydroxy chloroquine had been available for several decades, principally used for treatment of patients with rheuma toid arthritis, systemic lupus erythematosus, and other associ ated autoimmune disorders. 7, 8 the us health-care community began to focus on hydroxy chloroquine and its potential role in the treatment of sars-cov-2 infection, and the federal government and lay media were soon to follow. there was sufficient interest in hydroxychloroquine for health-care institutions to increase procurement of the drug, causing shortages in the supply chain that threatened the continued avail ability of the drug relied upon by millions of patients with rheumatological conditions. 9 unfortunately, reports began to surface questioning whether patients with active sars-cov-2 infection received benefit from hydrox ychloroquine, and safety concerns arose that focused on its propensity to induce prolonged qt arrhythmias. [10] [11] [12] these data led the us food and drug administration to revoke the emergency use authorisation for hydroxy chloro quine and chloroquine to treat active sars-cov-2 infection on june 15, 2020, only 11 weeks after initial issuance. 13 a small trial reported preliminary evidence of short-term post-exposure prophylaxis of hydroxychloroquine among 821 household and occupational contacts of patients with newly diagnosed sars-cov-2 infection. 14 the trial failed to demonstrate a difference between hydroxy chloroquine and placebo among all participants or among the 20 laboratory-confirmed cases of infection, although the authors noted that a marginal benefit of hydroxychloroquine could not be ruled out. randomised trials designed to evaluate interventions to prevent active infection have a unique set of challenges, including sparse event rates over time, which results in the need for substantial time and effort to achieve the power necessary to detect an effect. the results of large prevention trials will probably remain unavailable for several months. however, an alternative approach of compiling observa tional data from large clinical administrative databases might be useful to more rapidly identify preventive effects of an intervention. we aimed to examine whether patients with rheuma tological conditions receiving chronic hydroxy chloroquine therapy are at less risk of developing sars-cov-2 infection compared with a propensity-matched group of patients not receiving hydroxychloroquine. in this retrospective cohort study, de-identified information was obtained from across all us veterans affairs medical centers (vamcs) for eligible patients aged 18 years or older throughout the veterans health administration (vha). the vha is the largest inte grated health-care system in the usa, providing care in 1255 health-care facilities, including 130 health-care centres and 1074 outpatient sites, serving 9 million enrolled veterans each year. 15 a central clinical and adminis trative relational database, the corporate data warehouse, maintains all information from the vha's comprehensive electronic medical record sys tem and is accessible to vha clinical researchers following a rigorous approval process. the patient cohort consisted of all veterans in the vha system who were alive as of march 1, 2020, who had international classification of diseases (10th edition; icd-10) diagnostic code entries for rheuma toid arthritis, systemic lupus erythematosus, and associ ated rheumatologi cal conditions recorded from vha encounters between oct 1, 2016, and march 1, 2020 (appendix p 1). data were collected to determine the following: evidence of receipt of hydroxychloroquine to the equivalent of at least four 90-day supplies since april 1, 2019, and a medication possession ratio calculation of 80% or more from july 1, 2019, to june 30, 2020, with the most recent receipt within a timeframe to include the date of march 1, 2020; 16 baseline demographic data as of march 1, 2020, to determine age, race, sex, and any tobacco use; all icd-10 codes from oct 1, 2016, to march 1, 2020, to determine the presence of chronic comorbidities; labora tory variables to assess organ dysfunction and to char acterise classification and progression of the patient's rheumatological condition from april 1, 2019, to june 30, 2020, including c-reactive protein, erythrocyte sedi mentation rate, white blood cell count, haemo globin, haematocrit, platelet count, blood urea nitrogen, serum creatinine, aspartate amino transferase, alanine amino trans ferase, lactate dehydro genase, and alkaline phos phatase; out patient prescriptions for methotrexate, leflu no mide, sulfasa lazine, tofacitinib, prednisone, angiotensin-convert ing enzyme 2 inhibitors, angiotensin ii receptor blockers, and zinc, vitamin d, and vitamin c preparations where availability included the date of march 1, 2020; and outpatient prescriptions or infusion evidence before this study we searched pubmed, up to may 1, 2020, for published clinical trials assessing the effect of hydroxychloroquine to prevent laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. the search terms used were "covid-19", "sars-cov-2", and "hydroxychloroquine". we identified a lack of well powered studies to determine the preventive effect of hydroxychloroquine on the development of laboratory-confirmed sars-cov-2 infection. our study is the first to examine a large nationwide population of individuals with rheumatological conditions receiving long-term hydroxychloroquine with high adherence rates, comparing the population to a propensity-matched population not receiving hydroxychloroquine. the primary endpoint, the development of laboratory-confirmed sars-cov-2 infection, was not significantly different between the two propensity-matched cohorts. the findings of this study expand the knowledge base on the role of hydroxychloroquine in sars-cov-2 infection, supporting preliminary data from smaller studies suggesting that hydroxychloroquine might not be an effective agent to prevent sars-cov-2 infection. see online for appendix clinic orders for adalimumab, certolizumab, etanercept, golimumab, infliximab, abatacept, rituximab, belimumab, or tocilizumab where last dose administered would remain active (based on frequency given) up to the date of march 1, 2020. all univariate variables were assessed for their association with the use of hydroxychloroquine. those univariate variables with a standardised mean difference of more than 0·10 were entered into a nominal multivari ate logistic regression model to determine indepen dent variables associated with the use of hydroxychloro quine. this model computed a propensity formula, and a propensity score was calculated for each participant. each patient who was receiving hydroxychloroquine was matched to two patients who were not receiving hydroxy chloroquine (controls) with the next-nearest propensity score to the patient receiving hydroxychloro quine, stratified by the vamc and rural or urban status, sorted by area zip code. the resultant propensity population was assessed with the following data collection for data points between march 1 and june 30, 2020: hospital admission dates and discharge dates; ward locations associated with hospital admission for any reason; discharge diagnostic icd-10 codes associated with each admission; emergency or urgent care clinic encounters; any influenza tests done at the individual facilities; any pcr test results for sars-cov-2; and dates of death if applicable. this study was approved by the university of oklahoma health sciences center institutional review board and the oklahoma city va healthcare system research and development committee. the primary endpoint was the proportion of pcrconfirmed sars-cov-2 infection among those receiv ing chronic hydroxychloroquine versus the propensity-matched patients not receiving chronic hydroxy chloro quine between march 1 and june 30, 2020. secondary endpoints comparing patients receiving hydroxy chloroquine with those not receiving hydroxychloro quine within the same time period were: hospital admission associated with sars-cov-2 infection; intensive care requirement associated with sars-cov-2 infection; mortality associated with sars-cov-2 infection; and overall rates of any hospital admis sion and mortality for both propensity-matched groups. we did a univariate analysis to determine variables associated with the development of sars-cov-2 infection, including receipt of chronic hydroxychloroquine. variables with a p value of 0·05 or less were entered into a multivariate logistic regression model to determine variables independently associated with the development of sars-cov-2 infection; receipt of chronic hydroxychloroquine was included in the multivariate model regardless of p value. for all tests and analyses except where specified, the a-priori level of significance was set at a p value of 0·05 or less. standardised mean difference measurements were con sidered well balanced if they were less than 0·25. categorical variables were assessed using χ² test and fisher's exact test where appropriate. wilcoxon rank sum test was used to assess continuous vari ables. univariate and multivariate analyses were done as described. all analyses were done with jmp/sas statistical software (version 12). there was no funding source for this study. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. an icd-10 code for any rheumatological-associated condition was found for 194 900 patients. of these patients, 18 516 were excluded because they died before march 1, 2020. 100 639 patients were excluded because of the presence of only a non-specific icd-10 diagnostic code indicating arthritis otherwise unspecified, arthropathy, several univariate variables were found to be associated with the selection of hydroxychloroquine at a significant level (appendix pp 2-4). the resultant multivariate logistic regression model derived from these variables resulted in a good fit, and odds ratios (ors) and 95% cis for variables found to be independently associated with hydroxychloroquine selection are presented in table 1. the baseline characteristics of 10 703 patients who received hydroxychloroquine compared with 21 406 patients who did not receive hydroxychloroquine were largely similar in the propensity-matched analysis, although some small numerical differences were significant, including younger mean age in those receiving hydroxychloroquine (64·8 years [sd 12·9]) compared with those not receiving hydroxychloroquine (65·4 years [13·3], p<0·0001; table 2). the incidence of active sars-cov-2 infections during the study period did not differ between the two groups (31 [0·3%] of 10 703 vs 78 [0·4%] of 21 406; or 0·79, 95% ci 0·52-1·20, p=0·27; table 3), resulting in an overall rate of infection of 3·39 cases per 1000 patients. the figure shows no difference in time to positive sars-cov-2 pcr test between groups (p=0·27), with day 0 indicating march 1, 2020. sars-cov-2-related secondary outcomes showed no significant differ ence between the two groups among patients who developed active sars-cov-2 infection. overall hospital admission did not differ between the groups; however, overall mor tality was lower in patients receiving hydroxychloroquine than in data are n (%) or mean (sd) unless otherwise stated. csdmard=conventional synthetic disease-modifying antirheumatic drug. *csdmards include hydroxychloroquine, methotrexate, lefunomide, and sulfasalazine; other csdmard refers to agents other than hydroxychloroquine. univariate variables associated with the development of sars-cov-2 infection are presented in the appendix (pp 5-7). the resultant multivariate logistic regression model showed that the following variables were independently associated with sars-cov-2 infection: presence of polyarthritis (or 4·01, 95% ci 1·76-7·89), non-white race (1·65, 1·08-2·50), urban residence (1·78, 1·14-2·89), receipt of vitamin c (3·31, 1·37-6·83), receipt of an angiotensin-converting enzyme 2 inhibitor (1·74, 1·08-2·72), elevated erythrocyte sedimentation rate (1·69, 1·08-2·59), and baseline c-reactive protein greater than 10 μg/ml (2·14, 1·21-3·63). receipt of hydroxy chloro quine was placed into the model but was not independently associated with sars-cov-2 infection (or 0·79, 95% ci 0·51-1·42). our study examined a large nationwide population of patients with rheumatological conditions to determine whether chronic hydroxychloroquine might protect against the development of sars-cov-2 infection. in this study, the proportion of patients with laboratory-confirmed sars-cov-2 infection did not differ between people with rheumatological conditions who received hydroxychloroquine and those who did not, suggesting that chronic hydroxychloroquine might not have a role in the prevention of sars-cov-2 infection. the overall rate of infection of 3·39 cases per 1000 patients in this study appears to be relatively close to the rate of infection in the vha healthcare system, as 18 560 infections had been diagnosed-out of a total of close to 9 million enrolled veterans-by june 29, 2020. 17 although there were no differences between groups in infection-related secondary out comes among patients who developed active sars-cov-2 infection, overall mortality was lower in patients receiving hydroxychloroquine. given that our study's primary purpose was to investigate the association between a drug and prevention of a specific infection, we cannot make con clusions about the observed difference in overall mortality. the high adherence to hydroxychloroquine might result in prolonged survival in patients with sys temic lupus erythematosus and rheuma toid arthritis, 8 and individ uals in the hydroxychloroquine were slightly younger than those in the control group. reports of hydroxychloroquine's in-vitro activity against sars-cov-2 led to rapid inclusion of the drug in clinical studies and to clinical use in patients with active infection. 6, 18 by contrast, other early investigations sug gested that hydroxychloroquine might lead to a delay in mounting an initial antiviral response and increase the initial viral load. [19] [20] [21] one of the first open-label studies by gautret and colleagues 22 showed that patients infected with sars-cov-2 who received hydroxychloroquine and azithromycin had a higher frequency of nasal clearance of the virus compared with patients who did not receive the drug combination. a follow-up study by the same authors that did not include a control group showed rapid trans formation to negative pcr test for sars-cov-2 in patients receiving hydroxychloroquine and azithromycin. 23 both studies were limited because of the study design, small sample sizes, and questionable exclusion of some patients from data analysis. despite the scarcity of substantial evidence, use of hydroxychloroquine with and without azithromycin was rapidly and widely incorporated into treatment protocols for sars-cov2 infection in the usa and globally in early march, 2020. rheumatology associ ations such as the european league against rheumatism and the american college of rheumatology raised con cerns over possible hydroxychloroquine shortages, noting its effectiveness to treat joint pain, autoimmune rashes, and autoimmune thrombotic events, to prevent lupus flares, and to potentially prolong survival in patients with systemic lupus erythematosus and rheumatoid arthritis. 24 more recently, results of studies evaluating hydroxy chloroquine for the treatment of active sars-cov-2 infection have been inconclusive. a meta-analysis includ ing three studies did not show earlier or higher frequency of viral clearance in patents receiving hydroxychloro quine, and showed a two times increase in death in these patients. 25 in addition, a large observational study of 1376 patients in new york city (ny, usa) reported no signifi cant association between hydroxychloroquine use and a combined endpoint of intubation or death (hazard ratio 1·04, 95% ci 0·82-1·32). 10 effective antiviral pharmacological intervention could fill an important gap to prevent sars-cov-2 infection and is likely to play an important part even after effective vaccines become available. so far, there have been no reports of studies evaluating the preventive effects of pharmacological agents other than hydroxy chloroquine against sars-cov-2 infection. a 5-day high-dose course of hydroxychloroquine given to 821 household and occupational contacts of sars-cov-2-infected individuals as post-exposure prophylaxis failed to show a difference between hydroxychloroquine and placebo in the development of compatible symptoms of disease (49 [11·8%] of 414 individuals vs 58 [14·3%] of 407 individuals, p=0·35). 14 however, only 20 participants developed laboratoryconfirmed sars-cov-2 infection in the study (11 [2·7%] of 414 participants in the hydroxy chloroquine group vs nine [2·2%] of 407 in the placebo group, p=0·82). the trial was halted prematurely for futility before the a-priori level of power could be reached. 14 another study indirectly investigated usage of hydroxy chloroquine and colchicine in 1317 individuals who tested positive for sars-cov-2 infection in israel compared with individuals who tested negative. 26 in that study, five of six baseline variables were not balanced between individuals testing positive versus those testing negative, there was no baseline comparison of patients receiving or not receiving hydroxychloroquine, and there was no analysis of adherence among the patients deemed to be receiving hydroxychloroquine (ie, those who had one prescription dispensed between january, 2020, and the patient's first sars-cov-2 positive or negative test). the study showed no difference in the proportion of patients receiving hydroxychloroquine who had a positive test versus those who only had a negative test; however, only three (0·23%) patients in the infected group had received a hydroxychloroquine prescription. studies evaluating prolonged hydroxychloroquine use for prevention of sars-cov-2 infection might provide more useful evidence than short post-exposure regimens. hydroxychloroquine has a long terminal half-life of approximately 45 days and a large volume of distribution. 7 these pharmacokinetic characteristics result in substantial drug accumulation in plasma and tissues over time. our study takes advantage of a setting in which a specific group of patients has been receiving chronic hydroxy chloroquine over several months to years as a novel virus emerges among the population, setting up an ideal premise to test the hypothesis that hydroxychloroquine might be effective in preventing sars-cov-2 infection. the standard limitations of a non-randomised, observational retrospective study using a clinical administrative database apply to our study. however, a rigorous multivariate regression-derived, propensity-matching process was used to produce two comparable groups. march 1, 2020, was our baseline event date, just days before the first known entry of sars-cov-2 infection into the vha. to gather baseline data, we collected all chronic comorbidity data for the 4 preceding years, and we collected laboratory data from 1 year previously (using the most recent value for each) for comprehensiveness. women comprised only 24% of the study population; however, this proportion is much higher than that in most studies done using the vha, as only approximately 5-10% of the enrolled veterans are female. adherence to hydroxychloroquine was mea sured by a 12-month history of prescriptions filled to produce a medication possession ratio. this method does not ensure that patients are taking the medication appropriately, but our strict threshold of including only those with a medication possession ratio of 0·8 or above improves the chances that our population was adherent. no similar measure of adherence was undertaken to evaluate other medications that the patient was receiving, although each medication was documented to have a dispense date with supply that included march 1, 2020. if one extrapolates the high level of adherence of the included patients using hydroxychloroquine to other areas such as ove rall medi cation adherence or healthy lifestyle choices (eg, exercise and diet), a claim could be made that this high level of adherence might create an imbal ance between the included patients using hydroxy chloroquine and those not using hydroxychloroquine. any perceived imbalance could be corrected with appropriate multivariate analyses. as an alternative, investigators could choose to include patients with poor adherence to hydroxychloroquine (34% of patients receiving hydroxychloroquine were excluded in our study for poor adherence) to assess the endpoint of prevention of sars-cov-2 infection; this approach would invite much-deserved criticism, however, of diluting any actual preventive effect of hydroxy chloroquine. the primary endpoint was the development of sars-cov-2 infection during the initial 4-month period of the pandemic as recorded in the us vha system. although we present a time-to-event description in the figure, our study was not designed to provide sophisticated analysis of time-to-event data. many institutions' policies regarding sars-cov-2 pcr testing have depended on test supply availability and prevalence in a particular area; therefore, many people suspected of having sars-cov-2 infection might not have been tested, particularly early in march. thus, given that our outcome measures relied on the objective parameter of a positive sars-cov-2 pcr test, some patients with active infection might not have been included. it is also possible that veterans were tested and treated outside the vha, so we might have not been able to find these patients using the corporate data warehouse database. however, the vha system is used primarily for the majority of the health-care needs of its enrolled veterans, so its electronic database is quite comprehensive for each veteran's healthcare data. to control for the variabilities in testing practices and area prevalence, each propensity-matched patient not on hydroxychloroquine was matched to a patient on hydroxy chloroquine enrolled in the same vamc and by rural or urban residence, sorted by area zip code. the devastation of the sars-cov-2 pandemic and the absence of an available vaccine make it imperative that the research community prioritises pharmacological treatment or prevention strategies effectively and efficiently. the use of observational data drawn rapidly from large clinical administrative databases might be an effective strategy to identify promising agents for further research or to identify agents that might not merit more effort. our data adds to the expanding knowledge base that suggests that hydroxychloroquine might not be an effective agent in the battle against sars-cov-2. cag was responsible for the concept of the study. cag and skt contributed to drafting of the article and critical revisions. cag, skt, rjw, mbh, gk, and sch contributed to the study design, data analysis, and data interpretation, and approved the final version of the article. we declare no competing interests. the vha corporate data warehouse protects information of veterans within the electronic database and does not generally allow sharing of data to individuals or entities outside the vha. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges review of the 2019 novel coronavirus (sars-cov-2) based on current evidence covid-19) weekly epidemiologic update an interactive web-based dashboard to track covid-19 in real time remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology american college of rheumatology guideline for the treatment of rheumatoid arthritis hydroxychloroquine or chloroquine for treatment or prophylaxis of covid-19: a living systematic review observational study of hydroxychloroquine in hospitalized patients with covid-19 clinical efficacy of hydroxychloroquine in patients with covid-19 pneumonia who require oxygen: observational comparative study using routine care data association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid-19 in new york state fda cautions against use of hydroxychloroquine or chloroquine for covid-19 outside of the hospital setting or a clinical trial due to risk of heart rhythm problems a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 department of veterans affairs statistics at a glance standardizing terminology and definitions of medication adherence and persistence in research employing electronic databases us department of veterans affairs. department of veterans affairs covid-19 national summary hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro initial viral load and the outcomes of sars effects of hydroxychloroquine on immune activation and disease progression among hiv-infected patients not receiving antiretroviral therapy: a randomized controlled trial selective regulation of cytokine secretion by hydroxychloroquine: inhibition of interleukin 1 alpha (il-1-alpha) and il-6 in human monocytes and t cells hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study use of hydroxychloroquine and chloroquine during the covid-19 pandemic: what every clinician should know hydroxychloroquine in patients with covid-19: a systematic review and meta-analysis continuous hydroxychloroquine or colchicine therapy does not prevent infection with sars-cov-2: insights from a large healthcare database analysis no external funding was received for this study. this material is the result of work supported with the resources and use of facilities at the veterans affairs health care system (oklahoma city, ok, usa) and the vha corporate data warehouse. key: cord-272654-hh29olk7 authors: bošnjak, berislav; stein, saskia catherina; willenzon, stefanie; cordes, anne katrin; puppe, wolfram; bernhardt, günter; ravens, inga; ritter, christiane; schultze-florey, christian r.; gödecke, nina; martens, jörg; kleine-weber, hannah; hoffmann, markus; cossmann, anne; yilmaz, mustafa; pink, isabelle; hoeper, marius m.; behrens, georg m. n.; pöhlmann, stefan; blasczyk, rainer; schulz, thomas f.; förster, reinhold title: low serum neutralizing anti-sars-cov-2 s antibody levels in mildly affected covid-19 convalescent patients revealed by two different detection methods date: 2020-11-02 journal: cell mol immunol doi: 10.1038/s41423-020-00573-9 sha: doc_id: 272654 cord_uid: hh29olk7 neutralizing antibodies targeting the receptor-binding domain (rbd) of the sars-cov-2 spike (s) block severe acute respiratory syndrome coronavirus 2 (sars-cov-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ace2). we used a surrogate virus neutralization test (svnt) and sars-cov-2 s protein-pseudotyped vesicular stomatitis virus (vsv) vector-based neutralization assay (pvnt) to assess the degree to which serum antibodies from coronavirus disease 2019 (covid-19) convalescent patients interfere with the binding of sars-cov-2 s to ace2. both tests revealed neutralizing anti-sars-cov-2 s antibodies in the sera of ~90% of mildly and 100% of severely affected covid-19 convalescent patients. importantly, svnt and pvnt results correlated strongly with each other and to the levels of anti-sars-cov-2 s1 igg and iga antibodies. moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. compared to pvnt, svnt is less sophisticated and does not require any biosafety labs. since this assay is also much faster and cheaper, svnt will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy. within 6 months since its emergence, the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the cause of coronavirus disease 2019 (covid19) , has spread rapidly worldwide. covid-19 consists of a spectrum of clinical syndromes ranging from asymptomatic cases to mild, flu-like disease to severe illness requiring hospitalization mainly due to pulmonary complications. [1] [2] [3] although sars-cov-2 primarily targets the respiratory system, new data indicate that covid-19 also affects the vascular system, causing thrombotic microangiopathy and thrombosis in multiple organs, including the lungs. [4] [5] [6] it is not surprising, therefore, that patients with pre-existing cardiovascular diseases, hypertension, and other comorbidities are at particular risk. 7 sars-cov-2 utilizes angiotensin-converting enzyme 2 (ace2) as a receptor for entry into target cells and employs tmprss2, a cellular serine protease, for activation of the viral spike (s) protein. 8, 9 both ace2 and tmprss2 are abundant in the upper respiratory tract. 10 an early immune response against sars-cov-2 involves interleukin-6 and interferon signature gene expression in alveolar macrophages and infiltrating monocytes. 11 although this early immune response is accompanied by severe lymphopenia, 12, 13 increasing data indicate that successful recovery from covid-19 relies on antibody and t-cell responses. 12, [14] [15] [16] [17] importantly, there appears to be a strong correlation between circulating sars-cov-2-specific cd4 + and cd8 + t cells and igg antibodies against the nuclear (n) and/or the spike (s) protein of sars-cov-2. 16, 17 current data indicate that anti-sars-cov-2 igm antibodies appear within one week after infection and are present for a month before they gradually decrease. 18, 19 in contrast, anti-sars-cov-2 igg antibodies appear within 10-21 days after infection and appear to remain more-or-less stable for up to 3 months. 18, 19 it is not surprising, therefore, that antibody responses against sars-cov-2 have received attention as a method for accurate assessment of infection prevalence. 20, 21 particularly interesting are antibodies targeting the receptor-binding domain (rbd) of the s protein, as they can block virus entry into cells and thus prevent infection and spread. furthermore, these neutralizing antibodies may be used for passive antibody therapy, 20, 22 as approved by the united states food and drug administration on march 24th, 2020, as an emergency investigational new treatment for severe or lifethreatening covid-19. 23 in addition to general safety measures for plasma donation, a crucial parameter in convalescent plasma donor selection for covid-19 is an adequate neutralizing antibody titer. 24 however, the field is rapidly evolving, and there is still no consensus about the diagnostic value of divergent elisa-based antibody tests for sars-cov-2 seropositivity. 25 moreover, there is uncertainty regarding the durability of anti-sars-cov-2 antibody responses, especially as there are indications that antibody responses to other coronaviruses are variable and transient. [26] [27] [28] [29] it is also not clear whether all covid-19 patients, especially those with mild disease, will produce sufficient amounts of neutralizing antibodies against sars-cov-2 to prevent early reinfection. in the present study, we compared different experimental approaches to qualitatively and quantitatively assess antibody response to sars-cov-2 infection primarily in cohorts of convalescent individuals with mild covid-19 disease. we applied a sars-cov-2 s protein-pseudotyped-vesicular stomatitis virus (vsv) vector-based neutralization assay (pvnt) 8 that has relatively low throughput and relies on infectious viruses, with requirement of a biosafety-2 lab. as reported by others, 30 we also developed and applied a surrogate virus neutralization test (svnt) that detects antibodies interfering with the binding of the sars-cov-2 s protein rbd to ace2 in vitro. this assay is based on broadly available elisa techniques and allows highthroughput analysis. furthermore, we correlated the functional data obtained by svnt and pvnt to anti-sars-cov-2 s1 igg and iga levels measured using a commercial s1 protein elisa as well as to clinical parameters. we found low titers of neutralizing anti-sars-cov-2 s antibodies in 93% of convalescent patients with mild covid-19. importantly, only low levels of neutralizing anti-sars-cov-2 s titers could be detected in convalescent patients exhibiting clinical symptoms for a short period of time. in contrast, sera from convalescent patients with severe covid-19 contained significantly higher total and neutralizing antibody titers. thus, the svnt allows for high-throughput screening and will be valuable for epidemiological studies as well as for identifying suitable plasma donors for passive immunization. serum samples were collected from convalescent covid-19 individuals who volunteered to donate plasma at hannover medical school's (hms) institute of transfusion medicine and transplant engineering. all donors had pcr-diagnosed sars-cov-2 infection and showed only mild clinical symptoms. serum was also collected from inpatients with severe covid-19 symptoms and from healthy controls without any covid-19-related symptoms (tables 1, 2 , s1, and s2). the blood donors provided consent prior to blood donation and the inpatients at the time of hospital admission for their samples to be used for research purposes. written informed consent was obtained from all participants. studies investigating serum samples from healthy controls and covid-19 patients were approved by the hms institutional review board (#9001_bo_k2020, #8973_bo_k2020, and #7901_bo_k2018). elisa serum samples were analyzed in the clinical virology laboratory and clinic for rheumatology und immunology of hms using the ce-certified versions of euroimmun sars-cov-2 s1 igg and iga elisa (euroimmun, lübeck, germany) according to the manufacturer's recommendations. pseudotyped virus neutralization assay a pvnt was performed at hms's institute of virology and the primate center in göttingen as described earlier. 8 in brief, pseudotyped vsv particles were produced by calcium-phosphate transfecting hek293t cells carrying expression plasmids for the respective glycoproteins, either pcaggs-vsv-g 31 for expression of vsv-g of the control virus or pcg1-sars-2 sδ18 32 for the sars-cov-2 spike protein. eighteen hours later, the cells were infected with vsv*δg-fluc, a replication-deficient recombinant vsv in which the vsv-g open reading frame has been replaced by combined gfp and firefly luciferase expression cassettes. 33 this vsv*δg-fluc stock virus was propagated in bhk-21 g43 cells. 34 after incubating the transduced cells with the viral particles for 2 h at 37°c, the supernatant was removed, and the cells were washed twice with pbs. the cells were then supplied with medium containing an mab targeting vsv-g (supernatant from mouse hybridoma crl-2700; atcc) to neutralize residual vsv-g, a step that was omitted for the cells transfected with the vsv-g expression construct. twenty hours later, the pseudotype particle-containing supernatant was separated from the cells by centrifugation and used for neutralization assays. for the pvnt, vero76 cells were seeded at 1 × 10 4 cells per well in 96-well plates. the next day, complement in test sera was 35 ) by applying standard calcium-phosphate procedures. supernatants were collected and separated using a protein a-sepharose column (thermofisher). the bound recombinant protein was eluted with 0.1 m sodium citrate ph 3.5. the buffer was exchanged with pbsd, and integrity as well as purity was confirmed by analyzing 2 µg of protein on a 10% sds polyacrylamide gel. the surrogate virus neutralization assay was developed based on the hypothesis that virus neutralizing antibodies should also interfere with the binding of the rbd of sars-cov-2 (sars-cov-2 s rbd) to soluble, surface-immobilized ace2, as described by others, 30 with several modifications. depending on the amount of neutralizing antibodies present in convalescent sera, the binding of sars-cov-2 s rbd to ace2 should be blocked to various degrees that should correlate with the optical density of this enzyme-linked immune sorbent-based assay. in the assay reported herein, the hace2 protein (trenzyme or in-house produced) was coated with different concentrations in 100 mm carbonate-bicarbonate coating buffer (ph 9.6) on f96-maxisorp nunc-immuno plates (thermo scientific) at 4°c overnight. after washing in phosphate-buffered saline (pbs) with 0.05% tween 20 (pbst), the plates were blocked with 2% bovine serum albumin (bsa, sigma) and 0.1% tween 20 in 1× pbs for 1.5 h at 37°c. then, his-tag-conjugated sars-cov-2 s rbd (trenzyme) was added to the carrier buffer (1% bsa and 0.05% tween in 1× pbs) at different concentrations and incubated for 1 h at 37°c. unbound sars-cov-2 s rbd was removed by four pbst washes before an anti-his peroxidase-labeled monoclonal antibody (mab; clone 3d5, prepared in house) in carrier buffer was added for 1 h at 37°c. after the final wash, the colorimetric signal was developed by adding the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (sigma) and stopped by adding an equal volume of stop solution (0.2 m h 2 so 4 ). finally, absorbance readings at 450 and 570 nm were acquired using the spectramax id3 microplate reader (molecular devices). the k d values of the sars-cov-2 binding affinity to ace2 were calculated from binding curves based on their global fit using one-site specific binding analysis (graphpad prism). to test for the presence of neutralizing anti-sars-cov-2 s serum antibodies, 6 ng of sars-cov-2 s rbd was preincubated with test sera at final dilutions between 1:20 and 1:540, as indicated on the graphs, for 1 h at 37°c before adding them to plates coated with 150 or 300 ng/well ace2. for each reaction, the percent inhibition was calculated from optical density values after subtraction of background values as: inhibition (%) = (1 − sample od value/ average sars-cov-2 s rbd od value) × 100. neutralizing svnt titers were determined as the dilution with binding reduction > mean + 2sd of values from sera of healthy controls. statistical analysis linear data were analyzed using an unpaired t-test with welch's correction (for 2 groups) or welch's anova followed by dunnett's t3 multiple comparisons test (for 3 groups) and were correlated using the pearson r test. categorical data were analyzed using the χ 2 test or fisher's exact test for unpaired proportions, as indicated beneath each figure. correlation between linear and categorical data was assessed using ordinary one-way anova followed by the test for linear trend. all statistical analyses were conducted using graphpad prism 8.4 (graphpad software, usa). most individuals with mild covid-19 disease develop anti-sars-cov-2 antibodies between march 23 rd and may 11 th 2020, we enrolled as a first step 50 convalescent covid-19 patients diagnosed with sars-cov-2 infection by rt-pcr and 12 healthy control subjects who were not exposed to sars-cov-2. the samples from the convalescent covid-19 cases were split into two groups according to disease severity. eighty percent of the individuals (n = 40) had a mild clinical course, with an average symptom duration of 10 days (range, 0-25 days), and did not require an inpatient hospital stay (tables 1 and s1). ten patients had severe covid-19 and required hospital stays longer than 2 weeks and respiratory support. the patients with severe covid-19 had an average disease duration of 37 days (range from 19-71 days). to estimate overall antibody responses against sars-cov-2 in the serum of covid-19 convalescent individuals, we analyzed the presence of anti-sars-cov-2 igg and iga antibodies targeting the s1 protein by elisa. anti-sars-cov-2 s1-specific igg antibodies were detected in 35/37 (94.6%) of the mildly affected and in 10/ 10 severely affected covid-19 patients tested. one individual with mild disease was considered to have borderline serum positivity, and one patient was negative according to the manufacturer's classification (fig. 1a) . similarly, anti-sars-cov-2 s iga antibodies were present in 33/37 (89.2%) of the tested sera; two samples were diagnosed as borderline positive and two as negative fig. 1 qualitative analysis of serum total igg (a) and iga (b) antibodies against sars-cov-2 s1 in convalescent patients with mild or severe covid-19 and healthy controls (hc) determined by elisa. shaded area cutoff values to determine positive (above), borderline (within), and negative (below gray area) samples. dots, individuals; bars, mean, ***p < 0.001, welch's anova followed by dunnett's t3 multiple comparisons test low serum neutralizing anti-sars-cov-2 s antibody levels in mildly. . . b bošnjak et al. (fig. 1b ). all sera from tested healthy controls (8/8) were negative for sars-cov-2 s-specific igg and iga (fig. 1a, b) . a surrogate virus neutralization assay for the detection of neutralizing antibodies based on the notion that serum neutralizing antibodies might also reduce the binding of sars-cov-2 to ace2 in vitro, we developed an svnt, as reported by others. 30 we applied the svnt to determine the levels of neutralizing antibodies in the serum of covid-19 convalescents and compared them to healthy controls. at the outset, we coated microtiter plates with different amounts of ace2 obtained from a commercial vendor or produced inhouse and titrated soluble his-tagged sars-cov-2 s rbd. binding was revealed by a peroxidase-labeled anti-his mab and a chromogenic substrate ( fig. 2a, b) . the dissociation constant (k d ) in our assay was 5.9 ± 1.9 nm (mean ± sem) and thus similar to the values reported elsewhere. 36 based on the findings, we used 300 ng/well of commercially produced ace2 or 150 ng/well of in-house produced ace2 in combination with 6 ng/well sars-cov-2 s rbd-his to test sera for their ability to interfere with the binding of these two proteins. indeed, sera from convalescent patients interfered with s protein binding to ace2, though with different efficiencies, whereas sera from healthy controls did not (fig. 2c) . sera from convalescent patients were scored as "positive" at any tested dilution once binding reduction was > the mean + 2sd of values from sera of healthy controls. at serum dilutions of 1:20, this assay identified neutralizing antibodies in 38/40 (95.0%) of mildly affected convalescent patients and in 10/10 of severely affected convalescent patients but in none of the 12 tested healthy control samples (fig. 2c, d) . with increasing serum dilutions, the number of "positive" sera dropped constantly (fig. 2e-g) . the median svnt titer of the mildly affected convalescent cohort was 1:180, indicating that patients with mild covid-19 produce relatively low amounts of sasrs-cov-2 neutralizing antibodies (fig. 2h ). in contrast, severe covid-19 convalescent patients had a median svnt titer of ≥1:540 (fig. 2h) . importantly, data derived from testing several serum samples in assays using two different sources of ace2 correlated strongly (fig. s1 ). neutralizing antibodies determined by a pseudotyped virus neutralization test next, we determined levels of neutralizing antibodies by an established pvnt that relies on the use of replication-defective vsv particles carrying the sars-cov-2 s protein to reflect the entry of sars-cov-2 into host cells. 8 particles carrying the g-protein of vsv were used as a control. sera from healthy controls neither suppressed vsv-g nor sars-cov-2 s-driven transduction. similarly, sera from convalescents did not suppress transduction driven by vsv-g but in many cases transduction mediated by sars-cov-2 s was inhibited (fig. 3a) . we used this assay to determine the serum dilutions that reduce the transduction of pvsv by 90% (pvnt 90 ) and 50% (pvnt 50 ) (fig. 3b, c) . in contrast to the 10 tested serum samples from severely affected covid-19 convalescent patients who all had neutralizing antibodies, such molecules were detected in only 29 (pvnt 90 ) and 37 (pvnt 50 ) of the 40 tested serum samples from mildly affected convalescents but in none of the sera from healthy controls (fig. 3b, c) . of note, the median pvnt 50 in the mildly affected convalescent group was 1:100, and the median pvtn 90 was 1:25, further fig. 2 the surrogate virus neutralization test (svnt) detects neutralizing antibodies interfering with sars-cov-2 s rbd binding to human ace2. binding of sars-cov-2 s rbd to human ace2 from commercial vendor (a) and produced in-house (b). plates were coated with ace2 as indicated. his-tagged sars-cov-2 s rbd was titrated as indicated and detected with an anti-his peroxidase-labeled mab. representative assays performed in duplicate are presented as the mean ± sd. c inhibition of the interaction of sars-cov-2 s rbd with ace2 by the addition of sera from convalescent patients with mild (blue lines) or severe (red lines) covid-19 and healthy controls (hc; black lines). assay performed in duplicate; mean percentages of neutralization ± sd. d-g inhibition of the interaction of sars-cov-2 s rbd with ace2 at the serum dilutions indicated. individual values (dots) and means (line). shaded areas represent the mean ± 2sd of values from sera of healthy controls. *p < 0.05; ***p < 0.001, welch's anova followed by dunnett's t3 multiple comparisons test. h relative distributions of sars-cov-2 neutralizing serum titers determined as the dilution retaining binding reduction > mean + 2sd of hc. ***p < 0.001; fisher's exact test (hc vs. mild or severe) or the chi-squared test was used to assess the trend (mild vs. severe) low serum neutralizing anti-sars-cov-2 s antibody levels in mildly. . . b bošnjak et al. supporting the finding that patients with mild covid-19 only produce low amounts of sars-cov-2 neutralizing antibodies. in contrast, the median pvnt 50 and pvtn 90 in severely affected covid-19 convalescents were 1:1600 and 1:400, respectively, further indicating that patients recovering from severe disease produce higher neutralizing anti-sars-cov-2 antibody titers than patients with mild disease. positive correlation between total anti-s1 protein and neutralizing antibody levels in sera of convalescent individuals with mild covid19 we then analyzed the correlation between total levels of anti-s1 igg and iga and the amount of neutralizing antibodies in our cohort of mild covid-19 convalescent patients and healthy controls. as expected, an initial comparison showed a strong positive correlation between the levels of s protein-specific iga and igg antibody in sera (fig. s2 ). more importantly, there was a robust positive correlation between the percent inhibition of sars-cov-2 s rbd binding to ace2 at a 1:20 serum dilution (svnt 1:20 ) and pvnt 90 as well as pvnt 50 inhibitory titers (fig. 4a, b) . furthermore, there was a strong positive correlation between svnt and pvnt 50 as well as pvnt 90 inhibitory titers (fig. 4c, d) . similarly, a strong positive correlation between svnt 1:20 and levels of sars-cov-2 s1-specific igg and antibody levels in convalescents and healthy controls was observed, though levels of anti-s1 iga showed a weaker correlation (fig. 4e, f) . these data demonstrate that svnt reliably detects neutralizing serum antibodies against sars-cov-2. furthermore, a strong correlation with r 2 values between 0.64 and 0.75 was revealed when comparing sars-cov-2 s1-specific igg and iga antibody levels with pvtn 90 and pvtn 50 (fig. s3) . total and neutralizing anti-sars-cov-2 s antibody levels in sera correlate positively with symptom duration but not with the timing of sampling or patient age we next examined whether the level of the protective humoral response to sars-cov-2 in covid-19 patients correlated with disease duration, as defined by the number of days patients showed symptoms (mild cases) or until they were discharged from the hospital (severe cases). not surprisingly, severely affected patients had a 3.5 times longer disease duration, averaging 36 days, than the 10 days of the mildly affected patients (fig. 5a) . as severely ill patients produced higher neutralizing and total antibody titers, these data indicate that disease duration might directly influence antibody titers. this hypothesis is further supported by a positive correlation between the duration of symptoms and total anti-sars-cov-2 igg, but not iga, antibodies in convalescent patients with mild disease (fig. 5a, b) . although weaker, there was also a positive correlation between symptom duration and levels of neutralizing antibodies, as determined by svnt 1:20 , pvtn 90 , and pvtn 50 (fig. 5d, e) . these data are in agreement with data reported by robbiani et al. 37 and with a recent publication indicating that asymptomatic sars-cov-2 infection induces lower antibody levels than symptomatic infection. 38 altogether, our results and publicly available data suggest that a certain threshold of disease severity and/or duration might be required for successful mounting of neutralizing anti-sars-cov-2 humoral responses. conversely, in our cohort of convalescent covid-19 cases, we did not observe any correlation between levels of total and neutralizing anti-sars-cov-2 s antibodies with the timing of sampling after the occurrence of first symptoms (fig. s4) . similarly, the levels of neutralizing anti-sars-cov-2 s igg and iga antibodies did not correlate with the age of patients who had recovered from mild disease, even though convalescent patients with severe disease were on average older than those with mild covid-19 (fig. s5) . of note, our cohort of convalescent covid-19 patients with mild disease was selected based on the ability to donate blood and therefore included only three elderly patients (aged 60 or over). thus, there is not sufficient power to detect levels of total and neutralizing anti-sars-cov-2 s igg and iga antibodies in this age group. similarly, most of the severely affected patients investigated in this study were males, but the relatively small cohort size did not allow analysis of sex associations using our data. svnt allows rapid screening of sera of blood donors for the presence of neutralizing antibodies to validate our findings, we recruited a second cohort of 44 convalescent patients with mild covid-19 and analyzed their sera by elisa and svnt (tables 2 and s2 ). in this group of patients, elisa detected s1 protein-specific iga and igg antibodies in 38 of 44 analyzed serum samples (borderline counted as negative; fig. 6a ). as described above for the first group of mildly affected convalescent patients, levels of s protein-specific iga and igg antibodies showed a strong correlation (fig. s6) . similarly, applying the svnt confirmed that mildly affected covid-19 convalescent patients possess relatively low titers of neutralizing anti-s rbd antibodies (median 1:180). this assay revealed neutralizing antibodies in the sera of 40 of 44 (90.9%) individuals with mild covid-19 (fig. 6b ). along the same line, a strong positive correlation between the svnt 1:20 and total levels of sars-cov-2 s-specific igg and iga antibody levels was also identified (fig. 6c, d) . moreover, in this group of samples, we observed a weak positive correlation between symptom duration and svnt 1:20 or log-transformed sars-cov-2 s-specific igg but not iga levels (fig. s7a-c) . as expected, we did not detect any correlation between specific igg, iga, or svnt 1:20 and patient age or date of sampling (fig. s7d-f and data not shown) . as a final validation, we correlated the svnt 1:20 and total levels of sars-cov-2 s-specific igg and iga antibody levels, pooling the data from the initial and validation cohorts. as depicted in fig. s8 , the pooled set of data revealed a strong positive correlation between svnt 1:20 and total levels of sars-cov-2 s-specific igg and iga antibody levels as well as a weak positive correlation between symptom duration and svnt1:20 and log-transformed sars-cov-2 s-specific igg but not iga levels. altogether, the results from the validation cohort confirmed our initial results and further emphasize the usefulness of the svnt for rapid screening of a larger number of samples for the presence of neutralizing anti-sars-cov-2 s rbd antibodies. a detailed understanding of immune responses following sars-cov-2 infection will enable better treatment and diagnostic procedures, as well as the development of successful vaccines that will help to control the global covid-19 pandemic. in this regard, it is important to gain a better understanding of the presence of neutralizing anti-sars-cov-2 serum antibodies in the population, as they potentially prevent (re)infection and might be a treatment option. 39, 40 as reported by others, 30 we established an svnt that is based on elisa technology and thus can be adapted to allow for high-throughput analysis of samples. we validated this assay by comparing data from the svnt with those derived from a classical pvnt and found a strong correlation between the results obtained with these two tests, which is in line with the results of tan et al. 30 our experience confirms that svnt is technically less complicated, cheaper, and much faster than pvnt, making it more suitable for the rapid screening of a large number of samples. of note, tan et al. also showed that svnt, apart from a small degree of cross-neutralization with anti-sars-cov antibodies, is specific for sars-cov-2. 30 standardization of this test might, therefore, be important for the selection of convalescent plasma donors for the treatment of covid-19 patients. furthermore, as this assay does not rely on antispecies antibodies, it can also be applied to detect neutralizing antibodies in any animal species used for preclinical testing of sars-cov-2 vaccines. svnt might also be adapted for the detection of immunoglobulin classes that most efficiently neutralize sars-cov-2 s or used for rapid screening of neutralizing capacities of monoclonal sars-cov-2 s rbd-specific antibodies, accelerating the development of drugs based on recombinant neutralizing antibodies. on the other hand, a disadvantage of svnt, as compared to pvnt, is its intrinsic inability to detect neutralizing antibodies other than those binding to the sars-cov-2 s rbd. nevertheless, these non-rbdtargeting antibodies appear to have only a minor role in sars-cov-2 neutralization, 37, 41 which is supported by the robust correlation between svnt and pvnt reported in the present study and by others. 30 combining pvnt and svnt, we found that~90% of recovered patients with mild covid-19 possessed neutralizing serum antibodies. these findings are in line with an early report suggesting that recovered covid-19 patients have neutralizing sars-cov-2 s rbd antibodies in serum after discharge from the hospital; 16 other preliminary data indicate that neutralizing sars-cov-2 s rbd antibodies are undetectable in one-third of convalescent covid-19 patients. 37, 42 additional studies are therefore required to provide more detailed insight into the levels of neutralizing sars-cov-2 s antibodies in convalescent covid-19 individuals from different countries. those studies should also exclude false-positive pcr or covid-19 misdiagnosis as the possible reason for the lack of antibodies in certain suspected covid-19 patients. this would be particularly important, as convalescent covid-19 individuals without neutralizing antibodies might still be susceptible to reinfection and would not be able to provide plasma for the prevention and treatment of covid-19. our data also corroborate other studies indicating that levels of neutralizing antibodies in convalescent covid-19 individuals are generally low. 37, 42 interestingly, robbiani et al. reported recently that individual neutralizing antibodies against sars-cov-2 s rbd have a half-maximal inhibitory concentration against authentic sars-cov-2 ranging from 3 to 709 ng/ml. 37 together, these data suggest that a considerable proportion of covid-19 patients with mild disease can produce intermediate-to high-affinity igg antibodies. since such antibodies are not present in a certain proportion (5-30%) of covid-19 cases, these findings indicate that fig. 5 the duration of symptoms correlates with total and neutralizing sars-cov-2 s-specific antibody levels. a symptom duration of mild and severely affected covid-19 patients. dots, individuals; bars, mean, ***p < 0.001, welch's t test. weak positive correlation between duration of symptoms and levels of log-transformed sars-cov-2 s1-specific igg (b) and iga (c) antibodies, svnt 1:20 (d), pvnt 90 (e), or pvnt 50 (f) neutralizing antibody titers. dots, convalescent individuals with mild covid-19, outliers are marked with x, horizontal lines, means. b, c shaded areas indicate vendor-defined cutoff values to determine positive (above), borderline (within), and negative (below gray area) samples. d the shaded area indicates the mean ± 2sd range of inhibition of sera from hc. correlation, pearson r (b-d) or one-way anova followed by a test for the trend (e, f). an outlier was defined as a value with absolute residual value > 2sd of all residual values (d) or as a value > mean ± 2sd of values with the same titer low serum neutralizing anti-sars-cov-2 s antibody levels in mildly. . . b bošnjak et al. effective virus clearance does not rely exclusively on the humoral response but also includes cellular immune responses. 12, [14] [15] [16] [17] the present investigation was performed primarily on young and middle-aged convalescent covid-19 patients with mild disease fulfilling all criteria for blood donation. it is therefore not surprising that in the present study, patient age did not affect neutralizing anti-sars-cov-2 s igg titers, as has been reported by others. 37, 42 our data indicate that serum levels of total and neutralizing anti-sars-cov-2 s antibodies correlate positively with the duration of symptoms, which is also in line with the observations made by others. 42 this hypothesis is further supported by findings that significantly higher titers of neutralizing antibodies were present in sera of severely affected than in sera of mildly affected patients. however, as another study did not find a correlation between the duration of symptoms and neutralizing anti-sars-cov-2 s igg titers, 37 future studies are needed to evaluate how age or disease duration affects neutralizing antibody titers. the passive transfer of donor plasma seems to provide clinical benefit for severe but not critically ill patients. 24, 40 encouraged by the promising results from initial studies with small patient numbers, more than 50 larger randomized clinical trials are currently evaluating the effectiveness of plasma transfusion from convalescent to ill covid-19 patients. 24 the results of these studies will provide an answer as to whether the success of passive immunization by plasma transfusion correlates to total or neutralizing antibody levels in donor sera. moreover, the results will also be important for the development and potential clinical application of recombinant neutralizing anti-sars-cov-2 antibodies, such as the recently described 47d11. 43 in conclusion, this study reports a high-throughput svnt for sars-cov-2. the results obtained with this assay correlate highly with data obtained by classical but laborious and time-consuming pvnt. both assays revealed the presence of neutralizing anti-sars-cov-2 s antibodies, albeit at low titers, in the sera of many but not all convalescent covid-19 patients with mild disease. although these findings have implications for the selection of convalescent donors for passive immunization by plasma therapy, additional studies are required to understand why neutralizing anti-sars-cov-2 s antibodies do not develop in all patients and how long neutralizing antibodies are present in patients who have recovered from covid-19. clinical characteristics of 3062 covid-19 patients: a meta-analysis a novel coronavirus from patients with pneumonia in china prevalence of asymptomatic sars-cov-2 infection: a narrative review the science underlying covid-19: implications for the cardiovascular system pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 postmortem examination of covid-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov sars-cov-2 reverse genetics reveals a variable infection gradient in the respiratory tract pathological inflammation in patients with covid-19: a key role for monocytes and macrophages reappearance of effector t cells is associated with recovery from covid-19 functional exhaustion of antiviral lymphocytes in covid-19 patients breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 a serum anti-sars-cov-2 s1 antibodies determined by elisa. shaded areas, cutoff values to determine positive (above), borderline (within), and negative (below gray area) samples. dots, individuals; bars, mean. b relative distribution of sars-cov-2 neutralizing serum titers determined as the dilution retaining binding reduction > mean + 2sd of hc. correlation between svnt 1:20 and log-transformed sars-cov-2 s1-specific igg (c) and iga (d) levels measured by elisa. vertical shaded areas, 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generation of vesicular stomatitis virus pseudotypes the sequence of human ace2 is suboptimal for binding the s spike protein of sars coronavirus 2. biorxiv prepr key residues of the receptor binding motif in the spike protein of sars-cov-2 that interact with ace2 and neutralizing antibodies convergent antibody responses to sars-cov-2 in convalescent individuals clinical and immunological assessment of asymptomatic sars-cov-2 infections neutralizing antibodies against sars-cov-2 and other human coronaviruses deployment of convalescent plasma for the prevention and treatment of covid-19 the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications. ssrn electron a human monoclonal antibody blocking sars-cov-2 infection we thank all study participants for providing us with the samples. this work was supported by deutsche forschungsgemeinschaft, dfg excellence strategy exc 2155"resist" (project id39087428) and by funds of the state of lower saxony the online version of this article (https://doi.org/10.1038/s41423-020-00573-9) contains supplementary material.competing interests: the authors declare no competing interests. key: cord-275138-033r259v authors: hayden, frederick g; farrar, jeremy; peiris, j s malik title: towards improving clinical management of middle east respiratory syndrome coronavirus infection date: 2014-07-31 journal: the lancet infectious diseases doi: 10.1016/s1473-3099(14)70793-5 sha: doc_id: 275138 cord_uid: 033r259v nan a decade on from the 2002-03 outbreak of severe acute respiratory syndrome coronavirus (sars-cov) infections, the world is again confronted by the possible international spread of a novel coronavirus, the middle east respiratory syndrome coronavirus (mers-cov), which apparently originated in the arabian peninsula. 1 mers-cov is associated with severe respiratory tract infection, often renal failure, and mortality exceeding 40% in patients admitted to hospital. 2 similar to sars-cov, it is closely related to bat coronaviruses, but mers-cov has diff erent cellular receptor specifi city and a broader species range-camels seem to have a role as a natural host. saudi arabia has been most severely aff ected so far, but imported cases-either recent travellers or those transported for clinical care-have been seen in countries in europe, africa, asia, and north america. major nosocomial outbreaks have happened in the middle east, 3 and non-sustained human-to-human transmission events elsewhere, 4 and many more clinical cases are likely to have occurred. 5 there has been a recent surge in case reporting that could be the result of more human-to-human transmissions due to a change in exposure patterns, expansion of the virus in animal reservoir(s), seasonal variation, ongoing nosocomial clusters, or increased surveillance with reporting of mild or asymptomatic mers-cov detections, or both. 5 severe mers-cov disease has occurred primarily in older adults, particularly men, with comorbidities. 2 however, data on disease pathogenesis, particularly viral replication patterns and clinical manifestations, are scarce at present. despite more than 500 laboratory-confi rmed mers-cov cases so far, only a handful of patients have had systematic virological and biomarker sampling. 4, 6 consequently, there are many unanswered questions regarding sites of infection, pathogen dynamics, innate and adaptive immune responses, and host genetic factors. prolonged viral replication in the lower respiratory tract, extra pulmonary virus detection, severe lung injury with respiratory failure, and often renal failure are notable features, suggesting that an eff ective antiviral regimen, perhaps in combination with immunomodulatory agents, would provide clinical benefi t. although supportive care is central to clinical management of coronavirus infections, appropriate antiviral and immunomodulatory therapy for both sars 7 and mers-cov infections remain uncertain because of a scarcity of quality evidence. an absence of good animal models for mers-cov poses a major challenge. many agents have inhibitory activity in vitro for coronaviruses, including some licensed drugs, 7-9 but it is unclear whether their human pharmacology and tolerability would enable suffi cient doses to be given to exert antiviral eff ects in patients with mers-cov. one available drug that is inhibitory for coronaviruses in vitro at clinically achievable levels is the inosine-5´monophosphate dehydrogenase (impdh) inhibitor mycophenolic acid, 8 but animal data for this eff ect are scarce, and one patient developed infection while receiving mycophenolate mofetil. 4 ribavirin and interferon combinations are associated with modest centre for infections/public health england/science photo library antiviral eff ects in mers-cov inoculated rhesus macaques given high doses. 9 although the clinical relevance of these fi ndings is uncertain, the combined use of antiviral drugs to enhance inhibitory eff ects and reduce the potential for resistance emergence makes sense. at present, the strongest treatment evidence supports the use of convalescent plasma or other preparations that possess neutralising antibodies. 10, 11 convalescent plasma seemed to reduce duration of treatment in hospital and mortality when used early in patients with sars. 7 for mers-cov, low neutralising antibody responses and inability to acquire suffi cient convalescent plasma from survivors with comorbidities might restrict the eff ectiveness of this treatment, although these limitations might not apply to infected health-care workers. additionally, the availability of human neutralising monoclonal antibodies 12 or polyclonal immune globulin produced in transgenic cows or other hosts 13 could overcome these hurdles. the high seroprevalence of high-titre neutralising antibody to mers-cov or a closely related virus in dromedary camels in the region raises the possibility of using camel sera or engineered single domain camel antibodies for therapy. 13, 14 purifi ed immunoglobulins or immunoglobulin fragments (nanobodies) might off er a therapeutic option for severely ill patients until more defi ned, genetically engineered, antibodies become available. for any chosen intervention, we advocate that use must be accompanied by a prospective, protocolbased assessment of safety and eff ectiveness that includes sequential virological, clinical, and biomarker measurements. we wrote about the slow acquisition of such data in the 2009 h1n1 infl uenza pandemic. 15 one outcome from this circumstance was the formation of the international severe acute respiratory and emerging infection consortium (isaric), a global federation of academic clinical research networks. isaric has collaborated with who to develop biological sampling protocols that are applicable for patients with mers-cov. furthermore, working with colleagues in public health england, isaric experts have examined available data and ranked potential therapeutic options with regard to their priority for clinical study; 10 this information will be updated as new data become available. however, no mers-cov patients have yet been enrolled on therapeutic protocols incorporating systematic sampling through isaric or any other organisation. consequently, we are not learning what might benefi t or potentially harm such patients. whatever agent or agents are selected for testing, systematic harmonised data collection involving robust observational studies or, when possible, controlled trials, is needed to assess both disease pathogenesis and candidate therapeutics for mers-cov. clinicians and public health offi cials in aff ected middle eastern countries, particularly in saudi arabia, are uniquely positioned to undertake such studies. with support as needed from international partners like who and isaric, 11 regional governments, and funders, middle eastern colleagues have both the opportunity and the responsibility to undertake studies to advance the understanding of eff ective prevention and treatment strategies for mers-cov and any future novel cov outbreaks. thus far, mers-cov is yet another emerging infection threat for which the clinical research response has been too slow and uncoordinated. new investigative frameworks, possibly incorporating mandates into the international health regulations, are urgently needed. the virus has resurfaced in israel, and might be linked to use of intravenous inactivated polio vaccine (ipv). 5 transnational mobility also contributes to polio's persistence, and circulating vaccine-derived poliovirus (cvdpv) in yemen, mozambique, and madagascar is further complicating eradication eff orts. 6 we know that the spatial distribution of polio (where polio exists in some places, is contained in others, is detected but not virulent, has gained virulence through mutation, or is a threat) is very complex. the distribution is complex because the diff usion of poliovirus is associated with diff erent types of polio, bodies, ecologies, and geopolitical realities. polio can be biomedically engineered polioviruses (ipv), degraded versions of the virus (oral polio vaccine [opv]), mutating viruses, or the so-called wild polio virus and its various strains; bodies can have no poliovirus, wild polio resistance, symptomatic polio, ipv, opv, cvdpv, or be subclinical; ecologies vary across landscapes of built and natural environments; and geopolitical realities create diff erent regulatory structures, biomedical accessibilities, confl icts, migrations, and tensions. this complexity means that eradication of polio in some places for some people with certain forms of a vaccine might not be possible in the immediate future. we thus have to better imagine how diff erent types of viruses, bodies, built and natural ecologies, and geopolitical realities interact to produce the present landscape of infectious disease. 7 as health geographers, we argue that such complexity demands a diff erent spatial imaginary and concomitant vocabulary to understand polio. 8 a set of assumptions in standard epidemiological practice suggest that control can happen through geographical containment in particular places and bodies. 9 polio containment leads to the elimination of wild or mutated viruses in particular places-a process that provides the promise of biomedical science's capacity to eradicate and then extinguish these uncontrolled forms of life. although we are fully supportive of all eff orts to eliminate human suff ering, including vaccination, we also believe in the need to be more realistic about the capacities of the virus; 10 the assumptions embedded in vaccination eff orts do not appreciate the ontological position of viruses circulating through ecosystems. 11, 12 polioviruses are not bound to the humanly produced built and natural ecologies in which they exist nor the political or natural boundaries; the interest of polioviruses is survival, and this depends on their ability to fi nd a human host. polioviruses, therefore, do not rely on the ocularcentric spatial imagination of human beings. people need to see polioviruses to know how to eradicate and control them, including the viruses used in vaccines and laboratory studies. polioviruses know how to negotiate the negative spaces between human vision and the bodies and ecological land scapes that aff ord them their clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection severe acute respiratory syndrome and coronavirus broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques clinical decision making tool for treatment of mers-cov v.1.1 outbreak readiness workshop: clinical management and potential use of convalescent plasma potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein production of human polyclonal antibodies by transgenic animals formatted single-domain antibodies can protect mice against infection with infl uenza virus (h5n2) patient-oriented pandemic infl uenza research key: cord-275252-4e3cn50u authors: rad sm, ali hosseini; mclellan, alexander d. title: implications of sars-cov-2 mutations for genomic rna structure and host microrna targeting date: 2020-05-16 journal: biorxiv doi: 10.1101/2020.05.15.098947 sha: doc_id: 275252 cord_uid: 4e3cn50u the sars-cov-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. although the mutation rate is limited, recently introduced mutations in sars-cov-2 have the potential to alter viral fitness. in addition to amino acid changes, mutations could affect rna secondary structure critical to viral life cycle, or interfere with sequences targeted by host mirnas. we have analysed subsets of genomes from sars-cov-2 isolates from around the globe and show that several mutations introduce changes in watson-crick pairing, with resultant changes in predicted secondary structure. filtering to targets matching mirnas expressed in sars-cov-2 permissive host cells, we identified twelve separate target sequences in the sars-cov-2 genome; eight of these targets have been lost through conserved mutations. a genomic site targeted by the highly abundant mir-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. our results are compatible with a model that sars-cov-2 replication within the human host could be constrained by host mirna defence. the impact of these and further mutations on secondary structures, mirna targets or potential splice sites offers a new context in which to view future sars-cov-2 evolution, and a potential platform for engineered viral attenuation and antigen presentation. the sars-cov-2 virus has rapidly emerged as a zoonotic pathogen with broad cellular tropism in human, or zoonotic-host cells. host selection pressure on sars-cov-2 virus would be expected to have a major impact on the conservation of mutations that enhance viral fitness. of these selection pressures, the cellular-based adaptive and innate immune systems will be major constraints to viral evolution. intracellular detection and anti-viral pathways within infected cells are a critical frontline to control virus replication. the success of the sars coronaviruses is proposed to be due to their ability to suppress intracellular anti-viral pathways. for example, interference with dsrna detection and the interferon response is enabled through the activity of several nonstructural proteins (nsp). in addition, the sequestration of genomic viral rna into double membrane vesicles, and dsrna cleavage by nsp15, is inferred from the closely related sars-cov-1 virus, and likely acts to prevent intracellular detection of the virus [1] . in addition to encoded mechanisms of immune avoidance, the paucity of cpg runs in sars-cov-2 genome with unexpectedly low gc-content at codon position-three points to major selection pressure being placed on structural features of the genome [2] . as a recently emerged zoonotic pathogen, it might be expected that bat-adaptations will not be optimal for infection and replication in human cells. however, extensive mutation and strain-radiation has not yet been observed [3] . the low mutation rate in sars-cov-2 is reduced by the activity of the 3' -5' exonuclease nsp14 in the rna-dependent rna polymerase (rdrp) complex. an alternative possibility is that the observed mutation rate is lower than the actual mutation rate, but that deleterious mutations have already been lost through natural selection. the short time frame of sars-cov-2 evolution, coupled to a low mutation rate is consistent with a founder effect for geographical bias in mutation patterns [3] . a common primary focus of mutational analysis of emerging viruses is the alteration in amino acid sequence of viral proteins that may provide enhanced or new functions for virus replication, immune avoidance, or spread. however, nucleic acid secondary structure and sub-translational events have a critical impact on genome replication [4] , virus maturation and genome packaging [5] . in addition, the rna secondary structures of sars-cov-2 genes have been proposed to be druggable targets [6] [7] [8] . because little is known of the influence of sars-cov-2 mutations on the rna secondary structure, and its possible implications for inhibition by host mirna, we have modeled the impact of common mutations of the structure and susceptibility of the sars-cov-2 genome to interference from host mirna. the incident presence of host mirna targets within the sars-cov-2 genome may be pivotal for the action of host selection pressures to further shape further viral evolution. viruses not only alter host mirna expression, but may also produce mirnas to promote their infectivity [9] [10] [11] [12] . on the other hand, the host targets viral transcripts for inhibition of translation, or mrna destruction, through an mirna-mediated defence system. since mirna are extremely divergent between species, it would be expected that bat-adapted sars-cov-2 will undergo selection pressure derived from human mirna interference [9] [10] [11] 13, 14] . while perfect matches of mirna to target viral sequences results in mirna-induced silencing complex (mirisc)-mediate destruction of viral rna, imperfect matches interfere with translation [15] . a growing body of evidence suggests that human mirnas act as a critical host defense against coronaviruses. an interaction between human coronavirus oc43 nucleocapsid and mir-9 can enhance the type i interferon response necessary to clear viral infection [16] . several host mirnas (mir-574-5p, -14, -17, -9, -223 and -148a) bind to sars-cov encoded transcripts such as s, e, m, n and orf1a [17, 18] . on the other hand, sars-cov escapes from mirna-mediated defence through the manipulation of host mirna machinery [17, 18] . additionally, sars-cov and sars-cov-2 express short rnas that resemble mirnas and could impact upon host house-keeping or immune defence processes [19] [20] [21] . more recently, several studies have proposed that host mirnas bind sars-cov-2 transcripts [19, 21, 22] . however, host mirnas inhibition of viral replication is relevant only if the identified mirnas are expressed in target host cells. both dna viruses, and 'cytoplasmically-confined' rna viruses, use the host rna splicing-machinery to generate new viral transcripts, or to modify the host transcriptome in favor of their own replication [23] [24] [25] [26] [27] . it has been suggested that the fused leader sequence in 5′ end of the mouse hepatitis virus (betacoronavirus) mrnas is result of a non-canonical splicing process [28] . moreover, deep rna sequencing has identified several unknown sars-cov-2 viral rnas, possibly the result of non-canonical splicing events [29] . therefore, our study has additionally identified and mapped mutations to predicted mrna splice sites within the sars-cov-2 genome. no selective advantage of the identified sequence alterations in sars-cov-2 should be inferred by their inclusion here. however, the potential of these mutations to impact upon rna structure and mirna recognition provides a basis for ongoing monitoring of viral evolution at these sites in the sars-cov-2 genome. the interplay of viral genome sequences and host mirna might be veiwed as academic and refractory for translation into usable clinical outcomes. however, the inclusion of host mirna binding sites into the orf of conserved viral regions essential for the viral life cycle is a feasible mechanism for enhancing the attenuation of live vaccines [30] [31] [32] , or antigen availability of epiptopes for the adaptive immune response [33] [34] [35] [36] . geneious alignment tools were used to perform multiple sequence alignment. mutations with occurrence in multiple sequences originated from different countries were categorized as conserved mutation. potential splice donor / acceptor splice sites, exon splicing enhancer (ese), exon splicing silencer (ess), intron splicing enhancer (ise) and intron splicing silencer (iss) motifs were predicted using regrna2 [37] , hsf [38] and nipu [39, 40] tools. we used well-accepted methods to predict the rna secondary structure in both wild type and mutated sequences. minimum free energy (mfe) structures [41] and centroid structures [42] were calculated by rnafold program to predict rna secondary structures. to evaluate the impact of mutations on rna secondary setructure and basepair probability, we utilized rnafold, rnaalifold [43] , mutarna [40, 44] and rnasnp [45] programs. for identifying potential mirna binding sites, the sars-cov-2 genome was screened with regrna2 and mirdb [46] . we excluded mirnas that are not expressed in sars-cov-2 target cells such as lung, esophagus, kidney and small intestine [47, 48] . the expression level of mirnas in target cells were determined by tissueatlas [49] , imota [50] , or using published data. the impact of mutations on mirnas binding were visualized by regrna2, mirdb, intarna [51] and copomus [40] . we used intarna to illustrate mirna binding to its target. a total of 55 sars-cov-2 patient isolate sequences were collected from ncbi and gisaid databases were aligned against sars-cov-2 reference sequence (nc_045512.2). the mutations present in multiple sequences and in at least in three different countries were categorized as 'conserved mutations' [52] . in the line with previous reports, we confirmed the occurrence of conserved mutations at positions 1397 (nsp2), 3037 (nsp3), 8782 (nsp4), 11083 (nsp6), 14408 (nsp12), 23403 (s), 26143 (orf3a) and 28144/ 28881 (n) [52] [53] [54] [55] [56] . we also considered nine additional conserved mutations with occurrence in multiple countries (table 1 & s1). most of these mutations are substitutions of c/g to u. the high a/u content (u= 32.1%/ a= 29.9%/ g= 19.6%/ c=18.4%) and enrichment of codons in in pyrimidines is likely due to apobec editing of viral rna and the fact that nsp14 (proof-reading) does not remove u (the product of cytosine deamination) [57] . two mutations at 241 and 29742, are in the 5' and 3' untranslated regions (utrs). seven mutations are silent point mutations, including 313, 14805, 17247 and 28686, while the others result in amino acid changes ( table 1) . none of these amino acid changes are conservative substitutions. interestingly, the c27964u (s24l in orf8) exists only in 97 usa sequences, with the earliest isolated in march 9 th (mt325581.1), after usa underwent lockdown [58] ( figure s1 ). mutation amino acid change ggg to aac -nt28881-28884 r203k and g204r 3' utr g to u -nt29742 among all the mutations, only two mutations were predicted to have an impact on secondary structure of viral rna. first, a conserved mutation 1059 in nsp2 changed the secondary structure of nsp2 dramatically ( figure 1a ). this mutation also increased the watson-crick base-pair probability -predicted to result in more stable rna secondary structure ( figure 1b , c, d & e). this mutation had no effect on rna accessibility which is a consideration for rna-rna and rna-protein interactions ( figure 1f ). mutation 29742 occurs in a conserved region within 3′ utr known as the coronavirus 3' stem-loop ii-like motif (s2m). this mutation has significant effect on secondary structure of the 3′ utr (figure 2a & b) . it is wellknown that s2m present in most coronaviruses and plays a vital role in viral replication and invasion [60] [61] [62] . mutations in this region has been shown to increase the stability of 3′ utr and its interaction with 5′ utr [61] . translation, transcription and ubiquitination regulation [63, 64] . in addition, s2m interacts with viral and host proteins such as the polypyrimidine tract-binding protein (ptb), to regulate viral replication and transcription [61, 62] . collectively, these results suggest that 1059 and 29742 yield more stable rna structures. however, the relationship of changes in rna secondary structure of nsp2 and 3′ utr to viral replication or infectivity must be tested in adequate experimental assays. [59] . the mutated position is highlighted by a red line. in addition to previous studies, we have identified several human mirnas with potential binding sites across sars-cov-2 genome. we filtered our considered mirna to those with documented expression in sars-cov-2 target cells ( figure 3 & s2-11) and additionally focused on mirnas that have been reported as components of the anti-viral mirna-mediated defence system. we hypothesised that some mutations may represent escape mutations to avoid mirna-mediated defence. as shown in figure 4 , ten mutations occur in mirna binding site and abolish the binding of mirnas to their targets. only one mutation (c15293u) inside nsp12 did not destroy the mir-1273d binding site (gisaid: epi_isl_41922). mir-197-5p is upregulated in patients with cardiovascular disease and its detection has been proposed as a biomarker [65] [66] [67] . in addition, it is well-known that patients with cardiovascular disease are overrepresented in symptomatic covid-19 cohorts and have a higher mortality rate [68] . the c3037u conserved, but synonymous, mutation within nsp3 sequence abolished the mir-197-5p target sequence (figure 4 ). this mutation was introduced early january 2020 ( figure s1 ). this mirna was previously reported to act in defence against hepatitis viruses, such as hbv, hcv, hav and enterovirus 71 [69] [70] [71] . three mutations within nsp4 result in loss of mir-3935 and mir-18b-5p target sequences, although they are not conserved mutations. both mirna are expressed in sars-cov-2 target cells ( figure 3b , s4 & s5). nsp4 a9259g is in a sequence obtained from vietnam (gisaid: epi_isl_416429), nsp4 g9802u was reported in a chinese patient [72] and nsp4 c9803u was identified in the netherlands (gisaid: epi_isl_422940) ( figure 4 ). we identified five mirnas with perfectly-matched complementary sequences within the s-gene: mir-29b-3p, mir-338-3p, mir-4661-3p, mir-4761-5p and mir-4793-5p ( figure 3 ). as shown in figure 4 , four of these sites were altered by recently identified mutations in the s-gene. in particular, the mir-338-3p mirna is expressed at high levels in sars-cov-2 target cells ( figure 3b, s9) . the mir-338-3p target in the s-gene is removed by the c24034u conserved mutation identified early january in multiple countries ( figure s1 ). the mir-338-3p mirna acts as tumor suppressor in liver, lung and gastric cancers [73] [74] [75] . the expression level of mir-338-3p declines during hbv infection [76, 77] and mir-338-3p has a recognition site within vaccinia virus genome [78] . also, the g24057a mutation present in an sequence from brazil (gisaid: epi_isl_429691), destroys the complementarity of the mir-338-3p binding site. of interest, two more conserved mutations c22277a (q239k) and a23403g (d614g) occur in binding sites for mir-29b-3p and mir-4793-5p, respectively. lastly, g25311u in a patient sample isolated india (mt396242.1) removed the mir-4661-3p binding site within the s gene ( figure 4 ). in addition to the sites mentioned here, we identified an additional four host mirnas with perfect complementarity within the receptor binding domain (rbd) region of s gene ( figure 5 ). these mirna are not expressed by sars-cov-2 target cells (data not shown). however, because these sites exist within the critical ace-2 targeting region, these mirna target sequences may be relevant to mirna-mediated virus attenuation technology. for example, viral replication can attenuated in a species-specific and tissue-specific manner by host mirna machinery, which controls viral tropism, replication and pathogenesis [30] [31] [32] . atypical cytoplasmic rna splicing has been proposed to contribute to non-canonical viral transcripts, even for viruses that classically replicate in the cytoplasm [23] [24] [25] [26] [27] [28] . moreover, deep rna sequencing has identified several previously unidentified sars-cov-2 viral rnas that may be the result of non-canonical splicing events, or alternative transcriptional start sites [29] . we used regrna2 [37] , hsf [38] and nipu [39, 40] tools to identify the putative splice sites and motifs within sars-cov-2 genome. our computational prediction identified several 5′ donor and 3′ acceptor splice sites, as well as splice enhancer / inhibitor motifs [79] (table 2 ). however, none of conserved mutations introduced, or deleted, any potential splice sites. 10 at present there are nearly 200 mutations identified within global sars-cov-2 isolates. these mutations are mostly limited to point mutations, with little evidence for recombination events mediating the simultaneous transfer of multiple mutations. although mutations may be due rdrp / nsp12 infidelity, the predominance of c ® u and g ® a mutations is consistent with base-editing defence (e.g. apobec / adar) [57, 80] . the nsp14 exonuclease-based proof-reader is a critical counter-defence against host base-editor attack on the sars-cov-2 genome. it is also possible that the position of mutations within the genome could reflect accessibility of host base-editors to the sars-cov-2 genome upon uncoating, or during genome translation [57] . in our study, we filtered mutations to common / conserved events according to published sources [52] . there is little evidence that the existing mutations in sars-cov-2 have an impact on transmission, replication or viral load, but our study has flagged potential sites that could impact on viral fitness. it remains to be seen if these mutations undergo purifying selection in human populations over time. carriage of sars-cov-2 mutations through the rapid expansion into naive populations throughout the world is most likely due to a neutral founder effects, rather than from fitness gains. for example, the high ratio of non-synonymous to synonymous mutations is close to the expected value for an emerging virus that has not undergone purifying selection [3] . one of the mutations (a23403g; d614g) in the spike protein gene studied here defines the rapidly emerging gclade present in one-third of global sars-cov-2 isolates. this mutation has been extensively studied by korber et al. with respect to clinical outcomes and viral load [81] . the korber et al. study showed that the a23403g did not enhance spike protein binding to ace-2, however the mutation may be associated with higher viral load and poorer clinical outcomes. our own analysis demonstrated that this mutation abolished a potential interaction with mir-4793-5p, a mirna highly expressed in target cells including oesophagus, lung epithelium and small intestine. we propose that the a23403g mutation may represent an escape variant from the action of host mir-4793-5p. three mutations within nsp4 were predicted to abolish mir-3935 and mir-18b-5p targeting. the expression of mir-3935 and mir-18b is altered upon viral infections [82] [83] [84] [85] . the expression level of mir-3935 upregulates during h1n1, crimean-congo hemorrhagic fever virus, coxsackievirus a16 and enterovirus 71 infection [82, 84, 85] . on the other hand, mir-18b was reported to be downregulated during hbv and ebola virus infections [83, 86] . similar to what observed for mir-197-5p, both mir-18b and mir-3935 are upregulated in patient with cardiovascular disease [87] [88] [89] . it is not yet clear if anti-viral mirnas have evolved as host defense against viral infection, or are simply critical gene regulatory elements that assume an additional role for targeting viral transcripts -particularly when the human cellular defence machinery is confronted by an emerging zoonotic virus [9, 13, 14] . the possibility of including host mirna binding sites into the genome of live-attenuated viruses offers a further checkpoint for the further attenuation of live vaccines, in a host-cell specific manner. for example, the identification of mirna target sites in viral pathogens opens up opportunities for further study of viral host cell-tropism, or to create cellspecific or species-specific viral vaccines [30] [31] [32] . finally, mirna sites within the cds of viral genes may be critical for ribosomal stalling, leading to the production of pioneer translation products (ptp). enhanced production of ptp peptides may be critical for mhc-i loading for boosting the anti-viral ctl response [33] [34] [35] [36] . conflict of interest iran, taiwan, turkey, australia and hong kong iran, taiwan, sir lanka and turkey table s1 .newly identified common mutations with the counties of occurrence. sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon. current opinion in virology a comprehensive analysis of genome composition and codon usage patterns of emerging coronaviruses no evidence for distinct types in the evolution of sars-cov-2. virus evolution a g-quadruplex-binding macrodomain within the "sars-unique domain" is essential for the activity of the sars-coronavirus replicationtranscription complex structure of the sars 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and disease pathophysiology schnabel, r.b. mirna-197 and mirna-223 predict cardiovascular death in a cohort of patients with symptomatic coronary artery disease association of mir-197-5p, a circulating biomarker for heart failure, with myocardial fibrosis and adverse cardiovascular events among patients with stage c or d heart failure circulating micrornas as biomarkers and potential paracrine mediators of cardiovascular disease pathological findings of covid-19 associated with acute respiratory distress syndrome mir-197 expression in peripheral blood mononuclear cells from hepatitis b virus-infected patients host microrna mir-197 plays a negative regulatory role in the enterovirus 71 infectious cycle by targeting the ran protein reciprocal control of mir-197 and il-6/stat3 pathway reveals mir-197 as potential therapeutic target for hepatocellular carcinoma patient-derived mutations impact pathogenicity of sars-cov-2 the effect of mir-338-3p on hbx deletionmutant (hbx-d382) mediated liver-cell proliferation through cyclind1 regulation the egfr/mir-338-3p/eya2 axis controls breast tumor growth and lung metastasis epigenetic silencing of mir-338-3p contributes to tumorigenicity in gastric cancer by targeting ssx2ip differential plasma microrna profiles in hbeag positive and hbeag negative children with chronic hepatitis b. plos one the function of microrna in hepatitis b virus-related liver diseases: from dim to bright a computational analysis to construct a potential post-exposure therapy against pox epidemic using mirnas in silico silencer elements as possible inhibitors of pseudoexon splicing extreme genomic cpg deficiency in sars-cov-2 and evasion of host antiviral defense spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov-2 highly sensitive in vitro diagnostic system of pandemic influenza a (h1n1) virus infection with specific microrna as a biomarker hepatitis b virus x protein enhances hepatocarcinogenesis by depressing the targeting of nusap1 mrna by mir-18b. cancer biology & medicine systematic identification and bioinformatic analysis of micrornas in response to infections of coxsackievirus a16 and enterovirus 71 identification of potential microrna markers related to crimean-congo hemorrhagic fever disease circulating microrna profiles of ebola virus infection role of micrornas in cardiac hypertrophy, myocardial fibrosis and heart failure micrornas in heart disease: putative novel therapeutic targets? microrna-18b* induces apoptosis in cardiomyocytes through targeting topoisomerase 1 (top1) the authors declare that they have no conflicts of interest with the contents of this article. ah and am conceived of the study and wrote the paper. ah performed the data analysis and produced the figures. key: cord-270550-if748w2n authors: bailey, adam l.; dmytrenko, oleksandr; greenberg, lina; bredemeyer, andrea l.; ma, pan; liu, jing; penna, vinay; lai, lulu; winkler, emma s.; sviben, sanja; brooks, erin; nair, ajith p.; heck, kent a.; rali, aniket s.; simpson, leo; saririan, mehrdad; hobohm, dan; stump, w. tom; fitzpatrick, james a.; xie, xuping; shi, pei-yong; hinson, j. travis; gi, weng-tein; schmidt, constanze; leuschner, florian; lin, chieh-yu; diamond, michael s.; greenberg, michael j.; lavine, kory j. title: sars-cov-2 infects human engineered heart tissues and models covid-19 myocarditis date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.04.364315 sha: doc_id: 270550 cord_uid: if748w2n epidemiological studies of the covid-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. nonetheless, little is understood regarding sars-cov-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. here, we develop a human engineered heart tissue model and demonstrate that sars-cov-2 selectively infects cardiomyocytes. viral infection is dependent on expression of angiotensin-i converting enzyme 2 (ace2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. after infection with sars-cov-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. consistent with these findings, autopsy tissue obtained from individuals with covid-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. these findings establish human cardiomyocyte tropism for sars-cov-2 and provide an experimental platform for interrogating and mitigating cardiac complications of covid-19. to explore whether human cardiomyocytes might be susceptible to sars-cov-2 136 infection, we examined the expression of angiotensin converting enzyme 2 (ace2) within the 137 human heart. previous studies have established that ace2 serves as a cell-surface receptor for 138 sars-cov-2 through interactions with the spike protein in numerous human cell types 15, 16 . 139 immunostaining of human left ventricular myocardial tissue revealed evidence of ace2 140 expression in cardiomyocytes (fig. 1a) . we observed significant variation in ace2 expression 141 between individual cardiomyocytes within the same myocardial specimen. ace2 mrna was 142 abundantly expressed in the healthy human heart and further increased in the context of chronic 143 heart failure (fig. 1b) . rna sequencing of human pediatric and adult heart failure specimens 144 revealed robust expression of ace2 mrna within the human heart across the spectrum of age 145 (fig. 1c) . consistent with our immunostaining findings, primary human cardiomyocytes obtained 146 from the left ventricle and atria expressed ace2 mrna (fig. 1d) . these data are consistent with 147 prior single cell and bulk rna sequencing analyses of human myocardium and suggest that 148 cardiomyocytes might be permissive to sars-cov-2 infection 17 . 149 to ascertain whether human pluripotent stem cell-derived cardiomyocytes (hpsc-derived 150 cms) can serve as an appropriate model to study cardiac sars-cov-2 infection, we measured 151 ace2 mrna expression in hpsc-derived cms. quantitative rt-pcr revealed that hpsc-152 derived cms abundantly expressed ace2 mrna. in contrast, minimal ace2 mrna was detected 153 in human dermal fibroblasts, hpsc-derived cardiac fibroblasts, or human fetal cord blood derived-154 macrophages (fig. s1a-c) . human engineered heart tissues (ehts) self-assembled between two 155 deformable polydimethylsiloxane (pdms) posts after mixing cells in an extracellular matrix 156 composed of collagen and matrigel (fig. s1d) . ehts composed of either hpsc-derived cms and 157 fibroblasts or hpsc-derived cms, fibroblasts, and macrophages also expressed ace2 mrna 158 (fig. s1e) . immunostaining of ehts confirmed the presence of ace2 protein specifically in 159 hpsc-derived cms (fig. s1f) . these data suggest that hpsc-derived cms might be susceptible 160 to sars-cov-2 infection and serve as a suitable experimental model to study cardiac 161 manifestations of covid19 . (fig. s1g) . in contrast, two independent lines of hpsc-derived cardiomyocytes (hpsc-171 derived cms) were permissive to sars-cov-2 infection (fig. 1f) . undifferentiated hpsc lines did 172 not demonstrate evidence of infection (fig s3) . 173 to confirm cardiomyocyte tropism, we inoculated various combinations of hpsc-derived 174 cms, fibroblasts, and macrophages grown in monolayer culture with wild-type sars-cov-2 175 (usa_wa1/2019). we analyzed tissue culture supernatants for production of infectious virus 176 using a vero cell infection-based focus forming assay, and we measured intracellular viral rna 177 transcript levels using rt-qpcr at 3 days post-inoculation. these assays revealed the production 178 of infectious virus (fig. 2a) and viral rna (fig. 2b) selectively in cultures that contained hpsc-179 derived cms. cultures lacking hpsc-derived cms contained viral loads that were equivalent to 180 media-only controls. a time course of hpsc-derived cm infection showed that cardiomyocytes 181 rapidly produced infectious virus with peak titers observed at day 3 post-inoculation. these 182 kinetics were closely mirrored by sars-cov-2-mneongreen (fig. 2c) . 183 using sars-cov-2-mneongreen, we examined the relationship between viral replication 184 and cell death using flow cytometry. although the percentage of hpsc-derived cms that were 185 mneongreen-positive peaked at day 3 post-inoculation, significant levels of hpsc-derived cm 186 cell death were not observed until 4-5 days post-inoculation (fig. 2d) indicating that viral infection 187 precedes hpsc-derived cm cell death. sars-cov-2-infected cardiomyocytes also displayed 188 characteristics of cytopathic effects. cellular rounding, clumping, and syncytium formation first 189 were observed on day 3 post-inoculation. distortion of cellular morphology was evident by day 4 190 post-inoculation and cultures contained largely dead cells and debris by days 5-6 post-inoculation 191 (fig. 2e) . 192 to verify that cardiomyocytes are the primary target of sars-cov-2 in a simulated cardiac 193 environment, we infected two-dimensional tissues assembled with hpsc-derived cms (80%), 194 fibroblasts (10%), and macrophages (10%) with sars-cov-2-mneongreen. flow cytometry 195 performed 3 days following infection revealed mneongreen expression only in cd90 -cd14 -196 tnnt2 + cardiomyocytes. mneongreen was not detected in cd90 + fibroblasts or cd14 + 197 macrophages within infected two-dimensional tissues ( fig. 2f-g, fig. s4 ). these data to examine viral transcription and the host immune response to sars-cov-2 infection, 210 we performed rna sequencing. cultures containing either hpsc-derived cms, fibroblasts, or 211 macrophages were either mock-infected or inoculated with sars-cov-2. we also examined two-212 dimensional co-culture tissues assembled with 80% cardiomyocytes, 10% fibroblasts, and 10% 213 macrophages. cells and tissues were harvested on day 3 post-inoculation. principal component 214 analysis revealed separation between experimental groups consistent with their distinct cellular 215 composition (fig. 3a) . classification of transcript types demonstrated that infected hpsc-derived 216 cms and two-dimensional tissues comprised of hpsc-derived cms and fibroblasts or hpsc-217 derived cms, fibroblasts, and macrophages contained abundant viral transcripts (fig. s5a) . we 218 then assessed the expression of specific viral transcripts by aligning the rna sequencing data to 219 the sars-cov-2 genome and transcriptome. subgenomic rnas were identified based on the 220 presence of 5' leader sequences 21 . we observed robust expression of most sars-cov-2 221 genomic and subgenomic rnas in infected hpsc-derived cms and two-dimensional tissues with 222 the exception of orf7b (fig. 3b, fig. s5b) . 223 to facilitate differential expression analysis of host genes, we censored viral rnas from 224 the rna sequencing computational model. this was necessary given the asymmetric prevalence 225 of viral transcripts across samples. we identified numerous host genes that were differentially 226 regulated upon sars-cov-2 infection in each of the examined cell types and two-dimensional 227 tissues (fig. 3c) . conditions that supported viral replication (hpsc-derived cms and two-228 dimensional tissues) displayed the greatest overlap in differentially expressed genes. cell types 229 that did not support viral replication (fibroblasts and macrophages) also demonstrated numerous 230 differentially expressed host genes, indicating that sars-cov-2 might elicit changes in host gene 231 expression in the absence of direct viral infection. notably, host genes differentially expressed in 232 fibroblasts and macrophages exposed to sars-cov-2 were largely distinct (fig. 3d) . these 233 findings suggest that elements within or on the surface of sars-cov-2 virions may serve as 234 pathogen-associated molecular patterns (pamps) and stimulate distinct gene expression 235 programs in differing cell types. 236 go pathway analysis revealed that infected hpsc-derived cms and two-dimensional co-237 culture tissues showed upregulation of genes associated with immune cell activation, stress-238 induced transcription, and responses to pathogens including viruses. genes associated with 239 metabolism, oxidative phosphorylation, and mitochondrial function were downregulated by 240 infection. two-dimensional tissues displayed alterations in other pathways including upregulation 241 of cellular responses to cytokines and downregulation of genes involved in muscle contraction 242 ( fig. 3e-f) . host genes differentially expressed in macrophages and fibroblasts were associated 243 with pathways involved in innate immune cell activation, migration, and cytokine responses (fig. 244 3g-h). 245 examination of specific genes differentially regulated in infected hpsc-derived cms and 246 two-dimensional tissues (fig. 3i ) revealed marked reduction in components of the electron 247 transport chain (atp synthase, mitochondrial cytochrome c oxidase, nadph dehydrogenase) 248 and key upstream metabolic regulators (glycerol-3-phosphate dehydrogenase, pyruvate 249 dehydrogenase, succinate dehydrogenase complex). pdk4, an inhibitor of pyruvate 250 dehydrogenase was upregulated in infected hpsc-derived cms and two-dimensional tissues. we 251 also observed marked downregulation of numerous components and regulators of the contractile 252 apparatus including cardiac actin, troponin subunits, myosin light and heavy chains, desmin, 253 phospholamban, and calsequestrin in infected two-dimensional tissues. infected hpsc-derived 254 cms displayed similar changes, albeit to a lesser extent. ace2 expression was diminished in 255 infected cardiomyocytes and two-dimensional tissues. 256 infected hpsc-derived cms and two-dimensional tissues also displayed upregulation of 257 key regulators of innate immunity (fig. 3i) . type i interferon (ifn) activation was apparent by the 258 increased expression of ifnb1 and numerous ifn stimulated genes including ifit1, ifit2, ifit3, 259 isg15, mx1, and oas1. stress response programs (fos) and cytokine expression (tnf) were 260 similarly upregulated in these cell types. consistent with a greater innate immune response in 261 two-dimensional tissues, we found that several chemokines (ccl3, ccl4, ccl7, ccl8, and 262 cxcl8) and cytokines (il1b, il6, and csf3) were selectively upregulated in infected two-263 dimensional tissues. macrophages and fibroblasts contributed to enhanced chemokine and 264 cytokine responses in two-dimensional tissues. ccl3, ccl4, and ccl8 were selectively 265 expressed in infected macrophages and csf3, cxcl8, il1b, and il6 were induced in infected 266 fibroblasts (fig. s5c) . inhibitor of the sars-cov-2 rna-dependent rna polymerase 22-24 ( fig. 4a-b) . 277 after binding to ace2, the sars-cov (and sars-cov-2) spike protein must undergo 278 proteolytic activation to initiate membrane fusion 25 . host proteases located at the plasma 279 membrane (tmprss2) or within endosomes (cathepsins) most commonly perform this function. 280 the relative contributions of each of these protease families to sars-cov-2 infection varies by 281 cell-type 15,25 . rna sequencing data revealed that hpsc-derived cms express robust levels of 282 ace2 and multiple endosomal proteases including cathepsins and calpains (fig. 4c) . ace2 283 mrna was not abundantly expressed in either macrophages or fibroblasts. while tmprss2 284 expression was present at the lower limit of detection for rnaseq, we detected low, but 285 measurable levels of tmprss2 by rt-qpcr in hpsc-derived cms, but not in fibroblasts or 286 macrophages ( fig. 4c-d) . 287 to determine whether sars-cov-2 enters cardiomyocytes through an endosomal or 288 plasma membrane route, we inoculated hpsc-derived cms with sars-cov-2-mneongreen and 289 administered either the endosomal cysteine protease inhibitor e-64, which blocks cathepsins, or 290 the serine protease inhibitor camostat mesylate, which blocks tmprss2 (and possibly 291 tmprss4) 25 . notably, e-64 abolished sars-cov-2 infection of hpsc-derived cms as 292 demonstrated by reduced mneongreen expression and viral rna within the supernatant (fig. 293 4e-f). in contrast, camostat had no effect on cardiomyocyte infection over a range of doses ( fig. 294 4g-h). thus, sars-cov-2 enters cardiomyocytes through an endosomal pathway that requires 295 cathepsin but not tmprss2-mediated cleavage. 296 297 myocarditis is characterized by direct viral infection of cardiomyocytes and accumulation 299 of immune cells at sites of active infection or tissue injury 26,27 . to examine whether sars-cov-2 300 infection of cardiomyocytes in a three-dimensional environment mimics aspects of viral 301 myocarditis, we generated ehts containing either hpsc-derived cms and fibroblasts or hpsc-302 derived cms, fibroblasts, and macrophages. ehts were seeded in a collagen-matrigel matrix 303 between two pdms posts, infected with sars-cov-2, and harvested 5 days after inoculation. 304 hematoxylin and eosin (h&e) staining revealed evidence of tissue injury and increased interstitial 305 cell abundance within the periphery of sars-cov-2-infected ehts (fig. 5a) . immunostaining for 306 the viral nucleocapsid protein demonstrated evidence of prominent infection at the periphery of 307 the eht. nucleocapsid staining was localized within hpsc-derived cms. staining for cd68 308 demonstrated macrophage accumulation corresponding to sites of interstitial cell accumulation 309 and viral infection (fig. 5b, fig. s6 ). enrichment of nucleocapsid staining at the periphery of the 310 tissue suggests that viral diffusion might be limited by the three-dimensional eht environment. 311 consistent with our immunostaining results, infected ehts (with and without macrophages) 312 accumulated high levels of viral rna, as detected by quantitative rt-pcr (fig. 5c) . in situ 313 hybridization for viral spike sense and antisense rna was also indicative of viral replication within 314 ehts (fig. 5d, fig. s7) . ehts consisting of hpsc-derived cms and fibroblasts were assembled and allowed to 325 mature for 7 days prior to infection. ehts were inoculated with sars-cov-2, and contractile 326 function was analyzed daily. from days 0 to 3 post infection, the average maximal displacement 327 generated during beating did not differ between the mock and sars-cov-2-infected tissues. 328 however, on days 4 to 5 post infection, the sars-cov-2 inoculated tissues showed reduced 329 contraction relative to the mock-infected tissues ( fig. 5e-f ). on day 5 after inoculation, the 330 maximal displacement produced during contraction by the sars-cov-2 inoculated tissues was 331 markedly lower than mock infected-tissues. moreover, the tissues show reduced speed of 332 contraction and relaxation, consistent with systolic dysfunction (fig. 5g) . 333 334 to examine whether cardiomyocyte cell death might serve as a mechanism explaining 336 reduced eht contractility on days 4 to 5 post inoculation, we performed tunel staining. 337 consistent with the temporal course of sars-cov-2 cardiomyocyte infection and cell death in 338 our two-dimensional hpsc-cm cultures (fig. 2d) , we observed increased numbers of tunel 339 positive cardiomyocytes in sars-cov-2 infected ehts on day 5 post infection ( fig. 6a-b) . our 340 rna sequencing data suggest that other mechanisms also may contribute to reduced eht 341 contractility, including decreased expression of genes important for sarcomere function and 342 metabolism as well as activation of host immune responses (fig. 3i) . consistent with the 343 possibility that disrupted sarcomere gene expression might contribute to reduced eht 344 contractility, immunostaining of hpsc-derived cms infected with sars-cov-2 revealed evidence 345 of sarcomere loss 3 days following infection (fig. 6c) , a time point that preceded cell death. 346 furthermore, immunostaining of ehts demonstrated loss of troponin t expression in infected 347 cardiomyocytes. (fig. 6d-e) . thus, the reduction in contractile function may be multifactorial with 348 contributions from virus-induced cardiomyocyte cell death and loss of sarcomere elements. 349 we then examined the mechanistic relationship between cardiomyocyte infection, 350 inflammatory signaling, sarcomere breakdown, and cell death. inhibition of viral entry (ace2 351 neutralizing antibody) or viral replication (remdesivir) was sufficient to prevent type i ifn and tnf 352 expression following sars-cov-2 infection ( fig. 6f-g) . remdesivir similarly reduced 353 inflammatory gene expression in 3d ehts ( fig. s8a-b) . these data establish that viral infection 354 represents the upstream driver of inflammation in our model system. 355 to examine the impact of cardiomyocyte inflammatory signaling on cardiomyocyte cell 356 death, sarcomere gene expression, and sarcomere structure, we focused on inhibiting viral 357 nucleic acid sensing. tbk1 (tank-binding kinase 1) is an essential mediator of numerous nucleic 358 acid sensing pathways including rig-i, mavs, sting, and tlrs 29,30 . inhibition of tbk1 activity 359 was sufficient to reduce type i ifn activity (primary inflammatory signature identified in infected 360 cardiomyocytes, fig 3i) without impacting viral load or cardiomyocyte infectivity ( fig. 6f-h) . 361 inhibition of tbk1 activity during sars-cov-2 cardiomyocyte infection had no impact on 362 cardiomyocyte cell death (fig. 6i) . while tbk1 inhibition prevented reductions in tnnt2 and 363 myh7 mrna expression following cardiomyocyte sars-cov-2 infection, sarcomere breakdown 364 remained prevalent in infected cardiomyocytes treated with the tbk1 inhibitor. in contrast, 365 remdesivir prevented both reductions in tnnt2 and myh7 mrna expression and sarcomere 366 loss following sars-cov-2 infection ( fig. 6j-k, fig. s8c-d) . these data indicate that sars-cov-367 2 elicits an inflammatory response in cardiomyocytes that is at least partially dependent on viral 368 nucleic acid sensing and tbk1 signaling. however, tbk1-dependent cardiomyocyte 369 inflammation does not appear responsible for sarcomeric disassembly or cardiomyocyte cell 370 death. these findings do not rule out the possibility that other inflammatory pathways or cross-371 talk between infected cardiomyocytes and immune cells contributes to reduced eht contractility. 372 373 to validate the myocarditis phenotype generated by sars-cov-2 infection of the eht 375 model, we obtained autopsy and endomyocardial biopsy specimens from four subjects with 376 confirmed sars-cov-2 infection and clinical diagnoses of myocarditis. evidence of myocardial 377 injury (elevated troponin) and left ventricular systolic dysfunction were present in each case 378 (table 1) were noted accompanied by a mixed mononuclear cell infiltrate (fig. 7a) . these changes are 384 distinct from postmortem autolytic changes. examination of the coronary arteries from the covid-385 19 myocarditis autopsy cases demonstrated non-obstructive mild atherosclerotic changes, 386 consistent with the angiogram findings. there was no evidence of microvascular injury or 387 thromboembolic events. two autopsy heart samples from subjects with metastatic carcinoma and 388 an inherited neurodegenerative disease with similar tissue procurement times were included as 389 negative controls. 390 rna in situ hybridization for sars-cov-2 spike and nucleocapsid genes revealed 391 evidence of viral rna within the myocardium of each covid-19 myocarditis subject. viral 392 transcripts were located in cytoplasmic and perinuclear locations within cells that were 393 morphologically consistent with cardiomyocytes (fig. 7b, fig. s9a-b) . viral transcripts also were 394 identified in airway epithelial cells within the lung of this subject and other myocardial cell types 395 including perivascular adipocytes and pericytes (fig. s9c) . immunostaining for the nucleocapsid 396 protein further demonstrated presence of viral protein in cardiomyocytes fig. 7c) . the covid-19 397 myocarditis immune cell infiltrate was characterized by accumulation of a mixed population of 398 ccr2and ccr2 + macrophages within injured areas of the myocardium (fig. 7d) . minimal 399 evidence of t-cell infiltration was noted (fig. 7e) . macrophage abundance was highest in areas 400 that demonstrated evidence of cardiomyocyte injury as depicted by complement deposition (c4d 401 staining), a pathological marker of cardiomyocyte cell death 31-33 (fig. s9d) . together, these 402 observations provide initial pathological evidence that sars-cov-2 infects the human heart and 403 may contribute to cardiomyocyte cell death and myocardial inflammation that is distinct from 404 lymphocytic myocarditis. (4 mg/ml), 10% fbs, 1% non-essential amino acids, 1% glutamax supplement, and 1% pen-682 strep. all drug compounds were purchased from selleckchem (ruxolitinib, catalog number s8932; 683 mrt67307, catalog number s7948; e64, catalog number s7379; camostat, catalog number 684 s2874) and resuspended to a stock concentration of 10î¼m in pbs or dmso (depending on the 685 solubility profile), then diluted to working concentration in culture media (described above) and 686 sterile-filtered. 687 688 immunostaining was performed as previously described with a few modifications 56 . briefly, 690 cardiomyocytes were fixed for 20 minutes in 4% formaldehyde in phosphate buffered saline 691 (pbs). cells were then permeabilized with 0.4% triton x-100 for 20 minutes at room temperature. 692 the cells were blocked for 1 hour using a blocking solution containing 3% bovine serum albumin, 693 5% donkey serum, 0.1% triton x-100, and 0.02% sodium azide in pbs. primary antibodies (rabbit 694 anti troponin t, 1:400, abcam, ab45932) were added for 1-2 hours at room temperature or 695 overnight at 4 â°c. cells were then washed with pbs before incubating for 1 hour in secondary 696 antibody (cy3 donkey anti-rabbit, jackson immunoresearch, 711165152). 4â�²,6-diamidino-2-697 phenylindole (dapi) was used at a 1:50000 dilution to stain for nuclei. cells were visualized using 698 a nikon a1rsi confocal microscope (washington university center for cellular imaging). z-699 stacks of cells with 40x magnification were recorded in sequential scanning mode. images were 700 processed in imagej and z-stacks were converted to standard deviation projections 61 . 701 to test for changes in expression of the reported log 2 fold-changes reported by limma in each 806 term versus the background log 2 fold-changes of all genes found outside the respective term. 807 the r/bioconductor package heatmap3 71 was used to display heatmaps across groups of 808 samples for each go or msigdb term with a benjamini-hochberg false-discovery rate adjusted 809 p-value less than or equal to 0.05. perturbed kegg pathways where the observed log 2 fold-810 changes of genes within the term were significantly perturbed in a single-direction versus 811 background or in any direction compared to other genes within a given term with p-values less 812 than or equal to 0.05 were rendered as nnotated kegg graphs with the r/bioconductor package 813 to find the most critical genes, the raw counts were variance stabilized with the r/bioconductor 815 package deseq2 54 and then analyzed via weighted gene correlation network analysis with the 816 r/bioconductor package wgcna 73 . briefly, all genes were correlated across each other by 817 pearson correlations and clustered by expression similarity into unsigned modules using a power 818 threshold empirically determined from the data. an eigengene was created for each de novo 819 cluster and its expression profile was then correlated across all coefficients of the model matrix. 820 because these clusters of genes were created by expression profile rather than known functional 821 similarity, the clustered modules were given the names of random colors where grey is the only 822 module that has any pre-existing definition of containing genes that do not cluster well with others. 823 these de novo clustered genes were then tested for functional enrichment of known go terms 824 with hypergeometric tests available in the r/bioconductor package clusterprofiler 74 . significant 825 terms with benjamini-hochberg adjusted p-values less than 0.05 were then collapsed by similarity 826 into clusterprofiler category network plots to display the most significant terms for each module 827 of hub genes in order to interpolate the function of each significant module. the information for 828 all clustered genes for each module were combined with their respective statistical significance 829 results from limma to identify differentially expressed genes. figure 1 : ace2 is expressed in the human heart and in stem cell derived cardiomyocytes. clinical course and risk factors for mortality of adult inpatients with covid-19 china: a retrospective cohort study association of cardiac injury with mortality in hospitalized patients with 880 covid-19 and cardiac arrhythmias cardiac involvement in patients recovered from using magnetic resonance imaging outcomes of cardiovascular magnetic resonance imaging in 887 covid-19) cardiovascular magnetic resonance findings in competitive athletes 890 recovering from covid-19 infection elevated troponin in patients with coronavirus disease animal models of mechanisms of sars-cov-2 infection and covid-19 895 pathology functional assessment of cell entry and receptor usage 897 for sars-cov-2 and other lineage b betacoronaviruses coronavirus from wuhan: an analysis based on decade-long structural studies of sars 900 engineering approaches to modeling the mechanics of human heart failure for drug 903 engineering cardiac muscle tissue: a 905 maturating field of research cardiomyocyte maturation: advances in knowledge and implications for 907 regenerative medicine sars-cov-2 cell entry depends on ace2 and tmprss2 and is 909 the pathogenicity of sars-cov-2 in hace2 transgenic mice the ace2 expression in human heart 913 indicates new potential mechanism of heart injury among patients infected with sars-cov-914 2 an infectious cdna clone of sars-cov-2 ultrastructural characterization of sars coronavirus electron 920 microscopy of sars-cov-2: a challenging task the architecture of sars-cov-2 transcriptome sars-cov-2 infects and induces cytotoxic effects in human 950 cardiomyocytes myocardial localization of coronavirus in covid-19 cardiogenic shock multiorgan and renal tropism of sars-cov-2 association of cardiac infection with sars-cov-2 in confirmed autopsy cases sars-cov-2 productively infects human gut enterocytes inhibition of sars-cov-2 infections in engineered human tissues using 960 a human pluripotent stem cell-based platform to study sars-cov-2 tropism and model virus infection in human cells and organoids marked up-regulation of ace2 in hearts of patients with obstructive 965 hypertrophic cardiomyopathy: implications for sars-cov-2-mediated covid-19 angiotensin-converting enzyme 2 is an essential regulator of heart 968 function heart disease. titin mutations in ips cells define sarcomere 970 insufficiency as a cause of dilated cardiomyopathy comparison of the effects of a truncating and a missense mybpc3 972 mutation on contractile parameters of engineered heart tissue generation of quiescent cardiac fibroblasts from human induced 975 pluripotent stem cells for in vitro modeling of cardiac fibrosis efficient differentiation of human pluripotent stem cells to endothelial 978 progenitors via small-molecule activation of wnt signaling phosphomimetic cardiac myosin-binding protein c partially rescues a 981 cardiomyopathy phenotype in murine engineered heart tissue increased afterload augments sunitinib-induced cardiotoxicity in an 983 engineered cardiac microtissue model tmprss2 and tmprss4 promote sars-cov-2 infection of human small 985 intestinal enterocytes infection of bat and human intestinal organoids by sars-cov-2 covid-19 artic v3 illumina library construction and sequencing 989 protocol v4 (protocols.io.bgxjjxkn) human monoclonal antibody combination against sars coronavirus: 991 synergy and coverage of escape mutants growth, detection, 993 quantification, and inactivation of sars-cov-2 moderated estimation of fold change and dispersion for 995 rna-seq data with deseq2 epigenome-wide association study identifies cardiac gene patterning and 997 a novel class of biomarkers for heart failure disrupted mechanobiology links the molecular and cellular 999 phenotypes in familial dilated cardiomyopathy robust cardiomyocyte differentiation from human pluripotent stem cells via 1002 temporal modulation of canonical wnt signaling directed cardiomyocyte differentiation from human pluripotent stem cells by 1005 modulating wnt/î²-catenin signaling under fully defined conditions derivation of highly purified cardiomyocytes from human induced 1008 pluripotent stem cells using small molecule-modulated differentiation and subsequent 1009 glucose starvation directed differentiation of primitive and definitive 1011 hematopoietic progenitors from human pluripotent stem cells fiji: an open-source platform for biological-image analysis differentiation of cardiomyocytes and generation of human engineered 1016 heart tissue star: ultrafast universal rna-seq aligner featurecounts: an efficient general purpose program for 1020 assigning sequence reads to genomic features salmon provides fast and 1022 bias-aware quantification of transcript expression rseqc: quality control of rna-seq experiments edger: a bioconductor package for 1026 differential expression analysis of digital gene expression data limma powers differential expression analyses for rna-sequencing and 1029 microarray studies why weight? modelling sample and observational level variability improves 1031 power in rna-seq analyses gage: generally 1033 applicable gene set enrichment for pathway analysis advanced heat map and clustering analysis using 1035 heatmap3 pathview: an r/bioconductor package for pathway-based data 1037 integration and visualization wgcna: an r package for weighted correlation network 1039 analysis clusterprofiler: an r package for comparing 1041 biological themes among gene clusters rnascope 2.5 hd detection reagent -red the authors would like to acknowledge funding support from the national institutes of health 841 anti-human cd31 bv421 biolegend rrid: ab_2563810 a, immunohistochemistry of human heart tissue showing ace2 (red) expression in cardiomyocytes (green, sarcomeric actin). representative images from 5 analyzed specimens. b, rna sequencing demonstrating ace2 mrna expression in myocardial biopsies obtained from adult controls and heart failure patients. data is displayed as counts per million (cpm). n=18 pediatric, n=33 adult. each data point indicates an individual sample. n=60 controls, n=35 heart failure. c, rna sequencing demonstrating ace2 mrna expression in adult and pediatric heart tissue. data is displayed as counts per million (cpm insets are high magnification images of the boxed areas. representative images from 4 independent samples. b, immunostaining of mock or sars-cov-2 infected three-dimensional ehts for sarcomeric actin (cardiomyocytes, red), cd68 (macrophages, green), and nucleocapsid protein (white). ehts were harvested 5 days after inoculation. blue: dapi. images are representative of 4 independent experiments. representative images from 4 independent samples. c, quantitative rt-pcr of sars-cov-2 n gene expression in ehts consisting of hpsc-derived cardiomyocytes (cm) and fibroblasts (fb) or hpsc-derived cardiomyocytes, fibroblasts, and macrophages. ehts were either mock infected or inoculated with sars-cov-2 (moi 0.1) and harvested 5 days after inoculation. each data point represents individual samples/experiments. error bars denote standard error of the mean. bar height represents sample mean. dotted line: limit of detection. *p<0.05 compared to uninfected control (mock, mann-whitney test). d, in situ hybridization for sars-cov-2 orf1ab rna sense and anti-sense strands (red) in ehts 5 days after mock or sars-cov-2 infection (moi 0.1). hematoxylin: blue. representative images from 4 independent specimens. insets are high magnification images of the boxed areas. e, representative spontaneous beating displacement traces for an infected and an uninfected eht on day 5 post-infection. videos used to generate these traces can be found in supplemental videos 1 and 2. f, displacement (relative to uninfected mock condition) generated by spontaneous beating of ehts as a function of time following inoculation with sars-cov-2 (moi 0.1). each data point represents a mean value from 4-7 independent samples (2 independent experiments), error bars denote standard error of the mean. g, quantification of absolute displacement (left) and contraction speed (right) generated by spontaneous beating of ehts 5 days following mock or sars-cov-2 infection (moi 0.1). each data point denotes an individual eht, bar height corresponds to mean displacement, error bars represent standard error of the mean, *p<0.05 compared to mock (mann-whitney test). figure 6 . mechanisms of reduced eht contractility. a, combined immunostaining for cardiomyocytes (cardiac actin, green) and tunel staining (red) of ehts (cm+fb+mac) 5 days after mock or sars-cov-2 infection (moi 0.1). dapi: blue. representative images from 4 independent experiments. b, quantification of cell death (percent of tunel-positive cells) in areas of viral infection. each data point denotes an individual eht, bar height corresponds to the mean, error bars represent standard error of the mean, *p<0.05 compared to mock (mann-whitney test). c, immunostaining of hpsc-derived cardiomyocytes for troponin t (red) 3 days after inoculation with mock control or sars-cov-2-neongreen (moi 0.1). blue: dapi. arrows denote areas of sarcomere disassembly. d, immunostaining of ehts for troponin t (red) and sars-cov-2 nucleocapsid (green) 5 days after inoculation with mock control or sars-cov-2-neongreen (moi 0.1). blue: dapi. arrows denote sars-cov-2 nucleocapsid positive cells with reduced troponin t staining. e, quantification of troponin t staining in mock (white) and sars-cov-2 (red) infected ehts. np: nucleocapsid. data is presented as mean florescence intensity (mfi). mfi was measured in infected (np+) cardiomyocytes and uninfected (np-) cardiomyocytes located proximal or remote to areas of infection. each data point denotes an individual eht, bar height corresponds to the mean, error bars represent standard error of the mean, *p<0.05 compared to mock (mann-whitney test). f, quantitative rt-pcr measuring oas1, mx1, and tnf mrna expression in hpsc-derived cardiomyocytes 3 days after inoculation with mock control (white) or sars-cov-2 (green, moi 0.1). cells were treated with vehicle, ace2 antibody (ace2 ab) (20âµg/ml), remdesivir (10âµm), or tbk inhibitor (mrt67307, 10âµm). each data point denotes a biologically unique sample, bar height corresponds to the mean, and error bars indicate standard error of the mean. * p<0.05 compared to mock control. g, quantitative rt-pcr of sars-cov-2 n gene expression in hpsc-derived cardiomyocytes that were either mock infected (white) or inoculated with sars-cov-2 (green, moi 0.1) and harvested 3 days after inoculation. cells were treated with vehicle, ace2 ab (20âµg/ml), remdesivir (10âµm), or tbk1 inhibitor (mrt67307, 10âµm). each data point represents individual samples. error bars denote standard error of the mean. bar height represents sample mean. dotted line: limit of detection. *p<0.05 compared to uninfected control. ***p<0.05 compared to uninfected control and vehicle infected (mock, mann-whitney test). h-i, flow cytometry measuring the percent of infected (h) and viable (i) hpscderived cardiomyocytes following either mock infection (white) or inoculation with sars-cov-2 (green, moi 0.1). cells were harvested and analyzed 3 days after inoculation. cells were treated with vehicle, remdesivir (10âµm), or tbk1 inhibitor (mrt67307, 10âµm). each data point represents individual samples. error bars denote standard error of the mean. bar height represents sample mean. *p<0.05 compared to uninfected control. **p<0.05 compared to vehicle infected (mock, mann-whitney test). j, quantitative rt-pcr measuring tnnt2 mrna expression in hpsc-derived cardiomyocytes 3 days after inoculation with mock control (white) or sars-cov-2 (green, moi 0.1). cells were treated with vehicle, ace2 ab (20âµg/ml), remdesivir (10âµm), or tbk inhibitor (mrt67307, 10âµm). each data point denotes a biologically unique sample, bar height corresponds to the mean, and error bars indicate standard error of the mean. * p<0.05 compared to mock control. k, immunostaining of hpsc-derived cardiomyocytes for troponin t (red) 3 days after inoculation with mock control or sars-cov-2-neongreen (moi 0.1). hpsc-derived cardiomycoytes were treated with vehicle, remdesivir (10âµm) or tbk inhibitor (mrt67307, 10âµm). blue: dapi. arrows denote areas of sarcomere disassembly. merged images can be found in fig. s8. figure 7 . human autopsy and endomyocardial tissue from patients with suspected covid-19 myocarditis show evidence of sars-cov-2 cardiomyocyte infection. a, hematoxylin and eosin staining of cardiac autopsy (anterior left ventricular wall) and biopsy samples (right ventricular septum) from subjects without covid-19 (control case) and patients with a clinical diagnosis of covid-19 myocarditis (case 1-4) . b, in situ hybridization of cardiac autopsy and biopsy tissue for sars-cov-2 spike and nucleocapsid rna (red) showing evidence of viral infection. hematoxylin: blue. arrows denotes viral rna staining in cells with cardiomyocyte morphology. c, immunostaining of control and covid-19 myocarditis cardiac autopsy tissue for sars-cov-2 nucleocapsid (white) and cardiac actin (red). dapi: blue. arrows denotes nucleocapsid staining in cardiomyocytes .d, immunostaining of control and covid-19 myocarditis cardiac autopsy and biopsy tissue for cd68 (green) and ccr2 (red). dapi: blue. e, immunostaining of control and covid-19 myocarditis cardiac autopsy and biopsy tissue for cd3 (brown). hematoxylin: blue. key: cord-275690-83nrzfon authors: stanifer, megan l.; kee, carmon; cortese, mirko; triana, sergio; mukenhirn, markus; kraeusslich, hans-georg; alexandrov, theodore; bartenschlager, ralf; boulant, steeve title: critical role of type iii interferon in controlling sars-cov-2 infection, replication and spread in primary human intestinal epithelial cells date: 2020-04-24 journal: biorxiv doi: 10.1101/2020.04.24.059667 sha: doc_id: 275690 cord_uid: 83nrzfon sars-cov-2 is an unprecedented worldwide health problem that requires concerted and global approaches to better understand the virus in order to develop novel therapeutic approaches to stop the covid-19 pandemic and to better prepare against potential future emergence of novel pandemic viruses. although sars-cov-2 primarily targets cells of the lung epithelium causing respiratory infection and pathologies, there is growing evidence that the intestinal epithelium is also infected. however, the importance of the enteric phase of sars-cov-2 for virus-induced pathologies, spreading and prognosis remains unknown. here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of sars-cov-2 lifecycle in human intestinal epithelial cells. our results demonstrate that human intestinal epithelial cells fully support sars-cov-2 infection, replication and production of infectious de-novo virus particles. importantly, we identified intestinal epithelial cells as the best culture model to propagate sars-cov-2. we found that viral infection elicited an extremely robust intrinsic immune response where, interestingly, type iii interferon mediated response was significantly more efficient at controlling sars-cov-2 replication and spread compared to type i interferon. taken together, our data demonstrate that human intestinal epithelial cells are a productive site of sars-cov-2 replication and suggest that the enteric phase of sars-cov-2 may participate in the pathologies observed in covid-19 patients by contributing in increasing patient viremia and by fueling an exacerbated cytokine response. coronaviridae is a large family of single-stranded positive-sense enveloped rna viruses that can infect most animal species (human as well as domestic and wild animals). they are known to have the largest viral rna genome and are composed of four genera (cui et al., 2019) . generally, infection by coronaviruses results in mild respiratory tract symptoms and they are known to be one of the leading causes of the common cold (moriyama et al., 2020; paules et al., 2020) . however, in the last 18 years, we have witnessed the emergence of highly pathogenic human coronaviruses: the severe acute respiratory syndrome-related coronavirus (sars-cov-1), the middle east respiratory syndrome-related coronavirus (mers-cov) and, at the end of 2019, the severe acute respiratory syndrome-related coronavirus-2 (sars-cov-2) . sars-cov-2 is responsible for the coronavirusassociated acute respiratory disease or coronavirus disease 19 (covid-19) and represents a major global health threat and coordinated efforts are urgently needed to treat the viral infection and stop the pandemic. although sars-cov-2 primarily targets cells of the lung epithelium causing respiratory infection, there is growing evidence that the intestinal epithelium can also be infected. multiple studies have reported gastro-intestinal symptoms such as diarrhea at the onset of the disease and have detected the prolonged shedding of large amounts of coronavirus genomes in the feces even after the virus was not detectable in oropharyngeal swabs (wu et al., 2020b; xiao et al., 2020; xing et al., 2020; xu et al., 2020b) (wölfel et al., 2020) . although one study revealed the isolation of infectious virus particles from stool samples , to date, it remains unclear how many people shed infectious viruses in feces. most critically, it remains unknown whether or not there is a possibility for fecal transmission of sars-cov-2 but multiple health agencies worldwide have highlighted this possibility. the presence of such a large amount of coronavirus genomes in feces is hardly explainable by a swallowing virus replicating in the throat or by a loss of barrier function of the intestinal epithelium which will allow the release of viruses or genomes from the inside of the body (circulation or lamina propria) to the lumen of the gut. instead, it is likely due to an active replication in the intestinal epithelium. recently, intestinal biopsies of sars-cov-2 infected patients clearly show the presence of replicating viruses in epithelial cells of the small and large intestine (xiao et al., 2020) . sars-cov-2 infection of the gastrointestinal tract is supported by the fact that ace2, the virus receptor (hoffmann et al., 2020) , is expressed in intestinal epithelial cells (zhao et al., 2020) (lukassen et al., 2020) (wu et al., 2020a) (venkatakrishnan et al., 2020) and single cell sequencing analysis suggest that its expression is even higher on intestinal cells compared to lung cells (xu et al., 2020a) .this highlights that sars-cov-2 is not restricted to the lung but also infects the gastrointestinal tract. importantly, many animal coronaviruses are well known to be enteric and are transmitted via the fecal-oral route (wang and zhang, 2016) . additionally, the presence of human pathogenic coronaviruses in the gastrointestinal tract was previously reported for sars-cov-1 and mers-cov but remained seriously understudied (leung et al., 2003; wong et al.; zhou et al., 2017) . although it is now clear that human coronaviruses, particularly sars-cov-2, are found in feces and can infect the gastrointestinal tract, the importance of its enteric phase for viremia, pathogenesis and patient prognosis remains unknown. to combat the current pandemic of covid-19 and to prepare for potential future emerging zoonotic coronaviruses, we need to gain a better understanding of the molecular basis of sars-cov-2 infection, replication and spread in a tissue-specific manner. here, we engaged in studying sars-cov-2 infection of human intestinal cells. for this, we exploited both human intestinal epithelial cell lines and human organoid culture models to characterize how these cells support sars-cov-2 replication and spread and how they respond to viral infection. direct comparison of both primary and transformed cells show that human intestinal epithelial cells fully support sars-cov-2 infection and de-novo production of infectious virus particles. interestingly, viral infection elicited a robust intrinsic immune response where type iii interferon mediated response was shown to be significantly more efficient at controlling sars-cov-2 replication and spread as compared to type i interferon. importantly, human primary intestinal epithelial cells responded to sars-cov-2 infection by producing only type iii interferon. taken together, our data clearly highlight the importance of the enteric phase of sars-cov-2 and this should be taken into consideration when developing hygienic/containment measures, and antiviral strategies, and when determining patient prognosis. as there is growing evidence that the gastro-intestinal tract is infected by sars-cov-2, we engaged in studying virus infection, replication and spread in human intestinal epithelial cells (iecs). first, sars-cov-2 (strain bavpat1) was propagated in the green monkey cell line vero (see methods section). to detect viral infection, we used an antibody directed against a region of the nucleoprotein (np) which is conserved between of sars-cov-1 and sars-cov-2. additionally, we used the j2 antibody which detects double-stranded rna (dsrna) which is a hallmark of rna virus replication (targett-adams et al., 2008) . cells positive for np were found to be always positive for dsrna; the np signal was found to be dispersed within the cytosolic area whereas dsrna were found in discrete foci likely corresponding to replication compartments (harak and lohmann, 2015) (fig. s1a ). supernatants of infected vero cells were collected at 48 hours post-infection (hpi) and the amount of infectious virus particles present was measured using a tcid50 approach on vero cells (fig. s1b ). the colon carcinoma derived lines t84 and caco-2 cells were then infected with sars-cov-2 at a moi of 0.5 (as determined in vero cells) and, at different time post-infection, cells were fixed and immunostained using the anti-np and anti-dsrna antibodies (fig. 1a) . results show that sars-cov-2 infected caco-2 cells were readily detected as early as 4hpi and, by 24 hpi, most of the cells were infected (fig. 1b) . similar results were observed in the t84 cells, but detection of infection was slightly delayed compared to caco-2 cells and, although the same amount of sars-cov-2 was used to infect these cells, only around 20% of the cells were found infected at 24 hpi (fig. 1b) . these observations were in agreement with the increase in viral genome copy numbers over time (fig. 1c ) and the release of infectious virus particles in the supernatant of infected t84 and caco-2 cells (fig. 1d) . interestingly, infection of iecs by sars-cov-2 was associated with the generation of an interferon (ifn)-mediated intrinsic immune response. concomitant with the differences observed in virus replication and denovo virus production observed between t84 and caco-2 cells, t84 cells mounted a much stronger immune response compared to caco-2 cells (fig. 1e ) although much less t84 cells were infected (fig. 1b) . all together these results show that iecs are readily infected by sars-cov-2 and that infection of caco-2 cells lead to a weaker intrinsic immune response which is associated with more de-novo infectious virus production compared to t84 cells. this observation suggests that the ifn-mediated immune response controls sars-cov-2 infection in iecs. to directly test the function of ifns in controlling/restricting sars-cov-2 replication and spread in human iecs, t84 and caco-2 cells were mock-treated or pre-treated with type i (ifnb1) or type iii (ifnl) ifns. 24 hrs post-treatment, cells were infected with sars-cov-2 at a moi of 0.5 (as determined in vero cells), in the presence or absence of ifns ( fig. 2a) . 24 hpi, cells were immunostained using both the anti-np and anti-dsrna antibodies. results to address whether the endogenous levels of ifns generated by iecs controls sars-cov-2 replication and spread, we exploited our previously reported iecs depleted of either the type i ifn receptor (ifnar1) (ar-/-), the type iii ifn receptor (ifnlr1) (lr -/-) or depleted of both ifn receptors (dko). to control that our cells were functionally knocked-out for the type i ifn (ar-/-) and/or the type iii ifn receptor (lr-/-), t84 cells were treated with either ifn and the production of the ifn stimulated gene ifit1 was evaluated. as expected, type i ifn receptor knock-out cells (ar-/-) only responded to ifnl, whereas type iii ifn receptor knock-out cells (lr-/-) only responded to ifnb1 (fig. s2 ). the ifn receptor double knock-out cells (dko) did not respond to either ifn (fig. s2 ). wt or ifn receptor knock-out t84 cells were infected with sars-cov-2 at a moi of 0.1 (as determined in t84 cells). 24 hpi, cells were immunostained using the anti-np antibody and the number of infected cells was quantified using fluorescent microscopy (fig. 3a) . results showed that depletion of the type i ifn receptor (ar-/-) resulted in a slight increase in the number of infected cells. importantly, depletion of the type iii ifn receptor (lr-/-) resulted in a massive increase of cell infectivity by a factor of around seven. similar results were obtained when both the type i and the type iii ifn receptors were depleted (dko) (fig. 3b) . interestingly, this increase in the number of infected cells upon depletion of the type iii ifn receptor (lr-/-) was associated with a significant increase in viral genome copy numbers (fig. 3c ) and with a three orders of magnitude increase in de-novo infectious virus production (fig. 3d ). together these results suggest that the type iii ifn-mediated immune response actively participates in controlling sars-cov-2 infection in human iecs. to unambiguously address the importance of the ifn-mediated antiviral response, we used the pan-jak inhibitor (pyridone-6) to inhibit the stat1 phosphorylation activation and block the production of interferon stimulated genes (isgs). as we previously reported, treatment of t84 cells with the pan-jak inhibitor fully inhibits signal transduction downstream both the type i and type iii ifn receptors ( (pervolaraki et al., 2017) and data not shown). mock and pyridone-6 pre-treated t84 cells were infected with sars-cov-2 for 24hrs and analyzed using fluorescence microscopy following immunostaining using the anti-np antibody. results show both a significant increase in the number of infected cells (fig. 3e ) and an increase of viral genome copy number in cells treated with the pan-jak inhibitor (fig.3f ). importantly, and in agreement with the results observed in cells depleted of the type iii ifn receptor, this increase in infectivity was also associated with an increase in infectious denovo virus particle production ( fig. 3g ). all together, these results strongly support a model where the type iii ifn mediated signaling controls sars-cov-2 infection in human intestinal epithelial cells. to address whether primary human iecs can be infected by sars-cov-2 and support denovo virus production, we used human colon derived organoids from two distinct donors. intact ultrastructural organization and differentiation to all cell types (e.g. enterocytes, goblet cells, enteroendocrine cells, and stem cells) was confirmed using confocal fluorescent microscopy ( fig. 4a ) and quantitative-rt-pcr against cell type specific transcripts (fig. 4b ). as quantification of the number of infected cells in 3d organoids is very challenging, we exploited previously established protocols to differentiate and infect human intestinal organoids in two dimensions with viruses (ettayebi et al., 2016; stanifer et al., 2020) . non-differentiated organoids were seeded on human collagen-coated ibidi chambers. at 24 hrs post-seeding, differentiation was induced by removal of wnt3a and reducing the amounts of r-spondin and noggin for four days. upon full differentiation, organoids were infected with sars-cov-2. at 24 hpi, the infection was analyzed by immunostaining using the anti-np and anti-dsrna antibodies and by quantitative rt-pcr ( fig. 4c ). results show that independent of the donor, colon organoids were readily infected by sars-cov-2 as noted by the presence of cells positive for both np and dsrna (fig. 4d ). quantification revealed that around 8-13% of cells were infected in each donor (fig. 4e ). this infection was associated with an increase of viral genome copy number (fig. 4f ). interestingly, infection of organoids led to no type i interferon (ifnb1) production but an extremely large up regulation of type iii ifn (ifnl) ( fig. 4g and fig. s3 ). to determine if exogenously added ifns could prevent sars-cov-2 infection, colon organoids were mock or pre-treated with ifnb1 and ifnl and then subsequently infected with sars-cov-2 for 24 hrs. results show that pre-treatment of colon organoids with both ifnb1 and ifnl significantly impaired infection (fig. 4h ). this was associated with a reduction of sars-cov-2 genome copy numbers (fig. 4i ) and a decrease in infectious de-novo virus particle production (fig. 4j ). all together these results show that human colon organoids can support sars-cov-2 infection, replication and spread and that the type iii ifn response plays a critical role in controlling virus replication. human primary intestinal epithelial cells support robust replication of sars-cov-2 and secretion of infectious de-novo virus particles. we found that around 10% of the cells are infected in a human colon derived organoid leading to a modest but significant increase in viral genome copy number (fig. 4f ) and de-novo infection virus particles (fig. 4j ) over time. this modest replication of sars-cov-2 is explained by the fact that (i) only a small fraction of the cells is infected by sars-cov-2, and (ii) as shown in this work, ifns are potent inhibitors of sars-cov-2 replication ( fig. 2 and 3 ) and organoids are highly immunoresponsive upon viral infection (stanifer et al., 2020) (fig. 4g) . it is well known that in vivo intestinal epithelium cells are less immunoresponsive because of the gut microenvironment (e.g. microbiota and tissue specific immune cells) and as such they will very likely show a severely dampened immune response allowing for an even greater sars-cov-2 replication. importantly, the intestinal epithelium is the largest organ in the body and even if only a few percent of the cells are infected it will result in the generation of an extremely large amount of de-novo viruses. analysis of the single-cell rna sequencing data from the colon atlas revealed that only 3.8% of human colon epithelial cells express very low levels of the sars-cov-2 receptor ace-2 ( fig. s4a-e) . this is very different to the small intestine where ace-2 appears to be more expressed (qi et al., 2020) . this low ace-2 level could explain why we have a small percentage of infected cells in our colon organoids (fig. 4e) . on the contrary, tmprss2 seems to be not a limiting factor in the colon (fig. s4c-e) . interestingly, q-rt-pcr and western blot analysis do not support single cell analysis and clearly show that ace-2 is expressed in both our carcinoma derived lines and our colon organoids (fig. s4f-g) . the discrepancy between the single-cell rna sequencing and classical molecular and biochemical approaches is likely the results of (i) the sequencing being not deep enough to detect ace-2 in individual cells and (ii) rna expression not necessarily matching the protein expression levels. these observations highlight that although analyzing data from single-cell rna sequencing atlases could be very informative, their findings should be validated in tissues as they may be mis-leading or miss important sites of virus replication. the human colon carcinoma caco-2 cells produce very large amounts of infectious viral particles (between 10 8 and 10 9 infectious particles per ml). this is higher than the titers obtained in vero cells which are commonly used to isolate and propagate sars-cov-2 (harcourt et al., 2020) and where we routinely obtained titers of 10 5 and 10 6 infectious particles per ml in vero cells (fig. s1b) . interestingly, in a study comparing 13 different human cell lines, caco-2 cells were the only cell type found to support sars-cov-1 replication and, compared to green monkey cells, caco-2 cells were very efficient in producing infectious de-novo virus particles and did not show cytopathic effects (mossel et al., 2005) . these observations that caco-2 are excellent culture models supporting both sars-cov-2 and sars-cov-1 further highlight the potentially central role of intestinal epithelial cells in covid-19 patients. intriguingly, t84 cells, which are also colon-carcinoma derived cells supported sars-cov-2 infection, replication and spread but to a much lesser extent compared to caco-2 cells. analysis of the intrinsic immune response generated upon infection of both cell lines revealed that t84 cells are more immunoresponsive compared to caco-2 cells. in light of the results obtained by pretreating cells with exogenous ifns, we propose that t84 cells, by being able to mount a stronger and faster immune response compared to caco-2 cells can better restrict sars-cov-2 infection. this model is fully supported by our ifn receptor knock-out t84 cells, in which virus replication and de-novo virus production are drastically increased to levels similar to the ones observed in caco-2 cells. exogenously added ifns (both type i and type iii ifns) induce an antiviral state in our human intestinal epithelial cells, thereby restricting sars-cov-2 replication in these cells. similar observations were made with type i ifn in vero cells (mantlo et al., 2020) . interestingly, infection of the calu-3 human lung epithelial cells by sars-cov-2 seem to also mount an immune response (lokugamage et al., 2020) importantly, our work shows that, compared to the airway epithelium, the intestinal epithelium produces a typical antiviral response. this highlights that host/pathogen interaction should be considered in a tissue specific manner as different cellular responses and viral countermeasures might be established between the lung, the gut and other organs. interestingly, in human colon organoids we observed that only type iii ifn is made upon sars-cov-2 infection, although human intestinal organoids are capable of making both type i and iii ifn upon enteric virus infection (pervolaraki et al., 2018; stanifer et al., 2020) . the lack of type i induction appears to be specific to sars-cov-2 and it is likely that this virus encodes a specific antagonist which counteracts the production of type i ifn only. however, further studies are necessary to prove this novel concept. we propose that the gut is an active site of replication for sars-cov-2 and this could account for the viremia observed in covid-19 patients and for the presence of large amounts of sars-cov-2 genomes in the feces. the origin of the replicating sars-cov-2 in the intestinal epithelium is still not clear. to date only one paper reported the isolation of infectious virus from stool samples . further characterization of the sars-cov-2 enteric lifecycle is necessary to determine whether the viral infection observed in the gut is due to fecal/oral transmission or is a manifestation of virus spreading from the lung to the gut. in the context of the gut we foresee that at the onset of sars-cov-2 infection, human intestinal epithelial cells will mount an antiviral response through the type iii ifn signaling pathway. as immune cells participate in mounting an innate immune response, type i ifn will be secreted from these cells and will be able to act on intestinal epithelial cells further reinforcing their antiviral state against sars-cov-2. in respect to the severe pathologies observed in the lung, which are believed to be caused by a cytokine storm, the findings that lung epithelial cells mount a muted immune response upon sars-cov-2 infection suggests that the cytokines are coming from an alternative source. this source could be from local immune cells but also from the gastro-intestinal mucosa given the large immune response generated by primary human intestinal epithelial cells. as a matter of fact, many cytokines are released from epithelial cells towards the lamina propria (tissue side) (stanifer et al., 2016) , and will quickly enter the circulation to potentially fuel and promote the inflammation and pathology observed in the lung. in conclusion, the gastro-intestinal tract is an active site of sars-cov-2 replication and this should be considered when developing antiviral strategies as it may participate in the viremia and potentially reinfection. from a patient prognosis point of view, the severity of the disease should be correlated with the extent of the enteric replication. ifnl 2 (il28a) (#300-2k) and ifnl 3 (il-28b) (#300-2k) were purchased from peprotech and were used at a concentration of 100ng/ml each to make a final concentration of 300ng/ml, pyridone 6 (calbiochem #420099-500)was used at a final concentration of 2um. all sars-cov-2 infections were performed the moi indicated in the text. media was removed from cells and virus was added to cells for 1 hour at 37°c. virus was removed, cells were wash 1x with pbs and media or media containing inhibitors/cytokines was added back to the cells. 2d organoid seeding. 8-well ibidi glass bottom chambers were coated with 2.5% human collagen in water for 1 h prior to organoids seeding. organoids were collected at a ratio of 100 organoids/transwell. collected organoids were spun at 450g for 5 mins and the supernatant was removed. organoids were washed 1x with cold pbs and spun at 450g for 5 mins. pbs was removed and organoids were digested with 0.5% trypsin-edta (life technologies) for 5 mins at 37°c. digestion was stopped by addition of serum containing medium. organoids were spun at 450g for 5 mins and the supernatant was removed and organoids were re-suspended in normal growth media at a ratio of 250 µl media/well. the collagen mixture was removed from the ibidi chambers and 250 µl of organoids were added to each well. rna isolation, cdna, and qpcr. rna was harvested from cells using rnaeasy rna extraction kit (qiagen) as per manufactures instructions. cdna was made using iscript reverse transcriptase (biorad) from 250 ng of total rna as per manufactures instructions. q-rt-pcr was performed using itaq sybr green (biorad) as per manufacturer's instructions, tbp or hprt1 were used as normalizing genes. primer used: performed, and cells having fewer than 500 genes and more than 30% of umi count mapped to mitochondrial genes were discarded. consecutively, the resulting datasets were normalized, scaled and high-variance genes genes were selected. reciprocal pca-based data integration was done to merge the samples. afterwards, the resulting batch-corrected counts were used for calculating pca-based dimensionality reduction and unsupervised louvain clustering. furthermore, umap visualization was calculated using 50 neighboring points for the local approximation of the manifold structure. cell type annotation was based on the unsupervised clustering and the metadata provided by the colon atlas (smillie et al., 2019) . statistics. unless otherwise stated, statistical analysis was performed by a two-tailed unpaired t test using the graphpad prism software package. all samples were analyzed without blinding or exclusion of samples. zhou, j., li, c., zhao, g., chu, h., wang, d., yan, h.h.-n., poon, v.k.-m., wen, l., wong, b.h.-y., zhao, x., et al. (2017) . human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus. sci. adv. 3, eaao4966. sars-cov-2 launches a unique transcriptional signature from in vitro, ex vivo comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 type i and type iii interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia origin and evolution of pathogenic coronaviruses replication of human noroviruses in stem cell-derived human enteroids ultrastructure of the replication sites of positive-strand rna viruses isolation and characterization of sars-cov-2 from the first us covid-19 patient (microbiology) sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor enteric involvement of severe acute respiratory syndrome-associated 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coronaviruses intra-and inter-cellular rewiring of the human colon during ulcerative colitis reovirus intermediate subviral particles constitute a strategy to infect intestinal epithelial cells by exploiting tgf-β dependent pro-survival signaling asymmetric distribution of tlr3 leads to a polarized immune response in human intestinal epithelial cells visualization of double-stranded rna in cells supporting hepatitis c virus rna replication knowledge synthesis from 100 million biomedical documents augments the deep expression profiling of coronavirus receptors animal coronaviruses: a brief introduction emerging and re-emerging coronaviruses in pigs detection of sars-cov-2 in different types of clinical specimens virological assessment of hospitalized patients with covid-2019 covid-19 and the digestive system single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov prolonged presence of sars-cov-2 viral rna in faecal samples evidence for gastrointestinal infection of sars-cov-2 prolonged viral shedding in feces of pediatric patients with coronavirus disease high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding interferon-λ enhances adaptive mucosal immunity by boosting release of thymic stromal lymphopoietin single-cell rna expression profiling of ace2, the receptor of sars-cov-2 (bioinformatics) we would like to acknowledge vibor laketa and the infectious diseases imaging platform (idip) at the center for integrative infectious disease research, heidelberg, germany, for support with image acquisition and analysis. we also thank christian drosten at the charité, berlin and the european virus archive (evag) for the provision of the sars-cov-2 strain bavpat1. the authors declare no competing interests. key: cord-269275-b7xxk48t authors: tang, xiaojia; luo, yuhan; song, yuxia; fan, hongyang; dong, sisi; liu, peipei; chen, yingzhu title: neurological manifestations in covid-19 and its possible mechanism date: 2020-09-27 journal: aging (albany ny) doi: 10.18632/aging.103732 sha: doc_id: 269275 cord_uid: b7xxk48t in december 2019, the first cases of the acute respiratory illness now known as corona virus disease 2019 (covid-19) occurred in wuhan, hubei province, china. the main clinical manifestations of covid-19 are a fever, dry cough and general weakness, although in some patients, a headache, tight chest, diarrhea, etc. are the first clinical manifestations. neurological practice is involved in all aspects of medicine, from primary care for patients with migraines to consultations with patients in the intensive care unit. few disorders spare the nervous system, and newly emerging infections are no exception. as neurologists, we are concerned about the effects of sars-cov-2 infections on the nervous system. multiple neuropathy, rhabdomyolysis, cerebrovascular disease, central nervous system infections and other common neurological diseases require attention during this outbreak. aging viruses are prevalent -229e, oc43, nl63 and hku1and typically cause common cold symptoms in immunocompetent individuals [14] . the two other strains -sars-cov [15] and middle east respiratory syndrome coronavirus (mers-cov) [16] are zoonotic in origin, and have been linked to sometimes fatal illness. sars-cov-2 is the seventh member of the coronavirus family. zhou et al. found that sars-cov-2 was 96% identical at the whole-genome level to a bat coronavirus, so sars-cov-2 may have originated in bats [17] . an epidemic or outbreak can occur when the agent (pathogen), population (hosts) and environment create an ideal situation for spread [18] . the current evidence suggests that sars-cov-2 may have spread to humans via wild animals sold illegally in the huanan seafood wholesale market [19] . the extent of humanto-human transmission of sars-cov-2 was unclear at first, but now there is evidence of human-to-human transmission [5, 20, 21] . the main sources of infection are sars-cov-2-infected patients, including those who are asymptomatic [22] . the routes of transmission include droplet transmission, contact transmission and aerosol transmission [23, 24] . in a recent study, sars-cov-2 was detected in stool samples from patients with abdominal symptoms [20, 25] , so some scholars have proposed that sars-cov-2 could spread via fecal-oral transmission. further environmental studies will be needed to determine whether the virus remains viable under conditions that would favor fecal-oral transmission [26] . sars-cov-2 has not been confirmed to be transmitted vertically from mother to child [27] . based on the available data, a chinese team estimated a basic reproduction number (r0) of 3.77 for sars-cov-2, basically confirming that the new coronavirus is more contagious than sars [28] . sars-cov-2 employs a densely glycosylated, homotrimeric class i fusion spike (s) protein to enter host cells. the s protein exists in a metastable prefusion conformation that undergoes a dramatic structural rearrangement to fuse the viral membrane with the host cell membrane [29, 30] . epidemiological data indicate that the population is generally susceptible to sars-cov-2 [31] . therefore, it is necessary for individuals to wash or disinfect their hands frequently, go outside less, wear a mask, avoid group activities, stay away from patients with covid-19, maintain good living habits and keep an optimistic attitude [32] [33] [34] . sars-cov-2 has been reported to be associated with guillain-barré syndrome, rhabdomyolysis, acute cerebrovascular disease, central nervous system infections and other neurological diseases. four formal reports have described neurological problems in sars patients, including polyneuropathy [35] , myopathy and rhabdomyolysis [36] , large artery ischemic stroke [37] and central nervous system infections [38] . human coronaviruses (hcovs) can naturally reach the central nervous system, and could potentially cause neurological symptoms. among the coronavirus-induced animal diseases, feline infectious peritonitis virus, mouse hepatitis virus and hemagglutinating encephalomyelitis virus can all reach the central nervous system and induce different types of neuropathologies [10, 11] . the structure of sars-cov-2 is similar to that of the sars virus, and both viruses invade the human body through the angiotensin converting enzyme ii (ace2) receptor. thus, in this paper, we mainly describe the neurological diseases associated with sars-cov-2, but also briefly introduce the neurological diseases associated with sars. polyneuropathy, also known as peripheral neuropathy, is multiple-nerve damage of the extremities. the clinical manifestations are mostly distal symmetrical motor sensory dysfunction and autonomic nerve dysfunction [39] . the causes of polyneuropathic disorders include metabolic, toxic, infectious, inflammatory, autoimmune and genetic conditions [40] . zhao et al. reported a case of covid-19 initially presenting with acute guillain-barré syndrome (gbs). the female patient aged 61 years presented with acute weakness in both legs and severe fatigue. she received intravenous immunoglobulin, antiviral drugs of arbidol, lopinavir, and ritonavir, and supportive care. after 30 days of treatment, the muscle strength of the limbs returned to normal and the respiratory symptoms disappeared [41] . in a recently published article, two covid-19 patients were diagnosed with miller-fisher syndrome (mfs) and multiple cranial neuritis, respectively [42] . these cases suggest a possible link between gbs and sars-cov-2 infection. some patients with severe covid-19 progress rapidly and need to be transferred to an intensive care unit (icu) for further treatment [43, 44] . in such patients, the peripheral nerves could be particularly susceptible to peripheral microcirculation disturbances, since the vessels supplying them with blood lack autoregulation [45] . icu-acquired weakness, which can manifest as critical-illness polyneuropathy, critical-illness myopathy or both, is a frequent and disabling disorder in icu patients [46] . critical-illness polyneuropathy, an axonal sensory-motor polyneuropathy, is observed in up to a third of critically ill patients with systemic inflammatory response syndrome. critical-illness myopathy, an acute aging myopathy, develops in a similar setting, often in association with the use of corticosteroids and/or nondepolarizing neuromuscular-blocking agents [47] . tsai et al. [35] presented data from four patients with probable sars who developed axonal polyneuropathy, myopathy or both (2004). all of them had received intubation for respiratory distress and a high dose of steroid therapy for multiple organ failure. they developed distal-predominant weakness in all four limbs and a mild decrease in deep-tendon reflexes three to four weeks after the onset of sars. the most likely diagnoses were critical-illness polyneuropathy and/or critical-illness myopathy. some viruses, such as cytomegalovirus and varicella zoster virus, may cause peripheral neuropathy by directly attacking the nerves. it is not known whether direct attacks of the peripheral nervous system occur in hcov-associated neuropathy. rhabdomyolysis refers to the damage to striated muscle, the destruction of the muscle cell membrane integrity and the release of myoglobin, creatine kinase, other enzymes, small molecules and toxic substances into the systemic circulation due to various traumatic and non-traumatic factors, resulting in a group of clinical syndromes of organ damage [48, 49] . clinical examination, history evaluation, laboratory studies, muscle biopsies and genetic testing are useful tools for diagnosing rhabdomyolysis and differentiating acquired from inherited cases. acquired cases may be due to substance abuse, medication or toxic exposures, electrolyte abnormalities, endocrine disturbances and autoimmune myopathies [50] . in several recent studies on covid-19 [5, 12, 20] , a few patients exhibited varying degrees of myalgia, fatigue and elevated creatine and creatine kinase levels. in the study of guan et al., two patients clearly developed rhabdomyolysis as a complication of covid-19, while 14.90% (164/1099) exhibited myalgia or arthralgia symptoms and 13.7% (90/657) had creatine kinase levels ≥ 200 u/l [5] . tong's research group reported that a patient diagnosed with covid-19 had pain and weakness in both lower limbs and obvious tenderness after the ninth day of admission. laboratory examination indicated that the patient's myoglobin level was >12,000.0 μg/l (reference 0-140 μg/l), creatine kinase was 11,842 u/l (reference 38-174 u/l) and lactate dehydrogenase was 2,347 u/l (reference 109-245 u/l). the authors added hydration, alkalization, plasma transfusion, gamma globulin and symptomatic support therapy based on the patient's previous treatment with oxygen, antivirals, antibiotics and methylprednisolone [51] . creatine kinase and myoglobin are important indicators of rhabdomyolysis, but they are not routinely detected in the clinical practice. when patients have local muscle pain and weakness, rhabdomyolysis should be considered. in previous sars studies, some patients were clearly diagnosed with critical-illness myopathy [35] and rhabdomyolysis [50, 52] . in such patients, it cannot be ruled out that rhabdomyolysis may have developed due to the use of corticosteroids and/or nondepolarizing neuromuscular-blocking agents; however, the association of rhabdomyolysis with viruses such as influenza viruses a and b, human immunodeficiency virus, coxsackie virus, cytomegalovirus, west nile virus and dengue virus has also been well described [53] [54] [55] [56] . nevertheless, there is not yet sufficient evidence that hcovs can directly invade muscle cells. the population is generally susceptible to sars-cov-2, but the elderly are more susceptible (the median age of hospitalized patients in one study was 56 years [interquartile range, 42-68 years; range, 22-92 years] [20] ), and such patients are already at high risk for cerebrovascular diseases. viral infections are known to be associated with an increased risk of stroke [57] . in a study by mao et al., 214 patients diagnosed with covid-19 were enrolled, and six (2.80%) of them developed acute cerebrovascular disease (five cases of ischemic stroke and one case of cerebral hemorrhage). all but one of these patients (an ischemic stroke patient) died of respiratory failure [13] . in a study of 206 sars patients in singapore, large artery stroke was diagnosed in five patients, of whom four were critically ill and three died [58] . strokes are not uncommon in critically ill patients with multiple comorbidities, so sars-cov-2 infections in humans may increase the risk of stroke. central nervous system infections are among the most critical problems in public health, as patients frequently exhibit neurologic sequelae. the clinical manifestations include a fever, headache, vomiting, stiff neck, afebrile seizures and status epilepticus. hcovs cause a certain degree of nerve erosion, but their capacity to infect the central nervous system in humans has not been well characterized [10, 59] . moriguchi et al. described a patient with sars-cov-2-associated meningitis who was brought to the hospital by ambulance due to convulsions and a coma. interestingly, sars-cov-2 rna was not detected in the patient's nasopharyngeal swab, but was detected in the patient's cerebrospinal fluid [60] . zhao et al. [61] reported spinal cord involvement in a covid-19 patient one week after the onset of fever. after admission, his sars-cov-2 rna aging nasopharyngeal swab test was positive. based on the patient's acute flaccid myelitis of the lower limbs, urinary and bowel incontinence, and sensory level at t10, a diagnosis of acute myelitis was more likely. after the patient had been treated with high-flow oxygen, antiviral medication, steroids and human immunoglobulin, his body temperature returned to normal and two subsequent sars-cov-2 rna nasopharyngeal swab tests were negative. the muscle strength of both upper limbs recovered to grade 4/5, while the muscle strength of both lower limbs was grade 1/5. this study indicated that acute myelitis may be a neurological complication of covid-19. the above cases demonstrate the potential for neurological invasion of sars-cov-2. the presence of hcov in human central nervous system-related samples was detected as early as 1980 in autopsies of patients with multiple sclerosis [62] . in 2004, genetic material from sars-cov was detected in cerebrospinal fluid samples from a 32-year-old woman. the patient had a generalized tonic-clonic convulsions with loss of consciousness and up-rolling eyeballs lasting for one minute [38] . another patient, a doctor infected with the sars virus, had symptoms of restlessness, vomiting and confusion on the 33th day of illness. the patient died after treatment failed, and a brain biopsy was performed. a fragment specific for sars hcov was amplified from cultures of the brain suspension, and transmission electronic microscopy revealed the presence of an enveloped virus morphologically compatible with a coronavirus in the cultures [63] . since some covid-19 patients have complained of headaches, nausea etc, care providers should be alert for central nervous system infections caused by sars-cov-2 if such patients also exhibit symptoms such as a fever, epilepsy and disturbances of consciousness. in this section, we will explore various mechanisms that may explain the correlation between covid-19 and neurological disease. in a clinical retrospective study of 138 people, the most common complication of covid-19 during hospital admission was pneumonia, followed by acute respiratory distress syndrome (19.60%) and shock (8.70%) [20] . the patients in this study had varying degrees of hypoxia, accompanied by hypoxemia. most patients received oxygen inhalation (ordinary oxygen inhalation, 106 [76.81%]), and many received mechanical ventilation (non-invasive ventilation, 15 [10.09%]; intermittent mandatory ventilation, 17 [12.32%] ). more than 20% of the oxygen consumed by humans is used by the brain for atp production to generate the required membrane potential [64] . as soon as anoxia sets in, atp synthase begins to pump protons out of the mitochondrial matrix to maintain the mitochondrial membrane potential. continued lack of oxygen can eventually lead to the loss of high-energy phosphate esters, disturbances of neurotransmitter metabolism, the breakdown of the membrane, the failure of mitochondria and the accumulation of intracellular ca 2+ . the immediate consequence is irreversible neurological damage and even neuronal death [64, 65] . lack of oxygen increases the risk of stroke. for instance, the prolonged hypoxia of obstructive sleep apnea hypopnea syndrome can damage the sleep structure, increase blood pressure, reduce cerebral blood flow and promote microthrombosis and atherosclerosis, thus impacting the prognosis and recurrence of cerebral infarction [66, 67] . mao et al. reported that six covid-19 patients had acute cerebrovascular disease: five with severe infections (5/88) and one with a non-severe infection (1/126) (p=0.03) [13] . the symptoms of hypoxia in covid-19 patients are very obvious, and critical patients need ventilator support. covid-19 patients admitted to the icu tend to be older and have a greater number of comorbid conditions (e.g., hypertension, diabetes, cardiovascular and cerebrovascular diseases) than those not admitted to the icu [20] . this suggests that older age and these comorbidities may be risk factors for poor outcomes [68, 69] . the metallopeptidase ace2 has been confirmed to be the cell receptor for sars-cov-2, just as it is for sars-cov [70, 71] . however, sars-cov-2 cannot enter cells through other coronavirus receptors such as aminopeptidase n and dipeptidyl peptidase [71] . ace2 is highly expressed not only in the alveolar type ii cells of the lungs and the upper and stratified epithelial cells of the esophagus, but also in the absorptive enterocytes of the ileum and colon [72, 73] . the main physiological function of ace2 is to catalyze the conversion of angiotensin ii to angiotensin (1-7), with a vasodilator effect. in brain tissues, angiotensin (1-7) stimulates mas receptors to promote angiogenesis, and also inhibits oxidative stress, prevents neuroinflammation, improves cerebral blood flow, suppresses apoptosis and protects cerebral blood vessels [74] . enhancing the expression of ace2 may be an important strategy for treating cardiovascular and cerebrovascular aging diseases [75] . sars-cov-2 patients with cerebrovascular disease may be more likely to develop into severe patients with a higher risk of death, so more timely diagnosis is needed for such patients. acei and angiotensin ii receptor blocker antihypertensive drugs may increase the expression of the ace2 receptor [76] . in order to avoid aggravating sars-cov-2 infection symptoms, it is recommended that hypertensive patients on blood pressure control medications stop using acei and angiotensin ii receptor blocker antihypertensive drugs, and instead use calcium channel blocker diuretic antihypertensive drugs [77] . the responses of the immune system can be divided into innate immunity (also known as non-specific immunity) and adaptive immunity (also known as specific immunity, which can be further divided into humoral immunity and cellular immunity) [76, 78] . the immune mechanisms induced by sars-cov-2 are unclear. after sars-cov-2 enters the body through ace2, host factors trigger an immune response against the virus. the virus induces natural immunity, phagocytosis and phagocytic cell death, thus damaging tissues and organs. in four clinical retrospective studies that clearly identified the diagnosis of covid-19 [12, 19, 20, 79] , the absolute value of lymphocytes in most patients was reduced. these findings suggest that sars-cov-2 mainly attacks lymphocytes, especially t lymphocytes, similar to sars-cov. cd4+ t cells are well known to regulate or "assist" the functioning of other lymphocytes. cd8+ t cells are cytotoxic and can kill virus-infected cells [80] . barton et al. [81] reported that in two autopsies of covid-19 patients, immunohistochemistry revealed a small number of cd3+ t lymphocytes infiltrating the alveolar septum, while cd20+ b-lymphocytes were rare. cd8+ t cells were slightly more prevalent than cd4+ t cells, and cd68 detection revealed a few macrophages. some studies have suggested that the substantial decrease in the total number of lymphocytes in coronavirus patients may indicate that the virus consumes many immune cells and inhibits cellular immune function [82, 83] . after an antigen enters the body, the corresponding antigen-specific b cells are activated, induced to proliferate and eventually stimulated to differentiate into plasma cells. these plasma cells then produce specific antibodies that can enter the body fluid and exert immune effects. it is widely accepted that immunoglobulin m (igm) provides the first line of defense during viral infections, prior to the generation of adaptive, high-affinity igg responses that are important for long-term immunity and immunological memory [84] . li et al. successfully developed a rapid detection igg-igm combined antibody test kit for the diagnosis of covid-19. the kit has a sensitivity of 88.66% and a specificity of 90.63%, and can detect the infection within 15 minutes [85] . after the rehabilitation of most patients with the novel coronavirus, the body will produce specific antibodies that can kill and eliminate the virus. on february 8, 2020, with the pneumonia diagnosis and treatment program for novel coronavirus infection (trial version 5) [86] as a guide, the first people's hospital of jiangxia district carried out the first phase of a new convalescent plasma treatment on three critically ill patients. after 12 to 24 hours of convalescent plasma therapy, the patients' laboratory examination results, clinical signs and symptoms improved significantly. plasma therapy not only is safe and potentially effective, but also stimulates humoral immunity [87] . most covid-19 patients have a good prognosis, while a few patients have mild symptoms in the early stage and suddenly deteriorate in the later stage of the disease or during the recovery process. a large number of patients have exhibited a 'cytokine storm' (the rapid production of cytokines such as tumor necrosis factor alpha, interleukin-1, interleukin-6 and interferon gamma) due to the viral infection, which sometimes has progressed to acute respiratory distress syndrome and multiple organ failure [12, 19] . it is already known that hcov can spread from the respiratory tract to the central nervous system through transneuronal and hematogenous routes, resulting in encephalitis and neurological diseases [88] . the invasion of the blood-brain barrier by the coronavirus can destroy vascular endothelial connections, leading to blood-brain barrier dysfunction and enhanced permeability [89] . when the virus invades the human brain, it triggers immune damage, causing brain damage and acute or chronic inflammation, thus creating a vicious cycle. several current retrospective clinical studies have described covid-19 patients with abnormally low lymphocyte counts, prolonged prothrombin times and significantly increased lactate dehydrogenase levels. patients transferred to the icu had significantly higher white blood cell and neutrophil counts than those not transferred to the icu, as well as higher levels of d-dimer, creatine kinase and creatine [20] . the complications in severe cases have included rhabdomyolysis, shock, acute cardiac injury and acute kidney injury. several mechanisms are thought to link aging infections with acute vascular events, including the release of proinflammatory cytokines, the disruption of atherosclerotic plaques, physiological changes in the heart rate and vasoconstriction [90] . the inflammatory response in severe pneumonia is not limited to lung tissue; rather, the systemic inflammatory response is activated, and its amplification cascade impairs the function of distant organs [57, 91] . middle-aged and elderly patients account for the majority of covid-19 patients (especially critically ill patients) with abnormally increased d-dimer levels, and such patients are more prone to embolic vascular events and cerebrovascular disease [20] . umapathi et al. postulated that a hypercoagulable state predisposed a group of mainly critically ill sars patients to large cerebral arterial thromboembolism [58] . providers treating critically ill covid-19 patients with underlying diseases such as hypertension, diabetes, cancer, etc. should be alert to the potential for hypercoagulability and regularly assess routine blood coagulation. our research does not require an ethics statement. sars-cov-2 infection may involve the nervous system, and may cause diseases such as polyneuropathy, myopathy, cerebral infarction and central nervous system infections. cerebral infarction is the second most common cause of death and the leading cause of adult disability worldwide. patients with cerebrovascular diseases may face greater risks during infections, so it is necessary to strengthen protection to avoid infection, perform secondary prevention measures and monitor patients' symptoms and vital signs. during the period of high incidence of covid-19, neurologists need to pay great attention to the treatment of patients, especially those whose first symptoms are neurological symptoms. xiaojia tang, peipei liu and yingzhu chen conceived and designed the research. xiaojia tan wrote the manuscript, and all authors contributed to manuscript revision, read and approved the submitted version. and china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 who director-general's opening remarks at the media briefing on covid-19 and china medical treatment expert group for 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respiratory syndrome corona virus (mers-cov) the authors thank professor yingzhu chen and peipei liu, m.d., ph.d. no potential conflicts of interest were reported by the authors. this work was supported by the "six talent peaks" project of jiangsu province (no. wsw-246), "333 project" science program of jiangsu province (no. bra2015187) and the "thirteenth five-year" special fund for science, education and health of yangzhou (no. ljrc20187). key: cord-193133-puqcbf8t authors: piplani, sakshi; singh, puneet kumar; winkler, david a.; petrovsky, nikolai title: in silico comparison of spike protein-ace2 binding affinities across species; significance for the possible origin of the sars-cov-2 virus date: 2020-05-13 journal: nan doi: nan sha: doc_id: 193133 cord_uid: puqcbf8t the devastating impact of the covid19 pandemic caused by sars coronavirus 2 (sarscov2) has raised important questions on the origins of this virus, the mechanisms of any zoonotic transfer from exotic animals to humans, whether companion animals or those used for commercial purposes can act as reservoirs for infection, and the reasons for the large variations in susceptibilities across animal species. traditional lab-based methods will ultimately answer many of these questions but take considerable time. in silico modeling methods provide the opportunity to rapidly generate information on newly emerged pathogens to aid countermeasure development and also to predict potential future behaviors. we used a structural homology modeling approach to characterize the sarscov2 spike protein and predict its binding strength to the human ace2 receptor. we then explored the possible transmission path by which sarscov2 might have crossed to humans by constructing models of ace2 receptors of relevant species, and calculating the binding energy of sarscov2 spike protein to each. notably, sarscov2 spike protein had the highest overall binding energy for human ace2, greater than all the other tested species including bat, the postulated source of the virus. this indicates that sarscov2 is a highly adapted human pathogen. of the species studied, the next highest binding affinity after human was pangolin, which is most likely explained by a process of convergent evolution. binding of sarscov2 for dog and cat ace2 was similar to affinity for bat ace2, all being lower than for human ace2, and is consistent with only occasional observations of infections of these domestic animals. overall, the data indicates that sarscov2 is uniquely adapted to infect humans, raising questions as to whether it arose in nature by a rare chance event or whether its origins lie elsewhere. the devastating impact of covid-19 infections caused by sars-coronavirus 2 (sars-cov-2) has stimulated unprecedented international activity to discover effective vaccines and drugs for this and other pathogenic coronaviruses. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] it has also raised important questions on the mechanisms of zoonotic transfer of viruses from animals to humans, questions as to whether companion animals or those used for commercial purposes can act as reservoirs for infection, and the reasons for the large variations in sars-cov-2 susceptibility across animal species. [17] [18] [19] understanding how viruses move between species may help us prevent or minimize these pathways in the future. elucidating the molecular basis for the different susceptibilities of species may also shed light on the differences in susceptibilities in different sub-groups of humans. very recently, shi et al. published the results of experiments to determine the susceptibility to sars-cov-2 of ferrets, cats, dogs, and other domesticated animals. 20 they showed that sars-cov-2 virus replicates poorly in dogs, pigs, chickens, and ducks, but ferrets and cats are permissive to infection. other studies have reported the susceptibility of other animal species to sars-cov-2. 17, 20, 21 susceptible species such as macaques, hamsters and ferrets are used as animal models of sars-cov-2 infection. [22] [23] [24] in the absence of purified, isolated ace2 from all the relevant animal species that could be used to measure the molecular affinities to spike protein experimentally, computational methods offer considerable promise for determining the rank order of affinities across species, as a method to impute which species may be permissive to sars-cov-2. here we show how computational chemistry methods from structure-based drug design can be used to determine the relative binding affinities of the sars-cov-2 spike protein for its receptor, angiotensin converting enzyme (ace)-2, a critical initiating event for sars-cov-2 infection, across multiple common and exotic animal species. [25] [26] [27] the aim of these studies was to better understand the species-specific nature of this interaction and see if this could help elucidate the origin of sars-cov-2 and the mechanisms for its zoonotic transmission. to construct the three-dimensional structure of the sars-cov-2 spike protein, the sequence was retrieved from ncbi genbank database (accession number yp_009724390.1). a psi-blast search against the pdb database for template selection was performed and the x-ray structure of sars coronavirus spike template (refcode 6acc) was selected with 76.4% sequence similarity to sars-cov-2 spike protein. the protein sequences of the ace2 proteins for different species is summarized in table 11 and full sequence alignment in supplementary figure 1 . the phylogenetic tree for ace2 proteins from selected animal species is illustrated in supplementary figure 2 . the 3d-structures were built using modeller 9.21 (https://salilab.org/modeller/). 28 the quality of the generated models was evaluated using the ga341 score and dope scores, and the models assessed using swiss-model structure assessment server (https://swissmodel.expasy.org/assess). 29 the x-ray crystal structures of human ace2 (recode 3sci) and human sars spike protein (refcode 5xlr) were retrieved from protein data bank. protein preparation and removal of nonessential and non-bridging water molecules for docking studies were performed using the ucsf chimera package (https://www.cgl.ucsf.edu/chimera/). 30 these modelled structures were docked against sars-cov-2 spike protein structure using the hdock server (http://hdock.phys.hust.edu.cn/). 31, 32 molecular docking was performed on the homology modelled sars-cov-2 spike protein with human and animal ace2 proteins. the sars-cov spike protein was also docked with human ace2 protein to obtain the docking pose for binding energy calculations. the docking poses were ranked using an energy-based scoring function. the docked structures were analyzed using ucsf chimera software. the final models were optimized using the amber99sb-ildn force field in gromacs2020 (http://www.gromacs.org/). 33 docked complexes (sars-cov-2 spike with human ace2, human sars-cov spike with human ace2, sars-cov-2 spike with bat ace2 etc) were used as starting geometries for md simulations. simulations were carried out using the gpu accelerated version of the program with the amber99sb-ildn force field i periodic boundary conditions on an oracle cloud server. docked complexes were immersed in a truncated octahedron box of tip3p water molecules. the solvated box was further neutralized with na+ or cl− counter ions using the tleap program. particle mesh ewald (pme) was employed to calculate the long-range electrostatic interactions. the cutoff distance for the long-range van der waals (vdw) energy term was 12.0 å. the whole system was minimized without any restraint. the above steps applied 2500 cycles of steepest descent minimization followed by 5000 cycles of conjugate gradient minimization. after system optimization, the md simulations was initiated by gradually heating each system in the nvt ensemble from 0 to 300 k for 50 ps using a langevin thermostat with a coupling coefficient of 1.0/ps and with a force constant of 2.0 kcal/mol·å2 on the complex. finally, a production run of 100 ns of md simulation was performed under a constant temperature of 300 k in the npt ensemble with periodic boundary conditions for each system. during the md procedure, the shake algorithm was applied for the constraint of all covalent bonds involving hydrogen atoms. the time step was set to 2 fs. the structural stability of the complex was monitored by the rmsd and rmsf values of the backbone atoms of the entire protein. finally, the free energies of binding were calculated for all the simulated docked structures. calculations were also performed for up to 500 ns on human ace2 to ensure that 100ns is sufficiently long for convergence. duplicate production runs starting with different random seeds were also run to allow estimates of binding energy uncertainties to be determined for the strongest binding ace2 structures. the binding free energies of the protein-protein complexes were evaluated in two ways. the traditional method is to calculate the energies of solvated sars-cov-2 spike and ace2 proteins and that of the bound complex proteins and derive the binding energy by subtraction. δg (binding, aq) = δg (complex, aq) -(δg (spike, aq) + δg (ace2, aq) we also calculated binding energies using the molecular mechanics poisson boltzmann surface area (mm-pbsa) tool in gromacs that is derived from the nonbonded interaction energies of the complex. 34, 35 the method is also widely used method for binding free energy calculations. the binding free energies of the protein complexes were analyzed during equilibrium phase from the output files of 100 ns md simulations. the g_mmpbsa tool in gromacs was used after molecular dynamics simulations, the output files obtained were used to post-process binding free free energy decomposition analyses were also performed by mm-pbsa decomposition to get a detailed insight into the interactions between the ligand and each residue in the binding site. the binding interaction of each ligand-residue pair includes three terms: the van der waals contribution, the electrostatic contribution, and the solvation contribution. another estimate of the strength of the interaction between protein-protein complex can be obtained from the non-bonded interaction energy between the complex. gromacs has the ability to decompose the short-range nonbonded energies between any number of defined groups. to compute the interaction energies as a part of our analysis, we reran the trajectory files obtained during simulation to recompute energies using -rerun command. the interaction energy is the combination of short range coulombic interaction energy (coul-sr:protein-protein) and the short-range lennard-jones energy (lj-sr:protein-protein (see table 3 ). while this paper was being prepared, a paper by guterres and im described a substantial improvement in protein-ligand docking results using high-throughput md simulations. 36 they employed docking using autodock vina, followed by md simulation using charmm. the parameters they advocated were very similar to those used in our study. proteins were solvated in a box of tip3p water molecules extending 10 å beyond the proteins and the particle-mesh ewald method was used for electrostatic interactions. nonbonded interactions over 10 and 12 å were truncated. their systems were minimized for 5000 steps using the steepest descent method followed by 1 ns equilibration with an nvt setting. for each protein-ligand complex, they ran 3 × 100 ns production runs from the same initial structure using different initial velocity random seeds and an integration step size of 2 fs. the ancestry of sars-cov-2 traces back to the human, civet and bat sars-cov strains, which all use the same ace2 proteins for cellular entry. [37] [38] [39] the similarities and variations in sequences for both the sars-cov-2 spike protein and the sars spike protein were determined from sequences retrieved from ncbi genbank databank and aligned using clustalw. the spike protein receptor binding domain (rbd) region showed a 72% identity between the two viruses ( figure 1 ). conserved regions in black and non-conserved residues in red and blue. the three-dimensional structure of sars-cov-2 spike protein ( figure the ramachandran scores of the modeled ace2 structures for selected species are summarized in table 1 with the actual predicted ace2 structures and ramachandran plots for each selected species shown in supplementary figure 3 . figure 3) . the modelled structures were further assessed for quality control using ramachandran plot and molprobity scores in swissmodel structure assessment. the ramachandran plot checks the stereochemical quality of a protein by analyzing residue-by-residue geometry and overall structure geometry, is also a way to visualize energetically allowed regions for backbone dihedral angles ψ against φ of amino acid residues in protein structure. the ramachandran score of sars-cov-2 spike protein was 90% in the binding region and molprobity scores that provide an evaluation of model quality at both the global and local level was 3.17. further, the ace2 modelled structures from selected species were also assessed using swiss model structure assessment. ramachandran score or the percentage of amino acid residues falling into the energetically favored region for all species ranged from 96-99% (table 1) . these ramachandran and molprobity scores show that all the built structures were of good quality and were suitable for use in further studies. the ramachandran graphs are presented in supplementary figure 3 . the receptor binding domain (rbd) of sars-cov-2 spike protein was docked against ace2 receptor of various species using hdock server. the interacting residues of ace2 and sars-cov-2 spike protein are depicted in table 3 . we found certain key amino acids in the receptor binding motif (rbm) that were in accordance with previous studies 40 . certain amino acids including phe28, asn330, asp355 and arg357 were conserved in ace2 of most of the selected species and were observed to take part in the interaction with spike protein. tyr41, lys353, ala386 and arg393 also interacted with spike protein residues and were highly conserved across all species except bat, mouse, ferret and pangolin, respectively. spike protein interacting residues with ophiophagus hannah (king cobra) were least common amongst all the ace2 species included in the study, consistent with its low sequence similarity to human ace2. homo sapiens (human) the molecular dynamics simulation of complexes of sars-cov-2 spike protein and ace2 receptors of various species were performed for 100ns. all complexes became stable during simulation with rmsd fluctuations converging to a range of 0.5 to 0.8 nm from the original position. the calculated binding energies for the interactions of sars-cov-2 with ace2 from the species studied are presented in table 4 . the table 4 also includes observational in vivo data on sars-cov-2 infectivity and disease symptoms in the species where this has been reported. although bats carry many coronaviruses including sars-cov, a relative of sars-cov-2, direct evidence for existence of sars-cov-2 in bats has not been found. as highlighted by our data, the binding strength of sars-cov-2 for bat ace2 is considerably lower than for human ace2, suggesting that even if sars-cov-2 did originally arise from a bat precursor it must later have adapted its spike protein to optimise its binding to human ace2. there is no current explanation for how, when or where this might have happened. instances of direct human infection by coronaviruses or other bat viruses is rare with transmission typically involving an intermediate host. for example, lyssaviruses such as hendra are periodically transmitted from bats to horses and then to humans who contact the infected horse. similarly, sars-cov was shown to be transmitted from bats to civet cats and from them to humans. to date, a virus identical to sars-cov-2 has not been identified in bats or any other non-human species, making its origins unclear. to date, the most closely related coronavirus to sars-cov-2, is the bat coronavirus, batcov ratg1, which has 96% whole-genome identity to sars-cov-2. 50 hence other genetic factors could underlie the apparent lack of susceptibility of dogs to covid-19 clinical infection. it is known that gain of function (gof) mutations occur in viruses that can lead to pandemics. gof means viruses gain a new property e.g. in influenza virus gof has been associated with the acquisition of a new function, such as mammalian transmissibility, increased virulence for humans, or evasion of existing host immunity. 49 the conditioning of viruses to humans as pandemics progress is well recognized. however, the sars-cov-2 structures and sequences that we employed were from viruses collected very early in the pandemic. it is therefore not clear how sars-cov-2 could have developed such a high affinity for human ace2, notably higher than for those of putative zoonotic sources for sars-cov-2, unless it has been previously selected on human ace2 or an ace2 of another species bearing a closely homologous spike protein binding domain. interestingly, pangolin ace2 bears some similarities in its sbd to human ace2. this marries with the fact that pangolin-cov shares a highly similar rbd to sars-cov-2, although their remaining sequence has only 90% similarity. this could be consistent with a process of convergent evolution whereby human and pangolin coronaviruses infecting via ace2, have come to the same solution in respect of evolving an optimal spike rbd for binding of either human or pangolin ace2, respectively. our data does indicate that humans might be permissive to pangolin covs that use ace2 for cell entry, a fact that needs to be borne in mind in respect of future potential coronavirus pandemic sources. however, this does not mean that pangolin ace2 was the receptor on which the sars-cov-2 spike protein rbd was initially selected, with the strength of binding to pangolin ace2 lower than binding to human ace2. this makes it unlikely that pangolins are the missing intermediate host. if sars-cov-2 spike was selected on pangolin ace2, then given the higher affinity of sars-cov-2 for human ace2 than for bat ace2, sars-cov-2 would have to have circulated in pangolins for a long period of time for this evolution and selection to occur and to date there is no evidence of a sars-cov-2 like virus circulating in pangolins. another possibility would be a short term evolutionary step where a pangolin was recently coinfected with a bat ancestor to sars-cov-2 at the same time as it was infected by a pangolin cov allowing a recombination event to occur whereby the spike rbd of the pangolin virus was inserted into the bat cov, thereby conferring the bat cov with high binding for both pangolin and human ace2. such recombination events are known to occur with other rna viruses and can explain creation of some pandemic influenza strains 49 . nevertheless, such events are by necessity rare as they require coinfection of the one host at exactly the same time. most importantly, if such a recombination event had occurred in pangolins it might have been expected to have similarly triggered an epidemic spread of the new highly permissive sars-cov-2 like virus among pangolin populations, such as we now see occurring across the human population. currently there is no evidence of such a pangolin sars-cov-2 like outbreak, making this whole scenario less likely. indeed, pangolins might be protected from sars-cov-2 infection due to the existence of crossprotective spike rbd neutralising antibodies induced by exposure to pangolin cov, given the rbd similarity of these two viruses. another possibility which still cannot be excluded is that sars-cov-2 was created by a recombination event that occurred inadvertently or consciously in a laboratory handling coronaviruses, with the new virus then accidentally released into the local human population. given the seriousness of the ongoing sars-cov-2 pandemic, it is imperative that all efforts be made to identify the original source of the sars-cov-2 virus. in particular, it will be important to establish whether covid-19 is due to a completely natural chance occurrence where a presumed bat virus was transmitted to humans via an intermediate animal host or whether covid-19 has alternative origins. this information will be of paramount importance to help prevent any similar human coronavirus outbreak in the future. clinical trials for the treatment of coronavirus disease 2019 (covid-19): a rapid response to urgent need progress and prospects on vaccine development against sars-cov-2 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zoonotic aspects susceptibility of ferrets, cats, dogs, and other domesticated animals to sarscoronavirus 2 absence of sars-cov-2 infection in cats and dogs in close contact with a cluster of covid-19 patients in a veterinary campus infection and rapid transmission of sars-cov-2 in ferrets age-related rhesus macaque models of covid-19 simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility structural basis for the recognition of sars-cov-2 by full-length human ace2 ace2 the janus-faced protein -from cardiovascular protection to severe acute respiratory syndrome-coronavirus and covid-19 structural basis of receptor recognition by sars-cov-2 comparative protein modelling by satisfaction of spatial restraints toward the estimation of the absolute quality of individual protein structure models ucsf chimera--a visualization system for exploratory research and analysis hdock: a web server for proteinprotein and protein-dna/rna docking based on a hybrid strategy the hdock server for integrated protein-protein docking high performance molecular simulations through multilevel parallelism from laptops to supercomputers electrostatics of nanosystems: application to microtubules and the ribosome open source drug discovery, c. & lynn, a. g_mmpbsa--a gromacs tool for high-throughput mm-pbsa calculations improving protein-ligand docking results with high-throughput molecular dynamics simulations isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov predicting the angiotensin converting enzyme 2 (ace2) utilizing capability as the receptor of sars-cov-2 silico analysis of intermediate hosts and susceptible animals of sars-cov-2. chemrxiv identifying sars-cov-2 related coronaviruses in malayan pangolins an overview of sars-cov-2 and animal infection sars-cov-2 spike protein favors ace2 from bovidae and cricetidae the pathogenicity of 2019 novel coronavirus in hace2 transgenic mice comparative pathogenesis of covid-19, mers and sars in a nonhuman primate model risks and benefits of gain-of-function experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion a pneumonia outbreak associated with a new coronavirus of probable bat origin alignment of ace2 amino acid sequence from selected species. the sar-cov-2 spike protein binding region is highlighted in red we would like to thank harinda rajapaksha for assistance to optimise gromacs for this project. we would like to thank oracle corporation for providing their cloud computing resources through the oracle for research program for the modelling studies described herein. in particular, we wish to supplementary figure 4 . predicted and confirmed utilization of ace2 receptors by the sars-cov-2 spike protein based on sequence homology from qui et al. 41 key: cord-266444-rw94yls8 authors: dominguez andres, ana; feng, yongmei; campos, alexandre rosa; yin, jun; yang, chih-cheng; james, brian; murad, rabi; kim, hyungsoo; deshpande, aniruddha j.; gordon, david e.; krogan, nevan; pippa, raffaella; ronai, ze’ev a. title: sars-cov-2 orf9c is a membrane-associated protein that suppresses antiviral responses in cells date: 2020-08-19 journal: biorxiv doi: 10.1101/2020.08.18.256776 sha: doc_id: 266444 cord_uid: rw94yls8 disrupted antiviral immune responses are associated with severe covid-19, the disease caused by sar-cov-2. here, we show that the 73-amino-acid protein encoded by orf9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing il-6 signaling. furthermore, we showed that interfering with orf9c degradation by either proteasome inhibition or inhibition of the atpase vcp blunted the effects of orf9c. our study indicated that orf9c enables immune evasion and coordinates cellular changes essential for the sars-cov-2 life cycle. one-sentence summary sars-cov-2 orf9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection. sars-cov-2 is an enveloped, positive-sense single strand 29.9 kb rna virus (1, 2) that causes severe respiratory disease in humans . this coronavirus was first identified in wuhan, china, at the end of 2019 (3) . due to its easy human-to-human transmission and the lack of effective antiviral therapy, covid-19 has caused a pandemic with more than 19 million cases and over 740,000 deaths worldwide (https://covid19.who.int). mechanistically, the host protein ace2 serves as the viral receptor and host cellular proteases, such as tmprss2, play key roles in sars-cov-2 entry into host cells (4) (5) (6) (7) . ace2 expression is high in alveolar epithelial cells (8) , making the lung a highly vulnerable target for the virus. sars-cov-2 infection causes a wide range of disease, from asymptomatic to mild disease to severe disease that can lead to death (9) . sars-cov-2 is most similar to the coronaviruses sars-cov and mers-cov (10, 11) . however, neither of those became a global pandemic. current therapies are primarily palliative and supportive (9) . more than 2000 clinical trials are currently in progress worldwide (12) (https://clinicaltrials.gov/ct2/who_table). without effective vaccines or treatments, there is an urgent need to understand the pathology of sars-cov-2 infection, the roles of each of the 29 proteins encoded within the viral genome in the life cycle, virulence, and pathogenicity of the virus, and identify strategies for intervention or treatment. various therapeutic and vaccine strategies target viral entry mechanisms, such as vaccines or antibodies targeting on the spike (s) protein (13) (14) (15) (16) ; others target viral replication or assembly processes, such as the antiviral drug remdesivir, which interferes with rna replication and has emerged as superior to placebo in shortening recovery time in adults (17). another strategy for treatment is interfere with viral immune evasion mechanisms and thus enable the body's natural 4 antiviral responses to be more effective at clearing the virus. indeed, investigation of mechanisms of immune evasion by sars-cov-2 is an active area of translational research with immune evasion properties discovered for nonstructural protein 1 (nsp1) (18) . the sars-cov-2 genome contains 15 open reading frames (orfs), which encode 29 viral proteins (19) (20) (21) . orf1a and orf1ab encode polyproteins that are cleaved into 16 nonstructural proteins (nsp1 -nsp16) that comprise the replicase-transcriptase complex. spike (s) is encoded by orf2, envelope (e) by orf4, membrane (m) by orf5, and nucleocapsid (n) by orf9. an additional 9 orfs encode "accessory" proteins: orf3a, orf3b, orf6, orf7a, orf7b, orf8, orf9b, orf9c, and orf10. various studies have investigated the functions of the virally encoded proteins by performing interactome analysis in cells expressing individual viral proteins (19) or by evaluating the proteomic or transcriptomic changes associated with either viral infection (22) (23) (24) (25) (26) (27) . others have used computational approaches to investigate protein-protein interactions between sars-cov-2 viral proteins and host proteins (28) . the interactome and proteome studies identified cellular processes affected by sars-cov-2 infection or specific viral proteins, notably innate immune signaling (19, 20, 23, (28) (29) (30) , ubiquitin ligase activities (19, 20, 23, (28) (29) (30) , p38 mitogenactivated protein kinase (mapk) signaling (19, 20, 23, (28) (29) (30) . the transcriptomic studies identified interferon signaling (24) , cell death (27) , interleukin 1 (il-1), il-6, and chemokine signaling (22) . given the intense interest in catalytically active cov-2 proteins (31) (32) (33) (34) , we examined the lessstudied group of orfs encoding accessory proteins, which are largely thought to maintain viral 5 structural organization in replication organelles and within the viral particle (35, 36) . here, we showed that expression of only orf9c is sufficient to alter cellular networks in a manner that resembles full sars-cov-2 virus infection. seven of the 29 cov-2 proteins are orfs that lack catalytic activity and, in some cases, lack a known function (19) . each of these were tagged with strep at the n terminus and expressed them individually in the lung cancer epithelial cell line a549 in the presence or absence of the proteasome inhibitor mg132 (fig. 1a ). the protein encoded by orf9c was particularly unstable, with a profound increase in abundance evident in mg132-treated compared to that in vehicle-treated a549 cells (fig 1a) . orf9c is present in previously characterized strains of sars-cov (37), a conservation suggesting a function in coronavirus pathogenesis. phylogenetic analysis and alignment of the protein sequences showed that mutations are present in orf9c among different coronavirus strains with bat sars-like coronavirus orf14 as the closest ortholog sharing 94% sequence identity and only 77% identity with orf14 of sars-cov (fig. 1b, fig. s1a ). tmhmm analysis (38) of sars-cov-2 orf9c predicted a transmembrane sequence in the c-terminal domain, a motif not present in sars-cov-1 (or other human coronaviruses) orf9c sequence ( fig. 1b, fig. s1b ). additionally, a single nucleotide mutation in sars-cov-2 orf9c altered a termination codon, enabling the reading frame to extend by 3 amino acids (fig. 1c ). we assessed potential functions of orf9c by performing transcriptome, interactome, and proteome analysis of a549 lung cancer cells transfected with orf9c tagged at the n terminus with 2 copies of the strep tag (19) . to map the orf9c interactome, we conducted liquid chromatography tandem mass spectrometry (lc-ms/ms) of 2xstrep-tagged orf9c compared with control 2xstrep-tagged gfp) immunoprecipitated from a549 cells 24 h after transfection. orf9c interactome analysis revealed that most interacting proteins were classified as membrane proteins (fig. 1d , table s1) according to gene ontology cellular component. as a protein with a transmembrane domain, this was not surprising. however, we were surprised to find that the orf9c interactome was distributed throughout the membrane-bound organelles (fig. 1d ), including >30 proteins in the protein biosynthesis and transport systems of the endoplasmic reticulum (er) and golgi, >15 proteins in the mitochondria, and >30 other membrane-related proteins. given the instability of orf9c, we were not surprised to identify a group of membraneassociated proteins that function with the proteasome. comparison between orf9c and orf10, both expressed using the strep-tagged vector, confirmed that enrichment of membranal proteins as part of the interactome was selectively seen for the orf9c (fig. s1c). we conducted both label free quantification (lfq) and tandem mass tag (tmt) mass spectrometry analysis to identify changes in the cellular proteome in a549 cells expressing orf9c. we compared the proteomic changes associated with orf9c expression in the presence 7 or absence of proteasomal inhibition with mg132, using dmso as the vehicle control for comparison in each set. principle component analysis (pca) revealed that orf9c contributed to major variance in all data sets ( fig. s2a, s2b ). pairwise comparisons between orf9c and control untransfected samples identified differentially expressed proteins in both the dmso and mg132 groups (fig 2a) . in both the dmso and mg132 datasets, most changes induced by orf9c were a reduction in protein abundance (downregulation) ( fig. 2a, table s1 ). downregulated proteins identified using both approaches consistently showed ~60% overlap, while no overlap were identified among the upregulated proteins. thus, to maximize the discovery of orf9c dysregulated proteins, results from both technologies were combined. including the differentially regulated proteins identified by both the tmt and lfq analysis revealed 14 proteins were upregulated in common by orf9c expression in the presence or absence of the proteasome inhibitor and 144 proteins were downregulated in common (fig. 2b ). using the downregulated proteins and upregulated proteins identified for either the dmso or mg132 condition separately, we performed ingenuity pathway analysis (ipa) to assess signaling pathways deregulated in orf9c-expressing cells. in both the dmso and mg132 condition, interferon (ifn) signaling exhibited the greatest difference, both in terms of the intensity of the downregulation and the number of proteins significantly associated with this pathway, in response to orf9c (fig. 2c, table s1 ). other pathways affected by orf9c and of particular importance to virulence were antigen presentation and innate immune response pathways, such as irf/cytosolic pattern recognition receptors. we further examined potential upstream regulators of these pathways using ingenuity pathway analysis for proteins that exhibited a change in abundance in the orf9c-expressing cells. this analysis revealed that several components of the ifn machinery [interferons (ifnl1, ifna), interferon responsive 8 transcription factors (irf7, irf1), and an interferon receptor (infar)] were reduced, consistent with the impaired ifn signaling, and an increase in mapk1 (also known as erk2) abundance (fig. 2c, table s1 ). to assess if there were notable differences in the intensity of the changes in protein abundance in response to proteasome inhibition, we calculated relative changes in protein abundance between control and orf9c-expressing cells from both the dmso and mg132 conditions for proteins associated with ifn signaling or the ubiquitin proteasome (ubp) system and antigen presentation (fig. 2d ). the intensity of the changes was similar in the presence or absence of mg132, suggesting even small amounts of the unstable orf9c are sufficient to induce cellular changes including those that contribute to immune evasion. consistent with the ipa-based analysis (fig. 2b) , ifn signaling components, including ifi35, multiple ifit proteins, irf9, isg15, mx1, psmb8, and stat proteins, were downregulated in orf9c-expressing cells in both the presence and absence of mg132 (fig. 2d, left) . indicative of a decrease in antigen presentation capacity, multiple proteins involved in this process were decreased, including proteins involved in antigen loading and display [hla proteins, β2m, and antigen transporters (tap1 and tap2)] and proteins involved in ubp [ubiquitin-conjugating enzymes ube2i and ube2l6), deubiquitination enzymes (usp18 and ups41), and proteasome components (psmb and psme proteins)] (fig 2d, right) . these changes in the proteome indicated that the expression of only orf9c, even in the absence of proteasomal inhibition to stabilize this protein, is sufficient to elicit effective inhibition of ifn, immune recognition, and ubp components at the protein level. such a response suggested that orf9c contributes to immune evasion of sars-cov-2. we assessed transcriptional changes elicited by orf9c expression in a549 cells using rna-seq analysis. pca showed that changes induced by orf9c in both dmso-and mg132-treated cells cluster in distinct experimental groups (fig. s2c) . in contrast to the proteomic results that revealed predominant downregulation of proteins following orf9c expression, rna-seq analysis showed a similar number of transcripts were increased or decreased in the presence or absence of mg132 (fig. 3a, table s2 ). additionally, the number of differentially regulated transcripts was higher than that for the differentially regulated proteins. using ipa, we identified the pathways significantly enriched in differentially regulated transcripts. the same set of pathways were identified in the dmso and mg132 conditions, and similar to the proteomic results, most related to immune signaling (fig. 3b , upper, table s2). however, many of the specific pathways were different from those identified at proteomic level. at the transcriptional level, we detected the greatest effects on the complement system and several pathways involved in inflammatory signaling. thus, some components of antigen presentation and immune signaling pathways showed comparable changes at the protein and mrna levels; other changes elicited by orf9c expression were unique to the transcriptional level, such as induction of il-6 signaling and p38 mapk signaling, or the protein level, such as impairment of ifn signaling. we analyzed the orf9c-regulated transcripts for those encoding upstream regulators of the pathways altered at the transcriptional level by orf9c. this analysis identified the classic immune modulators tumor necrosis factor (tnf), il-1b, ifng, transforming growth factor β (tgfb), and nf-kb signaling components (fig. 3b , lower, table s2). we evaluated transcripts associated with the complement system or il-6 signaling in detail. in both the presence and absence of proteasome inhibition, complement system transcripts were mostly downregulated by orf9c expression in a549 cells (fig. 3c, left) . for il-6 signaling, we found some differences between the mg132 and dmso conditions (fig. 3c, right) . for several transcripts the intensity of the upregulation was greater in the absence of proteasome inhibition (map3k14 and map2k3, il6 and il6r, socs1 and socs3); for others the presence of the proteasome inhibitor resulted in a greater reduction in transcript abundance (il1b, tnfaip6, cd14, il1r2). thus, these results suggested that orf9c had a dose-dependent effect on some transcripts. we combined the results of the proteomic and transcriptomic (fig. 4a ), which revealed a small set of commonly upregulated or downregulated genes by orf9c at both the transcription and protein levels (fig. 4b ). we performed ipa canonical pathway analysis and found commonly altered pathways at both the transcript and protein levels (fig. 4c , table s1, s2). the direction of regulation (increased or decreased activity) was consistent between the transcripts and proteins. however, the number of components significantly enriched in most of the pathways differed between the protein and transcript levels. we compared our findings at the transcriptome, proteome, and interactome levels with those reported by stukalov et al. (39) for proteins with altered ubiquitination (ubiquitinome) in response to sars-cov-2 infection of a549 cells. within the top 10 enriched ipa canonical pathways, we noticed enrichment across all 5 protein ubiquitination data sets, sirtuin signaling, phagosome maturation, tight junction signaling, and caveolar-mediate endocytosis (fig. 4d , table s3). seven of the 10 were common between the ubiquitinome data (39) and our proteomic analyses by lfq or tmt mass spectrometry. we also compared the pathway enrichment across our transcriptomic data and those from blanco-melo et al. (22) reporting transcriptomic changes upon sars-cov-2 infection in human primary epithelial cells or ace-expressing a549 cells and with those from stukalov et al. (39) reporting transcriptomic changes in ace2-expressing a549 cells 12 and 24 hours after infection in the presence or absence of the proteasome inhibitor, we observed substantial overlap (55 -65%) in the orf9c-downregulated proteins ( fig. 2a) and 54 -72% overlap in orf9cupregulated transcripts and 45 -64% in the downregulated transcripts (fig. 3a) . thus, despite orf9c increasing in mg132-treated a459 cells, the persistent changes in mg132-treated cells suggested that even a low amount of orf9c is sufficient to elicit its cellular effects. however, some transcripts (399) and proteins (97) were downregulated by orf9c in cells not treated with mg132 and were upregulated in cells treated with mg132 ( fig. 5a ). pathway analysis on the transcripts and proteins showed discordant regulation in dmso-or mg132-treated cells in response orf9c. the most pronounced among all three datasets were components of the ubp and the unfolded protein response (upr) (fig. 5b, table s4 ). changes in events or pathways associated with the cell cycle was not surprising given the critical role ubp plays in their regulation. we thus hypothesized that both the ubp and upr were involved in degradation of orf9c. finding that upr signaling was also reversed upon mg132 treatment ( fig 5b) is consistent with the role of upr signaling in degradation of orf9c. to directly assess the importance of ubp and upr components in orf9c instability, we performed an sirna-based screen targeting over 1100 genes that encode components of both machineries in a549 cells stably expressing strep-tagged orf9c (fig. s3 ). the top hits independently validated as blocking orf9c degradation were sirnas targeting vcp [also known as p97, an atpase involved in export of unfolded proteins from the er for er-associated degradation (erad) and in er to golgi transport] (40, 41) , the proteasomal subunit psmd2, and the proteasome maturation factor pomp (42) , which has been also implicated in erad (43) 13 and in ifn-induced reorganization of proteasomes into immunoproteasomes (44) (fig. 5c , table 1 ). to assess if interfering with erad affected the cellular effects induced by orf9c, we compared the transcript abundance for 6 genes (ifngr1, igs15, irf9, socs1, psmb8, tap1) that were downregulated by expression of orf9c in dmso-treated cells with their abundance in cells treated with the vcp inhibitor mns-873, the heat shock protein 90 (hsp90) inhibitor geldanamycin, or the proteasome inhibitor bortezomib. although the hsp90 inhibitor and the proteasome inhibitor increased transcript abundance for some of the gens tested, vcp inhibition was the most consistently effective at enabling expression of each of these transcripts in the orf9c-expressing cells (fig. 5d ). these observations suggested that orf9c ability to attenuate key cellular signaling involved in antiviral responses, including antigen presentation, immune, and ifn pathways, requires the activity of vcp. the key to our ability to control spread of the sars-cov-2 virus is to understand its mechanism of action and how the concerted action of its 29 encoded proteins subvert cellular regulatory networks. one can divide viral "success" into two key phases: infection, which is the ability to enter a given cell type, and multiplication that enables continuous infection through viral replication and packaging, which exploits host cell machineries (45) . a third aspect to viral success is evasion from immune clearance. disruption of either the infection or replication phases should effectively inhibit the sars-cov-2 life cycle. accordingly, many efforts focus on neutralizing interaction of the viral s protein with ace2 (4, 46) . other efforts strive to interfere 14 with the viral life cycle after it invades target cells, and many focus on catalytically active proteins encoded by the sars-cov-2 genome (47). here, we analyzed one small, unstable sars-cov-2 protein, orf9c in the context of an epithelial lung cancer cell line. limited overlap between published studies of the orf9c interactome can be attributed to their use of different cell system (hek293 compared with a549 lung cancer cells used here, as the use of different filtering criteria (19) . however, many of the cellular changes elicited by orf9c in our study also occur following infection with full replicative sars-cov-2 virus (22) . those phenotypes included changes in ifn and other cytokine signaling, immune recognition (including antigen presentation; dendritic cell, t cell, and acute immune responses; and pattern recognition), cell cycle, and the complement system, all of which were downregulated by orf9c. additionally, similar to cells infected with the virus or expressing orf9c, il-1, il-6, and p38 mapk signaling pathways were upregulated. the primary change identified in our analysis was deregulation of the ifn system, coupled with changes in cytokines associated with tnf and stat signaling and factors implicated in innate immunity. in addition to mediating an antiviral response, aberrant ifn signaling is also critical for numerous pathological indications linked to covid-19 (48). thus, we concluded that sars-cov-2 orf9c elicits pathologies not seen with previously characterized coronavirus prototypes, primarily through effective modulation of ifn signaling. our findings suggested that orf9c enables cells to escape from immune surveillance through by reducing hla abundance and antigen presentation, while also slowing cell replication, which could viral replication of infected cells. strikingly, orf9c is predicted have a transmembrane domain and we found that the orf9c interactome was mostly comprised of membrane-associated proteins in multiple organelles, 15 including er, golgi, mitochondria, cell surface membrane, and peroxisomes. indeed, many of the cellular changes that we observed following orf9c expression are associated with membrane proteins or pathways mediated by proteins that associate with the membranes of various cellular compartments. importantly, sars-cov-2 orf9c is the first human coronavirus orf9c protein that has acquired this putative transmembrane sequence. mutations have been acquired along the course of evolution of orf9c, although ~80% of the sars-cov-2 orf9c sequence is identical to the ortholog in other coronaviruses, although greater similarity was identified with the bat sars-cov-2 sequence. the membranal anchoring capability identified in sars-cov-2 orf9c is novel feature that may mediate the effect on ifn signaling, antigen presentation, and immune evasion phenotypes, characteristics that make sars-cov-2 much more virulent and pathogenic than other coronaviruses. notably, 0.7-1.4% of patients were found to possess a mutation that is expected to impair transmembrane domain of sars-cov-2 orf9c (19) ; awaiting future assessment of clinical outcome, our data would predict a better clinical outcome, distinguishing these patients from those harboring the transmembrane domain. correspondingly, the interactome for the less virulence and pathogenic sars-cov-1 orf9c (49) did not overlap with that for sars-cov-2 orf9c. a notable signature that we identified is the upregulation of histone and histone deacetylaserelated factors, which suggested that histone modification may underlie the transcriptional repression. the increased transcription of ap1 family members (fosb, fosl1, creb, and atf3), which participate in the cellular stress response, may reflect the response to stress imposed by orf9c, which, in turn, can limit immune-related signaling identified in our study. another remarkable signature of orf9c expression in a459 cells was the association with ubp components. together with the observations on cellular immune pathways, this association with 16 the ubp suggested that orf9c induces changes in ubp components that alter the stability of cellular proteins implicated in cytokine signaling, antigen presentation, innate immunity, and the cell cycle. additionally, we identified upr components as important for orf9c instability, suggesting that this protein is misfolded or at least recognized as a misfolded protein by the host cell. in this scenario, we propose that misfolded orf9c engages upr (through vcp) and the ubp, which clears this protein. by engaging the ubp, orf9c promotes enhanced proteasome activity as suggested by our proteome analysis. our analysis revealed that interfering with vcp activity blunted the transcriptional repressive effects of sars-cov-2 orf9c on impact immune system components, such as irf9, infgr1, isg15, socs1 and tap1. proteasome inhibition with bortezomib was also effective, although not as consistently effective as mns-873, the vcp inhibitor. these findings suggested that inhibition of vcp or the proteasome, which has inhibitors currently in clinical trials for cancer (50, 51), may be considered among therapeutic measures to fight sars-cov-2 virulence and pathologies. however, we cannot exclude the possibility that orf9c stability and degradation mechanisms differ based on cell type or the activity of other viral proteins in infected cells. another potential therapeutic opportunity involves targeting the membrane association of orf9c, because this is a unique feature of the protein in the sars-cov-2 coronavirus. thus, identifying small molecules that could interfere with orf9c localization to the membrane could limit orf9c function and impede the ability of the virus to evade the immune response and reduce viral replication. given that orf9c is expected to affect immune evasion, virulence and pathogenesis, additional studies should assess the consequences of orf9c inhibition in vivo, using primates and possibly mouse models where sars-cov-2 shown to impact ifn signaling and immune response (48). secondary antibodies were used at 1:5000. immunoprecipitation of streptavidin-tagged cov-2 orfs was performed as previously described (19) . briefly, frozen cell pellets were thawed on ice for 15-20 minutes and suspended in 1ml lysis buffer with 50 mm tris-hcl, ph 7.4 at 4°c, 150 mm nacl, 1 mm edta and supplemented with 0.5% nonidet p-40 substitute, complete mini edta-free protease and phosstop phosphatase inhibitor cocktails (roche). samples were centrifuged 10 minutes at 4°c at 13,000g. protein quantification was performed using pierce's bca quantification kit as per the manufacturer's indications. supernatants (1 mg protein) were incubated 2h at 4°c with magstrep "type3" beads (30 μl; iba lifesciences) that had been previously equilibrated twice with 1 ml wash buffer (ip buffer supplemented with 0.05% np40). beads were washed five times with 1 ml wash buffer and then five times with 1 ml ammonium bicarbonate 50mm. raw fastq files were processed using cutadapt v1.18 (52) figure 4d , figure 5a and 5b were selected based on bh correct p <0.05 without fold change cutoff. differentially expressed proteins in figure 4d , figure 5a and 5b were selected based on p <0.01 without fold change cutoff. protein sequences similar to sars-cov-2 orf9c were retrieved through ncbi blastp using the nr database (58). sequence alignment was performed using clustalo (59). phylogenetic tree was built using phyml algorithm by 100 times bootstrap, and visualized using seaview (60) and geneious version 2020.2.2 (san diego, ca). transmembrane domain prediction was performed using tmhmm web server v2.0 (38) . transmembrane domain was predicted based on tmhmm posterior probability more than 0.5. statistical test results of rna-seq, proteomics and interactome data provided in supplementary tables. analyses of omics data in this study were performed using r customized scripts. statistical analysis of proteomics data sets was performed using msstats (label-free data) and mstatstmt (tmt data) bioconductor package. differential expression of rna-seq was performed using deseq2 bioconductor package following negative binomial distribution and wald test. pathway enrichment and upstream regulator analyses were performed using ipa following fisher's exact test and z-score calculation considering directional changes in ipa database. ana dominguez andres 1^, yongmei feng 1^, alexandre rosa campos 1^, jun yin 1^, chih-cheng beads were resuspended in 8m urea, 50 mm ammonium bicarbonate, and cysteine disulfide bonds were reduced with 10 mm tris (2-carboxyethyl) phosphine (tcep) at 30°c for 60 min. high confidence interacting proteins were selected using the following filtering criteria: log2fc > 3.3 (10x) and a p-value <0.025 (to include the p-value of proteins detected in at least 2 orf9c pulldown replicates but not detected in the negative controls). we also considered the 'crapomescore' < 0.5, which is the fraction of single affinity purification experiments a given protein-interacting candidate receives in the crapome database (crapome.org). a score of 1 means the candidate is identified in all experiments in that database. cells were lysed in uab buffer (8m urea, 50 mm ammonium bicarbonate (abc) and benzonase 24u/100ml) with vigorous shaking (20 hz for 10 min at room temperature using a retsch mm301 instrument). lysates were centrifuged at 14,000xg for 10 minutes to remove cellular debris, and protein concentration in supernatants was determined using bicinchoninic acid (bca) protein assay (thermo scientific fragmented precursors were detected in the ion trap as rapid scan mode with automatic gain control target set to 1 x 10 4 and a maximum injection time set at 35 ms. the dynamic exclusion was set to 20 seconds with a 10 ppm mass tolerance around the precursor. for processing label-free lc-ms/ms data, all raw files were processed with maxquant (version 1.5.5.1) using the integrated andromeda search engine against a target/decoy version of the curated human uniprot proteome without isoforms (downloaded in january of 2020) and the gpm crap sequences (commonly known protein contaminants). first search peptide tolerance was set to 20 ppm, and main search peptide tolerance was set to 4.5 ppm. fragment mass tolerance was set to 20 ppm. trypsin was set as the enzyme in specific mode, and up to two missed cleavages was allowed. carbamidomethylation of cysteine was specified as fixed modification and protein n-terminal acetylation and oxidation of methionine were considered variable modifications. in addition, the phosphopeptide-enriched samples were also searched with phosphorylation of serine, threonine or tyrosine considered as variable modification. the target-decoy-based false discovery rate (fdr) filter for spectrum and protein identification was set to 1%. statistical analysis of label-free proteomics data was carried out using in-house r script (version 3.5.1, 64-bit), including r bioconductor packages. first, peptide feature intensities (maxquant evidence table) were log2-transformed and normalized (loess normalization) across samples to account for systematic errors. then all non-razor peptide sequences were removed from the list. protein-level quantification and statistical testing for differential abundance were performed using msstats bioconductor package. cells were imaged with an ic200 high-content screening system (vala sciences) using a 20x objective to visualize strep-orf9c proteins (alexa 488) and nuclei (dapi). four images were obtained from different fields in each well for 384-well plates. images were analyzed with acapella high-content imaging and analysis software for valid cell numbers per field and to determine average alexa 488 intensity per cell. a549 cells expressing sars-cov-2 strep-orf9c were treated with dmso or mg132 (10um) served as negative and positive imaging controls, respectively. plate-to-plate variability was normalized using a control-based method; associated control samples were aggregated, and the mean and variance across wells were determined. the alexa488 mean intensity for all wells with sirna knockdown was normalized using unique non-targeting sirnas included in each plate as reference data points. the top 36 scoring hits were obtained using a threshold of > 1.46-fold increase in average intensity from duplicates (p-value <0.05). ten of the 36 sirna pools were selected for confirmation in a secondary deconvolution screen. for that screen, quantification data were converted to a z-score, and the average z-score from data in triplicate plates was determined. genes were defined as confirmed screen hits if they had 3 or more individual positive sirna score (cut-off of >3 sd). table s1 . interactome and proteome data analysis, pathway enrichment and upstream regulators analyses table s2 . transcriptome data analysis, pathway enrichment and upstream regulators analyses table s3 . canonical pathway comparison of different omics technologies and public data sets table s4 . canonical pathway analysis of mg132 reversed genes and proteins a novel coronavirus outbreak of global health concern a new coronavirus associated with human respiratory disease in china a novel coronavirus from patients with pneumonia in china sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor cell entry mechanisms of sars-cov-2 structural and functional basis of sars-cov-2 entry by using human ace2 a pneumonia outbreak associated with a new coronavirus of probable bat origin single-cell rna expression profiling of ace2, the receptor of sars-cov-2 sars-cov-2: a comprehensive review from pathogenicity of the virus to clinical consequences genome composition and divergence of the novel coronavirus (2019-ncov) originating in china systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov characteristics of registered studies for coronavirus disease 2019 (covid-19): a systematic review an alphavirus-derived replicon rna vaccine induces sars-cov-2 neutralizing antibody and t cell responses in mice and nonhuman primates structure, function, and antigenicity 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pathogenic pathways activated by the virus, proposes unique sex-specific differences and predicts tailored therapeutic strategies a dynamic immune response shapes covid-19 progression transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients interactome of sars-cov-2 / ncov19 modulated host proteins with computationally predicted ppis the global phosphorylation landscape of sars-cov-2 infection covid-19: viral-host interactome analyzed by network based-approach model to study pathogenesis of sars-cov-2 infection drug design targeting the main protease, the achilles' heel of coronaviruses crystal structure of sars-cov-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors structure of the rna-dependent rna polymerase from covid-19 virus structural and evolutionary analysis indicate that the sars-cov-2 mpro is a challenging target for small-molecule inhibitor design characterization of accessory genes in coronavirus genomes. biorxiv sars coronavirus accessory proteins insights into sars-cov-2 genome, structure, evolution, pathogenesis and therapies: structural genomics approach predicting transmembrane protein topology with a hidden markov model: application to complete genomes multi-level proteomics reveals host-perturbation strategies of sars-cov-2 and sars-cov. biorxiv ubiquilin and p97/vcp bind erasin, forming a complex involved in erad vcp/p97-mediated unfolding as a principle in protein homeostasis and signaling the proteasome maturation protein pomp facilitates major steps of 20s proteasome formation at the endoplasmic reticulum sirna silencing of proteasome maturation protein (pomp) activates the unfolded protein response and constitutes a model for klick genodermatosis ifn-gamma-induced immune adaptation of the proteasome system is an accelerated and transient response covid-19 infection: origin, transmission, and characteristics of human coronaviruses sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues competing interests: zar is a co-founder and serves as scientific advisor to pangea therapeutics. all other authors declare no competing interests.data and materials availability: all datasets will be deposited in publicly available data sets prior to publication; all reagents and study protocols are available by requests from the corresponding authors. key: cord-267887-ntwvquqz authors: yang, ren; huang, baoying; ruhan, a.; li, wenhui; wang, wenling; deng, yao; tan, wenjie title: development and effectiveness of pseudotyped sars-cov-2 system as determined by neutralizing efficiency and entry inhibition test in vitro date: 2020-08-21 journal: biosaf health doi: 10.1016/j.bsheal.2020.08.004 sha: doc_id: 267887 cord_uid: ntwvquqz with the development of the covid-19 epidemic, there is an urgent need to establish a system for determining the effectiveness and neutralizing activity of vaccine candidates in biosafety level 2 (bsl-2) facilities. previously, researchers had developed a pseudotyped virus system for sars-cov and mers-cov, based on hiv-1 core, bearing virus spike protein. during the development of a pseudotyped sars-cov-2 system, a eukaryotic expression plasmid expressing sars-cov-2 spike (s) protein was constructed and then co-transfected with hiv-1 based plasmid which containing the firefly luciferase reporter gene, into hek293t cells to prepare the pseudotyped sars-cov-2 virus (ppsars-2). we have successfully established the pseudotyped sars-cov-2 system for neutralization and entry inhibition assays. huh7.5 cell line was found to be the most susceptible to our pseudotyped virus model. different levels of neutralizing antibodies were detected in convalescent serum samples of covid-19 patients using ppsars-2. the recombinant, soluble, angiotensin-converting enzyme 2 protein was found to inhibit the entry of ppsars-2 in huh7.5 cells effectively. furthermore, the neutralization results for ppsars-2 were consistent with those of live sars-cov-2 and determined using the serum samples from convalescent patients. in conclusion, we have developed an easily accessible and reliable tool for studying the neutralizing efficiency of antibodies against sars-cov-2 and the entry process of the virus in a bsl-2 laboratory. the causative agent of the unprecedented global pandemic of coronavirus disease 2019 (covid-19) is a novel beta-coronavirus [1, 2, 3] , named as sars-cov-2 (also called as covid-19 virus in china) [4] . the control of the covid-19 pandemic is a great challenge because sars-cov-2 is a highly contagious virus, and covid-19 has diverse clinical manifestations (such as asymptomatic infection, common cold, and pneumonia) [5] . therefore, there is an urgent need to develop vaccines or therapeutics against covid-19. however, currently, sars-cov-2 live virus-associated experiments can only be conducted in a biosafety level 3 (bsl3) facility, which limits the development of sars-cov-2 vaccines and drugs to several scientific teams and local departments of disease control and prevention. hence, a reliable, rapid, and convenient neutralization assay, that can be handled in a bsl-2 laboratory is essential for screening and evaluation of antibodies and therapeutic agents against the sars-cov-2 infection. previous studies have suggested that the glycosylated spike (s) protein is the major surface protein responsible for receptor binding and entry of the virus into the host cell [6, 7, 8] and that both sars-cov-2 and sars-cov have the same main receptor, which is angiotensin-converting enzyme 2 (ace2) [9, 10] . pseudotyped antibodies at 37°c for 2 h. mouse anti-sars-cov-2 s pab and convalescent serum from covid-19 patients, diluted at 1:200 and 1:50, respectively, were used as primary antibodies. after the incubation period, the cells were washed with pbst 10 times and were incubated with goat anti-mouse igg fitc or anti-human igg fitc in 0.5% evans blue pbs at rt for 1 h. the cells were then washed with pbst, and mounted on cover slips using mounting buffer. fluorescence was observed using olympus ix50fl microscope and the fluorescent images were captured using the dp70 digital camera system. sars-cov-2 pseudotyped virus and mock virus were ultracentrifuged at 24,000 × g using 1 ml of 20% sucrose as a cushion. then, the medium was discarded, and the precipitated pseudoviruses were suspended in 200 μl of sds-page loading buffer. the samples were heated for 10 min at 95°c and then subjected to sds-page for immunoblotting, as previously described [16] . sars-cov-2 s protein was j o u r n a l p r e -p r o o f journal pre-proof identified using the mouse anti-sars-cov-2 s pab (sino biological, china), while hiv p24, used as reference, was identified using the rabbit anti-p24 polyclonal ab (sino biological). vero, vero e6, huh7, huh7.5, and bhk21 cells were seeded into 96-well plates one day before infection. when infected, the cells were approximately 60-80% confluent. following infection, the culture medium was removed, and 50 μl of serum-free dmem medium was added. the cells were stabilized for 30 min, following which 50 μl of pseudotype virus culture suspension was added to the cells. the culture medium was replaced after 12-16 h with fresh dmem supplemented with 2% fbs, and the culture was continued for 24 h. bright-glo luciferase assay system (promega co., us) was used to detect fluc expression in the cells, and the titer of pseudotype virus in different cell lines was obtained. thus, sensitive cell lines were identified. the sensitive cells were grown to 60-80% confluency for the experiment. the serum can be determined using the pseudotyped sars-cov-2 virus. various levels of neutralizing antibodies were detected in the serum samples from covid-19 convalescent patients, which exhibited 50% neutralization (nd 50 ) against ppsars-2 from less than 1:50 dilution to more than 1:2048 dilution. since hace2 has been reported as the sars-cov-2 entry receptor, we have utilized soluble hace2 as a binding competitor to inhibit the entry of pseudotyped sars-cov-2 [9] . ppsars-cov-2 entry was blocked using soluble ace2, as indicated by the significant reduction in reporter gene expression level ( figure 3b ). the ic50 of soluble hace2 was identified as 0.032 μg/ml to verify the true reliability of the system, serum samples of the sars-cov-2 convalescent patients were analyzed by microneutralization using live sars-cov-2 virus ( figure 4a) . results revealed the similar neutralizing potency between the pseudotyped-sars-cov-2 system and live-sars-cov-2. we then compared the results of the analysis conducted using the ppnt and the live virus ( figure 4b ) and found that the results from the ppnt were strongly correlated with those obtained using live virus (r 2 = 0.6931 and p < 0.005). sars-cov-2 s vsv-g without env a novel coronavirus genome identified in a cluster of pneumonia cases-wuhan genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a novel coronavirus from patients with pneumonia in china coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 receptor and viral determinants of sars-coronavirus adaptation to human ace2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b beta-coronaviruses production, concentration and titration of hiv-1-based lentiviral vectors pseudotyping viral vectors with emerging virus envelope proteins development and optimization of a sensitive pseudovirus-based assay for hiv-1 neutralizing antibodies detection using a3r5 cells receptor and viral determinants of sars-coronavirus adaptation to human ace2 potent human neutralizing antibodies elicited by sars-cov-2 infection a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 growth and quantification of mers-cov infection infection of bat and human intestinal organoids by sars-cov-2 highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig establishment and validation of a pseudovirus neutralization assay for sars-cov-2 key: cord-030934-t7akdu6x authors: bahrami, afsane; ferns, gordon a title: genetic and pathogenic characterization of sars-cov-2: a review date: 2020-08-26 journal: nan doi: 10.2217/fvl-2020-0129 sha: doc_id: 30934 cord_uid: t7akdu6x the first case of coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was reported in december 2019. this virus belongs to the beta-coronavirus group that contains a single stranded rna with a nucleoprotein within a capsid. sars-cov-2 shares 80% nucleotide identity to sars-cov. the virus is disseminated by its binding to the ace2 receptors on bronchial epithelial cells. the diagnosis of covid-19 is based on a laboratory-based reverse transcription polymerase chain reaction (rt-pcr) test together with chest computed tomography imaging. to date, no antiviral therapy has been approved, and many aspects of the covid-19 are unknown. in this review, we will focus on the recent information on genetics and pathogenesis of covid-19 as well as its clinical presentation and potential treatments. mers-cov s protein s1 s1 sars-cov s protein 55´3 a orf1a mers-cov ( constitute the viral envelope [36] . accessory proteins appear to promote the adaptation of covs to human host cells [37] . genomic analysis of ten genome of sars-cov-2 isolated from nine patients demonstrated 99.98% nucleotide identity [38] . another report found that 99.8-99.9% sequence similarity in sample of five infected patients [39] . phylogenetic analysis demonstrates that sars-cov-2 shares 50 and 80.0% nucleotide identity to mers-cov and sars-cov, respectively [8, 13, 38] . the sars-cov-2 constitutes a clade among the sub-genus sarbecovirus [40] . bioinformatics analysis of the viral genome from one covid-19 patient shared 89 and 82% sequence similarity with bat sars-like-covzxc21 and human sars-cov, respectively [41] . however, the external subunit of spike rbd of sars-cov-2 has only 40% amino acid (aa) identity with other sars-associated covs [41] . the s-protein of sars-cov-2 is longer (1282 aa) than for other viruses such as sars-cov (1255 aa) and bat sars-like covs (1246 aa). the s-glycoprotein of sars-cov-2 has been found to have three short insertions at the n-terminal end, with four variations in the receptor binding site within the rbd compared with sars-cov [42] . notably, sars-cov-2 orf3b codifies a new short protein. moreover, its novel orf8 sequence possibly encode a secreted protein with an α-helical structure with a β-sheet(s) consisting of six strands [41] . the high levels of genetic identity (96.3%) between the sars-cov-2 and bat-cov ratg13 does not indicate the precise variant that may have led to the outbreak in humans, although it has been suggested that the likelihood that the novel cov has derived from bats is very probable [43] . sars-cov-2 and ratg13 differ with respect to the number of major genomic properties, in which sars-cov-2 harbors a polybasic (furin) cleavage site insertion at the connection of the two subunits of the s-protein, s1 and s2 [44] . the n-protein is hidden within phospholipid bilayers and coated via two distinctive forms of s-proteins including the spike glycoprotein trimmer which is present in all covs, as well as the hemagglutinin-esterase (he) solely found in certain covs. for instance, sars-cov-2 does not appear to have the he gene. the m and e proteins are found inside the s-glycoproteins within the viral envelope [45] . the s, e, m, n and orf3a genes of sars-cov-2 are predicted to be 3822, 228, 669, 1260 and 828 nucleotides in length, respectively. moreover, sars-cov-2 has been predicted to contain an orf8 gene, of 366 nucleotide size, situated between the m and n corresponding orf genes [45] . the sars-cov sequence reveals serine substituting for glycine in the residue at position 543 of the nsp3 protein in bat sars-like and sars-cov. this aa substitution could promote local stiffness of the polypeptide chain for steric impact and potency of the serine side-chain to constitute h-bonds. beside, serine is a nucleophile that can establish structural environments, like those at active sites of enzyme. mutations in the nsp3 protein were reported to affect the replication of sars-cov-2 in infected cells [46, 47] . it has been reported that the single n501t variant in sars-cov-2's s-protein may enhance binding affinity for the ace2 cellular receptor [28] . furthermore, a single n439r mutation in sars-cov-2 rbd promotes its ace2-receptor binding and, thus potentially enhances human-to-human transmission [48, 49] . by studying the crystal structure of sars-cov-2 rbd binding to the human ace2 receptor has shown that the ace2 receptor-binding ridge in sars-cov-2 rbd results in a more compact conformation, leading structural alterations at the rbd/ace2 interface versus the sars-cov [48] . overall, sars-cov-2 binding affinity for ace2 is 10-20-times greater than for other sars-associated covs [50] . a missense mutation at the 614 position of s protein (aspartate to glycine, d614g mutation), in the spike protein of sars-cov-2, which has emerged as a predominant clade in europe (66% sequences) and is spreading worldwide (44% sequences). the d614g mutation promotes viral infectivity and transduction of multiple human cell types and mitigates neutralization sensitivity to individual convalescent sera [51] [52] [53] [54] . lipids play important roles at different stages in the covs life cycle. covs recruit intracellular membranes of the host cells to produce new compartments, or double membrane vesicles, which are used for the replication of the virion particle genome [55] . recently, an important lipid processing enzyme, known as cpla2 α has been reported to be related to the formation of double membrane vesicle and cov's amplification [56] . it has been demonstrated that the enzyme, phospholipase a2 group iid, is involved in anti-inflammation or proresolving lipid mediator regulation which may lead to worse outcomes in a sars-cov infection animal model by modulating the immune response [57] . it has been shown that there is a distinct insert that includes basic aas in the s1/s2 priming loop of sars-cov-2, which is not found in sars-cov or any sars-associated covs. it may substantially alter the entry pathway of sars-cov-2 compared with other viruses of the β-covs lineage b [58] . in a recent report it was shown that sars-cov-2's s-protein entry into 293/human ace2 receptor cells is primarily mediated via endocytosis, and that pikfyve, a tpc2 and cathepsin l are crucial for virus entry. pikfyve is the key enzyme in the early endosome involved in the synthesis of pi(3,5)p2 and its main downstream effector, tpc2. the s protein of sars-cov-2 could also stimulate syncytia in 293/human ace2 cells independently of exogenous protease [59] . in a study of 452 sars-cov-2 infected patients, it was found that severely affected cases had lower numbers of blood lymphocytes, percentages of monocytes, basophils and eosinophils as well as increased leukocytes numbers and neutrophil-lymphocyte-ratio. in most patients with unfavorable progression of covid-19, elevated concentrations of infection-associated markers and inflammatory cytokines was observed. the frequency of t cells was significantly lower, and less effective in severely affected subjects. both t helper (th) cells and suppressor t cell numbers in patients with covid-19 were below the reference range. the percentage of naive helper t cells was increased, and memory helper t cells and regulatory t cells reduced in severe conditions [60] . furthermore, simultaneous to the infection with sars-cov-2, cd4 + t lymphocytes are quickly over-activated to switch to the pathogenic th1 cells producing gm-csf. the cytokines environment activates inflammatory cd14 + cd16 + monocytes, leading to over-expression of il-6 and enhances the inflammatory response. regarding the increased infiltrations of inflammatory cells that have been found in lungs of severe sars-cov-2 infected patients [61, 62] , these population of abnormal and noneffective pathogenic th1 cells and inflammatory granulocytes may go to the pulmonary circulation and by immune stimulation, lead to functional impairment of the lungs and eventually death [63] . inflammasomes are very large intracellular poly-protein signaling complexes which are constitute in the cytosol as an inflammatory immune reaction to endogenous danger stimuli [64] . nlrp3 responds to wide spectra of pathogens and endogenous signals, and is involved in the molecular pathway of various auto-inflammatory disorders [65] . it has been reported that the sars-cov can induce the nlrp3 inflammasome in macrophages through orf8b. whereas sars-cov infects macrophages or monocytes, sufficient orf8b may be present to impact on the autophagy-lysosome pathway, and nlrp3 inflammasomes. sars-cov replicates efficiently in lung epithelial cells. these cells also amplify nlrp3 and support assembly of nlrp3 inflammasomes. in sars-cov patients, the full effect of the orf-8b on these inflammatory cascades was observed in the lung epithelium. interestingly, orf8b may be involved in the 'cytokine storm' or 'cytokine cascade' and inflammasome induction which happens within intensive sars-cov infection [66] . sars-cov-2 infection stimulates the immune response in two stages. in the early stages, a particular adaptive immune response is necessary to eradicate the virus and to impede progress to a more severe condition. the protective immune response at this phase requires that the host should have excellent general health and a suitable genetic context which provides antiviral immunity [67] . although, when the immune response protection is disabling, virus will disseminate and great damage to the affected tissues occurs, particularly in organs with a high levels of ace2 receptor expression. the injured cells activate innate inflammation within the lungs which is mainly mediated through pro-inflammatory macrophages/monocytes. lung inflammation is the major reason for the fatal respiratory disease at the severe stage of covid-19 [68] . in viral infections, host antiviral micrornas participate in the regulation of immune response to virus and are capable of targeting viral genes and interfere with replication, mrna expression and protein translation of virion particle gene. sardar et al. predicted the antiviral host-micrornas specifically for covid-19. they reported a list of six micrornas related to covid-19 including hsa-let-7a, hsa-mir101, hsa-mir126, hsa-mir23b, hsa-mir378 and hsa-mir98 which has been previously reported to be related to other viral infections, such as hiv [69] . virion particles spread from the respiratory mucosa, by binding to the ace2 receptors on ciliated bronchial epithelial cells, and after that may engage with other cells [70] . in one report from wuhan, the average incubation period of 425 sars-cov-2 infected patients was 5.2 days, but it this differed between individuals [71, 72] . until now, most patients with covid-19 have initially presented with mild manifestations in other words dry cough, sore throat and fever which spontaneously resolve. although, some patients have developed other more severe disease such as organ failure, septic shock, pulmonary edema, dyspnea, myalgia, fatigue and acute respiratory distress syndrome [73] . in contrast to sars-cov, patients infected with sars-cov-2, development of upper respiratory tract signs and manifestations are less common, suggesting that sars-cov-2 may target cells in the lower airway [74] . among cases with severe dyspnea, more than 50% have required intensive care. some covid-19 cases do not present with fever or radiologic abnormalities on admission, which makes initial diagnosis difficult [75] . the main characteristics of covid-19 on preliminary ct examination including bilateral multi-lobar groundglass opacities with a peripheral/posterior distribution and patchy consolidation, primarily in the lower lobes and fewer inside the right middle lobe [76] . the main reported laboratory test abnormalities in cases with severe covid-19 infection include: increased levels of liver enzymes (ldh, alt and ast), total bilirubin, creatinine, cardiac troponin, d-dimer, prothrombin time, procalcitonin and crp [77] . the histology of liver specimens of sars-cov infected patients have revealed a remarkable liver injury with an increase in mitotic cells, along with eosinophilic bodies as well as balloon-like hepatocytes [78] . cardiac involvement is another prominent manifestation of covid-19 and is closely related to a poor outcome [79] . in a recent systematic review, the incidence rate of diarrhea varied from 2 to 50% in covid-19 patients. it may develop earlier, or following the respiratory symptoms. findings of several studies showed that viral rna shedding is detect for a longer time period compared with nasopharyngeal swabs [50] . in an investigation on 1099 covid-19 patients, of whom 23.7% had severe disease with comorbidities of hypertension, 16.2% diabetes mellitus, 5.8% coronary heart diseases and 2.3% cerebrovascular disease [80] . another study, of 140 patients with covid-19, found that 30% and 12% had history of hypertension and diabetes, respectively [81] . analysis of 487 covid-19 cases, showed that older age (odds ratio [or] = 1.06; 95% ci: 1.03-1.1), male gender (or 3.7; 95% ci: 1.7-7.7) and hypertension as a comorbidity (or 2.7; 95% ci: 1.3-5.6) are related with more severe disease on admission [82] . moreover, patients with cancer were more vulnerable to severe events from covid-19 such as admission to the intensive care unit needing invasive ventilation, or death [83] . it has been reported that the highest viral load in throat swabs occurs at the time of development of symptoms. however, viral shedding was reported to occur before the onset of symptoms, and a major proportion of transmissibility happened before first symptoms in the index case [84] . furthermore, severe covid-19 cases tend to have an increased viral load and a long virus-shedding time [85] . at present, the diagnosis of covid-19 is largely based on guideline agreement that includes laboratory tests and chest ct imaging technique [75, 86] . pcr testing of asymptomatic or mild symptomatic contacts can be used in the evaluation of peoples who have been in contact with a covid-19 case [87] , and the who has not accepted the results of a chest ct without rt-pcr conformation in the diagnosis of covid-19 [88] . chest ct is a routine imaging tool for the diagnosis of pneumonia, which is relatively easy and rapid to perform. chest ct shows typical radiographic characteristics in almost all covid-19 cases, such as peripheral/posterior distribution and patchy consolidation, and/or interstitial alterations with a peripheral distribution, so may provide benefit for diagnosis of covid-19 [89] . respiratory tract samples were collected for the diagnosis and screening of patients with sars-cov-2 pneumonia; in the 5-6 days of the initiation of symptoms, patients with covid-19 have increased viral loads in their upper and lower respiratory tracts [90, 91] . for suspected cases, real-time fluorescence (rt-pcr) was performed to detect the positive nucleic acid of sars-cov-2 in sputum, throat swabs and secretions of the lower respiratory tract specimens [92] . a nasopharyngeal and/or an oropharyngeal swab are frequently recommended for screening or diagnosis of early infection [10, 93] . a single nasopharyngeal swab has become the preferable swab as it is welltolerated by the patient and safer for the operator. serological testing detects presence of igg, igm or both. a positive elucidation has been defined as a positive lgm, or convalescent sera with a higher lgg titer >four-times in comparison with the acute phase. sars-cov-2 igg and igm are detected in whole blood, plasma, serum or specimens. antibodies increase late in the course of illness; the mean duration of sars-cov-2 igm antibody detection was reported to be 5 days, whereas igg detection about 2 week following the appearance of symptoms [94] . in contrast to respiratory samples which may disturb from false-negative results because of the sampling factors, the presence of antibodies in blood uniformly is detectable. specimens are easier to gather versus respiratory samples, such as fewer risks to the operator. the serological assay is very easy, rapid, availability of elisa platforms, requires no instrumentation and can provide results in just 15 min [95] . based on the recommendation of who, covid-19 management protocols have mostly highlighted infection prevention, patient early detection and monitoring, and best supportive care [96, 97] . no specific antiviral treatment is currently recommended for covid-19 due to lack of evidence. many treatment regimens have been assessed for covid-19, some showing promising preliminary results. a total of 2531 trials on covid-19 have been registered to date in the clinicaltrial.gov (updated 10 july 2020). several pharmacotherapeutic agents have been used including lopinavir/ritonavir, hydroxychloroquine and ifn-β-1a (table 1) . results from several in vitro and clinical studies demonstrated that chloroquine phosphate, an old agent for the treatment of malaria, had significant efficacy and acceptable safety for treatment of covid-19 [98, 99] . findings of an open-label nonrandomized clinical trial among 22 infected patients indicated that hydroxychloroquine treatment significantly reduced viral load in covid-19 cases and its effectiveness is promoted by azithromycin [99] . in a systematic review including six published articles highlighting the potency of chloroquine in attenuation the replication of sars-cov-2-associated virus [100] . but several other studies demonstrated no evidence of a strong antiviral function, or clinical benefit of the hydroxychloroquine for the treatment of patients with severe covid-19 [101, 102] . the combination of lopinavir/ritonavir (lpv-r) is extensively used for treating hiv-infected patients. lpv-r has been suggested for treatment of covid-19. a total of 199 covid-19 patients were randomly assigned to receive lpv-r (n = 99) or standard-care (n = 100). treatment with lpv-r was not different from standard care regarding the time to clinical improvement, mortality rate at 28 days, as well as detection of viral rna at different time points [103] . arbidol as a wide-spectrum antiviral compound that can inhibit viral fusion of influenza. in one study, 50 patients with laboratory-confirmed sars-cov2 were randomly divided into two arms: 34 cases received lpv-r (400 mg/100 mg, two per day) and 16 patients were administrated arbidol (0.2 g a; three per day). no difference was observed concerning fever duration between the two arms. 2 weeks after the intervention, no viral load was found in cases received arbidol, while the viral load was detectable in 44.1% of lpv-r group patients. moreover, no adverse side effects were reported in either arm [104] . nelfinavir (nfv) is a potent hiv-1 protease inhibitor that received us fda approval in 1997 for treatment of hiv infection. the antiviral activity of nfv against sars-cov-2 was reported in vero e6 cells [105] . by using an integrative computational drug-discovery method, nfv was introduced as a potential inhibitor of sars-cov-2 main protease [106] . the main protease of covs (mpro) is an important protein necessary for the proteolytic maturation of the virion particle [107] . therefore, targeting mpro is considered to havepotential as a treatment for covid-19 through suppression of the polypeptide cleavage virus [108] [109] [110] . concerning the results of molecular docking, natural polyphenols such as hesperidin, rutin, diosmin, apiin and diacetyl-curcumin have been reported to have acceptable efficacy to target sars-cov-2 mpro than nfv [111] . cytokine-directed antagonists, in other words adalimumab (tnf-α) and cmab806 (il-6) against sars-cov-2 have been evaluated in clinical trials. the variety of cytokines such as type-i ifn-i contribute to the 'cytokine storm' and pathology of sars-cov-2. therefore, targeting the upstream origin of cytokine generation could be a promising therapeutic approach [112] . utilizing an in silico model, it has been shown that antipolymerase agents including sofosbuvir, idx-184, ribavirin (rbv) and remidisvir (gs-5734; rdv) can target rna-dependent rna polymerase of sars-cov-2 [113] . the first severe-infected patient with sars-cov-2 in the usa was cured by reception of intravenous rdv [114] . due to adverse side effects, the appropriate dose of rbv in clinical setting should be given with caution. in previous experience, for example in pandemic influenza a (h1n1), and avian influenza a (h5n1), passive immunization has been successful for treating of infectious complications [115] . a remarkable reduction in viral load and mortality was observed by using convalescent plasma therapy against severe acute viral respiratory infections, such as those created by covs [116] . patients who have recovered from sars-covs infection often have high titers of neutralizing antibody and may be a precious source of convalescent plasma. the fda has also approved the administration of plasma from recovered individuals for treatment of severe covid-19 patients [117] . monoclonal antibodies sars-cov-specific human monoclonal antibody (mab) can bind potently with sars-cov-2 region. but, some of the most powerful sars-cov-particular neutralizing antibodies (i.e., m396) that target the ace2 binding site of sars-cov did not bind to sars-cov-2 s-protein, indicating that the disparity in the rbd of sars-cov and sars-cov-2 has an important effect impact on the cross-reactivity of these mabs, and so novel mabs that specifically target sars-cov-2 rbd need to be designed [112] . effective sars-cov-2 vaccines are urgently needed in order to reduce infection severity, viral shedding as well as human-human transmission, so assisting the control of the cov outbreaks. because s-protein and associated fragments, in other words rbd of sars-and mers-covs are the main targets for designing vaccines, it is speculated that homologous regions of sars-cov-2 can also be applied as prime targets for designing vaccines against this novel covs [118] . in addition, other conserved regions of sars-cov-2 including two subunits of the s-protein, m-protein as well as n-protein, can be applied as another potential target for design and development of effective vaccines. antiviral vaccines can be categorized into two broad groups: dna-and rna-based vaccines, in which individuals are injected with genetically engineered plasmid containing the dna molecule encoding the antigen against which an immune response is eligible, thus the cells machinery creates the antigen, leading to immunological response; and peptide-or protein-based vaccines that include whole-inactivated virus, individual viral proteins or subdomains, and purified or recombinant proteinaceous antigens proteins from the virus, all of which are manufactured in vitro. the candidate vaccines that have recently entered clinical development include: mrna-1273, ad5-ncov, ino-4800 and lv-smenp-dc and pathogen-specific aapc ( table 2) . several platforms have progressed to development with potential for rapid development, including dna-and rna-based platforms, followed by those for developing recombinant-subunit vaccines. rna and dna vaccines can be made quickly because they do not require culture or fermentation, instead using synthetic processes [119, 120] . even with such promising platforms, sars-cov-2 vaccine development faces serious challenges. although the virus's s glycoprotein is a promising immunogen for protection, optimization of antigen design is crucial to obtaining an optimum host immune system response. another concern is the possible exacerbation of lung disease, either directly or because of antibody-dependent enhancement due to the type 2 helper t-cell response. furthermore, as with naturally acquired infection, the optimal duration of immunity is unknown; similarly, whether single-dose vaccines will confer lengthy immunity is doubtful. in the early phases of the epidemic, early detection assists management of the disease and preventive approaches such as masks, hand hygiene compliances, prevention of public contact, voluntary home quarantine, early diagnosis, contact tracing, intelligence social distance and travel restrictions have been recommended to decrease transmission. other approaches include limiting events that may facilitate superspreader potential including religious services (marriages and funerals) [121] . many dimensions of the sars-cov-2 and corresponding disease are unknown. for instance, the role of ace2 receptors in sars-cov-2 pathogenesis remains uncertain. future studies should be concentrate on profound understating of replication, pathogenesis and biological properties applying the relevant biological methods in other words reverse genetics and molecular techniques. genome wide association studies may provide an opportunity for the identification of potential genetic factors contributed in the development of covid-19. although host genetic studies are expensive and complex, more studies are required to determine the role of host genetics (such as variation in hla genes) in the immune response to covs, and the clinical outcome of covs-mediated disease. understanding of the sar-cov-2 viral genetics during the time and geography specially review regarding to the number and repetition of viral mutations and recombination rates and their association with viral infectivity, transmissibility, severity of disease and clinical manifestation, viral load and disease outcome are important knowledge gaps that navigate our research timetable. until now, no unique antiviral therapy has been approved; so treatment is mainly based on symptomatic therapy and best supportive care. the zoonotic link of sars-cov-2 infection has not been definitively proven; although, phylogenetic analysis shows that sars-cov-2 is very similar to sars-like bat covs. lessons from other human outbreaks from pathogenic viruses such as sars-cov, mers-cov and influenza viruses are very informative and valuable. different wide-spectra antivirals agents previously used for treatment of influenza, sars-and mers-covs are under assessment for repurposing either monotherapy or in combinations to treat covid-19 cases. sars-cov-2 is a novel human pathogen, and may interact with host antiviral defense via a specific pathway. altogether, the infection and development of sars-cov-2 relies on the interplay between the virus and the patient's immune response. investigations of the area of sars-cov-2-host interplay provide response to many crucial questions in virus pathogenesis, disease control and prevention. at present, covid-19 is leading to substantial global concerns. development of valid, accurate and appropriate serological tests is urgently needed. it will be essential to quickly design and develop effective therapeutic regimen and vaccines to prevent or stop infection of this novel covs. the covid-19 has caused more infections and deaths compared with either sars or mers. according to r0 values, it is deemed that sars-cov-2 is more infectious than sars or mers. as imposition of globalization, covs will cause spreads and outbreaks with various mutant strains similarly in the coming years. with promotion of scientific collaboration, which is as a consequence of globalization, we may have more powerful means of combating covs, in which we characterize the genome structure and pathogenesis of sars-cov-2 infection very well in the near future. a present treatment is mainly supportive, but trials of vaccines and antivirals are in progress. differences in the length of the spike as it is longer in sars-cov-2 are likely to play a major role in the pathogenesis and treatment of this virus. robust coordination and collaboration between researchers, vaccine developers, international regulators, policymakers, financiers, national public health institutes and governments will be required to ensure that potential late-stage vaccine candidates can be produced in adequate amount with high safety and efficacy as well as equitably provided to all affected areas, specially low-resource regions. ) and multiple lineage-specific accessory proteins at the 3 -end. • phylogenetic analysis demonstrates that severe acute respiratory syndrome coronavirus 2 (sars-cov-2) shares 50 and 80.0% nucleotide identity to middle east respiratory syndrome cov and sars-cov, respectively. • the s-protein of sars-cov-2 is longer than for other viruses such as sars-cov and bat sars-like covs. • virion particles spread from the respiratory mucosa, by binding to the ace2 receptors on ciliated bronchial epithelial cells, and after that may engage with other cells. • the s-glycoprotein mediates binding of the virus to the sensitive human cell surface receptors, followed by fusion of the virus and host cell membranes to assist viral entrance. • sars-cov-2 infection stimulates the immune response via two stages. at the incubation and nonalarming stages, a particular adaptive immune response is needed to eradicate the virus and to impede progress to severe condition. • the injured cells activate innate inflammation within the lungs, which is mainly mediated through pro-inflammatory macrophages/monocytes. lung inflammation is the major reason for the fatal respiratory disease at the severe stage of coronavirus disease 2019 (covid-19) infection. • the main characteristics of covid-19 on preliminary computed tomography (ct) examination including bilateral multi-lobar ground-glass opacities with a peripheral/posterior distribution and patchy consolidation. • at present, the diagnosis of covid-19 is largely based on laboratory tests pcr and chest ct imaging technique. although, no specific antiviral treatment for covid-19 is currently advised due to lack of evidence. • several pharmacotherapeutic agents have been used for treatment of covid-19 patients consisting lopinavir/ritonavir, hydroxychloroquine and ifn β-1a. • effective sars-cov-2 vaccines are urgently needed in order to decrease infection severity, viral shedding as well as human-human transmission. the most advanced candidates have recently moved into clinical development, including mrna-1273, ad5-ncov, ino-4800 and lv-smenp-dc, and pathogen-specific. all authors contributed to data collection, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work. this study was supported by birjand university of medical sciences. the authors have no other relevant affiliations or financial involvement with any organization or entity with 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treat critically ill patients an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies the covid-19 vaccine development landscape developing covid-19 vaccines at pandemic speed covid-19 and community mitigation strategies in a pandemic key: cord-254916-y1rw9q11 authors: ogando, natacha s.; dalebout, tim j.; zevenhoven-dobbe, jessika c.; limpens, ronald w.a.l.; van der meer, yvonne; caly, leon; druce, julian; de vries, jutte j. c.; kikkert, marjolein; bárcena, montserrat; sidorov, igor; snijder, eric j. title: sars-coronavirus-2 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology date: 2020-06-22 journal: j gen virol doi: 10.1099/jgv.0.001453 sha: doc_id: 254916 cord_uid: y1rw9q11 the sudden emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) at the end of 2019 from the chinese province of hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. here, we compared a variety of replication features of sars-cov-2 and sars-cov and analysed the cytopathology caused by the two closely related viruses in the commonly used vero e6 cell line. compared to sars-cov, sars-cov-2 generated higher levels of intracellular viral rna, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. immunofluorescence microscopy of sars-cov-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of sars-cov. electron microscopy revealed that the ultrastructural changes induced by the two sars viruses are very similar and occur within comparable time frames after infection. furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that sars-cov-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. an important difference between the two viruses is the fact that – upon passaging in vero e6 cells – sars-cov-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. these mutations change or delete a putative furin-like cleavage site in the region connecting the s1 and s2 domains and result in a very prominent phenotypic change in plaque assays. introduction for the first time in a century, societies and economies worldwide have come to a near-complete standstill due to a pandemic outbreak of a single rna virus. this virus, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] belongs to the coronavirus (cov) family, which is thought to have given rise to zoonotic introductions on multiple occasions during the past centuries. coronaviruses are abundantly present in mammalian reservoir species, including bats [2] , and should now be recognized definitively as a continuous zoonotic threat with the ability to cause severe human disease and explosive pandemic transmission. to date, seven covs that can infect humans have been identified, which segregate into two classes. on the one hand, there are four endemic human covs (hcovs), the first of which were identified in the 1960s, annually causing a substantial number of common colds [3, 4] . on the other hand, we now know of (at least) three zoonotic covs that recently have caused outbreaks in the human population: severe acute respiratory syndrome coronavirus (sars-cov) [5, 6] in [2002] [2003] , middle east respiratory syndrome-coronavirus (mers-cov) [7, 8] since 2012 (and probably earlier) and the current pandemic sars-cov-2 [9, 10] . the latter agent emerged near wuhan (pr china) in the fall of 2019 and its animal source is currently under investigation [11] [12] [13] . transmission to humans of sars-cov and mers-cov was attributed to civet cats [14] and dromedary camels [15] , respectively, although both species may have served merely as an intermediate host due to their close contact with humans. all three zoonotic covs belong to the genus betacoronavirus (beta-cov), which is abundantly represented among the covs that circulate in the many bat species on this planet [2, [16] [17] [18] [19] . the genetic diversity of bat covs and their phylogenetic relationships with the four known endemic hcovs (oc43, hku1, 229e and nl63; the latter two being alpha-covs) suggests that also these may have their evolutionary origins in bat hosts, for most of them probably centuries ago [20] . the potential of multiple covs from different genera to cross species barriers had been predicted and documented previously [2, 16-19, 21, 22] , but regrettably was not taken seriously enough to invest more extensively in prophylactic and therapeutic solutions that could have contributed to rapidly containing an outbreak of the current magnitude. compared to other rna viruses, covs possess an unusually large positive-sense rna genome with a size ranging from 26 to 34 kilobases [23] . the cov genome is single-stranded and its 5′-proximal two-thirds encode for the large and partially overlapping replicase polyproteins pp1a and pp1ab (4000-4500 and 6700-7200 amino acids long, respectively), with the latter being a c-terminally extended version of the former that results from ribosomal frameshifting. the replicase polyproteins are processed into 16 cleavage products (non-structural proteins, nsps) by two internal proteases, the papain-like protease (pl pro ) in nsp3 and the 3c-like or 'main' protease (m pro ) in nsp5 [24] . specific transmembrane nsps (nsp3, 4 and 6) then cooperate to transform intracellular membranes into a viral replication organelle (ro) [25] that serves to organize and execute cov rna synthesis, which entails genome replication and the synthesis of an extensive nested set of subgenomic mrnas. the latter are used to express the genes present in the 3′-proximal third of the genome, which encode the four common cov structural proteins [spike (s), envelope (e), membrane (m) and nucleocapsid (n) protein] and the 'so-called' accessory protein genes, most of which are thought to be involved in the modulation of host responses to cov infection [26] . the cov proteome includes a variety of potential targets for drug repurposing or de novo development of specific inhibitors of, e.g. viral entry (s protein) or rna synthesis [27] . the latter process depends on a set of enzymatic activities [24] including an rna-dependent rna polymerase (rdrp; in nsp12), rna helicase (in nsp13), two methyltransferases involved in mrna capping (a guanine-n7-methyltransferase in nsp14 and a nucleoside-2′-o-methyltransferase in nsp16) and a unique exoribonuclease (exon, in nsp14) that promotes the fidelity of the replication of the large cov genome [28] . other potential drug targets are the transmembrane proteins that direct the formation of the viral ro, several less well characterized enzymatic activities and a set of smaller nsps (nsp7-10) that mainly appear to serve as cofactors/modulators of other nsps. the newly emerged sars-cov-2 was rapidly identified as a cov that is relatively closely related to the 2003 sars-cov [9, 29, 30] . the two genome sequences are about ~80 % identical and the organization of orfs is essentially the same. the overall level of amino acid sequence identity of viral proteins ranges from about 65 % in the least conserved parts of the s protein to about 95 % in the most conserved replicative enzyme domains, prompting the coronavirus study group of the international committee on the taxonomy of viruses to classify the new agent within the species severe acute respiratory syndrome-related coronavirus, which also includes the 2003 sars-cov [1] . the close phylogenetic relationship also implies that much of our knowledge of sars-cov molecular biology, accumulated over the past 17 years, can probably be translated to sars-cov-2. many reports posted over the past months have described such similarities, including the common affinity of the two viruses for the angiotensinconverting enzyme 2 (ace2) receptor [9, 31] . this receptor is abundantly expressed in vero cells (african green monkey kidney cells). since 2003, vero cells have been used extensively for sars-cov research in cell-culture-based infection models by many laboratories, including our own. we set out to establish the basic features of sars-cov-2 replication in vero cells and compare it to the frankfurt-1 sars-cov isolate from 2003 [32, 33] . when requesting virus isolates (february 2020), and in spite of the rapidly emerging public health crisis, we were confronted -not for the first time -with administrative hurdles and discussions regarding the alleged 'ownership' of virus isolates cultured from (anonymous) clinical samples. from a biological and evolutionary point of view, this would seem a strangely anthropocentric consideration, but it ultimately forced us to reach out across the globe to australian colleagues in melbourne. after checking our credentials and completing a basic material transfer agreement, they provided us (within 1 week) with their first sars-cov-2 isolate (originally named 2019-ncov/victoria/1/2020 and subsequently renamed betacov/australia/vic01/2020 [34] , which will be used throughout this study. until now, this isolate has been provided to 17 other laboratories worldwide to promote the rapid characterization of sars-cov-2, in this critical time of lockdowns and other preventive measures to avoid a collapse of public health systems. in this report, we describe a comparative study of the basic replication features of sars-cov and sars-cov-2 in vero e6 cells, including growth kinetics, virus titres, plaque phenotype and an analysis of intracellular viral rna and protein synthesis. additionally, we analysed infected cells by light and electron microscopy, and demonstrated cross-reactivity of 13 available sars-cov-specific antisera (recognizing ten different viral proteins) with their sars-cov-2 counterparts. finally, we established the conditions for a mediumthroughput assay to evaluate basic antiviral activity and assessed the impact of some known cov inhibitors on sars-cov-2 replication. in addition to many anticipated similarities, our results also established some remarkable differences between the two viruses that warrant further investigation. one of them is the rapid evolution -during virus passaging in vero cells -of a specific region of the sars-cov-2 s protein that contains the so-called furin-like cleavage site. vero e6 cells and huh7 cells were grown as described previously [35] . sars-cov-2 isolate australia/vic01/2020 (genbank id: mt007544.1 [34] ) was derived from a positively testing nasopharyngeal swab in melbourne, australia, and was propagated twice in vero/hslam cells, before being shared with other laboratories. in leiden, the virus was passaged two more times at low m.o.i. in vero e6 cells to obtain a working stock (p2 stock) that was used in all experiments. sars-cov isolate frankfurt 1 [36] was used to compare growth kinetics and other features with sars-cov-2. infection of vero e6 cells was carried out in pbs containing 50 µg ml −1 deaedextran and 2 % fcs (bodinco). the inoculum was added to the cells for 1 h at 37 °c, after which cells were washed twice with pbs and maintained in eagle's minimal essential medium (emem; lonza) with 2 % fcs, 2 mm l-glutamine (paa) and antibiotics (sigma). viral titres were determined by plaque assay in vero e6 cells as described previously [37] . for plaque picking, plaque assays were performed using our p1 stock, while using an overlay containing 1 % of agarose instead of avicel (rc-581; fmc biopolymer). following neutral red staining, small and large plaques were picked and used to inoculate a 10 cm 2 dish of vero e6 cells containing 2 ml of emem-2%fcs medium, yielding p1 virus. after 48 h, 200 µl of the culture supernatant was used to infect the next dish of cells (p2), a step that was repeated one more time to obtain p3 virus. all work with live sars-cov and sars-cov-2 was performed in biosafety laboratory level 3 facilities at leiden university medical center, the netherlands. isolation of intracellular rna was performed by lysing infected cell monolayers with tripure isolation reagent (roche applied science) according to the manufacturer's instructions. after purification and ethanol precipitation, intracellular rna samples were loaded onto a 1.5 % agarose gel containing 2.2 m formaldehyde, which was run overnight at low voltage in mops buffer [10 mm mops (sodium salt) (ph 7), 5 mm sodium acetate, 1 mm edta]. dried agarose gels were used for direct detection of viral mrnas by hybridization with a 32 p-labelled oligonucleotide probe (5′-cacatggggatagcactac-3′) that is complementary to a fully conserved sequence located 30 nucleotides upstream of the 3' end of the genome as well as all subgenomic mrnas produced by sars-cov-2 and sars-cov. after hybridization, rna bands were visualized and quantified by phosphorimaging using a typhoon-9410 variable mode scanner (ge healthcare) and imagequant tl software (ge healthcare). in order to verify the amount of rna loaded, a second hybridization was performed using a 32 p-labelled oligonucleotide probe recognizing 18s ribosomal rna (5′-gatc cgag ggcc tcac taaac-3′). protein lysates were obtained by lysing infected cell monolayers in 4×laemmli sample buffer and were analysed by semi-dry western blotting onto hybond 0.2 µm polyvinylidene difluoride (pvdf) membrane (ge healthcare). membranes were incubated with rabbit antisera diluted in pbs with 0.05 % tween-20 containing 5 % dry milk (campina). primary antibodies were detected with a horseradish peroxidase-conjugated swine anti-rabbit igg antibody (dako) and protein bands were visualized using clarity western blot substrate (biorad) and detected using an advanced q9 alliance imager (uvitec cambridge). sars-cov-2 genomic rna was isolated from cell-culture supernatants using tripure isolation reagent (roche applied science) and purified according to the manufacturer's instructions. the total amount of rna in samples was measured using a qubit fluorometer and rna high sensitivity kit (thermo fisher scientific). for next-generation sequencing (ngs) library preparation, rna (25-100 ng) was mixed with random oligonucleotide primers using the nebnext first strand synthesis module kit for illumina (neb) and incubated for 10 min at 94 °c. ngs of samples was performed by a commercial service provider (genomescan, leiden, the netherlands) while including appropriate quality controls after each step of the procedure. sequencing was performed using a novaseq 6000 sequencing system (illumina). subsequently, sequencing reads were screened for the presence of human (grch37.75), mouse (grcm38.p4), e. coli mg1655 (embl u00096.2), phix (refseq nc_001422.1) and common vector sequences (univec and chlsab1.1). prior to alignment, reads were trimmed to remove adapter sequences and filtered for sequence quality. the remaining reads were mapped to the sars-cov-2 genbank reference sequence (nc_045512.2 [38] ). data analysis was performed using bowtie 2 [39] . raw ngs data sets for each virus sample analysed in this study are deposited in ncbi bioproject and available under the following link: http://www. ncbi. nlm. nih. gov/ bioproject/ 628043. only sars-cov-2-specific reads were included in these data files. to study evolution/adaptation of the s protein gene, we performed an in-depth analysis of reads covering the s1/s2 region of the s protein gene. this was done for the p2 stock and for the four virus samples of the plaque-picking experiment shown in fig. 1a . first, all reads spanning nt 23 576 to 23 665 of the sars-cov-2 genome were selected. next, reads constituting less than 1 % of the total number of selected reads were excluded from further analysis. the remaining number of reads were 3860 (p2 stock), 1924 (s5p1), 2263 (s5p2), 4049 (s5p3) and 3323 (l8p1). these reads were translated in the s protein orf and the resulting amino acid sequences were aligned, grouped on the basis of containing the same mutations/deletions in the s1/s2 region and ranked by frequency of occurrence (fig. 1b) . the sars-cov-specific rabbit or mouse antisera/antibodies used in this study are listed in table 1 . most antisera were described previously (see references in table 1 ), with the exception of three rabbit antisera recognizing sars-cov nsps 8, 9 and 15. these were raised using full-length (his) 6 -tagged table 1 . sars-cov-specific antisera used and their cross-reactivity with corresponding sars-cov-2 targets antigen type antibody type ifa signal* reference nsp3 (dgd7) transmembrane replicase protein, containing pl pro bacterial expression product rabbit polyclonal ++ [48] nsp4 (fgq4) transmembrane replicase protein synthetic peptide rabbit polyclonal ++ [109] nsp5 (due5) m pro bacterial expression product rabbit polyclonal + [48] nsp6 (gbz7) transmembrane replicase protein synthetic peptide rabbit polyclonal − [109] nsp8 (duk4) rna polymerase co-factor bacterial expression product rabbit polyclonal ++ [48] nsp8 ( * ++, strongly positive; +, positive; -, negative. following a plaque assay of the p1 virus stock, small and large plaques were picked and these virus clones were passaged three times in vero e6 cells, while their plaque phenotype was monitored. in contrast to the large plaque viruses (example l8; bottom row), the plaque phenotype of the small plaque viruses (example s5; top row) rapidly evolved within these three passages. (b) evolution/adaptation of the s protein gene during vero e6 passaging. overview of ngs data obtained for the p2 stock, s5p1/p2/p3 and s8p1 in the s1/s2 region of the sars-cov-2 s protein gene that encodes the so-called furin-like cleavage site. the analysis was based on ngs reads spanning nt 23 576 to 23 665 of the sars-cov genome (see methods for details) and their translation in the s protein orf. deletions are indicated with δ followed by the affected amino acid residues. bacterial expression products (nsp8 and nsp15) or a synthetic peptide (nsp9, aa 4209-4230 of sars-cov pp1a), which were used to immunize new zealand white rabbits as described previously [40, 41] . cross-reactivity of antisera to sars-cov-2 targets was evaluated microscopically by immunofluorescence assay (ifa) and for some antisera (nsp3 and n protein) also by western blot analysis. double-stranded rna was detected using mouse monoclonal antibody j2 from scicons [42] . cells were grown on glass coverslips and infected as described above [43] . at sars-cov-2 isolate betacov/australia/vic01/2020 was received as a stock derived from two consecutive passages in vero/hslam cells [34] . the virus was then propagated two more times at low m.o.i. in vero e6 cells, in which it caused a severe cytopathic effect (cpe). we also attempted propagation in huh7 cells, using the same amount of virus or a tenfold larger inoculum, but did not observe any cytopathology after 72 h (data not shown [38] and other field isolates [29] , isolate betacov/australia/vic01/2020 exhibits >99.9 % sequence identity. in addition to synonymous mutations in the nsp14-coding sequence (u19065 to c) and s protein gene (u22303 to g), orf3a contains a single non-synonymous mutation (g26144 to u). strikingly, the 3′ utr contains a 10 nt deletion (nt 29 750-29 759; cgaucgagug) located 120 nt upstream of the genomic 3′ end, which is not present in other sars-cov-2 isolates described thus far (>670 sars-cov2 sequences present in genbank on 17 april 2020). in about 71 % of the 95 173 p2 ngs reads covering this position, we noticed a g23607 to a mutation encoding an arg682 to gln substitution near the so-called s1/s2 cleavage site of the viral s protein (see discussion), with the other 29 % of the reads being wild-type sequence. as this ratio approximated the observed relative proportions between large and small plaques, we performed a plaque assay on the p1 virus stock (fig. 1a, leftmost well) and picked multiple plaques of each size, which were passaged three times in vero e6 cells while monitoring their plaque phenotype. interestingly, for several of the small-plaque virus clones (like s5; fig. 1a ) we observed rapid conversion to a mixed or large-plaque phenotype during these three passages, while large-plaque virus clones (like l8) stably retained their plaque phenotype (fig. 1a) . ngs analysis of the genome of a large-plaque p1 virus (l8p1) revealed that >99 % of the reads in the s1/s2 cleavage site region contained the g23607 to a mutation described above. no other mutations were detected in the genome, thus clearly linking the arg682 to gln substitution in the s protein to the large-plaque phenotype observed for the l8p1 virus. next, we also analysed the genomes of the p1, p2 and p3 viruses derived from a small-plaque (s5) that was picked. this virus clone retained its small-plaque phenotype during the first passage ( fig. 1a; s5p1 ), but began to yield an increasing proportion of large(r) plaques during subsequent passages. sequencing of s5p2 (fig. 1b) revealed a variety of lowfrequency reads with mutations near the s1/s2 cleavage site motif (aa 681-687; prrar↓sv), with g23607 to a (specifying the arg682 to gln substitution) again being the dominant one (in ~2.1 % of the reads covering nt 23 576 to 23 665 of the genome). at lower frequencies single-nucleotide changes specifying arg682 to trp and arg683 to leu substitutions were also detected. furthermore, a 10 aa deletion (residues 679-688) that erases the s1/s2 cleavage site region was discovered, as well as a 5 aa deletion (residues 675-679) immediately preceding that region. the amount of large plaques increased substantially upon the next passage, with ngs revealing the prominent emergence of the mutants containing the 10 aa deletion or the arg682 to gln point mutation (~22 and~12 % of the reads, respectively), and yet other minor variants with mutations in the prrar↓sv sequence being discovered. taken together these data clearly link the large-plaque phenotype of sars-cov-2 to the acquisition of mutations in this particular region of the s protein, which apparently provides a strong selective advantage during passaging in vero e6 cells. to our knowledge, a detailed comparison of sars-cov-2 and sars-cov replication kinetics in cell culture has not been reported so far. therefore, we infected vero e6 cells with the sars-cov-2/p2 virus stock at high m.o.i. to analyse viral rna synthesis and the release of infectious viral progeny (fig. 2a) . this experiment was performed using four replicates per time point and for comparison we included the sars-cov frankfurt-1 isolate [36] , which has been used in our laboratory since 2003. during the early stages of infection (until 8 h p.i.), the growth curves of the two viruses were similar, but subsequently cells infected with sars-cov clearly produced more infectious progeny (about 50-fold more) than sars-cov-2-infected cells, with both viruses reaching their plateau by about 14 h p.i. as shown in fig. 2b , despite its transition to a mainly large-plaque phenotype, the largest sars-cov-2/p3 plaques were still substantially smaller than those obtained with sars-cov frankfurt-1. in parallel, we analysed the kinetics of viral rna synthesis by isolating intracellular viral rna, subjecting it to agarose gel electrophoresis and visualizing the various viral mrna species by in-gel hybridization with a 32 p-labelled oligonucleotide probe recognizing a fully conserved 19 nt sequence located 30 nt upstream of the 3′ end of both viral genomes (fig. 3a) . this revealed the anticipated presence of the genomic rna and eight subgenomic mrnas, together forming the well-known 5′-and 3′-coterminal nested set of transcripts required for full cov genome expression. in general, for both viruses, the accumulation of viral rnas followed the growth curves depicted in fig. 2a . the relative abundance of the individual rnas was determined using the 12, 14 and 24 h p.i. samples (averages presented in fig. 3b ) and found to be largely similar, with the exception of sars-cov-2 mrnas 7 and 8, which accumulated to about four and twofold higher levels, respectively. strikingly, in spite of the ultimately lower yield of infectious viral progeny, sars-cov-2 rna synthesis was detected earlier and reached an overall level exceeding that of sars-cov. overall, we conclude that in vero e6 cells, sars-cov-2 produces levels of intracellular rna that are at least comparable to those of sars-cov, although this does not translate into the release of equal amounts of infectious viral progeny (fig. 2a) . to be able to follow virus replication in sars-cov-2-infected cells more closely, we explored cross-reactivity of a variety of antisera previously raised against sars-cov targets, in particular a variety of nsps. in an earlier study, many of those were found to cross-react also with the corresponding mers-cov targets [35] , despite the relatively large evolutionary distance between mers-cov and sars-cov. based on the much closer relationship with sars-cov-2, similar or better cross-reactivity of these sars-cov reagents was expected, which was explored using immunofluorescence microscopy. indeed, most antisera recognizing sars-cov nsps that were tested (nsp3, nsp4, nsp5, nsp8, nsp9, nsp13, nsp15) strongly cross-reacted with the corresponding sars-cov-2 target (fig. 4, table 1 ), the exception being a polyclonal nsp6 rabbit antiserum. likewise, both a polyclonal rabbit antiserum and mouse monoclonal antibody recognizing the n protein cross-reacted strongly (fig. 4b , table 1 ). the same was true for a rabbit antiserum raised against a c-terminal peptide of the sars-cov m protein (fig. 4e) . labelling patterns were essentially identical to those previously documented for sars-cov [45, 46] , with nsps accumulating in the perinuclear region of infected cells, where the elaborate membrane structures of the viral ros are formed (fig. 4a, c and d) . punctate structures in the same area of the cell were labelled using an antibody recognizing double-stranded rna (dsrna), which presumably recognizes replicative intermediates of viral rna synthesis [46, 47] . the n protein signal was diffusely cytosolic (fig. 4b) , whereas the m protein labelling predominantly showed the expected localization to the golgi complex (fig. 4e) , where the protein is known to accumulate [48] . we next used electron microscopy to investigate the ultrastructural changes that sars-cov-2 induces in infected cells, and focused on the membranous replication organelles (ros) that support viral rna synthesis and on the assembly and release of new virions (fig. 5) . compared to mock-infected control cells (fig. 5a-b) , various distinct membrane alterations were observed in cells infected with either sars-cov or sars-cov-2 ( fig. 5c-j) . at 6 h p.i., larger regions with membrane alterations were found particularly in cells infected with sars-cov-2 (data not shown), which may align with the somewhat faster onset of intracellular rna synthesis in sars-cov2-infected vero e6 cells (fig. 3a) . from 8 h p.i. onwards, sars-cov-and sars-cov-2-infected cells appeared more similar (fig. 5c-j) . double-membrane vesicles (dmvs) were the most prominent membrane alteration up to this stage (fig. 5d -e, h-i). in addition, convoluted membranes [46] were readily detected in sars-cov-infected cells, while zippered er [25, 49, 50] appeared to be the predominant structure in sars-cov-2-infected cells (fig. 5e , i, white arrowheads). as previously described for sars-cov [46] , sars-cov-2-induced dmvs also appeared to fuse through their outer membrane, giving rise to vesicle packets that increased in numbers as infection progressed (fig. 5f , k, white asterisks). virus budding near the golgi apparatus, presumably into smooth membranes of the er-golgi intermediate compartment (ergic) [45, 51, 52] , was frequently observed at 8 h p.i. (fig. 5k-l, o-p) . this step is followed by transport to the plasma membrane and release of virus particles into extracellular space. by 10 h p.i., released progeny virions were abundantly detected around all infected cells (fig. 5m -n, q-r). interestingly, whereas spikes were clearly present on sars-cov progeny virions, a relatively large proportion of sars-cov-2 particles seemed to carry few or no visible spike projections on their surface, perhaps suggesting a relatively inefficient incorporation of spike proteins into sars-cov-2 virions. this could potentially reduce the yield of infectious particles and may contribute to the lower progeny titres obtained for this virus (fig. 2a) . in order to establish and validate a cpe-based assay to identify potential inhibitors of sars-cov-2 replication, we selected four previously identified inhibitors of cov replication: remdesivir [53, 54] , chloroquine [55, 56] , alisporivir [57, 58] and pegylated interferon alpha (peg-ifn-α) [35, 59] [110, 111] respectively; fig. 6a ) than previously reported by others, but this may be explained by technical differences like a longer assay incubation time (72 h instead of 48 h) and the use of a different read-out (cell viability instead of qrt-pcr or viral load). based on the obtained half maximal cytotoxic concentration (cc 50 ) values of >100 µm, a selectivity index >22.5 was calculated. chloroquine potently blocked virus infection at low-micromolar concentrations, with an ec 50 value of 2.3±1.1 µm for both viruses (cc 50 >100 µm, si>45.5; fig. 6b ). alisporivir, a known inhibitor of different groups of rna viruses, was previously found to effectively reduce the production of cov progeny. in this study, we measured ec 50 values of 4.9±1.3 and 4.3±1.0 µm for sars-cov-2 and sars-cov, respectively ( fig. 6c ; cc 50 >100 µm, si>20). treatment with peg-ifn-α completely inhibited replication of sars-cov-2, even at the lowest dose of 7.8 ng ml −1 (fig. 6d ). in line with previous results [35, 59] , sars-cov was much less sensitive to peg-ifn-α treatment, yielding only partial inhibition at all concentrations tested (from 7.8 to 1000 ng ml −1 ). overall, we conclude that vero e6 cells provide a suitable basis to perform antiviral compound screening and select the most promising hits for in-depth mechanistic studies and further development. in this report, we describe a comparative analysis of the replication features of sars-cov-2 and sars-cov in vero e6 cells, one of the most commonly used cell lines for studying these two viruses. in contrast to the stable phenotype exhibited by sars-cov during our 17 years of working with this virus in these cells, sars-cov-2 began to exhibit remarkable phenotypic variation in plaque assays within a few passages after its isolation from clinical samples (fig. 1a) . in addition to the betacov/australia/vic01/2020 isolate used in this study, similar observations were made for a variety of other clinical isolates (data not shown). to establish the genetic basis for the observed plaque size heterogeneity, small and large plaques were picked and the resulting virus clones were passaged repeatedly and analysed using ngs. the consensus sequences obtained for s5p1 and l8p1, which differed by a single nucleotide substitution in the s protein gene, clearly established that a single s protein mutation (arg682 to gln) was responsible for the observed plaque size difference. this mutation is localized near the so-called furin-like s1/s2 cleavage site (fig. 1b) [61] in the s protein [62] . this sequence constitutes a (potential) processing site that is present in a subset of covs (including sars-cov-2 and mers-cov) but is lacking in others, like sars-cov and certain bat covs [61, 63] . this polybasic motif (prrar↓sv, in sars-cov-2) can be recognized by intracellular furin-like proteases during viral egress and its cleavage is thought to prime the s protein for fusion and entry [64] , which also requires a second cleavage event to occur at the downstream s2' cleavage site [61] . in general, the presence of the furin-like cleavage site does not appear to be critical for successful cov infection. using pseudotyped virions carrying mutant s proteins of sars-cov [65] or sars-cov-2 [66] , it was shown that its presence minimally impacts s protein functionality. in the sars-cov s protein, an adjacent sequence that is conserved across covs can be cleaved by other host proteases like cathepsin l or tmprss2 [67] [68] [69] , thus providing an alternative pathway to trigger viral entry. possibly, this pathway is also employed by our vero e6-cell adapted sars-cov-2 mutants that have lost the furin-like cleavage site, like clone l8p1 and multiple variants encountered in s5p3 (fig. 1a) . these variants contain either single point mutations or deletions of 5 to 10 aa (fig. 1b) , resembling variants recently reported by other laboratories [30, 70, 71] . interestingly similar changes were also observed in some clinical sars-cov-2 isolates that had not been passaged in cell culture [70] . it is currently being investigated why mutations that inactivate the furin-like cleavage site provide such a major selective advantage during sars-cov-2 passaging in vero e6 cells and how this translates into the striking large-plaque phenotype documented in this paper. an additional remarkable feature confirmed by our re-sequencing of the betacov/australia/vic01/2020 isolate of sars-cov-2 is the presence of a 10 nt deletion in the 3′ utr of the genome [34] . screening of other available sars-cov-2 genome sequences indicated that the presence of this deletion apparently is unique for this particular isolate, and likely represents an additional adaptation acquired during cell-culture passaging. this deletion maps to a previously described 'hypervariable region' in the otherwise conserved 3′ utr, and in particular to the so-called s2m motif [72] that is conserved among covs and also found in several other virus groups [73, 74] . the s2m element has been implicated in the binding of host factors to viral rnas, but its exact function has remained enigmatic thus far. strikingly, for the mouse hepatitis coronavirus the entire hypervariable region (including s2m) was found to be dispensable for replication in cell culture, but highly relevant for viral pathogenesis in mice [72] . although the impact of this deletion for sars-cov-2 remains to be studied in more detail, these previous data suggest that this mutation need not have a major impact on sars-cov-2 replication in vero e6 cells. this notion is also supported by the fact that the results of our antiviral screening assays (fig. 6) correlate well with similar studies performed with other sars-cov-2 isolates [54, 75, 76] . clearly, this could be different for in vivo studies, for which it would probably be better to rely on sars-cov-2 isolates not carrying this deletion in their 3′ utr. vero e6 cells are commonly used to isolate, propagate and study sars-cov-like viruses as they support viral replication to high titres [77] [78] [79] [80] [81] . this may be due to a high expression level of the ace-2 receptor [82] that is used by both sars-cov-2 and sars-cov [9] and/or the fact that they lack the ability to produce interferon [83, 84] . it will be interesting to evaluate whether there is a similarly strong selection pressure to adapt the s1/s2 region of the s protein when sars-cov-2 is passaged in other cell types. such studies are currently in progress in our laboratory and already established that huh7 cells may be a poor choice, despite the fact that they were used for virus propagation [9, 85] and antiviral screening in other studies [54, 86] . immunolabelling of infected huh7 cells (data not shown) revealed non-productive infection of only a small fraction of the cells and a general lack of cytopathology. while other cell lines are being evaluated, the monitoring of the plaque phenotype (plaque size and homogeneity) as illustrated above may provide a quick and convenient method to assess the composition of sars-cov-2 stocks propagated in vero e6 cells, at least where it concerns the evolution of the s1/s2 region of the s protein. given the ongoing sars-cov-2 pandemic, the detailed characterization of its replication cycle is an important step in understanding the molecular biology of the virus and defining potential targets for inhibitors of replication. the cross-reacting antisera described in this study (table 1) will be a useful tool during such studies. in general, the subcellular localization of viral nsps and structural proteins (fig. 4 ) and the ultrastructural changes associated with ro formation (fig. 5) were very similar for the two viruses. we also observed comparable replication kinetics for sars-cov-2 and sars-cov in vero e6 cells, although clearly lower final infectivity titres were measured for sars-cov-2 (~50-fold lower; fig. 2 ). nevertheless, rna synthesis could be detected somewhat earlier for sars-cov-2 and the overall amount of viral rna produced exceeded that produced by sars-cov (fig. 3) . this may be indicative of certain assembly or maturation problems or of virus-host interactions that are different in the case of sars-cov-2. these possibilities merit further investigation, in particular since our preliminary em studies suggested intriguing differences with sars-cov regarding the abundance of spikes on the surface of freshly released sars-cov-2 particles (fig. 5n, r) . our analysis of sars-cov-2 subgenomic mrna synthesis revealed an increased relative abundance of mrnas 7 and 8 (~four and ~twofold, respectively) in comparison to sars-cov. mechanistically, these differences do not appear to be caused by extended base-pairing possibilities of the transcription regulatory sequences that direct the synthesis of these two mrnas [24] . as in sars-cov, mrna7 of sars-cov-2 encodes for two proteins, the orf7a and orf7b proteins, with the latter presumably being expressed following leaky ribosomal scanning [32] . upon ectopic expression, the orf7a protein has been reported to induce apoptosis via a caspasedependent pathway [87] and/or to be involved in cell-cycle arrest [88] . the orf7b product is a poorly studied integral membrane protein that has (also) been detected in virions [89] . when orf7a/b or orf7a were deleted from the sars-cov genome, there was a minimal impact on the kinetics of virus replication in vitro in different cell lines, including vero cells, and in vivo using mice. in another study, however, partial deletion of sars-cov orf7b was reported to provide a replicative advantage in caco-2 and huh7 cells, but not in vero cells [90] . the sars-cov orf8 protein is membrane-associated and able to induce endoplasmic reticulum stress [91, 92] , although it has not been characterized in great detail in the context of viral infection. soon after the emergence of sars-cov in 2003, a conspicuous 29 nt (out-offrame) deletion in orf8 was noticed in late(r) human isolates, but not in early human isolates and sars-like viruses obtained from animal sources [93] [94] [95] . consequently, loss of orf8 function was postulated to reflect an adaptation to the human host. the re-engineering of an intact orf8, using a reverse genetics system for the sars-cov frankfurt-1 isolate, yielded a virus with strikingly enhanced (up to 23-fold) replication properties in multiple systems [96] . clearly, it remains to be established whether the increased synthesis of mrnas 7 and 8 is a general feature of sars-cov-2 isolates, and this indeed also translates into higher expression levels of the accessory proteins encoded by orfs 7a, 7b and 8. if confirmed, these differences definitely warrant an in-depth follow-up analysis as cov accessory proteins in general have been shown to be important determinants of virulence. they may thus be relevant for our understanding of the wide spectrum of respiratory disease symptoms observed in covid-19 patients [97] . based on the close ancestral relationship between sars-cov-2 and sars-cov [98] , one might expect that the patterns and modes of interaction with host antiviral defence mechanisms would be similar. however, our experiments with type-i interferon treatment of vero e6 cells (fig. 6 ) revealed a clear difference, with sars-cov-2 being considerably more sensitive than sars-cov, as also observed by other laboratories [76] . essentially, sars-cov-2 replication could be inhibited by similarly low concentrations of peg-ifn-alpha-2a that inhibit mers-cov replication in cell culture [35] . taken together, our data suggest that sars-cov-2 is less able to counteract a primed type-i ifn response than sars-cov [76, 99] . previously identified inhibitors of cov replication were used to further validate our cell-based assay for sars-cov-2 inhibitor screening. these compounds inhibited replication at similar low-micromolar concentrations and in a similar dose-dependent manner as observed for sars-cov (fig. 6 ). remdesivir is a prodrug of an adenosine analogue developed by gilead sciences. it was demonstrated to target the cov rna polymerase and act as a chain terminator [100] [101] [102] . the clinical efficacy of remdesivir is still being evaluated and, after some first encouraging results [103] , worldwide compassionate use trials are now being conducted. likewise, hydroxychloroquine and chloroquine have been labelled as potential 'game changers' and are being evaluated for treatment of severe covid-19 patients [104] . both compounds have been used to treat malaria and amebiasis [105] , until drug-resistant plasmodium strains emerged [106] . these compounds can be incorporated into endosomes and lysosomes, raising the ph inside these intracellular compartments, which in turn may lead to defects in protein degradation and intracellular trafficking [68, 107] . an alternative hypothesis to explain their anti-sars-cov activity is based 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a host factor for activity in vitro coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands genome organization of the sars-cov we thank various genomescan staff members for the pleasant and swift collaboration that facilitated the ngs and data analysis of the first sars-cov-2 samples. we are grateful to all members of the sections research and clinical microbiology of the lumc department of medical microbiology for their collaborative support and dedication during the current pandemic situation. in particular, we thank linda boomaars, peter bredenbeek, ien dobbelaar, martijn van hemert, sebenzile myeni, tessa nelemans, esther quakkelaar, ali tas, sjaak van voorden and gijsbert van willigen for their technical or administrative support, constructive discussions and/or scientific input. the authors declare that there are no conflicts of interest. five reasons to publish your next article with a microbiology society journal 1 . the microbiology society is a not-for-profit organization. 2. we offer fast and rigorous peer review -average time to first decision is 4-6 weeks. 3. our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as 'excellent' or 'very good'. 5. your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord-258595-bk35vxlr authors: westhaus, sandra; weber, frank-andreas; schiwy, sabrina; linnemann, volker; brinkmann, markus; widera, marek; greve, carola; janke, axel; hollert, henner; wintgens, thomas; ciesek, sandra title: detection of sars-cov-2 in raw and treated wastewater in germany – suitability for covid-19 surveillance and potential transmission risks date: 2020-08-18 journal: sci total environ doi: 10.1016/j.scitotenv.2020.141750 sha: doc_id: 258595 cord_uid: bk35vxlr abstract wastewater-based monitoring of the spread of the new sars-cov-2 virus, also referred to as wastewater-based epidemiology (wbe), has been suggested as a tool to support epidemiology. an extensive sampling campaign, including nine municipal wastewater treatment plants, has been conducted in different cities of the federal state of north rhine-westphalia (germany) on the same day in april 2020, close to the first peak of the corona crisis. samples were processed and analysed for a set of sars-cov-2-specific genes, as well as pan-genotypic gene sequences also covering other coronavirus types, using reverse transcription-quantitative polymerase chain reaction (rt-qpcr). additionally, a comprehensive set of chemical reference parameters and bioindicators was analysed to characterize the wastewater quality and composition. results of the rt-qpcr based gene analysis indicate the presence of sars-cov-2 genetic traces in different raw wastewaters. furthermore, selected samples have been sequenced using sanger technology to confirm the specificity of the rt-qpcr and the origin of the coronavirus. a comparison of the particle-bound and the dissolved portion of sars-cov-2 virus genes shows that quantifications must not neglect the solid-phase reservoir. the infectivity of the raw wastewater has also been assessed by viral outgrowth assay with a potential sars-cov2host cell line in vitro, which were not infected when exposed to the samples. this first evidence suggests that wastewater might be no major route for transmission to humans. our findings draw attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater. the current sars-cov-2 pandemic has far-reaching global consequences on public health, economic activities, and societies as a whole, which are unprecedented in many respects and cannot be fully assessed yet (who, 2020) . the number and proportion of persons infected with covid-19 are mainly determined based on individual testing and laboratory-based bio-molecular diagnostics using, e.g., reverse transcription-quantitative polymerase chain reaction (rt-qpcr). covid-19 cases are reported by regional health authorities and aggregated on the level of, e.g., the federal states in germany, as well as on a national level (rki, 2020) . it is expected that, based on the individual testing that is often triggered by symptoms of test candidates or their respective risk profile, the actual state of infection in a specific region can only be very roughly estimated (wurtzer et al. 2020) . oral swab samples (wu et al. 2020a) . therefore, it must be systematically assessed whether the virus might, in addition to respiratory droplets, also be transmitted via feces in wastewater (nemudryi et al. 2020 , wu et al. 2020a . it could be shown that the duration of viral shedding differed among patients between 14 and 21 days past the onset of the infection . furthermore, the magnitude of shedding varied from 10 2 to 10 8 rna copies per gram feces (lescure et al. 2020 , pan et al. 2020 . wastewater-based epidemiology (wbe) has been suggested as a potentially useful complementary tool to gain insights into the degree of infection spread in a population , choi et al. 2018 , rodriguez-manzano et al. 2010 . recently, various studies detected sars-cov-2 rna in wastewater worldwide (cf. (medema et al. 2020) , france (wurtzer et al. 2020) , usa , australia , and italy (la rosa et al. 2020) . wurtzer et al. (2020) reported the analysis of sars-cov-2 genes in the greater paris (france) area and were able to correlate trends in gene occurrence in the wastewater of different wastewater treatment plants (wwtp) with the number of infected individuals. medema et al. (2020) have shown a good correlation between the number of covid-19 cases and the gene concentration in the wastewater of different dutch cities. however, the studies differed in, e.g., the type of samples, processing procedure, and targeted genes (like n1, n2, n3, and orf1ab) in rt-qpcr analysis. only a few studies were complemented by infectivity tests of the genes to determine whether the genetic material was present in intact virus particles or as free nucleic acids. furthermore, only a few studies comprised phylogenetic analyses to better identify the genetic profile of the obtained material. an overview of recent studies conducted and reported in 2020 is given in table 1 . nine municipal wwtp operated by six different water boards were selected for analysis throughout north-rhine westphalia (germany). the plants differed in their design capacity, treatment processes, and connected catchment area characteristics (table 2 ). using installed autosampler devices, the operators of the wwtp collected 24 h flow-dependent composite samples on april 8 th , 2020, during dry-weather conditions, either midnight to midnight or between the morning of april 8 th , and the morning of april 9 th . in addition to raw wastewater (sewage) inflow samples collected after the sand trap, treated sewage was sampled at selected locations ( j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f frozen wastewater samples were thawed at 4°c, and a total of 45 ml were further processed. first, wastewater was centrifuged at 4,700x g for 30 minutes without break, and the clear supernatant was harvested. purified wastewater was then concentrated using centrifugal ultrafiltration units (amicon® ultra-15 centrifugal filter unit, sigma). therefore, 15 ml of wastewater was added to the filter unit and centrifuged for 15 minutes at 3,500x g, and the concentrated supernatant was harvested. this was repeated twice until 45 ml of wastewater were completely concentrated (final volume of concentrated supernatant approximately 450 µl). for the solid phaseof the wastewater sample, the pellet of the 45 ml sample was first washed with deionized water to remove aqueous remains from the sample and centrifuged at 4,700x g for five minutes before being resuspended in 150 µl deionized water and centrifuged at 4,700x g for five minutes again. a volume of 150 µl of the supernatant was then harvested for further rna extraction, and the mass was determined (approx. 150 mg in influent and 5-10 mg in effluent). contaminated equipment (e.g., reaction tubes, tips, filter units) were collected and autoclaved according to the daily cleaning program in the lab. rna was isolated using the nucleospin rna virus kit (macherey nagel) following the manufacturer's instructions. briefly, 150 µl supernatant was mixed with lysis buffer supplemented with carrier rna. after binding on silica membranes, samples were washed several times and eluted in 50 µl rnasefree water. isolated rna was stored at -80°c. probe-based luna universal one-step rt-qpcr kits, 2 µl of rna were subjected to reverse transcription performed at 55°c for 10 minutes. initial denaturation was performed for 1 min at 95°c j o u r n a l p r e -p r o o f table 3 : sequences of primers and probe established and verified for the detection of sars-cov-2 (in patient material) by rt-qpcr. e_sarbeco_f1 acaggtacgttaatagttaatagcgt figure s1 . purified wastewater samples that tested positive for sars-cov-2 (p2, p5, p11, p12) were investigated for replication-competent viruses following a recently published procedure (hoehl et al. j o u r n a l p r e -p r o o f all physicochemical and chemical parameters were analyzed immediately after sampling following din, en, or iso standard protocols. if this was not possible, the samples were chemically stabilized and then measured within the approved timeline of the standard protocols. the ph and conductivity were measured using a sension mm374 hach lange instrument (en iso 10523 (2012-04), en 27888 ). for the content of the dry residues, a defined volume of wastewater sample was dried at 105°c for 24 h (din 38409-1 (1987-01)), and the residue was weighed by a sartorius a 120 s balance. ready-to-use-cell test kits were used to analyze the chemical oxygen demand (cod) with a macherey & nagel pf12 and vario 4 (test kits 985022, 985033 and 98029, iso 15705 (2003-01)). total organic carbon (toc) and total bounded nitrogen (tn b ) were analyzed via combustion using a shimadzu carbon/nitrogen analyzer (en 1484 (en (1997 , en 12260 (2003-12)). for the analysis of ammonia, nitrate, nitrite, total organic nitrogen, ortho-phosphate, and other ions such as chloride and sulfate, a gallery discrete analyzer with photometric and turbidity methods were used after filtration through a 0.45-µm filter (thermo fisher scientific, iso 15923-1 (2014-07)). two different types of population size markers (exogenous and endogenous) were measured by liquid chromatography coupled with mass spectrometric detection (lc-ms) and photometric detection with a discrete analyzer (choi et al. 2018 j o u r n a l p r e -p r o o f acetic acid were added to the methanol and water. the separation was run after injection of 10 µl sample with a linear gradient starting with 20% up to 90% and ending again with 20% methanol. all data were collected in srm mode by using mass-labeled internal standards (supplementary table s1) and xcalibur 2.0.7. sp1 software for data acquisition and quantification. the loqs are in the range of 10 to 30 ng/l. as endogenous parameters, creatinine and urea were quantified after filtration via a 0.45µm filter by using a gallery discrete analyzer (thermo fisher scientific). creatinine was measured by photometry at 540 nm as quinonimine-chromogen after enzymatic reactions of creatine with creatinase, sarcosine oxidase, and ascorbate oxidase with a modified clinical method using thermo fisher scientific reagent kit (enzyme. colortest pap 981896) (dörner 2003) . the urea-method is based on a standardized bathwater method. in the first step, the contents of urea and ammonium are detected simultaneously. after enzymatic reaction of urea with urease to ammonium, ammonium reacts with nicotinamide adenine dinucleotide phosphate (nadph) in a buffered solution at ph 8. the loss of nadph is directly proportional to the ammonium content (thermo fisher scientific test kit 981820). the urea content is calculated by subtraction of the ammonium content (iso 15923-1 (2014-07) from the total content of urea + ammonium. in germany, local health authorities report the number of covid-19 cases confirmed by laboratory diagnosis after aggregation from the community to district level to state and federal authorities. whenever available, the cumulative prevalence and the cumulative number of covid-19 patients recovered and deceased were obtained from community-level reports published on the homepages of the responsible local health authorities on april 9 th , 2020. if community-level reports were not available, district-level data published by the state health ministry of north rhine-westphalia (mags) on april 9 th , 2020, was used. since the catchment areas of wwtps rarely correspond to administrative boundaries, the cases were estimated by calculating weighted sums relative to the j o u r n a l p r e -p r o o f residents of each community connected to that sewer network. acute prevalence was calculated by subtracting reported recovered and deceased patients (table 4) . nominal incidences (i nom ) were calculated by dividing the estimated number of cases (n nom ) in a catchment area by the nominal number of connected residents to the sewer (pe nom ). we note that these are estimates with considerable uncertainty since cases might be unevenly distributed between neighbouring districts, and recovered cases are often not based on laboratory diagnosis but the end of ordered quarantine. in addition, the actual number of persons staying in the catchment area on the day of sampling (pe actual ) may also differ from the nominal number of connected residents (pe nom ). treating the complete flow of the river emscher, wwtp klem is partly treating water that was treated upstreaming by other treatment plants. based on the assumption of poor removal of sars-cov-2 in conventional wwtp, reported cases for klem in table 4 are upper estimates covering the complete upstream catchment. showed a sequence identity above 90%. the similarity of c2 and c4 amplicons to the sars-cov-2 specific sequence was 50% and 47.3%, respectively ( figure 4) . therefore, we conclude that the retained samples were negative for sars-cov-2. in wastewater treatment, solid residues are largely removed. we thus compared the amount of viral rna in the aqueous and solid-phase of both inflow and effluent samples. during centrifugation in j o u r n a l p r e -p r o o f the processing of the wastewater samples, solid and aqueous phases are separated. to our understanding and as shown in other studies (kocamemi et al. 2020 ), viral rna of solid and liquid phase needs to be considered in wastewater surveillance. when comparing the aqueous and solid phase of the samples, we found a one log unit higher sars-cov-2 rna copy number per ml in the solid phase (25 copies/ml) compared to the aqueous phase of the inflow sample (1.8 copies/ml), respectively ( figure 5a ). the difference between the aqueous and solid phase was less evident in the effluent sample, with 8.8 and 13 gene equivalents per ml, respectively ( figure 5b ). when comparing the aqueous phase only, the effluent exhibited a higher gene equivalent concentrations than the influent, which we attribute to repartitioning of gene material from the solid-to the liquid phase during wastewater treatment. in contrast, a difference of total viral copy numbers per ml between untreated and treated wastewater was not detectable ( figure 5a and b). this might be explained by the sample conditions. while the residence time of the wastewater is about one day at the wwtp, sludge is continuously exchanged with an average sludge age of 10 to 14 days. we assume that solidphase concentrations are lower-estimates since we did not test whether further extraction steps could mobilize additional sars-cov-2 rna from the solid phase. results are shown as mean + sd of two independent pcr measurements from same sample material. rdrp rt-qpcr c t values for standard range between 18 (standard 1 = 10 8 ) and 37 (standard 6 = 10 2 ). values of tested wastewater above c t 38 were considered negative for sars-cov-2. standard curve was calculated using bio-rad cfx manager software with e = 96.5 %, r 2 = 0.990 and slope = -3.408. although the wastewater in the investigated plants was treated by different processes, viral rna is not eliminated, as shown above. to test the infectious potential of untreated and treated wastewater, a cell culture model was used to analyse that impact ( figure 6 ). infection of differentiated caco-2 cells with cell culture grown, replication-competent sars-cov-2 (moi 0.01) led to the induction of a cytopathic effect (cpe after 48-72 hours). the cpe is characterized by round and shrinking cells that detach from the culture surface. observation of cpe after two to three days at a low moi indicated rapid to moderate replication of the virus. inoculation of differentiated caco-2 cells for ten days with purified and concentrated wastewater (p2, p5, p11, and p12) did not result in the production of infectious sars-cov-2 particles (data not shown), which suggests that treated sewage appears to be non-infectious even though viral rna fragments can be detected. due to the lack of cpe, quantification and verification by rt-qpcr was not performed. the wastewater samples analysed showed typical wastewater characteristics for the different types of wwtps studied (brückner et al. 2018; metcalf and eddy 2012; supplementary j o u r n a l p r e -p r o o f 4. discussion during the sars-cov-2 pandemic, testing different sample material (e.g., throat swabs) was a crucial step in the detection of hotspots and limiting transmission. . the first sars-cov-2 pcr assays were developed against the e-gene and the n-gene to detect the virus in patient samples, and numerous studies analysing wastewater for sars-cov-2 surveillance used e-gene or n-gene specific pcrs (medema et al., ahmed et al., wu et al., 2020) . to a minor proportion, also pcr targeting sars-cov-2 rdrp was used (wurtzer et al., 2020) . in this study, wastewater samples were analysed with two different pcrs targeting sars-cov-2 rdrp and m-gene. in an initial analysis, a control sample when sequencing the different pcr amplicons obtained using wastewater samples taken during the sars-cov-2 pandemic and long before the outbreak (retained samples), distinct results for the test sample (p12) which fits sars-cov-2/human/usa/ca-czb-1525/2020 isolate from ncbi database was found, but not for the retained samples suggesting an unspecific signal in rt-qpcr, respectively. although rna samples used for pcr are highly purified, there might have been environmental contaminants affecting pcr. pcr is a highly sensitive method to detect nucleic acid and is strongly affected by ph, salt concentration, and contamination with extraneous nucleic acids that lead to false-negative or false-positive results, respectively (schrader et al. 2012; paul et al. 1991 which are complicated to sequence. establishing qualitative nested pcr could be an option to improve sequencing results. furthermore, extraneous nucleic acids can be accumulated during the concentration process of the wastewater sample. while single studies may avoid the accumulation of extraneous nucleic acids using filter units with 10 kda cut-off and therefore also could limit detection of sars-cov-2, most studies used filter units with 100 kda cut-off or other methods for concentrating wastewater samples medema et al. 2020; nemudryi et al. 2020; la rosa et al. 2020a) . although no false-positive results are described in these analyses, these studies did not use retained wastewater samples before the sars-cov-2 pandemic as control. nevertheless to establish sars-cov-2 wastewater-based epidemiology as a reliable tool for early-warning and surveillance of the current or future covid-19 outbreaks, the method´s detection limit, precision, accuracy, and reliability must meet certain criteria to be useful for public surveillance. for example, early-warning requires a low detection limit to detect the very first cases, while in later phases of the epidemics, high precision and accuracy are needed to survey whether certain thresholds are exceeded, for example, the threshold currently set in germany of 50 active cases per 100,000 residents. on the day of our sampling campaign, we estimate the nominal acute incidence of covid-19 to range between 30 and 174 cases per 100,000 residents in the nine catchment areas studied (table 4 ). based on surveillance results (figure 4 ), our findings indicate that the rt-qpcr employed is capable of resolving acute incidences of 50 cases per 100,000 residents in dry-weather conditions, although the method has not yet been optimized in terms of sensitivity and precision to detect even lower incidence. the nine catchment areas studied differ considerably in size, wastewater flow (table 2) , and nominal numbers of covid-19 cases at the day of sampling ranging from 36 to 1,037 acute cases and from 85 to 1924 cumulative cases (table 4) . inter-comparing these nine catchment areas, we plotted the estimated cumulative and the acute prevalence against the measured sars-cov-2 load (figure 8 ), the latter calculated from rt-qpcr measured m-gene copy concentration ( figure 4 ) and the actual wastewater flow q actual on the day of sampling (table 2) . clearly, the estimated nominal number of j o u r n a l p r e -p r o o f covid-19 cases increases with increasing measured sars-cov-2 load, resulting in correlations both for cumulative and acute prevalence ( figure 8a and b). scatter in the correlations might be attributed to various reasons, including variations in virus rna recovery and uncertainty in the estimated prevalence data. in contrast, plotting the incidence against sars-cov-2 concentration did not yield a conclusive correlation (not shown), likely because the precision of the qpcr employed was not sufficient to discriminate relatively minor differences in the incidence prevailing in the studied catchment areas at the time of sampling, ranging from 30 to 174 cases per 100,000 residents (less than an order of magnitude, figure 8c and d). although it cannot be evaluated based on the available data, if this correction is superior, it illustrates the potential of bioindicators to account for differences in wastewater composition. in contrast to our approach, medema et al. (2020) reported catchment-specific correlations plotting the increase in concentration (not load) of the n gene assays (and cycles of the e gene assays) against the increase in cumulative incidence from 0.1 to 100 cases per 100,000 residents (3 log 10 units) over a 7-weeks outbreak of the pandemics. while catchment-specific temporal correlations may be less susceptible to interference with other factors, we consider rna load-based evaluations more reliable to cope with variations in wastewater flow, for example, stormwater events in combined sewer systems or variations in industrial wastewater production during a lockdown of industry and businesses. table s5 . our results suggest that detected rna fragments appear to be non-infectious based on smallvolume laboratory studies testing about 1 to 10 gene copies in one reaction. however, given the large capacity and flow rate of wwtps (table 2) , we calculate that each of the studied wwtp emits 6*10 10 to 6*10 12 sars-cov-2 gene equivalents per day to the receiving water bodies, some of which serve as crucial water resources for drinking water production, cooling water abstraction, public swimming, irrigation, recreation, and natural habitats. this viral load can be very roughly compared to the potential viral shedding by infected persons in the catchment area, although many uncertainties come with such an appraisal: rose et al. (2015) report a mean amount of feces probably depending on factors such as the stage of infection, and are given in the range of 10 2 -10 8 gene copies per g feces . wölfel et al. (2020) showed the development of gene copy findings on the time course of the infection. with, e.g., 100 infected persons in a sampled catchment, these assumptions would yield a viral load in a range as broad as 10 6 -10 12 gene copies per d in the wastewater. the findings, as shown in figure 8 , are within but on the upper end of this broad range. few studies have evaluated the fate of coronaviruses and other enveloped viruses in wastewater treatment and surface water (gundy et al. 2009 , ye et al. 2016 . in a recent review by kitajima et al. (2020) , it is stated that currently no applicable dose-response models exist for sars-cov-2, which would allow for a better risk assessment. in a recent review by foladori et al. (2020) , the current knowledge about the fate of sars-cov-2 in wastewater treatment systems was summarized, and the few studies undertaken indicate that there is a significant reduction in viral load through the treatment process, but still, gene fragments are detectable in effluents. although it cannot be excluded with the current testing system of rt-qpcr targeting sars-cov-2 genes that virus emitted in large quantities is still infectious, studies analyzing sars-cov-2 environmental stability support the suggestion that infectivity and replication-competence of released viral particles are very unlikely , van doremalen et al. 2020 . based on our findings, we conclude the following:  sars-cov-2 rna was detected in the inflow of all 9 studied wwtp at concentrations similar to those reported in other studies. our screening of different target genes points to shortcomings in the selectivity of gene primers used in studies previously published by other authors that might also detect genes non-specific to sars-cov-2 viruses.  using sanger sequencing, we confirmed human sars-cov-2 for the investigated sample taken during the sars-cov-2 pandemic outbreak. in contrast, positive signals in rt-qpcr were not  quantification of sars-cov-2 gene concentrations and loads in wastewater needs to consider both aqueous and solid-phase sars-cov-2 detection methods and establish robust standard protocols for clean-up and rt-qpcr measurements.  we observed poor removal of sars-cov-2 in all three of the studied conventional activatedsludge wwtp. full-scale ozonation at one plant seemed to reduce sars-cov-2 fragments in the effluent. membrane-based wwtp planned to be included in future studies.  we found that the total load of gene equivalents in wastewater correlated with the cumulative and the acute number of covid-19 cases reported in the respective catchment areas. we consider load-based correlations superior to gene copy concentration-based approaches. analysis of suitable bioindicators in the wastewater may improve the assessment of sars-cov-2 loads data in catchment areas.  while our results indicate that rna fragments are not infectious to caco-2 cells in cell culturebased assays, further studies are needed to evaluate the risk sars-cov-2 may pose in the water cycle. it is recommended to further develop and implement the concept of wastewater-based epidemiology as a complementary measure to survey the outbreak of the sars-cov-2 pandemic and impose catchment-specific measures if necessary. j o u r n a l p r e -p r o o f highlights:  the first study that reports the detection of sars-cov-2 in wastewater in germany using rt-qpcr.  the presence of sars-cov-2 was confirmed by sequencing, but also the risk of false-positive results has been elucidated.  in raw wastewater, 3.0 to 20 gene equivalents per ml were found in raw wastewater.  the replication potential tests were negative for wastewater samples.  sanger sequencing was required to differentiate the genetic pattern clearly. first confirmed detection of sars-cov-2 in untreated wastewater in australia: a proof of concept for the wastewater surveillance of covid-19 in the community potential fecal transmission of sars-cov-2: current evidence and implications for public health regressing sars-cov-2 sewage measurements onto covid-19 burden in the population: a proof-of-concept for quantitative environmental surveillance status quo report on wastewater treatment plant, receiving water's biocoenosis and quality as basis for evaluation of large-scale ozonation process making waves: coronavirus detection, presence and persistence in the water environment: state of the art and knowledge needs for public health sentinel surveillance of sars-cov-2 in wastewater anticipates the occurence of covid-19 cases wastewater-based epidemiology biomarkers: past, present and future klinische chemie und hämatologie identification of a novel coronavirus in patients with severe acute 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sars-cov-2 in wastewater: potential health risk, but also data source presence of sars-coronavirus-2 rna in sewage and correlation with reported covid-19 prevalence in the early stage of the epidemic in the netherlands wastewater engineering : treatment and reuse editorial perspectives: 2019 novel coronavirus (sars-cov-2): what is its fate in urban water cycle and how can the water research community respond temporal detection and phylogenetic assessment of sars-cov-2 in municipal wastewater. preprint not yet peer reviewed asymptomatic cases in a family cluster with sars cov-2 infection concentration of viruses and dissolved dna from aquatic environments by vortex flow filtration sars-cov-2 rna titers in wastewater anticipated covid-19 occurrence in a low prevalence area presence and vitality of sars-cov-2 virus in wastewaters and rivers analysis of the evolution in the circulation of hav and hev in eastern spain by testing urban sewage samples the characterization of feces and urine: a review of the literature to inform advanced treatment technology pcr inhibitors-occurrence, properties and removal first detection of sars-cov-2 rna in wastewater in north america: a study in louisiana on the origin and continuing evolution of sars-cov-2 optimized rt-qpcr approach for the detection of intra-and extra-cellular sars-cov-2 rnas aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 water, sanitation, hygiene, and waste management for the covid-19 virus. interim guidance virological assessment of hospitalized patients with covid-2019 prolonged presence of sars-cov-2 viral rna in faecal samples sars-cov-2 titers in wastewater are higher than expected from clinically confirmed cases evaluation o flockdown impact on sars-cov-2 dynamics through viral genome quantification in paris wastewaters clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series survivability, partitioning, and recovery of enveloped viruses in untreated municipal wastewater we thank the water boards emschergenossenschaft und lippeverband (eglv), erftverband, linksniederrheinische entwässerungs-genossenschaft (lineg), niersverband, ruhrverband, and wasserverband eifel-rur (wver) for their participation in the sampling campaign on short notice and for ongoing support to fiw e.v. in difficult times. we acknowledge the work of all water-sector and health-care personnel for their continued service to society throughout the pandemics. m.b. was supported through the global water futures (gwf) program that is funded through the canada first research excellence fund (cfref). key: cord-272113-j82z4q8x authors: akaji, kenichi; konno, hiroyuki title: design and evaluation of anti-sars-coronavirus agents based on molecular interactions with the viral protease date: 2020-08-27 journal: molecules doi: 10.3390/molecules25173920 sha: doc_id: 272113 cord_uid: j82z4q8x three types of new coronaviruses (covs) have been identified recently as the causative viruses for the severe pneumonia-like respiratory illnesses, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and corona-virus disease 2019 (covid-19). neither therapeutic agents nor vaccines have been developed to date, which is a major drawback in controlling the present global pandemic of covid-19 caused by sars coronavirus 2 (sars-cov-2) and has resulted in more than 20,439,814 cases and 744,385 deaths. each of the 3c-like (3cl) proteases of the three covs is essential for the proliferation of the covs, and an inhibitor of the 3cl protease (3cl(pro)) is thought to be an ideal therapeutic agent against sars, mers, or covid-19. among these, sars-cov is the first corona-virus isolated and has been studied in detail since the first pandemic in 2003. this article briefly reviews a series of studies on sars-cov, focusing on the development of inhibitors for the sars-cov 3cl(pro) based on molecular interactions with the 3cl protease. our recent approach, based on the structure-based rational design of a novel scaffold for sars-cov 3cl(pro) inhibitor, is also included. the achievements summarized in this short review would be useful for the design of a variety of novel inhibitors for corona-viruses, including sars-cov-2. the term coronavirus (cov) is derived from the crown-like spikes on the surface of the virus. covs are enveloped positive-strand rna viruses that infect various vertebrates, including humans. in the 1960s, two human coronaviruses, the human alpha coronavirus 229e (hcov-229e) and the human beta coronavirus oc43 (hcov-oc43) were discovered as causative agents of disorders such as the common cold or respiratory illnesses of mild to moderate severity [1, 2] . additionally, two human coronaviruses, the alpha coronavirus nl63 [3] [4] [5] and the beta coronavirus hku1 [6, 7] , were then identified in 2004 and 2005. at the same time, a contrasting new human beta coronavirus causing life-threatening illnesses (severe acute respiratory syndrome (sars)-cov) was identified in 2003 [8] [9] [10] . sars spread rapidly worldwide from its likely origin in southern china; the subsequent sars epidemic involved approximately 8500 patients, with more than 800 fatalities. after nearly a decade, in 2012, a new respiratory illness similar to sars, named middle east respiratory syndrome (mers), affected more than 1800 patients with a fatality rate of 36% [11, 12] . even after these pandemics, no effective therapy has been developed for covs infections, and the present worldwide pandemic of corona-virus disease 2019 has resulted in more than 20,439,814 cases and 744,385 deaths [13] . the causative agent of covid-19 is a virus called sars-cov-2 since its single-stranded rna genome is 82% identical to that of sars-cov [14] [15] [16] . sars-cov recognizes angiotensin-converting enzyme 2 (ace2) [17] on the host cell membrane as a specific receptor using the spike (s) protein of the virus. the interaction of the viral s protein with the host cell receptor is followed by membrane fusion of the virus and host cell, which transports the virus rna into the host cell. thus, an agent such as a soluble ace2, or an antibody to this protein, could be a possible inhibitor of the virus-cell interactions. the viral genome injected into the host cell is then translated and processed to virus-derived structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, as well as nonstructural proteins that are used for the construction of viral particles. therefore, inhibition of the processing reaction, essential for the generation of viral proteins, is a promising approach for the suppression of viral proliferation. papain-like protease (pl pro ) and chymotrypsin-like protease (cl pro ) are the essential proteases for the processing reaction; pl pro cleaves the n-terminal region of the viral precursor protein at three sites whereas 3cl pro cleaves the c-terminal region of the precursor protein at 11 sites. the features of the sars-cov 3cl pro , which cleaves the precursor protein at three times more sites than pl pro and has no known homologs in the host cell [18, 19] , making it an ideal target for antiviral agents. in addition, the sequence of sars-cov-2 3cl pro shares 96% homology with that of sars-cov 3cl pro , initially identified from the sars causative coronavirus [20] . these findings indicate that the studies on the sars-cov 3cl pro are robust bases for designing therapeutic agents for covid-19. in this short review article, the efforts to develop therapeutic agents for sars focusing on the inhibitors of sars-cov 3cl pro are described. instead of an exhaustive survey of the inhibitors [21] , we provide an overview of several typical inhibitors, and our recent efforts for the rational design of new scaffolds are discussed based on the inhibitory mechanism and structural interactions with sars-cov 3cl pro . first, the protein chemistry of the target enzyme, sars-cov 3cl pro , is described in brief, as a basis for the structural analyses of protease-inhibitor interactions. the 29.7 kb positive-strand rna genome of sars-cov contains two open reading frames (orfs 1a and 1b) encoding two large replicative polyproteins, pp1a (486 kda) and pp1ab (790 kda) [22, 23] . the expression of the orf1b-encoded region of pp1ab is involved in the orf produced by ribosomal frameshifting into the -1 frame just upstream of the orf1a translation termination codon. the pp1a and pp1ab polyproteins are processed by two cysteine proteases: papain like protease (pl pro ) and 3c-like protease (3cl pro , also called main protease, m pro ). the name 3c-like is derived from picornavirus 3c proteases having a similar substrate specificity, which is encoded on the noncapsid region 3c [24] . the sars-cov 3cl pro cleaves 11 sites of the pp1ab [25] and is indispensable for viral replication, but is not found in the host cells, which makes the sars-cov 3cl pro an ideal target for antiviral agents [18, 19] . sars-cov 3cl pro consists of 306 amino acid residues and contains the catalytic dyad defined by his41 and cys145. the n-terminal part (1-184, domains i and ii) is composed of a two-β-barrel fold forming the chymotrypsin-like architecture as in picornavirus 3c pro . the substrate-binding site is located in a cleft between these two domains. the c-terminal part (201-303, domain iii), containing five α-helices, adopts a globular fold. domain iii, connected to domain ii via a long loop, is a globular cluster of five helices, which is an essential architecture to hold the active dimer-structure of the protease (figure 1a ) [26] . most cov-derived 3cl proteases recognize a conserved (leu/ile)-gln ↓ (ser, ala, or gly) core sequence as a canonical sequence (the cleavage site is indicated by ↓) at the active center ( figure 1b) [27] . a comparison of the cleavage efficiencies of synthetic substrates containing the 11 cleavage sites of sars 3cl pro confirmed that the most suitable substrate was the n-terminal site of sars 3cl pro itself, suggesting that sars 3cl pro cleaves itself most efficiently. a study using a fluorescent-dodecapeptide as an experimental substrate also revealed a similar tendency of sars-cov 3cl pro substrate recognition, as well as differences between sars-cov 3cl pro and mers-cov 3cl pro [28] . moreover, recent studies on the sars-cov polyprotein processing using native mass spectrometry (ms) combined with collision-induced dissociation (cid) revealed a dynamic reaction, including substrate consumption, the rise and fall of intermediate products and complexation [29] . sars 3cl pro is a cysteine protease in which cys145 and his41 form a catalytic dyad ( figure 2 ). the initial step of the hydrolysis is the deprotonation of cys145-thiol by the imidazole group of his41 to increase the nucleophilicity of the thiol group, which then attacks the substrate carbonyl carbon. therefore, sars 3cl pro shows the highest enzymatic activity at approximately ph 7, retaining the un-protonated imidazole form of his41. after the nucleophilic attack, the c-terminal substrate fragment is released from the enzyme, leaving a covalently modified enzyme by thioester formation between the enzyme thiol group and the carbonyl group at the substrate scissile site. the thioester is then hydrolyzed by the nucleophilic attack of a deprotonated water molecule, and the corresponding c-terminal fragment of the substrate is released to generate the free enzyme. thus, compounds containing a functional group, a so-called "warhead" which interacts with the thiol group of cys145, may be promising agents for inhibiting the catalytic potency of the cysteine protease. the first step of our inhibitor studies on sars-cov 3cl pro was the production of a mature protease on a scale of several milligrams per batch by a conventional expression procedure using e. coli to establish the assay protocol and crystallization procedure; however, the amount of the protease obtained from the initial trial was insufficient compared with the expected amount. careful examination of our expression procedure to yield mature sars-cov 3cl pro suggested that the mature 3cl pro was somehow susceptible to degradation, particularly considering arg188 located at the connecting loop between domains ii and iii. therefore, a mutated protease r188i sars-cov 3cl pro , with an ile instead of an arg at position 188, was produced using e. coli to yield the expected amount with high homogeneity [30] . of note, the mutation increased the stability and maintained almost the same three-dimensional structure (pdb code 3aw1) as that of native sars-cov 3cl pro . the use of the mutant r188i sars-cov 3cl pro allowed the evaluation of enzymatic activity via conventional high-performance liquid chromatography (hplc) without the need to use any specific substrate with fluorescent substituents. after the sars-cov pandemic in 2003, numerous studies have been conducted to identify inhibitors of sars-cov 3cl pro [31] [32] [33] [34] . these inhibitors are structurally classified into two types: peptide-mimetic inhibitors and nonpeptide small-molecule inhibitors. based on the inhibitory mechanism, these inhibitors can also be classified into irreversible (those that form a covalent bond with 3cl pro ), and reversible (noncovalent) inhibitors (those that compete with the substrate). a variety of first-generation inhibitors reported after the first outbreak of sars-cov provided valuable insights into further modifications through structure-based design. in the following sections, several typical inhibitory mechanisms, as well as our structural modification studies of peptide-mimetic to nonpeptide inhibitors, are described. a peptide-mimetic protease inhibitor generally contains a substrate-like sequence and a "warhead" interacting with the catalytic center of the target enzyme. in the inhibitors for sars-cov 3cl pro , the substrate-like sequence is designed by optimizing the specific interactions at the s1' to s4 sites of the substrate. the warhead interacting with the active center thiol of sars-cov 3cl pro should be an electrophilic functional group, considering the reaction mechanism of thiol protease described above. among the various electrophilic functional groups, a michael acceptor is one of the most commonly used warheads to effectively form a covalent bond by a nucleophilic attack with a thiol. a peptide-mimetic inhibitor containing a michael acceptor involves the replacement of a substrate's scissile amide bond with an appropriate michael acceptor. following the interaction with the active center of the sars-cov 3cl pro , the nucleophilic cys145 thiolate generated by a proton-withdrawing effect caused by his41 at the catalytic dyad promotes a typical 1,4-addition to the α,β-unsaturated structure of the michael acceptor ( figure 3 ). the resulting protonated his41 donates the proton to an unstable intermediate anion to form 3cl pro covalently bound to the inhibitor. thus, the michael acceptor type compound acts as a type of suicide substrate to abolish the catalytic activity of the enzyme by covalent modification. in a series of evaluations, using compound 1 targeting rhinovirus 3cl pro as a starting compound, optimizations of the side-chain structures at p1' to p4 sites of the sars-cov substrate were conducted to develop sars-cov 3cl pro specific inhibitors [35] [36] [37] (table 1 ). the interactions of the sars-cov 3cl pro with some of these inhibitors were analyzed by x-ray crystallography (pdb codes 2zu4 and 2zu5), which confirmed that cys145 sulfur and the α-carbon of the michael acceptor at the p1' site formed a covalent bond of 1.99 å. inhibitors with a five-membered lactam ring at the p1 site showed much stronger inhibitory activities than those with glutamine at the p1 site. for the p2 site substituent, a hydrophobic isopropyl substitute was preferred over a rigid and planar phenyl substitute (compounds 3 vs 2), indicating that the s2 pocket of sars-cov 3cl pro will accept rather large hydrophobic substituents. the p3 substituent is expected to be directed towards the bulk solvent and would have no interactions with the protease. unexpectedly strong inhibitory activities of compounds 4 to 3 were probably due to shifting of the n-terminal substituent toward the p4 site by a neighboring bulky tert-butyl group inducing hydrophobic interactions with 3cl pro . in another series of studies [38] , the effect of methylene insertion between the reactive michael acceptor and the sessile site was investigated. the results clearly showed that no elongation of the michael acceptor structure toward the prime site was tolerated (table 2) , which strongly suggests strict recognition of the prime-site structure. halomethyl ketone groups can form a covalent bond by an apparent alkylation caused by a thiolate anion, because the halomethyl group makes the adjacent ketone group more susceptible to a nucleophilic attack. the initial nucleophilic attack of a thiolate of cys145 of 3cl pro toward the carbonyl group of the halomethyl ketone leads to the reversible formation of a tetrahedral thiohemiketal that resembles the intermediate configuration in substrate cleavage ( figure 4 ). the subsequent intramolecular rearrangement leads to the final product, a covalently modified inactive enzyme. a direct mechanism, in which the thiolate ion directly attacks the halomethyl carbonyl group to yield the alkylated enzyme, is less conceivable than the above intramolecular rearrangement pathway based on the experimental kinetics data. an initial study of a series of inhibitors containing p1 site n,n-dimethyl glutaminyl fluoromethyl ketone combined with different p2 site substituents revealed two possibilities. one was the effectiveness of a halogenated methyl group as a warhead, and another was the remarkable contribution of the p2 site substituent on the inhibitory activity, probably due to the specific interactions with 3cl pro (table 3a ) [39] . the antiviral activity assessed by cytopathic effect (cpe) inhibition in sars-cov infected vero cell cultures, revealed that compound 10, with p2-leu, can protect the cells against sars-cov infection with an ec 50 of 2.5 µm, as well as the low toxicity in mice. the p2 leu of 10 can be replaced by ile or val, resulting in slightly lower ec 50 values (compounds 11 and 12) . in addition, these active compounds were inactive against rhinovirus type-2 in a cell-based assay, suggesting that they are specific for sars-cov. in contrast, removal of these p2-site substituents abolished the inhibitory activity (compound 13), indicating that the hydrophobic interactions at the p2 site were essential for potent inhibition. studies on another series of halomethyl ketone type inhibitors revealed additional possibilities regarding p1 site substituent and the inhibitory reaction mechanism (table 3b ) [40] . the results indicated that hydrophobic p1 substituents, such as simple aromatic groups (phenyl and naphthyl) or an aliphatic bulky group, were tolerated as those of a rather complexed lactam ring, keto-glutamine analogs, or an α,β-unsaturated ester structure. these data also indicate that the corresponding s1 pocket of the sars-cov 3cl pro might accept a simple ring structure containing heteroatoms at this specific interaction site, which provides a clue to our design of a potent substrate-based inhibitor described later in this review. additional information obtained from these series of inhibitors is the effect of the halogen atom in the halomethyl ketone group. the rate of the final irreversible step of the inhibition pathway (k 3 in figure 4 ) is directly related to the acceptability of a nucleophilic attack for the eventual alkylation. the values of k 3 of compound 14-16 (2.8 × 10 −2 /s-1.5 × 10 −2 /s) indicated that the chloromethyl ketone was effective as a warhead, although the k 3 value varied by two-fold depending on the bulkiness of the p1 site substituent. in contrast, the k 3 value of bromomethyl ketone inhibitor 17 was too small to be measured, indicating that the irreversible step was very slow. indeed, inhibitor 17 behaved as a reversible inhibitor for several hours and irreversible inhibition of the enzyme activity was only noticed after a 12 h incubation with inhibitor 17. thus, a time-dependent bimodal mode of inhibition for this inhibitor 17 was suggested, in which the initial formation of a reversible complex (e-i*) occurred followed by rearrangement to an irreversible complex (e-i) after at least 6 h incubation. in the crystal structure of sars-cov 3cl pro complexed with inhibitor 17 (pdb code 3d62), a thioether bond (1.7 å) between the carbon originally bound to bromine and the sulfur atom of cys145 was clearly detected supporting the above bimodal pathway. a trifluoromethyl group is another electron-withdrawing group that makes a neighboring carbonyl group susceptible to nucleophilic attack. initial studies of trifluoromethyl ketone type inhibitors ( table 4 ) yielded in an n-protected tetrapeptide compound 22 (ic 50 = 10 µm) [41] . a lineweaver-burk plot obtained from the inhibition reaction by 22 confirmed a competitive inhibition mode in the initial 4 h reaction, whereas a time-dependent decrease in the enzymatic activity as a function of the inhibitor concentration was observed in the prolonged reaction. the slow formation of a covalent adduct caused by the nucleophilic attack of the thiol on the carbonyl carbon was assumed to provide a rational explanation of the kinetic results ( figure 5 ). computational molecular modeling also rationalized the covalent bond formation, illustrating a transition state mimic in the substrate cleavage by sars-cov 3cl pro . table 4 . inhibitory activity of substrate-based trifluoromethyl ketone compounds against sars-cov 3cl pro . further studies on the related groups using the trifluoromethyl equivalent revealed that a thiazolyl ketone group could increase the inhibitory activity ten-fold due to its high electrophilicity ( figure 6 ). structural optimization at the p4 site combined with a benzothiazole warhead yielded inhibitors 24 and 25, which both had ic 50 values in the low nanomolar range [42, 43] . a nitrile group used as a warhead in an anti-diabetes dpp4 inhibitor is another functional group that has been incorporated in the inhibitor for sars-cov 3cl pro (figure 7) . among the nitrile-based tetrapeptide inhibitors with different n-protecting groups (mic:5-methylisoxazole-3-carboxyl, boc: tert-butyloxycarbonyl, and cbz: carboxybenzyl), cbz-avlq-cn 28 was ten-times more potent (ic 50 = 4.6 µm) than the other inhibitors [44] . interestingly, the longer cbz-hexapeptide inhibitor, cbz-tsavlq-cn, was less active than the tetrapeptide inhibitor 28. the results suggest that the cbz group of 28 might function as the p4 substituent interacting at the s4 pocket of sars-cov 3cl pro . the crystal structures of the sars-cov 3cl pro in complex with the nitrile-based inhibitor (pdb codes 3vb7, 3vb4, 3vb5, and 3vb6) demonstrated that the inhibitor was covalently bonded to the thiol group of cys145 via the nitrile warhead ( figure 8 ). in addition, the tetrapeptide inhibitor cbz-avlq-cn inhibited 3cl pro from human coronavirus strains such as 229e (ic 50 = 2.3 µm), nl63 (ic 50 = 2.8 µm), oc43 (ic 50 = 1.6 µm), and hku1 (ic 50 = 1.3 µm). in contrast, the same inhibitor had no observable inhibitory effect on caspases, which are common host cells proteins and effectors of apoptosis. these results suggest that the nitrile-based inhibitor is a specific and broad-spectrum inhibitor for coronavirus 3cl pro . peptide aldehyde with a substrate-like sequence has the potency to be an effective inhibitor for thiol proteases as an aldehyde group is another reactive electrophilic group involved in the nucleophilic addition of a thiol to yield hemithioacetal (figure 9 ). initial studies on aldehyde-type inhibitors led to a potent inhibitor 31 ( figure 10 ; k i = 53 nm) through the extensive structural optimization of a prototype inhibitor 30 based on the structural evaluation of the sars-cov 3cl pro complexed with inhibitor 30 (pdb code 3sn8) [38, 45] . the analyses of the crystal structure of sars-cov 3cl pro complexed with the resulting inhibitor 31 (pdb code 2gx4) revealed that the distance of the thiol sulfur atom of cys145 and the carbonyl carbon of the aldehyde was 1.24 å, an equivalent distance to a covalent c-s bond. in addition, an oxyanion hole is expected to be formed by the coordination of the n-h of cys145 and gly143 at the s1 pocket, which would stabilize a tetrahedral intermediate of the nucleophilic addition reaction. the p1 and p4 substituents of inhibitor 31 are held by hydrogen bonds at the corresponding s1 and s4 pockets of sars-cov 3cl pro , and the p2 site cyclohexyl group formed hydrophobic interactions at the s2 pocket of the protease. in our own studies on a series of substrate-based peptide aldehyde inhibitors, optimization of the p1 substituent was first conducted as the scissile site substituent generally shows the main influence on the enzyme specificity. as summarized in table 5 , a series of replacement functional groups at the p1 site of the substrate-based inhibitor 32 revealed imidazole was the most effective substituent (inhibitor 35, ic 50 = 5.7 µm), which showed more than six-fold stronger inhibitory activity compared with 32 (ic 50 = 37 µm). further structural analyses of the r188i sars-cov 3cl pro complexed with inhibitor 35 (pdb code 3aw0) revealed several noteworthy information regarding the interactions with 3cl pro : (i) the large hydrophobic s2 pocket was not fully occupied; (ii) the p3 substituent was directed outward of 3cl pro , resulting in no interactions with the protease; (iii) the s4 pocket was not fully occupied, and additional interactions via hydrogen bonds appeared feasible, and iv) the p5 substituent extended outside of 3cl pro and is not involved in the interactions with the 3cl pro . further structure optimization based on these inspections provided a potent tetra-peptide aldehyde inhibitor 37 (ic 50 = 98 nm) [40] . the x-ray crystal structure analysis of r188i sars-cov 3cl pro complexed with 37 (pdb code 3atw) confirmed the expected tight hydrophobic interactions formed by the p2 cyclohexyl group and hydrogen-bond interaction at the p4 site thr (figure 11 ). table 5 . optimization of a series of substrate-based peptide aldehyde inhibitors. in these x-ray structural analyses, the distance between the carbonyl carbon of the aldehyde and the thiol sulfur of the cys145 was 2.30 å (figure 11 ), and the electron density of the aldehyde group could be fitted to a carbonyl sp 2 carbon. these findings suggest that the aldehyde of inhibitor 37 interacts with the thiol of 3cl pro noncovalently. in addition, pre-incubation of inhibitor 37 with 3cl pro prior to the addition of the substrate caused no change in the ic 50 value compared with that obtained by simultaneous mixing of the inhibitor 37, 3cl pro , and the substrate, which suggests that no stable covalent bonds were formed between the inhibitor 37 and 3cl pro . kinetic data obtained from lineweaver-burk plots confirmed that aldehyde type inhibitor 37 functions as a competitive inhibitor without forming an irreversible covalent bond. figure 11 . interactions of inhibitor 37 with r188i sars-cov 3cl pro . although the substrate-based inhibitors described above showed strong inhibitory potency, the in vivo instability of these peptide-based compound was expected to be a major drawback preventing use as an oral therapeutic agent. the development of nonpeptide small-molecular sars-cov 3cl pro inhibitors would be an approach to overcome these drawbacks. to this end, three different procedures have been generally employed: evaluation of natural products, high-throughput screening of synthetic compound libraries, and the rational design of a new scaffold of inhibitor. although a few examples of the inhibitors derived from natural products [46] [47] [48] or virtual or high-throughput screening [49] [50] [51] [52] [53] have been reported, examples of the structure-based rational design of a new scaffold are very limited, and the inhibitory activities are still moderate. nevertheless, the rational approach contributes largely to the design of a variety of novel scaffolds with high potential as a therapeutic agent for sars-cov -related diseases. in the following section, two rational approaches for the design of nonpeptide inhibitors derived from a peptide aldehyde are described. as an approach for nonpeptide inhibitor, serine was selected as an attractive scaffold as this commercially available proteinogenic amino acid has three functional groups available for modification: an alcohol, an amino, and a carboxylic acid group [54] . each group of the serine can be orthogonally modified, which makes it feasible to introduce respective substituents corresponding to the p1 to p4 sites into the scaffold independently. as the parent inhibitor to be modified, the highly potent peptide aldehyde 37 was used and substituents at the p1, p2, and p4 sites, as those are the sites that interact closely with the respective mode of sars-cov 3cl pro , were selected as the substituents to be introduced on the serine scaffold ( figure 12, compound 38 ). an energetically favorable conformation mimicking the parent inhibitor 37 was then sought by molecular mechanic's calculation performed with spartan combined with docking simulations by gold. the results of the initial trial suggested an unexpected positioning of the substituent, in which the cyclohexyl group at the serine amid carbonyl occupied the s1 pocket instead of the expected s2 pocket of 3cl pro . similarly, it was reported by bai et al. that a cinnamoyl derivative was expected to interact with 3cl pro at the s1 , s1, and s2 pockets ( figure 12, compound 39) based on simulations using autodock 3.0. considering the contrasting results obtained from both simulations, a hybrid scaffold was designed combining bai's derivative and the serine derivative ( figure 12 ). extensive sar studies of the hybrid scaffold focusing on the p1' and p4 substituents resulted in an optimized compound 41 (ic 50 = 30 µm) as a novel small molecule inhibitor derived from serine. docking simulations of compound 41 with sars-cov 3cl pro confirmed the expected interactions at the s1', s1, and s4 pockets ( figure 12 ). another approach starting from the same peptide inhibitor 37 was based on closer inspection of the interactions with the sars 3cl pro [55] . previous analyses of the crystal structure of the sars-cov 3cl pro complexed with inhibitor 37 (pdb code 3atw) revealed that a cyclohexyl substituent of cyclohexylalanine (cha) at the p2 site was well packed in the hydrophobic s2 pocket and formed a critical interaction to make 37 a highly potent inhibitor. detailed structural evaluation of this hydrophobic pocket revealed that the p2 site cyclohexyl ring was rather close to the peptide backbone. in the crystal structure, the distance of the position 2 carbon (c2) of the cyclohexyl ring to the α-amide nitrogen of cha at the p2 site was estimated to be 3.48 å. the distance is approximately equal to the sum of two covalent bonds, and the connection of the two atoms by a methylene linker appears to be feasible, yielding a novel fused ring structure, a decahydroisoquinoline scaffold, that acts as a hydrophobic substituent at the p2 position ( figure 13 ). in addition, this fused ring scaffold can be a core scaffold to arrange the p1 site imidazole and active site functional aldehyde at the required positions. the acyl substituent on the nitrogen atom in the decahydroisoquinoline scaffold is expected to be a new substituent providing additional interactions with 3cl pro . to assess the validity of the above design, all possible configurations at the fused ring of the decahydroisoqunolin scaffold were separately synthesized by a combination of enantiomer resolution and pd-catalyzed stereoselective cyclization reaction. each synthesized derivative showed moderate but clear inhibitory activity, although the potency was different depending on the configuration. a specific configuration of inhibitor 42 (figure 13 ) was the most effective configuration, confirming the utility of the decahydroisoquinoline scaffold as a hydrophobic core. in addition, the acyl group on the nitrogen atom of the scaffold showed a limited effect on the inhibitory activity. x-ray crystal analyses of sars-cov 3cl pro complexed with inhibitor 42 and related compounds (pdb codes 4tww, 4twy, and 4wy3) rationalized the effective interactions causing these differences. the distance between the carbonyl carbon of the aldehyde of 42 and thiol sulfur of cys145 was 2.43 å, suggesting that the decahydroisoquinoline inhibitor was a competitive inhibitor like the parent peptide inhibitor 37 ( figure 14) . the p1 site imidazole of 42 located at the s1 pocket and the nitrogen atom of the imidazole formed hydrogen bonds, similar to the parent inhibitor 37. the decahydroisoquinoline scaffold of 42 adopted a trans-fused configuration and occupied most of the s2 pocket of 3cl pro directing the p1 site imidazole and warhead aldehyde into the active center. in contrast, the acyl substituent bound to the nitrogen of the fused ring was located on the surface of 3cl pro , where an additional interaction with the protease might be possible. to improve the moderate inhibitory activity of above decahydroisoquinoline-type inhibitor 42, the overall interaction mode of 42 was compared with that of the parent peptide inhibitor 37. an overlay of both interaction modes suggested that the nonprime site interactions of the parent inhibitor 37 were missing in the interaction mode of the decahydroisoquinoline-type inhibitor 42 ( figure 15 ). a detailed evaluation of the overlay of above both inhibitors complexed with 3cl pro revealed that the distance between the α-nitrogen of cha in the peptide aldehyde and 4-position of the decahydroisoquinoline scaffold was estimated to be 1.45 å, a distance equivalent to a covalent bond. thus, a dipeptide unit as a nonprime site substitute was introduced at the 4-position of the decahydroisoquinoline scaffold to yield a novel prototype inhibitor 43 ( figure 16 ) [56] . the synthesized inhibitor 43 showed 2.4 times greater inhibitory activity (ic 50 = 26 µm) than the initial decahydroisoquinoline-type inhibitor 42, which strongly suggested the positive effect of the 4-position substituent, probably through interaction with the nonprime site pocket of 3cl pro . structural optimization of this nonprime site substituent should be the next step to create a novel lead structure of nonpeptide small-molecule inhibitors based on a rational design. the inhibitor design briefly surveyed in this short review is a potential starting point for the development of anti-sars-cov agents; of note, most inhibitors described in this review have inhibitory potency against cov as well as good physicochemical and pharmacodynamics properties necessary for in vivo use. indeed, the inhibitory potencies against cov in cells were confirmed for several compounds, including peptide-based inhibitors and small-molecule inhibitors. the low toxicity for cells was also examined for a few compounds, although further in vivo studies are required. reevaluation of natural products such as flavonoids or indian medicinal plants is an alternative approach to design clinically useful inhibitors for sars-cov 3cl pro [57, 58] . enzymatic evaluations of the 3cl protease of sars and mers is another base for the development of therapeutic agents for sars related respiratory diseases such as covid-19. recent studies on the catalytic mechanism of the sars-cov 3cl pro and mers-cov 3cl pro revealed detailed insights regarding the difference in catalytic efficiencies between 3cl pro from sars-cov and mers-cov, and identified a potential allosteric site for inhibitor design. 30 these findings should contribute to the development of therapeutic agents based on sars-cov-2 3cl pro . in addition, the crystal structure of sars-cov-2 3cl pro revealed the potential effectiveness of an inhibitor designed on the basis of sars-cov 3cl pro ; of notes, this may be another basis for the development of therapeutic agents for covid-19 [59] . the rational design of a novel scaffold starting from a peptide-based inhibitor discussed in this review would be an alternative way to design novel cysteine protease inhibitors. although compounds showing antiviral activity can be discovered by screening a library composed of approved drugs or therapeutics in clinical development, as used in the present measures devised to combat covid 19, the development of novel and specific anti-sars cov inhibitors based on the achievements described in this review should be an alternative approach to consider, in the context of the treatment of sars-related infectious diseases. additionally, polymerase inhibitors and others should be considered, to target different vital routes, and effectively combat sars-cov-2 since multiple targets are useful to avoid resistance. author contributions: both authors (k.a. and h.k.) equally contributed to this article. all authors have read and agreed to the published version of the manuscript. funding: this work was supported, in part, by a grant-in-aid for scientific research 16h05104 given to ka from the japan society for the promotion of science. the authors declare no conflict of interest. cultivation of viruses from a high proportion of patients with colds a new virus isolated from the human respiratory tract identification of a new human coronavirus human coronavirus nl63 new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia a major outbreak of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome the newly emerged sars-like coronavirus hcov-emc also has an "achilles' heel": current effective inhibitor targeting a 3c-like protease assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors world health organization. covid-19 situation reprorts a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus coronavirus main proteinase (3cl pro ) structure: basis for design of anti-sars drugs biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors an overview of severe acute respiratory syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus cleavage of small peptides in vitro by human rhinovirus 14 3c protease expressed in escherichia coli mechanisms and enzymes involved in sars coronavirus genome expression dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors 3c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism comprehensive insights into the catalytic mechanism of middle east respiratory syndrome 3c-like protease and severe acute respiratory syndrome 3c-like protease processing of the sars-cov pp1a/ab nsp7-10 region evaluation of peptide-aldehyde inhibitors using r188i mutant of sars 3cl protease as a proteolysis-resistant mutant synthesis and evaluation of keto-glutamine analogues as potent inhibitors of severe acute respiratory syndrome 3cl pro design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors cinanserin is an inhibitor of the 3c-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro high-throughput screening identifies inhibitors of the sars coronavirus main proteinase inhibition of the severe acute respiratory syndrome 3cl protease by peptidomimetic alpha,beta-unsaturated esters synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent sars coronavirus 3cl protease inhibitor structural basis of inhibition specificities of 3c and 3c-like proteases by zinc-coordinating and peptidomimetic compounds structure-based design, synthesis, and evaluation of peptide-mimetic sars 3cl protease inhibitors design and synthesis of dipeptidyl glutaminyl fluoromethyl ketones as potent severe acute respiratory syndrome coronovirus (sars-cov) inhibitors development of broad-spectrum halomethyl ketone inhibitors against coronavirus main protease design, synthesis, and evaluation of trifluoromethyl ketones as inhibitors of sars-cov 3cl protease development of potent dipeptide-type sars-cov 3cl protease inhibitors with novel p3 scaffolds: design, synthesis, biological evaluation, and docking studies design, synthesis, and biological evaluation of novel dipeptide-type sars-cov 3cl protease inhibitors: structure-activity relationship study design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus 3c-like proteases peptide aldehyde inhibitors challenge the substrate specificity of the sars-coronavirus main protease synthesis, modification and docking studies of 5-sulfonyl isatin derivatives as sars-cov 3c-like protease inhibitors chalcones isolated from angelica keiskei inhibit cysteine proteases of sars-cov evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors virtual screening identification of novel severe acute respiratory syndrome 3c-like protease inhibitors and in vitro confirmation structure-based virtual screening against sars-3cl(pro) to identify novel non-peptidic hits identification of novel drug scaffolds for inhibition of sars-cov 3-chymotrypsin-like protease using virtual and high-throughput screenings discovery, synthesis, and structure-based optimization of a series of n-(tert-butyl)-2-(n-arylamido)-2-(pyridin-3-yl) acetamides (ml188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (sars-cov) 3cl protease discovery of n-(benzo[1,2,3]triazol-1-yl)-n-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (sars-cov) 3cl pro inhibitors: identification of ml300 and noncovalent nanomolar inhibitors with an induced-fit binding design and synthesis of a series of serine derivatives as small molecule inhibitors of the sars coronavirus 3cl protease fused-ring structure of decahydroisoquinolin as a novel scaffold for sars 3cl protease inhibitors evaluation of a non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold as a sars 3cl protease inhibitor inhibition of sars-cov 3cl protease by flavonoids covid-19: a promising cure for the global panic structural plasticity of sars-cov-2 3cl m pro active site cavity revealed by room temperature x-ray crystallography this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-264814-v4wnmg03 authors: flanagan, katie l.; best, emma; crawford, nigel w.; giles, michelle; koirala, archana; macartney, kristine; russell, fiona; teh, benjamin w.; wen, sophie ch title: progress and pitfalls in the quest for effective sars-cov-2 (covid-19) vaccines date: 2020-10-02 journal: front immunol doi: 10.3389/fimmu.2020.579250 sha: doc_id: 264814 cord_uid: v4wnmg03 there are currently around 200 sars-cov-2 candidate vaccines in preclinical and clinical trials throughout the world. the various candidates employ a range of vaccine strategies including some novel approaches. currently, the goal is to prove that they are safe and immunogenic in humans (phase 1/2 studies) with several now advancing into phase 2 and 3 trials to demonstrate efficacy and gather comprehensive data on safety. it is highly likely that many vaccines will be shown to stimulate antibody and t cell responses in healthy individuals and have an acceptable safety profile, but the key will be to confirm that they protect against covid-19. there is much hope that sars-cov-2 vaccines will be rolled out to the entire world to contain the pandemic and avert its most damaging impacts. however, in all likelihood this will initially require a targeted approach toward key vulnerable groups. collaborative efforts are underway to ensure manufacturing can occur at the unprecedented scale and speed required to immunize billions of people. ensuring deployment also occurs equitably across the globe will be critical. careful evaluation and ongoing surveillance for safety will be required to address theoretical concerns regarding immune enhancement seen in previous contexts. herein, we review the current knowledge about the immune response to this novel virus as it pertains to the design of effective and safe sars-cov-2 vaccines and the range of novel and established approaches to vaccine development being taken. we provide details of some of the frontrunner vaccines and discuss potential issues including adverse effects, scale-up and delivery. there are currently around 200 sars-cov-2 candidate vaccines in preclinical and clinical trials throughout the world. the various candidates employ a range of vaccine strategies including some novel approaches. currently, the goal is to prove that they are safe and immunogenic in humans (phase 1/2 studies) with several now advancing into phase 2 and 3 trials to demonstrate efficacy and gather comprehensive data on safety. it is highly likely that many vaccines will be shown to stimulate antibody and t cell responses in healthy individuals and have an acceptable safety profile, but the key will be to confirm that they protect against covid-19. there is much hope that sars-cov-2 vaccines will be rolled out to the entire world to contain the pandemic and avert its most damaging impacts. however, in all likelihood this will initially require a targeted approach toward key vulnerable groups. collaborative efforts are underway to ensure manufacturing can occur at the unprecedented scale and speed required to immunize billions of people. ensuring deployment also occurs equitably across the globe will be critical. careful evaluation and ongoing surveillance for safety will be required to address theoretical concerns regarding immune enhancement seen in previous contexts. herein, we review the current knowledge about the immune response to this novel virus as it pertains to the design of effective and safe sars-cov-2 vaccines and the range of novel and established approaches to vaccine development being taken. we provide details of some of the frontrunner vaccines and discuss potential issues including adverse effects, scale-up and delivery. the recent emergence of severe acute respiratory syndrome coronavirus-2 (sars-cov-2), the cause of coronavirus disease , is wreaking havoc due to widespread dissemination throughout the world. on 11 march 2020, who formally declared that a global pandemic and by the end of august 2020, almost 25 million cases and over 800,000 deaths had been reported worldwide involving all continents, except antarctica (1) . strategies to identify cases and limit spread by widespread testing and physical distancing have been challenging to implement, healthcare and public health systems have been overwhelmed, resulting in continued escalation in many countries and profound effects on lives and livelihoods. while the majority of people are either asymptomatic or experience a mild respiratory infection, ∼20% of cases are more severe and require hospital admission (2) . reported mortality rates vary by geographic region, ranging from almost 20% in france to <1% in many other countries (3) . this wide discrepancy suggests selection bias due to differences in local testing strategies and capacity, consistent with differences in health system capacity, population demographics and other health determinants. certain groups such as the elderly and those with particular comorbidities are more likely to die of covid-19 (3) . healthcare workers in particular have experienced significant morbidity and mortality from covid-19 (4), causing clear psychological impacts and threatening delivery of healthcare services (5) . there is no known effective treatment for this virus and currently no available vaccine, with the result being that sars-cov-2 continues to spread throughout the virus naïve population of the world. the urgent need for effective sars-cov-2 vaccines cannot be overstated. immunization can not only protect individuals but also, if provided to enough people in a timely way with (even) partially protective vaccines, induce sufficient herd immunity to curtail the spread of this virus and reduce morbidity and mortality across the globe. the race to develop safe and effective vaccines has seen sars-cov-2 candidate vaccines developed at a scale and pace never imagined before: currently almost 200 potential vaccines are in various stages of development (6) (7) (8) . a range of vaccine design approaches and platforms have been employed. however, since >95% of candidate vaccines typically fail it is expected that the eventual number of successful vaccines may be only be a handful. they may also become available in different time frames and suitable for use in different populations. most vaccine candidates are currently in preclinical trials, but a number have entered phase 1 or phase 1/2 studies, with plans to rapidly scale up to phase 2 and 3. trials are being conducted at "pandemic speed" (8) and using novel designs. this early success has already seen cooperation and collaboration as well as significant funding across the globe. for example, the coalition for epidemic preparedness innovations (cepi), a notfor-profit global coalition launched in 2017 to deal with the worldwide threat of epidemic outbreaks, is playing a pivotal role in supporting many of the frontrunner vaccines (9) . herein, we review what is currently known about the immune response to sars-cov-2 and the various vaccine platforms being used to develop the sars-cov-2 vaccines. understanding the mechanism of action of the various candidate vaccines is the key focus of this review. we also discuss potential challenges at the immunological level, assessment of vaccine safety and scale-up and delivery. coronaviruses are enveloped positive-sense single stranded rna viruses belonging to the coronaviridae family. they infect birds and mammals causing a range of symptoms from respiratory to gastrointestinal disease (10) . a number of relatively common seasonal coronaviruses are known to infect humans (11) , causing mild respiratory illness ("the common cold"). two previous lethal human coronavirus diseases, namely severe acute respiratory syndrome (sars caused by sars-cov-1) (12) and middle east respiratory syndrome (mers caused by mers-cov) (7) arose in 2002 and 2012, respectively. they have a high mortality rate of ∼9 and 40%, respectively, but fortunately, neither reached pandemic levels. sars-cov-1 was able to be contained by public health measures to prevent human-to human transmission and then disappeared before vaccine development had progressed significantly. the genome of sars-cov-2 encodes for the structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n) as well as a number of accessory and non-structural proteins (figure 1 ) (13) . the m and n proteins give the virion its shape, and the s proteins appear as spikes on the viral surface giving it a solar corona appearance. the s glycoprotein (spike) exists in trimeric form and is the structure by which binding to the host cells occurs. the virus receptor, angiotensin-converting enzyme 2 (ace2), expressed on the figure 1 | structure of sars-cov-2 and key antigenic components. illustration of sars-cov-2 which is a single stranded rna virus. the key antigenic components being targeted in vaccine design are shown on the right, consisting of the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. the main emphasis for human vaccines is based on the spike (s) protein, consisting of an s1 binding region and s2 fusion and cell entry region. the s1 domain contains the receptor binding domain (rbd) responsible for binding to the ace2 receptor on the surface of host cells. following fusion, the s protein sheds the s1 region and undergoes a dramatic structural change to its post-fusional state in order for the virus to enter the host cells. surface of multiple human cells is engaged via the receptor binding domain (rbd), which is part of the s1 subunit of the s glycoprotein (14) (figure 1) . the s2 subunit consists of two heptad repeat fusion regions, hr1 and hr2, responsible for membrane fusion and cell entry. the rbd domain can either be down and buried or rotated up and primed for ace2 binding (15) . the hidden down state is the predominant state of the s protein trimer in sars-cov-2, and likely a mechanism the virus uses to hide the entry epitopes and evade the host immune response (16) . two s trimers can concurrently bind to an ace2 dimer. following s protein binding in its prefusion state, the s1 subunit is cleaved and shed permitting the s2 dramatic conformational changes which constitute the post-fusion state required for viral entry (17, 18) . innate immunity to covid-19: protection or hyper-activation? sars-cov-2 stimulates an innate immune response via pattern associated molecular patterns (pamps) expressed by the virus, which in the case of sars-cov-2 consists of viral rna and its intermediates produced during replication (19) . these conserved pamps stimulate multiple immune response pathways via pattern recognition receptors (prrs), including sensing by endosomal toll-like receptors (tlrs) 3 and 7, cytosolic retinoic acid-inducible gene 1 (rig-1) and melanoma differentiationassociated protein 5 (mda5). local tissue damage in the lungs also releases damage-associated molecular patterns (damps) further contributing to local inflammation. the resultant inflammatory response provides immediate antiviral immunity via activation of antiviral type 1 and 3 interferon (ifn) pathways leading to an upregulation of inflammatory cytokines such as interleukin 6 (il-6) and il-1β, further recruiting neutrophils, other innate immune cells and stimulating anti-sars-cov-2 adaptive memory t cells and b cells (20) (figure 2) . there is still much to understand about the immune response to sars-cov-2 and the immunological differences in those with mild as compared to severe infection. emerging data suggest that the innate response to sars-cov-2 is aberrant (21, 22) . for example, the early type i and iii interferon responses are relatively suppressed by sars-cov-2, an immune evasion strategy employed by the virus, leading to early failure to control the virus. furthermore, uncontrolled local inflammation, or what has been described as "cytokine storm, " leading to tissue damage with inflammatory cell infiltration and the acute respiratory distress syndrome (ards) is thought to characterize late stage, severe manifestations of covid-19 (23) (24) (25) . for example, patients with severe covid-19 had higher levels of il-6, il-2r, il-10, and tumor necrosis factor alpha (tnf-α) than those with moderate disease, the former of which correlated with clinical severity and death (26, 27) . those covid-19 patients requiring icu admission demonstrated greater plasma levels of il-2, il-7, il-10, granulocyte colony-stimulating factor (gcsf), interferoninducible protein 10 (ip-10), monocyte chemoattractant protein-1 (mcp-1), macrophage inflammatory protein-1α (mip-1α) and tnf-α than the non-icu covid-19 patients, all supporting enhanced innate immunity in the sicker patients (28) . of note, the il-6 levels described in covid-19 patients are a log-fold lower than those described in classic cytokine storm (29) . furthermore, il-10 is an immunosuppressive cytokine suggesting dysregulated immune homeostasis rather than pure inflammation. upregulated chemoattractant chemokines further recruit inflammatory cells including neutrophils, macrophages, natural killer (nk) cells and t cells resulting in further immunopathology (21) (figure 2 ). antibodies (abs) produced by activated b cells play a key role in anti-viral immunity via several mechanisms including viral neutralization, antibody-dependent cellular cytotoxicity (adcc), antibody-dependent cellular phagocytosis (adcp), and antibody-dependent complement activation (adca) (figure 3) . it is thought that generation of high levels of neutralizing antibodies (nabs) against sars-cov-2 are required for a successful human vaccine (18) . however, some patients recover without producing high levels of nabs and those with severe disease may experience an early rise in nabs (30) . nevertheless, most vaccine efforts are focused on the induction of nabs to the s protein in order to block attachment of rbd to the ace2 receptor on host cells (31) . as mentioned before, this domain is hidden in the s protein's prefusion state, presenting challenges to the success of rbd-based vaccines (16) . for this figure 2 | key components of the innate immune response to sars-cov-2. antigen presenting cells (apcs), such as monocytes, macrophages, and dendritic cells (dcs), recognize pattern associated molecular patterns (pamps) expressed by sars-cov-2 via their pattern recognition receptors (prrs), such as toll-like receptor (tlr) 3 and 7. this activates intracellular signaling pathways leading to the expression of type 1 and 3 interferons (ifns), which in turn activate innate immune cells to produce pro-inflammatory cytokines and chemokines. this leads to an influx and activation of neutrophils, further apcs and other innate immune cells, such as natural killer (nk) cells. reason, some groups have focused on eliciting nabs to the less immunogenic s2 subunit of the spike protein (16) and sars-cov-2 vaccines based on other antigens, including the n protein, are also being developed. a recent report from the us describes outcomes among 35,322 covid-19 patients, many of whom had critical illness, transfused with the plasma from people who have recovered from covid-19 (convalescent plasma [cp]) (32) . early cp infusion within 3 days of illness had a lower 7-day mortality than those treated after 4 or more days (8.7% vs. 11.9%). in this uncontrolled study, those who received higher levels of igg abs in the transfused plasma had a better mortality outcome. these data, alongside results from another uncontrolled study showing recovery in severely unwell covid-19 patients treated with cp, lends support to the notion that naturally acquired abs can be protective (33) . however, other plasma factors such as cytokines, defensins and other non-specific abs may also play a protective role in these studies (34) . sars-cov-2-specific nabs recovered from infected humans also protected syrian hamsters and rhesus macaques in challenge studies (35, 36) . ideally, a sars-cov-2 vaccine would induce long-lasting abs, but it is not yet known how long specific abs persist in sars-cov-2 infected individuals or how long they would persist after vaccination. indeed, several recent studies indicate rapid waning of sars-cov-2 nabs in some individuals following natural infection, although others maintained high levels to 60-94 days (37, 38) , raising concerns about the persistence of nabs post-immunization; whether nabs plateau or continue to decline over time is yet to be determined. by contrast, nabs to sars and mers have been detected up to 2-3 years after infection in human survivors (39) and a recent study reports nabs 17 years after sars infection, suggesting that long-lasting coronavirus-specific nabs can be induced in some instances (40) so hopefully ab mediated immunity to sars-cov-2 will be equally long-lasting. importantly, class-switched igg memory b cells to s and rbd have been demonstrated in covid-19 patients confirming the generation of b cell memory which can provide a rapid recall response on subsequent sars-cov-2 exposure (41) . furthermore, we do not yet know what level of nabs are required for protection (42) . the standardization of a range of assays to support vaccine studies, such as viral neutralization assays, to enable comparison of different vaccine candidates in different populations will be key to facilitating vaccine development, an issue which represents a current focus of the who (43). t cell adaptive immunity: protection, immune suppression or disease enhancement? processing and presentation of sars-cov-2 epitopes by antigen presenting cells (apcs) via human leukocyte antigen (hla) class i leads to activation of naïve cd8 + t cells which differentiate into cytolytic effectors (cytotoxic t lymphocytes [ctl]) that kill virus-infected cells. activation of t helper 1 (th1) cd4 + t cells via viral peptides presented on hla class ii alleles further enhances the cd8 + t cell response, while hla class figure 3 | key components of the adaptive immune response to sars-cov-2. the adaptive immune response is activated following viral uptake and antigen processing by a range of apcs. the apcs present viral antigen to b cells which then differentiate into antibody producing plasma cells. the neutralizing antibodies (nabs) then bind to key viral proteins, such as the spike protein, and neutralize their activity. other ab-mediated antiviral functions include antibody dependent cellular cytotoxicity (adcc), antibody dependent cellular phagocytosis (adcp), and antibody dependent complement activation (adca). cytotoxic cd8 + t cells kill virally infected cells via the production of granzymes and perforin and the expression of fas ligand (fasl), all of which mediate cellular apoptosis. a series of cd4 + t cell populations are involved in the adaptive cellular response to sars-cov-2. follicular helper t cells (t fh ) and th2 cd4 + t cells both provide help for b cell antibody production. th1 and th17 cd4 + t cells are also thought to play a role in the inflammatory response and viral killing. cd4 + regulatory t cells have been implicated with an immunoregulatory role in sars-cov-2 infection via the production of anti-inflammatory cytokines and contact-mediated cellular suppression. whether cd8 + tregs and bregs play a role is not currently known. ii-restricted follicular helper (t fh ) and th2 cells enhance virusspecific antibody production (figure 3) . effective viral clearance therefore requires a combination of cd8 + ctl and cd4 + t cell mediated enhancement of b cell and cd8 + t cell responses (20) . pro-inflammatory th17 also play a role as evidenced by their high frequency in lung biopsy specimens of covid-19 patients (44) (figure 3) . severe viral disease, associated with an over exuberant t cell response, leads to local inflammation and toxicity, thus activation of immune modulatory factors or checkpoints is crucial in limiting the damage. indeed, regulatory t cells (tregs) play a key role in the control of inflammation and immunopathology in other coronavirus infections (45) , but this has yet to be investigated for sars-cov-2 (figure 3) . most activated t cells subsequently apoptose and die, but some persist and provide long-term virus-specific t cell immunity. natural infection with sars-cov-1 induced long-lasting memory t cells up to 6 years after infection (46) and memory t cells persisted to 2 years after mers infection (47) , supporting the potential for long-term persistence in sars-cov-2 infection. these should provide protective immunity on subsequent exposure to the pathogen, the ultimate goal of an effective vaccine. severe covid-19 patients exhibit immunological features associated with t cell anergy or exhaustion. for example, the lungs of severe covid-19 patients contain high levels of cd8 + t cells expressing classic markers and genes of exhaustion (29) , while patients who have recovered from moderate or severe covid-19 seem to have robust memory t cell formation (48) . these infiltrating cells also express high levels of markers of activation and cytotoxicity. other studies have similarly demonstrated that more activated and, in some cases, more exhausted t cells develop in symptomatic covid-19 patients compared to levels during the prodromal stage (49) . those who experience mild-moderate disease maintain their lymphocyte counts and have more polyfunctional t cells; while studies in those with severe disease are contradicting, reporting both lower and higher cd8 + t cell cytotoxicity (49) . severe disease is associated with profound lymphopaenia lending support to the theory that the disease also causes immunosuppression, although lymphocyte trafficking to the site of infection may also be responsible (49) . a biphasic t cell response has therefore been proposed, characterized by an early cd8 + cytotoxic t cell response to control the virus followed by t cell exhaustion and depletion as a result of viral persistence over many weeks in some patients. this model could account for poorer outcomes in the elderly who have diminished t cell repertoires and better outcomes in children who have a diverse and abundant naïve t cell pool (29) . nevertheless, the likely early protective role for cd4 + and cd8 + t cells in sars-cov-2 clearance suggests that their induction should be exploited in sars-cov-2 vaccine development strategies. no vaccine is licensed or available for sars or mers, however, considerable preclinical development studies have been performed. a range of vaccine development approaches were explored, including live attenuated vaccines and inactivated vaccines (such as inactivated whole virus), soluble protein vaccines, viral vectors, nanoparticles, and dna vaccines; most of these platforms are also being utilized for sars-cov-2, as discussed below (50, 51) . most of the sars and mers subunit candidate vaccines have targeted the s glycoprotein and been examined in studies conducted in animal models, although n, m, and e protein-based vaccines have also been tested (20) . however, the fact that animal models, including mice and nonhuman primates, display only limited clinical manifestations of sars and mers, that it is not usually lethal, severely limit the translation of results into humans. to overcome this, animal adapted strains and transgenic animal models of severe disease have been developed (20, 52) . in virus challenge studies in animals, high titers of nabs to both sars-cov-1 and mers virus correlated with protection (51, 53) . relatively few studies have investigated the protective role of t cells, and while some correlate cd4 + and cd8 + t cell responses with protection, adoptive transfer and t cell depletion assays in mice suggest they are not necessary for protection in mice immunized with a dna-based sars vaccine (54) . furthermore, vaccination of mice with sars and mers candidate vaccines has been shown to result in th2 lung pathology with eosinophilic infiltration following wild-type virus challenge (54) (55) (56) (57) . this immunopathological effect has been associated with whole virus vaccines with and without adjuvant and linked with responses to the n protein in particular; s protein-based vaccines may have a lesser effect. an n protein-based dna sars vaccine also elicited a delayed type hypersensitivity reaction in mice, further cautioning against using this protein for sars-cov-2 vaccines (57). furthermore, s-based vaccines appear more immunogenic and protective in these studies and the duration of protection has been shown in challenge studies to last 3-12 months in mice immunized with a vesicular stomatitis virus (vsv)-based sars vaccine and animals immunized with dna and/or protein-based mers vaccines (23, 58) . of note, in the former study, protection against viral challenge was complete in younger mice but limited in senescent mice, highlighting potential issues with successful vaccination in older individuals (59) . the only sars vaccines to reach human phase 1 clinical trials were based on inactivated virus, soluble s glycoprotein and dna approaches (52) . mers vaccines tested in human phase 1 clinical trials include a dna-based vaccine alone (60) ; dna in combination with adenovirus or modified vaccinia ankara (mva) viral vectors as a prime-boost strategy (52, 61) ; and a chimp adenovirus vaccine (62) . efficacy against disease has not been tested in humans since it has not been deemed ethical to perform challenge studies with lethal viruses for which there is no effective treatment. the first sars-cov-2 genome was isolated from patients with pneumonia from wuhan, china in early 2020 and identified as a novel human coronavirus (63, 64) . since that time, multiple viruses have been isolated and sequenced from patients throughout the world providing an understanding of the evolutionary capacity and diversity of this pathogen (63) . structural genomics and molecular interaction analysis (interactomics) can be used to rapidly identify putative functional sites, facilitating rational vaccine design by identifying potential b and t cell epitope regions of key viral proteins (65) . several groups have used computational programs and immunoinformatics to predict cd4 + and cd8 + t cell and b cell epitopes across multiple sars-cov-2 antigens from pathogen sequence data to design novel sars-cov-2 vaccines (66, 67) . the genetic diversity and stability of these regions over time can then be characterized, and cross-reactivity predicted and examined to ensure a vaccine provides cross-strain immunity. the s gene of human coronaviruses is well-known to undergo genetic drift which could compromise sars-cov-2 vaccines based on this region (68, 69) . reassuringly, comparisons between the sequences of known sars-cov-1 b and t cell epitopes from the n and s proteins have been shown that some of the epitopes map identically to proteins from sars-cov-2 (70). importantly, these epitopes showed no mutations among 120 available sars-cov-2 sequences suggesting that they reside in stable parts of the s protein. the t cell epitopes also covered a broad range of hla alleles and could therefore be future targets for vaccine design. one issue with this approach is that the epitopes will be specific for human hla alleles, making it difficult to test them for protective efficacy in animal models. one way to overcome this problem is to use humanized mouse models which have functional human immune systems (71) , bearing in mind that mice can also be modified to express the ace2 receptor (72) . also, there are often animal homologs of human cd4 and cd8 t cell epitopes so it may still be possible to screen for efficacy. however, it should be borne in mind that due to the urgent need to develop an effective sars-cov-2 vaccine, animal challenge data are not a mandatory pre-requisite to progression to clinical trials; nevertheless, ideally these should be conducted as part of the vaccine development strategy. the recent development of a laboratory-adapted sars-cov-2 strain that is pathogenic in mice, provides an ideal animal challenge model for testing the efficacy of sars-cov-2 candidate vaccines in mice (73) . vaccine technology has been advancing rapidly and there is a plethora of approaches to constructing sars-cov-2 vaccines. these range from the original technique employed centuries ago in the smallpox vaccine development of using modified or killed whole organism, to protein and peptide-based vaccines, nucleic acid dna and rna vaccines and nanoparticle constructs. each approach has unique advantages and disadvantages including varying time to development and scalability, cost, stability, as well as anticipated safety and immunogenicity profiles; all approaches are represented among preclinical and clinical trials (58-60) (figure 4 ). some vaccine candidates, particularly protein-based vaccines, require an adjuvant to boost their immunogenicity (74) . to date, very few adjuvants have been licensed for human use since the original adoption of aluminum salts (alum) from the early 1920s, which bias immunity toward a th2 response (75) . several of the newer adjuvants consist of toll-like receptor agonists to stimulate innate immunity combined with alum, such as as04 which is based on the tlr4 ligand monophosphoryl lipid a (mpl). oil-in-water emulsion adjuvants including mf59 and as03 are stronger adjuvants than alum and have been adopted in several licensed influenza vaccines. the adjuvant as01, present in the malaria vaccine rts,s, combines mpl and a saponin qs-21 (75) . a wide variety of adjuvant approaches are being taken with the various sars-cov-2 vaccines in development, including those mentioned above, some of which are described in the relevant sections below (76) . many vaccines are given via the intramuscular route, including the majority of the current covid-19 vaccines in development ( table 1) . however, since sars-cov-2 causes infection via the respiratory tract, a vaccine targeting the mucosal immune system might be preferable (77) . mucosal vaccines have the advantage of being needle-free and thus practical for mass-administration, but immune tolerance induction can be an issue. the formulation, including the use of mucosal adjuvants, is therefore important to ensure stimulation of adequate local and systemic immunity (78) . several groups have developed mucosal sars-cov-2 vaccines. a deoptimized live attenuated whole virus vaccine is being developed for intranasal (i.n.) administration, and several adenovirus-based vaccines will also be administered via the i.n. route ( table 1 ). an oral probiotic pill-based sars-cov-2 dna vaccine (bactrl) is also planned for clinical trials ( table 1) . the sublingual route is also considered an attractive route for inducing mucosal immunity (77) . an alternative approach is to combine parenteral vaccines with adjuvants such as retinoic acid and caf01 which are known to induce protective iga mucosal responses (79, 80) . researchers are also investigating self-administered mechanisms for sars-cov-2 vaccines to overcome the need for a healthcare worker to administer the vaccine. for example, inovio tm have developed a novel hand-held cellectra r 2000 intradermal delivery device which uses a brief electrical pulse to reversibly open skin cell pores to allow the entry of their ino-4800 dna plasmid vaccine (81) ( table 1 ). the university of pittsburgh school of medicine has developed an intradermal microneedle patch that can deliver sars-cov-2 protein antigens, either s or rbd (pittcovacc), through the skin which will enter human trials soon (82) ( table 1) . this approach delivers antigen and danger signals to the high-density dermal apcs stimulating a highly effective immune response, even in the absence of adjuvant. it requires only low doses of antigen thus reducing production costs. indeed, this method induced more potent s1-specific nabs in mice than the traditional needle injections, with or without the inclusion of tlr ligand sequences (83) . the development of live attenuated vaccines requires the organism to be modified via passage multiple times under unique conditions in the laboratory until it loses enough key virulent protein and peptide subunit vaccines are usually combined with an adjuvant in order to enhance immunogenicity. the main emphasis in sars-cov-2 vaccine development has been on using the whole spike protein in its trimeric form or components of it, such as the rbd region. multiple non-replicating viral vector vaccines have been developed, particularly focused on adenovirus; while there has been less emphasis on the replicating viral vector constructs. nucleic acid-based approaches include dna and mrna vaccines, often packaged into nanocarriers such as virus-like particles (vlps) and lipid nanoparticles (lnps). nanoparticle and vlp vaccines can also have antigen attached to their surface or combined in their core. the immune cell therapy approach uses genetically modified sars-cov-2-specific cytotoxic t cells and dendritic cells expressing viral antigens to protect against sars-cov-2 infection. each of these vaccine approaches has benefits and disadvantages in terms of cost and ease of production, safety profile and immunogenicity, and it remains to be seen which of the many candidates in development protect against factors so as to not cause disease, but retains its ability to replicate in the vaccine recipient, and stimulate a robust immune response that protects against infection (84) (85) (86) . these vaccines tend to be highly immunogenic, do not require an adjuvant and provide long-lasting immunity. however, they are usually contraindicated in those immunocompromised or pregnant. it is difficult to develop a live attenuated sars-cov-2 vaccine quickly because of the time and knowledge required to ensure that it is suitably attenuated and all virulence factors removed. reverse genetics can be employed for the rational design of the ideal attenuated sars-cov-2 vaccine virus, for example by determining key virulence factors, as has been done for other coronaviruses (87). long-term maintenance of consistent live vaccine stocks is also problematic (85, 86) . despite the challenges, three codon-pair deoptimized live attenuated vaccines are currently undergoing pre-clinical testing, but none have yet progressed to clinical trials (7) ( table 1) . these three candidates are being developed by codegenix/serum institute of india, indian immunologicals ltd/griffith university and mehment ali aydinlar university in turkey, respectively (7). this approach relies on using a single virus strain which may not cross-protect against other strains, particularly as the virus continues to spread worldwide and mutates as selection pressure increases once immunity becomes more widespread. inactivated vaccines are produced by growing the virus and then killing or "deactivating" it so that it can no longer replicate. the whole virus can be used, although alternative approaches include splitting the virus with a detergent to disrupt it or purifying antigenic components to create a subunit vaccine (88) . these vaccines are safer than live-attenuated vaccines but less immunogenic and often require an adjuvant. there are currently four inactivated vaccines in human clinical trials and a further nine in the preclinical stages of development (tables 1, 2 ). an alum adjuvanted purified formaldehyde inactivated whole virus vaccine developed by sinovac biotech., called picovacc, has been shown to be immunogenic in mice, rats and non-human primates (96) . it induced nabs to both vaccine and non-vaccine strains and provided partial or complete protection of macaques in challenge studies and has now entered human clinical trials in china ( pre-clinical data confirming induction of high levels of nabs in several animal models and protection against intra-tracheal sars-cov-2 challenge in rhesus macaques after 2 doses of vaccine (97) . the institute of medical biology/chinese academy of medical science has also developed an inactivated whole virus construct that is in a phase 1/2 trial; and bharat biotech international ltd also has one in a phase 1/2 trial ( table 2) . rather than using the whole or a split fragment of the virus as the vaccine, antigenic proteins (or peptides) of the virus can be generated using recombinant technology in the laboratory instead (98) . this is the most popular approach in the sars-cov-2 vaccine development field, with 50 protein/peptide constructs in preclinical trials and 8 that have entered clinical trials ( table 1) . they are relatively simple vaccines to make and thus cheap to produce. a major advantage over whole virus vaccines is their relative safety since unnecessary reactogenic components such as lipopolysaccharides and toxins can be excluded. however, protein/peptide vaccines often require adjuvants and multiple doses in order to stimulate an adequate immune response. peptide vaccines usually consist of 15-30 amino acid b cell and t cell epitope regions of key antigens allowing for a precise and targeted immune response. peptidebased vaccines have been developed as candidates against several viral pathogens, but few have been licensed except for use in veterinary practice (99) . this is in part due to their inherent limitations including a narrow breadth of immune response, potential for incorrect confirmation for epitope recognition, lack of b cell receptor cross-linking for b cell epitopes, hla restriction for t cell recognition, failure to induce cross-reactive immunity against different viral strains and failure to elicit longlasting immunity. the use of whole proteins in vaccines broadens their immunogenic potential, however the protein can also lose its native structure and thereby lose immunogenicity. in addition, as knowledge of the immune response to sars-cov-2 is still in its early stages, so too is the understanding of precisely what may be the most conformationally optimal, immunogenic and safe protein or peptide to utilize; this will be discovered as vaccine candidates using this approach are examined in clinical trials. the majority of sars-cov-2 candidate vaccines in development are based on the s protein or its rbd region (figure 1 ) (100) . indeed, five of the current protein/peptide vaccine candidates in clinical trials target the full s protein and the other two target rbd only ( table 2) . some novel technologies have been employed to overcome the issue of the s glycoprotein losing its immunogenic conformational form. the university of queensland has developed a molecular clamp technique that uses proteins to stabilize the s protein in its coiled shape. this seeks to ensure that functional nabs are produced that will tightly bind to virus in its native form and thus prevent cell entry (101) ( table 2) . other groups have focused on using powerful adjuvants to ensure immunogenicity, for example novavax's recombinant s protein sars-cov-2 nanoparticle vaccine, nvx-cov2373, combined with an adjuvant called matrix-m1 tm . this is a saponin-based adjuvant that enhances apc recruitment and action and stimulates high levels of nabs and cell-mediated immune responses (102) . the s protein in nvx-cov2373 is present its native trimeric form and has been shown to stimulate high levels of nabs in mice and baboons in preclinical studies. primary phase 1 clinical trial results have just been reported from a randomized, observer-blind, placebo-controlled trial of nvx-cov2373 conducted in 131 healthy adults (90) ( table 2) . safety and immunogenicity were assessed for 2 doses of vaccine (5 and 25 µg) with (n = 106) or without (n = 25) the matrix-m1 tm adjuvant (a 50 µg dose). a strong correlation was observed between nab titers and anti-spike igg to s protein with the adjuvanted, but not the unadjuvanted, vaccine. levels were comparable to those in convalescent sera (r = 0.958). both 5 and 25 µg adjuvanted doses elicited responses of similar magnitude and every participant seroconverted by both assays. t cell responses measured in 16 participants showed that nvx-cov2373/matrix-m1 induces antigen-specific polyfunctional cd4 + t cell responses (ifn-γ, il-2, and tnfα) in response to spike protein stimulation; there was a strong bias toward this th1 phenotype. the safety data are discussed in section comparative safety and tolerability results for current sars-cov-2 vaccine candidates in clinical trials and this vaccine is now progressing to phase 2 trials. clover biopharmaceuticals's protein trimer tag© vaccine is combined with as02 adjuvant; the uq vaccine uses mf59 adjuvant; the vaxine pty ltd. s protein vaccine has a special advax tm adjuvant; and the medigen s based construct is combined with the adjuvant cpg ( table 2) . generex biotechnology, in partnership with epivax, have developed a sars-cov-2 vaccine (epv-cov19) based on li-key technology with undisclosed sars-cov-2 peptides (103). this uses synthetic peptides chemically linked to the 4-amino acid ii-key to ensure robust immune activation of th1 and th2 cd4 + t cells in particular, which in turn facilitate antibody production (104, 105) . multiple viral vectors have been used for vaccine delivery due to their ability to infect cells and deliver the gene product encoding antigenic proteins for production inside the host cell following administration, usually via the intramuscular (i.m.) route (106) . vector viruses are usually genetically modified to reduce their virulence and render them replication defective. adenoviruses and poxviruses are the most widely used non-replicating viral vectors, but many other viruses can also be adapted for vaccine delivery. replicating viral vectors, such as measles virus and vesicular stomatitis virus (vsv), can also be used for sars-cov-2 vaccine design; they mimic natural infection, rendering them self-adjuvanting and therefore more potent, and can be used in lower doses (107) . while subunit vaccines are more focused on induction of humoral immunity, viral vector vaccines are able to induce robust cell mediated immunity (cmi) in addition to abs and have been shown in animal models to protect against challenge with pathogenic coronaviruses (51) . a modified vaccinia ankara (mva) vector expressing a mers-cov recombinant protein induced t cell responses and nabs in mice, while a rabies virus-based sars-cov-1 vaccine protected mice against sars-cov-1 challenge (13). human adenoviruses are the most common non-replicating viral vectors being used for sars-cov-2 vaccine development; constituting 3 of the 5 in clinical trials and 7 of the 19 in preclinical development ( table 1) . the three in clinical trials include cansino's ad5-ncov, janssen's ad26covs1 (108) and gamelaya's ad26-based gam-covid-vac lyo vaccine ( table 2) . all three encode the s protein. while adenovirus vectors are well-tolerated and highly immunogenic in most people, preexisting immunity to the viral vector can hamper the induction of immunity, particularly after repeated doses. animal adenoviruses can be used as vectors instead of human ones to overcome this problem, which is the approach used for the oxford university chimpanzee adenovirus-based candidate sars-cov-2 vaccine chadox1 ncov-19 (also called azd1222) and the simian adenovirus-based reithera vaccine grad, both of which are in clinical trials ( table 2) . anti-vector immunity can still develop after the first dose of vaccine even if a simian adenovirus vector is used. despite the concern about anti-vector immunity, 4 of the 5 adenovirus-based sars-cov-2 vaccines in clinical trials are planned to be given as a single dose, whereas almost all the other vaccines in clinical trials require at least 2 doses ( table 2) . phase 1 and 2 trials have now been completed and published for the cansino (ad5-ncov) and oxford (chadox1 ncov-19) , the safety aspects of which will be discussed later in this article. the ad5-ncov randomized, double-blind, placebo-controlled phase 2 trial results involving 603 volunteers ≥18 years of age showed >95% seroconversion by rbd-elisa, but only 59% seroconversion of nab responses in the higher dose group and 47% in the lower dose group after a single immunization (93) . fifty two percent of volunteers had pre-existing anti-ad5 nabs, and those with higher anti-ad5 immunity had approximately half the rbd-specific elisa and sars-cov-2 nabs as compared to those with low pre-existing anti-vector immunity, supporting interference with vaccine immunogenicity. preliminary findings from a phase 1/2, single-blind, randomized controlled chadox1 ncov-19 vaccine trial conducted in 1,077 healthy volunteers aged 18-55 years induced nabs in 91% of participants after a single dose of vaccine, rising to 100% after a booster dose (91). neutralization titers were comparable to those observed in convalescent plasma from people who had recovered from covid-19. both vaccine constructs elicited t cell responses which peaked at 14 days post-immunization, as determined by interferongamma (ifn-γ) enzyme linked immunospot (elispot) assay (91, 92). day 14 responses appeared higher with chadox1 ncov-19 (median 856 per million peripheral blood mononuclear cells [pbmc] ) as compared to the highest dose used of ad5-ncov (mean 580 per million pbmc) in the phase 1 trial (92); ad5-ncov elicited a median of 100-110 sfu/million pbmc at day 28 in the phase 2 trial (93). intracellular staining confirmed production of ifn-γ, tumor necrosis factor (tnf) and interleukin-2 (il-2) by both cd4 + and cd8 + t cells following ad5-ncov immunization, with more cytokine detected in cd4 + t cells as compared to cd8 + t cells (92) . institute pasteur replicating viral vector vaccine candidate tmv-083, based on measles virus, is the only viral vector vaccine that has entered phase 1 clinical trials to date ( table 2) , but there are 17 more at the pre-clinical stage of development. these include 5 each based on influenza virus or vsv, 3 more measles virusbased candidates, and 1 each based on avian paramyxovirus, horsepox (called tnx-1800), newcastle disease virus, and yellow fever virus ( table 1) . the majority target the s protein or rbd, although one measles virus-based candidate also targets the n protein. two of the influenza-based candidates are designed for intranasal administration. a live attenuated yellow fever vector-based vaccine (yfs0) expressing the prefusion form of the s protein recently showed that a single dose protected most hamsters in a sars-cov-2 challenge model (109) . nucleic acid-based vaccines (dna or mrna) offer a costefficient and scalable approach to sars-cov-2 vaccine development (110) . the major nucleic acid-based approaches are described below. dna-based vaccines are stable and safe to handle. however, while there are multiple dna vaccine constructs targeting numerous viral infections in animal and human studies, to date none have been licensed for human use. naked dna can be injected and taken up by apc or apc can be directly transfected with plasmid dna encoding the target antigen. subsequent expression and presentation of the target antigen leads to the induction of antigen-specific cd4 + and cd8 + t cells and antibody production. however, dna vaccines tend to be poorly immunogenic, necessitating various strategies to improve immunogenicity such as the use of viral promotors, immunostimulatory sequences and adjuvants. nanocarriers such a virus like particles (vlps) can also be used to improve dna delivery, avoid its degradation and be immunostimulatory. there are safety concerns about potential integration into the host dna causing dysregulated gene expression although the risk of this is thought to be very low. a series of animal studies with sars-cov-1 nucleic acidbased vaccines have shown induction of nabs and cellular immunity, with partial protection against turkey cov challenge (13) and vaccine-induced ab-mediated protection to the s protein in mice (51) . twelve dna-based sars-cov-2 vaccines are currently in preclinical development and 4 more have entered phase 1/2 clinical trials ( table 1) . two dna-based candidates in clinical trials are administered i.m., whereas the other 2 are administered via the intradermal (i.d.) route ( table 2) . inovio pharmaceuticals is testing their cellectra r electroporation i.d. delivery device to administer their s protein-based dna plasmid vaccine ino-4800 which induces nabs and t cells in mice and guinea pigs (111) ( table 2 ). an alternative approach currently in pre-clinical trials is being taken by symvivo with their dna b. longum vaccine bactrl which is administered as an oral pill (112) . messenger rna (mrna) vaccines consist of an rna strand that codes for a target antigen (113, 114) . the two main types are those packaged and delivered in non-replicating form or those packaged with other rna as an in vivo replicating vaccine. they offer a promising alternative to conventional vaccine approaches due to their high potency, capacity for rapid development and potential for low-cost manufacture, which could prove crucial for global accessibility for a covid19 pandemic vaccine. they also have a good safety profile, since they are not made with pathogen and do not integrate into host dna. several challenges encountered by mrna vaccines include the need for packaging, for example into particles or liposomes, as naked rna is otherwise rapidly broken down and the need for freezing or refrigeration for wide scale delivery. there are currently 16 rna-based vaccines in pre-clinical trials and 6 have entered clinical trials ( table 1) . the clinical trial candidates include two lipid nanoparticle encapsulated rna sars-cov-2 vaccines that have already progressed to phase 3 trials ( table 2) . moderna/niaid have published preliminary results from their phase 1 dose-ranging, safety and immunogenicity trial of 2 doses of mrna-1273 encoding the s protein conducted in 45 healthy adults aged 18-55 years (94) . the s protein in mrna-1273 is stabilized in its prefusion state by 2 proline substitutions in the s2 subunit; and the lipid nanoparticle coat consists of 4 lipids. all participants developed rbd abs by elisa and sars-cov-2-specific nabs by neutralization assay after 2 doses in the upper range of those who have recovered from covid-19 (convalescent serum). t cell responses measured by intracellular staining for th1 (tnf, il-2, ifn-γ) and th2 (il-4, il-13) cytokines following stimulation with an s protein peptide pool showed a th1 cytokine bias and low-level cd8 t cell responses. an interim report from the phase 1/2 dose-ranging study of 2 doses of biontech's mrna s protein vaccine candidate bnt162 administered to adults aged 18-55 years elicited rbdbinding igg and sars-cov-2 nabs in the order of 1.8-2.8fold that of convalescent human plasma. t cell responses were not reported in this paper. this vaccine encodes trimerized rbd which is modified by adding a "foldon" trimerization domain to increase immunogenicity (114) . both mrna-1273 and bnt162 are now in phase 3 clinical trials in adults ≥18 years ( table 2 ). the safety data for both will be reviewed in a later safety section. the remaining rna-based candidates in clinical trials include three in phase 1 and one in phase 1/2 ( table 2) . the imperial college london lnp-encapsulated rna vaccine consists of an s protein-based self-amplifying rna construct (lnp-ncovsarna); designed because sarna vaccines induce a more stable dna product and are more immunogenic than conventional nucleic acid vaccines (115) ( table 1) . nanoparticle-based vaccine approaches have received increasing interest in recent years due to their good safety profile and high immunogenic potential, with an ability to efficiently target dendritic cells (dcs) for processing and presentation, providing a clear advantage over less immunogenic dna and rna vaccines (116) . materials used to construct nanoparticle vaccines include self-assembling viruses (virus like particles [vlps]), lipids (as liposomes), proteins, metals (e.g., gold) and polymers which also act as their own adjuvant (117) . many nanoparticles are highly stable and less prone to degradation than other constructs such as "naked" protein, peptide, dna and rna vaccines (116, 118) . selected antigens, which may be proteins or nucleic acid, can either be attached to the surface of the nanoparticles or combined in the particle core. they can be synthesized in a variety of shapes and sizes to induce robust innate as well as adaptive immunity. other modifications include altering the nanoparticle surface to target certain cells or enhance immunogenicity and packaging with tlr ligands and other immune modulators. one of the most popular approaches for viral vaccine development is engineering vlps consisting of self-assembled viral membrane in a monomeric complex which display the viral epitopes but lack multiple key viral components, ensuring they have no replication capacity (119) . the widely used human papillomavirus (hpv) vaccines are an example of this approach. a nanoparticle polypeptide vaccine displaying sars-specific b cell epitope repeats from the c terminal heptad repeat region of the s protein in its native coiled formation was shown to elicit nabs in mice (120) . a number of the protein and nucleic acid-based sars-cov-2 vaccine candidates use nanocarriers to package the vaccine product to improve stability and ensure efficient antigen processing (118) . as mentioned above, novavax's full length s protein construct nvx-cov2373 is a nanoparticle vaccine and the majority of mrna-based sars-cov-2 vaccines are packaged lnps, including mrna-1273 and bnt162. there are 12 sars-cov-2 vlp vaccines in preclinical development and just one in a phase 1 clinical trial ( table 1) . the latter vaccine developed by mendicago inc. consists of plant-derived recombinant vlps made by transfecting viral genes into the cell nuclei of leaves permitting transient expression of viral proteins which form into vlps which are extracted and purified for clinical use (121) ( table 2) . sars-cov-2 vaccine approaches based on car-t cell concepts are also being tested in clinical trials. the shenzhen genoimmune medical institute in china is using a lentiviral vector system to create viral minigenes which express viral proteins (s, m, e and n proteins) and immune modulatory genes (p polyprotein protease) to modify dendritic cells (dcs). the lv-dc presenting sars-cov-2 specific antigens will activate cytotoxic t cells. novel dc (lv-smenp dc) and antigenspecific cytotoxic t cell vaccines are currently being tested by s.c. injection or i.v. infusion in a phase 1/2 multicenter therapeutic clinical trial in participants ranging from 6 months to 80 years of age (122) ( table 2) . the same company has also developed artificial dcs expressing sars-cov-2 mini-genes for various viral proteins which are being tested in a phase 1 clinical trial as a s.c. injection in sars-cov-2 infected individuals in the same age range ( table 2) . autologous dcs expressing sars-cov-2 antigens have also been developed by aivita biomed inc. in usa and are being tested as a s.c. administered vaccine in phase 1 clinical trials in healthy adults >18 years of age (123) ( table 2) . whilst not suitable for large-scale delivery and use, immune cell therapy might be used in the context of pre-emptive therapy in high-risk patients such as the elderly and immunocompromised. there is increasing evidence that immunization with live vaccines can improve survival against non-vaccine targeted infections, a phenomenon termed "non-specific, " "off-target, " or "heterologous" effects of vaccines (124, 125) . some of the best evidence comes from studies of immunization with the tuberculosis vaccine bacillus calmette-guérin (bcg); for example, bcg has been shown in three randomized trials to markedly reduce all-cause mortality in low birthweight neonates (126) . bcg causes epigenetic modification of innate immune cells (monocytes and natural killer (nk) cells) thereby enhancing innate responses in a process called innate immune training (127, 128) . experiments in both murine models and humans have further shown that bcg protects against certain viral pathogens (129) . reports that people living in countries where childhood bcg is routinely given have lower covid-19 mortality have been posited as further evidence for protective effects of bcg (130). however, these observations are fraught with confounders, including difficulty ascertaining covid-19 infection rates in these countries, the time the virus first entered the country and differences in national control strategies (131) . furthermore, other studies contradict these findings, such as a geographic regression discontinuity analysis along the east and west german border showing that the covid-19 infection rates do not vary according to the bcg vaccination strategies employed during the cold war (132) ; and a study from israel showing that sars-cov-2 infection rates in adults did not differ by bcg at birth status (133) . even if childhood bcg immunization does not protect against covid-19, a bcg dose as an adult may provide some short-term protection. indeed, it has been widely postulated that the nonspecific beneficial effects of bcg might protect against covid-19, resulting in an explosion of interest in this vaccine as a protective strategy in recent months (134, 135) . this has led to a number of clinical trials being established in north and south america, australasia, africa and europe, all testing the ability of bcg vaccine to protect against sars-cov-2 infection, mostly in high-risk exposed healthcare workers (136) ( table 2) . these trials collectively aim to recruit >20,000 participants, with the largest trial of 10,078 healthcare workers currently recruiting across multiple sites in australia and europe. non-specific protective effects of live vaccines have also been postulated for oral polio vaccine (opv) (137) and measles vaccine (138) . as a result, a study of the protective effects of opv against covid-19 is due to commence in usa in june (139) , and a clinical trial of measles-mumps-rubella (mmr) immunization and covid-19 protection is being conducted in egypt ( table 3) . a consistent feature of the off-target effects of live vaccines has been their sex-differential nature; females often showing greater susceptibility to these immunomodulatory effects (124, 125, (140) (141) (142) . indeed, sex differences in targeted vaccine immunogenicity and adverse events have also been widely described (141, 142) . this raises the possibility that any off-target protective effect of live vaccines may differ between the sexes and that the sars-cov-2 vaccine candidates may show sex differences in immunogenicity and reactogenicity. it is possible, but uncertain, that strategies to use these already licensed vaccines may provide a modest level of protection against covid-19, including those at highest risk, such as healthcare workers. it is important to recognize, however, that data from these studies will still need to accrue whilst we are waiting for sars-cov-2 targeted vaccines to come through the pipeline. given the unprecedented speed at which targeted vaccines are being developed, this approach may not be necessary or required; furthermore, these existing live vaccines are not currently recommended for this indication, so should only be utilized in this context in the setting of a clinical trial. even if an efficacious vaccine is developed there are a number of immunological challenges that need to be considered. furthermore, there is the enormous challenge of mass production and rapid and equitable delivery ( table 4) . it is extremely unlikely that any of the sars-cov-2 vaccines will be 100% effective; while they may not prevent becoming infected, it is hoped that they will prevent progression to severe disease. indeed, the seasonal influenza vaccine is generally about 50% protective against infection but does decrease disease severity and hospitalization rates (143) . a recent study in which macaques were vaccinated with the oxford university and astrazeneca adenovirus vaccine, chadox1 ncov-19, found that the primates were protected from sars-cov-2-induced pneumonia (144) . however, the macaques still had high levels of virus replicating in their upper respiratory tract. it is hoped that even if the vaccines do not prevent infection in the upper airways, they may reduce viral load and disease severity and in turn, the amount of virus a vaccinated person transmits to others. even a modestly efficacious sars-cov-2 vaccine could mitigate the severity of this pandemic and be highly beneficial in a world struggling to contain this novel virus and its devastating social and economic effects. most vaccines are tested in healthy young adult males and non-pregnant women and, if safe, they are then tested in healthy children prior to licensure. this therefore raises the issue that any vaccines may initially have less empiric data available on use in certain key vulnerable populations such as the elderly, immunocompromised groups and pregnant women. it is plausible that vaccines may be considerably less immunogenic in older and frail elderly who experience the most severe outcomes from covid-19, hence the importance of adjuvants in many of the vaccine candidates, both for dose sparing and enhancing immunogenicity (145) . efficacy in this population is also likely to vary by the type of vaccine construct and adjuvant used. this has been seen for other diseases such as influenza, where the adjuvanted and high dose vaccines are more immunogenic than the standard inactivated vaccines in the elderly (146, 147) . similarly, for herpes zoster, the efficacy of the live-attenuated vaccine in the elderly is approximately 60% compared with >90% for a subunit adjuvanted vaccine (glycoprotein e and liposomebased as01b adjuvant) (148) . thus, ensuring studies can be conducted in these priority target groups, either pre-or earlypost deployment, will be critical and special formulations may be required to ensure adequate immunogenicity. throughout life, humans are repeatedly exposed to the endemic human seasonal coronaviruses and develop human cov (hcov) antibody repertoires that can potentially cross-react with sars-cov-2-specific antigens (149) . this early immune imprinting leads to preferential expansion of cross-reactive abs when a related antigen is encountered, in this case sars-cov-2 (150). this long-recognized process is called original antigenic sin (oas) (151) and is known to occur with several common viruses, such as influenza and respiratory syncytial virus (152, 153) . oas can either lead to a less effective immune response or cause enhanced immunity and immunopathology (149, 150, 154) . oas is one of the mechanisms responsible for the phenomenon of antibody dependent enhancement (ade) of immunity which is thought to occur in covid-19, whereby non-neutralizing cross-reactive abs against other coronaviruses enhance host cell entry and viral infectivity and worsen disease severity (150, 154, 155) . this was seen to occur in cats vaccinated against feline infectious peritonitis coronavirus (156) (157) (158) and certain sars and mers vaccine platforms also appear to have shown worsened immunopathology in animal challenge studies compared with placebo (159) . recently, it was shown in vitro using human cells that nabs to coronavirus s protein can also trigger ade by causing a conformational change of the s protein (160) . antigen-experienced older individuals are more susceptible to the phenomenon of ade, while less antigen-experienced younger individuals mount more targeted antibody responses to viral neoantigens. this may in part account for the greater immunopathology of covid-19 in older individuals. indeed, it has been shown recently that covid-19-naïve children and elderly have quite different cross-reactive sars-cov-2-specific antibody signatures, which would play differing roles upon challenge with the wild-type virus (161) . theoretically, a sars-cov-2 vaccine could skew the immune system toward production of less effective or even harmful cross-reactive hcov abs via these processes of oas and ade, rather than induce highly-targeted sars-cov-2-specific abs as intended (162) . indeed, anti-spike abs taken from critically unwell covid-19 patients with severe lung injury skewed macrophages in vitro into a pro-inflammatory phenotype, implicating the abs in driving the surge in lung-resident proinflammatory macrophages and subsequent pro-inflammatory cytokine release in the lungs (163) . the authors attribute an aberrant igg glycosylation pattern in severe covid-19 patients with their more pro-inflammatory properties so hopefully the iggs induced by vaccination will not have this problem. indeed, none of the current sars-cov-2 vaccines in human clinical trials have reportedly caused ade to date, although the number of human subjects studied to date is still relatively small. since animal challenge data are not required to progress to clinical sars-cov-2 vaccine trials, the opportunity to screen for potential adverse events including ade at the pre-clinical stage of development may be missed. having said that, many of the sars-cov-2 vaccine candidates have undergone considerable pre-clinical testing, including challenge studies. there was no evidence of ade in pre-clinical sars-cov-2 challenge studies in rhesus macaques following immunization with 1 dose of chadox1 ncov-19 (144) or 2 doses of the inactivated whole virus vaccine candidate bbibp-corv (97), both of which were protective in these studies. it is postulated that basing the vaccine on the s1 rbd terminal subunit of the s glycoprotein should overcome this problem by inducing nabs only, although this has not been proven and, notably, only a few vaccine candidates have taken this approach. furthermore, the in vitro conformational changes causing ade described above occur in the rbd domain, so this approach may not be successful. aberrant manifestations of t cell responses were observed in the 1960s for other viral vaccine candidates, such as inactivated vaccines against measles and respiratory syncytial virus (rsv), resulting in vaccine associated enhanced respiratory disease (vaerd) upon subsequent wild-type pathogen exposure (164, 165) . a major driver of this was accentuation of th2 cytokines, with resultant allergic (eosinophilic) and airway hyperesponsiveness. this was compounded by another mechanism related to the conformation of the vaccine antigen, resulting in the generation of excessive non-neutralizing ab. having a high amount of binding, but not neutralizing, ab caused immune complex deposition and complement activation with local inflammation in the presence of a high viral load. it will therefore be important for vaccine candidates to mitigate the risk of vaerd by considering the t cell profile induced by vaccination, avoiding th2 biased cd4 + t cell immunity and biasing toward cd8 + cytotoxic t cell responses. the alum adjuvanted inactivated vaccine candidate picovacc could theoretically drive a th2 skewed immune response. while this was not observed in macaque studies when analyzing th1 and th2 cytokine profiles, it is not clear from the paper what samples were used to measure these cytokines, clarification of which will be crucial in vaccine safety assessment (96) . many of the other vaccine constructs favor th1 immunity, as demonstrated in the published human trial results for several of the viral vector (91-93) and mrna vaccines (94) and the novavax nanoparticle matrix m adjuvant vaccine (90) . original antigenic sin also applies to t cell responses and therefore pre-existing cov-specific t cell memory may be enhanced by immunization with a sars-cov-2 vaccine leading to either suboptimal t cell responses or immunopathology (151, 166) . vaccine safety assessment is integrated into all stages of the clinical development pipeline for candidate vaccines and continues once vaccines are deployed into the population. randomized controlled clinical trials examine safety, as well as immunogenicity and efficacy. decisions on the participant numbers to be included in phase 3 studies will likely be powered on efficacy endpoints, but these studies also need to be of sufficient size and planned duration to ensure comparison of injection site, systemic and unanticipated or "unsolicited" adverse events between groups. comprehensive safety studies are particularly critical because some candidate vaccines use platform technologies that have not been examined extensively in human subjects to date, including some of the viral vectors, mrna and nanoparticle constructs, and because of the potential for enhanced disease and adverse events related to aberrant immune responses to be seen upon infection pre-and post-licensure. a list of adverse events of special interest (known as aesi) across all body systems, including immunological, cardiovascular, neurological, musculoskeletal and dermatological manifestations, have been agreed by the brighton collaboration in conjunction with cepi and the who with input from regulatory agencies and other experts, as have the associated case definitions and surveillance strategies (167, 168) . listed conditions include anaphylaxis, vasculitides, myocarditis, generalized convulsions and meningoencephalitis, among many others. comprehensive data on safety are essential to ensure that the benefit:risk ratio of vaccination is favorable, to support decision-making by policy makers on wide-scale program implementation among healthy people, and to ensure that individuals are sufficiently confident to accept vaccination. some of these aesi will require post marketing (phase 4) studies but are also undergoing evaluation in some of the preclinical and early phase evaluations, particularly the larger phase 3 studies. interim phase 1/2 safety results have recently been published for two leading adenovirus-based vaccines, chadox1 ncov-19 (91) and ad5-ncov (92, 93) ; two mrna based vaccine candidates, mrna-1273 (94) and bnt162 (95) ; an aluminum adjuvanted inactivated whole virus vaccine (89) ; and an adjuvanted recombinant protein nanoparticle vaccine, nvx-cov2373/matrix-m1 (90) . all candidates exhibited acceptable safety profiles, and while local and systemic reactogenicity was common for the mrna, adenovirus and adjuvanted protein nanoparticle constructs, the inactivated whole virus vaccine was considerably less reactogenic ( table 5) . reactions were generally mild to moderate and resolved within days. importantly, there were no serious adverse events reported for any of these vaccine candidates. pain at the injection site was a particularly common feature with the mrna vaccines (approaching 100% in some groups), and while it was the commonest reaction to the inactivated vaccine, it was still lower for this candidate than for the other vaccines (table 5) . headache, myalgia and fatigue were the other most commonly reported symptoms for the mrna, adenovirus and adjuvanted protein nanoparticle candidate vaccines ( table 5) . documented fever was relatively common for mrna and adenovirus candidates but ≤5% for the inactivated virus and adjuvanted protein nanoparticle candidates ( table 5 ). the chadox1 ncov-19 trial underwent a protocol amendment during the trial allowing several sites to administer paracetamol prior to vaccination and 6 hourly for 24 h. this slightly reduced reports of pain, feeling feverish, chills, mylagia, headache and malaise but had no effect on confirmed fever. pre-existing adenovirus 5 immunity, increased age and male sex were all associated with decreased fever following immunization with ad5-ncov (93) . in terms of blood parameters, immunization with chadox1 ncov-19 was associated with transient neutropaenia and bnt162b1 with lymphopaenia. in the ad5-ncov phase 1 and 2 trials reactogenicity was dose-dependent for pain, headache and fever; while it was dosedependent for almost all parameters for the mrna vaccines ( table 5) . as a result of the high reactogenicity after a single dose of bnt162b1, it was decided not to give a second dose. interestingly, the ten participants given a booster dose of the chadox1 ncov-19 vaccine had less reactogenicity after the second dose. the opposite was found for the bnt-162b1 mrna and nvx-cov2373/matrix-m1vaccines for which reactogenicity was greater after the second dose (90, 95) . these favorable safety results (alongside the good immunogenicity reported earlier in this article) have allowed the progression of the adenovirus and mrna candidate vaccines to phase 3 clinical trials. whilst the sars-cov-2 vaccine pipeline is progressing at unprecedented speed, there is a concern that suppression of viral transmission in many countries will make evaluation of vaccine efficacy (ve) difficult, as phase 3 studies need sufficient infection rates to compare disease incidence in vaccinated with control individuals. reassuringly, planned studies are currently expanding to higher burden countries where infrastructure to conduct complex adaptive clinical trials exists. nevertheless, the time needed to accrue sufficient data on efficacy will be a critical determinant impacting vaccine availability. in the setting of this uncertainty "human challenge models" for sars-cov-2 have been proposed. this methodology involves the intentional infection of research participants, providing safety data and pointing to immune correlates of protection. this approach has recently advanced vaccine development for infections like salmonella typhi (typhoid vaccine) and group a streptococcus (169, 170) . the difference between these bacterial human challenge models and sars-cov-2 is that these pathogens have been researched for decades and have an effective antibiotic "rescue" treatment available. infecting a human with sars-cov-2 to test vaccine efficacy raises numerous ethical issues (171) , including those around informed consent of the healthy volunteer, noting that while severe covid-19 is less common in young adults, deaths have still occurred in this age group. strict infection control and personal protective equipment (ppe) measures would also be required to limit "third-party" risk to staff co-ordinating any studies. while it is uncertain if this approach will be required, the who is progressing a framework, including key criteria, that will be required by ethical review boards in order to facilitate "human challenge" trials (172). as noted by stanley plotkin, "extraordinary diseases require extraordinary solutions" (173) . it normally takes decades for a vaccine to be developed and licensed for use. however, the race is on to develop a sars-cov-2 vaccine for worldwide distribution in an unparalleled timeframe to vaccinate the world's population and provide widespread herd immunity. this is already being facilitated by rapid progression through the normally slow bureaucratic regulatory and approval processes and unprecedented worldwide collaboration between governments, universities, pharmaceutical companies and philanthropic organizations; such efforts must continue. in addition, ensuring good public communication regarding the safety and effectiveness of the vaccine will be key to gaining public trust and acceptance of a vaccine that could be seen as rushed. additionally, having carefully designed post-marketing (phase 4) surveillance is vital to detect rare or unexpected safety signals and aesi which may only be detected once the vaccine is rolled out on a mass scale. a key player in global access to a sars-cov-2 vaccine is the not-for-profit coalition for epidemic preparedness innovations (cepi) which is working with global health authorities and vaccine developers worldwide to support sars-cov-2 vaccine development (9, 174) . cepi was founded in 2017 with the consensus that new and sustainable models of partnership are needed to respond to worldwide infectious diseases threats. founding members include the bill & melinda gates foundation (bmgf), wellcome trust uk, world economic forum, india department of biotechnology and the government of norway. cepi's goal is not just to advance development, but to also advance licensing, manufacturing, delivery and stockpiling of vaccines once an effective vaccine has been developed (175). it has raised approximately $1.4 billion for sars-cov-2 vaccine development and initiated a series of partnerships in a bid to advance frontrunner candidate sars-cov-2 vaccines. cepi is investing across a range of vaccine technologies including novavax's nanoparticle vaccine (nvx-cov2373 and matrix-m tm ), clover biopharma's s-trimer protein-based vaccine (scb-2019), university of queensland's molecular clamp s protein vaccine, university of oxford's adenovirus vector vaccine (chadox1 ncov-19), inovio's dna plasmid vaccine (ino-4800) and moderna's and curevac's mrna vaccines (176) . many other government and philanthropic organizations are injecting large sums of money into the effort to develop effective sars-cov-2 vaccines. the european commission has registered an impressive $8 billion in donations toward the development and deployment of vaccines, treatments and diagnostics against sars-cov-2 (177). the us government agency biomedical advanced research and development authority (barda) has pledged almost $500 million to accelerate the development of moderna's mrna-1273 vaccine, with phase 2 trials expected to begin soon (178) . barda has also funded janssens's adenovirus 26 (ad26) i.n. vaccine, astra zeneca's azd1222 vaccine (formerly chadox1 ncov19), merck and iavi's rvsvg-cov2 and sanofi's recombinant sars-cov-2 protein vaccine and novavax's nanoparticle matrix m adjuvant vaccine (179) , demonstrating the range of vaccine strategies being supported. importantly, these phase 2/3 studies are being conducted in countries with high disease burden (e.g., north and south america and south africa), so the vaccine efficacy (ve) and safety results can be obtained as rapidly as possible whilst meeting the vaccine regulators' requirements. the logistics of scaling up to produce the billions of doses required to immunize the world's population is an onerous task. it is not yet clear how long immunity will last and therefore whether booster doses or annual immunization will be required. furthermore, multiple initial doses may be needed and the vaccine may have to change as the virus evolves naturally. barda have pledged that they will scale up to 300 million annual sars-cov-2 vaccine doses in the us each year under operation warp speed, while bmgf have recently awarded $5 million to inovio's dna based vaccine ino-4800 with the intention of providing over 1 million doses by the end of 2020. novavax has completed phase 1 studies and is progressing rapidly to phase 2 studies, with the aim of producing up to 100 million doses by the end of 2020 and entering large-scale manufacturing of billions of doses in 2021 (180) . curevac has a gmp-certified production facility that can produce 10 million doses of their mrna vaccine in a single production run and 300 million doses of azd1222 (chadox1 ncov-19) will be available by july 2021 (181) . even if sufficient sars-cov-2 vaccine doses can be manufactured, worldwide delivery presents another major logistic and financial hurdle. storage requirements will be enormous and the vaccine may need to be either frozen or refrigerated, presenting cold-chain issues. the decision about whether to prepare the vaccine in single-dose or multi-dose vials will impact manufacturing, storage, delivery and potential infection risks (182) . infrastructure and manpower will be required to distribute and administer the vaccine. equity will be a major issue since richer countries may procure the vaccine for their citizens while poorer countries may not be able to afford it. cepi is negotiating global access upfront in order to ensure equitable access, hopefully averting "vaccine nationalism" (183) . in early may, who launched the access to covid-19 tools (act) accelerator, which aims to handle the logistics of vaccine procurement and allocation (184) . covax, co-led by cepi, gavi and who, is the vaccine pillar of act charged with accelerating vaccine development and manufacture and guaranteeing fair and equitable worldwide access. in an open letter, more than 140 world leaders have united and called for a sars-cov-2 vaccine to be freely available to all people in all countries of the world in what they call "the people's vaccine, " which is surely the fairest way to tackle this unprecedented global pandemic (185) . by contrast, pneumococcal conjugate vaccine which has been available for 20 years, is still not included in the immunization programs of many countries due to lack of affordability, many children are left unprotected and about half a million deaths from pneumococcal disease each year. initially, as vaccine production commences, it will not be possible to deliver a sars-cov-2 vaccine to the entire world population and it is anticipated that key vulnerable populations will need to be targeted. these would likely include healthcare workers, the elderly and those with significant risk factors such as those with co-morbidities or the immunocompromised. it might even be used as a travel vaccine as borders re-open across the world, but is unlikely to be incorporated into infant schedules for some time given the very low risk of severe disease in that age group and time needed to conduct pediatric studies. while the world eagerly awaits effective sars-cov-2 vaccines as the solution to ending this pandemic, it should be borne in mind that there are many caveats even if robust sars-cov-2specific nabs, cd4 + and cd8 + t cell responses can be induced by vaccination. the number of doses and dosing frequency will need to be determined, and, particularly if repeated or annual vaccination is required, the financial burden and logistics of delivery will have to be supported. a better understanding of what level of nabs correlate with protection and development of standardized viral neutralization along with other assays to compare vaccine candidates is eagerly awaited. potential disease enhancement and other theoretical safety concerns related to each type of vaccine need to be understood and carefully monitored for, while potential suboptimal immunity may need to be overcome. certain vulnerable populations may respond poorly to vaccination, or the vaccine may predominantly protect against severe disease, rather than infection. if t cells prove critical for protection, it will be necessary to ensure that the included t cell epitopes are recognized by enough hla types to ensure worldwide coverage. scale-up of production to billions of doses and delivery to all regions of the world is a massive logistic challenge. in the short-term the strategy will likely need to be targeted vaccination toward those most at risk (e.g., healthcare workers) with the strategy reviewed as vaccine production increases. on the positive side, given the multiple candidates being developed, there is considerable optimism that some of the vaccines currently in trials will prove effective and be available for use in 2021 with delivery scaled-up thereafter. kf proposed the project and coordinated the study group. kf, nc, and fr wrote the first draft. eb, mg, ak, km, bt, and sw reviewed and edited the manuscript, provided comments, and suggested references substantially contributing to the content. all authors approved the final submitted version of the manuscript. this project was supported by the australasian society for infectious diseases (asid) vaccination special interest group (vacsig). covid-19 in the usa epidemiology of covid-19: a systematic review and meta-analysis of clinical characteristics, risk factors, and outcomes global covid-19 case fatality rates deaths in healthcare workers due to covid-19: the need for robust data and analysis factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019 the milken institute. 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(2020) available online at world leaders unite in call for a people's vaccine against covid-19 we would like to thank claudio rosa (www.claudiorosa.com) for medical illustration. key: cord-253844-y6xdcf20 authors: yesudhas, dhanusha; srivastava, ambuj; gromiha, m. michael title: covid-19 outbreak: history, mechanism, transmission, structural studies and therapeutics date: 2020-09-04 journal: infection doi: 10.1007/s15010-020-01516-2 sha: doc_id: 253844 cord_uid: y6xdcf20 purpose: the coronavirus outbreak emerged as a severe pandemic, claiming more than 0.8 million lives across the world and raised a major global health concern. we survey the history and mechanism of coronaviruses, and the structural characteristics of the spike protein and its key residues responsible for human transmissions. methods: we have carried out a systematic review to summarize the origin, transmission and etiology of covid-19. the structural analysis of the spike protein and its disordered residues explains the mechanism of the viral transmission. a meta-data analysis of the therapeutic compounds targeting the sars-cov-2 is also included. results: coronaviruses can cross the species barrier and infect humans with unexpected consequences for public health. the transmission rate of sars-cov-2 infection is higher compared to that of the closely related sars-cov infections. in sars-cov-2 infection, intrinsically disordered regions are observed at the interface of the spike protein and ace2 receptor, providing a shape complementarity to the complex. the key residues of the spike protein have stronger binding affinity with ace2. these can be probable reasons for the higher transmission rate of sars-cov-2. in addition, we have also discussed the therapeutic compounds and the vaccines to target sars-cov-2, which can help researchers to develop effective drugs/vaccines for covid-19. summary: the overall history and mechanism of entry of sars-cov-2 along with structural study of spike-ace2 complex provide insights to understand disease pathogenesis and development of vaccines and drugs. angiotensin-converting enzyme 2 ccl2 chemokine (c-c motif) ligand the sars-cov-2 is a single, positive-strand rna virus that causes severe respiratory syndrome in humans [1] . the coronavirus disease 2019 (covid-19) has emerged as a severe pandemic, claiming more than 0.8 million lives worldwide between december 2019 and august 2020 [2, 3] . compared to sars-cov, sars-cov-2 human-to-human infection is more readily transmitted and spread to almost all continents leading to the who's declaration of a public health emergency of international concern (pheic) on january 30, 2020 [3] [4] [5] . generally, coronaviruses can cause respiratory, gastrointestinal, and central nervous system diseases in humans and animals, threatening humans life and causing economic loss [6, 7] . these viruses also have the capacity to adapt to a new environment through mutations and are programmed to modify host tropism; thus, the threats are constant and long-term [6, 8, 9] . including sars-cov-2, other coronaviruses cross the species barrier into humans, which lead to outbreaks of severe and fatal respiratory diseases. the sars-cov was first identified in bats, and spread to other animals in different geographic regions. the sars-cov outbreak was first emerged in humans in 2003, through transmissions from animals in open-air markets in china [10, 11] . thereafter, a higher number of genetically related viruses were also identified in chinese horseshoes bats (rhinolophus sinicus) [11] [12] [13] . coronaviruses belong to the family coronaviridae and are divided into alpha (α-cov), beta (β-cov), gamma (γ-cov), and delta (δ-cov) coronaviruses. the alpha and betacoronaviruses can infect mammals, and the viruses found in humans are genetically similar to β-cov genus. the β-covs are further divided into different lineages (a, b, c, and d lineages): sars-cov and sars-cov-2 are grouped in lineage b, which has approximately 200 published virus sequences, whereas mers-cov belongs to lineage c, which has ~ 500 viral sequences [11] . the hcov-229e and hcov-nl63 belong to the alphacoronavirus family, whereas hcov-oc43, hcov-hku1 and sars-cov are betacoronaviruses [14] [15] [16] . the phylogenetic analysis shows that sars-cov-2 protein is firmly rooted in the β-genus lineage of bat coronaviruses [14] . the whole genome of sars-cov-2 shares 80% identity with that of sars-cov and is 96% identical to the bat coronavirus batcov-ratg13 [17] . the spike protein sequence similarity between sars-cov-2 and sars-cov is around 76-78%. the rbd alone shares a similarity of 73-76%, and rbm shares 50-53%. in contrast, the human mers-cov is related to tylonycteris bat coronavirus hku4, shares less sequence similarity (~ 54%) and recognizes dpp4 as their receptor. the sequence similarity between sars-cov-2 and sars-cov spike proteins explains the possibility of binding to the same receptor angiotensin converting enzyme 2 (ace2) in the host cell [14] . coronavirus is one of the largest genomes among all rna viruses ranging from 27 to 32 kb. receptor-mediated endocytosis is the main process of virus entry to the host cells. sars-cov-2 uses ace2, a cell-surface receptor that is present in the kidney, blood vessels, heart, and importantly, in the lung at2 alveolar respiratory tract epithelial cells for viral infection [18] . the spike protein, which is responsible for the viral entry, has n-terminal and c-terminal domains, and two major subunits s1 and s2 are present in almost all coronaviruses [6] . one of these s1 or s2 subunits binds with the host receptors and acts as a receptor-binding domain (rbd) (fig. 1a) . next-generation sequencing technology (ngs) revolutionized the biological sciences, including virus discovery. the ngs made it easy to recognize thousands of novel virus sequences from wild animal and human populations around the world. despite these vast coronavirus sequences are published, very little work has been performed for further studies. further, the lack of tools to test these novel viruses and their ability to infect humans hindered the efforts to predict the subsequent zoonotic viral outbreaks [11] . understanding the virology of coronaviruses and the methods to control their spread is currently a necessary task to maintain the global health and economic stability. in this review, we discuss the history of coronaviruses in both humans and animals, their transmissions, mechanism of host cell entry and the structural studies, explaining active and inactive receptor binding of spike protein and the key residues playing an important role in the receptor binding. the drug repurposing and the therapeutic targets for sars-cov-2 are also discussed. until the first identification of human coronaviruses 229e and oc43, in the late 1960s, coronavirus infections were witnessed as harmless for humans [14, 15] . the outbreak of sars-cov in southern china in the winter of 2002, took a fatality rate of 10% of the infected patients [18] [19] [20] . the virus had been rapidly spreading throughout the world, especially in asia, and controlled after july 2003 [21] . viral analysis of the outbreak of sars showed that bats are natural reservoirs for sars-covs, and civet cats and raccoon dogs are the intermediate hosts. in the year 2012, a novel highly pathogenic middle east respiratory syndrome coronavirus (mers-cov) was identified in humans, demonstrating that the coronaviruses are transmitted from animals to humans at any time and with unexpected consequences for the public health [22] . mers-cov, the slow-spreading virus, has affected ~ 1700 people with a fatality rate of ~ 36% [6, 22] . the animal sources of sars-cov-2 infections are bats and sars-cov-2 can be transmitted to cats, pangolins, and dogs [23] . other than humans, the coronaviruses have a big impact on the animal kingdom. animal coronaviruses can cause severe threat to their hosts; since 1984, an unrecognized infection massively spread among the swine population in europe, and later it was identified as porcine epidemic diarrhea coronavirus (pedv), which is derived from the porcine enteric coronavirus tgev [24] . in 2013, the same pedv in less than a year had caused a 100% fatality rate in piglets and rubbing out more than 10% of america's pig population [5, 25, 26] therefore, the total number of human coronaviruses identified has been increasing throughout the years. hcov-229e and hcov-oc43 are the first two discovered human coronaviruses in the 1960s, hcov-nl63 and hcov-hku1 human coronaviruses identified after the sars-cov outbreak in [22] . however, the story continues with the new identification of sars-cov-2 in december 2019 at the seafood wholesale market in wuhan, china. sars-cov-2 is the seventh member of the family of coronaviruses that infects humans and it is different from both mers-cov and sars-cov. although some infections caused by human coronaviruses are mild and associated with common colds, certain animal and human coronaviruses can make a severe impact on the human population. especially in young children, elderly people, and immune-deficient patients, the infections can be lethal [9, [27] [28] [29] . therefore, it is important to understand the mechanism of invading viruses to theirs hosts, transmission and prevention of these processes. the respiratory droplets are the main routes of transmissions; sars-cov-2 can be transmitted to a healthy person if he happens to have contact with the infected person or any of his belongings, including clothes, doorknobs, etc. studies have been reported that aerosol transmission (airborne transmission) is also possible for sars-cov-2, but there is no clear study on neonatal infections (mother to child) [30] [31] [32] [33] [34] [35] [36] . however, the transmission can be avoided by keeping a distance of 2 m between two people, wearing masks while going out, and the isolation of infected people. during the initial phase of the covid-19 outbreak, a dataset was obtained from 1099 patients with laboratoryconfirmed covid-19 from 552 hospitals in 30 provinces of china on january 29, 2020. only 2% of the patients had a history of contact with animals; more than three quarters have either visited the wuhan city or are residents. hence, the outbreak patterns or the source of infection could not be predicted from their study. the incubation period of the sars-cov-2 was from 1 to 12 days; however, the median incubation period was 4 days [32] . the most common symptoms are fever (43% on admission, and 88.7% during hospitalization), cough (67.8%), diarrhea (3.8%), and fatigue [32, 37] . the sars-cov-2 was detected in saliva, blood, sputum, and urine before the development of viral pneumonia, and some patients do not develop pneumonia at all. asymptomatic persons are potential sources of sars-cov-2 infection, which control the transmission dynamics of the current outbreak [32, 38] . the sars-cov-2 first identified in wuhan, china has spread all over the globe. as of august 20, 2020, more than 22 million confirmed infection cases and 0.8 million deaths had been reported across the world, including almost all the countries. the rate of infection or the average number of people getting infected by an individual (r0) was 2.75 in the case of sars pandemic in 2003. the r0 value of ebola 2014 was in the range of 1.51-2.53, and h1n1 influenza 2009 was from 1.46 to 1.48, and for mers, it was around 1. the sars-cov-2 r0 value was estimated to be in the range of 1.5-3.5. the comparison of r0 values of various coronaviruses shows that the difference is minimal. however, the difficulties arising for sars-cov-2 infection are due to the following: (1) basic properties of the viral infection and the infection periods are uncertain, (2) most of the infected individuals do not show symptoms, but are capable of spreading the infection, (3) changing susceptibility of the population in affecting the spread of infection remains unanswered. in addition, there are no control measures for this spread [39] . coronaviruses consist of four structural proteins: the nucleocapsid protein (n) forms the helical capsid to accommodate its genome. the whole structure is further surrounded by a lipid envelope, which is made of s (spike), e (envelope) and m (membrane) proteins. the membrane and the envelope proteins are needed for the virus assembly and the spike protein is for virus entry and host cell recognition [6] . the spike protein forms large protrusions (peplomers) on the virus surface (looks like the virus has crowns), and hence it is named as "corona" (corona is a latin word which means crown). it comprises three segments: (1) large ectodomain, (2) transmembrane domain and (3) intracellular tail. the receptor-binding subunits s1 and s2 are placed in the ectodomain region. during the infection, the s1 binds with the host receptor, and s2 fuses the host and viral membranes, thereby releasing the viral genome into the cell (fig. 1 ). the spike protein is a clove-shaped trimer with three s1 heads and a trimeric s2 stalk [6] . during viral infection, spike protein (~ 1300 amino acid residues) is cleaved by host proteases into receptor binding subunit s1 and membrane fusion subunit s2. during cell entry, the s1 subunit binds directly to the sugar receptors [40] and ace2 of the host cell surface, and the s2 subunit undergoes conformational changes and obtains post-fusion state [41] . during this state, the three pairs of heptad repeat region hr-n and hr-c in trimeric s2 form a six-helix bundle structure [42] . the buried hydrophobic fusion peptides become exposed and insert into the target host membrane. these fusion peptides and the transmembrane anchors are positioned at the end of a six-helix bundle structure, bringing the viral and host membranes to fuse [42, 43] . during this process, a large amount of energy is released, which accelerates the membrane fusion forward. along with this, receptor binding and low ph can also trigger this membrane fusion [6] . since the spike protein has a good binding affinity for sugar receptors of human cells, it uses them as a mechanism of cell entry [6] . notably, the sars-cov-2 has a higher affinity to human ace2 than the sars-cov virus strain. the ectodomain of the sars-cov-2 spike protein binds to the peptidase domain (pd) of ace2 with a k d (equilibrium dissociation constant) of ~ 15 nm [4] . spike protein priming is done by transmembrane protease serine 2 (tmprss2), which is also essential for the entry of sars-cov-2 [44] . generally, sars-cov enters host cells through endocytosis, where its spike protein is processed by cathepsin l and cathepsin b lysosomal proteases. the extracellular proteases, including elastase in the respiratory tract and tmprss2 on the surface of flung cells are also known to activate spike membrane fusion [6] . the viral entry to the host cells can be via: (1) the endocytic pathway and (2) non-endosomal pathway [45] (fig. 1b) . the endocytic pathway, especially clathrindependent endocytosis is extensively studied for sars-cov and mers-cov viral entry. since the sars-cov-2 also uses the same receptor as sars-cov, it is reported that sars-cov-2 also uses the same viral entry mechanism. wang et al. [46] reported clathrin-and caveolaeindependent endocytic pathways for sars-cov entry. despite the common use of endocytic pathway as a viral entry mechanism, the discrepancies about the same are unavoidable. thus, the exact nature of the viral entry is context-dependent, including the type of the virus and the host cells [47] . in addition, the macrophages can also act as a viral reservoir and support minimally for the sars-cov-2 entry and its replication. although the dendritic cells and other immune cells are not infected by sars-cov-2, they may serve as a transporter for the viruses, which is also responsible for the pathogenesis [48, 49] . the s1 subunit of sars-cov spike glycoprotein is predominantly composed of β-strand structures, composed of an n-terminal domain (ntd, residues 14-294) and three c-terminal domains (ctd1, residues 320-516; ctd2, residues 517-578 and ctd3, residues 579-663). the ntd is linked with ctd1 through the linker residues 295-319, where ctd1 acts as a potential rbd for sars-cov and binds explicitly with the ace2 receptor. gui et al. [43] reported that the sars-cov spike glycoprotein trimer could obtain different conformations, which are necessary for effective binding with ace2. all these conformations are termed based on the position of ctd1 of the spike glycoprotein (fig. 2) . the three spike glycoprotein monomers intertwine with each other and form densely packed homotrimer. the head portion of this trimer is taken place by ntds and ctd1s of s1 subunits, where the ctd1s are located at the center and the ntds are located outside of this triangular head. the s2 subunits represented as a stem for this trimer, which is further surrounded by ctd2s and ctd3s of the s1 subunits (fig. 2) . in the inactive state (fig. 2a, b) , s2 subunits (stem portion) are completely covered by the "down" position of ctd1s (head portion), which causes steric clashes for the binding between spike protein and ace2. in the active state (fig. 2c) , two ctd1s adapt the "down" conformation and one ctd1 rotates outward and obtains "up" conformation, which does not cover the s2 subunit and allows the interactions between spike protein and ace2. the "up" position also paves the way for the s2 subunit to expose and insert its fusion peptides into the host cell membrane [43] (fig. 2) . the sequence similarity between sars-cov-2 and sars-cov spike protein explains the possibility of having the same receptor ace2 in the host cell [14] . the sequence and the structural studies revealed the key residues, which are involved in spike-ace2 interactions. the key residue at position 493 in rbd of sars-cov-2 is gln, wherein sars-cov (479 is the corresponding residue in sar-cov) of civets and humans, it is lys and asn, respectively. since the residue 479 of rbd is near to the virus-binding hotspot residue lys31 of human ace2, the lys residue present in civet causes steric clashes and not favoring human ace2 receptor binding. however, the lys479asn mutation revealed that the asn present in 479 of human sars-cov enhances the viral binding with human ace2 receptors. the gln493 in sars-cov-2 rbd is compatible with the human ace2 receptor hotspot residue lys31, which explains its target cell identification [14] . similarly, the residue 501 in sars-cov-2 rbd is asn, wherein civet rbd, it is ser (487 is the corresponding residue in sars-cov), and for humans, it is thr. this residue also plays an important role in making interaction with the hot spot residue lys353 of receptor ace2. ser487thr mutation analysis shows encouraging results upon human ace2 receptor binding and plays a role in human-to-human transmission. thus, the interactions with lys353 will be favorable for threonine (human sars-cov) and asn (sars-cov-2) than serine (civet) [14] . the residues lue455, phe486, and ser494 in sars-cov-2 rbd (tyr442, leu472, and asp480 in human and civet sars-cov) are considered as important for the human ace2 receptor binding. tyr442 of human and civet sars-cov rbds shows unfavorable interactions with hotspot residue lys31 of human ace2; however, lue455 of sars-cov-2 provides favorable interactions. compared with leu472 of human civet sars-cov rbd, phe486 of sars-cov-2 provides better interaction with hotspot residue lys31 of human ace2. although ser494 provides positive support for the human ace2 receptor hotspot residue lys353, the sars-cov asp480 also makes favorable interaction with hotspot residue lys353. throughout this viral entry process, the lys31 and lys 353 of human ace2 receptors are termed as "hot spot" residues, which consist of a salt bridge buried in a hydrophobic environment and contribute critically for the virus and host cell receptor binding. thus, this key residue comparison of sars-cov-2 with the civet and human sars-cov explains how actively sars-cov-2 is choosing and binding with the human ace2 receptors, which is likely to cause the human-to-human transmission [14] . the heterogeneity of amino acids in ace2 receptors is also responsible for the wavering binding affinities between the host and the virus, which is associated with the viral transmission. however, the variants in the host cell receptors (ace2) can confer resistance against the invading pathogens. hussain et al. reported that the mutations s19p and e329g in ace2 disrupt the intermolecular interactions and have low binding affinity with viral spike protein. in addition to the variations in the viral spike protein, ace2 allelic variants can also drive the potential resistance against sars-cov-2 infection [50] . intrinsically disordered proteins (idps) and intrinsically disordered regions (idrs) also play major roles in a number of biological functions, including dna/rna binding, protein binding, and facilitating access to the binding sites between the binding partners [51, 52] . the rna-protein recognition often needs conformational changes in both rna and protein, which is facilitated by the structural flexibility of disordered residues [51] . also, the functional importance of intrinsically disordered regions in proteins includes transcription, translation, post-translational modifications, and cell signaling [51, 53] . the categorization of coronaviruses, based on the intrinsic disorder propensities, can represent useful identification for the viral life cycle and its pathogenicity. the idrs in sars-cov nucleocapsid protein comprise three segments, such as 1-44, 182-247 and 366-422 [54, 55] . the highly flexible intrinsic disordered linker region, which connects the ntd and ctd is rich in serine and arginine residues. an intrinsically disordered domain that flanks the ctd (c-terminal tail peptide) plays a significant role in dimer-dimer association in human coronavirus 229e (hcov-229e). likewise, the coronavirus hku1, has a partially disordered conserved linker loop (amino acids 428-587) structure [56] . hku1 s2 subunit also shows the presence of disordered residues at its protease cleavage site [57] . in sars-cov-2, the attachment of spike protein with the host cell is activated by the host cell enzymes trypsin, cathepsin l, furin and tmprss2 (fig. 1b) . the sequence comparison of sars-cov-2 against other lineage b betacoronaviruses shows that the unique amino acid pattern "rrar" is present at the s1/s2 junction of the spike protein, which is cleaved by the furin enzyme. however, the structure reported for sars-cov-2 spike protein (pdb code: 6vsb), shows that s1/s2 junction is in a disordered, solvent-exposed loop [4] . hence, it has been hypothesized that the unique amino acid sequence "rrar" present in sars-cov-2 is responsible for their effective transmission [58] [59] [60] . the binding with ace2 is governed by the intrinsically flexible receptor binding motif, and the binding interfaces along with the key residues are reported in the literature [61, 62] . in addition, based on our analysis (fig. 3) , the monomeric structure of the spike protein in the free form (pdb id: 6vsb; green color) [4] shows a number of missing residues in the structure. these missing residues of the spike protein attain a stable conformation upon binding to the ace2 receptor and hence, they are termed as disorderto-order transition (dot) residues (pdb id: 6lzg; light golden color). specifically, the regions, leu455 to pro491 and asn501 to val503 show disorder-to-order transitions [61] . these disordered-to-ordered residues are facilitating a better shape complementary and affinity between ace2 and spike protein. interestingly, the key residues responsible for transmission, and interaction with ace2 receptors (already discussed in section "key residues of sars-cov-2 and sars-cov") are overlapping with these mentioned disordered-to-ordered residues. based on these observations, the disorder-to-order conformational change is necessary to facilitate the spike protein binding with its receptor. thus, an in-depth analysis of these disordered residues will shed additional insights on the viral recognition and transmission mechanism. the studies by goh et al. [63] on 1918 h1n1 and h5n1 explain that it is very likely that disordered regions are important for host specificity and recognition, e.g., across species of birds. they also explained the changes created by disorder in crucial regions, increasing the virulence of both the h5n1 and the 1918 h1n1 viruses. therefore, the increasing reports for the intrinsically disordered regions in coronaviruses need to be pointed out. the importance and functional role of intrinsically disordered regions need to be thoroughly studied. this could identify additional targets for drugs to combat coronavirus through the disruption of their packing and assembly process. the receptor binding, along with its membrane fusion, is the initial and important step in the coronavirus infection and serves as primary targets for inhibiting the viral entry. the genome sequencing of sars-cov-2 and the comparison of the genomes of related virus proteins suggested the anti-hiv lopinavir plus ritonavir combination can be likely effective [15, 64, 65] . sars-cov-2 uses the ace2 receptor of at2 cells in the lung as its primary targets. since the viral entry is governed by receptor-mediated endocytosis, ap2-associated protein kinase 1 (aak1), a known regulator of endocytosis, can be a good target to interrupt the virus entry. the janus kinase inhibitor baricitinib, and its binding with the cyclin g-associated kinase (endocytosis regulator) is sufficient to inhibit aak1 [66, 67] . similarly, sunitinib and erlotinib, the oncology drugs, have been shown to inhibit viral infection of cells through the inhibition of aak1 [68] . however, these compounds bring serious side effects and cannot be considered for a safe therapy. fig. 3 structure of the monomeric spike protein (green)-ace2 receptor (blue) complex. the interface residues are shown in light golden. the disordered-to-ordered transition residues (leu455 to pro491 and asn501 to val503) have been marked in the figure based on the pathogenicity studies of sars-cov and mers-cov, an increased amount of inflammatory cytokines in serum is associated with the inflammation and extensive lung damage [69] . similarly, sars-cov-2 also has a high amount of il1b, ifnγ, ip10, and mcp1/ccl2, which lead to t-helper-1 (th1) activation. however, sars-cov-2 is shown to increase the t-helper-2 (th2) cytokines (il4 and il10) that are known to suppress inflammation, which differs from sars and mers-cov infection. in view of cytokines induced by sars-cov-2 and other sars-cov, mers-cov, corticosteroids were frequently used to reduce the inflammation-induced lung injury [2] . arabi et al. [70] examined the combination of interferon beta-1b, lopinavir, and ritonavir combination for the mers infection in saudi arabia. the antiviral nucleotide prodrug remdesivir showed a potent efficacy on mers-cov and sars-cov infections. since sars-cov-2 is an emerging virus, no effective treatment has been developed so far, yet the already available combination of lopinavir and ritonavir is being used [2] . since the sars-cov enters the cell through endocytosis and the lysosomal protease is priming the spike protein, targeting/inhibiting the endosomal acidification or lysosomal cysteine protease can block the sars-cov entry [6] . the view of the activation of sars-cov and sars-cov-2 by tmprss2 might also have therapeutic suggestions. a protease inhibitor, camostat mesylate, which is studied as a tmprss2 inhibitor and has been used to treat humans, is available and could be employed as a defense against the respiratory viruses [71, 72] . autophagy has been implicated in viral replications, which is also responsible for the formation of doublemembrane vesicles (dmv) in the host cells [73] . since the autophagosomes are degraded by lysosomes, inhibitors like lysosomotropic agents have been proposed for sars-cov-2 [74] . the antimalarial drugs hydroxychloroquine (hcq) and chloroquine (cq) have been demonstrated for sars-cov-2 antiviral activity. however, data to support the use of hcq and cq for covid-19 are incomplete [75] . these lysosomotropic agents are helpful for neutralizing the endosome-lysosomal acidic ph, thereby blocking protease activity and subsequently blocking the viral entry. however, how autophagy is implicated in the infection of covs is still under debate [47, 74, 76] . table 1 describes the different inhibitors used for blocking the viral entry. the most studied and the common pathway proposed for all coronaviruses is the endocytic pathway, so blocking that pathway is a big hallmark for treating the disease. identification of potential drugs for sars-cov-2 is a necessary task, so drug repurposing can also work for this scenario. the small molecules, which are already in clinical trials or used for some other diseases and the molecules from the expert's opinion are also listed in table 2 . the table is categorized into three groups: (1) compounds involving drug repurposing, (2) compounds in clinical and pre-clinical study, (3) compounds targeting the mechanism pathway. researchers in government and private sectors are making huge efforts to develop effective vaccines for sars-cov-2. the vaccine development approaches are mainly based on inactivated and attenuated viral protein particles, viral vectors and viral dna/rnas. a novel rna-based vaccine uses a part of the genetic code of spike protein mrna-1273 (clinicaltrials.gov: nct04283461) [139] . several other mrna-based vaccines by curevac (tübingen, germany), bnt162 by biontech (mainz, germany), pfizer (new york, ny, usa) and biontech mrna vaccine (mainz, germany) are in different stages of development [139, 140] . cansino biologics (tianjin, china), the company that developed the vaccine for ebola is also developing a vaccine named ad5-ncov for sars-cov-2. it is a spike protein-based vaccine that is undergoing phase i clinical trials in healthy individuals in wuhan china (clinicaltrials. gov: nct04313127) [141, 142] . the current status of the vaccines under development is available at the milken institute treatment and vaccine tracker: https ://milke ninst itute .org/sites /defau lt/files /2020-03/ covid 19%20tra cker%20032 020v3 -posti ng.pdf [141] . sars-cov-2 is a continuously growing life-threatening disease. this coronavirus has rapidly evolved and spread all over the world, having a mortality of more than 0.8 million lives so far. the exact origin and mechanism of attack and spatial distribution are still not completely explored. the viral and the host cell proteins assisting in invasion process can be a target for treating the infections. the available crystal structures and the binding mechanism proposed by other coronaviruses can help to study the sars-cov-2. the critical structural studies of the viral particles are also helpful to identify the drug targets. the positional changes (up and down conformation) of spike protein trimer determine the active to inactive state. thus, targeting these conformations and developing small molecules or peptides can also stop the viral entry. in addition, phylogenetic analysis and structural studies revealed that the hot spot residues, the role of gln493 in human ace2 receptor binding and the critical residue asn501 of rbd explain the human-tohuman transmission. the intrinsic disorder region and a precise furin-like cleavage site can be responsible for viral cycle and pathogenicity. however, in-depth studies are needed to address this issue, which may represent a potential antiviral strategy. the small molecules, which are in clinical trials, the drug compounds, which 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for entry into target cells identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-proteinbased cell-cell fusion assay oxazolidinones inhibit cellular proliferation via inhibition of mitochondrial protein synthesis sars-cov-2 vaccines: status report the pandemic pipeline sars-cov-2/covid-19: viral genomics, epidemiology, vaccines, and therapeutic interventions the covid-19 vaccine development landscape acknowledgements we thank indian institute of technology madras, key: cord-252767-as841xo0 authors: fischer, bastian; knabbe, cornelius; vollmer, tanja title: sars-cov-2 igg seroprevalence in blood donors located in three different federal states, germany, march to june 2020 date: 2020-07-16 journal: euro surveill doi: 10.2807/1560-7917.es.2020.25.28.2001285 sha: doc_id: 252767 cord_uid: as841xo0 most cases of coronavirus disease 2019 are mild or asymptomatic. therefore, many cases remain unrecorded. we determined seroprevalence of igg antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in 3,186 regular blood donors in three german federal states between 9 march and 3 june 2020. the igg seroprevalence was 0.91% (95% confidence interval (ci): 0.58–1.24) overall, ranging from 0.66% (95% ci: 0.13–1.19) in hesse to 1.22% (95% ci: 0.33–2.10) in lower-saxony. most cases of coronavirus disease 2019 are mild or asymptomatic. therefore, many cases remain unrecorded. we determined seroprevalence of igg antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in 3,186 regular blood donors in three german federal states between 9 march and 3 june 2020. the igg seroprevalence was 0.91% (95% confidence interval (ci): 0.58-1.24) overall, ranging from 0.66% (95% ci: 0. 13-1.19) in hesse to 1.22% (95% ci: 0.33-2.10) in lower-saxony. common symptoms of coronavirus disease 2019 (covid-19) include cough, fever and respiratory problems. while ca 80% of infected people only show mild or no symptoms, some develop severe pneumonia, multiple organ failure or even die [1] . current estimates assume a mortality rate of ca 2% in medically attended patients [2] . however, individuals with mild or no symptoms are not all included in these mortality estimates, and the number of unrecorded cases is unknown [3, 4] . although an acute infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is usually verified by pcr, a recent publication suggests a positive identification of anti-sars-cov-2 igg antibodies as an acceptable approach to confirm infection [5] . to determine an approximation of the actual rate of people who have recovered from covid-19, representative of the german population, we determined the anti-sars-cov-2 igg seroprevalence of regular blood donors resident in three different german federal states between march and june 2020. residual material leftover from routine diagnostics from 3,186 regular blood donors without any preselection (2,257 (70.84%) men and 929 (29.16%) women), donated in the period between 9 march and 3 june 2020, were screened for the presence of anti-sars-cov-2 igg directed against domain s1 of the sars-cov-2 spike protein using the anti-sars-cov-2 enzyme-linked immunosorbent assay (elisa) from euroimmun (lübeck, germany). in recent publications, this serological elisa showed a high specificity of 99-100% and a sensitivity of ca 65% [6] [7] [8] [9] . semiquantitative results were calculated as the ratio of the extinction of samples over the extinction of a calibrator. seropositive results were confirmed using the architect sars-cov-2 igg (abbott, wiesbaden, germany) targeting the viral nucleocapsid and the liaison sars-cov-2 s1/s2 igg assay (diasorin deutschland gmbh, dietzenbach, germany) targeting the sars-cov-2 spike protein. most samples (2,902/3,186; > 91%) were obtained between 23 march and 22 may 2020. samples were obtained from donors located in the three german federal states north rhine-westphalia (n = 1,700), lower saxony (n = 576) and hesse (n = 910). measurements were fully automated and processed according to the manufactures protocol using the euroimmun analyzer i system. overall, we found an anti-sars-cov-2 igg seroprevalence of 0.91% (29/3,186; 95% ci: 0.58-1.24) in our cohort; 24 male and five female donors. no statistical difference in seroprevalence was observed between men and women (p = 0.156). likewise, the seroprevalence did not differ statistically between the three federal states (p = 0.536), but incidence was highest in lower saxony (1.22%; 7/576; 95% ci: 0.33-2.10), followed by north rhine-westphalia (0.94%; 16/1,700; 95% ci: 0.49-1.39) and hesse (0.66%; 6/910; 95% ci: 0.13-1.19) (table) . all donors underwent a medical examination before donation, reported that they did not have current or recent diseases and had no physically detectable symptoms of infection such as fever or an increased leukocyte count. none of the seropositive blood donors reported a known positive medical history of sars-cov-2 infection. a second retrospective survey for sars-cov-2 related symptoms was not conducted. the figure shows the anti-sars-cov-2 igg distribution in blood donors with equivocal (ratio: ≥ 0.8 to < 1.1) and clearly seropositive (ratio: ≥ 1.1) test results. for clarity, values are presented in a histogram, choosing a binwidth of 0.2 (e.g. ratio 1.1-1.3). the 29 seropositive donors showed a broad spectrum of igg ratios ranging between 1.13 and 8.9. in addition, we identified nine blood donors with equivocal seropositive igg antibody ratios ranking between 0.8 and 1.08 who were not considered for the seroprevalence calculation. we used exclusively waste material from routine laboratory diagnostics, therefore the need for informed consent and ethical approval was waived. samples were collected in accordance with the german act on medical devices for the collection of human residual material. after a public festival in february 2020, a local covid-19 outbreak occurred in heinsberg, germany. according to estimations by streeck et al., this led to an infection rate of 15.5% (positive swab anamnesis and/or detection of anti-sars-cov-2 igg antibodies), whereby 22.2% of infected individuals were asymptomatic [10] . the seroprevalence in non-hotspot regions is currently open to question. in our study, we decided to measure overall anti-sars-cov-2 igg levels instead of neutralising antibodies, as not all individuals who have recovered from covid-19 seem to express detectable neutralising antibody levels [11] . this would potentially lead to a number of false-negative results. as false-positive measurements could account for a considerable number in populations with a low seroprevalence, initial seropositive measurements were verified with two additional assays (supplementary figure s1) . consequently, we revised one initially seropositive tested result. we determined an overall low igg seroprevalence of 0.91% (95% ci: 0.58-1.24) against sars-cov-2 in three german federal states. a recent study of the university hospital eppendorf revealed comparable values for hamburg. their data show that fewer than 1% of 914 tested regular blood donors expressed igg antibodies against the virus [12] . in addition, a national multicentre study, including four university children`s hospitals in baden-württemberg, revealed that 1.3 % of 4,932 individuals (children and their parents) tested between 22 april and 15 may expressed anti-sars-cov-2 igg antibodies [13] . it should be emphasised that the preselection of blood donors as study cohort is accompanied by limitations regarding representation of the population: blood donors are between 18 and 65 years-old, young healthy adults are usually overrepresented and other groups (e.g. children, hiv/hcv/hbv-infected patients, older people with underlying conditions, institutionalised people) are excluded or underrepresented. nevertheless, according to official data, only ca 0.2% of german citizens have so far (17 june 2020) been infected with the sars-cov-2 [14] . therefore, our findings suggest that there are a large number of unrecorded cases. compared with hotspot regions either in germany [10] or elsewhere in europe (e.g. lombardy, italy, with a seroprevalence of 23% [15] and madrid, spain, with a seroprevalence of 11.5% (95% ci: 9.9-13.3) [16] ), the low seroprevalence for sars-cov-2 determined in our study could be explained by the imposition of preventive, non-pharmaceutical, interventions at an early stage of the epidemic. a study by flaxman et al. indicates that these lockdowns contributed considerably to the containment of virus spreading and therefore may have saved many lives [17] . their model calculation estimating the current sars-cov-2 seroprevalence for different european countries revealed the lowest prevalence for germany (0.85%) and norway (0.46%), while higher values were estimated for belgium (8%) and spain (5.5%). all these rates are far too low to reach herd immunity, which would require ca 60% of the population to express protective antibodies against the virus [18] . in addition to the unambiguously seropositive blood donors, 0.3% of the individuals in our cohort showed equivocal levels of antibody. these donors may have been very recently infected and would subsequently have reached higher igg antibody levels against sars-cov-2. however, this assumes that they had an asymptomatic sars-cov-2 infection since blood donors represent a selection of apparently healthy individuals lacking any physically detectable symptoms. a longitudinal study on the antibody profile of sars-cov-1 patients in 2006 showed that infected persons did not produce detectable antibody titres within the first 7 days after the onset of symptoms; igg expression increased considerably on day 15 and reached a peak on day 60 [19] . first data for sars-cov-2 suggest a very similar timing of igg antibody formation [20] . in addition, scientists from the university hospital zurich showed that serum igg levels remained partially negative in covid-19 patients with a mild disease progression, whereas severe cases, independently of age, had significantly increased serum igg titres [21] . long et al. monitored 285 recovered covid-19 patients who tested positive for anti-sars-cov-2 igg antibodies within 19 days after symptom onset but also here, seroconversion was delayed in patients with milder symptoms [22] . it is also conceivable, that individuals with a weak humoral immune response (low antibody ratio) have a stronger cellular immune response. in this context, wu et al. recently presented a negative correlation between lymphocyte counts and neutralising antibody responses to sars-cov-2 in a cohort of covid-19 recovered patients [11] . interestingly, they also showed that 10 of 175 patients did not express detectable neutralising antibody titres at all. broad and comprehensive testing is required to better evaluate the number of people who have recovered from covid-19 and to elucidate the magnitude of unrecorded cases. it has to be taken into consideration that not all convalescents seem to express detectable levels of anti-sars-cov-2 igg antibodies and that there is missing evidence on antibody persistence. distribution of anti-sars-cov-2 igg ratios of blood donors with seropositive and equivocal test results, germany, march-june 2020, (n = 3,186) clinical characteristics and differential clinical diagnosis of novel coronavirus disease 2019 (covid-19) covid-19, a worldwide public health emergency covid-19: four fifths of cases are asymptomatic, china figures indicate covid-19: identifying and isolating asymptomatic people helped eliminate virus in italian village molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes united states food and drug administration (fda) evaluation of nine commercial sars-cov-2 immunoassays evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-sars-cov-2 antibodies validation of a commercially available sars-cov-2 serological immunoassay infection fatality rate of sars-cov-2 infection in a german community with a super-spreading event neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications nur geringe anzahl an blutspendenden weist antikörper gegen neuartiges corona-virus auf prevalence of covid-19 in children in covid-19: fallzahlen in deutschland und weltweit prevalence of sars-cov-2 specific neutralising antibodies in blood donors from the prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study estimating the effects of non-pharmaceutical interventions on covid-19 in europe what policy makers need to know about covid-19 protective immunity longitudinal profile of antibodies against sars-coronavirus in sars patients and their clinical significance patterns of igg and igm antibody response in covid-19 patients systemic and mucosal antibody secretion specific to sars-cov-2 during mild versus severe covid-19 antibody responses to sars-cov-2 in patients with covid-19 we gratefully thank birgit drawe, ricarda plümers, anika kleine, vanessa schmidt, janina tiemann, thanh-diep ly, christopher lindenkamp and christoph lichtenberg for their technical assistance. none declared. all authors have contributed significantly to the work and approved the final article. b. fischer and t. vollmer designed the study, analysed and interpreted the data and draft the manuscript. c. knabbe designed the study and revised the manuscript critically. this is an open-access article distributed under the terms of the creative commons attribution (cc by 4.0) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord-254968-czrgzyr3 authors: zhang, qiang; zhang, huajun; gao, jindong; huang, kun; yang, yong; hui, xianfeng; he, xinglin; li, chengfei; gong, wenxiao; zhang, yufei; zhao, ya; peng, cheng; gao, xiaoxiao; chen, huanchun; zou, zhong; shi, zheng-li; jin, meilin title: a serological survey of sars-cov-2 in cat in wuhan date: 2020-09-17 journal: emerging microbes & infections doi: 10.1080/22221751.2020.1817796 sha: doc_id: 254968 cord_uid: czrgzyr3 covid-19 is a new respiratory illness caused by sars-cov-2, and has constituted a global public health emergency. cat is susceptible to sars-cov-2. however, the prevalence of sars-cov-2 in cats remains largely unknown. here, we investigated the infection of sars-cov-2 in cats during covid-19 outbreak in wuhan by serological detection methods. a cohort of serum samples were collected from cats in wuhan, including 102 sampled after covid-19 outbreak, and 39 prior to the outbreak. fifteen sera collected after the outbreak were positive for the receptor binding domain (rbd) of sars-cov-2 by indirect enzyme linked immunosorbent assay (elisa). among them, 11 had sars-cov-2 neutralizing antibodies with a titer ranging from 1/20 to 1/1080. no serological cross-reactivity was detected between sars-cov-2 and type i or ii feline infectious peritonitis virus (fipv). in addition, we continuously monitored serum antibody dynamics of two positive cats every 10 days over 130 days. their serum antibodies reached the peak at 10 days after first sampling, and declined to the limit of detection within 110 days. our data demonstrated that sars-cov-2 has infected cats in wuhan during the outbreak and described serum antibody dynamics in cats, providing an important reference for clinical treatment and prevention of covid-19. in december, 2019, an outbreak of pneumonia of unknown cause occurred in wuhan, china. the pathogen was soon identified to be the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and the disease was designated coronavirus disease 2019 (covid-19) by world health organization (who) [1, 2] . the clinical symptoms of covid-19 mainly include asymptomatic infection, mild-tosevere respiratory tract illness, and even death [3] . compared with sars-cov, sars-cov-2 has the higher basic reproduction number, representing more transmissibility [4] . within a very short period of time, covid-19 has quickly become a very serious threat to travel, commerce, and human health worldwide [5] . by 24 july 2020, a total of 15,012,731 confirmed cases, including 619,150 deaths, involving 216 countries, areas, or territories, have been reported globally by who (https://www.who.int/emergencies/ diseases/novel-coronavirus-2019). the outbreak of covid-19 was first confirmed in wuhan, china, possibly associated with a seafood market. however, so far, there is no evidence that the seafood market is the original source of sars-cov-2 [6] . before sars-cov-2, four types of beta coronaviruses can infect humans, including sars-cov and mers-cov which are highly pathogenic and both originated from bats [7, 8] . genome analysis showed that sars-cov-2 has 96.2% overall genome sequence identity with bat cov ratg13, indicating that sars-cov-2 could also originate from bats [9] . the transmission of sars-cov-2 from bats to humans was suspected to via the direct contact between humans and intermediate host animals [6] . although several coronaviruses related to sars-cov-2 were isolated from pangolin, the molecular and phylogenetic analyses showed that sars-cov-2 hardly emerged directly from this pangolin-cov-2020 [10] . at present, it remains largely unknown which animals were the intermediate host of sars-cov-2. our previous study showed that sars-cov-2 uses the same cell entry receptor, angiotensin converting enzyme ii (ace2), as sars-cov [9] , suggesting that sars-cov-2 has the same host range as sars-cov. previous report demonstrated that sars-cov can infect ferrets and cats [11] , implying that they might be also susceptible to sars-cov-2. in fact, the recent reports have shown that sars-cov-2 can indeed infect cats, but not cause any obvious symptoms [12] [13] [14] . cat is one of the most popular pets and often has close contact with humans. thus, the prevalence of sars-cov-2 in cats is very important to investigate, especially in outbreak regions. here, we investigated the serological prevalence of sars-cov-2 in cats by an indirect elisa and virus neutralization tests (vnt), and monitored the serum antibody dynamics of cats infected sars-cov-2, providing a basis for further understanding the infection of sars-cov-2 in cats. a total of 102 cats were sampled in wuhan between jan. and mar. 2020 with three sources: (1) 46 abandoned cats were from 3 animal shelters, (2) 41 cats were from 5 pet hospitals, and (3) 15 cats were from covid-19 patient families. all cats in shelter and hospital were live in relatively close cages. blood samples were collected via leg venipuncture and sera were separated and stored at −20°c until further processing. nasopharyngeal and anal swabs were collected and put into tubes containing viral transport medium-vtm (copan diagnostics, brescia, italy) [15] . all samples were collected under full personal-protective equipment, including head covers, goggles, n95 masks, gloves, and disposable gowns. a set of 39 cat sera were retrieved from the serum bank in our lab, which were collected from wuhan between mar. and may, 2019. hyperimmune sera were obtained from neuropathy pathogen laboratory, huazhong agriculture university, with neutralization titres of 1/640 and 1/1280, respectively, against type i and ii feline infectious peritonitis virus (fipv). the convalescent serum of a covid-19 patient was collected from jiangxia tongji hospital with the consent of the patient and a neutralization titre 1/1280. sars-cov-2 (ivcas 6.7512) was isolated from a covid-19 patient as previously described [9] . vero e6 was purchased from atcc (atcc® crl-1586 ™ ). antibody was tested by indirect elisa with the sars-cov-2 rbd protein (sino biological inc., china) and peroxidase conjugated goat anti-cat igg (sigma-aldrich, usa). briefly, elisa plates were coated overnight at 4°c with rbd protein (1 μg/ml, 100 μl per well). after blocked with pbs containing 5% skim milk for 2 h at 37°c, the plates were added with sera at a dilution of 1: 40. after incubation for 30 min at 37°c, the plates were washed five times with washing buffer (pbs containing 0.05% tween-20). a 1:20,000 diluted anti-cat igg was added and incubated for an additional 30 min. after another 5 washes, tmb substrate (sigma-aldrich, usa) was added and incubated for 10 min. then the reaction was stopped, and optical density (od) was measured at 450 nm. as the judgment method described previously [16, 17] , those sera were considered positive if the od values were twice higher than the mean od of the 39 sera collected between mar. and may, 2019. for virus neutralization test, serum samples were heatinactivated by incubation at 56°c for 30 min. each serum sample was serially diluted with dulbecco's modified eagle medium (dmem) as two fold or three fold according to the od value and the sample quality, mixed with equal volume of diluted virus and incubated at 37°c for 1 h. vero e6 cells in 24-well plates were inoculated with the sera-virus mixture at 37°c; 1 h later, the mixture was replaced with dmem containing 2.5% fbs and 0.8% carboxymethylcellulose. the plates were fixed with 8% paraformaldehyde and stained with 0.5% crystal violet 3 days later. all samples were tested in duplicate and neutralization titres were defined as the serum dilution resulting in a plaque reduction of at least 50% [18] . the total protein concentration of purified and inactivated sars-cov-2 was determined by bradford protein assay [19] . 4 μg protein was subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred on to nitrocellulose membrane. then viral proteins were blotted with cat sera or human convalescent serum. protein bands were visualized by incubation with a goat anti-cat igg or mouse anti-human igg and then detected using the ecl system (amersham life science, arlington heights, il, usa). cat serum samples were detected with an indirect elisa based on recombinant rbd protein. from the 39 prior-to-outbreak sera, whose optical density (od) varied from 0.091 to -0.261, we set the cut-off as 0.32. the positive samples of 102 cat sera were screened according to this standard. as shown in table 1 and figure 1 , 15 (14.7%) cat sera collected during the outbreak were positive, with five strong positive ones with od more than 0.6. of which, cat#14 and cat#15 were from the same owner who was covid-19 patient. both type i and ii fipv hyperimmune sera showed no cross-reactivity with sars-cov-2 rbd protein. to further confirm the presence of sars-cov-2 specific antibody in cats, all of 15 elisa-positive sera were subjected to vnts for sars-cov-2. among them, 11 (10.8%) had sars-cov-2 neutralizing antibodies with a titre ranging from 1/20 to 1/1080 (table 1 and figure 2(a) ). however, 4 sera including #12, which was elisa strong positive with od of 0.852, showed no neutralizing activity, most likely because of recognition of non-neutralizing epitopes. another elisa strong positive one, #10, had very weak neutralizing activity. but strong neutralization was observed for the other three elisa strong positive sera, namely #4, #14 and #15, with neutralizing titre of 1/360-1/1080. consistent with the high neutralizing titre, the owners of cat#4, cat#14 and cat#15 were diagnosed as covid-19 patients. cat#1, cat#5∼9 was from pet hospitals, while cat#2, cat#10∼13 were initially abandoned cats and kept in animal protection shelters after the outbreak. again, both type i and ii fipv hyperimmune sera were negative for vnt. the sera of infected cats can specifically bind the s and n proteins of sars-cov-2 western blot assay was also performed to further verify the existence of sars-cov-2 specific igg in cat serum. as shown in figure 2 (b), s and n proteins of the purified sars-cov-2 were successfully detected with #4, #14 and #15 sera after diluted 100 folds, as well as human convalescent serum [20] . conversely, the elisa negative cat serum and healthy human serum did not probe the protein bands, thereby demonstrating the existence of sars-cov-2 specific igg in cat serum. fortunately, we had access to two cats, cat#14 and cat#15, for a long time, which gave us the opportunity to track the dynamic of antibody. we continuously sampled cat#14 and cat#15 every 10 days over 130 days. as shown in figure 3 (a), rbd antibodies of these two cats reached the peak at the second sampling, when both showed od>1.0 for elisa. after that, rbd antibodies turned down and decreased to detection limit in 110 days. accordingly, neutralizing antibodies showed similar trend (figure 3(b) ). in this study, we detected the presence of sars-cov-2 antibodies in cats in wuhan during the covid-19 outbreak with elisa, vnt and western blot. a total of 102 cats were tested, 15 (14.7%) were positive for rbd based elisa and 11 (10.8%) were further positive with vnt. these results demonstrated that sars-cov-2 has infected cats in wuhan, implying that this risk could also occur at other outbreak regions. in fact, it has been indeed successively reported that sars-cov-2 infected cats under natural conditions [14, 21] . retrospective investigation confirmed that all figure 2 . virus neutralization test and western blot assay of cat serum samples for sars-cov-2 (a) cat#14, cat#15 and cat#4 sera were 3-fold serially diluted and mixed with sars-cov-2; after incubated at 37°c for 1 h, the mixture was used to infect vero e6 cells, and replaced with semi-solid media 1 h later. the plates were fixed and stained 3 days later. all samples were tested in duplicate. (b) western blot of purified sars-cov-2 with cat or human sera. all sera were diluted 100 folds. c-n, negative cat serum. h-p, human convalescent serum. h-n, healthy human serum. of elisa positive sera were sampled after the outbreak, suggesting that the infection of cats could be due to the virus transmission from humans to cats. certainly, it is still needed to be verified via investigating the sars-cov-2 infections before this outbreak in a wide range of sampling. at present, there is no evidence of sars-cov-2 transmission from cats to humans. however, a latest report shows that sars-cov-2 can transmit between cats via respiratory droplets [12] . over all, some preventive measures are necessary for blocking the human-to-cat transmission or preventing the potential transmission risk of cats to other animals or humans. through analysing the background of the tested cats, we found that 4 of abandoned cats (9.8%), 4 of cats from pet hospitals (8.7%), and 3 of cats with patient owners (20%) were positive with vnt. although the positive rate among different source cats had no significant differences, the three cats with the highest neutralization titres (1/1080, 1/360, and 1/360, respectively) were owned by covid-19 patients. on the contrary, the sera collected from pet hospital cats and stray cats had neutralizing activity of 1/20-1/80, indicating that the high neutralization titres could be due to the close contact between cats and covid-19 patients. in addition, our data demonstrated that the duration of neutralizing antibody against sars-cov-2 is relatively transient in the infected cats. so, the low neutralization titres could also be due to the long-time interval between sample collection and actual infection date. although the infection in stray cats was not fully understood, it is reasonable to speculate that these infections are probably due to the contact with sars-cov-2 polluted environment, or covid-19 patients who fed the cats. the antibody-mediated humoral response is crucial for preventing viral infections, of which the neutralizing antibody can reduce the entry of the virus into an infected cell via blocking the interaction between virus and cell [22] . so, the neutralizing antibody is an important indicator that can reflect the host antiviral ability. although numerous reports have indicated that the infection of sars-cov-2 can induce the production of neutralizing antibody, the understanding about the dynamics of sars-cov-2 neutralizing antibody remains largely unknown. here, we continuously monitored the dynamics of binding antibody and neutralizing antibody against sars-cov-2 in the infected cats. we found that both these two antibodies can be induced with a relatively high level, however the duration of peak titre was very short, and decreased to the limit of detection within 110 days. it was worth noting that, these two cats were from the same owner who presented with fever and cough in mid-february, and was diagnosed and segregated as covid-19 patient on february 21. then these two cats were fostered in pet hospital and were also segregated. combined with the dynamic characteristic and timeline of antibody response, we speculated that these two cats should be infected at the same time. in addition, considering that the two cats were constantly in segregation, we believed that our data represented the antibody dynamic characteristic of primary infection. importantly, this transient antibody response induced by sars-cov-2 resembles those observed in seasonal coronavirus infections, implying that the convalescent cats after sars-cov-2 infection remain the risk of re-infection. in fact, this similar transient antibody response has also been observed in human antibody [23, 24] , suggesting that cat has a great potential as an animal model for assessing the characteristic of antibody against sars-cov-2 in human. our data provided a very important reference for the clinical treatment and prevention of covid-19. in addition, we also collected nasopharyngeal and anal swabs of each cat, and conducted sars-cov-2 specific qrt-pcr using a commercial kit which targeted orf1ab and n genes. seven samples from five cats were n gene single positive with ct ranging from 34.9 to 36.7, but no double gene positive sample was detected (according to the manufacture instruction in which ct value less than 37 was deemed as positive). the reason might be (1) that the viral rna load is too low to be detected; (2) the period that cat shed sars-cov-2 may be very short [21] , along with asymptomatic infection, we didn't catch the moment of acute infection; (3) there may be variants in the genomic sequences in cats, leading to the failure in amplification in cat samples. in conclusion, our study provided serological evidence for sars-cov-2 infection in pets, and described the dynamic characteristic of serum antibody in cats. further research is needed to investigate the transmission route of sars-cov-2 from humans to cats. in addition, some preventive measures should be implemented to maintain a suitable distance between covid-19 patients and companion animals such as cats and dogs, and hygiene and quarantine measures should also be established for those high-risk animals. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan the reproductive number of covid-19 is higher compared to sars coronavirus comparing sars-cov-2 with sars-cov and influenza pandemics the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status discovery of bat coronaviruses through surveillance and probe capturebased next-generation sequencing composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin are pangolins the intermediate host of the 2019 novel coronavirus (sars-cov-2)? virology: sars virus infection of cats and ferrets susceptibility of ferrets, cats, dogs, and other domesticated animals to sarscoronavirus 2 transmission of sars-cov-2 in domestic cats severe acute respiratory syndrome coronavirus 2-specific antibodies in pets in wuhan middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation detection of antibodies against dna polymerase of hepatitis b virus in hbsag-positive sera using elisa development of a novel rapid micro-neutralization elisa for the detection of neutralizing antibodies against chandipura virus vaccinia virus h3l envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice inactivation efficacy of nonthermal plasma-activated solutions against newcastle disease virus effectiveness of convalescent plasma therapy in severe covid-19 patients first detection and genome sequencing of sars-cov-2 in an infected cat in france perspectives on therapeutic neutralizing antibodies against the novel coronavirus sars-cov-2 the production of antibodies for sars-cov-2 and its clinical implication longitudinal evaluation and decline of antibody responses in sars-cov-2 infection we acknowledge jiangxia tongji hospital for providing the convalescent serum of covid-19 patient. we thank professor guiqing peng (huazhong agriculture university) for providing the hyperimmune sera against type i and ii fipv. we are particularly grateful to wuhan national biosafety laboratory running team, including engineer, biosafety, biosecurity, and administrative staff. no potential conflict of interest was reported by the author(s). key: cord-015503-j99cgsjt authors: tang, xiaolu; wu, changcheng; li, xiang; song, yuhe; yao, xinmin; wu, xinkai; duan, yuange; zhang, hong; wang, yirong; qian, zhaohui; cui, jie; lu, jian title: on the origin and continuing evolution of sars-cov-2 date: 2020-03-03 journal: natl sci rev doi: 10.1093/nsr/nwaa036 sha: doc_id: 15503 cord_uid: j99cgsjt the sars-cov-2 epidemic started in late december 2019 in wuhan, china, and has since impacted a large portion of china and raised major global concern. herein, we investigated the extent of molecular divergence between sars-cov-2 and other related coronaviruses. although we found only 4% variability in genomic nucleotides between sars-cov-2 and a bat sars-related coronavirus (sarsr-cov; ratg13), the difference at neutral sites was 17%, suggesting the divergence between the two viruses is much larger than previously estimated. our results suggest that the development of new variations in functional sites in the receptor-binding domain (rbd) of the spike seen in sars-cov-2 and viruses from pangolin sarsr-covs are likely caused by mutations and natural selection besides recombination. population genetic analyses of 103 sars-cov-2 genomes indicated that these viruses evolved into two major types (designated l and s), that are well defined by two different snps that show nearly complete linkage across the viral strains sequenced to date. although the l type (∼70%) is more prevalent than the s type (∼30%), the s type was found to be the ancestral version. whereas the l type was more prevalent in the early stages of the outbreak in wuhan, the frequency of the l type decreased after early january 2020. human intervention may have placed more severe selective pressure on the l type, which might be more aggressive and spread more quickly. on the other hand, the s type, which is evolutionarily older and less aggressive, might have increased in relative frequency due to relatively weaker selective pressure. these findings strongly support an urgent need for further immediate, comprehensive studies that combine genomic data, epidemiological data, and chart records of the clinical symptoms of patients with coronavirus disease 2019 (covid-19). the coronavirus disease 2019 (covid-19) epidemic started in late december 2019 in wuhan, the capital of central china's hubei province. since then, it has rapidly spread across china and in other countries, raising major global concerns. the etiological agent is a novel coronavirus, sars-cov-2, named for the similarity of its symptoms to those induced by the severe acute respiratory syndrome. as of february 28, 2020, 78,959 cases of sars-cov-2 infection have been confirmed in china, with 2,791 deaths. worryingly, there have also been more than 3,664 confirmed cases outside of china in 46 countries and areas (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/), raising significant doubts about the likelihood of successful containment. further, the genomic sequences of sars-cov-2 viruses isolated from a number of patients share sequence identity higher than 99.9%, suggesting a very recent host shift into humans [1] [2] [3] . coronaviruses are naturally hosted and evolutionarily shaped by bats [4, 5] . indeed, it has been postulated that most of the coronaviruses in humans are derived from the bat reservoir [6, 7] . unsurprisingly, several teams have recently confirmed the genetic similarity between sars-cov-2 and a bat betacoronavirus of the sub-genus sarbecovirus [8] [9] [10] [11] [12] [13] . the whole-genome sequence identity of the novel virus has 96.2% similarity to a bat sars-related coronavirus (sarsr-cov; ratg13) collected in yunnan province, china [2, 14] , but is not very similar to the genomes of sars-cov (about 79%) or mers-cov (about 50%) [1, 15] . it has also been confirmed that the sars-cov-2 uses the same receptor, the angiotensin converting enzyme ii (ace2), as the sars-cov [11] . although the specific route of transmission from natural reservoirs to humans remains unclear [5, 13] , several studies have shown that pangolins may have provided a partial spike gene to sars-cov-2; the critical functional sites in the spike protein of sar-cov-2 are nearly identical to one identified in a virus isolated from a pangolin [16] [17] [18] . despite these recent discoveries, several fundamental issues related to the evolutionary patterns and driving forces behind this outbreak of sars-cov-2 remain unexplored [19] . herein, we investigated the extent of molecular divergence between sars-cov-2 and other related coronaviruses and carried out population genetic analyses of 103 sequenced genomes of sars-cov-2. this work provides new insights into the factors driving the evolution of sars-cov-2 and its pattern of spread through the human population. for each annotated orf in the reference genome of sars-cov-2 (nc_045512), we extracted the orthologous sequences in human sars-cov, four bat sars-related coronaviruses (sarsr-cov: ratg13, zxc21, zc45, and bm48-31), one pangolin sarsr-cov from guangdong (gd) [17] , and six pangolin sarsr-cov genomes from guangxi (gx) [18] (table s1) . we aligned the coding sequences (cdss) based on the protein alignments (see materials and methods). most orfs annotated from sars-cov-2 were found to be conserved in other viruses, except for orf8 and orf10 (table 1 ). the protein sequence of sars-cov-2 orf8 shared very low similarity with sequences in sars-cov and bm48-31, and orf10 had a premature stop codon in both sars-cov and bm48-31 (fig. s1) . a one-base deletion caused a frame-shift mutation in orf10 of zxc21 ( fig. s1 ). to investigate the phylogenetic relationships between these viruses at the genomic scale, we concatenated coding regions (cdss) of the nine conserved orfs (orf1ab, e, m, n, s, orf3a, orf6, orf7a, and orf7b) and reconstructed the phylogenetic tree using the synonymous sites ( fig. 1a) . we also used codeml in the paml [20] 1a ). in parallel, we also calculated the pairwise dn, ds, and ω values between sars-cov-2 and another virus ( table 1) . the genome-wide phylogenetic tree indicated that sars-cov-2 was closest to ratg13, followed by gd pangolin sarsr-cov, then by gx pangolin sarsr-covs, then by zc45 and zxc21, then by human sars-cov, and finally by bm48-31 (fig. 1a) . notably, we found that the nucleotide divergence at synonymous sites between sars-cov-2 and other viruses was much higher than previously anticipated. for example, although the overall genomic nucleotides overall differ ~4% between sars-cov-2 and ratg13, the genomic average ds was 0.17, which means the divergence at the neutral sites is 17% between these two viruses (table 1) . this is because the nonsynonymous sites are usually under stronger negative selection than synonymous sites, and calculating sequence differences without separating these two classes of sites may underestimate the extent of molecular divergence by several folds. notably, the ds value varied considerably across genes in sars-cov-2 and the other viruses analyzed. in particular, the spike gene (s) consistently exhibited larger ds values than other genes (table 1 ). this pattern became clear when we calculated the ds value for each branch in fig. 1a for the spike gene versus the concatenated sequences of the remaining genes ( fig. s2 ). in each branch, the ds of spike was 2.22 ± 1.35 (mean ± sd) times as large as that of the other genes. this extremely elevated ds value of spike could be caused either by a high mutation rate or by natural selection that favors synonymous substitutions. synonymous substitutions may serve as another layer of genetic regulation, guiding the efficiency of mrna translation by changing codon usage [21] . if positive selection is the driving force for the higher synonymous substation rate seen in spike, we expect the frequency of optimal codons (fop) of spike to be different from that of other genes. however, our codon usage bias analysis (table s2 ) suggests the fop of spike was only slightly higher than that of the genomic average (0.717 versus 0.698, see materials and methods). thus, we believe that the elevated synonymous substitution rate measured in spike is more likely caused by higher mutational rates; however, the underlying molecular mechanism remains unclear. both sars-cov and sars-cov-2 bind to ace2 through the rbd of spike protein in order to initiate membrane fusion and enter human cells [1, 2, [22] [23] [24] [25] [26] . five out of the six critical amino acid (aa) residues in rbd were different between sars-cov-2 and sars-cov (fig. 1b) , and a 3d structural analysis indicated that the spike of sars-cov-2 has a higher binding affinity to ace2 than sars-cov [23] . intriguingly, these same six critical aas are identical between gd pangolin-cov and sars-cov-2 [16] . in contrast, although the genomes of sars-cov-2 and ratg13 are more similar overall, only one out of the six functional sites are identical between the two viruses ( fig. 1b) . it has been proposed that the sars-cov-2 rbd region of the spike protein might have resulted from recent recombination events in pangolins [16] [17] [18] . although several ancient recombination events have been described in spike [27, 28] , it also seems likely that the identical functional sites in sars-cov-2 and gd pangolin-cov may actually the result of coincidental convergent evolution [18] . if the functional aa residues in the sars-cov-2 rbd region were acquired from gd pangolin-cov in a very recent recombination event, we would expect the nucleotide sequences of this region to be nearly identical between the two viruses. however, for the cds sequences that span five critical aa sites in the sars-cov-2 spike (ranging from codon 484 to 507, covering five adjacent functional sites: f486, q493, s494, n501, and y505; fig. s3 originated from the gd pangolin-cov due to a very recent recombination event. alternatively, it seems more likely that a high mutation rate in spike, coupled with strong natural selection, has shaped the identical functional aa residues between these two viruses, as proposed previously [18] . although these sites are maintained in sars-cov-2 and gd pangolin-cov, mutations may have changed the residues in the ratg13 lineage after it diverged from sars-cov-2 (the blue arrow in fig. 1a ). in summary, it seems that the shared identity of critical aa sites between sars-cov-2 and gd pangolin-cov might be due to random mutations coupled with natural selection, and not necessarily recombination. the genome-wide ω value between sars-cov-2 and other viruses ranged from 0.044 to 0.124 (table 1) we downloaded 103 publicly available sars-cov-2 genomes, aligned the sequences, and identified the genetic variants. for ease of visualization, we marked each virus strain based on the location and date the virus was isolated with the format of "location_date" throughout this study (see table s1 for details; each id did not contain information of the patient's race or ethnicity). although sars-cov-2 is an rna virus, for simplicity, we presented our results based on dna sequencing results throughout this study (i.e., the nucleotide t (70/83) of nonsynonymous mutations), indicating either a recent origin [30] or population growth [31] . in general, the derived alleles of synonymous mutations were significantly skewed towards higher frequencies than those of nonsynonymous ones (p < 0.01, wilcoxon rank-sum test; fig. 2 ), suggesting the nonsynonymous mutations tended to be selected against. however, 16.3% (7 out of 43) synonymous mutations, and one nonsynonymous (orf8 (l84s, 28,144)) mutation had a derived frequency of ≥ 70% across the sars-cov2 strains. the nonsynonymous mutations that had derived alleles in at least two sars-cov-2 strains affected six proteins: orf1ab (a117t, i1607v, l3606f, i6075t), s (h49y, v367f), orf3a (g251v), orf7a (p34s), orf8 (v62l, s84l), and n (s194l, s202n, p344s). to detect the possible recombination among sars-cov2 viruses, we used haploview [32] to analyze and visualize the patterns of linkage disequilibrium (ld) between variants with minor alleles in at least two sars-cov-2 strains (fig. 3a ). since most mutations were at very low frequencies, it is not surprising that many pairs had a very low r 2 or lod value ( fig. 3b -c). consistent with another recent report [31] , we did not find evidence of recombination between the sars-cov2 strains. however, we found that snps at location 8,782 (orf1ab: t8517c, synonymous) and 28,144 (orf8: c251t, s84l) showed significant linkage, with an r 2 value of 0.954 (fig. 3b, red) and a lod value of 50.13 (fig. 3c, red) . among the 103 sars-cov-2 virus strains, 101 of them exhibited complete linkage between the two snps: 72 strains exhibited a "ct" haplotype (defined as "l" type because t28,144 is in the codon of leucine) and 29 strains exhibited a "tc" haplotype (defined as "s" type because c28,144 is in the codon of serine) at these two sites. thus, we categorized the sars-cov-2 viruses into two major types, with l being the major type (~70%) and s being the minor type (~30%). although we defined the l and s types based on two tightly linked snps, strikingly, the separation between the l (blue) and s (red) types was maintained when we reconstructed the haplotype networks using all the snps in the sars-cov-2 genomes ( fig. 4a ; the number of mutations between two neighboring haplotypes was inferred parsimoniously). this analysis further supports the idea that the two linked snps at sites 8,782 and 28,144 adequately define the l and s types of sars-cov-2. to determine whether l or s type is ancestral, we examined the genomic alignments of sars-cov-2 and other highly related viruses. strikingly, nucleotides of the s type at sites 8,782 and 28,144 were identical to the orthologous sites in the most closely related viruses ( fig. 4b) . remarkably, both sites were highly conserved in other viruses as well. hence, although the l type (~70%) was more prevalent than the s type (~30%) in the sars-cov-2 viruses we examined, the s type is actually the ancestral version of sars-cov-2. to further examine the relationship among the strains in the l and s types, we reconstructed a phylogenetic tree of all the 103 sars-cov-2 viruses based on their whole-genome sequences. our phylogenetic tree also clearly shows the separation of the two types (fig. 5) . viruses of the l type (blue) first clustered together, and likewise, viruses of the s type (red) were also more closely related to each other. therefore, our whole-genome comparisons further confirm the separation of the l and s types. thus far, we found that, although the l type is derived from the s type, l (~70%) is more prevalent than s (~30%) among the sequenced sars-cov-2 genomes we examined. this pattern suggests that l has a higher transmission rate than the s type. furthermore, our mutational load analysis indicated that the l type had accumulated a significantly higher number of derived mutations than s type (p < 0.0001, wilcoxon rank-sum test; fig. s5 ). we propose that, although the l type newly evolved from the ancient s type, it transmits faster or replicates faster in human populations, causing it to accumulate more mutations than the s type. thus, our results suggest the l might be more aggressive than the s type due to the potentially higher transmission and/or replication rates. to test whether the two types of sars-cov-2 had differences in temporal and spatial distributions, we stratified the viruses based on the locations and dates they were isolated ( fig. 6 and table s3 ). if the l type is more aggressive than the s type, why did the relative frequency of the l type decrease compared to the s type in other places after the initial breakout in wuhan? one possible explanation is that, since january 2020, the chinese central and local governments have taken rapid and comprehensive prevention and control measures. these human intervention efforts might have caused severe selective pressure against the l type, which might be more aggressive and spread more quickly. the s type, on the other hand, might have experienced weaker selective pressure by human intervention, leading to an increase in its relative abundance among the sars-cov-2 viruses. thus, we hypothesized that the two types of sars-cov-2 viruses might have experienced different selective pressures due to different epidemiological features. of note, the above analyses were based on very patchy sars-cov-2 genomes that were collected from different locations and time points. more comprehensive genomic data is required for further testing of our hypothesis. it is currently unclear how the l type specifically evolved from the s type during the development of sars-cov-2. however, we found that the sequence of viruses isolated from to further investigate the heteroplasmy of sars-cov-2 viruses in patients, we searched 12 deep-sequencing libraries of sars-cov-2 genomes that were deposited in the sequence read archive (sra) ( table s4 , materials and methods). we found 17 genomic sites that showed evidence of heteroplasmy of sars-cov-2 virus in five patients, but we did not find any other instances of the co-existence of l and s types in any patient (table 2) . these findings evince the developing complexity of the evolution of sars-cov-2 infections. further studies investigating how the different alleles of sars-cov-2 viruses compete with each other will be of significant value. in this study, we investigated the patterns of molecular divergence between sars-cov-2 and other related coronaviruses. although the genomic analyses suggested that sars-cov-2 was closest to ratg13, their difference at neutral sites was much higher than previously realized. our results provide novel insights into tracing the intermediate natural host of sars-cov-2. with population genetic analyses of 103 genomes of sars-cov-2, we found that sars-cov-2 viruses evolved into two major types (l and s types), and the two types were well defined by just two snps that show nearly complete linkage across sars-cov-2 strains. although the l type (~70%) was more prevalent than the s type (~30%) in the sars-cov-2 viruses we examined, our evolutionary analyses suggested the s type was most likely the more ancient version of sars-cov-2. our results also support the idea that the l type is more aggressive than the s type. since nonsynonymous sites are usually under stronger negative selection than synonymous sites, calculating sequence differences without separating these two classes of sites could lead to a potentially significant underestimate of the degree of molecular divergence. for example, although the overall nucleotides only differed by ~4% between sars-cov-2 and ratg13, the genomic average ds value, which is usually a neutral proxy, was 0.17 between these two viruses ( table 1) . of note, the genome-wide ds value is 0.012 between humans and chimpanzees [33] , and 0.08 between humans and rhesus macaques [34] . thus, the neutral molecular divergence between sars-cov-2 and ratg13 is 14 times larger than that between humans and chimpanzees, and twice as large as that between humans and macaques. the genomic average ds value between sars-cov-2 and gd pangolin-cov is 0.475, which is comparable to that between humans and mice (0.5) [35] , and the ds value between our analyses of molecular evolution and population genetics suggested that some amino acid changes might be favored by natural selection during the evolution of sars-cov-2 and other related viruses. however, negative selection appears to be the predominant force acting on these viruses. interestingly, the virus isolated from one patient in shenzhen on january 13, 2020 (sz_2020/01/13.a, gisaid id: epi_isl_406592) had c at both positions 8,782 and 28,144 in the genome, belonging to neither l nor s type ( fig. 4a and 5) . notably, this strain had one stop-gain mutation in orf1ab and had accumulated 20 silent and 5 nonsynonymous mutations after diverging from the ancestor haplotype (fig. 4a ). thus, it is possible that functional constraints on the genomic sequence were weakened after the disruption of orf1ab in this strain. notably, on viruses isolated from a patient living in south korean (skorea_2020/01.a, gisaid: epi_isl_411929), acquired six nonsynonymous mutations that were different from the most recent common ancestor of sars-cov-2: orf1ab (m902i and t6891m), s (s221w), orf3a (w128l and g251v), and e (l37h). if these changes are not due to sequencing errors, it would be interesting to test whether and how these mutations affect the transmission and pathogenesis of sars-cov-2. in this work, we propose that sars-cov-2 can be divided into two major types (l and s types): the s type is ancestral, and the l type evolved from s type. intriguingly, the s and l types can be clearly defined by just two tightly linked snps at positions 8,782 (orf1ab: t8517c, synonymous) and 28,144 (orf8: c251t, s84l). however, it is currently unclear whether l type evolved from the s type in humans or in the intermediate hosts. it is also unclear whether the l type is more virulent than the s type. orf1ab, which encodes replicase/transcriptase, is required for viral genome replication and might also be important for viral pathogenesis [36] . although the t8517c mutation in orf1ab does not change the protein sequence (it changes the codon agt (ser) to agc (ser)), we hypothesized this mutation might affect orf1ab translation since agt is preferred while agc is unpreferred (table s2 ). orf8 promotes the expression of atf6, the er unfolded protein response factor, in human cells [37] . thus, it will be interesting to investigate the function of the s84l aa change in orf8, as well as the combinatory effect of these two mutations in sars-cov-2 pathogenesis. in summary, our analyses of 103 sequenced sars-cov-2 genomes suggest that the l type is more aggressive than the s type and that human interference may have shifted the relative abundance of l and s type soon after the sars-cov-2 outbreak. as previously noted [19] , the data examined in this study are still very limited, and follow-up analyses of a larger set of data are needed to have a better understanding of the evolution and epidemiology of sars-cov-2. there is a strong need for further immediate, comprehensive studies that combine genomic data, epidemiological data, and chart records of the clinical symptoms of patients with sars-cov-2. the set of 103 complete genome sequences were downloaded from gisaid (global initiative on sharing all influenza data; https://www.gisaid.org/) with acknowledgment, genbank (https://www.ncbi.nlm.nih.gov/genbank), and nmdc (http://nmdc.cn/#/ncov). sequences and annotations of the reference genome of sars-cov-2 (nc_045512) and other related viruses were downloaded from genbank or gisaid (table s1 ). the genomic sequences of sars-cov-2 were aligned using muscle v3.8.31 [38] . the annotated cdss of other viruses were downloaded from genbank. to avoid missing annotations in other viruses, we also annotated the orfs using cdss annotated in sars-cov-2 using exonerate (--model protein2genome:bestfit --score 5 -g y) [39] . the protein sequences of sars-cov-2 and other related viruses were aligned with muscle v3.8.31 [38] , and the codon alignments were made based on the protein alignment with revtrans [40] . the codon alignments of the conserved orfs were further concatenated for down-stream evolutionary analysis. the phylogenetic tree was constructed by the neighbor-joining method in mega-x [41] using the parameters of kimura 2-parameter model, and only the third positions of codons were considered. yn00 from paml v4.9a [20] was used to calculate the pairwise divergence between sars-cov-2 and other viruses for each individual gene or for the concatenated sequences. the free-ratio model in codeml in the paml [20] package was used to calculate the dn, ds, and ω values for each branch. positive selection was detected using easycodeml [42] , a recently published wrapper of codeml [20] . the m7 and m8 models were compared. in the m7 model, ω follows a beta distribution such that 0⩽ω⩽1, and in the m8 model, a proportion p 0 of sites have ω drawn from the beta distribution, and the remaining sites with proportion p 1 are positively selected and have ω 1 >1. the lrts between m7 and m8 models were conducted by comparing twice the difference in log-likelihood values (2 ln δl) against a χ 2 -distribution (df=2). the positively selected sites were identified with the bayes empirical bayes (beb) score larger than 0.95. dnasp v6.12.03 [43] was used to generate multi-sequence aligned haplotype data, and popart v1.7 [44] was used to draw haplotype networks based on the haplotypes generated by dnasp. raxml v8.2.12 [45] was used to build the maximum likelihood phylogenetic tree of 103 aligned sars-cov-2 genomes with theparameters "-p 1234 -m gtrcat". we downloaded 12 sars-cov-2 metagenomic sequencing libraries (table s2) , and mapped the ngs reads to the reference genome of sars-cov-2 (nc_045512) using bwa (0.7.17-r1188) [46] with the default parameters. snp calling was done using bcftools mpileup (bcftools 1.9) [47] . we calculated the rscu (relative synonymous codon usage) value of each codon in the sars-cov-2 reference genome (nc_045512). the rscu value for each codon was the observed frequency of this codon divided by its expected frequency under equal usage among the amino acid [48] . the codons with rscu > 1 were defined as preferred codons, and those with rscu < 1 were defined as unpreferred codons. the fop (frequency of optimal codons) value of each gene was calculated as the number of preferred codons divided by the total number of preferred and unpreferred codons. the authors declare that they have no conflicts of interest. for each gene, the dn and ds values between sars-cov-2 and 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based on decade-long structural studies of sars cryo-em structure of the 2019-ncov spike in the prefusion conformation characterization of spike glycoprotein of 2019-ncov on virus entry and its immune cross-reactivity with spike glycoprotein of sars-cov identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin a new coronavirus associated with human respiratory disease in china homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human moderate mutation rate in the sars coronavirus genome and its implications origin time and epidemic dynamics of the 2019 novel coronavirus decoding evolution and transmissions of novel pneumonia coronavirus using the whole genomic data haploview: analysis and visualization of ld and haplotype maps the chimpanzee s, analysis c. initial sequence of the chimpanzee genome and comparison with the human genome evolutionary and biomedical insights from the rhesus macaque genome initial sequencing and comparative analysis of the mouse genome sars coronavirus replicase proteins in pathogenesis discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus muscle: multiple sequence alignment with high accuracy and high throughput automated generation of heuristics for biological sequence comparison revtrans: multiple alignment of coding dna from aligned amino acid sequences molecular evolutionary genetics analysis across computing platforms easycodeml: a visual tool for analysis of selection using codeml dnasp 6: dna sequence polymorphism analysis of large data sets popart: full-feature software for haplotype network construction raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools codon usage in regulatory genes in escherichia coli does not reflect selection for 'rare' codons the authors thank the researchers who generated and shared the sequencing data from note the derived alleles of synonymous mutations are skewed towards higher frequencies than those of nonsynonymous mutations. a. ld plot of any two snp pairs among the 29 sites that have minor alleles in at least two strains. the number near slashes at the top of the image shows the coordinate of sites in the genome. color in the square is given by standard (d'/lod), and the number in square is r 2 value. b. the r 2 of each pair of snps (y-axis) against the genomic distance between that pair (x-axis). c. the lod of each pair of snps (y-axis) against the genomic distance between that pair (x-axis). note that in both b and c, the red point represents the ld between snps at 8,782 and 28,144. a. the haplotype networks of sars-cov-2 viruses. blue represents the l type, and red is the s type. the orange arrow indicates that the l type evolved from the s type. note that in this study, we marked each sample with a unique id that starting with the geological location, followed by the date the virus was isolated (see table s1 for details). each id did not contain information of the patient's race or ethnicity. b. evolution of the l and s types of sars-cov-2 viruses. genome sequence alignments with the seven most closely related viruses indicated that the s type was most likely the ancient version of sars-cov-2. ".", the nucleotide sequence is identical; "-", gap. in our recent publication (https://doi.org/10.1093/nsr/nwaa036), we showed that among circulating sars-cov-2 (with 103 genomes analyzed) two different viral genomes co-exist. we identified them as lineages l and s. the concerned amino acid we used to define the l and s lineages is located in orf8 (open reading frame 8), which plays a yet undefined role in the viral life cycle. based on the finding that "l" lineage has a higher frequency than lineage s, we described the l lineage as aggressive. we now recognize that within the context of our study the term "aggressive" is misleading and should be replaced by a more precise term "a higher frequency". in short, while we have shown that the two lineages naturally co-exist, we provided no evidence supporting any epidemiological conclusion regarding the virulence or pathogenicity of sars-cov-2. by saying so, corrections will be made in the print version of this paper to avoid being misleading. key: cord-274834-24v2b509 authors: lima, rosiane; gootkind, elizabeth f.; de la flor, denis; yockey, laura j.; bordt, evan a.; d’avino, paolo; ning, shen; heath, katerina; harding, katherine; zois, jaclyn; park, grace; hardcastle, margot; grinke, kathleen a.; grimmel, sheila; davidson, susan p.; forde, pamela j.; hall, kathryn e.; neilan, anne m.; matute, juan d.; lerou, paul h.; fasano, alessio; shui, jessica e.; edlow, andrea g.; yonker, lael m. title: establishment of a pediatric covid-19 biorepository: unique considerations and opportunities for studying the impact of the covid-19 pandemic on children date: 2020-09-11 journal: bmc med res methodol doi: 10.1186/s12874-020-01110-y sha: doc_id: 274834 cord_uid: 24v2b509 background: covid-19, the disease caused by the highly infectious and transmissible coronavirus sars-cov-2, has quickly become a morbid global pandemic. although the impact of sars-cov-2 infection in children is less clinically apparent, collecting high-quality biospecimens from infants, children, and adolescents in a standardized manner during the covid-19 pandemic is essential to establish a biologic understanding of the disease in the pediatric population. this biorepository enables pediatric centers world-wide to collect samples uniformly to drive forward our understanding of covid-19 by addressing specific pediatric and neonatal covid-19-related questions. methods: a covid-19 biospecimen collection study was implemented with strategic enrollment guidelines to include patients seen in urgent care clinics and hospital settings, neonates born to sars-cov-2 infected mothers, and asymptomatic children. the methodology described here, details the importance of establishing collaborations between the clinical and research teams to harmonize protocols for patient recruitment and sample collection, processing and storage. it also details modifications required for biobanking during a surge of the covid-19 pandemic. results: considerations and challenges facing enrollment of neonatal and pediatric cohorts are described. a roadmap is laid out for successful collection, processing, storage and database management of multiple pediatric samples such as blood, nasopharyngeal and oropharyngeal swabs, sputum, saliva, tracheal aspirates, stool, and urine. using this methodology, we enrolled 327 participants, who provided a total of 972 biospecimens. conclusions: pediatric biospecimens will be key in answering questions relating to viral transmission by children, differences between pediatric and adult viral susceptibility and immune responses, the impact of maternal sars-cov-2 infection on fetal development, and factors driving the multisystem inflammatory syndrome in children. the specimens in this biorepository will allow necessary comparative studies between children and adults, help determine the accuracy of current pediatric viral testing techniques, in addition to, understanding neonatal exposure to sars-cov-2 infection and disease abnormalities. the successful establishment of a pediatric biorepository is critical to provide insight into disease pathogenesis, and subsequently, develop future treatment and vaccination strategies. conclusions: pediatric biospecimens will be key in answering questions relating to viral transmission by children, differences between pediatric and adult viral susceptibility and immune responses, the impact of maternal sars-cov-2 infection on fetal development, and factors driving the multisystem inflammatory syndrome in children. the specimens in this biorepository will allow necessary comparative studies between children and adults, help determine the accuracy of current pediatric viral testing techniques, in addition to, understanding neonatal exposure to sars-cov-2 infection and disease abnormalities. the successful establishment of a pediatric biorepository is critical to provide insight into disease pathogenesis, and subsequently, develop future treatment and vaccination strategies. keywords: covid-19, sars-cov-2, multisystem inflammatory syndrome in children (mis-c), viral transmission, viral susceptibility, biorepository, biobank, pediatric the global pandemic of covid-19, caused by the highly infectious and transmissible coronavirus, sars-cov-2, has become a leading cause of death in older adults [1] . while adults can develop life-threatening complications such as pneumonia, acute respiratory distress syndrome (ards), and sepsis from sars-cov-2 infection, its impact on children is less clinically apparent and needs to be studied. collecting high-quality biospecimens from infants, children and adolescents in a standardized manner during the covid-19 pandemic is essential for understanding the biologic consequences of sars-cov-2 infection in children. specific questions that must be addressed revolve around the role children play in viral transmission, differences in pediatric viral susceptibility and immune responses, which could guide potential therapies for adults, the impact of maternal sars-cov-2 infection on fetal development, and factors driving the development of severe hyperinflammatory shock and cardiac damage seen in multisystem inflammatory syndrome in children (mis-c). outlined here is a roadmap for establishing a biorepository of specimens obtained from infants, children and adolescents during the covid-19 pandemic. special attention is provided to pediatric-specific considerations in the establishment of a biorepository during the covid-19 pandemic. the goal is to enable pediatric centers world-wide to collect samples in a standardized manner to drive forward our understanding of covid-19. the impact of sars-cov-2 infection on infants and children is not well-defined. children are typically asymptomatic or mildly symptomatic during the acute infection, although some can develop significant complications requiring intensive care. in order to capture the full range of sars-cov-2 infection in the pediatric population, a covid-19 biospecimen collection study was designed and implemented, including patients seen in urgent care clinics and hospital settings, neonates born to sars-cov-2-infected mothers, and asymptomatic children. each study population required specific tailoring of study conduct to effectively and efficiently collect critical samples. cornerstones of the biorepository included open dialogue between research and clinical team members, a sensitivity to procedures required for specimen collection in children, and clear documentation of study participation and sample collection. close communication and collaborations with the adult covid-19 biorepository enable paralleled recruitment efforts and processing procedures and ensured consistency and harmonization across patient cohorts, facilitating high-quality comparisons between patient groups and with adult cohorts. central to the operation, physician-scientists in pediatrics, neonatology, medicine-pediatrics, and obstetrics-gynecology harmonized sample collection protocols, established clinical connections and provided clinical and scientific context to covid-19-related research in the neonatal and pediatric population. to establish a pediatric covid-19 biorepository during the surge of covid-19 cases locally, protocols were rapidly submitted to our institutional biosafety committee (ibc, mgh ibc#2020b000061) and the institutional review board (irb, mgh irb#2020p000955) for approval. during this initial wave of the covid-19 pandemic in late march 2020, covid-19 research proposals were prioritized by the ibc and irb. a biosafety protocol was submitted to the ibc to transition from a biosafety level 2 (bsl2) to an enhanced bsl2+ laboratory environment, allowing collection, processing, and storage of sars-cov-2-infected samples. ibc approval was obtained within 2 weeks. an expedited irb review facilitated irb approval 2 days following submission. figure 1 displays a timeline of study activity relative to the community surge of covid-19 cases in massachusetts. eligible participants in all cohorts were identified by screening outpatient clinic schedules or hospital admission lists, then discussing potential patients with care team members. if appropriate, parents/guardians or patients (if > 18 years of age) were called by phone to introduce the study and, if interested in participation, an informed consent was completed by phone. participants or their parent/guardian also selected which biospecimens they would provide to the biorepository. assent was completed by phone, when possible, with parents/ guardian present for children 7-17 years of age. inperson consent/assent was waived by the irb to avoid close contact between patients and research staff and to abide by the social distancing measures implemented by the state. witness requirements were also waived as a result of the restricted visitor hospital policy due to the pandemic. one copy of the signed consent form, and assent form if appropriate, was emailed to enrolled patients or parents/guardians, and another was uploaded into the electronic medical record flagging the patient as a research participant, clearly documenting research participation for the clinical care teams facilitating sample collection. paper copies were not provided to participants due to the covid-19 restrictions. upon enrollment, participants were assigned a unique study id number using redcap, a secure, centralized online database platform that allows simultaneous recruitment at multiple sites without risking assigning the same number to multiple patients. in order to include pediatric and neonatal patients from a range of clinical presentations for covid-19, we established 4 cohorts of patients from ages 0-25 years, reflecting the ages of patients cared for by the pediatric teams during the surge of covid-19 cases locally: 1) pediatric patients with mild-moderate covid-19, presenting to the mgh covid-19 urgent care clinics, 2) pediatric patients with severe covid-19 or mis-c requiring hospitalization, 3) newborns born to mothers infected with sars-cov-2 at any point during their pregnancy and infants born to non-infected mothers, and 4) asymptomatic children presenting to their wellvisits during the pandemic. each cohort presented unique challenges and required tailored strategies for a successful recruitment. in the pediatric urgent care units, challenges in enrollment included variability in patient volume and frequent rotation of nurses, medical assistants, and physicians. research coordinators had to be flexible to adapt to the frequently changing workflow within the clinic. for enrollment in the hospitalized cohort, the research team remained attentive to new admission lists and promptly completed enrollment protocols in order to obtain specimens prior to the initiation of treatment, as interventions such as intravenous immunoglobulin or steroids would interfere with the natural immune responses to sars-cov-2 infection. specimen collection from the hospitalized cohort was coordinated with clinical laboratory collections to minimize blood draws and collection procedures. for enrollment of newborns, the research coordinators coordinated with maternal arm of the adult covid-19 biorepository to allow simultaneous enrollment of mother and newborns prior to birth facilitating the recruitment process and limiting non-clinical interactions with the mother during the perinatal period. additionally, blood volumes collected for newborns were minimized to < 1 ml obtained by heal stick and coordinated with blood collection for newborn screening. enrolling well-visits was challenging as blood draws are not routinely obtained in all ages and children are often unwilling to undergo voluntary venipuncture. in each cohort, the research staff was mindful of the physical and [2] emotional stress the care teams were enduring while caring for patients with covid-19 and therefore sought to minimize disruptions in clinical care. figure 2 provides a schematic of the recruitment strategy. pediatric patients with mild-moderate covid-19 as most children did not require hospital-level care, significant efforts were made to enroll patients in the outpatient setting. covid-19 screening clinics, called respiratory infection control clinics, were established at massachusetts general hospital. as covid-19 symptoms are non-specific and current diagnostic reporting is time-delayed, all patients presenting to the pediatric covid-19 screening clinics were eligible to participate in the biorepository. during the surge of covid-19 cases locally, young adults up through 25 years of age were seen in the pediatric covid-19 clinics. for patients seen in the respiratory infection control clinics, the crc called eligible participants via telephone after acquiring approval from the lead physicians in the clinics. after acquiring verbal consent over the telephone, the crc alerted the clinical team of patient enrollment and the clinical teams obtained specimens for research. participants who provided informed consent could give nasopharyngeal, oropharyngeal swabs, and/or blood. stool and urine were not collected given time limitations of clinic visits and patient flow patterns established to minimize potential covid-19 exposures to clinical staff. blood was collected into one tube with an edta anticoagulant (edta tube) (bd), one serum separator tube (sst) (bd), and a paxgene rna tube (bd). blood volumes varied, depending on the age and weight of the patient, in accordance with limits established by the irb. the aerosolizing procedure of collecting nasopharyngeal and oropharyngeal swabs into 15 ml falcon tubes, containing 3 ml phosphate buffered saline (pbs) (gibco), was performed by clinical team members wearing n95 mask, face shield, protective outer gown, and disposable gloves. patients with severe covid-19 or multisystem inflammatory syndrome in children requiring hospitalization pediatric patients who were hospitalized with suspicion of sars-cov-2 exposure and/or symptoms concerning for sars-cov-2 infection or mis-c were identified by members of the research team, who subsequently requested approval from the clinical team to approach the patient. a member of the research team contacted the patient and family via phone to obtain informed consent, as described above, and coordinated with both the clinical and tcrc teams for specimen collection. sample collection was pre-planned with the clinical teams via emails and occurred every 2-3 days for this cohort of patients. hospitalized patients could opt to provide urine, stool, sputum, or if intubated, tracheal aspirates, in addition to blood, nasopharyngeal and oropharyngeal swabs. phlebotomy was aligned with clinical blood draws, when feasible, although participants had an option to undergo a separate venipuncture for research purposes. blood was collected into an edta tube, an sst tube, and a paxgene rna tube. repeat samples were collected on alternating days, as feasible. based on daily coordination between the research and clinical teams, discarded blood from clinical labs were also obtained from hospitalized patients. pregnant women with confirmed sars-cov-2 infection followed in the mgh obstetrics practice, presenting to the labor and delivery (l&d) unit, or hospitalized for sars-cov-2 illness, were approached to enroll their infant in the pediatric covid-19 biorepository following birth in collaboration with the maternal arm of the adult covid-19 biorepository. when universal screening for sars-cov-2 infection was initiated on all pregnant women admitted to l&d, asymptomatic sars-cov-2 positive patients were identified and offered enrollment. women who tested negative for sars-cov-2 were also approached as a control group. the pregnant mothers were simultaneously offered enrollment in the companion obstetric covid-19 biorepository, which included collection of placental biopsies, umbilical cord blood, and other maternal samples. the clinical team assessed the patient's interest in the biorepository, then a member of the research staff contacted the patient via telephone to obtain informed consent. parents could opt to have newborn blood, nasopharyngeal and oropharyngeal swabs, urine, stool, and (if intubated) tracheal aspirates collected. all samples were collected in the clinical setting by the clinical team members to accommodate covid-19 infection control guidelines, minimizing the risk of sars-cov-2 transmission and limit personal protective equipment (ppe) use. blood was collected via heel stick between 24 and 36 h of life, simultaneously with the heel stick for clinical newborn screening, into two edta microtainer tubes (bd). research nasopharyngeal and/or oropharyngeal swabs were obtained after 24 h of life, batched at the time of the nasopharyngeal swab for sars-cov-2 testing, if performed clinically. stool and urine were collected on day of life 0 and 2. stool was collected directly from the diaper. urine was collected by placing cotton balls in the diaper, then transferring the urine-soaked cotton balls into a specimen cup for transport. if the recruited newborn was intubated for clinical indications, tracheal aspirates were collected at the time of clinical suctioning. asymptomatic children presenting to their well-visit during the covid-19 pandemic children presenting for their 2-, 3-, or 4-year annual well-child visit with their pediatrician for planned phlebotomy were eligible to participate in this cohort. eligible patients were identified by study staff and clinicians. if appropriate, researchers contacted the parents via telephone prior to their visit to explain the research and obtain informed consent. blood and saliva were collected during their clinical phlebotomy. saliva collection is not considered an aerosolizing procedure; thus, these specimens could be collected in clinic without the need for n95 mask use. the specimens were immediately transported to the laboratory for processing. redcap databases were used to record all study data, including: 1) an enrollment log serving as the decoding log -study id numbers were assigned consecutively across all four patient groups; 2) a laboratory processing database with pertinent processing and freezer storage location information; 3) a chart review database, with demographic and clinical data, including covid-19 exposures, sars-cov-2 polymerase chain reaction (pcr) results, symptoms, and outcomes; 4) a questionresponse database about covid-19 exposures and risk factors, specifically for the well-visit cohort. in accordance with specimen transport guidelines, specimens were sealed in a leak-proof container labeled with subject's study id, then placed in a tight-sealed, biohazard-labeled, secondary container with a rigid outer container and lockable lid (e.g. igloo cooler) for transport to the laboratory. the entire research team was properly trained on bsl2+ procedures, as required for handling sars-cov-2 specimens. a coordinated effort by research personnel enabled successful and efficient troubleshooting, and processing of high influx of samples to the lab during the acute rise of covid-19 cases in the months of april-june 2020 (fig. 1) . scheduled shifts were implemented throughout the week to ensure the safety of all research staff and sample processing efficiency. three laboratory roles were created: 1) blood processing technician with extensive technical skill required for blood cell isolation, 2) biospecimen processing technician fully trained in bl2+ enhancement protocols, and 3) specimen labeling, quality control, and sample storage staff. these roles optimized processing workflow, safety precautions, and resources (including staff resource). paramount to the success of this biorepository included open communication via emails and the use of mobile group messaging outlets, frequent quality checks between staff regarding data and sample collection and processing, accessible leadership, and coordination with patients' clinical care teams. blood samples were processed following bsl2 safety guidelines, with a lab coat, nitrile/latex gloves, and a face shield or safety goggles. all other samples, including nasopharyngeal and oropharyngeal swabs, sputum, saliva, tracheal aspirates, stool, and urine were processed following bsl2+ safety guidelines. bsl2+ safety precautions require all samples to be processed in a certified biosafety cabinet (bsc), class ii a2, with intake airflow. well-trained laboratory personnel handling infectious specimens were required to wear closed-front water impermeable gowns, double nitrile/latex gloves, sleeve covers, and a face shield. outer gloves were removed when moving away from the bsc and replaced with a new glove when returning to work in the bsc. blood samples collected in tubes with an edta anticoagulant were stored at room temperature until processed, within 24 h of collection. tubes were spun at 1000 g for 10 min with brake activated. plasma was then collected, aliquoted, stored at − 80°c, and logged in the redcap database (fig. 3b) . immediately following the removal of plasma, samples with greater than 2 ml initial volume were processed for pbmc isolation using a ficoll density gradient [3] . briefly, blood was transferred into a 50 ml conical tube, then diluted 1:1 with hanks' balanced salt solution without calcium or magnesium (hbss minus) (gibco). this diluted blood was then gently layered on top of ficoll-paque plus (ge healthcare) at 2:1 ratio (2 volumes of blood diluted with hbss minus to 1 volume ficoll). careful attention was made to avoid any mixing of blood with the ficoll layer. the conical tube was then centrifuged at 1000 g for 30 min at room temperature with brake inactivated to allow adequate layering of cellular components. the cloudy ring below the plasma and above the ficoll (i.e. the pbmc layer) was collected and transferred to a new 15 ml conical tube, with hbss minus added to bring the volume to 15 ml (fig. 3a) . this tube was then centrifuged at 330 g for 10 min, with high brake activated. the supernatant was removed, the pbmc pellet was again washed with hbss minus, and then resuspended in 10 ml hbss minus for counting. cell count was obtained by diluting 10 μl of sample with 90 μl of trypan blue, mixed, and sampled on a hemocytometer. cells were then frozen in freshly-prepared freezing medium (rpmi 1640 medium with 1% penicillin-streptomycin, l-glutamine, 1% sodium pyruvate, 1% non-essential amino-acids, and 20% fetal bovine serum (fbs) (sigma)) with 10% dmso (sigma) for a goal concentration range of 5-10 million cells/vial, placed in a chilled mr. frosty filled with isopropanol, then immediately placed at − 80°c. final concentration (5-10 million cells per 1 ml of freezing medium) and number of aliquot vials were logged. the following day, pbmc cryovials were moved to a liquid nitrogen freezer for long term storage, and location was recorded in specimen log. pbmcs were isolated within 24 h of phlebotomy, although higher cell counts were obtained if isolated within 3-4 h of collection. if less than 5 ml blood was collected, a 15 ml conical tube, rather than a 50 ml conical tube could be used for ficoll layering. fresh freezing media were made throughout the day for each sample batch. neutrophils were extracted from the red blood cell layer that remained following the collection of pbmcs (fig. 3a) . neutrophils were isolated using easysep direct human neutrophil isolation kit (stemcell technologies). the remaining blood layer was incubated with easysep direct rapidspheres and easysep direct human neutrophil isolation cocktail, fig. 3 overview of laboratory blood processing procedures following bsl2 containment guidelines depicting steps for a) collection of plasma, isolation of pbmc and pmn, from blood collected into an edta tube and b) collection of serum from an sst blood tube (created with biorender.com) then diluted in easysep buffer. neutrophils were isolated by successive negative magnet selection using easysep magnets, then counted using a hemocytometer and aliquoted into eppendorf tubes for rna extraction (1 × 10 5 cells/tube) or dna analysis (5 × 10 6 cells/tube). neutrophils designated for rna extraction were resuspended in 100 μl of rna lysis buffer (tcl) (qiagen) with 1% β-mercaptoethanol (sigma), immediately stored at − 80°c and logged. neutrophils planned for dna analysis were pelleted then directly stored at − 80°c and logged. for rna extraction steps, a cleaning agent, such as rna-sezap should be used to remove rnase from the working surface. rna lysis buffer should be newly made for each sample using a 10:1 tcl to β-mercaptoethanol ratio. serum samples were collected from blood drawn into serum separator tubes without any anticoagulant (bd). blood was kept at room temperature, standing upright for 30-60 min, then spun at 1200 g for 10 min with brake activated. serum was then collected, aliquoted, stored, and logged (fig. 3b) . swab samples were delivered in phosphate buffered saline (pbs) [4] . samples were directly aliquoted into 1 ml aliquots, then immediately stored at − 80°c and logged (fig. 4a) . samples collected into a collection cup were mixed well at 1:1 ratio with 500 mm dl-dithiothreitol (dtt) (sigma)/pbs solution according to cdc recommendations. diluted samples were then divided into 1 ml aliquots, volume permitting, immediately stored at − 80°c and logged (fig. 4b) . aspirates collected into a sterile collection cup were divided into 1 ml aliquots (1 ml/vial), immediately stored at − 80°c and logged (fig. 4c) . stool samples collected from a diaper or specimen cup were divided using a micro spatula, volume permitting, into cryovials with 1 ml rnalater (invitrogen), empty cryovials without any additive/reagent, up to the 1.5 ml tube mark, and cryovials with 1 ml buffered glycerol saline (fisher). stool samples were fully submerged in rnalater or glycerol solution prior to immediate storage at − 80°c. samples were logged onto database (fig. 4d) . urine samples collected with cotton balls placed inside baby diapers were transferred using forceps, to a 10 ml syringe to dispense at most 1 ml of fluid into cryovials and immediately stored at − 80°c. samples collected into a tube or a sterile collection cup were aliquoted into cryovials (at 1 ml at most/vial) and immediately stored at − 80°c (fig. 4e) . supplies required for specimen collection and processing are listed in supplemental table 1 . sample labels, logging, storage, and quality control were performed by assigned lab #3 personnel. the pediatric covid-19 biorepository enrolled 327 pediatric and neonatal patients from a range of clinical presentations, including 178 patients from the urgent care/respiratory infection control clinic, 48 hospitalized children, 85 newborns born to mothers with or without sars-cov-2 infection, and 16 asymptomatic children presenting for their well-visits. the average age was 11 (± 8) years for enrolled children and adolescents and 1.3 (±1.3) days for newborns. equal gender distribution was seen, except more males were enrolled in the hospitalized cohort (60%, n = 29). sixty-four participants were positive for sars-cov-2 by clinical testing, most of whom were seen in the respiratory infection control clinic, while 21 patients were diagnosed with mis-c, all of whom were hospitalized. the one patient presenting to the respiratory infection control clinic with mis-c was ultimately hospitalized. table 1 characterizes total enrollment number, age, gender, sars-cov-2 infection status, and mis-c diagnosis within each enrollment site. a total of 972 biospecimens were collected. these biospecimens included 295 blood samples, 181 nasopharyngeal swabs, 145 nasopharyngeal swabs, 172 stool samples, 154 urine samples, 4 tracheal aspirate samples, and 21 sputum/saliva samples. hospitalized patients and newborns had the option of providing subsequent sampling. table 2 depicts the sample collection from each enrollment site. the goal of the biorepository is to provide high quality biospecimens for studies understanding how infants and children are impacted by and contribute to covid-19 pandemic. establishing a standardized biorepository collection protocol facilitates comparison of samples across institutions and with adult biorepositories. key neonatal and childhood factors of interest that will be studied using this biorepository focus on: 1) informing pediatric contribution to viral transmission, 2) teasing apart the dichotomy between pediatric and adult immune responses to covid-19, 3) ascertaining the impact of maternal sars-cov-2 infection on child fetal development, and 4) elucidating factors driving the mis-c. in this study, nasopharyngeal and oropharyngeal swabs were collected from pediatric patients presenting with symptoms concerning for sars-cov-2 infection in both the outpatient and hospitalized setting, from newborns born to mothers infected with sars-cov-2, and from healthy controls. additionally, saliva was collected from young children presenting for their annual well-visit. blood, tracheal aspirates, stool, and urine were also collected from the hospitalized patients and newborns for assessment of viral load. questions relating to the role of viral carriage in the pediatric population can be addressed using these samples. case reports and recent research studies suggests that asymptomatic children carry high viral loads despite lack of symptoms [5] [6] [7] . in adults, severe infection is not necessarily associated with a significant increase in viral loads by nasopharyngeal swab [8] , and asymptomatic individuals appear to have equal viral loads as symptomatic individuals [9] . the potential correlations between viral load, symptoms, and exposures have yet to be clarified in the pediatric population. age-stratification within adults show no differences in viral load across age-groups, although younger patients were less likely to develop severe disease [8] , thus a similar comparison among children would be informative. additional questions remain as to whether there are risk factors affecting viral load density in children, including household contacts or other environmental factors. viral studies are also needed to determine accuracy of viral testing techniques within the pediatric population. including infants born to covid-19 infected mothers will allow assessment of viral exposure in the airway, and through the meconium, giving important insight into neonatal exposure to sars-cov-2 infection. understanding how children are infected with sars-cov-2 will provide critical insight into how viral loads may impact disease severity in children, and how children may contribute to viral transmissibility driving this pandemic. these data will be critical to making decisions about risk factors for re-opening of schools and childcare as the pandemic progresses. covid-19 results in a major apparent dichotomy of immune response between children and adults [10] . children often develop mild infections whereas adults more commonly develop severe disease associated with high levels of mortality [11] . neonates appear to be unaffected, even when born to covid-19 positive mothers [12] . it has been postulated that children are less impacted by viral infection because they have fewer angiotensin-converting enzyme 2 (ace2) viral binding sites [7, 13] , although the research thus far remains conflicted. in this study, rna obtained from nasopharyngeal and oropharyngeal swabs, and/or saliva collected from neonates, children, and young adults, can be used to characterize ace2 expression, and potentially shed light on the availability of viral binding sites across the age span. further, prior research has shown immunosenescence in aged individuals, which affects t cell and b cell function, and cytokine production by innate immune cells [14] . it is yet to be evaluated as to whether this plays a central role in the age differences in the morbidity and mortality from covid-19. additionally, mis-c is shown to be driven by a cytokine storm and macrophage activation [15] . peripheral blood monocytes, plasma, serum, and neutrophil rna collected as part of this biorepository can be used to answer these questions. understanding the disease abnormalities may provide key insight into therapeutic targets. this study collects plasma, serum, pbmcs, and neutrophil rna from sars-cov-2 infected and uninfected children with a range of symptoms for comparison. neonatal development intimately depends on maternal health. prior infections and disease states causing maternal inflammatory activation and cytokine storm have resulted in increased risk of autism spectrum disorder, schizophrenia, cerebral palsy, cognitive delay, depression, and bipolar disorder in exposed children [16] [17] [18] . the effect of the sars-cov-2 hyperinflammatory milieu on the developing fetus is yet to be seen. this biorepository, partnered with the obstetric covid-19 biorepository will obtain placental tissue, cord blood, maternal and neonatal biospecimens to address these critical questions. following a mild or symptomatic infection of covid-19, children can develop a severe, post-infectious inflammatory response syndrome, termed multisystem inflammatory syndrome in children (mis-c), which is characterized by hyperinflammatory shock [19] , "kawasaki-like" cardiac damage, and possible death [20] . risk all enrolled participants provided clinical and demographic data. enrolled subjects had the option of selecting which biospecimens they would like to provide. stool and urine were not collected from participants enrolled in the urgent care and well-visit cohorts for logistic reasons, unless these individuals were later hospitalized for covid-19-related illness. enrolled subjects could also consent to provide specimens, then later decline any or all specimen collection. repeat biospecimen collection could occur if participants re-presented to care, or if hospitalized for multiple consecutive days factors for developing mis-c and biomarkers predicting severe complications need to be identified. these specimens collected through this pediatric covid-19 biorepository will be used to characterize the immune responses driving mis-c in hopes of mitigating this lifethreatening complication. although children were initially felt to be spared from covid-19, it has become clear that much needs to be learned as to how children and newborns are impacted by the pandemic. research is needed to address viral transmission by children, differences in pediatric viral susceptibility and immune responses, the impact of maternal sars-cov-2 infection on fetal development, and factors driving the mis-c. this pediatric covid-19 biorepository will serve as an important resource providing critical insight into disease pathogenesis, covid-19susceptibility, and future treatment and vaccination strategies. supplementary information accompanies this paper at https://doi.org/10. 1186/s12874-020-01110-y. additional file 1 supplemental table 1 . list of supplies required for specimen collection, processing, and storage in the pediatric covid-19 biorepository commonwealth of massachusetts covid-19 reporting isolation of lymphocytes, granulocytes and macrophages evaluation of swabs, transport media, and specimen transport conditions for optimal detection of viruses by pcr a well infant with coronavirus disease 2019 (covid-19) with high viral load viral rna load in mildly symptomatic and asymptomatic children with covid-19 pediatric sars-cov-2: clinical presentation, infectivity, and immune responses association of viral load with serum biomakers among covid-19 cases sars-cov-2 viral load in upper respiratory specimens of infected patients insight into the pediatric and adult dichotomy of covid-19: age-related differences in the immune response to sars-cov-2 infection coronavirus disease 2019 in children -united states vertical transmission risk of sars-cov-2 infection in the third trimester: a systematic scoping review nasal gene expression of angiotensinconverting enzyme 2 in children and adults immunosenescence: emerging challenges for an ageing population multisystem inflammatory syndrome related to covid-19 in previously healthy children and adolescents long-term risk of neuropsychiatric disease after exposure to infection in utero the fetal origins of mental illness schizophrenia and influenza at the centenary of the 1918-1919 spanish influenza pandemic: mechanisms of psychosis risk hyperinflammatory shock in children during covid-19 pandemic multisystem inflammatory syndrome in u.s. children and adolescents publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we are incredibly grateful for the selfless contributions by the entire pediatric and obstetrics covid19 received: 13 july 2020 accepted: 30 august 2020 we also acknowledge funding from the national heart lung and blood institute (5k08hl143183 to ly), the cystic fibrosis foundation (yonker18q0 to ly), and the eunice kennedy shriver national institute of child health and human development (1r01hd100022-01 to age). the funder/sponsor did not participate in the work.availability of data and materials a variety of pediatric samples collected during the covid-19 pandemic may become available to other researchers upon reasonable request to the correspondent author and compliance with the partners innovations office. the study received ethics approval from the partners/massachusetts general hospital institutional review board (irb#2020p000955) and the partners/ massachusetts general hospital institutional biosafety committee (ibc#2020b000061). verbal consent to participate was obtained from the participants or from parents/guardians (for children under 18 years of age). verbal assent was obtained, when possible, from children ages 7-18 years of age. all participants signed an irb-approved informed consent prior to participating. not applicable. the authors declare no competing interest. key: cord-261718-zqoggwnk authors: pietschmann, jan; vöpel, nadja; spiegel, holger; krause, hans-joachim; schröper, florian title: brief communication: magnetic immuno-detection of sars-cov-2 specific antibodies date: 2020-06-03 journal: biorxiv doi: 10.1101/2020.06.02.131102 sha: doc_id: 261718 cord_uid: zqoggwnk sars-cov-2 causes ongoing infections worldwide, and identifying people with immunity is becoming increasingly important. available point-of-care diagnostic systems as lateral flow assays have high potential for fast and easy on-site antibody testing but are lacking specificity, sensitivity or possibility for quantitative measurements. here, a new point-of-care approach for sars-cov-2 specific antibody detection in human serum based on magnetic immuno-detection is described and compared to standard elisa. for magnetic immuno-detection, immunofiltration columns were coated with a sars-cov-2 spike protein peptide. sars-cov-2 peptide reactive antibodies, spiked at different concentrations into pbs and human serum, were rinsed through immunofiltration columns. specific antibodies were retained within the ifc and labelled with an isotype specific biotinylated antibody. streptavidin-functionalized magnetic nanoparticles were applied to label the secondary antibodies. enriched magnetic nanoparticles were then detected by means of frequency magnetic mixing detection technology, using a portable magnetic read-out device. measuring signals corresponded to the amount of sars-cov-2 specific antibodies in the sample. our preliminary magnetic immuno-detection setup resulted in a higher sensitivity and broader detection range and was four times faster than elisa. further optimizations could reduce assay times to that of a typical lateral flow assay, enabling a fast and easy approach, well suited for point-of-care measurements without expensive lab equipment. the new severe acute respiratory syndrome coronavirus-2 (sars-cov-2), is causing ongoing 31 worldwide infections, leading to an unprecedented pandemic. according to world health 32 organization (who), it is estimated that up to 82% of people with coronavirus disease 19 (covid-33 19) are not aware that they are/were infected due to no or very mild symptoms [1] . 34 symptoms can be noted comparable to common cold cough, rhinitis or fever up to harsh symptoms, at a later point, serological assays would also be required to prove and monitor effectivity of 43 vaccination and longevity of the obtained immunity. fast, cheap and easily applicable on-site testing 44 solutions will thus become increasingly important, but currently only few rapid test systems are 45 available. lateral-flow-detection (lfd) approaches are easy to handle and results are gained after 10-46 15 min. however they are not quantitative, and their reliability, specificity and sensitivity is much 47 worse than that of lab-based assay formats based on enzyme-linked immunosorbent assay (elisa). in particular, specificity is a major challenge at currently available serological antibody tests. this 49 3 depends to a large extent on the antigen used in the test assays. enveloped positive-stranded rna 50 sars-cov-2 coronaviruses consist of five structural proteins, the spike glycoprotein (s), envelope 51 protein (e), membrane protein (m), the nucleocapsid protein (n) and a hemagglutinin esterase (he). the s-protein, a complex folded glycoprotein comprising two regions, s1 and s2, exhibits the highest 53 immunogenicity, has the most important role in host interaction, especially cell entry, and is also the 54 main target for neutralizing antibodies [5] . the proteins m, e and he are only weakly immunogenic 55 and less suitable as targets for antibody diagnosis. although the n protein is immunodominant, it is 56 not suitable for the specific analysis of the immune response against sars-cov-2 viruses due to its 57 high cross-reactivity with antibodies targeting related coronavirius strains [6, 7] . the company 58 euroimmun ag, lübeck, germany offers two elisa kits using a genetically modified n-protein 59 variant, which enables a more specific detection of antibodies already ten days after infection. however, for highly specific detection of immune response against sars-cov-2, typically the s1 61 subunit of s-protein should be used. currently, only few vendors offer these specific elisa formats 62 using the s1 subunit of s-protein for specifically detecting sars-cov-2 antibodies [8, 9] . nevertheless, specific, sensitive and quantitative rapid tests applicable for a decentralized point-of-64 care (poc) analysis are currently not available. magnetic immuno-detection (mind) could be a powerful tool for poc assay performance. mind as reference method to our poc mind approach, a typical laboratory-based elisa was performed 173 (fig 1) . after coating of elisa microtiter plate with sars-cov-2 spike protein peptide and blocking 174 8 with bsa, sars-cov-2 spike protein peptide specific antibody was diluted in the range from 175 1.22 ng·ml -1 to 5000 ng·ml -1 in pbs-buffer or serum and applied into wells. after addition of 176 biotinylated gar and subsequent labelling with streptavidin-ap, the elisa plate was read out at 177 405 nm and obtained measuring values were used to generate calibration curves for sars-cov-2 178 specific antibody concentrations in pbs (fig 1, black curve) and in human serum samples (fig 1, red 179 curve). blank values determined in pbs and serum were 0.085 au ± 0.005 au and same calibration measurements employing dilutions of sars-cov-2 specific antibody were 211 done with our poc mind-based setup (fig 2 and 3) . comparable to laboratory-based elisa, the same 212 dilutions of sars-cov-2 spike protein peptide specific antibody in pbs-buffer (fig 3, black in this proof-of-concept experiment, a commercially available sars-cov-2 spike protein 267 peptide with corresponding antibody was used. if using this peptide for testing of patient samples, funding: the author received no specific funding for this work. acknowledgments: the authors would like to thank max schubert for his helpful advices and support given in 308 discussions. competing interests: the authors declare no competing interests. 310 311 19; a narrative review clinical characteristics of 3,062 covid-19 patients: a meta-315 analysis sars-cov-2 and covid-19 in older 319 adults: what we may expect regarding pathogenesis, immune responses, and outcomes structure, function, and antigenicity of 322 the sars-cov-2 spike glycoprotein evaluation of 324 serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other 325 coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses reactivity between common human coronaviruses and sars-cov-2 using coronavirus antigen microarray antibody responses to sars-cov-2 in patients of novel 331 coronavirus disease 2019 comparison of four new 333 commercial serologic assays for determination of sars-cov-2 igg magnetic particle detection by frequency mixing for immunoassay applications immunofiltration columns for frequency mixing-based multiplex magnetic immunodetection. sensors (basel) sensitive and rapid 341 detection of cholera toxin subunit b using magnetic frequency mixing detection crp determination 343 based on a novel magnetic biosensor francisella 345 tularensis detection using magnetic labels and a magnetic biosensor based on frequency mixing magnetic biosensor for the detection 348 of yersinia pestis simple and portable magnetic 350 immunoassay for rapid detection and sensitive quantification of plant viruses sensitive aflatoxin b1 detection using 353 nanoparticle-based competitive magnetic immunodetection. toxins (basel) antibody and cytokine serum levels in 355 patients subjected to anti-rabies prophylaxis with serum-vaccination multiplex detection of different magnetic 358 beads using frequency scanning in magnetic frequency mixing technique key: cord-029813-o2uzcuai authors: rusconi, stefano; hayden, frederick g title: covid-19: studying the global pandemic – foreword date: 2020-07-27 journal: nan doi: 10.2217/fvl-2020-0211 sha: doc_id: 29813 cord_uid: o2uzcuai nan this special issue of future virology contains nine articles on diverse aspects of the covid-19 pandemic and its causative agent, sars-cov-2. the topics range from basic virology on coronavirus evolution and replication to identification of repurposed therapeutics for clinical testing to public health issues including the conundrums of asymptomatic viral transmission and risks to homeless populations. while several of the reports contain original data, others are reviews, special reports or commentaries. given the response of the global research community to the pandemic, we have received unprecedented submissions in this area and have therefore compiled these into one issue to provide a useful resource to those working in the field. while readers should be aware that all but one of the articles have undergone external peer review, as per the journal's usual policies, we should point out that the conclusions and opinions of some of the authors are speculative. the commentary by parvez [1] briefly reviews the detection of sars-cov-2 rna in fecal samples, including its persistence, and the finding of gastrointestinal complaints in a minority of hospitalized patients. others have also highlighted that some covid-19 patients present primarily or exclusively with gastrointestinal manifestations. given the presence of ace-2 receptors in intestinal epithelial cells and the documentation of sars-cov-1 infection involving the gastrointestinal tract, it is not surprising that the sars-cov-2 might also cause such infections and associated illness. of course, fecal aerosols were implicated in a very large sars outbreaks in hong kong in 2003. however, infectious sars-cov-2 has been only rarely isolated from fecal samples to date, and the possible risks of fecal-oral or fecal-aerosol transmission remain to be clarified. interestingly, parvez points out the greater expression of ace-2 receptor in cholangiocytes than hepatocytes in covid-19 patients and speculates about how this and other mechanisms, particularly immune-mediated ones, might be contributing to the substantial frequency of hepatic abnormalities seen in hospitalized patients. of course, other factors including hypoxemia, ischemia and medication side effects are also likely to be contributory. the latter concern has led to premature discontinuation of intravenous remdesivir therapy in hospitalized covid-19 patients. the immunopathologic effects of pro-inflammatory cytokines like il-6 are the subject of ongoing randomized, controlled trials in severe covid-19. the thoughtful commentary article by conway et al. [2] addresses the extraordinary challenges of mitigating covid-19 risks in homeless persons in central vancouver (bc, ca, usa). like many other inner city populations, the one in vancouver has high frequencies of psychiatric disorders, opiate drug dependence and overdose-related deaths. sadly, the latter increased temporally with loss of many supportive services in efforts at social distancing, including loss of daily directly observed drug administration. the authors describe the difficult choices faced by city administrators and private entities in balancing infection risks to staff and maintenance of essential services. they then propose specific strategies to improve current circumstances and also emphasize the long-term need for stable housing. these recommendations are highly relevant for essentially all urban centers that harbor such populations of high-risk persons and reflect more broadly the challenges in mitigating infection risks in other disenfranchised populations, like those in refugee camps. of course, once a safe and effective sars-cov-2 vaccine becomes available, immunization of such high-risk populations will be a priority, as has been shown by the deployment of hepatitis a virus vaccine to mitigate outbreaks in homeless populations in north america. yang et al. [3] report the case of a 57-year-old patient who had a positive rt-pcr for sars-cov-2 after three negative sputum tests in the context of mild illness. the patient was caring for her father from 1 to 6 february 2020, after her mother died from cardiac complications. her father was diagnosed with covid-19 on 5 february and she was admitted to the hospital on 7 february. the case is of interest because it points out at least two issues related to diagnostic testing. first, the sars-cov-2 rt-pcr is an imperfect test that depends on the type, quality and handling of the sample, duration and severity of illness and assay performance. in general, lower respiratory tract samples have higher yields than upper respiratory tract ones [4] . second, the low viral burden seen in some patients in the upper respiratory tract can make virologic diagnoses difficult, so that repeated sampling of multiple sites, testing of lower respiratory tract secretions, and, later in the course, detection of igm/igg antibodies may be necessary. another point raised by the authors is the importance of the epidemiological link, in other words, the likely exposure within her family, in leading to prompt intervention, along with clinical features and radiological findings. the patient was admitted quickly and started on two putative antivirals on the same day. whatever the chosen strategy, timely antiviral and supportive therapy is a major element for improving outcomes in sars-cov-2 illness. in parallel, some biomarkers, as illustrated in this patient, have demonstrated their value in evaluating the clinical course of this infection: lymphocyte count, cd4 and cd8 population subsets, ldh and crp. monitoring these laboratory measures, together with plasma d-dimer and il-6 levels that have implications for treatment and prognosis, can prove to be of help in clinical management. li et al. reported, both in a short communication [5] and in a special report [6] , some concern on the application of genomic evolutionary theories to sars-cov-2. the new coronavirus sars-cov-2, isolated from humans for the first time in december 2019, appears to have originated from bats. this was based on genetic analyses and comparisons with the sequences of other coronaviruses from different animal species. specifically, two bat coronaviruses share 88% of the genetic sequence with that of sars-cov-2. in comparison, sars-cov-2 shares approximately 79% genetic sequence homology with sars-cov and 50% with mers-cov. as with sars-cov and mers-cov, it is assumed that the transmission did not occur directly from bats to humans, but that there could be another animal yet to be identified that acted as an intermediate host in transmission of the virus to humans. from a molecular point of view, the fact that coronaviruses can infect different animal species and humans can depend on at least two factors: • mutations that lead to substitutions in the virus surface protein (the spikes or 'spikes' of the virus) that favors the attachment of the virus to the cell receptors of the new host and its entry into the cell to replicate. • possibility of entry into the cell independent of the link between the viral protein and receptor as an alternative route for transmission between the different animal species and humans. the point raised by li et al. is that the application of evolutionary theories to ssrna viruses -such as sars-cov-2 -could overestimate the divergence between a virus isolated in animal species and humans. sars-cov-2 is a positive-strand rna virus that reproduces its genomic rna through the action of an rna dependent rna polymerase and does not have an intermediate dna genome like hiv. moreover, rna viruses adapt to the host expression system, in other words, their codons adapt to the host system they are infecting. in detail, the authors point out that sars-cov-2 and ratg13, a bat coronavirus, were previously reported to have 17% synonymous changes at neutral sites [7] . through the alignment of coding sequences of both viruses, li et al. report that the rna modification system in host cells might be the cause of 87% of the synonymous substitutions between sars-cov-2 and ratg13 and conclude that an overestimation of virus divergence is a risk when comparing two rna viruses [5] . li et al. used several molecular techniques to investigate protein structures of sars-cov-2 [8] . the virus itself possesses five keys proteins: open-reading frame lab (orflab) in the nonstructural region and other four structural proteins, namely s (spike), m (membrane), e (envelope) and n (nucleoprotein) proteins. the s protein is part of the shell component and, as indicated above, a pivotal component for cell attachment. this protein is divided into two subunits, s1 and s2: s1 contains the receptor-binding domain (rbd) that binds to the angiotensin-converting enzyme 2 (ace2) cellular receptor and s2 mediates virus-cell membrane fusion. the authors obtained the complete sequences of sars-cov-2 and six other bat coronaviruses. they applied multiple techniques including the analysis of structure and, most interestingly, the back mutation of some codon substitutions within the rbd. all sarscov-2 five key proteins have a large homology with sars-cov from bats, although the lowest homology was with the s protein, which showed the highest amino acid homology with bat sars ratg13 reaching 97.71%. the authors concluded that it is very likely that sars-cov-2 developed from bat sars cov given this high similarity. moreover, the authors backmutated the changed three amino acid residues (glu 470 , gln 484 and asn 487 ) within the rbd structure and demonstrated that the mutated rbd structure has a stronger effect on ace2 binding. this report adds to the considerable data on the crystal structures and biophysical characterization of the ace2 interaction with the sars-cov-2 rbd that have been reported previously, including receptor conformational dynamics and other analyses highlighting similarities with the viruses from bats. hu et al. [9] have used a molecular docking software program to screen 109 plant-derived chemical agents for their ability to dock with the sars-cov-2 main protease (mpro). as they point out in this research article, the recent characterization of the crystal structure of mpro and its high degree of conservation across coronaviruses makes it an attractive target for drug discovery. they determined that the flavonoid rutin fits well into the mpro substrate-binding pocket and, in addition, interacts with pockets in the toll-like receptors (tlrs), tlr2, tlr6 and tlr7. the finding that remdesivir, which targets the viral rna-dependent rna polymerases of coronaviruses and several other rna virus families, also proved to be positive in the mpro docking model raises some questions about its specificity. the nature of the interactions with the tlrs, whether stimulatory or inhibitory of proinflammatory responses and antiviral effects, are unclear from the report. the authors went on to assess host target genes of rutin by another modeling system and found multiple hits involving cellular functions, some of which are involved with inflammation. the authors speculate that rutin might have the potential to provide both antiviral and antiinflammatory effects. while it is clear that this phytochemical has multiple pharmacological activities, as reviewed previously [10] , this in silico report does not provide biologic data on rutin's possible effects in sars-cov-2 infection. tan et al. discuss the critical public health issue of the extent to which asymptomatically infected persons are transmitting sars-cov-2 to others and contrast the currently available data with the epidemiologic findings in sars and mers [11] . detection of relatively high sars-cov-2 rna loads in upper respiratory tract samples has been reported in both presymptomatic (late incubation period) and truly asymptomatic infected persons. the proportion of asymptomatic infections has ranged widely from approximately 5-60% across various reports, but some studies have not had sufficient follow-up to document the development of later onset of symptoms. the factors that might impact on likelihood of asymptomatic infection (e.g., upper vs lower respiratory tract virus exposure, infectious virus inoculum size, pre-existing, cross-reactive immunity) have not been clarified. of note, a large portion of pandemic and seasonal influenza infections are asymptomatic or very mild and detected retrospectively in serologic studies. the extent to which such persons contribute to transmission remains uncertain. however, studies of covid-19 clusters have implicated the importance of pre-or asymptomatic persons in transmission, so that control measures clearly need to expand testing and isolation beyond those with symptoms. a recent modeling study estimated that 44% (95% ci: 25-69%) of secondary cases were infected during the presymptomatic stage of index cases [12] . such findings, again, highlight the importance of wide-scale, rapid detection of sars-cov-2 infections for effective application of traditional nonpharmaceutical control measures (e.g., masking, social distancing, quarantine of those possibly exposed). following a broad-ranging introduction, nemunaitis et al. focus on the strategy of repurposing a personalized cancer treatment with the proprietary name vigil r currently in phase iii trials to treat sars-cov-2 infections [13] . the cancer therapy involves harvesting tumor cells from a patient, transfecting with a dual shrna construct that both inhibit furin production and stimulate gm-csf expression to enhance immunogenicity of the tumor cells, and reintroducing them into the patient. the authors point out that the dual actions of their plasmid could result in both antiviral and anti-inflammatory effects in covid-19. however, the cancer treatment strategy is a very different proposition from in vivo transfection of lung epithelial cells by their proposed inhalation delivery of the plasmid. effective and safe aerosol delivery of small-molecular weight antivirals has proven very challenging in viral pneumonias to date, and the fragile nature of dna plasmids adds complexity to the process. depending on timing, there is also the concern that nonspecific gm-csf stimulation in lung might exacerbate ards. indeed, there is great interest in the use of immunomodulators in the subgroup of covid-19 patients who develop a hyperinflammatory state associated with lung injury and often other organ failure. a large number of clinical studies targeting excessively proinflammatory immune responses are currently in progress in covid-19. one agent of particular interest is baricitinib, an oral inhibitor of jak1 and jak2 and nak family members that is approved for the treatment of rheumatoid arthritis in adults. it has been proposed as a therapeutic option for covid-19 because of its anti-inflammatory and anticoronaviral properties in preclinical and limited clinical studies [14] . future science group 10 .2217/fvl-2020-0211 an obvious strategy is to combine antivirals with immunomodulators in severe covid-19, and a randomized, placebo-controlled trial of baricitinib's efficacy in combination with remdesivir is ongoing at present. in summary, this special issue addresses many different aspects of the ongoing pandemic. given the numerous unanswered questions regarding sars-cov-2 and covid-19, we trust that this collection of articles will stimulate both discussion and further investigation. we thank the authors, reviewers and the staff at future virology for making this special issue possible. financial & competing interests disclosure s rusconi received research grants to his institution from viiv healthcare, gilead sciences and janssen, outside the submitted work; he was also a paid consultant for viiv healthcare, gilead sciences, merck sharp and dohme, bristol-myers squibb, janssen and mylan. fg hayden reports personal fees from university of alabama antiviral drug discovery and development consortium, outside the submitted work; and being a noncompensated consultant to companies developing therapeutics and vaccines for novel coronavirus infections, including arcturus, cidara, fujifilm, gilead sciences, glaxosmithkline, regeneron, restorbio, ridgeback biotherapeutics, sab biotherapeutics, and vir. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. covid-19 and coronaviral hepatitis: evidence of collateral damage covid-19 in homeless populations: unique challenges and opportunities a suspected case of covid-19 turned into a confirmed case: a case report detection of sars-cov-2 in different types of clinical specimens the divergence between sars-cov-2 and ratg13 might be overestimated due to the extensive rna modification pros and cons of the application of evolutionary theories to the evolution of sars-cov-2 on the origin and continuing evolution of sars-cov-2 prediction and analysis of key protein structures of 2019-ncov possible sars-coronavirus 2 inhibitor revealed by simulated molecular docking to viral main protease and host toll-like receptor the pharmacological potential of rutin transmission and clinical characteristics of asymptomatic patients with sars-cov-2 infection temporal dynamics in viral shedding and transmissibility of covid-19 severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection: let the virus be its own demise mechanism of baricitinib supports artificial intelligence-predicted testing in covid-19 patients key: cord-266885-a5fdeuvv authors: dlotko, p.; rudkin, s. title: covid-19 clinical data analysis using ball mapper date: 2020-04-15 journal: nan doi: 10.1101/2020.04.10.20061374 sha: doc_id: 266885 cord_uid: a5fdeuvv in this note we provide a result of analysis of blood test data from patients with sars-cov-2 using ball mapper algorithm. we observe that patients with the virus and in particularly patients who end up in intensive care unit have quite narrow values of those parameters. please note that this is a preliminary work and it need to be validated on much larger dataset which we are trying to acquire at the moment. this is a work in progress. we share the results now hoping that they may be useful for people doing similar type of analysis to ours. if you have any questions or comments, please email us. please note that this paper will be edited, and it is far from its final version covid-19 rapidly becoming established as the modern global pandemics of the twenty first century. it is currently causing a large number of deaths, serious social distraction and major economical losses in the world especially in the western europe and the usa. to response this major treat word-wide scientific community is mobilising to use scientific methods to fight this pandemic. in this note we will analyse the clinical data made available by hospital israelita albert einstein in sao paulo, brasil. the anonymyzed dataset is provided at data4u (2020) . in the analysis we will use methods of topological data analysis. starting from pioneering works as in carlsson (2009) and nicolau et al. (2011) we will build up on the recent developement presented in d lotko (2019a) to localize clusters of patients that may be important from the perspective of a healthcare provider. we demonstrate that within the measurements of 16 major blood characteristics there is significant information to reliably classify those patients who will require icu treatment, isolate those spaces where covid-19 is most active and hence obtain a quick forecast of the likely care requirements of a presenting individual. the available dataset is composed of the following data collumns: 19. red blood cell distribution width (rdw), ranging between -1.598094344 and 6.982183933. in this work we will use variables from columns 1 as well as 6-20 as a characteristics/descriptor of a patient. our target will be to locate any clusters of patients with particularly high value of variable 2 (positively of sars-cov-2 result), patients that have been admitted to a regular ward, semi intensive or intensive care unit. for the purpose of this study we will use ball mapper algorithm d lotko (2019a) and its public domain implementation d lotko (2019b) available through cran repository. to make the obtained results reproducible, all the code that have been used, will be presented in this paper. in order to reproduce the results of this paper the reader should install r language r core team (2019) and the ballmapper package d lotko (2019b). subsequently the dataset is to be downloaded from the kaagle webpage data4u (2020) . this dataset is available in the format of microsoft exel. for the fuhrer processing it should be stored as a csv file. 2 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . the ball mapper algorithm, inspired by the work in carlsson (2009) , is a tool to produce a topological faithful representation of high dimensional dataset that can be visualized and explored in two dimensions. to achieve this aim, an input collection of points p together with a distance between points d, is covered with a collection of balls of a radius > 0. the centers of balls c 1 , . . . , c n are selected from the points of p in a greedy way as described in d lotko (2019a). the fact that the whole point cloud p is covered by balls of a radius centered in c 1 , . . . , c n implies that for every x ∈ p there exist i ∈ {1, . . . n} such that d(x, c i ) ≤ . given such a cover an abstract graph is constructed in the following way: vertices corresponds to the centers of balls c 1 , . . . , c n . an edge is placed between vertices c i and c j for i, j ∈ {1, . . . , n} if there exist p ∈ p such that d(p, c i ) ≤ and d(p, c j ) ≤ . in other words, the point p is covered jointly by the ball centered in c i and the ball centered in c j . the graph obtained as described above is called a ball mapper graph. its layout is a manifestation of the layout of the high dimensional point could p . sizes of vertices in its visualization will correspond to the number of points covered by the corresponding ball. in this experiment the variables 1 and 6-20 will constitute the point cloud p used to create the ball mapper graphs analyzed in this paper. a standard euclidean distance between points will be used. our task is to present how the predictive variables 2, 3, 4 and 5 (sars-cov-2 result, standard ward, semi intensive care and intensive care admission) changes over p by examining how they change over the obtained ball mapper graph. this will be achieved by the following procedure: every vertex of the ball mapper graph corresponds to a ball b(c i , ). let us take p i = b(c i , )∩p . the value of the ball, and therefore a point in the ball mapper graph, is determined as an average of value of the characteristic (one of the variables 1-5) in p i . this value will be presented using colour scale, where red colours means low values and violet means high values of the predictive variable. as one can observe in the section 2 there is no major variation between ranges of variables except from the first variable. therefore in this section we will use the original data from data4u (2020) without any normalization. in the first step the data are read into an array into r. we will also remove the rows with missing values and convert categorical variable in the column 2 to a numeric one. now, the data need to be re-arranged into a point cloud p and exploratory variables. this is achieved using the following code: cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . intensive_care <-data[,5] covid_and_icu <-covid_test*intensive_care pt_cloud<-cbind(data[,1],data[,6:19]) give the prepared datasets, we can create the initial ball mapper plots using the following command. the radius set to 7 was chosen to belance the level of detail in the plot with the need for a tangible discussion of the evidence. bm1 <-ballmapper::ballmapper(as.data.frame(pt_cloud),as.data.frame(covid_test),7) ballmapper::colorigraphplot(bm1,seed_for_plotting = 123) as a result the following plot presented in the figure 1 is obtained. the obtained ball mapper graph suggest that the patients, which are likely to have positive result for sars-cov2, have quite similar values coming from the blood tests. it is straightforward to give the values of all the used variables for clinical practitioners. as an example, we will consider all the balls 1 and 10 it can be achieved by the following instructions: pts <-ballmapper::points_covered_by_landmarks( bm1 , c(1,10) ) subset <-data [pts,] 4 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . av <-numeric( length(subset) ) stdev <-numeric( length(subset) ) for ( i in 1: the obtained values of average and standard deviations are summarized in the array below: in out second experiment we will use the value of the column 5 (determining if a patient was admitted to an intensive care unit). for that purpose the following code is used: bm2 <-ballmapper::ballmapper(as.data.frame(pt_cloud),as.data.frame(intensive_care),7) ballmapper::colorigraphplot(bm2,seed_for_plotting = 123) the results are presented in the figure 2 . as one can observe, the patients in the balls 14,23,24,24 but also 20 and 13 are likely to end up in the icu. it is worth noticing that those balls are far away in space, and therefore they represent patients with different values of blood parameters that are used to construct the ball mapper graph. once gain, it is possible to recover the precise values of be blood tests parameters and use them to assess the risk for newly admitted patients. let us not consider the patients which are tested positively for sars-cov-2 that required treatment in intensive care unit. to visualize their location the following code is to be used: bm3 <-ballmapper::ballmapper(as.data.frame(pt_cloud),as.data.frame(covid_and_icu),7) ballmapper::colorigraphplot(bm3,seed_for_plotting = 123) 5 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . 6 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. figure 3 is obtained. as one can observe, ball number 1 contains a lot of such patients, but also ball number 16 and 25 is clearly visible and majority of patients over there wil require icu. in our next exercise we will focus on the patients which have positive values of tests for sars-cov2. to obtain them the following code is used: let us observe which of those patients end up in normal ward, semi intensive and intensive care. for that purpose the following code is used. once again, the value = 5 is chosen to optimise the information tractability trade off. bm3 <-ballmapper::ballmapper(as.data.frame(covid_positive_pt_cloud), as.data.frame(covid_positive_regular_ward ),5) ballmapper::colorigraphplot(bm3,seed_for_plotting = 123) bm4 <-ballmapper::ballmapper(as.data.frame(covid_positive_pt_cloud), as.data.frame(covid_positive_semi_intensive_care ),5) ballmapper::colorigraphplot(bm4,seed_for_plotting = 123) bm5 <-ballmapper::ballmapper(as.data.frame(covid_positive_pt_cloud), as.data.frame(covid_positive_intensive_care ),5) ballmapper::colorigraphplot(bm5,seed_for_plotting = 123) the results are presented in the figure 4 as one can observe, the patients in the ball 10 almost entirely end up in the intensive care unit as well as 20% of patients from balls 2, 7, 8. patients from balls 1, 4, 11 will need at most semi intensive care. in the second version of the paper, guided by a medical expert, we will provide combinations of variables that is characteristic for patients with sars-cov-2 as well as the characteristics of patients that will need an intensive care unit. 8 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . unfortunately the dataset do not provide the information about the patients who died (except that there are such patients in the dataset). consequently we are not able to locate them in the global picture and we cannot assess the risk of death of a patient. consequently we cannot determine if there is a combination of blood results that is characteristics for those patients. in this section we will revisit the experiments from the section 4 with all the columns normalized to the interval [0, 1]. this will be performed by constructing a linear map that transform the minimal value in the column to 0 and maximal value in the column to 1. this normalization may be important for the following reason: as one can see by the ranges of variables described in section 2 it may be the case that not normalized data are skewed towards more senior patients as the value of the first columns is, for them, considerably larger than the values of the other columns. to normalize the data the following code is to be used: pt_cloud_normalized <-ballmapper::normalize_to_min_0_max_1(data) given the normalized data we can now see where the sars-cov-2 patients as well as the patients that will require a intensive care unit are located in this normalized dataset. note that, as usual, the value of the parameter is set to 1.2 in this instance. bm6 <-ballmapper::ballmapper(as.data.frame( pt_cloud_normalized ), as.data.frame(covid_test),1.2) ballmapper::colorigraphplot(bm6,seed_for_plotting = 123) bm7 <-ballmapper::ballmapper(as.data.frame( pt_cloud_normalized ), as.data.frame(intensive_care ),1.2) ballmapper::colorigraphplot(bm7,seed_for_plotting = 123) in the figure 5 we can see quite different picture compared to the one presented in figure 1 . this time the patients which are sars-cov-2 positive values are all located in balls 5,6, 10, 12, 18, while ball 10 contain only patients with positive values of tests. it is worth remarking that the patients with the positive values of tests are located close to a large ball number 1 that contain no patients with sars-cov-2. this observation may indicate some relation between blood results and sars-cov-2 that may be useful to locate high risk groups and speed up diagnosis using only standard blood tests. figure 6 show which patients required an intensive care unit (icu). as one can observe, amount the patients tested positively for sars-cov-2 the patients in the balls 10 and 18 have required icu, but patients from other balls did not. please note that the patients in balls 3, 4 and 15 also required icu, however those balls did not contain majority of sars-cov-2 patients. we believe that this is because the data describe all the patients in the hospital, not only ones with sars-cov-2. to locate the patients with sars-cov-2 that were admitted to icu the following code can be used: bm6 <-ballmapper::ballmapper(as.data.frame( pt_cloud_normalized ), as.data.frame(covid_and_icu),1.2) ballmapper::colorigraphplot(bm6,seed_for_plotting = 123) 9 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . figure 6 : ball mapper graph for normalized data colored by the patients who required intensive care unit. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. . the results can be found in the figure 7 . once again, we see clear patterns in balls 10 and 18 of patients tested positively for sars-cov-2 that require intensive care treatment. in the medical practice, they need to be given special attention. in the final part of this analysis we will therefore restrict only to patients diagnosed with sars-cov-2. to obtain them the following code is to be used: given those datasets let us locate the patients in regular ward, semi intensive and intensive care units. they can be localized using the following code. as usual, the value = 1.1 was obtained for the best balance of information and readability. bm8 <-ballmapper::ballmapper(as.data.frame( normalized_covid_positive_data ), . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 15, 2020. the results are presented in the figure 8 the analysis in the figure 8 indicate a hierarchy of patients: • those within ball 2 are very likely to stay in the regular ward. • patients in ball 4 and 5 will probably need semi-intensive care. • patients in balls 3 and 6, especially in 6, will need full icu. the presented analysis of the normalized data indicate that blood-based bio-markers may allow to determine, at early stage, patients with higher risk of being tested positively for sars-cov-2 as well as those, who may require icu. in the presented note we have analyzed the data provided in data4u (2020) using methods of topological data analysis, most notably ball mapper. the presented visualization indicate that the patients that may require special care (those with sars-cov-2 as well as those who may require icu) are located in distinct regions of space. given this, it seems to be possible to build a predictive system that would allow, for a given patient admitted to the hospital, to put him into appropriate risk factor groups. that may allow the health care providers more efficient use of resources and help the patient to optymize the treatment. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 15, 2020 . . https://doi.org/10.1101 /2020 topology and data diagnosis of covid-19 and its clinical spectrum ball mapper: a shape summary for topological data analysis ballmapper: create a ball mapper graph of the input data topology based data analysis identifies a subgroup of breast cancers with a unique mutational profile and excellent survival r: a language and environment for statistical computing. r foundation for statistical computing key: cord-261566-fn08b0y2 authors: mudgal, rajat; nehul, sanketkumar; tomar, shailly title: prospects for mucosal vaccine: shutting the door on sars-cov-2 date: 2020-09-15 journal: human vaccines & immunotherapeutics doi: 10.1080/21645515.2020.1805992 sha: doc_id: 261566 cord_uid: fn08b0y2 the sudden emergence of a highly transmissible and pathogenic coronavirus sars-cov-2 in december 2019 from china and its rapid global spread has posed an international health emergency. the rapid development of an effective vaccine is imperative to control the spread of sars-cov-2. a number of concurrent efforts to find an effective therapeutic agent or vaccine for covid-19 (coronavirus disease 2019) are being undertaken globally. oral and nasal mucosal surfaces serve as the primary portal of entry for pathogens like coronaviruses in the human body. as evidenced by studies on similar coronaviruses (sars-cov and mers-cov), mucosal vaccination can provide a safe and effective means for the induction of long-lasting systemic and mucosal immunity to confer protection against sars-cov-2. this article summarizes the approaches to an effective mucosal vaccine formulation which can be a rewarding approach to combat the unprecedented threat posed by this emerging global pandemic. in the 21st century, we have seen a worldwide spread of three previously unknown coronaviruses. the first outbreak of severe acute respiratory syndrome (sars) occurred in november 2002 in the guangdong province, china. the causative agent of the 2002 sars outbreak was identified as sars coronavirus (sars-cov). 1 another coronavirus, middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in 2012. 2 on december 31, 2019, several cases of pneumonia were reported in wuhan, china. 3 the etiological agent of the 2019 outbreak was later identified as sars coronavirus 2 (sars-cov-2) because the genomic sequence was closely related to that of the sars-cov from 2003. 4, 5 on february 12, 2020, the world health organization (who) named the disease caused by the novel coronavirus (sars-cov-2) as coronavirus disease 2019 . coronaviruses belong to the coronaviridae family. the family members are enveloped, positive-stranded rna viruses which appear as crown-like entities under the electron microscope due to spikes of glycoproteins protruding from their viral envelopes, thus exhibiting a corona-like appearance. 6 the coronaviridae family is divided into two subfamilies: letovirinae and orthocoronavirinae. the latter consists of the genera alphacoronvirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the pathogenic coronaviruses: sars-cov, mers-cov, and sars-cov-2 are all betacoronaviruses. 6 among known rna viruses, coronaviruses have the largest genomes size in the range of 26 to 32 kb in length. 7 two-third of the viral genome at 5ˊ end encodes up to 16 nonstructural replicase proteins which are translated as two polyproteins: pp1a and pp1ab. the genes encoding structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins are present at 3ˊ end of genomic rna. 7, 8 the s protein of coronaviruses is one of the most important targets for the development of sars vaccines and therapeutics because it is involved in receptor recognition, as well as virus attachment and entry. the s protein is made up of s1 and s2 subunits. s1 subunit has the receptor-binding domain (rbd) which binds with host receptor and then the s2 subunit mediates the fusion of viral and host membranes. 9 host receptor for sars-cov is angiotensinconverting enzyme 2 (ace2), whereas mers-cov recognizes dipeptidyl peptidase 4 (dpp4) as its receptor. 10, 11 the common symptoms of coronavirus infection are fever, cough, and sore throat. clinically, patients with sars suffer from atypical pneumonia. 12, 13 clinical presentation of sars-cov-2 patients is similar to patients infected with sars-cov. covid-19 manifests itself with symptoms including fever, dry cough, fatigue, and acute respiratory distress syndrome. 3, 14 these clinical features are a direct consequence of massive alveolar epithelial cell and vascular endothelial cell damage which is also accompanied by an exuberant release of proinflammatory cytokines and chemokines. 15 the disease severity and lung damage in the case of sars-cov-2 infection can be directly correlated with the dysregulated immune response at 7-10 days after symptom onset and is characterized by exuberant production of cytokines including il-2, il-7, il-10, mip-1a, ip-10, and tnf-α. 3, 15 this 'cytokine storm' was also reported in animal studies with sars-cov infection and was responsible for the dampening of adaptive immunity of patients in the later phase of infection. 16 as of now, the spread of sars-cov-2 is being quelled by strict policy measures such as travel restrictions, social distancing, patient isolation, and nationwide lockdown in several parts of the world. there are no approved vaccines available against any of the coronaviruses. this emerging global pandemic has instigated an unprecedented search for an effective prophylactic or therapeutic intervention against covid-19. [17] [18] [19] [20] in this review, we have discussed the potential and challenges for the development of a successful mucosal vaccine against sars-cov-2. vaccines have been one of the major contributors in the eradication of most of the infectious diseases in the last century. vaccination of a population interrupts the transmission chain of a communicable pathogen by not only protecting the immunized subjects but also by halting the transmission of the virus by 'breaking the chain' within a population by induction of herd immunity in the vaccine recipients. vaccines can be administered by either intramuscular or subcutaneous injection to introduce them into the systemic circulation. vaccination through systemic routes elicits strong systemic immune response but is not effective in generating efficient mucosal immunity. 21 mucosal vaccines are advantageous not just in evoking strong immune response at both mucosal sites and systemic circulation but also offer greater practicality in terms of cost and administration. 22 mucosal vaccines can be produced at considerably low cost for mass immunization, the administration is needle-free and convenient compared to systemic vaccines. 23 majority of mucosal vaccines are administered through oral or intranasal routes while rectal, vaginal, ocular, and sublingual routes can also be used. the selection of administration route depends on the nature of the antigen and the desired site for induction of immune response. upon oral immunization, immune responses are induced strongly in gastrointestinal (gi) tract, mammary glands, and salivary glands while intranasal vaccination induces marked antigen-specific immune response in respiratory, gi, and genital tracts. the primary mode of transmission of sars-cov is through mucosal membranes of the eyes, nose, or mouth. 24 studies with s protein of sars-cov-2 have revealed that it also recognizes human ace2 (hace2) receptor on the host cells similar to sars-cov. [25] [26] [27] ace2 is abundantly expressed in nasal and oral mucosa rendering them the primary targets for the viral entry and dissemination of sars-cov-2. 28 concomitant gastrointestinal symptoms are reported in confirmed covid-19 patients along with pulmonary pathology, characteristic of sars-cov-2 infection. the occurrence of acute hemorrhagic colitis in a few cases and the presence of sars-cov-2 rna in fecal samples of covid-19 patients indicate enteric involvement in covid-19 reasserting the similarity in tissue tropism with sars-cov. [29] [30] [31] considering the prominent role of nasal and gastric mucosa in the transmission and the clinical progression of sars-cov-2, mucosal immunization using oral or intranasal vaccine could be an effective strategy for immunoprophylaxis against sars-cov-2. vaccination at the entry site such as intranasal or oral immunization induces a strong local immune response in the case of sars-cov. 32, 33 consequently, vaccination at the mucosal sites reduces the risk of antibodydependent disease enhancement (ade) by blocking the virus at the entry site. administration of mucosal vaccine has also been shown to elicit strong systemic humoral immunity thereby neutralizing any virus particle that evades the primary immune response at the mucosal site. currently, several strategies are being examined for the development of a mucosal vaccine against sars-cov-2. 34 a unique mucosal system exists independent of the systemic immune system to protect exposed mucosal surfaces against environmental antigens and downregulation of systemic immune responses. it acts as the first line of defense against most of the antigens and prevents them from evoking systemic immune system. 35 the mucosal system serves as the most common portal for pathogen entry in the human body. the mucosal immune system consists of a complex network of tissues, non-lymphoid /lymphoid cells, and effector molecules including cytokines, chemokines, and antibodies. 36 the mucosal immune system is compartmentalized into immune inductive sites-where antigen sampling from mucosal surface occurs and then priming of b cells and t cells takes place, and immune effector site-where the activated immune effector cells move after extravasation and secrete cytokines to promote iga class-switch recombination. 37, 38 inductive site for the mucosal immune system is constituted by mucosa-associated lymphoid tissue (malt). malt comprises of highly organized structures such as appendix and peyer's patches in the intestine and tonsils in the upper airway. the effector sites of the mucosal immune system include lamina propria of various mucosae, surface epithelia, and exocrine glands. gut-associated lymphoid tissue (galt) and nasopharyngeal-associated lymphoid tissue (nalt) represent the major mucosal inductive sites. waldeyer's ring and oropharyngeal lymphoid tissues (paired palatine tonsils) in humans are considered anatomical equivalent of murine nalt. 39 larynx-associated lymphoid tissue (lalt), salivary duct-associated lymphoid tissue (sdalt), lacrimal ductassociated lymphoid tissue (ldalt), and conjunctiva-associated lymphoid tissue (calt) are also considered part of human malt. 40 these mucosal inductive sites are covered with a layer of enterocytes and microfold cells (m cells). m cells are specialized thin epithelial cells that move soluble antigen from the gut lumen to the underlying lymphoid tissues via transcytosis. exogenous antigens can activate t cells directly or are handed off to dendritic cells (dcs) that can act as antigen-presenting cells (apcs). 41, 42 these primed t cells move to the germinal centers and secrete cytokines to promote b-cell isotype switching to iga production. 43 secretory iga (siga) antibodies are the most essential effector molecule in the mucosa. it is recognized as the first line of protection against foreign toxins, pathogens, and overgrowth of commensal microbes. it is secreted as a dimer joined together by a joining chain and is actively transported exclusively across the mucosal surface via a polymeric iga receptor. 44 the activated t and b cells retain immunological memory and contribute to long-lasting protective immunity against the pathogen in systemic and mucosal systems. siga neutralizes toxins or pathogens in the mucosal environment using three mechanisms: immune exclusion, antigen excretion, and intracellular neutralization ( figure 1 ). 45 in addition to siga, transudated iga and igg, which are generated in response to both systemic and mucosal vaccination, also contribute to local surface defense in the genitourinary mucosa and in the lower respiratory tract which are more permeable to serum-derived antibodies than intestine. these antibodies employ a diverse range of effector functions to protect against pathogens. they neutralize toxins; can mediate opsonization and facilitate internalization of invading pathogens by phagocytes. transudated igg exert its immunopathological effect when siga-dependent elimination of pathogen is unsuccessful. 46 serum igg antibodies protect against viremia and are crucial for virus clearance from systemic circulation. a mucosal vaccine can be developed based on one of the several vaccine platforms including viral vectors, virus-like particle (vlp)-based, dnas, subunit, inactivated whole virus, or live-attenuated vaccine. 6, 47, 48 each of these platforms has its own advantages and disadvantages. vlp-based vaccines are composed of viral structural proteins capable of selfassembly. dna vaccine comprises viral immunogens encoded by a recombinant plasmid that is delivered to the site of administration where the plasmid expresses itself and elicits the desired immune response. 49 both these safe vaccine platforms preserve the inherent antigenic structure of viral immunogens and are noninfectious but often suffer from weak immunogenicity. viral vectors encode the antigenic protein and are delivered to the host cell where the antigen is expressed and is presented on the surface of apcs to induce a cellular and humoral immune response. 50 these vaccines are highly efficient but preexisting immunity against the viral vector might cause a harmful immune response in some recipients. live-attenuated vaccines or inactivated vaccines are based on pathogens that have been attenuated or inactivated by heat or chemical treatment. 46 as of now, all the mucosal vaccines licensed for human use are based on liveattenuated approach including oral polio vaccine (opv) and intranasal influenza vaccine (flumist®). safety concerns are often associated with attenuated vaccines due to the possibility of incomplete inactivation of harmful pathogens. subunit vaccines consist of specific antigenic fragments of virus capable of eliciting strong antibody-mediated and cellular immune response. 51 inactivated whole virus vaccines and subunit vaccines are relatively inexpensive, inert, and nontoxic but the possibility of alteration of immunogenicity of antigens needs to be confirmed consistently. few sars-cov and mers-cov vaccines based on the different platforms are summarized in table 1 . s protein is the main antigenic component of sars-cov, sars-cov-2, and mers-cov among four structural proteins (s, e, m, and n proteins). vaccines using s protein elicit a potent immune response and inhibit viral infection. 9, 53, 64 but the use of fulllength s protein as an antigen for vaccine design has raised few safety issues due to ade of viral infection in vaccinated subjects. a study on the chinese macaque models for sars-cov showed greater lung damage in vaccinated animals (vaccinated with fulllength s proteins) relative to unvaccinated subjects upon virus challenge. 65 anti-s protein igg antibodies have been implicated in this acute alveolar damage on exposure to the virus. similar symptoms have been observed in critical patients with sars-cov infection. 66 it is speculated that ade of viral infection is mediated through the binding of virus-neutralizing antibody complex to the fc receptors on the monocytes/macrophages leading to increased production of pro-inflammatory cytokines. additionally, this virus-antibody complex might activate the classical pathway of complement system or antibody-mediated cytotoxicity leading to cellular damage. 67 taking a cue from studies with sars-cov and mers-cov, caution must be exercised in selecting an antigen target that minimize ade while inducing a potent immune response against future exposure to sars-cov-2. the use of rbd of s protein as an antigen in the case of sars-cov or mers-cov has been extensively explored. 9,68 rbd of s protein is highly immunogenic and confers neutralizing capability against multiple strains of sars-cov and mers-cov. rbd-based vaccine minimizes the risk of ade upon exposure to virus in vaccinated individuals as it lacks the non-neutralizing immunodominant region of s protein. 69 other fragments of s protein of sars-cov and mers-cov that have been used for vaccine design include s1 and s2 fragments ( figure 2 ). 71,72 a mucosal vaccine based on n protein of sars-cov has shown the induction of both cellular and humoral immunity in mice. 61 several immunogenic domains of n protein are highly conserved in coronaviruses. 73 n protein of sars-cov-2 shares high sequence identity (~91%) with n protein of sars-cov and hence can be considered for the development of a broad spectrum coronavirus vaccine. 74 a challenging alternative that can warrant protection against future outbreaks of similar coronaviruses is to identify a universal epitope in the whole virus family or genera. this strategy is being undertaken for influenza viruses and can be extended to coronaviruses as well. 75 immunoinformatics can also be a powerful tool in prediction of epitopes on the viral surface proteins. 76 traditionally, mucosal immune induction needs a higher dose of antigen in comparison to parenteral immunization as antigenic preparation may get diluted in mucus in the nasal cavity or get expelled by mucus and ciliary movement in the respiratory tract. 77 effective intranasal vaccination requires the antigen to reach mucosal sites, cross the mucus layer, and induce local iga production. a vaccine administered through the oral route has to endure the low ph environment in the upper gi tract and a variety of nucleases and proteases present in the digestive tract before it can reach the immunological sites. mucosal antigens when administered alone lead to a weak induction of immune response. to overcome these physical and biochemical obstacles in priming mucosal immune cells, vaccines delivered through oral or nasal routes are often administered complemented with an adjuvant. 78 adjuvants are the supplementary materials (natural or synthetic) in a vaccine formulation that potentiates the immune-induction capacity of a vaccine. 79 adjuvant in a vaccine formulation plays a critical role in maintaining the structural integrity of the antigen, augmenting antigen bioavailability, and enhancing antigenic stimulation. adjuvants are broadly divided into two classes: adjuvants that can serve as carrier systems to facilitate antigen delivery to immune induction sites and adjuvants that can act as immunostimulators through enhanced internalization, presentation, and processing of the antigen in the apcs. 80 a number of polymers including chitosan and poly lacticco-glycolic acid (plga) have been used as carriers in various vaccine formulations for immunostimulation due to their high affinity toward mucosal surfaces. 81, 82 emulsions and liposomes are the other carriers routinely tested for mucosal vaccine. 48 dc and m cells are a major determinant for induction of mucosal immune response at immune inductive sites and thus represent an ideal site for antigen presentation. surface markers at the surface of epithelial cells and dcs can be strategically targeted for antigen delivery with specialized molecules such as lectins or nanoparticles for increased antigen uptake by apcs. 83, 84 pathogen recognition receptor agonists such as synthetic poly-(i:c), or toll like receptor agonists such as cpg oligodeoxynucleotides, engage these receptors present on the surface of apcs to potentiate the immunogenicity of the vaccine. 85, 86 other commonly used adjuvants including cholera enterotoxin (ct) and heat-labile enterotoxin (lt) from e. coli exert their adjuvanticity by interacting with gm1 gangliosides on the surface of follicular dendritic cells and in turn increase the induction of b-cell clones. 87 the use of immune-stimulating complexes (iscoms) has also proven to be highly effective for administration with mucosal vaccines. 88 one of the major factors influencing the rational design of a mucosal vaccine is immunotolerance at the mucosal sites. immunotolerance is the ingenious modulation of the mucosal microenvironment to avoid unnecessary induction of host immune cells against foreign antigens or commensal microbes. mucosal surfaces are exposed continuously to environmental, food, or self-antigens which lead to the development of a tolerogenic microenvironment, especially in gastric mucosa. 89 some vaccination strategies fail to develop effective immunity and can induce immunotolerance. this suppression of immune response is affected mainly by vaccine formulation, antigenic dose, and frequency of administration. administration of antigen at low doses for a long time leads to low-dose immunotolerance. 48 contrastingly, high dose of antigen administered at a low rate induces high-dose tolerance instead of immunostimulation. this hypo-responsiveness at the mucosal induction sites can be overcome by strategically determining the antigen dose, vaccine formulation, and timing of vaccine delivery. the design of a novel mucosal vaccine also aims at mimicking the kinetics of pathogen infection for increasing the clinical efficacy of the vaccine. optimal release timing of antigen at the inductive site helps in dodging the mucosal immunotolerance. to this end, the antigen is conjugated with adjuvants like tlr ligands or cd-40 specific antibodies. 90, 91 immunosenescence several issues are needed to be addressed for the design and development of an efficacious mucosal vaccine against sars-cov-2. it has been observed in human challenge studies that immunity acquired with coronavirus infection is often shortlived and in some cases, re-infection with the same virus was possible after an extended period. 92 it was reported in some cases that immunity acquired with sars-cov or mers-cov also declines considerably 2-3 years after viral infection. 93, 94 another concern for designing a vaccine against sars-cov-2 is that the viral infection is associated with severe pathology specifically in patients of higher age group (typically >50 years). 14 people in this age group do not respond very well to vaccination in terms of neutralizing antibody titers and require a higher amount of antigen to produce sufficient immunogenicity. specialized dose-regime in terms of the amount of antigen, use of specific adjuvants, or the immunization effects of follow-up doses after a single prime dose can be investigated in vulnerable age groups and immunocompromised people before extending vaccination to them. for most parenterally administered vaccines and mucosal vaccines, correlates and precise mechanism of their efficacy remain poorly defined. most of the licensed vaccines rely on the measurement of a single parameter that is statistically correlated with protection afforded by the vaccine. there is no absolute method of sampling, and the choice of sampling method varies according to the parameter to be evaluated. conventionally, quantification and qualification of secreted antibodies is performed using seroimmunoassays in body secretions such as saliva, tears, nasal, blood samples or genital secretions, and gut or organ lavages. 95 cellular correlates of immunity are analyzed using assays such as enzyme-linked immunospot assays (elispots), reverse-transcriptase pcr (rt-pcr) or cell-sorting techniques. 96 a modern system biology approach can also be used that utilizes functional genomics to identify molecular signatures that correlate well with traditional biological markers for evaluating vaccine efficacy. 97 correlates of protection differ greatly based on whether the objective of vaccination is to prevent a mucosal or a systemic infection. 96 several animal models including mice, ferrets, macaques, hamsters, and non-human primates have been used for evaluating safety and efficacy of sars-cov and mers-cov vaccines. 53, [98] [99] [100] [101] [102] ferrets are a suitable animal model for sars-cov vaccine evaluation as they support viral replication in the respiratory tract, develop similar disease symptoms, and display severe lung pathology. 103, 104 smaller animal models like rabbits and mice are a preferred choice for vaccine evaluation in animals because of inexpensive maintenance, ease of genetic manipulation, and standardized methods of testing. wild type mice are nonpermissive to sars-cov-2 viral replication. a transgenic mice model expressing hace2 has recently been developed by genetic manipulation to make the mice susceptible to sars-cov-2 infection. 105 this mice model mirrors the pathological features of sars-cov-2 infection in human patients albeit at a moderate level as compared to sars-cov. moreover, a young animal/mice model can effectively exhibit excellent neutralizing efficiency induced by a vaccine candidate but it cannot effectively mimic the clinical manifestations of covid-19 in an elderly population. hence, robust lethalchallenged mice models using senescent mice that can recapitulate the clinical disease in the aged human population are needed to be developed for assessing the efficacy of a covid-19 vaccine candidate. following animal modelbased preclinical studies, good manufacturing practices are employed for the scale-up of the vaccine production. currently, mucosal vaccines form a small proportion of licensed vaccines for humans. limited choice of adjuvants for human mucosal vaccines, immunotolerance, and differential degree of vaccine-induced immune response in different populations are some of the impediments associated with the development of mucosal vaccines. efficacies of different mucosal vaccines depend on myriad of factors such as age, environment, host genetics, the microbiome of the recipient, and the regimen of immunization. it ranges from 70% to 90% for rotavirus vaccines to 85%-to 90% for oral vaccines against influenza and polio viruses. 22 cholera, polio, and rotavirus oral vaccines are found to be less efficacious in developing countries. this weak induction of immune response can be attributed to nutritional deficiencies such as vitamin a or zinc, concomitant bacterial, helminth, or viral infections, and the presence of high levels of maternal antibodies in the breast milk. 106, 107 intranasal immunization using live attenuated or live vectors is also associated with the risk of the harmful antigens and adjuvants (such as ct and lt) accessing the central nervous system through the cribiform plate. 108 a number of infectious diseases have been successfully controlled with the use of parenterally administered vaccine that may or may not lead to the induction of mucosal immune response. administration of the vaccine through systemic circulation might not necessarily recapitulate the immune response generated by mucosal administration but is adequate for evoking immune response against mucosal pathogens such as human papilloma virus (hpv), influenza virus, or polio virus. all three licensed vlp-based hpv vaccines are administered intramuscularly and induce a high level of durable igg antibody response against the virus. 109, 110 these antibodies are also detectable at mucosal sites of infection such as oral cavity and cervix after vaccination and serve as the primary effectors of protection against hpv. 111, 112 currently, there are two types of licensed influenza vaccines: parenterally administered inactivated influenza vaccine and live attenuated influenza virus vaccine (laiv) which is delivered intranasally. inactivated influenza vaccine elicits some degree of local iga antibodies and high levels of systemic igg antibodies to mediate protection by diffusing into the mucosal sites. laiv, in turn, mimics natural infection and induce highly cross-reactive serum igg, mucosal iga, and cellular immunity in the recipients. 113, 114 yearly influenza vaccines are adjusted according to the prevalent strains in the coming flu season. in clinical settings, inactivated vaccine is found to be more effective than laiv in preventing symptomatic disease in healthy adults while laiv offers greater protection against influenza disease in children. [115] [116] [117] similarly, two types of vaccines are available for polio virus: an injectable polio vaccine (ipv) and opv. sabin opv, with its enormous impact in disease control worldwide, is considered to be the prototype oral vaccine. superiority of opv over ipv is attributed to the induction of high titers of mucosal iga antibodies. 118, 119 despite the generation of higher serum antibody titers, ipv provides limited intestinal immunity. 118 due to the risk of rare cases of vaccine associated paralytic polio, most developed countries have switched to either ipv or a sequential combination of ipv and opv. 120, 121 based on the substantial experience in control of mucosal pathogens using parenterally administered vaccines, several parenteral vaccines are being developed or evaluated against sars-cov-2. notably, dna-based vaccine by inovio pharmaceuticals (ino-4800), mrna-based vaccine by moderna (mrna-1273), adenovirus-vectored vaccine (chadox1), and an inactivated virus vaccine (picovacc) are currently under investigation in human clinical trials. [122] [123] [124] [125] rapid licensure and production of a mucosal vaccine time is the most valuable asset in the rapidly emerging covid-19 pandemic, especially in high-mortality areas. rapid development and testing of a viable vaccine can be achieved by adopting a number of novel strategies. instead of developing a novel vaccine development platform, utilizing an existing vaccine platform will accelerate the design and production of vaccines against sars-cov-2 and will enable a quicker future response against newly emerging viruses. since the outbreaks of sars-cov and mers-cov, several vaccine candidates were developed using different production platforms. these established platforms are being used for the development of vaccine candidate against sars-cov-2. 122, 124, 126 to expedite the process for licensure and use of the vaccine against sars-cov-2 in the wake of the current pandemic, eyal et al. have suggested an alternative model for accelerated rollout of an effective sars-cov-2 vaccine by skipping phase 3 clinical trials. 127 this study proposes a controlled human challenge model wherein nonsusceptible adult volunteers from a healthy group will be challenged with an increasing dose of live virus to determine a dose that induces clinical symptoms similar to the natural infection in the individuals of similar age (preparatory phase). after this dose for controlled human challenge model has been optimized, a randomized cohort of volunteers will receive the candidate vaccine or placebo before being challenged with the controlled dose of the live virus (phase 1). the successful vaccine candidates can then be administered to a large cohort of individuals for evaluation of shortterm safety prior to vaccine rollout (phase 2). this accelerated licensure of the vaccine can be followed up with post-approval surveillance to monitor the occurrence of rare side effects and long-term efficacy for future regulatory approvals. the overwhelming demand for a sars-cov-2 vaccine in the current pandemic by far exceeds the existing production capacity. to deliver required quantities of the successful vaccine, production process will have to be ramped up to an enormous scale. the necessary infrastructure can be developed simultaneously with the continued production on existing capacity. alternatively, rapid manufacturing of a successful vaccine can be facilitated by adopting a new pandemic paradigm, with a fast start and many steps of vaccine development and production executed in parallel before confirming a successful outcome of another step. 128 in recent years, rapid production of live attenuated vaccines is being facilitated by utilizing reverse genetics process that involves the precise deletion of specific genes of viral genome. 129 deletion of pathogenic components of the pathogen is advantageous over traditional methods of viral attenuation in terms of safety and efficacy of a vaccine. the current covid-19 pandemic has virtually brought most world economies to a halt and severely impacted the lives of a large proportion of the world population. development of a viable sars-cov vaccine is imperative to reduce mortality and morbidity associated with this novel virus outbreak. mucosal vaccines offer better patient compliance in terms of the physical and psychological comfort due to absence of needlestick injury and thus are highly compatible for mass immunization in a pandemic scenario. vaccine administration using injection involves the cost of injection device, it's safe disposal, and the employment of trained medical staff which adds a considerable cost for mass vaccination especially in developing countries. mucosally administered vaccines also abrogate the risk of transmission of infections by injection devices. despite our detailed understanding of mucosal system in mice, the complex cellular and molecular interplay of different component of innate immune response to mucosal vaccination in humans is still poorly deciphered. additionally, the emergence and rapid global spread of sars-cov-2 has provided a very small window for basic and translational studies that propel the development and evaluation of vaccine against a pathogen. while the knowledge gained from previous studies on sars-cov and mers-cov can be used for sars-cov-2 vaccine development, it is yet uncertain as to what extent it will work for sars-cov-2 or whether correlates of protection used will faithfully predict protective efficacy. potential ade and waning of vaccine-induced immune response represent other obstacles in the development of a mucosal vaccine against sars-cov-2. the currently licensed mucosal vaccines against viral pathogens are exclusively live-attenuated. the development of a safe and immunogenic live-attenuated vaccine is an attractive possibility for mucosal vaccine against sars-cov-2. live attenuated vaccines, by definition establish a mild infection at the immunization site and exhibit some level of in-built adjuvanticity thereby ensuring the delivery of a higher antigen dose and better targeting of mucosal inductive sites for immunostimulation. this candidate vaccine can be easily administered orally as encapsulated antigens or can be delivered through the intranasal route via droplets and aerosol spray. covid-19 pandemic demands for adopting nonconventional approaches for a viable mucosal vaccine development, hastening the vaccine licensure process, utilizing existing vaccine development and manufacturing platforms, and ensuring global distribution of the licensed vaccine in time to minimize the impact of covid-19 across the globe. it will also need an unprecedented scale-up of the manufacturing process and concerted efforts of the supply chains to make the vaccine available before this pandemic is over. learning from the covid-19 pandemic, sustained investments in vaccine development and production infrastructure should be made to save human lives and curtail the economic impact associated with a looming viral pandemic. moreover, continued global surveillance and vigilance in the post-pandemic scenario should be practiced to help the world counter a second wave of covid-19 or other possible future coronavirus outbreaks. the authors declare no potential conflict of interest. the compilation of this review was financially supported by department of science and technology, india science and engineering research board (serb) govt of india [ipa/2020/000054]. epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features of patients infected with 2019 novel coronavirus in wuhan the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 vaccines: status report origin and evolution of 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mucosal immunity induced by enhanced-potency inactivated and oral polio vaccines immunoglobulin response in serum and secretions after immunization with live and inactivated poliovaccine and natural infection vaccine policy changes and epidemiology of poliomyelitis in the united states polio vaccination: past, present and future immunogenicity of a dna vaccine candidate for covid-19 an mrna vaccine against sars-cov-2-preliminary report chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques development of an inactivated vaccine candidate for sars-cov-2. science. 2020 microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development human challenge studies to accelerate coronavirus vaccine licensure developing covid-19 vaccines at pandemic speed an improved reverse genetics system for influenza a virus generation and its implications for vaccine production key: cord-274399-cd7cmpoj authors: barzin, amir; schmitz, john l.; rosin, samuel; sirpal, rameet; almond, martha; robinette, carole; wells, samantha; hudgens, michael; olshan, andrew; deen, stephanie; krejci, patrick; quackenbush, eugenia; chronowski, kevin; cornaby, caleb; goins, janette; butler, linda; aucoin, julia; boyer, kim; faulk, janet; alston-johnson, devena; page, cristen; zhou, yijun; fiscus, lynne; damania, blossom; dittmer, dirk p.; peden, david b. title: sars-cov-2 seroprevalence among a southern u.s. population indicates limited asymptomatic spread under physical distancing measures date: 2020-09-29 journal: mbio doi: 10.1128/mbio.02426-20 sha: doc_id: 274399 cord_uid: cd7cmpoj characterizing the asymptomatic spread of sars-cov-2 is important for understanding the covid-19 pandemic. this study was aimed at determining asymptomatic spread of sars-cov-2 in a suburban, southern u.s. population during a period of state restrictions and physical distancing mandates. this is one of the first published seroprevalence studies from north carolina and included multicenter, primary care, and emergency care facilities serving a low-density, suburban and rural population since description of the north carolina state index case introducing the sars-cov-2 respiratory pathogen to this population. to estimate point seroprevalence of sars-cov-2 among asymptomatic individuals over time, two cohort studies were examined. the first cohort study, named screennc, was comprised of outpatient clinics, and the second cohort study, named screennc2, was comprised of inpatients unrelated to covid-19. asymptomatic infection by sars-cov-2 (with no clinical symptoms) was examined using an emergency use authorization (eua)-approved antibody test (abbott) for the presence of sars-cov-2 igg. this assay as performed under clia had a reported specificity/sensitivity of 100%/99.6%. screennc identified 24 out of 2,973 (0.8%) positive individuals among asymptomatic participants accessing health care during 28 april to 19 june 2020, which was increasing over time. a separate cohort, screennc2, sampled from 3 march to 4 june 2020, identified 10 out of 1,449 (0.7%) positive participants. importance this study suggests limited but accelerating asymptomatic spread of sars-cov-2. asymptomatic infections, like symptomatic infections, disproportionately affected vulnerable communities in this population, and seroprevalence was higher in african american participants than in white participants. the low, overall prevalence may reflect the success of shelter-in-place mandates at the time this study was performed and of maintaining effective physical distancing practices among suburban populations. under these public health measures and aggressive case finding, outbreak clusters did not spread into the general population. keywords covid-19, sars-cov-2, antibody, coronavirus, seroprevalence a cute virus infections typically induce a vigorous antibody response. the overwhelming majority of covid-19 patients also produce antibodies, initially of the igm isotype and shortly thereafter of the igg isotype. seroconversion rates approach 100% at 3 weeks after symptoms appear and persist for several months (1) (2) (3) (4) . the composition, quality, median and maximal duration of the igg response are unclear as the earliest cases occurred less than 7 months ago (5) . both presymptomatic and asymptomatic transmission events of sars-cov-2 have been reported (6) (7) (8) (9) (10) (11) . it is difficult, however, to define asymptomatic transmission a priori, as the full spectrum of sars-cov-2-associated clinical symptoms is still evolving (12, 13) . the degree to which asymptomatic transmission contributes to population seroprevalence and eventual herd immunity is unknown and the subject of intense debate. determining the extent to which sars-cov-2 can be transmitted asymptomatically shapes covid-19 disease modeling and informs public health interventions. asymptomatic transmission represents a key biological property of this virus. early serology studies for sars-cov-2 were conducted in densely populated, urban settings, such as wuhan, china; bergamo, italy; or boston, new york, los angeles, or seattle in the united states (14, 15) . these studies focused on patients who recovered after severe clinical disease (4). they were conducted at hot spots of transmission, in urban settings with multiple independent introductory events and extended opportunities for rapid spread, such as multiple modes of public transport and international travel (16) . north carolina (nc), in contrast, represents a largely suburban and rural population with limited public transport. the first case of covid-19 was reported on 2 march 2020. as in other locales, symptomatic cases were concentrated among vulnerable populations, such as residents of retirement homes, prison inmates, and workers in meatprocessing plants. "stay-at-home" directives were implemented 30 march to 8 may 2020. statewide, all schools were ordered to close 14 march and did not reopen during the study period. as of 19 june, nc reported 49,840 cases and 1,197 deaths (nc department of health and human services [dhhs]) for a population of 10,488,084 people (2019 census), based primarily on nucleic acid testing (nat) of suspected cases. thus, the fraction of infected persons based on case counts for this area and calendar time using viral nat is 0.5%. this study evaluated two cohorts of asymptomatic patients. screennc enrolled 2,973 asymptomatic participants across the university of north carolina health system (unc health), representing 267 different zip codes. these individuals sought medical care unrelated to covid-19. individuals with known covid-19 symptoms, at screening or in their recent past, were excluded. screennc2 tested 1,449 sera from patients in clinical care not related to covid-19 or recruited to the study. no patient was tested more than once. screennc enrolled 28 april to 19 june 2020, and screennc2 sampled patients 3 march to 4 june 2020. table 1 shows the demographic composition of the screennc cohort compared to patients accessing unc health sites during the same time period in 2020 as well as in 2019, i.e., pre-covid-19. table 2 shows the age distribution. there were no differences between overall patient populations accessing unc health in 2020 compared to 2019. when comparing the screennc population and patient populations accessing unc health in 2020, there were some notable differences. screennc enrolled fewer 80ϩyear-old patients who accessed care during the same calendar period (2.6% versus 7.4%), slightly more females (65.8% versus 63.6%), fewer black or african american participants (13.3% versus 23.3%, p յ 0.001 by fisher exact test), and fewer hispanic or latino participants than the comparator unc health population (2.6% versus 4.9%, p յ 0.001 by fisher exact test) during the same calendar period. these differences were used in a model of adjusted seroprevalence. sars-cov-2 igg antibody was detected using the abbott sars-cov-2 igg assay on the architect platform under emergency use authorization (eua). index values of ն1.4 were considered positive. this assay has a manufacturer reported analytical sensitivity of 100% and specificity of 99.6%. independent studies in the u.s. population report sensitivities/specificities of 100%/99.9% (17) , 92.9%/99.6% (18), 99.0%/99.8% (24) , and 100%/99.6% (25), respectively. on-site validation (n ϭ 317) established a sensitivity of 100% and a specificity of 98.9% at 3 weeks after symptom onset. intra-assay precision (% coefficient of variation [cv%]) was 1.1%, and interassay cv% was 0.92%. thus, in-house performance was comparable to the manufacturer's specification and to other studies in the united states. screennc identified 24 out of 2,973 (0.8%) positive participants, and screennc2 identified 10 out of 1,449 (0.7%) positive participants. black or african american participants had twice the unadjusted seropositivity rate of whites (1.5% versus 0.7%). the analysis of other demographic factors was not possible due to the small number of positive cases. the unadjusted seroprevalence remained constant over time for the screennc2 population but showed an upward, although imprecise, trend for the larger screennc cohort ( fig. 1 and table 3 ). the counts were adjusted for assay characteristics using the method of rogan and gladen (19) and cohort characteristics using direct standardization to yield a population this study identifies a very limited seroprevalence of sars-cov-2 among asymptomatic individuals accessing the unc health system. there was a suggestion of accelerating asymptomatic spread of sars-cov-2 during the study period and cohort, i.e., the transmission among persons who never subjectively felt ill. the seroprevalence of less than 1% was lower than estimates from earlier studies, but in line with recent studies using high-accuracy tests (15, 17) . the seroprevalence for this low-density community was lower than reported in a large study of convenience samples that focused around "hot spot" cities and did not explicitly exclude persons with past symptomatic infections (20) . this result may reflect the success of shelter-in-place state mandates and maintaining effective physical distancing among suburban populations during the time frame of our study. it may also reflect the low population density and delayed introduction of the virus compared to population centers and travel nodes. under these circumstances, early outbreak clusters did not expand far into asymptomatic patients, which indicates that continued viral nat and aggressive case finding can curb sars-cov-2 spread. the data suggest that members of the black community are disproportionately affected by the covid-19 epidemic, here as demonstrated by asymptomatic acquisition of infection and in other studies as demonstrated by increased incidence of symptomatic cases and deaths. significantly fewer members of the black and latinx communities participated in this study than accessed care in the same calendar time, which may have led to biased enrollment. there are many possible explanations for the variation in population seroprevalence among different preliminary reports and published studies (14, 17) . our study populations were derived from individuals accessing health care clinics and may not provide a population-representative study sample. it is very challenging to conduct random population sampling during a public health emergency when "stay-at-home" orders are in place. prevalence of transmission, patterns of behavior, and characteristics of the sample population were time varying during the sample period. logistical constraints, such as lack of personal protective equipment and the primacy of clinical care, limited study access. in nc, only persons with a valid medical reason and "essential workers" were exempted from the "stay-at-home" ordinance, and only those participated in the study. the degree to which underlying health conditions impact analytical assay sensitivity is currently unknown; for instance, screennc2 included patients with immune suppression and thus likely impairments in their ability to generate high-titer antibodies. defining "asymptomatic" is a challenge and a limitation, as it is based on selfreported clinical symptoms of infection, the exact definition of which continues to evolve. both symptom severity and awareness may differ among different communities. third, there exists a wide variety of antibody tests, each with differing performance characteristics (21, 22) . these serological tests are validated on symptomatic, hospitalized patients, not asymptomatic cases. this study employed an eua assay performed in a clia-certified laboratory on a venous blood sample, with demonstrated specificity to detect antibodies only to sars-cov-2, not to seasonal coronaviruses. in general, a robust igg response to sars-cov-2 is correlated with the presence of neutralizing antibodies for coronaviruses, but exactly which level of response and which antigen is a reliable predictor for protection has not been established (22) . this study only uses the presence of igg as a measure of exposure. the negative predictive value under any of the reported sensitivity/specificity characteristics is ͼ99.5% for assumed population seroprevalence of ͻ5%. the positive predictive value is 83.6% for an assumed seroprevalence of 5% and 48% for an assumed seroprevalence of 1%. this implies that perhaps even fewer individuals developed antibodies than reported. alternatively, it is possible that igg responses are lower in asymptomatic than symptomatic patients and decline over time (23) . in conclusion, based on the data presented in this study, it is unlikely (i) that "herd immunity" can be reached in the foreseeable future and (ii) that asymptomatic spread contributed a substantial number of infections. taken together, these data suggest that early and rigorous shelter-in-place policies and physical distancing measures are important tools to suppress sars-cov-2 infection in the majority of the population. enrollment. patients enrolled in the study were recruited at nine outpatient clinical sites and two emergency department sites across the state associated with the unc health network. upon arrival for sars-cov-2 seroprevalence in north carolina ® routine care or scheduled visits for enrollment into the study, patients performed a consent procedure that included reviewing recent covid-19 clinical history using unc irb-approved questionnaires. once consent was obtained, patients provided 5 ml of venous whole blood. this sample was collected by phlebotomists or nurses at each site and analyzed for the presence of sars-cov-2 antibody. results were reported in the unc medical record, and patients were notified of negative results via an electronic message. for those with a positive result, a physician was responsible for calling the patients and reviewing the results and answering follow-up questions. the screennc protocol was approved by the unc irb (unc irb 20-0937, nct04367740). screennc2 samples were sera obtained from unc patients for indications other than covid-19. this protocol was also approved by the unc irb, which allowed for waiver of hipaa and consent from these patients (unc irb 20-1308). the comparator cohorts (2019 and 2020) were composed of all patients with any procedure seen in the project testing sites. these include two emergency room sites and multiple outpatient locations. canceled appointments are excluded. nineteen departments in the unc health system were identified, as some study sites comprise multiple departments. the 2019 cohort covers 15 february 2019 to 15 june 2019, and the 2020 cohort covers 15 february 2020 to 15 june 2020. detection of igg antibody to sars-cov-2. igg antibody to sars-cov-2 was detected with a microparticle chemiluminescence assay (abbott laboratories) on the abbott architect i2000sr immunoassay analyzer according to the manufacturer's protocol under eua. the abbott sars-cov-2 igg assay utilizes microparticles coated with sars-cov-2 nucleocapsid protein. after washing, bound igg was detected via addition of anti-human acridinium-labeled second-step antibody. following a second wash step, pretrigger and trigger solutions were added and a chemiluminescent reaction was detected and reported in relative light units (rlu). the rlu generated is reflective of the amount of antibody bound to the microparticles. the sample rlu was compared to the assay-specific calibrator rlu to generate an index value (s/c). the presence of antibody was reflected by an index value of ն1.4. index values of ͻ1.4 were classified as negative for sars-cov-2 igg. the abbott sars-cov-2 igg assay was evaluated in-house to assess sensitivity, specificity, and reproducibility. sensitivity was determined by testing remnant serum samples from patients admitted to unc hospitals and confirmed to be infected by rt-pcr testing of nasopharyngeal swabs. sera collected in the first (n ϭ 22), second (n ϭ 26), third (n ϭ 17), and fourth (n ϭ 23) weeks after disease onset were tested. calculated sensitivities were 31.8%, 80.8%, 100%, and 100%, respectively. this pattern follows the known natural history of igg antibody development, where titers take 1 to 2 weeks to reach detectable levels after primary infection. specificity was determined by testing 277 sera. two hundred thirty-eight of these were collected prior to 1 january 2020 (predating widespread u.s. covid-19 cases), and 39 were collected after that date. twenty-three of these 39 patients had nasopharyngeal swabs tested by sars-cov-2 rt-pcr and were negative. additionally, 6 of the 39 samples were from individuals tested for endemic, seasonal coronavirus infection in respiratory secretions and were positive by pcr for the seasonal coronaviruses but not sars-cov-2. this group was important to include to establish the specificity of the igg elisa for sars-cov-2. the remaining 10 sera were from patients with igm antibodies to cytomegalovirus (cmv) or epstein-barr virus (ebv). this set of 39 sera was not "pristine" sera but reflective of the general population in that it included sera with potentially interfering antibodies to other infectious agents. of the 238 sera collected prior to 1 january 2020, 20 serum samples were from patients containing ige antibodies to common respiratory allergens, 10 were from individuals with positive antinuclear antibodies, and 9 were from individuals with igm antibodies to cmv or ebv. additional sera were obtained from healthy laboratory staff (n ϭ 9), pre-solid organ transplant waitlisted patients (n ϭ 55), hiv-positive persons (n ϭ 6), and a healthy platelet donor cohort (n ϭ 129). in total, three serum samples in this set of 277 sera had a positive result. all three were from samples collected prior to 1 january 2020. the estimated specificity of the assay was 98.9%. intra-assay precision (cv%), determined by performing the 10 replicate determinations of a positive sample, was 1.1%. the same sample was tested seven consecutive days and showed an interassay cv of 0.92%. statistical analysis. seroprevalence was estimated using the rogan and gladen estimator to adjust for assay sensitivity and specificity. direct standardization was used to estimate seroprevalence in the unc health population. confidence intervals were obtained using the nonparametric bootstrap percentile method. specifically, within each stratum of age group, gender, and race, sample seroprevalence was estimated and weighted by the stratum's proportion in the population. the rogan-gladen estimate can be negative, so all estimates of seroprevalence were constrained to be within the interval [0,1]. for comparison, demographic data for patients accessing the screening locations during the time period 15 february 2020 to 15 june 2020 were used. antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) antibody responses to sars-cov-2 in patients with covid-19 a new coronavirus associated with human respiratory disease in china presymptomatic ttransmission of sars-cov-2 -singapore potential presymptomatic transmission of sars-cov-2 asymptomatic cases in a family cluster with sars-cov-2 infection viral kinetics of sars-cov-2 in asymptomatic carriers and presymptomatic patients evidence of sars-cov-2 infection in returning travelers from wuhan, china presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility viral and host factors related to the clinical outcome of covid-19 clinical features of 69 cases with coronavirus disease seroprevalence of sars-cov-2-specific antibodies among adults in seroprevalence of immunoglobulin m and g antibodies against sars-cov-2 in china coast-to-coast spread of sars-cov-2 during the early epidemic in the united states performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in performance characteristics of four high-throughput immunoassays for detection of igg aantibodies against sars-cov-2 estimating prevalence from the results of a screening test seroprevalence of antibodies to sars-cov-2 in 10 sites in the united states severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients clinical and immunological assessment of asymptomatic sars-cov-2 infections sars-cov-2 seroprevalence and neutralizing activity in donor and patient blood from the san francisco bay area validation and performance comparison of three sars-cov-2 antibody assays we thank all the team members for their efforts and the participants of this study for their willingness to participate. we also thank the nurses and physicians who helped study enrollment.this project was funded by the unc school of medicine, unc health, and the university cancer research fund (ucrf). this work was also supported by public health service grants ca239583, ca016086, de02821, and es025124; the unc center for aids research (p30 ai050410); the unc lineberger cancer center (p30 ca016086); us epa (cr 83578501); and ul1tr002489 from the national center for advancing translational sciences (ncats) and from the national science foundation (grfp dge-1650116). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health.unc health data and further analysis were provided by marshall clark, james champion, kellie walters, and anna jojic at the north carolina translational and clinical sciences institute.the authors do not declare any conflicts of interest. key: cord-255883-mz6nyisw authors: asif, muhammad; saleem, mohammad; saadullah, malik; yaseen, hafiza sidra; al zarzour, raghdaa title: covid-19 and therapy with essential oils having antiviral, anti-inflammatory, and immunomodulatory properties date: 2020-08-14 journal: inflammopharmacology doi: 10.1007/s10787-020-00744-0 sha: doc_id: 255883 cord_uid: mz6nyisw coronavirus disease of 2019 (covid-19) has emerged as a global health threat. unfortunately, there are very limited approved drugs available with established efficacy against the sars-cov-2 virus and its inflammatory complications. vaccine development is actively being researched, but it may take over a year to become available to general public. certain medications, for example, dexamethasone, antimalarials (chloroquine/hydroxychloroquine), antiviral (remdesivir), and il-6 receptor blocking monoclonal antibodies (tocilizumab), are used in various combinations as off-label medications to treat covid-19. essential oils (eos) have long been known to have anti-inflammatory, immunomodulatory, bronchodilatory, and antiviral properties and are being proposed to have activity against sarc-cov-2 virus. owing to their lipophilic nature, eos are advocated to penetrate viral membranes easily leading to membrane disruption. moreover, eos contain multiple active phytochemicals that can act synergistically on multiple stages of viral replication and also induce positive effects on host respiratory system including bronchodilation and mucus lysis. at present, only computer-aided docking and few in vitro studies are available which show anti-sarc-cov-2 activities of eos. in this review, role of eos in the prevention and treatment of covid-19 is discussed. a discussion on possible side effects associated with eos as well as anti-corona virus claims made by eos manufacturers are also highlighted. based on the current knowledge a chemo-herbal (eos) combination of the drugs could be a more feasible and effective approach to combat this viral pandemic. [image: see text] the 2019 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has emerged as a new respiratory pathogen and is responsible for large-scale morbidities and mortalities around the globe. it is caused by a single positivestranded rna virus from the coronavirus (cov) family of coronaviridae (ludwig and zarbock 2020) . this family is composed of four genera out of which α-and β-cov can infect mammals including humans (ludwig and zarbock 2020) . these two strains are reported to be originated from rousettus leschenaultia, i.e. a bat species (lau et al. 2010; valencia 2020) . sars-cov-2 is identified as β-cov (valencia 2020) and is responsible for coronavirus disease 2019 . these viruses are wrapped in host cellsderived lipid membranes in which viral surface proteins are embedded. one of these surface proteins known as spike [s] protein protrudes out of membranes and give a characteristic crown/halo-like appearance to the virus when observed under the electron microscope hence, named coronavirus (latin word meaning: garland/crown) (ludwig and zarbock 2020) . once the virus gains entry into the respiratory tract, sars-cov-2 causes damage to epithelial cells of the airways making lungs unable to clear dirt and mucus which can lead to pneumonia. clinical symptoms of covid-19 include fever (approx. 99% of cases), chills, dry cough (observed in approx. 59% cases), sputum production (observed in approx. 27% cases), fatigue (observed in approx. 70% of cases), lethargy, arthralgia, myalgia (observed in approx. 35% cases), headache, dyspnoea (approx. in 31% of cases), nausea, vomiting, anorexia (approx. 40% cases), and diarrhoea (approx. 2% cases) (yang et al. 2020 ). in the extreme cases, patients experience a dramatic increase in the levels of pro-inflammatory chemokines and cytokines including il-6 and tnf-α, a condition known as "cytokine storm". this leads to the development of acute respiratory distress syndrome (ards), septic shock, metabolic acidosis, coagulation dysfunction, and even death (yang et al. 2020) . there is no clear, unified, and effective treatment plan for covid-19 but various approaches are being tried depending upon various sign and symptoms of individual patients. inhibition of virus entry into the host cell via affecting glycosylation of ace2 receptors in pulmonary cells as well as through inhibition of s protein priming and endocytosis (gao et al. 2020a; hoffmann et al. 2020) , blockage of rnadependent rna polymerase thus halting viral replication (gao et al. 2020b) , and increasing ph of pulmonary cells (alkalinisation) and endosomes thus disrupting viral replication as well as endosomes functions are among the few mechanisms through which currently studied drugs are known to act against sars-cov-2 (valencia 2020). use of convalescent plasma and il-6r blocker tocilizumab (a recombinant humanized anti-human il-6 receptor monoclonal antibody) either alone or in combination with different classes of drugs are also under clinical trials and have shown clinical improvements (girija et al. 2020; rothan and byrareddy 2020; yang et al. 2020) . currently, there is no vaccine available for sars-cov-2, but clinical trials are on the way to test the efficacies of multiple newly formulated vaccines; however, this will take some time to develop. an in vitro study conducted by hoffmann and colleagues revealed that sarc-cov-2 depends on cellular serine protease (tmprss2) for s proteins priming which are known to interact with human ace2 receptors in the lungs and facilitate entry into the cells. data of this study showed that blockage of tmprss2 by camostat mesylate inhibited infection of cells in in vitro assays. moreover, it was also found that sarc-cov-2 viruses also utilize endosomal cysteine proteases, cathepsin b and l (catb/l), for s protein priming (hoffmann et al. 2020 ). wang and colleagues used clinical isolates of sarc-cov-2 (2019-ncov) to evaluate the anti-sarc-cov-2 efficacy of five fda-approved drugs, i.e. ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine, and two well-known broad-spectrum antiviral drugs remdesivir (gs-5734) and favipiravir (t-705). african green monkey kidney cells (vero e6) were exposed to sarc-cov-2 in the presence of different concentrations of these drugs. rt-pcr techniques were used to quantify viral load by copy number estimation in the cell supernatants. among these drugs, remdesivir, an adenosine analogue and chloroquine, were found to be highly effective in blocking the infection of african green monkey kidney cells (vero e6, both drugs) and human lung cancer cells (huh-7, remdesivir only) by sarc-cov-2 (wang et al. 2020) . chloroquine (cq) and hydroxychloroquine (hcq) have been shown to block the release of the viral genome by inhibiting the transport of sars-cov-2 from early endosomes (ees) to endolysosomes (els). moreover, cq has been known to elevate the ph of the endosomes, thus halting endosomal maturation and ultimately failure in the transport and release of sarc-cov-2. hcq has also been proposed to halt the production of pro-inflammatory cytokines in covid-19 patients (liu et al. 2020) . essential oils (eos) are comprised of a complex mixture of volatile phytochemicals from diverse classes including monoterpenes, sesquiterpenes, and phenylpropanoids. numerous researchers have studied the antibacterial, antifungal, antioxidant, and antiviral properties of eos. these eos are found to be active against a wide variety of viruses, such as influenza virus (ifv), human herpesviruses (hsv), human immunodeficiency virus (hiv), yellow fever virus, and avian influenza (ma and yao 2020) . hsv (-1 and -2) are known to cause many life-threatening diseases in humans and are one of the major reasons for mortality in immunocompromised patients. hsv-1 is majorly responsible for hsv-induced lesions in the oral cavity and epidermis, while hsv-2 causes genital herpes, a sexually transmitted disease. an in vitro study conducted by schnitzler and colleagues showed that lemon balm oil inhibited plague formation of hsv-1 and hsv-2 viruses in a dose-dependent fashion. moreover, at higher concentrations it abolished the viral infectivity almost completely (schnitzler et al. 2008) . pre-treatment with eos obtained from illicium verum, melaleuca alternifolia, leptospermum scoparium, and matricaria recutita was found to inhibit the infective ability of acyclovir-sensitive and -resistant hsv stains, indicating the immense antiviral potential of eos (schnitzler et al. 2010) . anti-ifv properties of liquid and vapour forms of eos obtained from various plant species were studied using in vitro techniques. vapours of eos obtained from citrus bergamia, eucalyptus globulus, and their isolated compounds, i.e. citronellol and eugenol showed rapid anti-ifv actions. while in liquid form, eos obtained from cinnamomum zeylanicum, citrus bergamia, cymbopogon flexuosus, and thymus vulgaris showed better anti-ifv properties i.e. 100% inhibitory activity at 3.1 µl/ml as compared with others. vapour form of eos was also found to be safe against monolayers of epithelial cells. the study concluded that eos in vapour form could benefit people suffering from influenza (vimalanathan and hudson 2014) . carvacrol and its isomer thymol obtained from oregano have been shown to inhibit viral host cell fusion via depletion of viral cholesterol from the hiv-1 envelope membranes, thus blocking the entry of the virus into the host system (mediouni et al. 2020) . owing to the lipophilic nature of eos, these have the potential to intercalate into the lipid double layer of the viral envelope. subsequently, the fluidity of the membranes is changed and, at a higher concentration, the membranes are even ruptured (wink 2020) . major mechanisms through which eos induce antiviral actions are, direct actions on free viruses, inhibition of steps involved in virus attachment, penetration, intracellular replication, and release from host cells and inhibition of vital viral enzymes (ma and yao 2020; schnitzler et al. 2010) . keeping in view the diverse antiviral actions of eos, various claims were made by the eos manufacturers/suppliers as an effective therapy against covid-19. in this regard, research activities were conducted to check the anti-sarc-cov-2 efficacies of eos. the current review compiles available scientific information about the possible beneficial effects of eos in covid-19. the current review provides precise and comprehensive information on the essential oils and their possible contribution in the prevention and treatment of covid-19. all available information was collected via an electronic search of different scientific sources including pubmed (https ://www.pubme d.ncbi.nlm.nih.gov), sciencedirect (https ://www.scien cedir ect.com), google scholar (https ://www.googl escho lar.com), scientific electronic library online (scielo) (https ://www.sciel o.org), cochrane library (https ://www.cochr aneli brary .com/), and clinical trials database (https ://www.clini caltr ials.gov). the study database encompassed articles of peer-reviewed journals, books, thesis, dissertations, various patents, and supplementary reports covering anti-sarc-cov-2 properties of traditionally used essential oils. the authors opted the following keywords to find relevant studies: "essential oils", "antiviral", "covid-19", "sarc-cov-2", "bronchodilation", "immunomodulatory'', "anti-inflammatory'', "corona virus''. these terms were used alone or in combination using boolean operators ("and", "or", "not"). essential oils having scientifically established antiviral activities against sarc-cov-2 in in vitro, docking models, or in clinical settings were selected for reporting. essential oils having antiviral activities against other viruses and lacking any scientific evidence against sarc-cov-2 were excluded. enveloped viruses are known to respond sensitively to essential oils (schnitzler et al. 2010 ) which formed the basis of this work. essential oils obtained from eucalyptus (eucalyptus globulus) are traditionally used to treat various respiratory ailments including pharyngitis, bronchitis, and sinusitis. eucalyptus oil and its active constituent, 1,8-cineole have been shown to exhibit muscle relaxant effects by decreasing smooth muscle contractions of airways induced by different agents (bastos et al. 2009; coelho-de-souza et al. 2006) . moreover, clinical studies have indicated that inhalation of cineole (extracted from eucalyptus) exerted anti-inflammatory (by blocking cytokines release) and analgesic effects; hence, it can be effectively used in copd and asthmatic patients (juergens et al. 2020) . eucalyptus oil is reported to have in vitro antiviral activities against various strains of viruses including enveloped mumps viruses (mv) and herpes simplex viruses (hsv-1 and hsv-2) (lau et al. 2010 ). brochot and colleagues also reported the antiviral activities of eucalyptus oil and its active constituent, i.e. 1,8-cineole (eucalyptol) against influenza a (h1n1) virus in in vitro assays. both essential oil and 1,8-cineole were proposed to inactivate free influenza a virus and disrupt the envelope structures of virus (brochot et al. 2017 ). 1,8-cineole is also shown to protect mice against the hsv-2 virus (bourne et al. 1999) . having established the antiviral activity of eucalyptus oil and eucalyptol against respiratory viruses, multiple researchers have attempted to explore the antiviral efficacy of eucalyptus oil and its active ingredients against sarc-cov-2 using in vitro assays and molecular docking techniques. sharma and colleagues used molecular docking techniques to study the effects of jensenone, one of the active constituents of eucalyptus oil, on viral proteinase (mpro/3clpro). data obtained showed that jensenone formed complex with mpro via hydrophobic interactions with ala7, pro52, trp207, leu29, try126, and pro184; hydrogen bond interactions with m4, v18, l30, d10, and t16; and ionic interactions with lys3, asp34, arg38, and his163 (sharma and kaur 2020a). sharma and colleagues also predicted (preprints) the anti-proteinase efficacy of 1,8-cineole (eucalyptol), another active constituent of eucalyptus oil, using molecular docking techniques. data obtained showed that 1,8-cineole can bind with mpro and thus can inhibit viral reproduction. mpro/eucalyptol complexes were shown to form hydrophobic interactions, hydrogen bond interactions, and strong ionic interactions, respectively (sharma and kaur 2020b) . however, in vitro enzymes assays, and animal models are suggested to confirm the efficacy of jensenone/ 1,8-cineole against sarc-cov-2 proteinase. among these two compounds, 1,8-cineole is more extensively studied for its pharmacological potentials against various respiratory ailments (juergens et al. 2003) . 1,8-cineole (eucalyptol) is one of the components of vicks vaporub™ which is known to have nasal decongestant effects when applied to nose or inhaled as vapours in warm water. juergens and colleagues conducted a double-blind clinical trial to check the efficacy of 1,8-cineole in steroiddependent bronchial asthma patients. data of long-term studies showed 36% reduction of steroid dose in asthma patients receiving 1,8-cineole than placebo control group. 1,8-cineole was suggested to have profound bronchial antiinflammatory activity in severe asthmatic patients (juergens et al. 2003) . a recent review highlighted the favourable safety and efficacy profile of eucalyptol (1,8-cinoele) in numerous multi-centre, double-blinded, and randomized clinical trials conducted in germany in patients having acute and chronic respiratory conditions including rhinosinusitis, bronchitis, copd, and asthma, respectively (juergens et al. 2020) . a study conducted by merad and colleagues showed that almost all covid-19 positive patients have lung abnormalities. abnormal and overactive inflammatory responses to sars-cov-2 are proposed to be the major causes of disease severity and death in covid-19 patients. this hyper-inflammatory state is associated with increased levels of circulating cytokines, profound lymphopaenia, and substantial mononuclear cell infiltration in the lungs and other organs including heart, spleen, lymph nodes, and kidneys. the systemic cytokine profiles observed in patients showed increased production of cytokines such as il-6, il-7, and tumour necrosis factor (tnf) and many other pro-inflammatory cytokines (merad and martin 2020) . various in vitro and ex vivo studies were conducted to study the effects of eucalyptus oils and eucalyptol treatments on monocytes and macrophage recruitment in response to lung inflammation and infections. data of these studies demonstrate marked immunomodulatory properties of both eucalyptus oil and its active ingredient, i.e. eucalyptol. both treatments reduced the release of pro-inflammatory cytokines from monocytes and macrophages, but their phagocytic properties were not halted (juergens et al. 2020; sadlon and lamson 2010) . eucalyptol is also known to have mucolytic and bronchodilatory properties (juergens et al. 2020) . interestingly, eucalyptus oil has also been shown to have disinfection properties and inhibited the growth of viruses on various utensils and filter devices (usachev et al. 2013) . taken together, data from both preclinical and clinical trials point towards the promising therapeutic potential that resides in eucalyptus oil and its active constituent, i.e. eucalyptol in the prevention and treatment of covid-19. therefore, further studies are urgently warranted in this regard. garlic has been used as a medication to treat common cold, influenza, and other kinds of infections for centuries. garlic oil was chemically analysed by the gc-ms method and 18 compounds were identified, out of which allyl disulphide (28.4%), allyl trisulphide (22.8%), allyl (e)-1-propenyl disulphide (8.2%), allyl methyl trisulphide (6.7%), and diallyl tetrasulphide (6.5%) were identified as the main constituents of garlic essential oil. 17 compounds were studied for their activities against ace2 protein and viral main protease (mpro/6lu7) of sarc-cov-2. ace2 is involved in the viral invasion of host cells, while mpro is involved in viral replication. all the 17 compounds studied showed interactions with host protein (ace2) as well as with viral proteases, indicating that garlic oil has great potential to treat covid-19 patients (thuy et al. 2020) . virus-induced oxidative stress plays a critical role in the viral life cycle as well as in the pathogenesis of viral diseases. this leads to the activation of host antioxidant pathways including nuclear factor erythroid 2p45-related factor 2 (nrf2) (lee 2018) . nrf2 transcription factor is known to control the expression of various genes involved in antiviral actions (mccord et al. 2020) . a study conducted by mccord and colleagues showed that potent nrf2 activation by pb125 ® compound downregulated ace2 and tmprss2 mrna expression in human liver-derived hepg2 cells. both of these proteins are recognized to play a major role in the entry of sars-cov-2 into host cells. furthermore, treatment of primary human pulmonary artery endothelial cells with pb125 ® downregulated 36 genes controlling the expression of majority of cytokines identified in the "cytokine storm" during covid-19 severe cases. the authors suggested that nrf2 activation may significantly decrease the intensity of the cytokine storm in covid-19 patients (mccord et al. 2020) . diallyl sulphide (das), one of the active constituents of garlic, has been shown to induce nrf2 activation in lung mrc-5 cells. activated nrf2 after translocation into nuclei triggered p38/ erk-signalling pathways and is thus suggested to prevent oxidative stress-induced lung injury (ho et al. 2012; patel et al. 2018) . thus, on the basis of these docking and in vitro studies, it is proposed that garlic essential oils and their isolated constituents, especially das, have potential to prevent the entry of virus into host cells as well as to activate molecular antioxidant pathways that decrease the secretions of culprit pro-inflammatory cytokines. a study conducted by silva and colleagues screened the potencies of 171 essential oil components against different sarc-cov-2 proteins including main viral proteases (mpro), endoribonuclease (sars-cov-2 nsp15/nendou), adp-ribose-1-phosphatase (sars-cov-2 adrp), rnadependent rna polymerase (sars-cov-2 rdrp), spike proteins (sars-cov-2 rs), and human angiotensin-converting enzyme (hace2) protein using molecular docking techniques (silva et al. 2020 ). among the 171 screened compounds, (e,e)-α-farnesene, (e,e)-farnesol, and (e)-nerolidol showed better binding with sars-cov-2 mpro, indicating that these essential oil components when given alone and in a mixture can inhibit viral replication. non-structural protein 15 (nsp15), an endoribonuclease of sars-cov, is required for successful viral infection (bhardwaj et al. 2006) . (e,e)-αfarnesene, (e)-β-farnesene, (e,e)-farnesol, and (e)-nerolidol showed best binding scores; with nsp15. rna replication is catalysed by rna-dependent rna polymerase (rdrp) in rna viruses and is crucial step for viral replication; thus, making it a viable target for antiviral chemotherapy (shuai et al. 2006) . the best docking scores against rdrp were obtained for (e,e)-farnesol. the sars-cov-2 spike protein helps in the attachment of the viral cell to human cell via interaction with angiotensin-converting enzyme 2 (ace2) proteins present on host cells, making this interface a promising target to prevent binding of sars-cov-2 rs to human respiratory cells (zhang et al. 2020) . best binding with human ace2 was observed with α-bulnesene, eremanthin, (e,e)-α-farnesene, (e)-β-farnesene, (e,e)-farnesol, (e)-nerolidol, β-sesquiphellandrene, and (z)-spiroether, respectively. in case of sars-cov-2 spike proteins, comparatively better binding was observed with (e)-cinnamyl acetate, eremanthin, (e,e)-α-farnesene, (e)-β-farnesene, (e,e)-farnesol, and geranyl formate, respectively. overall, (e,e)-α-farnesene, (e)-β-farnesene, and (e,e)-farnesol showed better binding potentials with target proteins. these phytochemicals are present in variable quantities in essential oils obtained from different plants which can be used used to treat covid-19 but data from well-established preclinical and clinical studies is required. an in silico study conducted by kulkarni and colleagues evaluated the efficacy of a variety of eos present in diverse plant families to block the s1 (also called receptor binding domain, rbd) subunit of spike (s) proteins of sarc-cov-2. s1 protein is known to be involved in the interaction with host ace2 receptors. in silico study findings revealed that among the evaluated eos, anethole, cinnamaldehyde, carvacrol, geraniol, cinnamyl acetate, l-4-terpineol, thymol, and pulegone showed better potential to inhibit s1 subunit of s proteins. cinnamaldehyde was found to have more favourable binding properties as compared with other compounds (kulkarni et al. 2020 ). another molecular docking study conducted by elfiky evaluated the activity of cinnamaldehyde and thymoquinone against covid-19 and sars cov rna-dependent rna polymerases (rdrps). data obtained showed that both compounds have low binding affinities with rdrps (elfiky 2020). taken together, it is proposed that cinnamaldehyde may block the attachment of sarc-cov-2. further in vitro and in vivo studies, however, are required to establish this. the protective effects of cinnamaldehyde in lipopolysaccharide (lps)-induced acute lung injury (ali) mice model were evaluated by huang and colleagues. treatment with cinnamaldehyde was shown to markedly reduce lung wet/dry ratio and pulmonary oedema in mice. cinnamaldehyde also significantly inhibited neutrophils, macrophages, and total cell number in the bronchoalveolar lavage fluid. treatment with cinnamaldehyde decreased the levels of inflammatory cytokines such as tnf-α, il-6, il-13 and il-1β, respectively (huang and wang 2017) . this data along with findings of the above in silico studies give a clue about the possible beneficial role of cinnamaldehyde in covid-19, but detailed in vitro and in vivo studies are required to establish its efficacy. silva and colleagues used molecular docking techniques to screen the anti-sarc-cov-2 efficacies of eugenol, menthol, and carvacrol, major components of eos, against various proteins targets of sarc-cov-2. docking scores revealed that these compounds have binding affinities towards sarc-cov-2 spike protein, main protease (mpro), rna dependent rna polymerase and human ace-2 proteins, respectively (silva et al. 2020) . another in silico study conducted by kumar and colleagues evaluated the binding potential of carvacrol against sarc-cov-2 main protease (mpro) and showed that it has the potential to inhibit mpro and thus can halt viral replication (kumar et al. 2020) . plant extracts rich in menthol have been used in traditional medicine in asia for the treatment of respiratory ailments since centuries. menthol has been reported to provide symptomatic relief from nasal congestion associated with rhinitis and the sensation of dyspnoea associated with chronic obstructive pulmonary disease by its specific interaction with a cold-menthol-sensitive receptor (cmr1) located on trigeminal nerve endings (eccles 2003) . menthol has also been shown to have gastroprotective, anti-inflammatory, and immunomodulatory properties in rat models. treatment with menthol was found to significantly reduce the levels of pro-inflammatory cytokines, i.e. interleukin-1, interleukin-23, and tumour necrosis factor-α (tnf-α) in the treated rats (bastaki et al. 2018; rozza et al. 2014) . eugenol has been shown to have antiviral activities against hsv-1 and hsv-2, respectively (benencia and courrèges 2000) . besides, it has anti-inflammatory properties and has been shown to protect the lungs against lipopolysaccharide-(lps) induced acute injury. treatment with eugenol was also found to inhibit the recruitment of leukocytes into the lung and downregulated the expression of pro-inflammatory cytokines (il-6 and tnf-α) (barboza et al. 2018 ). an in vivo study conducted by games and colleagues evaluated the effects of three compounds including carvacrol in an elastase-induced pulmonary emphysema mice model (games et al. 2016) . results of the study showed that treatment with carvacrol reduced enlargement of alveoli, macrophages recruitment, and the levels of il-1β, il-6, il-8, and il-17 in the bronchoalveolar lavage fluid. the lung inflammation and emphysema were significantly less in the carvacrol-treated mice as compared with the disease control group. moreover, carvacrol has also been reported to have antiviral activities against hsv-1, acyclovir-resistant herpes simplex virus type 1, human respiratory syncytial virus (hrsv), and human rotavirus (rv) (kamalabadi et al. 2018; pilau et al. 2011) . in summary, data of in silico and in vivo animal models give a clue about the potential role of eugenol, menthol, and carvacrol in the treatment of covid-19 but further studies desinged to evaluate the anti-sarc-cov-2 efficacies of these eos are required. figure 1 depicts the effects of these discussed eos on the host respiratory system as well as on viral and hosts' pulmonary cells. after the emergence of shreds of preliminary scientific evidences about anti-sarc-cov-2 potentials of essential oils and their active components, various essential oils selling and extraction companies claimed about efficacy of their essential oils bearing products against covid-19. these claims were immediately noticed by the food and drug administration (fda) authority of usa and other fig. 1 the proposed anti-sarc-cov-2 actions of essential oils and their complementary effects on the human respiratory tract authorities, and warning letters were issued to the companies selling essential oils with these claims. a warning letter (marcs-cms 605752) was issued to a company by the center for drug evaluation and research, usa and was asked to withdraw the material about anti-corona efficacy of essential oils obtained from eucalyptus species, cinnamon, clove, frankincense, ginger, grapefruit, lemongrass, rosemary, tea tree, and lavender. another warning letter (marcs-cms 607753) was issued to a company claiming about immune-boosting and antiviral including anti-corona properties of a product named 'nobel laurel'. in addition to these sellers, fda has issued letters to various companies making false claims about their diagnostic products and other such materials (https ://www.fda. gov/consu mers/healt h-fraud -scams /fraud ulent -coron aviru s-disea se-2019-covid -19-produ cts). another issue associated with the use of essential oils is hypersensitivity reactions. essential oils containing pinene and linalool are known to cause wide variety of respiratory complications including seasonal asthma and rhinitis in allergic patients (gibbs 2019) . moreover, some individuals are sensitive/ allergic to specific components of eos and upon exposure may develop a wide range of allergic reactions including contact dermatitis (burfield 2000) . covid-19 has emerged as a very serious threat to global health. unfortunately, very few medications have been clinically shown to have efficacies against sarc-cov-2 and its inflammatory complications. drugs having different labelled uses are currently being tried in various combinations as supportive treatments. essential oils have long been known to have anti-inflammatory, antioxidant, immunomodulatory, and antiviral properties and are being proposed to have activity against sarc-cov-2. however, the existing information about these essential oils is very preliminary and the majority of claims are based on data obtained from computer-aided docking and preliminary in vitro studies. in this regard, well-planned in vitro and in vivo studies are warranted to establish the safe dose and clinical efficacy of essential oils against sarc-cov-2. moreover, keeping in view the multiple pharmacological attributes of essential oils, a combination approach whereby essential oils with established pharmacokinetic and pharmacodynamic properties are administered with synthetic drugs is suggested to combat this viral disorder and its associated complications. funding this research received no external funding. conflict of interest essential oil suppliers'/sellers' names mentioned in the manuscript were taken from fda letters and we do not intend to harm or damage their business repute. this review is purely for academic purposes. the authors declare no conflict of interest in the current work. an overview on the anti-inflammatory potential and antioxidant profile of eugenol menthol inhibits oxidative stress and inflammation in acetic acid-induced colitis in rat colonic mucosa inhibitory effect of 1,8-cineole on guinea-pig airway challenged with ovalbumin involves a preferential action on electromechanical coupling in vitro and in vivo activity of eugenol on human herpesvirus rna recognition and cleavage by the sars coronavirus endoribonuclease plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type 2 antibacterial, antifungal, and antiviral effects of three essential oil blends. microbiologyopen 6:e00459 safety of essential oils relaxant effects of the essential oil of eucalyptus tereticornis and its main constituent 1,8-cineole on guinea-pig tracheal smooth muscle menthol: effects on nasal sensation of airflow and the drive to breathe sars-cov-2 rna dependent rna polymerase (rdrp) targeting: an in silico perspective structurally related monoterpenes p-cymene, carvacrol and thymol isolated from essential oil from leaves of lippia sidoides cham. 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china angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-257105-vrwuaknf authors: davies, julie; randeva, harpal s.; chatha, kamaljit; hall, marcia; spandidos, demetrios a.; karteris, emmanouil; kyrou, ioannis title: neuropilin-1 as a new potential sars-cov-2 infection mediator implicated in the neurologic features and central nervous system involvement of covid-19 date: 2020-09-15 journal: mol med rep doi: 10.3892/mmr.2020.11510 sha: doc_id: 257105 cord_uid: vrwuaknf infection by the severe acute respiratory syndrome (sars) coronavirus-2 (sars-cov-2) is the cause of the new viral infectious disease (coronavirus disease 2019; covid-19). emerging evidence indicates that covid-19 may be associated with a wide spectrum of neurological symptoms and complications with central nervous system (cns) involvement. it is now well-established that entry of sars-cov-2 into host cells is facilitated by its spike proteins mainly through binding to the angiotensin-converting enzyme 2 (ace-2). preclinical studies have suggested that neuropilin-1 (nrp1), which is a transmembrane receptor that lacks a cytosolic protein kinase domain and exhibits high expression in the respiratory and olfactory epithelium, may also be implicated in covid-19 by enhancing the entry of sars-cov-2 into the brain through the olfactory epithelium. in the present study, we expand on these findings and demonstrate that the nrp1 is also expressed in the cns, including olfactory-related regions such as the olfactory tubercles and paraolfactory gyri. this furthers supports the potential role of nrp1 as an additional sars-cov-2 infection mediator implicated in the neurologic manifestations of covid-19. accordingly, the neurotropism of sars-cov-2 via nrp1-expressing cells in the cns merits further investigation. infection by the severe acute respiratory syndrome (sars) coronavirus-2 (sars-cov-2) is the cause of a new viral infectious disease, named as coronavirus disease 2019 (covid-19) (1). following the initial outbreak of covid-19 cases at the end of 2019, covid-19 has reached pandemic status within a few months with rapidly increasing rates of infection and mortality. thus, as of august 21st, 2020, over 21 million confirmed cases and over 760,000 deaths have been reported worldwide (2) . although covid-19 is asymptomatic or mild in most cases and manifests primarily as a lower respiratory tract infection which is transmitted mainly via air droplets (3) , emerging evidence indicates that sars-cov-2 can also invade and attack the central nervous system (cns) leading to a wide repertoire of neurological symptoms and complications (4) (5) (6) (7) . as such, increasing research focus has been placed on identifying the mediators that facilitate the sars-cov-2 infection in different human organs/tissues. it is now well-established that entry of sars-cov-2 into cells is facilitated by its spike proteins mainly through binding to the angiotensin-converting enzyme 2 (ace-2) (8, 9) . moreover, the sars-cov-2 spike viral proteins are primed/activated by the transmembrane protease serine 2 (tmprss2), which appears to also play a key role in this viral infection (8, 10, 11) . accordingly, there is now increasing interest in identifying additional molecular mediators that may also facilitate the sars-cov-2 infection of host cells and covid-19 symptoms, such as the receptor for advanced glycation end products (rage), angiotensin ii receptor type 2 (agtr2), cd147 and olfactory receptors (12) (13) (14) (15) . as such, recent data suggest that another protein/receptor termed neuropilin-1 (nrp1) may also be implicated in the sars-cov-2 infection (16) (17) (18) . nrp1 is a transmembrane receptor which, due to the lack of a cytosolic protein kinase domain, acts primarily as a co-receptor for various ligands and, thus, induces a multitude of effects, including cell proliferation, angiogenesis and axon control (19, 20) . as aforementioned, two preclinical studies have indicated that nrp1 may play a role as a new mediator of the sars-cov-2 infection (17, 18) . indeed, in the first of these studies, nrp1 was identified as a factor for sars-cov-2 infection, since, in addition to ace-2, the spike (s1) proteins of this coronavirus can also bind to nrp1 in vitro (18) . similar results were reported by a second study in which neuropathological analysis of covid-19 autopsies further revealed that sars-cov-2 infected nrp1-positive cells in the olfactory epithelium and bulb (17) . based on these findings, we hypothesise that nrp1 may be further implicated in the cns involvement and neurological features noted in patients with covid-19. to this aim, we explored in detail the expression of nrp1 in the human brain. to examine the detailed expression of nrp1 in the human brain, we employed a database which measured gene expression through single cell rna sequencing (rna-seq) and, thus, allowed to identify expression in cell types reported as fragments per kilobase of transcript per million mapped (fpkm) reads (21) . a second rna-seq dataset was also used to examine brain region specific gene expression using cap analysis of gene expression (cage) generated by the fantom5 project and reported as tags per million (22) (23) (24) . human protein expression of nrp1 in the brain was also extracted from the protein atlas database (proteinatlas. org) (23, 24) . visualisation of nrp1 expression in the brain was performed using the allen brain atlas with six human donors as assessed by microarray and presented as a heat map (25) . fig. 1a presents the single cell expression of nrp1 rna in specific cell types in the human brain. these data reveal that nrp1 expression is highest in mature astrocytes (5.8 fpkm), whilst also showing expression in endothelial cells (3.4 fpkm). moreover, fantom5 data analysis revealed nrp1 expression in the olfactory region (20.4 tags per million), which is higher than in other regions of the brain such as the cerebellum and thalamus (fig. 1b) . of note, highest expression of nrp1 was noted at both gene and protein level in the hippocampal formation (i.e., the region located in the temporal lobe of each cerebral cortex, medial to the inferior horn of the lateral ventricle) (fig. 1c) . to gain better insight, we further expanded our observations on single cell analysis of the hypothalamic arcuate and median eminence (arc-me) complex, using the single cell portal (26) . using t-distributed stochastic neighbor embedding (tsne), a machine learning algorithm for visualization, distinct cell sub-populations appear to express nrp1 ( fig. 2a and b ). more specifically, high expression was noted in endothelial cells, mural cells (vascular smooth muscle cells and pericytes), a specific cluster of neurons, perivascular macrophages (pvm) and microglia, as well as in vascular and leptomeningeal cells (vlmcs), as presented in fig. 2c . finally, nrp1 expression is also noted in the paraolfactory gyri of the human brain. indeed, six patient brains with global nrp1 expression are demonstrated in fig. 3 , as well as the olfactory specific regions of the brain; in humans the olfactory bulb inputs to the olfactory tubercle which in turn outputs to the limbic system (27, 28) . these data are displayed as heat maps with log 2 expression values presented in fig. 3 and table i , and show that nrp1 is expressed in the olfactory tubercles and paraolfactory gyri at reasonable levels in most brains. this study presents a detailed in silico analysis of the expression of nrp1 in the human brain, highlighting the potential role of nrp1 as an additional sars-cov-2 infection mediator in the cns via nrp1-expressing cells. our present findings further support the data of recent preclinical studies (16) (17) (18) , suggesting that nrp1 may be implicated in the neurologic features and cns involvement of covid19. it is known that human coronaviruses can lead to neurological sequelae, including vision alterations, seizures, motor coordination disturbances and a plethora of other neurological symptoms which may be evidenced with various imaging findings, including encephalopathy, oedema, haemorrhage and ischaemia (29-32). in accord with this, an increasing body of evidence now consistently indicates that sars-cov-2 infection may also be associated to a wide spectrum of neurological symptoms and complications from the cns (3-5,7). indeed, prominent neurologic features, including encephalopathy, agitation and confusion, as well as corticospinal tract signs, were present in a cohort of 58 consecutive patients (median age: 63 years) admitted to strasbourg university hospital with covid-19 related acute respiratory distress syndrome (ards) (33) . in addition, in this study single acute ischemic strokes were noted in two of the 13 patients who underwent magnetic resonance imaging (mri) of the brain. furthermore, in a retrospective case series study including 214 consecutive hospitalized patients (women/men: 127/87; mean age: 52.7 years) with laboratory-confirmed covid-19 from wuhan, china (the first epicentre of covid-19), almost half (45.5%) of the patients with severe covid-19 experienced impaired consciousness and other neurologic manifestations (e.g., acute cerebrovascular disease) (5) . overall, in this study neurologic symptoms were noted in more than one third (36.4%) of these 214 hospitalized patients with severe and non-severe covid-19. this observation is further supported by the findings of a more recent uk study which reported that patients with covid-19 may exhibit a wide spectrum of neurological syndromes, including encephalopathies with delirium/psychosis, cns inflammatory syndromes (e.g., encephalitis and encephalomyelitis) and stroke (34) . furthermore, a study from germany that reported the autopsy findings in six patients who died from covid-19 with sars-cov-2 pneumonia (women/men: 2/4, age range: 58-82 years) noted pronounced cns involvement with meningitis, panencephalitis and brainstem neuronal cell damage in all these cases (35) . of note, disruption to micro-structural and functional brain integrity in the recovery stages of covid-19 further suggests longer-term cns consequences/complications of sars-cov-2 infection (36). so far, it has been hypothesised that sars-cov-2 can enter into the cns through neural or haematogenous propagation, including through the olfactory epithelium of the nasal cavity and the olfactory bulb via retrograde transport along axons of olfactory sensory neurons, as well as via interaction with ace-2 of the capillary endothelium causing impairment of the blood-brain-barrier (bbb), or due to bbb disruption induced in the context of an underlying cytokine storm (5,37). in addition to these potential mechanisms, cantuti-castelvetri et al (17) recently showed that nrp1, which is highly expressed in the respiratory and olfactory epithelium, enhances sars-cov-2 entry into the brain, and that this can be inhibited by blocking nrp1 with a monoclonal antibody. interestingly, nrp1 has been shown to also play a role in mediating cell entry of other viruses, such as the epstein-barr virus (ebv) (38) . indeed, nrp1 interacts directly with the ebv glycoprotein b, whilst ebv infection is suppressed upon nrp1 knockdown (38) . given this newly identified role of nrp1 in enhancing sars-cov-2 entry into the cns, characterizing the precise expression of nrp1 in the human brain becomes important in the context of the neurologic involvement of covid-19. our present findings collectively show that nrp1 expression is present in the human brain, including olfactory regions, which corroborates the previously described data showing that nrp1 may mediate entry of sars-cov-2 into the brain through the olfactory epithelium/bulb (17) . this further highlights the potential importance of the olfactory tubercle for sars-cov-2 entry into the brain since in humans the olfactory bulb inputs to the olfactory tubercle which in turn outputs to the limbic system (27, 28) . finally, the parolfactory gyri which receive inputs from the olfactory bulb and provide input to the limbic system, also exhibit nrp1 expression, and so their potential involvement in the sars-cov-2 infection of the cns merits further research. in conclusion, in the present study we demonstrate that the nrp1, which is highly expressed in the respiratory and olfactory epithelium, is also expressed in the cns, including olfactory related regions such as the olfactory tubercles and paraolfactory gyri. given the data by cantuti-castelvetri et al (17) showing that nrp1 may mediate the entry of sars-cov-2 into the brain through the olfactory epithelium (17) , our findings warrant further investigation in order to elucidate the potential role of nrp1 as an additional sars-cov-2 infection mediator implicated in the neurologic manifestations of covid-19. indeed, the neurotropism of sars-cov-2 via nrp1-expressing cells in the cns should be investigated in preclinical and clinical studies, since clarifying how sars-cov-2 enters and spreads in the cns is vital for developing effective approaches to prevent this viral dissemination and its consequent complications. acknowledgements not applicable. funding no funding was received. all data generated or analysed during this study are included in this published article. jd, ek, ik consulted the literature, produced the figures and manuscript; mh and kc contributed to critical revision of the article; jd, hsr, ek, ik and das contributed to the writing of the manuscript and final edits. ik and ek contributed equally to the conception of the work, data and literature analysis as well as interpretation. all authors read and approved the final manuscript. jd is an alumnus of brunel university london. all authors read and approved the final manuscript. ethics approval and consent to participate not applicable. not applicable. das is the editor-in-chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article. the other authors declare that they have no competing interests. the four horsemen of a viral apocalypse: the pathogenesis of sars-cov-2 infection (covid-19). in: ebiomedicine world health organisation (who): coronavirus disease 2019 (covid-19): weekly epidemiological update organ-specific manifestations of covid-19 infection severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the central nervous system neurologic manifestations of hospitalized patients with coronavirus disease the covid-19 pandemic: consideration for brain infection neurological implications of covid-19 infections sars-cov-2 cell entry depends on ace2 and tmprss2 and is 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organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 neurologic features in severe sars-cov-2 infection zambreanu l, et al; ucl queen square national hospital for neurology and neurosurgery covid-19 study group: the emerging spectrum of covid-19 neurology: clinical, radiological and laboratory findings early evidence of pronounced brain involvement in fatal covid-19 outcomes xiao a, et al: cerebral micro-structural changes in covid-19 patients -an mri-based 3-month follow-up study quintero marzola id, ramos villegas y and moscote salazar lr: neurotropism of sars-cov 2: mechanisms and manifestations neuropilin 1 is an entry factor that promotes ebv infection of nasopharyngeal epithelial cells key: cord-266156-xmf4emln authors: miller, tyler e.; garcia beltran, wilfredo f.; bard, adam z.; gogakos, tasos; anahtar, melis n.; astudillo, michael gerino; yang, diane; thierauf, julia; fisch, adam s.; mahowald, grace k.; fitzpatrick, megan j.; nardi, valentina; feldman, jared; hauser, blake m.; caradonna, timothy m.; marble, hetal d.; ritterhouse, lauren l.; turbett, sara e.; batten, julie; georgantas, nicholas zeke; alter, galit; schmidt, aaron g.; harris, jason b.; gelfand, jeffrey a.; poznansky, mark c.; bernstein, bradley e.; louis, david n.; dighe, anand; charles, richelle c.; ryan, edward t.; branda, john a.; pierce, virginia m.; murali, mandakolathur r.; iafrate, a. john; rosenberg, eric s.; lennerz, jochen k. title: clinical sensitivity and interpretation of pcr and serological covid‐19 diagnostics for patients presenting to the hospital date: 2020-08-28 journal: faseb j doi: 10.1096/fj.202001700rr sha: doc_id: 266156 cord_uid: xmf4emln the diagnosis of covid‐19 requires integration of clinical and laboratory data. severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. our goal was to examine the clinical sensitivity of two most common sars‐cov‐2 diagnostic test modalities, polymerase chain reaction (pcr) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. we conducted a single‐center, retrospective study. to derive clinical sensitivity of pcr, we identified 209 pcr‐positive sars‐cov‐2 patients with multiple pcr test results (624 total pcr tests) and calculated daily sensitivity from date of symptom onset or first positive test. clinical sensitivity of pcr decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%‐71% from days 9 to 11, and 30% at day 21. to calculate daily clinical sensitivity by serology, we utilized 157 pcr‐positive patients with a total of 197 specimens tested by enzyme‐linked immunosorbent assay for igm, igg, and iga anti‐sars‐cov‐2 antibodies. in contrast to pcr, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after day 7, >80% after day 12, and 100% by day 21. taken together, pcr and serology are complimentary modalities that require time‐dependent interpretation. superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection. while many measures to mitigate the multifactorial impact of covid-19 are being implemented, one critical component of this strategy is the widespread testing and identification of individuals currently or previously infected by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the delivery of effective care and mitigation of infection depend on the performance of sars-cov-2 diagnostic testing and the clinical interpretation of results. the lack of a full understanding of the natural history and immunopathogenesis of covid-19 infection creates unique challenges in the implementation of diagnostic testing strategies. sars-cov-2 diagnostic assays have fixed technical performance metrics (eg, sensitivity and specificity). clinical sensitivity depends on more than technical performance and is also a function of pre-analytical variables and the disease state of the patient. interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. the goal of this study is to examine the clinical sensitivity and provide insights into the interpretation of the two most common sars-cov-2 diagnostic test modalities: polymerase chain reaction (pcr) and serology. laboratory-based diagnosis of active sars-cov-2 infection relies on the direct detection of virus-specific nucleic acids, most commonly obtained from the nasopharynx of infected patients. indirect markers of infection include the detection of sars-cov-2 specific antibodies, generated as part of the human immune response to the virus. serologic testing holds promise as a blood-based diagnostic aid, as a marker of viral exposure, and potentially as an indicator of protective immunity. understanding the presence of these biomarker in relationship to one another over the natural course of infection is required to effectively utilize these available diagnostic tests in clinical practice. [1] [2] [3] here, we share our experience of sars-cov-2 pcr sensitivity and separately obtained igm, iga, and igg sensitivity of an in-house enzyme-linked immunosorbent assay (elisa) during the natural course of disease in a cohort of patients presenting to the hospital. the project was conducted within the clinical laboratories of the massachusetts general hospital (mgh), a clinical laboratory improvement amendments-certified laboratory. the study was designed as a single-center, retrospective review of pcr results and serology data. pcr results were obtained between 3 march 2020 and 15 april 2020 and we superimposed serology data obtained from confirmed covid-19 positive patients as part of ongoing clinical validation studies of an elisa for regulatory approval. the study was conducted with approval from the mass general brigham institutional review board. we also used previously published data as a comparison dataset (wölfel et al 4 ). nucleic acid testing was performed as part of clinical care at mgh using three real-time pcr assays, each of which received eua by the fda. our laboratory-developed realtime pcr assay uses the centers for disease control and prevention (cdc) primers targeting regions of the n gene of sars-cov-2, the cobas sars-cov-2 test performed on the cobas 6800 (roche) targets regions of the orf1a and e genes, and the xpert xpress sars-cov-2 assay run on the genexpert infinity (cepheid) targets regions of the n and e genes. choice of which testing platform to use was determined by access to reagents available at the time of clinical testing provided for patient care. our laboratory-developed assay was validated to detect sars-cov-2 at or above 5 copies/µl with 100% technical sensitivity and specificity. for commercial assays, we internally validated the assays and found 100% technical sensitivity and specificity. within our validation cohort of known positive patients, we found 100% concordance between all three platforms. despite excellent (technical) performance characteristics, pre-analytical factors may decrease the performance of viral detection. these factors may include timing during the course of infection, improper sampling, specimen handling and others. an in-house elisa developed by massachusetts general hospital (boston, ma) and the ragon institute of mgh, mit, and harvard (cambridge, ma), was used to measure igg, iga, and igm antibodies that target the sars-cov-2 receptor binding domain (rbd) within the spike protein. the optical density (od) was read at 450 and 570 nm on a plate reader. od values were adjusted by subtracting the 570 nm od from the 450 nm od. to estimate antibody titers, we generated isotype-specific standard curves using anti-sars-cov-1/2 monoclonal igg, iga, and igm antibodies. we used this standard curve to calculate the concentration of anti-rbd igg, iga, and igm expressed in u/ ml. positive specimens were identified as those that had an u/ml three standard deviations above the mean of negative control specimens. (data not shown january 2020) when no detectable antibody responses would be expected. the date of symptom onset was determined by review of the electronic medical record by physician investigators. the onset date was determined in by one of two ways from the medical record: (a) as explicitly defined in the chart from an md-written note as "covid-19+ date of symptom onset" or (b) determined from md and non-md notes that stated date of symptom onset for any covid-19-related symptom that developed acutely and was new from baseline (fever, chills, loss of smell or taste, body aches, fatigue, runny nose, congestion, sore throat, cough, and shortness of breath). cases for which the date of symptom onset could not be determine were excluded from analysis (21/359, or 5.8%, of all pcr and serology cases). clinical laboratory test results are stored in a laboratory information management system connected to the electronic medical record. we performed two data queries with different end-dates: an initial pcr-query (3 march 2020 to 15 april 2020) and a second pcr-query (3 march 2020 to 4 may 2020). the initial pcr-query was performed to delineate clinical sensitivity over time, and analysis was restricted to patients with multiple pcr test results and at least one positive (ie, the most informative subset). these patients were considered confirmed covid-19 positive and taken as true positives. all pcr test results regardless of specimen type were used to confirm a patient as sars-cov-2 positive; however, only pcr test results from a np-swab specimen were used for sensitivity calculations. the resulting dataset consists of 624 pcr results from 209 unique subjects. in this subset, 83% were inpatients, 13% were patients from the ed, and 4% were outpatients. for each specimen, we manually mapped date of symptom onset and all test results on a daily scale and calculated: (a) the time (in days) from the date of symptom onset to the date of specimen collection, (b) the duration from the first positive pcr test result to any subsequent positive pcr test result, and (c) clinical detection rates (pcr-positive over total tests per day) at each day in relation to symptom onset or first pcr-positive, respectively. we modeled a linear daily regression trend after first positive pcr test, to estimate the time when pcr sensitivity reaches zero (foot-point analysis). the second pcr query was performed to capture hospital-wide testing metrics, and to assess whether the above subset analysis of sars-cov-2 patients with multiple pcr results is representative of the entire tested population in our setting. we extracted admission date, encounter, discharge date (when applicable), age, gender, and collection types and times, reporting dates and times along with results from all sars-cov-2 pcr tests. by clinical encounter, 55.5% of orders originated in the outpatient setting, 12.1% originated in the emergency department (ed), and 32.2% of orders originated from the inpatient setting. the overall pcr positivity rate was 27.0% (n = 3163/11 703) in unique individuals and 28.3% of all tests performed (n = 4320/15 251; table 1 ). all test results were used for calculations of test number over time, positivity rate and age as well as gender calculations ( figures s1 and s2) . to compare our data of mainly hospitalized patients to a population with mild disease, we used data derived from wölfel et al, 4 in which patients with a known exposure were instructed to present to the clinic at the first sign of symptoms. a positive pcr was necessary for inclusion into the study. we applied the validated limit of detection of our laboratory-developed assay (5 copies/µl) to the data derived from wölfel et al, 4 and used the same calculations for time-dependent clinical sensitivity for pcr. serologic analysis of igm, iga and igg status was performed in a subset of the above sars-cov-2 pcr-positive patients for which we had excess material in the mgh core laboratories for clinical validation studies. for each sample, we determined the days post symptom onset at the collection date and calculated daily sensitivity for each antibody isotype as well as detection rate of any isotype. although serology is currently considered a diagnostic aid (as opposed to a primary diagnostic test), we calculated sensitivity as the number of seropositive samples over the total number of tested samples from patients with the disease (ie, who tested positive by pcr). we plotted the sensitivity for both test modalities (pcr and serology) as percentages per overlapping 5-day leading intervals against the days since symptom onset. statistical analysis consisted of fisher's exact test (association of sars-cov-2 status with dichotomous factors), χ 2 with yates correction, or t test (comparison of means). sars-cov-2 viral rna levels decline over the course of infection. 4 this decline of rna levels clearly impacts the clinical sensitivity of pcr testing. it is not possible to determine the false negative rate of the pcr test from patients with a single pcr result. therefore, we identified patients | 5 with multiple pcr tests who had at least one positive test result (considered true positives). the resulting dataset is composed of 624 test results from 209 patients (6.6% of all pcr-positive patients, table 1 ). we compared this subset to all tested patients ( figure s1 , table s1 ) and contingency analysis of the multi-pcr vs. all single pcr-positive patient subset showed no significant differences in age, gender, and test type (table s1, figure s2 ). thus, we consider the multi-pcr subset demographically representative of our tested patient population. to derive clinical sensitivity over disease course, we needed to define an anchor point. we chose two different anchor points: symptom onset (subjective) and first positive test (objective). first, we examined the time course by anchoring test results to the day of symptom onset (figures 1 and s3) . clinical sensitivity remains above 90% for the first 5 days after symptom onset. in our cohort of patients presenting to the hospital, patients were admitted to the hospital a median of 8 days after symptom onset (interquartile range: 4-16 days; mean: 10 days), and between days 6 and 8, the clinical sensitivity of pcr ranged from 84% to 76%. on subsequent days the sensitivity decreases, and at day 18 the sensitivity decreases below 50%. we also compared sensitivity data from an earlier study of mildly symptomatic patients 4 and noted a steeper pcr sensitivity decline, consistent with viral levels dropping more quickly in this population ( figure s4 ). this data provide sensitivity estimates at the time of presentation (eg, a patient presents at day 10 after symptom onset). second, we also modeled how pcr sensitivity decreases over time after the first positive pcr test (figures s3 and s5 ). regression modeling and extension of pcr positivity decay (foot-point analysis) revealed that in our cohort, npswab specimens could stay pcr-positive beyond 20 and up to 40 days ( figure s5 ). seroconversion is also a dynamic response to the virus and assay sensitivity changes over time. to assess the sensitivity of our serology assay over time, we tested for igm, igg, and iga antibodies against the rbd of sars-cov-2 spike protein in 157 sars-cov-2 pcr-positive patients using an in-house elisa (table 1) . for some patients, we were able to assess serology at multiple time points (n = 591 total isotype tests on 197 total specimens). anchoring our serologic results to days after symptom onset shows that seroconversion starts as early as symptom onset, is detectable in 50% of subjects after day 7, and continues to increase with >80% of patients showing seropositivity after day 12 (tables 2 and s2 ; figures 1 and s4 ). in the subset of patients with multiple serological tests we saw isotype switching occur as short as 1-4 days, consistent with recent reports. 5 of note, we detected iga prior to igm or igg in a number of individuals, with two subjects having only detectable iga within 3 days of symptom onset. we also documented cases of igg positivity prior to igm or iga, highlighting the utility of measuring seroconversion using all three isotypes (table s3 , figure s6 ). the superimposition of serologic sensitivities with pcr sensitivities shows that, after day 7, seroconversion is a reliable diagnostic aid indicating recent or prior infection (figure 1 ). we present the dynamic clinical sensitivity of sars-cov-2 pcr and our serology platform in patients presenting to the hospital. we performed this single-center, retrospective analysis to share real-world evidence that can inform interpretation of pcr and serologic testing. we focused on assessing the clinical utility of both modalities in conjunction by direct superimposition of both sensitivity time courses. the direct superimposition shows that serology can function as a reliable diagnostic aid indicating recent or prior infection-in particular at times when pcr sensitivity is lower than 70%. our findings emphasize that understanding the specific sensitivity kinetics of both modalities is paramount for interpretation and effective utilization of sars-cov-2 diagnostics. there is considerable interest in moving sars-cov-2 diagnostics to evidence-based principles. [6] [7] [8] [9] while clinicians await formal guidance from large, prospective, multi-center studies-which will be challenging during the ongoing pandemic-there is considerable uncertainty surrounding sars-cov-2 diagnostics in clinical practice. 3,10-13 using available published data [3] [4] [5] [6] [7] 12, 14 and data presented here from our hospital, we offer the following five diagnostic principles for consideration: 1. in symptomatic patients, all interpretations are anchored on days post symptom onset. understanding performance characteristics of sars-cov-2 diagnostics over the course of the infection is key to interpretation of results. we provide two distinct approaches to anchor interpretation over the course of infection: subjective (date of symptom onset) and objective (first pcr-positive result). both approaches are valid and have limitations. for example, the quality of patient histories is variable and in many cases the day of symptom onset is unknown or cannot easily be reconstructed. as a practical suggestion, to make this data easily obtainable and searchable, we recommend placing the date of symptom onset (when available) in the front page of the (electronic) medical record of patients diagnosed with covid-19. 2. pcr is the diagnostic gold standard during acute infection. pcr testing using consensus primers has an estimated specificity of >99%. 15 based on early reports from wuhan 16 the overall clinical sensitivity is reported around 70%. we found a clinical sensitivity around 95% in the first 5 days after symptom onset and although pcr is an imperfect standard, concurrent igm/iga/igg antibody assessment in the first 5 days post symptom onset does not significantly aid in rendering a current diagnosis; at no point during active infection should serology replace pcr for diagnosis. 3. clinical sensitivity of pcr decreases with days post symptom onset. in clinical practice many symptomatic patients present to medical care after day 1 of symptom onset. our data show pcr sensitivity decreases with days post symptom onset ( figure 1 ) and with days post first pcr-positive test result ( figure s5) . notably, some patients may have an initial pcr-negative result at presentation ( figure s3 ). our data also indicate that severely ill patients (many patients in our cohort) remain pcrpositive for a longer period than mildly ill patients (patients in the wölfel et al 4 cohort figure s4 ). both time since symptom onset and disease severity may be key elements for the interpretation of pcr results. 4. serological assay sensitivity increases with days post symptom onset. by positivity alone, in our cohort, seropositivity surpasses pcr positivity after days 8-10 post symptom onset. remarkably, we find seroconversion does not follow the typical kinetics of igm antibodies followed by class-switched igg and iga antibodies. rather, all appear simultaneously at a cohort level, with igg or iga seropositivity preceding igm responses in some cases. supporting this are other studies that report overall low igm responses to sars-cov-2 that are often preceded by igg. 14, 17 these data highlight the benefit of measuring all three anti-sars-cov-2 antibody isotypes to maximize sensitivity. in the case of sars-cov-2, it is unknown for how long igm, iga, or igg antibodies remain detectable after infection. it is important to note that a positive serologic result for igm, iga, and/or igg does not conclusively indicate that a patient's presenting symptoms are due to a current sars-cov-2 infection. distinguishing a prior versus an acute infection by serology will require isotype-specific interpretation, serial serologic testing to demonstrate seroconversion, and integration with clinical data. 5. negative results do not completely preclude sars-cov-2 infection. ruling out sars-cov-2 infection remains challenging. supplementing pcr results with serologic assessment can increase sensitivity (serology as a diagnostic aid). however, our data clearly show that there is a window period (day 6-12 from symptom onset) when clinical sensitivity of pcr and serological assays are below 90%. in a symptomatic patient, if multiple pcr tests are negative and serological results after 8-12 days are also negative, we believe the likelihood of active sars-cov-2 infection to be low. clinical judgment is needed in this situation as there are rare scenarios where pcr negativity may be due to disease at a different anatomic site and/or serologic negativity may be due to an immunocompromised state. limitations in our study include relatively small numbers, a retrospective design, and selection bias due to the specific setting and testing practice. we evaluated symptomatic, mostly hospitalized patients and we cannot derive recommendations for asymptomatic or mildly symptomatic patients from our data. due to limited availability of tests and time constraints during an ongoing pandemic, we did not perform daily sampling. date of symptom onset is not consistently available, subjective, and affected by recall biases-yet, it represents a useful anchor point for disease time course in symptomatic patients. notably, the eclipse period ranges from 2 to 14 days 18-21 and some patients already mounted a serologic response at the time of presentation, which can be taken as an argument for the early y-axis deviation from zero in the serology curve ( figure 1 ) and a confirmation of date of symptom onset as an imperfect marker. we caution that the presented serology data are specific to our elisa, and we cannot extrapolate to anti-sars-cov-2 antibody responses in general. nonetheless, other publications indicate that the time courses are comparable. 14, 17, 22 our serological studies measured antibodies to the rbd of sars-cov-2. we chose this viral antigen because of its specificity to sars-cov-2, and because anti-rbd antibodies are typically neutralizing. plaque reduction neutralization tests are the gold standard for assessing neutralizing ability, [23] [24] [25] [26] and ongoing studies are in progress to confirm anti-rbd antibodies are neutralizing in sars-cov-2 infection. 27 some commercially available assays measure the more abundant nucleocapsid protein, which may increase sensitivity to detect a serologic response early in the course of infection, therefore, shifting the seroconversion curve to the left. however, antibodies to nucleocapsid protein are unlikely to provide protective immunity as nucleocapsid protein is inaccessible to antibodies in an intact virus. therefore, serology results also depend on the specific antigen employed in testing. finally, our multi-pcr cohort and serologic patient cohort are largely non-overlapping pcrpositive patients (n = 20/209). to enable additive sensitivity calculations from combined pcr and serology assays, prospective and systematically obtained repeated parallel pcr and quantitative serologic data will be necessary. our real-world data outline the strengths and weaknesses of two sars-cov-2 test modalities over the natural course of infection. we hope these data, in conjunction with the five diagnostic principles for consideration, will contribute to effective utilization and interpretation of covid-19-related laboratory data for patient care. diagnostic testing for severe acute respiratory syndrome-related coronavirus 2: a narrative review profiling early humoral response to diagnose novel coronavirus disease (covid-19) interpreting diagnostic tests for sars-cov-2 virological assessment of hospitalized patients with covid-2019 temporal dynamics in viral shedding and transmissibility of covid-19 evidence based management guideline for the covid-19 pandemic -review article covid-19 diagnosis and management: a comprehensive review laboratory diagnosis of covid-19: current issues and challenges covid-19 diagnostics in context sars-cov-2 serology: test, test, test, but interpret with caution covid-19 pandemic-a focused review for clinicians multitiered screening and diagnosis strategy for covid-19: a model for sustainable testing capacity in response to pandemic waiting for certainty on covid-19 antibody tests -at what cost? antibody responses to sars-cov-2 in patients with covid-19 us cdc real-time reverse transcription pcr panel for detection of severe acute respiratory syndrome coronavirus 2 detection of sars-cov-2 in different types of clinical specimens profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data antibody responses to sars-cov-2 in patients of novel coronavirus disease potent neutralizing antibodies from covid-19 patients define multiple targets of vulnerability identification of a critical neutralization determinant of severe acute respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model potent neutralization of sars-cov-2 by human antibody heavy-chain variable domains isolated from a large library with a new stable scaffold. mabs potent human neutralizing antibodies elicited by sars-cov-2 infection the authors thank all patients and laboratory staff for making this study possible. cr3022 reference sequences, cr3022-iga and cr3022-igm isotypes, and elisa protocols were kindly provided by stephanie fischinger, caroline atyeo, and matthew slein from the laboratory of dr galit alter (ragon institute). we thank yael heher md for excellent discussions. elisa protocols were developed and optimized with the laboratory of dr alejandro balazs (ragon institute). this work is also in part supported by nih (ro1 ca225655) to jkl, centers for disease control and prevention u01ck000490 (etr, rcc), nigms training grant t32 gm007753 (bmh, tmc), nih r01 ai146779 (ags). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institute of health or any other organization. dr gelfand reports personal fees from henry schein inc, outside the submitted work; dr turbett reports grants from centers for disease control and prevention, outside the submitted work; dr anahtar reports personal fees and other from day zero diagnostics, outside the submitted work; dr ryan reports grants from cdc, during the conduct of the study. dr. branda reports grants from zeus scientific, grants from biomerieux, grants from immunetics, personal fees from t2 biosystems, personal fees from diasorin, personal fees from roche diagnostics, grants from bay area lyme foundation, grants from lyme disease biobank foundation, outside the submitted work. key: cord-253457-gawn4s9g authors: yau, kevin; muller, matthew p.; lin, molly; siddiqui, naureen; neskovic, sanja; shokar, gagan; fattouh, ramzi; matukas, larissa m.; beaubien-souligny, william; thomas, alison; weinstein, jordan j.; zaltzman, jeffrey; wald, ron title: covid-19 outbreak in an urban hemodialysis unit date: 2020-07-15 journal: am j kidney dis doi: 10.1053/j.ajkd.2020.07.001 sha: doc_id: 253457 cord_uid: gawn4s9g rationale & objective: hemodialysis patients are at increased risk for covid-19 transmission due, in part, to difficulty maintaining physical distancing. our hemodialysis unit experienced a covid-19 outbreak despite following symptom-based screening guidelines. we describe the course of the covid-19 outbreak and the infection control measures taken for mitigation. study design: retrospective cohort study setting & participants: 237 maintenance hemodialysis patients and 93 hemodialysis staff at a single hemodialysis centre in toronto, canada. exposure: universal screening of patients and staff with severe acute respiratory syndrome coronavirus 2 (sars-cov-2). outcomes: the primary outcome was detection of sars-cov-2 in nasopharyngeal samples from patients and staff using reverse transcriptase-polymerase chain reaction (rt-pcr) . analytical approach: descriptive statistics were used for clinical characteristics and the primary outcome. results: eleven (4.6%) of 237 hemodialysis patients and 11 of 93 (12%) staff members had a positive rt-pcr test for sars-cov-2. among individuals testing positive, 12 of 22 (55%) were asymptomatic at time of testing, and 7 of 22 (32%) were asymptomatic for the duration of follow-up. one patient was hospitalized at the time of sars-cov-2 infection and four additional patients with positive tests were subsequently hospitalized. two patients (18%) required admission to the intensive care unit. after 30 days follow-up no patients had died or required mechanical ventilation. no hemodialysis staff required hospitalization. universal droplet and contact precautions were implemented during the outbreak. hemodialysis staff with sars-cov-2 infection were placed on home quarantine regardless of symptom status. patients with sars-cov-2 infection including asymptomatic individuals were treated with droplet and contact precautions until confirmation of negative sars-cov-2 rt-pcr testing. analysis of the outbreak identified two index cases with subsequent nosocomial transmission within the dialysis unit and in shared shuttle buses to the hemodialysis unit. limitations: single centre study. conclusions: universal sars-cov-2 testing and universal droplet and contact precautions in the setting of an outbreak appeared to be effective in preventing further transmission. hemodialysis patients are at increased risk for covid-19 transmission due, in part, to difficulty maintaining physical distancing. our hemodialysis unit experienced a covid-19 outbreak despite following symptom-based screening guidelines. we describe the course of the covid-19 outbreak and the infection control measures taken for mitigation. study design: retrospective cohort study. setting & participants: 237 maintenance hemodialysis patients and 93 hemodialysis staff at a single hemodialysis centre in toronto, canada. exposure: universal screening of patients and staff with severe acute respiratory syndrome coronavirus 2 (sars-cov-2). outcomes: the primary outcome was detection of sars-cov-2 in nasopharyngeal samples from patients and staff using reverse transcriptase-polymerase chain reaction (rt-pcr) . analytical approach: descriptive statistics were used for clinical characteristics and the primary outcome. conclusions: universal sars-cov-2 testing and universal droplet and contact precautions in the setting of an outbreak appeared to be effective in preventing further transmission. eleven of 237 (4.6%) hemodialysis patients and 11 of 93 (12%) of staff tested positive for covid-19. notably 55% of those testing positive were asymptomatic at the time of testing. this study demonstrates the importance of universal testing in stopping the spread of covid-19 during an outbreak. the coronavirus disease 2019 (covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has prompted widespread restrictions on ambulatory inperson healthcare encounters. however, patients with kidney failure who receive maintenance hemodialysis must continue to receive life-sustaining treatment, typically three times per week. 1 hemodialysis attendance, including travel to and from the centre, entails close interaction with individuals who may be infected with sars-cov-2. 2 concerns regarding viral acquisition are heightened by the fact that hemodialysis recipients have multiple risk factors for severe covid-19. 3 the us centers for disease control and prevention and the american society of nephrology have issued interim guidance to prevent covid-19 in outpatient hemodialysis units including screening protocols to identify symptomatic patients or healthcare workers. 4 however, a recent outbreak at a skilled nursing facility has led to increasing recognition of the role of asymptomatic individuals in disease transmission. 5 we report the dynamics and course of a recent covid-19 outbreak affecting patients and staff at an urban hemodialysis unit. st. michael's hospital is an academic medical centre in toronto, canada, where 240 patients receive maintenance hemodialysis. the hemodialysis unit is divided into two large rooms on the same floor down the hall from each other. each room is further subdivided into three clusters of 4-8 dialysis stations referred to as "pods". hemodialysis staff are assigned to work with patients in a specific pod although they may assist patients in other pods. hemodialysis patients typically dialyze three times a week on a morning, afternoon, evening, or overnight shift. prior to the outbreak, physical distancing was implemented in the waiting room and two layers of pre-screening for symptoms were conducted prior to dialysis: the first by telephone on the day prior to the scheduled dialysis session and the second, following the patient's arrival in the dialysis unit waiting area. dialysis pre-screening involved recording tympanic temperature, this study was approved by the unity health research ethics board. patient and staff consent was waived due to infection control measures with the exception of the two covid-19 patients admitted to the intensive care unit from whom informed consent was obtained. the reporting of this study followed the strengthening the reporting of observational studies in epidemiology (strobe) guidelines. 6 two patients were diagnosed with covid-19 on april 7, 2020. the detection of three additional cases on april 9, 2020 led to the declaration of an outbreak. investigation efforts were undertaken by an outbreak management team that was led primarily by the hospital infection prevention and control (ipac) team in collaboration with public health authorities, and the hemodialysis unit. between april 11, 2020 and april 22, 2020, all remaining patients and staff who interacted with hemodialysis patients were tested for sars-cov-2 by nasopharyngeal swabs. sars-cov-2 nasopharyngeal swabs were collected by personnel who had received instruction in the proper technique. nasopharyngeal swabs were performed by physicians, nurse practitioners, and staff from the hospital's covid-19 assessment centre under droplet and contact precautions in the hemodialysis unit with curtains drawn around the dialysis station at which the patient was being swabbed. sars-cov-2 testing was performed with the nuclisens easymag extractor/abi quantstudio5 platform using the altona realstar sars-cov-2 rt-pcr kit 1.0. hemodialysis unit staff who were sent for screening included physicians, nurse practitioners, nurses, dialysis support assistants, medical imaging technologists, allied health, porters, and environmental services staff. universal droplet and contact precautions including gloves, face shields, surgical masks, and isolation gowns were initiated on april 9, 2020 on the dialysis shift with known affected patients and expanded to the entire dialysis unit on april 10, 2020. contact tracing was a joint undertaking led by ipac and the hospital's occupational health department, with ipac taking the primary lead for patient contact tracing and occupational health for staff contact tracing. public health authorities conducted contact tracing for family members and community contacts. this included symptom screening of contacts and ongoing monitoring for 14 days post-exposure. all symptomatic contacts were referred for testing but asymptomatic household contacts were not routinely tested as per public health protocols at the time. hemodialysis staff testing positive for covid-19 had information recorded regarding dates worked, symptoms, date of symptom onset, duration of symptoms, locations worked while symptomatic, locations worked during 48 hours to 2 weeks prior to symptom onset, personal protective equipment usage, and recent contacts including patient interactions. universal sars-cov-2 testing, clinical characteristics and outcomes among 237 hemodialysis patients who agreed to testing (99%), 11 (4.6%) tested positive for sars-cov-2, while 11 of 93 (12%) staff tested were found to be positive (figure 1) . at the time of testing, six (55%) patients and six (55%) staff positive for sars-cov-2 were asymptomatic. three patients (27%) and four staff (36%) remained asymptomatic for the entire duration of follow-up (table 1) . among the 11 patients with covid-19 the median age was 66 (iqr, 63-72), six (55%) were male, and seven (64%) were dialyzed on the same shift ( table 2) . sharing the shuttle bus with other infected patients. these two patients took the same shuttle bus service to the same hemodialysis shift as other infected patients but dialyzed in different parts of the hemodialysis unit, suggesting that they acquired covid-19 outside of the hemodialysis unit. response to the outbreak following declaration of the outbreak, additional infection control measures were implemented. droplet and contact precautions were mandated for all patient contact until outbreak resolution. sars-cov-2-positive patients were cohorted in a dedicated waiting room that was subjected to thorough cleaning after the patient's departure. the number of environmental services staff was escalated to increase the frequency of unit cleaning. "safety coaches" were deployed to the hemodialysis unit to provide feedback to staff regarding proper usage of personal protective equipment. all inpatients were dialyzed in their hospital room regardless of covid-19 status. porters were required to utilize a face shield and mask when transporting dialysis patients, and universal masking of patients was implemented. patient movement between dialysis shifts was restricted and extra dialysis sessions (e.g. a saturday session for a patient who normally dialyzes on monday, wednesday, and friday) were put on hold in order to limit a given patient's exposure to additional cohorts of patients. patients with confirmed sars-cov-2 infection including asymptomatic individuals were dialyzed in a dedicated room separate from the main hemodialysis unit for the duration of their infection and maintained on droplet and contact precautions. repeat testing was performed following symptom resolution and a minimum of 14 days from symptom onset. two negative sars-cov-2 nasopharyngeal swabs within a 24 hour period were required prior to the patient being allowed to return to his/her regular station in the dialysis unit. among the six patients with a persistently positive sars-cov-2 nasopharyngeal swab, five (83%) were hospitalized ( table 3) . hemodialysis staff with confirmed sars-cov-2 including those who were asymptomatic, were asked to self-isolate at home. five hemodialysis staff were allowed to return to work following symptom resolution and documentation of two negative sars-cov-2 nasopharyngeal swabs performed 14 days from symptom onset. the return to work policy for hemodialysis staff was revised by ipac on may 7, 2020 to no longer require repeat sars-cov-2 testing. following this change in policy, the remaining six hemodialysis staff with sars-cov-2 infection were allowed to return to work 14 days from symptom onset assuming symptoms had resolved, without demonstration of a negative sars-cov-2 nasopharyngeal swabs. the outbreak was declared resolved on may 10, 2020 by ipac on the basis of no new cases being detected in the hemodialysis unit over a 14 day period. since the outbreak was declared over, only individuals who reported symptoms during the pre-dialysis screening process were tested. no additional patient or staff cases have been identified as of june 19, 2020. although droplet and contact precautions were rescinded in the hemodialysis unit, masks remain mandatory throughout the hospital and face shields must be worn by all hemodialysis staff in the course of patient care. infection control authorities concluded that sars-cov-2 transmission during an outbreak at the st. michael's hospital hemodialysis unit was likely to have originated from two index cases. patient #1 acquired the virus through an outbreak at a skilled nursing facility and hemodialysis staff #1 likely acquired the virus in the community. subsequent transmission likely occurred from patient-patient interactions or indirectly through staff. later transmission likely occurred through a shared bus shuttle service to and from dialysis despite implementation of universal droplet and contact precautions within the hemodialysis unit. our hemodialysis unit is divided into a two separate rooms, each which is further subdivided into three clusters of dialysis chairs referred to as pods. dates on the arrows reflect the day of hypothesized transmission. mitigating risk of covid-19 in dialysis facilities clinical characteristics of and medical interventions for covid-19 in hemodialysis patients in wuhan mild or moderate covid-19 covid-19 and dialysis units: what do we know now and what should we do? asymptomatic transmission, the achilles' heel of current strategies to control covid-19 strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies maintenance hemodialysis and coronavirus disease 2019 (covid-19): saving lives with caution, care, and courage epidemiology of covid-19 in an urban dialysis center serologic detection of sars-cov-2 infections in hd centers population false negative tests for sars-cov-2 infection -challenges and implications acknowledgements: we would like to thank all hemodialysis staff at st. michael's hospital for their dedication to patient care during the covid-19 pandemic.peer review: received may 25, 2020. evaluated by 2 external peer reviewers, with direct editorial input from a statistics/methods editor, an associate editor, and the editor-in-chief.accepted in revised form july 2, 2020. key: cord-029547-9ei1ram3 authors: li, jingwei; shao, jun; wang, chengdi; li, weimin title: the epidemiology and therapeutic options for the covid-19 date: 2020-05-28 journal: precis clin med doi: 10.1093/pcmedi/pbaa017 sha: doc_id: 29547 cord_uid: 9ei1ram3 an outbreak of coronavirus disease 2019 (covid-19), a disease caused by a novel pneumonia virus, has affected over 200 countries and regions worldwide. with the increasing number of patients and deaths, who have declared it as a global pandemic currently, indicating a third large-scale epidemic coronavirus has appeared since the emergence of severe acute respiratory syndrome coronavirus (sars) and middle-east respiratory syndrome (mers) in the twenty-first century. considering the great harm it has caused, researchers throughout the world have been chasing to exploit the pathophysiology, characteristics, and potential remedies for covid-19 to better battle the outbreak. therefore, the current study revisits advances of the virology, epidemiology, clinical features, therapeutic options, and prevention of covid-19. the features of asymptomatic carriers are also been explored. at the end of 2019, a sudden outbreak of pneumonia occurred in wuhan, china, which was later confirmed to be caused by a coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2). 1,2 soon after, the virus spread rapidly around the world and affected human health as well as causing huge economic losses to society. [3] [4] [5] on 12 february 2020, this novel disease was named coronavirus disease 2019 (covid-19) by who. 6 since then, medical experts around the world have been trying to find the control measures and treatment for it. because covid-19 is highly contagious, the number of patients has increased rapidly. thus, realizing its basic characteristics and finding appropriate methods to detect and cure covid-19 is necessary for outbreak control. the detection and rehabilitation judgment of covid-19 are mainly through reverse transcriptionpolymerase chain reaction (rt-pcr), 7 but experts have pointed out the low sensitivity will lead to missed diagnosis. so a combination of rt-pcr and other clinical features of covid-19 is important for diagnosis. 8 the treatments for covid-19 are still unclear and mainly contain administration of oxygen, drug therapy, and emergency treatments such as ecmo. 9 ,10 as we know, few studies have summarized and classified therapies for covid-19 systematically. therefore, we review basic characteristics such as virology and epidemiology of covid-19 and classified explore antiviral therapy, symptomatic treatment, and traditional chinese medicines for curing covid-19. considering no licensed vaccine exists for covid-19, we also summarize the research and development of its vaccine. notice that the recent emergence of asymptomatic carriers has brought great difficulties for prevention and control of this disease, 11 we highlight the characteristics of asymptomatic carriers and how to reduce their impact on the development of the covid-19 pandemic. in january 2020, after the outbreak of covid-19, scientists identified its pathogen and immediately confirmed it to be a novel coronavirus, which is found to be a β coronavirus of group 2b and was named 2019-ncov by who initially. 2 because 70% of its genetic sequences are similar to the sars-cov, it was later renamed sars-cov-2. 12 the origin of sars-cov-2 is unclear and is thought to be related to bat coronaviruses. zhou et al. compared gene sequence of sars-cov-2 with ratg13, a bat coronavirus, and found the whole-genome sequence of sars-cov-2 is 96.2% similar to ratg13, indicating that sars-cov-2 may originate from a bat coronavirus. 13 at the same time, wu and colleagues reported that the genome sequence of sars-cov-2 was closely linked with sl-covzc45, another bat coronavirus, with 82.3% similarity. 14 however, despite the high similarity of sars-cov-2 to the virus from bats, the genetic differences will take at least a few decades of evolution to make up. 15 thus, bats are unlikely to be the definitive hosts of it, and there may exist other intermediate hosts which brought the virus into humans. 16 the relative synonymous codon usage analysis of viruses found sars-cov-2 used the translation machinery of several snakes effectively. therefore, snakes are a potential reservoir of the novel virus. 17 apart from this, pangolins are thought to be the potential intermediate host of sars-cov-2 due to the sequence similarity of their spike receptor binding domains. 18 furthermore, liu et al. declared that turtles were another possible host of sars-cov-2. 19 thus, more studies are necessary for confirming the intermediate host of sars-cov-2. the pathogenic mechanism of sars-cov-2 is still unclear and thought to be closely related to viral sepsis, 20 but structural biology has explained how viruses enter cells. angiotensin converting enzyme ii (ace2) has been demonstrated to be the only receptor for sars-cov-2. 13 the s protein on the surface of coronavirus was confirmed to mediate virus entry, 21 which contains proteins s1 and s2 subunits. the receptor-binding domain (rbd) of s1 is responsible for the recognition of ace2 on the surface of human cells to complete the binding of viruses to cells. the s2 subunit mediates the fusion of the virus envelope and human cell membrane to complete the invasion process. there are two important conserved repeated amino acid sequences, hr1 and hr2, in the s2 subunit of sars-cov-2. together, they form a spiral structure called 6-hb, which is the key for the fusion of a coronavirus envelope with cell membrane. 22 the serine protease tmprss2 triggers the s protein and promotes its fusion with the cell membrane. 23 after that, the rna of the virus is released into the cytoplasm to complete its biosynthesis. sars-cov-2 is more contagious, although it has a similar structure of s protein to sars-cov, so virus-receptor-binding affinity is now being conducted in further studies. goh et al. found the rigidity and stability of sars-cov-2 shell are apparently higher than sars-cov, 24 which increase its adaption to the environment. moreover, the structural difference of hr1 contributes to the higher structural stability for the 6-hb of sars-cov-2, which has caused the better membrane fusion capacity of sars-cov-2. 22 thus, sars-cov-2 has a stronger infectious ability. it is worth noting that the expressions of ace2 in patients with some diseases such as diabetes are notably higher than those in healthy people, 25 indicating that people with underlying diseases are more likely to be infected. on 31 december 2019, wuhan municipal health commission reported that 27 pneumonia patients were associated with a south china seafood market, which was the earliest discovery of covid-19. 26 then, the quantity of covid-19 patients increased rapidly after chinese new year. up to 16 may 2020, covid-19 had affected more than 200 countries and regions, and the quantity of confirmed patients had reached 4 425 485 globally. 27 after the breakthrough of covid-19, experts began to predict the incubation stage of the sars-cov-2. according to the data collected in january 2020, the incubation stage of covid-19 patients ranges between 2 and 14 days, and its average is about 5 days (95% credible interval: 4.2-6.0) when implementing the best-fit lognormal distribution. thus, experts recommended that suspected patients are quarantined for at least 14 days. 28 the mode of sars-cov-2 transmission is also an important part of epidemiological investigation. it is clear that humanto-human transmission of covid-19 occurs mainly via droplet respiratory particles, 29 but whether sars-cov-2 can be transmitted via the eyes is still unclear. 30 it should be noted that several studies have showed that chief complaint of some covid-19 patients were digestive symptoms, and nucleic acids of sars-cov-2 were even found in fecal samples or anal swabs of some patients, demonstrating the possibility of an oralfecal route. [31] [32] [33] furthermore, mother-to-child transmission is also a potential route of covid-19. 34, 35 luckily, infection rates of children are relatively low, and most inpatient children only have mild symptoms even if they are infected, [36] [37] [38] thus less damage would occur to them. up until now, the largest case series data of china has shown that 86.6% of covid-19 patients were aged 30-79 years, 1% were aged under 1 year old, 1% aged 10-19 years, and 3% were aged above 80 years. among all patients, males account for 51%, and females account for 49%. furthermore, 4% of them are medical workers. 39 in the united states, the elderly (aged over 65) account for 31% of total covid-19 cases, 45% of inpatients, 53% of icu patients, and 80% of death cases. 40 in terms of epidemiology, the basic reproductive number and mortality rate are indispensable. basic reproductive number (r0) is thought to be an important index for predicting the development of an outbreak. the main method of calculating r0 is using the data of infected people, such as serial interval distribution and latent period, to build a proper mathematical model for predicting the trend of the epidemic. a big epidemiological data of 425 confirmed covid-19 patients reported that the basic reproductive number of sars-cov-2 was 2.2, which means each patient could infect 2.2 other people. 1 however, more recent research estimated that basic reproductive number was 5.8. 41 it is obvious that distinctions of r0 exist in these studies, and they result in data and model differences. the incubation period and serial interval distribution of former research are calculated only through 10 confirmed cases and six pairs of cases in clusters, respectively. 1 furthermore, the model of the former research is made merely on the basis of one region, 1 thus its r0 distinctly lacks universality. by contrast, the latter study gathered the information of incubation period, serial interval, and infectious period from the individual cases reported throughout china and designed two models to infer the growth rate of the outbreak in different perspectives. 41 so, its r0 better reflects the epidemic situation of china. similarly, the r0 of around 3.87 reported by imperial college covid-19 response team only represented the situation in europe. 42 according to the data analysis of the chinese center for disease control and prevention, the overall mortality of covid-19 patients was approximately 2.3%, 43 which is obviously lower than severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). 44 however, because of the lager quantity of covid-19 patients, the number of covid-19 deaths is still high. furthermore, the data from the epidemic area indicated that people aged 30-59 years had the highest death rate of any age group, and the risk of symptomatic infection increased year by year. 45 like most respiratory illnesses, covid-19 has many typical respiratory clinical symptoms. data from 1099 confirmed covid-19 patients have shown that 88.7% had a fever, 67.8% developed a cough, and 2.3% received invasive mechanical ventilation, 46 which is consistent with other studies. 47 for some severe patients, acute respiratory distress syndrome (ards), shock, and arrhythmia are common complications. 48, 49 apart from these symptoms, covid-19 patients sometimes have other non-respiratory symptoms such as fatigue, myalgia, headache, and digestive tract symptoms. 48, 50 moreover, the main changes in laboratory results of covid-19 are decreases in lymphocytes and eosinophils as well as increases in d-dimer, c-reactive protein, prothrombin time, and procalcitonin. 48, 51 computed tomography (ct) is one of the most efficient techniques for evaluating severity and differential diagnosis of pulmonary diseases, thus the ct characteristics ( fig. 1 ) of covid-19 are another important clinical feature. this clinical evidence indicates that ground glass opacity (ggo), consolidation, and interlobular septal thickening are the most common ct characteristics of covid-19, which occurs mainly in the posterior lung area and middle and lower lung regions. 52, 53 compared to patients with mild symptoms, signs such as consolidation, thickened bronchial wall, linear opacities, extrapulmonary lesions, and higher ct scores are marked ct features of critically ill patients. 54 therefore, ct is potentially used to diagnose and predict the prognosis of covid-19 patients. clinical symptoms are often used as indicators of clinically suspected cases. thus, when it comes to the clinical features above, it is easy to think about clinical diagnosis of covid-19 (table 2and fig. 2 ). according to the diagnosis and treatment program of novel coronavirus pneumonia, only a suspected case has one of the pieces of evidence of etiology or serology, such as positive nucleic acid, confirmation of gene sequencing, and virus specific antibody, to be confirmed to be covid-19 patient, 55 and the suspected cases were identified by a comprehensive analysis of epidemiological history and clinical manifestations. 56 however, there are also many problems with this program. the most obvious problem is that rt-pcr patients have any epidemiological history and conform to at least two clinical manifestations, or patients without clear epidemiological history conform to three clinical manifestations. epidemiological history: i. travel or residence history of an affected area or close contact with a suspected or confirmed case within 14 days before onset ii. have contact with covid-19 cases (nucleic acid positive) within 14 days prior to onset iii. have contact with patients with fever or respiratory symptoms from an affected area, or from communities with covid-19 cases iv. cluster onset (two or more cases of fever and/or respiratory symptoms within 2 weeks in a small area such as home, office, school, and class) clinical manifestations: i. fever and/or respiratory symptoms ii. with covid-19 imaging characteristics iii. normal or reduced number of white blood cells and/or lymphocytes in early covid-19 the suspected cases have at least one of the following etiological or serological evidences. i. the real-time fluorescent rt-pcr for specimens with a positive result of sars-cov-2 rna ii. virus gene sequences are highly homologous to sars-cov-2 iii. positive sars-cov-2 specific igm antibody and igg antibody in serum; the serum sars-cov-2 specific igg antibody changes from negative to positive or is four times higher in the recovery period than in the acute phase is the most common method for nucleic acid testing, but its sensitivity is not as high as expected, 57 thus it possibly causes misdiagnosis of covid-19. furthermore, although viral gene sequencing is an accurate diagnostic method and used on a small scale, 58 its complex operation and time-consuming process make it difficult for wider clinical use. sars-cov-2 specific antibody detection is a simple detection method with higher sensitivity than rt-pcr, 59 but its high sensitivity is only reflected after 5.5 days of symptom onset, 60 so it is just a supplementary means to rt-pcr. luckily, the fast and convenient method, ct, was found to have a higher diagnostic sensitivity for covid-19 than other diagnostic methods such as rt-pcr and can predict the prognosis of patients, 57,61 thus it is meaningful for the initial screening. zhang et al. developed a deep learning system for image recognition of covid-19 patients. they collected ct images from 4154 patients for validation and found this system precisely identified the covid-19 and other forms of pneumonia, and it also divided these patients into high-and low-risk groups correctly, 62 so rational use of artificial intelligence such as this may be better able to screen covid-19 patients and give them timely interventions to ensure a better prognosis. in addition, a crispr-cas12-based technique was confirmed to be a faster test for covid-19 with higher sensitivity, 63 thus it is possibly a replacement for the rt-pcr assay. the therapeutic schedule issued by the general office of national health committee of china on 3 march has shown that therapy of covid-19 should be combined with etiological treatment and symptomatic treatment, and symptomatic treatment is especially important for severe and critical patients. furthermore, traditional chinese medicine can be also used for the whole treatment cycle of covid-19 patients. 55 in the processes of covid-19 therapy, drugs and bioproducts play important roles (table 3) . here, we summarize the main treatments of novel pneumonia. the etiological treatment for covid-19 means antiviral therapy for it, which depends on antiviral drugs and bioproducts. the processes of virus infection could be described as attachment, penetration, uncoating, biosynthesis, assembly, and release. after that, the virus complete its proliferation and causes damage to host cells. similarly, the mechanism of antiviral therapy is through disturbing these processes. thus, the medicines for antiviral treatment of covid-19 are divided into penetration and uncoating inhibitors, biosynthesis inhibitors, and assembly inhibitors. penetration and uncoating inhibitors of sars-cov-2 mainly include chloroquine, arbidol, and camostat mesilate. chloroquine is an efficient drug for treatment of malaria as well as autoimmune rheumatic diseases. wang and colleagues treated vero e6 cells that were infected by sars-cov-2 with several drugs in vitro and found a low half-maximal effective concentration at 1.13 μmol and a high selectivity index of chloroquine, indicating that chloroquine probably inhibits the viral infection. 64 there have been tens of clinical trials to confirm the safety and efficiency of chloroquine in treating covid-19 patients, and its mechanism can be described as interfering with the glycosylation of ace2 or alkalizing the phagolysosome to inhibit viral replication, 65, 66 which prevents the sars-cov-2 entering the host cells. the in vitro study indicated that chloroquine only had an antiviral effect under high doses. however, the clinical data of chloroquine showed the mortality of highdosage group was apparently higher than the low-dosage group, but there was no significant difference in efficacy, 67 and the qt interval of some covid-19 patients was prolonged after hydroxychloroquine/azithromycin treatment, 68 thus it might not be an effective medicine for covid-19. arbidol is the other penetration inhibitor that suppresses the fusion of the virus lipid membrane and host cells to block the replication of the virus. arbidol also has the function of interferon (ifn) inducement, thus it has been widely used to treat a and b influenza viruses. it is promising that several studies of arbidol have shown a significant therapeutic effect for curing covid-19 patients. wang and colleagues found arbidol reduced the mortality and improved discharging rate by analysis of 67 discharged covid-19 patients. 69 furthermore, the other study compared the treatment effects between combination of arbidol and lopinavir/ritonavir, and simple lopinavir/ritonavir, finding that their ct scan improvement rates are 69% and 29%, and viral rna decrement rates are 75% and 35%, respectively. 70 thus, arbidol perhaps serves as a specific treatment for covid-19. however, another clinical report pointed out arbidol could not improve the symptoms or accelerate the clearance of sars-cov-2. 71 thus, further studies are needed to validate its antiviral efficiency. camostat mesylate is a specific drug for inhibiting tmprss2, a protease used for s protein priming to promote the entry of sars-cov-2, thus it is another potential penetration inhibitor. 23 however, there is still lack of clinical evidence to prove its effectiveness. the biosynthesis inhibitors for sars-cov-2 chiefly contain remdesivir, sofosbuvir, ribavirin, and lopinavir/ritonavir. remdesivir is as kind of broad spectrum antiviral medicine that suppresses the virus replication through inhibiting the activity of rnadependent rna polymerase, 72, 73 and its efficiency has been widely confirmed in coronaviruses such as sars-cov and mers-cov. recently, remdesivir has been found to be a potential drug to treat covid-19. a previous study showed that half-maximal effective concentration of remdesivir was 0.77 in an in vitro test treating covid-19 with a high selectivity index, demonstrating its therapeutic effect. 64 there, the first covid-19 case of america was reported to be cured completely after intravenous injection of remdesivir, 74 and clinical data showed that remdesivir dramatically alleviated clinical symptoms and reduced mortality for severe patients, 75 which demonstrated it is a potential drug for treating novel pneumonia. however, a patient died of hypotension and cardiac arrest after remdesivir treatment in a randomized controlled trial of ebola virus (ebov) treatment, 76 and 60% covid-19 patients had side effects after using remdesivir, 75 which indicated its security is worthy of further confirmation. up to now, several agencies have published conflicting clinical results from remdesivir. 77, 78 thus, more research should be conducted to verify its curative efficacy. furthermore, in vitro tests showed that other broad-spectrum antiviral medicines such as sofosbuvir and ribavirin interacted with rna-dependent rna polymerase of sars-cov-2, which prevents the replication of virus, thus they can also serve as antiviral drugs for covid-19. 79 lopinavir and ritonavir are suppressors of 3-chymotrypsin-like protease, a protease of coronavirus. furthermore, ritonavir suppresses the activity of cytochrome p450 isoenzymes and thus elevates plasma concentration of other medicines. therefore, combining lopinavir and ritonavir has a good inhibitory effect on virus biosynthesis, which was confirmed in the treatment of sars-cov and mers-cov. 80 recently, many covid-19 patients have received lopinavir/ritonavir therapy and to good effect. 81 moreover, the first report of the lopinavir/ritonavir clinical trial results have been published on 19 march 2020. in that study, 99 patients were assigned to the lopinavir/ritonavir group and 100 patients were treated with the routine therapy, and the median time of clinical improvement was advanced by 1 day when given the lopinavir/ritonavir treatment. 82 but it failed to accelerate clinical improvement significantly, and reduce mortality and viral rna detected in the throat. 82 similarly, the clinical trial in severe covid-19 patients indicated lopinavir/ritonavir was of no benefit compared to standard care. 83 therefore, further studies are needed to identify or exclude the possible benefits of lopinavir/ritonavir-based therapies. the most common antiviral products are ifns, which induce cells to synthesize antiviral proteins and thus inhibit all processes of the viral replication cycle. furthermore, it could also enhance immunity of patients, so it is widely used for therapy for multiple viruses such as mers-cov. 84 in this outbreak, ifns combined with antiviral drugs were recommended to treat covid-19, 85 which has achieved good clinical therapeutic effect. xu et al. reported that the combination of ifns and arbidol or lopinavir/ritonavir cured 62 covid-19 patients in the zhejiang province. 50 in addition, a combination use of ifn beta-1b, ribavirin, and lopinavir-ritonavir was found to be more effective than pure lopinavir-ritonavir. 86 these therapies have been initiated into multiple clinical trials. convalescent plasma therapy (cpt) is based on the principle of using a certain titer of viral-specific antibodies in the recovered plasma to obtain passive immunity, neutralize specific pathogens, and eventually clear the pathogens in blood circulation, thus achieving the treatment expectation. 87 luckily, key indicators of laboratory testing, clinical signs, and symptoms of several covid-19 patients were confirmed to improve significantly after cpt, 88 so cpt is recommended for covid-19 treatment. currently, clinical trials are under way to further evaluate the efficiency and safety of cpt to covid-19. the monoclonal antibody is a highly uniform antibody that is produced by a single b cell and specific to target the antigen epitopes, which have been confirmed to suppress viruses entering host cells extracellularly for many coronaviruses including sars-cov-2. 89 like most diseases, the main treatments of the complications of covid-19 are to strengthen supportive treatment, ensure adequate energy, and pay attention to water and electrolyte balance to maintain internal environment homeostasis. 55 for covid-19, hypoxia is a typical clinical symptom of covid-19, thus oxygen inhalation is the essential treatment for both mild and severe patients. 92, 93 it is worth noting that severe cases of covid-19 often develop severe inflammation, shock, combination of bacterial infection, and severe kidney damage as well as acute respiratory distress syndrome (ards), which often result in death. 94 thus, timely complications treatment is necessary for critical patients. glucocorticoids such as methylprednisolone and dexamethasone have strong anti-inflammatory as well as antishock effects, so they are usually used to save critical patients, especially patients with ards. 95 however, they suppress immunity, cause femoral head necrosis of patients, and cannot save patients with shock who have increased intrathoracic pressure, thus who did not recommend using glucocorticoids initially. 96 for these situations, physicians suggested that short-term administration of glucocorticoids should be adopted to decrease the side effects. 97 moreover, considering the immunosuppressive action, immune boosters such as α-ifn and thymosin are important for use in avoiding adverse events. for severe cytokine storms, simple glucocorticoids cannot suppress the inflammation efficiently, so potent blood purification measures such as an artificial liver system are essential. 98 the processes of urgent antishock therapy mainly contain complements of blood volume, improving cardiac contractility and vascular activity, thus vasoactive agents and positive inotropic drugs such as epinephrine, dopamine, and norepinephrine 99 are necessary for first aid of critical covid-19 patients. for patients with severe kidney damage, we should actively search for the primary cause and pay attention to the water, electrolyte, and acid-base balance. when homeostasis is decompensated and/or multiple organ failure occurs, continuous renal replacement therapy (crrt) should be adopted for treatment. 100 ards is one of most critical complications for patients, which is known for its high mortality rate, especially for pregnant patients. 101 for its therapy, ecmo is usually recommended and has a certain curative effect. 102 however, its complex operation and low cure rate make it difficult to perform in a primary hospital. luckily, stem cells and their extracellular vesicles are able to repair the damage and relieve lung symptoms, thus they have been used to cure ards. 103 currently, some covid-19 patients have been treated with umbilical cord mesenchymal stem cells and achieved good effects. 104 therefore, stem cells may be the hope for the treatment of severe covid-19 patients. tens of clinical trials of stem cell treatment have been registered in chinese clinical trial registry. recently, many covid-19 patients were found to have thrombotic risk, 105 therefore, antithrombotic treatments have potential application value in the treatment of covid-19. furthermore, patients with underlying diseases such as hypertension, diabetes, and cardiovascular disease have a potential bad prognosis, 106-108 so care should be taken to cure these basic diseases during treatment. covid-19 patients with low immunity are susceptible to hospital-acquired bacterial infections, 109 therefore, antibiotics such as amoxicillin are another symptomatic treatment medicine for covid-19. traditional chinese medicine has been confirmed to play a vital role in treating many respiratory viruses such as sars-cov. furthermore, the general office of national health committee pointed out that chinese medicines such as qingfei paidu decoction, qingfei touxie fuzheng recipe, and sheganmahuang decoction could be used for the whole therapeutic process of covid-19, 55 thus they are widely used for covid-19 treatment currently. the present clinical evidence has shown that a combination of western and chinese medicines apparently improved curative effect. 110 for example, wan et al. treated 124 patients with multiple traditional chinese medicines such as suhuang zhike capsule and xuebijing, and found their respiratory symptoms were significantly improved in a shorter time. 109 furthermore, four cases treated with antiviral drugs and shufeng jiedu capsule had an obvious improvement both in symptoms and blood biochemical indexes. 111 however, lack of a control group means these results are not reliable enough. although treatment based on syndrome differentiation and lack of scientific evidence on mechanisms are typical characteristics of traditional chinese medicines, many of them are gradually found to have antiviral potential for sars-cov-2. theaflavin is conventional ingredient of chinese medicine, which was found to have hydrogen bonds to rna-dependent rna polymerase with a binding energy of −8.8 kcal/mol, 112 thus it possibly inhibits the proliferation of sars-cov-2. lianhuaqingwen was also confirmed to significantly inhibit the replication of sars-cov-2 and suppress the inflammatory factors tnf-α, il-6, ccl-2/mcp-1, and cxcl-10/ip-10 in vero e6 cells, 113 so it is potentially a specific medicine for covid-19 both etiologically and symptomatically. the prevention and control of covid-19 can be divided into controlling the source of infection, cutting off the transmission route, and protecting susceptible people. controlling the source of infection in this outbreak mainly refers to timely isolation of suspected and confirmed cases, which greatly depends on wireless communication data. 114 isolation of convalescent patients is also necessary, because some of them have a positive rt-pcr test even after 13 days of recovery. 115 cutting off the transmission route includes sterilizing and wearing suitable masks such as n95, kn95, and medical surgical masks. 116 protecting susceptible people means self-segregation, improving constitution, and prophylactic vaccination. considering the strong infectivity of covid-19, the secondary outbreak of covid-19 can be prevented only if the public has immunity to sars-cov-2, thus vaccine is the key area among all prevention measures. therefore, scientists all over the world are working on the vaccine for sars-cov-2, aiming to completely control the outbreak. after a long period of effort, the vaccine targets of the virus had been found and shared with the world. 117, 118 soon afterward, medical institutions around the world adapted joint research and identified five development approaches, including a live attenuated vaccine, inactivated whole-virus vaccine, nucleic acid vaccine, protein vaccine, and viral vector-based vaccine (table 4 ), 119 and most of them used the s protein of sars-cov-2 as the main inducer of neutralizing antibodies directly, or inducing the expression of the whole s protein or receptor-binding domain of the s protein indirectly. 120 by interaction of the s protein and ace2 of host cells, uncoating and penetration of sars-cov-2 are induced. 23 thus, s-protein-based vaccines potentially inhibit the uncoating and penetration of the virus. whole-virus vaccines are conventional strategies to develop a vaccine for most viruses. by injecting artificially attenuated or inactivated viruses, pathogens trigger the body's immune response without causing disease. compared to other vaccines, these keep the inherent immunogenicity of sars-cov-2 and stimulate the body's innate immunity such as releasing toll-like receptors. however, live viruses have the potential to cause disease. furthermore, it has been reported that patients were more easily infected by sars-cov after immunization with a live attenuated vaccine or inactivated wholevirus vaccine. 121 thus, sufficient experiments are necessary to verify their safety. currently, three inactivated novel coronavirus pneumonia vaccines have entered the phase i/ii clinical trials. [122] [123] [124] nucleic acid vaccine nucleic acid vaccines, including dna vaccines and mrna vaccines, are to directly deliver foreign genes encoding antigenic proteins into host cells and synthesize the antigenic proteins by host cells to induce the immunity of hosts. nucleic acid vaccines of covid-19 mainly aim to induce the whole s protein or receptorbinding domain of s protein expression, and several of them have entered clinical trials. 125 for example, researchers of inovio pharmaceuticals are conducting the clinical trial of a dna vaccine. 126 in the meantime, the national institute of allergy and infectious diseases have developed a mrna vaccine, mrna-1273, which uses lipid nanoparticles to encapsulate the mrna encoding s protein of sars-cov-2. 127 interestingly, symvivo corporation have developed an oral dna vaccine to avoid invasive injection. it consists of live bifidobacterium longum, which contain the synthetic dna encoding s protein. 128 although patients with typical symptoms are easily diagnosed and isolated, we still need to pay attention to asymptomatic carriers. 135 in january 2020, a family cluster of three covid-19 patients with one symptomatic and two asymptomatic carriers drew great attention. 11 because any one of them could have the first one to transmit the virus to others, there may exist asymptomatic patients with high infectivity who have not been detected. at the same time, another family cluster of five patients was found to have contact with an asymptomatic carrier before onset of their symptoms. 136 according to time sequence, it is obvious that the virus was transmitted by an asymptomatic carrier. hereafter, the phenomenon of asymptomatic infection has been reported in several articles. 137, 138 on the basis of virology of sars-cov-2, quantity of shedding sars-cov-2 from pharyngeal sources is very high in the first week of symptoms, and they also replicate actively during this time, thus the first week after onset of symptoms is when the patients have the highest infectivity. 139 but in this time period, some patients only have mild symptoms or even no symptoms, so they might be invisible sources of infection. therefore, people who have traveled to endemic areas or had contact with patients should be isolated, monitored, and screened strictly, even if they are asymptomatic, to rule out the possibility of infection. covid-19, a disease that seriously endanger the safety of human life and properties, is caused by sars-cov-2, which attacks host cells through ace2. the spread of covid-19 mainly depends on airborne and droplet transmission. some researchers pointed out the possibility of fecal-oral transmission and vertical transmission, but these opinions need further confirmation. an accurate diagnosis of covid-19 is essential for containing disease, thus combnation of rt-pcr and clinical characteristics is widely used for its detection currently. the typical clinical features of covid-19 patients include fever, cough, and dyspnea. moreover, ggo, consolidation, and interlobular septal thickening are also typical ct signals of covid-19. using artificial intelligence to screen covid-19 early on is likely to be the future diagnostic directions of it. good treatment is necessary to reduce mortality, and drug therapy is the main treatment of covid-19. thus, we summarize the chief medicines and bioproducts for its treatment. according to the current reports, combined use of the drugs and bioproducts to treat covid-19 both etiologically and symptomatically has received great clinical effect. however, not all antiviral drugs for covid-19 have passed the clinical trials. a few clinical trial reports demonstrated that lopinavir-ritonavir, chloroquine, and remdesivir did not work as well as expected, 67, 75, 82 which reflects the fact that many antiviral drugs have gone through many extensive clinical trials with currently only modest effect. instead, some bioproducts such as monoclonal antibodies and stem cells are more likely to play important roles in treatment of severely affected patients in the future. furthermore, we should also focus on vaccine research and artificial intelligence-based studies for fighting the outbreak, both of which play vital roles in controlling the it. although antiviral effects of many drugs and vaccines have been obtained in vitro, it remains to be seen as to whether they have comparable activity in vivo. therefore, establishing efficient animal models as soon as possible is important for virology research, drug, and vaccine development. currently, rhesus macaques models have been established to study the transmission and distribution of sars-cov-2 as well as effects of viral infection on host prognosis, 140, 141 which is meaningful for virology study. furthermore, hace2 transgenic mice have been found to own typical covid-19 clinical symptoms, which could not be found in wild-type mice, 142 indicating this model may be used for development of 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declared that they had no conflict of interest. key: cord-261075-wqtxhiy8 authors: zhang, meng; zhou, lingyan; wang, jing; wang, kun; wang, yuan; pan, xudong; ma, aijun title: the nervous system——a new territory being explored of sars-cov-2 date: 2020-10-28 journal: j clin neurosci doi: 10.1016/j.jocn.2020.10.056 sha: doc_id: 261075 cord_uid: wqtxhiy8 in december 2019, covid-19 outbroke in wuhan, then sweeping the mainland of china and the whole world rapidly. on march 4, beijing ditan hospital confirmed the existence of sars-cov-2 in the cerebrospinal fluid by gene sequencing, indicating the neurotropic involvement of sars-cov-2. meanwhile, neurological manifestations in the central nervous system, peripheral nervous system and skeletal muscular were also observed, indicating the potential neuroinvasion of sars-cov-2. in particular, we focused on its neurological manifestations and specific pathogenesis, as well as its comparison with other viral respiratory infections.finally, we further summarized the significance of the neuroinvasion and the follow-up issues that need to be paid attention to by scientists, so as to help neurologists understand the influence of sars-cov-2 on nervous system better and promote the accurate diagnosis and efficient treatment of covid-19. in december 2019, a novel pneumonia of coronavirus, corona virus disease 2019 outbroke in wuhan, china, then sweeping the mainland of china and the whole world rapidly, seriously threatening individuals' life and health (1) (2) (3) (4) . due to the high 3 homology with the severe acute respiratory syndrome coronavirus (sars-cov), the pathogen was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2)(5). the main clinical symptoms involved by sars-cov-2 is pulmonary manifestations. however, there is growing evidence that sars-cov-2 can result in a broad spectrum of neurologic diseases (6) (7) (8) (9) , which is not surprising, as neurological manifestations have been reported in other respiratory viral infections, including coronavirus, but the nervous system manifestations of covid-19 are more common and disabling, raising the worldwide concerns about its potential long-term complications to humans (10, 11) . in particular, we focused on its neurological manifestations and specific pathogenesis, as well as its comparison with other viral respiratory infections.finally, we further summarized the significance of the neuroinvasion and the follow-up issues that need to be paid attention to by scientists, so as to help neurologists understand the influence of sars-cov-2 on nervous system better and promote the accurate diagnosis and efficient treatment of covid-19. sars-cov-2 is a non-segmented, large enveloped, positive single-stranded rna virus, which is the member of betacoronavirus genus, with an overall genomic sequence consistency of 96.2% and 79.5% sequence homology with the sars coronavirus (12) . it has been discovered that sars-cov-2 combined with a metalloproteinase, angiotensin-converting enzyme 2 (ace2) receptors to infect the target cells on their surfaces in line with sars-cov, which requires the participation of spike protein(s) and 4 transmembrane protein serine protease 2 (tmprss2) (12, 13) . therefore, neurons, endothelial cells, glial cells and arterial smooth muscle cells in the brain, which expressing ace2 receptors become vulnerable to it (14) (15) (16) . besides, recent researches found that sars-cov-2 has a higher affinity for human ace2 than sars-cov (17) , which further elucidate the reason for the strong pathogenicity and transmissibility of it. people are generally susceptible to sars-cov-2 (18) . it has shown the aged has higher susceptibility to develop severe sickness, especially with specific comorbidities. additionally, about half of the patients infected with sars-cov2 had chronic diseases, including cardiocerebrovascular disease as well as diabetes(3). a retrospective study (19) showed that among 214 patients diagnosed with covid-19, more than 30% of them had neurological manifestations. on march 4, beijing ditan hospital confirmed the existence of sars-cov-2 in the cerebrospinal fluid by gene sequencing (20) , indicating sars-cov-2 could invade the nervous system directly. moreover, it became the first evidence that sars-cov-2 may have the potential neuroinvasion. in fact, the earliest cases of encephalitis/meningitis occurred in japan (21) . a man in twenties was confirmed as a victim of sars-cov-2, who started with fever and fatigue at first, and he was found unconscious at home. during emergency treatment, he suffered from epileptic seizures several times. it was also found sars-cov-2 in his cerebrospinal fluid in 8 march, which is the further evidence that the virus is attacking the 5 nervous system. the central nervous system(cns) is essential to regulate the whole human body, but it has no immune function against most infections, especially the virus infection. the blood-brain barrier (bbb) is a natural cover for defence, preventing from the intracranial disease of the foreign pathogen. covs can enter the cns from the respiratory tract via hematogenous and transneuronal routes. increasing evidence shows that a variety of covs can not only infect the respiratory system but also have the neurotropism feature (21) (22) (23) (24) (25) (26) (27) (28) . the existing researches mainly focus on sars-cov. in 2004, the virus rna was detected in the cerebrospinal fluid of a sars patient, which led to the scientists' suspicion that sars-cov was neuroinvasive (29) . one year later, xu et al. isolated the virus from the brain tissue of a patient, and the neuropathologic manifestation was nerve cell damage. meanwhile, the patient showed neurological symptoms, making the hypothesis confirmed (30) . furthermore, extensive neuronal infections can be observed in animal models. furthermore, sars-cov entered human cells through a receptor named as angiotensin-converting enzyme 2 (ace2), which is consistent with sars-cov-2(31). therefore, sars-cov is neuroinvasive and neurotropic in both animals and humans, which may be related to the development of neurological diseases. given sars-cov2 has similar gene sequencing and pathologic anatomy with sars-cov, it could be 6 assumed that sars-cov-2 has the same neuroinvasion and pathogenesis. sars-cov-2 might cause a series of neurological damage by two mechanisms; one is the hypoxic brain injury. it is well known that hypoxia, hypercapnia, endocrine and metabolic disorders, and the accumulation of toxic substances caused by respiratory failure in severe covid-19 patients can cause neuronal swelling and brain edema, leading to brain damage (32) . the other is the parainfectious mechanisms, that is, immune-mediated brain damage. it is mainly referred to cytokine storm (33) . cytokine storm syndrome is a highly inflammatory state characterized by sharply elevated cytokine levels, overshooting immune responses and explosive multi-organ failure, leading to vasodilatory dysfunction, membrane leakage, coagulation dysfunction, multiple organ failure, and severe vasospasm shocks. besides, if ecmo is used, the contact between fluids in vivo and circuits in vitro might activate clotting and inflammatory pathways, leading to disseminated intravascular coagulation in severe cases(34,35). firstly, given that sars-cov-2 has the underlying neuroinvasion, carrying out the therapy to resist it as soon as possible can block the entry of the virus to the cns. moreover, the ability of antiviral agents to cross the blood-brain barrier is also an issue to consider in the future. although the progression of new antiviral drugs requires a lot of workforce and material resources, it can not benefit the general public a lot in the current epidemic. however, as the outbreak fades, the virus may mutate into a coronavirus, which is similar to the influenza virus, spreading from person to person over the long term. 7 therefore, it is necessary to accelerate the research and development of specific antivirus drugs and vaccines steadily. secondly, considering that sars-cov2 might be latent in some neurons to get away with immune surveillance, we can not guarantee that the virus has been eliminated. actually, sars-cov-2 is still can be detected; even the covid-19 patients are under the convalescent period (36) . if existing in the nervous system for the long term, it is likely to serve as a trigger for some human neurological diseases in those genetically predisposed individuals. thus the long-term follow-up of patients is an essential task in the future (37) . thirdly, the blood-brain barrier degrades gradually with age, which indicates that the nerve invasion ability of the virus to the elderly increases. once the virus invades the nervous system, demyelination, cell apoptosis and neurodegeneration will occur, then further aggravating the brain ageing and neurodegenerative diseases (38) . neurologists will face more age-related neurodegenerative diseases after we control the outbreak. therefore, more long-term neurological follow-up of the elderly is needed after the final concern is the immuno-therapies for covid-19 with autoimmune diseases of the nervous system. once diagnosed, neurologists must assess the risks and benefits of immunotherapy in light of the different conditions of the disease. for example, high doses or long-term use of steroid hormones may put patients at further risk for covid-19 infection. protective immunoglobulin is recommended if possible. additionally, given that the crucial step to control the pandemic is the application of sars-cov-2 8 vaccination, it is indispensable to weigh the safety and effectiveness of vaccination during immunotherapy. an increasing number of evidence indicated that sars-cov-2 might have potential neuroinvasion ， hence we summarized the existing related researches and data of covid-19, the neurological signs of it could be divided into three categories：symptoms involving the central nervous system, peripheral nervous system, and skeletal, muscular symptoms. the virus infections can cause vascular endothelial injury and vascular system damage, then leading to ischemic and hemorrhagic infarcts through overactive inflammation response, thrombosis and vasculitis (39) . a retrospective case series study in wuhan, china (19) , showed that the severe patients of covid-19 commonly had acute cerebrovascular diseases, and the pathophysiological changes during the infection may render the victims prone to it. firstly, in the latest study, 52% of patients of covid-19 had elevated il-6 levels, and 86% of them had elevated crp(3), suggesting a significant inflammatory response in their bodies. meanwhile, inflammation is also essential in the occurrence, development as well as prognosis of cerebrovascular diseases and may trigger cerebrovascular events(40). 9 secondly, according to the laboratory examination of the severe patients who had coagulation dysfunction and elevated d-dimer (41) , those people may have more possibility of venous thrombosis (42) and hemorrhagic stroke (43) . a 75-year-old woman infected by sars-cov-2 without any predisposing factors suffered from severe bilateral pneumonia and acute pulmonary embolism, suggesting the severe infections is a precipitant factor of acute venous embolism and stroke (44) . thirdly, sars-cov-2 readily attacks the lung, then causes dyspnea and decreased blood oxygen saturation (3) . hypoxemia attributes the alteration of consciousness, confusion or delirium, causing an acute or subacute stroke or intracerebral hemorrhage (45, 46) . additionally, overexpression of ace2 reduces the risk of ischemic stroke, which explains the reason why elderly covid-19 patients are more likely to have a stroke(47,48). although the virus seems to have a hard time penetrating the central nervous system, pathogens can be abnormally active in spreading and replicating, then may induce overreacting immune response and lead to fatal meningitis and encephalitis.the discovery of the positive rt-pcr of csf (20, 21) suggested that meningitis and encephalitis may associated with viral invasion of the cns. elevated temperature, headache, vomiting, and consciousness disorders are common signs, which are similar to acute encephalitis (49) . autopsy reports also showed that there was the edema of brain tissue and degeneration in the neurons of those victims (50) . presumably, sars-cov-2 may enter the cns from the 10 respiratory tract via hematogenous and transneuronal routes like other covs(51). acute necrotizing encephalopathy is a rare acute and severe explosive encephalopathy, which usually occurs after a viral infection. the most common hypothesis for its pathogenesis is the destruction of the blood-brain barrier caused by cytokine storm (52) radmanesh et al. observed two kinds of imaging characteristics by cerebral mri in 11 patients with severe covid-19: diffuse leukoencephalopathy and microhemorrhages, speculating that they were late complications in severe covid-19 patients (56) . given that similar involvement patterns have been described in patients after anoxic injury, it 11 was considered delayed post hypoxic leukoencephalopathy (dphl) which was associated with oligodendrocyte damage and demyelination (57, 58) a 66-year-old male in wuhan was diagnosed as covid-19 with acute myelitis(67), presenting with fever, pain, and acute delayed paralysis of the lower extremities along with urinary and fecal incontinence. the patients responded well to immunoglobulin and hormone. meanwhile, the high levels of c-reactive protein, inflammatory cytokines, and serum ferritin suggested that his myelitis was a result of a cytokine storm. there are also other central nervous system manifestations reported (19) such as headache, dizziness, ataxia, impaired consciousness, and epilepsy. dizziness and 12 headache are the most common complaints. a headache expert pointed out that there are two stages of covid-19-related headache. the first stage is an acute headache caused by a viral infection, primary cough, tension-type and heterophoria. the headache in the second stage(bounded by 7 to 10 days) could attribute to hypoxia, which could be used as a predictor of cytokine storm onset (68) . in the patients who had severe virus infection also showed agitation, corticospinal tract signs as well as a dysexecutive syndrome (69) . a covid-19 patient with meningitis/encephalitis also developed epilepsy (20) , and another present as focal status epilepticus as the first symptom (70) . therefore, it is reasonable to speculate that covid-19 patients may develop epilepsy under conditions of hypoxia, endocrine and metabolic disorders, and multiple organ damage. considering the different affinity of sars-cov-2 to different populations and the diversity of ace2 expression (73) , studies should focus more on the polymorphisms of ace2 in different races. guillain-barrésyndrome is a kind of neuroimmune disease secondary to respiratory or gastrointestinal infections. it is known that there is a molecular imitation mechanism. the invading virus has similar epitopes with some components of the nervous system, stimulating the immune system to produce antibodies which can not only bind to the virus, but also cross-react with components of the nervous system, then causing a series of neurological dysfunction (74) . (77) . consider that these are all observational studies, more epidemiological evidence is needed to support the potential relationship 14 between guillain-barré syndrome and covid-19 in the future. hyposmia is also a common symptom in covid-19 patients (78) . acute respiratory symptoms associated with coronavirus are characterized by hypoxia, one of the ways that coronavirus associated with the nervous system, causing endocrine and metabolic disorders, edema in the brain and subsequent neurological manifestations. besides, taste ,vision impairment, and facial pain have also been reported(19,79). skeletal muscular symptoms were also reported in covid-19 patients (19) . given the high levels of lactate dehydrogenase and creatine kinase, it is speculated that the symptoms resulted from skeletal muscle injury which might be associated with ace2 existing in it (79) . nevertheless, whether sars-cov-2 damages skeletal muscle through the receptor on the cells still needs further researches. moreover, the elevated pro-inflammatory cytokines, which indicated abnormal immune response mediated by infection, might be another reason for it. a great many viral infections could cause varying degrees of damage to the nervous system, including its the structure and function. such as acute disseminated encephalomyelitis, toxic encephalopathy and severe acute demyelinating diseases in connection with the post-infectious inflammatory response.the presence of the virus in 15 cerebrospinal fluid also confirms the nerve invasiveness of the virus (80, 81) . as sars-cov-2 has stronger binding ability to ace2, it is more pathogenic and transmissible as well as debilitating than sars-cov and mers-cov (82) . at the same time, covid-19 and acute respiratory distress syndrome patients had twice the rate of thrombosis complications compared to the ards from other causes, which explains why the rates of covid-19 associated d-dimer elevation and concurrent stroke appear to be much higher than other viruses. therefore, timely anticoagulant therapy is essential for the prognosis of patients(83,84). in conclusion, sars-cov-2 has potential neuroinvasion and may cause a series of nervous system diseases or symptoms. once involving the nervous system, it usually predicts poor prognosis. therefore, patient with covid-19 requires more physicians'attention to evaluate their neurologic manifestations as early as possible. paying attention to the patients who have neurological symptoms as the first manifestation other than the typical cough, vomiting, diarrhea, and fever is imperative for the control of the rapidly evolving epidemic. gene sequencing of cerebrospinal fluid and other neuroimaging examinations should also be taken in necessity. no funding. 16 the authors declare that they have no conflicts of interest. this study was based on publicly available data and did not require ethical approval. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating 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skeletal muscle clinical manifestations and organ-system complications: a mini review. pathog dis influenza type b-related encephalopathy. the 1971 outbreak of reye syndrome in chicago sars-cov-2 viral load in upper respiratory specimens of infected patients risk of ischemic stroke in patients with coronavirus disease 2019 (covid-19) vs patients with influenza neurological manifestations of sars-cov-2(cns and pns) key: cord-263945-yli5suxb authors: iancu, gabriela mariana; solomon, adelaida; birlutiu, victoria title: viral exanthema as manifestation of sars-cov-2 infection: a case report date: 2020-08-28 journal: medicine (baltimore) doi: 10.1097/md.0000000000021810 sha: doc_id: 263945 cord_uid: yli5suxb rationale: the clinical manifestations of the sars-cov-2 infection are mainly respiratory but the virus can cause a variety of symptoms. dermatological findings are less well-characterized. data is scarce on their timing, type and correlation with the immune response. patient concerns: we present the case of sars-cov-2 infection in a previously healthy woman who presented with respiratory symptoms and developed anosmia, diarrhea, and an erythematous maculo-papular rash on day 15 from symptom onset. diagnosis: the nasopharyngeal swab tested by real time pcr for covid-19 was positive. we interpreted this as a viral exanthema likely caused by an immune response to sars-cov-2 nucleotides. interventions: she was treated with hydroxychloroquine, azithromycin and lopinavir/ritonavir, and the rash with topical corticosteroids. outcomes: all symptoms resolved except for anosmia which persisted for 6 weeks. at the 4and 6-weeks follow-up the igg titers for sars-cov-2 were high. lessons: we must consider that sars-cov-2 has a multi-organ tropism. in our case, the sars-cov-2 infection had lung, nasopharyngeal, neurological, digestive, and skin manifestations. identifying the different manifestations is useful for understanding the extent of sars-cov-2 infection. we not only present a rare manifestation but also suggest that cutaneous manifestations may correlate with immunity. at the end of 2019, a novel coronavirus was identified as the cause of pneumonia cases in the city of wuhan, china. [1] the virus has spread to become a global pandemic. among the clinical features of the disease the most frequent manifestations are fever, cough, dyspnea, upper respiratory tract symptoms (rhinorrhea, sore throat), headache, myalgias, nausea, diarrhea, smell, and taste disorders confusion. [2] all these are nonspecific and can be encountered in other viral respiratory infections but the development of pneumonia (dyspnea and bilateral infiltrates on lung imaging) several days after the onset of symptoms is highly suggestive in the present context. [3] the course of the illness ranges from asymptomatic to severe. some of the complications that have been described are: acute respiratory distress syndrome, acute cardiac injury, thromboembolism (pulmonary or stroke), cytokine release syndrome with elevated inflammatory markers (d-dimer, ferritin), guillain-barre syndrome, secondary infections. [4] the recovery time is around 2 weeks for mild infections and between 3 and 6 weeks for severe disease. [5] various other symptoms have been associated with covid-19 but the predictive value of a single symptom in the diagnosis is uncertain. editor: maya saranathan. patient has provided informed consent for publication of the case. written informed consent was obtained from the patient for publication of this case report and accompanying images. dermatological findings have been reported (maculopapular rash, urticarial and chicken pox-like eruptions, transient livedo reticularis, chilblain lesions, etc.), but these are not well characterized. [6] pathogenetically, the appearance of cutaneous lesions during the sars-cov-2 infection can be explained by an immune response initiated by the viral nucleotides which activate langerhans cells with the secondary involvement of keratinocytes (maculopapular, urticarial and chicken pox-like rashes), by microthrombi formation and cutaneous vasculopathy (chilblain lesions, livedo reticularis, erythema multiforme-like rash, gangrene), or by reaction to the medication administered (urticaria, erythroderma, erythema multiforme, etc.). [7, 8] we report a case of disseminated exanthema that appeared after 15 days of treatment for sars-cov-2 infection in a patient without other medical and dermatological problems in the past. a 41 year old woman attended the emergency department for fever, myalgias, dysphagia, nasal congestion, headache, symptoms present for 2 days before presentation. she declares that she was in contact with a person who tested positive for sars-cov-2 a week before presentation. the patient is not a smoker and has no medical history of note. on physical examination she was found to be of normal weight, with a temperature of 38.3°c, pharyngitis, billateral submandibular microlymphadenopathy, oxygen saturation of 98% in ambient air, blood pressure of 110/60 mm hg, heart rate of 88/minute. the nasopharyngeal swab tested by rt-pcr for covid -9 was positive and the patient was admitted. the chest x-ray was unremarkable. the blood tests revealed an inflammatory syndrome: crp 16.35 mg/l (rv 0-5), fibrinogen 494.7 mg/dl (rv 170-420), esr 34 mm/hour (rv 0-20). the full blood count showed lymphopenia, with an absolute lymphocyte count of 1090/ml (rv 1500-4000) and a neutrophils to lymphocytes ratio of 2789. the d-dimers were slightly raised à603.32 ng/ml (rv 45-499). other blood tests performed were normal (ferritin, ldh, procalcitonin, blood cultures, nasal and pharyngeal swabs, urine culture). the routine renal and liver tests were also normal. no abnormalities were noted on fundoscopy. we initiated treatment (as per the national protocol) with hydroxychloroquine 400 mg twice a day on the first day, then 200 mg twice a day until day 10, azithromycin 500 mg/day for 5 days, with antifungal protection, and lopinavir/ritonavir 200/50 mg 2 tablets twice a day for 7 days. the patient became apyrexial on the second day of treatment. on the third day of treatment anosmia occurred (persisted for 6 weeks) and on the fourth day she reported dry cough, mild dyspnea, and diarrhea. crepitations were heard on auscultation of both lung bases. the oxygen saturation was within normal limits (96%-98%). systemic pulse corticosteroid therapy was given (methylprednisolone 120 mg intravenous daily for 3 days), under which the dyspnea and cough improved and no more rales are heard. the patient was discharged after a two-week hospital stay, following 2 negative sars-cov-2 tests (performed 24 hours apart). the diarrhea resolved 1 month after onset. one day after discharge an erythematous rash appeared initially on the trunk and disseminated over the next 5 days, centrifugally, to the proximal limbs. dermatological examination describes a disseminated erythematous maculopapular rash, purpuric in appearance, mildly pruritic, with a tendency to confluency. the clinical appearance suggests a viral exanthema in the context of sars-cov-2 infection (fig. 1a) . vitamin c and topical corticosteroids of medium potency were administered during the first days, followed by emollient, hydrating lotion thereafter. the rash extended centrifugally but spared the face, palms, and soles, as well as the mucous membranes. after 5 days of treatment the papule disappeared, the erythema improved but the purpuric appearance and mild pruritus persisted, along with the residual pink-brown macule (fig. 1b) . after 10 days, the exanthema disappeared almost completely, except for the persistence of discrete pink macule on the abdomen (fig. 1c) . follow-up at 1 month showed complete resolution of the rash, no respiratory symptoms but persistence of anosmia. the sense of smell returned partially 6 weeks after onset of anosmia. antibody testing for sars-cov-2 was undertaken at 4 and 6 weeks follow-up. igm were undetectable but igg were detectable -106.87 au/ml at 4 weeks and 117 au/ml at 6 weeks (rv under 10 au/ml). the patient donated plasma. the clinical manifestations of the sars-cov-2 infection are mainly respiratory, but also with multi-organ damage in variable proportions. in a study of 138 patients hospitalized in wuhan the clinical features identified at onset were: 99% fever (not as common in other studies), 70% fatigue, 59% dry cough, 40% anorexia, 35% myalgias, 31% dyspnea, 27% sputum production. [9] in a study of over 5000 patients in new york, only 31% had a temperature of over 38°c at presentation. [10] anosmia and dysgeusia have been identified as common symptoms in covid-19. in a survey of 202 outpatients from italy 64% reported alteration of smell or taste. [11] gastrointestinal symptoms (abdominal pain, nausea/vomiting, or diarrhea) were found in 18% of the patients in a systematic review. [12] in our case, the initial symptoms were predominantly respiratory, then digestive (diarrhea), neurological (anosmia), without any other organ dysfunction. mild lymphopenia was noted but the neutrophils to lymphocytes ratio of 2.789 was interpreted as a favorable prognostic factor. a neutrophils to lymphocytes ratio of over 3.3 is cited in literature as a negative prognostic factor. [13] using the online method suggested by liang et al for calculating the risk of progression of sars-cov-2 infection towards severe disease, we obtained a moderate risk in our case. [14] during the evolution of the case atypical cutaneous manifestations occurred, with rapid dissemination of an erythematous maculopapular, purpuric-like, mildly pruritic rash. exanthems are disseminated erythematous eruptions caused by medications (antibiotics, anticonvulsivants, tuberculostatics, antihypertensives, etc.), infections (respiratory viruses, enteroviruses, parvoviruses, herpesviruses, hiv, etc.), toxins released by some microorganisms (like staphylococcal scalded skin syndrome) or by autoimmune disease. [15, 16] viral exanthems secondary to sars-cov-2 infection are rarely described in literature. the first dermatological manifestations described in the context of sars-cov-2 infection were chilblain lesions on the plantar extremities and livedo reticularis. during the last months more types of cutaneous lesions were described in infected patients: urticarial, chickenpox-like, [17] vasculitic, morbilliform, [18] etc. a meta-analysis of 19 studies published by sachdeva et al, showed that cutaneous manifestations can precede respiratory ones by 3 days (12.50% of cases) or can appear after 13 days (69.40%) from the onset of respiratory manifestations in sars-cov-2. [8] the medium interval for the appearance of cutaneous lesions was 9.2 days and the medium interval to remission was 8.7 days, according to a study of 132 covid 19 positive patients with chilblain and erythema multiforme-like lesions, published by fernandez-nieto et al. [19] in our case the exanthema appeared 15 days from the onset of symtpoms and remitted after 10 days. although there was a procoagulation status in our case (slightly raised d-dimmers), there were no acro-ischemic or vasculitic lesions. the absence of vasculitic lesions in this case suggests the fact that the pathogenetic mecanism of the viral exanthema was an immune response to the viral nucleotides. we excluded a secondary drug reaction by the lack of "en cocarde" lesions, mucosal involvement and pruritus. in order to exclude other causes of viral exanthema we conducted serological tests (igm for parvovirus b19, human herpesvirus-6, 7, and epstein barr virus) which were negative. the pharyngeal swab was negative for streptococcus and the mildly elevated inflammatory markers along with the absence of the specific enanthema effectively excluded scarlet fever. particular about this case is the persistence of anosmia for 6 weeks after disease onset. aside from the respiratory tropism of the sars-cov-2 virus, which dominates the scientific interest at the moment, we must consider that sars-cov-2 has a multi-organ tropism. in our case the spectrum of manifestations included the respiratory, neurological, digestive, and skin lesions. identifying the manifestations known so far is useful to better understand the real extent of sars-cov-2 infection. we not only present a rare manifestation but also suggest that cutaneous manifestations may correlate with immunity. gabriela mariana iancu is responsible for the acquisition, analysis and interpretation of data, concept and design, investigation, methodology, resources, critically revising and validation the manuscript. adelaida solomon is responsible for the acquisition, analysis and interpretation of data, investigation, methodology, resources and validation the manuscript. victoria birlutiu is responsible for the concept, design, investigation, methodology, resources, critically revising and validation the manuscript. all authors contribute to writing, reviewing & editing the original draft. all authors read and approved the final manuscript. who director-general's remarks at the media briefing on 2019-ncov on 11 clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of coronavirus disease 2019 in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention who-director-general-s-opening-remarks-at-the-media-briefing-oncovid presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area uncomplicated drug-induced disseminated exanthemas molecular biology and clinical associations of roseoloviruses human herpes virus 6 and human herpes virus 7 classification of the cutaneous manifestations of covid-19: a rapid prospective nationwide consensus study in spain with 375 cases cutaneous manifestations in covid-19: lessons learned from current evidence cutaneous manifestations of covid-19: report of three cases and a review of literature clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china development and validation of a clinical risk score to predict the occurrence of critical illness in hospitalized patients with covid-19 varicella like exanthem as a specific covid-19-associated skin manifestation: multicenter case series of 22 patients alterations in smell or taste in mildly symptomatic outpatients with sars-cov-2 infection medicine (2020) 99:35 www.md-journal gastrointestinal manifestations of sars-cov-2 infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis characterization of acute acro-ischemic lesions in non-hospitalized patients: a case series of 132 patients during the covid-19 outbreak morbilliform exanthem associated with covid-19 the diagnostic and predictive role of nlr, d-nlr and plr in covid-19 patients the authors have no conflicts of interests to disclose.data sharing not applicable to this article as no datasets were generated or analyzed during the current study. key: cord-268206-ino9srb6 authors: hamed, manal a. title: an overview on covid-19: reality and expectation date: 2020-06-01 journal: bull natl res cent doi: 10.1186/s42269-020-00341-9 sha: doc_id: 268206 cord_uid: ino9srb6 recently, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), commonly known as coronavirus disease-2019 (covid-19) has rapidly spread across china and around the world. by the declaration of who, covid-19 outbreak considered as a public health problem of international concern. the aim of this study is to provide a comprehensive view on covid-19 and the future expectations to control virus progression. patients with liver disease, diabetes, high blood pressure, and obesity are more susceptible to the incidence of covid-19 infection. so, there is a rapid need for disease diagnosis, vaccine development, and drug discovery to detect, prevent, and treat this sudden and lethal virus. real-time polymerase chain reaction (rt-pcr) is considered as a rapid, accurate, and specific tool for disease diagnosis. under this emergency situation that the world facing against covid-19, there are about 15 potential vaccine candidates tested globally based on messenger rna, dna-based, nanoparticle, synthetic, and modified virus-like particle. certain drugs that are clinically approved for other diseases were tested against covid-19 as chloroquine, hydroxychloroquine, ivermectin, favipiravir, ribavirin, and remdesivir. convalescent plasma transfusion and traditional herbal medicine were also taken into consideration. due to the absence of effective treatment or vaccines against covid-19 so far, the precautionary measures according to who’s strategic objectives are the only way to confront this crisis. governments should adopt national medical care programs to reduce the risk of exposure to any future viral outbreaks especially to patients with pre-existing medical conditions. coronaviruses have been recognized for over 50 years. the word "corona" has many different meanings, but it was the sun that the virologists had in mind when they chose the name coronaviruses by comparing the characteristic projections on the outside of the virus with the solar corona. coronaviruses cause severe acute respiratory syndrome (sara) leading to death in most cases. coronaviruses are single-stranded rna viruses, about 120 nm in diameter. they are susceptible to mutation and are therefore highly diverse. they mainly infect human and non-human mammals and birds. the first coronaviruses found to infect humans were called 229e and oc43 in 1968, but they caused very mild infections until the outbreaks of severe acute respiratory syndrome (sars-cov-1) in 2003, mers (middle eastern respiratory syndrome) in 2012 and sars-cov-2 (covid-19) in 2019 which caused serious human infections (aronson 2020) . the virus that causes covid-19 (sars-cov-2) is thought to have originated in bats and then spread to humans through contamination of meat sold in china's meat markets with wild animals' wastes. the coronavirus syndrome is caused by spike glycoproteins, which are necessary for the viruses to enter host cells. the spike has two subunits: one subunit, s1, binds to a receptor on the surface of the host's cell; the other subunit, s2, fuses with the cell membrane. the cell membrane receptor is a form of angiotensin converting enzyme (ace-2). briefly, the s1 subunit of the spike binds to the ace-2 enzyme on the cell membrane surface, the host transmembrane serine protease (tmprss2) activates the spike and cleaves ace-2, and the tmprss2 acts on the s2 subunit, facilitating fusion of the virus to the cell membrane and then enters the cell. inside the cell, the virus is released from endosomes by acidification or the action of an intracellular cysteine protease (cathepsin), where it replicate and affect many organs especially the lung ( fig. 1) (zhong et al. 2003; zaki et al. 2012; aronson 2020; ecdc 2020a ecdc , 2020b chen et al. 2020; huang et al. 2020; zhu et al. 2020) . the aim of this study is to provide an overview on a newly coronavirus appeared in 2019 with future expectations to counteract its severity and progression. on 31 december 2019, a cluster of pneumonia cases of unknown etiology was reported in wuhan, hubei province, china. on 9 january 2020, china reported a novel coronavirus as the causative agent of this outbreak (coronavirus disease-2019) which is commonly known as covid-19 (ecdc 2020a) . the initial reported cases in wuhan mentioned that the majority of coronavirus cases were males with a median age of 55 years and linked to the huanan seafood wholesale market . most of these cases had similar symptoms like fever, cough, fatigue, myalgia, and diarrhea. many cases developed pneumonia, and some had severe and fatal respiratory diseases . on 25 march 2020, more than 416,916 cases of covid-19 were reported worldwide by more than 150 countries, where the latest country confirmed a covid-19 case outside of china is egypt (ecdc 2020a). on 8 april 2020, 1,391,890 cases of covid-19 have been reported, including 81,478 deaths (ecdc 2020a). the novel covid-19 coronavirus is a zoonotic β-coronavirus that belongs to the subgenus: sarbecovirus of the orthocoronavirinae subfamily . the severe acute respiratory syndrome coronavirus (sars-cov-1) and middle east respiratory syndrome coronavirus (mers-cov) are also zoonotic β-coronaviruses associated with the (zhong et al., 2003; zaki et al. 2012) . although the pathogenicity of covid-19, sars-cov-1, and mers-cov are 3%, 10%, and 40%, respectively (chen 2020), covid-19 has 1.4-5.5 high transmission rate than sars-cov-1 (2-5) and mers-cov (< 1) (chen 2020) . the mean incubation period for covid-19 is widely varies between individuals (5.2-14 days), so further investigations are needed to better understand the viral shedding time to inform optimal specimen collection for diagnosis . it was recorded that 2-10% of patients with covid-19 had sars-cov-2 rna in stool and blood samples which implicated the possibility of viral exposure in the liver . this was supported by the same authors through a marked elevation of liver function enzymes (aspartate and alanine aminotransferases) in covid-19 patients. in addition, gamma glutamyl transferase (ggt), a diagnostic biomarker for cholangiocyte injury, was elevated in 30 (54%) of 56 patients with covid-19. due to the ubiquitous distribution of the main viral entry receptor, namely, angiotensin converting enzyme 2 (ace2), sars-cov-2 causes a systemic disease, with possible involvement of the heart, the liver, the pancreas, and the kidneys, as well as determines alterations in circulating lymphocytes and the immune system . chai et al. (2020) stated that ace2 expression was enriched in cholangiocytes patients. this observation is indicating that sars-cov-2 might directly bind to ace2-positive cholangiocytes to dysregulate liver function. moreover, zhang et al. (2020) postulated that patients with liver cirrhosis or liver cancer might be more susceptible to sars-cov-2 infection because of their systemic immunocompromised status. zhao et al. (2020) reported a case of covid-19 with a history of co-infection of hiv-1 and hcv that showed delayed antibody response. this case highlights the possible influence of hiv-1-induced immune dysfunction on the immune responses to and clearance of sars-cov-2. one potential explanation is that he was taking anti-hiv-1 agents which had been reported to have anti-sars-cov-2 effects (martinez 2020) . another possibility is that the activated type i interferon (ifn-i) may help suppress sars-cov-2. previous study has shown that hiv-1 infection may induce high levels of ifn-i, which may to some extent clear sars-cov-2 infection, thus leading to persistently undetectable rna (tang et al. 2020b) . diabetes and uncontrolled glycaemia were reported as significant predictors of severity and deaths in patients infected with different viruses including the pandemic influenza a (h1n1), sars-cov, and mers-cov (yang et al. 2006; banik et al. 2016 ). in the current sars-cov-2 pandemic, wu and mcgoogan (2020) showed that patients with chronic diseases, including diabetes, were at higher risk for severe covid-19 infection and mortality. infection of sars-cov-2 in those with diabetes possibly triggers higher stress conditions, with greater release of hyperglycemic hormones as glucocorticoids and catecholamines, leading to increased blood glucose levels and abnormal glucose variability ). in another study, patients with type 2 diabetes and covid-19 were reported that they suffered from hypoglycemia (zhou and tan 2020) . hypoglycemia has been shown to mobilize pro-inflammatory monocytes, increase platelet reactivity, and therefore contributing to a higher cardiovascular mortality (iqbal et al. 2019 ). yet, it remains unknown how the inflammatory and immune response occurs in these patients, as well as whether hyper or hypoglycemia may alter the sars-cov-2 virulence, or the virus itself interferes with insulin secretion or glycemic control. wang et al. (2020b) explained that the metabolic disorders in diabetic patients reduce immune response by impairing macrophage and lymphocyte functions which might subsequently render individuals more susceptible to infectious viral disease complications. hypertension is a major risk factor of mortality worldwide, and its importance is further emphasized in the context of covid-19. patients with severe covid-19 infections commonly are older and have a history of hypertension. almost 75% of patients who have died in the pandemic in italy and china had hypertension (kreutz et al. 2020) . as previously mentioned that ace-2 level increased in sars-cov-2 patients to facilitate viral entry to host cells, wan et al. (2020) added that the expression of ace2 is also increased in patients with hypertension and diabetes due to treatment with ace-2 increasing drug that are at high risk of covid-19 and therefore should be treated with ace inhibitors or angiotensin ii type-i receptor blockers (arbs). leggio et al. (2017) and oyekale (2019) added that there is a significant association between obesity and hypertension. until now, there is no clear evident suggesting direct association between obesity and surveillance of covid-19. indirect association between obesity and covid-19 is monitored, where obesity is associated with decreased expiratory reserve volume, functional capacity, and respiratory system compliance. in patients with increased abdominal obesity, pulmonary function is further compromised by decreased diaphragmatic excursion, thus making ventilation more difficult. so, dietz et al. (2020) mentioned the impact of obesity on pulmonary function that increases the risk of covid-19 in obese patients. furthermore, increased inflammatory cytokines associated with obesity may contribute to the increased morbidity associated with obesity in covid-19 infections (dietz et al. 2020) . therefore, patients with obesity were more likely to need an intensive care unit for acute lung injury and might have prolonged mechanical ventilation and hospital stay, compared with patients with normal weight. in addition to the detrimental effects on lung function, obesity is a confirmed cause of diabetes and cardiovascular disease and higher overall mortality (qingxian et al. 2020) . by the declaration of the world health organization that covid-19 outbreak is a public health emergency of international concern, there is a rapid need of disease diagnosis, vaccine development, and drug discovery to detect, prevent, and treat covid-19. thereafter, realtime pcr (rt-pcr) is considered as a golden tool for diagnosing corman et al. 2020) . the period and type of specimen collected for rt-pcr play an important role in the diagnosis of covid-19. it was found that the respiratory specimens were positive for the virus, while serum was negative in the early period. it has also suggested that patients have high levels of virus in the early days of viral infection rather than in the late stage (pang 2020) . rapid collection and testing of specimens are important and should be guided by a laboratory expert ). there are seven rapid rt-pcr diagnostic kits available on the market for covid-19. six of these kits are for research purposes, while only one from beijing genome institute (bgi) (china) is approved for clinical diagnosis (pang et al. 2020) . additionally, there are two kits (bgi, china and veredus, singapore) that have the capability to detect covid-19 and another pathogens using sequencing and microarray technologies, respectively (pang et al. 2020) . with the emergency situation that the world facing against covid-19, there are about 15 potential vaccine candidates tested globally based on messenger rna, dna-based, nanoparticle, synthetic, and modified viruslike particle. coronavirus expresses several structural proteins, including nucleocapsid, membrane, envelope, and spike (s) proteins (peiris et al. 2004) . each one of them may serve as antigen to induce neutralizing antibodies and protective responses. prior to identification of the protein that contains the major neutralizing epitopes, the inactivated virus may be used as a vaccine because it is easy to generate whole killed virus particles. once the neutralizing epitopes are identified, the inactivated virus vaccine should be replaced by vaccines based on fragments containing neutralizing epitopes as they become more effective. the virus was inactivated by formaldehyde, uv light, and β-propiolactone to induce virus-neutralizing antibodies in animals qu et al. 2005 ). the first inactivated vaccine is being tested in the clinical trials in china. the safety of the inactivated vaccine production is limited due to the highly risk of infection exposure to workers during handling of concentrated live covid-19. additionally, the incomplete virus inactivation may cause virus outbreaks of the vaccinated people. moreover, some viral proteins may cause harmful immune or inflammatory responses and causing coronavirus-like diseases (wang and lu 2004) . the s protein of the virus, a type i transmembrane glycoprotein, is responsible for virus binding, fusion, and entry and is a major inducer of neutralizing antibodies (holmes 2003) . s protein consists of a signal peptide and 3 domains (extracellular, transmembrane, and an intracellular domain). its extracellular domain consists of 2 subunits: s1 and s2 (holmes 2003) . the s1 subunit is responsible for virus binding to the receptor, angiotensin-converting enzyme 2 . a fragment located in the middle region of the s1 subunit is the receptor-binding domain for angiotensin-converting enzyme 2 . the s2 subunit, which contains a putative fusion peptide and 2 heptad repeats, is responsible for fusion between the virus and the target cell membranes. infection by coronavirus is initiated by binding of the receptor binding domain in the viral s protein s1 subunit to angiotensin-converting enzyme 2 on target cells. this forms a fusogenic core between the 2 heptad repeats regions in the s2 domain that brings the viral and target cell membranes into close which results in virus fusion and entry tripet et al. 2004 ). this remark indicates that the s protein may be used as a vaccine to induce antibodies for blocking virus binding and fusion. in case of covid-19, tang et al. (2020a) postulated that one non-synonymous snp (single nucleotide polymorphism) was detected in gene encodes s (spike) protein in covid-19. this snp replaced serine amino acid by leucine, and for this reason, they called the two new strains: (s strain) and (l strain). the former (s) is the wild type which is milder while the latter (l) is the novel one which resulted in high binding affinity between sars-cov-2 virus with angiotensin-converting enzyme 2 receptor in human cells. the l strain is responsible for pandemic infection and never be seen in any previous version of corona (sars, mers) and also in bat, pangolin, civet, camel. many studies have recently confirmed the genetic similarity (96.2%) between covid-19 (sars-cov-2) and a bat sars-related coronavirus (sars-cov-1). it has also been confirmed that the sars-cov-2 uses the hamed bulletin of the national research centre (2020) 44:86 page 4 of 10 same receptor: the angiotensin converting enzyme 2, as the sars-cov-1 (gralinski et al. 2020; paraskevis et al. 2020) . bacillus calmette-guérin (bcg) is a well-known vaccine against tuberculosis (tb). hegarty et al. (2020) found that the bcg vaccinated healthy controls rechallenged with yellow fever virus showed improvement in anti-viral immunity and decrease in viral loads. accidentally, the same authors found that the incidence of covid-19 in 131 countries applied the national programs of bcg vaccination was 38.4 per million compared to 358.4 per million in countries not applied such a vaccination program. also, the death rate was 4.28/ million in countries with bcg programs compared to 40/million in countries without such a program. however, there is no clear evidence that the bcg vaccine protects people against infection with covid-19 virus. based on the current lack of an approved and effective vaccine for covid-19, it is important to evaluate certain drugs (fig. 2) that are clinically approved for other diseases against the new coronavirus. chloroquine (cq) and its derivative, hydroxychloroquine (hcq), are considered as prophylactic drugs against malaria and as treatments for autoimmune diseases ). previous studies have revealed that chloroquine has therapeutic activity against viruses, including human coronavirus in animal models and sars-cov-1 in cell culture studies (savarino et al. 2003; keyaerts et al. 2009 ). the mechanisms through which chloroquine may act to attenuate sars-cov-2 infections may be valuable for identifying new prophylactic and therapeutic agents. chloroquine is a weak base that becomes entrapped in membrane-enclosed low ph organelles, and interfering with their acidification (hempelmann 2007) . in case of viral infection, chloroquine may induced antiviral effects by inhibition of ph-dependent viral fusion/replication and prevention of viral envelope glycoprotein as well as host receptor protein glycosylation. chloroquine may also inhibit virion assembly in endoplasmic reticulum-golgi intermediate compartment-like structures. additionally, chloroquine may directly affect the host by attenuating the expression of pro-inflammatory factors and receptors that can induce acute respiratory syndrome, which is primarily responsible for coronavirusassociated mortality ). guastalegname and vallone (2020) concluded that no acute virus infection has been successfully treated by cq/hcq in human. chloroquine and hydroxychloroquine did not show any anti sars-cov-2 effect on in vivo. as the pathogenesis of covid-19 is still unknown, therefore, the immune effect provoked by chloroquine or hydroxychloroquine administration in covid-19 patients is unpredictable. ivermectin is an fda-approved broad spectrum antiparasitic agent. in recent years, it shows anti-viral activity against a broad range of viruses in vitro. studies on sars-cov proteins have revealed a potential role for importinα/β1 (impα/β1) that recognize nuclear localization sequences during infection in signal-dependent nucleocytoplasmic shutting of the sars-cov nucleocapsid protein (wulan et al. 2015) , and that may impact host cell division (wurm et al. 2001 ). in addition, the sars-cov accessory protein (orf6) has been shown to antagonize the antiviral activity of the signal transducer and activator of transcription (stat) factor by sequestering impα/β1 on the rough er/golgi membrane (frieman et al. 2007 ). therefore, the ivermectin nuclear transport inhibitory effect may be effective against sars-cov-2. caly et al. (2020) added that ivermectin acted as an inhibitor of sars-cov-2, with a single addition to vero-hslam cells 2 h post infection and showed~5000-fold reduction in viral rna at 48 h. favipiravir (t-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was discovered by chemical modification of a pyrazine analog initially screened by in vitro antiinfluenza virus activity in cells. favipiravir is an anti-viral agent that selectively and potently inhibits the rnadependent rna polymerase (rdrp) of rna viruses. favipiravir undergoes an intracellular phosphoribosylation to be an active form, favipiravir-rtp (favipiravir ribofuranosyl-5b-triphosphate), which is recognized as a substrate by rdrp, and inhibits the rna polymerase activity. the mechanism of action of favipiravir depends on inhibition of viral replication genome, which manifested in the middle of viral proliferation cycle. the antiviral activity of favipiravir was attenuated in the presence of purine bases, indicating its competition with purine rather than pyrimidine nucleosides. therefore, favipiravir with these unique profiles will be a promising therapeutic agents of rna viruses (furuta et al. 2017) . favipiravir was approved in japan as an influenza antiviral drug in 2014. the drug is considered to use when there is an outbreak of influenza virus infections in which other influenza drugs are either not effective or insufficiently effective. the drug may potentially have an antiviral effect on the novel coronavirus as it is classified into the same type of a single-stranded rna virus as the common influenza virus. however, the clinical application of favipiravir to treat coronavirus disease (covid-19) is under examination for clear evidence of its efficacy and safety (fujifilm 2020) . nitazoxanide is originally developed as an antiprotozoal agent treatment of intestinal infections caused by cryptosporidium parvum. nitazoxanide is a broad-spectrum antiviral agent undergoing clinical development for treatment of influenza and other viral respiratory infections (rossignol 2016) . it is presently undergoing phase 3 clinical development for treating acute uncomplicated influenza (rossignol 2014) . it exhibits in vitro activity against mers-cov and other coronaviruses by inhibiting expression of the viral protein. nitazoxanide also suppresses production of proinflammatory cytokines in peripheral blood mononuclear cells and interleukin 6 production. as it is used extensively in clinical trials, nitazoxanide may be tested as a treatment against sars-cov-2 (rossignol 2014). momattin et al. (2013) mentioned that there were seven reports of the use of ribavirin as a treatment in sars patients, while two studies showed improvements of symptoms in 71.4%-80% of patients. khalili et al. (2020) proved the utility of ribavirin in the treatment of covid-19 patients. the major problem with ribavirin was the incidence of hemolysis. interferon treatment led to improvements in clinical symptoms in sars patients, however, no advantage of ribavirin over interferon in patients with sars. the addition of lopinavir/ritonavir to ribavirin regimen was associated with improved clinical outcome and reduces the death rate comparing to ribavirin regimen alone (momattin et al. 2013) . remdesivir has been recently recognized as a promising antiviral drug against a wide array of rna viruses (including sars/mers-cov2) infection in cultured cells, mice, and non-human primate models (wang et al. 2020c) . remdesivir is an adenosine analogue, which incorporates into nascent viral rna chains and results in pre-mature termination (warren et al. 2016) . wang et al. (2020c) showed that remdesivir functioned at a stage post virus entry, which is in agreement with its putative antiviral mechanism as a nucleotide analogue. the same authors showed that the ec90 value of remdesivir against covid-19 in vero e6 cells was 1.76 μm, suggesting its working concentration is likely to be achieved as in non-human primates studied before. additionally, remdesivir also inhibited virus infection efficiently in a human liver cancer huh-7 cells, which is sensitive to covid-19. as previously mentioned, the novel coronavirus is a positive-stranded rna with structural proteins as spike protein (s), envelope protein (e), membrane protein (m), nucleocapsid phosphoprotein, and transcribed nonstructural proteins as orf1ab, orf3a, orf6, orf7a, orf10, and orf8 . the same authors showed that the orf8 and surface glycoprotein could bind to the porphyrin, respectively. at the same time, orf1ab, orf10, and orf3a proteins attack the heme on the 1-beta chain of hemoglobin to dissociate the iron to form the porphyrin. this attack causes less hemoglobin that carry oxygen and carbon dioxide. therefore, the lung cells have intense poisoning and inflammation due to its inability to exchange carbon dioxide and oxygen easily. according to these observations, chloroquine could prevent orf1ab, orf3a, and orf10 to attack the heme and inhibit the binding of both orf8 and surface glycoproteins to porphyrins that effectively relieve the symptoms of respiratory infection. favipiravir could inhibit the envelope protein and orf7a protein bind to porphyrin, prevents the virus from entering host cells, and catching free porphyrins. as liu and li (2020) confirmed this hypothesis that the novel coronavirus is dependent on porphyrins, it may lead scientists to discover new drugs or confirmed chloroquine and favipiravir for disease prevention or clinical treatment depending on inhibition of viral attach to heme to prevent the state of hypoxia. the therapeutic use of convalescent plasma donated by patients recovered from covid-19 might play a role in the efforts to find a possible treatment for covid-19 (ecdc 2020b) . the use of convalescent plasma was recommended before as an important treatment during outbreaks of ebola virus, middle east respiratory syndrome coronavirus, sars-cov-1, h5n1 avian influenza, and h1n1 influenza (zhou et al. 2007; hung et al. 2011; kraft et al. 2015) . in a study involving patients with pandemic influenza (h1n1) and sars virus, treatment of severe infection with convalescent plasma was associated with reduced respiratory viral load, serum cytokine response, and mortality (cheng et al. 2005; hung et al. 2011) . accordingly, these findings raise the hypothesis that use of convalescent plasma transfusion could be beneficial in patients infected with sars-cov-2. however, the risk of covid-19 transmission via plasma transfusion must be seriously consider through the uncertainties viraemia during the incubation period, an asymptomatic course of infection, viral plasma contamination, patients with liver or kidney transplantation, and the 14 days of recovery period of donor after symptom resolution with negative results of repeated antiviral tests . therefore, precautionary measures are suggested to mitigate these risks. the close similarity between covid-19 and sars-cov-1 through their rapid transmission, their genome sequences, and the common entrance of their spike protein to alveolar epithelial cells through binding with ace-2 enzyme receptor, all these encourage chinese researchers to treat covid-19 patients with traditional chinese medicine (tcm) used for sars-cov-1 before . as 3-chymotrypsin-like protease (3clpro) is a vital compound for virus replication, thereafter, water extract of houttuynia cordata, flavonoid extracted from litchi seeds, beta-sitosterol extracted from the root of isatis indigotica, naturally occurring sinigrin, indigo aloeemodin, hesperetin, quercetin, epigallocatechin gallate, gallocatechin gallate, herbacetin, rhoifolin, and pectolinarin were able to inhibit the sars 3clpro activity. moreover, flavonoids compounds as herbacetin, isobavaschalcone, quercetin 3β-d-glucoside, and helichrysetin had the potential to block the 3clpro activity of mers-cov (lin et al. 2005; lau et al. 2008; gong et al. 2008; luo et al. 2009; fung et al. 2011; nguyen et al. 2012; jo et al. 2019 and . in addition, many studies focused on the efficiency of tcm to target angiotensin converting enzyme 2 to prevent the infection of covid-19 like emodin from genus rheum and polygonum, baicalin from in scutellaria baicalensis, nicotianamine from soybean, tetra-o-galloyl-β-d-glucose from galla chinensis, and luteolin from veronicalina riifolia (yi et al. 2004; ho et al. 2007; deng et al. 2012; takahashi et al. 2015; wang et al. 2016) . moreover, the anti-inflammatory herbs as lonicerae japonicae flos, scutellariae radix, fructus forsythiae, and curcuma longa as well as the biologically active compounds hesperidin and naringenin isolated from citrus fruits could reduce the severity and mortality rate by their effect on the transcriptional and translational levels of inflammatory cytokines tnf-α, il-1β, and il-6 (chen et al. 2002; gao et al. 2014; el-rafie et al. 2014; hamed et al. 2019 and lu 2020; zou 2020) . due to the absence of effective treatment or vaccines against covid-19, the precautionary measures are the only way to confront this crisis. transmission of coronaviruses from contaminated dry surfaces has been postulated including self-inoculation of mucous membranes of the nose, eyes, or mouth (kampf et al. 2020) . human coronaviruses can remain infectious on inanimate surfaces for up to 9 days. so, various types of biocidal agents such as hydrogen peroxide, alcohols, sodium hypochlorite, or benzalkonium chloride are used worldwide for disinfection mainly in healthcare settings (kampf 2018) . social isolation, distancing, or quarantine of entire communities may be useful. nonetheless, these measures should be implemented in a prudent fashion while considering their cost efficiency. also, there is a real need to avoid the epidemic wave that would saturate the capacity of health services. moreover, it is important to note that collective infection control measures can actually reduce the frequency of infection (raoult et al. 2020) . the 78th situation report of who postulated the strategic objective control for covid-19 which are (1) interrupt human-to-human transmission including reducing secondary infections among close contacts and health care workers, preventing transmission amplification events, and preventing further international spread; (2) identify, isolate, and care for patients early, including providing optimized care for infected patients; (3) identify and reduce transmission from the animal source; (4) address crucial unknowns regarding clinical severity, extent of transmission and infection, treatment options, and accelerate the development of diagnostics, therapeutics and vaccines; (5) communicate critical risk and event information to all communities and counter misinformation; and (6) minimize social and economic impact through multisectoral partnerships (who 2020) . this can be achieved through a combination of public health measures, such as rapid identification, diagnosis and management of the cases, identification and followup of the contacts, infection prevention and control in health care settings, implementation of health measures for travelers, awareness-raising in the population, and risk communication (who 2020). covid-19 outbreak is a public health emergency of international concern. patients with liver disease, diabetes, high blood pressure, and obesity are more susceptible to the incidence of covid-19 infection. disease diagnosis, vaccines, and drug discovery are essential to control this pandemic situation. rt-pcr is considered as the most accurate and specific technique for 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patients with pneumonia in china coronavirus: chinese researchers claim tcm herbal remedy could 'inhibit' 2019-ncov i would like to acknowledge the national research centre for supporting this work.author's contributions i am the sole author of this review. the author read and approved the final manuscript. not applicable.availability of data and materials not applicable. this manuscript is a review article and does not involve a research protocol requiring approval by ethics committee. not applicable. the author declare that she has no competing interests.received: 23 april 2020 accepted: 20 may 2020 key: cord-031079-9lxhvyyb authors: chen, li; chen, haiyan; dong, shan; huang, wei; chen, li; wei, yuan; shi, liping; li, jinying; zhu, fengfeng; zhu, zhu; wang, yiyang; lv, xiuxiu; yu, xiaohui; li, hongmei; wei, wei; zhang, keke; zhu, lihong; qu, chen; hong, jian; hu, chaofeng; dong, jun; qi, renbin; lu, daxiang; wang, huadong; peng, shuang; hao, guang title: the effects of chloroquine and hydroxychloroquine on ace2 related coronavirus pathology and the cardiovascular system: an evidence based review date: 2020-07-27 journal: function (oxf) doi: 10.1093/function/zqaa012 sha: doc_id: 31079 cord_uid: 9lxhvyyb the ongoing pandemic of coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) poses a serious threat to global public health and there is currently no effective antiviral therapy. it has been suggested that chloroquine (cq) and hydroxychloroquine (hcq), which were primarily employed as prophylaxis and treatment for malaria, could be used to treat covid-19. cq and hcq may be potential inhibitors of sars-cov-2 entry into host cells, which is mediated via the angiotensin-converting enzyme 2 (ace2), and may also inhibit subsequent intracellular processes which lead to covid-19, including damage to the cardiovascular system. however, paradoxically, cq and hcq have also been reported to cause damage to the cardiovascular system. in this review, we provide a critical examination of the published evidence. cq and hcq could potentially be useful drugs in the treatment of covid-19 and other ace2 involved virus infections, but the antiviral effects of cq and hcq need to be tested in more well-designed clinical randomized studies and their actions on the cardiovascular system need to be further elucidated. however, even if it were to turn out that cq and hcq are not useful drugs in practice, further studies of their mechanism of action could be helpful in improving our understanding of covid-19 pathology. the coronavirus disease 2019 (covid-19) is due to infection by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). [1] [2] [3] the most common symptoms of covid-19 are fever and cough. 4, 5 both human-to-human and asymptomatic transmission have been reported. 6 the covid-19 pandemic has rapidly evolved into a global health crisis as there is currently no proven drug for treating coronavirus patients. however, the strategy of drug repurposing may offer hope for a new approach to covid-19 treatment. among the myriad existing drugs that are potential repurposing candidates for treating covid-19, the immunomodulatory agents chloroquine (cq) and hydroxychloroquine (hcq) have captured great attention. cq and its more soluble and less toxic metabolite hcq are primarily used for prophylaxis and treatment of malaria, but they have also been reported to effectively inhibit the effects of certain viruses, such as severe acute respiratory syndrome coronavirus (sars-cov) and influenza a h5n. [7] [8] [9] [10] recently, the possible use of cq/hcq as a repurposed therapeutic agent against covid-19 has been explored. 11 angiotensin-converting enzyme 2 (ace2), a new homolog of ace, can convert angiotensin ii (ang ii) to ang(1-7) 12, 13 . ang(1-7) binds and activates the g-protein coupled receptor mas (masr) 14 and acts as a natural damping mechanism for the activation of the classical renin-angiotensin system (ras), 12, 13, 15, 16 which plays a critical role in maintaining normal cardiovascular (cv) functions. apart from its crucial role in cv disease, ace2 has also been shown to be a functional host cellular entry receptor for coronavirus that directly binds the viral spike (s) protein, which is primed by the transmembrane serine protease 2 (tmprss2). [17] [18] [19] [20] the ongoing covid-19 pandemic, caused by sars-cov-2, poses a serious threat to global public health, and cross-sectional data suggest that sars-cov-2 infected patients have a high prevalence of cv disease. 21, 22 recent data indicated that cq and hcq (cq/hcq) may have a promising ability to inhibit sars-cov-2 and other ace2 related viral diseases [7] [8] [9] , but the effects of cq/hcq on the cv system seem paradoxical. cq/hcq shows cv benefits, including a reduction in the risk of developing hyperlipidemia and diabetes mellitus, but cv disorder has also been reported as one of the rare but severe side effects of cq/hcq. 23 in this review, we summarize and evaluate the published evidence concerning the actions and mechanisms of action of cq/hcq in treating sars-cov-2 and other ace2 related viral infections. we conclude that further mechanistic studies as well as well-designed clinical randomized trials are needed to investigate the molecular pathogenesis of sars-cov-2 infection and to examine the antiviral efficacy of cq/hcq against covid-19. furthermore, the effects and mechanisms of action of cq/hcq on the cv system should be further investigated. the ras is a humoral regulation cascade that elegantly orchestrates key vascular physiology in humans. sars-cov-2 infection has been proposed to interfere with ras through the ace2 receptor for host cell entry (figure 1) . 19, 24 severe covid-19 infection has many clinical characteristics which are strikingly similar to the effects of overactivation of the ras. it has been reported that coagulation is activated and accelerated in patients with sars-cov-2. 25 the complex entry process of coronavirus into susceptible cells requires multistep actions of receptor-binding and proteolytic processing of the s protein to promote virus-cell fusion. s protein cleavage occurs at the boundary between the s1 and s2 subunits, and s is further cleaved at the s2' site by host proteases to facilitate the fusion of viral and cellular membranes via extensive irreversible conformational changes. [26] [27] [28] [29] a recent study provided fresh evidence that sars-cov-2 exploits ace2 and tmprss2 for host cell entry. 24 like sars-cov entry into host cells, 17 the s glycoprotein domain b (s b ) of sars-cov-2 binds to the human ace2 (hace2) receptor and is subsequently primed by tmprss2. 30, 31 moreover, sars-cov-2 s has a similar or even higher (~10-to 20-fold) affinity for binding to hace2 as compared to sars-cov s. 30, 31 however, a novel and very important feature of sars-cov-2 s is that it harbors a furin cleavage site at the s1/s2 boundary, which is processed during biosynthesis. 31 therefore, the presence of the polybasic cleavage site in sars-cov-2 s, processed by furin-like proteases, may modulate tropism, transmissibility and pathogenicity of sars-cov-2, making it a highly pathogenic virus, like avian influenza viruses. 32 the relationship between the expression level of ace2 and susceptibly to sars-cov-2 infection still remains elusive. it will thus be interesting to determine whether sars-cov-2 interferes with ace2 expression and activity as well as to evaluate the functional consequence of the potential cleavage site used in sars-cov-2 and its impact on transmissibility and pathogenesis in animal models. in order to better understand the initial step of sars-cov-2 infection, elucidation of the interactional mechanism between the receptor-binding domain (rbd) of sars-cov-2 s and ace2 appears to be particularly important. two recent independent studies have reported the cryo-em structure of the sars-cov-2 spike trimer. 30, 31 moreover, another study presented the cryo-em structures of the full-length hace2-b 0 at1 (the neutral amino acid transporter) complex and a complex between the rbd of sars-cov-2 and the hace2-b 0 at1 complex as well as the hace2-rbd interface. 33 in addition, analytical modelling of structure predicted the potential residues of sars-cov-2 rbd that are recognized by ace2. 34 furthermore, x-ray crystallography data at a higher resolution showed the interaction between sars-cov-2 rbd and ace2, demonstrating that sars-cov-2 and sars-cov rbd share high structural similarity. 35 it remains to be investigated how sars-cov-2 alters the conformations of s glycoprotein trimers and the interactions between ace2 and s proteins in receptor-mediated endocytosis. interestingly, single-cell rna-sequencing data from multiple healthy human tissues discovered that the sars-cov-2 entry receptor ace2 and the viral entry-associated protease tmprss2 are highly expressed in nasal goblet and ciliated cells. 36 these new insights indicate that the primary viral sars-cov-2 transmission occurs through infectious droplets. although tmprss2 activity is essential for viral transmission, it still needs to be determined whether the endosomal cysteine proteases cathepsin b and l or other proteases, as reported in sars-cov and middle east respiratory syndrome coronavirus (mers-cov), 26, 28, [37] [38] [39] are involved in priming sars-cov-2 s. hence, further mechanistic studies are needed to elucidate the underlying detailed mechanism of sars-cov-2 entry into host cell and to test the potential of sars-cov-2 neutralizing antibodies. 40, 41 several studies have reported that 3% to 29% of covid-19 patients develop acute respiratory distress syndrome (ards) which is a common complication and cause of death as a result of sars-cov-2 infection. 4, 5, 21, 22 although the pathophysiology of covid-19 has not been completely unraveled, the potential main mechanism of covid-19-asscoiated ards would appear to be the immune-pathological event of the so-called cytokine storm. laboratory tests showed that patients infected with sars-cov-2 express high amounts of pro-inflammatory cytokines and chemokines, including interleukin (il)-1ꞵ, tumor necrosis factor α (tnfα), interferon-γ (ifn-γ), c-x-c motif chemokine ligand (cxcl)-10, and monocyte chemoattractant protein 1 (mcp1). 4 the evidence obtained from the postmortem biopsy study of a 50-year-old male patient suggested that the severe immune injury in covid-19-associated ards is related to over-activation of t cells, manifested by the elevation of t-helper-17 (th17) and high cytotoxicity of cd8 t cells. 42 sars-cov-2, sars-cov and mers-cov cause acute lethal disease characterized by dysregulated and excessive immune responses and lung damage during viral infection. it was reported that relative delayed type i interferon (ifn-i) signaling promoted inflammatory monocyte-macrophage accumulation in balb/c mice infected with sars-cov. 43 consequently, these accumulated mononuclear macrophages produce more monocyte chemoattractants through activating the ifn-α/β receptors and mononuclear macrophage-derived proinflammatory cytokines, such as tnfα, il-1β and il-6, induce apoptosis of t cells. signaling or tumor necrosis factor-related apoptosis-inducing ligand (trail)-death receptor 5 (dr5) signaling. this eventually results in the apoptosis of airway and alveolar epithelial cells. [44] [45] [46] the apoptosis of these endothelial and epithelial cells could potentially lead to vascular leakage and alveolar edema, which is regarded as playing a key role in the pathogenesis of virus infection-associated ards. it seems to be this deadly uncontrolled cytokine storm that triggers the frantic attack on the body by the immune system causing ards and finally unprecedented mortality in severe cases of sars-cov-2 infection. future work needs to investigate the details of the ifn signaling involved in sars-cov-2 infection, how the inflammatory response is triggered as well as the type of cell death that occurs during covid-19. also, further autopsy or biopsy studies, including more patients of different ages and backgrounds, would be needed to examine the histopathological changes and ace2 levels in different tissues. the acid milieu in endosomes and lysosomes (ph between 5 and 6) is due to a bafilomycin-sensitive pump that concentrates h + in the lumen of endosomes/lysosomes. 47 this low ph is essential for virus/cell membrane fusion. 8, [48] [49] [50] [51] . cq was reported to cause an increase in the intra-lysosomal ph of macrophages. 52 however, there is still no evidence showing the effect of hcq on the ph dynamics of endosomes/lysosomes. nevertheless, both cq and hcq are weak bases so they should both be able to elevate endosomal ph and could thereby inhibit virus/cell membrane fusion. it has recently been reported that cq is highly effective in the control of sars-cov-2 infection in vitro. 11 compared to remdesivir (gs-5734), the time-of-addition assay showed that cq functioned at entry as well as at post entry stages of the sars-cov-2 infection in vero e6 cells. 11 similarly, another in vitro study also found that hcq can efficiently inhibit sars-cov-2 infection via the same routes. 53 the therapeutic effect of both cq and hcq may be due to blockade of the transport of sars-cov-2 from early endosomes (ees) to endo-lysosomes (els), which seems to be the same viral genome releasing mechanism that operates in the case of sars-cov. however, the mode of actions of cq and hcq showed discrepancy in certain aspects such as in the morphology and ph values of endosomes/lysosomes. 53 another recent study found that hcq exhibited a smaller ec 50 than cq in in vitro anti-sars-cov-2 activity and physiologically-based pharmacokinetic models indicated that hcq is likely to be more effective than cq in the treatment of sars-cov-2 infection. 54 however, there are controversies about the effect of cq/hcq in altering endosomal /lysosomal ph and treating viral infection. it was reported that cq/hcq could directly bind to nucleic acids and inhibit the activation of the endosomal toll-like receptor (tlr) by masking tlr ligand-binding epitopes rather than increase the endosomal ph. 55 in addition, cq was shown to inhibit autophagy mainly through impairing autophagosome fusion with lysosomes rather than by increasing ph in this organelle. 48 furthermore, there was a study showing that cq could enhance porcine circovirus 2 infection of porcine epithelial cells via inhibition of endosome-lysosome system acidification. 56 the effect of cq/hcq may differ from cell type to cell type and between virus species. nevertheless, whether cq/hcq are able to affect the acidity of ees and els in sars-cov-2 infection should be examined carefully in the future. the progressive acidification that normally occurs from ees to els depends on a high ca 2+ concentration in the ees. the ph in the lumen of these organelles decreases in line with a decrease in the ca 2+ concentration. 56 ca 2+ signaling has been demonstrated to be involved in viral fusion into host cells of many viruses such as ebola virus (ebov), mers-cov and sars-cov. 57 ca 2+ release from intracellular stores within the endolysosomal system, via two-pore channels (tpc1, tpc2) and channels belonging to the mucolipin family (e.g. trpml1) can be evoked by nicotinic acid adenine dinucleotide phosphate (naadp) and phosphatidylinositol 3,5-bisphosphate (pip 2 ). 57, 58 in pancreatic acinar cells, antibodies against tpc2 are very effective, and much more effective than antibodies against tpc1, in reducing naadp-elicited ca 2+ release from acidic stores. 59 in this context it is of particular interest that it has very recently been shown that blocking tpc2 activity by tetrandrine decreases entry of sars-cov-2 s pseudovirions. 60 in contrast, a trpml1 inhibitor had no effect. 60 the endo-lysosomal ca 2+ level and ph, altered by tpc activity, 61-63 regulate the activity of furin required for proteolytic activation of the s protein and viral fusion. 64, 65 it was reported that inhibition of tpcs rather than trpml1 could block mers-cov infectivity. 66 moreover, it has been demonstrated that the endosomal calcium channels tpc1 and tpc2 are necessary for ebov infection. 57 interestingly, tetrandrine has been identified as a highly potent and low cytotoxic tpcs inhibitor. 57 because of its ability to disrupt tpcs function, tetrandrine can prevent ebov from escaping the endosomal network into the cell cytoplasm and thus block ebov infection. some other ca 2+ channel blockers such as amiodarone, verapamil, nimodipine, diltiazem, bepridil and lomerizine could effectively protect against filoviral entry into target cells. 67 there is much evidence indicating that sars-cov and sars-cov-2 infect host cells through ace2. 17, 18, 74, 75, 60 furthermore, cq could block sars-cov fusion with and entry into the host cell through interfering with the glycosylation of the ace2 receptor and the s protein. 8 cq/hcq have promising ability to inhibit sars-cov-2 and ace2related viral infection. it has been shown that cq is an effective inhibitor of the replication of the sars-cov in vero e6 cell culture. 76 another study further confirmed that cq is effective against sars-cov in frankfurt and urbani strains. 8 in addition, this study found that cq impaired the terminal glycosylation of ace2, suggesting that the variations in its glycosylation status might result in the ace2-sars-cov interaction being less efficient and therefore inhibit virus entry when the cells are treated with cq. also, it was shown that the recombinant sars-cov s protein downregulates ace2 expression. 77 in experimental mouse models, infection with avian influenza a h5n1 virus resulted in down-regulation of ace2 expression in the lung. 78 genetic inactivation of ace2 caused severe lung injury in h5n1-challenged mice, suggesting a role for ace2 in h5n1-induced lung pathologies. 78 cq was found to effectively inhibit autophagy in the lungs of avian influenza h5n1 mice and to ameliorate the acute lung injury and further, significantly improve the survival rate in mice infected with live avian influenza a h5n1 virus. 9 there is also evidence that cq had an inhibitory effect against the replication of human influenza a virus h1n1 and h3n2 in vitro. 7 ace2 could mediate the severe acute lung injury induced by influenza a (h7n9) virus infection in an experimental mouse model. moreover, ace2 deficiency worsened the disease pathogenesis markedly, mainly by targeting the angiotensin ii receptor type 1 (at1 receptor, at1r). 79 therefore, the potential effects and mechanism of cq/hcq against ace2 related viruses appears worth further investigation. cq/hcq shows cv benefits, including reductions in the risks of developing hyperlipidemia, diabetes mellitus, and thrombosis, as well as improving insulin sensitivity, glucose profiles, and hba1c, and decreasing cholesterol, triglycerides, and low-density lipoprotein-cholesterol (ldl-c). 80, 81 cq/hcq is extensively used in the treatment of rheumatic diseases, the patients of which are at higher risk of cv disease. 82, 83 a retrospective study of a cohort of 1,266 patients with rheumatoid arthritis (ra) found that hcq was associated with an approximately 72% reduction in the risk of cv disease. 84 another longitudinal registry showed that, compared to non-users, ra patients with hcq treatments had significantly lower levels of total and low-density cholesterol. 85 a longitudinal cohort study of 264 systemic lupus erythematosus patients found lowered serum cholesterol levels associated with hcq treatment. 86 a prospective, multicenter observational study of 4905 adults with ra also reported that use of hcq is associated with a 38% reduction of diabetes risk. 87 hcq is also found to reduce blood pressure variability among 899 systemic lupus erythematosus patients. 88 there are a few small clinical trials studying the effects of cq/hcq on cv risks in humans. a randomized controlled trial (rct) carried out among 116 patients with metabolic syndrome found that a one-year cq treatment decreased blood pressure, lipids, and the activation of c-jun n-terminal kinase (jnk). 89 another randomized, double-blinded, placebo-controlled crossover study found that a 8-week hcq treatment decreased insulin resistance, total cholesterol, and ldl-c among 23 ra patients. 90 a small open-label clinical trial administered hcq to 13 obese participants for six weeks, which significantly increased the insulin sensitivity index. 91 another rct with 135 patients with sulfonylurea-refractory type 2 diabetes proved that hcq could decrease glycated hemoglobin and improve glucose tolerance. 92 in animal studies, cq could lower blood pressure through toll-like receptor signaling and prevent the subsequent recruitment of immune cells to the vasculature in spontaneously hypertensive rats. 93 in rat hepatocytes, cq was shown to be an effective inhibitor of cholesterol synthesis. 94 it has also been reported that cq improved the cardiac diastolic function by inhibiting autophagy in streptozotocin-induced heart failure with preserved ejection fraction in mice. 95 evidence was also found that activation of ataxia telangiectasia mutated with low-dose cq decreased features of the metabolic syndrome including atherosclerosis in mice. 96 taken together, the claimed cv benefits of cq/hcq are mostly generated from animal studies or observational studies in humans. in rare cases, cq/hcq treatment presents cardiotoxicity including hypotension, arrhythmia, atrioventricular block 97 , cardiomyopathy 23, 98 and heart failure 23, 99 , which could be serious. 100, 101 the cardiotoxicity of cq/hcq may be under-recognized 23 . among the 25 episodes of intentional cq overdosage, 19% died, and 50% had cardiac arrest. 102 ex vivo acute cq treatment decreased heart function, and in vivo chronic lowdose cq treatment significantly decreased aortic output and total work in hearts. 103 the mechanisms underlying the effects of cq/hcq on the cv system are not fully understood (figure 2) . cq could improve insulin sensitivity by increasing the affinity of insulin receptors, inhibiting insulin degradation, and increasing insulin secretion. 104 cq/hcq could also increase the lipid clearance rate and expression of ldl receptors. 105 hcq is thought to protect against accelerated atherosclerosis, targeting toll-like receptor signaling, cytokine production, t-cell and monocyte activation, oxidative stress, and endothelial dysfunction. 106 hcq can also reduce the induction of endosomal nadph oxidase (nox) by tnfα, il-1β and antiphospholipid antibodies (apl) through the inhibition of the translocation of the catalytic subunit of nox2 into the endosome, which is involved in many inflammatory and pro-thrombotic signaling pathways. 107 however, chronic use of cq/hcq can result in an acquired lysosomal storage disorder, leading to cardiomyopathy characterized by concentric hypertrophy and conduction abnormalities associated with increased adverse clinical outcomes and mortality. 108 hcq is structurally and mechanistically similar to the class ia antiarrhythmic quinidine, 109 and may therefore inhibit voltage-gated sodium and potassium channels, prolonging the qt interval and increasing the risk of 'torsades de pointes' (a specific type of abnormal heart rhythm) and sudden cardiac death. 110 an animal study found that high-dose cq significantly impaired mitochondrial antioxidant buffering capacity and accentuated oxidative stress and mitochondrial dysfunction in pressure-overload hypertrophy. 103 in addition, cq may increase cv risk by impairing the terminal glycosylation of ace2, 8 which possibly amplifies ace/angii/at1 axis signaling and depresses ace2/ang1-7/masr axis signaling. the role of ace2 in the action of cq/hcq needs to be further studied. finally, it is challenging to interpret the extensive amount of covid-19 related research that has been published within a very short space of time. this is a highly unusual situation in the routine life cycles of any research topic. we therefore need to maintain a degree of healthy skepticism when interpreting the covid-19 related scientific literature. we carried out electronic searches using pubmed, web of science, researchgate and google. the search terms were "virus", "coronavirus", "angiotensin-converting enzyme 2", "chloroquine", "hydroxychloroquine", "cardiovascular system", and others, alone and in combination. many firstly identified references were investigated further to find the original primary research articles that were then cited in the review. conflict of interest: none declared. the initial entry of sars-cov-2 (an enveloped virus) 111 into host cells depends on ace2 and tmprss2. the s protein of sars-cov-2 binds to the functional receptor ace2 and employs tmprss2 for its priming. s protein is cleaved by tmprss2 at s2' site which results in virus/membrane fusion. 27 both ace2 and tmprss2 facilitate the virus transport into the target cell through the early and late endosomes where eventually the viral genome will be released into the cell cytoplasm. sars-cov-2 infection could influence the balance of ras, which leads to ang ii accumulation through the ace/angii/at1r axis and eventually causes acute lung injury. cq/hcq may block sars-cov-2 fusion with the host cell and entry into the target cell through elevating the ph in the endolysosomal system and/or by interfering with the glycosylation of the ace2 receptor and the s protein. 8 figure 2 the effects of chloroquine and hydroxychloroquine on the cardiovascular system. cq/hcq could protect against accelerated atherosclerosis targeting tlr signaling, cytokine production, t-cell and monocyte activation, oxidative stress, and endothelial dysfunction. however, cq/hcq interferes with the glycosylation of ace2 and this leads to dysregulation of the ras, which eventually causes imbalance of the ace/angii/at1 axis and the ace2/ang1-7/masr axis. meanwhile, cq/hcq can cause cardiotoxicity which may increase the risk of cv disease. therefore, cq/hcq may have a paradoxical effect on the cv system. china novel coronavirus i and research t. a novel coronavirus from patients with pneumonia in china a new coronavirus 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sulfonylureas--a randomized trial chloroquine suppresses the development of hypertension in spontaneously hypertensive rats inhibition of hepatic cholesterol biosynthesis by chloroquine chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice. drug design, development and therapy atm-dependent suppression of stress signaling reduces vascular disease in metabolic syndrome chloroquine cardiomyopathy: beyond ocular adverse effects hydroxychloroquineinduced restrictive cardiomyopathy: a case report chloroquine cardiomyopathy with conduction disorders cardiotoxicity of antimalarial drugs. the lancet infectious diseases cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature intentional chloroquine overdosage high-dose chloroquine is metabolically cardiotoxic by inducing lysosomes and mitochondria dysfunction in a rat model of pressure overload hypertrophy chloroquine augments the binding of insulin to its receptor cholesterol homeostasis is modulated by amphiphiles at transcriptional and post-transcriptional loci protective effects of hydroxychloroquine against accelerated atherosclerosis in systemic lupus erythematosus hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal nadph oxidase. annals of the rheumatic diseases hydroxychloroquine-induced cardiomyopathy: case report, pathophysiology, diagnosis, and treatment. the canadian journal of cardiology effects of quinine, quinidine, and chloroquine on alpha9alpha10 nicotinic cholinergic receptors risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease potential role of oral rinses targeting the viral lipid envelope in sars-cov-2 infection key: cord-265329-bsypo08l authors: van dorp, lucy; acman, mislav; richard, damien; shaw, liam p.; ford, charlotte e.; ormond, louise; owen, christopher j.; pang, juanita; tan, cedric c.s.; boshier, florencia a.t.; ortiz, arturo torres; balloux, françois title: emergence of genomic diversity and recurrent mutations in sars-cov-2 date: 2020-05-05 journal: infect genet evol doi: 10.1016/j.meegid.2020.104351 sha: doc_id: 265329 cord_uid: bsypo08l sars-cov-2 is a sars-like coronavirus of likely zoonotic origin first identified in december 2019 in wuhan, the capital of china's hubei province. the virus has since spread globally, resulting in the currently ongoing covid-19 pandemic. the first whole genome sequence was published on january 52,020, and thousands of genomes have been sequenced since this date. this resource allows unprecedented insights into the past demography of sars-cov-2 but also monitoring of how the virus is adapting to its novel human host, providing information to direct drug and vaccine design. we curated a dataset of 7666 public genome assemblies and analysed the emergence of genomic diversity over time. our results are in line with previous estimates and point to all sequences sharing a common ancestor towards the end of 2019, supporting this as the period when sars-cov-2 jumped into its human host. due to extensive transmission, the genetic diversity of the virus in several countries recapitulates a large fraction of its worldwide genetic diversity. we identify regions of the sars-cov-2 genome that have remained largely invariant to date, and others that have already accumulated diversity. by focusing on mutations which have emerged independently multiple times (homoplasies), we identify 198 filtered recurrent mutations in the sars-cov-2 genome. nearly 80% of the recurrent mutations produced non-synonymous changes at the protein level, suggesting possible ongoing adaptation of sars-cov-2. three sites in orf1ab in the regions encoding nsp6, nsp11, nsp13, and one in the spike protein are characterised by a particularly large number of recurrent mutations (>15 events) which may signpost convergent evolution and are of particular interest in the context of adaptation of sars-cov-2 to the human host. we additionally provide an interactive user-friendly web-application to query the alignment of the 7666 sars-cov-2 genomes. on december 31 2019, china notified the world health organisation (who) about a cluster of pneumonia cases of unknown aetiology in wuhan, the capital of the hubei province. the initial evidence was suggestive of the outbreak being associated with a seafood market in wuhan, which was closed on january 1 2020. the aetiological agent was characterised as a sars-like betacoronavirus, later named sars-cov-2, and the first whole genome sequence (wuhan-hu-1) was deposited on ncbi genbank on january 5 2020 (1) . human-to-human transmission was confirmed on january 14 2020, by which time sars-cov-2 had already spread to many countries throughout the world. further extensive global transmission led to the who declaring covid-19 as a pandemic on march 11 2020. betacoronaviridae comprise a large number of lineages that are found in a wide range of mammals and birds (2) , including the other human zoonotic pathogens sars-cov-1 and mers-cov. the propensity of betacoronaviridiae to undergo frequent host jumps supports sars-cov-2 also being of zoonotic origin. to date, the genetically closest-known lineage is found in horseshoe bats (batcov ratg13) (3) . however, this lineage shares 96% identity with sars-cov-2, which is not sufficiently high to implicate it as the immediate ancestor of sars-cov-2 (2) . the zoonotic source of the virus remains unidentified at the date of writing (april 23 2020). the analysis of genetic sequence data from pathogens is increasingly recognised as an important tool in infectious disease epidemiology (4, 5) . genetic sequence data sheds light on key epidemiological parameters such as doubling time of an outbreak/epidemic, reconstruction of transmission routes and the identification of possible sources and animal reservoirs. additionally, whole-genome sequence data can inform drug and vaccine design. indeed, genomic data can be used to identify pathogen genes interacting with the host and allows characterization of the more evolutionary constrained regions of a pathogen genome, which should be preferentially targeted to avoid rapid drug and vaccine escape mutants. there are thousands of global sars-cov-2 whole-genome sequences available on the rapid data sharing service hosted by the global initiative on sharing all influenza data (gisaid; https://www.epicov.org) (6, 7) . the extraordinary availability of genomic data during the covid-19 pandemic has been made possible thanks to a tremendous effort by hundreds of researchers globally depositing sars-cov-2 assemblies (table s1 ) and the proliferation of close to real time data visualisation and analysis tools including nextstrain (https://nextstrain.org) and cov-glue (http://cov-glue.cvr.gla.ac.uk). in this work we use this data to analyse the genomic diversity that has emerged in the global population of sars-cov-2 since the beginning of the covid-19 pandemic, based on a download of 7710 assemblies. we focus in particular on mutations that have emerged independently multiple times (homoplasies) as these are likely candidates for ongoing adaptation of sars-cov-measured via the site specific consistency index. for this analysis all ambiguous sites in the alignment were set to 'n'. to assess whether any particular open reading frame (orf) showed evidence of more homoplasies than expected given the length of the orf, an empirical distribution was obtained by sampling, with replacement, equivalent length windows and recording the number of homoplasies detected (table s3) . homoplasyfinder identified 1132 homoplasies (1042 excluding masked sites), which were distributed over the sars-cov-2 genome ( figure s5, table s4 ). of these, 40 sites have a derived allele at >1% of the total isolates. however, homoplasies can arise due to convergent evolution (putatively adaptive), recombination, or via errors during the processing of sequence data. the latter is particularly problematic here due to the mix of technologies and methods employed by different contributing research groups. we therefore filtered identified homoplasies using a set of thresholds attempting to circumvent this problem (filtering scripts and figures are available at https://github.com/liampshaw/cov-homoplasy-filtering). in summary, for each homoplasy we computed the proportion of isolates with the homoplasy pnn where the nearest neighbouring isolate in the phylogeny also carried the homoplasy (excluding identical sequences). this metric ranges between pnn=0 (all isolates with the homoplasy present as singletons) and pnn=1 (no singletons i.e. clustering of isolates with the homoplasy in the phylogeny). we reasoned that artefactual sequencing homoplasies would tend to show up as singletons, so excluded all homoplasies with pnn<0.1 from further analysis. to obtain a set of high confidence homoplasies, we then used the following criteria: ≥0.1% isolates in the alignment share the homoplasy (equivalent to >8 isolates), pnn>0.1, and derived allele found in strains sequenced from >1 originating lab and >1 submitting lab. we also required the proportion of isolates where the homoplasic site was in close proximity to an ambiguous base (±5 bp) to be zero. the application of these various filters reduced the number of homoplasies to 198 (table s5) . we also plotted the distributions of cophenetic distances between isolates carrying each homoplasy compared to the distribution for all isolates ( figure s6) , and inspected the distribution of all identified homoplasies in the phylogenies from our own analyses and on the phylogenetic visualisation platform provided by nextstrain. finally, we examined whether ambiguous bases were seen more often at homoplasic sites than at random bases(excluding masked sites), which was not the case ( figure s7 ). to further validate the homoplasy detection method applied to the alignment of the 7666 sars-cov-2 genome assemblies, we took advantage of the genome sequences for which raw reads were available on the short read archive (sra). a variant calling pipeline (available at https://github.com/damienfr/cov-homoplasy) was used to obtain high-confidence alignments for the 348 (out of 889 as of april 19 2020) sra genomic datasets both meeting our quality criterions and matching gisaid assemblies. the topology of the maximum likelihood phylogeny of these 348 samples was compared to that of the corresponding samples from the gisaid genome assemblies using a mantel test and the phytools r package (16) (figures s8-s9 , see supplementary text). ≥1%), 10 and 11 homoplasies were kept in the sra dataset and in the gisaid dataset, respectively. nine sites were detected in both datasets. for sites which failed the filtering thresholds, this was largely due to the low number of studied accessions, which increases the probability of an isolated strain displaying a homoplasy e.g. if n=2 isolates have a homoplasy, by definition they cannot be nearest neighbours, so pnn=0. the alignment was translated to amino acid sequences using seaview v4 (17) . sites were identified as synonymous or non-synonymous and amino acid changes corresponding to these mutations were retrieved via multiple sequence alignment. we assessed the change in hydrophobicity and charge of amino acid residues arising due to homoplastic non-synonymous mutations using the hydrophobicity scale proposed by janin (18) . the ten most hydrophobic residues on this scale were considered hydrophobic and the rest as hydrophilic. in addition, amino acid residues were either classified as positively charged, negatively charged or neutral at ph 7. the charge of each residue can either increase, decrease or remain the same (neutral mutation) due to mutation ( figure s10 ). sars-cov-1 and mers-cov are both zoonotic pathogens related to sars-cov-2, which underwent a host jump into the human host previously. we investigated whether the major homoplasies we detect in sars-cov-2 affect sites which also underwent recurrent mutations in these related viruses as these adapted to their human host. all coronaviridae assemblies were downloaded (ncbi taxid:11118) on 8th of april 2020 and human associated mers-cov and sars-cov-1 assemblies extracted. this gave a total of 15 assemblies for sars-cov-1 and 255 assemblies for mers-cov. following the same protocol (augur align) as applied to sars-cov-2 assemblies, each species was aligned against the respective refseq reference genomes: nc_004718.3 for sars-cov-1 and nc_019843.3 for mers-cov. this produced alignments of 29,751bp (187 snps) and 30,119bp (1588 snps) respectively. the 7666 sars-cov-2 genomes offer an excellent geographical and temporal coverage of the covid-19 pandemic (figure 1a-b) . the genomic diversity of the 7666 sars-cov-2 genomes is represented as maximum likelihood phylogenies in a radial (figure 1c ) and linear layout ( figure s1-s2 ). there is a robust temporal signal in the data, captured by a statistically significant correlation between sampling dates and 'root-to-tip' distances for the 7666 sars-cov-2 ( figure s3 ; r 2 =0. 20, p<0.001). such positive association between sampling time and evolution is expected to arise in the presence of measurable evolution over the timeframe over which the genetic data was collected. specifically, more recently sampled strains have accumulated additional mutations in their genome than older ones since their divergence from the most recent common ancestor (mrca, root of the tree). the origin of the regression between sampling dates and 'root-to-tip' distances ( figure s3 ) provides a cursory point estimate for the time to the mrca (tmrca) around late 2019. using treedater (12), we observe an estimated tmrca, which corresponds to the start of the covid-19 epidemic, of 6 october 2019 -11 december 2019 (95% cis) ( figure s4 ). these dates for the start of the epidemic are in broad agreement with previous estimates performed on smaller subsets of the covid-19 genomic data using various computational methods ( table 1) , though they should still be taken with some caution. indeed, the sheer size of the dataset precludes the use of some of the more sophisticated inference methods available. the sars-cov-2 global population has accumulated only moderate genetic diversity at this stage of the covid-19 pandemic with an average pairwise difference of 9.6 snps between any two genomes, providing further support for a relatively recent common ancestor. we estimated a mutation rate underlying the global diversity of sars-cov-2 of ~6×10 -4 nucleotides/genome/year (ci: 4x10 -4 -7x10 -4 ) obtained following time calibration of the maximum likelihood phylogeny. this rate is largely unremarkable for an rna virus (19, 20) , despite coronaviridae having the unusual capacity amongst viruses of proofreading during nucleotide replication, thanks to the non-structural protein nsp14 exonuclease, which excises erroneous nucleotides inserted by their main rna polymerase nsp12 (21, 22) . some of the major clades in the maximum likelihood phylogeny (figure 1c and figure s1 ) are formed predominantly by strains sampled from the same continent. however, this likely represents a temporal rather than a geographic signal. indeed, the earliest available strains were collected in asia, where the covid-19 pandemic started, followed by extensive genome sequencing efforts first in europe and then in the usa. the sars-cov-2 genomic diversity found in most countries (with sufficient sequences) essentially recapitulates the global diversity of covid-19 from the 7666-genome dataset. figure 2 highlights the proportion of the global genetic diversity found in the uk, the usa, iceland and china. in the uk, the usa and iceland, the majority of the global genetic diversity of sars-cov-2 is recapitulated, with representatives of all major clades present in each of the countries (figure 2a-c) . the same is true for other countries such as australia ( figure s2a ). this genetic diversity of sars-cov-2 populations circulating in different countries points to each of these local epidemics having been seeded by a large number of independent introductions of the virus. the main exception to this pattern is china, the source of the initial outbreak, where only a fraction of the global diversity is present (figure 2d ). this is also to an extent the case for italy (figure s2b) , which was an early focus of the covid-19 pandemic. however, this global dataset includes only 35 sars-cov-2 genomes from italy, so some of the genetic diversity of sars-cov-2 strains in circulation likely remains unsampled. the genomic diversity of the global sars-cov-2 population being recapitulated in multiple countries points to extensive worldwide transmission of covid-19, likely from extremely early on in the pandemic. the sars-cov-2 alignment can be considered as broken into a large two-part open reading frame (orf) encoding non-structural proteins, four structure proteins: spike (s), envelope (e), membrane (m) and nucleocapsid (n), and a set of small accessory factors (figure 3a ). there is variation in genetic diversity across the alignment, with polymorphisms often found in neighbouring clusters ( figure s5) . a simple permutation resampling approach suggests that both orf3a and n exhibit snps which fall in the 95 th percentile of the empirical distribution (table s3) . however, not all of these sites can be confirmed as true variant positions, due to the lack of accompanying sequence read data. however, we closely inspected those sites that appear to have arisen multiple times following a maximum parsimony tree building step. we identified a large number of putative homoplasies (n=1042 excluding masked regions), which were filtered to a high confidence cohort of 198 positions (see methods). these 198 positions in the sars-cov-2 genome alignment (0.67% of all sites) were associated with 290 amino acid changes across all 7666 genomes. of these amino acid changes, 232 comprised non-synonymous and 58 comprised synonymous mutations. two non-synonymous mutations involved the introduction or removal of stop codons were found (*13402y, *26152g). 53 of the remaining 101 non-synonymous mutations involved neutral hydrophobicity changes ( figure s10a ). in addition, 79 of the remaining 101 non-synonymous mutations involved neutral changes ( figure s10b ). both orf1ab and n had a four-fold higher frequency of hydrophilic → hydrophobic mutations than hydrophobic → hydrophilic mutations ( figure s10 ). in addition, neutral hydrophobic changes were clearly favoured in the s protein. lastly, 87 of the remaining 110 non-synonymous mutations involved neutral charge changes. amongst the strongest filtered homoplasic sites (>15 change points on the tree), three are found within orf1ab (nucleotide positions 11083, 13402, 16887) and s (21575). we exemplify the strongest signal and our approach using position 11083 in figure 3 and provide a full list of homoplasic sites, both filtered and unfiltered, in tables s4-5. the strongest hit in terms of the inferred minimum number of changes required (figure 3b -c) at orf1ab (11083 , codon 3606) falls over a region encoding the non-structural protein, nsp6, and is also observed in our analyses of the sra dataset (table s7) . we note that some of the hits also overlap with positions identified as putatively under selection using other approaches (http://virological.org/t/selection-analysis-of-gisaid-sars-cov-2data/448/3, accessed april 23 2020), with orf1ab consistently identified as a region comprising several candidates for non-neutral evolution. orf1ab is an orthologous gene with other humanassociated betacoronaviruses, in particular sars-cov-1 and mers-cov which both underwent host jumps into humans from likely bat reservoirs (23, 24) . we performed an equivalent analysis on human-associated virus assemblies available on the ncbi virus platform. we identified six putative homoplasic sites within sars-cov-1, two occurring within the 3c-like proteinase just upstream of nsp6 (10384, 10793) and a further two homoplasies within orf1ab at nsp9 and nsp13 ( figure s11 ). in addition, one homoplasy was identified in the spike protein and one in the membrane protein orfs. for mers-cov, multiple unfiltered homoplasies were detected, consistent with previous observations of high recombination in this species (25) , though only one invoked more than a minimum number of 10 changes on the maximum parsimony tree ( figure s12) . this corresponded to a further homoplasy identified in orf1ab nsp6(position 11631). it is of note that this genomic region coincides with the strongest homoplasy in sars-cov-2 which also occurs in the nsp6 encoding region of orf1ab. codon 3606 of orf1ab shares a leucine residue in mers-cov and sars-cov-2, though a valine in sars-cov. the exact role of these and other homoplasic mutations in human associated betacoronaviruses represents an important area of future work, although it appears that the orf1ab region may exhibit multiple putatively adapted variants across human betacoronavirus lineages. the genome alignment of the 7666 sars-cov-2 genomes can be queried through an open access, interactive web-application (https://macman123.shinyapps.io/ugi-scov2-alignmentscreen/). it provides users with information on every snp and homoplasy detected across our global sars-cov-2 alignment and allows visual inspection both within the sequence alignment and across the maximum likelihood tree phylogeny. figure 3 illustrates some of the functionalities of the web application using position 11083 in the alignment as an example. this particular homoplasy was observed 1078 times across the genomes and requires a minimum of 37 character-site changes to become congruent with the observed sars-cov-2 phylogeny (figure 3a and 3b ). pandemics have been affecting humanity for millennia (26) . over the last century alone, several global epidemics have claimed millions of lives, including the 1957/58 influenza a (h2n2) pandemic, the sixth (1899-1923) and seventh 'el tor' cholera pandemic (1961) (1962) (1963) (1964) (1965) (1966) (1967) (1968) (1969) (1970) (1971) (1972) (1973) (1974) (1975) , as well as the hiv/aids pandemic (1981-today). covid-19 acts as an unwelcome reminder of the major threat that infectious diseases represent in terms of deaths and disruption. one positive aspect of the current situation, relative to previous pandemics, is the unprecedented availability of scientific and technological means to face covid-19. in particular, the rapid development of drugs and vaccines has already begun. modern drug and vaccine development are largely based on genetic engineering and an understanding of host-pathogen interactions at a molecular level. the mobilisation to address the covid-19 pandemic by scientists worldwide has been remarkable. this includes the feat of the global scientific community who has already produced and publicly shared well over 11,000 complete sars-cov-2 genome sequences at the time of writing (april 23 2020), which we have used here with gratitude. further initatives in the united kingdom (https://www.cogconsortium.uk/data/) have already to date produced over 10,000 genomes, some of which overlap with those already available on gisaid. to put these numbers of sars-cov-2 genomes in context, it is interesting to consider parallels with the 2009 h1n1pdm influenza pandemic, the first epidemic for which genetic sequence data was generated in near-real time (27, 28) . the genetic data available at the time looks staggeringly small in comparison to the amount that has already been generated for sars-cov-2 during the early stages of the covid-19 pandemic. for example, fraser et al. considered 11 partial hemagglutinin gene sequences two months after the who had declared 2009 h1n1pdm influenza a pandemic (27) . this unprecedented genomic resource has already provided strong conclusions about the pandemic. for example, analyses by multiple independent groups place the start of the covid-19 pandemic towards the end of 2019 ( table 1 ). this rules out any scenario that assumes sars-cov-2 may have been in circulation long before it was identified, and hence have already infected large proportions of the population. extensive genomic resources for sars-cov-1 should in principle also be key to informing on optimal drug and vaccine design, particularly when coupled with knowledge of human proteome and immune interactions (29) . ideally, drugs and vaccines should target relatively invariant, strongly constrained regions of the sars-cov-2 genome, to avoid drug resistance and vaccine evasion. therefore ongoing monitoring of genomic changes in the virus will be essential to gain a better understanding of fundamental host-pathogen interactions that can inform drug and vaccine design. the vast majority of mutations observed so far in sars-cov-2 circulating in humans are likely neutral (32, 33) or even deleterious (34) . homoplasies, such as those we detect here, can arise by product of neutral evolution or as a result of ongoing selection. of the 198 homoplasies we detect (after applying stringent filters), some proportion are very likely genuine targets of positive selection which signpost to ongoing adaptation of sars-cov-2 to its new human host. indeed, we do observe an enrichment for non-synonymous changes (80%) in our filtered sites. as such, our provided list (table s5) contains candidates for mutations which may affect the phenotype of sars-cov-2 and virus-host interactions and which require ongoing monitoring. conversely, the finding that 78% of the homoplasic mutations involve no polarity change could still reflect strong evolutionary constraints at these positions (35, 36) . the remaining non-neutral changes to amino acid properties at homoplasic sites may be enriched in candidates for functionally relevant adaptation and could warrant further experimental investigation. one of the strongest homoplasies lies at site 11083 in the sars-cov-2 genome in a region of orf1a encoding nsp6. this site passed our stringent filtering cirteria and was also present in our analysis of the sra dataset (table s7) . interestingly, this region overlaps a putative immunogenic peptide predicted to result in both cd4+ and cd8+ t-cell reactivity (37) . more minor homoplasies amongst our top candidates, identified within orf3a (table s5) , also map to a predicted cd4 t cell epitope. while the immune response to sars-cov-2 is poorly understood at this point, key roles for cd4 t cells, which activate b cells for antibody production, and cytotoxic cd8 t cells, which kill virus-infected cells, are known to be important in mediating clearance in respiratory viral infections (38) . of note, we also identify a strong recurrent mutation in nucleotide position 21575, corresponding to the sars-cov-2 spike protein (codon 5). while the spike protein is the known mediator of host-cell entry, our detected homoplasy falls outside of the n-terminal and receptor binding domains. our analyses presented here provide a snapshot in time of a rapidly changing situation based on available data. although we have attempted to filter out homoplasies caused by sequencing error with stringent thresholds, and also used available short-read data to validate a subset of homoplasic sites in a smaller dataset, our analysis nevertheless remains reliant on the underlying quality of the publicly available assemblies. as such, it is possible that some results might be artefactual, and further investigation will be warranted as additional raw sequencing data becomes available. however, given the crucial importance of identifying potential signatures of adaptation in sars-cov-2 for guiding ongoing development of vaccines and treatments, we have suggested what we believe to be a plausible approach and initial list in order to facilitate future work and interpretation of the observed patterns. more data continues to be made available, which will allow ongoing investigation by ourselves and others. we believe it is important to continue to monitor sars-cov-2 evolution in this way and to make the results available to the scientific community. in this context, we hope that the interactive web-application we provide will help identify key recurrent mutations in sars-cov-2 as they emerge and spread. figure 1 . global sequencing efforts have contributed hugely to our understanding of the genomic diversity of sars-cov-2. a) viral assemblies available from global regions as of 19/04/2020. b) cumulative total of viral assemblies uploaded to gisaid included in our analysis. c) radial maximum likelihood phylogeny for 7666 complete sars-cov-2 genomes. colours represent continents where isolates were collected. green: asia; red: europe; purple: north america; orange: oceania; dark blue: south america according to metadata annotations available on nextstrain (https://github.com/nextstrain/ncov/tree/master/data). figure 1c .  phylogenetic estimates support that the covid-2 pandemic started sometimes around 6 october 2019 -11 december 2019, which corresponds to the time of the host-jump into humans.  the diversity of sars-cov-2 strains in many countries recapitulates its full global diversity, consistent with multiple introductions of the virus to regions throughout the world seeding local transmission events.  198 sites in the sars-cov-2 genome appear to have already undergone recurrent, independent mutations based on a large-scale analysis of public genome assemblies.  detected recurrent mutations may indicate ongoing adaptation of sars-cov-2 to its novel human host.  monitoring the build-up and patterns of genetic diversity in sars-cov-2 has 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acid replacements in human proteins a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 immunity to respiratory viruses transmission dynamics and evolutionary history of 2019-ncov the first two cases of 2019-ncov in italy: where they come from genomic epidemiology of sars-cov-2 in guangdong province 95% bci may 95% bci november 16 rate-estimated relaxed clock model 2020 (40) 54 6 95% ci october 28 95% ci november 16 unreported clock model (beast 95% hpd november strict clock model (beast v1.10) relaxed clock model (beast v1.10) 95% ci november 6 o analysed data and performed computational analyses l.v.d and f.b. acknowledge financial support from the newton fund uk-china nsfc initiative (grant mr/p007597/1) and the bbsrc (equipment grant bb/r01356x/1). computational analyses were performed on ucl computer science cluster and the south green bioinformatics platform hosted on the cirad hpc cluster. we thank jaspal puri for insights and assistance on the development of the alignment visualisation tool and nicholas mcgranahan and rachel rosenthal for their comments on the manuscript. we additionally wish to acknowledge the very large number of scientists in originating and submitting labs who have readily made available sars-cov-2 assemblies to the research community. key: cord-278522-e4qa19o6 authors: park, se yoon; yun, soon gyu; shin, jeong won; lee, bo young; son, hyo-ju; lee, seungjae; lee, eunjung; kim, tae hyong title: persistent severe acute respiratory syndrome coronavirus 2 detection after resolution of coronavirus disease 2019-associated symptoms/signs date: 2020-06-19 journal: korean j intern med doi: 10.3904/kjim.2020.203 sha: doc_id: 278522 cord_uid: e4qa19o6 there are limited data on the duration of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) rna in respiratory specimens after resolution of coronavirus disease 2019 (covid-19)-associated symptoms/signs. we determined duration of sars-cov-2 virus shedding in symptomatic patients after remission of symptoms. we investigated the duration of sars-cov-2 rna detection using real-time reverse transcriptase polymerase chain reaction for sars-cov-2 in nasopharyngeal/oropharyngeal swabs or sputum or saliva. six patients were included in the final analysis. the median (range) duration of sars-cov-2 viral detection after hospitalization was 34 days (22 to 67). after resolution of symptoms/signs, sars-cov-2 rna was detected for median (range) of 26 days (9 to 48). among the six patients, one had persistent detection of sars-cov-2 rna until day 67 of hospitalization, which was 30 days after symptom resolution. this case represents the longest duration of sars-cov-2 detection, and highlights the need for long-term follow up of covid-19 patients despite resolution of symptoms to confirm sars-cov-2 clearance. persistent severe acute respiratory syndrome coronavirus 2 detection after resolution of coronavirus disease 2019-associated symptoms/signs se yoon park 1 , soon gyu yun 2 , jeong won shin 2 , bo young lee 3 , hyo-ju son 1 , seungjae lee 1 , eunjung lee 1 , and tae hyong kim 1 since the first case of coronavirus disease 2019 (covid-19) was reported in wuhan, china in december 2019, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has infected more than three million people in 211 countries. by the end of april 2020, more than 208,000 people had died from covid-19 [1] . unlike other coronaviruses that have previously caused outbreaks in humans (sars-cov and middle east respiratory syndrome coronavirus [mers-cov]), sars-cov-2 is reported to have a higher viral load early in the disease process [2] . the virus can also be transmitted by patients who are asymptomatic or with mild symptoms [2, 3] . previous studies reported that the median duration of viral shedding is between 12-20 days, with the longest duration being 60 days [4] [5] [6] [7] . few studies have investigated the duration of sars-cov-2 detection during the patients' recovery phase after resolution of covid-19-associated symptoms/signs. therefore, we aimed to determine the duration of sars-cov-2 virus shedding in symptomatic patients after remission of symptoms. this study was conducted at the soonchunhyang university seoul hospital, which is a 738-bed university-affiliated hospital with an eight-bed airborne infection isolation room (aiir) for covid-19 patients. we included all patients with confirmed covid-19 admitted at the hospital from february 15 to march 26. the study was approved by the institutional review board of soonchunhyang university seoul hospital (approval number: 2020-04-039). the requirement for informed consent was waived due to the retrospective nature of the analysis. covid-19 was diagnosed by real-time reverse transcriptase polymerase chain reaction (rt-pcr) for sars-cov-2 using a powerchek tm 2019-ncov real-time pcr kit (kogenebiotech, seoul, korea) to determine virus through the identification of two genetic markers: envelope (e) gene and rna-dependent rna polymerase (rdrp) gene. samples were obtained from the nasopharyngeal/oropharyngeal swab, sputum, and saliva. demographic data collected included age, sex, and underlying illness. clinical data included initial symptom or sign associated with covid-19, treatment regimen, duration from admission to resolution of symptoms and signs, and time taken to resolution of lung findings on chest x-ray. time from resolution of covid-19-associated symptoms/signs to last positive result before consecutive negative results was also noted. of the seven patients admitted to the hospital, one was excluded from the analysis for being transferred to the hospital on day 6. four patients had pauci-symptoms of general weakness, anosmia/taste disorder, sore throat, and headache. two patients were diagnosed with pneumonia on chest computed tomography. the remaining two patients had productive cough and were diagnosed with sars-cov-2 pneumonia (table 1) . detailed laboratory test results on hospital admission are shown in supplementary table 1 . the median (range) time from onset of symptom/sign to admission was 5 days (2 to 11). the median (range) duration of symptom/sign in six patients and lung lesions on chest radiography in four patients after hospitalization was 5 days (4 to 38) and 18 (12 to 36), respectively. the total duration of sars-cov-2 viral shedding after hospitalization was median (range) 34 days (22 to 67). sars-cov-2 was detected after median 26 days (9 to 48) after resolution of symptoms/signs (table 1 and fig. 1 ). patients 1 and 2 were still hospitalized on may 4, 2020 while the others had been discharged. however, patient 5 was re-admitted on april 25, 2020 21 days after discharge due to positive result on nasopharyngeal and oropharyngeal swab testing (cycle threshold [ct] value for e gene 32.68; and rdrp gene 33.31). patient 5 was on scheduled adjuvant chemotherapy after breast cancer surgery. therefore, we repeated sars-cov-2 pcr test every 2 to 3 days prior to chemotherapy. she was re-discharged 2 days later after consecutive negative result on follow-up tests. patient 2 continued shedding sars-cov-2 in hospital day (hd) 67, with high viral titer in the sputum (e gene ct 28.23; rdrp gene ct 26.89). the patient had underlying comorbidities (schizophrenia and hypertension), and required 5 days of oxygen supplementation. she received 10 days of lopinavir/ritonovir, followed by hydroxychloroquine, but viral shedding persisted for more than 67 days (supplementary fig. 1) . sars-cov-2 shedding continued for about 4 weeks after resolution of covid-19-associated symptoms/signs, with the longest period being 48 days. in this study, four patients with pauci-symptoms had sars-cov-2 detected after admission for 22 to 52 days. this finding suggests that sars-cov-2 shedding may continue for more than 3 weeks regardless of presence of covid-19 symptoms. the shedding may be further prolonged as with patient 2, who to our knowledge, has the longest ongoing virus shedding among the reported covid-19 patients. data from a prospective cohort study of 137 patients with covid-19 indicated that the duration of viral detection was a median of 12 days, with a maximum of 45 days [6] . the risk factors for prolonged sars-cov-2 detection include older age, disease severity, lower lymphocyte, eosinophil, and cd8+ t cells count, and higher interleukin 6 (il-6) and il-10 levels [6] . patient 2, who had the longest duration of viral detection, was older (57 years) and had the lowest lymphocyte count (778/mm 3 ) on admission. in this present study, we also found that the virus can be detected for weeks after the resolution of clinical symptoms in younger patients who were mildly symptomatic and without lymphopenia (patients 3, 4, 5, and 6). in the present study, patient 5 was discharged after negative pcr test results. the patient was re-hospitalized after testing positive three weeks of discharge. case study from china report similar patterns [8] . the genetic material of dead viral particles remaining within epithelial cells can be detected as epithelial cells are desquamated. respiratory epithelial cells have a long halflife of up to months to years [9] . therefore, during the recovery phase, it is likely that the pcr could show the fluctuation. moreover, low nucleic acid content can reduce the reliability of the test (e.g., increasing results of false positives). to understand the best approaches and solutions (e.g., the necessity of re-isolation or self-isolation) to the fluctuations during their recoveries, further investigations are needed to review the display of the patients' symptoms and the presence of the live virus. this study had some limitations. first, we included only respiratory samples from only six patients. further studies, with a larger sample size, are needed to gather further information regarding viral shedding using various types of samples. second, viral culture, which is the gold standard for viral detection with infectivity, was not undertaken. it is possible that detection of sars-cov-2 with a comparatively lower viral titer could involve dead viral particles. it is necessary to confirm the presence of live virus for cases with trends similar to that patient 2 showed, considering that the patient showed increasing viral loads. in conclusion, we found that sars-cov-2 virus shedding can persist for more than three weeks after resolution of covid-19-associated symptoms/signs. due to prolonged viral shedding after resolution of covid-19-associated symptoms/signs and limited number of aiirs, the securing and distribution of aiirs at the national level should be decided considering these characteristics. no potential conflict of interest relevant to this article was reported. world health organization. coronavirus disease 2019 (covid-19) situation reports world health organization, c2020 sars-cov-2 viral load in upper respiratory specimens of infected patients asymptomatic and human-to-human transmission of sars-cov-2 in a 2-family cluster clinical course and risk factors for mortality of adult inpatients with covid-19 china: a retrospective cohort study duration of sars-cov-2 viral shedding during covid-19 infection early risk factors for the duration of sars-cov-2 viral positivity in covid-19 patients case report: viral shedding for 60 days in a woman with novel coronavirus disease (covid-19) recurrence of positive sars-cov-2 rna in covid-19: a case report ciliated epithelial cell lifespan in the mouse trachea and lung this research was supported by the soonchunhyang university research fund. key: cord-270533-s2d3q4ob authors: lau, yu-lung title: sars: future research and vaccine date: 2004-11-05 journal: paediatr respir rev doi: 10.1016/j.prrv.2004.07.005 sha: doc_id: 270533 cord_uid: s2d3q4ob severe acute respiratory syndrome (sars) is a new infectious disease of the 21st century that has pandemic potential. a novel coronavirus (cov) was identified as its aetiological agent and its genome was sequenced within months of the world health organisation issuing a global threat on sars. the high morbidity and mortality of this potentially pandemic infection demands a rapid research response to develop effective antiviral treatment and vaccine. this will depend on understanding the pathogenesis and immune response to sars cov. further understanding of the ecology of sars cov in human and animals will help prevent future cross species transmission. likewise for the super-spreading events, clarification of the underlying reasons will be important to prevent a large scale outbreak of sars. lastly it is of utmost importance that international research collaboration should be strengthened to deal with sars and any other emerging infectious disease that can seriously threaten our future. severe acute respiratory syndrome (sars), a newly emerged infectious disease of humans in the 21st century, appeared in guangdong province in southern china in november 2002 and spread to 26 countries on five continents along international air travel routes, causing large scale outbreaks in hong kong, singapore and toronto in early 2003. 1 the world health organisation (who) issued a global alert on sars on 12 march 2003 . with support of the who, authorities in affected regions implemented epidemiologic surveillance and adherence to infection-control procedures, including patient isolation and quarantine for contacts, which helped to contain the sars outbreak by mid-july 2003. however, there were a total of 8098 sars cases and 774 associated deaths. the aetiologic agent of sars was identified as a coronavirus (cov) [2] [3] [4] [5] and the genome sequence established it as a novel member of the family 6, 7 . this novel cov has satisfied koch's postulates for causation by its consistent isolation from sars patients, viral isolation, reproduction of disease in non-human primates after inoculation and the presence of specific antibody response against the virus in both patients and experimentally infected primates 8 . all of these remarkable findings were accomplished within a few months of the issue of the who global alert on sars, testifying to the rapid response of the collaboration of the international research effort to deal with such emerging pandemics. the origin of sars remains uncertain despite closely related covs that were recovered from civet cats and other animals in guangdong province, suggesting the sars-cov could have originated from such animals and implicating sars as a zoonotic disease. 9 other members of the cov family can cause fatal diseases in poultry and laboratory rodents. the two previously known human covs cause only mild upper respiratory infections. 10 despite the 2002/2003 sars epidemic being eventually controlled by case isolation, there is still neither an effective treatment for sars nor an efficacious vaccine to prevent infection. 1 the high morbidity and mortality of sars, as well as the potential of reemergence, make it paramount to focus on future research to develop effective means to treat and prevent the disease should it reappear. indeed, sporadic reemergence of cases have been reported in guangdong province as well as from research laboratories summary severe acute respiratory syndrome (sars) is a new infectious disease of the 21st century that has pandemic potential. a novel coronavirus (cov) was identified as its aetiological agent and its genome was sequenced within months of the world health organisation issuing a global threat on sars. the high morbidity and mortality of this potentially pandemic infection demands a rapid research response to develop effective antiviral treatment and vaccine. this will depend on understanding the pathogenesis and immune response to sars cov. further understanding of the ecology of sars cov in human and animals will help prevent future cross species transmission. likewise for the super-spreading events, clarification of the underlying reasons will be important to prevent a large scale outbreak of sars. lastly it is of utmost importance that international research collaboration should be strengthened to deal with sars and any other emerging infectious disease that can seriously threaten our future. ß 2004 elsevier ltd. all rights reserved. in singapore, taiwan and beijing since the conclusion of the 2003 sars epidemic. on 30 may 2003, the national institute of allergy and infectious diseases (niaid) convened a colloquium entitled 'sars: developing a research response' on the national institute of health campus, with over 500 participants that included physicians, scientists and policymakers from the united states, china, canada, europe and elsewhere, to coordinate a robust research response to the sars threat in each of the following five areas: (1) clinical research; (2) epidemiology; (3) diagnostics; (4) therapeutics; and (5) vaccines. 11 one year has gone by since this colloquium, with intense research activities conducted in many laboratories worldwide addressing those issues raised. despite over 1900 published papers in pubmed on various aspects of sars, many questions are still awaiting answers. this review will attempt to highlight some of the important issues that have only been partially addressed, pointing to areas of clinical interest rather than covering all aspects comprehensively. careful description of the clinical manifestations of sars, correlating with virologic and immunologic parameters, has suggested that there is an initial viral replicative phase of about 10 days, possibly followed by an immunopathological phase. 12 it has been hypothesised that the immunopathological response is triggered by the viral antigens, hence the most strategic treatment is to stop the viral replication at the initial phase of the disease so that the peak viral load and the subsequent damage is minimised. 13 therefore, an effective antiviral treatment has a window of opportunity of several days after disease onset to modify the peak viral load, thereby, in theory, decreasing the morbidity and mortality. at present, the treatment is largely empirical and therefore controversial, ranging from supportive therapy without intervention to a combination of antivirals and steroids. a recent report on an open trial of a combination of a protease inhibitor and a nucleoside analogue against a historical control suggests a favourable clinical response using lopinavir/ritonavir and ribavirin. 13 however, controversy remains as no controlled studies have been undertaken, and there is a need to set up international clinical trial networks to develop and perform clinical protocols should sars reappear. the future research priorities in sars therapeutics should focus on antiviral drug screening as no clinically proven candidates exist. a high-throughput screening assay has to be developed and applied to the existing drug candidate libraries. 11 some in vitro activity against sars cov was observed with certain preparations of interferona as well as with glycyrrhizin. 14, 15 in vivo activity has also been demonstrated with interferon-a in a non-human primate model but there have been no studies in humans. 16 molecular studies of sars cov and other coronaviruses suggest several potential molecular targets for antiviral drugs. these include viral binding, fusion and other activities mediated by the glycoprotein spike on the cov surface, as well as the rna-dependent rna polymerase and the cysteine protease. 11 the identification of angiotensin-converting enzyme 2 (ace2) as a functional receptor for the sars cov has opened up possibilities of treatment strategies such as interference of binding or fusion of sars cov with target cells. 17, 18 the sars cov has surface spike (s) proteins which contain the s1 and s2 domains; the ace2, which is a metallopeptidase, binds the s1 domain of the sars cov s protein efficiently and anti-ace2 has been shown to block sars cov replication in vitro using vero e6 cells as an experimental model. 17 a number of antibodies, peptides and small compounds can bind to ace2 and it is possible that some of these can be useful in the treatment of sars, 19 either by blocking the s-protein-binding site or by inducing a conformation in ace2 that is not favourable to binding or fusion. a soluble form of the ace2 may slow viral replication in an infected individual. 17 recently, a human monoclonal antibody 80r, against s1 domain of the sars cov, has been developed that can potently neutralise sars cov infection and efficiently inhibit syncytia formation through blocking of receptor binding. 20 this monoclonal antibody can be used as an immediate treatment strategy for emergency prophylaxis and treatment of sars, while the more time-consuming development of vaccines and new drugs is underway. during the winter and spring months, the diagnostic challenges of differentiating sars from other respiratory infections can be great, making a rapid, simple and accurate diagnostic assay for sars cov an imperative public health tool to control any future sars outbreak as well as the initiation of treatment protocol. the first generation pcr assays for sars cov were far from satisfactory but realtime pcr, coupled with an improved rna extraction method, has allowed detection of viral rna in nasopharyngeal aspirates with 80% specificity. 21 knowing where in the body the virus can be found at different stages of the disease might lead to new diagnostic strategies. 22 for example, the report that sars cov can replicate in peripheral blood cells in sars patients shortly after onset indicates that blood could be used as an appropriate clinical specimen for diagnosis. 23, 24 nevertheless, availability of a highly sensitive and specific direct detection method of sars cov in readily obtained clinical specimens such as nasopharyngeal secretions will be most ideal, obviating the need of the laborious steps of rna extraction. serologic assays are not useful for early diagnosis as igg antibodies do not appear for 7-10 days after onset of symptoms. it has been stated that igm antibodies typically appear earlier, but detection of igm antibodies does not appear to permit earlier diagnosis. 1, 11 since a few sars patients have had late seroconversion, it is best to test the convalescent serum collected at least 21 days and preferably 28 days after onset of symptoms, to rule out sars. 1 at present, the most widely used methods for detection of antibodies against sars cov are indirect immunofluorescence assay and elisa with cell-culture extract, which are difficult to standardise. 25 therefore, recombinant-antigenbased elisa assays are being developed using highly immunogenic nucleocapsid protein of sars cov, which can be used for a large scale epidemiological study of seroprevalence. 25 the characterisation of immune responses to the sars cov, including the impact of sars on immune function and any immunopathological responses the virus may trigger, is crucial in helping the development of new therapeutic strategies and vaccines. 11 there is a rapid and generalised lymphopaenia in patients with sars during the acute phase of infection, which is in distinct contrast to the proliferative response seen in hiv-, cmv-or ebv-infected patients. 26 in patients who recovered from sars, an equally rapid and dramatic restoration of t cell, b cell and nk cell counts was seen in peripheral blood. the mechanism through which the sars cov precipitates such lymphopaenia so rapidly is unclear but could be related to immunologic trigger of apoptosis of uninfected lymphocytes. elucidation of the underlying mechanism may help design treatment strategies. the initial increase in viral load in the first 10 days of disease 12 also suggests that the role of innate immunity as a first-line defence is important and may influence the subsequent disease progression. it is important to identify the host innate immune response genetics that are predictors of disease susceptibility and progression. this would help to improve prognostic capabilities and allow identification of patients likely to benefit from aggressive interventions. such information is also of value in the development of entry criteria for interventional studies. 11 since immune response might play a role in sars pathogenesis, one must be cautious of the possibility of enhancement of the disease in immunised subjects. 27 this has indeed occurred for experimental vaccine directed against feline infectious peritonitis virus, which is also a coronavirus. therefore caution has been urged on developing sars vaccines. 27 however, given the urgent need for a safe and effective sars vaccine, multiple strategies for vaccine development have been pursued simultaneously. at least 10 candidate sars vaccines are at different phases of development. [27] [28] [29] [30] [31] these include the development of live-attenuated and inactivated virus vaccine, in addition to other strategies that elicit strong t cell responses, such as dna-based vaccine and engineered adenovirus vectors, as well as those that elicit production of neutralising antibodies. 11 subunit vaccines based on s protein fragments and peptides are also being developed. sars cov s protein, expressed by attenuated vaccinia virus, has been shown to immunise mice protectively. 30 similarly, a dna vaccine encoding the sars cov s protein can induce t cell and neutralising antibody responses, as well as protective immunity, in a mouse model. 31 viral replication can be reduced by more than six orders of magnitude in the lungs of mice vaccinated with these s plasmid dna expression vectors and protection is mediated by a humoral but not a t-cell-dependent immune mechanism. 31 all the experimental testing of candidate sars vaccines would require an animal model that reproduces sars symptoms and pathology as in humans. however, no animal model described to date can reliably mimic the respiratory symptoms seen in humans with sars. 27 even the macaques model, used initially to fulfil the last of koch's postulates in confirming sars cov as the etiologic agent, 8 does not always reproduce the sars-like symptoms when infected with sars cov. 27 this could be due to macaques not being inbred like mice. finding a consistent sars animal model which can be used for testing potential drugs and vaccines is certainly a top research priority. monkey, mouse and ferret have all been used as animal models for answering different research questions. the origin of sars cov and its ecological relationship with other animal cov will certainly be an important topic of research, as the animal reservoir of such cov will be the constant potential source of another sars outbreak. understanding of the ecology will help to implement measures to minimise transmission across species taking place again. nevertheless, the recent sporadic reemergence of sars was due to lapses of security in research laboratories in handling sars cov, testifying to the urgent need to maintain and monitor laboratory safety in research institutes that deal with such pathogens. another mystery in the 2003 sars epidemiology is the so-called super-spreading events, such as the community outbreak in the amoy garden housing complex of hong kong, which affected more than 300 residents of this private housing estate. 32 tentative evidence of airborne transmission of the sars cov has been suggested by an epidemiologic analysis of this outbreak, coupled with airflow simulations and experimental studies. 32 better understanding of these super-spreading events will be crucial in preventing similar events from happening again. sporadic cases of sars may continue to appear but if vigorous public health measures can be maintained another major outbreak is unlikely, making it impossible to perform controlled clinical studies of either candidate antivirals or vaccines for sars cov. 27 nevertheless, the global community needs to be prepared for such emerging and reemerging infectious diseases if the human race is to have a future. understand the pathogenesis and immune responses to sars cov. develop effective antiviral therapeutics and efficacious vaccines. develop rapid and accurate diagnostic tests for early diagnosis of sars cov infection. clarify the ecology of human and animal sars cov to prevent cross species transmission. understand the reasons for super-spreading events. the severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus koch's postulates fulfilled for sars virus isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars coronavirus: a new challenge for prevention and therapy sars research working group co-chairs.severe acute respiratory syndrome: developing a research response clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings severe acute respiratory syndromerelated coronavirus is inhibited by interferon-alpha glycyrrhizin an active component of liquorice roots, and replication of sars-associated coronavirus pegylated interferon-a protects type 1 pneumocytes against sars coronavirus infection in macaques angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus expression cloning of functional receptor used by sars coronavirus novel peptide inhibitors of angiotensin-converting enzyme 2 potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association early diagnosis of sars coronavirus infection by real time rt-pcr detection of sars coronavirus in patients with suspected sars sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome caution urged on sars vaccines generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus effects of a sars-associated coronavirus vaccine in monkeys severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice a dna vaccine induces sars coronavirus neutralization and protective immunity in mice evidence of airborne transmission of the severe acute respiratory syndrome virus further reading key: cord-275565-xerr4vki authors: kumar, manish; kuroda, keisuke; patel, arbind kumar; patel, nidhi; bhattacharya, prosun; joshi, madhvi; joshi, chaitanya g. title: decay of sars-cov-2 rna along the wastewater treatment outfitted with upflow anaerobic sludge blanket (uasb) system evaluated through two sample concentration techniques date: 2020-09-15 journal: sci total environ doi: 10.1016/j.scitotenv.2020.142329 sha: doc_id: 275565 cord_uid: xerr4vki for the first time, we present, i) an account of decay in the genetic material loading of sars-cov-2 during upflow anaerobic sludge blanket (uasb) treatment of wastewater, and ii) comparative evaluation of polyethylene glycol (peg), and filtration as virus concentration methods from wastewater for the quantification of sars-cov-2 genes. the objectives were achieved through tracking of sars-cov-2 genetic loadings i.e. orf1ab, n and s protein genes on 8th and 27th may 2020 along the wastewater treatment plant (106 million liters per day) equipped with uasb system in ahmedabad, india. peg method performed better in removing materials inhibiting rt-qpcr for sars-cov-2 gene detection from the samples, as evident from constant and lower ct values of control (ms2). using the peg method, we found a reduction >1.3 log10 in sars-cov-2 rna abundance during uasb treatment, and the rna was not detected at all in the final effluent. the study implies that i) conventional wastewater treatment systems is effective in sars-cov-2 rna removal, and ii) uasb system significantly reduces sars-cov-2 genetic loadings. finally, peg method is recommended for better sensitivity and inhibition removal during sars-cov-2 rna quantification in wastewater. wastewater-based epidemiology (wbe) has already proved its capability as a tool of environmental surveillance of epidemic and pandemic in a given community through viral load detection in the wastewater, shredded both from symptomatic and asymptomatic covid-19 patients (bivnis et. al., 2020; xagoraraki and o'brien, 2020; choi et al. 2018; yang et al., 2015; hellmér et al., 2014; asghar et al., 2014; ahmed et al., 2020a , hata et al., 2020 wölfel et al., 2020a; zhang et al., 2020; randazzo et al 2020) . the second quarter of 2020 has been exceptional in discovering several new knowledge pertaining to sars-cov-2 genetic material loading, its analytical methods, and various strong implications pouring around the world (ahmed et al., 2020a; bar-or et al., 2020; haramoto et al., 2020; la rosa et al., 2020; medema et al., 2020; nemudryi et al., 2020; randazzo et al., 2020a; rimoldi et al., 2020; wurtzer et al., 2020a; wurtzer et al., 2020b kumar et al., 2020a . while most of the studies could explicitly prove the correlation of sars-cov-2 genetic loading with the covid-19 patients in the area, some compared the methods to improve srs-cov-2 rna extraction (ahmed et al., 2020b) and some studies traced back the sars-cov-2 genes in the wastewater long before any covid-19 patient were declared, implying high early warning capability of wbe (randazzo et al 2020a , prevost,et al 2015 , lodder and husman 2020 , prussin et al 2020 , ahmed et al., 2020c , sherchana et al., 2020 . however, the great majority of the existing studies are based on analysis of raw wastewater only, or just a comparison of influent and final treated effluent samples from wastewater treatment plants (wwtps). thus, there still remains questions pertaining to: i) capability of conventional wwtps to reduce the abundance of sars-cov-2 rna, ii) better understanding of the protocol, virus j o u r n a l p r e -p r o o f journal pre-proof precipitation through peg and filtration which one is better methods for concentrating the samples before rna isolation. further, while in wbe surveillance is being accelerated in india upon phasing out of lockdown, several questions are raised around its capability owing to incomplete sewer systems, significant wastewater leak, high ambient temperature, open defecation, strong seasonality component, and common sewer overflow (cso) situations in india. overall, there are several apprehensions about infectivity through genetic material present in the wastewater become a pertinent question. these all warrants a study that can track the genetic loading after each wastewater treatment stage in indian settings and highlight the effects of wastewater treatment on rna decay of the corona virus. such study will help curing the commonly perceived fear of the commons pertaining to the effectiveness of wwtps. further, the number of confirmed cases in india has passed 0.5 million as of the last day of june, 2020 (ministry of health and family welfare, india) with >16000 official casualties. in gujarat province, which is a hotspot and our study site, the confirmed cases will soon reach to 25,000 in ahmedabad city. (ministry of health and family welfare, india) indicating the need of immediate attention. at this juncture keeping the pulse of wbe progression as mentioned above, we focused on three major objectives i.e.: i) tracking the conventional treatment system for genetic loading decay of sars-cov-2 along the treatment process and evaluate its effectiveness, ii) appraising the genetic loading reduction through upflow anaerobic sludge blanket (uasb) systems, and iii) comparing the performances between peg and filtration as virus concentration methods in terms of sars-cov-2 rna sensitivity and inhibition removal. we thus hereby present the first ever data pertaining to uasb performances in sars-cov-2 rna j o u r n a l p r e -p r o o f journal pre-proof removal; and comparisons of two most frequently used techniques of virus concentration in wastewater samples for sars-cov-2 rna detection. we collected wastewater samples on 8 and 27 may, 2020 from old pirana wwtp at ahmedabad, gujarat that receives wastewater of 106 million liters per day (mld), including the wastewater from hospitals treating covid-19 patients. confirmed cases of covid-19 patients in ahmedabad was 56,352 and 150,857 respectively on 7 th and 26 th may 2020 (kumar et. al., 2020a) . the wwtp employed upflow anaerobic sludge blanket (uasb) after the primary treatment of raw sewage water ( figure 1 ). three separate streams join three inlet chambers (7.5 m x 5 m x 2.5 m) that uses six grit chambers (10.2 m × 10.2 m x 1.0m) i.e. viral rnas were isolated from sewage samples using the following steps: precipitation of viral particle; viral rna isolation and quality checking of rna; rt-pcr analysis of viral rna for the presence of sars-cov-2. the procedure was followed as described in an earlier report with minor modifications (hjelmsø mh et al., 2017) . the sewage samples (50 ml) were centrifuged at 4500×g (model: sorvall st 40r, thermo scientific) for 30 min to remove the sludge particles. the supernatants were filtered with 0.22 micron filters (mixed cellulose esters syringe filter, himedia) to remove bacterial and eukaryotic cells. further each sewage filtrate was concentrated (for viral precipitation) using two methods: 1) using 96 well filter plate and 2) j o u r n a l p r e -p r o o f poly ethylene glycol (peg) method. for the first method filtrate was concentrated using the 96 well filter plate (acroprep tm advance 350 10k omega tm ; pall corporation) with a capacity to filter less than 10kd molecules and samples were concentrated 30 times for before rna isolation. for the second method, peg 9000 (80 g/l) (make: srl) and nacl (17.5 g/l) (make: vetec) were mixed in 25 ml filtrate and incubated at 10°c, 100 rpm (model: incu-shaker tm 10lr, benchmark) overnight. the next day the mixture was centrifuged at 13000×g (model: kubota 6500, kubota corporation) for 90 mins. after centrifugation supernatant was discarded and the remaining pellet was resuspended in 300µl rnase free water. this was further used as a sample for rna isolation. rna isolation was carried out using a commercially available kit ( quantitation of sars-cov-2 viral rna was carried out by real-time pcr with an instrument control), one negative control (from extraction run spiked with ms2), and no template control (ntc) were included. the real-time pcr thermal profile was a primary ung incubation step of 1 cycle of 25°c 2 minutes, 1 cycle of reverse transcription 53°c 10 minutes, 1 cycle of activation 95°c 2 minutes which was followed by 40 cycles of amplification including denaturation at 95°c for 03 seconds and extension 60°c for 30 seconds. interpretation of the result was performed by the applied biosystems interpretive software. in the process, the probes anneal to three specific sars-cov-2 target gene sequences: orf1ab, n protein, s protein, ms2 (internal process control). all control wells must pass for the real-time rt-pcr plate to be considered valid. if all genes show amplification then the sample will be considered as the positive. detailed procedures were carried out as described in the product manual and interpretations of results were analysed as instructed in manual. although there is no direct correlation of the c t value to copy numbers as the kit j o u r n a l p r e -p r o o f used for the detection is absent present assay but considering 500 copies of sars-cov-2 genes taken as positive control with c t values of average 26 for all the three genes i.e. orf1ab, n and s, the same was extrapolated to compare it with sample c t values and derive approximate copies of genes in the wastewater sample, using the well-established principle of 3.3 c t change corresponding to a 10-fold gene concentration change. in this semiquantitative assay, the amount of rna used as template was multiplied with the enrichment factor to derive copy numbers for each waste water sample i.e. the enrichment factor with peg method i.e. 80x and filtration methods (30x) was taken into the account to maintain the equivalence. we analyzed orf1ab, n protein genes and s protein gene from the raw wastewater, influent of uasb, effluent of uasb, water in the aeration pond, and the final effluents from wwtp old pirana after treatment by polishing pond, sampled on may 8 and may 27, 2020. the obtained amplification cycles (c t ) and concentrations of sars-cov-2 using peg for virus concentration are shown in table 1 , and those using filtration method in table 2 . the positive control sample had c t values of the three sars-cov-2 genes ranging 27.92 to 29.52, while the sars-cov-2 genes were not detected from the negative control sample. the limit of quantification (loq) of the overall method was defined as sample concentration equivalent to 1 copy per reaction tube, which was 1.7×10 2 copies/l. our results showed that peg was more suitable than filtration as a concentration method of sars-cov-2 rna in terms of sensitivity and inhibition removal. ms2 was added in each superior performance over filtration method in terms of sars-cov-2 rna sensitivity and inhibition removal. in the following sections, we evaluated sars-cov-2 rna reduction based on results with peg method. we observed a reduction of sars-cov-2 rna both during uasb treatment and during treatment at the aeration tank and the polishing pond. on may 8, all the samples were detected but inconclusive (only 1 out of 3 sars-cov-2 genes was positive) and/or not quantifiable (concentration below the loq of 1.7×10 2 copies/l). on may 27, on the other hand, raw wastewater and uasb inlet samples were detected above the loq at 1.8×10 3 copies/l and 3.5×10 3 copies/l, respectively. these sars-cov-2 rna abundance of raw wastewater in old pirana wwtp were comparable to those of untreated wastewater samples in istanbul, turkey (kocamemi et al 2020) infection density of surveyed catchment. on may 27, the sars-cov-2 rna concentration was reduced to a level with inconclusive detection after uasb treatment. when the loq of 1.7×10 2 copies/l was used as a maximum concentration after uasb, the viral reduction during uasb treatment was more than 1.3 log 10 . comparison of sars-cov-2 rna abundance during various wastewater treatment processes are provided in table 3 . the reduction of sars-cov-2 rna during wastewater treatment processes have been observed for treatments including secondary treatment (activated sludge/a2o/extended aeration) and tertiary treatment (decantation, coagulation, flocculation, sand filtration, disinfection and naclo/uv; randazzo et al 2020a, balboa et al 2020) in spain and an unspecified wastewater treatment process in paris (wurtzer et al 2020 , borchardt et al 2007 . in those studies, the log 10 reduction of sars-cov-2 rna was inferred to be 2 in wurtzer et al (2020) and from >0.1 to >0.8 in randazzo et al (2020) . in the present study, the resulting log 10 reduction of >1.3 during uasb was well within the ranges above. to our knowledge, this is the first report on sars-cov-2 rna during uasb treatment. in the case of sewage sludge digestion, anaerobic treatment has shown to be less effective in inactivation of poliovirus 1, echovirus 1 and rotavirus sa-11 in sludge (scheuerman et al 1991) . anaerobic wastewater treatment has been employed for treating variety of wastewaters, such as industrial, agricultural and municipal wastewater (mccarty 1981; mccarty and smith 1981) . therefore, reduction of sars-cov-2 rna during various anaerobic wastewater treatment, such as uasb, must be investigated in more details in the future. in the case of treatment at the aeration tank and the polishing pond, the sars-cov-2 rna was detected but inconclusive and/or not quantifiable in the aeration tank water, but the final effluents were negative with all three genes on both may 8 and may 27, potentially suggesting a reduction of sars-cov-2 rna in the aeration tank and the polishing pond. in wastewater treatment ponds, viruses are removed through various mechanisms, including adsorption, predation and sunlight inactivation (verbyla and mihelcic, 2015) . in the study j o u r n a l p r e -p r o o f site, significant degradation by sunlight and constant high temperature (~40 o c in average) in the polishing pond is very likely, but further study needs to be substantiated through high data resolution. in summary, we have successfully evaluated peg and filtration as concentration methods for sars-cov-2 rna detection. we also demonstrated the removal of sars-cov-2 during uasb treatment process. note that our results are based on small number of samples and semiquantitative analytical method, therefore our results must be substantiated through more thorough investigation in the future. we tracked the sars-cov-2 genetic loading i.e. orf1ab, n protein genes and s protein genes along the conventional treatment system outfitted with uasb system. we have found a gradual decrease in rna copies of sars-cov-2 from the raw wastewater to influent of uasb after primary treatment, effluent of uasb, aeration pond, and the final effluents after polishing pond at the study cite of 106 mld wwtp of pirana, ahmedabad, india. higher rna loading detected in the influent of 27 th may, 2020 owing to higher covid-19 active cases in ahmedabad than that on 8 th may, 2020 directly translated into higher decay along the treatment. on may 27, the sars-cov-2 rna concentration was reduced to a level with inconclusive detection after uasb treatment owing to a reduction >1.3 log 10 . to our knowledge, this is the first report on sars-cov-2 rna during uasb treatment, yet a detailed research pertaining to the reduction of sars-cov-2 rna during various anaerobic wastewater treatment, such as uasb, is further required. as we could not detect any genes j o u r n a l p r e -p r o o f in the final effluents on both may 8 and may 27, a remarkable reduction of sars-cov-2 rna in the aeration pond followed by polishing pond is evident. among the concentration methods of wastewater sample, our results explicitly indicated that peg has an advantage over filtration in terms of sensitivity and inhibition removal for rt-qpcr run and gene detection. we conclude this on the basis of c t values of ms2 which was nearly constant for peg method but varying in nature with filtration method, particularly for the raw wastewater samples. it implies that filtration method was not capable of sufficiently removing sample matrix in the sample water, and thus resulted in greater inhibitory effect of rt-qpcr than peg method. overall, implications of our study can be expressed through three major findings i.e.: i) conventional treatment system seems to be effective in reducing the sars-cov-2 genes, ii) uasb system enhances the decay of genetic loading, and iii) on this first-time comparison of peg method and filtration method, peg method has shown superior performance over filtration method in terms of sars-cov-2 rna sensitivity and inhibition removal. the authors declare no competing financial interest. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f gbrc is highly appreciated. we acknowledge the financial assistance from kiran c patel centre for sustainable development 2020) first confirmed detection of sars-cov-2 in untreated wastewater in australia: a proof of concept for the wastewater surveillance of covid-19 in the community first confirmed detection of sars-cov-2 in untreated wastewater in australia: a proof of concept for the wastewater surveillance of covid-19 in the community comparison of virus concentration methods for the rt-qpcr-based recovery of murine hepatitis virus, a surrogate for sars-cov-2 from untreated wastewater recycled water safety: current status of traditional and emerging viral indicators environmental surveillance for polioviruses in the global polio eradication initiative the fate of sars-cov-2 in wastewater treatment plants points out the sludge line as a suitable spot for incidence monitoring wastewater-based 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faecal samples prolonged presence of sars-cov-2 viral rna in faecal samples time course quantitative detection of sars-cov-2 in parisian wastewaters correlates with covid-19 confirmed cases evaluation of lockdown impact on sars-cov-2 dynamics through viral genome quantification in paris wastewaters wastewater-based epidemiology for early detection of viral outbreaks community sewage sensors for monitoring public health molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes. emerging microbes & infections viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province acknowledgement: key: cord-032222-i6gfp4me authors: xue, ling; li, jiao; wei, lin; ma, cuiqing title: a quick look at the latest developments in the covid-19 pandemic date: 2020-09-10 journal: j int med res doi: 10.1177/0300060520943802 sha: doc_id: 32222 cord_uid: i6gfp4me in december 2019, a new respiratory disease manifesting as viral pneumonia emerged in wuhan, china. isolation and identification of the virus showed that the pathogen causing this disease was a novel coronavirus. on january 12, 2020, the world health organization named the novel coronavirus causing the outbreak 2019 novel coronavirus (2019-ncov). the disease caused by the virus was named coronavirus disease 2019 (covid-19). later, the coronavirus study group of the international committee on taxonomy of viruses formally named this virus severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the virus shows strong infectivity and high lethality, arousing widespread concern. as an emerging virus, a comprehensive understanding of sars-cov-2 is missing. to provide a reference and a theoretical basis for further study of sars-cov-2, recent advances in our understanding of the virus are summarized in this review. coronaviruses are widespread in nature, only infect vertebrates and can cause respiratory, enteric, hepatic and neurologic system diseases in humans and animals. 1 coronaviruses can be divided into four genera: alpha, beta, gamma, and delta coronaviruses. 2 alpha and beta coronaviruses mainly infect mammals, while gamma and delta coronaviruses mainly infect birds. 3 seven coronaviruses can infect humans including the alpha coronaviruses, hcov-229e and hcov-nl63, and the beta coronaviruses, hcov-oc43, hcov-hku1, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and sars-cov-2. [4] [5] [6] [7] hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1 infections are relatively common in human populations. these viruses generally cause only mild respiratory symptoms similar to the common cold. 8 however, sars-cov, mers-cov, 9 and sars-cov-2 have higher infection and death rates. structural characteristics sars-cov-2 is an enveloped coronavirus. the virion is round or elliptic, often pleomorphic, and has a diameter between 60 nm and 140 nm. 10, 11 the virion can look like a crown when observed under electron microscopy. the viral spike protein present on the envelope is the most important surface protein of the virus and mediates infection. the spike protein contains two subunits: s1 and s2. s1 comprises the receptor binding domain (rbd) that recognizes cellular receptors, and s2 plays an important role in the process of membrane fusion. 12 the spike protein determines the specificity and host range of the virus, and its sequence can be altered through gene recombination or mutation of the rbd. 13 in addition, the spike protein is an important target of host neutralizing antibodies and plays a vital role in vaccine design. sars-cov-2 is a positive-sense, singlestranded rna virus, and is prone to mutation. the viral genome is about 30 kb in length, 14 encoding several open reading frames including orf1ab and the s, e, m, and n genes. 10 the genome encodes nonstructural proteins, structural proteins and helper proteins. the s, e, m, and n genes encode the spike protein, envelope protein, membrane protein and nucleoprotein, respectively. the sars-cov-2 genome shares high homology with the genomes of bat coronaviruses, 10, 15, 16 suggesting a likely origin of the virus in bats. sars-cov-2 has difficulty surviving at high temperatures and in low humidity environments. 17 the virus can be effectively inactivated at 56 c for 30 minutes. 18 at least one study demonstrated a significant positive correlation between ambient average relative humidity levels (23.33%-82.67%) and confirmed cases of covid-19. 17 coronaviruses are enveloped lipophilic viruses that are easily dissolved and destroyed by lipid solvents. most approved disinfectants including ether, 75% ethanol, chlorinated disinfectants, peroxyacetic acid, and chloroform can effectively kill coronaviruses. in addition, the virus is also sensitive to ultraviolet light. 18 pathogenic mechanism sars-cov-2 is an emerging coronavirus whose effects on the human body are not yet fully understood. preliminary studies have suggested that the pathogenic mechanism of sars-cov-2 is similar to that of sars-cov. both viruses bind to the cell surface receptor angiotensin converting enzyme 2 (ace2) in human airway epithelial cells via the s protein on the envelope and enter the host cell. 16, [19] [20] [21] [22] ace2 is highly expressed in alveolar cells type 2 as well as in the esophagus, ileum, colon, heart, kidney, bladder and testis. [23] [24] [25] [26] [27] [28] these findings suggest that ace2, which is highly expressed in esophageal epithelial cells, intestinal epithelial cells, cardiomyocytes, and bladder epithelial cells, may allow sars-cov-2 to invade several human organs and lead to multiple organ damage and failure. human populations are generally susceptible to sars-cov-2. as of june 15, 2020, there were 84,823 confirmed cases and 4,645 deaths in china, with 7,823,334 confirmed cases and 431,541 deaths globally. 29 currently, the mortality rate is about 5.5% in china and globally, lower than that of sars (10%) and mers (36%). 30 based on current epidemiological data, the incubation period of sars-cov-2 appears to be long, ranging from 1 to 14 days but lasting for 3 to 7 days in most cases. 11 the clinical manifestations of sars-cov-2 are similar to those of sars-cov. both viruses primarily cause lower respiratory symptoms, but the clinical features associated with sars-cov-2 are milder than those associated with sars-cov. 14, 31 the symptoms of most patients are nonspecific: fever, cough, and fatigue are the main manifestations. a few patients have symptoms such as nasal congestion, runny nose, and diarrhea. severe cases can develop into acute respiratory distress syndrome. 31-35 sars-cov-2 can be detected in sputum, nose and throat swabs, alveolar lavage fluid and other samples by real-time reverse transcription polymerase chain reaction. 36 specific igm and igg antibodies can be detected in the sera of covid-19 patients. 11 laboratory examinations showed that lymphocytes were decreased while levels of c-reactive protein, aspartate aminotransferase, and alanine aminotransferase were increased in most patients. on imaging examination, bilateral pneumonia can be seen with patchy shadows or ground-glass opacity. cases with milder symptoms may not show pneumonia. 31, 32, 34, 35 most patients have a good prognosis, while a few progress to critical condition. the elderly, especially those with a history of chronic diseases and surgeries, are more susceptible to infection and have higher mortality rates and worse prognoses. 37, 38 transmission and prevention sars-cov-2 can infect many animals as well as humans, and entry into human populations most likely represents a zoonotic transmission event. some groups suggested that the natural host of the virus is the bat, 16, 39, 40 although the intermediate hosts remain unclear. other wild animals may also be involved in transmission such as minks 39 and pangolins. 41, 42 sars-cov-2 is highly infectious and pathogenic. the affinity of the sars-cov-2 s protein for ace2 is approximately 10-to 20-fold higher than that of sars-cov, which may explain why sars-cov-2 is highly infectious. 43 in the face of this outbreak, we need to raise our awareness of personal protection and do all we can to reduce the spread of the virus. since the middle of december 2019, there has been persistent human-to-human transmission. [44] [45] [46] sources of infection are primarily patients with covid-19 and asymptomatic cases. [47] [48] [49] the modes of transmission include droplet, contact, and aerosol spread as well as potential transmission via the fecal-oral route. 11 no evidence of vertical transmission was observed in women who acquired sars-cov-2 infection in late pregnancy. 50 thus, we should avoid eating wild animals, improve our knowledge of sars-cov-2, and enhance awareness of personal protection strategies. to prevent or reduce the spread of the pandemic, measures should be taken to maintain social distance, avoid gatherings, wear masks when going out, cut off routes of transmission, and protect susceptible populations. sars-cov-2 is a novel coronavirus, and an effective vaccine has yet to be developed. 51 a recombinant adenovirus type 5 vector vaccine, developed by chen wei's team, showed good safety and immunogenicity in a phase i clinical trial, rapidly inducing both humoral and t-cell responses against sars-cov-2 in most participants. 52 currently, this vaccine is in phase 2 clinical trials, where its safety, tolerability and immunogenicity will be further evaluated. detection of sars-cov-2 nucleic acid is the gold standard for diagnosis of sars-cov-2 infection. methods for antigen detection, antibody detection and simpler nucleic acid detection are also being developed. igm/igg antibody rapid detection assays have appeared on the market and have been applied for clinical case detection. 11 compared with nucleic acid detection, antibody detection is often faster and more convenient. antibody detection also has good sensitivity and specificity, and can be used as a supplement to nucleic acid detection. 53 in general, diagnosis of covid-19 is based on comprehensive analysis of epidemiological risk factors, clinical manifestations, laboratory examinations, imaging examinations and pathogen detection results. there are no specific antiviral drugs against sars-cov-2, 54 and recovery from covid-19 basically depends on immunity and complementary clinical therapy. because of the urgency of the sars-cov-2 pandemic, current treatments for sars-cov-2 are largely based on clinical experience with sars and mers. the inhibitory effects of antiviral agents approved or under development on sars-cov-2 are being tested, including agents developed to treat human immunodeficiency virus or influenza virus infections. 55 potential covid-19 therapies include antiviral drugs (interferon-a, lopinavir/ritonavir, ribavirin, and hydroxychloroquine) as well as antimicrobial agents. 11 respiratory support is provided in severe cases including oxygen therapy, non-invasive mechanical ventilation, invasive mechanical ventilation, and extracorporeal membrane oxygenation. 56, 57 antibodies obtained from the plasma of convalescent patients have been used in clinical therapy. passive antibody transfer was previously used to treat sars 58 and influenza infection [59] [60] [61] and achieved good results. however, this therapy has some limitations, including limited availability of specific antibodies and the existence of certain safety risks. thus, safe and effective specific therapeutic antibodies must be developed for clinical treatment. clinical case studies showed that the antiviral drug remdesivir, which has broad spectrum antiviral effects, [62] [63] [64] [65] is effective in inhibiting sars-cov-2. [66] [67] [68] however, data from a clinical trial by a chinese research team showed that remdesivir does not provide significant clinical or antiviral effects in severe cases of covid-19. 69 some clinical trials of remdesivir are still ongoing to determine whether the drug is effective for treatment of patients with covid-19. in addition, baricitinib, 70 chloroquine 68 and some chinese patent drugs 11 with antiviral activity were also found to have therapeutic effects on covid-19. our understanding of the etiology, transmission route and pathogenic mechanism of sars-cov-2 is still preliminary and not comprehensive. some countries, including china, have controlled the covid-19 epidemic well, but the virus is still spreading in other parts of the world at an alarming rate. covid-19 may become a seasonal disease coexisting with humans for a long time. 71 there are currently no safe and specific antiviral drugs for treatment of patients with covid-19, and vaccine development will take time. therefore, the basic biological characteristics, transmission mode, and strategies for prevention and treatment of sars-cov-2 need to be further studied. in addition, broadspectrum anti-coronavirus drugs and specific monoclonal antibodies against sars-cov-2 need to be developed. in this review, we aimed to provide a reference and theoretical basis for further studies of the prevention and treatment of covid-19. the authors declare that there is no conflict of interest. this work was supported by grants from the national natural science foundation of china (81971474) and the scientific and the key r&d projects of hebei province (20277738d). ling xue https://orcid.org/0000-0001-6910-4228 coronavirus pathogenesis interspecies transmission and emergence of novel viruses: lessons from bats and birds discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian 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randomised, double-blind, placebo-controlled, multicentre trial baricitinib as potential treatment for 2019-ncov acute respiratory disease potential impact of seasonal forcing on a sars-cov-2 pandemic key: cord-280662-gakayv6e authors: bian, jingwei; li, zijian title: angiotensin-converting enzyme 2 (ace2): sars-cov-2 receptor and ras modulator date: 2020-10-13 journal: acta pharm sin b doi: 10.1016/j.apsb.2020.10.006 sha: doc_id: 280662 cord_uid: gakayv6e the coronavirus disease 2019 (covid-19) outbreak is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). angiotensin-converting enzyme 2 (ace2) was rapidly identified as the critical functional receptor for sars-cov-2. ace2 is well-known as a counter-regulator of the renin-angiotensin system (ras) and plays a key role in the cardiovascular system. given that ace2 functions as both a sars-cov-2 receptor and a ras modulator, the treatment for covid-19 presents a dilemma of how to limit virus entry but protect ace2 physiological functions. thus, an in-depth summary of the recent progress of ace2 research and its relationship to the virus is urgently needed to provide possible solution to the dilemma. here, we summarize the complexity and interplay between the coronavirus, ace2 and ras (including anti-ras drugs). we propose five novel working modes for functional receptor for sars-cov-2 infection and the routes of ace2-mediated virus entering host cells, as well as its regulatory mechanism. for the controversy of anti-ras drugs application, we also give theoretical analysis and discussed for drug application. these will contribute to a deeper understanding of the complex mechanisms of underlying the relationship between the virus and ace2, and provide guidance for virus intervention strategies. the coronavirus disease 2019 outbreak is caused by a novel coronavirus, which was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). globally, as of 16 may 2020, there have been 4,396,392 confirmed covid-19 cases and 300,441 deaths, which has already posed a great threat to global public health security 1 . although respiratory symptoms are predominant, multi-organ dysfunction occurs in response to sars-cov-2 infections, including acute cardiac and kidney injuries, arrhythmias and liver function abnormalities 2 . at present, most patients with covid-19 have a good prognosis; however, patients with underlying disorders have greater severity and higher mortality. 25 .2% of all patients had at least one underlying disease, particularly, hypertension (14.9%) and coronary heart disease (2.5%) 3 . therefore, integrated treatment for covid -19 patients with underlying diseases is key to reduce mortality. sars-cov-2 has been shown to share the same functional receptor, angiotensin-converting enzyme 2 (ace2), with severe acute respiratory syndrome coronavirus (sars-cov) 4, 5 . ace2 is a carboxypeptidase and a negative regulator of the renin-angiotensin system (ras) through balancing its homology angiotensin-converting enzyme (ace). ace mediates angiotensin (ang) ii production to activate ras that plays a key role in cardiovascular diseases, especially hypertension 6 . thus, ace inhibitor (acei) is used widely for treatment of hypertension, which reduces ang ii levels. since ace2 is a homologue of ace, disputes have arisen about whether acei can upregulate ace2 and thus the risk and severity of coronavirus infection increase. it calls into question whether to continue using of acei in virus-infected patients with hypertension. at present, some experts suggest that covid-19 patients with hypertension should stop using acei 7, 8 . on the other hand, other experts believe that not only does acei not increase the risk of sars-cov-2 infection, but acei could reduce lung injury and cardiovascular damage in covid-19 patients 9 . thus, a comprehensive was the functional receptor of sars-cov-2. excitingly, the structures of sars-cov-2 and ace2 were obtained by cryo-electron microscopy (cryo-em) quickly. firstly, the cryo-em structure of the sars-cov-2 s-protein was determined in the prefusion conformation. the predominant state of the s-protein trimer has one of the three rbds rotated up in a receptor-accessible conformation. the biophysical and structural evidence showed that the sars-cov-2 s-protein bound ace2 with higher affinity than the sars-cov s-protein (approximately 10-to 20-fold) 30 , which might explain why sars-cov-2 is more contagious than sars-cov. then, the cryo-em structure of full-length human ace2 in complex with a neutral amino acid transporter b 0 at1 was revealed 31 . immediately, the crystal structure of the rbd of sars-cov-2 s-protein bound with ace2 was determined 32 . this series of work strongly confirmed that ace2 is the functional receptor of sars-cov-2. notably, the structure of full-length human ace2 in complex with b 0 at1 revealed a novel mode that showed sars-cov-2 bound to ace2 in a homodimer manner (fig. 1b) . in this mode, b 0 at1 might be a key factor in determining the formation of an ace2 homodimer. thus, these results suggest that sars-cov-2 binds with ace2 by two working modes: i) sars-cov-2 binds directly with an ace2 monomer without the b 0 at1 protein (fig. 1a) and ii) sars-cov-2 binds with ace2 homodimer when with the existing of b 0 at1 protein (fig. 1b) . however, the binding affinity and biological function of these two different modes are still unclear. ace2 is widely expressed across a variety of organs and its expression is higher in many organs (such as heart, kidney, etc.) than that in lung. however, the lung is the major organ infected by sars-cov-2 33 . in addition, although ace2 knockout mice showed a significant decrease in sars-cov infection, it did not completely prevent virus infection from occurring 34 . those data suggested that there could be other receptors involved in a viral invasion. more recently, cd147, a transmembrane glycoprotein that belongs to the immunoglobulin superfamily, was identified as a receptor for sars-cov-2 17, 35 . interestingly, previous studies have shown that cd147 played a key role in sars-cov invasion into host cells, while cd147 antagonistic peptides have an inhibitory effect on sars-cov 36 . these results further suggested that cd147 might be a novel receptor for sars-cov-2. another possible receptor is cd209l (l-sign), a type transmembrane glycoprotein in the c-type lectin family 37, 38 , which has been identified as the receptor of sars-cov from in vitro studies 25 . thus, given that sars-cov-2 is similar to sars-cov, cd209l could be another potential receptor for sars-cov-2. therefore, besides ace2, there are several other potential receptors for sars-cov-2. sars-cov-2 might invade cells through an alternative j o u r n a l p r e -p r o o f receptor mode (fig. 1c ) or a co-receptors mode (fig. 1d) . interestingly, viruses are very tricky, as they can also infect host cells in a detour or bait-and-switch strategy. for example, sars-cov can first attach to the surface of dendritic cells (dc) through the dc-sign receptor, which is a dc-specific intercellular adhesion molecule-3-grabbing non-integrin, a homologue of cd209l. then the virus is presented to host cells by the dc cells and binds to ace2 of host cell, which eventually leads to infection 26 . similarly, sars-cov-2 might also use this subterfuge to infect a host cell. since the dc-sign receptor plays only a transmitting role, it could be called the "transmissive receptor" (fig. 1e ). in summary, we propose five distinctive working modes of sars-cov-2 receptors: monomer receptor, homodimer receptor, alternative receptors, co-receptors and transmissive receptor. the five modes are distinct but also interconnected. ace2 was discovered as a homologue of ace in 2000 39, 40 . it plays a physiological and pathological role in cardiovascular, renal, intestinal and respiratory systems [41] [42] [43] [44] . ace2 is a type i transmembrane protein, which consists of 805 amino acids with an extracellular n-terminal domain and a short to be internalized into cytoplasm upon virus binding 47, 48 . in addition to the membrane-bound form, the soluble form of ace2 can be detected in the plasma and urine through its extracellular domain shedding 49, 50 . a disintegrin and metallopeptidase domain 17 (adam17) and type ii transmembrane serine proteases (tmprss2) are considered as the proteases for ace2 proteolytic cleavage 51-53 . identifying the distribution of ace2 in organs has major implications for understanding the pathogenesis and treatment options for sars-cov-2. ace2 was initially only detected in the heart, kidneys and testes 39, 41, 54 . then, ace2 mrna expression was revealed in virtually all organs (72 human tissues) by real-time pcr 55 . in 2004, hamming et al. 56 investigated the localization of the ace2 protein in human organs by immunohistochemistry and ace2 protein expression was found in various organs, including oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain. more recently, single-cell rna-seq datasets revealed that the abundance of ace2 expression from high to low is in the ileum, heart, kidney, bladder, respiratory tract, lung, esophagus, stomach and liver 57 . here, we present a map of ace2 distribution and expression in major organs based on the previous results and the biogps website (http://biogps.org) (fig. 3 , left panel). given that ace2 is the functional receptor of sars-cov-2, its expression is associated with the organic vulnerability to sars-cov-2. indeed, the tissue expression of ace2 shows some correlation with the sites of sars-cov-2 infection. for example, ace2 protein is abundantly expressed in the lung and heart which are the vulnerable organs for sars-cov-2 33 . however, the level of ace2 expression is not completely related with the vulnerability to sars-cov-2. here, we present a comparative map of ace2 expression and vulnerability to sars-cov-2 in different organs (fig. 3 ). based on the comparative map, the ace2 expression is the highest in ileum, but the ileum is not the most vulnerable organ, suggesting other complicated mechanisms might be involve in virus infection. the possible mechanisms for inconsistency between ace2 expression and virus vulnerability include: i) there might be other unknown receptor-mediated virus infection; ii) ace2 alone is not enough to mediate virus infection, which needs another co-receptor's assistance (such as angiotensin ii type 2 receptor (at2r), a potential receptor for sars-cov-2 58 ); iii) the function of the ace2 receptor is regulated by some protein as yet unidentified. for example, b 0 at1, a neutral amino acid transporter also expressed in the ileum 59 , can bind ace2 and promotes ace2 to form a homodimer. the formation of ace2 homodimer hides the cleavage-sites for ace2 ectodomain shedding, which j o u r n a l p r e -p r o o f reduces ace2 endocytosis and further decreases virus infection in the ileum at least 31 ; and/or iv) some proteases, notably tmprss2, are also involved in the virus infection. tmprss2 can promote the membrane fusion between virus and host cell upon ace2 engagement and sars-cov-2 s-protein activation 60 . in summary, the inconsistency between ace2 expression and virus vulnerability reflects the complex mechanisms involved in virus infection and also further supports the proposed working modes shown in fig. 1 . understanding how ace2-mediated sars-cov-2 entering cell will provide valuable information for virus pathogenesis and drug target. after sars-cov-2 binds to ace2, there are two routes that sars-cov-2 enters host cell: endocytosis and membrane fusion. coronaviruses, such as sars-cov and mers-cov, have been shown to enter host cells via receptor-mediated endocytosis 48, 61 . recently, using sars-cov-2 s-protein pseudovirus system, it was found that, after binding with ace2, sars-cov-2 was also endocytosed into cell (fig. 4a) 62 . receptor-mediated endocytosis, including both clathrin-dependent pathways and caveolae-dependent pathways, is the most important mechanism for virus internalization 48, 63 . clathrin-dependent endocytosis is considered the primary endocytic route for the coronavirus. the infection of both sars-cov and mers-cov have been shown to occur by clathrin-dependent endocytosis 48, 61 . as an alternative to clathrin, caveolae-dependent endocytosis has been also described as the endocytic route for some viruses, such as simian virus 40 (sv40) 63 . however, the caveolae-dependent endocytosis does not seem to involve ace2 endocytosis after sars-cov infection. additionally, lipid raft-dependent endocytosis was found in sars-cov as a novel pathway, which is both clathrin-and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluids 47, 64 . therefore, it is reasonable to assume that sars-cov-2 binds to ace2 and might be able to either initiate clathrin-dependent and/or non-caveolae lipid raft-dependent endocytosis to enter host cell (fig. 4a ), but these need to be further verified by biological experiments. in addition to ace2-mediated virus endocytosis, tmprss2-mediated direct membrane fusion is another important way for sars-cov-2 entry. unlike the route that ace2 binds with sars-cov-2 j o u r n a l p r e -p r o o f to mediated virus endocytosis together, during the process of tmprss2-mediated membranes fusion, ace2 plays a role in arresting and fixing the sars-cov-2 at the surface. after the viruses arrested and fixed, tmprss2 induces direct membrane fusion between virus and host cell 60 (fig. 4b) . notably, the route of tmprss2-mediated membrane fusion is also crucial for sars-cov and mers-cov entry 65 . the proteolytic shedding of a transmembrane protein like ace2 can result in release of the soluble extracellular domain (ectodomain) from the membrane and a fragment that remains bound to the membrane. the shedding is an important regulatory mechanism to control the function and distribution of membrane proteins, which can terminate the function of a full-length membrane protein or release the biologically active ectodomain to activate the membrane protein. in addition, the shedding contributes to membrane protein endocytosis 66, 67 . ace2 shedding can increase sars-cov entry [51] [52] [53] , but the exact molecular mechanism responsible for increased virus entry is unclear at present. some researchers have suggested that ace2 shedding promoted ace2-mediated virus internalization and increased virus uptake into target cells 53 . here, we summarize two distinct modes for ace2 shedding: adam17-dependent shedding and tmprss2-dependent shedding (fig. 5 ). adam17, a disintegrin and metalloproteinase, has an important established role in the regulation of ace2 shedding 68 . adam17 functions in the shedding of ace2 via arginine and lysine residues within ace2 amino acids 652 to 659 53 (fig. 5) . sars-cov s-protein binding facilitates adam17-dependent ace2 shedding and has been shown to induce viral entry into the cell 52 . on the other hand, there is also another research has suggested that only type ii transmembrane serine proteases tmppss2, but not adam-17, promotes sars-cov entry via ace2 shedding 53 . different from adam17, tmppss2 requires arginine and lysine residues within ace2 amino acids 697 to 716 for ace2 cleavage 53 . notably, the neutral amino acid transporter b 0 at1 might inhibit tmprss2-dependent ace2 shedding (fig. 5b) since b 0 at1 binds ace2 to form a protein complex. the structure of the complex of ace2/b 0 at1 reveals that the tmprss2 cleavage sites (residues of 697-716) are hidden in the dimeric interface of ace2 31 . it indicates that b 0 at1 may block the access of tmprss2 to the cleavage sites on ace2 which would result in decrease of ace2 shedding. in addition, the proprotein convertase furin has been also identified as an important factor for regulation of ace2-mediated virus entering host cells. furin mainly preactivates sars-cov-2 s-protein during viral packaging, and enhances virus entry into target cells 69, 70 . however, recent study showed that furin reduced sars-cov-2 entry into vero cells 61 shows that the increase of adam-17-dependent ace2 shedding is associated with myocardial hypertrophy and fibrosis 73 . in addition, elevated soluble ace2 activity is associated with severer of myocardial dysfunction and is an independent predictor of adverse clinical events 73 . however, the question whether soluble ace2, produced by ace2 shedding, can bind with sars-cov-2 s-protein to clear away virus is confusing, which will be the future direction for research on soluble ace2. ras plays a critical role in maintaining blood pressure homeostasis, as well as fluid and salt balance. therefore, ras is intimately connected the pathophysiology of heart and kidney diseases 74, 75 . production of angiotensins from angiotensinogen requires the participation and coordination of many j o u r n a l p r e -p r o o f on ang ii, because the affinity of ace2 with ang ii is 400 times higher than that with ang i 80 . ang (1) (2) (3) (4) (5) (6) (7) binds to the mas receptor (masr), which is a seven transmembrane g-protein-coupled receptor, and forms an ace2-ang (1-7)-masr axis to mediate vasodilation and decreases blood pressure 81, 82 . in summary, current knowledge of the ras has been transformed from a linear hormonal system to a complex counter-regulatory system. the counter-regulatory ras axis, ace2-ang (1-7)-masr axis, opposes the effect of the ace-ang ii-at1r axis and has been show to reverse organ damage in renal or cardiovascular diseases 78 (fig. 6) . insert fig. 6 led to significant down-regulation of ace2 expression 34 . in post-mortem autopsy heart tissues from 20 patients who succumbed to sars-cov, seven heart samples had detectable viral sars-cov genome. these patients were also characterized by reduced myocardial ace2 expression 83 , which further confirmed that ace2 expression was decrease after virus infection. given that ace2 functions as a counter-regulator of ras, the decrease of ace2 expression leads to the weakened ace2-ang (1-7)-masr axis, mainly manifested as the increase of ang ii and decrease of vasodilator ang (1-7) level. at present, a cohort study of 12 covid-19 patients have partly confirmed this view. it was found that the level of ang ii in the plasma sample from sars-cov-2 infected patients was significantly higher than that from uninfected individuals 84 since ace2 has been identified as the functional receptor of sars-cov-2, some disputes have at present, it is not clear whether acei and arb increase ace2 expression at the protein level. for example, perindopril (acei) was able to increase hepatic ace2 expression at the protein level under conditions of liver fibrosis 89 . however, another acei drug ramipril decreased ace2 protein expression after myocardial infarction 90 . for arb drugs, olmesartan up-regulated ace2 protein expression in the carotid arteries after balloon injury. but there were no changes in ace2 protein expression in uninjured carotid arteries in olmesartan-treated rats 91 . therefore, from an ace2 ace2 functions as both a sars-cov-2 receptor and ras modulator, which presents the dilemma of j o u r n a l p r e -p r o o f how to limit virus entry while protecting its physiological function. therefore, it is of great significance to understand fully the function and mechanism of ace2 and the relationships among virus, ace2 and ras. although ace2 has been identified as a sars-cov-2 receptor, there might be other receptors or co-receptors for this virus that are yet to be discovered. in this review, we propose five working modes of functional receptors for sars-cov-2: monomer receptor, homodimer receptor, alternative receptors, co-receptors and transmissive receptor. we summarize the routes of ace2 receptor-mediated virus entering host cells (ace2-mediated virus endocytosis via clathrin-dependent pathways and non-caveolar lipid raft dependent pathways, and tmprss2-mediated membrane fusion upon ace2 engagement) and its regulatory mechanism. in addition, a comparative map of ace2 expression and vulnerability to sars-cov-2 in different organs was described. moreover, the complex relationship among coronavirus, ace2 and ras (including anti-ras drugs) is also summarized and discussed. these will contribute to a deeper understanding of the complex mechanisms and intervention strategies for virus infection and target organ damage. these raise further important implications for therapeutic targets for sars-cov-2. effective therapies against sars-cov-2 are urgently required due to the severity of the outbreak. based on the above theoretical summary, five steps are important anti-viral targets, including 1) the binding between coronavirus and ace2; 2) virus entry mediated by ace2; 3) virus replication; 4) virus assembly, and 5) virus exit (fig. 7) . one of important strategies to control viral infections is to block the initial binding of virus to its in addition, ace2 interference is another way to block the binding between the sars-cov-2 s-protein and ace2. for example, chloroquine can impact terminal glycosylation of ace2, thereby preventing it from binding to the s-protein and inhibiting the sars-cov-2 infection [103] [104] [105] . furthermore, interfering with other receptors (such as cd147) proposed in fig. 1 also can be considered as the therapeutic targets 17 . for example, meplazumab, an anti-cd147 humanized antibody, significantly inhibited the viruses from invading host cells 35 . j o u r n a l p r e -p r o o f â�¡). ace2 receptor-mediated endocytosis is a complex process regulated by a variety of proteins, which are potential target for interference. for example, ap2-associated protein kinase 1 (aak1) is a well-known positive regulator of receptor endocytosis. an inhibitor of aak1 (baricitinib) is predicted to reduce the ability of the sars-cov-2 entry by inhibiting ace2 receptor-mediated endocytosis 106 . in addition, camostat mesylate, a tmprss2 inhibitor, has been shown to inhibit sars-cov-2 entry into cells 60 . of course, in addition to the steps related to ace2 (the binding between coronavirus and ace2, and virus entry mediated by ace2), virus replication has been the hotspot for antiviral drug research. some drugs have entered clinical practice (fig. 7 â�¢) . covid-2019 situation reports sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2 clinical characteristics of coronavirus disease 2019 in china evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural 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molecule-3-grabbing integrin the authors acknowledge funding support from the national natural science foundation of china (91939301, 81820108031, 91539123, and 81471893) and beijing municipal natural science foundation (7172235, china). all authors researched data for the article and discussed its content. jingwei bian wrote the manuscript. zijian li designed, reviewed and edited this manuscript before submission. on behalf of all authors, the corresponding author states that there is no conflict of interest. key: cord-266987-ikt8r2o1 authors: loeffelholz, michael j.; tang, yi-wei title: laboratory diagnosis of emerging human coronavirus infections – the state of the art date: 2020-03-30 journal: emerg microbes infect doi: 10.1080/22221751.2020.1745095 sha: doc_id: 266987 cord_uid: ikt8r2o1 the three unprecedented outbreaks of emerging human coronavirus (hcov) infections at the beginning of the twenty-first century have highlighted the necessity for readily available, accurate and fast diagnostic testing methods. the laboratory diagnostic methods for human coronavirus infections have evolved substantially, with the development of novel assays as well as the availability of updated tests for emerging ones. newer laboratory methods are fast, highly sensitive and specific, and are gradually replacing the conventional gold standards. this presentation reviews the current laboratory methods available for testing coronaviruses by focusing on the coronavirus disease 2019 (covid-19) outbreak going on in wuhan. viral pneumonias typically do not result in the production of purulent sputum. thus, a nasopharyngeal swab is usually the collection method used to obtain a specimen for testing. nasopharyngeal specimens may miss some infections; a deeper specimen may need to be obtained by bronchoscopy. alternatively, repeated testing can be used because over time, the likelihood of the sars-cov-2 being present in the nasopharynx increases. several integrated, random-access, point-of-care molecular devices are currently under development for fast and accurate diagnosis of sars-cov-2 infections. these assays are simple, fast and safe and can be used in the local hospitals and clinics bearing the burden of identifying and treating patients. cov, mers-cov and sars-cov-2). in temperate regions endemic hcovs usually display a winter seasonality, although hcov-229e has been detected sporadically throughout the year [12] . endemic hcovs are globally distributed and are maintained in the human population. the sars-cov pandemic came to an end in 2003 (https://www.who.int/csr/resources/ publications/cds_csr_aro_2004_2.pdf?ua=1. accessed 3 february 2020), less than a year after the first reported case. in contrast, human cases caused by mers-cov continue to be reported at the time of writing, more than seven years after the first reported case. most laboratory-confirmed mers cases have occurred in the eastern mediterranean region, and the majority of those in saudi arabia. unlike the endemic hcovs, sars-cov and mers-cov are maintained in zoonotic reservoirs. the sars and mers outbreaks were driven in part by super-spreading events in which individuals directly infected a disproportionally large number of contacts [13] . the sars-cov-2-caused coronavirus disease 2019 (covid19) epidemic originated in a wuhan, china market that sold exotic animals for consumption. based on genetic relatedness to other betacoronaviruses, sars-cov-2 likely has a zoonotic reservoir. however, the precise source of sars-cov-2 that initially infected humans remains to be confirmed. the sars-cov-2 appears to be substantially more contagious than sars-cov ( table 1 ). the distribution of sars-cov-2 in different mammalian species is unknown. an interesting question is the susceptibility of farm animals and pets, and their role in the epidemiologic cycle as their angiotensin-converting enzyme 2 (ace2) receptor shares similarity with human ace2 [14] . infections caused by endemic hcovs have an incubation period of 2-5 days and are associated with mild upper respiratory symptoms (the "common cold"). endemic hcovs are among the most frequent cause of upper respiratory tract infections. lower respiratory tract infections (bronchiolitis, pneumonia) are rare. following an incubation period of usually 4-5 days, patients infected with sars-cov often present with symptoms of fever, headache, and myalgias. respiratory symptoms including cough and dyspnoea usually develop from several days to a week after illness onset. atypical pneumonia and respiratory deterioration occur in 20-30% of cases. the incubation period and clinical course of mers are similar to that of sars, the exception being a higher proportion of cases progressing to respiratory deterioration and distress. the incubation period and clinical course of sars-cov-2 infection are probably similar to that of sars. li et al. first reported a mean incubation period of 5.2 days [15] . fever and cough are frequently reported early in the course of illness [16, 17] . infections are also characterized by dyspnoea, respiratory distress and positive chest x-ray [10] . lower respiratory symptoms often develop about 1 week from the onset of initial symptoms [16] . globally over 8000 cases and over 900 deaths due to sars-cov were reported, with a case-fatality ratio of approximately 11% (https://www.who.int/csr/sars/en/ whoconsensus.pdf. accessed 3 february 2020). between september 2012 and november 2019, there were 2494 laboratory-confirmed cases of mers, with 858 deaths (https://www.who.int/emergencies/merscov/en/. accessed 4 february 2020). the mers casefatality rate of 34.4% is about triple that of sars, and persons in the 50-59 year age group are at highest risk for primary cases. in the short time from its emergence in december 2019 to 15 march 2020, the sars-cov-2 has been reported in 134 countries. at the time of writing, the situation was evolving rapidly, with over 142,000 confirmed cases reported globally (over 81,000 in china) and 3194 deaths in china (3.9% case-fatality rate) and over 2100 deaths outside of china. of countries and continents outside of china, south korea, iran, and europe (particularly italy) have experienced a high number of covid-19 cases (https://www.who.int/emergencies/diseases/novel-coro navirus-2019/situation-reports/. accessed 15 march 2020). mortality rates vary widely, and depend on the age of patients, underlying risk factors, and the denominator definitionhospitalized cases, all symptomatic cases, only moderate to severe cases, etc. in a study of adult patients (mean age 59.7 y; 40% with chronic illnesses) with sars-cov-2 pneumonia admitted to the intensive care unit (icu), 61.5% died within 28 days [18] . in contrast, a study of hospitalized patients (median age 47.5 years) across beijing showed [8, 20, 22, 59] 18% of cases to be severe and 73% mild, with a fatality rate of 0.9% [19] . mortality is highest in older persons, with a median age of 59-75 years [15, 17] . treatment for all severe hcov infections is supportive although a randomized, double-blinded, control clinical trial has been conducted on a gilead drug remdesivir [20] based on one study focused on children, a total of 28 children aged from 1 month to 17 years have been reported in china. all paediatric cases with laboratory-confirmed sars-cov-2 infection were mild cases with no deaths reported [21] . during the first 2 months of the current outbreak, covid-19 spread rapidly throughout china and caused varying degrees of illness with a death rate of 1.3%. patients often presented without fever, and many did not have abnormal radiologic findings [22] . the chinese centers for disease control and prevention team analysed more than 72,000 patient records, of which 44,672 were laboratory-confirmed cases, 16,186 suspected cases, 10,567 clinically diagnosed cases, and 889 asymptomatic cases. of the confirmed cases, about 14% of the illnesses were severe, which included pneumonia and shortness of breath, and about 5% have the critical disease, marked by respiratory failure, septic shock, and multi-organ failure. the overall case-fatality rate was 2.3%, and of 1023 deaths included in the study, the majority were in people age 60 and older or those with underlying medical conditions http://www.cidrap.umn.edu/news-perspective/ 2020/02/more-outbreak-details-emerge-covid-19-casestop-70000 (accessed 18 february 2020). it must be appreciated that no matter how accurate and fast laboratory testing methods are, the diagnosis of viral pneumonias such as caused by sars-cov-2 involves collecting the correct specimen from the patient at the right time. the endemic hcovs have been detected from a variety of upper and lower respiratory sources including throat, nasal nasopharyngeal (np), sputum, and bronchial fluid [12, 23, 24] . wang et al have just reported that oropharyngeal (op) swabs (n = 398) were used much more frequently than np swabs (n = 8) in china during the covid-19 outbreak; however, the sars-cov-2 rna was detected only in 32% of op swabs, which was significantly lower than that in np swabs (63%) [25] . the us centers for disease control and prevention (cdc) recommends collecting the upper respiratory np swab. collection of an op specimen is a lower priority, and, if collected, should be combined in the same tube as the np swab (https://www.cdc.gov/coronavirus/ 2019-ncov/lab/guidelines-clinical-specimens.html. accessed 16 march 2020). swab specimens should be placed in a universal or viral transport medium. nasopharyngeal aspirates are also suitable specimens for the detection of hcovs. for the most sensitive detection of sars-cov, mers-cov, and sars-cov-2, the collection and testing of both upper and lower respiratory samples [sputum, bronchoalveolar lavage fluid (bal)] is recommended [26] . however, the collection of sputum and particularly bal via bronchoscopy increases biosafety risk to healthcare workers through the creation of aerosol droplets. proper use of personal protective equipment (ppe) by healthcare workers is important. bronchoscopy is a highly technical procedure requiring well-trained staff and may not be available in many parts of the world. upper respiratory specimens are easy to collect, thereby increasing access to testing for patients with mild symptoms, and in the resource limited settings. sars-cov and mers-cov rna are also detected from stool, urine and blood specimens, although generally less reliably than from respiratory specimens [26] [27] [28] ]. an exception is sars-cov rna which is consistently detected in feces at about two weeks after symptom onset [26, 29] . for the most sensitive detection of endemic hcovs, upper respiratory specimens should be collected within the first few days of symptom onset. the dynamics of rna shedding in mers and sars patients may reflect the specimen source, severity of illness, as well as underlying risk factors. among hospitalized patients who did not require ventilator support, mers-cov rna levels in the upper respiratory tract usually peaked in the first week after symptom onset. among eventual fatal cases requiring ventilation, rna levels in lower respiratory tract specimens peaked between weeks 2 and 3 [27] . similar shedding patterns were seen for sars-cov: rna positive rates peaked in upper respiratory tract specimens at 7-10 days after symptom onset and then steadily declined after that, while rna positive rates in lower respiratory tract specimens remained higher throughout 3 weeks after onset of illness [26] . in one study, diabetes was associated with prolonged mers-cov rna shedding in the respiratory tract [27] . viral pneumonias typically do not result in the production of purulent sputum. thus, a nasopharyngeal swab/wash is usually the collection method used to obtain a specimen for testing. nasopharyngeal specimens may miss early infection; a deeper specimen may need to be obtained by bronchoscopy. alternatively, repeated testing can be used because over time, the likelihood of the sars-cov-2 being present in the nasopharynx increases. self-collected saliva specimens were tested positive in 11 of 12 covid-19 patients, suggesting it is a promising non-invasive specimen for diagnosis, monitoring, and infection control in sars-cov-2 infections [30] . at the time of writing there was little data on the performance of upper vs. lower respiratory tract specimens for the detection of sars-cov-2 [16] . serum is another source for the detection of sars-cov-2. however, only 15% of patients hospitalized with pneumonia had detectable rna in serum [16] . specimens collected for laboratory testing of hcovs should be maintained at refrigerated temperature for up to 72 h, or frozen at −70°c or below (https://www.cdc.gov/coronavirus/ 2019-ncov/lab/guidelines-clinical-specimens.html. accessed 15 march 2020). rectal specimens have been reported positive in patients infected with sars-cov-2 [20] . if the patient's travel or exposure history or symptoms suggest possible infection with a high-risk, novel agent, sars-cov, or mers-cov, then the initial handling of the specimen should be performed under biosafety level 3 (bsl-3) conditions until the specimen or an aliquot is rendered noninfectious by lysis or another method. virus isolation should not be routinely performed in this situation (https://www.asm.org/ articles/policy/laboratory-response-network-lrn-sentinel-level-c. accessed 4 february 2020). the u.s. cdc biosafety guidelines state that routine diagnostic testing of specimens from suspected or confirmed sars-cov-2 patients, can be handled in a bsl-2 laboratory using standard precautions (https://www.cdc. gov/coronavirus/2019-ncov/lab/lab-biosafetyguidelines.html. accessed 21 march 2020). isolation of hcovs in cell culture is not routinely performed for diagnostic purposes due to the lack of permissive cell lines, time to results, labour and expertise requirements, and the lack of commercial antisera for culture confirmation (table 2) . sars-cov and mers-cov and sars-cov-2 will grow in primary monkey cells and cell lines such as vero and llc-mk2, but cell culture should not be performed for suspect cases in routine diagnostic laboratories for biosafety reasons [2, 6, 31, 32] . however, virus isolation in cell cultures is critical to obtain isolates for characterization and to support the development of vaccines and therapeutic agents. rapid antigen tests would theoretically provide the advantage of fast time to results and low-cost detection of hcovs but are likely to suffer from poor sensitivity based on the experience with this method for influenza (flu) viruses [33] [34] [35] [36] [37] (table 2 ). in a pre-peer reviewed article, diao et al. reported that a fluorescence immunochromatographic assay is an accurate, rapid, early and simple method for detecting nucleocapsid protein of sars-cov-2 in np swab for diagnosis of covid-19 (https://www.medrxiv.org/content/10.1101/2020.03. 07.20032524v2. accessed 15 march 2020). the incorporation of colloidal gold-labeled immunoglobulin g (igg) as the detection reagent is an approach that may increase the sensitivity of rapid antigen tests for respiratory viruses [38] . monoclonal antibodies specifically against sars-cov-2 have been under preparation. novel approaches to concentrate antigen, or to amplify the detection phase are needed if these methods are to have clinical utility. sona nanotech (halifax, canada) is developing a quick-response lateral-flow test to screen covid-19 patients targeting to produce results in 5-15 min (https://sonanano.com/sona-develops-rapidscreening-test-for-coronavirus/. accessed 15 february 2020). timing of specimen collection, when viral titres are highest, may improve the diagnostic sensitivity of rapid antigen tests for hcovs [39] . serological assays are not routinely used for diagnosis of hcov infections due to the lack of commercial reagents, let alone commercial reagents that have been vetted by clinical trials and the regulatory review process [40, 41] (table 2 ). serological assays, on the other hand, are important for understanding the epidemiology of emerging hcovs, including the burden and role of asymptomatic infections. it has been particularly important for antibody detection in the diagnosis of cases of novel and emerging hcovs, such as sars-cov and mers-cov [2, 3] . in these situations, affected patients may not test positive for viral rna, particularly in the early phase of the disease, but retrospectively can be shown to have developed an immune response. when sars-cov-2 was identified, especially when rapid antigen testing and/or molecular assays are neither available nor stable, serology can be used as a supplementary diagnostic tool. a recent study demonstrated that both igm and igg antibodies were detected 5 days after onset in all 39 patients infected with sars-cov-2 infection. the authors recommended to use serology to facilitate the diagnosis of sars-cov-2 infections when an np swab specimen was collected inappropriately and the molecular assays were performed unsatisfactorily [42] . in china, six serology devices have just received urgent approval from the national medical products administration (nmpa) by 12 march 2020 (table 3) . proper specimen handling and storage are important to maintain the integrity of specimens and the performance of serologic tests. random-amplification deep-sequencing approaches played a critical role in identifying mers-cov and sars-cov-2 [6, 11, [43] [44] [45] [46] [47] . for the clinical diagnostic application, the genetic heterogeneity of hcovs precludes a single "pan-hcov" molecular assay [48] [49] [50] [51] ( table 2 ). some pan-cov assays use degenerate primers [52] , some utilize multiple primer sets [53] , and others employ a single set of nondegenerate primers [54] . current molecular respiratory panels that detect the endemic hcovs (hcov-nl63, hcov-hku1, hcov-oc43, and hcov-229e) require multiple sets of pcr oligonucleotides [12, [55] [56] [57] . sars-cov-2 cases tested negative for endemic hcovs included in molecular respiratory panels [10] . in china, at the time of revising, eleven molecular devices from shanghai zj bio-tech, shanghai geneodx biotech, bgi biotech (wuhan), mgi tech, da an gene, sansure biotech, shanghai biogerm medical biotech capitalbio (chengdu), beijing applied biological technologies, maccura biotechnology, and wuhan easydiagnosis biomedicine have received urgent approval from nmpa and their characteristics are contrasted in table 3 . variable performance has been reported on these devices [47, 58] . in their registration certificates, it was clearly indicated that the certificate was for urgent and supplemental diagnosis of pneumonia caused by sars-cov-2. additional multi-centre clinical trial data are needed for extension after one year. among them, one (mgi tech) uses its ngs technique to detect all pathogens in a given specimen including sars-cov-2 and the other one (innovita) uses its isothermal amplification followed by chip detection. the other nine devices incorporated real-time pcr technique with hydrolysis probes. after nucleic acids get extracted (separated reagents and systems), the extracts are transferred to a real-time pcr thermocycler (e.g. abi 7500 fast dx real-time pcr instrument) for nucleic acid amplification and detection. several rt-pcr protocols for detection of sars-cov-2 rna have been posted by the world health organization at https://www.who.int/emergencies/ diseases/novel-coronavirus-2019/technical-guidance/ laboratory-guidance. (accessed 15 march 2020). three of these protocols are listed below. the us cdc developed developed a rt-pcr diagnostic panel for universal detection of sars-like betacoronaviruses and specific detection of sars-cov-2 [20] . three separate rt-pcr reactions target the n gene. one primer/probe set detects all betacoronaviruses, while two sets are specific for sars-cov-2. all 3 assays must be positive to report presumptive positive for sars-cov-2 (https://www.fda.gov/ media/134922/download. accessed 15 march 2020). specimen types included upper and lower respiratory specimens (such as np or op swabs, sputum, lower respiratory tract aspirates, bal, and nasopharyngeal wash/aspirate or nasal aspirate). it the charité algorithm (berlin, germany) begins with two rt-pcr assays that detect e and rdrp genes of subgenus sarbecovirus (sars-cov, sars-cov-2, and bat-associated betacoronaviruses). both assays must be positive to advance to the next step in the testing algorithm. the second step consists of a [52] [53] [54] naat, monoplex, specific-hcov high sensitivity and specificity for special species, potential quantification 1-8 h diagnosis (detection, differentiation, and limited typing) and research [69, 70] naat, multiplex high sensitivity and specificity, covering other pathogens, filmarray rp ez is clia-waived 1-8 h diagnosis (detection, differentiation, and limited typing) and research [12, [55] [56] [57] naat, poct rapid and safe, good sensitivity and specificity, some are clia-waived diagnosis (detection and limited differentiation) and research [63, 67] note: eia, enzyme immunoassay; ifa, immunofluorescent assay; naat, nucleic acid amplification test; clia, clinical laboratory improvement act. sars-cov-2 specific rt-pcr that targets rdrp [59, 60] . exclusivity testing showed that alphacoronaviruses (cov-nl63 and −229e) and betacoronaviruses hcov-oc43, hcov-hku1 and mers-cov were not detected (https://www.who.int/docs/default-source/ coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2. accessed 8 february 2020). the university of hong kong li ka shing faculty of medicine protocol uses two assays (n gene screening assay followed by orf1b assay for confirmation) to detect subgenus sarbecovirus [30, 61] . since sars-cov is not circulating in humans currently, cases that are positive should be considered as sars-cov-2 infected cases. exclusivity testing showed that 229e, oc43 and mers, 229e, hku1, nl63, oc43 yielded negative results (https://www.who.int/docs/defaultsource/coronaviruse/peiris-protocol-16-1-20.pdf? sfvrsn=af1aac73_4. accessed 8 february 2020). all three novel coronaviruses are highly contagious. fast, safe, simple to use diagnostic devices performed at or near the point of care (poc) (figure 1 ) which have been shown to impact patient management and control of infectious disease epidemics [62], are extremely desirable in poc when biosafety facility is limited (table 3) . several manufactures have been spending efforts to generate devices for poc testing (poct) [63] . the id now™ (previously alere i) influenza a & b assay (abbott, san diego, ca) was cleared by the us fda for direct use on np swabs as the first-ever clinical laboratory improvement amendments (clia)-waived nucleic acid-based test in january 2016 [64, 65] . similarly, the xpert® xpress flu/rsv (cepheid, sunnyvale, ca) and cobas® liat® flu a/b & rsv (roche molecular systems, pleasanton, ca) assays are integrated nucleic acid extraction-independent devices that have recently received fda clearance and clia-waiver for simultaneous detection and identification of flua, flub, and rsv in nasopharyngeal swabs [66] . the filmarray® respiratory ez panel (biofire, salt lake city, ut) so far so far is the only clia-waived syndromic panel that covers a set of 14 respiratory viral and bacterial pathogens including classical coronavirus species [67] . considering the increased levels of mortality and infectivity associated with three novel-coronavirus outbreaks, these random-access, safe and simple tests, which offer fast and accurate detection and identification, are likely to have an immediate impact on prompt clinical and epidemiological decisions [7, 63] . lysis buffer can be used to inactivate the infectivity of specimens so the testing can be run at poc when a biosafety cabinet is not available. fast near-patient and poct could help more efficiently triage of suspected cases of novel coronavirus, helping to focus limited resources on enabling appropriate use of quarantine. a handful of diagnostics developers are now striving to bring fast sars-cov-2 tests to market as soon as possible, with hopes of ultimately assisting with the ongoing outbreak in china. molecular diagnostic tests for use at the are in development from cepheid and hibergene (dublin, ireland). cepheid has some advantages in the molecular poct space because it already has instruments placed in china. mobidiag, meanwhile, may offer additional benefits with a multiplex test for coronavirus and flu viruses (https://www. genomeweb.com/pcr/diagnostics-firms-rush-developrapid-point-care-tests-novel-coronavirus#.xkea3sgzy 2x. accessed 15 february 2020). mjl and y-wt are employees of cepheid, the commercial manufacturer of the xpert xpress sars-cov-2 test. figure 1 . evolutions in molecular testing procedures. the point-of-care test (poct) devices incorporate nucleic acid extraction, amplification and detection together into an integrated and sealed cartridge making it simple, rapid and safe. during end-point pcr, dna is detected or measured at the completion of pcr amplification, requiring post-pcr processing. real-time pcr is a closed-tube system in which dna is detected or measured during the exponential phase of amplification. epidemiology, genetic recombination, and pathogenesis of coronaviruses a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory 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a mobile laboratory in liberia: impact on outbreak response, case management and laboratory systems strengthening point-of-care testing for infectious diseases: past, present, and future evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b profile of the alere i influenza a & b assay: a pioneering molecular pointof-care test parallel validation of three molecular devices for simultaneous detection and identification of influenza a and b and respiratory syncytial viruses performance and impact of a clia-waived, point-of-care respiratory pcr panel in a pediatric clinic identification of a new human coronavirus development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays key: cord-268034-7id7sfsu authors: auerswald, heidi; yann, sokhoun; dul, sokha; in, saraden; dussart, philippe; martin, nicholas j.; karlsson, erik a.; garcia-rivera, jose a. title: assessment of inactivation procedures for sars-cov-2 date: 2020-05-28 journal: biorxiv doi: 10.1101/2020.05.28.120444 sha: doc_id: 268034 cord_uid: 7id7sfsu severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of coronavirus disease 2019 (covid-19), presents a challenge to laboratorians and healthcare workers around the world. handling of biological samples from individuals infected with the sars-cov-2 virus requires strict biosafety and biosecurity measures. within the laboratory, non-propagative work with samples containing the virus requires, at minimum, biosafety level-2 (bsl-2) techniques and facilities. therefore, handling of sars-cov-2 samples remains a major concern in areas and conditions where biosafety and biosecurity for specimen handling is difficult to maintain, such as in rural laboratories or austere field testing sites. inactivation through physical or chemical means can reduce the risk of handling live virus and increase testing ability worldwide. herein we assess several chemical and physical inactivation techniques employed against sars-cov-2 isolates from cambodian covid-19 patients. this data demonstrates that all chemical (avl, inactivating sample buffer and formaldehyde) and heat treatment (56°c and 98°c) methods tested completely inactivated viral loads of up to 5 log10. to determine if any viable virus remained post inactivation, 50% polyethylene glycol 8000 133 (sigma-aldrich, st. louis, usa) in pbs was added (1/5 of total sample volume) to an aliquot 134 from each sample condition and incubated overnight at 4°c. following incubation, virus was 135 recovered by centrifugation at 1,500 rpm for 1h. precipitates were washed twice with sterile pbs, 136 re-constituted with infection medium, and used for infecting the tcid50 on vero e6 cells and 137 recovery cultures on vero cells. negative controls were treated the same way to examine 138 cytotoxicity of possible remaining traces of inactivation solutions. 139 140 sars-cov-2 real-time rt-pcr 141 following inactivation, rna from one aliquot per condition per virus isolate and negative control 142 was immediately extracted with the qiaamp viral rna mini kit (qiagen) and stored at -80°c 143 until further processing. real-time rt-pcr assays for sars-cov-2 rna detection were 144 performed in duplicate using the charité virologie algorithm (berlin, germany) to detect both e 145 and rdrp genes [9] . in brief, real-time rt-pcr was performed using the superscript™ iii one-146 step rt-pcr system with platinum™ taq inc., la jolla, ca,, usa). analysis of variance was performed comparing mean ct values for each 157 inactivation method. difference between standard (avl) and each specific inactivation method 158 was determined using dunnett's test for many-to-one comparison. a p-value of less than 0.05 was 159 considered to indicate statistical significance. agreement, including bias and 95% confidence interval, between ct values following inactivation by avl and other methods was assessed using all chemical and thermal inactivation methods resulted in the reduction of viable sars-cov-2 to 186 undetectable levels. untreated virus isolates had a concentration of viable virus up to 6.67 x 10 5 187 (isolate 2310) before treatment (table 1) previous studies have been conducted on the effectiveness of chemical inactivation 220 techniques on sars-cov-2 [11, 12] , the majority of these based on infectious agents of concern 221 such as ebola [13] and sars and mers coronaviruses [14] . as with other viruses, the primary 222 step in the molecular detection of sars-cov-2 is viral lysis to begin the extraction of nucleic 223 acids. the buffers used in this lysis step yield varying results [11, 13, 15, 16] ; however, unlike 224 previous studies [11] , this study found that avl buffer alone was successfully able to fully 225 inactivate up to 5 log10 of virus from three different primary isolates of sars-cov-2. apart from 226 differences in isolates utilized and a slight reduction in titer, it is unclear as to the reasons why 227 avl buffer fully inactivated in this study versus others, but further work is warranted to determine 228 the exact effectiveness of this step alone. inactivating sample transport media, either made in-house or commercially available, also 230 presents an attractive way to inactivate samples at the point of sampling to ensure safe handling 231 along the transport chain and within the laboratory. these inactivating transport media include the 232 key components of many viral lysis buffers including chaotropic agents (gitc), detergents (triton 233 x-100) and buffering agents (edta, tris-hcl) to inactivate a preserve viral rna. previous 234 studies have shown that gitc-lysis buffers are able to inactivate sars-cov-2 samples [11, 12] ; 235 however, the addition of triton-x may be necessary for complete inactivation [11] . in line with 236 these studies, commercial sample transport media containing both gitc and triton-x was 237 successfully able to inactivate up to 5 log10 of virus with no loss of molecular diagnostic sensitivity. 238 apart from sample media and buffers utilized for diagnostic testing, various disinfectant 239 and inactivating chemicals are available for sample treatment. formaldehyde has a long history of 240 use for inactivation against a number of viruses and in a number of fixation techniques, including 241 vaccine preparations [17, 18] . formaldehyde has been shown to successfully inactivate both sars 242 and mers [14, 19, 20] and has been suggested to be a viable alternative for disinfection and 243 inactivation of 20] . formaldehyde treatment did successfully inactive up to 5 244 log10 of virus; however, this treatment severely impacted viral detection in subsequent molecular 245 testing. this decreased detection is not unexpected as formaldehyde treatment results in rna 246 degradation and modification [21] . therefore, formaldehyde treatment does not appear to be a 247 solution for increased molecular sars-cov-2 testing; however, it does remain a viable alternative 248 for sample inactivation or disinfection. 249 perhaps the most studied technique thus far regarding sars-cov-2 has been thermal 250 inactivation at various times and temperatures [11, [22] [23] [24] . several previous studies have shown 251 heat to be an effective inactivation technique against other coronaviruses, including sars, mers, and human seasonal strains [14, 23, 25] . similar to previous studies, 56 o c heat treatment for 30 or 253 60 minutes was fully able to inactivate up to 5 log10 of sars-cov-2 from three different isolates 254 [11, 22] . interestingly, while other studies utilized 95 o c for 5 to 10 minutes for inactivation, heat 255 treatment at 98 o c for only 2 minutes was also able to completely inactivate up to 5 log10 of virus. 256 these results are very promising as high heat treatment is extremely rapid and may be a vital 257 addition to the testing arsenal, as rt-pcr can possibly be performed directly from these samples 258 without the need for nucleic acid extraction [26, 27] . interestingly, the shortened time period of 259 high heat treatment may mitigate some of the reduction in detection seen in previous studies and 260 make this technique more employable [11] . 261 overall, the agreement and retained sensitivity amongst rt-pcr results, combined with 262 the fact that all methods resulted in 100% virus inactivation up to a viral load of 5 log10, suggests 263 that any of the tested methods, except formaldehyde, are useful to inactivate sars-cov-2 264 samples. given the who recommendation to "test, test, test," these data can help to optimize 265 sample inactivation for austere or remote areas. indeed, it may be possible to use basic tools such 266 as a stopwatch and boiling water to achieve 100% virus inactivation without compromising sample 267 integrity, significantly decreasing possible exposure during sample transportation and handling, 268 allowing for dissemination of testing to labs with decreased biosafety and biosecurity capacity, world health organization. coronavirus disease (covid-19) outbreak an interactive web-based dashboard to track covid-303 19 in real time. the lancet infectious diseases guidelines for handling and processing specimens associated with coronavirus disease world health organization. laboratory testing for coronavirus disease 2019 (covid-309 19) in suspected human cases: interim guidance un list of least developed countries developed-countries.aspx emerging infectious diseases and public health policy: insights 316 from cambodia, hong kong and indonesia determination of 50% endpoint titer using a simple formula statistical methods for assessing 325 agreement between two methods of clinical measurement evaluation of heating and chemical protocols for inactivating sars-328 validation of a lysis buffer containing 4 m guanidinium thiocyanate (gitc)/ triton x-100 for extraction of sars-cov-2 rna for covid-19 comparison of formulated lysis buffers containing 4 to 6 m gitc, roche 332 external lysis buffer and qiagen rtl lysis buffer. biorxiv buffer avl alone does not inactivate ebola virus in a 335 representative clinical sample type inactivation of the coronavirus that induces severe acute 338 respiratory syndrome, sars-cov virus inactivation by nucleic acid extraction reagents unreliable inactivation of viruses by commonly used lysis buffers inactivation methods for whole influenza vaccine production formaldehyde treatment and safety testing of experimental poliomyelitis 346 vaccines. american journal of public health and the nation's health coronavirus disinfection in histopathology persistence of coronaviruses on inanimate surfaces and their 351 inactivation with biocidal agents the effect of formaldehyde fixation on rna: optimization of 353 heat inactivation of the severe acute respiratory syndrome 356 inactivation of coronaviruses by heat resilient sars-cov-2 diagnostics workflows including viral heat 360 inactivation heat inactivation of the middle east respiratory syndrome 362 coronavirus. influenza and other respiratory viruses extraction-free covid-19 (sars-cov-2) diagnosis by rt-pcr to 364 increase capacity for national testing programmes during a pandemic an alternative workflow for molecular 367 detection of sars-cov-2 -escape from the na extraction kit-shortage key: cord-254446-yxqbe1dj authors: ren, yunzhao r.; golding, amit; sorbello, alfred; ji, ping; chen, jianmeng; bhawana, saluja; witzmann, kimberly; arya, vikram; reynolds, kellie s.; choi, su‐young; nikolov, nikolay; sahajwalla, chandrahas title: a comprehensive updated review on sars‐cov‐2 and covid‐19 date: 2020-05-29 journal: j clin pharmacol doi: 10.1002/jcph.1673 sha: doc_id: 254446 cord_uid: yxqbe1dj this literature review aims to provide a comprehensive current summary of the pathogenesis, clinical features, disease course, host immune responses, and current investigational antiviral and immunomodulatory pharmacotherapies, in order to facilitate the development of future therapies and measures for prevention and control. this article is protected by copyright. all rights reserved the disease name -covid-19‖ and the associated virus name -sars-cov-2‖ were coined by the world health organization (who) and the coronavirus study group of the international committee on virus taxonomy, respectively, on february 11 1, 2 . currently no specific drug has been approved by the fda for treating covid-19, and the current management of patients is mainly supportive. fda has issued only emergency use authorizations (eua) to permit the emergency use of chloroquine phosphate, hydroxychloroquine sulfate, and remdesivir. therapeutic development for covid-19 includes repurposing existing medications and developing investigational candidates. the first reported confirmed covid-19 case was presented as atypical pneumonia on december 8, 2019 in wuhan, china. 3 the patient was among a cluster of 41 cases reported to who on january 11, 2020 4 . as of may 19, 2020, sars-cov-2 has infected over 4.6 million people worldwide 5 . covid-19 had resulted in a global death toll of more than 319,000. an epidemiology study noted that of 44,672 confirmed covid-19 cases in china through february 11, 13 .8% and 4.7% of patients were in severe and critical condition, respectively 6 . the reported median incubation period of covid-19 is 4 days 7 , and the median period from symptom onset to hospital admission ranges from 7 to 10 days 8, 9 . median time from onset of the first symptom to dyspnea is 4-5 days 7, 8 ; to pneumonia, 3 days 7 ; to icu admission, 6 days 10 ; and to acute respiratory distress syndrome (ards), 8 days 8 . the median duration of hospitalization is 12 days 7 to 22 days 11 ; median length of icu stay, 18 days 10 ; median time from admission to invasive mechanical ventilation, 14.5 days 11 ; and median time from admission to death was 18.5 days 11 . the currently estimated reproductive number (r 0 ) of sars-cov-2, the average number of people to which one infected individual will pass the virus, ranges from 2.2 to 5.7 [12] [13] [14] , whereas the reported r 0 for sars-cov is approximately 3 15 . sars-cov-2 is an airborne virus which can be transmitted by aerosol 16 . a hospital survey detected the maximum transmission distance of sars-cov-2 aerosol might be 4 meters from the covid-19 patients 17 . in the contaminated area of the hospital, the viral nucleic acid positive rate was 75% for computer mice, 70% for floor swabs, 60% for trash cans, 43% for sickbed handrails, and 8% for doorknobs. in a virus viability test, authors used a nebulizer to generate artificial aerosols with small particle size (<5 μm) containing sars-cov-2 18 . the results showed that sars-cov-2 remained viable in the artificial aerosols for at least 3 hours. the virus is most stable on plastic and stainless steel surfaces, on which viable virus had been detected for up to 72 hours. no viable virus was detected after 4 hours on copper and after 24 hours on cardboard. the who, therefore, advises the public not to touch their eyes, nose, or mouth with their hands to limit self-contamination 19 . in addition, the centers for disease control and prevention (cdc) recommends wearing cloth face coverings in public settings where other social distancing measures (6 feet) are difficult to maintain 20 . a case study reported that 5 of 39 passengers on a coach bus contracted the virus from an infected patient who did not wear a protective face mask. however, the same patient bought a mask and wore it before transferring to a mini-bus, and none of the 14 passengers on the mini-bus contracted the virus 21 . live sars-cov-2 was isolated from nasal/pharyngeal swabs and sputum, but not from stool, in patients with covid-19 22 . the live viral copies peaked during the early stage of symptom onset (≤ 4 days); and could not be detected after day 8 in samples from 9 mild cases of infection. this discovery suggests that the viral transmission occurs primarily through the airborne route rather than the fecal-oral route during early stages of the disease. indeed, there have been sporadic case reports on the human-to-human transmission from asymptomatic or pre-symptomatic subjects [23] [24] [25] . one study estimated that the transmissibility of the asymptomatic cases is comparable to that of symptomatic cases 26 . an epidemiology report from china indicated that 1.2% (889) of 72,314 tested/suspected/diagnosed cases were asymptomatic 27 . identification and isolation of asymptomatic subjects has helped reduce the pandemic in an italian village 28 . these reports suggest that a panpopulation screening for viral exposure is an effective way to critically contain the spread of the disease. two meteorology models consistently found that higher relative humidity favored sars-cov-2 transmission 29, 30 . the two models differed regarding the trend of temperature effect on viral transmission, probably due to the different ranges of temperature studied in china (winter) and brazil (autumn). an epidemiology study from china found that although the proportion of male patients (51.4%) was comparable to that of females (48.6%), male patients (63.8%) comprised almost two-thirds of the total deaths 6 . data from the cdc website as of may 14, showed that among approximately 111,000 covid-19 cases in the u.s., 3% were in children (<17 years old); 23% were in the elderly (≥65 years old), and most (74%) were in patients between 18 and 64 years old 31 . of 580 hospitalized patients in 14 u.s. states from march 1-30, 261 (45%) were non-hispanic white; 192 (33.1%) were non-hispanic black; 47 (8.1%) were hispanic; 32 (5.5%) were asian, two (0.3%) were american indian/alaskan native, and 46 (7.9%) were of other ethnic origins or unknown 32 . culture of bronchoalveolar lavage fluid collected from early wuhan cases identified the etiology of the virus 33 to date, the virus phylogenetically closest to sars-cov-2 by genetic homogeneity is a coronavirus isolated from the horseshoe bat (bat cov ratg13) with an overall genome sequence identity of 96.2%, which is higher than that of sars-cov (<80%) 34 . angiotensinconverting enzyme 2 (ace2) was identified as a shared receptor required for cell entry both for sars-cov and sars-cov-2, with higher binding affinity for sars-cov-2 35 . sequence comparison of spike (s) protein, the viral ligand of ace2, identified three short insertions located at the n-terminus region that are conserved in sars-cov-2 and bat cov ratg13, but not in sars-cov 34 . examination of the receptor-binding domain (rbd) of s protein surprisingly identified that a malayan pangolin coronavirus had a higher degree of similarity (97.4%) than bat cov ratg13 (89.2%), indicating that recombination may have occurred during the evolution of sars-cov-2 36 . variation analysis based on 95 sequences of sars-cov-2 up to february 14 revealed very high homology (>99.9%) among different strains 37 . another group estimated that the evolution rate of sars-cov-2 is approximately 1.8 × 10 -3 per base per year 38 , which indicates that sars-cov-2 transmission in humans is a recent event. the sars-cov-2 genome sequence can be found at https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/. the direct diagnosis of covid-19 requires detection of sars-cov-2-specific rna from patients' samples. reverse transcription-polymerase chain reaction (rt-pcr) is the most widely used technique for diagnosis. a commercial rt-pcr test kit usually uses 2 to 3 pairs of primers detecting the different regions of sars-cov-2 genomic rna to increase the test specificity. the sensitivity of this method is not optimal. one paper noted that the sensitivity of rt-pcr (59%), even after 25% of patients had multiple tests, was lower than that of a ct scan (88%) 39 . a test report of 4,880 wuhan cases with typical covid-19 symptoms and history of close patient contact demonstrated that the positive rate was about 40% for nasal and pharyngeal swabs, 50% for sputum samples, and 80% to 100% for bronchoalveolar lavage fluid 40 . another study screened 353 subjects in wuhan and found that the positive rate from nasopharyngeal swabs was 2.5-fold higher than that of oropharyngeal swabs 41 . interestingly, pharyngeal swab viral nucleic acid screening results of 2,510 patients between january 23 and february 25 from a hospital fever clinic in hunan province (a neighboring province of hubei) demonstrated that the positive rate of sars-cov-2 (1.3%) was lower than that of influenza a (2.3%) and influenza b (3.3%) 42 . it is unclear whether the lockdown status of hubei province or the sensitivity of the detection methods between different viruses contributed to the result. the disease course also affects viral nucleic acid detection results. one study closely followed throat swab samples or deep nasal cavity swab samples from 56 hospitalized covid-19 patients and found that the positive rate was the highest (100%) within week 1 since the symptom onset 43 . however, the positive rate reduced to about one-third at week 3. similar results were obtained from another study, in which the positive rate of throat swabs from 43 patients was >90% when tested within 1-3 days since symptom onset, but decreased to <80% on day 5, and <50% after day 14 44 . other than the traditional rt-pcr, other viral rna detecting methods such as loop-mediated isothermal amplification (lamp) were expeditiously developed and approved by the fda 45 . the apparent advantage of lamp is the much shorter waiting time for the results (<15 minutes) compared to the traditional rt-pcr (3 hours). crispr, the powerful gene editing technique, premiered in this pandemic and was also approved by fda, though the commercial kit requires an isothermal amplification step 46 . reports on the relationship between viral load in respiratory tracts and disease severity showed conflicting results. one study (n=12) reported that the high viral load from a patient's respiratory tracts is moderately associated with a high murray score for acute lung injury and low pao 2 /fio 2 47 . the same study also reported that the high viral load is associated with high plasma angiotensin ii concentration. however, two other studies (n=23 and n=11) did not find significant differences in viral load between mild and severe cases 48, 49 . one study demonstrated that the speed of viral clearance differs significantly in mild and severe cases 10 . the average time of viral nucleic acid turning positive to negative was about 10 days in mild cases and 18 days in severe cases. in non-survivors, persistent viral rna was detected until death 11 . however, another study with intensive testing was able to detect viral nucleic acid in throat/deep nasal cavity swab samples from 3 of 56 hospitalized patients with mild-to-moderate confirmed covid-19 5 weeks after symptom onset 43 . sars-cov-2 was detected in the whole blood and serum 50, 51 . more studies are needed to investigate the correlation between viremia with blood viral load and disease severity. patients on admission, and discovered that the cutoff of 2.0 µg/ml had a sensitivity of 92% and a specificity of 83% in predicting in-hospital mortality 69 . higher levels of hypersensitive troponin i in patients with severe covid-19 (table 3) indicates an association of sars-cov-2 infection and cardiomyopathy 8, 11 . a review summarized that cardiovascular complications associated with covid-19 include myocardial injury, myocarditis, acute myocardial infarction, heart failure, dysrhythmias, and thromboembolic events 70 two covid-19-associated kawasaki disease cases were reported in a 6-month-old girl and a 5-year-old boy, respectively 84 il-4, il-6, tnf-α and ifn-γ were found between the two groups. whether the age differences between the two groups also contributed to these observed differences is unclear. two rsv-infected children (12.5%) and one sars-cov-2-infected child (2.5%) developed severe pneumonia. all children survived. various case reports, case series, retrospective and case-controlled studies of pregnant women with covid-19 presented clinical and laboratory data on maternal and neonatal manifestations and outcomes [89] [90] [91] [92] [93] . reviews and analyses of published reports provided additional insights [94] [95] [96] [97] . most of the pregnant women in these studies and reports were in their third trimester, and many of their babies were delivered by caesarean section. in general, they experienced signs and symptoms of covid-19 much like those in non-pregnant women. no maternal deaths were reported. fetal distress, premature births, premature rupture of membranes, respiratory difficulties, and low birth weight were observed among some babies born to mothers with covid-19 89, 95, 98 . no definitive cases of vertical transmission of the sars coronavirus 2 (sars-cov-2) from mother to fetus have been identified, although two highly suspect cases have been reported. wang and colleagues described a male infant delivered by emergency cesarean section to a 34-year-old woman with covid-19 confirmed by pharyngeal swab 99 . the infant had a viral nucleic acid detected from pharyngeal swab approximately 36 hours after birth. tests of the cord blood, placenta, and the mother's breast milk were negative for sars-cov-2. both mother and infant recovered. alzamora and colleagues reported on a 41-year-old diabetic woman with covid-19-induced respiratory failure whose neonate was positive for sars-cov-2 nucleic acid from nasopharyngeal swab 16 hours after a cesarean section delivery 100 . serologies for sars-cov-2 were negative for the mother and the baby at the time of delivery. the mother converted to seropositive status on postpartum day 4; whereas the neonate, who had not been breastfed, remained seronegative at that time. in relation to neonatal survival, zhu and colleagues reported one neonatal death in which a male baby born to a mother with confirmed covid-19 developed refractory shock, gastric bleeding, multi-organ failure, and disseminated intravascular coagulation approximately 8 days after birth 98 three publications reported findings with inconsistent results from systematic reviews or meta-analyses. a systematic review by vardavas and nikitara found an association between smoking and covid-19 illness progression 115 . emami and colleagues in a meta-analysis reported a high prevalence of smoking (7.6%) in hospitalized patients with covid-19 116 . however, a meta-analysis by lippi and henry found no association between smoking and covid-19, although they acknowledged the findings reported by liu above 117 . the inconsistent results on smoking may be attributed to lack of data on smoking quantity and duration, small population size and/or few smokers in certain studies, and the presence of other concurrent comorbid conditions. future research should consider including documentation of nicotine exposures through vaping and ecigarettes. the scientific community recently has debated a possible therapeutic role for nicotine in treating covid-19. some epidemiologic data has shown lower numbers of smokers among patients with covid-19, indicating that nicotine may mediate the viral transmission by lowering ace2 levels 118, 119 . a randomized clinical study is being planned in france to more formally assess if nicotine could reduce the risk of contracting the disease 120 . an analysis of 92 deceased patients with covid-19 revealed that 91 deaths were due to complications directly related to the viral infection 121 . among them, ards was most prevalent (76%), followed by myocardial injury (34%), liver injury (16%), and renal insufficiency (15%). multiple organ dysfunction syndrome occurred in 15% of cases. autopsy and biopsy of covid-19 cases have been sporadically reported. a report of complete autopsy in 12 consecutive covid-19-positive deaths (8 males and 4 females with median age of 73 years) in germany found that half of the cases had coronary heart disease and a quarter of the cases had respiratory diseases (asthma/copd) 122 . the cause of death was found within the lungs or the pulmonary vascular system in all 12 cases. deep venous thrombosis was identified in 7 of 12 patients (58%) in whom venous thromboembolism was not suspected before death. pulmonary embolism was the direct cause of death in 4 patients. the histopathology examination of lungs found diffuse alveolar damage in 8 cases. the lesions included hyaline membranes, activated pneumocytes, microvascular thromboemboli, capillary congestion, and protein-enriched interstitial edema, which were consistent with ards diagnosis. however, another immunohistology investigation of lung tissues from 2 covid-19 patients who died with respiratory failure found that the pattern of covid-19 pneumonitis was predominantly a pauci-inflammatory septal capillary injury with significant septal capillary mural and luminal fibrin deposition and permeation of the inter-alveolar septa by neutrophils without hallmarks of classic ards 123 three clinical studies recorded the baseline cytokine plasma concentrations in 21 124 , 43 125 , and 274 126 patients with covid-19 on admission or from initial tests. the median time from symptom onset to admission in these three papers was 7-8 days 124 , 6 days 125 , and 9-10 days 126 , respectively. however, the cytokine examination date from a fourth study may be even later 4 , since the median time for blood sample collection in this paper was 4 days since subjects were transferred to a designated hospital. one study reported that ifn-γ, tnf-α, il-1β, and il-8 plasma concentrations in covid-19 patients were significantly higher compared to results from four healthy in addition to the consistent trend of lower lymphocyte counts observed in severe cases of covid-19 (table 3 ) from different studies, total t cells, cd4 + t cells, and cd8 + t cells also were significantly lower in severe/critical covid-19 cases than in non-severe cases 124, 127, 128 . subgroups of cd4 + t cells did not show significant proportion changes of cd45ra + naïve t cells and cd45ro + memory t cells between severe cases and moderate cases 124 . however, the proportion of cd45ra + regulatory t cells in severe cases (0.5%) was only half the value of that in moderate cases (1.1%). one study noted a slight improvement of mean t cell counts (including cd4 + or cd8 + subpopulations) in comparison to baseline values after 5-14 days of in-hospital treatment 128 . the count improvement appears baselineproportional, regardless of disease severity. several published studies have observed features of cellular exhaustion in t cells analogous to that described for nk cells 129 . in two related studies, the authors showed that healthy individuals could be distinguished from mild and severe covidt cells; specifically, the severe group had much lower levels of non-exhausted (pd-1 -ctla-4 -tigit -) cd8 + t cells 127, 131 . they also found that cd8 + t cells in covid-19 patients exhibit many aspects of exhaustion and reduced function, such as once in the cell, the virus is not killed or neutralized; instead, it may continue to replicate, and/or stimulate or kill the target cells, causing more inflammation and damage. of note is that ade is not necessarily associated with past humoral response to a related pathogen, such as dengue virus 144 , and can occur during the primary humoral response when the neutralizing antibody is at a suboptimal level. this was documented in some severe cases following sars-cov infection 145, 146 . in a study reporting 75 patients with sars, the lung radiographic worsening of some severe cases was correlated with the time of igg seroconversion 145 . the pattern is consistent with that of covid-19, in which the disease in some severe cases suddenly worsened around one to two weeks 62, 63 , when seroconversion of anti-sars-cov-2 occurred (around 5 to 10 days after the onset of the first symptom) 48 . in addition, the recovered sars patients had higher and sustainable or steadily increasing levels of both anti-n antibody and anti-s neutralizing antibody since the seroconversion 146 . however, the titer of anti-n antibody in the sras non-survivors was low, and the titer of anti-s antibody decreased rapidly approximately 5 days after the peak, an observation much like that in some patients with severe cases of covid-19 48 . in vitro studies demonstrated that human anti-s serum enhanced sars-cov infection in human monocyte-derived macrophages (mdm) 147 . the infection mechanism is very different from that of the ace2-mediated, endosomal/lysosomaldependent pathway, and can be blocked by anti-fcγr ii antibody 148 on april 21, the national institutes of health (nih) issued general treatment guidelines for covid-19 150 , with the recommendations based on scientific evidence and expert opinion. we do not intend to repeat the previously well-documented work in our review, but prefer to focus on two important topics: antiviral and immunomodulatory pharmacotherapies. because results from clinical trials currently are being generated at such a blazing pace, this review was up to date at the time it was written. siddiqi hk et al. 159 many viral cellular adherence/endocytosis blocking reagents were proposed. the cellular infection mechanism of sars-cov-2 is believed to be the same as sars-cov, which is an ace2-mediated, endosomal-dependent pathway. chloroquine 160 and hydroxychloroquine 161 were first identified through in vitro drug screening to reduce viral titers from the supernatant of infected cell cultures. the mechanism probably is through interference with viral entry/endocytosis by increasing the ph of the endosome 162 . two series of open-label, non-randomized studies in france reported only one death in 80 patients with relatively mild disease treated with hydroxychloroquine sulfate (200 mg tid for 14 days) and azithromycin (500 mg on day 1 followed by 250 mg qd) 163, 164 . the authors justified the use of azithromycin because it had been shown to be effective against zika and ebola viruses in vitro. of note is that azithromycin also prolongs the qt interval. based on limited scientific information, it is reasonable to believe that hcq may be an effective treatment. fda issued an eua on march 28 to permit the emergency use of chloroquine phosphate and hydroxychloroquine sulfate supplied from the strategic national stockpile to treat adults and adolescents who weigh 50 kg or more and are hospitalized with covid-19 for whom a clinical trial is not available, or participation is not feasible 165 . it should be noted that the fda's typical process for eua is to review its circumstances and appropriateness periodically. the review would include regular assessments, based on additional information from the sponsor, regarding progress on the unapproved product's -or unapproved use of an approved product's-approval, licensure, or clearance. an observational study in 1376 patients from new york city did not find a significant difference in the rate of intubation or mortality between patients who received hydroxychloroquine and those did not 166 o intravenous immunoglobulin (ivig) has been used to treat sars patients [188] [189] [190] the covid-19 pandemic is still ongoing, and a long time may pass before we can fully grasp the complete picture of the pathogen's characteristics; including its vulnerabilities, which can be used to inform development of effective and efficient treatments. development of antiviral therapeutics, led by dna/rna polymerase and protease inhibitors, has been streamlined since their invention in combatting hiv. given worldwide extensive efforts, we are hopeful that anti-sars-cov-2 replication drugs will be discovered. it is unclear whether overreactive immune response and/or css play important roles in patients with severe covid-19. however, some case reports suggest the efficacy of immunomodulatory agents in treating patients with severe covid-19, which could pave the way for large-scale randomized, blinded, and controlled clinical trials. last, but not least, the antibody profiles and timelines in recovered covid-19 patients are encouraging. these should inform and guide the development of the ultimate antiviral weapon, the vaccine, for preventing covid-19 in the 21st century. symptom wang d et al. 8 deng y et al. 9 zhou f et al. 11 wu j et al. 10 8 deng y et al. 9 zhou f et al. 11 wu j et al. 10 1 p≤0.001 2 counted as endocrine system disease 3 counted as heart disease 4 counted as coronary heart disease 5 patients experienced severe acute respiratory syndrome 6 the 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doctors and scientists covid-19: european drugs agency to review safety of ibuprofen renin-angiotensin-aldosterone system inhibitors and risk of covid-19 risks of ace inhibitor and arb usage in covid-19: evaluating the evidence updated approaches against sars-cov-2 controversial treatments: an updated understanding of the coronavirus disease review of emerging pharmacotherapy for the treatment of coronavirus disease sars cov-2: recent reports on antiviral therapies based on lopinavir/ritonavir, darunavir/umifenovir, hydroxychloroquine, remdesivir, favipiravir and other drugs for the treatment of the new coronavirus covid-19illnessin native and immunosuppressed states: a clinical-therapeutic staging proposal. the journal of heart and lung transplantation remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro insights 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plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis treatment of 5 critically ill patients with covid-19 with convalescent plasma guidance for industry: investigational covid-19 convalescent plasma we would like to thank joanne berger, fda library, and karen valentine, fda center for devices and radiological health, for editing the manuscript. procalcitonin (ng/ml) 22% vs. 75% 1, 3 n/a 1% vs 25% key: cord-276630-qci7khki authors: lima, william gustavo; brito, júlio césar moreira; overhage, joerg; nizer, waleska stephanie da cruz title: the potential of drug repositioning as a short-term strategy for the control and treatment of covid-19 (sars-cov-2): a systematic review date: 2020-06-08 journal: arch virol doi: 10.1007/s00705-020-04693-5 sha: doc_id: 276630 cord_uid: qci7khki the novel human coronavirus (sars-cov-2), the causative agent of covid-19, has quickly become a threat to the public health and economy worldwide. despite the severity of some cases, there are no current pathogen-specific antivirals available to treat the disease. therefore, many studies have focused on the evaluation of the anti-sars-cov-2 activity of clinically available drugs. here, we conducted a systematic review to describe the drug repositioning strategy against sars-cov-2 and to discuss the clinical impact of this approach in the current pandemic context. the systematic review was performed on march 23, 2020, using pubmed/medline, scopus, cochrane library, and biblioteca virtual de saúde (bvs). the data were summarized in tables and critically analyzed. after the database search, 12 relevant studies were identified as eligible for the review. among the drugs reported in these studies, 57 showed some evidence of antiviral activity. antivirals, especially antiretrovirals, are the main class of therapeutic agents evaluated against covid-19. moreover, studies have reported the anti-sars-cov-2 activity of antitumor (16%; 9/57), antimalarial (7%, 4/57), and antibacterial (5%; 3/57) agents. additionally, seven pharmacological agents (chloroquine, tetrandrine, umifenovir (arbidol), carrimycin, damageprevir, lopinavir/ritonavir) are in phase iv of clinical trials. due to the evidence of the anti-sars-cov-2 activity of various clinically available agents, drug repositioning stands out as a promising strategy for a short-term response in the fight against the novel coronavirus. electronic supplementary material: the online version of this article (10.1007/s00705-020-04693-5) contains supplementary material, which is available to authorized users. in late 2019, a cluster of pneumonia cases reported in wuhan (china) was associated with a novel coronavirus, initially called the 2019 novel coronavirus (2019-ncov) [1] . posteriorly, the sequence of the 2019-ncov genome revealed high similarity to sars-cov, the causative agent of the epidemic of severe and acute respiratory syndrome (sars) between 2002 and 2003 in asia. then, the international committee on taxonomy of viruses (ictv) renamed 2019-ncov as sars-cov-2, and the world health organization (who) defined that this pathogen causes the coronavirus disease of 2019 (covid-19) [2] [3] [4] [5] . sars-cov-2 is responsible for a respiratory infection that can progress to severe pneumonia. covid-19 has an estimated mortality rate of approximately 2-3.5%, which increases with age and the presence of comorbidities (e.g., hypertension, cardiac insufficiency, diabetes, and asthma). by april 15, 2020, the novel coronavirus had affected 2,033,406 people and caused more than 130,000 deaths worldwide [6] . the public health calamity caused by covid-19 has led to the exhaustion of health systems worldwide, forcing countries to adopt extreme measures, such as the closure of their land borders and initiating social distancing policies to slow down the spread of the disease [7] . currently, laboratories and medical teams worldwide have focused on the repurposing of food and drug administration (fda)-approved drugs to treat the most severe cases of covid-19, since there are no specific chemotherapeutic agents to treat this infection [1] . indeed, drug repositioning might be a short-term alternative to fight this disease. since the efficacy, safety, and toxicity of these drugs are already well known, the initial phases of clinical trials could be skipped, which would reduce the cost and duration of the process [8] . in general, drug repurposing is a cheaper, faster, and accessible way to make drugs available to the clinic [9, 10] . in this context, several clinical and preclinical studies have searched for new pharmacological alternatives against covid-19 in clinically available drugs. however, current studies remain decentralized, and no recent review has been able to summarize the available evidence of the anti-sars-cov-2 activity of these fda-approved drugs. thus, in this systematic review, we aim to describe the drug repositioning strategy against sars-cov-2 and its clinical impact in the current context of the covid-19 pandemic. we performed a systematic review according to the cochrane handbook [11] . the search and selection of articles, as well as extraction, analysis, and interpretation of data, were conducted according to the preferred reporting items for systematic reviews and meta-analyses (prisma) statement [12] . pubmed/medline, scopus, cochrane library, and biblioteca virtual de saúde (bvs) were searched for articles investigating the antiviral activity of clinically available drugs published until march 23, 2020. we aimed to select articles describing clinical and pre-clinical tests (in vitro, in vivo, and in silico) to include the largest amount of data in this review. additionally, we searched the clinicatrial.gov website to identify ongoing trials with potential candidates for the drug repositioning strategy against sars-cov-2. indexed keywords from medical subject headings (mesh) were used to build search strategies. the terms "antiviral agents" or "drug repositioning" or "drug repurposing" were combined with the keywords "covid-19" or "sars-cov-2" or "2019-ncov" by the use of boolean and between the terms, as in the example: "drug repositioning" and "covid-19". all details about the search strategies can be found in the supplementary file. to avoid losing any possible study, the reference list of all included studies and relevant reviews regarding this topic were also screened. two authors (w.g.l. and j.c.m.b.) independently screened the databases and extracted relevant information following the prisma flowchart. the degree of agreement between the evaluators was determined by the kappa coefficient (performed with a 95% confidence interval) [13] . discrepancies about the relevance of the sources were resolved by a third researcher (w.s.c.n.). finally, all data of interest were summarized in tables (tables 1, 2, 3 and 4) for further critical analysis and interpretation. as shown in figure 1 , we identified 33 articles during the initial search (30 from pubmed/medline, two from biblioteca virtual de saúde, and one from scopus). after the exclusion of repeated records and the selection of articles by the inclusion criteria ( fig. 1) , 15 relevant studies were preselected. of these, 11 were excluded following the criteria described in figure 1 , and four were selected for extraction of variables of interest [14] [15] [16] [17] . the reference lists of included articles were analyzed, and eight new studies were identified [18] [19] [20] [21] [22] [23] [24] [25] , totaling 12 papers [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . the degree of agreement between the two authors was considered substantial (kappa = 0.825) [13] . all selected studies were published in 2020, and they describe a total of 57 drugs that showed some evidence of antiviral activity against sars-cov-2 ( fig. 2 and tables 1, 2, 3 and 4) [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . as shown in figure 2a , 22 different classes of drugs showed a potential therapeutic effect against covid-19. antivirals, especially antiretrovirals, were the most frequently studied class of therapeutic agents (30%; 17/57). however, the activity of antitumor (16%; 9/57), antimalarial (7%, 4/57), antibacterial (5%; 3/57), anticoagulant (3.5%; 2/57), anti-inflammatory (3.5%; 2/57), phosphodiesterase (pde)-inhibiting (3.5%; 2/57), anti-rheumatic (3.5%; 2/57), sedative-hypnotic (3.5%; 2/57), and anti-venous insufficiency (3.5%; 2/57) agents was also investigated. other classes of drugs have also been studied against sars-cov-2 (i.e., anthelmintic, antiallergic, antiemetic, antiepileptic, antifungal, antihypertensive, antipsychotic, anti-hemolyticuremic syndrome, anti-angioedema, lipid-lowering, immunomodulatory, and anti-pulmonary-hypertension drugs); however, only one agent of each class was evaluated (1/57, 1.7%) ( fig. 2a) . most of the drugs with potential activity against covid-19 were identified by molecular docking (53%; 30/57) ( fig. 2b ) [20] [21] [22] [23] [24] using the main protease (3clpro) of sars-cov-2 as the molecular target (table 3) . moreover, 39% (22/57) (fig. 2b ) of the drugs showed an anti-covid-19 effect in clinical trials (table 4) , and 10% of them showed evidence of action in clinical practice (10%; 6/57) [14] [15] [16] [17] (table 1) . additionally, 19% (11/57) [18, 19] ( fig. 2b ) of the drugs studied showed antiviral activity in vitro. for instance, remdesivir showed the highest activity against sars-cov-2, inhibiting 50% of the virus at a concentration of 0.77 μm [18] , while ribavirin showed a less pronounced effect (109.50 μm) [18] (table 2) . therefore, the antiviral activity of only 27 compounds (47%; 27/57) has been experimentally evaluated. the remaining 30 reported drugs were identified only by theoretical methods (in silico) and need proof of antiviral efficacy in further studies (table 3) . regarding the clinical trials of new therapeutic options against covid-19, most drugs are in phase ii (36%; 8/22) or iii (27%; 6/22) (table 4 ). only seven drugs (chloroquine, tetrandrine, umifenovir (arbidol), carrimycin, table 1 clinical evidence of potential candidates for drug repositioning against covid-19 (sars-cov-2) *lopinavir (400 mg) + ritonavir (100 mg), q12h, orally; associated with umifenovir (200 mg), q12h, orally. the duration of antiviral treatment was 6-15 days **solution containing umifenovir (20.0%), lopinavir + ritonavir (17.4%) and interferon (19.4%) administered through inhalation ***in this study, 99 patients were assigned to receive lopinavir/ritonavir (400 mg and 100 mg, orally), and 100 patients were assigned to the standard of care (oxygen supplementation, noninvasive and invasive ventilation, antibiotic agents, vasopressor support, renal-replacement therapy, and extracorporeal membrane oxygenation) # in these studies, the therapeutic scheme used was not clearly defined treatment with lopinavir/ritonavir did not improve the clinical condition of patients compared to the standard of care*** (table 4 ). interestingly, two of these agents in advanced clinical studies, umifenovir (arbidol) and the association lopinavir/ritonavir, have demonstrated previous evidence of action. wang et al. [14] reported that the use of arbidol in combination with lopinavir/ritonavir inhibits the aggravation of pneumonia caused by sars-cov-2 and promotes a virus-negative conversion in patients from china. arbidol has also shown a potent in vitro effect against sars-cov-2, inhibiting the virus up to 60 times compared to the untreated control at concentrations ranging from 10 to 30 μm [26] . the novel coronavirus (sars-cov-2), the causative agent of covid-19, has quickly become a threat to the public health and economy worldwide [7, 27] . recent clinical reports have shown that sars-cov-2 causes mild, selflimiting respiratory tract illness as well as severe progressive pneumonia, which can progress to multiorgan failure and death [1] . despite the severity of some cases, there are no current pathogen-specific antivirals available to treat this disease [1] . therefore, many studies have focused on the evaluation of the anti-sars-cov-2 activity of clinically available drugs [1] . after the analysis of the selected studies, we identified 57 molecules with potential antiviral activity against sars-cov-2. of these, only six drugs (lopinavir/ritonavir, umifenovir (arbidol), remdesivir, chloroquine, and hydroxychloroquine) have shown promising results in preclinical trials and have clinically lessened the symptoms of covid-19. since the other fda-approved drugs reported in this review showed weak activity against covid-19, we have chosen to discuss only the most promising molecules. lopinavir and ritonavir are antiretrovirals widely used as a combination drug to treat human immunodeficiency virus (hiv) infections. lopinavir is an hiv type 1 aspartate protease inhibitor, and ritonavir increases its plasma half-life through the inhibition of cytochrome p450 [28] . previous studies showed that lopinavir inhibits the 3-chymotrypsinlike protease, which is involved in viral replication and is highly conserved among members of different viral species [29] . lopinavir inhibited the replication of mers-cov and sars-cov in huh7 cells (human liver strain) in a dosedependent manner, with an ec 50 of 8.0 μm for both viruses [29] . moreover, lopinavir/ritonavir significantly reduced the mortality rate of mice infected with mers-cov, suggesting a strong in vivo antiviral effect [30] . little is known about the activity of lopinavir/ritonavir against sars-cov-2 and its effectiveness (table 1) . for instance, wang et al. [14] showed that the use of lopinavir/ ritonavir considerably improved the clinical condition of patients with covid-19. however, arbidol, a broad-spectrum antiviral with activity against several enveloped and non-enveloped viruses (e.g., influenza virus, adenovirus, avian coronavirus, and hepatitis b and c viruses) [31] , was also administered to the patients (n = 4). the combination of lopinavir/ritonavir with arbidol makes it difficult to associate the observed antiviral effect to lopinavir/ritonavir or to its synergistic effect with arbidol. in contrast, mo et al. [16] showed that the clinical improvement of patients with covid-19 was more associated with supportive measures, such as mechanical ventilation and oxygen supplementation, than with the use of lopinavir/ritonavir. likewise, a study of 199 hospitalized adult patients with confirmed sars-cov-2 infection showed that the use of lopinavir/ritonavir does not increase the effectiveness of the standard treatment [15] . these findings confirm that lopinavir/ritonavir has low clinical efficacy and is not a therapeutic option to treat covid-19. however, the efficacy related to the use of these antiretrovirals as a combination therapy with other drugs should be elucidated in future studies. arbidol (umifenovir) is an anti-influenza agent that has been used in china and russia for many years. it interacts with the viral hemagglutinin (ha) and inhibits the fusion of the viral particle with the plasma membrane [32] . this drug inhibited crucial stages of the sars-cov-2 replication cycle in vitro in a concentration ranging from 10 to 30 μm [26] . also, a study has indicated that arbidol significantly lessened pneumonia associated with covid-19 [14] . these results have stimulated the initiation of clinical trials with this medicine (table 4) . for instance, a chinese group of the ruijin hospital is currently conducting a phase iv study to evaluate the efficacy and safety of arbidol hydrochloride tablets in treating pneumonia in 380 patients with covid-19 (clinicaltrial.gov, nct04260594). previous in vitro studies have indicated that arbidol shows significant activity against other coronaviruses. herein, a research group showed that this compound negatively affects the early stages of viral replication of sars-cov at a concentration of 50 μg/ml. this study also highlighted the stronger antiviral activity of arbidol mesylate compared to arbidol as an alkaline preparation [33] . these preliminary results indicate that arbidol is a promising candidate for drug repositioning against sars-cov-2. however, further studies are required to assess the difference in the effectiveness of arbidol and arbidol mesylate in patients with covid-19. remdesivir is a broad-spectrum prodrug developed to treat infections caused by ebola virus and marburg virus. its active ingredient (gs-441524) decreases the production of rna by interfering with viral rna polymerase and evading proofreading by viral exonuclease [34] . recent studies have shown that remdesivir inhibits the replication of sars-cov and mers-cov in human lung cells [35] . wang et al. [18] showed that remdesivir has potent activity against sars-cov-2 in kidney cells, with an ec 50 of 0.77 μm. additionally, this drug induced the clinical remission of the symptoms of covid-19 in the first reported case in the usa (table 1 ) [17] . due to these promising results, a phase iii clinical trial has evaluated the antiviral activity of remdesivir and its safety in patients with severe covid-19. this study is assessing the ability of remdesivir to normalize body temperature and oxygen saturation in 400 hospitalized adult patients (clinicaltrial.gov, nct04292899). therefore, the results of this clinical trial can guide the prescription of remdesivir as an anti-sars-cov-2 agent in the future. chloroquine and hydroxychloroquine are antimalarial agents widely used to treat rheumatic diseases and have also shown promising activity against covid-19 [36] . the first evidence of their antiviral effect against coronavirus was reported in 2004 when keyaerts and collaborators showed that chloroquine inhibited the in vitro replication of sars-cov on vero cells e6. in this study, chloroquine negatively affected sars-cov-2 at a concentration of 8 μm [37] . interestingly, chloroquine inhibited the virus when the cells were treated with the drug before or after exposure to sars-cov, suggesting prophylactic and therapeutic effects [38] . this drug also affects the entry and replication of sars-cov-2, with an ec 50 of 1.13 μm [18] . moreover, hydroxychloroquine, a less toxic derivative of chloroquine, is also able to inhibit the entry and replication of sars-cov-2 with an ec 50 of 4.51 μm ( table 2 ) [19] . additionally, the preclinical results have suggested that this antimalarial blocks the transport of sars-cov-2 from endosomes to endolysosomes, which is a process required to release the viral genome [18, 19] . the promising in vitro results of chloroquine and hydroxychloroquine have motivated the initiation of clinical studies of these substances ( table 4) . one of these studies evaluates the prophylactic effect of the oral use of chloroquine in 10,000 patients against sars-cov-2 (clinicaltrial. gov, nct04303507). additionally, several clinical trials have evaluated the prophylactic use of hydroxychloroquine (clinicaltrial.gov, nct04318015, and nct04318444), its therapeutic use in monotherapy (clinicaltrial.gov, nct04315896), or the effect of its combination with other antivirals (clinicaltrial.gov, nct04303299, nct04321278 and nct0430127, and nct0430127 and nct0430127) against covid-19. the rapid popularization of these preliminary results has led to a massive and irrational demand for chloroquine and hydroxychloroquine. in brazil, for example, after the first reports of the clinical effectiveness of hydroxychloroquine against covid-19, this drug quickly sold out in pharmacies, which has compromised patients who use this drug continually to treat autoimmune diseases. this irrational demand caused the agência nacional de vigilância sanitária (anvisa) to include these two drugs in the list of controlled substances to prevent their use to treat covid-19 without prescription or proof of effectiveness. this situation highlights the importance of controlling the release of preliminary results, especially in the panic scenario created by the pandemic. although several studies have identified clinically available agents that are active against sars-cov-2 infections, supportive therapy remains essential. for instance, mechanical ventilation and oxygen supplementation have been critical to the survival of patients with severe covid-19. mo et al. [16] showed that that oxygen supplementation and noninvasive or invasive ventilation have generated similar results to the use of an antiretroviral with known in vitro activity against sars-cov-2. additionally, wang et al. [14] showed that the clinical recovery of patients with covid-19 is more associated with supportive therapies than with the use of antiviral agents. however, the identification of available drugs with anti-sars-cov-2 activity and their use in association with supportive therapies should be considered. also, the development of faster diagnostic tools to test for covid-19 might be crucial, since some of the candidates for drug repositioning must be administered early in the course of infection. thus, better testing methodologies could lead to the early administration of drugs and improve the treatment of covid-19. the rapid spread of sars-cov-2 worldwide has put pressure on research centers to develop effective therapies and vaccines for the treatment of sars-cov-2. drug repositioning is a promising short-term strategy in the fight against the novel coronavirus. however, supportive measures are essential mainly for severe covid-19 patients, and the implementation of drug repositioning should be done only if the efficacy of the drugs have been proven. although lopinavir/ ritonavir had low anti-sars-cov-2 activity, arbidol, remdesivir, and chloroquine/hydroxychloroquine showed promising effects against this coronavirus. therefore, the outcomes of the ongoing clinical trials are urgently needed to evaluate the best treatment options for covid-19. furthermore, additional studies of the antiviral activity of molecules that have shown a promising in silico effect may increase the therapeutic arsenal against the novel coronavirus. a novel coronavirus from patients with pneumonia in china sars and mers: recent insights into emerging coronaviruses host factors in coronavirus replication epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study coronavirus infections-more than just the common cold coronavirus cases the global macroeconomic impacts of covid-19: seven scenarios new uses for old drugs drug 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coronavirus infection and spread publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank ufmg/pharmacy school-ppgcf for the key: cord-267134-5gz2dotn authors: sallenave, jean-michel; guillot, loïc title: innate immune signaling and proteolytic pathways in the resolution or exacerbation of sars-cov-2 in covid-19: key therapeutic targets? date: 2020-05-28 journal: front immunol doi: 10.3389/fimmu.2020.01229 sha: doc_id: 267134 cord_uid: 5gz2dotn covid-19 is caused by the severe acute respiratory syndrome (sars) coronavirus (cov)-2, an enveloped virus with a positive-polarity, single-stranded rna genome. the initial outbreak of the pandemic began in december 2019, and it is affecting the human health of the global community. in common with previous pandemics (influenza h1n1 and sars-cov) and the epidemics of middle east respiratory syndrome (mers)-cov, covs target bronchial and alveolar epithelial cells. virus protein ligands (e.g., haemagglutinin or trimeric spike glycoprotein for influenza and cov, respectively) interact with cellular receptors, such as (depending on the virus) either sialic acids, dipeptidyl peptidase 4 (dpp4), or angiotensin-converting enzyme 2 (ace2). host proteases, e.g., cathepsins, furin, or members of the type ii transmembrane serine proteases (ttsp) family, such as transmembrane protease serine 2 (tmprss2), are involved in virus entry by proteolytically activating virus ligands. also involved are toll like receptor (tlr) family members, which upregulate anti-viral and pro-inflammatory mediators [interleukin (il)-6 and il-8 and type i and type iii interferons among others], through the activation of nuclear factor (nf)-kb. when these events (virus cellular entry and innate immune responses) are uncontrolled, a deleterious systemic response is sometimes encountered in infected patients, leading to the well-described “cytokine storm” and an ensuing multiple organ failure promoted by a downregulation of dendritic cell, macrophage, and t-cell function. we aim to describe how the lung and systemic host innate immune responses affect survival either positively, through downregulating initial viral load, or negatively, by triggering uncontrolled inflammation. an emphasis will be put on host cellular signaling pathways and proteases involved with a view on tackling these therapeutically. covid-19 is a respiratory disease whose aetiologic agent is a novel beta coronavirus (cov) called severe acute respiratory syndrome (sars)-cov-2/2019-ncov. the initial outbreak of the pandemic began in december 2019, and it is currently affecting the health and safety of the global community. indeed, on may 12, 2020, 4.5 million worldwide cases were confirmed (probably a significant under-estimation given the number of untested asymptomatic subjects), with a death toll exceeding 286,000. before the sars-cov-2 outbreak, two related highly pathogenic covs viruses, middle east respiratory syndrome (mers)-cov (1) and sars-cov (2) , provoked catastrophic epidemics and pandemics, respectively. unfortunately, no drugs nor vaccines have currently been approved to prevent or treat these viral episodes. the first anatomical/histological reports from the lungs of severely sars-cov-2-affected patients experiencing acute respiratory disease syndrome (ards) revealed excessive inflammatory activation and destruction of the bronchial and alveolar epithelium, features already observed during the first sars pandemics in 2003 (3, 4). indeed, in the latter pandemic, lung alveolar epithelial cells were identified as the most likely site of virus replication, and it was suggested that alveolar macrophages may be responsible for the dissemination of viruses within the lungs (3). in accordance, initial histological analyses of lung biopsies from patients positive for sars-cov-2 have shown exfoliation of the bronchial epithelium, which may induce altered mucociliary clearance and affect host immune responses (5). indeed, there is no doubt that the latter are involved in modulating disease onset and progression. for example, early studies report that, similarly with what was observed with sars-cov, lymphopenia [sometimes equivalent or more severe than that observed in human immunodeficiency virus (hiv) infection] is often observed in severely affected patients progressing to ards. despite, or maybe correlated with this, aberrant non-effective innate immune host responses seem associated with severe lung disease during sars (6) (7) (8) (9) (10) (11) (12) . the following sections will give an overview of the molecular and cellular mechanisms underpinning sars-cov virus infections and how lung and systemic host innate immune responses affect survival either positively, through downregulating the initial viral load, or negatively, by triggering uncontrolled inflammation. a particular emphasis will be put on the description of the host cellular signaling pathways and proteases involved with a view on tackling these therapeutically. covs are enveloped viruses with a positive-polarity, singlestranded rna genome encoding four structural proteins: the transmembrane trimeric spike glycoprotein (s, composed of two subunits s1 and s2), envelop (e), matrix (m), and nucleocapsid (n) (13) . the entry of cov viruses into host epithelial cells is mediated by the interaction between the viral envelope s protein homotrimers and the cell surface receptors. following proteolytic cleavage of the cov s protein ("priming"), the s1 ecto-domain recognizes a membrane receptor [angiotensinconverting enzyme 2 (ace2) for sars-cov and sars-cov-2 as well as dipeptidyl peptidase 4 (dpp4) for mers-cov], whereas the s2 c-terminal domain is involved in cell fusion and viral entry (14) (15) (16) . this mechanism of action is very similar to that used by influenza, except that the latter use sialic acids as the cognate receptor for its hemagglutinin (ha) ligand. importantly, many viruses (influenza, mers, cov, and paramyxoviruses such as hendra and nipah viruses) use similar host proteolytic enzymes for cleaving their ligands (ha and s), namely, mostly lysosomal (cathepsins b, l), furin, or trypsin-like proteases (17, 18) . indeed, it is believed that it is the cellular source of these proteases that may determine the infectivity spectrum of these viruses, with the lung and the gastro-intestinal tract being high producers (19, 20) . although a variety of these proteases have been studied and shown to be involved to varying degrees in virus activation, including neutrophil elastase (21) , proteases of the type ii transmembrane serine proteases (ttsp) family [hat, transmembrane protease, serine (tmprss)2, and tmprss4] have recently been demonstrated to be particularly important, albeit probably at different stages of the virus cell cycle (19, 20, 22, 23) . in particular, recent research on sars-cov-2 has focused on tmprss2 and has shown it to be important (although mostly using cell lines infected with pseudotyped virus particles bearing sars-cov-2 s protein) for virus entry (24, 25) . in that context, it has also been demonstrated that the serine protease inhibitor camostat (see also below section on therapeutic targets and conclusion) was protective (24, 25) . in contrast, dpp4 which is necessary for the entry of mers-cov (26, 27) is not involved in sars-cov-2 entry (24) . unlike other sars-covs, the s protein of sars-cov-2 has a furin cleavage site at the boundary between the s1 and s2 subunits, which is processed during biogenesis and which may explain cov-2 high infectivity (28) . although mechanistic studies are obviously still in their infancy, it is very likely that sars-cov and sars-cov-2 target mainly respiratory epithelial cells with similar mechanisms. indeed, as indicated above, initial work has shown that ace2 is the s receptor for both sars-cov (29) and sars-cov-2 viruses (24, 28, 30) , and structural studies using cryo-electron microscopy suggest a binding of two s protein trimer to an ace2 dimer (28, 30) . whether this is strictly dependent on ace2/protease expression is debatable since ace2 is present in other tissues in humans [such as the intestine, kidney, and testis (31) ]. indeed, "seasonal" low pathogenic covs (e.g., cov-229e, cov-oc43) infect mostly upper airways, whereas pathogenic covs (sars-cov/sars-cov-2 and mers) have a tropism for the distal lung and can cause severe pneumonia and ards (32) , as currently demonstrated again in the present pandemic. indeed, potentially explaining this is the fact that seasonal coronaviruses do not use ace2 as a receptor. in vitro, primary nasal and tracheobronchial epithelial cells as well as the calu-3 bronchial cell line were shown to express ace2 (the latter not colocalizing with cilia), and their infection with sars-cov was shown to be highly cytotoxic (33, 34) . in the distal lung, as hinted above, primary alveolar type ii epithelial (atii) cells are also permissive to sars-cov infection (35, 36) . sars-cov-2 has also been shown to infect various respiratory epithelial cell lines including a549 (alveolar origin), beas2-b (bronchial origin), calu-3 cells, as well as primary human bronchial epithelial cells (24) . besides the lung, ace2 is also highly expressed in the intestine (37) , and gastrointestinal symptoms have been recorded with covid-19 (38) . it was shown that sars-cov2 is able to infect enterocytes as well as intestinal organoids and induces a viral response characterized by the expression of mediators related to type i and iii ifn (39) . even if sars-cov2 is thought to originate from bats, the intermediate host between bats and humans is still unknown. sars-cov was previously shown to infect various wild and domestic animals, including cats, ferrets and pigs (40) (41) (42) . similarly, recent work reveals that domestic animals, including ferrets and cats, are permissive to sars-cov-2 infection. in contrast, the virus replicates poorly in pigs, ducks, chickens, and dogs (43) . given the described importance of host proteases in mediating infectivity of a number of viruses, it is no surprise that, upon virus infection, murine knock-out (ko) for some of these molecules has shown some protection. for example, tmprss2-ko mice were protected from pulmonary disease and death following h1n1 and h7n9 influenza infection, but not from that of the influenza h3n2 subtype, demonstrating some specificity and showing also that other ttsp proteases [such as desc1 (tmprss11e) and mspl (tmprss13)] or other factors may be important (44) (45) (46) (47) . similarly, tmprss2 ko mice showed reduced body weight and viral loads compared to wt mice in animals infected with sars-cov (48) . also, it was demonstrated that over-expression of the human dpp4 in mice promoted mers-cov infection, causing lethal disease (49) , and that tmprss2 was instrumental in that context (48) . the control of viral infection requires an optimal and innate coordinated host antiviral immunity. this response is activated by various sensors, including pattern recognition receptors (prr), which recognize pathogen-associated molecular patterns (pamps). although for many viruses, viral rna is a pamp classically detected by different sensors, including toll-like receptors (tlr)3 (which senses double stranded (ds)rna), tlr7 and tlr8 [which sense single stranded (ss)rna], rig-i (which senses short dsrna and ssrna specific motifs), and mda-5 (which senses long dsrna) (50), the sensors potentially recognizing sars-cov genomic material are still elusive. in addition, although, as mentioned above, distal peripheral lung alveolar epithelial cells seem to harbor sars-cov infection in vivo, and although respiratory epithelial cells are known to express tlr3, tlr7, and tlr8 (51, 52) and initiate innate immunity in the lung (53), the study of these cells in anti-cov responses has been hampered by their general poor permissibility to the virus in vitro (except for intestinal caco-2 and hek293 kidney epithelial cells) (54) . in that respect, although the specific prr involved was not identified, the m protein of sars-cov was indeed shown to induce interferon (ifn)-β in a tlrrelated-traf3-independent mechanism in hek293 cells (55) . regarding the lung, the differentiated calu-3 cell line [when cultured at the air-liquid interface (ali)] is the model of choice: in that set-up, sars-cov infection triggered an inflammatory response characterized by increased production of interleukin (il)-6, il-8, gamma interferon (ifn-γ), inducible protein 10 (ip-10), and activation of the transcription factor nf-κb (56) . however, the kinetics of this response was extremely slow, and importantly, type i ifn, an important mediator of anti-viral responses, was undetected. also, another study involving a549 cells demonstrated that the trimeric spike s glyprotein and virus-like particles were able to modestly upregulate ccl2, an important monocytic chemokine (57) . in addition to lung epithelial cells cultured at ali, precisioncut lung slices could also be an interesting tool to study sars-cov2-cells interactions (58) , as demonstrated in influenza infections with human (59) or animal-derived material (60). as mentioned above, ttsps can activate virus-ligands (ha and s protein), but they are also able to modulate cell signaling pathways. for example, recombinant hat is able to activate mucin gene expression in nci-h292 lung epithelial cells (61) . relatedly, we have shown both in vitro in epithelial cells and in a murine model that influenza h3n2 is able to upregulate mucin expression and that this is dependent on human (or mouse) hat upregulation and tace activity (62) . interestingly, haga et al. have shown that inhibiting tace prevents sars-cov cellular entry (63) . strengthening the signaling potential of the receptors, iwata-yoshikawa et al. demonstrated in vivo that poly ic (tlr3 ligand) induces the expression of a variety of pro-inflammatory mediators (ccl2, kc, and il-1) through the expression of tmprss2 (48) . in addition, although unclear as whether it is beneficial or detrimental to the host cell, sars-cov have been shown to activate host stress response, apoptosis, and autophagy (13) . these are also various pathways that may also need to be evaluated therapeutically in the context of the current pandemic. relatedly, we have shown that chloroquine, which also inhibits the autophagic cellular flux by decreasing autophagosomelysosome fusion, can inhibit influenza-mediated ccl5 production (64) . importantly, after having established a foothold in the epithelial compartment, sars-cov can disrupt the epithelial polarity, thereby getting access to the parenchyma tissue: for example, it has been shown that the virus membrane protein e binds to pals1 (protein associated with lin seven 1), a junction protein involved in epithelial polarity, and modifies its cellular distribution at the surface of hek-293 cells (65) . myeloid cells, e.g., alveolar and interstitial macrophages or dendritic cells (dcs), elicit different immune responses toward influenza viruses, according to their subtypes (66) . it is thus predictable that specificities may also exist with respect to sars-cov-2 infections. indeed, although studies are scant, these cells have generally been shown to be poorly permissive to sars-cov replication (54, 67, 68) . however, a few studies have shown that myeloid cells can respond to sars-cov infection. indeed, dosch et al. showed that the s protein could, through tlr2, trigger nf-κb activation and inflammatory responses in peripheral blood mononuclear cells (pbmc) (69) . also, in common with epithelial cells, it was shown that pbmcs and dcs infected with sars-cov produced cytokines and chemokines such as and c-c motif chemokine ligand (ccl)-2 and/or c-x-c motif chemokine ligand (cxcl)-10/rantes/tumor necrosis factor (tnf)/il-8/il-6, but, importantly, not ifn-β (67, 68) . by contrast, a study performed mostly on thp-1 macrophages suggest that mers s protein suppresses macrophages pro-inflammatory responses through dpp4-induction of irak-m and pparγ (70) . furthermore, in an interesting "2-way" system involving differentiated sars-permissive lung calu-3 cells and monocytederived macs and dcs, it was shown that mediators produced by calu-3 cells activate cytokine production by macrophages (il-1β, g-csf, mip-1, and tnf-α) and dcs (il-12p40, mip-1, ifn-γ, il-6, il-8, and mcp-1) but that some of these calu-3 derived mediators (in particular il-6 and il-8) compromised the ability of dcs and macs to activate naïve t cells and phagocytosis (4, 56). this echoes data obtained from patients suggesting that sars may in fact be partly caused by a "paralysis" of the adaptive immune system, characterized by a diminished number of immune cell types including t lymphocytes, dcs and macs (4). demonstrating that sars-cov can induce tlr-dependent host responses in vivo, tlr4, tlr3, and tram ko mice were shown to be more susceptible to mouse-adapted sars-cov, albeit without exhibiting extra mortality (71) . in comparison, mice deficient for the signaling molecule trif were highly susceptible to cov infections, exhibited diminished lung function, aberrant inflammatory responses, and importantly, higher mortality (71) . in addition, a mouse genetic study revealed that the tlr adaptor protein ticam2 was a susceptibility gene to sars-cov (72); mice ko for ticam2 (72), but also myd88 (73), another tlr adaptor protein, were highly susceptible to a mouse-adapted sars-cov lung infection. since polymorphisms of tlrs and myd88 have been associated in humans with heightened sensitivity to a variety of pathogens (74) , these studies, in addition to demonstrating the role of tlr pathways in the sars-cov infection, suggested a human genetic predisposition to sars-cov, and this could explain the variability of severity in patients with covid-19 disease. forthcoming human genetic studies from international collaborative efforts (https://www. covid19hg.org) could reveal genetic variants associated with sars-cov2 susceptibility, as in the gene encoding ace2 as recently suggested (75) . indeed, ace2 genetic variants may be associated with a modulated ace2 protein expression, the sars-cov-2 receptor, which may explain in part patients' susceptibility to infection. genes associated with tlr pathways also represent good candidates, as demonstrated in other respiratory viral infection (e.g., influenza) where tlr3 variants (76) were shown to modulate its virulence. maladaptive activation of innate immune responses (see figure 1b) as already mentioned above, aberrant maladaptive innate immune host responses, including "cytokine storm" events, have been associated with severe lung disease and the development of ards during sars and the covid-19 current episode. mechanistically, these events usually occur at a late stage of the disease, and several mechanisms have been proposed. in particular, a murine study has shown that a prolonged (albeit delayed, as demonstrated also in vitro, see above) type i ifn signaling was instrumental in triggering over-exuberant innate inflammatory monocytes-macrophages immune responses and an impaired virus-specific t-cell response (77) . in complement to the mechanism proposed above, increased lung inflammatory protease (neutrophil elastase and metalloprotease) activity has been demonstrated in ards (78, 79) , with a concomitant imbalance between protease and protease inhibitors activity (80) . in addition, although not yet measured, to our knowledge, in sars murine models, we and others have shown increased protease-mediated lung damage in mice infected with influenza (81-83). additionally, in a mers-cov murine model, it was shown that excessive complement activation was partly responsible for exacerbated lung inflammation (84) . lastly, "cytokines storm" may also results from socs (suppressors of cytokine signaling) inhibition (85) . indeed, upon influenza infection, socs1 and socs3 were shown to reduce type i ifn antiviral responses in human bronchial epithelial cells (86) . also, socs4-deficient mice exhibited heightened sensitivity to influenza infection (87) . studies about socs involvement during coronavirus infections are currently lacking and should therefore bring new interesting information. on may 12, 2020, using the term "covid, " an unbiased search of already registered trials on https://clinicaltrials.gov/ retrieved 1,409 hits, and, when refined with "double blind/placebo, " 119 hits were found. although the number of trials that are ongoing or "under recruitment" is expectedly very high, the range of molecules tested is relatively narrow and aimed at targeting mainly antivirals. these include remdesivir (21 hits), lopinavir/ritonavir (also used in aids), as well as interferons (46 hits) . also falling in that category are trials testing molecules aiming to block viral entry at the cellular surface by targeting ace-inhibitors (32 hits) or the membrane proteases of the ttsp family (see above) using camostat mesilate (5 hits). repurposing of non-antiviral drugs may offer new promising options, such as with ivermectin-an fda-approved anti-parasitic drug widely available and recently shown to inhibit sars-cov-2 in vitro (88) . because the virus load is not necessarily correlated with symptoms deterioration in sars (the latter being often caused by worsening of inflammation at day 7-10 post onset of clinical signs), it follows that anti-inflammatory drugs could/should be prescribed during that stage of the disease (8) . in that context, "classical" anti-inflammatory drugs are indeed currently being tested against covid-19 [e.g., methylprednisolone, budesonide, hydrocortisone, azithromycin, and non-steroïdal anti-inflammatory drugs (nsaids)]. in addition, more specific agents are also being investigated, targeting either il-β (anakinra, 13 hits), il-6 signaling (siltuximab/3 hits, tocilizumab/42 hits, sarilumab/13 hits), or cd24 (cd24fc) with the main objective to modulate the "cytokine storm." however, chloroquine/hydroxychloroquine has, so far, undoubtedly taken the lion's share (178 hits), and it has attracted a lot of media attention. in that respect, the results from an initial pan-european endeavor ("discovery"), now conducted largely in france because of enrollment difficulties, are eagerly awaited. this drug has a "mixed" mode of action. indeed, it acts as an anti-viral (presumably through inhibition of lysosomal enzymes requiring an acidic ph and of activation of endolysosomes, see above section "mechanisms of entry") and as an anti-inflammatory molecule, and it has notably been used in inflammatory rheumatic diseases (89) . despite a relative safe profile, having been administered to millions of people over the years, worries have nevertheless arisen about cardiac issues in many individuals with severe covid-19, and this will have to be properly assessed (90) . regardless, the ultimate prize in the fight against covid-19 (or further sars-cov infections) undoubtedly lies with the future generation of effective vaccines and the development of neutralizing antibodies (91, 92) . unfortunately, coronavirus vaccines in general have attracted less attention compared to the effort dedicated to vaccines against other potential pandemic viruses such as influenza. for example, from 2012 onwards, few sars-cov vaccines reached phase 1 clinical trials for lack of interest from the pharmaceutical industry when it became evident that the virus was not making a "comeback" after its initial appearance. however, although probably too late for affecting the current "first wave" of sars-cov-2 pandemic, many pharmaceutical companies and research laboratories are now working on a plethora of vaccine formulations [for a review, see (91) and https://clinicaltrials.gov, the latter reporting so far 83 clinical trials on vaccines]. indeed, in pre-clinical studies, the determination of cryo-em structures of the sars-cov-2 s ectodomain trimer is providing a blueprint for the design of vaccines and inhibitors of viral entry (28) . in this context, promising results show that murine polyclonal antibodies against s protein of sars-cov are able to elicit polyclonal antibody responses, preventing sars-cov-2 entry into cells, and thus indicating that cross-neutralizing antibodies targeting conserved s epitopes can be elicited upon vaccination (28) . in addition to testing the best sars-cov-2 specific epitopes from the most suitable proteins (s, n, etc.) and way of administration (best vectors, etc.), it is important to select the best animal models. although convincing murine studies are still pending, as indicated above in the section "mechanisms of entry. . . ", studies in other animals investigated the virus susceptibility of chickens, ducks, dogs, pigs, cats, and ferrets, with the latter two being the most permissive (43) . further up in the phylogenetic scale, a recent study reported that an inactivated vaccine candidate for sars-cov-2 was protective in macaques (93) . finally, large epidemiological studies have demonstrated that bacille calmette-guerin (bcg) can heterologously protect against virus infections [e.g., yellow fever virus (94) , probably by tapping on trained immunity mechanisms (95, 96) ]. using such adjuvant-mediated strategies against sars-cov viruses may therefore be an exciting avenue worthwhile pursuing (97) (98) (99) . all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. lg and j-ms received grants from the faculté de médecine sorbonne université (aap covid19 and université de paris (fonds d'urgence sars-cov-2 et covid19) , respectively. this 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neutralizing antibodies against sars-cov-2 and other human coronaviruses rapid development of an inactivated vaccine candidate for sars-cov-2 bcg vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity trained immunity and local innate immune memory in the lung induction of autonomous memory alveolar macrophages requires t cell help and is critical to trained immunity effects of toll-like receptor stimulation on eosinophilic infiltration in lungs of balb/c mice immunized with uv-inactivated severe acute respiratory syndrome-related coronavirus vaccine could bcg vaccination induce protective trained immunity for sars-cov-2? front immunol innate immune memory of tissue-resident macrophages and trained innate immunity: re-vamping vaccine concept and strategies host signaling and proteolytic pathways in the resolution or the exacerbation of coronavirus (cov-2) infection in covid-19 disease: what therapeutic targets? the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 sallenave and guillot. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-258312-3v5t4k8d authors: majachani, nicole; francois, jean luc m.; fernando, ashen k.; zuberi, jamshed title: a case of a newborn baby girl infected with sars-cov-2 due to transplacental viral transmission date: 2020-10-25 journal: am j case rep doi: 10.12659/ajcr.925766 sha: doc_id: 258312 cord_uid: 3v5t4k8d patient: female, 31-year-old final diagnosis: covid-19 • sars-cov-2 symptoms: asymptomatic medication:— clinical procedure: — specialty: pediatrics and neonatology objective: unusual clinical course background: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a highly infectious virus and is responsible for the current pandemic. it mainly infects cells of the lower respiratory tract and has been linked to severe respiratory complications. although multiple routes of transmission have been reported in the literature, there is no definitive evidence for transplacental transmission. we present a case of neonatal sars-cov-2 likely due to transplacental transmission. case report: 31-year-old hispanic woman in the final week of pregnancy developed mild respiratory symptoms of covid-19 pneumonia and tested positive for sars-cov-2 infection. she had a history of human immunodeficiency virus (hiv) infection and gestational diabetes. two days later, she gave birth to a baby girl who tested positive for sars-cov-2 on the first day after birth. she was delivered via elective cesarean section adhering to a strict infection control protocol. conclusions: this report presents a case of a 31-year-old mother with mild symptoms of covid-19 pneumonia who was positive for sars-cov-2 infection and who gave birth to a baby girl who was also positive for sars-cov-2. this case supports the possibility of transplacental transmission of sars-cov-2. systemic acute respiratory syndrome coronavirus 2 (sars-cov-2) is a highly infectious virus from the coronaviridae family and is responsible for the current pandemic which began late december 2019 [1] . the virus predominantly affects the respiratory system and can lead to severe respiratory complications such as respiratory failure [2] . at the moment, possible routes of transmission mentioned in the literature include droplet, fomite, and aerosol transmission [3] . recent studies have investigated the possibility of vertical transmission of sars-cov-2 from an infected mother to her fetus; however, there is limited evidence in support of transplacental transmission. in response to the potential risks to both the mother and fetus, the american college of obstetricians and gynecologists, the american academy of pediatrics, and the centers for disease control have developed guidelines which provide a framework for detecting infections early and preventing potential transmission of sars-cov-2. pregnant women admitted for suspected sars-cov-2 or who develop symptoms during admission are isolated and should be prioritized for testing [4, 5] . furthermore, delivery should be performed in a negative-pressure isolation ward whenever possible, with strict precautionary measures to reduce the risk of droplet transmission [6, 7] . neonates born to mothers infected with sars-cov-2 should be screened at 24 and 48 h of age using nasopharyngeal swabs for polymerase chain reaction (pcr) analysis. any positive result requires repeated testing at 48-to 72-h intervals until there are 2 consecutive negative results [6] . furthermore, asymptomatic infants with positive or pending sars-cov-2 tests may be discharged home with plans of outpatient observation through 14 days after birth [6] . clinical staff caring for the mother and infant should use droplet and contact precautions, including the use of personal protective equipment such as gown, gloves, eye protection, and standard surgical mask [4] [5] [6] [7] in addition to separating the mother and newborn to minimize the risk of postnatal infection [6, 7] . a 1-day-old hispanic girl was born via elective cesarean section at 34 weeks due to oligohydramnios and maternal history of eclampsia. her mother was a 31-year-old g2p2 woman with a past medical history of hiv and type a2 gestational diabetes. two days prior to giving birth, her mother tested positive for sars-cov-2 via nasopharyngeal swab pcr after experiencing shortness of breath and myalgia. upon admission to the hospital, initial laboratory studies revealed a white blood cell count (wbc) of 10 000 cells/mm 3 (table 1 ) and a chest x-ray showed bibasilar opacities, supporting her diagnosis of sars-cov-2 ( figure 1 ). initial examination of the neonate was normal and the apgar score was 9/10 at 1 and 5 mins. vital signs were within normal limits and a complete blood count revealed a normal wbc of 9000 cells/mm 3 ( table 2 ). due to the above circumstances, the patient was admitted to the neonatal intensive care unit (nicu) for hypoglycemia monitoring and continuous cardiac, respiratory, and pulse oximetry monitoring. the patient was also started on prophylactic oral zidovudine. as per guidelines, the neonate was tested for sars-cov-2 at 24 h of age via nasopharyngeal swab with the becton-dickinson (bd max) automated polymerase chain reaction system (becton-dickinson, franklin lakes, nj, usa), which yielded a positive result. at the time, the patient's lungs were clear to auscultation bilaterally with no signs of respiratory distress and 95% oxygen saturation on room air. she continued to be monitored for any signs of respiratory deterioration. over the next 10 days, the patient continued to be monitored and remained asymptomatic. laboratory results remained within normal limits, and testing for sars-cov-2 remained positive when repeated at 48 h and at 7 days of age. she continued to feed well and grow appropriately during her stay. no imaging studies were required and she was subsequently discharged from the nicu on day 10 with instructions for follow-up in 2 weeks. vertical transmission is the passage of infectious agents from an infected mother to her offspring. this may occur across the placenta, through breastmilk, or from direct contact during or after birth [8] . concerning transplacental vertical transmission, disease-causing organisms overcome the protective barrier to infect the offspring during the antenatal, perinatal, or postnatal periods of gestation [8] . the result often increases fetal morbidity and mortality from complications like organ injury, morphological abnormalities, intrauterine growth restriction, and intrauterine fetal demise [9] . as a result, trimester-specific pregnancy screenings are done for infections with known vertical transmission. these include parvovirus, human immunodeficiency virus, zika, varicella-zoster, rubella, cytomegalovirus, and toxoplasmosis [8, 9] . due to the novelty of sars-cov-2, there is insufficient evidence on transplacental transmission of this virus. nonetheless, there are proposed mechanisms by which it may take place. understanding the structure and function of the placenta is essential when considering mechanisms of transplacental transmission. the placenta is the interface between the maternal and fetal circulatory systems and is composed of fetal-derived progenitor cells which differentiate into cytotrophoblasts and syncytiotrophoblasts [8] . it is needed for fetal nourishment and protection against pathogens that may be present in the maternal circulation. it is believed that the syncytiotrophoblast layer confers a broad non-specific resistance to microbial infections [8] . however, some pathogens are able to overcome this barrier through unique mechanisms. for instance, the zika virus, which causes congenital microcephaly, may be able to penetrate the placenta due to its ability to infect different cells of the placenta as well as cells of the maternal immune system [8] . with regards to transplacental transmission of sars-cov-2, it is essential to understand how the virus infects cells of the body. sars-cov-2 infects host cells by binding to angiotensinconverting enzyme 2 (ace2), a membrane-bound aminopeptidase [10, 11] . this leads to receptor-mediated endocytosis of the virus, which is necessary for replication [11] . while ace2 is predominantly expressed in the heart, lungs, kidneys, and gastrointestinal tract, recent studies uncovered ace2 expression in various placental tissues and fetal organs [10] [11] [12] . while this expression is low in early gestational ages, it increases significantly in later stages of pregnancy [11] , thereby increasing variables complete blood count and differential the risk of fetal infection. although similar viruses like severe acute respiratory syndrome coronavirus 1 have not demonstrated the ability to cause fetal infection, sars-cov-2 is able to bind ace2 with much higher affinity [11] , thus increasing the probability of transplacental transmission. while the presence of ace2 in the placenta provides a plausible mechanism for transplacental transmission, only 3 studies have found evidence in support of its occurrence. dong et al. described a case of a newborn that tested positive for anti-sars-cov-2 immunoglobulin m (igm) 2 h after birth [13] . this class of antibody dominates the early phase of infection and is unable to cross the placenta. although this neonate tested negative for sars-cov-2, the positive igm antibodies suggest an immune response of fetal origin. furthermore, intrapartum exposure was unlikely to be the cause since igm may take up to 3 to 7 days to be detectable [13] . additionally, other studies detected sars-cov-2 in placental tissue using electron microscopy and pcr [14, 15] . this provides evidence that sars-cov-2 is able to infect the placenta and possibly cause fetal infection. however, in a recent study, researchers were unable to detect viral particles in products of conception from infected mothers [16] . furthermore, all babies born to these mothers complete blood count and differential base excess (mmol/l) -2.0-3.0 -2.8 table 2 . the results of the laboratory investigations of a baby girl on the first day of birth. * severe acute respiratory syndrome coronavirus 2; ** human immunodeficiency virus-ribonucleic acid; *** partial pressure of co 2 ; **** partial pressure of o 2 . tested negative for sars-cov-2. these findings suggest that, in most cases, the placenta is able to protect the fetus from sars-cov-2 infection. this also explains the paucity of reported cases suspicious for transplacental transmission of sars-cov-2. at this time, the literature identifies 3 other cases of neonates testing positive for sars-cov-2 as early as 16 h after birth from infected mothers [17] . similar to our case, a strict infection control protocol was followed and the babies were delivered via cesarean section. although no products of conception in these cases were tested for viral particles, the positive pcr results within the first 24 h and strict infection control protocol make transplacental transmission more likely than peripartum transmission. a case report of neonatal covid-19 infection in china outcome of coronavirus spectrum infections (sars, mers, covid 1-19) during pregnancy: a systematic review and meta-analysis transmission of covid-19 virus by droplets and aerosols: a critical review on the unresolved dichotomy practice advisory: novel coronavirus 2019 centers for disease control: interim considerations for infection prevention and control of coronavirus disease 2019 (covid-19) in inpatient obstetrics healthcare settings. centers of disease control american academy of pediatrics committee on fetus and newborn section of neonatal perinatal medicine and committee on infectious disease. initial guidance: management of infants born to mothers with covid-19 expert consensus for managing pregnant women and neonates born to mothers with suspected or confirmed novel coronavirus (covid-19) infection [corrected and republished in microbial vertical transmission during human pregnancy conclusions we presented the case of a 31-year-old mother with mild symptoms of covid-19 pneumonia who was positive for sars-cov-2 infection and who gave birth to a baby girl who was also positive for sars-cov-2. this case supports the possibility of transplacental transmission of sars-cov-2. sars-cov-2 can likely overcome the placental barrier though its high affinity for ace-2 expressed in the placenta and fetal organs iugr and infections covid-19) pandemic and pregnancy the sars-cov-2 receptor ace2 expression of maternal-fetal interface and fetal organs by single-cell transcriptome study evidence and possible mechanisms of rare maternal-fetal transmission of sars-cov-2 possible vertical transmission of sars-cov-2 from an infected mother to her newborn visualization of severe acute respiratory syndrome coronavirus 2 invading the human placenta using electron microscopy detection of sars-cov-2 in placental and fetal membrane samples clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records key: cord-263532-q044i7ym authors: goyal, bhupesh; goyal, deepti title: targeting the dimerization of the main protease of coronaviruses: a potential broad-spectrum therapeutic strategy date: 2020-05-13 journal: acs comb sci doi: 10.1021/acscombsci.0c00058 sha: doc_id: 263532 cord_uid: q044i7ym [image: see text] a new coronavirus (cov) caused a pandemic named covid-19, which has become a global health care emergency in the present time. the virus is referred to as sars-cov-2 (severe acute respiratory syndrome-coronavirus-2) and has a genome similar (∼82%) to that of the previously known sars-cov (sars coronavirus). an attractive therapeutic target for covs is the main protease (m(pro)) or 3-chymotrypsin-like cysteine protease (3cl(pro)), as this enzyme plays a key role in polyprotein processing and is active in a dimeric form. further, m(pro) is highly conserved among various covs, and a mutation in m(pro) is often lethal to the virus. thus, drugs targeting the m(pro) enzyme significantly reduce the risk of mutation-mediated drug resistance and display broad-spectrum antiviral activity. the combinatorial design of peptide-based inhibitors targeting the dimerization of sars-cov m(pro) represents a potential therapeutic strategy. in this regard, we have compiled the literature reports highlighting the effect of mutations and n-terminal deletion of residues of sars-cov m(pro) on its dimerization and, thus, catalytic activity. we believe that the present review will stimulate research in this less explored yet quite significant area. the effect of the covid-19 epidemic and the possibility of future cov outbreaks strongly emphasize the urgent need for the design and development of potent antiviral agents against cov infections. coronaviruses (covs) have been known since 1947, when the first prototype murine strain jhm was reported. 1, 2 covs are enveloped viruses consisting of single positive-strand rna, and they infect various vertebrates (bats, pets, livestock, poultry, and humans). among humans, covs are responsible for respiratory, gastrointestinal, and neurological problems. 3, 4 covs belong to subfamily coronavirinae of the family coronaviridae. the coronavirinae is further subdivided into four genera (α, β, γ, and δ). each genus is further divided into four lineage subgroups. a new coronavirus resulted in the outbreak of a pneumonialike illness in wuhan, china, in late december 2019, and has become a life-threatening concern worldwide in the present time. 5, 6 the virus has been termed sars-cov-2 (severe acute respiratory syndrome-cororavirus-2), 7 as the rna genome is ∼82% similar to that of the sars coronavirus (sars-cov). 5, 6 sars-cov-2 belongs to the β-coronavirus group. the pneumonia-like illness caused by sars-cov-2 was named as covid-19. many patients infected with covid-19 suffer from fever, dry cough, tiredness, and breathing difficulty under severe conditions; others may be just silent carriers of the virus. the world health organization (who) declared covid-19 a pandemic on march 11, 2020. as of 2:00 am cest, may 6, 2020, there were more than 3.5 million confirmed cases globally with 245,150 deaths due to the sars-cov-2. 8 the figures clearly indicate that covid-19 imposes a huge health care crisis globally. the scientific and medical fraternity across the world have been working tirelessly and at record-breaking speed to find a solution to bring this virus outbreak under control; however, no success has been achieved at the time of publication of this review. similar to sars and mers (middle east respiratory syndrome), the genome of sars-cov-2 encodes non-structural proteins [sars-cov-2 m pro (main protease), also known as 3chymotrypsin-like cysteine protease (ccp or 3cl pro ), papainlike protease, and rna-dependent rna polymerase (rdrp)], helicase, structural proteins (spike glycoprotein), and accessory proteins. the non-structural proteins play a key role during the virus's life cycle, and spike glycoprotein is necessary for the interactions of the virus with the host cell receptors during viral entry. 3 the non-structural and structural proteins were recognized as promising targets for the design and development of antiviral agents against sars and mers. 3 sars-cov-2m pro plays a key role in polyprotein processing and is active in a dimeric form. 9 the m pro offers a promising target for the development of broad-spectrum anti-coronaviral therapeutic agents due to its highly conserved three-dimensional structure among various covs (figures 1 and 2) . 10 the covs are subject to extensive mutagenesis; however, key proteins are highly conserved, as mutations in key proteins are often lethal to the virus. 11 thus, drugs targeting conserved m pro are usually capable of preventing the replication and proliferation of the virus and display broad-spectrum antiviral activity. in addition, drugs targeting m pro can reduce the risk of mutation-mediated drug resistance in future deadly viral strains. the individual monomers of sars-cov m pro are enzymatically inactive, and two strategies have been employed to develop inhibitors against this enzyme: (i) molecules targeting the substrate binding pocket to block the catalytic activity, and (ii) dimerization inhibitors. numerous reports on the inhibitor design against sars-cov m pro are based on the substrate binding pocket. 12, 13 however, no inhibitor targeting the substrate binding pocket has reached clinical trials to date. an alternative potential therapeutic strategy is to inhibit the dimerization of m pro , and there are a few reports on inhibitors targeting the dimerization of sars-cov m pro . 14, 15 in the present review, literature reports highlighting the effect of mutations and n-terminal deletion of residues of sars-cov m pro on its dimerization and, thus, catalytic activity are compiled. to the best of our knowledge, this review is the first compilation of the various studies focusing on the dimerization of sars-cov m pro . a number of inhibitors targeting the substrate binding pocket of sars-cov m pro are reported in the literature, and they can be found discussed elsewhere. 12, 13 ■ structural and functional details of sars-cov-2 m pro enzyme sars-cov-2 m pro is a dimer consisting of two monomers that are arranged almost perpendicular to one another. 9 each monomer comprises three domains and possesses a catalytic dyad (his41 and cys145) situated in a cleft between domains i and ii (residues 10−99 and 100−182, respectively). the catalytic residues are situated in the chymotrypsin-like double βbarrel fold consisting of domain i and ii. the catalytic domains are connected by a long loop region to the c-terminal domain iii (residues 198−303) composed of five antiparallel α-helices. a contact interface (∼1394 å 2 ) was formed between domain ii of monomer a and the nh 2 -terminal residues ("n-finger") of monomer b in the dimeric structure of sars-cov-2 m pro . the dimerization is necessary for enzymatic activity as the n-finger of each of the two monomers interact with glu166 of the other monomer, which assist in the correct orientation of the s1 pocket of the substrate binding site. the c-and n-terminus of the monomers constitute the dimer interface and are closely held in the dimer than in the monomeric state where mobility of these termini are higher. the structural design of sars-cov-2 m pro was found to be similar to the crystal structure of sars-cov m pro (figure 1a,b) . only 12 out of 306 residues are the homology models of sars-cov-2 m pro were found to be very much similar to sars-cov m pro . 16 thus, inhibitors targeting sars-cov m pro may also block the enzymatic activity of sars-cov-2 m pro . previous studies highlighted that many sars-cov m pro inhibitors displayed efficacy against mers-cov. 17, 18 the sars-cov m pro exists as a homodimer in the crystal structure, 19 and the important residues along with their key roles are listed in table 1 . the key residues that stabilize the dimeric structure of sars-cov m pro are shown in figure 3 . the m pro play a vital role in cleaving the polyproteins translated by the virus rna. 32 the m pro cleave the large polyprotein 1ab (replicase 1ab, ∼790 kda) at 11 different sites and the recognition sequence at most sites was found to be leu− gln↓(ser/ala/gly) (↓ shows the cleavage site). the replication of the virus can be efficiently blocked by inhibiting m pro activity. as no human proteases with an analogous cleavage specificity were reported, the inhibitors against m pro are not likely to be toxic. the various mutation analyses, n-terminal truncation studies, and md simulation studies that highlighted key residues of sars-cov m pro involved in the stabilization of the catalytically active dimeric structure of the enzyme are listed in table 2 and are arranged in chronological order. in 2004, bacha et al. identified a cluster of conserved serine residues (ser139, ser144, and ser147) situated in the close proximity of the active site of sars-cov m pro that can be targeted to inhibit the protease activity. 33 the alanine substitution of ser139, ser144, and ser147 had a devastating impact on the sars-cov m pro catalytic activity. the serine cluster (ser139, ser144, and ser147) is highly conserved in proteases among various covs, which, in turn, highlight that targeting this site will provide broad-spectrum therapeutic agents against cov protease. in a later report, barrila et al. highlighted that ser147 of sars-cov m pro play a key role in stabilizing the dimeric structure and mutation of conserved ser147 to ala lead to dimer instability. 25 the backbone of ser147 forms hydrogen bonds with the backbones of ser144 and his163 residues. a 150-fold reduction in the catalytic efficiency and complete loss of dimerization was observed in s147a mutant as compared to wild-type (wt) enzyme. in another study, chou et al. reported that high salt concentration and low ph led to a decrease in the m pro dimerization and activity. 24 the observed decrease in activity of m pro was attributed to the salt bridge interaction between arg4 and glu290 residues. the study highlighted that e290a mutation led to a complete loss of catalytic activity and 22 hsu et al. 23 arg4, ser10, gly11, glu14, asn28, ser139, phe140, ser147, glu290, arg298 dimerization chou et al., 24 barrila et al., 25 chen et al., 26 chen et al., 27 lin et al., 28 shiet al., 29 hu et al., 30 barrila et al. 31 . s139a, s144a, s147a s139a, s144a, and s147a mutations have a devastating effect on the catalytic activity of sars-cov m pro . bacha et al. 33 2. a 150-fold reduction in the catalytic efficiency and complete loss of dimerization were observed in s147a mutant as compared to wild-type (wt) enzyme. barrila et al. 25 3. r4a, e290a e290a mutation led to a complete loss of catalytic activity and dimerization, whereas r4a mutation resulted in an approximately 5-fold decrease in the dimerization and a modest loss in the enzymatic activity. chou et al. 24 4. n-terminal truncated (residues 1−7) sars-cov m pro n-terminal truncated protease dimer adopts a different state as compared to the full-length protease dimer; md simulations depicted that the angle between the two monomers increased and the dimension of the substrate binding pocket was reduced in the n-terminal truncated protease dimer, which is not appropriate for the substrate binding. hsu et al. 35 6. sars-cov m pro size and conformation of the substrate binding pocket s1 are linked to the protonation states of the histidine residues (his163 and his172) comprising the pocket. the n-terminus of another monomer in the protease dimer plays a critical role in the catalytic activity by sustaining the correct conformation of the oxyanion loop and substrate binding pocket s1 through hydrogen bonds. tan et al. 36 7. analytical ultracentrifugation experiments depicted that a tight dimer was formed in the mature enzyme (k wei et al. 15 10. hybrid sars-cov m pro between the wt enzyme and the inactive mutant c145a the simulation and experimental results concluded that (i) dimerization was a mandatory requirement for the enzymatic activity of the protease, and (ii) only one monomer in the protease dimer displayed catalytic activity. chen et al. 37 11. sars cov m pro sars cov m pro exists as a homodimer in its active form. the biochemical and biophysical data depicted a monomer−dimer equilibrium with a dissociation constant k d ≈ 6 μm. graziano et al. 38 12. md simulations of dimeric and monomeric forms of sars-cov m pro md simulations highlighted that the interactions between the n-terminus of one monomer and another monomer of the protease helped to maintain the dimer's enzymatic activity. zheng et al. 39 13. s1a, f2a, r4a, s10a, e14a, s139a, f140a the ser10 and glu14 residues located in the α-helix a′ of domain i of sars-cov m pro are highly conserved among various covs proteases and contribute significantly in the monomer− monomer interactions. the individual mutations of ser10 and glu14 to ala resulted in weak dimerization and no enzymatic activity. al. 26 14. g11a g11a mutation led to a complete loss in the enzymatic activity of sars-cov m pro . the g11a mutant structure was the first reported crystal structure of the monomeric sars-cov m pro , and the structure provided a better understanding of the dimerization and catalytic mechanism of the protease. chen et al. 27 15. s123a, s123c, s139a, and double mutants s123a/r298a, s139a/q299a deletion of gln299 or arg298 significantly decreased the catalytic activity to only 1−2% of wt enzyme, and the enzyme existed predominantly in the monomeric form. the point mutants of gln299 and arg298 depicted that these residues are involved in dimerization and play a key role in fixing the catalytically active conformation of the enzyme. lin et al. 28 16. r298a r298a mutation leads to disruption of the dimeric structure as well as irreversible inhibition of the catalytic activity of the enzyme. zhong et al. 41, 42 19. s139a and f140a s139a and f140a on the dimer interface of sars-cov m pro resulted in different conformational changes in the crystal structure of the enzyme. ser139 of monomer a was involved in the hydrogen-bond interaction with gln299 of monomer b, and s139a mutation resulted in the complete loss of dimerization. hu et al. 30 20. sars-cov m pro substrate-induced dimerization is necessary for the enzymatic activity of sars-cov m pro in the polyprotein. li et al. 43 the mutagenesis studies highlighted that glu166 plays a linking role between the dimer interface and substrate binding site. 34 md and docking simulations highlighted that nterminal truncated protease dimer adopts a different state as compared to the full-length protease dimer. md simulations depicted that angle between the two monomers increased and the dimension of the substrate binding pocket reduced in the nterminal truncated protease dimer, which is not appropriate for the substrate binding. additionally, surface plasmon resonance highlighted that n-terminal truncated protease does not bind with the model substrate. in 2005, hsu et al. reported that n-terminal truncated (residues 1−3) protease exists predominantly as dimer with 76% enzymatic activity; however, n-terminal truncated (residues 1− 4) protease exists mostly as monomer with very little enzymatic activity. 35 the study indicated that arg4 have an influential effect on the catalytic activity and dimeric structure of the protease. the last c-terminal helically truncated protease also displayed a higher tendency to exist as a monomer and displayed little activity. these observations highlighted that both n-and c-terminal regions affect the dimerization and enzymatic activity of the sars-cov m pro . the study provided key insights for the novel design of inhibitors targeting the dimer interface of sars-cov m pro . in another study, the conformational flexibility of the sars-cov m pro was investigated by analyzing several crystal structures and md simulations. 36 the size and conformation of the substrate binding pocket s1 are linked to the protonation state of his163 and his172 comprising the pocket. the study highlighted that the n-terminus of another monomer in the protease dimer plays a critical role in the catalytic activity in sustaining the correct conformation of the oxyanion loop and substrate binding pocket s1 through hydrogen bonds. in 2005, hsu et al. reported the crystal structure of the product-bound c145a mutant protease and suggested the maturation mechanism of the enzyme. 23 the analytical ultracentrifuge experiments depicted that a tight dimer was formed in the mature enzyme (k d = 0.35 nm) as compared to c145a mutant possessing 10 additional n-terminal (k d = 17.2 nm) or c-terminal residues (k d = 5.6 nm). the inhibitors targeting the dimer interface may block the maturation of protease as both n and c termini are near to the sars-cov m pro active site in the product-bound c145a structure. in 2005, ding et al. studied the interaction between sars-cov m pro and a dimerization inhibitor n8 (sgfrkmaf) by affinity capillary electrophoresis. 14 the thermodynamic analysis highlighted that hydrophobic contacts and electrostatic interactions play major roles in the binding of dimerization inhibitor with sars-cov m pro . in a later report by the same research group, n8 and its mutants were evaluated for their ability to act as dimerization inhibitors of sars-cov m pro . 15 the peptide cleavage assay highlighted that n8 inhibited the dimerization of protease enzyme with a dimerization inhibition constant (k i ) of 2.20 mm. the comparison between the inhibitory activities of n8 and its mutants indicated that hydrophobic contact of met6 and electrostatic interaction of arg4 of n8 contributed significantly in its binding with the enzyme. in 2006, chen et al. employed md simulations and mutational studies to investigate that why dimer is catalytically active as compared to the monomer and whether both monomers in the dimer are active. 37 the md simulations depicted that the monomers are always catalytically inactive, two monomers comprising the dimer are asymmetric, and only one monomer display catalytic activity at a time. md simulations also highlighted that the correct conformation required for the catalytic activity in one monomer can be induced by the formation of dimer. the simulation and experimental results concluded that (i) dimerization was a mandatory requirement for the enzymatic activity of the protease, and (ii) only one monomer in the protease dimer displayed catalytic activity. in another study, graziano et al. employed chemical crosslinking, enzyme kinetics and small-angle x-ray scattering techniques to investigate the oligomeric state of sars cov m pro . 38 the sars cov m pro exists as a homodimer in its active form. the biochemical and biophysical data depicted a monomer−dimer equilibrium with a dissociation constant, k d , of ∼6 μm. in 2007, zheng et al. performed md simulations of the dimeric and monomeric form of a sars-cov m pro to get insight into the activity of the enzyme. 39 the key interactions between the two monomers in the dimer were investigated and how these interactions help in maintaining the function of the dimer was studied. the study highlighted that the interactions between the n-terminus of one monomer with another monomer of the protease helped to maintain the dimer enzymatic activity. the key insights obtained from md simulations will be beneficial in the design of specific protease inhibitors targeting the dimer interface of sars-cov m pro enzyme. chen et al. identified critical residues involved in the sars-cov m pro dimerization and activity by systematic mutation analysis. 26 a total of seven residues on the dimer interface of the enzyme were selected to assess their influence on the catalytic activity and dimer stability by employing biophysical and biochemical techniques. the ser10 and glu14 residues located in the α-helix a′ of domain i of sars-cov m pro are highly conserved among various covs proteases and contribute significantly in the monomer−monomer interactions. the individual mutations of ser10 and glu14 to ala resulted in weak dimerization and no enzymatic activity. the results of the study will be beneficial in the better understanding of the dimerization activity relationship of sars-cov m pro and will provide key insights for the design of antiviral compounds targeting the dimer interface of the sars-cov m pro . further chen et al. reported that mutation of gly11 residue situated at the dimer interface to ala led to a complete loss in the enzymatic activity of sars-cov m pro . 27 a complete dimer dissociation in the crystal structure of g11a mutant was observed. the g11a mutation might shorten the α-helix a′ (ser10−gly15) of domain i, which led to the misorientation of the n-finger of the enzyme. as a result, n-finger could not properly squeeze into another monomer pocket during dimerization; thus resulting in the destabilization of the dimer structure. the hydrogen bond interactions between two helices a′ [ser10a···ser10b and gly11a/b···glu14b/a (ser10a and ser10b indicate ser10 of monomer a and b, respectively)] play a major role in the stability of the dimer interface. the g11a mutant structure was the first reported crystal structure of the monomeric sars-cov m pro and provided a better understanding of the dimerization and catalytic mechanism of the protease. in 2008, grum-tokars et al. reviewed the literature related to different sars-cov m pro expression constructs and assays used to calculate the enzymatic activity. 45 the enzymatic activity of sars-cov m pro was significantly reduced in two cases: (i) on adding affinity-tags or non-native sequences to the n-or cterminus of the protease enzyme, and (ii) when the concentration of the enzyme used in assays was below the equilibrium dissociation constant of the sars-cov m pro dimer. in 2008, lin et al. analyzed the quaternary structure of the cterminal truncated mutants of sars-cov-m pro enzyme by employing sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques. 28 the deletion of c-terminus from 306 to 300 does not affect the structure and catalytic activity of the enzyme. however, deletion of gln299 or arg298 significantly decreased the catalytic activity to only 1− 2% of wt enzyme, and the enzyme existed predominantly in the monomeric form. the point mutants of gln299 and arg298 depicted that these residues are involved in dimerization and played a key role in fixing the catalytically active conformation of the enzyme. the monomeric crystal structure of the sars-cov m pro r298a mutant was reported by shi et al. 29 the study highlighted that arg298 play an important role in maintaining the dimer structure of the enzyme. the authors tried to solve two puzzles: (i) how the dimer−monomer switch was controlled, and (ii) why dimerization was necessary for the enzymatic activity. the results highlighted that r298a mutation leads to disruption of the dimeric structure as well as irreversible inhibition of the catalytic activity of the enzyme. in 2013, wu et al. presented the crystal structure of r298a mutant of sars-cov m pro in the presence of a peptide substrate. 40 the r298a mutant undergoes a reversible substrate induced dimerization with minute changes in the relative position of the domain iii of each monomer as compared to wt m pro . as indicated by active enzyme centrifugation (aec) experiments, the kinetic parameters of the r298a mutant were identical with that of wt m pro . the study provided key insights into the mechanisms that governed monomer−dimer switch during m pro maturation process. in 2008, zhong et al. reported that c-terminal domain [m pro -c (residues 187−306)] of sars-cov m pro exist as a monomer and dimer, and m pro -c dimer possess a novel dimerization interface. 41 the n-finger deleted sars-cov m pro does not maintain the active dimer structure; however, form a new dimer that is not active. thus, the n-finger of sars-cov m pro play a critical role in the formation of catalytically active dimer of sars-cov m pro . later in 2009, the authors reported stable m pro -c dimer as the 3d domain-swapped dimer. 42 the n-finger deleted m pro also undergo 3d domain swapping of the cterminal domains and form a stable dimer. next, hu et al. reported that two adjacent mutations (s139a and f140a) on the dimer interface of sars-cov m pro resulted in the different conformational changes in crystal structure of the enzyme. 30 the s139a mutation resulted in the complete loss of dimerization. the ser139 of monomer a was involved in the hydrogen bond interaction with gln299 of monomer b. the study suggested that the cooperativity among all the key elements control the dimerization of sars-cov m pro and the stability of the dimer greatly depends on the integrity of the dimer interface. in 2010, li et al. presented the maturation mechanism of sars-cov m pro and concluded that substrate-induced dimerization is essential for the enzymatic activity of sars-cov m pro in the polyprotein. 43 a modified model for the m pro maturation process was proposed in the study. in 2010, cheng et al. demonstrated the significance of substrate-induced dimerization of m pro to its catalytic mechanism. 44 the results of the experimental studies highlighted that dimerization of m pro was necessary for the protease activity. the mutagenesis studies highlighted that glu166 plays a linking role between dimer interface and substrate binding site. the authors mentioned that the connection between dimer interface and substrate binding site by glu166 may be universal in all proteases among various covs. the another study highlighted that asn28 was essential for the enzymatic activity and dimerization of sars-cov m pro . 31 the n28a mutation led to a complete inactivation of the enzyme and a decrease of 19.2-fold in the dimerization k d . the interactions between asn28 and cys117 play a key role in the dimer stability and enzymatic activity of sars-cov m pro . the residue asn28, a buried residue, display interactions with catalytic loop and βsheet region of sars-cov m pro where cys117 is present. the conformational switch of residues (ser139, phe140, and leu141) in the catalytic loop region from standard loop conformation to a short 3 10 -helical conformation lead to a diminished dimerization of m pro . the n28a mutant crystal structure revealed about the critical role of asn28 in preserving the structural integrity of the active site and in positioning critical residues that are involved in binding at the dimer interface and substrate catalysis. covid-19, a novel infectious disease caused by a singlestranded positive-sense rna virus, has presented a serious worldwide public health care emergency. the whole world remains unprepared to efficiently control this infectious disease, despite the lessons learned from the previous coronavirus (cov) infections that caused sars and mers. scientists around the world are looking for effective and promising therapeutic antiviral agents as well as vaccine candidates for combating covid-19. however, no specific antiviral drugs or effective vaccines for covid-19 have been discovered to date. the genetic reshuffling, mutations, and interspecies transmission of the rna viruses highlight the urgent need for the design and development of broad-spectrum antiviral drugs. thus, a coherent effort is required to develop effective drugs and vaccines against cov infections and other highly pathogenic viruses to decrease the devastating impact on human life. as the clinical drug discovery and development process is costly, time extensive, and difficult, the design and development of broadspectrum antiviral drugs are of paramount importance. thus, drugs targeting highly conserved proteins such as main protease (m pro ) among various covs will provide two advantages: (i) the potential for broad-spectrum antiviral activity, and (ii) reduced risk of mutation-mediated drug resistance. the cas data highlighted that sars-cov m pro has drawn significantly more attention than other targets, and a large number of compounds with therapeutic potential have been identified against sars-cov m pro . a total number of 49 patents and 2178 potential drug candidates have been listed in the cas registry of chemical substances for m pro . cov-infected patients administered with the hiv drug combination of lopinavir/ ritonavir (kaletra), a m pro inhibitor, have shown considerable improvement (nct04307693 46 and nct04255017 47 ), highlighting m pro as a high-value target for the development of drug candidates against covs. 3, 48 another clinical trial study on acs combinatorial science pubs.acs.org/acscombsci review different combinations of protease inhibitors such as oseltamivir, favipiravir, and hydroxychloroquine (hcq) 49 is presently underway for treatment of covid-19. 50 twenty-seven clinical trial studies registered with the u.s. national library of medicine database were underway on the protease inhibitors in cov infections as of may 6, 2020. 51 a number of reports have been published on the design of small-molecule and peptidomimetic inhibitors targeting the substrate binding pocket of sars-cov m pro . 12, 13 however, no such inhibitor has advanced to clinical trials to date. an alternative approach includes the combinatorial design of peptide-based inhibitors that target the dimerization of sars-cov m pro as a potential therapeutic strategy. dimerization inhibitors have been successfully employed against hiv protease and other viral enzymes. 52−57 the various mutation analyses listed in the present review highlight the key residues of sars-cov m pro that are crucial for the dimerization and thus catalytic activity of the enzyme. the studies provide future directions for the design of potential dimerization inhibitors against m pro . the md studies of m pro compiled in the review will act as molecular guide for the structure-based design of potent dimerization inhibitors. in addition, the already reported dimerization inhibitors of m pro will provide the framework for further modifications to design potent antiviral agents. the peptide-based interface inhibitors may provide better therapeutic options than small molecules, as the large surface area of the dimer interface of m pro will be better targeted with peptide inhibitors. the peptide-based inhibitors have additional advantages over small molecules as drug candidates due to their greater chemical diversity, high specificity, low toxicity, possibility of rational design, low accumulation in tissues, and stability toward proteolytic cleavage (peptidomimetics). 54,56,58−60 in parallel with the drug development, scientists around the globe are actively involved in developing rapid point-of-care diagnostic methods for sars-cov-2. we believe that the combinational design of peptide-based dimerization inhibitors of m pro provide an attractive approach to combat covs and that this review will stimulate research in this less explored yet highly relevant area. the previous research efforts toward the design of potent antiviral agents against sars and mers should be used to draw the line of defense more quickly against the novel deadly sars-cov-2. the present review provides strong groundwork for the design and development of novel dimerization inhibitors of sars-cov-2 m pro for combating this mysterious and rapidly evolving virus on an invisible battlefield. chemical abstracts service; ccp, 3-chymotrypsin-like cysteine protease; cest cov, coronavirus; covid-19, coronavirus disease hiv, human immunodeficiency virus k d , dissociation constant; k i , dimerization inhibition constant; mers-cov, middle east respiratory syndrome coronavirus md, molecular dynamics; m pro rna-dependent rna polymerase; sars-cov, severe acute respiratory syndrome coronavirus; sars-cov-2, severe acute respiratory syndrome coronavirus-2 world health organization; wt, wild-type ■ references a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin: i. isolation and biological properties of the virus a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin: ii. pathology coronaviruses-drug discovery and therapeutic options recent insights into emerging coronaviruses a new coronavirus associated with human respiratory disease in china sidorov, i. a.; sola, i.; ziebuhr, j. severe acute respiratory syndrome-related coronavirus: the species and its viruses−a statement of the coronavirus study group main protease provides a basis for design of improved α-ketoamide inhibitors learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov evolutionary selection associated with the multi-function of overlapping genes in the hepatitis b virus an overview of severe acute respiratory syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy characterization and inhibition of the main protease of severe acute respiratory syndrome coronavirus. chembioeng rev the interaction between severe acute respiratory syndrome coronavirus 3c-like proteinase and a dimeric inhibitor by capillary electrophoresis the n-terminal octapeptide acts as a dimerization inhibitor of sars coronavirus 3c-like proteinase middle east respiratory syndrome coronavirus (mers-cov): an updated overview and pharmacotherapeutics repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection coronavirus main proteinase (3cl pro ) structure: basis for design of anti-sars drugs 3c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism a novel auto-cleavage assay for studying mutational effects on the active site of severe acute respiratory syndrome coronavirus 3c-like protease sars-cov 3cl protease cleaves its c-terminal autoprocessing site by novel subsite cooperativity mechanism of the maturation process of sars-cov 3cl protease quaternary structure of the severe acute respiratory syndrome (sars) coronavirus main protease long-range cooperative interactions modulate dimerization in sars 3cl pro residues on the dimer interface of sars coronavirus 3c-like protease: dimer stability characterization and enzyme catalytic activity analysis mutation of gly-11 on the dimer interface results in the complete crystallographic dimer dissociation of severe acute respiratory syndrome coronavirus 3c-like protease: crystal structure with molecular dynamics simulations correlation between dissociation and catalysis of sars-cov main protease mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3c-like protease two adjacent mutations on the dimer interface of sars coronavirus 3c-like protease cause different conformational changes in crystal structure mutation of asn28 disrupts the dimerization and enzymatic activity of sars 3cl pro crystallographic studies on coronaviral proteases enable antiviral drug design identification of novel inhibitors of the sars coronavirus main protease 3cl pro severe acute respiratory syndrome coronavirus 3c-like proteinase n terminus is indispensable for proteolytic activity but not for enzyme dimerization: biochemical and thermodynamic investigation in conjunction with molecular dynamics simulations critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease hilgenfeld, r. ph-dependent conformational flexibility of the sars-cov main proteinase (m pro ) dimer: molecular dynamics simulations and multiple x-ray structure analyses only one protomer is active in the dimer of sars 3c-like proteinase cov main proteinase: the monomer-dimer equilibrium dissociation constant insight into the activity of sars main protease: molecular dynamics study of dimeric and monomeric form of enzyme mechanism for controlling the monomer-dimer conversion of sars coronavirus main protease without its n-finger, the main protease of severe acute respiratory syndrome coronavirus can form a novel dimer through its c-terminal domain xia, b. c-terminal domain of sars-cov main protease can form a 3d domain-swapped dimer maturation mechanism of severe acute respiratory syndrome (sars) coronavirus 3c-like proteinase blocks the substrate-induced dimerization of sars coronavirus main protease evaluating the 3c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery role of lopinavir/ ritonavir in the treatment of sars: initial virological and clinical findings hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro hydroxychloroquine for treatment of covid-19 : a randomized control trial (thdms-covid-19).https:// d = protease+inhibitors+covid-19&draw=2&rank=1 (accessed 2020-05-06). (51) 31 studies found for: lopinavir/ritonavir | coronavirus. https:// cli nical tri als .gov/ct 2/res ult s? structure-based discovery of antiviral inhibitors targeting the e dimer interface of japanese encephalitis virus dimer disruption and monomer sequestration by alkyl tripeptides are successful strategies for inhibiting wild-type and multidrug-resistant mutated hiv-1 proteases strategies in the design of antiviral drugs targeting the dimerization interface of hiv-1 protease: inhibition with cross-linked interfacial peptides hiv-1 reproduction is inhibited by peptides derived from the n-and c-termini of hiv-1 protease peptide-based molecular strategies to interfere with protein misfolding, aggregation and cell degeneration the current state of peptide drug discovery: back to the future? rationally designed peptides and peptidomimetics as inhibitors of amyloid-β (aβ) aggregation: potential therapeutics of alzheimer's disease acs combinatorial science pubs.acs.org/acscombsci review key: cord-258268-7ypq0t3d authors: zanin, luca; saraceno, giorgio; panciani, pier paolo; renisi, giulia; signorini, liana; migliorati, karol; fontanella, marco maria title: sars-cov-2 can induce brain and spine demyelinating lesions date: 2020-05-04 journal: acta neurochir (wien) doi: 10.1007/s00701-020-04374-x sha: doc_id: 258268 cord_uid: 7ypq0t3d sars-cov-2 can attack the central nervous system in the early stages of infection. headache, anosmia, and dysgeusia are common symptoms. disturbance of consciousness and seizures can occur as complications in case of severe covid-19. we described the case of a covid-19 patient admitted for interstitial pneumonia and seizures. mri showed newly diagnosed demyelinating lesions. high-dose steroid treatment allowed neurological and respiratory recovery. we speculated a delayed immune response induced by sars-cov-2. the virus may lead to a sirs-like immune disorder or play a role of infective trigger. prompt invasive treatment should be adopted to avoid hypoxic neurotoxicity and prevent cns injuries. on january 24, 2020, a new virus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been identified, quickly gaining worldwide attention [21] . more than one third of patients with sars-cov-2 develop neurological manifestations [10] . similarly to other coronavirus, sars-cov-2 can attack the olfactory bulb and then affect the central nervous system (cns) through the olfactory tract in the early stages of infection [5] . neurological impairment and demyelinating reaction appear as complications in case of severe coronavirus disease 2019 (covid-19) [10] . we described the case of a covid-19 patient with newly diagnosed demyelinating lesions. a 54 years old women, with a past medical history of anterior communicating artery (acoma) aneurysm treated surgically 20 years before, was found unconscious at home. when the rescue arrived, she regained consciousness and became unrest. at the emergency department, a brief neurological examination revealed a gcs of 12 (e3 m6 v3), without focal sensorimotor deficits. no signs of both tongue biting and incontinence were reported by the familiars. anosmia and ageusia were referred by several days. head ct scan was normal fig. 1) . chest x-ray ( fig. 2 ) revealed an interstitial pneumonia (ip), and real-time polymerase chain reaction (rt-pcr) for sars-cov-2 was positive. the patient was admitted to our neurosurgical unit and complete blood tests showed moderate lymphocytopenia with mild elevation of inflammatory indices (wbc 8.81/mm 3 , ly 0.3/mm 3 , crp 41.3 mg/l, fibrinogen 520 mg/dl). both blood and urinary cultures were negative. antiretroviral and hydroxychloroquine were started. no abnormalities at arterial blood gas (abg) analysis were detected (po2 89, pco2 41, ph 7.43). after few hours, the patient clinically deteriorated. body temperature was normal, and no electrolyte disorders were found. abg revealed a severe normocapnic hypoxia. therefore, she was intubated. subsequent head ct scan was unchanged. electroencephalography showed two seizures starting from right frontotemporal region and diffusing in homologous contralateral hemisphere. antiepileptic therapy with lacosamide, levetiracetam, and phenytoin was started with seizures control. brain mri revealed alterations of the periventricular white matter, hyperintense in t2wi, without restriction of diffusion nor contrast enhancement ( fig. 3a-f ). similar lesions were found at the bulbo-medullary junction and in both the cervical and dorsal spinal cord (fig. 3g) . chemical-physical cerebrospinal fluid (csf) examination was normal, and further analysis ruled out multiple sclerosis. the csf rt-pcr for neurotropic viruses, including sars-cov-2, was negative. high-dose steroid treatment (dexamethasone 20 mg/die for 10 days and 10 mg/die for 10 days) allowed a progressive recovery of the pulmonary impairment. the patient was tracheostomized on the 7th day. after 15 days, ventilator weaning was performed, and the patient was discharged from the intensive care unit (icu) and addressed to our neurosurgical unit. the patient was transferred to rehabilitation without sensorimotor deficits after 12 days. the family of coronaviruses shows a potential neurotropism that can induce neurological disorders like polyneuropathy, encephalopathy, demyelinating lesions, and ischemic stroke [8, 15] . the main clinical manifestations are headache, disturbance of consciousness, paralysis, paraesthesia, and seizures [14] . the neurological complications could appear delayed to respiratory symptoms [8] . sars-cov-2 shows a genetic similarity to sars-covand mers-cov [4, 17] and presents an analogous neurotropism. previous articles showed that a large number of patients report anosmia and dysgeusia. moreover covid-19 may lead to symptoms similar to intracranial infections [2] . our patient showed symptoms consistent with a neurological involvement consequent to sars-cov-2 infection. anosmia and dysgeusia appeared early, while seizures occurred as covid-19 complication. moriguchi explains seizures as results of sars-cov-2 encephalitis [12] . otherwise, we observed demyelinating lesions related to the neurological impairment. the presence of demyelination, as well as sars-cov virus particles and genome sequences, in the brain has been detected in autopsy studies [6, 19] . our patient's brain and spine mri showed new onset of multiple, non-enhancing demyelinating lesions. previous cerebral mri controls performed as follow-up after the acoma aneurysm surgery were normal. multiple sclerosis (ms), viral encephalitis, and bacterial infections were excluded. therefore, we speculated a pathogenesis sars-cov-2 related. neurotropism may occur via trans-lamina cribrosa that enables sars-cov-2 to reach the brain through the olfactory tract [1] . the interaction between the spike protein s1 and the host ace-2 receptor, expressed in the capillary endothelium, allows the virus to penetrate into the neuronal cells [16] . the viral particles budding lead to the onset of symptoms such as anosmia and dysgeusia in the early phase of infection [10] . the delayed cns damage appears mediated by the immune system [9] . as previously demonstrated, the pathogenesis of severe viral infections is closely linked to the development of virus induced systemic inflammatory response syndrome (sirs) or sirs-like immune disorders [3] . for sars-cov-2 infection, the pro-inflammatory state induced by the cytokine storm, mainly sustained by il1, il-6, and tnf α, may be responsible of the activation of glial cells with subsequent demyelination [11] . a possible alternative could be the production of antibodies against glial cells triggered by the virus, as a para-infective or post-infective phenomenon. zhao described a case report of guillan-barrè syndrome during sars-cov-2 infection [20] . moreover, sars-cov-2 may play a role of infective trigger, similar to the one of epstein barr virus in ms. in sars-cov-2 infection, neurological impairment was observed only in case of severe covid-19 [10] . as a matter of fact, our patient showed an ip that required icu. therefore, we supposed that a severe pneumonia with subsequent cns hypoxia that leads to an increased anaerobic metabolism is required to trigger a neurological damage. sars-cov-2 was not detected in the csf probably because the neurological damage was sustained by a delayed immune response that occurred after the viremia. moreover, as reported by different authors, csf clearance, low sensibility of the method, and delayed sampling could explain this occurrence [7, 13, 18] . sudden neurological impairment with seizures in covid-19 patients may be sustained by cns involvement and demyelinating lesions. early csf collection is suggested. prompt invasive treatment should be adopted to avoid hypoxic neurotoxicity and prevent cns injuries. conflict of interest the authors declare that they have no conflict of interest. informed consent the patient's consent has been obtained for this publication. cervical and dorsal mri t2wi sagittal view (g): numerous focal hyperintense intramedullary signal alterations in t2 and without contrast enhancement, located at the bulb-medullary junction, at c2 and from c3 to th 6 evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms could sars-coronavirus-2 trigger autoimmune and/or autoinflammatory mechanisms in genetically predisposed subjects advances in the research of cytokine storm mechanism induced by corona virus disease 2019 and the corresponding immunotherapies detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? viruses multiple organ infection and the pathogenesis of sars neurologic features in severe sars-cov-2 infection neurological complications during treatment of middle east respiratory syndrome infectious immunity in the central nervous system and brain function neurologic manifestations of hospitalized patients with coronavirus disease covid-19: consider cytokine storm syndromes and immunosuppression a first case of meningitis/ encephalitis associated with sars-coronavirus-2 sars-cov-2: "three-steps" infection model and csf diagnostic implication clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia neurological manifestations in severe acute respiratory syndrome cryo-em structure of the 2019-ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china encephalitis as a clinical manifestation of covid-19 detection of severe acute respiratory syndrome (sars)-associated coronavirus rna in autopsy tissues with in situ hybridization guillain-barrè syndrome associated with sars-cov-2 infecion: causality or coincidence? lancet neurol a novel coronavirus from patients with pneumonia in china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-277399-0w8is9xm authors: esteves, sandro c.; lombardo, francesco; garrido, nicolás; alvarez, juan; zini, armand; colpi, giovanni m.; kirkman‐brown, jackson; lewis, sheena e. m.; björndahl, lars; majzoub, ahmad; cho, chak‐lam; vendeira, pedro; hallak, jorge; amar, edouard; cocuzza, marcello; bento, fabiola c.; figueira, rita c.; sciorio, romualdo; laursen, rita j.; metwalley, ahmad m.; jindal, sunil k.; parekattil, sijo; ramasamy, ranjith; alviggi, carlo; humaidan, peter; yovich, john l.; agarwal, ashok title: sars‐cov‐2 pandemic and repercussions for male infertility patients: a proposal for the individualized provision of andrological services date: 2020-05-22 journal: andrology doi: 10.1111/andr.12809 sha: doc_id: 277399 cord_uid: 0w8is9xm the prolonged lockdown of health facilities providing non‐urgent gamete cryopreservation—as currently recommended by many reproductive medicine entities and regulatory authorities due to the sars‐cov‐2 pandemic will be detrimental for subgroups of male infertility patients. we believe the existing recommendations should be promptly modified and propose that the same permissive approach for sperm banking granted for men with cancer is expanded to other groups of vulnerable patients. these groups include infertility patients (eg, azoospermic and cryptozoospermic) undergoing medical or surgical treatment to improve sperm quantity and quality, as well as males of reproductive age affected by inflammatory and systemic auto‐immune diseases who are about to start treatment with gonadotoxic drugs or who are under remission. in both scenarios, the “fertility window” may be transitory; postponing diagnostic semen analysis and sperm banking in these men could compromise the prospects of biological parenthood. moreover, we provide recommendations on how to continue the provision of andrological services in a considered manner and a safe environment. our opinion is timely and relevant given the fact that fertility services are currently rated as of low priority in most countries. severe acute respiratory syndrome-coronavirus 2 (sars-cov-2) is a novel coronavirus and causative agent of covid-19, a disease with potentially dangerous implications for human health. the remarkable increase in the number of infections by sars-cov-2 worldwide raised the prospect of massive hospitalizations that few healthcare systems would be able to deal with. on this basis, governments across the globe have announced the most far-reaching restrictions on personal freedom in modern history. the urgent need to avoid a collapse in the healthcare system has been the justification for the implemented measures, and reproductive medicine societies, as well as regulatory authorities, decisively followed by issuing guidance based on expert best judgment. the key recommendations for practitioners include suspension of initiation of new fertility treatment and non-urgent gamete cryopreservation, as well as suspension of elective surgery and non-urgent diagnostic procedures. 1,2 sperm banking has been rated as of low priority, indicating that clinical harm is very unlikely if postponed for six months. 3 exceptions are oncological patients who require urgent fertility preservation. taking the above mentioned into account, we would like to raise a viewpoint hardly voiced so far. our concerns are that, first of all, a prolonged lockdown of andrological services will be detrimental to subgroups of male infertility patients. secondly, the andrological community is uneasy about how to provide optimal care to our patients without compromising safety. we, therefore, propose remedies to mitigate the consequences of a prolonged cessation of andrological services. the aim is to help authorities and healthcare providers identify which patients might be prioritized for the continuation of andrological services in a safe environment. at the time of writing (april 21), the global deaths caused by sars-cov-2 represent approximately one percent of total deaths expected to occur worldwide over the first three months of the current year, with a wide variation in the reported death rates per country (www.world omete rs.info/coron avirus). in total, more than 2.5 million infections by sars-cov-2 have been reported, 95% of which have been defined as mild. among the severe or critical cases, the overwhelming majority affects people aged 50 and above. by contrast, the reported death rate among individuals of reproductive age remains low, ranging from 0.2% in china to 0.8% in the united states, with an estimated 1.5:1 male to female ratio, mainly affecting those individuals with pre-existing conditions, including cardiovascular disease, diabetes, chronic respiratory disease, hypertension, obesity, and cancer. 4 while it is prudent to advocate temporary social distancing and closure of non-emergency health services, we do not know how long this pandemic will last. estimates ranging from 3 to 12 months have been projected, depending on how effective governments implement quarantine measures and how long it takes to achieve herd immunity. thus, we would like to consider what a prolonged lockdown of clinics providing andrological services might mean for infertility patients. this consideration will focus primarily on priority recommendations for sperm banking and diagnostic semen analysis for patients seeking fertility rather than donors. the "time" variable is crucial in specific subgroups of infertile males. besides reproductive-age oncological patients, loss of time is particularly consequential among patients under medical treatment aimed at improving sperm quantity or quality and in those with inflammatory or auto-immune diseases who will either start treatment-with potentially gonadotoxic drugs-or are under the "remission window" of such treatment, as explained in more detail below. in both scenarios, the "fertility window" may be transitory and, therefore, the implications of postponing diagnostic semen analysis and sperm banking in these men could permanently compromise the prospects of biological parenthood. hence, the provision of andrological services cannot be considered a low priority. our opinion is particularly important given the fact that healthcare providers are reluctant to recommend assisted conception in most cases-using either fresh or frozen-thawed spermatozoa-as pregnancy might act as a comorbidity in women affected by sars-cov-2. 5,6 up to 30% of male cancer survivors lose their fertility potential after anti-cancer therapy. 7 chemotherapy, radiotherapy, and radical surgical procedures might irreversibly impair spermatogenesis and/ or ejaculation. cancer itself can also affect fertility directly (eg, testicular cancer and hodgkin's lymphoma). 8 currently, the only reliable method of fertility preservation in reproductive-aged men with cancer is sperm banking. 9 sperm banking must be ideally completed before the start of gonadotoxic therapy. specimens are usually collected by masturbation and ejaculated spermatozoa are cryopreserved using slow or rapid freezing protocols. before cryopreservation, the specimen undergoes semen analysis, which is used to both assess the baseline sperm variables (eg, count, motility, and morphology) and to plan. after thawing, it is inevitable that sperm parameters are overall reduced, and such specimens have to be used with intrauterine insemination (iui) or assisted reproductive technology (art) to allow these patients to have biological children. 10 the costs associated with sperm banking are relatively low and most cancer patients who banked sperm were found to be pleased by having taken that decision. 11 the most vulnerable male infertility patient during the sars-cov-2 pandemic is probably the non-obstructive azoospermic (noa) or cryptozoospermic patient being medically treated to restore or improve spermatogenesis. an example is the patient with hypogonadotropic hypogonadism (hh), in whom azoospermia results from the lack of adequate testicular stimulation by pituitary gonadotropins. 12 in pre-pubertal and post-pubertal hh males, gonadotropin treatment increases testicular size, promotes virilization, and restores spermatogenesis (to varying degrees) in up to 90% of patients, with reported pregnancy rates-either by intercourse or with the aid of iui or art-of up to 65%. [12] [13] [14] however, the treatment duration is long-typically six months or longer-and expensive as well. moreover, the follow-up during treatment requires monitoring serum levels of pituitary and sexual hormones, as well as semen parameters. sperm banking should be considered in men with hh who respond to therapy, that is, have viable spermatozoa in the ejaculate, in particular, when the continuation of gonadotropin therapy during the sars-cov-2 pandemic is neither possible (eg, due to economic or logistic reasons), nor desired. in patients who have not responded yet and have economic constraints to continue therapy, the medication dose and regimen could be adjusted (eg, decrease hcg dose and suspend fsh injections) to keep intratesticular as well as serum testosterone levels within lower normal limits. another example refers to males with noa due to spermatogenic failure, including those with rare numbers of spermatozoa occasionally found in the ejaculate (cryptozoospermia), accounting for 60% of the azoospermia cases. 15 although the condition is untreatable, medical therapy has been explored as a way to optimize or induce spermatogenesis and, thus, increase the likelihood of having spermatozoa retrieved surgically or ejaculated. a few cohort studies have shown that spermatozoa can be occasionally found in the ejaculate after the use of medication for boosting intratesticular testosterone production, like hcg injections-alone or combined with fsh injections-, and estrogen receptor modulators, such as tamoxifen. [16] [17] [18] [19] similar to hh patients, the continuation of gonadotropin therapy is not always possible nor desired in men with noa due to spermatogenic failure who require sperm utilization or banking. moreover, immediate art might not be an option in some countries with strict lockdown measures during the current sars-cov-2 pandemic. thus, sperm banking is urged in noa patients who respond to medical therapy and present sperm in the ejaculate as a way to preserve fertility and allow future art. naturally, semen analyses are required to monitor treatment results and identify who is eligible for sperm banking. patients achieving cryptozoospermia or severe oligozoospermia after treatment may have a short window for sperm cryopreservation as their semen quality could deteriorate. 20 if the opportunity of sperm banking is lost, surgical sperm retrieval will be required, which could inflict both clinical and financial burdens on patients. nevertheless, sperm retrieval and cryopreservation of testicular spermatozoa should be considered in specific situations of persistent azoospermia when a narrow window of opportunity exists. sperm retrieval can be performed on an outpatient basis under local/intravenous anesthesia; the procedure is associated with minimal postoperative complications. 21 along the same lines, varicocelectomy has been used as an attempt to improve spermatogenesis in noa men with a coexistent varicocele. spermatogonia type b, pachytene spermatocytes, and early spermatids are vulnerable to heat stress associated with varicocele. 22 in a systematic review comprising 468 noa patients with varicocele, 44% of the treated patients had viable ejaculated sperm postoperatively, suitable for icsi or cryopreservation. 23 these patients should also be monitored with semen analyses, and sperm cryopreservation recommended for those with ejaculate spermatozoa due to the risk of relapse to azoospermia. 24 lastly, loss of fertility due to a late obstruction has been reported in up to 12% and 50% of men with obstructive azoospermia subjected to vasovasostomy and vasoepididymostomy, respectively. 25, 26 this situation can also occur after the transrectal resection of the ejaculatory ducts. in both scenarios, semen analysis is used to monitor the patency status postoperatively, and sperm banking should be offered to those patients who experience a continuous decrease in sperm count/quality during the follow-up as a way to avoid future sperm retrievals. 26, 27 infertile men of advanced paternal age (eg, >50 years) have occasionally used sperm banking for planning of medically assisted reproduction. 28, 29 given that advanced age is a risk factor for sars-cov-2 complications, and severe sars-cov-2 illness might be treated with non-specific anti-viral drugs with possible gonadotoxic effects, 30 sperm banking could be offered to those patients who are concerned about acquiring the infection. at present, the prevailing consensus is to allow gamete cryopreservation to continue for oncological patients. however, males at reproductive age affected by non-oncological conditions (ie, inflammatory bowel diseases and autoimmune disorders) may also need immediate sperm banking. 31 gonadotoxic drugs (eg, cyclophosphamide, methotrexate, mycophenolate mofetil, and mtor inhibitors) are commonly used to control the inflammatory process in such patients. inflammatory bowel disease (eg, crohn's disease and ulcerative colitis) mainly affects young adults, and drugs used for treatment (eg, sulfasalazine, azathioprine, and methotrexate) appear to harm sperm quality. 32 the sulfapyridine metabolite of sulfasalazine impairs semen parameters and increases the production of reactive oxygen species. 32,33 moreover, pregnancy-related complications and the risk of congenital abnormalities might increase when the father had used azathioprine before conception. [34] [35] [36] also, methotrexate (mtx) is an immunosuppressive agent used to treat inflammatory and auto-immune diseases with known teratogenic effects. besides, the antifolate mechanism of mtx decreases dna synthesis and inhibits cellular proliferation, possibly resulting in oligozoospermia. 37 likewise, young men may be affected by systemic autoimmune diseases (sads) (eg, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, ankylosing spondylitis, dermatomyositis, behçet disease, psoriasis, among others). 38 in these patients, the chronic inflammation could adversely affect the hypothalamic-pituitary-testicular axis and the testicles directly, causing impairment of sperm quality and quantity. however, gonadal dysfunction is primarily related to the effects of immunosuppressive therapy (eg, alkylating agents, methotrexate, and mycophenolate mofetil). 39 among patients with inflammatory bowel disease or sad considering fertility preservation, sperm banking is usually conditioned to a temporary discontinuation of therapy for at least 3-4 months. 35 several patients might have been planning for this "fertility window" for an extended time, which unfortunately occurred during the sars-cov-2 pandemic. sperm banking is, therefore, an option for these patients who are concerned about establishing a pregnancy during the sars-cov-2 pandemic, in particular, those with semen abnormalities who are candidates for art. on this basis, we would argue that the same permissive approach that has been granted for men with cancer to enable gamete preservation should be extended to male patients with inflammatory and autoimmune diseases. we need to consider the health and psychological consequences of not offering the above patients andrological services. the lockdown of andrological services may have a devastating psychological impact on men undergoing fertility-related treatment. like women, men undergoing fertility treatment may also experience anxiety and stress. 40, 41 this psychological distress can aggravate the feeling of fear and uncertainty imposed by the sars-cov-2 pandemic, 42 which might have negative consequences for the reproductive outcome. the damage to the affected patients is difficult to measure, and it will take months, perhaps years before we can assess the broader implications of the current restrictive measures for patients as well as healthcare providers. while we believe that the various lockdowns will slow the spread of sars-cov-2, a strict lockdown is unlikely to last too long due to its practicality and pitfalls on other aspects of society, mainly economical. thus, a certain level of risk of infections by sars-cov-2 is expected because there will be new cases when measures are relaxed, and no vaccine is likely to be available soon. therefore, not only urgent short-term responses, but also long-term measures are essential. hence, in this time of uncertainty, denying andrological services from those who need it most might be even worse than the risks of providing them. we, therefore, propose some remedies that we believe might offer fertility providers and patients alike greater autonomy, and that could be used to alleviate the adverse impact of the coronavirus pandemic in the months to come (figure 1 ). • before any service is provided, active sars-cov-2 infections and suspected cases should be excluded. testing patients with the use of polymerase chain reaction (pcr) and/or blood antibody testing is recommended before starting sperm banking. ideally, only samples from patients with negative results or who have acquired herd immunity should be cryopreserved. • andrological services (eg, diagnostic semen analysis and sperm cryopreservation) should not only be available for oncological patients, but also for the group of patients listed below. a. patients with severe male factor infertility under medical or surgical treatment aiming at improving sperm quantity or quality (eg, patients with noa or cryptozoospermia/severe oligozoospermia, including post-varicocele repair, and those with evidence of loss of patency after successful surgical reconstruction of the reproductive tract). b. men at reproductive age affected by inflammatory diseases or sads, that is, before initiation of gonadotoxic therapy or if under the "fertility window" achieved after temporary (at least three months) discontinuation of therapy. during the coming weeks, we should continue to look critically and objectively at the sars-cov-2 evidence. although our recommendations are unlikely to create any further burden to the already overwhelmed medical infrastructure, we acknowledge that patients might be reluctant to use andrological services on the basis of fear of being infected or economic reasons. we also realize that much is unknown about sars-cov-2 and its implication on male reproductive health. the existing data indicate that a subject can be infectious 3-5 days before the onset of actual symptoms of the viral infection, and the risk of such cases spreading the infection has not been rigorously researched. 46 while testing patients and staff with the use of pcr and/or antibody kits is recommended, the majority of clinics lack prompt access to these tests. moreover, some countries face a short supply of test kits, which have been made available for symptomatic patients and frontline health providers only. besides, the accuracy of these tests has been questioned, with some reports suggesting that many of the sars-cov-2 kits in the market have a false-negative rate of 30%-40%. 47 thus, it remains to be determined how clinics can screen patients and healthcare providers optimally. likewise, it remains to be decided who-patients or clinics-will assimilate the costs related to testing, ppe, and reduced patient volume due to extra measures instituted to avoid infections. along these lines, clinics and hospitals providing andrological services have to determine ways of protecting themselves from potential liability issues. although the overall mortality rate among men at reproductive age remains low, it should be considered that contamination of patients and staff could occur with sars-cov-2 in the context of asymptomatic shedding. for this reason, it seems sound to advise postponing medical therapy in azoospermic men who had planned to initiate it and who have no pressing concerns (eg, no maternal factors such as advanced maternal age) until it is deemed safe to obtain regular semen analyses, hormone profiles, and banking of spermatozoa. the same reasoning applies to semen analysis and sperm banking in men under therapy who opt to continue on medication till the pandemic ends. at present, limited data exist about potential routes of sars-cov-2 infection in respiratory, cardiovascular, digestive, urinary, and reproductive systems. in this regard, data of virus load in semen or testicular biopsies of sars-cov-2 infected patients is minimal. [48] [49] [50] nevertheless, angiotensin-converting enzyme 2 (ace2) receptors, used by the virus to enter host cells, exist in spermatogonia, sertoli cells, and leydig cells. 51, 52 also, previous reports suggested that other coronaviruses, like the sars coronavirus, could cause orchitis. 53 as for pregnancy with the use of banked or fresh ejaculate spermatozoa during the sars-cov-2 pandemic, it has been suggested that pregnant women might be at a higher risk of developing complications, including miscarriage, preeclampsia, and preterm birth. 5, 6 however, the evidence is still limited, and we, therefore, abstain from making recommendations about the use of fresh or banked spermatozoa for assisted conception during the pandemic until more data are available. naturally, the use of spermatozoa for assisted conception-either fresh or frozen-thawed-would not be recommended in most cases if it is confirmed that pregnancy acts as an important comorbidity factor. notwithstanding these observations, it should be acknowledged that serology testing, once properly validated and widely available, will be helpful to identify immune patients that could be allowed for treatment. 54 these patients have little risk of either pregnancy complications or propagating the disease when attending fertility clinics. nevertheless, the provision of andrological services should only be undertaken if the medical infrastructure can support them. we reiterate the above recommendations that care should only be restarted if social distancing can be maintained, areas regularly disinfected, and screening for signs and symptoms of the infection undertaken before allowing patients into the facility in accordance with guidance issued by health regulatory authorities. we propose remedies to mitigate the consequences of a prolonged cessation of andrological services due to the sars-cov-2 pandemic to vulnerable subgroups of male infertility patients. in a moment when the reorganization of healthcare services is focused on supporting sars-cov-2 patients who might need critical care, limiting burdens for national health systems could still represent a relevant issue. we advocate that correct identification of the more "time-sensitive" cases is crucial for regulating the continuation of andrological services, including diagnostic semen analysis and sperm banking. moreover, we provide recommendations on how to most optimally provide care to our patients-without compromising safety-once andrological services are resumed. the aim is to help authorities and healthcare providers identify which patients might be prioritized during the sars-cov-2 pandemic for the continuation of andrological services in a safe environment. none. american society for reproductive medicine patient management and clinical recommendations during the coronavirus coronavirus covid-19: eshre statement on pregnancy and conception guidelines office rapid reaction group: an organization-wide collaborative effort to adapt the eau guidelines recommendations to the covid era covid-10 coronavirus pandemic clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records sperm banking for fertility preservation: a 20-year experience genitourinary cancer patients have worse baseline semen parameters than healthy sperm bankers contemporary and future insights into fertility preservation in male cancer patients sperm banking and assisted reproductive outcome in men with cancer: a 10 years' experience sperm banking and the cancer patient hypogonadotropic hypogonadism revisited application of hormonal treatment in hypogonadotropic hypogonadism: more than ten years experience clinical use of fsh in male infertility clinical management of infertile men with nonobstructive azoospermia beneficial effect of tamoxifen on sperm recovery in infertile men with nonobstructive azoospermia the effect of human chorionic gonadotropin-based hormonal therapy on intratesticular testosterone levels and spermatogonial dna synthesis in men with non-obstructive azoospermia optimization of spermatogenesis-regulating hormones in patients with non-obstructive azoospermia and its impact on sperm retrieval: a multicentre study hormonal stimulation of spermatogenesis: a new way to treat the infertile male with non-obstructive azoospermia? optimal management of extreme oligozoospermia by an appropriate cryopreservation programme methods of surgical sperm extraction and implications for assisted reproductive technology success insight into oxidative stress in varicocele-associated male infertility: part 1 outcome of varicocele repair in men with nonobstructive azoospermia: systematic review and meta-analysis induction of spermatogenesis in azoospermic men after varicocelectomy repair: an update partial obstruction, not antisperm antibodies, causing infertility after vasovasostomy the kinetics of sperm return and late failure following vasovasostomy or vasoepididymostomy: a systematic review surgical treatment of male infertility in the era of intracytoplasmic sperm injection -new insights management and counseling of the male with advanced paternal age paternal age and assisted reproductive technology: problem solver or trouble maker antivirals and male reproduction immunosuppressive therapy and fertility preservation: indications and methods inflammatory bowel disease in subfertile men and the effect of mesalazine on fertility sulfasalazine induced oxidative stress: a possible mechanism of male infertility outcome of pregnancies when fathers are treated with 6-mercaptopurine for inflammatory bowel disease inflammatory bowel diseases and human reproduction: a comprehensive evidence-based review the risk of congenital abnormalities in children fathered by men treated with azathioprine or mercaptopurine before conception review article: the safety of therapeutic drugs in male inflammatory bowel disease patients wishing to conceive increased risk of autoimmune disorders in infertile men: analysis of us claims data male fertility potential alteration in rheumatic diseases: a systematic review anxiety and sexual stress in men and women undergoing infertility treatment infertility-related stress in men and women predicts treatment outcome 1 year later immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china implementation of air quality control in reproductive laboratories in full compliance with the brazilian cells and germinative tissue directive quality management in art clinics: a practical guide what is a clean room? in: esteves sc, varghese a, worrilow kc eds. clean room technology in art clinics: a practical guide, 1st edn sars-cov-2 and covid-19: the most important research questions covid-19 testing: the threat of false-negative results detection of 2019 novel coronavirus in semen and testicular biopsy specimen of covid-19 patients no evidence of sars-cov-2 in semen of males recovering from covid-1. fertil steril. ahead of print study of sars-cov-2 in semen and urine samples of a volunteer with positive naso-pharyngeal swab angiotensin-converting enzymes play a dominant role in fertility scrna-seq profiling of human testes reveals the presence of the ace2 receptor, a target for sars-cov-2 infection in spermatogonia, leydig and sertoli cells orchitis: a complication of severe acute respiratory syndrome (sars) developing antibody tests for sars-cov-2 sars-cov-2 pandemic and repercussions for male infertility patients: a proposal for the individualized provision of andrological services key: cord-252600-bvh1o64r authors: galasiti kankanamalage, anushka c.; kim, yunjeong; damalanka, vishnu c.; rathnayake, athri d.; fehr, anthony r.; mehzabeen, nurjahan; battaile, kevin p.; lovell, scott; lushington, gerald h.; perlman, stanley; chang, kyeong-ok; groutas, william c. title: structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element date: 2018-04-25 journal: european journal of medicinal chemistry doi: 10.1016/j.ejmech.2018.03.004 sha: doc_id: 252600 cord_uid: bvh1o64r abstract there are currently no approved vaccines or small molecule therapeutics available for the prophylaxis or treatment of middle east respiratory syndrome coronavirus (mers-cov) infections. mers-cov 3cl protease is essential for viral replication; consequently, it is an attractive target that provides a potentially effective means of developing small molecule therapeutics for combatting mers-cov. we describe herein the structure-guided design and evaluation of a novel class of inhibitors of mers-cov 3cl protease that embody a piperidine moiety as a design element that is well-suited to exploiting favorable subsite binding interactions to attain optimal pharmacological activity and pk properties. the mechanism of action of the compounds and the structural determinants associated with binding were illuminated using x-ray crystallography. proteins, s (spike glycoprotein), e (envelope protein), m (membrane glycoprotein), and n (nucleocapsid protein), which play a critical role in virion-cell receptor binding, replication and virion assembly, are located at the 3 0 end of the genome [1, 12] . coronavirus entry is initiated by the binding of the spike protein (s) to cell receptors, specifically, dipeptidyl peptidase 4 (ddp4) and angiotensin converting enzyme 2 (ace2) for mers-cov and sars-cov, respectively [1e5] . entry into cells requires host proteases for cleavage at two sites in the s protein, in the case of most cov [13, 14] translation of the genomic mrna of orf1a yields polyprotein pp1a, while a second polyprotein (pp1b) is the product of a ribosomal frame shift that joins orf1a together with orf1b. orf1a encodes a papain-like cysteine protease (plpro) and a 3c-like cysteine protease (3clpro). polyproteins pp1a and pp1b are processed by 3clpro (11 cleavage sites) and plpro (3 cleavage sites) resulting in sixteen mature nonstructural proteins, including rna-dependent rna polymerase (rdrp) and helicase, which play important roles in the transcription and replication of coronaviruses (fig. 1 ). both proteases are essential for viral replication, making them attractive targets for drug development [9,10,15e17] . mers-cov 3clpro is a chymotrypsin-like cysteine protease having a catalytic cys148-his41 dyad and an extended binding site [18e21] . the protease displays a stringent primary substrate specificity for a p 1 gln residue [22] and has a strong preference for a p 2 leu residue. the p 3 residue side chain is oriented toward the solvent while the s 4 subsite is shallow, preferring a small hydrophobic p 4 residue (ala). functional and structural studies have delineated the similarities between the 3clpro of coronaviruses that can be exploited in the design of broad-spectrum inhibitors [23] . we have recently reported the first demonstration of clinical efficacy of a coronavirus protease inhibitor (a dipeptidyl aldehyde bisulfite adduct inhibitor designated gc376) [24, 25] . specifically, administration of gc376 to cats infected with fipv, a coronavirus that is 100% fatal in cats, reversed the progression of fatal fip and resulted in clinical remission in a majority of animals (>90%). since fip disease progression is quite rapid and its pathogenesis primarily immune-mediated, features shared by mers-cov, we hypothesized that a viral protease inhibitor could reverse the pathogenesis of mers-cov in affected hosts. interrogation of this hypothesis entailed, as a first step, the design of a new and versatile class of peptidomimetic inhibitors of mers-cov 3cl protease. we describe herein the structure-guided design of inhibitors of mers-cov 3clpro that embody a piperidine moiety as a novel design element, as well as pertinent structural and biochemical studies. these inhibitors were also examined against other coronaviruses, including sars-cov, fipv and mhv to evaluate the spectrum of activity against multiple coronaviruses. the structure-guided design of inhibitor (i) encompassed the following steps: (a) we first determined a high resolution x-ray crystal structure of mers-cov 3clpro in complex with gc376 ( fig. 2/panel a) . examination of the active site of the complex revealed that the aldehyde bisulfite adduct had reverted to the precursor peptidyl aldehyde, which subsequently formed a tetrahedral hemi-thioacetal upon reaction with the active site cys148. notably, the electron density at this stereocenter was consistent with the formation of both r and s enantiomers at the covalent binding site (also observed for the other structures described in the following sections). the structure reveals a network of backbone hydrogen bonds which ensure correct positioning of the inhibitor to the active site, as well as two critical hydrogen bonds with the p 1 gln surrogate [26] side chain. the inhibitor p 2 leu side chain is ensconced in the hydrophobic s 2 subsite of the enzyme. importantly, the structure shows a hydrophobic-driven interaction between the benzyl group of the inhibitor and the g-lactam ring of the gln surrogate side chain; (b) based on the forgoing, we reasoned that extending the "cap" would allow the inhibitor to assume an extended conformation and orient the phenyl ring toward the hydrophobic s 4 pocket of the enzyme. validation of this idea was obtained by synthesizing extended inhibitor gc813 and determining a high resolution x-ray crystal structure of the mers-cov 3clpro:gc813 complex ( fig. 2/panel b) . the m-cl phenethyl side chain is clearly shown to occupy the hydrophobic s 4 subsite. in addition to an array of h-bonds with gln192, glu169, and gln167 and the backbone of the inhibitor, which serve to correctly position the inhibitor at the active site, the inhibitor interacts with the s 1 , s 2 and s 4 subsites, but not the s 3 subsite; (c) we hypothesized that the attachment of a piperidine ring to the peptidyl component would yield a structurally novel peptidomimetic (i) capable of (1) orienting recognition elements r 3 and r 4 in a correct vector relationship for optimal interactions with the s 3 and s 4 subsites, (2) rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor with potentially diminished pk liabilities and, (3) providing a flexible means for the structure-guided parallel optimization of admet/pk and physicochemical properties using diversity sites r 3 and r 4 in inhibitor (i) (fig. 3) . in summary, the piperidine-based design strategy is a hitherto unrecognized effective means of rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor capable of engaging in optimal binding interactions with all four s 1 -s 4 subsites but which, however, is anticipated to display diminished pk liabilities due to its reduced peptidyl character. furthermore, the aforementioned piperidine-based design strategy has wide applicability and can be extended to any protease with an extended binding site. preliminary evidence in support of this approach is provided by the results of enzyme and cell-based screening of derivatives of (i) (tables 1 and 2) , as well as the results of structural studies (vide infra) (see table 3 ). the synthesis of final compounds 9(a-f) and 10(a-f) is outlined in scheme 1. 1-boc-4-piperidinone was reacted with different grignard reagents to yield the corresponding 1-boc-4-piperidinol derivatives (1c and 1e). refluxing (l) leucine methyl ester hydrochloride with trichloromethyl chloroformate yielded the isocyanate which was reacted with (1c and 1e, or commercially-available nsubstituted 4-piperidinol 1a) to form the corresponding carbamate adducts (4a, 4c and 4e) that were hydrolyzed to the corresponding acids (5a, 5c and 5e) with lithium hydroxide in aqueous thf. subsequent coupling with glutamine surrogate methyl ester hydrochloride 11 afforded the desired dipeptidyl esters (6a, 6c and 6e) which were either treated with lithium borohydride directly or were first treated with dry hcl in dioxane followed by reaction with an alkyl sulfonyl chloride or alkyl chloroformate, to yield esters (7b, 7d and 7f) prior to reduction with lithium borohydride, to yield alcohols 8 (a-f). dess-martin oxidation, followed by flash chromatography, yielded pure aldehydes 9(a-f). the enantiomeric purity of the aldehydes was consistently high, with the amount of epimerized aldehyde ranging between 0 and 10%. the corresponding bisulfite adducts 10(a-f) were readily obtained as white solids by stirring the aldehydes with sodium bisulfite in an ethyl acetate/ water mixture. the synthesized compounds are listed in table 1 . the inhibitory activity of the synthesized compounds against 3clpro of mers-cov, sars-cov or fipv, and the antiviral activity of two representative compounds (compounds 10a and 10c) in a cellbased system including mers-cov, fipv and mhv were evaluated as described in the experimental section. the ic 50, ec 50, and cc 50 values, are listed in tables 1 and 2 these are the average of at least two determinations. it is evident that derivatives of (i) function as highly potent inhibitors of all tested coronaviruses in enzyme (table 1) and cell based assays (table 2 and fig. 4 ). more importantly, representative aldehyde bisulfite adduct compounds 10a and 10c display potent inhibition toward mers-cov in both enzyme and cell-based systems, with low cytotoxicity (cc 50 > 100 mm) ( table 2 and fig. 4 ). for example, compound 10a has a selectivity index (si ¼ cc 50 /ec 50 ) of >250. with the exception of compounds 9e-10e, the aldehyde and aldehyde bisulfite adducts were found to have comparable in vitro potency toward mers-cov 3clpro. furthermore, pharmacological activity was found to be dependent on the nature of the r 3 group (compounds 9e-10e are 10-fold less active toward mers-cov 3clpro than compounds 9a-d, 10a-d and 9f-10f). in order to establish the mechanism of action of (i), as well as obtain structural information that can be used to guide the optimization of pharmacological activity, the high resolution x-ray crystal structures of several derivatives of (i) bound to mers-cov 3clpro were determined, including the cocrystal structure of the mers-cov 3clpro:inhibitor 10c complex (fig. 5a) . the formation of a tetrahedral adduct via the reaction of the aldehyde, generated from aldehyde bisulfite adduct 10c under the crystallization conditions used [27, 28] , with the active site cysteine (cys148) is clearly evident, confirming the mechanism of action of (i). inspection of the structure reveals the presence of prominent electron density consistent with the structure of inhibitor 10c; however, the n-bocpiperidinyl moiety was disordered. the position and orientation of the benzyl group suggest that the piperidine ring is likely projecting toward the s 4 subsite. inhibitor 10c is bound to the active site of the enzyme via a network of backbone h-bonds with gln192, gln167, and glu169 (fig. 5b ). additionally, a h-bond with his41 serves to stabilize the hemi-thioacetal tetrahedral adduct. also clearly evident are three critical h-bonds involving the p 1 gln surrogate ring oxygen and nitrogen with glu169, his166 and phe143. the h-bonding interactions are near identical to those of inhibitor gc813 (fig. 2/panel b) . the structural complementarity of inhibitors 10c and gc813 is also evident in the electrostatic surface representation of the enzyme with the two inhibitors nestled in the active site (fig. 6) . the cocrystal structure of the mers-cov 3clpro:aldehyde bisulfite adduct 10e complex also showed that, under the crystallization conditions used, the aldehyde bisulfite adduct reverted to the precursor aldehyde, which subsequently formed a tetrahedral adduct with the active site cysteine (cys148) (fig. 7a ). the piperidinyl moiety was disordered and consequently its precise location could not be discerned. however, inhibitor 10e is engaged in the same h-bonding interactions as inhibitor 10c (fig. 7b ). mers-cov constitutes a global public health concern. there are currently no licensed vaccines or antiviral drugs for the prevention and treatment of coronavirus infections. we disclose herein for the first time the design and utilization of a general class of piperidinebased peptidomimetic inhibitors of coronavirus 3cl proteases. attachment of the piperidine moiety to a dipeptidyl component permits the resultant hybrid inhibitor to engage in favorable binding interactions with the s 3 and s 4 subsites of the enzyme. more importantly, the approach disclosed herein can be extended to other proteases of medical relevance. finally, the disclosed compounds potently inhibit mers-cov, and their mechanism of action and mode of binding to mers-cov 3cl protease have been illuminated using x-ray crystallography. reagents and dry solvents were purchased from various chemical suppliers (aldrich, acros organics, chem-impex, tci america, and bachem) and were used as obtained. silica gel (230e450 mesh) used for flash chromatography was purchased from sorbent technologies (atlanta, ga). thin layer chromatography was performed using analtech silica gel plates. visualization was accomplished dropwise under a n 2 atmosphere to a solution of 1-boc-4piperidinone (10 mmol) in dry thf (15 ml) in an ice bath kept at 0 c. the reaction mixture was stirred for 3 h at room temperature under a n 2 atmosphere while monitoring completion of the reaction by tlc. the reaction mixture was diluted with water (25 ml) and the solution was acidified to ph~3 using 5% hydrochloric acid. the solvent was removed on the rotary evaporator and the residue was extracted with ethyl acetate (75 ml) and the layers separated. the organic layer then washed with brine (40 ml) and dried over anhydrous sodium sulfate, filtered and concentrated to yield a colorless oily product which was purified by flash chromatography to yield 1c and 1e. 4.1.2. synthesis of (l) leucine methyl ester isocyanate 3 (l) leucine methyl ester hydrochloride 2 (100 mmol) was placed in a dry 500-ml rb flask and then dried overnight on the vacuum pump. the flask was flushed with nitrogen and dry dioxane (200 ml) was added followed by trichloromethyl chloroformate (29.67 g, 150 mmol), and the reaction mixture was refluxed for 10 h. the solvent was removed on the rotary evaporator and the residue was vacuum distilled to yield pure isocyanate 3 as a colorless oil [27, 28] . table 3 crystallographic data for mers-cov 3clpro in complex with compounds gc376, gc813, 10c and 10e. mers-cov 3clpro: gc376 mers-cov 3clpro: compound 10c mers-cov 3clpro: compound 10e hkl. c r factor ¼ ʃ hkl jjf obs (hkl) j -jf calc (hkl) jj/ʃ hkl jf obs (hkl)j; rfree is calculated in an identical manner using 5% of randomly selected reflections that were not included in the refinement. d r meas ¼ redundancy-independent (multiplicity-weighted) r merge. [42, 43] r pim ¼ precision-indicating (multiplicity-weighted) r merge. [44, 45] . e cc 1/2 is the correlation coefficient of the mean intensities between two random half-sets of data [46, 47] . 4.1.3. synthesis of substituted piperidine-derived carbamates 4a, 4c and 4e. general procedure a solution of substituted or unsubstituted 1-boc-4-piperidinol (1c, 1e or 1a) (20 mmol) in dry acetonitrile (15 ml) was treated with triethylamine (4.05 g, 40 mmol) followed by the amino acid methyl ester isocyanate 3 (20 mmol). the resulting solution was refluxed for 2 h and then allowed to cool to room temperature. the solution was concentrated and the residue was taken up in ethyl acetate (75 ml). the organic layer was washed with 5% hcl (2 â 20 ml) and brine (20 ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, leaving compounds 4a, 4c and 4e as colorless oils. 4.1.4. synthesis of acids 5a, 5c and 5e. general procedure a solution of ester (4a, 4c or 4e) (20 mmol) in tetrahydrofuran (30 ml) was treated with 1 m lioh (40 ml). the reaction mixture was stirred for 3 h at room temperature and the disappearance of the ester was monitored by tlc. most of the solvent was evaporated off and the residue was diluted with water (25 ml). the solution was acidified to ph~3 using 5% hydrochloride acid (20 ml) and the aqueous layer was extracted with ethyl acetate (3 â 100 ml). the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to yield the corresponding compounds 5a, 5c and 5e as colorless oils. 4.1.5. synthesis of compounds 6a, 6c and 6e. general procedure edci (2.40 g, 12.5 mmol, 1.25 eq) and hobt (1.92 g, 12.5 mmol, 1.25 eq) were added to a solution of compound (5a, 5c or 5e) (10 mmol) in dry dmf (20 ml) and the mixture was stirred for 30 min at room temperature. in a separate flask, a solution of deprotected glutamine surrogate 11 (2.23 g, 10 mmol) in dmf (15 ml) cooled to 0e5 c was treated with diisopropylethylamine (diea) (9.5 g, 40 mmol, 4 eq), stirred for 30 min, and then added to the reaction mixture containing the acid. the reaction mixture was stirred for 12 h while monitoring the reaction by tlc. the solvent was removed and the residue was partitioned between ethyl acetate (100 ml) and 10% citric acid (2 â 40 ml). the layers were separated and the ethyl acetate layer was further washed with saturated aqueous nahco 3 (40 ml), followed by saturated nacl (50 ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield a yellow-colored oily product. purification by flash chromatography yielded esters 6a, 6c and 6e as white solids. 4.1.6. synthesis of compounds 7b, 7d and 7f. general procedure 4 m hcl in dioxane (8 ml) was added to a solution of compound (6a, 6c and 6e) (10 mmol) in dry dcm (5 ml) and the mixture was stirred for 1 h at room temperature. the solvent was removed and the residue was dried under high vacuum for 2 h before the product was dissolved in dry thf (20 ml). an appropriate alkyl sulfonyl chloride or alkyl chloroformate derivative (11 mmol/1.1 eq) was added to the solution with stirring. the reaction mixture was stirred for 12 h at room temperature and the residue was dissolved in ethyl acetate (50 ml) and washed with 5% hcl (2 â 20 ml). the ethyl acetate layer was further washed with saturated nacl (20 ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield a crude product. purification by flash chromatography yielded the corresponding esters 7b, 7d and 7f as white solids. lithium borohydride (2 m in thf, 7.5 ml, 15 mmol) was added dropwise to a solution of ester (6 or 7) (5 mmol) in anhydrous thf (30 ml), followed by absolute ethyl alcohol (15 ml) and the reaction mixture was stirred at room temperature overnight. the reaction mixture was then acidified by adding 5% hcl and the ph adjusted tõ 2. removal of the solvent left a residue which was taken up in ethyl acetate (100 ml). the organic layer was washed with brine (25 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to yield compounds 8 (a-f) as white solids. compound 8 (a-f) (5 mmol) was dissolved in anhydrous dichloromethane (50 ml) under a nitrogen atmosphere and cooled to 0 c. dess-martin periodinane reagent (3.18 g, 7.5 mmol, 1.5 eq) was added to the reaction mixture with stirring. the ice bath was removed and the reaction mixture was stirred at room temperature for 3 h (monitoring by tlc indicated complete disappearance of the starting material). a solution of 10% aqueous sodium thiosulfate (20 ml) was added and the solution was stirred for another 15 min. the aqueous layer was removed and the organic layer was washed with 10% aqueous sodium thiosulfate (20 ml), followed by saturated aqueous sodium bicarbonate (2 â 20 ml), water (2 â 20 ml) and brine (20 ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. the yellow residue was purified by flash chromatography (silica gel/methylene chloride/ ethyl acetate/methanol) to yield a white solid 9 (a-f). absolute ethanol (12 ml) was added to a solution of aldehyde 9 (a-f) (5 mmol) in dry ethyl acetate (20 ml) with stirring, followed by a solution of sodium bisulfite (540 mg; 5 mmol) in water (5 ml) and the reaction mixture was stirred for 3 h at 50 c. the reaction mixture was allowed to cool to room temperature and then vacuum filtered. the solid was thoroughly washed with absolute ethanol and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to yield a yellowish oil. the oily product was treated with ethyl ether (2 â 50 ml) to form a white solid. the white solid was stirred with ethyl ether (30 ml) and ethyl acetate (15 ml) for 5 min. careful removal of the solvent using a pipette left the corresponding aldehyde bisulfite adducts 10 (a-f) as white solids. the fret protease assay was performed by preparing stock solutions of the substrate (dabcyl-ktsavlq/sgfrkme-edans derived from the cleavage sites on the viral polyproteins of sars-cov) and inhibitor in dmso and diluting into assay buffer which was comprised of 20 mm hepes buffer, ph 8, containing nacl (200 mm), edta (0.4 mm), glycerol (60%), and 6 mm dithiothreitol (dtt). the expression and purification of the 3clpro of mers-cov, sars-cov or fipv was performed by a standard method described previously by our lab [24, 29] . the protease (3clpro of mers-cov, sars-cov or fipv) was mixed with serial dilutions of each compound or with dmso in 25 ml of assay buffer and incubated at 37 c for 30 min, followed by the addition of 25 ml of assay buffer containing substrate. fluorescence readings were obtained using an excitation wavelength of 360 nm and an emission wavelength of 460 nm on a fluorescence microplate reader (flx800; biotec, winoosk, vt) 1 h following the addition of substrate. relative fluorescence units (rfu) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously [29] . the dosedependent fret inhibition curves were fitted with a variable slope by using graphpad prism software (graphpad, la jolla, ca) in order to determine the ic 50 values of the inhibitors. the effects of compounds 10a and 10c on the replication of mers-cov, fipv or mhv-a59 were examined in vero81, crfk or ccl9.1 cells, respectively [30] . briefly, confluent and semi-confluent cells were infected at an moi of 0.01 pfu/cell. following adsorption, cells were incubated with medium containing dmso (<0.1%) or each compound (up to 100 mm) for 48 h. after incubation, viral titers were determined with a tcid 50 (fipv or mhv) or plaque assay (mers-cov). ec 50 values were determined using graphpadprism software [31]. the cytotoxic dose for 50% cell death (cc 50 ) for compounds 10a and 10c was determined in vero81, crfk or ccl9.1 cells. confluent cells grown in 96-well plates were treated with various concentrations (1e100 mm) of each compound for 72 h. cell cytotoxicity was measured by a cytotox 96 nonradioactive cytotoxicity assay kit (promega, madison, wi). the in vitro therapeutic index was calculated by dividing the cc 50 by the ec 50. purified mers-cov 3clpro, in 100 mm nacl, 20 mm tris ph 8.0, was concentrated to 8 mg/ml (0.5 mm). stock solutions of 100 mm gc376, gc813, compound 10c or compound 10e were prepared in dmso and the complex with mers 3clpro was prepared by mixing the concentrated protein supplemented with 3 mm compound and incubating overnight at 4 c. all crystallization experiments were conducted using compact 300 (rigaku reagents) sitting drop vapor diffusion plates at 20 c using equal volumes of protein and crystallization solution equilibrated against 75 ml of the latter. crystals of mers 3clpro in complex with gc813, compound 10c and compound 10e that displayed a prismatic morphology were obtained from the index ht screen (hampton research) condition g10 (25% (w/v) peg 3350, 100 mm bis-tris ph 5.5, 200 mm mgcl 2 ) in 1e2 days. crystals of the gc376 complex were obtained from the index ht screen (hampton research) condition e6 (30% (v/v) peg 550 mme, 100 mm bis-tris ph 6.5, 50 mm cacl 2 ). samples were transferred to a fresh drop containing 80% crystallant and 20% (v/v) peg 200 before storing in liquid nitrogen. x-ray diffraction data were collected at the advanced photon source beamline 17-id using a dectris pilatus 6 m pixel array detector. intensities were integrated using xds [32, 33] using autoproc [34] and the laue class analysis and data scaling were performed with aimless [35] , which suggested that the highest probability laue class was 2/m and space group c2. structure solution was conducted by molecular replacement with phaser [36] using a previously determined isomorphous structure of mers 3clpro (pdb: 4rsp [37] ) as the search model. structure refinement and manual model building were conducted with phenix [38] and coot [39] , respectively. disordered side chains were truncated to the point for which electron density could be observed. structure validation was conducted with molprobity [40] and figures were prepared using the ccp4mg package [41] . coordinates and structure factors for the mers 3clpro inhibitor complexes were deposited to the worldwide protein data bank (wwpdb) with the accession codes: 5wkj (gc376), 5wkk (gc813), 5wkl (inhibitor 10c) and 5wkm (inhibitor 10e). coronaviridae in field's virology coronaviruses: important emerging human pathogens sars and mers: recent insights into emerging coronaviruses update on human rhinovirus and coronavirus infections middle east respiratory syndrome and severe acute respiratory syndrome pathogenesis of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease middle east respiratory syndrome coronaviruses: drug discovery and therapeutic options antiviral drugs specific for coronaviruses in preclinical development mers-cov vaccine candidates in development: the current landscape role of the spike glycoprotein of human middle east syndrome coronavirus (mers-cov) in virus and syncytia formation middle east respiratory syndrome infection mediated by the transmembrane serine protease tmprss2 host cell entry of middle east respiratory distress syndrome coronavirus after two-step, furin-mediated activation of the spike protein an overview of severe acute respiratory distress syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy the sars-coronavirus papainlike protease: structure, function and inhibition by designed antiviral compounds identification, synthesis, and evaluation of sars-cov and sars-cov 3c-like protease inhibitors structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus inhibitor recognition specificity for mers-cov papain-like protease may differ from that of sars-cov 157e162 where s 1 , s 2 , s 3 , …. s n and s 1 ', s 2 ', s 3 ', …. s n ' correspond to the enzyme subsites on the n-terminus and c-terminus side, respectively, of the scissile bond structure of main protease from human coronavirus nl63: insights for wide spectrum anti-coronavirus drug design reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor efficacy of a 3c-like protease inhibitor in treating various forms of acquired feline infectious peritonitis tripeptide aldehyde inhibitors of human rhinovirus 3c protease: design, synthesis, biological evaluation, and cocrystal structure solution of p1 glutamine isosteric replacements structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3cl protease. structure-activity relationships and biochemical, x-ray crystallographic, cell-based, and in vivo studies inhibition of norovirus 3cl protease by bisulfite adducts of transition state inhibitors broad-spectrum antivirals against 3c or 3c-like proteases of picornaviruses, noroviruses and coronaviruses alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sarscoronavirus infection in a mouse model automatic indexing of rotation diffraction patterns data processing and analysis with the autoproc toolbox an introduction to data reduction: space-group determination, scaling and intensity statistics phaser crystallographic software ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot molprobity: all-atom structure validation for macromolecular crystallography developments in the ccp4 molecular graphics project scaling and assessment of data quality improved r-factors for diffraction data analysis in macromolecular crystallography global indicators of x-ray data quality linking crystallographic model and data quality resolving some old problems in protein crystallography towards automated crystallographic structure refinement with phenix.refine supplementary data related to this article can be found at https://doi.org/10.1016/j.ejmech.2018.03.004. key: cord-270116-r2rnnsfh authors: lippi, giuseppe; mattiuzzi, camilla; bovo, chiara; plebani, mario title: current laboratory diagnostics of coronavirus disease 2019 (covid-19) date: 2020-05-11 journal: acta biomed doi: 10.23750/abm.v91i2.9548 sha: doc_id: 270116 cord_uid: r2rnnsfh laboratory medicine provides an almost irreplaceable contribution to the diagnostic reasoning and managed care of most human pathologies. the novel coronavirus disease 2019 (covid-19) is not an exception to this paradigm. although the relatively recent emergence does not allow to draw definitive conclusions on severe acute respiratory syndrome coronavirus 2 (sars-cov-2) diagnostics, some standpoints can be conveyed. first and foremost, it seems now clear that we will be living together with this virus for quite a long time, so that our vigilance and responsiveness against the emergence of new local outbreaks shall be maintained at the highest possible levels. the etiological diagnosis of covid-19 is, and will remain for the foreseeable future, deeply based on direct identification of viral rna by means of molecular biology techniques in biological materials, especially upper and lower respiratory tract specimens. whether other materials, such as blood, urine, stools, saliva and throat washing, will become valid alternatives has not been unequivocally defined so far. as concerns serological testing, promising information can be garnered from preliminary investigations, showing that the vast majority of covid-19 patients seem to develop a sustained immune response against the virus, characterized especially by emergence of anti-sars-cov-2 igg and iga, 1 to 2 weeks after the onset of fever and/or respiratory symptoms. whether these antibodies will have persistent neutralizing activity against the virus is still to be elucidated on individual and general basis. the availability of rapid tests for detecting either viral antigens or anti-sars-cov-2 antibodies are a potentially viable opportunity for purposes of epidemiologic surveillance, though more information is needed on accuracy and reliability of these portable immunoassays. (www.actabiomedica.it) a new viral outbreak, sustained by a member of the coronaviridae family that has been finally defined severe acute respiratory syndrome (sars) coronavirus 2 (sars-cov-2) has recently emerged in wuhan, china at the end of 2019 (1) . the virus has since then spread all around the world, persuading the world health organization (who) to declare this infectious disease as the very last pandemic, 10 years after the h1n1 swine flu outbreak, in 2009-2010 (2) . covid-19 has already affected millions of people worldwide, causing such a high mortality that it may be responsible of over 50 million deaths if timely and appropriate measures, such as nationwide lockdown and social distancing (3), will not be undertaken by national health agencies and governments (1) . as other coronaviruses, sars-cov-2 is an enveloped virus with positive-sense, single-stranded rna genome, containing four main structural proteins known as spike (s, which contains the receptor-binding domain, known as rbd), envelope (e), membrane (m), and nucleocapsid (n), along with additional genes such as orf1a/b, orf3a, orf6, orf7a/b, orf8, and orf10, which encode accessory proteins, including the rna-dependent rna polymerase (figure 1 and table 1 ) (4, 5) . this microorganism has likely emerged due to bats spillover, probably through another intermediate animal (pangolin, perhaps) (6) . human transition has been largely fostered by emergence of mutations in the s protein, which has amplified the affinity of this protein moiety (within a furin-cleavage site) for angiotensin converting enzyme 2 (ace2) (7) , its natural receptor at the surface of cells of a vast array of organs and tissues, especially alveo-lar type 2 cells in the lung (at2), but also lymphocytes and cells of the heart, kidney and gastrointestinal system (8, 9) . binding of sars-cov-2 to ace2 is fostered by s protein priming catalyzed by transmembrane serine protease 2 (tmprss2) (10) . the large and widespread diffusion of ace2 at cell surface clearly explains the frequent lung involvement with interstitial pneumonia, occasionally evolving into acute respiratory distress syndrome (ards), along with possible injury of many other organs and tissues, thus justifying the risk of developing multiple organ failure (mof), which is then associated with an extremely high death rate (11) , especially in certain susceptible populations (12) . the histological examination of lung tissue frequently shows diffuse alveolar damage, characterized by the presence of cellular fibromyxoid exudates, desquamation of pneumocytes and hyaline membrane formation, which is consistent with ards (13) . although it has now been convincingly established that covid-19 has an almost favorable clinical course in as many as 80-85% of infected patients, who can be totally asymptomatic or may only display mild respiratory symptoms, in 10-15% of sars-cov-2 positive patients the disease evolves into severe or even critical forms, needing mechanical ventilation, sub-intensive or even intensive care (14, 15) . this is probably dependent on some demographic (advanced age, male sex) and clinical risk factors (hypertension, diabetes, cardiovascular disease, chronic respiratory disorders, cancer, obesity) (12) , but also on the presence of polymorphisms in the sequence of the ace2 gene, which may variably influence virulence and pathogenecity of sars-cov-2 by influencing receptor binding (16) . despite many biological aspects of this severe infectious disease remain largely obscure, it has now been clearly acknowledged that early management is associated with much better outcome, with lower progression towards systemic complications, including immunosuppression, development of a "cytokine storm" and severe inflammatory response syndrome (sirs) (17, 18) . in this perspective, it is now almost unquestionable that laboratory diagnostics plays an essential, almost vital, role in covid-19 as in many other human disorders (19) , as will be further discussed in the following parts of this article. before specifically discussing the current armamentarium for etiological diagnosis, it is worthwhile mentioning here that the who currently defines a "confirmed case" of covid-19 as patient who has received laboratory confirmation of sars-cov-2 infection, regardless of the presence of clinical signs and symptoms (20) . the almost logical consequence of this straightforward connotation is that the etiological diagnosis of covid-19 is only possible by detecting nucleic acid material (i.e., rna) of sars-cov-2 in biological samples. according to the who and the us centers for disease control and prevention (cdc), the material to be collected for initial covid-19 testing include upper respiratory specimens (nasopharyngeal and oropharyngeal swab, or wash in ambulatory patients) and/or lower respiratory specimens (sputum and/or endotracheal aspirate or bronchoalveolar lavage) (21) (22) (23) . additional biological samples that may be tested include blood, stool, urine, saliva and throat washing, though the significance of identifying the virus in these matrices remains undetermined (24,25) (table 2). once appropriately and accurately collected, the biological specimens (especially nasopharyngeal and oropharyngeal swabs) shall be placed into separate sterile tubes, containing 2-3 ml of viral transport media, and must be kept refrigerated at 2-4°c for less than 4 days, or frozen at -70°c (or below) until testing is carried out (26) . processing specimens not fulfilling these stringent pre-analytical requirements may be associated with generation of "false negative" tests results, and shall hence be avoided. the definitive diagnosis of sars-cov-2 infection, as endorsed by both the who and cdc, shall then be performed using molecular biology techniques on upper and lower respiratory materials. therefore, the diagnostic strategy encompasses the use of realtime reverse-transcription polymerase chain reaction (rrt-pcr) assays, targeting one or more genes in the sars-cov-2 genome. a typical rt-pcr procedure for detecting this coronavirus encompasses, in sequence, rna isolation, its purification, reverse transcription to cdna, cdna amplification with rt-pcr instrumentation, followed by (fluorescent) signal detection (25) . a validated diagnostic workflow, which has been endorsed by the who, and is hence now largely used in europe, entails a first-line screening assay with amplification of e gene, followed by a confirmatory assay with amplification of rdrp (rna-dependent rna polymerase) gene, and then an additional potential confirmatory assay, entailing amplification of n gene (27) . the cdc has also developed a molecular biology assay, that has been defined "centers for disease control and prevention (cdc) 2019-novel coronavirus (2019-ncov) real-time reverse transcriptase (rt)-pcr diagnostic panel" (28) . according to the cdc, the primers and probes for detecting sars-cov-2 have been identified from genetic regions belonging to n gene, encompassing the usage of two primer/probe sets. an additional primer/probe set can then be used for amplifying human rnase p gene (rp) in control specimens. importantly, a recent study which has assessed the comparative performance of multiple primer/probe sets, revealed that the who and cdc protocols display exceptional sensitivity compared to other assays (29) . importantly, regardless of the technique that will be used, the identification of sars-cov-2 by molecular biology techniques in either upper or lower respiratory specimens enables the diagnosis of active infection from this coronavirus, but does not rule out any co-infection by other microorganisms (e.g., bacteria, fungi, viruses, and so forth) (27) . the accuracy and reliability of rt-pcr for diagnosing sars-cov-2 infection depends on many biological and technical variables (30) . beside the influence of procedures used for collecting, transporting and storing the specimens, as well as from concomitant antivishowed that the rate of rt-pcr detection of sars-cov-2 in patients diagnosed with covid-19 is as high as 93% in bronchoalveolar lavage fluid, but then decreases to 72% in sputum and 63% in nasal swabs, respectively, whilst it is only 32% in pharyngeal swabs and 29% in stool (21) . to et al also reported that the positive rate of rt-pcr for sars-cov-2 is 15-30% in blood and 14-38% in rectal swabs, respectively (31) . the suboptimal diagnostic accuracy of nasopharyngeal and oropharyngeal swabs has been confirmed in some other published studies. for example, zhao et al (32) and yang et al. (33) , reported that the positive rate of rt-pcr for sars-cov-2 in these materials is only 70%, decreasing to approximately 60% in the study of ai et al. (34) . a major influence of the analytical techniques used for detecting viral rna has also been recently highlighted by wang et al, who showed that the limit of detection (i.e., the lowest detectable amount of virus) displayed by six commercial rt-pcr kits is extremely heterogeneous, so that the use of some of these tests may potentially generate false-negative results due to inadequate analytical sensitivity (35) . this information is noteworthy, whereby would shed some light on the fact some symptomatic patients who were not originally diagnosed as having sars-cov-2 infection by rt-pcr (or who have then been diagnosed as reinfected after two consecutive negative rt-pcr tests) may have been misclassified due to the use of methods with inadequate analytical sensitivity. it is also important to mention here that some of these initially falsenegative test results may then turn later positive, when swabs are re-collected some days after initial testing since the incubation of the virus is generally between 3-7 days (36) . interesting evidence has been published by zhang et al (37) , who showed that 14.1% patients who are later diagnosed with covid-19 may have negative test results initially, but this rate would then decreases in parallel with the number of repeated tests on follow-up, from 6.9% to 0.3% from 2 up to 5 consecutive ensuing swab tests, respectively. another interesting aspect that emerged from this study, is that the risk of progressing towards more severe disease stages was almost double in patients with initially positive swab test than in those with initially negative result (44.6% vs. 24.4%; p=0.015). recent studies have also been published on the possibility to use rapid reverse transcription loop-mediated isothermal amplification (rt-lamp) assays for sars-cov-2 detection, but additional evidence is needed at this point in time for validating their routine usage in covid-19 diagnostics (38, 39) . importantly, fully-automated commercial rt-pcr have also been recently introduced in the diagnostic market, which are characterized by high-throughput and fast turnaround time, thus enabling to reduce by nearly 90% the bench time per sample and allowing to analyze larger volume of patients in a shorter timeframe (40) . serological testing is conventionally defined as a diagnostic procedure used for identifying the presence of an immune response against an infectious agent (41) . inherent to this definition is the origin of many misunderstanding and misconceptions regarding the use of serological testing in covid-19, whereby this type of testing is not meant to replace the identification of viral rna for etiological diagnosis of covid-19, but rather for establishing as to whether individuals have been infected by the virus and/or have developed an immune response. the cdc endorses a highly reasonable conception underlying serological testing in covid-19, that is a strategy used mostly for epidemiological and surveillance purposes (28) . to put this in the context of covid-19, serology testing encompasses the identification (by qualitative assays) and/or measurement (using quantitative assays) of different classes of immunoglobulins (typically iga, igm, igg) against sars-cov-2 for establishing whether a person has been infected by sars-cov-2, and has then developed antibodies which, if possessing neutralizing effects, may prevent future re-infection. although the emergence of covid-19 is still too recent to enable us presenting definitive data on the individual response against this new coronavirus, some useful information has been published. guo et al have first shown that the median time of antibodies appearance in serum or plasma of covid-19 patients begins 3-6 days after the onset of symptoms for both igm and iga, whilst it is delayed to 10-18 days for igg (42) . the posi-tive rate for the different classes of antibodies is 85.4% for igm, 92.7% for iga and 77.9% for igg, respectively. in another recent study, padoan et al studied the kinetics of anti-covid-19 antibodies (43), concluding that igm and igg tend to appear 6-7 days after symptoms onset. notably, although 100% of covid-19 patients seem to develop anti-sars-cov-2 igg antibodies 12 days after the onset of symptoms, igm could only be found in <90% of this same group of patients. these important findings have been confirmed in a subsequent study, in which we showed that the rate of anti-sars-cov-2 antibody positivity up to two weeks after the onset of symptoms is as high as 100% for both iga and igm, whilst igm could only be measured in 60% of covid-19 patients after the same period (44) . similar data were published by jin et al (45) , who also showed that positivity for anti-sars-cov-2 igm and igg antibodies is 50% and 95%, respectively, and by du et al, who reported that the rate of detectable anti-sars-cov-2 igm and igg antibodies in convalescent patients is 78% and 100%, respectively (46) . in a more recent investigation, pan et al also observed that the cumulative rate of positivity for anti-sars-cov-2 igm and igg antibodies 15 days from symptom onset is about 74% and 97%, respectively (47) . an interesting aspect, recently highlighted, is that sars-cov-2 may trigger efficient generation of secretory iga even in asymptomatic or mild infections, so that their assessment both in blood and saliva may complement and perhaps improve the diagnostic process (48) . one of the major unresolved issues, almost entirely attributable to the very recent emergence of this novel coronavirus disease, is establishing whether anti-sars-cov-2 antibodies shall be considered neutralizing (i.e., effective to neutralize virulence and/or pathogenicity), as well as their persistence in blood. encouraging data on the former aspect have emerged from a recent publication, showing that human anti-sars-cov-2 antibodies seem to specifically target nucleocapsid and spike proteins, and thus possess neutralizing effect against the virus (49) . in a separate investigation, okba et al confirmed that serum collected from covid-19 patients is capable to neutralize sars-cov-2 infection (50) . as concerns the persistence of neutralizing antibodies in the circulation, some information can be translated from earlier findings on the former and relatively similar coronavirus disease sars, whereby the titer of anti-sars-cov-1 neutralizing antibodies was found to be stably high for 16 months after infection, but progressively declined afterwards, falling to 50-75% after 4 years and ~10% after 6 years, respectively (51) . a final issue that will need to be clarified is the possible crossreaction of current anti-sars-cov-2 immunoassays with previous coronaviruses such as sars-cov-1, mers-cov, hcov-hku1, hcov-oc43, hcov-nl63, and hcov-229e. the first serological strategy entails qualitative (or semi-quantitative) assessment by means of the socalled "rapid tests", which are basically portable devices to be used singly, with non-automated procedures, for producing rapid test results (i.e., around 5-20 min). since the leading advantages of these membranebased immunoassays encompass low sample volume (a drop of blood may generally be sufficient), little operator training, low cost, easy performance and relatively simple interpretation, their usage is mostly reserved to bedside or near-to-patient rapid testing (52) . these tests could conventionally entail two strategies, the former encompassing direct detection of sars-cov-2 antigens, the latter based instead on anti-sars-cov-2 antibodies identification. a comprehensive description of this technology has been provided, and is regularly updated, by the european center for disease control and prevention (ecdc) (53) . major concern has been recently raised on the analytical and diagnostic performance of these tests, especially after spain and some other european countries complained that many rapid test kits are inaccurate and do not allow to obtain a reliable diagnosis and surveillance of covid-19 (54). additional emphasis has then been provided by the recent publication of a study by cassaniti et al (55) , who claimed that the sensitivity of one of these rapid tests was <20%, thus potentially leading to under-diagnosing covid-19 in a large subset of patients. this would persuade us to conclude that the general paradigm that "one-size-fits-all" does not (and shall not) apply here, and that each single device must be adequately validated before entering routine clinical usage. the underlying problem is the fact that some of these tests underwent quick com-mercialization, without adequate analytical and clinical validation. our straightforward suggestion, also endorsed by the ecdc, is that scientific publications shall be made urgently available for clarifying performance and limitations of each single rapid diagnostic test before its introduction into routine diagnostics, clinical management and public health or epidemiologic surveillance (53) . it shall also be clear, that the most reasonable placement of these tests within the clinical decision making is for supporting decentralized testing capacity, but they shall not be considered a replacement of central laboratory diagnostics. the second serological option encompasses centralized testing within microbiological and clinical laboratories, by using fully-automated immunoassays (56) . although this alternative strategy is more expensive, requires the collection of whole blood samples by venipuncture rather than capillary blood, and is essentially dependent on availability of specific laboratory analyzers, it has some important advantages. these basically include better accuracy and reliability, the possibility to generate quantitative data (which are essential for longitudinal titer monitoring), performance by skilled laboratory personnel (thus inherently lowering the risk of errors and subjective interpretation), permanent storage of test results within the laboratory information system (lis), along with more stringent quality monitoring as enabled by performance of internal quality control and, hopefully in a near future, external quality assessment (eqas) schemes. the modern generation of laboratory analyzers is characterized by exceptional throughput and very limited turnaround time (i.e., they can perform hundreds tests per hour). the use of centralized laboratory diagnostics shall hence be considered a robust and viable strategy for epidemiological surveillance purposes. importantly, the university hospitals of padova and verona (italy) have been forerunners worldwide in conceiving and developing a project, which has been approved by the scientific committee of the veneto region and is now underway, entailing a vast epidemiological screening by means of validated fully-automated immunoassays of all healthcare personnel working in the veneto region (i.e., between 50,000-70,000 people). phase 2 of this project encompasses the possibility to broaden this epidemiological analysis to the nearly 5 million inhabitants of the entire veneto region (57). since the current epidemiological figures contribute to raise several doubts that the pandemic will cease soon, it becomes imperative to identify reliable predictors of disease severity, which may enable earlier clinical interventions and more appropriate usage of healthcare resources within a system of care whose responsive capacity has been literally overwhelmed by this unprecedented and virtually unpredictable epidemiological crisis (58, 59) . therefore, the possibility to identify a subset of subjects which will be more likely to progress towards severe/critical disease is an additional and almost essential contribution provided by laboratory medicine. this group of patients can be identified by discretional use of laboratory resources, whereby unfavorable clinical course has been associated with lymphopenia, thombocytopenia, neutrophilia, increased concentration of biomarkers of cardiac injury (i.e., cardiac troponins), c reactive protein and other inflammatory cytokines, liver and kidney function tests (60, 61) , as well as of d-dimer (62) and procalcitonin (63) . the fairly recent emergence of covid-19, the third coronavirus outbreak after sars in 2002-2003 and middle east respiratory syndrome (mers) in 2012, does not allow drawing definitive conclusions on sars-cov-2 diagnostics. nevertheless, some standpoints can be conveyed (figure 2) . first and foremost, it seems now rather clear that we will be living together with this virus for a quite a long time, so that our vigilance and responsiveness against the emergence of new local outbreaks must be maintained at the highest possible levels. that said, the etiological diagnosis of covid-19 is, and will remain for long, deeply based on direct identification -by means of molecular biology techniques -of viral rna in biological materials, especially upper and lower respiratory specimens. whether other biological matrices, such as blood, urine, stools and saliva, will represent valid alternatives has not been unequivocally defined, so far. as concerns serological testing, promising information can be garnered from preliminary investigations, showing that the vast majority of covid-19 patients seem to develop a sustained immune response against the virus, characterized by emergence of anti-sars-cov-2 igg and iga, 1 to 2 weeks after the onset of fever and/or respiratory symptoms. whether these antibodies will have persistent neutralizing activity against the virus is still to be elucidated on an individual and general basis. the availability of rapid tests for detecting either viral antigens or anti-sars-cov-2 antibodies shall then be seen as a potentially viable opportunity for purposes of epidemiologic surveillance, though more information is needed on accuracy and reliability of the many portable immunoassays that are now widely available in the market. one final consideration shall be clearly 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conflict of interest in connection with the submitted article key: cord-262119-s6hc7fxs authors: ostaszewski, marek; niarakis, anna; mazein, alexander; kuperstein, inna; phair, robert; orta-resendiz, aurelio; singh, vidisha; aghamiri, sara sadat; acencio, marcio luis; glaab, enrico; ruepp, andreas; fobo, gisela; montrone, corinna; brauner, barbara; frischman, goar; monraz gómez, luis cristóbal; somers, julia; hoch, matti; gupta, shailendra kumar; scheel, julia; borlinghaus, hanna; czauderna, tobias; schreiber, falk; montagud, arnau; de leon, miguel ponce; funahashi, akira; hiki, yusuke; hiroi, noriko; yamada, takahiro g.; dräger, andreas; renz, alina; naveez, muhammad; bocskei, zsolt; messina, francesco; börnigen, daniela; fergusson, liam; conti, marta; rameil, marius; nakonecnij, vanessa; vanhoefer, jakob; schmiester, leonard; wang, muying; ackerman, emily e.; shoemaker, jason; zucker, jeremy; oxford, kristie; teuton, jeremy; kocakaya, ebru; summak, gökçe yağmur; hanspers, kristina; kutmon, martina; coort, susan; eijssen, lars; ehrhart, friederike; rex, d. a. b.; slenter, denise; martens, marvin; haw, robin; jassal, bijay; matthews, lisa; orlic-milacic, marija; senff ribeiro, andrea; rothfels, karen; shamovsky, veronica; stephan, ralf; sevilla, cristoffer; varusai, thawfeek; ravel, jean-marie; fraser, rupsha; ortseifen, vera; marchesi, silvia; gawron, piotr; smula, ewa; heirendt, laurent; satagopam, venkata; wu, guanming; riutta, anders; golebiewski, martin; owen, stuart; goble, carole; hu, xiaoming; overall, rupert w.; maier, dieter; bauch, angela; gyori, benjamin m.; bachman, john a.; vega, carlos; grouès, valentin; vazquez, miguel; porras, pablo; licata, luana; iannuccelli, marta; sacco, francesca; nesterova, anastasia; yuryev, anton; de waard, anita; turei, denes; luna, augustin; babur, ozgun; soliman, sylvain; valdeolivas, alberto; esteban-medina, marina; peña-chilet, maria; helikar, tomáš; puniya, bhanwar lal; modos, dezso; treveil, agatha; olbei, marton; de meulder, bertrand; dugourd, aurélien; naldi, aurelien; noel, vincent; calzone, laurence; sander, chris; demir, emek; korcsmaros, tamas; freeman, tom c.; augé, franck; beckmann, jacques s.; hasenauer, jan; wolkenhauer, olaf; wilighagen, egon l.; pico, alexander r.; evelo, chris t.; gillespie, marc e.; stein, lincoln d.; hermjakob, henning; d’eustachio, peter; saez-rodriguez, julio; dopazo, joaquin; valencia, alfonso; kitano, hiroaki; barillot, emmanuel; auffray, charles; balling, rudi; schneider, reinhard title: covid-19 disease map, a computational knowledge repository of sars-cov-2 virus-host interaction mechanisms date: 2020-10-27 journal: biorxiv doi: 10.1101/2020.10.26.356014 sha: doc_id: 262119 cord_uid: s6hc7fxs we hereby describe a large-scale community effort to build an open-access, interoperable, and computable repository of covid-19 molecular mechanisms the covid-19 disease map. we discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. we highlight the role of relevant databases and text mining approaches in enrichment and validation of the curated mechanisms. we describe the contents of the map and their relevance to the molecular pathophysiology of covid-19 and the analytical and computational modelling approaches that can be applied to the contents of the covid-19 disease map for mechanistic data interpretation and predictions. we conclude by demonstrating concrete applications of our work through several use cases. the coronavirus disease 2019 (covid-19) pandemic due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] has already resulted in the infection of over 40 million people worldwide, of whom one million have died 1 . the molecular pathophysiology that links sars-cov-2 infection to the clinical manifestations and course of covid-19 is complex and spans multiple biological pathways, cell types and organs [2, 3] . to gain the insights into this complex network, the biomedical research community needs to approach it from a systems perspective, collecting the mechanistic knowledge scattered across the scientific literature and bioinformatic databases, and integrating it using formal systems biology standards. with this goal in mind, we initiated a collaborative effort involving over 230 biocurators, domain experts, modelers and data analysts from 120 institutions in 30 countries to develop the covid-19 disease map, an open-access collection of curated computational diagrams and models of molecular mechanisms implicated in the disease [4] . to this end, we aligned the biocuration efforts of the disease maps community [5, 6] , reactome [7] , and wikipathways [8] and developed common guidelines utilising standardised encoding and annotation schemes, based on community-developed systems biology standards [9] [10] [11] , and persistent identifier repositories [12] . moreover, we integrated relevant knowledge from public repositories [13] [14] [15] [16] and text mining resources, providing a means to update and refine contents of the map. the fruit of these efforts was a series of pathway diagrams describing key events in the covid-19 infectious cycle and host response. we ensured that this comprehensive diagrammatic description of disease mechanisms is machine-readable and computable. this allows us to develop novel bioinformatics workflows, creating executable networks for analysis and prediction. in this way, the map is both human and machine-readable, lowering the communication barrier between biocurators, domain experts, and computational biologists significantly. computational modelling, data analysis, and their informed interpretation using the contents of the map have the potential to identify molecular signatures of disease predisposition and development, and to suggest drug repositioning for improving current treatments. covid-19 disease map is a collection of 41 diagrams containing 1836 interactions between 5499 elements, supported by 617 publications and preprints. the summary of diagrams available in the covid-19 disease map can be found online 2 in supplementary material 1. the map is a constantly evolving resource, refined and updated by ongoing efforts of biocuration, sharing and analysis. here, we report its current status. in section 2 we explain the set up of our community effort to construct the interoperable content of the resource, involving biocurators, domain experts and data analysts. in section 3 we demonstrate that the scope of the biological maps in the resource reflects the state-ofthe-art about the molecular biology of covid-19. next, we outline analytical workflows that can be used on the contents of the map, including initial, preliminary outcomes of two such workflows, discussed in detail as use cases in section 4. we conclude in section 5 with an outlook to further development of the covid-19 map and the utility of the entire resource in future efforts towards building and applying disease-relevant computational repositories. the covid-19 disease map project involves three main groups: (i) biocurators, (ii) domain experts, and (iii) analysts and modellers: i. biocurators develop a collection of systems biology diagrams focused on the molecular mechanisms of sars-cov-2. ii. domain experts refine the contents of the diagrams, supported by interactive visualisation and annotations. iii. analysts and modellers develop computational workflows to generate hypotheses and predictions about the mechanisms encoded in the diagrams. all three groups have an important role in the process of building the map, by providing content, refining it, and defining the downstream computational use of the map. figure 1 illustrates the ecosystem of the covid-19 disease map community, highlighting the roles of different participants, available format conversions, interoperable tools, and downstream uses. the information about the community members and their contributions are disseminated via the fairdomhub [17] , so that content distributed across different collections can be uniformly referenced. the biocurators of the covid-19 disease map diagrams follow the guidelines developed by the community, and specific workflows of wikipathways [8] and reactome [7] . the biocurators build literature-based systems biology diagrams, representing the molecular processes implicated in the covid-19 pathophysiology, their complex regulation and the phenotypic outcomes. these diagrams are main building blocks of the map, and are composed of biochemical reactions and interactions (further called altogether interactions) taking place between different types of molecular entities in various cellular compartments. as there are multiple teams working on related topics, biocurators can provide an expert review across pathways and across platforms. this is possible, as all platforms offer intuitive visualisation, interpretation, and analysis of pathway knowledge to support basic and clinical research, genome analysis, modelling, systems biology, and education. table 1 lists information about the created content. for more details see supplementary material 1. communicating to refine, interpret and apply covid-19 disease map diagrams. these diagrams are created and maintained by biocurators, following pathway database workflows or standalone diagram editors, and reviewed by domain experts. the content is shared via pathway databases or a gitlab repository; all can be enriched by integrated resources of text mining and interaction databases. the covid-19 disease map diagrams, available in layout-aware systems biology formats and integrated with external repositories, are available in several formats allowing a range of computational analyses, including network analysis and boolean, kinetic or multiscale simulations. both interactions and interacting entities are annotated following a uniform, persistent identification scheme, using either miriam or identifiers.org [18] , and the guidelines for annotations of computational models [19] . viral protein interactions are explicitly annotated with their taxonomy identifiers to highlight findings from strains other than sars-cov-2. moreover, tools like modelpolisher [20] , sbmlsqueezer [21] or memote 3 help to automatically complement the annotations in the sbml format and validate the model (see also supplementary material 2). the knowledge on covid-19 mechanisms is rapidly evolving, as demonstrated by the rapid growth of the covid-19 open research dataset (cord-19) dataset, a source scientific manuscript text and metadata on covid-19 and related coronavirus research [28] . cord-19 currently contains over 130,000 articles and preprints, over four times more than when it was introduced 10 . in such a quickly evolving environment, biocuration efforts need to be supported by other repositories of structured knowledge about molecular mechanisms relevant for covid-19, like molecular interaction databases, or text mining resources. contents of such repositories may suggest improvements in the existing covid-19 disease map diagrams, or establish a starting point for developing new pathways (see section "biocuration of database and text mining content"). interaction and pathway databases contain structured and annotated information on protein interactions or causal relationships. while interaction databases focus on pairs of molecules, offering broad coverage of literature-reported findings. pathway databases provide detailed description of biochemical processes and their regulations of related interactions, supported by diagrams. both types of resources can be a valuable input for covid-19 disease map biocurators, given the comparability of identifiers used for molecular annotations, and the reference to publications used for defining an interaction or building a pathway. table 2 text-mining approaches can help to sieve through such rapidly expanding literature with natural language processing (nlp) algorithms based on semantic modelling, ontologies, and linguistic analysis to automatically extract and annotate relevant sentences, biomolecules, and their interactions. this scope was recently extended to pathway figure mining: decoding pathway figures into their computable representations [30] . altogether, these automated workflows lead to the construction of knowledge graphs: semantic networks incorporating ontology concepts, unique biomolecule references, and their interactions extracted from abstracts or full-text documents [31] . the covid-19 disease map project integrates open-access text mining resources, indra [32] , biokb 15 , ailani covid-19 16 , and pathwaystudio 10 . all platforms offer keyword-based search allowing interactive exploration. additionally, the map benefits from an extensive protein-protein interaction network (ppi) 17 generated with a custom text-mining pipeline using opennlp 18 and gnormplus [33] . this pipeline was applied to the cord-19 dataset and the collection of medline abstracts associated with the genes in the sars-cov-2 ppi network [34] using the entrez gene reference-into-function (generif). for detailed descriptions of the resources, see supplementary material 3. molecular interactions from databases and knowledge graphs from text mining resources discussed above (from now on called altogether 'knowledge graphs') have a broad coverage at the cost of depth of mechanistic representation. this content can be used by the biocurators in the process of building and updating the systems biology focused diagrams. biocurators can use this content in three main ways: by visual exploration, by programmatic comparison, and by direct incorporation of the content. first, the biocurators can visually explore the contents of the knowledge graphs using available search interfaces to locate new knowledge and encode it in the diagrams. moreover, solutions like covidminer project 19 , pathwaystudio and ailani offer a visual representation of a group of interactions for a better understanding of their biological context, allowing search by interactions, rather than just isolated keywords. finally, indra and ailani offer assistant bots that respond to natural language queries and return meaningful answers extracted from knowledge graphs. second, programmatic access and reproducible exploration of the knowledge graphs is possible via data endpoints: sparql for biokb and application programming interfaces for indra, ailani, and pathway studio. users can programmatically submit keyword queries and retrieve functions, interactions, pathways, or drugs associated with submitted gene lists. this way, otherwise time-consuming tasks like an assessment of completeness of a given diagram, or search for new literature evidence, can be automated to a large extent. finally, biocurators can directly incorporate the content of knowledge graphs into sbml format using biokc [35] . additionally, the contents of the elsevier covid-19 pathway collection can be translated to sbgnml 20 preserving the layout of the diagrams. the sbgnml content can then be converted into other diagram formats used by biocurators (see section 2.3 below). the biocuration of the covid-19 disease map is distributed across multiple teams, using varying tools and associated systems biology representations. this requires a common approach to annotations of evidence, biochemical reactions, molecular entities and their interactions. moreover, the interoperability of layout-aware formats is needed for comparison and integration of the diagrams in the map. the covid-19 disease map diagrams are encoded in one of three layout-aware formats for standardised representation of molecular interactions: sbml 21 [36] [37] [38] , sbgnml [27] , and gpml [24] . these xml-based formats focus to a varying degree on user-friendly graphical representation, standardised visualisation, and support of computational workflows. for the detailed description of the formats, see supplementary material 1. each of these three languages has a different focus: sbml emphasizes standardised representation of the data model underlying molecular interactions, sbgnml provides standardised graphical representation of molecular processes, while gpml allows for a partially standardised representation of uncertain biological knowledge. nevertheless, all three formats are centered around molecular interactions, provide a constrained vocabulary to encode element and interaction types, encode layout of their diagrams and support stable identifiers for diagram components. these shared properties, supported by a common ontology 22 [39] , allow cross-format mapping and enable translation of key properties between the formats. therefore, when developing the contents of the map, biocurators use the tools they are familiar with, facilitating this distributed task. the covid-19 disease map community ecosystem of tools and resources (see figure 1 ) ensures interoperability between the three layout-aware formats for molecular mechanisms: sbml, sbgnml, and gpml. essential elements of this setup are tools capable of providing cross-format translation functionality [40, 41] and supporting harmonised visualisation processing. another essential translation interface is a representation of reactome pathways in wikipathways gpml [42] and sbml. the sbml export of reactome content has been optimised in the context of this project and facilitates integration with the other covid-19 disease map software components. the contents of the covid-19 disease map diagrams can be directly transformed into inputs of computational pipelines and data repositories. besides the direct use of sbml format in kinetic simulations, celldesigner sbml files can be transformed into sbml qual [43] using casq [44] , enabling boolean modelling-based simulations (see also supplementary material 3). in parallel, casq converts the diagrams to the sif format 23 , supporting pathway modelling workflows using simplified interaction networks. notably, the gitlab repository features an automated translation of stable versions of diagrams into sbml qual. finally, translation of the diagrams into xgmml format (the extensible graph markup and modelling language) using cytoscape [45] or ginsim [46] allows for network analysis and interoperability with molecular interaction repositories [47] . thanks to the community effort discussed above supported by a rich bioinformatics framework, we constructed the covid-19 disease map, focussing on the mechanisms known from other coronaviruses [48] and suggested by early experimental investigations [pmid:32511329]. then, we applied the analytical and modelling workflows to the contributed diagrams and associated interaction databases to propose initial map-based insights into covid-19 molecular mechanisms. the covid-19 disease map is an evolving repository of pathways affected by sars-cov-2. figure 2 . it is currently centred on molecular processes involved in sars-cov-2 entry, 22 http://www.ebi.ac.uk/sbo/main/ 23 http://www.cbmc.it/fastcent/doc/sifformat.htm replication, and host-pathogen interactions. as mechanisms of host susceptibility, immune response, cell and organ specificity emerge, these will be incorporated into the next versions of the map. the covid-19 map represents the mechanisms in a "host cell". this follows literature reports on cell specificity of sars-cov-2 [3, [49] [50] [51] [52] [53] . some pathways included in the covid-19 map may be shared among different cell types, as for example the ifn-1 pathway found in cells such as dendritic, epithelial, and alveolar macrophages [54] [55] [56] [57] [58] . while at this stage, we do not address cell specificity explicitly in our diagrams, extensive annotations may allow identification of pathways relevant to the cell type of interest. the sars-cov-2 infection process and covid-19 progression follow a sequence of steps ( figure 3 ), starting from viral attachment and entry, which involve various dynamic processes on different time scales that are not captured in static representations of pathways. correlation of symptoms and potential drugs suggested to date helps downstream data exploration and drug target interpretation in the context of therapeutic interventions. human host ilc1, ilc-2, ilc3 natural killer renin-angiotensinaldosterone system (raas) granulocytes nasal mucosa disease map golgi er cd8+ cd4+ ace2 tmprss2 integrative stress response dendritic cells transmission of sars-cov-2 primarily occurs through contact with respiratory drops, airborne transmission, and through contact with contaminated surfaces [66] [67] [68] . upon contact with the respiratory epithelium, the virus infects cells mostly by binding the spike surface glycoprotein (s) to angiotensin-converting enzyme 2 (ace2) with the help of serine protease tmprss2 [69] [70] [71] [72] . importantly, recent results suggest viral entry using other receptors of lungs and the immune system [73, 74] . once attached, sars-cov-2 can enter cells either by direct fusion of the virion and cell membranes in the presence of proteases • dendritic cells. • nk cells. • monocytes and macrophages. • t cells, th1 and th2 response. • b cells, antibody production. asymptomatic/pre -symptomatic. vaccine? pre-exposure prophylaxis? antivirals? sirs, shock. shortness of breath. anosmia, ageusia, cough, fever, diarrhea. multiple organ dysfunction ards, complications. host response • cellular stress. • apoptosis. systemic and ventilation support oxygen therapy host raas ards; acute respiratory distress syndrome. raas; renin-angiotensin-aldosterone system. sirs; systemic inflammatory response syndrome. pathophysiology virus-host cell interactions and host response disease map critical asymptomatic (lung, heart, kidney) (nasal and respiratory epithelium, alveoli, vascular endothelial) tmprss2 and furin or by endocytosis in their absence. regardless of the entry mechanism, the s protein has to be activated to initiate the plasma or endosome membrane fusion process. while in the cell membrane, s protein is activated by tmprss2 and furin, in the endosome s protein is activated by cathepsin b (ctsb) and cathepsin l (ctsl) [71, 75] . activated s promotes the cell-or endosome-membrane fusion [76] with the virion membrane, and then the nucleocapsid is injected into the cytoplasm. these mechanisms are represented in the corresponding diagrams of the map 25 . within the host cell, sars-cov-2 hijacks the rough endoplasmic reticulum (rer)-linked host translational machinery. it then synthesises viral proteins replicase polyprotein 1a (pp1a) and replicase polyprotein 1ab (pp1ab) directly from the virus (+)genomic rna (grna) [48, 77] . through a complex cascade of proteolytic cleavages, pp1a and pp1ab give rise to 16 non-structural proteins (nsps) [78] [79] [80] . most of these nsps collectively form the replication transcription complex (rtc) that is anchored to the membrane of the double-membrane vesicle [78, 81] endoplasmic reticulum stress and unfolded protein response as discussed above, the virus hijacks the er to replicate. production of large amounts of viral proteins exceeds the protein folding capacity of the er, creating an overload of unfolded proteins. as a result, the unfolded protein response (upr) pathways are triggered to assure the er homeostasis, using three main signalling routes of upr via perk, ire1, and atf6 [87]. their role is to mitigate the misfolded protein load and reduce oxidative stress. the resulting protein degradation is coordinated with a decrease in protein synthesis via eif2alpha phosphorylation and induction of protein folding genes via the transcription factor xbp1 [88] . when the er is unable to restore its function, it can trigger cell apoptosis [89, 90] . the results are er stress and activation of the upr. the expression of some human coronavirus (hcov) proteins during infection, in particular the s glycoprotein, may induce activation of the er stress in the host cells [91] . based on sars-cov results, this may lead to activation of the perk [92], ire1 and in an indirect manner, of the atf6 pathways [93] . processes of degrading malfunctioning proteins and damaged organelles, including the ubiquitin-proteasome system (ups) and autophagy [94] are essential to maintain energy homeostasis and prevent cellular stress [95, 96] . autophagy is also involved in cell defence, including direct destruction of the viruses via virophagy, presentation of viral antigens, and inhibition of excessive inflammatory reactions [97, 98] . sars-cov-2 directly affects the process of ups-based protein degradation, as indicated by the host-virus interactome dataset published recently [34] . this mechanism may be a defence against viral protein degradation [99] . the map describes in detail the nature of this interaction, namely the impact of orf10 virus protein on the cul2 ubiquitin ligase complex and its potential substrates. interactions between sars-cov-2 and host autophagy pathways are inferred based on results from other covs. a finding that covs use double-membrane vesicles and lc3-i for replication [100] may suggest that the virus induces autophagy, possibly in atg5-dependent manner [101] , although some evidence points to the contrary [102] . also, the cov nsp6 restricts autophagosome expansion, compromising the degradation of viral components [103] . recently revealed mutations in nsp6 [104] indicate its importance, although the exact effect of the mutations remains unknown. based on the connection between autophagy and the endocytic pathway of the virus replication cycle [105] , autophagy modulation was suggested as a potential therapy strategy, either pharmacologically [96, [105] [106] [107] , or via fasting [108] . apoptosis, a synonym for programmed cell death, is triggered by virus-host interaction upon infection, as the early death of the virus-infected cells may prevent viral replication. many viruses block or delay cell death by expressing anti-apoptotic proteins to maximize the production of viral progeny [109] . in turn, apoptosis induction at the end of the viral replication cycle might assist in viral dissemination while reducing an inflammatory response. for instance, sars-cov-2 [110] and mers [111] are able to invoke apoptosis in lymphocytes, compromising the immune system. apoptosis follows two major pathways [112] , called extrinsic and intrinsic. extrinsic signals are transmitted by death ligands and their receptors (e.g., fasl and tnf-alpha). activated death receptors recruit adaptors like fadd and tradd, and initiator procaspases like caspase-8, leading to cell death with the help of effector caspases-3 and 7 [113, 114] . in turn, the intrinsic pathway involves mitochondria-related members of the bcl-2 protein family. cellular stress causes bcl-2 proteins-mediated release of cytochrome c from the mitochondria into the cytoplasm. cytochrome c then forms a complex with apaf1 and recruits initiator procaspase-9 to form the apoptosome, leading to the proteolytic activation of caspase-9. activated caspase-9 can now initiate the caspase cascade by activating effector caspases 3 and 7 [114] . the intrinsic pathway is modulated by sars-cov molecules [115, 116] . as intrinsic apoptosis involves mitochondria, its activity may also be exacerbated by sars-cov-2 disruptions of the electron transport chain, mitochondrial translation, and transmembrane transport [34] . the resulting mitochondrial dysfunction may lead to increased release of reactive oxygen species and pro-apoptotic factors. another vital crosstalk is that of the intrinsic pathway with the pi3k-akt pro-survival pathway. activated akt can phosphorylate and inactivate various pro-apoptotic proteins, including bad and caspase-9 [117] . sars-cov uses pi3k-akt signalling cascade to enhance infection [118] . moreover, sars-cov could affect apoptosis in a cell-type-specific manner [119, 120] . sars-cov structural proteins s, e, m, n, and accessory proteins 3a, 3b, 6, 7a, 8a, and 9b have been shown to act as crucial effectors of apoptosis in vitro. structural proteins seem to affect mainly the intrinsic apoptotic pathway, with p38 mapk and pi3k/akt pathways regulating cell death. accessory proteins can induce apoptosis via different cascades and in a cellspecific manner [114] . sars-cov e and 7a protein were shown to activate the intrinsic pathway by blocking anti-apoptotic bcl-xl localized to the er [121] . sars-cov m protein and the ion channel activity of e and 3a were shown to interfere with pro-survival signalling cascades [114, 122] . the viral replication and the consequent immune and inflammatory responses cause damage to the epithelium and pulmonary capillary vascular endothelium and activate the main intracellular defence mechanisms, as well as the humoral and cellular immune responses. resulting cellular stress and tissue damage [123, 124] impair respiratory capacity and lead to acute respiratory distress syndrome (ards) [61, 125, 126] . hyperinflammation is a known complication, causing widespread damage, organ failure, and death, followed by a not yet completely understood rapid increase of cytokine levels (cytokine storm) [127] [128] [129] , and acute ards [130] . other reported complications, such as coagulation disturbances and thrombosis are associated with severe cases, but the specific mechanisms are still unknown [64, [131] [132] [133] , although some reports suggest that covid-19 coagulopathy has a distinct profile [134] . the sars-cov-2 infection disrupts the coagulation cascade and is frequently associated with hyperinflammation, renin-angiotensin system (ras) imbalance and intravascular coagulopathy [132, [135] [136] [137] . hyperinflammation leads in turn to detrimental hypercoagulability and immunothrombosis, leading to microvascular thrombosis with further organ damage [138] . importantly, ras is influenced by risk factors of developing severe forms of covid-19 [139] [140] [141] . ace2, used by sars-cov-2 for host cell entry, is a regulator of ras and is widely expressed in the affected organs [142] . the main function of ace2 is the conversion of angii to angiotensin 1-7 (ang1-7), and these two angiotensins trigger the counter-regulatory arms of ras [143] . the signalling via angii and its receptor agtr1, elevated in the infected [142, 144] , induces the coagulation cascade leading to microvascular thrombosis [145] , while ang1-7 and its receptor mas1 attenuate these effects [143] . the innate immune system detects specific pathogen-associated molecular patterns (pamps), through pattern recognition receptors (prrs). detection of sars-cov-2 is mediated through receptors that recognise double-stranded and single-stranded rna in the endosome during endocytosis of the virus particle, or in the cytoplasm during the viral replication. these receptors mediate the activation of transcription factors such as ap1, nfkappab, irf3, and irf7, responsible for the transcription of antiviral proteins, in particular, interferon-alpha and beta [146, 147] . sars-cov-2 reduces the production of type i interferons to evade the immune response [49] . the detailed mechanism is not clear yet; however, sars-cov m protein inhibits the irf3 activation [148] and suppresses nfkappab and cox2 transcription. at the same time, sars-cov n protein activates nfkappab [149] , so the overall impact is unclear. these pathways are also negatively regulated by sars-cov nsp3 papain-like protease domain (plpro) [150] . the map contains the initial recognition process of the viral particle by the innate immune system and the viral mechanisms to evade the immune response. it provides the connection between virus entry (detecting the viral endosomal patterns), its replication cycle (detection cytoplasmic viral patterns), and the effector pathways of pro-inflammatory cytokines, especially of the interferon type i class. the latter seems to play a crucial but complex role in covid-19 pathology: both negative [151, 152] and positive effects [3, 153] of interferons on virus replication have been reported. interferon type i signalling interferons (ifns) are central players in the antiviral immune response of the host cell [55] , specifically affected by sars-cov-2 [154] [155] [156] [157] . type i ifns are induced upon viral recognition of pamps by various host prrs [48] as discussed earlier. the ifn-i pathway diagram represents the activation of tlr7 and ifnar and the subsequent recruiting of adaptor proteins and the downstream signalling cascades regulating key transcription factors including irf3/7, nf-kappab, ap-1, and isre [48, 158] . further, the map shows irf3mediated induction of ifn-i, affected by the sars-cov-2 proteins. sars-cov nsp3 and orf6 interfere with irf3 signalling [159, 160] and sars-cov m, n, nsp1 and nsp3 act as interferon antagonists [48, 150, 158, 161, 162] . moreover, coronaviruses orf3a, orf6 and nsp1 proteins can repress interferon expression and stimulate the degradation of ifnar1 and stat1 during the unfolded protein response (upr) [163, 164] . another mechanism of viral rna recognition is rig-like receptor signalling [58] , leading to sting activation [165] , and via the recruitment of traf3, tbk1 and ikkepsilon to phosphorylation of irf3 [56] . this in turn induces the transcription of ifns alpha, beta and lambda [166] . sars-cov viral papain-like-proteases, contained within the nsp3 and nsp 16 proteins, inhibit sting and the downstream ifn secretion [167] . in line with this hypothesis, sars-cov-2 infection results in a unique inflammatory response defined by low levels of ifn-i and high expression of cytokines [58, 168] . the ifnlambda diagram describes the ifnl receptor signaling cascade [169] , including jak-stat signaling and the induction of interferon stimulated genes, which encode antiviral proteins [170] . the interactions of sars-cov-2 proteins with the ifnl pathway are based on the literature [171] or sars-cov homology [58] . metabolic pathways govern the immune microenvironment by modulating the availability of nutrients and critical metabolites [172] . infectious entities reprogram host metabolism to create favourable conditions for their reproduction [173] . sars-cov-2 proteins interact with a variety of immunometabolic pathways, several of which are described below. heme catabolism is a well-known anti-inflammatory system in the context of infectious and autoimmune diseases [174, 175] . the main effector of this pathway, heme oxygenase-1 (hmox1) was found to interact with sars-cov-2 orf3a, although the nature of this interaction remains ambiguous [34, 176] . hmox1 cleaves heme into carbon monoxide, biliverdin (then reduced to bilirubin), and ferrous iron [pmid:31396090]. biliverdin, bilirubin, and carbon monoxide possess cytoprotective properties, and have shown promise as immunomodulatory therapeutics [177, 178] . importantly, activation of hmox1 also inhibits the nlrp3 inflammasome [178] [179] [180] , which is a pro-inflammatory and prothrombotic multiprotein system [181] highly active in covid-19 [182] [183] [184] . it mediates production of the pro-inflammatory cytokines il-1b and il-18 via caspase-1 [181] . the sars-cov orf3a, e, and orf8a incite the nlrp3 inflammasome [185] [186] [187] [188] . still, the potential of the hmox1 pathway to fight covid-19 inflammation remains to be tested [176, 189, 190] despite promising results in other models of inflammation [176, 178, [191] [192] [193] . the tryptophan-kynurenine pathway is closely related to heme metabolism. the ratelimiting step of this pathway is catalysed by the indoleamine 2,3 dioxygenase enzymes (ido1 and ido2) in dendritic cells, macrophages, and epithelial cells in response to inflammatory cytokines like ifn-gamma, ifn-1, tgf-beta, tnf-alpha, and il-6 [194] [195] [196] . crosstalk with the hmox1 pathway also increases the expression of ido1 and hmox1 in a feed-forward manner. metabolomics analyses from severe covid-19 patients revealed enrichment of kynurenines and depletion of tryptophan, indicating robust activation of ido enzymes [197, 198] . depletion of tryptophan [173, 199, 200] and kynurenines and their derivatives affect the proliferation and immune response of a range of t cells [176, [201] [202] [203] [204] [205] . however, despite high levels of kynurenines in covid-19, cd8+ t-cells and th17 cells are enriched in lung tissue, and t-regulatory cells are diminished [206] . this raises the question of whether and how the immune response elicited in covid-19 evades suppression by the kynurenine pathway. the sars-cov-2 protein nsp14 interacts with three human proteins: gla, sirt5, and impdh2 [34] . the galactose metabolism pathway, including the gla enzyme [207] , is interconnected with amino sugar and nucleotide sugar metabolism. sirt5 is a naddependent desuccinylase and demalonylase regulating serine catabolism, oxidative metabolism and apoptosis initiation [208] [209] [210] . moreover, nicotinamide metabolism regulated by sirt5 occurs downstream of the tryptophan metabolism, linking it to the pathways discussed above. finally, impdh2 is the rate-limiting enzyme in the de novo synthesis of gtp, allowing regulation of purine metabolism and downstream potential antiviral targets [211, 212] . the pyrimidine synthesis pathway, tightly linked to purine metabolism, affects viral dna and rna synthesis. pyrimidine deprivation is a host targeted antiviral defence mechanism, which blocks viral replication in infected cells and can be regulated pharmacologically [213] [214] [215] . it appears that components of the dna damage response connect the inhibition of pyrimidine biosynthesis to the interferon signalling pathway, probably via sting-induced tbk1 activation that amplifies interferon response to viral infection, discussed above. inhibition of de novo pyrimidine synthesis may have beneficial effects on the recovery from covid-19 [215] ; however, this may happen only in a small group of patients. covid-19 pathways featured in the previous section cover mechanisms reported so far. still, certain aspects of the disease were not represented in detail because of their complexity, namely cell-type-specific immune response, and susceptibility features. their mechanistic description is of great importance, as suggested by clinical reports on the involvement of these pathways in the molecular pathophysiology of the disease. the mechanisms outlined below will be the next targets in our curation roadmap. cell type-specific immune response covid-19 causes serious disbalance in multiple populations of immune cells. some studies report that covid-19 patients have a significant decrease of peripheral cd4+ and cd8+ cytotoxic t lymphocytes (ctls), b cells, nk cells, as well as higher levels of a broad range of cytokines and chemokines [128, [216] [217] [218] [219] . the disease causes functional exhaustion of cd8+ ctls and nk cells, induced by sars-cov-2 s protein and by excessive pro-inflammatory cytokine response [217, 220] . moreover, the ratio of naïve-to-memory helper t-cells, as well as the decrease of t regulatory cells, correlate with covid-19 severity [206] . conversely, high levels of th17 and cytotoxic cd8+ t-cells have been found in the lung tissue [221] . pulmonary recruitment of lymphocytes into the airways may explain the lymphopenia and the increased neutrophil-lymphocyte ratio in peripheral blood found in covid-19 patients [216, 222, 223] . in this regard, an abnormal increase of the th17:treg cell ratio may promote the release of pro-inflammatory cytokines and chemokines, increasing disease severity [224] . sars-cov-2 infection is associated with increased morbidity and mortality in individuals with underlying chronic diseases or a compromised immune system [225] [226] [227] [228] . groups of increased risk are men, pregnant and postpartum women, and individuals with high occupational viral exposure [229] [230] [231] . other susceptibility factors include the abo blood groups [232] [233] [234] [235] [236] [237] [238] [239] [240] and respiratory conditions [241] [242] [243] [244] [245] [246] . importantly, age is one of the key aspects contributing to the severity of the disease. the elderly are at high risk of developing severe or critical disease [227, 247] . age-related elevated levels of pro-inflammatory cytokines (inflammation) [247] [248] [249] [250] , immunosenescence and cellular stress of ageing cells [125, 227, 247, 251, 252] may contribute to the risk. in contrast, children are generally less likely to develop severe disease [253, 254] , with the exception of infants [125, [255] [256] [257] . however, some previously healthy children and adolescents can develop a multisystem inflammatory syndrome following sars-cov-2 infection [258] [259] [260] [261] [262] . several genetic factors have been proposed and identified to influence susceptibility and severity, including the ace2 gene, hla locus, errors influencing type i ifn production, tlr pathways, myeloid compartments, as well as cytokine polymorphisms [156, 235, [263] [264] [265] [266] [267] [268] [269] . we aim to connect the susceptibility features to specific molecular mechanisms and better understand the contributing factors. this can lead to a series of testable hypotheses, including the role of vitamin d counteracting pro-inflammatory cytokine secretion [270] [271] [272] in an age-dependent manner [247, 273] , and modifying the severity of the disease. another example of a testable hypothesis may be that the immune phenotype associated with asthma inhibits pro-inflammatory cytokine production and modifies gene expression in the airway epithelium, protecting against severe covid-19 [245, 246, 274] . in order to understand complex and often indirect dependencies between different pathways and molecules, we need to combine computational and data-driven analyses. standardised representation and programmatic access to the contents of the covid-19 disease map enable the development of reproducible analytical and modelling workflows. here, we discuss the range of possible approaches and demonstrate preliminary results, focusing on interoperability, reproducibility, and applicability of the methods and tools. our goal is to work on the computational challenges as a community, involving the biocurators and domain experts in the analysis of the covid-19 disease map and rely on their feedback to evaluate the outcomes. in this way, we aim to identify approaches to tackle the complexity and the size of the map, proposing a state-of-the-art framework for robust analysis, reliable models, and useful predictions. visualisation of omics data can help contextualise the map with experimental data creating data-specific blueprints. these blueprints could be used to highlight parts of the map that are active in one condition versus another (treatment versus control, patient versus healthy, normal versus infected cell, etc.). combining information contained in multiple omics platforms can make patient stratification more powerful, by reducing the number of samples needed or by augmenting the precision of the patient groups [275, 276] . approaches that integrate multiple data types without the accompanying mechanistic diagrams [277] [278] [279] produce patient groupings that are difficult to interpret. in turn, classical pathway analyses often produce long lists mixing generic and cell-specific pathways, making it challenging to pinpoint relevant information. using disease maps to interpret omics-based clusters addresses the issues related to contextualised visual data analytics. footprints are signatures of a molecular regulator determined by the expression levels of its targets [280] . for example, a footprint can contain targets of a transcription factor (tf) or peptides phosphorylated by a kinase. combining multiple omics readouts and multiple measurements can increase the robustness of such signatures. nevertheless, an essential component is the mechanistic description of the targets of a given regulator, allowing computation of its footprint. with available sars-cov-2 related omics and interaction datasets [281] , it is possible to infer which tfs and signalling pathways are affected upon infection [282] . combining the covid-19 disease map regulatory interactions with curated collections of tf-target interactions like dorothea [283] will provide a contextualised evaluation of the effect of sars-cov-2 infection at the tf level. the virus-host interactome is a network of virus-human protein-protein interactions (ppis) that can help understanding the mechanisms of disease [34, [284] [285] [286] . it can be expanded by merging virus-host ppi data with human ppi and protein data [287] to discover clusters of interactions indicating human mechanisms and pathways affected by the virus [288] . these clusters first of all can be interpreted at the mechanistic level by visual exploration of covid-19 disease map diagrams. in addition, these clusters can potentially reveal additional pathways to add to the covid-19 disease map (e.g., e protein interactions or tgfbeta diagrams) or suggest new interactions to introduce into the existing diagrams. computational modelling is a powerful approach that enables in silico experiments, produces testable hypotheses, helps elucidate regulation and, finally, can suggest via predictions novel therapeutic targets and candidates for drug repurposing. mechanistic models of pathways allow bridging variations at the scale of molecular activity to variations at the level of cell behaviour. this can be achieved by coupling the molecular interactions of a given pathway with its endpoint and by contextualising the molecular activity using omics datasets. hipathia is such a method, processing transcriptomic or genomic data to estimate the functional profiles of a pathway conditioned by the data studied and linkable to phenotypes such as disease symptoms or other endpoints of interest [289, 290] . moreover, such mechanistic modelling can be used to predict the effect of interventions as, for example, the effect of targeted drugs [291] . hipathia integrates directly with the diagrams of the covid-19 map using the sif format provided by casq (see section 2.3), as well as with the associated interaction databases (see section 2.2). the drawback of approaches like hipathia is their computational complexity, limiting the size of the diagrams they can process. an approach to large-scale mechanistic pathway modelling is to transform them into causal networks. carnival [292] combines the causal representation of networks [13] with transcriptomics, phosphoproteomics, or metabolomics data [280] to contextualise cellular networks and extract mechanistic hypotheses. the algorithm identifies a set of coherent causal links connecting upstream drivers such as stimulations or mutations to downstream changes in transcription factor activities. analysis of the dynamics of molecular networks is necessary to understand their dynamics and deepen our understanding of crucial regulators behind disease-related pathophysiology. discrete modelling framework provides this possibility. covid-19 disease map diagrams, translated to sbml qual (see section 2.3), can be directly imported by tools like cell collective [293] or ginsim [46] for analysis. preserving annotations and layout information ensures transparency and reusability of the models. importantly, cell collective is an online user-friendly modelling platform 26 that provides features for real-time in silico simulations and analysis of complex signalling networks. the platform allows users without computational background to simulate or analyse models to generate and prioritise new hypotheses. references and layout are used for model visualisation, supporting the interpretation of the results. the mathematics and code behind each model, however, remain accessible to all users. in turn, ginsim is a tool providing a wide range of analysis methods, including efficient identification of the states of convergence of a given model (attractors). model reduction functionality can also be employed to facilitate the analysis of large-scale models. viral infection and immune response are complex processes that span many different scales, from molecular interactions to multicellular behaviour. the modelling and simulation of such complex scenarios require a dedicated multiscale computational architecture, where multiple models run in parallel and communicate among them to capture cellular behaviour and intercellular communications. multiscale agent-based models simulate processes taking place at different time scales, e.g., diffusion, cell mechanics, cell cycle, or signal transduction [294] , proposed also for covid-19 [295] . physiboss [296] allows such simulation of intracellular processes by combining the computational framework of physicell [297] with maboss [298] tool for stochastic simulation of logical models to study of transient effects and perturbations [299] . implementation of detailed covid-19 signalling models in the pysiboss framework may help to better understand complex dynamics of multi-scale processes as interactions and crosstalk between immune system components and the host cell in covid-19. in this case study, we combine computational approaches discussed above and present results derived from omics data analysis on the covid-19 disease maps diagrams. we measured the effect of covid-19 at the transcription factor (tf) activity level by applying viper [300] combined with dorothea regulons [283] on rna-seq datasets of the sars-cov-2 infected cell line [168] . then, we mapped the tfs normalised enrichment score (nes) on the interferon type i signalling pathway diagram of the covid-19 disease map using the sif files generated by casq (see section 2.3). as highlighted in figure 4 , our manually curated pathway included some of the most active tfs after sars-cov-2 infection, such as stat1, stat2 , irf9 and nfkb1. these genes are well known to be involved in cytokine signalling and first antiviral response [301, 302] . interestingly, they are located downstream of various viral proteins (e, s, nsp1, orf7a and orf3a) and members of the mapk pathway (mapk8, mapk14 and map3k7). sars-cov-2 infection is known to promote mapk activation, which mediates the cellular response to pathogenic infection and promotes the production of proinflammatory cytokines [281] . altogether, we identified signaling events that may capture the mechanistic response of the human cells to the viral infection. in this use case, the hipathia [289] algorithm was used to calculate the level of activity of the subpathways from the covid-19 apoptosis diagram, with the aim to evaluate whether covid-19 disease map diagrams can be used for pathway modelling approach. to this end, a public rna-seq dataset from human sars-cov-2 infected lung cells (geo gse147507) was used. first, the rna-seq gene expression data was normalized with the trimmed mean of m values (tmm) normalization [303] , then rescaled to range [0;1] for the calculation of the signal and normalised using quantile normalisation [304] . the normalised gene expression values were used to calculate the level of activation of the subpathways, then a case/control contrast with a wilcoxon test was used to assess differences in signaling activity between the two conditions. the activation levels have been calculated using transcriptional data from gse147507 and hipathia mechanistic pathway analysis algorithm. each node represents a gene (ellipse), a metabolite (circle) or a function (square). the pathway is composed of circuits from a receptor gene/metabolite to an effector gene/function, with interactions simplified to inhibitions or activations (see section 2.3, sif format). significantly deregulated circuits are highlighted by color arrows (red: activated in infected cells). the color of the node corresponds to the level of differential expression of each node in sars-cov-2 infected cells vs normal lung cells. blue: down-regulated elements, red: up-regulated elements, white: elements with not statistically significant differential expression. hipathia calculates the overall circuit activation, and can indicate deregulated interaction even if interacting elements are not individually differentially expressed. results of the apoptosis pathway analysis can be seen in figure 5 and supplementary material 5. importantly, hipathia calculates the overall activation of circuits (series of causally connected elements), and can indicate deregulated interactions resulting from a cumulative effect, even if interacting elements are not individually differentially expressed. when discussing differential activation, we refer to the circuits, while individual elements are mentioned as differentially expressed. the analysis shows an overactivation of several circuits, specifically the one ending in the effector protein bax. this overactivation seems to be led by the overexpression of the bad protein, inhibiting bcl2-mcl1-bcl2l1 complex, which in turn inhibits bax. indeed, sars-cov-2 infection can invoke caspase8-induced apoptosis [305] , where bax together with the ripoptosome/caspase-8 complex, may act as a pro-inflammatory checkpoint [306] . this result is supported by studies in sars-cov, showing bax overexpression following infection [121, 307] . overall, our findings recapitulate reported outcomes. with evolving contents of the covid-19 disease map and new omics data becoming available, new mechanism-based hypotheses can be formulated. in the covid19 disease map community we strive to produce interoperable content and seamless downstream analyses, translating the graphic representations of the molecular mechanisms to executable models. we are also aware of parallel efforts towards modelling of covid-19 mechanisms, which we plan to include as a part of our ecosystem. these efforts are not yet directly interoperable with the covid 19 disease map content as they use either different notation schemes or require parameters not covered by our biocuration guidelines at the same time, they provide a complementary source of information and the opportunity to create an even broader toolset to tackle the pandemic. the modified edinburgh pathway notation (mepn) scheme [308] allows for the detailed visual encoding of molecular processes using the yed platform but diagrams are constructed in such a way as to also function as petri nets. these can then be used directly for activity simulations using the biolayout network analysis tool [309] . the current mepn covid-19 model details the replication cycle of sars-cov-2, integrated with a range of host defence systems, e.g. type 1 interferon signalling, tlr receptors, oas systems, etc. simulations of altered gene expression, interactions with drug targets or changes to interaction kinetics can be represented by introducing relevant transitions or nodes directly in the diagram. currently, models constructed in mepn can be saved as sbgn.ml files, however is a loss of information and the features associated computationally are not compatible with other covid-19 disease map diagrams (not modelled as petri nets). the covid-19 disease map can support dynamic kinetic modelling to quantify the behaviour of different pathways and evaluate the dynamic effects of perturbations. however, it is necessary to assign a kinetic equation or a rate law to every reaction in the diagram to be analysed. this process is challenging because any given reaction depends on its cellular and physiological context, which makes it difficult to parameterise. software support of tools like sbmlsqueezer [21] and reaction kinetics databases like sabio-rk [310] are indispensable in this effort. nevertheless, the most critical factor is the availability of experimentally validated parameters that can be reliably applied in sars-cov-2 modelling scenarios. the covid-19 disease map is both a knowledgebase and a computational repository. on the one hand, it is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. on the other hand, it is a computational resource of curated content for graph-based analyses and disease modelling. it offers a shared mental map for understanding the dynamic nature of the disease at the molecular level and also its dynamic propagation at a systemic level. thus, it provides a platform for a precise formulation of models, accurate data interpretation, monitoring of therapy, and potential for drug repositioning. the covid-19 disease map spans three platforms and assembles diagrams describing molecular mechanisms of covid-19. these diagrams are grounded in the relevant published sars-cov-2 research, completed where necessary by mechanisms discovered in related beta-coronaviruses. this unprecedented effort of community-driven biocuration resulted in over forty diagrams with molecular resolution constructed since march 2020. it demonstrates that expertise in biocuration, clear guidelines and text mining solutions can accelerate the passage from the published findings to a meaningful mechanistic representation of knowledge. the covid 19 disease map can provide the tipping point to shortcut research data generation and knowledge accumulation, creating a formalized and standardized streamline of well defined tasks. this approach to an emerging pandemic leveraged the capacity and expertise of an entire swath of the bioinformatics community, bringing them together to improve the way we build and share knowledge. by aligning our efforts, we strive to provide covid-19 specific pathway models, synchronize content with similar resources and encourage discussion and feedback at every stage of the curation process. with new results published every day, and with the active engagement of the research community, we envision the covid-19 disease map as an evolving and continuously updated knowledge base whose utility spans the entire research and development spectrum from basic science to pharmaceutical development and personalized medicine. moreover, our approach includes a large-scale effort to create interoperable tools and seamless downstream analysis pipelines to boost the applicability of established methodologies to the covid-19 disease map content. this includes harmonisation of formats, support of standards, and transparency in all steps to ensure wide use and content reusability. preliminary results of such efforts are presented in the case studies. the covid-19 disease map community is open and expanding as more people with complementary expertise join forces. in the longer run, the map's content will help to find robust signatures related to sars-cov-2 predisposition or response to various treatments, along with the prioritization of new potential drug targets or drug candidates. we aim to provide the tools to deepen our understanding of the mechanisms driving the infection and help boost drug development supported with testable suggestions. we aim at building armor for new treatments to prevent new waves of covid-19 or similar pandemics. a pneumonia outbreak associated with a new coronavirus of probable bat origin covid-19 and the kidney: from epidemiology to clinical practice receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues covid-19 disease map, building a computational repository of sars-cov-2 virus-host interaction mechanisms systems medicine disease maps: 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self-organization of multicellular tissues rapid community-driven development of a sars-cov-2 tissue simulator physiboss: a multiscale agent-based modelling framework integrating physical dimension and cell signalling physicell: an open source physics-based cell simulator for 3-d multicellular systems maboss 2.0: an environment for stochastic boolean modeling conceptual and computational framework for logical modelling of biological networks deregulated in diseases functional characterization of somatic mutations in cancer using network-based inference of protein activity stat2 and irf9: beyond isgf3 ifnβdependent increases in stat1, stat2, and irf9 mediate resistance to viruses and dna damage edger: a bioconductor package for differential expression analysis of digital gene expression data a comparison of normalization methods for high density oligonucleotide array data based on variance and bias sars-cov-2 triggers inflammatory responses and cell death through caspase-8 activation bax/bak-induced apoptosis results in caspase-8-dependent il-1β maturation in macrophages over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in vero e6 cells the mepn scheme: an intuitive and flexible graphical system for rendering biological pathways a graphical and computational modeling platform for biological pathways sabio-rk: an updated resource for manually curated biochemical reaction kinetics key: cord-267458-uofy7jyx authors: jiang, xiao-lin; zhang, xiao-li; zhao, xiang-na; li, cun-bao; lei, jie; kou, zeng-qiang; sun, wen-kui; hang, yang; gao, feng; ji, sheng-xiang; lin, can-fang; pang, bo; yao, ming-xiao; anderson, benjamin d; wang, guo-lin; yao, lin; duan, li-jun; kang, dian-ming; ma, mai-juan title: transmission potential of asymptomatic and paucisymptomatic sars-cov-2 infections: a three-family cluster study in china date: 2020-04-22 journal: j infect dis doi: 10.1093/infdis/jiaa206 sha: doc_id: 267458 cord_uid: uofy7jyx data concerning the transmission of sars-cov-2 in asymptomatic and paucisymptomatic patients are lacking. we report a three-family cluster of infections involving asymptomatic and paucisymptomatic transmission. eight (53%) of 15 members from three families were confirmed with sars-cov-2 infection. of eight patients, three were asymptomatic and one was paucisymptomatic. an asymptomatic mother transmitted the virus to her son, and a paucisymptomatic father transmitted the virus to his three-month-old daughter. sars-cov-2 was detected in the environment of one household. the complete genomes of sars-cov-2 from the patients were >99.9% identical and were clustered with other sars-cov-2 sequences reported from china and other countries. m a n u s c r i p t severe acute respiratory syndrome coronavirus 2 (sars-cov-2), that causes coronavirus disease 2019 (covid-19), emerged in december 2019 in wuhan, china [1] . it has since been declared a global pandemic with over 1,000,000 cases reported as of april 3, 2020 [2] . person-to-person transmission has been established [3] [4] [5] [6] [7] [8] , and asymptomatic transmission of sars-cov-2 has been reported [9] . however, studies on the potential transmission of sars-cov-2 by asymptomatic persons and those with mild illness have been limited [10] . herein, we report a 3-family cluster study of eight patients associated with asymptomatic and pauciasymptomatic (one mild symptom only) sars-cov-2 transmission in shandong province, china. the first positive sars-cov-2 patients in this cluster were identified on january 21, 2020 triggering an epidemiological investigation by the local center for disease control and prevention. to identify the possible infective source, the epidemiological investigation focused on exposure history before the onset of illness, such as travel history to wuhan or hubei province, visiting live animal markets, and contact history with febrile persons. medical records were also closely reviewed to verify the timelines of events and clarify clinical progressions. to examine possible environmental contamination of sars-cov-2 in households, select surfaces that may be frequently touched by family members were sampled in the bedroom (door handle, bedside light switch, and sliding of wardrobe door), kitchen (door handle, faucet switch, light switch, rice cooker plug), and bathroom (door handle, handrail, the surface of the toilet bowl, sink). one swab per site (room) with multiple surfaces was collected. all close contacts of sars-cov-2 positive patients were traced, including family members who lived with the patients and individuals who had contact with the patients within 1 meter without wearing proper personal protection. close contacts were quarantined at home and monitored for fever (≥38°c) and symptoms. in addition, nasopharyngeal swabs of close contacts were collected every 24 a c c e p t e d m a n u s c r i p t 5 hours from day 1 to 14 to detect sars-cov-2 by molecular assay. if any close contact had positive detection of sars-cov-2, they were sent to a hospital for isolation and treatment. all collected environmental and patient samples were stored at −80°c before being transported using cold chain to a biosafety level 2 enhanced laboratory to perform molecular detection of sars-cov-2. a real-time reverse transcriptase pcr (rrt-pcr) test kit (gz-d2rm, shanghai geneodx biotech co., ltd) targeting the orf1ab and n genes of sars-cov-2 was used. a cycle threshold (ct) value less than 37 was interpreted as positive for sars-cov-2 rna and a ct value of 40 or more was defined as a negative test. a medium load (weakly positive), defined as a ct value of 37 to less than 40, required confirmation by retesting. positive samples were sequenced directly from the original specimens as previously described [11] . the maximum likelihood phylogenetic tree of the complete genomes was conducted by using raxml software (version 8.2.9) [12] with 1000 bootstrap replicates, employing the general time-reversible nucleotide substitution model. patients 1 (62-year-old woman) and 2 (65-year-old man) were a couple who lived with their son (patient 3), daughter-in-law (patient 4), and two grandchildren. patient 1 presented with cough, rhinorrhea, and sputum on january 12, 2020 ( figure 1 ). on january 15, she visited a health clinic and was diagnosed with a common cold. she was prescribed intravenous infusions of ampicillin and sulbactam, ribavirin, and traditional medicine for five days. on january 16, she developed a fever (38°c). on january 17, patient 2 reported symptoms of fever (37.8°c), cough, sputum, earache, and upset stomach. he was also diagnosed with a common cold at the health clinic and received the same prescription as patient 1 for three days. however, their symptoms did not resolve at the conclusion of the treatment regimen leading both to seek care at a local hospital on january 21. nasopharyngeal swabs were collected from both patients at the hospital and confirmed positive for the infant had no clinical symptoms before, during, or after hospitalization. the chest ct images on admission or hospitalization showed that patients 1-6 had ground-glass opacities. however, no significant abnormalities were observed for patients 7 and 8 (supplemental a c c e p t e d m a n u s c r i p t 9 we report a unique three-family cluster of infection with sars-cov-2, in which eight of 15 members were confirmed with sars-cov-2 infection. particularly interesting is that of 6 secondary patients, two were asymptomatic, one was paucisymptomatic, and three were symptomatic. our findings show that the transmission of sars-cov-2 by individuals with asymptomatic or paucisymptomatic infections is possible. patients 1 and 2 were likely first exposed to sars-cov-2 after visiting their hometown in xiaogan hubei province, china. their son (patient 3) and daughter-in-law (patient 4, asymptomatic), whom they live with, were later found to be infected with sars-cov-2. patient 5 (asymptomatic) was identified to be infected with sars-cov-2 after frequent contact with patients 3 and 4 during work and home visits. she transmitted the virus to her son (symptomatic) whom she lives with. patient 7 (paucisymptomatic) was found to be infected with sars-cov-2 after frequent contact with patient 3 during work. he likely transmitted the virus to his daughter (patient 8, asymptomatic). in addition, consistent with previous studies [5] [6] [7] [8] , the transmission of sars-cov-2 during the incubation period of patient 3 likely occurred. patients 5 and 7 were infected after their exposures to a presymptomatic patient 3 during working or home visits. these findings may help explain the rapid spread of sars-cov-2 between person-to-person. the currently available evidence shows that sars-cov-2 is transmitted between people through droplets and close contact [13] . a recent study showed extensive environmental contamination by a sars-cov-2 patient [14] , suggesting the contaminated environment as a potential medium of transmission. in this study, we detected sars-cov-2 in two environmental swabs from the household of patient 3. such detection of sars-cov-2 in contaminated environments of the household may provide an additional contribution to virus transmission among family members as the virus can remain viable and infectious on the surface up to seven days [15] . however, the direct researchbased evidence describing exactly how sars-cov-2 is transmitted is limited, and further studies are required. we cannot rule out the possibility of unknown covid-19 patients (e.g., asymptomatic carriers) transmitting the virus. however, according to screening protocols implemented by the provincial, municipal, and county-level center for disease control and prevention, all close contacts were a c c e p t e d m a n u s c r i p t 10 traced, and all patients with positive rrt-pcr results in this study were confirmed by whole-genome sequencing, including those who were asymptomatic or pauciasymptomatic (patients 4, 5, 7, and 8). m a n u s c r i p t 11 we thank all patients involved in the study. we also thank dr. hong-guang ren from beijing institute of biotechnology for generating phylogenetic tree. a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster novel coronavirus pneumonia emergency response epidemiology t transmission of 2019-ncov infection from an asymptomatic contact in germany a familial cluster of infection associated with the 2019 novel coronavirus indicating potential person-to-person transmission during the incubation period potential presymptomatic transmission of sars-cov-2 presymptomatic transmission of sars-cov-2 -singapore presumed asymptomatic carrier transmission of covid-19 covert coronavirus infections could be seeding new outbreaks genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies advice on the use of masks in the community, during home care and in healthcare settings in the context of the novel coronavirus (covid-19) outbreak surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 a c c e p t e d m a n u s c r i p t key: cord-252389-xrdbmosj authors: kumar, mukesh; thakur, ajit kumar title: neurological manifestations and comorbidity associated with covid-19: an overview date: 2020-10-14 journal: neurol sci doi: 10.1007/s10072-020-04823-6 sha: doc_id: 252389 cord_uid: xrdbmosj first in 2002, severe acute respiratory syndrome coronavirus (sars-cov), second in 2012, middle east respiratory syndrome coronavirus (mers-cov), and now the third in the december 2019, emergence of tremendously pathogenic and large-scale epidemic novel coronavirus (sars-cov-2) has brought the worst conditions into the human inhabitants of the twenty-first century. the sars-cov-2 uses the resembling receptor, angiotensin-converting enzyme 2 (ace2) as that for sars-cov, and mainly feasts through the respiratory tract. the ace2 receptor appearances have been also detected upon glial cells and neurons, which makes them a potential target of sars-cov-2 disease (covid-19). consequently, cells expressing ace2, apart from lung and cardiovascular tissue, neurons and glial cells may act as targets and are thus vulnerable to sars-cov-2 systemic infection as well as its central nervous system (cns) comorbidities. investigation of the neurological manifestations of covid-19 is a step towards better understanding the sars-cov-2 infections, inhibiting the additional spread and treating patients affected by this pandemic. in this concern, more clinical examinations for cns involvement of sars-cov-2 are warranted. in this article, we have reviewed the neurological characteristic features of covid-19 patients, latent neurotropic mechanisms of sars-cov-2 involvement in the comorbidity associated with cns disorders, and neurological manifestations associated with covid-19. therefore, in the perspective of covid-19 pandemic, clinicians and healthcare workers should be aware of a wide spectrum of neurological manifestations associated with covid-19 along with their signs and symptoms for initial diagnosis and isolation of the patients. one of the most contagious viruses is coronavirus (family: coronaviridae), primarily targeting the human respiratory system and infecting the epithelial cells of the respiratory tract, but it also has neuroinvasive capabilities to spread from the respiratory tract to the central nervous system (cns) [1, 2] . earlier epidemics or pandemics of coronaviruses comprise the severe acute respiratory syndrome (sars-cov) during 2002 and middle east respiratory syndrome (mers-cov) during 2012 [3, 4] . the present pandemic is an acute respiratory disease, caused by a novel coronavirus (sars-cov-2, earlier known as 2019-ncov); the sars-cov-2 disease 2019 (covid-19) was spread throughout china and rapidly acknowledged the global attention. on 30 january 2020, the world health organization (who) officially stated the covid-19 epidemic as a public health emergency of global concern. the symptoms of covid-19 infection habitually appear after an incubation period most commonly around 5 days (range from 1 to 14 days) [5] . the most common symptoms of covid-19 illness are fever, cough, and fatigue; other symptoms include headache, hemoptysis, and dyspnea, among others. in the most severe cases, patients may develop pneumonia, acute respiratory distress syndrome, acute cardiac problems, and multi-organ failure [2, 6] . the first cases of covid-19 were reported in december 2019 [7] . the appearance of sars-cov-2 marked the third introduction of extremely pathogenic and large-scale epidemic coronavirus into the human inhabitants after sars-cov and mers-cov [8] . the elderly and people with underlying diseases are more susceptible to infection and prone to serious outcomes, which may be related with acute respiratory distress syndrome (ards) and cytokine storm [9, 10] . the covid-19 pandemic is of great global public health concern in the twenty-first century. special attention and efforts should be applied to prevent and reduce the transmission in susceptible populations, including children, elderly people, and healthcare providers [6] . recently, several reports on meningitis, encephalitis, myelitis, and peripheral nerve affection in the context of covid-19 were published, suggesting that sars-cov-2 can directly or indirectly infect structures and functions of the nervous system [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . coronavirus infections have been associated with the neurological manifestations, viz., febrile seizures, convulsions, change in mental status, and encephalitis [21] . in a recently published systemic review, the neurotropic and neuroinvasive abilities of coronaviruses in humans have been described [2] . a growing body of evidence shows that neuroinvasion and neurotropism is a common feature of human coronaviruses. the neuroinvasive human viruses, respiratory viruses, may damage the cns as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to cns cells (virus-induced neuropathology) [1, 2] . coronavirus enters through nasal infection and reached to the cns through the olfactory bulb, causing neuro-inflammation and demyelination of neuronal cells [9] . it has also been reported and correlated that the patients infected with sars-cov-2 show encephalopathy and neuro-inflammation resulting from a cytokine storm [22] . therefore, exploring the neurologic manifestations associated with covid-19 is urgently required for better understanding the sars-cov-2 brain infections, inhibiting the additional spread and treating patients affected by this pandemic. in this review, we have explored the epidemiology and pathophysiology of covid-19, their comorbidity in brain disorders, and neurological manifestations reported. coronaviruses are enveloped, pleomorphic, spherical particles, 150 to 160 nm in size, positive-sense single-stranded viruses ((+) ssrna virus) belonging to the family coronaviridae, and containing 8-10 open reading frames (orfs) [23] . orf1a and orf1b are translated into polyprotein 1a and polyprotein 1ab, which are processed by viral proteases to produce 16 nonstructural proteins containing rna-dependent rna polymerase enzyme (rdrp). the viral rna is replicated via transcription of a minus-strand template by rdrp. coronaviruses generate 6-9 subgenomic mrnas (sgmrnas) during replication, which lead to translation of accessory and physical proteins from downstream orfs. spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins are required for completion of a viral replication cycle, and these proteins translated from sgmrnas [24] [25] [26] [27] . the antibodies generated against the n protein of sars-cov may cross counter with covid-19; however, the heterophilic antibodies of sars-cov may not afford cross protection to covid-19, though it can be used for diagnostic purposes [23] . an additional possible role of sars-cov-n protein is its capability to counter host immune response as a viral suppressor protein of rnai (vsr) [28] . the vsrs suppress the rnai at the pre-dicer or post-dicer level to overcome the host defense to establish infection [23] . in a clustal w analysis of n-protein of sars-cov and covid-19 by ncbi amino acid blast, it is demonstrated and reported that more than 90% sequence identity were similar in sars-cov and covid-19. so, the n-protein of covid-19 may act in an analogous fashion to sars-cov as a vsr to counter the host defense mechanism [23] . covid-19 has significant consequences for morbidity and mortality worldwide since december 2019. to explore the effects of comorbid chronic diseases on clinical outcomes of covid-19, it was found that diabetes present in 10%, coronary artery disease/cardiovascular disease (cad/cvd) was in 8%, and hypertension was in 20%, which were much higher than that of 3% chronic pulmonary disease [29] . the occurrence of a core and receptor-binding domain (rbd) from the crystal structure of sars-cov-2 has exposed that is more closely involved in recognizing the hace2. although sars-cov and sars-cov-2 both exploit the same receptor hace2 in humans to gain entry into the host, the sars-cov-2 binding is more compacted by a four-residue motif from 482 to 485 in the hace2 ridge, thus improving the binding affinity of sars-cov-2 over sars-cov for hace [30] . moreover, the two viral hot spots, namely, hot spot-31 and hot spot-353 on hace2, are stabilized more by the sars-cov-2 rbd as compared to sars-cov. all this clearly indicates why sars-cov-2 has a selective advantage over sars-cov in causing infection and is a more evolved and lethal strain [22] . the key clinical manifestation of sars-cov-2 is severe pneumonia causing immense respiratory distress in the patients, especially the aged or those with already pre-existing conditions. sars-cov-2 leads to chronic inflammation of the lungs, severe dyspnea, fever, dry cough, and cyanosis and in more vulnerable patients a complete lung failure. ace2, which is the entry point for sars-cov-2, has almost an ubiquitous presence in human organs including lung parenchyma, gastrointestinal tract, nasal mucosa, renal and urinary tract, human airway epithelia, vascular endothelium, lymphoid tissues, reproductive organs, as well as in brain [31] . the virus is expected to enter chiefly through the nasal mucosa or the gastrointestinal tract due to their higher expression of protein hace2. the exciting part though is that recently reported studies have noted altered mental health in some covid-19 patients (n = 214) showing symptoms like anosmia (5.1%) and ageusia (5.6%) without nasal obstruction or other rhinitis symptoms, thereby indicating a neuroinvasive nature of the virus [16, [32] [33] [34] . specifically, pre-existing chronic conditions are strongly correlated with disease severity and being admitted to intensive care unit also, compared to covid-19 patients with no pre-existing chronic diseases. covid-19 patients who present with diabetes, hypertension, cad/cvd, or chronic pulmonary disease have a higher risk of developing severe disease, respectively. surprisingly, the authors found no correlation between chronic conditions and increased risk of mortality (or 2.09, 95% ci 0.26 to 16.67) [29] . engaged together, cardio-metabolic diseases, such as diabetes, hypertension, and cad/cvd, were more common than chronic pulmonary disease in covid-19 patients. however, each comorbid disease was correlated with increased disease severity, and after an active treatment, increased risk of mortality in patients with pre-existing chronic diseases may reduce [29] . the ace2 receptor expression in the brain has been also reported and detected over glial cells and neurons, which makes them a potential target of covid-19 systemic infection as well as its cns comorbidities. thus, cells exposing ace2, such as neurons and glial cells, may act as targets and are thus susceptible to sars-cov-2 infection [35] . it is possible that sars-cov-2 could infect the brain through olfactory nerves located in the nasal cavity, like other neurotropic coronaviruses. moreover, it is also suggested that respiratory failure in severe covid-19 cases might be treated from the perspective of the cns [36] . in an independent prospective clinical study, it was concluded that neurological symptoms are often seen in patients with covid-19. moreover, the headache was the most commonly seen neurological symptom in this disease along with dizziness, impaired consciousness, smell and gustation impairments, cerebrovascular disorders, epileptic seizures, and myalgia [37] . the psychiatric disorders are common in several neurological disorders, e.g., alzheimer's disease, parkinson's disease, epilepsy, migraine, essential tremor, and stroke. these comorbidities increase disease load and may complicate the treatment of mutual disorders. early studies of the comorbidity of psychiatric and neurological disorders were cross-sectional, and the time order of the relatives was not possible to elucidate [38] . the latest work has clarified time associations between psychiatric disorders and neurological disorders, particularly in epilepsy and stroke where epidemiological evidence recommends that there is a bidirectional relationship [39] . though these associations are understood in many neurological disorders, repetitive screening for psychiatric disorders in such cases is infrequent, mostly due to the lack of partnerships between psychiatrists and neurologists and the paucity of neuropsychiatrists [38] . various evidences have been reported and support the possible cns comorbidities associated with covid-19 pathophysiology [1, 2, [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . therefore, the healthcare provider should be aware of the wide spectrum of neurological sign and symptoms associated with covid-19 for early diagnosis and isolation of the patients [40] . apart from these, much more prerequisites should be done to improve the finding and treatment of covid-19 patients suffering from neurological and psychiatric disorders and their comorbidities after sars-cov-2 infection. there is also a need to understand the prospect of these comorbidities which may motivate alliances to improve the lives of the publics affected by neurological and psychiatric disorders associated with covid-19. the neurological manifestations of sars-cov-2 have been recently recognized from computed tomography (ct) and magnetic resonance imaging (mri) images of the brain of a patient who contracted covid-19 and showed symptoms of necrotizing hemorrhagic encephalopathy [13] . a rare disorder such as acute necrotizing encephalopathy (ane) is leading to brain dysfunction generally caused by viruses, which outcomes in liver problems, seizures, mental disorientation, and subsequent infection. this sickness is categorized by multifocal symmetric lesions in the brain that influence the brain stem, thalami, cerebellum, and cerebral white matter. the causes of ane is neuro-inflammation resulting from a cytokine storm characterized primarily by the creation of the interleukin-6 (il-6), secreted by the macrophages, which have been activated by the granulocyte-macrophage colony-stimulating factor (gm-csf) produced by the helper t cells [22] . the resultant cytokine storm may also cause a surge in interleukin (il)-2, il-7, interferon-γ, inducible protein 10, monocyte chemo attractant protein 1, macrophage inflammatory protein 1-α, and tumor necrosis factor-α leading to hyperinflammation [10] . the systemic inflammation causes severe encephalopathy in the patient, and that may lead even to stroke. in the patient, specifically infected with sars-cov-2 showing ane, the mri images displayed clear evidence of hemorrhage through hypointense signal intensity in the susceptibility-weighted images and increase in the rim on the post contrast images [22] . as a pandemic virus, no effective treatment has been established until date for this disease resulting from sars-cov-2. thus, awareness of the possible entry and pathogenicity of sars-cov-2 into the cns will have important guiding significance for the prevention and treatment of covid-19. to identify the possible routes by which this virus invades the cns, several studies have been postulated for the olfactoryhematogenous pathway, trans-neuronal machinery, and lymphatic pathway, as putative routes of coronavirus entry into the cns [1, 19] . if the neuroinvasion of sars-cov-2 gets a part in the extension of respiratory failure in covid-19 patients, the precaution with masks will absolutely be the most efficient measure to defend against the possible entry of the virus into the cns [41] . it might be estimated that the indication of the patients infected via conjunctiva will be lighter than those infected intranasally. the probable neuroinvasion of sars-cov-2 may also partially explain why some patients developed respiratory failure, while others not. as sars-cov-2 may conceal itself in the neurons from the immune recognition, complete clearance of the virus may not be sure even the patients have recovered from the acute infection [41] . in sustain of this, there is evidence that sars-cov-2 is still detectable in some patients during the convalescent period. so, given the probable neuroinvasion, the risk of sars-cov-2 infection may be currently underestimated [31] . it is also a prerequisite to find effective antiviral drug therapies that can cross the blood-brain barrier to prevent and treat cns infections from sars-cov-2. currently, as encephalopathy also has been identified as one of the symptoms of covid-19 [13] , therefore, neuroinvasiveness of sars-cov-2 needs to be evaluated to fully understand the neurological implications of covid-19. the brain reportedly, like most other organs, expresses the hace2 considered to be the entry point of the sars-cov-2 viruses in humans and is therefore not immune to viral infection [22] . though sars-cov-2 is yet to be detected in cerebrospinal fluid, sars-cov with similar structural and functional features has been detected in the cerebrospinal fluid of patients, indicating the ability of the virus to breach the extremely rigid blood-brain barrier [31] . if prior studies with other covs are taken into consideration, then sars-cov-2 like its other family members will first infect the peripheral nerve terminals and then slowly crawl its way through the synapse-connected route into the cns [42] . the associated field with sars-cov and mers-cov earlier studies observed that they infiltrate the brains of transgenic mice when administered intranasally. the virus through infiltrates into the brain took place via the olfactory nerves, ultimately affecting the thalamus and the brain stem [31] . the brain stem has been observed and reported as worst infected in covid-19 patients. several reports warranted for subsequent investigational studies along with the hematologic spread of sars-cov-2 in cns as well as retrograde neuronal transport from the lungs into the cns through vagal nerve afferents must be taken into consideration [31, [42] [43] [44] . also, with reports appealing infection of the gastrointestinal tract by sars-cov-2, the virus could even use the enteric nervous system and its sympathetic afferent neurons to reach the cns. now, with the reports of spontaneous breathing, hyposmia, and ageusia in covid-19 patients, scientists have started to speculate that not only does sars-cov-2 infects lungs but it has severe implications in neurons, specifically those in the medulla oblongata, which regulates breathing, lung, and heart functions, and any damage to it can result in chronic respiratory distress as reported in covid-19 patients [22] . it has been put forward that the latency period of the virus may be enough to destroy the neurons in the medullary region of the brain and can lead to coma or death. as reports of sars-cov-2 reaching the blood-brain barrier through the circulating blood and breaching it by attacking the endothelial layer to gain access to cns emerge, the virus might just be using an alternating route in the form of the olfactory bulb instead of the common hematological route [1, 19] . the neuronal cells infected with the virus, immune systems (microphase, t cells, and monocytes) triggered, and inflammatory system activated leads to cytokine storm and oxidative stress (fig. 1 ). if this is to be measured, the virus might just be making its way into olfactory mucosa, mostly consisting of olfactory neurons along with blood vessels and epithelial cells [22] . the olfactory mucosa relates to the olfactory bulb through the cribriform plate, which is found at the very base of the frontal lobes of the brain [16] . this very much elucidates the hyposmia and other neurological symptoms that are being increasingly observed in covid-19 patients. the fact to note here is that the long-term effects of the neuroinvasive nature of the virus may result in an increased risk of neurodegenerative diseases with involvement in the pathogenesis of neurological disorders like parkinson's disease and multiple sclerosis [42] . further, the conditions are more likely to be worsened in covid-19 patients with pre-existing neurological disorders [22] . neurological manifestations in covid-19 patients generally arise between 1 and 14 days after the beginning of sars-cov-2 infectious symptoms, and it has been predicted on average of 5-day incubation period (time between infection and symptoms onset) [45] . the sars-cov-2 is a singlestranded rna coronavirus, the genome of which has 89.1% similarity in its nucleotide sequence to a group of sars-like coronaviruses. it has been classified as the genus betacoronavirus, subgenus sarbecovirus, and is the seventh member of the coronavirus family that can infect humans [35] . naturally, common cold symptoms are caused by these four human coronaviruses, viz., nl63, hku1, 229e, and oc43, whereas the three others are accountable for several pandemic infectious diseases, such as sars in 2002 (sars-cov), mers in 2012 (mers-cov), and covid-19 (sars-cov-2) in 2019 [7, 46] . the neurological manifestations reported by sars patients, sars-cov was detected in samples/specimens of sars patients. csf samples of a sars patient who presented generalized tonic-clonic convulsion tested positive for sars-cov, suggesting possible infection of the cns by sars-cov [18] . the sars-cov was isolated from a sample of brain tissue of one patient with sars that had presented severe cns symptoms, and pathological examination of the brain tissue showed neuronal necrosis and glial cell hyperplasia [15, 35] . furthermore, sars viral particles and its genomic sequence were detected in the neurons in the brains of all eight confirmed cases of sars autopsies. of these, six confirmed cases presented edema and scattered red degeneration of the neurons [47] . the reported prevalence of neurologic manifestations associated with covid-19 patient is 36.4% in china and 57.4% in europe [32, 48] . in a recent published report from hospital network in chicago, illinois, out of total 509 patients, neurologic manifestations were present at covid-19 onset in 215 (42.2%), at hospitalization in 319 (62.7%), and at any time during the disease course in 419 patients (82.3%). furthermore, it was explained and reported in an independent study that the most frequent neurologic manifestations in covid-19 patients were myalgias (44.8%), headaches (37.7%), encephalopathy (31.8%), dizziness (29.7%), dysgeusia (15.9%), and anosmia (11.4%) [49] . the aspect for these differences in frequencies of neurologic manifestations might be due to genetic factors including polymorphism in expression of the viral receptor ace2 in the nervous system and, possibly, sars-cov-2 strain variations [50] . the first case of meningitis/encephalitis associated with sars-cov-2 was reported from the yamanashi university hospital, japan [11] . the sars-cov-2 rna was detected in the csf specimen that confirms direct evidence of the fig. 1 neurodegenerations and cns comorbidities associated with covid-19. sars-cov-2 reaching the blood-brain barrier through the circulating blood and breaching it by attacking the endothelial layer to gain access to cns emerges. the neuronal cells infected with virus, immune systems (microphase, t cells, and monocytes) triggered, and inflammatory system activated leads to cytokine storm, oxidative stress, and associated neurological manifestations neuroinvasiveness of sars-cov-2 [11, 35] . in addition to this, the pathological findings in brain tissue of covid-19 patients, diagnosed on the 13th day of hospital admission, have been reported in the gross anatomy of a patient admitted because of cerebral infarction. however, only non-specific brain edema and atrophy without further histopathological observations were found by gross anatomical examination of this patient and without any signs of infection. thus, detailed neurological examination and attempts to separate sars-cov-2 from the neuronal tissue are required to provide direct neurotropic evidence of sars-cov-2 [16] . in a recent review [51] , authors have categorized the reported neurological findings related to covid-19 into three categories: a) central (headache, dizziness, impaired consciousness, acute cerebrovascular disease, ataxia, seizures, and special senses) b) peripheral (hypogeusia, hyposmia) c) musculoskeletal (ischemic or hemorrhagic) apart from the above, increasing evidence indicated that coronaviruses may invade the cns, causing neurological disorders. though headache has been the most common neurological manifestations reported by covid-19 patients [52, 53] , a systematic review of covid-19 patients concluded that the mean prevalence of headache was only 8% (95% ci 5.7-10.2%) [54] . apart from headache, dizziness is also a common neurological manifestations of covid-19, with a reported prevalence of between 7 and 9.4% [55, 56] . in an independent study, authors have reported that out of total 138 admitted patients in hospital, those who required icu care (n = 36) were more likely to report dizziness than those who did not (n = 102) (icu 8/36 [22.2%] vs. non-icu 5/102 [4.9%], p = 0.007). confusion and headache were the fifth (9/99 [9%]) and sixth (8/99 [8%] ) most common symptoms of the covid-19 patients (n = 99) at admission, respectively [57] . furthermore, it has recently been reported that several cases of covid-19 have concurrent severe neurological symptoms, such as acute cvd (acute ischemic stroke, cerebral venous sinus thrombosis, cerebral hemorrhage, subarachnoid hemorrhage), meningitis/encephalitis, acute necrotizing hemorrhagic encephalopathy, and acute guillain-barré syndrome [11, 58] . in [32, 35] . an observational study was done with 6147 covid-19 patients in fars province, iran, for the occurrence of seizures in patients with covid-19 and to clarify the circumstances of the occurrence of seizures in these patients. although authors have found seizure rate 0.08% ( as recognition of the disease has developed, the reported incidence of chemo-sensitive impairment (loss of taste and smell) in covid-19 has been considerably higher than in the initial stages, ranging from 19.4 to 88% [33, 59] ; also, as stated by bookstaver et al., the clinical symptoms of viral infection are usually nonspecific and sometimes may be mild or symptomless [17] . therefore, the prevalence of neurological manifestations was very likely underestimated in the early stages of the covid-19 outbreak [17, 33, 60] . however, cvd is among the most prevalent comorbidities of covid-19 patients, especially in severe cases. chen et al. reported that cardiovascular and cerebrovascular diseases were the most prevalent (40/99; 40%) chronic underlying conditions [57] . they have reported that out of total 138 hospitalized patients, cvd (7/138, 5.1%) ranked fifth among the most common comorbidities, while the first to fourth comorbidities were hypertension ( [55] . furthermore, coronary heart disease as well as cvd has been confirmed to be independent risk factors associated with fatal outcomes [61] . most of the reported covid-19 patients from these observations are explainable as have been elderly (age ≥ 50 years old, 70%) who are statistically more likely to have comorbidities and thus more likely to progress into severe cases [62, 63] . additionally, elderly patients' cns comorbidities might not be underestimated rather more studies are warranted through clinical examinations for cns involvement of sars-cov-2. it has been reported that sars-cov-2-infected children tend to have a milder covid-19 disease with lower mortality [64] . however, the occurrence of underlying neurological disorder or cns comorbid condition may predispose them for severe covid-19 manifestations [65] . furthermore, children with neuromuscular disorders are high-risk patients for severe covid-19 disease. neuromuscular disorders exuberated the conditions related to respiratory muscle weakness, tracheostomy, non-invasive or invasive ventilation, weak cough, and compromised airway clearance and predispose for severe covid-19 conditions [66] . in a case-series study, abdel-mannan et al. found that out of the 27 children with covid-19 pediatric multisystem inflammatory syndrome, 4 patients (14.8%) who were previously healthy had new-onset neurological symptoms. symptoms included encephalopathy, headaches, brainstem and cerebellar signs, muscle weakness, and reduced reflexes. authors have concluded that children with covid-19 presented with new neurological symptoms involving both the central and peripheral nervous systems and splenial changes on imaging, in the absence of respiratory symptoms. however, they have warranted additional research to assess the association of neurological symptoms with immune-mediated changes among children with covid-19 as well as close neurodevelopmental surveillance is required to assess the neurological and cognitive outcomes in these patients [67] . therefore, in view of the above reported neurological manifestations associated with covid-19, further studies should be carried out to gather evidences on the best therapeutic and management options of covid-19 and associated comorbidities. however, at present, prevention aimed on reducing transmission in the community remains the only proven efficient option to combat covid-19, until the pharmacotherapies and/or vaccines are developed. during the current context of the covid-19 pandemic, clinicians and healthcare workers should be aware of a wide spectrum of neurological manifestation associated with covid-19 along with their signs and symptoms for initial diagnosis and isolation of the patients. there are increasing evidences that indicated that coronaviruses may invade the cns and exuberated neurological disorders. an increasing number of reports and inclusive clinical data have been emerged out for covid-19 patients of different age groups with severe neurological symptoms. moreover, further clinical studies still needed to bring a reasonable concern of sars-cov-2, being a new neuropathogenic agent that 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study. key: cord-252049-rgdynmla authors: tomar, sakshi; johnston, melanie l.; st. john, sarah e.; osswald, heather l.; nyalapatla, prasanth r.; paul, lake n.; ghosh, arun k.; denison, mark r.; mesecar, andrew d. title: ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3cl(pro)): implications for nsp5 regulation and the development of antivirals date: 2015-06-08 journal: journal of biological chemistry doi: 10.1074/jbc.m115.651463 sha: doc_id: 252049 cord_uid: rgdynmla all coronaviruses, including the recently emerged middle east respiratory syndrome coronavirus (mers-cov) from the β-cov subgroup, require the proteolytic activity of the nsp5 protease (also known as 3c-like protease, 3cl(pro)) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. kinetic studies indicate that in contrast to 3cl(pro) from other β-cov 2c members, including hku4 and hku5, mers-cov 3cl(pro) is less efficient at processing a peptide substrate due to mers-cov 3cl(pro) being a weakly associated dimer. conversely, hku4, hku5, and sars-cov 3cl(pro) enzymes are tightly associated dimers. analytical ultracentrifugation studies support that mers-cov 3cl(pro) is a weakly associated dimer (k(d) ∼52 μm) with a slow off-rate. peptidomimetic inhibitors of mers-cov 3cl(pro) were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that mers-cov 3cl(pro) undergoes significant ligand-induced dimerization. kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. primary sequence comparisons and x-ray structural analyses of two mers-cov 3clpro and inhibitor complexes, determined to 1.6 å, reveal remarkable structural similarity of the dimer interface with 3cl(pro) from hku4-cov and hku5-cov. despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control mers-cov 3cl(pro) dimerization. activation of mers-cov 3cl(pro) through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of mers-cov 3cl(pro) inhibitors as antiviral agents. states (23) , there is an urgent need to study and characterize the properties of important drug targets of mers-cov for the development of effective therapeutics. coronaviruses express a ͼ800-kda replicase polyprotein, which is processed by viral 3cl pro protease (or nsp5) at 11 distinct cleavage sites to yield intermediate and mature nonstructural proteins (nsp) responsible for many aspects of virus replication (3, 24 -26) . because of its indispensable role in the virus life cycle, 3cl pro is an important target for therapeutic intervention against coronavirus infections (27) (28) (29) (30) (31) (32) (33) . a number of kinetic, biophysical, and x-ray structural studies have demonstrated that sars-cov 3cl pro is only active in vitro as a tightly associated dimer with a dimer dissociation constant (k d ) in the low nanomolar range (34 -38) . the addition or deletion of amino acids, e.g. his 6 affinity tags, at either the n or c terminus drastically reduces the enzymatic rate and decreases the ability of sars-cov 3cl pro to dimerize (37) . although cellular evidence for the auto-cleavage mechanism (cis versus trans) of 3cl pro is lacking, models for how 3cl pro cleaves itself from the polyprotein to form the mature dimer have been proposed based on in vitro studies using purified 3cl pro (34, 39, 40) . a current model posits that two inactive 3cl pro molecules within two separate polyproteins recognize each other and form an immature dimer capable of cleaving the nsp42nsp5 and nsp52nsp6 sites in trans, followed by formation of an active and mature dimer that can then rapidly process other cleavage sites and multiple polyproteins. it has also been proposed that substrate-induced dimerization regulates the enzymatic activity of sars-cov 3cl pro during virus replication; however, no experimental evidence of this has ever been demonstrated in infected cells (40) . although our knowledge of sars-cov 3cl pro is extensive, the dimerization properties of 3cl pro from mers-cov and other coronaviruses, as well as the factors regulating their enzymatic activity, remain largely unknown. to understand the properties of mers-cov 3cl pro , we conducted a series of kinetic, biophysical and x-ray structural studies. here, we report a detailed kinetic and biophysical analysis of mers-cov 3cl pro activity and dimerization. these kinetic and biophysical studies provide evidence for a weakly associated mers-cov 3cl pro dimer. in addition, we utilized our previous knowledge on the design of potent sars-cov 3cl pro peptidic inhibitors to design a series of inhibitors of mers-cov 3cl pro that exhibit low micromolar potency. we demonstrate that mers-cov 3cl pro requires the binding of a ligand for dimer formation, indicating that ligand-induced dimerization is likely a key mechanism in the regulation of mers-cov 3cl pro activity during virus infection. construct design and expression of mers-cov 3cl pro -the gene encoding 3cl pro protease of mers-cov (amino acid residues 3248 -3553 in the replicase polyprotein, genbank tm accession number ahc74086.1) was codon-optimized for optimal expression in e. coli (biobasic inc). the gene was subcloned into pet-11a expression vector with an n-terminal his 6 tag followed by the nsp42nsp5 auto-cleavage site using the forward primer 5ј-atatacatatgcaccaccaccac-caccacagcggtgttctgcagtctggtc-3ј and the reverse primer 5ј-gacggatccttactgcatcacaa-cacccatgatctgc-3ј. the construct was verified by dna sequencing at the purdue university genomics core facility. this construct results in the expression of mers-cov 3cl pro without any n-or c-terminal extensions. mers-cov 3cl pro was expressed through auto-induction in escherichia coli bl21-de3 cells in the presence of 100 g/ml carbenicillin as described previously (41) . cells were harvested by centrifugation at 5000 ϫ g for 20 min at 4°c, and the pellets were stored at ϫ80°c until further use. mers-cov 3cl pro purification-frozen pellets from 4 liters of bacterial cell culture were thawed on ice and resuspended in 250 ml of buffer a (20 mm tris, ph 7.5, 0.05 mm edta, 10% glycerol, and 5 mm ␤-mercaptoethanol (bme)), containing 500 g of lysozyme and a small amount of dnase. cells were then lysed using a single pass through a french press at 1200 p.s.i., and cell debris was removed from the cleared lysate by centrifuging at 29,000 ϫ g for 30 min. solid ammonium sulfate was added to the cleared lysate to a final concentration of 1 m through gradual mixing on ice. hydrophobic interaction chromatography-the cleared lysate, mixed with ammonium sulfate, was loaded at a flow rate of 3 ml/min onto a 60-ml phenyl-sepharose 6 fast-flow highsub column (amersham biosciences) equilibrated with buffer b (50 mm tris, ph 7.5, 1 m ammonium sulfate, 0.05 mm edta, 10% glycerol, and 5 mm bme). the column was then washed with 5ϫ column volume (300 ml) of buffer b at a flow rate of 4 ml/min. protein was eluted using a 5ϫ column volume (300 ml) linear gradient to 100% buffer a. fractions (12 ml) were collected, and those containing mers-cov 3cl pro , as judged through sds-page analysis and specific activity measurements, were pooled (120 ml) and exchanged into 2 liters of buffer a via overnight dialysis in a 10,000 molecular weight cutoff snakeskin dialysis tubing (thermo scientific). deae anion-exchange chromatography-dialyzed sample from the previous step was loaded at a flow rate of 3 ml/min onto a 120-ml deae anion-exchange column (amersham biosciences) equilibrated with buffer a. the column was then washed with 2ϫ column volume (240 ml) of buffer a at a flow rate of 4 ml/min. a linear gradient (total volume 480 ml) to 40% buffer c (50 mm tris, ph 7.5, 1 m nacl, 0.05 mm edta, 10% glycerol, and 5 mm bme) was used to elute the protein. fractions (6 ml) were collected, and those containing mers-cov 3cl pro were pooled (66 ml) and dialyzed for 4 h in 4 liters of buffer d (20 mm mes, ph 5.5, 0.05 mm edta, 10% glycerol, and 5 mm bme). mono s cation-exchange chromatography-following dialysis, the ph of the sample was manually adjusted to 5.5 using 1 m solution of mes, ph 5.5, and any precipitated protein was removed by filtering through a 0.22-m pore size millex-gp filter (millipore). the filtered sample was then loaded at a flow rate of 2 ml/min onto an 8-ml mono s 10/100 column (amersham biosciences) equilibrated in buffer d. the column was then washed with 5ϫ column volume (40 ml) of buffer d at a flow rate of 2 ml/min. protein was eluted using a 25ϫ column volume (200 ml) and a linear gradient to 50% buffer e (50 mm mes, ph 5.5, 1 m nacl, 0.05 mm edta, 10% glycerol, and 5 mm bme). fractions (2 ml) were collected, and those containing mers-cov 3cl pro were pooled (22 ml) and concentrated to ϳ5 mg/ml. gel filtration chromatography-as the final purification step, the concentrated protein sample was loaded onto the preparation grade superdex 75 26/60 gel filtration column (amersham biosciences) equilibrated with buffer f (25 mm hepes, ph 7.5, 10% glycerol, 2.5 mm dithiothreitol (dtt)). protein was eluted isocratically at a flow rate of 1 ml/min with buffer f. fractions (2 ml) containing mers-cov 3cl pro were pooled (total volume of 34 ml) and concentrated to ϳ5 mg/ml. for final storage of the purified mers-cov 3cl pro enzyme, 300-l protein aliquots were placed into 1-ml screw-cap vials, flash-frozen under liquid nitrogen, and then stored at ϫ80°c until further use. purification of sars-cov, hku4-cov, and hku5-cov 3cl pro -sars-cov 3cl pro and hku5-cov 3cl pro with authentic n and c termini were expressed and purified as described previously (37, 42) . hku4-cov 3cl pro was purified utilizing a modified protocol from ref. 42 . final protein yield was calculated based on the measurement of total activity units (m product/min), specific activity (units/mg), and milligrams of protein obtained (bio-rad protein assay) after each chromatographic step. synthesis of compounds 1-11-the peptidomimetic compounds with michael acceptor groups (compounds 1-9, table 3 ) were synthesized via very similar methods to those published previously (30, 43) . synthesis of noncovalent peptidomimetic compounds 10 and 11 (table 3 ) has been described previously (33) . fluorescence-based kinetic assays-the enzymatic activity of 3cl pro was measured using the following custom-synthesized peptide: hilytefluor tm -488-esatlqsglrkak-(qxl tm -520)-nh 2 (anaspec, inc.). the hilytefluor tm -488 fluorescence group was internally quenched by qxl tm -520 dye. this substrate works as a generic peptide substrate for 3cl pro enzymes and was designed based on the nsp42nsp5 cleavage sequence for many coronavirus 3cl pro enzymes. the rate of enzymatic activity was determined at 25°c by following the increase in fluorescence ( excitation ϭ 485 nm, emission ϭ 528 nm, bandwidths ϭ 20 nm) of hilyte fluor tm -488 upon peptide hydrolysis by the enzyme as a function of time. assays were conducted in black, half-area, 96-well plates (corning glass) in assay buffer (50 mm hepes, ph 7.5, 0.1 mg/ml bsa, 0.01% triton x-100, and 2 mm dtt) using a final reaction volume of 100 l. the resulting florescence was monitored using a biotek synergy h1 plate reader. the rate of the reaction in arbitrary fluorescence units/s (afu/s) was determined by measuring the initial slope of the progress curves, which were then converted to units of micromolars of product produced per min (m/min) using experimentally determined values of fluorescence "extinction coefficient" as described previously (37) . all reactions were carried out in triplicate. determination of enzymatic efficiency-the apparent enzymatic efficiency for each of the 3cl pro enzymes was determined by measuring the rate of enzymatic activity as a function of varying substrate concentration in 100-l reactions. reactions were initiated by the addition of enzyme to the wells of an assay plate containing varying concentrations of substrate. the final substrate concentrations varied over a range from 0 to 2 m. the final enzyme concentrations for each 3cl pro studied were as follows: mers-cov 3cl pro at 1 m, sars-cov 3cl pro at 100 nm, hku5-cov 3cl pro at 250 nm, and hku4-cov 3cl pro at 200 nm. because 3cl pro enzymes cannot be saturated with this substrate at a substrate concentration that would still allow accurate fluorescent measurements without the inner filter effect, only the apparent k cat /k m values can be determined from the slope of the line that results from a plot of the enzymatic activity (y axis), normalized for the total enzyme concentration, against the substrate concentration (x axis). influence of dimerization on the activity of 3cl pro enzymes-the dependence of the enzymatic activity on the total enzyme concentration was determined using the fret-based assay described above. the final enzyme concentrations were varied over a concentration range from 2 m to 100 nm for mers-cov 3cl pro , 500 to 10 nm for sars-cov 3cl pro , 250 to 0.6 nm for hku5-cov 3cl pro , and 200 to 10 nm for hku4-cov 3cl pro . reactions were initiated by the addition of substrate, at a final concentration of 2 m, to the assay plates containing varying enzyme concentrations in the assay buffer. initial rates were determined from the initial slopes of the progress curves at each enzyme concentration. the rates of the 3cl pro -catalyzed reactions measured over a range of enzyme concentrations can be fit to either equation 1 or 2 to determine the values of the dissociation constant for the monomer-dimer equilibrium as well as the turnover numbers. nonlinear regression and the program tablecurve 2d version 4.0 were used to fit the data to either equation 1 or 2 below (44) . in equation 1, v max is the rate of the enzymatic activity calculated at each enzyme concentration (c t ); k d is the monomerdimer equilibrium dissociation constant, and k cat, m and k cat, d are the turnover numbers for the monomer and the dimer, respectively. in equation 2, v max , c t , and k d have been described previously, and k cat is the turnover number for the dimer only. inhibition assays-to determine the percent inhibition for compounds 1-9, the total concentration of the substrate was fixed at 1.0 m, and the enzymes were fixed at 250 nm for sars-cov 3cl pro , hku5-cov 3cl pro , hku4-cov 3cl pro , and at 500 nm for mers-cov 3cl pro . dmso stocks (100ϫ) of the compounds were diluted a hundred-fold to a final concentration of 50 m in 80 l of the enzyme solution and incubated for 20 min. after 20 min, the enzymatic activity was measured as initial slope of the progress curve, obtained by initiating the reaction with 20 l of 5 m substrate. % inhibition was calculated using equation 3 . in equation 3, rate sample is the initial slope of the progress curve in afu/s measured in the presence of the compound; rate pos is the initial slope measured in the absence of any compound, and rate neg is the baseline substrate hydrolysis calculated in the absence of enzyme. all the reactions were carried out in triplicate and contained a final dmso concentration of 1%. for compounds displaying more than 50% inhibition, a more extensive characterization of the inactivation kinetics was performed through progress curve analysis. to the reaction well, 20 l of 5 m substrate was added to a final concentration of 1 m, and the total inhibitor concentration [i] total was varied from 0 to 50 m. the reaction was initiated with the addition of 80 l of mers-cov 3cl pro to a final concentration of 500 nm. fluorescence intensity was then measured over time as afu t for a period of 70 min. equation 4 describes the resulting time course of reaction. in equation 4 , v i is the initial velocity of the reaction; k obs is the observed first-order rate constant for the reaction in the absence and presence of inhibitor; t is the time in minutes; [p] t is the concentration of product produced at time t, and [p] i is the initial product concentration, which is zero. product concentrations were calculated from the values of afu t , using the experimentally determined fluorescence extinction coefficient. the resulting values of [p] t were then plotted against time t, and the data were fit to equation 4 with [p] i ϭ 0 using the nonlinear regression program tablecurve 2d to derive the fitted parameters v i and k obs and their associated errors ⌬v i and ⌬k obs . values for each k obs were then plotted against [i] total and the data were fit to equation 5 . in equation 5, k inact defines the maximum rate of inactivation at infinite inhibitor concentration, and k i defines the concentration of inhibitor that yields a rate of inactivation equal to 1 ⁄ 2k inact . the half-life of inactivation at infinite inhibitor concentration, which is a measure of inactivation efficiency, is defined as t 1 ⁄2 ∞ ϭ 0.693/k inact . auc analysis-to determine the oligomeric state of mers-cov 3cl pro , sedimentation velocity experiments were performed at 20°c on the beckman-coulter xla ultracentrifuge using varying concentrations of mers-cov 3cl pro (4 -23 m) in 25 mm hepes, ph 7.5, 50 mm nacl, and 1 mm tris(2-carboxyethyl)phosphine at 50,000 rpm. to characterize the effect of the ligand on the monomer-dimer equilibrium of mers-cov 3cl pro , sedimentation velocity experiments were conducted on the beckman-coulter xli instrument using different stoichiometric ratios of mers-cov 3cl pro with compounds 6 and 10. samples were prepared by mixing 25 m mers-cov 3cl pro with 25, 50, and 100 m compound 6 or 10 and incubating the mixture overnight at 4°c before performing the experiments. absorbance optics (280 nm) and interference optics were utilized for protein detection. solvent density, viscosity, and partial specific volumes were calculated using sednterp. sedphat was used to fit the data to the monomer-dimer self-association model to estimate the sedimentation coefficients (s), apparent molecular weights, and k d and k off values from size distribution analysis. to obtain exact molecular weights, sedimentation equilibrium experiments were performed at concentrations of 3 and 17 m mers-cov 3cl pro . the experiments were done at 20°c utilizing a twochannel centerpiece and run at multiple speeds (8100, 13,800 and 24,000 rpm) in a an-60 ti rotor. the rate sample , rate pos , and rate neg are as described above for equation 3 . mers-cov 3cl pro crystallization, x-ray data collection, and structure determination-purified mers-cov 3cl pro was concentrated to 1.6 mg/ml in 25 mm hepes, ph 7.5, and 2.5 mm dtt. inhibitor complexes of mers-cov 3cl pro with compounds 6 and 11 were formed by incubating mers-cov 3cl pro with the compounds in a 1:3 stoichiometric ratio at 4°c overnight. after iterative rounds of optimization of the crystallization conditions based on the initial hits obtained from high throughput screening of qiagen nextel screens, crystals of mers-cov 3cl pro inhibitor complexes suitable for x-ray diffraction were grown by the hanging-drop, vapor diffusion method at 20°c in 0.2 m sodium acetate, 0.1 m bistris, ph 7.0, and 20% peg-3350 for the mers-cov 3cl pro and 6 complex, and 0.2 m ammonium acetate, 0.1 m bistris, ph 5.5, 12% peg-3350 for the mers-cov 3cl pro and 11 complex. for x-ray data collection, crystals were flash-cooled in liquid nitrogen after dragging the crystals through a cryo-solution that contained the crystallization solution supplemented with 15% 2-methyl-2,4-pentanediol. x-ray diffraction data were collected for mers-cov 3cl pro and 6 and mers-cov 3cl pro and 11 complexes at the lilly research laboratories collaborative access team (lrl-cat) sector 31 and the life sciences collaborative access team (ls-cat) sector 21 at the advanced photon source, argonne national laboratory, respectively. data were processed and scaled using mosflm version 7.0.5 (45) and hkl2000 version 706 (46) . the method of molecular replacement was used to obtain initial phases using the program phaser-mr in phenix suite version 1.8.4 (47) . for mers-cov 3cl pro and 6 complex, the x-ray structure of sars-cov 3cl pro (pdb code 3v3m) was used as a phasing model (32) . the final mers-cov 3cl pro and 6 complex structure was then used to calculate the initial phases for the mers-cov 3cl pro and 11 complex model. automated model building using autobuild in phenix was initially used to build a preliminary model of the mers-cov 3cl pro and 6 inhibitor complex. each structure was then refined using iterative cycles of refinement using phenix refine coupled to manual model building using coot (48) based on f o ϫ f c and 2f o ϫ f c maps. coordinates and molecular library files for inhibitor molecules were built using the program elbow in the phenix suite. water molecules were added to peaks in residual (f o ϫ f c ) density maps that were greater than 3 using the "find water" function in coot. molprobity was used to assess structural quality of the final model (49) . the measured structure factor amplitudes and the atomic coordinates for the final structures were deposited in the protein data bank with accession codes 4rsp (mers-cov 3cl pro and 6 complex) and 4ylu (mers-cov 3cl pro and 11 complex), respectively. structural superposition was performed using the method of least squares fitting of c-␣ atoms in coot. pymol was used to generate figures of all the structures (50) . termini-insertion of the nsp42nsp5 cleavage site between the n-terminal his 6 tag and the coding region for mers-cov 3cl pro results in autoprocessing of the his tag and overexpres-sion of mers-cov 3cl pro without any n-terminal extension in e. coli bl21-de3 cells. mers-cov 3cl pro was purified to high purity and an overall yield of 10% using four sequential chromatographic steps. a summary of the percent enzyme yield, total activity units, and the fold-purification after each chromatographic step is summarized in table 1 . approximately 12 mg of highly pure mers-cov 3cl pro can be obtained per liter of bacterial cell culture. to verify the production of the enzyme with correct n and c termini, the molecular mass of purified mers-cov 3cl pro was determined by maldi to be 33.4 kda, which is close to the theoretical molecular mass of 33.3 kda for the authentic/mature mers-cov 3cl pro monomer. western blot analysis of purified mers-cov 3cl pro using an anti-his 6 antibody also confirmed the absence of the n terminus his 6 tag associated with the expression plasmid (data not shown). these results demonstrate that the n-terminal his 6 tag is auto-catalytically removed by mers-cov 3cl pro during its expression in e. coli, indicating mers-cov 3cl pro is enzymatically active when expressed in e. coli. fret-based peptide substrate was used to measure the enzymatic activity of mers-cov 3cl pro as a function of substrate concentration over a substrate concentration range from 0 to 2.0 m (fig. 1a) . we observed that mers-cov 3cl pro cannot be saturated by the substrate over this concentration range, which is typical for other coronavirus 3cl pro enzymes because the k m values for peptide substrates approach 1 mm (51) (52) (53) (54) . therefore, the slope of the kinetic response of mers-cov 3cl pro to increasing substrate concentration was determined to derive an apparent (k cat /k m ) value, which is a measure of enzymatic efficiency. we also determined and compared the apparent (k cat /k m ) values for 3cl pro enzymes from sars-cov, hku5-cov, and hku4-cov under similar experimental conditions (fig. 1b) . mers-cov 3cl pro is able to hydrolyze the peptide substrate; however, the enzymatic efficiency of mersis noticeably lower than other 3cl pro enzymes tested. specifically, mers-cov 3cl pro was 5-fold less efficient at processing the peptide substrate when compared with sars-cov 3cl pro . even among the ␤-covs from the same 2c genogroup (mers, hku5, and hku4), mers-cov 3cl pro was the least efficient enzyme. mers-cov 3cl pro is a weakly associated dimer-because a dimer has consistently been shown to be the catalytically active form of all 3cl pro enzymes studied to date, we tested the hypothesis that the lower enzymatic efficiency of mers-cov 3cl pro is a result of the reduction in its ability to dimerize. therefore, we determined the dependence of the enzymatic activity of mers-cov 3cl pro on the total enzyme concentration and compared it with other 3cl pro enzymes from hku4, hku5, and sars coronaviruses (fig. 2) . it is immediately apparent from the data plotted in fig. 2 that the response of mers-cov 3cl pro enzymatic activity to an increasing enzyme concentration is nonlinear. the strong curvature suggests that a dimer is either the most active form or the only active form of mers-cov 3cl pro . to determine the mechanism of dimerization, the data in fig. 2 were first fit to equation 1 (see "experimental procedures"), which describes a model where both the monomer and the dimer are active. a fit of the data to equation 1 yielded a negative turnover value for the monomer (k cat, m ), suggesting the monomer is inactive and that the dimer is the only active form of the enzyme. therefore, the data were fit to equation 2 (see "experimental procedures"), which considers only the dimer as the active form of the enzyme. the kinetic data for all four 3cl pro enzymes, mers-cov, hku4-cov, hku5-cov, and sars-cov, fit well to this model, and the resulting values for the monomer-dimer equilibrium dissociation constant, k d , and apparent turnover number, k cat , for each enzyme are provided in table 2 . the lower k cat value for mers-cov 3cl pro , when compared with other coronavirus 3cl pro enzymes, indicates a moderate reduction (2-4-fold) in its ability to turn over the substrate, which is consistent with the observed lower apparent (k cat /k m ) value. in contrast, there is a substantial reduction in the ability of mers-cov 3cl pro to dimerize compared with the other 3cl pro enzymes. based on the k d values, the capacity of mers-cov 3cl pro to dimerize is ϳ78 -130-fold weaker than the other enzymes ( table 2 ). these results indicate that the mers-cov 3cl pro dimer is much more weakly associated than the other coronavirus 3cl pro enzymes studied, and these results raise questions as to the structural and mechanistic differences among the 3cl pro enzymes that ultimately regulate protease activity during coronavirus replication. mers-cov 3cl pro inhibition by designed peptidomimetic compounds-in an effort to develop potent inhibitors of mers-cov 3cl pro , we designed and synthesized nine peptidomimetic compounds containing a michael acceptor group, i.e. an ␣,␤-unsaturated carbonyl, capable of irreversibly reacting with the active site cysteine of mers-cov 3cl pro (table 3) . these compounds were designed and synthesized based on our understanding and knowledge of the interactions of similar inhibitor molecules with sars-cov 3cl pro (30, 31) . at a concentration of 50 m, compounds 6 -9 displayed more than 50% inhibition of mers-cov 3cl pro and were further evaluated for their ability to inactivate the enzyme in a time-and concentration-dependent manner (fig. 3) . data from the kinetic progress curve for compound 6 (fig. 3) , as well as for table 2 . final enzyme concentrations varied over the concentration ranges of 2 m to 100 nm for mers-cov 3cl pro , 500 to 10 nm for sars-cov 3cl pro , 250 to 0.6 nm for hku5-cov 3cl pro , and 200 to 10 nm for hku4-cov 3cl pro . final substrate concentration was fixed at 2 m. experiments were done in triplicate. error bars represent the standard deviation for triplicate data. shaded box represents the data that are plotted in b. b, enlarged view of the fitted data at low total enzyme concentrations, marked in shaded box in a, illustrating the nonlinear dependence of enzymatic activity on the total concentrations of 3cl pro from sars-cov, hku5-cov, and hku4-cov. the michael acceptor group for compound 1 is shaded to highlight this group for all the compounds. the stereochemistry at the benzyl stereocenter of compound 5 is a 1:1 mixture of enantiomers (racemic); therefore, the compound was tested as a mixture of diastereomers. * % inhibition was measured as the % loss in enzymatic activity after 20 min of incubation of 500 nm mers-cov 3cl pro with 50 m of the compound. a as compounds 1-5 showed ͻ50% inhibition of mers-cov 3cl pro , values of k inact , t 1/2 ∞ and k i were not determined (nd) for these compounds. 50 compounds 7-9 (data not shown), were fit to the appropriate equations (see under "experimental procedures") to obtain the kinetic parameters, k inact , t 1 ⁄2 ∞ , and k i , and the resulting values are provided in table 3 . we identified four compounds, 6 -9, as micromolar inhibitors of mers-cov 3cl pro with k i values less than 10 m ( table 3) . analysis of structure-activity relationships of these compounds suggests that the s 2 subsite pocket of mers-cov 3cl pro is small and can only accommodate a smaller p 2 -isobutyl substituent (compounds 6-9) but not bigger substituents such as p 2 -benzyl or p 2 -isobutylenyl (compounds 1-5). it was also observed that replacing the p 4 -ethoxy (compound 6) with p 4 -isopropoxy (compounds 7 and 8) had no effect on the inhibitory activity of the compounds. finally, these compounds provide an excellent chemical scaffold to study the molecular details of interactions of substrate-like compounds with the enzyme and to develop more potent inhibitors of mers-cov 3cl pro for therapeutic intervention. to evaluate broad spectrum specificity of these compounds, we also calculated % inhibition of sars-cov 3cl pro , hku5-cov 3cl pro , and hku4-cov 3cl pro after 20 min of incubation in the presence of 50 m compounds 6 -9. except for compound 9, which inhibited sars-cov 3cl pro by 76%, we observed 100% inhibition of all other enzymes in the presence of compounds 6-9. furthermore, we performed progress curve analysis of hku5-cov 3cl pro and hku4-cov 3cl pro in the presence of varying concentrations of compounds 6 -9. the k i values of compounds 6 -9 for hku5-cov 3cl pro are 0.49 ϯ 0.16, 0.60 ϯ 0.21, 1.30 ϯ 0.53, and 0.47 ϯ 0.06 m, respectively. the k i values of compounds 6 -9 for hku4-cov 3cl pro are 0.39 ϯ 0.14, 0.50 ϯ 0.17, 0.85 ϯ 0.33, and 0.64 ϯ 0.25 m, respectively. these data suggest that peptidomimetic compounds 6 -9 have the potential to be developed as coronavirus 3cl pro inhibitors with broad spectrum specificity. weak association of the mers-cov 3cl pro dimer is supported by auc studies-to further explore the mechanism of mers-cov 3cl pro dimerization, we performed analytical ultracentrifugation sedimentation velocity (auc-sv) studies at varying concentrations of mers-cov 3cl pro (fig. 4a) . unlike enzyme kinetics, auc allows determination of the monomer-dimer equilibrium constant (k d ) in the absence of substrate. mers-cov 3cl pro displayed a continuous size distribution at different protein concentrations. two distinct peaks corresponding to monomer (2.9 s) and dimer (3.9 s) species are observed, with the dimer peak becoming more pronounced at higher enzyme concentrations (fig. 4a) . we fit the auc data to a monomer-dimer equilibrium model to determine the values for k d and k off , where k d is the equilibrium dissociation constant for a monomer from the dimer, and k off is the rate constant for dissociation of the monomer from the dimer. the resulting best fit value for k d is 52 ϯ 5 m and that for k off is 10 ϫ4 s ϫ1 . the k d value of 52 m for mers 3cl pro is dramatically different from sars-cov 3cl pro , which has reported k d values ranging from low nanomolar up to 10 m depending on the enzyme construct used and the experimental conditions and methods utilized to determine the dissociation constant (37) . the dimer affinity of mers-cov 3cl pro is substantially weaker than that for sars-cov 3cl pro , when comparing the same enzyme construct, i.e. the enzyme without any n-or c-terminal modifications. the auc-sv calculated k d value for mers-cov 3cl pro is ϳ150,000 times higher than the value of 0.35 nm determined for sars-cov 3cl pro (34) . the auc results (fig. 4a) show that the monomer peak at ϳ2.9s does not gradually shift peak position toward the dimer peak at ϳ3.9s with increasing concentrations of mers-cov 3cl pro ; rather, the two peaks change in area, which is indicative of very slow monomer-dimer exchange rate (k off ϳ10 ϫ4 s ϫ1 ) and the formation of hydrodynamically stable monomer and dimer species (55) . this k off value is 1000 times slower than the k off value (10 ϫ1 s ϫ1 ) reported for sars-cov 3cl pro indicating that the sars-cov enzyme has a significantly more rapid monomer-dimer exchange rate (56) . these observations support a model whereby the mers-cov 3cl pro dimer is weakly associated, suggesting the enzyme exists mainly as a monomer in solution. mers-cov 3cl pro undergoes extensive ligand-induced dimerization-the weak association of mers-cov 3cl pro monomers engenders the following questions. "are higher levels of expression of 3cl pro in mers-cov-infected cells necessary to allow formation of active dimer?" "are other mechanisms such as substrate-or ligand-induced dimerizations involved in activating 3cl pro ?" to explore the latter question of ligand-induced dimerization of mers-cov 3cl pro , we performed auc experiments in the presence of compound 6, which acts as a substrate mimetic and mechanism-based inhibitor, also known as a suicide substrate. peptidomimetic compounds such as compound 6, which contains a michael acceptor group, interact and react with the active site cysteine of cysteine proteases to covalently modify them. we utilized compound 6 to form a covalent mers-cov 3cl pro and inhibitor 6 complex that is stable over long periods of time, making it amenable to analysis by auc-sv experiments. in contrast, incubation of a normal peptide substrate with the enzyme would lead to immediate hydrolysis of the substrate and dissociation of the products from the enzyme, confounding auc experiments and subsequent data analysis. mers-cov 3cl pro was incubated with varying concentrations of compound 6 in stoichiometric ratios of 1:1, 1:2, and 1:4. the modified enzyme was then subjected to auc studies to determine the influence of compound 6 on the monomer-dimer equilibrium (fig. 4b) . a significant shift in the area under 2.9s peak (monomer) to 4.1s peak (dimer) is detected upon addition of increasing concentrations of compound 6. we obtained similar results when auc studies were performed utilizing a complex of mers-cov 3cl pro with a noncovalent peptidomimetic inhibitor (compound 10, figs. 4c). the transition of mers-cov 3cl pro from monomer to dimer in the presence of compounds 6 and 10 suggests that the enzyme undergoes extensive dimerization upon substrate binding. the observed ligand-induced dimerization of mers-cov 3cl pro , as demonstrated through auc studies, prompted us to investigate whether or not the enzymatic activity of mers-cov 3cl pro could be increased at low concentrations of a compound via ligand-induced dimerization. to do so, we chose to use a noncovalent peptidomimetic compound (compound 10, fig. 5a ) that we previously identified as an inhibitor of sars-cov 3cl pro . because of the time-dependent, irreversible nature of the reaction between compound 6 and mers-cov 3cl pro , use of compound 6 was not ideal for these kinetic studies as it would further complicate kinetic data analysis. the kinetic response of mers-cov 3cl pro to increasing concentrations of compound 10 was first measured at a single enzyme concentration of 1.0 m (fig. 5a) . interestingly, an increase in the activity of mers-cov 3cl pro , as high as 195%, was observed in the presence of low inhibitor concentrations (0.1 to 20 m). inhibition of enzymatic activity was observed only at higher inhibitor concentrations (40 m or greater). these results suggest that at low concentrations, compound 10 binds to a monomer and induces the formation of a dimer. the resulting dimer then has one free active site that is capable of processing the substrate. at higher concentrations of inhibitor, the substrate and inhibitor directly compete for the free active site. the model of activation and inhibition suggested by the data at 1 m enzyme would predict that at higher enzyme concentrations less activation by a compound would be observed at lower inhibitor concentrations, and the inhibition of activity would be detected at lower inhibitor concentrations because the equilibrium would be pushed toward dimer formation. in contrast, lower enzyme concentrations would result in higher activation by compounds, and inhibition by the compound would occur at significantly higher compound concentrations. therefore, we further measured the activity of mers-cov 3cl pro at two additional enzyme concentrations (0.5 and 2.0 m) in the presence of varying concentrations of compound 10. remarkably, we observed that the activation effect was most pronounced at the lowest mers-cov 3cl pro concentration tested (0.5 m), and the effect decreased as the enzyme concentration was increased (1.0 and 2.0 m) (fig. 5a ). moreover, inhibition by compound 10 occurred at lower compound concentrations when higher concentrations of enzyme were used. these observations further support a model whereby enzyme activation can occur through ligand-induced dimerization. the activation and inhibition of mers-cov 3cl pro by compound 10 can be explained by a simple kinetic model depicted in fig. 5b . the mers-cov 3cl pro monomer exists in equilibrium with the dimer, and their relative concentrations depend on the total enzyme concentration. in the absence of substrate or compound, the k d value is 52 m, and the equilibrium is represented by the gray spheres (blue box) in fig. 5b . the monomer is unable to hydrolyze the substrate and is therefore inactive. binding of inhibitor (fig. 5b, green triangle) to the monomer results in monomer to dimer switch leading to the formation of a dimer that contains inhibitor bound in one of the active sites. once the dimer is formed, the substrate binds in the second active site and catalysis takes place. under high inhibitor concentrations, however, the inhibitor molecule directly competes with substrate for the free dimer active site, and inhibition of the enzymatic activity is observed as a result. we would also expect to observe induced dimerization and activation in the presence of the substrate. indeed, the monomer-dimer kinetic studies performed in fig. 2 were performed at a fixed concentration of substrate at 2 m. in this experiment, the k d value for the mers-cov 3cl pro dimer was determined to be 7.8 m, which is lower than the k d value determined in the absence of substrate using auc, thereby supporting substrateinduced dimerization. given the high k m value of 3cl pro for the peptide substrate (51-54), even higher substrate concentrations would be required to observe substrate activation in a plot of catalytic activity versus substrate concentration. however, we are limited to use our fret-based substrate only at low concentrations due to a significant inner filter effect at higher concentrations of substrate. therefore, a compound that both mimics substrate and has higher binding affinity can act as a useful surrogate for the substrate, allowing the observation of ligand-induced dimerization and activation even at low substrate concentrations. x-ray structure of mers-cov 3cl pro in complex with compound 6-to gain atomic level detail and molecular insight into the mechanism for substrate-induced dimerization of mers-cov 3cl pro , we attempted to crystallize and determine the x-ray structures of the unliganded mers-cov 3cl pro monomer and the mers-cov 3cl pro covalently modified with compound 6. unfortunately, we were unable to crystallize the unliganded mers-cov 3cl pro monomer after multiple attempts, but we were able to crystallize and determine the x-ray structure of mers-cov 3cl pro in complex with compound 6 to a resolution of 1.6 å. the statistics for x-ray data collection, processing, and refinement are summarized in table 4 . the mers-cov 3cl pro and 6 complex crystallized as a biologically relevant, symmetrical dimer in space group c2 with one monomer in the asymmetric unit. electron density for the entire protein was clearly visible and strong electron density (f o ϫ f c ͼ4) was present for compound 6 within the active site (fig. 6a) . mers-cov 3cl pro has a smaller s 2 pocket than sars-cov 3cl pro -the active site of mers-cov 3cl pro bound with compound 6 is shown in fig. 6, a and b. compound 6 is covalently bound to the active site cysteine (cys-148) via a 1.8 å bond between the ␥-sulfur and the electrophilic ␤-carbon of the michael acceptor. the pј 1 -ethyl ester carbonyl, which mimics the carbonyl of the scissile bond in a substrate, forms a hydro-shaded box) . b, kinetic model describing the equilibrium between different species of mers-cov 3cl pro that are formed in the absence (blue box) and presence (green box) of a ligand is shown. based on the auc-calculated k d value of ϳ 52 m, mers-cov 3cl pro primarily exists as a monomer in solution in the absence of a ligand. upon ligand binding (inhibitor i in our case) to the monomer, the monomer-dimer equilibrium shifts toward dimer formation. next, under lower inhibitor concentrations (cyan-shaded box), substrate (s) binds in the second active site and catalysis takes place. however, under higher inhibitor concentrations (yellow-shaded box), inhibitor directly competes with the substrate for the second active site, and inhibition of the enzymatic activity is observed. gen bond with the backbone nh of gly-146 that forms part of the oxyanion hole (fig. 6b) . within the s 1 subsite, the p 1 -lactam carbonyl, which is a surrogate for the amide of p 1 -glutamine of substrates, participates in a hydrogen bonding interaction with the imidazole ring of his-166, and the p 1 -lactam nh forms a hydrogen bond with the carboxylate oxygen of glu-169. the p 2 -backbone amide nh forms a hydrogen bond with the side chain carbonyl of gln-192 (fig. 6b ). the p 2 -leucine side chain atoms of the inhibitor make hydrophobic contacts with the side chains of met-168 and leu-49 that line the s 2 subsite pocket. moreover, compared with the equivalent residue thr-25 in sars-cov 3cl pro , met-25 in the s 2 pocket of mers-cov 3cl pro is expected to reduce the size of the hydrophobic pocket, which is supported by our observed sar described above. the smaller size of the s 2 pocket in mers-cov 3cl pro is also consistent with the preference for a smaller leucine residue at the p 2 position of cleavage sites instead of a bulkier phenylalanine or methionine residue. indeed, analysis of the preference for leucine or phenylalanine at the p 2 position for the 11 3cl pro cleavage sites within the polyprotein of mers-cov shows that none of the 11 cleavage sites contain a phenylalanine residue at this position (fig. 6c) . leucine is the predominantly favored residue at this position followed by methionine. analysis of the cleavage sites from sars-cov, hku4-cov, and hku5-cov shows that none of the 11 cleavage sites from group 2c members (mers-cov, hku4-cov, and hku5-cov) contain a phenylalanine residue at the p 2 position; however, the sars-cov nsp52nsp6 cleavage site contains a phenylalanine residue at this position. other interactions are also observed to play a significant role in stabilizing the mers-cov 3cl pro -compound 6 complex. the p 3 -carbonyl and p 3 -nh participate in hydrogen bonding interactions with the backbone nh and carbonyl of glu-169. the p 4 -serine side chain is within hydrogen bonding distance of the side chain carboxamide of gln-195 and the backbone carbonyl of lys-191. x-ray structure of mers-cov 3cl pro in complex with a noncovalent inhibitor-we were also able to obtain diffraction quality crystals of mers-cov 3cl pro in complex with compound 11, which has an almost identical chemical structure as that of compound 10 (fig. 6d) . we previously showed that compounds similar to 10 and 11 act as potent noncovalent inhibitors of 3cl pro from sars-cov (33) . the x-ray structure of compound 11 bound to mers-cov 3cl pro was determined to a resolution of 2.1 å and the x-ray data collection, processing, and refinement statistics are summarized in table 4 . the mers-cov 3cl pro and 11 complex crystallized in space group p2 1 with two biologically relevant dimers in the asymmetric unit. the overall root mean square deviation between the c-␣ atoms of the four chains was less than 1 å, with the highest c-␣ root mean square deviation of 0.719 å between chains c and d. strong electron density (f o ϫ f c ͼ4) was present for compound 11 within all the four active sites of the two dimers (fig. 6d) . the binding orientation for compound 11 in the active site of mers-cov 3cl pro is similar to the binding orientation of related compounds in the active site of sars-cov 3cl pro (pdb code 4mds). the benzotriazole group binds in the s 1 subsite; phenyl propionamidyl occupies the sј 1 -s 2 subsite, and the thiophene group binds in the s 2 subsite. compound 11 also forms two direct and one water-mediated hydrogen bond interactions with amino acids in the mers-cov 3cl pro active site (fig. 6e) . the n3 of the benzotriazole ring forms a hydrogen bond with the side chain ⑀-nitrogen of conserved his-166, and the central acetamide oxygen forms a hydrogen bond with the backbone nh of conserved glu-169. the nh of the phenyl propionamidyl group interacts with backbone carbonyl oxygen of the catalytic his-41 residue through a water-mediated hydrogen bond, and the imidazole ring of his-41 engages with the phenyl ring of phenyl propionamidyl group through t-shaped stacking. the phenyl ring also form hydrophobic contacts with leu-49. interactions at the 3cl pro dimer interface-analysis of the mers-cov 3cl pro and 6 and mers-cov 3cl pro and 11 crystal structures reveals key differences between the dimer interface of mers-cov and sars-cov 3cl pro (pdb code 2alv) figure 6. x-ray crystal structure of mers-cov 3cl pro in complex with inhibitors. a, solvent-accessible surface (gray-shaded surface) of mers-cov 3cl pro and compound 6 complex. compound 6 is displayed in ball and stick model with atoms colored as follows: carbons (orange), nitrogens (blue), and oxygens (red). electron density associated with compound 6 is shown as an f o ϫ f c electron density difference map contoured to 3 (green mesh). substrate binding pockets s 4 -sј 1 are labeled, where asterisk indicates the electrophilic carbon of compound 6 that forms a c-s covalent bond with the active site cysteine cys-148. b, mers-cov 3cl pro and compound 6 complex with the mers-cov 3cl pro backbone represented as a ribbon model and relevant amino acids that interact with compound 6 represented as ball and sticks. mers-cov 3cl pro carbon atoms are colored blue, and compound 6 carbon atoms are colored orange. nitrogen atoms are colored blue, and oxygen atoms are colored red. catalytic residues cys-148 and his-41 are also shown. hydrogen bonds are depicted as red dashed lines. c, sequence logos showing amino acid conservation for the 11 polyprotein cleavage sites of different 3cl pro enzymes (mers-cov, hku5-cov, hku4-cov, and sars-cov), generated using the weblogo server (63) . residues p 2 -pј 1 are shown. height of each letter corresponds to the amino acid conservation at that position. d, solvent-accessible surface (gray-shaded surface) of mers-cov 3cl pro and compound 11 complex. compound 11 is displayed in ball and stick model. electron density associated with compound 11 is shown as a 2f o ϫ f c electron density difference map contoured to 1.5 (green mesh). functional groups of compound 11 with their corresponding binding pockets are highlighted in yellow, green, and blue ellipses. chemical structure of compound 11 is shown in the inset. e, interactions between mers-cov 3cl pro and compound 11 are illustrated. catalytic residues cys-148 and his-41 are also shown. hydrogen bonds are depicted as red dashed lines. ( fig. 7) (30) . two arginine residues, arg-4 and arg-298 (fig. 7 , a-c), form some of the key interactions at the dimer interface of sars-cov 3cl pro , and mutation of either of these amino acids results in a drastic loss of dimerization in sars-cov 3cl pro (36, 38) . interestingly, these two arginine residues (arg-4 and arg-298) are substituted in mers-cov 3cl pro by two hydrophobic residues (val-4 and met-298) that are unable to participate in the formation of hydrogen bonds or salt bridges. therefore, we initially thought that the loss of these key interactions might simply explain the ͼ100,000-fold weaker dimerization observed for mers-cov 3cl pro compared with sars-cov 3cl pro . surprisingly, however, structural analysis of the dimer interface from the available x-ray structure of hku4-cov 3cl pro (pdb code 2ynb; fig. 7, b and c) , and primary sequence alignment of 3cl pro from mers-cov, hku5-cov, hku4-cov and sars-cov (fig. 8) revealed that val-4 and met-298 are conserved between all the ␤-cov 2c members studied here. substantial differences between the ability of mers-cov 3cl pro and hku4/hku5-cov 3cl pro to dimerize, despite their high sequence identity, led us to the hypothesis that nonconserved residues between mers-cov and other ␤-cov 2c members that are remote from the dimer interface may play a significant role in dimer formation. analysis of nonconserved residues of mers-cov 3cl pro -analysis of our current crystal structures does not reveal a clear mechanism for the monomer to dimer switch of mers-cov 3cl pro upon ligand binding. therefore, we attempted to identify the nonconserved residues in mers-cov 3cl pro that might affect enzymatic activity due to their proximity to key residues involved in substrate binding and/or dimer formation. based on a sequence alignment, mers-cov 3cl pro contains ϳ24 nonconserved amino acids (pink arrows in fig. 8 ). upon analyzing the position of these amino acids in the crystal structure, we observed that a remarkable number of these amino acids are present in the loop regions. fig. 9a illustrates the nonconserved residues present in the loop regions as gray (monomer a) and pink (monomer b) spheres. interestingly, we also observed that there are hot spots in the protein structure where most of these amino acids are clustered. these hot spots include the n-terminal region, the active site region, the interdomain loop (loop between the catalytic fold and domain iii), and the domain iii. in mers-cov 3cl pro , nonconserved amino acid his-8, which forms van der waals contacts with lys-155 of the same monomer and thr-128 of the other monomer, is present at the end of the n-terminal finger (fig. 9 , b and c), whereas amino acids asp-12 and ala-15 are part of the n-terminal helix (fig. 9b) . additionally, amino acids thr-128, lys-155, and ser-158 are present within 6 å of the n-terminal region (fig. 9b ). substitution to these amino acids in mers-cov 3cl pro might have changed the protein dynamics in a way that only ligand binding populates the monomer conformation, which is more amenable to dimer formation. we also observe that some of the nonconserved residues in mers-cov 3cl pro are located in proximity to the substratebinding site and might contribute toward ligand-induced dynamic changes favorable for dimer formation. for example, nonconserved amino acid met-61 forms hydrophobic interactions with met-43, which in turn is in close proximity to the catalytic residue his-41 (fig. 9d) . residue ala-171 is present on a loop, and this loop, along with conserved residues his-166 and his-175, forms the s 1 subsite for binding the p 1 amino acid of the substrate (fig. 9e ). in addition to its influence on substrate binding, ala-171 may also contribute toward dimer formation upon substrate binding due to its close proximity with glu-169. this glutamate residue in sars-cov 3cl pro (glu-166) has been established as a key residue linking the substrate-binding site to the dimer interface (56) . val-132 forms hydrophobic interaction with other nonconserved residue ala-114 within domain ii (fig. 9f) . additionally, val-132 is present within van der waals contact distance of glul-290 from extrahelical domain iii (fig. 9f) . it is noteworthy that glu-290 forms a salt bridge with arg-4 across the dimer interface in sars-cov 3cl pro . however, this interaction is not formed in mers-cov 3cl pro due to the substitution of arg-4 with val-4. tyr-137 forms hydrophobic contacts with the conserved residue tyr-185 (fig. 9g) . besides amino acid val-132 that connects domains ii and iii, residue tyr-185, along with two other nonconserved residues, thr-183 and met-189, is present on the inter-domain loop that connects the catalytic fold (domains i and ii) with the extra-helical domain iii (fig. 9g) . flexibility within these residues might affect the orientation of domain iii required for dimer formation. model for regulation of the enzymatic activity of mers-cov 3cl pro during polyprotein processing-enzymatic activity of coronavirus 3cl pro is required for the processing of viral polyproteins at 11 distinct cleavage sites, allowing the release of nonstructural proteins that subsequently form a replication complex for virus genome replication. because of its indispensable role in the virus life cycle, regulation of the enzymatic activity of 3cl pro is instrumental for efficient replication of coronaviruses. based on our experimental results, we propose a model to explain the mechanism for regulating the enzymatic activity of mers-cov 3cl pro in the context of polyprotein processing during virus infection (fig. 10) . a number of in vitro studies performed on sars-cov 3cl pro have established the mechanism for 3cl pro auto-release from the polyprotein (34, 39, 40) . based upon these studies and our data on mers-cov 3cl pro , we propose the polyprotein processing model in fig. 10 . the steps proposed for auto-release of mers-cov 3cl pro from the polyprotein (steps 1-4, fig. 10 ) have been adapted from chen et al. (39) , where it is suggested that the n-terminal auto-processing does not require the formation of a mature 3cl pro dimer for sars-cov. based on the differences between the properties of sars-cov 3cl pro and mers-cov 3cl pro , as highlighted in our studies, we added two additional steps (steps 5 and 6, fig. 10 ) that mers-cov 3cl pro may need to utilize for efficient polyprotein processing. in fig. 10, step 1 , two immature mers-cov 3cl pro monomers in the polyprotein approach each other and form an immature dimer via interactions between domain iii, which allows each of the monomers to insert their n termini into the active site of the other monomer. in step 2, the n termini are cleaved, and the dimer with uncleaved c termini adopts a conformation similar the n and c termini are labeled, and the yellow cylinder labeled s represents a ligand that can be a peptide inhibitor, peptide substrate, or 3cl pro cleavage sites in the polyprotein. various steps required for the auto-release of 3cl pro from the polyprotein and subsequent processing of the polyprotein cleavage sites are described in the text. suggested by our auc and kinetic studies, the shaded region (steps 5 and 6) highlights the additional steps mers-cov 3cl pro would undertake during polyprotein processing and have been described in the kinetic model depicted in fig. 5b. to the mature dimer. our observation of auto-cleavage of the n-terminal his 6 tag from mers-cov 3cl pro during expression in bacterial cells supports steps 1 and 2, where formation of an immature dimer capable of auto-processing the n terminus occurs. in step 3, two dimers with uncleaved c termini approach each other, followed by insertion of the c terminus from one dimer into one of the active sites of the other dimer. in step 4, the c termini are cleaved and mature dimer is released from the polyprotein. for sars-cov, the 3cl pro dimer formed in step 4 continues to process cleavage sites in the polyprotein, effectively skipping steps 5 and 6 (red arrow in fig. 10 ) because the dimer is tightly associated. however, the high k d value of mers-cov 3cl pro dimer suggests that the active and mature dimer may dissociate into inactive, mature monomers in the absence of any ligand (step 5). in order for polyprotein processing to proceed, another step (step 6) must occur. in step 6, a substrate s, e.g. one of the 11 polyprotein cleavage sites, would induce dimer formation and hence activate catalysis and cleavage at the substrate recognition sites. our auc results and the kinetic activation studies performed in the absence and presence of inhibitors support steps 5 and 6 where the inactive but mature monomers require binding of a ligand to undergo ligand-induced dimerization and formation of an active, mature dimer that can then process the polyprotein cleavage sites. nonconserved amino acids of mers-cov 3cl pro may regulate the dimer formation-long range interactions have been reported to modulate dimerization and activity of 3cl pro enzymes. barrila et al. (57) demonstrated that mutation of a conserved amino acid ser-147, which is distant from the dimer interface, results in a total loss of dimerization and enzymatic activity of sars-cov 3cl pro . although ser-147 does not form direct interactions at the dimer interface, disruption of the dimer upon mutation stems from the fact that ser-147 makes several interactions with other residues involved in forming a hydrogen bonding network within sars-cov 3cl pro . site-directed mutagenesis studies on domain iii of sars-cov 3cl pro , where n214a and s284a/t285a/i286a mutants were characterized, revealed that despite being present on an entirely different domain, these residues affect catalysis through a network of residues undergoing correlated motions across the entire protease (58, 59) . utilizing 3cl pro temperature-sensitive mutants of mhv, stobart et al. (60) have also demonstrated that second-site mutation physically distant from the temperature-sensitive mutation suppresses the temperature-sensitive phenotype through long range interactions, thereby regulating 3cl pro enzymatic activity during polyprotein processing and virus replication. our studies also suggest that long range interactions among the nonconserved residues can significantly alter the properties of mers-cov 3cl pro . a detailed analysis of nonconserved residues of mers-cov 3cl pro among ␤-cov 2c members identified hot spots, including the n-terminal finger and helix, the active site region, the inter-domain loop, and the domain iii, where these residues are clustered. several studies done on sars-cov 3cl pro have demonstrated that amino acids from the n-terminal finger, the n-terminal helix, and domain iii significantly contribute toward dimer formation. in addition to the direct interactions at the dimer interface, correct orientation between the catalytic fold and domain iii is also crucial for dimer formation. wu et al. (61) showed that the mostdramaticdifferencebetweenthecrystalstructuresofmonomer and the ligand-bound dimer of the r298a mutant of sars-cov 3cl pro was a 33°rotation of domain iii (38) . this rotation results in a steric clash between domain iii from two monomers and would essentially block dimer formation. however, upon addition of a ligand, domain iii of the r298a mutant adopts the correct orientation and results in the formation of a dimer structure. similar to the sars-cov 3cl pro r298a mutant, ligand binding into the active site of the mers-cov 3cl pro monomer possibly stabilizes the inter-domain loop conformation that maintains domain iii in the correct orientation for dimer formation. most of the nonconserved residues within domain iii are present on the surface and also are distant from the dimer interface. these residues may be involved in providing the flexibility required for conformational changes during the monomer to dimer switch. we have identified several amino acids in mers-cov 3cl pro that may contribute to the dimer formation upon ligand binding. however, single amino acid mutagenesis alone is unlikely to reveal significant differences in the dimerization properties. as demonstrated by myers et al. (62) for ornithine decarboxylase, the response of single amino acid to ligand binding may be limited to only local conformational changes and may not have significant contribution toward dimer stability. however, local conformational changes in a network of residues may propagate larger effects that stabilize dimer formation upon ligand binding. analysis of the nonconserved residues of mers-cov 3cl pro discussed here sets forth a framework to perform systematic single or multiple mutagenesis studies to gain insights into the mechanism for ligand-induced dimerization of the enzyme. development of 3cl pro inhibitors with broad spectrum specificity-insights into the mechanistic and structural similarities as well as differences between 3cl pro enzymes from different coronavirus subgroups are instrumental for the development of 3cl pro inhibitors with broad spectrum specificity. to evaluate the broad spectrum specificity of our peptidomimetic compounds, we determined their inhibitory activity against 3cl pro from mers-cov, sars-cov, hku5-cov, and hku4-cov. our inhibitory data and k i values clearly show that compounds 6 -9 inhibit all the 3cl pro enzymes tested here. the x-ray structure of mers-cov 3cl pro in complex with compound 6 revealed that out of eight direct hydrogen bonds formed between compound 6 and mers-cov 3cl pro , four of these hydrogen bonds involve interactions with conserved structural elements of the peptide backbone of the enzyme. furthermore, the amino acids that form hydrogen bonds with compound 6 through side chain interactions are conserved in all the coronavirus 3cl pro enzymes evaluated here, as well as 3cl pro enzymes from other ␤-coronaviruses like mhv, oc43, and hku1. these results suggest that canonical structural features exist among the 3cl pro enzymes that can be exploited for structure-based design of broad spectrum inhibitors. for the noncovalent inhibitor compound 11, the x-ray structure reveals two direct hydrogen bonding interactions between the compound and mers-cov 3cl pro . one of the hydrogen bonds forms with the side chain ⑀-nitrogen of conserved his-166, and the second involves the backbone nh of conserved glu-169. we speculate these interactions remain conserved in other 3cl pro enzymes as well, because his-166 and glu-169 amino acids are conserved in all 3cl pro enzymes. in fact, the crystal structure of sars-cov 3cl pro in complex with an inhibitor similar to compound 11 (pdb code 4mds) reveals that the interactions of the inhibitor with the amino acids his-166 and glu-169 are conserved. the identification of 3cl pro -inhibitor interactions utilizing conserved elements of the protein structure, including the peptide backbone and conserved side chains of active site residues, suggests that the development of broad-spectrum inhibitors of coronavirus 3cl pro is feasible. our studies here demonstrate the unique properties of mers-cov 3cl pro among ␤-cov 2c members, evident from the requirement for a ligand to induce dimerization. although the peptidomimetic compounds containing a michael acceptor group (for example, compounds 6 -9) induce dimer formation of mers-cov 3cl pro , the irreversible nature of their reaction with the active site cysteine ensures complete inhibition of the enzyme at stoichiometric ratios in a time-dependent manner. on the contrary, noncovalent peptidomimetic compounds (for example, compounds 10 and 11) inhibit the enzymatic activity of mers-cov 3cl pro only at high compound concentrations. based on these observations, compounds that irreversibly modify the 3cl pro active site may serve as better candidates for the development of inhibitors for mers-cov 3cl pro . potential complexity in the development of mers-cov 3cl pro inhibitors as antiviral agents-induced dimerization of mers-cov 3cl pro , as seen in the presence of peptidomimetic inhibitors, has significant implications in the development of antiviral agents targeting mers-cov 3cl pro . as a consequence of enzyme activation, the development of an effective antiviral agent may necessitate the development of a compound that can inhibit the mers-cov 3cl pro monomer and stabilize it without inducing dimerization and/or inhibit the active sites of the dimer at low doses, ensuring inactivation of both the active sites within the dimer. on the contrary, it is also possible that the presence of an inhibitor could enhance the activity of mers-cov 3cl pro to an extent that results in a complete loss of the temporal and spatial regulation of the enzymatic activity, thereby disrupting viral genome replication. ramifications of ligand-induced dimerization and activation of mers-cov 3cl pro , as seen in the presence of lower concentrations of inhibitor, will need to be further explored in virus-infected cells. bovine coronavirus as the causative agent of winter dysentery: serological evidence disease in a dairy herd associated with the introduction and spread of bovine virus diarrhoea 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emc/2012-related viruses in bats middle east respiratory syndrome coronavirus in bats middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart hospital outbreak of middle east respiratory syndrome coronavirus interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 the molecular biology of coronaviruses virus-encoded proteinases and proteolytic processing in the nidovirales coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor drug design targeting the main protease, the achilles' heel of coronaviruses design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors ) design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived sars-cov 3clpro inhibitors discovery, synthesis, and structure-based optimization of a series of n-(tert-butyl)-2-(n-arylamido)-2-(pyridin-3-yl) acetamides (ml188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (sars-cov) 3cl protease discovery of n-(benzo[1,2,3]triazol-1-yl)-n-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (sars-cov) 3clpro inhibitors: identification of ml300 and noncovalent nanomolar inhibitors with an induced-fit binding mechanism of the maturation process of sars-cov 3cl protease quaternary structure, substrate selectivity and inhibitor design for sars 3c-like proteinase residues on the dimer interface of sars coronavirus 3c-like protease: dimer stability characterization and enzyme catalytic activity analysis evaluating the 3c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3c-like protease liberation of sars-cov main protease from the viral polyprotein: n-terminal autocleavage does not depend on the mature dimerization mode maturation mechanism of severe acute respiratory syndrome (sars) coronavirus 3c-like proteinase x-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases a mouse model for betacoronavirus subgroup 2c using a bat coronavirus strain hku5 variant structurebased design, synthesis, and biological evaluation of peptidomimetic sars-cov 3clpro inhibitors is dimerization required for the catalytic activity of bacterial biotin carboxylase? processing diffraction data with mosflm processing of x-ray diffraction data collected in oscillation mode phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot molprobity: all-atom structure validation for macromolecular crystallography the pymol molecular graphics system biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors high-throughput screening identifies inhibitors of the sars coronavirus main proteinase a new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods characterizing proteinprotein interactions by sedimentation velocity analytical ultracentrifugation mutation of glu-166 blocks the substrate-induced dimerization of sars coronavirus main protease long-range cooperative interactions modulate dimerization in sars 3clpro dynamically-driven inactivation of the catalytic machinery of the sars 3c-like protease by the n214a mutation on the extra domain dynamically-driven enhancement of the catalytic machinery of the sars 3c-like protease by the s284-t285-i286/a mutations on the extra domain temperaturesensitive mutants and revertants in the coronavirus nonstructural protein 5 protease (3clpro) define residues involved in long-distance communication and regulation of protease activity mechanism for controlling the monomer-dimer conversion of sars coronavirus main protease long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function we-blogo: a sequence logo generator multiple sequence alignment with hierarchical clustering deciphering key features in protein structures with the new endscript server key: cord-281529-2rec51xg authors: haagmans, bart l; al dhahiry, said h s; reusken, chantal b e m; raj, v stalin; galiano, monica; myers, richard; godeke, gert-jan; jonges, marcel; farag, elmoubasher; diab, ayman; ghobashy, hazem; alhajri, farhoud; al-thani, mohamed; al-marri, salih a; al romaihi, hamad e; al khal, abdullatif; bermingham, alison; osterhaus, albert d m e; alhajri, mohd m; koopmans, marion p g title: middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation date: 2013-12-17 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70690-x sha: doc_id: 281529 cord_uid: 2rec51xg background: middle east respiratory syndrome coronavirus (mers-cov) causes severe lower respiratory tract infection in people. previous studies suggested dromedary camels were a reservoir for this virus. we tested for the presence of mers-cov in dromedary camels from a farm in qatar linked to two human cases of the infection in october, 2013. methods: we took nose swabs, rectal swabs, and blood samples from all camels on the qatari farm. we tested swabs with rt-pcr, with amplification targeting the e gene (upe), nucleocapsid (n) gene, and open reading frame (orf) 1a. pcr positive samples were tested by different mers-cov specific pcrs and obtained sequences were used for phylogentic analysis together with sequences from the linked human cases and other human cases. we tested serum samples from the camels for igg immunofluorescence assay, protein microarray, and virus neutralisation assay. findings: we obtained samples from 14 camels on oct 17, 2013. we detected mers-cov in nose swabs from three camels by three independent rt-pcrs and sequencing. the nucleotide sequence of an orf1a fragment (940 nucleotides) and a 4·2 kb concatenated fragment were very similar to the mers-cov from two human cases on the same farm and a mers-cov isolate from hafr-al-batin. eight additional camel nose swabs were positive on one or more rt-pcrs, but could not be confirmed by sequencing. all camels had mers-cov spike-binding antibodies that correlated well with the presence of neutralising antibodies to mers-cov. interpretation: our study provides virological confirmation of mers-cov in camels and suggests a recent outbreak affecting both human beings and camels. we cannot conclude whether the people on the farm were infected by the camels or vice versa, or if a third source was responsible. funding: european union projects emperie (contract number 223498), antigone (contract number 278976), and the virgo consortium. an emerging betacoronavirus-middle east respiratory syndrome coronavirus (mers-cov)-causes illness characterised predominantly by mild-to-severe respiratory complaints, with most patients requiring admission to hospital because of pneumonitis or acute respiratory distress syndrome. old age and the presence of comorbidities or immunosuppression seem to increase the risk of infection and are associated with severe forms of the disease. however, some patients can remain asymptomatic or mildly symptomatic and atypical presentations such as gastroenteritis have occurred. 1 as of dec 2, 2013, 163 laboratory-confi rmed cases, including 71 deaths, have been reported to who. 2 all of these cases were directly or indirectly linked to the middle east region, including saudi arabia, qatar, united arab emirates, kuwait, oman, and jordan. coronaviruses reside in many animal hosts and can adapt to diff erent species, including people. mers-cov belongs to the lineage c betacoronaviruses, which are associated with bats 3 and the closest relatives of mers-cov have been identifi ed in vesper bats from europe, asia, and south africa. [4] [5] [6] notably, this coronovirus is able to replicate in various bat cell lines. 7 molecular clock dating of epidemiologically unlinked human mers-cov isolates estimated their divergence from a common ancestor in mid-2011, with a cluster of isolates from the eastern parts of the arabian peninsula diverging in late 2012. these fi ndings suggest that the reported mers-cov diversity in human beings is the result of several independent, geographically structured, zoonotic events from an unknown reservoir in the middle east. 8, 9 human-to-human transmission has been noted, especially in health-care settings and households, but is thought to be relatively ineffi cient. 1 to determine how circulation of mers-cov in human beings is maintained and to mitigate the chain of transmission, identifi cation of possible animal reservoirs and modes of transmission is needed. 10 limited available exposure history of patients suggest that contact with livestock, including dromedary camels, might have a role. 1, 9, 11, 12 in addition, our recent investigations have provided evidence for the circulation of mers-cov or a related virus in dromedary camels. 13 both mers-cov spike protein binding antibodies and virus neutralising antibodies were reported in dromedary camels from diff erent regions, including oman and egypt, but no virus shedding could be detected and, therefore, the signifi cance of these observations remained an issue of debate. 13, 14 in this study we aimed to assess presence of mers-cov in dromedary camels from a farm in qatar that was linked to two human cases of mers-cov. in october, 2013, the qatar supreme council of health recorded two cases of laboratory confi rmed mers-cov. case 1 involved a 61-year-old man who was the owner of a farm that he visited regularly. the patient had substantial contact with animals including camels, sheep, pigeons, and hens, and no history of travel outside of qatar in the 2 weeks before becoming ill. mers-cov infection was diagnosed by e gene (upe) pcr on a sputum sample collected on oct 13, 2013, in the national virology laboratory of qatar. case 2 involved a 23-year-old male employee of the farm owned by case 1 and was identifi ed in the close-contact investigation of that case. he was reported on oct 17, 2013, and had no underlying medical conditions and no recent travel history, but had regular contact with the animals on the farm. mers-cov infection was diagnosed in a throat swab taken on oct 17, 2013, by the national virology laboratory of qatar. diagnosis for both cases was confi rmed (orf1b and n gene pcr) by public health england. 15, 16 qatari authorities, with support from who, did an epidemiological investigation at the farm within 1 week of diagnosis of the fi rst case, which included the collection of various clinical samples from the farm's dromedary camels. full details of the outbreak investigation will be described elsewhere. we collected serum, rectal swabs, and nasal swabs from all camels present on the premises where the two men had been in contact with the animals. after an experienced animal handler fi xated the camel by nose pinching, samples were obtained through swabbing with fl ocked swabs (floqswabs, copan improve diagnostics, brescia, italy) that were inserted deep into one of the nostrils of the camel. after sampling, swabs were put into tubes containing viral transport medium-utm (copan diagnostics, brescia, italy). in addition, we obtained fi ve stool samples from three diff erent cages. the sample collection was done jointly by the communicable disease control outbreaks rapid response team of the public health department and the animal health team, which used full personal-protective equipment including n95 masks, goggles, disposable gowns, gloves, and head covers during the handling of the animals, and during the environmental and human sampling. serum samples were stored at -20°c and all other samples were stored at -80°c and were shipped to the netherlands on dry ice. we tested nose swabs with two independent rt-pcr targets specifi c for mers-cov (upe and nucleocapsid [n gene]) in one laboratory (department of viroscience, erasmus medical center, rotterdam, netherlands), and did rt-pcr testing with a third target (orf1a) in another laboratory (national institute of public health and the environment, bilthoven, netherlands). we inoculated vero and huh-7 cells for 1 h with cellculture medium containing samples from camel nose or rectal swabs, or with mers-cov (emc isolate) as a positive control. after washing, the cells were incubated with medium containing 1% fetal bovine serum at 37°c. we stained formaldehyde-fi xed huh-7 cells infected with mers-cov by use of heat-inactivated (at 56°c for 30 min) camel sera or a mers-cov patient's serum according to standard protocols with fl uorescein isothiocyanateconjugated antibodies as a second step. we isolated rna from 140 μl of swab medium or culture supernatant with the qiaamp viral rna minikit (qiagen, hilden, germany) and eluted it in 50 μl. camel mers-cov rna was quantifi ed on the abi prism 7700 with the taqman fast virus 1-step master mix (applied biosystems, bleiswijk, netherlands), with 10 μl isolated rna, one taqman mix, 0·5 u of uracil-n-glycosylase, primer, and probes targeting the n gene, upe, or orf1a as described elsewhere. 17, 18 we used rna dilutions isolated from a mers-cov isolate emc stock as a standard control. 10 μl of rna was reverse transcribed with the superscript iii fi rst strand synthesis system (invitrogen, bleiswijk, netherlands) with random hexamers. cdna was used to amplify six diff erent camel mers-cov fragments, including a partial spike gene by use of pfu ultra ii fusion hs dna polymerase (agilent technologies, santa clara, ca, usa). we did the pcr as follows: one initial denaturation step of 95°c for 5 min, followed by 39 cycles of 95°c for 20 s, 50°c for 20 s, 72°c for 2 min, and a fi nal extension of 72°c for 5 min. the amplifi ed camel mers-cov fragments were sequenced directly on both strands with the bigdye terminator version 3.1 cycle sequencing kit on an abi prism 3100 genetic analyser (applied biosystems). we generated phyml phylogenetic trees with seaview 4 software with the approximate likelihood-ratio test based on a shimodaira-hasegawa-like procedure, which used a general time reversible as substitution model. we used nearest neighbour interchange and subtree pruning and regrafting-based tree search algorithms to estimate the tree topologies. 19 rna from human samples (sputum for qatar 3 case and throat swab for qatar 4 case) was extracted with the nuclisens easymag system (biomérieux, basingstoke, uk). viral sequences from the two human cases were isolated (with the same techniques as for camel cases) in the reference department, public health england, london, uk. we used primers designed against sequences of mers-cov jx869059_emc/2012 and bat coronaviruses hku4 and hku5 to amplify the entire genomes from human cases for sequencing on an illumina miseq sequencer, subsequently indicated as qatar_3_2013 (case 1) and qatar_4_2013 (case 2). the sequences obtained in this study were deposited in genbank under the following accession numbers: camel_mers-cov/qatar_1_2013_orf1a_1_ partial, kf933380; camel_mers-cov/qatar_1_2013_ orf1a_3_partial, kf933381; camel_mers-cov/ qatar_1_2013_orf1b_partial, kf933382; camel_ mers-cov/qatar_1_2013_spike_partial, kf933383; camel_mers-cov/qatar_1_2013_orf4b_full, kf933384; camel_mers-cov/qatar_1_2013_orf1a_2_ partial, kf933385; qatar_3_2013, kf961221; and qatar_4_2013, kf961222. we tested all sera for the presence of igg antibodies reactive with mers-cov (residues 1-747), severe acute respiratory syndrome coronavirus (sars-cov; residues 1-676), and human coronavirus oc43 (residues 1-760) s1 antigens as described previously. 13, 20 we report results as the relative mean fl uorescent intensity (rfu) for each set of quadruplicate spots per antigen. we did igg antibody testing only because anti-camel igm conjugates were not available. for virus neutralisation, serum samples were heatinactivated by incubation for 30 min at 56°c. we prepared twofold serial dilutions with 96 well plates, starting dilution at 1/10. mers-cov was diluted in iscove's modifi ed dulbecco's medium (imdm) supplemented with penicillin, streptomycin, and 1% fetal bovine serum, to a dilution of 2000 tcid 50 per ml. we then added 50 μl of virus suspension to the plates and the plates were incubated at 37°c for 1 h. next, we incubated virus-serum mixtures on 96 well plates containing vero cells for 1 h, and then washed them with phosphate buff er saline and incubated them with imdm and 1% fetal bovine serum for 5 days. the sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. samples from 14 camels were obtained on oct 17, 2013 (table, fi gure 1). nose swab specimens from fi ve camels were positive for all three assays (upe, n gene, and orf1a), one sample reacted in two assays, and fi ve camels tested positive in only one rt-pcr assay. subsequently, all samples were subjected to a pcr assay targeting the spike gene of mers-cov (nucleotides 22449-22806) to obtain a fragment for sequencing as defi nitive confi rmation. such sequencing confi rmed the presence of sequences specifi c to mers-cov in three camels. alignment of these partial spike gene sequences with known human mers-cov sequences, including those determined from the two related human cases and some other human mers-cov isolates such as hafr-al-batin_1_2013 (kf600628) and riyadh_3_2013 (kf600613), showed 100% identity, but the sequences diff ered from emc mers-cov, which is used routinely in the erasmus mc laboratory, by one nucleotide. the three camel mers-cov sequences obtained by this approach were identical. virus culture attempts were not successful, although one sample tested for camel 7 yielded a positive rt-pcr of the supernatant (table) and staining of the fi xed virus-infected cells (data not shown). further passage of the virus on vero cells, however, was not successful. in addition, serum from camel 9 showed low levels of mers-cov as determined by rt-pcr, and all other camel sera tested negative (data not shown). rectal swabs and faecal materials were negative for mers-cov when tested for reactivity in the upe and n gene rt-pcrs. further sequencing of the virus from camel 5 with primers specifi c to mers-cov rendered six diff erent fragments covering 4·2 kb across the mers-cov genome (fi gure 1). fragments 1 (nucleotides 6708-7459), 2 (8095-9034), and 3 (10073-10658) were located in orf1a, fragment 4 (18792-19584) in orf1b, fragment 5 (22449-22806) in the spike gene, and fragment 6 (26074-26846) in orf4b. the sequences obtained from the human cases from the same farm diff ered by one nucleotide diff erence in orf1a and one in orf4b. the mers-cov emc isolate diff ers by eight nucleotides from the camel mers-cov in orf1a. alignment of the divergent 940 nucleotide orf1a fragment (data not shown) and a 4·2 kb concatenated sequence fragment was used for phyml phylogenetic analysis. the camel mers-cov clustered with viral sequences obtained from the two human cases related to the farm and with a sequence from hafr-al-batin as the next closest relative (fi gure 1). less stringent methods of analysis do show limited bootstrap support (data not shown), in line with relatively limited sequence variation within this fragment. all camel sera were positive in the immunofl uorescence assay when tested in a 1/200 dilution (fi gure 2). when serum samples from camels were tested at diff erent dilutions on the microarray, we noted reactivity with mers-cov antigen, but not the sars-cov antigen. reactivity to human coronavirus oc43 antigen, as a proxy to betacoronavirus, varied between negative (less than the cutoff value of 4000) and saturating signals (data not shown). consistent with the array data, all serum samples had mers-cov neutralising capacity, with titres varying between 160 and 5120 (fi gure 2). we also noted a strong correlation between microarray titres and virus neutralising antibody titres (fi gure 2). the two camels with the highest mers-cov load in the swabs as shown by taqman assay (camels 5 and 7) had a relatively low serum neutralisation titre of 640. we present, to our knowledge, the fi rst virological confi rmation of mers-cov in dromedary camels (panel). we and others previously reported mers-cov neutralising antibodies in dromedary camels from the canary islands, oman, and egypt, suggesting circulation of mers-cov or a mers-cov-like virus in camels. 13, 14 high prevalences and antibody titres were reported in the camels from oman and egypt suggesting widespread circulation. however, virological testing was unable to detect mers-cov viral sequences in camels, probably because only faecal and serum samples were analysed. in addition, shedding kinetics of mers-cov in camels (and human beings) are unknown, and therefore, inter pretation of negative rt-pcr results from screening is diffi cult because positive and negative predictive values remain to be determined. 22 in the outbreak reported, we showed evidence for presence of the virus in six camels according to internationally recommended criteria of two independent rt-pcr targets. furthermore, we used sequence confi rmation as an additional test to increase the level of certainty-during an emerging disease outbreak such as this, options for validation of assays for clinical testing are limited. 22 discrepancies between assays can be explained by diff erences in detection limits, or by diff erences in specifi city of the primer sets. in our study, n gene rt-pcr yielded four additional weak positive results that could not be confi rmed by any other method, and an individual orf1a rt-pcr identifi cation was made for only one animal. these cases could have resulted from mispriming, or represent presence of levels of virus around the detection limit, as can be expected at the end of the infection cycle. nevertheless, our results suggest a widespread and recent outbreak in this herd, coinciding with infection in two people. our data should not be taken as evidence for infection of people from the camels. comparison of sequence data from the orf1a gene and a concatenated 4·2 kb sequence from the human and camel viruses showed that these viruses are very similar, but distinct. notably, samples from camels and people were analysed in diff erent laboratories in the netherlands and the uk. the data provided show proof that camels can be infected with mers-cov. the sequence diff erence between human and animal viruses from the farm, based on the available overlapping sequence, is so small that we cannot conclude whether the people on the farm were infected by the camels or vice versa. another possibility is that people and camels were infected from a third as yet unknown source. although additional sequencing might provide improved resolution, it probably will not provide conclusive evidence. the most important unknown is the exact timing of infections, both in the infected people and camels. if the virus entered the farm through either the people or the camels, the infections observed would probably have resulted from a chain of transmissions that introduced mutations. because we did not identify any seronegative animals that were pcr positive, and because all animals had mers-cov neutralising antibodies, our sampling probably took place relatively late in the outbreak on this farm. a more detailed analysis of the outbreak, including testing of additional animals and environmental samples is ongoing, as are attempts to obtain full mers-cov genomes of the human and animal specimens. we cannot exclude the possibility that other common livestock species including cattle, sheep, and goats, or other animals including wild species were involved in the spread of mers-cov. while confi rmation of the source is awaited, we recommend that a detailed case history is taken of any cases of mers-cov, including review of any animal exposures (including animal products), and targeted (prospective) serosurveys to determine what risk factors are associated with human infection. we searched pubmed for articles published in english up to dec 1, 2013, with the search terms "novel coronavirus emc" or "mers-cov". we identifi ed 121 reports linked to the middle east respiratory syndrome coronavirus (mers-cov). one report 21 described the detection in one bat specimen of a single 181 bp fragment in a highly conserved area of the coronavirus genome, identical to the human mers-cov laboratory isolate emc. our report describes the fi rst detection of mers-cov in dromedary camels on a farm in qatar that had been linked to human cases of the disease. our study provides proof that mers-cov has infected dromedary camels in qatar. our results suggest the simultaneous occurrence of a mers-cov outbreak in people and camels. on the basis of the present data, we cannot conclude whether the people on the farm were infected by the camels or vice versa. another possibility is that people and camels could have been infected from a third as yet unknown source. we recommend that detailed case histories be taken, including review of any animal exposures including animal products, and targeted (prospective) serosurveys to determine what risk factors-other than contact with an individual with the infection-are associated with human infection. blh wrote the report, drew the fi gures, and generated, analysed, and interpreted data. shsad, mma, and mpgk coordinated the study, wrote the report, and interpreted data. cbemr wrote the report, drew the fi gures, did the literature search, and analysed and interpreted data. vsr drew the fi gures, and generated, analysed, and interpreted data. mg, rm, and ab generated and interpreted data. gjg generated, analysed, and interpreted data. mj generated and analysed data. ef led the fi eld investigation, obtained samples, and collected and interpreted data. ad supervised fi eld outbreak management and collected and interpreted data. hg and fa supervised animal health fi eld investigations. ma-t coordinated the study and interpreted data. saa-m coordinated the study. hear led outbreak management and collected and interpreted data. aak coordinated the study and interpreted data. admeo wrote the report and interpreted data. blh, admeo, and vsr hold a pending patent for mers-cov. admeo is scientifi c adviser of viroclinics biosciences. all other authors declare that they have no confl icts of interest. doi:10.1371/ currents.outbreaks.0bf719e352e7478f8ad85fa30127ddb8. 2 who. global alert and response (gar): novel coronavirus infection-update ecology, evolution and classifi cation of bat coronaviruses in the aftermath of sars close relative of human middle east respiratory syndrome coronavirus in bat human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart recovery from severe novel coronavirus infection family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt alert and response (gar): novel coronavirus infection-update alert and response (gar): novel coronavirus infection-update detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections seaview version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building specifi c serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronavirus: data gaps for laboratory preparedness we thank who and the food and agriculture organization of the united nations for their generous help in organisation of the study, theo bestebroer for providing middle east respiratory syndrome coronavirus specifi c primer sets, berend jan bosch for providing antigens for the microarray, and jeroen cremer for technical support. contributions to the study were funded through the european union fp7 projects emperie (contract number 223498; to blh and admeo) and antigone (contract number 278976; to mpgk and admeo). key: cord-268561-vq1uhj5i authors: da silva, severino jefferson ribeiro; silva, caroline targino alves da; guarines, klarissa miranda; mendes, renata pessôa germano; pardee, keith; kohl, alain; pena, lindomar title: clinical and laboratory diagnosis of sars-cov-2, the virus causing covid-19 date: 2020-08-04 journal: acs infect dis doi: 10.1021/acsinfecdis.0c00274 sha: doc_id: 268561 cord_uid: vq1uhj5i [image: see text] in december 2019, a novel beta (β) coronavirus eventually named sars-cov-2 emerged in wuhan, hubei province, china, causing an outbreak of severe and even fatal pneumonia in humans. the virus has spread very rapidly to many countries across the world, resulting in the world health organization (who) to declare a pandemic on march 11, 2020. clinically, the diagnosis of this unprecedented illness, called coronavirus disease-2019 (covid-19), becomes difficult because it shares many symptoms with other respiratory pathogens, including influenza and parainfluenza viruses. therefore, laboratory diagnosis is crucial for the clinical management of patients and the implementation of disease control strategies to contain sars-cov-2 at clinical and population level. here, we summarize the main clinical and imaging findings of covid-19 patients and discuss the advances, features, advantages, and limitations of different laboratory methods used for sars-cov-2 diagnosis. t he recent emergence of a novel coronavirus in the human population has caused dramatic and unprecedented impact of the economy and prompted mobilization of public health authorities around the world to counter the rapid spread of the virus. coronaviruses (covs) are members of the coronaviridae family and are an important group of viruses that infect a large number of animals including mammalian and avian species. 1 the coronavirinae subfamily is divided into four genera based on genetic features: alphacoronavirus (α-covs), betacoronavirus (β-covs), gammacoronavirus (γ-covs), and deltacoronavirus (δ-covs). the α-covs (hcov-229e and hcov-nl63) and β-covs (hcov-oc43 and hcov-hku1) cause human infection and have been associated with mild respiratory diseases. 2 in the 21st century, however, three β-covs have emerged from animal reservoirs to cause severe disease in humans: severe acute respiratory syndrome coronavirus (sars-cov), 3 the middle east respiratory syndrome coronavirus (mers-cov), 4 and the pandemic severe acute respiratory syndrome coronavirus 2 (sars-cov-2). 5,6 the genome of covs consists of a single-stranded positive sense (+ssrna) of around 30 kb in size. the genomic rna is capped at the 5′ end and has a poly(a) tail at the 3′ end, allowing it to act as an mrna for translation of the replicase polyproteins. 1,7 the 5′ terminal region of the genome encodes a polyprotein that is cleaved into 16 nonstructural proteins involved in the transcription and replication process, and the 3′ terminal region encodes viral structural proteins. 8 in december 2019, the world was on alert due to a cluster of severe pneumonia cases of unknown origin in wuhan, hubei province, china. this outbreak was epidemiologically linked to a wholesale animal and seafood market where live and freshly slaughtered animals were kept and sold. 9 of the initial 41 patients hospitalized with pneumonia, two-thirds had a history of direct exposure to this market. 10 on the basis of the clinical presentation and the link with the animal market, similar to sars epidemiology, a cov was suspected as the causative agent and therefore pan-cov pcr primers were used to test the samples followed by sequencing. 11 the causative agent was identified as a novel cov, eventually named sars-cov-2, and the respiratory syndrome associated with the infection was designated as coronavirus disease-2019 (covid-19) by the world health organization (who). the sars-cov-2 genome has about 80% sequence identity to sars-cov (with whom it is classified into the species severe acute respiratory syndrome-related coronavirus) 12 and 50% to mers-cov. the most closely related virus to sars-cov-2 found so far is a cov isolated from bats, named ragt13 cov, whose nucleotide identity is 96%, suggesting that sars-cov-2 is also of bat origin. however, it is not clear whether sars-cov-2 jumped to humans directly from bats or through an intermediate host. 13 the rapidly increasing numbers of covid-19 prompted who to declare first a public health emergency of international concern (pheic) on january 30, 2020 and then a pandemic on march 11, 2020. 14 as of july 31, 2020, more than 17 million cases of covid-19 and 677 549 deaths have been reported in 213 countries and territories around the world. most of the cases have been reported by the usa, followed by brazil, india, russia, south africa, mexico, and peru. 15 different from the other highly pathogenic covs, sars-cov-2 has acquired the ability to establish sustained humanto-human transmission. its basic reproductive number (r0), i.e., the number of secondary infections generated from one infected individual, is estimated to be between 1.4 and 6.49, with a mean of 3.28. 16 ultimately, this metric will require further investigations and may vary across settings and locations. on the basis of the travel history and symptom onset of patients in china, the mean incubation period of covid-19 has been calculated to be 6.4 days, ranging from 2 to up to 14 days. 17 clinically, the spectrum of covid-19 manifestations ranges from asymptomatic and mild to severe infections requiring oxygen therapy and ventilation support. 9,18, 19 since its emergence, a wide variety of methods have been developed for the purpose of the rapid and accurate diagnosis of covid-19. on the basis of clinical criteria alone, sars-cov-2 cannot be reliably distinguished from infections with other pathogens that cause similar symptoms, including influenza, seasonal cov, adenovirus, bocavirus, human metapneumovirus, parainfluenza, respiratory syncytial virus rhinovirus, bordetella pertussis, legionella pneumophila, myco-plasma pneumonia, 20,21 and even the mosquito borne dengue virus. 22 in this context, the laboratory-based diagnosis assumes a role for the clinical management of patients and the implementation of disease control measures. here, we review the clinical features, laboratory methods, and imaging findings that are used for covid-19 diagnosis. in addition, we explore the next steps of the methods under development for covid-19 diagnosis. a rapid presumptive diagnosis based on clinical assessment and epidemiological characteristics is critical to ensuring appropriate patient care and controlling viral transmission, thus contributing to disease control. as mentioned and as with other respiratory viral infections, the signs and symptoms of covid-19 are nonspecific and the clinical spectrum of disease can range from no symptoms to severe pneumonia and death. 23 asymptomatic infection has been reported in many settings, but some patients develop clinical disease at a later stage of infection. the proportion of truly asymptomatic infections is unclear, but some estimates indicated that up to 80% of infections do not result in overt clinical signs of disease. 24 a recent mathematical model suggested that undocumented infections might be major drivers of sars-cov-2 spread in the world. 25 according to who criteria, a person is suspected of being infected with covid-19 in three scenarios: (i) a patient with acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g., cough, shortness of breath) and a history of travel to or residence in a location reporting community transmission of covid-19 disease during the 14 days prior to symptom onset; (ii) a patient with any acute respiratory illness who has been in contact with a confirmed or probable covid-19 case in the last 14 days prior to symptom onset; and (iii) a patient with severe acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g., cough, shortness of breath, and requiring hospitalization) and in the absence of an alternative diagnosis that fully explains the clinical presentation. if a patient then tests positive with a laboratory diagnostic, then the infection becomes a confirmed overall, 81% of laboratory-confirmed covid-19 patients develop mild to moderate disease, which includes nonpneumonia and pneumonia cases, 14% have severe disease (dyspnea, tachypnea, blood oxygen saturation ≤93%, and pao2/fio2 ratio of 50% of the lung field within 24−48 h), and 5% of cases reach critical state (respiratory failure, septic shock, and/or multiple organ dysfunction/failure). 27 risk factors for severe covid-19 include age ≥65 years and people with preexisting concurrent conditions such as cardiovascular disease, hypertension, diabetes, chronic respiratory disease, immunodeficiencies, cancer, and obesity. 28−30 accurate estimates of the covid-19 case fatality rate are still lacking, but it is believed to be around 3% 9 and can be as high as 14.8% in patients >80 years of age. 27 however, this number is dependent on the level of testing in countries, testing accuracy, and death-reporting policies. interestingly, men have a much higher risk of death than women. 27, 31 table 1 summarizes the clinical signs and symptoms of covid-19 patients seen in several studies. in general, the most common clinical manifestations are fever, dry cough, fatigue, sputum production, dyspnea, sore throat, headache, myalgia or arthralgia, and chills ( figure 1 ). less common symptoms include nausea or vomiting, nasal congestion, diarrhea, hemoptysis, and conjunctival congestion. 23 in children, sars-cov-2 infection is generally mild and in some cases asymptomatic; however, when presented, the main symptoms include fever (about 41%), cough (48%), and pharyngeal erythema (46.2%). 32 more recently, several studies have been suggested that covid-19 infection was associated with cutaneous manifestations in patients. 33−35 major manifestations observed in covid-19 patients including different types of lesions such as purpuric, papulovesicular, livedoid, urticarial, maculopapular, and thromboticischemic. 36 a recent study in european covid-19 patients reported that 85.6 and 88.0% of patients had olfactory and gustatory dysfunctions, respectively. these disorders persisted after the resolution of other symptoms. 37 finally, a single retrospective case series in wuhan, china, reported neurologic manifestations in 78 of 214 hospitalized patients (36.4%) with a laboratory-confirmed diagnosis of covid-19. these symptoms were more common in severe cases, along with acute cerebrovascular events, headache, dizziness, and impaired consciousness. it is unclear whether the neurologic manifestations were caused by sars-cov-2 directly or by pulmonary disease or other organ damage indirectly or by cytokines. 38 since this study included only hospitalized patients in a single location, the true percentage of neurological manifestations in covid-19 needs further evaluation. imaging techniques such as chest x-rays, pulmonary computed tomography (ct) scans, and lung ultrasounds are important tools in the early diagnosis of pneumonia in patients with covid-19. although chest x-rays are less expensive and more convenient for follow up in pneumonia cases, the technique has low-resolution and projection overlapping, which could lead to many false-negative covid-19 cases. 39 reports indicate that covid-19 patients submitted to chest ct scans on admission presented abnormal results (about 90%), showing bilateral multiple ground-glass and patchy opacity. 40 a clinical laboratory findings and biomarkers in covid-19 patients. several studies have reported hematologic and blood chemistry alterations in patients infected by sars-cov-2. 43, 44 major laboratory findings in covid-19 patients identified by meta-analysis include leukopenia, leukocytosis, decreased albumin levels, increased levels of creactive protein, lactate dehydrogenase (ldh), creatinine kinase, and biliarubin, and a high erythrocyte sedimentation rate (esr). 45 increased levels of creatine kinase and lactate dehydrogenase were associated with myalgia. 38 a growing body of evidence suggests that sars-cov-2 infection can trigger the overproduction of cytokines in some patients, known as a cytokine storm, which is associated with poor outcomes. 10,46−49 as for other severe viral infections, the exacerbated production of proinflammatory cytokines may be involved in some of the pathophysiology of covid-19, including pulmonary edema and lung failure and damage to the liver, heart, and kidneys. compared to healthy adults, covid-19 patients had higher levels of il-1β, il-1ra, il-7, il-8, il-9, il-10, basic fgf, g-csf, gm-csf, ifn-γ, ip-10, mcp-1, mip-1a, mip-1b, pdgf, tnf-α, and vegf. serum biomarkers associated with severe disease included il-2, il-7, il-10, g-csf, ip-10, mcp-1, mip-1a, and tnf-α. 10 a recent retrospective study of 150 confirmed covid-19 cases (68 fatal and 82 discharged cases) in wuhan, china, identified several serological markers that were more elevated in lethal cases than in survivors: elevated ferritin, il-6, myoglobin, creactive protein, and cardiac troponin. 50 together, these findings suggest that covid-19 mortality might be due to infection-driven hyperinflammation. diagnostic virology of covid-19. laboratory virology tests are essential for a correct diagnosis and the population level prevalence of covid-19, given the number of asymptomatic cases or nonspecific clinical symptoms. results from these tests guide clinicians and health officials in the management, control, and prevention of covid-19. several analytical parameters are used to determine the performance of covid-19 assays, including clinical sensitivity, clinical specificity, positive predictive value (ppv), negative predictive value (npv), and overall accuracy. 51 the confirmation of a sars-cov-2 infection in the laboratory can be achieved by direct and indirect virology methods. while direct detection is more specific, indirect methods allow a greater opportunity for virus detection after the acute phase of the disease. in direct tests, the clinical sample is examined directly for the presence of particles, virus antigens, or viral nucleic acids, whereas indirect methods detect the serological response against the infection (figure 2 ). samples. the world health organization (who) recommends that all procedures performed with covid-19 be done only by trained professionals and should take place in a laboratory with an appropriate level of biosafety. nonpropagative diagnostic assays, such nucleic acid amplification tests (naats), sequencing, and some serological tests (e.g., elisa type assays) can be performed in biosafety level 2 (bsl-2) laboratories provided that the initial processing (before virus inactivation) of samples takes place in a validated biological safety cabinet. however, procedures that involve propagative virus work, such as virus culture, isolation, or neutralization assays, should be performed in laboratories equivalent to bsl-3 using validated practices. the work team must use appropriate personal protective equipment including eye protection (goggles or a disposable face shield), a respirator or facemask, a long-sleeved gown, and gloves and follow all standard operating procedures regarding sample collection and handling. respirators that offer a higher level of protection such as n95 respirators or powered air-purifying respirators should be used instead of a face mask when performing an aerosol-generating procedure. samples from patients with a suspected or confirmed case must be transported to un3373, a code referring to "biological substance category b"; however, virus-infected cultures or isolates must be transported as category a -un2814, "an infectious substance that affects humans". 52,53 specimens for diagnosis. choosing the correct sample for diagnosis tests is an essential step in a reliable diagnosis. sars-cov-2 infection can be detected in a variety of clinical specimens, such as nasopharyngeal or oropharyngeal aspirates or washes, nasopharyngeal or oropharyngeal swabs, sputum, tracheal aspirates, and bronchoalveolar lavage. 54−58 the median duration of sars-cov-2 shedding in respiratory samples is 24 (irq, 18−31) days in survivors, but shedding can last for up to 42 days. 59 given the invasiveness and requirements for equipment and skilled labor, the collection of specimens other than sputum from the lower respiratory tract should be considered only in special situations. a nasopharyngeal swab collected with a fiberplastic shaft swab is the preferred choice for swab-based sars-cov-2 testing because it provides reliable results while not being too invasive. calcium alginate swabs and wooden shaft swabs are not recommended because they may contain substances that inactivate some viruses and inhibit pcr testing. when collection with a nasopharyngeal swab is not possible, oropharyngeal swabs, nasal midturbinate swabs, or anterior nares (nasal swab) swabs are also acceptable alternatives. swabs should be placed immediately after collection into sterile tubes containing 2 to 3 ml of a viral transport medium to preserve viral integrity. upon collection and with the appropriate storage medium, specimens can be stored at 2−8°c for up to 72 h. in cases of delayed testing or shipping, samples should be stored at −70°c or below. 57 inadequate sample collection, handling, and storage are important variables that may result in false-negative test results. in addition to respiratory tract specimens, sars-cov-2 can also be detected in other samples such as stool, anal swabs, and blood but not in urine. 54,60,61 sars-cov-2 detection in blood is not frequent and is associated with disease severity. 60, 61 the detection rate of sars-cov-2 rna in fecal specimens is similar to that of pharyngeal swabs; 54,62 however, importantly, fecal viral shedding appears to occur for a longer period of acs infectious diseases pubs.acs.org/journal/aidcbc review time. 63 sars-cov-2 detection in stool is not associated with the presence of gastrointestinal symptoms or the severity of illness. 64−67 despite high virus rna levels in feces, successful isolation of sars-cov-2 from patient stool has been reported. 68 the isolation of sars-cov-2 from urine and ocular secretions from infected patient has been described. 69, 70 viral shedding in human breast milk and semen has been detected, 71,72 while in tears it has either been undetected 73 or acs infectious diseases pubs.acs.org/journal/aidcbc review detected at very low frequency. 74 serum and plasma samples are used for serological assays, especially 1 week postinfection. the median seroconversion time for total anti-sars-cov-2 antibodies, igm and then igg, were days 11, 12, and 14, respectively. 75 virus isolation. sars-cov-2 was first isolated from bronchoalveolar lavage from a patient with pneumonia in vero e6 and huh7 cells. the viral identity was confirmed by immunofluorescence using the now-known cross-reactive anti-sarsr-cov rp3 n antibody, rt-qpcr, and metagenomics sequencing. the cellular infectivity of the isolated virus was confirmed by virus neutralization assay using sera from convalescing patients. 11 culture-based methods for sars-cov-2 detection have been used in research and public health laboratories in different parts of the world, but virus isolation is not recommended as a routine diagnostic procedure because it has low sensitivity, it is time-consuming, and it requires bsl-3 containment. 56 sars-cov-2 is also culturable in several cell lines, including human airway epithelial, 5,76 vero e6, vero ccl-81, and huh-7 cells. 11,58,77,78 vero e6 cells express high levels of angiotensin-converting enzyme 2 (ace2), 79 which has been identified as a key a cell receptor for sars-cov-2 infection. 80 similar to sars-cov-1 and mers-cov, sars-cov-2 isolation is enhanced in an engineered vero e6 that expresses tmprss2 (transmembrane serine protease 2) levels that are 10-fold higher than in human normal lung tissue and other human cell lines, suggesting that tmprss2 protease is important for sars-cov-2 infection. 81 recently, zang and colleagues demonstrated that tmprss2 and tmprss4 promote sars-cov-2 entry into enterocytes. 82 harcourt and co-workers studied the susceptibility of several cell lines such as vero ccl-81, vero e6, hek-293t, a549, and huh-7 as well as the big brown bat kidney cell line (efk3b) to support the productive replication of sars-cov-2. no cytopathic effect (cpe) was observed in any of the cell lines except in vero e6 and vero ccl81 cells, in which the virus grew to >10 7 pfu/ml at 24 h postinfection. huh-7 and hek-293t cells showed only modest viral replication, whereas no virus replication was detected in either a549 or efk3b cells. sars-cov-2 produced distinct plaques in vero e6 cells, but plaques in vero ccl-81 cells were not as clear, suggesting that vero e6 cells might be the best choice for the propagation, quantification, and study of plaque phenotypes of different sars-cov-2 strains. 78 however, should virus isolation be attempted, vero e6 cells clearly stand out as a cell line that supports infection and virus production. a recent cross-sectional study used 90 sars-cov-2 rt-qpcr confirmed specimens and evaluated their ability to infect vero cell lines. 83 for the study, 90 patient samples were incubated on vero cells (ccl-81) during 4 days, and then the cytopathic effect was evaluated. of these, 26 samples (28.9%) demonstrated viral growth, but there was no growth in samples with a ct value >24, 83 which suggests using samples with low ct values for successful viral isolation. electron microscopy. human cov was first isolated in 1965 from a patient with a common cold. 84 later, the virus was named "coronavirus" due to the virus' appearance under the electron microscope that resembles the solar corona. 85 cov particles are pleomorphic, and their surfaces are covered with a distinct layer of projections, which corresponds to the long spike protein. 80 given the virus' distinctive morphology, electron microscopy has been used to observe and identify virus particles after isolation in culture systems in the initial outbreak in wuhan 11 and subsequently by others. 11, 86, 87 the first attempts to recover sars-cov-2 virions with the characteristic fringe of surface spike proteins from the first covid-19 case in australia failed, but adding trypsin to the cell culture medium immediately allowed the visualization of the virus with characteristic cov morphology, with particles of 90−110 nm diameter displaying prominent spikes (9−12 nm) on the surface. 88 real-time rt-qpcr. the gold standard diagnostic test for sars-cov-2 infection is viral rna detection by reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr) given its sensitivity, specificity, and speed. rt-qpcr shows better performance than serology because it can identify positive cases in the early stage of infection, even during the incubation period of the disease and after symptoms have disappeared. 58 many laboratory-developed tests based on the rt-qpcr assay have been published or are in development for sars-cov-2 detection. some of these developed assays are specific to the pandemic strain of the virus, and others also recognize genetically similar strains such as sars-cov. molecular targets for sars-cov-2 rt-qpcr include the genes that encode the nucleocapsid (n), envelope (e), spike (s), and rna-dependent rna polymerase proteins given their high degree of genetic conservation (figure 3) . 56, 89 most assays use two targets in the viral genome, but in the event of reagent shortages, rt-qpcr screening with only one set of primers instead of two may be considered after thorough validation in the individual laboratory. 90 performing a sample preheating step instead of rna extraction can also be considered during a shortage of viral nucleic acid extraction kits. in a study with 87 oropharyngeal swab samples (65 positive and 22 negative for sars-cov-2) in denmark, it was found that heating for 5 min at 98°c resulted in a sensitivity, specificity, and accuracy of 97.4, 100.0, and 98.3%, respectively, as compared with magna pure 96 rna-extracted samples. 91 although some laboratories have recommended heat inactivation of the virus (56−60°c for 30−60 min) before rna extraction to protect laboratory personnel, this procedure adversely affects the efficacy of rt-qpcr for sars-cov-2 detection. 92 high rates of false-negative sars-cov-2 rt-qpcr results and prolonged nucleic acid conversion have been reported in some studies, which have implications for returning to normal activities and covid-19 control. 93−95 in addition to in-house tests, many molecular assay kits have been approved by the food and drug administration's (fda) and have been made commercially available for the detection and amplification of the sars-cov-2 rna genome (https:// www.fda.gov/medical-devices/emergency-situations-medicaldevices/emergency-use-authorizations). however, diagnostic laboratories should rigorously validate these commercial kits before routine use since the analytical sensitivity of some commercial rt-qpcr tests differs substantially, which could lead to false-negative results. 96 two of the most widely used assays in the western world have been developed by charite, hospital germany 97 and the u.s. cdc. 98 nalla and co-workers compared the performance of the charité(n, polymerase, and e targets) and the cdc (n1, n2, and n3 targets) primer/probe sets side-by-side using clinical samples. it was found that all assays were highly specific to sars-cov-2, with no cross-reactivity with other respiratory viruses. the cdc n2 and the charitéhospital e-gene primer/probe sets performed equally well and were the most sensitive assays for detecting sars-cov-2. 99 we anticipate acs infectious diseases pubs.acs.org/journal/aidcbc review that this list will grow as several new rt-qpcr assays are being developed to promote the accurate and rapid detection of sars-cov-2. 97 59 the results demonstrated that the median duration between the onset of symptoms to nucleic acid conversion was 24 days (iqr, 18−31) and that the longest duration was 42 days after the onset of symptoms. 59 it was found that higher viral loads (inversely proportional of the ct value) can be observed in upper respiratory samples soon after symptom onset 55 and that the peak occurs within the first week of illness onset of covid-19 confirmed patients. 55, 103 in another study, the authors evaluated the viral shedding patterns demonstrated in patients with mild and severe covid-19 disease using specimens from 76 patients, including 46 individuals classified as mild cases and 30 classified as severe cases. 104 the results showed that the viral load of severe cases was around 60 times higher when compared with that of mild cases, indicating that higher viral loads might be associated with severe clinical findings. in addition, it was revealed that the ct values of severe cases were significantly lower than those of mild cases in infected patients at the time of admission and that early viral clearance was observed in patients classified as mild cases (10 days after onset). 104 sars-cov-2-positive rt-qpcr detection has also been demonstrated even after the resolution of covid-19 symptoms. 58, 111 for this reason, the cdc has recommended obtaining at least two negative upper respiratory tract samples, collected in intervals of 24 h or longer, to document sars-cov-2 clearance. 112 on the other hand, the viral shedding in asymptomatic patients with covid-19 also has been investigated. 113, 114 zhou and co-workers analyzed the viral shedding pattern in 31 patients that were confirmed to have covid-19 using rt-qpcr but were asymptomatic during admission to the hospital. 113 subsequently, the study divided the 31 patients into 2 groups: 9 patients who remained asymptomatic during hospitalization (aps) and 22 patients who presented symptoms after admission to the hospital (apis). the results demonstrated that the median ct value of aps (39.0, interquartile range [iqr] 37.5−39.5) was higher than when compared to apis (34.5, iqr 32.2−37.0), showing a lower viral load in aps. also, it was found that the duration of viral shedding remained similar in the two patient groups, which reflects the possibility of sars-cov-2 transmission in the community during the asymptomatic period. in addition, the study findings revealed that the viral load of aps peaked during the first week after admission to the hospital while that of apis peaked during the second week of covid-19 infection. these findings alert professionals and health authorities to the possibility of transmission during the asymptomatic period of patients with covid-19 disease. however, further scientific studies with more patients are required to elucidate the real role of asymptomatic patients in the transmission chain. reverse transcription loop-mediated isothermal amplification (rt-lamp). the recent increase in the number of covid-19 cases in the world has encouraged a global effort to develop point-of-care platforms for diagnosing sars-cov-2. reverse transcription loop-mediated isothermal amplification (rt-lamp) is perhaps one of the most promising platforms for rapid development and accessible sars-cov-2 testing and has many advantages, compared to rt-qpcr, such as high specificity and sensitivity, simple operation, rapid amplification, and low cost,. 115, 116 rt-lamp assays have been developed for other covs of the same genus (betacoronavirus), including sars-cov 117,118 and mers-cov. 119, 120 not surprisingly, several studies have already demonstrated the use of rt-lamp for sars-cov-2 detection. 121−125 crispr/cas-based diagnostic methods. the clustered regularly interspaced short palindromic repeats (crispr)/cas machinery has recently been adapted as a poc tool for the rapid detection of nucleic acids (dna or rna). 126, 127 overall, this crispr machinery is programmed to cleave specific sequences in the dna/rna target where the results can be easily observed by combination with a lateral-flow strip. initially, zhang's team developed a crispr/cas-based platform called specific high-sensitivity enzymatic reporter unlocking (sherlock) that, combined with isothermal preamplification to detect strains of single-strand rna viruses, identifies mutations and human genotype dna, and distinguishes pathogenic bacteria. 126, 127 more recently, using the same knowledge, they adapted a protocol using the sherlock system for sars-cov-2 detection. 128 on the other hand, mammoth bioscience company developed another platform based on the crispr/cas system named the endonuclease-targeted crispr trans reporter (de-tectr) to detect any rna or dna target, which has now been used to detect the sars-cov-2 rna genome from respiratory swab rna extracts. 129 the suitability of detectr technology for the detection of sars-cov-2 was evaluated using 78 patient specimens, including 36 patients with covid-19 infection and 42 patients with other viral respiratory infections, and then compared with the cdc rt-qpcr as a reference method to confirm covid-19 infection. the sars-cov-2 detectr test had 95% positive predictive agreement and 100% negative predictive agreement when compared with rt-qpcr results. despite these promising results, crispr/cas-based diagnostic methods are not widely used by diagnostic laboratories and need further implementation. taken together, these results highlight the great potential of crispr-based diagnostic methods as a rapid, specific, portable, and accurate detection platform for the detection of the sars-cov-2 genome in patient samples. sensors. sensors represent another alternative detection method with rapid and high throughput. since the emergence of sars-cov-2, several research groups from different parts of the world have also focused on alternative sensing modalities based on sensors for the diagnosis of sars-cov-2 that reduce the use of expensive laboratory equipment, trained laboratory personnel, and extensive sample preparation and provide fast and accurate results. 130 in this context, qiu and co-workers developed a dual-functional plasmonic biosensor combining the plasmonic photothermal (ppt) effect and localized surface plasmon resonance (lspr) to detect sars-cov-2. 131 in another study, the authors used a field-effect transistor (fet)based sensor to detect sars-cov-2 from patient samples. 132 in general, these recent research findings are based on technologies previously used for the detection of other viral pathogens that now can be adapted for the detection of sars-cov-2. for instance, due to its high sensitivity and specificity, simple operation, low cost, and other advantages, the sensors using programmable biomolecular components named the toehold switch developed in response to previous ebola and zika outbreaks represent another powerful platform for detecting the sars-cov-2 genome. 133−135 genome sequencing. whole genome sequencing was used to identify potential etiological agents involved in the index cases of the covid-19 pandemic in wuhan. 11 in addition to unequivocally confirming the diagnosis of a sars-cov-2 infection, regular sequencing of a percentage of patient samples from clinical cases can be used to monitor changes in the viral genome over time and trace transmission patterns. for this purpose, several sequencing protocols based on sanger and next-generation sequencing (ngs) are being applied to rapidly generate the genome sequences. 5,136,137 lu and colleagues used a combination of sanger, illumina, and oxford nanopore minion sequencing technologies to obtain the whole-genome sequences of sars-cov-2 from six patient specimens from wuhan, china. 5 holshue and colleagues reported the first case of the novel coronavirus in the united states and used a combination of the sanger method, illumina, and minion to generate the whole-genome sequences. 136 whole-genome sequencing using minion technology coupled with phylogenetic analyses and travel history allowed the identification of sars-cov-2 entry routes into latin america during its emergence in brazil. 138 as of july 31, 2020, 13 303 genome sequences had been deposited in genbank (https://www.ncbi. nlm.nih.gov/genbank/sars-cov-2-seqs/), including complete genome sequences from different countries around the world and partial genome sequences. sars-cov-2 has evolved continuously since its emergence. the binding regions of primers and probes should be monitored continuously for matching to the virus genome as more sequence information becomes available. in this scenario, artesi and colleagues reported that changes within the sars-cov-2 primer binding region negatively affected the performance of commercial rt-qpcr assays (unpublished data, https://www.medrxiv.org/content/10.1101/2020.04. 28. 20083337v1 ). in another study, rana and co-workers analyzed the whole genome sequence of 93 patients with covid-19 and found variations in the primer and probe binding sites that could produce false-negative results. 139 a comprehensive bioinformatics analysis of 17 000 sars-cov-2 sequences from around the world identified the presence of mutations/ mismatches in the primer/probe binding regions of 7 assays out of 27 rt-qpcr assays studied, including the charite-orf1b reverse primer and the us-cdc-n-1 probe. 140 however, it should be noted that except at the 3′ end, rt-qpcr can tolerate mismatches at the 5′ extremity or in the middle of a primer. thus, oligonucleotide binding regions should be continuously monitored by bioinformatics and also by wet-laboratory experiments in order to identify changes that may influence rt-qpcr performance. 139, 141, 142 taken together, whole genome sequencing and bioinformatics will be important in global efforts to combat the pandemic, with the resulting analyses being used to guide a wide range of studies, including diagnostics, 97,121,122 molecular epidemiology studies, 5 viral evolution, 143−145 elucidatation of possible hosts of the virus, 5,146−148 identification of targets for drugs and vaccines, 149,150 molecular determinants involved in virulence and pathogenicity, 151, 152 and factors related to the host's immune response to the virus. 153, 154 serology. serological methods are being increasingly used and can be used for diagnosis, contact tracing, and herd immunity assessment, and vaccine efficacy evaluation. seroepidemiologic studies can assist in the investigation of the ongoing pandemic and the retrospective assessment to determine the attack rate or the progress of the pandemic through antibody detection. these studies can assist health authorities and governments in making sound decisions with respect to the implementation of public health measures during the course of the current pandemic. population-based serosurveys for sars-cov-2 has been applied in several sites to assess the burden of covid-19 and more accurately determine sars-cov-2 prevalence and transmission dynamics. 155−157 moreover, serology can also be useful in situations where rt-qpcr is negative and there is a strong epidemiological link to covid-19 infection. 94 in these cases, paired serum samples collected in the acute and convalescent phase can be of diagnostic value. the adaptive immune response to sars-cov-2 infection has been studied, 75,94,158,159 despite several knowledge gaps in this area. long and co-workers studied the antibody responses to sars-cov-2 in 285 patients with covid-19 in china. seroconversion for igm and igg occurred simultaneously or sequentially, and the median day of seroconversion for both immunoglobulins was 13 days after covid-19 onset. the seroconversion rate for igg reached 100% 19 days after symptom onset. 94 in a related work, zhao and colleagues showed that the median seroconversion times for total antibodies, igm, and igg were 11, 12, and 14 days after sars-cov-2 infection. 75 severe patients tend to have higher antibodies titers than patients with mild covid-19. 94, 160 to investigate the antibody responses to sars-cov-2 in convalescing individuals, robbiani and co-workers analyzed 149 plasma samples collected an average of 39 days after the onset of symptoms from convalescing individuals. most convalescent plasmas obtained from recovered patients did not contain high concentrations of neutralizing activity. 161 a related study with convalescing patients detected serum neutralizing antibodies in 13 out of 14 patients, and there was a strong correlation among neutralization antibody titers, the numbers of virus-specific t cells, and anti-s-rbd igg but not with anti-np igg. 159 notably, the antibody response and viral clearance can be delayed in immune-compromised individuals and people posteriorly infected with sars-cov-2. 162 in the past few months, a variety of serological tests have been designed to detect sars-cov-2, mainly enzyme-linked immunosorbent assays (elisa), immunofluorescence assays (ifa), chemiluminescence enzyme immunoassays (clia), and lateral flow assays (lfa). 163 of these serological methods, elisa, clia, and lfa are used for the first-line screening of patient samples with covid-19. most serological assays are based on the sars-cov-2 nucleocapsid protein (n), transmembrane spike protein (s), or s receptor-binding domain (rbd) because of their high antigenicity. 164−166 as mentioned before, sars-cov-2 enters host cells using its spike protein and represents the main target of neutralizing antibodies produced by the host. 167 in this context, several studies have demonstrated that serological tests using the s antigen are more sensitive than those using n-antigen-based antibody assays. 163 instead of a single antigen, some companies have combined n-and s-based antigens to develop serological assays for sars-cov-2 detection. 163 cross-reactivity between sars-cov-2 and sars-cov-1 has been reported against the n protein but not against the s1 subunit of the s protein. 94 cross-reactive antibodies between sars-cov-2 and sars-acs infectious diseases pubs.acs.org/journal/aidcbc review cov-1 have been detected in both the rbd region located in the s1 subunit and also in non-rbd regions. 168 anderson and colleagues analyzed patient samples with sars-cov-1 and sars-cov-2 infections and showed that there is no detectable cross-neutralization by sars-cov-1 patient serum against sars-cov-2, despite significant cross-reactivity between the n proteins from the two viruses 169 many elisa-based assays have been developed to evaluate the human antibody response (iga, igm, and igg) against sars-cov-2. 11,170−172 cross reactivity to other coronaviruses has been reported, and therefore test results should be interpreted in light of the local epidemiological scenario. 11 for example, it has been shown that igm elisa allows a higher detection efficacy than rt-qpcr after 5.5 days of symptom onset, and the combined use of igm elisa and rt-qpcr increases the positive detection rate to 98.6% when compared with a single rt-qpcr test (51.9%). 171 the median time of appearance of igm and iga antibodies in plasma was 5 days, while igg was detected 14 days after symptom onset. in the same study, sars-cov-2 n elisa did not show any cross-reactivity with other covs, with the exception of sars-cov-1, a virus that has not been in circulation since the 2002 to 2003 outbreak. 171 perera and co-workers developed an elisa based on the receptor-binding domain (rbd) of the sars-cov-2 s protein to detect igg and igm antibodies in human sera and compared the elisa results with confirmatory microneutralization and 90% plaque reduction neutralization tests (prnt 90 ). they found that prnt 90 were more sensitive than microneutralization to detecting antibodies against sars-cov-2, indicating that elisa should be used for screening and prnt 90 should be used for confirmation in large-scale seroepidemiological studies. 173 a magnetic chemiluminescence enzyme immunoassay (mclia) has also been developed for virus-specific antibody detection. in a study with 285 covid-19 patients, sera from these patients did not cross react with the sars-cov spike antigen, but some cross-reactivity was found with the sars-cov nucleocapsid antigen. 94 many lateral flow assays (lfa) have been developed to detect sars-cov-2 174−177 and have obtained emergency use authorization from the fda (https://www.fda.gov/medicaldevices/emergency-situations-medical-devices/emergency-useauthorizations). a combined igm/igg assay seems to be a better choice in terms of performance and sensitivity to either antibody alone (igm or igg). recently, li and colleagues developed a rapid lfa to simultaneously detect igm and igg antibodies against sars-cov-2 within 15 min in human blood. 178 the assay uses the receptor binding domain of the sars-cov-2 s protein as the antigen. they validated the assay with 525 blood samples previously screened by rt-qpcr, including 397 positive and 128 negative samples. the assay had a sensitivity of 88.66% and a specificity of 90.63% compared to rt-qpcr. in addition, several studies have been used to evaluate the clinical sensitivity of serological tests with covid-19 patient samples collected on different days after the onset of symptoms. 179−183 pan and colleagues evaluated a commercial lateral flow immunochromatographic test targeting viral igm or the igg antibody and compared it with rt-qpcr. the sensitivity of the assay for sars-cov-2 detection was 11.1% in early-stage patients (1−7 days after onset), 92.9% in intermediate-stage patients (8−14 days after onset), and 96.8% in late-stage patients (more than 15 days after onset). 180 in similar study, tang and co-workers compared the diagnostic performance of two sars-cov-2 commercial serologic tests using 103 specimens from pcr-confirmed sars-cov-2 patients and 153 control specimens from different days after disease onset (<3, 3−7, 8−13, and ≥14 days). both assays had poor diagnostic sensitivity during the first 14 days of symptoms, generating a high rate of false-negative results. 179 the time of sampling since the onset of symptoms must be taken into account when performing the serological diagnostic test, and false-negative results can lead to the false assumption that a person is not infected, consequently bringing about serious challenges and implications for the spread or containment of the disease. most patients will seroconvert only in the second week after infection, and thus negative serological test results obtained during the first 14 days of the disease should be interpreted with caution. 75 as opposed to classical serum neutralization assays such as the microneutralization and plaque reduction neutralization test (prnt), pseudovirus neutralization assays for sars-cov-2 can be performed in biosafety level 2 facilities. pseudovirusbased neutralizing assays for sars-cov-2 have been developed using vesicular stomatitis virus (vsv) and lentiviral pseudovirus systems. 184, 185 such systems will facilitate the study of covid-19 humoral immunity and the development and evaluation of vaccines and therapeutics against sars-cov-2 once the reliability and cross reactivity have been assessed. antigen detection tests represent another alternative to detecting sars-cov-2. antigen detection assays are developed to directly detect viral particles in biological samples such as nasopharyngeal secretions from infected patients. in this context, some rapid antigen assays have been proposed to detect sars-cov-2. 172,186−189 a fluorescence lfa antigen test based on the n protein has been developed and validated using nasopharyngeal and oropharyngeal samples from 127 suspected covid-19 cases (82 positive by rt-qpcr). compared with rt-qpcr, the test achieved a sensitivity of 93.9% and 100% specificity. 186 however, some commercially available point-of-care tests have very low sensitivity, which hinders their use in covid-19 diagnosis. 187, 190 for the validation of serological tests, robust test validation with adequate clinical samples representative of a real-world scenario (e.g., timing from disease onset, different disease severities, different geographical locations, and patient age) is of paramount importance. particular attention should be paid to the low specificity of some assays and the high possibility of false-positive results. 181 a low specificity of serological assays may have important consequences in terms of both diagnosis and population surveillance of covid-19 patients at the individual level and population level. 163, 190 at the individual level, the risks posed by false-positive results can be of great concern. for instance, people who have never been infected may return to work or travel because they are considered to be immune to sars-cov-2 infection. at the population level, false-positive results may increase the prevalence of covid-19 disease and provide a distorted picture of the higher population immunity and lower mortality rate than what is actually happening, which can negatively affect seroepidemiological surveillance studies. thus, the independent validation of covid-19 serological tests using samples from different stages of the disease is urgently needed to encourage health care professionals and governments to make sound decisions. differential diagnosis. since the clinical manifestations of covid-19 are similar to those of other respiratory diseases, differential diagnosis is of paramount importance in assisting physicians in the therapeutic management of patients and health officials in establishing disease control policies. in an effort to do so, respiratory pathogens in patients suspected of covid-19 in italy have been investigated. 20 here, researchers tested the nasopharyngeal swabs of 126 suspected causes using sars-cov-2 rt-qpcr and a commercial multiplex respiratory cartridge that detects and differentiates viral and bacterial pathogens. only 3 patients were confirmed to be infected with sars-cov-2, and 67 (53.2%) were positive for other respiratory pathogens, including 26 (20.6%) positive for influenza a and 10 (7.9%) positive for influenza b. other pathogens detected in the patient samples were common cold cov (h-cov 229 e, h-cov nl63, and h-cov hku1), rhinovirus, enterovirus, metapneumovirus, adenovirus, mycoplasma pneumoniae, streptococcus pneumoniae, and legionella pneumophila. 20 these results highlight the diagnostic differential and demonstrate the importance of using a spectrum of molecular kits for the rapid detection of respiratory pathogens in order to improve the clinical management and treatment of patients infected with covid-19. in another study, yan and co-workers reported the cases of two patients in singapore who initially had moderate symptoms including myalgia, a mild cough, and diarrhea and presented with thrombocytopenia and a normal chest radiograph. 22 dengue was suspected, and a rapid serological test for dengue produced false-positive results. as patient symptoms worsened, they were later diagnosed with covid-19 by rt-qpcr. taken together, these findings suggest that special attention is needed in the differential diagnosis between arboviruses and sars-cov-2 infection, especially in countries where is there a circulation of denv, zikv, and chikv. moreover, coinfection with sars-cov-2 and other respiratory viruses, such as common cold cov, influenza, and metapneumovirus, have been reported, highlighting the need for repeat testing based on clinical indications. 191−194 the rapid spread of sars-cov-2 around the world, with mounting cases of fatal pneumonia and the economic crisis, is a global concern. diagnostics represent one of the most powerful tools in mitigating these effects until a vaccine can be established. this needed is further highlighted by overlapping symptoms from other pathogens that can confound diagnosis based only on clinical criteria. as we have reviewed, many approaches have been established for the diagnosis of sars-cov-2, and we anticipate the coming months to bring about many more innovative strategies. critically, the development of inexpensive point-of-care diagnostic platforms will accelerate the global response to the current pandemic, especially in countries with health systems devoid of adequate laboratory infrastructure. it will be important that widespread diagnostic development and validation continue in the coming months and that the large-scale implementation of these sars-cov-2 diagnostic platforms is successful as this pandemic evolves. reliable, easy-to-use assays will be absolutely critical, especially as the disease moves through low-and middle-income countries. 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sensitive and simultaneous detection of igg/igm/antigen of sars-cov-2 within 15 min 2020) diagnostic accuracy of serological tests for covid-19: systematic review and metaanalysis coinfection with covid-19 and coronavirus hku1 -the critical need for repeat testing if clinically indicated coinfection of influenza virus and severe acute respiratory syndrome coronavirus 2 (sars-cov-2) coinfection with sars-cov-2 and human metapneumovirus sars-cov-2 and influenza virus co-infection epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in emerging 2019 novel coronavirus (2019-ncov) pneumonia china: a descriptive, cross-sectional, multicenter study prevalence and clinical presentation of health care workers with symptoms of coronavirus disease 2019 in 2 dutch hospitals during an early phase of the pandemic clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (covid-19) in the united states clinical features, laboratory characteristics, and outcomes of patients hospitalized with coronavirus disease 2019 (covid-19): early report from the united states key: cord-253862-jl1zhg13 authors: khalaf, khalil; papp, natalia; chou, jadzia tin-tsen; hana, doris; mackiewicz, andrzej; kaczmarek, mariusz title: sars-cov-2: pathogenesis, and advancements in diagnostics and treatment date: 2020-10-06 journal: front immunol doi: 10.3389/fimmu.2020.570927 sha: doc_id: 253862 cord_uid: jl1zhg13 the emergence and rapid spread of sars-cov-2 in december 2019 has brought the world to a standstill. while less pathogenic than the 2002–2003 sars-cov, this novel betacoronavirus presents a global threat due to its high transmission rate, ability to invade multiple tissues, and ability to trigger immunological hyperactivation. the identification of the animal reservoir and intermediate host were important steps toward slowing the spread of disease, and its genetic similarity to sars-cov has helped to determine pathogenesis and direct treatment strategies. the exponential increase in cases has necessitated fast and reliable testing procedures. although rt-pcr remains the gold standard, it is a time-consuming procedure, paving the way for newer techniques such as serologic tests and enzyme immunoassays. various clinical trials using broad antiviral agents in addition to novel medications have produced controversial results; however, the advancement of immunotherapy, particularly monoclonal antibodies and immune modulators is showing great promise in clinical trials. non-orthodox medications such as anti-malarials have been tested in multiple institutions but definitive conclusions are yet to be made. adjuvant therapies have also proven to be effective in decreasing mortality in the disease course. while no formal guidelines have been established, the multitude of ongoing clinical trials as a result of unprecedented access to research data brings us closer to halting the sars-cov-2 pandemic. coronaviruses are widely known virulent pathogens affecting mammalian and avian species. previously, six globally distributed species of the virus have been identified to cause illness in humans. they are: human coronavirus oc43 (hcov-oc43), human coronavirus hku1 (hcov-hku1), human coronavirus 229e (hcov-229e), human coronavirus nl63 (hcov-nl63), severe acute respiratory syndrome coronavirus (sars-cov), and middle east respiratory syndrome coronavirus (mers-cov) ( table 1) . sars-cov first emerged in [2002] [2003] in china, presenting as an atypical pneumonia with febrile state, headaches, and a marked cough that may rapidly deteriorate into respiratory failure. the sars-cov outbreak was the first to be declared a pandemic, and ultimately infected 8,000 persons in 29 countries, with a mortality rate of 10% (5). epidemiological analysis posited an infectious zoonotic agent, and further serological studies identified the intermediate host to be the palm civet (paguma larvata). spillover from animals to humans was hypothesized to be via the horseshoe bat (genus rhinolophus). an adaptation of the sars-cov virus was later proven to be due to interspecies dwelling (6) . a combination of ribavirin and pulse prednisolone showed positive results early in the outbreak and was adopted as the standard protocol. eventually, ribavirin was demonstrated to have increased cytotoxicity and lacked efficient antiviral action in vitro, and while pulse prednisolone showed efficacy in managing critically ill patients, its high dose administration was associated with disseminated fungal infections (7) . later, the combination of interferon-alpha1 (inf-α1) and corticosteroids was found to yield a better prognosis, but did not prove beneficial for patients in the late stage of the disease (8) . protease inhibitors produced an outcome similar to that of inf-α1 (9) . a novel coronavirus later named mers-cov emerged in saudi arabia in 2012. similar to sars, infected patients presented with a variety of clinical courses, from mild upper respiratory symptoms to fulminant pneumonia and multi-organ system failure. phylogenic and sequencing studies have proposed a bat origin, and surface protein modification was found to be derived from the intermediate hosts, dromedary camels. from 2012 to 2015, more than 2,000 confirmed cases were reported, mainly in middle eastern countries, with a mortality rate of 35% (10) . as with sars-cov, no definitive protocol was implemented, and most patients were treated with supportive therapy to preserve organ integrity (11) . in a cohort study conducted by omrani et al., a treatment protocol involving ifn-α2 and ribavirin was initiated, and while survival improved significantly at 14 days after treatment, this was not seen at day 28, necessitating further treatment options (12) . sporadic outbreaks occur to this day, with patients undergoing largely supportive therapy. the sars-cov-2 outbreak began in december 2019 in wuhan, china where clusters of atypical pneumonia yielded evidence of a novel strain of coronavirus. as of august 24, 2020, 16,036,308 cases had been reported worldwide, with a fatality rate of 5%. although this novel virus is less severe than the first sars-cov outbreak, human-to-human transmission remains very high and the number of cases continues to rise exponentially in major urban areas, highlighting the urgent need to develop new containment, diagnostic, and treatment protocols. as time is of the essence, the pathogenic mechanism cannot be studied with the rigor and comprehension otherwise expected, and the opportunities to compare therapeutic protocols are few. origin of the virus during the initial outbreak, liu et al. obtained samples of blood, bronchoalveolar lavage fluid, and anal and oral swabs from patients in wuhan, china suffering from a pneumonialike illness resembling the 2002-2003 sars-cov. a pan-cov pcr was performed on samples from five patients, and were positive for a pathogen from the coronaviridae family. this was compared with a full-length sequence of viral rna from a bat coronavirus (bat-covratg13), and demonstrated 96.2% similarity. thus, it is probable that the bat is the main reservoir of the novel coronavirus. identification of the intermediate host is an essential step in controlling the spread of disease, and became a priority for research teams. unfortunately, this was complicated by the many species of wild animals sold at the huanan seafood market, where the first cases were reported to have had contact. in 2019, a sars-cov-like pathogen known to be widely distributed in the malayan pangolin samples was discovered. the receptorbinding domain (rbd) present on the spike protein (s) is a crucial determinant in host range, as its interaction with the host receptor is responsible for the infection. rbd sequences from bat-covratg13, pangolin-sars-like cov and the novel sarslike pathogen were aligned. ninety three percentage similarity was demonstrated between the novel sars-like pathogen and the pangolin sars-like cov, and 89% similarity was demonstrated between the novel sars-like pathogen and the bat-covratg13. thus, on the basis of the rbd, the pangolin-sars-like cov is determined to be more likely than the bat-covratg13 to infect humans, making this the possible intermediate host (13) . xiao et al. conducted another study in which the pangolin-sars-like cov was isolated and amino acid sequence was compared to sars-cov-2. this yielded 100, 98.6, 97.8, and 90 .7% similarity with the s, m, e, and n proteins, respectively, of the novel sars-cov, strengthening the previous assumption that the pangolin was the intermediate host (14) . pathogenic classification is used to determine whether the pathogen is new or recurring in order to best implement safety and treatment protocols. while serological reactivity to viral proteins had been the mainstay of viral classification in the past, the process today now depends on replicated protein sequences. the international committee on taxonomy of viruses (ictv) maintains a study group for each viral family (15) . after analysis, the novel virus was assigned to the order nidovirales on the basis of the following domains: polyprotein protease (3clpro), catalytic domain of rna polymerase (rdrp), nidovirus-associated rdrp (niran), zinc binding domain (zbd), and helicase (hel1) (16) . subsequent next generation sequencing and phylogenic analysis placed the novel pathogen within the subgenus sarbecovirus of the genus betacoronavirus (17) ( table 2) . like other coronaviruses, the viral envelope is composed of a lipid bilayer derived from host cellular material, and the structural proteins are spike proteins composed of a trimeric glycoprotein projecting from the envelope like a crown. cryo-electron microscopy was used to compare the structural differences between the spike (s) proteins of sars-cov and sars-cov-2. unlike the previous sars-cov which possessed a single arginine targeted by a trypsin protease, sars-cov-2 possesses an s1/s2 protease cleavage site which is recognized by furin proteases. neuman et al. demonstrated the high level of protein organization and interaction within the envelope: the s protein was shown to be aligned with the npc, a crucial component of viral organization in protein-protein interactions that ensures high specificity and effectiveness for host invasion (19) . sars-cov-2 is an enveloped virus with a 29,904 bp positivesense non-segmented rna genome. the non-structural proteins (nsps) are described in table 3 . the remainder of the genome is responsible for accessory and structural proteins such as s, m, e and n. host cell recognition is the first and most essential step in viral pathogenesis. studies on the 2002-2003 sars-cov outbreak uncovered key interactions between the spike (s) protein, the rbd and angiotensin-converting enzyme 2 (ace-2). due to the previously mentioned similarities between sars-cov-2 and its predecessor, a viral infectivity study was performed using hela cells with and without the ace-2 receptor, which showed that only cells possessing the ace-2 receptor were infected (21) . the spike trimer is a class i fusion protein; upon infection the spike is cleaved by host proteases at the s1/s2 site for division of the two domains. the s1 domain possesses the rbd which recognizes and binds the ace-2 receptor in a prefusion state. structural rearrangement of the s protein subsequently exposes a furin cleavage site on the s2 domain which enables viral entry by means of fusion after the s1 domain is shed (22) . viral replication was hypothesized to occur via a process called autophagy: an evolutionary cellular process in which cytoplasmic proteins create isolation membranes surrounding materials destined for degradation (23) . evidence of coronavirus autophagy was first demonstrated by prentice et al., who showed that in coronavirus mouse hepatitis viruses, atg5-induced autophagy was required for the formation of double membrane vesicles (dmv) and for replication (23) . another study confirmed the use of atg5dependent autophagy of betacoronaviruses through nsp 6 (24). chen et al. previously showed that both sars-cov and mers-cov employ the use of papain-like proteases (plpro) to induce the formation of autophagosomes, but their fusion to lysosomes is inhibited which promotes the replication process (25) . however, other studies have demonstrated that key autophagy proteins atg5 or atg7 were not required for coronavirus mouse hepatitis or sars-cov viral replication (26) . reggiori et al. also yielded comparable results showing that the replication and release processes are not dependent on autophagy, but that the dmv's were coated with (lc3)-i (non-lipidated microtubule associated protein i light chain 3), thus showing the importance of this protein for viral replication (27) . a recent study by benveunto et al. has shown that sars-cov-2 induced mutations within the nsp6 protein, which in turn induced autophagosome formation by inhibiting phagosomelysosome fusion (28, 29) . sars-cov-2 may take advantage of this autophagy mechanism, as the acidic ph required for endosome maturation also functions to release the virion at the appropriate site. once all necessary proteins are translated and replication has taken place, virion assembly occurs in the golgi apparatus before release from the cell (30) . symptoms attributed to sars-cov-2 shereen et al. determined a mean incubation period of 5 days in the first 425 patients testing positive for sars-cov-2 (31). the time from infection to death varied from 6 to 41 days, with a mean of 14 days (32) . in january of 2020, huang et al. examined all patients exhibiting pneumonia-like symptoms in order to determine the clinical features of sars-cov-2; importantly, this study did not discriminate between previously healthy patients and patients suffering from chronic illnesses. data was collected from the 41 positive cases, and analysis yielded the following: fever was the first and most common primary symptom (98%), followed by dry cough (76%), lymphopenia (63%), dyspnea (55%), and fatigue/myalgia (44%). less common symptoms included sputum production (28%), headache (8%), hemoptysis (5%), and diarrhea (1%) (33) . a recent systematic review of 148 studies spanning nine countries disputes the above-mentioned data. in this analysis by grant et al., the most common symptoms were as follows: fever (78%), dry cough (57%), fatigue (31%), anosmia (25%), and difficulty breathing (23%) (34) . another study by clemency et al. assessed the likelihood that sars-cov-2 infection presents with symptoms. the positive likelihood ratio of having symptoms with disease from highest to lowest was as follows: anosmia and ageusia, followed by loss of appetite, fever, muscle pain, fatigue, dry cough, productive cough, diarrhea, difficulty breathing, and sore throat (35) . the stability of sars-cov-2 on various surfaces was compared to that of its predecessor sars-cov, and it was found that both viruses exhibit similar stability. the differences in epidemiological spread are therefore most likely to be dependent upon other factors such as high viral load and viral shedding in asymptomatic persons. the study is highly suggestive of an aerosolized pathogen as it remains viable and infectious in droplet form for up to 3 h (36). while resembling the previous sars-cov outbreak, this data confirms that sars-cov-2 is far more transmissible. kinetic studies employed to compare the two strains showed that sars-cov-2 binds to the ace-2 receptor with a 10-to 20-fold higher affinity than sars-cov, suggesting that this could be another main factor explaining the ease of transmission (37) . to further support this hypothesis, computational structural predictions were established to differentiate between the rate of association between the ace-2 receptor and the spike protein of both sars-cov and sars-cov-2. by incorporating the computer simulation into a mathematical model, it was shown that the novel pathogen has slower binding capacity than sars-cov, consistent with the varying life cycles of the two strains. in other words, the longer incubation period of sars-cov-2 was associated with the slower interaction between the spike protein and the ace-2 receptor. this allows for maintenance of an increased viral load in the body, thus contributing to increased human-to-human transmission (38) . the renin-angiotensin-aldosterone system (raas) is responsible for inducing a coordinated cascade regulating both cardiovascular and renal functions. angiotensin-converting enzyme (ace) is responsible for converting angiotensin i to angiotensin ii, with the latter functioning as a pro-sympathetic molecule throughout the body to regulate blood pressure. wakahara et al. measured ace and ace-2 expression under various conditions. in addition to the ace-2 role in local regulation of raas through the conversion of angiotensin ii into vasodilators and anti-trophic peptide, it acts and is expressed in synergy with ace concentrations and is believed to possess counter-regulatory effects to ace (39) . as the expression of the ace-2 receptor is vital for viral entry, it is essential to establish where this gene is expressed throughout the body to identify possible routes of infection and/or the extent of organ damage during infection. zou et al. employed the dataset systems of single-cell rna sequences from different body tissues to identify organs expressing the ace-2 gene in high concentrations. the symptoms reported to be associated with covid-19 (dyspnea, cardiac injury, kidney failure, diarrhea) were compared to ace-2 expression on target cells. it is on this basis that the organs at high risk of damage during viremia were recognized to be the lungs, heart, kidney, and upper respiratory tract (40) . a minority of patients is seen to present with gastrointestinal involvement, and a further study by wong et al. on 140 patients in wuhan demonstrated this complaint in 39% of their cohort. the virus was detected in stool by means of rt-pcr. staining of the n protein showed presence of the virus in the cytoplasm of duodenal, rectal and gastric epithelium thus indicating another possible mode of transmission and another sign of possible infection (41) . zhao et al. also compared the symptoms and disease characteristics of covid-19 patients with that of other pneumonias, indicating that liver damage was more prominent in covid-19 patients. however, it was not clear whether liver enzyme elevation was due to drug toxicity or viral shedding (42) . the ace-2-receptor mapping experiment, while important, provides little information on the broad expressions of the receptor, as ace-2 concentration may differ due to various clinico-pathologies such as hypertension, diabetes, heart failure, smoking and kidney injury (43, 44) . remuzzi and remuzzi in italy demonstrated that two-thirds of patients who died from sars-cov-2 were elderly, diabetic or suffered from cardiovascular disease (45) . their first line drug of choice was angiotensin receptor blockers (arbs). ferrario et al. demonstrated in 2005 that selective blockade of angiotensin ii synthesis or activity has an up-regulatory effect of ace-2 receptors in the kidneys and heart (46) . this ace-2 upregulation in the aforementioned comorbidities could therefore be responsible for the severity of infection in these patients. guo et al. demonstrated that in 187 sars-cov-2-positive patients, 28 .7% had an increase in troponin-t and crp levels that correlated with myocardial injury. sars-cov-2 patients who presented with an increase in cardiac markers exhibited a 59.6% mortality rate compared to those with normal cardiac markers (8.9% mortality rate). sars-cov-2 genomic material has also been isolated from cardiac biopsies. while the pathophysiologic development of myocardial injury is not yet fully understood, it is believed to be caused by either cytokine storm or direct myocardial damage through viral integration (47) . to further understand the organ damage and hemostatic changes, han et al. assessed the change in coagulation proteins in both covid-19 patients and healthy patients. they found that antithrombin levels were lower in covid-19 patients, whereas fibrinogen, fibrinogen/fibrin degradation products and d-dimer were high (48) . a recent cohort study by wichmann et al. reported a possible connection between covid-19 and incidence of thromboembolism. autopsies of 12 covid-19 patients revealed the incidence of deep vein thrombosis (dvt) in 58% of cases, and a third of patients suffered a fatal pulmonary embolism. further studies should be conducted to further elucidate the formation of thromboembolism in the course of covid-19 (49) . cd147, also known as emmprin (extracellular matrix metalloproteinase inducer) is a highly glycosylated transmembrane glycoprotein shown to be involved in facilitating sars-cov endocytosis. this presents an alternative route of viral invasion (50) . like the ace-receptor, cd147 is also present in various tissues such as lymphoid (51) , testes, liver, cerebral, and renal tissues, as well as cardiac and skeletal muscle (52) . in addition to enabling viral entry, cd147 has been linked to other pathologic conditions such as cancer (53, 54) , heart disease (55) and alzheimer's disease (56) . furthermore, the speed of invasion may be increased as cd147 has been shown to be upregulated upon t-cell activation (57) . wang et al. has since shown that the spike protein possesses high affinity for cd147, thus mediating viral invasion and facilitating viral replication. it acts as a negative regulator of the immune system which may contribute to the significant cd4+ decline during covid-19 (58) . under hypoxic conditions, this receptor is involved in the regulation of cell proliferation and apoptosis. the cd147 receptor, a marker of early-stage disease, can be useful for the assessment of pathological progression because it is often expressed on the surface of activated regulatory t cells (59) . lung epithelium is the largest area of the body in constant contact with the external environment, and is responsible for processing inhaled air containing large volumes of bacteria and viruses. the immune response to sars-cov was widely studied, and sars-cov-2 is believed to induce the same responses. innate immunity is the primary countermeasure against infection, and is maintained by a variety of cell types found in airway epithelium, including dendritic cells, innate lymphoid cells and alveolar macrophages. the protective signaling cascade begins as the pattern recognition receptors (prrs) of innate cells recognize pathogen-associated molecular patterns (pamps) of viruses. after recognition, type i and iii interferon (ifn) and other proinflammatory cytokines undergo transcription. autocrine and paracrine signals in both healthy and infected cells subsequently ensure the translation of these cytokines (60) . passive evasion refers to any mechanism of immune avoidance that does not directly interfere with the host immune system. many non-structural proteins (nsp) are translated in order to shield viral genomic material from the human immune system, as shown in table 3 . nsp 3, 4, and 6 are believed to function as modifiers of the intracellular membranes of organelles, where the virus may safely replicate. another passive mechanism is the conformational change of the viral mrna through either the addition of a 5'-cap (via formation of n-7-methyltransferase in nsp 14), or a 2'-o-methyl-transferase as with nsp 16, which marks the viral mrna to be of host origin. in another example, nsp 15 induces endoribonuclease activity against viral mrna at certain stages of its development to avoid detection (20) . nsp 1 was shown to inhibit host mrna translation through the activation of exonucleases. these induce degradation of the host genome and prevent the ifn signal transduction that is necessary to activate viral clearance. nsp 3 expresses plpro, which greatly resembles host cellular de-ubiquitinase action. this induces disruption of ubiquitin-mediated degradation and may also inhibit the innate immune response (61). as with the previous outbreak, immune clearance of sars-cov-2 relies mostly on the activity of th1 cells via the extensive secretion of il-2, il-10, ifn-γ, and tnf-α/β. this cytokine microenvironment activates macrophages and causes cytotoxic t-cell proliferation, initiating pathogen clearance. following degradation and the presentation of viral antigens on apcs, the humoral response acts to limit future infections through antibody neutralization (62) . in the lungs, alveolar macrophages are the first cells to make contact with microorganisms. their main function is to destroy any invaders without overstimulating the adaptive immune system, since foreign pathogens are ever-present. protective measures employed by alveolar macrophages include the surface expression of cd200 and tgf-β receptors, which function as negative regulators of dendritic cells and t cells (63, 64) . this inactivation is exploited by sars-cov, leading to an increase in viral load. additionally, law et al. demonstrated that macrophages and dendritic cells are targeted by sars-cov, resulting in a very low expression of antiviral cytokines. this inhibits apoptosis and hinders the bridging of the innate and adaptive immune systems, leading to lymphopenia (65) . in a cohort study by qin et al. of patients with confirmed sars-cov-2 infection, lymphopenia and an increase in neutrophil-to-lymphocyte ratio was routinely observed among infected patients and was especially evident in severe cases (66) . it has been shown that these two parameters are indicative of systemic inflammation, and were associated with the worst prognosis (67, 68) . because qin and colleagues did not observe any changes in either cd8+ or b cells, it was then hypothesized that sars-cov-2 plays a major role in indirectly damaging lymphocytes, with the release of proinflammatory cytokines and chemokines that results in the consumption of the lymphocytes necessary to prevent innate overactivation (69). qin et al. also noted that the increase in neutrophil-to-lymphocyte ratio was accompanied by an increase in procalcitonin, indicating bacterial co-infection (66). zhao et al. showed that mice depleted of alveolar macrophages and subsequently infected with a mouse-adapted sars-cov strain quickly developed activation of dendritic cells, which in turn activated cytotoxic t cells and initiated viral clearance (70) . while most viral infections end with eradication and development of immunological memory, adaptive immunity does on occasion fail to develop adequately. callow et al. demonstrated in 1990 that patients previously infected with human coronavirus 229e showed a decline in antibody concentration and were capable of being re-infected after 1 year (71) . other studies involving mers-cov have come to the same conclusion, determining that the concentration of virusneutralizing antibody was dependent upon disease severity (72, 73) . it is important to note that the failure of adaptive immunity could be due to either insufficient antibody response or decrease in t-cell durability (74, 75) . appropriate adaptive immunity requires early cd8+ and cd4+ responses. in the case of sars-cov-2, viral evasion of the innate immune system leads to an increase in cytokine production and late cd4+/cd8+ response, which then leads to pathogenic inflammation in patients with high viral loads. due to rapid and unopposed sars-cov-2 replication, cd4+ t lymphocytes are quickly activated to differentiate into th1 cells and are responsible for releasing pro-inflammatory cytokines il-6, gm-csf, and ifn-γ. gm-csf activates to further produce inflammatory monocytes (cd14+ and cd16+) which release more il-6. this disrupts the homeostasis of the immune system leading to cytokine storm (76) . sars-cov-2-related hyperinflammation involves very high levels of il-1-β, il-6, and tnf-α (77). sars-cov-2 is thought to bind to the toll-like receptor (tlr), activating inflammasomes and resulting in the cleavage of pro-il-1β to form il-1β, a mediator of inflammation, fever and lung injury (78) . the pathological immune response has a wide variety of clinical presentations, from mild symptoms to pulmonary failure and death. extensive lung damage is associated with neutrophil and macrophage infiltration while cd4+ and cd8+ count in peripheral blood are simultaneously lowered (79) . croft et al. performed serological analyses in both healthy and sars-cov-2-positive individuals. the cd4+ t cells in infected individuals demonstrated very high levels of cd69, cd44, and cd38, indicating a hyperactive state compared to the healthy group. further analysis showed high levels of the t-cell activation marker ox40 on cd4+ cells, which has been proven to be crucial for cell expansion, survival and cytokine production in both t cells and innate cells (80) . it is important to note that expression levels of ox40 also varied depending on the severity and progression of the disease, and may possibly be a marker of poorer prognosis. cd8+ t cells were also found in a hyperactive state, with higher expressions of cd69, cd44, and cd38. in various deteriorating icu patients, there was an increase in expressions of pd-1 and tim-3 in both cd4+ and cd8+ t cells, indicating immune exhaustion (76, 81) . aside from exhaustion antigens such as pd-1, bellesi et al. (82) revealed upregulated expression of the cd95 antigen on both cd4+ and cd8+ t cells. when cd95 apoptosis-related antigen is observed to be highly expressed, this is accompanied by a lower cd4+ and cd8+ count, which may partially explain the loss of lymphocytes in sars-cov-2-positive patients. the loss of naive cells appears to be particularly important in this context. complement activation can be initiated via the alternative pathway, classical pathway or lectin pathway. innate immunity recognizes pathogen-associated molecular patterns (pamps) in host cells and initiates a destructive response. the mannosebinding lectin (mbl) pathway has been shown to be the first line of defense against sars-cov (83). it is composed of pattern recognition molecules associated with serine protease from the mbl-associated serine proteases (masps), which circulate as zymogens and become active after recognition, binding to carbohydrate-based pamps (84) . to further understand the triggers of the excessive immune response to sars-cov-2, gao et al. showed that as with sars-cov, masp-2 contact with the n protein led to cleavage of c4 and c2 into c4a/c4b and c2a/c2b, respectively, and the mbl pathway proceeded as normal. none of the other pathways triggered complement activation (85) . due to the high infectivity and severity of the virus, the world health organization (who) has been prompted to develop new tools to ensure the fast and accurate diagnosis of the viral infection. many governments have given an unprecedented level of freedom to nominated laboratories to establish reliable diagnostic tests. the increased demand for rapid testing has fueled research in sequencing, characterizing, and understanding sars-cov-2, in order to definitively diagnose it in human samples. as of august 2020, the recommended sampling sites are as follows: the upper respiratory tract (nasopharynx, oropharynx, anterior nares), lower respiratory tract (sputum, bronchoalveolar lavage, pulmonary tissue biopsy) as well as urine, feces or whole blood. according to centers for disease control (cdc) guidelines, material from the upper and lower respiratory tracts are preferred, and should be sampled if possible. material should be collected by an experienced specialist to ensure high standards, and should be tested as soon as possible; exceptions are serum samples which can be stored for up to 2-4 weeks, to be referenced in future follow-up (86) . the overwhelming number of infected cases compounded with the shortage of healthcare personnel has led to the prioritization of testing of affected individuals. due to limited access and resources, the cdc has been compelled to define specific criteria for testing all 2019-ncov patients under investigation (pui) . in order to be tested, the pui needs to manifest with: -"fever and symptoms of lower respiratory illness (cough, difficulty breathing) after 14 days of travel to wuhan city or contact with a 2019-ncov pui within the last 14 days, " or -"fever or symptoms of lower respiratory illness (cough, difficulty breathing) after contact with a patient with a confirmed case of sars-cov-2 infection within 14 days" (87) . the introduction of restrictions not only guarantees good quality patient care and prioritizes acute and severe cases, but maintains the integrity of an already strained healthcare system. the high volume of cases demands fast and efficacious testing regimes for a variety of settings, including the hospital. depending on the type of technology and the personnel available, a few specialized diagnostic protocols have proven to be useful in the field. sars-cov-2 is an rna virus which sheds detectable genetic material in almost all excretions of an infected individual. this material can be detected by a simple nucleic acid test which is capable of identification and characterization of nucleotide sequences. in blood, sputum and other samples, the amount of genetic material is very sparse thus necessitating an additional amplification step in order to reach a particular detection threshold. this method is known as the nucleic acid amplification test (naat). another technique currently available is the polymerase chain reaction (pcr). real-time polymerase chain reaction (rrt-pcr) is the gold-standard molecular technique for detection of sars-cov-2 viral rna in all recommended samples. it is a primary diagnostic test that targets the following sequences that code for structural viral proteins: spike (s), membrane (m), envelope (e), nucleocapsid (n), and rnadependent rna polymerase (rdrp). the available literature suggests that the spike protein (s) may be the primary pivot in intracellular interactions with host cells, thus demonstrating high immunogenic character (88) . high infectivity of sars-cov-2 has compelled the cdc to publish rrt-pcr primers and probes together with all relevant literature for public access (87) . such advances in research were possible thanks to past experience with the previous betacoronavius epidemic. primer design was based on the nucleotide sequences that matched sars-cov and mers-cov with 80 to 90% accuracy (21) . the wide availability of protocols has accelerated progress in research and diagnostic measures. nevertheless, it should be noted that the high mutation rate and large genetic variability of the virus may negatively affect the performance of the assay, and may lead to an increasing number of false-negative results (89) . additionally, the difficulty of the assay, complexity of the logistic analysis, and protocol duration (45 min to a few hours) (90) confer some limitations to this diagnostic tool (62) . the full rna extraction protocol should be implemented in a biosafety cabinet at bsl-2 security level by trained and skilled personnel. it is recommended that none of the samples be heat-treated before rna extraction, which means that samples pose a high risk of infection to laboratory technicians (86) . false-positive results may also be obtained in cases where the amount of viral material in a collected sample is too low for detection (89) . based on a published summary report by the fda, the analytical sensitivity of rt-pcr is 80% with a limit of detection of 6.25 cp/ul. specificity showed no crossreactivity with the most common pathogens: bacterial (legionella pneumophilia, mycobacterium tuberculosis and m. pneumoniae, and streptococcus pneumoniae and s. pyogenes) and viral (adenovirus, parainfluenza, rhinovirus, rsv, etc.). the only detected cross-reactivity corresponded to the severe acute respiratory syndrome coronavirus (sars-cov), but this is not surprising since the n3 target gene in sars-cov-2 demonstrates more than 80% genetic similarity to other betacoronaviruses (91) . the diagnostic protocol formulated by the cdc clearly states the process of confirmation of sars-cov-2 infection, though the criteria depend on the area where the pui is being diagnosed. in areas where the concentration of sars-cov-2 virus is high, a single positive naat result is required to diagnose the patient. in areas with low viral circulation more than one of the following is required: -positive naat for at least two different targets on the sars-cov-2 genome (one specific for the virus) -1 positive naat for betacoronavirus (sars-cov, mers-cov, sars-cov2) with sars-cov-2 genome sequencing. it is indicated that the sequenced fragment must be longer or different from the fragment used in the naat assay (86) . in cases where all tested controls are positive, with all sars-cov-2 markers below growth thresholds, the naat result is negative. however, cases with high clinical suspicion may still produce a negative rt-pcr result. this may be due to a number of factors, such as poor quality and handling of specimens, specimen collection too early or late in the disease course, contamination, and/or technical errors (86) . in other words, the lack of a positive result does not exclude covid-19 disease. in such instances identification of the infected individual is based on clinical observation, patient history and epidemiological information. given the number of tests done to date, in the case of discordant opinions, re-sampling and use of different markers is advised (86) . the limitations of naat diagnostic techniques for sars-cov-2 have generated increasing demand for quicker and simpler tests based on serology. rt-pcr, while very reliable and precise, is only capable of identifying the presence of viral load, and cannot inform on the state or progress of the infection in a given patient. in contrast, serological testing and enzyme-based testing measure immunological responses to the virus, allowing for differentiation between exposed asymptomatic, acutely or mildly sick, and recovered cases. additionally, it is able to quantify the number of cases within a short period of time, and this is crucial for modeling the population-scale of infection, determining the level of prophylaxis, and has implications for the development of a vaccine against sars-cov-2. there are many immunoassay techniques currently approved or pending. their versatility and creativity augment the number of ways with which one can detect the pathogen in a studied sample. they can be divided into serologic tests and enzyme-based immunoassays. serologic tests exploit the natural responses of the human immune system, while enzyme-based immunoassays detect the antigen with specifically manufactured monoclonal antibodies. all tests in this category detect either the antigen shed by the virus or the antibody that is produced by b-cells in response to the pathogen. the rapid diagnostic test (rdt) is a serologic type of diagnostic tool that allows for detection of the target after 10-30 min (90) . it is a qualitative lateral flow type assay that resembles the standard rapid chromatographic test used for the detection of beta-hcg hormone in the urine of pregnant women. as a field-based test, it requires a drop of blood or other body fluid for identification of igg and/or igm antibodies, or the viral antigen itself (90) . antigen based-rdts target the structural viral proteins (s, n, etc.) mainly found in secretions of the upper respiratory tract, and react to the antibodies that are produced 10-30 days after the initial infection. sufficient time is required for the concentration of antibodies to reach the threshold of detection (92) . it is also advised to measure igg and igm titer baselines before or during the first few days after exposure (90) . the non-governmental organization find recognizes 10 cemarked rapid tests ready for use in the field (93, 94) . another report by john hopkins center for health and security lists a few rdts currently approved for diagnostic use worldwide, but the antibody-rdt lateral flow assay by cellex inc. is the only rdt approved in the united states. this test was developed in the usa and china, with a sensitivity and specificity equal to 93.8 and 95.6%, respectively (90) . the statistical significance was determined from studies in two chinese hospitals on 128 sars-cov-2-positive patients and 250 sars-cov-2-negative patients (90) . the test is currently available for purchase with a disclaimer from the fda that the test alone cannot be used for definitive diagnosis. in regards to the april 8 2020 who statement concerning immunoassays, the antibody-/antigen-detecting rdts are only to be used for research purposes (95) . according to recent data, these diagnostic tests do not produce results with appropriate reliability. bruning et al. (96) claims that immunodiagnostic testing for influenza infections in cases with a viral load comparable to sars-cov-2 showed test sensitivity that varied between 34 and 80% (96) . also, due to their abrupt development, the majority of available rapid tests appear not to have been properly validated by relevant institutions and may pose a serious risk to diagnostic efficacy. the simplicity of the rdt means it is neither capable of quantifying the number of antibodies and therefore the phase of infection, nor is it able to ascertain whether these antibodies are part of long-term immunity to the virus. as of august 2020, it is unknown whether exposure imparts immunity after recovery. a 10 day or more delay in diagnosis is a serious diagnostic limitation and may not be of increased benefit to acute-state patients. another type of serologic testing for sars-cov-2 is a neutralization assay. it is a valuable method that detects antibodies capable of clearing the infection. the procedure consists of the infection of a special type of cell (e.g., veroe6) with sars-cov-2, followed by incubation for 3-5 days at variable concentrations. during this time cells are titrated with human serum antibodies in order to detect the quantity of antibodies necessary to stop viral replication (90) . a study performed by zhou et al. demonstrated that 1:40-1:80 dilutions of igg-positive sample were capable of neutralizing 100tcid 50 (50% tissueculture-infective dose) in sars-cov-2 cultures (21) . other than this result, it remains a relatively novel area of research, and information regarding its usefulness in the diagnosis of covid-19 is sparse (90) . table 4 lists a number of exemplary testing kits currently available for diagnostic use. enzyme-linked immunosorbent assay (elisa) is currently one of the most popular commercially used enzyme based immunoassays. as indicated by the name, it is an assay using an enzymatic reaction to indicate a positive diagnostic result. plates covered with viral antigens, prepared by the manufacturer, are incubated with the patient's serum. in cases where the specific igg and igm antibodies are present, the antibody-antigen binding complex will be visualized through an enzymatic reaction (colorimetric change, fluorescence, etc.) the whole procedure may take up to 5 h and requires a large sample of blood, plasma, or serum. it is both a qualitative and quantitative type of assay, thus has great potential for future diagnoses of sars-cov-2 infections. in a study of sars-cov-2-specific antibody responses in covid-19 patients, nisreen et al. used elisa to demonstrate that severe viral infection resulted in higher antibody levels. additionally, they claim that igg seroconversion can be confirmed by applying the same technique in the second week of symptom onset (97) . another team proved that the accuracy of elisa is dependent on the timing of the infection. on days 0-10 after symptom onset, elisa showed <60% positive rate; the rate rapidly increased beyond that time frame for both igm and igg antibodies. liu et al. evaluated immunoassays for detection of antibodies against sars-cov-2, and demonstrated that viral protein s (spike)-based igm elisa had statistically higher sensitivity than n (nucleocapsid)based elisa (p < 0.05) in detecting igm antibodies. it is suspected that the level of immunogenicity of the s protein is the reason for the relatively higher specificity compared to the n protein (98) . based on this data, the antigen-based elisa will be a valuable diagnostic method for the fight against covid-19. currently, the ngo find lists 66 different immunoassay kits already commercialized, with 16 additional test kits in development (94) . with supplemental research, these have the potential to become a powerful rapid diagnostic tool for the hospital setting. however, due to their complexity they, unlike rdts, may not be used in the field. to reiterate, there is an insufficient amount of evidencebased research regarding elisa's specificity and sensitivity for use in diagnostic confirmation, to be comparable with rrt-pcr assays. a lesser known enzyme-based immunoassay that is nevertheless noteworthy is the chemiluminescent immunoassay (clia). very similar to elisa, it uses enzyme-labeled antibodies which activate an enzymatic reaction upon contact with their target. the photon of light emitted-luminescencecan then be quantified and directly corresponded to the volume of reagents. the benefit of this technique is its high sensitivity and the possibility of enhancing the reaction to allow for a larger threshold in samples with higher substrate concentration. clia can be used to detect versatile targets including igm, igg, and iga, and there seems to have been a recent increase in trust for this technique among clinicians. a systematic review by bastos et al. compared lfia, elisa, and clia tests, and the results suggest that clia exhibits the highest sensitivity and specificity for igm and igg in patients with covid-19 (99) . however, bastos and colleagues also demonstrated that due to excessive discrepancies in test rests, none of the three techniques tested were reliable enough to be recommended for large-scale diagnostic purposes. after the 2002-2003 sars outbreak, chandwani and shuter conducted an in vitro study using an engineered prototype of sars-cov to test lopinavir/ritonavir, protease inhibitors indicated for dual-therapy prophylaxis and treatment of hiv-1 (100). lopinavir has higher potency but is less bioavailable, thus co-administration with ritonavir, which additionally inhibits cytochrome p450 34a, leads to prolongation of its action (101) . they showed that this dual therapy inhibited viral cytopathic activity. furthermore, chu et al. conducted a non-randomized trial in 2004, placing two groups of patients on different regimens: the first group received ribavirin, a nucleoside inhibitor, in dual therapy with a reducing dose of corticosteroids; and the second group received lopinavir/ritonavir/ribavirin. it was shown that treatment group two had a decrease in viral load, fewer nosocomial infections and an increase in circulating lymphocytes, indicating a favorable outcome (102) . after the emergence of sars-cov-2, a randomized trial was conducted involving 199 severe cases. ninety nine of these patients received lopinavir/ritonavir while 100 received standard supportive care. detectable viral load in both groups was the same, and time to improvement after lopinavir/ritonavir was only decreased by 1 day, as compared to the group receiving standard care. the authors concluded that the treatment benefits of lopinavir/ritonavir were not established for severe illness (103) . similarly, an open-label randomized control trial by cao et al. (chictr2000029308), involving severe sars-cov-2 cases, compared lopinavir/ritonavir treatment with standard care alone, and they showed that the antivirals yielded no clinical benefits. further trials are thus recommended to establish any possible benefits for patients suffering from less severe illness (104) . favipiravir, another rdrp inhibitor, is a broad antiviral with a mechanism of action that is hypothesized to either incorporate within viral rna leading to chain termination, or bind to a conserved region of the rdrp and prevent nucleotide addition (105) . interferon-alpha (ifn-α) is an antiviral that binds to interferon receptors and activates signal modulators (jak1/2). the phosphorylated interferon receptor binds to the signal modulators, resulting in immune modulation and antiviral protein transcription. in an open-label control study conducted by cai et al., the antiviral activity of favipiravir + ifn-α was compared to that of lopinavir/ritonavir + ifn-α in patients with confirmed sars-cov-2 infection. they demonstrated that patients receiving favipiravir + ifn-α exhibited faster viral clearance and radiological improvement, compared to the other study group. although yielding positive results, this was not a double-blind placebo-controlled randomized study, and more trials must be implemented before definitive conclusions can be drawn (106) . umifenovir is a broad spectrum antiviral possessing dual properties: direct antiviral and virucidal action, as well as demonstrating virustatic effect through impedance of various stages of the viral life cycle (107) clinical trials-mechanism of action unknown. uprifosbuvir, setrobuvir, balaprevir, 2'-c-methylcytidine, valopectibine bms-986094, mk0608, r7128, r1479, idx-184, yak, psi-6130 and psi-6206 apparent in the umifenovir group, and no side effects were observed in either group (111) . an open-label randomized control trial (chictr2000030254) was conducted by chen et al. in which adult sars-cov-2 patients were administered either favipiravir or umifenovir. they showed that favipiravir significantly improved symptoms associated with cough and pyrexia. however, no clinical benefit could be observed when comparing viral clearance between the two therapies (112) . after isolating the genomic sequence of sars-cov-2, in particular that pertaining to rna dependent rna-polymerase (rdrp), elfiky showed that the rdrp of sars-cov-2 exhibits 90.18% similarity with that of sars-cov. an rdrp model was engineered from the ncbi nucleotide protein database using the sars-cov rdrp genome as a template to test several antiviral drugs. to ensure high reliability of the model, a nucleotide comparison between the rdrp model and the sars-cov-2 rdrp was established, and was shown to yield 97.08% homology. the study sought to establish which of 24 compounds (table 5) could bind to rdrp active sites and elicit inhibitory activity. drug docking site analyses were interpreted using protein-ligand-interaction-profiler (plip). five fda-approved drugs showed very high affinity for rdrp, and could prove to be beneficial against sars-cov-2. additionally, three drugs from the clinical trial by elficky showed high affinity for rdrp, and these include idx-184, setrobuvir and yak. drug side effects and toxicity are yet to be disclosed (117) . additionally, one antiviral drug that sparked interest was remdesivir. in a small cohort study, a 10 day course of remdesivir was administered to 53 patients with severe infection, and clinical improvement was observed in 68%. fewer clinical benefits were observed in patients who received invasive ventilator support, and 13% of the patients, especially those who received invasive ventilation, died after the treatment course. multiple factors impede the accurate measurement of remdesivir efficacy and these include preexisting conditions and duration of intubation. as a result, any clinical benefits need to be further investigated in future placebo-controlled trials (118) . in a double blind placebo-controlled randomized trial (ntc04280705) biegel et al. administered remdesivir to hospitalized sars-cov-2 patients presenting with lower respiratory tract involvement to assess its efficacy and safety. remdesivir was shown to be superior to placebo in decreasing recovery time (119) . however, a multicenter, randomized, placebo-controlled trial conducted by wang et al. to assess the efficacy of remdesivir in patients with severe sars-cov-2 (ntc04257656) showed that remdesivir was of no clinical benefit compared to placebo (120) . lastly, a multicenter analysis involving a double-blind placebo-controlled trial (ntc04280705) was implemented to assess the efficacy and safety of remdesivir in the treatment of hospitalized sars-cov-2 patients. preliminary results from this trial showed that patients receiving remdesivir had a faster time to recovery and a lower mortality rate when compared to the placebo group, and it is on the basis of this that the fda issued authorization of remdesivir for emergency use on may 1, 2020. interferons (ifns) are important cytokines with critical antiviral activity. infected innate immune cells produce ifn which enable the jak-stat pathway, leading to recruitment of more nk cells and macrophages. as with sars-cov and mers-cov, nsp 1 of sars-cov-2 exhibits anti-ifn activity by targeting proteins of the jak-stat signaling pathway (121) . ifn inhibition has been correlated to disease severity (122) . furthermore, ifn treatment has already proved effective against ss-rna viruses, and is widely used in the treatment of hcv and hbv. zhang et al. combined ifn-α and ifn-γ in a study conducted in vivo and in vitro. this combination demonstrated synergy, and smaller dosages were required than in ifn monotherapy. this suggests that reduction in unwanted side effects may be possible with the use of dual therapy (123) . it was shown that sars-cov-2 evades detection from cytosolic rig-i and mda5, preventing the activation of ifn-i and the subsequent stimulation of innate cells. it is at this point that the importance of toll-like receptors (tlrs) should be emphasized. negishi et al. demonstrated that viruses that evade cytosolic safeguards can be inhibited by tlr-3 action. tlr-3 functions to activate ifn-ii, which in turn elicits an antiviral response (124) . the use of tlr-3 agonists in mice by shahabi nezhad et al. showed promising results; they were able to increase levels of ifn-α/β/γ, which compensates for the inhibitory actions of sars-cov on signaling pathways (105) but further research is needed to determine if this may result in toxic overstimulation of the immune system (125) . in an open-label randomized phase ii trial by hung et al., a triple combination of ifn-β-1b, lopinavir/ritonavir and ribavirin was administered to covid-19 patients with mild to moderate disease. this combination proved to be safe and superior to lopinavir/ritonavir + ribavirin, with patients showing alleviation of symptoms, shortened duration of viral shedding, and shortened hospital stay. these promising results mean that future trials utilizing ifn-β-1b are warranted (126) . anti-c5a-antibody, eculizumab, and bevacizumab the discovery of the sars-cov-2 specificity to masp-2 of the mbl pathway opened the potential for prophylactic treatment against cytokine-mediated lung damage. it was previously shown in the literature that acute lung injury due to viral infection could be prevented by the use of anti-c5a-antibody treatment (127) , and on the basis of this, gao et al. used a recombinant c5a-antibody in an open-label trial involving severely ill patients the outcome of the first two recipients of the monoclonal antibody was described. both patients showed improved oxygen saturation, increased lymphocyte count, decrease in inflammatory proteins, improvement in liver function, and alleviation of pneumonia. although the use of anti-c5a antibody shows great promise, the trial is still ongoing and the final efficacy is yet to be disclosed (85) . another trial (ntc0428713) is currently assessing the potential of eculizumab to reduce mortality in 19 patients. in a similar mechanism of anti-inflammatory action, eculizumab inhibits c5 cleavage thus preventing the release of c5a. for the treatment of ards, a clinical trial (nct04275414) is comparing the therapeutic potential and side effects of the monoclonal antibody bevacizumab for critical covid-19 patients. by targeting vascular endothelial growth factor (vegf), an angiogenic factor, bevacizumab may prevent the disruption of the vascular barrier that causes edema and lung injury (128) . a preliminary study conducted by wang et al. (129) identified potential antibodies with the capacity to neutralize sars-cov-2. the s protein ectodomains of both sars-cov and sars-cov-2 were expressed in hek-293t cells using plasmid transduction. similarly, hek-293t cells were transfused with plasmids containing sars-cov1/2 in s protein subdomains tagged with either the mice or human fc portion of igg. h2l2 mice antibodies were produced through gradual immunization with the sars-cov s protein ectodomain. the spleen and lymph nodes were then harvested to produce hybridomas. of the 51 samples, only one of the hybridomas (47d11) exhibited cross-reactivity sars-cov and sars-cov-2 s proteins. the chimeric antibody was reformatted and expressed as a fully human igg. it was shown that this novel igg tightly binds the conserved rbd of the sars-cov-2 s protein in infected cells and neutralized the virus in veroe6 cells. this represents the very first human monoclonal antibody that is able to fully neutralize sars-cov-2 (129) . this cross-neutralizing antibody, due to a conserved epitope region on the spike protein, could be the key to preventing future betacoronavirus outbreaks. 47d11 has recently completed phase i clinical trials (nct04411628) to establish dosing in hospitalized patients with sars-cov-2 under longterm follow up. now undergoing phase ii trials (nct04427501), the monoclonal antibody will be tested on ambulatory patients, with results estimated to be available in september. as stated earlier, the ace-2 receptor is crucial for viral attachment and entry. an in vitro experiment conducted by monteil et al. exposed veroe6 cells to varying concentrations of plaque forming units (pfu) from sars-cov-2 in the presence and absence of human recombinant soluble ace-2 (hrsace-2). the infection was inhibited 15 h after introduction of the virus. the experiment was repeated using human capillary organoids and human kidney organoids, and the same inhibitory actions of hrsace-2 were observed. it is important to note the dosedependent nature of this inhibitory action. hrs-ace-2 has already undergone phase i and ii clinical trials for the treatment of ards (130) , and monteil et al.' findings suggest that hrs-ace-2 could be a potential therapeutic agent against sars-cov-2 and phase ii trials (nct04335136) are currently underway in various european countries (131) . in an open label trial by the recovery collaborative group, 2,014 hospitalized patients received either low dose dexamethasone or standard care alone by random assignment. it was observed that the group of patients on mechanical ventilation who received dexamethasone exhibited lower mortality rates compared to those receiving standard care alone (132) . while other studies further support the role of glucocorticoids in the reduction of mortality (133) some reported conflicting results and showed no clinical benefit or even harm to the patient (134) . a systematic review of 15 studies concluded that while critically ill patients are more likely to benefit from glucocorticoid therapy, their use was associated with increased mortality as it resulted in longer hospital stays and increased tendency toward serious nosocomial infections (135) . clinical trials are currently ongoing to assess the risk vs. benefit for the use of glucocorticoids in the treatment of covid-19. cytokine storm remains the main cause of acute lung injury and organ damage in the course of sars-cov-2 infection. it is chiefly caused by gm-csf and il-6. il-6 receptors exist in two forms: the soluble form (sil-6) and the membrane bound (mil-6). to initiate pro-inflammatory action, il-6 binds to sil-6 and the complex binds to gp130 to complete signal transduction. due to this, the therapeutic use of the il-6 antagonist tocilizumab, which binds to both sil-6 and mil-6, was suggested. xu et al. qualified 21 critical patients in a trial with tocilizumab. clinical symptoms, radiological findings and laboratory values all improved after treatment, and 19 patients were successfully discharged (136) . in an open label study by morena et al., however, 51 critically ill sars-cov-2 patients were treated with tocilizumab. all patients presented with decreased oxygen saturation and an increase in plasma il-6. while positive results were observed with tocilizumab rapidly decreasing inflammatory markers, no clinical benefit was reported as patients quickly developed life-threatening bacterial and fungal infections (137) . jordan et al. conducted another study administering 27 critical patients with a single dose of tocilizumab. this resulted in significant decrease in inflammatory proteins and reduction in fever. twenty two patients receiving mechanical ventilation were able to be extubated and vasopressors discontinued. two patients died, and the authors report that these patients were already in severe septic shock due to sars-cov-2-related pneumonia, and were unresponsive to vasopressors. four patients did not respond to the medication and had poorer outcomes. while the results are promising and in line with the findings by xu et al., limitations of this work include the lower-thanrecommended dose of tocilizumab, chosen by the authors due to drug shortage, and the absence of a control group (138) . a placebo-controlled randomized clinical trial is still necessary before recommendations can be made. historically, il-1 has been shown to act as a pro-inflammatory cytokine with actions on several immune cells (139, 140 (144) . convalescent plasma has been employed and has shown promise in the treatment of sars, mers and influenza (145) (146) (147) . in a study by duan et al., 10 severely ill patients were transfused with 200 ml of convalescent plasma harvested from donors who have recovered from sars-cov-2 and had antibody titers above 1:640. within 1 to 3 days, symptoms had disappeared in all 10 patients, and radiological investigation showed improvement after seven days. viral load was undetectable by rt-pcr in 7 patients and no adverse side effects were detected (148) . shen et al. treated five covid-19 patients with convalescent plasma. all five patients were critically ill and intubated, presenting with ards and pneumonia and experiencing high viral load despite treatment with antivirals. the outcome was largely positive: ards was resolved in four patients within 12 days, and three patients were able to be weaned off mechanical ventilation within 2 weeks. three patients were discharged while two remained stable in recovery (149) . joyner et al. recently made an assessment of the safety of convalescent plasma in 20,000 covid-19 patients. the incidence of serious adverse events including transfusion reactions, cardiac events and thrombotic events was low. mortality rates were shown to be higher in critical patients receiving mechanical ventilation or those in septic shock. the conclusion drawn from the study provided evidence that the administration of convalescent plasma in a hospital setting was safe and that early administration is more likely to reduce fatality rates (150) . it has been previously shown that sars-cov induces viroporin production in the host cell membrane to facilitate virion release. viroporin 3a has been associated with nlrp3 (nod-like r family, pyrin domain 3) inflammasome activation (151) , which induces the production of pro-inflammatory cytokines such as il-1-β and il-18 via the gasdermin d (gsmd) pathway (151, 152) . like sars-cov, sars-cov-2 induces the production of large amounts of il-1-β (33) . on the basis of this, a possible treatment could be il-38, a member of the il-1 family. when placed with activated peripheral mononuclear cells, il-38 demonstrates suppressive and anti-inflammatory effects through the inhibition of il-1, il-6, and tnf production (153) . another member of the il-1 family is il-37. both in vitro and in vivo studies showed that il-37 acts as a negative regulator of inflammation, aiding in the protective actions exhibited by tgfβ on dendritic cells and thus attenuating the t cell response (154) . both il-38 and il-37 could potentially be valuable in the treatment of covid-19 (155) . baricitinib is a janus kinase (jak) 1/2 inhibitor used to treat rheumatoid arthritis. jak 1/2 inhibition prevents activation of pro-inflammatory cytokines such as gm-csf, il-2, il-6, il-12, and il-23. adaptor-related protein complex 2 (ap2)-associated protein kinase i (aak1) induces receptor-mediated endocytosis. baricitinib has been shown to have very high affinity for aak1, thus could feasibly inhibit both cytokine storm and viral entry to the cell (156) . in a small cohort study, titanji et al. administered baricitinib and hydroxychloroquine to 15 patients with moderate to severe covid-19. twelve of the 15 patients recovered, and vitals and inflammatory markers were seen to improve after baricitinib was initiated. two patients however developed serious bacterial or fungal infections due to prolonged icu stay (157) . a phase iii double blind placebo-controlled randomized trial involving baricitinib (nct04421027) is currently ongoing to assess the efficacy and safety of the drug as a potential immune inhibitor preventing cytokine storm and viral entry. sars-cov-2 is the most structurally and genetically similar to sars-cov, thus findings from monoclonal studies on sars-cov have been utilized to target the shared aspects between the strains. the monoclonal antibodies shown in table 6 are engineered to specifically bind to different domains on s1 or s2. though none have progressed to clinical trials, they mechanism of action 80r binds s1 and prevents interaction with ace-2 (158) synergistic action; bind s1 and prevent interaction with ace-2; prevent immune escape (159, 160) f26g18 f26g19 m396 bind linear epitope of s1; bind conformational epitope of s1; inhibit interaction of s1 with ace-2 (161) binds hdr domain of s2 and prevents interaction of receptor in vitro (162) 201 binds s1 and prevents interaction with ace-2 (163) monoclonal antibodies studied for the treatment of sars-cov-2 function igg that binds to a conserved region in the rbd of the s protein and fully neutralizes sars-cov and sars-cov-2 (129) anakinra il-1-inhibitor used to reduce effects of the cytokine storm in sars-cov-2 (143) show promise in vitro and in vivo against sars-cov and sars-cov-2 (164) . the immunomodulatory potential of mesenchymal stem cells (msc) was first identified by luk thromboembolism as a result of endothelial injury in the course of infection is a serious and fatal complication in critically ill patients (168) (169) (170) . tang et al. enrolled 449 covid-19 patients with severe disease for a study in which 99 patients who received low molecular weight heparin for a week or more. these patients were associated with better prognosis than the patients who were not administered anticoagulant therapy (171) . helms et al. followed 150 icu patients in a multicenter prospective cohort study. thromboembolic events were observed in 16.7% of patients despite prophylactic and therapeutic use of anticoagulants. the authors noted that pulmonary embolism was diagnosed a few days after icu admission and was more common in ards patients. they conclude that although other papers have reported the effectiveness of heparin, a higher anticoagulant target should be implemented with other anticoagulants such as anti-xa (172) . the pathogenesis of thromboembolism in the course of covid-19 is still unclear. one of the proposed pathways implicates ards: the profound hypoxemia and vasoconstriction may lead to vascular occlusion (173) . another proposed mechanism is that unlike in a healthy lung, a diseased lung is unable to maintain the balance between fibrinolysis and coagulation, thus resulting in decreased action of tissue plasminogen activators (tpa) (174) . more randomized clinical trials are currently ongoing to assess the efficacy of various anticoagulants. in a 2014 pilot study by fowler et al., critically ill septic patients were given either a vitamin c infusion or a placebo. a dramatic decline in inflammatory markers was observed in the vitamin c group, and their sequential organ failure assessment (sofa) score was decreased compared to the placebo group. there were also no adverse events observed with the vitamin c group (175) . in 2019, fowler et al. published on the use of vitamin c vs. placebo in septic patients with ards, and they concluded that the vitamin c infusion did not improve either inflammatory markers or organ dysfunction score (176) . several clinical trials are ongoing verifying the benefits of vitamin c in the treatment of covid-19. widely used as an antimalarial drug, hydroxychloroquine has been shown to possess broad antiviral action, including effectiveness against hiv-1 and influenza type a and b. its antiviral activity has been tested on sars-cov-2. in preventing the glycosylation of the ace-2 receptor, hydroxychloroquine effectively prevents viral entry (177) . furthermore, it has been shown to alkalinize the organelle, which serves to prevent the formation of mature endosomes required to shield the virus from immune cells, and for replication (178) . apart from its broad antiviral activity, hydroxychloroquine has proven to be adequately anti-inflammatory, interfering with nlrp3 activation and impairing the production of pro-inflammatory cytokines, specifically il-1-β (179) . in an open-label nonrandomized clinical trial by gautret et al., sars-cov-2-positive patients were divided into three groups: group 1 receiving hydroxychloroquine, group 2 receiving hydroxychloroquine + azithromycin (antibiotics were given at the discretion of the physician to prevent opportunistic infections), and group 3 not receiving hydroxychloroquine. the primary outcome in group 1 and 2 was viral clearance within 3 to 6 days, but greater results were achieved when hydroxychloroquine was combined with azithromycin (180) . azithromycin, a bacteriostatic agent, has shown antiviral activity against zika virus, ebola virus (181) and rsv. its antiviral mechanism of action, with regards to rsv, is hypothesized to be in decreasing the expression of fusion proteins in airway epithelium (182) . in a study conducted by million et al., patients suffering from early sars-cov-2 infection were treated with hydroxychloroquine and azithromycin; this combination proved to efficaciously reduce the viral load and was deemed safe with minimal risk of complications (183) . chen et al. also showed that hydrochloroquine was safe and efficacious in the treatment of mild disease (184) . the efficacy of hydrochloroquine with or without azithromycin has since been disputed. in a recent open-label randomized control trial, the efficacy and safety of hydroxychloroquine + standard care vs. standard care alone was assessed by wei et al. in patients with mild to moderate covid-19. the group receiving hydroxychloroquine was shown to suffer more adverse effects, and there was no observed clinical benefit compared to patients given standard care alone (185) . molina et al. also concluded that while hydroxychloroquine proved to be beneficial in past studies, the combination of hydroxychloroquine and azithromycin for severe sars-cov-2 infections resulted in inadequate viral clearance, and the clinical benefits previously seen in patients with mild to moderate illness were not observed in hospitalized patients with severe disease (186) . this broad spectrum antiparasitic agent has been shown to possess antiviral activity. in vitro studies indicate that ivermectin prevents non-structural proteins of both dengue virus (187) and hiv-1 (188) from interacting with the importin α/β1 on the host cell, which prevents viral integration. it has been shown that the sars-cov nucleocapsid (n) protein integrates with the nucleus and nucleolus, and prevents cytokinesis of the host cell via importin-α/β1. the exact role of the n protein in the cell cycle is not known, but it is postulated that this structural protein enters the nucleolus to promote viral replication and encourage suitable conditions for viral packaging (189) . an in vitro experiment by caly et al. showed that a single dose of ivermectin administered to inoculated vero cells effectively controlled viral replication within 1 to 2 days, which may prove beneficial for newly infected patients. it was hypothesized that like in other viruses, ivermectin's antiviral action against sars-cov-2 is derived from the inhibition of importin-α/β1. (190) . alam et al. treated 100 mild to moderately ill patients with a combination of ivermectin and doxycycline. symptom improvement was observed after 72 h following treatment, and no side effects were noted. this study however makes no conclusion on the efficacy and safety of this therapy as it is not a place-controlled randomized clinical trial (191) . similarly, caly et al.' results, while promising, cannot be applied until safety margins have been established in further clinical trials. this glycopeptide antibiotic used in the treatment of grampositive bacterial infections has been shown to possess antiviral activity against ebola, mers, sars and hiv-1. it is believed that teicoplanin interferes with endosome formation through alkalization. cleavage of the s protein by cathepsin in the late endosome is inhibited, which in turn prevents the release of viral rna (192) . baron et al. showed that the cathepsin l sequence is conserved in sars-cov, suggesting that teicoplanin could be a key treatment in patients who are diagnosed early with sars-cov-2 (193) . there have been tremendous advancements in the field of immunotherapy since the development of chimeric antigenreceptor t cells (car-t). these cells differentiate from our basic t-cells by overcoming t-cell control safeguards and therefore express fewer exhaustion markers pd-1, tim3, and lag3. furthermore, they are capable of differentiating into terminal effector t-cells responsible for pathogen and tumor destruction. car-t cell treatment has already shown great advances in oncology, inducing long-term remission in patients suffering from acute lymphoblastic leukemia (194) . it is currently being investigated for the treatment of viral infections such as hiv-1, hbv, and hcv. development of car-t cells specific to hiv-1 infection has now entered clinical trials (195) . crispr-cas (clustered regularly interspaced short palindromic repeats), a mechanism evolved to protect against bacteriophages has shown great promise as a genetic editing tool. in vitro studies have used lentiviral vectors consisting of cas9 (crispr-associated proteins) and sgrna specific ccr5 (single guided rna responsible for the detection of the genome of interest) against cd4+ t cells susceptible to hiv-1 infection. the viral vector was able to disrupt the ccr5 gene in the cd4+ cells thus inhibiting hiv-1 entry (196) . in a novel study, the use of the crispr-cas system against human lung epithelium infected with sars-cov-2 yielded positive results: crispr-cas was able to be transfected in the lung epithelium to degrade the virus (197) . this method was shown to possess protective actions against the known pathogenic coronaviruses. gene editing and car-t cell therapy open a new frontier in future treatment modalities. the rapid spread of sars-cov-2 poses a threat of global proportions. time is of the essence, and the discovery of accurate diagnostic methods and treatment protocols are imperative in preventing further spread of this pathogen. similarities between sars-cov-2 and its predecessor have formed the framework upon which diagnostic and treatment approaches to the novel virus are based. rt-pcr primers, based on sars-cov and mers-cov, have proven to be highly sensitive and specific, though not without their flaws. time consuming and prone to producing false negative results, this has led to the employment of more efficient testing methods such as serologic tests and enzyme-based assays, capable of quantifying infected patients on a large scale. to date, there are still no therapies specifically targeting sars-cov-2. while many fda-approved antivirals on the market have had success in patients presenting with differing degrees of illness severity, the development of specific antivirals remains an area of active research. on the other hand, immunotherapy has been shown to be effective, particularly with the discovery of hrs-ace-2 and other promising immune modulators, the development of the 47d11 monoclonal antibody capable of neutralizing sars-cov-2, as well as msc therapy. the non-traditional use of anti-malarial agents had previously showed great promise but have now proven to lack adequate antiviral action and have been associated with severe complications. until guidelines are updated following the multitude of ongoing clinical trials, standard care remains the main treatment modality. rigorous research with regards to this pandemic not only adds to the scientific literature, but is critical for public health policy surrounding future outbreaks. only with collaborative research efforts and dissemination of knowledge may we interrupt exponential transmission of disease and maintain human losses at the minimum. kk and jc: conceptualization. kk, np, and dh: investigation and resources. kk, np, jc, and dh: writing-original draft preparation. kk, jc, and mk: writing-review and editing. jc and mk: visualization. mk and am: supervision and project administration. all 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endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) teicoplanin: an alternative drug for the treatment of covid-19? t cells expressing cd19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial car t cells beyond cancer: hope for immunomodulatory therapy of infectious diseases ccr5 gene disruption via lentiviral vectors expressing cas9 and single guided rna renders cells resistant to hiv-1 infection development of crispr as a prophylactic strategy to combat novel coronavirus and influenza key: cord-282338-u01qv3uc authors: cherry, james. d. title: the chronology of the 2002–2003 sars mini pandemic date: 2004-11-05 journal: paediatr respir rev doi: 10.1016/j.prrv.2004.07.009 sha: doc_id: 282338 cord_uid: u01qv3uc severe acute respiratory syndrome (sars) was a new human disease in the autumn of 2002. it first occurred in southern china in november 2002 and was transported to hong kong on february 21, 2003 by an infected and ill patient. ten secondary cases spread the infection to two hospitals in hong kong and to singapore, toronto and hanoi. in march 2003 a novel coronavirus (sars-cov) was found to be the causative agent. within 11 weeks from the first sars case in hong kong it had spread to an additional 27 countries or special administrative regions. the mini pandemic peaked during the last week of may 2003 and the last new probable case was on july 13, 2003. there were a total of 8096 probable cases and 774 deaths. sixty-six per cent of the cases occurred in china, 22% in hong kong, 4% in taiwan and 3% in both singapore and canada. twenty-one per cent of all cases occurred in healthcare workers. it is an honour to have been asked to contribute the lead paper in this symposium on paediatric sars. of all the participants in this symposium i have the distinction of having had no first-hand experience with sars. nevertheless, i do have some credentials. in march of 2003 i travelled to and through hong kong, shanghai, taipei, seoul and narita and realised on my trip back to los angeles on march 17 th that i needed a chapter on sars for the 5 th edition of our book 'textbook of pediatric infectious diseases' which was soon to be published. so, with the help of four hong kong colleagues this chapter was written in under 2 weeks and is the most complete reference to date relating to paediatrics. 1 sars was apparently a new human disease which first occurred in southern china in the autumn of 2002 and spread to 29 countries or regions with a total of 8096 cases and 774 deaths. [1] [2] [3] the aetiologic agent of sars (a previously unrecognised coronavirus; sars-cov) was isolated and its genome sequenced in record time. [3] [4] [5] [6] [7] [8] following an incredible global public health effort, the mini pandemic was brought under control within 7 months of its initial occurrence. 9 the overall effort demonstrated unprecedented international cooperation and this was facilitated by electronic transmission of information. in general, worldwide media coverage was unusually accurate and provided pictures to augment scientific data. as of june 1 st , 2004 there were approximately 2000 citations related to sars in the medical literature. however, of these publications only about 0.1% are related to children. during the third week of march 2003 both the who and the cdc published preliminary case definitions for severe acute respiratory syndrome (sars). table 1 contains the who initial definitions of suspect and probably cases and table 2 the cdc original definition of a suspected case. 10, 11 following the rapidly expanding knowledge about sars paediatric respiratory reviews (2004) (table 3) . 12 the cdc case definition was updated on four occasions throughout the epidemic as understanding of the clinical, laboratory, and transmission characteristics of sars associated coronavirus increased. 13 the most recent version from the cdc, which is exceedingly complex, is presented in table 4 . transmission of sars apparently first occurred in fosham city, guangdong province, china. 1, 9 the primary case may have been a 46-year-old man who had a fever for 9 days and respiratory distress. he was hospitalised and treated in an intensive care unit. he recovered but his wife, aunt, and aunt's daughter all became ill. 14 [15] [16] [17] [18] viral particles were seen on electron microscopic examination of swabs and sputum specimens from sars patients. the sars virus grew in vero cell and fetal rhesus kidney cell tissue cultures. cytopathic effect was noted in the vero cell cultures in 5 to 6 days and in the fetal rhesus kidney cell cultures in 2 to 4 days. 16, 17 this virus, which was a novel coronavirus (sars-cov), was rapidly identified using both conventional virologic methods and cutting edge molecular biology. the genome was completely and rapidly sequenced in several laboratories. [4] [5] [6] [7] [8] this was a significant accomplishment when one realises that coronaviruses have the largest genomes (30 000 bases of positivestrand rna) found in any rna virus. sequence data allowed the rapid development of highly specific diagnostic tests and also helped in the epidemiologic study of the mini pandemic. the genome sequence data from human isolates assisted in the search for the origin of the disease when similar viruses were isolated from himalayan palm civets and raccoon dogs in a live animal market in shenzhen, china. 19 the viral genomes recovered in nasal swabs from the palm civets were 99.8% homologous to the human sars-cov isolates. early in the epidemic orf8 reading frame sequences from human isolates were identical to those from the palm civets, which suggested animal to human transmission. as noted above, sars originated in southern china in november 2002. presumably the first case or cases were the result of transmission from palm civets to humans. subsequently, human to human transmission occurred. apparently, human to human transmission did not occur outside of china until february 21, 2003 when the adult physician from guangdong province, whose illness commenced on february 15, stayed at the metropole hotel in hong kong. 15 the chronology of events related to the metropole hotel is presented in fig. 1 . case a (the primary case) stayed on the ninth floor of the hotel on february 21, 2003 and was then admitted to hospital #2 the following day and died on february 23, 2003. secondary cases related to case a, in addition to cases b to k, were four healthcare workers at hospital #2 and two of the patient's family members. and -cough or breathing difficulty and one or more of the following exposures during the 10 days prior to onset of symptoms: -close contact b with a person who is a suspect or probable case of sars -history of travel to an area with recent local transmission of sars -residing in an area with recent local transmission of sars 2. a person with an unexplained acute respiratory illness resulting in death after 1 november, 2002, a but on whom no autopsy has been performed and one or more of the following exposures during to 10 days prior to onset of symptoms: -close contact, b with a person who is a suspect or probable case of sars -history of travel to an area with recent local transmission of sars -residing in an area with recent local transmission of sars a case should be excluded if an alternative diagnosis can fully explain their illness. as sars is currently a diagnosis of exclusion, the status of a reported case may change over time. a patient should always be managed as clinically appropriate, regardless of their case status -a case initially classified as suspect or probable, for whom an alternative diagnosis can fully explain the illness, should be discarded after carefully considering the possibility of co-infection -a suspect case who, after investigation, fulfils the probable case definition should be reclassified as 'probable' -a suspect case with a normal cxr should be treated as deemed appropriate and monitored for 7 days. those cases in which recovery is inadequate should be re-evaluated by cxr -those suspect cases in whom recovery is adequate but whose illness cannot be fully explained by an alternative diagnosis should remain as 'suspect' -a suspect case who dies, on whom no autopsy is conducted, should remain classified as 'suspect'. however, if this case is identified as being part of a chain transmission of sars, the case should be reclassified as 'probable' -if an autopsy is conducted and no pathological evidence of rds is found, the case should be 'discarded' a the surveillance period begins on 1 november 2002 to capture cases of atypical pneumonia in china now recognized as sars. presence of two or more of the following features: fever (might be subjective), chills, rigors, myalgia, headache, diarrhea, sore throat, or rhinorrhea mild-to-moderate respiratory illness temperature of >100.4 8f (> 38 8c) * and one or more clinical findings of lower respiratory illness (e.g. cough, shortness of breath or difficulty breathing) severe respiratory illness meets clinical criteria of mild-to-moderate respiratory illness and one or more of the following findings: --radiographic evidence of pneumonia or --acute respiratory distress syndrome or --autopsy findings consistent with pneumonia or acute respiratory distress syndrome without an identifiable cause possible exposure to sars-associated coronavirus (sars-cov) one or more of the following exposures in the 10 days before onset of symptoms: travel to a foreign or domestic location with documented or suspected recent transmission of sars-cov a or close contact b with a person with mild-to-moderate or severe respiratory illness and history of travel in the 10 days before onset of symptoms to a foreign or domestic location with documented or suspected recent transmission of sars-cov a likely exposure to sars-cov one or more of the following exposures in the 10 days before onset of symptoms: close contact b with a person with confirmed sars-cov disease or close contact b with a person with mild-to-moderate or severe respiratory illness for whom a chain of transmission can be linked to a confirmed case of sars-cov disease in the 10 days before onset of symptoms tests to detect sars-cov are being refined and their performance characteristics assessed c ; therefore, criteria for laboratory diagnosis of sars-cov are changing. the following are general criteria for laboratory confirmation of sars-cov: detection of serum antibody to sars-cov by a test validated by cdc (e.g. enzyme immunoassay) or isolation in cell culture of sars-cov from a clinical specimen or detection of sars-cov rna by a reverse transcription polymerase chain reaction test validated by cdc and with subsequent confirmation in a reference laboratory (e.g. cdc) information about the current criteria for laboratory diagnosis of sars-cov is available at http://www.cdc.gov/ncidod/sars/labdiagnosis.htm a case may be excluded as a sars report under investigation (sars rui), including as a cdc-defined probable sars-cov case, if any of the following apply: an alternative diagnosis can explain the illness fully d or antibody to sars-cov is undetectable in a serum specimen obtained > 28 days after onset of illness e or the case was reported on the basis of contact with a person who was subsequently excluded as a case of sars-cov disease; then the reported case is also excluded, provided other epidemiologic or laboratory criteria are not present case classification sars rui reports in persons from areas where sars is not known to be active sars rui-1: cases compatible with sars in groups likely to be first affected by sars-cov f if sars-cov is introduced from a person without clear epidemiologic links to known cases of sars-cov disease or places with known ongoing transmission of sars-cov 8c) is expected. however, clinical judgment may allow a small proportion of patients without a documented fever to meet this criterion. factors that might be considered include: patients' self-report of fever; use of antipyretics; presence of immunocompromising conditions or therapies; lack of access to healthcare; or inability to obtain a measured temperature. initial case classification based on reported information might change, and reclassification might be required. a types of locations specified will vary (e.g. country, airport, city, building, or floor of building). the last date a location may be a criterion for exposure is 10 days (one incubation period) after removal of that location from cdc travel alert status. the patient's travel should have occurred on or before the last date the travel alert was in place. transit through a foreign airport meets the epidemiologic criteria for possible exposure in a location for which a cdc travel advisory is in effect. information about cdc travel alerts and advisories and assistance in determining appropriate dates are available at http://www.cdc.gov/ncidod/sars/travel.htm. b close contact is defined as having cared for or lived with a person with sars or having a high likelihood of direct contact with respiratory secretions and/or body fluids of a person with sars (during encounters with the patient or through contact with materials contaminated by the patient) either during the period the person was clinically ill or within 10 days of resolution of symptoms. examples of close contact include kissing or embracing, sharing eating or drinking utensils, close (i.e. <3 feet) conversation, physical examination, and any other direct physical contact between persons. close contact does not include activities such as walking by a person or sitting across a waiting room or office for a brief time. c the identification of the aetiologic agent of sars (i.e. sars-cov) led to the rapid development of enzyme immunoassays and immunofluorescence assays for serologic diagnosis and reverse transcription polymerase chain reaction assays for detection of sars-cov rna in clinical samples. these assays can be very sensitive and specific for detecting antibody and rna, respectively, in the later stages of sars-cov disease. however, both are less sensitive for detecting infection early in illness. the majority of patients in the early stages of sars-cov disease have a low titre of virus in respiratory and other secretions and require time to mount an antibody response. sars-cov antibody tests might be positive as early as 8-10 days after onset of illness and often by 14 days after onset of illness, but sometimes not until 28 days after onset of illness. information about the current criteria for laboratory diagnosis of sars-cov is available at http://www.cdc.gov/ncidod/sars/labdiagnosis.htm. d factors that may be considered in assigning alternate diagnoses include the strength of the epidemiologic exposure criteria for sars-cov disease, the specificity of the alternate diagnostic test, and the compatibility of the clinical presentation and course of illness with the alternative diagnosis. e current data indicate that > 95% of patients with sars-cov disease mount an antibody response to sars-cov. however, health officials may choose not to exclude a case on the basis of lack of a serologic response if reasonable concern exists that an antibody response could not be mounted. f consensus guidance is in development between cdc and cste on which groups are most likely to be affected first by sars-cov if it re-emerges. sars-cov disease should be considered at a minimum in the differential diagnoses for persons requiring hospitalisation for pneumonia confirmed radiographically or acute respiratory distress syndrome without identifiable aetiology and who have one of the following risk factors in the 10 days before the onset of illness: (1) travel to mainland china, hong kong, or taiwan, or close contact with an ill person with a history of recent travel to one of these areas, or; (2) employment in an occupation associated with a risk for sars-cov exposure (e.g. healthcare worker with direct patient contact or worker in a laboratory that contains live sars-cov) or; (3) part of a cluster of cases of atypical pneumonia without an alternative diagnosis. guidelines for the identification, evaluation, and management of these patients are available at http://www.cdc.gov/ncidod/sars/absenceofsars.htm. g during the 2003 sars epidemic, cdc case definitions were the following: suspect case; (1) meets the clinical criteria for mild-tomoderate respiratory illness and the epidemiologic criteria for possible exposure to sars-cov but does not meet any of the laboratory criteria and exclusion criteria or; (2) unexplained acute respiratory illness that results in death of a person on whom an autopsy was not performed and that meets the epidemiologic criteria for possible exposure to sars-cov but does not meet any of the laboratory criteria and exclusion criteria; probable case; (3) meets the clinical criteria for severe respiratory illness and the epidemiologic criteria for possible exposure to sars-cov but does not meet any of the laboratory criteria and exclusion criteria. was investigating the outbreak in hanoi became ill and returned to bangkok. patients c, d, and e were the primary cases in singapore and their illnesses led to 70 cases in singapore and three cases in germany. patients f and g were the initial cases in canada and case f was subsequently linked to a cluster of 16 cases in toronto. patients h and j were linked to outbreaks among healthcare workers in hong kong hospitals #1 and #3. patients i, l, and m were suspect cases in the united states. case k was a potential primary case in ireland. on february 28, 3003 the hanoi office of the world health organization was contacted by officials at the vietnam french hospital. 20 they were concerned about the unusual influenza-like illness that patient b had which they suspected could have been due to an avian influenza virus. dr. carlo urbani from the who office answered the call and rapidly determined that the illness which had spread throughout the hospital was unusual. he carried out an extensive on-site investigation and worked with the hospital staff to set up quarantine procedures. his efforts led to the containment of sars in hanoi. unfortunately, he became ill with sars on march 11, 2003 and died in an isolation room in a bangkok hospital on march 29, 2003. he did not live to see the worldwide successes of sars control which resulted to a great extent from his early detection of sars. following fig. 2 were india, japan and mongolia. of these countries, no secondary transmission occurred in india, japan was later removed from the list and in mongolia there were nine cases with eight of these being importations. as of may 12, 2003 new countries or administration regions included bulgaria, china, macao sar, columbia, finland, poland and the republic of korea. of these countries or regions only macao sar and the republic of korea had accepted cases and in both regions the cases were imported. in the june 2nd report one case in the russian federation was noted but this was subsequently removed from the data set. eighty-seven per cent of all probably sars cases occurred in hong kong or china and information relating to these cases in presented elsewhere in this symposium. only five other countries (canada, taiwan, singapore, the united states and vietnam) had more than 20 probable cases. in canada there were 257 cases with the first case occurring on february 23, 2003 and the last case on june 12, 2003. the vast majority of cases in canada (98%) were secondary to five primary importations and 43% of the cases occurred in healthcare workers. in taiwan 6% of the cases were importations and 20% occurred in healthcare workers. in singapore 3% of the cases were importations and 41% of the locally acquired cases were in healthcare workers. in singapore the epidemic was controlled within 13 weeks of the initial importation. all 27 probable cases in the united states were importations. however, only eight of these were laboratory confirmed. in vietnam, 62 of the 63 cases were the result of local transmission. the outbreak was controlled within 7 weeks of the first and only imported case. in summary, the sars mini pandemic of 2002-2003 probably does not deserve to be called a pandemic. however, the death rate in adults was sobering. in reality, significant epidemic disease occurred in only five countries and the special administrative region of hong kong. nevertheless, sars was an eye opener in regard to the number of healthcare workers who became infected. hopefully the clinical lessons relating to isolation practices, personnel protection and the effective use of quarantine will be retained. personally i will be less cavalier in my approach to patients with fever and respiratory illness. severe acute respiratory syndrome (sars) world health organization. summary of probable sars cases with onset of illness from 1 world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) the genome sequence of the sars-associated coronavirus identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome the sars coronavirus: a postgenomic era world health organization. severe acute respiratory syndrome (sars) outbreak news: severe acute respiratory syndrome (sars) trends in tuberculosis morbidity -united states world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) tracing the path of sars: a tale of deadly infection update outbreak of severe acute respiratory syndrome -worldwide coronavirus as a possible cause of severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada world health organization. summary on major findings in relation to coronavirus by members of the who multi-centre collaborative network on sars aetiology and diagnosis isolation and characterization of viruses related to the sars coronavirus from animals in southern china key: cord-256888-tdx12ccj authors: bradley, benjamin t; maioli, heather; johnston, robert; chaudhry, irfan; fink, susan l; xu, haodong; najafian, behzad; deutsch, gail; lacy, j matthew; williams, timothy; yarid, nicole; marshall, desiree a title: histopathology and ultrastructural findings of fatal covid-19 infections in washington state: a case series date: 2020-07-16 journal: lancet doi: 10.1016/s0140-6736(20)31305-2 sha: doc_id: 256888 cord_uid: tdx12ccj background: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the cause of an ongoing pandemic, with increasing deaths worldwide. to date, documentation of the histopathological features in fatal cases of the disease caused by sars-cov-2 (covid-19) has been scarce due to sparse autopsy performance and incomplete organ sampling. we aimed to provide a clinicopathological report of severe covid-19 cases by documenting histopathological changes and evidence of sars-cov-2 tissue tropism. methods: in this case series, patients with a positive antemortem or post-mortem sars-cov-2 result were considered eligible for enrolment. post-mortem examinations were done on 14 people who died with covid-19 at the king county medical examiner's office (seattle, wa, usa) and snohomish county medical examiner's office (everett, wa, usa) in negative-pressure isolation suites during february and march, 2020. clinical and laboratory data were reviewed. tissue examination was done by light microscopy, immunohistochemistry, electron microscopy, and quantitative rt-pcr. findings: the median age of our cohort was 73·5 years (range 42–84; iqr 67·5–77·25). all patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease including diabetes and obesity. the major pulmonary finding was diffuse alveolar damage in the acute or organising phases, with five patients showing focal pulmonary microthrombi. coronavirus-like particles were detected in the respiratory system, kidney, and gastrointestinal tract. lymphocytic myocarditis was observed in one patient with viral rna detected in the tissue. interpretation: the primary pathology observed in our cohort was diffuse alveolar damage, with virus located in the pneumocytes and tracheal epithelium. microthrombi, where observed, were scarce and endotheliitis was not identified. although other non-pulmonary organs showed susceptibility to infection, their contribution to the pathogenesis of sars-cov-2 infection requires further examination. funding: none. in december, 2019, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was identified in wuhan, china from a cluster of severe pneumonia cases. 1 the virus and the disease it causes (covid-19) have now spread globally and are responsible for an ongoing pandemic that has claimed hundreds of thousands of lives. in the months after its emergence, the community of health-care workers and researchers acted quickly to sequence the virus, establish transmission chains, elucidate the receptor, and test therapeutics. 2, 3 these efforts have revealed similarities and differences between sars-cov-2 and the related virus, severe acute respiratory syndrome coronavirus (sars-cov). both viruses have similar clinical presentations, with the highest viral load identified in lower respiratory samples. 4, 5 viral rna has also been detected in blood, stool, and urine samples, suggesting the potential for extrapulmonary spread and multiorgan involvement. 6, 7 the viruses also share a common cellular entry receptor, angiotensin converting enzyme 2 (ace2). 3 despite their similarities, sars-cov was responsible for a restricted disease outbreak with high mortality, whereas sars-cov-2 has caused a greater number of infections with relatively lower mortality. 8 post-mortem studies have shown pulmonary, renal, and small vessel injury, with particles resembling virus observed in the kidney by electron microscopy. [9] [10] [11] [12] one study described two complete autopsies without tissue-based methods for detection of sars-cov-2. 9 our study expands the literature by documenting a series of 14 fatal covid-19 cases that occurred in washington state during february and march, 2020. systematic evaluation of all major organs was done by a combina tion of light microscopy, immunohistochemistry, electron microscopy, and quantitative rt-pcr. our findings serve as a basis for generating further discussion related to the tropism, mechanisms of dissemination, and pathophysiology of severe sars-cov-2 infection. in this case series, patients with a positive antemortem or post-mortem sars-cov-2 result were considered eligible for enrolment. preliminary testing for sars-cov-2 was done at the washington state department of health public health laboratory (shoreline, wa, usa). confirmatory testing was done by the us centers for disease control and prevention (cdc) in atlanta (ga, usa). both locations used the cdc-designed 2019 ncov real-time rt-pcr assay for virus detection. autopsies were done at the king county medical examiner's office (seattle, wa, usa) and snohomish county medical examiner's office (everett, wa, usa) in negative-pressure isolation suites during february and march, 2020. given safety concerns, in-situ dissection was done for patients 2, 3, 4, 5, 6, 7, and 9. patients 1, 8, 10, 11, 12, 13 , and 14 were examined by standard autopsy procedure. three to 23 blocks were submitted per autopsy case. limited autopsies submitted two blocks containing sections from the left and right lung lobes, whereas complete autopsies submitted at least four blocks of lung-containing sections from central and peripheral locations of both lungs. resource limitations led to fresh tissue collection for patients 8, 13 , and 14 only. institutional review board approval was requested and waived for this study (study00009856). autopsy material was fixed in 10% neutral buffered formalin and submitted for standard processing with haematoxylin and eosin staining. select kidney sections were stained with periodic acid schiff and jones methenamine silver. evaluation of haematoxylin and eosin sections was done by consensus agreement by four board-certified forensic pathologists (ny, tw, jml, and dam) with expert guidance provided in cardiothoracic pathology (hx and gd) and renal pathology (bn). immunohistochemical staining was done on formalinfixed, paraffin-embedded 5-µm sections following citrate ph 6·0 antigen retrieval, endogenous biotin, and peroxidase block. immunohistochemistry for sars-cov-2 was done using a monoclonal antibody to the spike protein (1:250; genetex; irvine, ca, usa) on the ventana benchmark ultra ihc (roche diagnostics; basel, switzerland). images were visualised and captured with a digital camera mounted on a nikon eclipse 80i microscope using nis-elements advanced research software version 4.13 (nikon instruments; tokyo, japan). samples from patients 8 and 13 were placed in halfstrength karnovsky fixative. tissue was then post-fixed in 1% osmium tetroxide, processed according to standard transmission electron microscopy procedures, and embedded in polybed 812 (polyscience; warrington pa, usa). suitable sections were identified by toluidine blue staining. thin sections were examined using a tecnai g2 spirit bio-twin transmission electron microscope (fei; hillsboro, or, usa) and digital images and measurements were acquired using amt image capture software (version 602.446). rna was extracted from 0·5 µg of tissue using the direct-zol rna miniprep plus kit (zymo research; irvine, ca, usa). complementary dna was synthesised using the iscript cdna kit (biorad; hercules, ca, usa). samples were tested in triplicate using itaq evidence before this study we searched pubmed and medline for peer-reviewed articles published between database inception and may 1, 2020, that described the histopathological features of severe covid-19 infections, with the search terms "sars-cov-2", "covid-19", "autopsy", "postmortem", and "histology". our search was restricted to studies published in english. of the eight studies identified by our search, one documented complete histopathological findings from all major organs in two autopsies. other series showed evidence of diffuse alveolar damage, endothelial injury, and viral particles within renal cells. tissue quantitative rt-pcr (rt-qpcr) was used as an ancillary technique to identify virus in one study. this study provides crucial information related to the natural history of fatal covid-19 from early in the us outbreak. our analysis used multiple methods, including clinical chart review, histopathological evaluation, electron microscopy, immunohistochemistry, and quantitative rt-qpcr to examine all major organ systems. to our knowledge, no previous studies have used all these techniques simultaneously. our results support previous studies, which suggested that diffuse alveolar damage is the major source of pulmonary injury in covid-19; however, we found no evidence of widespread microvascular injury. additional investigations raised the possibility of extrapulmonary involvement in renal, intestinal, cardiac, and lymphoid tissues. in addition to the results of previous studies, our findings provide a histological explanation for physiological derangements observed by clinicians in patients who died with covid-19. further investigation is required to characterise the extent of extrapulmonary injury caused by severe acute respiratory syndrome coronavirus 2 infection. universal probes supermix (biorad) with the cdc 2019 ncov n1 and n2 primer/probe set and sarbeco e gene primer/probe set (idt; coralville, ia, usa). cycle threshold values less than 40 were considered positive. the limit of detection was estimated to be 700 copies per reaction based on serial dilutions of a positive control plasmid (cov019; exact diagnostics; fort worth, tx, usa). negative control reactions included on each plate repro ducibly showed no amplification. pcr was done on patients 8, 13, and 14. tissues examined included lung, trachea, subcarinal lymph node, heart, spleen, liver, large intestine, kidney, and whole blood. there was no funding source for this study. the median age of our cohort was 73·5 years (range 42 to 84; iqr 67·5-77·25). seven members of the cohort were part of a single cluster from a long-term care facility. 13 all patients had clinically significant comorbidities, the most common being hypertension, chronic kidney disease, obstructive sleep apnoea, and metabolic disease, including diabetes and obesity (table 1) . the most frequent presenting symptoms were respiratory distress, fever, and cough. less commonly encountered initial symptoms included altered mental status and gastrointestinal symptoms (nausea, vomiting, or diarrhoea). during hospital admission, six patients had acute kidney injury shown by elevated creatinine and three patients had elevated troponins. additional respiratory pathogens were isolated from two patientspatient 8 had influenza a and meticillin-sensitive staphylococcus aureus and patient 7 had sputum cultures positive for pseudomonas aeruginosa. excluding those who declined resuscitative measures (six patients), all patients were intubated. patient 12 was electively extubated less than a day before death. the median time from symptom onset to intubation was 3·5 days (iqr 2-5; eight patients), with most intubations occurring at the time of admission. time to death after symptom onset varied from 1 day to 23 days, with a median time of 7 days (iqr 2-13). for patients 8, 13, and 14, fresh tissue was collected at the time of autopsy for molecular and ultrastructural examination. patient 8 was a 76-year-old woman who presented with cough and malaise for 3 days after returning from international travel. on admission, the patient was hypoxic with elevated troponins and leukocytosis. her x-ray showed extensive bilateral opacities and testing was positive for sars-cov-2, influenza a, and meticillin-sensitive s aureus. the patient had increasing oxygen demands and died 7 days after admission. patient 13 was a 71-year-old man with a complex cardiovascular history, end-stage renal disease, and chronic pulmonary fibrosis who presented with shortness of breath, delirium, and new onset atrioventricular node block. given his respiratory symptoms and lymphopenia, sars-cov-2 testing was done and returned a positive result. 4 days after admission, the patient developed worsening hypoxia followed by sudden cardiac arrest and died. patient 14 was a 73-year-old woman who presented after 2 days of progressive shortness of breath while travelling abroad. on admission, she had clinically significant respiratory distress with hypoxia and signs of shock with elevated lactate, leukocytosis, and initial normal troponin. her chest x-ray showed diffuse lung disease and sars-cov-2 testing was positive. the patient's clinical course was complicated by multifactorial encephalopathy, new atrial fibrillation, and presumed ventilator-associated pneumonia and she died 21 days after admission. all seven decedents examined by gross standard autopsy had heavy, oedematous lungs, with an average weight of 975 g for the right lung and 700 g for the left lung. intraparenchymal haemorrhage was observed in patient 6 and pulmonary consolidation was present in patient 14. the volume of pleural fluid was highly variable and ranged from 0 ml to 450 ml per pleural space. patients 10 and 12 had evidence of central pulmonary emboli (figure 1). splenomegaly was observed in patients 8 and 13 (350 g and 505 g, respectively), whereas splenic atrophy was observed in patient 13 (63 g). scattered punctate subarachnoid haemorrhages were observed in the brain of patient 8. additional gross findings showed changes including varying degrees of cardiac and atherosclerotic disease, hypertensive renal surface changes, and hepatic congestion in most patients. organ weights and gross obser vations are available in the appendix (p 1). histopathological examination of the pulmonary system showed a spectrum of diffuse alveolar damage in 12 (86%) of 14 patients, which was evidenced by the presence of intra-alveolar fibrin, hyaline membranes, or loosely organising connective tissue in the alveolar septal walls. 11 (92%) of 12 patients with diffuse alveolar damage showed acute or acute and organising diffuse alveolar damage ( figure 2a, b) . for patient 2, only organising diffuse alveolar damage was observed. airways and alveolar spaces contained large, reactive multinucleated cells that stained positive for the epithelial marker ttf-1 and negative for the macrophage marker cd68 (figure 2c, d). microscopic haemorrhage was identified cardiac findings were mostly non-specific and associated with pre-existing comorbidities. the most common changes observed were fibrosis in 14 (100%) patients and myocyte hypertrophy in 13 (93%) patients. in patient 8, myocarditis was present with aggregates of lymphocytes surrounding necrotic myocytes ( figure 3c, d) . sars-cov-2 s protein immunohistochemistry was negative in patient 8. amyloid was identified in small vessels in patient 7 and within the myocardium in patient 13. patients 5, 9, and 12 had splenic white pulp depletion, which was confirmed by cd45 staining in patient 9 (figure 3e). patient 8 had a small sub-centimetre splenic infarction. subcarinal lymph nodes showed normal follicular architecture with haemophagocytosis observed in patients 8, 11, and 14. sars-cov-2 immunohistochemical staining was negative in the subcarinal lymph node from patient 12 and spleen from patient 10. lymphoid tissue from additional patients was not available for testing. the kidneys showed mild to severe arterione phrosclerosis and diabetic nephropathy. although restricted by autolysis, features suggestive of acute tubular injury, including extensive tubular epithelial vacuolisation, were identified in 11 patients. in patient 14, chronic inflammation of the renal parenchyma and focal segmental glomerulosclerosis were present. sars-cov-2 immunohistochemical testing in patients 8 and 10 showed patchy, granular cytoplasmic staining of the renal tubular epithelial cells (figure 3f). virus was not visualised in the endothelial cells or glomeruli by immunohistochemistry. pathological findings in the liver showed predominantly chronic changes associated with pre-existing comorbidities, with variable degrees of acute congestion. centrilobular necrosis consistent with hypoperfusion injury was identified in four patients (patients 3, 8, 12, and 14). liver inflammation was not prominent, although some patients had mild periportal lymphocytic inflammation. weak sars-cov-2 immunohistochemical staining in the liver was indistinguishable from background. sam ples of the thyroid gland, pituitary gland, adrenal glands, and pancreas were largely unremarkable. post-mortem autolysis prevented thorough investigation of the gastro intestinal system and assessment by sars-cov-2 immunohistochemical staining. brain examination was done in five patients (patients 1, 8, 10, 11, and 12). patient 8 had acute pathology with scattered punctate subarachnoid haemorrhages and rare microhaemorrhage in the brainstem. neuropathological examinations were otherwise unremarkable. focal pulmonary micro thrombi were identified in five patients (patients 3, 8, 9, 13, 14; figure 4) . patient 3 and patient 9 also had microthrombi in the trachea, although in patient 3 this was qualified by chronic tracheostomy. an organising thrombus was identified within a small renal vein in patient 13. microscopic involvement by grossly observed pulmonary emboli was seen in patient 10 and patient 12. pathological findings for individual patients are provided in table 2. by electron microscopy, aggregates of uniform, round enveloped particles ranging in size from around 70 nm to 100 nm with peripheral spike-like projections consistent with the morphology described for sars-cov-2 were observed in the lung, trachea, kidney, and large intestine of patient 8 and patient 13. 14,15 when confined to vesicles or in the extracellular space, structures with these characteristics were designated coronavirus-like particles. for simplicity, we refer to these coronavirus-like particles as viral particles. viral particles were present both inside tracheal epithelial cells and the extracellular space adjacent to the cell membrane or mixed with the luminal mucus ( figure 5a, b) . in the lung, extensive sloughing of pneumocytes into alveolar spaces was observed. some of these pneumocytes contained abundant autophagosomes, and occasionally viral particles were observed within the vesicles. viral particles were seen both in type 2 and type 1 pneumo cytes ( figure 5c, d) . aggregates of viral particles were found in enterocytes; however, the membranes of the surrounding vesicles were often disrupted and the tissue affected by post-mortem artefact (figure 5e, f). in the kidney, viral particles were observed in tubular epithelial cells, and more rarely in endothelial cells ( figure 5g, h) . some viral particles were associated with double membranes, and resembled double membrane vesicles (figure 5g). 16 rare ill-defined round particles were also observed in podocytes. definitive viral particles were not observed in the other examined organs, including the heart, spleen, and liver. sars-cov-2 rna was detected in the lung, trachea, subcarinal lymph node, kidney, large intestine, and spleen of all three tested patients (patients 8, 13, and 14) . viral rna was also detected in the liver, heart, and blood for patient 8 and patient 13. in all three patients, the lungs and trachea had the lowest cycle threshold values. the spectrum of pathological findings in people who die from covid-19 is only beginning to emerge. [9] [10] [11] [12] we present a case series of autopsy findings in 14 patients who died after sars-cov-2 infection. our results show the central role of lung damage and provide evidence that suggests extrapulmonary involvement of sars-cov-2 during severe infection. the major histopathological observation in our series of patients who died with covid-19 was diffuse alveolar damage-type lung injury in the acute or organising phases (12 [86%] of 14 patients). lung tissue from most decedents showed pulmonary oedema, prominent reactive type 2 pneumocytes, intra-alveolar fibrin, and hyaline membranes. these findings are similar to those described during the 2002-03 sars-cov outbreak and more recent covid-19 studies. 9, 11, [17] [18] [19] in contrast to sars-cov, in which organising diffuse alveolar damage was predominantly observed in those with longer hospitalisations (>10 to 14 days), we found evidence of organising diffuse alveolar damage in a patient with covid-19 who died 2 days after symptom onset (patient 2) and observed acute and organising diffuse alveolar damage in patients 8 and 11, who died within a week of symptom onset. 20 compared with the study by franks and colleagues, 20 our patient cohort also had a shorter interval from symptom onset to death (median 7 days vs 12·3 days). we hypothesise that there was a subclinical period during which lung injury was occurring in covid-19 patients with organising diffuse alveolar disease who died in the week after symptom onset. the hypothesis that lung injury might occur in covid-19 patients before symptom onset is supported by evidence of abnormal pulmonary ct findings in asymptomatic patients. 21 these findings could have implications for screening patients at the time of admission to identify and aggressively manage those with pre-existing diffuse alveolar disease-type injury. additionally, we noted mostly focal sars-cov-2 immunohistochemical staining in the lungs of tested patients. in areas with less severe diffuse alveolar damage the virus was more readily visualised in pneumocytes. the absence of strong diffuse viral staining might indi cate that most cytotoxic damage caused by sars-cov-2 occurs early on in infection, with diffuse alveolar damage seen later as part of an exuberant host response. whether covid-19 patients are at increased risk for endothelial injury causing pulmonary microthrombi has become central to the discussion of patient management. 22 this theory is based on the observed mismatch between lung compliance and oxygen saturation in ventilated patients. 23 these results have led to the proposal that covid-19 acute respiratory distress syndrome (ards) might represent a novel type of lung injury. an early pathology report documenting three patients with covid-19 found active endotheliitis and endothelial cells containing coronavirus-like particles, supporting the claims of microvascular damage during infection. 15 however, the observation of virally infected endothelial cells has been called into question. 24 although our findings document coronavirus-like particles in the endothelial cells of the kidney, we did not observe endothelial cell infection in other organs surveyed by electron microscopy or immunohistochemistry. additionally, no histological evidence of endotheliitis was observed in our cohort. given the scarcity of these findings, we propose that the lung injury observed in our cohort presented the typical ards lung phenotype and not a novel type of injury. infection of endothelial cells by sars-cov-2 is hypothesised to cause dysregulation of the clotting system, which particularly affects small vessels and leads to pulmonary microthrombi and altered ventilatory patterns in intubated patients. 22, 23 around a third of our cohort (five patients) had infrequent microthrombi. as no formal grading scale for the severity of microthrombi in tissue exists, we would classify the presence of microthrombi in our cohort as less than one per low power (10×) field and within the scope of what is seen in other causes of diffuse alveolar damage. most microthrombi were seen when a full autopsy was done, and we suspect rare microthrombi might also be present in our cohort of limited autopsies. however, this factor is unlikely to have changed our overall findings or final cause of death. macroscopic pulmonary arterial thrombi were identified in two patients (patient 10 and patient 12). patient 10 showed evidence of organisation with adherence to the pulmonary artery wall, indicating an event that probably predated covid-19 infection. an organising thrombus was also found in a small renal vein on microscopic evaluation of patient 13 and was considered probably unrelated to covid infection. cardiac injury in covid-19 is common, although our results do not provide direct evidence of myocardial injury by sars-cov-2. in a study that documented the clinical course of patients in the intensive care unit in kirkland, wa, 25 33% of patients had cardiomyopathy of unclear cause. previous post-mortem examinations have detected viral rna in cardiac tissue from a single patient, although histopathological evidence of myocarditis was not present. 10 in our cohort, patients 8, 11, and 13 had elevated troponin; however, only patient 8 had histologically apparent lymphocytic myocarditis. myocardial tissue from this patient was positive for viral rna by pcr, but immunohistochemistry and electron microscopy results were negative. as the patient was also viraemic, the low rna level detected in the cardiac tissue might represent contamination by circulating virus rather than direct infection. patient 8 also tested positive for influenza a, a known cause of viral myocarditis, before death. 26 contamination by viral rna could also explain the results in the liver and spleen, where no definitive virus was identified by electron microscopy or immunohistochemistry. tubular epithelial cells, endothelial cells, and podocytes express ace2, making kidneys a candidate target for sars-cov-2 infection. 27 direct infection of kidney cells by the virus has been proposed as a mechanism for acute kidney injury observed during sars-cov-2 infection. 28 two studies reported sars-cov-2 in tubular epithelial cells and podocytes. 14, 15 these studies depended on ultrastructural findings, which carry a risk of confusion with cellular structures if used without immunolabelling. therefore, we labelled the observed structures as coronavirus-like particles, although positive pcr (three of three patients) and immunohistochemistry (two of four patients) data support our assertion that infection of renal cells occurs by covid-19. multiple orthogonal approaches, including pcr, immunohistochemistry, and electron microscopy were used to help identify tissues that harboured viral particles. the pattern of virus distribution seen in our cohort raises questions about the mechanism of viral dissemination during severe infections, specifically whether sars-cov-2 can be transported by lymphocytes. current data support this hypothesis, with viral rna detected in blood samples and in vitro data showing pseudotyped sars-cov-2 capable of infecting lymphocytes. 6, 29 in our study, two of three whole blood samples and three of three lymph nodes tested positive by pcr, in addition to sars-cov-2 immunohistochemistry highlighting lymphocytes in the tracheal submucosa of patient 8. if sars-cov-2 is capable of productive infection in lymphocytes, this could provide a mechanistic explanation for the poor survival and cytokine derangements of severe covid-19 cases with lymphopenia. 30 the extent to which our findings of extrapulmonary involvement of sars-cov-2 are generalisable to nonsevere covid-19 infections is uncertain. as sars-cov-2 has been detected in urine and stool of non-severe covid-19 cases, there is evidence suggesting productive infection of non-pulmonary sites. 7,31 these questions raise concerns for susceptible groups, including those with chronic renal injury or inflammatory bowel disease. whether these patients are at increased risk for more serious complications during sars-cov-2 infection requires close monitoring. another patient group that requires close monitoring are those undergoing organ transplantation. although extrapulmonary infection might be less common in mild or subclinical disease, it is unclear whether active extrapulmonary infection can exist in a patient without concurrent respiratory infection. our study has several limitations. electron microscopy was limited to patients 8 and 13, pcr to patients 8, 13, and 14, and immunohistochemical testing to patients 8, 10, 11, and 12. future studies will reveal whether the relatively consistent findings in this small number of patients are borne out in larger samples. as safety limitations in place during february and early march, 2020, precluded complete autopsies of seven patients, our ability to detect rare findings was restricted for part of the cohort. in some situations, there was an extended postmortem interval before examination. as post-mortem autolysis reduces the sensitivity of electron microscopy and pcr, patient samples collected several days after death might generate false negative results. however, we could detect viral particles and viral rna from specimens up to 140 h post mortem (patient 13). extended postmortem intervals should not be an absolute criterion for exclusion in future studies. in conclusion, our findings show the central role of diffuse alveolar damage-type lung injury in patients with severe covid-19. microthrombotic disease and endothelial injury were not as pronounced as reported in previous studies. we found broad tropism for sars-cov-2 with coronavirus-like particles identified in the pulmonary system, kidneys, and gastrointestinal tract. our results also raise the question as to whether sars-cov-2 can cause direct myocardial injury and whether direct infection of lymphocytes promotes viral dissemination and immune dysregulation. these findings provide histological context for clinical observations and help characterise the pathophysiology of sars-cov-2, hopefully leading to novel treatment strategies. btb conceived and designed the study. hm, rj, and ic contributed to clinical data collection. hm, bn, and btb contributed to figure design. ny, tw, jml, dam, rj, ic, and btb did the post-mortem examinations. ny, tw, jml, dam, rj, ic, hm, hx, bn, gd, and btb contributed to histopathological evaluation of tissue. gd contributed to immunohistochemical studies. bn and btb contributed to electron microscopy images. btb and slf contributed to molecular testing. btb and hm wrote the manuscript. all authors contributed to data analysis, data interpretation, and editing the manuscript. we declare no competing interests. a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention quantitative detection and viral load analysis of sars-cov-2 in infected patients detection of sars-cov-2 in different types of clinical specimens persistence and clearance of viral rna in 2019 novel coronavirus disease rehabilitation patients a major outbreak of severe acute respiratory syndrome in hong kong covid-19 autopsies pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies pathological findings of covid-19 associated with acute respiratory distress syndrome complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases epidemiology of covid-19 in a long-term care facility in king county, washington renal histopathological analysis of 26 postmortem findings of patients with covid-19 in china endothelial cell infection and endotheliitis in covid-19 autophagy during viral infection-a double-edged sword pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer multiple organ infection and the pathogenesis of sars lung pathology of fatal severe acute respiratory syndrome lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study management of covid-19 respiratory distress covid-19 pneumonia: different respiratory treatments for different phenotypes? electron microscopy of sars-cov-2: a challenging task characteristics and outcomes of 21 critically ill patients with covid-19 in washington state detection of viruses in myocardial tissues by polymerase chain reaction. evidence of adenovirus as a common cause of myocarditis in children and adults tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis acute kidney injury in covid-19: emerging evidence of a distinct pathophysiology sars-cov-2 infects t lymphocytes through its spike protein-mediated membrane fusion longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients prolonged viral shedding in feces of pediatric patients with coronavirus disease 2019 we thank the technical staff of king county medical examiner's office (seattle, wa, usa) and snohomish medical examiner's office (everett, wa, usa) for their assistance with autopsy procedures. we thank jennifer swicord, gianni niolu, and the electron microscopy laboratory at the university of washington (seattle, wa, usa) for preparation of electron microscopy specimens. key: cord-266511-g5h4tazp authors: deslandes, a; berti, v; tandjaoui-lambotte, y; alloui, chakib; carbonnelle, e; zahar, jr; brichler, s; cohen, yves title: sars-cov-2 was already spreading in france in late december 2019 date: 2020-05-03 journal: int j antimicrob agents doi: 10.1016/j.ijantimicag.2020.106006 sha: doc_id: 266511 cord_uid: g5h4tazp abstract the covid-19 epidemic is believed to have started in late january 2020 in france. we report here a case of a patient hospitalized in december 2019 in our intensive care, of our hospital in the north of paris, for hemoptysis with no etiological diagnosis and for which rt-pcr was performed retrospectively on the stored respiratory sample which confirmed the diagnosis of covid-19 infection. based on this result, it appears that the covid-19 epidemic started much earlier. after its onset in december 2019 in china, the new coronavirus (sars-cov-2) spreads widely in several countries, causing covid-19 illness. 1 world health organization (who) declared covid-19 a pandemic on march 11, 2020. 3 france reported the first cases of sars-cov-2 related infection on january 24, 2020. 5 both cases had a history of travel to wuhan. 6 to the best of our knowledge, these 2 cases are believed to be the first confirmed cases in france. covid-19 most commonly present with influenza-like illness (ili). 7 while china was facing covid-19 outbreak, european countries were struggling with seasonal influenza. 8 clinical symptomatology between covid-19 and iliis similar,we therefore decided retrospectively to look for sars-cov2 in respiratory samples collected in the intensive care units (icus) of our hospital near paris. we reviewed medical record of icus patients admitted for ili between december 2, 2019 and january 16, 2020, with a negative rt-pcr performed at admission. every respiratory sample collected in our hospital are frozen at -80°c in thermoscientific -86°c freezer and stored for four years in case of a need for further analysis. samples taken from patients with both ili symptoms (fever higher than 38.5°c, cough, rhinitis, sore throat or myalgia) and ground glass opacity according to their medical record underwent sars-cov-2 rt-pcr. a description of sample selection is available in figure 1 . sars-cov-2 rt-pcr was performed on the 14 selected biobanks between april 6 and 9, 2020. rt-pcr was performed strictly according to the charité protocol 11 targeting the e gene, coding for envelope protein, pangenomic of sars-cov-1 and sars-cov-2, on a quantstudio7 (thermofisher) device. positive result (figure 2 ) was confirmed with the gene finder® covid19 plus realamp kit (ifmr-45) according to the manufacturer's recommandations. this test targets 3 viral genes (rdrp, e and n coding respectively for the viral rna-dependant rna polymerase, envelope and nucleocapsid protein), and the cellular rnase p gene, in order to confirm the quality of the respiratory sample. during the study period, 14 (24%) patients out of 58 admitted for ili were included in our analysis (table 1) .one sample was positive taken from a 42 years old manborn in algeria, who lived in france for many years, and worked as a fishmonger. his last trip was in algeria during august 2019. one of his child presented with ili prior to the onset of his symptoms. his medical history consisted in asthma, type ii diabetes mellitus. he presented to the emergency ward on december 27 2019 with hemoptysis, cough, headache and fever, evolving for 4 days. initial examination was unremarkable and the performed ct scan revealed bilateral ground glass opacity in inferior lobes (figure 3 ). at admission he had a lymphopenia, an elevated c-reactive protein and fibrinogen while pro calcitonin was in normal range value. no pathogen was identified on sputum sample collected in the emergency ward. the patient was admitted to the icu with antibiotic therapy, and evolution was favorable until discharge on december 29,2019 we report an observation of a patient infected with covid-19 one month before the first reported cases in our country. on admission, the patient presented clinical signs and radiological patterns frequently seen previously in the chinese 12 and italian 13 cohorts. identifying the first infected patient is of great epidemiological interest as it changes dramatically our knowledge regarding sars-cov-2 and its spreading in the country. moreover, the absence of a link with china and the lack of recent travel suggest that the disease was already spreading among the french population at the end of december, 2019. further studies are required to evaluate sars-cov-2's actual onset on french territory, the actual extent of sars-cov-2 contamination in the population during late 2019 and january 2020, and explore the potential unnoticed deaths that could have happen at the time. covid-19 is considered to be responsible for 86334 cases and 12210 deaths as of april 10, 2020 4 in france, but our findings suggest that these numbers could be underestimating the actual burden of covid-19. two recent studies suggested that around 18 to 23% infected with sars-cov-2 were asymptomatic 16 and that around 55% of infected were caused by unidentified infected persons. 17 our results strongly support these two assumptions, suggesting that many asymptomatic patients were not diagnosed during january 2020 and contributed to the spread of this epidemic. furthermore, since these results change our understanding of the dynamic of the epidemic, it also means that several models used to predict the evolution and outcomes of the sars-cov-2 propagation might be based on biased data and would need to be adjusted to the actual profile of the epidemic. 18 our study presents several limitations. firstly, due to the retrospective nature of the analyses carried out, medical records were not exhaustive and some relevant information might have been missing. secondly, we are not able to rule out false negative results due to the sensitivity of rt-pcr 19 and a technique of storage that possibly damage the quality of the samples. 20 to avoid any false positive result we have taken all the usual precautions and we also confirmed it by two different, techniques and staff. thirdly we restricted our analyses to only a few samples and we chose to limit the selected records to icu patients with compatible symptoms and ct, even though most patients actually have mild symptoms. fourthly, we restricted our analyses to patients with a negative multiplex pcr at the time even though cross contamination has been described in literature. 9 finally, we conducted a monocentric study in the northern paris area, which faced a particularly high burden in this epidemic. 21 these limitations could explain why we were only able to identify one person infected with sars-cov-2, in our population. a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia who director-general's opening remarks at the media briefing on covid-19 -11 situation update worldwide clinical and virological data of the first cases of covid-19 in europe: a case series first cases of coronavirus disease 2019 (covid-19) in france: surveillance, investigations and control measures clinical characteristics of coronavirus disease 2019 in china early release -co-infection with sars-cov-2 and influenza a virus in patient with pneumonia acute respiratory distress syndrome: the berlin definition detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study chest ct features of covid-19 in clinical features of patients infected with 2019 novel coronavirus in wuhan novel coronavirus (covid-19) pneumonia with hemoptysis as the initial symptom: ct and clinical features estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship estimation of covid-19 outbreak size in italy. the lancet infectious diseases 0 prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal detection of sars-cov-2 in different types of clinical specimens impact of long-term storage on stability of standard dna for nucleic acid-based methods cartes, données et graphiques figure 1. selection process for testing sars-cov-2 : severe acute respiratory syndrome coronavirus-2 figure 2. result of charite protocol rt-pcr assay. patient's curb in yellow, positive and negative test sample in purple) ethical approval: not required key: cord-009476-4emc4o6n authors: madani, tariq a title: case definition and management of patients with mers coronavirus in saudi arabia date: 2014-09-22 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70918-1 sha: doc_id: 9476 cord_uid: 4emc4o6n nan the threat to global health security from emerging and re-merging respiratory tract infections will be ever present because of the genetic adaptability of microbes, and their ability to resist clinical interventions and public health measures aimed at their elimination. although much has been learned from previous outbreaks, present surveillance systems have their inherent weaknesses, and recent experiences with mers-cov 14 show that pandemic preparedness still faces major political and scientifi c challenges. 15 an important priority for control of infectious disease is to ensure that scientifi c and technological advances in molecular diagnostics and bioinformatics are well integrated into public health. more eff ective and wider partnerships based on equity and best ethical practice, across governments, health care, academia, industry, and with the public, are essential to eff ectively galvanise economic, political and scientifi c measures required to develop core capacities, including legislation, national focal points, and pandemic planning to reduce risk of global spread and reduce the burden of respiratory tract infectious diseases. an urgent need exists to establish trusting and eff ective meaningful collaborations between countries to tackle new emerging microbial threats. this will facilitate early and rapid detection of potential pandemic infectious diseases through public health actions within the framework of the international health regulations. 16 outbreak and prevent human-to-human and animalto-human transmission; an appropriate management algorithm, including best-practice guidelines for accurate diagnosis, infection control, intensive care, emergency medicine, and treatment; prioritise research related to the mers-cov outbreak such as case-control and cohort studies, seroprevalence studies, and clinical trials; and to eff ectively monitor outbreak control activities. a continously operating command and control centre was established in the minister's office. in addition to the advisory council, nine further platforms were established: interministerial to coordinate efforts between the ministry of health (moh) and other concerned ministries; capacitybuilding to recruit and mobilise qualified staff to work in hospitals treating patients with mers-cov, increase the number of beds in intensive care units, and provide state-of-the-art machines such as extracorporeal membrane oxygenation to treat patients with respiratory failure refractory to conventional ventilation; public relations to communicate relevant information to the public, health-care workers, and local and international media; clinical operation to coordinate management of patients and transfers between hospitals; public health to collect data related to patients and their contacts; data analysis to enter and analyse data; epidemiological to provide consultations on data analysis and interpretation; laboratory to ensure fast and reliable diagnostic testing; and, infection control to oversee infection control practice and staff training activities. a mers referral hospital run by well trained staff was designated in riyadh, jeddah, and dammam to receive and manage all patients infected with mers-cov. the moh enforced strict infection prevention as new clinical information became available, a revision of the mers-cov case defi nition seemed appropriate. 2 the new case defi nition (appendix) was developed based on reported health-care-associated mers-cov pneumonia (added as category 2 in the new case defi nition) and non-respiratory characteristics of patients with confi rmed infection who fi rst presented with acute febrile dengue-like illness with body aches, leucopenia, and thrombocytopenia (added as category 3). the new case defi nition added a fourth category for contacts of people with mers-cov who present with not only lower respiratory tract but also isolated upper respiratory tract features. this defi nition classifi ed the status of patients into three categories of suspect, probable, or confi rmed infection. the new mers-cov case defi nition was revised and approved by the advisory council after seeking external cdc expert opinion. an algorithm for mers-cov case management was developed (fi gure). according to this algorithm, patients with confi rmed mers-cov who have no evidence of pneumonia or who recover from pneu monia but remain positive for mers-cov, can be isolated at home after careful assessment of the home situation and suitability for isolation by the treating physician, highly trained social workers, or other health-care professionals by telephone or home visits. the cdc has released recommendations on how to assess the home situation and the advice to be given to patients on home isolation and his or her caregivers and household members, 3 and also released guidance for the public, clinicians, and public-health authorities in the usa on control of the mers-cov infection. 4 ministry of health, riyadh, saudi arabia; and department of medicine, faculty of medicine, king abdulaziz university, jeddah, saudi arabia tmadani@kau.edu.sa tim uyeki, and ray arthur for their critical review of the case defi nition and the mers-cov case management algorithm, and fadwa mushtaq for her secretarial assistance world health organization. who experts probe middle-eastern respiratory syndrome coronavirus (mers-cov world health organization. who revised interim case defi nition for reporting to who-middle east respiratory syndrome coronavirus (mers-cov): as of us centers for disease control and prevention. interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus first confi rmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities health-care-associated bacterial infections are an important cause of morbidity and mortality in critically ill patients, especially patients needing mechanical ventilation. decolonisation with topical antibiotics, such as selective digestive tract decontamination (sdd) or selective oropharyngeal decontamination (sod), eradicates potentially pathogenic bacteria, preventing ventilator-associated pneumonia and bacteraemia. in two dutch studies, 1,2 sdd and sod reduced mortality, intensive-care unit (icu) length of stay, icu-acquired bacteraemia, and carriage with antibiotic-resistant bacteria. accordingly, both measures were deemed cost eff ective. 3 only studies that assessed the unit-wide implementation of sdd or sod provide evidence of a key: cord-256737-ptjng78b authors: mcbride, corrin e.; machamer, carolyn e. title: palmitoylation of sars-cov s protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with m protein date: 2010-09-01 journal: virology doi: 10.1016/j.virol.2010.05.031 sha: doc_id: 256737 cord_uid: ptjng78b coronaviruses are enveloped rna viruses that generally cause mild disease in humans. however, the recently emerged coronavirus that caused severe acute respiratory syndrome (sars-cov) is the most pathogenic human coronavirus discovered to date. the sars-cov spike (s) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. coronavirus s proteins are palmitoylated, which may affect function. here, we created a non-palmitoylated sars-cov s protein by mutating all nine cytoplasmic cysteine residues. palmitoylation of sars-cov s was required for partitioning into detergent-resistant membranes and for cell-cell fusion. surprisingly, however, palmitoylation of s was not required for interaction with sars-cov m protein. this contrasts with the requirement for palmitoylation of mouse hepatitis virus s protein for interaction with m protein, and may point to important differences in assembly and infectivity of these two coronaviruses. coronaviruses are enveloped positive strand rna viruses that infect many avian and mammalian species, including humans. these viruses target a variety of tissues and generally cause mild disease. in humans, coronaviruses are responsible for approximately 20% of common cold cases (larson et al., 1980) . however, in 2002, a novel human coronavirus, severe acute respiratory syndrome coronavirus (sars-cov), emerged in the guangdong province of china (kuiken et al., 2003; rota et al., 2003) . sars-cov is unlike any other human coronavirus to date, causing severe respiratory disease and death in 10% of infected patients (who, 2003) . while many enveloped viruses assemble at the plasma membrane, coronaviruses assemble intracellularly and bud into the lumen of the endoplasmic reticulum golgi intermediate compartment (ergic) (klumperman et al., 1994) . to produce infectious virus, the envelope proteins must be targeted to the ergic for virus assembly. many coronavirus envelope proteins localize near the assembly site when exogenously expressed alone (corse and machamer, 2000; klumperman et al., 1994) ; however, others rely on lateral interactions with other envelope proteins to localize to the virus assembly site (mcbride et al., 2007; nguyen and hogue, 1997; opstelten et al., 1995) . like other coronaviruses, sars-cov encodes 3 envelope proteins, spike (s), envelope (e) and membrane (m) (marra et al., 2003; rota et al., 2003) . the s protein is the second most abundant protein in the virion envelope. it is a type-i membrane protein that determines host cell tropism and is responsible for virus-cell as well as cell-cell fusion (cavanagh, 1995; gallagher and buchmeier, 2001) . the sars-cov s glycoprotein is large (approximately 180 kda) and heavily glycosylated with 23 potential n-linked glycosylation sites (marra et al., 2003; rota et al., 2003) . the cytoplasmic tail of the sars-cov s is palmitoylated (petit et al., 2007) and contains a weak endoplasmic reticulum retrieval signal that helps it localize to the virus assembly site when co-expressed with sars-cov m (mcbride et al., 2007) . the m protein is the most abundant protein in the virion envelope. m can form homo-oligomers and acts as a scaffold for virus assembly, interacting with s, e and the viral nucleocapsid hogue and machamer, 2008) . sars-cov m has an n-linked glycosylation site, three transmembrane domains and a long cytoplasmic tail (voss et al., 2006) . the e protein is the least abundant protein in the virion envelope although it has an important role in virion budding and release (dediego et al., 2007; fischer et al., 1998; kuo and masters, 2003; machamer and youn, 2006; ortego et al., 2007 ortego et al., , 2002 . although the topology and glycosylation of sars-cov e is controversial, it has a single hydrophobic domain and is palmitoylated on its cytoplasmic tail (reviewed in (liu et al., 2007) . virology 405 (2010) [139] [140] [141] [142] [143] [144] [145] [146] [147] [148] protein palmitoylation is a common post-translational modification that occurs on cytoplasmic cysteine residues. protein palmitoylation occurs by the addition of the fatty acid palmitate to a protein via a thioester linkage (resh, 2006a) . protein palmitoylation can greatly increase the hydrophobicity of a protein, which can in turn affect protein activity. transmembrane proteins are commonly palmitoylated on cysteine residues that are at or near the lipid bilayer (resh, 2006a) . cytoplasmic proteins can also be palmitoylated, inducing membrane association (greaves and chamberlain, 2007) . palmitoylation is generally reversible and can be highly dynamic (linder and deschenes, 2007) . addition of palmitate to proteins can greatly influence a protein's trafficking, stability, localization and interaction with other proteins (delandre et al., 2009; mccormick et al., 2008; van itallie et al., 2005) . because of the great versatility of protein palmitoylation, it can be used to dynamically regulate protein function (baker et al., 2003; iwanaga et al., 2009) . palmitoylation of viral proteins can play an important role in virus assembly and infection (grantham et al., 2009; majeau et al., 2009; rousso et al., 2000) . the s and e proteins of several coronaviruses have been shown to be palmitoylated (boscarino et al., 2008; corse and machamer, 2002; liao et al., 2006; lopez et al., 2008; petit et al., 2007; thorp et al., 2006) ; however, not much is known about the role of this modification. coronavirus s protein palmitoylation appears to be important for cell-cell fusion (petit et al., 2007) , infectivity and virus assembly (thorp et al., 2006) . in addition, for mouse hepatitis virus (mhv), it has been shown that s protein palmitoylation is important for interaction with the m protein (thorp et al., 2006) . coronavirus e protein palmitoylation is important for e protein stability, virus assembly and production (boscarino et al., 2008; lopez et al., 2008) . although many important roles for palmitoylation of coronavirus envelope proteins have been suggested, few studies have analyzed all of the possible palmitoylated cysteine residues with regard to function (petit et al., 2007) . pharmacological inhibition of palmitoylation is commonly used (thorp et al., 2006) , although this has deleterious effects on general cellular functions since palmitoylation is a common modification (mikic et al., 2006; resh, 2006b) . in this study, we addressed the role of sars-cov s protein palmitoylation without the use of general palmitoylation inhibitors by mutating the 9 potentially palmitoylated cytoplasmic cysteine residues. we show that palmitoylation is not necessary for sars-cov s protein stability, localization and trafficking. we confirm previous reports that suggest a role for sars-cov s protein palmitoylation in cell-cell fusion and also provide evidence for the importance of palmitoylation in sars-cov s partitioning into detergent-resistant membranes (drms). importantly, we show that sars-cov s palmitoylation is not necessary for efficient interaction with sars-cov m, which differs from published experiments for mhv (thorp et al., 2006) and suggests a significant difference between the two viruses that may have important implications for virus assembly and infectivity. results from other labs have shown that coronavirus spike proteins are palmitoylated (bos et al., 1995; petit et al., 2007) ; however, there is no information about the compartment in which this post-translational modification occurs. since the protein acyltransferases responsible for protein palmitoylation are localized throughout the entire secretory pathway (tsutsumi et al., 2008) , it is possible that coronavirus spike proteins could be palmitoylated at the er, golgi or the plasma membrane. after glycoproteins are synthesized, their sugars become modified as the protein traffics through the secretory pathway. in the medial golgi, glycoproteins become resistant to digestion with endoglycosidase h (endo h) (herscovics, 1999) . to determine if sars-cov s becomes palmitoylated in a pre-medial golgi compartment, hek293t cells exogenously expressing sars-cov s were labeled for 30 min with 35 s-methionine/ cysteine to measure total protein expression or 3 h-palmitic acid to measure palmitoylated protein. while 35 s-methionine/cysteine labels newly translated proteins, 3 h-palmitic acid labels both newly made and pre-existing proteins. after radiolabeled cells were lysed, s protein was immunoprecipitated, denatured and digested with endo h. as expected, upon endo h treatment, there was a population of newly made 35 s-labeled s protein that had not yet trafficked past the medial golgi and was thus sensitive to endo h digestion (fig. 1a, left) . there was also a population of 3 h-palmitic acid labeled s protein that was sensitive to digestion with endo h (fig. 1a, right) , indicating that s can be palmitoylated before it traffics through the golgi en route to the plasma membrane. this result suggests that at least a portion of sars-cov s is palmitoylated in a pre-medial golgi compartment but does not rule out the possibility of additional palmitoylation in a postmedial golgi compartment. palmitoylation is reversible and proteins can be palmitoylated and de-palmitoylated rapidly (linder and deschenes, 2007) . generally, viral proteins are stably palmitoylated, but there are examples of proteins containing palmitate chains that are rapidly turned over (rocks et al., 2005) . to determine if sars-cov s palmitoylation was a stable or dynamic modification, we performed a pulse-chase assay. hek293t cells expressing sars-cov s were pulse labeled with 35 smethionine/cysteine or 3 h-palmitic acid and chased for various times. the 3 h-palmitate label (fig. 1b , right) was similar in stability to the total 35 s-methionine/cysteine labeled population (fig. 2, left) . this suggests that palmitoylation of sars-cov s is a stable posttranslational modification. like other coronaviruses spike proteins, sars-cov s has multiple cysteine residues in its cytoplasmic tail that could be palmitoylated. sars-cov s, like mhv s, has 9 cytoplasmic cysteines that are putative palmitoylation sites ( fig. 2a ) (petit et al., 2007) . to determine the role of sars-cov s palmitoylation, we mutated all 9 of the cytoplasmic cysteine residues to alanines. to ensure that the mutant sars-cov s was not palmitoylated, we radiolabeled hek293t cells expressing wild-type or mutant s with 35 s-methionine/cysteine or 3 h-palmitic acid. while wild-type and mutant sars-cov s proteins were expressed at similar levels, only wild-type sars-cov s was protein palmitoylation can have dramatic effects on protein turnover. palmitoylation often increases protein stability (ochsenenbauer-jambor et al., 2001) ; in support of this, mhv e palmitoylation was reported to increase its half-life (lopez et al., 2008) . to determine if palmitoylation affects the stability of s, we calculated the half-lives of sars-cov s and s pn . hek293t cells expressing sars-cov s or s pn were pulse labeled and chased for various times. wild-type and mutant s proteins were immunoprecipitated and analyzed. the percentage of protein remaining relative to the zero chase time was calculated for each time of chase. both sars-cov s and s pn had similar half-lives of approximately 2.75 h (fig. 3) . this result suggests that palmitoylation of sars-cov s does not affect turnover or stability when s protein is exogenously expressed. sars-cov s localizes to the plasma membrane when exogenously expressed alone in cells (bisht et al., 2004; hofmann et al., 2004; schwegmann-wessels et al., 2004; simmons et al., 2004) . transmembrane protein trafficking as well as soluble protein translocation to membranes can both be regulated by protein palmitoylation (resh, 2006a) . in fact, palmitoylation of different cysteines on the same protein can lead to dramatic differences in subcellular localization (roy et al., 2005) . to determine if palmitoylation alters the subcellular localization of sars-cov s, we performed indirect immunofluorescence microscopy on hek293t cells expressing sars-cov s or s pn . hek293t cells expressing s or s pn were fixed, permeabilized and costained with antibodies to sars-cov s and golgin-160 (a golgi marker). sars-cov s and s pn were both present throughout the secretory pathway, with staining at the cell surface and some internal concentration at the golgi complex co-localizing with golgin-160 ( fig. 4a ). to further assess the localization of sars-cov s and s pn at the plasma membrane, we performed surface staining on nonpermeabilized cells with mouse anti-sars-cov s. subsequently, cells were washed, fixed, permeabilized and then stained for total s protein with rabbit anti-sars-cov s. surface staining of intact cells revealed that sars-cov s and sars-cov s pn were both present at the plasma membrane (fig. 4b ). these results suggest that blocking palmitoylation does not affect the subcellular localization of sars-cov s when exogenously expressed. although both sars-cov s and s pn were present at the cell surface, it is possible that there could be a difference in the amount of protein at the plasma membrane at steady state if palmitoylation affects a post-golgi trafficking step. to determine if palmitoylation affects the steady-state levels of s protein at the plasma membrane, we performed a cell surface biotinylation assay. hek293t exogenously expressing sars-cov s or s pn were surface biotinylated at 0°c with a membrane-impermeable biotinylating reagent. biotinylated s proteins were captured with streptavidin agarose resin and analyzed by sds-page followed by western blotting. sars-cov s and s pn were both present in similar amounts at the plasma membrane, with 8-9% of the total protein expressed surface biotinylated at steady state ( fig. 4c ). previous data from our lab and others have shown that coronavirus s and m proteins can interact directly (godeke et al., 2000; hsieh et al., 2008; huang et al., 2004; mcbride et al., 2007; nguyen and hogue, 1997; youn et al., 2005) . palmitoylation has been shown to regulate protein-protein interactions (shmueli et al., 2010) . using the palmitoylation inhibitor 2-bromopalmitate (2-bp) (thorp et al., 2006) or truncation analysis (bosch et al., 2005) , it has been indirectly shown that palmitoylation of mhv s protein is necessary for interaction with mhv m. when the sars-cov s protein is exogenously expressed alone, it localizes to the cell surface. however, when co-expressed with sars-cov m, sars-cov s localizes to the golgi complex, co-localizing with m (mcbride et al., 2007) . to determine if sars-cov s palmitoylation is necessary for interaction with m, we performed indirect immunofluorescence microscopy. hek293t cells exogenously co-expressing sars-cov m and sars-cov s or s pn were fixed, permeabilized and co-stained with antibodies to sars-cov s and sars-cov m. upon co-expression with sars-cov m, s was retained intracellularly at the golgi complex and co-localized completely with m (fig. 5a) . surprisingly, sars-cov s pn was also retained intracellularly at the golgi when co-expressed with sars-cov m (fig. 5a) . palmitoylation did not affect the steady-state localization of sars-cov s when co-expressed with m by indirect immunofluorescence microscopy but might influence the trafficking of s in the absence or presence of sars-cov m. to determine if palmitoylation affects the trafficking of sars-cov s, we performed a pulse-chase assay and measured acquisition of endo h resistance. hek293t cells expressing sars-cov s or s pn in the absence or presence of sars-cov m were pulse labeled with 35 s-methionine/cysteine and chased for various times. s protein was immunoprecipitated, denatured and digested with endo h. s and s pn had similar trafficking kinetics through the secretory pathway. after 40 min of chase, 70-80% of both sars-cov s and s pn were resistant to digestion with endo h, implying that 70-80% of the pulse-labeled protein had moved past the medial golgi, presumably en route to the plasma membrane (fig. 5b) . interestingly, when co-expressed with sars-cov m, carbohydrate processing of both s and s pn was dramatically reduced and only 10-20% of the labeled protein was resistant to endo h digestion after 40 min of chase (fig. 5b) . thus, both sars-cov s and s pn interacted with m and were retained in a pre-medial golgi compartment, which is consistent with the previously published cis-golgi localization of some coronavirus m proteins (swift and machamer, 1991) . taken together, these results and previously published in vitro binding data (mcbride et al., 2007) suggest that palmitoylation of sars-cov s is not essential for interaction with sars-cov m. this reveals a potentially important difference between assembly of sars-cov and mhv. one of the best known functions of protein palmitoylation is directing incorporation into specific membrane domains that are resistant to extraction with cold triton x-100. detergent-resistant membranes (drms) are sometimes referred to as lipid rafts and are regions of membrane that are enriched in cholesterol and glycosphingolipids (brown, 2006) . drms may represent important signaling centers at the plasma membrane although they can also be found earlier in the secretory pathway at the golgi (simons and van meer, 1988) . mhv s is enriched in drms and enrichment was reduced by treating cells with 2-bp prior to isolation (thorp et al., 2006) . to determine if sars-cov s partitions into drms and if that partitioning is dependent on s protein palmitoylation, we isolated drms by floatation on a sucrose step gradient after cold triton x-100 extraction. fractions were collected and 10% of each fraction was analyzed by sds-page and western blotting with antibodies to sars-cov s. to identify which fractions contained drms, a small portion of each fraction was spotted onto nitrocellulose and incubated with hrp-cholera toxin b which binds to gm1, a glycolipid enriched in drms (brown, 2006) . gm1 was primarily present in fraction 4 for both samples (fig. 6, bottom panel) . sars-cov s was also present in fraction 4 (fig. 6 top panel) while s pn was completely absent from fractions that contained drms (fig. 6 middle panel) . amido black staining of the membrane ensured that similar amounts of total protein were present in each lane for both s and s pn (data not shown). it is important to note that the upper fully glycosylated mature form of sars-cov s was enriched in drms while the immature partially processed form of sars-cov s was present in the more dense fractions. these results demonstrate that sars-cov s palmitoylation is necessary for s partitioning into drms. approximately 8% of total s protein is at the plasma membrane at steady state by biotinylation (fig. 4c) , and an average of 15% of total s is present in detergentresistant membranes at steady state. thus, it is likely that all s at the plasma membrane is present in drms. a role for coronavirus s protein palmitoylation in membrane fusion has been suggested (bos et al., 1995; chang et al., 2000; petit et al., 2007; shulla and gallagher, 2009) . however, these studies have either used global palmitoylation inhibitors like 2-bp, which can be toxic to cells (mikic et al., 2006) , or only partial mutagenesis of palmitoylated cysteines. to determine a role for sars-cov s cytoplasmic cysteine residues in cell-cell fusion, we compared the fusion activities of sars-cov s and s pn in vero cells, which express the functional sars-cov receptor, ace2. vero cells expressing sars-cov s or s pn were briefly trypsinized to activate the protein for fusion (simmons et al., 2004) . the number of nuclei per syncytia (≥3 nuclei) was counted 24 h after trypsinization. vero cells expressing sars-cov s had extensive syncytium formation with approximately 5 nuclei per syncytia, but syncytium formation was absent in cells expressing sars-cov s pn (fig. 7) . these results suggest a critical role for s protein palmitoylation in cell-cell fusion. in conclusion, we have shown that palmitoylation is dispensable for the stability, subcellular localization and trafficking of sars-cov s. however, sars-cov s palmitoylation is important for partitioning into drms and for cell-cell fusion. most importantly, sars-cov s palmitoylation is not important for interaction with sars-cov m. sars-cov m retained non-palmitoylated s in the golgi and reduced the extent of non-palmitoylated s trafficking through the secretory pathway as well as wild-type sars-cov s. these results demonstrate a significant difference between sars-cov and mhv that may have significant implications for virus assembly and infection. protein palmitoylation is a common post-translational modification where a 16 carbon fatty acid chain is added to cysteine residues on proteins. palmitoylation can be dynamic and plays an important role in regulating protein function and activity. in fact, alterations in protein palmitoylation have been shown to affect protein stability, trafficking and subcellular localization as well as protein-protein interactions (linder and deschenes, 2007; lopez et al., 2008; resh, 2006a; roy et al., 2005; thorp et al., 2006; van itallie et al., 2005) . transmembrane proteins can be palmitoylated throughout the secretory pathway and cytoplasmic proteins can be palmitoylated at different secretory pathway membranes. additionally, many important regulatory proteins, signaling molecules and trafficking components have been shown to be palmitoylated (linder and deschenes, 2007) . in line with the important role of palmitoylation in cellular processes, global inhibition of palmitoylation has deleterious effects on cellular function (mikic et al., 2006; resh, 2006b) . in addition to the palmitoylation of endogenous cellular proteins, many viral proteins are palmitoylated. perhaps the most well-known examples of palmitoylated viral proteins are the influenza virus ha and m2 proteins (holsinger et al., 1995; sugrue et al., 1990; veit et al., 1991) . however, there are many more examples of viral protein palmitoylation including those encoded by hepatitis c virus (majeau et al., 2009 ), human immunodeficiency virus (rousso et al., 2000) , herpes simplex virus (nozawa et al., 2003) and some coronaviruses (bos et al., 1995; corse and machamer, 2002; petit et al., 2007) . inhibition or disruption of viral protein palmitoylation can have negative effects on important protein-protein interactions in the virion envelope (yu et al., 2006) , virus assembly (boscarino et al., 2008; lopez et al., 2008; majeau et al., 2009; thorp et al., 2006; ye et al., 2004) , infectivity (boscarino et al., 2008; lopez et al., 2008; rousso et al., 2000; yan et al., 2004) , subcellular protein localization (nozawa et al., 2003) and even protein stability (lopez et al., 2008) . two of the coronavirus envelope proteins, s and e, contain conserved palmitoylated cysteine residues in their cytoplasmic tails. palmitoylation of the coronavirus e protein does not seem to be important for golgi localization (boscarino et al., 2008; corse and machamer, 2002; lopez et al., 2008) or virus entry (lopez et al., 2008) but has been implicated in protein stability and efficient virus growth (boscarino et al., 2008; lopez et al., 2008) . e proteins have 2-3 potentially palmitoylated cytoplasmic cysteine residues. the work performed to determine the role of coronavirus e protein palmitoylation used mutagenesis of all potentially palmitoylated cysteine residues, which conclusively implicated or eliminated possible roles for e protein palmitoylation. coronavirus s proteins contain a cysteine-rich domain in their cytoplasmic tails with at least 7 cysteine residues (hogue and machamer, 2008) . coronavirus s protein palmitoylation has been implicated in cell-cell fusion, virus-cell fusion, virus assembly and infectivity; however, studies focusing on the role of s protein palmitoylation have so far mutated only some of the palmitoylated cysteines (petit et al., 2007; shulla and gallagher, 2009) or used pharmacological inhibitors of protein palmitoylation (thorp et al., 2006) . these methods and results led to only a partial disruption of protein palmitoylation and partial phenotypes. here, we mutated all 9 cytoplasmic cysteine residues to conclusively determine the role of sars-cov s palmitoylation. we constructed a palmitoylation-null mutant protein, sars-cov s pn , with all 9 cytoplasmic cysteine residues mutated to alanines, which was not palmitoylated (fig. 2) . the compartment in which envelope proteins become palmitoylated has not been determined for any coronavirus. when sars-cov s was exogenously expressed in cells, we identified a population of palmitoylated, endo h sensitive sars-cov s (fig. 1a) . this suggests that at least some sars-cov s protein is palmitoylated in a pre-medial golgi compartment. mhv s can also be palmitoylated in an early compartment (van berlo et al., 1987) . however, it will be important to determine if s protein palmitoylation occurs similarly during an infection when other viral proteins are present. also, we determined that palmitoylation of sars-cov s is a relatively stable post-translational modification (fig. 1b) , which appears to be common among palmitoylated viral proteins (linder and deschenes, 2007) . this does not rule out the possibility that different cysteines may have differential rates of palmitate turnover; however, it does suggest that there is always a population of s protein that is palmitoylated to some extent. unlike the mhv e protein, disruption of sars-cov s palmitoylation did not affect the stability of the protein since sars-cov s and s pn had similar half-lives when expressed exogenously (fig. 3) . however, like the e protein, sars-cov s palmitoylation does not appear to be important for subcellular localization (fig. 4) (boscarino et al., 2008; corse and machamer, 2002; linder and deschenes, 2007; lopez et al., 2008) . we confirmed previous results published for mhv and sars-cov that s palmitoylation is important for cell-cell fusion. however, we show a complete inhibition of cell-cell fusion (not a partial reduction) when cells expressed s pn (fig. 7) . this abolition of cell-cell fusion was not due to a reduction in the amount of sars-cov s pn at the plasma membrane ( fig. 4c) but possibly due to an exclusion of sars-cov s pn from drms (fig. 6) . in fact, drms have been implicated in cell-cell fusion events at the plasma membrane (mukai et al., 2009; teissier and pecheur, 2007) . although it is possible that mutating all 9 cysteine residues to alanines dramatically disrupted the conformation of the sars-cov s protein, this seems unlikely since s pn was stable, trafficked through the secretory pathway properly (fig. 5b) , had the correct subcellular localization and could still interact with the m protein (fig. 5a) . most importantly, we show that palmitoylation of sars-cov s is not necessary for interaction with the m protein. previous results from our lab suggested sars-cov s and m could interact in vitro using a recombinant sars-cov s cytoplasmic tail purified from bacteria. this recombinant s protein was not palmitoylated, yet it was able to interact with in vitro transcribed and translated sars-cov m (mcbride et al., 2007) . in that in vitro experiment, only the tail of the sars-cov s protein was used; here we confirm those results using different in vivo assays using the full length protein. we showed that sars-cov m was able to retain s pn at the golgi complex similarly to wild-type s (fig. 5a) . also, sars-cov m was able to reduce the amount of s pn carbohydrate processing (fig. 5b ) and the amount of s pn at the plasma membrane (data not shown) similar to wild-type sars-cov s. these results suggest a significant difference between mhv and sars-cov. for mhv, very low concentrations of 2-bp, which only slightly reduced the amount of mhv s palmitoylation, had dramatic results on the ability of mhv s to form a complex with m. this resulted in reduced mhv s incorporation into virions, which led to a reduction in infectious virus (thorp et al., 2006) . mhv s appears to be extremely sensitive to changes in palmitoylation levels, where as sars-cov s can better tolerate disruptions in palmitoylation. an interesting possibility is that wild-type fully palmitoylated s and palmitoylation-null s both interact equally well with m, but only partially palmitoylated s protein does not interact efficiently with m protein. it is also possible that an endogenous protein fig. 6 . sars-cov s palmitoylation is necessary for partitioning into detergent-resistant membranes. at 24 h post-transfection, detergent-resistant membranes (drms) were extracted from hek293t cells expressing sars-cov s or s pn using cold triton. drms were isolated using discontinuous density ultracentrifugation, and fractions were collected from the top. s protein was identified by western blotting (upper and middle panels) and drms were identified using hrp-cholera toxin b, which binds ganglioside gm1 (bottom panel). a representative image of 3 independent experiments is shown. was sensitive to low concentrations of 2-bp in the studies using mhv; this could have subsequently affected mhv s trafficking, localization and/or stability since none of these variables were tested after 2-bp treatment. we attempted to use virus-like particle (vlp) production to determine if sars-cov s palmitoylation was important for virus assembly, but the ability of s and s pn to be released from membranous vesicles when expressed alone in 293t cells precluded our ability to determine the contribution of palmitoylation to assembly using this assay. our results suggest that there would be no difference between s and s pn assembly into virions since both can interact equally well with m; however, it is possible that spike incorporation into virions could depend on more than interaction with m protein. even if sars-cov s pn is well incorporated into assembling virions, overall infection may be limited by the cell-cell fusion defect seen with s pn . also, recently published work suggests an unexpected role for mhv s cytoplasmic cysteine residues in virus-cell fusion (shulla and gallagher, 2009 ). here we examined the ability of palmitoylation-null sars-cov s to interact with m without complications from virus infection; however, it will also be important to determine the role of s protein palmitoylation in infected cells by inserting the mutations into an infectious clone. there is no clear consensus sequence for protein palmitoylation. palmitate adducts at different cysteines can have different dynamics (linder and deschenes, 2007) and with the large number of potentially palmitoylated cysteines in coronavirus s proteins, there could be many possibilities for differential s protein regulation based on which cysteines are palmitoylated and when. identifying palmitoylated sars-cov s cysteine residues will prove to be difficult due to the 9 potentially palmitoylated cysteines; a large number of combinatorial cysteine mutations would have to be made to fully uncover the role of each. also, mutagenesis of a palmitoylated residue could induce palmitoylation of residues that are not usually modified. although s and s pn are both present at the plasma membrane in similar amounts, s pn is excluded from drms. while this is interesting, a more important observation revolves around the amount of sars-cov s at the cell surface. based on our calculations, only 7-8% of sars-cov s protein expressed in cells is present at the plasma membrane at steady state; however, 15% of total s is present in drms. this suggests that all s present at the plasma membrane is in drms. since drms form in the late golgi, it is likely that s is enriched in these domains before trafficking to the plasma membrane. it seems that coronaviruses have evolved multiple mechanisms to control the amount and distribution of s at the plasma membrane. these mechanisms include er retrieval (lontok et al., 2004; mcbride et al., 2007) , endocytosis (lontok et al., 2004; petit et al., 2005; youn et al., 2005) , lateral protein-protein interactions (mcbride et al., 2007; opstelten et al., 1995) and sequestration in drms (thorp et al., 2006) . it is possible that too much s at the cell surface may compromise a productive infection. in support of this idea, previous work in our lab where the er retrieval and endocytosis signals of the infectious bronchitis virus (ibv) s protein were mutated in an infectious clone showed massive syncytia formation early after transfection but failed to generate any infectious virus (youn et al., 2005) . this suggests that too much s at the plasma membrane is detrimental to infection, possibly due to premature cell death. anchoring in drms insures that any s that is present at the plasma membrane is functional and fully fusion competent. thus, only small amounts of s protein at the cell surface are required to ensure efficient cell-cell fusion. it is also possible that mhv relies heavily on s protein palmitoylation for interaction with m because mhv s does not contain an er retrieval signal. the sars-cov s er retrieval signal presumably increases the possibility of s-m interaction by promoting cycling of s through the budding compartment. without this mechanism, mhv may rely on palmitoylation to present the s protein cytoplasmic tail properly for interaction with m. it will be interesting to determine the role of ibv s palmitoylation in s-m interaction since ibv s contains a canonical er retrieval signal. in conclusion, our results suggest that sars-cov s palmitoylation is important for s partitioning into drms and cell-cell fusion. however, sars-cov s palmitoylation was not necessary for s protein stability, trafficking or subcellular localization. additionally, we conclude that sars-cov s palmitoylation is not necessary for efficient interaction with m protein, which is different from previously published results for mhv (thorp et al., 2006) . this suggests there are differences in the requirements for coronavirus assembly that could translate into important differences in virus infection and spread. hek293t and vero cells were maintained in dulbecco's modified eagle's medium (dmem) (invitrogen/gibco, grand island, ny) supplemented with 10% fetal bovine serum (atlanta biologicals, lawrenceville, ga) and 0.1 mg/ml normocin (invivogen, san diego, ca) at 37°c and 5% co 2 . expression plasmids pcaggs/sars-cov s and pcaggs/sars-cov m were previously described (mcbride et al., 2007) . pcaggs/sars-cov s pn was generated using quikchange (stratgene, la jolla, ca) site-directed mutagenesis to introduce the following mutations sequentially into pcdna 3.1/sars-cov s: c1217a, c1218a, c1222a, c1223a, c1225a, c1230a, c1232a, c1235a and c1236a. the mutated region of sars-cov s pn was excised from pcdna3.1 and subcloned into pcaggs-mcs expression vector (niwa et al., 1991) using ecorv and xhoi. transient transfections were performed using fugene6 transfection reagent (roche, indianapolis, in) as per the manufacturer's instructions. when co-expressed with sars-cov m, there was a decrease in the expression level of sars-cov s and s pn . to counteract this decrease, we co-transfected s and m at a 3:1 ratio. briefly, 1 day after plating cells, 50% confluent 35 mm dishes of hek293t or vero cells were transfected with a total of 1.5 μg of dna per dish when expressing sars-cov s or s pn alone. when sars-cov s or s pn was coexpressed with sars-cov m, 2 μg of dna was used per dish: 1.5 μg pcaggs/sars-cov s or s pn and 0.5 μg sars-cov m. for detergentresistant membrane isolation, a 60% confluent 10 cm dish of hek293t cells was transfected with 12 μg of dna. rabbit anti-sars-cov s (mcbride et al., 2007) , rabbit antisars-cov m (mcbride et al., 2007) and rabbit anti-golgin 160 (hicks and machamer, 2002) polyclonal antibodies were previously described. mouse anti-sars-cov s monoclonal antibodies were from biodefense and emerging infections (bei) research resources (manassas, va). alexa 488-conjugated donkey anti-mouse igg was from invitrogen/ molecular probes (eugene, or), and texas red-conjugated donkey anti-rabbit igg was from jackson immunoresearch (westgrove, pa). horseradish peroxidase-conjugated (hrp) anti-rabbit igg was from amersham/ge healthcare (piscataway, nj). at 24 h post-transfection, hek293t cells were pulse labeled and chased as previously described (mcbride et al., 2007) . briefly, cells were starved in methionine/cysteine-free dmem, labeled for 20 min with 50 μci of 35 s methionine/cysteine, expre 35 s 35 s (perkin elmer, waltham, ma) in methionine/cysteine-free dmem per 35 mm dish and then chased for the times indicated. cells were lysed in detergent solution (50 mm tris-hcl [ph 8.0], 1% np-40, 0.4% deoxycholate, 62.5 mm edta) containing protease inhibitor cocktail (sigma, st. louis, mo). lysates were clarified for 10 min at 4°c at 16,000 ×g and sds was added to a final concentration of 0.2%. sars-cov s and s pn were immunoprecipitated with rabbit anti-sars-cov s polyclonal antibodies (mcbride et al., 2007) sds, 20% glycerol, 0.025% bromophenol blue and 5% 2-mercaptoethanol) and samples were subjected to 8% sds-page. to reduce variation in the sars-cov s and s pn half-life experiment, cells were first seeded on a 10-cm dish. the following day, cells were transfected with 12 μg of pcaggs/sars-cov s or s pn . 20 h post-transfection, cells were trypsinized and seeded onto 35 mm dishes. cells were allowed to reattach for 4 h and were then labeled as described above. labeled proteins were visualized by molecular imager fx phosphoimager (biorad) and quantified using quantity one software. at 24 h post-transfection, hek293t cells were labeled with 3 hpalmitic acid as previously described (corse and machamer, 2002) . briefly, hek293t cells were washed and incubated for 20 min in serum-free dmem. cells were labeled for 30 min at 37°c with 250 μci of 3 h-palmitic acid ([9,10-3 h(n)]-) dried under n 2 and resuspended in dmem supplemented with 10% fbs, 50 mm hepes [ph 7.2] and 1x non-essential amino acids (invitrogen/gibco, grand island, ny). a parallel dish was labeled for 30 min with 50 μci 35 s-methionine/ cysteine as described above to detect total s protein. cells were chased for various times and lysed and immunoprecipitated as described above. for endo h assays, samples were eluted and digested as described above. for all other assays, samples were eluted in 1x laemmli sample buffer. samples were subjected to 8% sds-page, gels were impregnated with 2,5 diphenyloxazole (ppo) and processed by fluorography at −80°c. hek293t cells were prepared for indirect immunofluorescence microscopy as previously described (mcbride et al., 2007) . cells were seeded onto glass coverslips treated with 1 mg/ml poly-l-lysine, mol wt n300,000 (sigma, st. louis, mo) to improve cell adherence during processing. briefly, at 24 h post-transfection hek293t cells were washed in pbs and fixed for 10 min in 3% paraformaldehyde in pbs. fixative was quenched in 10 mm glycine in pbs (pbs/gly) and cells were permeabilized for 3 min in 0.5% tx-100 in pbs/gly. cells were washed in pbs/gly and co-stained with primary antibodies diluted in 1% bovine serum albumin (bsa) in pbs/gly as follows: mouse anti-sars-cov s (1:100) and rabbit anti-golgin 160 (1:500), mouse anti-sars-cov s (1:100) and rabbit anti-sars-cov s (1:400), or mouse anti-sars-cov s (1:100) and rabbit anti-sars-cov m (1:400). cells were washed in pbs/gly and co-stained for 15 min with secondary antibodies as follows: alexa 488 donkey anti-mouse (1:500) and texas red donkey anti-rabbit (1:400). cells were washed in pbs/gly and mounted in 0.1 m n-propylgallate in glycerol. images were obtained with an axioscop microscope (zeiss, thornwood nj) equipped for epifluorescence using a sensys charge-coupled device camera (photometric, tucson, az) and ip lab software (scanalytics, vienna, va). cells were seeded onto dishes treated with 1 mg/ml poly-l-lysine mol wt n300,000 (sigma, st. louis, mo) to improve cell adherence during processing. at 24 h post-transfection, hek293t cells were washed with pbs and biotinylated in 1 mg/ml sulfo-nhs-lc-biotin (pierce/thermoscientific, rockford, il) in pbs for 30 min at 0°c. after washing in pbs, the biotinylation reaction was quenched for 3 min with pbs containing 50 mm glycine. cells were lysed in lysis buffer (10 mm hepes [ph 7.2], 0.2% np-40, 150 mm nacl) containing protease inhibitor cocktail at 0°c for 10 min. lysates were clarified for 10 min at 16,000 × g at 4°c. 10% of the sample was reserved for quantification of total s protein. biotinylated proteins were isolated overnight at 4°c using washed streptavidin agarose resin (pierce/ thermoscientific, rockford, il). streptavidin beads were washed in lysis buffer and biotinylated proteins were eluted in 1x laemmli sample buffer for 3 min at 100°c. samples were subjected to 8% sds-page then transferred to polyvinylidene di-flouride membrane (pvdf), (millipore, bedford,ma) for western blotting. pvdf membranes were blocked for 30 min in 5% non-fat dry milk in tris buffered saline with tween (tbst, 150 mm nacl, 10 mm tris-hcl [ph 7.4], 0.05% tween-20). membranes were incubated overnight at 4°c with rabbit anti-sars-cov s polyclonal antibody diluted 1:5,000 in 5% non-fat dry milk made in tbst. membranes were washed in tbst and then incubated at room temperature for 1 h with hrp-conjugated anti-rabbit igg diluted 1:10,000 in 5% non-fat dry milk made in tbst. membranes were washed in tbst then treated with hyglo chemiluminescence reagent (denville scientific, metuchen, nj) as per the manufacturer's instructions. membranes were analyzed using a versa doc imaging station (biorad, hercules, ca) and quantified using quantity one software (biorad). detergent-resistant membranes (drms) were isolated by using discontinuous density ultracentrifugation. at 24 h post-transfection, hek293t cells were washed with cold pbs and lysed in 1 ml cold 1% triton x-100 in tne (10 mm tris-hcl [ph 7.4], 150 mm nacl, 5 mm edta) for 1 h at 0°c, with occasional mixing. ten percent of the lysate was reserved for quantification of total s protein. all of the following steps were performed at 0°c: 1 ml of the lysate was mixed with 1 ml of 85% sucrose in tne and placed in a pre-cooled sw41 ultracentrifuge tube (beckman, palo alto, ca), 4 ml of 35% sucrose in tne was overlaid on the lysate containing 85% sucrose, 1 ml of 5% sucrose in tne was overlaid on the 35% sucrose and 5 ml of tne was overlaid on the 5% sucrose. samples were centrifuged for 18 h at 285,000×g at 4°c. after ultracentrifugation, drms were visible floating near the 5%/35% sucrose interface. after removal of 3.5 ml of tne from the top of the gradient, 600-μl fractions were collected. to identify fractions containing drms, 3 μl of each fraction was dot blotted onto nitrocellulose membrane and dried. after blocking for 30 min in 5% non-fat dry milk in tbst, the membrane was washed in tbst and incubated for 1 h with hrpcholera toxin b (1:25,000) (molecular probes/invitrogen) in 3% bsa in tbst. to identify fractions containing sars-cov s protein, 10% of each fraction was subjected to 8% sds-page and western blotting after the addition of concentrated laemmli sample buffer to 1×. membranes were treated with hyglo chemiluminescence reagent as per the manufacturer's instructions and analyzed using a versa doc imaging station. at 48 h post-transfection, vero cells were washed with pbs, briefly trypsinized with 0.02% trypsin (invitrogen/gibco, grand island, ny) for 3 min at room temperature and 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glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection palmitoylation and polymerization of hepatitis c virus ns4b protein this work was supported by national institutes of health grant r21 a1072312 (to c.e. machamer) and a ford foundation pre-doctoral diversity fellowship (to c.e. mcbride).monoclonal anti-sars-cov s protein (similar to 341c), nr-617 was obtained through the nih biodefense and emerging infections research resources repository, niaid, nih.we also thank the machamer lab for helpful discussion and comments on the manuscript. key: cord-255815-5d9bqji0 authors: malik, ajamaluddin; alsenaidy, mohammad a. title: mers‐cov papain-like protease (pl(pro)): expression, purification, and spectroscopic/thermodynamic characterization date: 2017-05-30 journal: 3 biotech doi: 10.1007/s13205-017-0744-3 sha: doc_id: 255815 cord_uid: 5d9bqji0 within a decade, mers-cov emerged with nearly four times higher case fatality rate than an earlier outbreak of sars-cov and spread out in 27 countries in short span of time. as an emerging virus, combating it requires an in-depth understanding of its molecular machinery. therefore, conformational characterization studies of coronavirus proteins are necessary to advance our knowledge of the matter for the development of antiviral therapies. in this study, mers-cov papain-like protease (pl(pro)) was recombinantly expressed and purified. thermal folding pathway and thermodynamic properties were characterized using dynamic multimode spectroscopy (dms) and thermal shift assay. dms study showed that the pl(pro) undergoes a single thermal transition and follows a pathway of two-state folding with t (m) and van’t hoff enthalpy values of 54.4 ± 0.1 °c and 317.1 ± 3.9 kj/mol, respectively. an orthogonal technique based on intrinsic tryptophan fluorescence also showed that mers-cov pl(pro) undergoes a single thermal transition and unfolds via a pathway of two-state folding with a t (m) value of 51.4 °c. our findings provide significant understandings of the thermodynamic and structural properties of mers-cov pl(pro). frequent fatal coronavirus outbreaks in humans and animals have caused serious concerns in the healthcare sector, scientific community, and animal husbandry. first human outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in 2002 caused life-threatening atypical pneumonia in more than 8000 people in 26 countries with a case fatality rate (cfr) of 10% (pillaiyar et al. 2015; al-tawfiq et al. 2014; who 2016b) . another lethal coronavirus outbreak emerged in the arab peninsula countries in 2012 which was caused by what is now known as the middle east respiratory syndrome coronavirus (mers-cov) . from september 2012 to december 2016, 1864 laboratory-confirmed mers-cov cases of infection in 27 countries with 659 mortalities (nearly four times higher cfr than sars-cov) have been reported (al-tawfiq et al. 2016; who 2016a) . coronavirus survivors after acute infections suffer from many health issues and require long-term medical assistance (han et al. 2003; ong et al. 2005; chan et al. 2003; leow et al. 2005; siu 2016; cha et al. 2016) . in addition to sars-cov and mers-cov, at least four other pathological coronaviruses (hcov-oc43, hcov-229e, hcov-hku1, and hcov-nl63) are continuously circulating in humans causing relatively mild respiratory conditions that may in some instances escalate to severe pathological illnesses (mackay et al. 2012; carbajo-lozoya et al. 2012; simon et al. 2007) . coronaviruses have also caused deadly diseases in animals, leading to huge economic losses in the animal husbandry sector (vlasova et al. 2014; lee and lee 2014; sun et al. 2016) . moreover, high mutation and recombination rates in coronaviruses allow them to cross species barriers and adapt to new hosts more easily (denison et al. 2011; lau and chan 2015) . viral proteases are essential for pathogenesis and virulence. like all coronaviruses, mers-cov contains two cysteine proteases (main protease and papain-like protease) which processes viral nonstructural polypeptides (kilianski et al. 2013; hilgenfeld 2014) . mers-cov main protease (m pro , also called the 3c-like protease, 3cl pro ) cleaves at eleven sites, while mers-cov papain-like protease (pl pro ) cuts at three sites on the nonstructural polypeptides and releases mature nonstructural proteins (hilgenfeld 2014) . thus, mers-cov proteases make up a suitable target for antiviral therapies. mers-cov open-reading frame 1 (orf1) encodes two large polyproteins (pp1a and pp1b). mers-cov pl pro domain is encoded on the pp1a proteins (residue 1484-1800) (yang et al. 2014; hilgenfeld 2014; kilianski et al. 2013) . like other coronaviruses, mers-cov pl pro contains a catalytic triad and exhibits similar proteolytic, deubiquitination, and isg15-linked isgylation properties (lin et al. 2014; chen et al. 2007; clementz et al. 2010; yang et al. 2014; zheng et al. 2008) . in this study, we expressed and purified mers-cov pl pro . thermal stability was studied by thermal shift assay using intrinsic fluorescence and dynamic multimode spectroscopy (dms). mers-cov pl pro was found to unfold via a single thermal transition and follows a pathway of two-state folding. this study will not only help in the understanding of the folding and stability of mers-cov pl pro but also could help shed some light on other deubiquitinating enzymes with the similar folding scaffold. the orf of mers-cov pl pro (1484-1800 polyprotein residues, genbank accession number nc_019843.2) was cloned into pet28a plasmid under t7 promoter as published before (lin et al. 2014) . the codon was optimized (genscript, usa) and cloned between ncoi and xhoi sites which was in frame of c-terminal his tag present on the vector. e. coli bl21 (de3) plyss was used for the expression of recombinant protein. low-molecular weight protein markers, prepacked ni-nta, and superdex 75 columns were from amersham biosciences (united kingdom). chicken egg lysozyme was from usb corporation, usa. benzonase, ans, and kanamycin from sigma. iptg was purchased from bio basic, canada. all other chemicals used in this study were of reagent grade. cary 60 spectrometer and cary eclipse spectrofluorometer were from agilent technologies, usa. akta purification system was from amersham biosciences (united kingdom) and sds-page assembly from bio-rad (usa). thermomixer and benchtop cooling centrifuge were from eppendorf, germany. innova 44r shaking incubator was from new brunswick, germany. chirascan-plus spectropolarimeter was from applied photophysics, united kingdom. expression and purification of mers-cov pl pro in e. coli bl21 (de3) plyss e. coli bl21 (de3) plyss harboring pet28a-mpl pro was used for expression of mers-cov pl pro . protein expression and soluble protein extraction were performed as described in lin et al. (2014) . purification of mers-cov pl pro was performed with minor modification of an earlier published protocol (lin et al. 2014) . briefly, 1 mm dtt was used throughout unless described. cleared crude lysate was passed through a 1-ml ni-nta column pre-equilibrated with 20 mm tris, ph 8.5, 500 mm nacl, 10 mm imidazole, and 1 mm dtt and washed with 20 cv equilibration buffer. bound protein was eluted with a linear gradient of 0-50% buffer b (equilibration buffer containing 500 mm imidazole) at 1 ml/min flow rate on akta purification system. the purity of eluted fractions was analyzed on sds-page. the prepacked superdex 75 equilibrated with 20 mm tris, ph 8.5, 100 mm nacl, and 1 mm dtt was calibrated with proteins of known molecular weight. subsequently, ni-nta-purified mers-cov pl pro was further purified using superdex 75 column. the purity of eluted fractions was analyzed on sds-page. highly pure fractions were pooled, aliquoted, and stored at -80°c. before analysis, the frozen aliquots were thawed and centrifuged at 13,000 rpm for 15 min at 4°c. protein concentration was determined spectrophotometrically at 280 nm using a molar extinction coefficient of 42,400 m -1 cm -1 . tryptophan fluorescence spectra (50 lg/ml) of mers-cov pl pro were recorded using a cary eclipse fluorescence spectrophotometer in a 10-mm-path length cuvette. to measure tryptophan fluorescence, mers-cov pl pro sample was excited at 295 nm and emission spectra were collected between 305 and 400 nm (5 nm excitation and 5 nm emission bandwidth). temperature melting studies of mers-cov pl pro were performed at 10°c increments as well as a linear increase of 1°c/min. mers-cov pl pro incubated at different temperatures from 20 to 90°c in peltier-controlled cary eclipse fluorometer. the temperature of the protein samples was monitored using an internal temperature probe. when the desired temperature was reached, the sample was allowed to equilibrate for 2 min before tryptophan fluorescence spectra were measured. the maximum fluorescence intensity (i max ) and maximum fluorescence wavelength (k max ) were plotted with respect to temperature. in a similar experiment, mers-cov pl pro was gradually heated from 20 to 80°c at a rate of 1°c/min during which tryptophan fluorescence was measured by exciting at 295 nm and collecting at 330 and 350 nm to obtain the temperature melting curve. to study the secondary structure of mers-cov pl pro in terms of conformational and thermal stability, dynamic multimode spectroscopy was applied. the measurement was performed using chirascan-plus spectrophotometer, calibrated with (1s)-(?)-10-camphorsulfonic acid. in this study, 0.2 mg/ml of mers-cov pl pro was gradually heated from 20 to 94°c at 1°c/ml rate. internal thermal probe was inserted in the 0.1-cm-path length cuvette to precisely monitor the actual temperature of the samples. far-uv cd spectra from 200 to 250 nm were recorded at each temperature. thermal transition data were processed using manufacturer's global 3 software. the steady-state kinetics of mers-cov pl pro was measured as described in lin et al. (2014) , with slight modification. briefly, 50 lm fluorogenic peptidyl substrate, dabcyl-frlkggapikgv-edans (genscript), was mixed with different concentrations of mers-cov pl pro (6.8-0.1 lm, in twofold serial dilution) using 50 mm phosphate at ph 6.5 as a buffer at room temperature. the fluorescence signal was measured for 30 min at 60-s intervals in a hidex chameleon plate reader using 340 nm (excitation) and 535 nm (emission filter) with 20% gain. expression and purification of recombinant mers-cov pl pro in this study, mers-cov pl pro was overexpressed in e. coli bl21 (de3) plyss. mers-cov pl pro was purified in two-step chromatography as described in lin et al. (2014) . ni-nta elute contained minor impurities (data not shown). when ni-nta elute passed through gel filtration column, one major symmetrical sharp peak was obtained (fig. 1a) . sds-page analysis of the pooled fractions showed the yield of highly pure mers-cov pl pro (fig. 1b) . we obtained nearly 10 mg of mers-cov pl pro from a 1-l shake flask culture. in an earlier study, the yield of soluble mers-cov pl pro was strain dependent and the best yield (*52 mg purified protein from 1 l shake flask culture) was obtained with e. coli bl21 (de3) star strain (lin et al. 2014) . the difference in the yield of mers-cov pl pro was due to the different strain's genetic background. intrinsic fluorescence spectroscopy is a highly sensitive tool, which provides information about the microenvironment of trp and tyr residues within proteins. tyrosine emission maximum is less sensitive to its local environment compared to tryptophan. indole ring in the tryptophan residues undergoes two isoenergetic transitions which causes polarity sensitivity, while tyrosine undergoes through a single electronic state (ghisaidoobe and chung 2014) . during the course of protein unfolding, the polarity of fluorophore microenvironment changes, which in turn leads to changes in the maximum fluorescence intensity (i max ) as well as maximum fluorescence wavelength (k max ). therefore, intrinsic tryptophan fluorescence emission is sensitive to the tertiary structure and detects subtle protein conformational changes in solution. it has been frequently used for the characterization of protein's conformational changes under different stress conditions (kumar et al. 2005; xiao et al. 2015) . a previous study analyzing the quaternary structure of mers-cov pl pro using analytical ultracentrifugation technique has shown that pl pro is found in the monomeric state (lin et al. 2014) . mers-cov pl pro contains ten tyrosine and five tryptophan residues (fig. 2a) . mers-cov pl pro consists of two domains: n-terminal ubiquitinlike (ubl) domain and a catalytic core domain. the n-terminal ubl domain consists of 62 residues and contained one a-helix, one 3 10 -helix and five b-strands. the substrate-binding region is solvent exposed and comprised of the right-hand scaffold (lei et al. 2014) . three tryptophan residues (w93, 243 and 303) are buried, while w187 and w190 are surface exposed (fig. 2b) . figure 3a shows the decrease in intrinsic fluorescence of mers-cov pl pro with increasing temperature. at low temperature (20°c), highest fluorescent intensity (i max ) was observed with maximum fluorescence wavelength (k max ) at 343 nm, indicating an overall localization of all five tryptophans in the partially hydrophobic environment. as the temperature increases gradually from 20 to 90°c, i max decreased with major transitions occurring between 50 and 60°c (fig. 3a, b) . initially, k max increased from 343 to 344 nm when the temperature was increased from 20 to 30°c. as the temperature was increased further, a blue-shift in k max with increasing temperature was observed, indicating conformational rearrangements in different temperature regimes. the major blue-shift in k max was found between 50 and 60°c (fig. 3c) . the 14-nm blue-shift of tryptophan fluorescence spectrum indicated that the microenvironment of the tryptophan residues is becoming more hydrophobic during thermal denaturation. mers-covpl pro thermal unfolding is different from most other proteins. commonly, protein unfolding fluorescence spectra are characterized by a long wavelength shift ''red-shift.'' but some proteins, fig. 2 a sequence of c-terminal his-tagged mers-cov pl pro showing ten tyr and five trp residues, which are highlighted in green and blue, respectively. potential internal protease site is highlighted in red and his-tagged residues are underlined. b three-dimensional structure of mers-cov pl pro (pdb id: 4r3d) with the side chains of the five trp residues being numbered and colored in blue including mers-cov pl pro , exhibit blue-shift upon denaturation (slutskaya et al. 2015; duy and fitter 2006; pattanaik et al. 2003) . pig pancreatic a-amylase showed red-shifted fluorescence spectra when chemically unfolded and showed blue-shifted spectra during thermal unfolding (duy and fitter 2006) . equine lysozyme first exhibits a blue-shift transition at lower temperature and red-shift above 50°c (morozova et al. 1991) . to obtain temperature melting curve, mers-covpl pro was gradually heated from 20 to 80°c at 1°c/min and the ratio of 330/350 nm tryptophan fluorescence was plotted with respect to temperature (fig. 4) . data were fitted according to the equation f = y 0 ? a/(1 ? exp(-(xx 0 )/ b)) with an r 2 value of 0.9910. our results showed that mers-cov pl pro was moderately stable and unfolds via a single transition with a t m value of 51.4°c. since mers-cov pl pro is a protease, it may undergo autolysis during thermal shift assay. it contains one potential autolysis site (lkgg) as shown in fig. 2a . to evaluate the extent of autolysis as well as the extent of irreversible thermal unfolding and aggregation, mers-cov pl pro (0.2 mg/ml) was incubated on a thermomixer from 20 to 70°c with 10°c intervals. six samples were gradually heated and equilibrated at the respective temperatures for 3 min. when the desired temperatures were attained, the samples were removed, kept on ice, and centrifuged at 13,000 rpm for 15 min to remove any forming aggregates. equal volumes of the supernatant were analyzed (fig. 5) . if mers-cov pl pro would undergo autolysis during the course of thermal incubation, we would expect to see the appearance of two or more bands of mers-cov pl pro fragments on sds-page gels or at least a decrease in the band intensity of mers-cov pl pro . we found that the intensity of mers-cov pl pro band was apparently unchanged, indicating that autolysis was not occurring during the thermal shift assays applied in this study. in another scenario, irreversible unfolding aggregates may form during thermal shift assay. if this is the case, then aggregated protein will be pelleted down upon centrifugation and band intensity would have decreased in the supernatant sample. our result showed that the band intensity of the supernatant samples incubated from 20 to 70°c was apparently unchanged (fig. 5) , indicating fig. 3 thermally induced structural changes in mers-cov pl pro as monitored by the intrinsic tryptophan fluorescence spectroscopy. a to monitor tryptophan fluorescence at different temperatures, mers-cov pl pro was slowly heated and allowed to equilibrate for 2 min at the respective temperatures. the sample was excited at 295 nm and the emission spectra were collected from 305 to 400 nm (5 nm excitation and 5 nm emission bandwidth). b effect of temperature on the tryptophan emission intensity showing the decrease of i max with increasing temperature. major transition occurred between 50 and 60°c. c effect of temperature on the tryptophan maximum emission wavelength (k max ). blue-shift was observed during thermal unfolding of mers-cov pl pro . initially, red-shift was observed when the temperature was increased from 20 to 30°c. further increase in temperature leads to blue-shift with major changes occurring between 50 and 60°c no or insignificant aggregation occurring during thermal shift assays. information about thermal stability, unfolding pathway, and secondary structure of mers-cov pl pro was obtained using an orthogonal method. we employed dms, a newly developed information-rich experimental technique, to obtain spectroscopic and thermodynamic data of melting temperature (t m ) and van't hoff enthalpy (dh vh ) (john and weeks 2000; greenfield 2006; al-ahmady et al. 2012) . temperature-induced secondary structural changes in mers-cov pl pro in the far-uv region were monitored to study thermodynamic parameters. moreover, dms also characterized thermal unfolding pathway and identified the number of folding intermediate species and their relative concentrations during the unfolding process (malik et al. 2015 (malik et al. , 2016 . mers-cov pl pro was gradually heated from 20 to 94°c at 1°c/min increments and far-uv cd spectra were recorded from 200 to 250 nm. far-uv cd spectra at several wavelengths were plotted as a function of temperature (fig. 6a) . mers-cov pl pro underwent a single thermal transition as a function of temperature, suggesting two-state folding. similar transition was also observed in the thermal shift assay using fluorescence spectroscopy. in an earlier study, secondary structure content was calculated and it was found that b-sheet structure (31%) is dominant in mers-cov pl pro (lin et al. 2014) . similarly, our results showed that mers-cov pl pro presented a single negative minimum at *218 nm suggesting a predominant b-sheet structure as well. when mers-cov pl pro was gradually heated, secondary structure was lost and became irregularly disorder structure (fig. 6b) . the relative concentrations of folded and unfolded species as a function of temperature are shown in fig. 6c . the thermal melting point (t m ) and van't hoff enthalpy (dh vh ) of mers-cov pl pro calculated using global 3 analysis software were 54.4 ± 0.1°c and 317.1 ± 3.9 kj/mol, respectively. the thermal melting points (t m ) of mers-cov pl pro calculated by thermal shift assay and dms were found to be in close proximity, reflecting tertiary-secondary structure unfolding events, respectively. in a recent study, papainlike protease of murine coronavirus also underwent a single thermal transition with a t m value of *46°c (mielech et al. 2015) . a three-dimensional model of the thermal transitions of mers-cov pl pro was generated using global 3 analysis software (fig. 6d) . a single transition is clearly evident at lower wavelengths in the far-uv spectra region. mers-cov pl pro was expressed in soluble state in e. coli and purified to homogeneity in two-step chromatography. two orthogonal techniques were used for studying unfolding pathway that include the utilization of dms and thermal shift assay. the results showed that mers-cov pl pro unfolds via a single thermal transition and follows a two-state unfolding pathway. thermal shift assay calculated a t m value of 51.4°c and dms method calculated a t m value of 54.4 ± 0.1°c, in agreement with sequential unfolding events of tertiary and secondary structures, respectively. similar folding behavior and thermal melting point were also observed in papain-like protease of murine lipid-peptide vesicle nanoscale hybrids for triggered drug release by mild hyperthermia in vitro and in vivo travel implications of emerging coronaviruses: sars and mers-cov middle east respiratory syndrome coronavirus: current situation and travelassociated concerns replication of human coronaviruses sars-cov, hcov-nl63 and hcov-229e is inhibited by the drug fk506 a case report of a middle east respiratory syndrome survivor with 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acute respiratory syndrome heat-induced unfolding of apo-cp43 studied by fluorescence spectroscopy and cd spectroscopy proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production acknowledgements the authors extend their appreciation to the deanship of scientific research at king saud university for funding this work through the research project no r5-16-02-05. conflict of interest to the best of our knowledge, no conflict of interest, financial or others, exists. all authors are fully aware of this submission. key: cord-277487-jgbjxgh1 authors: graham, simon p.; mclean, rebecca k.; spencer, alexandra j.; belij-rammerstorfer, sandra; wright, daniel; ulaszewska, marta; edwards, jane c.; hayes, jack w. p.; martini, veronica; thakur, nazia; conceicao, carina; dietrich, isabelle; shelton, holly; waters, ryan; ludi, anna; wilsden, ginette; browning, clare; bialy, dagmara; bhat, sushant; stevenson-leggett, phoebe; hollinghurst, philippa; gilbride, ciaran; pulido, david; moffat, katy; sharpe, hannah; allen, elizabeth; mioulet, valerie; chiu, chris; newman, joseph; asfor, amin s.; burman, alison; crossley, sylvia; huo, jiandong; owens, raymond j.; carroll, miles; hammond, john a.; tchilian, elma; bailey, dalan; charleston, bryan; gilbert, sarah c.; tuthill, tobias j.; lambe, teresa title: evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored covid-19 vaccine candidate chadox1 ncov-19 date: 2020-06-20 journal: biorxiv doi: 10.1101/2020.06.20.159715 sha: doc_id: 277487 cord_uid: jgbjxgh1 clinical development of the covid-19 vaccine candidate chadox1 ncov-19, a replication-deficient simian adenoviral vector expressing the full-length sars-cov-2 spike (s) protein was initiated in april 2020 following non-human primate studies using a single immunisation. here, we compared the immunogenicity of one or two doses of chadox1 ncov-19 in both mice and pigs. whilst a single dose induced antigen-specific antibody and t cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in sars-cov-2 neutralising titres. as sars-cov-2 began to spread around the world at the beginning of 2020 several vaccine platform technologies were employed to generate candidate vaccines. several use replicationdeficient adenoviral (ad) vector technology and express the sars-cov-2 spike (s) protein. the first phase i clinical study of an ad5-vectored vaccine has been reported 1 , chadox1 ncov-19 (azd1222) phase i trials (nct04324606) began in april 2020 with phase ii and iii trials (nct04400838) started soon thereafter, and an ad26-vectored vaccine is expected to enter phase i shortly. typically, only one dose of ad-vectored vaccines has been administered in early preclinical challenge studies or clinical studies against emerging or outbreak pathogens 2-5 . rhesus macaques immunised with a single dose of chadox1 ncov-19 were protected against pneumonia but there was no impact on nasal virus titers after high dose challenge to both the upper and lower respiratory tract 6 . to increase antibody titres and longevity of immune responses, a booster vaccination may be administered. homologous prime-boost immunisation resulted in higher antibody titres including neutralising antibodies and a trend towards a lower clinical score in a mers-cov challenge study 7 . here, we set out to test the immunogenicity of either one or two doses of chadox1 ncov-19 in mice and pigs, to further inform clinical development. 'prime-boost' vaccinated inbred (balb/c) and outbred (cd1) mice were immunised on 0 and 28 days post-vaccination (dpv), whereas, 'prime-only' mice received a single dose of chadox1 ncov-19 on day 28. spleens and serum were harvested from all mice on day 49 (3 weeks after boost or prime vaccination). analysis of sars-cov-2 s protein-specific murine splenocyte responses by ifnγ elispot assay showed no statistically significant difference between the prime-only and primeboost vaccination regimens, in either strain of mouse ( figure 1a ). intracellular cytokine staining (ics) of splenocytes ( figure 1b) showed, in both mouse strains, that the response was principally driven by cd8 + t cells. the predominant cytokine response of both cd8 + and cd4 + t cells was expression of ifn-γ and tnf-α, with negligible frequencies of il-4 + and il-10 + cells, consistent with previous data suggesting adenoviral vaccination does not induce a dominant th2 response 8, 9 . there were no signficant differences in cd4 + and cd8 + t cell cytokine responses between prime-only and primeboost mice. prime-only and prime-boost pigs were immunised on 0 dpv and prime-boost pigs received a second immunisation on 28 dpv. blood samples were collected weekly until 42 dpv to analyse immune responses. ifn-γ elispot analysis of porcine peripheral blood mononuclear cells (pbmc) showed responses on 42 dpv (2 weeks after boost) that were significantly greater in the prime-boost pigs compared to prime-only animals (p < 0.05; figure 1c ). the prime-boost 42 dpv responses were greater than responses observed in either group on 14 dpv, but inter-animal variation meant this did not achieve statistical significance. ics analysis of porcine t cell reponses showed a dominance of th1-type cytokines (similar to the murine response) but with a higher frequency of s-specific cd4 + t cells compared to cd8 + t cells ( figure 1d ). however, cd4 + and cd8 + t cell cytokine responses did not differ significantly between vaccine groups or timepoints (14 vs. 42 dpv). sars-cov-2 s protein-specific antibody titres in serum were determined by elisa using recombinant soluble trimeric s (fl-s) and receptor binding domain (rbd) proteins. a significant increase in fl-s binding antibody titres was observed in prime-boost balb/c mice compared to their prime-only counterparts (p < 0.01), however, the difference between vaccine groups for cd1 mice was not significant (figure 2a ). antibody responses were evaluated longitudinally in pig sera by fl-s and rbd elisa. compared to pre-vaccination sera, significant fl-s specific antibody titres were detected in both prime-only and prime-boost groups from 21 and 14 dpv, respectively (p < 0.01; figure 2b ). fl-s antibody titres did not differ signifcantly between groups until after the boost, when titres in the prime-boost pigs became significantly greater with an average increase in titres of > 1 log10 (p < 0.0001). rbd-specific antibody titres showed a similar profile with significant titres in both groups from 14 dpv (p < 0.05) and a further significant increase in the prime-boost pigs from 35 dpv onwards which was greater than the prime-only pigs (p < 0.0001; figure 2c ). sars-cov-2 neutralising antibody responses were assessed using a virus neutralisation test (vnt; figure 2d ) and pseudovirus-based neutralisation test (pvnt; figure 2e ). after the prime immunisation, sars-cov-2 neutralising antibody titres were detected by vnt in 14 and 28 dpv sera from 2/3 prime-boost and 2/3 prime-only pigs. two weeks after the boost (42 dpv), neutralising antibody titres were detected and had increased in all prime-boost pigs, which were significantly greater than the earlier timepoints and the titres measured in the prime-only group (p < 0.01). in agreement with this analysis, serum assayed for neutralising antibodies using the pvnt revealed that antibody titres in 42 dpv prime-boost pig sera were significantly greater than earlier timepoints and the prime-only group (p < 0.001). statistical analysis showed a highly significant correlation between pvnt and vnt titres (spearman's rank correlation r = 0.86; p < 0.0001). in this study, we utilised both a small and a large animal model to evaluate the immunogenicity of either one or two doses of a covid-19 vaccine candidate, chadox1 ncov-19 (now known as azd1222). small animal models have variable success in predicting vaccine efficacy in larger animals but are an important stepping stone to facilitate prioritisation of vaccine targets. in contrast, larger animal models, such as the pig and non-human primates, have been shown to more accurately predict vaccine outcome in humans [10] [11] [12] . the mouse data generated in this study suggested that the immunogenicity profile was at the upper end of a dose response curve, which may have saturated the immune response and largely obscured our ability to determine differences between prime-only or prime-boost regimens. we have developed the pig as a model for generating and understanding immune responses to vaccination against human influenza [13] [14] [15] and nipah virus 16, 17 .the inherent heterogeneity of an outbred large animal model is more representative of immune responses in humans. extensive development of reagents to study immune responses in pigs in recent years has extended the usefulness and applicability of the pig as a model to study infectious disease. these data demonstrate the utility of the pig as a model for further evaluation of the immunogenicity of chadox1 ncov-19 and other covid-19 vaccines. we show here that t cell responses are higher in pigs that received a prime-boost vaccination when compared to prime only at day 42, whilst comparing responses 14 days after last immunisation demonstrates the prime-boost regimen trended toward a higher response. in addition, chadox1 ncov-19 immunisation induced robust th1-like cd4 + and cd8 + t cell responses in both pigs and mice. this has important implications for covid-19 vaccine development as virus-specific t cells are thought to play an important role in sars-cov-2 infection [18] [19] [20] [21] [22] . while no correlate of protection has been defined for covid-19, recent publications suggest that neutralising antibody titres may be correlated with protection in animal challenge models 23, 24 . a single dose of chadox1 ncov-19 induces antibody responses, but we demonstrate here that antibody responses are significantly enhanced after homologous boost in one mouse strain and to a greater extent in pigs. however, it is likely that a combination of neutralising antibodies and antigen-specific t cells would act in synergy to prevent and control infection, as we have recently shown in the context of influenza vaccination 13, 25 . whilst human immunogenicity and clinical read-outs are a critically meaningful endpoint, studies in small animals and pigs will help prioritise candidates to be tested in humans. further clinical studies are needed to assess immunogenicity after prime-boost vaccination and the impact on clinical efficacy and durability of the immune response. mouse and pig studies were performed in accordance with the uk animals (scientific procedures) act 1986 and with approval from the relevant local animal welfare and ethical review body (mice -project license p9808b4f1, and pigs -project license pp1804248). the principles of the 3r's were applied for the duration of the study to ensure animal welfare was not unnecessarily compromised. vero e6 cells were grown in dmem containing sodium pyruvate and l-glutamine (sigma-aldrich, poole, uk), 10% fbs (gibco, thermo fisher, loughborough, uk), 0.2% penicillin/streptomycin (10,000 u/ml; gibco) (maintenance media) at 37 °c and 5% co2. sars-cov-2 isolate england-2 stocks were grown in vero e6 cells using a multiplicity of infection (moi) of 0.0001 for 3 days at 37 °c in propagation media (maintenance media containing 2% fbs). sars-cov-2 stocks were titrated on vero e6 cells using mem (gibco), 2% fcs (labtech, heathfield, uk), 0.8% avicel (fmc biopolymer, girvan, uk) as overlay. plaque assays were fixed using formaldehyde (vwr, leighton buzzard, uk) and stained using 0.1% toluidine blue (sigma-aldrich). all work with live sars-cov-2 virus was performed in acdp hg3 laboratories by trained personnel. the propagation, purification and assessment of chadox1 ncov-19 titres were as described previously 7 . a synthetic dna, encoding the spike (s) protein receptor binding domain (rbd; amino acids 330-532) of sars-cov-2 (genbank mn908947), codon optimised for expression in mammalian cells (idt technology) was inserted into the vector popinttgneo incorporating a c-terminal his6 tag. recombinant rbd was transiently expressed in expi293™ (thermo fisher scientific, uk) and protein purified from culture supernatants by immobilised metal affinity followed by a gel filtration in phosphate-buffered saline (pbs) ph 7.4 buffer. a soluble trimeric s (fl-s) protein construct encoding residues 1-1213 with two sets of mutations that stabilise the protein in a pre-fusion conformation (removal of a furin cleavage site and the introduction of two proline residues; k983p, v984p) was expressed as described 26 . the endogenous viral signal peptide was retained at the nterminus (residues 1-14), a c-terminal t4-foldon domain incorporated to promote association of monomers into trimers to reflect the native transmembrane viral protein, and a c-terminal his6 tag included for nickel-based affinity purification. similar to recombinant rbd, fl-s was transiently expressed in expi293™ (thermo fisher scientific) and protein purified from culture supernatants by immobilised metal affinity followed by gel filtration in tris-buffered saline (tbs) ph 7.4 buffer. for analysis of t cell responses in pigs, overlapping 16mer peptides offset by 4 residues based on the predicted amino acid sequence of the entire s protein from sars-cov-2 wuhan-hu-1 isolate (ncbi reference sequence: nc_045512.2) were designed and synthesised (mimotopes, melbourne, australia) and reconstituted in sterile 40% acetonitrile (sigma-aldrich) at a concentration of 3 mg/ml. three pools of synthetic peptides representing residues 1-331 (pool 1), 332-748 (pool 2) and 749-1273 (pool 3) were prepared for use to stimulate t cells in ifn-γ elispot and intracellular cytokine staining (ics) assays. for analysis of t cell responses in mice, overlapping 15mer peptides offset by 11 residues were designed and synthesised (mimotopes) and reconstituted in sterile 100% dmso (sigma-aldrich) at a concentration of 100 mg/ml. two peptide pools spanning s1 region (pool 1: 1 to 77 and 317-321, pool 2:78-167) and 2 peptide pools spanning s2 region (pool 3:166 to 241, pool 4:242 to 316) were used for stimulating splenocytes for ifn-γ elispot analysis, and single pools of s1 (pool 1 and pool 2) and s2 (pool 3 and pool 4) were used to stimulate splenocytes for ics. mice: inbred female balb/colahsd (balb/c) (envigo) and outbred crl:cd1 (cd1) (charles river) of at least 6 weeks of age were randomly allocated into 'prime-only' or 'prime-boost' vaccination groups (balb/c n=5 and cd1 n=8). prime-boost mice were immunised intramuscularly with 10 8 infectious units (iu) (6.02x10 9 virus particles; vp) chadox1 ncov-19 and boosted intramuscularly four weeks later with 1 × 10 8 iu chadox1 ncov-19. prime-only mice received a single dose of 10 8 iu chadox1 ncov-19 at the same time prime-boost mice were boosted. spleens and serum were harvested from all animals a further 3 weeks later. pigs: six 8-10-week-old, weaned, female, large white-landrace-hampshire cross-bred pigs from a commercial rearing unit were randomly allocated to two treatment groups (n = 3): 'prime-only' and 'prime-boost'. both groups were immunised on day 0 with 1 × 10 9 iu (5.12 × 10 10 vp) chadox1 ncov-19 in 1 ml pbs by intramuscular injection (brachiocephalic muscle). 'prime-boost' pigs received an identical booster immunisation on day 28. blood samples were taken from all pigs on a weekly basis at 0, 7, 14, 21, 28, 35 and 42 dpv by venepuncture of the external jugular vein: 8 ml/pig in bd sst vacutainer tubes (fisher scientific) for serum collection and 40 ml/pig in bd heparin vacutainer tubes (fisher scientific) for peripheral blood mononuclear cell (pbmc) isolation. mice: antibodies to sars-cov-2 fl-s protein were determined by performing a standardised elisa on serum collected 3-weeks after prime or prime-boost vaccination. maxisorp plates (nunc) were coated with 100 ng/well fl-s protein overnight at 4°c, prior to washing in pbs/tween (0.05% v/v) and blocking with blocker casein in pbs (thermo fisher scientific) for 1 hour at room temperature (rt). standard positive serum (pool of mouse serum with high endpoint titre against fl-s protein), individual mouse serum samples, negative and an internal control (diluted in casein) were incubated for 2 hours at rt. following washing, bound antibodies were detected by addition of alkaline phosphatase-conjugated goat anti-mouse igg (sigma-aldrich), diluted 1/5000 in casein, for 1 hour at rt and detection of anti-mouse igg by the addition of pnpp substrate (sigma-aldrich). an arbitrary number of elisa units were assigned to the reference pool and od values of each dilution were fitted to a 4-parameter logistic curve using softmax pro software. elisa units were calculated for each sample using the od values of the sample and the parameters of the standard curve. pigs: serum was isolated by centrifugation of sst tubes at 1300 × g for 10 minutes at rt and stored at -80°c. sars-cov-2 rbd and fl-s specific antibodies in serum were assessed as detailed previously 26 with the exception of the following two steps. the conjugated secondary antibody was replaced with goat anti-porcine igg hrp (abcam, cambridge, uk) at 1/10,000 dilution in pbs with 0.1% tween20 and 1% non-fat milk. in addition, after the last wash, a 100 µl of tmb (one component horse radish peroxidase microwell substrate, biofx, cambridge bioscience, cambridge, uk) was added to each well and the plates were incubated for 7 minutes at rt. a 100 µl of biofx 450nmstop reagent (cambridge bioscience) was then added to stop the reaction and microplates were read at 450nm. end-point antibody titres (mean of duplicates) were calculated as follows: the log10 od was plotted against the log10 sample dilution and a regression analysis of the linear part of this curve allowed calculation of the endpoint titre with an od of twice the average od values of 0 dpv sera. the ability of pig sera to neutralise sars-cov-2 was evaluated using virus and pseudovirus neutralisation assays. for both assays, sera were first heat-inactivated (hi) by incubation at 56 °c for 2 hours. virus neutralization test (vnt): starting at a 1 in 5 dilution, two-fold serial dilutions of sera were prepared in 96 well round-bottom plates using dmem containing 1% fbs and 1% antibiotic-antimycotic (gibco) (dilution media). 75 μl of diluted pig serum was mixed with 75 μl dilution media containing approximately 64 plaque-forming units (pfu) sars-cov-2 for 1 hour at 37 °c. vero e6 cells were seeded in 96-well flat-bottom plates at a density of 1 × 10 5 cells/ml in maintenance media one day prior to experimentation. culture supernatants were replaced by 100 µl of dmem containing 10% fcs and 1% antibiotic-antimycotic, before 100 µl of the virus-sera mixture was added to the vero e6 cells and incubated for six days at 37 °c. cytopathic effect (cpe) was investigated by brightfield microscopy. cells were further fixed and stained as described above, and cpe scored. each individual pig serum dilution was tested in quadruplet on the same plate and no sera/sars-cov-2 virus and no sera/no virus controls were run concurrently on each plate in quadruplet. wells were scored for cytopathic effect and neutralisation titres expressed as the reciprocal of the serum dilution that completely blocked cpe in 50% of the wells (nd50). researchers performing the vnts were blinded to the identity of the samples. pseudovirus neutralisation test (pvnt): lentiviral-based sars-cov-2 pseudoviruses were generated in hek293t cells incubated at 37 °c, 5% co2. cells were seeded at a density of 7.5 x 10 5 in 6 well dishes, before being transfected with plasmids as follows: 500 ng of sars-cov-2 spike, 600 ng p8.91 (encoding for hiv-1 gag-pol), 600 ng csflw (lentivirus backbone expressing a firefly luciferase reporter gene), in opti-mem (gibco) along with 10 µl pei (1 µg/ml) transfection reagent. a 'no glycoprotein' control was also set up using carrier dna (pcdna3.1) instead of the sars-cov-2 s expression plasmid. the following day, the transfection mix was replaced with 3 ml dmem with 10% fbs (dmem-10%) and incubated at 37 °c. at both 48 and 72 hours post transfection, supernatants containing pseudotyped sars-cov-2 (sars-cov-2 pps) were harvested, pooled and centrifuged at 1,300 x g for 10 minutes at 4 °c to remove cellular debris. target hek293t cells, previously transfected with 500 ng of a human ace2 expression plasmid (addgene, cambridge, ma, usa) were seeded at a density of 2 × 10 4 in 100 µl dmem-10% in a white flat-bottomed 96-well plate one day prior to harvesting of sars-cov-2 pps. the following day, sars-cov-2 pps were titrated 10-fold on target cells, with the remainder stored at -80 °c. for pvnts, pig sera were diluted 1:20 in serum-free media and 50 µl was added to a 96-well plate in quadruplicate and titrated 4fold. a fixed titred volume of sars-cov-2 pps was added at a dilution equivalent to 10 6 signal luciferase units in 50 µl dmem-10% and incubated with sera for 1 hour at 37 °c, 5% co2. target cells expressing human ace2 were then added at a density of 2 x 10 4 in 100 µl and incubated at 37 °c, 5% co2 for 72 hours. firefly luciferase activity was then measured with brightglo luciferase reagent and a glomax-multi + detection system (promega, southampton, uk). pseudovirus neutralization titres were expressed as the reciprocal of the serum dilution that inhibited luciferase expression by 50% (ic50). mice: single cell suspension of mouse spleens were prepared by passing cells through 70 μm cell strainers and ack lysis (thermo fisher) prior to resuspension in complete media (mem supplemented with 10% fcs, pen-step, l-glut and 2-mercaptoethanol). for analysis of ifn-γ production by elispot assay, splenocytes were stimulated with s peptide pools at a final concentration of 2g/ml on ipvh-membrane plates (millipore) coated with 5g/ml anti-mouse ifn-γ (clone an18; mabtech). after 18-20 hours of stimulation, ifn-γ spot forming cells (sfc) were detected by staining membranes with anti-mouse ifn-γ biotin mab (1 µg/ml; clone r46a2, mabtech) followed by streptavidin-alkaline phosphatase (1 µg/ml) and development with ap conjugate substrate kit (bio-rad). for analysis of intracellular cytokine production, cells were stimulated with 2 μg/ml s peptide pools, media or cell stimulation cocktail (containing pma-ionomycin, biolegend), together with 1 μg/ml golgiplug (bd biosciences) and 2 μl/ml cd107a-alexa647 for 6 hours in a 96-well u-bottom plate, prior to placing at 4 o c overnight. following surface staining with cd4-buv496, cd8-percp-cy5.5, cd62l-bv711, cd127-bv650, cd44-apc-cy7 and live/dead aqua (thermo fisher), cells were fixed with 10% neutral buffered formalin (containing 4% paraformaldehyde) and stained intracellularly with tnf--af488, il-2-pe-cy7, il-4-bv605, il-10-pe and ifn-γ-e450 diluted in perm-wash buffer (bd biosciences). sample acquisition was performed on a fortessa (bd) and data analysed in flowjo v9 (treestar). an acquisition threshold was set at a minimum of 5000 events in the live cd3 + gate. antigen-specific t cells were identified by gating on live/dead negative, doublet negative (fsc-h vs fsc-a), size (fsc-h vs ssc), cd3 + , cd4 + or cd8 + cells and cytokine positive. total sars-cov-2 s specific cytokine responses are presented after subtraction of the background response detected in the media stimulated control spleen sample of each mouse, prior to summing together the frequency of s1 and s2 specific cells. pigs: pbmcs were isolated from heparinised blood by density gradient centrifugation and cryopreserved in cold 10% dmso (sigma-aldrich) in hi fbs 17 . resuscitated pbmc were suspended in rpmi 1640 medium, glutamax supplement, hepes (gibco) supplemented with 10 % hi fbs (new zealand origin, life science production, bedford, uk), 1% penicillin-streptomycin and 0.1% 2-mercaptoethanol (50 mm; gibco) (crpmi). to determine the frequency of sars-cov-2 s specific ifn-γ producing cells, an elispot assay was performed on pbmc from 0, 14, 28 and 42 dpv. multiscreen 96-well plates (mahas4510; millipore, fisher scientific) were pre-coated with 1 µg/ml anti-porcine ifn-γ mab (clone p2g10, bd biosciences) and incubated overnight at 4 °c. after washing and blocking with crpmi, pbmcs were plated at 5 × 10 5 cells/well in crpmi in a volume of 50 µl/well. pbmcs were stimulated in triplicate wells with the sars-cov-2 s peptide pools at a final concentration of 1 µg/ml/peptide. crpmi alone was used in triplicate wells as a negative control. after 18 hours incubation at 37 °c with 5% co2, plates were developed as described previously 17 . the numbers of specific ifn-γ secreting cells were determined using an immunospot ® s6 analyzer (cellular technology, cleveland, usa). for each animal, the mean 'crpmi only' data was subtracted from the s peptide pool 1, 2 and 3 data which were then summed and expressed as the mediumcorrected number of antigen-specific ifn-γ secreting cells per 1 x 10 6 pbmc. to assess intracellular cytokine expression pbmc from 14 and 42 dpv were suspended in crpmi at a density of 2 × 10 7 cells/ml and added to 50 µl/well to 96-well round bottom plates. pbmcs were stimulated in triplicate wells with the sars-cov-2 s peptide pools (1 µg/ml/peptide). unstimulated cells in triplicate wells were used as a negative control. after 14 hours incubation at 37 °c, 5% co2, cytokine secretion was blocked by addition 1:1,000 bd golgiplug (bd biosciences) and cells were further incubated for 6 hours. pbmc were washed in pbs and surface labelled with zombie nir fixable viability stain (biolegend), cd4-percp-cy5.5 mab (clone 74-12-4, bd bioscience) and cd8β-fitc mab (clone ppt23, bio-rad antibodies). following fixation (fixation buffer, biolegend) and permeabilization (permeabilization wash buffer, biolegend), cells were stained with: ifn-γ-af647 mab (clone cc302, bio-rad antibodies, kidlington, uk), tnf-α-bv421 mab (clone mab11, biolegend), il-2 mab (clone a150d 3f1 2h2, invitrogen, thermo fisher scientific) and il-4 bv605 mab (clone mp4-25d2, biolegend) followed by staining with anti-mouse igg2a-pe-cy7 (clone rmg2a-62, biolegend). cells were analysed using a bd lsrfortessa flow cytometer and flowjo x software. total sars-cov-2 s specific cytokine positive responses are presented after subtraction of the background response detected in the media stimulated control pbmc sample of each pig, prior to summing together the frequency of s-peptide pools 1-3 specific cells. graphpad prism 8.1.2 (graphpad software, san diego, usa) was used for graphical and statistical analysis of data sets. anova or a mixed-effects model were conducted to compare responses over time and between vaccine groups at different time points post-vaccination as detailed in the results. neutralising antibody titre data were log transformed before analysis. neutralising antibody titre data generated by the vnt and pvnt assays were compared using spearman nonparametric correlation. p-values < 0.05 were considered statistically significant. were immunised on day 0 and 28 with chadox1 ncov19 (prime-boost) or chadox1 ncov19 on day 28 (prime-only); pigs (n=3) were immunised with chadox1 ncov-19 on days 0 and 28 (primeboost), or only on day 0 (prime-only). to analyse sars-cov-2 s-specific t cell responses, all mice were sacrificed on day 49 for isolation of splenocytes and pigs were blood sampled longitudinally to isolate pbmc. following stimulation with sars-cov-2 s-peptides, responses of murine splenocytes (a) and porcine pbmc (c) were assessed by ifn-γ elispot assays. using flow cytometry, cd4 + and cd8 + t cell responses were characterised by assessing expression of ifn-γ, tnf-α, il-2, il-4 and il-10 (mice; b) and ifn-γ, tnf-α, il-2 and il-4 (pigs; d). each data point represents an individual mouse/pig with bars denoting the median response per group/timepoint. : sars-cov-2 s protein-specific antibody responses following chadox1 ncov-19 primeonly and prime-boost vaccination regimens in mice and pigs. inbred balb/c (n=5) and outbred cd1 (n=8) were immunised on day 0 and 28 with chadox1 ncov19 (prime-boost) or chadox1 ncov19 on day 28 (prime-only), whereas, pigs were immunised with chadox1 ncov-19 on days 0 and 28 (prime-boost), or only on day 0 (prime-only). to analyse sars-cov-2 s protein-specific antibodies in serum, all mice were sacrificed on day 49 and pigs were blood sampled weekly until day 42. antibody units or end-point titres (ept) were assessed by elisa using recombinant sars-cov-2 fl-s for both mice (a) and pigs (b), and recombinant s protein rbd for pigs (c). sars-cov-2 neutralising antibody titres in pig sera were determined by vnt, expressed as the reciprocal of the serum dilution that neutralised virus infectivity in 50% of the wells (nd50; d) , and pvnt, expressed as reciprocal serum dilution to inhibit pseudovirus entry by 50% (ic50; e). each data point represents an individual mouse/pig sera with bars denoting the median titre per group. a single dose of chadox1 mers provides broad protective immunity against a variety of mers-cov strains. biorxiv chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques. biorxiv a single dose of chadox1 mers provides protective immunity in rhesus macaques antigen encoded by vaccine vectors derived from human adenovirus serotype 5 is preferentially presented to cd8+ t lymphocytes by the cd8α+ dendritic cell subset immunization with an adenovirus-vectored tb vaccine containing ag85a-mtb32 effectively alleviates allergic asthma the pig: a model for human infectious diseases large animal models for vaccine development and testing the contribution of non-human primate models to the development of human vaccines comparison of heterosubtypic protection in ferrets and pigs induced by a single-cycle influenza vaccine immunogenicity and protective efficacy of seasonal human live attenuated cold-adapted influenza virus vaccine in pigs aerosol delivery of a candidate universal influenza vaccine reduces viral load in pigs challenged with pandemic h1n1 virus vaccine development for nipah virus infection in pigs bovine herpesvirus-4-vectored delivery of nipah virus glycoproteins enhances t cell immunogenicity in pigs. vaccines (basel) targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients clinical and immunological features of severe and moderate coronavirus disease 2019 transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients presence of sars-cov-2 reactive t cells in covid-19 patients and healthy donors. medrxiv sars-cov-2 infection protects against rechallenge in rhesus macaques dna vaccine protection against sars-cov-2 in rhesus macaques vaccination with viral vectors expressing np, m1 and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice a serological assay to detect sars-cov-2 seroconversion in humans this study was supported by engineering sarah gilbert and teresa lambe are named on a patent application covering chadox1 ncov-19. the remaining authors declare no competing interests. the funders played no role in the conceptualisation, design, data collection, analysis, decision to publish, or preparation of the manuscript. correspondence and material requests to professor simon p. graham (simon.graham@pirbright.ac.uk) and professor teresa lambe (teresa.lambe@ndm.ox.ac.uk). key: cord-253905-zknmfgsh authors: li, xingguang; zai, junjie; zhao, qiang; nie, qing; li, yi; foley, brian t.; chaillon, antoine title: evolutionary history, potential intermediate animal host, and cross‐species analyses of sars‐cov‐2 date: 2020-03-11 journal: j med virol doi: 10.1002/jmv.25731 sha: doc_id: 253905 cord_uid: zknmfgsh to investigate the evolutionary history of the recent outbreak of severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) in china, a total of 70 genomes of virus strains from china and elsewhere with sampling dates between 24 december 2019 and 3 february 2020 were analyzed. to explore the potential intermediate animal host of the sars‐cov‐2 virus, we reanalyzed virome data sets from pangolins and representative sars‐related coronaviruses isolates from bats, with particular attention paid to the spike glycoprotein gene. we performed phylogenetic, split network, transmission network, likelihood‐mapping, and comparative analyses of the genomes. based on bayesian time‐scaled phylogenetic analysis using the tip‐dating method, we estimated the time to the most recent common ancestor and evolutionary rate of sars‐cov‐2, which ranged from 22 to 24 november 2019 and 1.19 to 1.31 × 10(−3) substitutions per site per year, respectively. our results also revealed that the betacov/bat/yunnan/ratg13/2013 virus was more similar to the sars‐cov‐2 virus than the coronavirus obtained from the two pangolin samples (srr10168377 and srr10168378). we also identified a unique peptide (prra) insertion in the human sars‐cov‐2 virus, which may be involved in the proteolytic cleavage of the spike protein by cellular proteases, and thus could impact host range and transmissibility. interestingly, the coronavirus carried by pangolins did not have the rrar motif. therefore, we concluded that the human sars‐cov‐2 virus, which is responsible for the recent outbreak of covid‐19, did not come directly from pangolins. december 2019 in the chinese city of wuhan, causes a respiratory illness called covid-19, which can spread from person to person. 1 cases and 858 deaths in 27 countries during the 2012 mers outbreak. 7, 8 coronaviruses are known to circulate in mammals and birds. previous studies revealed that both sars-cov and mers-cov are zoonotic in origin, originally coming from bats, [9] [10] [11] [12] with sars-cov spreading from bats to palm civets to humans, [13] [14] [15] and mers-cov spreading from bats to camels to humans. 16, 17 recent research has also reported that the sars-cov-2 virus likely originated in bats, a proposal based on the similarity of its genetic sequence to that of other known coronaviruses. 18 however, like sars-cov, mers-cov, and many other coronaviruses, the sars-cov-2 virus may have been transmitted to humans by an intermediate animal host. 19 therefore, the identity of the animal source of sars-cov-2 remains a key and urgent question. furthermore, to stem future outbreaks of this type and preventing the transmission of zoonotic diseases to humans should be a top research priority. the existence of an intermediate animal host of sars-cov-2 between a probable bat reservoir and humans is still under investigation. the discovery of a virus closely related to the newly emerged sars-cov-2 in a data set from pangolins sampled more than a year ago illustrates that the sampling of other mammals handled or consumed by humans could uncover even more closely related viruses. 20 during a press conference on 7 february 2020, two researchers (shen yongyi and xiao lihua) from the south china agricultural university in guangzhou identified the pangolin as a potential source of the sars-cov-2 virus based on genetic comparison of coronaviruses taken from pangolins and from humans infected during the recent outbreak. by analyzing more than 1 000 metagenomic samples and using molecular biology testing, they found that the positive rate of β coronavirus in pangolins was 70% and that the genome sequence of an isolated virus strain was 99% similar to that of the sars-cov-2 virus. thus, whether pangolins acted as a direct intermediate animal host of the sars-cov-2 virus is worth further investigation. in the present study, we performed analyses of the transmission dynamics and evolutionary history of the virus based on 70 genomes of sars-cov-2 strains sampled from australia (n = 4), belgium (n = 1), china (hubei province, n = 19; guangdong province, n = 16; zhejiang province, n = 4; taiwan, n = 1), finland (n = 1), france (n = 4), germany (n = 1), japan (n = 1), korea (n = 1), singapore (n = 3), thailand (n = 2), united kingdom (n = 2), and united states (n = 10) with sampling dates between 24 december 2019 and 3 february 2020. we reanalyzed two of the 21 pangolin metagenome samples from previously published data 20 (table s1 ). to investigate the potential intermediate hosts of sars-cov-2 (between originating animal and human hosts), two samples (srr10168377 and srr10168378) obtained from previously reported malayan pangolin (manis javanica) viral metagenomic sequencing data (bio project prjna573298) were downloaded from the ncbi sra public database. 20 after assembly, the srr10168377 and srr10168378 genomes were 16 999 and 6 392 bp in length, respectively. we defined another data set ("dataset_6") composed of six genome sequences of coronavirus strains. betacov/wuhan-hu-1/2019 (epi_isl_402125) was grouped as "clade a," one (betacov/bat/yunnan/ratg13/2013; epi_isl_402131) and two (bat-sl-covzc45; mg772933 and bat-sl-covzxc21; mg772934) sars-related coronaviruses were grouped as "clade b" and "clade d," respectively. the two assembled genomes from srr10168377 and srr10168377 were grouped into "clade c." the two data sets ("dataset_70" and "dataset_6") were aligned using mafft v7.222 22 and then manually curated using bioedit v7.2.5. 23 to assess the recombination of "dataset_70," we employed the pairwise homoplasy index (phi) to measure the similarity between closely linked sites using splitstree v4.15.1. 24 the best-fit nucleotide substitution models for the two data sets were identified according to the bayesian information criterion (bic) method with 3 (24 candidate models) or 11 (88 candidate models) substitution schemes in jmodeltest v2.1.10. 25 to evaluate the phylogenetic signals of "dataset_70" and "dataset_6," we performed likelihood-mapping analysis 26 using tree-puzzle v5.3, 27 with 25 000 to 175 000 randomly chosen quartets for the two data sets. for "dataset_70," split network analysis was performed using kishino-yano-85 distance transformation with the neighbornet method, which can be loosely thought of as a "hybrid" between the neighbor-joining (nj) and split decomposition methods, implemented in tree-puzzle v5.3. for "dataset_70," nj 28 phylogenetic trees were constructed using the kimura 2-parameter method 29 implemented in mega v7.0.26. 30 for "dataset_6," nj 28 phylogenetic trees were constructed using the maximum composite likelihood (mcl) method, 31 tempest v1.5. 35 we also estimated the evolutionary rate and time to the most recent common ancestor (tmrca) for "dataset_70" using ml dating in the treetime package. 36 the hiv transmission cluster engine (hiv-trace; www.hivtrace.org) 44 was employed to infer transmission network clusters for sars-cov-2 "dataset_70." all pairwise distances were calculated and the putative linkages between each pair of genomes were considered whenever their divergence was ≤0.0001 (0.01%) or ≤0.00001 (0.001%) substitutions/site (tn93 substitution model). multiple linkages were then combined into putative transmission clusters. clusters that comprised of only two linked nodes were identified as dyads. this approach detects transmission clusters in which the clustering strains are genetically similar, implying a direct or indirect epidemiological connection. to investigate the putative parents of sars-cov-2, we performed similarity plot analysis based on the kimura two-parameter method 29 with a window size of 200 bp and step size of 20 bp using simplot v.3.5.14. 45 we divided "dataset_6" into four clades (ie, clade a, clade b, clade c, and clade d), with clade a designated as the query group. for "dataset_70" and "dataset_6," hky and gtr + g were the models of best-fit, respectively, across the two different substitution schemes (ie, 24 and 88 candidate models) according to the bic method, and were thus used in subsequent likelihood-mapping and phylogenetic analyses for the two data sets. the phi tests of "dataset_70" did not find statistically significant evidence of recombination (p = 1.0). likelihood-mapping analysis of "dataset_70" revealed that 69.7% of li et al. | 3 the quartets were distributed in the center of the triangle, indicating a strong star-like topology signal reflecting a novel virus, which may be due to exponential epidemic spread ( figure 1a) . likewise, 25.9% of the quartets from "dataset_6" were distributed in the center of the triangle, indicating a strong phylogenetic signal ( figure 1b) . the split network generated for "dataset_70" using the neighbornet method was highly unresolved, and the phylogenetic relationship of "data-set_70" was probably best represented by a network rather than a tree ( figure 1c ). the existence of polytomies indicated-in contrast to that expected in a strictly bifurcating tree-an explosive, star-like evolution of sars-cov-2. both the nj and ml phylogenetic analyses of sars-cov-2 "dataset_70" also showed star-like topologies, in accordance with the likelihood-mapping results (figures 2 and s1 ). the ml phylogenetic tree showed greater star-like topology than the nj phylogenetic tree, indicating that the ml method was more reasonable for "dataset_70." root-to-tip regression analyses between genetic divergence and sampling date using the best-fitting root showed that (table 1) . furthermore, based on bayesian time-scaled phylogenetic analysis using the tip-dating method, the estimated tmrca dates and evolutionary rates from "dataset_70" ranged from (table 1) . thus, the estimated tmrca dates and evolutionary rates for sars-cov-2 from "dataset_70" were consistent among the different clock models (strict and relaxed) but were distinct among the different dating methods (constrained-dating and tip-dating). the estimated tmrca dates and evolutionary rates for sars-cov-2 from "data-set_70" using the tip-dating method exhibited much narrower 95% bcis than the constrained-dating method. in addition, the estimated tmrca dates and evolutionary rates for sars-cov-2 from "data-set_70" were consistent between the different coalescent tree models (ie, constant and exponential) when using the tip-dating method but were distinct when using the constrained-dating method. for each f i g u r e 1 likelihood-mapping and split network analyses of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). likelihoods of three tree topologies for each possible quartet (or for a random sample of quartets) are denoted by data points in an equilateral triangle. distribution of points in seven areas of the triangle reflects tree-likeness of data. specifically, three corners represent fully resolved tree topologies; center represents an unresolved (star) phylogeny; and sides represent support for conflicting tree topologies. results of likelihoodmapping analyses of two data sets ("dataset_70," a; and "dataset_6," b) and split network analyses of "dataset_70" (c) are shown we considered individuals as genetically linked when the genetic distance between sars-cov-2 strains was <0.01% substitutions/site. based on this, we identified one large transmission cluster that included 66 of 70 (94.29%) genomes, thus suggesting low genetic divergence for "dataset_70" (figure s3 ). we also considered individuals as genetically linked when the genetic distance between sars-cov-2 strains was <0.001% substitutions/site. based on this, we identified 6 transmission clusters that included 37 of 70 (52.86%) genomes for "dataset_70" (figure 5 ). clusters ranged in size from 2 to 23 genomes. the nj and ml phylogenetic topologies of "dataset_6" were consistent with each other (figure s4 ), indicating that the use of a small number of sequences could show similar topological results. homology plot analysis of "dataset_6" also revealed that betacov/bat/yunnan/ ratg13/2013 was more similar to the sars-cov-2 virus than the coronavirus obtained from the two pangolin samples (srr10168377 and srr10168378), consistent with phylogenetic analysis ( figure s5 ). of note, "clade d" (bat-sl-covzc45 and bat-sl-covzxc21) had higher similarity to the sars-cov-2 virus in the first 12 000 bp region of the full alignment than to the pangolin coronavirus ( figure s5 ). we also found that a unique peptide (prra) insertion region in the spike protein at the junction of s1 and s2 junction in the human sars-cov-2 virus ("clade a") induced a furin cleavage motif (rrar), which could be a predicted polybasic cleavage site, and thus a unique feature of sars-cov-2, in comparison with the other three clades ("clade b," "clade c," and "clade d") ( figure s6 ). on the basis of "dataset_70," our likelihood-mapping analysis confirmed additional tree-like signals over time compared with our previous results. 46, 47 this result implies increasing genetic divergence of sars-cov-2 in human hosts ( figure 1a) , consistent with the findings of our earlier studies. 46, 47 split network analysis for sars-cov-2 "dataset_70" human-to-human transmission ( figure 1c ). these results are consistent with the ml phylogenetic analyses, which showed polytomy topology from "dataset_70" (figure 2 ). however, nj phylogenetic analyses showed a more bifurcating tree topology compared with the ml phylogenetic analyses ( figure s1 ). this is a good example showing the differences between nj and ml phylogenetic construction methods. "dataset_70" had a minor strong positive temporal signal based on root-to-tip regression and ml dating analyses (figures 3 and s2) , with the estimated tmrca dates and evolutionary rates for sars-cov-2 found to be nearly identical using both analyses ( table 1 ). the estimated tmrca dates and evolutionary rates for sars-cov-2 were very similar across different clock models and coalescent tree priors using the tip-dating method. the estimated tmrca dates and evolutionary rates for sars-cov-2 were also very similar across different clock models using the constraineddating method, but highly distinct across the different coalescent tree priors ( table 1 ). the tmrca estimated by the tip-dating method was relatively narrower than that determined by the constrained-dating method, consistent with our previous studies. 46, 47 bayesian analyses with the tip-dating method using a strict clock as well as constant size coalescent tree prior indicated that sars-cov-2 is evolving at a rate of 1.24 × 10 −3 substitutions per site per year (table 1 ), in accordance with our prior research 46, 47 and similar to that found for other human f i g u r e 4 estimated maximum-clade-credibility tree of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) using tip-dating method. colors indicate different sampling locations. nodes are labeled with posterior probability values. estimated maximum-clade credibility (mcc) tree of "dataset_70" are shown f i g u r e 5 transmission clusters of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). structure of inferred sars-cov-2 transmission clusters from "dataset_70" using genetic distances of <0.001% substitutions/site is shown. nodes (circles) represent connected individuals in overall network, and putative transmission linkages are represented by edges (lines). nodes are color-coded by sampling locations coronaviruses. 41 our results also suggest that the virus originated on 24 november 2019, which is in further agreement with our earlier studies. 46, 47 we identified eight phylogenetic clusters (number of sequences 2 to 7) with posterior probabilities between 0.99 and 1.0 using bayesian inference ( figure 4) . we also identified six transmission clusters (number of sequences 2 to 23) when the genetic distance between the sars-cov-2 strains was <0.001% substitutions/site ( figure 5) . however, our conclusions should be considered preliminary and explained with caution due to the limited number of sars-cov-2 genome sequences presented in this study. as more genome sequences become available, there may be stronger among-lineage rate variation over time as to warrant using a relaxed clock model, but we anticipate that the evolutionary rates and tmrca dates will be broadly similar to those estimated here. as the number of substitutions is still small, it is tempting to speculate that sequencing errors could have a considerable impact on the evolutionary rate and tmrca date estimates. we removed three sars-cov-2 genome sequences (ie, betacov/wuhan/ipbcams-wh-02/2019, epi_isl_403931; betacov/shenzhen/szth-001/2020, epi_isl_406592; betacov/shenzhen/szth-004/2020, epi_isl_406595) with potential sequencing errors, but these may have less impact on the above estimates when more substitutions of sars-cov-2 are accumulated over time. we also expect that as more sars-cov-2 genome sequences become available, the estimated 95% bcis of the evolutionary rates and tmrca dates will be narrower. we found that the pangolin-cov virus from the two pangolin samples was clustered with the sars-cov-2 virus with 100% bootstrap support; however, betacov/bat/yunnan/ratg13/2013 was more similar to the sars-cov-2 virus than to the pangolin coronavirus and the human sars-cov-2 virus ("clade a") showed a unique peptide (prra) insertion not found in the other three clades ("clade b," "clade c," "clade d"). this insertion constitutes an rrar motif in the spike protein at the junction of s1 and s2 junction in the human sars-cov-2 virus, after considering the next amino acid (r) of the unique peptide (prra) ( figure s6 ). of note, the highly favored motifs for furin cleavage are arg-x-(arg/lys)-arg (rxrr or rxkr), and the minimal motifs for furin cleavage can be rxxr. 48 we also note that some of the other coronaviruses have a furin motif in almost the same location in their spike proteins. 49, 50 lentiviruses have an rkxr (r, arginine; k, lysine; x, any amino acid) site between gp120 and gp41, cleaved by furin to convert gp160 into subunits. [51] [52] [53] [54] therefore, it is tempting to speculate that cleavage or lack of cleavage of the spike protein at this site could significantly impact host range and transmissibility. taken together, the pangolin coronavirus samples (srr10168377 and srr10168378) were less similar to the sars-cov-2 virus than to the betacov/bat/yunnan/ ratg13/2013 virus and did not have the rrar motif. therefore, we concluded that the human sars-cov-2 virus, which is responsible for the current outbreak of covid-19, did not come directly from pangolins. however, due to the limited viral metagenomic data obtained from pangolins, we cannot exclude that other pangolins from china may contain coronaviruses that exhibit greater similarity to the sars-cov-2 virus. in conclusion, our results emphasize the importance of further epidemiological investigations, genomic data surveillance, and experimental studies of the role of the unique furin cleavage motif (rrar) of sars-cov-2 in the spike protein at the junctions of s1 and s2. such work could positively impact public health in terms of guiding prevention efforts to reduce sars-cov-2 transmission in real time, and to stem future outbreaks of zoonotic diseases. this study was supported by a grant from the national natural science foundation of china (no. 31470268) to prof. yi li. this study was sponsored by the k.c. wong magna fund in ningbo university. we gratefully acknowledge the authors and originating and submitting laboratories for their sequences and meta-data shared through gisaid, 21 on which this study is based. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia epidemiology, genetic recombination, and pathogenesis of coronaviruses identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated 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functional analysis of potential cleavage sites in the mers-coronavirus spike protein structural investigation of the hiv-1 envelope glycoprotein gp160 cleavage site 3: role of sitespecific mutations maturation of hiv envelope glycoprotein precursors by cellular endoproteases processing and routage of hiv glycoproteins by furin to the cell surface the convertases furin and pc1 can both cleave the human immunodeficiency virus (hiv)-1 envelope glycoprotein gp160 into gp120 (hiv-1 su) and gp41 (hiv-i tm) evolutionary history, potential intermediate animal host, and cross-species analyses of sars-cov-2 the authors declare that there are no conflict of interests. xl conceived and designed the study and drafted the manuscript. xl, bf, and ac analyzed the data. xl, qn, jz, qz, yl, bf, and ac interpreted the data and provided critical comments. all authors reviewed and approved the final manuscript. http://orcid.org/0000-0002-3470-2196 key: cord-260729-b12v3c8c authors: de lang, anna; baas, tracey; teal, thomas; leijten, lonneke m; rain, brandon; osterhaus, albert d; haagmans, bart l; katze, michael g title: functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov–infected macaques date: 2007-08-10 journal: plos pathog doi: 10.1371/journal.ppat.0030112 sha: doc_id: 260729 cord_uid: b12v3c8c the pathogenesis of severe acute respiratory syndrome coronavirus (sars-cov) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. using functional genomics, we analyzed early host responses to sars-cov infection in the lungs of adolescent cynomolgus macaques (macaca fascicularis) that show lung pathology similar to that observed in human adults with sars. analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (il)-6, il-8, and ip-10, which corresponds to the host response seen in acute respiratory distress syndrome. as opposed to many in vitro experiments, sars-cov induced a wide range of type i interferons (ifns) and nuclear translocation of phosphorylated signal transducer and activator of transcription 1 in the lungs of macaques. using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. our studies emphasize that the induction of early ifn signaling may be critical to confer protection against sars-cov infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of sars. infection with sars-cov causes lower respiratory tract disease with clinical symptoms that include fever, malaise, and lymphopenia [1] . approximately 20%-30% of sars patients require management in intensive care units, and the overall fatality rate has approached 10%. interestingly, children seem to be relatively resistant to sars, but the reason for this restriction is not known [2] [3] [4] . the clinical course of sars follows three phases [5, 6] . in the first phase, there is active viral replication and patients experience systemic symptoms. in the second phase, virus levels start to decrease while antibodies, which are effective in controlling infection, increase. however, pneumonia and immunopathological injury also develop in this phase. ultimately, in the third phase, fatal cases of sars progress to severe pneumonia and acute respiratory distress syndrome (ards), characterized by the presence of diffuse alveolar damage (dad) [1, 7] . it has been hypothesized that the pathological changes are caused by a disproportional immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such as cxcl10 (ip-10), ccl2 (mcp-1), interleukin (il)-6, il-8, il-12, il-1b, and interferon (ifn)-c [8] [9] [10] [11] [12] [13] . these in vivo data have been confirmed with in vitro experiments, demonstrating that sars-cov infection induces a range of cytokines and chemokines in diverse cell types [14] [15] [16] [17] [18] [19] . in contrast, production of type i ifns seems to be inhibited or delayed by sars-cov in vitro [14] [15] [16] [17] [18] [20] [21] [22] . moreover, no ifn-a or ifn-b has been detected in the sera of sars patients or in lungs of sars-cov-infected mice [23] [24] [25] . recent in vitro studies demonstrated that type i ifn inhibition or delay may be orchestrated by sars-cov proteins orf 3b, orf 6, and n [26] . the inhibition of ifn production would benefit sars-cov replication, since pretreatment of cells with ifn before sars-cov infection efficiently prevents replication in these cells [21, [27] [28] [29] [30] . furthermore, prophylactic treatment of macaques with pegylated ifn-a reduces sars-cov replication in the lungs [31] . although ifn production was absent in clinical samples, gene and protein expression profiles in these patients were likely impacted by clinical treatments and concurrent preexisting disease. in addition, most if not all virus-host response information is from clinical blood/sera samples that were taken relatively late during infection-little is known about what happens early during infection. animal studies are of great value to decipher the host's initial innate immune response, without confounding clinical treatment (steroid and mechanical ventilation) or underlying co-morbidity. in order to elucidate early host responses during the acute phase of sars-cov infection, we infected cynomolgus macaques with sars-cov and used macaque-specific microarrays and real-time (rt)-pcr techniques to study host gene expression profiles. adolescent cynomolgus macaques infected with sars-cov develop dad similar to sars patients, but clear most of the virus in the lungs by day 6 [7] . because sars-cov replicates predominantly in the lower respiratory tract of macaques, the virus infects a range of cells, including type 1 and type 2 pneumocytes, that are different from those analyzed in vitro. the ability to simultaneously examine virus replication and host response gene expression profiles in the lungs of these animals during the acute phase of sars offers the opportunity to further unravel the pathogenesis of sars. six cynomolgus macaques were inoculated with sars-cov strain hku-39849 and lung tissues were collected at day 1 (n ¼ 2, 1a and 1b) or day 4 (n ¼ 4, 4a-4d). no lesions or clinical symptoms were detected on day 1 after sars-cov infection, whereas on day 4, three out of four monkeys were lethargic, with one of these animals showing mildly labored breathing. pathological changes at day 4 post infection included dad, characterized by flooding of the alveoli with edema fluid, infiltration of neutrophils, damage to the alveolar and bronchial epithelia, and occasional type 2 pneumocyte hyperplasia, as described earlier [31] . four mock-infected animals were included in the study to serve as a reference for host response without viral challenge and to examine outbred inter-animal variation. our previous experience with a/ texas/36/91 influenza virus demonstrated that viral mrna was detected in representative samples of the lung rather than throughout the whole lung [32] . based on this experience, the level of infection in separate lung samples was evaluated using rt-pcr. sars-cov mrna was detected in all animals, and 13 pieces out of the total of 16 lung pieces from infected animals contained high levels of virus, while the three remaining pieces of lung contained very low levels of virus (;3-4 logs lower, figure 1a) . no viral rna could be detected in the samples from the mock-infected animals. for gene expression experiments, lung samples from sars-cov-infected animals were compared to a reference lung sample from mockinfected animals. the three samples with lower virus levels (1a-low, 4a-low, and 4d-low) were analyzed individually so as not to dilute the gene expression of pooled pulmonary samples with higher sars-cov levels and also to potentially further define pulmonary infection. samples from animals with high viral mrna levels showed greater gene expression changes (;2,000 genes day 1, ;800 genes day 4) compared to samples from animals with low levels of viral mrna (;400 genes), indicating a response of lung tissue to the virus ( figure 1b) . additionally, the two day 1 animals showed higher numbers of differentially expressed genes than the day 4 animals. in contrast, gene expression analysis of the separate mock samples revealed limited differentially expressed genes. in order to examine how gene expression would be influenced by presence of virus, timing after inoculation, and individual animal variation, global expression profiling was performed. hierarchical clustering methods were used to order rows (genes) and columns (samples) to identify groups of genes or samples with similar expression patterns [33, 34] . these data were plotted as a heat map in which each matrix entry represents a gene expression value (figure 2a ). red corresponds to higher gene expression than that of the controls; green corresponds to lower gene expression. this analysis yielded 2,050 genes with day 1 samples on one side of the heat map and day 4 samples on the other side of the heat map, indicating an influence of timing after inoculation. there are two major roots to the hierarchical dendrogram, with the larger root composed of all the day 1 samples and the three day 4 samples with the highest virus levels. the smaller root is composed of the remaining day 4 samples with the lowest sars-cov levels. although transcriptional profiling shows some variation when comparing samples from the same animal, the underlying gene expression is similar with a reduction in fold change in the ''low'' samples. these comparisons suggest that both individual animal variation and the ''asynchronous'' nature of the infection in the animals' lungs are factors involved in determining transcription of cellular genes. to validate that the host response from infected animals comprises a stronger transcriptional profile than individual variation from mock-infected animals, differential gene expression patterns in the separate mock samples were investigated, but only 38 genes were differentially expressed ( figure 2b ). these results suggest that underlying basal levels of gene transcription do not confound expression levels after infection. even in a basal state, some low-level lung-to-lung variations were identified within the same animal but not enough to disrupt segregation of lung pieces based on mock-infected animals. severe acute respiratory syndrome coronavirus (sars-cov) infection causes a progressive atypical pneumonia. in typical cases, largely confined to adult and elderly individuals, acute respiratory distress syndrome develops, and admission to an intensive care unit is required. although these complications can be fatal, most sars patients recover, suggesting that protective immune responses are operational. in this study, we simultaneously examined virus replication and host-response gene expression profiles in macaque lungs during the acute phase of sars to gain more insight into the early events that take place after sars-cov infection. we show that a strong host response is induced in the lungs of sars-cov-infected macaques, illustrated by the induction of several pathogenic cytokines and chemokines. interestingly, antiviral pathways are activated as well, demonstrated by the presence of phosphorylated signal transducer and activator of transcription 1 (stat1) transcription factors throughout the lung, but not in sars-cov-infected cells. a subset of cells was shown to produce interferon-b, a cytokine involved in the resistance to many viral infections and able to activate stat1. activation of this antiviral pathway upon sars-cov infection may be an important escape route of the host to withstand the devastating effects of sars-cov. different time points after inoculation, a venn diagram was generated with each set (circle) holding to the parameters of an absolute fold change . 2 and p , 0.0001 in at least two animals ( figure 3a ). the day 1 set contained 1,278 genes and the day 4 set contained 950 genes. when examining host responses that were similar throughout the course of the infection, the intersection of the day 1 and day 4 sets indicates that 597 genes show shared responses. the heat map of these 597 genes is shown in figure 3b . if more stringent criteria were used to find common responses in all six animals, using the 1,278 genes from the day 1 set and the 129 genes that are differentially expressed in all day 4 animals, a subset of 97 genes was identified. this subset included ifnstimulated genes (isgs), like ifits, mx1, gbp1, and g1p2, and also various chemokines and cytokines, such as cxcl10 (ip-10), ccl2 (mcp-1), il-6, and il-8 ( figure s1 ). these same cytokines and chemokines have been reported to be upregulated in human sars cases [9] [10] [11] [12] . this set also included cathepsin l (ctsl), which has been shown to be required for sars-cov entry into a cell [35] . even though only 97 genes were commonly regulated in all animals, indicated with blue bars in figure 3b , the heat map highlights that the other 500 genes show similar expression trends. both sets of commonresponse genes showed similar functionality: cellular growth and proliferation, cell death, cellular movement, immune response, and cell-to-cell signaling. next, we analyzed genes that were differentially expressed exclusively on either day 1 or day 4 in order to find signature gene expression patterns for each day. genes identified as unique responses at day 1 (681 genes) and at day 4 (353 genes) in the venn diagram showed unique functionality ( figure 3c ). the gene expression profile at day 1 shows a prominent innate host response to viral infection; top functional categories on day 1 are the immune response, the hematological system, and the immune and lymphatic system. genes like ifn-c, ccl4 (mip-1-b), csf3, il1a, and tnf are included in these categories. at day 4, a smaller panel of unique differentially expressed genes that play a role in cell cycle, cellular assembly, and dna repair were identified like ccnb2, ccne1, cdca5, cenpa, chaf1a, and prc1. in order to investigate genes that are most strongly regulated after sars-cov infection, genes included in the venn diagram ( figure 3a ) that also held to an absolute fold change . 5 were queried ( figure s2 ). from this set, genes that were involved in the immune response and lung repair processes were used to generate a heat map ( figure 4) . a number of genes that have been reported to be up-regulated in sars patient sera, such as ccl2 (mcp-1), cxcl10 (ip-10), il-6, and il-8, were strongly (;20-fold) induced in all animals. many cell cycle and matrix genes indicative of tissue repair processes were also highly differentially expressed at day 4 (e.g., anln, areg, cdc2, cdkn3, cks2, fosl1, and kif2c). likewise, tissue factor pathway inhibitor 2 (tfpi2), an anticoagulant, was strongly up-regulated during infection in all animals (averaging ;20-fold), as well as plscr1, ser-pine1 (pai1), and thbs1, all genes involved in procoagulation and platelet activation, were induced. concomitant expression of tfpi2 with these pro-coagulation genes might function as an inhibitory response to restrain the activation of the coagulation pathway during acute inflammation. surprisingly, expression of diverse ifn-a genes and expression of ifn-b was up-regulated ;10to 20-fold in the day 1 samples. furthermore, ifn-c, a type ii ifn, was efficiently transcribed on day 1 after sars-cov infection (;5-fold). other genes associated with the induction of ifns like ddx58 (rig-i), irf-7, and signal transducer and activator of transcription 1 (stat1), were also highly induced (;8fold). up-regulation of type i ifns in these sars-cov infected macaques is remarkable, since sars-cov inhibits ifn production in many in vitro studies. we did not detect induced ifn-b mrna expression using ma104 cells or caco2 cells and the sars-cov-hku virus (unpublished data). not only ifns, but also several ifn-responsive genes (e.g., g1p2, gbp1/2, ifi/ifits, mx1/2, isg20, and oas1/2/l) were highly transcribed, showing a persistent activation of the innate immune response. furthermore, suppressor of cytokine signaling 1(socs1) is induced at the onset of infection, presumably to establish negative feedback to attenuate cytokine signaling. of note, ifit1 (isg56/ifi56), often used to gauge ifn induction, was up-regulated an average of ;13fold. to further explore some of the pathogenic and antiviral pathways that are induced after sars-cov infection, we investigated the transcription of various cytokines, chemokines, ifns, isgs, and transcription factors involved in the jak/stat pathway. as can be seen in figure 5a , a wide range of chemokines and cytokines are differentially expressed after sars-cov infection in macaque lungs, especially on day 1 after infection. besides previously mentioned chemokines, we detected monocyte chemotactic protein genes like ccl8 (mcp-2) and ccl7 (mcp-3), but also ccl11 (eotaxin), a chemotactic protein for eosinophils. in the samples with low sars-cov mrna levels, the induction of chemokines is less evident, suggesting that the presence of these molecules is restricted to areas in the lung where virus is present. furthermore, sars-cov-infected macaques showed a stronger induction of ifns (14 unique genes) and isgs (20 unique genes) on day 1 than day 4 and when virus was present at high levels. note that besides ifn-a, ifn-b, and ifn-c, the ifn-ks (il-29, il-28a, il-28b), which are type i ifns, were induced in samples with high sars-cov levels. in the absence of viral rna, no ifns, but interestingly, a number of isgs (17 unique genes) were detected, suggesting paracrine stimulation ( figure 5b ). differential expression of a selection of strongly upregulated genes, cxcl10 (ip-10), il-6, il-8, and ifn-b, was confirmed using rt-pcr ( figure 6 ). in accordance with the microarray data, the rt-pcr data showed that cxcl10 (ip-10), il-6, il-8, and ifn-b were all expressed at levels that were approximately 100 times higher in the sars-cov-infected animals at day 1 than in the uninfected control animals and were still elevated on day 4 after infection. as can be seen in figure 6 , the induction of ifn-b was strongly correlated to the presence of virus (r spearman ¼ 0.88, p , 0.0001). for cxcl10 (ip-10), il-6, and il-8 the correlation is less evident, which is not surprising since these cytokines can be induced by other factors than the virus itself. in order to visualize the host response in the lungs of sars-cov-infected macaques, ifn-b production and translocation of phosphorylated stat1 was studied using immunohistochemistry. in the lungs of the sars-cov-infected macaques, a modest number of cells stained positive for ifnb at day 1 post infection, whereas no ifn-b-positive cells could be detected in mock-infected macaques ( figure 7a -7c). notably, most of the cells that stained positive for ifn-b were located very close to blood vessels, but not in the alveoli where most sars-cov antigen-positive cells (mainly type 2 pneumocytes at 1 day post infection) are located. to examine whether the ifns that are produced in the lungs of these sars-cov-infected macaques are biologically active and able to induce stat1 phosphorylation and translocation, lung sections of the infected macaques were stained with antibodies against phosphorylated stat1. as shown in figure 7d and 7e, no phosphorylated stat1 could be detected in the lungs of pbs-infected macaques, while in the lungs of sars-cov-infected macaques, cells with phosphorylated stat1 in their nucleus were abundantly present. subsequently, the same pieces of lung from sars. genes were included if they met the criteria of a 2-fold change or more (p 0.0001). a two-of-nine strategy allowed samples to cluster together if profile similarities existed based on timing of inoculation (n ¼ 2 samples for day 1). (b) the number after pbs refers to the animal (i.e., pbs 1), while the number after the dash refers to the lung piece (i.e., pbs 1-1). thirty-eight genes are displayed with an absolute fold change . 2 and p ,0.0001 in at least two animal samples. up-regulated genes are indicated in bold underline. only one gene, hla-dqa1, was down-regulated . 5. no up-regulated genes met these criteria in mock-infected animals. separate mock samples (i.e., pbs 1-1) were compared to the total mock pool. doi:10.1371/journal.ppat.0030112.g002 cov-infected macaques at day 1 were double stained for phosphorlylated stat1 and sars-cov (figure 7 f) . notably, phosphorylated stat1 was not detected in the nucleus of sars-cov-infected cells (type 2 pneumocytes), while cells directly adjacent to these sars-cov-infected cells stained for phosphorylated stat1 in many, but not all, foci containing sars-cov-positive cells. thus, type i ifns are produced in the lungs of sars-cov-infected macaques, and are able to activate the jak/stat pathway. however, translocation of stat1 does not occur in sars-cov-infected pneumocytes. although recent studies indicate that the sars-cov orf6 protein is able to inhibit nuclear translocation of stat1 in vitro, this was not demonstrated in experiments using infectious sars-cov [26] . in order to assess whether sars-cov inhibits phosphorylation and translocation of stat1, ma104 cells were infected with sars-cov for 24 h and then either fixed directly or treated with type i ifn. cells infected with sars-cov, but not treated with ifn, stained positive for sars-cov (unpublished data), but lacked staining for phosphorylated stat1, indicating that sars-cov or other soluble mediators are not able to induce stat1 phosphorylation ( figure 8 ). after treatment of the ma104 cells with ifn, phosphorylated stat1 could be detected in the nucleus of most cells, but not in the nucleus of sars-cov-infected cells (figure 8 ). this demonstrates that sars-cov inhibits the translocation of phosphorylated stat1 to the nucleus, confirming our in vivo data. besides inhibiting translocation of phosphorylated stat1, sars-cov also seems to reduce stat1 phosphorylation, as the majority of sars-covinfected cells contained low levels of phosphorylated stat1 in their cytoplasm. pathogenic viruses escape the antiviral action of the ifn system by inhibiting both ifn production and signaling pathways. here, we report that even though production and signaling of type i ifns is inhibited by sars-cov in vitro as well as in sars-cov-infected cells in vivo, high levels of type i ifns are induced in the lungs of sars-cov-infected macaques. these ifns are able to activate stat1, followed by the transcription of numerous isgs. using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. our results emphasize the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of sars-cov infection in cynomolgus macaques. to our knowledge, this study represents the first functional genomics investigation of sars-cov infection in cynomolgus macaques. all experimental animals showed signs of infection because viral mrna could be detected in random samples from the lung, indicating that the virus had spread throughout the whole lung at the time of necropsy. furthermore, pathological examination of sars-cov-infected macaques at day 4 post infection revealed multifocal dad [31] . unlike 10% of humans with sars, which are mainly restricted to the elderly, adult macaques used in this study do not succumb to sars-cov infection. however, the sars-cov-induced pathology in these macaques likely resembles the pathological changes seen in the majority of human sars patients that recover from the disease. although none of the current animal models has fully reproduced all features of sars, the most important aspects of this disease are observed in experimentally infected macaques, providing valuable insights into the initial innate immune response after infection without confounding clinical treatment or underlying co-morbidity. using macaque-specific microarrays, we were able to observe that with early infection, high levels of viral mrna corresponded to a strong cellular host response. this strong host response is dominated by genes involved in the immune response and includes a wide range of genes corresponding with what is seen in human ards. during the acute phase of human ards, activated neutrophils and macrophages enter the alveoli and produce a number of cytokines and chemokines such as il-6, il-8, and cxcl10 (ip-10) [36] , as were found in the lungs of our sars-cov-infected macaques. researchers have postulated that these genes also predict adverse sars patient outcome [37] . during the chronic phase of human ards, type 2 pneumocytes start to proliferate and differentiate in order to repair the damaged lung. at day 4, [14, 38] . we also detected a strong presence of genes involved in the coagulation pathway, including tfpi2, serpine1, and timp1. the idea of a pro-coagulation profile mimics the clinical-pathological observations of sars patients that showed unusually disseminated small vessel thromboses in the lungs [5, 39] . additionally, cathepsin l was up-regulated in all sars-cov-infected macaques. induction of this gene after sars-cov infection is quite interesting because cathepsin l is an endosomal protease that is necessary for sars-cov to infect a cell [35] . remarkably, sars-cov infection in macaques leads to a strong transcription of ifns. not only ifn-a, ifn-b, and ifnk (all type i ifns), but also ifn-c, a type ii ifn, were all highly up-regulated, especially on day 1 after infection. the expression of ifn-b, which strongly correlated to the amount of virus present, continued throughout day 4 and was confirmed using immunohistochemistry; ifn-b-positive cells could be detected in the lungs of the sars-cov-infected macaques. the induction of ifn-b in these sars-covinfected macaques is surprising, because several reports have shown that sars-cov inhibits or delays type i ifn production in a number of cell types [14] [15] [16] [17] [18] 20, 22] . for example, sars-cov blocks a step in the activation of irf-3, a transcription factor that is required for ifn-b induction [21] . in addition, the sars-cov proteins orf3b, orf6, and nucleocapsid have been shown to function as ifn antagonists, as has the sars-cov nsp1 gene that prevents the production of sendai virus-induced ifn-b in 293 cells [26, 40] . interestingly, it was recently shown that plasmacytoid dendritic cells (pdcs) are able to produce ifn-a and ifn-b after sars-cov infection, while conventional dcs did not produce these type i ifns [41] . pdcs are known for their ability to produce very high amounts of ifn-a and ifn-b and are considered firstline sentinels in immune surveillance in the lung [42] [43] [44] [45] [46] . we speculate that the ifn-b-producing cells detected in the lungs of sars-cov-infected macaques are pdcs. future studies may address the nature of these ifn-producing cells once technical difficulties in detecting pdcs in macaque tissues have been tackled. these studies may also shed light on whether decreasing numbers of pdcs observed in clinical blood samples from human sars patients are caused by sequestering of pdcs by the lungs, destruction of pdcs by sars-cov, or destruction or suppression of pdcs by steroid treatment [47] . when ifns are produced, they bind to their receptors on the cell membrane, after which stat1, a key member of the jak/stat pathway, is phosphorylated and subsequently translocated to the nucleus, followed by the production of a wide range of ifn-stimulated genes. in vitro, sars-cov inhibited translocation of stat1 to the nucleus, and phosphorylation of stat1 was strongly reduced. however, the inhibition of stat1 phosphorylation was not absolute because cells with low levels of phosphorylated stat1 in their cytoplasm were also detected. in accordance with our data, kopecky-bromberg et al. recently showed that the sars-cov protein orf6 is able to inhibit stat1 translocation [26] . this strategy is not unique to sars. other viruses have been shown to be able to block signaling of ifns by affecting phosphorylation and/or translocation of the stat proteins. for example, measles virus v protein inhibits translocation of stat1, but does not affect phosphorylation, whereas measles virus p protein blocks both of these processes [48] . other paramyxoviruses, like rinderpest virus, nipah virus, hendra virus, and mumps virus, as well as flaviviruses like west nile virus and japanese encephalitis virus, are able to block activation of stat1 and stat2 [49] [50] [51] [52] . inhibition of stat1 phosphorylation is not always complete. for example, sendai virus suppresses tyrosine phosphorylation of stat1 during the early stages of infection, but this block becomes leaky after a couple of hours with phosphorylated stat1 accumulating in the cytoplasm [53] . in contrast to these in vitro data, we observed phosphorylated stat1 in the nuclei of numerous cells in the lungs of sars-cov-infected macaques, indicating that these cells had been activated by the ifns produced in the lung. however, phosphorylated stat1 was not detected in sars-covinfected cells. the observations made in this study indicate that sars-cov-infected macaques produce ifns in response to virus infection and are further capable of activating the stat1 pathway in cells surrounding the sars-cov-infected cells. the importance of ifns in controlling sars-cov infection has been suggested in several animal studies. mice clear sars-cov in the absence of nk cells, t cells, or b cells, suggesting that innate immune responses are sufficient to limit sars-cov infection in these animals [23] . indeed, stat1 knock out mice, which are resistant to the effects of ifns, to some extent show a worsening of pulmonary disease and an increase in viral replication in the lungs compared to normal mice after infection with sars-cov [54] . although ifn treatment was not conducted in sars-cov infection mouse studies, prophylactic treatment of macaques with pegylated ifn-a protects type 1 pneumocytes from infection with sars-cov [31] . in addition, potent antiviral activity is observed in vitro when cells are treated with ifns before they are infected with sars-cov [27, 29, 30] . although we cannot determine the effect of neutralizing ifn-b in sars-covinfected animals, based on the experiments utilizing recombinant ifns in these animals, we postulate that type i ifns are partly responsible for the relatively mild clinical symptoms that are seen in sars-cov-infected macaques. in addition, a recent study again demonstrated the importance of ifns in viral infections, as macaques infected with the highly pathogenic and fatal 1918 influenza virus showed limited induction of type i ifns (only ifna4 reached fold changes . 5) and delayed induction of isgs, while macaques infected with the low-pathogenic k173 influenza virus showed a strong induction of these antiviral molecules early during infection [55] . notably, ifn-b was not up-regulated (absolute fold change , 2) in any of the influenza virusinfected animals, even in those animals that recovered, unlike sars-cov-infected macaques that showed a very strong presence of ifn-b. in conclusion, our study demonstrates that cynomolgus macaques can be infected with sars-cov, as indicated by presence of viral mrna at different locations throughout the lung at day 1 and day 4, with gross pathology becoming noticeable at day 4. furthermore, we show that infection of cynomolgus macaques with sars-cov leads to a strong immune response, including the induction of various cytokines and chemokines, resembling the host response seen in human sars patients. strikingly, despite the fact that sars-cov infection blocks the production of ifns in vitro, type i ifns are strongly induced in the lungs of sars-covinfected macaques. the production of ifn early during infection leads to widespread activation of stat1 and the production of isgs. this suggests that, although sars-cov blocks ifn signaling in infected cells, locally produced ifns are capable of activating non-infected cells and possibly can prevent infection of these cells. thus, sars-cov infection in macaques leads to the differential activation of both pathogenic and antiviral signaling pathways in vivo, and the outcome may be determined by the relative contribution of these signaling pathways. were infected intratracheally with 1 3 10 6 tcid 50 sars-cov (hku-39849) as described earlier [31] . virus stocks were generated in vero e6 cells that were defective in ifn production. two animals were euthanized on day 1 after infection and four animals were euthanized on day 4. in addition, four animals were mock (pbs) infected and euthanized on day 4, serving as a negative control group. one lung from each monkey was fixed in 10% formalin for histopathology and immunohistochemistry while the other was used for real-time pcr and microarrays. lung samples were randomly excised from three different lung areas (cranial, medial, caudal) and stored in rnalater (ambion, http://www.ambion.com/). sixteen pieces of lung were taken from the sars-cov-infected animals, two to three pieces of lung per animal. twelve pieces of lung were taken from the mock-infected animals, three pieces of lung per animal. individual lung samples in rnalater were transferred to trizol reagent (invitrogen, http:// www.invitrogen.com/), homogenized using polytron pt2100 tissue grinders (kinematica, http://www.kinematica.ch), and then processed to extract rna. all experiments were executed under a biosafety level 3, and approval for animal experiments was obtained from the institutional animal welfare committee. oligonucleotide microarray analysis. infected macaque lung samples were co-hybridized with a reference mock-infected macaque lung sample on macaque oligonucleotide arrays containing 131 viral probes, corresponding to 26 viruses, and 22,559 rhesus probes, corresponding to ;18,000 rhesus genes. the reference mock-infected sample was created by pooling equal mass quantities of total rna extracted from the 12 individual lung pieces from mock-infected animals. an agilent 2100 bioanalyzer was used to check the purity of the total rna prior to crna probe production with the agilent low rna input fluorescent linear amplification kit (agilent technologies, http://www.agilent.com/). arrays were scanned with an agilent dna microarray scanner, and image analysis was performed using agilent feature extractor software (agilent technologies). each microarray experiment was done with two technical replicates using dye reversal [56] . all data were entered into a custom-designed database (expression array manager) and analyzed with resolver 4.0 (rosetta biosoftware, http://www.rosettabio.com/) and spotfire deci-sionsite for functional genomics (spotfire, http://www.spotfire.com/). in our data analysis, genes were selected to be included for transcriptional profile based on two criteria: a greater than 99.99% probability of being differentially expressed (p 0.0001) and an expression level change of 2-fold or greater. ingenuity pathway analysis (ingenuity systems, http://www.ingenuity.com/) was used to functionally annotate genes according to biological processes and canonical pathways. in accordance with proposed miame standards, primary data are available in the public domain through expression array manager at http://expression.microslu.washington.edu/ expression/index.html [57] . quantitative real-time rt-pcr. rt-pcr was performed to detect sars-cov mrna and to validate cellular gene expression changes as detected with microarrays. each reaction was run in triplicate using taqman 2x pcr universal master mix (applied biosystems, http:// www.appliedbiosystems.com/) with primers and probe specific for the sars-cov nucleoprotein gene [7] , or for macaque cellular genes (sequences shown in table 1 ). differences in gene expression are represented as the fold change in gene expression relative to a calibrator and normalized to a reference, using the 2 àddct method [58] . gapdh (glyceraldehydes-3-phosphate dehydrogenase) or 18s rrna were used as endogenous controls to normalize quantification of the target gene. the samples from the mock-infected macaques were used as a calibrator. immunohistochemistry. formalin-fixed, paraffin-embedded lung samples from sars-cov-infected and mock-infected macaques were stained for sars-cov, phosphorylated stat1, and ifn-b using mouse-anti-sars-nucleocapsid (clone ncap4, mouse igg2b; imgenex, http://www.imgenex.com/), mouse-anti-phospho-stat1 (clone st1p-11a5, mouse igg2a-j; zymed laboratories, http://www. invitrogen.com/), and rabbit-anti -ifn-b (chemicon, http://www. chemicon.com/), respectively. after deparaffinization, antigen retrieval was performed using a citrate buffer for the sars-cov and stat1 staining. no antigen retrieval was performed when staining for ifn-b. goat-anti-mouse igg2a hrp, goat-anti-mouse igg2b ap (southern biotech, http://www.southernbiotech.com/), and anti-rabbit igg-hrp (dako, http://www.dako.com/) were used as secondary antibodies. signals were developed with fast red and dab (sigma, http://www.sigmaaldrich.com/) and counterstained with mayer's hematoxylin. in vitro sars-cov and stat1 staining. ma104 cells (african green monkey foetal kidney cells, ecacc) were cultured in eagle's minimal essential medium (emem; cambrex, http://www.cambrex. com/) supplemented with 2 mm glutamine, 1% non-essential amino acids and 10% foetal bovine serum. cells were seeded in 96-well plates and infected with sars-cov (moi 0.5), and 24 h after infection, selected wells were treated with universal type i ifn (5,000 u/ml, sigma) for 30 min at 37 8c. subsequently, cells were fixed with 10% neutral-buffered formalin and treated with 70% ethanol. sars-cov-infected cells were visualized using purified human igg from a convalescent sars patient (csl), followed by staining with an antibody to human igg, linked to alexa fluor 594 (invitrogen). phosphorylated stat1 was visualized using mouse-anti-phospho-stat1 (zymed), followed by staining with a fitc-linked antibody to mouse igg. lung pathology of fatal severe acute respiratory syndrome severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update clinical presentations and outcome of severe acute respiratory syndrome in children clinical picture, diagnosis, treatment and outcome of severe acute respiratory syndrome (sars) in children clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study the severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe 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processes of stat1 resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus statistical design and the analysis of gene expression microarray data minimum information about a microarray experiment (miame)-toward standards for microarray data analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method we thank s. smits for her assistance with sars-cov infections and rna isolations. author contributions. adl, tb, ado, blh, and mgk conceived and designed the experiments. adl, tb, tt, lml, and br performed the experiments. adl, tb, and blh analyzed the data. adl, tb, ado, blh, and mgk wrote the paper.funding. this work was supported by the us national institutes of health, r01 grant hl080621-01a1, and by the european union, grant sp-22-ct-2004-511060.competing interests. the authors have declared that no competing interests exist. key: cord-275926-rj23z7po authors: fontanella, marco m.; de maria, lucio; zanin, luca; saraceno, giorgio; terzi di bergamo, lodovico; servadei, franco; chaurasia, bipin; olivi, alessandro; vajkoczy, peter; schaller, karl; cappabianca, paolo; doglietto, francesco title: neurosurgical practice during the sars-cov-2 pandemic: a worldwide survey date: 2020-05-05 journal: world neurosurg doi: 10.1016/j.wneu.2020.04.204 sha: doc_id: 275926 cord_uid: rj23z7po abstract background and objective the sars-cov-2 pandemic has consistently changed medical practice throughout specialties, regardless of their contribution in facing the disease itself. we surveyed neurosurgeons worldwide to investigate the situation they are experiencing. design and participants a 17-question, web-based survey was administered to neurosurgeons worldwide through the wfns and the neurosurgery cocktail from march 28 to april 5, 2020 by web link or e-mail invitation. questions were divided into three subgroups: general information, health system organization, and institutional plans for the sars-cov-2 outbreak. collected data was initially elaborated using survey monkey® software. country specific data were extracted from the who website. statistical analysis was performed using r version 3.6.3. results of the 446 respondents, most were from italy (20%), india (19%), and pakistan (5%). surgical activity was significantly reduced in most centers (79%) and dedicated in-hospital routes were created for sars-cov-2 patients (58%). patient screening was performed only when there were symptoms (57%) and not routinely before surgery (18%). the preferred methods included a nasopharyngeal swab and chest x-ray. health professionals were rarely screened (20%) and sometimes, even if sars-cov-2 positive, were asked to work if asymptomatic (26%). surgical planning was changed in most institutions (92%), while indications were modified for non-urgent procedures (59%) and remained unchanged for subarachnoid hemorrhages (85%). conclusions most neurosurgeons worldwide reported work reorganization and practices that respond to current international guidelines. differences in practice might be related to the perception of the pandemic and significant differences in the health systems. sharing data and experiences will be of paramount importance to address the present moment and challenges in the near future. we are in the midst of a pandemic caused by a novel coronavirus, sars-cov-2, first detected in wuhan (china) in december 2019. since then, covid-19 has spread quickly, with more than 2,000,000 confirmed cases and more than 100,000 deaths on april 19, with 213 countries involved worldwide. 1 given the serious public health risk, medical practice has consistently changed during sars-cov-2 pandemic. the impact of the covid-19 outbreak might change in relation to the diffusion of the virus, as well as the health system of the individual country; furthermore, this pandemic is influencing different medical specialties in a variety of ways. 2 most surgical subspecialties are not primarily involved in fighting the disease itself, but they must still change their organization, as most national and international societies suggest stopping all elective activity, maintaining only emergent and urgent procedures. [2] [3] [4] [5] [6] [7] [8] neurosurgeons might feel fairly useless during the sars-cov-2 pandemic. however, international guidelines have been introduced calling for a tailored triage according to the degree of emergency, 3, 9, 10 and we believe that sharing information about the organization of neurosurgical activity throughout the world might be helpful at this time. furthermore, it might be interesting to investigate whether there is any association between the level of infection and consequent country reorganization of the neurosurgical system, as well as the neurosurgeons' practice. we, therefore, conducted an online survey that was submitted to neurosurgeons worldwide between march 28 and april 5, 2020 through the world federation of neurosurgical societies (wfns) 11 and the neurosurgery cocktail. [12] [13] [14] the questions were divided into three subgroups: 1. information on the country and its involvement by neurosurgeons were asked about their country of practice, its involvement by the pandemic, and duration of the emergency; 2. health system organization and screening for health professionals: national and regional measures adopted to face the outbreak were queried, as well as the screening rate and precautions undertaken for sars-cov-2 positive health professionals; 3. institutional plans for the sars-cov-2 outbreak: any special measures adopted for sars-cov-2 positive neurosurgical patients were investigated, i.e. their screening rate and method, any changes in surgical indications, planning and activity for oncologic procedures, non-emergency surgeries, and subarachnoid hemorrhages (sahs). most question were closed-ended, multiple choice. some allowed also an open answer (q7,15 and 17 -see supplementary material). the primary goal was to collect data on neurosurgeons' perceptions of the health emergency, the national/regional measures undertaken for health professionals and throughout neurosurgical departments, and the changes in neurosurgical indications, planning, and activity. the secondary aim was to investigate correlations between the data collected and the epidemiological scenario in each country. the third aim was to look into differences among regions, nations and territories along with possible causes and consequences of diversities. data were initially elaborated using survey monkey ® software online; country specific historical data were extracted from the who website. 1 for open answers, the most recurring terms were rendered as a "word cloud", in which a population of words is represented with different sizes according to their frequency. survey's responses to q3 were first converted into estimates in days (<1 week: 3 days; >1 week to <1 month: 15 days; >1 month to <3 month: 45 days; >3 months: 100 days). pearson's correlation was used to estimate the correlation between the number of answers in the surveys with the number of new cases in each country and the duration of the pandemic. the latter was calculated as the difference in days from the first confirmed case to the last day of the survey. where a i is the observed number of answers registered for country i. for each country, the median of the differences between each survey's entry date and perception date was calculated. statistical analysis was performed using r version 3.6.3. 15 on april 5, 2020 the survey was closed and 446 responses had been collected. the skipping rate for each question ranged from 0% (q1) to 89% (q17). most responses (85%) were filled during only 3 days in march (saturday 28, sunday 29, and tuesday 31). a total of 66 countries worldwide responded to the survey (fig. 1) . most respondents were from italy (20%), followed by india (19%) and pakistan (5%). statistical analysis did not reveal a significant correlation between the incidence of disease and number of responses by country (fig. 2 ). for the majority of respondents (97%), the nation was facing a sars-cov-2 outbreak. the duration of the health emergency was between 1 week and 1 month for most respondents (63%), between 1 month and 3 months for 32% respondents, shorter than 1 week for 3% respondents, and longer than 3 months for 2% respondents. figure 3 shows correlations between disease activity during the survey and time-length perception of respondents from some countries. regarding the special measures adopted in neurosurgical departments to face the sars-cov-2 outbreak, in most cases there was a reduction of surgical activity without centralization (79%), while there was a centralization of surgery in high-volume centers in 9% cases and full closure of neurosurgical departments in 5%. no special measures to face the outbreak were reported in 7% of centers. figure 4b shows the categorizations of special measures undertaken by countries in relation to the incidence of disease. the overall reported screening rate of health professionals for sars-cov-2 was 20%; 26% respondents reported that sars-cov-2 positive health professionals were asked to keep working if asymptomatic. with respect to the precautions adopted worldwide for sars-cov-2 positive neurosurgical patients, in most cases hospitals reserved dedicated routes for them (58%), in 27% cases specific operating rooms were dedicated to sars-cov-2 patients, and in 21% cases neurosurgical units were reserved for sars-cov-2 patients. other respondents (21%) replied with open answers and among them, no special measures were usually undertaken. the overall screening rate for sars-cov-2 was 57% for symptomatic patients and only 18% for patients undergoing surgery. the preferred methods for screening was nasopharyngeal swab (86%), followed by chest ct scan (26%) and chest x-ray (25%). surgical planning was globally changed in most institutions (92%): only urgent or emergency procedures were performed in 49% cases; urgencies/emergencies and procedures that could not be postponed were performed in 45% cases. oncologic procedures were preserved in 71% cases. the resulting reduction rate in number of surgical procedures was >70% for almost half of respondents (47%). surgical indications for sars-cov-2 patients were modified in 59% cases for pathologies such as chronic subdural hematomas (cshs) and tumors, while the modus operandi in treating aneurysmal sahs did not change in 85% of centers. this survey, dedicated to neurosurgery and sars-cov-2 worldwide, demonstrated a number of interesting findings. a high number of responses (n=446) was received, suggesting a relevant global impact of covid-19 on the neurosurgical community, even though it is a surgical specialty that is not primarily involved in fighting the disease. 16 italy and india were the countries with the most respondents ( fig. 1 ). this finding is independent of the incidence of disease, as shown in figure 2 . conversely, the us was the country with least number of respondents in relation to the incidence of disease during the study period. although it might be tempting to relate the number of answers to perception of the health emergency, we should point out that the survey circulated widely among neurosurgeons, but we cannot state that the percentage of respondents (i.e. respondents/non respondents) was the same among the different nations. the same correlation was found with regards to the medical perception of disease activity (q2) in different countries, and only few respondents (3%) claimed their country was not facing the outbreak during the time period studied: among them, neurosurgeons from germany were probably the most "wrong", since their country had between 10 4 to 10 5 sars-cov2 patients during the study period (fig. 4a) . notably, reactions and perceptions of covid-19 impact on a country may be consistently driven by government actions, as happened in india and pakistan, where most strict lockdown measures were undertaken with respect to other world countries, 17, 18 possibly influencing general and health professional awareness of the health emergency. nonetheless, the differences in number of responses might be due to a heterogeneity of the survey distributions among different countries. furthermore, the perception of the emergency might be related to the health system, with germany having the highest rate of icu beds/population. 19 regarding the time-length perception of covid-19, italian and iranian respondents perceived the start of the health emergencies much earlier than the actual one (fig. 3) ; chinese neurosurgeons, instead, located the start of the health emergency almost at the inflection point of decrease in incidence rate, when the sars-cov-2 pandemic was about to reach the plateau phase (fig. 3) . the perception corresponded well to reality among the other respondents. it is tempting to interpret these data as the consequence of the strain that physicians are experiencing in countries with the longest disease involvement at the moment of the study: some might perceive that the emergency is longer than reality due to the continuous stress, or because of media pressure about other countries (i.e. china and iran) experiencing the outbreak. on the other hand, accumulating evidence shows that sars-cov-2 might have been circulating in italy well before february 21st, thus explaining the significant outbreak that took place in lombardy in northern italy. 20 others may perceive that the emergency is shorter than reality due to epidemiology for complex reasons: chinese people outside wuhan experienced the outbreak at a later stage of the epidemic and individuals emigrating from wuhan was the main infection source for other provinces, causing a rapid increase in case load when wuhan was already in the plateau phase; the general perception in china about the national involvement might have been in reality delayed. 21 health system organization with respect to health system organization, the most frequent action undertaken globally was reduction of surgical activity without centralization (79%, yellow bars on fig. 4b ). significant high rates were registered in india (81%) and pakistan (85%). centralization of surgery to high-volume centers was reported in only 9% cases and italy was the country with the highest number of positive respondents (23%), followed by germany (6%) and india (5%). only 7% of respondents report that their country did not undertake any special measure. these data show how most countries acted according to international guidelines in the management of elective procedures. 5 india and pakistan have been reported to be the world's best respondents to the sars-cov-2 pandemic, 22-24 thus reflecting high rates of neurosurgical activity reorganizations. neurosurgical centers should undertake national and regional measures to meet patients' needs with logistical capabilities, as reported by guidelines. 3 interestingly, health reorganization may vary signficantly even within the same country. more than 100,000 positive cases were confirmed in italy by april 19 with more than 30,000 in lombardy alone. 20 in this region, which is still at the center of the health emergency in italy, neurosurgical departments were urgently reorganized and centralization of surgery in highvolume centers was decided. [6] [7] [8] other italian regions are still facing the health emergency, but at lower levels with incidences that tend to decrease from the north to the south. 20 the same incidence disproportion between regions within a country is clearly visible in even smaller european countries such as switzerland, where the cantons of vaud and geneva account for more than 4,000 cases each, while the canton of schaffhausen has not yet reached 100 cases. 25 these significant variations in a single country justify the different regional reorganizations. guidelines for risk assessment and management of exposure of healthcare workers vary according to the risk of sars-cov-2 infection (categorized as high or low) and recommend covid-19 testing only for workers at a high risk of infection. 26 in this sense, the global attitude did not deviate significantly from recommendations, 3, 16 as only 21% of respondents reported ongoing screening for health professionals, mainly from brazil (50%), mexico (42%), and germany (28%). a minority of respondents (26%) declared that sars-cov-2 positive health professionals kept working if asymptomatic and a large portion of these respondents were from italy (36%). indeed, no clear national guidelines are available for sars-cov-2 positive health professionals, 27 resulting in heterogeneity of recommendations throughout the country. covid-19 positive italian health professionals have reached more than 13,000, with more than 100 deaths of physicians (most of them are general practitioners) and almost 30 nurses. 28 at spedali civili hospital, in brescia in northern italy, sars-cov-2 positive health professionals are not allowed to work and daily temperature screening procedures are undertaken at the hospital entrance for both health professionals and visitors. 29 nonetheless, there is a general perception that health professionals might have been asymptomatic carriers of the disease. 30 regarding precautions adopted worldwide for sars-cov-2 positive neurosurgical patients, the most widely undertaken measure globally was to reserve dedicated routes to sars-cov-2 patients (58%), while specific operating room and entire neurosurgical units were created in a minority of cases. some respondents (21%, mainly from austria, germany, and uk) reported not taking any special measures for sars-cov-2 patients. however, guidelines clearly state that sars-cov-2 positive patients should be cohorted in a separate location from sars-cov-2 negative patients and specific hospital policy for management of known or suspected sars-cov-2 positive patients in the operating room should be developed. [31] [32] concerning the screening of neurosurgical patients, facilities should use portable radiography when chest x-rays are considered necessary, thus avoiding the need to bring patients into radiography departments; chest ct scan has been recently reported to have a high sensitivity (97%) for covid-19 screening, but lower specificity and accuracy. 33, 34 a recently published paper in jama analyzed the sensitivity of different rt-pcr screening sources demonstrating that bronchoalveolar lavage fluid is the most sensitive specimen (93%), followed by sputum (72%), nasal swab (63%), fibrobronchoscope brush biopsy (46%), pharyngeal swabs (32%), feces (29%), and blood (1%); the authors underline that multiple testing from different sites improve sensitivity and reduces false-negative results. 35 most guidelines at present recommend a single upper respiratory nasopharyngeal swab for suspect cases. 2 in this survey, most respondents referred that nasopharyngeal swab was the preferred method for screening (86%), followed by ct scan (26%), and chest x-ray (25%). some respondents indicated more than one screening method, especially those from italy (57%) and india (19%), where the most common combination was the nasopharyngeal swab with chest x-ray. the covid-19 outbreak had a relevant impact on surgical planning, with most respondents reporting a significant change in surgical activity in their institutions (92%). the majority (94%) performed only procedures that could not be postponed (i.e. tumors with evident mass effect) and/or urgent/emergency procedures, while in a few cases (6%) the entire neurosurgical department was closed. this obviously resulted in a significant reduction of the overall number of surgical procedures: most respondents claimed more than 70% reduction of surgical interventions. procrastinating elective procedures has been one of the crucial indications delivered by international societies 3-5 with many important aims: a. to contain the spread of sars-cov-2, by reducing visits to hospitals by people with no urgent medical issue; b. to reduce the patient load on intensive care units with non-covid-19 patients; c. to reduce the possibility of treating asymptomatic sars-cov-2 patients, who would be at high risk of deteriorating due to the surgical stress and would increase the risk of infecting health professionals. surgical indications for sars-cov-2 non-emergency patients (i.e. cshs and tumors) have been modified in only 59% cases, while 41% neurosurgeons worldwide referred that their institutions continued operating on elective neurosurgical patients in the same way as the pre-outbreak era; international guidelines clearly state that non-emergency procedures should be delayed. [3] [4] [5] [6] [7] studying correlations between incidence of disease and actions undertaken by various countries (fig. 4c) , middle-eastern nations (i.e. turkey, egypt, saudi arabia, etc.) were the most reactive to the health emergency, followed by european countries (i.e. italy, spain, austria, etc.), and the americas (i.e. us, mexico, brazil, etc.). as for aneurysmal sahs, most respondents (85%) did not change their indications and treatment (fig. 4d) . even if some of these findings might seem against guidelines, the word cloud resulting from the open answers puts "patient" at the center and sums up what international societies have been suggesting: "postpone surgery and be conservative as much as possible, delay elective procedures, but, as for emergency symptomatic patients, try to operate with all recommended precautions" (fig. 5) . we must indeed stress that all medical efforts, institutional plans, and health system organization would be useless without the appropriate and recommended use of personal protective equipment (ppe). 26,36,37 although india is the world's second most populous country, the incidence rate of sars-cov-2 infections has risen less than other countries since the beginning of the outbreak. 38 the reason might be found in the earlier government actions that india undertook while the virus was spreading out from china. 17, 18, 39 limits of the study our study has many limits. first, it is not an epidemiological study and does not allow drawing conclusions about the actual prevalence and incidence of the variables investigated. it does allow, though, to draw conclusions regarding the perception of neurosurgeons about the covid-19 health emergency with respect to the actual epidemiology data. second, although this survey spread out widely among neurosurgeons, respondents were mostly from italy, india and pakistan, while the rest of the world was represented with lesser numbers. heterogeneity of the survey's percentage of respondents (i.e. respondents/non-respondents) among different countries might have biased some responses. notwithstanding, this is the first survey conducted on the impact of covid-19 on the neurosurgical community and we believe that data from this study can help neurosurgeons and global health organizations to tackle this health emergency. sars-cov-2 pandemic has consistently changed medical practice, with an enormous impact on all specialties, regardless of their contribution in facing the disease itself. neurosurgeons worldwide have changed their surgical planning and activity, in most cases following national and international guidelines. dedicated routes were put in place for sars-cov-2 patients in most cases and surgical activity was limited to procedures that could not be postponed, resulting in an overall reduction of surgeries by more than 70%. the lockdown will be soon followed by the rebuilding phase, when delayed elective procedures will need to be performed, thus opening a new challenge that to be addressed, possibly by sharing current knowledge and experience worldwide. deviation for each country of registered number of answers to the survey from the expected value. blue bars indicate an excess of answers; red bars indicate a lack of answers. covid-19) covid-19 in neurosurgery news, guidelines and discussion forum covid-19: recommendations for management of elective surgical procedures. american college of surgeons neurosurgery in the storm of covid-19: suggestions from the lombardy region, italy (ex malo bonum) hub and spoke' lombardy neurosurgery group. may we deliver neuro-oncology in difficult times (e.g. covid-19)? neurosurgery during the covid-19 pandemic: update from lombardy, northern italy. acta neurochir (wien) 19 / société française de neurochirurgie world federation of neurosurgical societies telegram: join group chat the r project for statistical computing the coronavirus disease 2019 global pandemic: a neurosurgical treatment algorithm india is enforcing the harshest and most extensive covid-19 lockdown in the world the variability of critical care bed numbers in europe focolaio di infezione da nuovo coronavirus sars-cov-2: la situazione in italia distribution of the covid-19 epidemic and correlation with population emigration from wuhan, china coronavirus disease (covid-19 pakistan's response to coronavirus among world's best, says who country head how india is responding to covid-19: quarantine, travel limits and tests embed=y&:showvi zhome=no&:host_url=https%3a%2f%2fpublic.tableau.com%2f&:embed_code_version=3&: tabs=no&:toolbar=yes&:animate_transition=yes&:display_static_image=no&:display_spinner= no&:display_overlay=yes&:display_count=yes&publish=yes&:loadorderid=0. accessed covid-19 -raccomandazioni per gli operatori sanitari coronavirus in italia, morti altri sette medici: il totale sale a 116. la repubblica am i part of the cure or am i part of the disease? keeping coronavirus out when a doctor comes home maintaining trauma center access and care during the covid-19 pandemic: guidance document for trauma medical directors. american college of surgeons congress of neurological surgeons -cns.org chest ct features of covid-19 in correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases | radiology detection of sars-cov-2 in different types of clinical specimens guidance for wearing and removing personal protective equipment in healthcare settings for the care of patients with suspected or confirmed covid-19 guidance for health system contingency planning during widespread transmission of sars-cov-2 with high impact on healthcare covid-19 dashboard /profiles/julia-hollingsworth">julia h <a href="/profiles/brettmckeehan">brett mckeehan</a> and tara john. coronavirus live news and updates from around the world do you modify surgical indications if the patient is ncov positive for non-emergency surgery (e.g. chronic subdural hematoma, tumors?) you modify your modus operandi in treating aneurysmal subarachnoid hemorrhage? * abbreviations list sars-cov-2: severe acute respiratory syndrome coronavirus 2 wfns: world federation of neurosurgical societies who: world health organization ncov: novel coronavirus sahs: subarachnoid hemorrhages cshs: chronic subdural hematomas icu: intensive care unit rt-pcr: reverse transcription polymerase chain reaction survey administered to neurosurgeons through the wfns and the neurosurgery cocktail from march 28 to april 5 2020 by web link or e-mail invitation.how have we changed our neurosurgical activity in the storm of 2019-ncov pandemic?as neurosurgeons we might feel fairly useless during the 2019-ncov pandemic. we think, though, that it is important to share information on the organization of the neurosurgical activity in this moment. your participation to this brief survey is highly appreciated. key: cord-252456-971d0sir authors: hemida, maged gomaa; ba abduallah, mohammed m. title: the sars-cov-2 outbreak from a one health perspective date: 2020-03-16 journal: one health doi: 10.1016/j.onehlt.2020.100127 sha: doc_id: 252456 cord_uid: 971d0sir the sars-cov-2 is a new human coronavirus candidate recently detected in china that is now reported in people on inhabited continents. the virus shares a high level of identity with some bat coronaviruses and is recognised as a potentially zoonotic virus. we are utilizing the one health concept to understand the emergence of the virus, as well as to point to some possible control strategies that might reduce the spread of the virus across the globe; thus, containment of such virus would be possible. the severe acute respiratory syndrome-2 was named the coronavirus infectious diseses-19 . however, it was called the novel coronavirus at the beginning of this outbreak. this virus was provisionally named 2019-ncov at the very beginning of its emergence in china, after emerging in china in late 2019 [1] . since that time, there has been an escalation in the number of confirmed cases, notably from china, but also in more than 113 countries and territories around the globe. historically four coronaviruses candidates (hcov-229e, hcov-oc43, hcov-nl63, and hcov-uku1), causing common cold or flu-like symptoms were known. patients infected by these viruses usually recover spontaneously in a short time [2] . the sars-cov-2 is the seventh coronavirus known to infect humans, appearing in less than ten years since the emergence of middle east respiratory syndrome coronavirus (mers-cov) in late 2012 and sars-cov in 2003 [3, 4] . this virus belongs to the family coronaviridae; a large group of viruses that have the potential to infect and cause diseases to a large number of mammals, birds as well as humans. members of this family cause a wide variety of clinical signs in their affected hosts, including respiratory, enteric, nervous, and systemic health problems. the viral genomes of coronaviruses have many unique features that make them prone to frequent coding changes, thus generating new candidates in a short period [5] . the reasons behind this rapid mutational frequency are the poor proofreading capability of the viral rna polymerase and the possibility of recombination between various members of this family [6] . recent studies showing the (angiotensin-converting enzyme a, (ace-2)), ace-2 is the primary receptors for the sars-cov-2 and other candidates of the b lineage of the beta coronaviruses [7] . high expression levels of some potential viral receptors in many hosts from mammals, birds in addition to humans make them susceptible to j o u r n a l p r e -p r o o f the virus infection. in recent studies, it is apparent that sars-cov-2 uses the mechanism that sars-cov used for cell entry. the mechanism of the virus entry to the cell resulting from the interaction between the virus receptors and the spike glycoprotein, after priming of that viral protein by the serine protease tmprss2 [8] . further, inhibition of tmprss2, blocked cell entry by sars-cov-2, indicating similar or identical pathways for cell invasion and possible strategies for therapeutics [8] . the who declared the virus as a global health emergency on jan 31, 2020 [9] . as of 10 am cet 03 march 2020, there are 128920 laboratory-confirmed cases reported to the who from 113 countries and territories across the globe with a total fatality of 4747 [10] . currently, the case fatality rate is relatively low (⁓3.6%) compared to infections with severe acute respiratory syndrome coronavirus (sars-cov, (10%) and mers-cov (32%) [11] . the numbers of infected and dead persons are much higher than the latter viruses. however, these numbers will be subject to changes with the newly reported cases and the outcomes of the ongoing active human cases. during mid-february 2020, a new criterion for the patient identification has been launched considering the chest computed tomography (ct) scans to identify patients suffering from respiratory distresses. [12] the reasons behind the switch from the detection of the viral nucleic acids by real-time pcr to ct scanning at that time for patient identification are (i) to give a chance to all patients to get the proper care at the right time (ii) the variation in the incubation period of the viral infection among various people (iii) the possibility of super-spreader of the virus who shed the virus to the environment in large numbers and poses a great risk of infection to the close contact people. this phenomenon have been reported previously in ebola, virus, sars-cov, mers-cov, and recently in sars-cov-2 [13] [14] [15] . using some mathematical tools models late feb, 2020, the basic reproduction (r0) to the humanto-human transmission was around 3.58. however, the same study found the r0 for the bat-to-j o u r n a l p r e -p r o o f human was 2.30 [16] . based on these findings, the r0 of sars-cov-2 was almost close to sars-cov but higher than mers-cov, with the exception of the mers-cov outbreak in south korea [16] . the mers-cov and the sars-cov-2 are both considered airborne pathogens [17] with a tendency to transmit through the aerosol of infected patients. the human-to-human nosocomial transmission was recently reported, which is contributing substantially to the spreading and sustainability of the virus. some family clusters were also recently reported inside china [18] . the incubation period of the diseases ranges from 2-14 days post-infection [19, 20] . the asymptomatic individuals may play essential roles in the spread of the virus within a specific community [21] . however, there are ongoing debates about the possibility of infected persons to shed the virus early during the course of an infection with this virus even before the appearance of the clinical signs [20, 22] . the emergence of the virus was believed to be linked with some human cases who suffered from severe pneumonia, which have a relation with a wet animal huanan seafood wholesale market in wuhan, china (1). these observations, along with the sequence identity of the virus, have drawn attention to the likely zoonotic origin of the virus. the sars-cov-2 infection is potentially another important example of the one health concept, after sars-cov, mers-cov, and ebola virus, in which there is an excellent overlapping in human, animal, and environmental health [23] . the full-length genome of the virus was recently made available to the scientific community [20] . this sequence will have a significant impact on the development of novel diagnostic assays, antiviral drugs, and vaccines against the virus soon. interestingly, the virus genome and its spike glycoprotein showing ‫9‬ 6.11 % and 92.86 identities to the rhinolophus affinis bat coronavirus, respectively (table 1) , from wuhan, china (ratg13, j o u r n a l p r e -p r o o f journal pre-proof accession number mn996532.1) recently deposited in the genbank [24] . bats are representing almost one-fourth of the mammalian population [25] . they are widely distributed all over the world in all continents except antarctica [25] . bats are classified under the order chiroptera that are found more than 50 million years ago. this order includes more than 1300 species of bats [25] . several studies suggested that bats are the common reservoir for the sars-cov were the sequences for viruses isolated from chinese horseshoe bats shared a high degree of identity with sars-cov [26] . metagenomics paved the way for the discovery of a large number of viruses in different species, including bats. there are more than 200 coronaviruses have been identified in various species of bats [27] . the main bat reservoirs of the sars-cov was identified in 2003 [28] . meanwhile, several species of bats including, taphozous perforatus, rhinopoma hardwickii, and pipistrellus kuhlii were believed to be the ancestors for the mers-cov [29] . based on the previous emergence history of sars-cov, the presence of a large number of mammals and birds overcrowded in one place may give a chance for pathogens, particularly those with rna genomes such as coronaviruses and influenza viruses, to emerge. however, it remains uncertain whether a similar emergence for sars-cov-2 can be postulated. one potential reservoir of sars-cov was the palm civet cat (paguma larvata), a member of the viverridae, a group of mammals found in asia [30] . sars-cov was able to infect and replicated efficiently in these animals [31] . serosurveillance for the antibodies against sars-cov in workers from one wild animal market in guangzhou was conducted in 2006 [32] . this study showed the presence of specific igg antibodies against sars-cov in these workers where the civet cats were available [32] . this same study confirmed the hypothesis of the implication of civet cats in the emergence and sustainability of sars-cov at that time. the identification of this reservoir was one of the milestones in control and containment of sars-cov (9), and j o u r n a l p r e -p r o o f subsequent banning of the trade of civet cats in the wild animal market was a significant step toward the containing of sars-cov [33] . this approach highlights the roles of one health in controlling such zoonotic pathogens. based on the evidence gained from studies of sars-cov, and from the genetic identity of sars-cov-2, it could be postulated that the virus responsible for the recent outbreak is transmit ted from bats to humans either directly or indirectly through adaptation in an unidentified host [34, 35] . the cone health concept could be the most logical approach in case of fighting and control some zoonotic pathogens, especially those who do not have available medication or vaccines during an epidemic or outbreak. there are many aspects by which the one health concept may contribute substantially to the control of the current sars-cov-2 outbreak. adoption of some of one health-based control strategies was of great success in controlling mers-cov, contributing at least in part to the decline in the case fatality rates from 52% in 2012 to 32% in 2020 in case of mers-cov [23, 36] . similar approaches were also successful in containing some of the highly pathogenic avian influenza viruses [37] . in the following sections, we are suggesting some one health-based control strategies for the containment of sars-cov-2 ( figure 2 ). coronaviruses are enveloped viruses like influenza, hiv, rabies etc, their envelope composed of a lipid bilayer, which the virus acquires from the host cell membrane while releasing from the infected cells [38] . using lipid solvents and detergents was proved to inactivate sars-cov through permeant damage of the lipid components of the viral envelope [39] . this study showed j o u r n a l p r e -p r o o f a drastic drop in the viral titer after one-minute treatment [39] . this raises the mandate of hand as well as personal hygiene as one of the gold standard control measures for not only coronaviruses but also form most enveloped viruses. coronaviruses are small, approximately 100-120 nm in diameter. this is making them able to pass the pores of the surgical masks very easily. thus, wearing special masks with minute pore sizes like n95 could be one of the preventive measures against respiratory pathogens, including influenza and coronaviruses, in comparison to another type of masks [40, 41] . however, the who already indicated certain rules for wearing the n95 masks, particularly for the health care workers and people in close contact with active cases [42] . the genome of coronaviruses is composed of single-strand positive-sense rna molecules. the virus genome act as a mrna and is infectious. the ultraviolet light has harmful effects on the sars-cov genome when used at 254 nm wavelength for 60 minutes [43, 44] . meanwhile, the effect of temperature on the virus infectivity/survival has great impact on the control of this class of respiratory viruses. sars-cov may live in the environment at average room temperature about 22 c for 2hrs without losing its infectivity. however, the virus infectivity is greatly affected by exposure to 56ºc for at least 30 min [44] . these parameters have great roles in the virus control as we discuss below. as mentioned above, the corona viral genomes are prone to frequent changes due to many factors; a new study highlighted the presence of two variants (l and s) of the sars-cov-2. this study confirmed that variant l (more severe) was dominant during the early outbreak in china, which was responsible for at least 70% of the cases at that time. however, the implementation of drastic control measures against the virus posed some selective pressure on this variant then the s variant becomes the predominant at this time [45] . thus, frequent monitoring of the virus on the genomic level is highly encouraged. meanwhile, j o u r n a l p r e -p r o o f the preparation of vaccine and antiviral drugs should be done from the most recent generation and seeds of the circulating strains and variants of the virus to ensure high efficacy rates. meanwhile, there is an urgent need for the development of novel diagnostic assays that enable the early detection of the virus, even in low copy numbers in the patient specimens. detection of cases during the early stage of the infection could be a crucial step in the early identification of the infected person; thus, proper treatment and control should be in place as early as possible. furthermore, the development of multiplex diagnostic assays that enable the detection of the most common respiratory pathogen in one reaction is highly recommended. it would be of high value to develop novel diagnostic tests that distinguish the seven human coronavir uses in one reaction per sample. this unique approach may help in screening a large number of people and animals and will have a significant impact on the fast track testing of travelers from at-risk regions. both sars-cov and mers-cov have zoonotic origins in which the civet cats and dromedary camels played important roles in the sustainability and transmission of these viruses in the community as well as spell over to the human [30, 46] . to identify the animal reservoirs in the context of sars-cov and mers-cov, scientist screened a large number of animals and birds to identify the main reservoir for those two viruses [47, 48] . sas-cov-2 was believed to be originated in people who came in close contact with the live wet market in wuhan, china, late 2019 [22] . in the case of sars-cov-2, recent studies used the informational spectrum tools to identify and predict the actual reservoir, the virus receptors as well as potential therapeutic and vaccine targets [49] . the wuhan wet market was hosting a large number of wild animals, birds, reptiles, and amphibians which never exist in one location in nature [50] . simply, there is a close j o u r n a l p r e -p r o o f human-animal contact in the supply chain of these wild animal markets starting from hunting, transporting, selling, processing, including slaughtering, and cooking. this is in addition to the exposure of people to their blood, secretions, and execrations. this chain poses a great risk for the contact between human and these wild animals, which may put the handlers at risk of infection not only for coronaviruses but also for other pathogens that these animals and birds may harbor. in the light of the one health concept, control of this emerging virus requires the reduction of the viral shedding to the environment from the main reservoirs, for animal-person transmission and from humans for person-person transmission [35] . based on the previous experience from the other emerging diseases, particularly sars-cov and influenza viruses, avoiding the mixing of various species of animals, birds, and mammals, is highly suggested [51, 65, 66] . this strategy will minimize the possibility of recombination among not only these pathogens but also for other common pathogens in the near future. a large number of bats may also be available in this particular market, including the rhinolophus affinis. the viral genome of the bat coronavirus from this particular species was close to the sars-cov-2 (table 1) . this result is suggesting these bats are the main ancestor of this virus. about 49% of the first reported human cases had a various levels of contact with this market [22] . a recent study showed that second, if seroconversion is proved in any of these animals or birds, testing specimens and tissues from these animals or birds must be carried out to detect the virus in these animals. the live poultry markets were responsible for the transmission of many viral pathogens to humans, especially various types of avian influenza viruses [37, 51, 52] . several studies reported that banning the storage of live poultry in live markets at least for a short period of time for overnight drastically reduced the ability to isolates the avian influenza viruses by 84% compared with the standard procedures [51] . another study showed a drastic decrease in the number of reported human cases of avian influenza h7n9 by almost 97% after the closure of live poultry markets in four major provinces in china [53] . thus, banning the live wild animal trading could have substantial effects on the control strategies not only for coronaviruses but also for other pathogens such as avian influenza viruses. regular monitoring of the dynamic changes of coronavirus in different species of bats is highly recommended. the environment serves as the intermediate vessels between the animals and humans. the environmental factors that may contribute to the spread of the virus include air, water, soil, etc. coronaviruses are airborne viruses that produce nosocomial infections [54] . they are mainly transmitted through droplet infection [55] . when the virus released from the animals or humans, it passes through the air and may drop on some services or objects. some respiratory pathogens with pandemic potential such as h1n1, sars-cov, and mers-cov may remain viable in the environment for a longer time up to months on these objects until picked up with another person to contaminate the mucus membranes through touching the nose, mouse or eyes [56] . there are several factors that control the virus's survival in the environment on these objects, such as the j o u r n a l p r e -p r o o f journal pre-proof temperature, the relative humidity, the strain of the virus [56] . some coronaviruses such as hcov-229e and mers-cov were detected on some environmental surfaces during some outbreaks [57, 58] . one of the critical control measures in the case of airborne infection is to develop novel assays that enable us to detect and estimate the virus concentration in the air. this approach will have great implications, especially in the health care settings. the process of decontamination of the virus-contaminated surfaces by the appropriate disinfectants or virucidal agents was successful in case of other respiratory viruses such as sars-cov and avian influenza [59] . the hydrogen peroxide vapor showed great success in the decontamination of surfaces contaminated with sars-cov and avian influenza viruses [59] . the who suggested using two the significant burden in controlling the respiratory viruses is how to manage the person-toperson transmission. this could be adopted through many strategies. first, personal and hand hygiene are the gold standard approach toward minimizing the possibility of infection for j o u r n a l p r e -p r o o f individuals. this can be achieved by washing the hands with the antiseptics and or soaps and water for at least 30 seconds as per the who guidelines [61] . this approach will reduce the virus load in the contaminated hands from virus droplets, which might touch the nose or mouse of persons. second, practice extreme caution by applying a social distance of at least one meter between you and anyone who is coughing or sneezing [61] . third, educate the public on how to avoid the frequent touching of the mucous membranes, especially those of nose, mouse, and eyes [61] . fourth, respiratory protection and hygiene through wearing the most appropriate personal protective equipment, as suggested earlier, according to the who's standards [61] . fifth, vomiting and diarrhea were reported in many sars-cov-2 patients [62] . this is suggesting the possibility of the fecal oral route as a mode of transmission of the virus [62] . this stresses out the importance of hand hygiene to avoid any possible transmission of the virus through the fecal oral route. sixth, one of the main pillars in the control of virus spreads is the identification of the infected personnel. it is one of the crucial steps toward reducing the virus spread in a community. the earlier the identification and detection of the positive cases, the better the control of these cases. thus, they will receive the better health care treatment and avoid virus spread particular ly during the very early stage of the infection. seventh, special attention should be paid to how to protect the health care workers dealing with active cases. they should provide by the standard originated from bats [25, 65] . this highlights the mandate for continuous monitoring of the coronaviruses population in bats as an alarm to the emergence of new novel coronaviruses in the future. second, there is ongoing research to develop antiviral therapy that might help in the treatment of the active human cases of the sars-cov-2. each approach mainly depends on a unique strategy to halt the virus replication or disrupt it at a certain point; thus, the virus cannot complete its replication cycle in the host. therefore, it reduces the possibility of virus shedding from the infected patients to its close contacts. a recent study used some serine protease tmprss2 inhibitors to prevent the virus from entry to the cell. these compounds are clinically approved and potentially will reduce the virus replication [8] . some fda approved anthelminthic compounds such as niclosamide have potent antiviral effects not only for sars-cov-2 but also for other viruses, including sars-cov, mers-cov, zika virus, and hcv [63] . a recent study showed the great values of the remdesivir (gs-5734) for prophylactic as well as treatment for the mers-cov in the rhesus macaque model [64] . this approach could be a promising trend for the prevention and treatment of sars-cov-2; however, further studies are required to confirm these findings. another method was to use the nucleotide analog inhibitors, such as the remdesivir, which targets the viral polymerase (rna dependant rna polymerase). this approach diminished the ability of the virus to copy its genome; thus, the replication cycle j o u r n a l p r e -p r o o f may be interrupted. this compound gave promising results in the cell culture model, which requires further testing in laboratory animals before the clinical trials [65] . although sars-cov-2 is rapidly transmitting across the globe, it may be contained if sincere containment measures are implemented. the drastic drop in the number of reported cases in china, along with a reduction in the reported deaths could be an indicator of an early containment of the virus. implementing the one health concept from all aspects involving the animal, environment, and humans could contribute substantially to the control of sars-cov-2 in the near future. the authors declare there is no conflict of interest. we wish to thank king abdul-aziz city for science and technology (kacst) for their generous funding through the mers-cov research grant program (number 20-0004), which is a part of targeted research program (trp). aetiology: koch's postulates fulfilled for sars virus isolation of a novel coronavirus from a man with pneumonia in saudi arabia another decade, another coronavirus molecular evolution and emergence of avian 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possible role of dry surface contamination isolation and identification of human coronavirus 229e from frequently touched environmental surfaces of a university classroom that is cleaned daily extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards evaluating the virucidal efficacy of hydrogen peroxide vapour virucidal activity of world health organization-recommended formulations against enveloped viruses, including zika, ebola, and emerging coronaviruses covid-19) advice for the public clinical features of patients infected with 2019 novel coronavirus in wuhan broad spectrum antiviral agent niclosamide and its therapeutic potential prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus key: cord-274141-vujx538o authors: chinsembu, kazhila c. title: coronaviruses and nature’s pharmacy for the relief of coronavirus disease 2019 date: 2020-10-06 journal: rev bras farmacogn doi: 10.1007/s43450-020-00104-7 sha: doc_id: 274141 cord_uid: vujx538o [image: see text] coronaviruses (covs) cause acute and chronic respiratory, enteric, and central nervous system diseases in many species of animals including humans. while the history of covs dates as far back as the 1940s, identification of the first human covs (hcovs) as causative agents of mild respiratory infections was reported in the 1960s. these hcovs were named human cov 229e (hcov-229e) and hcov-oc43 (pillaiyar et al. 2020) . the replication and pathogenesis of covs were actively studied by virologists in the 1970s, leading to the discovery of four other human covs, namely hcov-hong kong university 1 (hcov-hku1), human coronavirus-nl63 (hcov-nl63), severe acute respiratory syndrome (sars)-cov, and middle east respiratory syndrome (mers)-cov (pillaiyar et al. 2020) . sars is a highly contagious and often fatal respiratory infection. hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1 are ubiquitous and cause approximately onethird of common cold in humans. hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1 strains are usually associated with mild, self-limiting upper respiratory tract infections, such as the common cold (andersen et al. 2020) . however, subtypes hcov-229e, hcov-oc43, hcov-hku1, and hcov-nl63 can cause life-threatening pneumonia and bronchitis in the elderly and immunologically compromised individuals including those undergoing chemotherapy and persons living with hiv/aids. indeed, before the discovery of sars-cov, two coronaviruses, hcov-229e and hcov-oc43, were known to infect humans, but they caused only selflimiting upper respiratory tract infections (30% of the common colds), and had never been reported to cause severe illness (kuba et al. 2005) . sars-cov caused a global epidemic from november 2002 to 2003; about 8422 people were infected in 32 countries, 916 persons died, and the fatality rate was 10-15%. analysis of the full sequence of sars-cov, which is about 29.7 kb in length, reveals that it is a large, single-stranded, positivesense rna virus . in 2013, mers-cov also triggered an epidemic in the middle east and a major outbreak in south korea occurred in may-june 2015 (kim et al. 2017a) . with a case-fatality rate of 35.5%, mers-cov is heavily endemic in dromedary camels and causes lower respiratory tract infections in humans (kim et al. 2017a) . although the sars-cov epidemic had been under control for years, wang et al. (2017) warned that re-emergence of this threatening infection posed a possible global risk as potentially new strains of sars-cov would be more dangerous than the previous ones. toward the end of 2019 and the beginning of 2020, multiple human cases of a new viral infection were reportedly linked to the huanan seafood wholesale "wet" market (south china seafood city food market) in wuhan, china zhang et al. 2020 ). on 7 january 2020, the new virus was identified as a novel coronavirus and officially named by the world health organization as 2019-ncov ). on 20 january 2020, the national health commission of china confirmed the human-to-human transmission of 2019-ncov with its presenting common symptoms such as fever, tiredness, dry cough, shortness of breath, and pneumonia (elfiky 2020) , and less common signs as diarrhea, conjunctivitis, headache, loss of taste or smell, and rash on skin or discoloration of fingers and toes. the world health organization also recognized 2019-ncov as the causative agent of the coronavirus disease 2019 . the 2019-ncov was later named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the international committee on taxonomy of viruses, coronaviridae study group (ling 2020) . a phylogenetic analysis found a bat origin for the sars-cov-2 (lu et al. 2020; wan et al. 2020) . yang et al. (2020) reported that sars-cov-2, formerly known as 2019-ncov, the causative pathogen of covid-19, had rapidly spread across china and around the world, causing an outbreak of acute infectious pneumonia. the spread of sars-cov-2, a new highly contagious and lethal strain of hcovs, has now caused a global pandemic (remuzzi and remuzzi 2020) . in the morning of 7 september 2020, johns hopkins university reported that there were over 26,994,442 total confirmed cases and 880,994 deaths globally; over 6,189,488 cases and 187,541 deaths were in the united states of america (usa). covid-19 has spread more rapidly due to increased global travel and adaptation of sars-cov-2 in every environment (vellingiri et al. 2020) . currently, the seven identified human coronaviruses (hcovs) are sars-cov-2, mers-cov, sars-cov, hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1 strains (andersen et al. 2020 ). molecular evolutionary analysis shows that all hcovs originate in animals; sars-cov, mers-cov, hcov-nl63, and hcov-229e are derived from bats; hcov-oc43 and hcov-hku1 are sourced from rodents (forni et al. 2017) . the 229e and nl63 strains of hcovs belong to alpha coronaviruses while oc43, hku1, sars, mers, and sars-cov-2 belong to beta coronaviruses (elfiky 2020) . most people infected with sars-cov, mers-cov, and sars-cov-2 present with severe respiratory distress, and many of the infected succumb to coronavirus infection (andersen et al. 2020) . pillaiyar et al. (2020) reviewed recent developments in the discovery and development of synthetic drugs to treat hcovs. however, these drugs have not achieved extended distribution for clinical application because they have not yet been tested in rigorous randomized controlled clinical trials (ford et al. 2020) . zhou et al. (2020a, b) reported that there were no effective drugs targeting sars-cov-2. yang et al. (2020) also stated no efficacious and safe antiviral drugs or vaccines were available for the treatment of this sudden and lethal disease. supportive care and non-specific treatment to ameliorate covid-19 symptoms of the patient are the main options available. remdesivir, produced by the company gilead, shows ec 90 of 1.76 μm against sars-cov-2 in vitro (elfiky 2020). on 1 may 2020, remdesivir was granted approval by the us food and drug administration for emergency use in the treatment of covid-19 patients. an inhibitor of the enzyme rnadependent rna polymerase (rdrp) involved in hcov replication, the drug reduced mortality from 11 to 8% and duration of symptoms from 15 to 11 days. however, in comparison with the placebo, remdesivir's reduction of patient mortality was not significantly different. due to the absence of conventional therapies, yang et al. (2020) reported that more than 85% of sars-cov-2-infected patients in china were treated with traditional chinese medicine (tcm). during the 2002 to 2003 outbreak, tcm was also used as a supplementary treatment of severe laboratoryconfirmed conditions of sars (hsu et al. 2006a) . along this trajectory, the aim of the current review is to provide an update on the bioactivity functions of medicinal plants and other natural products that have previously been used to treat coronaviruses. in the short term, knowledge of these natural products may be significant in the relief of covid-19 symptoms. in the long term, natural products that alleviate covid-19 symptoms may be used in the search for novel drugs against sars-cov-2. in the face of current challenges to the treatment of covid-19, data presented in this review may help to reimagine a new future where natural products are the new frontier for more efficacious and low-cost sars-cov-2 inhibitors. the goal is to create a strong bioprospecting pipeline from which pharmaceutical companies can advance the discovery and repurposing of covid-19 drugs from current medicinal plants and other natural products. to prevent author bias, this review was carried out using a comprehensive and systematic data mining approach similar to that used by chinsembu (2019) . to obtain pertinent literature, the key words "sars" and "medicinal plants" were concomitantly searched in google scholar, elsevier's payto-view service science direct, scopus, scielo, and pubmed central, the us national library of medicine's digital archive of biomedical and life sciences journal literature. literature sources included peer-reviewed journal articles and refereed books. medicinal plants, mushrooms, algae, and sponges with anti-sars activity were included. wherever possible, active compounds and anti-sars profiles mainly half maximal inhibitory concentration (ic 50 ) and effective concentration (ec 50 ) were recorded. inclusion criteria included plants, fungi, and marine organisms, namely algae and sponges. exclusion criteria included animal products. countries reported in the review were those where primary data were collected; hence, geographical scope was not deliberately selected. the international plant names index (http://www.ipni.org) and the plant list (http://www.theplantlist.org) were used to verify names of plant species. the spike protein, a type i membrane-bound protein, is essential for sars-cov attachment to the host cell receptor angiotensin-converting enzyme 2 (ace2). in general, sars-cov entry starts with the attachment of the virus to its specific receptor ace2 on the host cell surface (keyaerts et al. 2007 ). binding of ace2 then induces the viral envelope protein to undergo conformational changes that mediate fusion between sars-cov and host cell membranes. the spike protein is responsible for these two steps in the coronavirus entry process (keyaerts et al. 2007) . a member of the c-type lectin superfamily, dendritic cell (dc)-specific icamgrabbing non-intergrin (dc-sign), a molecule designated as cd209, binds to surface proteins of sars-cov, consequently facilitating viral entry (liu et al. 2015) . sars-cov spike protein contains a number of n-linked glycosylation sites that modulate binding to the dc-sign receptor on dc (zhang et al. 2007 ). sars-cov binding to dc-sign is required for dc infection and target cell trans-infection. dc-sign plays an important role in mediating dc adhesion and internalization of sars-cov. antiviral agents from plants generally block virus entry and membrane fusion (hsieh et al. 2004 ). de clercq (2005 suggested that it was feasible to develop sars-cov fusion inhibitors analogous to enfuvirtide, a linear 36-amino acid synthetic peptide marketed under the trade name fuzeon, an approved anti-hiv drug that inhibits the entry of the virus into cells. sars-cov entry inhibitors fall into several categories. the first group consists of inhibitors that bind to the ace2 receptor, the second class comprises entry inhibitors that bind to the virus and prevent it from interacting with its receptors, and the third set of inhibitors can obstruct the conformational changes thus thwarting sars-cov fusion with the target cells (keyaerts et al. 2007 ). an effective inhibitor of sars-cov, the medicinal basidiomycete ganoderma lucidum (curtis) karst. contains ganoderic acid f, a lanostane triterpene that acts as an inhibitor of ace2 receptor (ic 50 4.7 × 10 −6 m) (morigiwa et al. 1986 ). lee (2019) asserts that lectins have emerged as a new class of antiviral biologic drugs or biologics by taking advantage of a unique glycosylation pattern only found on the surface of viruses. lectins, a group of proteins with carbohydrate recognition activity, recognize the sars-cov spike protein (liu et al. 2015) . keyaerts et al. (2007) found prominent anti-sars-cov activity among mannose-specific plant lectins. one of the most potent lectins against the sars-covinduced cytopathicity is the mannose-specific plant lectin isolated from leek (allium porrum l.) with an ec 50 of 0.45 μg/ml and a selectivity index of > 222. the n-acetyl glucosamine-specific lectins isolated from the stinging nettle (urtica dioica l.) and from the tobacco plant (nicotiana tabacum l.) were also markedly active against the sars-cov with a selectivity index of > 77 and > 59, respectively (kumaki et al. 2011) . a mannose-specific lectin (agglutinin) of hippeastrum striatum (lam.) h.e.moore prevented the penetration of sars-cov into vero e6 cells (ec 50 2.5 μg/ml at 4°c) (keyaerts et al. 2007 ). sars-cov spike protein contains 12 n-glycosylation sites and the sugars attached to four of these n-glycosylation sites have been identified (zhou et al. 2010) . two of the four sugars were found to be high-mannose-type glycans and the other two showed complex glycan structures . the potent anti-sars-cov activity of mannose-specific plant lectins is therefore attributable to the presence of high-mannose-type glycans in the sars-cov spike protein (keyaerts et al. 2007; zhou et al. 2010 ). many lectins from algae have antiviral activity. a highly potent broad-spectrum antiviral, griffithsin is a 121-amino acid red alga-derived lectin that binds to the terminal mannose residues of the asparagine(n)-linked man 5-9 glcnac2 structures found on the envelope of sars-cov (lee 2019). initially identified as an anti-hiv-1 agent with ec 50 of 0.43-0.98 nm, griffithsin isolated from the aqueous extract of the red algae griffithsia corallinoides (l.) trevisan found off the eastern shore of chatham island, new zealand, is a 12.7-kda lectin that also inhibits sars-cov (ec 50 0.048-960 nm), various avian cov subtypes (ec 50 0.032-0.57 nm), bovine cov (ec 50 0.057 nm), puffinosis cov (ec 50 0.57 nm), and hcov mutants with ec 50 of 0.16 nm (mitchell et al. 2017 ). some viruses such as ebola and sars-cov use cathepsins, a family of proteases, to infect host cells (huang et al. 2006) . inhibitors of cathepsin l prevent entry of sars-cov into target cells (simmons et al. 2005) . unfortunately, many cathepsin inhibitors are non-selective (zhou et al. 2016) . leupeptin (1), also known as n-acetyl-l-leucyl-l-leucyl-l-argininal, is protease inhibitor produced by actinomycetes and isolated from a strain of streptomyces exfoliatus (waksman and curtis) waksman and henrici, which can inhibit cysteine, serine, and threonine peptidases. simmons et al. (2005) showed that inhibitors of proteases such as leupeptin act as potent inhibitors of sars-cov entry into host cells. sars-cov spike protein-driven virus-cell fusion is inhibited by 1 , which exhibited a dose-dependent inhibition of sars-cov infection ) (ic 95 15.2 μm). this inhibition is the result of an absolute requirement in cell lines for endosomal processing of the sars-cov spike protein by cathepsin l during entry into the target cell ). the oligopeptide antipain (2), an inhibitor of trypsin and papain, isolated from actinomycetes inhibited sars-cov entry into host cells (simmons et al. 2005 ). (dong et al. 2016; zhou et al., 2020a, b) , and cassia tora l. emodin inhibited sars-cov via the blockage of viral entry by binding to the spike proteins and interfering with sars-cov 3cl pro activity (hyun et al. 2009 ). in assays involving sars-cov and hcov-oc43, emodin in a dose-dependent manner significantly blocked the interaction between the sars-cov spike protein and ace2 (a functional sars-cov receptor), inhibited the 3a ion channel (k 1/2 value of about 20 μm), and stopped new coronavirus release (dong et al. 2016; ho et al. 2007 ). sars-cov-2 spike protein directly binds to the host cell surface ace2 receptor facilitating virus entry and replication . corollary, emodin can be considered a potential lead therapeutic agent in the treatment of sars-cov-2 infection. experimental data now suggest that combining emodin and the drug toremifene, a first-generation non-steroidal selective estrogen receptor modulator for the treatment of metastatic breast cancer, provides a potential therapeutic approach for sars-cov-2 (zhou et al., 2020a, b) . terpenoids isolated from medicinal plants have attracted attention because many of them exhibit specific in vitro antiviral effect against sars-cov (wen et al. 2007 ). triterpenoids inhibit the entry of ebola, marburg, hiv, and influenza a viruses with distinct structure-activity relationships (si et al. 2018 ). these natural agents modulate a broad range of virus-host fusion functions via wrapping the heptad repeat 2 domain prevalent in viral envelopes. euphorbia neriifolia l. produces 22 triterpenoids with stronger activity against hcovs than actinomycin d (chang et al. 2012) . saikosaponins represent a group of oleanane derivatives, usually as glucosides, found in medicinal plants in the genera bupleurum l., heteromorpha cham. & schltdl., and scrophularia l. (cheng et al. 2006) . saikosaponins a, b 2 , c, and d demonstrated anti-hcov-229e activity at concentrations of 0.25-25 μm, with the strongest activity being noted for saikosaponin b 2 (4) (ic 50 1.7 ± 0.1 μm) (cheng et al. 2006 ). since saikosaponin b 2 blocks hcov-229e attachment and penetration, it could be a novel lead for the development of a potential chemo-preventive agent for sars-cov infection (cheng et al. 2006) . yang et al. (2020) reported that saikosaponins and glycyrrhizin from toona sinensis (juss.) m.roem. had potent anti-sars-cov effects by inhibition of viral cellular entry, adsorption, and penetration. the licorice plant, glycyrrhiza glabra l., was reported to have specific anti-sars effects (polansky and lori 2020) . constituents of licorice include triterpenoids such as glycyrrhizin or glycyrrhizic acid (5). glycyrrhizin (5) exerted very high antiviral activity and in vitro inhibition of sars-cov (ec 50 300 μg/ml) (cinatl et al. 2003) . glycyrrhizin and its derivatives inhibited sars-cov replication in vitro (baltina et al. 2015) . ryu et al. (2010a) reported that 95% methanolic extracts of the bark of tripterygium regelii sprague & takeda remarkably inhibited sars-cov 3cl pro activity (> 70% inhibition at 30 μg/ml) (ryu et al. 2010a) . the active compounds of t. regelii are four quinone-methide triterpenoid derivatives: celastrol, pristimerin, tingenone, and iguesterin (6) with ic 50 values of 10.3, 5.5, 9.9, and 2.6 μm, respectively, against sars-cov 3cl pro (ryu et al. 2010a ). the seeds of aesculus turbinata blume, a medicinal plant found in china, japan, and south korea, contain a natural saponins called escins, constituents with strong virucidal effects against sars-cov (ec 50 of 6 μm, selectivity index value of 2.5) (kim et al. 2017b; salinas et al. 2019) . betulin and betulinic acid, the main antiviral components of the betula l. plants, inhibit hiv fusion and one of the steps in hiv maturation (chinsembu 2016a) . a methanolic extract of betula papyrifera marshall exerted antiviral activity against bovine coronavirus (rastogi et al. 2015) . many natural polyphenols, with antioxidant properties, possess antiviral activity and some of them, for example, myricetin (7), an inhibitor of coronavirus helicase nsp13 (ic 50 value of 2.71 μm), are already being used against coronaviruses (semwal et al. 2016; yu et al. 2012) . naturally occurring flavonoids such as quercetin, naringin, hesperetin, and catechin, the most abundant polyphenols in the human diet, are usually found in fruit and vegetables as glycosides and sometimes as acylglycosides. luteolin and quercetin can interfere with adsorption of the virus to its host cells. it was hypothesized that specific flavonoids, such as quercetin, hesperetin, and myricetin (7) and their glycosylated derivatives, may play an effective role in inhibiting sars-cov entry into host cells, specifically by binding with high affinity to the spike protein, helicase, and protease sites on the ace receptor (ngwa et al. 2020) , in addition to acting against fatty acid synthase (wen et al., 2011) . extracted from tcm herbal formulae used to treat sars, the following active polyphenols inhibited various steps during sars-cov entry and replication: baicalin, emodin, epigallocatechin, gallate, gallocatechin gallate, herbacetin, isobavaschalcone, kaempferol derivatives, luteolin, myricetin, quercetin, 3-β-d-glucoside, rhoifolin, pectolinarin, scutellarein, and tetra-o-galloyl-β-d-glucose ). quercetin (8) is an aglycone present at high concentration in onions. this compound has virucidal activity against enveloped viruses such as mengovirus, herpes simplex, parainfluenza type 3, pseudorabies, respiratory syncytial, and sindbis viruses ). quercetin is able to inhibit h + -atpase of lysosomal membrane and thus prevents virus coat removal and blocks viral replication and competitively inhibits sars-cov 3cl pro with ic 50 of 42.79 ± 4.97 μm (chen et al. 2006) . torreya nucifera (l.) siebold & zucc. is a traditional medicinal plant in asia. the ethanol leaf extract of t. nucifera exerts good sars-cov 3cl pro inhibitory activity (62% at 100 μg/ml) (ryu et al. 2010b ). following the bioactivityguided fractionation of t. nucifera, the biflavone amentoflavone (9) showed the most potent sars-cov 3cl pro inhibitory effect with ic 50 of 8.3 μm (ryu et al. 2010b) . apigenin, luteolin, and quercetin inhibited sars-cov 3cl pro activity with ic 50 values of 280.8, 20.2, and 23.8 μm, respectively (ryu et al. 2010b ). zhong et al. (2013) found that jinchai, a capsule of tcm consisting of plants such as lonicera japonica thunb., bupleurum chinense dc., astragalus membranaceus (fisch.) bunge, and codonopsis pilosula subsp. tangshen (oliv.) d.y.hong inhibited sars-cov. jinchai blocks sars-cov infection by weakening adsorption of viruses to cells and reduces the ability of sars-cov to infect surrounding tissues (zhong et al. 2013) . the main active constituents of jinchai are chlorogenic acid and baicalin (10) whose ec 50 (μg/ml) at 48 h was 12.5 to 25, ec 50 (μg/ml) at 72 h was 25 to 50, cc 50 (μg/ml) was > 100, and si calculated as cc 50 /ec 50 at 48 h was > 4 to 8 . camellia sinensis (l.) kuntze (black tea) polyphenol epigallocatechin gallate (egcg) inhibited bovine coronavirus propagation; egcg interfered with the adsorption of bovine coronavirus to madin-darby bovine kidney cells (bansal et al. 2013) . although sanguisorba officinalis l. extract inhibits hiv-1 infection by binding to the viral envelope and blocking entry of the virus, it did not inhibit sars-cov entry (liang et al. 2013) . in oriental herbal medicine, the bark of cinnamon plant cinnamomum cassia (l.) j. presl has been used as a spice, infusion, and prime component of herbal remedies for common cold, gastrointestinal infections, cancer, chronic cardiovascular disease, and gynecological disorders. zhuang et al. (2009) found that cinnamon bark extract has anti-rna viral effects. the cinnamon extract inhibited wild-type sars-cov infection in vitro with an ic 50 of 43 μm; and the proposed mechanism of action for cinnamon bark extract was blocking cell entry via endocytosis (polansky and lori 2020) . whereas the antiviral, anti-bacterial, and anti-cancer effects were attributable to essential oils such as cinnamaldehyde (11), the anti-inflammatory actions of cinnamon water extract were due to the presence of polyphenols such as flavonoids and tannins. molecular docking studies show that 11 may block the attachment of sarc-cov-2, though detailed in vitro and in vivo studies are required to confirm its efficacy (asif et al. 2020) . compound 11 had the lowest electronegativity value among the phytochemicals with a score of − 4.34; binding affinity in kilocalorie per mole was − 5 (asif et al. 2020; kulkarni et al. 2020 ). stilbene derivatives displayed antiviral activities against sars-cov ). the stilbenoid resveratrol (12) exists widely in different plants including grape vitis vinifera l., polygonum cuspidatum, and cranberry vaccinium macrocarpon aiton (lin et al. 2017) . resveratrol inhibits various viral infections, including the inhibition of hiv and sars-cov replication . compound 12 was a potent in vitro inhibitor of sars-cov (at ≤ 0.5 mg/ml) and mers-cov, at ≤ 62.5 μm within 24 h of infection lin et al. 2017) . it was predicted that resveratrol (12) could be a potential agent against new hcovs in the near future (lin et al. 2017 ). polysaccharides isolated from tcm herb astragalus mongholicus bunge have been widely used to boost immunity against viruses. a study by zhang et al. (2018) showed that supplementation with a. mongholicus polysaccharides can inhibit the replication of avian coronaviruses. extracts of acanthopanax gracilistylus var. trifoliolatus c.b. shang, the root of sophora flavescens aiton, and the dried root of sanguisorba officinalis and torilis elata spreng. reduced the in vitro replication of sars-cov more potently than the ribavirin drug control (kim et al. 2010) . cimicifuga racemosa (l.) nutt., melia azedarach l., coptis chinensis franch., phellodendron chinense c.k.schneid., and sophora tonkinensis var. polyphylla x.c.huang & z.c.zhou decreased mouse hepatitis virus a59 (mhv-a59) production and intracellular viral rna and protein expression with ec 50 values ranging from 2 to 27.5 μg/ml (kim et al. 2008) . cimicifuga racemosa, m. azedarach, c. chinensis, p. chinense, and s. tonkinensis were good candidates for the treatment of coronaviral infections in both humans and animals (kim et al. 2008 ). the underlying antiviral mechanism was most likely due to the inhibition of rna-dependent rna polymerase or proteases that are crucial for coronavirus rna replication. melia azedarach and c. chinensis may also affect virus assembly or release. silvestrol (13), a natural cyclopenta[b]benzofuran, isolated from the fruits and twigs of aglaia foveolata pannell, at 0.6-2 μm, inhibited cap-dependent viral mrna translation of hcov-229e with an ic 50 of 40 nm (müller et al. 2018) . ouabain (14), also known as g-strophanthin, is a toxic cardiac glycoside that was traditionally used as an arrow poison for both hunting and warfare in eastern africa. however, this substance, in lower doses, can be used medically to treat hypotension and some arrhythmias. in addition, ouabain diminishes sars-cov titers and the number of sars-cov rna copies at 0-3000 nm (yang et al. 2018) . wu et al. (2020) described drug names, chemical structures, pharmacological functions, and sources of 20 potential rdrp inhibitors from an in-house natural product database. their results were based on computer virtual screening because no in vitro and in vivo experiments were conducted. andrographolide has been studied for its effects on cell signaling, immunomodulation, and stroke. using computational approaches, enmozhi et al. (2020) showed that andrographolide (15), an extremelly bitter labdane diterpenoid isolated from andrographis paniculata (burm.f.) wall. ex nees, docked successfully in the binding site of sars-cov-2 m pro . the molecule obeys lipinski's rule making it a promising compound to treat covid-19 (enmozhi et al. 2020) . omacetaxine mepesuccinate (trade names synribo), formerly named as homoharringtonine (16), is a plant alkaloid derived from cephalotaxus fortunei hook that was approved by the us fda in october 2012 for the treatment of chronic myeloid leukemia in adults. homoharringtonine has been reported to exhibit potent inhibitory activity against coronaviruses, namely porcine epidemic diarrhea virus, murine hepatitis virus (dong et al. 2018; andersen et al. 2019) , and sars-cov-2 with ec 50 at 2.10 μm (choy et al. 2020 ). tylophorine (17), a phenanthraindolizidine alkaloid, has anti-hcov activity in the nanomolar concentrations, up to 1000 nm . tylophorine derivatives, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines, have been identified as potent in vitro inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (tgev). this class of potent compounds showed ec 50 values ranging from 8 to 1468 nm as determined by immunofluorescent assay of the expression of tgev spike and nucleocapsid proteins and by real-time qrt-pcr analysis of viral yields. tylophorine compounds could be novel leads for further drug development on their own or as templates for drug design . tylophorine and related alkaloids, isolated from tylophora indica merr., inhibit tgev replication for anti-coronavirus activity and suppress nitric oxide production in raw264.7 cells, a measure of antiinflammation (lee et al., 2012a, b) . additionally, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of tylophorine (65.7%) in rats (lee et al., 2012a, b) . the chymotrypsin-like protease (3cl pro ) is vital for sars-cov replication and is therefore a promising drug target. the active site of sars-cov 3cl pro has a catalytic dyad with the sulfur of cys145 as a nucleophile and the imidazole ring of his41 as a general base (ryu et al. 2010a) . also called main protease (m pro ), the 3cl pro of sars-cov mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins; thus, it is an important target for drug development . curcumin inhibits sars-cov 3cl pro (ic 50 value of 23.5 μm) (ryu et al. 2010a ). the extracts of cibotium barometz (l.) j.sm. and dioscorea batatas decne. also inhibited sars-cov 3cl pro activity with ic 50 values of 39 μg/ml and 44 μg/ml, respectively (cuong et al. 2009; wen et al. 2011) . tannic acid, 3-isotheaflavin-3-gallate, and theaflavin-3,3′digallate, three phenolic compounds from black tea, inhibit sars-cov 3cl pro with ic 50 values of 3, 7, and 9.5 μm, respectively . isatis indigotica fortune root extracts were frequently used as a remedy during the 2002/2003 sars outbreak in china. a water extract of i. indigotica had anti-sars-cov 3cl pro activity . the root extract of i. indigotica contains indigo, indirubin, indican (indoxyl-β-d-glucoside), β-sitosterol, γ-sitosterol, and sinigrin (18). the ic 50 in the cell-free assays was 115 μm for β-sitosterol, 121 μm for sinigrin, and 300 μm for indigo. the cell-based assay indicated that the antioxidant sinigrin (ic 50 217 μm) was more efficient in blocking the cleavage processing of the 3cl pro than indigo (ic 50 752 μm) and β-sitosterol (ic 50 1210 μm) . compound 18 is a glucosinolatetype compound also found in some plants of the family brassicaceae such a brussels sprouts, broccoli, and the seeds of black mustard (brassica nigra (l.) andrz. echinaforce® is a standardized preparation extracted from freshly harvested herb (herba tintura 2580 mg/125 drops) and roots (radix tintura 135 mg/125 drops) of echinacea purpurea (l.) moench plants (purple coneflower) with a 65% alcoholic solution (schapowal 2020) . echinaforce® reduces the infectivity of hcov-229e in a dose-dependent manner. it inhibits hcov-229e infection of respiratory epithelial cells (schapowal 2020) . hcov-229e was irreversibly inactivated when exposed to echinaforce® (ic 50 of 3.2 μg/ml) (schapowal 2020) . echinaforce® showed a dosedependent reduction of hcov 229e infectivity with an ic 50 = 9 ± 3 μg/ml (engler et al. 2017) . complete neutralization was achieved with 50 μg/ml (engler et al. 2017) . similar inhibition was observed for mers-cov when 10 μg/ml of echinaforce® reduced infectivity by 99.9% and 50 μg/ml of echinaforce fully blocked infectivity (engler et al. 2017 ). extracts of echinacea purpurea have been used traditionally in north america for the treatment of various infections and wounds, and they have become very popular herbal medicines globally (hudson 2012) . rondanelli et al. (2018) found that a combination of e. purpurea, vitamin d, vitamin c, and zinc was useful in the treatment of the common cold. vitamin d supplementation also lessens the risk of covid-19 infection and deaths (grant et al. 2020) . echinacea angustifolia dc., echinacea pallida (nutt.) nutt., and echinacea purpurea are the most common echinacea moench species recognized for treating common cold and sars (hudson 2012) . coronavirus infection in humans is characterized by uncontrolled replication of the virus and a prominent proinflammatory response (wong and yuen 2008) . still, in the management of coronavirus disease, the role of immunomodulators to decrease excessive inflammation remains elusive (wong and yuen 2008) . the immunomodulatory compounds tomentins a-e (19-23), geranylated flavonoids, isolated from paulownia tomentosa (thunb.) steud. lower the concentration of pro-inflammatory cytokines interleukin-1β and tumor necrosis factor alpha . these tomentins could be useful in allaying the multisystem inflammatory syndrome, also known as the cytokine storm, seen in many covid-19 patients. a n t i -i n f l a m m a t o r y a n d a n t i n e o p l a s t i c bisbenzylisoquinoline alkaloids isolated from stephania tetrandra s.moore, namely cepharanthine (24), fangchinoline, and tetrandrine, acted as immunomodulators and inhibited the expression of hcov-oc43 spike and nucleocapsid proteins (kim et al. 2019) . after the cells were infected with hcov-oc43, each compound (5 μm) was added to the wells, and cytokine mrna was qantified by real-time qrt-pcr. compound treatment following virus infection reduced the mrna expression levels of ifn-α1 by almost 20-fold at 4 days post infection, and about 100-fold reduction for ifn-β1 at 3 days post infection (kim et al. 2019 ). according to islam et al. (2020) , natural products with anticoronavirus activity are major constituents of common dietary supplements and can therefore be used to improve the immunity of the general population in pandemics such as covid-19. according to kwon et al. (2013) , five phlorotannins of the edible brown algae ecklonia cava kjellman competitively inhibit the binding of sars-cov spike protein to sialic acids at a concentration of less than 36.6 μm. two phlorotannins caused the inhibition of sars-cov rna and sars-cov protein synthesis in late stages, with ic 50 values of 12.2 ± 2.8 and 14.6 ± 1.3 μm, respectively . the results suggest that compounds isolated from e. cava have strong antiviral activity and may be developed into natural therapeutic drugs against sars-cov infection . a commercially available e. cava extract, seapolynol™, was approved as a new dietary ingredient (ndi) by the us food and drug administration in 2008 (fda-1995-s-0039-0176) (lee et al., 2012a, b) . an ethanol extract of the edible brown algae ecklonia cava, laminariaceae, contained eight phlorotannins that exhibited a dose-dependent (competitive) inhibitory effect on sars-cov 3cl pro with ic 50 values ranging from 2.7 to 164.7 μm . dieckol showed the most potent sars-cov 3cl pro trans/cis-cleavage inhibitory effects. isolated from the marine alga halimeda tuna (j.ellis & solander) j.v.lamouroux, halitunal is a novel diterpene aldehyde with a unique cyclopentadieno[c]pyran ring system. halitunal displayed antiviral activity against murine coronavirus a59 in vitro (gustafson et al. 2004) . dercitin, an acridine alkaloid isolated from a marine sponge in the genus dercitus gray, inhibited the coronavirus a59 strain (ec 50 1.8 μm). mycalamides a and b, protein synthesis inhibitors and antiviral compounds isolated from a sponge mycale incrustans burton, also inhibited coronavirus a59 (laport et al. 2009 ). in vivo activity against coronavirus a59 was observed in mice treated with a 2% mycalamide mixture at a dosage of 0.2 μg/kg daily with 100% survival over a 2-week period (gallimore 2017) . triterpenes (thyrsiferol, thyrsiferol acetate, venustatriol) isolated from the red alga laurencia venusta yamada inhibit coronaviruses, and orthosterol disodium sulfates from the sponge petrosia weinbergi van soest inhibit coronavirus a59 (el sayed 2000) . spirulina platensis (gomont) geitler is a microscopic filamentous alga rich in proteins, vitamins, essential amino acids, minerals, and essential fatty acids like γ-linolenic acid. it is produced commercially and sold as a food supplement in health food stores around the world. spirulina platensis (15 g) enhances the immune status and inflammatory and oxidative markers of covid-19 patients (mccarty and dinicolantonio 2020). china xiao et al. (2003) provided leading insights into the use of fourteen chinese herbal medicines in the prevention and treatment of sars. tcm played an important role in the fight against sars and it was reported by practitioners to be very effective (lau et al. 2005) . the state administration of tcm of the people's republic of china formed a panel of tcm specialists to draw up a technical scheme for the prevention and treatment of sars-cov using tcm . scutellaria baicalensis is one of the most widely used tcms and its roots are used to treat inflammation, cancer, and viral and bacterial infections. baicalin, baicalein, wogonin, and oroxylin a are the main active components in s. baicalensis. the most abundant active compound, baicalin, exhibits anti-sars-cov properties. yeh et al. (2013) affirmed that licorice (glycyrrhiza uralensis fisch. ex dc.) is a common ingredient in prescriptions of tcm. licorice has antiviral activity against various dna and rna viruses including hiv and sars-cov. chinese multi-plant remedy consisting of houttuynia cordata thunb., chrysanthemum morifolium ramat., artemisia scoparia waldst. & kit., eupatorium fortunei turcz., and amomum tsao-ko crevost & lemarié was effective in the prevention and treatment of sars-cov (zhang and chen 2008) . extracts of lonicera japonica are a common tcm used to treat sars-cov (wang et al. 2014) . to control sars-cov in 2003, l. japonica was widely used in prescriptions publicly published by the state administration of tcm. panax ginseng c.a.mey., commonly known as ginseng, is a known natural antiviral agent with protective effects against sars-cov (im et al. 2016) . extracts of t. sinensis inhibit sars-cov replication wu et al. 2014 ). extracts of plants lycoris radiata miq., artemisia annua l., pyrrosia lingua (thunb.) farw., and lindera aggregata (sims) kosterm. displayed anti-sars-cov actions with ec 50 values of 2.4 ± 0.2, 34.5 ± 2.6, 43.2 ± 14.1, and 88.2 ± 7.7 μg/ml, respectively (li et al. 2005 ). an ethanol stem cortex extract of l. radiata was very potent against sars-cov (ec 50 of 2.1 ± 0.2 nm against the bj-006 viral strain) (li et al. 2005) . the active compound lycorine was identified as the anti-sars-cov component (ec 50 value of 15.7 ± 1.2 nm). lycorine shows moderate to potent antiviral activity and reduces viral titers of sars-cov (roy et al. 2018) . jinchai is a widely used tcm that deters sars-cov entry, replication, and lung inflammation induced by viral infection (zhong et al. 2013) . roots and rhizomes of glycyrrhiza glabra, a natural sweetener that contains glycyrrhizin, inhibit virus growth and inactivate virus particles. glycyrrhizic acid, a major triterpene glycoside isolated from glycyrrhiza species, has activity against several enveloped viruses including sars-cov and hiv . diammonium glycyrrhizinate, extracted and purified from g. uralensis, inhibited sars-covs . ho et al. (2007) found that two widely used tcm plants in the family polygonaceae inhibited the interaction of sars-cov spike protein and ace2; the ic 50 values for extracts of the root tubers of rheum officinale baill. and the root tubers/vines of polygonum multiflorum ranged from 1 to 10 μ/ml (ho et al. 2007 ). chen et al. (2020) stated that the mortality of covid-19 patients in china was markedly reduced because of effective combination therapy of tcm and western medicines. at least 20 patent tcms were recommended by government guidelines in the treatment of covid-19 patients in china . several tcm plant formulae for treating sars-cov and covid-19 were reported by yang et al. (2020) . in lebanon, the oil of laurus nobilis cav. exerted remarkable activity against sars-cov with an ic 50 value of 120 μg/ml and a selectivity index of 4.16 (loizzo et al. 2008 ). an ornamental tree and a member of the cupressaceae family, cupressus sempervirens l. contains flavonoid derivatives, dit e r p e n e s , c a t e c h i n s a n d f l a v o n o l i c o l i g o m e r s , proanthocyanidins, phenolic acids, fatty acids, and essential oils (orhan and tumen 2015) . the essential oil of c. sempervirens had a mild inhibitory outcome on sars-cov activity (700 ± 2.3 μg/ml) (orhan and tumen 2015; loizzo et al. 2008) . volatile oils are potential agents for treatment of sars-cov (pasdaran et al. 2016 ). an ethanolic extract of thymus vulgaris sm. found in lebanon inhibits sars-cov (bekut et al. 2018) . lebanese essential oils have also been evaluated for their inhibitory activity against sars-cov replication in vitro by visually scoring the virus-induced cytopathogenic effect post infection. the papain-like protease (pl pro ) encoded by sars-cov cleaves the viral replicase polyprotein. since this enzymatic activity is important for viral infection, pl pro is a target of interest in the development of antiviral therapies. nine diarylheptanoids from the bark of alnus japonica (thunb.) steud. were evaluated for their inhibitory activities against sars-cov pl pro . hirsutenone was found to be the most potent inhibitor of sars-cov pl pro (ic 50 of 4.1 ± 0.3 μm). seven tanshinones isolated from salvia miltiorrhiza bunge. were selective inhibitors of sars-cov 3cl pro and pl pro viral cysteine proteases with ic 50 values from 0.8 to 30 μm (park et al. 2012b) . cryptotanshinone was the most potent inhibitor of sars-cov pl pro . cho et al. (2013) obtained 12 flavonoids from the fruits of paulownia tomentosa that function as sars-cov pl pro inhibitors. these flavonoids resulted in significant inhibition of sars-cov pl pro in a dose-dependent manner. tomentin e exhibited the highest inhibitory effect with an ic 50 of 5 ± 0.06 μm . tomentin isolated from sphaeralcea angustifolia (cav.) g.don inhibited pl pro with ic 50 values ranging from 5 to 14.4 μm. singapore chiow et al. (2016) conducted in vitro virus neutralization assays where an ethyl acetate (ea) fraction of houttuynia cordata and three of its constituent flavonoids were tested on murine coronavirus. with no cytotoxicity, the ea fraction of h. cordata, added before the viral adsorption stage, inhibited murine coronavirus (ic 50 0.98 μg/ml) (chiow et al. 2016) . the flavonoid quercetin weakly inhibited murine coronavirus with a minimum inhibitory concentration of 125 μg/ml, cytotoxicity cc 50 value of 116.50 μg/ml, and selectivity index of 0.93. the ea fraction and quercetin yielded a long-lasting anti-murine coronavirus effect of up to 6 days. overall, this study demonstrated that the ea fraction of h. cordata and its flavonoid component, quercetin, neutralizes murine coronavirus in vitro (chiow et al. 2016 ). the eps® 7630 liquid herbal drug umckaloabo®, a 100 μg/ml extract of pelargonium sidoides dc., exerts antiinfluenza virus activity in vitro and in vivo (theisen and muller 2012) . discovered in south africa, umckaloabo, a decoction of the roots of pelargonium sidoides, was originally used by zulu traditional healers as a remedy for acute bronchitis. umckaloabo® is known to inhibit the entry and replication of hcov-229e (michaelis et al. 2011) . it is a common medication for acute bronchitis in south africa and germany. although uncontrolled human trials for sars-cov anti-infectives have been reported, no randomized controlled trials with a specific anti-coronavirus agent had been conducted with respect to therapy or prophylaxis (wong and yuen 2008) . there had not been any clinical trials for drugs of infections caused by hcov-oc43, hcov-229e, hcov-nl63, and hcov-hku1 (wong and yuen, 2008) . urged that randomized placebo control trials should assess the efficacy of intravenous baicalin (10) for the treatment of sars especially in developing countries where such formulations were available and affordable. in a randomized double-blinded placebo controlled trial, quercetin supplementation in doses of 500 and 1000 mg/day for 12 weeks significantly increased plasma quercetin levels with no reported side effects, but neither decreased total number of upper respiratory tract infection sick days nor reduced symptomatology in all subjects (heinz et al. 2010) . liu et al. (2012) reviewed 12 randomized controlled trials and one quasirandomized controlled trial where 12 herbal remedies were administered on 640 sars patients. case reports, case series, controlled observational studies, and randomized clinical trials provided compelling data that tcm had beneficial effects in the treatment or prevention of sars (lau et al. 2005) . for example, the rate of fatality in hong kong and singapore was approximately 18%, while the rate for beijing was initially more than 52% until the 5th may 2003 when it decreased to 4%, and then to 1% after 20th may 2003 . the dramatic reduction in fatality in beijing was associated with the use of tcm as a supplement to conventional therapy (chen and nakamura 2004) . lau et al. (2005) reported that during the sars outbreak of 2002/2003, 1063 volunteers including 926 hospital workers and 37 laboratory technicians working in high-risk virus laboratories used tcm, namely sang ju yin plus yu ping feng san. compared with the 0.4% of infection in the placebo group, none of the tcm users was infected. in a controlled clinical study, hsu et al. (2006b) reported that supplementary treatment with tcm resulted in marked relief of symptoms and truncated the disease course. the clinical benefits of tcm were supported by laboratory studies especially for the use of glycyrrhizin (5), baicalin (10), and mol376, an inhibitor of cathepsin l that could become a lead compound for sars therapy . after a review of eight randomized controlled trials, liu et al. (2004) resolved that a combination of tcm with conventional medicine had beneficial effects such as decrease of mortality, relief of symptoms, and control of fungal infections in patients with sars. however, the trial evidence was not sufficient enough due to the lack of methodological rigour. in a review of 90 peer-reviewed chinese publications after the sars epidemic of 2002 , leung (2007 concluded that tcm used together with conventional treatment had positive effects including better control of fever and quicker amelioration of chest infection. wu et al. (2008) urged a rerun of clinical trials involving tcm for the treatment of sars because the validity of results was jeopardized by questionable trial designs and experimental biases. tcm should be evaluated in carefully designed clinical trials, either used alone or integrated with western medicine, to cover the prevention and treatment of patients suffering from covid-19 pneumonia (ling 2020) . by mid-march 2020, at least 14 tcm clinical trials with a total of 2714 patients were ongoing for the treatment of sars-cov-2 infection . chinese treatment data showed that patented tcm had good therapeutic efficacy in the treatment of covid-19, with minimal adverse reactions (zhuang et al. 2020 ). empirical data on sars-cov treatments are scarce (wong and yuen 2008) , and the majority of viral diseases do not have targeted drugs or vaccines (mahapatra et al. 2019) . although the ongoing sars-cov-2 global pandemic should remind scientists that current options for treating life-threatening zoonotic coronavirus infections are very limited , medicinal plants offer a strong pipeline for the discovery of novel lead compounds that can be converted into new drugs to treat covid-19. medicinal plants hold great promise for drug development against sars-cov-2, but there is paucity of research on the development of anti-sars-cov-2 drugs from natural products. in addition, most of the data on natural products with activity against hcovs are from non-clinical and pre-clinical studies . potential anti-coronavirus therapies can be divided into two categories depending on their target: those that act on the human immune system or human cells, and those that interfere with the coronavirus itself . the unmet need for pharmacologically potent and safe antivirals ought to be fulfilled by thinking out of the box and indigenous knowledge of medicinal plants may be a masterstroke against deadly infectious diseases such as covid-19 (mahapatra et al. 2019) . vellingiri et al. (2020) postulated that medicinal plants are potential sources of drugs for the treatment of covid-19. ling (2020) also submitted that traditional plant medicines are a good source of natural compounds for the discovery and development of drugs against sars-cov-2. so, the mortality of covid-19 patients in china was limited by the use of tcm. as the growing covid-19 global pandemic reformats the classical norms of pharmaceutical and clinical interventions, this review is a poignant reminder that repurposing of current medicinal plants and other natural products to treat covid-19 should become part of our new vernacular health system. comparative genomics lends credence to the repurposing of current anti-sars natural products for the treatment of covid-19. this is because the genome sequence of sars-cov is very similar to that of sars-cov-2 (tahir ul qamar et al. 2020) . phylogenetic analysis of whole genomes reveals that sars-cov-2 shares 79.7% nucleotide sequence identity with sars-cov zhou et al., 2020a, b) . compared with sars-cov, the envelope and nucleocapsid proteins of sars-cov-2 share sequence identities of 96% and 89.6%, respectively (zhou et al., 2020a, b) . the virion of sars-cov-2 consists of a similar structure as sars-cov and mers-cov. like sars-cov, sars-cov-2 also uses ace2 as its cellular receptor to enter host cells (zhou et al., 2020a, b) . the chymotrypsin-like protease (3cl pro ) of sars-cov is conserved and shares 99.02% sequence identity with sars-cov-2 3cl pro (tahir ul qamar et al. 2020 ). these comparative genomic similarities signify one epic biomedical prospect: if medicinal plants used against sars-cov and mers-cov are carefully repurposed, they could be effective against sars-cov-2. in this new normality, natural products should become part of the covid-19 survival kit. in the absence of an effective and safe drug that inhibits sars-cov-2, the use of antimalarial plants and synthetic drugs has gained traction in the treatment of covid-19. the notion that antimalarial plants in the genus artemisia l. can be repurposed to prevent and treat covid-19 has often been associated with experimental data that chloroquine inhibits the replication of viruses. chloroquine inhibits chikungunya virus replication in vero a cells (ic 50 7 μm, ic 90 15 μm, selectivity index = 37.14) in a dose-dependent manner. chloroquine, whose chemical structure mimics nucleoside analogues, has in vitro activity against sars-cov and sars-cov-2 ). an antimalarial drug known since 1934, chloroquine blocks hiv-1 and hcovs at a very early stage; it also inhibits the replication of mers-cov in a dose-dependent mode (ec 50 of 3 μm) (de wilde et al. 2014 ). chloroquine exerts antiviral as well as immunomodulatory properties. chloroquine (ec 50 of 1.13 μm against sars-cov-2) blocks viral infection by increasing the endosomal ph required for viral fusion (vellingiri et al. 2020) . a 9-aminoquinoline, chloroquine inhibits sars-cov replication and downregulates ifn-γ and tnf-α production in mice (prinsloo et al. 2018 ). in the first half of 2020, clinical t r i a l s ( n c t 0 4 2 6 1 5 1 7 a n d n c t 0 4 3 0 7 6 9 3 ) o f hydroxychloroquine treatment for covid-19 were underway in china (wang et al. 2020b ). the world health organization announced it would resume its halted randomized controlled trials to find out whether hydroxychloroquine was able to prevent covid-19. this was after the lancet on 4 june 2020, due to concerns with respect to the veracity of the data and analyses, retracted an influential article which had earlier reported that hydroxychloroquine increased the mortality of covid-19 patients. on 15 june 2020, the us food and drug administration revoked the emergence use of hydroxychloroquine to treat covid-19 patients after reports that the drug increases the risk of cardiovascular side effects including cardiomyopathy and cardiac arrest. taken together, these developments signify that many countries should carefully evaluate the efficacy and safety of natural antimalarial agents for the possible treatment of covid-19. rich in flavonoids such as acacetin, genkwanin, and 7-methoxyacacetin, leaf extracts of artemisia afra jacq., a common antimalarial remedy in kenya, rwanda, and zambia (chinsembu 2015) , should be carefully evaluated in covid-19 patients. the need for medicinal plants and other natural products to treat covid-19 patients is urgent especially in africa where health systems grapple with high caseloads of hiv/aids and malaria in addition to being heavily underresourced in terms of funding and staff. overall, plants with antiplasmodial (chinsembu 2015) , anti-tuberculosis (chinsembu 2016b) , and anti-hiv replication (chinsembu 2019 ) activities may present good prospects for the discovery of new active compounds with anti-sars-cov-2 action. pechuel-loeschea leubnitziae o. hoffm. (asteraceae), an indigenous plant used to "steam" patients suffering from pneumonia and coughs in namibia mofolo et al. 2020) , should also be evaluated for anti-covid-19 function. high-throughput screening of these plants should be conducted, and computational techniques can quicken the process. moreover, plant active compounds should be subjected to absorption, distribution, metabolism, and excretion (adme) evaluation to verify that oral administration would be effective ). analysis of structure-activity relationships may be helpful in understanding the mechanisms of certain active compounds such as myricetin (5), baicalein (6), and quercetin. further studies including in silico molecular docking simulations should be done to investigate the binding affinities of plant active compounds to sars-cov-2. a docking analysis can test whether the active compound has the potential to directly interact with sars-cov-2 proteins ). application of structure-based drug design strategies may aid in the development of novel sars-cov-2 inhibitors from natural compounds. to predict the general in vivo effects of the natural compounds, network pharmacology analysis can be carried out. only 0.0004% plant samples may directly lead to a commercial drug (chinsembu 2016a) . thus, there are few plantderived anti-sars agents in clinical trials. from 2012 to 2017, only twelve new antivirals were approved by the us food and drug administration; eight were for the treatment of pathologies related to hepatitis c virus and two were combinations of anti-hiv drugs (mercorelli et al. 2018) . since less than 15% of plant diversity has been explored for pharmaceutical purposes (chinsembu, 2016a) , the transition from natural product to anti-sars-cov-2 drug prototypes is a daunting reality as much as it is a beautiful prospect. this review shows that plants, fungi, and marine organisms have anti-sars-cov properties. many of these plants, fungi, and marine organisms have other pharmacological benefits because structural diversity and adaptation to various environmental conditions induce them to synthesize defensive compounds with assorted biological activities (prinsloo et al. 2018) . corollary, diverse plant secondary metabolites are an important library of novel lead compounds that may be functional against sars-cov-2. bio-assay-guided fractionation of medicinal plant extracts may provide a quicker pipeline for the discovery of novel compounds against sars-cov-2. an examination of the research publications on anti-sars natural products cited in this review shows that about 8 out of 10 are authored by the chinese. since the chinese represent about 80% of all research efforts and publications on anti-sars natural products, there is 80% chance that the next big discovery of novel natural products to treat covid-19 will be from tcm. china is the world leader in research and development for innovative anti-sars agents from natural products. by all intents and purposes, the first wellresearched and clinically proven herbal remedy for covid-19 will most likely be discovered and developed in china. the most notable gap identified in the current literature is that many countries in africa, asia (except china), australia/ oceania, europe, north america, and south america lag behind in terms of research efforts on anti-coronavirus remedies from medicinal plants and other natural products. in many countries all over the world, there is need to conduct detailed ethnobotanical studies to establish putative anti-sars-cov-2 medicinal plants and their active compounds. the search for new anti-sars-cov-2 agents should be rigorously extended to marine organisms. this review shows that there is a dearth of experimental data on posology and cytotoxicity of plant extracts and active compounds with in vitro activity against sars-cov-2. more studies on pan-assay interference compounds (pains) and sars-cov-2 immunomodulatory agents from plants and other natural products should be conducted. there is also lack of pharmacological and rigorous human clinical trial data in relation to natural products for treating covid-19. the world is in the middle of a covid-19 pandemic. although the deadly threat posed by the current covid-19 pandemic requires the development of new therapeutic agents, there are currently no efficacious and safe vaccines or drugs to prevent or treat this highly infectious disease. even in developed nations, blockbuster vaccines or drugs have not yet been developed or approved for the prevention and treatment of covid-19. only one synthetic drug (remdesivir) has been approved for emergency use in the usa. compared with placebo, remdesivir did not significantly reduce the mortality of covid-19 patients in clinical trials. against this backdrop, this review provides a modest but insightful snapshot of medicinal plants, fungi, and marine organisms that contain putative active compounds that inhibit sars-cov-2. data in this review show two recurring motifs. the first one is that the search for herbal remedies against covid-19 is more intense in china than in any other part of the world. the second is that most putative anti-sars-cov-2 remedies contain active compounds that inhibit hcovs at the cellular and molecular levels. since structure dictates function, many of these compounds inhibit sars-cov replication because their chemical structures mimic nucleoside analogues. while many of the natural products possess anti-sars-cov properties, their extracts and active compounds should be evaluated for human cytotoxicity and dosage. putative anti-sars-cov-2 extracts and active compounds should be rigorously tested in animal and randomized placebo clinical trials. due to the current lack of effective and safe drugs, natural products presented in this review may form part of the health beliefs, self-medication choices, and selfefficacy practices that people may use to manage covid-19. natural products presented in this paper 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in the treatment of coronavirus disease 2019 (covid-19) in china procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection acknowledgments the author is grateful to the anonymous reviewers and to professor rogelio pereda-miranda, editor-in-chief of revista brasileira de farmacognosia, for their help in improving the original manuscript. key: cord-259603-bh198xgl authors: snijder, e.j.; decroly, e.; ziebuhr, j. title: the nonstructural proteins directing coronavirus rna synthesis and processing date: 2016-09-14 journal: adv virus res doi: 10.1016/bs.aivir.2016.08.008 sha: doc_id: 259603 cord_uid: bh198xgl coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like sars and mers. they have polycistronic plus-stranded rna genomes and belong to the order nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key rna-synthesizing enzymes. coronavirus genomes (~ 26–32 kilobases) are the largest rna genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral rna polymerases. the primary functions that direct coronavirus rna synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. coronavirus replicase functions include more or less universal activities of plus-stranded rna viruses, like an rna polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mrna capping (nsp14, nsp16) and fidelity control (nsp14). several smaller subunits (nsp7–nsp10) act as crucial cofactors of these enzymes and contribute to the emerging “nsp interactome.” understanding the structure, function, and interactions of the rna-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies. coronaviruses (covs) are the best-known and best-studied clade of the order nidovirales, which is comprised of enveloped plus-stranded (+rna) viruses and currently also comprises the arteriviridae, roniviridae, and mesoniviridae families (de groot et al., 2012a,b; lauber et al., 2012) . in addition to including various highly pathogenic covs of livestock (saif, 2004) and four "established" human covs causing a large number of common colds (pyrc et al., 2007) , covs have attracted abundant attention due to their potential to cause lethal zoonotic infections (graham et al., 2013) . this was exemplified by the 2003 outbreak of severe acute respiratory syndrome-coronavirus (sars-cov) in southeast asia and the ongoing transmission-since 2012-of the middle east respiratory syndromecoronavirus (mers-cov), which causes $35% mortality among patients seeking medical attention. both these viruses are closely related to covs that are circulating in bats (ge et al., 2013; menachery et al., 2015) and other potential reservoir species. they may be transmitted to humans either directly or through intermediate hosts, like civet cats for sars-cov (song et al., 2005) and dromedary camels for mers-cov (reusken et al., 2013) . formally, the family coronaviridae now includes about 30 species, divided into the subfamilies torovirinae and coronavirinae, the latter being further subdivided in the genera alpha-, beta-, gamma-, and deltacoronavirus. sars-cov and mers-cov are betacoronaviruses, and the same holds true for one of the best-characterized animal cov models, murine hepatitis virus (mhv). this explains why the bulk of our current knowledge of cov molecular biology is betacoronavirus based, even more so for the replicative proteins that are the central theme of this review, which will mainly summarize data obtained studying sars-cov proteins. despite their unification in the same virus order, nidoviruses cover an unusually broad range of genome sizes, ranging from $13-16 kilobases (kb) for arteriviruses, via $20 kb for mesoniviruses, to $26-32 kb for covs (nga et al., 2011) . together with the genomes of roniviruses, which infect invertebrate hosts, cov genomes are the largest rna genomes known to date . the common ancestry of these extremely diverse virus lineages was inferred from their polycistronic genome structure, the common principles underlying the expression of these genomes, and-most importantly-the conservation of an array of "core replicase domains," including key enzymes required for rna synthesis. while retaining this conserved genomic and proteomic blueprint, nidovirus genomes are thought to have expanded gradually by gene duplication and acquisition of novel genes (lauber et al., 2013) , most likely by rna recombination. in addition to the high mutation rate that characterizes all rna viruses, these genomic innovations appear to have enabled nidoviruses to explore an unprecedented evolutionary space and adapt to a wide variety of host organisms, including mammals, birds, reptiles, fish, crustaceans, and insects. whereas the poor replication fidelity generally restricts rna virus genome sizes, it has been postulated that nidovirus genome expansion was enabled by the acquisition of specific replicative functions that counter the error rate of the rna polymerase (deng et al., 2014; eckerle et al., 2010; snijder et al., 2003) (discussed in more detail later). as in all nidoviruses, at least two-thirds of the cov genome capacity is occupied by the two large open reading frames (orfs) that together constitute the replicase gene, orf1a and orf1b (fig. 1 ). these orfs overlap by a few dozen nucleotides and are both translated from the viral genome, with expression of orf1b requiring a -1 ribosomal frameshift to occur just fig. 1 outline of the cov genome organization and expression strategy, based on sars-cov. the top panel depicts the sars-cov genome, including various regulatory rna elements, and the 5 0 -and 3 0 -coterminal nested set of subgenomic mrnas used to express the genes downstream of the replicase gene. utr, untranslated region; trs, transcription-regulatory sequence. below the rnas, the 14 open reading frames in the genome are indicated, i.e., the replicase orfs 1a and 1b, the four common cov structural protein genes (s, e, m, and n) and the orfs encoding "accessory proteins." the bottom panel explains the organization and proteolytic processing of the pp1a and pp1ab replicase polyproteins, the latter being produced by -1 ribosomal frameshifting. the nsp3 (pl pro ) and nsp5 (3cl pro ) proteases and their cleavage sites are indicated in matching colors. the resulting 16 cleavage products (nonstructural proteins (nsps)) are indicated, as are the conserved replicase domains that are relevant for this review. domain abbreviations and corresponding nsp numbers: pl pro , papain-like proteinase (nsp3); 3cl pro , 3c-like proteinase (nsp5); tm, transmembrane domain (nsp3, nsp4, and nsp6); niran, nidovirus rdrp-associated nucleotidyl transferase (nsp12); rdrp, rna-dependent rna polymerase (nsp12); zbd, zinc-binding domain (nsp13); hel1, superfamily 1 helicase (nsp13); exon, exoribonuclease (nsp14); n7-mt, n7-methyl transferase (nsp14); endou, uridylate-specific endoribonuclease (nsp15); 2 0 -o-mt, 2 0 -omethyl transferase (nsp16). upstream of the orf1a termination codon (brierley et al., 1989) . the efficiency of this highly conserved frameshift event, which may approach 50% in the case of covs (irigoyen et al., 2016) , is promoted by specific primary and higher-order rna structures. as a result, in cov-infected cells, the replicase subunits encoded in orf1a are overexpressed in a fixed ratio relative to the proteins encoded in orf1b. the primary translation products of the cov replicase are two huge polyproteins, the orf1a-encoded pp1a and the c-terminally extended pp1ab frameshift product (fig. 1) . the former is roughly 4000-4500 amino acids long, depending on the cov species analyzed. the size of the orf1b-encoded extension is more conserved (around 2700 residues), resulting in pp1ab sizes in the range of 6700-7200 amino acids. probably already during their synthesis, either two or three orf1aencoded proteases initiate the proteolytic cleavage of pp1a and pp1ab to release (sometimes) 15 or (mostly) 16 functional nonstructural proteins (nsps; fig. 1 ). the highly conserved nsp5 protease has a chymotrypsin-like fold (3clike protease, 3cl pro ) (anand et al., 2002 (anand et al., , 2003 gorbalenya et al., 1989) and is the viral "main protease" (therefore sometimes also referred to as m pro ). the 3cl pro cleaves the nsp4-nsp11 part of pp1a and the nsp4-nsp16 part of pp1ab at 7 and 11 conserved sites, respectively. these sites can be summarized with the p4-p2 0 consensus motif (small)-x-(l/i/v/f/m)-q#(s/a/g), where x is any amino acid and # represents the cleavage. the processing of three sites in the nsp1-nsp4 region is performed by one or two papain-like proteases (pl pro ) residing in the very large nsp3 subunit (mielech et al., 2014) . whereas alphacoronaviruses and most betacoronaviruses (though not sars-cov and mers-cov) have two pl pro domains in their nsp3, presumably the result of an ancient duplication event, gamma-and deltacoronaviruses have only a single pl pro . the cleavage sites (lxgg# or similar) resemble the c-terminal lrgg# motif of ubiquitin, which explains why cov pl pro domains were found capable to also act as deubiquitinases (ratia et al., 2006) . this secondary function has been implicated in the disruption of host innate immune signaling by removing ubiquitin from certain cellular substrates. more than any other cov-encoded enzyme, the cov 3cl pro and pl pro domains have been characterized in exquisite structural and biochemical detail, both in their capacity of critical regulators of nsp synthesis and as two of the primary drug targets for this virus family. space limitations unfortunately prevent us from summarizing these studies in more detail, but a variety of excellent reviews is available to compensate for this omission (baez-santos et al., 2015; hilgenfeld, 2014; mielech et al., 2014; steuber and hilgenfeld, 2010) . once released from pp1a and pp1ab, most covs nsps studied thus far assemble into a membrane-bound ribonucleoprotein complex that drives the synthesis of different forms of viral rna (see later) and is sometimes referred to as the replication and transcription complex (rtc) . while viral rna production takes off, peculiar convoluted membrane structures, spherules tethered to zippered endoplasmic reticulum, and doublemembrane vesicles begin to accumulate in cov-infected cells (gosert et al., 2002; knoops et al., 2008; maier et al., 2013) . as for other +rna viruses, they have been postulated to serve as scaffolds, or perhaps even suitable microenvironments, for viral rna synthesis. nevertheless, many questions on their biogenesis and function remain to be answered, and the exact location of the metabolically active rtc still has to be pinpointed "beyond reasonable doubt" for covs and other nidoviruses (hagemeijer et al., 2012; neuman et al., 2014a; van der hoeven et al., 2016) . three orf1a-encoded replicase subunits containing transmembrane domains (nsp3, nsp4, and nsp6; fig. 1 ) have been implicated in the formation of the membrane structures that are induced upon cov infection and with which the rtc is thought to be associated (angelini et al., 2013; hagemeijer et al., 2014) . in addition to actively engaging in host membrane remodeling, they may serve as membrane anchors for the rtc by binding the nsps that lack hydrophobic domains, like all of the orf1b-encoded enzymes. for more details, the reader is referred to the numerous recent reviews of the "replication organelles" of covs and other +rna viruses (den boon and ahlquist, 2010; hagemeijer et al., 2012; neuman et al., 2014a; romero-brey and bartenschlager, 2016; van der hoeven et al., 2016; xu and nagy, 2014) . the common ancestry of nidovirus replicases is not only reflected in their conserved core replicase domains but also in the synthesis of subgenomic (sg) mrnas that are used to express the genes located downstream of orf1b ( fig. 1) . although some nidoviruses (e.g., roniand mesoniviruses) have only a few of these genes, they are much more numerous in arteriviruses and covs, their number going up to about a dozen orfs for some covs. in addition to the standard set of four cov structural protein genes (encoding the spike (s), envelope (e), membrane (m), and nucleocapsid (n) protein), genomes in different cov clusters contain varying numbers of orfs encoding so-called "accessory proteins" narayanan et al., 2008) . the proteins they encode are often dispensable for the basic replicative cycle in cultured cells, but highly relevant for cov viability and pathogenesis in vivo, for example, because they enable the virus to interfere with the host's immune response. most of the genes downstream of orf1b are made accessible to ribosomes by positioning them at the 5 0 end of their own sg transcript. occasionally, two or even three genes are expressed from the same sg mrna, usually by employing ribosomal "leaky scanning" during translation initiation. nidoviral sg mrnas are 3 0 -coterminal with the viral genome, but in most nidovirus taxa, including covs, the sg transcripts also carry common 5 0 leader sequences ($65-95 nucleotides in covs), which are identical to the 5 0 -terminal sequence of the viral genome ( fig. 1) (pasternak et al., 2006; sawicki et al., 2007; sola et al., 2011) . the joining of common leader and different sg rna "body" sequences occurs during minus-strand rna synthesis (sawicki and sawicki, 1995; sethna et al., 1989) . this step can be either continuous, to produce the full-length minus strand required for genome replication, or interrupted (discontinuous) to produce a subgenome-length minus-strand rna that can subsequently serve as the template for the synthesis of one of the sg mrnas. the polymerase jumping that is the basis for leader-to-body joining occurs at specific "transcriptionregulatory sequences" (trss). these conserved sequence motifs are comprised of up to a dozen nucleotides, and are found in the genome at the 3 0 end of the leader sequence and at the 5 0 end of each of the sg mrna bodies. quite likely, also higher-order rna structure and transcription-specific protein factors play a role in the interruption of minus-strand rna synthesis at a body trs, after which the nascent minus strand (with a body trs complement at its 3 0 end) is translocated to the 5 0 -proximal part of the genomic template. guided by a base-pairing interaction with the leader trs, the synthesis of the subgenome-length minus-strand rna is resumed and completed with the addition of the complement of the genomic leader sequence. in this manner, a nested set of subgenome-length templates for sg mrna synthesis is produced, providing a mechanism to regulate the abundance of the different viral proteins by fine-tuning the level at which the corresponding sg mrna is generated (nedialkova et al., 2010) . the cov transcription strategy allows the rtc to use the same 3 0 -terminal recognition/initiation signals in both full-and subgenome-length templates of either polarity. moreover, the presence of the common 5 0 leader sequence may be important for mrna capping or other translation-related features. during the past two decades, studies on the cov enzyme complex that controls this elegant replication and transcription mechanism have been accelerated by four important developments. first, using bioinformatics, expression systems, and virus-infected cells, the replicase polyprotein processing scheme and the proteases involved were elucidated, thus defining the boundaries of the 16 mature nsps (fig. 1 ) that are working together during cov replication (ziebuhr et al., 2000) . second, using this information and promoted by rapidly advancing methods in structural biology, x-ray or nmr structures were obtained for numerous (recombinant) full-length cov nsps or domains thereof, in particular for sars-cov (neuman et al., 2014b) . third, multiple techniques for the targeted mutagenesis of cov genomes were developed and refined, which was a specific technical challenge due to the exceptionally large size of the cov rna genome (almazan et al., 2014) . by launching engineered mutant genomes in susceptible cells, the rna and protein players in the cov replication cycle can now be interrogated directly, to reveal their importance, function(s) and/or interactions in vivo. finally, in vitro biochemical assays were developed for a variety of cov replicative enzymes, including many of those involved in rna synthesis and processing. for the purpose of this review, we have chosen to focus on these latter functions, as performed by the cov nsp7 to nsp16 products nga et al., 2011; sevajol et al., 2014; subissi et al., 2014a) . these subunits include several replicative enzymes that are more or less universal among + rna viruses, such as rna polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in, e.g., mrna capping, cap modification, and promoting the fidelity of cov rna synthesis. several smaller subunits, in particular nsp7 to nsp10, have been identified as crucial cofactors of these enzymes and contribute to the emerging cov "nsp interactome," which will likely need to be advanced considerably to achieve a more complete understanding of the intricacies of cov rna synthesis. making that step will obviously be key to understanding the evolutionary success of covs, and nidoviruses at large. moreover, this knowledge will lay the foundation for the development of improved strategies to combat current and future emerging covs, including targeted antiviral drug development. 2. coronavirus nsp7-10: small but critical regulatory subunits? the 3 0 -terminal part of orf1a, the approximately 1.7 kb separating the nsp6-coding sequence and the orf1a/1b ribosomal frameshift site, encodes a set of four small replicase subunits, named nsp7 to nsp10 (fig. 1) . although highly conserved among coronavirinae, these proteins seem to lack enzymatic functions. instead, they have emerged as (putative) interaction partners and modulators of orf1b-encoded core enzymes like nsp12 (rna-dependent rna polymerase, rdrp), nsp14 (exoribonuclease, exon), and nsp16 (ribose 2 0 -o-methyl transferase, 2 0 -o-mtase). furthermore, several of them have been predicted or shown to interact with rna. additionally, a fifth, very small cleavage product is assumed to be released from this region of pp1a: the nsp11 peptide resulting from cleavage of pp1a at the nsp10/11 junction (fig. 1) . in the pp1ab frameshift product, the n-terminal sequence of nsp11 (encoded between the nsp10/11 junction and orf1a/1b frameshift site) equals the n-terminal part of the nsp12 subunit. depending on the cov species, nsp11 consists of 13-23 residues and its actual release, function (if any), or fate in cov-infected cells have not been established. in cell culture models, for some (infectious bronchitis virus (ibv)) but not other (mhv) covs, the nsp10/11 and nsp10/12 cleavages were found to be dispensable for virus replication (deming et al., 2007; fang et al., 2008) , even though the conservation of this cleavage site suggests that it is generally required for full replicase functionality. processing of the nsp7-nsp10 region of pp1a/pp1ab has been studied in some detail for mhv (bost et al., 2000; deming et al., 2007) , human cov 229e (hcov-229e) (ziebuhr and siddell, 1999) , and ibv (ng et al., 2001) , confirming the release of these subunits in infected cells and the use of the predicted 3cl pro cleavage sites. processing at these sites was found to be critical for mhv replication, the exception being inactivation of the nsp9/10 cleavage site, which yielded a crippled mutant virus. depending on antibody availability, the subcellular localization of nsp7 to nsp10 has been studied for several covs using immunofluorescence microscopy. without exception, and in line with their role as interaction partner of key replicative enzymes, these subunits localize to the perinuclear region of infected cells (bost et al., 2000) , where the membranous replication organelles of covs accumulate (gosert et al., 2002; knoops et al., 2008; maier et al., 2013) . it should be noted, however, that these labeling techniques cannot distinguish between fully processed nsps and polyprotein precursors or processing intermediates. the structure of the 83-amino acid sars-cov nsp7 was determined using both nmr (peti et al., 2005) and x-ray crystallography (zhai et al., 2005) , with the latter study resolving the structure of a hexadecameric supercomplex consisting of recombinant nsp7 and nsp8 (see later; fig. 2 ). in both structures, the nsp7-fold includes four helices, but their position and spatial orientation is quite different, suggesting that the protein's conformation is strongly affected by the interaction with nsp8, in particular, where it concerns helix α4 (johnson et al., 2010) . reverse-genetics studies targeting specific residues in sars-cov nsp7 confirmed the protein's importance for virus replication (subissi et al., 2014b) , although the impact of single point mutations was smaller than anticipated on the basis of the biochemical characterization of the rna-binding properties of nsp7-containing protein complexes in vitro (see later). the $200-amino-acid-long nsp8 subunit initially took center stage due to two studies, the first describing a fascinating hexadecameric structure consisting of eight copies each of nsp7 and nsp8 (fig. 2) (zhai et al., 2005) , and the second reporting an nsp8-specific "secondary" rna polymerase fig. 2 crystal structure of the sars-cov nsp7-nsp8 hexadecamer (pdb 2ahm) (zhai et al., 2005) . purified recombinant sars-cov nsp7 and nsp8 were found to self-assemble into a supercomplex of which the structure was determined at 2.4 å resolution. (a) the complex forms a doughnut-shaped hollow structure of which the central channel is lined with positively charged side chains (in blue) and was postulated to mediate doublestranded rna binding. the outside of the structure is predominantly negatively charged (red) surface shading). (b and c) sars-cov nsp8 resembles a "golf club"-like shape that can adopt two conformations, as presented here in orange and green. these nsp8 conformations are integrated into a much larger, hexadecameric structure that is composed of eight nsp8 subunits and eight nsp7 subunits, of which one is shaded pink. in (b), the hexadecamer is depicted against the background of the surface plot presented in (a). activity (imbert et al., 2006) that was implicated in the mechanism of initiation of cov rna synthesis. this template-dependent activity was reported to depend on the presence of mn 2+ or mg 2+ and to typically generate products of up to six nucleotides (for more details, see section 3.2). around the same time, purified recombinant sars-cov nsp7 and nsp8 were found to self-assemble into the hexadecameric supercomplex of which the structure was determined at 2.4 å resolution (zhai et al., 2005) . the complex was described, and also visualized by electron microscopy, as a doughnut-shaped hollow structure of which the central channel is lined with positively charged side chains ( fig. 2a) . a combination of structural modeling, rna-binding studies, and site-directed mutagenesis led to the hypothesis that the complex may slide along the replicating viral rna together with other viral proteins, possibly as a processivity factor for the rdrp (nsp12; see later). within the nsp7-nsp8 hexadecamer, sars-cov nsp8 was found to adopt two different conformations ( fig. 2b and c). these were named "golf club" and "golf club with a bent shaft" (zhai et al., 2005) , with the globular head of the golf club being considered a new fold. although the structures of feline coronavirus (fcov) nsp7 and nsp8 were found to resemble their sars-cov equivalents, they were found to assemble into a quite different higher-order complex, with two copies of nsp7 and a single copy of nsp8 forming a heterotrimer (xiao et al., 2012) . biochemical and reverse-genetics studies pointed toward an important role in rna synthesis for sars-cov nsp8 residues k58, p183, and r190, whose replacement was lethal to sars-cov. of these residues, p183 and r190 were postulated to be involved in interactions with nsp12, whereas k58 may be critical for nsp8-rna interactions (subissi et al., 2014b) . reverse-genetics studies targeting the 3 0 -proximal rna replication signals in the mhv genome provided strong evidence for an interaction between nsp8 and these rna structures (a so-called "bulged stemloop" and rna pseudoknot). when making a particular 6-nucleotide insertion in the rna pseudoknot, which strongly affected mhv replication, multiple suppressor mutations evolved, of which several mapped to the genomic region encoding nsp8 and nsp9 (z€ ust et al., 2008) . these interactions were postulated to be part of a molecular switch that controls minus-strand rna synthesis, or its initiation from the 3 0 end of the viral genome (te velthuis et al., 2012; z€ ust et al., 2008) . using screening approaches based on yeast two-hybrid and glutathione s-transferase (gst) pull-down assays, sars-cov nsp8 was reported to be an interaction partner of many other viral proteins (including nsp2, nsp3, and nsp5 to nsp16), although most of these interactions remain to be verified in the infected cell (von brunn et al., 2007) . the cov nsp9 subunit is about 110 amino acids long and was the second replicase cleavage product, after nsp5, for which crystal structures were obtained (egloff et al., 2004; sutton et al., 2004) . the biologically active form of the protein is believed to be a dimer that is capable of binding nucleic acids in a nonsequence-specific manner, with an apparent preference for single-stranded rna (egloff et al., 2004; ponnusamy et al., 2008; sutton et al., 2004) . several nsp9 point mutations that block cov replication have now been described (chen et al., 2009a; miknis et al., 2009 ), but the protein's exact function has remained enigmatic thus far. the nsp9 monomer consists of a β-barrel, composed of seven β-strands, and a c-terminal domain formed by a single α-helix. the latter domain plays a key role in the formation of the parallel helix-helix dimer conformation that-based on sequence conservation, structural considerations, and experimental data (miknis et al., 2009 )-is thought to be the biologically most relevant state of sars-cov nsp9. nevertheless, multiple alternative structures were described, including a sars-cov form that is stabilized by β-sheet interactions (sutton et al., 2004) and, for hcov-229e nsp9, an antiparallel helix-helix dimer that is stabilized by a disulfide bond (ponnusamy et al., 2008) . replacement of the hcov-229e cys residue involved in dimerization (cys-69) resulted in conversion to the parallel helix-helix dimer described for sars-cov nsp9. whereas wild-type hcov-229e nsp9 is organized as a trimer of dimers, the cys-69 ! ala mutant and sars-cov nsp9 both form rod-like polymers (ponnusamy et al., 2008) . disulfide bonding of the latter protein could not be detected (miknis et al., 2009) . although sars-cov and other betacoronaviruses do contain an equivalent cys residue, the feature is not conserved in alphacoronaviruses that are much more closely related to hcov-229e. thus, it cannot be excluded that the disulfide-bonded form of hcov-229e nsp9 is an artifact of recombinant protein purification and crystallization, although it was suggested that oxidative stress due to viral infection may favor its formation in cov-infected cells (ponnusamy et al., 2008) . we are not aware of experiments directly addressing the existence of such a disulfide-linked nsp9 dimer in covinfected cells. the importance of nsp9 dimerization for sars-cov and ibv viability was demonstrated in reverse-genetics studies (chen et al., 2009a; miknis et al., 2009 ) that also independently confirmed the importance of dimerization of the α-helical domain and in particular a putative gxxxg proteinprotein interaction motif. although rna binding in vitro was not disrupted in dimerization-incompetent sars-cov nsp9 variants, their affinity for ssrna 20-mers was reduced by 5-to 12-fold compared to the wild-type protein (miknis et al., 2009) . replacement of some of the basic residues (e.g., lys-10, lys-51, and lys-90) in the β-barrel domain of ibv nsp9 also significantly reduced the protein's capability to bind rna in vitro, but these mutations only modestly affected virus replication upon reverse engineering (chen et al., 2009a) . it remains to be studied how nsp9 dimerization and mutagenesis may affect interactions with other replicase subunits, like nsp8 and nsp12-rdrp. these proteins were identified as nsp9 interaction partners using different technical approaches (brockway et al., 2003; sutton et al., 2004; von brunn et al., 2007) and colocalize with nsp9 on the membranous replication organelles (bost et al., 2000) . at present, the available data suggest that, for efficient cov replication, nsp9 homodimerization is a more critical feature than the protein's affinity for rna per se. alternatively, the correct positioning of rna on larger protein complexes consisting of (or containing) nsp9 may be important for the protein's correct functioning in viral rna synthesis (miknis et al., 2009) . currently, the fact that suppressor mutations arose in mhv nsp9 (and nsp8) after mutagenesis of 3 0 -proximal mhv replication signals (see earlier) is the most compelling evidence for the involvement of nsp9-rna interactions in a critical step of cov replication. the protein may be part of a molecular switch (z€ ust et al., 2008) and/or possess features that are relevant to viral pathogenesis, as mutations in nsp9 were found to contribute to increased sars-cov pathogenesis in an animal model employing young mice infected with a mouse-adapted virus strain (ma-15) (frieman et al., 2012) . the small nsp10 subunit (139 residues in the case of sars-cov) is among the more conserved cov proteins and is thought to serve as an important multifunctional cofactor in replication. using yeast two-hybrid assays, nsp10 was shown to interact with itself, as well as with nsp1, nsp7, nsp14, and nsp16. these interactions were confirmed by coimmunoprecipitation and/or gst pull-down assays (brockway et al., 2004; imbert et al., 2008; pan et al., 2008; von brunn et al., 2007) . the important role of nsp10 in replication was first inferred from the phenotype of temperature-sensitive mutants of mhv in which an nsp10 mutation was responsible for a defect in minus-strand rna synthesis (sawicki et al., 2005) . in addition, the protein was implicated in the regulation of polyprotein processing since an engineered mhv nsp10 double mutant (asp-47 and his-48 to ala) was partially impaired in the processing of the nsp4-nsp11 region (donaldson et al., 2007) . when nsp10 was characterized in biochemical and structural studies, the protein was found to bind two zn 2+ ions with high affinity, suggesting the presence of two zinc-finger motifs (matthes et al., 2006) . additionally, in in vitro assays, nsp10 displayed a weak affinity for single-and doublestranded rna and dna, although no obvious sequence specificity could be established, suggesting that the protein may function as part of a larger rna-binding complex. crystal structures of monomeric and dodecameric forms of sars-cov nsp10 were solved by different laboratories, but obvious structural rearrangements between the two forms were not detected (joseph et al., 2007; su et al., 2006) . the structures revealed a new fold in which the zn 2+ ions are coordinated in a unique conformation and in which a cluster of basic residues on the protein's surface probably contributes to the rna-binding properties of nsp10. more recent biochemical studies revealed that nsp10 interacts with nsp14 and nsp16 and regulates their respective exon and ribose-2 0 -o-mtase (2 0 -o-mtase) activities (bouvet et al., 2010 (bouvet et al., , 2012 . both these cofactor functions will be discussed in more detail later, in section 5. although a virus-encoded rdrp is at the hub of the replication of all rna viruses, special properties have long been attributed to the cov rdrp. these ideas find their origin in a combination of cov features, like the exceptionally long rna genome , the complex mechanism underlying subgenomic rna synthesis pasternak et al., 2006; sawicki et al., 2007; sola et al., 2011) , the reported high rna recombination frequency (graham and baric, 2010; lai and cavanagh, 1997) , and the size and positioning of the rdrpcontaining subunit, nsp12, within the replicase polyprotein. it remains to be elucidated to which extent features like polymerase processivity, fidelity, and template switching (during either genomic recombination or subgenome-length negative-strand rna synthesis) are determined by the properties of the nsp12-rdrp subunit itself or by some of its protein cofactors, such as nsp7 and nsp8 (see earlier). in fact, some cofactors have been studied more extensively than nsp12 itself, and the same holds true for some of the specific rna signals employed by the rdrp during, e.g., replication and subgenomic mrna synthesis. protein subunits of the larger rnasynthesizing complex, like nsp7-nsp8, the nsp13-helicase, and the nsp14-exon, likely exert a strong influence on rdrp behavior and performance. on the other hand, a recent study employing homology modeling and reverse genetics of the mhv rdrp domain described the first two nsp12 mutations that can induce resistance to a mutagen and reduce the mhv rdrp error rate during virus passaging (sexton et al., 2016) . so, not unexpectedly, also features within nsp12 itself contribute to properties like nucleotide selectivity and fidelity regulation. all of the currently identified nsp12 cofactors, and most other cov nsps, assemble into membrane-associated enzyme complexes (see earlier). the large number of viral subunits in these complexes (subissi et al., 2014a) , the likely requirement for host factors (van hemert et al., 2008) , and the concept of rna synthesis occurring in a dedicated microenvironment in the infected cell (knoops et al., 2008; v'kovski et al., 2015) complicate the straightforward characterization of the cov rdrp. to reconstitute the enzyme's activities in vitro, purified recombinant nsp12 is a key reagent but, for many years, such studies were hampered by poor nsp12 expression in escherichia coli. the first in vitro activity assays have only been developed recently (subissi et al., 2014b; te velthuis et al., 2010) , and the same technical issues with protein production explain the current lack of an nsp12 crystal structure. consequently, structural information is restricted to sequence comparisons and some homology-based structure models of the c-terminal rdrp domain of the $930-residue-long nsp12 (xu et al., 2003) . moreover, most of what we have learned so far is based on the characterization of a single nsp12 homolog only, that of the sars-cov. the nsp12-coding sequence includes the orf1a/1b ribosomal frameshift site and a programmed -1 frameshifting event directs orf1b translation to yield the pp1ab polyprotein that includes nsp12. the 3cl pro -driven cleavage required to release the n-terminus of nsp12 is the same that separates nsp10 and nsp11. about 925-940 amino acids downstream (932 in the case of sars-cov), the nsp12/nsp13 cleavage site separates the cov rdrp subunit from the helicase-containing cleavage product, whichuniquely among +rna viruses-resides downstream of the rdrp domain for reasons that are poorly understood thus far . nsp12 consists of at least two domains, the recently described n-terminal "nidovirus-wide conserved domain with nucleotidyl transferase activity" (nidovirus rdrp-associated nucleotidyltransferase (niran); see later) (lehmann et al., 2015a ) and the c-terminal canonical rdrp domain (gorbalenya et al., 1989) . the latter possesses the common motifs and structural features found in other rna polymerases, which are often summarized as a "cupped right hand" with subdomains called fingers, palm, and thumb each playing specific roles in binding of templates and ntps, initiation, and elongation (te velthuis, 2014; xu et al., 2003) . in simplified form, the reaction catalyzed by the rdrp comes down to selecting the appropriate ntp to match with the template and the formation of a phosphodiester bond to extend the 3 0 end of the nascent rna chain with this incoming nucleotide (ng et al., 2008; van dijk et al., 2004) . reconstituting these activities in vitro using a purified rdrp preparation can be relatively straightforward, but sometimes is a huge technical challenge depending-among other factors-on the efficiency of recombinant rdrp expression and purification, the existence of specific template requirements (e.g., recognition signals), and the need for protein cofactors. the initiation mechanism of the cov rdrp, primer dependent or de novo, continues to be a much-debated issue, with important implications for the question of how covs maintain the integrity of the crucial terminal sequences of their genome. compared to a de novo-initiating rdrp, the enzyme's active site, which is enclosed by the thumb and fingers domains, needs to be more accessible when a primer-template duplex has to be accommodated. de novo initiation, on the other hand, requires specific structural elements (so-called "priming loops") that serve to properly position the initiating ntps for catalysis, thus creating an initiation platform for rna synthesis. bioinformatics analyses grouped the cov rdrp with primer-dependent rdrps, as found in, e.g., picornaviruses and caliciviruses, in part based on the identification of a specific sequence motif (motif g) that is thought to mediate primer recognition ( fig. 3a ) (beerens et al., 2007; fig. 3 comparison of coronavirus nsp12 and arterivirus nsp9, containing the highly conserved niran and rdrp domains. (a) similarity density plot derived from a multiple sequence alignment including rdrp subunits from all nidovirus lineages. to highlight local deviations from the average, areas displaying conservation above and below the mean similarity are shaded in black and gray, respectively. conserved sequence motifs of niran (subscript n; see also b) and rdrp (subscript r) are labeled. domain boundaries used for bioinformatics analyses and uncertainty with respect to the niran/rdrp domain boundary are indicated with vertical and by dashed horizontal lines, respectively. below each plot, the predicted secondary structure elements are presented in gray for α-helices and black for β-strands. gorbalenya et al., 2002; xu et al., 2003) . this prediction appeared to be further supported by the identification of sars-cov nsp8 as a de novoinitiating second rna polymerase (see earlier), capable of synthesizing products of up to six nucleotides in length that could serve to prime rna synthesis by the nsp12-rdrp (imbert et al., 2006) . support for a direct interaction between nsp8 and nsp12 was obtained using different technical approaches subissi et al., 2014b; von brunn et al., 2007) . however, although a similar primer-independent rdrp activity was reported for the fcov nsp8 (xiao et al., 2012) , other studies have called into question this concept of a primase-main rdrp (i.e., nsp8/nsp12) tandem working in concert to achieve initiation of processive cov rna synthesis (see later). using recombinant sars-cov nsp12, preliminary evidence for primer-dependent rdrp activity on poly(a) templates was first obtained using a gst-nsp12 fusion protein, although these efforts were hampered by protein instability, which also led to the conclusion that the n-terminal domain of nsp12 is required for activity (cheng et al., 2005) . subsequently, a c-terminally his 6 -tagged sars-cov nsp12 was found to mediate homopolymeric rna synthesis in a primer-dependent manner (te velthuis et al., 2010) . both these activities must probably be considered relatively weak and nonprocessive compared to the activity observed when a sars-cov nsp12 rdrp assay was supplemented with nsp7 and nsp8 (subissi et al., 2014b) . however, at the same time, this study reinvigorated the debate on the initiation mechanism of the coronavirus rdrp, as the nsp7-8-12 tripartite complex displayed both primer-dependent and de novo initiation of rna synthesis, whereas no de novo-initiating rdrp activity could be detected for nsp8 or the nsp7-nsp8 complex alone (subissi et al., 2014b) . to add to the confusion, other studies reported de novo initiation by sars-cov nsp12 alone (ahn et al., 2012) and primer-dependent rdrp activity of sars-cov nsp8, when expressed without affinity tags commonly used to facilitate purification (te velthuis et al., 2012). technical differences between these studies and those summarized earlier (e.g., regarding expression constructs and templates used) may have contributed to the contradictory results obtained on the rdrp activities of nsp8 and nsp12. thus far, five different laboratories addressed the two (putative) coronavirus rdrps in seven independent studies, none of which succeeded in exactly reproducing the results of any of the other studies (ahn et al., 2012; cheng et al., 2005; imbert et al., 2006; subissi et al., 2014b; te velthuis et al., 2010 te velthuis et al., , 2012 xiao et al., 2012) . nidovirus rdrps appear to be technically challenging and sensitive proteins that may respond to minute changes in purification protocols or assay conditions. clearly, both the role of nsp8 (primase or processivity factor?) and the initiation mechanism employed by the nsp12-rdrp require further study. although the bioinformatics-based prediction that nsp12 uses a primer-dependent initiation mechanism is compelling, it lacks the direct support of an nsp12 crystal structure. at the same time, the question of the nature and source of the primer that would be used by nsp12 seems to be wide open again. as for other rna viruses, the nsp12-rdrp of covs is a primary drug target that may, in principle, be inhibited without major toxic side effects for the host cell. nucleoside analogs constitute an important class of antiviral drug candidates that can target viral rdrps, but efforts to use them to inhibit cov replication were not very successful thus far (chu et al., 2006; ikejiri et al., 2007) . moreover, it remains to be established that their target in the infected cell is indeed the nsp12-rdrp. the mismatch repair capabilities attributed to the nsp14-exon domain (see later) (bouvet et al., 2012) may pose an additional hurdle, as the efficacy of a nucleoside analogue with anticoronavirus activity may be determined by the balance between its propensity to be incorporated by the nsp12-rdrp and its tendency to resist excision by the mismatch repair mechanism mediated by nsp14-exon. similar considerations apply to ribavirin, a guanosine analog with broadspectrum antiviral activity that is used to treat patients infected with a variety of rna viruses. its mechanism of action appears to differ on a case-by-case basis, but may include the induction of lethal mutagenesis by increasing the rdrp error rate, inhibition of viral mrna capping, and reduction of viral rna synthesis by inhibition of the cellular enzyme inosine monophosphate dehydrogenase (impdh), which decreases the availability of intracellular gtp (crotty et al., 2000 (crotty et al., , 2002 smith et al., 2013 smith et al., , 2014 . although ribavirin was used to treat small numbers of sars and mers patients, high doses were used and the benefits of the treatment remained essentially unclear (zumla et al., 2016) . experiments with different covs in animal models falzarano et al., 2013) and infected cell cultures (ikejiri et al., 2007; pyrc et al., 2006) also established its poor activity and strongly suggested that ribavirin does not target the cov rdrp directly or is targeted (itself ) by the nsp14-exon activity (smith et al., 2013) . innovative nucleoside inhibitors continue to be identified or developed (peters et al., 2015; warren et al., 2014) and the recently described in vitro rdrp assay (subissi et al., 2014b) may prove very useful for establishing their mechanism of inhibition more precisely. a better understanding of nsp12-rdrp structure and function will also be required to design strategies that minimize the impact of drug resistance-inducing mutations, which are a common problem when targeting enzymes of rapidly evolving rna viruses. since the delineation of the borders of the cov rdrp-containing replicase cleavage product (boursnell et al., 1987; gorbalenya et al., 1989) , which is now known as nsp12, it had been clear that the protein must be a multidomain subunit, with the canonical rdrp domain roughly occupying its c-terminal half (fig. 3a ). only recently, first clues to some of the properties and possible functions of the n-terminal part of nsp12 were obtained (lehmann et al., 2015a) . a renewed bioinformatics analysis across the (still expanding) order nidovirales revealed that the nidoviral rdrp-containing replicase subunit contains a conserved n-terminal domain of 200-300 residues ($225 residues in cov nsp12; fig. 3b ). in cov nsp12, about 175 residues separate the niran and rdrp domains, leaving space for the presence of an additional domain between the two. based mainly on biochemical data obtained with the arterivirus homolog (see later), the n-terminal domain was concluded to possess an essential nucleotidylation activity and hence it was coined nidovirus rdrp-associated nucleotidyltransferase (niran) (lehmann et al., 2015a) . niran conservation was found to be lower than that of the downstream rdrp domain (fig. 3a) , but the analysis suggested that the evolutionary constraints on niran have been similar in different nidovirus lineages, which would be in line with a conserved function. gorbalenya and colleagues identified three key niran motifs (a-b-c) containing seven invariant residues (fig. 3b) , with domains b and c being most conserved (lehmann et al., 2015a) . the identification of the niran domain was further supported by the conservation of its predicted secondary structure elements in different nidovirus families (fig. 3a) . extensive database searches did not reveal potential niran homologs in either the viral or the cellular world, although it cannot be excluded that the domain has diverged from cellular ancestors to a level that prevents their identification with the currently available sequences and tools. nevertheless, its unique presence in nidoviruses and its association with the important rdrp domain suggest that niran may be a crucial regulator or interaction partner of the downstream rdrp domain that must have been acquired before the currently known nidovirus lineages diverged. niran and the zinc-binding domain (zbd) that is associated with the nsp13-helicase protein (see later) are the only unique genetic markers of the order nidovirales identified thus far. mainly due to the lack of sufficient amounts of recombinant cov nsp12, the preliminary biochemical characterization of niran was restricted to its arterivirus homolog, using recombinant nsp9 of equine arteritis virus (eav) (lehmann et al., 2015a) . for both eav and sars-cov, it could be shown that replacement of conserved niran residues can cripple or completely block virus replication in cultured cells. a combination of biochemical assays revealed that in vitro the niran domain exhibits a specific, mn 2+ -dependent enzymatic activity that results in the self-nucleotidylation of eav nsp9. the activity was abolished upon mutagenesis of conserved key residues in niran motifs a, b, and c. although utp was found to be the preferred substrate for niran's in vitro nucleotidylation activity, also gtp could be used, albeit less efficiently. the conserved lysine residue in motif a (the eav equivalent of lys-73 in sars-cov nsp12) was concluded to be the most likely target residue for nucleotidylation via formation of a phosphoamide bond. although the importance of the niran domains of arterivirus nsp9 and coronavirus nps12 was supported by the outcome of reverse-genetics studies (lehmann et al., 2015a) , the role of the produced protein-nucleoside adducts in viral replication remains unclear at present. in fact, the unique dual specificity for utp and gtp seems to argue against two initially considered potential niran functions (lehmann et al., 2015a) . the first of these was a role as an rna ligase, a type of activity however that commonly is atp dependent. the second was its involvement in synthesizing mrna cap structures. one of the four enzymes required for this process, the crucial guanylyl transferase (gtase), still remains to be identified for covs (see later). however, niran's substrate preference for utp over gtp is difficult to reconcile with this hypothesis and has not been observed for other gtases involved in mrna capping. the third hypothesis that was put forward links back to the open question of the initiation of coronavirus rna synthesis, which presumably is a primer-dependent step (see earlier). nsp12 nucleotidylation could be envisioned to play a role in protein-primed rna synthesis, a strategy used by, e.g., picornaviruses and their relatives, which covalently attach an oligonucleotide to a viral protein (called vpg in the case of picornaviruses) that subsequently mediates the initiation of rna synthesis (paul et al., 2000) . the first step in the synthesis of the "protein primer" is a nucleotidylation step during which a nucleotide monophosphate is covalently attached to the vpg. niran could be involved in a similar mechanism either directly or indirectly, by transferring the bound nucleotide to another protein player. although such a mechanism would definitely revolutionize the concept of the initiation of cov rna synthesis, it is clearly not very compatible with some of the currently available data, such as the reported presence of a 5 0 cap structure (rather than a vpg-like molecule) on cov mrnas. evidently, the further in-depth characterization of niran is needed to fill the current knowledge gaps, starting with the biochemical characterization of a cov niran domain, which may confirm and extend the features now deduced from the analysis of its distantly related arterivirus homolog. helicases are versatile ntp-dependent motor proteins that play a role in cellular nucleic acid metabolism in the broadest possible sense, including processes like dna replication, recombination and repair, transcription, translation, as well as rna processing. helicases are also encoded by all +rna viruses with a genome size exceeding 7 kb, suggesting they are required for the efficient replication of +rna viral genomes above this size threshold. given the large size of the genomes of covs and related nidoviruses, they may depend on the function(s) of a replicative helicase even more than other +rna virus taxa. however, despite their abundance and conservation, the specific role of helicases in +rna virus replication remains poorly understood. for an extensive recent review of nidovirus helicases, the reader is referred to lehmann et al. (2015c) . currently, helicases are classified into six superfamilies (sfs) (singleton et al., 2007) , with +rna viral helicases belonging to sf1 (e.g., alphaviruses and nidoviruses), sf2 (e.g., flaviviruses), or sf3 (e.g., picornaviruses). the presence of a sf1 helicase (hel1) domain in the cov replicase polyprotein was discovered upon the early in-depth analysis of the first full-length cov genome sequence that became available (ibv) (gorbalenya et al., 1989) . the hel1 domain maps to the c-terminal part of the replicase cleavage product that is now known as nsp13, which is about 600 residues long. the cov hel1 domain contains all characteristic sequence motifs of the sf1 superfamily. the n-terminal part of nsp13 is formed by a multinuclear zbd, one of the most conserved domains across the order nidovirales (gorbalenya, 2001; nga et al., 2011) . this qualification also applies to the helicase-containing subunit as a whole, despite considerable size differences between, e.g., cov nsp13 and its arterivirus homolog (designated nsp10) (lehmann et al., 2015c) . the zbd and hel1 domains occupy a conserved position downstream of the rdrp domain in all nidovirus replicase polyproteins studied so far. sf1 helicases contain at least a dozen conserved motifs that direct the binding of ntps and nucleic acids. of these, motifs i and ii (also known as the walker a and b boxes) are common to helicases of all sfs as well as ntpases. structurally, the catalytic core of sf1 helicases like the cov hel1 domain is formed by two reca-like domains, designated 1a and 2a (fig. 4) , that bind to nucleic acids through stacking interactions of aromatic residues with the bases of their nucleic acid substrates (velankar et al., 1999) . cyclic conformational changes of the reca-like domains mediate the conversion of the energy from hydrolysis of the phosphodiester bonds of ntps into directional movement along the nucleic acid substrate, with the so-called "inchworm" model now widely being considered as best supported by the available experimental data (lehmann et al., 2015c; velankar et al., 1999; yarranton and gefter, 1979) . additional domains, located upor downstream of 1a and 2a, or inserted internally, can mediate supplemental protein-protein and protein-nucleic acid interactions or enzymatic activities, thus contributing to the functional versatility and specificity of the enzyme (lehmann et al., 2015c; singleton et al., 2007) . within helicase sf1, the cov hel1 domain belongs to the upf1-like family (sf1b) which is characterized by moving in the 5 0 -to-3 0 direction along the nucleic acid strand to which they bind. upf1-like helicases may unwind either dna or rna and, in some cases, also both substrates without a clear preference, as was readily observed during the in vitro characterization of different nidovirus helicases. the cov hel1 activity was first demonstrated in vitro using recombinant hcov-229e nsp13 (seybert et al., 2000a) . bacterially expressed nsp13 from hcov-229e and sars-cov, and also the homologous helicase (nsp10) of the arterivirus eav, displayed 5 0 -to-3 0 unwinding activity on double-stranded rna or dna substrates containing single-stranded 5 0 overhangs (ivanov and ziebuhr, 2004; ivanov et al., 2004b; seybert et al., 2000a,b; tanner et al., 2003) . following the biochemical characterization of sars-cov nsp13, it was calculated that unwinding occurs in discrete steps of 9.3 base pairs each, with a catalytic rate of 30 steps per second (adedeji et al., 2012a) . the nsp13 ntpase activity can use all four natural ribonucleotides and nucleotides as substrate, with atp, datp, and gtp being hydrolyzed most efficiently, and utp being the least preferred substrate (ivanov and ziebuhr, 2004; ivanov et al., 2004b; tanner et al., 2003) . replacement of a conserved lys in motif i, the walker a box (walker et al., 1982) , kills the in vitro ntpase activity of all nidovirus helicases tested thus far and, when introduced by reverse genetics, this mutation also abolished replication of the arterivirus eav (seybert et al., 2000b) . the substrate preferences summarized earlier support a threedimensional model of the sars-cov hel1 core domains (1a and 2a) that was based on structural information available for multiple cellular helicases (hoffmann et al., 2006) . the model predicts both the existence of multiple hydrogen bonding interactions with the βand γ-phosphates of the ntp and a lack of specific interactions with the nucleobase. thus, the mere presence of a 5 0 triphosphate group appears to be the main determinant for ntp/dntp binding. since nidovirus helicases are presumed to unwind double-stranded rna intermediates that are formed during viral replication, considerable attention was given to the in vitro characterization of their nucleic acid substrate preferences (seybert et al., 2000a,b) . the hcov-229e and eav helicases could not unwind substrates with 3 0 single-stranded tails or blunt-ended substrates. in contrast, rna and dna substrates with one or two 5 0 single-stranded regions were unwound efficiently, suggesting that the nidovirus helicase must bind to a single-stranded region before initiating unwinding in the 5 0 -to-3 0 direction. however, the in vitro assays did not yield any clear indications for the preferred recognition of specific sequences or higher-order structures in the substrate (lehmann et al., 2015c) . also a more in-depth biochemical characterization, performed with sars-cov nsp13, confirmed that the cov helicase does not discriminate between rna and dna substrates (adedeji et al., 2012a) . consequently, it cannot be excluded that the enzyme, in addition to being engaged in viral rna synthesis, may also target host dna. nuclear translocation of nidovirus helicases has not been reported thus far, but the light microscopy techniques used to study the protein's subcellular distribution would not suffice to detect the nuclear import of only a small fraction of the protein. as a final caveat it should be stressed that the biochemical properties summarized earlier are all derived from in vitro studies using recombinant helicases, expressed in different systems and sometimes containing substantial foreign sequences. the in situ characterization of the helicase as one of the key enzymes of the nidovirus rna-synthesizing machinery remains to be addressed. in that context, sequence specificity, for example, could be conveyed by other subunits of the replicase complex, which may target the helicase to, e.g., the initiation sites for viral genome or antigenome synthesis, or to signals controlling the production of subgenomic mrnas. as summarized by lehmann et al. (2015c) , other important helicase features that could be dramatically different in the setting of the infected cell are (the need for) helicase oligomerization, cooperativity between multiple helicase molecules binding to the same substrate, and-consequentlythe overall processivity of the enzyme, which in vitro appeared to be quite low given the large cov genome size (adedeji et al., 2012a) . the nidovirus helicase subunit domain is unique among its +rna virus homologs in having a conserved n-terminal domain of 80-100 residues that contains 12 or 13 conserved cys/his residues (den boon et al., 1991; van dinten et al., 2000) . the domain was recognized as a potential zbd (gorbalenya et al., 1989) and early in vitro studies with the recombinant hcov-229e and eav helicases confirmed that zn 2+ ions are essential for retaining the protein's enzymatic activities, suggesting that zbd modulates nidovirus helicase function (seybert et al., 2005) . a recent structural study of the arterivirus nsp10-helicase (deng et al., 2014) will be discussed in more detail later. this first nidovirus helicase structure confirmed the binding of three zinc ions by the zbd, which adopts a unique fold that combines a ring-like module with a so-called "treble-clef" zinc finger. zbd and hel1 interact extensively (deng et al., 2014) but, in the helicase primary structure, they are separated by a variable and uncharacterized domain that essentially explains the size difference of about 130 residues between cov and arterivirus helicase subunits (seybert et al., 2005) . using the arterivirus prototype eav, the functional importance of zbd was probed extensively by combining biochemistry and reverse genetics (seybert et al., 2005; van dinten et al., 2000) . this yielded a variety of phenotypes for nsp10-zbd mutants, the most striking being mutants deficient in subgenomic mrna synthesis while remaining capable of (and even enhancing) viral genome replication (van dinten et al., 1997 (van dinten et al., , 2000 (see later). most replacements of conserved zbd cys and his residues profoundly impacted the helicase activity of eav nsp10, even when performed in a semiconservative manner that could preserve zinc binding. in reverse-genetics studies, most of these zbd mutations rendered the virus nonviable. recently, the impact of these mutations on zbd integrity and zbd-hel1 interactions could be rationalized with the help of the nsp10 crystal structure (deng et al., 2014) . despite its importance as a potential drug target, a cov nsp13 or hel1 crystal structure has not been obtained thus far due to technical complications with recombinant protein production and crystallization. instead, several cov helicase models have been described, mainly based on cellular helicase structures (bernini et al., 2006; hoffmann et al., 2006; lehmann et al., 2015c) . given this limitation, and despite the large evolutionary distance between the two enzymes, it is interesting to have a closer look at the recently published eav nsp10-helicase structure (deng et al., 2014) . the overall structure of eav nsp10 (fig. 4) consists of the n-terminal zbd, a new domain designated 1b, the two reca-like hel1 domains (1a and 2a), and a short c-terminal domain, which is not conserved among nidoviruses and needed to be deleted to allow nsp10 crystallization. this 65-amino-acid c-terminal truncation did not affect the helicase core domains and only modestly influenced levels of atpase and helicase activity compared to the full-length protein (deng et al., 2014) . compared to cellular representatives of the sf1 helicase superfamily, nsp10 is most similar to upf1 and its close homolog ighmbp2, which also contain an n-terminal zbd. moreover, the location and orientation of the newly discovered 1b domain of nsp10 (residues 83-137) resembles that of the domain with the same name found in the upf1-like helicase subfamily. the nsp10 zbd uses 12 conserved cys and his residues to coordinate three zinc ions and folds into two zinc-binding modules that are connected by a disordered region. the n-terminal ring-like structure of nsp10 coordinates two zinc ions and the closest similarity that was found for this module was with the n-terminal zinc-binding ch-domain of upf1. both proteins have a second zinc-binding module downstream (a so-called treble-clef zinc finger in the case of nsp10), but these are structurally different, suggesting that the nidoviral zbd represents a new kind of complex zinc-binding element. the previous suggestion of zbd codetermining hel1 function was strengthened by the presence of an extensive interface of 1019 å 2 that was proposed to be involved in intramolecular signaling (deng et al., 2014) . a second crystal structure was obtained for nsp10 in complex with a partially double-stranded dna substrate, revealing possible nucleic acid-binding clefts at the protein's surface that are formed by the zbd + 1b and zbd + 1a domains. although the exact path of the nucleic acid strands could not yet be determined, it became clear that the positively charged zbd, and in particular its n-terminal ring-like module, must be involved in nucleic acid binding. in line with the biochemical data summarized earlier, most of the nsp10-substrate contacts identified are not base-specific and occur with the nucleic acid backbone. whereas the hel1 core domains were found to be quite similar in the absence or presence of bound substrate, a remarkable 29 degree rotation was observed for domain 1b, enlarging the dimensions of the nucleic acid substrate channel formed by domains 1a and 1b, but not allowing it to accommodate a duplex substrate. consequently, it was postulated that an element near the entrance of the substrate channel may destabilize the duplex and facilitate the entrance of one of the strands into the channel. since the double-stranded region of the duplex could not be modeled, additional studies are needed to verify the existence and molecular details of this proposed unwinding mechanism (deng et al., 2014; lehmann et al., 2015c) . likewise, direct structural information on cov nsp13 is needed to be able to assess to which extent the structural observations made for arterivirus nsp10 can be translated to distantly related (and larger) nidovirus helicases (fig. 4) . in general, however, the analysis of nsp10 provided a clear basis for a model in which the function of the common reca-like core domains of nidovirus helicases is modulated by specific extension domains, presumably to facilitate the involvement of the nidovirus helicase in multiple processes in the infected cell (see later). as outlined earlier, the biochemical characterization of purified recombinant nidovirus helicases, the functional probing of (in particular) the eav nsp10-helicase using reverse genetics, the eav nsp10 structure, and advanced bioinformatics analyses together have painted a picture of an enzyme that is involved in multiple critical steps of the viral replicative cycle. space limitations do not allow an in-depth discussion of all of these (putative) functions, which-based on eav nsp10 studies-may include a poorly characterized role in virion biogenesis (van dinten et al., 2000) , not unlike what was uncovered for, e.g., the helicase-containing ns3 protein of flaviviruses (liu et al., 2002; ma et al., 2008) . likewise, we will not discuss the first reports on possible interactions with host proteins, such as the ddx5 helicase (for sars-cov nsp13; chen et al., 2009b) and polymerase δ (for ibv nsp13; xu et al., 2011) . instead, we will focus on the most significant findings related to nidovirus helicase functions in rna synthesis and processing, specifically (i) genome replication, (ii) transcription of subgenomic mrnas, (iii) mrna capping, and (iv) posttranscriptional mrna quality control. the presumed "default" function of +rna viral helicases is to cooperate with the viral rdrp to achieve the efficient amplification of the genome. in this context, helicases are presumed to promote rdrp processivity by opening up the double-stranded rna intermediates of viral replication, and possibly also by removing rna secondary structures in single-stranded template strands (kadare and haenni, 1997) . in this light, reports on molecular interactions between the cov rdrp (nsp12) and helicase (nsp13) were not unexpected von brunn et al., 2007) . the same holds true for the observation that sars-cov nsp12 can stimulate the in vitro helicase activity of nsp13 (adedeji et al., 2012a) and for the fact that both nsp12 and nsp13 (like most other cov replicase cleavage products) associate with the membranous replication organelles in nidovirus-infected cells (denison et al., 1999; ivanov et al., 2004b; knoops et al., 2008) . in spite of all these indications for rdrp-helicase interplay, the polarity of nidovirus helicase-mediated unwinding (5 0 -to-3 0 ) remains a major conundrum, as it is opposite to the polarities of the rdrp and many other +rna helicases, which move in a 3 0 -to-5 0 direction on the rna strand they initially bind to (seybert et al., 2000a) . this strongly suggests that the two enzymes cannot simply operate as a tandem that moves along the same template strand while copying it, a consideration that also applies to the sf1b helicase employed by alphaviruses. this problem could be resolved if rdrp and helicase would move along different strands of the same rna duplex, which might allow the helicase to separate the two strands and provide a single-stranded template to be copied by the rdrp (lehmann et al., 2015c) . also, it is tempting to speculate that the helicase, by trailing along the nascent strand (following the rdrp at a certain and, possibly, somewhat flexible distance), provides (i.e., leaves behind) a single-stranded template, thus facilitating initiation and elongation of rna synthesis by the next rtc. this, for example, could occur in cases when multiple rtcs act simultaneously/consecutively on the same template to produce multiple plus-strand rnas from the same minus-strand template, a process that is generally thought to add to the large excess of plus-over minus-strand rnas in nidovirus-infected cells. clearly, significantly more work is needed to explore this possibility. nidovirus sg mrnas are each produced from their own subgenomelength minus-strand template (see section 1). in the case of arteri-and coronaviruses, these derive from a process of discontinuous minus-strand rna synthesis and this unique mechanism likely requires specific functional interactions between (among others) rdrp and helicase. these interactions may contribute to maintaining a proper balance between continuous and discontinuous minus-strand rna synthesis, and thus between the production of new genomes and sg mrnas. the serendipitous identification of an arterivirus nsp10-zbd mutation (ser-59 ! pro) that essentially inactivated transcription while leaving replication unaffected was an early indication for the involvement of the nidovirus helicase in the control of sg mrna synthesis (van dinten et al., 1997) . such control functions could also be related to the recognition of trss (fig. 1) , the frequency with which each of the trss is used to produce a subgenome-length minus strand, or mechanistic aspects of the stalling and reinitiation of rna synthesis or the transfer of the nascent strand to an upstream position on the template (see earlier), which must occur during the discontinuous step in sg rna synthesis (lehmann et al., 2015c) . recently, the eav nsp10 ser-59 ! pro mutation, which selectively reduces transcription of all subgenomic mrnas to below 1% of their normal levels, was reanalyzed in the context of the nsp10 crystal structure. as postulated when this virus mutant was first described, its phenotype appears to be based on the special structural properties of the pro residue in combination with the position of residue 59 in a "hinge" region that connects zbd to the rest of the protein (deng et al., 2014) . although residue 59, located just downstream of the second zinc-binding module of the zbd, is fairly distant from the rna-binding surface, it resides in a region that connects the zbd treble-clef zinc finger to a helix that interacts with domains 1a and 1b and with the nucleic acid. thus, specific mutations affecting the flexibility of this hinge region may drastically influence the long-distance signaling within nsp10, apparently preventing the rnasynthesizing machinery to work in "transcription mode" and dedicating it exclusively to full-length minus-strand rna synthesis and genome amplification. since this kind of mutations barely affected nsp10s in vitro ntpase and helicase activities (seybert et al., 2005) , it may well be that changed interactions with specific protein partners will turn out to be the key to explaining the transcription-negative phenotype of the corresponding virus mutants (lehmann et al., 2015c) . for the coronavirus ibv, a point mutation in a somewhat comparable position of nsp13 (arg-132 ! pro; just downstream of zbd) was reported to cause a similar block of sg mrna synthesis (fang et al., 2007) but, thus far, this observation has not been followed up in more detail for ibv or confirmed for nsp13 of another cov. in addition to its role in rna synthesis, the nidovirus helicase is also assumed to be involved in the capping pathway of viral mrnas by exhibiting an rna 5 0 -triphosphatase (rtpase) activity that can remove the 5 0 -terminal triphosphate from the rna substrate. for sars-cov and hcov-229e nsp13, this first step in viral cap synthesis was shown to rely on the same ntpase active site that provides the energy for the protein's helicase activity (ivanov and ziebuhr, 2004; ivanov et al., 2004b) . the cov capping pathway is discussed in section 5 of this review. finally, the remarkable similarities between eav nsp10 and the cellular helicase upf1 (deng et al., 2014) have given rise to the intriguing but still speculative hypothesis that the nidovirus helicase may be involved in the posttranscriptional quality control of viral rna. common features of the two helicases include their 5 0 -to-3 0 polarity of unwinding, their lack of substrate specificity and striking similarities in terms of domain organization and fold (fig. 4) (lehmann et al., 2015c) . using several pathways, including nonsense-mediated mrna decay, upf1 mediates rna quality control in eukaryotic cells (cheng et al., 2007; clerici et al., 2009) , while its activity can be modulated through interactions of its n-terminal zbd. it was postulated that a similar function in nidovirus replication, e.g., detection and elimination of defective viral mrnas (including the genome), could explain the conservation of the unique zbd across the nidovirus order, which stands out for containing members with very large + rna genomes. such a function would prevent the synthesis of defective viral polypeptides, which might interfere with the proper functioning of full-length viral proteins. in this manner, not unlike the nsp14-exon domain (see earlier), the nidovirus helicase may have contributed to genome expansion by providing a form of compensation for the relatively poor fidelity of genome replication by the nidoviral rdrp (deng et al., 2014) . clearly, this is just one of the scenarios for the involvement of the helicase in the posttranscriptional fate of viral rna products. further experimental work will be needed to explore these possibilities in more detail, as they are compatible with the much broader realization that the functions of rna helicases can extend far beyond merely the unwinding of rna structure. due to its multifunctionality and involvement in several key processes in viral rna synthesis and processing, the nidovirus helicase is an important target for antiviral drug development, which was mainly explored for covs following the sars-cov outbreak. the highly conserved nature of the helicase offers the interesting perspective of developing inhibitors with a potential broad-spectrum activity. on the other hand, avoiding toxicity resulting from inhibition of the abundant cellular ntpases/helicases poses a serious challenge, which is why-as in the case of the rdrp-obtaining a crystal structure for a cov helicase should be considered a research priority. in theory, a variety of helicase properties may be targeted with specific inhibitors, ranging from the active and nucleic acid-binding sites of the enzyme to interaction surfaces for multimerization and modulation by protein cofactors (kwong et al., 2005) . several compound families were found to target the atpase of nsp13, and thus also its helicase activity. these include naturally occurring flavonoids (yu et al., 2012) , chromones (kim et al., 2011) , and bananins (kesel, 2005; tanner et al., 2005) , all exhibiting in vitro ic 50 values in the low-micromolar range. other compounds appear to target the helicase of nsp13 activity more specifically, like the triazole ssya10-001, which was found to inhibit the replication of multiple beta-covs (sars-cov, mers-cov, and mhv) in cell culture-based infection models (adedeji et al., 2012b (adedeji et al., , 2014 . the ic 50 value in in vitro helicase assays was about 5.5 μm, whereas ec 50 values in cell cultures assays were in the range of 7-25 μm, depending on the cov analyzed, suggesting that broad-spectrum activity may indeed be achieved. to our knowledge, the antiviral potential and toxicity of ssya10-001 in animal models remain to be tested. another interesting group of helicase-directed antiviral hits are bismuth complexes, which were postulated to inhibit the ntpase and helicase functions by competing for zinc ions with the zbd. in sars-cov-infected cell cultures the determined ec 50 and cc 50 values were 6 μm and 5 mm, respectively (yang et al., 2007) . nsp10-13-14-16 cap structures consists of a 7-methylguanosine ( m7 g) linked to the first nucleotide of the rna transcript through a 5 0 -5 0 triphosphate bridge (for a review, see decroly et al., 2012) . in eukaryotic cells, the synthesis of the cap structure is a multistep process that occurs in the nucleus and is coupled to rna pol ii-driven transcription (shatkin, 1976) . capping begins with the hydrolysis of the 5 0 γ-phosphate of the nascent rna transcript by an rna 5 0 -triphosphatase (rtpase). subsequently, a gmp molecule is transferred to the 5 0 -diphosphate of the rna by a gtase, leading to the formation of gpppn-rna. the cap structure is methylated at the n7 position of the guanosine by an (adomet)-dependent n7-mtase, yielding cap-0 ( m7 gpppn). the cap-0 structure is then converted into cap-1 ( m7 gpppn 2 0 -om ) or cap-2 by an adomet-dependent 2 0 -o-mtase that methylates the 2 0 -o position of the ribose of the first or first and second rna nucleotide, respectively. due to the cytoplasmic localization of their mrna synthesis, nidoviruses, and all other +rna viruses of eukaryotes, cannot rely on the standard capping pathway outlined earlier, which is executed by host cell enzymes in the nucleus. cap structures can protect viral mrnas from degradation by the cellular 5 0 -to-3 0 exoribonuclases involved in rna turnover (liu and kiledjian, 2006) . cap methylation is critical for mrna recognition by translation initiation factor eif4e (filipowicz et al., 1976; ohlmann et al., 1996) , and thus for viral translation and replication as a whole . in addition to promoting mrna stability and securing their recognition by host cell ribosomes, capping of viral mrnas also promotes escape from certain antiviral responses of the host cell. the retinoic acidinducible gene 1 and melanoma differentiation-associated protein 5 (mda5) were shown to detect either uncapped triphosphorylated rna or cap-0-containing rna (devarkar et al., 2016; hyde and diamond, 2015; hyde et al., 2014; schuberth-wagner et al., 2015; z€ ust et al., 2011) , resulting in the expression of antiviral interferon-stimulated genes (isgs) in infected and neighboring cells. interferon-induced proteins with a tetratricopeptide repeat 1 (ifit1/56) and ifit2/54 (ifit2) have been shown to recognize miscapped rnas, in order to restrict viral translation (daffis et al., 2010; pichlmair et al., 2011) . a subsequent study identified ifit1 as the only interferon-induced protein whose rna-binding affinity was affected by the ribose-2 0 -o methylation state of the 5 0 cap structure (cap-0/cap-1). the data support a model in which ifit1 efficiently binds and sequesters capped mrna that lacks a ribose-2 0 -o-methyl group. consistent with this, viral mrna translation was shown to be reduced in cells infected with 2 0 -o-mtase-deficient covs (habjan et al., 2013) . other studies suggested that 2 0 -o methylation of cap structures prevents or delays the mda5-dependent recognition of viral mrnas as "nonself." this mrna cap modification thus limits the antiviral response launched upon infection, thereby affecting viral pathogenesis (daffis et al., 2010; schuberth-wagner et al., 2015; z€ ust et al., 2011) . the genomic and sg mrnas of nidoviruses are presumed to be capped at their 5 0 end and polyadenylated at their 3 0 end (fig. 1) . the presence of a cap structure was first suggested based on studies using 32 p-labeled mhv rna that was digested with rnase t1 and t2 and subjected to deaecellulose chromatography (lai and stohlman, 1981) . the presence of a cap was further substantiated by immunoprecipitation experiments using a cap-specific monoclonal antibody that was shown to specifically bind to equine torovirus mrnas (van vliet et al., 2002) . although the (presumed) capping machinery of arteriviruses has remained essentially uncharacterized thus far (lehmann et al., 2015b) , three conserved putative capping enzymes were identified in the conserved orf1b-encoded part of the replicase of coronaviridae, roniviridae, and mesoniviridae, which all have substantially larger genomes. these enzymatic activities, which were proposed to participate in the synthesis of a cap-1 structure ( n7m gpppn 2 0 om ), are the following: (i) the nsp13 helicase/rtpase (ivanov and ziebuhr, 2004) , (ii) the nsp14 n7-mtase (chen et al., 2009c; ma et al., 2015) , and (iii) the nsp16 2 0 -o-mtase (bouvet et al., 2010; decroly et al., 2008; snijder et al., 2003; von grotthuss et al., 2003; zeng et al., 2016) . at present, structural and functional studies, which were mainly performed with purified recombinant sars-cov nsps, suggest that covs follow a capping pathway that is very similar to that of eukaryotic cells. cap synthesis is presumed to start by hydrolysis of the 5 0 end of a nascent rna by the rtpase function of nsp13 to yield pp-rna (ivanov and ziebuhr, 2004) . then, a still elusive gtase must transfer a gmp molecule onto the pp-rna to yield gppp-rna. the cap structure is then methylated at the n7 position by the n7-mtase domain of the bifunctional nsp14 (bouvet et al., 2010; chen et al., 2009c) . cap modification is completed by the conversion of the cap-0 into a cap-1 structure, which involves the nsp10/nsp16 complex (bouvet et al., 2010; chen et al., 2009c) in which nsp16 possesses 2 0 -o-mtase activity. in the following paragraphs, we will describe the different cov enzymes involved in mrna capping in more detail. as described earlier, the nsp13 helicase domain is thought to unwind double-stranded rna in a 5 0 -to-3 0 direction, an activity that energetically depends on the nucleotide triphosphatase (ntpase) function of the enzyme (ivanov and ziebuhr, 2004; ivanov et al., 2004b; seybert et al., 2000a) . additionally, using the same active site, the protein was found to exhibit rtpase activity (ivanov and ziebuhr, 2004) , which was found to be abolished if the conserved active-site lys residue of the walker a box motif was replaced with ala (ivanov and ziebuhr, 2004; ivanov et al., 2004b) . specific rtpase activity on viral mrna species would require specific recruitment of nsp13 to the 5 0 end of viral mrnas, which has not been demonstrated for cov helicases but may involve yet other factors. in this context, it remains to be studied if the common leader sequence present on cov mrnas contributes to the recruitment of nsp13 and/or other proteins involved in 5 0 capping reactions. several other +rna virus helicases were shown to possess an activity that can target the phosphodiester bond between the β and γ phosphate groups of the 5 0 -terminal ntp of diverse substrate rnas, suggesting that this dual function of helicase and capping rtpase is a common feature in this group of viruses ivanov and ziebuhr, 2004) . even though experimental evidence has been obtained to suggest an nsp13-associated 5 0 rtpase activity, coronavirus nsp13 homologs proved to be unable to transcomplement the yeast strain ybs20, which lacks the cet1 locus encoding the yeast rtpase involved in mrna capping (chen et al., 2009c) . the lack of trans-complementation by nsp13 could be due to technical reasons, such as inappropriate subcellular localization of nsp13, misfolding of the protein, or a functional mismatch with other players of the distantly related yeast capping machinery. alternatively, it could indicate specific substrate requirements for coronavirus nsp13-associated rtpase activities. in any case, a possible involvement of nsp13 in the first step of cov mrna capping remains to be corroborated in further studies, for example, by in vitro reconstitution experiments of the entire cov capping pathway. the cov nsp involved in the second step of rna capping, gtase, remains to be identified. bioinformatics analysis of cov genome sequences failed to identify replicase gene-encoded domains that may perform this activity. eukaryotic rna capping enzymes belong to the ligase family and have been shown to form a gtase-gmp adduct upon incubation with gtp . a substantial number of sars-cov nsps were expressed and purified (nsp7, nsp8, nsp10, and nsp12-16), but covalent linkage of gmp to any these proteins could not be demonstrated (jin et al., 2013) . in addition, nsps were screened for gtase activity in a yeast trans-complementation system using the ybs2 strain lacking the gene (ceg1) encoding the yeast gtase (chen et al., 2009c) , but also this powerful approach failed to identify the cov gtase. consequently, several hypotheses remain to be explored. first, it is possible that the n-terminal niran domain of nsp12 (see earlier) forms a covalent adduct with gtp, as observed for its arterivirus homolog nsp9 (lehmann et al., 2015a) . another possibility is that the cov capping pathway is unconventional and, for example, resembles that of alphaviruses in which the gtp molecule needs to be methylated at its n7 position before the gtase-m gmp adduct can be formed (ahola and ahlquist, 1999) . interestingly, this second possibility might explain nsp14s capability to convert gtp into m gtp (see later) (jin et al., 2013) . finally, the involvement of a host gtase remains an interesting possibility, in particular since cytoplasmic forms of cellular capping enzymes have been described recently (mukherjee et al., 2012; schoenberg and maquat, 2009 ). further work is needed to explore these various hypotheses and resolve this important question. coronavirus nsp14 is a bifunctional protein that plays a crucial role in viral rna synthesis. its n-terminal exonuclease (exon) domain (minskaia et al., 2006; snijder et al., 2003) , which is thought to promote the fidelity of cov rna synthesis, will be discussed in more detail in section 6. the c-terminal part of nsp14 carries an adomet-dependent guanosine n7-mtase activity. interestingly, as in the case of the gtase (see earlier), bioinformatics analyses of cov genome sequences failed to identify proteins or protein domains related to cellular and/or viral n7-mtases, again illustrating the significant divergence of nidoviruses from other viral and cellular systems. however, using a trans-complementation assay and a yeast strain lacking the abd1 (n7-mtase) gene, guo et al. discovered a sars-cov nsp14-associated n7-mtase activity (chen et al., 2009c) by demonstrating that the protein was able to restore the growth of the △abd1 yeast mutant. they also showed that a range of alphacoronavirus nsp14 homologs were able to complement the defect of the △abd1 yeast strain. the n7-mtase activity of nsp14 was subsequently confirmed and characterized using purified recombinant enzymes (bouvet et al., 2012; chen et al., 2009c) . the sars-cov n7-mtase was shown to methylate 5 0 cap structures in a sequenceindependent manner using a range of rnas and it also proved to be active on cap analogues and gtp (jin et al., 2013) , corroborating the trans-complementation experiments in yeast in which rescue required efficient methylation of a wide range of cellular rnas. in contrast to the exon activity, the in vitro n7-mtase activity was not found to be affected by interactions with nsp10 (bouvet et al., 2010) . the n7-mtase domain was further characterized by alanine scanning mutagenesis and key residues for enzymatic activity were identified (chen et al., 2009c) including 10 crucial residues distributed throughout the domain and two clusters of residues essential for mtase activity (fig. 5 ). the first cluster (nsp14 residues 331-336) corresponds to the dxgxpxa motif of the adomet-binding site. in cross-linking experiments, mutations in this motif strongly decreased the binding of 3 h-labeled adomet. the role of the second cluster, between residues 414 and 428, was revealed by x-ray structure analysis of a sars-cov nsp10/nsp14 complex expressed in e. coli (fig. 6) (ma et al., 2015) . these residues form a constricted pocket that holds the cap structure (gpppa) between two β-strands (β1 and β2) and helix 1, placing the n7 position of the guanine in close proximity of adomet and ready for methyl transfer using an in-line mechanism. , and ibv (genus gammacoronavirus). the alignment was generated using clustal omega (sievers et al., 2011) and rendered using espript version 3.0 (robert and gouet, 2014) . conserved exon motifs i, ii, and iii and clusters of residues involved in sam binding and n7-mtase activity (1 and 2) are highlighted in gray. catalytic residues of exon and residues involved in the formation of zinc fingers are indicated by asterisks and arrowheads, respectively. also shown are the secondary structure elements of sars-cov nsp14 (pdb 5c8s) and the border between the n-terminal exon and c-terminal n7-mt (nmt) domains. the structure also revealed that the nsp14 n7-mtase domain exhibits a noncanonical mtase fold. whereas the canonical fold contains a 7-strand β-sheet that is commonly present in the rossman fold, nsp14 contains only a 5-strand β-sheet and an insertion of a 3-strand antiparallel β-sheet between β5 and β6. in line with previous mutagenesis data (chen et al., 2009c) , the fig. 6 surface representation of the three-dimensional structure of the nsp10/nsp14 complex (pdb 5c8s). the nsp10 ribbon structure is shown with conserved residues colored in blue (using a scale from dark to light blue). the coloring of the nsp14 surface is based on the conservation of the respective residues among covs (using a scale from dark to light red). the upper panel shows the surface containing the exon catalytic site with one mg 2+ ion bound in the active site (green sphere). the lower panel shows the opposite side of the structure with the n7-mtase active site. the cap analog gpppa and sah are shown in stick representation. the figures were generated using ucsf chimera (pettersen et al., 2004) . the degree of conservation of specific residues was determined using an alignment of nsp10 and nsp14 sequences of eight coronaviruses representing the four genera of the coronavirinae subfamily (sars-cov, mers-cov, mhv, tgev, fcov, hcov-229e, ibv, and bulbul coronavirus hku11). nsp14 x-ray structure revealed functionally relevant interactions between the n-terminal (exon) and c-terminal (n7-mtase) domains, with three α-helices of exon stabilizing the base of the n7-mtase substrate-binding pocket. the specific role of n7-mtase activity in virus replication was supported by reverse genetics. nsp14 mutations were introduced in sars-cov rna replicons expressing a luciferase reporter gene. a d331a mutation in the adomet-binding site, which blocks the n7-mtase activity of nsp14, was shown to reduce the luciferase expression (by 90%), indicating that viral rna replication and/or transcription were impaired in this in vitro system (chen et al., 2009c) . the nsp14 n7-mtase is an attractive target for antiviral strategies, especially because it exhibits a range of features that are distinct from host cell mtases . the druggability of the enzyme was explored using a small set of previously documented mtase inhibitors. these in vitro assays revealed that adohcy (the coproduct of the methylation reaction), sinefungin (another adomet analog), and ata efficiently inhibited nsp14 n7-mtase activity with ic 50 values of 12 μm, 39.5 nm, and 2.1 μm, respectively (bouvet et al., 2010) . ata was also shown to limit sars-cov replication in infected cells (he et al., 2004) . in the yeast-△mtase trans-complementation assay mentioned earlier, micromolar concentrations of sinefungin were reported to effectively suppress the nsp14 n7-mtase activity of sars-cov, mhv, transmissible gastroenteritis virus (tgev), and ibv (sun et al., 2014) . however, other compounds, such as ata and adohcy, did not exert an inhibitory effect in the context of yeast cells. these discrepancies may reflect differences in cell penetration of the compounds between yeast and (virus-infected) mammalian cells. the yeast system was also applied to screen a library of 3000 natural product extracts, and three hits were obtained displaying potent inhibitory effects on the cov n7-mtase (sun et al., 2014) . further work is needed to optimize these hits and test their inhibitory activities in assays using cov-infected cells. the presence of a 2 0 -o-methyl transferase (2 0 -o-mtase) domain in cov nsp16 was first inferred using bioinformatics (snijder et al., 2003; von grotthuss et al., 2003) . computational threading produced a model containing a conserved k-d-k-e catalytic tetrad that is characteristic of adomet-dependent 2 0 -o-mtases and a conserved adomet-binding site ( fig. 7) (von grotthuss et al., 2003) . the 2 0 -o-mtase activity was then confirmed by in vitro biochemical assays using purified fcov nsp16 (decroly et al., 2008) . the recombinant protein was shown to specifically recognize short, cap-0-containing rnas and to transfer a methyl group from adomet to the 2 0 -o position of the first nucleotide of the n7-methylated substrate. in contrast, a recombinant form of the sars-cov nsp16 homolog was inactive under similar experimental conditions. since yeast two-hybrid experiments and gst pull-down assays had revealed that sars-cov nsp16 strongly interacts with nsp10 pan et al., 2008) , a possible involvement of the latter in regulating or supporting the 2 0 -o-mtase activity was tested. it was shown that purified nsp10 interacts with nsp16 in vitro and thereby triggers a robust 2 0 -o-mtase activity (bouvet et al., 2010) . effective methyl transfer was demonstrated using synthetic capped n7-methylated rna and longer rna mimicking the 5 0 end of the sars-cov genome (bouvet et al., 2010) . in contrast, rna with a nonmethylated cap structure (gppp-rna) was not bound by the nsp10/ nsp16 complex and, consequently, could not serve as a substrate. these observations suggest that sars-cov mrna capping follows a strict order of reaction steps: after gtp transfer by the still elusive gtase, the cap is methylated by the nsp14 n7-mtase at the guanosine-n7 position to produce a cap-0 structure that, in a subsequent reaction, is bound by the nsp10/ nsp16 complex and converted to a cap-1 structure employing the 2 0 -o-mtase activity of nsp16. mutagenesis of sars-cov nsp10 and nsp16 confirmed the importance of the catalytic tetrad of the nsp16 2 0 -o-mtase (decroly et al., 2011) and showed that the nsp10-nsp16 interaction is absolutely required for activity (decroly et al., 2011; lugari et al., 2010) . crystallographic studies of the nsp10/nsp16 complex revealed the molecular basis for the stimulation of the nsp16 2 0 -o-mtase activity by nsp10 (fig. 7) (chen et al., 2011; decroly et al., 2011) . the cov 2 0 -o-mtase belongs to the rrmj/fibrillarin superfamily of ribose 2 0 -o-methyl transferases (feder et al., 2003) which have a number of viral orthologs in flaviviruses, alphaviruses, and other nidoviruses. as mentioned earlier, this family contains a conserved k-d-k-e catalytic tetrad (fig. 7) that is located in close proximity to the substrate-binding pocket accommodating the rna substrate. the structure revealed that nsp10 is an allosteric regulator that stabilizes nsp16. moreover, structural and biochemical analyses indicated that nsp10 binding extends and narrows the rna-binding groove that accommodates the rna substrate, thereby promoting the rnaand adomet-binding capabilities of nsp16 (fig. 7) . ) , and ibv (genus gammacoronavirus). the alignment was generated using clustal omega (sievers et al., 2011) and rendered using espript version 3.0 (robert and gouet, 2014) . residues of the catalytic tetrad k-d-k-e are indicated by asterisks and secondary structure elements of sars-cov nsp16 (pdb 2xyv) are shown. (b) surface representation of the three-dimensional structure of the nsp10/nsp16 (continued) the functional relevance of 2 0 -o-mtase regulation by nsp10 for virus replication is not yet understood. the nsp16-associated 2 0 -o-mtase activity (itself ) is highly conserved across the coronaviridae family and its functional relevance has been supported by reverse-genetics studies using genetically engineered alpha-and betacoronavirus mutants that lack 2 0 -o-mtase activity (devarkar et al., 2016; hyde and diamond, 2015; schuberth-wagner et al., 2015; z€ ust et al., 2011) . furthermore, the phenotype of temperature-sensitive mhv mutants suggested nsp16 to play a role in rna synthesis or in the stability of plus-strand rna (sawicki et al., 2005 ). an early sars-cov study was able to show that the insertion of a stop codon immediately upstream of the nsp16-coding sequence blocked viral rna synthesis (almazan et al., 2006) . subsequently, for hcov-229e, mhv, and sars-cov, several mutants with reduced or ablated 2 0 -o-mtase activity were described that generally retained robust viral replication in cell culture (menachery et al., 2014; z€ ust et al., 2011) . the studies also revealed that the impact of nsp16 mutations may depend on the cell types used and that the lack of 2 0 -o-mtase activity causes more profound effects in primary cells and immune cells. the sars-cov nsp16 mutants were further characterized in infected mice and showed a robust attenuation as judged by viral titers, weight loss, lung histology, and respiratory function. the nsp16 mutants also displayed increased sensitivity to treatment with type i interferon. this was also observed for the corresponding mhv and hcov-229e nsp16 mutants (z€ ust et al., 2011) . however, in contrast to the latter study, the sars-cov nsp16 mutant was not found to induce type i interferons, either in vitro or in vivo (menachery et al., 2014) . this observation suggests that sars-cov may have a larger repertoire of functions for preventing induction of type i interferons following cellular sensing of "nonself" rnas, such as viral rnas with incompletely methylated 5 0 cap structures. together, these data established that the highly conserved nsp16 2 0 -o-mtase plays an important role in limiting the detection of viral fig. 7 -cont'd complex (pdb 2xyv). nsp10 is shown in ribbon representation with conserved residues colored in dark to light blue according to their conservation among covs. zinc molecules are shown as spheres and zinc-coordinating residues are shown in stick representation. the surface of nsp16 is colored in dark to light red according to the conservation of the respective residues among coronaviruses. sah is depicted in a stick model. the figure was generated using ucsf chimera (pettersen et al., 2004) . the degree of conservation of specific residues was determined using an alignment of nsp10 and nsp16 sequences of eight coronaviruses (see fig. 6 ). rna by the host's antiviral sensors, but that the specific role of this activity in escaping host innate immune responses may differ to some extent among covs. the mechanism underlying the induction of the antiviral immune response following infection with 2 0 -o-mtase knockout mutants was studied using specific knockout mice in which viral replication was found to be restored. the absence of mda5, a cap-0 sensor, restored mhv replication, whereas the nuclear translocation of irf3 and interferon induction were reduced (z€ ust et al., 2011) . this suggested mda5 to function in the primary recognition of the rna produced by cov 2 0 -o-mtase mutants and to initiate the isg cascade that restricts viral replication. among the isg products, the ifit family of proteins was shown to be critically involved in reducing the replication of cov nsp16 mutants. the replication of mhv and hcov-229e nsp16 mutants and wild-type controls was similar in ifit1à/à knockout mice (habjan et al., 2013; z€ ust et al., 2011) . likewise, replication of a sars-cov δnsp16 mutant was increased in both ifit1 and ifit2 knockdown mice (menachery et al., 2014) , suggesting that ifit family proteins mediate the primary attenuation of sars-cov 2 0 -o-mtase knockout mutants. the earlier results indicate that the nsp16 2 0 -o-mtase constitutes a new and attractive target for the development of antiviral drugs against sars-cov and hcov-229e, as well as newly emerging covs like mers-cov and porcine epidemic diarrhea virus (pedv). for example, the nsp10-nsp16 interface may be targeted to limit viral 2 0 -o-mtase activity and thus restore the antiviral responses mediated by mda5 and ifit1 (menachery and baric, 2013) . interestingly, the nsp10 residues involved in the nsp10/nsp16 interaction are quite conserved within the cov family and it was recently demonstrated that nsp10 of different covs (fcov, mhv, sars-cov, mers-cov) is functionally interchangeable in the stimulation of nsp16 2 0 -o-mtase activity . thus, molecules or peptides blocking this interface may have broad-spectrum anti-cov effects, a concept that was explored and supported using synthetic peptides that mimic the nsp10 interface and suppress nsp16 2 0 -o-mtase activity in vitro (ke et al., 2012; wang et al., 2015) . the antiviral effect of the mhv tp29 peptide, for example, was first demonstrated in mhv-infected cells and was subsequently confirmed to limit mhv replication in mice and to enhance the type i interferon response . the same peptide was also effective in blocking the replication of a sars-cov replicon. the cov exon domain was identified on the basis of comparative sequence analyses (snijder et al., 2003) that suggested a distant relationship of the nsp14 n-terminal domain (and equivalent polyprotein regions of other nidoviruses) with cellular dedd exonucleases, a large protein superfamily containing rna and dna exonucleases from all kingdoms of life (zuo and deutscher, 2001) . the designation "dedd" alludes to four invariant asp/glu residues that are part of three sequence motifs, i-iii, that are conserved in members of this superfamily (fig. 5) . the dedd superfamily is also referred to as dnaq-like family because it includes dnaq, the ε subunit of e. coli dna polymerase iii, a well-characterized proofreading enzyme scheuermann et al., 1983) . conservation of a fifth residue, his, located four positions upstream of the conserved asp in motif iii identifies exon as a member of the deddh subgroup, while members of the deddy exonucleases contain tyr at the equivalent position. the acidic residues are required to form two metal-binding sites. based on catalytic models initially developed for cellular exonucleases and catalytic rna (beese and steitz, 1991; steitz and steitz, 1993) , the conserved his and the site a metal ion are thought to activate a water molecule that launches a nucleophilic attack on the phosphorus group of the 3 0 -terminal phosphodiester, while the site b metal ion is thought to stabilize the transition state (derbyshire et al., 1991) . exon is conserved in covs and all other known nidoviruses with genome sizes of >20 kb minskaia et al., 2006; nga et al., 2011; snijder et al., 2003; zirkel et al., 2011) . the correlation between genome size and exon conservation suggests that, in nidoviruses with medium-and large-size genomes, the correct nucleotide selection and recognition of properly formed base pairs by the rdrp is not enough to accomplish the necessary replication fidelity and, therefore, requires additional functions suitable to detect and remove misincorporated nucleotides. recently, biochemical evidence has been provided to suggest that exon may have exactly this function (bouvet et al., 2012) . cov mutants that lack exon activity provided additional evidence for exon being involved in mechanisms that keep the cov mutation rate at a relatively low level (<10 à6 mutations per site per round of replication for mhv and sars-cov) (eckerle et al., 2007 (eckerle et al., , 2010 , while other rna viruses have much higher mutation rates, ranging from 10 à3 to 10 à5 mutations per site per round of replication (drake and holland, 1999; sanjuan et al., 2010) . exon knockout mutants of sars-cov and mhv were shown to display a mutator phenotype with significantly increased mutation frequencies approaching those of other rna viruses (eckerle et al., 2007 (eckerle et al., , 2010 . considering that these studies were performed under selection pressure favoring genotypes with high replication efficiency, the total number of mutations in rnas produced by exon-deficient viruses may be even higher than calculated in that study, especially in genome regions that are not subject to selection in in vitro cell culture systems. while inactivation of exon activity was tolerated by mhv and sars-cov (albeit with reductions in replication efficiency), stable exon-deficient mutants of the alphacoronaviruses hcov-229e and tgev could not be recovered (becares et al., 2016; minskaia et al., 2006) , supporting the critical role of this activity in cov replication. taken together, the available information provides compelling evidence for exon playing a key role in high-fidelity replication of covs. consistent with this hypothesis, genetically engineered exon knockout mutants were shown to be significantly more sensitive to rna mutagens such as ribavirin and 5-fluorouracil (up to 300-fold). furthermore, compared to wild-type virus, the exon knockout mutants were shown to accumulate a much higher number of mutations when propagated in the presence of mutagens (smith et al., 2013) . the lack of exon activity was also shown to have profound effects on viral replication and pathogenesis in vivo (graham et al., 2012) . exon-negative mutants displayed a stable mutator phenotype in a number of mouse models of human sars, providing a promising approach for the stable attenuation of highly pathogenic covs with important implications for vaccine development (graham et al., 2012) . coronavirus nsp14 is a bifunctional protein comprised of an n-terminal exon domain and a c-terminal n7-mtase domain. surprisingly, the latter domain is not conserved in the torovirinae (genera torovirus and bafinivirus), representing the other subfamily of the coronaviridae, but in other (more distantly related) nidovirus branches (lauber et al., 2013; nga et al., 2011) . this conservation pattern suggests that a common ancestor of the corona-, mesoni-, and roniviridae contained the two-domain exon/n7-mtase structure while some lineages lost the n7-mtase domain at a later stage. although nsp14 does not require other proteins for activity minskaia et al., 2006) , its ribonucleolytic (but not the n7-mtase) activity was shown to be stimulated significantly in the presence nsp10. in line with this, nsp10 variants carrying amino acid substitutions that prevent the interaction with nsp14 failed to stimulate exon activity (bouvet et al., 2012 (bouvet et al., , 2014 . to date, there is no evidence to suggest a direct role for nsp10 in catalysis. most likely, interactions between the nsp10 and the n-terminal domain of nsp14 stabilize the exon active site in a catalytically competent conformation. mutagenesis data and a recent x-ray structure analysis of a sars-cov nsp10/nsp14 complex (see fig. 6 ) revealed that the nsp10 surface required for interaction with nsp14 overlaps with the surface involved in the interaction and activation of the nsp16 2 0 -o-mtase activity (see earlier) (bouvet et al., 2014; ma et al., 2015) . coronavirus exon activities were first demonstrated using recombinant forms of sars-cov nsp14 expressed in e. coli (minskaia et al., 2006) . the protein was shown to require mg 2+ or mn 2+ ions for activity and to degrade a range of single-stranded (ss) synthetic rnas with 3 0 -to-5 0 directionality to yield reaction products of about 8-12 nucleotides. the data further suggested that rna secondary structure affects exon activity (minskaia et al., 2006) . mutational analysis of predicted active-site (asp/glu) residues confirmed their critical involvement in catalysis (minskaia et al., 2006) . in a subsequent study, using nsp10/nsp14 complexes, the substrate specificity was characterized in more detail and revealed dsrna with a terminal mismatch to be the preferred substrate for exon activity. excision efficiencies using different mismatched base pairs (a:g, a:a, a:c, u:g, u:c, u:u) were found to be similar, suggesting that the mismatch rather than the nature of the nucleotide misincorporated at the 3 0 end determines exon activity (bouvet et al., 2012) . in a recent study, the structures of unliganded, sam-bound, and sah-gpppa-bound sars-cov nsp10-nsp14 complexes were determined by x-ray crystallography (ma et al., 2015) . the structures provide important insight into the two-subdomain structure of nsp14, the two catalytic sites of exon and n7-mtase, critical substrate-binding residues, the contribution of nsp10 to enhancing exon activity, and the roles of as many as three zinc fingers present in nsp14 (fig. 6) . in the structure, one molecule of nsp14 was found to bind one molecule of nsp10. given that nsp10 tends to form multimers , it is tempting to speculate that, in infected cells, the nsp10-nsp14 complex may form even larger complexes, for example, by interacting with nsp10-nsp16 complexes that might be stabilized by nsp10-nsp10 interactions. in this way, consecutive methylation reactions of the 5 0 cap structure could be spatially coordinated. comparison of the nsp10 surfaces that interact with nsp14 and nsp16, respectively, revealed a significant overlap (bouvet et al., 2014) , with a substantially larger surface being involved in interactions between nsp10 and nsp14. buried solvent accessible areas for interactions with nsp14 and nsp16 were determined to be 2236 and 938 å 2 , respectively (decroly et al., 2011; ma et al., 2015) . the structure of the nsp10-nsp14 complex also helps to explain the observed stimulation of exon activity by nsp10 (see earlier). two regions of nsp10 interact extensively with different structural elements of the nsp14 n-terminal domain, most likely to maintain the structural integrity of the exon domain. interestingly, the observed interaction of n-terminal residues of nsp10 with nsp14 led to interpretable electron density for these residues that had not been observed in previous structures of nsp10 (joseph et al., 2007; su et al., 2006) or the nsp10-nsp16 complex (chen et al., 2011; decroly et al., 2011) , consistent with the proposed role of the n-terminal loop of nsp10 in stabilizing interactions with nsp14. the structure of the exon domain is essentially comprised of a twisted β-sheet that is formed by five β-strands and flanked by α-helices on either side. it resembles that of other dedd superfamily exonucleases, such as the ε subunit of e. coli dna polymerase iii, but also has unique features. these include two segments (residues 1-76 and 119-145) that are involved in the interaction with nsp10 and two zinc fingers in the exon domain (see fig. 5 ). the second zinc finger was found in close proximity to the catalytic site. both its position in the structure and mutagenesis data support a role for this zinc finger in catalysis (ma et al., 2015) . the other zinc finger appears to be required to maintain the structural integrity of nsp14. consistent with this, nsp14 variants containing substitutions in zinc finger 1 proved to be insoluble when expressed in e. coli. five residues predicted to coordinate two mg 2+ ions, , were found in the catalytic site, with one mg 2+ ion being coordinated by asp-90 (exon motif i) and glu-191 (motif ii) (fig. 5) . the second mg 2+ ion expected to be involved in the two-metalion-assisted catalytic mechanism of exon (beese and steitz, 1991; chen et al., 2007; ulferts and ziebuhr, 2011) was not identified, presumably due to the lack of an rna substrate and/or product in this structure. with one exception, metal ion-coordinating residues identified in the active site corresponded well to those identified in related cellular proofreading exonucleases (beese et al., 1993; hamdan et al., 2002) and previous predictions for nidovirus homologs (snijder et al., 2003) . the structure clearly revealed to be involved in catalysis, thus revising previous predictions on the identity of exon motif ii in the cov nsp14 primary structure and making nsp14 a "deed outlier" in the dedd superfamily of exonucleases. the combined structural and functional information obtained for nsp14 including its subdomains and the nsp10 cofactor provides an excellent basis for studies using even larger multisubunit complexes to obtain insight into the coordinated action of key replicative enzymes involved in rna synthesis, quality control, capping, methylation, and other functions (subissi et al., 2014a) . the nsp15-associated endou domain is one of the most conserved proteins among covs and related viruses (fig. 8) , suggesting important functions in the viral replicative cycle. already in the first sequence analyses of torovirus and arterivirus replicase genes published more than 25 years ago (den boon et al., 1991; snijder et al., 1990) , the identification of a conserved sequence in the 3 0 -terminal orf1b region, including the (at the time unknown) endou domain, was key to establishing phylogenetic relationships between corona-and toroviruses and, subsequently, also arteriviruses (cavanagh and horzinek, 1993; snijder et al., 1993) . outside the nidovirales, no viral homologs of endou have been identified to date. together with the helicase-associated zbd (see earlier), the nidoviral endou has therefore been proposed to be a unique and universally conserved genetic marker common to all nidoviruses (ivanov et al., 2004a; snijder et al., 2003) . only recently, with the identification of the first nidoviruses in insects (now classified in the family mesoniviridae) and reanalysis of the ronivirus replicase gene, it was found that endou is not conserved in those nidovirus branches that replicate in invertebrate hosts (mesoniviridae, roniviridae) (lauber et al., 2012 (lauber et al., , 2013 nga et al., 2011; zirkel et al., 2013) , suggesting specific roles in vertebrate hosts. to date, these functions and, more specifically, the biologically relevant substrates of endou have not been identified. characterization of mhv endou knockout mutants revealed only minor effects on viral rna synthesis in infected cells, with all rna species being equally affected, and caused a slight reduction in virus titers, which was most evident at later time points post infection (kang et al., 2007) . for the arterivirus eav, substitutions of several conserved residues in the endou domain were found to cause more profound effects, both in virus reproduction and viral rna accumulation, with sg rna being more affected than genome replication in several mutants. in some cases, reduction in virus titers by up to 5 log was observed (posthuma et al., 2006) . the (limited) information obtained in these studies suggests that endou activity is not strictly required for nidovirus rna synthesis, at least in cell culture. however, the strong conservation clearly suggests an important in vivo function that remains to be identified in suitable model systems. initial evidence for specific functions of nidovirus endou domains in infected cells was obtained in studies showing that sars-cov nsp15, but surprisingly not the homologous proteins from hcov-nl63 and hcov-hku1, counteracts mavs-induced apoptosis (lei et al., 2009) . further experiments will be necessary to confirm and assess the significance of these functions for virus replication and/or virus-host interactions. nidovirus-encoded endou activities have been characterized using recombinant forms of cov nsp15 (sars-cov, hcov-229e, mhv-a59, ibv) and arterivirus nsp11 (eav, prrsv) (bhardwaj et al., 2004; ivanov et al., 2004a; kang et al., 2007; nedialkova et al., 2009) . in a number of studies, recombinant nidovirus endous were shown (i) to have endonucleolytic activity, (ii) to cleave 3 0 of pyrimidines, preferring uridine over cytidine, and (iii) to release reaction products with 2 0 ,3 0 -cyclic phosphate and 5 0 -oh ends. using suitable test substrates, rna structural features were shown to affect endou cleavage efficiency, with unpaired pyrimidines being processed more efficiently (bhardwaj et al., 2006; nedialkova et al., 2009 ). the role of metal ions in endou activity is not entirely clear, with somewhat contradictory data being reported for different homologs. while the activities of cellular and cov homologs were shown to require (or to be significantly stimulated by) mn 2+ ions (bhardwaj et al., 2004; ivanov et al., 2004a; laneve et al., 2003 laneve et al., , 2008 , arterivirus endou activities proved to be less dependent on metal ions. low concentrations of mn 2+ were found to stimulate only marginally the arterivirus endou activities, whereas higher concentrations (previously shown to be required for optimal nucleolytic activities in cov and cellular homologs) inhibited the activities of eav and prrsv endous (nedialkova et al., 2009) . metal ion requirements are commonly used to distinguish between the two basic catalytic mechanisms employed by ribonucleases: the metal-independent mechanism that, for example, is employed by rnase a and results in products with 2 0 ,3 0cyclic phosphate ends (as described earlier for endou), and the metaldependent mechanism in which catalysis is aided by two divalent cations coordinated by conserved acidic residues and generates products with 3 0 -oh and 5 0 -phosphate ends (as described earlier for exon). the critical (or supportive) role of metal ions observed for several cellular and viral endou homologs is inconsistent with an rnase a-like (metal-independent) reaction mechanism. also, metal ions were not detected in any of the structures determined for coronavirus nsp15s or xendou, arguing against a direct role of metal ions in catalysis (renzi et al., 2006; ricagno et al., 2006) . however, mn 2+ ions were found to change the intrinsic tryptophan fluorescence of sars-cov nsp15, suggesting conformational changes that, potentially, may affect activity and were shown to be unrelated to protein multimerization (bhardwaj et al., 2004; guarino et al., 2005) . also, regarding the role of mn 2+ in rna binding, contradicting data have been reported, with rna binding by sars-cov nsp15 being enhanced in the presence of mn 2+ or not affected by metal ions in the case of xendou (bhardwaj et al., 2006; gioia et al., 2005) . structural information has been obtained by x-ray crystallography and cryoelectron microscopy studies for several cov and cellular endou homologs (bhardwaj et al., 2008; renzi et al., 2006; ricagno et al., 2006; xu et al., 2006) . sars-cov and mhv nsp15s were shown to form homohexamers comprised of a dimer of trimers. the nsp15 monomers have an α + β structure with three subdomains, a small n-terminal, an intermediate-sized middle, and a large c-terminal domain, the latter basically representing the "conserved domain" of the cov-like superfamily (see earlier). one side of this domain contains two β-sheets that line the positively charged active-site groove, while the other side is formed by five α-helices. the structures of the catalytic domains are largely conserved among cov endous and xendou, supporting the proposed common phylogeny of viral cellular members of this large endoribonuclease family (bhardwaj et al., 2008; renzi et al., 2006; snijder et al., 2003) . the endou hexamer reported for sars-cov nsp15 (ricagno et al., 2006) has dimensions of 80-96 â 110 å , forming a three-petal-shaped surface that surrounds a small, predominantly negatively charged central channel with an inner diameter of $15 å . the hexamer has six-independent active sites located on the surface of the molecule. interactions between individual protomers are predominantly mediated by residues located in the n-terminal and middle domains. database searches failed to identify closely related structural homologs, suggesting that endous diverged profoundly from other ribonucleases (renzi et al., 2006; ricagno et al., 2006) . nevertheless, the presumed endou catalytic residues (his/his/ lys, fig. 8 ) could be superimposed with the catalytic his/his/lys residues of bovine rnase a, the prototype of a large superfamily of pyrimidinespecific ribonucleases (ricagno et al., 2006; ulferts and ziebuhr, 2011) . the superposition also includes a number of conserved substrate-binding residues. a comparison of viral and cellular endou structures with that of rnase a and related nucleases using the pdbefold server revealed similarities between the structural cores of these enzymes that may be described as "interrelated by topological permutation," providing initial evidence for a common ancestry of the two endonuclease families (ulferts and ziebuhr, 2011) . further studies are required to substantiate this hypothesis (see later). the hexameric form is thought to be the fully active form of cov nsp15. this is supported by the exponential increase of activity with increased protein concentrations, the reduced activities determined for protein variants that do not multimerize and the increased rna-binding activities observed for hexameric forms of endou (bhardwaj et al., 2006; guarino et al., 2005; xu et al., 2006) . consistent with this, hexamerization has been confirmed for different cov endou homologs and residues confirmed to be involved in intersubunit interactions are highly conserved among covs. in the structure of a truncated, monomeric form of sars-cov nsp15 that lacks 28 n-terminal and 11 c-terminal residues, two loops of the catalytic domain were found to be displaced compared to their location in the hexamer, resulting in the destruction of the active site (joseph et al., 2007) . in hexameric structures, the two loops pack against each other and are stabilized by intermonomer interactions, suggesting that hexamerization may induce an allosteric switch. furthermore, cross-linking and cryoelectron microscopy studies support a specific role of hexamerization in rna binding (bhardwaj et al., 2006 (bhardwaj et al., , 2008 . in the structure model, the proposed active-site his and lys residues (fig. 8) identified by comparative sequence analysis and site-directed mutagenesis (bhardwaj et al., 2004; gioia et al., 2005; guarino et al., 2005; ivanov et al., 2004a; kang et al., 2007; snijder et al., 2003) were found to be embedded in a positively charged groove of the catalytic domain. other residues identified in the active site and proposed to be involved in binding to the substrate phosphate include the side chain of a conserved thr (fig. 8 ) and the main chain amide of a conserved gly (fig. 1) (bhardwaj et al., 2008; ricagno et al., 2006; xu et al., 2006) . the proposed functional role of the latter residues has also been corroborated by mutagenesis data for several endou homologs (kang et al., 2007; renzi et al., 2006; ricagno et al., 2006) . as mentioned earlier, endou and rnase a share a number of features in their active sites. in rnase a, pyrimidine binding primarily involves thr-45 and phe-120. while thr-45 forms hydrogen bonds with the pyrimidine base, phe-120 interacts with the base through stacking interactions (raines, 1998) . the ser-293/tyr-342 residues of sars-cov nsp15 and the thr-45/phe-120 residues of rnase a occupy similar positions in the active-site clefts of the two enzymes (ulferts and ziebuhr, 2011) . the ser/thr residue is conserved in viral and cellular domains, while tyr-342 is conserved in viral endou homologs while conservative substitutions (phe, trp) are occasionally found in cellular endou homologs (renzi et al., 2006; ricagno et al., 2006) . the role of sars-cov endou ser-293 and tyr-342 (and equivalent residues in related enzymes) in substrate binding received strong support by molecular modeling and site-directed mutagenesis data, with the conserved ser/thr residue being confirmed to have a critical role in the differential cleavage of uridine-and cytidinecontaining substrates, respectively (bhardwaj et al., 2008; nedialkova et al., 2009; ricagno et al., 2006) . similarities in their active-site structures and reaction products containing 2 0 ,3 0 -cyclic phosphate ends suggest that endous and rnase a-like endoribonucleases employ similar catalytic mechanisms (nedialkova et al., 2009; ricagno et al., 2006) . for rnase a, it has been established that two his residues in the active site act as general base and acid, respectively. the his residue that acts as a general base attracts a hydrogen from the ribose 2 0 -hydroxyl group that subsequently attacks the 5 0 p-o bond. the second his donates a hydrogen to the 5 0 -o, thus facilitating displacement of this group and subsequent product release (raines, 1998) . in a second step, the 2 0 ,3 0 -cyclic phosphate is hydrolyzed, resulting in a 3 0 -phosphomonoester product and recovery of the enzyme. the latter is essentially a reverse reaction of the transphosphorylation reaction, with the protonated his now acting as an acid and the other his acting as a base. in both reaction steps, lys interacts with the phosphate to stabilize the pentavalent reaction intermediate. although the reaction mechanisms employed by endous have not been studied in detail, the enzymes are thought to use an rnase a-like catalytic mechanism. this is supported by several lines of evidence, including (i) the conserved spatial positions in the structure and the critical functional role of two his and one lys residue(s) (ricagno et al., 2006) , and (ii) the release of 2 0 ,3 0 -cyclic phosphate-containing reaction products (bhardwaj et al., 2004; ivanov et al., 2004a) that are converted to products with 3 0 -hydroxyl ends (nedialkova et al., 2009 ). since the first in-depth analysis of a cov replicase in 1989 (gorbalenya et al., 1989) , significant progress has been made in terms of its structural and functional characterization. a multitude of enzymatic functions has been identified and characterized in vitro, although mainly using artificial substrates so far. protein structures were obtained for most of the subunits from the nsp7-16 region (neuman et al., 2014b) , but unfortunately two prominent remaining "blank spots" on this map concern two key enzymes in cov rna synthesis, rdrp and helicase. filling those gaps would constitute an important step forward, to address basic questions like the priming mechanism employed by the rdrp and the function of the niran domain, and to accelerate targeted drug discovery, for example, in the area of nucleoside inhibitors of cov rna synthesis, which has received little attention thus far. clearly, where cov mrna capping is concerned, identification of the elusive gtase remains a research priority (subissi et al., 2014a) . for other nsps, potential functions (nsp9, the n-terminal domain of nsp15) or substrates (nsp15-endou) remain to be found. as highlighted by several nsp-wide interaction screening studies pan et al., 2008; von brunn et al., 2007) and more specifically by the in vitro data on the interplay between nsp7-8-12-14 (subissi et al., 2014b) and nsp10-14-16 (bouvet et al., 2014) , cov nsps need to work together in many ways. the further characterization of the "nsp interactome," now also inside the cov-infected cell, will undoubtedly provide more clues as to how specific functions are switched on and off or modulated. likewise, attention should be given to defining the interactions of cov nsps with the specific rna signals for genome replication (madhugiri et al., 2014; yang and leibowitz, 2015) , discontinuous minus-strand rna synthesis (attenuation at body trss, nascent minus-strand transfer, and reinitiation; pasternak et al., 2006; sawicki et al., 2007; sola et al., 2011) , and the transcription, capping, and polyadenylation of sg mrnas (fig. 1) . these rna sequences, several of which may be cis-acting, could provide starting points for improved biochemical assays, ultimately paving the way for the complete in vitro reconstitution of some of these multi-nsp-driven processes. the analysis of cov rtc structure and function in the living infected cell remains an enormous technical challenge, requiring continued toolbox development. it is likely that several functional riddles can only be solved by studying infected cells. for example, the endoribonuclease activity of the nsp15 endou domain, a potential "suicide enzyme" for an rna virus, must be controlled tightly in the infected cell. whereas the enzyme is highly active and displays only very limited substrate specificity in vitro (ivanov et al., 2004a; nedialkova et al., 2009) , it may be confined to a specific compartment in the infected cell and/or its activity may be modulated by interactions with other nsps or host factors. such differences between in vitro and in vivo activities will surely emerge for other nsps as well, and they may be better understood following the further characterization (including their lipid composition) of the membranous replication organelles with which the metabolically active cov rtc presumably is associated (hagemeijer et al., 2012; neuman et al., 2014a; van der hoeven et al., 2016) . these studies should also answer the question of how both nsps and viral rna substrates are targeted to or recruited by the membrane-bound cov rtc, in particular also during the earlier stages of infection when viral rna synthesis appears to be taking off in the absence of the prominent membrane rearrangements observed later in infection. specific mutations in cov genomes can now be reverse engineered, but many of the functions encoded by nsp7-nsp12 are so basic that their inactivation will merely result in dead virus mutants that do not provide many deeper insights into nsp function. this in part explains why most progress thus far has been made for some of the functions that can-fortunatelybe inactivated without such lethal consequences, like the nsp14 exon and nsp16 2 0 -o-mtase enzymes (eckerle et al., 2007 (eckerle et al., , 2010 graham et al., 2012; menachery et al., 2014; smith et al., 2013; z€ ust et al., 2011) . for this reason, the field should continue to also employ "traditional" (forward) genetic methods to characterize (and produce more) conditionally defective covs, like temperature-sensitive mutants (sawicki et al., 2005) . thanks to the advent of next-generation sequencing technologies, tracing the evolution of crippled virus mutants and (pseudo) revertants has become much more straightforward than before, and this approach (letting the virus do the work) likely is among the most economical ones in uncovering previously unknown interactions between the protein and rna players in cov replication (z€ ust et al., 2008) . furthermore, it may be possible to develop cell-based assays for the analysis of cov nsp functions that do not rely on having a replication-competent virus to start with. unraveling the molecular mechanisms underlying the presumed mismatch excision function (bouvet et al., 2012) of the nsp14-exon, which is uniquely encoded by rna virus genomes larger than 20 kb (nga et al., 2011) , connects to the mechanisms driving the evolution of nidoviruses at large (lauber et al., 2013) . also, replicases from other nidovirus branches will need to be studied to fully understand the basic principles governing the profound divergence and genome expansion of this exceptional order of +rna viruses. the error rate and genomic plasticity of rna viruses are among their most fascinating features, and also form the basis for the many problems caused by rna virus mutation and adaptation, including successful zoonotic transfer. as exemplified by the viable cov mutants lacking the exon or 2 0 -o-mtase functions (graham et al., 2012; habjan et al., 2013; menachery et al., 2014; z€ ust et al., 2011) , the functional characterization of cov replicative enzymes can be key to the development of conceptually new live attenuated vaccine prototypes. likewise, it will contribute to the development of broad-spectrum and highly effective antiviral drugs targeting essential enzyme functions, critical interactions with nsp cofactors, or "nonessential" nsp functions that promote efficient viral replication and/or pathogenesis. as highlighted by the sars and mers outbreaks of the past 15 years, having such compounds available would definitely strengthen our first line of defense against cov infections in humans. with great respect, we acknowledge the many ground-breaking contributions of our longterm collaborator dr. alexander gorbalenya, founding father of the functional characterization of the replicase of coronaviruses and nidoviruses at large. we are also grateful for the scientific and technical contributions over many years by numerous past and present lab members and colleagues in leiden (e.j.s.), marseille (e.d.), and giessen/ belfast/w€ urzburg (j.z.). specifically, we would like to acknowledge the input of our long-term collaborators clara posthuma, bruno canard, bruno coutard, isabelle imbert, volker thiel, and stuart siddell. we thank aartjan te velthuis for his assistance with preparing fig. 2 . mechanism of nucleic acid unwinding by sars-cov helicase severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses. antimicrob biochemical characterization of a recombinant sars coronavirus nsp12 rna-dependent rna polymerase capable of copying viral rna templates putative rna capping activities encoded by brome mosaic virus: 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cord-268483-joiajgs4 authors: shah, vibhuti kumar; firmal, priyanka; alam, aftab; ganguly, dipyaman; chattopadhyay, samit title: overview of immune response during sars-cov-2 infection: lessons from the past date: 2020-08-07 journal: front immunol doi: 10.3389/fimmu.2020.01949 sha: doc_id: 268483 cord_uid: joiajgs4 after the 1918 flu pandemic, the world is again facing a similar situation. however, the advancement in medical science has made it possible to identify that the novel infectious agent is from the coronavirus family. rapid genome sequencing by various groups helped in identifying the structure and function of the virus, its immunogenicity in diverse populations, and potential preventive measures. coronavirus attacks the respiratory system, causing pneumonia and lymphopenia in infected individuals. viral components like spike and nucleocapsid proteins trigger an immune response in the host to eliminate the virus. these viral antigens can be either recognized by the b cells or presented by mhc complexes to the t cells, resulting in antibody production, increased cytokine secretion, and cytolytic activity in the acute phase of infection. genetic polymorphism in mhc enables it to present some of the t cell epitopes very well over the other mhc alleles. the association of mhc alleles and its downregulated expression has been correlated with disease severity against influenza and coronaviruses. studies have reported that infected individuals can, after recovery, induce strong protective responses by generating a memory t-cell pool against sars-cov and mers-cov. these memory t cells were not persistent in the long term and, upon reactivation, caused local damage due to cross-reactivity. so far, the reports suggest that sars-cov-2, which is highly contagious, shows related symptoms in three different stages and develops an exhaustive t-cell pool at higher loads of viral infection. as there are no specific treatments available for this novel coronavirus, numerous small molecular drugs that are being used for the treatment of diseases like sars, mers, hiv, ebola, malaria, and tuberculosis are being given to covid-19 patients, and clinical trials for many such drugs have already begun. a classical immunotherapy of convalescent plasma transfusion from recovered patients has also been initiated for the neutralization of viremia in terminally ill covid-19 patients. due to the limitations of plasma transfusion, researchers are now focusing on developing neutralizing antibodies against virus particles along with immuno-modulation of cytokines like il-6, type i interferons (ifns), and tnf-α that could help in combating the infection. this review highlights the similarities of the coronaviruses that caused sars and mers to the novel sars-cov-2 in relation to their pathogenicity and immunogenicity and also focuses on various treatment strategies that could be employed for curing covid-19. the whole world is currently confronting a crisis situation that first appeared in late december 2019 as merely a few cases of pneumonia in wuhan, china. the patients were exhibiting common symptoms like fever, dry cough, sore throat, breathlessness, and fatigue. sample swabs from the oral cavity and anal region were collected along with the blood and bronchoalveolar lavage fluid (balf) from all seven of the patients, irrespective of their age and gender, which were then sent to the wuhan institute of virology for further examination. as the outbreak initiated at the seafood market with the onset of winter, similar to that of the previous severe acute respiratory syndrome (sars) infection, the scientists first screened the samples using pan-cov qpcr primers. surprisingly, five samples were reported positive for coronavirus. thorough investigation employing next-generation sequencing and phylogenetic analysis led to the identification of the causative agent of this respiratory disease, a novel coronavirus (2019-ncov) (1) . as more cases started to appear around the world, on february 11, 2020, the world health organization assigned a name, corona virus disease 2019 or covid-19, to the disease and declared it a pandemic on march 11, 2020. the virus was renamed from 2019-ncov to sars-cov-2 by the international committee on taxonomy of viruses on the basis of its genetic similarity to a previously known coronavirus, severe acute respiratory syndrome coronavirus (sars-cov) (2) . transmission of sars-cov-2 occurs when a healthy individual inhales or comes into contact with respiratory droplets from an infected person. the average incubation period before patients exhibit disease symptoms ranges from 2 to 14 days (3) . before the spread of covid-19, sars emerged as an epidemic in 2003, followed by middle east respiratory syndrome (mers) in 2012, both caused by a novel coronavirus of zoonotic origin and assigned to the genus betacoronavirus (4) . the worldwide outbreak of sars-cov-2 has put life on hold, having a major impact on the world's economy, and has claimed ∼436,167 lives globally as of june 15, 2020 (5, 6) . unlike previous episodes of coronavirus spread, where it took months to identify the cause of infection and perform genome sequencing (7) , advancement in science and technology made it possible to identify the causative organism swiftly. within a few weeks of the outbreak, different laboratories across the world had sequenced the whole viral genome and had also provided structural and functional insights into the essential proteins required by the virus for its survival. these immediate scientific inputs helped with developing diagnostic kits and defining treatment strategies for effective prognosis and prevention (8) (9) (10) . in this review, we are emphasizing the immunological aspect of sars-cov-2 pathogenesis by taking into consideration the previous experimental and clinical knowledge obtained from the coronaviruses that were responsible for causing sars and mers. this approach will assist in utilizing immunotherapies, repurposing the previously approved antiviral drugs, and developing therapeutic vaccines specific to novel coronavirus more effectively. initial genome sequencing and phylogenetic analysis of novel coronavirus sars-cov-2 has shown that it is genetically similar to previously known coronavirus sars-cov and hence is placed under the family coronaviridae. coronavirus contains positivesense single-stranded rna (+ve ssrna) as its genetic material, which can be about 30 kb in length and is mostly protected by an outer fatty layer of an envelope that also helps the virus to evade host immune response and assists its entry inside the host cell (11, 12) . the subfamily coronavirinae is further subdivided into four genera, namely alpha-, beta-, gamma-, and delta-coronavirus (α-cov, β-cov, γ-cov, and δ-cov). viruses having the potential to infect humans are placed under the genus α-cov and β-cov (sars-cov & mers-cov), whereas viruses of γ-cov and δ-cov genera are mostly known to infect avians and pigs (13) . the novel coronavirus, sars-cov-2 falls under the genus β-cov, as it shares 88% sequence identity with sars-cov-like coronaviruses (derived from bat) but is only 79% identical to sars-cov and 50% identical to mers-cov (3) . thus, it can be deduced by its genome identity that the immediate host of this virus could be a bat, which then transmitted it to some unknown intermediate host that acted as a source for the transmission of the virus to humans. like those of sars-cov and mers-cov, the sars-cov-2 genome comprises of 12 open reading frames (orfs) in number. at the 5 ′ end of the viral genome, overlapping orfs 1a and 1b are present that encode the rna polymerase and other nonstructural proteins of the virus and occupy approximately twothirds of the genome. genes encoding structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n), are present in the remaining one-third of its genome spanning from the 5 ′ to the 3 ′ terminal, along with several genes encoding non-structural proteins (nsps) and accessory proteins scattered in between, as shown in figure 1 . despite being in the same serogroup, there is a slight difference in the nucleotide number, sequence, gene order, and expression method among previously known coronaviruses and the novel sars-cov-2 (1, 14, 15) . recent reports highlight that a few amino acid substitutions have occurred in the novel coronavirus genes encoding the s protein, nsp2, nsp3, and receptor-binding domain (rbd). these mutations in the nsp2 & nsp3 are also believed to impart the enhanced infection abilities of the novel coronavirus (16, 17) . rna viruses are prone to acquiring genetic mutations that eventually help them to escape the host immune system and develop drug resistance. researchers have also found minor mutations in sars-cov-2 genotype in different covid-19 patients (18) . one such hotspot of mutation in the sars-cov-2 genome is the rna-dependent rna polymerase gene. on analyzing 220 sequences across the globe, eight repetitive novel point mutations were observed. viral genetic sequences accessed from europe exhibited five mutation hotspots, whereas the remaining three point mutations were solely present in the sequences from north america. these unique mutations suggest that the viral strains are continuously evolving across the globe figure 1 | schematic representation of the coronavirus structure and genomic comparison of coronaviruses. (a) representation of coronavirus showing different components of the particle, which is 100-160 nm in diameter. the single-stranded rna (ssrna) genome, covered with the envelope and membrane proteins, gains access into the host cell and hijacks the replication machinery. (b) the ssrna of sars-cov-2 is about 30 kb and has similarities with the genomes of sars-cov and mers-cov. translation of this ssrna results in the formation of two polyproteins, namely pp1a and pp1ab, that are further sliced to generate numerous non-structural proteins (nsps). the remaining orfs encode for various structural and accessory proteins that help in assembly of the viral particle and evading immune response. and that the strains from europe, north america, and asia might have co-existed the whole time (19) . another similar report analyzed 7,666 global viral genomic sequences and found 198 unique mutation sites on sars-cov-2 genome that encodes nsps and s protein, suggesting that the virus is trying to adapt to its new host (20) . as numerous drugs are currently being designed to target the proteins that are essential for the survival of the virus, rapid genetic mutation occurring in these proteins might not prove to be a potential candidate for drug design. therefore, the invariable region of the virus could be a better target to avoid drug failures. interestingly, sars-cov-2, similar to sars-cov, exploits the angiotensin-converting enzyme 2 (ace2) receptor to gain access inside human cells, whereas mers-cov binds specifically to dipeptidyl peptidase 4 (dpp4) receptor (21, 22) . binding of the virus particle to the specific receptor on the host cell plays a key role in governing its pathogenicity. functional evaluation was carried out to reveal the potential receptors for different betacoronaviruses (β-cov) including sars-cov-2, and it was found out that the entry of the virus particle was enhanced in human cells expressing ace2 receptor instead of dpp4 or aminopeptidase n (apn) in the case of the novel coronavirus (23) . recent structural insights provided by cryo-em studies of s protein in prefusion conformation highlighted that the binding efficiency of ace2 and s protein of sars-cov-2 is 10-20 times greater than for the previously known sars-cov (24, 25) . the latest reports suggest that the trimeric s protein of sars-cov-2 is sliced by transmembrane protease serine 2 (tmprss2), similar to sars-cov (26, 27) . hence, profound knowledge of the potential receptors to which the virus particle can bind and its associated proteases will help us in designing specific antiviral drugs and neutralizing antibodies and will lead us to foresee whether particular coronaviruses of zoonotic origin could be able to adapt and infect humans. all coronaviruses initiate entry inside the target cell by engaging the host receptor with the s glycoprotein present on their surface so as to gain entry inside the target cell. the region of s protein containing the rbd is present on the s1 subunit. in a few coronaviruses, rbd is present at the n-terminus region of s1, whereas in sars-cov, it is situated at the c-terminus region (28, 29) . the fusogenic activity of virus-cell membrane is governed by two tandem domains, heptad repeats (hr1,2) that are present on the s2 region of s protein (30, 31) . initially, it was believed that sars-cov enters the target cell merely by virtue of cell membrane integration of virus particle and host cell membrane (32) . later, it was discovered that an essential proteolytic cleavage event takes place in the s protein at the s2 position of sars-cov that results in membrane fusion and facilitates virus entry inside the cell (33) . once the coronavirus is inside the host cell via membrane fusion, it releases its +ve ssrna genome into the cytoplasmic compartment, where the translation of orf-1a and orf-1b begins resulting in the formation of two large polyproteins (pp1a and pp1ab). three functional proteases then cleave the polyproteins into 16 non-structural proteins (nsp1-16), which eventually create the viral rna polymerase and other accessory proteins for virus assembly (34) (35) (36) . an uninterrupted replication-transcription event results in the formation of various nested sets of subgenomic (sg) mrnas that eventually translate into numerous structural and accessory proteins (37) . the e glycoproteins after synthesis are incorporated into the rough endoplasmic reticulum or golgi membrane. the +ve ssrna combines with capsid protein to form the nucleocapsid, followed by budding of assembled virus particles in the er-golgi intermediate compartment (ergic) (38) . lastly, the virus particle-loaded vesicles are fused with the cell membrane for effective shedding of the virus (4). these new virions are now accessible to infect the neighboring healthy cells and are also released into the surrounding environment via respiratory droplets that are highly contagious and hence potentially spread the disease to healthy individuals. the path followed by sars-cov-2 to reach the lungs is via the naso-oral cavity. once the virus is inhaled, it enters the epithelial cells of the nasal cavity by engagement of ace2 receptor with the viral rbd and initiates its replication (27, 39, 40) . this initial asymptomatic phase lasts for about 1-2 days, during which the virus multiplies in the upper respiratory tract, where no major hindrance is caused by the innate immune cells. within 2-14 days of initial encounter, the common symptoms of covid-19 start to appear, which are similar to those of sars and mers, i.e., fever, dry cough, pharyngitis, shortness of breath, joint pain, and tiredness. numerous problems arise during this phase of the disease, including nosocomial and fomite transmission of infection, which enhances the chances of community spread (41) . soon, the virus begins to move toward the lower respiratory tract via airways, and this triggers a strong innate immune response. patients at this stage start exhibiting enhanced pro-inflammatory response that leads to viral sepsis accompanied by other complications, including pulmonary edema, acute respiratory distress syndrome (ards), different organ failures, and death in the worst scenarios (42) . the infected individuals rarely show the intestinal symptoms like diarrhea that were evident in other coronavirus infections. patients are recommended to be quarantined to prevent community spread of this pandemic virus (43) . the severity of covid-19 has been found to be greater in aged individuals and in people with a health history, such as those immune-compromised by hiv infection or by chemotherapy for cancer. diabetic and asthma patients, along with individuals with hypertension, obesity, or heart, kidney, or liver disorders, are also at higher risk if they acquire the disease (44) . autopsy reports of individuals who died due to sars show multi-organ dysfunction, with the highest viral titers in the lungs and immune cells in circulation, thus damaging the pulmonary and immune system (45, 46) . as opposed to adults, only a very small population of children has been infected with sars-cov-2. in one study, the symptoms displayed by children above 15 years were found to be milder as compared to those of younger children, who showed severe symptoms but with rare deaths and better prognosis (47) . the study speculated two major possibilities related to covid-19 severity in children among different age groups. one of these rests on the finding that ace2 activity is higher in children aged 4-13 years; after this age, it starts to decline until adolescence. this could be one of the reasons why lung fibrosis is observed mainly in younger children. secondly, differential cd4 + and cd8 + t cell populations have been seen in children as compared to adults (48, 49) . a large number of clinical and epidemiological criteria were defined to assess probable pediatric cases of covid-19 (50) . a preliminary report from a cross-sectional study of children admitted to us and canadian pediatric intensive care units (picus) during march 14-april 3, 2020, revealed that the 48 children were admitted in the usa whereas no covid-19 cases were reported in canadian picus. the study revealed that there are fewer covid-19 cases in children as compared to adults and that there is a median picu time of 5 days (51). a recent preprint from paris reports that 11 children (age 3.7-16.6) were admitted experiencing symptoms similar to kawasaki disease (kd) along with gastrointestinal issues and elevated inflammatory markers. further investigation suggested that they were also sars-cov-2-positive, speculating that this could be the reason for kd shock syndrome (52) . similar cases have been observed in new york, where four otherwise healthy sars-cov-2-positive children started displaying symptoms similar to kd and toxic shock syndrome, thereby needing intensive care (53) . therefore, medical practitioners should be prepared to tackle such sudden post-infection complications to avoid the associated risks. once the virus gains access inside the target cell, the host immune system recognizes the whole virus or its surface epitopes, eliciting the innate or adaptive immune response (figure 2 ). pathogen recognition receptors (prrs) present on immune cells, mainly toll-like receptors 3, 7, and 8, are the first to identify the virus, which leads to enhanced interferon (ifn) production. the function of host innate immune cells is impaired during sars-cov and mers-cov infection by their non-structural proteins, which affects the overall cytokine production (54) (55) (56) . humoral response against sars-cov-2 has been found to be similar to that against other coronavirus infections, involving the characteristic igg and igm production. at the onset of sars-cov infection, b cells elicit an early response against the n protein, while antibodies against s protein could be detected after 4-8 days from the appearance of initial symptoms (57, 58) . although n protein is smaller than s protein, it is highly immunogenic, and the absence of glycosylation sites on it results in n-specific neutralizing antibody production at an early stage of acute infection (59) . sars-cov-specific iga, igg, and igm antibodies were detected after the onset of symptoms at different time points in infected patients. a persistent level of igg was detected for a longer period, whereas igm levels started to decline after 3 months (60, 61) . in an observational case study of 16 sars-cov-2 patients, anti-s-rbd igg was detected in all of the subjects, whereas anti-n igg and anti-s-rbd igm were detected in 15 patients and anti-n igm in 14 patients (62) . an elisa-based time kinetics study to detect the covid-19 specific humoral immune response showed that the patients produced igm and igg antibodies that did not cross-react with other human coronaviruses except sars-cov. igm and iga antibodies were detected 5 days after the onset of initial symptoms, whereas igg was detected after 14 days (63) . another kinetic study of viral shedding and antibody detection was published in a preprint and reported the presence of higher igg and igm antibody titers in severe patients. they also observed that weak responders for igg antibody had higher viral clearance than strong responders. this observation suggests that robust antibody response leads to disease severity while feeble response is associated with the elimination of virus (64) . a case study on pediatric patients reports that 5 out of 6 children showed a protective humoral response, with neutralizing igg and igm antibodies targeting the n and s-rbd proteins of sars-cov-2 (65) . these studies propose that igm-based elisa can be used for early diagnosis of patients along with qpcr techniques to improve the sensitivity and specificity of the technique. in addition to neutralizing antibodies, which are defensive and useful, there are numerous non-neutralizing antibodies in the system that aid the infection of immune cells and apcs. previously existing sars-cov antibodies may promote the viral infection in fcr-expressing cells (66) . this ace2-independent pathway of viral entry does not result in viral replication; rather, viral shedding by macrophages enhances inflammation and tissue injury by myeloid cell activation. this mechanism of viral entry through non-neutralizing antibody that results in aberrant activation of immune cells is called ade (antibody-dependent enhancement) (66, 67) . ade has been observed in a number of viral infections, including sars and mers. in the case of sars, anti-s antibodies were observed to be involved in ade to gain entry into fcr-expressing cells (68) , while in mers, a neutralizing mab (mersmab1) targeting rbd aided in mers pseudo-virus entry via the dpp4 pathway (69) . although there is no clear evidence regarding ade in sars-cov-2 infection, it is still necessary to consider all of the odds in the pursuit of developing vaccines and treatment regimens involving antibodies (70) . during viral infection, t cells also recognize the viral antigens presented by mhc class i [mhc; human leukocyte antigen (hla) in humans], which in turn promotes the cytokine release and cytotoxic activity of cd8 + t cells (71) . but in some other cases, mhc class ii is also found to present sars-cov peptides to cd4 + t cells. due to the genetic polymorphism of hla, some haplotypes, like hla-b * 07, hla-b * 46, hla-drb1 * 12 (72) , and hla-cw * 08 (73) , are found to be more susceptible to coronavirus infection, whereas the hla-drb1 * 03, hla-a * 02, and hla-cw * 15 haplotypes are protected from sars-cov infection (74) . similarly, hla-drb1 * 11 and hla-dqb1 * 02 were found to be vulnerable to mers-cov infection (75) . additionally, mhc expression is also found to be reduced during the infection due to epigenetic modifications of downstream molecules (76, 77) . so far, hla association is not very wellidentified for sars-cov-2 infection, and this could be crucial for the prevention and treatment of covid-19. however, in a recent report, blood plasma from covid-19 patients was able to block the expression of hla-dr on cd14 + monocytes, which was restored effectively on inhibiting il-6, suggesting that decreased hla-dr expression in sars-cov-2 patients is due to the buildup of hyper-inflammatory conditions (78) . decrease in mhc expression is also evident in cancer cells, which is a mechanism by which they evade the immune response by epigenetically modifying calnexin promoter. but infection with influenza virus in these cancer cells results in enhanced mhc-i presentation due to the increased expression of chromatin remodeling proteins, which stabilizes p53 expression and hence augments the immune surveillance of cancer cells (79) . therefore, molecules that can upregulate chromatin regulators and increase the mhc-i expression could potentially be used for covid-19. most of the t-cell epitopes presented by mhc complex are derived from structural proteins such as the s and n proteins of the coronavirus in both humans and animal models, while the nsps have regulatory effects on the signaling cascade (80, 81) . t cells can be stimulated by 14 epitopes, most of which are observed to be located on orf3 and the s protein in sars patients (61) . in a large cohort study during sars-cov infection, s protein was the only immuno-dominant epitope for cd8 + t-cell activation (61) , whereas, in mers, cd8 + response was against the s and n proteins along with some of the m/e epitopes (82) . these t-cell epitopes have been tested in animal models by assessing the lung pathology and t-cell response upon infection in balb/c and c57bl/6 mice (80, 83). the sequence of sars-cov-2 being more similar to sars-cov than to mers-cov, with no mutation in 19 epitopes, provides a prospective subunit vaccine for stimulating a strong t-cell response in covid 19 patients (84) . in a recent study, samples from 20 convalescing covid-19 patients were analyzed to check the development of adaptive immune response during infection. the results highlighted that helper t cells were eliciting a robust immune response against s, m, and n protein. the effect of adaptive immune response on humoral immunity was also compared, where a strong cd4 + t-cell response against sars-cov-2 eventually resulted in an increase in anti-s-rbdspecific igg and iga antibody titer. along with cd4 + t cells, immunogenic epitopes on s, m, and n proteins were also able to activate cd8 + t cells. however, such t-cell response was not specific to recovered patients only but was also present in 40-60% of the individuals who were not exposed to sars-cov-2. further analysis showed that they had pre-existing cross-reactive cd4 + t cells, which might have been generated in response to some previous coronavirus infection. hence, these t-cells could impart protective immunity in such individuals against sars-cov-2 to some extent (85) . these epitopes could be a promising factor in developing immunotherapy by small molecules that can increase the presentation of viral epitopes. a rapid and coordinated immune response during viral infection leads to enhanced secretion of various cytokines, which acts as a defense mechanism against the virus. numerous reports suggest that individuals affected with sars-cov or mers-cov have dysregulated cytokine production from both innate and adaptive immune cells. in the case of sars, infected hematopoietic cell, monocyte-macrophages, and other immune cells trigger enhanced secretion of pro-inflammatory cytokines like tnf-α, il-6, and ifn-α/-γ, with reduced anti-inflammatory cytokines (86) (87) (88) . similarly, mers-cov infection leads to delayed but increased production of ifn-α and pro-inflammatory cytokines like il-6, il-8, and il-1β (89) (90) (91) . such elevated levels of cytokines were associated with multi-organ dysfunctional syndrome (mods) and ards due to the accumulation of numerous immune cells like macrophages, neutrophils, and dendritic cells in the lungs causing alveolar damage and edema (56, 92, 93) . similarly, in covid-19 patients, secretion of cytokines and chemokines, which attract the immune cells to the lungs, was increased, hence causing ards, which is fatal to critically ill individuals (94, 95) . signature cytokines in severely ill covid-19 patients were consistent with those in sars and mers, i.e., enhanced expression of il-6, tnf-α, macrophage inflammatory protein 1-α (mip-1α), mcp3, gm-csf, il-2, and ip-10 along with elevated chemokines (ip-10, ccl2/mcp1, cxcl1, cxcl5) were also detected in sars-cov-2 infection (96) (97) (98) (99) . in children, the increased inflammatory markers include il-6, il-1, and c-reactive protein along with procalcitonin in serum (52) . in a case study, a 14-year-old child with cytokine storm was treated with anakinra (il-1 receptor antagonist) in order to stabilize the respiratory illness and other clinical symptoms (100) . transcriptomic analysis of pbmc and balf showed that a number of immune regulators were upregulated, particularly cxcl10, with respect to balf. this study also reported that several apoptotic genes and p53 signaling molecules were upregulated, suggesting a possible reason for lymphopenia in these patients (101) . therapeutic measures to control such cytokines involve neutralizing antibodies or small molecular drugs that can stop the signaling cascade for cytokine production. the most potent antiviral machinery acquired by immune cells is the secretion of interferons that act as secondary messengers stimulating the neighboring cells. most innate immune cells are efficient in producing ifns that are involved in obstructing cell proliferation, apoptosis, and immunomodulation (54, 102) . as an escape mechanism, sars-cov or mers-cov uses several ways to overcome the host immune response, one of which is by severe leukopenia and lymphopenia (103) (104) (105) . after gaining entry to the cell, these viruses encode different proteins that interact with downstream signaling molecules of tlrs and the jak-stat pathway. mers-cov encoded matrix protein, accessory proteins from orf 4a, 4b, and 5, which directly inhibits the ifn promoter and nuclear localization of irf3 (106) . plpro, encoded by sars-cov and mers-cov, prevents the dissociation of nf-κb from iκbα, whereas nonstructural proteins of sars-cov, i.e., plpro and orf3b, inhibit irf3 phosphorylation and hence its translocation to the nucleus (4, 107, 108). these viral accessory proteins also inhibit the jak-stat pathway, resulting in inhibition of genes by isre promoters (109) (110) (111) (figure 3) . a new investigation revealed that sars-cov-2 infection leads to an overall decrease in the transcription of antiviral genes because of the lower production of type i and iii interferons with sufficient isg expression, along with elevated chemokine secretion. results obtained from in-vivo and ex-vivo covid-19 experiments were in tune with the in-vitro findings. therefore, a decrease in the innate antiviral response, along with hyper-inflammation, could be one of the causes of covid-19 severity (112) . in addition to reduction in t cells, sars-cov-2 infection also enhances the exhaustion of effector t cells, decreasing the immune response against the virus (94, 113) . exhaustion and loss in function of effector t cells is the result of increased expression of inhibitory receptors like pd-1, tim-3, and tigit on its surface as a result of cytokines like il-6, il-10, and tnf-α or by decreasing the regulatory t-cell population (114, 115) . following viral/antigen clearance, most of the effector t cell undergoes apoptosis in the contraction phase. subsequently, a pool of memory t cells are generated that are programmed to fight against re-infection. cd4 + memory t cells, upon restimulation, trigger b cells and other immune cells by cytokine production, while cytotoxic memory t cells help in destroying the infected cells during subsequent infection (116, 117). case studies in recovered sars patients showed that both cd4 + and cd8 + memory t cells were efficient in eliciting immune response from 3 months to 6 years without the presence of any antigens (118) . in a case study of 23 recovered sars-cov patients, the patients showed very low frequencies of memory b cells, while memory t cells elicited a response against the s protein in 60% of recovered individuals (119) . considering the memory t-cell subset, n-specific helper t cells had more of central memory markers (cd45ra − , ccr7 + , cd62l − ) while the cd8 + t cell population had the effector memory (cd45ra + , ccr7 − , cd62l − ) phenotype in a steady-state manner (120) . the study suggests that an effective vaccine or t cell epitopes could be used to target a particular population for rapid viral clearance. in recent reports, covid-19 subjects have shown reduced regulatory t cell populations and memory t cells, which may aggravate the inflammatory response leading to cytokine storm and hence enhance the tissue damage and organ failure (114) . in a mouse model, the use of cd4 + memory t cells as a vaccine by the intranasal, but not the subcutaneous, route imparted a protective response against the human coronavirus. the infused cd4 + memory t cell, upon re-stimulation, produces ifn-γ and recruits cd8 + t cells for rapid clearance in response to sars-s366 peptide (121) . recently, a human ace-2-expressing mouse model has been developed by crispr/cas9 technology that recapitulates the human symptoms upon infection with sars-cov-2 through the intra-nasal route. this tool will be beneficial for evaluating the efficacy of vaccines for covid-19 and also to study its transmission and pathogenesis (122) . just like sars and mers, there are no specific clinically approved drugs available for covid-19 as of june 15, 2020 (123) . currently, the treatment regime focuses mainly on providing intensive care in order to alleviate the symptoms and discomfort associated with covid-19. conservative fluid therapy accompanied by broad-spectrum antibiotics are also given to the patients as a protective measure to avoid opportunistic bacterial infections. however, ventilator support for respiration is provided to the patient under extreme conditions (124) . numerous fda-approved antiviral drugs, vaccines, and immunotherapies that are already being used to treat other diseases have also been considered as a possible approach for treating covid-19 ( table 1) . but this approach may reduce the availability of these drugs and vaccines for the intended diseases and for the patients with the greatest need. the molecular, structural, and functional relationships of lopinavir viral protease involved in immature, noninfectious hiv virus particle, and inhibits plpro or 3clpro in sars-cov-2. favilavir viral rna polymerase purine analog blocking viral rna synthesis. remdesivir (141) ribavirin guanosine nucleoside binds to nucleoside binding pocket of the enzyme. (133, 140, 142) galidesivir adenosine analog, effective against ebola, zika, and other rna viruses. chloroquine/hydroxychloroquine heme polymerase and ace2 increases endosomal ph and terminal glycosylation of ace2, inhibiting sars-cov-2 entry. (144, 145) nitazoxanide glutathione-s-transferase alters ph and inhibits viral maturation. reported against tb, helminthic, and protozoan infection. umifenovir/arbidol n/a interacts with aromatic residues of viral glycoproteins. is being trialed for prophylactic action against covid-19. sars-cov-2 with sars-cov might define the use of existing anti-viral drugs against covid-19 (147, 148) , considering the total time it takes to perform clinical trials and get fda approval for the use of novel drugs and vaccines. the increasing knowledge of the genetic, immunological, and molecular mechanisms behind its enhanced pathogenicity might help in developing specific treatment approaches for covid-19 in the future. considering the studies on the molecular mechanism of coronavirus infection (147) , several antiviral drugs could be repurposed for the treatment of covid-19. remdesivir is a nucleotide analog that acts as an antiviral agent for a wide variety of viruses and has been tested widely against previous epidemics of coronavirus infections in both in-vitro and in-vivo models (138, (149) (150) (151) . this adenosine analog gets incorporated into the newly synthesized viral rna, which inhibits the addition of further nucleotides by viral rnadependent rna polymerase and hence terminates the ongoing transcription. administration of intravenous remdesivir was found to be effective in treating the first known patient of covid-19 in the usa (152) . a randomized double-blinded clinical trial on 1,059 adult hospitalized covid-19 patients was sponsored by the national institute of allergy and infectious diseases, usa, to further test the potency of intravenously administered remdesivir. the preliminary outcomes of the trial reported that remdesivir treatment decreased the median recovery time in the treatment group (11 days) as compared to the placebo group (15 days). the mortality rate was also less in the treatment group (7.1%) in contrast to the placebo group (11.9%) (153) . numerous clinical studies, similar to this, are required so as to validate the proposed drugs for covid-19. favipiravir, ribavirin, and galidesivir are also potential nucleoside analogs that might be useful against novel coronavirus infection (154) . the combinatorial therapy approach of using remdesivir along with chloroquine, a well-known anti-malarial drug, has also been tested in vitro so as to study its effectiveness against sars-cov-2 (141, 155) . it has been reported that chloroquine immuno-modulates the host microenvironment and also interferes with the replication of the virus and its interaction with the receptor (156, 157) . in a randomized clinical trial (nct04308668) involving 821 asymptomatic individuals across the us and canada who had come into close contact with potential covid-19 patients, the individuals were given either hydroxychloroquine or placebo as a prophylactic measure. the results revealed that hydroxychloroquine treatment had the same effect as did the placebo group. the usage of hydroxychloroquine resulted in minor side effects (40.1%) as compared to the placebo treatment (6.8%). however, no cardiovascular disorder or treatment-related major complications were observed (158) . based on the putative function of hydroxychloroquine on the endosomal acidification, whereby it is presumed to hinder viral uncapping, it can be observed that it has a great potential for prophylaxis, not to prevent infection but to reduce effective viral load in patients and thus lead to milder disease. numerous clinical trials to further explore the usage of hydroxychloroquine in different combinations are in the pipeline and will finally provide a better understanding of the efficacy of this drug for covid-19. a few anti-hiv drugs, such as lopinavir/ritonavir in combination with interferon beta (ifn-β), have been tested in vivo for treating coronavirus infections (sars-cov, mers-cov) and have also been used in the case of covid-19 (138, 139, 159) . various complementary therapies could also be employed as a preventive measure against viral infections. many essential proteases, such as chymotrypsin (3c-like protease) and plpro, which are required by coronavirus for completing the replication process, can also be targeted using drugs. cinanserin, flavonoids, and some small molecules are known to inhibit 3clpro, whereas diarylheptanoids are used to inhibit plpro (160) (161) (162) . in a recent study, 16 potential anti-hcov drugs were identified through a systems biology-based approach, such as melatonin, mercaptopurine, sirolimus, dactiomycin, and toremifene, which are to be tested further for their potency (163) . in the absence of any dependable vaccine or drugs with tested efficacy and when the pandemic onslaught is ongoing, a worthy therapeutic approach is passive immunization using purified antibodies. the source of such antibodies could be the sera of convalescing individuals, mabs, or genetically modified antibodies from an animal host, which can efficiently neutralize the virus. this is an age-old practice, with pioneering work having been done by the nobel laureate, emil behring, who applied this approach for diphtheria, and has been used whenever there are sudden outbreaks of viral diseases like sars, mers, h1n1, h5n1, ebola, and many others (61, 164, 165) . as opposed to active vaccination, plasma therapy is the only means to provide immediate immunity for viral clearance, as in the case of sars-cov-2. as in other epidemic diseases, convalescent sera are currently being employed for covid-19 in a number of countries (166, 167) . although a randomized controlled trial is yet to be reported, limited studies in 10 patients have been documented with no remission of severe respiratory afflictions on receiving neutralizing antibodies from 39 convalesced donors with antibody titers of 1:160, along with drugs and oxygen support (168) . a report from hong kong suggested that this therapy had poor outcome in sars patients, with a number of limitations in their study (169) . as with transfusion of any blood products, precautionary screening of infectious agent is warranted in plasma transfusion. recently, the fda in the usa has approved trials of convalescent plasma therapy in covid-19 under specific guidelines; plasma donation is advised 3 weeks after a patient becomes virus-negative on pcr. the major challenge in this therapy is obtaining donors with similar blood antigens with a high antibody titer of sars-cov-2 (170) . another potential adverse effect of this approach is ade of infection, which is common in so many other viruses. but, to date, the incidence of ade has not been reported in the case of sars-cov-2. another major point of contention is the selection of patients for this therapeutic approach. in most clinical trials, patients with severe diseases are being recruited, while the presumed mechanism of action of convalescent plasma, based on its content of virus-neutralizing antibodies, rather points to plausible favorable outcomes in earlier phases of the disease because in the later, more severe phases, the hyperimmune response, rather than the viral load, becomes the more critical pathology. finally, there are no available data on the heterogeneity of response to convalescent plasma transfusion, which may further illustrate the importance of careful evidencebased patient selection, as heterogeneity of response may result from both virus and host-intrinsic factors which are, to date, not revealed. researchers around the world are working hard to develop a potential vaccine candidate so as to stop the deadly pandemic caused by sars-cov-2. however, vaccine development is not an easy task, as a number of successful clinical trials are required before approval for patients. different approaches are being utilized for designing a specific vaccine targeting either the structural proteins or viral replication process, which eventually results in the inhibition of viral growth and its further transmission. the common strategies involve the use of live attenuated vaccine (lav), inactivated virus, subunit vaccines, monoclonal antibody vaccine, virus vectors, protein vaccines, and dna/rna-based vaccines (171) (172) (173) (174) . there are numerous subunit vaccines targeting all or a part of s protein that have already been tested for sars and mers in animal models (175) and could be potential candidates for testing against sars-cov-2. a recent pilot study with a purified inactivated sars-cov-2 virus vaccine displayed very promising outcomes in different animal models. the neutralizing antibodies generated after vaccination were able to effectively target 10 different strains of sars-cov-2 without developing any ade of infection (176) . various randomized controlled trials (nct04327206, nct04328441) are also underway to evaluate the effectiveness of the bcg vaccine against sars-cov-2 for healthcare professionals. an adenovirus vector-based vaccine candidate, chadox1 (presently azd1222), developed by oxford university (licensed to astrazeneca) for use against sars-cov-2 has been reported to activate both the humoral and cell-mediated immune response when tested in rhesus monkey (177) . the phase i clinical trial to confirm its potency is also in progress (nct04324606). another group has followed a similar approach by using a recombinant adenovirus type 5 (ad5-ncov) vectorbased vaccine for covid-19. the full report from the phase i clinical trial (nct04313127) of ad5-ncov shows that it is very effective in generating both humoral and rapid t-cell response post immunization. the group is now ready for the next clinical trial phase to further strengthen the effectiveness of the ad5-ncov vaccine (178) . it should be noted that there are potential risks associated with the usage of live attenuated viruses, for example, complications resulting in lung damage by infiltrating eosinophils, as seen in in vivo models (179, 180) . however, eosinophil immunopathology due to sars-cov vaccine could be reduced by using tlr4 agonist as an adjuvant (181) . viral neutralizing antibodies specifically targeting various regions of s, i.e., s1-rbd, s1-ntd, or the s2 region, and blocking the interaction of virus with the receptor are well-known for sars and mers (182) . these neutralizing antibodies could prove to be the best and potential candidate for cross-neutralization of sars-cov-2. despite being structurally related, some of the sars-cov neutralizing monoclonal antibodies failed to interact with the s-protein of sars-cov-2, which could be attributable to the substantial differences in their rbd (183) . a recent study reported the presence of high titres of neutralizing anti-s-rbd igg antibodies, but no antibodies were detected against the n protein in recovered covid-19 patients, suggesting that anti-s igg persists longer than does anti-n igg. along with the humoral immune response, they also observed an s protein-specific t cell-population producing ifn-γ, which further contributes to conferring protective immunity against sars-cov-2 infection (184). recently, a monoclonal antibody (47d11) has been identified from 51 sars-spike hybridomas that targets the conserved s-rbd region (residue 338-506) and therefore can very effectively neutralize sars-cov-2 along with sars-cov (185). on similar lines, a group has isolated a single-domain antibody from a phage display library targeting the s-rbd region of sars-cov-2. the fully humanized singledomain antibody was able to neutralize the virus by interacting with a cryptic epitope in s protein (186) . these mab and singledomain antibodies could be used to treat as well as to design quick diagnostic kits for covid-19. the new technology of the microneedle array (mna) has been employed for delivering sars-cov-2 s1 subunit vaccine, which could be really helpful in the treatment of the emerging covid-19 outbreak (187) . the transfer of s1 subunit by mna elicited a strong virus specific-antibody response in sars-cov-2 (187) . a novel encapsulated mrna vaccine candidate developed by modernatx, inc. that encodes full length s protein of sars-cov-2, is also under clinical trial (nct04283461). there is an urgent need to develop more such specific vaccines that could neutralize the novel coronavirus effectively (188) . the host innate immune system encounters upcoming infections, and this results in elevated production of various cytokines and type i interferons (ifns). in the case of prolonged infection, hyperactivation of the immune system may also result in the development of a pro-inflammatory microenvironment, leading to adverse outcomes and even death. the induction of numerous lymphokines, such as il-6, il-1β, tnf-α, and ccl2, that are pro-inflammatory in nature has also been observed in the case of covid-19 (189) (190) (191) . a previous study in a mers animal model showed that treatment with recombinant type-1 ifn (rifn) decreased the viral rna level in lungs with a decrease in ifn-stimulating gene expression. early treatment with rifn resulted in a dampening of cytokine and chemokine release that lowered the migration of neutrophils and other cells in lung (91) . an allogenic mesenchymal stem cell-based (remestemcel-l) therapy developed by mesoblast, which has been previously used for inflammatory conditions and graft vs. host disease in children and adults, is now being assessed for covid-19 (192) (193) (194) . in this therapy, bone marrow-derived mscs from the donor are grown in vitro and are then transfused to the recipient patients. upon infusion, these cells exhibit antiinflammatory activity by reducing pro-inflammatory cytokine production via the recruitment of anti-inflammatory cells in the affected tissue (195) . currently, a randomized placebo-controlled trial (nct04371393) with 300 patients is ongoing for treating ards caused by covid-19. treatment with rifn, inhibitors of the pro-inflammatory pathway, cytokine inhibitors such as tocilizumab, lenzilumab, and many others are still to be used in combination with other drugs for treating covid-19. so far, there is not much evidence from clinical trials of such inhibitors with which to predict the outcome of these anticytokine therapies. considering the current situation of more than 8 million people being infected, with ∼436,167 deaths as of june 15, 2020, there is an urgent need to control the sars-cov-2 pandemic. the fatality rate of sars-cov-2 in lower than those of other coronaviruses that caused catastrophes in the past, but the higher infectivity rate makes it worse. raising awareness of this contagious virus is one of the many ways by which its spread can be prevented. the governing authorities concerned in every country have approved guidelines and taken necessary action to quarantine infected people and break the chain of community spread. antibodies, vaccines, and drugs developed for previously emerged coronaviruses could potentially be used for treating sars-cov-2. the combination of various neutralizing antibodies against s protein could enhance the effectiveness of viral clearance. among various antivirals and other small molecules that are fda approved, chloroquine/hydroxychloroquine has shown better positive outcome in covid-19 patients. in clinical trials, some of the combinational antiviral drugs like lopinavir + ritonavir and blockers like angiotensin receptor blocker that were thought to be effective, have failed in curing the disease (139, 196) . cytokine storm being one of the symptoms of infected individuals, anticytokine therapy for tnf and il-6 should be attempted to determine the efficacy of these antibodies in the treatment of sars-cov-2 infection. clinical trial chictr2000029765 with tocilizumab, a monoclonal humanized antibody against il-6 receptor, has shown some efficacy, but this still needs to be tested in a larger cohort. with the increasing number of deaths, there is an immense need to accelerate the development of rapid and sensitive diagnostic kits and to commence clinical trials of the readily available and safe drugs to reduce the rising infections and covid-19-related deaths so as to bring life back on track. vs and pf contributed equally in writing the review. conception of idea was done by sc, vs, and pf. manuscript writing and editing was done by all the authors. we are thankful to csir-indian institute of chemical biology, jadavpur, kolkata and national centre for cell science (nccs), pune for providing infrastructure facilities. we would also like to acknowledge department of biotechnology-systems medicine cluster (dbt-symec) grant and j c bose fellowship-serb (science and engineering research board) to sc, for the financial support. a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor 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adult human mesenchymal stromal cells for the treatment of pediatric patients who failed to respond to steroid treatment for acute graft-versus-host disease remestemcel-l: human mesenchymal stem cells as an emerging therapy for crohn's disease transplantation of ace2-mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia mesoblast to evaluate anti-inflammatory cell therapy remestemcel-l for treatment of covid-19 lung disease are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? key: cord-150183-zzzyewjb authors: phillips, j. c. title: synchronized attachment and the darwinian evolution of coronaviruses cov-1 and cov-2 date: 2020-08-27 journal: nan doi: nan sha: doc_id: 150183 cord_uid: zzzyewjb cov2019 has evolved to be much more dangerous than cov2003. experiments suggest that structural rearrangements dramatically enhance cov2019 activity. we identify a new first stage of infection which precedes structural rearrangements by using biomolecular evolutionary theory to identify sequence differences enhancing viral attachment rates. we find a small cluster of mutations which show that cov-2 has a new feature that promotes much stronger viral attachment and enhances contagiousness. the extremely dangerous dynamics of human coronavirus infection is a dramatic example of evolutionary approach of self-organized networks to criticality. it may favor a very successful vaccine. the identified mutations can be used to test the present theory experimentally. the standard tool for comparing two or more proteins is blast, which compares different protein sequences site-by-site. it is not enough to profile ψ(aa) site by site for two proteins, as this produces hundreds of oscillations, with no clear differences between the two proteins. instead we plot ψ(aa,w), where w is a rectangular box of length w = 35 over which ψ(aa) is averaged . this w value is chosen to maximize the hydropathic shape differences between cov-1 and cov-2, as measured by their variance ratio, which is dominated by extrema [11] . smoothing the profile over w enhances resolution by reducing the number of extrema to the level which corresponds to critical domain motion differences. choosing the best value of w to enhance resolution of evolutionary differences is similar to adjusting the focal plane of a microscope. our main result is shown in fig. 1 , which displays the 400-800 central region of the spikes. this region is part of the n-terminal domain (s1) responsible for cellular attachment [14] . fig. 1 shows the ψ(aa,w) hydropathic profiles of for cov-1 and cov-2, using the ψ(aa) values of mz [4] . while the two ψ(aa, 35) profiles are similar, there are important differences in their extrema. the maxima are most hydrophobic and are located in the globular interior, while the minima are most hydrophilic and are located on the globular surface. the two cleavage sites s1/s2 and s 2' [2] of cov-1 have moved lower (hydrophilically, further outside) in cov-2 ( fig. 1) , consistent with the very accurate mz scale. when a cleavage segment is further outside, there is more space for cleavage and reassembly, which will occur more rapidly. the insertion prra in cov-2 was identified [2] with blast (w = 1) as unique to cov-2, but with blast alone one cannot show that this change has made cov-2 more dangerous. a characteristic feature of second order phase transitions is that they break what physicists often call a "hidden" symmetry, here the key symmetry of extrema [15] . proteins are typically composed of domains of ~ 100 amino acids, and these domains can rotate from the resting state to the functional state, and then return reversibly to the resting state. this phase transition requires a balance between stability and flexibility [16, 17] . to maximize the reversibility and extend protein life one can synchronize this domain motion by leveling the pivotal hydrophobic (inside) extrema forming level sets [10, 11, [18] [19] [20] [21] . viruses must act rapidly before being destroyed by antibodies, and they could do this through synchronized motion differently, by leveling their hydrophilic (outside) extrema. as shown in fig. 1 , such a leveling of minima 1-3 occurs in cov-2, while it is absent from cov-1. the change in minimum 2 is especially striking: it is caused by a cluster of four critical mutations from cov-1 to cov-2. these are (cov-1 site numbering from uniprot p59594): 546gln to leu; 556 and 561ser to ala; and 568ser to leu. the differences associated with each of these mutations are hydropathically large (~50-100 in the mz scale [4] ; all 20 amino acids span a range from most hydrophilic to most hydrophobic of 170). how important is the fractal mz scale? in fig. 2 the mz panoramic profiles of cov-1 and cov-2 are compared across the entire 1255 amino acid spike. we see immediately a strong hydrophobic peak above 1200, which is responsible for anchoring the spike to the central cushion; it is almost unchanged from cov-1 to cov-2. the central hydrophilic level set, absent from cov-1 and present in cov-2, is our main result ( there is an excellent review of the principles of self-organized criticality in living matter [3] . these can be compared to relaxation of homogeneous glass alloys, whose composition has been adjusted to bring the glass network to a critical point. there the glass (commercial gorilla glass) is strong and flexiblenot brittle [21] . another broad and deep review includes fractals and their many implications for quantifying protein dynamic criticality [22] . in the genomic age abstract tools have many applications to analyzing the evolution of protein functions. some readers may be interested in the connections between hydropathic scaling theory of proteins and the more general synchronization of complex networks. the analogy is closest for networks that are scale-free because they are near a second-order phase transition. social networks contain many such examples, usually described by one or two fractals [23]. generally it is found that synchronizability is robust against random removal of nodes, but it is fragile to specific removal of the most highly connected nodes. in proteins the most highly connected nodes are hydrophobic extrema (deeply interior), where the number of van der waals contacts is largest. in coronavirus spikes, it is the hydrophilic extrema that are most exposed to water and most likely to attach to protein targets. thus the symmetry of extrema is broken between hydrophobic (proteins) and hydrophilic (cov-2). one can also regard darwinian selection as a kind of percolation [24] . further extensions to multiphase hydrodynamic level sets require a special theory [15] . our primary focus here has been on enhanced attachment caused by the synchronization of covthese grouped sites are all close to the hydrophilic minimum 1 in fig. 1 (mz scale) and fig. 3 (kd scale). fragmental binding is a feature common to both scales, and thus not sensitive to whether the phase transition is first-or second-order. instead, by synchronizing minima 2 and 3 with minimum 1, cov-2 is able to greatly increase its attachment to ace2 (angiotensinconverting enzyme 2) and probably other receptors as well; synchronization is specifically sensitive to allosteric interactions and is definitely a second-order effect. in conclusion, the answer to the question posed in [1] is that, compared to cov-1, cov-2 has moved closer to its functional critical point [3] . cov-2 has an added synchronized feature in its central attachment region that contributes to its extreme contagiousness in an asymptomatic phase [26] . the attachment of the s1 region precedes cleavage [2] , and it is likely that both mechanisms enhance the infectiveness of cov-2. an early estimate of the basic contagious reproductive number (r0) of coronavirus 2019 was 2.2-2.7, but this was later revised to 5.7 [27]. by comparison, the numbers for h1n1 flu (1918, 1.8; 2009, 1.5) were much smaller [28] the uncontrolled r0 for cov-1 was 3.6, and this dropped to 0.7 after the rapid implementation of control measures [29] . the new feature explains the unexpected yet often asymptomatic behavior of early stages of cov-2 infection, when the new feature is acting alone. it should be possible to desynchronize this added attachment feature and reduce r0 by attaching a small molecule in the region 530-575. in any case, the "hidden" symmetry of the cov-2 sequence should be of interest to both biologists and physicists. the effects of the four critical mutations on cov contagiousness could be tested on mice. if the predictions of their importance should prove to be correct, then the general principle of darwinian evolution of proteins would be confirmed. the level hydrophilic extrema of the spikes of cov-2 may be unique among viruses. these level extrema bring cov-2 contagion very close to a critical point. there very small differences in dna can cause large fluctuations in infection levels, not only between individuals, but also even in neighboring countries [30] . the quasi-linear spike morphology, with its large surface/volume ratio, is ideal for strong protein-water interactions, explaining [1] . the oxford vaccine is based on the cov-2 spike protein hydrophobically anchored to an adenovirus vaccine vector [31] [32] [33] . it seems likely that this vaccine will be more effective for cov-2 than for cov-1 because the synchronized attachment mechanism is stronger for cov-2 and thus more easily disrupted by induced antibodies. thus vaccines based on the entire spike are expected to be more effective than vaccines based on only part of the spike [34] . the most dangerous type of flu virus is h 3 n 2 , and it does not exhibit level sets [35] . flu viruses mutate significantly annually, while cov-2 has shown only a few mutations [36] . the spike stability against mutations is consistent with maximized attachment. there are many examples of scale-free networks. readers unfamiliar with the general properties of scale-free networks, such as (often only 1 or 2) fractals, preferential attachment, and synchronization, may find early reviews interesting [37, 38] . the sequences used here for cov-1 and cov-2 are the ones used in [2] , that is, aap13441.1 and yp_009724390.1. many other cov sequences are aligned (w = 1) in their supp. table [2] . similar w = 1 sequence alignments have been reported by others [39, 40] , but the genetic origin of cov-2 remains mysterious [1, 40] . historically it has long been the case that the amount of information that could be obtained from hydropathic scales was limited, because the fractals describing second-order phase transitions were not known. now that we are in the genomic age, with a very large sequence data base available for many proteins and many species, the discovery of these 20 fractals [5, 13] opens a new biophysics field of accurate thermodynamic analysis of small but medically important evolutionary differences. the methods used here on cov-1 and cov-2 do not require elaborate calculation to obtain new results. the hydropathic scaling methods are easily implemented on a spread sheet, but are still not routine, and vary between proteins. the modular nature of protein domains responsible for protein assembly is well known [41, 42] . here we maximized small and otherwise mysterious evolutionary differences by using hydropathic scaling to optimize w at 35. this enables us to focus on domain-scale motion responsible for stronger attachment in cov-2. evidence for large-scale motion has been found in the evolution of many proteins [4] [5] [6] [7] 9] , including molecular motors [11] , while it is not accessible to traditional w = 1 blast-based alignment methods [2, [43] [44] [45] . from detailed genomic studies it has been suggested that "a more comprehensive theory of molecular evolution must be sought" [46] . readers wishing to explore these novel scaling methods can obtain copies of sample excel spread sheets from the author. many physicists have long suspected that proteins must function near a critical point very near thermodynamic equilibrium [3] . however, to implement this idea in practice with protein sequences, one needs to compress the existing vast array of three-dimensional structural data into a one-dimensional form. if you are one in a million, namely among the small community of coauthors of the 500+ references of [3] , what comes next may not surprise you. as discussed above, this near-miracle compression has been achieved in the 21 st century by two brazilian physicists [13] , located far from the main stream. [13] described the discovery of 20 (not just one) universal amino-acid specific fractals in the solvent accessible surface areas (sasa) of proteins. this amazing 21 st century discovery was achievable only for proteins. it has made possible the quantitative analysis of darwinian evolution of many different proteins [4] [5] [6] [7] [8] [9] [10] [11] , which brings us to the present application. inserted at 681 in cov-1 (the s1/s2 cleavage interface) [2] , has an average mz hydropathicity 108.5. this lowers ψ(aa,35) to 140.9 at the 3 minimum, aligning it with ~140 minima 1 and 2. the three minima span ~ 250 amino acids sites, which makes their water-driven synchronization for cov-2, but not cov-1, outside the range of most simulation or modeling methods. why the coronavirus has been so successful structure, function, and antigenicity of the sars-cov-2 spike glycoprotein criticality and dynamical scaling in living systems scaling and self-organized criticality in proteins: lysozyme c 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impacts of control measures virus batters some areas, why does it spare others? oxford covid-19 vaccine begins human trial stage future prospects for the development of cost-effective adenovirus vaccines atomic structure of human adenovirus by cryo-em reveals interactions among protein networks subunit vaccines against emerging pathogenic human coronaviruses prediction (early recognition) of emerging flu strain clusters controlling the sars-cov-2 outbreak, insights from large scale whole genome sequences generated across the world collective dynamics of 'small-world' networks statistical mechanics of complex networks the proximal origin of sars-cov-2 a genomic perspective on the origin and emergence of sars-cov-2 assembly of cell regulatory systems through protein interaction domains arrangements in the modular evolution of proteins immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop the neutral theory in light of natural selection with the mz scale panoramic spike profiles of cov-1 and cov-2 reveal a set of hydrophilic level extrema in cov-2, but not in cov-1. the new level set was identified with a even on the panoramic picture, it is clear that with the kd scale the three minima that were aligned in cov-2 are not level. this is shown in more detail in fig key: cord-278618-7tu5c7m1 authors: romano-bertrand, sara; aho-glele, ludwig-serge; grandbastien, bruno; gehanno, jean-françois; lepelletier, didier title: sustainability of sars-cov-2 in aerosols: should we worry about airborne transmission? date: 2020-06-12 journal: j hosp infect doi: 10.1016/j.jhin.2020.06.018 sha: doc_id: 278618 cord_uid: 7tu5c7m1 nan sars-cov-2 is predominantly transmitted by respiratory droplets and contact with contaminated surfaces but the role of aerosol is debated. the usual personal protective equipment (ppe) recommended for healthcare workers (hcws) include gown or apron, gloves, protective goggles, head cover and face mask. for the later, the world health organization recommends wearing of an anti-projection or surgical mask when caring for covid-19 patients, and a respirator (filtering facepiece particles (ffp) or n95 mask) only in case of aerosol-generating procedures (agps) (https://apps.who.int/iris/bitstream/handle/10665/331498/who-2019-ncov-ipcppe_use-2020.2eng.pdf). this is based on previous knowledge [1] and the doctrine that: a patient positive for sars-cov-2 is contagious by respiratory secretions (>10μm in size) that disseminate only on short distance (<1m); sars-cov-2 carried on large droplets settles onto local surfaces and is not stable in the air; sars-cov-2 aerosol dispersion is possible during agps which extensively expose hcws and therefore hcws need to wear a respirator for a higher respiratory protection during agps. however, an experimental study of van doremalen et al, [2] assessed the sustainability of sars-cov-2 in aerosols (<5μm at 65% of hygrometry (expressed in %rh for relative humidity)) performed using a high-powered machine that does not reflect normal cough conditions (https://www.who.int/publications-detail/modes-of-transmission-of-virus-causing-covid-19-implicationsfor-ipc-precaution-recommendations). they showed that sars-cov-2 remained viable and infective at least 3 hours in aerosols, which opened the debate on sars-cov-2 transmission through longdistance aerosols (>1m), and questioned the appropriateness of respiratory protection for hcws. an individual who is well, emits 10 to 10 4 particles per liter of expired air, including 95% of <1μm-size particles [3] . when speaking, the rate of emitted particles can reach 5,000 particles per minute, with a size up to 60μm. a cough generates 10 3 to 10 4 particles of size between 0.5 and 30μm (with a predominance of < 2μm particles size) [3] , while a sneeze produces around 10 6 particles between 0.5 and 16μm in size (https://www.anses.fr/fr/system/files/air2006et0003ra.pdf). however, the particles size evolves based on temperature and humidity. large particle size can rapidly desiccate at high temperature and low humidity, subsequently remaining suspended in the air [4] . models assessing viral infectivity in aerosols and droplets, focused mainly on influenza virus, showed that respiratory droplets, usually of size between 10 to 100μm at their emission, can rapidly shrink even more when poorly concentrated in organic substances, depending on humidity [5] . droplets containing mixtures mimicking respiratory mucus decrease from 10μm to 1.9μm under 64 rh%, which significantly extended their time to settle from a height of 1.5m, from 8min to 216min [5] . on the other hand, even if the humidity appears as an important parameter to consider in the potential distance and time of droplets dissemination, it may not modulate the stability of respiratory viruses in aerosols in presence of organic material. a study comparing infectivity of both fine aerosols and stationary droplets containing pandemic influenza a (h1n1) according to seven different conditions of humidity at 25°c showed no significant differences of infectivity for both aerosols and droplets containing respiratorylike secretions [6] . furthermore, even if influenza is considered as a droplet-mediated disease, a study showed that 43% of viral rna emitted from patients was carried on particles of <1μm of diameter, suggesting the potential for airborne transmission [7] . under real-life conditions in healthcare settings, a study assessed the extended sars-cov-2 dispersion within the hospital environment, by sampling air and surfaces in room of three covid-19 patients, before routine cleaning for one of them (patient 3) [8] . all environmental samples collected after cleaning in rooms of patients 1 and 2 were negative, while sars-cov-2 was retrieved from 61% of surfaces sampled in the room of patient 3. the authors were not able to identify sars-cov-2 in air but detected it onto air exhaust outlets, suggesting that the virus in droplets was displaced by airflows and reached the vents [8] . aerodynamic analysis of sars-cov-2 in two hospitals caring for covid-19 patients in wuhan showed that sars-cov-2 was present in air (including small particles size) especially in confined spaces [9] . but the important limitation of these two studies is that the authors did not demonstrate that sars-cov-2 was viable and infectious [8, 9] . another study of the indoor air in healthcare settings caring for covid-19 patients in iran did not detected sars-cov-2 in air samples collected 2 to 5 m from the patients' beds with confirmed covid-19 in ventilated rooms [10] . however, these results are not transposable to poorly ventilated environments, and airborne transmission may occur in confined spaces in absence of respiratory protection. the important issues of clinical relevance that remain to be addressed are: what is the infective dose to contaminate a person by inhalation? what is the impact of air ventilation on indoor contamination during the patient hospitalization? the viral load detected by rt-pcr in respiratory specimens can be highly variable between patients [11] and the relationship between viral load in respiratory tract and emitted droplets from patient, along with the viability of sars-cov-2 in patient-generated aerosols remain to be demonstrated. furthermore, the increased infective risk for hcws seems limited to when agps are performed compared to normal ward based care, provided the hcw is wearing a mask [12] . by considering all these elements, a potential airborne transmission of sars-cov-2 cannot be excluded, especially in confined spaces, and needs further investigation. this was already the conclusion of roy should be considered as droplet preferentially even if airborne transmission under rare specific circumstances has been described [14] . to date, implementing both droplets and contact precautions for hcws seems adequate in significantly reducing the risk of infection by sars-cov-2 during clinical care, as previously demonstrated for sars-cov-1 [1] . to efficiently prevent from contamination, wearing a face mask must be combined with other ppe [1, 15] . the growing anxiety regarding the availability of ppe, especially face masks, urges to rationalize their indications. in order to prevent a supply shortage, the requirement of face mask may be argued on the level of required effectiveness. respirator (ffp or n95 masks) must be only reserved to hcws when performing agps [15] . a precise list of agps should be dressed in order to strictly address the indications of respiratory masks. funding: no funding to report. conflict of interest: none to declare. hospital hygiene and infection control team epidemiology and infection control department. dijon university hospital, 14 rue paul gaffarel service de médecine préventive hospitalière hygiène, prévention et contrôle de l'infection (hpci), centre hospitalier universitaire vaudois physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 study on transport characteristics of saliva droplets produced by coughing in a calm indoor environment how far droplets can move in indoor environments--revisiting the wells evaporation-falling curve mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence influenza virus infectivity is retained in aerosols and droplets independent of relative humidity measurements of airborne influenza virus in aerosol particles from human coughs air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient aerodynamic analysis of sars-cov-2 in two wuhan hospitals a field indoor air measurement of sars-cov-2 in the patient rooms of the largest hospital in iran viral load of sars-cov-2 aerosol generating procedures and infective risk to healthcare workers: sars-cov-2 -the limits of the evidence airborne transmission of communicable infection -the elusive pathway viral load distribution in sars outbreak escalating infection control response to the rapidly evolving epidemiology of the coronavirus disease 2019 (covid-19) due to sars-cov-2 in hong kong key: cord-283966-eln8ljjj authors: meyer, benjamin; müller, marcel a.; corman, victor m.; reusken, chantal b.e.m.; ritz, daniel; godeke, gert-jan; lattwein, erik; kallies, stephan; siemens, artem; van beek, janko; drexler, jan f.; muth, doreen; bosch, berend-jan; wernery, ulrich; koopmans, marion p.g.; wernery, renate; drosten, christian title: antibodies against mers coronavirus in dromedary camels, united arab emirates, 2003 and 2013 date: 2014-04-17 journal: emerg infect dis doi: 10.3201/eid2004.131746 sha: doc_id: 283966 cord_uid: eln8ljjj middle east respiratory syndrome coronavirus (mers-cov) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the arabian peninsula since 2012. dromedary camels have been implicated as possible viral reservoirs. we used serologic assays to analyze 651 dromedary camel serum samples from the united arab emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. recombinant spike protein–specific immunofluorescence and virus neutralization tests enabled clear discrimination between mers-cov and bovine cov infections. most (632/651, 97.1%) camels had antibodies against mers-cov. this result included all 151 serum samples obtained in 2003. most (389/651, 59.8%) serum samples had mers-cov–neutralizing antibody titers >1,280. dromedary camels from the united arab emirates were infected at high rates with mers-cov or a closely related, probably conspecific, virus long before the first human mers cases. cov) is an emerging pathogen associated with severe respiratory symptoms and renal failure in infected patients (1, 2) . globally, 156 laboratory-confirmed cases of infection with mers-cov, including 65 deaths, were reported as of early november 2013. all human cases were linked to the arabian peninsula (saudi arabia, jordan, oman, qatar, kuwait, and the united arab emirates). imported cases were detected in countries in europe and africa (united kingdom, germany, italy, france, and tunisia) (3) . transmission patterns, including the putative zoonotic source of the virus, remain unclear. hypotheses include frequent zoonotic infections with limited subsequent human-to-human transmission chains and existence of a self-sustained epidemic in humans (4) . a recent study found evidence to support the existence of epidemiologically unlinked cases in a large outbreak in the al-hasa region, saudi arabia (5) . it was speculated that zoonotic introductions of mers-cov from an unknown reservoir might occur at high rates, in addition to obvious human-tohuman transmission. coronaviruses (cov) are positive-sense rna viruses. viruses in the genera alphacoronavirus and betacoronavirus are associated with mammals and show a particularly high level of diversification in bats. viruses in the genera gammacoronavirus and deltacoronavirus are mostly avian-associated viruses (6, 7) . mers-cov belongs to betacoronavirus phylogenetic lineage c that, in addition to mers-cov, contains 2 distinct bat-associated cov species (hku4 and hku5) (1, 8) . insectivorous bats of the family vespertilionidae were recently shown to carry viruses that are probably conspecific with mers-cov (9) . however, the limited rate of contact between humans and insectivorous bats makes a continuous and frequent acquisition of mers-cov from bats an unlikely scenario. in a manner similar to observations regarding severe acute respiratory syndrome cov (sars-cov), an intermediate reservoir host might exist from which human infections are acquired. dromedaries from different regions in africa and the arabian peninsula have been shown to have antibodies against mers-cov (10, 11) . animals from the arabian peninsula had high neutralizing serum activities overall and reciprocal antibody titers <320-1,280, which support recent infection with mers-cov or a highly related virus. thus, dromedaries might serve as intermediate hosts. however, detailed serologic studies in countries with actual incidence of mers-cov infections in humans have not been conducted. serologic analysis of covs is challenging because of cross-reactivity between covs infecting the same host and the broad distribution of covs in diverse mammalian species (6, 7, (12) (13) (14) . antibodies directed against some of the major antigens of different covs are known to crossreact in standard serologic assays (15, 16) . potential crossreactivity is a diagnostic challenge because camelids are known to be infected with bovine cov (bcov), a distinct betacoronavirus of phylogenetic lineage a unrelated to the mers-cov (17, 18) . as an additional challenge, camel immunoglobulins lack a light chain peptide, which affects specific physical properties, such as altered size and stability, compared with immunoglobulins of other mammals (19, 20) . the influence of this feature on serologic assays has not been thoroughly investigated. thus, serologic assays should be applied with caution, and different assay formats should be tested concurrently. we reported a 2-staged approach for mers-cov serologic analysis in humans (15, 16) . expanding upon these studies, we used in the present study a recombinant mers-cov spike protein immunofluroescence assay (rifa) augmented by a validated protein microarray (10, 21) , followed by mers-cov-specific neutralization assay, to screen 651 dromedary serum samples from the united arabian emirates. cross-reactivity against clade a betacoronaviruses was assessed by using a immunofluorescence assay (ifa) and a bcov-specific neutralization assay. serum samples obtained in 2003 and 2013 were compared to obtain information for the time in which mers-related cov has been circulating in camels. a total of 651 dromedary (camelus dromedarius) serum samples were systematically sampled in dubai, united arab emirates and the surrounding area in 2003 (collection 4, n = 151) and in 2013 (collections 1a, 1b, 2, and 3; n = 500). the total number of camels in that area was 360,000 in 2010 (22) . fecal samples were also available for collections 1a and 1b (n = 182), all obtained in 2013. animals in collection 1b were born and raised at the dubai central veterinary research laboratory, which tests ≈70,000 camels per year (23) and had no contact with other camels. camels in collection 2 were racing camels (age range 2-8 years), and camels in collection 3 were adult livestock camels originally purchased from saudi arabia, sudan, pakistan and oman. dromedary blood was obtained for routine health screening by jugular vein puncture according to standard veterinary procedures by trained personnel. for most serum samples, animal owners requested sample codes to be anonymous. all samples obtained during 2003 and 2013 were stored at -80°c until further analysis. for comparison, 16 serum samples from c. bactrianus camels in zoologic gardens in germany were included in the study. all serum samples were shipped in agreement with german import regulations. for screening purposes, an rifa was used (15, 24) . in brief, vero b4 cells were transfected with pcg1 eukaryotic expression vector that contained the complete spike sequence of mers-cov or human cov-oc43. cells were fixed 24-h post-transfection with ice-cold acetone/ methanol and stored dry at 4°c. serum samples were applied at a dilution of 1:80 for 1 h at 37°c, which was optimal for reducing nonspecific reactions and maintaining sensitivity. secondary detection was conducted by using a goat anti-llama igg fluorescein isothiocyanate-conjugated antibody. for some negative serum samples, dilutions of 1:20 and 1:40 were also tested. a confirmatory assay based on a protein microarray was performed as described (10, 21) by using the spike s1 subunits of mers-cov, human cov-oc43, and sars-cov. serum samples were used at 1:20 dilutions on microarray chips. relative light units were determined by using secondary cyanine 5-conjugated goat anti-llama igg. a mers-cov ifa with infected vero cells was conducted as described (15) by using commercially available mers-cov ifa slides (euroimmunag, lübeck germany). serum samples were used at dilutions of 1:20-1:5,120. secondary detection was conducted by using goat anti-llama fluorescein isothiocyanate-labeled igg (1:200 dilution; agrisera, vännas, sweden). serum neutralization tests were conducted as described (10) with camel serum diluted in 25 ml serum-free dulbecco minimum essential medium. the starting dilution was 1:40. after incubation for 1 h at 37°c, each well was infected for 1 h at 37°c with a 50 ml virus-serum mixture. supernatants were removed and fresh complete dulbecco minimum essential medium was added. assays were terminated by fixation with 8% paraformaldehyde for 30 min and stained with crystal violet after 3 days. neutralization titers were defined as serum dilutions reducing cytopathic effects in 2 parallel wells. viral rna was extracted from serum and fecal samples by using the magna pure system (roche, basel, switzerland) and an input volume of 100 μl of serum or fecal material suspended 1:10 in phosphate-buffered saline buffer. the elution volume was 100 μl for serum and fecal suspensions. to identify cov-specific nucleic acids, 2 generic cov pcrs were performed as described (25) (26) (27) , followed by subsequent sanger sequencing of the amplified dna. to characterize reactivity of camel serum samples with mers-cov in different assay formats, we chose 11 camel serum samples with weak and strong reactivity predetermined by using a simple ifa. the 11 serum samples were titrated in a 2-fold dilution series in all applied assays. the reactivity pattern of the mers-cov spike protein (mers-s) was compared against that of the human cov-oc43 spike protein (oc43-s). as in our previous study (10) , human cov-oc43 was used instead of bcov in these initial experiments because it is serologically indistinguishable from bcov and is not subject to handling restrictions of german animal diseases protection act (28) . overall titers against mers-s were higher than those against oc43-s, and several serum samples reacted exclusively against 1 of the 2 viruses (table 1) , suggesting the absence of general cross-reactivity between spike proteins of both viruses by ifa. typical patterns of reactivity observed for camel serum samples are shown in the figure, panel a. a previously published microarray-based assay that used the receptor-binding s1 spike subunit of mers-cov (mers-s1), human cov-oc43 (oc43-s1), and sars-cov (sars-s1) was also evaluated. in contrast to our previous studies (10,21) we chose a lower fluorescence intensity cutoff of 4,000 instead of 20,000 relative fluorescence units (rfu) to maximize the sensitivity and thereby challenge the target specificity. all 3 mers ifanegative serum samples had signal intensities <4000 rfu at serum dilutions of 1:20 (table 1 ). all rifa-positive serum samples had saturated signals >65,535 rfu. the oc43-s1 reactivity pattern across the serum panel was comparable with that for the oc43-s rifa. as expected, all serum samples were negative against the sars-s1 control antigen. a comparison of typical reactivity patterns in the microarray with those of the ifa is shown in the figure, panel b. results for the rifa and protein microarray were highly congruent. the panel of camel serum samples was additionally tested in a commercially available ifa that used cells infected with mers-cov (vifa) (euroimmun ag). the use of whole virus provides additional structural and nonstructural protein antigens, including envelope, membrane, nucleocapsid, and diverse replicase proteins. however, because of conserved features of nonstructural proteins among even distantly related covs (7,12) cross-reactivity was possible with this assay (15) . in the tested panel of camel serum samples, vifa titers corresponded well to titers determined by rifa and generally equal to or higher than titers in the rifa (table 1) to confirm results from affinity assays with results from a functional test, we determined endpoint virus neutralization titers by using a microneutralization test against mers-cov and bcov. in most animals mers-cov serum neutralization titers were higher than titers against bcov (serum samples 4-11) ( table 1) . high ifa titers generally corresponded with high neutralization titers, with exceptions for some bcov antibody-positive serum samples. divergence between affinity and neutralization assays can result from waning neutralizing antibody activity for infections that occurred long ago. neutralization assays confirmed the absence of cross-neutralization between mers-cov and bcov antibodies in either direction even at low dilutions, such as 1:40. however, sample no. 1 (table 1) neutralized bcov at a dilution of 1:40 despite showing negative results in all other serologic assays. this finding indicates that nonspecific neutralization activities might be encountered with camel serum samples, suggesting that higher serum dilutions should be used when conducting critical investigations such as viral reservoir studies. on the basis of the validation studies, we investigated 4 collections of serum samples from dromedaries from the united arab emirates that were sampled in 2003 and 2013. for initial screening, we chose the rifa because of its proven sensitivity and decreased chances of generating false-positive results. all 667 camel serum samples from the united arab emirates and germany were initially screened at dilutions of 1:80. a total of 89.0%-100.0% of serum samples in 4 collections showed positive results ( table 2) . seroprevalence was higher for collections from exclusively adult animals (collections 3 and 4) than for a collection from young racing camels (2-8 in the rifa. re-testing at lower dilutions of 1:20 and 1:40 confirmed absence of reactivity in these serum samples. subcollection 1b contained serum samples from 5 animals that were born in, and had never left, a closed animal research facility in dubai; these animals were seronegative. a confirmatory microneutralization test was conducted at dilutions of 1:640 and 1:1,280 for all ifa-reactive serum samples. these high dilutions were chosen on the basis of our observation of high levels of neutralizing serum activity in camels (10) . most (59.8%, 389/651) serum samples had high neutralizing titers >1,280 (table 2 ). in 18.4% (120/651) of all serum samples, neutralization titers ranged from 640 through 1,280, and 21.8% (142/651) of rifa-positive serum samples had neutralizing titers <640. to rule out cross-reactivity and to study additional exposure of mers-cov-positive camels with bcov (17, 18) , all serum samples having mers-cov neutralizing titers >640 were tested by using a bcovspecific microneutralization assay. at a dilution of 1:640, a total of 19.2% (23/120) of mers-cov-neutralizing serum samples had concomitant neutralizing activities against bcov (table 3 ). of serum samples that had mers-cov neutralizing antibody titers >1,280, a total of 24.2% (94/389) had concomitant neutralizing activities against bcov. fecal samples were available for 182 dromedaries in collection 1. all samples were tested by using a subfamily coronavirinae-specific broad-range reverse transcription pcr (rt-pcr) and a highly sensitive rt-pcr specific for genus betacoronavirus phylogenetic lineage c. both assays were specific for the viral rnadependent rna polymerase gene. two positive fecal samples were identified by both assays. sequencing of amplified cdna fragments of 182 nt and 404 nt identified sequences 99% identical with bcov strain mebus (genbank accession nos. kf894801 and u00735.2). to further confirm virus identity, we amplified a region within the spike protein gene (positions 24303-24702 in bcov strain mebus) by using rt-pcr and sequencing it. amplicons from both animals were 97% identical at nucleotide level with bcov strain mebus, indicating the presence of bcov in camels as reported (10) . we tested all serum samples in the same way by rt-pcr and obtained uniformly negative results. we have shown that dromedaries from the united arab emirates, a country with human cases of mers-cov infection, have antibodies that can neutralize mers-cov at high rates. antibodies were detected in serum samples obtained in 2013 and in serum samples obtained >10 years earlier, which indicated the longstanding presence of mers-cov or a closely related virus in dromedaries in that region. our data add to previous studies in which our group and others have reported wide antibody prevalence in camels in various regions, including oman, egypt, and the canary islands (10, 11) . a 10% lower seroprevalence in collection 2, which contained young racing camels, suggests that animals might be infected as juveniles. however, because only limited data were made available by owners, a definite statement awaits confirmation. the absence of antibodies in a control cohort from germany might be explained by the fact that these animals belonged to a different camelid species (c. bactrianus vs. c. dromedarius). however, because mers-cov has a highly conserved receptor structure, we did not assign high priority to the hypothesis that the closely related camel species c. bactrianus, should be less susceptible than c. dromedarius camels to mers-cov (29, 30) . differences in antibody prevalence rates might reflect a restricted geographic distribution of the virus, which corresponds to our previous finding of a relatively lower prevalence of antibodies against mers cov in camels from the canary islands, which have been isolated from their point of origin in africa for many years (10) . therefore, mers-cov-like viruses in camelids might be spreading across a region covering at least the eastern arabian peninsula, including oman, the united arab emirates, egypt, and morocco from where some of the antibody-positive camels described by reusken et al. originated (10) . the high rates of antibody prevalence in contemporary serum samples and samples from 2003 suggest that the virus has spread in camelids for some time. however, recognition of camelids as the bona fide reservoir for mers-cov has to await sequencing of camelid-associated mers-related cov. in this context, only animals infected with conspecific viruses can be regarded as reservoirs for a given virus. although neutralization assays can provide evidence of infection with a virus belonging to the same serotype, no systematic studies have defined whether serotypes correlate with covs species. nevertheless, for several cov clades, serotypes defined by neutralization assay will not include >1 viral species. members of the species betacoronavirus 1, including cov-oc43 and bcov, show cross-neutralization with each other, but the closely related sister species (human cov-hku1) does not show cross-neutralization (31) . feline cov (fcov) comprises 2 subserotypes that show limited cross-reactivity but are considered 1 virus species. transmissible gastroenteritis virus of swine shows more efficient cross-neutralization with 1 of these fcov subserotypes than the other and is classified as 1 species with fcov even though it is carried by a different host (32) . human covs 229e and nl63, which form 2 closely related sister taxa, do not show cross-neutralization and concordantly form 2 different species by genetic criteria (33) . therefore, our finding of high neutralizing antibody titers in camelids is suggestive (but not evidentiary) of the presence of viruses conspecific with mers-cov in camelids. final confirmation will depend on the identification of virus sequences in camelids, which should expectably be closely related to human-specific mers-cov sequences. camels probably acquired mers-cov at some unknown time. potential sources include bats of the family vespertilionidae, in which a virus with a close phylogenetic relationship with mers-cov has been detected (9). this virus, which is carried by vespertilionid bats of the genus neoromicia, has been confirmed to be conspecific with mers-cov. lineage c betacoronaviruses in other bat taxa have also been proposed to be related to mers-cov (34, 35) . however, although these viruses cluster phylogenetically with mers-cov, they are not conspecific with mers-cov on the basis of sequence distance criteria, such as that were proposed by drexler et al. (36) . in vespertilionid bats, including those in the genus neoromicia, virus conspecific with mers-cov differs from human mers-cov, even if formally a member of the same species. the observed degree of sequence divergence between this virus and mers-cov makes any direct and recent transmission from bats to humans seem unlikely. nevertheless, it cannot be excluded from available data that the virus source population in bats has not been detected. for example, a recent investigation of rhinolophus bats in china identified viruses with close relationships to the bona fide ancestor of sars-cov, and viruses described in many studies yielded only conspecific yet less related viruses (37) . in that study, viruses from civet cats, which are deemed to be intermediary hosts in the transition of sars-cov from bats to humans, were still more closely related to human sars-cov than even the closest bat-borne virus. if camelids should function as intermediary hosts in a similar manner, we should expect a virus in camelids that has a closer phylogenetic relationship with any bat-borne cov and thus should be easily detectable with available rt-pcrs. larger studies to confirm the presence of mers-cov in camelids should receive high priority so as to define the animal reservoir of mers-cov and possibly control it by such measures as vaccination or control of animal movement. however, before implementation of any control measures, whether camelids are a continuous source of infection for humans needs to be firmly established. isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov)-update transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a 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distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats detection and prevalence patterns of group i coronaviruses in bats, northern germany contact investigation of a case of human novel coronavirus infection treated in a german hospital investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughtethouse workers in jeddah and makkah, saudi arabia, fall 2013 analysis of the genome sequence of an alpaca coronavirus enteric coronavirus infection in a juvenile dromedary (camelus dromedarius) comparison of physical chemical properties of llama vhh antibody fragments and mouse monoclonal antibodies naturally occurring antibodies devoid of light chains specific serology for emerging human coronaviruses by protein microarray cvrl. 26th annual report detection of a novel human coronavirus by realtime reverse-transcription polymerase chain reaction human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray antigenic and biological relationships between human coronavirus oc43 and neonatal calf diarrhoea coronavirus human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc examination of seroprevalence of coronavirus hku1 infection with s protein-based elisa and neutralization assay against viral spike pseudotyped virus antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry coronaviruses in bats from mexico group c betacoronavirus in bat guano fertilizer genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor we thank tobias bleicker for providing excellent technical assistance.the work was supported by a european research project on emerging diseases detection and response (emperie; www.emperie.eu/emp/) (contract no. 223498) and antigone (contract no. 278976). c.d. has received infrastructural support from the german centre for infection research, the german ministry for research and education, and the german research council (grants 01kio701 and dr 772/3-1).mr meyer is a doctoral student at the bonn institute of virology, bonn, germany. his research interests are spike protein-mediated entry of bat-borne coronaviruses into cells and advancement of specific serologic tests for antibodies against coronaviruses. key: cord-267770-ik1ib3zb authors: koo, hyun jung; choi, sang-ho; sung, heungsup; choe, jooae; do, kyung-hyun title: radiographics update: radiographic and ct features of viral pneumonia date: 2020-06-05 journal: radiographics doi: 10.1148/rg.2020200097 sha: doc_id: 267770 cord_uid: ik1ib3zb editor’s note.—articles in the radiographics update section provide current knowledge to supplement or update information found in full-length articles previously published in radiographics. authors of the previously published article provide a brief synopsis that emphasizes important new information such as technological advances, revised imaging protocols, new clinical guidelines involving imaging, or updated classification schemes. articles in this section are published solely online and are linked to the original article. human coronaviruses are positive-sense rna viruses belonging to the coronaviridae family, and human coronavirus infections have become global concerns since the outbreak of severe acute respiratory syndrome coronavirus (sars-cov-1) in 2002-2003 and middle east respiratory syndrome coronavirus (mers-cov) in 2012. recently, a new coronavirus, novel coronavirus (2019-ncov), which was initially identified in wuhan, hubei province, china, in early december 2019, has rapidly spread worldwide and has been subsequently named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the disease caused by this organism is called coronavirus disease 2019 (covid-19). the genetic sequences of this virus are at least 70% identical to those of sars-cov-1 and 50% similar to those of mers-cov, and it is the seventh member of the coronaviridae family that can infect humans (1) . sars-cov-2 may have originated from horseshoe bats, a reservoir for sars-cov-1, and is responsible for rapid human-to-human transmission worldwide. as of 12:22 am gmt on april 8, 2020, a total of 1 428 428 confirmed cases of covid-19 and 82 020 deaths across 184 countries have been reported (2) . respiratory symptoms such as fever, cough, and dyspnea are common, although the potential transmission of viruses by asymptomatic patients remains a problem. approximately 20% of sars-cov-2 infections were reported as severe and the mortality rate was 3% (3) . while droplet and contact transmission are the main modes of disease transmission, airborne transmission is also occasionally possible, consistent with the findings for sars-cov-1 and mers-cov infections. the incubation period of this virus ranges from 2 to 14 days. early detection of sars-cov-2 infection and immediate isolation of those patients from the naive population are important steps to prevent an epidemic spread of the infection. in this updated review, we expand on the information presented in our 2018 article (4) and focus on the clinical features and chest ct findings of sars-cov-2 pneumonia to help radiologists detect the disease at its early stage. editor's note.-articles in the radiographics update section provide current knowledge to supplement or update information found in full-length articles previously published in radiographics. authors of the previously published article provide a brief synopsis that emphasizes important new information such as technological advances, revised imaging protocols, new clinical guidelines involving imaging, or updated classification schemes. articles in this section are published solely online and are linked to the original article. this copy is for personal use only. to order printed copies, contact reprints@rsna.org nome (e gene, orf1ab region [rna-dependent rna polymerase (rdrp) gene], and n gene) are performed as first-line, confirmatory, and additional confirmatory assays. the pathogenesis of sars-cov-2 is still under investigation. in an in vitro study, sars-cov-2 inoculation into the human airway epithelial layers induced cytopathic effects (1). angiotensin-converting enzyme 2 (ace2) receptors on the surface of sars-cov-2 anchor onto the respiratory cells, as well as onto the pneumocytes present in the nasopharyngeal mucosa, and consequently induce viremia. high levels of plasma cytokines and chemokines were noted in severely ill patients infected with sars-cov-2 (table 1) (8). these results suggest that an immunopathologic mechanism may be responsible for the progression of disease severity. chest radiographs may show bilateral infiltrates, but the findings may be nonspecific or normal (fig 1) during the early stage of sars-cov-2 infection. ct findings of pneumonia caused by sars-cov-2 are similar to those findings of to prevent the spread of sars-cov-2 to health care workers or other patients, laboratory test sampling should be performed in a dedicated isolated location, where contact is strictly limited between others and patients with suspected sars-cov-2 infection. most patients with sars-cov-2 infection have normal white blood cell and neutrophil counts, as well as a normal or reduced lymphocyte count (5) . c-reactive protein and erythrocyte sedimentation rate (esr) levels can be slightly high, but the procalcitonin level is usually normal. a high procalcitonin level indicates a bacterial coinfection. a reverse transcription-polymerase chain reaction (rt-pcr) test of an upper respiratory tract specimen (obtained with nasopharyngeal swab and/or oropharyngeal swab) and/or sputum sample is the standard diagnostic tool for determining hospitalization and isolation of patients with sars-cov infection. however, the positivity rate for rt-pcr tests on throat samples is reportedly 59%-71% (6, 7) , possibly owing to a low viral load, specimen error, and/or laboratory error. assays for the target sites of the virus genote. -ggo = ground-glass opacity. percentage of the area of lung involvement: + = 10%-25%, +++ = 50%-75%. *the pathogenesis of sars-cov-2 is still under investigation. rt-pcr test results were positive for sars-cov-2, and she was transferred to our hospital and admitted to a containment zone. initial chest ct findings (not shown) were normal. a, posteroanterior (pa) chest radiograph obtained 9 days after initial symptom onset shows no definite abnormality. b-e, follow-up axial chest ct images obtained on the same day as the chest radiograph show multifocal ground-glass opacities (arrows), predominantly located in the peripheral areas of both lungs. after 13 days of conservative management, her respiratory symptoms ameliorated, and a negative rt-pcr test result for sars-cov-2 was obtained. rt-pcr was repeated twice to confirm this negative result. mild cough and hemoptysis in a 64-year-old woman who was a caregiver in a hospital where sars-cov-2-infected patients were present. she underwent a screening test for sars-cov-2, and rt-pcr test results were positive. a, anteroposterior supine chest radiograph obtained 7 days after symptom onset shows ill-defined consolidation (arrows) in the peripheral areas of the right middle to lower lung zones. b-e, axial chest ct images obtained on the same day as the chest radiograph show irregular consolidation, with fine reticulation (arrows) in the peripheral subpleural areas of both the middle to lower lung zones, mainly in the right lung. she recovered within 14 days with conservative management and was discharged uneventfully after a negative rt-pcr test result. pneumonia caused by other human coronaviruses, which are characterized by ground-glass opacities and consolidations (4, 9, 10) . comparable with pneumonia caused by the two other coronaviruses, sars-cov-1 and mers-cov, the most common ct finding for sars-cov-2 pneumonia is ground-glass opacity (figs 1, 2) . a reversed halo sign is an uncommon ct feature but could be visualized in the early stage of sars-cov-2 pneumonia. during the sars-cov-2 epidemic situation in china, the sensitivity of chest ct for identifying sars-cov-2 pneumonia cases has been reported to be 97% (95% confidence interval: 95%, 98%) (6). the high sensitivity of chest ct was considered in part a result of imaging patients who were at a relatively advanced stage of sars-cov-2 pneumonia. consolidation is noted as the second most common finding during the first 11 days of symptom onset (11) . approximately half of the patients were diagnosed with multifocal groundglass opacity and consolidation, and reticulation or nodules are rare (6) . serial follow-up chest ct can help indicate the evolution of the disease and help monitor therapeutic effects (12, 13) . bilateral and peripheral ground-glass opacities are predominant patterns after symptom onset (11) , and the extent of ground-glass opacity and consolidation occasionally manifesting as a "crazy-paving" pattern increases as the disease progresses and peaks at 6-11 days (12) . bilaterality was noted among 76% of cases 3-5 days after initial symptom onset (11, 12) . severe cases manifest as acute respiratory distress syndrome, with diffuse haziness of the bilateral lungs (fig 3) . images obtained in most patients (75%) who are in the recovery phase after 2 weeks from initial symptom onset show a gradual resolution of consolidation, with residual ground-glass opacity (fig 4) (13) . although more than 70% of sars-cov-2 pneumonia cases exhibit typical ct findings, not every case of sars-cov-2 pneumonia is distinguishable from other types of viral pneumonia. chest ct findings sometimes result in a false-positive diagnosis owing to overlapping ct manifestations. the differential diagnosis of sars-cov-2 pneumonia includes other pneumonia-causing viral pathogens, and their ct figure 3 . fever, cough, sputum production, and anorexia for 1 week in a 77-year-old woman with asthma, which was managed daily with a long-acting β-agonist salmeterol and corticosteroid inhaler, who presented to a sars-cov-2 screening clinic. the test results confirmed sars-cov-2 infection, and the patient was transferred to a containment zone at our hospital. a, anteroposterior chest radiograph obtained 14 days after symptom onset shows bilateral ill-defined groundglass opacity and consolidation (arrows). b, c, anteroposterior chest radiograph, b, obtained 19 days after symptom onset shows an increase in the extent of consolidation (arrows) after the patient's symptoms worsened. despite the use of extracorporeal membrane oxygenation (ecmo) in the intensive care unit, diffuse consolidation continued to increase until 4 weeks after the admission period, as depicted on the anteroposterior radiograph in c. d, axial chest ct image obtained 5 weeks after symptom onset shows diffuse consolidation with air bronchogram and pneumothorax (arrowheads), clinically indicating acute respiratory distress syndrome. the patient died that day owing to sepsis and multiorgan failure. findings are briefly described in table 2 . detailed ct findings and relevant images have been provided in a previous article (4) . a study involving the use of chest ct for differentiating sars-cov-2 pneumonia from other types of viral pneumonia found that radiologists who were blinded to rt-pcr results could correctly distinguish cases of sars-cov-2 infection with 67%-97% sensitivity (7) . peripheral distribution and ground-glass opacity were significantly noted characteristics of sars-cov-2 pneumonia, and fine reticular opacity and vascular thickening were also a more commonly noted pattern in half of the sars-cov-2 pneumonia cases, as opposed to other viral pneumonia cases. pleural effusion and lymphadenopathy were uncommon. as an initial laboratory test, a respiratory virus nucleic acid test is commonly performed to detect other common respiratory viruses such as adenovirus, parainfluenza virus, respiratory syncytial virus, and influenza virus. in addition, considering the potential false-negative results of laboratory tests and highly contagious nature of the viruses, chest ct could prove to be a fast diagnostic tool in combination with clinical manifestations for diagnosing sars-cov-2 infection. ct images are helpful to distinguish sars-cov-2 pneumonia in suspicious cases with initially negative molecular test results, and some of these cases could show positive results at repeat molecular testing during the follow-up monitoring period. chest ct is useful for diagnosis and establishing the next management strategies, including patient isolation. however, radiation exposure, the risk of the virus spreading to health care workers, medical costs, and the time required for disinfect-ing imaging examination rooms are considerable factors that make us consider the appropriateness of performing imaging. as droplet and contact transmissions are the main modes of spread for sars-cov-2, all patients with suspected sars-cov-2 infection who undergo radiologic imaging should be masked and imaged using a separated dedicated imaging machine. the imaging location should be disinfected after each imaging examination. health care workers who manage patients with suspected sars-cov-2 infection should wear a medical mask and goggles, and the use of an isolation gown is recommended. a multidisciplinary panel of experienced radiologists and pulmonologists from 10 countries suggested the utility of imaging within clinical scenarios representing community situations, medical resources, and various risk factors (14) . on the basis of this suggestion and experience managing patients with covid-19, we provide a brief clinical setting to show the use of chest ct to facilitate the diagnosis of sars-cov-2 pneumonia (fig 5) . for the management of patients with respiratory symptoms in endemic areas, an rt-pcr test is the primary diagnostic tool used for discriminating sars-cov-2-positive cases. after confirming sars-cov-2 infection, chest ct could be used to evaluate disease severity (fig 5) . clinical judgment regarding the use of chest ct is dependent on the patient risk factors related to covid-19, such as age greater than 65 years and any comorbidities. the availability of local medical resources, including medical team members, personal protection devices, rt-pcr tests, hospital containment beds, and ventilators, has also affected the process of patient management. in the situation of significant medical resource constraints, a patient with mild symptoms of covid-19 without significant respiratory dysfunction does not require chest ct examination. chest ct is considered in patients with moderate to severe symptoms with pulmonary dysfunction or damage. if a patient has contact history with a confirmed sars-cov-2-infected patient, chest ct could be performed as the next step for excluding a potential false-negative outcome from a negative rt-pcr test result. in cases with typical ct findings, bilateral groundglass opacity, consolidation, and fine reticulation, sars-cov-2 infection is highly suspected. therefore, a repeat rt-pcr test is warranted, even if the first rt-pcr test result was negative. in a case with atypical ct findings (fig 4) , there could be a probability of infection by other pathogens, but a repeat rt-pcr test or follow-up ct is still necessary, especially for patients with underlying comorbidities. the ct findings could be subtle during the early stage of infection. if no abnormalities are detected on chest ct images, patients are still recommended to maintain a 2-week isolation period in their personal space or home to prevent community transmission owing to a potential false-negative result. in a scenario wherein rt-pcr tests for sars-cov-2 are unavailable, chest ct could be used as a screening tool for identifying potential patients with sars-cov-2 pneumonia. in an endemic area, it is not only the management of a patient with respiratory symptoms that needs attention. because an asymptomatic patient in the early stage of covid-19 is also a source of infection transmission, taking a careful history and performing rt-pcr tests are necessary when radiologists incidentally detect imaging findings suggestive of covid-19 at chest ct in routine practice. although various trials for developing treatment strategies for sars-cov-2 infection are ongoing, there are no approved therapeutic drugs or vaccines for this disease to date. patients with sars-cov-2 infection need to be isolated in a designated area to prevent further spread of the virus, and intensive care is critical for severely ill patients. current management methods for sars-cov-2 pneumonia are based on optimal conservative care, including respiratory support. similar to plasma transfusion used as treatment of mers-cov infection, the blood plasma of patients who recovered from sars-cov-2 infection, which contained neutralizing antibodies, was transfused in several patients with sars-cov-2 infection (15) . however, further tests for the validation of this strategy are necessary. chest ct can be used as a rapid diagnostic tool in combination with clinical manifestations to diagnose sars-cov-2 infection. a novel coronavirus from patients with pneumonia in china johns hopkins coronavirus resource center a novel coronavirus outbreak of global health concern radiographic and ct features of viral pneumonia emerging 2019 novel coronavirus (2019-ncov) pneumonia correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases performance of radiologists in differentiating covid-19 from viral pneumonia on chest ct clinical features of patients infected with 2019 novel coronavirus in wuhan, china radiographicclinical correlation in severe acute respiratory syndrome: study of 1373 patients in hong kong ct correlation with outcomes in 15 patients with acute middle east respiratory syndrome coronavirus temporal changes of ct findings in 90 patients with covid-19 pneumonia: a longitudinal study chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia the role of chest imaging in patient management during the co-vid-19 pandemic: a multinational consensus statement from the fleischner society treatment of 5 critically ill patients with covid-19 with convalescent plasma key: cord-193489-u6ewlh16 authors: wang, rui; hozumi, yuta; yin, changchuan; wei, guo-wei title: decoding sars-cov-2 transmission, evolution and ramification on covid-19 diagnosis, vaccine, and medicine date: 2020-04-29 journal: nan doi: nan sha: doc_id: 193489 cord_uid: u6ewlh16 tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). much of this development has been based on the reference genome collected on january 5, 2020. based on the genotyping of 6156 genome samples collected up to april 24, 2020, we report that sars-cov-2 has had 4459 alarmingly mutations which can be clustered into five subtypes. we introduce mutation ratio and mutation $h$-index to characterize the protein conservativeness and unveil that sars-cov-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while sars-cov-2 nucleocapsid protein, spike protein, and papain-like protease are relatively non-conservative. in particular, the nucleocapsid protein has more than half its genes changed in the past few months, signaling devastating impacts on the ongoing development of covid-19 diagnosis, vaccines, and drugs. the ongoing pandemic of coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) poses crucial threats to the public health and the world economy since it was detected in wuhan, china in december 2019 [1] . as of april 24, 2020, more than 2.6 million cases of covid-19 have been reported in 185 countries and territories, resulting in more than 184,000 deaths [2] . tragically, there is no sign of slowing down nor relief at this monument partially due to the fact there is no specific anti-sars-cov-2 drugs and effective vaccines. sars-cov-2 is a positive-strand rna virus and belongs to the beta coronavirus genus. the genomic information underpins the development of antiviral medical interventions, preventative vaccines, and viral diagnostic tests. the first sars-cov-2 genome was reported on january 5, 2020 [3] . it has a genome size of 29.99 kb, which encodes for multiple non-structural and structural proteins. the leader sequence and orf1ab encode non-structural proteins for rna replication and transcription. among various nonstructural proteins, viral papain-like (pl) proteinase, main protease (or 3cl protease), rna polymerase, and endoribo-nuclease are common targets in antiviral drug discovery. however, it typically takes more than ten years to put an average drug to the market. the downstream regions of the genome encode structural proteins including spike (s) protein, envelope (e) protein, membrane (m) protein, and nucleocapsid (n) protein. notably, s-protein uses one of two subunits to bind directly to the host receptor angiotensinconverting enzyme 2 (ace2), enabling the virus entry into host cells [4] . the nucleocapsid (n) protein, one of the most abundant viral proteins, can bind to the rna genome and is involved in replication processes, assembly, the host cellular response during viral infection [5] . the e protein is a small integral membrane protein, a virulence factor, regulating cell stress response and apoptosis, and promoting inflammation [6] . the structural proteins, especially, the spike protein and the n protein, are the candidate antigens for vaccine development. developing safe and effective vaccines is urgently needed to prevent the spread of sars-cov-2. however, it typically takes over one year to design and test a new vaccine. furthermore, the sars-cov-2 genome undergoes rapid mutations partially stimulated as a response to the challenging immunological environments arising from the covod-19 patients of different races, ages, and medical conditions. sars-cov-2 exists as heterogeneous and dynamic populations because of their error-prone replication [7] . the vaccine developed at one time may not be effective for mitigating the infection by new mutated virus isolates. an alarming fact is that many of these mutations may devastate the on-going effort in the development of effective medicines, preventive vaccines, and diagnostic tests. accurate identification of the antigens and their mutations represents the most important roadblock in developing effective vaccines against covid-19. for example, different vaccines are needed for different geographic locations due to predominant mutations in the corresponding regions. in covid-19 diagnosis, the diagnosis kits are designed using two major methods, i.e., specific serological tests and molecular tests. serological tests are to detect specific covid-19 proteins. molecular diagnoses test specific covid-19 pathogenic genes, which usually rely on the polymerase chain reaction (pcr).because of the fast mutations of the sars-cov-2 genome, genotyping analysis of sars-cov-2 may optimize the pcr primer design to detect sars-cov safely and reduce the risk of false-negatives caused by genome sequence variations. in addition, the genotyping analysis may also reveal those regions that are highly conserved with very few mutations, which can be selected as a target sequence for reliable drug therapy and general diagnosis. the evolution pattern through the highly frequent mutations of sars-cov-2 can be observable on short time scales. in the early infection period (i.e., february 2020), the sars-cov-2 variants were clustered as s and l types [8] . recent genotyping analysis reveals a large number of mutations in various essential genes encoding the s protein, the n protein, and rna polymerase in the sars-cov-2 population [9] . monitoring the evolutionary patterns and spread dynamics of sars-cov-2 is of grace importance for covid-19 control and prevention. although mutations occur randomly, most preserved mutations can be regarded as virus responds to the host immune system surveillance. as a result, the faster and the wider the sars-cov-2 spread is, the more frequent and diverse the mutations will be. the tracking and analysis of covid-19 dynamics, transmission, and spread is of paramount importance for winning the on-going battle against covid-19. genetic identification and characterization of the geographic distribution, intercontinental evolution, and global trends of sars-cov-2 is the most efficient approach for studying covid-19 genomic epidemiology and offer the molecular foundation for region-specific sars-cov-2 vaccine design, drug discovery, and diagnostic development [10] . for example, different vaccines for the shell can be designed according to predominant mutations. this work provides the most comprehensive genotyping to reveal the transmission trajectory and spread dynamics of covid-19 to date. based on genotyping 6156 sars-cov-2 genomes from the world as of april 24, 2020, we trace the covid-19 transmission pathways and analyze the distribution of the subtypes of sars-cov-2 across the world. we use k-means methods to cluster sars-cov-2 mutations, which provides the updated molecular information for the region-specific design of vaccines, drugs, and diagnoses. our clustering results show that globally, there are at least five distinct subtypes of sars-cov-2 genomes. while, in the u.s., there are at least three significant sars-cov-2 genotypes. we introduce mutation hindex and mutation ratio to characterize conservative and non-conservative proteins and genes. we unveil the unexpected non-conservative genes and proteins, rendering an alarming warning for the current development of diagnostic tests, preventive vaccines, and therapeutic medicines. tracking the sars-cov-2 transmission pathways and analyzing the spread dynamics are critical to the study of genomic epidemiology. temporospatially clustering the genotypes of sars-cov-2 in transmission provides insights into diagnostic testing and vaccine development in disease control. in this work, we retrieve and genotype 6156 sars-cov-2 isolates from word as of april 24, 2020. there are 4459 single mutations in 6156 sars-cov-2 isolates. based on these mutations, we classify and track the geographical distributions of 6156 genoytype isolates by k-means clustering. the sars-cov-2 genotypes, represented as snp variants, are clustered as five groups in the world table 2 . the genotypes in the u.s. are clustered as three groups table 2 . the optimal clustering groups are established using the elbow method in the k-means clustering algorithm (see supporting material). the detailed distribution of the snp variants from the world for each cluster is provided in the supporting material. the snp variant clusters from 11 countries that have the highest number of cases recorded in are listed in table 2 figure 1 . the geographic distribution of the snp variant clusters reflects the approximate transmission pathways and spread dynamics across the world. several findings can be read from the table 2: 1. two early subtypes of sasr-cov-2 (cluster i and ii) are epidemic in the asian countries (cn, jp, kr). 2. the subtypes of sars-cov-2 in cluster iii is not spreading in the european countries (uk, de, fr, it). 3. all of the subtypes of sars-cov-2 in five different clusters can be found in the us, au, and canada. moreover, we analyze the statistic of snp variants located in different states of the united states. in table 3 , we list the number of cases in three different clusters with respects to the west coast states we note that cluster c in the u.s. is derived from cluster iii in the world, with an additional mutation at the leader sequence 241. the high spread in new york is consistent with the high transmission of sars-cov-2 in the european countries, where the subtype in cluster iii is predominated. table 5 presents the statistics of single mutations on various sars-cov-2 proteins that occurred in the recorded genomes between january 5, 2020, and april 24, 2020. the papain-like protease has the highest number of mutations of 599 while the envelope protein has the lowest number of mutations of 13. since the sizes of proteins vary dramatically from 1945 for the papain-like protease to 75 for the envelope protein, it is useful to consider the mutation ratio, i.e., the number of mutations per residue. in this category, the envelope protein still has the lowest score of 0.17, whereas the nucleocapsid protein has the highest score of 0.56, i.e, 235 mutations on its 419 residues. note that 3cl protease has the second-lowest mutation ratio of 0.22, indicating its conservative nature. another relatively conservative protein judged by the mutations ratio is the rna-dependent rna polymerase. it has 223 mutations over its 932 residues. counting the number of single mutations and mutation ratio does not reflect the fact some mutations occur numerous times over genome samples while other mutations may happen only on a few genome samples. to account for the frequency effect of mutations, we introduce a mutation h-index to measure both the number of mutations and the frequency of mutations of a given protein or genetic section. it is defined as the maximum value of h such that the given protein genetic section has h single mutations that have each occurred at least h times. it is very interesting to note from table 5 that the mutation h-index correlates very well with the number of mutations per residue. specifically, nucleocapsid protein has both the highest number of mutations per residues of 0.56 and the highest h-index of 27, suggesting that it is the most non-conservative protein in sars-cov-2 genomes. in contrast, the envelope protein has the lowest number of mutations per residues of 0.17 and the lowest h-index of 5, indicating its relatively conservative nature. by combining the number of mutations per residue and the mutation h-index, we report that the three most conservative sars-cov-2 proteins are 1) the envelope protein, 2) the main protease, and 3) the endoribonuclease. it is found that the most non-conservative sars-cov-2 proteins are 1) the nucleocapsid protein, 2) the spike protein and 3) the papain-like protease. real-time rt-pcr (rrt-pcr) is routinely used in the qualitative detection of nucleic acid from sars-cov-2 for diagnostic testing covid-19 [11, 12] . the primers used in the rrt-pcr are critical for the precise diagnosis of covid-19 and the discovery of new strains. the primer sequences are specially designed for amplifying the conserved regions across the different existing strains for high specificity and sensitivity, and also are subject to genotype changes as the sars-cov-2 coronavirus evolves. in diagnostic testing covid-19, many rrt-pcr primers are designed to detect for three perceived conservative sars-cov-2 regions: (1) rna-dependent rna polymerase (rdrp) gene in orf1ab region, (2) the e protein gene, and (3) the n protein gene [11] . our genotyping statistics given in table 5 indicates that the nucleocapsid protein is the worst choice. among four structural proteins of sars-cov-2, the spike surface glycoprotein (s) of 1273 amino acid residues, nucleocapsid protein (n) of 419 amino acid residues, membrane protein (m) of 222 amino acid residues, envelope protein (e) of 75 amino acid residues, the s protein is the most divergent with 385 unique mutations among the 6156 sars-cov-2 genomes. the n protein has 235 unique mutations, the e protein has 13 mutations. considering the lengths of the proteins, all the four structural proteins undergo high mutations. the rdrp gene, which is often used in diagnostic testing covid-19, also has 223 mutations. therefore, all the three regions in routine rrt-pcr target, namely rdrp, the n protein gene, and the e protein genes, have significant mutations. precise and robust diagnosis tools must be re-established according to the conserved regions and predominated mutations in the sars-cov-2 genomes detailed in the supporting material. notably, sars-cov-2 has a unique furin cleavage site, where four amino acid residues (prra) are inserted into the s1-s2 junction region 681-684 of the s protein [13] . the furin cleavage site is crucial for zoonotic transmission of sars-cov-2 [14] . this study reveals crucial mutations near the s1-s2 junction region in the s protein, including 23403a>gmoreover, these mutations of the s protein sars-cov-2 are located at the epitope region, corresponding to the regions 469-882 and 599-620 in sars-cov) [15] . additionally, many mutated amino acids are on the surface of the s protein as shown in fig. 5 . unfortunately, the s protein is the second most non-conservative protein in the genome based on the number of mutations per residue and mutation h-index. in fact, about half of the receptor-binding domain residues of the s proteins have had mutations in the past few months as shown in fig. 6 . because the surface accessibility of epitope is also important for the interaction of antibody and antigen, these mutations are critical for the antigenicity of the s protein. the convalescent covid-19 patients show a neutralizing antibody response after infection, which are directed against the s protein or the n protein [16] . the neutralizing antibody responses against sars-cov-2 could give some defense against sars-cov-2 infection and thus, having implications for preventing sars-cov-2 outbreaks. the divergence of spike proteins, the non-conserved regions of the spike proteins might contribute to the antigenicity. the high frequent mutations identified in the s protein and the n proteins must be considered when designing a vaccine. unfortunately, there is no specific effective drug for sars-cov-2 at this point. much of the drug discovery effort focuses on sars-cov-2 non-structural proteins. among the major non-structural proteins of sars-cov-2, the main protease of 306 amino acids has 68 mutations with 0.22 mutations per residue and the mutation h-index of 9, rna polymerase of 932 amino acids has 223 mutations with 0.24 mutations per residue and the mutation h-index of 13, and papain-like protease of 1945 amino acids has 599 mutations with 0.31 mutations per residue and the mutation h-index of 15. in fact, the main protease is the most popular drug target because there are no similar known genes in the human genome, which implies sars-cov-2 main protease inhibitors will be likely less toxic [17] . the present study suggests that the main protease is the second most conservative protein. therefore, it remains the most attractive target for drug discovery. the sars-cov-2 spike glycoprotein, or s protein, comprised of two subunits, s1 and s2, of very different properties [13] , see fig. 5 . among them, the s1 subunit, as shown in fig. 5 , contains the receptor-binding domain (rbd) responsible for binding to the host cell receptor angiotensinconverting enzyme 2 (ace2). the rbd is also the common binding domain for antibodies. the s2 subunit offers the structural support of the s protein and mediates fusion between the viral and host cell membranes. after the fusion, the virus releases the viral genome into the host cell. the s1 rbd protein plays key parts in the induction of neutralizing-antibody and t-cell responses, as well as protective immunity. however, s2 and extracellular domain (ecd) of spike protein and their combination are commonly used in recombinant proteins in sars-cov-2 antibody development. table 5 , the s protein is the most heterogeneous structural protein with a significant number of mutations as shown in figs. 5 and 6 and table 6 . the divergence of the spike protein, the non-conserved regions of the spike protein might contribute to the antigenicity difference in sars-cov-2 isolates. we found that most of the high frequent mutations of the s protein are located in the s1 subunit. figure 6 indicates that near half of the amino acid residues have had mutations since january 5, 2020. one of the important mutations at s1 is 23010 (v483a) within the rbd for ace2 binding. the structural study revealed that the amino acids 442-487 in the s1 subunit may impact viral binding to human ace2 [18, 19] . the mutations identified in this study imply the change in ace2 binding affinity and the transmissibility of sars-cov-2 as well as negative impacts in preventive vaccine and diagnostic test development. top1 10323 k90r 52 1 8 43 0 0 top2 10097 g15s 51 0 1 0 50 0 top3 10851 a266v 44 0 2 16 0 26 top4 10582 d176d 19 0 0 5 0 14 top5 10771 y239y 15 15 0 0 0 0 top6 10507 n151n 11 0 10 1 0 0 top7 10948 r298r 11 0 0 0 11 0 top8 10265 g71s 9 0 0 0 9 0 top9 10870 l272l 9 0 0 1 4 4 top10 10319 l89f 8 0 1 4 0 3 top11 10450 p132p 8 main protease sars-cov-2 main protease, or 3cl protease, is essential for cleaving the polyproteins that are translated from the viral rna [17] . it operates at multiple cleavage sites on the large polyprotein through the proteolytic processing of replicase polyproteins and plays a pivotal role in viral gene expression and replication. sars-cov-2 main protease is one of the most attractive targets for anti-cov drug design because its inhibition would block viral replication and it is unlikely to be toxic due to no known similar human proteases. another reason for the focused drug discovery efforts in developing sars-cov-2 main protease inhibitors is that this protein is relatively conservative as shown in table 5 . figure 7 illustrates the main protease mutation patterns. figure 8 further highlights the inhibitor binding domain (bd). indeed, the main protease is relatively conservative compared to the spike protein. table 7 lists top 11 mutations and their frequency in our dataset. it is interesting to see that many mutations, such as y239y, n151n, r298r, l272l, and p132p, are degenerate ones. one possible explanation is that nondegenerate may be non-silent and likely cause unsurvivable disruption to the virus. note that mutation g15s mostly occurs in cluster iv. mutation y239y is restricted to cluster i. some other mutations, such as r298r, g71s, and p132p, are specific to certain clusters. nonetheless, some mutations at the bd shown in fig. 8 are worth noting. they can undermine the ongoing drug discovery effort. top1 3037 f106f 3889 0 80 1800 964 1045 top2 2891 a58t 120 0 119 0 1 0 top3 3177 p153l 72 0 69 2 1 0 top4 4540 y607y 60 0 60 0 0 0 top5 7011 a1431v 45 0 43 2 0 0 top6 6312 t1198k 44 0 42 1 1 0 top7 7438 y1573y 34 0 9 21 4 0 top8 3373 d218d 29 0 3 0 26 0 top9 4002 t428i 26 0 1 0 25 0 top10 6040 f1107f 26 0 10 12 0 4 figure 9 : illustration of sars-cov-2 papain-like protease mutations using 6w9c as a template [20] . papain-like protease sars-cov-2 papain-like protease (plpro) is a cysteine cleavage protein located within the non-structural protein 3 (ns3) section of the viral genome [20] . like, the main protease, plpro activity is required to cleave the viral polyprotein into functional, mature subunits and, thereby, contributes to the biogenesis of the virus replication. additionally, plpro possesses a deubiquitinating activity. the sars plpro is also a major therapeutic and diagnostic target. as shown in table 5 , the sars plpro is prone to mutations. figure 9 shows that mutations are all over the places in plpro. table 8 lists top ten mutations in plpro. five of these mutations are degenerate ones, including one of the highest frequented mutations. note that none one of the top mutations occurred in cluster i. on the contrast, cluster ii has many different mutations. [21] . as one of the non-structure proteins, rdrps located in the early part of orf1b section. like most other rna viruses, sars-cov-2 rdrps are considered to be highly conserved to maintain viral functions and thus targeted in antiviral drug development as well as diagnostic tests. on the other hand, the sars-cov-2 rna polymerase lacks proofreading capability and thus its mutations are deemed to happen as shown in table 5 . figure 10 illustrates the sars-cov-2 rdrp mutations since january 5, 2020. surprisingly, there are many mutations in sars-cov-2 rdrp. table 9 describes the top ten mutations. as in other cases, five of these mutations are degenerate ones. cluster i has no nondegenerate mutations. endoribo-nuclease (nendou) protein is a nidoviral rnauridylate-specific enzyme that cleaves rna [22] . it contains a c-terminal catalytic domain belonging to the endou family rna processing. the nendou protein is presented among coronaviruses, arteriviruses, and toroviruses. the many aspects of the detailed function and activity of sars-cov-2 nendou protein are yet to be revealed. figure 11 depicts sars-cov-2 nendou protein mutations. like in most other sars-cov-2 proteins, mutations have occurred over different parts. table 5 shows that nendou is relatively conservative. table 10 lists the top twelve high-frequency mutations of the sars-cov-2 nendou protein that occurred in the past few months. three of these mutations are degenerate ones. the frequencies of these mutations range from 38 to 6. note that cluster i do not have any of these mutations. total frequency cluster i cluster ii cluster iii cluster iv cluster v top1 26319 v25v 8 0 7 1 0 0 top2 26340 a32a 7 0 5 2 0 0 top3 26326 l28l 5 0 5 0 0 0 top4 26256 f4f 4 0 0 2 2 0 top5 26301 l19l 3 0 2 1 0 0 top6 26433 k63k 2 0 0 1 0 1 top7 26370 y42y 1 0 1 0 0 0 top8 26392 s50g 1 0 1 0 0 0 top9 26313 f23f 1 the sars-cov-2 envelope (e) protein is one of sars-cov's four structural proteins. as a transmembrane protein, it involves in ion channel activity, and thus facilitates viral assembly, budding, envelope formation, pathogenesis, and release of the virus [23] . the e protein may not be essential for viral replication but it is for pathogenesis. figure 12 illustrates e protein as a very small pentamer with a few mutations. table 11 shows its top thirteen mutations. note that the first 7 mutations are degenerate ones. all other mutations have very low frequencies. as shown in table 5 , the sars-cov-2 e protein is very conservative. total frequency cluster i cluster ii cluster iii cluster iv cluster v top1 28881 r203k 989 1 20 4 964 0 top2 28882 r203r 983 0 18 1 964 0 top3 28883 g203r 983 0 18 1 964 0 top4 28657 d128d 125 1 124 0 0 0 top5 28311 p13l 102 0 101 1 0 0 top6 28688 l139l 91 0 90 1 0 0 top7 29045 p258t 67 0 65 2 0 0 top8 29046 p258r 67 0 65 2 0 0 top9 29047 p258p 67 0 65 2 0 0 top10 29049 r259l 67 0 65 2 0 0 top11 29050 r259r 67 0 65 2 0 0 top12 29051 q260e 67 0 65 2 0 0 top13 29052 q259r 67 0 65 2 0 0 top14 29053 q260h 67 0 its primary function is to encapsidate the viral genome. to do so, it is heavily phosphorylated (or charged) and thereby, can bind with rna. additionally, sars-cov-2 n protein confirms the viral genome to replicasetranscriptase complex (rtc) and plays a crucial role in viral genome encapsulation. therefore, it may function completely differently at different stages of the viral life cycle. sars-cov-2 n protein is considered to be one of the most conservative sars-cov-2 proteins in the literature and is a popular target for diagnosis of vaccine development [11] . the present works shown in table 5 indicates the sars-cov-2 n protein is the worst target of any drug, vaccine, and diagnostic development. table 12 presents the top fourteen mutations of the sars-cov-2 n protein since january 5, 2020. note that only three out of fourteen top mutations are degenerate ones, which is a significantly lower ratio than that of other proteins. the frequency of 14th mutation is 67, which suggests there are many mutations associated with these mediate-sized proteins. most top mutations occurred to clusters ii and iv. cluster v has none of the top fourteen mutations. membrane protein sars-cov-2 membrane (m) protein is another structural protein and plays a central role in viral assembly and viral particle formation. it exists as a dimer in the virion and has certain geometric shapes to enable certain membrane curvature and binding to nucleocapsid proteins. similar to other sars-cov proteins, m protein is also a popular target for viral diagnosis and vaccines. table 5 gives sars-cov-2 m protein the meddle ranking for its conservation. table 13 details the top eleven mutations in sars-cov-2 m protein occurred in the past few months. seven of these mutations are degenerate. clusters i and v have relatively a few of these mutations. on january 5, 2020, the complete genome sequence of sars-cov-2 was first released on genbank (access number: nc 045512.2) by zhang's group at fudan university [3] . since then, there has been a rapid accumulation of sars-cov-2 genome sequences. in this work, 6156 complete genome sequences with high coverage of sars-cov-2 strains from the infected individuals in the world are downloaded from the gi-said database [25] (https://www.gisaid.org/) as of april 24, 2020. all the records in gisaid without the exact submission date will not take into considerations. to rearrange the 6156 complete genome sequences according to the reference sars-cov-2 genome, multiple sequence alignment (msa) is carried out by using clustal omega [26] with default parameters. snp genotyping measures the genetic variations between different members of a species. establishing the snp genotyping method for the investigation of the genotype changes during the transmission and evolution of sars-cov-2 is of great importance. by analyzing the rearranged genome sequences, snp profiles which record all of thesnp positions in teams of the nucleotide changes and its corresponding positions can be constructed. the snp profiles of a given genome of a covid-19 patient capture all the differences from a complete reference genome sequence and can be considered as the genotype of the individual sars-cov-2. the jaccard distance measures dissimilarity between sample sets. the jaccard distance of snp variants is widely employed in the phylogenetic analysis of human or bacterial genomes [9] . in this work, we utilize the jaccard distance to compare the difference between the snp variant profiles of sars-cov-2 genomes. the jaccard similarity coefficient, also known as the jaccard index, is defined as the intersection size divided by the union of two sets a, b [27] : the jaccard distance of two sets a, b is scored as the difference between one and the jaccard similarity coefficient and is a metric on the collection of all finite sets: therefore, the genetic distance of two genomes corresponds to the jaccard distance of their snp variants. in principle, the jaccard distance measure of snp variants takes account of the ordering ofsnp positions, i.e., transmission trajectory, when an appropriate reference sample is selected. however, one may fail to identify the infection pathways from the mutual jaccard distances of multiple samples. in this case, the dates of the sample collections offer useful information. additionally, clustering techniques, such as kmeans described below, enable us to characterize the spread of covid-19 onto the communities. k-means clustering is one of the fundamental unsupervised algorithms in machine learning which aims at partitioning a given data set x = {x 1 , x 2 , â· â· â· , x n , â· â· â· , x n }, x n â�� r d into k clusters {c 1 , c 2 , â· â· â· , c k }, k â�¤ n such that the specific clustering criteria are optimized. more specifically, the standard k-means clustering algorithm starts to pick k points as cluster centers randomly and then allocates each data to its nearest cluster. the cluster centers will be updated iteratively by minimizing the within-cluster sum of squares (wcss) which is defined by: where âµ k = i â�� c k x i /n k is the mean of points located in the k-th cluster c k and n k is the number of points in c k . here, â· 2 denote the l 2 distance. the algorithm above only provides a way to obtain the optimal partition for a fixed number of clusters. however, we are interested in finding the best number of clusters for the snp variants. therefore, the elbow method is applied. by varying the number of clusters k, a set of wcss can be calculated in the k-means clustering process, and then the plot of wcss according to the number of clusters k can be carried out. the location of the elbow in this plot will be considered as the optimal number of clusters. to be noticed, the wcss measures the variability of the points within each cluster which is influenced by the number of points n . therefore, as the number of total points of n increases, the value of wcss becomes larger. additionally, the performance of k-means clustering depends on the selection of the specific distance. in this work, we propose to implement k-means clustering with the elbow method for analyzing the optimal number of the subtypes of sars-cov-2 snp variants. the jaccard distance-based and locationbased representations are considered as the input features for the k-means clustering method. suppose we have a total of n snp variants concerning a reference genome in a sars-cov-2 sample. the location of the mutation sites for each snp variant will be saved in the set s i , i = 1, 2, â· â· â· , n . the jaccard distance between two different sets (or samples) s i , s j is denoted as d j (s i , s j ). therefore, the n ã�n jaccard distance-based representation will be: suppose we have n snp variants with respect to a reference genome in a sars-cov-2 sample. among them, m different mutation sites can be counted. for i-th snp variant, v i = [v 1 i , v 2 i , â· â· â· , v m i ], i = 1, 2, â· â· â· , n is a 1 ã� m vector which satisfies: therefore, an n ã� m location-based representation will be: hundreds of complete genome sequences are deposit to the gisaid every day, which results in an evergrowing massive quantity of high dimensional data representations for the k-means clustering. for example, if the dataset of an organism involves 10,000 snps, the initial representation will be a 10,000dimensional vector for each sample, which can be computationally difficult for a simple k-means clustering algorithm. therefore, a dimensionality reduction method is used to pre-process the data. the essential idea of pca-based k-means clustering is to invoke the pca to obtain a reduced-dimensional representation of each sample before performing the k-means clustering. in practice, one can select a few lowest dimensional principal components as the k-means input for each sample. in ref. [28] , the authors proved that the principal components are the continuous solution of the cluster indicators in the k-means clustering method, which provides us a rigorous mathematical tool to embed our high-dimensional data into a low-dimensional pca subspace. the rapid global transmission of coronavirus disease 2019 (covid-19) has offered some of the most heterogeneous, diverse, and challenging mutagenic environments to stimulate dramatic genetic evolution and response from severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this work provides the most comprehensive genotyping of sars-cov-2 transmission and evolution up to date based on 6156 genome samples and reveals five clusters of the covid-19 genomes and associated mutations on eight different sars-cov-2 proteins. we introduce mutation h-index and mutation ratio to qualify individual protein's degree of non-conservativeness. we unveil that sars-cov-2 envelope protein, main protease, and endoribonuclease protein are relatively the most conservative, whereas, sars-cov-2 nucleocapsid protein, spike protein, and papain-like protease are relatively the most non-conservative. we report an alarming fact that all of the sars-cov-2 proteins have undergone intensive mutations since january 5, 2020, and some of these mutations may seriously undermine ongoing efforts on covid-19 diagnostic testing, vaccine development, and drug discovery. the nucleotide sequences of the sars-cov-2 genomes used in this analysis are available, upon free registration, from the gisaid database (https://www.gisaid.org/). eighteen tables are provided in the supporting material for snp variants of 6156 sars-cov-2 samples across the world, snp variants of 1625 sars-cov-2 samples in the us, snp variants in five global clusters, snp variants in three us clusters, and mutation records for eight sars-cov-2 proteins. the acknowledgments of the sars-cov-2 genomes are also given in the supporting material. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 who. coronavirus disease 2019 (covid-19) situation report 93. coronavirus disease (covid-2019) situation reports a new coronavirus associated with human respiratory disease in china the sars-cov s glycoprotein: expression and functional characterization the coronavirus nucleocapsid is a multifunctional protein severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis the population genetics and evolutionary epidemiology of rna viruses origin and evolution of the 2019 novel coronavirus genotyping coronavirus sars-cov-2: methods and implications models of rna virus evolution and their roles in vaccine design detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr diagnosing covid-19: the disease and tools for ddtection structure, function, and antigenicity of the sars-cov-2 spike glycoprotein furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry a strategy for searching antigenic regions in the sars-cov spike protein coronavirus infections: epidemiological, clinical and immunological features and hypotheses structure of mpro from covid-19 virus and discovery of its inhibitors. biorxiv sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus the crystal structure of papain-like protease of sars cov-2 structure of the rna-dependent rna polymerase from covid-19 virus structural model of the sars coronavirus e channel in lmpg micelles crystal structure of rna binding domain of nucleocapsid phosphoprotein from sars coronavirus 2. center for structural genomics of infectious diseases (csgid) gisaid: global initiative on sharing all influenza data-from vision to reality clustal omega. current protocols in bioinformatics distance between sets k-means clustering via principal component analysis this work was supported in part by nih grant gm126189, nsf grants dms-1721024, dms-1761320, and iis1900473, michigan economic development corporation, bristol-myers squibb, and pfizer. the authors thank the ibm tj watson research center, the covid-19 high performance computing consortium, and nvidia for computational assistance. key: cord-256508-ce59ovan authors: asselah, tarik; durantel, david; pasmant, eric; lau, george; schinazi, raymond f. title: covid-19: discovery, diagnostics and drug development date: 2020-10-08 journal: j hepatol doi: 10.1016/j.jhep.2020.09.031 sha: doc_id: 256508 cord_uid: ce59ovan an epidemic of acute respiratory syndrome (covid-19) started in humans in wuhan in 2019, and became a pandemic. groups from china identified and sequenced the virus responsible for covid-19, named sars-cov-2, and determined that it was a novel coronavirus (cov) that shared high sequence identity with batand pangolin-derived sars-like covs, suggesting a zoonotic origin. sars-cov-2 is a member of coronaviridae, a family of enveloped, positive-sense, single-stranded rna viruses that infect a broad range of vertebrates. the rapid release of the sequence of the virus has allowed the development of diagnostic tools (e.g., rt-pcr). additionally, serological tests can allow identification of persons who have been infected. in humans, covs tend to cause mild to moderate upper respiratory tract infections. the fatality rate is around 1-3% for infected persons. an acute respiratory distress syndrome (ards) likely due to an uncontrolled immune activation (“cytokine storm”) occurs in patients with severe disease and poor prognosis. risk factors for mortality include: advanced age, obesity, diabetes, hypertension and other comorbidities. drug repurposing has been used to rapidly identify potential treatment for covid-19, which could move quickly to phase-3. better knowledge of the virus, its enzymes, will be mandatory to develop more potent and specific direct-acting antiviral agents (daa). in the long term, a vaccine to prevent infection would be crucial; however even if successful it might not be available before 2021-22. to date, with the exception of intravenous remdesivir and dexamethasone, which have modest effects in moderate to severe covid-19, no strong clinical evidence supports the efficacy and safety of any other drugs against sars-cov-2. the aim of this review is to provide insights on the discovery of sars-cov-2, its virology, the diagnostic tools, and the ongoing drug discovery effort. the world health organization (who) announced on march 11 th 2020, that the outbreak of "coronavirus disease 2019" (covid19) , which initially started in asia, has become pandemic. on september 4 th 2020 the etiologic agent "severe acute respiratory syndrome (sars)-cov-2 has spread all over the world with a global estimation of around 26 million confirmed cases and around 865,000 deaths [1] . the rapid availability of the virus rna genome sequence was instrumental in the development of diagnostic tools and for the identification of experimental treatments. in this review, we will clarify aspects related to the discovery of sars-cov-2, virological features, diagnostic tools, complex pathogenesis, including a focus on the liver and gastro-intestinal lesions, and of course drug discovery. the causative agent of covid-19 is a novel coronavirus (cov) officially named sars-cov-2. it was named after sars-cov-1, because of a genomic homology [2] . coronaviruses are enveloped, large positive-sense single-stranded rna viruses (+ssrna), gathered in the coronaviridae family. covs can infect a broad range of vertebrates, including bats, birds, pangolins, snakes, mice, and humans. due to sequence similarities with ratg13 bat and pangolin cov strains, it is currently thought that sars-cov2 has a zoonotic origin and has secondary acquired human-to-human spreading capacity [3] . in particular, the acquisition of i) mutations in the receptor binding area, ii) a polybasic furin cleavage site (rrrar) at the junction of subdomain 1 and 2 of the spike protein and iii) a site of o-linked glycosylation in the same area, has enabled the virus to efficiently interact with high affinity (via its spike protein) with its bona fide cellular receptor (angiotensin-converting enzyme 2 (ace-2)) [4] , to become more virulent and pathogenic, while potentially evading immune response by o-glycan epitope masking [3] . where does the virus replicate? following replication and sub-genomic rna synthesis, the viral structural proteins, are translated and inserted into the endoplasmic reticulum (er). these proteins move along the secretory pathway into the er-golgi intermediate compartment. in infected cells, the cov rna-synthesizing machinery associates with modified er membranes that are transformed into the viral replication organelle; double-membrane vesicles appear to be the central hub for viral rna synthesis [5] . otherwise, the duration of sars-cov-2 is significantly longer in stool samples than in respiratory samples [6] . disrupted cell membrane [9] . viral particles were observed in the epithelial cells by electron microscopy suggesting that these lesions might be partially caused by a direct cytotoxicity. a better understanding of the functions/roles of viral proteins, as well as of the virus replication cycle, with a particular attention on host-cell/virus interactions, will allow the identification of novel or a better characterization of targets for antiviral agent development. the success of drug development for hepatitis c virus (hcv) has inspired scientists to achieve the same for other viruses [10] . entry process. many cell types could express ace2 and transmembrane serine protease 2 (tmprss2), the two cellular factors important for virus entry [11] , including nasal and lower airway epithelial cells (pneumocytes), lung resident immune cells, endothelial cells, as well as neurons, enterocytes, cardiomyocytes, hepatocytes, kidney cells [12] [13] [14] . but the presence of mrna in these cell-types is not sufficient. study of the expression at protein levels of these entry factors, as well as the demonstration of bona fide viral entry and active replication in all these cell-types is yet missing. interestingly, it was very recently shown that ace2 is an interferon-stimulated-gene (isg) [15] , meaning that the presence of ifns in the microenvironment of the virus replication site could further enhance the spreading of the virus. the molecular details of the entry process, involving the spike protein and host receptor/coreceptor, have already been studied [16] . polybasic furin cleavage site at the junction of subdomain 1 and 2 of the spike protein, may explain the large number of cell types that may be infected by the virus with the consequent diverse organ manifestations, including, possibly the thrombotic complications as a result of possible endothelial cell infection. this research will facilitate the identification of neutralizing antibodies or small molecules, which could target this step of the life cycle. covs encode several enzymes that are crucial for the replication of the virus and ideal target for antiviral development, amongst which two proteases/proteinases (plpro and 3clpro), the rdrp, a helicase, an mrna-cap-methyltransferase, and an exoribonuclease. these enzymes have been well studied for sars-cov, and thanks to the high homology between the two sars covs, we could expect functional similarities and the possible repurposing of drugs [3] [4] 11] . the rdrp (also identified as nsp12) bears the main enzymatic activity of the replicase complex. recent advance in antiviral research against hcv [10, 17] , has confirmed that rdrp are major target for very specific antiviral discovery. similarly to hcv, sars-cov2 genome is characterized by a positive-sense single-strand rna and share a similar replication cycle requiring a rdrp. this polymerase displays resembling catalytic mechanisms and key conserved amino acids in the active site. the rdrp 3d structure has been readily characterized [18] [19] . interestingly it has a large nterminal extension containing a kinase-like fold. the polymerase domain, like hcv, is composed of three subdomains; a fingers subdomain, a palm subdomain, and a thumb subdomain. moreover, j o u r n a l p r e -p r o o f also the 3-chymotrypsin-like protease (3clpro) is vital to virus replication and the 3clpro cleavage sites are highly conserved, so it could be a promising drug target [18] . plpro and 3clpro/mpro are essential enzymes for the proteolytic processing of cov replicase polyprotein; their activities are needed very early in the infection process to step-by-step release other viral enzymatic activities. they are also attractive targets for specific antiviral discovery. sars-cov1 and sars-cov2 share high amino-acid sequence identities for these two proteases and 3d structures are also available. moreover biochemical assays are also available for functional testing, at least for the sars-cov proteins [20] . plpro is a cysteine protease, encoded by nsp3, and involved in the release of nsp1 to 3, as well as in the regulation of host innate immunity functions to allow viral escape [20] . although the similarity between plpro of sars-covs is not very high, the catalytic domain, around the triad cyst-his-asp, is well conserved; therefore drug already in the pipeline for sars-cov1 might be repurposed. 3ctpro/mpro is encoded by nsp 5, forms a functional homodimer, utilizes a catalytic dyad cys-his, and is involved in the release of nsp4 to 16 from polyprotein. its activity is key for cov replication-cycle and its inhibition very efficient at stopping viral replication. due to the dimeric nature of this protease, not only catalytic inhibitors, but also allosteric ones can be developed increasing possibilities of success. moreover the very high similarity of 3ctpro/mpro sars-covs allow repurposing of drugs [20] . some specific antiviral screenings have been promptly started on 3ctpro of sars-cov-2 and several drug candidates already identified [21] [22] . beside virologic aspects of covid-19, it is also important to better understand immunologic ones, as well as their mutual amplification responsible for an increased pathogenesis in patients. if the virus can be studied in cell culture model, immunological aspects of covid-19 can only be studied either in relevant animal model or during clinical studies, using patient samples. it is now rather well established that in patients with poor outcome there is an uncontrolled "cytokine storm", featuring a local and systemic production of pro-inflammatory cytokines such as il-6, tnf-α and il-1β [23] [24] [25] [26] [27] . recently, it has been reported that ace2 is a human interferon-stimulated gene (isg) in vitro using airway epithelial cells and extend the findings to in vivo viral infections. these data suggest that sars-cov-2 could exploit species-specific interferon-driven upregulation of ace2, a tissue-protective mediator during lung injury, to enhance infection [28] . more studies are needed to clarify the origin of this massive and uncontrolled cytokine production. covid-19 tests can be grouped as nucleic acid, serological, antigen, and ancillary tests, all of which play distinct roles in hospital, point-of-care, or large-scale population testing [29] . methods for the detection of viral nucleic acid. pcr tests for sars-cov2 have been available since january 2020. rt-qpcr-based assays performed on respiratory specimens has j o u r n a l p r e -p r o o f emerged as the cornerstone of covid-19 diagnostic testing. the usa centers for disease control and prevention (cdc) has developed a widely used sars-cov2 rt-qpcr assay [30] . the kit contains pcr primer-probe sets for two regions of the viral nucleocapsid gene (n1 and n2), and for the human rnase-p gene to ensure the rna extraction was successful. this assay differs from the who's one, which target sars-cov2 rdrp and e genes [31] . to avoid potential cross-reaction with other endemic covs, as well as potential genetic drift, at least two molecular targets should be included in the assay. evolution and potential mutations in sars-cov-2 genome strengthens the need to continue optimizing the oligonucleotides by global updated sharing of sars-cov-2 genomes [32] . theoretical specificity of most rt-qpcr assays is 100% because the primer design is specific to the sars-cov-2 genome. occasional false positive results may occur due to technical errors or reagent contamination [33] . a cycle threshold (ct) value of rt-qpcr less than 40 is generally interpreted as positive when results are interpreted as qualitative [34] [35] . quantitative interpretation of ct as indicators of the copy number of sars-cov-2 rna in specimens needs an appropriate standard curve with adequate limit of detection [36] . a rigorous assessment of the diagnostic accuracy of the many newly introduced sars-cov-2 assays is hampered [37] [38] . the sensitivity of viral rna testing varies with timing of testing relative to exposure. a false positive result erroneously labels a person infected, with consequences including unnecessary quarantine and contact tracing. false negative results are more consequential, because infected persons may not be isolated and can infect others. one modeling study estimated that the probability of a falsenegative result in an affected patient decreases from 100% on day 1 to 67% on day 4 [40] . on the day of symptom onset, the median false-negative rate estimation was 38%. sample pooling strategy was suggested to offer a viable alternative to detect community transmission at a time when tests are in short supply globally [41] [42] [43] . one potential limitation of pool testing is that the false-negative rate may increase owing to dilution of positive samples. point-of-care pcr kits can shorten the turnaround time for screening and diagnosing patients with suspected sars-cov-2. these rapid tests typically have lower throughput and are generally more expensive than other tests. time efficient methods that do not require thermal cycling have been designed and are evaluated [44] . crispr-cas12/cas13-based assay are also currently in development for point-of-care use [45] [46] . nature of samples tested. the current diagnostic strategy to identify patients with covid-19 is to test samples taken from the respiratory tract to assess for the presence of sars-cov-2 specific nucleic acid targets [47] . a nasopharyngeal specimen is the preferred choice for testing, but oropharyngeal, mid-turbinate, or anterior nares samples are also acceptable [48] . a pharyngeal virus shedding was shown to be very high during the first week of symptoms [49] . infectious virus was readily isolated from throat-and lung-samples, but not from stool ones. serum and urine were usually negative for the presence of viral nucleic acid [50] [51] . the viral load in nasopharyngeal samples peaks within the first few days after symptom onset, before declining [48, [51] [52] . for nasopharyngeal specimen, samples should be obtained by using a flocked swab to enhance the j o u r n a l p r e -p r o o f collection and release of cellular material [53] [54] . samples taken from sputum, endotracheal aspirates, and bronchoalveolar may have greater sensitivity than upper respiratory tract specimens [50] . inadequate sample collection may result in a false-negative test. bronchoalveolar lavage fluid specimens had the highest positive rates of sars-cov2 rt-qpcr assay [50] . a single nasopharyngeal swab has become the preferred swab, as it is well tolerated and safe. saliva may also be an alternative specimen source that requires less personal protective equipment and fewer swabs, but requires further validation [55] [56] . serologic testing. if rt-qpcr-based molecular assays for detecting sars cov-2 in respiratory specimens remain the current reference standard for diagnosis, point-of care technologies, and serologic immunoassays have also rapidly emerged [57] [58] [59] . serologic tests that identify antibodies to sars-cov-2 from clinical specimens may be less complex than molecular tests [60] . as antibody responses to infection take days to weeks to be reliably detected [60] , their utility for diagnosing acute infections is limited [48] . serologic assays might be more relevant in surveying for asymptomatic infection or in scenarios in which patients present to medical care with late complications of disease, when rt-qpcr may be falsely negative [55, 61] . seroconversion in most cases of sars occurs during the second week of symptoms [49] . for sars-cov-2 infection, timing of seroconversion appears similar to or slightly earlier than in sars-cov-1 infection [62] . in a study of 285 patients with covid-19, 100% of patients were tested positive for antiviral igg within 19 days after symptom onset, with seroconversion for igg and igm occurring simultaneously or sequentially [61] . negative results would not exclude covid-19 infection, particularly among those with recent exposure to the virus. the viral spike protein is perceived as the clear candidate for inclusion in an immunoassay that detects whether antibodies are present [58; 63] . the other protein that appears to be important antigen for the development of serological assays is the n protein (structural component of the nucleocapsid). indeed, antibodies to this protein are frequently detected in covid-19 patient [64] [65] , suggesting that n protein may be one of the immunodominant antigens in the early diagnosis of covid-19 [60, [66] [67] . it is now established that sars-cov-2 pre-existing immune reactivity can exist in the general population. serum samples from patients with covid-19 showed some cross-reactivity for the sars-cov-1 nucleocapsid antigens [61, [68] [69] . a recent study detected sars-cov-2-reactive cd4+ t cells in 50% of unexposed individuals, suggesting cross reactive t cell recognition between circulating ''common cold'' coronaviruses and sars-cov-2 [66] . t cell reactivity was highest against proteins other than the coronavirus spike protein, but t cell reactivity was also detected against spike. several monoclonal antibodies have been described that target the s glycoprotein of sars-cov-2 from memory b cells of an individual who was infected with sars-cov-1 in 2003 [68] . one antibody (s309) potently neutralizes sars-cov-2 by engaging the receptor-binding domain of the s glycoprotein. enzyme-linked immunosorbent assays (elisa) and clia are common laboratory platforms that can measure antibody titers (igg and igm). a variation of these tests can use magnetic, proteincoated microparticles, known as a chemiluminescent microparticle immunoassay. being able to quantify antibodies will be important for identifying convalescent plasma donors with abundant titers and studying how the immune system responds to the virus. neutralizing antibodies (nabs) play important roles in virus clearance and have been considered as a key immune product for protection or treatment against viral diseases. in covid-19, transfusion of convalescent plasma or serum from recovered patients was also considered as a promising therapy [71] . the neutralization assay is a laboratory-based test that uses live virus and cell culture methods to determine if patient antibodies can prevent viral infection in vitro [72] . because immunofluorescence assay is a labor-intensive method, a substantial number of the new commercial covid-19 antibody tests developed as screening tests are not elisa-based. they are lateral flow immunoassays (lfia), which provide no quantitative information. these qualitative lfia represent typically small, portable rapid diagnostic tests (rdt), and can be used at point of care. conclusions on serologic testing. antibody testing is ramping up quickly, with a growing list of commercial kits and test protocols from academic researchers [57] . many questions will have to be answered. the first and most urgent is the validation of serologic tests. a recent meta-analysis showed wide range sensitivities from 66% in lfias to 98% in the clias [73] ; sensitivities were higher with increased time after symptom onset. the specificities are excellent (99%). assays must be optimized further, independently validated, and used in an algorithm format to achieve the highest possible accuracy for decision making [74] [75] . second, there is insufficient data of the magnitude and duration of antibody responses after infections. although data suggest that neutralizing titers correlate with severity of infection [61] , it remains elusive, whether this effect is caused by ongoing somatic hypermutation or ongoing production of highly potent antibodies that were initially generated. moreover, any documentation that limits individual freedoms on the basis of biology risks becoming a platform for restricting human rights [76] . physiopathology. several potential pathogenic mechanisms may be involved including coagulopathy, endothelial dysfunction, and excessive release of pro-inflammatory cytokines. the endothelial dysfunction caused by infection activates an excessive thrombin generation and inhibits fibrinolysis, which designates hypercoagulability [77] . lymphopenia is frequent in patients with covid-19 [78] . the cytokine release syndrome could have a major role in patients with severe covid-19 as in acute respiratory distress syndrome (ards) [79] . the pathological features of covid-19 related ards are diffuse alveolar damage with hyaline membrane formation with fibrin deposition and a few multinucleated enlarged cells [7] [8] . in patients who died from covid-19associated respiratory failure, the histologic pattern in the peripheral lung was diffuse alveolar j o u r n a l p r e -p r o o f damage with perivascular t-cell infiltration [9] . the lungs also showed distinctive vascular features, consisting of severe endothelial injury, but also widespread thrombosis with microangiopathy. alveolar capillary microthrombi were frequent, with a high amount of new vessel growth (intussusceptive angiogenesis). transmission by asymptomatic carriers. several findings are consistent with person-to-person transmission of this novel coronavirus in hospital and family settings . a case of sars-cov-2 infection acquired outside asia in which transmission appears to have occurred during the incubation period [81] . furthermore, in a previously reported family cluster, some of the family members had positive rt-qpcr results without any symptoms [47] . clinical characteristics. among 1,099 patients from china with laboratory-confirmed covid-19, 5.0% were admitted to the intensive care units (icu), 2.3% underwent invasive mechanical ventilation, and 1.4% died [78] . the most common symptoms were fever and cough. the median incubation period was 4 days. in another study including 191 patients, with 54 who died in hospital, half of the patients had a comorbidity, with hypertension being the most common, followed by diabetes and coronary heart disease [52] . in-hospital death was associated with older age, higher sequential organ failure assessment (sofa) score, and d-dimer greater than 1 μg/ml on admission. in another study of the 1,591 patients infected with sars-cov-2 admitted to icu in italy, the median age was 63 years and 82% were male [82] . among 1,300 patients with available respiratory support data, 99% needed respiratory support, including 88% who received mechanical ventilation and 11% who received non-invasive ventilation. finally, in this case series of critically ill patients admitted to icus, the majority were older men and icu mortality was 26%. moreover, data from previous coronavirus infections such as severe acute respiratory syndrome and middle east respiratory syndrome, as well as emerging data from the covid-19 pandemic, suggest there could be substantial fibrotic consequences following sars-cov-2 infection [83] . imaging findings. the hallmarks of covid-19 were bilateral and peripheral ground-glass and consolidative pulmonary opacities [84] . notably, 56% of patients with early disease had a normal ct. with a longer time after the onset of symptoms, ct findings were more frequent, including consolidation, bilateral and peripheral disease, greater total lung involvement, linear opacities, "crazy-paving" pattern and the "reverse halo" sign. bilateral lung involvement was observed in 28% in early phase and 88% in late phase of the disease. ct scans at the time of symptoms may increase diagnosis rate since rt-pcr sensitivity may be as low as 60% [85] . also, chest x-ray findings in covid-19 patients frequently showed bilateral lower zone consolidation [86] . coagulopathy disorders. sars-covid-19-induced infection can be associated with a coagulopathy, findings consistent with infection-induced inflammatory changes as observed in patients with disseminated intravascular coagulopathy (dic) [87] . the initial coagulopathy of covid-19 presents with elevation of d-dimer and fibrin/fibrinogen-degradation products. covid-19-j o u r n a l p r e -p r o o f associated coagulopathy should be managed as it would be for any critically ill patient, using thromboembolic prophylaxis and standard supportive care measures for those with sepsis-induced coagulopathy or dic. current data do not suggest the use of high anticoagulation doses [87] . among all the numerous clinical manifestations associated with covid-19 infection, we can recall cardiological lesions with acute myocardial injury [88] ; neurological lesions with encephalitis and myalgia [89] [90] , cutaneous manifestations with rash and urticaria [91] , and acute kidney injury [92] . (figure 3) . elevation of liver enzyme occurs in 5 to 50 % of patients. the pattern of liver injury is mainly at hepatocellular rather than cholestatic level [93] [94] , with hepatocyte degeneration, focal necrosis, capillary bile duct cholestasis and inflammation in the portal area, but interestingly sars-cov-2 cannot be detected in samples [95] . frequently, the severity of liver injury had been correlated to the severity of covid-19. the presence of underlying chronic liver diseases could render covid-19 patients at higher risk of severe liver injury, such as acute-on-chronic liver failure [96] , with data suggesting that nafld/madld could be an independent risk factors for severe covid-19 [97] [98] . the virus was found in stool samples in around 50% of patients with covid-19, with around 18% of them complaining of abdominal pain and diarrhea [99] . it was demonstrated that sars-cov2 is capable to productively replicate in ace2-positive enterocytes [12] . due to the abundance of the virus in the small intestine, liver cell exposure through the hepatic reticular system is expected. hepatic default immune status might play a critical role in covid-19 infection. indeed, it has been shown that in patients with mafld, the polarization status of macrophage might be skewed due to metabolic stimuli such as fatty acids and thus affecting host-inflammatory response to signals generated from the gut-liver axis [97] . in covid-19, the "cytokine storm" bears resemblance to sars caused by the sars-cov-1, where cytokine storm has been associated with disease [100] [101] [102] . on the other hand, direct cytopathic damage by sars-cov-2 is also possible as there are entry receptors ace-2 in cholangiocytes [103] . also, learning from sars experience, the use of antibiotics, antivirals, together with possible secondary bacterial infection, might lead to liver injury in covid-19 patients [104] . moreover, tocilizumab is evaluated for the treatment of covid-19 patients with serious lung damage and accompanying elevated blood levels of il-6 [105] . prophylactic nucleoside analogs against hepatitis b virus had been recommended for those hepatitis b surface antigen positive covid-19 patients planned for immunosuppressive therapy [102] . liver damage, which lead to drug withdrawal, has been reported in patients treated with remdesivir. accordingly, it is not recommended for those covid-19 patients with alt> 5 times uln or with liver decompensation to receive remdesivir [106] . lastly, hypoxia and shock induced by covid-19related complications may also cause hepatic ischemia [107] . to manage liver injury related to covid-19, several guidelines have been issued [100] [101] [102] 108] . suffered from gastrointestinal symptoms such as nausea or vomiting, diarrhea and anorexia [109] , with similar incidence among adults and children [110] . patients with gastrointestinal symptoms may require longer duration of hospitalization [78] [79] 111] . in some patients, gastrointestinal without respiratory symptoms might be the presenting clinical features [112] [113] . the underlying mechanism may be related to the abundant expression of ace2 mrna and receptor protein in the enterocytes [112] [113] . histological changes with the presence of plasma cell and lymphocytes infiltration in patients' lamina propria of enterocytes suggested an immune-mediated response [114] . the capability of sars-cov2 to infect enterocytes has also been demonstrated in human intestinal organoids [12] . one of the major concerns of enteric infection is whether fecal source can lead to fomite transmission, especially when infective aerosols are generated from the toilet plume. indeed, cluster of cases infected with covid-19, in analogy to "amoy garden" during the sars in 2003, has recently been suggested in hong kong [115] . in accordance to the surface stability study on plastic and different materials, sars-cov-2 could remain viable up to 72 hours [116] . in one study, in 20% of patients, sars-c0v2 rna remained positive in feces despite clearance in the respiratory specimen [114] . taking together, it is of great importance that the presence of sars-cov-2 in the stool need to be determined for the epidemiology control of covid-19. there is a major concern with the potential concomitant infection of sars-cov-2 with influenza or other respiratory diseases such a respiratory syncytial virus, or tuberculosis or even bacterial infections or mycoplasma. co-infection with sars-cov-2 and influenza a virus in a patient with pneumonia has been reported in china [117] . covid-19 might be underdiagnosed because of false-negative tests for upper respiratory specimens or co-infection with other respiratory viruses. prevention and transmission control measures. washing hands frequently, using mask and social distancing are important. china banned travel to and from wuhan city on 23 january 2020, and this shutdown was associated with the delayed arrival of covid-19 in other cities by approximately 3 days [118] . suspending intra-city public transport, closing entertainment venues and banning public gatherings were associated with reductions in case incidence. early on, the spatial distribution of covid-19 cases in china was explained well by human mobility data [119] . following the implementation of control measures, this correlation dropped and growth rates became negative in most locations. a contact-tracing application, which builds a memory of proximity contacts and immediately notifies contacts of positive cases could achieve epidemic control if used by enough people [120] . much like with influenza, antiviral drugs to be effective likely need to be started early in infection course. in turn, this represents a burden to identify drugs that are indeed effective against the virus in clinical trials. patients with early disease may benefit from antiviral agents to reduce viral load, patients with severe and late disease may benefit from anti-inflammatory j o u r n a l p r e -p r o o f drugs. furthermore, in the beginning of the disease, anti-inflammatory drugs might be harmful by increasing viral load. drug repurposing. drug repurposing (also called drug repositioning or reprofiling) is a strategy for identifying new uses for approved or investigational drugs that are outside the scope of the original medical indication [121] . this strategy offers various advantages over developing an entirely new drug, with a reduction risk of failure because safety has already been evaluated. but also the time frame and the cost can be reduced, because most of the preclinical testing and safety assessment have been done. an extensive repositioning activity of approved drugs has been embarked for the covid pandemic. a selection of drugs being tested for covid-19 are represented in table 1 . for example of large randomized ongoing trial the design of "solidarity", is provided in table 2 . has also been combined with azithromycin, an antibiotic. hcq would inhibit sars-cov-2 entry into cells. few data are coming from reports and small studies [122] . a systematic review on the efficacy and safety of hcq for the treatment of covid-19 concluded that there is currently no evidence from rcts to inform on hcq efficacy [123] . in a multicenter, open label, randomized controlled trial, 150 patients admitted to hospital with laboratory confirmed covid-19 were included in the intention to treat analysis (75 patients assigned to hcq plus standard of care (soc), 75 to soc [124] . there was no difference in term of efficacy between the 2 arms. adverse events were higher in hcq recipients than in non-recipients. lopinavir is an antiretroviral protease inhibitor used in combination with ritonavir in hiv therapy, which has shown some antiviral activity against sars-cov [125] . a randomized, controlled, openlabel trial involving hospitalized adult patients with confirmed sars-cov-2 infection and severe respiratory illness covid-19 was performed [126] . patients were randomly assigned to receive either lopinavir-ritonavir, in addition to soc, or soc alone. there were no differences between groups (virologic aspects, duration of disease, mortality), indicating that there is no benefit in hospitalized adult patients with severe covid-19. cell culture data suggest that this compound demonstrates activity with an ec 50 of 26.6 µm [127] . one wonders why it was selected for clinical trials with such a weak activity. evaluating in humans repurposed drugs that are essentially ineffective in culture against sars-cov-2 is being repeated over and over again wasting time and resources. remdesivir is a prodrug of a nucleotide analog that is intracellularly metabolized to an analogue of adenosine triphosphate that inhibits viral rna polymerases. remdesivir has broad-spectrum activity against members of several virus families, including filoviruses (e.g., ebola) and coronaviruses (e.g., sars-cov and mers-cov [128] . six large studies are ongoing ( table 3) . unfortunately remdesivir must be given intravenously for at least 5 days, although an aerosol formulation is being developed. were reported [129] . from the 53 patients whose data were analyzed, clinical improvement was observed in 36/53 patients (68%). a randomized, double-blind, placebo-controlled, multicenter trial was performed in china [106] . mortality at day 28 was similar between the two groups (14% died in the remdesivir group vs 13% in the placebo group). there was no difference in the two groups regarding clinical improvement and viral load decreased. this trial did not attain the predetermined sample size because the outbreak of covid-19 was brought under control in china, therefore, it is difficult to reach a definitive conclusion. gilead is conducting two randomized, open-label, multicenter, phase 3 clinical studies to evaluate the safety and efficacy of two dosing durations -5 days and 10 days -of remdesivir in adults diagnosed with covid-19 (the simple studies). the first simple study is involving hospitalized patients with confirmed sars-cov-2 infection, oxygen saturation of 94% or less while they were breathing ambient air, and radiologic evidence of pneumonia [130] . in the second simple study patients were randomized to receive open-label remdesivir for 5 or 10 days or soc alone. at day 11, a higher proportion of patients in the 5-day treatment group achieved improvement in clinical status versus the soc group, achieving statistical significance for a ≥ 1-point improvement in ordinal scale (p=0.026)(gilead press release). however, most clinician would have preferred to see a decrease in mortality on treatment. clearly another controlled study will have to be performed soon. among other antiviral drugs being tested for covid-19 we can quote arbidol [131] [132] , favipiravir [133] , famotidine [134] [135] , and camostat (tmprss2 inhibitor) [11] . dexamethasone. glucocorticoids may modulate inflammation-mediated lung injury and thereby reduce progression to respiratory failure and death. in a controlled, open-label trial of patients hospitalized with covid-19, patients were randomly assigned to receive oral or intravenous dexamethasone (6 mg once daily) for up to 10 days or to receive soc alone [136] . in the dexamethasone group, the incidence of death was lower than that in the soc group among patients receiving invasive mechanical ventilation (29.3% vs 41.4%) and among those receiving oxygen without invasive mechanical ventilation (23.3% vs 26.2%) but not among those who were receiving no respiratory support at randomization (17.8% vs 14.0%). in a recent trial involving patients with ards who were undergoing mechanical ventilation, mortality at 60 days was 15 percentage points lower among those receiving dexamethasone than among those receiving soc [137] . in the early phase of the infection, anti-inflammatory drugs may not be efficient (maybe harm-full) increasing viral load. viral shedding in sars-cov-2 appears to be higher early in the illness and declines thereafter [49, 54, 138] . the greater mortality benefit of dexamethasone in patients with covid-19 who are receiving respiratory support and among those recruited after the first week of their illness suggests that at that stage the disease may be j o u r n a l p r e -p r o o f dominated by inflammation, with active viral replication playing a secondary role. clearly a trial of the combination remdesivir and dexamethasone may yield interesting results. early triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate covid-19. future clinical study with interferon beta-1b as a backbone is warranted [139] . tocilizumab (actemra), also known as atlizumab, and sarilumab (kevzara) are both immunosuppressive drugs, mainly for the treatment of rheumatoid arthritis. they are both humanized monoclonal against the interleukin-6 receptor (il-6r), and are given by injection. clinical trials are ongoing. moreover other monoclonal antibodies or agents targeting other inflammatory cytokines (tnf-α, il-1β...) should be soon tested. kinase inhibitors. the janus activating kinase (jak)-signal transducer and activator of transcription (stat) pathway has been implicated as a key driver in many inflammatory diseases. with the onset of small molecule inhibitors that can selectively and specifically target key jaks involved in controlling downstream inflammation, exploration for their utility across a broad range of diseases has become a rapidly expanding field [139] [140] , including viral infections (eg. hiv; [141] [142] [143] ). baricitinib and ruxolitinib are two known jak inhibitors. recently, artificial intelligence enabled the identification of a group of approved drugs that could inhibit clathrin-mediated endocytosis and thereby inhibit viral infection of cells [144] [145] . the drug targets are members of the numbassociated kinase (nak) family. baricitinib was identified as a nak inhibitor, with a particularly high affinity for aak1, a pivotal regulator of clathrin-mediated endocytosis. this drug is also known to target jak and could have a dual action against the virus and inflammation [146] . the nih/niaid sponsored actt-2 study is still ongoing and compares remdesivir to remdesivir plus baricitinib in moderate to severe patients. in a small uncontrolled cohort of veterans affairs patients with moderate-severe covid-19, treatment with baricitinib plus hydroxychloroquine was associated with recovery in 11 of 15 patients [147] . two other kinase inhibitors, namely imatinib mesylate and dasatinib, could also be envisaged to treat covid-19 [148] . furthermore, ruxolitinib (another jak inhibitor-incyte) is being evaluated in a multicenter phase-ii clinical trial [149] . convalescent plasma. the immediate use of convalescent plasma provides a promising treatment. in a preliminary uncontrolled case series of 5 critically ill patients with covid-19 and ards, administration of convalescent plasma containing neutralizing antibody was followed by improvement in their clinical status [71] . the limited sample size of this study precludes a definitive statement about the efficacy of this treatment. vaccines are the most effective strategy for preventing infectious disease since they reduce morbidity and mortality, and they are more cost-effective than treatment. despite previous coronaviruses epidemics, there is still no approved vaccine for human coronaviruses [150] . we will have to improve our understanding and knowledge regarding immune response to sars-cov2. interestingly, in rhesus macaques, comparing the humoral and cellular immunity between primary infection and re-challenge revealed notably enhanced neutralizing antibody and immune responses [151] . these results suggest that primary sars-cov-2 exposure protects against subsequent reinfection in rhesus macaques. in human, in a large study of the icelandic population observed that humoral response did not decline within 4 months after infection, that 44% of persons who had been infected had not been diagnosed with pcr, and that the fatality rate was 0.3% [152] . we have also to recall that few cases of sars-cov-2 re-infection were reported. epidemiological, clinical, serological and genomic analyses confirmed that the patient had reinfection instead of persistent viral shedding from first infection [153] . this case lead to several open questions: how frequent is reinfection? are reinfections less severe than the first? will vaccine protect against reinfections? these results suggest sars-cov-2 may continue to circulate among the human populations despite herd immunity due to natural infection or vaccination. further studies of patients with re-infection will shed light on protective correlates important for vaccine design. regarding vaccine development, among the different strategies, we can recall the use of recombinant subunit vaccine, dna or mrna vaccine. subunit vaccines are believed to be highly safe because they are expected to induce the immune system without introducing infectious viruses [154] . a better knowledge of sars-cov-2 s or/and n protein organizations will be require to develop such vaccines. the sars-cov-2 spike (s) glycoprotein mediates host cell attachment and is required for viral entry; it is the primary vaccine target for many candidate sars-cov-2 vaccines. dna vaccines are based on direct injection of plasmids encoding the antigens, followed by a large range of immune responses. mrna-based vaccines contain mrnas encoding the antigens, which are translated at the host cellular machinery by vaccination [155] . mrna vaccines have advantages over conventional vaccines, by the absence of genome integration, the improved immune responses, the rapid development, and the production of multimeric antigens [155] [156] . preliminary report of an mrna vaccine against sars-cov-2 was published [157] . the candidate [158] . long-term assessment will be relevant given that natural history studies suggest that sars-cov may not generate long-lived antibody responses [159] . furthermore safety evaluation will be mandatory, since there have been concerns about the potential for vaccineassociated enhanced respiratory disease. of the three doses evaluated, the 100-μg dose elicits high neutralization responses and th1-skewed cd4 t cell responses, coupled with a reactogenicity profile that is more favorable than that of the higher dose. the results of two early phase covid-19 vaccine trials were reported, one at oxford university (uk), with support from astrazeneca [160] , and the second supported by cansino biologics in china [161] . both groups used an adenoviral vector, and both report the vaccine achieving humoral responses to the sars-cov-2 spike glycoprotein receptor binding domain by day 28 as well as tcell responses. both report local and systemic mild adverse events such as fever, fatigue, and injection site pain. in neither trial was a severe adverse event reported. although these preliminary data are encouraging sars-cov-2 is a novel pathogen in humans, and many of the technologies being used to build vaccines are relatively untested. there is still a long way to go and phase 3 trials of these vaccines will require thousands of subjects in order to confirm efficacy and safety. the rapid sequencing of the virus has allowed the development of diagnostic tools. table 4 summarize future directions. a test and trace programs will be essential. later, "test, trace and treat (t3)" programs will become mandatory once effective drugs would have been identified and safe therapies developed (figure 4) . several issues will be important to understand. it will be important to precise how transmissible and pathogenic is sars-cov-2 in the ongoing and future epidemic. furthermore, it will be important to improve diagnostic tools. ideally a single or combined test that provides virological and serological output would be ideal. in many countries, at the end of containment, strict recommended measures will be important to avoid new waves of contamination. however, few innovative treatment modalities have been discovered since the bulk of the effort to date has been focused on a vaccine. vaccines might not be enough to quell this pandemic. open-reading-frames (orfs), allowing structural, non-structural and accessory viral protein synthesis [87] . sars-cov2 is 29,903 base-long and contains 6 majors orfs, as well as additional accessory genes; the reference sequence is registered in genbank with id: mn908947.3 [1] . (a and b) up to 28 different polypeptides are potentially produced in fine from the different orfs and after polyprotein processing by viro-encoded proteases [87] . if the rna genome contained in virions can already serve, after cell entry, as a template for the synthesis of non-structural proteins, which are to achieve sars-cov-2 elimination there will be a need to improve protection, testing, treating and preventing strategies. a test and trace programs will be essential. later, test, trace and treat (t3) programs will become mandatory once effective and safe therapies are developed j o u r n a l p r e -p r o o f immunoassays: detection/quatification of seroconversion: patient igm and igg specific to sars-cov-2 spike or nucleocapsid proteins. -neutralization assay: quantitative information on antibodies able to inhibit virus growth ex vivo, -enzyme-linked immunosorbent assay (elisa): quantification of antibodies specific to the virus, -immunochromatography assay: qualitative lateral flow assay (rapid diagnostic test) : detection of antibodies specific to the virus. rapid diagnostic tests (rdt): -works with venous whole blood, serum, or plasma; -rapid test (15 min); no instrument required, -only qualitative: aid in the screening and diagnosis in combination with rt-pcr, -aid in risk stratification, and cohort study. who covid-19 situation report 181 a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a unifying structural and functional model of the coronavirus replication organelle: tracking down rna synthesis viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province, china pathological findings of covid-19 associated with acute respiratory distress syndrome a novel coronavirus from patients with pneumonia in china pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 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sars-cov-2 infection protects against rechallenge in rhesus macaques disappearance of antibodies to sarsassociated coronavirus after recovery safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial immunogenicity and safety of a recombinant adenovirus type-5-vectored covid-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial define mechanisms determining establishment of sars-cov-2 infection: characterize all steps of the virus replication cycle 2. define structure and function of the sars-cov-2 enzymes and their interractions 3. understand physiopathology and immune response 4. improve methods for study of the replication cycle and virus-host interactions to reveal new targets for therapeutic approaches develop and validate diagnostic tools improving sensibility and specificity (serology, rapid diagnostic test) supported in part by nih grant r01-ai-141327, the center for aids research/nih grant p30-ai-050409 and national science foundation grant 2032273 (to rfs).j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f key: cord-271243-8cfyen86 authors: xiao, y.; meng, q.; yin, x.; guan, y.; liu, y.; li, c.; wang, m.; liu, g.; tong, t.; wang, l.-f.; kong, x.; wu, d. title: pathological changes in masked palm civets experimentally infected by severe acute respiratory syndrome (sars) coronavirus date: 2008-05-31 journal: journal of comparative pathology doi: 10.1016/j.jcpa.2007.12.005 sha: doc_id: 271243 cord_uid: 8cfyen86 summary masked palm civets are highly susceptible to infection with the severe acute respiratory syndrome coronavirus (sars-cov). infected animals become less aggressive and develop pyrexia, lethargy and diarrhoea. the present study describes the spectrum of histopathological changes in the lung, spleen, lymph node, liver, small intestine, kidney and cerebrum of civets infected experimentally with sars-cov. in-situ hybridization (ish) with probes specific for the rna polymerase gene demonstrated viral rna in the lung, small intestine and cerebrum only. in-situ labelling was employed in order to demonstrate cellular apoptosis in the cerebrum, but there was no evidence of apoptosis within the myocardium. these results indicate that sars-cov causes multi-organ pathology in civets, similar to that observed in human sars patients. these parallels suggest that civets may be used as an animal model of this infection to gain insight into the pathogenesis of sars and for evaluation of candidate vaccines and antiviral drugs. the aetiological agent of this syndrome was a newly emerged and previously unrecognized coronavirus, now known as sars coronavirus (sars-cov) ksiazek et al., 2003) . sars is an acute pulmonary syndrome characterized by atypical pneumonia, progressive respiratory failure and death in up to 10% of infected individuals (poon et al., 2004) . although the sars epidemic has subsided, many authorities, including the world health organization (who) and the us centers for disease control and prevention (cdc), have warned of the possible re-emergence of this highly infectious disease. it is therefore imperative that effective measures to prevent and treat the disease are developed and evaluated. to achieve this goal, animal models will play an essential role in studying the pathogenesis of sars-cov infection and in developing effective vaccines and therapeutics. a wide range of animal species has been confirmed to be susceptible to experimental infection with sars-cov, including rodents (mice and hamsters), carnivores (ferrets and cats) and non-human primates (wang et al., 2006) . adult mice infected with sars-cov via the respiratory tract show no clinical signs of disease and only mild respiratory tract inflammation . aged mice, hamsters and ferrets do show signs of clinical disease such as weight loss and ruffled fur but do not develop lung pathology (martina et al., 2003; roberts et al., 2005a,b) . two groups of investigators have studied sars-cov infection in african green monkeys and common marmosets mcauliffe et al., 2004; greenough et al., 2005) . others have evaluated cynomolgus monkeys and rhesus macaques as potential models, but clinical disease is inconsistently induced in these latter species mcauliffe et al., 2004; rowe et al., 2004) . our laboratory has evaluated the suitability of guinea-pigs, hamsters, albino hamsters, chickens and rats as experimental models following inoculation of sars-cov strain bj01. no clinical signs or tissue histopathological changes were observed in any of these animals post-infection. by contrast, all cynomolgus monkeys and rhesus macaques inoculated in this manner developed interstitial pneumonia of variable severity but no other tissue changes . this pulmonary pathology was similar in nature to that seen in sars patients, but the lesions were less severe than in infected humans. therefore, there remains a requirement for an animal model of human sars that closer approximates the tissue pathology that occurs in the human disease. in october 2003 it was reported that sars-covlike viruses had been isolated from masked palm civets from a live animal market in guangdong, china (guan et al., 2003) . this report prompted us to examine the feasibility of using civets as a better animal model for sars. in a preliminary infection study, two groups of civets were inoculated with two different strains of sars-cov. strain gz01 was isolated during the early stages of the sars epidemic and strain bj01 was isolated during the middle phase of the epidemic (wu et al., 2005) . both strains were shown capable of infecting civets and inducing clinical signs including pyrexia, lethargy and loss of aggression (wu et al., 2005) . the aim of the present study was to extend these observations by characterizing the tissue pathology in the infected civets and determining the distribution of viral rna in tissues from those animals. in addition, analysis of cellular apoptosis in selected tissues was also conducted in an attempt to understand the pathogenesis of this viral infection in civets and to further determine the suitability of this species to model the human disease. the animals and groups used in this study correspond exactly to those described in a previous publication (wu et al., 2005) . sars-cov isolates were propagated in vero e6 cells for two additional passages to generate virus stocks with titres of 1 â 10 6 50% tissue culture infective doses (tcid 50 )/ml. ten one-yearold masked palm civets were housed in individual biosafety isolators and were divided into two groups (n ¼ 5 per group). animals in groups a and b were inoculated with 3 ml of virus solution containing 3 â 10 6 tcid 50 of bj01 and gz01 isolates, respectively, with 2 ml instilled into the trachea and 1 ml given intranasally. a control civet was mock-infected in an identical fashion with 3 ml of vero e6 cell culture supernatant. all work with infectious virus was performed inside a biosafety cabinet, in an approved animal biosafety level 3 laboratory. serology and polymerase chain reaction (pcr) analysis were conducted to confirm that the civets had not been previously exposed to sars-cov. animal experiments were conducted in accordance with the animal ethics guidelines and approved protocols issued by the harbin veterinary research institute, chinese academy of agricultural sciences. one animal from each group was sacrificed at 3, 13, 23, 34 and 35 days post-infection (dpi), and lung, spleen, lymph node, small intestine, kidney, trachea, cerebrum, pancreas, sex glands, stomach and heart were collected from each animal. these samples were fixed in 10% neutral buffered formalin and embedded in paraffin wax. sections taken from these blocked samples were stained by haematoxylin and eosin (he). two 3-digoxigenin-labelled oligonucleotide probes (probe 1: 5 0 -acc ctg cta aag cat ata agg att acc tag-3 0 ; probe 2: 5 0 -caa tgg ctg att tag tct atg ctc tac gtc-3 0 ), which target the rna polymerase gene of sars-cov, were purchased from invitrogen (carlsbad, california, usa). ish was performed on the full range of tissues collected from each animal with ready-to-use reagents purchased from boster biological technology co. ltd. (wuhan, china) following the manufacturer's instructions. briefly, tissue sections were de-waxed in xylene and rehydrated in gradient ethanol. endogenous peroxidase activity was quenched by incubation in 3% h 2 o 2 . the tissues were digested with 3 mg/ml pepsin in 0.14 m citric acid at 37 c for 20 min and incubated at 37 c for 4 h with pre-hybridization buffer containing 35% deionized formamide, 5 â standard saline citrate, 2% blocking reagent, 0.1% n-lauroylsarcosine, 0.02% sodium dodecyl sulphate and 100 ng/ml salmon sperm dna. the tissue sections were hybridized with digoxigenin-labelled oligonucleotide probes (10 mg/ml) at 37 c for 18 h. the slides were rinsed in a series of graded salt solutions (â2, â0.5 and â0.2 standard saline citrate). after blocking at 37 c for 30 min, the sections were incubated with biotin-labelled mouse anti-digoxigenin antibody at 37 c for 60 min, followed by incubation with avidin-peroxidase conjugate at 37 c for 20 min. the signals were developed using the substrate diaminobenzidine tetrahydrochloride (dab) and slides were counterstained with mayer's haematoxylin. the same organs from the uninfected control civet were used as negative controls. cerebrum and heart from all infected animals and one uninfected control animal were used for detection of cellular apoptosis. apoptotic cells were identified using an in-situ cell apoptosis detection kit (boster biological technology co. ltd.) according to the manufacturer's instructions. briefly, cerebral and cardiac tissues embedded in paraffin wax were de-waxed in xylene and rehydrated in gradient ethanol. endogenous peroxidase activity was quenched in 3% h 2 o 2 for 10 min. the sections were washed three times, 5 min for each wash, with tris-buffered saline (tbs; 0.01 m tris, 0.15 m nacl, ph 7.4) after digestion with proteinase k. the sections were exposed to biotin-16-dutp and terminal deoxynucleotidyl transferase in labelling buffer (1 in 10) at 37 c for 2 h, followed by the same washing process as described above. after blocking at 37 c for 30 min, the sections were incubated with anti-digoxigenin antibody and avidin-peroxidase conjugate, followed by signal development using identical procedures to those described above. as previously described (wu et al., 2005) , from 3 dpi, all infected animals became lethargic and less aggressive. febrile episodes also commenced around 3 dpi and temperatures remained elevated for up to 7 days. diarrhoea developed between 3 and 14 dpi in infected civets. leucopenia was also observed, with white blood cell counts reaching a minimum at approximately 3 dpi and returning to normal from 13 dpi onwards. the uninfected control animal remained clinically normal throughout the experiment. no gross pathological changes were observed in any of the infected animals or in the uninfected control animal. in comparison to the control animal, various histopathological changes were observed in civets infected with sars-cov and these were similar in the animals infected with the two different sars-cov isolates. the histopathological changes for different organs are summarized below. lungs. at 3 dpi, acute interstitial inflammatory infiltrates and oedema were evident and there was congestion of the alveolar septae. the lumina of alveoli and bronchioles were variably filled with proteinrich oedema fluid, erythrocytes, cellular debris and lymphocytes (fig. 1a) . at 13 dpi, the pathological changes remained severe. the alveolar septae were thickened with infiltration of macrophages and lymphocytes and congestion of vessels (fig. 1b) . the lesions of interstitial pneumonia were even more distinct at 23 dpi, but by 34 and 35 dpi only foci of interstitial pneumonia remained. spleen. at 3 dpi there was extensive patchy necrosis of the splenic tissue, with atrophy of white pulp lymphoid aggregates (fig. 1c) . the atrophy of white pulp decreased in severity from 13 dpi but was still evident at 35 dpi. lymph nodes. at 13 dpi there was depletion of lymphoid follicles within the lymph nodes (fig. 1d) and this change was also observed at 23 dpi. liver. at 3 dpi there was diffuse congestion of the liver (fig. 1e) . focal aggregations of lymphocytes were present in hepatic lobules and portal areas at days 13 and 23 dpi. at later time points there were no microscopical abnormalities in the liver. cerebrum. within the cerebrum there was evidence of neuronal degeneration and mild neuronophagia from 3 to 13 dpi (fig. 1f ). in two cases there was mild oedema around the small veins and nerve cells. no microscopical changes were observed in this tissue after 13 dpi. kidneys. there was focal haemorrhage within the renal cortex at 3 dpi, but no changes were found after 23 dpi. small intestine. mild focal haemorrhage was observed in the lamina propria of the small intestine at 13 dpi. localization of viral rna ish was employed to locate sars-cov in tissue sections collected from infected civets. as expected, the tissue sections from organs of the control civet were negative for ish. positive signals were detected in the lungs of infected animals at 3 dpi in both groups. in these animals sars-cov was also isolated by cell culture and the presence of viral rna was confirmed by reverse transcriptase polymerase chain reaction (rt-pcr; table 1 and wu et al., 2005) . positive signals were also found in the small intestine (3 dpi in both groups) and cerebrum (3 and 13 dpi in both groups). these signals were localized to the cytoplasm of pneumocytes lining the alveolar septae and macrophages within the alveolar spaces (fig. 2a) . in the small intestine, the positive signal was mainly localized to macrophages (fig. 2b) . in the cerebrum, positive signals were mainly associated with glial cells at 3 dpi (fig. 2c ) and neurons at 13 dpi (fig. 2d) . no positive signal was found in any other tissue (table 1) . at 3 dpi, glial cells of the cerebrum in both groups of infected animals underwent apoptosis (fig. 3a) and at 13 dpi there was also evidence of neuronal apoptosis (fig. 3b) . no apoptosis was noted in the myocardium at 3 dpi. by 13 dpi, no apoptosis was found either in the myocardium or in the cerebrum (table 1) . the causative agent of sars has been identified as a new coronavirus not previously endemic in humans . this finding is supported by the lack of serological evidence of previous infection in healthy humans, which suggests that sars-cov has recently emerged in the human population. it has been proposed that animal-to-human interspecies transmission is the most probable explanation for emergence of the sars-cov. recent epidemiological investigations by two independent groups suggest that bats may be a natural reservoir of sars-cov. bats are among the live animals sold in markets in southern china, providing a possible explanation for the occurrence of infection among civets and other animals in these markets (lau et al., 2005; li et al., 2005a) . the isolation of sars-cov from palm civets was a significant finding in identifying this species as a source of the human sars-cov, but its role as a potential natural reservoir has not been established (guan et al., 2003) . in one study it was shown that sars-cov genome sequences from civets underwent rapid evolution before transmission to humans, suggesting that civets were a relatively new host to those viruses, rather than a natural reservoir host that had adapted to sars-cov (song et al., 2005) . in another study it was shown that while approximately 80% of civets in one market in guangzhou had serological evidence of infection by sars-cov, animals on farms were largely free from infection. this suggested that infection of civets was associated with trading activities under conditions of overcrowding and mixing of various animal species, which provided an environment for efficient interspecies transmission (tu et al., 2004) . furthermore, it has been shown that the s proteins of sars-cov isolated from human patients in the early stage of the outbreaks bound to and utilized the civet ace2 receptor molecule, but s proteins of the civet isolates used human ace2 markedly less efficiently (li et al., 2005b) . these findings provide evidence that civets have served as an efficient amplification host for sars-cov and that the sars-cov has undergone rapid evolution after transmission to the human population. while it is now commonly believed that civets are unlikely to be the natural reservoir of sars-cov, the high susceptibility of this species to infection and the efficient amplification of sars-cov during the sars outbreaks remain focal points for further investigation. it is important to know why civets display higher susceptibility to infection by sars-cov in table 1 detection of sars-cov and apoptosis in tissue from infected civets days post-infection tissue examined the market environment and whether civets have similar clinical and pathological changes as humans post-infection. in the present study, multi-organ pathological changes were found in experimentally infected civets. the lungs were the main target organs, but changes were also identified within the spleen, lymph node, cerebrum, liver, kidney and small intestine. interstitial pneumonia and diffuse alveolar damage were observed in lungs throughout the experiment. these lesions were similar to those described for sars-cov-infected macaques kuiken et al., 2003; qin et al., 2005) , but were milder than in humans with sars (ding et al., 2003; franks et al., 2003; nicholls et al., 2003; tse et al., 2004) . the observed follicular depletion in lymph nodes and atrophy of splenic white pulp also occur in human sars (zhao et al., 2003) . we have also demonstrated the presence of sars-cov rna in the cytoplasm of alveolar epithelia, macrophages infiltrating the pulmonary interstitium, macrophages of the small intestine and glial cells and neurons of the cerebrum. this distribution is again similar to that observed in human sars patients (ding et al., 2004; to et al., 2004; nicholls et al., 2006) . there is currently little information available regarding the distribution of sars-cov rna in other experimental animals with the exception of detection of viral rna in bronchiolar epithelial cells of mice at 2 dpi . in-situ apoptosis analysis revealed that for two civets, glial cells and neurons in the cerebrum displayed apoptosis at 3 and 13 dpi, respectively. this finding suggests that sars-cov may cause direct damage to those cells, providing useful information for future study of disease pathogenesis. the same set of probes and methods were applied to sars-cov infected monkey tissues obtained from previous studies, but no positive results were obtained, suggesting that civets and monkeys respond differently to sars-cov infection. in summary, the data presented in this study further corroborate previous findings (wu et al., 2005) in demonstrating that civets are more susceptible to sars-cov infection than other 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acute respiratory syndrome coronavirus infection of golden syrian hamsters cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) antibodies to sars coronavirus in civets. emerging infectious diseases review of bats and sars civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates clinical pathology and pathogenesis of severe acute respiratory syndrome we thank qingyu zhu of the institute of microbiology and epidemiology, academy of military medical sciences, beijing, china, for providing sars-cov isolates bj01 and gz01 and the vero e6 cell line. this project was funded by a special grant for identifying the animal reservoir of the sars-cov-like virus (2003a02) from the ministry of science and technology of china. key: cord-265366-vmuqbpkk authors: leibowitz, jill; krief, william; barone, stephen; williamson, kristy a.; goenka, pratichi k.; rai, shipra; moriarty, shannon; baodhankar, prachi; rubin, lorry g. title: comparison of clinical and epidemiologic characteristics of young febrile infants with and without sars-cov-2 infection date: 2020-10-09 journal: j pediatr doi: 10.1016/j.jpeds.2020.10.002 sha: doc_id: 265366 cord_uid: vmuqbpkk objective: to determine features that distinguish febrile young infants with sars-cov-2 infection. study design: retrospective single-center study included febrile infants <57 days evaluated in the emergency department of cohen children’s medical center of northwell health, new hyde park, new york during march 1-april 30 of 2018, 2019, and 2020. sociodemographic and clinical features were compared between those seen during the 2020 covid-19 pandemic and previous years, as well as between sars-cov-2 infected infants and sars-cov-2 uninfected infants (sars-cov-2 negative or evaluated during 2018 and 2019). results: in all, 124 febrile infants <57 days of age were identified; 38 during the 2-month study period in 2018, 33 in 2019, and 53 in 2020. during 2020, fewer febrile infants had a serious bacterial infection (sbi) or a positive respiratory viral panel (rvp) than in prior years (6% versus 21%, p = .02; 15% versus 53%, p<.001, respectively). sars-cov-2 was the most frequent pathogen detected in 2020; of 30 infants tested, 20 tested positive. infants with sars-cov-2 were more likely to identify as hispanic (p=.004), have public insurance or were uninsured (p=.01), exhibited lethargy (p=.02), had feeding difficulties (p=.002), and had lower white blood cell (p=.001), neutrophil (p<.001), and lymphocyte counts (p=.005) than the 81 infants without sars-cov-2 infection. none of the infants with sars-cov-2 had concurrent sbi or detection of another virus. overall, disease in infants with sars-cov-2 was mild. conclusions: during the peak of the pandemic, sars-cov-2 was the predominant pathogen among febrile infants. socioeconomic, historical, and laboratory features differed significantly between sars-cov-2 infected and uninfected infants. none of the 20 infants with sars-cov-2 infection had an identified co-viral or serious bacterial infection. based on data from the centers for disease control and prevention through july 2020, infants < 3 months of age accounted for 18.8% of hospitalized pediatric covid-19 patients in the us, and children younger than one year of age accounted for 10% of fatalities associated with sars-cov-2 in us children, however, the proportion of children younger than 2-3 months of age was not specified. 4, 5 although an early study from wuhan, china reported 10.6% of covid-19 cases in children less than one year of age to be critical or severe, of these cases, not all were laboratory confirmed sars-cov-2 infection and other viral etiologies of illness were not excluded. 6 since then, a number of case reports and small series describing infants less than 2 months of age have been published, most reporting mild disease, with many infants coming to medical attention due to fever, lethargy and poor feeding, but without respiratory manifestations as seen in adult patients. 7, 8, 9, 10, 11, 12, 13 the largest case series to date describes 18 infants younger than 90 days of age who tested positive for sars-cov-2, 14 of whom were febrile. 9 the objective of this study was to compare the clinical and demographic characteristics and hospital course of febrile infants who presented to cohen children's medical center (ccmc) during march and april of 2020, the time period of peak covid-19 incidence in our region, to febrile infants treated in ccmc during march and april of previous years. 14 particular emphasis was placed on infants in whom sars-cov-2 was detected in order to provide relevant clinical data for clinicians evaluating infants with fever during the pandemic. we conducted a single centered, retrospective study of febrile infants evaluated in the york, which was the epicenter of covid-19 in the us during the study period. the investigation included both infants who were evaluated and discharged from the ccmc ed and those admitted to an inpatient unit. per institutional policy, all febrile infants < 28 days are hospitalized, but for infants 29-56 days of age a decision to hospitalize is based on clinical criteria. for infants with an ed revisit or readmission to ccmc within 7 days, only the first visit was included in the study. patients were classified as having sars-cov-2 when a nasopharyngeal or combined nasopharyngeal/oropharyngeal swab tested positive by one of several nucleic acid amplification (naa) assays for sars-cov-2 in northwell health laboratories. as of march 24, 2020 all infants admitted to ccmc underwent sars-cov-2 testing but prior to march 24, testing for sars-cov-2 was not universal because of limited testing capacity. during the study period sars-cov-2 testing of febrile infants who were discharged from the emergency department was not universally applied. cases were ascertained through review of icd-10 billing codes and discharge diagnosis in the electronic medical record. clinical, laboratory and sociodemographic data were abstracted from the electronic health record and managed with the use of a redcap electronic database. 15 collected data included age, sex, ethnicity, race, primary language, insurance, length of hospital stay, history of sick contacts or known covid-19 exposure, history of prematurity or underlying medical condition, disposition from the ed (discharged home versus hospital admission), and need for admission to the pediatric intensive care unit (picu). the presence or absence of symptoms such as fever, cough, rhinorrhea, feeding difficulties (defined as decreased oral intake), emesis, diarrhea, irritability, lethargy and rash, and vital signs and ascultatory lung examination were also recorded. laboratory results included sars-cov-2 naa assays test results, complete blood count (cbc) and white blood cell differential, urinalysis, hepatic assays, cerebrospinal fluid parameters, bacterial cultures (urine, blood, cerebrospinal fluid), respiratory viral panel (rvp); genmark diagnostics, carlsbad, ca), erythrocyte sedimentation rate and c-reactive protein, stool assays (stool culture, rotavirus antigen, multiple pathogen gastrointestinal panel by naa (gi naa) assay, and herpes simplex virus (hsv) testing (naa testing of cerebrospinal fluid, whole blood, surface specimens and vesicles). days of antimicrobial therapy, measured from date of first to last dose, and type and duration of respiratory support (e.g., supplemental oxygen, non-invasive ventilation or mechanical ventilation), measured from date of initiation to discontinuation, were recorded. neutropenia was defined as an absolute neutrophil count (anc) of < 1000 cells/µl, lymphopenia was defined as an absolute lymphocyte count (alc) of < 3000 j o u r n a l p r e -p r o o f blood, urine, or cerebrospinal fluid that was deemed pathogenic. 18, 19 febrile infants were classified based on the year of presentation (eg, 2018, 2019, 2020). febrile infants were also categorized based on their sars-cov-2 status: (1) infants who were sars-cov-2 test positive during march-april 2020 were classified as sars-cov-2 infected; (2) infants who were treated between march and april 2020 but were not tested for sars-covsixteen of the sars-cov-2 infected infants were reported in previous studies. 13, 14 this study was approved by the northwell health institutional review board. we categorized and analyzed the data according to the patients' sars-cov-2 status (infected or uninfected) and year of presentation. for purposes of analysis, we combined the 2018 and 2019 cohorts as a single cohort. we performed descriptive and bivariable analyses using fisher exact test for categorical data, student t test for continuous variables, and the wilcoxon rank-sum test j o u r n a l p r e -p r o o f for ordinal data. all tests of significance were 2-sided with an α value of 0.05. statistical analysis was performed by using spss 25 (ibm corp, armonk, ny). overall, the age distribution was as follows: 12 (10%) were 0-14 days, 29 (23%) were 15-28 days, and 83 (67%) were 29-56 days; 69 (56%) were male; 80(65%) were admitted to general inpatient unit, 2 (2%) to the picu, and 42 (34%) were discharged home from the ed. patient demographics are summarized in table i . compared with febrile infants presenting in 2018 and 2019, febrile infants in 2020 were similar in age and by sex, but were more likely to identify as hispanic (p=.04) ( table 1 ). in 2020, there were significantly fewer infants with a sbi, as sbi was detected in three of 53 (6%) infants in demographic and clinical variables of 20 sars-cov-2 positive infants were compared with 81 uninfected infants (71 seen 2018 and 2019 and the 10 sars-cov-2 negative infants seen in 2020) ( table 2 and table 3) . a significantly higher proportion of infants in the covid-19 group had public insurance or were uninsured and identified as hispanic (p=.01 and p=.004, respectively). the racial distribution of the groups differed significantly with a higher proportion of patients identified as "multiracial/other" and a smaller proportion identified as asian or black in the sars-cov-2 group (p=.04). infants with covid-19 were significantly younger (p=.03), had a higher proportion with reported feeding difficulty (p=.002) and had a higher proportion with reported lethargy (p=.02) than the sars-cov-2 negative group. infants the key findings of our study are that during the peak of the covid-19 pandemic in new york, sars-cov-2 was the predominant pathogen identified among febrile infants younger than 57 days of age, and the disease was self-limited in all infants with covid-19. none of the infants infected with sars-cov-2 had an additional pathogen identified. febrile young infants with sars-cov-2 infection differ from other febrile infants in that they are younger, are more likely to be lethargic or exhibit feeding difficulties, have lower mean wbc, anc, and alc and have a higher likelihood of neutropenia and lymphopenia despite performing sars-cov-2 testing on only 57% of the 2020 cohort of febrile infants due to limitations on availability of testing during this time, sars-cov-2 was detected in 38% of the entire cohort and 67% of those tested. in contrast, although the entire 2020 cohort was tested for other respiratory viruses, a virus was detected in only 15% of febrile infants, similar to a study that demonstrated a decrease in influenza rates while covid-19 was prevalent and while local school closures and stay-at-home orders were in place. 20 the majority of febrile infants with covid-19 had a relatively mild infections, with only 2 of 20 infants requiring supplemental oxygen, one of whom also required high flow nasal cannula, findings similar to those described in other case reports and small case series. 8, 9, 10, 11, 12 however, severe disease with respiratory failure has been reported in a 4-weekold infant with covid-19 who was born after a 33-week gestation and in previously healthy infants with covid-19 who developed pneumonia and evidence of myocardial involvement. 21, 22, 23 the significantly higher proportion of infants with sars-cov-2 infection of hispanic ethnicity and with public health insurance compared with sars-cov-2 uninfected infants may reflect the more pronounced impact of covid-19 in hispanic persons than those of other ethnicities and in persons from lower socioeconomic groups that has been observed in adults. 24, 25 the sars-cov-2 infected infants were younger than the sars-cov-2 uninfected infants, and were also younger than the subgroup with other respiratory viral infections. this may in part be an artifact of our practice of admitting infants younger than 29 days of age hospital and preferential testing of admitted patients over ambulatory patients. this finding also could reflect the high prevalence of sars-cov-2 among women presenting in labor during this pandemic with transmission from the infant's mother or another household member. 26 infants with covid-19 presented with lethargy and feeding difficulty more with sars-cov-2 infection among infants younger than 90 days of age. 9, 12 the uncommon occurrence of sbi or a second viral pathogen strongly support the assertion that sars-cov-2 infection is the cause of the febrile illness in most or all of these infants rather than that detection reflects an asymptomatic infection. there were several limitations to this study. this was a single center study and findings may not be generalizable. additionally, as the initial phase of the covid-19 pandemic in the united states took place in march and april of 2020, we compared febrile infants seen during this time period with febrile infants evaluated in the ed during corresponding months of 2018 and 2019 so generalizability to other time periods is not possible. lastly, due to variable testing for sars-cov-2 at the initial stages of the pandemic, 23 of 53 infants evaluated in the ed because of fever were not tested; therefore we may have underestimated the prevalence of covid-19 in this patient population during this time period. sars-cov-2 should be considered as a cause of fever in young infants, particularly when the infant has poor feeding and/or lethargy and when leukopenia, lymphopenia, or neutropenia is present. the infection generally is self-limited and has a relatively rapid clinical resolution. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f .14 j o u r n a l p r e -p r o o f .09 mean wbc x 5 defined as presence of any of the following: abnormal lung exam (crackles, rhonchi or wheeze), hypoxia, parenchymal infiltrate on chest x-ray 6 defined as an anc of < 1.0x10/ 9 l 7 defined as an alc of < 3.0x10/ 9 l j o u r n a l p r e -p r o o f clinical features of patients infected with 2019 novel coronavirus in wuhan who declares covid-19 a pandemic novel coronavirus in the united states hospitalization rates and characteristics of children aged <18 years hospitalized with laboratoryconfirmed covid-19 -covid-net, 14 states sars-cov-2 associated deaths among persons aged <21 years -united states epidemiology of covid-19 among children in china mmwr morb mortal wkly rep febrile infant: covid-19 in addition to the usual suspects sars-cov-2 infection in infants less than 90 days old sars-cov-2 infection (covid-19) in febrile infants without respiratory distress fever without a source in a young infant due to sars-cov-2 novel coronavirus infection in febrile infants aged 60 days and younger a case series of the 2019 novel coronavirus (sars-cov-2) in 3 febrile infants in new york early experience of covid-19 in a us children' hospital research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support screening for severe combined immunodeficiency in neonates lanzkowsky's manual of pediatric hematology and oncology 6 th ed the changing epidemiology of serious bacterial infections in young infants a clinical prediction rule to identify febrile infants 60 days and younger at low risk for serious bacterial infections comparison of clinical features of covid-19 vs seasonal influenza a and b in us children infant with sars-cov-2 infection causing severe lung disease treated with remdesivir a 55 day old female infant infected with covid 19: presenting with pneumonia, liver injury and heart damage sars-cov-2 infection in febrile neonates disparities in incidence of covid-19 among underrepresented racial/ethnic groups in counties identified as hotspots during hospitalization and mortality among black patients and white patients with covid-19 universal screening for sars-cov-2 in women admitted for delivery neonates hospitalized with community-acquired sars-cov-2 in a colorado neonatal intensive care unit clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiology, clinical features, and disease severity in patients with coronavirus disease children's hospital in new york city risk of bacterial coinfections in febrile infants 60 days old and younger with documented viral infections key: cord-277253-vy0mvzeb authors: liu, hongbo; ye, fei; sun, qi; liang, hao; li, chunmei; lu, roujian; huang, baoying; tan, wenjie; lai, luhua title: scutellaria baicalensis extract and baicalein inhibit replication of sars-cov-2 and its 3c-like protease in vitro date: 2020-04-11 journal: biorxiv doi: 10.1101/2020.04.10.035824 sha: doc_id: 277253 cord_uid: vy0mvzeb covid-19 has become a global pandemic that threatens millions of people worldwide. there is an urgent call for developing effective drugs against the virus (sars-cov-2) causing this disease. the main protease of sars-cov-2, 3c-like protease (3clpro), is highly conserved across coronaviruses and is essential for the maturation process of viral polyprotein. scutellariae radix (huangqin in chinese), the root of scutellaria baicalensis has been widely used in traditional chinese medicine to treat viral infection related symptoms. the extracts of s. baicalensis have exhibited broad spectrum antiviral activities. we studied the anti-sars-cov-2 activity of s. baicalensis and its ingredient compounds. we found that the ethanol extract of s. baicalensis inhibits sars-cov-2 3clpro activity in vitro and the replication of sars-cov-2 in vero cells with an ec50 of 0.74 μg/ml. among the major components of s. baicalensis, baicalein strongly inhibits sars-cov-2 3clpro activity with an ic50 of 0.39 μm. we further identified four baicalein analogue compounds from other herbs that inhibit sars-cov-2 3clpro activity at microm concentration. our study demonstrates that the extract of s. baicalensis has effective anti-sars-cov-2 activity and baicalein and analogue compounds are strong sars-cov-2 3clpro inhibitors. coronaviruses (covs) are single stranded positive-sense rna viruses that cause severe infections in respiratory, hepatic and various organs in humans and many other animals [1, 2] . within the 20 years of the 21st century, there are already three outbreaks of cov-causing global epidemics, including sars, mers, and covid-19. the newly emerged cov infectious disease (covid-19) already caused more than 1.5 million confirmed infections and 89 thousands deaths worldwide up to april 9, 2020 (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports). there is an urgent call for drug and vaccine research and development against covid-19. covid-19 was confirmed to be caused by a new coronavirus (sars-cov-2), whose genome was sequenced in early january 2020 [3, 4] . the genomic sequence of sars-cov-2 is highly similar to that of sars-cov with 79.6% sequence identity [5] and remain stable up to now [6] . however, the sequence identities vary significantly for different viral proteins [7] . for instance, the spike proteins (s-protein) in covs are diverse in sequences and even in the host receptors that bind due to the rapid mutations and recombination [8] . although it has been confirmed that both sars-cov and sars-cov-2 use ace2 as receptor and occupy the same binding site, their binding affinities to ace2 vary due to subtle interface sequence variations [9] . on the contrary, the 3clike proteases (3cl pro ) in covs are highly conserved. the 3cl pro in sars-cov and sars-cov-2 share a sequence identity of 96.1 %, making it an ideal target for broad spectrum anti-cov therapy. although many inhibitors have been reported for sars-cov and mers-cov 3cl pro [10] [11] [12] [13] , unfortunately none of them has entered into clinical trial. inspired by the previous studies, several covalent inhibitors were experimentally identified to inhibit the 3cl pro activity and viral replication of sars-cov-2, and some of the complex crystal structures were solved [14, 15] . in addition, a number of clinically used hiv and hcv protease inhibitors have been proposed as possible cure for covid-19 [16] and some of them are now processed to clinically trials [17] . several computational studies proposed potential sars-cov-2 3cl pro inhibitors by virtual screening against the crystal or modeled three-dimensional structure of sars-cov-2 3cl pro as well as machine intelligence [18] [19] [20] [21] [22] [23] . highly potent sars-cov-2 3cl pro inhibitors with diverse chemical structures need further exploration. traditional chinese medicine (tcm) herbs and formulae have long been used in treating viral diseases. some of them have been clinically tested to treat covid-19 [24] . anti-microbial and anti-inflammatory activities have been reported [27] . remarkably, the extracts of s. baicalensis have exhibited broad spectrum anti-viral activities, including zika [28] , h1n1 [29] , hiv [30] and denv [31] . in addition, a multicenter, retrospective analysis demonstrated that s. baicaleinsis exhibits more potent antiviral effects and higher clinical efficacy than ribavirin for the treatment of hand, foot and mouth disease [32] . several s. baicalensis derived mixtures or pure compounds have been approved as antiviral drugs, such as baicalein capsule (to treat hepatitis) and huangqin tablet (to treat upper respiratory infection) in china. most of the s. baicaleinsis ingredients are flavonoids [33] . flavonoids from other plants were also reported to mildly inhibit sars and mers-cov 3cl pro [34, 35] . here we studied the anti-sars-cov-2 activity of s. baicalensis and its ingredients. we found that the ethanol extract of s. baicalensis inhibits sars-cov-2 3cl pro activity and the most active ingredient baicalein exhibits an ic50 of 0.39 m. in addition, the ethanol extract of s. baicalensis effectively inhibits the replication of sars-cov-2 in cell assay. we also identified four baicalein analogue compounds from other herbs that inhibit sars-cov-2 3cl pro activity at microm concentration. we prepared the 70% ethanol extract of s. baicalensis and tested its inhibitory activity against sars-cov-2 3cl pro . we expressed sars-cov-2 3cl pro and performed activity assay using a peptide substrate (thr-ser-ala-val-leu-gln-pna) according to the published procedure of sars-cov 3cl pro assay [11, 36] . the inhibitory ratio of s. baicalensis extract at different concentrations on sars-cov-2 3cl pro activity were 6 shown in figure 1a . the crude extract exhibits significant inhibitory effect with an ic50 of 8.5 g/ml, suggesting that s. baicalensis contains candidate inhibitory ingredients against sars-cov-2 3cl pro . we tested the inhibitory activity of four major ingredients from s. baicalensis: baicalein, baicalin, wogonin and wogonoside in vitro. baicalein showed the most potent anti-sars-cov-2 3cl pro activity with an ic50 of 0.39 m ( figure 1b and table 1 ). baicalin inhibited sars-cov-2 3cl pro activity for about 41% at 50 μm, while wogonin and wogonoside were not active at this concentration. a b we performed molecular docking to understand the inhibitory activity of s. baicalensis ingredients. in the docking model, baicalein binds well in the substrate binding site of sars-cov-2 3cl pro with its 6-oh and 7-oh forming hydrogen bond interactions with the carbonyl group of l141 and the backbone amide group of g143, respectively ( figure 2a ). in addition, the carbonyl group of baicalein is hydrogen bonded with the backbone amide group of e166. the catalytic residues h41 and c145 are well covered by baicalein, accounting for its inhibitory effect. as the 7-oh in baicalin is in close contact with the protein, there may not be enough space for glycosyl modification, explaining the low activity of baicalin. as for wogonin, the absence of 6-oh together with its additional 8-methoxyl group alters the binding orientation and weakens the binding strength ( figure 2b ). hydrogen bond is observed between its 5-oh and the backbone carbonyl group of l141, while the interaction with e166 by its 8-methoxy group is weaker than that formed by the carbonyl group in baicalein. we searched for baicalein analogues from available flavonoid suppliers and selected 8 flavonoids and 2 glycosides for experimental testing. four flavonoid compounds were found to be potent sars-cov-2 3cl pro inhibitors. among them, scutellarein is mainly distributed in genus scutellaria and erigerontis herba (dengzhanxixin or dengzhanhua in chinese) in its glucuronide form, scutellarin. scutellarin has long been used in cardiovascular disease treatment for its ability to improve cerebral blood supply [37] . for all the active flavonoid compounds that we found, the introduction of glycosyl group, as in the case of baicalein and baicalin, decreased the inhibition activity, probably due to the steric hindrance of the glycosyl group, which is also true for scutellarein/scutellarin, and myricetin/myricetrin. as glycosides and their a b c d corresponding aglycones are often interchangeable in vivo, for instance, baicalin was reported to be metabolized to baicalein in intestine [39] , while baicalein can be transformed to baicalin by hepatic metabolism [40] , we expect that both the flavonoid form of the active compounds and their glycoside form will function in vivo. we suggest that these compounds can be further optimized or used to search for other tcm herbs containing these compounds or substructures for the treatment of covid-19. targetmol. the dna of sars-cov-2 3cl pro (referred to genbank, accession number mn908947) was synthesized (hienzyme biotech) and amplified by pcr using primers n3clp-nhe (5'-catggctagcggttttagaaaaatggcattccc-3') and n3clp-xho (5'-cactctcgagttggaaagtaacacctgagc-3'). the pcr product was digested with nhe i/xho i and cloned into the pet 21a dna as reported previously [41] . the resulting sars-cov-2 pet 3cl-21x plasmid encodes a 35 064 da sars-cov-2 3cl pro with a c-terminal 6xhis-tag. the sars-cov-2 pet 3cl-21x plasmid was further transformed to e. coli bl21<de3> for protein expression as reported [41] . the recombinant protein was purified through a nickelnitrilotriacetic acid column (ge healthcare) and subsequently loaded on a gel filtration column sephacryl s-200 hr (ge healthcare) for further purification as previously described [42] . a colorimetric substrate thr-ser-ala-val-leu-gln-pna (gl biochemistry ltd) and assay buffer ( the structure of sars-cov-2 3cl pro (pdb id 6lu7) [14] and s. baicalensis concentrations for 48h. the supernatant was collected and the rna was extracted and analyzed by relative quantification using rt-pcr as in the previous study [3, 43] . viral rna was extracted from 100 μl supernatant of infected cells using the automated nucleic acid extraction system (tianlong, china), following the manufacturer's recommendations. sars-cov-2 virus detection was performed using the one step system (roche, rotkreuz, switzerland). orf 1ab was amplified from cdna and cloned into ms2-ncov-orf1ab and used as the plasmid standard after its identity was confirmed by sequencing. a standard curve was generated by determination of copy numbers from serially dilutions (10 3 -10 9 copies) of plasmid. the following primers used for quantitative pcr were 1ab-f: 5ʹ-agaagattggttagatgatgatagt-3ʹ; 1ab-r: 5ʹ-ttccatctctaattgaggttgaacc-3ʹ; and probe 5ʹ-fam-tcctcactgccgtcttgttg acca-bhq1-3ʹ. the individual ec50 values were calculated by the origin 2018 software. coronaviruses -drug discovery and therapeutic options antiviral drugs specific for coronaviruses in preclinical development a novel coronavirus from patients with pneumonia in china 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phytochemistry, pharmacology, and flavonoid biosynthesis of scutellaria baicalensis baicalein and baicalin as zika virus inhibitors anti-h1n1 virus, cytotoxic and nrf2 activation activities of chemical constituents from scutellaria baicalensis inhibition of hiv replication by baicalin and s. baicalensis extracts in h9 cell culture extract of scutellaria baicalensis inhibits dengue virus replication efficacy of scutellaria baicalensis for the treatment of hand, foot, and mouth disease associated with encephalitis in patients infected with ev71: a multicenter, retrospective analysis a targeted strategy to analyze untargeted mass spectral data: rapid chemical profiling of scutellaria baicalensis using ultra-high performance liquid chromatography coupled with hybrid quadrupole orbitrap mass spectrometry and key ion filtering inhibition of sars-cov 3cl protease by flavonoids characteristics of flavonoids as potent mers-cov 3c-like protease inhibitors 3c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism therapeutic effects of breviscapine in cardiovascular diseases: a review identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 metabolism of constituents in huangqin-tang, a prescription in traditional chinese medicine, by human intestinal flora hepatic metabolism and disposition of baicalein via the coupling of conjugation enzymes and transporters-in vitro and in vivo evidences biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase maturation mechanism of severe acute respiratory syndrome (sars) coronavirus 3c-like proteinase in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) key: cord-271781-cfv0ta10 authors: patel, kishan p.; vunnam, srinivas r.; patel, puja a.; krill, kaleigh l.; korbitz, parker m.; gallagher, john p.; suh, jane e.; vunnam, rama r. title: transmission of sars-cov-2: an update of current literature date: 2020-07-07 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-020-03961-1 sha: doc_id: 271781 cord_uid: cfv0ta10 severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the etiologic agent for the 2019 coronavirus disease (covid-19) pandemic, has caused a public health emergency. the need for additional research in viral pathogenesis is essential as the number of cases and deaths rise. understanding the virus and its ability to cause disease has been the main focus of current literature; however, there is much unknown. studies have revealed new findings related to the full transmission potential of sars-cov-2 and its subsequent ability to cause infection by different means. the virus is hypothesized to be of increased virulence compared with previous coronavirus that caused epidemics, in part due to its overall structural integrity and resilience to inactivation. to date, many studies have discussed that the rationale behind its transmission potential is that viral rna has unexpectedly been detected in multiple bodily fluids, with some samples having remained positive for extended periods of time. additionally, the receptor by which the virus gains cellular entry, ace2, has been found to be expressed in different human body systems, thereby potentiating its infection in those locations. in this evidence-based comprehensive review, we discuss various potential routes of transmission of sars-cov-2—respiratory/droplet, indirect, fecal-oral, vertical, sexual, and ocular. understanding these different routes is important as they pertain to clinical practice, especially in taking preventative measures to mitigate the spread of sars-cov-2. sars-cov-2, previously known as the 2019 novel coronavirus, is an enveloped non-segmented positive-sense rna virus responsible for the 2019 coronavirus disease (covid19) pandemic. it is classified as a beta coronavirus, rendering mammalian hosts susceptible to infection. six other coronaviruses have been identified to infect human hosts, resulting in epidemics, including severe acute respiratory syndrome-related coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [1] . as of may 7, 2020, the global count has reached 3,672,238 confirmed cases with 254,045 covid-19-related deaths [2] , causing growing public health concern. r t , the effective reproduction number, approximates the potential for a virus to spread given a specific measure of time in relation to control measures in place. as suggested by inglesby in an article in jama, without measures such as social distancing in place, the r t is estimated to be similar to that of its value in january 2020 ranging between 2 and 4 [3] . the transmission potential of the virus may be due in part to its structure and overall tenacity. sars-cov-2 was found to have one of the hardest outer protective shells among all coronaviruses. this is believed to result in more stable viral particles, resulting in its greater resilience in bodily fluid [4] . in a nejm study, sars-cov-2 was found to remain viable, in aerosolized form for up to 3 h, and stable on plastic and stainless steel surfaces for up to 72 h [5] . the virus was found to be more stable on smooth surfaces [6] . however, it should be noted that the findings presented are in the absence of in vivo data and based solely on a limited number of studies, potentially creating a gap in our understanding. studies have demonstrated that the mechanism by which sars-cov-2 gains cellular entry in the respiratory tract is through the angiotensin-converting enzyme 2 (ace2) receptor [7] . the first step involves viral attachment to ace2 to enable host cell entry; the second step involves activation of the virus internalization process by proteases, particularly tmprss2 [8] . ace2 has been noted to be expressed in various tissues and organ systems throughout the body, including the central nervous system, gastrointestinal system, heart, lung, testes, and kidney [9] [10] [11] . it is hypothesized that this ace2-mediated mechanism impacts the tropism of sars-cov-2 and its ability to invade different organ systems and cause damage [9, 10] . studies have shown the detection of viral rna in various bodily fluid samples. wang [12] . a preprint study reported a covid-19 patient with no urinary symptoms having tested positive for sars-cov-2 in the urine [13] . one case report depicted a patient with meningitis/encephalitis associated with covid-19, whereby sars-cov-2 rna was detected in cerebrospinal fluid (csf) [14] , whereas another study of pediatric covid-19 patients showed that four patients with neurological symptoms had negative csf samples for viral rna [15] . a prospective study by sun j et al. examining 49 covid-19 patients in china noted that persistent prolonged viral rna shedding has been detected in body fluids, particularly that of nasopharyngeal and fecal samples [16] . additionally, while viral shedding has been widely noted, a study by xiao et al. had successfully isolated infectious sars-cov-2 from fecal matter [17] . it is important to note that although quantitative viral rna has been detected from various body sources, the ability to recover viable virus is of critical importance to understand transmission routes. as some of the studies included in this review lack this data, it may be very difficult to draw reasonable conclusions. in this review, the latest literature pertaining to the potential for various routes of transmission of sars-cov-2 will be discussed. this narrative review serves to provide information on the latest literature available pertaining to the transmission potential of sars-cov-2. this paper was not systematically conducted, and the data presented was made available according to the authors' preference and may be subject to bias or missing data. as the covid-19 pandemic is evolving, research becomes progressively available, keeping in mind that some of the studies collected thus far may consist of case reports or series with lowquality methodology as they were obtained when this review was written. therefore, the conclusions of this review may be limited in evidence as further research becomes readily available. similar to sars-cov and mers-cov, sars-cov-2 is predominantly characterized as a respiratory tract infection. the virus commonly presents with nonspecific lower respiratory tract infection symptoms, such as fever, cough, and shortness of breath [18] . the first cases of covid-19 took place in wuhan, china, where a group of individuals were evaluated for pneumonia of unknown etiology [19] . following the outbreak, it was suspected that the virus propagated by means of community and intrafamilial spread [20] . the transmission is thought to be predominately person to person, either by direct contact or through droplets originating from infected individuals [21] . droplets can be formed through coughing, sneezing, singing, breathing, and speaking [22] . droplet transmission involves exposing an entry point, such as mucosa (nose and mouth) or conjunctiva, to potentially infective respiratory droplets (typically > 5-10 μm in diameter) produced by someone having respiratory symptoms within a 1 m proximity [23] . the inhalation of small, exhaled respiratory droplets containing infectious virions is thought to occur; there is a growing concern for long-range human-to-human infection should these droplets remain airborne and viable [24] . another medium to consider is airborne dust, as inhalation of virus-laden fine particles may transport the virus in deeper bronchial and alveolar regions [24] . a study analyzing covid-19 patients from three hospitals in china demonstrated higher positive rates of viral rna in bronchoalveolar lavage fluid, sputum, and nasal swabs [12] . furthermore, in a study evaluating environmental contamination in symptomatic patients, one of the three subjects had 87% of his tested room sites, including air outlets, return positive for viral rna despite only having mild upper respiratory tract involvement. environmental contamination through airflow may perpetuate viral transmission through infectious droplets [25] . compared with direct human-to-human spread, the role of indirect transmission is less understood. airborne transmission occurs when larger respiratory droplets, which have evaporated, or dust particles harbor microbes in the droplet nuclei with a size < 5 μm in diameter. this allows microbes to remain in the air for longer periods of time and travel greater than 1-m distance. airborne transmission becomes a threat when aerosolization of particles takes place, especially in essential procedures such as endotracheal intubation, bronchoscopy, and cardiopulmonary resuscitation [23] . liu et al. investigated the aerodynamic nature of the virus based on findings from hospitals in china. the concentration of viral rna in aerosols detected in isolation wards and ventilated patient rooms was very low but elevated in toilet areas. additionally, airborne viral rna levels in the majority of public areas were undetectable with the exception of a few areas prone to crowding [26] . in addition to aerosolization of microbe particles, infection secondary to contact with contaminated objects or exposure to fomites may occur. a study from the nejm characterized the viability of the virus in multiple surfaces of inoculated samples, with their results indicating that indirect transmission by means of fomite and aerosolization was plausible [5] . in an early study by cai et al., covid-19 patients were tracked to identify their environmental exposures within a shopping mall. the findings suggested that viral spread was unlikely due to respiratory droplet transmission alone. direct contact with other subjects, asymptomatic individuals, or exposure to contaminated environments accounted for transmission in some subjects [27] . moriarty et al. highlighted the transmission potential of the virus on cruise ships totaling more than 800 confirmed cases; viral rna was discovered on multiple surfaces of cabins in symptomatic and asymptomatic individuals despite quarantining efforts, questioning the role of transmission by fomites [28] . additionally, a preprint study conducted by santarpia et al. highlights environmental viral shedding observed in 13 quarantined individuals in the usa; of 163 surface and aerosol samples collected, 126 (77.3%) tested positive by reverse transcriptase-polymerase chain reaction (rt-pcr) for sars-cov-2. furthermore, 80.4% of all room surface samples tested positive for the presence of viral rna, supporting significant indirect contamination and likely airborne transmission [29] . it should be noted that to the best of our knowledge, no viable virus has been isolated from fomite samples indicating limited data on its transmissibility. thus far, there have been many studies analyzing the possibility of fecal-oral transmission of covid-19. potential sars-cov-2 infection in the gastrointestinal tract has been discussed in regard to the expression of ace2 and tmprss2 in the epithelium [10, 30] . additionally, studies have noted that its fecal-oral transmission potential may lie in the fact that prolonged viral shedding can occur in fecal matter-one case reported an asymptomatic covid-19 patient experiencing viral detection in the stool for up to 42 days, while nasopharyngeal sampling was negative [31] . prolonged viral detection in stool samples has also been detected in pediatric patients following the recovery of covid-19 pneumonia [32] . in another study involving pediatric covid-19 patients postdischarge by zhang et al., all subjects had positive stool rt-pcr results after 10 days, without positive throat swabs, clinical symptoms, or imaging findings [32] . similar findings were demonstrated in adults, whereby patients had positive rt-pcr results in anal swabs after recovery, yet still met criteria for hospital discharge due to negative nasopharyngeal testing [33] . while prolonged viral shedding in the stool has been noted, whether or not these particles are infectious and have the potential of being spread fecal-orally is questionable. supporting its potentiation, one study by zhang et al. has characterized the presence of live virus in stool; they were able to successfully culture sars-cov-2 in vero cells which was isolated from a stool specimen of a patient with severe covid-19 pneumonia [34] . additionally, a study published in jama by ong et al. supported the possibility of this transmission by demonstrating extensive environmental contamination from a symptomatic covid-19 patient. samples were collected from the room of a patient whose fecal matter tested positive for sars-cov-2 by rt-pcr prior to routine cleaning, including from the surface of the toilet bowl, inside of the bowl, and door handle-all of which tested positive. however, samples obtained post-cleaning were negative, implying that current decontamination measures are effective. these findings suggested that viral shedding in the stool could be contributing to a possible route of transmission [25] . yeo et al. discussed the clinical implications that fecal-oral transmission of covid-19 may have in infection control, especially in areas with poor sanitation [35] . with new findings, it was recommended that when handling stool of covid-19 patients, strict precautions be practiced [35] . in fact, the detection of sars-cov-2 in untreated wastewater was confirmed in australia [36] . yeo et al. also discussed the need for hospital-directed recommendations regarding proper management and disinfection of sewage due to growing concerns for the existence of fecal-oral transmission [35] . although vertical transmission of covid-19 has been studied, there still remains a need for further conclusive evidence. certain studies have suggested evidence for vertical transmission on the basis that some neonates born to covid-19positive mothers had elevated igm antibodies following birth [37] [38] [39] . in a study by dong et al., a neonate born to a covid-19-positive mother was found to have elevated igm antibodies 2 h after birth but tested negative for covid-19 on nasopharyngeal specimens. typically, igm antibodies do not appear until 3-7 days after infection in part due to its molecular structure, but this elevation was present soon after birth in the setting of negative maternal vaginal secretions for sars-cov-2 [38, 39] . another study conducted by zeng et al. examined 6 pregnant covid-19 patients and highlighted that two infants had elevated igm levels [39] . in a study by parazzini et al., covid-19 mothers with both vaginal and cesarean deliveries were assessed (6 and 31, respectively). two neonates tested positive for sars-cov-2 on rt-pcr testing; three neonates had elevated sars-cov-2 igg and igm levels but tested negative on rt-pcr. it was concluded that the rate of vertical or peripartum transmission of covid-19 is low to none for cesarean delivery, but no data was available for vaginal delivery [40] . additionally, a study involving 31 covid-19 pregnant mothers reported no vertical transmission in their neonates or placentas [41] . zamaniyan et al. reported a pregnant woman with severe covid-19 pneumonia having given birth to a preterm baby at 32 weeks gestation with no evidence of sars-cov-2 infection. however, testing for covid-19 by rt-pcr was positive in both an amniotic sample and a second nasal and throat test the neonate underwent 24 h after birth via cesarean delivery; testing was negative in the vaginal secretion sample, umbilical cord blood, and first neonate test. since the amniotic fluid and the neonate tested positive, it may suggest that the newborn was affected intrauterine by sars-cov-2 [42] . to oppose, in a retrospective review of nine covid-19 pregnant mothers who underwent cesarean section, six patients had samples of amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples tested for sars-cov-2, and all were negative [43] . furthermore, there has been a case of neonate in china who tested positive for covid-19 via rt-pcr of pharyngeal swabs 36 h after birth; however, vertical transmission in the case was not confirmed [44] . the impact that covid-19 pregnancies can have on neonates is largely unknown. in a clinical analysis of 10 neonates born from nine covid-19 mothers by zhu et al., it was found that perinatal infection of sars-cov-2 may lead to adverse effects in newborns, as some of the neonates experienced fetal distress, premature labor, respiratory distress, thrombocytopenia, abnormal liver function tests, and even death [45] . a recent study in jama by baud et al. depicted a case of miscarriage during the second trimester in a covid-19 female related to placental infection with sars-cov-2. the fetal surface of the placenta tested positive for sars-cov-2; histopathology revealed mixed inflammatory infiltrates and funisitis. although vertical transmission was not proven, no other etiology of fetal demise was identified [46] . nevertheless, further research is needed to determine whether sars-cov-2 crosses the placental barrier. in a cross-sectional study examining 34 adult males recovering from covid-19, sars-cov-2 was not detected in any of the subjects' semen approximately 1 month after initial disease confirmation. however, the findings were unable to definitively rule out the presence of sars-cov-2 in seminal fluid due to a lack of testing during the acute phase of infection [47] . however, a recent cohort study by li et al. demonstrated that semen testing for sars-cov-2 resulted positive for six of the 38 male covid-19 patients, four of which were in the acute stage of infection and two being in the recovery phase [48] . the study by pan et al. also characterized that there was low overlapping gene expression of ace2 and tmprss2 in human testes, suggesting that the sars-cov-2 would not likely be able to gain testicular cellular entry via this mechanism [47] . however, as stated by sama et al., ace2 has been noted to be highly expressed in the testis [11] . to et al. noted the detection of sars-cov-2 in selfcollected saliva specimens in 91.7% (11/12) of covid-19 patients [49] . the need for safe sexual intercourse behavior may be heightened due to the fact that the virus has been detected in high concentrations in saliva and nasal mucosa and may have potential receptor binding in rectal and anal epithelial cells. lastly, the potential for an ocular route of transmission has recently been explored. certain studies have noted the detection of viral rna in conjunctival specimens, while others demonstrated limited evidence of its presence. a case study of an ocular manifestation of covid-19 presenting as bilateral conjunctivitis 13 days after onset of illness suggested that viral shedding occurs in the eyes; rt-pcr of conjunctival samples on days 14 and 17 following the onset of symptoms tested positive. the conjunctiva was confirmed to be a site of viral replication when he tested positive via rt-pcr of consecutive conjunctival swabs. on day 19, the conjunctival swab specimen resulted negative [50] . moreover, 38 covid-19 patients at a hospital center in china were retrospectively reviewed for ocular manifestations; the study concluded that about one-third of patients with covid-19 had ocular abnormalities, which frequently occurred in patients with more severe disease [51] . in a case series of 38 covid-19 patients published in jama ophthalmology, 31.6% (95% ci, 17.5-48.7) of subjects had ocular manifestations consistent with conjunctivitis such as conjunctival hyperemia, chemosis, or increased secretions. similar to the prior study, this more commonly occurred in patients with severe systemic manifestations or abnormal laboratory tests. there was a low prevalence of viral nucleotides in conjunctival specimens of 5.2% (95% ci, 0.6-17.8) [51] . a recent case report of the first patient in italy to be diagnosed with covid-19 depicted bilateral conjunctivitis, in addition to fever, respiratory symptoms, nausea, and vomiting. sars-cov-2 rna was detected via conjunctival swab on day 3 of hospitalization while still having persistent conjunctivitis-it remained positive until day 21 and again on day 27 (conjunctivitis resolved on day 20). in order to demonstrate that the ocular samples contained infectious virus, an rna-positive ocular sample was inoculated in vero e6 cells, with a cytopathic effect being observed 5 days later. viral replication was confirmed via rt-pcr from purified rna in the cell growth medium. implications of these findings are that ocular fluid of covid-19 patients may contain infectious virus and serve as a possible source of infection [52, 53] . however, a few studies have demonstrated limited evidence for an ocular route of transmission. in a study of 17 covid-19 patients, tears were sampled and all samples resulted negative for the presence of sars-cov-2, while nasopharyngeal samples were positive. likewise, patients with upper respiratory tract infections did not demonstrate any evidence of viral shedding in tears, suggesting that transmission through tears is likely low [54] . a couple of preprint studies have arrived at similar conclusions. in a study by sun et al. involving 72 patients with laboratory-confirmed covid-19, only two patients had conjunctivitis, but sars-cov-2 rna fragments were found in the ocular discharge of one of the two [55] . another preprint study which was a retrospective cohort study enrolled 67 confirmed and suspected covid-19 patients, of which 63 were laboratory confirmed. of the 63 laboratory-confirmed patients, conjunctival specimens from one patient were positive on pcr testing and two patients had probable positive results-none of these three had ocular symptoms [56] . the outbreak of covid-19 and evolving pandemic warrant the need for better infection control by means of identifying viral sources. emerging studies have detected the presence of viral rna in multiple regions of the human body, raising concern for its ease of transmissibility. thus far, covid-19 was originally thought to be spread by respiratory droplets; however, newer studies have highlighted the potential for alternative routes. detection of viral rna shedding in multiple bodily fluids/samples suggests the potential for additional modes of transmission, such as bloodborne, urinary, and fecal-respiratory. transmission by such means remains controversial given the limited supporting data for each mode. clinicians should be aware that the detection of viral rna in numerous bodily fluids can potentiate new routes of transmission as more data on sars-cov-2 emerges becoming more readily available. these findings emphasize the importance of control measures as they could impact daily practices, from precautions taken by the general public to prevent asymptomatic spread of disease to the isolation precautions practiced in healthcare facilities. limitations to consider when interpreting certain studies discussing transmission routes are availability of research, sample size of patients, and testing capacity in relation to diagnostic supply shortages. the role of whole genome sequencing in characterizing the presence of sars-cov-2 has been discussed as a comprehensive detection method. the limitations with this method include cost, time, and complications; however, it may play a part in elucidating further understanding of viral transmission pathways. as covid-19 is not completely understood, the need for further research confirming and/or refuting these potential routes of transmission remains a priority. the origin, transmission and clinical therapies on coronavirus disease 2019 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coronavirus disease 2019 (covid-19) patients the infection evidence of sars-cov-2 in ocular surface: a single-center cross-sectional study. medrxiv ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva. medrxiv publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions the authors contributed to and reviewed all aspects of the article. competing interests the authors declare that they have no competing interests. key: cord-290792-ggcz1zfw authors: qutob, n.; awartani, f.; salah, z.; asia, m.; abu khader, i.; herzallah, k.; balqis, n.; sallam, h. title: seroprevalence of sars-cov-2 in palestine: a cross-sectional seroepidemiological study date: 2020-09-01 journal: nan doi: 10.1101/2020.08.28.20180083 sha: doc_id: 290792 cord_uid: ggcz1zfw seroprevalence rates are important indicators to the epidemiology of covid-19 and the rates vary according to the population. in palestine, there is lack of data on the the percentage of undiagnosed population with previous mild or asymptomatic covid-19. the purpose of this study is to assess the seroprevalence rate in the palestinian population residing in the west bank. blood samples were collected from1319 adults from palestinian households in the west bank and 1136 individuals visiting laboratories for a routine checkup. serological tests for the 2455 serum samples were done using an immunoassay for the qualitative detection of antibodies against sars-cov-2 .the random sample of palestinians living in the west bank yielded 0% seroprevalence with 95% ci [0,0.0036], while the lab referrals sample yielded 4 positive cases. hence the estimated seroprevalence in this sample is 0.354% with 95% ci [0.0011,0096]. our results indicate that seroprevalence persist low and is inadequate to provide herd immunity and emphasize the need to maintain health measures to keep the outbreak under control. coronavirus disease 2019, known as covid-19, is an infectious respiratory disease caused by novel coronavirus sars-cov-2 1 . since its emergence in wuhan, china in december 2019 2 , sars-cov-2 has spread rapidly around the globe, ultimately being declared by the world health organization (who) as a global pandemic 3, 4 and new cases and deaths are being reported daily 5 . most authorities rely on pcr testing results to estimate number of covid-19 cases and make up-to-date decisions 6 . thus, numbers of patients tested positive for sars-cov-2 through pcr testing, symptomatic patients, those admitted to hospitals, or deceased from cobvid-19 are updated on a daily basis. however, the data may exclude a fraction of the population with previous mild or asymptomatic has not been tested by pcr . the proportion of the population who have overcome the infection without being noticed can probably be approximated by testing for antibodies against sars-cov-2. antibodies may confer immunity to repeat infection and a high proportion of immune individuals can attenuate the epidemic. measures of anti-sars-cov-2 seroprevalence can also be used to estimate the clinical impact of covid-19. to this effect, several serological surveys of sars-cov-2 have been done worldwide [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . there is lack of data on the the percentage of undiagnosed palestinian population with previous mild or asymptomatic covid-19. prevalence of covid-19 among palestinian remains unknown and many are concerned about this uncertainty. to this end, we conducted a population cross sectional based seroepidemiological study to assess the spread of sars-cov-2 throughout the west bank. the study included 2491 individuals (1355 from randomly selected households and 1136 from laboratory referrals; was designed to be representative by cities and used elecsys ® anti-sars-cov-2 testing. here, we describe the study design and the results of the first wave of the study. the study conducted is a cross-sectional serologic testing study aimed to investigate seropositivity for sars-cov-2 in the non-institutionalized palestinian population residing in the west bank. the study involved 1355 participants from 11 governorates, including 112 localities (supplementary table 1). 1395 households were selected using 3-stage cluster sampling. the cluster of households or census track is considered to be a geographic location that is comprised of approximately 100 households. the process for conducting cluster sampling was carried as follows : (1) selecting a cluster of households, (2) selecting 10 households randomly from each cluster and (3) selecting a person at random from the selected household. the clusters were selected using probability proportional to size (pps) sampling (table 1) . to select the number of clusters within each population location: (1) we calculated the sampling interval which equals the total number of households divided by the total number of clusters need to be selected by the sample say for example (m). so the sampling interval si= n/m, where n is the total number of households. (2) selected a random number r0 between 0 and si. (3) calculated ri as r0+i*si, a cluster is selected in li if ri belongs to the interval [ ci-1, ci]. field work was carried out between 15th th june 2020 and 30 th june 2020 by a team of registered nurses, laboratory technicians, nursing students and laboratory technician students from the arab american university following standardized health protocols (world health, 2020) . the study also included 1136 participants from medicare laboratory referrals between 1 st may 2020 and 9 th july 2020 in 16 branches in the west bank (supplementary table 2 ) . participants donated a blood sample for antibodies detection. blood samples were centrifuged and serum was separated, labelled, stored at -20c at aaup laboratory until it was used. approval from national ethical committee was obtained (phrc/hc/737/20). written informed consent was obtained from the 1355 study participants and approvals were obtained from medicare laboratories for samples to be tested. serological tests for 2491 serum samples were done using an immunoassay for the qualitative detection of antibodies against sars-cov-2 (elecsys ® anti-sars-cov-2) in human serum by using the cobas analyzer cobas e 411 (roche) . the assay uses a recombinant protein representing the nucleocapsid (n) antigen for the determination of antibodies against sars-cov-2 with testing time of 18 minutes. we included 6 samples from recovered cases with detected sars-cov2 antibodies as a positive control. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . symptomatic patients with a pcr confirmed sars-cov-2 infection. one or more consecutive specimens from these patients were collected after pcr confirmation at various time points. we estimated seroprevalence as the proportion of individuals who had a positive result in the total sars-cov-2 antibodies in the immunoassay. we used wilson method with continuity correction and boundary truncation (wccbt) to construct 95% ci for the population parameter of seroprevalence 17 . of 1395 eligible individuals, 5.8% declined to participate. of the 1355 participants, the proportion of testing was lower in females compared to males (137, 1218 respectively) including 349 in age group (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) , 314 tests in age group (25-34), 377 tests in age group (35-49) and 315 tests in age group (50+). none of the tested specimens revealed presence of antibodies against sars-cov-2. a 95% ci for the population parameter of seroprevalence was [0,0.0036]. of the1136 participants from medicare laboratory referrals in 16 branches. the proportion of testing was lower in males than females (395, 741 respectively) including 71 in age group lower less than 15 , 173 in age group (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) , 297 tests in age group (25-34), 290 tests in age group (35-49) and 305 tests in age group (50+) . out of the 1215 tested participants, 3 males, ages 38, 58, 59 and 1 female, age 40 revealed antibodies against sars-cov-2, with 95% ci [0.0011,0096]. to our knowledge, this is the first population-based sars-cov-2 seroprevalence study in palestine. the findings from this seroprevalence study for sars-cov-2 indicate that the estimated seroprevalence of the total sars-cov2 antibodies is 0% in the west bank in palestine. seroprevelance in palestine is very close to that identified in jordan (0% prevalence) 15 , a neighboring country, which was explained to be due to the strict closures implemented by the jordanian government and the eventual curtailing of infections in jordan. in comparison to other countries like spain, italy. japan india, los angeles, germany, switzerland the seroprevalence in palestine is low [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . its is, however, noteworthy that a comparison with other countries may be problematic due to the timing and the stage of the pandemic which may vary affecting the seroprevalence estimates. a key strength of our study is the random selection of households residents collected between 15 th june 2020 and 30 th june 2020. the clusters were selected using probability proportional to size (pps) sampling. this technique ensures getting self-weighting sample which can be used to produce unbiased estimators for the parameters of interest. however, samples from female were more difficult to obtain due to a cultural inhibition regarding allowing nurses to enter the all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted september 1, 2020. . households. also, random sample did not include children under the age of 15 to avoid anxiety due to fear of needles in the study to eliminate personal fears due to blood withdrawl. as for the lab referals sample , it represents the population of the lab referals between the 1 st may 2020 and 9 th july 2020. the seroprevalence in this sample was 4 positive cases out of the 1136 samples tested. since the seroprevalence within the lab referral population is close to zero , we used wislon method with continuity correction and truncation (wccbt) to construct a 95% confidence intervale for the population parameter of seroprevalence [0.0011,0096]. our study only detected sars-cov2 antibodies. however, it is important to recognize that cellular immunity may play a role in providing immunity against sars-cov-2 reinfection 18 . further studies aimed at testing cellular immunity are important. it is also noteworthy that previous studies have indicated that asymptomatic individuals were reported to have a weaker immune response to sars-cov-2 infection and a higher percentage of asymptomatic individuals became seronegative when compared to symptomatic individuals in the early recovering phases. the reduction in neutralizing antibody levels may have implications for immunity strategy and serological surveys 19 . in conclusion, our study provides estimates of sars-cov-2 seroprevalence in palestine. our estimate is low indicating that as of july 2020 the population does not have herd immunity. in this situation, health measures have to be taken to keep the outbreak under control. in order to monitor the sars-cov-2 seroprevalence in palestine and inform policy makers about the efficacy of their survalance system, conducting population-based seroprevalence studies on a regular basis is important. outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle world health organization. novel coronavirus(2019-ncov) the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan covid-19 dashboard by the center for systems science and engineering case-fatality rate and characteristics of patients dying in relation to covid-19 in italy prevalence of sars-cov-2 infection in the luxembourgish population the con-vince study serological tests facilitate identification of asymptomatic sars-cov-2 infection in wuhan performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in repeated seroprevalence of anti-sars-cov-2 igg antibodies in a population-based sample from sars-cov-2 seroprevalence trends in healthy blood donors during the covid-19 milan outbreak seroprevalence of covid-19 virus infection in guilan province prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup seroprevalence rate in healthy blood donors from a community under strict lockdown measures seroprevalence of sars-cov-2 -specific antibodies among adults everything or nothing -a better confidence intervals for binomial proportion in clinical trial data analysis robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19 clinical and immunological assessment of asymptomatic sars-cov-2 infections we thank the participants for their cooperation.we thank the registered nurses, laboratory technicians, nursing students and laboratory technician students from the arab american university: ahmad hodrub, mohammad faisal, wajdi tu`ma , adam maraw`a, sharhabeel nasrallah, hisham zahran and mohammad barakatwe thank medicare labs for providing blood samples of individuals visiting their laboratories. no other authors reported disclosures. the study was funded by the arab american university. key: cord-282142-76jr4p7n authors: wang, yun; wang, yiliang; han, xiaoxue; ye, jiazhuo; li, ruiman title: potential effect of covid-19 on maternal and infant outcome: lesson from sars date: 2020-08-07 journal: front pediatr doi: 10.3389/fped.2020.00511 sha: doc_id: 282142 cord_uid: 76jr4p7n the coronavirus disease 2019 (covid-19), caused by sars-cov-2, is highly infectious and its ongoing outbreak has been declared a global pandemic by the who. pregnant women are susceptible to respiratory pathogens and the development of severe pneumonia, suggesting the urgent need to assess the potential maternal and infant outcome of pregnancy with covid-19. the intrauterine vertical transmission potential of sars-cov-2 also remains controversial. herein, we discuss the potential effect of covid-19 on maternal and infant outcomes based on current studies, including those published in chinese, in a total of 80 mothers with covid-19 and 80 infants. we also comprehensively explored the mother-to-child transmission routes of sars-cov-2, in particular the route of intrauterine vertical transmission. given sars-cov-2 is a sister to sars-cov, of the sars-related coronavirus species, we made a comprehensive comparison between them to learn from experiences with sars. although there is no evidence supporting the intrauterine vertical transmission of sars-cov-2, our comprehensive analysis suggests that the adverse maternal and infant outcomes caused by covid-19 cannot be underestimated. further, we speculated that the inconsistency between nucleic acids and serological characteristics igm to sars-cov-2 of infants' specimens may be caused by the disruption of the amniotic barrier by the inflammatory factors induced by sars-cov-2 infection. our review is beneficial to understand the effect of sars-cov-2 on maternal and infant outcomes. at the beginning of december 2019, a cluster of pneumonia cases with unknown causes were reported (1, 2) . a subsequent high-throughput sequencing revealed that the pneumonia epidemic resulted from a novel beta coronavirus tentatively named "2019 novel coronavirus" (2019-ncov) that was subsequently termed "severe acute respiratory syndrome coronavirus 2" (sars-cov-2) (3) (4) (5) . sars-cov-2 is a sister to sars-cov, of the sars-related coronavirus species (4) (5) (6) (7) (8) . pneumonia caused by sars-cov-2 was correspondingly termed "coronavirus disease 2019" (covid-19) (4, 5) . the ongoing outbreak of covid-19 has posed a great threat to global human health, which has been declared by the who as a global public health emergency (1, 2) . as shown by the center for systems science and engineering at johns hopkins university (last updated on 07/07/2020), the global cumulative number of confirmed cases has reached 11,779,263, with 6,758,547 cures, and 540,948 deaths (9) . due to the high transmissibility of covid-19, the prevention, and control of covid-19 infection has become a major concern (4, 5) . pregnant women are more susceptible to respiratory pathogens and the development of severe pneumonia than the general population, especially so for those with chronic diseases or maternal complications (4) . the physiologic changes in pregnancy, including altered cell-mediated immunity, and alterations in pulmonary function, may confer the susceptibility and severity of pneumonia to pregnant women (6, 10, 11) . pneumonia arising from infectious etiology is the most common non-obstetric infectious condition that occurs in pregnant women (6, 12, 13) . in particular, universal sars-cov-2 screening for women admitted for delivery found that all women with positive test results were asymptomatic at the time of testing (14, 15) . therefore, the effect of sars-cov-2 infection on maternal and infant outcomes needs to be explored, especially the intrauterine vertical transmission potential of covid-19. moreover, in the use of a reverse transcriptase-polymerase chain reaction (rt-pcr) and the specific antibody to sars-cov-2 of neonate samples remains controversial (4, 5, (16) (17) (18) (19) (20) . given sars-cov-2 is a sister of sars-cov, it is important for us to learn from the experience of preventing and controlling sars-cov among pregnant people. in this review, we made a comprehensive comparison of sars-cov-2 and sars-cov in genetic, infection, transmission, and clinical characteristics. based on such a comparison, we summarized the potential maternal, and infant outcomes from pregnancy with covid-19 or sars. further, we discuss the potential of mother-tochild transmission of sars-cov-2, in particular the possibility of intrauterine vertical transmission. the guidelines for those women with sars-cov-2 infections during pregnancy and puerperium prepared by numerous experts were also briefly presented (21) . considering the ongoing global public health emergency, we believe that our review is important for understanding the mother-to-child transmission potential of sars-cov-2 and its implication for the safe management of covid-19 in pregnancy. the pathogen contributing to the covid-19 epidemic was tentatively named "2019-ncov" (3, 22) . based on phylogeny, taxonomy, and established practice, the coronavirus study group (csg) of the international committee on taxonomy of viruses formally recognizes this virus as a sister to sars-cov and designates it as sars-cov-2 (8) . a coronavirus is spherical, enveloped, and the largest of the positive-strand rna abbreviations: sars-cov-2, severe acute respiratory syndrome coronavirus 2; sars-cov, severe acute respiratory syndrome coronavirus 1; sars, severe acute respiratory syndrome; covid-19, 2019 novel coronavirus disease; ace2, angiotensin-converting enzyme 2; lga, large-for-gestational-age; sga, smallfor-gestational-age; ttn, transient tachypnea of the newborn; ncpap, nasal-continuous positive airway pressure. virus and sars-cov-2 is the seventh member of enveloped rna coronaviruses with the ability to infect humans (2, 6) . the coronaviruses currently known to infect humans include hcov-229e and hcov-nl63 (alphacoronavirus genus), hcov-oc43, hcov-hku1, mers-cov, sars-cov, and sars-cov-2 (betacoronavirus genus) (23) . sars-cov is the pathogen that caused the sars epidemic from 2002 to 2003 (24) . there were 8,422 cases and 916 deaths in 29 countries, with most of them having occurred in mainland china by 31 july 2003 (24) . given the great similarity between sars-cov and sars-cov-2, a comprehensive comparison between these two viruses can help us learn from the sars epidemic to control and prevent covid-19. their comparisons were mainly presented according to their clinical and viral characteristics ( table 1 ). in general, there was a 79.5% similarity in the whole genome between sars-cov and sars-cov-2, while only a 74.9% similarity in the gene coding spike glycoprotein (3, 22, 25) . both sars-cov-2 and sars-cov spread rapidly from humanto-human transmission (7, (28) (29) (30) (31) (32) . sars-cov can be spread via respiratory droplets, secretions, nosocomial contacts, and mechanical aerosols, such as the aerosols arising from the flushing of toilets (33) (34) (35) . sars-cov-2 seems to spread more easily among humans than sars-cov, which may result from the various modes of transmission and its high affinity with its receptor angiotensin-converting enzyme 2 (ace2) ( table 1) . the latest pilot experiment confirmed that 4 out of 62 stool specimens (6.5%) tested positive to sars-cov-2, and another 4 patients who tested positive toward sars-cov-2 in rectal swabs also had sars-cov-2 detected in their gastrointestinal tract, saliva, or urine (7) . the results suggest the possibility of transmission via aerosols arising from the flushing of toilets. in particular, sars-cov-2 can be detected in esophageal erosion and bleeding sites in cases with severe peptic ulcers after symptom onset (7) . although these results only suggest the existence of sars-cov-2 nucleotides fragments in these samples, sun et al. (36) reported that urine samples of covid-19 patients can isolate sars-cov-2 with the infectious ability, suggesting the existence of infectious viral particles in these samples. moreover, the gastrointestinal tract highly expressed ace2 as indicated by the human protein atlas, which may explain the existence of sars-cov-2 in urine and stool specimens (7) . indeed, the 20-30fold higher affinity of sars-cov-2 spike glycoproteins binding to ace2 than the sars-cov spike protein may also enable the rapid transmission of covid-19 (25) (26) (27) . collectively, the mounting routes of transmission and high affinity with ace2 might jointly contribute to the rapid spread of sars-cov-2. however, covid-19 exhibited a lower-case fatality rate than sars (7) . the median incubation period of sars-cov is also longer than sars-cov (7) . despite the high phylogenetic homogeneity between sars-cov-2 and sars-cov, there are still some clinical characteristics differentiating covid-19 from sars. the symptoms of those infected with sars-cov have been more common in respiratory out-patient clinics and wards (37, 38) . after analyzing the 1,099 covid-19 patients, guan et al. (7) found that the typical radiological finding on chest computed tomographies is groundglass opacity with a ratio of 50.00%. consistent with previous publications (1, 32, 39) , the most common clinical characteristics of covid-19 are fever (87.9%) and cough (67.7%), but not the gastrointestinal symptom that is more frequently observed in sars (7). the absence of fever in covid-19 seems to be more frequent than in sars-cov (1%), as fever occurred in only 43.8% of covid-19 patients on initial presentation (40) , implying the limitation of focusing on fever detection in defining surveillance cases (41) . although there were relatively few cases of patients infected with sars during pregnancy based on previous clinical studies and case reports, there were more than 100 cases of sars-cov infections that occurred in pregnant women as estimated by the who (24) . sars-cov infection during pregnancy was associated with a risk of adverse maternal and neonatal complications, including intrauterine growth restriction, preterm delivery, spontaneous miscarriage, severe maternal illnesses, such as, admission to the intensive care unit (icu), renal failure, and disseminated intravascular coagulopathy, and death (4, 6, 13, (42) (43) (44) (45) (46) . in detail, a case-control study found that the clinical characteristics of sars in pregnant women were similar to those reported for non-pregnant patients with sars (47) . however, all the pregnant women with sars required endotracheal intubation, and six were admitted to the icu, whereas the intubation rate and icu admission rate in the non-pregnant group was only 17.5 and 12.5%, respectively (47) . there were three deaths among pregnant women with sars, while no deaths obstetrical ultrasounds revealed a low-lying placenta (placenta previa), but the pregnancy was otherwise normal. the cesarean section was performed at 38 weeks gestation due to the placenta previa and a healthy baby girl was delivered (48, 49) . antibodies against sars-cov were tested positive from the maternal serum, umbilical cord blood, and breast milk. no viral rna was detected in specimens of maternal serum and whole blood, or in swabs from the maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk. however, no clinical specimens were available for testing from the infant in this study (48) . another 38-year-old woman was exposed to sars-cov in the same hotel as the aforementioned patients (50) . the serum samples taken on days 28 and 64 post-onset of illness tested positive for antibodies against sars-cov. her pregnancy continued and was unremarkable except for developing elevated glucose levels. due to the preterm rupture of membranes and fetal distress, this patient underwent a cesarean section at 36 weeks gestation and obtained a healthy baby boy. the mother's serum samples at the time of delivery were positive for antibodies against sars-cov, but both umbilical cord blood and placenta were negative. also, breast milk sampled 12 and 30 days after delivery were negative for sars-cov antibodies. the specimens, including maternal blood, stool, nasopharynx samples, and umbilical cord blood of the infant, were negative for sars-cov rna. consistently, the stool samples from the neonate obtained on days-of-life 12 and 30 were negative for sars-cov rna. yudin et al. (51) reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks' gestation due to sars. following a 21-day stay in the hospital, the antibody against sars-cov tested positive, while she had a normal labor delivery. together, there were no cases of vertical transmission identified among the pregnant women with sars-cov infection (24, 43, 45, 52) . however, the effect of sars on maternal outcomes seems to be associated with the stage of pregnancy when the onset of sars-cov occurs (44, 53) . wong et al. (44) found that the sars-cov infections present during the first trimester of pregnancy was more likely to cause spontaneous miscarriages, while infections present after 24 weeks of pregnancy developed into delivered preterm. current research involving pregnancy with covid-19 were listed in table 2 . results seems to be inconsistent between antibody-based serological characteristics and rt-pcr-based virologic evidence of infants. specifically, a retrospective study published in the lancet from (5) reported that the clinical characteristics of sars-cov-2 infection in pregnancy were similar to those reported for non-pregnant adults with a sars-cov-2 infection. in brief, the typical symptoms, including fever (in seven of nine patients), cough (in four), myalgia (in three), malaise (in two), and sore throat (in two), were observed in these patients, while none of them developed severe covid-19 pneumonia or died. all patients underwent a cesarean section and their live births had a 1-min apgar score of 8-9 and a 5min apgar score of 9-10 (5). the samples of amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples from six patients tested negative for sars-cov-2 (5), suggesting no intrauterine vertical transmission of sars-cov-2 in the nine pregnant covid-19 patients. however, this study enrolled only nine pregnant women with covid-19, and sample collection was successful in only six infants (5) . another study from chen et al. reported four pregnant women with covid-19 (16) . all mothers recovered from covid-19 and had no critical maternal illness, although one mother suffered severe dyspnea after delivery which required respiratory support, and one developed anemia and dyspnea after admission. of note, none of the three infants whose parents provided consent to be diagnosed tested positive for sars-cov-2 from throat swab samples or developed serious clinical symptoms such as fever, cough, or diarrhea. however, two newborns had a rash, which disappeared spontaneously without treatment; a newborn from the mother with placenta previa was considered to suffer from transient tachypnea of the newborn and was supported by non-invasive mechanical ventilation for 3 days. of note, a study published in jama pediatrics indicated three neonates born to a pregnant woman with covid-19 tested positive for sars-cov-2 by qrt-pcr (20) . however, as indicated by the medical record, the throat swab sample of the neonate was collected at more than 48 h after delivery. no direct testing of intrauterine tissue samples, such as amniotic fluid, cord blood, or placenta, was collected to detect sars-cov-2, which is critical for confirming that the sars-cov-2 infection in the neonate was due to intrauterine transmission (20) . therefore, intrauterine sars-cov-2 infection remains uncertain. recently, two studies published in jama from separate research teams in china reported that three neonates may have acquired sars-cov-2 in utero from mothers with covid-19 based on the elevated igm antibodies to sars-cov-2 in neonates (17, 19) . specifically, the study from zeng et al. made a retrospectively review for six pregnant women with covid-19 (19) . all these mothers had mild clinical manifestations and performed cesarean deliveries in their third trimester. of note, all six newborn babies had a normal 1-and 5-min apgar score and none of them presented any symptoms of covid-19. however, serological characteristic results indicated that two infants had sars-cov-2-specific igg and igm concentrations higher than the normal level (<10 au/ml). given that igm is not usually transferred from mother to fetus because of its larger macromolecular structure under normal conditions (57), the author speculated that the neonates may have been infected with sars-cov-2 in utero from mothers with covid-19. however, all neonatal throat swabs and blood samples had negative rt-pcr test results. moreover, this study is limited by the small sample size, lack of cord blood, placenta, amniotic fluid, mother's vaginal secretions, and breast milk and by incomplete information on the outcome of the infants (19) . similar to the case mentioned above, another study from lan et al. reported that an infant girl born to a mother with covid-19 (34 weeks, 2 days of gestation) may have acquired sars-cov-2 in utero due to the elevated igm antibodies to sars-cov-2 (17). however, both the infant's nasopharyngeal swabs and breast milk sampled 3 days after delivery had a negative rt-pcr test result of sars-cov-2. moreover, all neonates had a normal 1-and 5-min apgar score. the mother's vaginal secretions obtained at delivery also tested negative for sars-cov-2. however, this study is limited by the single case, and the lack of amniotic fluid and placenta. there was also no detailed information regarding the pregnancy stage of the onset of covid-19. in summary, there was no positive rt-pcr result in the neonate specimens obtained within 24 h post-birth (5, 14, 16, 17, 19) , implying no virologic evidence for congenital infection. however, the serological characteristics of infants reported three neonates with elevated igm antibodies to sars-cov-2 born to a mother with covid-19, suggesting a possible vertical transmission of sars-cov-2 from mother to newborn (17, 19) . indeed, the virologic evidence for supporting the utero transmission should be diagnosed based on rt-pcr test results of the samples from neonates but not igm detection with a high incidence of its false-positive and false-negative results (58, 59) . a reasonable explanation for such inconsistency may be the disruption of the placenta or amniotic barrier caused by the inflammatory mediators from mothers that, induced by sars-cov-2, facilitates the cross of igg and igm. in detail, lga, large-for-gestational-age; sga, small-for-gestational-age; ttn, transient tachypnea of the newborn; ncpap, nasal-continuous positive airway pressure. the placenta is a barrier to viral infection (60) . the damage of the placenta by sars-cov-2 may represent an important link in the vertical transmission according to the experience from sars-cov. the two placentas from women who were recovering from sars-cov infection in the third trimester of pregnancy had abnormal weights and pathologies (53) . by contrast, in the case of covid-19, whether the placentas from those pregnant while infected with covid-19 were abnormal or damaged in most of these studies are unknown (5, 16, 17, 19) . indeed, a study reported that there were various degrees of fibrin deposition inside and around the villi with local syncytial nodule increases in three placentas from those pregnant while infected with covid-19, especially a placenta with massive infarction (55) . however, these three placenta samples tested negative for the nucleic acid of sars-cov-2, suggesting no virologic evidence in the placenta (55) . however, another study revealed that sars-cov-2 invasion of the placenta in a woman with covid-19 in the second trimester through molecular and immunohistochemical assays and electron microscopy (61) . moreover, the public antibody-protein profiles resident in human protein atlas (hpa) revealed enrichment of the sars-cov-2 receptor ace2 in the placenta and ovary (62) . collectively, the possibility of sars-cov-2 infection acquired from the uterus cannot be excluded, highlighting the potential for severe morbidity among pregnant women with covid-19. an editorial published in jama holds that sars-cov-2 can theoretically be transmitted in the uterus, especially given that virus' nucleic acid has been detected in blood samples (59) . however, nucleic acids do not represent infectious particles. indeed, it had been revealed that inflammatory mediators, including il-6, il-1î², and tnf-î±, cause severe dysfunction of the amniotic barrier via decreasing the expression of tight junctions-associated factors claudin-3 and claudin-4 and inducing apoptosis of the amniotic epithelial cells (63) . of note, il-6 has prominent pro-inflammatory properties (64) . il-6 was significantly increased in all infants from mothers with covid-19 (19) and the clinical and immunological features suggested that both the concentration of il-6 and tnf-î± are higher in severe covid-19 patients than in moderate patients (65) . the elevated igm antibody to sars-cov-2 in the blood was not observed in all neonates, which may be associated with the different levels of inflammatory mediators among them. collectively, in addition to the possibility of false-positive and false-negative results of igm (59), disruption of the placenta barrier and amniotic barrier caused by inflammatory mediators causing the elevated igm concentration also needs to be further investigated. however, determination of the level of ordinary igg but not specific to sars-cov-2 in neonate blood would be a crucial indicator explaining the disruption of the placenta and amniotic barrier. in general, the routes of mother-to-child transmission of sars-cov-2 mainly include intrauterine vertical transmission, birth, or breastfeeding. there is currently no evidence supporting the intrauterine vertical transmission of both sars-cov and sars-cov-2 based on the discussion above (4-6, 42, 51) . however, all the pregnant women recruited in these studies were in their third trimester. of note, the effect of sars-cov-2 on the infant and maternal outcome may be closely associated with their pregnancy stage during the virus infection, which was observed in both sars-cov and rubella (44, 66) . therefore, the possibility of intrauterine transmission in pregnancy with sars-cov-2 infection in the first or second trimester of pregnancy cannot be overlooked. the potential damage caused by inflammatory factors (above) also needs to be assessed. for the transmission during birth, most of the people pregnant while infected with covid-19 discussed above underwent a cesarean section to deliver the live births in current studies, three neonatal from which exhibited early-onset infection with sars-cov-2 (20) . by contrast, there were ten patients with covid-19 who performed vaginal delivery, all infants from which tested negative for sars-cov-2 (16, 20, 54) . of note, such low transmitted cases were greatly based on the comprehensive protective methods. indeed, the samples of vaginal mucosa and shedding in birth canals are crucial samples indicating whether sars-cov-2 could be transmitted during vaginal delivery. there were few studies that collected vaginal secretion (1/80) or infant blood (12/80); all tested negative for sars-cov-2 (5). further, as revealed by hpa tissue atlas, vaginal secretion expresses virtually no ace2 (62) , implying that sars-cov-2 may not infect the tissue. together, the risk of sars-cov-2 transmission by vaginal delivery seems low, although more definitive evidence is required. finally, to determine the potential of sars-cov-2 transmission via breastfeeding, several studies collected and analyzed breast milk samples (7/63) from patients with covid-19 pneumonia after their first lactation (5, 17) . however, these samples tested negative for sars-cov-2, suggesting no evidence supporting the breastfeeding transmission of sars-cov-2 (5). of note, such results were similar to pregnancies with sars-cov infection. no viral rna was detected in the specimens of umbilical cord blood, amniotic fluid, and breast milk from those pregnant while infected with sars-cov (48) (49) (50) . indeed, the antibody against sars-cov can be tested from the umbilical cord and breast milk (48) (49) (50) . based on such experiences from sars-cov, the antibody against sars-cov-2 derived from pregnancy may penetrate the placental barrier to orchestrate antiviral defense in the fetus to combat sars-cov-2, which needs to be further determined. in summary, there was a low possibility for mother-to-child transmission of sars-cov-2 if adequate protective measures were taken. however, the most crucial point is the potential effect of covid-19 on maternal and fetal outcomes, rather than whether sars-cov-2 can be acquired from the uterus; however, the determination of mother-to-child transmission potential is also important. that said, the effect of covid-19 on maternal and fetal outcomes should be paid considerable attention. according to the experience from sars, although no mother-to-child transmission was observed in sars, sars-cov infection was associated with a high risk of severe maternal illness, maternal death, and spontaneous miscarriages (4, 6, 13, (42) (43) (44) (45) (46) . indeed, maternal pneumonia is closely associated with a high incidence of various adverse obstetrical outcomes, including the premature rupture of membranes, preterm labor, intrauterine fetal demise, intrauterine growth restriction, and neonatal death (67) (68) (69) . further, although observed in a few cases, covid-19 may be related to the adverse maternal and infant outcome, including premature births, fetal distress, abnormal fetal liver function, rapid heart rate, etc. ( table 2) . to address the safety issues for the obstetrical management and delivery of pregnant women with covid-19, the advice for those women with sars-cov-2 infections during pregnancy and puerperium was prepared by numerous experts from the fields of obstetrics and gynecology, pediatrics, infectious diseases, and critical care (21, (70) (71) (72) (73) (74) (75) . similar to the recommendations for the non-pregnant, early isolation, early diagnosis, and early management are still the core criteria of prevention and control transmission for pregnant women with suspected and probable sars-cov-2 infection. these recommendations mainly include: 1. at times of covid-19 outbreaks, all pregnant patients should be assessed for travel history or contact with people from the worst-hit areas of the epidemic within 2 weeks. the definition of a case with suspected covid-19 should be focused on the clinical symptoms of covid-19; 2. pregnant women with labor-confirmed sars-cov-2 infection should be treated centrally according to the designation by the department of medical administration. the corresponding risk of adverse pregnancy outcomes contributed by covid-19 should be informed to the patients; 3. a chest radiograph, especially the computed tomography, is crucial for evaluating the development of covid-19; 4. pregnant women with suspected or probable covid19 should be informed to the cdc and placed in an isolation room or a negative pressure room if it is available; 5. prenatal examination and delivery of pregnant women with a sars-cov-2 infection should be carried out in negative pressure isolation or on an isolation ward. the management medical staff should wear protective equipment; 6. the timing of childbirth should be based on the specific conditions of the mother and child, the gestational week, and the childbirth conditions. the delivery mode depends on obstetric indication; 7. the specific anesthesia method for sars-cov-2-infected pregnant women who require surgical delivery can be general anesthesia and regional anesthesia, which should be performed based on the professional anesthesiologist; 8. given that the possibility of the intrauterine vertical transmission of sars-cov-2 cannot be excluded, all newborns from pregnant patients with suspected or confirmed covid-19 should be isolated for at least 14 days and should not be breastfed during this period until a sars-cov-2 infection is ruled out or cured. the mothers should squeeze milk regularly to ensure lactation. an expert team consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anesthesiologists should jointly manage pregnant women with covid-19 and their newborn baby; 9. pregnant women with covid-19 should be managed by fixed staff, including obstetrics, neonatal, and other related professionals. the healthcare workers caring for pregnant covid-19 patients should not care for other patients. all healthcare workers should be daily monitored for fever and cough symptoms of covid-19. such individuals should be isolated if they were confirmed or suspected of covid-19; 10. all health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent the spread of sars-cov-2. yuw, yiw, and jy: conception and design, collection and/or assembly of references, data analysis, interpretation, and manuscript writing. xh and rl: conception and design, manuscript writing, and final approval of manuscript. all authors read and approved the final manuscript. clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding what are the risks of covid-19 infection in pregnant women? clinical characteristics 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coronavirus disease-19 brief clinical recommendations: management tactics for pregnant women and women in labor and childbirth with suspected or confirmed covid-19 infection funding this work was supported by grants from the research and industrialization of medical devices for digital childbirth (no. 2015b020233010). key: cord-277841-7sp8ftbc authors: kumari, pratibha; singh, archana; rinchui ngasainao, moses; shakeel, ilma; kumar, sanjay; lal, seema; singhal, anchal; singh sohal, sukhwinder; kumar singh, indrakant; imtaiyaz hassan, md. title: potential diagnostics and therapeutic approaches in covid-19 date: 2020-08-12 journal: clin chim acta doi: 10.1016/j.cca.2020.08.013 sha: doc_id: 277841 cord_uid: 7sp8ftbc abstract the most important aspect of controlling covid-19 is its timely diagnosis. molecular diagnostic tests target the detection of any of the following markers such as the specific region of the viral genome, certain enzyme, rna-dependent rna polymerase, the structural proteins such as surface spike glycoprotein, nucleocapsid protein, envelope protein, or membrane protein of sars-cov-2. this review highlights the underlying mechanisms, advancements, and clinical limitations for each of the diagnostic techniques authorized by the food and drug administration (usa). significance of diagnosis triaging, information on specimen collection, safety considerations while handling, transport, and storage of samples have been highlighted to make medical and research community more informed so that better clinical strategies are developed. we have discussed here the clinical manifestations and hospital outcomes along with the underlying mechanisms for several drugs administered to covid-19 prophylaxis. in addition to favourable clinical outcomes, the challenges, and the future directions of management of covod-19 are highlighted. having a comprehensive knowledge of the diagnostic approaches of sars-cov-2, and its pathogenesis will be of great value in designing a long-term strategy to tackle covid-19. the ongoing coronavirus pandemic is the most catastrophic global crisis after the second world war [1] . covid-19 is a contagious disease, caused by a novel severe acute respiratory syndrome coronavirus (sars-cov-2). the genome analysis of this new virus showed ~79.5% similarity with sars-cov and middle east respiratory syndrome coronavirus (mers-cov) [2] . however, the highest sequence similarity (~96%) was observed for the bat coronavirus. therefore, it has been speculated that covid-19 was transmitted from bats to humans. recent studies suggest an intermediary animal host, which potentially could be pangolin [3] or dog [4] . earlier studies on covid-19 suggest that this disease spreads through close contact through the infected person and with small respiratory droplets released while coughing, sneezing, or talking [5] . tiny droplets of saliva or sputum released from the mouth may carry heavy loads of viruses that are known to stay in the air for a long time and hence potentially can act as carriers of infection [6] . the inhalation of these minute droplets leads to the spreading of viral infection from the sick to a healthy person even when a person is not in direct physical contact with the infected person. even though covid-19 is not an airborne disease, some medical practices such as cardiopulmonary resuscitation and intubation may trigger respiratory secretions that can get aerosolized and hence resulted in its spread [7] . the virus gets its way inside the human body through eyes, nose, and mouth and thus spread through contacting the virus from contaminated surfaces and then touching these body parts [8] . however, the extent of viral transmission through contaminated surfaces depends upon environmental conditions such as temperature and humidity [9] . the entry of sars-cov-2 into host cells is facilitated by the binding of homo-trimer spike protein (s) that is on the surface of the virus to ace2 on the host's cell membrane [10] . the recognition of the host cell receptor is a key determinant of the tissue tropism and pathogenicity of the virus [11] . a schematic representation of the life cycle of sars-cov-2 is illustrated in figure 1 . the life cycle of sars-cov-2 is similar to the sars-cov, mers-cov [12] . sometimes, animal viruses get accidentally transferred to humans and can evolve itself causing sickness. thus, it becomes a human host-virus, such as sars-cov, mers-cov, and sars-cov-2. the current covid-19 pandemic has intensified the interest of scientists all over the world for developing treatment options to mitigate the impact of the disease on human life [12, 13] . more than 300 clinical trials are currently in progress for the treatment of covid-19. different strategies have been adopted to combat covid-19. however, there is no approved treatment for the same till date. in this article, we evaluated literature for reports informing various diagnostic methods, potential antiviral chemical therapeutics, and effective treatment strategies towards clinical management of covid-19 patients. diagnosis of covid-19 is a critical step in tracing the virus and understanding its epidemiology. this helps to block the transmission and ensure proper patient care. the preliminary step of covid-19 diagnosis is through observation of signs and symptoms such as initial loss of smell or taste or both [14] , cough, mild to high fever, myalgia, or fatigue [15] , etc. in addition, several gastrointestinal symptoms of vomiting, diarrhoea, and nausea in some individuals are observed [16] . however, inconsistencies in the manifestation of symptoms from asymptomatic to severe cases like, septic shock, imbalanced metabolic acidosis, coagulation dysfunction, and acute respiratory pneumonia-like syndrome are mostly reported [17] . the presentation of signs and symptoms should be considered as the basis for further tests and not for diagnostic purposes. the prime consideration in the diagnosis of covid-19 is the detection of symptoms in clinical situations. conventionally, samples for pathology are collected from upper and lower respiratory regions (throat, oropharyngeal, nasopharyngeal, bronchoalveolar fluid, and sputum) through swabs for rt-pcr test. in the blood and urine of the infected, there is still an absence of the virus; hence, these are not considered a useful clinical specimen. reports of inconsistency in rt-pcr test results for cov-sars-2 in various tissues [18] and temporal variation of test results from the same tissues [19] suggests that the inter-link between the temporal surge of viral load and its bio-distribution in different tissues of the body would have a critical implication on the accuracy of various tests for diagnosis. many assays or tools have been commercialized for the diagnosis of covid-19 and many more are currently in development. various molecular assays and immunological assays are currently validated in different laboratories. we discuss here some techniques for the diagnosis of sars-cov-2 pathogenesis: the knowledge of the viral genome sequence has made it possible for us to use molecular techniques in the detection of sars-cov-2. molecular diagnostic methods target to detect either specific regions of the viral genome or rna-dependent rna polymerase (rdrp) and/or structural proteins of sars-cov-2 (table 1) . currently, the rt-pcr test is a single discrete standard method for the diagnosis of sars-cov-2. the rt-pcr protocol involves the extraction of rna from the specimen and reverses the transcription to cdna followed by pcr amplification of a specific region with sars-cov-2 specific primers and detection through specific probes. evaluation of rdrp and envelope (e) genes was originally devised [20] which was adopted by charité germany for the diagnosis of covid-19. the assay screens the e gene which is a pan-sars-betacoronavirus gene. the confirmatory test is done by targeting the rdrp gene using specific primers and probes listed in table 2 . the limit to detection is 3.6 copies (rdrp gene) and 3.9 copies (e gene) per reaction and cycle threshold value of less than 37.0 is treated as a positive test. specific probes and primers target the 1ab gene (orf1 ab gene or transcriptase/replicase gene) as a confirmatory assay. while the level of e gene confirms the presence of sars related virus. the minimum limit of detection is taken as 1000 copies/ml [18] . the cycle threshold value of less than 40 is set as positive confirmation test criteria. three nucleocapsid protein gene (n gene) regions are targeted using primer and probe ( table 2 ). the assay uses individual probe and primers for three n genes (n1, n2, n3 gene) with an additional primer and probe for detecting human rnase p. n3 gene primer and probe sets are for detection of all sars-like coronaviruses. the sensitivity of this assay is lower than other assays as it has a limit of detection of 8.3 copies per reaction. [21] . for the qualitative detection of the specific gene sequence of sars-cov-2, the sample is generally collected as nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage, or nasopharyngeal wash/aspirate as recommended by the fda. in addition, swabs of upper respiratory specimens including nasopharyngeal, nasal swabs, or mid-turbinate are collected from an individual, with or even without symptoms of covid-19. despite the great advantages of these methods, a well-trained technical person is required to carry out such diagnostic procedures. potential of these molecular tools are restricted to the samples obtained from the respiratory tracts of the suspected individuals. sputum, nasopharyngeal aspirates, bal fluid, nasal aspirates, nasopharyngeal or oropharyngeal swabs can only be tested through this approach. also, the chances of falsenegative results become high when the lab reagents are contaminated, used past their expiry date, or samples are not timely collected from the right region. false-negative results are also obtained with improper storage and transport of specimen, the presence of amplification inhibitors in samples, and if the mutation rate of the virus is high during the pcr cycle [63]. detector assay is an rna-sensing assay that uses synthetic sars-cov-2 rna fragments to recognize the signature of e and n gene sequences of sars-cov-2. viral rna targets are reversed transcribed to cdna and amplified which subsequently transcribed back to rna isothermally. the rna fragments in the reaction combine with cas13a protein (ribonuclease) that binds with the amplified rna product forming a sherlock. ribonuclease (cas13a) then cleaves the surrounding fluorophore-quencher probes emitting signals. this crispr-cas13 is a working protocol for sars-cov-2 detection that has been rolled out for clinical trials [22] . for the diagnosis of covid-19 by immunological technique, viral antigen (antigen test) present in the specimen or antibodies or immunoglobulins produced (antibody test) against specific components of sars-cov-2 are detected. these are blood-based tests commonly used to detect antibodies generated against sars-cov-2 after the infection. antibody response to sars-cov-2 infection is similar to any typical viral infection [23] . therefore, the chance of antibody cross-reactivity from infections of other pathogens can exist and sometimes be misinterpreted. like most immunological diagnostic protocols, enzyme-linked immunosorbent assay (elisa) for covid-19 detection uses igm and igg antibody against nucleocapsid (n) and receptor binding domain spike proteins (s) of sars-cov-2. the immunochromatographic test is a rapid chromogenic double antigen/double antibody sandwich elisa developed in the wake of the sars-cov outbreak during 2004 [24] . recombinant n protein of sars-cov-2 was taken as an antigen which is immobilized on a nitrocellulose strip with igg coupled to colloidal gold particles. anti-sars-cov-2 n protein antibody igg binds to the sars-cov-2 antigen to form red-coloured antibody-antigen-gold complex ( figure 2 ). the specificity of the immunochromatographic test is around 98.2% in various kits. most tests for rn are done through an indirect elisa method using igg where n protein is bounded with igg/igm antibody to a microplate or nitrocellulose membrane. a secondary antibody conjugated with horseshoe reddish peroxidase (hrp) binding to the complex is detected by the generation of colour on the addition of substrate tmb (3,3'5, 5' tetramethylbenzidine). the sensitivity for the igg tests is reported to be 99.0% [23] . ab-elisa is a double-antigen sandwich immunoassay for the detection of total antibodies that binds to the sars-cov-2 spike protein receptor domain (rbd) in plasma or serum ( figure 3 ). in this method, immobilized mammalian cell-expressed recombinant antigen containing the receptor-binding domain of the virus spike proteins and hrp-conjugate antigen were used. the specificity of the ab-elisa test kit is 99.1% [23] . igm specific for recombinant spike protein rbd detection is done using hrp-conjugate and tmb as a substrate for the colorimetric test following igm µ-chain capture method. the specificity of igm elisa is 98.2% [23] . a sensitivity value of above 68% is taken as sars-cov-2 nucleocapsid protein test positive [25] . in principle, the detection of viral antigen would be the virus-specific marker for the diagnosis of covid-19. these diagnostic methods are having a great advantage of convenience and rapidness but limited by inadequate sensitivity and specificity compared to standard rt-pcr. due to these reasons, results from these methods need to be interpreted with caution. change in physio-pathological parameters such as hematological and histopathological can act as a measure to detect sars-cov-2. a blood sample of suspected patient is taken for hematological diagnosis. many studies have shown that individuals with sars-cov-2 exhibit clinical characteristics of systemic inflammation such as lymphopenia, neutrophilia, thrombocytopenia, lowered serum albumin and increased c-reactive proteins (crps), lactate dehydrogenase, aspartate aminotransferase, alanine transferase, cardiac troponin, erythrocyte sedimentation rate, d dimer, ferritin, creatine kinase, etc [26] . until now, a well-established hematological parameter/ pathophysiological novel marker for covid-19 is still lacking. however, monitoring of hematological parameters could serve as a clinical indicator of severity and prognostication of the disease for early intervention of treatment, but not as a diagnostic tool. computed tomography (ct) is serving as a valuable technique for the diagnosis and progression of the disease. ct scan results of covid-19 infected individual shows a variety of abnormalities that are unique from other viral pneumonia infections of sars, mers, and adenoviruses [27] . ct scan was found to be more appropriate in providing insights about pneumonia but the results were variable in different patients highlighting the different stages the virus might have been at that time [28] . the scan manifestation unique to covid-19 patients includes patchy lesion-that are nodular to irregularly shaped have ground-glass opacity (ggo) and are consolidated in the subpleural region of the central lung lobes [29] . they have distinct reversed halo sign and pulmonary nodules with halo sign [27] . a followup ct scan revealed that the lesions were migratory with the phenomenon of absorption of the primary lesion to the emergence of new lesions in the pleural and sub-pleural regions [30] . since the varied representation of pneumonia is obtained by cxr and ct scan in different patients, therefore it was difficult to conclude the actual pathogenic mechanism [31] . also, certain asymptomatic patients showed early changes under ct scan which is an excellent approach to identify the patients without symptoms however; this was not always the case [32] . the crucial factors of diagnosis and treatment of virus-related diseases can be effectively addressed through nanotechnological platforms. the nanomaterials and microfabrication technologies offer valuable diagnostic tools for viral detection in ultralow concentration in blood, serum, and plasma ( figure s1) . nanomaterials respond to a minor stimulus and provide a signal even to ultra-low concentrations. plasmonic metal nanoparticles, such as gold nanoparticles (aunps), offer high surface area, which immobilizes various analytes and induces a significant change in plasmonic signal. a colorimetric detection methodology has been reported for the detection of influenza virus a (iva) using gold nanoparticles (nps) modified with monoclonal anti-hemagglutinin antibodies (mab). the aunps-mab probes are sensitive for surface recognition sites of the virus and induce the plasmon effect in the attached aunps, leading to visual detection of viral strains [33] . the enveloped virus proteins like hemagglutinin primarily may be utilized for detection parameters for the rapid and easy detection of viral infections. zhao et al. [34] reported the fluorescent quantum dots (qds) and superparamagnetic nanoparticle beads functionalized with mab for diagnosis of avian influenza virus (aiv). the detection of infectious viruses using various other nanomaterials such as sinps [35] , agnps [36] , cunps [37] , and cnts [38] despite the challenges associated with covid-19 therapy, there are still several approaches currently being undertaken which show significant outcomes [40] . in this section, we shall discuss the positive impacts of some of the clinically used medications for the treatment of severe covid-19 patients ( figure s2, table s1 ). several drugs are in clinical trials and some of them have shown significant promise in addressing covid-19 patients [13, 41, 42] . the site of action of these drugs is illustrated in figure 4 . in addition, to drugs under clinical trials, several vaccines are in the pipeline which is expected to play a significant role in controlling the covid-19 pandemic (table s2) . this section provides a brief discussion of some of the promising drugs which are used to fight sars-cov-2 infection. remdesivir (gs-5734™) is a novel small molecule, a broad-spectrum antiviral nucleoside analog that is now being investigated as a potential treatment option for covid-19 [43] . it belongs to the class of polymerase inhibitors [44] . it is reported to show activity against different rna viruses, including sars-cov, mers-cov, nipah virus, hendra viruses, and ebola viruses. remdesivir is a prodrug of its parent adenosine analog, gs-441524. both of these medicines metabolized into the active component as nucleoside triphosphate (gs-443902) after ingestion and they show antiviral activity against sars-cov [45] . being a nucleoside analog, remdesivir targets the viral genome replication process by acting as an rdrp inhibitor. yin et al. [46] established the structural basis leading to inhibition of the rnadependent rna polymerase of sars-cov-2 using remdesivir. on metabolization of remdesivir into active nucleoside triphosphate (ntp) which competes with atp for incorporation into nascent rna strand resulting in premature rna synthesis leading to termination and ceasing of growth of rna strands [45] . it has been reported that remdesivir and chloroquine are highly effective in the control of sars-cov-2 infection in-vitro. recently, who has claimed that antiviral drug remdesivir failed in the first trials for the treatment of covid-19 disease [47] . however, the drug now has become a matter of debate between the clinical experts, scientists, and us-based pharmaceutical companies. there are still controversies regarding the results obtained from this trial, as clinicians suggest no benefit in covid-19 treatment using remdesivir; whereas, the company claims it as a promising drug for the same [48]. lopinavir/ritonavir has been used in combination with other anti-retroviral medicinal products for the treatment of hiv-1 infected people. during the present pandemic of covid-19, this combination of drugs has shown potential for the treatment, and many trials are under progress. lopinavir/ritonavir act as a protease inhibitor drug and inhibit the action of 3clpro, chymotrypsin-like protease enzyme, that plays a vital role in the processing of virus and thus interfere with the process of viral replication and its release from host cells. in an adaptive, randomized controlled clinical trial, lopinavir/ritonavir has been administered twice orally as tablets (200 mg and 50 mg) for 14 days to patients. while the others who cannot swallow, 5 ml oral suspension is provided twice daily, followed by a daily assessment. the study is in phase 2 clinical trial [50] . in another phase-2/phase-3 clinical trial, a random, double-blind, multi-centric investigation is being carried out on covid-19 patients. four different groups of patients are receiving lopinavir/ritonavir and hydroxychloroquine. the study started on the 6 th of april 2020 and due completion in april 2021 [51]. novaferon has shown great potential as an antiviral drug against covid-19 [52] . it is a chloroquine is a widely used antimalarial drug that was observed to have a broad-spectrum antiviral activity [53] . it has been reported to block sars-cov-2 infection at low micromolar concentrations [54] . chloroquine has the inhibition capability of replication of several intracellular micro-organisms, including sars-cov-2. the use of chloroquine increases endosomal ph and thereby interferes with the glycosylation of the cellular receptor of sars-cov. therefore, it has the potential to block viral infection. hydroxychloroquine is a 4aminoquinoline with immunosuppressive, anti-autophagy with antimalarial effects [55] . it has been purposed that hcq interferes with the processing and presentation of antigens and production of cytokines and hence suppresses immune functions. during the pandemic of covid-19, hcq has been studied and under numerous trials for the treatment of coronavirus disease [56] . in two small, uncontrolled studies, hcq and its congener, chloroquine, were reported to be effective against covid-19, however, the publishing journal later declared that the trial did not meet the society's expected standard [57] . due to severe complications observed in the covid-19 patients, who recently discouraged the use of both cq and hcq [58] . auranofin is an fda approved lipophilic organogold compound which has been used to treat rheumatoid arthritis. it exhibits anti-inflammatory and potential antineoplastic activities. these famotidine is a competitive histamine h2-receptor antagonist that inhibits gastric secretion. famotidine is the active compound in the heartburn drug, pepcid. according to a report, on the 27 th of april 2020, northwell health in the new york city area began a clinical trial to test famotidine against sars-cov-2. the trials have been underway since 7 th of april with 1,174 patients, including 187 who were critically ill. famotidine is structured in such a way that it could prevent the replication of sars-cov-2 protease [61] . huaier granule is under phase 2 and phase 3 clinical trial for the treatment of covid-19, in a study started by chen xiaoping at tongji hospital on 1 st of april 2020, over 550 patients are under investigation primarily for their mortality rate, and icu stays [62] . this study is likely to be completed by september 2020. another example of nucleoside analog used to stop viral rna synthesis and viral mrna capping with broad-spectrum antiviral activity is ribavirin which has been analysed for its action against sars-cov-2 [65] . it is a prodrug, which when metabolized resembles purine rna nucleotides that interferes with rna metabolism required for viral replication. it has been observed during a comparative study on sars-cov-2 patients treated with combined therapy of lopinavir/ritonavir and ribavirin; that they are at lower risk of death than when receiving ribavirin monotherapy [66] . ivermectin is an fda-approved anti-parasitic drug having broad-spectrum antiviral activity in vitro. a laboratory level study demonstrated that ivermectin has antiviral action against the sars-cov-2 clinical isolate in vitro in the vero-hslam cells. researchers have claimed that a single dose of ivermectin was able to cause ~5000-fold reduction in viral rna at 48 h. it has led to the hypothesis that this is likely through inhibition of impα/β1-mediated nuclear import of viral proteins [67] . however, on the 10 th of april 2020, the fda issued guidance not to use ivermectin intended for animals as a treatment for covid-19 in humans [68]. it is an antiviral widely used to treat the influenza virus. arbidol can effectually prevent sars-cov-2 infection in vitro [41] . lopinavir/ritonavir and arbidol have been recently recommended by the national health commission and national administration of traditional chinese medicine for dealing with covid-19 [69] . a recent study, data suggest that arbidol monotherapy is more effective than lopinavir/ritonavir in treating covid-19. no viral load was observed in the arbidol group than the 44.1% viral load in patients having lopinavir/ritonavir on the 14 th day of treatment. additionally, no apparent side effects were found in both groups [70] . darunavir is a second generation of hiv-1 protease inhibitors used to prevent sars-cov-2 infection in-vitro [71] by inhibiting viral replication at 300 μm and this inhibition efficiency was 280-fold compared to the untreated group. carmofur is a pyrimidine analog used as an antineoplastic agent. it is an orally delivered sars-cov-2 is more contagious, sporadic, and deadly as compared to previously known two tests-a rapid assay for detection of antibodies reactive to recombinant viral protein and neutralization assay wang lab currently in use in 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role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro management of corona virus disease-19 (covid-19): the zhejiang experience arbidol monotherapy is superior to lopinavir/ritonavir in treating covid-19 spica = solid phase immunochromatographic assay, na = neutralization assay) table 2: primers and probes for targeting sars-cov-2 genes in an rt-pcr test for covid-19 diagnosis. the sequence of the primers and probes are written in 5՜ to 3՜-end pattern (left to right). probes labelled at the 5'-end = reporter (fam = 6-carboxyfluorescein, hex = hexachloro-fluorescein) and at 3'-end = quencher (bhq1 = black hole quencher1 the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper key: cord-289716-nleql08z authors: tsitsilonis, ourania e.; paraskevis, dimitrios; lianidou, evi; pierros, vassilios; akalestos, athanasios; kastritis, efstathios; moutsatsou, paraskevi; scorilas, andreas; sphicopoulos, thomas; terpos, evangelos; thomaidis, nikolaos; tsakris, athanassios; voulgaris, nikolaos; daskalaki, christina c.; evangelakou, zoi; fouki, christina; gianniou, despoina d.; gumeni, sentiljana; kostaki, evangelia-georgia; kostopoulos, ioannis v.; manola, maria s.; orologas-stavrou, nikolaos; panteli, chrysanthi; papanagnou, eleni-dimitra; rousakis, pantelis; sklirou, aimilia d.; smilkou, stavroula; stergiopoulou, dimitra; trougakos, ioannis p.; tsiodras, soritios; sfikakis, petros p.; dimopoulos, meletios-athanasios title: seroprevalence of antibodies against sars-cov-2 among the personnel and students of the national and kapodistrian university of athens, greece: a preliminary report date: 2020-09-21 journal: life (basel) doi: 10.3390/life10090214 sha: doc_id: 289716 cord_uid: nleql08z due to early implementation of public health measures, greece had low number of sars-cov-2 infections and covid-19 severe incidents in hospitalized patients. the national and kapodistrian university of athens (νκua), especially its health-care/medical personnel, has been actively involved in the first line of state responses to covid-19. to estimate the prevalence of antibodies (igs) against sars-cov-2 among nkua members, we designed a five consecutive monthly serosurvey among randomly selected nkua consenting volunteers. here, we present the results from the first 2500 plasma samples collected during june–july 2020. twenty-five donors were tested positive for anti-sars-cov-2 igs; thus, the overall seroprevalence was 1.00%. the weighted overall seroprevalence was 0.93% (95% ci: 0.27, 2.09) and varied between males [1.05% (95% ci: 0.18, 2.92)] and females [0.84% (95% ci: 0.13, 2.49)], age-groups and different categories (higher in participants from the school of health sciences and in scientific affiliates/faculty members/laboratory assistants), but no statistical differences were detected. although focused on the specific population of nkua members, our study shows that the prevalence of anti-sars-cov-2 igs for the period june–july 2020 remained low and provides knowledge of public health importance for the nkua members. given that approximately one in three infections was asymptomatic, continuous monitoring of the progression of the pandemic by assessing ig seroprevalence is needed. the worldwide outbreak of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which emerged in wuhan, china in december 2019 rapidly spread to more than 200 countries and as of 21 august 2020, over 22 million confirmed cases and nearly 800,000 deaths from the acute respiratory disease named coronavirus infectious disease 2019 (covid-19) were recorded [1] . due to the ease of viral transmission primarily through person-to-person contact, crowded and confined indoor spaces or indoor occurring events have been directly associated with higher infection rates [2] . the risk of transmission from symptomatic and pre-symptomatic patients is in general considered to be higher. however, asymptomatic infection frequently occurs and the potential of viral transmission from asymptomatic individuals bearing similar viral loads in their respiratory and gastrointestinal samples with symptomatic cases has also been reported [3] [4] [5] . molecular techniques based on rt-qpcr are used to diagnose acute sars-cov-2 infection in principle in nasopharyngeal swabs and recovery from infection in most cases correlates with pcr negativity [6] . within 2-3 weeks after the onset of symptoms, the immune system of infected individuals reacts by producing antibodies (igs) to sars-cov-2 proteins, which can be detected in peripheral blood. as already established for other viral infections, determining anti-sars-cov-2 igs is important for evaluating recovery of hospitalized patients from covid-19, but is also the method-of-choice for evaluating sars-cov-2 seroprevalence in a given population [7] . since the emergence of the pandemic, numerous serology tests were developed and marketed, although only some of them pursued food and drug administration (fda) approval. the performance of many serology tests, as defined by their sensitivity (true positive rate) and specificity (true negative rate), has been overestimated, as they were validated using samples from hospitalized, often critically ill covid-19 patients with high anti-sars-cov-2 ig titers [8] . thus, to accurately evaluate disease epidemiology, highly sensitive and specific tests which determine sars-cov-2 serostatus in outpatients, asymptomatic individuals and contact persons must be preferentially applied. due to the early nationwide lock-down and additional public health measures, greece was among the european countries less affected by the sars-cov-2 pandemic, with nearly 8000 confirmed cases and 238 deaths [1] . as of april 2020, the estimated prevalence of anti-sars-cov-2 igs in the general population was 0.23% [9] , although higher percentages were sporadically recorded in overpopulated or sars-cov-2-exposed niches, due to differences in infection rates [10] . the national and kapodistrian university of athens (nkua) is amongst the biggest educational -research institutes in the larger area of the balkans and eastern mediterranean basin and the most highly populated institution in greece, with more than 70,000 students (undergraduates and postgraduates) and over 4000 personnel (faculty members/laboratory assistants, scientific affiliates and administrative officers). due to the structure of its curriculum, students and personnel are often placed in indoor lecture halls, laboratories and clinics, thus increasing the probability of sars-cov-2 transmission. moreover, because of the significant number of its covid-19-dedicated medical clinics, a significant proportion of nkua personnel, especially health-care workers, had a high exposure risk. as with all educational institutions in greece, nkua complied with the decision of the greek government and suspended its operation since 10 march 2020. to ensure safe reopening of nkua institutional facilities, we pursued to assess sars-cov-2 seroprevalence among nkua members over a period of five consecutive months (june-october 2020) in order to estimate key epidemiological parameters, namely the epidemic growth rate and the fraction of subclinical/asymptomatic infections, as well as the proportion of nkua members who may remain susceptible to sars-cov-2 infection. following analysis of the first plasma samples from 2500 nkua members collected in june-july 2020, we present herein the results, which may eventually assist the management of the available nkua human resources. the study was conducted at selected nkua locations in athens, greece during june-july 2020 and samples were collected from nkua members, comprising faculty members/laboratory assistants, scientific affiliates, administrative officers, undergraduate and postgraduate students. the protocol was approved by the ethics and bioethics committee of the school of medicine, nkua (protocol # 312/02-06-2020) and all volunteers agreed to participate in the study via signing a written inform consent. peripheral blood was collected by venipuncture in bd vacutainers with spray-coated k2edta (bd biosciences, san jose, ca, usa) and centrifuged at 500× g for 20 min. blood plasma was transferred in dna-rna free cryovials (corning, ny, usa) and frozen at −20 • c until ig measurement, performed no later than 20 days post blood collection. plasma samples were analyzed using the ce-ivd roche cobas elecsys ® anti-sars-cov-2, an electrochemiluminescence immunoassay (eclia) for the qualitative detection of total igs (igg, igm and iga; pan-ig) generated against sars-cov-2 (roche diagnostics, indianapolis, in, usa). the test was performed according to the manufacturer's instructions. the assay uses the recombinant nucleocapsid (n) protein of sars-cov-2 as antigen and the method is based on the well-established "double-antigen sandwich" format between donors' plasma, biotinylated sars-cov-2-specific n antigen and sars-cov-2-specific recombinant n antigen labeled with a ruthenium complex. after concomitant incubation of reagents, streptavidin-coated microparticles are added; the immune-complex binds to the solid phase via a biotin-streptavidin interaction and is further aspirated into the measuring cell of a roche cobas e411 analyzer, where microparticles are magnetically captured onto the electrode surface. unbound material is washed out and the emission of chemiluminescence induced by the specific electrical current at the electrode is measured by a photomultiplier. test results are generated by interpolating the eclia signal with that of a threshold generated during calibration. a cut-off index (coi) of 1.0 or higher classifies a plasma sample as "reactive" (i.e., anti-sars-cov-2 positive). the total procedure requires 20 µl of plasma and the duration of the assay is 18 min. according to the manufacturer's package insert, elecsys ® anti-sars-cov-2 exhibits high overall clinical specificity of 99.81% with no cross-reactivity to the common cold coronaviruses; clinical sensitivity, determined by testing a total of 204 samples from 69 symptomatic patients with a pcr-confirmed sars-cov-2 infection, is 100% for samples collected ≥14 days after pcr confirmation in this collective; these values were verified in our study by measuring 25 rt-qpcr sars-cov-2 positive and 25 negative samples. the prevalence of antibodies against sars-cov-2 was initially computed by calculating the unweighted proportions of positive tests (unweighted prevalence). then, the final estimation of the prevalence was obtained by following a two-step approach: firstly, the unweighted proportions of positive tests was adjusted for the sensitivity and specificity of the test according to the manufacturer's specification, as implemented in the epir package (r version 3.6.3, r foundation for statistical computing, vienna, austria); and secondly, the prevalence was estimated after weighting for the age distribution (18-74 years old) of the population in the attica region (data from the 2011 census). prior to the analysis of the nkua donors' samples, plasma from 50 individuals was analyzed in exactly the same way using the roche cobas elecsys anti-sars-cov-2 ® . of these, 25 were archival samples, collected and kept frozen at −80 • c prior to the sars-cov-2 pandemic (between april-july 2019) and thus, expected to be negative; indeed, they were all classified as negative with the roche cobas elecsys anti-sars-cov-2 ® (coi < 1.0). the remaining 25 plasma samples were collected from convalescence plasma donors and were prior tested for anti-sars-cov-2 iggs with the commercially available euroimmun anti-sars-cov-2 enzyme-linked immunosorbent assay (elisa; euroimmun ag, luebeck, germany); 23/25 were classified as positive with the roche assay (coi ≥ 1.0). two samples tested negative with the roche assay, were also negative with the euroimmun elisa, suggesting that the two sars-cov-2-infected individuals likely did not develop a measurable (by the two assays) humoral response. we then analyzed the first cohort of 2500 samples from nkua donors collected during june-july 2020 using the roche cobas elecsys anti-sars-cov-2 ® . demographic characteristics and distribution of the participants into categories depending on occupation are shown in table 1 . a total of 25 cases were tested positive for antibodies against sars-cov-2; thus, the unweighted seroprevalence of anti-sars-cov-2 igs was 1.00%. after adjusting for age and the indicated sensitivity (100%) and specificity (99.81%) of the test, the weighted overall seroprevalence was 0.93% (95% ci: 0.27, 2.09) ( in addition, a non-significant increasing trend was observed for the weighted prevalence with age (p = 0.157). our statistical analysis revealed no significant associations between seroprevalence and the selected parameters (age, gender, school, position). in more detail, 14/25 (56%) of seropositive cases reported travelling mostly to european union (eu) countries during the past 5-8 months. similarly, 14 seropositive donors (56%) reported known contact with suspected covid-19 cases. among the seropositive nkua members, only 6/25 cases (24%) stated rt-qpcr-confirmed sars-cov-2 infection. the most common sars-cov-2-related symptoms were fever and headache, whereas 12/25 (48%) individuals reported smell and/or taste loss. finally, 9/25 (36%) of sars-cov-2-infected individuals were asymptomatic. emerging seroprevalence studies are currently implemented worldwide as they are considered a valuable tool to reveal the extent of sars-cov-2 infection via estimating the proportion of the population exposed to the virus. as of now, serological surveys reported that viral spread ranges from <1.0% in countries less affected by the pandemic (e.g., germany, greece, norway, iceland) to higher levels in countries that did not implement early public health measures (5.5% in spain, 6.0% in belgium, 8.5% in the uk). in some heavily affected areas in italy, brazil and some states in the usa, seroprevalence was found to be as high as 25% [9] [10] [11] [12] [13] [14] [15] . nevertheless, it was reportedly suggested that sars-cov-2 infections are potentially underdiagnosed and asymptomatic cases account for up to 41% [3] and in specific populations up to 59% [16] of infections. in our preliminary results, the weighted overall seroprevalence of igs against sars-cov-2 was 0.93% (95% ci: 0.27, 2.09) among 2500 donors from the nkua sampled in june-july 2020. our estimate is similar to the seroprevalence (0.85%) of igs against sars-cov-2 found in large urban areas in greece in a leftover igg serosurvey using samples tested during march and april 2020 [9] . we also showed that seroprevalence varied between gender (higher in males), age groups (higher in the 55-74 years age group) and position at the nkua (higher in faculty members/laboratory assistants), although the differences recorded were not statistically significant. as for occupation, higher prevalence was estimated in affiliates of the school of health sciences (1.43%) and this is in agreement with the anti-sars-cov-2 prevalence (1.07%) among health-care workers in two hospitals in athens, as tested between 13 april and 15 may 2020 [10] . although the study population and sampling periods were not identical, all previous analyses including ours, suggest that the seroprevalence of anti-sars-cov-2 is low in greece. the current study included students and personnel of the largest academic institution in the attica region and to our knowledge is the first study in greece comprising samples collected until end of july 2020. we should note that flight bans were lifted together with the opening of land borders at the beginning of july. therefore, it is of importance to investigate the impact of tourism and increased mobility to the populations' exposure to sars-cov-2 in the forthcoming months. our study provided an estimate of the exposure of the members of the nkua to sars-cov-2; this knowledge is useful for the design of public health measures for the upcoming semester. our findings that the overall seroprevalence is low and that there is no difference among members of different schools or age groups, suggest that that there is no need for tailored measures among the members of the nkua. this knowledge is of public health importance. the low seroprevalence found in our study was due to the early implementation of public health measures in greece, including the lock-down and the postponement of all in person educational activities in academic institutions, including the nkua. noteworthy, a significant number of nkua volunteers work in or are in close contact with nkua hospitals, clinics and laboratories, being at higher risk of contracting covid-19. indeed, affiliates of the school of health sciences showed the highest prevalence (1.43%) among tested groups; however, this difference was not statistically significant, thus suggesting the low levels of sars-cov-2 infection among health-care workers and affiliates. moreover, a substantial number of positively tested donors (>50%) were undergraduate or postgraduate students reporting recent mobility mainly to and from eu countries, probably as part of the exchange educational visits between higher education european universities and institutions. as expected in countries where the burden of covid-19 cases was low and therefore sars-cov-2 prevalence is less than 1%, high false positive rates are to be encountered with antibody tests. a suggested practice to overcome this limitation is to verify seropositive results with a different immunoassay [7, 11, 15] and this is eventually planned, upon collection of the 5000 nkua donors' samples. moreover, in the elegant study of gudbjartsson et al. [15] , pan-ig assays instead of separately measuring iggs, igms or igas, were shown to more accurately detect ig levels in qpcr-positive cases, with no decrease even at four months after diagnosis. the test we used was a pan-ig assay, as we also considered that not all potentially infected donors mount the same type, qualitatively and/or quantitatively, of humoral immune response. indeed, post-infection kinetics of seroconversion as well as persistence of anti-sars-cov-2 igs in the peripheral blood of non-hospitalized covid-19 patients was shown to vary between individuals [17, 18] . however, seronegativity in cases of symptomatic sars-cov-2-infected individuals who, for yet unclear reasons, develop low or even undetectable ig titers, as well as the possibility of low antigenic stimulation in mildly infected or asymptomatic people, should be utterly considered [19] . our analysis was based on a non-representative sample and this is a limitation of our study. nevertheless, the results presented herein in conjunction with available results from national or regional seroepidemiological studies in other countries, suggest that seroprevalence rates are far too low to reach protective (herd) immunity, which as estimated requires the presence of antibodies against sars-cov-2 in approximately 60% of the population [19, 20] . our preliminary report on 2500 nkua volunteered members clearly reveals low seroprevalence of detectable igs to sars-cov-2 during june-july 2020, in accordance with the low burden of sars-cov-2 in greece, as compared to other eu and non-eu countries. although limited to nkua members, to our knowledge this is the first serosurvey using samples collected until the end of july 2020, i.e., after the lifting of travel restrictions in greece and the eu. our results further suggest that broader testing for the presence of anti-sars-cov-2 igs is necessary to define the extent of non-recorded sars-cov-2-infected cases. they also highlight that nkua members should comply with public health measures, including maintenance of social distancing and avoidance of indoor crowding. indoor transmission of sars-cov-2. medrxiv 2020 estimating the extent of true asymptomatic covid-19 and its potential for community transmission: systematic review and meta-analysis asymptomatic cases in a family cluster with sars-cov-2 infection rapid asymptomatic transmission of covid-19 during the incubation period demonstrating strong infectivity in a cluster of youngsters aged 16-23 years outside wuhan and characteristics of young patients with covid-19: a prospective contact-tracing study viral dynamics in mild and severe cases of civid-19 prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study antibody responses to sars-cov-2 in patients with covid-19 repeated leftover serosurvey of sars-cov-2 igg antibodies antibodies against sars-cov-2 among health care workers in a country with low burden of covid-19 sars-cov-2 igg seroprevalence in blood donors located in three different federal states prevalence of sars-cov-2 specific neutralising antibodies in blood donors from the lodi red zone remarkable variability in sars-cov-2 antibodies across brazilian regions: nationwide serological household survey in 27 states seroprevalence of antibodies to sars-cov-2 in 10 sites in the united states humoral immune response to sars-cov-2 in iceland asymptomatic and symptomatic sars-cov-2 infections in close contacts of covid-19 patients: a seroepidimiological study long-term sars-cov-2 rna shedding and its temporal association to igg seropositivity a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease what policy makers need to know about covid-19 protective immunity a mathematical model reveals the influence of population heterogeneity on herd immunity to sars-cov-2 the authors would like to thank sotirios roussos for valuable discussions, roche diagnostics for kindly donating laboratory testing kits used in the study, nkua-sarg for financial support and all nkua members who voluntarily participated to the study. the authors declare no conflict of interest for this paper. roche diagnostics and nkua-sarg had no role in the design, execution, interpretation or writing of the study. key: cord-024317-w1ep0wq8 authors: ku, zhiqiang; ye, xiaohua; toa salazar, georgina; zhang, ningyan; an, zhiqiang title: antibody therapies for the treatment of covid-19 date: 2020-04-30 journal: antib ther doi: 10.1093/abt/tbaa007 sha: doc_id: 24317 cord_uid: w1ep0wq8 an outbreak of covid-19, the disease caused by infection of the coronavirus sars-cov-2, that began in december 2019 in wuhan, china has caused more than 2,990,559 confirmed human infections and 207,446 deaths as of april 27, 2020 (coronavirus covid-19 global cases by the center for systems science and engineering (csse) at johns hopkins university). scientists are working quickly on multiple aspects of the pandemic. genetic analyses are conducted to reveal the source and evolution of sars-cov-2, providing knowledge that can be used to contain it and to avoid future outbreaks. epidemiological studies which incorporates lessons learned from outbreaks of previous related viral diseases can guide development of public health measures effective to contain the current and future outbreaks. basic virology studies reveal viral structure and function. pathology studies inform development of strategies to interfere with infection. covid-19 prevention and treatment strategies are being developed in preclinical and clinical studies. antibody-based therapy is one viable treatment option. here, we discuss some of the most active areas of developing strategies to treat covid-19, focusing on approaches to generate neutralizing antibodies against sars-cov-2 for prophylactic and therapeutic treatment of covid-19. significance: development of sars-cov-2 neutralizing antibodies with the desired efficacy and safety profile is a critical part of the toolbox of therapies for the treatment of covid-19. we discuss in this review the current state of discovery and development of such antibodies. and nucleocapsid (n) proteins 4 . the spike protein comprises an n-terminal s1 subunit responsible for receptor binding and a c-terminal s2 subunit responsible for membrane fusion ( figure 1c ) 5 . the s1 subunit is further divided into the n-terminal domain (ntd), the receptor-binding domain (rbd), subdomain 1 (sd1) and subdomain 2 (sd2). the s2 subunit is further divided into the fusion peptide (fp), the heptad repeat 1 (hr1) and heptad repeat 2 (hr2) ( figure 1c ) 6 . like sars-cov, sars-cov-2 enters cells through binding of the host cellular receptor angiotensin-converting enzyme 2 (ace2) via its spike protein 2 . in contrast, mers-cov enters cells through binding of the host receptor dipeptidyl peptidase 4 (dpp4) via its spike protein 7 . these host receptors are indispensable for virus infection 2, 4 . receptor binding triggers a conformational change of the spike protein to an activated state 8 . the activated spike is cleaved by a protease (tmprss2 for sars-cov and sars-cov-2) at the s1/s2 site, releasing the s1 subunit and exposing the fp on the s2 subunit 9 . the fp inserts into target cell membrane, hr1 and hr2 refold to form a postfusion conformation that drives viral membrane fusion with target cells ( figure 1d ) 8, 10 . the cryo-em structure of the sars-cov-2 spike in the prefusion conformation was recently published 11 . the interaction between sars-cov-2 spike rbd and the full-length human ace2 has also been revealed by cryo-em or crystallization 6, [12] [13] [14] . similar to the spike protein of sars-cov, the sars-cov-2 spike protein also possesses extensive glycosylation, which may be important for virus binding to cells and facilitate immune evasion through epitope masking 15, 16 . the non-structural proteins encoded by coronaviruses are essential for virus replication inside cells 1 figure 1d ). however, the coronavirus protein nsp14 has a unique exoribonuclease (exon) function, which provides the proofreading capability. this mechanism may affect the susceptibility of coronaviruses to nucleoside analogues 19 . approved viral protease inhibitors, such as the hiv aspartic protease inhibitors lopinavir and ritonavir, have shown efficacy in a clinical trial 20 . it is hypothesized that lopinavir and ritonavir exhibit their efficacy by inhibiting the 3-chymotrypsin-like protease 20 ( figure 1d ). in addition to repurposing existing antiviral drugs, drugs used to modulate the host the spike protein of sars-cov-2 plays an essential role in virus entry into host cells and is a primary target of neutralizing antibodies 5, 9 (figures 1c,d) . due to the functionality and high immunogenicity of the coronavirus s1 subunit, most neutralizing antibodies characterized for coronaviruses to date target s1, in particular the s1-rbd [44] [45] [46] . two mers-cov neutralizing mabs, g2 and 7d10, target the s1-ntd region and function by blocking spike protein interaction with the host receptor dpp4 47, 48 . compared to the s1 subunit, the coronavirus s2 subunit is more conserved and bears epitopes that could potentially be targeted by broadly neutralizing antibodies 10, 49 . generation of antibodies with broad neutralizing activity against different coronaviruses, or at least sars-related coronaviruses, would be of great value for confronting future waves of coronavirus-related disease. however, broadly neutralizing antibodies against different human coronaviruses are very rare, probably related to the sequence variance of spike protein and cryptic nature of the highly conserved epitopes 28, 50 . the s2 conformation is highly dynamic during membrane fusion, presenting a major challenge in preparing antigens for discovery of antibodies against this protein 10 . antigen stabilizing strategies used in discovery of antibodies against hiv and rsv proteins may be explored in the design of stable coronavirus s2 proteins 51-53 . to target the whole spike protein or domain proteins, several well-established methods have been used for isolating sars-cov-2 monoclonal antibodies (table 1) . xiong et al. a structural study indicates that cr3022 recognizes a highly conserved cryptic epitope on rbd that is distinct from the receptor-binding site 50 . antibodies that bind to the spike protein with high affinity can be first tested for their ability to block spike interaction with ace2 or directly tested in cell based viral neutralization assays in vitro. two in vitro assay systems are commonly used to evaluate the neutralization activity of antibodies against coronavirus 56 monkey and mouse models have been reported for testing sars-cov-2-neutralizing antibodies [58] [59] [60] . in the monkey study, researchers found that rhesus macaques infected with sars-cov-2 through the intratracheal route had mild illness, and their lungs showed signs of pneumonia similar to those in humans with covid-19 58 . after 3 or 6 days of infection, the virus could be isolated from bronchus, lung tissues, and oropharyngeal swabs. however, no monkey developed severe symptoms during the study 58 . in a study using a mouse model, transgenic mice with human ace2 expression, but not wild-type mice, were found to be susceptible to sars-cov-2 infection 59 . mice inoculated with sars-cov-2 through the intranasal route had a 5~10% loss of body weight and histopathology in the lung, such as interstitial pneumonia, and infiltration of immune cells. no mice died during the study 59 . to establish a model that can mimic more severe human infections, different animal models and other experimental factors must be considered and tested. antibody-dependent enhancement (ade) is the phenomenon of non-neutralizing or subneutralizing antibodies facilitating virus infection, leading to more severe disease. the most widely known example is dengue ade, which has various mechanisms 61, 62 . in one mechanism, certain immune cells, which don't express the receptor for virus entry but should take the risk of ade into consideration. the unprecedented impact of the current covid-19 pandemic on the human race calls for unprecedented response from all aspects of society to combat the disease. to address the immediate need for therapies, repurposing existing drugs and strategies is a logical first step. this includes testing antivirals, drugs used to modulate the host immune system, and cp transfusion as therapies for covid-19. it is likely that sars-cov-2 will stay with us as a seasonal infection 68 subunit. the s1 subunit is further divided into the n-terminal domain (ntd), the receptorbinding domain (rbd), subdomain 1 (sd1), and subdomain 2 (sd2). the s2 subunit is further divided into the fusion peptide (fp), the heptad repeat 1 (hr1) and heptad repeat 2 (hr2). 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elucidates activation of coronavirus fusion molecular mechanism for antibody-dependent enhancement of coronavirus entry potential impact of seasonal forcing on a sars-cov-2 pandemic china medical treatment expert group for c: comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis. the european respiratory viral load of sars-cov-2 in clinical samples the authors declare no potential conflicts of interest. key: cord-264031-0y7xbgun authors: wierbowski, shayne d.; liang, siqi; chen, you; andre, nicole m.; lipkin, steven m.; whittaker, gary r.; yu, haiyuan title: a 3d structural interactome to explore the impact of evolutionary divergence, population variation, and small-molecule drugs on sars-cov-2-human protein-protein interactions date: 2020-10-13 journal: biorxiv doi: 10.1101/2020.10.13.308676 sha: doc_id: 264031 cord_uid: 0y7xbgun the recent covid-19 pandemic has sparked a global public health crisis. vital to the development of informed treatments for this disease is a comprehensive understanding of the molecular interactions involved in disease pathology. one lens through which we can better understand this pathology is through the network of protein-protein interactions between its viral agent, sars-cov-2, and its human host. for instance, increased infectivity of sars-cov-2 compared to sars-cov can be explained by rapid evolution along the interface between the spike protein and its human receptor (ace2) leading to increased binding affinity. sequence divergences that modulate other protein-protein interactions may further explain differences in transmission and virulence in this novel coronavirus. to facilitate these comparisons, we combined homology-based structural modeling with the eclair pipeline for interface prediction at residue resolution, and molecular docking with pyrosetta. this enabled us to compile a novel 3d structural interactome meta-analysis for the published interactome network between sars-cov-2 and human. this resource includes docked structures for all interactions with protein structures, enrichment analysis of variation along interfaces, predicted δδg between sars-cov and sars-cov-2 variants for each interaction, predicted impact of natural human population variation on binding affinity, and a further prioritized set of drug repurposing candidates predicted to overlap with protein interfaces†. all predictions are available online† for easy access and are continually updated when new interactions are published. † some sections of this pre-print have been redacted to comply with current biorxiv policy restricting the dissemination of purely in silico results predicting potential therapies for sars-cov-2 that have not undergone thorough peer-review. the results section titled “prioritization of candidate inhibitors of sars-cov-2-human interactions through binding site comparison,” figure 4, supplemental table 9, and all links to our web resource have been removed. blank headers left in place to preserve structure and item numbering. our full manuscript will be published in an appropriate journal following peer-review. the ongoing global covid-19 pandemic caused by the infection of sars-cov-2 has to date infected impact of mutations on interaction binding affinity and performed a comparison of protein-protein and 96 protein-drug binding sites. we compile all results from our structural interactome into a user-friendly 97 web server allowing for quick exploration of individual interactions or bulk download and analysis of the 98 whole dataset. further, we explore the utility of our interactome modeling approach in identifying key 99 interactions undergoing evolution along viral protein interfaces, highlighting population variants on 100 human interfaces that could modulate the strength of viral-host interactions to confer protection from or 101 susceptibility to covid-19, and prioritizing drug candidates predicted to bind competitively at viral-102 human interaction interfaces. enrichment of divergence between sars-cov and sars-cov-2 at spike-ace2 binding interface to highlight the utility of computational and structural approaches to model the sars-cov-2-human 106 interactome, we first examined the interaction between the sars-cov-2 spike protein (s) and human 107 angiotensin-converting enzyme 2 (ace2) (fig 1.a) . this interaction is key for viral entry into human 108 cells 3 and is the only viral-human interaction with solved crystal structures available in both sars-cov 47 109 and sars-cov-2 [48] [49] [50] . comparison between sars-cov and sars-cov-2 revealed that sequence 110 divergence of the s protein was highly enriched at the s-ace2 interaction interface (fig 1.a; log2oddsratio=2.82, p=1.97e-5), indicating functional evolution around this interaction. to explore the 112 functional impact of these mutations on this interaction, we leveraged the rosetta energy function 51 to 113 estimate the change in binding affinity (δδg) between the sars-cov and sars-cov-2 versions of the 114 s-ace2 interaction (fig 1.b and 1.c) . the predicted negative δδg value of -14.66 rosetta energy units 115 (reu) indicates an increased binding affinity using the sars-cov-2 s protein driven by better optimized 116 solvation and hydrogen bonding potential fulfillment along the ace2 interface. our result is consistent 117 with the hypothesis that increased stability of the s-ace2 interaction is one of the key reasons for 118 elevated transmission of sars-cov-2 52 . moreover, recent experimental energy kinetics assays have 119 shown that sars-cov-2 s protein binds ace2 with 10-20-fold higher affinity than that of sars-cov s 120 protein 53 supporting the conclusions from our computational modeling. among individuals 6, 7, 54 . several hypotheses for genetic predisposition models have been proposed 123 including that expression quantitative trait loci (eqtls) may up-or down-regulate host response genes 124 and that functional coding variants may alter viral-human interactions 55, 56 . for instance, a recent rna-in order to add a structural component to our interactome map, and thereby enable modeling of 149 the binding affinity for these interactions, we additionally performed docking in pyrosetta using our 150 eclair interface likelihood predictions to refine the search space (supplemental after constructing the 3d interactome between sars-cov-2 and human, we first looked for evidence 162 of interface-specific variation by mapping both gnomad 58 reported human population variants 163 (supplemental table 3 ) and sequence divergences between sars-cov and sars-cov-2 164 (supplemental table 4 ) onto the predicted interfaces. in general, conserved residues have been shown 165 to cluster at protein-protein interfaces 63 , and a recent analysis of sars-cov-2 structure and evolution 166 likewise concluded that highly conserved surface residues were likely to drive protein-protein 167 interactions 64 . consistent with these prior findings at an interactome-wide level, we observed significant 168 depletion for both viral and human variation along the predicted interfaces comparable to that observed 169 on solved human-human interfaces (fig 2.a) . nonetheless, considering each interaction individually, our analysis uncovered a 13 interaction 171 interfaces enriched for human population variants (fig 2.b) , and 7 enriched for recent viral sequence 172 divergences (fig 2.c) . a breakdown of variant enrichment on each interface is provide in supplemental 173 table 5 . the individual viral interfaces showing an unexpected degree of variation may-like the 174 previously discussed s-ace2 interface-be indicative of recent functional evolution around the viral-human interaction. considering the slower rate of evolution in humans, enrichment of population variants along the human interfaces is unlikely to be a selective response to the virus. rather, these 177 interfaces with high population variation along the interfaces may represent edges in the interactome whose strength may fluctuate among individuals or between populations. alternatively, enrichment and depletion of variation along the human-viral interfaces could help distinguish viral proteins that bind 180 along existing-and therefore conserved-human-human interfaces from those that bind using novel 181 interfaces-that would be less likely to be under selective pressure. to further explore the functional impact of naturally occurring variants on the human interactors 183 of sars-cov-2, we considered variants with phenotypic associations as reported in hgmd 65 , clinvar 66 184 or the nhgri-ebi gwas catalog 67 . interactors of sars-cov-2 were significantly more likely than the 185 rest of the human proteome to harbor phenotypic variants in each of these databases (fig 2.d) . notably, 186 among the individual disease categories enriched in this gene set, several were consistent with reported 187 comorbidities including heart disease, respiratory tract disease, and metabolic disease 68, 69 (fig 2. e; 188 supplemental table 6 ). disruption of native protein-protein interactions is one mechanism of disease 189 pathology, and disease mutations are known to be enriched along protein interfaces 70, 71 . human 190 population variants on the predicted human-viral interface were more likely to be annotated as 191 deleterious by sift 72 and polyphen 73 but showed identical allele frequency distributions compared to 192 those off the interfaces (supplemental figure 4) . however, mapping annotated disease mutations onto 193 the protein interfaces only revealed significant enrichment along known human-human interfaces; no 194 such enrichment was found on human-viral interfaces (fig 2.f) . this is likely because unlike with human-195 human interactions, mutations disrupting human-viral interactions would not disrupt natural cell function, 196 and therefore would be unlikely to be pathogenic. our finding that disease mutations and viral proteins 197 affect human proteins at distinct sites is consistent with a two-hit hypothesis of comorbidities whereby 198 proteins whose function is already affected by genetic background may be further compromised by viral 199 infection. we next sought to explore the impact of sequence divergence in sars-cov-2 relative to sars-cov on 202 viral-human interactions. mutations between the two viruses were identified by pairwise alignment and the 203 impacts of these mutations on the binding energy (δδg) for 250 interactions amenable to docking were 204 predicted using a pyrosetta pipeline 46, 59, 60 . although the binding energy for most interactions was 205 unchanged-either because no mutations occurred near the interface or because the mutations that did 206 had marginal effect-we observed an increased likelihood of the divergence from sars-cov to sarscov-2 resulting in decreased binding energy (i.e. more stable interaction) (fig 3. a; supplemental table 208 7). the significant outliers in these δδg predictions can help pinpoint key differences between the viral-209 human interactomes of sars-cov and sars-cov-2. we further note a wide range of affinity impacts 210 among various human interactors of a single viral protein (fig 3.d) and hypothesize that these differences 211 may help identify the most important interactions. to further explore the significance of these changes in interaction affinity, we considered those 213 interactions with the largest decrease in binding energy; corresponding to the largest predicted increase in 214 affinity. specifically, we highlight the interaction between coronavirus orf9c and human mitochondrial nadh scanning mutagenesis along all docked interfaces in pyrosetta. we identified as binding energy hotspot 254 mutations all mutations with a predicted δδg at least one standard deviation away from the mean for identical amino acid substitutions across the rest of the interface. in total, out of 2,241 population variants 256 on eligible interfaces, 161 (7.2%) were identified as hotspots predicted to disrupt interaction stability, and 257 116 (5.2%) were identified as hotspots predicted to contribute to interaction stability (fig 3.b) . most of the 258 hotspot mutations were predicted to be driven by solvation or repulsive forces, with disruptive hotspots 259 primarily being driven by repulsive forces and stabilizing hotspots primarily being driven by solvation forces 260 (fig 3.c) . results summarizing the predicted impact of all 2,241 population variants on the docked 261 interfaces are provided in supplemental table 8 . the current version of the sars-cov-2 human structural interactome web server describes 332 291 viral-human interactions reported by gordon et al. 20 . we will continue support for the web server with 292 periodic updates as additional interactome screens between sars-cov-2 and human are published. as we update, a navigation option to select between the current or previous stable releases of the web 294 server will be provided. overall, we present a comprehensive resource to explore the sars-cov-2-human protein-protein 297 interactome map in a structural context. analysis through this framework allows us to consider the recent 298 evolution of sars-cov-2 in the context of its interactome map and to prioritize for further functional 299 characterization key interactions. likewise, our consideration of underlying variation in the human 300 proteins that interact with sars-cov-2 may be valuable in explaining differences in response to 301 infection. we particularly note that our observation that perturbation from underlying disease mutations 302 and viral protein binding occur at distinct sites on the protein is of clinical interest. further investigation 303 into the combined role of these two sources of perturbation to better understand the mechanisms linked 304 to comorbidities is warranted. however, our work is not without limitation. firstly, we note that although structural coverage 306 from our homology modelling of sars-cov-2 proteins was robust (supplemental figure 1) , the same done to orient the most likely interface residues on each structure towards each other, protein-protein 309 docking using incomplete protein models introduces some bias and low coverage may exclude some 310 true interface residues. for this reason, the initial eclair interface annotations-which are less subject 311 to structural coverage limitations-may provide orthogonal value. we additionally note that direct perhaps most importantly, we emphasize the importance of further experimental 323 characterization to confirm the predictions made here. nonetheless we believe our 3d structural sarscov-2-human interactome web server will prove to be a key resource in informing hypothesis driven 325 exploration of the mechanisms of sars-cov-2 pathology and host response. the scope, and potential 326 impacts of our webserver will continue to grow as we incorporate the results of ongoing and future 327 interactome screens between sars-cov-2 and human. additionally, we note that our 3d structural 328 interactome framework can be rapidly deployed to analyze future viruses. homology-based modeling of all 29 sars-cov-2 proteins was performed in modeller 90 using a multiple 332 template modeling procedure. in brief, a list of candidate template structures for each protein to be 333 modelled was obtained by running blast 91 against a reference containing all sequences in the protein data bank (pdb) 92 . templates were filtered to only retain those with at least 30% identify to the protein 335 to be modelled, and the remaining templates were ranked using a weighted combination of percent 336 identity and coverage as described previously 93 . to compile the final set of overlapping templates for 337 modeling, first the top ranked template was selected as a seed. overlapping templates were iteratively 338 added to the set so long as 1) the new template increased the overall coverage by at least 10%, and 2) 339 the new template retained a total percent identity no more than 25% worse than the initial seed template. pairwise alignments between the protein to be modelled and the template set were generated using a regions with large gaps (at least 5 gaps in the alignment in a 10 residue window). finally, alignment was to accommodate predictions between sars-cov-2 and human, slight alterations were made. using the predicted interface probabilities reported by eclair, we set up the initial docking 379 conformation to explore a restricted search space for each docking simulation. in cases where multiple 380 structures were available for the human protein, all structures were weighted based on the eclair 381 scores for the covered residues in each structure to maximize both coverage age inclusion of likely 382 interface residues. for each protein in the interaction, we performed a linear regression classification in scikit-learn 102 to optimally separate the likely interface residues from likely non-interface residues. the plane defined by this linear regression served as a reference to orient the structures along the y-axis the x-z plane and separated a distance of 5 å along the y-axis. for each docking attempt, a series of 387 random perturbations from these initial conformations were made to search the nearby space. first, the 388 human protein was rotated up to 360° along the y-axis to allow full exploration of different rotations of 389 the two interfaces relative to each other. second, to apply some flexibility to the plane predicting the 390 interface vs. non-interface sides of each protein, up to 30° of rotation along the x-and z-axis were 391 allowed for both the viral and human proteins. finally, a random translation up to 5 å in magnitude was 392 applied to the human protein along the x-z plane so that the docking could explore contact points other 393 than the center of masses along these axes. after initializing these guided starting conformations, docking was simulated in pyrosetta 46 395 using a modified version of the protein-protein docking methodology provided by gray 2006 103 . the 396 initial demo (https://graylab.jhu.edu/pyrosetta/downloads/scripts/demo/d100 docking.py) takes two 397 chains from a co-crystal structure, applies a random perturbation, and re-docks them. because 398 randomized initial orientation was already handled as described previously, these steps were removed 399 from our docking runs. in brief, the protein models were converted to centroid representation, slid into 400 contact using the "interchain_cen" scoring function, and converted back to full atom representation, 401 before having their side-chains optimized using the predefined "docking" and "docking_min" scoring table 2 . to annotate interface residues from atomic resolution docked models, we used a previously described 407 and established definition for interface residues 45 . in brief, the solvent accessible surface area (sasa) 408 for both bound and unbound docked structures was calculated using naccess. 100 we define as accessibility) and 2) in contact with the interacting chain (defined as any residue whose absolute 411 accessibility decreased by ≥ 1.0 å 2 ). the full list of sars-cov-2 mutations is reported in supplemental table 4 . human population variants in all 332 human proteins shown to interact with sars-cov-2 430 proteins were obtained from gnomad 58 reported as the most general term with no more significant ancestor term (supplemental table 6 , sheet 465 1). raw enrichment values for all terms are also predicted (supplemental table 6 , sheet 2). for curation of disease and trait associations from nhgri-ebi gwas catalog (http://www.ebi.ac.uk/gwas/) 67 the scoring function used for these calculations is as described previously 59 using the following weights; protein-ligand docking using smina the previous viral-human interactome screen by gordon et al. 20 supplemental table 9 . list of 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with smina from the csar 2011 benchmarking exercise sars-cov-2 that contain disease annotations in hgdm (log2or=0.57, p=1.70e-4) sars-cov-2 proteins were significantly more likely to harbor disease mutations than non-interactors error bars indicate ± se. e, a sample of individual disease terms enriched in human genes targeted by 603 comparison of the enrichment of hgdm, clinvar, and gwas annotated mutations on human-vial 605 interfaces or human-human interfaces for the same gene set. although disease mutations were enriched 606 on human-human interfaces (hgmd, log2or=0 24), no 607 enrichment was observed on human-viral interfaces (hgmd, log2or=0.21, p=0.13; clinvar the gwas category was removed from this analysis because most lead gwas 609 snps occurred in non-coding regions. error bars indicate ± se predicted changes in binding affinity from sequence divergences between sars-cov and sars-613 an overall representation of 614 these δδg predictions is reported (mean=-1.40 reu, std=6.16 reu) with interactions sorted from those 615 with the largest decrease in binding energy (most stabilized relative to sars-cov) to those with the 616 largest increase in binding energy (most destabilized relative to sars-cov) < z-score ≤ -1, n=85), or strongly stabilizing (z-score ≤ -2, n=31) score < 1, n=1,964) showed minimal impact of binding affinity. c, breakdown of the contribution of each 623 term in the pyrosetta energy function used for in-silico scanning mutagenesis for all population variants a breakdown of which term contributed most heavily 625 to the classification of all 277 interface hotspot population variants is shown on the right. d, individual 626 sars-cov-2-human interactions involving the same viral protein can have distinct interfaces with 627 distinct predicted changes in binding affinity between sars-cov and sars-cov-2 versions of the 628 protein. an example involving orf9b is highlighted where some interactions (e.g. tomm70 and ptbp2) 629 are predicted to be more stabilized in sars-cov-2 whereas others (e.g. bag5, slc9a3r1, and 630 mark2) are predicted to me unaffected. e, docked structure for the interaction between sars-cov-2 631 orf9c and human ndufaf1 sars-cov-2 (bottom) orf9c. interface residues are colored by their predicted energy contribution 633 from blue (stabilizing) to white (no impact) to red (destabilizing) -2 are labeled in red, while other residues with a major contribution to the binding 635 affinity are labeled in green (ndufaf1) or blue (orf9c). the overall predicted change in binding energy 636 (δδg=-21.7 reu) suggests the interaction is more stable (lower energy) in the sars-cov-2 version of 637 the interaction supplemental figure 4. summary of human population variant frequency and deleteriousness summary of allele frequency for human population variants either on or off the predicted human-700 viral interface presented as either a raw distribution or a cumulative density respectively. variants in 701 either category had roughly identical allele frequency distributions. c, d, summary of the sift 702 deleteriousness score for human population variants either on or off the predicted human-viral interface 703 presented as either a raw distribution or a cumulative density respectively population variants on the 706 interface were significantly more likely to be classified deleterious. f, g, summary of the polyphen 707 deleteriousness score for human population variants either on or off the predicted human-viral interface 708 presented as either a raw distribution or a cumulative density respectively. plots are colored based on 709 the split between polyphen benign, possibly damaging, and probably damaging categories. e, pie chart 710 breakdown of these categories. pie char outlines distinguish interface (green) from non-interface 711 (orange) for each sars-cov-2-human interaction with 3d structure available for both proteins, 50 independent 680 guiding docking trials were used to select a final docked configuration. the structure for the viral protein 681 is colored from white to blue with darker blue corresponding to higher eclair prediction. the structure 682 for the human protein is colored similarly using a green to white gradient. initial semi-random docked 683 configurations were generated using five steps. first a plane separating eclair predicted likely 684 interface from likely non-interface residues was drawn to divide each protein. second, the two protein 685 chains were separated 5 å apart on the y-axis using the previously defined plane to orient the likely 686 interface sides of each protein towards each other. third, the human protein was randomly rotated up 687 to 360° along the y-axis to sample different orientations of the two interfaces relative to each other. key: cord-285168-qkadqohe authors: delatorre, edson; mir, daiana; graf, tiago; bello, gonzalo title: tracking the onset date of the community spread of sars-cov-2 in western countries date: 2020-04-23 journal: nan doi: 10.1101/2020.04.20.20073007 sha: doc_id: 285168 cord_uid: qkadqohe the sars-cov-2 rapidly spread around the world during 2020, but the precise time in which the virus began to spread locally is currently unknown for most countries. here, we estimate the probable onset date of the community spread of sars-cov-2 from the cumulative number of deaths reported during the early stage of the epidemic in western europe and the americas. our results support that sars-cov-2 probably started to spread locally in all western countries analyzed between the middle of january and early february 2020, thus long before community transmission was officially recognized and control measures were implemented. a novel betacoronavirus, designated sars-cov-2, was identified as the causative agent of a severe acute respiratory disease (now known as in wuhan, hubei province, china, in december 2019. (1) (2) in the following weeks, sars-cov-2 rapidly spread around the world, infecting more than 2 million people and causing more than 135,000 deaths as of april 15th, 2020. (3) the exponential growth of the covid-19 pandemic has overloaded hospitals and governments' response measures have disrupted social contacts for >1 billion inhabitants worldwide. the first infections of sars-cov-2 identified in europe and north america in january 2020, were related to travelers returning from china. (4) (5) since february 2020, community transmission of sars-cov-2 was already documented in the usa and multiple european countries. (6) however, the precise start time of sustained sars-cov-2 local transmission in most of these countries is currently unknown. the presence of a substantial fraction of asymptomatic, but infectious individuals probably facilitated the undocumented dissemination of the novel coronavirus within and among countries before its detection by public health systems. (7) this suggests that when domestic infections with sars-cov-2 were reported, epidemics were probably already growing exponentially in most countries for some time. genomic epidemiology could be a useful tool to track the geographic spread of sars-cov-2 over time and to infer the timing of community transmission. its implementation has enabled to trace back the time of the most recent common ancestor (t mrca ) of sars-cov-2 to late november 2019, (8) consistent with epidemiological findings that show viral local transmission in wuhan by the middle december 2019. (9) within this same framework, a recent analysis of sars-cov-2 genomes from 21 neighborhoods in new york city (nyc) revealed that the nyc epidemic has been mainly sourced from untracked transmission between the usa and europe and also pointed that the virus was likely circulating in the city weeks before the first confirmed sars-cov-2 infection case. (10) at the moment, however, it is challenging to estimate the onset date of local transmission clusters within countries (or cities) based solely on genetic data. due to the short period of time since the beginning of the outbreak and to the uneven geographic sampling of sars-cov-2 genomes, in most cases, there's still not enough phylogenetic signal to define location-specific clusters. (8, 11) . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 23, 2020. . here, we aimed to develop a simple inference method to estimate the onset date of the community spread of sars-cov-2 in different countries from the cumulative number of reported deaths during the early stage of the epidemic. the cumulative number of deaths could be a reliable tracker of the sars-cov-2 epidemic's progress within a country. (12) the lack of testing and the great proportion of asymptomatic subjects profoundly restrain the capacity of most countries to count the true number of sars-cov-2 infections, while the smaller number of deaths is less prone to such degree of underestimation. furthermore, the cumulative number of reported deaths represents a time-delayed tracker of the sars-cov-2 epidemic and thus provides valuable information on early epidemic dynamics even when data is obtained after implementation of national control measures that reduce the viral spread. current estimates support an infection fatality ratio of sars-cov-2 of around 1% and a median time between infection and death of around three weeks. (13) (14) to infer the probable onset date of the community spread of sars-cov-2 in a given location, we assumed that: a) as soon as the virus starts spreading locally, the epidemic starts to grow exponentially; b) the cumulative number of deaths starts to increase exponentially 20 days later than the beginning of the epidemic exponential growth; and c) the rate of exponential growth of the number of deaths remains roughly constant during the epidemic early weeks. for this study we selected china and those countries from western europe (belgium, france, germany, italy, netherlands, spain, united kingdom [uk]), north america (new york, usa) and south america (brazil) that were most heavily affected until 5th april 2020. (3) to capture the early period of exponential growth of virus transmission, before national control measures were implemented, we set the start point to the day when the cumulative number of deaths was above one and then counted the cumulative number of deaths during the following three weeks. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 23, 2020. . the cumulative number of reported deaths has grown exponentially during the selected period in all countries analyzed with mean estimated epidemic doubling times ranging from 2.6 days to 3.8 days, consistent with previous estimations [supplementary data (figure 1) ]. (15) (16) (17) linear regression analyses of the log number of the projected total infected individuals over time resulted in a high coefficient of determination (r 2 ≥ 0.95) and significant increase (p < 0.0001) for all analyzed countries (figure 1 ). to validate our approach, we first estimated the beginning of community transmission of sars-cov-2 in locations with previous molecular clock estimates. using our framework, we traced the onset date of community transmission of sars-cov-2 to early december 2019 in china and to early february 2020 in new york (figure 1 and table 1 ). these estimates were consistent with molecular clock calibrations from sars-cov-2 genomic data (table 1) (8, 10) , and suggest that both approaches might provide roughly similar results. we next used our approach to estimate the onset date of local transmission of sars-cov-2 in western europe. our findings suggest that community transmission might have started around middle january 2020 in italy and between late january and early february 2020 in the remaining countries (figure 1 ). in some countries (italy and netherlands) community transmission was traced long before (2-4 weeks) the first confirmed sars-cov-2 infection case; while in others (spain, france, united kingdom, germany, and belgium) the onset date roughly coincides with the time of detection of the first imported cases (figure 1 ). in all countries analyzed, however, sars-cov-2 probably started to spread locally long before community transmission was officially recognized and control measures for social distancing and air travel restrictions were implemented (table 1) . of note, our estimation of the onset date of community transmission of sars-cov-2 in european countries is consistent with the estimated t mrca of some european and european/american clades like the a1a (28 th january: 19 th january -17 th february) and the a2a (25 th january: 19 th january -2 nd february). (10) these results suggest that early community transmission of sars-cov-2 in europe could have been fueled by the rapid dissemination of few regional clades. south america was one of the last geographic regions in the world to declare community transmission of sars-cov-2. our study, however, supports that the virus started to spread in brazil at around the early february 2020 (figure 1 ), thus coinciding with the epidemic expansion . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 23, 2020. . https://doi.org/10.1101/2020.04. 20.20073007 doi: medrxiv preprint in north america and western europe and preceding by more than 20 days the first confirmed sars-cov-2 infection case in the country (table 1) . according to a recent report of the infogripe (http://info.gripe.fiocruz.br) -a system of the fundação oswaldo cruz (fiocruz) that weekly monitor the number of hospitalizations of patients who have had symptoms such as fever, cough or sore throat and difficulty breathing in brazil -the number of such cases started to increase above levels observed in 2019 since the middle february 2020. (18) more importantly, of the retrospective cases that tested positive for some respiratory virus, nine cases were identified as sars-cov-2 in the eight epidemiological week, between 16th and 22nd february (https://covid.saude.gov.br/). these epidemiological data fully agree with our estimation and clearly support that sars-cov-2 was already circulating in the brazilian population for some weeks before being detected on 26th february 2020. our findings support that, except italy, sars-cov-2 started to spread locally in several countries from western europe and the americas at around the same time. despite this, early epidemiological dynamics seem to be quite different across countries. among those countries where sars-cov-2 started to spread in late january, spain passed the milestone of 1,000 deaths earlier (20th march) than france (24th march), the uk (27th march), netherlands (31th march), and germany (3rd april). (3) similarly, among those regions where sars-cov-2 started to spread in early february, new york (30th march) and belgium (2nd april) reached 1,000 deaths much earlier than brazil (10th april). (3) some studies support that mortality rate might vary across regions according to the availability of health-care facilities, underlying demography and environmental factors (average temperature, precipitation seasonality and levels of air pollution). (13, (19) (20) (21) (22) early dynamics in the reported number of deaths may also be influenced by the time when local social distancing measures were implemented (table 1 ) and by the percentage of deaths with laboratory confirmation within each country. the narrow confidence intervals of our estimates should be interpreted with caution because our method does not account for uncertainty and geographic variability in the mortality rate and lag-time between infection and death. (13) (14) as more clinical and epidemiological data becomes available, it will be possible to refine these estimates by using more accurate regionally-specific parameters . despite this, data from countries that implement wide-scale testing for sars-cov-2 since the beginning of the outbreak, including people who have mild or . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 23, 2020. . no symptoms, supports that the mortality rate will probably not exceed 1% of the total number of infected individuals. (23) hence, the estimates presented here should be closer to the upper limit of the likely time range when virus community transmission has started. another limitation is that our method is sensitive to underestimation of the true number of deaths from sars-cov-2 and should thus be used with caution in countries with limited capacity for testing and/or substantially delayed in the report of deaths. in summary, our results support that community transmission of sars-cov-2 probably started in many western countries between middle january to early february 2020, thus long before control measures to restrict air travels and promote social distancing were implemented. that quite long period of cryptic community transmission (> 4 weeks) in all analyzed countries draws attention to the great challenge of tracking the early global spread of sars-cov-2 and supports that control measures should be adopted at least as soon as first imported cases are detected in a new geographic region. this is especially important in the light of studies showing that sars-cov-2 very likely will enter in a regular circulation after the initial pandemic wave, causing recurrent outbreaks in the next years whose frequency and intensity are dependent upon virus' biological features still not well understood, like the duration of immunity that sars-cov-2 can induce. (24) in this scenario, intense virological surveillance is pivotal to early detect the virus re-emergence, informing systems of contact tracing and providing evidence to carry out appropriate control measures. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 23, 2020. . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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implications. biorxiv why daily death tolls have become unusually important in understanding the coronavirus pandemic estimates of the severity of coronavirus disease 2019: a model-based analysis incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2. emerg infect dis estimating the growth rate and doubling time for short-term prediction and monitoring trend during the covid-19 pandemic with a sas macro. medrxiv challenges in control of covid-19: short doubling time and long delay to effect of interventions. medrxiv brazilian ministry of health exposure to air pollution and covid-19 mortality in the united states. medrxiv relationship between average daily temperature and average cumulative daily rate of confirmed cases of covid-19. medrxiv global covid-19 transmission rate is influenced by precipitation seasonality and the speed of climate temperature warming. medrxiv temperature significantly change covid-19 transmission in 429 cities. medrxiv making sense of the global coronavirus data: the role of testing rates in understanding the pandemic and our exit strategy. medrxiv. 2020 projecting the transmission dynamics of sars-cov-2 through the postpandemic period the authors declare no conflict of interest. key: cord-262786-otxpc46a authors: mohammadi, soheil; moosaie, fatemeh; aarabi, mohammad hadi title: understanding the immunologic characteristics of neurologic manifestations of sars-cov-2 and potential immunological mechanisms date: 2020-09-01 journal: mol neurobiol doi: 10.1007/s12035-020-02094-y sha: doc_id: 262786 cord_uid: otxpc46a similar to its predecessors, coronavirus disease 2019 (covid-19) exhibits neurotrophic properties, which lead to progression of neurologic sequelae. besides direct viral invasion to the central nervous system (cns), indirect cns involvement through viral-mediated immune response is plausible. aberrant immune pathways such as extreme release of cytokines (cytokine storm), autoimmunity mediated by cross-reactivity between cns components and viral particles, and microglial activation propagate cns damage in these patients. here, we review the currently available evidence to discuss the plausible immunologic pathways that may contribute to the development of covid-19 neurological complications, namely alzheimer’s disease, parkinson’s disease, stroke, multiple sclerosis, guillain-barre syndrome, seizure, and brainstem involvement. in december 2019, the outbreak of novel coronavirus disease 2019 (covid-19) emerged in wuhan, china. to date, the outbreak has turned into a pandemic, infecting millions of people globally [1] . although the virus, also known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), mainly manifests as an acute respiratory infection [2] , recent evidence suggests that 36% of affected patients exhibit neurological sequelae [3] . neurologic symptoms are more common among severe cases of the disease, regarding that 84% of the intensive care unit (icu)-admitted sars-cov-2 patients exhibited at least one neurologic symptom [4] . in line with these, brain magnetic resonance imaging (mri) investigations in sars-cov-2 patients show multifocal hyperintense white matter lesions and cortical signal abnormalities (particularly in the medial temporal lobe) on fluid-attenuated inversion recovery (flair), along with intracerebral hemorrhagic and microhemorrhagic lesions, a n d l e p t o m e n i n g e a l e n h a n c e m e n t [ 5 , 6 ] . electroencephalogram (eeg) studies demonstrate high amplitude monomorphic delta waves, indicating the central nervous system (cns) involvement in sars-cov-2 patients [7] . also, functional magnetic resonance imaging (fmri) is recently proposed as an independent predictor of neurologic outcomes [8] . sars-cov-2 patients present with elevated plasma levels of neurofilament light chain protein (nfl) and glial fibrillary acidic protein (gfap), which are known as biochemical indicators of neuronal injury and glial activation, respectively [9] . also, postmortem brain autopsies demonstrate virus invasion to different brain regions, including the hypothalamus and olfactory bulb, accompanied by neural death and demyelination [10, 11] . several pathogenic mechanisms have been proposed for the neurological deficit in sars-cov-2: indirect cns involvement through systemic inflammation, direct invasion of the virus into the cns, multi-organ failure, hypoxia, sepsis, etc. [12, 13] . in this article, we aim to revisit the possible pathomechanisms that may contribute to development of neurologic complications in patients afflicted with this virus with a substantial focus on immunological pathways. angiotensin-converting enzyme 2 (ace-2) [19] . further investigations in rodents show that ace-2 is expressed in brain regions like cortex and raphe and brainstem, predominantly in neurons [19] . also, spatial distribution analysis reveals that ace-2 is highly expressed by neurons and glial cells in different human brain regions, including middle temporal gyrus, cingulate gyrus, substantia nigra, choroid plexus, and, to a lesser extent, hippocampus [20] . a c e -2 i s a t y p e i t r a n s m e m b r a n e metallocarboxypeptidase that acts as a receptor for sars-cov as well as sars-cov-2 [21] . binding of viral spike (s) protein to ace-2 receptors accompanied by proteolytic cleavage of viral spike protein mediated by transmembrane serine protease 2 (tmprss2) facilitates cell entry ( fig. 1 ) [21] . systemic inflammatory response syndrome (sirs) is defined as excessive host immune response against noxious stimuli (e.g., viral infection), through which the primary protective role of cytokine release turns into a detrimental response against host tissues, leading to impaired integrity of capillary walls and end-organ dysfunction [22] . sirs accounts for the neurologic sequelae found in sars-cov patients [23] . sars-cov infects macrophages, monocytes, and dendritic cells and upregulates the expression of proinflammatory cytokines (e.g., tumor necrosis factor-α (tnf-α), interleukin-6 (il-6)), and inflammatory chemokines fig. 1 three phases of cns infection by sars-cov-2. first, the virus directly invades the brain through vascular or retrograde axonal transport within the cribriform plate, and thus the viral load increases in the csf. second, the virus infects, replicates in, and kills neural cells through ace-2 receptors and tmprss2, a facilitator of viral entry. finally, in the third phase, the immune-mediated response against the virus can indirectly involve the brain, although the viral replication declines in this last phase. cns central nervous system, ace-2 angiotensin-converting enzyme 2, tmprss2 transmembrane serine protease 2. adopted from https://www.dpz.eu/de/infothek/wissen/coronaviren.html, by markus hoffmann. adopted with permission (c-c motif chemokine ligand 2 (ccl2), c-c motif chemokine ligand 3 (ccl3), c-c motif chemokine ligand 5 (ccl5), c-x-c motif chemokine 10 (cxcl10)) [24] . complicated cases of sars-cov exhibit higher levels of proinflammatory cytokines like il-6 and interferon-γ (ifn-γ), making them more susceptible to neurologic complications [25] . studies on postmortem cases indicate that lymphocytes and monocytes infiltrate in brain vessel walls, exacerbating the neuronal degeneration and demyelination process [17] . last but not least, indirect immunofluorescence staining and cell-based enzyme-linked immunosorbent assay (elisa) study in the serum of sars-cov patients reveal the presence of igg and igm autoantibodies as well as complement cytotoxicity against human epithelial and endothelial cells [26] . this finding implies a role for autoimmunity in postinfectious complications of sars-cov. middle east respiratory syndrome (mers), another respiratory infection from the cov family, was first reported in saudi arabia in 2012. nearly 20% of mers-cov patients develop neurologic manifestations during the disease course [17] . however, the neurologic sequelae do not always occur in the infection process and might manifest after a delay of 2 or 3 weeks [17] . stroke, encephalopathy, seizure, gbs, and brainstem involvement could accompany mers infection [16, 17] . brain mri in neurologically affected mers-cov patients reveal hyperintense lesions in white matter and subcortical frontal, parietal, and temporal lobes, along with basal ganglia and corpus callosum [27] . the mechanisms of neurological involvement in mers-cov are almost similar to sars-cov, with a surge in circulating cytokine levels and a role for autoreactive autoantibodies [28, 29] . sars-cov-2 invasion throughout the cns can be divided into three phases: first, the virus directly invades the brain through vascular or retrograde axonal transport within the cribriform plate, and thus, the viral load increases in the csf [4] . second, the virus infects, replicates in, and kills neural cells through ace-2 receptors [30] . third, the immunemediated response against the virus can indirectly involve the brain, although the viral replication declines in this final phase [31] (fig. 1) . recent evidence shows that the third phase (also known as sirs) accounts for the majority of cns disturbances mediated by sars-cov-2 [31] . sars-cov-2 invades astrocytes, macrophages, and microglia in cns and induces a proinflammatory state characterized by increased levels of inflammatory mediators including interleukin-1β (il-1 β), interleukin-2 (il-2), il-2 receptor (il-2r), interleukin-4 (il-4), il-6, interleukin-10 (il-10), interleukin-18 (il-18), ifn-γ, tnf-α, granulocyte colony-stimulating factor (gcsf), monocyte chemoattractant protein 1 (mcp-1), macrophage inflammatory protein 1-α (mip-1α), cxcl10, ccl2, and c-reaction protein (crp) [25] . also, sars-cov-2 induces in vitro expression of inflammatory cytokines such as il-6, interleukin-12 (il-12), interleukin-15 (il-15), and tnf-α in glial cells [17] . severe cases of the disease compared with non-severe cases tend to increase in circulatory levels of il-2, il-2r, il-6, il-10, ifn-γ, tnf-α, ccl2, procalcitonin (pct), crp, erythrocyte sedimentation rate (esr), and white blood cell (wbc) and neutrophil counts and decrease in total lymphocyte, cd4+ t lymphocyte, and cd8+ t lymphocyte counts, while b cell count remains in the normal range [24, 32, 33] . also, antibodies (namely igg, igm, and iga) against viral particles rise in the disease course [34] . seroconversion of igm and igg antibodies begins 4 days after the symptom onset [35] . although the median time for seropositivity against the receptor-binding domain of the virus in the serum is equal for different isotypes, igg is estimated to remain positive in the serum for 75 days, that is the longest period comparing with iga and igm, lasting for 51 and 47 days, respectively [34] . of note, the social isolation and the resultant stress exacerbate the aforementioned cytokine release in affected patients [25] . cytokine storm is a term used for the extreme release of interferons, tumor necrosis factors, chemokines, interleukins, and other mediators in a hyperactive and injurious manner against host tissues [36] . cytokine storm is attributed to sars-cov-2 as well as sars-cov infection and accompany with a spectrum of sirs manifestations, including hypotension, tachycardia, tachypnea, and fever, leading to adverse clinical outcomes [36] . cytokine storm involves the cns, regarding the fact that most cytokines are either produced in the brain tissue or able to cross the blood-brain barrier in the condition that released cytokines (namely, il-1β, il-6, and tnf-α) disrupt the integrity of blood-brain barrier [37] . so, brain autopsy studies of postmortem sars-cov-2 patients demonstrate the infiltration of monocytes, macrophages, and t-lymphocytes into the vessel walls ( fig. 2) [15] . also, interferon-α2 (ifn-α2) and ifn-γ upregulate the expression of ace-2 receptors and, thus, facilitate the entry of the virus into host cells in-vitro [38] . cytokine storm results in neural death, activation of microglia, disruption of synaptic plasticity, and impairment in neurotransmitter metabolism. cytokine storm is also a predictor of hippocampal atrophy [12] . cytokine storm stimulates the secretion of glucocorticoids through manipulation of the hypothalamic-pituitary-adrenocortical axis; nevertheless, the resultant increase in glucocorticoids is not able to suppress the inflammation due to impaired negative feedback mechanism [25] . last but not least, the cytokine storm might lead to delayed immune dysregulation after the disease course, manifested by persistent inflammation or immunosuppression, which might cause delayed complications [25] . cns features of sars-cov, mers-cov, and sars-cov-2 are summarized in table 1 . regarding the widespread influence of sars-cov-2mediated immune response on cns, we discuss the inflammatory correlates of neurologic sequelae of sars-cov-2 in the following sections. brain areas like the hippocampus and midbrain are vulnerable to direct sars-cov-2 invasion and induction of alzheimer's disease (ad) and parkinson's disease (pd), respectively [18] . the aberrant immune response characterized by a surge in cytokine levels (e.g., il-6) derived by sars-cov-2 accelerates the process of neurodegeneration that may contribute to the development of neurodegenerative diseases [42] . the mentioned aberrant immune response may be a consequence of infection of intestinal mucosa and modulation of the gut microbiome by sars-cov-2, which can trigger neuroinflammation and neurodegeneration [24] . interestingly, modulation of the gut microbiome plays a role in the pathophysiology of ad and pd [43, 44] . seemingly, other coronaviruses like human coronavirus oc43 (hcv-oc43) may trigger delayed neural degeneration in animal models [45] . alzheimer's disease ad, the most prevalent neurodegenerative disorder, is characterized by the accumulation of intraneuronal aggregates of hyperphosphorylated tau and extracellular beta-amyloid plaques [37, 46, 47] . considering the role of cholesterolbinding protein (apolipoprotein e (apoe)), a potential link between ad and sars-cov-2 infection is implicated. serum cholesterols bind to apoe receptors and induce ace-2 receptor transport to the cell surface [48] . super-resolution imaging study demonstrates that high cholesterol levels confer a 2-fold increase in sars-cov-2 entry sites and thus facilitate the viral entry [48] . apoe and ace-2 are highly expressed in alveolar type ii cells, and the apoe e4 allele modulates the pro-/anti-inflammatory state in macrophages [49] . on the other hand, apoe, acting in conjunction with apoc1 and apoj, transports cholesterol to maintain the myelin and neural membranes, dendritic reorganization, and synaptic turnover [50] . apoe functions as a beta-amyloid chaperone through the transport of beta-amyloids to the lysosomes [50] . apoe e4 allele associates with increased formation and deposition of beta-amyloid, and apoe e4-positive mice exhibit increased accumulation of tau aggregates [50] . in line with these, homozygote apoe e4e4 not only acts as a risk factor for severe sars-cov-2 infection but also confers a 14-fold increase in susceptibility to ad [49] . a growing body of evidence implicates that neuroinflammation may be an origin of ad. persistent systemic inflammation can activate glial cells like microglia and astrocytes. activated microglia secrete pro-inflammatory cytokines (e.g., il-1β, il-6, il-12, tnf-α) [37, 51] . we hypothesize that not only the persistent systemic inflammation caused by sars-cov-2 may act as a trigger for microglial activation but also large amounts of pro-inflammatory cytokines secreted in response to this viral infection may aggravate neurodegeneration leading to ad. elevated levels of tnf-α associate with a 4-fold increase in cognitive dysfunction [37] . increased il-1β and il-6 mitigates microglial phagocytosis of β-amyloid plaques and thus impairs synaptic plasticity and memory [19, 25] . ifn induces synaptic degeneration as a mediator of post-viral inflammatory response. it is worth knowing that βamyloids can encircle viral particles and enhance the aforementioned ifn-mediated response [52] . on the other hand, nod-, lrr-, and pyrin domain-containing protein 3 (nlrp3) inflammasomes play a role in microgliamediated il-1β release in ad. sars-cov-2 open reading frame 3a (orf3a) protein stimulates nlrp3 inflammasomes, activation of which accelerates the neurodegeneration process in ad [12] . pd, the second most common neurodegenerative disorder, is characterized by the degeneration of dopaminergic neurons of substantia nigra pars compacta, along with microscopic findings of intracellular α-synuclein aggregates [53] [54] [55] [56] . autoantibodies against α-synuclein, gangliosides, pigment neuromelanin ma1/ma2, leucine-rich glioma inactivated 1 (lgi1), and glutamic acid decarboxylase (gad65) are found in the serum of pd patients, which implicate a role for autoimmunity in the pathogenesis of pd [53] . knowing that sars-cov-2 may remain latent in neurons, we hypothesize that autoimmunity in sars-cov-2 infection due to crossreactivity mediated by antiviral antibodies may lead to delayed progression of pd [28] . this hypothesis is supported by the fact that anti-cov antibodies are found in the csf of pd patients [28] . we also postulate that aberrant cytokine release into the csf of sars-cov-2 patients may increase the risk of progression into pd by aggravating the neurodegeneration process, considering the fact that pd patients exhibit elevated levels of pro-inflammatory cytokines (including il-1β, il-6, tnf-α, and ifn-γ) in csf. cytokine storm activates microglia, which are implicated in the pathophysiology of pd [57] . moreover, the extreme release of cytokines into the csf mediated by sars-cov-2 dysregulates the balance between production, release, and reuptake of neurotransmitters including dopamine, which possibly contributes to the development of pd [25] . moreover, the common finding of anosmia (total loss of sense of smell) in pd and sars-cov-2 reinforces their association. anosmia is a common finding in neurodegenerative diseases [58] , and over 90% of pd patients develop anosmia or hyposmia (partial loss of sense of smell). anosmia may precede motor symptoms of pd and thus can be used as a marker of underlying neurodegeneration [59] . although mri studies of pd patients show that volume and sulcus depth of olfactory bulb decrease, biopsy samples of olfactory epithelium are normal in these patents [60] . these findings suggest that the observed anosmia in these patients is due to a disturbance in central and not peripheral processing of smell [60] . knowing that autopsy studies of postmortem pd patients suffering from anosmia show abundant deposition of lewy bodies in the olfactory bulb, anosmia is attributed to the distribution of lewy bodies from the medulla in early stages of pd [61] . however, in later stages, cognitive deficits and cholinergic denervation may underlie the development of anosmia in these patients [60] . on the other hand, a recent review reports a nearly 23% prevalence for anosmia in sars-cov-2 patients [62] . several explanations are suggested to shed light on the underlying cause of anosmia in sars-cov-2 patients. at first, it was hypothesized that the local inflammation and the resulting coryza and sinusitis account for anosmia in these patients. however, recent imaging studies demonstrate no signs of sinusitis in sars-cov-2 patients. moreover, the presence of anosmia in the absence of other symptoms suggests that coryza cannot be the reason for olfactory dysfunction in sars-cov-2 [15] . indeed, milder inflammation and inflammatory infiltrations in sars-cov-2 infection comparing with sars suggest a different route of infection through which the virus transmits via infected macrophages to the supporting cells of the olfactory epithelium and olfactory bulb, leading to impairment of olfactory receptors [63] . several findings support this latter hypothesis: first, olfactory epithelial cells (particularly sustentacular cells) and olfactory bulb cells express ace-2 and tmprss2 [64, 65] . second, postmortem studies show that the virus can invade the brain through the olfactory epithelium and olfactory bulb [66] . third, positron emission tomography-computed tomography (pet-ct) study in sars-cov-2 patients with anosmia demonstrate declined orbitofrontal cortex activity, suggesting neural dysfunction [67]. forth, the genome sequence of the virus was found in the olfactory mucosa and olfactory bulb, indicating axonal transport [66] . the latter hypothesis, also known as post-viral olfactory dysfunction, is reported in a case of sars-cov as well and is more prevalent among patients with a history of the neurologic disease [64] . maintenance of viral load in the olfactory epithelium in post-viral olfactory dysfunction can disrupt the regenerative capacity of neurons [64] . both ischemic and hemorrhagic strokes are being increasingly reported in sars-cov-2 patients and are the most common findings in neuroimaging studies (table 2) [40, 75] . the prevalence of stroke is estimated to be between 0.2 and 1% of all infected patients [76] , often proposed as the most common neurologic manifestation in sars-cov-2 patients [77] . high incidence of stroke in sars-cov-2 patients under 50 years old without cardiovascular risk factors accompanied by worse outcomes in sars-cov-2-mediated stroke comparing with other strokes indicates that this viral infection precipitates the pathways leading to stroke [76] . postmortem autopsy studies of sars-cov-2-mediated stroke demonstrate hyperemic and edematous brain areas [76] . also, micro-and macrothrombosis are found in other organs such as lung and kidney [76] . these findings indicate that coagulopathy may underlie the occurrence of stroke in these patients. severe inflammatory response mediated by sars-cov-2 can upregulate pro-coagulative factors [17] . recent investigations suggest that blood d-dimer, crp, fibrinogen, and wbc count increases in sars-cov-2 patients, which propagate the hypercoagulable state. moreover, a group of sars-cov-2 patients present positive lupus anticoagulant, anti-β2-glycoprotein-1, and anticardiolipin (marker of antiphospholipid syndrome) antibodies, which may precipitate stroke [15] . another key contributor to stroke is the vascular wall injury, which induces the release of tissue factor [17] . damage to vascular walls may occur as a consequence of direct viral invasion and replication in the vascular epithelium, also known as endotheliitis [66] . endotheliitis due to sars-cov-2 is reported in other organs such as lung, heart, kidney, and bowel but not yet investigated in cns [15] . also, the aberrant immune response mediated by sars-cov-2 may provide the driving force for vascular wall damage [23] . this process takes place as a component of secondary hemophagocytic lymphohistiocytosis, characterized by inflammatory-mediated tissue damage through hyperactivation of macrophages and lymphocytes, leading to microangiopathy [15] . several other mechanisms may increase the risk of stroke in sars-cov-2 infection. the cytokine storm mediated by sars-cov-2 precipitates microthrombosis [41] . in fact, elevated expression of pro-inflammatory cytokines (namely il-1β, il-6, and tnf-α) increases the risk of ischemic and hemorrhagic strokes [78] . also, anti-cytokine therapies against these mediators protect from recurrence of stroke in experimental models [78] . moreover, systemic inflammation can ignite atrial fibrillation and rupture the carotid plaques, leading to ischemic stroke [15] . also, sars-cov-2 downregulates the expression of ace-2 receptors, which degrade angiotensin ii. so, the increase in angiotensin ii and the resulting activation of the renin-angiotensin-aldosterone system (raas) system promotes sympathetic tone, oxidative stress, and inflammation and triggers fibrotic events [79] . multiple sclerosis (ms) is a chronic disabling disorder characterized by demyelination of white matter and, to a lesser extent, gray matter of cns [51] . recently, the first casereport of ms after sars-cov-2 infection was presented in the literature [80] . a sars-cov-2 patient without a history of in flair images along with abnormalities in frontal, [5] neurological symptoms developed visual acuity and field defects and hyperreflexia. orbital and brain mri investigations demonstrated enhancement of left optic nerve accompanied by demyelinating lesions in supratentorial periventricular areas of occipital and temporal lobes (table 2) . interestingly, csf pcr analysis of sars-cov-2 was negative, while csf oligoclonal igg and igm were positive, conferring a postinfectious immunological response as an etiology for this phenomenon [80] . an autoimmune inflammation mediated by activated microglia and cytotoxic t-cells against oligodendrocytes underlies the myelin degeneration in ms [51] . elevated levels of pro-inflammatory cytokines (e.g., tnf-α, il-6, ifn-γ, il-1β) are found in the csf of ms patients suffering from either white matter or gray matter deficits [24] . tnf-α is isolated from active areas of neurodegeneration in postmortem ms brains [57] . overexpression of tnf-α stimulates apoptosis of oligodendrocytes and infiltration of immune cells into the csf, while intrathecal injection of tnf-α monoclonal antibodies prevents from the development of ms in animal models of experimental autoimmune encephalomyelitis (eae) [57] . il-6 maintains the balance between cytotoxic and regulatory t-cells. overproduction of il-6 inhibits the differentiation of t-cells into regulatory t-cells and thus promotes the inflammatory state and demyelination in mice [57] . in line with this, il-6 deficient mice are resistant to ms, and administration of il-6 receptor (il-6r) antibodies inhibits the myelin degeneration in eae mice [57] . moreover, the aberrant release of il-6 disrupts the blood-brain barrier integrity and facilitates the infiltration of cytotoxic t-cells [57] . last but not least, il-1β receptor (il-1βr) antibodies antagonize the demyelination process in rat models of eae, which suggests a role for il-1β in the pathogenesis of ms [81] . after all, knowing that the aforementioned cytokines surge as a result of sars-cov-2 mediated inflammation, we postulate that persistent inflammation in sars-cov-2 may propagate myelin destruction and lead to delayed progression of ms. moreover, it is hypothesized that an autoimmune reaction mediated by the cross-reaction between viral particles and myelin basic protein may provide the driving force for neural demyelination. this hypothesis is supported by the fact that genome of other coronaviruses like cov-oc43 and cov-229e, as well as their antibodies, are isolated from cns of ms patients, and coronavirus-like particles are found in perivascular cuffs of human ms brain [82] . in fact, the virus might lie dormant in astrocytes and oligodendrocytes and trigger the autoimmunity mediated by molecular mimicry. interestingly, intracranial inoculation of murine coronavirus (mouse hepatic virus (mhv)), induces optic neuritis and focal encephalitis leading to chronic demyelination in mice [83] . in this case, both innate and adaptive immune responses comprising of activation of microglia, b cells, cd4 t cells, and [74] cd8 t cells along with the direct viral toxicity are proposed to mediate myelin stripping [68, 70] . gbs i s a pos t-in fectiou s autoimmune caus e of polyneuropathy in predisposed individuals that leads to flaccid paralysis of lower extremities, along with autonomic and sensory deficits [69] . autoimmunity is the milestone of gbs pathophysiology. neurotrophic macrophages present bacterial or viral particles to lymphocytes in peripheral nervous system (pns) [69] . the resultant immune response (i.e., complement and macrophage activation, cytokine release, and free radical production) accompanied with the molecular mimicry between pathogen epitopes and host gangliosides such as n-acetylgalactosaminyl gd1a (galnac-gd1a), ganglioside d1a (gd1a), ganglioside (gm1), and ganglioside m1b (gm1b) and myelin proteins result in acute neural damage [69] . gbs is reported in other post-viral infections, including influenza, enteroviruses, zika, sars-cov, mers-cov, and sars-cov-2 [13] . several cases of gbs due to sars-cov-2 infection are presented in the literature with the varying onset of gbs symptoms from a day before to 3 weeks after presentation of sars-cov-2 symptoms [71, 84] . enhancement of cervical and cranial roots (e.g., optic, oculomotor, and facial nerves) in mri studies along with presence of axonal and demyelinating patterns in nerve conduction studies are suggestive of gbs in sars-cov-2 patients (table 2 ) [71, [84] [85] [86] . several findings indicate that the occurrence of gbs in sars-cov-2 is mediated by an autoimmune response mediated by molecular mimicry and not the direct viral invasion. binding of sars-cov-2 spike protein to cell surface occurs not only through ace-2 receptors but also through sialic acid-containing glycoproteins and gangliosides. considering the fact that anti-ganglioside antibodies are found in the serum of sars-cov-2-triggered gbs patients, a cross-reactivity between sars-cov-2-binding gangliosides and peripheral nerve glycolipids are implicated [87] . also, recent experiments are not able to detect the viral genome in csf of a high proportion of post-sars-cov-2 gbs patients [23, 39, 72] . moreover, the administration of intravenous immunoglobulin (ivig) resolves symptoms and improves the outcomes in post-sars-cov-2 gbs patients [59] . other plausible theories may underlie the development of gbs in sars-cov-2. a shift in immune response toward pro-inflammatory cytokines (e.g., il-1β, il-6, tnf-α, and ifn-γ) occurs in acute stages of gbs and mediate neural degeneration [69] . similarly, elevated levels of il-1β and il-6 are found in the csf of gbs patients [69] . so, the release of pro-inflammatory cytokines in sars-cov-2 may propagate the neurodegeneration leading to gbs. also, the sars-cov-2-mediated cytokine storm may result in disruption of blood-brain barrier integrity and infiltration of lymphocytes leading to gbs [73] . recent retrospective, case series, and case report studies indicate that sars-cov-2, as well as sars-cov and mers-cov patients, is prone to encephalopathy and seizure [88] . brain mri studies of these patients demonstrate multifocal involvement of cortex and white matter (including periventricular areas), along with multiple hyperintense areas in t2-weighted and flair images in temporal, frontal, and occipital lobes and hippocampus (table 2 ) [74, 89] . consequences of sars-cov-2 infection, including organ failure, hypoxia, metabolic disturbance, and direct viral invasion into the cns, are proposed to explain the observations of encephalopathy and seizure in these patients [88] . however, most findings are in favor of immune-mediated encephalopathy and seizure. except for two case reports, all the remaining studies are not able to detect the viral genome in the csf of these patients [41] . seemingly, most patients exhibit a dramatic response to plasmapheresis and steroid therapy, which indicates a role for autoimmune-mediated injury [23] . as mentioned before, the aberrant release of pro-inflammatory cytokines like il-1β, il-6, and tnf-α disrupts blood-brain barrier permeability, and the resulting infiltration of wbcs and activation of microglia and astrocytes damages neurons and glial cells [24, 41] . also, it is speculated that the viralmediated production of autoantibodies against glial cells might be responsible for neural injury [74] . transgenic mice infected by sars-cov and mers-cov exhibit brainstem involvement due to trans-synaptic projection of the virus throughout the olfactory epithelium, olfactory bulb, thalamus, and finally the brainstem [16] . the virus might target the primary respiratory oscillator (known as pre-bötzinger complex) in the brainstem and result in respiratory failure in these animal models [16] . seemingly, acute respiratory failure due to brainstem dysfunction is reported in sars-cov-2 patients [90] . also, a recent case study reports post-infectious brainstem involvement presenting with involuntary movements, myoclonus, and hyperekplexia after gradual respiratory symptom relief in a case of sars-cov-2 [91] . postmortem autopsy studies elucidate areas of hyperemia and edema and neurodegeneration in the brainstem [90] . mri findings in a case of brainstem involvement demonstrate high signal lesions in bilateral pons in t2-flair images ( table 2 ) [92] . these findings are in favor of brainstem involvement due to direct viral invasion. however, the absence of viral genome in csf along with the presence of inflammatory mediators (namely tnf-α and interleukin-8 (il-8)) and pleocytosis in csf in a group of patients with brainstem dysfunction suggests that the aberrant immune response might underlie the brainstem dysfunction in sars-cov-2 [90] . sars-cov-2 pandemic, beginning in december 2019, has now become a global concern. although the acute respiratory manifestations of the disease are greatly considered, reports of neurologic sequelae are dramatically increasing. a recent cohort study of sars-cov-2 patients reported a 4.3% prevalence for neurologic symptoms, other than anosmia and headache [93] . also, para-clinic investigations support the idea of post-sars-cov-2 cns involvement ( table 2) . although the cns complications might be a consequence of direct viral invasion into the cns, recent findings are in favor of immune-mediated cns involvement [31] . sars-cov-2 genome sequences are frequently undetectable in the csf [93] . also, recent studies suggest that blood-brain barrier integrity is disrupted, and lymphocytic pleocytosis and wbc infiltration into the cns occur in these patients [93] . interestingly, the presence of auto-antibodies against gangliosides in sars-cov-2 patients suggests that an autoimmune phenomenon mediated by the virus might underlie the neurologic symptoms [93] . as a result, we propose that the aberrant immune response mediated by sars-cov-2 is responsible for the majority of acute or chronic neurological manifestations. in fact, sars-cov-2 infection increases the risk of alzheimer's disease, parkinson's disease, stroke, multiple sclerosis, guillain-barre syndrome, seizure, encephalopathy, and brainstem involvement through different immune pathways including fig. 3 the pathomechanisms of immune-mediated cns involvement in sars-cov-2 infection. sars-cov-2 induces neurodegeneration through cytokine storm, autoimmunity, microglial and inflammasome activation, and gut microbiome modulation. these pathways lead to development of alzheimer's disease in susceptible individuals and worsen the severity of parkinson's disease in affected patients. inflammatorymediated vascular wall injury, atrial fibrillation, carotid plaque rupture, and upregulation of pro-coagulative factors may increase the risk of stroke in these patients. cytokine storm and autoimmunity induced by cross-reactivity between viral particles and cns components may contribute to the development of autoimmune disorders of cns, like guillain-barre syndrome. also, the aforementioned mechanisms exacerbate symptoms in multiple sclerosis patients. cytokine storm induces microglial activation and wbc infiltration into the cns and, thus, precipitates seizure and encephalopathy. cns central nervous system, wbc white blood cell. adopted from https://www.dpz.eu/de/infothek/wissen/ coronaviren.html, by markus hoffmann. adopted with permission cytokine storm, autoimmunity, microglial and inflammasome activation, gut microbiome modulation, wbc infiltration into the cns, inflammatory-mediated vascular wall injury, and upregulation of pro-coagulative factors (fig. 3) . precise investigation and follow-up of sars-cov-2 patients is warranted in this issue, as the delayed presentation of these complications may lead to underdiagnosis. immunomodulatory drugs such as cytokine-blocking therapies (e.g., il-6r monoclonal antibodies, tnf-α inhibitors, il-1β antagonists), antibodies against viral spike proteins and viral binding receptors (ace-2), anti-inflammatory drugs like nonsteroidal antiinflammatory drug (nsaid), and prophylactic antibodies are under investigation in the prevention and treatment of sars-cov-2 [94, 95] . however, the efficiency of these 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claims in published maps and institutional affiliations the authors declare that they have no conflict of interest. key: cord-282058-it0ojdk3 authors: yu, yuanqiang; chen, pingyang title: coronavirus disease 2019 (covid-19) in neonates and children from china: a review date: 2020-05-15 journal: front pediatr doi: 10.3389/fped.2020.00287 sha: doc_id: 282058 cord_uid: it0ojdk3 at the end of 2019, a novel coronavirus began to spread in wuhan, hubei province, china. the confirmed cases increased nationwide rapidly, in part due to the increased population mobility during the chinese lunar new year festival. the world health organization (who) subsequently named the novel coronavirus pneumonia coronavirus disease 2019 (covid-19) and named the virus severe acute respiratory syndrome coronavirus-2 (sars-cov-2). soon, transmission from person to person was confirmed and the virus spread to many other countries. to date, many cases have been reported in the pediatric age group, most of which were from china. the management and treatment strategies have also been improved, which we believe would be helpful to pediatric series in other countries as well. however, the characteristics of neonatal and childhood infection still have not been evaluated in detail. this review summarizes the current understanding of sars-cov-2 infection in neonates and children from january 24 to may 1, as an experience from china. from 2002 to 2003, the outbreak of severe acute respiratory syndrome (sars) in guangzhou, china, caused a global epidemic, which brought widespread concern about a coronavirus epidemic (1) . later, another zoonotic coronavirus pathogen, known as the middle east respiratory syndrome coronavirus (mers-cov), spread in the middle east from 2012, and the disease was named middle east respiratory syndrome (mers) (2) . a new type of coronavirus was recently reported in wuhan, hubei province, china, which also causes severe respiratory disease. the outbreak of the disease began in china, and has brought a heavy burden on the whole world (3) . considering that newborns and children are susceptible to infectious diseases, the prevalence of the disease among them is the subject of much attention. the strategy in dealing with the cases in neonates and children, as well as a healthy pediatric age group, form elaborate plans in fighting against the novel coronavirus disease. such experience from the chinese government and hospitals may also benefit the rest of the world. here, we review the advances of current research from january 24 to may 1 in the epidemiology, clinical manifestations, management, and treatment of this disease in newborns and children. cases and recommendations in the pediatric age group from china, published either in english or chinese, are included. references for this review were identified through searches of pubmed for articles published from january 1, 2003, to may 1, 2020, by use of the terms "coronavirus, " "neonate, " "children, " "covid19, " and "sars-cov-2." relevant articles published between 2003 and 2020 were identified through searches in the authors' personal files. we further searched the recent online articles from the covid-19 academic research communication platform of chinese medical journal network, where the latest relevant chinese articles are published. some news and policies from who are also involved for the latest information of covid-19. articles published in english and chinese were included. articles resulting from these searches and relevant references cited in those articles were reviewed. in late december 2019, wuhan, hubei province, china reported for the first time a large cluster of patients with unexplained pneumonia associated with the wholesale huanan seafood market (4) . subsequently, the chinese center for disease control and prevention (china cdc) sent a rapid response team to identify the source of the pneumonia virus cluster, and then isolated and sequenced a new coronavirus, named 2019 novel coronavirus (2019-ncov) (5) . the world health organization (who) subsequently named the novel coronavirus pneumonia coronavirus disease 2019 (covid-19) and named the virus severe acute respiratory syndrome coronavirus-2 (sars-cov-2). since the virus was transmitted to additional family members by a family (including a 10-year-old asymptomatic child) returning to shenzhen from wuhan, widespread transmission had quickly emerged from person to person (6) . as the chinese lunar new year festival approached, population mobility had increased and the virus had spread rapidly throughout the country (7) . although the incubation period values are very similar to sars or mers, the transmission of covid-19 may be more rapid, because of the possibility of transmission during the incubation period (8, 9) . specifically, some patients may be completely asymptomatic carriers who have passed symptombased screening, but rt-pcr was positive for sars-cov-2 (10, 11) . who then identified the incident as a public health emergency of international concern (pheic) on january 30, and on march 11 assessed that covid-19 could be characterized as a pandemic (12, 13) . among the previously diagnosed family from shenzhen, the 10year-old asymptomatic boy was the first child confirmed infected with the virus (6) later, on january 19, 2020, a 7-year-old boy with a fever and cough was reported in shanghai after visiting his grandfather in wuhan (14) . the symptoms of covid-19 appear to be less severe in infants and children than in adult patients, similar to the sars-cov infection (15) (16) (17) . the first case series report in children showed that the interval between symptom onset and exposure to index symptomatic case ranged from two to 10 days (mean 6.5 days), which suggests a longer incubation period for sars-cov-2 infection in children (18) . furthermore, the mean number of secondary symptomatic cases in a household exposure setting was 2.43, similar to the basic reproductive number in earlier research on adults (18, 19) . most cases in children were likely to expose themselves to family members or other children with covid-19, and linked directly or indirectly to hubei province, indicating that extra protection of children in families is urgently needed, especially those linked to wuhan (16, 20) . a 13-month-old child was reported as the first severe case on february 8 (21) . furthermore, a 17 day-old newborn was reported as the first neonatal infection on february 5, testing positive with sars-cov-2 in pharyngeal swabs and anal swabs (22) . in another case, pharyngeal swab testing was positive 36 h after birth (23) . china cdc reported that, as of february 8, 2020, of 2,135 pediatric patients <18-years old, 728 (34.1%) were laboratory-confirmed with covid-19 and 1,407 were (65.9%) suspected (16) . nearly 1% of the total population of patients reported were children under 10-years old (24) . two deaths were reported in children. one was a 14-year-old boy and the other was a 10-month-old child (16, 25) . seven neonates were reported with a positive nucleic acid test, and three with elevated igm antibodies to sars-cov-2 and negative nucleic acid tests (22, 23, (26) (27) (28) (29) (30) . therein, no death but one severe case was involved (26) . therefore, we call for preventive and protective measures for pregnant women, newborns, and children against the spread of the disease as soon as possible. the rapid and close collaboration between epidemiologists, virologists, biologists, clinicians, and drug researchers during the covid-19 outbreak is commendable. early in the disease outbreak, different models estimated the basic reproduction number r0 of sars-cov-2, calling for public health interventions and preparation plans (31-34). the chinese government had taken emergency measures, such as organizing medical teams to support wuhan, controlling population movements, establishing more hospitals for the treatment of covid-19, and developing specific vaccines (35) . a nationwide school closure had also been ordered, and children were confined in their homes with online courses offered (36) . based on the epidemiological data, different countries have adopted different measures to limit the spread of the novel coronavirus as well, such as issuing travel warnings, interrupting flights, prohibiting nationals from going to severely affected countries, and adopting 14 day quarantine rules for nationals from affected areas (37, 38) . coronaviruses (covs) are pathogens that can infect humans, domestic animals, and much wildlife, and can invade multiple organ systems such as the respiratory, gastrointestinal, liver, and central nervous systems. this subfamily includes four genera: alpha-coronavirus, beta-coronavirus, gamma-coronavirus, and delta-coronavirus (39) . sars-cov-2 is the seventh cov known to infect humans and cause respiratory diseases. it belongs to the clade 2 of the subgenus sarbecovirus, orthocoronavirinae subfamily of beta-coronavirus, and is different from sars-cov and mers-cov (5, 40) . the novel coronavirus was first isolated from human airway epithelial cells and observed under a transmission electron microscope (5) . electron micrographs showed the distinctive spikes(s) (about 9-12 nm) and corona of the virus particles. in ultrathin sections of the human airway epithelium, virus particles were filled in membrane-bound vesicles in the cytoplasm or distributed in the extracellular matrix (5) . researchers had found that the genome had 89% nucleotide homology with bat sarslike covzxc21, and even 96.2% sequence identity with batcov ratg13 (41, 42) . another study also suggests that pangolins may be possible hosts of sars-cov-2 (43) . in addition, the sars-cov-2 genomic sequence is far from sars-cov (about 79%) and mers-cov (about 50%) (40, 41) . the amino acids in different proteins have also been replaced accordingly, which further explains the structural and functional differences between sars-cov-2 and sars-cov (44) . however, sars-cov-2 has a similar receptor-binding domain structure to sars-cov, which is located in the s1 conserved domain and critical for determining host tropism and transmission capabilities (40) . they may use the same cell-targeted receptor angiotensin-converting enzyme 2 (ace2), and cryo-em showed that sars-cov-2 s had 10-to 20-fold higher affinity to bind with ace2 than sars-cov s (41, 45, 46) . further research and understanding of the structure of sars-cov-2 would better facilitate the development of vaccines as well. it has to be mentioned that the specimens from the respiratory and gastrointestinal tracts were detected as sars-cov-2, which indicates the potential multiple ways of sars-cov-2 transmission, including fecal-oral transmission, and the possibility of targeting different organs (47) . cases in adults with active virus replication in the upper respiratory tract display a shed pattern that resembles patients with influenza (48, 49) . furthermore, from biopsy samples taken from the lung, liver, and heart tissues of infected and dead adult patient, similar pathological features to sars and mers coronavirus infections have been found (50, 51) . the lungs showed evidence of acute respiratory distress syndrome (ards), while the liver showed moderate microvascular steatosis and mild lobular and portal activity. the heart tissue was infiltrated with mononuclear inflammatory cells, without substantial damage (50) . a recent study also found highly expressed ace2 in proximal and distal enterocytes (52) . in human small intestinal organoids (hsios), enterocytes were readily infected by sars-cov-2 (53) . these all reflect the complexity of this novel virus, and we still need more data on transmission dynamics and pathology in neonates and children to further explain the virologic characteristics. during the rapid spread of covid-19 in china and other countries, sars-cov-2 infection in pregnant women seems inevitable. however, there are only several reports of infection in pregnant women and of neonates born to infected mothers in china. of the 34 pregnant women who were confirmed with the sars-cov-2 infection in multiple hospitals in wuhan, including one pregnant woman with a negative nucleic acid test result, 30 had a fever and 16 had a cough (54) (55) (56) (57) . other symptoms included diarrhea in eight patients, myalgia in seven, fatigue in six, sore throat in five, shortness of breath in five, chest pain in three, headache in three, and rashes in two (54) (55) (56) (57) . among them, 30 were in their third trimester and the other four were in the second trimester. fetal distress was monitored in eight of the pregnant women. one case had vaginal bleeding during the third trimester, and six had premature rupture of membranes (prom). in addition, one patient had gestational hypertension and another had preeclampsia (55) . other comorbidities included hypothyroidism and polycystic ovary syndrome (57) . all patients had an epidemiological history and had been exposed to covid-19. most patients showed typical features of chest ct images, such as multiple plaque-like ground glass shadows in the lungs, plaque consolidation, and blurred borders (54, 55) . finally, 26 of the pregnant women delivered their babies by cesarean section, and three of them delivered vaginally. one case with a gestational age of 28 weeks had a benign outcome and did not give birth, with conserved treatment to prolong gestation (56) . furthermore, there was one miscarriage at 26 gestational weeks within the onset of bipolar disorder, and the woman required the termination of her pregnancy. it was unknown whether the outbreak of covid-19 influenced her onset of bipolar disorder. noticeably, four of these 34 patients developed severe pneumonia, in which one developed worse and was transferred into icu (55, 56) . the clinical characteristics of covid-19 in pregnant women appear to be similar to those reported in non-pregnant adult patients with covid-19, which could be further confirmed with recent cases outside wuhan (55, (58) (59) (60) (61) . according to the recent report of 118 pregnant women with covid-19 in wuhan, the risk of severe disease compared favorably with the risk in the general population of patients in mainland china (62) . no maternal death has been reported. comparably, the clinical outcome of pregnant women during sars in hong kong was worse than that of infected women who were not pregnant (63) (64) (65) . pregnant women infected with mers-cov might also develop serious diseases, and even the maternal outcome was fatal (66) . considering the relationship between sars-cov-2 and sars-cov or mers-cov, more cases need to be observed, and covid-19 in perinatal pregnant women needs treatment with more caution. of the 30 pregnant women in the third trimester mentioned above, 29 of them gave birth to 30 babies, including one set of twins (54) (55) (56) (57) . of these, 12 were premature infants (gestational age ranging from 31 weeks to 36 weeks plus 3 days), among them three were low-birth-weight infants, and two were smallfor-gestational-age (sga) infants (54) (55) (56) . the 1-and 5-min apgar scores of all live births were 8-10, except for one lga infant who had a 1-min apgar score of 7-and a 5-min apgar score of 8. pharyngeal swab specimens were collected from 22 of the 30 neonates, and only one was positive at 36 h after birth (54) (55) (56) (57) . six of the newborns developed shortness of breath, in which five were premature and intrauterine fetal distress was found in mothers of four neonates, but no severe neonatal asphyxia was observed. other symptoms included vomiting, moaning, edema and skin damage, fever, milk rejection, and gastrointestinal bleeding (54) . the newborn with positive sars-cov-2 had no fever and cough, with only mild shortness of breath (23, 57) . so far, three patients developed disseminated intravascular coagulation (dic) in two case series, possibly because of immature immune function of the neonates and suspected sepsis (26, 54) . one of them eventually died, one improved with antibiotic treatment, and the other also improved after receiving intravenous immunoglobulins (ivig) transfusion (26, 54) . it suggests that gamma-globulin may be effective in severe cases. however, the dose of ivig was not mentioned in the case and needed further exploration (54) . radiographic findings were non-specific. within the 33 neonates born to affected mothers reported recently, chest radiographic images in the three with positive sars-cov-2 showed pneumonia (26) . recently, another case of neonatal death within 2 hours of birth was reported because of severe neonatal asphyxia. the mother developed severe pneumonia and septic shock after admission (60) . therefore, the severity of neonatal symptoms is closely related to the maternal condition (54) . moreover, maternal chronic illness or complications and effective treatment of the newborns may also affect their outcome (58) . however, there is no evidence that the emergence of covid-19 in the third trimester of pregnancy may result in severe adverse outcomes in neonates, which is caused by vertical transmission in the womb (55) . amniotic fluid, umbilical cord blood, neonatal throat swabs, and even breast milk samples were collected and tested, but no sars-cov-2 was found (55) . pathological analysis has also showed no evidence of viral infection or chorioamnionitis in placental tissue (67) . in addition, one study used public single-cell rna sequencing databases to analyze mrna expression profiles and found that the expression of ace2 in different cell types in the early maternal-fetal interface was very low, which may provide an explanation of low risk of vertical transmission in covid-19 and sars (68) . however, at least five neonates born to covid-19 pregnant women tested positive for sars-cov-2 (23, 26, 27) . three infants born to mothers with covid-19 had elevated igm antibodies to sars-cov-2 (29, 30) . they were delivered in negative-pressure isolation rooms, and the mothers wore masks in delivery. these results remind us that more evidence is still needed to evaluate whether vertical transmission could be a possible way of coronavirus transmission (58, 63) . in addition, a neonate was diagnosed as having covid-19 17 days after birth and he had a history of close contact with two confirmed cases (parents of the newborn) (22, 58) . the patient's early clinical symptoms were mild, such as transient fever and diarrhea, without any severe complications. x-ray imaging of the lungs showed inflammatory changes. repeated positive nucleic acid test results of pharyngeal and anal swabs indicated that the virus could appear in the respiratory and digestive tracts of newborns (22) . this case also indicates that there is a possibility that family members or the community may be a source of neonatal infection. another case recently reported was a 19 day-old baby boy, who also showed gastrointestinal symptoms (28) . although the symptoms could be mild, protection of the newborns still needs to be strengthened. they may show different symptoms from adults, therefore, either the parents or the doctors should be more aware of any abnormal conditions when breastfeeding. additionally, no cases of sars-cov-2 infection have been reported in women in the first trimester of pregnancy. given that the fetus of a mother infected with sars-cov in the first trimester of pregnancy would develop intrauterine growth restriction (iugr), more attention should be paid on the prevention of covid-19 in the first trimester of pregnancy (63) . the proportion of infants and children diagnosed with covid-19 is currently small, which may be related to the lack of pathogen detection among them. it may be because they have a lower risk of exposure, or that they either have mild symptoms or are asymptomatic, which is not easily identified, rather than them being less susceptible than adults (16, 25, 69) . the early stages of the covid-19 epidemic mainly involve adults over the age of 15, indicating confirmed childhood cases are more likely transmitted from family members or the community (19) . in addition, the ability of children to transmit the virus may be limited, and no clear report has been found that children can be the source of infection in adults (70) . the most common symptoms of covid-19 in children were a fever and cough. other symptoms included fatigue, myalgia, nausea, vomiting, and diarrhea, which seems to be milder than adults with covid-19 (table 1) (20, 25, 81, 82) . within 2,135 pediatric patients <18-years old who reported with covid-19, groups of all ages were susceptible (16) . the median age of all patients was 7-years, and no statistically significant difference was shown in gender (16) . among both confirmed and suspected cases, 94 (4.4%), 1088 (51.0%), and 826 (38.7%) were diagnosed as asymptomatic, mild, or moderate, respectively (16) . another report in 171 children with sars-cov-2 infection showed the median age was 6.7-years (25) . fever was present in 41.5% of the children at any time of the illness (25) . specifically, symptoms could be mild in infants (28 days to 1-year), with only fever or mild upper respiratory symptoms (15, 71) . however, the proportion of severe and critical cases amongst pediatric groups was highest in infants <1-year old, which reveals that young children, particularly infants, were vulnerable to sars-cov-2 infection (16) . according to the case of a 55 day-old female infant, multiple organ damage affecting the lungs, liver, and heart may be present (72) . both the nasopharyngeal swab and stool specimen tested positive for sars-cov-2. the symptoms were initially mild but progressed rapidly later. therefore, frequent and careful monitoring, as well as timely and appropriate treatment, are important in infant cases (72) . similarly, children with sars-cov-2 infection may also have severe symptoms. the first severe case of childhood infection was reported on january 27, 2020, in wuhan (21). he was a 13-month-old child, with frequent vomiting and diarrhea at first, which rapidly progressed to other acute symptoms including shortness of breath and oliguria 6 days later, which turned to ards, septic shock, and acute renal failure at last. he had no comorbidities. nucleic acid tests were not positive until it was performed for the third time. given that his immune system may be overreacted, and it was necessary to maintain acid-base balance and improve organ function in the critically ill patient, continuous renal replacement therapy (crrt) was used and finally improved his symptoms. in severe or critically ill pediatric patients, the most common symptom is shortness of breath, and invasive mechanical ventilation may be indicated for effective respiratory support (73) . children with cancer could also be exposed to sars-cov-2 infection. an 8-year-old boy with acute lymphoblastic leukemia was confirmed with covid-19 recently (73) . the symptoms included pancytopenia and fever. the conditions turned critical regardless of assisted ventilation. therefore, development of standardized guidance for prevention in children with cancer and collaboration among the pediatric oncology community are urgently required (83) . in addition, another situation also needs to be paid attention to. this was a case of a child diagnosed with covid-19 with acute appendicitis (75) . he was initially prepared for abdominal surgery for "acute appendicitis, " but he developed a fever before the operation. his mother told the doctor that he had dinner with his grandmother before, who was earlier confirmed to be sars-cov-2 positive. therefore, it has to be considered that children and infants may not cooperate with the examination, and the description may be unclear. respiratory symptoms and physical signs are not obvious among them as well. when the emergence of surgical related symptoms happens, such as acute abdominal pain as the first manifestation, the possibility of sars-cov-2 infection needs to be discussed, and more concern is also needed on the reasonable arrangements for surgical operations during the epidemic. in addition to atypical clinical symptoms, early radiographic findings of children with pulmonary infections were also milder than those of adults, and most were nodular ground-glass changes or unilateral patchy lesions (18, 25, 76, 84) . the ct characteristics were atypical, with a more localized ground glass opacity (ggo) extent, lower ggo attenuation, and relatively rare interlobular septal thickening (85) . furthermore, the ct imaging of severe cases of covid-19 may be similar to the findings of adults, such as pulmonary parenchymal groundglass lesions and consolidative pulmonary opacities in the lung (21, 86, 87) . on the other hand, laboratory tests of the 13-month-old severe case mentioned above showed similar characteristics to adult cases. in the acute phase of the disease, c-reactive protein was significantly increased, cd3 + t cells and natural killer cells were significantly reduced, and c3 and c4 levels were also significantly reduced (21) . the child's t cell activation was inhibited, but the body's immune system can be over-activated, indicating the complex role of the immune system in the progression of covid-19. other abnormal laboratory findings in common and severe cases are elevated creatine kinase mb, decreased lymphocytes, and elevated procalcitonin and alanine aminotransferase, which indicates possible damage of multiple organs (20, 88) . noticeably, the older children may have significantly decreased lymphocytes, elevated procalcitonin, and decreased creatine kinase compared with the younger patients, such as children under 5-years old (20) . the reliability of real-time reverse transcription pcr (rt-pcr) for the detection of sars-cov-2 has been demonstrated, particularly in collected patient saliva or pharyngeal swabs (89, 90) . recommendations from china for the diagnosis of covid-19 also suggest the use of real-time fluorescent rt-pcr to detect sars-cov-2 nucleic acid (80, (91) (92) (93) . it is important especially in children with atypical symptoms (6) . another method suggested is metagenomic next-generation sequencing (mngs) of rna extracted from bronchoalveolar lavage fluid (balf) or other specimens (80, 94) . however, it has to be mentioned that the first two pharyngeal swab nucleic acid tests of the severe 13-month-old child abovementioned were negative, and they were not positive until the third nucleic acid test on the 13th day of onset (21) . the delay in diagnosis and treatment of children may be fatal, since there have been two deaths in children (16, 25) . therefore, other samples should be actively explored and evaluated for the diagnostic value of sars-cov-2 infection in children as in recommendations, such as the upper or lower respiratory tract, blood, stool, and urine, in order to increase the positive rate of nucleic acid detection (80, 92, 93, 95) . given that neonates seem to manifest gastrointestinal symptoms more commonly, persistent anal swab tests might be more useful (22, 28, 52) . however, there are still some atypical cases with epidemiological history, respiratory or gastrointestinal symptoms, and positive chest ct manifestations that may have negative rt-pcr results for sars-cov-2 in adults (96) (97) (98) . in the diagnosis of patients with suspected covid-19, the positive rate of chest ct imaging may be even higher than that of rt-pcr analysis. the patients may first show a positive chest ct, and the improvement of the chest ct can be reflected earlier in recovery, indicating its better sensitivity in diagnosis of covid-19 (99) . given that chest radiographic images could also reflect abnormalities in most cases of neonates and children, the combination of imaging and nucleic acid tests may be a better method for comprehensive evaluation of pediatric patients with covid-19 (25, 26) . additionally, chest x-rays and cts should be performed with more caution in pediatric patients for protection to this vulnerable population from the risk of radiation (85) . moreover, application of pulmonary ultrasounds in neonates may show pulmonary abnormalities of covid-19 with better sensitivity and safety than chest x-rays and cts, which provides more chances in monitoring and evaluation of the disease (100). in addition, specific antibody tests are available for retrospective diagnostic and epidemiological studies, which have already been used as one of the methods for diagnosis of covid-19 according to the latest version of new coronavirus pneumonia prevention and control protocol from national health commission of the people's republic of china (china nhc) (80) . igm antibodies to sars-cov-2 in neonates may also have indication in vertical transmission (29, 30) . recently, a new platform called cas13-assisted viral expression and read restriction (carver) was developed for rapid diagnosis of ssrna viruses. it mainly uses cas13 to detect and destroy viral rna (101). the crispr system seems to illustrate the unique and comprehensive prospect of virus infection diagnosis and treatment in future (101, 102) . finally, the additive effect of seasonal influenza on the covid-19 epidemic may interfere with doctors' clinical decisions, so more tests should be considered to distinguish covid-19 from other acute respiratory infections with similar symptoms in order to strengthen management of covid-19 (77) . in the prevention and management of covid-19, pregnant women, neonates, and children should be considered as the main high-risk population (58) . china nhc has provided prevention and control protocols for covid-19 and updated these during the epidemic. the latest 7th version provided on march 3 covered all populations in china (80) . furthermore, specific recommendations for neonates and children were also provided as national consensus guidelines (91) (92) (93) . the guidelines define the suspected and confirmed cases in different populations, as well as the criteria for discharge (80, 92, 93) . figure 1 is extracted from these guidelines as a concise protocol for management in pregnant women, neonates, and children. according to the management plan in pregnant women and neonates, newborns of mothers suspected or diagnosed with sars-cov-2 infection in delivery should be well-rescued and cared for via the cooperation of the department of obstetrics and neonatology (92) . all neonates with suspected or laboratory-confirmed covid-19 should be admitted to neonatal intensive care units (nicus) (91, 92) . high-risk neonates should be placed in a designated room for medical observation for at least 14 days (91) . if a pregnant woman or newborn is diagnosed or suspected of infection, breastfeeding should be avoided (91) . recently, a global guideline for pregnant women with suspected sars-cov-2 infection has also been provided (103) . moreover, recent research found that there may be potential risks of sars-cov-2 transmission in hospital settings, hence pediatricians and neonatologists should be more careful in treating the patients in nicus and pediatric intensive care units (picus) (104, 105) . home confinement and online courses may have a psychological impact on children and adolescents, emphasizing the importance of the awareness and guidelines provided for students from the government (36, 106) . finally, it must be noted that rt-pcr-positive results may still be found in pediatric patients recovered from covid-19 (47, 71) . in infants and young children, negative pharyngeal swab results may have already been detected, but viral nucleic acid can still be detected in fecal specimens (78) . a contingency plan for nicus recently suggested sars-cov-2 negative results of respiratory specimens or anal swabs should be obtained at least 48 h before discharge (91) . further isolation and long-term follow-up of discharged children with positive results of anal swabs should be considered for their potential transmission in public health (107) . the treatment of neonates and children is similar to that of adults, but it also has its own characteristics. to date, there are no specific drugs that can cure covid-19, and vaccines are still being studied. the purpose of treatment is to improve the patient's symptoms and provide better support. the most effective treatment is oxygen therapy, which is important in treating symptomatic newborns and critically ill children. it is closely related to the children's final outcome, and early treatment can reduce complications, such as ards or respiratory failure (108) . in adult covid-19 cases, severe ards is always associated with high mortality (109) . therefore, timely ventilation might be vital in preventing ards or respiratory failure in pediatric covid-19 cases. secondly, the effect of antiviral treatment in covid-19 is still uncertain. the first reported case in the united states benefited from an investigational antiviral drug called remdesivir, which has also proved to have a clinical benefit in the rhesus macaque model of mers-cov infection (110, 111) . lopinavir-ritonavir treatment reported no benefit in adult severe cases (112) . in 36 pediatric cases, mild cases received interferon alfa by aerosolization twice a day, while most moderate cases were given interferon alfa with lopinavir/ritonavir syrup twice a day (20) . however, no specific improvement of such antiviral treatment has been analyzed in pediatric cases, and it would be helpful to provide more clinical trials in the future. in addition, the use of corticosteroids remains controversial. who's current interim guidelines recommended against the use of corticosteroids unless indicated for another reason (113) . different studies have shown that it could be either beneficial or unfavorable for patients with coronavirus infection (such as sars and mers) (114, 115) . recently, expert consensus in china has advised against the use of corticosteroids in children under 18-years-old (116) . moreover, traditional chinese medicine may have a therapeutic effect on covid-19, but it is not fully recommended for children as well, because childhood toxicity is uncertain (117, 118) . intravenous immunoglobulin (ivig) is used to rescue newborns and critically ill children and may improve the disease (21, 54) . finally, recent studies on the structure of sars-cov-2 spike glycoprotein and cell entry have provided possible solutions for vaccine design and the application of protease inhibitors (119, 120) . the blocking effect of crossneutralizing antibodies may also indicate the feasibility of developing convalescent plasma therapy from healthy donors as a clinical trial in china, which has already been used in severe and critically ill pediatric and adult cases (77, (119) (120) (121) (122) . neonates with epidemiology history such as being born to sars-cov-2 infected mothers within 14 days before and 28 days after delivery, or direct exposure to family members, caregivers, medical staff, or visitors with covid-19 should be suspected with infection, whether with or without symptoms. suspected cases with both negative nucleic acid tests at least 24 h interval and negative igm and igg to sars-cov-2 within 7 days will be suspended quarantine. the management plan in perinatal pregnant women, neonates, and children from the community is from the latest new coronavirus pneumonia prevention and control protocol from china nhc (80) . the management plan in neonates born to the mothers is from the national guideline of perinatal and neonatal management plan of sars-cov-2 infection (92). in elderly patients with covid-19 (>65-years), especially those with comorbidities, clinical outcomes are usually poor (123) . however, to date, only two neonates born to mother with covid-19 and two children with covid-19 have been reported to have died in china, and most newborns and children have eventually recovered. some patients were still isolated in hospital for further observation (81) . further analysis is needed to better understand the prognosis of covid-19 in neonates and children. neonates born to mothers with covid-19 in the first and second trimester need close monitoring and further assessment. in addition, follow-up studies have shown that some children with sars had deficiencies in lung function assessment and decreased exercise capacity (124) . therefore, we call for long-term follow-up and comprehensive assessment of infected newborns and children after discharge to determine the prognosis of covid-19. since 2003, the chinese government has gained many lessons from the sars outbreak. in the covid-19 epidemic, besides china, the global response has been more timely, including coordination among different countries, sharing of disease information and cases, government and media reports, and public response (24, 125) . the chinese government has taken effective measures to control the epidemic. the experts also made recommendations for high-risk groups including pregnant women, newborns, and children. in addition, compared to adults, children have milder conditions, a faster recovery, and a better prognosis (126) . a series of improvements to date have been applied to prevent the prevalence of covid-19 in the global community. however, given that the symptoms of covid-19 in neonates and children are atypical, and transmission within family members is quite common, more effort should be made to protect this high-risk population. although there is still no direct evidence of vertical transmission, the rescue of newborns of infected pregnant women in delivery should not be delayed. furthermore, development of vaccines and effective treatments like novel antiviral drugs is also urgent and necessary. current outbreak will be restricted only if the whole world stands together and cooperates constantly. yy and pc contributed to the conception of the review. yy contributed to the literature search and writing of the manuscript. final integration and editing were done by pc. the table and figure were drafted by yy. a novel coronavirus associated with severe acute respiratory syndrome middle east respiratory syndrome a new coronavirus associated with human respiratory disease in china the first disease x is caused by a highly transmissible acute respiratory syndrome coronavirus a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan incubation period of 2019 novel coronavirus (2019-ncov) 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the editorial board clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment structure, function and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor convalescent plasma as a potential therapy for covid-19 treatment of 5 critically ill patients with covid-19 with convalescent plasma clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study severe acute respiratory syndrome (sars) in neonates and children sars to novel coronavirusold lessons and new lessons clinical and transmission dynamics characteristics of 406 children with coronavirus disease 2019 in china: a review the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright â© 2020 yu and chen. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-292025-dr611nse authors: kam, kai-qian; yung, chee fu; maiwald, matthias; chong, chia yin; soong, han yang; loo, liat hui; tan, natalie woon hui; li, jiahui; nadua, karen donceras; thoon, koh cheng title: clinical utility of buccal swabs for severe acute respiratory syndrome coronavirus 2 detection in coronavirus disease 2019–infected children date: 2020-06-13 journal: j pediatric infect dis soc doi: 10.1093/jpids/piaa068 sha: doc_id: 292025 cord_uid: dr611nse severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was detected from at least 1 buccal specimen in 9 of 11 coronavirus disease 2019 (covid-19)–infected children (81.8%). viral loads in buccal specimens were substantially lower than those in nasopharyngeal specimens. buccal swabs are not good as covid-19 screening specimens in children. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative novel coronavirus for coronavirus disease 2019 (covid-19), has been found in various clinical specimens of infected individuals [1] . nasopharyngeal and oropharyngeal swabs are the recommended upper respiratory tract specimens for detection of sars-cov-2 [2] ; however, the method of collection comes with risks and challenges. nasopharyngeal and oropharyngeal specimen collection requires healthcare workers to be in close contact with individuals who may be infected with covid-19, posing a risk of nosocomial viral transmission to the healthcare workers [3] . nasopharyngeal specimen collection is uncomfortable, and young children are often uncoop-erative during the procedure, thus increasing the risk of nasal trauma. collection of nasopharyngeal samples is potentially aerosol-generating, with an inherent requirement for negativepressure isolation facilities. recent studies showed evidence of consistent detection of sars-cov-2 in saliva specimens of infected adults, raising the possibility of using saliva as a form of screening for sars-cov-2 [4] [5] [6] . the use of saliva specimens to detect the presence of zika virus in children has been reported [7] . although children are often unable to produce saliva specimens spontaneously, buccal swabs can be performed to obtain saliva for testing. buccal swabs are less invasive and cause less discomfort for children. buccal swabs are also unlikely to trigger a sneezing or coughing response, in contrast to nasopharyngeal specimen collection. the concern regarding sneezing and coughing in response to the procedure of nasopharyngeal specimen collection is negated when buccal swabs are done instead. hence, the collection of buccal swabs does not require negative-pressure isolation facilities as it is not aerosol-generating. in addition, if the virus could be detected in saliva, this suggests a potential route of viral transmission in children, especially in infants who tend to drool and place objects in their mouths. it is important to understand the viral shedding pattern in buccal specimens of children in order to predict the routes of viral transmission. we conducted a study to evaluate the presence of sars-cov-2 in buccal specimens in covid-19-infected children. kk women's and children's hospital is an 830-bed hospital that provides care for approximately 500 children's emergency daily attendances and 12 000 deliveries per year. it is the primary hospital for evaluation and isolation of covid-19 in the pediatric population in singapore. from 23 march 2020 to 3 april 2020, all inpatient pediatric confirmed covid-19 cases diagnosed via positive sars-cov-2 polymerase chain reaction (pcr) from nasopharyngeal swabs using the real-time reverse transcription (rrt)-pcr assay for the e gene were included in this study. in addition to daily nasopharyngeal swabs for sars-cov-2 pcr taken for these children, daily buccal swabs were also taken on bilateral buccal mucosa to assess for viral shedding in saliva. buccal specimen collection was stopped if there were negative buccal sars-cov-2 specimens on 2 consecutive days. the cycle threshold (ct) values of all nasopharyngeal and buccal swabs for sars-cov-2 for each child were obtained. the ct values were reported in relation to the day of illness or day of diagnosis for symptomatic and asymptomatic patients, respectively. the study was approved by the institutional ethics review board. written informed consent was waived in light of the need to inform public health outbreak control policies. nasopharyngeal and buccal swabs were collected using mini utm kits (copan, brescia, italy) with flocked swabs and 1 ml of universal transport medium. from this medium, 200 µl was used for extraction of viral nucleic acids using the ez1 virus mini kit v2.0 (qiagen, hilden, germany) into 60 µl of eluate. rrt-pcr targeting the sars-cov-2 e gene was performed according to the method described by corman et al [8] . all reactions were run on a quantstudio 5 instrument (thermofisher applied biosystems, foster city, ca). a volume of 5 µl was used in pcr assays; with conversion factors, this represented 1/60 of the swab contents per reaction. the positive control consisted of a plasmid with a sars-cov-2 e gene insert, adjusted to 1000 copies per reaction. during the study period, the mean ct value of all positive control pcr assays was 29.86 and the standard deviation was ±0.68. thus, with the conversion factor, a ct value of 29.86 corresponded to approximately 6 × 10 4 virus genome copies per swab. continuous variables were expressed as median (range), and the mann-whitney u test or t test was used for analysis. categorical variables were expressed as percentage proportions. the spearman rank correlation was used to analyze the association between 2 continuous variables. statistical significance was set at p < .05. all analyses were done using the spss software, version 23 (ibm, armonk, ny). eleven covid-19-infected children were included in this study. six (54.5%) children were asymptomatic, and 5 symptomatic children (45.5%) had a mild course of illness. the asymptomatic children remained well throughout the admission, with no subsequent development of symptoms. the median ages of asymptomatic and symptomatic children were 8.4 years (range, 2.1-12.5) and 3.8 years (range, 0.3-11.8), respectively. however, the difference was not statistically significant (p = .24). sars-cov-2 was detected from at least 1 buccal specimen in 9 of 11 children (81.8%). one asymptomatic child with nasopharyngeal ct values of 33.0 and 30.0 on days 1 and 2 of diagnosis, respectively, had undetectable buccal sars-cov-2. another symptomatic child with nasopharyngeal ct values of 26.9 and 32.6 on days 2 and 3 of illness, respectively, had undetectable buccal sars-cov-2. in the 9 infected children with detectable sars-cov-2 in buccal specimens, the mean difference of ct values between buccal and nasopharyngeal specimens for all infected patients was 10.7 (range, 6.1-16.1), and this was statistically significant (p < .001). there was a general trend for buccal specimens to contain lower sars-cov-2 viral loads (higher ct values) compared with nasopharyngeal specimens, as shown in figure 1 . the sensitivity of buccal swabs compared with nasopharyngeal swabs ranged from 25% to 71.4% on different days of collection during the first week of illness/diagnosis. buccal sars-cov-2 was undetectable by day 8 of admission/diagnosis, although the nasopharyngeal sars-cov-2 was still detectable. there was no statistically significant association between buccal or nasopharyngeal ct values with age (p > .05). our findings confirm that sars-cov-2 can be detected in buccal specimens of infected children and that the viral load is the highest in the first week of illness or diagnosis. the detection of virus in buccal specimens from children suggests a high possibility that, as with adults [4] [5] [6] , sars-cov-2 is present and potentially transmissible via the saliva of children. in our study, the average viral loads of buccal sars-cov-2 were consistently lower than the respective nasopharyngeal specimens, with substantial differences between the average ct values. some studies have shown that no growth of the virus in culture was obtained in specimens that yielded positive sars-cov-2 pcr results with higher ct values [9, 10] . thus, saliva may not be a major route of viral transmission for sars-cov-2. two covid-19-infected children had negative buccal specimens despite detectable nasopharyngeal viral load. although there are advantages with buccal specimen collection compared with nasopharyngeal swabs, buccal specimen collection does not appear to be a good screening modality for covid-19 due to the reduced sensitivity and higher ct values. in our cohort, buccal sars-cov-2 was undetectable by day 8 of illness/diagnosis, despite continued detection of the virus in the nasopharynx of all patients. hence, buccal swabs similarly do not appear to be a good alternative to document viral clearance in infected children. however, in resource-limited places where isolation facilities are unavailable for nasopharyngeal specimen collection from children, buccal swabs would appear to be an alternative, albeit less sensitive, screening procedure. our study is limited by a small sample size of 11 children with covid-19. no additional buccal specimens were collected after 3 april 2020; hence, we could not extend our observations beyond that time. although our pcr assay was not set up to be quantitative, viral loads can be estimated from ct values using the laboratory's dilution factors and the fact that positive controls contained 1000 copies of target per reaction. in conclusion, we confirm the detection of sars-cov-2 from buccal specimens of children with covid-19. however, buccal specimens yielded substantially lower viral loads and had poor sensitivity compared with nasopharyngeal specimens. buccal swabs for sars-cov-2 are not good as screening specimens for covid-19 in children. detection of sars-cov-2 in different types of clinical specimens interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (covid-19) safety management of nasopharyngeal specimen collection from suspected cases of coronavirus disease 2019 consistent detection of 2019 novel coronavirus in saliva temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study saliva is a reliable tool to detect sars-cov-2 detection of zika virus in saliva detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr virological assessment of hospitalized patients with covid-2019 presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility key: cord-267782-4pjfnund authors: lan, fan-yun; suharlim, christian; kales, stefanos n; yang, justin title: association between sars-cov-2 infection, exposure risk and mental health among a cohort of essential retail workers in the usa date: 2020-10-30 journal: occup environ med doi: 10.1136/oemed-2020-106774 sha: doc_id: 267782 cord_uid: 4pjfnund objectives: to investigate sars-cov-2 (the virus causing covid-19) infection and exposure risks among grocery retail workers, and to investigate their mental health state during the pandemic. methods: this cross-sectional study was conducted in may 2020 in a single grocery retail store in massachusetts, usa. we assessed workers’ personal/occupational history and perception of covid-19 by questionnaire. the health outcomes were measured by nasopharyngeal sars-cov-2 reverse transcriptase pcr (rt-pcr) results, general anxiety disorder-7 (gad-7) and patient health questionnaire-9 (phq-9). results: among 104 workers tested, 21 (20%) had positive viral assays. seventy-six per cent positive cases were asymptomatic. employees with direct customer exposure had an odds of 5.1 (95% ci 1.1 to 24.8) being tested positive for sars-cov-2 after adjustments. as to mental health, the prevalence of anxiety and depression (ie, gad-7 score >4 or phq-9 score >4) was 24% and 8%, respectively. after adjusting for potential confounders, those able to practice social distancing consistently at work had odds of 0.3 (95% ci 0.1 to 0.9) and 0.2 (95% ci 0.03 to 0.99) screening positive for anxiety and depression, respectively. workers commuting by foot, bike or private cars were less likely to screen positive for depression (or 0.1, 95% ci 0.02 to 0.7). conclusions: in this single store sample, we found a considerable asymptomatic sars-cov-2 infection rate among grocery workers. employees with direct customer exposure were five times more likely to test positive for sars-cov-2. those able to practice social distancing consistently at work had significantly lower risk of anxiety or depression. who declared covid-19 as a pandemic on 11 march 2020. 1 since then, accumulating evidence has shown the transmission capability of sars-cov-2, the virus causing covid-19, not just from symptomatic patients but from asymptomatic carriers. [2] [3] [4] interventions have been implemented worldwide to minimise transmission, including social distancing, travel bans, stay-at-home orders and school and non-essential business closures. 5 6 all measures are intended to reduce contact and to prevent transmission, especially when the index patients are in subclinical stage of sars-cov-2 infection. 7 while most community residents benefit from these risk reduction policies, certain essential employees, such as healthcare workers (hcws), first responders and retail workers, continue to experience potential sars-cov-2 exposure risk due to the nature of their job. 8 furthermore, once essential workers are infected with sars-cov-2, they may become a significant transmission source for the community they serve. 9 the psychological stress associated with working during the covid-19 pandemic is also of great public interest. 10 studies have indicated pandemic awareness, infection fear and family concerns what is already known about this subject? ► the health of essential workers during the covid-19 pandemic is of great public and media interests. ► research, however, has largely focused on healthcare workers with relatively limited literature investigating non-healthcare essential workers. ► previous studies suggested essential workers are not able to benefit from mitigation policies. ► their occupational exposures increase their own risk to sars-cov-2 infection, and increase the risk of secondary transmissions to their colleagues, families and communities. what are the new findings? ► the present study fills in the knowledge gap of covid-19 impacts on grocery/retail market workers during the pandemic, from both physical and psychological perspectives. ► in this single store sample (n=104), we found an alarming infection rate of 20% positive sars-cov-2 rt-pcr assay result among these workers and the majority (76%) of them were asymptomatic at the time of testing. ► furthermore, employees with direct customer exposure were five times more likely to test positive for sars-cov-2. ► our study also found the inability to practice social distancing consistently at work was a significant risk factor for anxiety and depression. ► at the same time, commuting to work by public transportation/shared rides was significantly associated with depressive state. workplace contribute significantly to essential workers' mental distress during an emerging disease pandemic. 11 12 pioneering covid-19 studies on essential workers have largely focused on hcws. studies showed the attack rates of sars-cov-2 among hcws in early outbreaks ranged from 0% to 14%, with fever and loss of smell/taste being the best predictors of the disease. 13 14 in terms of mental health, about half of the hcws included in one study reported anxiety and depressive symptoms with psychological stress risk factors including living in areas with higher prevalence or being frontline hcws. 15 while hcws have been widely discussed in covid-19related research, there are relatively limited studies investigating other essential workers. a recent publication looking at six asian countries showed that various non-hcws were also affected during early covid-19 transmission, with service and sales workers comprising 18% of possible work-related cases. 9 while previous studies have reported sars-cov-2 cluster infections in supermarket settings, 16 17 no study has examined the sars-cov-2 exposure risks or psychological stress among grocery retail essential employees. therefore, we conducted this study aiming to investigate: 1) sars-cov-2 infection rate, transmission and exposure risks among grocery retail employees, 2) their use of personal protective equipment (ppe) and perception on covid-19 and 3) their mental health state during the covid-19 pandemic. this cross-sectional study is reported according to the strengthening the reporting of observational studies in epidemiology guideline. 18 we used secondary data from a covid-19 testing tent site that included information collected from 104 adults employed at one grocery retail store in the greater boston area of massachusetts, usa as part of a city-mandated group testing. clinical evaluation and nasopharyngeal swab sampling were conducted on each individual over three consecutive days in early may 2020. all workers older than 18 years sent by the store and presented for testing were included in this study (100% response rate). the specimens were collected using nasopharyngeal swab inside the designated covid-19 testing tent. a trained physician performed the swabbing procedure and transferred each specimen to a 3 ml vial with viral transport media. the samples were then transported to quest diagnostic laboratory in marlborough, massachusetts, where real-time, reverse-transcriptase-pcr (rt-pcr) diagnostic panels were conducted to detect sars-cov-2. all sampling, specimen storage, transportation and testing procedures followed the guidelines of the us centers for disease control and prevention. 19 as part of the group testing procedure, participants' basic demographic information, sars-cov-2-related exposure information, ppe usage and mental health surveys were collected through a paper-based questionnaire completed on site prior to testing. the basic information section of questionnaire included age, sex, race/ethnicity and medical history including past medical problems, prescription medication history, smoking status, alcohol intake, recreational drug use history and primary care physician information. for past medical issues, participants responded to a checklist which included the following diseases: chronic obstructive pulmonary disease/emphysema, asthma, heart disease, high cholesterol, high blood pressure, diabetes, hiv, hepatitis c, cancer and other(s). the following questions were included for employment history: most recent job position(s) at the store in the past month, full-time/part-time employment status, work hours per week (<20 hours, 20-39 hours, 40 hours and above), average length of shifts, additional employment(s) outside this retail store and transportation method(s) to work (by foot or bike, private car, public transportation, shared rides or others). workers selected their job position(s) from the following choices: cashier, front end associate, cart attendant, janitorial crew, stocker, backroom, receiving, sales associate, fresh food associate, supervisor and/or specialised roles. participants were given the choice to answer with free text for some other position if not listed as above. employees were asked to identify any additional employment(s) in the following categories: healthcare, drivers and transport, services and sales, cleaning and domestic, public safety, restaurant/fast food, others. 9 as to covid-19-related information, participants indicated new-onset symptoms within the past 1-2 weeks as a yes or no to a checklist of 11 common covid-19 symptoms, including fever/chills, headache, running nose, sore throat, cough (acute, new onset, dry or productive), shortness of breath, loss of taste or smell, diffuse body ache, fatigue/ feeling run down, nausea, diarrhoea. if participants answered yes to any of the above symptom(s), they were asked to indicate symptom onset. participants were asked if they had been exposed to anyone that has confirmed sars-cov-2 in the past 14 days. if they answered yes, they were asked of whom the exposure was (colleague, friend, family/relatives) and how many days ago the exposure occurred. information on mental health was recorded using two validated screening tools on depression and anxiety: patient health questionnaire-9 (phq-9) 20 and general anxiety disorder-7 (gad-7). 21 for phq-9, a total score of no higher than 4 indicates no or minimal depression, with a total phq-9 score ranging from 0 to 27. the score of gad-7 ranges from 0 to 21. a gad-7 score of no higher than 4 indicates no or minimal anxiety. participants were also asked to self-identify any history of depression and/or anxiety. how might this impact on policy or clinical practice in the foreseeable future? ► this is the first study to demonstrate the significant asymptomatic infection rate, exposure risks and associated psychological distress of grocery retail essential workers during the pandemic, which supports the policy recommendations that employers and government officials should take actions on implementing preventive strategies and administrative arrangements, such as methods to reduce interpersonal contact, repeat and routine sars-cov-2 employee testing, to ensure the health and safety of essential workers. ► our significant mental health finding calls for action in providing comprehensive employee assistance services to help essential workers cope with the psychological distress during the covid-19 pandemic. social distancing, ppe usage, covid-19 prevention knowledge score and covid-19 pandemic perception score participants answered a likert scale, from never (one) to always (five), for four questions that assessed employee's practice of social distancing and ppe use. participants answered another likert scale with six statements, from completely disagree (one) to completely agree (five), which captured the workers' knowledge on ppe and self-perceptions toward covid-19 pandemic. both employee's ppe knowledge and covid-19 perception were then tabulated to a score ranging from 3 to 15. a complete list of questions is included in online-only supplement 1. employees' job position was classified into two categories: those with significant face-to-face, direct exposure to customers and those without significant customer exposure. employees with direct customer exposure include cashier, front end associate, sales associate, fresh food associate, cart attendant, janitorial crew, supervisor and manager of all levels. those without direct customer exposure include stocker, backroom, receiving and maintenance. the covid-19 testing was conducted as part of a city-mandated group testing, independent to this research. the existing medical records collected for the city testing were de-identified at the primary clinical site prior to analysis. therefore, the study of de-identified data received a non-human research determination by the management sciences for health (sc#0012020). we performed univariate analyses to compare the workers' characteristics by their sars-cov-2 rt-pcr testing results, anxiety and depression status. for binary variables, pearson's χ 2 test with yates' continuity correction was performed, while for variables with at least one cell count less than five, fisher's exact test was conducted instead. as to continuous variables, data were examined by q-q plots and determined if they followed normal distribution beforehand. then we performed parametric t-test or non-parametric wilcoxon rank sum test, as appropriate. logistic regression models and models adjusting for potential confounders were further built. due to the small sample size and event numbers, we used the inverse probability weighting (ipw) method to avoid inflated ses of the parameter estimates. 22 the ipw was calculated based on the selected variables determined from the univariate analyses results. extreme weights (below the 5th and above the 95th percentile) were truncated as an additional sensitivity analysis. ors with 95% cis were presented. we performed secondary sensitivity analysis according to employees' job titles. employees' job position(s) were initially categorised into positions with greater direct customer exposure versus those without. in the sensitivity analysis, we categorised the jobs into supervisory positions vs non-supervisory positions. all p values reported are two-tailed. a p value <0.05 was considered statistically significant. we used r software (v.3.6.3) to conduct statistical analyses. in table 1, we presented the characteristics of all tested employees stratified by sars-cov-2 rt-pcr assay results. among the 104 workplace grocery retail employees that underwent testing and completed the survey, 47% were female with an average age of 49 years. the majority (62%) of employees in this retail store were non-caucasian minorities. twenty-one out of 104 employees tested positive for sars-cov-2 indicating a point prevalence of 20%. among these sars-cov-2-positive employees, 91% of them had a job position with significant direct customer exposure compared with 59% among the sars-cov-2-negative employees (p=0.009). seventy-six per cent of workers with positive tests were asymptomatic. among the 25 smokers, only one tested positive for sars-cov-2 (p=0.022). we did not observe statistical difference of sars-cov-2 status associated with protective behaviour (social distancing, use of gloves and/or masks and avoid commuting by public transportation or shared rides), nor did we find significant differences in ppe knowledge, covid-19 perception and mental health status between sars-cov-2-positive and sars-cov-2-negative employees. table 2 shows the distributions of workers' characteristics, comparing those with at least mild anxiety versus those reporting no or minimal anxiety. ninety-nine out of 104 workers (95%) completed the gad-7 questionnaire, with 24 workers (24%) reporting at least mild anxiety. we observed no statistical differences to anxiety by age, gender, smoking, alcohol consumption, marijuana use, possible sars-cov-2 exposure, job position, commuting method and ppe use. only 46% of workers with anxiety reported they were able to practice social distancing consistently at work, whereas the majority (76%) of those without reported anxiety were able to do so at work (p=0.009). employees screening positive for anxiety also reported less consistent mask use (63%) comparing with those screened negative for anxiety (84%), although this result did not reach statistical significance (p=0.072). the covid-19 pandemic perception score, which mainly evaluated the extent of worries on getting oneself and one's family infected due to work, were equally high among employees who screened positive for anxiety by gad-7 and those who did not (median score 13 vs 12, p=0.09). as to depression, there were 8 out of 99 (8%) who screened positive for at least mild depression (table 3) . workers who reported at least mild depression recorded higher proportion of possible sars-cov-2 exposure in the past 14 days compared with those without depression (63% vs 21%, p=0.019). workers who screened positive for depression by phq-9 were less likely to practice social distancing consistently at work and more likely to commute by public transportation or shared rides, compared with those without depression (25% vs 73% and 50% vs 11%, p=0.010 and p=0.013, respectively). employees with direct customer exposure were five times more likely to test positive on sars-cov-2 rt-pcr assay comparing with those without direct customer exposures (or 5.1, 95% ci 1.1 to 24.8) after adjusting for age, gender, smoking and sars-cov-2 community prevalence in workers' residential cities (table 4). while cigarette smokers had an 90% risk reduction in having positive sars-cov-2 rt-pcr assay result in the crude analysis (or 0.1, 95% ci 0.01 to 0.6), this finding was not statistically significant after ipw adjustments. in addition, those reporting possible exposure in the past 14 days had an or of 5.0 (95% ci 1.0 to 25.1) in screening positive for depression, after adjusting for age, gender, smoking, customer-facing jobs, sars-cov-2 community prevalence in workers' residential cities and workers' self-reported history of anxiety and depression. the ability to practice social distancing consistently at work was inversely associated with both anxiety and depression, with adjusted or 0.3 (95% ci 0.1 to 0.9) and 0.2 (95% ci 0.03 to 0.99), respectively. moreover, those commuting to work by foot, bike or private car demonstrated a 90% risk reduction in screening positive for depression (or 0.1, 95% ci 0.02 to 0.7) after accounting for potential confounders. in the sensitivity analysis using truncated ipw, all significant results remained robust. in further sensitivity analysis, we categorised the workers' jobs into supervisory positions and non-supervisory positions. there were 7 out of 21 (33%) sars-cov-2-positive employees with supervisory positions, while among those tested negative for sars-cov-2 only 7.2% held a supervisory position (p=0.005). after using truncated ipw to adjust for age, gender, smoking and sars-cov-2 community prevalence, those with supervisory positions had an or of 6.0 (95% ci 1.5 to 24.9) of having positive sars-cov-2 testing results. table 2 characteristics of retail essential employees in a single grocery store in massachusetts, usa presented for sars-cov-2, the virus causing covid-19, rt-pcr assay testing by generalised anxiety disorder 7-item scale (gad-7) screening score for anxiety our current study presents multiple valuable covid-19-related associations in a group of essential workers during the pandemic. first, the infection rate of 20% positive sars-cov-2 rt-pcr assay results at this grocery retail store was significantly higher than the surrounding communities. in addition, most of these employees were asymptomatic at time of testing. after ipw adjustments, employees with direct exposure to customers had more than five times increased odds to have a positive sars-cov-2 rt-pcr assay result. we also found the ability to practice social distancing at workplace was inversely correlated to workers' anxiety and depression status. lastly, having a confirmed sars-cov-2 exposure history in past 14 days and commuting to work by public transportation or shared rides was strongly associated with depressive mood. to the best of our knowledge, this study is the first to report the above associations in a cohort of grocery retail essential employees. there is limited research discussing non-hcws essential workers in this pandemic, particularly retail employees and their exposure to customers. 9 the sars-cov-2 infection rate among these retail employees was significantly higher than of the local community around similar time period, which was 0.9%-1.3%. 23 previous studies on hcws suggested covid-19 infections among hcws were consistent with community exposure rather than work-related exposure, with the prevalence ranging from 0% to 14%. 13 14 in fact, a pioneering study conducted in the table 3 characteristics of retail essential employees in a single grocery store in massachusetts, usa presented for sars-cov-2, the virus causing covid-19, rt-pcr assay testing by patient health questionnaire-9 (phq-9) screening score for depression these are in contrast to positions mainly dealing with consumer goods or the environment, such as stocker, backroom, receiving and maintenance. †ipw adjusting for age, gender, smoking and sars-cov-2 community prevalence in workers' residential cities. ‡ipw adjusting for age, gender, job positions with direct customer exposure, and sars-cov-2 community prevalence of workers' residential cities. §ipw adjusting for age, gender, smoking, customer-facing jobs, sars-cov-2 community prevalence of workers' residential cities and self-reported history of anxiety and/or depression. rt-pcr, reverse transcriptase pcr; truncated ipw, ipw with extreme weights (below 5th and above 95th percentile) truncated. netherlands investigated the viral genetic sequences of affected hcws and found the infection was more likely to be acquired from the communities. 24 in our current study, we did not observe a difference in sars-cov-2 community prevalence among those tested positive versus negative employees, indicating the possibility of a true work-related sars-cov-2 exposure. in terms of exposure risk, >90% of employees with positive assay result had a position with significant direct exposure to customers. we also found that employees in supervisory positions, with exposure from both customers and colleagues, had increased sars-cov-2 exposure risk. employees in supervisory positions may have more exposure due to frequent interpersonal contacts, therefore leading to their higher infection rates. notably, most of the sars-cov-2-positive assay workers were asymptomatic at time of testing. as evidence has shown probable transmission from asymptomatic or mildly symptomatic carriers, 3 25 26 these workers as a cluster carries significant risk to their customers, colleagues and families. our findings further strengthens the retail cluster transmission observed in a previous study from china, which involved supermarket employees, clients and the families of affected cases, resulting in a infection rate of 9.2% among the market workers. 17 in this cohort, cigarette smoking was found to be a protective factor of sars-cov-2 rt-pcr assay result in the crude analysis. despite a lack of statistical significance after ipw adjustment, our finding echoes a recently published systematic review indicating lower smoking prevalence among patients with covid-19 in comparison with the general population. 27 in that review, the authors pooled 13 chinese studies on hospitalised patients with covid-19 and found a prevalence of 6.5% of current smokers, which was around one-fourth of the smoking prevalence among the general population. the potential biological mechanism involving nicotinic receptors has been proposed in another study. 28 in fact, research has shown nicotinic receptor activity can promote sars-cov-2 transmission through co-expression of ace2 receptor, the host receptor for the virus. therefore, the competitive nature of nicotine and sars-cov-2, as a nicotinic agent, for the receptor may serve as a key to prevent the infection. 28 our finding of fewer current smokers with a positive sars-cov-2 assay result, while in agreement with recent epidemiological studies, contradicts common perception and clinical recommendation on risks and effects of cigarette smoking on lung health warranting further research investigations. 29 while previous research has raised concerns on psychological distress due to covid-19 in addition to physiological threats on essential workers, 11 most of them were focused on hcws. 10 15 30-32 the prevalence of anxiety among hcws in other countries ranged from 20% to 65% during the covid-19 pandemic. 15 30 32 in our study, 24% of these workers had at least mild anxiety, suggesting non-hcws essential employees experience similar level of psychological distress. contrary to common beliefs on the association between sufficient ppe and employees' psychological distress, 33 34 the inability to practice social distancing consistently at work was a significant risk factor for anxiety and depression in this essential worker cohort. while we are unable to discern the direction of the effect due to the cross-sectional nature of this study, these mental health findings support the need to implement further preventive strategies and to provide additional mental health assistance to essential employees. our current study has several limitations. first, our limited sample size may prevent identification of certain associations that may require larger statistical power, and incidental findings may by chance be observed in a small sample-sized study. however, the large effect sizes (ie, ors) are unlikely to be entirely biassed by unmeasured confounding factors. second, this is a cross-sectional study and therefore causal relationship could not be inferred. at the same time, survey collection was conducted prior to sars-cov-2 rt-pcr sampling, suggesting our major findings should be free of reverse causation and any recall bias would be minimised. third, while a majority of the employees from this retail store were tested at this designated location, some employees received testing at other clinics due to insurance, scheduling and/or location convenience. as this was a city-mandated testing, employees were assigned by the retail headquarter to be tested at this location if they had not received or scheduled to receive sars-cov-2 testing. selection was neither based on their exposure risk nor health outcome and therefore the current study should be free of selection bias. lastly, since our data collection was largely based on selfreported questionnaire, we incur unavoidable risk of measurement error, misclassification and related information bias. at the same time, our study enjoys several strengths. first, the sars-cov-2 rt-pcr assay samples were collected by nasopharyngeal approach which provides the highest test sensitivity among all methods 35 and the outcomes of interest were assessed by validated screening tools including gad-7 and phq-9. the possibility of outcome misclassification was therefore minimised. second, our secondary sensitivity analysis results were in accordance with the main analysis which further strengthened our findings. third, our study participants were restricted to grocery retail employees from one store and such restriction could eliminate potential confounding factors such as socioeconomical status. lastly, we included all workers that were scheduled and presented to the testing tent during group testing days without any exclusion criteria. as a result of our strengths, findings in this study may be generalised to grocery store employees working during the covid-19 pandemic in similar settings. in conclusion, in this cohort of grocery retail essential workers, 20% had a positive sars-cov-2 rt-pcr assay result and the majority (76%) of them were asymptomatic at time of testing. employees with direct customer exposure were five times more likely to have a positive sars-cov-2 assay result. the ability to social distance consistently at work was a significant protective factor for anxiety and depression. commuting to work by public transportation/shared rides and having an exposure to a confirmed case within the past 14 days were positively associated with depression. further research is warranted to investigate these associations and their public health implications among essential employees. contributors jy designed the study and collected the data. f-yl conducted data analysis and drafted the manuscript. f-yl, cs, snk and jy all contributed to the interpretation of the data, revising the manuscript and final approval. jy supervised the project. the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests snk has received covid-19-related consulting fees from open health. provenance and peer review not commissioned; externally peer reviewed. data availability statement de-identified data are available on reasonable request. supplemental material this content has been supplied by the author(s). it has not been vetted by bmj publishing group limited (bmj) and may not have been peer-reviewed. any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by bmj. bmj disclaims all liability and responsibility arising from any reliance placed on the content. where the content includes any translated material, bmj does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. justin yang http:// orcid. org/ 0000-0002-9743-2074 covid-19: who declares 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assessing generalized anxiety disorder: the gad-7 evaluation of the propensity score methods for estimating marginal odds ratios in case of small sample size massachusetts department of public health covid-19 dashboard -wednesday covid-19 in health-care workers in three hospitals in the south of the netherlands: a cross-sectional study clinical course and outcomes of patients with severe acute respiratory syndrome coronavirus 2 infection: a preliminary report of the first 28 patients from the korean cohort study on covid-19 presumed asymptomatic carrier transmission of covid-19 systematic review of the prevalence of current smoking among hospitalized covid-19 patients in china: could nicotine be a therapeutic option could the smoking gun in the fight against covid-19 be the (rh)ace-2? world health organization. who statement: tobacco use and covid-19 anxiety and depression in health workers and general population during covid-19 epidemic in iran: a web-based cross-sectional study development of health care workers' mental health during the sars-cov-2 pandemic in switzerland: two cross-sectional studies mental health outcomes among frontline and secondline health care workers during the coronavirus disease 2019 (covid-19) pandemic in italy the mental health of medical workers in wuhan, china dealing with the 2019 novel coronavirus saving the frontline health workforce amidst the covid-19 crisis: challenges and recommendations detection of sars-cov-2 in different types of clinical specimens key: cord-278370-fuu20ae7 authors: palao, m.; fernández-díaz, e.; gracia-gil, j.; romero-sánchez, c.m.; díaz-maroto, i.; segura, t. title: multiple sclerosis following sars-cov-2 infection date: 2020-07-07 journal: mult scler relat disord doi: 10.1016/j.msard.2020.102377 sha: doc_id: 278370 cord_uid: fuu20ae7 sars-cov-2 infection can produce neurological features. the most common are headache, anosmia and dysgeusia but patients may also develop other central nervous system (cns) injuries. we present a patient affected by covid-19 who initially consulted for decreased visual acuity. the mri showed inflammation in the right optic nerve and demyelinating lesions in the cns. we speculate that an immune mechanism induced by sars-cov-2, which can activate lymphocytes and an inflammatory response, plays a role in the clinical onset of the disease. this pathogen may be associated with either the triggering or the exacerbation of inflammatory/demyelinating disease. all the authors read and approved the final manuscript. sars-cov-2 infection can produce neurological features. the most common are headache, anosmia and dysgeusia but patients may also develop other central nervous system (cns) injuries. we present a patient affected by covid-19 who initially consulted for decreased visual acuity. the mri showed inflammation in the right optic nerve and demyelinating lesions in the cns. we speculate that an immune mechanism induced by sars-cov-2, which can activate lymphocytes and an inflammatory response, plays a role in the clinical onset of the disease. this pathogen may be associated with either the triggering or the exacerbation of inflammatory/demyelinating disease. at the end of 2019, the first case of sars-cov-2 virus infection was reported in wuhan (china) with a subsequent worldwide expansion due to a high rate of infectivity, becoming a pandemic with more than 4 million patients affected to date 1 . it is a viral infection with a wide spectrum of severity and prognosis. although the respiratory system is principally involved, the infection can also produce complications in other anatomical locations. currently, there is little information regarding the neurological manifestations associated with covid-19 2 . with respect to demyelinating diseases, several cases of peripheral nervous system involvement in the form of guillain-barré syndrome have been reported 3, 4 . however, available information about demyelinating complications of the central nervous system (cns) is limited with only one report of acute disseminated encephalomyelitis (adem) in a severe covid-19 patient being published to date 5 and a single case of meningo-encephalitis 6 , the latter with the presence of sars-cov-2 in the cerebrospinal fluid (csf) confirmed by pcr. as viral infections have been linked to the development of demyelinating diseases 7 it would be interesting to know if this relationship also exists in the case of sars-cov-2. we present a case of first presentation of demyelinating disease in the form of optic neuritis following sars-cov-2 infection. this case was included and partially described in a previously published registry of neurological complications of covid-19 2 and here we fully discuss all the clinical and radiological details. our patient is a 29-year-old woman, with a medical history of asthma and rhinoconjunctivitis. she consulted on april 14 th for a 10-day history of decreased visual acuity in the right eye. at the beginning, she presented with an upper altitudinal defect, followed by further worsening in both visual field defect and visual acuity. this was associated with continuous retro-ocular pain exacerbated by eye movements as well as colour desaturation. she had not previously experienced any neurological symptoms. at the time of her assessment, she was living with her father who had confirmed sars cov2 infection. symptoms had started 2-3 weeks earlier with the patient complaining of anosmia and dysgeusia associated with asthenia and proximal myalgias in her limbs which disappeared within a week. on neurological examination, she presented with a visual acuity of 20/200 and a relative afferent pupillary defect in her right eye. fundoscopic examination showed papillitis in the right eye. in addition, there were signs of pyramidal tract dysfunction seen as hyperreflexia in the lower limbs with a right-sided predominance, ipsilateral ankle clonus, right mute plantar response and bilateral hoffmann's sign. orbital mri (fig.1) confirmed a right-sided optic nerve lesion with significant contrast enhancement. brain mri (fig.2) showed sparse supratentorial periventricular demyelinating lesions, only one of them with gadolinium enhancement indicating activity. no infratentorial or cortical/juxtacortical lesions were detected. the mri of the whole spinal cord was normal. laboratory results showed the presence of oligoclonal igg bands in the csf. anti-igg-nmo-aqp4 and anti-mog antibodies were not identified in serum samples. sars-cov-2 pcr analysis of nasopharyngeal exudate and csf was negative whereas immunological testing was positive for both igm and igg, compatible with past infection. the autoimmune and serological studies in blood and csf ruled out other aetiologies. the patient was treated with three intravenous pulses of methylprednisolone at a daily dose of 1 g, followed by a tapering oral regimen with progressive improvement of ocular pain and visual acuity. on discharge the patient was able to distinguish letters in near vision optotype at 20/30. we report a first presentation of cns demyelinating disease shortly after covid-19 disease. to date, there is little evidence previously described in the literature 5 . in our case, the patient presented symptoms attributed to covid-19 infection (anosmia and dysgeusia) prior to the visual manifestations. the brief temporal gap leads us to consider the existence of a causal relationship. there is plenty of evidence to support the association between viral infections and the development of demyelinating diseases. for example, epstein-barr virus infection is considered an important risk factor for the development of multiple sclerosis 7 . other viruses that cause common infections in humans like varicella-zoster, influenza or adenovirus are associated with more frequent and severe relapses in patients with ms 8, 9 . indeed, some studies show that viral respiratory tract infections may be linked to most of the exacerbations of ms 10 . if we focus on the coronavirus (cov) family, there is clear evidence of its neurotropic character. in previous epidemics caused by other coronaviruses the presence of human cov in the brain tissue of patients with ms, as well as its rna has been demonstrated 11 . in addition, the presence of intrathecal synthesis of human anti-cov antibodies in csf obtained from ms patients has also been observed 12 . further evidence is provided by an animal model of coronavirus, mhv-59 (mouse hepatitis virus), which has been previously used for the creation of viral-induced inflammation models. when inoculated intracranially, it can induce meningitis, acute focal encephalitis and most importantly, optic neuritis 13 . such demyelination does not seem to be produced as a result of direct infection by the virus itself, but rather by the activated t-lymphocytes being responsible for the damage, through activation of the microglia and inflammatory mediators 14 . immunopathological mechanisms for demyelination include autoimmunity, direct immune cytotoxicity, and indirect damage 15 . the proposed mechanism by which these viruses access the cns are either through haematogenic spread or via the neurogenic pathway 16 . in addition, it has been postulated that the virus can spread locally through the cribiform plate of the ethmoid bone where it is believed to cause the olfactory symptoms 12 . since our patient met the criteria for multiple sclerosis diagnosis and exhibited non-enhancing periventricular lesions, we assume that the pathogenic process had already started prior to covid-19 disease, due to genetic or previous environmental factors. in this case, sars-cov-2 may have acted as a precipitating factor rather than multiple sclerosis being a direct consequence of the infection. however, we cannot rule out the possibility of the result for the sars-cov-2 pcr in csf being a false negative because the test has not been properly validated and its sensitivity is unknown. our own experience along with the animal models described earlier, reinforce the idea of sars-cov-2, as with other viral infections, being a trigger of neurological autoimmunity. further evidence derived from both experimental and clinical studies are warranted. we thank dr. j ahmad md. for the expert advice in the translation process. written informed consent was obtained from the patient for the publication of this case report. this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. the author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this articles. covid-19 map -johns hopkins coronavirus resource center neurologic manifestations in hospitalized patients with covid-19: the albacovid registry guillain-barré syndrome related to covid-19 infection guillain-barré syndrome associated with sars-cov-2 infection: causality or coincidence? sars-cov-2 can induce brain and spine demyelinating lesions a first case of meningitis/encephalitis associated with sars-coronavirus-2 viral infections and multiple sclerosis viral infections trigger multiple sclerosis relapses: a prospective seroepidemiological study factors associated with onset, relapses or progression in multiple sclerosis: a systematic review the role of infections in multiple sclerosis detection of coronavirus rna and antigen in multiple sclerosis brain should we expect neurological symptoms in the sars-cov-2 epidemic? experimental optic neuritis induced by a demyelinating strain of mouse hepatitis virus viral-induced suppression of self-reactive t cells: lessons from neurotropic coronavirus-induced demyelination pathogenesis of mouse hepatitis virus-induced demyelination key: cord-274841-rcdoewwv authors: tay, matthew zirui; poh, chek meng; rénia, laurent; macary, paul a.; ng, lisa f. p. title: the trinity of covid-19: immunity, inflammation and intervention date: 2020-04-28 journal: nat rev immunol doi: 10.1038/s41577-020-0311-8 sha: doc_id: 274841 cord_uid: rcdoewwv severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the causative agent of the ongoing coronavirus disease 2019 (covid-19) pandemic. alongside investigations into the virology of sars-cov-2, understanding the fundamental physiological and immunological processes underlying the clinical manifestations of covid-19 is vital for the identification and rational design of effective therapies. here, we provide an overview of the pathophysiology of sars-cov-2 infection. we describe the interaction of sars-cov-2 with the immune system and the subsequent contribution of dysfunctional immune responses to disease progression. from nascent reports describing sars-cov-2, we make inferences on the basis of the parallel pathophysiological and immunological features of the other human coronaviruses targeting the lower respiratory tract — severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). finally, we highlight the implications of these approaches for potential therapeutic interventions that target viral infection and/or immunoregulation. the first cases of coronavirus disease 2019 (covid19) likely occurred from a zoonotic transmission in china in december 2019, linked to a large seafood market that also traded in live wild animals. the causative virus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is capable of human-to-human transmission and spread rapidly to other parts of china and then to other locations. by 24 march 2020, sars-cov-2 had infected more than 381,000 people across 195 countries/regions and killed more than 16,000: a pandemic as declared by the world health organization 1 . daily reports of sharp rises in the number of new cases continue to emerge from many countries/regions, but efforts to overcome the virus are hampered by a lack of knowledge of several important aspects of sars-cov-2 infection, ranging from pathogen biology to host response and treatment options. therefore, there is an urgent need to better understand the host-pathogen biology of covid-19 as this will offer important insights into treatment and management of the disease, including identification of new therapies. here, we review the literature on sars-cov-2 pathophysiology, its interaction with target cells and the immune response to the virus, including the contribution of dysfunctional immune responses to disease progression. specifically, we highlight the implications of specific features of the infection for promising therapeutic interventions that could target the virus or the dysfunctional immune response. moreover, we discuss how studies focused on the adaptive immune response will be crucial in informing the development of vaccines and therapeutic monoclonal antibodies. coronaviruses are known to cause disease in humans and animals. among these, four (human coronaviruses 229e, nl63, oc43 and hku1) typically infect only the upper respiratory tract and cause relatively minor symptoms 2 . however, there are three coronaviruses (severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and sars-cov-2) that can replicate in the lower respiratory tract and cause pneumonia, which can be fatal. sars-cov-2 belongs to the betacoronavirus genus. its closest relative among human coronaviruses is sars-cov, with 79% genetic similarity 3 . however, among all known coronavirus sequences, sars-cov-2 is most similar to bat coronavirus ratg13, with 98% similarity 4 , and coronavirus sequences in the pangolin (a scaly anteater) also share high similarity 5 . like the other respiratory coronaviruses, sars-cov-2 is transmitted primarily via respiratory droplets, with a possible, but unproven, faecal-oral transmission route. on infection, the median incubation period is approximately 4-5 days before symptom onset [6] [7] [8] [9] , with 97.5% of symptomatic patients developing symptoms within 11.5 days 8 . at the point of hospital admission, patients with covid-19 typically exhibit a fever and dry cough; less commonly, patients also experience difficulty in breathing, muscle and/or joint pain, headache/dizziness, diarrhoea, nausea and the coughing up of blood 6, [10] [11] [12] [13] [14] [15] . within 5-6 days of symptom onset, sars-cov-2 viral load reaches its peak -significantly earlier than that of the related sars-cov, where viral load peaks at about 10 days after symptom onset [16] [17] [18] [19] . severe covid-19 cases progress to acute respiratory distress syndrome (ards), on average around 8-9 days after symptom onset 11, 20 . the pathophysiology of sars-cov-2 infection closely resembles that of sars-cov infection, with aggressive inflammatory responses strongly implicated in the resulting damage to the airways 21 . therefore, disease severity in patients is due to not only the viral infection but also the host response. the pattern of increasing severity with age is also broadly consistent with the epidemiology of sars-cov and mers-cov 6, 11, 14 . ards seen in severe covid-19 is characterized by difficulty in breathing and low blood oxygen level 22 . as a result, some patients may succumb to secondary bacterial and fungal infections 14 . ards may lead directly to respiratory failure, which is the cause of death in 70% of fatal covid-19 cases 22 . in addition, the vast release of cytokines by the immune system in response to the viral infection and/or secondary infections can result in a cytokine storm and symptoms of sepsis that are the cause of death in 28% of fatal covid-19 cases 22 . in these cases, uncontrolled inflammation inflicts multi-organ damage leading to organ failure, especially of the cardiac, hepatic and renal systems (fig. 1 ). most patients with sars-cov infection who progressed to renal failure eventually died 23 . the first step in infection is virus binding to a host cell through its target receptor. earlier work on sars-cov demonstrated that this virus principally targets airway epithelial cells, alveolar epithelial cells, vascular endothelial cells and macrophages in the lung, all of which express the angiotensin-converting enzyme 2 (ace2) host target receptor used by sars-cov [24] [25] [26] (fig. 2) . as sars-cov-2 uses the same entry receptor, these cell subsets are likely targeted by this virus 4, 27, 28 . sars-cov infection reduces ace2 expression in lung cells. because loss of pulmonary ace2 function is associated with acute lung injury, virus-induced ace2 downregulation may be important for disease pathology [29] [30] [31] [32] . ace2 has been shown to regulate the renin-angiotensin system (ras) 32 . therefore, a reduction in ace2 function after viral infection could result in a dysfunction of the ras, which influences blood pressure and fluid/electrolyte balance, and enhance inflammation and vascular permeability in the airways. covid-19 shows a difference in fatality rate between males (2.8%) and females (1.7%) 33 . as ace2 is located on the x chromosome, there may be alleles that confer resistance to covid-19, explaining the lower fatality rate in females. alternatively, the oestrogen and testosterone sex hormones have different immunoregulatory functions, which could influence immune protection or disease severity 34 . sars-cov-2 shares 79% genome sequence identity with sars-cov 4 . the spike (s) protein is expressed on the surface of the virus particles, giving the characteristic 'crown' appearance. the s protein comprises two subunits: s1 and s2. the s1 subunit consists of an amino-terminal domain and a receptor-binding domain (rbd), which in sars-cov spans from amino acid residue 318 to amino acid residue 510 (refs [35] [36] [37] ). the rbd binds to ace2 as its host cell target receptor, which starts the infection process 4 . rbd binding to ace2 triggers endocytosis of the sars-cov-2 virion and exposes it to endosomal proteases 38 . the s2 subunit consists of a fusion peptide (fp) region and two heptad repeat regions: hr1 and hr2 (refs 39, 40 ). within the endosome, the s1 subunit is cleaved away, exposing the fusion peptide, which inserts into the host membrane. the s2 region then folds in on itself to bring the hr1 and hr2 regions together. this leads to membrane fusion and releases the viral package into the host cytoplasm. there is 72% similarity in the amino acid sequence of the rbds of sars-cov and sars-cov-2, with highly similar tertiary structures. computational modelling and biophysical measurements indicate that the sars-cov-2 rbd binds to ace2 with higher affinity than that of sars-cov 41, 42 . in addition, the sars-cov-2 s protein contains a furin-like cleavage site, similarly to mers-cov and human coronavirus oc43, which is not found in sars-cov 43 . these characteristics could contribute to the increased infectivity of sars-cov-2 relative to sars-cov. in addition to furin precleavage, the cellular serine protease tmprss2 is also required to properly process the sars-cov-2 spike protein and facilitate host cell entry 44 . one pathway for the development of therapeutics against sars-cov-2 is to block the host target ace2 receptor or tmprss2 (fig. 3) . currently, there are compounds that target these molecules that have been clinically approved for other indications. for example, machine learning algorithms predicted that baricitinib, a janus kinase (jak) inhibitor approved for treatment of rheumatoid arthritis, could inhibit ace2-mediated endocytosis 45 . another jak inhibitor, ruxolitinib, will be tested in clinical trials for treatment of covid-19 later this year 46 . an alternative strategy is to deliver high concentrations of a soluble form of ace2 that could potentially reduce virus entry into target host cells. this principle is being tested with apn01, a recombinant form of ace2 developed by apeiron that is currently in clinical trials 47 . monoclonal antibodies targeting the when severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infects cells expressing the surface receptors angiotensin-converting enzyme 2 (ace2) and tmprss2, the active replication and release of the virus cause the host cell to undergo pyroptosis and release damageassociated molecular patterns, including atp, nucleic acids and asc oligomers. these are recognized by neighbouring epithelial cells, endothelial cells and alveolar macrophages, triggering the generation of pro-inflammatory cytokines and chemokines (including il-6, ip-10, macrophage inflammatory protein 1α (mip1α), mip1β and mcp1). these proteins attract monocytes, macrophages and t cells to the site of infection, promoting further inflammation (with the addition of ifnγ produced by t cells) and establishing a pro-inflammatory feedback loop. in a defective immune response (left side) this may lead to further accumulation of immune cells in the lungs, causing overproduction of pro-inflammatory cytokines, which eventually damages the lung infrastructure. the resulting cytokine storm circulates to other organs, leading to multi-organ damage. in addition, non-neutralizing antibodies produced by b cells may enhance sars-cov-2 infection through antibody-dependent enhancement (ade), further exacerbating organ damage. alternatively , in a healthy immune response (right side), the initial inflammation attracts virus-specific t cells to the site of infection, where they can eliminate the infected cells before the virus spreads. neutralizing antibodies in these individuals can block viral infection, and alveolar macrophages recognize neutralized viruses and apoptotic cells and clear them by phagocytosis. altogether, these processes lead to clearance of the virus and minimal lung damage, resulting in recovery. g-csf, granulocyte colony-stimulating factor; tnf, tumour necrosis factor. ◀ nature reviews | immunology s protein may also inhibit virus entry or fusion and are further discussed in the section entitled b cell immunity. nafamostat mesylate 48, 49 and camostat mesylate 44 are known inhibitors of tmprss2 and are currently approved in several countries/regions to treat other conditions. while there are no clinical trials specifically testing these drugs against covid-19 at the time of writing, when camostat mesylate was tested on sars-cov-2 isolated from a patient, it prevented entry of the virus into lung cells 44, 50 . if this approach is validated, rapid repurposing of these drugs will be effective and timely in the fight against covid-19. inflammatory immunopathogenesis sars-cov-2 infection and the destruction of lung cells triggers a local immune response, recruiting macrophages and monocytes that respond to the infection, release cytokines and prime adaptive t and b cell immune responses. in most cases, this process is capable of resolving the infection. however, in some cases, a dysfunctional immune response occurs, which can cause severe lung and even systemic pathology. cytopathic viruses, including sars-cov-2 (ref. 51 ), induce death and injury of virus-infected cells and tissues as part of the virus replicative cycle. viral infection and replication in airway epithelial cells 52 could cause high levels of virus-linked pyroptosis with associated vascular leakage, as seen in patients with sars-cov 53 . pyroptosis is a highly inflammatory form of programmed cell death that is commonly seen with cytopathic viruses 54 . this is a likely trigger for the subsequent inflammatory response 55 . il-1β, an important cytokine released during pyroptosis, is elevated during sars-cov-2 infection 11 . using a variety of pattern-recognition receptors (prrs), alveolar epithelial cells and alveolar macrophages detect the released pathogen-associated molecular patterns (pamps), such as viral rna, and damage-associated molecular patterns (damps), including atp, dna and asc oligomers. a wave of local inflammation ensues, involving increased secretion of the pro-inflammatory cytokines and chemokines il-6, ifnγ, mcp1 and ip-10 into the blood of afflicted patients 11, 22 . these cytokines are indicators of a t helper 1 (t h 1) cell-polarized response, which parallels observations made for sars-cov and mers-cov 56 . secretion of such cytokines and chemokines attracts immune cells, notably monocytes and t lymphocytes, but not neutrophils, from the blood into the infected site 57, 58 . pulmonary recruitment of immune cells from the blood and the infiltration of lymphocytes into the airways may explain the lymphopenia and increased neutrophil-lymphocyte ratio seen in around 80% of patients with sars-cov-2 infection 6, 59 . in most individuals, recruited cells clear the infection in the lung, the immune response recedes and patients recover. however, in some patients, a dysfunctional immune response occurs, which triggers a cytokine storm that mediates widespread lung inflammation. it was observed that patients with severe covid-19, requiring intensive care in hospitals, exhibited higher blood plasma levels of il-2, il-7, il-10, granulocyte colony-stimulating factor (g-csf), ip-10, mcp1, macrophage inflammatory protein 1α (mip1α) and tumour necrosis factor (tnf) 11 . il-6 levels in these patients continue to increase over time and are relatively more elevated in non-survivors than survivors 60 . notably, there exists a highly inflammatory monocyte-derived fcn1 + macrophage population in the bronchoalveolar lavage fluid of patients with severe but not mild covid-19 (ref. 61 ). also, patients with severe disease show a significantly higher percentage of cd14 + cd16 + inflammatory monocytes in peripheral blood than patients with mild disease 62 . these cells secrete inflammatory cytokines that contribute to the cytokine storm, including mcp1, ip-10 and mip1α (fig. 1) . the mechanisms by which sars-cov-2 subverts the body's innate antiviral cytokine responses are yet to be studied, but research on sars-cov shows that multiple viral structural and non-structural proteins antagonize interferon responses. antagonism occurs at various stages of the interferon signalling pathway, including by preventing prr recognition of viral rna 63-65 , by preventing prr signalling through tbk1/inhibitor of nuclear factor-κb kinase subunit-ε (ikkε), traf3 and irf3 (refs 63,66 ), by preventing downstream interferon signalling through stat1 (ref. 67 ) and by promoting host mrna degradation and inhibiting host protein translation 68 . it is very likely that at least some of these pathways are conserved in sars-cov-2. antagonism of the interferon response aids viral replication, resulting in increased release of pyroptosis products that can further induce aberrant inflammatory responses. unrestrained inflammatory cell infiltration can itself mediate damage in the lung through excessive secretion of proteases and reactive oxygen species, in addition to the direct damage resulting from the virus. together, these result in diffuse alveolar damage, including desquamation of alveolar cells, hyaline membrane formation and pulmonary oedema 57, 58 . this limits the www.nature.com/nri efficiency of gas exchange in the lung, causing difficulty in breathing and low blood oxygen levels. the lung also becomes more vulnerable to secondary infections. in addition to local damage, cytokine storm also has ripple effects across the body. elevated levels of cytokines such as tnf can cause septic shock and multi-organ failure. these may result in myocardial damage and circulatory failure observed in some patients 69 . older people (those aged over 60 years) and people with co-morbidities are more likely to develop such a dysfunctional immune response that causes pathology and also fails to successfully eradicate the pathogen. the exact reasons for this are unclear, although one reason may be an ageing lung microenvironment causing altered dendritic cell maturation and migration to the lymphoid organs 70 , and thereby defective t cell activation. in contrast, children tend not to develop severe disease despite being capable of experiencing high viral titres 71 . across all age groups younger than 18 years, more than 50% of children experienced mild symptoms or were asymptomatic, with less than 6% of children developing severe symptoms 72 . thus, while the aforementioned studies represent important inroads, a full picture of the critical host immune factors that underlie the development of severer inflammatory responses in some patients remains poorly defined. it remains controversial whether virus persistence is necessary to drive the ongoing damage. the peak of viral titres in respiratory tract samples might occur even before symptom onset of pneumonia in sars-cov and sars-cov-2 infections 17, 19 . however, a large retrospective cohort study showed that viral rna was detectable in non-survivors up until the point of death, suggesting a correlation between virus persistence and poor disease outcome 60 . as viral rna may linger even after active infection, and is not representative of the infectivity of the virus, whether the poor disease outcome is directly due to large amounts of infectious particles is speculative at this moment. furthermore, earlier studies of sars-cov found that the virus may infect other targets besides lung cells. notably, virus was found in t lymphocytes 73 , macrophages [74] [75] [76] and monocyte-derived dendritic cells 77 can be passed through customized columns that are specially designed to trap pro-inflammatory cytokines, before the purified blood is passed back into patients. 79, 80 . a clinical trial of the il-6 antagonist tocilizumab is also under way to test its efficacy 81 , and sarilumab is also being explored 82 . other clinical trials are also testing the effects of targeting granulocytemacrophage colony-stimulating factor (gm-csf), including the use of gimsilumab 83 , lenzilumab 84 and namilumab 85 . another novel adjunctive therapy is cytosorb 86 , which acts by absorbing a broad spectrum of cytokines, damps and pamps in order to reduce their circulating levels and ameliorate immunopathology. thalidomide, an agent with immunomodulatory properties, has also been successfully administered to a single patient with covid-19 (ref. 87 ). as a result, two clinical trials have now been initiated to test its potential to reduce lung injury 88,89 . tnf antagonism was suggested but not tested in the context of sars-cov infection, and it has not yet been tested in patients with covid-19 (ref. 90 ). a small open-label, non-randomized study suggested that a combination of hydroxychloroquine (a known antimalarial agent) and azithromycin (a common antibiotic) may be beneficial for treating patients with severe covid-19 (ref. 91 ). although hydroxychloroquine's effect on direct inhibition of the virus 92 and its anti-inflammatory and immunomodulatory activities are known 93 , whether these mechanisms play a role against covid-19 remains to be determined 94 . nature reviews | immunology t cell immunity both t and b cell responses against sars-cov-2 are detected in the blood around 1 week after the onset of covid-19 symptoms. cd8 + t cells are important for directly attacking and killing virus-infected cells, whereas cd4 + t cells are crucial to prime both cd8 + t cells and b cells. cd4 + t cells are also responsible for cytokine production to drive immune cell recruitment. the first autopsy of a patient with covid-19 revealed an accumulation of mononuclear cells (likely monocytes and t cells) in the lungs, coupled with low levels of hyperactive t cells in the peripheral blood 57 . together with reports of lymphopenia and reduced peripheral t cell levels in patients 6,95-97 , these findings suggest that t cells are attracted away from the blood and into the infected site to control the viral infection. in patients with covid-19, increased t cell exhaustion and reduced functional diversity predicted severe disease 98 . despite the impaired response, patients who recovered from sars-cov infection developed coronavirus-specific memory t cells, which were found up to 2 years after recovery 99, 100 . sars-cov-specific cd4 + t cells express ifnγ, tnf and il-2, which suggests that patients with sars-cov infection exhibit a t h 1 cell response and mainly use cellular immunity to control the infection 101, 102 . although this pro-inflammatory profile may be an aggravating factor for immunopathogenesis, cd4 + t cells have been hypothesized to control sars, as depletion of these cells in mice resulted in slower clearance of the virus from the host and severer lung inflammation 103 . with the use of a mouse-adapted strain of sars-cov, immunization with dendritic cells bearing sars-cov peptides resulted in higher numbers of virus-specific cd4 + and cd8 + t cells that accumulated in the lungs and increased survival 104, 105 . also, transfer of sars-cov-specific cd4 + and cd8 + t cells into immunodeficient mice resulted in better protection against a mouse-adapted strain of sars-cov 105 . despite evidence for an important role of t cells in controlling infection, several vaccine formulations against sars-cov previously tested in animal models showed signs of immunopathology associated with t h 2 cellmediated eosinophil infiltration 106, 107 . in particular, aged mice that were vaccinated seemed to display increased immunopathology rather than protection 108 . further study of the nature of protective versus detrimental t cell responses is critically needed to determine the optimal t cell engagement strategies for vaccines 109 . coronavirus-specific t cells are clearly important in eliminating the virus and controlling disease development and should be considered in vaccine strategies. however, whether t cell responses alone are capable of preventing infection in human settings remains to be investigated. this knowledge will be important for vaccine development. b cell immunity b cell responses in patients with covid-19 occur concomitantly with t follicular helper cell responses, from around 1 week after symptom onset 110 . in patients with sars-cov infection, b cell responses typically arise first against the nucleocapsid (n) protein. within 4-8 days after symptom onset, antibody responses to s protein are found 111, 112 . neutralizing antibody responses, likely to the s protein, begin to develop by week 2, and most patients develop neutralizing antibodies by week 3 (refs 113, 114 ). given that viral titres peak earlier for sars-cov-2 than for sars-cov [16] [17] [18] [19] , antibody responses may also arise earlier. it seems that a subset of patients may not develop longlasting antibodies to sars-cov-2 (ref. 115 ). it remains unknown whether these patients are susceptible to reinfection, of which there are sporadic reports 116, 117 . antibodies are likely to be effective against sars-cov-2: convalescent serum samples have been applied with apparently good clinical results in covid-19 (ref. 118 ) and were also previously used successfully in the treatment of sars [119] [120] [121] . while mechanistic correlates of protection have not yet been identified in humans, neutralization of the virus is presumed to be an important mechanism of action for antibodies, although the specific titre and specificity of the antibody repertoire required (for protection) remain undefined. in sars-cov, the primary target of neutralizing antibodies is the rbd 122 , comprising a 193 amino acid region (amino acids 318-510) in the s protein, which can independently bind to the host target ace2 receptor [35] [36] [37] . although a few previously identified monoclonal antibodies to sars-cov also bind to or neutralize sars-cov-2 (ref. 123 ), the majority do not 124 . this could be due to significant differences in the rbds of sars-cov-2 and sars-cov (fig. 4) . in particular, of the 33 amino acids in the region (amino acids 460-492) in the sars-cov s protein that contains the critical residues that contact ace2 (ref. 125 ), less than half (15/33) are conserved in sars-cov-2. nevertheless, mouse antiserum raised against sars-cov protein can cross-neutralize sars-cov-2 pseudovirus, indicating overlapping neutralizing epitopes between the two viruses 28, 126 . in china, hospitals have initiated the use of convalescent plasma as a source of therapeutic polyclonal antibodies for treatment of covid-19, and early data suggest a positive impact on respiratory viral load and mortality 127, 128 . efforts are under way to develop therapeutic monoclonal antibodies to sars-cov-2, using approaches including phage library display, traditional mouse immunization and hybridoma isolation, and cloning of b cell sequences from convalescent human patients [129] [130] [131] [132] . sars-cov does not appear to have strong mechanisms to escape or prevent antibody neutralization, such as glycan shielding of the receptor-binding site against antibody binding 133 . this is further corroborated by the fact that patients with sars-cov infection were generally capable of developing neutralizing antibodies. a recombinant s protein fragment that included the rbd of sars-cov showed the highest immunogenicity relative to other recombinant s protein fragments tested, suggesting that the immune system is capable of targeting neutralizing epitopes effectively 134 . thus, if sars-cov-2 behaves like sars-cov in this respect, it is likely that these efforts will be successful in developing neutralizing monoclonal antibodies. it is possible that alterations in the s protein will render sars-cov-2 resistant to some monoclonal antibodies, especially as it spreads and mutates. as of now, the entire rbd remains conserved, and there are only four www.nature.com/nri known rare non-synonymous alterations in the s protein: v483a, l455i, f456v and g476s 135 . the v483a alteration maps to a similar natural alteration found in mers-cov, i529t, where it reduced viral protein binding to the host receptor target and also increased resistance to antibody neutralization from serum samples from patients with mers 136 . f456v and g476s alterations also map to similar alteration positions in sars-cov (l443r and d463g), which were found in a panel of neutralization escape mutants 137 . however, the selection of therapeutic antibody candidates should include careful consideration of potential unwanted side effects. for example, pre-existing antibodies to other coronaviruses may exacerbate sars-cov infections through antibody-dependent enhancement [138] [139] [140] . also, previous studies in animal models showed that in sars-cov infection, neutralizing antibodies fig. 4 | sequence alignment and structural comparison of sars-cov and sars-cov-2 spike proteins. a | sequence alignment of severe acute respiratory syndrome coronavirus (sars-cov) spike protein and severe acute respiratory syndrome coronavirus 2 (sars-cov-2) spike protein, with conserved amino acid residues shown in black and non-conserved residues shown in colours. b | the 3d structure of sars-cov-2 (protein data bank id 6vsb 42 , peach ribbon) is superimposed on the sars-cov receptor-binding motif (rbm) complex with the neutralizing antibody (nab; red ribbon) interfacing with the rbm (protein data bank 2dd8 (ref. 151 ), purple ribbon). peach and purple spheres denote the rbms of sars-cov-2 and sars-cov, respectively. magenta spheres denote non-synonymous alterations in the sars-cov-2 spike protein that have been reported 135 . to s protein can potentially augment severe lung injury by exacerbating inflammatory responses 141 . in addition, a correlation has been observed where development of ards coincides with antiviral igg seroconversion in 80% of patients 19 . patients who developed neutralizing antibodies to s protein earlier in infection had a higher rate of disease; it took an average of only 14.7 days for patients who died of infection to reach their peak levels of neutralizing antibody activity, as opposed to 20 days for patients who went on to recover 142 . similarly, for mers, patients with severer disease appear to have higher antibody titres than those with mild disease 143, 144 , although one study argues that it is a delay in the development of antibody responses that is associated with disease 145 . the binding of antibody-virus immune complexes to activating fc receptors on alveolar macrophages could induce the expression of pro-inflammatory factors, including il-8 and mcp1, which add to the immunostimulatory milieu 146 . such complexes may also activate the complement system and lead to further unwanted inflammation 141 . as a result, it is important to consider engineering therapeutic antibodies with little or no pro-inflammatory activity but that retain their virus-neutralizing capacity 147 . for instance, alterations could be made to the fc region and/or its glycosylation to change its 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severe acute lung injury by skewing macrophage responses during acute sars-cov infection antibody responses against sars coronavirus are correlated with disease outcome of infected individuals feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia transmission of mers-coronavirus in household contacts kinetics of serologic responses to mers coronavirus infection in humans fcgamma receptors as regulators of immune responses the role of fc-fcgammar interactions in igg-mediated microbial neutralization anti-inflammatory activity of immunoglobulin g resulting from fc sialylation clinical trial analysis of 2019-ncov therapy registered in china structural basis of receptor recognition by sars-cov-2 structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody the authors thank insight editing london for its inputs on an early draft. this work was supported by core funds at the singapore immunology network (sign) through the biomedical medical research council (bmrc), a*star. the authors contributed equally to all aspects of the article. the authors declare no competing interests. nature reviews immunology thanks a. barrett, a. hosmalin, k. ishii and h. wei for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-260508-z11exbyu authors: wang, hongru; pipes, lenore; nielsen, rasmus title: synonymous mutations and the molecular evolution of sars-cov-2 origins date: 2020-10-12 journal: biorxiv doi: 10.1101/2020.04.20.052019 sha: doc_id: 260508 cord_uid: z11exbyu human severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is most closely related, by average genetic distance, to two coronaviruses isolated from bats, ratg13 and rmyn02. however, there is a segment of high amino acid similarity between human sars-cov-2 and a pangolin isolated strain, gd410721, in the receptor binding domain (rbd) of the spike protein, a pattern that can be caused by either recombination or by convergent amino acid evolution driven by natural selection. we perform a detailed analysis of the synonymous divergence, which is less likely to be affected by selection than amino acid divergence, between human sars-cov-2 and related strains. we show that the synonymous divergence between the bat derived viruses and sars-cov-2 is larger than between gd410721 and sars-cov-2 in the rbd, providing strong additional support for the recombination hypothesis. however, the synonymous divergence between pangolin strain and sars-cov-2 is also relatively high, which is not consistent with a recent recombination between them, instead it suggests a recombination into ratg13. we also find a 14-fold increase in the dn/ds ratio from the lineage leading to sars-cov-2 to the strains of the current pandemic, suggesting that the vast majority of non-synonymous mutations currently segregating within the human strains have a negative impact on viral fitness. finally, we estimate that the time to the most recent common ancestor of sars-cov-2 and ratg13 or rmyn02 based on synonymous divergence, is 51.71 years (95% c.i., 28.11-75.31) and 37.02 years (95% c.i., 18.19-55.85), respectively. the covid19 pandemic is perhaps the biggest public health and economic threat that the world 23 has faced for decades (li, guan, et al. 2020; ; zhou, yang, et al. 2020) . it is 24 caused by a coronavirus (lu, et al. 2020 ; zhang and holmes 2020), severe acute respiratory the genome of human coronavirus can effectively recombine with other viruses to form a 1 chimeric new strain when they co-infect the same host (forni, et al. 2017; boni, et al. 2020 ). 2 complicated recombination histories have been observed in the receptor binding motif region of 3 the spike protein xiao, et al. 2020; zhang, et al. 2020 ) and several other 4 regions (boni, et al. 2020 ) of the sars-cov-2, it is thus important to exhaustively search along 5 the viral genome for other regions potentially of recombination origin and identify possible 6 donors associated with them. to identify possible viral strains that may have contributed, by 7 recombination, to the formation of human sars-cov-2, we searched ncbi and embl virus 8 entries along with gisaid epiflu and epicov databases for similar sequences using blast in 9 100bp windows stepping every 10bp (fig. 1b) . the majority of the genome (78.1%, 2330/2982 10 of the windows) has one unique best hit, likely reflecting the high genetic diversity of the 11 coronavirus. 21.9% of the genomic regions has multiple best hits, which suggests that these 12 regions might be more conserved. among the windows with unique best hits, 97.0% (2260/2330) 13 of them were the ratg13 or rmyn02 bat strains and 1.9% of them, including the ace2 14 contact residues region of the s protein, were the pangolin sars-cov-2 virus. these 15 observations are consistent with previous results that ratg13 and rmyn02 are the most 16 closely related viral strains, while the region containing the ace2 contact residues is more 17 closely related to the pangolin virus strain table 4 ). the mosaic pattern that different regions of 23 the genome show highest identity to different virus strains is likely to have been caused by the 24 rich recombination history of the sars-cov-2 lineage (boni, et al. 2020 ; li, giorgi, et al. 2020; in some genomic regions may suggest recombination between the ancestral lineage of sars-1 cov-2 and distantly related virus lineages, although more formal analyses are needed to 2 determine the recombination history (see also boni, et al. 2020 for further discussion). 3 searching databases with blast using the most closely related viral strains, ratg13 and 4 rmyn02, we observe a very similar pattern, as that observed for sars-cov-2, in terms of top 5 hits across the genome (fig. 1b) , suggesting that these possible recombination events with 6 distantly related lineages are not unique to the sars-cov-2 lineage, but happened on the 7 ancestral lineage of sars-cov-2, ratg13, and rmyn02. a notable exception is a large region 8 around the s gene, where rmyn02 show little similarity to both sars-cov-2 and ratg13. 9 10 sequence similarity and recombination 11 we focus further on studying the synonymous evolution of sars-cov-2, and analyzing wuhan-12 hu-1 as the human ncov19 reference strain we performed recombination analyses across the five viral genomes based on the 23 concensus of the seven recombination-detection methods implemented in rdp5 (see methods). 24 we identified nine recombination regions affecting at least one of the sequences incongruence when compared with genome-wide trees ( fig. 2 and supplementary figure 1-3) . 1 particularly, a recombination signal is found in a region encompassing the rbd of the s protein, 2 suggesting that the human sars-cov-2 (wuhan-hu-1) sequence is a recombinant with the 3 pangolin-cov (gd410721) as the donor (supplementary table 5 ). phylogenetic analyses also 4 support that wuhan-hu-1 and gd410721 form a clade relative to ratg13 (supplementary 5 figure 1c , 1d). phylogenetic analyses (fig. 2 ) in genomic regions with all recombination tracts 6 (supplementary table 5 ) masked using maximum-likelihood (fig. 2a) and neighbor-joining 7 based on synonymous (fig. 2b ) or non-synoymous (fig. 2c ) mutation distance metrics, 8 consistently support rmyn02 as the nearest outgroup to human sars-cov-2, in contrast to 9 previous analyses before the discovery of rmyn02, which instead found ratg13 to be the 10 nearest outgroup ). this observation is also consistent with the 11 genome-wide phylogeny constructed in previous study (zhou, chen, et al. 2020). 12 we plot the overall sequence similarity (% nucleotides identical) between sars-cov2 13 and the four other strains analyzed in windows of 300 bp (fig. 1) . notice that the divergences 14 between human sars-cov-2 and the bat viral sequences, ratg13 and rmyn02, in most 15 regions of the genome, are quite low compared to the other comparisons. a notable exception is 16 the suspected recombination region in rmyn02 that has an unusual high level of divergence 17 recombination event with more distantly related viral strains (fig. 1e) . the other four sequences 1 are all highly, and approximately equally, divergent from rmyn02 in this large region (fig. 1e) , 2 suggesting that the rmyn02 strain obtained a divergent haplotype from the recombination 3 event. when blast searching using 100-bp windows along the rmyn02 genome, we find no 4 single viral genome as the top hit, instead the top hits are found sporadically in different viral 5 strains of the sars-cov lineage (fig. 1f) , suggesting that the sequence of the most proximal 6 donor is not represented in the database. 7 8 while the overall divergence in the s gene encoding the spike protein could suggest the 10 presence of recombination in the region, previous study ) reported that the tree 11 based on synonymous substitutions supported ratg13 as the sister taxon to the human sars-12 cov-2 also in this region. that would suggest the similarity between gd410721 and human 13 sars-cov-2 might be a consequence of convergent evolution, possibly because both strains 14 adapted to the use of the same receptor. an objective of the current study is to examine if there 15 are more narrow regions of the spike protein that might show evidence of recombination. we 16 investigate this issue using estimates of synonymous divergence per synonymous site (ds) in 17 sliding windows of 300 bp. however, estimation of d s is complicated by the high levels of 18 divergence and extremely skewed nucleotide content in the 3rd position of the sequences 19 (table 1) which will cause a high degree of homoplasy. we, therefore, entertain methods for 20 estimation that explicitly account for unequal nucleotide content and multiple hits in the same 21 site such as maximum likelihood methods and the yn00 method (yang and nielsen 2000) . it is 22 shown that for short sequences, some counting methods, such as the yn00 method, can 23 perform better in terms of mean squared error (mse) for estimating d n and d s (yang and 24 nielsen 2000). however, it is unclear in the current case how best to estimate d s . for this 25 reason, we performed a small simulations study (see methods) for evaluating the performance of the maximum likelihood (ml) estimator of d n and d s (as implemented in codeml (yang 2007) ) 1 under the f3x4 model and the yn00 method implemented in paml. in general, we find that 2 estimates under the yn00 are more biased with slightly higher mse than the ml estimate for 3 values in the most relevant regime of d s < 1.5 (fig. 3) . however, we also notice that both 4 estimators are biased under these conditions. for this reason, we perform a bias correction 5 calibrated using simulations specific to the nucleotide frequencies and d n /d s ratio observed for 6 sars-cov-2 (see methods). the bias corrections we obtain are ^* = ^ + 0.455^2 -0.824^3 7 + 0.264^4, for the ml estimator and ^* = ^+ 1.492^2-3.166^3 + 1.241^4 for yn00. notice 8 that there is a trade-off between mean and variance ( fig. 3 ) so that the mse becomes very 9 large, particularly for the for yn00 method, after bias correction. for d s >2 the estimates are 10 generally not reliable, however, we note that for d s <1.5 the bias-corrected ml estimator tends 11 overall to have slightly lower mse, and we, therefore, use this estimator for analyses of 300 bp 12 regions. by recombination with more divergent strains. we, therefore, also estimate d n and d s for the 20 regions with inferred recombination tracts (supplementary table 5 ) removed from all 21 sequences (table 3) respectively. this confirms that rmyn02 is the virus most closely related to sars-cov-2. the 24 relative high synonymous divergence also shows that the apparent high nucleotide similarity between sars-cov-2 and the bat strains (96.2% (zhou, yang, et al. 2020) and 97.2%(zhou, 1 chen, et al. 2020)) is caused by conservation at the amino acid level (d n / d s = 0.0410 and 2 0.0555) exacerbated by a high degree of synonymous homoplasy facilitated by a highly skewed 3 nucleotide composition at the third position of codons (with an at content >72%, table 1 ). 4 the synonymous divergence to the pangolin sequences gd410721 and gx_p1e in 5 genomic regions with inferred recombination tracts removed is 0.5095 (95% c.i., 0.4794-0.5396) 6 and 1.0304 (95% c.i., 0.9669-1.0939), respectively. values for other comparisons are shown in 7 tables 2 and 3. in comparisons between sars-cov-2 and more distantly related strains, d s will 8 be larger than 1, and with this level of saturation, estimation of divergence is associated with 9 high variance and may be highly dependent on the accuracy of the model assumptions. this 10 makes phylogenetic analyses based on synonymous mutations unreliable when applied to 11 these more divergent sequences. nonetheless, the synonymous divergence levels seem differences between estimates larger than 2.0 should not be interpreted strongly, as these 24 estimates have high variance and likely will be quite sensitive to the specifics of the model 25 assumptions. we find that d s (sars-cov-2, gd410721) approximately equals d s (gd410721, ratg13) 1 and is larger than d s (sars-cov-2, ratg13) in almost the entire genome showing than in these 2 parts of the genome gd410721 is a proper outgroup to (sars-cov-2, ratg13) assuming a 3 constant molecular clock. one noticeable exception from this is the rbd region of the s gene. 4 in this region the divergence between sars-cov-2 and gd410721 is substantially lower than 5 between gd410721 and ratg13 (fig. 4a,4c) . the same region also has much smaller 6 divergence between sars-cov-2 and gd410721 than between sars-cov-2 and ratg13 7 (fig. 4a,4c) . the pattern is quite different than that observed in the rest of the genome, most 8 easily seen by considering the ratio of d s (sars-cov-2, gd410721) to d s (sars-cov-2, 9 ratg13) (fig. 2b, 2d) . in fact, the estimates of d s (sars-cov-2, ratg13) are saturated in this 10 region, even though they are substantially lower than 1 in the rest of the genome. this strongly 11 suggests a recombination event in the region and provides independent evidence of that 12 previously reported based on amino acid divergence (e.g.,(zhang, et al. 2020)). 13 the combined evidences from synonymous divergence and the topological 14 recombination inference, provide strong support for the recombination hypothesis. however, 15 these analyses alone do not distinguish between recombination into ratg13 from an unknown 16 source as previously hypothesized (boni, et al. 2020 ) and recombination between sars-cov-2 17 and gd410721 as proposed as one possible explanation by lam et al. . to 18 distinguish between these hypotheses we searched for sequences that might be more closely 19 related, in the rbd region, to ratg13 than sars-cov-2 and we plotted sliding window 20 similarities across the genome for ratg13 (fig. 1c) . we observe relatively low sequence 21 identity between ratg13 and all three other strains in the ace2 contact residue region of the 22 spike protein, which is more consistent with the hypothesis of recombination into ratg13, as 23 proposed in (boni, et al. 2020 ). moreover, our blast search analyses of ratg13 in this region 24 show highest local sequence similarity with gx pangolin virus strains which is the genome-wide with the hypothesis of recombination from a virus related to gx pangolin strains, than with 1 recombination between sars-cov-2 and gd410721. 2 unfortunately, because of the high level of synonymous divergence to the nearest 3 outgroup, tree estimation in small windows is extremely labile in this region. in fact, synonymous 4 divergence appears fully saturated in the comparison with gx_p1e, eliminating the possibility to 5 infer meaningful trees based on synonymous divergence. however, we can use the overall 6 maximum likelihood tree using both synonymous and nonsynonymous mutations (fig. 2d ). the 7 ml tree using sequence from the ace2 contact residue region supports the clustering of sars-8 cov-2 and gd410721, but with unusual long external branches for all strains except sars-9 cov-2, possibly reflecting smaller recombination regions within the ace2 contact residue region. 10 11 the use of synonymous mutations provides an opportunity to calibrate the molecular clock 13 without relying on amino acid changing mutations that are more likely to be affected by selection. 14 the rate of substitution of weakly and slightly deleterious mutations is highly dependent on 15 ecological factors and the effective population size. weakly deleterious mutations are more 16 likely to be observed over small time scales than over long time scales, as they are unlikely to 17 persist in the population for a long time and go to fixation. this will lead to a decreasing dn/ds 18 ratio for longer evolutionary lineages. furthermore, changes in effective population size will 19 translate into changes in the rate of substitution of slightly deleterious mutations. finally, 20 changes in ecology (such as host shifts, host immune changes, changes in cell surface receptor, 21 etc.) can lead to changes in the rate of amino acid substitution. for all of these reasons, the use 22 of synonymous mutations, which are less likely to be the subject of selection than 23 nonsynonymous mutations, are preferred in molecular clock calculations. for many viruses, the 24 use of synonymous mutations to calibrate divergence times is not possible, as synonymous 25 sites are fully saturated even at short divergence times. however, for the comparisons between sars-cov-2 and ratg13, and sars-cov-2 and rmyn02, synonymous sites are not 1 saturated and can be used for calibration. we find an estimate of ï� = 0.0391 between sars-2 cov-2 and ratg13, excluding just the small rdb region showing a recombination signal in 3 sars-cov-2 (supplementary table 5 providing an approximate 95% confidence interval of (0.0332, 0.0464). also, using 59 human 7 strains of sars-cov-2 from genbank and national microbiology data center (see methods) 8 we obtain an estimate of ï� = 0.5604 using the f3x4 model in codeml. notice that there is a 14-9 fold difference in d n /d s ratio between these estimates. assuming very little of this difference is 10 caused by positive selection, this suggests that the vast majority of mutations currently 11 segregating in the sars-cov-2 are slightly or weakly deleterious for the virus. 12 13 to calibrate the clock we use the estimate provided by (http://virological.org/t/phylodynamic-15 analysis-of-sars-cov-2-update-2020-03-06/420) of =1.04ã�10 -3 substitutions/site/year (95% ci: 16 0.71x10-3, 1.40x10-3). the synonymous specific mutation rate can be found from this as 17 d s /year = s = /(ps +ï�pn), where ï� is the d n /d s ratio, and pn and ps are the proportions of 18 nonsynonymous and synonymous sites, respectively. the estimate of the total divergence on 19 the two lineages is then ^= ( + )â�� . inserting the numbers from table 3 for the 20 divergence between sars-cov-2 and ratg13 and rmyn02 ,respectively, we find a total 21 divergence of 96.92 years and 74.05 years respectively. taking into account that ratg13 was 22 isolated july 2013, we find an estimated tmrca between that strain and sars-cov-2 of 23 times. the estimate for sars-cov-2 and ratg13 is compatible with the values obtained using 1 different methods for dating (boni, et al. 2020) . the variance in the estimate in d s is small and 2 the uncertainty is mostly dominated by the uncertainty in the estimate of the mutation rate. we 3 estimate the s.d. in ^ using 1000 parametric simulations, using the ml estimates of all 4 parameters, for both ratg13 vs. sars-cov-2 and for rmyn02 vs. sars-cov-2, and for each 5 simulated data also simulating values of and from normal distributions with mean 1.04ã�10 -3 6 and s.d. 0.18ã�10 -3 , and mean 0.5604 and s.d. 0.1122, respectively. we subject each 7 simulated data set to the same inference procedure as done on the real data. our estimate of 8 the s.d. in the estimate is 11.8 for ratg13 vs. sars-cov-2 and 9.41 for rmyn02 vs. sars-9 cov-2, providing an approximate 95% confidence interval of (28.11, 75.31) and (18.19, 55 .85), 10 respectively. for ratg13, if including all sites, except the 244-bp in the rbd of the s gene 11 (supplementary table 5 ), the estimate is 55.02 years with an approx. 95% c.i. of (29.4, 80.7). 12 as more sars-cov-2 sequences are being obtained, providing more precise estimates of the 13 mutation rate, this confidence interval will become narrower. however, we warn that the 14 estimate is based on a molecular clock assumption and that violations of this assumption 15 eventually will become a more likely source of error than the statistical uncertainty quantified in 16 the calculation of the confidence intervals. we also note that, so far, we have assumed no 17 variation in the mutation rate among synonymous sites. however, just from the analysis of the 18 300 bp windows, it is clear that is not true. the variance in the estimate of ds among 300 bp 19 windows from the ratg13-sars-cov-2 comparison is approximately 0.0113. in contrast, in 20 the simulated data assuming constant mutation rate, the variance is approximately 0.0034, 21 suggesting substantial variation in the synonymous mutation rate along the length of the 22 genome. alternatively, this might be explained by undetected recombination in the evolutionary 23 history since the divergence of the strains. 24 the highly skewed distribution of nucleotide frequencies in synonymous sites in sars-cov-2 1 (kandeel, et al. 2020), along with high divergence, complicates the estimation of synonymous 2 divergence in sars-cov-2 and related viruses. in particular, in the third codon position the 3 nucleotide frequency of t is 43.5% while it is just 15.7% for c. this resulting codon usage is not 4 optimized for mammalian cells (e.g, (chamary, et al. 2006) ). a possible explanation is a strong 5 mutational bias caused by apolipoprotein b mrna-editing enzymes (apobecs) which can 6 cause cytosine-to-uracil changes (giorgio, et al. 2020). 7 a consequence of the skewed nucleotide frequencies is a high degree of homoplasy in 8 synonymous sites that challenges estimates of d s . we here evaluated estimators of d s in 300 bp 9 sliding windows and found that a bias-corrected version of the maximum likelihood estimator 10 tended to perform best for values of d s < 2. we used this estimator to investigate the 11 relationship between sars-cov-2 and related viruses in sliding windows. we show that 12 synonymous mutations show shorter divergence to pangolin viruses, than the otherwise most 13 closely related bat virus, ratg13, in part of the receptor-binding domain of the spike protein. 14 this strongly suggests that the previously reported amino acid similarity between pangolin 15 viruses and sars-cov-2 is not due to convergent evolution, but more likely is due to 16 recombination. in the recombination analysis, we identified recombination from pangolin strains 17 into sars-cov-2, which provides further support for the recombination hypothesis. however, 18 we also find that the synonymous divergence between sars-cov-2 and pangolin viruses in this 19 region is relatively high, which is not consistent with a recent recombination between the two. it 20 instead suggests that the recombination was into ratg13 from an unknown strain, rather than 21 between pangolin viruses and sars-cov-2, as proposed in (boni, et al. 2020 ). the alternative 22 explanation of recombination into sars-cov-2 from the pangolin virus, would require the 23 additional assumption of a mutational hotspot to account for the high level of divergence in the 24 region between sars-cov-2 and the donor pangolin viral genome. to fully distinguish between 25 these hypotheses, additional strains would have to be discovered that either are candidates for introgression into ratg13 or can break up the lineage in the phylogenetic tree between 1 pangolin viruses and ratg13. 2 the fact that synonymous divergence to the outgroups, ratg13 and rmyn02, is not 3 fully saturated, provides an opportunity for a number of different analyses. first, we can date the 4 time of the divergence between the bat viruses and sars-cov-2 using synonymous mutations 5 alone. in doing so, we find estimates of 51.71 years (95% c.i., 28.11-75.31) and 37.02 years 6 (95% c.i., 18.19-55.85), respectively. most of the uncertainty in these estimates comes from 7 uncertainty in the estimate of the mutation rate reported for sars-cov-2. as more data is being 8 produced for sars-cov-2, the estimate should become more precise and the confidence 9 interval significantly narrowed. we note that the mutation rate we use here are estimated based 10 on the entire genome, which may differ from that in non-recombination regions. to address this 11 problem, we downloaded all the sars-cov-2 sequences that are available until 2020-0817 12 from gisaid, and obtained an estimate of 1:0.81 for the ratio of mutation rates in the 13 recombination and non-recombination regions, using the "gtrgamma" model implemented in 14 the raxml (stamatakis 2014). given the length ratio between the two partitions is 1:4, the 15 difference between the partitions will cause a slight overestimate of the mutation rate by ~5%, 16 which is relatively small compared to the confidence intervals and the potential for other 17 unknown sources of uncertainty. however, we warn that a residual cause of unmodeled 18 statistical uncertainty is deviations from the molecular clock. variation in the molecular clock 19 could be modeled statistically (see e.g., (drummond, et al. 2006 ) and (lartillot, et al. 2016) ), but 20 the fact that synonymous mutations are mostly saturated for more divergent viruses that would 21 be needed to train such models, is a challenge to such efforts. on the positive side, we note that 22 the estimates of d s given in table 3 very different approach based on including more divergent sequences and applying a relaxed 25 molecular clock. we see the two approaches as being complimentary. in the traditional relaxed molecular clock approach more divergent sequences are needed that may introduce more 1 uncertainty due to various idiosyncrasies such as alignment errors. furthermore, the relaxed 2 molecular clock uses both synonymous and non-synonymous mutations and is, therefore, more 3 susceptible to the effects of selection. our approach allows us to focus on just the relevant in-4 group species and to use only synonymous mutations. the disadvantage is that we cannot 5 accommodate a relaxed molecular clock. however, the fact that both approaches provide 6 similar estimates is reassuring and suggests that neither idiosyncrasies of divergent sequences, 7 natural selection, or deviations from a molecular clock has led to grossly misleading conclusions 8 another advantage of estimation of synonymous and nonsynonymous rates in the 9 outgroup lineage, is that it can provide estimates of the mutational load of the current pandemic. 10 the d n /d s ratio is almost 14 times larger in the circulating sars-cov-2 strains than in the 11 outgroup lineage. while some of this difference could possibly be explained by positive 12 selection acting at a higher rate after zoonotic transfer, it is perhaps more likely that a 13 substantial proportion of segregating nonsynonymous mutations are deleterious, suggesting a 14 very high and increasing mutation load in circulating sars-cov-2 strains. 15 16 we are grateful to dr. e.c holmes for providing the genome sequence of rmyn02. we thank 18 dr. we also thank 2. replicates. the associated distance matrix for (b) and (c) can be found in table 3 . basic local alignment search tool evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 an exact nonparametric method for inferring mosaic 10 structure in sequence triplets hearing silence: non-neutral evolution at 12 synonymous sites in mammals relaxed phylogenetics and dating with 14 confidence phylip (phylogeny inference package) version 3.7a. distributed by the 16 author mafft multiple sequence alignment software version 7: 4 improvements in performance and usability identifying sars-cov-2 related coronaviruses in malayan pangolins a mixed relaxed clock model emergence of sars-cov-2 through recombination and strong purifying 14 selection phylogeny-aware alignment with prank genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus 18 origins and receptor binding rdp: detection of recombination amongst aligned sequences evaluation of methods for detecting recombination from dna 3 sequences: computer simulations identification of breakpoints in 5 intergenotypic recombinants of hiv type 1 by bootscanning analyzing the mosaic structure of genes detection of novel coronaviruses in bats in myanmar coronavirus from wuhan: an analysis based on decade-long structural studies of sars 13 a new coronavirus associated with human respiratory disease in china isolation of sars-cov-2-related coronavirus from malayan pangolins paml 4: phylogenetic analysis by maximum likelihood the mean of d s estimates using different methods; ml.corr and yn00.corr are the bias corrected versions of the ml and yn00 methods, respectively. (b) errors in d s estimates as measured using the ratio of square root of mean squared error (mse) to true d s . all the estimates are based on 10,000 simulations. ml: maximum-likelihood estimates using the f3x4 model in codeml; ml.corr, maximumlikelihood estimates with bias correction; yn00, count-based estimates in (yang and nielsen 2000); yn00.corr, yn00 estimates with bias correction key: cord-268718-tt07cwrf authors: tan, heng wee; xu, yan‐ming; lau, andy t. y. title: angiotensin‐converting enzyme 2: the old door for new severe acute respiratory syndrome coronavirus 2 infection date: 2020-06-30 journal: rev med virol doi: 10.1002/rmv.2122 sha: doc_id: 268718 cord_uid: tt07cwrf coronavirus (cov) disease 2019 (covid‐19) is an ongoing pandemic caused by severe acute respiratory syndrome cov 2 (sars‐cov‐2). the highly contagious sars‐cov‐2 belongs to the genus betacoronavirus, and it is phylogenetically closely related to sars‐cov, a human cov that caused an outbreak back in 2002 to 2003. both sars‐cov‐2 and sars‐cov enter human cells via the interactions between viral crown‐like spike protein and human angiotensin‐converting enzyme 2 (ace2) receptor. here, we aim to review the involvement of ace2 in human cov infections by discussing the roles of ace2 in cov evolution, cross‐species transmissibility, and covid‐19 susceptibility. we also provide our perspectives on covid‐19 treatment and prevention. hcov-oc43, and hcov-hku1 are regularly circulating in the human population and are responsible for respiratory infections. 6, 7 people infected with these covs generally show mild symptoms of upper respiratory disease such as fever, sore throat, and coughing, and only on rare occasion, they may cause lower respiratory tract infections and severe pneumonia. 8 11 all these incidences signify that the covs have continually posed a serious threat to the well-being of humans and animals. 9, 10 genome sequencing and phylogenetic analysis of the newly emerged sars-cov-2 has placed it under the genus betacoronavirus, making it a close relative to another two notorious covs: sars-cov and mers-cov. 4 the betacoronavirus also contains a range of bat cov strains, including several strains that show high % sequence similarity to the sars-cov-2, suggesting that sars-cov-2 is likely originated from the bats. [12] [13] [14] however, the intermediate reservoir of sars-cov-2 remains unclear. the surface of all covs has a characteristic crown-like structure known as the spike protein (commonly referred to as s protein). the spike protein can bind to a specific cell membrane receptor and it is the key mediator for cov entry into host cells. 15 previously, angiotensin-converting enzyme 2 (ace2) was identified as the target receptor for sars-cov. [16] [17] [18] current studies have indicated that the novel sars-cov-2 also uses the ace2 receptor. 14, 19 here, we review the involvement of ace2 in human cov infections by discussing the significances of ace2 in relation to cov evolution, cross-species transmissibility, and covid-19 susceptibility. lastly, we provide our perspectives on covid-19 treatment and prevention. also, since covid-19 is an ongoing pandemic, some of the first-hand data discussed in this review are sourced from non-peer-reviewed preprints. ace2, a homologue of ace, was firstly described 20 years ago. 20, 21 both ace2 and ace are zinc metalloproteases that play crucial roles in the renin-angiotensin system (ras), a system that regulates blood pressure, fluid, and electrolyte homeostasis. 22, 23 human ace2 is a protein with 805 aa encoded by the ace2 gene (hgnc: 13557) while ace is a larger protein consists of 1306 aa encoded by the ace gene (hgnc: 2707). ace2 and ace share approximately 40% identity and 61% similarity in their aa sequences. 21 despite the similarity, ace and ace2 do not share the same substrate specificity. 24 also, ace inhibitors that commonly used for treating high blood pressure or cardiovascular and kidney diseases, such as captopril, enalaprilat, and lisinopril, are ineffective against ace2. 24 in the ras, ace2 acts as a potent counter-regulator against ace. 25 physiologically, ace converts inactive decapeptide angiotensin (ang) i into vasoconstrictor ang ii and degrades vasodilator bradykinin, leading to increased blood pressure. 20 ace2, on the other hand, decreases blood pressure by competing with ace to hydrolyze ang i into the nonapeptide ang-(1-9), and at the same time degrades ang ii into ang-(1-7) and promote the release of vasodilator bradykinin. 20, 26 ace2 and ace are mainly expressed in the cell membrane of vascular endothelial cells found in various organs. generally, ace is more widespread than ace2 with highest levels of expression observed in, but not limited to, gastrointestinal tract, kidney, and lung. 21, 27 for ace2, gallbladder, gastrointestinal tract, heart, kidney, and testis are the primary organs of expression. 27, 28 both ace2 and ace can be secreted from the cell surface into the circulation or urine. 20, 29, 30 aberrant expression of ace or ace2 is associated with many diseases, including hypertension, lung injury, and cardiovascular, renal, and liver diseases. [31] [32] [33] ace2 is also known to be involved in humanand animal-cov infections. the high-resolution cryogenic electron microscopy (cryo-em) structure of full-length human ace2 was recently revealed, and its interactions with sars-cov or sars-cov-2 were determined. 34 the interactions between spike protein and host receptor are critical for cov pathogenesis. the spike protein is a crown-shaped class i viral membrane fusion protein distributed throughout the surface of all covs. 35 it is made up of a short intracellular tail and a large ectodomain connected by a single-pass transmembrane anchor. 36 the ectodomain consists of two subunits: three s1 subunit heads resting above a trimeric s2 subunit stalk. 37 the s1 subunit is responsible for host receptor-binding while the s2 subunit is accountable for creating an entrance for the viral genomes to invade the host cells by fusing the viral and host membranes. 35, 38 structural studies on the s1 subunit have revealed two receptor-binding domains (rbds) that can interact with a variety of receptors. specifically, the n-terminal domain mainly binds sugar receptors and ceacam1 in mouse hepatitis cov [39] [40] [41] [42] whereas the c-terminal domain appears to bind protein receptors (eg, apn, ace2, and ddp4) more exclusively. 38, [42] [43] [44] [45] [46] in order to bind a host-cell receptor, the rbd undergoes hinge-like conformational movements that either buried (lying state; receptorinaccessible state) or exposed (standing state; receptor-accessible state) its receptor-binding regions. 47 some covs, such as lineage a betacoronavirus, have a shorter spike-like envelope-associated hemagglutinin-esterase that acts as a receptor-destroying enzyme. 48, 49 structures and functions of cov spike proteins are reviewed extensively in li. 2 so far, three covs (hcov-nl63, sars-cov, and sars-cov-2) have shown to utilize human ace2 receptor. however, the rbds of these three ace2-utilizing human covs do not share identical sequences. previously, the interactions between ace2 and s1 domain of sars-cov spike protein was firstly reported by li et al, 16 and the structure of ace2-spike protein complex had been determined. 50 research indicated that the ace2 protein could bind directly with the extended tyrosine-enriched loop on the rbd (residues 424-494) of sars-cov spike protein 50, 51 and that the distantly-related ace protein did not have the same function as ace2. 16 interestingly, although hcov-nl63 also interacts with ace2, its rbd shares no sequence and structural similarity with the sars-cov. 52 in fact, unlike sars-cov that binds the ace2 through a continuous subdomain, hcov-nl63 binds the receptor with three discontinuous beta-loops. 52 research has indicated that both hcov-nl63 and sars-cov bind overlapping regions of ace2 and that mutations in the ace2 protein generally resulted in the same binding properties of these two strains. 17 however, some mutations in the ace2 protein only hinder the interactions of either hcov-nl63 or sars-cov, indicating that both strains engage ace2 differently. 17, 53 the engagement of sars-cov-2 with ace2 is similar to sars-cov, and important features of the spike protein in sars-cov-2 and its most related covs are compared and summarized in andersen et al. 54 virus infectivity study has indicated that the sars-cov-2 is able to utilize ace2 of human, chinese horseshoe bats, civet, and pig but was not able to use mouse ace2. 14 the cryo-em structure of sars-cov-2 spike protein was recently determined. 55 results indicated that the spike proteins of sars-cov and sars-cov-2 were highly homologous and structurally similar, with minor differences in the position of their rbds in receptor-inaccessible states: rbd of sars-cov packs tightly against the n-terminal domain of the neighboring protomer whereas rbd of sars-cov-2 angles closer to the central cavity of the trimer. 55 notably, the affinity between ace2 and the spike protein of hcov-nl63 is weaker compared to sars-cov and sars-cov-2, and although sars-cov and sars-cov-2 share similar spike protein and rbd sequences, the affinity between the spike protein of sars-cov-2 and ace2 appears to be 10 to 20 folds higher than sars-cov and ace2, suggesting that sars-cov-2 has a much higher human-to-human transmissibility compared to sars-cov and hcov-nl63. 6, 52, 53, 55 in addition, both sars-cov and sars-cov-2 use the same host transmembrane protease serine 2 (tmprss2) and possibly other proteases (eg, cathepsin b and cathepsin l) to cleave their spike protein and enhance viral entry. 56, 57 therefore, other host factors in addition to ace2 are likely also contributed to human cov pathogenesis and required further study. sequences in the spike proteins determine the tissue and species tropism of covs. mutations in the rbd of spike protein not only could affect the virulence of covs, but also could alter viral host spectrum, allowing cross-species infection to happen. 36, 37, 51 rna viruses can rapidly evolve and adapt to new environments due to their high mutation rates associated with the infidelity of rna polymerase. 58 this feature of rna viruses increases the probabilities of covs to invade a new host population. in fact, a substantial number of emerging pathogens that caused major epidemics in the past few decades are by rna viruses. 59 recognizing the origin and host of covs is extremely critical for disease control and prevention. understanding the underlying molecular mechanism of human receptor usage by different cov strains is equally important. bats are likely the ancestors of five out of seven human covs. 3, 15, 60, 61 they are the natural reservoirs for covs and many other viruses such as ebola, hendra, marburg, and nipah. 62 studies have suggested that the diverse viral inhabitants within and between bat species may perhaps promote co-evolution of these zoonotic viruses, further increasing the chances of cross-species transmission. 62 china's border are currently the prime suspect of being its intermediate host. 69 the spike protein rbd of pangolin cov strains showed ≈99% similarity to sars-cov-2, with only one aa difference. the binding capacity of pangolin rbd was tested, and it showed that the spike proteins of pangolin cov and sars-cov-2 could potentially bind to both pangolin and human ace2. 69 however, despite the high % similarity in the rbd region of pangolin cov and sars-cov-2, they shared only a mere ≈90% similarity of their overall genomes. 69 a few more similar studies also analyzed cov strains isolated from pangolin in china, and although pangolin covs are considered phylogenetically closer to sars-cov-2, the % genome similarity was only 92.4% at best. [70] [71] [72] since closely related covs should have higher genome similarity, like those observed between sars-cov and covs isolated from generally, higher ace2 affinity is correlated with higher infectibility of covs, and those strains with a higher ace2 affinity may eventually be the dominant circulating strains in human. few months into the covid-19 outbreak, more than a hundred mutations were already detected in the sars-cov-2 genomes isolated from 103 covid-19 patients. 74 these sars-cov-2 strains could be subdivided into two major types: the highly contagious and faster-growing l-type that found in ≈70% of samples and the evolutionarily older and less aggressive s-type that found in ≈30% of samples. 74 similarly, sars-cov strains previously isolated from sars patients showed vary ace2 affinities: some strains had a moderate affinity for human ace2 whereas some strains, especially those isolated during the late phase of the sars-cov outbreak, had a higher affinity. 75,76 sars-like cov strains isolated from palm civets during and after sars-cov outbreak showed high affinity for civet ace2 but lower affinity for human ace2, and consequently, higher infectivity in civet cells compared with human cells. 73, 77 the spike protein rbds of sars-cov and civet cov only differ by two residues, and these differences are enough to cause sars-cov to bind human ace2 more effectively than civet cov. 78 thus, the host range, tissue tropism, and receptor-binding property of covs can change dramatically simply due to a few mutations on the spike protein rbd. previously, aa positions of human ace2 that may play a role in host range and cross-species infection of covs were identified: k31, e35, d38, y41, m82, and k353. 10 chinese horseshoe bats are considered the native host for sars-cov even though the overall genetic similarity between sars-cov and sars-like civet covs is greater than that between sars-cov and sars-like bat covs. 79,80 sars-like bat covs share identical genome f i g u r e 1 phylogenetic tree of human and animal angiotensin-converting enzyme 2 (ace2). amino acid sequences were obtained from uniprot or national center for biotechnology information and aligned by clustalw using mega x (v10.1.7). 88 maximum likelihood tree was constructed using jones-taylor-thornton model with 100 bootstrap replications. numbers at the branches represent bootstrap probability values ≥50%. the known and suspected hosts of human coronaviruses that utilized human ace2 receptors (hcov-nl63, sars-cov, and sars-cov-2) are shown. solid arrows represent interspecies transmission with substantial evidence; broken arrows represent suspected interspecies transmission. *human ace, a homologue of ace2, was used as an outgroup organizations and high sequence similarity with sars-cov, and these sars-like covs can be found in diverse bat populations but not in other mammals. 81, 82 despite being phylogenetically related, the spike protein rbds of sars-like bat covs were not identical to sars-cov, and bat covs were generally not able to utilize human ace2 or any ace2 ortholog tested. 83 on the other hand, sars-cov was able to infect palm civet but was inefficient against bats. 83, 84 however, in other studies, several bat cov strains that had the ability to utilize human ace2 were identified, suggesting that the direct spillover of novel covs from bat to human is certainly possible. 12, 85, 86 the relationships between human covs and ace2 of other different animal species have not been systematically assessed. previously, sars-cov was not able to bind rat ace2, but it could interact with mouse ace2 with low affinity. 10, 84 comparing the ace2 sequences of human and other mammalians may give us some clues on crossspecies receptor usage of sars-cov-2. 87 here, we performed a phylogenetic analysis of ace2 from a range of mammals and found that human ace2 are most closely related to other species in the same family hominidae (chimpanzee and orangutan; figure 1 ). 88 furthermore, evolutionary inference suggests that species from the family cercopithecidae (macaque and monkey) also have ace2 similar to human ace2 (figure 1 ). previously, human cov strain oc43 was detected in fecal samples of macaque and chimpanzee, signifying the potential of cov spillovers among human, macaque, chimpanzee, and monkey. 89, 90 unfortunately, research regarding covs in the abovementioned primates is scarce, and further studies are required to examine if one of these species could be the intermediate host for sars-cov-2. furthermore, there is a rising concern regarding the transmission of covid-19 in companion pets such as cats and dogs. although so far there is no evidence that household pets can spread covid-19, it appears that some animals, especially those in the family felidae (eg, cat and malayan tiger), may contract sars-cov-2 from humans. 91, 92 at present, the roles of ace2 in domestic animals in connection to their infection with sars-cov-2 plausibly transmitted from humans have remained unclear. the roles of ace2 expression in sars-cov-2 pathogenesis and human covid-19 susceptibility are largely unknown. because sars-cov-2 uses ace2 as the receptor, it is logical to assume that all human cells that express ace2 are the potential targets for sars-cov-2. therefore, the following questions regarding ace2 expression in relation to covid-19 susceptibility can be asked: (a) which parts of the body express ace2? (b) which ace2-expressing organs are prone to sars-cov-2 infection? (c) is higher expression level of ace2 equal to greater covid-19 susceptibility? ace2 is expressed in a tissue-and species-specific pattern. [93] [94] [95] in human, ace2 is expressed in virtually all organs, with gastrointestinal tract, heart, and kidney among the highest expressed organs. 27, 28, 96 lung is the main target for sars-cov-2, but the overall expression of ace2 in human lung vary significantly according to different studies-some studies indicated that lung expressed high levels of ace2 94 while some suggested otherwise. 27, 97 using single-cell rna sequencing, zhao et al showed that the lung ace2 explicitly expressed in a small population of type ii alveolar cells, and these ace2-expressing cells also highly expressed certain genes that involved in promoting viral reproduction and transmission. 98 additionally, they found that age or smoking was not associated with ace2-expressing lung cell number but revealed that asian had higher lung ace2 expression than white and african american, suggesting asian might be more susceptible to covid-19. 98 however, the above study only tested on eight samples (with only one asian). in another study, 224 samples were collectively analyzed for ace2 expression in healthy lungs, and results indicated that lung ace2 expression was not significantly associated with age, gender, or race. 99 this study, however, did find that current and former smokers had higher ace2 expression compare with nonsmokers. additionally, ace2 was specifically expressed in different lung cell types of smokers and nonsmokers, suggesting that not only smokers might be more susceptible to covid-19, but the viral infection path could be different between smokers and nonsmokers. 99 a recent study also found increased ace2 expression in lower airways of smokers and individuals with chronic obstructive pulmonary disease. 100 further investigation is required to verify if higher lung ace2 expression is associated with greater covid-19 susceptibility. individuals with pre-existing disease may be more prone to covid-19. in vitro study showed that human lung cells exposed to inflammatory cytokines, rhinovirus, h1n1 influenza, or cov (sars-cov or mers-cov) resulted in increased expression of ace2. 101 these data suggest that people with viral infection and/or high inflammatory cytokine levels could be more susceptible to covid-19. also, increased cytokines during virus infection may further accelerate covid-19 infection. another two potential risk factors for covid-19 are obesity and cancers. 102 research has shown that human adipose tissues express high levels of ace2. 102 in diabetic mice, the ace2 expressions in the serum, liver, and pancreas were up-regulated. 103 poorer prognosis was observed in cancer patients. 105 in addition, using similar methods as described previously, 106 we compared the ace2 expression in lung cancer patients of various smoking statuses. we found that smokers, including ex-smokers, generally have upregulated ace2 ( figure 2b ). these results are to some extent in agreement with cai's and leung's studies 99,100 that smoking may increase lung ace2 expression. sars-cov was previously shown not only able to affect the lung but also other organs, including brain, gastrointestinal tract, kidney, liver, and more. 107, 108 there is no doubt that the lung is the main target for sars-cov-2. however, many covid-19 patients also showed f i g u r e 2 cancer tissues and the lungs of smokers generally contained higher levels of angiotensin-converting enzyme 2 (ace2), and these tissues can be potentially more susceptible to covid-19. a, ace2 expression data of 41 types of cancers obtained from the cancer genome atlas (tcga) database 104 ; mrna expression z-score threshold was set at ±1.5. b, ace2 expression in lung cancer patients with different smoking histories analyzed using similar methods as described previously 106 other symptoms in addition to respiratory symptoms, suggesting that sars-cov-2 could perhaps infect other organs (figure 3 ). sars-cov-2 might infect other ace2-expressing tissues via blood circulation as a recent report showed that sars-cov-2 rna had been detected in the blood of some covid-19 patients. 109 intriguingly, it is recently indicated that higher circulating ace2 is associated with milder symptoms of covid-19, suggesting the protective role of secreted ace2, probably by minimizing the direct contact between sars-cov-2 and ace2-expressing tissues. 110 this may also explain why women and children with confirmed covid-19 generally respond better to the disease because they appear to have higher levels of ace2 in the blood. 110 with a great proportion of covid-19 patients developed renal abnormalities and acute kidney injury, the kidney seemed to be a major organ affected by the disease. 97, 111, 112 a consecutive cohort study of covid-19 patients carried out by cheng et al showed that older patients and/or those with kidney impairments had higher risks of in-hospital death. 113 the liver is another organ that may be prone to sars-cov-2 infection as studies have shown that ace2 is enriched in the cholangiocytes, and liver injury is observed in some of the covid-19 patients. 114, 115 it is worth noting that although kidney and liver generally have higher ace2 expressions than the lung, there is so far no evidence that the sars-cov-2 can directly infect the liver and kidney cells in the human body. the gastrointestinal tract, especially the duodenum and small intestine, expressed high levels of ace2. 27, 96 since about up to 10% of covid-19 patients have shown gastrointestinal symptoms such as diarrhea, it is possible that sars-cov-2 can infect the gastrointestinal tract. 116 clinically, the detection of viral rna based on oral swab was used for confirmation of covid-19 because the sputa of covid-19 patients contained sars-cov-2 rna during infection (before the onset of symptoms). 117 interestingly, it was shown that in some cases, viral rna could be detected in the sputum even after 2 weeks of clinical recovery. 118 in addition to sputum, sars-cov-2 rna has been detected in the stools of a covid-19 patient, 119 f i g u r e 3 tissue distribution of angiotensin-converting enzyme 2 (ace2) expression and potential covid-19 susceptibility. organ highlighted in red represents positive ace2 expression. human images and ace2 expression data were obtained from the human protein atlas (http://www.proteinatlas.org) 27, 96 a healthy lifestyle is essential to keep the immune system active, and a balance host immune response is important to overcome virus infection. 1 supplementations such as vitamin a and vitamin d could be used to strengthen the immune system and provide benefits in defending virus infection. 130, 131 specifically, regular intake of vitamin d is correlated with higher protection against respiratory tract infections. 132 moreover, it is shown that zinc, an essential micronutrient, can inhibit viral rna-dependent rna polymerase of sars-cov and other viruses. 133 selenium, another essential micronutrient involved in ras and immune system, 134 has been shown to increase the immune responses of se deficient patients and animal models upon cov and other viral infections. 135, 136 in the century-long of war against viruses, vaccination is considered one of the most effective proactive measures to prevent the spread of viral diseases. previously, multiple attempts have been made to create cov vaccines, but these vaccines mainly only focused on sars-cov or mers-cov and have not yet undergone clinical trials. 56, 137 however, in just less than 2 months after covid-19 is declared a global pandemic, a few sars-cov-2 vaccines are already being trialed in humans as scientists in different countries raced to create the first effective covid-19 vaccine. spike protein of cov is a major inducer of host immune responses and the main target of neutralizing antibodies during infection. 37 recently, potential t-and b-cell epitopes of sars-cov-2 for vaccines and antibody-based neutralization have been identified using structural bioinformatic and machine learning techniques-the spike protein rbd region of 494-508 appear to be the best vaccine candidate. 138 dna/rna vaccines will likely be the most cost-effective form of vaccination against the fast mutating covs, if successfully developed. the fundamental idea of dna-or rna-based vaccine is to induce host primary immune response by injecting dna/rna that encodes a viral protein (eg, spike protein of cov) into the body, and let the body cells produce the mimic protein for antibody stimulation and immunological memory. 139, 140 based on the past success with hiv-1 treatment, gene therapybased strategy to combat covid-19 could also be considered. 141 for instance, lung cells that targeted by sars-cov-2 could be geneedited to become covid-19 resistant by knockdown or knockout of sars-cov-2 pathogenesis-related host gene(s) or permanent gene disruption of the cov genome. also, peptide mimetics or smallmolecule inhibitors have been developed to block viral entry or to suppress viral infection, and similar strategies can also be applied to covid-19. 142 particularly, targeted immunotherapy using smallmolecule inhibitors to prevent cytokine storm syndrome has emerged as a potent treatment option for covid-19 patients. 143 the ultimate objective of targeted immunotherapy is to optimize the cytokine productions and inflammatory responses upon viral infections. 1 previously, ace2 inhibitors have been tested in sars-cov and hcov-nl63, but their antiviral activity against ace2-utilizing covs has remained unknown. 144, 145 although it is possible to create an ace2 inhibitor that targeting the ace2 binding region of covs or create a recombinant human ace2 (rhace2) to trap sars-cov-2, the effects of these inhibitors or rhace2 on the ras should also be evaluated carefully. studies showed that the use of serine protease inhibitors to block tmprss2 could increase survival in mice upon sars-cov infection and reduce the entry of sars-cov and sars-cov-2 from entering the human cells. 57, 146 however, inhibiting host proteases will likely lead to adverse side effects, and therefore, this approach is considerably less attractive than targeting viral proteases. recently, the crystal structure of sars-cov-2 main protease was determined, and this would facilitate the development of viral protease inhibitors specifically targeting sars-cov-2. 147 lastly, reports have suggested that convalescent plasma collected from recovered covid-19 patients could be used to treat patients in critical conditions, and this method should be further explored. 148 to conclude, there are still many unknowns regarding covid-19. coronavirus infections and immune responses structure, function, and evolution of coronavirus spike proteins origin and evolution of pathogenic coronaviruses emerging coronaviruses: genome structure, replication, and pathogenesis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene 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wuhan, china: a retrospective case series study self-reported olfactory and taste disorders in patients with severe acute respiratory coronavirus 2 infection: a cross-sectional study high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes sars-cov-2: recent reports on antiviral therapies based on lopinavir/ritonavir, darunavir/umifenovir, hydroxychloroquine, remdesivir, favipiravir and other drugs for the treatment of the new coronavirus treatment efficacy analysis of traditional chinese medicine for novel coronavirus pneumonia (covid-19): an empirical study from wuhan remdesivir in covid-19 vitamin effects on the immune system: vitamins a and d take centre stage vitamin d and influenza-prevention or therapy? vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data zn 2+ inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture selenium species: current status and potentials in cancer prevention and therapy review: micronutrient selenium deficiency influences evolution of some viral infectious diseases selenium, selenoproteins and viral infection a decade after sars: strategies for controlling emerging coronaviruses potential t-cell and b-cell epitopes of 2019-ncov rapid-response vaccines-does dna offer a solution? protective efficacy of in vitro synthesized, specific mrna vaccines against influenza a virus infection fight fire with fire: gene therapy strategies to cure hiv design and synthesis of small molecule-sulfotyrosine mimetics that inhibit hiv-1 entry covid-19: immunopathology and its implications for therapy structurebased discovery of a novel angiotensin-converting enzyme 2 inhibitor ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis protease inhibitors targeting coronavirus and filovirus entry crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors the use of convalescent plasma to treat emerging infectious diseases: focus on ebola virus disease angiotensin-converting enzyme 2: the old door for new severe acute respiratory syndrome coronavirus 2 infection key: cord-254395-tu4aqczj authors: froggatt, heather m.; heaton, brook e.; heaton, nicholas s. title: development of a fluorescence-based, high-throughput sars-cov-2 3cl(pro) reporter assay date: 2020-10-27 journal: j virol doi: 10.1128/jvi.01265-20 sha: doc_id: 254395 cord_uid: tu4aqczj in late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), emerged, likely from a zoonotic reservoir. this virus causes covid-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. while the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. at this time, only one fda-approved anti-sars-cov-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. thus, the identification of new and efficacious antivirals is of the highest importance. in order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. with respect to drug targets, most attention is focused on either the viral rna-dependent rna polymerase or the main viral protease, 3cl(pro). 3cl(pro) is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. in this work, we report a new assay to identify inhibitors of 3cl(pro). our reporter is based on a green fluorescent protein (gfp)-derived protein that fluoresces only after cleavage by 3cl(pro). this experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (bsl2) with high-throughput compatible protocols. using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress sars-cov-2 replication and spread. importance the covid-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. however, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. in this work, we present a method for identifying inhibitors of the sars-cov-2 main protease, 3cl(pro). this reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (bsl2) with high-throughput compatible sample processing and analysis. this assay may help identify novel antivirals to control the covid-19 pandemic. antiviral therapeutics aim to disrupt the replication cycle and reduce viral load in infected individuals. therapeutic development efforts have led to a number of candidate antiviral compounds focused mainly on two essential viral enzymes, the rnadependent rna polymerase (rdrp) and the main viral protease. remdesivir (gs-5734), recently fda approved as an antiviral for sars-cov-2, targets the polymerase to suppress hcov replication by inducing termination of rna polymerization (8) ; however, the benefits of this drug in clinical trials and early use appear limited (9) . another nucleoside analogue, ␤-d-n 4 -hydroxycytidine (nhc; eidd-1931) , also inhibits sars-cov-2 polymerase activity, likely via inducing lethal mutagenesis of the viral genome (10) . in addition to the rdrp, the viral proteases, which are critical to liberate individual viral proteins from the polyprotein produced by initial genome translation, present another attractive drug target. for sars-cov-2, lopinavir/ritonavir, a protease inhibitor combination, is shown to interact with the main coronavirus protease, known as 3cl pro or m pro (11) ; however, early clinical trial results with these compounds have shown no significant benefits to sars-cov-2 patients (12) . more recently, structure-based design has enabled the rapid development of new antivirals targeting the sars-cov-2 protease 3cl pro (13) (14) (15) . at this time, these newly designed compounds are in the early stages of testing. thus, the discovery of additional effective sars-cov-2 antiviral drugs remains of high importance. the identification (and subsequent improvement) of novel drugs targeting sars-cov-2 will require robust and high-throughput screening approaches. here, we report the development and validation of a fluorescent reporter optimized to detect sars-cov-2 3cl pro activity. this assay is performed in human cell culture and does not require biosafety level 3 (bsl3) containment. our reporter is based on flipgfp, which fluoresces only after protease-mediated activation (16) . we generated and tested three reporter constructs with distinct cleavage target sequences for activation by the sars-cov-2 3cl pro . we also show that the reporter with the best signal-to-noise ratio for sars-cov-2 is also activable by other coronavirus 3cl pro proteins across subgroups (betacoronavirus, alphacoronavirus, and gammacoronavirus) and host species (human, rodent, and bird). finally, we used this reporter to test the inhibition of sars-cov-2 3cl pro with a known coronavirus 3cl pro inhibitor, gc376 (17) , and then validated the correlation between reporter inhibition and inhibition of sars-cov-2 replication. these experiments together demonstrate the utility of this approach for the identification of novel antiviral drugs that target the sar-cov-2 main protease, 3cl pro . generation of a fluorescent sars-cov-2 3cl pro activity reporter. in order to develop a fluorescent reporter responsive to the sars-cov-2 main protease, we started with the flipgfp protein (16) . flipgfp is used to detect protease activity by expressing the green fluorescent protein (gfp) 10th and 11th beta-strands, ␤10-11, separately from, and in a conformation incompatible with, the rest of the gfp beta-barrel, ␤1-9 (fig. 1a, top) . a linker containing a cleavage site holds the two gfp beta-strands, ␤10-11, in an inactive, parallel conformation. when the appropriate protease is present, the linker containing the cleavage site is cut. this cleavage allows gfp ␤11 to reorient such that gfp ␤10-11 are antiparallel and able to fit into gfp ␤1-9, inducing fluorescence ϳ100-fold over background (16) . sars-cov-2 generates two proteases that cleave the viral polyprotein, a papain-like protease (pl pro ) and a chymotrypsin-like protease (3cl pro ). 3cl pro , also known as the main protease or m pro , is the more conserved viral protease, with only 5 amino acid changes between sars/sars-like covs and sars-cov-2, compared to the 102 differences found in pl pro (18) . 3cl pro cleaves at a consensus sequence, lq2, which is highly conserved across the coronavirus family (19) . additionally, 3clpro has been shown to effectively cleave luciferase-based protease biosensors (20, 21) and fluorescence resonance energy transfer (fret)-based probes (13, 17, (22) (23) (24) (25) (26) (27) (28) . with the aim of generating a protease reporter compatible with sars-cov-2 and other present and future coronaviruses to support viral inhibitor screening, we selected cov 3cl pro as our protease target. although cov 3cl pro requires the minimal consensus sequence lq2 for cleavage, the cleavage site context influences cleavage efficiency (29) . among the cov polyprotein cleavage sites, 3cl pro targeted sites surrounding the 3cl pro sequence itself are generally cleaved most efficiently (30) . additionally, different covs have distinctive optimal cleavage site sequences (24) . in order to develop an efficiently cleaved cov 3cl pro reporter, we tested three different cleavage sequences predicted to be highly compatible with the sars-cov-2 3cl pro (fig. 1a, bottom) . cov reporter 1 contains the conserved nsp4-5 cleavage site present in the sars-cov and sars-cov-2 polyproteins (31) . cov reporter 2 contains an optimized cleavage sequence for the sars-cov 3cl pro (32) . cov reporter 3 contains an optimized sequence shown to be highly cleaved by many cov family members (24) . as a negative control, we included a construct harboring the tobacco etch virus (tev) protease cleavage site, as in the initial flipgfp (16) with coronavirus cleavage sequences. flipgfp splits gfp into ␤1-9 and ␤10-11, with ␤11 held in parallel to ␤10 by heterodimerized coiled coils e5/k5 and a linker sequence containing a coronavirus cleavage site. the cov main protease, 3cl pro , cuts at the cleavage site, allowing ␤11 to "flip" antiparallel to ␤10, enabling self-assembly of the complete gfp beta-barrel and resulting in detectable fluorescence. the pan-coronavirus 3cl pro consensus sequence, lq, is in bold. (b) quantification of fluorescence from 293t cells 48 h after transfection with each flipgfp reporter or superfolder gfp (sfgfp) individually and without a protease. statistical analysis is relative to sfgfp. (c) quantification of fluorescence from 293t cells 48 h after transfection with each flipgfp reporter and either the sars-cov-2 3cl pro or an influenza virus protein (a/pr8/1834 np). statistical analysis is relative to np control. (d) images corresponding to panel c. green, cleaved flipgfp; blue, nuclei. data are shown as means ϯ sds (n ϭ 3). p values were calculated using unpaired, two-tailed student's t tests (*, p ͻ 0.05; **, p ͻ 0.001; ns, not significant). experiments were performed twice. construct (16) . to observe whether these flipgfp constructs background fluoresced without cov 3cl pro activity, we transfected cells with each reporter or a superfolder gfp (sfgfp) expression plasmid. compared to sfgfp, which is properly folded and fluorescing, the inactive flipgfp constructs produced significantly lower fluorescence, from a 10-to 100-fold reduction depending on the reporter ( our goal was to identify a construct that showed minimal background fluorescence while still being efficiently cleaved by sars-cov-2 3cl pro , allowing strong fluorescence for detection via microscopy, plate reader, or flow cytometry. to test our 3cl pro reporters, we cotransfected each reporter with a sars-cov-2 3cl pro expression plasmid. at 48 h posttransfection, we could detect gfp-positive cells with each of the three cov reporters transfected with the sars-cov-2 3cl pro ( fig. 1c and d) . in contrast, with transfection of a negative control, nucleoprotein from an h1n1 influenza virus (a/pr8/ 1934 np), we did not detect any signal above background levels of fluorescence. further, the reporter containing the tev cleavage site was not activated by sars-cov-2 3cl pro . fluorescent signal across the treatment conditions demonstrated that while all three cov reporters showed significant induction of gfp signal when coexpressed with the sars-cov-2 3cl pro , cov reporter 2 had substantial background and the level of induction with cov reporter 1 reached only half that of the other two cov reporters. with a 100-fold change in fluorescence and minimal background, we selected cov reporter 3 for further testing ( fig. 1c and d) . many cov 3cl pro proteins activate the flipgfp cov 3cl pro reporter. cov 3cl pro proteins are reasonably conserved across coronavirus groups ( fig. 2a) (33) . further, cov reporter 3 was based on an optimized cleavage sequence for cov 3cl pro s from each coronavirus group (24) . to test whether this protease reporter was compatible with a variety of cov 3cl pro proteins, we expressed cov reporter 3 with four other coronavirus proteases from different groups (alphacoronavirus, betacoronavirus, and gammacoronavirus) and host species (human, mouse, and bird). at 48 h after transfection, all cov 3cl pro s induced visible fluorescence compared to the control influenza virus nucleoprotein with cov reporter 3 (fig. 2b) . expression of the cov 3cl pro or pr8 np constructs was quantified using quantitative pcr (qpcr) (fig. 2c ). quantification with a plate reader demonstrated that sars-cov (betacoronavirus, human) and avian infectious bronchitis virus (ibv; gammacoronavirus, avian) resulted in levels of fluorescence similar to that with sars-cov-2 (betacoronavirus, human) (fig. 2c ). murine hepatitis virus (mhv; betacoronavirus, murine) and human coronavirus 229e (hcov-229e; alphacoronavirus, human) were less compatible with cov reporter 3, while still producing 12-and 80-fold changes in fluorescence, respectively, over background (fig. 2c) . these experiments show that our flipgfp 3cl pro reporter is generally compatible with many cov 3cl pro proteins across coronavirus groups and host species, potentially enabling protease inhibitor screening for a variety of covs in addition to sars-cov-2. development of a flipgfp cov 3cl pro reporter-based assay for protease inhibitor screening in human cells. to develop an assay for protease inhibitor screening using our cov reporter 3, we first needed to optimize the experimental conditions. we performed a transfection time course with sars-cov-2 3cl pro to determine an early, appropriate time point for sample collection (fig. 3a) . at 12 h posttransfection, only a few gfp fluorescing cells were visible and fluorescent signal was just above background (fig. 3b) . at 24 h posttransfection, green cells were visible without appreciable background signaling (fig. 3b) . at 48 h postinfection, most cells produced a high gfp signal, with some background fluorescence detectable (fig. 3b) . we therefore selected the 24-h posttransfection time point. to increase the sensitivity of our assay, we titrated the level of sars-cov-2 3cl pro transfected with cov reporter 3; our goal was to maximize activation of the reporter while minimizing the amount of protease available in the cell. we transfected cells with five ratios of reporter to protease: 1:1, 1:0.8, 1:0.4, 1:0.2, and 1:0. at 24 h posttransfection, we observed significant decreases in reporter activation at reporter-to-protease ratios of 1:0.4 and 1:0.2 (fig. 3c) . however, a 1:0.8 reporter-to-protease ratio resulted in no significant loss of fluorescence compared to that with a 1:1 ratio (fig. 3c ). based on these experiments together, we selected a 1:0.8 reporter-to-protease ratio for transfection and a 24-h posttransfection endpoint as the optimal conditions for our protease inhibitor assay using flipgfp 3cl pro reporter 3. finally, we wanted to verify that our assay could detect drug inhibition of the sars-cov-2 3cl pro with a known inhibitor. therefore, we selected a recognized pancoronavirus 3cl pro inhibitor, gc376, to test our assay (17) . four concentrations of gc376, that did not significantly impact cell viability compared to vehicle alone, were applied to cells at the time of transfection with cov reporter 3 and sars-cov-2 3cl pro . as expected, reporter activity levels were maintained at the lower protease inhibitor concentrations, while fluorescence was reduced at the higher concentrations of gc376 (fig. 3d) . thus, our assay successfully detected inhibition of sars-cov-3 3cl pro by the protease inhibitor gc376. however, it is also important to verify that inhibition of our reporter is strongly correlated with inhibition of sars-cov-2. we infected veroe6 cells with sars-cov-2 at a multiplicity of infection (moi) of 0.01 before applying protease inhibitor at the same four concentrations as tested with the protease reporter. at 24 h postinfection, we collected rna and performed reverse transcription (rt)-qpcr to detect sars-cov-2 rna; similar to the case with the reporter, viral rna levels were suppressed in a dose-dependent manner (fig. 3e) . our observed inhibition of the virus is consistent with reports of inhibition of sars-cov-2 by gc376 in the literature (22, 23) . together, these experiments demonstrate feasibility of using our flipgfp cov 3cl pro reporter assay to identify protease-targeting inhibitors of sars-cov-2. our goal for this study was to develop a cell-based assay to screen for novel sars-cov-2 antiviral drugs at bsl2; to our knowledge, no such assay optimized for sars-cov-2 currently exists. therefore, we generated a reporter requiring a coronavirus protease, 3cl pro , for activation of a gfp fluorescent signal. we showed that this reporter is responsive to the sars-cov-2 3cl pro , in addition to many different coronavirus 3cl pro proteins. after optimizing screening conditions, we demonstrated that our reporter was sensitive to treatment with a known coronavirus protease inhibitor, gc376. these experiments illustrate the utility of our approach to identify, and subsequently optimize, novel protease inhibitors of sars-cov-2. to meet the demands of virus research during the sars-cov-2 pandemic, reporter assays need to be flexible and high-throughput. our reporter is activated with expression of a single cov protein, 3cl pro , allowing for sars-cov-2 drug testing at bsl2. additionally, the reporter is compatible with many cov 3cl pro proteins, supporting rapid testing of inhibitors against a variety of coronaviruses, present or future, and without synthesis of protease substrates or purification of viral proteins (13, 17, (22) (23) (24) (25) (26) (27) (28) . further, as our assay is performed in living cells, our system enables the discovery of protease inhibitors while simultaneously evaluating effects on cellular viability. our assay is scalable, and the analysis requires only a basic fluorescent plate reader, supporting high-throughput screening. in addition to applications in drug discovery pipelines, this assay could be deployed to determine targets of antivirals identified via viral screening. reporter assays, including ours, also have limitations. our reporter utilizes cov 3cl pro expressed alone; during a cov infection, the protease is only one of many viral proteins present, and any inhibitors that may affect cross-viral protein interactions would be missed. additionally, cov infection induces significant cellular membrane rearrangements that transfection of the protease alone does not. thus, the subcellular access of therapeutic compounds to the viral protease may fail to be reflected in our assay, and the effects of an identified protease inhibitor could significantly differ when applied to authentic viral infection. finally, although this plasmid-based expression presents many advantages, it also necessitates further screen hit testing in the context of coronavirus infection. although the correlation between our reporter and inhibition of viral infection was appreciable with the drug gc376, testing of more inhibitors is required to make generalizable correlations between our reporter assay and viral infection readouts. to have the greatest impact on the covid-19 pandemic, an effective sars-cov-2 antiviral needs to be identified as early as possible. countries around the world have taken drastic and necessary steps to limit the spread of virus, yet infection rates continue to rise in some. an antiviral treatment is unlikely to stop the spread of infection, but it is likely to limit the mortality associated with sars-cov-2 infection. it is our hope that this reporter assay facilitates the identification of sars-cov-2 protease inhibitor candidates to be rapidly optimized and translated to clinical use. cell culture. all cells were obtained from the atcc and grown at 37°c in 5% co 2 . 293t cells were grown in dulbecco's modified eagle medium (dmem) supplemented with 5% fetal bovine serum (fbs), glutamax, and penicillin-streptomycin. veroe6 cells were grown in minimum essential medium (mem) supplemented with 10% fetal bovine serum, pyruvate, nonessential amino acids (neaa), and penicillinstreptomycin. for transfection, plates were polylysine treated and seeded with 293ts. twenty-four hours later, plasmid dna, opti-mem, and transit-lt (mirus) were combined using pipetting and incubated at room temperature for 20 min before being added to cells by droplet. plasmids. constructs (excluding cov reporters 1, 2, and 3) were cloned into the plex expression vector using the bamhi and noti restriction sites and dna assembly (new england biolabs [neb]). coronavirus 3cl pro expression plasmids (sars-cov-2, sars-cov, mhv, ibv, and hcov-229e) were generated using codon-optimized gblocks (idt). the tev control flipgfp (addgene; number 124429) was designed to include a silent nhei restriction site ahead of the tev cleavage sequence and generated using a gblock (idt) (fig. s1 ) with primers flipgfp for and flipgfp rev (table s1 ) for insertion into bamhi-and noti-digested plex. the coronavirus 3cl pro flipgfp reporters (reporters 1, 2, and 3) were generated using forward primers containing the cleavage sequences (cov rep 1 for, cov rep 2 for, and cov rep 3 for) along with the flipgfp rev primer (table s1 ) and assembled into nhei-and noti-digested tev control flipgfp plasmid (table s1 ). dna was transformed into neb 5-alpha high-efficiency competent cells (neb). insert size was verified with pcr, and purified plasmids were sequenced using sanger sequencing. journal of virology imaging and quantification. cells were fixed with 2% paraformaldehyde (pfa) at room temperature for 20 min before incubation in 1:10,000 hoechst 33342 (life technologies) in phosphate-buffered saline (pbs) at 4°c overnight. images were obtained using the zoe fluorescent cell imager (bio-rad). quantification was performed using the cellinsight cx5 platform (thermo scientific). cov 3cl pro and pr8 np qpcr. at 48 h after transfection of 293ts, rna was prepped according to the rneasy 96 kit (qiagen). one-step rt-pcr was performed using primers targeting the respective cov 3cl pro (sars-cov-2, sars-cov, mhv, ibv, or 229e) or pr8 np and a housekeeping gene (18s) with the itaq universal sybr green one-step kit (bio-rad) on an applied biosystems quantstudio3 instrument. cytotoxicity assays. at 24 h before treatment, plates were polylysine treated and seeded with 293t or veroe6 cells. the next day, medium was replaced with complete medium containing gc376 (medkoo) at the desired concentrations using a constant level of a vehicle (dimethyl sulfoxide [dmso]). after 24 h of treatment, cells were collected according the celltiter-glo (promega) protocol and luminescence levels assessed using a luminometer. sars-cov-2 infections and viral qpcr. at 24 h before infection, plates were polylysine treated and seeded with veroe6 cells. the next day, the cells were washed with pbs before infection with sars-cov-2 isolate usa-wa1/2020 from bei resources in 2% fbs-mem infection medium at an moi of 0.01 for 1 h. virus was removed and cells were then placed in infection media containing gc376 (medkoo) at the desired concentrations using a constant level of a vehicle (dmso). at 24 h postinfection, cells were collected in trizol (invitrogen), followed by rna isolation. one-step rt-qpcr was performed with primers targeting the sars-cov-2 n region (bei) using the express one-step superscript qrt-pcr kit (thermo fisher) on an applied biosystems quantstudio3 instrument. rna was normalized using an endogenous 18s rrna primer/probe set (applied biosystems). supplemental material is available online only. the views, opinions, and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the u.s. government. the following reagent was deposited by the centers for disease control and prevention and obtained through bei resources, niaid, nih: sars-related coronavirus 2, isolate usa-wa1/2020, nr-52281. biocontainment work was performed in the duke regional biocontainment laboratory, which received partial support for construction from the national institutes of health, national institute of allergy and infectious diseases (uc6-ai058607). we thank clare smith for help establishing sars-cov-2 infection assays at bsl3 and laura froggatt for designing the flipgfp diagram. duke university may file for intellectual property protection for the technology described in this report. china novel coronavirus investigating and research team. 2020. a novel coronavirus from patients with pneumonia in china feng z. 2020. early transmission dynamics in a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the species severe acute respiratory syndromerelated coronavirus: classifying 2019-ncov and naming it sars-cov-2 johns hopkins coronavirus resource center. 2020. covid-19 map physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis the current and future state of vaccines, antivirals and gene therapies against emerging coronaviruses broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice why are lopinavir and ritonavir effective against the newly emerged coronavirus 2019? atomistic insights into the inhibitory mechanisms a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 structure-based design of antiviral drug candidates targeting the sars-cov-2 main protease crystal structure of sars-cov-2 main protease provides a basis for design of improved ␣-ketoamide inhibitors structure of mpro from sars-cov-2 and discovery of its inhibitors designing a green fluorogenic protease reporter by flipping a beta strand of gfp for imaging apoptosis in animals broad-spectrum antivirals against 3c or 3c-like proteases of picornaviruses, noroviruses, and coronaviruses genome composition and divergence of the novel coronavirus (2019-ncov) originating in china coronavirus 3clpro proteinase cleavage sites: possible relevance to sars virus pathology identification of novel proteolytically inactive mutations in coronavirus 3c-like protease using a combined approach assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors boceprevir, gc-376, and calpain inhibitors ii, xii inhibit sars-cov-2 viral replication by targeting the viral main protease feline coronavirus drug inhibits the main protease of sars-cov-2 and blocks virus replication profiling of substrate specificities of 3c-like proteases from group 1, 2a, 2b, and 3 coronaviruses coronaviruses resistant to a 3c-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance high-throughput screening identifies inhibitors of the sars coronavirus main proteinase reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor inhibition of sars-cov 3cl protease by flavonoids conservation of substrate specificities among coronavirus main proteases biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase prediction of the sars-cov-2 (2019-ncov) 3c-like protease (3clpro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates the substrate specificity of sars coronavirus 3c-like proteinase chimeric exchange of coronavirus nsp5 proteases (3clpro) identifies common and divergent regulatory determinants of protease activity ncbi viral genomes resource key: cord-281679-xmbnpawj authors: meekins, david a.; morozov, igor; trujillo, jessie d.; gaudreault, natasha n.; bold, dashzeveg; artiaga, bianca l.; indran, sabarish v.; kwon, taeyong; balaraman, velmurugan; madden, daniel w.; feldmann, heinz; henningson, jamie; ma, wenjun; balasuriya, udeni b. r.; richt, juergen a. title: susceptibility of swine cells and domestic pigs to sars-cov-2 date: 2020-08-16 journal: biorxiv doi: 10.1101/2020.08.15.252395 sha: doc_id: 281679 cord_uid: xmbnpawj the emergence of sars-cov-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. the susceptibility of different animal species to sars-cov-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. in the current study, we determined the ability of sars-cov-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. sars-cov-2 was able to replicate in two different porcine cell lines with cytopathic effects. interestingly, none of the sars-cov-2-inoculated pigs showed evidence of clinical signs, viral replication or sars-cov-2-specific antibody responses. moreover, none of the sentinel pigs displayed markers of sars-cov-2 infection. these data indicate that although different porcine cell lines are permissive to sars-cov-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. pigs are therefore unlikely to be significant carriers of sars-cov-2 and are not a suitable pre-clinical animal model to study sars-cov-2 pathogenesis or efficacy of respective vaccines or therapeutics. the emergence of sars-cov-2, the causative agent of covid-19, has resulted in a global pandemic with over 20 million cases and 740,000 deaths as of august 13, 2020 [1, 2] . sars-cov-2 causes a respiratory disease in humans with a broad clinical presentation, ranging from asymptomatic or mild illness to severe fatal disease with multi-organ failure [3] [4] [5] [6] . sars-cov-2 is rapidly transmissible via contact with infected respiratory droplets and can also be transmitted by asymptomatic carriers [6] [7] [8] . to curb viral spread, countries have instituted varying levels of social distancing policies, which have significant negative economic and social impacts [9] . mitigating the effects of this unprecedented pandemic will necessitate the development of effective vaccines and therapeutics, which will require well-characterized and standardized preclinical animal models. sars-cov-2 is a member of the betacoronavirus genus that includes the pathogenic human viruses sars-cov-1 and mers-cov [2, [10] [11] [12] . while details of the origin of sars-cov-2 are unknown, evidence indicates it emerged from a zoonotic spillover event, with bats and perhaps pangolins as probable origin species [2, [13] [14] [15] . the potential for a reverse zoonotic event, i.e. human-to-animal transmission, is possible and of significant concern to animal and public health [16] [17] [18] . instances of natural human-to-animal transmission of sars-cov-2 have been reported with covid-19 patients in domestic settings (dogs and cats), zoos (lions and tigers), and farms (mink) [18] [19] [20] . therefore, investigations into the infectivity of sars-cov-2 in various animal species with human contact are essential to assess and control the risk of a spillover event and to establish the role these animals may play in the ecology of the virus. several studies have determined the susceptibility of different animal species to sars-cov-2 via experimental infection [20, 21] . cats, hamsters, and ferrets are highly susceptible to sars-cov-2 infection, demonstrate varying clinical and pathological disease manifestations, readily transmit the virus to naïve animals, and mount a virusspecific immune response [22] [23] [24] [25] [26] [27] [28] . dogs are mildly susceptible to experimental sars-cov-2 infection, with limited viral replication but with clear evidence of seroconversion in some animals [22] . poultry species seem to be resistant to sars-cov-2 infection [22, 26] . these findings establish the respective utility of different animal species as pre-clinical models to study sars-cov-2. several lines of evidence suggest that pigs could be susceptible to sars-cov-2 infection. pigs are susceptible to both experimental and natural infection with the related betacoronavirus, sars-cov-1, and demonstrate seroconversion [29, 30] . structure-based analyses predict that the sars-cov-2 spike (s) protein receptor binding domain (rbd) binds the pig angiotensin-converting enzyme 2 (ace2) entry receptor with similar efficiency compared to human ace2 [31] . single-cell screening also indicated that pigs co-express ace2 and the tmprss2 activating factor in a variety of different cell lines, and sars-cov-2 replicates in various pig cell lines [2, 26, 32, 33] . despite these preliminary data indicating that pigs could be susceptible to sars-cov-2 infection, two recent studies revealed that intranasal inoculation of three and twelve pigs, respectively, with 10 5 pfu or tcid50 of sars-cov-2 did not lead to any detectable viral replication or seroconversion [22, 26] . however, the single route of intranasal inoculation used in these studies suggests that additional investigations are necessary before definitive conclusions can be made regarding susceptibility of pigs to sars-cov-2. in the present study, we determined the susceptibility of swine cell lines and domestic pigs to sars-cov-2 infection. two different porcine cell lines were found to be permissive to sars-cov-2 infection showing cytopathic effects (cpe). domestic pigs were challenged via simultaneous oral/intranasal/intratracheal inoculation with a 10 6 tcid50 dose of sars-cov-2. sars-cov-2 did not replicate in pigs and none of them seroconverted. furthermore, the virus was not transmitted from sars-cov-2 inoculated animals to sentinels. the present findings, combined with the other studies [22, 26] , confirm that pigs seem resistant to sars-cov-2 infection despite clear susceptibility of porcine cell lines. pigs are therefore unlikely to play an important role in the covid-19 pandemic as a virus reservoir or as a pre-clinical animal model to study sars-cov-2 pathogenesis or develop novel countermeasures. sars-cov-2 usa-wa1/2020 isolate (genbank accession # mn985325) [34] was eighteen pigs (mix of males and females, five weeks of age) were used in the study. pigs were acquired from a source guaranteed free of swine influenza virus (siv), porcine circovirus-2 (pcv-2), and porcine reproductive and respiratory syndrome virus (prrsv) infection. the study outline is illustrated in figure 1 . upon arrival, pigs were acclimated for 3 days prior to sars-cov-2 inoculation. nine pigs were designated as uninfected negative controls and housed in separate bsl-2 facilities. three of these uninfected negative control pigs were humanely euthanized at 3 days post challenge (dpc) to provide negative control clinical and tissue samples. the nine principal infected pigs were housed in the same room in two separate groups (4 or 5 pigs each; gross pathological examinations on major organs were performed and respiratory tissue samples were collected and either stored in 10% neutral-buffered formalin or stored as fresh samples at -80˚c. blood and swab samples were all filtered using a 0.2µm filter prior to storage at -80˚c. rna was isolated from blood, swabs, and tissue samples, using a magnetic bead-based protocol in a bsl-3+ laboratory at the bri at ksu. lung tissue homogenates (200 mg per 1ml dmem; 20% w/v) were prepared by thawing tissue, mincing it into 1mm sections, followed by lysis in a 2 ml sure-lock tube containing 5 mm stainless steel homogenization beads using the tissuelyser lt (qiagen, germantown, md, usa) for 30 seconds at 30 hz followed by 1 min of 30 hz while keeping the sample cold. following clarification via a 3-minute centrifugation (3,000xg; room temperature), supernatants were mixed with an equal volume of rlt lysis buffer. blood and clinical swabs were directly mixed with an equal volume of rlt lysis buffer. 200 µl of each sample lysate was used to extract rna using a magnetic bead-based during post mortem examinations, the upper and lower respiratory tract, central nervous system, lymphatic and cardiovascular systems, gastrointestinal and urogenital systems, and integument were evaluated. lungs were removed in toto and the percentage of the lung surface that was affected by macroscopic lesions was estimated by single veterinarian experienced in evaluating gross porcine lung pathology as previously described [36, 37] . lungs were evaluated for gross pathology such as edema, congestion, discoloration, atelectasis, and consolidation. tissue samples of interest were collected and either fixed in 10% neutral-buffered formalin for histopathological examination or frozen at -80˚c for rt-qpcr testing. tissues were fixed in formalin for 7 days, then transferred to 70% ethanol (thermofisher scientific, waltham, ma, usa) prior to trimming and paraffin embedding following standard automated protocols used in the histology section of the kansas state veterinary diagnostic laboratory. following embedding, tissue sections were cut and stained with hematoxylin and eosin and evaluated by a board-certified veterinary pathologist who was blinded to the treatment groups. to detect sars-cov-2 antibodies in sera, indirect elisas were performed observed. neutralizing sera from sars-cov-2-infected cats from a separate study [38] was used as a positive control. to determine the consensus sequence of the usa-wa/1/2020 virus and to analyze if there were any nucleic acid substitutions in the sars-cov-2 virus after passage in porcine cell lines, rna was extracted from cell culture supernatant as described above. the rna was then subjected to rt-pcr amplification using a tiledprimer approach to amplify the entire sars-cov-2 genome as described previously [39] . briefly, the pcr amplicons were pooled and subjected to library preparation for next generation sequencing using the nextera xt library prep kit (illumina, san diego, ca, usa). the library was normalized and sequenced using a miseq nano v2 2x250 sequencing kit. the sequence was then analyzed by mapping reads to the parent sequence (genbank accession # mn985325) [34] to generate a consensus sequence. the sars-cov-2 usa-wa1/2020 isolate, which was isolated from a human patient in washington state, usa, was used as the parent stock for the study [34] . the to determine the effect of sars-cov-2 infection in domestic pigs, nine sixweek-old sars-cov-2 seronegative piglets were inoculated with a total of 1 x 10 6 tcid50 of the usa-wa1/2020 isolate, which was passaged once in swine st cells ( figure 1 ). the challenge material (total 4 ml) was administered orally (1 ml), intranasally (1 ml; 0.5 ml each nostril) and intratracheally (2 ml) after sedation of the animals. at 1-day post challenge (dpc), six uninoculated sentinel contact pigs were comingled with the principal inoculated animals (3 animals per pen). daily rectal temperatures were recorded for each pig and clinical signs were monitored daily, including observations for signs of lethargy, hyporexia, respiratory distress (coughing, labored breathing, nasal discharge), and digestive issues (diarrhea or vomiting). no significant temperature elevation or change in rectal temperature was observed in the principal inoculated nor sentinel contact pigs throughout the study (figure 3) . moreover, no obvious clinical signs were observed for any of the principal inoculated nor sentinel pigs throughout the 21-day observation period. to detect viral replication in the principal and sentinel pigs, clinical samples were subjected to rt-qpcr to detect the sars-cov-2 n gene ( table 2) table 2 ). the only the exception was a low suspect positive result in a nasal swab at 1 dpc in a principal inoculated pig #161, for which one of two qpcr replicates yielded a low fluorescent amplification curve with a ct of 37 (table 2) . moreover, viral rna was not detected in any lung sample collected at post-mortem examination on 4, 8 and 21 dpc (table 2 ). in addition, gross and histopathological analysis of trachea and lung from the principal challenged pigs did not reveal the presence of any obvious pathological lesions ( table 3 , figure 4 ). these results indicate that sars-cov-2 failed to replicate in the respiratory and digestive tract as well as the blood in orally/intranasally/intratracheally inoculated pigs throughout an observation period of 21 days. this is confirmed by the fact that the principal infected pigs failed to transmit sars-cov-2 to co-mingled sentinel animals. to determine whether the orally/intranasally/intratracheally inoculated pigs sars-cov-2 is a zoonotic agent, and a detailed understanding of the susceptibility of various animal species to sars-cov-2 is central to controlling its spread [16, 17] . in addition, the development of animal models that emulate covid-19 in humans is essential for pre-clinical testing of novel vaccines and therapeutics [20] . in this study, we inoculated nine pigs with a high dose of sars-cov-2 that was passaged once in porcine cells. simultaneous oral/intranasal/intratracheal inoculation did not result in any detectable viral rna in the blood, the oral/nasal/rectal cavities, or the lungs. also, none of the co-mingled, sentinel contact pigs shed viral rna. moreover, a virus-specific immune response characteristic of infection was not observed within the 21-day study period in the principal infected or sentinel pigs. the transient nature of the igm and igg response observed in pig #848 could indicate cross-reactivity of antibodies directed against a porcine coronavirus such as porcine epidemic diarrhea virus [40] . such antibodies could be maternally derived and therefore transient as the lack of sars-cov-2 specific reactivity by the end of the study might suggest. in contrast to previous sars-cov-2 swine studies [22, 26] , the present study used a more stringent inoculation procedure (intratracheal and oral, in addition to intranasal) and 1 log higher titer of virus inoculum (10 6 vs 10 5 ). in addition, the inoculum in the present study was passaged once in porcine st cells. these results, combined with previous intranasal pig inoculation studies [22, 26] , indicate that pigs seem to be resistant to sars-cov-2 infection, are unlikely to be a sars-cov-2 carrier animal species, and are also not suitable as an animal model for research. the results of the present and previous sars-cov-2 inoculation studies in pigs are intriguing in light of the findings that the porcine ace2 receptor seems highly compatible with the sars-cov-2 rbd, suggesting that pigs could be susceptible to sars-cov-2 infection [2, 31] . pigs are susceptible to both experimental and natural infection with sars-cov-1 [29, 30] . however, the experimental sars-cov-1 infection was via simultaneous intranasal/oral/intraocular/intravenous inoculation [29] , thus the actual route(s) of sars-cov infection cannot be determined. recently, several porcine cell lines have been shown to be permissive to sars-cov-2 infection [26, 33] ; in addition, single-cell screening studies showed that porcine ace2/tmprss2 expression are compatible with infection [32] . in contrast to previous reports that some porcine cell lines are susceptible to sars-cov-2 infection, but show no cpe [26, 33] , we found that both st and pk-15 cell lines are susceptible to infection and observed cpe after two or four passages, respectively. the absence of sars-cov-2 replication and transmission in the present and two previous pigs studies [22, 26] seems to lessen the need to monitor pig populations for sars-cov-2 during the ongoing pandemic. however, the evidence described above suggests pig susceptibility should not be disregarded, because all pig studies to date have used rather young pigs and commercially available pig breeds/genetics. we also have to be aware that unforeseen genetic changes in the sars-cov-2 genome may result in a better compatibility of the virus for pigs in the future. pigs are considered to be an excellent model for studying human infectious diseases based on their relatedness to humans in terms of anatomy and immune responses and they have been found to be much more predictive for the efficacy of therapeutics when compared to rodent models [41] . however, the results presented here indicate that pigs are not a suitable preclinical model for sars-cov-2 pathogenesis studies and the development and efficacy testing of therapeutics and/or vaccines. a recently available article indicates that while pigs are not susceptible to sars-cov-2 infection, neutralizing antibody responses were detected in pigs infected via intramuscular or intravenous inoculation routes [42] ; this indicates that pigs could be used for immunogenicity studies related to sars-cov-2. however, the use of pigs to monitor sars-cov-2 immune responses must be careful to screen for cross-reactive maternal antibodies derived from other coronaviruses [43] . alternate pre-clinical animal models, namely non-human primates, syrian hamsters, transgenic or transduced mice expressing human ace2, ferrets, or even cats need to be considered to gain additional insights into sars-cov-2 pathogenesis and virulence. comprehensive characterization of sars-cov-2 pathogenesis in preclinical animal models and the establishment of standardized infection and testing protocols will be crucial for the development of much-need countermeasures to combat covid-19. 1 swabs/blood were tested from samples on 0, 3, 5, 7, 10, and 14 dpc. lung tissue was collected 4, 8, and 21 dpc. 2 swabs/blood were tested from samples on 0, 3, 5, and 10 dpc. lung tissue was collected on 21 dpc 3 swabs/blood were tested on 0 dpc. lung tissue was collected on 3 dpc for these uninfected controls. *one pig (#161) had a ct signal of 37.72 (3.82x10 4 copy number/ml) for 1/2 of rt-qpcr wells on 1 dpc. magnification is 10x for main images and 20x for inserts. coronavirus disease (covid-19) situation report -206 a pneumonia outbreak associated with a new coronavirus of probable bat 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associated with the covid-19 outbreak possible bat origin of severe acute respiratory syndrome coronavirus 2. emerg infect dis is covid-19 the first pandemic that evolves into a panzootic? vet ital covid-19 and veterinarians for one health, zoonotic-and reverse-zoonotic transmissions a critical needs assessment for research in companion animals and livestock following the pandemic of covid-19 in humans. vector borne zoonotic dis are animals a neglected transmission route of sars-cov-2? pathogens evidence for sars-cov-2 infection of animal hosts. pathogens infectivity, virulence, pathogenicity, host-pathogen interactions of sars and sars-cov-2 in experimental animals: a systematic review susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2. science transmission of sars-cov-2 in domestic cats infection and rapid transmission of sars-cov-2 in ferrets sars-cov-2 is transmitted via contact and via the air between ferrets simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility pathogenesis and transmission of sars-cov-2 in golden hamsters susceptibility of pigs and chickens to sars coronavirus sars-associated coronavirus transmitted from human to pig. emerg infect dis receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus single-cell screening of sars-cov-2 comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study. lancet microbe severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, united states. emerg infect dis division of viral diseases. real-time rt-pcr panel for detection -2019-novel coronavirus pathogenic and antigenic properties of phylogenetically distinct reassortant h3n2 swine influenza viruses cocirculating in the united states comparison of pathogenicity and transmissibility of influenza b and d viruses in pigs. viruses sars-cov-2 infection, disease and transmission in domestic cats ncov-2019 sequencing protocol v2 v.2 lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts the pig: a model for human infectious diseases pigs are not susceptible to sars-cov-2 infection but are a model for viral immunogenicity studies emerging and re-emerging we gratefully thank the staff of ksu biosecurity research institute, the the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. key: cord-270396-3bcnnyfq authors: karacin, cengiz; bilgetekin, irem; b basal, fatma; oksuzoglu, omur b title: how does covid-19 fear and anxiety affect chemotherapy adherence in patients with cancer date: 2020-07-17 journal: future oncology doi: 10.2217/fon-2020-0592 sha: doc_id: 270396 cord_uid: 3bcnnyfq aim: to investigate how covid-19 fear and anxiety (cov-fa) affects chemotherapy adherence in patients with cancer. materials & methods: the records of 3661 patients with chemotherapy (ct) appointments were retrospectively reviewed. results: the ct postponement rates before and after covid-19 were 11.6% and 14.2%, respectively (p = 0.017). the rate of cov-fa-related ct postponement after telemedicine was lower than that before (4.6% vs 17.4%, p = 0.012). the median time to come back to treatment of the cov-fa group was 47 days (range 19–72 days). advanced age (≥60 years) was found to be the independent factor that was predictive of time to come back to treatment (p = 0.043). conclusion: the ct postponement rate increased after covid-19. cov-fa-related ct postponement decreased after telemedicine. advanced age could be predictive of time to come back to treatment. patients with appointments between 17 january 2020 and 10 may 2020 were retrospectively reviewed on the appointment list of patients who were going to receive ct at our healthcare center. patients who did not come for ct on the date of their appointment were identified by reviewing the patient records. the ct postponement rates were calculated for the 60-day periods before and after 10 march 2020, when the first covid-19 case was diagnosed in turkey. we planned to include all adult patients (>18 years of age) with postponed ct and histopathologically diagnosed cancer. illiterate patients, those with symptomatic brain metastases that could disrupt cognitive functions, those with psychiatric disorders and those with anxiety disorders were excluded from the study. to obtain the most accurate information about the psychiatric and anxiety disorders of the patients, we made a two-step evaluation. firstly, we examined the follow-up notes and histories in our patient follow-up forms in detail and hospital patient electronic data file were retrospectively screened for any information about psychiatric problems. secondly, we asked each patient whether they had a history of psychiatric or anxiety disorder. data on patients' age, sex, comorbidities, history of smoking, marital status, number of children, educational background, place of residence and household, cancer type, disease stage, ct regimen and date of ct postponement were obtained from the hospital records. a patient questionnaire (prepared using the google survey program) that involved the beck anxiety inventory (bai) and questions about covid-19 fear was sent to patients on whatsapp, along with the informed consent form. a database was created using the data obtained from the hospital records and the patients' responses in the questionnaire. the reasons given by the patients who postponed ct were first investigated using the hospital records. patients who reported that they did not come to their ct appointment due to cov-fa according to the hospital records were called by phone to confirm their reason for ct postponement and the cov-fa group was formed. patients who had postponed their ct but had no record of the reason for the postponement in the hospital records were called; those who stated they postponed their ct due to cov-fa were also included in the cov-fa group. during the telephone conversations with patients in the cov-fa group, the patients were invited to participate in the survey study and all of these patients (30 patients) agreed. those with postponed ct due to neutropenia, thrombocytopenia, acute renal failure, drug-related side effects like skin toxicity and non-cov-fa reasons such as infection and thrombosis were also invited to participate in the survey study and formed the 'other' group (80 patients). the demographic and clinical characteristics, questionnaire responses and bai scores of both groups (30 patients in the 'cov-fa' and 80 patients in the 'other' group) were compared. factors associated with ct postponement due to cov-fa were identified. we started the telemedicine practice 35 days after the first covid-19 case in turkey for patients who had been receiving treatment at the medical oncology chemotherapy day unit. as part of this practice, patients with a booked ct appointment were called by a medical oncology specialist doctor a few days before their appointment. during these phone calls, patients were asked whether they had any symptoms of covid-19 (fever, cough, sputum and shortness of breath), whether they had been in contact with someone infected with covid-19 and whether they had traveled abroad in the last 14 days. the patients were also informed about whether there were covid-19 cases in our healthcare center as of the day the phone call was made. any questions the patients asked about covid-19 were answered. follow-up of the cov-fa group & termination of the study the hospital records of the patients who postponed their ct appointments due to cov-fa on the day of freezing the database for the present study were reviewed again and a record of whether they had returned to our clinic to receive ct again was added to the database. the dates when patients returned to receive ct again were recorded. those who had not yet been readmitted for ct administration were interviewed by phone and the last contact date for these patients was recorded as may 28, 2020, the date of freezing the study database. the bai is a multiple-choice self-report inventory used for measuring the severity of anxiety in children and adults [11] . it was developed by beck et al. and a study of its reliability and validity in turkey was carried out by ulusoy et al. [12] the questions used in this measure ask about common symptoms of anxiety that the subject has had during the past week (including the day on which the test is taken), such as numbness and tingling, sweating not due to heat and fear of the worst happening [11] . the bai contains 21 questions, each answer being scored on a scale value of 0 (not at all) to 3 (severely) [11] . higher total scores indicate more severe anxiety symptoms [11] . data analysis was performed using ibm ssps statistics for windows v.20.0 software (ibm, ny, usa). qualitative variables were expressed as frequencies and percentage, whereas the quantitative variables were expressed as mean (± standard deviation) or median. the conformity of the numerical data to the normal distribution was assessed using the kolmogorov-smirnov test. the student t test was used to compare the parametric data and the mann-whitney u test was used to compare the nonparametric data. pearson's chi-square or fisher's exact test was performed to compare the categorical data. tcbt was defined as the elapsed time from ct postponement until either return for treatment or the last contact. tcbt was determined using the kaplan-meier method and the log-rank test was used for univariate comparison. a multivariate cox regression model was used to identify independent predictive factors on tcbt. all statistical analyses were two-way and the level of statistical significance was set at p < 0.05. of 2112 patients with ct appointments in the 60-day period preceding the first covid-19 case (10 march 2020), 245 (11.6%) had their ct appointment postponed; of a total of 1549 patients with a ct appointment in the 60-day period following the first covid-19 case, 220 (14.2%) had their ct appointment postponed (χ 2 = 5.729, p = 0.017). the three most common reasons for postponing ct after covid-19 were neutropenia (23.1%), thrombocytopenia (20.9%) and cov-fa (13.6%; table 1 ); 27 patients (17.4%) postponed ct due to cov-fa before the introduction of the telemedicine practice, but only three patients (4.6%) postponed ct for this reason (p = 0.012; figure 1 ) after the telemedicine practice was initiated. the median age of the 110 patients who participated in the study was 63 years (range 54-70); 36.4% were male and 63.6% were female. the demographic and clinical characteristics of all patients in the cov-fa group and the other group are shown in table 2 . the percentage of female patients in the cov-fa group was significantly higher than that in the other group (80.0 vs 57.5%, p = 0.049). there was no significant difference between the two groups in terms of age, disease stage or ct administration modality. in total, 110 patients who responded to the questionnaire, including those with cov-fa-related ct postponement or ct postponement due to other reasons, were compared in terms of their social characteristics, survey results and bai. the groups were similar in terms of marital status, place of residence, educational background, comorbidity, history of smoking and access to the hospital. the cov-fa group had a higher rate of 'yes' responses to the questions 'are you afraid of covid-19?', 'does it bother you to think about covid-19?', 'does the concern of getting covid-19 disrupt your sleep?', 'do you worry about covid-19 news and stories you read on social media?', 'does the thought of getting covid-19 give you palpitations?' than the other group ( table 3 ). the mean bai of the cov-fa group was significantly higher than that of the other group (18.9 vs 3.3, p < 0.001). as of the date when the study analyses were performed, 16 of the 30 patients in the cov-fa group returned to receive ct again, while 14 were not admitted by our clinic for ct again. we contacted those 14 patients and they stated they did not want to return to their treatment for a while due to the pandemic process. the median ct postponement time of the patients in the cov-fa group was 47 days (range 19-72; figure 2 ). following univariate analysis of the factors that may affect the tcbt of the patients, advanced age (≥60 years) and advanced-stage disease were associated with longer tcbt.. the median tcbt of patients with stage ii, iii and iv disease were 33, 53 and 70 days, respectively (p = 0.048; table 4 ). the median tcbt was found to be longer in those aged ≥60 years than in those aged <60 years (70 vs 43 days, p = 0.003; figure 3 ). the median tcbt of those receiving palliative treatment was 70 days and the mean for those who did not receive palliative treatment was 45 days (p = 0.072). as a result of the univariate analysis, a cox regression model was created based on the factors whose p-value was <0.20 (age, stage and treatment modality; table 5 ). as a result of the multivariate analysis, advanced age (≥60 years) was found to be an independent factor predicting tcbt (hazard ratio = 0.291, p = 0.043). as far as we know, ours is the first study to investigate cov-fa within the context of ct adherence in patients with cancer receiving active ct. in the present study, it was seen that the ct postponement rate increased significantly following the first covid-19 case in turkey and cov-fa was identified as the third most frequent reason for ct postponement. cov-fa-related ct postponement was more frequent in women than in men. patient age was also found to be an independent factor that predicted tcbt. patients aged ≥60 years had longer tcbt than those aged <60 years. studies have shown that anxiety disorders have negative effects on adherence to ct in patients with cancer [4, 5] . in their study involving 135 patients with colon cancer, zhu et al. investigated the causes that led to postponement of adjuvant ct initiation; they concluded that anxiety is an independent predictive factor for postponing ct [5] . in a prospective study that included 50 patients with advanced lung cancer, greer of the factors that negatively affects adherence to chemotherapy [4] . the present study also investigates how elevated anxiety and fear in society during the pandemic can affect the chemotherapy process. the significant difference found in the ct postponement rates in our oncology center before and after the first covid-19 case (11.6 vs 14.2%) suggested that it could be due to cov-fa. cov-fa was the third most common cause among the reasons for postponing ct after the first covid-19 case. patients who have had covid-19 infection may be more likely to develop fear and anxiety; however, because there were no covid-19 infected patients in our study, we could not comment on ct postponement rates in these patients. the present study revealed that female sex can be a risk factor for cov-fa, which is consistent with the literature. many studies have proven that anxiety disorders occur more often in women [13] . in the study conducted by braamse et al. to investigate factors associated with the causes of anxiety and depression in patients with colorectal cancer, women were twice as likely as men to have anxiety [14] . the relationship between anxiety and female sex has been found to be similar in studies of the covid-19 pandemic, including one in turkey [7, 15] . a meta-analysis showed that covid-19 anxiety was particularly more common in female healthcare workers than in male healthcare workers [15] . one study showed that chronic diseases and living in the city center also increase the risk of anxiety [13] . in the present study, there was no significant difference between the patients with ct postponement due to cov-fa and those with ct postponement due to other reasons in terms of chronic diseases and place of residence. telemedicine provides an important advantage in protecting patients with cancer from covid-19 infection because these patients already have immunosuppression [16] ; 35 days after the first covid-19 case in turkey, we started interviewing follow-up patients at our oncology center using telemedicine. patients' follow-up, examination results and treatment responses were all evaluated during video calls. moreover, the patients were questioned about whether they had symptoms of covid-19, thereby ensuring the early diagnosis of a possible covid-19 infection and reducing the risk of these patients infecting other patients in the ct day unit. during the interviews, we shared up-to-date information about whether covid-19 cases were seen in our healthcare center and encouraged patients to continue their treatment by informing them about the undesired consequences of treatment discontinuation. these interviews using telemedicine might have yielded positive results, especially in terms of ct postponement rates. ct postponement rates related to cov-fa decreased after the telemedicine application had started; however, it would not be correct to associate this decrease in cov-fa-related ct postponement rate to telemedicine alone. we have not encountered any study in the literature that examines the tcbt in patients with postponed ct. in the present study, the median tcbt of the patients who postponed ct due to cov-fa was 47 days (range . the multivariate analysis showed that only advanced age is an independent factor that predicts tcbt. studies have shown that advanced age is an important prognostic factor in terms of covid-19 mortality and this information has been frequently covered in the news and social media [17, 18] . we believe that the long tcbt of the elderly patients herein may be related to the coverage of this information in the news. as a matter of fact, one of the questions the patients were asked as part of the present study was whether they were following news or social media concerning covid-19; the majority (76.7%) of the patients who postponed their ct due to cov-fa responded 'yes' to this question. repeated media exposure to community crisis can lead to increased anxiety and studies have shown that media and social media exposure might increase covid-19 anxiety [19, 20] . people have been exposed to news of covid-19 in different ways and have different ideas of the severity of the pandemic. our study did not measure media exposure or social media exposure by an objective method; however, rates of following the news or social media concerning covid-19 were similar between the 'cov-fa' and 'other' groups in our study. rural residents may have lower access to and use of certain health information sources relative to urban residents [21] . considering that the difference between rural and urban residents might affect covid-19 anxiety, we compared the 'cov-fa' and 'other' groups in terms of the regions where the patients live but did not find any difference between the groups. the present study has some limitations. patients included in the present study were very heterogeneous in terms of their cancer diagnoses and only a small number of patients were included. another limitation arising from the retrospective design of the study is that the elapsed time from the date of survey delivery to the patients and the date of ct postponement was not the same for all patients. patients with psychiatric disorders were excluded from the study; however, cancer-related anxiety that we have not yet noticed (especially in patients at the beginning of the diagnosis and treatment process) might be confounding some of the results. our study was conducted within a short period of time after the first covid-19 case. it would be useful to support our findings using longitudinal studies to generalize our results, because different results can be obtained with long-term studies. the present study concludes that cov-fa increases ct postponement rates, telemedicine can contribute to reducing cov-fa-related ct postponement rates, female sex can be a risk factor for cov-fa-related ct postponement and advanced age is a factor that could predict increased tcbt. • we aimed to investigate how covid-19 fear and anxiety (cov-fa) affects chemotherapy (ct) adherence in patients receiving active ct for cancer, and to identify the characteristics of patients who postponed their ct due to cov-fa, as well as the time to come back to treatment (tcbt) and the factors affecting tcbt in these patients. • the records of 3661 patients with ct appointments during the 60-day periods preceding and following the date when the first covid-19 case was seen in turkey were reviewed retrospectively. • cov-fa-related ct postponement rates were calculated before and after telemedicine. • the ct postponement rates before and after the first covid-19 case were 11.6 and 14.2%, respectively (p = 0.017). • the rate of cov-fa-related ct postponement after telemedicine was lower than that before (4.6 vs 17.4%, p = 0.012). • the median tcbt of the cov-fa group was 47 days (range 19-72). advanced age (≥60 years) was found to be the independent factor that was predictive of tcbt (hazard ratio = 0.291, p = 0.043). c karacin contributed to the study concept, study design, data analysis and interpretation and manuscript writing. i bilgetekin contributed to the study concept, data analysis and interpretation. f bugdayci basal enrolled patients and contributed to data interpretation. ob oksuzoglu contributed to the study concept and revised the manuscript. future science group 10.2217/fon-2020-0592 progress in adjuvant chemotherapy for breast cancer: an overview cytotoxic chemotherapy in the contemporary management of metastatic castration-resistant prostate cancer (mcrpc) association of delayed adjuvant chemotherapy with survival after lung cancer surgery the relationship with delayed chemotherapy and survival in cancer patients behavioral and psychological predictors of chemotherapy adherence in patients with advanced non-small cell lung cancer • the relationship between anxiety and chemotherapy adherence high level of unmet needs and anxiety are associated with delayed initiation of adjuvant chemotherapy for colorectal cancer patients • the relationship between anxiety and delaying initiation of adjuvant chemotherapy who declares covid-19 a pandemic levels and predictors of anxiety, depression and health anxiety during covid-19 pandemic in turkish society: the importance of gender •• health anxiety during covid-19 sars-cov-2 infection anxieties and general population restrictions delay diagnosis and treatment of acute haematological malignancies covid-19 and cancer: a comprehensive review mental health, risk factors and social media use during the covid-19 epidemic and cordon sanitaire among the community and health professionals in wuhan, china: cross-sectional survey an inventory for measuring clinical anxiety: psychometric properties the beck anxiety inventory: psychometric properties anxiety and mood: a review of nomenclature, comorbidity and epidemiology factors associated with anxiety and depressive symptoms in colorectal cancer survivors prevalence of depression, anxietyand insomnia among healthcare workers during the covid-19 pandemic: a systematic review and meta-analysis telemedicine for cancer patients during covid-19 pandemic: between threats and opportunities • the impact of telemedicine in cancer patients the impact of social media on panic during the covid-19 pandemic in iraqi kurdistan: online questionnaire study predictors of mortality for patients with covid-19 pneumonia caused by sars-cov-2: a prospective cohort study media exposure to mass violence events can fuel a cycle of distress mental health problems and social media exposure during covid-19 outbreak we thank all our patients who participated in the study. we would also like to thank ozan yazici, yakup ergun, tulay eren, ali alkan and ismail erturk, who made very important contributions in the preliminary evaluation of the article. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.no writing assistance was utilized in the production of this manuscript. the authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the declaration of helsinki for all human or animal experimental investigations. in addition, for investigations involving human subjects, informed consent has been obtained from the participants involved. a special permission was obtained from the ministry of health for this study. key: cord-267115-6jqdi417 authors: giobbe, giovanni giuseppe; bonfante, francesco; zambaiti, elisa; gagliano, onelia; jones, brendan c.; luni, camilla; laterza, cecilia; perin, silvia; stuart, hannah t.; pagliari, matteo; bortolami, alessio; mazzetto, eva; manfredi, anna; colantuono, chiara; di filippo, lucio; pellegata, alessandro; li, vivian sze wing; eaton, simon; thapar, nikhil; cacchiarelli, davide; elvassore, nicola; de coppi, paolo title: sars-cov-2 infection and replication in human fetal and pediatric gastric organoids date: 2020-06-24 journal: biorxiv doi: 10.1101/2020.06.24.167049 sha: doc_id: 267115 cord_uid: 6jqdi417 coronavirus disease 2019 (covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection is a global public health emergency. covid-19 typically manifests as a respiratory illness but an increasing number of clinical reports describe gastrointestinal (gi) symptoms. this is particularly true in children in whom gi symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. by contrast, fetuses seem to be rarely affected by covid-19, although the virus has been detected in placentas of affected women. these observations raise the question of whether the virus can infect and replicate within the stomach once ingested. moreover, it is not yet clear whether active replication of sars-cov-2 is possible in the stomach of children or in fetuses at different developmental stages. here we show the novel derivation of fetal gastric organoids from 8-21 post-conception week (pcw) fetuses, and from pediatric biopsies, to be used as an in vitro model for sars-cov-2 gastric infection. gastric organoids recapitulate human stomach with linear increase of gastric mucin 5ac along developmental stages, and expression of gastric markers pepsinogen, somatostatin, gastrin and chromogranin a. in order to investigate sars-cov-2 infection with minimal perturbation and under steady-state conditions, we induced a reversed polarity in the gastric organoids (rp-gos) in suspension. in this condition of exposed apical polarity, the virus can easily access viral receptor angiotensin-converting enzyme 2 (ace2). the pediatric rp-gos are fully susceptible to infection with sars-cov-2, where viral nucleoprotein is expressed in cells undergoing programmed cell death, while the efficiency of infection is significantly lower in fetal organoids. the rp-gos derived from pediatric patients show sustained robust viral replication of sars-cov-2, compared with organoids derived from fetal stomachs. transcriptomic analysis shows a moderate innate antiviral response and the lack of differentially expressed genes belonging to the interferon family. collectively, we established the first expandable human gastric organoid culture across fetal developmental stages, and we support the hypothesis that fetal tissue seems to be less susceptible to sars-cov-2 infection, especially in early stages of development. however, the virus can efficiently infect gastric epithelium in pediatric patients, suggesting that the stomach might have an active role in fecal-oral transmission of sars-cov-2. severe acute respiratory syndrome coronavirus 2 is responsible for a pandemic that has proven catastrophic, due to the lack of immunity in the human population and the range of pathological features associated with infection, including severe and often life-threatening respiratory syndromes causing major health, social and economic consequences. the virus has been shown to infect respiratory epithelial cells and to spread mainly via the respiratory tract 1 . as governments and international health agencies seek effective policies to minimise infections, maintain health care delivery, and eventually ease movement restrictions, understanding the pathogenesis and the various mechanisms for transmission is of the utmost importance. it is well established that adults are more likely than children to develop symptoms upon sars-cov-2 infection, but little is known on the role of children in transmission of the disease. a growing body of literature suggests that replication at the level of the gastrointestinal (gi) tract not only occurs in a large proportion of confirmed cases 2 , but it also extends the overall duration of shedding, after viral clearance from the respiratory tract has occurred 3 . interestingly, infected children have been shown to be particularly prone to develop gi symptoms which can be moderate-to-severe, leading to intensive care unit (icu) admission and mimicking, in some cases, symptoms of appendicitis 4 . additionally, sars-cov-2 was detected by means of electron microscopy in stool samples, elevated concentrations of sars-cov-2 rna were detected in air samples collected in patients' toilet areas 5 and rectal swabs from mildly symptomatic pediatric patients persistently tested positive, even after viral clearance from the upper respiratory tract had occurred 6 . this evidence, together with the recent demonstration of a high receptor density at the level of the oral cavity and tongue 7 , raises important questions about the likelihood of fecal-oral transmission and whether therapeutic interventions to reduce gastrointestinal infection will play a role in the control of the disease. however, a recent report of 244 consecutive covid-19 positive children from wuhan did not find any difference in fecal nucleic acid rt-pcr between children with or without gi symptoms, suggesting that rt-pcr detection of the virus was not due to gut infection but coming instead from the respiratory tract from swallowed sputum 8 . defining the role of the gi tract in sars-cov-2 infection may also help understand the risks of vertical transmission during gestation since amniotic fluid is swallowed by the fetus during gestation and viral contamination has been isolated from the placenta of an affected woman 9 . while samples from affected mothers have so far failed to prove that amniotic fluid, cord blood, and breast milk contain sars-cov-2 10 , very limited data are available at this stage for pregnant women with covid-19, and even fewer data are available on intrauterine vertical transmission 11 . while severe symptoms and death have been recorded in infants as young as 10 months of age 12 , very few cases of pathology in neonates have been associated with infection 13 and when 6 newborns from infected mothers were screened for sars-cov-2, they tested negative for the virus 10 . however, this limited cohort cannot exclude the possibility of infection of the fetuses. reliable human in vitro gi model systems that faithfully reproduce infection dynamics and disease mechanisms will prove key to advance our understanding of sars-cov-2 replication and pathology in the gi tract. little information is available with respect to the distribution of the viral receptor angiotensinconverting enzyme 2 (ace2) at the level of the gi tract of humans 14 . in particular, we lack fundamental information regarding which region of the gi system is the target of replication and primarily associates with the prolonged shedding of sars-cov-2 in both pediatric and adult patients. organoids have attracted great attention, enabling in vitro disease modeling and providing an ideal tool for studying infectious pathogens, particularly of the gi tract 15 . recent studies have demonstrated how sars-cov-2 can efficiently infect human intestinal enteroids 16, 17 , providing evidence in support of the hypothesis that sees sars-cov-2 as a fecaloral transmissible pathogen. however, it remains to be elucidated whether access to the duodenum depends on passive transport of infected oral fluids across the stomach, or on active viral replication in the gastric mucosa. human gastric organoids derived from adult patients and induced pluripotent stem cells have proved to be instrumental for the generation of reliable in vitro models for the characterization of infectious agents 18, 19 . organoid derivation from human fetal organs has been shown for the intestine 20 , liver 21 and pancreas 22 . here, we describe the novel derivation of proliferative progenitors from human fetal stomach and their expansion in vitro as enterospheres. furthermore, we provide insight into the ability of sars-cov-2 to infect an organoid-based model of the gastric mucosa at both fetal and pediatric ages. this work aims to unravel the susceptibility of the stomach to sars-cov-2 infection through the development of an innovative expandable in vitro model that faithfully reproduces the gastric microenvironment. a deeper understanding of the susceptibility of the human stomach to sars-cov-2 infection and replication could lay the foundations for the development of therapeutic options to reduce gastrointestinal infection. organoids are organized three dimensional structures that can be grown from isolated stem cells found in adult and fetal tissues. in order to derive a novel in vitro gastric model of fetal origin, we firstly characterized the tissues isolated from human fetuses and compared them to gastric mucosa obtained from pediatric patients undergoing surgery (fig. 1a) . developing stomach structures are shown in fig. 1b from carnegie stage (cs) 23 (corresponding to mid-week 8) to post conception week (pcw) 21. gastric crypts start to invaginate between pcw 11 and pcw 12 and form a clearly defined crypt at around pcw 20 (fig. 1c) . we characterized the appearance of gastric markers during fetal development. mucin 5ac positive pit mucous cells were evident at pcw 11, while pepsinogen c (marking chief cells) started to emerge at around pcw 20 (fig. 1d) . mucin 6, a gland mucous cell marker, was constitutively expressed from early week 8 (cs 20), together with enteroendocrine cells marked by chromogranin a that were present from mid-week 8 (cs 23) (fig. 1e) . we then defined three distinct groups of gastric epithelial tissues based on gland maturity: 1) early fetal stomachs from pcw 8 to pcw 15; 2) late fetal stomachs from pcw 17 to pcw 21; 3) pediatric stomachs. real time quantitative pcr (qpcr) was performed on gastric tissues obtained from these three groups to examine the gene expression changes of stem cell and differentiated cell markers. a significant correlation between developmental stage and mrna expression was observed for axin2, mucin 5ac (muc5ac), pepsinogen a5 (pga5), with a similar trend for chromogranin a (chga) and atpase h+/k+ transporting subunit beta (atp4b) (fig. 1f) . on the other hand, expression of leucine-rich repeat-containing g-protein coupled receptor 5 (lgr5) and somatostatin (sst) were significantly higher in the late fetal stomachs. following gastric tissue characterization, we efficiently extracted glandular crypts from fetal and pediatric stomachs utilizing chelating buffers and mechanical stress. to improve compatibility with subsequent clinical application of this organoid system, isolated fetal cells were expanded in a chemically defined medium, without the use of animal serum or conditioned media. each gastric cytokine, based on previous work 19 was screened and selectively removed from the organoids split to single cells and grown for 10 days to allow clonal organoid formation. while r-spondin 1,wnt-3a and noggin withdrawal led to more differentiated morphology, chir99021 (gsk-3 inhibitor) proved to be essential in the formation of fetal gastric organoids starting from single cells (fig. 2a) . no difference was found among fetal and pediatric organoid growth in the medium. we then performed isolation of several gastric organoid lines (supplementary table 1 ). the isolation protocol proved to be highly efficient and we obtained a biobank composed of 5 lines of early fetal stage (from cs23 to pcw 11), 6 lines of late fetal stage (from pcw 18 to pcw 21), and 4 lines of pediatric stage organoids (from 4 months-to 11 years-old). expanding organoids were stained for the epithelial marker ezrin (ezr) and luminal polarized f-actin fig. 2c . muc5ac was present on the luminal side of the organoids of all stages, with a relatively lower expression in the early pcw 11 (fig. 2c ). organoids were expanded and counted for several months, showing higher rate of expansion for earlier fetal stages (fig. 2d) . no plateau was reached in any of the curves even after several months, showing the possibility to obtain stable fetal gastric organoid lines ( supplementary fig. 1 ). after weekly passaging for more than 10 weeks, we further characterized the organoid lines to evaluate genomic stability. single nucleotide polymorphism (snp) arrays on early fetal, late fetal and pediatric organoids showed no chromosomal duplications, no large deletions, nor other karyotype aberrations, demonstrating the organoids are genetically stable after prolonged in vitro culture (fig. 2e) . real time pcr was performed on organoids grouped in early fetal (cs 23 to pcw 11), late fetal (pcw 18 to pcw 20) and pediatric. stem cell crypt markers lgr5 and axin2 were expressed in these organoids, indicating the presence of proliferating cells. muc5ac showed a pattern of increased expression along differentiation comparable to the tissue of origin in fig. 1f . the expression patterns of muc6 and sst were also comparable to the tissue of origin. on the other hand, chga showed an inverted pattern of expression, while transcript expression of proton pump transporter atp4b, responsible for gastric acid secretion, was lost in the organoid model (fig. 2f) . next, we characterized the transcriptomics of gastric epithelial tissues and gastric-derived organoids, at three developmental stages. rna-seq was performed on the three groups of early fetal, late fetal and pediatric samples. principal component analysis (pca) showed smaller heterogeneity in the organoid groups derived at different stages of fetal and pediatric development with respect to the primary tissues analyzed at the same stages, which may also include some heterogeneity from the surrounding cells as a result of the isolation procedure (fig. 3a) . when pca was performed including only organoid samples, the overall variability due to the different developmental stage was comparable to that between biological replicates within the same group ( supplementary fig. 2a ). this analysis suggests that transcriptional differences related to the developmental stage of the tissue of origin could be more subtle than those captured at pca level. we then analyzed the expression of typical gastric markers in organoids derived from tissues at different stages 23 . the only differentially expressed gene (deg) was muc5ac, which was more highly expressed in organoids from tissues at later developmental stages (fig. 3b) , confirming the qpcr results above (fig. 2f) . we did not observe processes of "intestinalization" of the organoids in culture, as cdx2 expression was negligible ( supplementary fig. 2c ). consistent with the qpcr results in fig. 2f , expression of atp4a and atp4b proton transporters were not detectable in the rna-seq, confirming the absence of the parietal cells in the organoids ( supplementary fig. 2d ). on the other hand, most putative genes identifying gastric crypt stem cells (sox9, olfm4, procr, mki67, tacstd2) were expressed at all developmental stages ( supplementary fig. 2e ). rna-seq analysis on the gastric primary tissues showed a significant increase in transcript levels of the functional markers along the developmental stage (fig. 3c) , confirming that the temporal trend shown by pca (fig. 3a) is related to specific gastric developmental stages. when we performed hierarchical clustering analyses of the previously reported genes representing the six stomach cellular subtypes 23 , most of these genes from the analysis were not degs (fig. 3d) . indeed, these six cell types are known to be all co-present at different stages of embryo development from pcw 7 to 25 23 . furthermore, we clustered degs between pair of conditions to reproduce a pseudo-temporal profile between the three developmental stages considered (fig. 3e) . we highlighted in fig. 3f the results of a pathway enrichment analysis from selected clusters that displayed gastric-related functions. full results are reported in (supplementary file 1). in order to validate both fetal and pediatric gastric organoids as functional in vitro models of sars-cov-2 infection and replication, we optimized the culture condition for viral infection in a 3d system (fig. 4a) . standard organoids of endodermal organs have a luminal polarity facing the internal portion of the structure, with an apical (inner) f-actin and zonula occludens-1 (zo-1), and basal (external) lamina marked by b-4 integrin (b4-int) (fig. 4b) . such inner polarity might be an obstacle to an efficient viral infection in vitro, given that the apical side is luminal. in addition, matrigel might impede efficient diffusion of the virus, thus affecting the likelihood of establishing an infection, and subsequent detection and quantification of the viral progeny released from the infected organoids. to maximize the efficiency of infection and the effective quantification of viral progeny released, we reverted the polarity of the gastric organoids 24 to expose the apical side of the cells on the outer side. organoids were removed from the surrounding extracellular matrix and cultured in suspension for 3 days, resulting in the exposure of the apical f-actin on the outer side, accompanied with muc5ac secretion externally (fig. 4c) . conversely, zo-1 and b4-int expression was inverted compared to standard organoids in fig. 4b . full 3d deconvolution images of reversed organoids are shown in supplementary fig. 3a . we then analyzed the absolute expression of ace2 and transmembrane protease serine 2 (tmprss2) sars-cov-2 receptors in our gastric models to evaluate the sars-cov-2 infection potential. rna-seq data analysis showed that expression of ace2 was significantly lower in early fetal stomachs compared to the pediatric ones, while late fetal samples' higher variability prevents drawing a final conclusion. on the other hand, tmprss2 mrna expression was consistently high throughout the stomach samples ( fig. 4d) . we further performed rna sequencing on rp-gos to evaluate the transcriptional changes. pca analysis showed similar clustering among the different stages between rp-gos ( supplementary fig. 3b ) and normal polarity organoids ( supplementary fig. 2a) . a comparable pattern of expression for ace2 and tmprss2 was observed also in rp-gos, with ace2 significantly higher expressed in pediatric organoids (fig. 4e ). protein expression of ace2 was further confirmed by immunofluorescence staining in all the rp-gos derived at pcw11, pcw 20 and pediatric stages (fig. 4f) . to investigate the susceptibility of organoids to sars-cov-2 infection, we selected a sars-cov-2 isolate obtained from the pharyngeal swab of a 14-year-old pediatric patient. purity of the isolate was confirmed by means of molecular testing comprising an extended panel of bacterial and viral respiratory agents. reversed-polarity gastric organoids derived at pcw11, pcw 20 and pediatric stages and normalpolarity organoids from the same stages were infected by trained virologists in a biosafety level 3 (bsl3) laboratory. after a 2-hour infection, the organoids were cultured up to 96 hours in suspension and checked for structural integrity and viability by visual examination on a daily basis (fig. 5a) . vero e6 cells were used as a susceptible substrate for sars-cov-2, to validate the infection in vitro (supplementary fig. 4a ). in next, we performed rna-seq analysis on non-infected and infected organoids samples at each developmental stage. interestingly, we identified significantly degs in samples from pcw 20 (19 degs) and pediatric organoids (13 degs), but not in pcw11 samples (fig. 6a ). all the degs in both developmental stages were up-regulated after the infection, among which 10 genes (cmpk2, ddx58, dhx58, herc5, ifi44, ifit2, ifit3, irf7, mx1, rsad2) were in common. we further performed an analysis to find the degs associated with the infection irrespective of the developmental stage, identifying a further 8 degs (bst2, eif2ak2, herc6, ifi44l, lamp3, slc1a3, stat1, usp18), for an overall number of degs equal to 30. intriguingly, approximately 63% of the degs identified in this study as respondent to the infection were previously found to be degs in a literature survey of transcriptomic data on sars-cov, where 38 genes were identified as degs at the intersection of at least 9 studies (fig. 6b ). among the common degs, some were first responders of the infection process, like the ddx58 and ifih1 encoding the viral rna sensors rig-i and mda5 respectively, and their regulators, such as dhx58; others more downstream players of the response, such as oas2 that is activated by detection of dsrna to inhibit viral replication, ifit2 that inhibits the expression of viral mrnas, and bst2 that limits viral secretion. all the 30 degs identified in this study, except slc1a3, showed an up-regulation in response to the infection in samples from pcw 20 and the pediatric patient (fig. 6c) . ifi44l showed the highest fold change both in pcw 20 and in pediatric samples. this gene was previously found to be a marker of viral infection compared with to bacterial infection 25 and more recently described as a negative modulator of innate immune responses induced after virus infections 26 . type i, ii and iii interferon (ifn) transcripts were not differentially expressed between non-infected and infected rp-gos. we then performed an enrichment analysis within the reactome database to understand the functional implications of the degs up-regulation after the infection in pcw20 and pediatric samples (fig. 6d ). interestingly, the majority of degs fell within pathways associated with the innate response to viral infection, particularly those involved in the regulation of type i ifn alpha/beta by cytoplasmic patternrecognition receptors (prrs) such as rig-i and mda5, and the expression of ifn stimulated genes (isgs). moreover, to capture more subtle differences between non-infected and infected samples that do not emerge in the deg analysis, we performed a quantitative set analysis for gene expression (qusage) 27 within the gene ontology database (supplementary file 2). categories related to the ifn response to the infection were identified in all three sample groups (pcw11, pcw20, and pediatric), other categories were reported for completeness, but require further studies to understand their relevance. reliable in vitro models capable of reproducing complex in vivo systems are becoming increasingly important in life sciences and play a crucial role in the investigation of emerging pathogens of devastating sanitary and economic impact like sars-cov-2. in the context of the covid-19 pandemic, it is still unclear how gastrointestinal virus replication might affect the clinical outcome of infection, the development of immunity and the transmission dynamics in the population. while it has been shown that sars-cov-2 is frequently detected in rectal samples of affected children and adults, it remains to be determined if the virus is able to produce a primary infection throughout the entire gi tract, or if its presence could be related in part to a passive transport of contaminated sputum coming from the upper respiratory tract. most importantly, the ability of sars-cov-2 to persist in the gi tract after respiratory clearance, has not yet been fully elucidated in terms of viral infectivity, possibly impairing important public health and policy measures for the control of the disease. these concerns are particularly relevant in children who appear on average to suffer a less severe respiratory illness compared to adults, despite recording more prominent gi symptoms with clinical pictures mimicking appendicitis 28 , a hyperinflammatory shock syndrome (paediatric multisystem inflammatory syndrome -temporally associated with sars-cov-2, pims-ts) 29 , or acting as relatively asymptomatic carriers of the virus. those risks have prompted clinical guidelines recommending the avoidance of aerosol producing procedures, including upper gi endoscopies, in children with confirmed or suspected covid-19 for the safety of frontline clinical staff and other patients. susceptibility of the different portions of the gi tract to sars-cov-2 infection has not been fully characterized and due to a paucity of autopsy reports targeting the gastric compartment 2 , the capacity of sars-cov-2 to infect the gastric mucosa is still unclear. two recent studies show that the sars-cov-2 receptor angiotensin converting enzyme 2 (ace2) is highly expressed on differentiated enterocytes and that intestinal organoids derived from the small intestine can be easily infected by sars-cov-2 16, 17 . interestingly, intestinal organoids derived from both human and horseshoe bats are fully susceptible to sars-cov-2 infection and sustain robust viral replication. although vertical transmission of sars-cov-2 seems to be anecdotal, it is still unclear if this lack of infection relates to the inability of the virus to migrate through the placenta 30 , to the low susceptibility of the fetal cells to infection, or simply on low viremic loads. whereas human fetal intestinal organoids have already been reported 20 , a reliable 3d culture in vitro model of human gastric mucosa at different developmental stages has been challenging to achieve. in this study, we describe successful derivation of human gastric organoids from both fetal and pediatric samples and we demonstrate that gastric cells are susceptible to sars-cov-2 infection. furthermore, we describe how a reversed-polarity organoid model can help expose the apical domains, in direct contact with the surrounding microenvironment, so that pathogens can easily access surface receptors on the cells. past studies showed laborious pathogen infection in human gastric organoids by microinjection of helicobacter pylori solution into the lumen of each organoid 18, 19 . other studies showed disruption of 3d organoid organization in favor of a 2d monolayer culture to overcome inner polarity problems in h. pylori infection 31 . recently, in sars-cov-2 infection studies, intestinal organoid 3d structures were sheared to expose the apical viral receptors and then reaggregated in ecm hydrogel droplets 16, 17 . in these studies, infection of organoids upon shearing and embedding was attained, as proved by the immunofluorescent staining of viral antigens and detection of viral rna. however, release of the infectious progeny in the culture supernatants differed greatly, ranging from positive titers of around 10 1-2 tissue culture infectious dose 50% (tcid50)/ml 16 to 10 4-5 tcid50/ml 17 . such discrepancy could depend on multiple factors, but we believe that the laborious nature of this approach might increase inter-operator and inter-laboratory variability, resulting in the generation of less-reproducible data. to overcome these complications, we decided to prevent the system perturbation and infect human gastric organoids under steady-state conditions. taking advantage of a polarity reversion study 24 , we generated fetal and pediatric cultures of rp-gos in suspension. in this condition of exposed apical polarity and absence of surrounding matrigel, we could infect organoids and readily titrate the infectivity of the progeny virus, recording infection level comparable to those shown by zhou et al. 17 , taking into account an ffu-to-tcid50 conversion factor of 0.28 (data not shown, from previous validation of assays). interestingly, when we infected gastric organoids through shearing and re-embedding in matrigel, infection was achieved but failed to detect virus in the supernatants, indicating this approach as suboptimal for our purposes. we demonstrated that the rp-gos are fully susceptible to sars-cov-2 infection, with an efficiency of replication that correlates directly with the developmental stage of origin. quantification of gene transcripts coding for the viral receptors ace2 and tmprss2 suggested that the observed levels of replication are not dependent on difference in the density of receptors, given their statistically comparable expression across the three stages. nonetheless, variation in the protein-to-mrna ratio across organoids of different developmental stage should be taken into account and receptivity investigated in future work. immunofluorescence staining for the nucleocapsid indicated a clear cytosolic localization of this protein that in some cells was associated with the presence of the cleaved caspase 3, confirming the occurrence of apoptosis in the gi compartment 16 . apoptosis in infected gi mucosal cells might account at least in part for the frequent abdominal pain, vomit and diarrhea described in covid-19 patients 32 , in particular in pediatric populations. apoptosis is one of the key mechanisms of cells to restrict viral infections by destruction of the cellular machinery indispensable for virus replication; on the other hand, selected viruses have evolved diverse adaptative strategies to control this phenomenon in their favor 33 . to this respect, sars-cov was shown to replicate in vitro to high titers in cells undergoing apoptosis and to low titers in cells where cytopathic effect was limited and a persistent infection was established 34 . interestingly, induction of apoptosis for sars-cov was proved to be caused by a nuclear localization of the nucleocapsid protein that in turn resulted in its cleavage by caspases 6 and 3. the precise mechanism underpinning nucleocapsid cleaving, apoptosis and the replication efficiency of sars-cov remains unexplored. we reckon that similar mechanistic studies are of great interest to decipher the pathology of sars-cov-2 in the gi system and its implications on virus shedding and transmissibility. rp-go of late fetal and pediatric age infected with sars-cov-2 shared a transcriptional footprint surprisingly similar to those described for infected human small intestine enteroids 16, 17 in which type i ifn genes were either poorly expressed or undetectable, despite enterocytes and gastric cells displaying moderate levels of isgs primarily involved in the recognition of viral rna. moreover, our transcriptional data are in considerable agreement with clinical and experimental profiles derived from covid-19 patients, infected normal human bronchial epithelial cells and in vivo studies in ferrets 35 that highlighted a negligible expression of genes of the ifn family but a robust expression of chemokines and isgs. our data provide novel evidence in support of the hypothesis that pathogenesis of covid-19 is at least in part dependent on a reduced innate antiviral response and an unbalanced cytokine production. nevertheless, in our model chemokines were not differentially expressed, whereas in small intestine organoids, zhou et al 17 reported degs coding both chemokine receptors and ligands. since we conducted a bulk rna-seq analysis and the number of infected cells in our organoids were still a minority, we speculate that many processes specific of infected cells, most likely did not reach a statistically significance level and might have gone undetected, hence imposing a cautionary approach in our interpretation of data. interestingly, a large overlap of degs with previous transcriptomic studies of sars-cov infection was found, including the peculiar feature of a limited/absent type i ifn induction and the recruitment of a subset of cytoplasmic prrs. similarly to sars-cov, in which orf3b and orf6 are the main antagonists of ifn, a recent study currently under peerrevision 36 indicates the sars-cov-2 orf3b protein as a potent ifn inhibitor, supporting ours and the published transcriptomic data herein discussed. our gastric organoid system offers a unique tool to characterize the replication of viruses and some of the associated pathological consequences of infection. this innovative model could represent an in vitro scalable platform for the development and testing of antiviral drug candidates targeting the gi system. a deeper understanding of the pathogenic mechanism underpinning the viral colonization of the gi system will potentially expand the available therapeutic options for the inhibition and preventing of gi infection, in an attempt to suppress viral shedding and halt spreading of the disease. the clinical importance of our findings relates to the worrisome phenomenon of prolonged shedding of sars-cov-2 from the gi tract and calls for further research to assess the risk of vertical transmission in infected women. defining the susceptible age and the target anatomical sites will prove of crucial importance for the implementation of sensitive and sustainable diagnostic screening for the identification of contagious asymptomatic patients. human fetal stomachs were dissected from tissue obtained immediately after termination of pregnancy from 8 to 21 pcw (post conception week), in compliance with the bioethics legislation in the uk. human pediatric gastric surgical biopsies were collected after informed consent, in compliance with all relevant ethical regulations for work with human participants, following the guidelines of the licenses 08nd13 and 18ds02. fetal stomachs and pediatric biopsies were collected in ice-cold sterile phosphate buffered solution (pbs -sigma-aldrich) and processed within a few hours of collection. gastric crypt stem cells were isolated from specimens following a well-established dissociation protocol 19 . briefly, fetal stomachs where cut open longitudinally along the lesser curvature, while @ 0.5 cm 2 pediatric biopsies where processed as they were obtained. specimens were cold-washed in a plate with chelating buffer (sterile milli-q water (merck millipore) with 5.6 mmol/l na2hpo4, 8.0 mmol/l kh2po4, 96.2 mmol/l nacl, 1.6 mmol/l kcl, 43.4 mmol/l sucrose, 54.9 mmol/l d-sorbitol, 0.5 mmol/l dl-dithiothreitol, ph 7, all from sigma-aldrich). mucus was removed with a glass coverslip and mucosa was stripped from muscle layer. tissue was cut in small pieces, transferred in a 15 ml tube in new chelating buffer and pipetted repeatedly. supernatant was discarded and 10 ml of 10 mm edta was added and incubated for 10 min at room temperature. edta was discarded and mucosa pieces were washed in ice cold pbs with ca 2+ /mg 2+ (sigma-aldrich). tissue was transferred to a new 10 cm plate on ice and pressure was applied on top with a sterile 3.5 cm plate, to release the crypts from the mucosa. table 2 . cell were passaged every 6-8 days. to passage the organoids, matrigel droplets were disrupted by pipetting in the well and transferred to tubes on ice. cells were washed with 10 ml of cold basal admem+++ and centrifuged at 200 g at 4â°c. (first method) for single cell dissociation, supernatant was discarded, and the pellet resuspended in 1 ml of tryple (thermo fisher) and incubated for 5 min. after incubation organoids were disaggregated by pipetting, and 10 ml of ice-cold admem+++ was added to dilute and inhibit tryple. (second method) for standard organoid passage during expansion, the organoid pellet was resuspended in 1.5 ml of ice-cold admem+++ and organoids were manually disrupted by narrowed (flamed) glass pipette pre-coated in bsa 1% in pbs, to avoid adhesion to the glass. cells were washed, pelleted and supernatant discarded. almost-dry pellets of disaggregated organoids (or single cells) were thoroughly resuspended in cold liquid matrigel, aliquoted in 30 âµl droplets in pre-warmed multi-well plates and incubated at 37â°c for 20 min to form a gel. rho-kinase inhibitor (tocris) was added to single cell dissociated organoids. medium was added and changed every 3 days. fully grown gastric organoids at day 7 after single cell disaggregation were removed from surrounding extracellular matrix using a modified published protocol 24 . matrigel was dissolved with 60 min treatment of the droplets with cell recovery solution (corning) at 4â°c. organoids were retrieved from the plates using 1% bsa-coated cut-end tips and transferred to 1% bsa-coated 15 ml tubes. cells were extensively washed with ice-cold pbs and centrifuged at 200 g for 5 min at 4â°c. supernatant was discarded, the pellet was resuspended in complete medium and transferred to non-tissue culture treated low-adhesive multiwell plates (pre-coated in 1% bsa). organoids were cultured in suspension for 3 days to allow reversion of polarity, before use in infection experiments. for rna isolation from stomach tissues, i) pediatric stomach biopsies consisted of only mucosal layer from surgical samples; ii) late fetal stomachs were cut open and mucosal layer was isolated; iii) early fetal stomachs were processed with no layer isolation, given the small size of the samples. mucus was removed from all the samples with a glass coverslip to prevent rna loss during the isolation protocol, and tissues washed in ice-cold pbs. then the tissues were finely cut with a scalpel on a petri-dish on ice and transferred to 1.5 ml tubes. recovery solution (corning) at 4â°c. cells were then washed in ice cold pbs to remove matrix leftovers that could interfere with rna isolation. organoids were centrifuged at 200 g at 4â°c and supernatant discarded. dry pellets of tissues and organoids were lysed with rlt buffer (qiagen). rna was isolated with rneasy mini kit (qiagen) following manufacturer's instructions. total rna was quantified using the qubit 2.0 fluorimetric assay (thermo fisher scientific). rna reverse transcription was performed using the high-capacity cdna reverse transcription kit (thermo fisher), according to the manufacturer's instructions. reverse transcription was done using the t100 thermal cycler (bio-rad). the qrt-pcr was performed with taqman gene expression assay probes (thermo fisher) according to the manufacturer's instructions. the following probes (all from thermo fisher) were used: gapdh (glyceraldehyde 3-phosphate dehydrogenase), lgr5 (leucine-rich repeat-containing gprotein coupled receptor 5), axin2 (axin-like protein), muc5ac (mucin 5ac), muc6 (mucin 6), pga5 (pepsinogen a5), sst (somatostatin), gast (gastrin), chga (chromogranin a), atp4b (atpase h+/k+ transporting subunit beta). reactions were performed on step one plus real-time pcr system (applied biosystems) and results were analyzed with stepone (version 2.3) software (life technologies). gapdh expression was used to normalize ct values for gene expression, and data were shown as relative fold change to controls (early fetal stage), using â��â��ct method, and presented as mean â± sem. for rna-seq data of original tissues and organoids with spontaneous polarity, total rna (100 ng) from each sample was prepared using quantseq 3' mrna-seq library prep kit (lexogen gmbh) according to manufacturer's instructions. the amplified fragmented cdna of 300 bp in size were sequenced in singleend mode using the nova seq 6000 (illumina) with a read length of 100 bp. illumina novaseq base call (bcl) files were converted into fastq files through bcl2fastq (version v2.20.0.422) following software guide. sequence reads were trimmed using bbduk software (bbmap suite 37.31), following software guide, to remove adapter sequences, poly-a tails and low-quality end bases (regions with average quality below 6). alignment was performed with star 2.6.0a 37 on hg38 reference assembly obtained from cellranger website (ensembl 93), following online site guide. the expression levels of genes were determined with htseq-count 0.9.1 by using cellranger pre-build genes annotations (ensembl assembly 93). all transcripts having <1 cpm in less than 4 samples and percentage of multimap alignment reads > 20% simultaneously were filtered out. for rna-seq data of non-infected and infected rp-gos, a total of 600 pg of rna was used as input for the synthesis of cdna with the smart-seq v4 ultra low input rna kit for sequencing (takara bio usa, mountain view, ca, usa). manufacturer suggested protocol was followed, with minor modifications. 75 pg of dna generated with smart-seq v4 kit were used for preparation of library with nextera xt dna library preparation kit (illumina inc., san diego, ca, usa), following suggested protocol. libraries were sequenced in pair-end mode using a nova seq 6000 sequencing system on an sp, 100 cycles flow cell (illumina inc., san diego, ca, usa). illumina novaseq base call (bcl) files were converted into fastq files through bcl2fastq (version v2.20.0.422) following software guide. alignment was performed with star 2.6.0a34 on hg38 reference assembly obtained from the gencode website (primary assembly v. 32). transcripts estimated counts were determined with rsem 1.3.0 38 by using the gencode v.32 genes annotations. all genes having <1 cpm in less than 2 replicates of the same condition were filtered out. differentially expressed genes (degs) were computed with edger 39 , using a mixed criterion based on p-value, after false discovery rate (fdr) correction by benjamini-hochberg method, lower than 0.05 and absolute log2(fold change) higher than log2(1.5). this analysis was paired between non-infected and infected samples derived from the same original sample. for rna-seq data of organoids with spontaneous polarity, degs were clustered according to a flat, increasing, or decreasing profile according to the differential expression analysis between pairs of time points. principal component analysis was performed by singular value decomposition (svd) on log2(cpm+1) data, after centering, using matlab r2017a (the mathworks). degs over-representation analysis of gene ontology (go) and reactome categories was performed using cluego (version 2.5.4) 40 . reactome hierarchy was visualized using cluego within cytoscape 41 . hierarchical clustering of degs was performed on median-centered log2(cpm+1) data in matlab, using euclidean distance and complete linkage. log-normalized expression data were analyzed by the quantitative set analysis for gene expression (qusage) 27 bioconductor package. vero e6 cells (atccâ® crl 1586â�¢) were maintained in dulbecco's modified eagle's medium (dmem, thermo fisher) supplemented with 10% fetal calf serum (fcs), penicillin (100 u/ml) and streptomycin (100u/ml) (all from thermo fisher) at 37â°c in a humidified 5% co2 incubator. the sars-cov-2 isolate was obtained from a nasopharyngeal swab collected from a 14-year-old boy in italy. briefly, the swab viral transport medium was filtered through a 0.22 âµm filter, serially diluted and incubated onto a confluent layer of vero e6 cells, for 5 days. to ensure purity of the viral isolate, the supernatant of the highest dilution in which cytopathic effect was visible was tested for the presence of 21 human respiratory pathogens including sars-cov-2, using the qiastat-dx respiratory sars-cov-2 panel (qiagen). viral stocks were produced infecting at a multiplicity of infection (moi) of 1 vero e6 cells cultured in dmem supplemented with 2% fcs, penicillin (100 u/ml) and streptomycin (100u/ml) and incubating the cells for 48 hours. supernatants were collected when 80% cells exhibited cytopathic effect and cleared by low-speed centrifugation before being stored at -80â°c. all infections in this paper were performed using the third culture passage of the original isolate. intact organoids were embedded in two 3 âµl-drops of matrigel per well, in 24-well plates. embedded organoids were washed once in dmem and infected at a moi of 0.5 by incubation with 250 âµl of an expansion medium viral suspension for 2 hours. after removal of the inoculum, organoids were washed twice with a dmem solution and 400 âµl of complete medium were added to each well to maintain the culture at 37 â°c with 5% co2. reversed-polarity organoids were infected at a moi of 1 by incubation with 250 âµl of an expansion medium viral suspension for 2 hours. after infection, organoids were washed twice in dmem to remove unbound virus. rp-gos were dispersed in a 400 âµl expansion medium at 37 â°c with 5% co2. for all organoid cultures 50 âµl of supernatant were harvested at 0, 24, 48, and 72 hours post infection. an equal volume of expansion medium replaced the sampled supernatant at each collection time. an extra sample at 96 hours post infection was collected for the rp-gos. samples were stored at -80â°c before titration through the ffa. supernatants of organoid cultures and aliquots of viral stocks were serially diluted and incubated on confluent monolayers of vero e6 cells, in 96-well plates, for 1 hour. culture medium formulation was the same used for virus propagation. after infection, the inoculum was removed and an overlay of mem, 2% fbs, penicillin (100 u/ml) and streptomycin (100u/ml) and 0.8% carboxy methyl cellulose was added. after 27 hours, the overlay medium was removed and cells were fixed in a 4% paraformaldehyde (pfa) phosphate buffered solution (pbs), for 30 minutes at 4â°c. upon removal, cells were permeabilized by incubation with a 0.5 % triton x-100 solution for 10 minutes. immunostaining of infected cells was performed by incubation of the j2 anti-dsrna monoclonal antibody (1:10,000; scicons) for 1 hour, followed by 1-hour incubation with peroxidase-labeled goat anti-mouse antibodies (1:1000; dako) and a 7 min incubation with the true blueâ�¢ (kpl) peroxidase substrate. solution of 1% bovine serum albumin and 0.05% tween-80 in pbs was used for the preparation of working dilutions of immuno-reagents. after each antibody incubation, cells were washed 4 times through a 5 min incubation with a 0.05% tween-80 pbs solution. focus forming units (ffu) were counted after acquisition of pictures at a high resolution of 4800 x 9400dpi, on a flatbed scanner. human gastric tissues were fixed in 4% paraformaldehyde (pfa -sigma-aldrich) for 2 hours and embedded in paraffin wax, then cut at 7 âµm on a microtome. hematoxylin and eosin (h&e) tissue slides were stained according to manufacturer's instructions with hematoxylin and eosin (h&e) (thermo fisher). immunostaining was performed by blocking and permeabilizing the tissue slides with pbs + triton x-100 0.1% with bsa 0.5%. organoid whole-mounts were blocked and permeabilized with pbs + triton x-100 0.5% with bsa 1% for 2 h at room temperature in rotation. primary antibodies were incubated in blocking buffer for 24h at 4â°c in rotation and extensively washed in pbs + triton x-100 0.1%. secondary antibodies were incubated overnight at 4â°c in rotation and extensively washed. slides were mounted in mounting medium, while floating organoids were moved to a glass-bottomed petri dish and blocked with a coverslip on top. the full list of primary and secondary antibodies is presented in supplementary table 3. organoids were imaged in bright field using a zeiss axio observer a1. immunofluorescence images of whole-mount staining and sections were acquired on a confocal microscope zeiss lsm 710. infected organoid immunofluorescence images were acquired on a leica tcs sp5. statistical analyses were performed using the following software: matlab (v. r2017a) for pca, pie plot, bar plot, hierarchical clustering with proteomic and rna-seq data. graphpad prism mac (v. 6.0h) was used with all other graphs and charts. 42 . c) hierarchical clustering of deg genes included in (b), data were median-centered for each pair of non-infected and infected conditions. d) results from an enrichment analysis within reactome database of degs highlighted in (a). symbol size is proportional to number of genes. p-value < 10 -5 . white symbols were not enriched and were added to highlight the hierarchy between categories within reactome structure. virological assessment of hospitalized patients with covid-2019 evidence for gastrointestinal infection of sars-cov-2 prolonged presence of sars-cov-2 viral rna in faecal samples gastrointestinal features in children with covid-19: an observation of varied presentation in eight children aerodynamic analysis of sars-cov-2 in two wuhan hospitals characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa comparative study of the clinical characteristics and epidemiological trend of 244 covid-19 infected children with or without gi symptoms first case of placental infection with sars-cov-2 clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records vertical transmission of coronavirus disease 2019: severe acute respiratory syndrome coronavirus 2 rna on the fetal side of the placenta in pregnancies with coronavirus disease 2019-positive mothers and neonates at birth sars-cov-2 infection in children epidemiology of covid-19 among children in china digestive system is a potential route of covid-19: an analysis of single-cell coexpression pattern of key proteins in viral entry process organoids as an in vitro model of human development and disease sars-cov-2 productively infects human gut enterocytes. science (80-. ) infection of bat and human intestinal organoids by sars-cov-2 modelling human development and disease in pluripotent stem-cell-derived gastric organoids in vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury long-term expansion of functional mouse and human hepatocytes as 3d organoids extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell rna-sequencing controlling epithelial polarity: a human enteroid model for host-pathogen interactions a qpcr expression assay of ifi44l gene differentiates viral from bacterial infections in febrile children novel functions of ifi44l as a feedback regulator of host antiviral responses quantitative set analysis for gene expression: a method to quantify gene set differential expression including gene-gene correlations clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 hyperinflammatory shock in children during covid-19 pandemic does the human placenta express the canonical cell entry mediators for sars-cov-2? a novel human gastric primary cell culture system for modelling helicobacter pylori infection in vitro review article: gastrointestinal features in covid-19 and the possibility of faecal transmission caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis cell type-specific cleavage of nucleocapsid protein by effector caspases during sars coronavirus infection imbalanced host response to sars-cov-2 drives development of covid-19 sars-cov-2 orf3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant sequence analysis star: ultrafast universal rna-seq aligner rsem: accurate transcript quantification from rna-seq data with or without a reference genome edger: a bioconductor package for differential expression analysis of digital gene expression data cluego: a cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks cytoscape: a software environment for integrated models of biomolecular interaction networks interferon-induced transmembrane protein (ifitm3) is upregulated explicitly in sars-cov-2 infected lung epithelial cells dc is founder, shareholder, and consultant of next generation diagnostic srl. all the other authors of the study declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. the authors declare that all data supporting the findings of this study are available within the article, its supplementary information, attached files, and online deposited data (gastric rna-seq data: gse_xxxx, it will be deposited during revision), or from the authors upon reasonable request. key: cord-273182-djb0ozrt authors: díez, josé maría; romero, carolina; vergara-alert, júlia; belló-perez, melissa; rodon, jordi; honrubia, josé manuel; segalés, joaquim; sola, isabel; enjuanes, luis; gajardo, rodrigo title: cross-neutralization activity against sars-cov-2 is present in currently available intravenous immunoglobulins date: 2020-09-09 journal: immunotherapy doi: 10.2217/imt-2020-0220 sha: doc_id: 273182 cord_uid: djb0ozrt background: cross-reactivity against human coronaviruses with flebogamma(®) dif and gamunex(®)-c, two available intravenous immunoglobulins (ivig), has been reported. in this study, these ivig were tested for neutralization activity against severe acute respiratory syndrome coronavirus 2 (sars-cov-2), sars-cov and middle east respiratory syndrome cov (mers-cov). materials & methods: neutralization capacity of lots of ivig manufactured prior to covid-19 pandemic was assessed against these viruses in cell culture. infectivity neutralization was quantified by percent reduction in plaque-forming units and/or cytopathic/cytotoxic methods. results: all ivig preparations showed neutralization of sars-cov-2 isolates. all ivig lots produced neutralization of sars-cov. no ivig preparation showed significant neutralizing activity against mers-cov. conclusion: the tested ivig contain antibodies with significant in vitro cross-neutralization capacity against sars-cov-2 and sars-cov, but not mers-cov. these preparations are currently under evaluation as potential therapies for covid-19. the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which causes the respiratory disease covid-19 was declared a pandemic by the who in march 2020. most infected patients (80%) have mild symptoms. however, about 20% of covid-19 patients can progress to severe pneumonia and to acute respiratory distress syndrome which is associated with multi-organ failure and death [1] . the current critical situation demands an effective and reliable therapy that is immediately available to control the progression of the disease [2] . convalescent plasma or plasma-derived immunoglobulin (ig; either polyvalent ig prepared from healthy donors or hyperimmune ig prepared from donors with high antibody titers against a specific antigen) have been historically used as a readily available therapeutic option in outbreaks of emerging or re-emerging infections [3] . to date, seven human coronaviruses (hcov) have been identified. four of them (hcov-229e, hcov-nl63, hcov-oc43 and hcov-hku1) are globally distributed [4] and are associated with about 15% of common colds, typically causing mild symptoms [5] . in contrast, sars-cov, middle east respiratory syndrome cov (mers-cov), and sars-cov-2 are zoonotic epidemic viruses [6] that can cause severe respiratory infections and fatalities. sars-cov emerged in china in 2002 with the last reported case in 2014. mers-cov emerged in saudi arabia a decade later, in 2012, and led to an outbreak in south korea in 2015. mers-cov still emerges sporadically in humans from its reservoir in camelids [7] [8] [9] . more recently (december 2019), the novel coronavirus sars-cov-2 emerged in china and because of its extraordinary human-to-human transmissibility is currently causing an unprecedented pandemic [10] . coronaviruses share some morphological and functional properties that may be associated with cross-reactive immune responses which may have important therapeutic implications [11] . sars-cov, sars-cov-2 and mers-cov are classified within the family coronaviridae, genus betacoronavirus, subgenera sarbecovirus (sars-cov, sars-cov-2) and merbecovirus (mers-cov). sars-cov-2 has four main structural proteins including spike (s) glycoprotein, small envelope (e) glycoprotein, membrane (m) glycoprotein and nucleocapsid (n) protein [12] . s protein is the main determinant of the coronavirus entry into the host cell and is also the major target of neutralizing antibodies [13, 14] . spikes are formed by trimers of protein s, which is in turn formed by subunit (s1) that mediates the binding to the cell receptor, and a membrane-anchored subunit (s2) that mediates the fusion of the virus with cell membranes [15] . the receptor-binding domain (rbd) is a key functional component within the s1 subunit that is responsible for virus binding to host cell [16] . potent neutralizing antibodies often target rbd. however, the s1 subunit shows a higher variability than s2. antibodies targeting s1 are often virus-specific making s2 a better target for cross-neutralizing antibodies [17, 18] . the amino-acid sequence identity among the s proteins of human betacoronaviruses causing mild (hcov-oc43 and hcov-hku1) and severe (sars-cov, sars-cov-2 and mers-cov) respiratory infections varies between 22 and 33% [14] . however, the s proteins of sars-cov and sars-cov-2 share 77% amino-acid identity [19] . shared protein homologies among coronaviruses can cause cross-reactivity and/or cross-neutralization antigenic responses (i.e., antibodies able to recognize a coronavirus, but that have generated in response to prior infection of other different circulating coronavirues). cross-reactivity has been described among hcovs of the same genus, particularly betacoronaviruses. cross-reactivity between sars-cov, mers-cov and other endemic hcovs has been reported in some studies [20] [21] [22] . however, it is uncertain whether such cross-reacting antibodies among coronaviruses have also the capacity of reducing viral infectivity by a cross-neutralization effect. recently, we reported cross-reactivity in elisa binding assays against antigens of sars-cov, sars-cov-2 and mers-cov with flebogamma r dif 5 and 10% and gamunex r -c, two currently available intravenous igs (ivig) [23] . as a continuation of this study, here we evaluated the neutralization capacity of those same ivig products against these epidemic hcovs. experimental products ivig products used in this study were flebogamma r dif 5% and 10% (instituto grifols s.a., barcelona, spain) and gamunex r -c 10% (grifols therapeutics inc., nc, usa), two highly purified (≥98-99% immunoglobuin g [igg]), unmodified human igs. each product is manufactured from plasma collected from thousands of donors in the usa and/or several european countries. igg concentrations in flebogamma dif products were 50 and 100 mg/ml (5 and 10%) and in gamunex-c, the concentration was 100 mg/ml (10%). to ensure a virus-free product, both ivig manufacturing processes contain dedicated steps with high pathogen clearance capacity, such as solvent/detergent treatment, heat treatment, caprylate treatment and nanofiltration (planova™, asahi kasei, brussels, belgium). the plasma used to manufacture the ivig lots tested was collected from march 2018 to october 2019. six different lots of flebogamma dif and gamunex-c were tested at several dilutions for cross-reactivity against sars-cov, sars-cov-2 and mers-cov by: elisa techniques; and well-established neutralization assays in cell cultures. lots were identified as f1 and f2 for flebogamma 5% dif, f3 and f4 for flebogamma 10% dif and g1 and g2 for gamunex-c. each experiment was performed in duplicate. handling of viruses and cell cultures was carried out at the level 3 biosafety laboratories in the centro nacional de biotecnología -consejo superior de investigaciones científicas (cnb-csic; madrid, spain) and the institut de recerca i tecnologia agroalimentàries -centre de recerca en sanitat animal (irta-cresa; barcelona, spain), following the centers' biohazard safety guidelines and under authorizations #a/es/00/i-8 and #sa-10430-20, respectively. recombinant sars-cov was generated from urbani strain using a previously described reverse genetic technique [24] . two different sars-cov-2 isolates, collected from nasopharyngeal swab from covid-19 patients, were tested: sars-cov-2 mad6 isolated from a 69-year-old male patient from madrid (spain); and sars-cov-2 (accession id epi isl 418268 at gisaid repository: http://gisaid.org) isolated from a an 89-year-old male patient from badalona (spain). both stock viruses were prepared by collecting the supernatant from vero e6 cells, as previously described [25] . recombinant mers-cov was generated using a previously described reverse genetic system [26] from the reference sequence of mers-cov isolated from the index patient emc/2012 (genebank jx869059) [27] . huh7 is a well differentiated human hepatocyte-derived carcinoma cell line, kindly provided by dr l carrasco (centro de biología molecular severo ochoa -consejo superior de investigaciones científicas [cbmso-csic], madrid, spain). huh7 is composed of epithelial-like cells susceptible to infection by mers-cov [28] . vero e6 is a cell line isolated from kidney epithelial cells extracted from an african green monkey. vero e6 is composed of epithelial-like cells susceptible to infection by sars-cov and sars-cov-2 [29] . at cnb-csic, vero e6 cell lines were kindly provided by dr e snjider (university of leiden medical center, the netherlands). both huh7 and vero e6 cell lines were cultured in dulbecco's modified eagle medium (dmem) supplemented with 25 mm hepes buffer, 2 mm l-glutamine (sigma-aldrich, mi, usa), 1% nonessential aminoacids (sigma-aldrich), 10% fetal bovine serum (fbs; biowhittaker, inc., md, usa). in the post infection semisolid medium, the percentage of fbs was reduced to 2%, and diethylaminoethyl (deae)-dextran (sigma-aldrich) was added to a final concentration of 0.08 mg/ml. at irta-cresa, vero e6 cells were obtained from the atcc (atcc crl-1586) and cultured in dmem (lonza, basel, switzerland) supplemented with 5% fbs (euroclone, pero, italy), 100 u/ml penicillin, 100 μg/ml streptomycin and 2 mm glutamine 8 (all from thermofisher scientific, ma, usa). in the post infection medium, the percentage of fbs was 2%. igg elisa testing procedures qualitative determination of igg class antibodies cross-reactivity against antigens of the tested coronaviruses was performed using elisa techniques. ivig samples were serially diluted using the buffer solutions provided in each igg elisa kit. the following kits were used for the qualitative determination of igg class antibodies in the experimental ivig lots: sars coronavirus igg elisa kit (creative diagnostics, ny, usa), against virus lysate; human anti-sars-cov-2 virus spike 1 [s1] igg elisa kit (alpha diagnostic intl. inc., tx, usa), against s1 subunit spike protein; rv-402100-1, human anti-mers-np igg elisa kit (alpha diagnostic intl inc.), against n protein; rv-402400-1, human anti-mers-rbd igg elisa kit (alpha diagnostic intl inc.), against rbd of s1 subunit spike protein (s1/rbd); rv-402300-1, human anti-mers-s2 igg elisa kit (alpha diagnostic intl. inc.), against s2 subunit spike protein; rv-405200 (formerly rv-404100-1). in all cases, the determinations were carried out following the manufacturer's instructions. reactivity was rated as negative if no reaction was observed with neat ivig or positive if the lowest ivig dilution demonstrated reactivity. this neutralization assay was based on the reduction in plaque forming units (pfu) after exposing a given amount of virus to the product to be characterized and comparing with the untreated control. this assay is performed in cell cultured plates with a semisolid ovarlay to allow plaque formation. for this assay, ivig samples were serially diluted (factor 10 dilutions: 1:10 2 , 1:10 3 , 1:10 4 and 1:10 5 ) in dulbecco's phosphate-buffered saline (gibco, thermo fisher scientific, ma, usa). samples of each ivig dilution were incubated for 1 h (37 • c; 5% co 2 ) with 300 pfus of sars-cov, sars-cov-2 or mers-cov. aliquots of 50 μl of each ivig dilution-virus complex were added in duplicate to confluent monolayers of vero e6 cells (for sars-cov and sars-cov-2) or huh7 (for mers-cov), seeded in 12-well plates and incubated for 1 h (37 • c; 5% co 2 ). after this adsorption time, the igg-virus complex inoculum was removed and a semi-solid overlay was added (dmem 2% fbs + 0.6% agarose). cells were incubated for 72 h at 37 • c. the semi-solid medium was removed, the cells were fixed with 10% neutral buffered formaldehyde (sigma-aldrich) for 1 h at room temperature, and stained with 0.2% aqueous gentian violet for 10 min, followed by plaque counting. the sensitivity threshold of the technique was 20 pfu per ml. the neutralization potency of the ivig products was expressed in two ways: percent reduction in pfu calculated from the pfu count after neutralization by ivig relative to initial pfu count inoculated onto the cells; and plaque reduction neutralization test (prnt 50 ) value, calculated as the -log 10 of the reciprocal of the highest ivig dilution to reduce the number of plaques by 50% compared with the number of plaques without ivig. neutralization assay for sars-cov-2 (epi isl 418268 isolate) this neutralization assay measured the cytopathic/cytotoxic virus-induced effect by detecting cellular enzymatic activity after incubation with a given amount of the relevant virus and comparing this with the relevant untreated control. for this assay, a fixed concentration of a sars-cov-2 stock (10 1.8 tcid 50 /ml, a concentration that achieves 50% cytopathic effect) was mixed with decreasing concentrations of the ivig samples (range 1:10 to 1:5120), each mixture was incubated for 1 h at 37 • c, and added to vero e6 cells. to assess potential plasma-induced cytotoxicity, vero e6 cells were also cultured with the same decreasing concentrations of plasma in the absence of sars-cov-2. uninfected cells and untreated virus-infected cells were used as negative and positive infection controls, respectively (see supplementary figure 1 ). plasma from a covid-19 positive patient with a high half-maximal inhibitory concentration (ic 50 ) was included as an active positive control (expressed as the -log 10 of the reciprocal of the dilution). all the cultures were incubated at 37 • c and 5% co 2 for 3 days. cytopathic or cytotoxic effects of the virus or plasma samples were measured at 3 days post-infection, using the cell titer-glo luminescent cell viability assay (promega, wi, usa). luminescence was measured in a fluoroskan ascent fl luminometer (thermo fisher scientific). neutralization curves are shown as nonlinear regressions. ic 50 values were determined from the fitted curves as the plasma dilutions that produced 50% neutralization. details of the technique are available elsewhere [25] . cross-reactivity studies (elisa-binding assays) ivig products showed consistent reactivity to antigens of sars-cov (culture lysate) at 10-100 mg/ml igg, sars-cov-2 (s1 subunit protein) at 100 μg/ml igg and mers-cov (n protein, s1 subunit/rhd protein and s2 subunit protein) at 50 μg/ml igg (table 1) . all the assayed ivig preparations had neutralizing activity against sars-cov ranging from 39 to 61% (figure 1 ). all 10% igg ivig preparations (f3, f4, g1 and g2) showed prnt 50 neutralization titers between 2.0 and 3.3, corresponding to 50-61% pfu reduction ( figure 1b & c) . the highest pfu reductions, 59.3 and 61.9% (prnt 50 neutralization titers of 3.2 and 3.3), were observed with lots f4 and g1, respectively, at 1 and 0.1 mg/ml igg (dilution factors 2 and 3). the f1 and f2 lots, (5% igg) showed a lower neutralization capacity with pfu reductions of 39.5 and 43.3%, respectively ( figure 1a ). for sars-cov-2 mad6 isolate, all ivig lots, except f1 (inconclusive results) showed a significant neutralizing activity and reached prnt 50 titers ranging from 4.5 to >5 (figure 2 ). pfu reductions ranging from 78.2 to 82.5% were observed with lots f2, f3 and f4 at a dilution factor of one. even at the highest dilution factor (5 = 0.5 and 1 μg/ml), the pfu reduction ranged from 38.5 to 50.9% corresponding to prnt 50 titers of 4.5-5.0 (figure 2a & b) . for lots g1 and g2, the pfu reduction was even higher, ranging from 88.5 to 89.5% at a dilution factor of one to 61.7-62.5% at a dilution factor of five with prnt 50 titers greater than five ( figure 2c ). for the sars-cov-2 epi isl 418268 isolate, f4 and g1 lots neutralized 58.4 and 64.7%, respectively, tcid 50 counts at a dilution factor of one (figure 3 ). one replicate of f4 product failed to demonstrate neutralization. no ivig lot showed any significant pfu reduction (i.e., >10%) on mers-cov even at the lowest dilution factor (10 mg/ml igg). the results presented here demonstrate for the first time significant cross-neutralization activity against sars-cov and especially sars-cov-2 in two therapeutic ivig concentrates (flebogamma r dif and gamunex r -c). this neutralizing activity correlates with the cross-reactivity to different coronavirus antigens observed in elisa-binding assays with ivig, as shown in a previous study [23] . the plasma used to manufacture the tested ivig lots was collected prior the detection of sars-cov-2 in europe and the usa. therefore, these results should be ascribed to cross-reactivity against sars-cov-2 by antibodies against endemic hcovs in the human population at large. similar results have been reported for sars-cov and mers-cov [20] [21] [22] . ivig are polyclonal igg antibodies reacting to a broad range of different antigens. antibody titers and specificities may vary slightly among different lots and manufacturers, depending on the plasma donor population [30] . our neutralization studies showed that the studied ivig products contain antibodies with cross-neutralizing capacity against sars-cov (40-60%) and sars-cov-2 (80-90%), but not against mers-cov (<10%). these results suggest that the cross-neutralizing antibodies target antigenic regions more conserved in sars-cov and sars-cov-2 than in mers-cov. no significant differences in the neutralizing capacity were observed among ivig lots regardless the country of origin for the plasma. this reinforces the broad applicability of these results. two different neutralization techniques were used for sars-cov-2 and both techniques showed not only the ivig neutralization capacity, but also the reliability of the results. in addition, results obtained with two different sars-cov-2 isolates confirm that the neutralization capacity is not dependent on the isolate. this was not unexpected since no significant sequence differences have been observed among sars-cov-2 isolates currently circulating throughout the world. the percentage of sars-cov-2 cross-neutralization was higher in the pfu reduction technique than in the cytopathic effect/cytotoxic technique with very low or negative values in some few cases (inconclusive for lot f1 by the pfu study and cytopathic effect in one replicate of lot f4). this suggests that the technique used and/or slight variations in methodology may significantly influence the nature or magnitude of the results. therefore, further evaluation this cross-neutralizing activity should be carried out. cross-neutralization is gaining attention as a protective mechanism against viral infection in the context of the covid-19 health emergency. the results of this study are in agreement with recent studies that describe cross-neutralization of sars-cov-2 by monoclonal antibodies from memory b cells of an individual who was infected with sars-cov [31] . furthermore, sars-cov-2-reactive cd4 + t cells have been detected in around half of unexposed individuals, suggesting that there is cross-reactive t-cell recognition between circulating common cold coronaviruses and sars-cov-2 [32] . however, the levels of cross-neutralizing antibodies against sars-cov-2 in the sera of sars-cov patients can be highly variable [33] . ivig products are prepared using plasma from thousands of different donors, hence containing a broad representation of the state of immunity in the population at that time. this is consistent with the low rate of variability found among the different lots of ivig products tested. nevertheless, greater variability is expected among individuals with respect to infection by a given endemic human coronavirus. therefore, it has been hypothesized that the diversity of symptoms observed in sars-cov-2infected individuals and even the potential for getting infected may depend on pre-existing cross-immunity due to previous exposure to other endemic hcovs. in this regard, a detailed study of the state of immunity in the general population distinguishing those affected and not affected by the sars-cov-2 may be warranted. the higher cross-neutralizing capacity of the tested ivig preparations against sars-cov and sars-cov-2 than mers-cov may be explained by higher sequence identity of the s proteins of circulating mild hcovs (hcov-oc43 and hcov-hku1) with sars-cov and sars-cov-2 compared with mers-cov (32-33% vs [23] [24] [25] [19, 34] . additionally, differences in specific domains of the s protein between sars-cov and sars-cov-2 might explain higher cross-reactivity of the tested ivig against sars-cov-2 compared with sars-cov (80-90% vs 40-60%). the absence of cross-neutralization against mers-cov despite the cross-reactivity observed in elisa assays suggests that these antibodies are not neutralizing. however, this does not necessarily indicate that such antibodies are not functional by another mechanism. for example, these non-neutralizing antibodies could be labeling the virion for identification by immune cells and subsequent destruction [35] . despite the limitations of the in vitro nature of this study, the clinical implications of the findings are encouraging, and the results may support the use of ivig as a therapeutic option for covid19 . in vitro neutralization studies should be deemed as a partial characterization of a more complex response that can take place in vivo where the host's response mechanisms can include antibody dependent cellular phagocytosis, antibody-dependent cellular cytotoxicity [36] , as well as viral mechanisms such as antibody-dependent enhancement [37] . nevertheless, positive results with the administration of ivig (immunomodulatory dose) to counteract hyper inflammation in patients with severe covid-19 [38] have already been reported in case studies [39, 40] . ivig use is being tested in an ongoing clinical trial [41] . further studies looking at the functionality of these antibodies could improve our understanding the human coronavirus acquired immunity. this could pave the way for ivig (and other igg products such as intramuscular or subcutaneous preparations) as a potential therapeutic/prophylactic approach to fight current and future epidemics due to emerging hcovs. under the experimental conditions of this study, flebogamma r dif and gamunex r -c ivig contained antibodies with significant neutralization capacity against sars-cov and sars-cov-2, but not against mers-cov. additional research is warranted to advance ivig toward clinical use for covid-19. • intravenous immunoglobulin products were tested against severe acute respiratory syndrome coronavirus 2 in cell culture neutralization assays. • for plaque forming unit method, viral neutralization ranged from 79 to 89.5%; prnt 50 titers ranged from 4.5 to >5. • for cytopathic method, viral neutralization ranged from 47 to 64.7%; ic 50 was around 1. • there was also neutralization of sars-cov, ranging from 39.5 to 55.1%; prnt 50 ; 2.0-3.3. • results support current trials assessing intravenous immunoglobulin as potential therapy for covid-19. any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no outsourced writing assistance was utilized in the production of this manuscript. this work is licensed under the attribution-noncommercial-noderivatives 4.0 unported license. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ strategies for the prevention and management of coronavirus disease 2019 sars-cov-2 causing pneumonia-associated respiratory disorder (covid-19): diagnostic and proposed therapeutic options use of human immunoglobulins as an anti-infective treatment: the experience so far and their possible re-emerging role •• a comprehensive review on the role of intravenous immunoglobulin (ivig) as anti-infective treatment in community and emerging diseases epidemiology, genetic recombination, and pathogenesis of coronaviruses update on human rhinovirus and coronavirus infections the 2019 novel coronavirus disease (covid-19) pandemic: a zoonotic prospective. asian pac sars: prognosis, outcome and sequelae sars-cov-2/covid-19: viral genomics, epidemiology, vaccines, and therapeutic interventions middle east respiratory syndrome european centre for disease prevention and control. covid-19. 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profiling early humoral response to diagnose novel coronavirus disease (covid-19) protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts antibody-dependent cellular phagocytosis in antiviral immune responses molecular mechanism for antibody-dependent enhancement of coronavirus entry covid-19: consider cytokine storm syndromes and immunosuppression high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease • the first clinical study to evaluate the efficacy of ivig in the treatment of severely ill covid-19 patients effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with covid-19 key: cord-283956-zgrtux7i authors: amin, sk. abdul; jha, tarun title: fight against novel coronavirus: a perspective of medicinal chemists date: 2020-06-12 journal: eur j med chem doi: 10.1016/j.ejmech.2020.112559 sha: doc_id: 283956 cord_uid: zgrtux7i the ongoing novel coronavirus disease (covid-19) pandemic makes us painfully perceive that our bullet shells are blank so far for fighting against severe human coronavirus (hcov). in spite of vast research work, it is crystal clear that the evident does not warrant the commercial blossoming of anti-hcov drugs. in this circumstance, drug repurposing and/or screening of databases are the only fastest option. this study is an initiative to recapitulate the medicinal chemistry of severe acute respiratory syndrome (sars)-cov-2 (sars-cov-2). the aim is to present an exquisite delineation of the current research from the perspective of a medicinal chemist to allow the rapid development of anti-sars-cov-2 agents. coronaviruses (covs) typically cause mild to severe respiratory and intestinal infections in mammals, including humans [1] [2] . it belongs to the family coronaviridae which comes under the order nidovirales [3] [4] . covs are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. the first two genera (i.e., alpha-and betacoronavirus mainly infect mammals, whereas, gammacoronaviruses and deltacoronaviruses foul avian species. more than 60 years have passed since the identification of first human cov (hcov) was documented as a respiratory tract modulator [5] [6] . in december 2019, several clusters (epidemiologically associated with a seafood and animal market in wuhan, china) of patients suffering fever, illness, severe respiratory tract infections and pneumonia of unknown origin were reported [7] [8] [9] [10] [11] [12] . this finally leaded to the isolation of a novel coronavirus (2019-ncov) and the disease recently named as covid-19. world health organization (who) already characterized covid-19 as world pandemic [13] . this infection has spread over to 216 countries and territories [14] . before covid-19 outbreak, there were six species of hcovs that were reported for their association with respiratory tract infections ( table 1) . [ table 1 [15] . the seventh strain of hcov is novel coronavirus (2019-ncov aka sars-cov-2) which is taxonomically belongs to the betacoronavirus genre and possesses high nucleotide sequence similarity with sars-cov and mers-cov [16] [17] [18] [19] [20] . sars-cov-2 is a positive-sense single-stranded rna viruses surrounded by an envelope (figure 1 ). sars-cov-2, about 30,000 bp single-stranded rna virus, utilizes host cellular components to accomplish its physiological affairs such as viral entry, the assembly and budding of virions, genomic replication, and protein synthesis, subsequently executes pathological damage to the host [21] [22] [23] . thus, punctuating any juncture of viral life cycle by small molecules, peptides, vaccines or physical elements may potentially gain therapeutic benefit to host. depending on several viral targets (figure 1 ) related to the stages of viral life cycle, novel anti-viral agents may be designed and discovered. nonetheless, different structure-based modeling techniques and numerous ligandbased computational techniques may be fruitful strategy to design newer inhibitors against sars-cov-2 [24] [25] [26] . meanwhile, the hefty menace posed by current outbreak of covid-19, it is obvious that the scientific community is looking for effective drugs within plausible time. the coherent development and well organised strategies remains the only hope to triumph the battle against partially known sars-cov-2. now, repurposing of existing anti-viral drugs based on previous ground work of closely related coronavirus and rapid screening of drug databases is one of the strategic and economic ways to eradicate covid-19 pandemic [27] [28] [29] . the traditional bioinformatics and chemo-informatics approaches readily generated new data into sars-cov-2 research at an explosive pace. considering the severity of the spread of covid-19, this study is in-line with the concept of finding the chemo-types to expedite the process of anti-hcov drug discovery. here, an exquisite picture of the recent research including target-based and biological screening is provided. we includes virtual (in silico) as well as experimental (in vitro) screening approaches in response to sars-cov-2 reported until april 2020. the main aim is to provide the scientific community with an overview of the medicinal chemistry of sars-cov-2 to allow the rapid development of antiviral agents. this work, a part of our rational drug design and discovery [30] [31] [32] [33] [34] [35] [36] , is an initiative to pave the way of anti-sars-cov-2 drug discovery paradigm that could help to facilitate the global efforts to fight against covid-19. the details structural biology of the sars-cov-2 virus is yet to be discovered. it contains a 30,000 bp, single-stranded positive sense rna genome encapsulated within a membrane envelope ( figure 1 ) [37] [38] [39] . it recruits multi-subunit replication machinery [40] . the genome of sars-cov-2 comprises about 30,000 nucleotides with ten open reading frames (orfs). the 3′ terminal regions encodes for several structural proteins including spike (s), membrane envelope (e) and nucleocapsid (n) proteins (figure 1) whereas, the 5′ terminal orf1ab responsible for two viral replicase polyproteins pp1a and pp1b. upon proteolytic cleavage of these two viral replicase polyproteins fabricate sixteen non-structural proteins (nsp) (figure 2 ) [37] . [ table 2 may be placed here] the spike glycoprotein of coronavirus is the main conciliator of entry into the host cells [45] [46] [47] [48] [49] [50] [51] [52] . this spike glycoprotein contains (i) a large ecto-domain, (ii) a single-pass transmembrane anchor, and (iii) a short c-terminal intracellular tail [44] . the ecto-domain consists of a receptor-binding unit s1 and a membrane-fusion s2 stalk. basically, the receptor-binding unit s1 binds to a specific cell surface receptor via its receptor binding domain (rbd), whereas the trimeric s2 fuses the viral membranes and host cell to enable the entry of viral genomes into host cells (figure 2 ). researchers already identified angiotensin converting enzyme 2 (ace2) as a functional receptor for sars-cov [48] [49] [50] [51] . the crowned shaped spike glycoprotein of covs binds directly to ace2 on the host cells surface and plays critically in virus infection. ace2 is expressed widely with conserved primary structures throughout the animal kingdom. ace2 from fish, amphibians, reptiles, birds, to mammals can potentially interact with rbd of sars-cov-2 [44]. therefore, blocking of the rbd and ace2 interaction is an obvious therapeutic intervention to treat diseases caused by covs. specific antibodies or small molecular inhibitors can disrupt the interaction of rbd with ace2. among the set of sixteen non-structural proteins, nsp5 is identified to play a pivotal role in the life cycle of sars-cov-2 replication as well as maturation (figure 2) . being a key component, the nsp5 is termed as main protease (mpro). like other rna viruses, the functional significance of this mpro or chymotrypsin-like protease (3clpro) of sars-cov-2 emerges as an attractive drug target for the development of anti-viral agents. recently, the 3d structure of 3clpro of sars-cov-2 was reported [1] . like other coronaviruses mpro, it also consists of three domains. the domain i (comprising of 8-101 amino acids) and ii rdrp active site is appointing two successive aspartate residues projected from a beta-turn structure making them surface accessible through the nucleotide channel. as configured to sars-cov, the robson [54] performed a preliminary bioinformatics studies to propose a synthetic vaccine and peptidomimetic antagonist against the spike glycoprotein of sars-cov-2. the author employed q-uel language to perform the bioinformatics approach. krsfiedllfnkv was identified as a well conserved sequence motif that corresponds to the known cleavage sites of the sars virus. this sequence motif formed the basis for design of specific synthetic vaccine epitope and peptidomimetic agent [54] . a group of scientists from the cairo university, egypt predicted covid-19 spike binding site to a cell-surface receptor namely glucose regulated protein 78 (grp78) by employing structural bioinformatics in combination with protein-protein docking [55] . notably, the region iv was supposed to be a pivotal driving force for grp78 binding (predicted binding affinity: -9.8 kcal/mol). prediction of this binding site sheds light on the mode of envelope protein recognition by the grp78 substrate-binding domain for future endeavours [55] . in a molecular modeling study to explore potential inhibitors of rna binding to n terminal domain (ntd) of nucleocapsid protein (n protein), sarma et al [56] pointed out two potential hits namely zinc000003118440 and zinc000000146942 (figure 3 ). the authors employed two ntd structures of n proteins namely 2ofz and 1ssk. firstly, a set of diverse compounds from asinex and maybridge library were docked. then 15 compounds for each of the targets were prioritized with significant docking scores. further mm-gbsa binding free energy, pharmacokinetic properties (qikprop), drug-likeness (swissadme) and molecular dynamics (md) studies were performed to screen the compounds. out of these two potential hits, one compound was a theophylline derivative. since theophylline derivative is commonly used as a bronchodilator, hence, the author further screened approved bronchodilators against the n protein 8 rna binding site of covid-19 [56] . the approved bronchodilators showed mm-gbsa binding affinity in the following order: formeterol> terbutaline > ipratropium bromide >tiotropium bromide > theophylline > salbutamol. recently, native or non-native protein-protein interactions (ppis) are emerged as a target for structure-based screening of small molecule. it may be an alternative drug design paradigm which could accelerate anti-viral drug discovery against various pathogens [57] [58] [59] . since the orthostatic/allosteric stabilization of non-native ppis of sars-cov-2 nucleocapsid protein results abnormal protein oligomerizaion and finally leading to loss of viral activity. scientists from national chung hsing university, taiwan acclaimed non-native ppis of n-terminal domain of the mers-cov nucleocapsid protein (mers-cov n-ntd) [60] . they reported a crystal structure of mers-cov n-ntd in a non-native dimeric configuration which turned a target for virtual screening of orthosteric stabilizers from acros and zinc drug databases. this finding provides valuable insight and further motivations into the design of new anti-virals based on stabilizing a non-native protein interaction interface of n protein [60] . gupta and co-workers [61] employed computational techniques to explore the best possible structure of the sars-cov2 e protein present in the pdb database. the author reported that e protein of sars-cov-2 is a pentameric protein comprised of 35 α-helices and 40 loops. near about 8,000 compounds were docked whereas 700 compounds were prioritized with significant affinities or docking scores. the docking study with e protein of sars-cov-2 and phytochemicals like belachinal, macaflavanone e &vibsanol divulged that amino acids such as v25 and f26 play crucially in the binding interactions. the functional behaviour of e protein of sars-cov-2 after 200ns molecular dynamics studies revealed that α-helix and loops of e protein escapades random movement and modulates normal ion channel activity to succour the pathogenesis in human and other vertebrates. unlikely the random manoeuvre of the e protein of sars-cov-2 gets reduced after binding with inhibitors [61] . khan et al [62] pinpointed three fda-approved drugs such as remdesivir, saquinavir and darunavir) and two small molecules (flavone and coumarin derivatives) as possible inhibitors of 3clpro by target-based virtual screening. a number of 8,000 compounds were docked and top 700 primary hits were distinguished by significant affinities or docking scores. then the binding interaction between the active site residue of 3clpro and the selected compounds were extensively scrutinized using the plif module in moe. further, md simulation and binding free energy calculations were recorded to evaluate the dynamic behaviour, stability of protein-ligand contact, and binding affinity of the hit compounds [62] . kandeel and al-nazawi [63] reported statistics of pairwise sequence comparison matrix among the main protease (mpro) of covs. a high pairwise sequence alignment identity (96.08%) was found between sars-cov-2 and sars-cov-1 mpro, whereas only 51.61% identity was exposed for sars-cov-2 and mers-cov mpro. in consequence, the number of amino acid differences between sars-cov-2 and sars-cov-1 mpro was only 12, while it was 153 between sars-cov-2 and mers-cov mpro. an early virtual screening (vs) study of fda approved drugs (retrieved from selleckchem inc.) against the first resolved sars-cov-2 mpro crystal structure (pdb: 6lu7) was performed. that helps the repurposing of already approved drugs to eradicate covid-19 [63] . detailed scanning of the binding mode of these drugs with sars-cov-2 mpro conferred that hydrophobic and hydrogen bonding interactions were the main imperators for binding. interestingly, telbivudine (figure 3) , an anti-hepatitis b virus agent, bind with the sars-cov-2 mpro through hydrogen bond interactions with amino acid residues s49 and q189. moreover, a broad spectrum antiviral agent, ribavirin (figure 3) , interacted with the sars-cov-2 mpro by forming hydrogen bonds with side chain amino acid residue q189 and the backbone amino acid residue t25. ribavirin is officially approved against respiratory syncytial virus (rsv) infection. it is also used in combination with interferon α2b against hepatitis c virus. moreover, it also exhibited potency against sars-cov infection [63] . in a study to distinguish potential active herbs against 2019-ncov, ma et al. leading to viral eradication [52] . in another study, elfiky [67] reported sars-cov-2 rdrp targeted molecular docking study of some anti-polymerase drugs which have been approved for use against various viruses. not surprisingly, ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovirexhibited promising binding affinity against sars-cov-2 rdrp. these results were also consistence with previous one [52] . guanosine derivative (idx-184), setrobuvir, yak has potential to block the sarscov-2 strain. since these drugs have already passed the adme and toxicity measurements these may be used as a new therapeutic drug candidate against sars-cov-2. lung and co-workers [68] identified theaflavin, a polyphenolic constituent present in black tea, hydrophobic interactions were found to be the driving force in binding. theaflavin formed hydrogen bonds with d452, r553 and r624 of sars-cov-2 rdrp. additionally, a π-cation interaction was found with r553 [68] . calligari et al [69] autodockvina scoring −10.0 kcal/mol) was recognised as the best autodock vina scoring drug. in spite of hcv main protease measure very low identity with the sars-cov-2 homolog (only 7.5%), thus this finding was thunderbolt [69] . therefore, it may be inferred that the similar topology of active site of both proteases was the main driving force in binding of simeprevir. it fitted well into the two hydrophobic pockets fringed the catalytic dyad h41-c145 of sars-cov-2 mpro. it was also fitted into the hydrophobic loop f181-f185. moreover, the binding of simeprevir to sars-cov-2 protease was anchored by three hydrogen bonding interactions with e166, g143 and n142 [69] . in the same article, the homology modelling of sars-cov-2 s protein by the aid of itasser server was done by using sars-cov spike (pdb: 5wrg, 5 × 58, 5xlr) as a template. global pairwise sequence alignment measured that sars-cov-2 s protein shares about 76% of its primary sequence with its homolog in addition an overall similarity of 87%. autodock vina molecular docking of the homology modelled structure of s protein predicted umifenovir (db13609), enfuvirtide (db00109), and pleconaril (db05105) as potential inhibitors [69] . in a study to repurposing existing drugs against current pandemic covid-19, wu et al [70] predicted some potential drugs acting on a certain target or multiple targets of sars-cov-2. bioinformatics based homology modelling was utilised to build possible targets such as viral mpro, plpro, rdrp, helicase, spike, etc. next, these modelled proteins and human relative proteins including human ace2 and type-ii transmembrane serine protease enzymes were forwarded to systematically analyse and screen zinc drug database (zdd) along with database of traditional chinese medicine and natural products and the database of commonly used 78 anti-viral drugs [70] . this study seems to be very interesting due to its deep discussions and vast target predictability. the lead candidates emerged from this study required in vitro and in vivo studies for further conformations. the new drug discovery and development takes more than five to ten years of investigations as well as cost billions of dollars. thus, drug repositioning is the only cheap strategy to respond immediately. fda-approved drugs justify safe alternatives if at least modest anti-sars-cov-2 activity can be achieved. currently, academia and industry personnel are involved in the testing of -(i) approved drugs and/or (ii) drug candidates in clinical trials. the in vitro screenings of fda approved drugs as well as the compounds which are currently under clinical trials phases were well documented [71] [72] [73] [74] [75] [76] [77] and the need for further in vivo testing to facilitate drug discovery efforts against sars-cov-2. nafamostat ( figure 5) is a potent membrane fusion inhibitor of mers-cov [78] . now, it has been found to inhibit the 2019-ncov infection (ec 50 besides, this is agent marketed as an anti-protozoal drug [79] and also reported to possess antiviral against a broad range of viruses [80] . in the same article, wang et al [72] remdesivir have been illustrated few years ago [81] . now, it has been found to effectively block sars-cov-2 infection at low-micromolar concentration [72] . it exhibited half-maximal effective concentration (ec 50 ) of 0.77 μm against 2019-ncov in vero e6 cells with half cytotoxic concentration (cc 50 ) > 100 μm; selectivity index (si) > 129.87). remdesivir also inhibited virus infection in sars-cov-2 sensitive human liver cancer huh-7 cells [72] . a widely-used anti-malarial drug chloroquine (cq, figure 6 ) has been used for more than 70 years [82] , now, found to shows clinical potency against covid-19. [ figure 6 may be placed here] the molecular mechanism of chloroquine against sars-cov is known [83] [84] . very recently, wang et al [72] reported time-of-addition assay that explained the function of cq (ec 50 = 1.13 μm; cc 50 > 100 μm; si > 88.50) at both entry as well as at post-entry stages of the novel corona virus infection in vero e6 cells. the same group of researches further evaluated the in vitro anti-sars-cov-2 effect of hydroxychloroquine (hcq, figure 6 ) sulphate, a derivative of cq, in comparison to cq [73] . hydroxychloroquine sulphate [85] was introduced long before, first synthesized in 1946 [73] . upon introduction of a hydroxyl group into cq resulted in about 40% less toxic agent than cq in animals. both cq and hcq are weak bases and restrict the virus infection by-(i) triggering endosomal ph which is essential for virus/cell fusion and (ii) interfering with the glycosylation of ace2 receptor and spike protein of coronavirus [73] . additionally, both cq and hcq obstructed the sars-cov-2 transport from early endosomes (ees) to endolysosomes (els). interestingly, it exhibited very distinct binding mode than the other covalent or peptidomimetic 3clpro inhibitors [43] . in vitro screening of 48 drugs was screened against hcovs infection [71] . immunofluorescence analysis with an antibody specific for the viral n protein of sars-cov-2 was scored for each drug treated cells. the dose-response curve (drc) was developed after analysing the confocal microscope images of both viral n protein and cell nuclei. remdesivir (sars-cov-2 ic 50 = 11.41 µm), lopinavir (sars-cov-2 ic 50 = 9.12 µm) and chloroquine (sars-cov-2 ic 50 = 7.28 µm) were used as reference drugs. consequently, 24 drugs showed good activities with ic 50 ranges of 0.1 to 10 μm. an anti-helminthic drug, niclosamide, and a corticosteroid used to treat asthma and allergic rhinitis, ciclesonide, emerged as sars-cov-2 inhibitors with ic 50 of 0.28 μm and 4.33 μm, respectively. notably, niclosamide reduces mers-cov replication by inhibiting skp2 activity leading to enhancement in autophagy [86] . thus, a similar mechanism may be introduced by niclosamide to hamper sars-cov-2 infection [71] . zhang et al [87] reported structure-based design, synthesis and activity assessment of α-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication. these showed good inhibitory properties against the isolated proteases, viral replicons, virus-infected huh7 cells. nearequipotency against the enteroviruses, alphacoronaviruses, and betacoronaviruses was observed upon optimization of the p2 substituent of the α-ketoamides. the cyclopentylmethyl and cyclohexylmethyl at the p2 substituent disposed low-micromolar ec 50 values against the three virus genera in cell cultures. compound 11r was found promising against mers-cov in virus-infected huh7 cells [87] . by dint of high similarity among the 3clproteases of coronavirus, it is awaited that compound 11r is expected to display good anti-viral activity against covid-19 in near future. meanwhile, the clinical trial studies of some molecules have been started against covid-19 mainly through drug repurposing [88] ; those are depicted in table 2 . the medicinal chemistry of sars-cov-2 infection is still in its infancy, with target specific lead molecules are yet to identify. this study deals with the information currently available on potential targets for therapeutic invention and screening of new compounds or drug repurposing against sars-cov-2 (figure 8 ). as the 3d structure of the sars-cov-2 3c-like protease bears 96% identity with its ortholog from (sars-cov). interestingly, the residues involved in the catalysis, substrate binding and dimerization of 3clpro are 100% conserved. in addition, the polyprotein pp1ab sequences are highly similar (86% identity). depending upon the alike substrate specificities and high identities, we are of the opinion that the previous progress of specific sars-cov inhibitors development can undertaking a course of action on the design and discovery of inhibitors against sars-cov-2. our group have already explored the structural properties important for sars-cov viral 3clike protease inhibitors [30] . recently in a collaborative work, our research team suggested the implications of naphthyl derivatives against sars-cov-2 plpro enzyme though in-depth ligandreceptor interaction analysis [89] . we have already predicted some in-house glutamine-based molecules to use as a seed for drug design and optimization against plpro of sars-cov-2 [90]. in fact, some other anti-viral drugs can also be taken into consideration. in this regards, target-based vs is one of the most important approaches used for drug repurposing. the computational analyses are not subordinate but are a right choice to enrich the basal knowledge during the long process leading to drug development (figure 8) . until any clear-cut treatment approach is prescribed for covid-19, the use of already approved drugs is only alternative strategy. howbeit, relatively limited computational resource or biased in silico screening may scattered the linearity of drug discovery of novel coronavirus. in the near future, the virtual hits may serve as a promising drug like molecule against sars-cov-2 after details in vitro and in vivo laboratory investigations. moreover, the availability of x-ray crystal structures of the important viral proteins will trigger more exhaustive docking calculation of diverse chemotypes. it is crystal clear that the prevention of covid-19 requires strong and sustainable global collaborative work [91] . data sharing is exigent to fill the knowledge gaps on this global pandemic. further progress of the scientific understanding regarding the structural and molecular biology of sars-cov-2 will legitimate the shape of lead compounds to achieve therapeutic goals. the development of medicinal chemistry through bioinformatics and chemo-informatics studies remains indispensable with a bit of savoir faire. the authors have no conflict of interests. sk. abdul amin is a senior research fellow at department of pharmaceutical technology, jadavpur university, kolkata, india. he is working under the guidance of tarun jha. his research area includes design and synthesis of small molecules with anti-cancer and anti-viral properties, computational chemical biology, and large-scale structure-activity relationship analysis. he has published sixty two research/review articles in different reputed peer-reviewed journals and three book chapters. he enjoys a good conversation on science, regional history, contemporary art and books. tarun jha, a professor at department of pharmaceutical technology, jadavpur university, kolkata, india, has supervised 16 ph.d. students and guided nine research projects funded by different organizations. he has published more than 150 research articles in different reputed peer-reviewed journals. his research area 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inhibit the recently emerged novel coronavirus (2019-ncov) in vitro hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro fda approved drugs with broad anti-coronaviral activity inhibit sars-cov-2 in vitro the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro teicoplanin potently blocks the cell entry of 2019-ncov in vitro screening of a fda approved chemical library reveals potential inhibitors of sars-cov-2 replication identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay nitazoxanide: a new thiazolideantiparasitic agent nitazoxanide: a first-in-class broad-spectrum antiviral agent current knowledge about the antivirals remdesivir (gs-5734) and gs-441524 as therapeutic options for coronaviruses. one health chloroquine: mechanism of drug action and resistance in plasmodium falciparum new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? chloroquine is a potent inhibitor of sars coronavirus infection and spread current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review rein t (2019) skp2 attenuates autophagy through beclin1-ubiquitination and its inhibition reduces mers-coronavirus infection endoribonuclease from sars cov-2. 6vww deposited diffraction resolution: 2.20 å the crystal structure of papainlike protease of sars cov-2 6w9c deposited it is a challenging task to identify effective anti-covid-19 drugs urgently. an exquisite picture of the recent research including target-based and biological screening is provided. experimental screening approaches in response to 2019-ncov is highlighted. it is an initiative from the perspective of a medicinal chemist to provide information about potential targets and drug repurposing against covid-19. the authors have no conflict of interests. key: cord-286269-vrjyj2y1 authors: sagheb, setareh; lamsehchi, ameneh; jafary, mohamadreza; atef-yekta, reza; sadeghi, kourosh title: two seriously ill neonates born to mothers with covid-19 pneumoniaa case report date: 2020-09-21 journal: ital j pediatr doi: 10.1186/s13052-020-00897-2 sha: doc_id: 286269 cord_uid: vrjyj2y1 background: coronavirus disease 2019 (covid-19), a highly contagious viral disease has spread from wuhan, hubei province, china to all over the world from its first recognition on december 2019. to date, only a few neonatal early-onset sepsis by sars-cov-2 has been reported worldwide. case presentation: in this report, we present two seriously ill neonates who were born from mothers with stablished covid-19 pneumonia. laboratory tests showed lymphopenia with high ldh and hypocalcemia right after the birth. they had fever for days without responding to antibiotics and despite ruling out other potential causes. both patients had positive rtpcr for sars-cov-2 in the second round of testing but the first assay tested was negative. hydroxychloroquine was used to treat both patients; the first patient was treated with it over a period of 14 days before showing signs of improvement. the second patient responded to the treatment over a period of 5 days. conclusion: although based on the available evidences, vertical transmission of covid-19 is less likely, many aspects of pathogenesis and transmission of this novel virus are still unclear. therefore we cannot rule out the vertical transmission totally. further investigations are warranted to determine the exact mechanisms and routes of transmission. since december 2019, the coronavirus disease (covid-19), which is a highly contagious virus, has been rapidly spreading worldwide. accordingly, it was firstly appeared in wuhan, hubei province, china [1] . coronavirus is an rna virus, which can cause a wide spectrum of clinical manifestations in human from a common cold to severe acute respiratory syndrome (sars) known as the sarsassociated coronavirus [2] . it was reported that severe acute respiratory syndrome coronavirus 2 (sars-cov-2) similar to sars-cov and middle east respiratory syndrome coronavirus (mers-cov), does not have vertical transmission from mother diagnosed with covid-19 infection to her fetus [3, 4] . however, reports of 3 neonates with early-onset covid-19 infection in china pointed out that not much is understood on intrauterine transmission, so further investigations are required. to date, few neonates infected by covid-19 have been reported [5] . in the following sections, we presented two neonates with covid-19 positive reverse transcription polymerase chain reaction (rt-pcr) assay in iran. these two affected neonates' parents provided us with a written consent to report on their neonates. this survey was approved by the medical ethics committee of tehran university of medical sciences (ir.tums.vcr.rec 1399.46). a preterm (gestational age = 31 weeks) infant boy was born on march 3, 2020, in shariati hospital, tehran-iran. his apgar scores were 7 and 8 in 1 and 5 min after birth, respectively. moreover, the weight at the time of birth was 1550 g. his 36-year-old mother (gravid: 2, abortion: 1) had shortness of breath and fever since about 7 days before delivery. in addition, she had positive rt-pcr test result for sars-cov-2. unfortunately, she died by passing 3 days from cesarean delivery. the baby had tachypnea and retraction after birth so he was transferred to nicu immediately, without performing skin to skin contact, with early initiation of continuous positive airway pressure (cpap) therapy. at the first step, we washed the baby with lukewarm water to prevent probable transmission of covid-19 from amniotic fluid to baby. afterward, we isolated him in a separate room. also, droplet and contact precautions were performed during transferring him to the nicu, and we also had strict precaution protocols for nurses who were taking care of him. subsequently, he was intubated due to progressive severe respiratory distress. then, he was injected by surfactant through endotracheal tube (et) after intubation. pharyngeal swab for sara-cov-2 was sent to the laboratory within an hour after admission. moreover, the first chest x-ray was performed after the injection of surfactant due to progressive respiratory distress. it showed a good aeration due to early intervention and ventilator support (fig. 1a) . his first pharyngeal swab rt-pcr test for covid-19 was negative. two days after birth, his axillary temperature reached 38.5°c (101.4°f), and chest radiography performed for him, showed a diffuse opacification of both lungs with air bronchograms and hypoaeration (fig. 1b) . antibiotic regimen was upgraded and meningitis was also ruled out by performing lumbar puncture and negative cerebrospinal fluid (csf) culture. moreover, we checked serum & csf herpes simplex viruses (hsv) pcr which were also negative. the results of nasopharyngeal multiplex pcr tests (for enterovirus, adenovirus, and h1n1) were also negative. a massive pulmonary hemorrhage complicated his situation on day 4 after his birth. in such case, we decided to treat him by 5 mg/kg/ day of hydroxychloroquine that was administered via feeding tube, considering the probable covid-19 infection. due to the persistent high fever despite administrating antibiotics for 5 days (excluding (hemophagocytic lymphohistiocytosis (hla)), viral and bacterial sepsis as explained earlier), we conducted a pharyngeal swab test again for covid-19 rt-pcr assay on day 7 after his birth time, and the rt-pcr test result for covid-19 was positive at this time ( table 1) . as he was still suffering from fever (39°c axillary), we continued the administration of fig. 1 a chest x-ray of case 1 after surfactant therapy (2 h after birth). b chest x-ray of case 1 in 2 days after birth. image date: 2020/03/05 hydroxychloroquine until the fever stopped on day 16 after his birth (i.e. treatment lasted for a 14-day period). hydroxychloroquine was well-tolerated by the patient without any significant clinical or laboratory adverse event. on his first laboratory tests, the baby had mild metabolic acidosis (ph: 7.24, pco2:41 mmhg, po2:60 mmhg, hco3:17.6 meq/l, be:− 9.4 meq/l), lymphopenia (2.4 × 10 9 /l), the elevated ldh (1645 u/l normal: ≤450 u/l), and normal crp (6 mg/dl). the results of renal and liver functions' tests were within a normal range. hyponatremia (124 meq/l) with a high amount of urine sodium (na: 128 meq/l) and normal urine specific gravity (usg: 1008) demonstrated the presence of syndrome of inappropriate antidiuretic hormone secretion (siadh) on day 2 of his life. moreover, he also had hypocalcemia (6.6 mg/dl) ( table 1) . his normal brain sonography changed to grade ш, ivh (intraventricular hemorrhage) through the first week. during hospitalization, both blood and urine culture tests' results were negative. he was diagnosed with prolonged pulmonary hemorrhage lasted for 14 days; however, his massive pulmonary hemorrhage has well recovered. furthermore, because of his mother death, we was not breastfed. therefore, he was fed with formula, and during hospitalization, only his father could come to visit him with respect to all precautions and having at least 1.5 m distance from him. he was discharged in may with negative covid-19 rt-pcr test's results that was performed twice. in follow up, he has a cerebroventricular shunt because of hydrocephalous. on march 5, 2020, a pregnant woman had emergency cesarean, because of her respiratory distress, and then she gave birth to a baby boy. she was suspected to having covid-19 infection as she had fever and tachycardia, and also because she came from the city of qom where the outbreak was started from in iran. she had one other child (gravid: 2, living child: 1, abortion 0). lung hrct was performed for her and the ct scan pattern was similar to those found in sars-cov − 2; however, her covid-19 rt-pcr test was negative. the baby's gestational age was 33 week + 4 days. moreover, his apgar scores were 7 and 8 in 1 and 5 min after birth respectively. also, his weight at the time of birth was 1930 g. the baby had tachypnea and retraction soon after birth, without performing skin to skin contact; therefore he was transferred to nicu with early cpap and then admitted in an isolated room. similar to case 1, we performed all the instructions for droplet and contact precautions during transferring him to the nicu, and the standard nursing protocols for droplet and contact transmission were controlled during hospitalization. subsequently, he was intubated due to progressive severe respiratory distress, so he received surfactant through et. surprisingly, the baby had fever at birth time, immediately when he was delivered. two days later, his fever stopped and we could extubate him. by performing chest x-ray, his lung showed a good aeration after receiving surfactant (fig. 2) . his o2 saturation was 98% without oxygen therapy. the result of the first covid-19 rt-pcr assay (within 1 h of birth) was negative. notably, one nurse fed him with milk, which was prepared with an electric pump from his mother. precautions for milking process were done, including washing breast, hands, and electric pump. the baby displayed no other symptoms until 12 days after his birth. by passing 12 days from birth, he had apnea and fever, so we performed sepsis workup while trying to exclude other causes of apnea. in this regard, the patient did not need oxygen support. while investigating the potential causes such as performing lumbar puncture, changing antibiotics, and giving caffeine, a second rt-pcr nasopharyngeal specimen for covid-19 test was taken for testing purposes. the result of test performed on day 12 was positive for covid-19. therefore he was subsequently treated by hydroxychloroquine 5 mg/kg/day via a feeding tube for 5 days, hence after which the fever stopped. also, chest x-ray displayed no particular pattern during the fever period. similar to case 1, hydroxychloroquine was welltolerated by the infant with no significant clinical or laboratory adverse event. in his first blood tests, arterial blood gas analysis showed ph: 7.31, pco2:34 mmhg, po2:178 mmhg, hco3:17.1 meq/l, and be:-8.2 meq/l. moreover he had lymphopenia (1.9× 10 9 /l), the elevated ldh (963 u/l normal: ≤450 u/l), and low crp (1.9 mg/dl). he also had hypocalcemia 7.7 mg/dl and csf, blood, and urine culture tests were negative during admission ( table 2) . we discharged him with two negative sars-cov-2 rt-pcr test results, which were performed with 24 h space. to date, he has not shown any abnormal symptoms after his recovery. for this case, follow up neurological testing will also be done in future. presently, there is no specific guideline for the management of covid-19 infection in neonates. as a result, usually, expert opinions and protocols for adult infection treatment and control are followed [6] . in our experience, hydroxychloroquine was used for both patients; and for the first case, a treatment course of 14 days appeared to be helpful. according to the lack of reliable scientific research on early-onset infection of covid-19 in neonates, it may be assumed that the symptoms in the patients were caused by prematurity or sepsis, rather than sars-cov-2 infection. nevertheless, it is clear that our data on early-onset infection of covid-19 is incomplete from many aspects. recently, two reports [3, 4] announced that intrauterine transmission of sars-cov-2 is not probable. they evaluated cord blood, amniotic fluid and even breast milk samples of mothers diagnosed with covid-19 pneumonia, but sars-cov-2 tests were negative in all cases. furthermore, zhu et al. observed ten neonates born from mothers infected by covid-19 and suggested that sars-cov-2 may cause several harmful effects such as premature labor, respiratory distress, thrombocytopenia or abnormalities in liver function tests or maybe death on neonates however, all their neonate's covid-19 rt-pcrs were negative [1] . in this report, our neonates had lymphopenia, the elevated ldh, and hypocalcemia immediately after birth time. the first case developed sign and symptoms of siadh on the second day after his birth with onset of running a high temperature. furthermore he developed a high anion gap metabolic acidosis on the 3rd day. after which, he suffered from the prolonged pulmonary hemorrhage on the 4th day of his birth. normal apgar scores of both cases in the absence of severe metabolic acidosis after birth, beside normal neurologic examination for gestational age ruled out asphyxia. consequently, because of performing all the aforementioned droplet and contact precautions during hospitalization, having high ldh, lymphopenia and siadh soon after birth may be due to early-onset infection of sars-cov-2. one study in china reported that, 3 neonates born from 33 patients infected by sars-cov-2 had an early-onset infection [5] . in our study, chest x-ray patterns were unremarkable in term of covid-19 infection; however, the patients were too unstable to go through a ct scan during admission. furthermore, another study conducted on a limited number of patients showed a high level of sars-cov-2 igm in neonates born from covid-19 infected mothers within 2 first hours of their birth [7] , which may indicate infection transmission from mother to fetus. also, we believe that we had strict operational procedures ensuring the prevention of infection transmission during labor and after transferring neonates to the isolated ward. however, since the birthdays are close to each other (march 3 and march 5 in 2020), a common denominator for infection can be possible in this short timeframe, and horizontal transmission in these cases cannot be ruled out. it is worth noting that, although our neonates' rt-pcr tests' results for sars-cov-2 were negative 1 hour after their birth, they tested positive on day 7 and 12. furthermore, zou et al. in china [8] surveyed the relationship between viral loads of sars-cov-2 in rt-pcr test, and the timing of swab sampling after symptoms were also detected. it seems that sensitivity of rt-pcr test depends on the source of sampling (e.g. throat, nose, etc.), time of sampling and the health care provider's skill for performing an accurate sampling. to the best of our knowledge, only a few neonatal early-onset sepsis by sars-cov-2 have been reported worldwide. despite older children usually show mild symptoms related to covid-19 infection, neonates with covid-19 infection typically exhibit an expanding range of clinical features. moreover, it seems that, preterm neonates may have more severe symptoms compared to term neonates in this infection, which can be attributed to a weak immune system of the preterm neonates. considering limited evidence, the priority should be given to clinical features of neonates, especially fever; once overriding rt-pcr test results. during the current coronavirus pandemic, we suggest considering the treatment of sars-cov-2 infection in seriously ill neonates with persistent fever and history of covid-19 pneumonia in their mothers. obviously, other potential causes must also be considered and excluded beforehand. it can be concluded that determining the preventive strategies and early detection of covid-19 in neonates along with implementation of standard protocols for treatment, require further in-depth investigations. also, the exact possibility of vertical transmission of covid-19 during pregnancy still needs to be determined. clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia coronavirus pathogenesis clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records an analysis of 38 pregnant women with covid-19, their newborn infants, and maternal-fetal transmission of sars-cov-2: maternal coronavirus infections and pregnancy outcomes neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china managing neonates with respiratory failure due to sars-cov-2 possible vertical transmission of sars-cov-2 from an infected mother to her newborn sars-cov-2 viral load in upper respiratory specimens of infected patients publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all staff nurses who have a critical role in caring these patients. received: 27 april 2020 accepted: 9 september 2020 authors' contributions ss and al prepared the primary draft of the manuscript. ray reviewed and edited the text. mj followed the laboratory tests and prepared pictures and tables and also edited the text. ks reviewed and made necessary changes and final edition. all the authors read and approved the submitted manuscript and have critical roles in caring and treatment of the patients. there was no funding resource. all data including patients' medical records, images and laboratory data are kept in our hospital for the minimum of 5 years based on the local regulations. parents of the two affected neonates provided us with a written consent to report on their neonates which the copies are available by request. all the authors declare no competing interest in this manuscript. key: cord-214854-ck61ja2t authors: zhong, jing; roesch, enja laureen; viereck, thilo; schilling, meinhard; ludwig, frank title: rapid and sensitive detection of sars-cov-2 with functionalized magnetic nanoparticles date: 2020-10-08 journal: nan doi: nan sha: doc_id: 214854 cord_uid: ck61ja2t the outbreak of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) threatens global medical systems and economies, and rules our daily living life. controlling the outbreak of sars-cov-2 has become one of the most important and urgent strategies throughout the whole world. as of october, 2020, there have not yet been any medicines or therapies to be effective against sars-cov-2. thus, rapid and sensitive diagnostics is the most important measures to control the outbreak of sars-cov-2. homogeneous biosensing based on magnetic nanoparticles (mnps) is one of the most promising approaches for rapid and highly sensitive detection of biomolecules. this paper proposes an approach for rapid and sensitive detection of sars-cov-2 with functionalized mnps via the measurement of their magnetic response in an ac magnetic field. experimental results demonstrate that the proposed approach allows the rapid detection of mimic sars-cov-2 with a limit of detection of 0.084 nm (5.9 fmole). the proposed approach has great potential for designing a low-cost and point-of-care device for rapid and sensitive diagnostics of sars-cov-2. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is an rna based virus that causes coronavirus disease 2019 (covid-19) [1] . sars-cov-2 with a dimeter ranging from about 60 to 140 nm has spike, envelope, membrane and nucleocapsid proteins [2, 3] . spike protein is the one for allowing the virus to bind to the receptor ace2 of a host cell and to enter the host cell [4] [5] [6] [7] [8] . a patient infected with sars-cov-2 may suffer from severe pneumonia and acute respiratory distress syndrome [3, 9, 10] . since the first infection case of sars-cov-2 reported in december, 2019 [10, 11] , sars-cov-2 has spread across the whole world, which causes severe problems in medical systems, economics and other social issues. till early october 2020, world health organization (who) has reported more than 35 million cases of infection and 1 million deaths in the world [12] . in addition to other control measures, e.g. social distance and hand washing, who has strongly recommended a large amount of testing not only of symptomatic persons and their contacts but also of asymptomatic contacts. therefore, large amounts of tests are one of the most important measures to control the outbreak of sars-cov-2. to date, polymerase chain reaction (pcr) based tests with high sensitivity and specificity are the gold standard for the diagnostics of sars-cov-2 infection [13, 14] . pcr tests include several complex experimental procedures, which costs at least a few hours to get the test results. in a pcr test, most experimental procedures require sophisticated and very expensive instrumentation and should be handled by experienced experts. otherwise, there is a high chance for a false positive/negative result. it leads to huge efforts for medical staffs in the clinics, especially during the exponential increase in infection cases. in developing and underdeveloped countries, the situation for the diagnostics of sars-cov-2 with pcr is even worse due to the lack of equipped labs, instrumentation and experienced experts. therefore, it is of extreme importance and necessity to develop new methods and/or instrumentation for rapid diagnostics of sars-cov-2 with reasonable costs. who highly encourages researches on the performance of rapid diagnostics approaches and potential diagnostic utilities. however, page 3 of 21 the development of new approaches for rapid diagnostics, in addition to pcr-based tests, is still a challenging research topic. homogeneous biosensing based on magnetic nanoparticles (mnps) is one of the most promising approaches for rapid and sensitive detection of specific biomolecules, e.g. protein, dna/rna and virus. it employs the change in the magnetization dynamics and consequent magnetic response of the mnps exposed to time-varying magnetic fields [15] . the binding behavior of the biomolecules to functionalized mnps, dominated by brownian relaxation, increases their hydrodynamic size or forms cross-linking structures, which significantly changes the brownian relaxation time of the mnps and their dynamic magnetization in timevarying magnetic fields [16] [17] [18] . for instance, for mnps bound with biomolecules, the harmonics in magnetic particle spectroscopy (mps) dominated by brownian relaxation decrease when exposed to a sufficiently strong ac magnetic field. especially, higher harmonics decrease faster than the fundamental harmonic, thus showing a decrease in the harmonic ratio that is independent of the mnp concentration [16, 19] . thus, the measurement of the mnp magnetization and its susceptibility/spectra in ac magnetic fields can be used to detect the quantity of specific biomolecules. an mps system has been demonstrated to be a low-cost, versatile and sensitive tool to measure mnp magnetization and dynamics for biomolecule detection [16, 20] . for instance, a mixing-frequency mps system was designed to measure the mixing components of protein g-functionalized mnp magnetization for antibody detection [21] . zhang et al. reported on the measurement of the harmonic ratio of anti-thrombin dna aptamers-functionalized mnps for the detection of thrombin [22] . zhong et al. investigated the effect of binding behavior on mnp relaxation time and harmonic ratio for the detection and imaging of biotinylated igg using streptavidin functionalized mnps [16, 19] . yang et al. demonstrated the feasibility of wash-free, sensitive and specific assays for the detection of different viruses, e.g. orchid and influenza viruses, with antibody-functionalized mnps by measuring the reduction in the ac susceptibility in mixed-frequency ac magnetic fields [23] [24] [25] . tian et al. reported on homogeneous detection of sars-cov-2 utilizing an opto-magnetic measurement system for the detection of the rnd-dependent rna polymerase coding page 4 of 21 sequence of sars-cov-2 [26] . all these approaches have demonstrated that mnp-based homogeneous biosensing is a wash-free and mix-and-measure approach for rapid and sensitive detection of specific biomolecules. therefore, it is of great interest and importance to investigate mnp-based biosensing for rapid and sensitive detection of sars-cov-2. in this paper, we propose the detection of sars-cov-2 via the measurement of the mps in this paper exposed to a sufficiently strong ac magnetic field with excitation frequency f0, the mps signal consists of not only the fundamental harmonic at frequency f0 but also higher harmonics at frequency i×f0. the custom-built smps was used to measure the 1 st and 3 rd harmonics (m1 and m3) of the output voltage from the gradiometric detection coils on experimental samples in ac magnetic fields with different excitation frequencies f0, where the harmonic at frequency i×f0 is defined as i th harmonic mi. figure 4 shows the limit of detection (lod) is one of the most important measures for sensitive detection. to evaluate the lod, the measurement sensitivity  and noise level on the harmonic ratio are calculated. the measurement sensitivity, defined as the relative change in r3rd/1st to cmv page 13 of 21 dr3rd/1st/dcmv, is calculated by a linear fitting of the r3rd/1st vs. cmv curves shown in fig. 5 . figure 6a shows the measurement sensitivity vs. frequency curve for different excitation frequencies. it indicates that with increasing frequency from 140 to 1418 hz the measurement sensitivity  decreases from 0.017 nm -1 to 0.004 nm -1 , which quantitatively fits well with published results [16] . the noise level is calculated with the standard deviation  of 10-repeated measurements on the harmonic ratio of an mnp sample with cmv = 0 nm. in principle, with increasing the excitation frequency, the signal-to-noise ratio (snr) of the measured harmonic gets improved, which would improve the standard deviation in measured harmonic ratio due to faraday's law. however, the data in frequencies of 299 hz and 459 hz show higher noise levels than that in 180 hz, which may be caused by instabilities of the measurement system. figure 6b shows the lod , estimated from the measurement sensitivity  and standard deviation  by  = [30, 31] . it indicates that the lod is in the range from about 0.10 to 0.37 nm. note that the estimated lod is obtained for a single measurement. with n-time repeated measurements, the lod can be further improved by a factor of √n. the current approach employs the measurement of magnetic signal of the functionalized mnps, which does not have depth limitation. when combining with multi-color magnetic particle imaging [33] [34] [35] , the present approach can be extended to visualize the spatial distribution of sars-cov-2 in vivo, which is of great significance and interest not only to control the outbreak of sars-cov-2 but also to fundamental researches, e.g. understanding the underlying mechanisms of the infection process and virus proliferation. this paper investigated rapid and sensitive detection of sars-cov-2 with functionalized mnps. for a proof-of-concept, functionalized mnps were used as sensors to detect a mimic virus consisting of 100 nm-polystyrene beads conjugated with sars-cov-2 spike proteins. experiments on acs spectra and mps signal of samples with different mimic virus concentrations were performed. experimental results showed that the binding behavior between mimic sars-cov-2 and functionalized mnps increases the effective brownian relaxation time and changing the mps signal. the change in the ratio of the 3 rd to 1 st harmonics with the mimic virus concentration was used to analyze the measurement sensitivity and limit of detection. we believe that the proposed approach is of great promise to highly sensitive and rapid detection with a low cost, easy handling of the sample to be detected (mix-and-measure). we envisage that the present work is of great interest and significance to develop new methods and design point-of-care devices for rapid diagnostics of sars-cov-2 to control its outbreak, as well as fundamental researches on the virus 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magnetically enhanced high-specificity virus detection using bioactivated magnetic nanoparticles with antibodies as labeling markers homogeneous circle-to-circle amplification for real-time optomagnetic detection of sars-cov-2 rdrp coding sequence sars-cov-2 (covid-19) by the numbers, elife magnetic nanoparticle temperature imaging with a scanning magnetic particle spectrometer fluxgate based detection of magnetic nanoparticle dynamics in a rotating magnetic field methods for the determination of limit of detection and limit of quantitation of the analytical methods, chronicles of young scientists harmonized guidelines for single-laboratory validation of methods of analysis rapid tests for sexually transmitted infections (stis): the way forward detecting the coronavirus (covid-19) tomographic imaging using the nonlinear response of magnetic particles first experimental evidence of the feasibility of multi-color magnetic particle imaging dual-frequency magnetic particle imaging of the brownian particle contribution the authors declare no conflicts of interest. key: cord-268540-wrjzr3ws authors: park, you jeong; farooq, jeffrey; cho, justin; sadanandan, nadia; cozene, blaise; gonzales-portillo, bella; saft, madeline; borlongan, maximillian c.; borlongan, mia c.; shytle, r. douglas; willing, alison e.; garbuzova-davis, svitlana; sanberg, paul r.; borlongan, cesar v. title: fighting the war against covid-19 via cell-based regenerative medicine: lessons learned from 1918 spanish flu and other previous pandemics date: 2020-08-13 journal: stem cell rev rep doi: 10.1007/s12015-020-10026-5 sha: doc_id: 268540 cord_uid: wrjzr3ws the human population is in the midst of battling a rapidly-spreading virus— severe acute respiratory syndrome coronavirus 2, responsible for coronavirus disease 2019 or covid-19. despite the resurgences in positive cases after reopening businesses in may, the country is seeing a shift in mindset surrounding the pandemic as people have been eagerly trickling out from federally-mandated quarantine into restaurants, bars, and gyms across america. history can teach us about the past, and today’s pandemic is no exception. without a vaccine available, three lessons from the 1918 spanish flu pandemic may arm us in our fight against covid-19. first, those who survived the first wave developed immunity to the second wave, highlighting the potential of passive immunity-based treatments like convalescent plasma and cell-based therapy. second, the long-term consequences of covid-19 are unknown. slow-progressive cases of the spanish flu have been linked to bacterial pneumonia and neurological disorders later in life, emphasizing the need to reduce covid-19 transmission. third, the spanish flu killed approximately 17 to 50 million people, and the lack of human response, overcrowding, and poor hygiene were key in promoting the spread and high mortality. human behavior is the most important strategy for preventing the virus spread and we must adhere to proper precautions. this review will cover our current understanding of the pathology and treatment for covid-19 and highlight similarities between past pandemics. by revisiting history, we hope to emphasize the importance of human behavior and innovative therapies as we wait for the development of a vaccine. [figure: see text] hindsight is 20/20 global shutdowns, widespread reduction in economic activity, and stalls in the workforce and education systems are just a few of the major historic events that have amalgamated in 2020 due to the outbreak of the covid-19 pandemic. according to the data collected by johns hopkins university, there are more than 8 million confirmed coronavirus cases and 437,500 deaths worldwide as of june 15, 2020 [1]. the us currently leads the world with more than 2.1 million confirmed cases and 116,130 deaths-steadily reaching dr. anthony fauci of the national institute of allergy and infectious disease projected number of 100,000 to 240,000 nation-wide deaths back in march of 2020. history repeats itself and when assessing the proper responses to the current pandemic, it is important to learn from the past. in a historical review of viral pandemics, the 1918 spanish flu stands out which infected 500 million people and led to an estimated 17 to 50 million deaths [2] . like the current pandemic, the spanish flu began from a zoonotic transmission, progressed rapidly from infection to symptom onset with viral consolidation in the lung, and lead to death primarily by pneumonia in severe cases [3, 4] . the main catalyst of the high mortality rate of the spanish flu was lack of initial response, overcrowding, and poor hygiene, emphasizing the critical role human behavior plays in the spread of infectious diseases. three important takeaways stand out from the 1918 pandemic. first, those who survived the first wave developed immunity against the virus and gave these individuals immunity during the second wave. this highlights the potential of passive immunity-based treatments using antibodies collected from the plasma of recovered covid-19 patients. the second lesson is that the end of spanish flu cases in 1920 marking the eradication of the pandemic may not capture the full picture of the pandemic. evidence of slow-progressive cases of spanish flu has been associated with secondary bacterial pneumonia linked to neurological disorders like pediatric encephalitis lethargica in the 1920 s [5] , which was suggested to manifest in adulthood as parkinson's disease [6] . similarly, the potential long-term neurological and cardiac repercussions of covid-19 warrant critical attention as the research surrounding this pandemic unfolds. third, the overcrowding in military camps and lack of sanitation due to poverty during world war i contributed to the escalation of the spanish flu, emphasizing the importance of human behavior on containing the current pandemic. as states reopen in response to the current economic crisis and restrictions continue to ease over the next few months, the security blanket of disillusioned comfort-or ignorance of the dangers of the virus-may be the biggest threat to the future health of our society. this review will cover our current understanding of the pathology of covid-19 and highlight similarities between the spanish flu and past pandemics' pathology, social behavior, and treatments. by focusing on these disease overlaps, in particular convalescent plasma and stem cells targeting the virus entry point, we may gain insights on the best treatments and human behavior to address the current pandemic. furthermore, we will discuss the latest preclinical and clinical data of available therapies and by highlighting critical gaps in knowledge, we hope to emphasize the importance of human behavior and innovative therapies as we wait for the development of a vaccine. binding of spike protein and human angiotensinconverting enzyme 2: the shot heard around the world the term coronavirus refers to more than 40 species of viruses within the family coronaviridae that cause respiratory tract infections in humans. coronaviruses classify into either alpha and beta subtypes that infect humans, or into gamma and delta subtypes, which mainly infect avians. severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome-related coronavirus (mers-cov), are two betacoronaviruses that precipitated the sars and mers epidemics in 2003 and 2012, respectively. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a new genetically-related betacoronavirus species responsible for the covid-19 pandemic. sars-cov-2 is a single-stranded, non-segmented, positive-sense rna virus that measures 65-125 nm in diameter and 29.9 kb in length [7, 8] . it is composed of a 5'-untranslated region (utr), 3'-utr, replicase complex, membrane protein gene, nucleocapsid gene, envelope protein gene, and spike protein gene [9] . the trimeric spike protein (sp) of sars-cov-2 mediates the entry of the virus into host cells and is therefore critical to the pathogenesis of the disease (fig. 1) . sp is a class one dual subunit transmembrane fusion glycoprotein exclusively expressed by viruses that binds to human angiotensin-converting enzyme 2 (ace2), an endogenous extracellular receptor. this initiates cleavage events that facilitate sars-cov-2 fusion and entry into the host cells [10] . the s1 and s2 subunits of sp are non-covalently bound, host-processed proteins that mediate the binding and fusion capabilities of sars-cov-2, respectively [10] [11] [12] . the s1 subunit recognizes and binds to ace2 with high affinity, implicating the interaction of viral sp and human ace2 as the key intermediary for host-cell entry [13] . ace2 is a type 1 integral membrane protein that functions as an extracellular receptor attached to the plasma membrane of cells in the lungs, heart, brain, and other vital organs [14, 15] . it is primarily expressed on type 2 pneumocytes in the lungs and on enterocytes in the small intestine. ace2 normally modulates blood pressure through the renin-angiotensin system by cleaving circulating angiotensin ii into the vasodilator angiotensin. sars-cov-2 leverages the ubiquitous expression and physiologic importance of ace2 to enter and replicate within healthy cells throughout the body. this leads to an increase in viral load and systemic adverse multi-organ effects. the potent interaction between ace2 and sp mediates the efficient entry of the virus into host cells. electrostatic interactions between the negatively charged ridges that flank the catalytic domain of ace2 and the positively charged receptor-binding domain (rbd) of sp enable sars-cov-2 to localize to its receptor [16] . ace2 receptors contain a peptidase domain that binds to the rbd of sps through polar interactions [17] . following this docking, two proteolytic cleavage events take place. first, furin protease cleaves sp into s1 and s2 subunits. second, the serine protease tmprss2 cleaves sp on the s2 subunit, initiating a series of conformational changes that prime the virus to enter the cell [18, 19] . tmprss2 also cleaves ace2 between amino acids 697 to 716, and this cleavage is crucial to enhancing viral uptake [20] . the metalloprotease adam17 also acts on ace2, but this does not augment viral uptake [20] . therefore, tmprss2 both activates sars-cov-2 for uptake and simultaneously amplifies the rate of its uptake by cleaving ace2. after binding and proteolytic processing, the final step for infection is the viral entry into the host cell. it is not entirely clear how sars-cov-2 enters the cell, as there is evidence for both fusion and endocytosis. in a fusion-based entry model, sars-cov-2 fuses its envelope with the plasma membrane of the host cell and empties its genetic contents into the cytosol, where it hijacks the host machinery to replicate its genes and produce more virus. in the endocytosis model, sp interacts with ace2 to cause a ph-dependent internalization of the virus-receptor complex [21] . on the other hand, the endocytic-uptake of sars-cov-2 is independent of clathrin and caveolae-based pathways [21] . clathrin-mediated endocytosis involves the formation of vesicles coated by the protein clathrin that form following internal budding of the plasma membrane. caveolar endocytosis occurs when oligomerized caveolin membrane form scaffolds that invaginate into endocytic vesicles. the precise type of endocytosis that results in the internalization of sars-cov-2 is unknown but does not involve these two mechanisms. the sp gene of sars-cov-2 determines its affinity for ace2, in contrast to the sps of related coronaviruses with slightly different sequences that favor binding to other targets. each cycle of viral replication within the host cell produces additional copies of the viral rna genome, including the regions that encode this specific sp variant. reversetranscriptase polymerase chain reaction (rt-pcr) recognizes the sp of sars-cov-2 and measures total viral load. this technique utilizes a primer that binds to regions of the sars-cov-2 rna genome, including the sp region, and amplifies the amount of virus present such that the fewer cycles it takes to reach a threshold value indicates a higher initial concentration of sars-cov-2. sp expression and viral load are highest at the onset of infection and lowest during recovery [22] . elevated viral load thus indicates increased infectivity due to the additional sp binding ace2. sp is integral for sars-cov-2 to recognize and internalize into cells. viral binding and fusion with host cells are obliterated in the absence of sp expression. after uptake, the virus uses host cell ribosomes to replicate; however, the cell will eventually undergo apoptosis or immune system-mediated destruction [23] . therefore, sars-cov-2 must continuously infect new cells using sp-mediated uptake mechanisms. sp is also crucial for sars-cov-2 to infect other tissues beyond the original site in the respiratory tract. the virus travels to other organs through the bloodstream and, upon sp binding, fuses with ace2-expressing organs to establish secondary sites of infection. thus, sp and ace2 must co-exist for the virus to propagate its growth and remain contagious. the critical interaction of sp and ace2 as the gatekeeper of sars-cov-2 entry into host cells can be confirmed and exploited using human recombinant soluble ace2 (hrsace2). ace2 is a transmembrane protein and is not present endogenously in a soluble form; therefore, sp always binds to cell-surface ace2. however, the administration of hrsace2 introduces soluble ace2 into the circulation that can complex with free sars-cov-2, thus blocking its ability to bind to cell-associated ace2 [24] . the virus still binds to these functionally inactive receptors in a dose-dependent manner but does not undergo proteolytic activation or entry into the cell [24] . importantly, sp binds to hrsace2, but not to mouse recombinant soluble ace2 [24] . to this end, the hrsace2 proves the selective binding of sp to ace2 and may be therapeutically valuable as an option to minimize the potential uptake of the virus. the specific structural features and amino acid sequences of sp and ace2 underlie the selectiveness of their binding and explain the preferential binding of sars-cov-2 to ace2 and rbsace2. sp makes direct contact with ace2 at three different points. in the first region of interaction, gln 498 , thr 500 , and asn 501 on the rbd interact with tyr 41 , gln 42 , lys 353 , and arg 357 on ace2 [17] . in the second region, lys 417 and tyr 453 on the rbd associate with asp 30 and his 34 on ace2 [17] . the final point of contact is between gln 474 and phe 486 on the rbd and gln 24 and met 82 on ace2 [17] . substitutions of these amino acids may disrupt the affinity of the binding between sp and ace2. indeed, certain variants of the ace2 allele exhibit weakened intermolecular interactions, pointing to the possibility of covid-19resistance [25] . similarly, the level of expression of ace2 may be an indicator of resistance to sars-cov-2 infection or a prognostic marker whereby increased expression correlates with higher infectivity. leveraging this to identify highrisk populations could help slow the spread of the virus. in contrast, alterations in the amino acid sequence may also increase the ability of the virus to enter the cell by further stabilizing the sp-ace2 interaction [26] . in addition to the rbd, amino acids 450-650 of sp are arranged in two anti-parallel β-sheets, β5 and β6. these sp structures, especially β6, bind with high affinity to ace2, further emphasizing sps' importance as the ligand for ace2 [27] . although there are animal reservoirs of the virus, sars-cov-2 is most strongly associated with pathological effects in humans. transgenic animal models expressing human ace2 recapitulate its specificity and affinity for sp. upon inoculation with sars-cov-2, mice expressing human ace2 rapidly display signs of lethal infection in the lungs and brain, whereas mice expressing murine ace2 do not develop infection [28] . in the transgenic mice, there is a rapid accumulation of immune cells, particularly macrophages, in the epithelia of the lungs and brain where they release proinflammatory cytokines and initiate a full immune response [28] . the capacity of sp to utilize human ace2 to enter cells ensures the viability of the virus in several tissues. ace2 expression is high in the small intestines, kidneys, heart, and lungs, although it is also present in key organs such as the nerves and brain [29] . rt-pcr analysis demonstrates the highest viral load in the lungs and intestines, with 3.6 × 10 5 and 2.7 × 10 5 copies per gram of tissue, respectively [30] [31] [32] . the prevalence of sp-ace2 binding in these organs explains their substantial capacity to harbor the virus. it also justifies the elevated viral loads in those other organs such as the heart and brain. the highly specific interaction between sp and ace2 is well defined and is a critical target for therapeutic strategies that attempt to prevent infection. a variety of vital organs express the ace2 receptor and are thus susceptible to spbinding and the lethal damage induced by viral infection. therefore, it is crucial to evaluate the effects of sars-cov-2 in the most commonly affected organ, the lungs, but also in other ace2-and sp-expressing organs such as the brain, heart, intestines, and liver. the lungs are the primary site of infection and nearly all patients with covid-19 present with some form of respiratory impairment. the most common complaints upon hospitalization are cough and shortness of breath, with 69% and 66% of patients reporting these symptoms, respectively. sars-cov-2 spreads through person-to-person contact or via small droplets, measuring less than 10 µm in diameter, that enter the respiratory tract after inhalation [33, 34] . as the virus traverses the airways, it binds to apical (luminal) ace2 receptors via sp, then enters cells where it initiates replication. it preferentially infects cells of the alveoli, although it can also infect cells of the upper respiratory tract. viral infiltration of the lung parenchyma directly precipitates the onset of severe acute respiratory syndrome (sars). despite the crucial role the respiratory tract plays in the transmission of sars-cov-2, airborne transmission is not an appreciable route of spread. the lungs are the primary organ of the respiratory system and oversee numerous critical functions for survival and homeostasis, including acid-base balance and thermoregulation. they perform their primary role of gas exchange by drawing air into alveoli lined with pneumocytes. oxygen diffuses across type 1 pneumocytes, which comprise 95% of the surface area of the alveoli. type 2 pneumocytes are the most common cells in the alveoli, although they are small and do not constitute a large proportion of the total surface area. they secrete the pulmonary surfactant that prevents the alveoli from collapsing, and they can divide into type 1 or type 2 pneumocytes. the coexpression of ace2 receptors and tmprss2 transmembrane proteases by type 2 pneumocytes explains the susceptibility of the lungs to sp-mediated sars-cov-2 infection [35] . well-differentiated type 2 pneumocytes express ace2 more abundantly than poorly differentiated cells [36] . the targeting of these critical lung cells by sp has important functional consequences that manifest as sars. sars initially presents as fever, lethargy, pain, and cough, although it rapidly advances to shortness of breath and pneumonia. macrophages of the innate immune system in the lungs likely detect sars-cov-a using rna viral genome-recognizing rig-like receptors, a specific type of toll-like receptor (tlr) present on dendritic cells and macrophages designed to complex with foreign molecules. binding of the viral rna to rig-like receptors induces the recruitment of myd88 and mavs [37] . these two proteins promote nf-kb and type 1 interferon production culminating in the release of il-6 and tnf alpha cytokines [37] . this immune cascade results in extensive tracheobronchial inflammation, diffuse alveolar damage, alveolar edema, pneumocyte desquamation, fibrin deposition, pneumocyte hyperplasia, and recruitment of alveolar macrophages (fig. 2) [38] . furthermore, a less severe form of pneumoconiosis known as anthracosis develops due to carbon accumulation [38] . sp interacts with ace2 receptors on alveolar macrophages, both types of pneumocytes in the lungs, and the bronchial and submucosal gland epithelium of the upper airways [38] . after replication, sars-cov-2 preferentially exits cells via the apical surface, returning it to the lumen where it can continue to propagate through lung tissues [36] . the pathogenesis of sars-cov-2 produces severe respiratory outcomes within a few weeks of initial infection, even in the absence of preexisting respiratory comorbidities such as chronic obstructive pulmonary disease (copd) [39] . in half of the critically ill patients, the development of sars-cov-2induced pneumonia currently results in death within 28 days of diagnosis and 7 days of admission to the intensive care unit (icu) [40] . patients that require ventilators or who develop acute respiratory distress syndrome (ards), characterized by fluid accumulation in the alveoli, are more likely to die [40] . approximately 67% and 71% of critically ill patients develop ards or require ventilator therapy, respectively [40] . one of the highest concentrations of ace2 in the body is in the lung tissue. the potent binding of sp to ace2 and subsequent cellular infection may explain the prevalence of respiratory symptoms in covid-19 patients. transgenic mice expressing hace2 in the lungs exhibit a very similar pathology in the lungs compared to human patients, including pneumonia and macrophage infiltration; however, these respiratory symptoms are absent in wild-type mice [41] . thus, the propensity of sars-cov-2 to elicit lung-tissue damage and induce respiratory symptoms relies upon successful uptake into type 2 pneumocytes. sars-cov-2 produces more severe symptoms in elderly patients than young patients, implicating an age-based loss of protection. approximately 90% of infected pediatric patients are asymptomatic or exhibit mild symptoms [42] . given the critical role of the lungs as the primary site of infection, this suggests there exist underlying physiological differences between adult and adolescent lung tissue that either predisposes adults to infection or protect adolescents. while the severity of respiratory symptoms is useful in predicting outcomes, several non-invasive methods also allow tracking of the extent of covid-19-induced lung damage. computer tomography (ct) analysis is a diagnostic imaging procedure useful for evaluating the pathology of the lungs. ct image-calculated well-aerated lung volume predicts the risk of icu admission and death [43] . thus, a more widespread sars-cov-2 lung infection correlates with worse outcomes. ultrasound is another valuable imaging technique to explore the extent of lung damage. covid-19 pneumonia increases the presence of b-lines, consolidation, and pleural line abnormalities in the posterior regions of the lungs [44] . combined with its safety and efficiency, ultrasound imaging is valuable as both a diagnostic tool and as a follow-up measure to track changes in the extent of covid-19 pneumonia in the lungs. the interaction of sars-cov-2 with the lungs directly explains its tremendous infectivity potential. not only is the respiratory tract is the primary point of entry of the virus into the body, but it also contains the highest concentration of ace2 receptors for sp to complex with. furthermore, sars-cov-2 survives within the respiratory tract more effectively than it can in other tissues, highlighting the imminent need for therapeutics targeting the lungs [45] . sp mediates this enhanced survival by downregulating ace2 in the lungs [46] . specifically, sp infects bronchoalveolar stem cells (bascs) and controls basc growth, inflammation, and ace2 production [47] . sp downregulates ace2 to self-limit its own infectivity. excess virus production results in catastrophic lung damage and effectively destroys the tissues within which sars-cov-2 lives. sp-induced downregulation of ace2 expression thus enhances viral survival, which increases the probability that sars-cov-2 will successfully transmit to another host. simultaneously, ace2 is also upregulated in the presence of lung tissue damage due to interferon activity [48] . this contradictory mechanism highlights the complexity of sars-cov-2 infection in the respiratory tract. ideal survival conditions depend on the interplay of sp-mediated downregulation of ace2 and interferon-mediated upregulation of ace2. the lungs are a conduit through which sars-cov-2 accesses the systemic circulation to infect other vital organs and tissues. as the virus travels from the lungs to the rest of the body, it propagates widespread damage and dysfunction by promoting inflammation that culminates in a cytokine storm [49] . this worsens patient outcomes and results in long-term tissue damage [50] . therefore, it is essential to continue to investigate the pathology and treatments for sars-cov-2 infection in the lungs as the primary target of the virus. fig. 2 the coexpression of ace2 receptors and tmprss2 transmembrane proteases by type 2 pneumocytes explains the susceptibility of the lungs to spmediated sars-cov-2 infection. infection activates the immune cascade resulting in extensive tracheobronchial inflammation, diffuse alveolar damage, alveolar edema, pneumocyte desquamation, fibrin deposition, pneumocyte hyperplasia, and recruitment of alveolar macrophages notwithstanding, the recognition that ace2 is also highly expressed in other organs equally warrants examination of the role of this enzyme/receptor in the downstream consequences of sars-cov-2 beyond lung infection. the neuroinvasive capacity of sars-cov-2 has been demonstrated in both humans and animals. intensive care patients display neurological symptoms such as nausea, headaches, and vomiting [51] . a recent study on sars-cov-2 found that 78 out of 214 patients demonstrated infection of the central nervous system (cns). more severe patients manifested acute cerebrovascular diseases, impaired consciousness, and skeletal muscle injury [52] . although the exact route sars-cov-2 takes to infect the cns is not known, sars-cov-2 is very similar to sars-cov and mers-cov. looking at the mechanisms of entry of sars-cov and mers-cov may provide key insight into the route sars-cov-2 takes to infect the cns. sars-cov, like sars-cov-2, utilizes the ace2 receptor for entry to the cell while mers-cov enters cells through dipeptidyl peptidase 4 (dpp4). the presence of ace2 alone is not conclusive of a cell's vulnerability to infection. interestingly, human intestinal endothelial cells expressing ace2 were not infected by sars-cov, and hepatocytes, non-detectable-ace2 expressing cells, were infected by sars-cov [53] . sars-cov and mers-cov were found to infect the brains of patients in the early 2000 s, and most viral material was found in neurons [54] . transgenic mice inoculated intranasally with sars-cov and mers-cov displayed a high mortality rate. mice administered low concentration of mers-cov intranasally only displayed viral infection of the cns, not the lungs, indicating that the cause of death may be directly due to cns infection. for both sars-cov and mers-cov infected mice the brain stem was most concentrated with viral particles. evidence now suggests coronaviruses enter the cns via synaptic avenues upon infection of peripheral nerve terminals [55] . swine hemagglutinating encephalomyelitis virus (hev) particles, a coronavirus, were enclosed in smooth surface vesicles within the axons of mice [55] . vesicles containing hev were transported via microtubules to perikaryons and dendrites, where the viral material was later found in high concentrations. upon replication in these cells, virions were packed in spinule-coated vesicles in the trans-golgi networks. it is hypothesized that coated vesicles mediate endo-and exocytosis of the virus into and out of the cns. extracellular virion accumulation may have been the causing factor of dilated synaptic clefts, consequently allowing the trans-synaptic transfer of the coated virus-containing vesicles to other host neurons [55] . this phenomenon must now be explored with sars-cov-2 to attenuate brainstem-induced cardiovascular and pulmonary complications. further elucidation regarding the neuroinvasive capacity of sars-cov-2 may provide insight to better mitigate symptoms and prevent infection in humans. cardiac involvement in sars-cov-2 infection is prevalent among patients [56] . as noted above, the acute disease progression begins by infiltrating the lungs via sp-ace2 interaction and is then followed by collateral tissue injury and inflammation. along with inflammation arises vasodilation, endothelial permeability and, leukocyte recruitment, which contribute to the exacerbation of pulmonary injury, hypoxemia, and cardiovascular distress [56] . severe patients present with systemic inflammation which has the propensity to damage the heart without direct viral infection of the tissue [57] . direct sars-cov-2 infection, hypoxemia, and respiratory failure all also contribute to myocardial injury seen amongst patients. out of these 3 factors, it remains unclear which is the main contributor to myocardial complications. cell populations in the heart most vulnerable to infection may be distinguished by expression of ace2. for instance, myocardial pericytes express ace2, and infection or disruption caused by inflammation may lead to ischemic injury and disruption of the microvasculature [58] . biomarkers troponin i and brain-type natriuretic peptide were both present at augmented levels in severely ill patients [59] . blood pressure elevation and arrhythmias are common symptoms in patients however this may be due indirectly by systemic inflammation. acute coronary syndromes are present with sars-cov-2 infection. the risk of this instance is significantly increased when an underlying disease is present creating a systemic prothrombotic state [56] . further investigation on the cause of cardiac complications that come with sars-cov-2 must be completed to effectively treat patients and minimalize debilitating injury or death. gastrointestinal abnormalities were seen in 2%-10.1% of patients positive for sars-cov-2 [60] . symptoms including diarrhea and vomiting indicate that the virus can interact with cell populations in the gut. the ace2 receptor is present in the gastrointestinal tract at significant amounts [61] . recently discovered, viral material was found to be present in infected individuals' fecal material [62] . the phenomenon of gut-lung communication has been seen previously in other diseases, such as asthma [63] . further investigation is imperative to possibly mediate sars-cov-2 through probiotics or antibiotics [60] . although only a small percentage of patients experience gastrointestinal symptoms with sars-cov-2, this should be taken seriously as it may exacerbate pulmonary complications caused by the virus. liver injury is linked to multiple etiologies such as alcohol consumption, toxins, bile duct dysfunction, and viral infections. epidemiologists reported that 75 out of 148 covid-19 patients had varying degrees of liver dysfunction [64] . the exact mode of sars-cov-2 entry to the liver is unknown, and further research is needed to differentiate whether injuries are due to drug treatments, systemic inflammation caused by pulmonary infection, or viral infiltration of liver tissue [65] . sars-cov-2, the cause of viral pneumonia spreading across the globe, can infect many vital organs via sp-ace2 mediated cellular infection. sp is critical to extrapulmonary organ infection due to its ability to promote viral uptake in these tissues. furthermore, certain mechanisms in the gastrointestinal tract and heart bolster sp-mediated viral uptake. in the gastrointestinal system, the high concentration of the tmprss2 protease enhances the ability of sars-cov-2 to enter intestinal epithelial cells [66] . in the heart, pericytes are robust targets of sp binding due to their elevated ace2 expression, and pericyte infection results in microvascular dysfunction that worsens cardiac symptoms [58] therefore, infection of non-lung organs may further exacerbate pulmonary infection and other comorbidities such as cardiovascular and cerebrovascular diseases. establishing effective treatments to mitigate the effect of sars-cov-2 on non-lung organs will be very beneficial for sequestering the spread of the virus outside of the lungs, which will likely treat the noted comorbidities. although covid-19 is primarily a respiratory virus, it may have acute and chronic consequences on non-lung organ systems. delineating how quickly covid-19 progresses, particularly in patients who develop dire respiratory, cardiovascular, and cerebrovascular complications, is key to understanding the virus's multi-organ pathology and establishing appropriate treatment. the incubation period for covid-19 is approximately 5.1 days. for 97.5% of symptomatic patients, the estimated number of days before symptom onset is 11.5 [67] . acute respiratory distress syndrome occurs approximately 8 to 9 days after the incubation period [68] . regarding patients who develop myocardial injury, a median of 18.5 days passed between symptom onset and death [69] .covid-19 patients with cardiovascular complications typically suffer from acute cardiac injury, arrhythmias, and heart failure [70] . according to the national health commission of china (nhc), 58% of covid-19 patients with dire symptoms had hypertension. 44% of these patients suffered from arrhythmia and 25% suffered from heart disease [71] . the nhc stated that 11.8% of patients who died from covid-19 endured severe heart injury due to high amounts of ctnl or cardiac arrest without having a pre-existing heart condition [72] . interestingly, the nhc reported that some patients initially showed symptoms such as chest tightness and heart palpitations rather than the common respiratory symptoms suggesting covid-19 may lead to chronic malady [71] . some patients previously infected by sars-cov later developed chronic cardiovascular damage [71] . for instance, cardiovascular disorders emerged in 44% of 25 sars-cov patients 12 years postinfection. hyperlipidemia progressed in 68% of patients [71] . sars-cov spurred a substantial elevation of free fatty a c i d s , l y s o p h o s p h a t i d y l c h o l i n e , lysophosphatidylethanolamine, and phosphatidylglycerol in the blood serum of these patients [71] . the cardiovascular repercussions of covid-19 infection may be primarily due to the presence of the ace2 receptor in cardiac tissue. covid-19 internalization via the ace2 receptor leads to a loss of the ace2 peptidase pathway, which can generate a rise in blood pressure, fibrosis, and inflammation [72] . infected patients have substantially greater angiotensin ii (ang ii) levels than healthy individuals, indicating the loss of ace2 activity [72] . in addition, ace2 plays a role in the progression of diabetes mellitus and hypertension [71] . individuals with diabetes and hypertension are at higher risk from covid-19 because of increased ace2 expression. furthermore, covid-19's affinity for the ace2 receptor may have detrimental effects on the cardiovascular system, potentially causing acute and chronic cardiac injury in patients. although the expression of the ace2 pathway may be a prominent agent of cardiovascular complications in covid-19 patients, there may be other signaling pathways that lead to covid-19-induced cardiac complications. covid-19 can cause vascular inflammation by spurring the secretion of the following inflammatory factors: troponin, natriuretic peptides, and il-6 cytokines. inflammation in the cardiovascular system can lead to diffuse microangiopathy, thrombosis, myocarditis, cardiac arrhythmias, heart failure, and acute coronary syndrome [73] . regarding the initial 41 confirmed covid-19 cases in wuhan, 5 of those patients suffered myocardial affliction shown by the rapid escalation of troponin i level in the cardiac region [71] . covid-19 can cause myocardial trauma through an increase in inflammatory factors, such as il-6, lactate dehydrogenase, ferritin, and ddimer, which indicates the formation of a cytokine storm [69] . the virus can also impair myocardial tissue through hypoxemia and respiratory damage [71] . covid-19 may also impact the heart directly, inducing myocardial injury through viral myocarditis and stress cardiomyopathy rather than via inflammatory factors [69] . the multi-organ pathology of covid-19 also includes the cerebrovascular system since the virus may have neurotrophic capabilities [74] . the prospect of complications associated with covid-19 increases for patients with pre-existing neurological conditions [75] . covid-19 may manifest in a multitude of acute and chronic neurological diseases, ranging from acute ischemic stroke to encephalitis, guillain-barré syndrome, and acute necrotizing hemorrhagic encephalopathy [75] . the incidence of stroke in covid-19 patients exemplifies the need for vigilance on non-respiratory diseases that may present with devastating symptoms as that produced by the primary lung pathology. cerebrovascular disease appeared in 5.7% of covid-19 patients with dire symptoms during the late onset of the virus [74] . on average, stroke occurred 12 days post covid-19 diagnosis [74] . in italy, ischemic stroke appeared at a rate of 2.5% in a group of hospitalized patients. this percentage was 5% in china and 3.7% with dutch covid-19 patients [75] . the risk of ischemic stroke in covid-19 patients increases with cardiovascular complications, such as hypotension, heart failure, shock, and arrhythmogenic cardiomyopathy [76] . cerebral ischemia in covid-19 patients has been linked to temporary hypercoagulability in some dire cases [77] . since ace2 receptors are prominent in the endothelial lining of blood vessels, covid-19 may spur vascular endothelial damage, increasing the chance of thrombogenesis and cerebrovascular ischemia [77] . covid-19 may also incite stroke by intensifying inflammation through an increase in d-dimer and crp [74] . however, the exact mechanism behind covid-19-induced ischemic stroke is unknown, so further examination is warranted [77] . along with ischemic stroke, other acute cerebrovascular diseases in the clinical setting of covid-19 have been explored. in wuhan, 36% of 214 covid-19 patients suffered from neurological complications, and 6% of the patients developed acute cerebrovascular disease [77] . covid-19 may injure the neurological system through direct impairment to particular receptors, secondary hypoxia, cytokine storm generation, and retrograde nervous transport [75] . starting in the lung, the virus can retrogradely move across neuronal synapses to infiltrate the brainstem, demonstrating how covid-19 has also caused brainstem-induced cardiovascular and pulmonary complications [75] . covid-19 can spur glial cell activation and heightened inflammation in the cns through the escalation of il-6, il-12, and il-15, as well as tumor necrosis factor-alpha (tnf-α) [75] . the virus may also bind to the ace2 receptors in endothelial cells along the blood-brain barrier, allowing the virus to infiltrate the central nervous system [75] . moreover, covid-19 can potentially impair the bloodbrain barrier, leading to acute necrotizing encephalopathy [75] . 20% of hospitalized patients developed hypoxic ischemic encephalopathy after the onset of covid-19 [75] . additionally, the pathology behind covid-19-induced encephalitis can be linked to inflammation and edema [75] . pcr has revealed the presence of covid-19 in cerebrospinal fluid, which may also stimulate encephalitis [74] . in some cases, covid-19 has incited the development of guillain-barré syndrome (gbs), where the body's immune system attacks nerves, leading to flaccid paralysis. the virus has been associated with 5 gbs cases in italy and 2 gbs cases in wuhan [75] . in addition to acute repercussions, covid-19 infection may have chronic neurological consequences. although a direct link between covid-19 and demyelinating disease, such as multiple sclerosis, has not yet been established, the association between human coronaviruses and multiple sclerosis (ms) has been explored. an oc43 coronavirus detection test was conducted on human brain autopsy samples for patients with ms. the ms samples demonstrated a substantially higher level of oc43 than the control group [76] . in addition, ms mice models developed acute encephalomyelitis and a chronic demyelinating condition when infected with the jhmv coronavirus strain [76] . although the exact mechanism mediating the chronic manifestations of sars-cov-2 infection is currently unknown, the interaction of sp and ace2 contributes significantly. sp-ace2 binding results in host-cell dysfunction that precipitates the development of these acute and chronic symptoms, including host-cell death. therefore, the serious long-term repercussions of covid-19 should be closely monitored in infected patients with high sp and ace2 expression. human behavior like the current pandemic, the 1918 spanish flu pandemic disrupted the daily life of the plagued countries [78] . when the first article surrounding this influenza was published in a newspaper in madrid, only a handful of cases were present. however, this occurred during the season of local holidays in madrid which entailed people gathering in ballrooms and large parties. these events put people at high risk for exposure and eventually the number of infected individuals was slowly rising [78] . eventually, towns began to take the pandemic more seriously and various measures were taken. schools and universities were beginning to shut down for the rest term, yet public gatherings such as church services or cinemas remained open [78] . public health measures were later adopted including the disinfection of public areas using phenolic oil or creoline which was a popular disinfectant at the time. cafeterias, churches, tramway wagons, and other public areas were being disinfected. even mail began to be disinfected. in some cities, the streets were cleaned with a mixture of water and sodium hypochlorite, and spitting was banned. after seeing how quickly the virus was able to spread through public gatherings, public health officials imposed new safety measures for the citizens of spain [78] . these measures included avoiding public gatherings in closed environments, avoiding any direct contact with those who were ill, and ventilating homes. they also recommended that individuals clean and disinfect their mouth and nostrils with hydrogen peroxide [78] . covid-19 has disrupted the lives of people worldwide in an eerily similar fashion. the virus originated in wuhan, china with the first case documented in december 2019. after only a few months, the virus began spreading worldwide. by march, covid-19 was declared a pandemic by the world health organization (who). many schools and universities shut down beginning in the middle of march and transitioned into virtual classes taken online at home. adults have also been quarantined at their homes with jobs and businesses shutting down around the same time as schools in march with an exception to essential workers such as healthcare workers and grocery store workers. having individuals stay home largely limits the number of people gathering in public. these measures, more commonly known to the public as social distancing, are implemented to reduce the number of interactions between individuals. this prevents individuals who are infected with the virus but have not been diagnosed with covid-19 or are asymptomatic to interact with healthy individuals. covid-19 is transmitted by one individual to another when in close proximity [79] . social distancing aims to reduce transmission of the virus by physically separating individuals. this includes the closure of office buildings, a ban on large public gatherings, and the separation of individuals in public [79] . guidelines created by the cdc recommend that people avoid any close contact with individuals who are sick and maintain 6 feet of distance from others. another method used to prevent the spread of covid-19 is isolation. patients who are diagnosed with covid-19 or are suspected to have been exposed are isolated in hospitals to prevent the transmission of the disease to non-infected individuals [79] . patients are typically isolated for the full duration of the virus's incubation period to ensure that they are no longer infected when leaving isolation. isolation can occur individually or in groups. individuals have also started to self-isolate themselves at their own homes. when individuals begin to feel ill, they refrain from any public activity such as their job and will isolate themselves at home. individuals also practice this when they have been exposed to another individual who has covid-19 [79] . the cdc also recommends avoiding any mass gatherings and crowded areas and covering the mouth and nose with a mask or face cover when around others. quarantine is an old yet effective tool in controlling disease outbreaks. this practice was commonly employed in fourteenth-century italy when ships coming from a plagueinfected port arrived at a port in italy [79] . the ships were required to anchor and wait for 40 days before passengers were allowed to disembark. these 40 days allowed for asymptomatic passengers to begin showing symptoms and be identified. this allowed for only healthy passengers to be allowed off the ship while infected individuals were sent to get treatment. this also prevented the asymptomatic passengers from spreading the disease to people in the port city. this quarantine method was also implemented during the sars epidemic in 2003. this measure was thought to be successful and effective [79] . quarantine means restricting the movement of individuals who have been exposed to a disease but are not ill or are simply asymptomatic. by restricting the movement of these individuals, the number of interactions they have with other individuals is decreased which is crucial if they are infected with a disease. during the quarantine period, individuals are carefully monitored for signs of any symptoms [79] . quarantine methods have been used during the covid-19 pandemic, especially on those individuals who have traveled to countries with high rates of covid-19. many travelers who returned from a country with high covid-19 rates such as china were subjected to a mandatory 2-week quarantine. some countries used isolation tents to conduct the 2-week quarantine while others simply strongly recommended individuals to self-isolate at home. quantitative assessments of the effectiveness of quarantine may provide empirical evidence for the recommended guidelines. the susceptible-exposed-infectious-recovered (seir) model shows the speed at which individuals move from the susceptible state onto the exposed, infectious, and recovered state [80] . preventive measures taken in china such as travel bans, home quarantine, and extended vacations have been successful in decreasing the transmission of infection. there were decreases in the transmission of covid-19 after these measures, however, they are only effective if the isolation period is long enough [80] . good hygiene also plays a key role in preventing the spread of disease. hand hygiene has been proven to be especially significant. appropriate handwashing can break the transmission cycle and reduce the risk of spreading disease remarkably [81] . health officials recommend frequent handwashing and avoiding touching the nose, mouth, or eyes with unwashed hands to prevent the spread of disease. an equally essential hygiene protocol requires cleaning and disinfecting any surface that is touched daily like a phone or a light switch. additionally, mask-wearing has been recommended by the center for disease control (cdc) to block pathogens from entering the respiratory tract. it is recommended to wear a mask whenever in public and in direct contact with other individuals regardless of whether they are ill or not. stronger masks such as n95 masks can block 91% of pathogens [81] . while wearing a mask has been recommended by health officials, the need for better equipment to avoid the spread of the virus intranasally may require innovations in the use of air purifiers and nasal protective devices. finally, it is critical to monitor one's health and be alert for any possible symptoms of covid-19. for those experiencing the symptoms of covid-19, urgent medical care is highly recommended. if the covid-19 test comes back positive, the cdc recommends staying at home except to receive medical care. it is essential to stay away from others while ill and to disinfect any surfaces that come into contact with the patient. the best methods to stay proactive about covid-19 are to maintain good hygiene and follow the cdc behavioral guidelines. washing hands frequently, remembering to wear a face mask in public areas, and practicing social distancing guidelines are all efficient ways to protect from covid-19. when the spanish flu began spreading to other countries such as the u.s., finding a vaccine became a tantamount goal for scientists and clinicians alike. during the peak of this epidemic spanish flu "vaccines" were developed and distributed throughout the u.s. health officials informed the public that these vaccines would protect from the influenza. however, the specific cause of influenza was yet to be discovered [83] . these vaccines targeted multiple types of bacteria that had been isolated from victims of the spanish flu, suggesting that influenza was a bacterial disease. these spanish flu vaccines were widely used but still had little to no supporting evidence for their benefits. only a few attempts to test their effectiveness were made once the vaccines were distributed [82] . eventually, the surgeon general, american public health association, and editors of the journal of the american medical association made a public statement that there was currently no serum or any means of curing influenza and that there was no vaccine to prevent it. they informed the public that all claims made by newspapers and other sources were unofficial and experimental. this editorial warned health officials and physicians to stop making false promises without evidence to support them. without knowing the causative agent of the spanish flu there was no evidence to suggest the effectiveness of a vaccine [82] . the scientific community was failing to find the cause of the influenza which led researchers to believe that influenza was a viral disease. virology was still in its early stages during this time. however, by the 1930 s there was an improvement in the process of isolating and identifying pathogens [84] . these research advances aided in the search for the spanish flu vaccine. the spanish flu vaccine was not developed until 2005, more than 80 years after the beginning of the outbreak. the virus causing the spanish flu, known as the h1n1 virus, is considered an avian influenza virus that underwent multiple mutations. another avian influenza virus known as h5n1 began to infect humans and had a mortality rate of approximately 60% [83] . although human to human transmission was not possible at the time of this bird flu pandemic, researchers were worried that this virus could mutate and have the capability to transfer from human to human posing a dangerous threat to public health. the live attenuated influenza vaccine elicits an immune response that triggers the production of chemokines and cytokines associated with t-cell activation that clears the virus rapidly [83] . currently, there is no vaccine that prevents covid-19. while human behaviors, including social distancing and proper hygiene that plagued the spanish flu, remain a challenge in battling covid-19, there is much clamor for evidence-based and science-supported treatments in the current pandemic. in particular, databases for registered clinical trials have been established allowing a much better translation of safe and effective approaches for covid-19. the genome of the sars-cov-2 was sequenced in mid-january triggering researchers around the world to begin developing a vaccine [84] . the first covid-19 vaccine candidate began human clinical trial in mid-march (clinicaltrials.gov: nct04283461). as of april 8, 2020, there were 115 global covid-19 vaccine candidates, 73 of which were currently at preclinical stages [84] . some of the more advanced candidates targeting sp is crucial to developing a potent vaccine that blocks the virus from binding to ace2 and entering host cells, thus preventing infection from occurring [84] . polyclonal murine antibodies against sp already effectively inhibit sars-cov-2 uptake in mice [13] . knowledge of the exact molecular structure of sp will facilitate the development of human antibody-based vaccines that potently inhibit sp-mediated sars-cov-2 cell entry. most of the covid-19 vaccines that are being developed are in north america, while there is also development activity in china, other parts of asia, australia, and europe. the global vaccine effort has been exceptional in terms of scale and speed. projects indicate a vaccine may be available as soon as early 2021. this would be a massive change from traditional vaccine development which takes on average over 10 years. even the accelerated development of the ebola vaccine took 5 years [84] . to assess the efficacy of these vaccines, covid-19-specific animal models are being developed. these include ace2transgenic mice, hamsters, ferrets, and non-human primates [84] . sars-cov-2 vaccine development poses some obstacles. it is crucial to optimize antigen design to ensure we receive an optimal immune response. some researchers argue the best approach is targeting the full-length protein, while others believe that targeting only the receptor-binding domain is more efficient [85] . researchers are also worried about exacerbating lung diseases as a result of antibody-dependent enhancement as seen previously with sars and mers vaccine candidates. this effect may be associated with a type 2 helper t-cell response. this is why it is critical to test all vaccines with a proper animal model and to safely monitor all clinical trials [85] . adjuvants may be required to generate a sufficient immune response. adjuvants triggering a th1 response and showing a high neutralizing antibody response are more suited to be protective (nct04368988). although we can infer the duration of protection received from a vaccine by looking at vaccine experience with sars and mers, we do not know the duration of protection from a covid-19 vaccine. more research is required to find the duration of immunity as well if vaccines will be potent in single or multiple doses [85] . vaccine development has been moving at a rapid pace since the genetic sequence for sars-cov-2 was released in january. moderna's mrnabased candidate entered clinical trials only 10 short weeks after the sequence was published. since then many other developers have begun the clinical trial phase (nct04283461). some candidates have even begun to manufacture additional materials for clinical trials in phase 2 studies. after phase 2, commercial manufacturing will take place. facilities capable of producing large quantities of product will need to be identified, proper technology will need to be transferred, and manufacturing processes will need to be adapted. all of the research and development procedures need to be performed without knowing the clinical efficacy of the vaccine candidate [85] . in situations like this covid-19 outbreak, it is inefficient to conduct randomized and controlled trials with placebo groups. although this approach is feasible, it is not nearly as fast, and results are often harder to interpret [85] . a possible method moving forward could be to test several different vaccines simultaneously in a trial designed using one shared control group. this allows more participants to receive an active vaccine. this method is advantageous in terms of the number of participants receiving the vaccine and the speed of the trial, however, developers often avoid trials that generate comparative data [85] . currently, most of the leading vaccine developments are still in phase i of clinical trials. further data and investigation on these possible vaccines are necessary to assess the efficacy of these vaccines. if any of these vaccines prove to be viable in their later phases, developers will be able to manufacture vaccines as early as the start of next year. in response to the influenza virus, the global influenza surveillance network was established in 1947 and is responsible for the continuous monitoring of antiviral drugs for clinical treatment [86] . two antiviral compounds, m2 ion channel blockers (adamantanes) and na inhibitors are the current options available to combat infection and counteract the spread of viruses. adamantanes are commonly used to inhibit iav virus replication and block entry and na inhibitors block the release of virions after budding from the host cell. however, the rapid emergence of drug-resistant viral strains has placed limitations on the use of na inhibitors and m2 blockers [87] . therefore, more initiatives should be put forth for the development of new drugs and clinical assessment. the antiviral medication amantadine is no longer recommended due to the increase in resistance to h1n1 and h3n2 and it only inhibits influenza a. oseltamivir is continuously used, but the recent development of new targetoriented drugs such as zanamivir, favipiravir, baloxavir, and pimodivir sheds new light on the treatment for influenza. there is currently no proven effective antiviral treatment for covid-19. however, many antiviral treatments have demonstrated efficacy for the treatment of covid-19 includi n g l o p i n a v i r a n d r i t o n a v i r , c h l o r o q u i n e , a n d hydroxychloroquine that may help to alleviate symptoms in patients. with over 300 clinical trials currently testing various antiviral drugs, treatment has focused on the uses of chloroquine, hydroxychloroquine, lopinavir/ritonavir, favipiravir, remdesivir, and oseltamivir. the focus has been primarily on repurposing the available therapeutic drugs to treat patients with the sars-cov-2 infection. hydroxychloroquine and chloroquine are antiviral drugs that possess similar chemical structures and have been commonly used for the treatment of rheumatoid arthritis and malaria [88] . their mechanism and action involve targeting lysosome to control graft-versus-host disease in humans. chloroquine can inhibit the entry of sars-cov-2 and prevent virus-host cell fusion by interfering with glycosylation of the ace2 receptor and its binding with sp. this suggests the potential use of chloroquine in the early stages of infection before the virus can reduce ace2 expression and activity [89] . both hydroxychloroquine and chloroquine can inhibit certain cellular functions and molecular pathways involved in immune activation and are both being tested in in vitro studies for effectiveness [88] . the u.s. food and drug administration (fda) has authorized the use of chloroquine and hydroxychloroquine for emergency treatment of covid-19 and has the potential to decrease symptoms and progression of the disease. however, both drugs have been associated with cardiac risks in patients and more evidence is required to determine the safety and effectiveness of these medications in treating covid-19. another antiviral drug being explored for the treatment of covid-19 is lopinavir/ritonavir (lpv/r). lopinavir is a protease inhibitor and ritonavir is a booster and they are commonly used to treat hiv infection in combination. in vitro studies have demonstrated the ability of lopinavir in inhibiting sars-cov, reducing symptoms such as diarrhea and recurrence of fever, and lowering the risk of acute respiratory distress syndrome with lpv/r and ribavirin [88] . the use of lpv/r for treatment of covid-19 has proven effective in combined therapy for reducing serious complications such as acute kidney injury and secondary infections [90] . a combination of lopinavir and ritonavir has significantly improved the clinical condition of sars-cov patients and serves as a viable treatment option in covid-19 infections [91] . favipiravir (brand name avigan) has been developed and used for the treatment of avian influenza or other types of influenza that are resistant to na inhibitors. favipiravir inhibits the rna-dependent rna polymerase of rna viruses such as ebola, influenza, and norovirus and induces lethal rna transversion mutations to produce a nonviable virus phenotype [90] . favipiravir is converted to an active phosphoribosylated form in cells that can be recognized by viral rna polymerase and inhibiting rna polymerase activity [92] . repurposed favipiravircan be used as an experimental agent against single-strand rna virus sars-cov-2 and clinical trials have demonstrated that patients treated with favipiravir have a superior recovery rate [89] . in mildmoderate covid-19 patients, favipiravir reduced the time of fever reduction and cough relief, but concerns exist relating to adverse effects and not enough knowledge has been produced to recommend favipiravir as an effective treatment [88] . remdesivir is another novel antiviral drug that was originally developed for the treatment of the disease ebola. remdesivir can inhibit viral rna polymerases and has widespread activity usage against filoviruses and coronaviruses. in vitro testing has developed to demonstrate remdesivir activity against sars-cov-2 and demonstrates clinical improvement. in animal experiments, the drug has proven to reduce viral load in lung tissue of mice with mers-cov, improve lung function, and alleviate the lung damage. additionally, remdesivir yields promising results for covid-19 patients in recovering from pneumonia. in a study of patients using the drug in the united states, 70% of patients had improvement in regards to oxygen requirements and were extubated from mechanical ventilation [89] . currently, there are four clinical trials in the united states, and two additional trials in china registered on clinicaltrials.gov [88] . remdesivir serves as a promising therapeutic treatment for covid-19. additionally, oseltamivir (tamiflu), which is approved for the treatment of influenza a and b, is used to target the neuraminidase distributed on the surface of the influenza virus to inhibit the spread throughout the body. several clinical trials are studying the effectiveness of oseltamivir against covd-19 and also in combinations with chloroquine and favipiravir. however, a study in wuhan reported no positive outcomes after administering oseltamivir and it does not serve as a recommended treatment for covid-19 [88] . a potential target for drug development for covid-19 also involves inhibition of ace2, the host cell receptor for the s protein of sars-cov-2 that is primed by tmprss2 protease and may prevent the entry of the virus. the antimalarial drugs, chloroquine, and hydroxychloroquine have been shown to inhibit terminal phosphorylation of ace2 and serve as candidate drugs against the covid-19 disease [90] . chloroquine has inhibition activity against sars-cov-2 at relatively high doses but includes a potential risk of side effects. additionally, the triple combination of cepharanthine/ selamectin/mefloquine hydrochloride serves as a candidate drug combination against the sars-cov-2 infection with access through ace2 [90] . the ability to weaken the binding of the virus to ace2 may prevent entry and replication of the virus and can be blocked by experimental and established drugs. further studies are required to determine the benefits of such medications and their therapeutic potential in inhibiting the binding of the virus to ace2. with over 300 clinical trials targeting various antiviral medications for the treatment of covid-19, there are still no final verified antivirals specific to covid-19. further testing is still needed to explore the efficacy and safety of these antiviral drugs. research regarding sars-cov-2 has demonstrated the potential of repurposing drugs with appropriate pharmacological effects and therapeutic efficiencies in the treatment of covid-19 patients [89] . the antiviral drugs being explored including hydroxychloroquine, chloroquine, remdesivir, favipiravir, and lopinavir and ritonavir may be able to limit the spread of the virus and reduce morbidity and mortality caused by the covid-19 pandemic. since a vaccine is still not available, more research is required to approve antiviral drugs against sars-cov-2. from recent clinical trials, the antiviral drugs remdesivir, favipivir, and lopinavir and ritonavir can be administered for the treatment of severe covid-19 pneumonia and to lower the mortality rate of the disease [90] . to this end, antiviral treatment of covid-19 is promising, with several potential drug candidates that demonstrate the capacity to perturb the growth and development by interfering with sp and ace2. azithromycin targets sp directly, preventing viral uptake, while hydroxychloroquine interferes with ace2 terminal glycosylation, thus weakening sp-ace2 binding [93, 94] . overall, the repurposing of available drugs for immediate use of treatment for covid-19 could improve the currently available clinical management. plasma therapy is the process of extracting therapeutic molecules, such as antibodies and plasma, from the immunized or recovering individual and transferring the extracted molecule to ill individuals [95] . this type of regenerative medicine was first developed to battle the spanish flu during the early 20th century [96, 97] . during the spanish flu pandemic, transfusion of blood from influenza survivors significantly decreased mortality rates [97] . another notable discovery was made during a latter viral outbreak. during the arenavirus outbreak, victims often experienced severe hemorrhage [96] . in vivo studies during this outbreak highlighted the protective abilities of plasma therapy. specifically, transfusing blood from immune species into healthy species decreased the severity of the symptoms. these discoveries opened the possibility of utilizing plasma therapy to battle against viral outbreaks. tremendous advancements were made in the molecular techniques and production of human immunoglobin during the years after each subsequent pandemic or epidemic, including measles, hiv/aids, and 2003 sars-cov. after several past experiences with viral outbreaks worldwide, there was one constant observation: plasma therapy must be applied as soon as possible after the first onset of disease. clinical trials have reported that healthy and infected patients who received plasma therapy early displayed a decrease in symptom severity and mortality rate [96] . based on previous pandemics and discoveries on plasma therapy, if plasma therapy became an available treatment option for covid-19 patients, convalescent plasma therapy should be applied at the earliest, either as a preventive measure or early therapeutic option, to maximize the efficiency of the treatment. plasma therapy may be used to harvest antibodies from individuals who have been exposed to the sars-cov-2 to treat covid-19 patients with pulmonary, neural, or cardiovascular dysfunctions. the transplanted antibodies may bind to sp or ace2 sites, preventing the coronavirus from fusing with the cell membrane and injecting its viral genome. by blocking this crucial entry point, inflammatory symptoms and infections significantly decreased compared to patients without antibodies [98] . this occurs due to the correlation between inflammatory symptoms and infections and the expression of ace2. if no blockage is present, the virus binds to pulmonary ace2 and transfers its viral genome into the cell, causing cell death [99] . the increased cell mortality and loss of ace2 expression stimulate inflammatory and immune activity [99] . inflammatory response and overreaction of the immune system to the increased infection and cell death would further cause pulmonary damage, possibly worsening respiratory distress, post-viral infection, and severe acute respiratory failure [99, 100] . conversely, blocking ace2 or viral sp with antibodies would preserve ace2 expression and lower cell mortality, decreasing inflammatory symptoms and infections. viral infections may also damage neural and cardiovascular cells, decreasing ace2 expression in the brain and heart, respectively [99, 100] . similar to pulmonary damage, neural and cardiovascular cell mortality induces inflammatory activity, further causing structural damage and cognitive and cardiovascular dysfunctions [99, 100] . on the other hand, blocking ace2 or sp via antibodies prevents cell death from viral infection, which consequently preserves ace2 expression and increases anti-inflammatory activity. based on previously mentioned research advancements in plasma therapy, its utility for covid-19 patients has reached clinical testing. in addition to 152 publications of review and commentary articles on convalescent plasma therapy for covid-19, there are 173 ongoing clinical trials that are investigating the use of convalescent plasma for covid-19. small clinical studies conducted in the us and east asian countries have displayed an increase in antibody titers and a decrease in the viral activity of plasma-treated patients [101] [102] [103] [104] [105] . the viral load in covid-19 patients was nearly undetectable and assumed to be eliminated after treatment [102, 104] . despite different dosage of plasma, ranging from 200 ml to 500 ml, no findings on adverse events of convalescent plasma therapy were reported [101] [102] [103] . additionally, significant recovery in respiratory functions was demonstrated seven days after convalescent plasma infusion [102] . infusion of convalescent plasma also stabilized the health conditions of critically ill covid-19 patients, removing the need for ventilators over time [101] . patients who fully recovered after receiving convalescent plasma therapy were discharged from hospitals [103] . current evidence favors the effective use of convalescent therapy for covid-19. to further encourage the idea of plasma therapy as a possible covid-19 treatment, direct comparisons of protocols, safety, and efficacy in ongoing or proposed clinical trials should be made to assess the potential of convalescent plasma for covid-19. in one clinical trial, 10 chinese patients with severe cases of covid-19 received convalescent plasma treatment after approximately 16 days of contraction [102] . 200-ml convalescent plasma was transfused into each patient. patients were able to tolerate this level of dosage without displaying any symptoms [102] . clinical symptoms significantly improved in all patients 3 days after receiving treatment followed by no traces of sars-cov-2 after one week. improvement in pulmonary functions, such as oxygen saturation and lymphocyte counts, due to antibodies obtained from the treatment highlight decreased inflammation and hyperactivity of the immune system [102] . in another clinical study in south korea, two patients received convalescent plasma treatment. similar to the previously described study, both patients were diagnosed with severe pneumonia and acute respiratory distress syndrome [101] . both patients received a total of 500 ml dose of convalescent plasma, which was administered twice in 12hour intervals. patients received treatment at different times: one receiving 22 days after onset of symptoms and the other after 7 days [101] . no adverse effects of the transfusion were present in either patient. both patients demonstrated a significant recovery in respiratory functions and levels of lymphocytes increased almost immediately after plasma therapy [101] . both clinical studies presented findings that parallel previous discoveries from earlier pandemics and studies regarding convalescent plasma treatment. earlier treatment after exposure to sars-cov-2 was more effective compared to later treatments [102] . as seen in previous studies, inflammatory activity decreased and normal pulmonary and respiratory functions returned after convalescent plasma therapy [101, 102] . additionally, plasma therapy does not present any detrimental or adverse reactions to patients even when administered large dosages [101, 102] . these results suggest that plasma therapy is safe and effective for covid-19 patients, especially when treated early. however, some limitations are present in the two clinical trials. both clinical trials had a small number of subjects. furthermore, some patients received other sources of standard care while also receiving plasma therapy, such as antiviral treatment [102] . indeed, this opens the possibility of antiviral treatment contributing to the recovery of the patients. however, this also raises uncertainty regarding the true therapeutic effects of plasma therapy in human subjects. additionally, some patients were treated much later after exposure to sars-cov-2 compared to other patients [101, 102] . for patients who received plasma treatment late, it is difficult to determine whether their recovery was due to convalescent plasma or natural recovery. the varying times of treatment along with limited human trials limit the potential of convalescent plasma infusion. future clinical studies should increase the number of test subjects and apply plasma therapy as early as the first onset of symptoms. as noted above, plasma therapy involves the use of convalescent plasma to transfer antibodies and boost the patient's antiinflammatory response against covid-19 [106, 107] . with the advancements in purification technology over the past several decades, human immunoglobulin for intravenous use have been used as the second line of passive immunotherapies against these diseases using manufactured plasma-derived immunoglobulins (ig) [106, 107] . in addition to immunoglobulins, the blood contains a heterogeneous population of cells, one of which are stem cells, that may confer therapeutic benefits. if the stem cells stand as active components rendering the therapeutic inflammatory response in convalescent plasma, then isolating these stem cells, as opposed to the whole plasma treatment, may afford equally or more robust functional outcomes in covid-19 patients. stem cell therapy stands as the prominent regenerative approach in a wide range of diseases, which interestingly include covid-19 primary symptoms (i.e., lung disease) and comorbid diseases such as but not limited to diabetes, cardiovascular and cerebrovascular disorders [108] . besides their regenerative capabilities, stem cells release immune system modulators which highlight their applicability in covid-19's aberrant immune and inflammatory response. to this end, stem cells can impart protection by attenuating inflammation and are being explored as treatment options for covid-19 in clinical trials (table 1 ). preclinical studies have consistently shown the therapeutic effects of stem cell therapy on animal models of diseases plagued with impaired immune and inflammatory reactions [112] [113] [114] , and the benefits are thought to be due to the release of modulators that attenuate the deleterious immune and inflammatory response that leads to secondary cell death. prominent sources of stem cells for regenerative therapy are embryonic stem cells (escs), fetal stem cells, induced pluripotent stem cells (ipsc), and adult stem cells. pluripotent escs generated from the blastocyst can differentiate into all three germ layers [110] . their great differential ability elucidates their therapeutic potential for regenerative therapy, as they can evolve into specialized cells [115] . however, ethical concerns surrounding the destruction of embryos and high risk for tumorigenicity limit the use of escs in clinical applications. fetal stem cells are produced from fetal tissues, such as the placenta, amniotic fluid, and fetal membranes [116] . they have great proliferative capabilities, can differentiate into a variety of progenitor cells, and are less likely to spur immune rejection [117] . ipscs are genetically reprogrammed somatic cells that behave similarly to escs [110] . they show promise in the treatment of degenerative disorders due to their self-renewal and differentiative capabilities [118] . however, they are likely to be rejected by the host and have the highest tumorigenicity of other stem cell types. adult stem cells are generated from adult tissue and include mesenchymal stem cells (mscs), hematopoietic stem cells, and resident adult stem cells [119] . they show significant therapeutic potential for damaged tissue repair as they can be engineered to differentiate into specialized cells, replacing impaired cells, as well as secrete anti-inflammatory agents [110, 119] . mscs derived from bone marrow and the human umbilical cord demonstrate significant therapeutic efficacy for regenerative therapy and have a long track record of safety in hematologic disorders [120] . mscs carry significant restorative capabilities, as they can differentiate into specialized cells like chondrocytes, adipocytes, and osteoblasts and secrete paracrine and autocrine factors that promote tissue rehabilitation and ameliorate inflammation [121] . clinical trials using mscs to treat hematologic disorders have confirmed their safety profile, expediting their application in nonhematological disorders, and the use of mscs for brain disorders associated with a destructive immune response like stroke and tbi have reached clinical trials with promising results [122, 123] . because mscs possess the ability to regulate the immune and inflammatory activity, msc therapy can potentially be used to treat covid-19. as previously described, the intermolecular interaction between the viral sp and human ace2 phase ii castem cells will be intravenously injected into patients with or without acute respiratory distress syndrome (ards) induced by covid-19. patients will receive doses of either 3, 5 or 10 million cells/kg, and dose escalation will ensue if initial cell infusion proves to be safe. adverse events, mortality rate, time it takes for rt-pcr to be negative for sars-cov-2, and changes in blood oxygen levels will be assessed. in addition, levels of il-1 beta, il-2, il-6, and il8 will be evaluated to further elucidate castem efficacy. leng et al. [109] mesenchymal stem cells (mscs) complete post intravenous msc administration, covid-19 patients demonstrated ameliorated pulmonary function two days after treatment, upregulation of peripheral lymphocytes, reduction in c-reactive protein, and elimination of cxcr3+ cd4+ t cells, cxcr3+cd8+ t cells, and cxcr3+ nk cells 3-6 days following treatment. nct04313322 wharton-jelly derived mesenchymal stem cells (wj-mscs) phase i covid-19 patients will be administered wj-mscs intravenously at a dosage of 1x10e6/kg. they will be given three doses three days apart. clinical improvement will be evaluated over three weeks in addition to the conduction of a ct scan and rt-pcr for viral rna. nct04473170 autologous non-hematopoietic peripheral blood stem cells (nhpbscs). phase ii survival rate and clinical improvements for covid-19 patients are to be monitored after treatment with (nhpbscs). patient immune profile will be evaluated, measuring levels of immune biomarkers, such as cd3, cd4, cd8. number of acute phase proteins and inflammatory markers (e.g. crp, esr, il-6) will also be examined. ye et al. [110] allogeneic human dental pulp stem cells (dpscs) phase ii 20 patients with severe pneumonia induced by covid-19 were administered intravenous dpscs at a dosage of 3.0x107 human dpscs in 30ml saline solution. the trial began on april 6th and will continue until december 31st. neutrophil, t lymphocyte, b lymphocyte, natural killer cell, and macrophage levels, along with alterations in serum cytokine levels (il-1 β, il-2, tnf-a, itn-γ, il-4, il-6, il -10) will be examined. nct04366323 allogeneic and expanded adipose tissue-derived mesenchymal stem cells phase ii allogeneic and expanded adipose tissue-derived mscs administered in two doses (80 million cells) to patients with severe pneumonia caused by sars-cov-2 infection. safety and efficacy will be measured through the frequency of adverse events and mortality rate. nct04346368 bone-marrow derived mesenchymal stem cells phase ii bm-mscs will be delivered intravenously at a dose of 1*10e6 /kg to severe covid-19 patients. safety and efficacy of cell-based treatment assessed through clinical symptom amelioration, frequency of adverse events, and mortality rate. improvement of pneumonia will be evaluated through alterations in pao2/fio2 ratio and ct scan. viral density, changes in cd4+, cd8+ cells, and cytokine levels will also be analyzed. nct04416139 umbilical cord derived mesenchymal stem cells (uc-mscs) phase ii patients with acute respiratory distress syndrome caused by covid-19 will be treated with intravenous uc-mscs at a dose 1 million xkg. patient improvement will be evaluated over three weeks, along with the assessment of the immune profile, investigating the stem cells' effect on the cytokine storm. changes in tnfa, il-10, il-1, il-6, and il-7 cytokine levels in plasma will be noted. nct04437823 umbilical cord derived mesenchymal stem cells phase ii covid-19 patients will receive intravenous treatment of uc-mscs at a dose of 5 x 10^5 cells/kg on days 1, 3, 5. the frequency of adverse events and mortality rate will be assessed to determine clinical efficacy, along with the conductance of ct scans, pcr tests, and sequential organ failure assessment (sofa). sengupta et al. [111] bone-marrow derived mesenchymal stem cells complete exosomes (exoflo™) isolated from allogeneic bm-mscs were administered intravenously (15ml) to 24 severe sars-cov-2 patients. the clinical condition and oxygenation state of patients were significantly ameliorated and an 83% survival rate was detected. the average amount of neutrophils decreased and mean numbers of cd3+, cd4+, and cd8+ lymphocytes were upregulated. nct04315987 nestacell® (msc) phase ii covid-19 patients will be treated with intravenous nestacell® at a dose of 2x10^7 cells on days 1, 3, 5 and 7. mortality rate and respiratory improvement will be assessed over 10 days. oxygen saturation will be measured through hypoxia status and pao2/fio2 ratio. cd4+ and cd8+ t cell levels will also be evaluated to determine the immune profile. initiates the infection of host cells [124] . once the virus enters the host, macrophages of the immune system detect and bind to the foreign molecule to fend off the virus, recruiting proteins and initiating the immune cascade in the process [39] . the viral-induced immune cascade causes extensive tracheobronchial inflammation, further causing pulmonary damage in covid-19 patients and promoting sars and ards symptoms, such as pulmonary edema, hypoxia, respiratory distress, and lung damage [40] . furthermore, replicated sars-cov-2 utilizes the lungs to circulate within the patient's body. similar to the pathology of the pulmonary infection, sars-cov-2 can infiltrate and infect ace2-harboring cardiac or neural tissue. the viral activity prompts an increased immune response, leading to inflammation and further causing damage in the heart or brain [36, 39] . since covid-19 causes pulmonary damage as well as cardiovascular and cerebrovascular infection by overreacting the immune system and increasing inflammation, msc transplantation may be a suitable approach against covid-19. in addition to its anti-immune and anti-inflammatory mechanisms, mscs may possess the ability to interfere with viral docking via ace2-sp interaction. as previously discussed, mscs regulate immune and inflammatory activity by inhibiting inflammatory factors and cytokine storms. mscs are able to bind to various surface receptors to activate these mechanisms, such as il-1r, tnfi, and iir [125] . due to its immune-sensing adaptability, mscs may also bind to ace2, an entry receptor for sars-cov-2. if mscs possess this ability, the stem cell may competitively inhibit sars-cov-2 from entering and infecting the cell while also promoting anti-inflammatory mechanisms. future studies should investigate this potent interaction between ace2 and mscs or other compatible stem cells that can interfere with ace2-sp docking of sars-cov-2 and modulate immune and inflammatory responses. preclinical studies have investigated the use of mscs in treating lung diseases with similar dysregulation of regular immune and inflammatory responses as covid-19. in one notable study, the endotoxin model of acute lung injury was used to observe the therapeutic effects of msc [126] . mice were treated with msc 4 h after severe lung damage. msc treated mice demonstrated significant pulmonary recovery. significant survival and histological improvements were also observed in msc-treated groups compared to the control. additionally, proinflammatory activity was replaced with an anti-inflammatory response to endotoxin after msc doses were introduced, highlighting the immunomodulatory effects of msc [126] . several clinical trials have investigated the therapeutic effects of msc on lung diseases, including ards. phase 1 dose-escalation study showed no safety issues for the treatment of ards with bmscs (nct 01775774) [127] . furthermore, high-dose mscs improved daily sequential organ failure assessment (sofa) scores when compared to lower doses. however, when the same group rolled out the phase iia study, there were no statistical differences between treatment and placebo groups in mortality and the number of ventilator-free days [128] . this discrepancy may have been due to the substantial variation in cell viability observed at the time of injection [128] . another phase i, double-blind, placebo-controlled trial assessing the safety of human adipose tissue-derived msc transplantation in patients with ards revealed short-term improvement in oxygenation, but phase i in conjunction with standardized treatment (oseltamivir + azithromycin), patients will be treated with intravenous uc-mscs at a dose of 1x10^6 cells/kg. clinical amelioration will be assessed through the presence of dyspnea and sputum, fever, ventilation necessity, monitoring of blood pressure, heart rate and respiratory rate, and oxygen saturation. cxcr3, cd4, cd8, and cd56 cell counts, along with il-6 and il-10 levels will be analyzed to distinguish the anti-inflammatory capabilities of uc-mscs. nct04486001 adipose-derived allogeneic mesenchymal stem cells phase i adipose-derived allogeneic mscs will be intravenously delivered to covid-19 patients. clinical improvement following stem cell treatment will be assessed via frequency of adverse incidents, mortality rate, the number of ventilator and icu free days, total hospital and icu days, and improvement in oxygenation. nct04390152 wharton-jelly mesenchymal stem cells phase ii patients will receive two doses of wj-mscs (50*10e6 cells) along with standardized treatment of hydroxychloroquine and lopinavir/ritonavir or azithromycin and ventilator support. mortality rate, sequential organ failure assessment (sofa), and frequency of adverse events will be evaluated. in addition, wj-msc efficacy against the cytokine storm will be assessed by monitoring levels of il-6, il-8, il-10, and tnf alpha. ventilator-free days, icu-free days and duration of hospital stay remained unchanged (nct01902082) [129] . although the efficacy of the treatment of lung diseases with mscs is not yet conclusive, its safety and promising preclinical data warrant further exploration to covid-19-especially in face of the high morbidity and mortality of the current pandemic. because of the overlapping pathology of neurological diseases stemming from a harmful inflammatory response and covid-19-induced pulmonary, cardiovascular, and cerebrovascular disorders, mscs may provide therapeutic benefits to covid-19 patients where multiple organ systems are affected. the envisioned msc therapy for covid-19 entails a minimally invasive approach. mscs injected intravenously (iv) display the propensity to migrate to the lungs making iv administration favorable to treat pulmonary diseases [130, 131] . another route to consider is intranasal administration. intranasal administration of mscs has been tested with neurological diseases, such as stroke, highlighting the homing capability of mscs [132] . mscs delivered through the nose have also been shown to be able to restore alveolar growth and vascular development in rat models of bronchopulmonary dysplasia [133] . these relevant findings from other disease indications highlight the many modes of delivery for stem cell therapy for covid-19, which must be taken into consideration in future clinical trials. furthermore, because inflammation is an evolving, progressive, and chronic pathology, prolonged treatment through repeated administration of mscs may be necessary for covid-19 patients. in this case, less invasive methods like iv injection or intranasal administration are favorable. the potent immunomodulatory capacity of mscs renders it a promising option as a novel treatment method for covid-19. although there are currently 43 proposed or ongoing clinical trials employing stem cell therapy against sars-cov-2 infection, there are no approved msc-based treatments or therapies. preliminary results from the us and china demonstrate improved immune function without adverse events in covid-19 patients treated with mscs [134, 135] . intravenous msc transplantation increases the levels of peripheral lymphocytes and regulatory dendritic cells, with a simultaneous reduction in overactive cytokine-secreting leukocytes, c-reactive protein, and tnf-a [134] . additionally, delivery of bone marrow-derived msc exosomes improves patient oxygenation, increases neutrophil and t-lymphocyte counts, and decreases acute phase reactant production [135] . treatment with human umbilical cord wharton's jelly-derived mscs (hwjcs) similarly improves pulmonary function, normalizes leukocyte counts, and abates acute phase reactant release [136, 137] . msc-based therapy is also effective in reducing mortality rates among h7n9-induced ards patients. the similarities in systemic multi-organ complications between h7n9 and sars-cov-2 infections, along with direct evidence of the benefits of mscs transplantation for covid-19, further supports the potential of stem cells as an effective treatment [138] . stem cells are at the forefront of innovative regenerative medicine-based strategies targeting tissue repair and immunomodulation of covid-19 [139] [140] [141] [142] . the use of umbilical cord-derived stem cells [136, 137, [143] [144] [145] [146] and bone marrow-derived mscs [134, 135, [147] [148] [149] [150] for targeting neuroinflammation has been documented. the validated safety and efficacy of stem cells in treating respiratory conditions, including ards and lung damage, predicts their value for ameliorating covid-19 outcomes. furthermore, stem cells effectively repair and regenerate extra-pulmonary tissues damaged by sars-cov-2 infection, notably the heart and brain. perhaps most compelling is that mscs cannot be infected by sars-cov-2, as they are ace2 negative. therefore, sp-mediated viral uptake is blocked, allowing healthy mscs to directly counter inflammation and tissue damage induced by sars-cov-2. this ensures that patients treated with mscs will continue to benefit through the full life cycle of the transplanted stem cells, particularly the sustained anti-inflammatory and regenerative effects. the future of stem cell therapy for covid-19 is promising, with anticipated positive clinical trial results to come over the next few months. since the report of dozens of cases of pneumonia of unknown causes in wuhan, hubei province in december of 2019, covid-19 has infiltrated across the globe and shaken the lives of the vast majority of the human population. despite the us leading the world at 2.1 million confirmed cases and 116,130 deaths, many states have begun reopening in response to an economic crisis [1]. georgia was the first state to begin reopening businesses on april 24th and since then, all 50 states are now in the process of easing restrictions. amidst this pandemic, another equally devastating threat to the future health of our society is rising: "disillusioned comfort", or our ignorance of the dangers of the virus that give us a false blanket of security that we may continue to live life as it is. when looking back on the history of pandemics, the 1918 spanish flu reminds us that human response, or lack thereof, stands as the major determinant for the spread of infectious diseases. whether the reopening of this country was premature, only time will tell. although covid-19 primary triggers a respiratory disease, pervasive myocarditis and fatal arrhythmia cases in infected patients suggest covid-19's deleterious effects on the cardiovascular system. moreover, a subset of patients also manifests neurologic symptoms likely due to the virus' ability to retrogradely travel from the lung to the brainstem cardiorespiratory center via neuronal synapses. this multi-organ, heart-brain infection may exacerbate respiratory failure. the critical entry receptor for sars-cov-2, the virus responsible for covid-19, is the ubiquitous ace2 receptor to which viral sp docks. the ace2 may reveal acute and chronic multiorgan effects of covid-19 which warrants further investigation and continued action to lessen the spread of 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critical discussions on covid-19. conflict of interest the authors declare no conflict of interest.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. key: cord-033780-184e64tr authors: smith, rasheid; geary, sean m; salem, aliasger k title: implications of current and future approaches to coronavirus disease 2019 testing date: 2020-10-13 journal: nan doi: 10.2217/fvl-2020-0318 sha: doc_id: 33780 cord_uid: 184e64tr nan the relentless spread of severe acute respiratory syndrome-associated coronavirus-2 (sars-cov-2), the cause of the coronavirus disease 2019 and the resulting pandemic has challenged the global economy, as well as impacting on the efficacy of governmental and medical systems to the serious detriment of not only those vulnerable to the potentially fatal consequences of becoming infected with sars-cov-2, but to the broader populace. since the first reports of covid-19 in wuhan, china in december 2019, the scientific community has been in a race against time to elucidate the minutiae of the virus to inform public policy and implement appropriate strategies to maximize public safety. according to the weekly epidemiology report by the world health organization (who), as of 24 august 2020, 23 million people have contracted covid-19 worldwide and this has led to 800,000 reported deaths though actual figures maybe higher due to inefficiencies in testing and reporting from individual nations. while sars-cov-2 has a case death rate in the usa of 3.1%, it can vary from as high as 28.9% (yemen) to as low as 0.2% (qatar) [1] . socioeconomic factors greatly influence this case mortality as countries with resources and proper planning and implementation have reduced this to near zero in some cases. analyzing the facts garnered to date about the sars-cov-2 virus, a major route of transmission is via droplets and aerosols emitted from infected individuals by coughing, sneezing or breathing [2] . transmission through surfaces (fomite transmission) is assumed to be possible due to reports demonstrating that viable viruses can be found on surfaces such as plastic and stainless steel for up to 72 h after being administered with aerosols [3] . however, the applicability of this study in real life scenarios has been called into question due to high initial viral administration [4] . nevertheless, transmission of sars-cov-2 via fomites has been listed as possible by who and the centers for disease control and prevention (cdc). sars-cov-2 has an incubation period of approximately 14 days with 97.5% of patients who will develop symptoms doing so within 11.5 days, on average [5] . there is an age-related disparity with respect to a patient's susceptibility to infection with estimates that individuals over 20 years being approximately twice as likely to become infected as children (0-19 years) [6] . additionally, sars-cov-2 infections in children/adolescents (ages 0-19 years) are often less severe [7, 8] , although cases of serious medical conditions such as hyperinflammatory shock have been documented [9] . additionally, a significant portion of patients (adults/children) may present as asymptomatic (or subclinical presentation) [10] [11] [12] [13] , although the prevalence of this and how it relates to presymptomatic patients (patients who have not yet but will develop symptoms in a reasonable time) are not well understood. the complexities of covid-19-related symptoms are still revealing themselves, as a growing body of research suggests that covid-19 presentation is not limited to the respiratory system but symptoms may manifest as neurological (headaches, stroke) [14] [15] [16] , gastrointestinal (diarrhea, nausea) [17, 18] dermatological (erythematous rashes, widespread urticaria, chickenpox-like vesicles) [1, 19] , renal (proteinuria, hematuria), cardiovascular (myocardial ischemia, myocarditis) or endocrinal (diabetic ketoacidosis, hyperglycemia) [20] . this broad array of clinical presentations can be explained by the pathophysiology of sars-cov-2. the spike protein which facilitates cellular entry of sars-cov-2, binds to ace-2 after priming by the host serine protease, tmprss2 [21] . ace-2 is expressed by a broad array of tissues including cardiomyocytes, vasculature, gastrointestinal epithelia, and renal tubules [22] resulting in multiple potential viral entry ways. the presentation of multiorgan involvement is hypothesized to result from five mechanisms: direct viral toxicity, endothelial cell damage and thromboinflammation, dysregulation of the immune response and dysregulation of the renin-angiotensin-aldosterone system [20] . the current reality is that sars-cov-2 is a highly transmissible airborne disease with a broad presentation of symptoms and leaves lasting damage in severe cases, and for which there is a scarcity of effective medications to treat it. this coupled with the gaps in knowledge has resulted in a dire situation where implementing sound policy and planning has been the only reliable vanguard of public safety. integral to ultimately ensuring public safety is the implementation of effective testing in order to: define what percentage of individuals are testing positive; identify trends in disease prevalence; identify potential epicenters of disease spread and facilitate swift measures to mitigate spread of the virus. the importance of testing has been aptly demonstrated by countries such as south korea and germany where increased availability of sars-cov-2 tests has resulted in a containment of viral outbreaks. under current us food and drug administration (fda) guidelines, covid-19 testing is performed using an in vitro diagnostic device (a device used to diagnose a disease or condition from human samples). such a device is granted emergency use authorization (eua) in laboratories that are certified and meet guidelines governed under clinical laboratory improvement amendments of 1988 (clia) to perform high complexity (h), moderate complexity (m) testing or testing in a patient care setting (w) that has received an clia certificate of waiver. testing thus can be performed in a centralized (i.e., samples are collected then transported to a facility certified to perform tests) or point of care (poc; i.e., samples are tested immediately after sample collection often within the same vicinity of the patient) setting. similar to other coronaviruses, the sars-cov-2 genome comprises positive sense single stranded rna and is approximately 30 kb in length [23] . as such the gold standard for sars-cov-2 detection is through the reverse transcriptase polymerase chain reaction (rt-pcr). rt-pcr involves a two-step process converting rna from samples (from nasopharyngeal swabs or saliva) to complimentary dna and then using fluorescently labeled primers to amplify and detect the presence of targeted genes. the sars-cov-2 genes targeted for detection so far include: rdrp, orf1ab, the n gene, the e gene and the s gene; with sensitivities ranging from 3.8 to 10 rna copies present per reaction dependent on the gene used [24] . currently, a large array of rt-pcr-based tests are available to detect covid-19 as summarized in other publications [24, 25] and there are currently 158 tests that have received eua by the fda [26] . despite being the current gold standard, rt-pcr tests are not without their limitations, as many testing protocols are currently only using cycle threshold values (number of amplification cycles required for the signal to exceed the background levels) to determine infection status. using the cycle threshold value in this manner only informs as to the presence of the virus and may not reveal disease progression, severity and viral load in the sample; and as such the results are largely qualitative despite the inherent quantitative nature of real-time rt-pcr [27] . additionally, comparing the plethora of tests available is challenging due to inter-test variability due to specific viral genes targeted, primer design; differences in stated limit of detection (lod) parameters (copies/ml, 50% tissue culture infective dose, copies/μl, copies per reaction volume). an additional limitation is the turnaround time for rt-pcr tests with average run times being approximately 4 h. in august 2020, the fda gave eua to two saliva-based tests (saliva direct, yale university; i-covid university of illinois, urbana champaign, il, usa) for sars-cov-2, which alleviates issues of the uncomfortable nature of the nasopharyngeal swabs [28] . additionally, saliva-based tests limit the risk of infection to medical personnel as well as time required to collect samples as medical personnel are not required to collect samples. these saliva tests will provide the opportunity to lower the cost of collecting samples as they are generally easier to collect from patients. chest computed topography (ct) images have revealed characteristics common among covid-19 patients including reverse-halo sign, consolidative opacities, ground glass opacities, interlobular septal thickening and the crazy-paving pattern [29] [30] [31] . as of the drafting of this manuscript chest ct images were not recommended by the cdc or the fda for diagnosis of covid-19 due to overlapping similarities with viral pneumonia, h1n1 and sars cov thus the possibility of false positive cases being reported with chest ct diagnosis is evident [24, 32] . nevertheless, initial studies have demonstrated that chest ct imaging is more accurate than rt-pcr at detecting sars-cov-2 patients [32] with 97.2% versus 83% in the early stages of infection [33] . other drawbacks of implementing chest ct imaging as a mass diagnosis tool include processing costs and the technical skill required to accurately read chest ct images. resultantly, chest ct imaging may be better used as a complimentary technology to molecular detection techniques such as rt-pcr and antibody serum tests. immunoassays (antibody serum tests), such as enzyme-linked immunosorbent assays (elisas), are used to detect the presence of serum antibodies (either iga, igg or igm) to viral proteins and can indicate when a person has developed an immune response to sars-cov-2. the antibody response to any of sars-cov-2 four major structural proteins: spike (s) glycoprotein, small envelope (e) glycoprotein, membrane (m) glycoprotein and nucleocapsid (n) protein may vary with intensity and affinity during the course of an infection [34] . infected individuals also go through antibody maturation and antibody class switching, refining the affinity and class, respectively, of antibodies produced to various epitopes, generally transitioning from (igm to iga or igg) [35] . the sensitivity of immunoassays is dependent on the time of testing, viral antigen epitope used [36] and isotype of antibodies being detected [37] . the greatest sensitivity is observed when a mixture of antibodies is being detecting. overall the elisa technique is unsuitable for poc testing due to the amount of labor and extended time it takes to generate results; however, in a centralized setting it may offer the advantages of determining antibody titers and selective isotype detection to monitor disease progression and the type/quality of immune responses [38] . direct chemiluminescence immunoassays (dclis) may have advantages over elisas with run times of 1-2 h versus 4 h for elisas; and being automated, dclis are less labor intensive [38] . while covid-19 serology tests are not used to diagnose covid-19, the importance of serological testing in general is coming into focus currently with attempts to tackle unanswered questions about covid-19. questions such as the issue of post infection immunity and how it relates to antibody production and isotype switching in asymptomatic patients. the aforementioned tests can generally be considered as centralized testing approaches where samples are collected and then results are generated by a certified laboratory that has expertise in consistently performing the test (e.g., chest ct, serology or rt-pcr). under certain circumstances, such as during a pandemic, this system may be overwhelmed by the sheer volume of tests, resulting in significant lag times between testing and results [39] . important tools that may alleviate the burden of these centralized approaches are poc tests. poc devices generally generate results more quickly and usually in a qualitative fashion. these technologies are based on a lateral flow assay, which is a paper-based platform for the detection of analytes (proteins and nucleic acids in complex mixtures), where the sample is placed on a test device and the results are displayed within 5-30 min [40] . however, issues as to the sensitivity of lateral flow immunoassays (lfias) have been raised when compared with centralized approaches such as dclis or elisas (e.g. 66% [lfias] vs 97% [dclis] vs 84.3% [elisas]) [41] . notable advances in biosensing have allowed the rapid and accurate detection of sars-cov-2; the clusters of regularly interspaced short palindromic sequences (crispr)-based diagnostic product, sherlock biosciences' 1h test for sars-cov-2, is one such example. this test achieves detection of rna (or dna) virus signatures through two consecutive reactions: amplification of the viral rna using an isothermal amplification reaction and detection of the resulting amplicon using crispr-mediated cleavage of the rna based reporter (crispr-mediated collateral reporter unlocking) [42, 43] . this test has also been validated to be 100% specific and 100% sensitive when using a fluorescence readout and 100% specific and 97% sensitive when using a lateral-flow readout [44] . emerging technologies such as graphene field-effect transistors have been used to detect as low as 1 fg/ml of sars cov-2 spike protein in transport medium for nasopharyngeal swabs and detect sars-cov-2 in clinical samples with readings obtained within 1 min [45] . additionally, qui et al. used the plasmonic photothermal effect and a localized surface plasmon resonance sensing unit to attain real-time and label-free detection of sars-cov-2 viral sequences including rdrp, orf1ab and e genes with an lod of 0.22 ± 0.08 pm for the rdrp gene [46] . it is prudent to note that this study was performed in nuclease free water and therefore its compatibility with medical samples will still have to be evaluated. silicon nanowire field-effect transistors have been previously used to provide labelfree, real-time detection of proteins [47] , dna [48] and even single virions [49] ; however, issues with sensitivity in physiologically relevant substrates and high manufacturing costs has hampered furthering this technology [50] . these issues have largely been solved in a variety of ways on a laboratory scale [51] [52] [53] , thus employing these approaches toward sars-cov-2 detection (protein or genetic) is possible and could result in technologies that fill an important void in the current spectrum of testing methodologies. equally important to detecting positive cases is identifying the cases that will most likely transition to severe respiratory conditions and therefore requiring admission into intensive care units. this requires testing for additional biomarkers that adequately indicate disease severity and onset of symptoms such as acute inflammation. the elecsys r il-6 immunoassay, which detects il-6, an early marker from acute inflammation, is the only in vitro diagnostic test to have received eua to manage covid-19. other cytokines and proteins can be implemented which are associated with increased severity of covid-19 cases such as il-2, il-7, il-10, g-csf, ip-10 and cardiac troponin among others [13] . park et al. have demonstrated that tgfβ-induced protein and its acetylated by-product are consistently elevated in the blood of sars-cov-2 pneumonia patients and tracks the severity of the sars-cov-2 infection [54] . ideally, a sars-cov-2 biosensor should provide accurate, sensitive, reproducible measurements of its target analyte in real-time and be cost-effective to the patient [50] . the present pandemic demonstrates the important impact that such effective biosensors could play in public health. however, the current iteration of tests seems to be divided into two camps: accurate/expensive methods of detection that have the potential for quantitative results but are associated with significant lag times and are only applicable in a centralized testing environment with trained personnel; or inexpensive poc testing that offers qualitative results but may sacrifice sensitivity for ease of implementation and manufacturability. the current situation calls for the mobilization of both tactics to effectively help in dampening the spread of sars-cov-2. qualitative tests are easy to produce en masse and accurately screen the population for sars-cov-2, thus helping to track the spread of the virus. robust, quantitative tests are also required that offer results quickly to elucidate more information on the progression of sars-cov-2 and the physiological response to it. advances in testing may therefore contribute to filling the current gaps in knowledge with regards to the 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respiratory syndrome coronavirus 2 detection multiplexed electrical detection of cancer markers with nanowire sensor arrays silicon nanowire biosensor for highly sensitive and rapid detection of dengue virus electrical detection of single viruses silicon nanowires and their impact on cancer detection and monitoring surface modifying doped silicon nanowire based solar cells for applications in biosensing enhanced sensing of nucleic acids with silicon nanowire field effect transistor biosensors ultrasensitive label-and pcr-free genome detection based on cooperative hybridization of silicon nanowires optical biosensors acetylated k676 tgfbip as a severity diagnostic blood biomarker for sars-cov-2 pneumonia key: cord-293056-kz3w0nfh authors: indes, jeffrey e.; koleilat, issam; hatch, ayesha nzeribe; choinski, krystina; jones, davis brent; aldailami, hasan; billett, henny; denesopolis, john m.; lipsitz, evan title: early experience with arterial thromboembolic complications in patents with covid-19 date: 2020-08-28 journal: j vasc surg doi: 10.1016/j.jvs.2020.07.089 sha: doc_id: 293056 cord_uid: kz3w0nfh introduction: little is known about the arterial complications and hypercoagulability associated with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. we sought to characterize our experience with arterial thromboembolic complications in patients with hospitalized for coronavirus disease 2019 (covid-19). methods: all patients admitted from march 1 to april 20, 2020 and who underwent carotid, upper, lower and aortoiliac arterial duplex, ct angiogram or mra for suspected arterial thrombosis were included. a retrospective case-control study design was used to identify, characterize and evaluate potential risk factors for arterial thromboembolic disease in sars-cov-2 positive patients. demographics, characteristics and laboratory values were abstracted and analyzed. results: during the study period, 424 patients underwent 499 arterial duplex, ct angiogram or mra imaging studies with overall 9.4% positive for arterial thromboembolism. of the 40 patients with arterial thromboembolism, 25 (62.5%) were sars-cov-2 negative or admitted for unrelated reasons and 15 (37.5%) were sars-cov-2 positive. the odds ratio for arterial thrombosis in covid-19 was 3.37 (95% ci 1.68 – 6.78, p=0.001). although not statistically significant, in patients with arterial thromboembolism, patients who were sars-cov-2 positive compared to those testing negative or not tested tended to be male (66.7 % v. 40.0%, p=0.191), have a less frequent history of former or active smoking (42.9% vs 68.0%, p=0.233) and have a higher white blood cell count (wbc 14.5 vs. 9.9, p=0.208). while the sars-cov-2 positive patients trended toward a higher the neutrophil-to-lymphocyte ratio (8.9 vs. 4.1, p=0.134), cpk level (359.0 vs. 144.5, p=0.667), crp level (24.2 vs. 13.8, p=0.627), ldh level (576.5 vs. 338.0, p=0.313) and ferritin level (974.0 vs. 412.0, p=0.47), these did not reach statistical significance. patients with arterial thromboembolic complications and sars-cov-2 positive when compared to sars-cov-2 negative or admitted for unrelated reasons were younger (64 vs. 70 years, p=0.027), had a significantly higher body mass index (bmi) (32.6 vs. 25.5, p=0.012), a higher d-dimer at the time of imaging (17.3 vs. 1.8, p=0.038), a higher average in hospital d-dimer (8.5 vs. 2.0, p=0.038), a greater distribution of patients with clot in the aortoiliac location (5 vs. 1, p=0.040), less prior use of any antiplatelet medication (21.4% vs. 62.5%, p=0.035) and a higher mortality rate (40.0 % vs. 8.0%, p=0.041). treatment of arterial thromboembolic disease in the covid-19 positive patients included open thromboembolectomy in 6 patients (40%), anticoagulation alone in 4 (26.7%) and 5 (33.3%) did not require or their overall illness severity precluded additional treatment. conclusions: patients with sar-cov-2 are at risk for acute arterial thromboembolic complications despite a lack of conventional risk factors. a hyperinflammatory state may be responsible for this phenomenon with a preponderance for aortoiliac involvement. these findings provide an early characterization of arterial thromboembolic disease in sars-cov-2 patients. the exact relationship between the occurrence of arterial thrombotic events and sars-cov-2, 13 remains unclear. we therefore set out to identify and characterize patients in the early stage of 14 the epidemic with arterial thromboembolic disease and its relationship to sars-cov-2 infection 15 at our institution. 7 imaging studies performed during the study period were reviewed. these studies included 1 computed tomography angiography (cta), magnetic resonance angiography (mra), and/or 2 arterial duplex. the choice of imaging modality (cta, mra or arterial duplex) was obtained at 3 the provider's discretion and was based on the presenting clinical complaints. for patients 4 requiring duplex testing, the majority of these studies were conducted as portable bedside tests. patients presenting with neurologic events underwent mra in addition to carotid duplex 6 evaluation as part of the stroke protocol. sars-cov-2 status was obtained from the medical record. patients were tested for sars-cov-9 2 based on clinical suspicion and in those requiring surgical treatment as part of their pre-10 procedural evaluation. nasopharyngeal swab specimens were tested for sars-cov-2 by in-11 house polymerase chain reaction (pcr) testing. confirmatory repeat testing was obtained in 12 patients with high clinical suspicion for covid-19 and negative initial testing. patients were 13 excluded if sars-cov-2 test results were pending at the time of data abstraction. patients were 14 divided into one of three groups: sars-cov-2 positive, sars-cov-2 negative, and patients not 15 tested. the sars-cov-2 positive group included patients with a range of covid-19 16 symptomatology (mild to severe) as well as those tested as part of routine preoperative 17 preparation. the sars-cov-2 negative group included patients who presented with respiratory 18 symptoms or other influenza-like illness symptoms and who were found to have a negative pcr 19 result. patients admitted for other reasons and without suspicion for covid-19 were not tested 20 and categorized as such. of the 25 patients who are sars-cov-2 negative or not tested 5 were 21 negative, 19 were not tested (admitted for unrelated reasons) and 1 patient was under patients with imaging that confirmed acute thrombosis were further evaluated. sars-cov-2 2 positive patients were compared to the combined sars-cov-2 negative and not tested groups. retrospective chart review abstracted demographic variables including age, race, body mass 4 index (bmi), gender, ethnicity, medical comorbidities and treatment for further evaluation. the 5 presence of metabolic syndrome was defined as patients with three or more of the following: 6 bmi greater than 30, serum triglyceride level 150 mg/dl or greater within the last 6 months, 7 serum high-density lipoprotein (hdl) level less than 40 mg/dl in men or 50 mg/dl in women 8 within the last 6 months, systolic blood pressure greater than 130 mmhg or diastolic blood 9 pressure greater than 85 mmhg, and fasting serum glucose greater than 100 mg/dl. additional missing data was assumed to be missing at random and excluded from the analysis for that 2 variable. the odds ratio for acute arterial thrombosis in covid-19 was evaluated with a two-3 tailed fisher's exact test. this study was approved by the institutional review board of 4 mmc/aecom with a waiver of informed consent for this observational review (#2020-11452). (table i) . patients positive for sars-cov-2 had significantly higher d-dimer levels at the time of imaging 4 (217.3 vs.1.8, p=0.038) and average in-hospital d-dimer levels (8.5 vs. 2.0, p=0.043) than 5 sars-cov-2 negative or not tested patients (table ii) . sars-cov-2 positive patients exhibited 6 a higher white blood cell count (wbc) despite lack of statistical significance (14.5 vs. 9.9, 7 p=0.208). there were no significant differences in prothrombin time (pt), aptt, platelet count, (table iii) . the sars-cov-2 positive patients were treated with a left ventricular assist device. amputation was required in 3 of the patients in the sars-cov-2 1 negative or untested group. there were significantly more sars-cov-2 positive patients with aortoiliac involvement 4 compared to the sars-cov-2 negative or not tested patients (33.3% vs. 4.0%, p=0.040). there 5 was a trend towards less tibial/pedal (13.3% vs. 40.0%, p=0.154) and more upper 6 extremity(20.0% vs. 8.0%, p=0.537) involvement in the sars-cov-2 positive patients when 7 compared to sars-cov-2 negative or not tested patients . carotid and femoropopliteal 8 involvement was relatively equal between the two groups (table iii) . the covid-19 specific severity of disease on presentation was documented (table iv) . most of 11 the patients with arterial thrombosis had critically severe disease (60.0 %). three patients (20.0% 12 ) were asymptomatic, and one each had mild, moderate and severe covid-19 presentation 13 (6.7% each). there was no observed correlation between the severity of covid-19 disease and 14 the degree of arterial thrombosis. none of the patients in the study had bleeding complications. 15 30 day outcomes resulted in 6 deaths due to covid 19 complications, 4 major amputations, 3 16 patients were discharged to skilled nursing facilities (snf) and one to home. ranged from mild to severe in intensity. patients with arterial thrombosis who were sars-cov-10 2 positive had significantly higher d-dimer levels, bmi, were younger, and less often on 11 antiplatelet medications as compared to patients who were sars-cov-2 negative or not tested. in addition, we noticed that sars-cov-2 positive patients with arterial thromboembolic 13 complications exhibited a trend towards higher wbc count and neutrophil to lymphocyte ratio 14 which may indicate an increased inflammatory response. other markers of increased 15 inflammation such as crp and cpk were also greater in the sars-cov-2 positive group when 16 compared to their negative counterparts, but these did not reach statistical significance. all 17 patients with acute arterial ischemia are placed on a heparin drip upon diagnosis. we did not observe a typical cardioembolic pattern in the sars-cov-2 positive group but rather 20 a picture that was more consistent with in situ thrombosis. the arterial thrombotic events figure 1c ). the majority of arterial emboli originate in the heart and travel to the extremities, with the lower 13 extremities being affected more frequently than the upper extremities and carotid arteries. there remains much to learn about the sars cov-2 and the disease it causes. in addition to an 2 early lack of comprehensive testing for both active disease as well as prior infection, the 3 reliability of many of these tests have been called into question. false negative rates ranging 4 from 20% to as high as 50% for diagnostic tests have been quoted and more recently it has been 5 suggested that antibody tests may not be as indicative of prior infection as first believed. 11,12 6 whether these are issues with the tests themselves or a peculiarity of the virus is unclear. we do 7 not know whether the virus can remain dormant and "reactivate" in an individual nor whether 8 individuals develop immunity and if so to what degree and for how long. we also do not know 9 whether the impact of inflammatory changes and possible endothelial damage will resolve with 10 convalescence or whether the changes may persist and lead to symptomatic vascular disease in 11 the future at an undetermined time point. if the latter is the case, we will need to anticipate this 12 eventuality and investigate therapies to help delay or prevent these occurrences. clinical characteristics of 2 coronavirus disease 2019 in china clinical characteristics 4 of 113 deceased patients with coronavirus disease 2019: retrospective study hospitalized patients with 2019 novel coronavirus-infected pneumonia in covid-19 pathophysiology a review table 1: demographic factors and clinical characteristics for patients with covid-19. iqr, interquartile range. dvt, deep venous thrombosis. pe, pulmonary embolism. bmi, body mass index htn, hypertension. dm, diabetes mellitus. hld, hyperlipidemia. cad, coronary artery disease pvv, peripheral vascular disease. copd, chronic obstructive pulmonary disease. chf, congestive heart failure. ckd, chronic kidney disease. covid-19, coronavirus disease 2019. ali, acute limb ischemia table 2: laboratory values and treatment characteristics for patients with covid-19. iqr, interquartile range. wbc, white blood cell. aptt, activated partial thromboplastin time. scr, serum creatinine. cpk, creatine phosphokinase. crp, c-reactive protein. ldh, lactate dehydrogenase anatomic distribution, treatment details and outcomes for patients with acute arterial thrombosis. ac, anticoagulation. covid-19, coronavirus disease. los, length of stay covid-19, coronavirus disease. ards, acute respiratory distress syndrome, cia, common iliac artery, eia, external iliac artery, sfa, superficial femoral artery, cca, common carotid artery, sca, subclavian artery, ica, internal carotid artery, hcq, hydrochloroquine, rle right lower extremity key: cord-296986-8fuj072z authors: kumar, manish; taki, kaling; gahlot, rohit; sharma, ayushi; dhangar, kiran title: a chronicle of sars-cov-2: part-i epidemiology, diagnosis, prognosis, transmission and treatment date: 2020-05-15 journal: sci total environ doi: 10.1016/j.scitotenv.2020.139278 sha: doc_id: 296986 cord_uid: 8fuj072z abstract in order to benefit the public, community workers and scientific community, we hereby present a chronicle of sars-cov-2 that leads to the unseen precedent of social distancing and lockdown owing to coronavirus disease (covid-19). information on this life-threatening pandemic of covid-19 is sparse and discrete; and the urgency is such that the dissemination of information is increasing with numerous daily publications on the topic. therefore, we developed a comprehensive review on various aspects of sars-cov-2 and covid-19. we scientifically compiled published research, news, and reports from various sources to comprehend and summarize the information and findings on coronaviruses. the review explicitly covers the aspects like genome and pedigree of sars-cov-2; epidemiology, prognosis, pathogenesis, symptoms and diagnosis of covid-19 in order to catalog the right information on transmission route, and influence of environmental factors on virus transmissions, for the robust understanding of right strategical steps for proper covid-19 management. we have explicitly highlighted several useful information and facts like: i) no established relationship between progression of sars-cov-2 with temperature, humidity and/or both, ii) the underlying mechanism of sars-cov-2 is not fully understood, iii) respiratory droplet size determines drop and airborne-based transmission, iv) prognosis of covid-19 can be done by its effects on various body organs, v) infection can be stopped by restricting the binding of s protein and ae2, vi) hydroxychloroquine is believed to be better than chloroquine for covid-19, vii) ivermectin with vero-hslam cells is able to reduce infection by ~5000 time within 2 days, and viii) nafamostat mesylate can inhibit sars-cov-2 s protein-initiated membrane fusion. we have also suggested future research perspectives, challenges and scope. j o u r n a l p r e -p r o o f 8 overall, who defines covid-19 patient as: i) an individual with acute respiratory illness like fever and/or any sign of respiratory ailments like cough, sore throat, and/or has a travel history to locations reported community covid-19 spread during the 14 days prior to the onset of symptoms. or (2) an individual with any acute respiratory illness and/or been in contact with a confirmed or probable covid-19 individual in the last 14 days. or (3) an individual with severe acute respiratory illness with unexplained clinical diagnosis of alternate reasons for the problem. the central idea is to consider even a slightest probable case as a suspected patient based on above points with inconclusive or awaiting test results. while, a laboratory confirmed case is a person with a laboratory confirmation i.e. tested positive for covid-19, irrespective of clinical signs and symptoms (asymptomatic patient). the trickiest feature of covid-19 has lately been identified as being the asymptomatic (up to 60% in india). the risk is so heavy, spread is so fast and symptoms are of so varying nature that even travelling to a community spread country or being a physical contact of an infected person are being treated as symptoms. diagnostic testing for the sars-cov-2 is currently undertaken using two approaches: whole genome sequencing and real-time reverse transcriptase-polymerase chain reaction (rrt-pcr, nucleic acid testing). table 1 compiles the emerging diagnostics techniques being used/developed for sars-cov-2. the sequencing approach was used primarily in the early days of the outbreak, almost all diagnostic testing for sars-cov-2 is done using rrt-pcr . many companies also launched rt-qpcr test kits to assist clinical diagnosis chu et. al. (2020) described two 1-step rtqpcr assays to detect two different regions (orf1b and n) of the viral genome. saliva serves as a promising non-invasive specimen for the j o u r n a l p r e -p r o o f 9 diagnosis of covid-19 patients with a high positive rate of 91.7% (11/12) in determining the disease using rt-qpcr (non-probes sybr based fluorescence signal) . though this technique has a sensitivity of 50%-79% of detection, depending on the protocol used and the sample type; the number of clinical specimens collected (yam et. al., 2020) , safety hazards and detection time are some issues. ct scan is suggested as an auxiliary diagnostic method to avoid reporting false results and to increase sensitivity . early diagnosis and evaluation of disease severity of covid-19 patients can be performed using high-resolution ct (hrct) scan of the chest . however, inability to differentiate from other viral pneumonia makes the interpretation of the hysteresis of abnormal ct imaging tedious and inadequate . nevertheless, the chest ct had a very low misdiagnosed rate (3.9%, 2/51) in comparison with any other method. in previous studies on sars or mers, lung parenchymal irregularity was reported in the central and bilateral upper lobes of lungs (das et. al., 2016; paul et. al., 2004) . however, li and xia (2020) showed that the peripheral (sub-pleural), and middle and lower zones of lungs showed an abnormality at the initial stages, followed by affecting the upper lobes, and in severe cases, all five lobes of both lungs were affected. recently, the center for disease control and prevention (cdc) is working on serologic (antibody) immunoassays (ias) testing i.e. protein testing, for diagnosis of covid-19 (vashist, 2020 (vashist, 2020) . further, wastewater based epidemiological (wbe) surveillance for the genetic material of sars-cov-19 has drawn loads of attention that can be helpful in early detection as well as resurgence of covid-19 (kumar et al., 2019a,b; kumar et al., 2020a,b,c; kitajima et al., 2020) . wbe surveillance are expected to become handy for the potential eradication of the virus in a community. sars-cov-2 moves from the back of the throat to the lungs and then ultimately into the blood. all coronaviruses contain specific genes in downstream regions of orf1 that encode proteins for viral replication, nucleocapsid, and spikes formation (van et. al., 2012) . sars-cov-2 and sars-cov binds with a receptor called angiotensin-converting enzyme 2 (ace-2) found on the respiratory cells with the help of s protein, which helps in entry and replication for further spreading the infection. the receptor-binding domain (rbd) is loosely attached among viruses leading to infection of multiple host (raj et. al., 2013; perlman and netland, 2009 ). fig. 4 (a) demonstrates the interaction of viral s protein with ace-2 on the host cell surface which is highly significant being an initiation step of infection/pathogenicity. cryogenic electronic microscopic structure analysis revealed that the binding affinity of s protein to ace-2 is about 10−20 times higher for sars-cov-2 than that of sars-cov, which is the reason for the higher transmissibility and contagiousness of sars-cov-2 as compared to sars-cov. after , however, there still exists a lack of information about the antigen presentation of sars-cov-2. therefore, till date, no sars-cov-2 specific antiviral agents could be discovered for a possible treatment to save lives and to produce an effective vaccine for future prevention. demonstrates the progression of sars-cov-2 inside the host body. inflammatory response of ards is mainly caused by the release of large amounts of pro-inflammatory cytokines known as 'cytokine storm' . sars-cov-2 infection undergoes 'cytokine storm' that triggers viral sepsis and inflammatory-induced lung injury leading to complications like pneumonitis, respiratory failure, ards, shock, organ failure, and potentially death , prompetchara et. al., 2020 . during the cytokine storm mechanism in the body a wide variety of cytokines (ifn-α, ifn-γ, il-1β, il-6, il-12, il-18, il-33, tnf-α, tgfβ, and others) and chemokines (ccl2, ccl3, ccl5, cxcl8, cxcl9, cxcl10, and j o u r n a l p r e -p r o o f 12 others) are released william and chambers, 2014; cameron et al., 2008) . although, the detailed beneficial role of cytokines is unclear currently due to the recent progression of the disease, there are already clinically tested drugs available which may add value to the current treatment process of cytokine storm. interleukin-6 inhibitors, or il-6 inhibitors drugs, which were found to be helpful in patients of france, china, and new york works based on blocking a specific cytokine associated with inflammation. further, supplementary table-2 shows various supporting statements given by doctors and researchers regarding 'cytokine storm' mechanisms and response in our body. overall, understanding of immuno-pathological mechanism of sars-cov-2 infection has great potential to develop therapeutic strategies to contain the pandemic. the prognosis of covid-19 can be done by its effects on various body organs. the recovery rate from the infection of covid-19 is varying based on its severity. people with mild condition (with few or no symptoms having a resemblance to some upper respiratory diseases such as common cold) are recovering within two weeks, the recovery duration can go up to six weeks for severe or critical disease cases. two to eight weeks of duration from the symptoms onset has 16-24 february 2020). various complications and health issues associated with covid-19 have been reported, like pneumonia affecting lungs, ards which may lead to respiratory and multiorgan failure (cascella et. al., 2020; heymannand and shindo, 2020) , abnormal clotting, sepsis and damage to kidney, liver, and heart . in order to understand the severity of the virus outbreak, it is crucial to focus on the population groups which are at high risk, which will allow them to direct the resources towards the most vulnerable. high variance among the age groups. the age group of >80 years is considered a vulnerable age group due to highest death risk associated with it in all the countries. however, the age group below 50 years, and children can have mild symptoms, but the risk of death is low. the cfr caused by covid-19 is distinctive than the other historical pandemics like malaria and spanish flu, where the most vulnerable age group was children or young adults. however, in the case of covid-19, along with the age group the underlying health conditions also have a significant impact on the death risk ( fig. 5(b) ). the early-stage cfr data of china shows that people with underlying health conditions such as cardiovascular disease, cancer, hypertension, chronic respiratory disease, and diabetes, are at higher risk (who, 23 rd january 2020). the cfr due to covid-19 was highest for those underlying cardiovascular disease (10.5%), followed by diabetes (7.3%), chronic respiratory disease (6.3%), hypertension (6.0%), and cancer (5.6%). a comparison suggests that people with no previous underlying health condition had ten times lesser cfr (0.9%) and thus the elderly age group with pre-existing health conditions are more. j o u r n a l p r e -p r o o f 14 overall, the susceptibility and recovery of the disease is highly dependent upon the innate immunity of the person. transmission. however, to date, no such study has been conducted that could establish a relationship between the fecal−oral transmission of the sars-cov-2. zhou et. al., 2020b reported that viral shedding is one of the most significant ways of transmission and observed that the viral shedding was found to last for 37 days in survivors. the amount of viral load contributed to the environment by asymptomatic and symptomatic patients was conducted by bai et. al., 2020 and zou et. al., 2020a . the authors reported that transmission was possible even in the case of asymptomatic patients, but in the case of symptomatic patients increase in viral loads was observed. as of now, the environmental factors that has been studied to understand the covid-19 transmissions, are the ambient environmental temperature, humidity but still, no authenticity on the relationship is confirmed yet kumar et al., 2020a ; tobias and molina 2020). apart from climatic conditions (temperature and humidity), the transmission may be further triggered by population and medical facility (dalziel et. al., 2018; hemmes et. al., 1960) . based on the assumption that the sars-cov-2 is similar to sars-cov, it has been hypothesized that the sars-cov-2 may not survive at high temperature and humidity (chan et. al. 2011 , kumar et al., 2020 tobias and molina, 2020) the underlying hypothesis of decrease in the spread of viruses during warmer seasons is higher vitamin d levels, resulting in better immune responses (aranow, 2011; kumar et al., 2020) . interestingly, a common man notion is that in cold weather, people tend to stay inside, often in a closed room, at times close to each other, and that enhances the infection probability. a study confirmed a strong relationship does exist between the reported cases, temperature, and absolute with the findings for the influenza virus (park et. al., 2020; steel et. al., 2011; lipsitch and viboud, 2009; shaman and kohn, 2009) , where the high temperature and high humidity leads to a significant reduction in transmission of the influenza virus. this might be due to the following reasons: (i) cold and dry weather might cause weakness in the immunity of the host (kudo et. al., 2019; eccles, 2002) , (ii) unlike influenza virus they might be more stable in lower temperature and within the respiratory droplets (lowen and steel, 2014; tellier, 2009) . also, the observations made for sars cov for temperature and humidity (chan et. al., 2011; yuan et. al., 2006) were inconsistent with the findings of wang et. al., 2020. li et. al., 2020 reported at present, there is no proven antiviral agent that can control the covid-19 outbreak and inactivate sars-cov-2. by 29 th april 2020, no drug has been approved by us food and drug administration that can prevent covid-19. currently, management strategies for covid-19 patients with mild symptoms include infection prevention by using oseltamivir/ intravenous antibiotics, clinical supportive measures for severe patients such as oxygen and mechanical ventilator support . some of the therapeutics drugs and therapy reduction in viral rna (caly et. al., 2020) . another drug, nafamostat mesylate (fusan) as demonstrated by japanese researchers can inhibit the fusion of sars-cov-2 (s) protein and initiated membrane at achievable and safe concentrations in the patients (hannah, 2020) . remdesivir is an antiviral drug that is intravenous and inhibits the synthesis of viral rna by preventing the replication of rna by early termination of rna transcription . lo et. al. (2017) successfully demonstrated that remdesivir is a potent antiviral towards sars and mers-cov. as per the cdc, "remdesivir has in-vitro activity against sars-cov-2 and in-vitro and in-vivo activity against related beta coronaviruses". hydroxychloroquine and chloroquine are anti-malarial drugs that are taken through oral administration, and both of the drugs belong to the quinolone family. yazdany and kim (2020) demonstrated that both the medicines have a potent antiviral property that can control sars-cov-2 in-vitro. roque (2020) reported that the use of hydroxychloroquine is much safer and has more potential of inhibiting sars-cov-2. hydroxychloroquine has been proven more successful than chloroquine (inhibition rate did not exceed 50% ) at inhibiting sars-cov-2 . paton et. al. (2011 ), ooi et. al. (2006 reported negative results of hydroxychloroquine and chloroquine during random testing for influenza in random patients. henceforth, there is lack of report, facts and figures to support the use of hydroxychloroquine and chloroquine as an efficient treatment mechanism (yazdany and kim, 2020) . j o u r n a l p r e -p r o o f 19 early signs are that convalescent plasma therapy can reduce the mortality rate in sars-cov-2 patients (cheng et. al., 2005; lai, 2005; soo et. al., 2004) . mair-jenkins et. al., 2015 showed recovery from sars-cov-2 at early-stage of treatment with convalesced plasma therapy. unlike sars-cov (mair-jenkins et. al., 2015) and mers-cov (koenig et. al., 2015 , many patients are donating plasma with sars-cov-2 antibodies to control covid-19. duan et. al., 2020 demonstrated the potential of convalescent plasma therapy to treat the severe covid-19 patients. 10 patients were treated with plasma therapy, a 200 ml of convalescent plasma neutralize with antibody titers above 1:640 was given to the patients who showed rapid improvement in symptoms with three days of convalescent plasma transfusion. however, treatment by convalescent plasma therapy is still questionable . covid-19 is a severe global health issue which is caused by sars-cov-2. the genomic study revealed that the phylogeny of the sars-cov-2 is very similar to sars-like bat/pangolin. the disease result in respiratory illness like sars-cov and mers-cov and may cause death in severe cases. the mortality is significantly higher in the elderly age group, mostly having preexisting health conditions. at the initial stage the disease may be identified by symptoms such as fever, dry cough, muscle pain and fatigue but challenge of identifying the asymptomatic patient is huge. the transmission is mainly through the respiratory droplets and their diameters, and through direct contact with an infected surface. in the case of airborne transmission, the probability of being infected is very less and case-specific. the higher transmissibility and contagiousness of sars-cov-2 may be attributed to the high binding affinity of sars-cov-2, s protein to ace2. the rapid transmission is due to the weak linkage between the receptor-j o u r n a l p r e -p r o o f 20 binding domain (rbd) of sars-cov2 and the host cell. some of the profound drugs available to control cytokine storm are interleukin-6 inhibitors or il-6 inhibitors, but for older people or people with past medical histories inhibiting the immune system may result in severe health issues. one of the probable ways to curb the infection is to restrict the binding between s protein of sars-cov-2 to ace2. though the cause and effects are known still the transmission mechanism is not fully understood. 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factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin sars-cov-2 viral load in upper respiratory specimens of infected patients we acknowledge the moral support of various initiatives started by the administration of iit gandhinagar. we also acknowledge the help received from ms. payal mazumder, dr. arbind patel, mr alok k thakur and anonymous referees. serum gold-coated antibodies produce colorimetric signal on paper in presence of target. 2 bosch et. al., 2017; 3 amer et. al., 2013; 4 martel et. al., 2013; 5 rissin et. al., 2010; 6 wat et. al., 2008; 7 imai et. al., 2007; 8 shirato et. al., 2007; 9 rowe et. al., 1999 key: cord-290904-ngvhk0qy authors: zheng, zhiqiang; monteil, vanessa marthe; maurer-stroh, sebastian; yew, chow wenn; leong, carol; mohd-ismail, nur khairiah; cheyyatraivendran arularasu, suganya; chow, vincent tak kwong; lin, raymond tzer pin; mirazimi, ali; hong, wanjin; tan, yee-joo title: monoclonal antibodies for the s2 subunit of spike of sars-cov-1 cross-react with the newly-emerged sars-cov-2 date: 2020-07-16 journal: euro surveill doi: 10.2807/1560-7917.es.2020.25.28.2000291 sha: doc_id: 290904 cord_uid: ngvhk0qy background: a novel coronavirus, sars-cov-2, which emerged at the end of 2019 and causes covid-19, has resulted in worldwide human infections. while genetically distinct, sars-cov-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (sars) in 2002–2003, utilises the same host cell receptor as sars-cov-2 for entry: angiotensin-converting enzyme 2 (ace2). parts of the sars-cov-1 spike glycoprotein (s protein), which interacts with ace2, appear conserved in sars-cov-2. aim: the cross-reactivity with sars-cov-2 of monoclonal antibodies (mabs) previously generated against the s protein of sars-cov-1 was assessed. methods: the sars-cov-2 s protein sequence was aligned to those of sars-cov-1, middle east respiratory syndrome (mers) and common-cold coronaviruses. abilities of mabs generated against sars-cov-1 s protein to bind sars-cov-2 or its s protein were tested with sars-cov-2 infected cells as well as cells expressing either the full length protein or a fragment of its s2 subunit. quantitative elisa was also performed to compare binding of mabs to recombinant s protein. results: an immunogenic domain in the s2 subunit of sars-cov-1 s protein is highly conserved in sars-cov-2 but not in mers and human common-cold coronaviruses. four murine mabs raised against this immunogenic fragment could recognise sars-cov-2 s protein expressed in mammalian cell lines. in particular, mab 1a9 was demonstrated to detect s protein in sars-cov-2-infected cells and is suitable for use in a sandwich elisa format. conclusion: the cross-reactive mabs may serve as useful tools for sars-cov-2 research and for the development of diagnostic assays for covid-19. the severe acute respiratory syndrome coronavirus (sars-cov-1), a virus considered to have a zoonotic origin, is the aetiological agent for the infectious disease, sars, which first emerged in 2002-2003 [1, 2] . in december of 2019, another novel coronavirus (sars-cov-2), which causes coronavirus disease , appeared to have crossed species barriers to infect humans and was effectively transmitted from person to person, leading to an outbreak in wuhan, china [3] [4] [5] . this virus subsequently spread worldwide, leading the world health organization (who) to declare a pandemic on 11 march 2020 [6] . to date, sars-cov-2 continues to pose a high global health and economy burden, and as at 3 may 2020, covid-19 had affected 215 countries with over 3.35 million confirmed cases. to tackle the problems caused by sars-cov-2, improving its detection and knowledge of its infection mechanism is important. in this respect, the viral surface spike glycoprotein (s protein) has been demonstrated to play key role in host cell selectivity and binding. the s protein is functionally divided into two subunits, with the s1 subunit containing the receptor binding domain (rbd), which allows attachment to host cells, and the s2 subunit mediating fusion between viral and host membranes (reviewed by li, f.) [7] . phylogenetic analysis revealed that like sars-cov-1 and bat-derived sars-like coronaviruses (sl-covs), sars-cov-2 belongs to lineage b of the betacoronavirus genus [8, 9] . a study of 56 complete and partial sars-cov-2 genomes isolated from covid-19 patients showed very high sequence conservation of more than 99%, indicating a recent introduction of the virus into the human population [10] . although the animal source of sars-cov-2 is not clear, sars-cov-1 is believed to have originated from sl-covs residing in bats [11] [12] [13] [14] . for the majority of sl-covs, the s1 subunit has low sequence identity to that of sars-cov-1, which suggests species-dependent receptor binding [14, 15] . on the other hand, the high amino acid sequence identity of more than 90% in the s2 subunit suggests that the fusion mechanism during virus infection is well-conserved [14, 15] . while sars-cov-2 shares higher whole-genome sequence identity with bat-sl-covzc45 and bat-sl-cov-zxc21 (88-89%) than with sars-cov-1 (79-82%), the rbd of sars-cov-2 is more similar to sars-cov-1 rbd [8, 9] . in line with this, several research groups have demonstrated that sars-cov-2 utilises the same host receptor, angiotensin-converting enzyme 2 (ace2), as sars-cov-1 for viral entry [3, [16] [17] [18] . due to its role in virus entry, the s protein has been the target for the generation of monoclonal antibodies (mab). in our previous work, we used five different fragments of sars-cov-1 s protein to immunise rabbits. a fragment corresponding to residues 1029 to 1192 in the s2 subunit of sars-cov-1 was found to stimulate neutralising antibodies against sars-cov-1 [19] . this fragment was subsequently used to generate a panel of murine mabs with their respective binding domains characterised and described in lip et al. [20] . one of them, mab 1a9, which binds to the s protein through a recently identified epitope within the s2 subunit at amino acids 1111-1130, has the ability to bind and cross-neutralise pseudotyped viruses expressing the s protein of human sars-cov-1, civet sars-cov and bat sl-cov strains [21] . in this study, we aim to verify if the sequence of the immunogen used to generate mab 1a9, as well as three other mabs, is conserved in different coronaviruses and if these mabs bind to the s protein of sars-cov-2 expressed in mammalian cell lines. importantly, mab 1a9 is investigated for its ability to detect the s protein in sars-cov-2 infected cells and purified s protein in a sandwich elisa format when paired with another mab binding to the s1 subunit of sars-cov-2. the name of the viruses, for which sequences are being compared figure on the left side of the alignment, together with the respective sequences' genbank accession numbers. colour schemes represent the following categories of amino acids: blue -hydrophobic, cyan -aromatic, green -polar, magenta -negative charge, orange -glycines, pink -cysteines, red -positive charge, yellow -prolines, white -unconserved. and grown in dmem supplemented with 10% fbs, 100 units/ml penicillin, 100 µg/ml streptomycin and 500 µg/ml geneticin (thermo fisher scientific). cells were maintained at 37 °c with 5% co 2 . the hybridoma for mab 1a9 was previously generated [20] . all mabs were purified from cell culture supernatants using hitrap protein g hp affinity columns (ge healthcare, chicago, il, united states) and stored at −80 °c. the purity of the mab was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoretic (sds-page) analysis. the concentration of the purified mab was determined using the coomassie plus protein assay reagent (thermo fisher scientific). sars-cov-2 s-protein-expressing plasmids were codon-optimised and generated by gene synthesis (bio basic asia pacific, singapore) according to genbank accession number: qhd43416.1. one plasmid is for expressing untagged full-length s protein while the other is for expressing a myc-tagged s-protein fragment consisting of residues 1048-1206 (sars-cov-2 numbering). the pxj40-myc expression vector was used as an empty vector control and pxj40-myc-hbcag plasmid expressing myc-tagged hepatitis b virus core antigen (hbcag) was used as a negative control. 293ft cells were seeded onto 6-cm dishes 24 hours before transient transfection using x-tremegene hp dna transfection reagent (roche, basel, switzerland) according to the manufacturer's protocol. at 24 hours post-transfection, cells were harvested, spun down by centrifugation and washed with cold phosphate buffered saline (pbs) twice. cells were then resuspended in 2× laemmli sample buffer, boiled and sonicated. clarified supernatant containing the protein of interest was obtained by spinning down the cell lysate at 13,000 rpm at 4 °c to remove the cell debris and further analysed by western blot (wb) analysis. equal amounts of total cell lysates were loaded per lane and resolved using electrophoresis on sds-page gels and transferred onto nitrocellulose membrane (bio-rad, hercules, ca, united states). the membrane was blocked in 5% skimmed milk in tris-buffered saline with 0.05% tween 20 (tbst) for 1 hour at room temperature (rt) and incubated with primary antibodies at 4 °c overnight. after the membrane was washed with tbst, it was incubated with a horseradish peroxidase (hrp)-conjugated secondary antibody (thermo fisher scientific) at rt for 1 hour. the membrane was then washed with tbst again and bound antibodies visualised with enhanced chemiluminescence substrate (thermo fisher scientific) using chemidoc mp imaging system (bio-rad). mers: middle east respiratory syndrome; sars-cov-1: severe acute respiratory coronavirus; sars-cov-2: severe acute respiratory coronavirus; sb: shown below. high to low pairwise amino-acid identity are coloured coded respectively by contrasting green to red backgrounds. the sequence identity is not affected by the order in which paired sequences are compared so only one-way comparisons are shown to avoid redundancies; the abbreviation 'sb' is used when the pairwise amino-acid identity in question is already shown in a further cell of the table. for immunofluorescence (if) analysis, cos-7 cells on glass coverslips were transfected as above and fixed at 24 hours post-transfection in 4% paraformaldehyde for 10 min at rt followed by permeabilisation with 0.2% triton x-100 ( b. the abilities of 2b2, 1a9, 4b12 and 1g10 monoclonal antibodies to bind to sars-cov-2 s protein was determined by elisa. individual wells were coated with 20 ng of sars-cov-2 s protein and incubated with serially diluted mabs as indicated. a representative plot from three independent experiment is show for each antibody and error bars correspond to standard deviations of each mab experiment carried out in triplicates. c. each photo depicts an immunofluorescence assay using either no primary antibody, or the primary antibody indicated below it (myc, 2b2, 1a9, 4b12, or 1g10). immunofluorescence analysis was performed on empty vector-transfected cos-7 cells (top photos) and cells expressing full-length sars-cov-2 s protein (bottom photos). the indicated primary antibodies were used followed by alexa fluor 488-conjugated secondary antibody (green). cell nuclei were counterstained with dapi (blue). scale bar = 50 µm. number: 40589-b08v1) was diluted with coating buffer (0.1 m nahco 3 , 34 mm na 2 co 3 ) and a total of 20 ng of protein was loaded into individual wells of a 96 well plate (nunc, roskilde, denmark) and allowed to coat overnight at 4 °c. plates were then washed four times with 0.05% tween 20 in pbs (pbst) and blocked with 5% bovine serum albumin (bsa)/pbst for 30 min before murine antibodies serially diluted with blocking buffer were added to desired wells for 1 hour. plate were washed four times with pbst before incubation for 1 hour with hrp-conjugated goat anti-mouse igg (thermo fisher scientific) secondary antibodies diluted in blocking buffer, and washed four times with pbst. visualisation of bound secondary antibodies was done by the addition of 3,3',5,5'-tetramethylbenzidine (tmb) substrate (thermo fisher scientific) for 5 min in the absence of light and the reaction was stopped with 2 m sulphuric acid. optical density at 450 nm (od450nm) was determined by a tecan (männedorf, switzerland) infinite m1000 reader and normalised od450nm was obtained by subtracting background absorbances determined in bsa coated wells. the human mab cr3022 was expressed in a similar manner as previously described [22] . the variable heavy (vh; genbank accession number: dq168569) and variable light (vl; genbank accession number: dq168570) genes of cr3022 were generated by gene synthesis (bio basic asia pacific) and cloned into pfusess-chig-higg1 and pfuse2ss-clig-hk cloning vectors (invivogen, san diego, ca, united states) respectively. transfection of suspension freestyle 293 cells (thermo fisher scientific) and purification of antibodies by fast protein liquid chromatography is as described in our previous study [23] . mab 1a9 was diluted with coating buffer (0.1 m nahco 3 , 34 mm na 2 co 3 ) and 0.1 µg of antibody was coated onto individual wells of a maxisorp flat-bottom plate (nunc) overnight at 4 °c. the plate was washed three times with pbst before blocking was done using 5% bsa/ pbst at 37 °c for 60 min. dilutions of his-tagged full length sars-cov-2 s protein (sino biological inc., catalogue number: 40589-b08v1) and his-tagged h7n7-ha (sino biological inc., catalogue number: 11082-v08b) were added to desired wells and incubated at 37 °c for 90 min followed by three washes with pbst. 100 µl of cr3022 antibody was added at a concentration of 1 µg/ml and incubated at 37 °c for 60 min followed by three pbst washes before hrp-conjugated goat antihuman igg (thermo fisher scientific) was added for 60 min at 37 °c. finally, after three pbst washes, tmb (sigma-aldrich) was added for 5 min and the reaction was stopped by 2 m sulphuric acid. the od450nm was determined by a tecan infinite m1000 reader. statistical analyses were performed using an unpaired, one-tailed student's t-test with welch's correction for unequal variances. p values < 0.05 were considered statistically significant. all works with live virus were performed in the biosafety level (bsl)3 facility at the public health agency of sweden. vero-e6 cells were infected with sars-cov-2 (sars-cov-2-iso/01/human/2020/swe; genbank accession number: mt093571) at a multiplicity of infection (moi) of one in dmem 2% fbs (thermo fisher scientific). at 24 hour post-infection, cells were fixed with chilled methanol/acetone and the cells were kept at −20 °c overnight. cells were then stained using mab 1a9 at 5 µg/ml at 37 °c for 30 min in if buffer (bsa 0.2%, triton ×100 0.1% in pbs, ph 7.4). the cells were washed three times with pbs and incubated, subsequently with alexa fluor 488-conjugated goat antimouse igg antibody (thermo fisher scientific) in if buffer containing dapi for an additional 30 min. cells were washed three times with pbs before visualisation and image acquisition with fluorescent microscopy. s protein reference sequences for sars-cov-1, sars-cov-2, batratg13, middle east respiratory syndrome (mers) and human common-cold coronaviruses 229e, nl63, oc43 and hku1 were downloaded from the national center for biotechnology information (ncbi). a multiple sequence alignment was created with multiple alignment using fast fourier transform (mafft) using the slow but accurate l-ins-i parameter settings [24] and the alignment curated, cut to the target region 1029-1192 (sars-cov-1 numbering) and visualised with jalview [25] . we used molecular evolutionary genetics analysis (mega) x [26] to calculate the number of amino-acid differences for all sequence pairs in the alignment of the mab target region and the full s protein normalised by the length of the aligned sequence of the respective reference protein to obtain per cent amino acid identities. to determine sars-cov-2 sequence diversity in the s protein within the current pandemic, 230 human and environmental viral sequences were downloaded from gisaid's epicov database on 1 march 2020. we gratefully acknowledge the authors, originating and submitting laboratories of the sequences on which this part of the research is based. the list is detailed in supplementary table 1 . the nt sequences were searched with basic local alignment search tool (blast)x against the reference s protein. 174 hits covered the full length of the s protein and amino-acid mutations were counted and tabulated using a custom perl script (supplementary table 2 ). ethical approval was not required for this study. sequence alignment of the s2 fragment corresponding to residues 1029 to 1192 shows that this fragment, which encompasses the heptad repeat (hr)2 but not hr1, is highly conserved in sars-cov-1 and sars-cov-2 ( figure 1 ). when compared with additional reference sequences from bat ratg13 (closest bat precursor), mers and human common cold coronaviruses 229e, nl63, oc43 and hku1 (figure 1) , it becomes apparent that the amino-acid identity between sars-cov-2 and sars-cov-1 is much higher in this region (93% , table) than over the full protein length (78%, table) and the similarity drops sharply (< 40% in this region) when considering mers and the other coronaviruses infecting humans regularly. we also studied the sequence diversity across 174 sars-cov-2 s proteins derived from nt sequences shared via the gisaid platform [27] . only four aminoacid mutations were found within the putative antibody-binding region compared with 30 mutations over the full length protein (supplementary table 2 ). two of these four amino-acid mutations are from a sequence flagged in gisaid's epicov database as lower quality due to many undetermined bases. four mabs with distinct binding profiles to sars-cov-1, as previously mapped by internal deletion mutagenesis study, were selected for testing to determine if they cross-react with sars-cov-2. a fragment containing residues 1048 to 1206 of sars-cov-2 s protein was expressed in 293ft cells via transient transfection and wb analysis was performed using the four mabs, namely 2b2, 1a9, 4b12 and 1g10. as shown in figure 2a , all four mabs detected this fragment of sars-cov-2, which is consistent with the sequence alignment shown in figure 1 . due to the easy detachment of 293ft cells, cos-7 cells were used for if assay instead. if analysis performed on transiently transfected cos-7 cells showed binding of the four mabs to this s protein fragment of sars-cov-2 ( figure 2b ). these interactions are also specific for the sars-cov-2 s protein (1048-1206) fragment as all four mabs did not show binding to the negative control hbcag. next, the full-length s protein of sars-cov-2 was overexpressed in 293ft and cos-7 cells and detected with each of the mabs using wb and if analyses. as shown in figure 3 , all four mabs bound to the full-length s protein of sars-cov-2 ( figure 3a ). the binding of these mabs to recombinant purified s protein was also determined using indirect elisa where different concentrations of antibodies were used for binding. binding to s protein was observed for all four mabs with 1a9 showing the strongest binding ( figure 3b ). similarly, all four mabs bound to the full-length s protein of sars-cov-2 when tested via if ( figure 3c ). collectively our data demonstrates the ability of all four mabs to bind full-length s protein in both its native and denatured forms. utility of monoclonal antibody 1a9 for detection of s protein in a sandwich elisa format and in sars-cov-2 infected cells based on indirect elisa data, mab 1a9 has the strongest binding to s protein when compared with the other three mabs. hence, a sandwich elisa was performed to determine if it can be paired with the human mab cr3022 which is known to bind to the s1 subunit of sars-cov-2. as shown in figure 4a , recombinant s protein was detected at 15.6 ng/ml and above when 1a9 was used as a capture antibody and cr3022 was used as a detector antibody. since the s protein was his-tagged, a his-tagged haemagglutinin (ha) protein of influenza a virus was used to check for specificity of binding. the absorbance readings in the presence of s protein were significantly higher than that in the presence of ha for protein concentrations of 15.6 ng/ml and above. next, 1a9 was tested on sars-cov-2-infected vero-e6 cells. as shown in figure 4b , mab 1a9 stained a considerable number of sars-cov-2-infected cells at 24 hours post-infection showing that it is sensitive enough to detect the expression of s protein during infection. numerous mabs against the s protein of sars-cov-1 have been generated for research and diagnostic assay development. some of these may be able to cross-react with the s protein of sars-cov-2 and serve as tools to aid research on this newly emerged virus. in this current study, an immunogenic domain in the s2 subunit of sars-cov-1 was found to be highly conserved in multiple strains of sars-cov-2 ( figure 1 and table) . consistently, wb and if analyses showed that four different mabs generated using this sars-cov-1 domain were cross-reactive against the s protein of sars-cov-2 (figures 2 and 3) . recent cross-reactivity studies have evaluated sars-cov-1 neutralising antibodies that bind to the rbdcontaining s1 subunit. although both sars-cov-1 and sars-cov-2 use ace2 as a receptor for viral entry [3, 16] , several sars-cov-1 rbd-directed mabs did not cross-react with sars-cov-2 rbd [28, 29] . interestingly, cr3022, which was isolated from a sars convalescent patient [22] , showed cross-reactivity to sars-cov-2 rbd and recognises an epitope that does not overlap with the ace2 binding site [28] . among the four mabs tested in this study, indirect elisa showed that 1a9 binds strongest to the s protein of sars-cov-2 ( figure 3b ). to determine if 1a9 is useful for detection of s protein in a sandwich elisa, it was paired with cr3022 since 1a9 binds to s2 subunit while cr3022 binds to s1 subunit. as would be expected, these two antibodies can be paired to detect s protein from 15.6 ng/ml ( figure 4a ). in addition, mab 1a9 stained a considerable number of sars-cov-2-infected cells at 24 hours post-infection showing that it is sensitive enough to detect the expression of s protein during infection ( figure 4b ). thus, mabs 1a9 will be useful for studying the kinetics of sars-cov-2 replication in vitro and development of diagnostic assays for covid-19. it is noteworthy that cytotoxic t-lymphocyte (ctl) epitopes also reside at residues 884-891 and 1116-1123 within the s2 subunit of sars-cov-1 [30] . interestingly, the latter ctl epitope overlaps with the epitope recognised by mab 1a9 [21] . hence, the s2 subunit may serve as an important antigen for inducing both humoral as well as cell-mediated immunity against sars-cov-1 and sars-cov-2. to our knowledge, this is the first study showing that mabs targeting the s2 domain of sars-cov-1 can cross-react with sars-cov-2 and this observation is consistent with the high sequence conservation in the s2 subunit. the ability of these antibodies, particularly 1a9, to detect sars-cov-2 s protein in indirect and sandwich elisas demonstrate their utility for detection of sars-cov-2 infections in a public health setting. whether or not the current sensitivity of these antibodies are sufficient for robust detection of sars-cov-2 infections in a clinical setting and how they compare to existing pcr-based detection remains to be determined. successful development of these antibodies into a point of care diagnostic kit will provide a complementary approach to existing detection methods. besides the mabs characterised here, several other mabs have been reported to bind to epitopes in the s2 subunit of sars-cov-1 [31] [32] [33] . thus, it will be important to determine if these mabs can also cross-react with sars-cov-2. maurer-stroh, s -designed experiments, performed experiments, analysed and organised data, wrote the manuscript. chow vtk -analysed and organised data. lin rtp -analysed and organised data. mirazimi a-designed experiments, performed experiments, analysed and organised data, wrote the manuscript. hong wj-analysed and organised data. tan yj-designed experiments, performed experiments, analysed and organised data, wrote the manuscript. identification of a novel coronavirus in patients with severe acute respiratory syndrome sars working group. a novel coronavirus associated with severe acute respiratory syndrome a pneumonia outbreak associated with a new coronavirus of probable bat origin an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies early 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mechanisms of coronavirus cross-species transmission angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses amino acids 1055 to 1192 in the s2 region of severe acute respiratory syndrome coronavirus s protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents monoclonal antibodies targeting the hr2 domain and the region immediately upstream of the hr2 of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus substitution at aspartic acid 1128 in the sars coronavirus spike glycoprotein mediates escape from a s2 domaintargeting neutralizing monoclonal antibody human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants contribution of fc-dependent cell-mediated activity of a vestigial esterase-targeting antibody against h5n6 virus infection parallelization of mafft for large-scale multiple sequence alignments jalview version 2--a multiple sequence alignment editor and analysis workbench molecular evolutionary genetics analysis across computing platforms disease and diplomacy: gisaid's innovative contribution to global health potent binding of 2019 novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody cryo-em structure of the 2019-ncov spike in the prefusion conformation characterization of cytotoxic t-lymphocyte epitopes and immune responses to sars coronavirus spike dna vaccine www.eurosurveillance.org expressing the rgd-integrin-binding motif fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (sars) coronavirus s protein neutralizes the virus in a rhesus macaque sars model b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s2 domain of the surface spike glycoprotein a human sars-cov neutralizing antibody against epitope on s2 protein license, supplementary material and copyright the work performed in nus/nuhs was supported by nuhs research office under project number nuhsro/2020/033/ ro5+5/coronavirus/loa (wbs r-571-000-071-733). the work performed in imcb and bii was also supported by a*star through intramural funding and an a*cruse gap funding (accl/19-gap064-r20h-f). wjh and yjt declare that they are involved in the licensing of mabs 1a9, 1g10, 4b12 and 2b2 to commercial companies as research or diagnostic reagents. the other authors have declared that no competing interests exist. key: cord-254469-7q6xi2xx authors: wang, fuzhou; kream, richard m.; stefano, george b. title: an evidence based perspective on mrna-sars-cov-2 vaccine development date: 2020-05-05 journal: med sci monit doi: 10.12659/msm.924700 sha: doc_id: 254469 cord_uid: 7q6xi2xx the first outbreak of coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) occurred in wuhan, hubei province, china, in late 2019. the subsequent covid-19 pandemic rapidly affected the health and economy of the world. the global approach to the pandemic was to isolate populations to reduce the spread of this deadly virus while vaccines began to be developed. in march 2020, the first phase i clinical trial of a novel lipid nanoparticle (lnp)-encapsulated mrna-based vaccine, mrna-1273, which encodes the spike protein (s protein) of sars-cov-2, began in the united states (us). the production of mrna-based vaccines is a promising recent development in the production of vaccines. however, there remain significant challenges in the development and testing of vaccines as rapidly as possible to control covid-19, which requires international collaboration. this review aims to describe the background to the rationale for the development of mrna-based sars-cov-2 vaccines and the current status of the mrna-1273 vaccine. in late 2019, in wuhan, hubei province, china, an outbreak occurred of coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this outbreak was followed by the release of the first situation report from the world health organization (who) on january 20, 2020, and since then, a global pandemic has resulted in an increasing number of deaths [1] [2] [3] . the pandemic has resulted in uncertainty regarding when or whether the pandemic will end, and whether covid-19 will follow the same clinical course as severe acute respiratory syndrome (sars) did 17 years ago. however, the current view is that this novel coronavirus infection will persist, and without a treatment, a vaccine is urgently required [4] . review of the epidemiology of the sars epidemic of 2003 shows that it disappeared suddenly without resulting in a pandemic, and this was also the reason for the lack of development of a sars vaccine [5] . however, covid-19 is different from sars based on its ability to spread globally. currently, antiviral pharmaceuticals and immunotherapies are two principal therapeutic approaches for covid-19 [6] , whereas vaccines are still considered the most promising way to eradicate this virus [7] . in general, dna-based and protein-based vaccines have been the major approaches for the development of stable and effective vaccines with less innate immunogenicity [8, 9] . however, mrna-based vaccines have become a potentially preventive approach, as they are safer, more efficient, and easier to manufacture [10] . due to the rapid spread of covid-19, mrnabased vaccine development seems to be an approach to prevent infection and to prevent a second wave of this pandemic. typically, at least 12 to 18 months is required to develop a new vaccine, using current processes for vaccine development, clinical trials, and regulatory approval [11] . therefore, it is unlikely that an effective vaccine will be developed in time to control the current pandemic. however, on march 16 2020, the first phase i clinical trial of a novel lipid nanoparticle (lnp)-encapsulated mrna-based vaccine, mrna-1273, which encodes the spike protein (s protein) of sars-cov-2, began in the united states (us), conducted by moderna and the vaccine research center (vrc) of the national institute of allergy and infectious diseases (niaid) [12, 13] . the first patient enrolled in this phase 1 study was a 43-year-old woman at the kaiser permanente washington health research institute in seattle, washington, usa [12] . the phase i study of mrna-1273 is being conducted to evaluate the safety and immunogenicity, with the following trials currently being planned to evaluate the efficacy and effective dose of the new mrna vaccine [13] . this review aims to describe the background to the rationale for the development of mrna-based sars-cov-2 vaccines and the current status of the mrna-1273 vaccine. the terminology for the rna virus that causes covid-19, sars-cov-2, has been established by the international committee on taxonomy of viruses (ictv) [14] , due to its extensive homology with the 2003 sars coronavirus. the sars-cov-2 coronavirus belongs to the subfamily of coronavirinae, with a genomic structure of (+)ss-rna of 30 kb in length that includes a 5'-cap structure and 3'-poly-a tail [15] . from the viral rna, polyprotein 1a/1ab (pp1a/pp1ab) is synthesized in the host to form 16 non-structural proteins (nsps) that organize the replication-transcription complex (rtc) in doublemembrane vesicles (dmvs). the nrtc synthesizes a set of minus-strand subgenomic rnas (sgrnas) discontinuously [16] . between open reading frames (orfs), transcription terminates, and then a subsequent acquisition of a leader rna occurs. during this process, subgenomic mrnas need these sgrnas as the templates [14, 17] . at least six orfs exist for a typical cov, including sars-cov-2. the first orfs (orf1a/b) with over 65% of the whole genome length encode 16 nsps. of note, two polypeptides (pp1a and pp1ab) come from a -1 frameshift between orf1a and orf1b. for the other orfs on the 35% of the genome close to the 3'-terminus encode at least four main structural proteins, including spike (s), membrane (m), envelope (e), and nucleocapsid (n). all these structural and non-structural proteins are translated from the sgrnas [14, 16, 17] . currently, more than 200 complete and partial genome sequences of sars-cov-2 have been decoded and deposited in the global initiative on sharing all influenza data (gisaid) database (https://www.gisaid.org/) and in the national institutes of health (nih) genbank database (https://www.ncbi.nlm.nih.gov/nuccore/?term=covid-19). phylogenetic analysis showed that sars-cov-2 was closely related to two sars-like coronaviruses present in bats, including bat-sl-covzc45 and bat-sl-covzxc21, with 88% identity, and showed 79% homology with sars-cov, and 50% with mers-cov [18] . however, homology modeling disclosed that sars-cov-2 had a similar rbd structure to that of sars-cov, despite amino acid variation at some key residues [19, 20] . these findings suggest that sars-cov-2 emerged from a single animal source within a short period [21] . however, because the sequence similarity between sars-cov-2 and its close relatives bat-sl-covzc45 and bat-sl-covzxc21 is less than 90%, the bat-derived viruses may not be the direct origins of sars-cov-2. vaccines based on the cytoplasmic expression of chimeric mrnas containing curated orf viral sequences possess great potential to translate directly in the cytoplasm and block chromosomal integration [22] . once injected, the delivered mrna can be processed by immune cells and start to produce targeted protein directly through translation, of which is being followed by activating other immune cells to recognize the newly produced viral protein to make antibodies. two types of rna vaccine exist against infectious pathogens, or non-replicating mrna vaccine and self-amplifying or replicon rna vaccine [10, 23] . due to different delivery methods, non-replicating mrna vaccines are further divided as ex vivo loading of dendritic cell and direct in vivo injection into various anatomical sites [10] . the penetration of the lipid membrane barrier is the first step for exogenous mrna to reach the cytoplasm before the translation of functional protein happen [24] . also, the uptake mechanisms of mrna vaccines show cell specificity [25] , and the physicochemical properties of the mrna may significantly influence its cellular delivery and organ distribution [26] . all these factors must be considered when designing an effective mrna-based vaccine. even so, an mrna vaccine is still considered the most promising candidate because it can be scaled rapidly, which can save time when the rapidly spreading covid-19 emerged and started to infect millions of people worldwide [7, 27] . as a (+)ss-rna virus, sars-cov-2 possesses self-amplifying rna that can realize extreme rna replication in the cytosol [28] . this finding supports the role of mrna-based vaccine development. however, the safety and efficacy of mrna vaccines for use in humans remain unknown. the hypothetical benefits of mrna vaccine seem strong, whereas limitations such as the delivery and stability issues related to rna degradation, and the safety concerns due to immunogenicity hinder its development [29] . the results from the phase i trial of the mrna-1273 vaccine are awaited [13] . the mrna-based vaccines actively induce activation of both b cell responses and t cell cytotoxicity. first, the mrna vaccines use the mrna sequence of the target protein that recombine in vitro, rather than the sequence of the target antibody. subsequently, the recombinant target protein mrna sequence is carried by lnps and enters the somatic cytoplasm to achieve direct translation and encoding the target protein. when the target protein is released from the host cell, the antigen-presenting cells will quickly capture and process the heterologous protein. then, the presentation of mhc i and mhc ii on the surface of the antigen-presenting cell membrane [30] . this step is important for the subsequent activation of b cells and t cells and is also the key to the humoral and cytotoxic response. figure 1 is a schematic representation of mrnabased active immunization. targeting the sars-cov-2 s protein sequence in mrna vaccine development finding the most suitable target site for sars-cov-2 vaccine development is extremely important. the spike glycoprotein (s protein) is now a key target for vaccine development, therapeutic antibody generation, and the clinical diagnosis of covid-19. sars-cov-2 enters the host cell by using highly glycosylated homotrimeric s protein to achieve fusion with cell membranes through its structural changes. this process includes: the s1 subunit binds to the host cell receptor, which triggers trimeric instability that is followed by the separation of the s1 subunit from the s2 subunit to form a highly stable fusion structure [19] [20] [21] . to access host cell receptors, rbd in the s1 subunit undergoes hinge-like conformational changes to hide or expose key sites for receptor binding, which is very similar to sars-cov [19] [20] [21] . this high homology of rbd suggests that the covid-19 virus shares the same host cell receptor ace2 as sars-cov [19] [20] [21] . although there are similarities, covid-19 has its own characteristics. the most significant change is the rrar amino acid sequence with a s1/s2 protease cleavage site, which is consistent with the characteristics of a furin recognition site. this common phenomenon occurs more frequently in influenza viruses rather than in sars viruses that only have a single arginine [31] . also, sars-cov-2 and ratg13 s proteins have 29 amino acid residues that differ, 17 of which are located at the rbd site. the rbd of sars-cov-2 is much closer to the center of the trimeric s protein. one of the three rbds in the s protein will spiral upwards to form a spatial conformation that helps the virus bind to the host receptor ace2 easily, which suggests that sars-cov-2 would be more infectious than sars [32] . a cross-reactivity test of rbd of sars-cov-2 was performed using the rbd monoclonal antibody of sars, and it was found that this antibody did not cross-react with sars-cov-2 [33] . these results provide an important structural biological basis for designing vaccines more accurately and discovering antiviral drugs. s protein helps the virus to enter target cells, but this endocytosis simultaneously depends on both the binding of s protein to membrane ace2 receptors and the initiative activation of s protein by cellular proteases [34] . therefore, a vaccine against s protein provides an approach for preventing the proliferation and spread of sars-cov-2. the vaccine can prevent the initial activation of the s protein by blocking the s protein binding e924700-3 to ace2. sars-cov-2 infects cells in a transmembrane serine protease 2 (tmprss2) protein-coding gene-independent manner, and cathepsins b and l directly activate s protein in cells that do not express tmprss2, and sars-cov-2 infects cells in a tmprss2-dependent manner. recently, hoffmann et al. showed that the use of tmprss2 inhibitors significantly blocked the entry of sars-cov-2 into cell lines expressing tmprss2 and that promoting tmprss2 expression antagonized this inhibition [35] . therefore, if the antibody titer of s protein is high enough to prevent the virus being engulfed into endosomes or undergoing fusion at the host cell surface, then virus infection associated with s protein activation and the intracellular release of the virus particles will be effectively inhibited. given the bioavailability of ace2 as a key determinant of the spread and infectious capacity of sars-cov-2, if enough s protein figure 1 . schematic diagram of the mrna-based vaccine targeted to the spike protein (s protein) of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the mrna-based vaccine targeted to the s protein of sars-cov-2 works by active immunization. this technique will not use part of the virus but only recombine mrna of the s protein in vitro according to the gene sequence, which is coated with lipid nanoparticles for effective delivery. once injected into the muscle, the myocytes take up the lipid nanoparticle (lnps) and then release the mrnas into the cytoplasm for translation into the s proteins. these endogenously synthesized s proteins will be secreted to activate both humoral and cellular immune responses. s protein -spike protein; im -intramuscular, lnp -lipid nanoparticle; dc -dendritic cell; mhc -major histocompatibility complex; ag -antigen. antibodies are formed in the body, then two therapeutic effects may occur. firstly, the s protein-antibody complex will be quickly cleared by the immune system, which will subsequently result in clearance of the virus itself. secondly, ace2 bioavailability will be significantly reduced, which helps mitigate the spread and infectivity of the virus [36] . although empirical data from both sars-cov and sars-cov-2 research have identified ace2 as the primary portal for cellular entry of these two viruses [19] [20] [21] , the probability of a membrane-associated co-receptor complex remains. therefore, targeting of ace2/sars-cov-2 binding may not be the rate-limiting step for inhibiting viral infection. even with hopes of an mrna-based vaccine targeting the s protein, it is important to note that the stoichiometric relationship between s protein and the immune response occurs in several ways. first, the intrinsic ratio of outer surface s protein per sars-cov-2 virion (s/v) may predetermine the preference of induced igg in immunological responses. the inherent ratio of nucleotides to the s protein (n/s) may determine whether there are redundant s proteins when igg effectively neutralizes the s protein, which reduces the immune efficacy of igg. therefore, both ratios will finally determine how high the antibody titer should be and how many vaccine boosters are needed so that it can effectively neutralize the virus without concerns about the existence of redundant s protein complexes that may effectively remove igg antibodies to leave unblocked s protein available for processing and binding to ace2. although several previous studies have investigated this finding in human immunodeficiency viruse (hiv) infection and concluded that low spike density with wide spacing of the envelope spikes is unhelpful for the activation of b cells [37, 38] , this remains unclear for sars-cov-2, and it is a challenging factor for vaccine development against sars-cov-2. an important consideration relates to mutations in the s protein that may affect the degree of viral pathogenesis [39] . direct evidence of functionally meaningful s protein mutations affects the s protein sars-cov-2 and the host cell ace2, which appears to mediate a higher binding affinity when compared with sars-cov [40] . the reason for this increased s protein/ace2 affinity in sars-cov-2 (-15.7 kcal/mol) versus that of sars-cov (-14.1 kcal/mol) is mainly attributed to three main factors [40] . first, the emergence of two loops around the rbds in sars-cov-2 may promote its chemical interaction with ace2 by increasing the number of atoms needed. second, more amino acid residues in the s protein of sars-cov-2 determine a higher number of protein-protein contacts between s protein and ace2. third, a longer capping loop in the s protein of sars-cov-2 favors its interaction with the receptor. therefore, it is difficult to ensure that the new vaccine that is targeted to s protein can be used long term or in the near future, it there is the possibility that the vaccine will not be effective because of mutations in the s protein. for protein-based immunization, repeated vaccine administrations due to the clearance and indication may be required to maintain a therapeutically effective level [41] . furthermore, some non-specific effects may result from these typical vaccines, such as cross-reactive tcrs and antibodies, bystander activation of pre-existing effector or memory cells, and classical cell-mediated immunity may function as either beneficial or unfavorable to the public health of the population [42] . also, although there have been previous studies on the safety of live attenuated or killed vaccines, and the safety of these vaccines used in clinics is very reliable, the potential for inducing infection still exists [43] . there are four main safety and efficacy advantages of the use of mrna-based antiviral vaccines over traditional approaches. first, mrna-based antiviral vaccines minimize the potential risk of infection and insertion-induced mutagenesis due to natural degradation of mrna in the cellular microenvironment [44] . second, the immunogen's high efficacy due to engineered mrna structural modifications improves its stability and translation efficacy. third, the high potency of mrna-based vaccines capable of generating potent antiviral neutralizing immunoglobulins with only one or two low-dose immunizations [23, 45] may induce strong immune responses by activating both cd8+ and cd4+ t cells [46] . fourth, engineered mrna production facilitates large-scale production of sufficient vaccine doses required to treat mass populations [29, 47] . all these factors make the mrna vaccine more suitable for a rapid response to the emerging covid-19 pandemic. although these beneficial features of mrna vaccines provide some hope for the development of the first clinically applicable sars-cov-2 mrna vaccine, recent reports regarding rare cases of moderate or severe reactions for different mrna vaccines have raised concerns about safety and immunogenicity, including in the primary outcome findings of the phase i trial on mrna-1273 [20, 48] . therefore, it is important to clearly understand the potential risks of this type of mrna-based vaccine, which include local and systemic inflammatory responses, the biodistribution and persistence of the induced immunogen expression, possible development of autoreactive antibodies and toxic effects of any non-native nucleotides and delivery system components [48] [49] [50] . glycosylation of viral envelopes is a very common biological phenomenon. glycosylation refers to the process in which proteins or lipids are linked to sugar chains by the action of enzymes. glycosylation is one of the important forms of posttranslational modification of proteins and is an essential way to regulate protein localization, function, persistence, and e924700-5 diversity [51, 52] . for viruses, their replication and invasion into host cells are closely associated with the glycosylation of their structural proteins [53] . the main purpose of viral vaccine development is to evoke an immune response to fight the virus. however, the high glycosylation of proteins on the surface of the viral envelope works like invisible camouflage, which can help the virus to successfully escape the detection of the human immune system and achieve the purpose of survival [54, 55] . therefore, the higher the degree of glycosylation, the greater the probability that the virus will escape the immune response and the lower the success rate of vaccine development. therefore, overcoming the highly glycosylated viral envelope is another challenge of sars-cov-2 vaccine development. sars-cov-2 includes highly glycosylated spherical particles and has a large structure containing at least 66 n-linked glycan sites, of which 54 sites show similarity with sars-cov [56] . in contrast, the glycan sites of hiv are between three times and six times that of the influenza virus, which is one of the major reasons why an hiv vaccine has not been successfully developed at this time [57] . however, sars-cov-2 has more than twice the glycan sites of hiv [58] . this unusual degree of glycosylation means that sars-cov-2 may mutate quickly, making the development of a vaccine extremely difficult. however, vaccine development failures caused by glycosylation remains significant, because vaccine design is based on the traditional inactivated or attenuated live vaccines, and different traditional vaccines may induce various glycosylation patterns of antibodies that subsequently affect the protective role of the antibodies [59] . however, the use of mrna-based vaccine technology and targeting only the s protein, rather than the entire virus particle, may lead to the human immune system producing s protein antibodies without the influence of viral glycosylation. the successful delivery of oligonucleotide-based therapies [60] has provided new opportunities to develop mrna-based vaccines. given their beneficial properties, which include lack of persistence, lack of integration into the genome, the absence of induction of autoantibodies, the ability to produce mrna vaccines in large quantities, and their high purity [61] , an mrna-based vaccine has become a hopeful choice for the development of a sars-cov-2 vaccine. currently, no clinically applicable vaccine against covid-19 is available, and even one of the first vaccines, mrna-1273, is undergoing a phase i safety and immunogenicity trial [13] . the recently developed encapsulated rna delivery and self-amplifying rna technique assists in the ease of use of the mrna vaccine with an increased success rate. however, several issues still need to be clarified for mrna-based vaccines. concerning the worldwide spread of covid-19, the development of an effective and safe vaccine in time has become the most promising way to control the current viral pandemic. if currently available technology can be used to develop a sars-cov-2 vaccine, which may be clinically applicable, this will give hope for controlling the covid-19 pandemic. however, further research and development should continue to ensure that an effective sars-cov-2 vaccine is developed. there are several challenges to address as new formulations are required to stabilize the mrna vaccine at higher temperatures during its distribution. a more suitable packaging formula for the mrna vaccine within the nanoparticles is required to ensure vaccine stability. the best way should be sought to anchor the rnase inhibitor into the mrna vaccine co-formulation and to develop new ways to purify or remove the mrna vaccine-related reaction components. studies are still required to investigate the mechanisms for reducing the innate immune response to the delivered mrna vaccine. also, novel in vivo delivery methods requires development to maximize the uptake efficiency of the mrna vaccine, and to determine which immune signaling pathways are the most effective in response to the exogenous mrna vaccine. in addition to the described technical difficulties related to mrna vaccines, the difficulties associated with vaccine development need to be resolved. even though clinical trials of some vaccines have begun [13] , there remain several technical issues that affect vaccine development and efficacy that require further study. first, strict bioinformatics analysis of the probability of a membrane-associated co-receptor complex is required to find the rate-limiting pattern of antibodies that affect ace2/sars-cov-2 binding. the stoichiometric relationship between the s protein and the strength of the immune response requires studies on the intrinsic ratio of the outer surface s protein per sars-cov-2 virion (s/v) and nucleotides to the s protein (n/s). the precise function of viral self-defense proteins and the spherical configuration of the virion require further research, as does the mutation probability of the s protein that affects viral tropism and receptor affinity. there is also the potential hindrance of the high glycosylated state of the viral envelope to vaccine development. therefore, further in-depth studies are needed to elucidate the structure and corresponding physiological and immunological properties of the s protein and also other structural proteins that have a potential role in vaccine development, including mrna based vaccines. this review aimed to describe the background to the rationale for the development of mrna-based sars-cov-2 vaccines e924700-6 and the current status of the mrna-1273 vaccine. although mrna vaccines are commencing human clinical trials, due to the rapid global spread of this new viral pandemic, it may not be possible to develop a safe and effective vaccine for sars-cov-2 in time to prevent the increasing number of deaths due to this novel rna virus. the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status world health organization (who) world health organization (who) base medicine task force: covid-19: facts and recommendations from a to coronaviruses -drug discovery and therapeutic options discovering drugs to treat coronavirus disease 2019 (covid-19) immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic dna vaccine delivery and improved immunogenicity aiming at the heart: the capsid protein of dengue virus as a vaccine candidate mechanism of action of mrna-based vaccines principles of vaccine potency assays moderna doses first patient with mrna-1273 in coronavirus vaccine trial safety and immunogenicity study of 2019-ncov vaccine (mrna-1273) to prevent sars-cov-2 infection coronaviridae study group of the international committee on taxonomy of viruses: the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 the reproductive number of covid-19 is higher compared to sars coronavirus improved translation efficiency of therapeutic mrna naming the coronavirus disease (covid-19) and the virus that causes it understanding of covid-19 based on current evidence crystal structure of sars-cov-2 main protease provides a basis for design of improved a-ketoamide inhibitors structural basis for the recognition of the sars-cov-2 by full-length human ace2 the proximal origin of sars-cov-2 new vaccine technologies to combat outbreak situations nucleoside modified mrna vaccines for infectious diseases lipid-based mrna vaccine delivery systems developing mrna-vaccine technologies introduction to rna vaccines sars-cov-2 and covid-19: the most important research questions features, evaluation and treatment coronavirus (covid-19) improved translation efficiency of therapeutic mrna mrna vaccine delivery using lipid nanoparticles the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade novel coronavirus 2019) -recent trends cryo-em structure of the 2019-ncov spike in the prefusion conformation identification of novel proteolytically inactive mutations in coronavirus 3c-like protease using a combined approach sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor covid-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics. hum vaccin immunother modeling how many envelope glycoprotein trimers per virion participate in human immunodeficiency virus infectivity and its neutralization by antibody estimating the stoichiometry of human immunodeficiency virus entry structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry role of changes in sars-cov-2 spike protein in the interaction with the human ace2 receptor: an in silico analysis pharmacokinetics and immunogenicity of broadly neutralizing hiv monoclonal antibodies in macaques non-specific effects of vaccines illustrated through the bcg example: from observations to demonstrations risk of transmission associated with live attenuated vaccines given to healthy persons caring for or residing with an immunocompromised patient stability of the osmoregulated promoter-derived prop mrna is posttranscriptionally regulated by rnase iii in escherichia coli mrna as novel technology for passive immunotherapy modified tumour antigen-encoding mrna facilitates the analysis of naturally occurring and vaccine-induced cd4 and cd8 t cells in cancer patients inhibition of the inflammatory pathway enhances both the in vitro and in vivo transfection activity of exogenous in vitro-transcribed mrnas delivered by lipid nanoparticles complexities of viral mutation rates induction of an ifn-mediated antiviral response by a self-amplifying rna vaccine: implications for vaccine design type i interferons (alpha/beta) in immunity and autoimmunity new insights into the role of glycosylation in lipoprotein metabolism functional roles of o-glycosylation exploitation of glycosylation in enveloped virus pathobiology effects of glycosylation on the properties and functions of influenza virus hemagglutinin virus glycosylation: role in virulence and immune interactions emerging wuhan (covid-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd26 glycosylation profiling to evaluate glycoprotein immunogens against hiv-1 site-specific analysis of the sars-cov-2 glycan shield opportunities to exploit antibody glycosylation in vaccination oligonucleotide therapies: the past and the present vaccination with messenger rna (mrna) status of mrna sars-cov-2 vaccines key: cord-274396-l611eisi authors: park, su-jin; yu, kwang-min; kim, young-il; kim, se-mi; kim, eun-ha; kim, seong-gyu; kim, eun ji; casel, mark anthony b.; rollon, rare; jang, seung-gyu; lee, min-hyeok; chang, jae-hyung; song, min-suk; jeong, hye won; choi, younho; chen, weiqiang; shin, woo-jin; jung, jae u.; choi, young ki title: antiviral efficacies of fda-approved drugs against sars-cov-2 infection in ferrets date: 2020-05-22 journal: mbio doi: 10.1128/mbio.01114-20 sha: doc_id: 274396 cord_uid: l611eisi due to the urgent need of a therapeutic treatment for coronavirus (cov) disease 2019 (covid-19) patients, a number of fda-approved/repurposed drugs have been suggested as antiviral candidates at clinics, without sufficient information. furthermore, there have been extensive debates over antiviral candidates for their effectiveness and safety against severe acute respiratory syndrome cov 2 (sars-cov-2), suggesting that rapid preclinical animal studies are required to identify potential antiviral candidates for human trials. to this end, the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir for sars-cov-2 infection were assessed in the ferret infection model. while the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (pbs)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the pbs-treated control group. only the emtricitabine-tenofovir-treated group showed lower virus titers in nasal washes at 8 days postinfection (dpi) than the pbs-treated control group. to further explore the effect of immune suppression on viral infection and clinical outcome, ferrets were treated with azathioprine, an immunosuppressive drug. compared to the pbs-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (sn) antibody titers. taken together, all antiviral drugs tested marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers. despite the potential discrepancy of drug efficacies between animals and humans, these preclinical ferret data should be highly informative to future therapeutic treatment of covid-19 patients. low sn titers, resulting in a prolonged infection. as several fda-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial. keywords covid-19, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), antiviral therapeutics, immunosuppression, serum neutralization, ferrets i n december of 2019, a novel coronavirus (cov) disease , identified in wuhan, hubei province, china, in patients with pneumonia, was found to be caused by a previously unknown betacoronavirus. the outbreak rapidly spread to other provinces in mainland china, and despite great efforts, the epidemic continued to spread from china to europe, north america, and other asian countries. the world health organization (who) announced that the severe acute respiratory syndrome cov 2 (sars-cov-2) epidemic was a public health emergency of international concern on 30 january 2020, and on 11 march 2020, the who director general characterized covid-19 as a pandemic (http://www.euro.who.int/en/health-topics/health -emergencies/coronavirus-covid-19/news/news/2020/01/2019-ncov-outbreak-is-an -emergency-of-international-concern; https://www.who.int/dg/speeches/detail/who -director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march -2020). currently, the number of people diagnosed with sars-cov-2 infection is increasing by approximately 10,000 cases a day. as of 17 april 2020, approximately 2,074,529 cases had been diagnosed as covid-19 and 139,378 deaths had occurred (1) . further, recent studies reported the detection of sars-cov-2 rna in various clinical specimens, suggesting other transmission routes for this virus besides respiratory secretions (2) (3) (4) . unfortunately, to date, no vaccines or antiviral drugs have been approved for the treatment of sars-cov-2 infection by regulatory agencies. researchers are fervently working to develop vaccines specifically for this virus, as well as potential treatments for covid-19. although many clinical trials are ongoing, there are no specific therapeutic agents approved for covid-19. developing sars-cov-2-specific antiviral drugs from scratch could take years; drugs that have already been approved by the u.s. food and drug administration (fda) have the potential to reach patients more quickly. during the sars outbreak in 2003, screening of approved drugs identified lopinavir-ritonavir, a human immunodeficiency virus type 1 (hiv-1) aspartate protease inhibitor, as effective against sars-cov replication (5) . the antiviral activity of lopinavir-ritonavir against middle east respiratory syndrome coronavirus (mers-cov) both in vitro (6) and in an animal model (7) has been reported, and case reports suggest that the combination of lopinavir-ritonavir with ribavirin and interferon alpha results in virologic clearance and survival (8, 9) . chloroquine (cq), a widely used antimalarial with immunomodulatory effects (10), was found in a recent study to inhibit the growth of sars-cov-2 in vitro (11) . however, this finding has not been strongly supported by clinical studies of approximately 100 sars-cov-2-infected patients (12, 13) . a derivative of chloroquine, hydroxychloroquine (hcq) sulfate, was first synthesized in 1946 by adding a hydroxyl group to cq, resulting in a compound found to be much less toxic than cq in an animal study (14) . in autoimmune diseases, hcq sulfate works by reducing inflammation (15) . however, recent reports have also shown heart risk concerns with the use of cq and hcq sulfate for covid-19 treatment. emtricitabine-tenofovir (truvada) is a prescription medicine for hiv approved by the u.s. fda for preexposure prophylaxis to reduce the risk of hiv infection in adults and adolescents. as a nucleotide analogue, it is reported that the active triphosphate form of this tenofovir diphosphate inhibits activity for rna-dependent rna polymerase (rdrp) of hiv and hepatitis b virus (hbv) (16, 17) . still, even these existing drugs will need rigorous testing for efficacy and safety and ultimately ramped-up production before they can be deployed widely against covid-19. generally, immunocompromised patients are more susceptible to bacterial, fungal, viral, and parasitic infections than healthy persons due to their inability to mount successful immune responses. this can be caused by impairment or weakening of the immune system by a number of conditions, including diseases (e.g., diabetes or hiv infection), malnutrition, and the use of certain medications. it has become apparent that sars-cov-2 infection also affects immunocompromised individuals more severely. a majority of covid-19 patients who were clinically diagnosed are older than ϳ60 years and have underlying complications, including heart disease, diabetes, hypertension, or cancer, indicating that age and reduced immune activity are the critical risk factors or determinants for covid-19 morbidity and mortality. we have recently established a ferret model for sars-cov-2 infection and transmission that highly recapitulates aspects of the human infection (18) . elevated body temperatures and virus replication were readily detected in sars-cov-2-infected ferrets. sars-cov-2-infected ferrets shed the virus through nasal washes and in saliva, urine, and fecal specimens. sars-cov-2 was transmitted readily to naive direct-contact ferrets but less efficiently to naive indirect-contact ferrets (18) . further, acute bronchiolitis was observed in infected lungs. in this report, we evaluated the efficacy of oral administration of lopinavir-ritonavir, hcq sulfate, and emtricitabine-tenofovir for sars-cov-2 infection in ferret infection models. we also treated ferrets with azathioprine, an immunosuppressive drug, and evaluated the replication kinetics of sars-cov-2. while most drug treatments reduced clinical symptoms (cs), none of them led to a significant reduction of in vivo virus titers in ferrets. thus, a drug candidate study in a robust preclinical animal model should greatly facilitate testing the efficacies and safety of therapeutic treatments for covid-19 patients. clinical features of sars-cov-2-inoculated ferrets treated with antivirals. in order to determine the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine (hcq) sulfate, or emtricitabine-tenofovir for treatment of sars-cov-2 infection, sars-cov-2 antibody-free ferrets (10/group) were inoculated with 10 5.8 50% tissue culture infective doses (tcid 50 )/ml of an nmc-ncov02 strain through the intranasal (i.n.) route ( fig. 1 ). at 1 day postinfection (dpi) with sars-cov-2, ferrets were administered lopinavir (24 mg/kg of body weight)-ritonavir (6 mg/kg), hydroxychloroquine sulfate (12.5 mg/kg), or emtricitabine (6 mg/kg)-tenofovir (9 mg/kg) daily via oral gavage for 14 days (fig. 1 ). in addition, to test the effect of immunosuppression on viral infection and clinical outcome, a group (n ϭ 10) of ferrets was also treated with phosphatebuffered saline (pbs) (as a control) or azathioprine, an immunosuppressive drug (10 mg/kg), for 7 days prior to sars-cov-2 infection (fig. 1) . while all groups of sars-cov-2-infected ferrets showed elevated temperatures at 4 to 6 dpi, the lopinavirritonavir-or emtricitabine-tenofovir-treated group exhibited mild fever compared with the pbs-treated group ( fig. 2a) . as with the pbs-treated group, the hydroxychloroquine sulfate-or azathioprine-treated group showed ϳ7% body weight loss at 8 dpi, while the lopinavir-ritonavir-or emtricitabine-tenofovir-treated group showed a ͻ4% change in body weight on average (fig. 2b ). to compare clinical features of sars-cov-2 infection following treatment with each drug, we developed an arbitrary scoring method to generate clinical symptom values based on 20-min observations of cough, rhinorrhea, and reduced activity and compared these cs values among the ferret groups as described in table 1 . the average cs value of pbs-treated control ferrets was 3.5 at 2 dpi, remained relatively high (ͼ5) for 3 to 6 dpi, and returned to normal (less than 1) by 11 dpi (table 1 ). the lopinavir-ritonavir-treated ferrets showed the reduced overall cs values (less than 5) that peaked at 4 to 5 dpi and resolved by 10 dpi (table 1) . the hcq sulfate-treated ferrets also showed reduced cs values at 6 to 8 dpi compared with those of the pbs-treated group, but overall clinical symptoms were similar to those of the pbs-treated control group (table 1) . although the emtricitabine-tenofovir-treated ferrets initially demonstrated clinical symptoms similar to those of the pbstreated control group, their overall cs values were relatively low at 3 to 8 dpi, and clinical symptoms were ultimately resolved by 9 dpi. finally, the azathioprine-treated immunosuppressed ferrets also showed cs values similar to those of the pbs-treated control group, but this group's clinical symptoms lasted slightly longer than those of the pbs-treated control group (table 1) . these results collectively showed that the lopinavir-ritonavir-, hcq sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the pbs-treated control group. the overall clinical symptoms of immunosuppressed ferrets were similar but persisted slightly longer than those of pbs-treated control ferrets. comparisons of virus titers and shedding periods in antiviral-drug-treated ferrets. to evaluate the antiviral activity of each drug against sars-cov-2, we measured infectious virus titers in nasal washes from drug-treated ferrets (fig. 2c ). sars-cov-2 was isolated from all infected ferrets regardless of drug treatment from 2 dpi to 6 dpi, with similar virus titers (2.75 to 3.2 log 10 tcid 50 /ml). at 8 dpi, the emtricitabinetenofovir-treated ferrets exhibited reduced virus titers compared with those of the pbs-treated control group. although infectious virus was not detected in ferrets of the pbs-or antiviral-drug-treated groups at 10 dpi, three of four azathioprine-treated ferrets were positive for virus even at 10 dpi (fig. 2c) , suggesting delayed virus clearance in the upper respiratory tracts of immunocompromised ferrets. because gastrointestinal involvement has been documented in coronavirus infections of animals and humans (19) , we also collected fecal specimens and performed quantitative real-time pcr (qrt-pcr) to determine whether any of the drug treatments affected sars-cov-2 shedding in the gastrointestinal system (fig. 2d) . the results showed that the viral rna was present in fecal specimens of all groups from 2 to 8 dpi, with peak viral rna copy numbers observed at 4 to 6 dpi. however, there was no statistical difference in viral rna copy numbers among the groups during the experimental period. by 10 dpi, viral rna copy numbers declined in all drug-treated ferrets. to further evaluate virus titers in tissues, three ferrets from each group were euthanized at 4 and 8 dpi, and virus titers were measured in nasal turbinate and lungs. at 4 dpi, all groups of ferrets showed high virus titers of more than 3.0 log 10 tcid 50 /g in nasal turbinate tissues, and the virus was also isolated from their lung tissues (fig. 3) . at 8 dpi, while all nasal turbinate tissues were positive for virus, the azathioprinetreated group showed a much higher virus titer (ϳ3.0 log 10 tcid 50 /g) than those of the other groups (fig. 3a) . at 8 dpi, the azathioprine-treated group still had detectable virus titers in their lung tissues, whereas the rest of groups were negative for the virus (fig. 3b) . to compare the serum neutralization (sn) antibody titers among drug-treated groups, blood was collected from each group of ferrets at 10, 14, and 21 dpi. at 10 dpi, the pbs-treated control and drug-treated groups demonstrated sn titers greater than 40 (fig. 4) . the antiviral-treated groups showed sn titers similar to those of the pbs-treated control group until 14 dpi, but they exhibited lower sn titers at 21 dpi than the pbs-treated control group (fig. 4) . it is noteworthy that the azathioprine-treated immunosuppression group showed geometric mean sn titers of 33.6 and 47.5 at 10 and 21 dpi, respectively, suggesting the continuously reduced sn antibody response of the immunosuppressed group. in this study, we evaluated the antiviral efficacies of three fda-approved drug candidates against sars-cov-2 infection using a ferret infection model which has previously proven to be highly susceptible to sars-cov-2 infection (18, 20) . although several clinical trials continue to evaluate these drug candidates, most of the enrolled patient populations are considered heterogeneous with regard to the duration and severity of illness at enrollment. further, given the rapid spread of covid-19 around the efficacies of fda-approved drugs against sars-cov-2 ® world, there are relatively higher mortality rates in some regions and in certain age groups. therefore, the use of highly susceptible animal models and controlled experimental settings should be an effective approach prior to human clinical trials to evaluate the in vivo antiviral effects of potential therapeutics. ferrets treated with antivirals showed relatively reduced overall clinical scores compared with those of the pbs-treated control group, which displayed high overall clinical scores. of the three drugs, the emtricitabine-tenofovir-treated group showed a noticeable reduction in overall clinical scores (յ4) and a shorter duration (8 dpi) of clinical symptoms. although attenuation of the overall clinical scores was observed in lopinavir-ritonavir-and hcq-treated groups at some time points, their clinical durations were comparable with those of the pbs-treated control group. these results suggest that treatment with emtricitabine-tenofovir, a nucleotide analogue that inhibits rnadependent rna polymerase activity, may be the most likely candidate to reduce clinical symptoms, including the cough and morbidity of sars-cov-2-infected hosts. while the lopinavir-ritonavir protease inhibitor and a cq/hcq sulfate autophagy inhibitor have been shown to be effective against sars-cov, mers-cov, or sars-cov-2 in in vitro culture (5), several reports have already described no benefit for clinical improvement of covid-19 patients. furthermore, hqc has been reported to be associated with a number of side effects, including a heart rhythm problem, severely low blood pressure, and muscle or nerve damage. thus, these existing fda-approved drugs still need rigorous testing for efficacy and safety in an animal model prior to clinical trials of covid-19 patients. since the emergence of sars-cov-2 in china in december 2019, the number of cases has rapidly increased as the disease has spread globally. the increase in the number of cases is alarming and specially compounded by the possibility of viral transmission from asymptomatic individuals. several studies indicate that asymptomatic patients can transmit the virus to persons in close contact (21, 22) . therefore, although clinical symptoms were attenuated in ferret groups treated with antiviral candidates, we also evaluated virus titers in respiratory and gastrointestinal tracts using nasal washes and stool samples, respectively, from sars-cov-2-infected ferrets. regardless of antiviral candidate treatment, sars-cov-2 was detected at more than 2 log 10 tcid 50 /ml until 6 dpi, and there was no statistical difference between the pbs-treated control group and the antiviral-drug-treated groups. however, the emtricitabine-tenofovir-treated group showed relatively low virus tiers and shortened periods of virus shedding in nasal wash specimens compared with those of the other groups. currently, the role of sn antibody in the pathogenesis and disease clearance of sars-cov-2 is unclear. wu et al. (23) recently reported that the sn antibody levels in covid-19 patients were variable, depending on the immune status of the patient, and that about 30% of patients failed to develop high sn titers after sars-cov-2 infection, although the disease durations of these patients were comparable to those of others. this suggests that the sn antibody titer may be closely associated with immune activity rather than with the virus titer and disease duration in patients. interestingly, we found that the antiviral-treated groups showed lower sn antibody titers than the pbs-treated control group. it is possible that as the antiviral treatment reduced the overall clinical symptoms of sars-cov-2-infected ferrets, it might evoke weak immune responses and thereby lead to reduced neutralizing antibody responses. nevertheless, further immunological studies are needed to understand the detailed mechanisms of the low sn antibody titers in antiviral-treated ferrets. efficacies of fda-approved drugs against sars-cov-2 ® while covid-19 is typically characterized by respiratory symptoms, gastrointestinal symptoms have been reported in some cases. moreover, there is evidence of viral rna in the stools of sars-cov-2 patients. emtricitabine-tenofovir has reportedly shown antiviral efficacy in the gastrointestinal tract as well as in the respiratory tract (24) . however, qrt-pcr analysis of stools revealed no statistical difference in virus titers in stools among the pbs-treated control and the antiviral-treated groups, suggesting that none of the tested antiviral candidates significantly diminished gastrointestinal sars-cov-2 replication in infected ferrets. in conclusion, although there may be some discrepancies in drug efficacy between animals and humans, these results of a preclinical ferret infection study should aid in the selection of antiviral treatments of covid-19 patients. this also suggests that a robust preclinical animal model for sars-cov-2 infection is valuable in order to identify antiviral drugs for future human efficacy trials. a sars-cov-2 strain, nmc-ncov02, was propagated in vero cells in dulbecco's modified eagle medium (dmem; gibco, grand island, ny) supplemented with 1% penicillin-streptomycin (gibco) and tpck (tosylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin (0.5 g/ml; worthington biochemical, lakewood, nj) in a 37°c incubator supplemented with 5% co 2 for 72 h. propagated virus was stored at ϫ80°c as the working virus stock for animal studies. the 50% tissue culture infective dose (tcid 50 ) was determined through fixation and crystal violet staining. immunosuppression and antiviral-drug candidate treatments. ten ferrets were treated orally with azathioprine (10 mg/kg) (celltrion) daily for 7 days prior to sars-cov-2 infection, and treatment continued until 14 days postinfection (dpi) to reduce the immune response. as a control, pbs in the same volume was administered to 10 ferrets. to confirm the immunosuppressed status of ferrets, blood samples were collected from azathioprine-treated ferrets, and the percentage of lymphocytes was assessed at 7 and 4 days preinfection and at 7, 14, and 21 dpi. the reduction in lymphocyte numbers in azathioprine-treated ferrets compared with lymphocyte numbers in the pbs control group was confirmed (see fig. s1 in the supplemental material). for treatment with candidate antiviral drugs, groups of ferrets (10/group) were administered lopinavir (16 mg/kg)-ritonavir (4 mg/kg) (abbott), hydroxychloroquine sulfate (25 mg/kg) (elyson), or emtricitabine (6 mg/kg)-tenofovir (7.35 mg/kg) (gilead) daily via oral gavage starting at 1 dpi of sars-cov-2 infection and continuing until 14 dpi. experimental infection of ferrets. groups of 10-to 12-month-old female ferrets (10/group), seronegative for sars-cov-1 and sars-cov-2, were intranasally inoculated with 10 5.8 tcid 50 /ml of nmc-ncov02 under anesthesia. the body weights and temperatures of infected ferrets were monitored every other day until 14 dpi. nasal washes and stool specimens were collected every other day from the inoculated ferrets. blood was collected at 10, 14, and 21 dpi to measure the serum neutralization titer. three ferrets per group were euthanized at 4 and 8 dpi, and nasal turbinate and lungs were collected to measure tissue virus titers and examine lung histopathology. virus titers in nasal washes and tissues were determined by 50% tcid 50 assessment in vero cells, while the virus titers in stool specimens were measured with quantitative real-time pcr (qrt-pcr). briefly, total rna was extracted using trizol reagent (thermo fisher scientific) or an rneasy kit (qiagen), and cdnas were generated with a sars-cov-2specific primer by reverse transcription using quantitect reverse transcription (qiagen). qrt-pcrs were performed using a sybr green supermix (bio-rad) and a cfx96 touch real-time pcr detection system (bio-rad) with a spike gene-based, sars-cov-2-specific primer set as previously described (18) , and virus rna copy numbers were calculated as a ratio with respect to the standard control. clinical scoring methods of sars-cov-2-infected ferrets. the ferrets were monitored daily over a 14-day period for temperature change, weight loss, clinical symptom, and movement and activity change. briefly, the frequency of cough and rhinorrhea was assessed in each group of ferrets and scored on the basis of the following criteria: no evidence of cough (score, 0), occasional cough (score, 1), and frequent cough (score, 2) and no nasal rattling or sneezing (score, 0), moderate nasal discharge on external nares (score, 1), and severe nasal discharge on external nares (score, 2). a change in a ferret's activity was assessed and scored on the basis of the following criteria: normal movement and activity (score, 0), mildly reduced movement and activity (score, 1), and considerably reduced movement and activity (score, 2) for at least 20 min. neutralizing assay. sera were collected from each group of ferrets to detect the serum neutralization titer. heat-inactivated 10-fold-diluted serum samples were serially diluted by 2-fold. an equal volume of sars-cov-2 at 100 tcid 50 was added to all diluted samples. the mixture of serum and virus was incubated at 37°c for 1 h and then added to vero cells in a 96-well tissue culture plate for 90 min. the mixture of serum and virus was then removed, followed by two washes with cold pbs. fresh medium was added to infected cells, and cells were incubated at 37°c in 5% co 2 for 4 days. supernatants were removed, fixed with a 10% formalin solution, and stained with crystal violet to determine the titer. statistical analysis. to assess significant differences in values for weight loss, temperature, viral titers, and serum neutralization titers, statistical analyses were done. asterisks indicate the statistical significance between pbs-administered and treated ferrets determined by two-way analysis of variance (anova) and a subsequent dunnett test (*, p ͻ 0.05; **, p ͻ 0.001; and ***, p ͻ 0.0001). all statistical analyses were performed using graphpad prism version 8.20 for windows (graphpad software, la jolla, ca). park et al. who. 2020. coronavirus disease 2019 (covid-19) situation report 88. who detection of sars-cov-2 in different types of clinical specimens viral load of sars-cov-2 in clinical samples temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treatment with lopinavir/ritonavir or interferon-␤1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-␣ of middle east respiratory syndrome virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen antimalarial drug chloroquine counteracts activation of indoleamine (2,3)-dioxygenase activity in human pbmc hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro hydroxychloroquine for sars-cov-2 infection: how did we get here? connecticut hospitals using anti-malaria drug chloroquine 'out of desperation' to treat covid-19 animal toxicity and pharmacokinetics of hydroxychloroquine sulfate hydroxychloroquine for autoimmune diseases advances in nucleotide antiviral development from scientific discovery to clinical applications: tenofovir disoproxil fumarate for hepatitis b tenofovir alafenamide (taf) as the successor of tenofovir disoproxil fumarate (tdf) infection and rapid transmission of sars-cov-2 in ferrets enteric involvement of severe acute respiratory syndromeassociated coronavirus infection susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 transmission of efficacies of fda-approved drugs against sars-cov-2 ® 2019-ncov infection from an asymptomatic contact in germany a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications drug-susceptible hiv-1 infection despite intermittent fixed-dose combination tenofovir/emtricitabine as prophylaxis is associated with low-level viremia, delayed seroconversion, and an attenuated clinical course all animal experiments were approved by the medical research institute, a member of the laboratory animal research center of chungbuk national university (larc) (approval number cbnur-1352-20), and were conducted in strict accordance with and adherence to relevant policies regarding animal handling as mandated under the guidelines for animal use and care of the korea centers for disease control (kcdc). the handling of virus was performed in an enhanced biosafety level 3 (bsl3) containment laboratory as approved by the korea centers for disease control and prevention (protocol kcdc-14-3-07). supplemental material is available online only. key: cord-280774-r2xm164s authors: gallizzi, romina; sutera, diana; spagnolo, alessandra; bagnato, anna maria; cannavò, serafinella patrizia; grasso, loredana; guarneri, claudio; nunnari, giuseppe; mazza, francesca; pajno, giovanni battista title: management of pernio‐like cutaneous manifestations in children during the outbreak of covid‐19. date: 2020-09-19 journal: dermatol ther doi: 10.1111/dth.14312 sha: doc_id: 280774 cord_uid: r2xm164s background: during the outbreak of covid‐19 many pernio‐like lesions have been increasingly reported. the aim of the study is to describe our management of these skin manifestations and to evaluate a possible correlation to sars‐cov‐2 infection. methods: all patients underwent clinical and laboratory tests to detect a possible underlying connective disease and also to specific sars‐cov‐2 investigations such as oropharyngeal swab and igg‐igm serology. results: nine patients aged between five and fifteen years old were evaluated. skin lesions observed were purplish, erythematous and oedematous, in some cases painful and itchy. six out of nine had respiratory and systemic symptoms (cough, nasal congestion, chills, fever, asthenia) that preceded cutaneous findings of approximately two weeks. concerning blood exams, three out of nine had d‐dimer weakly increased, four had ana positivity: two with a title 1:160, one with 1:320 and one with 1:5120 and a speckled pattern. the latter patient had also ena ss‐a positive and rf positivity, confirmed at a second check, so as to allow us to make a diagnosis of connective tissue disease. four out of nine had apl positivity (igm). reactants acute phase were all negative. oropharyngeal swabs and serology tests for sars‐cov‐2 was negative (borderline in one patient for igm). no treatment was needed. conclusions: even if we do not have enough data to prove it, we hypothesize a correlation between pernio‐like lesions and sars‐cov‐2 infection for an increased number of these lesions described during the pandemic and also because such manifestations appeared when temperatures were mild and patients were at home in isolation for the lockdown. many questions remain open about interaction host‐virus. this article is protected by copyright. all rights reserved. during the outbreak of covid-19 many skin manifestations have been reported, and among these, in very significant numbers, newly vascular eruptions and peculiar pernio-like skin lesions have been described in observational studies 1, 2, 3, 4 . pernio, also referred to as chilblains, is a rare inflammatory condition. chilblains derives from two old english words "chill" (cold) and "blegen" (sore). most commonly, pernio affects acral skin and develops among susceptible individuals who are exposed to cold, the lesions usually appear in fall or winter and disappear in spring or early summer. it is typically idiopathic and acute, nevertheless chronic forms also exist 5 . the diagnosis of pernio is largely clinical and based on a thorough history and physical exam. the differential diagnosis must exclude diseases that can often be confused with other forms of pernio or vasculitis processes like systemic lupus this article is protected by copyright. all rights reserved. erythematous (sle) or other conditions as raynaud phenomenon, acrocyanosis, cryoglobulinemia, cold panniculitis and interferonopathies. the prognosis of pernio is good with minimal chronic sequelae. single or multiple erythematous, purplish, edematous lesions appear, accompanied by intense pain, itching, or burning. usually, pernio affects the toes and dorsum of the proximal phalanges 6 . the mainstay of treatment is the avoidance of the cold and, in some cases, drugs as nifedipine and other calcium channel blockers are needed for the resolution of existing lesions 5 . since march 2020, children with acral red and painful skin lesions, referable to chilblain have come to our attention. the increased number of cases of pernio-like lesions compared to the cases per year we usually observe, the mild temperatures of those months in southern italy and the concomitant lockdown, led us to hypothesize a possible correlation with sars-cov-2 infection. we evaluated the personal history and photographs of skin lesions of 26 patients, sent to us by their pediatrician, through multidisciplinary telematic meetings with dermatologists, rheumatological pediatricians, infectious disease specialist. we only included patients with pernio-like skin lesions (nine patients). patients who could not perform the oropharyngeal swab for sars-cov-2 were not admitted to the hospital. we collected informed consent to obtain clinical information and photos of patients and to perform blood chemistry sampling. therefore, we evaluated 9 cases of children who in our group of patients no significant difference in gender was detected (5 females and 4 males). the median age was 11,4 years (from 5 to 15 years). two patients were siblings. all patients were from the south of italy coming from the town of messina and surrounding. their family histories and their personal histories were negative for autoimmune disorders, raynaud's phenomenon, acrocyanosis, chilblains or photosensitivity except for one child who had suffered from an episode of raynaud's phenomenon a few years earlier. no family member of these patients presented symptoms attributable to sars-cov-2 infection but, for work reasons, their parents were in contact with public. only two siblings had both parents with compatible symptoms and confirmed sars-cov-2 infection by positive nasal swab. the cutaneous manifestations observed were purplish, erythematous and edematous, four children reported subjective symptoms, painful and pruritus localized to the sole of the feet or to the toes and/or fingers or heels ( fig. 1,2,3 ). feet alone were mostly affected (6 out of 9), hands alone (3 out of 9) (fig.1) . six patients had respiratory and systemic symptoms (cough, nasal congestion, chills, fever, asthenia) that preceded the skin lesions by about two weeks (table1). this article is protected by copyright. all rights reserved. we evaluated 9 cases of children who presented pernio-like lesions, since march to april 2020 during the outbreak of covid-19. we performed first level tests, all negative except for d-dimer weakly increased in three of our patients. d-dimer is a marker of activation of coagulation and fibrinolysis and it provides a rapid evaluation of thrombotic activity. its level correlated to coagulopathy have been described as prognostic factors in the evolution of sars-cov-2 infection, especially in more severe patients. zhang's study developed a triage, testing d-dimer levels at the admission, on the first and third day to predict survival in a cohort of patients and to evaluate management and follow up. the result was that a regulatory level of d-dimer at the presentation is highly predictive for survival 7 . this is useful to highlight, as in our case, the d-dimer of our patients was weakly increased, a condition perfectly correlated with the mild symptoms of sars-cov-2 putative infection presented. we have also performed autoimmunity tests, three out of nine had ana positivity (speckled pattern): two with a title of 1:160, one 1:320 not confirmed at a subsequent check after two months, another asymptomatic cases generated specific antibody responses for sars-cov-2 9 . the case of our family is emblematic: two parents presented striking symptoms of sars-cov-2 infection, high fever and this article is protected by copyright. all rights reserved. difficulty in breathing, and three positive swabs; the father had positive serology with high igg, such as to allow donation of his plasma for therapeutic purposes and the mother was surprisingly negative. sons who came to our observation, both with chilblain, presented mild respiratory symptoms and they were negative for both swabs and igg serology, while in one of the two cases, the igm were borderline. despite the negativities of diagnostic test for sars-cov-2, we are still convinced that there is a correlation between this infection and the development of pernio-like lesions, as described nasopharyngeal swab for sars-cov-2 gave negative results 4 . in a report of 19 adolescent patients with a clinical diagnosis of pernio-like lesions nasopharyngeal swab and igg serology for sars-cov-2 nucleocapsid protein were negative. importantly, iga serology for s1 domain of sars-cov-2 spike protein was positive in 6 patients and borderline in 3 patients 10 . in a study performed in sicily (italy) via teledermatology a total of 22 patients complaining of perniosis-like lesions were screened, mainly in the pediatric age (≤18yrs). all of them were tested with rhino-pharyngeal swabs and sars-cov-2 was detected in 6 patients, 5 of whom were children 11 . the understanding of the immunopathogenetic mechanisms of interaction between the sars-cov-2 infection and children is very intriguing but current knowledge does not seem to be sufficient. why some children who come into contact with the sars-cov-2 do not develop striking respiratory symptoms but present pernio-like lesions with negativity on diagnostic tests? matricardi et al developed the first model of interaction between the human immune system and sars-cov-2, as an attempt to produce a synthesis of the actual knowledge 12 . what emerges is that innate immunity represents the first line of defense against the new sars-cov-2 and this first comparison establishes the natural history of the pathology: either the infection will effectively block in the upper airways or if the virus manages to reach the lungs. innate immunity is essential for controlling virus replication before an adaptive immune response is generated 13 . type i interferones (ifn-i) are major components of the innate immune system and it represents critical antiviral molecules 14 . it is hypothesized that the ifn-i response may induce microangiopathic changes, producing chilblains and lupus-like erythematous eruption. a mechanism has recently been considered to explain the appearance of autoimmune phenomena following the whole sars-cov-2 infection: molecular mimicry 16, 17, 18 . lucchesi and floel 19 have hypothesized a molecular mimicry mechanism between neuronal proteins present in the brain stem respiratory pacemaker neurons (dab1, aifm and surf1) and viral epitopes of sars-cov-2 treated antigenic. the same mechanism, according to angileri et al., could be responsible of anosmia, leukopenia and multi-organ failure caused by vascular damage, assuming they are associated with the molecular mimicry 20 . a type 3 hypersensitivity therefore occurs with the deposition of antibody antigen complexes precipitating inside the tissues, in particular the blood vessels, inducing a serious inflammatory state by the action of the complement anaphylatoxins (c3a and c5a), which in turn stimulate the release of histamine from mast cells and the recruitment of phagocytes, first in neutrophils, the main cause of tissue damage and following «leukocytoclastic vasculitis» (lcv), also reported in the english medical literature from the term «hypersensitivity vasculitis 21, 22 ». this pathogenic mechanism could explain the appearance of pernio-like lesions due to sars-cov-2 infection. in conclusion, we think there is a correlation between pernio-like lesions and sars-cov-2 infection, but further studies are needed to prove it. this, for the increased number of these lesions described during this short time, as in our experience, and because such manifestations appeared when temperatures were mild in southern italy and patients were at home for the lockdown. to our knowledge, this is one of the few studies that collects a series of pediatric patients with perniolike lesions, evaluating the possible association with covid 19 (oropharyngeal swab and serology test) but also for rheumatological diseases. this article is protected by copyright. all rights reserved. chilblain-like lesions during covid-19 epidemic: a preliminary study on 63 patients chilblain-like lesions on feet and hands during the covid-19 pandemic acral cutaneous lesions in the time of covid-19 pernio (chilblains) statpearls treasure island (fl): statpearls publishing d-dimer levels on admission to predict in-hospital mortality in patients with covid-19 autoantibodies in nonautoimmune individuals during infections yuxin chen & bing gu (2020) different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid-19 patients a clinical, histopathological and laboratory study of 19 consecutive italian paediatric patients with chilblain-like lesions: lights and shadows on the relationship with covid-19 infection diversity of clinical appearance of cutaneous manifestations in the course of covid-19 the first, holistic immunological model of covid-19: implications for prevention, diagnosis, and public health measures type i interferons (a/b) in immunity and autoimmunity covid-19) infection-induced chilblains: a case report with histopathologic findings covid-19 a proteiform disease inducing also molecular mimicry phenomena? guillain barre syndrome associated with covid-19 infection: a case report covid-19 and molecular mimicry: the columbus' egg? molecular mimicry between sars-cov-2 and respiratory pacemaker neurons molecular mimicry may explain multi-organ damage in covid-19 immunobiology: the immune system in health and disease leukocytoclastic vasculitis list of abbreviations acl: anti-cardiolipin antibodies aifm: apoptosis-inducing factor 1, mitochondrial ana: antinuclear antibodies anti-β2gp1: anti β-2-glycoprotein1 antibodies apl: antiphospholipid antibodies c3a: complement factor 3 anaphylotoxin c5a: complement factor 5 anaphylotoxin cbc: complete blood count clia: chemiluminescent microparticle immunoassay covid key: cord-293578-yu2i0u2h authors: kusadasi, nuray; sikma, maaike; huisman, albert; westerink, jan; maas, coen; schutgens, roger title: a pathophysiological perspective on the sars-cov-2 coagulopathy date: 2020-08-10 journal: hemasphere doi: 10.1097/hs9.0000000000000457 sha: doc_id: 293578 cord_uid: yu2i0u2h recent evidence is focusing on the presence of a hypercoagulable state with development of both venous and arterial thromboembolic complications in patients infected with sars-cov-2. the ongoing activation of coagulation related to the severity of the illness is further characterized by thrombotic microangiopathy and endotheliitis. these microangiopathic changes cannot be classified as classical disseminated intravascular coagulation (dic). in this short review we describe the interaction between coagulation and inflammation with focus on the possible mechanisms that might be involved in sars-cov-2 infection associated coagulopathy in the critically ill. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has crossed the species barriers and is currently causing lethal illness throughout the world. the clinical course in the critically ill infected with sars-cov-2 is defined by the development of severe hypoxia based on acute respiratory distress syndrome and multiple organ failure necessitating intensive care support. 1 from a system biology point of view, at least 4 pathophysiological mechanisms are possibly involved in the pathogenesis of the critical illness associated with sars-cov-2 infection (fig. 1) . these comprise (1) the renin-angiotensin, (2) the kallikrein-kinin, (3) the complement and (4) the coagulation system. [2] [3] [4] [5] [6] angiotensin converting enzyme-2 is considered as the functional receptor for sars-cov-2 involved in cell attachment and entry. 7 the kallikrein-kinin system is currently deemed as a possible route involved in the development of pulmonary edema via the production of bradykinin and upregulation of bradykinin receptors. 6 finally, the interaction between nucleocapsid (n) proteins of sars-cov-2 and masp-2, the key serine protease in the lectin pathway of complement activation, resulting in aberrant complement factor 5 (c5) over-activation and deteriorating inflammatory lung injury has been emphasized. 2 besides these important mechanisms, recent evidence is focusing on the presence of a hypercoagulable state with development of both venous and arterial thromboembolic events such as pulmonary embolism, deep vein thrombosis and ischemic stroke. [8] [9] [10] [11] the ongoing activation of coagulation related to the severity of the illness in sars-cov-2 infected patients is further characterized by thrombotic microangiopathy and endotheliitis sars-cov-2 ace2 figure 1 . the 4 interacting pathophysiological mechanisms in sars-cov-2 infection. the potential role of plasma kallikrein in case of sars-cov-2 infection may be summarized as (1) the activation of fxii with the end-product thrombin (coagulation system) (2) the generation of bradykinin with subsequent vascular permeability and leakage (kallikrein-kinin system), (3) the activation of the renin-angiotensin system through conversion of renin from pre-renin leading to a pro-inflammatory state through increased angiotensin 1 receptor activation and (4) the activation of c5, in part through activation of c1 via fxii and in part through activation of c3 via plasmin (complement system). [2] [3] [4] [5] [6] powered by eha review article as described in autopsies. 12, 13 there are however important reasons to not classify these microangiopathic changes as classical disseminated intravascular coagulation, possibly in part based on local plasma leakage in the lungs. 6,13-15 based on evidence from clinical course, sars-cov-2 infection associated coagulopathy occurs in two distinct phases. the ongoing coagulation might be caused by prekallikrein dependent contact activation, which is exacerbated by gradually increasing intravascular (micro)angiopathy caused by ongoing endothelial activation. in this short review we describe the interaction between coagulation and inflammation emphasizing the possible mechanisms that might be involved in sars-cov-2 infection associated coagulopathy in the critically ill. these mechanisms are summarized in figure 2 and will be discussed below. the haemostatic system is based on consecutive activation of precursor proteins. 16 tissue factor (tf) plays a pivotal role as initiator of this system in case of vascular injury, known as the extrinsic pathway. 17 tf expressed after the endothelial cell damage activates coagulation locally. 18 coagulation factor (f) vii, present in the blood circulation, contacts with tf forming the extrinsic tenase complex. 19 this complex initiates thrombin generation but is inhibited by tissue factor pathway inhibitor (tfpi) after sufficient formation of activated fx (fxa). the amplicification loop, known as the intrinsic pathway, 20 can be activated through feedback amplification of thrombin via fxi, which in turn activates the intrinsic tenase complex (fviiia:fixa: fx) leading to amplification of thrombin generation. the intrinsic pathway can also be triggered directly through contact activation, in which fxii is the initiator. fxii is activated spontaneously when negatively charged surfaces contact the blood 21 and directly activates fxi. thrombin in turn stimulates several other factors (fv, fviii and fxiii) in order to accelerate prothrombin generation and convert fibrinogen to fibrin. subsequently fibrin clot is formed. although the role of fxii in bleeding complications seems not to be clinically relevant, its role in inflammation is apparent in acute and chronic inflammation. [22] [23] [24] [25] a systemic infection, like sepsis, is commonly associated with systemic inflammation and activation of coagulation which can result in microthrombosis, a process called immunothrombosis or thromboinflammation. [26] [27] [28] the crosstalk between inflammation and coagulation in sepsis takes place at different levels and is bidirectional with the endothelium being at the center of the process. the interaction between coagulation and activated negatively charged particles and activated platelets activate fxii after sars-cov-2 infection. pk is converted to kallikrein independent of fxiia when endothelial cell bounded hk bounds pk. pk generates fxiia on the surface of the endothelial cells. the binding of fxiia, pk and hk to endothelial cells results in production of kallikrein. fxiia activates plasma coagulation pathway via fxia on the surface of platelets. together with low adamts13, high vwf and high fviii levels, the intrinsic tenase complex (fviiia:fixa:fx) leads to increased thrombin generation. meanwhile, the extrinsic tenase complex (tf:fviia), together with the hypoxia induced interactions through hif-1 and hif-2 augment thrombin generation. the hif-1 and hif-2 stimulate the fibrin clot maintenance by inhibiting pai-1. kallikrein not only leads to production of the vasoactive peptide bradykinin, but also stimulates the conversion of plasminogen to plasmin, leading to increased d-dimer production. the feedback amplification of thrombin occurs through fv, fviii, fxi and platelet activation (green). the natural feedback inhibition by activated protein c (apc) and antithrombin is given in red. a = activated, adamts13 = a disintegrin and metalloprotease with thrombospondin type 1 motifs 13, annexin a2 = co-receptor of plasminogen and t-pa, apc = activated protein c, f = factor, fdp = fibrin degradation product, hif = hypoxia inducible factor, hk = high molecular weight kininogen, pai-1 = plasminogen activator inhibitor-1, pk = prekallikrein, pl = phospholipids, polyp = polyphosphate, tafi = thrombin activatable fibrinolysis inhibitor, tfpi = tissue factor pathway inhibitor, tpa = tissue plasminogen activator, upa = urok. endothelial cells in sepsis is well defined and is essential for a properly functioning haemostatic system. [29] [30] [31] the extrinsic tenase complex (tf: fviia) formed after endothelial cell damage initiates thrombin generation by activating fx or fix. this tenase complex connects the extrinsic and intrinsic route via fix. the expression of tf on monocytes, macrophages and endothelial cells is induced by proinflammatory cytokines (il-1, il-6 and tnf-a). 32, 33 increased levels of these proinflammatory cytokines have been observed in severely ill patients infected with sars-cov-2. 34, 35 thrombin results in the recruitment of leucocytes and platelets and stimulates the production of proinflammatory cytokines. [36] [37] [38] [39] [40] [41] second crosstalk is at the level of the protein c system. thrombin binds to thrombomodulin on the endothelial cell surface which in turn enhances the generation of activated protein c (apc). 42 the apc acts as anticoagulant agent by inactivating fv and fviii and has been suggested to act as anti-inflammatory factor by downregulation of proinflammatory cytokine production and decreasing leucocyte adherence and apoptosis. 43 decreased levels of apc results in decreased inactivation of fv and fviii, which in particular might cause a limited anticoagulant response. the anticoagulant effect of apc is well defined, however its use in microangiopathic patients, such as in sepsis, did not improve outcome. 44, 45 third crosstalk is at the level of fibrinolysis where apc has been suggested to neutralize fibrinolysis inhibitors tafi and pai-1 resulting in the activation of the fibrinolysis. 46 decreased levels of apc might in turn limit the fibrinolytic response. the haemostatic system is a complex and dynamic process and continuously subject to changes based on inflammation related factors, especially in critically ill. there is plethora of evidence indicating fxii being at the heart of the development of sars-cov-2 related coagulopathy in critically ill. it has been shown that negatively charged domain parts are related to the pathogenesis of sars-cov-2 47-50 which in turn might lead to activation of fxii. prekallikrein (pk) generates fxiia on the surface of activated endothelial cells, in a receptordependent manner. the binding of fxiia, pk and high molecular weight kininogen to endothelial cells results in generation of kallikrein (fig. 2 ). in addition, the release of polyphosphate (polyp) by platelet activation at the site of endotheliitis can be an important driver of coagulation by activating fxii. 51 in turn, activated fxii initiates the kallikrein-kinin and intrinsic coagulation system resulting in the end-products bradykinin and thrombin, respectively. 52, 53 therefore, the hypercoagulable state in sars-cov-2 might be due to ongoing fxii activation by, amongst others, negatively charged virus particles, activated platelets and the plasma kallikrein-kinin system. the role of kallikrein in a variety of proteolytic systems, such as the intrinsic pathway of coagulation and fibrinolysis, has sufficiently been emphasized. 54, 55 since prekallikrein is seen as a potential regulator in the fibrin clot formation through fxii activation and is involved in the pathogenesis of thrombosis, there might be an important role for controlling the hypercoagulable state of the patients infected with sars-cov-2 as a therapeutic target. 56, 57 recently, shatzel and colleagues emphasized the available evidence, obtained from inflammation models, for the inhibition of the contact activation route as a potential pharmacological target in patients with sars-cov-2 infection. 58 the observed massive coagulopathy in critically ill patients infected with sars-cov-2 may also be played by the activation of endothelial cells. the very high plasma levels of fviii and von willebrand factor (vwf) and the histological post-mortem analysis of different organs indicate the profound endothelial activation in sars-cov-2 associated coagulopathy in critically ill. 9,59 fviii and vwf have also been emphasized as being strongly associated with risk of venous thrombosis. 60 moreover, a relatively low adamts13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs 13, a plasma metalloprotease cleaving vwf) activity in relation to high vwf antigen may contribute to microangiopathic changes in critically ill patients infected with sars-cov-2. 61 an additional important mechanism contributing to the sars-cov-2 associated coagulopathy might be the sars-cov-2 infection induced severe hypoxia. hypoxia has been linked to augmentation of thrombosis. [62] [63] [64] hypoxia-inducible factor-1 (hif-1) is activated under hypoxic conditions resulting in upregulation of plasminogen activator inhibitor-1 (pai-1) and tf as well as inhibition of protein s. [62] [63] [64] hypoxia inducible factor-2 (hif-2) is also upregulated and stimulates pai-1 and inhibits tfpi. 65, 66 finally, from another angle obesity influences the risk of a hypercoagulable state. obese subjects have shown to have enhanced coagulation by elevated levels of vwf, tf, fvii, fviii and fibrinogen, and impaired fibrinolysis by increased secretion of plasminogen activator inhibitor (pai)-1 and thrombin activatable fibrinolysis inhibitor (tafi). 67-69 a significant number of patients infected with sars-cov-2 has been described to be obese indicating that being obese may be a predisposing risk factor for the hypercoagulable state. taken together, all these mechanisms may contribute to the complex hypercoagulable state of the critically ill patients infected with sars-cov-2 having severe hypoxia and ongoing endothelial activation. surprisingly, the platelet counts, antithrombin (at), fibrinogen and protein c levels ranged between normal to slightly increased levels in sars-cov-2 infected patients who are severely ill. 5, 9, 70 these data are in contrast to what is known about evolving disseminated intravascular coagulation (dic) where platelet counts, at, protein c and fibrinogen levels are clearly diminished. as the focus of this perspective is restricted to sars-cov-2 infection associated coagulopathy in critically ill, the reader is referred to several recent articles emphasizing the differences between dic and sars-cov-2 associated coagulopathy. 71, 72 moreover, several at-binding proteins (cd13, cd300f and lrp-1) on human monocytes have been identified and indicated to be involved in blocking the activity of nuclear factor-kb. 73 at also seemed to possess antiviral activity against hiv, hsv and hcv. [74] [75] [76] [77] therefore, normal to high levels of at might hypothetically be caused by sars-cov-2 infection itself and be due to its possible role in anti-inflammation. another interesting observation is the increased level of d-dimer during the severe clinical course of sars-cov-2 infection. 1,5,9 d-dimer is a fibrin degradation product. factors enhancing the degradation of fibrin clot by plasmin might influence the production of d-dimer. annexin a2, a calcium-and phospholipid-binding protein, is an endothelial surface co-receptor for tissue plasminogen activator (tpa) and plasminogen that stimulates the profibrinolytic state. 78 there is increasing evidence for a role of annexin a2 at the first phase of viral life cycle known as cell attachment and entry in a variety of viral infections. 79 sars-associated cytokines (il-6 and ifn-gamma) have been shown to upregulate the expression of annexin a2. 80 the plasma kallikrein is involved in many proteolytic activities. one of the additional functions of kallikrein (2020) 4:4 www.hemaspherejournal.com is to stimulate the production of plasmin. 81 plasmin in turn can generate d-dimer from fibrin clots. although available data is limited, this may imply that factors interfering either with this co-receptor and/or stimulating plasmin might modulate the production of d-dimer. the use of pharmacological thrombosis prophylaxis decreases the risk of thrombotic events in covid-19. 82 however, despite the use of thrombosis prophylaxis and therapeutic anticoagulation, the risk of thromboembolic events remained high and appeared not to be sufficient. 8, 9 pharmacological targeting of the different pathophysiological mechanisms other than routine anticoagulation therapies might improve the severe clinical course seen in patients with sars-cov-2 infection at several of them are currently clinically being tested. at the level of platelets, anti-polyp antibodies have the potential to ameliorate the hyperinflammatory and prothrombotic state. 83 although successful in animal models, 84 no human drug currently exists. blocking fxi or fxii is an appealing option and both have been shown to prevent contact activated thrombosis in animal models. considering its role in the bradykinin pathway, fxii is the attractive target in sars-cov-2 related pathology. from this point of view, blocking fxiia by its strong inhibitor c1-esterase inhibitor (c1-inh) is of interest and is currently been explored by osthoff and colleagues at the university hospital basel in switzerland. the potential role of plasma kallikrein in case of sars-cov-2 infection may be summarized as (1) the activation of fxii with the end-product thrombin (2) the generation of bradykinin with subsequent vascular permeability and leakage, (3) the activation of the renin-angiotensin system through conversion of renin from prerenin leading to a pro-inflammatory state through increased angiotensin 1 receptor activation and (4) the activation of c5, in part through activation of c1 via fxii and in part through activation of c3 via plasmin. the inhibition of plasma kallikrein might therefore play a central role as target treatment, especially as 2 drugs are already on the market (lanadelumab and ecallantide). the use of these agents, alone or in combination with other agents acting on complement, coagulation and/or renin-angiotensin systems, might be an attractive treatment strategy. at the level of the complement system, the risk of thrombotic complications in patients with paroxysmal nocturnal hemoglobinuria, a rare life threatening disease manifesting with hemolytic anemia, bone marrow failure and thrombosis, has well been established. 85 even more so, treatment with the c5 blocking agent eculizumab decreases these thrombotic risk significantly indicating the inhibition of complement activation as a possible target to reduce the risk of thrombotic complications. 86 finally, the interplay between inflammation and thrombosis has already being stressed. interfering with anti-inflammatory agents, such as tocilizumab, anti-il-6, anti-il-1 and others, could have a beneficial effect on the level of coagulopathy as well. future research to prevent or treat the sars-cov-2 infection related coagulopathy should therefore focus on targets other than regular antithrombotic treatment. the severe critical illness as seen in patients infected with sars-cov-2 is characterized by multiple organ failure and a deep hypoxia related to ards and pulmonary embolism. central in the pathogenesis are the activation of the renin-angiotensin, the kallikrein-kinin, the complement and the coagulation system. pharmacological interventions aimed at these mechanisms may alter the clinical course in these patients. clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a singelcentered, retrospective, observational study highly pathogenic coronavirus n protein aggravates lung injury by 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baolin title: covid-19 update: the race to therapeutic development date: 2020-10-24 journal: drug resist updat doi: 10.1016/j.drup.2020.100733 sha: doc_id: 280068 cord_uid: rszu1c48 the covid-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), represents an unprecedented challenge to global public health. at the time of this review, covid-19 has been diagnosed in over 40 million cases and associated with 1.1 million deaths worldwide. current management strategies for covid-19 are largely supportive, and while there are more than 2000 interventional clinical trials registered with the u.s. national library of medicine (clinicaltrials.gov), results that can clarify benefits and risks of candidate therapies are only gradually becoming available. we herein describe recent advances in understanding sars-cov-2 pathobiology and potential therapeutic targets that are involved in viral entry into host cells, viral spread in the body, and the subsequent covid-19 progression. we highlight two major lines of therapeutic strategies for covid-19 treatment: 1) repurposing the existing drugs for use in covid-19 patients, such as antiviral medications (e.g., remdesivir) and immunomodulators (e.g., dexamethasone) which were previously approved for other disease conditions, and 2) novel biological products that are designed to target specific molecules that are involved in sars-cov-2 viral entry, including neutralizing antibodies against the spike protein of sars-cov-2, such as regn-cov2 (an antibody cocktail) and ly-cov555, as well as recombinant human soluble ace2 protein to counteract sars-cov-2 binding to the transmembrane ace2 receptor in target cells. finally, we discuss potential drug resistance mechanisms and provide thoughts regarding clinical trial design to address the diversity in covid-19 clinical manifestation. of note, preventive vaccines, cell and gene therapies are not within the scope of the current review. fusion peptide (fp) and heptad repeat (hr) regions within the s2 subunit mediate membrane fusion and release of viral rna. therapeutic potential of these regions gained attention during previous efforts towards treatments of respiratory illness caused by the closely related coronavirus, sars-cov. it is important to note that the s2 subunit is less exposed than s1 and may only be accessible following conformational changes (coutard et al., 2020; xia et al., 2020) . nevertheless, examining virus binding and entry as a critical checkpoint within the virus life cycle reveals several targets that are likely beneficial to reducing virus spread within the host system. after releasing viral rna into the cytoplasm, the virus hijacks the host translational machinery to translate the rna into two large polyproteins, which will ultimately produce the replicasetranscriptase complex (rtc) through viral protease cleavage . the importance of viral replication proteins, particularly the viral proteases, main protease (mpro), papain-like protease (plpro), as well as rdrp activity of the rtc is evidenced by the functionality conservation across coronaviruses (li and de clercq, 2020) . blockade of these proteins with inhibitors, such as boceprevir (an inhibitor of mpro) and antivirals that block viral rna polymerase activity may alleviate sars-cov-2 infection (choy et al., 2020; . the rtc drives production of both negative sense rna and positive sense rna with rdrp and helicase functionality thiel et al., 2003) . discontinuous transcription of negative sense rna yields the subgenomic rna (sgrna) to be translated into viral accessory and structural proteins, including the s, m, n, and e proteins. most of these proteins localize to the endoplasmic reticulum (er) where they are folded and post-translationally modified before being transported to the er-golgi intermediate compartment (fehr and perlman, 2015) . mature virions are an assembly of nucleocapsid-coated genomic rna and structural proteins that are released from the cell via exocytosis. once released, virion particles may interact with other ace2 expressing cells in various organs, including the heart, liver, and kidney . a critical approach to treating covid-19 patients, particularly those with severe respiratory or cardiovascular complications may be through regulating the virus-mediated immunopathology and host immune response to the viral infection. the initial host immune response involves the activation of the type i and iii interferon (ifn) responses, followed by activation of ifn-stimulated j o u r n a l p r e -p r o o f genes (isgs) as well as later release of pro-inflammatory cytokines and chemokines, which mount an antiviral defense against the viral infection (cardone et al., 2020) . however, coronaviruses, including the novel sars-cov-2, have been documented to employ strategies towards blocking and evading this response, resulting in dampening of antiviral mechanisms and increases in viral replication, titer, and spread (blanco-melo et al., 2020; park and iwasaki, 2020) . given the critical role of the ifn system in viral defense, exogenous ifn as a therapeutic has been explored and will be discussed in greater detail in the following sections. other mechanisms employed by the immune system to combat the virus can also play a role in disease severity (cardone et al., 2020) . with continuing viral replication, infected cells can become apoptotic, releasing increased levels of pro-inflammatory cytokines or damage-associated molecular patterns (damps) , which may lead to increased tissue damage (cardone et al., 2020) . in some settings, the release of high levels of pro-inflammatory cytokines may be associated with severe symptoms and virus persistence (fig. 1) . elevations have included il-6, il-1β, and tnfα, with high levels of il-7, il-8, il-9, il-10, ifn-γ, tnf, mcp1, mip1a, mip1b, g-csf, gm-csf having also been observed in covid-19 patient plasma . in some patients, this event is believed to promote cytokine release syndrome (crs) or "cytokine storm" resulting in the recruitment of hyperactive macrophages and monocytes to sites of infection (fig. 1) . the continuous activation of immune cells leaves damaging effects ranging from capillary damage to multiorgan failure. in severe patients, initial lung injury can progress into ards, a major contributor to covid-19 mortality . thus, to mitigate the harmful effects of the host immune response, a considerable portion of therapeutic programs are aimed towards reducing the overproduction of pro-inflammatory cytokines and associated cytokine storm. the current review highlights the potential therapeutic strategies for the treatment of covid-19, including small molecule drugs and therapeutic proteins to target the sars-cov-2 viral entry, viral amplification or the host immune responses. however, preventive vaccines, cell and gene therapies are not within the scope of this review. while treatment options that prevent infection or viral replication of sars-cov-2 are yet to be approved, the availability of therapeutics with antiviral, anti-inflammatory, or immunomodulatory j o u r n a l p r e -p r o o f activities provide many potential candidates (table 1) . using drugs previously approved for other indications takes advantage of existing detailed information on human pharmacology and toxicology to expedite clinical trials and regulatory review. indeed, the majority of ongoing clinical trials for covid-19 are aimed to evaluate the safety and efficacy of the existing drugs, previously one way these drugs are being used is to target the endolysosomal pathway that sars-cov-2 uses to enter target cells. chloroquine (cq) and hydroxychloroquine (hcq), the antimalarial drugs that increase ph in the endosome, lysosome, and golgi apparatus have also been reported to alter ace2 terminal glycosylation, impacting sars-cov binding to ace2 (fox, 1993; mauthe et al., 2018; vincent et al., 2005; yao et al., 2020) . azithromycin, a broad-spectrum macrolide antibiotic that induces lysosomal alkalization, has been reported to have potential in vitro activity against h1n1 influenza virus by interfering with its internalization (renna et al., 2011; tran et al., 2019) . to prevent s priming, camostat, a pancreatitis drug that inhibits tmprss2, was found to inhibit sars-cov-2 infection in human lung cells . while camostat and azithromycin are currently under investigation, the potency of cq/hcq in covid-19, which initially showed promise, was later challenged due to a lack of efficacy by larger studies (boulware et al., 2020; horby et al., 2020b; kupferschmidt, 2020; world health organization, 2020d ). an emergency use authorization (eua) for cq and hcq, which had been authorized by fda to allow certain uses of those drugs in the context of initial reports of potential benefit and widespread expert interest, was revoked based on fda's ongoing assessment of available scientific information associated with the emergency use of those products (hinton, 2020) . specifically, fda determined that cq and hcq are unlikely to be effective in treating covid-19 for the authorized uses in the eua. additionally, in light of ongoing serious cardiac adverse events and other serious side effects, fda determined that the known and potential benefits of cq and hcq no longer outweigh the known and potential risks for the authorized use. many clinical strategies to inhibit viral replication and release involve the use of repurposed antivirals (artese et al., 2020; drożdżal et al., 2020; pawar, 2020) . drugs that have been under study for potential uses in covid-19 include remdesivir, a broad-spectrum rdrp inhibitor that was previously studied for ebola treatment, and favipiravir (obtained conditional marketing approval in japan for the treatment of influenza), and the approved hiv protease inhibitor combination lopinavir/ritonavir (beigel et al., 2020; furuta et al., 2017; grein et al., 2020; mcmahon et al., 2020; sheahan et al., 2017; . preclinical studies have shown that remdesivir inhibited replication of sars-cov, mers-cov, and sars-cov-2 in vitro (agostini et al., 2018; brown et al., 2019; sheahan et al., 2017; sheahan et al., 2020; , which prompted several clinical studies including the actt trial sponsored by the u.s. national institute of allergy and infectious diseases (nct04280705) and the solidarity trial sponsored by who (world health organization, 2020c). preliminary report from randomized, double-blind, placebo-controlled phase-3 actt trial showed that remdesivir shortened the recovery time in hospitalized patients with covid-19 (beigel et al., 2020) . as a result, remdesivir was granted eua in the us (u.s. food and drug administration, 2020a), special approval for emergency in japan (pharmaceuticals and medical devices agency, 2020), and conditional marketing authorization in the eu (european medicines agency, 2020), where the safety and efficacy will be continuously evaluated through ongoing trials. however, the interim results from the randomized open-label solidarity trial reported little or no effect of remdesivir on overall mortality, initiation of ventilation and duration of hospital stay among hospitalized patients (pan et al., 2020) . moreover, the lopinavir/ritonavir combination was reportedly ineffective in hospitalized covid-19 patients also based on results from randomized open-label trials recovery collaborative group, 2020; world health organization, 2020d) . exogenous ifn treatments, which have been previously approved for multiple sclerosis, are being tested for their antiviral and immunomodulatory properties. clinical trials are focused on testing subcutaneous administration or nebulized ifn β, with direct inhalation into the lungs of patients, to stimulate an immune response. ifn β is an endogenously produced cytokine protein with antiviral effects, which may be deficient in lung cells of covid-19 patients. therefore, restoring j o u r n a l p r e -p r o o f type i ifn levels might reduce the viral load in covid-19 patients (mantlo et al., 2020) . however, the interim results from the who solidarity trial reported that ifn-β1a was ineffective in hospitalized patients (pan et al., 2020) . this has only been tested in small trials, with larger trials being necessary to fully understand the treatment results. pegylated ifn λ, which is known for its antiviral activity independent of type i interferons, is being tested in clinical trials to prevent infection and to slow viral replication while reducing inflammatory damage (prokunina-olsson et al., 2020) . ifn λ, may have a narrow therapeutic window due to the heightened risk of bacterial infections, impacting the duration or timing of administration (broggi et al., 2020). as described above, covid-19 patients with severe illness often present increased levels of cytokines, chemokines, t lymphocytes, nk cells, and growth factors in plasma (huang et al., 2020b) . the use of immunomodulators to reduce serum levels of pro-inflammatory cytokines might be helpful in mitigating immune-related symptoms. severe covid-19 cases are also linked to high levels of serum il-6, which can be a component of cytokine release syndrome (filocamo et al., 2020; . several clinical trials are evaluating the potential use of the existing anti-inflammatory therapeutic proteins (guaraldi et al., 2020) including (i) il-6r blockers tocilizumab (nct04381936), indicated for treatment of rheumatoid, giant cell, and juvenile idiopathic arthritis, as well as cytokine release syndrome, and sarilumab (nct04315298), indicated for rheumatoid arthritis (ra); (ii) il-1r blocker anakinra (nct04330638), a recombinant protein drug also treating ra; (iii) tnf-α blocker infliximab (nct04344249), another biologic for ra; and (iv) ifn-γ blocker emapalumab (nct04324021) approved for primary hemophagocytic lymphohistiocytosis treatment (filocamo et al., 2020; spinelli et al., 2020) . however, recent results from a phase-3 trial of tocilizumab (roche, 2020) and sarilumab (sanofi, 2020) were both disappointing. additional therapies that are commonly used for allergies or heartburn are being tested to potentially reduce cytokine storm in pulmonary tissues (hogan et al., 2020) . histamine blockers (the h1 receptor antagonist cetirizine and h2 receptor antagonist famotidine) may control the hyper-immune response . several small molecule drugs with anti-inflammatory activity are also being tested for covid-19. among these, the corticosteroid dexamethasone has been reported to decrease the risk of death in covid-19 patients with severe respiratory complications (horby et al., 2020a) . the fda recently added dexamethasone to the list of drugs for temporary compounding by outsourcing facilities and pharmacy compounders during the covid-19 public health emergency to help prevent demand j o u r n a l p r e -p r o o f and supply interruptions (u.s. food and drug administration, 2020d; university of oxford, 2020a). who also strongly recommends corticosteroids (i.e. dexamethasone, hydrocortisone or prednisone) for the treatment of patients with severe and critical covid-19 (world health organization, 2020b) . other anti-inflammatory small molecule drugs include the anti-microtubule agent colchicine (nct04322682) to block elongation of microtubules and interfere with cytokine and chemokine secretion, and the janus kinase (jak) inhibitors ruxolitinib and baricitinib (nct04345289) to inhibit the release of pro-inflammatory cytokines (spinelli et al., 2020) . heparin, previously approved as a blood thinner, was originally used in covid-19 clinical trials to limit lung injury and prevent blood clot formation (ayerbe et al., 2020; . due to the affinity of the spike protein to cell surface glycans, heparin, which is a secretory glycosaminoglycan (gag), is now being tested as a decoy protein with administration in a spray and nebulized formulation (dixon et al., 2020; kim et al., 2020) . by binding to the spike protein within the blood, heparin may prevent the interaction of s protein with ace2 preventing initial infection or viral spread. the development of novel biotechnology products mainly focuses on preventing viral entry through targeting the interaction between the s protein and the ace2 host cell receptor (fig. 2c, table 2 ). several strategies are under clinical investigation, including recombinant proteins and monoclonal antibodies against the key molecules involved in sars-cov-2 viral entry. recombinant human soluble ace2 (rhsace2) protein represents a novel approach to preventing binding of sars-cov-2 to ace2, by introducing a soluble form in excess to compete with endogenous membrane-bound ace2 for s protein binding (table 2) . upon entering the bloodstream, the exogenous rhsace2 is intended to bind and neutralize circulating sars-cov-2 virions, serving as a scavenger or decoy (monteil et al., 2020; tai et al., 2020; . rhsace2 proteins were previously explored to treat acute lung injury, pulmonary hypertension, and ards, as a soluble additive protein to replace deficient ace2 and as a potential soluble decoy protein to sars-cov (hemnes et al., 2018; imai et al., 2005) . in addition to inhibiting virus infection, treatment with rhsace2 may reduce interleukins and tnfα production, and supplement endogenous ace2 enzymatic activity, contributing to the regulation of the renin-j o u r n a l p r e -p r o o f angiotensin system (ras) to prevent further lung injury (hemnes et al., 2018; . rhsace2 was reported to be safe in healthy subjects and in patients diagnosed with ards, which led to multiple rhsace2 therapies advancing to later phase clinical trials (table 2) (haschke et al., 2013; ju et al., 2020; khan et al., 2017) . a similar protein to rhsace2 in development is a recombinant bacterial carboxypeptidase (b38-cap), an ace2 receptors-like enzyme that was explored previously for hypertension and cardiac dysfunction (minato et al., 2020) . expanding on soluble ace2, fc-ace2 fusion proteins are being designed with igg1 fc portion to prolong the circulating half-life of the rhsace2 and modulate the immune system response designing a therapy to target multiple epitopes of the sars-cov-2 spike protein may increase the potential of binding and neutralizing the virus. the ace-mab bispecific fusion protein has two functional arms, with one arm being a fully human antibody targeting the spike protein, to prevent binding to ace2, and the other arm being a truncated rhsace2 protein (cel-sci, 2020a). through targeting three parts of the spike protein, the anti-covid-19 darpin® fusion protein candidates are proposed to potentially block the binding of the spike protein to ace2, block protease activation of the spike protein, and "cuff" the protein to prevent conformational changes (balfour, 2020). many of these fc fusion proteins aim to not only be used as a treatment but to have prophylactic potential. small peptide-based therapies are also being developed that target the n protein and elicit cytolytic t cell response (amawi et al., 2020; cel-sci, 2020a, b) . by targeting n protein antigens, those agents may have a broader antiviral potential as the n protein shares 90% amino acid homology with sars-cov and 48% with mers-cov (grifoni et al., 2020) . masking the sialic acid glycans on the host cell receptors in a patient's airway may reduce the binding of the spike protein and j o u r n a l p r e -p r o o f lower the risk of sars-cov-2 infection, potentially offering prophylactic activity (pneumagen, 2020) . preliminary data reported a reduction in sars-cov-2 infectivity with the multivalent carbohydrate binding molecule (mcbms) neumifil. additional targeted therapies are under development, not specifically to prevent the viral infection, but to treat downstream pathology of covid, such as acute lung injury, pneumonia, and ards. recombinant human plasma gelsolin (rhu-pgsn) is under investigation to treat covid-19-induced pneumonia (bioaegis therapeutics, 2020) and has been reported to reduce inflammation (dinubile et al., 2020; yang et al., 2015) . controlling inflammation to decrease potential organ injury may be important to improve recovery from covid-19 infection. neutralizing antibodies employ the same strategy as the decoy ligands, aiming to block the interaction of the virus with the ace2 host cell receptor, thereby inhibiting viral entry . antibodies can also alter the conformation of a protein needed for viral entry or destroy infected cells through effector functions. several antibodies are being developed against the spike protein (table 3) , with the aim of blocking receptor binding to the host cell membrane (eli lilly, 2020; wong, 2020) . such antibodies have been designed based on convalescent serum isolated from patients who have recovered from severe sars-cov-2 infection, from phage display libraries, transgenic mice engineered to express the trimeric sars-cov-2 spike protein, b cell isolation, and 3d printed lymph nodes (3dprint, 2020; businesswire, 2020; hansen et al., 2020; ju et al., 2020; snyder, 2020; zimmer, 2020) . anti-sars-cov-2 antibodies are being isolated from wide populations of patients who have recovered from infection, with the hope of identifying a strong candidate that will effectively bind and neutralize the virus. using antibodies from patients who have recovered from sars-cov-2 infection as a molecular template follows a similar strategy as using convalescent serum from recovered patients; to provide a potential boost to the immune system or to provide a prophylactic immunity prior to infection. (table 3 ). these cocktails have demonstrated an activity to reduce viral load in vitro as well as in animal models with lung inflammation and injury (hansen et al., 2020; regeneron, 2020; snyder, 2020; zimmer, 2020) . antibody therapy development is advancing towards cocktails against all coronavirus strains, projecting past development for sars-cov-2 (taylor, 2020) . several antibody products are also being developed to control cytokine storm and treat covid-19-induced pneumonia. these therapies include targeting of il-6r (levilimab) (nct04397562), tlr4 (eb05) (nct04401475), cxcl10 (eb06) and angiopoietin-2 (ang2) (ly3127804) (nct04342897). targeting tlr4, which is upstream of il-6, il-8 and type i and type iii interferons, or il-6r may control the proinflammatory response in patients and suppress cytokine storm (sallenave and guillot, 2020) . eb05 and levilimab both aim to inhibit cytokine release through blocking the inflammatory signaling cascade. anti-cxcl10 aims to reduce the high expressions of the chemokine cxcl10, which has been associated with long-term lung injury (oliviero et al., 2020) . controlling excessive inflammation and cytokine release may reduce progression to ards in infected patients. meplazumab, an anti-cd147 mab, was developed to prevent the binding of the sars-cov-2 spike protein to cd147 host cells and to regulate cytokine secretion in patients with covid-19 acquired pneumonia (bian et al., 2020). cd147 was believed to be an additional host cell receptor to ace2, which the virus utilized for internalization, though recent studies have demonstrated a lack of binding of cd147 to the spike protein (shilts and wright, 2020) . modulation of the immune system to selectively remove cells infected by sars-cov-2 may have potential in reducing the spread of the virus. a tri-specific nk cell engager (trike) antibody is being developed, with the aim to engage nk cells and eliminate sars-cov-2 infected cells, before further virions are released into the blood stream (gt biopharma, 2020). a similar approach involves a bi-specific antibody targeting the spike protein and the nkp46 nk cell receptor (cytovia therapeutics, 2020). while most of the antibody therapeutics being developed are igg, development of igm and iga therapies may potentially increase binding power as these antibodies have a greater number of j o u r n a l p r e -p r o o f binding domains than igg (igm bio, 2020). igm and iga antibodies are based on naturally occurring antibodies following severe sars-cov-2 infection produced from single b cells. these antibodies can undergo transport across mucosal membranes, which may have potential for treating infected lung tissue. single-domain antibodies (sdabs), or nanobodies, may potentially be used to neutralize the spike protein, through targeting a cryptic epitope in the trimeric spike protein interface (table 3 ) (wrapp et al., 2020; wu et al., 2020c) . these nanobodies, designed from screening nanobody libraries and shark variable domain of immunoglobulin new antigen receptor (vnar) phage display libraries, are composed of the complementary determining regions with molecular weights of 12-15 kda (jaimes et al., 2020; kupferschmidt, 2020; wu et al., 2020c) . nanobodies are being developed against s protein and n protein to prevent viral protein-ace2 receptor interactions. nanobodies have demonstrated successful blocking of ace2 binding and were shown to recognize rbd in different structural conformations (huo et al., 2020) . these therapies have been proposed for use as inhalation agents and may potentially neutralize the virus in a dose-dependent manner. the concerted effort of the global scientific community to address the covid-19 pandemic has been remarkable. this includes the rapid launch of a large volume of clinical trials to investigate potential therapeutics for covid-19 treatment, as well as preventive vaccines (not included in this review). to mount an immediate response to the health crisis, many existing drugs are being tested for use in covid-19. these include antiviral drugs and immunomodulators that were previously approved for the treatment of other disease conditions. these drugs have a regulatory record and established safety profiles which help expedite the development process. the novel targeted therapies, including neutralizing antibodies and fc-fusion proteins, are mainly targeting the sars-cov-2 viral entry step. some of these products may prove to be helpful in preventing the spread of the virus in the body, thereby accelerating recovery after infection or providing a means of prophylaxis. still, the sars-cov-2 virus remains novel and many aspects of transmission, infection, and treatment are yet to be unraveled. the current strategies under development mainly target ace2 and s protein interaction for viral entry. other potential therapeutic targets may include the sugar j o u r n a l p r e -p r o o f moieties as additional receptors on the host cells (qing et al., 2020) , mediation of viral entry processes by accessory proteins, secondary infection to organs outside the respiratory system, and the dynamic immune response to the infection liu et al., 2014) . further research into the pathophysiology of covid-19 is needed to understand the mechanisms that result in severe manifestation of the disease. since the beginning of the sars-cov-2 pandemic, the virus has been undergoing various mutations potentially impacting the effectiveness of the repurposed drugs or novel therapeutics, with constant surveillance identifying mutations in the rdrp and spike proteins (pachetti et al., 2020; van dorp et al., 2020) . therapeutic development for covid-19 will need to monitor the changing viral genome to stay ahead of potential drug resistance and understand how the virus evades the human immune system (pachetti et al., 2020; van dorp et al., 2020) . clinical trial design constitutes a critical component in the development of therapeutics against covid-19. the fda has established provisions for timely interaction and responses to sponsor inquiries and submissions to facilitate early discussion of efficient approaches to product development (u.s. food and drug administration, 2020c). the course of covid-19 disease is diverse, ranging from asymptomatic to fatal respiratory failure. it is therefore important to evaluate therapeutics in adequate and well-controlled clinical trials with defined populations to generate a safety and efficacy database that will best inform the clinical use of the drug or biologic. this article reflects the views of the authors and should not be construed to represent fda's views or policies. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. this work was funded by the u.s. food and drug administration. the funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. as there is a huge volume of therapeutic approaches for covid-19, we may not cover all available therapeutic development programs. in addition, research results are dynamic and change as new evidence emerges. second, the clinical trial information was derived from the u.s. national library of medicine clinicaltrials.gov, which was last accessed on 17 october 2020. third, only articles/publications/translations from english were analyzed so some relevant international data might be missing. 1) sars-cov-2 attachment to host receptor ace2, and release of genomic rna, through cleavage with tmprss2 or endo-/lysosomal proteases characterizes initial infection. several agents can be employed to inhibit these interactions including neutralizing antibodies, recombinant human soluble ace2 (rhsace2), protease inhibitors, and endosomal ph modulators (fehr and perlman, 2015) . 2) replication and translation of genomic rna into structural proteins to assemble mature virions for release results in viral amplification and increases in infection. antivirals inhibiting viral proteases and rdrp are being evaluated against this checkpoint (fehr and perlman, 2015) . 3) due to viral replication, host immune responses are triggered that can cause hyperactivation of immune cells and constant production of pro-inflammatory cytokines, resulting in more severe disease presentation (cardone et al., 2020; huang et al., 2020b) . novel and repurposed immunomodulators are under investigation to mitigate harmful immune responses. angiotensin-converting enzyme 2 (ace2); transmembrane protease serine 2 (tmprss2); rna-dependent rna polymerase (rdrp); main protease (mpro); papain-like protease (plpro); il (interleukin); tumor necrosis factor α (tnfα) prellis biologics pursues bioprinted lymph nodes for production of covid-19 antibodies coronavirus susceptibility to the antiviral remdesivir is mediated by the viral polymerase and the proofreading exoribonuclease the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade cytovia therapeutics, 2020. cytovia therapeutics and macromoltek to develop dual-acting natural killer immunotherapy against sars cov2 (covid-19) recombinant human plasma gelsolin (rhu-pgsn) improves survival and attenuates lung injury in a murine model of multi-drug resistant pseudomonas aeruginosa pneumonia, open forum infectious diseases can nebulised heparin reduce time to extubation in sars cov 2 the charter study protocol fda approved drugs with pharmacotherapeutic potential for sars-cov-2 (covid-19) therapy lilly begins world's first study of a potential covid-19 antibody treatment in humans first covid-19 treatment recommended for eu authorisation coronaviruses: an overview of their replication and pathogenesis use of anakinra in severe covid-19: a case report mechanism of action of hydroxychloroquine as an antirheumatic drug understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 tm) therapeutic for treatment of covid-19 infection tocilizumab in j o u r n a l p r e -p r o o f patients with severe covid-19: a retrospective cohort study pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects a potential therapeutic role for angiotensin-converting enzyme 2 in human pulmonary arterial hypertension food and drug administration (fda) revoke the emergency use authorization (eua) for emergency use of oral formulations of chloroquine phosphate (cq) and hydroxychloroquine sulfate (hcq) sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor dualhistamine receptor blockade with cetirizine -famotidine reduces pulmonary symptoms in covid-19 patients dexamethasone in hospitalized patients with covid-19 -preliminary report effect of hydroxychloroquine in hospitalized patients with covid-19 clinical features of patients infected with 2019 novel coronavirus in clinical features of patients infected with 2019 novel coronavirus in neutralizing nanobodies bind sars-cov-2 spike rbd and block interaction with ace2 igm bio, 2020. atreca, beigene, and igm biosciences agree to collaborate on novel antibody treatment for covid-19 angiotensin-converting enzyme 2 protects from severe acute lung failure novel ace2-igg1 fusions with improved activity against sars-cov2, biorxiv phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and j o u r n a l p r e -p r o o f proteolytically sensitive activation loop neutralizing antibodies against sars-cov-2 and other human coronaviruses covid-19 dashboard by the human neutralizing antibodies elicited by sars-cov-2 infection a.i. bayliffe and a.l. lazaar, a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (sars-cov-2) spike glycoprotein binding interactions three big studies dim hopes that hydroxychloroquine can treat or prevent covid-19 therapeutic options for the 2019 novel coronavirus (2019-ncov) expression of the sars-cov-2 cell receptor gene ace2 in a wide variety of human tissues accessory proteins of sars-cov and other coronaviruses hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro boceprevir, gc-376, and calpain inhibitors ii, xii inhibit sars-cov-2 viral replication by targeting the viral main protease treatment of severely ill covid-19 patients with anti-interleukin drugs (cov-aid): a structured summary of a study protocol for a randomised controlled trial antiviral activities of type i interferons to sars-cov-2 infection chloroquine inhibits autophagic flux by decreasing autophagosomelysosome fusion remdesivir for the treatment of covid-19 -preliminary report b38-cap is a bacteria-derived ace2-like enzyme that suppresses hypertension and cardiac dysfunction inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 covid-19 pulmonary and olfactory dysfunctions: is the chemokine cxcl10 the common denominator? characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant repurposed antiviral drugs for covid-19 type i and type iii interferons -induction, signaling, evasion, and application to combat covid-19 combating devastating covid-19 by drug repurposing special approval for emergency on remdesivir for covid-19 pneumagen ltd announces positive anti-viral activity for novel glycan approach in preventing covid-19 infections covid-19 and emerging viral infections: the case for interferon lambda distinct roles for sialoside and protein receptors in coronavirus infection lopinavir-ritonavir in patients admitted to hospital with covid-19 (recovery): a randomised, controlled, open-label, platform trial regeneron announces start of regn-cov2 phase 3 covid-19 prevention trial in collaboration with national institute of allergy and infectious diseases (niaid) azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection roche provides an update on the phase iii covacta trial of actemra/roactemra in hospitalised patients with severe covid-19 associated pneumonia innate immune signaling and proteolytic pathways in the resolution or exacerbation of sars-cov-2 in covid-19: key therapeutic targets? sanofi and regeneron provide update on kevzara® (sarilumab) phase 3 u.s. trial in covid-19 patients a small molecule oxocarbazate inhibitor of human cathepsin l blocks sars and ebola pseudotype virus infection into hek 293t cells broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov no evidence for basigin/cd147 as a direct sars-cov-2 spike binding receptor vanderbilt hijaking sars-cov-2? the potential role of jak inhibitors in the management of covid-19 effect of remdesivir vs standard care on clinical status at 11 days in patients with moderate covid-19: a randomized clinical trial systimmune, 2020. covid-19 pandemic: development of recombinant human ace2 fusion protein drug (si-f019) for the treatment of patients with novel coronavirus pneumonia characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy celltrion plans july covid-19 trial, advances 'super antibody mechanisms and enzymes involved in sars coronavirus genome expression azithromycin, a 15-membered macrolide antibiotic, inhibits influenza a(h1n1)pdm09 virus infection by interfering with virus internalization process coronavirus (covid-19) update: fda issues emergency use authorization for potential covid-19 treatment fda coronavirus treatment acceleration program (ctap) food and drug administration, 2020c. fda guidance for industry -covid-19: developing drugs and biological products for treatment or prevention food and drug administration, 2020d. temporary policy for compounding of certain drugs for hospitalized patients by outsourcing facilities during the covid-19 public health emergency dexamethasone reduces death in hospitalised patients with severe respiratory complications of covid-19 this national clinical trial aims to identify treatments that may be beneficial for people hospitalised with suspected or confirmed covid-19 emergence of genomic diversity and recurrent mutations in sars-cov-2, infection chloroquine is a potent inhibitor of sars coronavirus infection and spread a human monoclonal antibody blocking sars-cov-2 infection remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro phase i start marks first for anti-sars-cov-2 mabs, first for abcellera world health organization, 2020a. coronavirus -world health organization coronavirus disease (covid-19): dexamethasone solidarity" clinical trial for covid-19 treatments world health organization, 2020d. who discontinues hydroxychloroquine and lopinavir/ritonavir treatment arms for covid-19 structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods identification of human single-domain antibodies against sars-cov-2 fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein plasma gelsolin improves lung host defense against pneumonia by enhancing macrophage nos3 function in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) rational use of tocilizumab in the treatment of novel coronavirus pneumonia perspectives on therapeutic neutralizing antibodies against the novel coronavirus sars-cov-2 first antibody trial launched in covid-19 patients fda reissued emergency use authorization with revisions to allow for remdesivir to be used only to treat adults and children with suspected or laboratory confirmed covid-19 administered in an in-patient hospital setting footnote: selected clinical trials may have expanded or been withdrawn since publication as this is a rapidly expanding field. mab, monoclonal antibody ribonucleic acid; tmprss2, transmembrane serine protease 2 hiv, human immunodeficiency virus; il, interleukin; tnf, tumor necrosis factor the endosome, lysosome, and golgi apparatus interferes with ace2 glycosylation, virus-ace2 interaction, and virus endocytosis who solidarity trial (international, >12000), nct04381936/recovery (uk, 4716) , nct04334148 (us, 2000) , nct04303507 (uk/thailand, 40000) (hinton, 2020; horby et al., 2020b; us national library of medicine, 2020; world health organization, 2020c, d) camostatsmall molecule footnote: listed therapeutics and clinical trials may have expanded or been withdrawn since publication as this is a rapidly expanding field. therapeutics have been identified from clinicaltrials.gov and publicly available literature searches. ace2, angiotensin converting enzyme 2; mabs, monoclonal antibodies; igm, immunoglobulin m; iga, immunoglobulin a; sdabs, single domain antibodies; il-6r, interleukin-6 receptor; tlr4, toll-like receptor 4 protein; cxcl10, c-x-c motif chemokine 10; c5a, complement component 5a; trike, tri-specific nk cell engager; n/a indicates that a clinical trial has not been registered with clinicaltrials.gov key: cord-283709-y59h5bw8 authors: chan, renee w y; hemida, maged g; kayali, ghazi; chu, daniel k w; poon, leo l m; alnaeem, abdelmohsen; ali, mohamed a; tao, kin p; ng, hoi y; chan, michael c w; guan, yi; nicholls, john m; peiris, j s malik title: tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study date: 2014-08-28 journal: lancet respir med doi: 10.1016/s2213-2600(14)70158-4 sha: doc_id: 283709 cord_uid: y59h5bw8 background: middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic infection causing severe viral pneumonia, with index cases having resided in or recently travelled to the arabian peninsula, and is a global concern for public health. limited human-to-human transmission, leading to some case clusters, has been reported. mers-cov has been reported in dromedary camels but phenotypic characterisation of such viruses is limited. we aimed to compare mers-cov isolates from dromedaries in saudi arabia and egypt with a prototype human mers-cov to assess virus replication competence and cell tropism in ex-vivo cultures of human bronchus and lung. methods: we characterised mers-cov viruses from dromedaries in saudi arabia and egypt and compared them with a human mers-cov reference strain. we assessed viral replication kinetics and competence in vero-e6 cells (rhesus monkey), tissue tropism in cultures of ex-vivo human bronchial and lung tissues, and cytokine and chemokine induction, gene expression, and quantification of viral rna in calu-3 cells (human respiratory tract). we used mock-infected tissue as negative controls for ex-vivo experiments and influenza a h5n1 as a positive control for cytokine and chemokine induction experiments in calu-3 cells. findings: we isolated three dromedary strains, two from saudi arabia (dromedary/al-hasa-kfu-hku13/2013 [ah13] and dromedary/al-hasa-kfu-hku19d/2013 [ah19d]), and one from egypt (dromedary/egypt-nrce-hku270/2013 [nrce-hku270]). the human and dromedary mers-cov strains had similar viral replication competence in vero-e6 cells and respiratory tropism in ex-vivo cultures of the human respiratory tract, and had similar ability to evade interferon responses in the human-respiratory-tract-derived cell line calu-3. interpretation: the similarity of virus tropism and replication competence of human and dromedary mers-cov from the arabian peninsula, and genetically diverse dromedary viruses from egypt, in ex-vivo cultures of the human respiratory tract suggests that dromedary viruses from saudi arabia and egypt are probably infectious to human beings. exposure to zoonotic mers-cov is probably occurring in a wider geographical region beyond the arabian peninsula. funding: king faisal university, egyptian national research centre, hong kong food and health bureau, national institute of allergy and infectious diseases, and european community seventh framework program. middle east respiratory syndrome coronavirus (mers-cov) is a disease of public health concern globally, with 837 laboratory-confi rmed cases and 291 deaths reported to who as of july 23, 2014. 1 index-case patients have all resided in or recently travelled to the arabian peninsula or adjacent countries. travel-associated cases or secondary transmission arising from such cases have been reported in europe, north america, africa, and asia. 2 some case clusters have been associated with limited human-to-human transmission occurring within family or health-care settings, and the remaining sporadic cases are presumed to be zoonotic in origin. [3] [4] a recent increase in reported cases from saudi arabia associated with health-care facilities is a particular cause of concern. 3 establishment of the proximate zoonotic source of human infection and modes of transmission from animals to human beings remains crucial for public health. mers-cov has been detected in dromedary camels (camelus dromedarius), in association with and preceding human infection, and in dromedary slaughterhouses and farms. [5] [6] [7] [8] [9] viruses from most of the recent human cases from the arabian peninsula phylogenetically cluster within clade b, but the earliest human mers-cov strains from 2012 are genetically distinct and are denoted as clade a. 10 dromedary mers-cov strains detected in the arabian peninsula in 2012-13 were clade b viruses, phylogenetically related to viruses from human cases from the same regions, but genetically diverse (non-clade a/b) viruses were reported from egypt. 11 researchers have also reported serological evidence collected between 2009 and 2013 of mers-cov infection in dromedary camel serum samples from tunisia, nigeria, ethiopia, and egypt, suggesting that mers-cov or antigenically related viruses might be geographically more widespread than is appreciated at present. 6, 12 findings from two studies of 226 people in saudi arabia and 179 people in egypt working in dromedary abattoirs showed that all remained seronegative for mers-cov, suggesting that trans mission to humans is uncommon 11, 13 and raising the question of whether dromedary mers-cov strains have the capacity to infect human beings. full genome sequences of dromedary viruses suggest that these viruses are similar to viruses in humans. 11, 14 there were some aminoacid diff erences between the human and dromedary viruses, including in the genetic sequence of the spike protein (a major determinant of host specifi city), but they did not aff ect the receptor binding interface with the cell receptor dpp4. 11, 14 however, because a single aminoacid change can profoundly aff ect host range and because the host range determinants of mers-cov are not well defi ned, dromedary viruses need to be phenotypically characterised in a physiologically relevant manner. 15 so far, few virus isolates from dromedaries have been available for biological characterisation and direct evidence for replication competence of dromedary mers-cov in human respiratory tissues has been lacking. the only biological characterisation of a dromedary mers-cov virus so far was the fi nding of dpp4-receptor-dependent virus replication in huh-7 cells, 16 a transformed human hepatoma cell line physiologically unrelated to the human respiratory tract. we aimed to compare mers-cov isolates from dromedaries in saudi arabia and egypt with the prototype human mers-cov emc strain to assess virus replication competence and cell tropism in ex-vivo cultures of human bronchus and lung. we used a human mers-cov strain emc (hmers-cov) provided by r a m fouchier (erasmus university medical center, rotterdam, netherlands). we prepared the emc virus stock as previously described. 17 three dromedary camel strains, two from saudi arabia (dromedary/al-hasa-kfu-hku13/2013 [ah13] and dromedary/al-hasa-kfu-hku19d/2013 [ah19d]) and one from egypt (dromedary/egypt-nrce-hku270/2013 [nrce-hku270]), were prepared. the dromedary camel mers-cov isolates ah13 and ah19d had been isolated in vero-e6 cells (atcc, crl-1586, manassas, va, usa) from nasal (ah13) and faecal (ah19d) swabs, collected from dromedaries in al-hasa, saudi arabia, as previously described. 14 the detection of dromedary camel mers-cov nrce-hku270 from a nasal swab of a dromedary in an abattoir in egypt has been previously reported. 11 for this study, we successfully isolated the virus from this specimen in vero-e6 cells as previously described. 14 viruses were grown, aliquoted, and stored at -80°c until used. all the dromedary mers-cov strains used in this study were passage 3 after isolation in vero-e6 cells. we had previously compared the full genome sequence of the ah13 virus in the original nasal swab with the vero-e6 passaged virus used in this accession numbers of virus genomes retrieved for this analysis are jx869059, kc776174, kc164505, kc667074, kf192507, kj156881, kj156949, kj156944, kj556336, kj156952, kf600613, kf186564, kf600627, kf186567, kf600651, kj156866, kf600634, kf600632, kf600644, kf186565, kf186566, kf600645, kf600647, kf600630, kf600652, kf745068, kj156869, kj650297, kj650296, kj650295, kf600628, kf961221, kf961222, kj156874, kj156910, kj156934, methods for viral titrations are described in the appendix. the methods we used for ex-vivo organ cultures and infection, quantifi cation of viral rna and host cytokine and chemokine mrnas, and quantifi cation of protein and immunohistochemistry have been previously described 17 and are also detailed in the appendix. we did experiments with live mers-cov in a biosafety level 3 biocontainment facility at the university of hong kong. this study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw-13-104). we cultured vero-e6 and calu-3 cells using dulbecco's modifi ed eagle's medium. cells were seeded at 1 × 10⁵ cells per well in 24-well tissue-culture plates and infected with the mers-cov strains at a multiplicity of infection of 0·01 or 2 as indicated. after 1 h of virus adsorption at 37°c, we removed the virus inoculum, washed the cells with phosphate-buff ered saline (pbs) three times to remove unbound virus, and replenished with fresh culture medium. we measured virus replication kinetics by titrating infectious virus in infected culture supernatants. the human-respiratory-tract-derived cell line calu-3 (atcc htb-55, manassas, va, usa) was cultured and infected with virus, as for the vero-e6 cells. rna from the infected cells was also collected at 6, 24, and 30 h post infection for analysis of gene expression and quantifi cation of viral rna. we harvested 1 ml of culture supernatants from calu-3 cells at 30 h post infection to measure cytokine and chemokine protein concentrations using elisa (r&d systems, minneapolis, mn, usa) and cytometic bead array (bd bioscience, san jose, ca, usa), using methods specifi ed by the manufacturer. as a positive control for assessment of cytokine and chemokine induction in calu-3, we used a highly pathogenic avian infl uenza h5n1 virus, a/hong kong/483/1997. infl uenza virus was prepared in madin-darby canine kidney cells as previously described. we created thermal inactivation curves of the coronaviruses at 37°c, using culture wells inoculated with virus dilutions in the absence of permissive cells, or with an ex-vivo culture of mouse lung prepared from c57bl/6n mice. the mouse lung culture was prepared similar to the ex-vivo organ culture of human samples. we added 1 ml of virus dilution into 24-well plates with diff erent starting concentrations (10⁴, 10³, and 10² tcid 50 /ml). we collected 130 μl of supernatant at 1, 24, 48, and 72 h post incubation to measure viral titration. to assess tissue tropism in ex vivo experiments, we infected bronchial and lung tissues with 1 ml of each mers-cov virus dilution at a titre of 10⁶ 50% tissue culture infective dose per ml (tcid 50 /ml). after incubation for 1 h at 37°c, we washed the cultures three times with 5 ml of warm pbs to remove unbound virus. mock-inoculated tissue served as controls (1 ml of dulbecco's modifi ed eagle's medium without virus). we collected culture supernatants from the infected cultures at 1, 24, 48, and 72 h post infection and titrated them in parallel for infectious virus using the tcid 50 assay. samples for experiments on the ex-vivo cultures of human bronchus and lung were provided by fi ve independent donors. we assessed the extent of the overall viral replication with area-under-curve analysis using the trapezoid rule. we used the area calculated from 24 to 72 h post infection above the limit of detection of infectious virus titre for this analysis. the results of the four viruses were compared with the non-parametric friedman test. we did the experiments with vero-e6 and calu-3 cells with three biological replicates of each cell line and calculated mean and standard error of the mean (sem). we compared diff erences in log 10 transformed viral titres by a two-way anova followed by a bonferroni multiple-comparison test. we compared the quantitative cytokine and chemokine mrnas between viruses and over time with a two-way anova followed by a bonferroni multiple-comparison test. the protein concentrations of the cytokines and chemokines between viruses at 30 h post infection were compared by a oneway anova followed by a bonferroni multiplecomparison test. see online for appendix we used p less than 0·05 to indicate statistical significance. statistical analyses were done with graphpad prism version 6.0 for mac. the funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. figure 1 shows the phylogeny of mers cov strains used in this study within the global context of mers-cov. only mers-cov with full genome sequences were included in this analysis. most viruses cluster within clade b mers-cov, including the dromedary camel virus isolates from saudi arabia used in this study (ah13 and ah19d). the prototype human mers-cov virus (emc) is a clade a virus. the dromedary mers-cov isolates, nrce-hku270 and dromedary/egypt-nrce-hku205/2013, have a long branch in the phylogenetic tree separating them from clade a and clade b viruses and also from each other, suggesting that they are genetically diverse from previously described mers-cov. we used the dromedary mers-cov isolate nrce-hku270 in this study. the nucleotide and aminoacid similarity between these human and dromedary camel viruses is provided the appendix. all four mers-cov strains replicated effi ciently in vero-e6 cells at multiplicities of infection of 0·01 and 2 (fi gure 2, table). egyptian dromedary mers-cov nrce-hku270 replicated at least as well as did human mers-cov emc, and reached peak titres even faster than human mers-cov at 48 h post infection when infected at low multiplicity of infection. although the two saudi dromedary mers-cov strains (ah13 and ah19d) replicated less effi ciently than did human mers-cov during the fi rst 24 h post infection, the viral titre of ah13 was similar by 72 h post infection. replication of dromedary mers-cov nrce-hku270 was signifi cantly higher than that of the other two dromedary mers-cov strains at 24 h and 48 h post infection at multiplicity of infection of 0·01. immunohistochemistry of ex-vivo cultures of human bronchus and lung infected with human and dromedary mers-cov strains suggested that all four viruses could infect these tissues (fi gure 3). because of their threedimensional nature, infection in ex-vivo cultures was non-uniform throughout the histological specimen (appendix). figure 4 shows viral replication kinetics of the viruses in the ex-vivo cultures of human bronchus and lung. in the absence of permissive cells, the thermal inactivation curves of each of these mers-cov strains showed virus infectivity largely decreasing to undetectable levels by 24 h post infection (fi gure 4a). we noted similar thermal inactivation kinetics when we substituted mouse lung tissue (non-permissive to mers-cov) for the human ex-vivo tissues (data not shown). although the extent of virus replication varied from donor to donor, comparison with the thermal inactivation curves provided convincing evidence of virus replication of all four viruses in the ex-vivo human bronchial (fi gure 4b) and lung (fi gure 4c) cultures from each donor over the 72 h experiment period. two replicates of ex-vivo cultures per donor from three donors, infected with the same virus (human mers-cov emc) on diff erent occasions, showed that the change of area-under-curve from the same tissue donor in bronchus ranged between 0 to 1·36-fold and in lung ranged between 0 to 1·46-fold, providing an indication of the range of technical variation in these ex-vivo assays (appendix). by contrast, the diff erence of area-undercurve for human mers-cov emc between donor 3 and donor 1 was 5·3-fold for the bronchus (area under curve above detection limit 112·8 for donor 1 patchy nature of the infection, the variation between donors, and lack of adequate statistical power, statistical comparisons between viruses have to be interpreted with caution. within these limitations, we noted no signifi cant diff erence in the replication competence of the four viruses in either the bronchus (p=0·151) or the lung (p=0·270) as assessed by the area-under-the-curve using the friedman test (appendix). immunohistochemical analysis of virus-infected bronchial tissues showed that the types of cells infected by human and dromedary mers-cov were very similar (fi gure 5). co-staining of viral antigen with the goblet cell marker muc5ac (fi gure 5a) and ciliated cell marker β-tubulin (fi gure 5b) lent support to the notion that mers-cov infects non-ciliated bronchial epithelium other than goblet cells. co-staining of viral antigen and alveolar epithelial cell marker ae1/3 showed that all four viruses infected alveolar epithelial cells (fi gure 5c), and co-staining with the alveolar type ii epithelial cell marker prosurfactant protein c showed that type ii alveolar epithelium was infected (fi gure 5d). we noted no evidence of infection of alveolar macrophages (fi gure 5e). all four viruses infected lung endothelium (appendix). thus, overall, the respiratory tropism of the human and dromedary mers-cov was similar. all three mers-cov strains (emc, ah13, nrce-hku270) replicated effi ciently in calu-3 cells (ah19d was not included because it is in the same clade as ah13). to quantify the viral rna and cytokine and chemokine gene induction and expression, the humanrespiratory-tract-derived cell line calu-3 was cultured and infected with virus as for the vero-e6 cells. rna from the infected cells was also collected at 6, 24, and 30 h post infection for analysis of gene expression and quantifi cation of viral rna. we harvested 1 ml of culture supernatants from calu-3 cells at 30 h post infection to measure cytokine and chemokine protein concentrations using elisa (r&d systems, minneapolis, mn, usa) and cytometric bead array (bd bioscience, san jose, ca, usa), using methods specifi ed by the manufacturer. as a positive control for assessment of cytokine and chemokine induction in calu-3, we used a highly pathogenic avian infl uenza h5n1 virus, a/hong kong/483/1997. infl uenza virus was prepared in madin-darby canine kidney cells as previously described. 17 despite effi cient replication, none of the mers-cov strains induced signifi cant gene expression or protein secretion of tumour necrosis factor α (tnfα), cxcl10, or interferon β (fi gure 6). we used infl uenza a (h5n1) virus, a potent inducer of interferon β and cxcl10 in epithelial cells, for comparison. we noted evidence of interleukin 6 induction by all three mers-cov strains; although ah13 and nrce-hku270 induced greater production of interleukin 6 mrna than the human emc strain, the human emc strain induced higher concentrations of interleukin 6 protein than did either dromedary strain (fi gure 6f). we compared the viral replication competence and respiratory tropism of three mers-cov isolates from dromedary camels with the prototype human clade a mers-cov isolate emc (panel). the two dromedary mers-cov isolates from al-hasa, ah13 and ah19d, are phylogenetically closely related to recent clade b human viruses from saudi arabia. 18 as shown by the long-branch in the unrooted phylogenetic tree (fi gure 1) and genetic similarity tables (appendix), egyptian dromedary mers-cov nrce-hku270 and dromedary mers-cov nrce-hku205 are genetically divergent from all other mers-cov detected to date, and are also divergent from each other, suggesting that the global diversity of mers-cov is wider than previously appreciated. the lack of a representative human clade b mers-cov isolate is a limitation of this study. comparison of viral genetic data can provide clues, but is not of itself fi nal evidence for the competence of an animal virus to infect human beings. with sars coronavirus, two aminoacid changes in the spike protein strikingly altered the ability of that virus to transmit effi ciently in human beings. 15 because all the crucial determinants of host tropism with mers-cov are not fully understood, especially those outside of the spike-receptor binding domain, phenotyping of such viruses provides essential complementary information to the genetic analysis. the three mers-cov isolates from dromedary camels (ah13, ah19d, and nrce-hku270) were similar to the human mers-cov emc virus in replication competence in the rhesus monkey cell line vero-e6. all four viruses replicated in ex-vivo cultures of the human bronchus and lung and infected the same cell types in the bronchus (non-ciliated bronchial epithelium) and lung (alveolar epithelium, including type ii alveolar epithelial cells). we noted infection of lung endothelial cells with all four viruses, suggesting that potential for dissemination beyond the respiratory tract was also comparable. overall, the tropism of human mers-cov emc for the human respiratory tract was similar to that previously reported. 17 phenotypically, human and dromedary mers-cov have much the same tropism and replication competence in human respiratory ex-vivo cultures. to our knowledge, this report provides the fi rst comparative data from living human respiratory tissue in situ. therefore, our study provides new and crucial information to the emerging understanding of a disease with global importance for public health. the heterogeneity of virus replication between individual donors in our ex-vivo infection experiments might result from diff erences in host susceptibility, which are physiologically relevant to the reported epidemiology of human mers and could imply that individual variations might be an important determinant of disease susceptibility and severity. however, this question needs more systematic investigation. thus, overall, our data support and complement the viral genetic sequence data of the spike proteins of the dromedary viruses, which seem similar to the spike proteins of human mers-cov strains, 11, 13, 16 and strongly suggest that dromedary mers-cov strains have the capacity to infect human beings. infection of ex-vivo cultures provided useful information of virus cell tropism in the absence of autopsy data for mers. ex-vivo organ cultures provided information about virus tropism at early stages of the infection, which is not usually available in autopsies or from patients with late-stage disease (often after lengthy mechanical ventilation). thus, our fi ndings emphasise the usefulness of ex-vivo cultures for risk assessment of animal viruses for human health. mers-cov is reported to be a weak inducer of type i and type iii interferons. 17, 19 immune evasion mechanisms are relevant to pathogenesis and interspecies transmission, and diff erences might aff ect virus replication competence in new hosts. we therefore investigated whether human mers-cov and dromedary mers-cov diff ered in their ability to evade eliciting type i interferon and other innate immune responses. because the multiplicity of infection cannot be accurately controlled in experiments with ex-vivo cultures of the human bronchus and lung, we used calu-3, an epithelial cell line derived from human lung adenocarcinoma that has been used as a cell line to study viral infections of the human respiratory tract. 20 human mers-cov emc, dromedary mers-cov ah13, and dromedary mers-cov nrce-hku270 replicated effi ciently in these cells, although the dromedary mers-cov strains replicated signifi cantly more effi ciently in these cells (appendix). despite this effi cient virus replication, no virus elicited any tnfα, cxcl10, or interferon β mrna or protein responses after virus infection (fi gure 6). by contrast, infl uenza a h5n1 virus was a potent inducer of interferon β and cxcl10. we noted some induction of interleukin 6 by dromedary mers-cov strains ah13 and nrce-hku270 and human mers-cov at late timepoints post infection (30 h; fi gure 6e and 6f). importantly, the human and dromedary mers-cov behaved similarly by not eliciting interferon and cxcl10 responses, suggesting that these viruses do not diff er in evasion of type i interferon responses. in view of the high prevalence of infection in dromedaries [5] [6] [7] [8] 10 and implied frequent human exposure, the rarity of human infection and disease remains a puzzle and is reminiscent of the epidemiology of avian infl uenza a h5n1. 21 the exact mode of transmission of mers-cov from dromedaries to people remains unclear and whether unusual modes of exposure or host susceptibility have a role should be investigated. serological and virological evidence of mers-cov in dromedaries has been reported from countries in east and north africa outside of the arabian peninsula, 11,12 but zoonotically acquired mers has not yet been diagnosed in human beings in the african continent. our fi nding that the genetically divergent dromedary mers-cov isolated in egypt is phenotypically similar to human mers-cov emc in explant cultures from human respiratory tissue underscores the possibility that zoonotic mers might be occurring, unrecognised, in northeast africa and beyond. at present, many countries only test for mers-cov in patients with a recent travel history to the arabian peninsula, and locally acquired mers could be missed. alternatively, diff erences in cultural practices of camel husbandry, modes of animal contact, and consumption of animal products (eg, fresh camel's milk) 22 might account for diff erences in occurrence of zoonotic disease in diff erent geographical regions. in conclusion, our fi ndings suggest that dromedary mers-cov has competence to infect the human respiratory tract and does not diff er phenotypically from human mers-cov in this respect. this evidence strengthens the contention that dromedary camels are the proximate source of infection for human beings. our fi ndings also emphasise the need to consider mers in the diff erential diagnosis of viral pneumonia in a much wider geographical region in northeast africa and beyond. rwyc planned and did the experiments with ex-vivo cultures, analysed the data, and contributed to drafting of the report. mgh, gk, aa, and maa did the fi eld studies and detection of virus in dromedary specimens, and planned the experiments. dkwc, llmp, and yg did the genetic sequencing and phylogenetic analysis. kpt did the in-vitro culture and quantitative pcr assays. hyn isolated the mers-cov strains. mcwc was involved in study design and obtained research funding. jmn did and interpreted the histology and immunohistochemistry studies. jsmp obtained research funding, planned and coordinated the study, analysed the data, and wrote the report. all authors contributed to the data interpretation, analysis, and provided critical comments on the draft report. we declare no competing interests. to assess the infection potential of dromedary camel middle east respiratory syndrome coronavirus (mers-cov) strains for humans, genetic analysis should be complemented with phenotypic characterisation in physiologically relevant invitro cell cultures. we searched pubmed for reports published up to july 5, 2014, with the terms "mers coronavirus" and "dromedary" and "culture" or "isolate". we applied no date or language restrictions. three papers were retrieved, only two of them related to mers-cov isolates from dromedary camels. one other recent paper not yet cited in pubmed was identifi ed through search of the literature. only one of these studies pertained to the phenotypic characterisation of dromedary mers-cov in cell cultures in vitro. 16 this study used a transformed human hepatoma cell line, not physiologically relevant to the human respiratory tract. our fi ndings show similar virus tropism and replication competence of human and dromedary mers-cov in ex-vivo cultures of the human respiratory tract. these fi ndings suggest that dromedary mers-cov from the arabian peninsula, in addition to genetically diverse dromedary viruses from egypt, can be infectious to human beings. exposure to zoonotic mers-cov might be happening in a wider geographical region beyond the arabian peninsula. virological evidence of mers-cov needs to be sought in patients with severe unexplained viral pneumonia in africa in addition to the middle east. middle east respiratory syndrome coronavirus (mers-cov)-update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update who. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: quantifi cation of the extent of the epidemic, surveillance biases, and transmissibility middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-tohuman transmission of mers coronavirus spread, circulation, and evolution of the middle east respiratory syndrome coronavirus mers coronaviruses in dromedary camels geographic distribution of mers coronavirus among dromedary camels investigation of antimiddle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 mers coronavirus in dromedary camel herd, saudi arabia structural biology: adaptation of sars coronavirus to humans isolation of mers coronavirus from dromedary camel tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia effi cient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential evaluation of the calu-3 cell line as a model of in vitro respiratory syncytial virus infection avian infl uenza virus (h5n1): a threat to human health stability of middle east respiratory syndrome coronavirus in milk key: cord-287847-rmhvc5n5 authors: miles, brett a.; schiff, bradley; ganly, ian; ow, thomas; cohen, erik; genden, eric; culliney, bruce; mehrotra, bhoomi; savona, steven; wong, richard j.; haigentz, missak; caruana, salvatore; givi, babak; patel, kepal; hu, kenneth title: tracheostomy during sars‐cov‐2 pandemic: recommendations from the new york head and neck society date: 2020-04-20 journal: head neck doi: 10.1002/hed.26166 sha: doc_id: 287847 cord_uid: rmhvc5n5 the rapid spread of sars‐cov‐2 in 2019 and 2020 has resulted in a worldwide pandemic characterized by severe pulmonary inflammation, effusions, and rapid respiratory compromise. the result of this pandemic is a large and increasing number of patients requiring endotracheal intubation and prolonged ventilator support. the rapid rise in endotracheal intubations coupled with prolonged ventilation requirements will certainly lead to an increase in tracheostomy procedures in the coming weeks and months. performing tracheostomy in the setting of active sars‐cov‐2, when necessary, poses a unique situation, with unique risks and benefits for both the patient and the health care providers. the new york head and neck society has collaborated on this document to provide guidance on the performance of tracheostomies during the sars‐cov‐2 pandemic. the rapid spread of sars-cov-2 in 2019 and 2020 has resulted in a worldwide pandemic. [1] [2] [3] [4] the dramatic proinflammatory effects of sars-cov-2 result in a wide variety of clinical presentations; however, severe pulmonary inflammation, effusions and rapid respiratory compromise are a hallmark of this disease. [5] [6] [7] subsequent pneumonia, acute respiratory distress syndrome and death have been reported not infrequently. the result of this pandemic is a large and increasing number of patients requiring endotracheal intubation and prolonged ventilator support. [8] [9] [10] [11] [12] [13] certainly, the rapid rise in endotracheal intubations coupled with prolonged ventilation requirements will lead to an increase in tracheostomy procedures in the coming weeks and months. 14, 15 although a generally well-tolerated and safe procedure, the risks and benefits of tracheostomy in terms of outcomes, pulmonary care and risks to the health care team remain unknown. 16, 17 fortunately, although not perfect, rapid testing protocols have allowed us to be able to detect active infection in patients who are affected by sars-cov-2. [18] [19] [20] [21] what is clear is that the upper aerodigestive tract, the nasopharynx and the trachea harbor a high viral load during the acute stages of the infection. [22] [23] [24] therefore, performing a tracheostomy in the setting of active sars-cov-2, when necessary, poses a unique situation, with unique risks and benefits for both the patient and the health care providers. the risk of this procedure has to be balanced with the known risks of prolonged intubation, primarily tracheal and subglottic stenosis, the management of which can be problematic if significant mucosal injury and subsequent stenosis occur. the new york head and neck society is a nonprofit organization founded in 1979, which encourages the exchange and advancement of scientific knowledge relative to the management of head and neck cancer and includes several member institucertainly, adequate pressure to avoid cuff leakage and aerosolization is critical when managing sars-cov-2 patients, but it should be recognized that unnecessarily high cuff pressures are also problematic. the minimum cuff pressure required to create an adequate seal should be individualized for each patient and verified frequently by care providers. certainly, high peak pressures, or issues with ventilation may make the appropriate cuff pressure a moving target. this is a dynamic process, and frequent adjustments may be indicated depending on ventilation parameters. prevention of tracheal mucosal pressure necrosis, resulting tracheal and cricoid chondritis and subsequent stenosis is critical in the sars-cov-2 population. 25, 26 sars-cov-2 testing via the reverse transcriptionpolymerase chain reaction (rt-pcr) detection platform for sars-cov-2 and pan-sarbecovirus detection is recommended for all patients who are being considered for tracheostomy. it should be remembered that data surrounding accuracy of the test during the pandemic is forthcoming, and false negatives are a real possibility. 27 in addition, rt-pcr may not be reliable when determining infectivity/active virus vs the mere presence of viral dna and therefore while levels detected by rt-pcr do tend to correlate with the active viral load, robust data are lacking to support the utilization of a testing protocol for viral load and decision making in sars-cov-2 positive patients. the test may be performed a second time if clinical suspicion or institutional policy warrants repeat testing for the presence of sars-cov-2 prior to high-risk procedures. when determining the appropriate time of tracheostomy in the sars-cov-2 patient, several factors are considered, and individual cases may certainly have mitigating circumstances that lead to the decision to perform tracheostomy. however, for the majority of patients, health care teams should seek to capitalize on the intersection of the risk of contamination/infection and decreasing viral load in the upper and lower airways over time with the risks of prolonged intubation (ie, tracheal stenosis, weaning issues, pulmonary toilet, etc.). although the overall risk of tracheal stenosis secondary to prolonged intubation depends on a variety of factors, reported rates of severe, symptomatic stenosis are generally in the 1% to 2% range when modern low-pressure cuffs are utilized. [28] [29] [30] [31] [32] therefore, in light of the relatively low risk of clinically relevant stenosis, and despite the traditional 10-day cutoff for increased stenosis risk used by many practitioners in the general population, when dealing with a sars-cov-2 patient, the risk for symptomatic or severe tracheal stenosis is acceptable in light of the significant risks of tracheostomy in the acute phase of the infection during periods of the highest viral loads. the decreasing viral load, although logarithmic in nature, is somewhat variable, and high viral loads have been observed somewhat late in the course of the infection in critically ill patients (figures 1 and 2) . 23, 24, 33 therefore, when feasible, waiting until approximately 21 days after the onset of symptoms is recommended prior to consideration of tracheostomy for the majority of cases in order to avoid exposing health care teams to increased risk. earlier tracheostomy may certainly be medically indicated in some situations depending on the clinical situation, or health care system issues during a pandemic situation, and we recognize the potential need to perform tracheostomy more urgently. tracheostomy should not be delayed regardless of sars-cov-2 status in life-saving situations or in situations in which the tracheostomy would significantly improve the prognosis of the patient. certainly the timing of tracheostomy remains a controversial issue and data is limited related to sars-cov-2. 53 alternative emerging strategies in the management of sars-cov-2 critically ill patients, such as extracorporeal membrane oxygenation, antiviral therapy and convalescent plasma therapy, may also be considered by the team, but the available data and decision making regarding this is beyond the scope of these recommendations. [34] [35] [36] [37] [38] clearly, these are multidisciplinary decisions that will be individualized depending on the patient and institutional expertise. in addition, it should be noted that avoiding tracheostomy in patients at high risk of mortality is critical. if the primary team managing the patient determines that there is an extremely high risk of mortality in the near future or that the patient has a high likelihood of withdrawal of care, the risks of tracheostomy should be avoided in this situation. patients with significant medical comorbidities, acute respiratory distress syndrome/severe respiratory failure and a low chance of recovery who are infected with sars-cov-2 should be carefully evaluated, and discussions with family members, consultants, institutional ethics committees and the treating team should focus on overall prognosis and goals of care prior to performing tracheostomy as a routine matter of care. these decisions are highly individualized and rely on solid communication among team members managing these highrisk patients. although the exact technical details regarding tracheostomy will depend on the situation and procedural protocols and technical expertise, there are some specific technical aspects related to the sars-cov-2 (and other viral pandemics) that should be considered. ideally, the procedure should be performed at bedside in the intensive care unit in a negative pressure room or using a portable high efficiency particulate air (hepa) filtration system to avoid patient transportation and contamination of other areas in the medical center. if it is necessary to perform the procedure in the operating room (or), a specific or cluster should be designated to avoid contamination of additional or resources for noninfected patients. in addition to standard airborne and droplet precautions, techniques to minimize aerosolization of the virus during the procedure include the following: paralysis to prevent coughing; consider glycopyrrolate to reduce secretions; preoxygenation and cessation of ventilation during the tracheostomy procedure; utilization of closed suctioning systems; avoiding monopolar electrocautery, or harmonic technology, and using cold instrumentation when feasible; minimizing suctioning and bronchoscopy during the procedure; and ensuring the cuff is inflated prior to resuming ventilation, so the circuit is closed. in addition to standard open tracheostomy with an apneic technique, percutaneous/dilational tracheostomy techniques have been evaluated extensively in the literature and have been shown to be a safe alternative to traditional open surgical tracheostomy. [39] [40] [41] understandably, the techniques utilized when performing tracheostomy will vary based on patient characteristics, provider expertise and institutional experience. although data are limited, techniques that avoid opening the airway and are closed, such as a percutaneous dilational technique, may be preferential in the setting of active sars-cov-2 infection. 42, 43 therefore, if there are no anatomical or other contraindications, percutaneous dilational tracheostomy may be considered if the expertise is available. it should be remembered that the decrease in aerosolization during percutaneous tracheostomy only holds true if airway manipulation (ie, bronchoscopy with cuff deflation or concurrent ventilation) is not performed, and although there have been some associated higher complication rates with blind percutaneous tracheostomy compared to bronchoscopic technique, ultrasound-guided techniques have been shown to be noninferior to bronchoscopic techniques. [44] [45] [46] therefore, when considering a percutaneous tracheostomy, a closed ultrasound-guided or "cuff-up" apneic technique is recommended for sars-cov-2 patients. although there are limited data on the current pandemic to fully inform personal protective equipment (ppe) recommendations, performing tracheostomy in an actively infected sars-cov-2 patient is certainly a high-risk procedure for health care workers. 47 health care personnel performing the tracheostomy should wear at minimum: waterproof cap, goggles with an antimist screen, impermeable operating room surgeon's gown and gloves and a transparent plastic facial shield worn outside the goggles and n95 that is effective in filtering 99.5% particles larger than 0.75 μm and 95% effective in the .1 to .3 μm range. 48 the minimum number of health care workers required to perform the procedure should be present to prevent unnecessary exposures. the effectiveness of the n95 mask in the prevention of sars-cov-2 infection during tracheostomy procedures remains unknown, but given the high risk and the reported size of thevirus ranging from 70-90 nm, consideration for power air-purifying respirator (papr) systems for personnel performing tracheostomy should be entertained. 54 these systems should be used when available in situations of acute active infection, or suspicion of high viral loads, as there is some evidence of superior protection (papr provides 2.5-100 times greater protection than the n95 when staff are appropriately trained). 47, 49, 50 the effectiveness of n95 and papr in this situation has certainly not been compared in a head-tohead trial, and therefore, the use of papr vs n95 will depend on institutional resources and policy and the clinical situation until further data is available. techniques to manage the acute airway with endotracheal intubation, video laryngoscope for example, should be utilized if possible to avoid emergent tracheostomy in sars-cov-2 patients due to the high risk of unsafe conditions and health care worker contaminations. 48 similarly, intubation techniques (ie, rapid sequence intubation) that avoid mask ventilation, such as prolonged open airway manipulation, are recommended when appropriate. when life-threatening airway obstruction occurs in a setting in which intubation is not possible, health care workers should perform the tracheostomy with the above-noted ppe, keeping in mind that papr respirator use is often not feasible or available in emergent situations. in situations in which cardiopulmonary resuscitation is being performed, chest compressions should be avoided at the time the airway is entered until the airway is secured and the cuff is inflated on the device to minimize health care worker exposure. posttracheostomy management should also be mentioned as, in addition to routine tracheostomy care, there are some considerations for sars-cov-2 patients. securing circuits properly and avoiding unnecessary humidification systems may reduce the risk of unexpected circuit disconnection and aerosolization leading to exposure. the circuit should remain closed as much as possible, and closed-line suctioning should be used. heat moister exchangers with viral filters and hepa filtration should be used when possible. tracheostomy tube changes should be avoided and should only be performed in cases of cuff failure or emergent situations. although the members of the health care team performing tracheostomy vary across institutions, team members may include surgeons, medicine/intensivists, anesthesiologists, respiratory therapists, nurses and other ancillary staff required during these procedures. the importance of appropriate ppe/papr training and usage cannot be overstated in the setting of active sars-cov-2 infection. teams that perform the procedure regularly will be more efficient and less likely to be unfamiliar with the procedure or appropriate health care protective measures and infection control. the inclusion of trainees such as residents and fellows during these procedures requires careful consideration and will vary based on institutional policies. currently, there are limited data on the host innate immune status of sars-cov-2 infected patients. 51 consideration of the inclusion of health care workers who have previously been exposed and subsequently recovered from documented sars-cov-2 infection may be warranted. although the exact timing of immunity and subsequent safety for the return of health care providers infected with sars-cov-2 remains unknown, sufficient antibody responses have been documented to occur between 15 and 20 days, or approximately 2 weeks, after the onset of symptoms ( figure 3 ). 23 inclusion of these individuals on these teams may allow for high-risk procedures to be performed by health care workers who have mounted an immune response to the virus depending on institutional quarantine policies. similarly, these individuals should not be involved in tracheostomy procedures or other airway procedures in noninfected patients due to the risk of iatrogenic infection with sars-cov-2 due to limited available data about the risks. 52 tracheostomy in the sars-cov-2 infected patient represents a unique situation, with a unique set of risks and implications. when compared to traditional tracheostomy procedures in the setting of prolonged ventilation, sars-cov-2 represents a unique entity in terms of timing, indications and infection control considerations that must be remembered when performing these procedures and managing patient's posttracheostomy. additional resources are listed below. • careful consideration of "who" and "when" when tracheostomy is planned is recommended. • careful consideration of the location and technique to avoid unnecessary risks to health care providers is recommended. • when clinically appropriate, delay of tracheostomy procedures is recommended to allow for reduced viral load and to decrease the risk of nosocomial infection to critical health 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soumelis, vassili title: sars-cov-2 induces activation and diversification of human plasmacytoid pre-dendritic cells date: 2020-07-10 journal: biorxiv doi: 10.1101/2020.07.10.197343 sha: doc_id: 255997 cord_uid: oer5lxxr several studies have analyzed antiviral immune pathways in severe covid-19 patients. however, the initial steps of antiviral immunity are not known. here, we have studied the interaction of isolated primary sars-cov-2 viral strains with human plasmacytoid pre-dendritic cells (pdc), a key player in antiviral immunity. we show that pdc are not permissive to sars-cov-2 infection. however, they efficiently diversified into activated p1-, p2-, and p3-pdc effector subsets in response to viral stimulation. they expressed checkpoint molecules at levels similar to influenza virus-induced activation. they rapidly produced high levels of interferon-α, interferon-λ1, il-6, ip-10, and il-8. importantly, all major aspects of sars-cov-2-induced pdc activation were inhibited by hydroxychloroquine, including p2and p3-pdc differentiation, the expression of maturation markers, and the production of interferon-α and inflammatory cytokines. our results indicate that pdc may represent a major player in the first line of defense against sars-cov-2 infection, and call for caution in the use of hydroxychloroquine in the early treatment of the disease. severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) is the third zoonotic coronavirus that emerged in the last two decades. sars-cov-2 is the causative agent of coronavirus disease 2019 that appeared in late 2019 in wuhan, hubei province in china (nandakumar, 2020; sheahan and frieman, 2020) . sars-cov-2 became rapidly pandemic, and infection have now been detected in 216 countries and territories, and is responsible for approximatively 10 million confirmed cases and 500,000 deaths as of 26 of june 2020 (who weekly update).  sars-cov-2 infection may lead to a diversity of clinical presentations, ranging from asymptomatic or mild "flu-like" syndrome, to severe and life-threatening acute respiratory failure. disease aggravation usually occurs after 8 to 10 days following initial symptoms (tang et al., 2020) . at this late stage, three main factors were shown to contribute to the progression and severity of the infection (tang et al., 2020) : 1) viral persistence was evidenced in the lung and systemic circulation, although it is not constant (tang et al., 2020) , 2) an excess production of pro-inflammatory cytokines, such as il-1b and il-6 (tay et al., 2020; arnaldez et al., 2020) , 3) a defect in type i interferon (ifn) production, especially in critically ill patients (tay et al., 2020; acharya et al., 2020) . although these abnormalities were confirmed in several studies, their origin and underlying mechanisms remain mostly unknown. in particular, it is not known whether an imbalance between inflammatory cytokines and type i ifn occurs early in the disease, at the stage of the primary infection, and whether the virus itself may be responsible. to fill this gap of knowledge, it becomes essential to investigate the early innate immune response to sars-cov-2. among the immune cells that are involved in innate anti-viral immunity, plasmacytoid predendritic cells (pdc) play a particularly important role as the major source of type i ifn in response to viral infection (liu, 2005) . pdc can sense a large array of viruses including the coronaviruses murine hepatis virus (mhv) and the middle east respiratory syndrome coronavirus (mers) (scheuplein et al., 2015; cervantes-barragan et al., 2007) , and respond by producing innate cytokines, including all forms of type i ifns (α and β), type iii ifn, and inflammatory cytokines, in particular tnf-α and il-6 (liu, 2005; yin et al., 2012; gilliet et al., 2008) . however, different viruses may induce different cytokine patterns (thomas et al., 2014) , possibly creating an imbalance between ifn versus inflammatory cytokine response. additionally, some viruses were shown to subvert pdc functions through different mechanisms not necessarily related to productive infection. this is the case for hiv, which may induce pdc apoptosis in vitro (meyers et al., 2007) and pdc depletion in vivo (soumelis et al., 2001; meera et al., 2010) . human hepatitis c virus can inhibit ifn-α production by pdc through the glycoprotein e2 binding to bdca-2 (florentin et al., 2012) . human papillomavirus induces very low ifn response in pdc (bontkes et al., 2005) , which may be due to impaired tlr-7 and -9 signaling (hirsch et al., 2010) . whether sars-cov-2 induces efficient pdc activation, or may interfere with various biological pathways in pdc is currently unknown. in this study, we have systematically addressed the interactions between clinical sars-cov-2 isolates and primary human pdc in order to reproduce the early stages of the infection. we showed that pdc are resistant to productive infection with sars-cov-2 strains but still mount substantial ifn responses upon viral challenge. interestingly, pdc responded to sars-cov-2 by a complete activation program, including diversification into effector subsets, production of type i and type iii ifn, as well as inflammatory cytokines. we also showed that hydroxychloroquine, an antimalarial drug proposed for treatment of covid-19 patients (das et al., 2020; mahévas et al., 2020) , inhibits sars-cov-2-induced pdc activation and ifn production in a dose-dependent manner. our results establish pdc as a potential key player in innate immunity to sars-cov-2, and raise caution regarding pharmacological manipulation that could inhibit pdc effector functions. in order to efficiently recapitulate sars-cov-2-pdc interactions, we used two strains of sars-cov-2 primary isolates. their viral genome sequences were nearly identical with 99,98% identity. sequence comparison with reference strain wuhan-hu-1 (ncbi accession number nc_045512.2) showed that both strains contain a subset of mutations (c241t; c30307t; a23403g and g25563t), characteristic of the gh clade based on gisaid nomenclature. human primary pdc were purified from healthy donor peripheral blood mononuclear cells (pbmc) by cell sorting. first, we asked whether sars-cov-2 was able to induce pdc activation, and diversification into ifnproducing and/or t cell stimulating effectors, as we previously described for influenza virus a (flu) (alculumbre et al., 2018) . after 24 hours of culture, sars-cov-2activated pdc efficiently diversified into p1 (pd-l1 + cd80 -), p2 (pd-l1 + cd80 + ), and p3 (pd-l1 + cd80 + ) pdc subsets, similar to flu stimulation ( fig 1a) . p1-, p2-, and p3-pdc were all significantly induced by sars-cov-2 and flu, as compared to medium control ( fig 1b) . in parallel, we observed a sharp decrease in non-activated p0-pdc (pd-l1 -cd80 -) (fig 1a and b) . sars-cov-2-induced pdc activation was comparable with magnetically-versus facs-sorted pdc (fig s1a and s1b ), confirming that both methods are suitable for subsequent experiments. all main findings were confirmed on at least three independent experiments using facssorted pdc, with a protocol that excluded as-dc, a rare dendritic cell (dc) subset that shares some markers and functional features with pdc (villani et al., 2017) , based on cd2, cd5 and axl expression ( fig s1a) . pdc activation and diversification was observed with two independent primary sars-cov-2 strains (fig 1c) , which both induced similar proportions of p1-p3 subsets. pdc diversification was also observed by co-culturing of pdc with sars-cov-2-infected vero e6 cells with a similar efficiency than free sars-cov-2 ( fig s1c) . sars-cov-2 improved pdc viability as compared to medium condition ( fig 1d) , which is compatible with subsequent effector functions. next, we asked whether sars-cov-2 -induced pdc activation was dependent on productive infection. we first checked whether pdc express at their cell surface ace2, the major sars-cov-2 entry receptor (hoffmann et al., 2020, 2) . no significant expression was detected using an anti-ace2-specific antibody, as compared to a low and high expression on vero e6 and 293t-ace2 cell lines, respectively ( fig 1e) . the ability of pdc to replicate sars-cov-2 was then assessed. human pdc were challenged with sars-cov-2 strain 220_95 at moi of 2, and cultured for 2h, 24h or 48 h. our results showed that pdc were refractory to sars-cov-2 infection, as evaluated by quantifying 1) the intracellular production of the nucleoprotein antigen (n) (fig 1f) , or the accumulation of viral rna in sars-cov-2-infected cells (fig s1d) , and 2) the release of infectious progeny virus in the supernatants of infected cells using plaque assays ( fig 1g) . as positive control, the permissive vero e6 cells produced high level of the n antigen, increased viral rna overtime ( fig s1d) , and high viral titers following sars-cov-2 incubation ( fig 1g) . similar results were obtained with pdc isolated from three independent donors ( fig s1e) . overall, these results show that pdc are resistant to sars-cov-2 infection, and are efficiently activated by the virus independently of ace2 expression. their viability was not affected by sars-cov-2 challenge. activating immune checkpoints play a key role in t cell stimulation, and serve as surrogate markers of dc differentiation (guermonprez et al., 2002) . we first assessed the dose-dependent effect of sars-cov-2 on cd80 expression and subset diversification. cd80 was induced in a dose-dependent manner by sars-cov-2 at moi 0.04 to 1 (fig 2a) . this was accompanied by an increase in p3-pdc subset, and a slight decrease in p1-pdc ( fig 2b) . a detailed phenotypic analysis was subsequently performed on pdc after 24 and 48 hours of culture with sars-cov-2 ( fig 2c and fig s2a) . diversification was observed at both time points, with a slight increase in p3-pdc at 48 hours ( fig s2a) . p2-and p3-pdc significantly upregulated cd80, cd86, ccr7, and ox40l, as compared to non-activated p0-pdc, in both sars-cov-2 and flu conditions ( fig 2c) . pd-l1, and cd62l, an integrin that promotes lymph node homing, were both higher on p1-and p2-pdc ( fig 2c) . expression of checkpoint molecules persisted at 48h, especially the higher cd80 and cd86 expression on p3-pdc ( fig s2b) . a key and defining function of pdc is their ability to produce large amounts of type i ifn (gilliet et al., 2008) . we measured the production of several cytokines at the protein level after 24 hours of culture. both sars-cov-2 and flu induced high levels of ifn-α2 and ifn-λ1, both being critical anti-viral effector cytokines ( fig 3a) . ifn-α levels following sars-cov-2 activation reached up to 80 ng/ml, indicating a very efficient activation. the chemokine ip-10 was also significantly induced ( fig 3a) , possibly due to an autocrine ifn loop (blackwell and krieg, 2003) . inflammatory cytokines il-6 and il-8 were comparably induced by sars-cov-2 and flu ( fig 3a) . however, tnf-α levels were marginally induced by sars-cov-2 as compared to flu activation ( fig 3a) . cytokine production was maintained after 48 hours of viral activation ( fig 3b) . secreted protein levels were similar to 24h levels for most cytokines. interestingly, ifn-α levels raised by 3-fold between 24h and 48h for one donor ( fig 3a and b) , indicating the possibility of increased production. such strong ifn producer suggests a potential virus controller. because the oropharyngeal mucosa is an entry site for sars-cov-2, we aimed at validating our results using pdc purified from tonsils. sars-cov-2 induced a marked diversification of tonsillar pdc into all three activated subsets ( fig s2c) . tonsillar pdc efficiently produced ifn and inflammatory cytokines in response to sars-cov-2 ( fig s2d) . overall, our results establish sars-cov-2 as a very efficient inducer of type i and type iii ifn responses. inflammatory cytokines were induced at similar level than flu activation, without any significant imbalance that would be suggestive of excessive inflammatory response. given that sars-cov-2 did not infect pdc, and did not interfere with pdc activation, we asked whether pharmacological agents could modulate pdc diversification and cytokine production. hydroxychloroquine (hcq) is known to inhibit endosomal acidification which may diminish pdc activation (kuznik et al., 2011, 9; sacre et al., 2012) . additionally, it is being tested in several clinical studies as a potential treatment for covid-19 (das et al., 2020; mahévas et al., 2020) . hence, we addressed its role in sars-cov-2-induced pdc activation. following 24 hours of culture, we found that hcq inhibited pdc diversification in response to sars-cov-2, which is similar to the decrease observed with flu, used as a positive control ( fig 4a) . in particular, p2-and p3-pdc differentiation were almost completely inhibited ( fig 4b) . inhibition of sars-cov-2-induced pdc diversification by hcq was dosedependent ( fig s3a) . the significant decrease in p3-pdc was paralleled by a decrease in cd80, cd86, and ccr7 expression (fig 4c and d) . ox40-ligand expression was not significantly affected by hcq (fig 4c and d) . however, hcq inhibited the appearance of an ox40-ligand high pdc population ( fig s3b and s3c ), which may impact subsequent t cell activation. last, we assessed the effect of hcq on innate pdc functions. we measured cytokine production after 24 hours of sars-cov-2-induced pdc activation in the presence or absence of hcq. we found that ifn-α and λ levels were decreased by hcq (fig 4e) . this was also the case for il-6 and il-8, with a much lesser impact on ip-10 ( fig 4e) . together, these results show that hcq inhibits sars-cov-2-induced pdc diversification and cytokine production. type i ifns are critical cytokines that control viral replication. several chronic viral infections were associated to poor type i ifn responses (lee et al., 2013; snell et al., 2017; marsili et al., 2012; dolganiuc et al., 2006) . in covid-19 patients, decreased serum levels of type i ifn were associated with severity in late stage infection, and increased viral load (tay et al., 2020; acharya et al., 2020) . this raised the question as to whether sars-cov-2 was intrinsically capable of inducing a robust ifn response, or on the contrary could interfere with ifn production and other antiviral immune pathways. in this study, we have used primary sars-cov-2 isolates and human primary pdc in order to increase the relevance to a naturally occurring infection. our results demonstrate that sars-cov-2 is a strong ifn inducer by efficiently stimulating primary pdc. viral sensing was independent of the expression of the ace2 entry receptor or the ability of the virus to replicate in pdc. however, the precise molecular mechanisms involved remain to be investigated. both type i and type iii ifns were induced at high levels upon sars-cov-2 stimulation. this strongly suggests that the defects observed in critically ill covid-19 patients are acquired during disease evolution through secondary events, not necessarily associated to direct effect of the virus. possible mechanisms could be related to inflammatory cytokines, such as tnf, and endogenous glucocorticoid response, both being able to promote pdc apoptosis ( (abe and thomson, 2006) . however, additional mechanisms may be involved and need to be explored in the context of severe covid-19. an excessive production of inflammatory cytokines, such as il-1 β, il-6 and tnf, was associated to covid-19 severity (tay et al., 2020; arnaldez et al., 2020; vabret et al., 2020) . their cellular source and the underlying mechanisms are currently unknown. our results indicate that sars-cov-2-induced pdc activation promotes a balanced production of innate cytokines, including large amounts of type i and type iii infs, without any significant excess in inflammatory cytokines. this suggests that pdc activation may not be a causal factor of covid-19 aggravation. in support to that, recent studies indicated that endothelial cells may be a target of sars-cov-2 infection, and could be at the origin of the systemic and multi-organ production of inflammatory cytokines (pons et al., 2020) . bronchial epithelial cells could also be involved in the production of high levels of il-6, which were not detected in the serum and in pbmc transcriptomic studies in severe covid-19 (wilk et al., 2020) . this supports the view of pdc as being protective through an early and efficient production of antiviral cytokines, with later defects due to currently unknown mechanisms, associated with late stage aggravation. on the contrary, nonprofessional innate immune cells such as endothelial cells and bronchial epithelial cells may be involved in the secondary worsening of covid-19 through the excessive and uncontrolled production of inflammatory cytokines. several therapeutic approaches have been explored and are currently being tested in clinical trials on covid-19 patients (vabret et al., 2020; tay et al., 2020) . these include antiviral agents (yang et al., 2020) , immune-modulatory molecules, such as glucocorticoids (fernández cruz et al., 2020) , and anti-inflammatory molecules, such as hcq (touret and de lamballerie, 2020; lecuit, 2020) . this latter drug was additionally shown in in vitro studies to interfere with sars-cov-2 replication (wang et al., 2020) . continued efforts in mapping and dissecting immune effector pathways to sars-cov-2 will be of major importance in order to design efficient treatment strategies adapted to each patient and stage of the infection. buffy coats from healthy human donors were obtained from etablissement français du sang, paris, saint-antoine crozatier blood bank. peripheral blood mononuclear cells (pbmcs) were isolated through ficoll density gradient centrifugation (ficoll-paque; ge healthcare). pdc were isolated through a first step of pdc magnetic sorting (human plasmacytoid dc enrichment kit; stemcell), and subsequent flow cytometric sorting on the basis of live, lineage -(cd16, cd14, cd19, cd20, cd56 and cd3), cd11c -cd4 + , and cd2 -cd5cells to a 98% purity. due to some logistic issues, alternatively frozen pbmcs from etablissement français du sang, paris, saint louis blood bank, were thawed and placed at 37°c for 2h for cell recovery. sars-cov-2 viruses were isolated from nasopharyngeal swab specimens collected at service de virologie (hospital saint louis, paris). samples were centrifugated at 4,000 x g for 10 min then filtered using a 0.45 μm filter, and diluted 1:1 with dmem-4% (dmem supplemented with 4% fbs, 1% penicillin-streptomycin, 1% glutamax and 25 mm hepes). vero e6 cells were seeded in 96-well cell culture plate (15,000 cells/well), and incubated at 37°c with 200μl of inoculum and observed daily for cytopathogenic effects (cpe) by light microscopy. substantial cpe were seen at 72-96 hours post inoculation. culture supernatants were then collected, clarified by centrifugation, filtered using a 0.45 μm filter and kept at -80°c. we confirmed sars-cov-2 replication by rt-qpcr and whole viral genome sequences were obtained by next generation sequencing using illumina misseq sequencers. strains sequences have been deposited in the global initiative of sharing all influenza data (gissaid) database with accession id epi_isl_469284 (220_95) and epi_isl_469283 (211_61). all viruses belong to the gh clade. sars-cov-2 strains were further propagated on vero e6 in dmem-2% (dmem supplemented with 2% fbs, 1% penicillin-streptomycin, 1% glutamax and 25 mm hepes). viruses were passaged three times before being used for experiments. for the last passage, viruses were purified through a 20% sucrose cushion by ultracentrifugation at 80,000 x g for 2 hours at 4°c. pellets were resuspended in hne 1x ph7.4 (hepes 5 mm, nacl 150 mm, edta 0.1 mm), aliquoted and stored at viruses titer was ascertained by plaque assays in vero e6 cells and expressed as pfu per ml. cells were incubated for 1 hour with 10-fold dilution of viral stocks. the inoculum was then replaced with avicel 2.4% mixed at equal volume with dmem supplemented with 4% fbs, 2% glutamax, 50mm mgcl 2 , 0.225 % of nahco 3 , and incubated 3 days at 37°c before plaque counting. vero cells were plated (50,000 cell per well) in p24-well plates 4 hours before being incubated with sars-cov-2 diluted in dmem-2%. freshly purified pdc were seeded in p96-well plates (50,000 cells per well) and incubated with sars-cov-2 diluted in pdcs culture medium (rpmi 1640 medium with glutamax, 10% of fbs, 1% of mem neaa, 1% of sodium pyruvate, and 1% of penicillin/streptomycin). at 2, 24 and 48 hour post-inoculation, vero cells were trypsinized and transferred to p96-well plates. to quantify infectious viral particle released in the supernatants of infected cells, pdc and vero cells were inoculated with sars-cov-2 as described above and incubated at 37°c for 72-hour. at indicated time points, supernatants were collected and kept at -80°c. virus titer were then determined by plaque assay on vero e6 cells as described above. vero e6 and pdc were inoculated with sars-cov-2 as described above. at the indicated time points, cells were washed thrice with pbs. vero e6 were further incubated with trypsin 0.25% for 5 min at 37°c to remove cells surface bound particles. total rna was extracted using the rneasy plus mini kit (qiagen) according to manufacturer's instruction. cdnas were generated from 80 ng total rna by using the maxima first strand synthesis kit following manufacturer's instruction (thermo fisher scientific). amplification products were incubated with 1 unit of rnase h for 20 min at 37 °c, followed by 10 min at 72°c for enzyme inactivation, and diluted 10fold in dnase/rnase free water. real time quantitative pcr was performed using a to sort pdc, cells were stained with zombie violet or buv fixable viability dye pdc cytokine production of ifn-α2, il-8, il-6, ip-10 and tnf-α, was measured in culture supernatants using bd cytometric bead array (cba), according to the manufacturer's protocol, with a 20pg/ml detection limit. acquisitions were performed on a lsr fortessa (bd biosciences), and cytokine concentrations were determined using fcap array software (bd biosciences). the concentration of secreted ifn-λ1 was measured by enzyme-linked immunosorbent assay (elisa) (r&d systems, duoset dy7246), according to the manufacturer's instructions. the manufacturer reported no cross-reactivity nor interference with ifn-α, ifn-β 1a, il-10rβ, ifn-λ2 and λ3, and il-28rα. the optical density value (od) of the supernatants was defined as its absolute od value, minus the od absorbance from blank wells. the detection limit was 85pg/ml and all samples were run in duplicates. statistical analyses were performed with one-way anova, kruskall wallis's test with dunn's multiple comparison post-test or mann whitney's test, in prism (graphpad software). 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coronaviruses type iii ifns are produced by and stimulate human plasmacytoid dendritic cells key: cord-290796-x9xqqcj6 authors: stefanelli, p.; bella, a.; fedele, g.; fiore, s.; pancheri, s.; benedetti, e.; fabiani, c.; leone, p.; vacca, p.; schiavoni, i.; neri, a.; carannante, a.; simmaco, m.; santino, i.; zuccali, m. g.; bizzarri, g.; magnoni, r.; benetollo, p. p.; brusaferro, s.; rezza, g.; ferro, a. title: longevity of seropositivity and neutralizing titers among sars-cov-2 infected individuals after 4 months from baseline: a population-based study in the province of trento date: 2020-11-13 journal: nan doi: 10.1101/2020.11.11.20229062 sha: doc_id: 290796 cord_uid: x9xqqcj6 background. there are conflicting results about the duration of antibodies induced by sars-cov-2, but several studies show a rapid decay in a few months after infection. to evaluate antibody decline, we re-evaluated the presence of anti-sars-cov-2 antibodies among individuals found seropositive in a first population survey conducted 4 months before. methods. all individuals above ten years of age resident in 5 municipalities of the autonomous province of trento, northern italy, who resulted igg positive for anti-sars-cov-2 nucleocapsid (nc) antibodies in a serosurvey conducted on may 2020 were retested after 4 months. anti-sars-cov-2 antibodies were detected using the abbott sars-cov-2 igg assay (abbott diagnostics, usa) detecting anti-nc antibodies. samples that gave a negative result were re-tested using the same test plus liaison sars-cov-2 igg assay (diasorin, italy) to assess anti-spike (s) s1/s2 igg antibodies. seroprevalence was calculated as the proportion of positive people on the total number of tested. a neutralizing assay was performed on a subgroup of formerly positives sera using fifty-percent tissue culture infective dose (tcid50) as endpoint dilution to produce a cytopathic effect in 50% of inoculated vero e6 cells culture. in all the analyses a p value < 0.05 were considered statistically significant. statistical analysis was performed by stata version 16.1 (stata corp., college station, texas, usa). findings. overall, 1159 out of 1402 initially anti-nc seropositive participants were enrolled in the study. of them, 480 (41.1%) became seronegative for anti-nc igg antibodies. when 479 negative sera were tested for anti-s igg, 373 samples (77.9%) resulted positives. a functional neutralization assay was performed on 106 sera showing high concordance with anti-s antibodies positivity. interpretation. a decline of anti-nc igg values was recorded 4 months after the first evaluation. worth of note, a high proportion of anti-nc seronegative individuals were positive for anti-spike igg antibodies, which appear to persist longer and to better correlate with neutralization activity. the presence of neutralizing antibodies induced by natural infection or by an effective vaccine is likely to be predictive of protection. thus, whether antibody response to the administration of vaccines may confer protection is still undefined, and cases of reinfection by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) have been sporadically reported1. the duration of the immune response after infection is also under investigation. the duration of protection against infection with common human coronaviruses appears to be rather short 2, 3 , and there are studies showing declines in igg antibodies against sars-cov-2 among both symptomatic and asymptomatic individuals 4, 5 . whether memory-b-cell and t-cell responses may still confer protection in individuals experiencing antibody decline to undetectable levels is unknown 6 . in general, controversies exist on the possibility that sars-cov-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic covid-19 [7] [8] [9] . the type of antibody response may also play a role. experimental vaccination against sars-cov with nc can induce strong antibody responses that were found to be non-neutralizing 10 . while nonneutralizing antibodies might still exert antiviral activity, for example via the fc-fc receptor-based effector function, non-neutralizing nc antibodies may lead to enhanced disease for some vaccine candidates in animal models when neutralizing antibodies are absent 10 . instead, studies conducted on sars-cov-2 and other coronaviruses have shown that the spike protein is the main target for neutralizing antibodies [11] [12] [13] . thus, assessing the duration of detectable antibody response and changes in the titer of neutralizing antibodies is an important step in order to better understand their dynamics and to predict the duration of protection against sars-cov-2 infection. in order to evaluate the persistence of sars-cov-2 antibodies, we repeated a serosurvey in five municipalities of the autonomous province (ap) of trento, italy, recruiting those individuals who had resulted positive in a large population-based seroprevalence study conducted 4 months before 14 . moreover, in a subsample of seropositive participants, the antibody neutralizing titre was also evaluated. as already reported 14 , the study was conducted in 5 municipalities of the ap of trento, in the northern of italy, with the highest incidence of covid-19 confirmed cases. the department of prevention of the azienda provinciale per i servizi sanitari (apss) sent a letter of invitation to participate at a second study to all the citizens who resulted to be positive for anti-sars-cov-2 antibodies in the serosurvey conducted 4 months before, between may 5 and 15, 2020. blood samples (5 ml) were collected in serum separator tubes (bd diagnostic systems, franklin lakes, nj, usa) and centrifuged at room temperature at 1600 rpm for 10 min. aliquots were transferred to 2ml polypropylene, screw cap cryotubes (sorfa, zhejiang, china) and immediately frozen at -20 °c. frozen sera were then shipped to the istituto superiore di sanità (iss) as national reference laboratory for covid-19, in dry ice following biosafety shipment condition. upon arrival serum samples were immediately stored at -80 °c. 14 two commercial clia assays, employing either nc or s antigens and designed for high throughput in healthcare settings, were used. in particular, all the serum samples were evaluated by using the abbott sars-cov-2 igg assay (abbott diagnostics, chicago, il, usa); sera resulting negative were retested using the diasorin liaison sars-cov-2 igg assay (diasorin, italy). the abbott diagnostics anti-nc igg assay was performed on the architect i2000sr automated analyser. the analyser automatically calculates sars-cov-2 nc igg antibody concentration expressed as an is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229062 doi: medrxiv preprint index value. according to the manufacturer's instructions, the results were interpreted considering as positive an index of ≥1.4 and as negative an index of <1.4. the diasorin sars-cov-2 igg assay is also a two-step clia assay for the detection of igg antibodies against s1/s2 antigens of sars-cov-2. the assay was performed on the liaison® xl fully automated chemiluminescence analyzer. the analyser automatically calculates sars-cov-2 s1/s2 igg antibody concentrations expressed as arbitrary units (au/ml). the assay range is up to 400 au/ml. according to manufacturer's instructions, values ≥ 15 au/ml were interpreted as positive, and values ≤ 12 au/ml as negative; in case of results falling within an equivocal zone in between 12 au/ml and 15 au/ml, the test was repeated. in vitro neutralising activity provides quantitative results as a measure of a functional humoral immune response against sars-cov-2. a known amount of sars-cov-2 (code 77iii, isolated and cultivated at iss, titre 1x10 5,4 ; gisaid accession id: epi_isl_412973) was incubated with different dilutions of the serum sample to determine the dilution at which cytopathic effect on vero e6 cells (atcc® crl-1586) is observed in 50% of infected wells (mn 50%). the detailed protocol is described below: two-fold serial dilutions of serum samples starting at 1:8 dilution up to 1:512 in cell culture medium emem (sigma) supplemented with 1x pen/strep and 2% fetal bovine serum (fbs; corning) were added to 96-well plates. the mixture of virus (100 tcid50) and serum was incubated at 37ºc for 1 hour for a total 100 µl. after this incubation period, a solution of 22,000 cells per well in a total volume of 100 µl was added and incubated at 37ºc for 5 days. finally, the neutralization titer was calculated and expressed as the serum dilution capable of reducing the cytopathic effect to 50% (mn 50%). positive and negative sera samples and cell culture control together with the virus were added in each test. the igg values were summarized by the median and interquartile range (iqr). the differences among igg values between the first and the second survey were evaluated by the wilcoxon test. the is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229062 doi: medrxiv preprint < 0.20 = "poor", 0.20-0.40 = "fair", 0.40-0.60 = "moderate", 0.60-0.80 = "good", and 0.80-1.00 = "very good"). a multivariable logistic regression model was used to determine the relationship between persistent anti-nc igg in the second serosurvey (positive versus negative) and a set of explanatory variables. the following variables that were significantly associated (p< 0.10) at the univariate analysis were included in the multivariable model: gender, age group, geographical area, presence of symptoms, working in contact with the public and household size, igg positivity group (weak, medium, high) olfactory and gustatory dysfunctions, fever, weakness, cough, dyspnea, arthralgia, diarrhoea, and abdominal pain and vomit. the likelihood ratio test was used to compare different models. a subset of anti-nc igg positive samples was tested with the neutralization test. assuming a positive proportion of 95% and precision of 4%, 106 samples are required with an alpha error of 5%. in all the analyses a p-value < 0.05 was considered statistically significant. statistical analysis was performed by the stata version 16.1 (stata corp., college station, texas, usa). informed consensus for blood collection was obtained from all the participants. the study was approved by the ethical committee of the iss (prot. pre bio ce n.15997, 04.05.2020), overall, 1159 individuals of the 1402 individuals who resulted seropositive in the first survey (82.7%) were enrolled in the study. all age groups were well represented. the proportion of those who were retested ranged between 72.6% in the age group 20-29 years and 93.1% in the age group 60-69 years. of the 1159 individuals who resulted initially seropositive 480 (41.4%) seroreverted at the second evaluation. as shown in figure 1 , a statistically significant reduction in the median value was observed in the second survey, from a median of 5.7 (iqr = 3.5) to 1.9 (iqr = 2.8) (p-value < 0.0001 using the non-parametric wilcoxon signed-rank test). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; as shown in figure 2 , when the participants were stratified in three groups in accordance with their anti-nc igg level at the baseline [i.e., weak positive (with a value between 1.4 and 3), medium positive (between 3 and 5), and high positive (greater than 5)], the median value of the weakly and moderately positive groups decreased below the assay cut-off after 4 months, while the median of the highly positives remained above the cut-off. the samples resulting negative for antibodies against nc in the second study were tested to evaluate the presence of antibodies against the s protein. since for one sample the available amount of serum was not sufficient for the analysis, 479 available serum samples were tested, and 373 of them (77.9%) resulted positive (figure 3 ). a subgroup of 106 sera that were positive for anti-nc-igg at the first testing were tested for anti-nc igg, anti-s igg, and their neutralizing activity 4 months after the baseline. of the 106 sera, 97 (91.5%) showed neutralizing activity (tcid50 ≥ 1/8), and 9 sera (8.5%) had a tcid50 titer <1/8; 57 (53.8%) were anti-nc positive and 93 (87.7%) were anti-s positive. (figure 4 ). table 1 high and significative agreement (94,3%) was found between anti-s and tcid50 (k=0.70; p<0.0001) ( table 1 ). to further confirm the concordance, when igg levels were considered, a good correlation between anti-s and ticd50 was observed (rho-spearman: 0.84, p < 0.0001) compared with anti-nc/anti-s (rho-spearman: 0.61, p < 0.0001) and anti-nc/ticd50 (rho-spearman: 0.56, p < 0.0001). the multivariable logistic regression model showed that age group, gender, anti-nc igg level in the first serosurvey, and cough were factors associated with the persistence of anti-nc seropositivity ( table 2 ). in particular, the individuals with high anti-nc igg levels in the first serosurvey had the highest probability to be seropositive after four months (or=69.2). age above 70 years and cough, as reported during the first survey, were also strongly associated with persistent anti-nc igg levels. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229062 doi: medrxiv preprint hereby, we report the results of a repeated serosurvey conducted in five municipalities in the ap of trento, located in northern italy 14 these results appear to be consistent with those obtained for other human coronaviruses, such as nl63, 229e, oc43, and hku1, showing a rapid decay of antibodies directed against the nucleocapsid protein 16 . however, other studies showed different results, with high igg levels after several months [7] [8] [9] 17 . the inconsistency in the results of previous studies could be explained by differences in the study populations (i.e., patients with mild vs moderate or severe disease) or in the use of different methods (i.e., detection of antibodies directed against the nucleocapsid vs whole spike or the receptor binding domain of the spike) 4 . more recently, seow et al. 18 in a longitudinal study of rt-pcr confirmed covid-19 cases, the participants showed a wide range of antibody responses, and a decline in antibodies levels and virus neutralization was observed within three months of the onset of symptoms 18 . for those who developed a low neutralizing antibody response (id50 100-300) the titers could return to baseline over a relatively short period, whereas those who developed a robust neutralizing antibody response maintained titers >1000 despite the . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229062 doi: medrxiv preprint initial decline 18 . the decline of protective antibodies might be explained, to some extent, by the sporadic covid-19 reinfection that have been reported [19] [20] [21] . in a more recent study, detectable neutralizing antibody responses were detected for several months after infection 17 . to this regard, animal models show that sars-cov-2 infection protect from re-infection for at least some time 22, 23 . this protection appears to be more pronounced in the lower respiratory tract rather than in the upper respiratory tract 23 . the types of antibody response may also play a role. atyeo et al 24 , showed that a predominant humoral response to nucleocapsid protein is associated with poor outcome in patients admitted to hospital, compared to response to spike protein. most vaccine candidates elicit responses to s rather than nc protein. measuring antibodies to s will therefore indicate whether there has been a good response. to this regard, our findings are in good agreement with the study by wajnberg is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229062 doi: medrxiv preprint funds: apss sustained the expenses for the igg assays on collected sera. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229062 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229062 doi: medrxiv preprint figure 3 percentage of anti-spike (s1/s2) igg antibodies on retested anti-nc igg negative sera. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; table 1 . concordance between igg against nc and s proteins and neutralization activity. anti-s -p-value* is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted november 13, 2020. ; https://doi.org/10.1101/2020.11.11.20229062 doi: medrxiv preprint is recurrence possible in coronavirus disease 2019 (covid-19)? case series and systematic review of literature t cell-mediated immune response to respiratory coronaviruses duration of antibody responses after severe acute respiratory syndrome. emerg infect dis rapid decay of anti-sars-cov-2 antibodies in persons with mild covid-19 clinical and immunological assessment of asymptomatic sars-cov-2 infections targets of t-cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals sars-cov-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic covid-19 loss of anti-sars-cov-2 antibodies in mild covid-19 loss of anti-sars-cov-2 antibodies in mild covid-19. reply vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants sars-cov-2 vaccines: status report the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients a human monoclonal antibody blocking sars-cov-2 infection prevalence of sars-cov-2 igg antibodies in an area of north-eastern italy with a high incidence of covid-19 cases: a population-based study immunological considerations for covid-19 vaccine strategies seasonal coronavirus protective immunity is short-lasting robust neutralizing antibodies to sars-cov-2 infection persist for months longitudinal evaluation and decline of antibody responses in sars-cov-2 infection serum antibody profile of a patient with covid-19 reinfection asymptomatic reinfection in two healthcare workers from india with genetically distinct sars-cov-2 genomic evidence for reinfection with sars-cov-2: a case study sars-cov-2 infection protects against rechallenge in rhesus macaques syrian hamsters as a small animal model for sars-cov-2 infection and countermeasure development distinct early serological signatures track with sars-cov-2 survival the authors would like to thank the study's participants. the authors declare no conflict of interest related to this study. key: cord-287501-7it4kh0e authors: roh, changhyun title: a facile inhibitor screening of sars coronavirus n protein using nanoparticle-based rna oligonucleotide date: 2012-05-03 journal: int j nanomedicine doi: 10.2147/ijn.s31379 sha: doc_id: 287501 cord_uid: 7it4kh0e hundreds of million people worldwide have been infected with severe acute respiratory syndrome (sars), and the rate of global death from sars has remarkably increased. hence, the development of efficient drug treatments for the biological effects of sars is highly needed. we have previously shown that quantum dots (qds)-conjugated rna oligonucleotide is sensitive to the specific recognition of the sars-associated coronavirus (sars-cov) nucleocapsid (n) protein. in this study, we found that a designed biochip could analyze inhibitors of the sars-cov n protein using nanoparticle-based rna oligonucleotide. among the polyphenolic compounds examined, (−)-catechin gallate and (−)-gallocatechin gallate demonstrated a remarkable inhibition activity on sars-cov n protein. (−)-catechin gallate and (−)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by qds-conjugated rna oligonucleotide on a designed biochip. at a concentration of 0.05 μg ml(−1), (−)-catechin gallate and (−)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based rna oligonucleotide biochip system. severe acute respiratory syndrome (sars) is an infectious disease that began in guandong, china in november 2002. it has caused serious infections in many nations, such as asia, europe and canada. [1] [2] [3] [4] [5] [6] according to the world health organization (who), the mortality of patients afflicted with sars is 15% on average and 50% or higher in patients aged 65 years and over. 7 sars-associated coronavirus (sars-cov) is an enveloped and positively single-stranded rna virus with a typical genome size of 29.7 kb. it encodes rna-directed rna polymerase and structural proteins, including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. [8] [9] [10] [11] the n protein is a 422 amino acids alkaline protein with a short lysine-rich region suggested as the nuclear localization signal. it plays an important role in the process of virus particle assembly by enveloping the entire genomic rna. 12 the sars-cov n protein is a major pathological determinant in the host and might cause host cell apoptosis, upregulate the proinflammatory cytokine production, and block innate immune responses. moreover, the n protein of the sars-cov is an important antigen for both the early diagnosis of sars and the detection of diseases. 13 since sars broke out in 2003, researchers have made great efforts to develop fast and accurate analytical methods for its early diagnosis. [14] [15] [16] [17] [18] in addition, due to its essential role in sars replication, the sars-cov n protein is mainly regarded as a prime target for anti-sars therapy. for this reason, the sars-cov n protein is an attractive and crucial target for anti-sars therapeutic drug discovery. polyphenolic compounds are phytochemicals found in numerous plants and fruits. [19] [20] [21] they have been reported to act as antioxidants, free radical scavengers, metal chelators, and to be antiallergic, anticancer, antioxidant, anti-inflammatory, antifungal, and antiviral and antibacterial agents. in general, these polyphenolic compounds are known to have medicinal and chemopreventive activity in human health. [22] [23] [24] [25] in particular, (-)-catechin gallate and (-)-gallocatechin gallate are known as a type of catechin. (-)-catechin gallate and (-)-gallocatechin gallate are the most abundant catechins, particularly in tea and other plants, and they are a potent antioxidants 26 with possible therapeutic properties for many disorders, including cancer. 27, 28 researchers reported the benefit of catechin gallate from green tea in the treatment of human immunodeficiency virus (hiv) infection, where (-)-catechin gallate has been shown to reduce plaques related to acquired immunodeficiency syndrome-related dementia. 29, 30 there have also been reports showing that catechin gallate can be beneficial in treating brain, 31 prostate, 32 and other types of cancers. 33 in this study, we report a novel approach for the inhibitor screening of sars-cov n protein using a quantum dots (qds)-conjugated oligonucleotide system with wide applicability for facile and sensitive imaging analysis on a biochip. we elucidated the inhibitor on sars-cov n protein identified through a high-throughput screening strategy using an optical nanoparticle-based rna oligonucleotide. to the best of our knowledge, this is the first report on the inhibition effects of (-)-catechin gallate and (-)-gallocatechin gallate on sars-cov n protein using an optical nanoparticle-based rna oligonucleotide platform. the production of designed rna oligonucleotide and primer with t7 promoter sequence were synthesized by bioneer co ltd (seoul, republic of korea) and amplified by polymerase chain reaction (pcr). template rna was prepared by in vitro transcription using t7 rna polymerase (promega, madison, wi). all rna transcript products were separated by 8 m urea 6% polyacrylamide gel electrophoresis (page) after phenol extraction and ethanol precipitation procedures. the produced rna oligonucleotide was solved in 0.1% diethylpyrocarbonate (depc) solution and stored at -70°c for further experiments. the gene was amplified by a pcr with the primer set in the direction of sense at 5′-agtggatccatgtctgataatggacccca-3′ and in the direction of antisense at 5′-gccgtcgacttatgcctgagttgaatcagc-3′, containing restriction enzyme sites of bamhi/sali. pcr was run with the following conditions on a thermal cycler: denaturation at 94°c for 1 minute; annealing at 60°c for 30 seconds; and an extension step at 72°c for 2.5 minutes. the sequence was repeated 35 times and followed by a 7 minute final extension step at 72°c. the pcr product was digested with bamhi/sali, and then ligated into bamhi/sali digested expression vector pet 24a+ (novagen, madison, wi), and transformed into escherichia coli dh5α (stratagene, la jolla, ca). the colony with insert gene was transformed into e. coli bl21 (de3) (stratagene). it was then plated on luria-bertani (lb) agar containing 50 µg ml -1 kanamycin. groes/el expressing plasmid from e. coli and sars-cov n-expressing plasmid, which possessed ampicillin-and kanamycin-resistant markers, were cotransformed into e. coli bl21 (de3) according to biotransformation procedures. the transformant was grown in a 250 ml flask containing 50 ml lb medium supplemented by 50 µg ml -1 of kanamycin and ampicillin at 37°c until the cell concentration reached od 600 nm of 0.6 and isopropyl-thio-β-d-galactopyranoside (iptg) of a final concentration of 0.1 mm. it then was left to grow overnight at 25°c with shaking. the cells were harvested by centrifugation at 4000 rpm for 30 minutes at 4°c and resuspended in 100 mm potassium phosphate-buffered saline (ph 7.5) containing 1 mm phenylmethylsulfonyl fluoride (pmsf). the cells were lysed by sonicator ® (f60 sonic dismembrator; fisher scientific, fair lawn, nj). the cell debris was removed by centrifugation at 13,000 rpm for 30 minutes. the supernatant was collected and the recombinant sars-cov n protein was purified with ni-nitrilotriacetic acid (ni-nta) affinity chromatography column (qiagen, germany). the supernatant was equilibrated with buffer a (10 mm tri-hcl, 500 mm nacl, 50 mm imidazole, 1 mm pmsf, ph 8.0). the bound protein was eluted with buffer b (10 mm tris-hcl, 500 mm nacl, 250 mm imidazole, 1 mm pmsf, ph 8.0) at 4°c. the purity of the purified protein was estimated by sodium dodecyl sulfate (sds)-page in the eluted fractions using 12% polyacrylamide running gels. 34 the purity of the enzyme was estimated by sds-page. the protein concentration was determined as described by the bradford method. 35 the purified sample was supplemented with 50% glycerol and stored at -20°c until use. the amine group of rna oligonucleotide was first covalently conjugated onto the surface of the carboxyl terminated qd605 (10 pm). that is, 10 pm of qd605 were conjugated with 400 pm of rna oligonucleotide with the coupling reagent edc (n-(3-dimethylaminopropyl)-n-ethyl-carbodiimide hydrochloride, 40 nm), which was used to activate an amide bond formation to produce qds-conjugated rna oligonucleotide (qds-based sars-cov n rna oligonucleotide) at a qds:rna oligonucleotide molar ratio of 1:40 for 1 hour at room temperature. qds-rna oligonucleotide conjugate was then collected using centrifugal filtration at 15 000 rpm for 30 minutes followed by several washing steps with a tris buffer (50 mm tris-hcl [ph 7.4], 5 mm kcl, 100 mm nacl, 1 mm mgcl 2 , and 0.1% nan 3 ). after centrifugal filtration and washing, the pellet of qds-rna oligonucleotide was dispersed by brief sonication (22 khz, amplitude 12 µm, and sonication time 120 seconds) using a sonic dismembrator model f60 (fisher scientific). the recombinant sars-cov n protein was directly immobilized onto the functional prolinker tm -terminated surface. for the binding of the specific rna oligonucleotide, the conjugated qds-conjugated rna oligonucleotide was facilitated by spotting on an immobilized sars-cov n protein chip. subsequently, the polyphenolic compound used as inhibitor was spotted on the conjugated rna oligonucleotide and the sars-cov n protein. after incubation for 1 hour at 25°c, the chip was then washed three times with phosphate-buffered saline (ph 7.2) for 1 minute. the chip was analyzed by a confocal laser scanning microscope lsm 510 meta (carl zeiss, jena, germany). the signal intensity was determined by software for the lsm 510 (lsm image browser; carl zeiss). a histogram of the intensity was achieved from the region of the spotted chip. the value of signal intensity was achieved by calculating and expressing it as the mean intensity. for the inhibitor screening of the sars-cov n protein, we designed the qds-conjugated specific rna oligonucleotide for specific sars-cov n protein targeting: first, the submit your manuscript | www.dovepress.com sars-cov n protein (1 µl) was immobilized on a glass chip; second, qds-conjugated rna oligonucleotide conjugates (1 µl) were bound on an immobilized chip; third, inhibitor treatment was performed on the conjugated rna oligonucleotide and sars-cov n protein; fourth, washing and unspecific binding removal was done; fifth, detection was achieved to show directly the specific recognition of the inhibition effect of sars-cov n protein on the biochip. the schematic design of the inhibitor screening for effective monitoring of sars-cov n protein is illustrated in figure 1 . to accomplish the feasibility of targeting and imaging, we used qd605 conjugates having an rna oligonucleotide for sars-cov n protein with an emission wavelength as the optical imaging probe. secondary structure of rna oligonucleotide and expression and purification of sars-cov n protein figure 2a presents the secondary structure of rna oligonucleotide that binds to sars-cov n protein. the rna secondary structure of the used rna oligonucleotide was analyzed using the mfold program. 36 in order to improve the solubility of the overexpressed recombinant sars-cov n protein, the coexpression of n protein with e. coli molecular chaperone groes/el was performed. the sars-cov n protein was purified by a single chromatography step on a ni 2+ affinity column. the c-terminal his-tagged sars-cov n protein was visualized with a molecular mass of approximately 48 kda on a sds-page ( figure 2b ). the sequence is 5′-gggagagcggaagcgugcugggccugucgguucgcugucuugcuacguuacguuacacgguuggcauaacccagaggucgauggaucccccc-3′. the qds-supported rna oligonucleotide was conjugated in the reaction for the amide formation from the coupling of 5′-end-amine-modified rna oligonucleotide at the surface of qds displaying carboxyl groups via standard edc coupling. the qds-conjugated rna oligonucleotide was confirmed on a 2.5% agarose gel at 100 v in tae buffer. agarose gel electrophoresis showed a band with mobility having a different band pattern between the free qds and the qdsconjugated rna oligonucleotide, which confirmed the formation of qds-conjugated rna oligonucleotide. the mobility shifts were compared (data not shown). on agarose gel, the qds-conjugated rna oligonucleotide showed a lower mobility shift than free qds, thus demonstrating the amide formation between qds and rna oligonucleotide for conjugation. in table 1 , the effects of polyphenolic compounds on the inhibition of the sars-cov n protein used in this study are described. among the polyphenolic compounds screened, (-)-catechin gallate and (-)-gallocatechin gallate showed high anti-sars-cov n protein activity. the chemical structures of (-)-catechin gallate and (-)-gallocatechin gallate are shown in figure 3 . figure 4 shows our elucidation of (-)-catechin gallate; (-)-gallocatechin gallate showed high inhibition activity in a concentrated manner against sars-cov n protein. (-)-catechin gallate and (-)-gallocatechin gallate, at 0.005 µg ml -1 or more, concentrationdependently attenuated the binding affinity as evidenced by qds-rna oligonucleotide on the designed biochip ( figure 4a and b) . at a concentration of 0.05 µg ml -1 , (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a qds-rna oligonucleotide biochip platform. as shown in figure 4a and b, (-)-catechin gallate and (-)-gallocatechin gallate showed a similar pattern when comparing the concentration-dependent anti-sars activity. the half-maximal inhibitory concentration (ic 50 ) values of (-)-catechin gallate and (-)-gallocatechin gallate were found to be approximately 0.05 µg ml -1 , respectively ( figure 4a and b). other polyphenolic compounds to the inhibition of sars-cov n protein on the nanoparticle-based rna oligonucleotide biochip system were detected as nearly similar to the background signal, due to the high affinity with the qds-conjugated aptamer-sars-cov n protein (data not shown). to perform high-throughput screening of the inhibitors, it would be efficient to be able to measure the anti-sars activity from optical images of a biochip containing multiple reaction compounds. to demonstrate the feasibility of the inhibitor assays, the latter were carried out on a biochip within 60 minutes at 37°c, and optical images were obtained from the reaction mixtures on a plate using a qds-based imaging system. the inhibition of the anti-sars activity from (-)-catechin gallate and (-)-gallocatechin gallate were clearly illustrated, and dose dependency was distinctly observable in the optical images. we demonstrated inhibitor screening on a biochip platform using a qds-conjugated rna oligonucleotide. we discovered a novel function of (-)-catechin gallate and (-)-gallocatechin gallate as anti-sars agents. the discovery of anti-sars agents has been of considerable interest in developing an efficient and effective methodology for high-throughput screening. our main goal in this study is to demonstrate a proof-of-concept that sars-cov n protein can be inhibited and detected with remarkable simplicity and speed. regarding its application, this designed platform for a novel inhibitor assay possesses significant potential as a target screening. in particular, it is a promising method for inhibitor screening because of its high sensitivity, low cost, rapid response, compatibility for miniaturization, and low labor-intensity. in addition, this platform is expected to be applicable to the inhibitor screening of other types of diseases. quantitative and sensitive detection of sars coronavirus nucleocapsid protein using quantum dots-conjugated rna aptamer on chip coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the spike protein of sars-cov -a target for vaccine and therapeutic development are drugs for sars on the horizon? severe acute respiratory syndrome (sars): multi-country outbreak the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (n) and membrane (m) proteins of sars coronavirus a major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein identification of a novel coronavirus in patients with severe acute respiratory syndrome detection of sars coronavirus in plasma by real-time rt-pcr rapid detection of severe acute respiratory syndrome (sars) coronavirus by a loop-mediated isothermal amplification assay detection of specific antibodies to severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein for serodiagnosis of sars coronavirus pneumonia detection of sars coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay molecules of interest genistein effects of flavanoids on immune and inflammatory cell functions metabolism of isoflavones in human subjects flavonoids from carnation (dianthus caryophyllus) and their antifungal activity therapeutic potential of inhibition of the nf-κb pathway in the treatment of inflammation and cancer antimicrobial activity of flavonoids flavonoids: modulators of brain function? green tea and skin cancer: photoimmunology, angiogenesis and dna repair the unfolded protein response regulator grp78/bip as a novel target for increasing chemosensitivity in malignant gliomas neurodegeneration in autoimmune demyelination: recent mechanistic insights reveal novel therapeutic targets epigallocatechin gallate, the main polyphenol in green tea, binds to the t-cell receptor, cd4: potential for hiv-1 therapy how can (-)-epigallocatechin gallate from green tea prevent hiv-1 infection? mechanistic insights from computational modeling and the implication for rational design of anti-hiv-1 entry inhibitors flavonoids activated caspases for apoptosis in human glioblastoma t98g and u87mg cells but not in human normal astrocytes targeting cwr22rv1 prostate cancer cell proliferation and gene expression by combinations of the phytochemicals egcg, genistein and quercetin induction of apoptosis in human bladder cancer cells by green tea catechins cleavage of structural proteins during the assembly of the head of bacteriophage t4 a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding structural analysis by energy dot plot of a large mrna this research was supported by the basic science research program through the national research foundation of korea (nrf), which is funded by the ministry of education, science and technology (grant no 2011-0010634). the author reports no conflicts of interest in this work. key: cord-279443-2e4gz2bo authors: khan, suliman; liu, jianbo; xue, mengzhou title: transmission of sars-cov-2, required developments in research and associated public health concerns date: 2020-06-09 journal: front med (lausanne) doi: 10.3389/fmed.2020.00310 sha: doc_id: 279443 cord_uid: 2e4gz2bo severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is rapidly spreading across the world to cause thousands of mortalities each day. poor responses from the authorities to the spread of infection, lack of effective measures for prevention, unavailability of promising treatment options, and sufficient diagnostic options have created an alarming for the world. the transmission routes from human to human of sars-cov-2 can be the direct transmission, droplet inhalation transmission, contact transmission, transmission through saliva, and transmission via fecal–oral routes. due to the asymptomatic spread of sars-cov-2's, developing control and prevention measures is challenging. implementing proper strategies addressing the infection control and clinical supplies, understanding the mechanism associated with pathogenesis, advancing in preventive measures and effective treatment and diagnostic options are necessary to control the ongoing pandemic. in this article, we briefly discuss the features, entry mechanism, infectiousness, and health consequences related to the covid-19 outbreak. introduction sars-cov-2 has infected over five million people worldwide after its emergence in wuhan, china (1) . the world has witnessed that this virus can spread rapidly to cause the death-causing covid-19 disease. although the rate of recovery is higher in people with strong immune responses, however, the immune-compromised individuals are at higher risks to be readily killed by the infection (2) . the major reasons for higher morbidity and mortality rates are rapid human-human transmission, unavailability of promising diagnostic and therapeutic options, scarcity of clinical supplies, shortage of medical and clinical staff, and lack of effective preventive measures (3) . besides the physical illness, the covid-19 epidemic has also increased the risk of psychological problems among healthcare workers, infected individuals, and the general public (3, 4) , due to the fear of treatment failure, higher morbidity and mortality, lack of psychological interventions, and infodemia (3, 5, 6) . during the early days of the epidemic in china, a number of countries suspended travel to and from china, evacuated their nationals from the epicenter, and placed them in quarantine to curb the risks of pandemic (6) . these responses were not sufficient to prevent the spread of covid-19, therefore, it became a global pandemic (7) . considering the seriousness of this situation scientists and medical researchers came forward and extended their services to the development of therapeutic strategies, preventive measures, and strategies to control the unfolding pandemic. until now, researchers have unveiled some of the important biological and clinical features for covid-19 infection, including the characterization of the whole genome (8) and spike glycoproteins (9) , investigation of clinical features and evaluation of different broad-spectrum antiviral drugs in combination with either antibacterial, antimalarial and/or traditional chinese medicines (10) . nevertheless, more research work is required to further investigate the sources of transmission, the biology of viral incubation and reemergence, and the potential of vertical transmission from mothers to neonates. in this article, we discuss the features of coronaviruses, the mechanism of infectiousness of sars-cov-2, and its medical consequences. we also describe the populations at higher risk and challenges in research progress. this narrative review article will benefit the public and scientific community regarding the current progress and the need for further work. to identify and select the papers in this review we searched the published research and review articles relevant to origin and outbreaks of three human coronaviruses, and features, transmission, spread, entry mechanisms, infectiousness, control strategies, and animals hosts for sars-cov-2. we also search the papers published on sars and mers coronaviruses in the aspects of animal models and sources of transmission. we reviewed the world health organization, u.s. centers for disease control and prevention, nature reports, medline, pubmed central, embase, google scholar, and sciencedirect, according to the relevancy as explained earlier, until april 20, 2020. the search terms "novel coronavirus, sars-cov-2 and covid-19, sars and mers" were broadly used. studies conducted in laboratory and clinical based observations, and/or conducted through bioinformatics techniques were included. pneumonia is one of the most frequent manifestations of covid-19 infection, which is characterized by fever, bilateral infiltrates on chest imaging, cough, and dyspnea (11) . the period from infection to symptoms appearance ranges from 2 to 14 days, while the average period reported so far is ∼5 days (12) . one of the previous studies reported the onset of fever and respiratory symptoms ∼3-6 days in a family cluster of infections (13) . similarly, in an analysis of 10 patients with confirmed covid-19 pneumonia, the estimated mean incubation period was 5 days (11) . furthermore, the majority of the individuals showed moderate symptoms whereas 20% of the infected patients showed severe illness of respiratory failure and septic shock and gastrointestinal complications (11, 13) . common laboratory abnormalities associated with covid-19 are lymphopenia and elevated aminotransferase levels (10) . creactive protein (crp) levels have been reported to alter with the development of symptoms, such that patients with severe pneumonia present high crp levels (10, 14) . in a recent study, wang (14) reported that crp levels at the early stage of covid-19 are positively correlated with lung lesions and symptoms development, which can be used as one of the key indicators for disease development and severity. wang et al. (10) investigated 138 patients [median age; 56 years, interquartile range; 42-68 years] with covid-19 pneumonia in wuhan and reported that 136 patients developed fever, 82 patients had a dry cough and 96 patients had fatigue. besides lymphopenia, parenchymal lung abnormalities were also common among all patients as depicted from computed tomography of the chest, including bilateral patchy shadows or ground-glass opacities. nonetheless, some people have been reported to be initially asymptomatic and may remain asymptomatic or go on to develop disease on later stages (who; march 23, 2020). although it is important to know about the symptoms' appearance and severity, however, understanding the transmission of the infection to healthy individuals from covid-19 patients and zoonotic sources can be of great importance in the aspects of developing strategies to prevent and control the spread of covid-19. during november 2002, a novel coronavirus caused sars epidemic in guangdong, china (15), followed by subsequent outbreaks in hong kong (15, 16) . this outbreak was reported to be caused by sars-cov, originated from market civets before its transmission and infection in humans (17) . by the end of the epidemic, sars-cov infected 8,098 people and caused 774 fatalities in 29 different countries (16) . later on, during june 2012 a patient infected with mers-cov developed severe pneumonia and died in jeddah, saudi arabia (16, 18) , following by series of clustered outbreak in the middle east and several other countries (16, 19) . before transmitting into humans, mers-cov originated and replicated in dromedary camels (17) . until 2020, mers-cov infected 2,468 individuals and caused 851 fatalities worldwide (20, 21) . in december 2019, clusters of patients reported with covid-19 caused by sars-cov-2 were epidemiologically found linked to animals and the seafood selling market in wuhan, china (22) . the zoonotic source of its origin and transmission is still debatable, however, some reports suggested bats (23) as the possible sources of transmission (9) . the human-to-human transmission of sars-cov-2 is thought to occur mainly via respiratory droplets produced by coughing or sneezing from an infected individual (24) . the rapid increase in suspected as wellconfirmed cases has also been inferred with viral transmission through the fecal-oral route and aerosol formation. the halflife on the surfaces of stainless steel, copper, and cardboard is ∼5.8 h, while that on the plastic surface is 6.8 h (25) . moreover, several reports have confirmed the asymptomatic transmission while there is a chance for the animal to humans transmission (26) . overall, these observations indicate that appropriate care is necessary while handling both confirmed and suspected individuals. moreover, the surfaces of potentially viruscontaminated places, objects, and containers should be cleaned with effective disinfectants. the sars-cov-2 contains a single-stranded rna with 29,891 nucleotides, encoding for 9,860 amino acids (27) . the spike glycoproteins of sars-cov-2 contain two subunits (s1 and s2) (8) . the s2 subunit contains transmembrane and cytoplasmic domains along with fusion peptide. novel coronavirus has over 80% identity with sars-cov. however, spike receptor-binding domains (rbd) are only 40% identical (28) , while structural elements open reading frame (orf)3b and orf8 were found with no homology (29) . coronaviruses contain six orfs regions which serve as templates for the production of sub-genomic mrnas and encode protein, spike, nucleocapsid, and membrane proteins. orfs are responsible for the production of pp1a and pp1ab polypeptides (30) . both sars-cov and mers-cov infect bronchial epithelial cells and type ii pneumocytes through ace2 and cd26 receptors, respectively (17, 31, 32) . the mechanism associated with the infectiousness of sars-cov-2 is yet to investigate, however, it likely infects the bronchial cells through ace2. in general, a virus entry to the host cell comprises a series of fundamental interactions; (i) binding to a target host cell via cellular receptors; (ii) fusing the envelope with a cellular membrane; and (iii) forking over its genetic material inside the cell (figure 1 ). the process of viral genomic delivery of nucleic acids into the host cell is highly dependent upon binding specificity to receptors, proteolytic activation, and endocytosis efficiency (33, 34) . coronaviruses demonstrate a great degree of plasticity regarding the entry pathways, which can occur at the plasma membrane or through the endocytic pathway (35) (figure 2) . the entry to the host cell process of sars-cov-2 is regulated by glycosylated spike (s) fusion protein and host receptor known as ace2. the s proteins is capable of significant structural rearrangement thus, play a crucial role in fusing the viral membrane with the host cell membrane (36) . this fusion process sparks off with binding of the s1 subunit to ace2 and is linked with the accessibility of receptor determined by hingelike conformational movements of the receptor-binding domain (rbd) of s1. thus, rbd can transiently hide or expose the determinants of receptor binding through receptor-inaccessible state or receptor-accessible state, respectively (37) . once the virus has entered to the host cell, the replication-transcription complex (rtc) is organized in double-membrane vesicles to initiate transcription of polyprotein 1a/1ab (pp1a/pp1ab). these pp1a/pp1ab proteins encode chymotrypsin-like protease (3clpro), main protease (mpro), and papain-like proteases for the production of non-structural proteins (nsps) (28) . transmembrane helical segments in the orf1ab region encodes for nsp2 and nsp3 (38) . the structural proteins and nsps play a role in the pathogenicity of sars-cov-2 by blocking the innate immune response and assembly and release of newly synthesized virions (39) . during the first days of the wuhan epidemic, two strains of novel coronavirus were reported namely s strain and l strain. observations suggested that l strain was more aggressive and more fatal as compared to s strain. a group of researchers from pasteur institute shanghai and peking university reported that the rate of infection for l strain was as high as 70%, while that of s was ∼30% as indicated by the analyzed samples. on the other hand, s type strain was found to be the ancestral version and was closely related to viruses like tg13. further analysis based on population genetics indicated that these strains mainly differed at orf1ab and orf8 regions. interestingly, the development of new variations of the spike protein in sars-cov-2 variants is linked to mutations, and natural selection (40) . therefore, further studies should evaluate the combinational impacts of genomic data, epidemiological data, and chart records of the clinical symptoms of patients with covid-19. after the identification of sars-cov-2, debates started among scientists on its sources of origination and zoonotic source of transmission to humans (29) . the identity of the animal source of sars-cov-2, is still one of the key missing gaps that scientists are being racing to investigate. it is a known fact that coronaviruses circulate in mammals and birds (17) , and researchers have already suggested bats to be the source of origination for sars-cov-2 (23). however, an intermediate animal was probably the source of transmission of the virus to humans. early claims came figure 2 | the sars-cov-2 transmission from bats via unknown intermediate to humans causes infectiousness known as covid-19 disease. the binding of s protein to ace2 receptor initiates the life cycle which is then followed by conformational changes in the s protein, which further facilitates the fusion of viral envelope and host cell membrane. following the fusion through endosomal pathway, sars-cov-2 then releases rna into the host cell, which is translated into pp1a and pp1ab. next, viral proteinases cleave the translated proteins into small products, meanwhile a series of sub-genomic mrnas are produced by polymerase enzyme through discontinuous transcription, which are then translated into specific viral proteins. these viral proteins and genome rna are assembled to form virions in golgi and endoplasmic reticulum, which are later transported out of the cell via vesicles. this figure was designed by updating and modifying the information from our previously published paper (29) . frontiers in medicine | www.frontiersin.org from researchers related to intermediate sources of transmission faced controversies (9) . a recent report discredited an earlier statement that pangolin could be the possible intermediate source that might have received the virus from the bat and transferred it to humans (40) . according to more recent study on molecular and phylogenetic analyses, it is unlikely that sars-cov-2 emerged directly from the pangolin coronaviruses (41) , suggesting that pangolins may not be responsible for the transmission of sars-cov-2 to humans. with further spread of the virus after its outbreak in wuhan, more people became infected, thus, human to human transmission became more evident. one of the reasons for the high rate of infectiousness in humans is thought to be the higher affinity of rbd for binding to ace2 receptors (29, 42) . in addition, the determination of host range and binding to the ace2 are highly dependent on six rbd amino acids "l455, f486, q493, s494, n501, and y505 in sars-cov-2" in sars-cov-2, thus, rbd can also bind to ace2 from ferrets and cats (42) . on the other hand, the highaffinity of rbd to human ace2 is thought to be linked with natural selection on a human ace2, indicating that sars-cov-2 was not produced with purposeful manipulation (42) . these observations support the hypothesis that sars-cov-2 was transmitted from a yet unknown intermediate zoonotic source to humans. spike glycoproteins have been well-documented in the aspects of transmission and entry of sars-cov-2 into host cells (13, 28) . it is notable that the polybasic cleavage site of sars-cov-2 at the junction of s1 and s2 allows cleavage by proteases such as furin, which plays a crucial role in infectiousness and determining host range. despite the unknown functional consequence, the higher genetic variation in spike indicates that sars-cov-2 with polybasic cleavage sites may be discovered in several other species (42, 43) , which can be the possible source of transmission for sars-cov-2 to humans. interestingly, the mutation found in the polybasic cleavage site was not related to that of the bat and pangolin viruses (42) , therefore, it may be linked with the virus's ability for transmission and infection in humans. the determination of polybasic cleavage and predicted o-linked glycans further suggest that the virus was most likely transferred from an animal with ace2 to humans, as these are not possible in cell cultures (42) . further research to determine the impact of polybasic cleavage and predicted o-linked glycans on transmissibility and pathogenesis is necessary. although investigating the mechanisms underlying entry to host cell, transmission, polybasic cleavage, and predicted olinked glycans are required to determine the research gaps associated with transmission and origination, however, this work requires suitable animal models. unfortunately, there is no promising model while the non-human primates tested for sars and mers were unable to develop severe diseases in response to the infectiousness (44) . nevertheless, the models developed for the expression of human ace2 and dpp4 (16) can be further modified and used to study the transmission and infectiousness of sars-cov02. moreover, crispr-interceded genetically modified small animals can be also utilized for the study of the pathogenicity of sars-cov-2. nevertheless, it is important to investigate the ultimate source of viral transfer to humans, as even if the virus is eradicated with social distancing, other sources including zoonotic and environmental sources can again cause the transfer into humans, and thus another outbreak will be the result. the ability of rapid human to human transmission of covid-19 infection especially through asymptomatic infected individuals and aerosol, has paralyzed life across the globe (29, 43) . although the covid-19 infection primarily affects physical health, however, it can also affect mental health through the fear of transmission from unknown sources and high mortality rate that can further paralyze life (5) . it is deemed necessary that timely effective services should be provided to the vulnerable populations as reported by khan et al. (5) . the adverse impacts of covid-19 are specific to the populations, therefore, we discuss the most vulnerable populations, the current evidence on known vulnerable groups and the associated health risks in response to the covid-19 infection. rapidly increasing mortalities and morbidities in healthcare workers are causing serious medical concerns and adversely affecting healthcare services worldwide (3) . the fear of being infected due to close contacts with infected symptomatic and asymptomatic patients, and prolonged working schedules may decrease the working efficiency in current doctors and nurses (3, 7) . a large number of medical and clinical staff are likely to be infected with covid-19 infection. only in wuhan, more than 15 hundred persons from healthcare settings were reported infected (3) . in addition to the high risk of contracting infection due to direct interaction with infected and suspected individuals (3), healthcare workers have also been reported to develop severe mental conditions including stress, anxiety, and related mental illnesses (3, 4) . to mitigate the risk of contracting infection the medical staff should adhere to standard precautions while providing patient care (45, 46) . according to the cdc report on coronavirus disease, individuals with underlying chronic medical conditions are at higher risk for contracting covid-19 infection. huang et al. reported that 32% of sars-cov-2 infected individuals had diabetes, hypertension, and cardiovascular disease (11) . the fatality rate was also high in individuals who had diabetes mellitus, chronic lung disease (47), cerebrovascular diseases (48) , and hypertension (47) . furthermore, covid-19 infection in patients with lung cancer can develop severe covid-19 disease that can lead to death (49) . luo et al. (49) reported that more than half of the covid-19 infected individuals who had lung cancer, needed hospitalization, whereas nearly a quarter of them died. however, people living with human immunodeficiency virus do not present excess morbidity and mortality among symptomatic covid-19 patients (50) . the higher risk of disease and death in individuals with underlying diseases might be linked with weaker or comprised immune responses. the elder individuals are comparatively more affected by covid-19 infection; however, individuals of any age can acquire the infection (51) . according to the previous reports, 87% of infected individuals were between 30 and 79 years old. moreover, the mortality rate was higher in older people. the case fatality rate of 8% was observed among individuals having age between 70 and 79 years, while 15% fatality rate was reported in people with 80 years or older (47) . covid-19 infection in pregnant women is of serious concern, as it might have detrimental effects not only on mother's health but also on neonatal health can be at risk (52) . in a recent study, covid-19 infection was found to cause adverse neonatal outcomes. two of the neonates were tested positive, and for covid-19 while, five were found with neonatal pneumonia, suggesting the possibility of a link between adverse pregnancy outcomes and covid-19 infection (52). dong et al. (53) reported a newborn with elevated igm antibodies to sars-cov-2, who was born to a mother with covid-19, suggesting the possibility of vertical transmission. therefore, further investigations should focus on adverse pregnancy outcomes and the possibility of vertical transmission. the approach to prevention, evaluation, diagnosis, and treatment of pregnant women with suspected covid-19 should be similar to that in non-pregnant individuals, with the consideration that pregnant women with other potentially severe respiratory infections, such as influenzaappear to be more vulnerable to developing severe sequelae. moreover, pregnant women should be given attention and provided with the utmost facilities in terms of treatment and diagnosis. controlling the spread and transmission of infection is one of the major issues that authorities are currently considering with serious attention. world health organization (who) and u.s. centers for disease control and prevention (cdc) recommend face and eye protection for droplet and contact precautions. during aerosol-generating procedures, such as non-invasive ventilation, tracheotomy, cardiopulmonary resuscitation, tracheal intubation, bronchoscopy, and manual ventilation before intubation, additional precautions are warranted such as airborne infection isolation room and wearing the appropriate personal protective equipment (ref 1 and ref 2) . to control the transmission requires the identification and isolation of the infected individuals. samples from the nasopharyngeal swab, oropharyngeal swab, sputum, tracheal aspirate, or bronchoalveolar lavage should be tested for the detection of the virus (54) . the symptoms of covid-19 pneumonia are primarily similar to influenza and seasonal allergies (10, 55, 56) , therefore using thermo-scanners and physical observations are not are not able to adequately differentiate between those conditions. although quantitative real time polymerase chain reaction (qrt-pcr) is the major confirmatory test however, to provide further testing support developing additional testing kits that could rapidly detect sars-cov-2 with maximum accuracy in suspected, confirmed, and asymptomatic patients may be useful. to control the ongoing pandemic and risk of future epidemics, the development of safe and effective vaccines is necessary, that should be available for individuals at high risk of contracting covid-19 infection. until now, effective vaccine against covid-19 is not available, however, some vaccines with preventive potential against covid-19 infection are in pipeline. such as the mrna-based vaccine developed by national institute of allergy and infectious diseases in usa, is being trialed (57) . while the ino-4800-dna based vaccine is currently being developed (57) . moreover, center for disease control and prevention (ccdc) in china has started working on inactivated virus vaccine that may be used widely if found promising (57, 58) . stermirna therapeutics has reported the development of mrna-based vaccines that can soon be available for trials (57, 58) . nevertheless, more work is required; sars-cov specific live-attenuated (16) and rhesus θ-defensin 1 and protein cage nanoparticles based vaccines can be evaluated for covid-19 infection (59, 60) . moreover, monoclonal antibodies should be considered that are effective in inhibiting virus-cell receptor binding and virus-cell fusion (16) . sars-cov-2 is likely originated in bats and introduced to the world through a yet unknown intermediate. without finding the missing intermediate, sars-cov-2 may reemerge even if the current spread is controlled completely through social distancing and isolation. an earlier report that indicated pangolins as the possible source of transmission of sars-cov-2 has been discredited, therefore, further work is required to identify the unknown intermediate animal source that caused the transmission of the virus to humans. based on their role in transmission and infectiousness, spike glycoproteins, rbd binding to ace2 and mutations in polybasic cleavage sites related to different animals should be studied further. given the importance of the current outbreak in wuhan, further studies are necessary to provide deep understating of replication, pathogenesis, and biological properties using the relevant biological techniques such as reverse genetics and molecular techniques. to unveil pathogenesis and entry mechanisms further investigations should focus on structural elements orf3b and orf8 in novel coronavirus. these regions may play an important role in high human to human spread and may be linked to the severity of the disease. these investigations will help the control and prevention of covid-19 mediated pneumonia and novel emerging diseases in the future. the covid-19 outbreak has affected millions of people around the globe by causing mortalities and morbidities. thus, curbing covid-19 and preventing it from spreading further requires the 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for the 2019 novel coronavirus outbreak -united states clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records first case of 2019 novel coronavirus in the united states covid-19: learning from lessons to guide treatment and prevention interventions. msphere rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 khan, liu and xue. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-284498-54j6ys8s authors: ihsanullah, ihsanullah; bilal, muhammad; naushad, mu. title: coronavirus 2 (sars-cov-2) in water environments: current status, challenges and research opportunities date: 2020-10-16 journal: nan doi: 10.1016/j.jwpe.2020.101735 sha: doc_id: 284498 cord_uid: 54j6ys8s the outbreak of covid-19 has posed enormous health, social, environmental and economic challenges to the entire human population. nevertheless, it provides an opportunity for extensive research in various fields to evaluate the fate of the crisis and combat it. the apparent need for imperative research in the biological and medical field is the focus of researchers and scientists worldwide. however, there are some new challenges and research opportunities in the field of water and wastewater treatment concerning the novel coronavirus 2 (sars-cov-2). this article briefly summarizes the latest literature reporting the presence of sars-cov-2 in water and wastewater/sewage. furthermore, it highlights the challenges, potential opportunities and research directions in the water and wastewater treatment field. some of the significant challenges and research opportunities are the development of standard techniques for the detection and quantification of sars-cov-2 in the water phase, assessment of favorable environments for its survival and decay in water; and development of effective strategies for elimination of the novel virus from water. advancement in research in this domain will help to protect the environment, human health, and managing this type of pandemic in the future. the current global pandemic of covid-19 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been growing briskly [1, 2] . although the major transmission routes of sars-cov-2 are through respiratory droplets and direct contact [3] [4] [5] , recent studies have reported the presence of viral rna of sars-cov-2 in untreated and treated wastewater and human feces [3, [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . in most of the studies, the samples were collected from wastewater treatment plants (wwtps), while one study reported in the presence of sars-cov-2 in wastewater from a cruise ship and commercial passenger aircraft [22] . all the reported samples were collected during the period from janruary 2020 to july 2020. recent studies conducted in j o u r n a l p r e -p r o o f the netherlands and france confirmed that a reasonably high viral load of sars-cov-2 rna is found in the sewage/wastewater [19, 23] . the presence of sars-cov-2 in feces and municipal wastewater poses a severe threat to the environment due to its potential spread via these routes [24] [25] [26] [27] [28] . therefore, there are serious concerns regarding the spread of sars-cov-2 through virus-laden aerosols-borne and fecal-oral routes [8, [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] . it is important to take the necessary precautions to limit the spread of the virus in the environment [39] . nevertheless, it creates some new environmental challenges that demand an imperative need for research. development of effective standard techniques for the detection and quantification of sars-cov-2 in water, assessment of the existing water purification technologies and development of novel advanced water treatment systems are major challenges and open research opportunities. furthermore, careful surveillance of water and wastewater to be used as an early warning tool for such outbreaks in future, understanding the survival and decay mechanism of the novel virus in water and wastewater, analysis of potential pathways of sars-cov-2 into water bodies are other potential research opportunities for environmental researchers [40] [41] [42] [43] [44] . the major challenges and opportunities in the field of water and wastewater research are presented in fig. 1 . the objective of this article is to highlight the potential opportunities and challenges in the field of water research concerning the novel sars-cov-2 virus. the directions for future research to safeguard human health, environment, predict and manage this type of pandemic in the future, are also provided. j o u r n a l p r e -p r o o f numerous studies have reported the presence of pathogenic viruses that enter into the water bodies through different sources [45, 46] . previous studies have demonstrated that sars-cov-2 can enter into the water bodies from hospital wastewater and sewage [5, [47] [48] [49] . the waste and wastewater discharged from the quarantine facilities, airports/seaports, residential buildings of infected humans and animals, are the potential sources of sars-cov-2 than can enter into the water bodies. the sars-cov-2 may also find its ways to groundwater through possible leaching and infiltrations of effluents from health care facilities, sewage, solid landfill and drainage water. another important source of viral contamination to the water environment is the leakage from sewage pipes. the possible sources and pathways of sars-cov-2 in water systems are depicted in fig. 2 . due to the fast-growing number of patients worldwide, the virus concentration in wastewater/ sewage is expected to increase rapidly. the potential pathways of sars-cov-2 into wastewater are via shedding in the stool (for the viable virus), while through urine and stool (for the nonviable virus) [50] . a careful and extensive investigation of all the potential sources of water contamination by sars-cov-2 is needed that potentially includes wastewater dispatches from hospitals and greywater from washing personal protective equipment (ppe), surfaces and floors [51] . table 1 summarizes the literature reporting the presence of sars-cov-2 rna in wastewater, and untreated water in various parts of the world [3, 17, 19, 42] . the concentrations of sars-cov-2 rna in untreated wastewater was in the range of 5.6 × 10 to 4.6 × 10 8 copies per liter [6, 7, 14, 16, 52, 53] . some studies also reported the detection of sars-cov-2 in treated water samples [3, 16] . previous reports suggested that sars-cov-2 survival in an aqueous environment is strongly dependent on the characteristics of water/wastewater [54] [55] [56] . the ph, temperature, presence of antagonistic bacteria, organic matter, sunlight and oxidants might affect the survival of sars-cov-2 in aqueous environmmnet [54, 57, 58] . a previous study reported that the inactivation of coronaviruses in the water was highly dependent on the level of organic matter, temperature, and presence of antagonistic bacteria [54] . the suspended solids and organic matter present in water can provide protection for viruses that adsorb to these particles, and they can survive up to several days [54] . however, some published reports suggested that coronaviruses are very sensitive to high temperature and oxidants, such as chlorine [57] . studies have reported that surrogate coronaviruses remained infectious in water and sewage for days to weeks [46] . a recent study estimated that the half-life of sars-cov-2 in wastewater is in the range of 4.8 and 7.2 h [44] , while another study reported that the virus causing covid-19 could survive in untreated wastewater from hours to days [59] . it is essential to understand the stability and decay mechanism of sars-cov-2 in water and wastewater. the significant parameters that can predict the reduction kinetics need to be determined, to establish the favorable and unfavorable conditions for the survival of sars-cov-2 in aqueous environment. detailed investigations of various water/wastewater samples with different characteristics are required to assess the potential exposure risk of water contaminated with this virus. launching a monitoring program is vital to determine the fate of sars-cov-2 water cycle [49] . theoretical and computational analysis might help to provide a basis for experimental research. until now, there is no evidence and enough data to confirm if the water and wastewater containing sars-cov-2 could be the potential source of its transmission. although some studies predicted a low risk of sars-cov-2 transmission via wastewater (especially the treated water) [55, 60] , still extensive investigations need to be performed to validate these predictions and inial findings. the information regarding the viability of the closely related sars-cov-1 virus in wastewater may provide useful information about the survival of sars-cov-2 in an aqueous environment [54] . as a precaution, the wastewater/sewage treatment plants should be considered as the potential transmission routes. a recent study demonstrated the quantitative microbial risk assessment (qmra) approach to investigate the potential health risks of sars-cov-2 in sewage to wwtps workers [61] . results revealed that the viral loads in wastewater/sewage at the entrance of wwtps were above the who benchmark of tolerable risk used for virus infection of 10 -3 [6, 7, 14, 16, 52, 53] . it is essential to evaluate bioaerosol and airborne particle risks for nearby communities and wwtp workers [62] . in general, it can be made mandatory for the wwtp workers that perform manual cleaning of screening use face masks and face shields. the aerosol formation during wastewater treatment needs to be carefully examined as a potential source of virus transmission [62] . water-related exposures may occur in communities with weak sewage infrastructure, or that use wastewater for irrigation [63] . viral aerosols from a leaking sewage pipe at amoy gardens complex in hong kong were identified as the transmission route for coronaviruses during the sars-cov-1 outbreak in 2003 [47] . the virus can also make its way into the drinking water distribution systems from the accidental contamination of drinking water with raw sewage and can enter individual homes. the presence j o u r n a l p r e -p r o o f of sars-cov-2 in water may also affect the water supply system by forming the biofilms that can compromise the water quality and endanger human health. there is also an imperative need for research to investigate the fecal-oral transmission potential of sars-cov-2 [33, 64] . it is crucial to examine if the sars-cov-2-contaminated wastewater has adverse impacts on aquatic life, soil, and wildlife. this needs careful examinations, risk analysis and thorough investigations to reach a conclusion. furthermore, monitoring and control measures are essential along the wastewater treatment route to avert coronavirus spread. it is recommended that the wwtp managers and stakeholders develop risk management strategies for the protection of wwtp workers and nearby communities [61] . the disinfection steps in both wwtps and drinking water plants need to be developed carefully. the quantitative disinfection kinetics need to be established, and an optimum dose for inactivation of sars-cov-2 need to be determined. decentralized virus inactivation treatment for waster discharged from wwtps and drinking water plants can also be beneficial in reducing the environmental load of sars-cov-2. detection and quantification of sars-cov-2 in water and wastewater is an essential but challenging step to track the infectious disease [50, 65] . due to complex nature of wastewater matrix, it is essential to develop new biomarker extraction techniques as well as selective, sensitive, and cost-effective tools for the analysis of wastewater samples containing the novel virus [66] . to avoid exposure to dangerous virus sars-cov-2 and to protect the safety of the lab analysts, it is recommended to use virus surrogates instead of harmful sars-cov-2 [34] . wastewater-based epidemiology (wbe) has been recognized as a useful tool for evaluating, predicting and managing the disease outbreaks [40, 43, 44, 67] . the concept is mainly based upon the detection, extraction, and analysis of biological and chemical compounds (referred to as biomarkers). recent studies have reported the successful detection of the novel virus in municipal wastewater and human stool [6, 7, 15, 42, 51, [68] [69] [70] . surveillance of relative changes in concentrations of sars-cov-2 rna at the inlet of wwtp over time can serve as a useful tool for early warning for virus spread in the population [19] . both upstream sampling (i.e., at sewerage maintenance holes) and downstream sampling (i.e., at the wwtp) approach can be used; however, upstream sampling is more appropriate due to variability in downstream samples. a gis-based sewerage map and flow rates would aid in the selection of upstream sampling locations [71] . quantification of sars-cov-2 rna in wastewater settled solids in wwtps may also be used as a reliable and sensitive target for wbe [72] . moreover, monitoring of international airports and seaports sewage would allow very early detection of the entrance of the virus into a country. it could also help the relevant authorities in deciding the implementation or removal of lockdown [34] . it is essential to develop an integrated wastewater surveillance programs by considering both privacy and inequality concerns of the public [71] . wurtzer et al. [73] performed the time-course quantitative analysis of sars-cov-2 in paris sewage by rt-qpcr to investigate virus circulation in humans. however, these studies are at the initial stage, and a large pool of data from different parts of the world is necessary to develop a reliable and sensitive detection method for sars-cov-2 detection in wastewater/sewage [74] . a multidisciplinary research approach, including engineers, microbiologists, chemists, and public health experts, will be productive to nurture more effective techniques for the quantification of sars-cov-2 in water. since sars-cov-2 has a presumably short half-life in water, the detection technique must be valid for both viable and non-viable sars-cov-2 in water. the virus can be degraded into other products; it is essential to develop strategies that could use these degraded products as target materials for detection and quantification [50] . the lack of standardized and optimized protocol for the detection and quantification of sars-cov-2 in wastewater is another major challenge [59, 75, 76] . this may lead to discrepancies in the results obtained by different laboratories, as indicated by a recent study [3] . currently, the reverse transcription-quantitative polymerase chain reaction (rt-qpcr) has been employed widely for detection of sars-cov-2 in water samples [77] . there is an imperative need to develop a standard operating procedure for accurate detection and quantification sars-cov-2 in water and wastewater. furthermore, a standard sampling procedure must be developed to extract/isolate, detect and quantify the virus accurately. this is an essential step for the development of commercial laboratories in various parts of the world that can accurately detect and quantify the virus with reproducible results. advancements in the field of microbiology might play a key role in developing a low cost, efficient method for detection and quantification of viral rna [78] . recently, various innovative detection techniques have been reported in the literature [79] [80] [81] . novel nanomaterials-based sensors were useful for the detection of waterborne pathogens [82] . there is a research potential to develop techniques using a similar approach for the detection of sars-cov-2 by utilizing j o u r n a l p r e -p r o o f different novel nanomaterials. a reliable widely-accepted surveillance system needs to be developed for the accurate quantification of sars-cov-2 in water samples. the persistence of sars-cov-2 in the water environment is assumed to be short due to the enveloped nature of the virus [49, 60, 83] . the half-life of sars-cov-2 in hospital wastewater is in the range of 4.8 and 7.2 h at 20 °c [44] . however, little is known about the vitality of sars-cov-2 in water [16] . furthermore, the accurate survival period and concentration of sars-cov-2 in water are still indefinite, and these are open areas for research. it is essential to understand the decay mechanism and breakdown products of sars-cov-2 in water and wastewater. it will not only help in developing effective techniques for accurate detection and quantification but also will be useful for proposing an efficient disinfection technique. a recent study reported the transcriptomic architecture of sars-cov-2 [84] . the sars-cov-2 may present in the water phase either in a viable state or in the non-viable form of viral debris [50] . however, the actual degradation of products and accurate decay mechanism of sars-cov-2 in water phase need to be explored yet. the traditional treatment techniques for the removal of viruses from wastewater include sand filters, chlorine treatment, uv inactivation, ozone treatment, microbial treatment, membranes and pond systems [85] [86] [87] [88] [89] [90] . though it is assumed that the current techniques used for wastewater treatment might be helpful to eliminate the novel virus; due to the global impact of this j o u r n a l p r e -p r o o f pandemic, experimental results are needed to validate this assumption [62, [91] [92] [93] . likewise, experimental shreds of evidence are required to confirm the effectiveness of household filtration systems, chlorination, densification and boiling for the elimination of the novel virus from water. it might be needed to upgrade the existing water and wastewater treatment systems or develop new treatment techniques to treat the sars-cov-2-contaminated water. the more stringent treatment above the current level is needed for virus reduction to ensure the safety of recycled water [94, 95] . who has highlighted guidelines on the safe management of water in july 2020 [62] . a recent study stated that the current disinfection strategy recommended by who might not be adequate to deactivate sars-cov-2 in water [96] . the existing wastewater treatment system might need necessary alternation or additional pretreatment steps to deactivate the sars-cov-2. it should also be investigated if the presence of sars-cov-2 in wastewater affect the treatment process for the removal of other pollutants. recent advancement in nanotechnology, biotechnology and material sciences have opened many doors of applications and exhibited tremendous potential in water purification [97] [98] [99] [100] [101] [102] . the development of effective techniques for the inactivation of sars-cov-2 is vital to limit its presence in wastewater and lessen its potential adverse effects on human health and the environment. significant treatment steps are essential to upgrade both the wastewater and drinking treatment pants to eradicate sars-cov-2 or its rna fragments. recent years have witnessed tremendous progress in the applications of various novel materials in water treatment [103] [104] [105] [106] . nanomaterials and their composites might play a critical role in the development of an efficient method for the eradication of sars-cov-2 from water [106] [107] [108] [109] [110] . advances in membranes systems provide an effective route for the removal of viruses from water [111] [112] [113] [114] [115] . for drinking water, a potable water straw or uv based system can be developed to filter/kill the virus and to reduce the risk of waterborne viral infection [116] . a recent study reported that there is a negative correlation between sunlight uv dose and percent positive of sars-cov-2 [117] . other techniques such as oxidation, coagulation and photocatalytic killing of the virus must also be explored to determine the effective treatment option for the deactivation of sars-cov-2 in water. the efficiency of emerging disinfection technologies for sars-cov-2 inactivation need to be appraised. the water distribution systems may potentially host the novel virus due to biofilm growth and presence of bacterial colonies. innovations and improvements in the water distribution and plumbing systems are vital to minimize the transmission of sars-cov-2 through the water [118] . as a general rule, it is the combination of different techniques in primary, secondary and tertiary treatments that would allow enough virus removal to limit the possibility of environmental contamination. the sars-cov-2 pandemic should develop a global awareness that solutions have to be found for improving the wastewater treatment even in developing countries, due to the possibility of its spread through the aquatic environment from inadequately treated wastewater. the presence of sars-cov-2 in wastewater may have consequences for public health in developing countries with poor water and sewage infrastructure, and inadequate institutional treatment/disinfection facilities. future research in these lines is essential for the protection of public health and the environment. in addition, robust policy intervention is essential to ensure reasonable compliance regarding the discharge of wastewater in various parts of the world. j o u r n a l p r e -p r o o f one of the major environmental concern associated with covid-19 pandemic is the excessive use of disinfectants (such as alcohol-based hand sanitizers and disinfecting soaps). the usage of disinfectants during the covid-19 pandemic increases environment and energy footprints significantly. it is important to carefully monitor the wastewater for possible commination and mitigate the spread of sars-cov-2. a recent report reveals that 2000 tons of disinfectants are dispensed at in sewage systems in wuhan city of china from january 29 to february 18, 2020 [119, 120] . these discharges not only poses a potential threat to the marine environment but can also contaminate drinking water resources. another study estimated that excessive use of disinfectants and frequent handwashing could increase consumption of drinking water by 20% and lead to the generation of 15-18% more wastewater [121] . the alcohol-based hand sanitizer is not only associated with some health issues, but it may also result in adverse impacts on the environment [122, 123] . a recent study highlighted that the extensive use of disinfectants poses a significant threat to urban wildlife [124] . these disinfectants may find their ways to reach water bodies and pollute them. it is, therefore, necessary to assess the exact hostile impacts of the disinfectants that discharge into water bodies and mitigate them to the maximum possible extent. in addition, the use of masks, gloves and other personal protective equipment (ppe) resulted in the generation of a massive amount of wastes in the environment. strategies for proper management of waste must be developed to lessen its adverse environmental impacts. in summary, besides severe threats to humanity, the ongoing pandemic offers the opportunity to environmental engineers and scientists to play a responsible role to control the potential spread multidisciplinary research, including engineers, microbiologists, chemists, and public health experts are needed to address these challenges and to safeguard the aquatic 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covid-19 surveillance and potential transmission risks postlockdown detection of sars-cov-2 rna in the wastewater of montpellier, france, one heal the authors declare that there is no conflict of interest during and after the study.the authors declare that there is no conflict of interest regarding the publication of this article j o u r n a l p r e -p r o o f (february-april) 2020 wastewater from wwtpsmilan and rome (italy) [17] april 2020 wastewater from wwtps istanbul (turkey) [18] june 2020 river water quito (ecuador) [20] (april-may) 2020 aircraft and cruise ship wastewater passenger aircraft flight from los angeles -brisbane hongkong -brisbane new delhi-sydney cruise ship (australia) [22] march 2020wastewater from the treatment facility massachusetts (usa) [42] (february-march) 2020sewage/wastewater from wwtps netherlands [51] may 2020 wastewater from wwtps gujarat (india) [53] april 2020raw and treated wastewater samples from wwtpsmilano metropolitan area italy) [60] (march-april) 2020 wastewater from wwtps paris (france) [73] february 2020 sewage from hospital sewage disinfection pool hospital of zhejiang university, china [92] may 2020 wastewater from wwtps, influent pump stations, or interceptor lines new york (usa) [125] (march-april) 2020 wastewater from wwtps different localities in israel [126] (february-april) 2020 wastewater from wwtps valencia (spain) [127] (march-may) 2020 secondary-treated wastewater yamanashi prefecture (japan) [128] (march-april) 2020 wastewater from wwtps ishikawa and toyama prefectures (japan) [129] april 2020 wastewater from wwtps ourense (spain) [130] (may-june) 2020 wastewater from wwtps jaipur (india) [131] (march-april) 2020wastewater from the drainage of covid-19 infected areas and quarantine centervarious districts in pakistan [132] (january-april) 2020 wastewater from wwtps southern louisiana (usa) [133] (april-june 2020) untreated wastewater from wwtps czech republic [134] april 2020 wastewater from wwtps north-rhine westphalia (germany) [135] (may-july) 2020 wastewater from wwtps montpellier (france) [136] key: cord-287658-c2lljdi7 authors: lopez-rincon, alejandro; tonda, alberto; mendoza-maldonado, lucero; mulders, daphne g.j.c.; molenkamp, richard; perez-romero, carmina a.; claassen, eric; garssen, johan; kraneveld, aletta d. title: classification and specific primer design for accurate detection of sars-cov-2 using deep learning date: 2020-09-10 journal: biorxiv doi: 10.1101/2020.03.13.990242 sha: doc_id: 287658 cord_uid: c2lljdi7 in this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in sars-cov-2. a convolutional neural network classifier is first trained on 553 sequences from available repositories, separating the genome of different virus strains from the coronavirus family with considerable accuracy. the network’s behavior is then analyzed, to discover sequences used by the model to identify sars-cov-2, ultimately uncovering sequences exclusive to it. the discovered sequences are first validated on samples from other repositories, and proven able to separate sars-cov-2 from different virus strains with near-perfect accuracy. next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets on existing datasets, obtaining competitive results. finally, the primer is synthesized and tested on patient samples (n=6 previously tested positive), delivering a sensibility similar to routine diagnostic methods, and 100% specificity. in this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in sars-cov-2. a convolutional neural network classifier is first trained on 553 sequences from ngdc, separating the genome of different virus strains from the coronavirus family with accuracy 98.73%. the network’s behavior is then analyzed, to discover sequences used by the model to identify sars-cov-2, ultimately uncovering sequences exclusive to it. the discovered sequences are validated on samples from ncbi and gisaid, and proven able to separate sars-cov-2 from different virus strains with near-perfect accuracy. next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets, obtaining competitive results. finally, the primer is synthesized and tested on patient samples (n=6 previously tested positive), delivering a sensibility similar to routine diagnostic methods, and 100% specificity. the proposed methodology has a substantial added value over existing methods, as it is able to both identify promising primer sets for a virus from a limited amount of data, and deliver effective results in a minimal amount of time. considering the possibility of future pandemics, these characteristics are invaluable to promptly create specific detection methods for diagnostics. the coronaviridae family presents a positive sense, single-strand rna genome. these viruses have been identified in avian and mammal hosts, including humans. coronaviruses have genomes from 26.4 kilo base-pairs (kbps) to 31.7 kbps, with g + c contents varying from 32% to 43%; human-infecting coronaviruses belonging to this family include sars-cov, mers-cov, hcov-oc43, hcov-229e, hcov-nl63 and hcov-hku1 1 . in december 2019, sars-cov-2, a novel, human-infecting coronavirus was identified in wuhan, china, using next generation sequencing (ngs) 2 . as of the 12 th august of 2020, the new sars-cov-2 has 20,162,474 confirmed cases across almost all countries, with 3,641,603 cases in the european region 3 . in addition, sars-cov-2 has an estimated mortality rate of 3-4%, and it is spreading faster than sars-cov and mers-cov 4 . as a typical rna virus, new mutations appears every replication cycle of coronavirus, and its average evolutionary rate is roughly 10-4 nucleotide substitutions per site each year 2 . in the specific case of sars-cov-2, rt-qpcr testing using primers in orf1ab and n genes have been used to identified the infection in humans 5 . this method has come into question; yang et al. in a study from 866 respiratory specimens showed that for 0-7 days after onset of illness, the sputum samples had a negative rate of 11.1% in severe and 17.8% in mild cases, follow by 26.7% and 27.0% in nasal swabs and finally 40% and 38.7% for throat swabs 6 . zhao et al. reports that 35.2% of 173 patients did not show positive in rt-pcr test 7 , which has been further explored by arevalo et al. 8 and woloshin et al. 9 . these problems could be the result of the variation of viral rna sequences within virus species, and the viral load in different anatomic sites 10 . it has been noted that, population mutation frequency of site 8,872 located in orf1ab gene and site 28,144 located in orf8 gene gradually increased from 0 to 29% as the epidemic progressed 11 . apart from the false negative test problems, sars-cov-2 assays can yield a small portion of false positives through nonspecific detection of other coronaviruses, as the virus is closely related to other coronavirus organisms 12 . in addition, sars-cov-2 may be present with other respiratory infections, hindering its identification 13, 14 . thus, it is fundamental to improve existing diagnostic tools to contain the spread. for example, diagnostic tools combining computed tomography (ct) scans with deep learning have been proposed, achieving an improved detection accuracy of 82.9% 15 . another solution being used for studying sars-cov-2, is sequencing of the viral complementary dna (cdna). for example, we can use this sequencing data with cdna, resulting from the pcr of the original viral rna; e,g, real-time pcr amplicons to identify the sars-cov-2 16 . classification using viral sequencing techniques is mainly based on alignment methods such as fasta 17 and blast 18 . these methods rely on the assumption that cdna sequences share common features, and their order prevails among different sequences 19, 20 . however, these methods suffer from the necessity of needing base sequences for the detection 21 . nevertheless, it is necessary to develop innovative improved diagnostic tools that target the genome to improve the identification of pathogenic variants, as sometimes several tests, are needed to have an accurate diagnosis. therefore, as an alternative, deep learning methods have been suggested for classification of dna sequences. the advantage of these methods are that they do not need pre-selected features to identify or classify dna sequences. deep learning has been efficiently used for classification of dna sequences, using one-hot label encoding and convolution neural networks (cnn) 22, 23 , albeit the examples in literature are featuring dna sequences of length up to 500 bps, only. in particular, for the case of viruses, ngs genomic samples might not be identified by blast, as there are no reference sequences valid for all genomes, as viruses have high mutation frequency 24 . alternative solutions based on deep learning have been proposed to classify viruses, by dividing sequences into pieces of fixed length, ranging from 300 bps 24 to 3,000 bps 25 . however, this approach has the negative effect of potentially ignoring part of the information contained in the input sequence, that is disregarded if it cannot completely fill a piece of fixed size. the global impact of sars-cov-2 prompted researchers to apply effective alignment-free methods to the classification of the virus: for example, in 26 the authors propose the use of machine learning digital signal processing for separating the virus from similar strains, with remarkable accuracy. nevertheless, there is no human-readable information that can be extracted from their black-box procedure, so the biological insight provided by their approach is limited. given the impact of the world-wide outbreak, international efforts have been made to simplify the access to viral genomic data and metadata through international repositories, such as the national genomics data center (ngdc) repository 11 , the national center for biotechnology information (ncbi) repository 27 and the global initiative on sharing all influenza data (gisaid) repository 28 , expecting that the easiness to acquire information would make it possible to develop medical countermeasures to control the disease worldwide, as it happened in similar cases earlier [29] [30] [31] . thus, taking advantage of the available information of international resources without any political and/or economic borders, we propose an innovative system based on viral gene sequencing. using a cnn to separate coronaviruses belonging to different strains 32 , including sars-cov-2, we apply techniques inspired by explainable ai in computer vision to discover representative cdna sequences that the network uses to classify sars-cov-2. we then validate the discovered sequences on datasets not used during the training of the cnn, and show how to exploit them to create a novel, highly informative set of sequence features (e.g. viral sequences). such sequences can be later inspected and analyzed by human experts. experimental results show that the new set of sequence features leads traditional, simple classifiers, to correctly assess sars-cov-2 with remarkable accuracy (> 99%). a few of the discovered sequences also possess the correct characteristics for potentially becoming primers, as just checking for their presence in samples is enough to specifically identify sars-cov-2 ( fig. 1) . laboratory testing on the most promising sequences identified showed that the primers found by our approach can be a viable alternative to the commonly adopted primers at the time of writing. figure 1 . overall procedure to find the specific sars-cov-2 21-bps rna sequences to create a primer set. summarizing the results of experiments 1-4 ( fig. 3) , we discovered 12 meaningful 21-bps sequences that best characterize sars-cov-2. for all the analyzed data, these sequences appear only in sars-cov-2 samples and not in any other viruses, as summarized in table 1 . remarkably, our results outperform earlier publications using machine learning for identifying sars-cov-2 (see for example 26 ) , with the added benefit of producing human-readable results instead of a plain black box classifier. we calculated the frequency of appearance of different primer sets' sequences used in sars-cov-2 rt-pcr tests developed by who referral laboratories and compared it to our primer design in the dataset from the gisaid ( table 2) repository. all of the sequences have a frequency of appearance of > 99%, with the exception of china-cdc-n-f with a 68.52%. this is consistent with the percentage of genomes with mutation in the primer region in the gisaid latest update summary of august 11 th28 . in the analysis of specificity in silico, we compared all the primers sets' sequences with the ncbi-b and ngdc dataset, the results show that hku-n-f, hku-n-r, charite-e-f, charite-e-r and us-cdc-n2-f are not specific to sars-cov-2 as they detect sars-cov-1 too. the rest of the sequences, including our design, only appear in sars-cov-2. thus, in summary from 8 different primer sets, 3 of them are not specific to sars-cov-2, and from the remaining 5, considering frequency of appearance only, our design is in 3rd best option calculated with the lower limit. 99.59% china-cdc-nto validate the data obtained in silico by laboratory methods a conventional pcr was performed on cdna obtained from rna from sars-cov-2 and other human coronaviruses. in addition, rnas from nasopharyngeal swabs from six patients previously diagnosed with sars-cov-2 infection and four patients negative for sars-cov-2 by routine diagnostic method 5 were analyzed with the same conventional pcr (fig. 2) . different dilutions of sars-cov-2 rna were detected with similar sensitivity compared to the diagnostic reference assay. (fig. 2 lanes 1-8) . our candidate primer set exclusively detected sars-cov-2 and did not amplify rna from other human coronaviruses (figure 9, lanes 9-14) . the candidate primer set was able to detect sars-cov-2 rna from patient samples previously found positive for sars-cov-2, but not in patients previously found negative (fig. 2, lanes 15-24) . although further validation will be required to develop this candidate primer set into a diagnostic assay, our results clearly demonstrate the power of our method to select potential sequences for further validation. being able to reliably identify sars-cov-2 and distinguish it from other similar pathogens is important to contain its spread. the time of processing samples and the availability of reliable diagnostic tests is a challenge during an outbreak. developing innovative diagnostic tools that target the genome to improve the identification of pathogens, can help reduce health costs and time to identify the infection, instead of using unsuitable treatments or testing. moreover, it is necessary to perform an accurate classification to identify the different species of coronavirus, the genetic variants that could appear in the future, and the co-infections with other pathogens. given the high transmissibility of the sars-cov-2, the proper diagnosis of the disease is urgent, to stop the virus from spreading further. considering the false negatives given by the standard rt-qpcr detection, better implementations such as using deep learning are necessary in order to properly detect the virus. while the accuracy of current rt-qpcr testing is around 70%, and ct scans with deep learning go up at 83%, we believe that the use of the sequences detected by a cnn-based methodology has the potential to improve the accuracy of the diagnosis. our results, show that by targeting one out of the 12 selected 21-bps specific sequences, we are able to distinguish sars-cov-2, from any other virus (> 99%). further testing is necessary to confirm these promising results so it is essential to create multidisciplinary groups that work to stop the outbreak. finally, as an interesting remark, by comparing the discovered sequences against other hosts, we noticed that from the 12 sequences exclusive to sars-cov-2, one of them appears in 13 of 17 samples from manis javanina. in contrast, 5 of the sequences of sars-cov-2 appear in the only sample available from rhinolophus affinis and 11 out of 12 in 2 canine samples (table 1) . this is consistent with the findings of zhang et al. 33, 34 , and could point to the zootonic origin of the virus. nevertheless, more data is necessary. as a result of the high density populations, and ever growing interaction between people, it is possible that other pandemics may occur. we believe that our methodology has a substantial added value over traditional methods, because it is a fast method and only limited set of viral sequencing data is needed. moreover, this procedure led to a primer set with a very high specificity for sars-cov-2 with at least the same accuracy as the best primers sets in the world developed by who referral laboratories. thus, thinking forward, our methodology can be applied in future viral pandemics to speed up the development of accurate detection methods for diagnosis and thereby contribute to limit the spread of a virus. the cnn used during all the experiments is composed of one convolutional layer with 12 different filters or weights (each with window size 21) with maxpooling (with pool size and stride 148), a fully connected layer (196 rectified linear units with dropout probability 0.5), and a final softmax layer with 5 units, to differentiate the different classes of coronavirus strains. the optimized used is adaptive momentum (adam) 35 , with learning rate 10 −5 and a batch size of 50 samples, run for 1,000 epochs 32 . the convolutional layer of the network, in simple terms, is analyzing subsequences of 21 base pairs that can appear in different points of the virus genome. we selected 21 as designed primers for rt-pcr tests have a length of 18-22 bps normally. the pool size of the maxpooling represents the interval in which a specific 21-bps sequence can be recognized (in this case, 148 positions). through the training process, the convolutional layer is de-facto learning new features to characterize the problem, directly from the data. in this specific case, the new features are 21-bps sequences that can more easily separate different virus strains. by analyzing the result of each filter in a convolutional layer, and how its output interacts with the corresponding max pooling, it is possible to detect human-readable sequences of base pairs that might provide domain experts with relevant information. it is important to notice that these sequences are not bound to specific locations of the genome; thanks to its structure, the cnn is able to detect them and recognize their importance even if their position is displaced in different samples. we downloaded 583 sequences (*.fasta files) from the ngdc on march 15 th ,2020 (table 3) . we left out 30 sars-cov-2 sequences and then, we divided the rest of the data into 80% training, 10% validation, 10% testing. the trained cnn described above obtained a mean accuracy of 98.73% in a 10-fold cross-validation. once the network is trained, in a first analysis, we plot the inputs and outputs of the convolutional layer, to visually inspect for patterns. as an example, in fig. 4a we report the visualization of the first 1,250 bps of each of the 553 samples from the ngdc 11 repository . each filter slides a 21-bps window over the input, and for each step produces a single value. the output of a filter is thus a sequence of values in (0, 1). the output of the max pooling for each of the 12 filters is then further inspected for patterns. it is noticeable how samples belonging to different classes can be already visually distinguished. at this step, we identify filter 0 as the most promising, as it seems to focus on a few relevant points in the genome, that could correspond to meaningful cdna sequences. given this data, it is now possible to identify the 21-bps sequences that obtained the highest output values in the max pooling layer of filter 0, in a section of 148 positions. this process results in 210 (31,029 divided by 148) max pooling features, each one identifying the 21-bps sequence that obtained the highest value from the convolutional filter, in a specific 148-position interval of the original genome: the first max pooling feature will cover positions 1-148, the second will cover position 149-296, and so on. we graph the whole set of max pooling features for the complete data 4,410 (210*21), fig. 4b . the cnn architecture, and the visualization of the filter, and max pooling are available in the supplementary material section 1. analyzing the different sequence values appearing in the max pooling feature space, a total of 3,827 unique 21-bps cdna sequences, that can potentially be very informative for identifying different virus strains. for example, sequence agg taa caa acc aac caa ctt is only found inside the class of sars-cov-2, in 59 out of 66 available samples. sequence cac gag taa ctc gtc tat ctt is present again only in sars-cov-2, in 63 out of the 66 samples. the combination of the convolutional and max pooling layer allows the cnn to identify sequences even if they are slightly displaced in the genome (by up to 148 positions). thus, we create a table of feature appearance of each of the sequences selected from the previous step. this results, in just a set of feature to differentiate sars-cov-2 from other viruses. the experiments presented in the following subsections to validate our method have different objectives and make use of different datasets. a summary of all the experiments and datasets used is shown in figure 3 . table 3 . organism, assigned label, and number of samples in the unique sequences for the ngdc repository (left) and query: gene="orf1ab" and host="homo sapiens" and "complete genome" in the ncbi repository (right). we use the ncbi organism naming convention 36 we downloaded the dataset from the ngdc repository 11 on march 15 th 2020. we removed repeated sequences and applied the procedure to translate the data into the sequence feature space. this leaves us with a frequency table of 3,827 features (21-bps sequences) with 583 samples (table 3 (left) ). next, we ran a state-of-the-art feature selection algorithm 37, 38 , to reduce the sequences needed to identify different virus strain to the bare minimum. remarkably, we are then able to correctly differentiate all the coronavirus (mers-cov, sars-cov-2, sars-cov-1, etc) samples using only 53 of the original 3,827 sequences, obtaining a 100% accuracy in a 10-fold cross-validation with a simpler and more traditional classifier, such as logistic regression. the list of the 53 features is available in the supplementary material section 2. we downloaded data from ncbi 27 on march 15 th 2020, with the following query: gene="orf1ab" and host="homo sapiens" and "complete genome". the query resulted in 407 non-repeated sequences (table 3 (right)). we call this dataset ncbi-a, where 68 sequences belong to sars-cov-2. then, we applied the procedure to translate the data into the set of sequence features, and we run the same state-of-the-art feature selection algorithm 37 . the result is a list of 10 different sequences (table 4) , for which just checking for their presence is enough to differentiate between sars-cov-2 and other viruses in the dataset, with a 100% accuracy. each of the sequences, in fact, only appears in sars-cov-2 samples. , for a total of more than 900 viruses. then, we applied the procedure to translate the data into the sequence feature space and run the feature reduction algorithm 37 . this results in 2 extra sequences of 21 bps: just by checking for their presence, we are able to separate sars-cov-2 from the rest of the samples with a 100% accuracy. the sequences are: aat aga aga att att cta ttc and cga taa caa ctt ctg tgg ccc. from the gisaid repository 28 , we downloaded 53,183 sequences available on august 10 th , for sars-cov-2, from different countries, from there 52,645 have as < 1% ns, high coverage and host="homo sapiens". then, we calculated the frequency table of the 21-bps sequences obtained from experiments 2 and 3, to verify which sequences remain and could be used for detection. the appearance frequency of the target sequences among the samples in the gisaid dataset is reported in table 1 , second column. in addition, we downloaded 26 sequences from gisaid repository of other hosts (manis javanica, rhinolophus affinis, canine and felis catus) to make a comparison in the sequences from experiment 2 and 3. experiment 5: design of the candidate primer set. after the analysis carried out on the deep learning model, we ran an analysis with primer3plus 39 , to see which of the sequences could be used as a forward primer, using sample ncbi nc045512.2 as the reference sars-cov-2 sequence. we uncover the sequence tag cac tct cca agg gtg ttc that shows a frequency of appearance of 99.57% in viral genomes available from different countries in gisaid 28 and 100.0% in the ncbi 27 datasets. using the reference sars-cov-2 sequence, we identify that this discovered sequence is located between nucleotides 25,604 and 25,624 in the orf3a gene. in sars-cov, this gene encodes a protein of 274 aa, that is related with necrotic cell death 40 , chemokine production like interleukin 8 (il-8) and rantes/ccl5, nfκb activation resulting in an inflammatory response 41 and may play an important role in the virus life cycle 42 . we design a specific primer set for detection of sars-cov-2 using primer3plus 39 . we use tag cac tct cca agg gtg ttc as forward primer and gca aag cca aag cct cat ta as reverse primer, obtaining an amplicon size of 179 bps. then, we run an in-silico pcr test using fastpcr 6.7 43 with default parameters in nc045512.2 used as a reference sars-cov-2 sequence, this yields t m = 56.2 • c for the forward primer, t m = 53.1 • c for the reverse primer and ta = 58 • c. in addition, we calculated the frequency of appearance of different primers sets' sequences used in sars-cov-2 rt-qpcr tests developed by who referral laboratories and compared it to our primer design sequences in 52,645 sequences from the gisaid repository and the 583 samples of different coronaviruses from the ngdc dataset from experiment 1. the used primers set are developed by university of hong kong (hku-n); charite, berlin, germany (charite-e); us-cdc, united states (us-cdc-n1,us-cdc-n2,us-cdc-n3) and china cdc, china (china-cdc-orf1ab, china-cdc-n) ( table 9) . we selected this primers as they are the ones more commonly used as stated in the gisaid status update of august 11, 2020. we do not consider degenerate primer sets. coronavirus genomics and 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covid-19: a systematic review and meta-analysis clinical diagnosis of 8274 samples with 2019-novel coronavirus in wuhan a deep learning algorithm using ct images to screen for corona virus disease (covid-19). medrxiv the first case of 2019 novel coronavirus pneumonia imported into korea from wuhan, china: implication for infection prevention and control measures rapid and sensitive sequence comparison with fastp and fasta basic local alignment search tool applications of alignment-free methods in epigenomics alignment-free sequence comparison-a review phylogenetically diverse tt virus viremia among pregnant women dna sequence classification by convolutional neural network a deep learning approach to dna sequence classification viraminer: deep learning on raw dna sequences for identifying viral genomes in human samples identifying viruses from metagenomic data by deep learning machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: covid-19 case study dbsnp: the ncbi database of genetic variation global initiative on sharing all influenza data-from vision to reality how ownership rights over microorganisms affect infectious disease control and innovation: a root-cause analysis of barriers to data sharing as experienced by key stakeholders managing severe acute respiratory syndrome (sars) intellectual property rights: the possible role of patent pooling threats to timely sharing of pathogen sequence data accurate identification of sars-cov-2 from viral genome sequences using deep learning a genomic perspective on the origin and emergence of sars-cov-2 extreme genomic cpg deficiency in sars-cov-2 and evasion of host antiviral defense a method for stochastic optimization genbank: the nucleotide sequence database. the ncbi handb automatic discovery of 100-mirna signature for cancer classification using ensemble feature selection machine learning-based ensemble recursive feature selection of circulating mirnas for cancer tumor classification primer3plus, an enhanced web interface to primer3 sars-coronavirus open reading frame-8b triggers intracellular stress pathways and activates nlrp3 inflammasomes augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/x1 and 7a/x4 proteins through nf-κb activation severe acute respiratory syndrome coronavirus orf3a protein interacts with caveolin fastpcr software for pcr primer and probe design and repeat search viral rna was isolated from cell-cultured sars-cov-2, sars-1, mers-cov, hcov-nl63, hcov-oc43, hcov-229e, and from nasopharyngeal swabs from n = 10 patients by magna pure lc (roche diagnostics, the netherlands) using the total nucleic acid isolation kit. the rna was converted into cdna using superscriptiii (thermo-fisher scientific, usa) and random hexamers. subsequently, conventional pcr was performed on the cdna using hotstar taq dna polymerase (qiagen, the netherlands) with 400nm forward primer (5'-ag cac tct cca agg gtg ttc-3') and 400nm reverse primer (5'-gca aag cca aag cct cat ta-3') and the following cycling conditions: 15 min at 95 • c, followed by 40 cycles of 1 min. at 95 • c , 1 min. at 5 • cc and 1 min. at 72 • c. the pcr products were visualized by electrophoresis. the same rna was used in a diagnostics reference assay by corman et al. 5 and the cycle threshold values form this reference assay were used for estimating sensitivity. the study was approved by the medical ethical commission of the erasmus mc (mec-2015-306). lmm, cap made the biological analysis, and primer design. alr and at made the programming, data collection and experiments in silico. dm and rm made the pcr validation. ec, adk and jg made the experiment and study design. all the authors contributed to the writing. key: cord-296362-9vi8xwu7 authors: wang, jian-min; wang, lin-fa; shi, zheng-li title: construction of a non-infectious sars coronavirus replicon for application in drug screening and analysis of viral protein function date: 2008-09-12 journal: biochemical and biophysical research communications doi: 10.1016/j.bbrc.2008.06.129 sha: doc_id: 296362 cord_uid: 9vi8xwu7 abstract severe acute respiratory syndrome virus (sars-cov) was the causative agent of the sars outbreaks in 2002–2003. a safer in vitro system is desirable for conducting research on sars-cov and to screen for antiviral drugs against the virus. based on the infectious cdna clone of rsars-cov-δe, in which the e gene has been deleted, a safe non-infectious replicon was constructed by replacing the s gene with the enhanced green fluorescent protein (egfp) gene. successful replication was achieved as evident from continuous expression of egfp detected by both fluorescence and western blot. treatment with antiviral drugs demonstrated that the replication could be significantly inhibited by 0.4mg/ml of cysteine proteinase inhibitor e-64d, but not by ribavirin. the same replicons containing further deletion of the coding regions for non-structural proteins (nsp) 1, 2 or 16 confirmed previous observation that nsp16, but not nsp1 or nsp2, was essential for efficient viral replication or transcription. severe acute respiratory syndrome virus (sars-cov) was the causative agent of the sars outbreaks in [2002] [2003] . a safer in vitro system is desirable for conducting research on sars-cov and to screen for antiviral drugs against the virus. based on the infectious cdna clone of rsars-cov-de, in which the e gene has been deleted, a safe non-infectious replicon was constructed by replacing the s gene with the enhanced green fluorescent protein (egfp) gene. successful replication was achieved as evident from continuous expression of egfp detected by both fluorescence and western blot. treatment with antiviral drugs demonstrated that the replication could be significantly inhibited by 0.4 mg/ml of cysteine proteinase inhibitor e-64d, but not by ribavirin. the same replicons containing further deletion of the coding regions for non-structural proteins (nsp) 1, 2 or 16 confirmed previous observation that nsp16, but not nsp1 or nsp2, was essential for efficient viral replication or transcription. ó 2008 elsevier inc. all rights reserved. severe acute respiratory syndrome (sars) illness which was caused by a novel coronavirus (sars-cov) [1] first arose in guangdong province, china, in late 2002. the virus spread rapidly to 26 countries and killed 916 people out of 8422 infected patients with a fatality rate of 11% [2] . though sars epidemic was finally controlled by patient isolation and the ban of wild animal trading, the significant morbidity and mortality, especially the potential threat from sars reemergence, reminds us the continuous risk facing the public health system [1] . moreover, the wide distribution and genetic diversity of sars-like covs discovered in china [3] [4] [5] also serve as a warning for potential human infection by related bat-borne covs. sars-cov resembles to members of the three previously known groups of coronavirus and was classified as a member of group 2b in the family coronaviridae based on phylogenetic analysis of genome sequences [6, 7] . the sars-cov virion contains a singlestranded 29,700-nt rna genome of positive polarity. the first two-thirds of the genome encode two overlapping polyproteins, orf1a and orf1b. like other coronaviruses, these two polyproteins are processed into 16 mature replicase proteins by viral specific proteinases and most of them are believed to associate with a poorly characterized replication/transcription complex that mediates the synthesis of genome rna (replication) and subgenomic mrnas (transcription) [7] [8] [9] [10] . recently, the functions of several non-structural proteins (nsps) have been determined, including a non-canonical rdrp (nsp8), a single-stranded rna-binding protein (nsp9), a rna-dependent rna polymerase (nsp12), a superfamily 1-like helicase (nsp13), a 3 0 ? 5 0 exonuclease (nsp14), a uridylate-specific endoribonuclease (nsp15), and a 2 0 -o-ribose methyltransferase activities (nsp16) [11] [12] [13] [14] . the complicated interactions network among these nsps makes it difficult, if not impossible, to fully understand their functions [15, 16] . the rest of 3 0 end genome codes for four main structural proteins, the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. the s protein, which mediates both cell attachment and membrane fusion, is responsible for virus host range and tissue tropism. proteins e, m, n can assemble into non-infectious virus like particles (vlps) in the absence of s [17] . besides the nsps and major structural proteins, sars-cov possesses several group-specific proteins and their functions during virus life cycle remain elusive and deletion of genes for those proteins seems to have no effect virus replication and/or transcription [18] . recent studies on these group-specific proteins revealed that some of them may be associated with virus pathogenesis and virus-host interaction [19] [20] [21] [22] . sars-cov is highly virulent and classified as a biosafety level 3 (bsl3) agent, which can only be handled in a bsl3 laboratory. for in-depth study of functions of different viral proteins and regulatory sequence elements by reverse genetics, full-length infectious cdna clones have been established using various techniques including bacterial artificial chromosome (bac) vector [23] [24] [25] and sars-cov replicon cell lines [26] , which can also be used for antiviral drug screening. among them, the sars-cov replicon cell lines which retain the genes necessary for replication and transcription represent the safest system available. however, these cell lines need to be continuously selected with antibiotics and is not readily amenable to introduction of mutations in different protein coding genes. here, we report the construction of a plasmidbased replicon system which can be introduced into cells by transfection and can be easily manipulated for introduction of mutations. the usefulness of this replicon was demonstrated by its application in testing known antiviral drugs and in confirming the essential function of nsp16 in virus replication. cells and viruses. 293t cell line was used in this study and maintained in dmem medium (gibco) supplemented with 10% (vol/vol) foetal bovine serum (fbs, gibco). the plasmid rsars-cov-de encodes an infectious attenuated virus derived from sars-cov urbani strain (genbank accession no. ay278741) [25] and was kindly provided by dr. luis enjuanes (cnb, csic, madrid). pcr amplification of egfp gene and modification of pbluescriptii. primers egfpf 5 0 -actagtatggtgagcaagggcga-3 0 and egfpr 5 0 -actagtcttgtacagctcgtccatg-3 0 (spei site is underlined) were used to amplify the egfp gene from a vector pegfp-n1 (clontech). the pcr products were purified from agarose gels and cloned into pgem-t easy vector (promega) for sequencing. authentic clone with no mutation was used for subsequent cloning. the plasmid pbluescriptii (clontech) was modified by inserting a 35 bp sequence (5 0 -tatggtaccgtttaaacgacggatcctctagac tg-3 0 ) containing polycloning sites using the vector sites kpni and xbai (underlined), which resulted in the introduction of the pmei (gtttaaac) site and the deletion of the spei from the original vector. construction of replicon pbac-sars-cov-deds/egfp. the previously constructed infectious cdna clone (plasmid rsars-cov-de) [25] was digested with pmei and bamhi producing a 7.5 kb fragment containing 3 0 end of pp1b, s and 3a gene. this fragment was subcloned into a modified pbluescriptii vector (described above), generating the plasmid pbs-sars-cov1b-3a. the egfp gene was then inserted to replace s gene with single enzyme spei, generating pbs-sars-cov1b-3ads/egfp. this gfp containing fragment was cloned back into pbac-sars-cov-de digested by pmei and bamhi, generating pbac-sars-cov-deds/egfp (fig. 1) . in-frame deletion of nsp coding regions. to delete the junction region of nsp1-nsp2 which contains the c-terminus of nsp1 (139 aa) and the n-terminus of nsp2 (107 aa), pbac-sars-cov-deds/egfp was digested by pmli and self-ligated to generate pbac-sars-cov-dedsdnsp1-2j/egfp. to delete the n-terminus of nsp2, firstly, primer pair nsp2f 5 0 -gcagtcgatcatcagcataccta-3 0 (212-234 nt, forward) and nsp2r 5 0 -caggacatggcattttcactacag-3 0 (1355-1378 nt, reverse) were used to amplify a fragment containing partial nsp1 and nsp2 sequences. the amplified product was cloned into pgem-teasy to generate clone pgt-nsp1-2. then, the n-terminus of nsp2 (107 aa) was further deleted by the following primers: dnsp2f 5 0 -tgcggatccccacgtgttgaaaaga a-3 0 (1120-1136 nt) and dnsp2r 5 0 -ataggatccacctccattgag ctcac-3 0 (788-804 nt) (bamhi site is underlined). the amplified fragment was ligated through bamhi site to generate a clone pgt-dnsp2. last, the plasmid pgt-dnsp2 was digested with pmli and cloned back into the plasmid pbac-sars-cov-deds/egfp. using the similar deletion strategy for nsp2, based on the plasmid pbs-sars-cov1b-3ads/egfp, core sequence of nsp16 (from 20,631 to 21,422) was deleted by pcr amplification with primers dnsp16f 5 0 -gtggtcgacaggcttatcattagagaaaac-3 0 (21,423-21,443 nt) and dnsp16r 5 0 -agagtcgaccaagttaggcatcgcaacacc-3 0 (20,610-20,630 nt) (sali site is underlined), with a new sali site as a genetic marker. the amplified fragment was ligated through the sali site to generate a clone pbs-sars-cov1b-3adnsp16/egfp. the generated plasmid was cloned back into the plasmid pbac-sars-cov-deds/egfp between pmei and bamhi sites to generate pbac-sars-cov-dedsdnsp16/egfp. plasmid purification and transfection. the above constructed plasmids were purified with the qiagen large-construct kit (qia-gen) according to the manufacturer's protocol. transfection was performed when 293t cells were grown to 85% confluence with fu-gene6 transfection reagent (roche) according to manufacturer's instructions. the transfected cells were incubated at 37°c with 5% co 2 for 12 h and then cultured with fresh dmem containing 10% fbs at 37°c with 5% co 2 either until the observation at 72 h.p.t. or used for antiviral drug test with 0.4 mg/ml ribavirin (sigma) or 0.4 mg/ml e-64d (sigma). western blot analysis. cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page). proteins were transferred onto immobilon-p transfer membranes (milipore) by semi-dry electrophoresis transfer (bio-rad). the membrane were blocked overnight in tbs (0.2 m nacl, 50 mm tris-hcl, ph 7.5) with 3% (w/v) bsa (biosharp) and then probed with rabbit polyclonal antiserum against sars-cov n protein (produced at the australian animal health laboratory) or gfp (beyotime, china) diluted 1:2000 in immunoreaction enhancer solution i (toyobo) for 1 h at 37°c, followed by washing in tbs-t (0.1% tween 20 in tris-buffered saline) for three times. after incubation with the alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (diluted 1:2000 in immunoreaction enhancer solution ii (toyobo) for 1 h at 37°c, the signal was developed a bcip/nbt substrate mix (sino-american). quantitative real-time pcr analyses. total cellular rnas from transfected 293t cells were extracted with rnaprep cell kit (tian-gen) at 72 hpt. rq1 rnase free dnase (promega) was used to digest dna at 37°c for 30 min followed by incubating at 65°c for 10 min. reverse transcription was performed in a 25-ll volume was conducted at 42°c for 60 min, followed by heating to 70°c for 5 min. primers used for rt and quantitative pcr were as follows: qsars-n-f: 5 0 -taccgaagagctacccgacgagtt-3 0 ; qsars-n-r: 5 0 -gcacggtggcagcattgttattag-3 0 (for n gene detection); qbactin-f: 5 0 -caactgggacgacatggagaaaat-3 0 ; qb-actin-r: 5 0 -cc agaggcgtacagggatagcac-3 0 (for b-actin detection). real-time quantitative pcr assays were conducted in a dnaengine opticon machine (mj). sybr green real-time pcr master mix (toyobo) was mixed with forward and reverse primers (0.4 lm each) in a final pcr mixture of 25 ll. the thermal cycling conditions were as follows: 1 cycle at 95°c for 1 min, followed by 40 cycles of 95°c for 15 s, 55°c for 20 s, 72°c for 20 s, 80°c for 3 s. fluorescence was measured after each cycle. a melting curve was generated after the last extension step from 55°c to 95°c. the experiment was repeated three times and the data represent the means of triplicates. as described in materials and methods, the egfp gene was inserted to replace the s gene in the previously published construct rsars-cov-de to generate the non-infectious replicon plasmid pbac-sars-cov-deds/egfp (fig. 1) . in this construct, none of the transcription regulation signals (trss) of sars-cov was interrupted and most orfs were kept intact with the exception of the e and s genes. the egfp gene was cloned under the control of the s gene trs, retained the sequence of s gene at 5 0 and 3 0 end (30 and 102 nt, respectively). it is expected that the replicon can express most sars-cov orfs at the same level as the original infectious cdna clone. the resulting bac clone was stably propagated in escherichia coli dh10b cells. when the purified pbac-sars-cov-deds/egfp was transfected into 293t cells, egfp expression was observed at 72 h post-transfection ( fig. 2a, left panel) , indicating that this protein could be expressed from the s gene trs. western blot further confirmed the expression of egfp and n proteins (fig. 2b) . these results demonstrated that the pbac-sars-cov-deds/egfp replicon could replicate and transcribe in these cells. to explore the feasibility of using the replicon system as a tool for drug screening, an inhibition assay with two known antiviral drugs, ribavirin and e-64d, was conducted at 12 h post-transfection. the results showed a significant reduction in fluorescence in the cells at 60 h after treatment with 0.4 mg/ml e-64d. (fig. 2a, right panel) . however, cells treated with ribavirin, a widely used broad-spectrum replicase inhibitor, did not have obvious effect on the fluorescence signal compared with those of the untreated cells ( fig. 2a, middle panel) . it is worth noting that no obvious cytotoxic effect was observed for either ribavirin or e-64d at the concentrations used in this study. quantitative real-time rt-pcr was used to evaluate the inhibition of sars-cov n gene messenger rna production. the n gene copy number was reduced by more than 2 logarithmic units after treatment with 0.4 mg/ml e-64d (fig. 2c) , indicating an efficient inhibition of sars-cov replication. in contrast, the mrna level for the house keeping gene from both treated and untreated cells remained at a similar level. while the fluorescence intensity detected by microscope showed no significant difference between control and ribavirin treated cells, real-time rt-pcr revealed that ribavirin might have a small, but invisible, inhibitory effect on sars-cov replication. functional analysis of the sars-cov nsp1, nsp2 and nsp16 sars-cov polyprotein is processed into 16 non-structural proteins which form the replication/transcription complex during viral life circle. in previous studies, nsp16, but not nsp1 and nsp2, was reported to be required for virus replication [27, 28] . in this study, we constructed three recombinant variants to test whether the same conclusion could be obtained using this new replicon system, the first one with a deletion in the junction domain between nsp1 and nsp2 genes (pbac-sars-cov-dedsdnsp1-2j/egfp), the second one with a deletion within nsp2 n-terminus (pbac-sars-cov-dedsdnsp2n/egfp), and a third in which nsp16 was deleted (pbac-sars-cov-dedsdnsp16/egfp) (see materials and methods). the results presented in fig. 3 indicate that fluorescence intensity did not change for the first two deletion constructs compared with the original replicon, but there was a sharp reduction for the third construct with the nsp16 deletion. this was further confirmed by real-time rt-pcr evaluation of the n gene transcript, which revealed a reduction of close to 3 logs (fig. 3b) . in this study, a non-infectious sars-cov replicon was generated based on the sars-cov-de bac clone. the replicon retains most sars-cov genes except the s and e genes. the insertion of egfp gene renders it convenient to monitor virus replication by measuring fluorescence intensity. the deletion of s gene allows the manipulation of the replicon in a conventional laboratory instead of using a bsl3 facility. our results indicated that the replicon could transcribe and express heterogeneous genes in vitro and can be used for antiviral drug screening and analysis of viral protein functions. moreover, our newly constructed replicon is a stable bac system and can be easily amplified in e. coli and manipulated by reverse genetics. as indicated in the results, it will be possible to insert any mutation in this replicon for gene function analysis. ribavirin has been widely used as viral replicase inhibitor including sars-cov [29] . however, our results showed that ribavirin is not an effective inhibitor of sars-cov replication at a noncytotoxic concentration at least in the cell line used in this study. our observation is in agreement with previous published studies [26, 30, 31] . in contrast, e-64d was found quite effective against sars-cov infection, again in consistence with previous studies based on live viruses [23, 26] . bats were identified as the natural reservoirs of sl-covs. bat sl-covs and sars-cov have a high similarity in genome organization, gene and gene product sequences, particularly in replicase proteins (96-98% amino acid identity) [3] [4] [5] , suggesting that the function of most of these replicase proteins is conserved among different sars and sars-like covs. even though the pathogenesis of bat sl-covs in human is unknown, there is a high potential of other related covs of bat origin to infect and cause diseases in human. in this regard, it is important to stress that our replicon system can be used to screen for drugs with equal effect to sars-cov or other unknown but closely related bat covs which could potentially emerge in the future. the severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses full-length genome sequences of two sars-like coronaviruses in horseshoe bats and genetic variation analysis severe acute respiratory syndrome coronavirus phylogeny: toward consensus unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mrna synthesis sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2 0 o)-methyltransferase activity the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded rnabinding subunit unique in the rna virus world a second, non-canonical rna-dependent rna polymerase in sars coronavirus discovery of an rna virus 3 0 ->5 0 exoribonuclease that is critically involved in coronavirus rna synthesis analysis of intraviral protein-protein interactions of the sars coronavirus orfeome the sars-coronavirus plnc domain of nsp3 as a replication/transcription scaffolding protein efficient assembly and release of sars coronavirus-like particles by a heterologous expression system severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/x1 and 7a/x4 proteins through nf-kappab activation severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release severe acute respiratory syndrome coronavirus orf3a protein interacts with caveolin reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo derivation of a novel sars-coronavirus replicon cell line and its application for anti-sars drug screening the nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells development of a standard treatment protocol for severe acute respiratory syndrome inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs small molecules targeting severe acute respiratory syndrome human coronavirus this work was jointly funded by the state key program for basic research grant (2005cb523004) from the chinese ministry of science and technology, the knowledge innovation program key project administered by the chinese academy of sciences (kscx1-yw-r-07). we thank drs. luis enjuanes and marta l. dediego for kindly providing the rsars-cov-de infectious cdna clone and technical advice. key: cord-288731-x2cwyvb7 authors: puenpa, jiratchaya; suwannakarn, kamol; chansaenroj, jira; nilyanimit, pornjarim; yorsaeng, ritthideach; auphimai, chompoonut; kitphati, rungrueng; mungaomklang, anek; kongklieng, amornmas; chirathaworn, chintana; wanlapakorn, nasamon; poovorawan, yong title: molecular epidemiology of the first wave of severe acute respiratory syndrome coronavirus 2 infection in thailand in 2020 date: 2020-10-06 journal: sci rep doi: 10.1038/s41598-020-73554-7 sha: doc_id: 288731 cord_uid: x2cwyvb7 the coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a major global concern. several sars-cov-2 gene mutations have been reported. in the current study associations between sars-cov-2 gene variation and exposure history during the first wave of the outbreak in thailand between january and may 2020 were investigated. forty samples were collected at different time points during the outbreak, and parts of the sars-cov-2 genome sequence were used to assess genomic variation patterns. the phylogenetics of the 40 samples were clustered into l, gh, gr, o and t types. t types were predominant in bangkok during the first local outbreak centered at a boxing stadium and entertainment venues in march 2020. imported cases were infected with various types, including l, gh, gr and o. in southern thailand introductions of different genotypes were identified at different times. no clinical parameters were significantly associated with differences in genotype. the results indicated local transmission (type t, spike protein (a829t)) and imported cases (types l, gh, gr and o) during the first wave in thailand. genetic and epidemiological data may contribute to national policy formulation, transmission tracking and the implementation of measures to control viral spread. in december 2019 patients presenting with viral pneumonia of unknown cause were reported in wuhan, china. in january 2020 a novel human coronavirus provisionally named '2019 novel coronavirus' was identified via next-generation sequencing 1, 2 . the international committee on taxonomy of viruses subsequently changed the official name of the virus to 'severe acute respiratory syndrome coronavirus 2′ (sars-cov-2) 3 , and the disease it caused was dubbed 'coronavirus disease 2019′ . on 30 january 2020 the world health organization declared the sars-cov-2 outbreak a so-called 'public health emergency of international concern' , and on 11 march 2020 it declared the outbreak a pandemic 4 . as of 31 may 2020 more than 6 million people had been infected with sars-cov-2, and there had been more than 373,000 deaths 5 . sars-cov-2 belongs to the family coronaviridae, the subfamily coronavirinae, and the order nidovirales. the genome of coronaviruses consists of positive-stranded rna of approximately 27 to 32 kb in length, including 7 to 10 open reading frames (orfs) and untranslated regions at the 5′ and 3′ ends of the rna 6 . based on their genomic diversity coronaviruses are divided into four genera; alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. prior to the emergence of sars-cov-2, in the last two decades six other coronaviruses have been detected in humans. two are alphacoronaviruses (human coronavirus nl63 and scientific reports | (2020) 10:16602 | https://doi.org/10.1038/s41598-020-73554-7 www.nature.com/scientificreports/ human coronavirus 229e) that usually cause mild upper respiratory disease 7 . the other four are betacoronaviruses, including the weakly pathogenic human coronavirus oc43 and human coronavirus hku1, and the highly pathogenic severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). sars-cov-2 is the seventh human betacoronavirus, and its genome is closely related to sars-cov which emerged in 2002 and 2003 1 . as of 30 may 2020 there were more than 36,000 sars-cov-2 genome sequences in the gisaid database (https ://www.gisai d.org/), contributed by numerous laboratories around the world. during the early period of the outbreak genome sequencing revealed two types of viruses based on differences in two single nucleotide polymorphisms in orf1ab and orf8; l type and s type 8 . another analysis categorized sars-cov-2 into three types, a, b, and c, based on amino acid changes 9 . as of 30 may 2020 there were seven clades identified in the gisaid database, s, l, v, g, gr, gh, and o. sars-cov-2 genetic variation may be associated with differences in viral replication 10 , though more evidence is needed to verify any putative associations between mutations and pathogenesis in humans. in thailand the ministry of public health reported the first laboratory-confirmed case of sars-cov-2 in a 61-year-old chinese traveller who had arrived from wuhan on 12 january 2020. this was reportedly the first recorded case outside of china 11 . by the end of january two confirmed cases had been reported, both thai nationals. of those, one was a 73-year-old woman from nakhon pathom province who had recently returned from china. the first unequivocally domestically contracted sars-cov-2 infection in thailand was documented on 31 january 2020 when a taxi driver who had not travelled outside thailand tested positive. in january the percentage of confirmed cases in which the patients were travellers from other countries was 89.5% (17/19) , but the corresponding percentage in february was 65.2% (15/23). on 29 february 2020 sars-cov-2 was designated a dangerous communicable disease under the communicable disease act. the act stipulates that all infected people must be hospitalized. on 15 march 2020 covid-19 spread within a boxing stadium and drinking venues in bangkok, then it spread throughout thailand 12 . notably however, most cases confirmed in april were people who had returned from a mass religious meeting in indonesia, thai workers from malaysia, and immigrants at the detention centre in songkla province 13, 14 . similar to china, the incidence of covid-19 in thailand decreased dramatically when the thai government prohibited social gatherings after the first wave of the rapid spread of sars-cov-2. on 16 march 2020 the thai government announced that the thai new year's national holiday (songkran) between the 13 th and 15 th of april 2020 would be postponed indefinitely 15 . on 18 march 2020 the thai government began implementing a social distancing policy, including the mandatory closure of all schools and universities, entertainment and sporting venues, and all stores except food markets 16 . subsequently the thai government announced that a nationwide 10 p.m. to 4 a.m. curfew would commence on 02 april 2020. as of 6 june 2020 there had been 3,104 hospitalizations and 58 deaths in thailand, equating to a fatality rate of < 2% 17 . in the current study the genotypes of the sars-cov-2 strains involved in the outbreak in thailand during the first wave from february to april 2020 were investigated. based on the genome sequences available in giasid, nucleotide variation in four regions of the sars-cov-2 genome was used to conduct viral tracking and identify sites of origin of outbreaks in thailand. ethics statement. the research proposal was approved by the institutional review board of the ethics committee of the faculty of medicine, chulalongkorn university, thailand (irb number 301/63). the institutional review board of the ethics committee for human research waived the need for consent because all samples were anonymous. all methods were performed in accordance with the relevant guidelines and regulations. study population and sample collection. the study was conducted using anonymized sars-cov-2-positive specimens collected from a diagnostic service except for the first specimens from the chinese traveller from wuhan (provided from the department of medical science, ministry of public health, thailand). patient identifiers including personal information and hospitalization number were removed from the samples to ensure patient confidentiality. the demographic data recorded included sex, age, vital signs and exposure history. the specimens were collected from bangkok, nonthaburi, samut prakan, songkla, ubon ratchathani and yala ( figure s1 ). the samples included a record of the date they were procured, and the putative location of infection (boxing stadium, specific drinking venues, a mass religious meeting from indonesia, thai workers from malaysia, and an immigrant detention centre, among others) (fig. 1 ). a total of 40 positive nasopharyngeal and/or throat swab samples (except the first specimens from the chinese traveller from wuhan) were confirmed to be sars-cov-2-positive via two separate multiplex real-time reverse transcription polymerase chain reaction (rt-pcr) assays. in the first multiplex assay the allplex 2019-ncov (seegene, seoul, republic of korea) incorporating primers and probes specifically targeting rdrp, n and e genes was used. the second assay used the lightmix modular sars and wuhan cov (tib-molbiol, berlin, germany) incorporating primers and probes corresponding to the rdrp and e genes. viral rna was extracted from 200 µl of sample using a maglead 12gc instrument (precision system science, chiba, japan) with a maglead consumable kit (precision system science, chiba, japan) in accordance with the manufacturer's instructions. all rna specimens were transferred to the center of excellence in clinical virology at chulalongkorn university for conventional pcr and sequencing, and sars-cov-2 rdrp, s, n and e genes were amplified via a primer set specific for sars-cov-2 (table s1 ). www.nature.com/scientificreports/ the one-step rt-pcr reactions were conducted using the superscript iii platinum one-step rt-pcr system with platinum taq dna polymerase (invitrogen, carlsbad, ca). briefly, the pcr reaction mixture contained 2-3 μl of rna, 0.5 μm of each primer, 12.5 μl of 2x reaction mix (invitrogen) and 1 μl of ssiii rt/platinum taq mix, and was adjusted to a final volume of 25 μl with nuclease-free water. amplification was conducted in a thermal cycler (eppendorf, hamburg, germany) via a protocol including reverse transcription at 45 °c for 30 min, initial denaturation at 94 °c for 3 min, 40 cycles of 30 s of denaturation at 94 °c, 30 s of primer annealing at 53 °c, 90 s of extension at 68 °c, and further extension for 7 min at 68 °c. pcr products were separated on a 2% agarose gel with a 100-base pair dna ladder and visualized on an ultraviolet trans-illuminator. pcr products were gel-purified using the hiyield gel/pcr dna fragment extraction kit (rbc bioscience co, taipei, taiwan). dna sequencing was performed by first base laboratories sdn bhd, selangor, malaysia. www.nature.com/scientificreports/ phylogenetic analysis. the seqman ii component of the dnastar software (v. 6.0) was used for nucleotide sequence assembly. genome sequences were aligned using clustalw, implemented via the bioedit program (v. 7.2.0). the mega program (v. 6.06) was used for phylogenetic tree construction, which was performed via the neighbour-joining method with 1,000 bootstrap replicates. evolutionary distances were calculated using the maximum composite likelihood method. representative sequences from different areas of the world available in the genbank and gisaid databases were utilized in phylogenetic analysis. sequences were deposited in gen-bank (accession no. mt502900-mt503099; table s2 ). statistical analysis. statistical analysis was conducted using the statistical package for social sciences v. 19 .0 (spss inc., chicago, il). the chi-square test was used to analyse demographic patient factors, and p < 0.05 was deemed to indicate statistical significance. first wave of sars-cov-2 outbreak in thailand. the first wave of sars-cov-2 outbreak started in early march 2020 and peaked between the 22th and 29th of march 2020. as the thai government implemented incremental public health measures to mitigate viral transmission, there was a marked overall decline in the sars-cov-2 cases since 20 march 2020 (fig. 1) . ) were selected to analyse genetic variations. phylogenetic analysis of concatenated sequences of worldwide sars-cov-2 isolates revealed five main clusters (fig. 2a) . based on genetic variations and amino acid changes the clusters were defined as l, s, g, v and o types. the types and patterns of nucleotide substitution are shown in fig. 2b . type l originated in china during the first period of the outbreak. wuhan-hu-1 (nc_045512) is the reference type l strain. type s was detected during the early period of the outbreak, and it has nucleotide substitutions at position 8,782 in orf1ab (c8782t) and 28,144 (t28144c) in orf8. types g and v evidently branched off from type l. type g has single nucleotide changes in orf1b (c14408t) and s (a23403g). there is also a gr strain in the present study wu5 was identified as type l, which was closely related to wuhan-hu-1. ten strains were identified as type g, including seven gh strains and three gr strains. two strains were identified as type o. most of the samples (27/40) were clustered within the distinct type s branch. this branch was defined as type t based on a nucleotide substitution in the s gene (g24047a) and an amino acid change in the spike protein (a829t). therefore, type t was the most prevalent in the samples collected in thailand in the present study. variation patterns and exposure history. in the current study exposure history was divided into three categories; imported, boxing stadium, and nightclub. 'imported' was defined as any cases in which the patient had returned from an endemic area or had been in contact with travellers who had returned from an endemic area. it also included the aforementioned migrant worker and religious pilgrimage cohorts. 'boxing stadium' was defined as cases in which the patient contracted sars-cov-2 from the boxing stadium in bangkok, or had been in contact with anyone who was most likely infected at that boxing stadium. the boxing stadium group was the largest cluster in the march 2020 outbreak in bangkok. 'nightclub' was defined as cases in which the patient most likely contracted sars-cov-2 from a nightclub, entertainment venue, or restaurant in bangkok, or had been in contact with a person who was most likely infected at a nightclub. the wu5 strain was clustered with the l type detected in january 2020 during the first period of the outbreak in thailand. this isolate was closely related to wuhan-hu-1. the likely source of exposure was an imported case from wuhan, china, the first endemic area. samples in the imported group also belonged to types gh, gr, and o. two samples isolated in march 2020 were classified as o types. the travel histories of the patients these samples were derived from indicated that one of them had recently returned from outside of thailand, and the other was a member of the cohort from the southern part of thailand who had recently undertaken a religious pilgrimage. the sars-cov-2 samples derived from the above-described group of migrant workers in may 2020 were identified as type gr. all samples in the boxing stadium group were identified as type t. these samples were obtained during the large outbreak in march 2020 in thailand. the sars-cov-2 samples derived from the nightclub included gh, gr and t types. most of the type t samples in the nightclub group were collected in march 2020, and the gh and gr type samples in that group were collected in march and april 2020. thus, there were multiple sars-cov-2 types circulating during this period. to assess associations between sars-cov-2 types and clinical signs, the results of genetic variation analysis were compared with clinical data. clinical data pertaining to 4 samples was missing, and these samples were excluded from this analysis. by way of this, the one type l sample identified in the present study was excluded from this component of the analysis. clinical symptoms and sars-cov-2 types are shown in table 1 . most samples were derived from patients with common symptoms of upper respiratory infection such as fever, coughing, and a sore throat. eight of the patients had pneumonia, and 7 of the samples from these 8 patients were sars-cov-2 type t. one patient with type t sars-cov-2 required admission to the intensive care unit (icu). one patient reported diarrhoea, and none of the patients died. there were no significant associations between sars-cov-2 type and clinical symptoms. table 1 . clinical symptoms and the type of viral variation. there were four samples of which no clinical data were available. the missing data were excluded from this table. age (average age) 34 viral genome sequencing data have been used to investigate viral transmission and factors associated with it, in a field known as 'genomic epidemiology' 18 . several reports describe the use of genomic data to track viral transmission [19] [20] [21] . many reports have described sars-cov-2 genome variation and the use of complete genome data to track its transmission 8, 9, 22 . however, analysis of polymorphic viral genome segment may be helpful in rapidly identifying patterns of epidemiology and viral genetic cluster. in the current study four regions of the sars-cov-2 genome were sequenced to identify genetic variants. in january and february 2020 the confirmed sars-cov-2 cases were identified as having been imported from china. genetic variations of l and s types were identified during the early period of the outbreak in china 8 . one sample in the current study collected in january 2020 was closely related to the sars-cov-2 strain circulating in china at that time identified as type l. the first outbreak in thailand was evidently associated with a boxing stadium and entertainment venues in bangkok during march 2020 12 . all the samples associated with that outbreak analysed in the present study were type t. type t branched off from type s, which originated from china, but type t has not been identified in other countries. this indicated local transmission in bangkok. interestingly, after the first outbreak in march 2020 type t was detected less frequently. this may have been a result of intervention policies such as mandatory closure of sporting and entertainment venues (fig. 1) . the mandatory closure of public places may help to control local transmission. notably however, there were several cases of patients who had recently returned from outside of thailand testing positive for sars-cov-2. they included multiple genetic variants such as types gh, gr and o. in march 2020 the patients classified as imported cases-including returned travellers and the group who had undertaken a religious pilgrimage from the southern part of thailand 23 -were identified as having type o. after the land border closure and suspension of all international flights, the number of cases decreased (fig. 1) . these interventions may help to limit imported cases. a new cohort of imported cases identified in may 2020 included a group of migrant workers in the southern part of thailand 24 with type g2 sars-cov-2. this indicated multiple introductions of sars-cov-2, and that there may be an outbreak in the southern part of thailand. in the current study no specific clinical signs were significantly associated with any specific sars-cov-2 types. upper respiratory infection, fever, coughing, sore throat, and runny nose were the most common symptoms in covid-19 patients, as has been frequently previously reported [25] [26] [27] [28] . clinical outcomes may associate with host factors such as age, lymphocytopenia, and cytokine responsiveness rather than sars-cov-2 genetic factors 29 . as of 22 may 2020 only one of the forty patients involved in the present study had been admitted to the icu, and all had been discharged from hospital. in the 40 patients analysed in the present study the clinical course of covid-19 was generally mild. the percentage of patients admitted to the icu was 2.8%, the percentage with concurrent pneumonia was 22.2%, the percentage who were asymptomatic was 36.1%, and there were no fatalities, suggesting that sars-cov-2 does not usually lead to severe disease, unlike sars-cov and mers 30, 31 . these clinical data are similar to reports derived from china in which approximately 80% of confirmed cases were considered mild, 15% of confirmed cases were diagnosed as severe with pneumonia, and approximately 5% were deemed critical cases 32 . the reported case-fatality rate of covid-19 in thailand (1.9%) is lower than that of sars (22%) 30 , as it is in several countries including italy (9.3%), iran (7.8%), spain (6.2%), the uk (4.9%), the netherlands (4.3%), france (4.2%), china (4.0%), and the usa (1.3%) 33 . reports suggest that elderly covid-19 patients are at higher risk of hospitalization, pulmonary complications, and death 25, 28, 34 , as are elderly sars and mers patients 35, 36 . the multiple origins of sars-cov-2 transmission into thailand identified in the current study via phylogenetic analysis are similar to the pattern identified in shanghai 29 . our study had some limitations. we did not analyse the whole-genome sequence for all samples due to time and cost. therefore, we may have missed some genetic polymorphisms, which could potentially be of interest and reveal novel subclades to sars-cov-2. due to the small sample size, a significant correlation between clinical symptoms and the viral genetic variation was not obtained. the samples we obtained encompassed the peak outbreak activity in thailand at that time, so although they are limited in numbers, we believe that they may be sufficiently representative despite the relatively low sample size compared to other studies. in summary, in the present study sars-cov-2 tracking and sites of origin were investigated in thailand via genetic analysis. most patients exhibited mild febrile illness without sequelae, but multiple origins of sars-cov-2 were evident. understanding viral genetic and transmission patterns may facilitate more accurate 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characteristics and prognostic factors based on 4-week follow-up the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update j.p. collected data and drafted the manuscript. k.s. analyzed data and drafted the manuscript. j.c. performed sequence analysis and edited the manuscript. p.n., r.y. and c.a. performed genome sequencing and analyzed data. r.k., a.m. and a.k. collected and provided specimens. n.w., c.c. and y.p. supervised the study and finalized the manuscript. all authors reviewed the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-73554 -7.correspondence and requests for materials should be addressed to y.p.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-290056-x74cq2k5 authors: delgado-roche, livan; mesta, fernando title: oxidative stress as key player in severe acute respiratory syndrome coronavirus (sars-cov) infection date: 2020-04-30 journal: arch med res doi: 10.1016/j.arcmed.2020.04.019 sha: doc_id: 290056 cord_uid: x74cq2k5 abstract the emergence of viral respiratory pathogens with pandemic potential, such as severe acute respiratory syndrome coronavirus (sars-cov), the pathogenic agent of covid-19, represent a serious health problem worldwide. respiratory viral infections are, in general, associated with cytokine production, inflammation, cell death, and other pathophysiological processes, which could be link with a redox imbalance or oxidative stress. these phenomena are substantially increased during aging. actually, severity and mortality risk of sars-cov-2 infection or covid-19 disease have been associated with the age. the aim of the present work was to contribute with the understanding of the possible link between oxidative stress and the pathogenesis, severity and mortality risk in patients affected by sars-cov infection. respiratory viral infections represent a group of diseases that affect millions of people worldwide, mainly kids and elderly people. respiratory viruses, which comprises influenza (iv), human respiratory syncytial (hrsv), human rhinovirus (hrv), human metapneumovirus (hmpv), parainfluenza, and adenovirus and coronavirus (cov), can infect the upper and/or lower respiratory tract in humans. many of them cause common clinical signs and symptoms, including nasal congestion, cough, sore throat, and fever, or more specific and severe manifestations, such as bronchiolitis, pneumonia and severe acute respiratory syndrome (1) . respiratory viral infections are, in general, associated with cytokine production, inflammation, cell death, and other pathophysiological processes, which could be link with a redox imbalance or oxidative stress (os). it is known that an overproduction of reactive oxygen species (ros) and antioxidant mechanisms deprivation are crucial for viral replication and the subsequent virus-associated disease (2). infection is characterized by fever, severe pneumonia, rnaaemia, combined with the incidence of ground-glass opacities, and acute cardiac injury. of note, a significant high blood levels of cytokines and chemokines have been observed in patients with covid-19 infection. the "cytokine storm" trigger a proinflammatory environment which is strongly associated with a severe tissue damage, contributing with the fatal outcomes of covid-19 patients (7) . it is well established the link between inflammation and os (8) . thus, the aim of the present work was to review the role of os in the pathogenesis and progression of respiratory diseases caused by sars-covs. os has been defined as a disturbance in the prooxidant-antioxidant balance in favor of the prooxidants, leading to cell damage (sies, 1991) . however, the current knowledge on the redox signaling pathways has been reconceptualized os, including two main mechanisms: (1) macromolecular damage, and (2) disruption of thiol redox circuits, which leads to aberrant cell signaling and dysfunctional redox control (10, 11) . the disruption of redox circuits caused by specific infections have been associated with inhibition of nrf2 pathways and/or nf-kb signaling activation, leading to inflammation and oxidative damage (12) . while there is a clear correlation between os markers and the severity of many viral diseases such as hepatitis c (hcv), for sars-cov clinical data is limited. however, in the preclinical setting, many lines of evidence suggest that overproduction of ros and a deprived antioxidant system plays a major role in the pathogenesis of sars-cov infection, as well as in the progression and severity of the respiratory disease. experimental animal models of severe acute respiratory syndrome have shown an enhanced ros levels and disturbance of antioxidant defense during sars-cov infection (13) . some authors suggest that the onset of severe lung injury in sars-cov infected patients depends on activation of the oxidative stress machinery that is coupled with innate immunity and activates transcription factors, such as nf-κb, resulting in an exacerbated proinflammatory host response (14) . lin and coworkers (15) , showed that sars-cov 3cl pro (a viral protease) caused a arch med res e20_477 5 significant increase in ros production in hl-cz cells, which in turn, is involved in 3cl pro -induced cell apoptosis. in this study, the authors demonstrated that sars-cov 3cl pro activates nf-kbdependent reporter gene, which was correlated with the increase of ros levels in hl-cz cells. consensus nf-kb sites exist in the promoters of apoptosis related genes and proinflammatory genes. therefore, the authors suggest that ros-activated nf-kb signal transduction pathway, induced by sars-cov 3cl pro , might be considered a key player in sars-cov pathophysiology. in addition, other sars-cov´s protease, the 3a protein, has been associated with the activation of mitochondrial cell death pathways (intrinsic and extrinsic signaling). the proposed mechanism involves bax oligomerization and higher levels of p53 in 3a protein-expressing huh7 cells, which depended on the p38 mapk activation in these cells (16) . mitogen-activated protein kinase (mapk) are a family of serine/threonine that are activated in response of environmentally stresses including oxidative stress, dna damage, carcinogenic stimuli and viral infections. activated (phosphorylated) forms of all mapk members has been detected in cells infected with sars-cov (2). techniques, in which hallmarks of apoptosis were observed in sars-cov-infected lung, spleen, and thyroid tissues. also, activation of apoptosis induced by sars-cov, in particular hcovs, was described in previous in vitro studies and animal models (17) . apoptosis can be induced by multiple mechanisms in hcov-infected cells. cov was shown to induce caspase-dependent apoptosis, needed for viral replication (18) . literature reports suggested that sars-cov replicates in vero-e6 cells inducing a weak akt signaling pathway activation, which cannot prevent apoptosis induced by sars-cov infection (19) . activation of the pi3k/akt signaling pathway by a variety of viruses is thought to be involved in the establishment of latent and chronic infections by allowing virus-infected cells to escape from apoptosis. activation of the pi3k/akt signaling pathway may lead to a delay in oxidative stress -nf-kb -toll-like receptor (mainly tl4) signaling pathways, triggered by viral pathogens like sars-cov, may further amplify the host inflammatory response, ultimately leading to acute lung injury. tlr4-trif-traf6 signaling was identified as a pathogenic pathway that can mediate the severity of acute lung injury. the oxidized phospholipid, generated by lung macrophages, was able to induce cytokine overproduction and lung injury via tlr4-trif. oxidized phospholipids have been previously identified in human and animal lungs infected with sars virus. in in vivo models, loss of tlr4 or trif expression protected mice from h5n1-induced acute lung injury (ali). in addition, deletion of ncf1, which can regulate ros generation, improves the severity of ivinduced ali. thus, these authors suggest that oxidative stress and innate immunity play key roles in the severity of ali caused by respiratory viruses (22) . in the clinical setting, shao and coworkers (23) observed an upregulation of mitochondrial genes and genes responding to oxidative stress in peripheral blood mononuclear cells (pbmc) of convalescent sars-cov patients. some of these genes, including prdx1, fth1, and fos are sensitive to oxidative stress, showed a remarkable elevation. in addition, stress response protein dnajb1, differentiation-associated gene ifrd1, cytokine il-1b, and other genes were overexpressed in pbmc of sars-cov infected patients. these results support the association between oxidative stress, inflammation and the pathogenesis of sars-cov infection (23) . the severity and mortality risk of sars-cov-2 infection or covid-19 disease have been associated with the age (24) . based on current data, the mean case fatality rate for adults aged under 60 is estimated to be less than 0.2%, compared with 9.3% in those aged over 80. furthermore, comorbidities such as diabetes, obesity, and hypertension increased mortality risk by five times, however the risk seems to be lower for younger patients than older subjects (25) . in a large study analyzed 4021 confirmed cases of cov pneumonia, the results showed that 1 052 (26.2%) patients were aged at 60 years or older. the observed mortality rate of patients aged 60 years and over (5.3%) was significantly higher than that of patients under 60 years (1.4%) (26) . on the other hand, a retrospective study of patients with covid-19, who were hospitalized in hainan provincial people's hospital during january-february 2020, showed that elderly patients (32.14%) were more likely to progress to severe disease than youngers (27) . it is well established that antioxidant mechanisms deprivation together with oxidative damage accumulation occur during ageing process (28, 29) . it has been observed that in aged mice there are more severe sars-cov-induced lung lesions than in young-adult mice. the transcription profile in aged mice generally indicated a stronger proinflammatory environment than in young animals. it is suggested that age related accumulated oxidative damage and a weakened antioxidant defense system cause a disturbance in the redox balance, resulting in increased reacting oxygen species. therefore, ageing is not only associated with alterations in the adaptive immune response, but also with a proinflammatory state in the host. in macaques, a stronger host response to virus infection than young adult macaques, with an increase in differential expression of genes associated with inflammation, arch med res e20_477 8 with nf-kb as central player, has been documented (30) . in conclusion, literature evidence suggests that oxidative stress and chronic low-grade inflammation in the lungs are associated with aging and may contribute to age-related immune dysfunction and mortality risk in aged patients affected by respiratory virus infections, such as sars-cov-2. the authors declare no conflict of interests. pathophysiology of clinical symptoms in acute viral respiratory tract infections redox biology of respiratory viral infections pathology and pathogenesis of severe acute respiratory syndrome coronavirus pathogenesis. 1 st ed. advances in virus research defining the epidemiology of covid-19 -studies needed coronavirus covid-19 global cases by the center for systems science and engineering clinical features of patients infected with 2019 novel coronavirus in wuhan, china oxidative stress: a concept in redox biology and medicine oxidative stress: from basic research to clinical application redefining oxidative stress radical-free biology of oxidative stress respiratory viral infections and subversion of cellular antioxidant defenses the pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models low dose ionizing radiation produces too few reactive oxygen species to directly affect antioxidant concentrations in cells severe acute respiratory syndrome coronavirus 3c-like protease-induced apoptosis severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 map kinase activation severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase r but is resistant to its antiviral activity human coronavirus: host-pathogen interaction importance of akt signaling pathway for apoptosis in sars-cov-infected vero e6 cells inhibition of akt kinase activity suppresses entry and replication of influenza virus anti-influenza a virus activity of rhein through regulating oxidative stress, tlr4, akt, mapk, and nf-κb signal pathways identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury upregulation of mitochondrial gene expression in pbmc from convalescent sars patients covid-19 -navigating the uncharted covid-19: risk factors for severe disease and death the reproductive number of covid-19 is higher compared to sars coronavirus clinical features of covid-19 in elderly patients: a comparison with young and middle-aged patients oxidative stress in the aging process: fundamental aspects and new insights the oxygen paradox, oxidative stress, and ageing exacerbated innate host response to sars-cov in aged non-human primates the authors would like to acknowledge all the healthcare professionals which are in the first line against covid-19 disease worldwide. key: cord-279940-i2rgjpxf authors: comentale, giuseppe; manzo, rachele; pilato, emanuele title: sars-cov-2 interference in heme production: is it the time for an early predictive biomarker? date: 2020-06-29 journal: j mol med (berl) doi: 10.1007/s00109-020-01945-4 sha: doc_id: 279940 cord_uid: i2rgjpxf nan sars-cov-2 is a new single-stranded rna virus that seems to have brought the entire planet to its knees in the last months. first discovered in a seafood market of wuhan, it causes a particular kind of interstitial pneumonia and respiratory failure that results in patient's urgent need of respiratory mechanical support [1] . this has a high impact on the hospitals congestion conditioning and in turn also its mortality rate. sars-cov-2 preferentially infects alveolar epithelial cells through its spike protein causing the disruption of the alveolocapillary membrane that leads to hypoxemic respiratory failure and radiological "ground-glass" pattern [2] . infection is further characterized by high c-reactive protein and ferritin levels, anaemia and diffuse thrombosis [3, 4] . in particular, thrombosis seems to drive the entire disease course: sars-cov-2 infection triggers a large thrombophilic response that results in diffuse occlusion of the smaller vessels, especially in the lungs where the result is a wide thrombotic microangiopathy [5] explaining the radiologic "ground glass" pattern. this hypothesis is confirmed using clinical and autoptic evidence of diffuse arterial and vein thrombosis among covid-19 patients [6] who benefit greatly from anticoagulant therapy [7] . this thrombophilic state could be triggered by the wide and powerful inflammatory response to the sars-cov-2 infection and its cytokine storm [8] . accordingly, covid-19 patients have high serum levels of il-6 correlated with the severity of the respiratory failure [9] . a multicentre trial is actually ongoing to test whether tocilizumab, a well know il-6 blocker routinely used to treat chronic inflammation of rheumatoid arthritis, can be used to mitigate the negative effects of the inflammation and to reduce the pulmonary damage seen in these patients (clinicaltrials. gov identifier: nct04317092). not only il-6 but also the proinflammatory cytokine il-1 was proposed to have a role in the overwhelming inflammatory response of infected patients [10] . furthermore, as il-1 was shown to be a major culprit in the development of many cardiovascular diseases [11] , its involvement in the sars-cov-2 infection could explain the high mortality and morbidity rate among cardiopathic patients. one of the most important lessons learned from previously treated covid-19 patients is that early diagnosis and treatment can drastically improve the prognosis [12] . in view of this, a strategy focused on screening would guarantee the identification of asymptomatic patients where an early appropriate treatment could be the key to fighting this virus. this approach, however, needs a low cost, fast, and accurate screening method which, unfortunately, today is yet to be developed. identifying the mechanisms by which sars-cov-2 damages the human body, focusing on the structural proteins, could be a possible strategy to finding an effective solution. recently, liu et al. reported the binding capacity of some structural virus proteins, such as orf8 and orf10, on the porphyrins and heme group [13] . the authors showed that the binding of these proteins could inhibit the iron inclusion in the porphyrins, causing an increase in their concentrations and a reduction in haemoglobin synthesis. in addition, orf 10 was shown to attack the 1-β chain of the haemoglobin causing dissociation of the heme group and the loss of its iron ions resulting in increased serum iron and in ferritin production. free iron ions lead to an increased cellular oxidative stress and could be one of the possible mechanisms through which sars-cov-2 triggers the inflammatory response that destroys the lung alveolar cells. on the other hand it could stimulate ferritin production, aminolevulinic acid (ala) synthetase transcription and, so, increased levels of porphobilinogen and ala. even if its involvement is not yet well described, ala excess could explain, like in acquired acute porphyria, extrapulmonary symptoms of covid-19 patients like gastrointestinal, neurological and neuropsychiatric ones. therefore, sars-cov-2, like hepatitis c virus and human immunodeficiency virus, could lead to a particular form of porphyria. this mechanism could also explain why traditional molecules such as chloroquine show promising activity in treating covid-19 patients, even if no clear evidence is available regarding the true effectiveness and safety of this drug. severe and life-threatening side effects can usually occur, but the lack of a standardized and useful treatment makes the risk acceptable compared with the acute respiratory distress syndrome or multiorgan failure related death. it is hypothesised that chloroquine could act via an interference with the endocytosis mechanism of the virions. furthermore, according to the identified side effects including cardiac ones (i.e., qt prolongation), probably, there are other collateral pharmacodynamic mechanisms occurring involving specific ions channels. if demonstrated, this mechanism could be a tool to open the way to new strategies in early detection of infected individuals. from this point of view, sars-cov-2 seems to be very similar to malaria: many clinical and scientific reports have shown that covid-19, not only can be successfully treated with chloroquine but also, like malaria, appears to be diagnosed much more frequently in blood group a patients [14] . based on these results, it is reasonable to assume that low functional heme group levels can increase their production through a positive feedback loop. in conclusion, if an increase in heme production pathway brings increased precursor synthesis, could we routinely dose marker for lead exposure or porphyria, such as serum porphobilinogen and aminolaevulinic acid, as biomarkers in the stages of infection? to date, there seem to be no further published or ongoing studies about the link between sars-cov-2 and porphobilinogen and ala synthesis. these two molecules, instead of others such as ferritin or bilirubin that can increase in many conditions, could represent not only a sensitive biomarker of sars-cov-2 infection but also a specific and low-cost diagnostic test. voided urine collection can be easily done at home by patients, and, unlike the nasopharyngeal swab specimen, it usually does not require healthcare intervention. furthermore, it allows obtaining a lot of samples on which ala and porphobilinogen can be tested. this could be useful for two reasons: it could allow reducing the impact of the infection on the health system congestion, but, on the other hand, it could also provide a useful method to screen and to follow up very quickly the population without the need of the healthcare professional intervention. in addition, if these indicators would reflect the damaging activity of the virus, they could potentially be used as a marker to monitor the response to the treatments. further investigations are maybe necessary to assess this role, but these hypotheses could be also a food for thought to better understand the sars-cov-2 pathogenetic mechanisms and to find new methods to fight against it. epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid-19) during the early outbreak period: a scoping review coronavirus (covid-19) outbreak: what the department of radiology should know hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19): a meta-analysis covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and followup incidence of thrombotic complications in critically ill icu patients with covid-19 venous thromboembolism in covid-19 patients the versatile heparin in covid-19 will complement inhibition be the new target in treating covid-19 related systemic thrombosis? the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality precision medicine in covid-19: il-1β a potential target imbalance between interleukin-1β and interleukin-1 receptor antagonist in epicardial adipose tissue is associated with non st-segment elevation acute coronary syndrome early antiviral treatment contributes to alleviate the severity and improve the prognosis of patients with novel coronavirus disease (covid-19) covid-19: attacks the 1-beta chain of hemoglobin and captures the porphyrin to inhibit human heme metabolism relationship between the abo blood group and the covid-19 susceptibility conflict of interest the authors declare that they have no conflict of interest.ethical approval ethical approval is not required. key: cord-253665-1dn3ek34 authors: vishnubalaji, radhakrishnan; shaath, hibah; alajez, nehad m. title: protein coding and long noncoding rna (lncrna) transcriptional landscape in sars-cov-2 infected bronchial epithelial cells highlight a role for interferon and inflammatory response date: 2020-07-07 journal: genes (basel) doi: 10.3390/genes11070760 sha: doc_id: 253665 cord_uid: 1dn3ek34 the global spread of covid-19, caused by pathogenic severe acute respiratory syndrome coronavirus 2 (sars-cov-2) underscores the need for an imminent response from medical research communities to better understand this rapidly spreading infection. employing multiple bioinformatics and computational pipelines on transcriptome data from primary normal human bronchial epithelial cells (nhbe) during sars-cov-2 infection revealed activation of several mechanistic networks, including those involved in immunoglobulin g (igg) and interferon lambda (ifnl) in host cells. induction of acute inflammatory response and activation of tumor necrosis factor (tnf) was prominent in sars-cov-2 infected nhbe cells. additionally, disease and functional analysis employing ingenuity pathway analysis (ipa) revealed activation of functional categories related to cell death, while those associated with viral infection and replication were suppressed. several interferon (ifn) responsive gene targets (irf9, ifit1, ifit2, ifit3, ifitm1, mx1, oas2, oas3, ifi44 and ifi44l) were highly upregulated in sars-cov-2 infected nbhe cell, implying activation of antiviral ifn innate response. gene ontology and functional annotation of differently expressed genes in patient lung tissues with covid-19 revealed activation of antiviral response as the hallmark. mechanistic network analysis in ipa identified 14 common activated, and 9 common suppressed networks in patient tissue, as well as in the nhbe cell model, suggesting a plausible role for these upstream regulator networks in the pathogenesis of covid-19. our data revealed expression of several viral proteins in vitro and in patient-derived tissue, while several host-derived long noncoding rnas (lncrnas) were identified. our data highlights activation of ifn response as the main hallmark associated with sars-cov-2 infection in vitro and in human, and identified several differentially expressed lncrnas during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to sars-cov-2 remains to be investigated. coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was declared a global pandemic by the world health organization (who) on phenomenal changes in ncrna expression are also seen within host cells, which can play a major role in respiratory virus pathogenesis, with long non-coding rnas (lncrnas) exhibiting higher tissue specificity than coding genes [30] . lncrnas are around 200 base pairs in length and have been predicted to play a role in innate immune responses via their association with ifn mechanistic pathways [31] . understanding the effects of their differential expression and modes of action will immensely impact the fields of immunology and infectious diseases. whole transcriptome analysis of host response to sars-cov in mouse strains highlighted over 500 differentially expressed annotated lncrnas, which clearly showed association with innate immune signaling and pathogenesis regulation through signal transducer and activator of transcription 1 (stat1) [31] . in our current study, we employed modern computational genomics tools and performed more in-depth analysis of transcriptome data from primary normal human bronchial epithelial cells (nhbe) during the course of sars-cov-2 infection and lung biopsies derived from covid-19 patients from the blanco-melo et al. study, revealing multiple affected mechanistic networks and functional categories related to innate immunity, interferon activation and cellular response to viral infection [32] . additionally, we characterized the lncrna transcriptional portrait in response to sars-cov-2 infection in nhbe cells, as well as in lung biopsies derived from covid-19 patients. to our knowledge, this is the first study highlighting systemic alterations in cellular host response to sars-cov-2 infection in the context of lncrnas, adding to previously published data, in efforts to further grasp their potential utilization as disease markers or therapeutic targets. raw rna sequencing data were retrieved from the sequence read archive (sra) database under accession no. (prjna615032) [32] . detailed experimental procedures are explained in the aforementioned reference. single-end fastq files for mock (srx8089292, srx8089293, and srx8089294) and sars-cov-2 infected (srx7990869, srx7990870, and srx7990871) nhbe cells (24 h) as well as fastq files for lung biopsies from healthy (srx8089341 and srx8089342) and from patients with covid-19 (srx8089343 and srx8089344) were retrieved using the sra toolkit version 2.9.2 as previously described [33] . fastq files were subsequently mapped and aligned to the hg38 reference genome (including both protein coding and non-coding rnas) using built in rna-seq analysis module in clc genomics workbench 20.0 with default settings as we described before [34] . to detect viral genome transcriptome, raw sequence data were aligned to the ncbi viral genome reference sequence in clc genomics workbench 20.0 and subsequently mapped reads to severe acute respiratory syndrome coronavirus 2 isolate wuhan-hu-1, complete genome (ncbi reference sequence: nc_045512.2) was estimated. all expression values for mrna, lncrna, and viral genes from the current study are provided in supplementary table s1 . normalized expression data (tpm (transcripts per million) mapped reads) for protein coding were separated from those of lncrnas using biomart ensembl tool and were sequentially imported into altanalyze v.2.1.3 software for differential expression analysis using 2.0-fold change and <0.05 p-value cut-off. transcripts with raw expression values < 1.0 tpm were excluded from the analysis. hierarchical clustering was performed using cosine for columns and cosine for rows, and marker finder prediction as described before [35, 36] . cell type and gene expression explanations were allotted using a custom gene-set enrichment approach. cell type predictions were created from the software gene ontology (go)-elite in altanalyze using its previously defined cell and tissue marker gene database. this database encompasses markers for lots of nominated cell types and bulk tissue samples. the databases are created mainly from the ensembl database that includes all external id systems related to ensembl as well as supported platforms (e.g., illumina). relationships to numerous biological ontology (go, disease and phenotype), pathway and gene set resources are conserved. marker finder analysis was achieved within each experimental condition to predict specific markers based on gene-set enrichment by go-elite algorithm as described before [37] . upregulated genes were imported into the ingenuity pathways analysis (ipa) software (ingenuity systems; qiagen, redwood city, ca, usa) www.ingenuity.com/) and were subjected to functional annotations and regulatory network analysis using upstream regulator analysis (ura) to analyze upstream molecules, which are connected to genes in the dataset via a set of either direct or indirect relationship based on changes in expression. mechanistic networks (mn) analysis was performed using ipa to generate signaling cascades which connect upstream regulators to help visualize how they connect to explain the observed changes in gene expression. downstream effector analysis (dea) identifies the biological processes (disease) and functions, which are casually affected by deregulation of genes in datasets and predicts their activation state (z score). ipa uses precise algorithms to predict functional regulatory networks from gene expression data and provides a significance score for each network according to the fit of the network to the set of focus genes in the database. the score represents the negative log of the p-value for the probability that focus genes in the network are found together by chance [34] . statistical analyses and graphing were performed using microsoft excel 2016 and graphpad prism 8.0 software (graphpad, san diego, ca, usa). two tailed t-test was used for comparative groups. p-values ≤ 0.05 (two-tailed t-test) were considered significant. for ipa analyses, a z score (−2.0 ≥ z ≥ 2.0) was considered significant. to highlight the changes in cellular host genes in response sars-cov-2 infection, we employed recent rna-seq datasets utilizing the nbhe cell model. analysis of three biological triplicates of nhbe cells mock treated (control) or infected with sars-cov-2 (usa-wa1/2020) instigated differential expression of 377 upregulated and 3012 downregulated mrnas (figure 1a and supplementary table s2 ). a volcano plot (scatterplot) that shows statistical significance (log p value; y-axis) vs. magnitude of change (log fold change; x-axis) is depicted in figure 1b , with selected genes being displayed. the red colors (right) and blue colors (left) represent genes with significantly upregulated or downregulated expression in nhbe sars-cov-2 vs. mock-infected cells, respectively ( figure 1b) . transcriptome data were subsequently mapped and aligned to the ncbi viral genome reference sequence which revealed the expression of several sars-cov-2 viral genes, particularly open reading frame (orf) that codes for viral non-structural proteins (nsp), orf1ab_8; s_14; orf3a_9; e_10; m_51; orf6_14; orf7a; orf8_8; n_65; and orf10_9, except orf7b which was not expressed (figure 1c) . we subsequently employed the marker gene finder algorithm to identify genes associated with the sars-cov-2, employing the nhbe model. modules of co-expressed mrnas (120 genes) were associated with specific biological condition, (i.e., sars-cov-2), where the heatmap highlights differentially expressed mrnas that are positively correlated with an idealized cluster-specific expression profile. we found putative markers that are selectively expressed in sars-cov-2 vs. control infected nbhe cells. the text on the left side of the y-axis indicates enriched cell-type markers from the default gene-set enrichment analysis (gene ontology-elite) along with top default gene markers for each go term displayed on the right side of the y-axis on the heatmap. the color scale displays differential gene expression (log2). in sars-cov-2 infected cells, top modules of co-expressed genes were associated with acute inflammatory response, response to tnf and interferon gamma (infg), immune response, cell adhesion, leukocyte migration, cell projection, lipid transport, transcription from rna polymerase ii promotor, cytosol and extra cellar space, and cell projection, respectively. whereas cell cycle, mitosis, vascular membrane and mrna transport, and cellular development go classifications were enriched in the mock control group (figure 1d and supplementary table s3 ). canonical pathway analysis on the differentially expressed genes in sars-cov-2 nhbe cells using ingenuity pathway analysis (ipa) revealed downregulation of several canonical pathways, including ephrin receptor signaling (figure 2a and supplementary table s4 ). ipa upstream regulator analysis provides a powerful tool to predict the deregulated functional activities that are possibly affected by the transcriptome data. figure 2b horizontal bars denote the different upstream regulators (top 10 up and down regulated genes) in the sars-cov-2 group based on the z-scores. this analysis revealed significant enrichment in several functional classifications, those predicted to be activated (top 10) are associated with type i and iii interferon groups that play important roles against viral infection, mainly ifnl1 (il-29; ifn-lambda 1), ifng cytokines and igg complex, and lonp1, cst5, spi1, trap1, kdm5b, dap3, and let-7 microrna (mir/mirna). conversely, functional categories that were predicted to be inhibited (top 10) are interrelated to p53 binding and cell cycle (esr1, cab39l, syvn1, rabl6, tp63, brd4, mapk1, myc, mitf and erbb2 (figure 2b and supplementary table s5 ). igg complex and ifnl were observed among the top predicted upstream regulators in the sars-cov-2 group and were chosen for mechanistic network analysis to provide better understanding of the upstream regulators leading to downstream effector molecules and subsequent changes in gene expression. the predicted active state of igg complex was anticipated to activate the nfkb and rela complex through inhibiting erk directly. although mechanistic network analysis illustrates that the igg complex plays a role in the downregulation of tp63 and activation of rela through activation of tnf, the effect of this relationship is still under evaluation (figure 2c) . likewise, the predicatively active ifnl and its associated network molecules, are illustrated in figure 2d downstream effector analysis in ipa predicts the differences in the downstream biological functions that are presumed to be affected by the changes in the transcriptome. tree map (hierarchical heat map) portrays the affected downstream functional groups based on differentially expressed mrnas, where the major boxes represent a category of diseases and functions in the sars-cov-2 group. each individual rectangle (blue and orange colors) indicates the decreasing and increasing state, while rectangle dimensions using fisher exact test (fet) p-value is associated with increasing overlap significance, either up or down as a group, and color intensity stipulates higher absolute z-scores (figure 3a ). disease and function analysis on the differentially expressed genes revealed the most significant enrichment in pathways related to reactive oxygen species, induction of apoptosis and necrosis, as well as activation of neutrophils in sars-cov-2 infected nhbe cells (figure 3a,b) . on the other hand, the most suppressed functional category in sars-cov-2 infected nhbe cells was the infectious disease category, which includes replication and infection by viruses (supplementary table s6 ). ipa analysis revealed the suppression of viral infection and replication as the main affected functional category in sars-cov-2 nhbe cells. therefore, we sought to elucidate the expression of selected genes with known role in combating viral infection. figure figure (figure 4c ). the role of those lncrnas in the cellular response to sars-cov-2 remains to be investigated. transcriptome data of lung tissue from patients with covid-19 compared to healthy subjects from the blanco-melo study [32] were subjected to differential and biomarker discovery analysis (figure 5a ). differentially expressed mrnas and lncrnas in covid-19 compared to healthy control are listed in supplementary table s8 . functional enrichment studies show differentially expressed genes that positively correlate with an idealized cluster-specific expression profile in covid-19 lung tissue, while enriched gene ontology (go) associated with enriched go terms are indicated on the y axis. color scales display marker gene correlation and differential gene expression (log2). interestingly, several enriched go classifications in the covid-19 group were associated with immune response, negative regulation of viral genome replication, activation of jun, and regulation of nfkb pathways (figure 5a ). mapping and aligning rna-seq data to the ncbi viral genome reference sequence revealed expression of orf1ab_8, s_14, m_51, orf67a, orf8_8 and n_65 viral genes (figure 5b ). there was no detected expression of orf3a_9, e_10, orf6_14, orf7b and orf10_9 viral genes in covid-19 lung tissue (figure 5b) . in order to identify common genes and lncrnas in the context of sars-cov-2 infection, we compared the transcriptome of sars-cov-2 nhbe cells and lung tissue from patients with covid-19, and identified 27 common upregulated and 867 common downregulated genes (figure 5c) . similarly, comparing the lncrna transcriptome also revealed 5 common upregulated and 57 common downregulated lncrnas in sars-cov-2 nhbe and covid-19 patient-derived lung tissue (figure 5d ), suggesting a plausible role for these genes and lncrnas in host response to sars-cov-2 infection. the list of common altered mrnas and lncrnas in nhbe sars-cov-2 and covid-19 is provided in supplementary table s9. the top ten activated upstream regulator networks (cst5, ifng, ifnl1, ifna2, spi1, rny3, prl, tgm2 , mir-122 and mir-122-5p) in lung tissue derived from covid-19 patient based on transcriptome and ipa analyses, revealed the enrichment of functions related to immune system associated jak-stat cascade, type 1 interferon receptor binding, cytokine receptor binding, and mhc 1 biosynthesis (figure 6a and supplementary table s10 ). likewise, the ten most inhibited upstream regulator networks were mapk1, aspscr1-tfe3, ehmt1, syvn1, il1rn, btk, erg, tp53, uhrf2, and gper1, respectively (figure 6a) . when comparing affected upstream regulator networks in sars-cov-2 nhbe and lung tissue from covid-19 patients, we observed 14 common activated (ifnl1, cst5, spi1, trap1, ifng, nupr1, tgm2, smarcb1, rny3, stat1, irf9, prl, ifna2, and interferon α) and 9 common suppressed networks (tap1, gper1, tfeb, uhrf2, aspscr1-tfe3, il1rn, btk, syvn1, and mapk1, figure 6b the topmost enriched functional category based on rna-seq from lung-derived tissue from covid-19 patient using the regulator effect network analysis was the suppression of viral infection and replication (figure 7 ). the network combined differentially expressed potential upstream regulators (18, including 14 activated (orange; eif2ak2, ifna2, ifng, ifnl1, interferon α, irf9, jak, paf1, prl, rny3, sgpl1, spi1, stat1 and tgm2), four inhibited (blue; btk, mapk1, il1r1 and usp10)) and 18 mediator genes (including 17 increased and 1 decreased) in the middle of the hierarchy, which are involved in the inhibition of 5 major downstream effector functions such as viral infection and replication, and infection of epithelial cells. among the 18 upstream regulators, irf9, interferon α, ifna2 and eif2ak2 suppress downstream functions directly, as well as through the mediator genes, which collectively implies a massive ifng response and subsequent predicted suppression of viral replication (figure 7) . concordantly, disease and function analysis revealed viral infection as the most affected functional category in lung tissue from covid-19 patients (figure 8 and supplementary table s11 ). cumulative complex network depicts the viral infection functional category according to subcellular localization. the majority of the green color associations indicate the down regulation and the minority in red indicate the upregulation of appropriate molecules in the extracellular space, plasma membrane, cytoplasm and nucleus (figure 8 ). since the recent outbreak of covid-19, the number of affected individuals is rising expeditiously, claiming hundreds of thousands of lives thus far. such a crisis calls for prompt responses from research communities to better understand and contain this rapidly spreading pandemic. in our current study, we explored recent in vitro and patient-derived transcriptome datasets to dissect the cellular and molecular changes in response to sars-cov-2 infection using state-of-the art bioinformatics pipelines. in vitro and patient-derived data revealed a central role for ifn signaling in the host response to sars-cov-2 infection. as part of our innate anti-viral defense, host cells release interferons as the first warning to a suspected infection. interferons are a large group of signaling proteins (cytokines) that facilitate interference with viral replication as a defense mechanism, classed based on receptor signaling receptors and subsequent pathways, with some overlap. our data shows a number of differentially expressed genes within the interferon type i and type iii pathways in the sars-cov-2 infected cells compared to control nhbe cells. these genes (supplementary table s2 ) include stat2 and irf9, translocate to the nucleus and stimulate additional transcription of genes related to anti-viral responses, common to interferon type i and iii [39, 40] . in addition, other aberrantly expressed genes in response to viral infection include vav1, which is member of interferon type i and iii pathway, leading to camp response element-binding protein (creb) mediated chromatin remodeling, also contributing to gene specific repression [41] . furthermore, our data identified irs2, mtor genes, 4ebp1, eif4a3, and rps6, (components of type i and ii signaling pathways) to be differentially expressed, affecting mrna translation in response to interferon receptor signaling [42] . blanco-melo et al., also found a high level of chemokines induction, in concordance with our data, as well as high levels of type i and type iii interferon response in human adenocarcinoma alveolar basal epithelial (a549) cells expressing ace2, and in calu-3 host cells infected with sars-cov-2 [32] . in these models, a significant interferon i and iii response signature is seen, which is concordant with our findings. looking into changes in gene expression in lung biopsies from covid-19 patients revealed substantial induction of immune response (p = 5.7 × 10 −35 ), including innate immune response (p = 5.3 × 10 −29 ). in particular, we observed the activation of antiviral defense (p = 4.7 × 10 −19 ) mechanisms through upregulation of genes that inhibit viral replication (oas1, oas2, oasl, isg15, mx1, apobec3a, c19orf66, eif2ak2, ifit1, ifitm1, ifitm2, ifitm3, ltf, tnf, and zc3hav1). several of the upregulated genes are also known to play a role in preventing viral entry into host cells (fcn1, ifitm1, ifitm2, and ifitm3). a number of receptors which facilitate viral entry into host cells have been described. interestingly in a patient with active viral gene transcription, we observed upregulation of several surface receptors implicated in viral entry into host cells (cd4, cd86, cd80, ace2, cldn1, cd55, cr1, and clec5a). concordant with our findings, recent data revealed sars-cov-2 cell entry to depend on ace2 and tmprss2 [7, 24] . while ace2 was upregulated in covid-19 lung tissue, tmprss2 was severely downregulated (−7.1 fc, p = 0.007) suggesting downregulation of tmprss2 as a plausible mechanism through which the host counteracts sars-cov-2 infection. it is not clear why ace2 expression was unregulated in lung tissue from covid-19 patient. it is possible that ace2 expression is induced in response to interferon signaling. in fact, analyzing data from nhbe cells treated with infb revealed a time-dependent increase in ace2 expression in response to infb treatment (supplementary figure s2) . looking into commonalities in upstream analysis between the nhbe and covid-19 data, our data revealed 14 common activated (ifnl1, cst5, spi1, trap1, ifng, nupr1, tgm2, smarcb1, rny3, stat1, irf9, prl, ifna2, and interferon α) and 9 common suppressed (tap1, gper1, tfeb, uhrf2, aspscr1-tfe3, il1rn, btk, syvn1, and mapk1) networks. interestingly, our data highlighted a role for the activation states of paf1, ifnl1, ifng, stat1, rny3, spi1, jak, prl, sgpl1, tgm2, eif2ak2, ifna2, interferon α, and irf9 as the upstream regulators leading to subsequent activation of cybb, ifitm3, zc3hav1, ifitm1, ifitm2, mx1, tnf, oas3, isg15, apobec3a, ifit1, oas1, oasl, and stat2 activation, collectively leading to inhibition of viral genome replication. our data also highlights a central role for cst5 and ifng activation and suppression of mapk1 and interleukin-1 receptor antagonist (il1rn) in the host response against sars-cov-2 infection. downregulation of the mitogen-activated protein kinase 1 (mapk1) network was observed in sars-cov-2 infected nhbe as well as in covid-19 lung tissue. microrna translation of ifn-stimulated genes is additionally regulated through the mitogen-activated protein kinase (mapk) pathway [43] , as well as a variety of essential cell processes including mitosis, cell survival, apoptosis, metabolism and cell differentiation [44] . mapk1, or erk2, was found to be suppressed in the in vitro model and patient data in our current study. upon phosphorylation, mapk1 subsequently phosphorylates a number of transcription factors including fos, myc, egr-1, elk-1, and jun [45] . therefore, its suppression will ultimately affect transcriptional activation of further downstream genes. research into the ebola virus by zampieri et al., show glycoprotein mediated cytotoxicity as a result of mapk signaling cascade. this inhibition of erk2 was found to negatively affect cellular viability and integrin expression [46] . this is also in concordance with other studies that show inhibition of the erk pathway as a result of hepatitis c virus (hcv) and hiv type 1 infection [47, 48] . in addition to protein coding genes, our data also highlighted a number of lncrnas which were differentially expressed in covid-19 patient-derived lung tissue. for instance, using marker finder algorithm, upregulation of ac131011. 2 interestingly, we observed upregulation of metastasis-associated lung adenocarcinoma transcript 1 (malat1) and nuclear-enriched autosomal transcript 1 (neat1) lncrnas in nhbe cells in response to sars-cov-2 infection. despite the emergence of lncrnas as critical mediators of various biological processes, mechanistic roles for many lncrnas in viral infection are still poorly understood. studies on malat1 have shown this lncrna to be overexpressed in several cancer tissues, associated with high rates of metastasis, and poor prognosis in lung cancer [49] , breast cancer [50] , colon cancer [51] and esophageal cancer [52] , as well as several other cancer types. malat1 has also been implicated in the regulation of histone acetylation [53] , endothelial to mesenchymal transition (emt) [54] , and cardiac inflammation and dysfunction [55] . emerging research has highlighted yet another role for malat1 lncrna in viral infection and innate immune processes, affirming the crucial roles it plays in many biological processes. wei et al., investigates the role of malat1 in inflammatory injury following lung transplant, which could give plausible indicators for the role of malat1 in inflammation injury following sars-cov-2 infection. interestingly, silencing malat1 alleviated inflammatory injury by inhibiting neutrophil chemotaxis and immune cell infiltration to the site of infection [56] . they suggest this could regulate the progression of acute lung injury through nf-kb and p38 mapk pathways [57] , however is it also plausible that the inhibition of neutrophil chemotaxis lightens the burden of cytokine storms in lung inflammation injury. bhattacharyya et al., describe the role of malat1 in two flaviviruses; japanese encephalitis virus (jev) and west nile virus (wnv). neuro2a cells treated with these viruses show malat1 overexpression, promoting inflammatory response [58] . malat1, along with lncrna neat1, have been shown to be potential biomarkers for hiv infection, after the detection of high levels of both lncrnas in peripheral blood mononuclear cells (pbmcs) upon infection [59] . qu et al., found malat1 to promote hiv-1 transcription and infection by alleviating the epigenetic silencing of hiv-1 transcription via its interaction with enhancer of zeste homolog 2 (ezh2), which binds the hiv-1 promoter [60] . neat1 knockout mice present enhanced inflammation through the activation of nlrp3 and nlrc4 inflammasomes, promoting inflammatory mediated cell death in vivo [61] . in addition to this, neat1 has also been shown to be involved in hiv-1 replication in infected cells. the knockdown of neat1 enhanced viral production by promoting nucleus-to cytoplasm export of hiv-1 mrna transcripts in hela cells [62] . the current data provides us with plausible indicators surrounding the involvement of such lncrnas in the progression of sars-cov-2 infection. the specific mechanisms, whether it be via the regulation of inflammation, epigenetic silencing of target genes or dysregulation of gene expression are yet to be deciphered. further research into the roles of malat1 and neat1 in the context of covid-19 will be valuable in further understanding the mechanism behind disease progression in the perusal of potential biomarkers and therapeutic intervention. our results highlighted activation of ifn and inflammatory response as the main hallmark associated with sars-cov-2 infection, and identified several differentially expressed lncrnas during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to sars-cov-2 remains to be investigated. interferon and inflammatory response to sars-cov-2 infection might provide explanation to cytokine storms associated with severe covid-19 cases. supplementary materials: the following are available online at http://www.mdpi.com/2073-4425/11/7/760/s1. figure s1 : expression of selected antiviral host response genes in calu-3 cells infected with sars-cov-2. figure s2 : expression of ace2 in nhbe cells response to ifnb based on rnaseq analysis. table s1 . mapped reads expression values (tpm) for mrna, lncrna, and viral genes in all samples analyzed in current study. table s2 : differentially expressed genes in sars-cov-2 infected vs. control nhbe cells. table s3 : clustering marker gene correlations for sars-cov-2 infected and control nhbe cells. table s4 : affected canonical pathways based on ipa analysis of differentially expressed mrnas in sars-cov-2 infected nhbe cells. table s5 : upstream regulator analysis based on ipa analysis of differentially expressed mrnas in sars-cov-2 infected nhbe cells. table s6 : disease and function analysis based on ipa analysis of differentially expressed mrnas in sars-cov-2 infected nhbe cells. table s7 : differentially expressed lncrna genes in nhbe sars-cov-2 infected cells. table s8 : differentially expressed genes in lung tissue from covid-19 compared to healthy subject. table s9 . common upregulated and downregulated lncrnas in sars-cov-2 nhbe and covid-19 patient. table s10 : affected upstream regulator networks in lung tissue from covid-19 patient compared to healthy subject 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transcript 1 (neat1) and metastasis associated lung adenocarcinoma transcript 1 in the peripheral blood of hiv-1-infected patients long noncoding rna malat1 releases epigenetic silencing of hiv-1 replication by displacing the polycomb repressive complex 2 from binding to the ltr promoter the lncrna neat1 promotes activation of inflammasomes in macrophages neat1 long noncoding rna and paraspeckle bodies modulate hiv-1 posttranscriptional expression this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this work was supported by startup grant from qatar biomedical research institute (qbri) for nehad alajez (qb13). the authors declare no conflict of interest. key: cord-304792-8sdxqmkb authors: khan, md. abdullah-al-kamran; islam, abul bashar mir md. khademul title: sars-cov-2 proteins exploit host’s genetic and epigenetic mediators for the annexation of key host signaling pathways that confers its immune evasion and disease pathophysiology date: 2020-05-08 journal: biorxiv doi: 10.1101/2020.05.06.050260 sha: doc_id: 304792 cord_uid: 8sdxqmkb the constant rise of the death toll and cases of covid-19 has made this pandemic a serious threat to human civilization. understanding of host-sars-cov-2 interaction in viral pathogenesis is still in its infancy. in this study we aimed to correlate how sars-cov-2 utilizes its proteins for tackling the host immune response; parallelly, how host epigenetic factors might play a role in this pathogenesis was also investigated. we have utilized a blend of computational and knowledgebase approach to elucidate the interplay between host and sars-cov-2. integrating the experimentally validated host interactome proteins and differentially expressed host genes due to sars-cov-2 infection, we have taken a blend of computational and knowledgebase approach to delineate the interplay between host and sars-cov-2 in various signaling pathways. also, we have shown how host epigenetic factors are involved in the deregulation of gene expression. strikingly, we have found that several transcription factors and other epigenetic factors can modulate some immune signaling pathways, helping both host and virus. we have identified mirna hsa-mir-429 whose transcription factor was also upregulated and targets were downregulated and this mirna can have pivotal role in suppression of host immune responses. while searching for the pathways in which viral proteins interact with host proteins, we have found pathways like-hif-1 signaling, autophagy, rig-i signaling, toll-like receptor signaling, fatty acid oxidation/degradation, il-17 signaling etc significantly associated. we observed that these pathways can be either hijacked or suppressed by the viral proteins, leading to the improved viral survival and life-cycle. moreover, pathways like-relaxin signaling in lungs suggests aberration by the viral proteins might lead to the lung pathophysiology found in covid-19. also, enrichment analyses suggest that deregulated genes in sars-cov-2 infection are involved in heart development, kidney development, age-rage signaling pathway in diabetic complications etc. might suggest why patients with comorbidities are becoming more prone to sars-cov-2 infection. our results suggest that sars-cov-2 integrates its proteins in different immune signaling pathway and other cellular signaling pathways for developing efficient immune evasion mechanisms, while leading the host to more complicated disease condition. our findings would help in designing more targeted therapeutic interventions against sars-cov-2. though several human coronaviruses outbreaks caused severe public health crisis over the 79 past few decades, the recent coronavirus disease (covid-19) outbreak caused by the severe 80 acute respiratory syndrome coronavirus 2 (sars-cov-2) has beaten the records of the 81 previous and still the case counts are still on the upswing. about 210 countries and territories 82 around the globe has been affected by this outbreak and around a total of 2 millions of people 83 are already infected with sars-cov-2 and the number is steadily rising till the date of 84 writing this article (worldometer, 2020) . out of the closed cases, almost 20% of the patients 85 have suffered death and about 5% of the active cases are in critical conditions (worldometer, 86 2020) . though the death rates from covid-19 was estimated to be as small as 3.4% (who, 87 2020), at present the global fatality rate is changing very rapidly; therefore, more 88 comprehensive studies needto be done for the efficient controlling to overturn this pandemic. 89 coronaviruses are single stranded positive sense, enveloped rna virus having ~30kb 90 genome (lu et al., 2020) . amongst the four genera, sars-cov-2 (accession no. 91 nc_045512.2) belong to the betacoronavirus genus and it has ~29.9kb genome encoding 11 92 genes (ncbi-gene, 2020 between sars-cov and sars-cov-2; as in sars-cov-2, there have been 380 amino acids 98 substitution, deletion of orf8a, elongation of orf8b, and truncation of orf3b observed 99 (lu et al., 2020) . 100 though the overall mortality rate from sars-cov is higher than that of sars-cov-2, 101 several unique features of sars-cov-2 like-increased incubation period and dormancy 102 inside the host, thus spreading more efficiently (lauer et al., 2020) . this suggests that sars-103 cov-2 might be using some immune evasion strategies to maintain its survival and essential 104 functions within its host. 105 upon viral infection, host innate immune system detects the virion particles and elicits the 106 first sets of antiviral responses (katze et al., 2008) to eliminate the viral threats. however, 107 viruses itself have generated various modes of actions to evade those immune response by 108 modulating the host's intracellular signaling pathways (kikkert, 2020) . this arm-wrestling 109 between the host and the infecting virus results in the immunopathogenesis. different human 110 coronaviruses also show similar features of host-pathogen interactions, which ranges from the 111 viral entry, replication, transcription, translation, assembly to the evasion from host innate 112 immune response (fung and liu, 2019) . not only this but also different antiviral cellular 113 responses like-autophagy (ahmad et al., 2018) , apoptosis (barber, 2001) etc. can also be 114 moderated by the virus to ensure its survival inside the host cells. apart from these, several 115 other host-virus interactions are also observed like-modulation of the activity of host 116 transcription factors (lyles, 2000) , host epigenetic factors (e.g. histone modifications, host 117 mirnas etc.) (adhya and basu, 2010) . all of these multifaceted interactions can lead to the 118 ultimate pathogenesis and progression of the disease. 119 the interplay between different human coronaviruses and host was previously reported (fung 120 and liu, 2019), however, sars-cov-2 interactions with the host immune response and its 121 outcome in the pathogenesis are still to be elucidated. gordon we have obtained the transcription factors (tfs) which bind to the given promoters from 181 cistrome data browser (zheng et al., 2018 ) that provides tfs from experimental chip-seq 182 data. we utilized "toolkit for cistromedb", uploaded the 5kb upstream promoter with 1kb 183 downstream from transcription start site (tss) bed file of the deregulated genes and fixed 184 the peak number parameter to "all peaks in each sample". 185 we extracted the experimentally validated target genes of human mirnas from mirtarbase 187 database (huang et al., 2019a) . 188 we have downloaded the experimentally validated tfs which bind to mirna promoters and 190 module it. we have considered those tfs that are expressed itself and that can 'activate' or 191 'regulated' mirnas. 192 we used epifactors database (medvedeva et al., 2015) to find human genes related to 194 epigenetic activity. 195 we have utilized kegg mapper tool (kanehisa and sato, 2020) for the mapping of 197 deregulated genes sars-cov-2 interacting host proteins in different cellular pathways. we 198 then searched and targeted the pathways which are found to be enriched for sars-cov-2 199 deregulated genes. from these pathway information, we have manually sketched the 200 pathways to provide a brief overview of the interplay between sars-cov-2 and host 201 immune response, their outcomes in the viral pathogenesis. 202 differentially expressed genes showed that deregulated genes of sars-cov-2 infection can 214 exert biological functions like-regulation of inflammatory response, negative regulation of 215 type-i interferon, response to interferon-gamma, interferon-gamma mediated signaling, 216 nik/nf-kappab signaling, regulation of apoptotic process, cellular response to hypoxia, 217 angiogenesis, negative regulation of inflammatory response, zinc ion binding, calcium ion 218 binding etc. which were not enriched for sars-cov infection ( figure 1a , 1b). also, 219 different organ specific functions like-heart development, kidney development etc. were only 220 enriched for differentially expressed genes in sars-cov-2 infection ( figure 1a ). 221 deregulated genes of sars-cov-2 infection were found to be related to pathways like-nf-222 kappab signaling, jak-stat signaling, rig-i-like receptor signaling, natural killer cell 223 mediated cytotoxicity, phagosome, hif-1 signaling, calcium signaling, gnrh signaling, 224 arachidonic acid metabolism, insulin signaling, adrenergic signaling in cardiomyocytes, 225 ppar signaling etc. ( figure 1c , supplementary figure 1, 2) which were found to be absent 226 for sars-cov infection. 227 cov 3p, hsa-mir-146a-5p, hsa-mir-155-5p, hsa-mir-146b-5p, hsa-mir-27a-3p, hsa-mir-146b-260 3p, hsa-mir-141-5p) were found to be targeting in both infections. 261 although there are some similarities between sars-cov and sars-cov-2 genetic 286 architecture, it is yet to know if they modulate common host pathways. also, it is largely 287 unknown how sars-cov-2 uniquely exhibit some unique clinical features even having 288 much similarities of the viral genes. 289 as now the probable genetic and epigenetic regulators behind the differential gene expression 290 have been identified, we aimed to explore how these deregulated genes are playing a role in 291 the battle between virus and host. to obtain a detailed idea of the outcomes resulting from 292 viral-host interactions and how sars-cov-2 uses its proteins to evade host innate immune 293 response, we have mapped the significantly deregulated genes and host interacting protein in 294 different overrepresented functional pathways using kegg mapper ( phagosome ( figure 13a ), arachidonic acid metabolism ( figure 13b ), pvr signaling ( figure 302 14) etc., aberration of these pathways might provide sars-cov-2 an edge over the host 303 immune response. also, sars-cov-2 can prevent the relaxin downstream signaling ( figure 304 15) which plays a crucial role in lung's overall functionality and its abnormal regulation 305 might results in the respiratory complications found in covid-19. 306 from previous studies we have compiled information on deregulated genes (blanco -melo et 307 al., 2020) and virus-host interactome (gordon et al., 2020) in sars-cov-2 infection, 308 however, to get detailed pictures of the affected pathways, which is still remained obscure, 309 we have investigated how our identified host genetic and epigenetic factors are playing a role 310 and how viruses are utilizing those. giving a closer look we have found some pathways 311 which sars-cov-2 might be using but not sars-cov. figure 13a) . 357 apoptosis is one important intracellular host immune response to reduce further spread of 360 viruses from the infected cells (barber, 2001) . several signaling pathways are involved to 361 elicit this apoptotic response inside the infected cells which can be suppressed by the viral 362 proteins, for example-nsp12 is found to target rip1, thus it might fail to relay signaling to 363 casp8/fadd mediated apoptosis, and necrosis by rip1/rip3 complex ( figure 8) ; nsp5 364 might block glutathione peroxidase which is involved in 15(s)-hete production and 365 ultimately 15-oxoete production, thus apoptotic induction by these metabolites through 366 pparγ signaling axis will not take place ( which in turn results in vascular remodeling and leakage of inflammatory cytokines (powell 400 and rokach, 2015). 5-oxoete another arachidonic acid metabolism product that can also 401 induce inflammation (powell and rokach, 2015) , production of both compounds might be 402 hindered by sars-cov-2 nsp5 as it interacts with an upstream metabolic enzyme 403 glutathione peroxidase ( figure 13b ). 404 previously it was reported that il-17 signaling enhances antiviral immune responses (ma et 405 al., 2019). sars-cov-2 nsp13 can bind il-17 receptor and inhibit the downstream 406 signaling from il-17 receptor to traf6 for activating nfκb/mapks/cebpb signaling axis, 407 thus decreasing the antiviral inflammatory responses (figure 11 ). 408 during acute viral infections toll-like receptor 4 (tlr4) signaling plays important roles in 409 eliciting inflammatory responses (olejnik et al., 2018) . sars-cov-2 protein nsp13 interacts 410 with tbk1 which might reduce the signaling from irf7, resulting in less ifn-i productions; 411 while nsp12 interacts with rip1; as a result, the activation of downstream nfκb and 412 mapks (p38 and jnk) pathways and induction of inflammatory responses from these 413 pathways will be stalled ( figure 12) . from the enrichment analysis, we have found that deregulated genes were also involved in 482 processes and functions like-heart development, kidney development, age-rage signaling 483 pathway in diabetic complications, zinc ion binding, calcium ion binding etc ( figure 1a, 1b flicek epigenetic modulation 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calcium flux in immune cells and 776 impaired immune responses re: who director-general's opening remarks at the media briefing on 779 covid-19 -3 re: coronavirus cases micrornas in autophagy and their emerging 782 roles in crosstalk with apoptosis characterization 784 of the lipidomic profile of human coronavirus-infected cells: implications for lipid 785 metabolism remodeling upon coronavirus replication mir-146a inhibits 788 oxidized low-density lipoprotein-induced lipid accumulation and inflammatory 789 response via targeting toll-like receptor 4 endothelial cell control of thrombosis parp9-794 dtx3l ubiquitin ligase targets host histone h2bj and viral 3c protease to enhance 795 interferon signaling and control viral infection cistrome data browser: 798 expanded datasets and new tools for gene regulatory analysis key: cord-292152-gmru83ac authors: makrinioti, heidi; mac donald, alexander; lu, x; wallace, s; mathew, jobson; zhang, f; shao, j; bretherton, j; tariq, mehmood; eyre, e; wong, a; pakkiri, l; saxena, amulya k; wong, g w title: intussusception in two children with sars-cov-2 infection in children date: 2020-08-08 journal: j pediatric infect dis soc doi: 10.1093/jpids/piaa096 sha: doc_id: 292152 cord_uid: gmru83ac this report compares intussusception as likely associated with sars-cov-2 infection in infants that presented in wuhan and london. while the intussusception was reduced by enemas in wuhan, the outcome was fatal. whereas the intussusception was not reduced by enemas in london and required surgery, the outcome was favourable. based on the data so far, severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection in children is shown to run a milder course with lower reported mortality rates (1, 2) . both chinese and italian epidemiological studies have shown that approximately 10% children may present with gastrointestinal symptoms, and the presence of these symptoms is associated with increased disease severity (3) . however, as the definition of suspected cases does not include presentations with gastrointestinal symptoms only, there is still no clear answer to the question "should we screen for sars-cov-2 infection in children who require admission to hospital with gastrointestinal symptoms?". the current advice from public health of england points against this direction (https://www.gov.uk/government/publications/wuhan-novel-coronavirus-initialinvestigation-of-possible-cases/investigation-and-initial-clinical-management-ofpossible-cases-of-wuhan-novel-coronavirus-wn-cov-infection). in addition to the fact that most of the presentations of the infection in children are atypical, there is very recent evidence showing that the highest viral shedding is taking place 2 to 3 days before onset of symptoms (https://www.nature.com/articles/s41591-020-0869-5). this evidence could potentially add on existing stress to front-line pediatricians not only around diagnosis and timely management, but also around their protection from contracting the disease. there is mounting evidence around increasing number of deaths in healthcare professionals (4) . therefore, data clarifying the definition of suspected cases is eagerly anticipated. case studies could be valuable in providing this information while wider epidemiological data is underway. this brief report describes two cases of intussusception in infants found to be positive with sars-cov-2. the first case was reported at wuhan children's hospital in wuhan which had a lethal outcome and the second case is being reported from west middlesex-chelsea and westminster hospital in london with a favourable outcome. on 2nd february 2020, a previously healthy 10-month-old girl was brought to the emergency department (ed) of the wuhan children's hospital. she presented with a history of 30 hours paroxysmal crying, vomiting, and passing red currant jelly stool. ileocolic intussusception was confirmed by ultrasound examination 2 hours after admission and pneumatic reduction was successful. abdominal x-ray after pneumatic reduction did not reveal any evidence of perforation. two days after, due to development of progressive respiratory distress, a chest computed tomography (ct) scan was performed showing small dense infiltrates in both lower lung fields. her throat swab and antibody testing were positive for sars-cov-2. the infant's clinical condition continued to deteriorate with progressive distention of the abdomen and absence of bowel sounds. abdominal ct showed signs of peritonitis, ascites, and swelling of the small intestinal wall. two days later, a laparotomy was performed with the placement of a defunctioning ileostomy. despite maximum support with antibiotics, inotropes, and mechanical ventilation, there was a deterioration in her condition; and with the development of disseminated intravascular coagulation, the infant passed away on 8th march. the infant had no other underlying disease, but her father had fever and cough 5-days prior to her presentation and had recovered without having any specific investigations. on 30th of march 2020, an otherwise fitting well 10-month old girl presented at the ed of the west middlesex hospital in poor condition. she had been unwell for 2 weeks with intermittent coryzal symptoms and bilateral conjunctivitis and followed by abdominal symptoms for which she had visited the ed twice before the latter presentation. she presented with 2 days of bilious vomiting and red currant jelly stool. during her last presentation she was lethargic, had poor peripheral perfusion, and was unresponsive to pain (including interosseous access). following resuscitation, and suspicion of intussusception she was transferred to chelsea and westminster hospital. due to failure of air enema reduction management, a surgical reduction was undertaken during which a malrotation was also identified for which a ladd's procedure was performed. after full recovery, the infant was discharged on 5th of april. her nasopharyngeal and throat swabs on 30th of march and on 4th of april tested positive in sars-cov-2. extended virus detection panel by pcr did not reveal co-infection with other viral agents. in the history, mother was identified to have a flu-like illness 3 weeks before. the family isolated as per public health of england advice, but mother was never tested for sars-cov-2 due to unavailability of mass testing in the united kingdom. one week after the onset of the mother's illness, the other sibling also got unwell with mild respiratory symptoms and fever. blood investigations performed on admission were not suggestive of any abnormalities, consistent with epidemiological data described in most children in the chinese cohort (1). inflammatory markers were not raised (c-reactive protein level was 13.8 mg/l in case 1 and below 5 mg/l in case 2, white cell counts level was 5.68x 109/l and 13.2x 109/l in case 2). there was no derangement in results of renal and liver investigations. lymphopenia, that has been described in cases of children who deteriorated and required extracorporeal membrane oxygenation (ecmo), was not noted in any of the two cases (5) . the absolute number of lymphocytes was 4.57x 109/l in case 1 and 5.6x 109/l in case 2. this report describes two infants with intussusception and sars-cov-2 infection. there is a case report recently published highlighting similar association (https://journals.lww.com/peconline/citation/2020/06000/covid_19_infection_is_a_diagnostic_challenge_in.24.aspx). the first case developed respiratory symptoms during hospitalization and underwent a chest ct scan that revealed changes, whilst the second case had preceding mild respiratory symptoms, therefore did not undergo chest imaging as per current standards of care. abnormal ct findings have been reported even in children with no respiratory symptoms, but this did not alter their management m a n u s c r i p t course (6) . although both infants presented with symptoms suggestive of intussusception, their disease course differed. whereas, the wuhan case had successful reduction of intussusception but was troubled by respiratory and non-respiratory complications, the outcomes in the london case on the other hand was favourable though this infant had failure of air enema reduction and required urgent surgery for the reduction of the intussusception. these cases describe possible association between sars-cov-2 infection and intussusception and the first case is closer to describe causality as rectal swabs tested positive for sars-cov-2. however, this finding needs to be described with caution, as recent data show that although sars-cov-2 can be detected up to 9 days in stool samples, there is no potential for viral shedding (7, 8) . rectal swab for detection of sars-cov-2 was not part of the standard of care provision at the national health service (nhs) at that time of the pandemic. in the second case, the child was incidentally found to have undiagnosed malrotation of the bowel. waugh's syndrome has been described in toddlers who present with signs of chronic intussusception and malrotation of the bowel. in these cases, malrotation of the bowel consists a structural risk factor rather than a trigger for intussusception (9, 10) . the suggested mechanisms underlying include either gut epithelial cells' infection with local reactive mesenteric adenitis that can trigger intussusception or mesenteric adenitis secondary to upper respiratory tract infection with sars-cov-2. back in 1992, the association between adenovirus and intussusception has been documented (11). adenovirus-2 (adv-2) serotype, most frequently described as a respiratory pathogen, was the most detected pathogen in children with intussusception (12) . angiotensin-converting enzyme 2 (ace2) receptor is the functional receptor for sars-cov-2. immunohistochemistry in human tissues revealed the surface expression of ace2 protein on enterocytes of the small intestine, functioning as a co-receptor for nutrient uptake such as amino acid resorption from food (13) . these finding highlights that the gut might also be an important entry site for sars-cov-2 infection. however, there is still no evidence of viral replication in gut epithelial cells (14) . many asymptomatic patients undergoing procedures are being found to be sars-cov-2 positive and association with underlying problem (intussusception, appendicitis) is unclear and requires further study (15) . in adults, evidence of asymptomatic infection on admission for elective surgical procedure was associated with evidence of increased inflammatory response and more adverse outcomes (4). in conclusion, this brief report aims to urge front-line pediatricians to consider sars-cov-2 infection when managing a child with intussusception and to consider taking appropriate precautions, being also aware of any possible respiratory deterioration. for future management and prevention strategies, it is also crucial to understand whether sars-cov-2 can infect gut epithelial or endothelial cells and whether susceptibility to gastrointestinal infection is associated with differential innate immune responses of the host. also, a probabilistic model like the one set up for the associations between rotavirus vaccine and incidence of intussusception, would be particularly helpful (16) . sars-cov-2 infection in children characteristics of and important lessons from the covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention 2019-ncov: polite with children! pediatr rep clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection covid-19, ecmo, and lymphopenia: a word of caution clinical and ct features in pediatric patients with covid-19 infection: different points from adults viral rna in feces of three children during recovery period of covid-19 pneumonia virological assessment of hospitalized patients with covid-2019 waugh's syndrome: a report of six patients chronic intussusception associated with malrotation in a child: a variation of waugh's syndrome? case rep surg molecular epidemiology of adenovirus isolates from patients diagnosed with intussusception in melbourne, australia tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis master regulator analysis of the sars-cov-2/human interactome asymptomatic cases in a family cluster with sars-cov-2 infection potential intussusception risk versus health benefits from rotavirus vaccination in latin america a c c e p t e d m a n u s c r i p t key: cord-266348-tbr2ynx0 authors: stroemer, a.; grobe, o.; rose, r.; fickenscher, h.; lorentz, t.; krumbholz, a. title: diagnostic accuracy of six commercial sars-cov-2 igg/total antibody assays and identification of sars-cov-2 neutralizing antibodies in convalescent sera date: 2020-06-17 journal: nan doi: 10.1101/2020.06.15.20131672 sha: doc_id: 266348 cord_uid: tbr2ynx0 the reliable detection of immunoglobulin g (igg) or total antibodies directed against the novel severe acute respiratory syndrome coronavirus type 2 (sars-cov-2) is important for clinical diagnostics and epidemiological studies. here, we compare the diagnostic accuracy of six commercially available sars-cov-2 igg (abbott sars-cov-2 igg; diasorin liaison sars-cov-2 s1/2 igg; epitope edi novel coronavirus covid-19 igg elisa kit; euroimmun anti-sars-cov-2 elisa (igg); mikrogen recomwell sars-cov-2 igg) or total sars-cov-2 antibody assays (roche elecsys anti-sars-cov-2). the test sensitivities were analyzed with a set of 34 sera obtained from 26 patients after pcr-confirmed sars-cov-2 infection and varied from 76.9% (euroimmun) to 96.2% (abbott). the majority of assay results were confirmed in a laboratory-developed plaque reduction neutralization test and by a sars-cov-2 igg-specific line assay including measurement of generally low igg avidities (mikrogen recomline coronavirus igg [aviditaet], prototype). moreover, 100 stored sera collected during summer 2018 (n = 50) and winter season 2018/2019 (n = 50) were included to demonstrate test specificities. these varied from 96.0% (diasorin) to 100% (epitope edi). a subset of sera were retested with a lateral flow test (standard q covid-19 igm/igg duo) and a considerably lower sensitivity was noted. overall, the diagnostic accuracy of the six sars-cov-2 igg/total antibody assays was good and varied from 92.9% (euroimmun) to 98.4% (abbott). due to the different specificities, results of commercially available sars-cov-2 antibody tests should be interpreted with caution. a high proportion of antibody-positive patient sera demonstrated neutralizing capacity against sars-cov-2. at the end of the year 2019, chinese local health authorities reported the occurrence of a cluster of 41 pneumonia cases in wuhan, a megapolis in the hubei province [1] . shortly after, a novel beta coronavirus 42 -which is now designated as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [2] -was 43 discovered in the bronchoalveolar lavage fluid of a patient with pneumonia [3, 1] . this virus emerged more recently, the measurement of immune response against sars-cov-2 came into the focus of clinical 51 diagnostics, particularly by the detection of virus-specific antibodies. the use of sars-cov-2 antibody tests 52 could clarify the etiology of the disease in patients who present late, after two weeks from onset of 53 symptoms. moreover, these tests can demonstrate the viral spread in the community and may even 54 identify individuals who are potentially protected from re-infection by neutralizing antibodies [4] . 55 it is believed that the majority of developed antibodies is directed against the abundant viral nucleocapsid 56 protein while antibodies directed against the spike protein are considered more specific and may possess 57 neutralizing capacity [4] . the diagnostic value of serological tests, however, is limited due to their potential 58 cross-reactivity with other human coronaviruses. furthermore, it is still unclear, how long sars-cov-2 59 antibodies will persist and whether they are protective at all [4] . 60 in the following, we present data on the diagnostic performance of six commercially available sars-cov-61 2 igg or total sars-cov-2 antibody tests. variations in their diagnostic sensitivity and specificity were 62 observed. the latter could lead to a considerable number of false positive results in countries with a 63 currently low sars-cov-2 prevalence such as germany. moreover, we demonstrate sars-cov-2 64 neutralizing antibodies in a marked proportion of convalescent sera. 65 66 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. between four and 60 days (median 19 days) after a positive real-time rt-pcr result in which parts of the 69 sars-cov-2 e or n genes [5] were detected in respiratory samples. only sera from individuals that 70 exhibited a cycle threshold (ct) ≤ 35 (median ct 26.8) in the initial real-time rt-pcr were included. these 71 samples should contain sars-cov-2 igg/total antibodies and, therefore, were considered to determine 72 serological assay sensitivities. seven subjects with repeated sample entries were included in the study 73 group. among them were two patients, each with a serum taken 0 and 9 days before the diagnosis of germany) were included to ensure that cross-reactivity is not occurring. none of these 102 sera were 79 expected contain sars-cov-2 antibodies. 80 test results were also used to calculate the accuracy of the assays, i.e. the proportion of correctly identified 81 samples from all samples. 82 twelve sera that were sent to us for routine sars-cov-2 antibody tests without a previous sars-cov-2 83 pcr result were also included to show test performance. 84 the ethics committee of the medical faculty of the kiel university approved the setting of this study (az 85 d467/20). all sera were stored at -20°c until testing. 86 the testing was done with five sars-cov-2 igg assays (abbott sars-cov-2 igg; diasorin liaison® sars-87 cov-2 s1/2 igg, diasorin, dietzenbach, germany; epitope edi™ novel coronavirus covid-19 igg elisa kit, (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint in general, borderline results were counted as positive. in order to allow a better comparison of the test 97 results, the raw data were converted to relative indices according to the decision limits specified by the 98 manufacturer. a signal/cut-off value of <1 was valued as negative and ≥1 as positive, which corresponds 99 to a previous study [6] . 100 a subset of sera was tested with and without avidity reagent in an igg line assay ( to an untreated cell-control and a virus control. a serum dilution ≥ 1:20 that prevented the formation of 126 plaques by 50% compared to the virus control (prnt50) was considered likely to be protective. 127 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint thirty-seven samples taken from 26 patients with a pcr-confirmed sars-cov-2 infection were analyzed 129 with six sars-cov-2 igg or total antibody assays. samples #1, #7, and #9 were taken nine days before to 130 four days after pcr and were all found to be free of sars-cov-2 antibodies by the assays. these samples 131 were not considered for calculation of sensitivity but clearly demonstrated seroconversion (figure 1 ). out 132 of the remaining 34 samples, only one serum (#20; figure 1 ) which was obtained ten days after a positive 133 rt-pcr was tested negative for sars-cov-2 igg/total antibodies in the six assays. however, this sample 134 exhibited a prnt50 1:10 to 1:20. all other samples were reactive in at least one assay (figure 1 overall, ten sera were found to be reactive in the six assays, and four of them were found to be reactive 146 in one assay. none contained sars-cov-2 neutralizing antibodies (figure 2, supplementary material) . 147 thus, assay specificities ranged from 96.0% to 100.0% (table 1) . two samples with a typical constellation 148 of an acute ebv infection did not show cross reactivity with any of the sars-cov-2 igg/total antibody 149 assays (data not shown). 150 taken together, assay accuracies varied from 92.5% to 98.5% (table 1) . 151 twelve routine samples obtained from individuals who were interested in their sars-cov-2 antibody 152 status were re-evaluated by all six assays (figure 3 ). six of them -including three family members of a 153 confirmed covid-19 case (#22; figure 1 ) -were classified sars-cov-2 igg/total antibody positive by the 154 majority of the tests. isolated reactivity was observed in two sera (#2, #10; figure 3 ). 155 thirty of the 37 sera obtained from covid-19 cases were retested in the igg recomline assay. among 156 them, 27 (90%) were confirmed as sars-cov-2 igg positive (figure 1, supplementary material) . one out 157 of the ten archived sera that were reactive in at least one of the six sars-cov-2 igg/total antibody tests 158 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint was also classified as sars-cov-2 igg positive by the blot results. the underlying sample was collected in 159 winter 2018/2019 and was found to be reactive for sars-cov-2 igg/total antibodies by the mikrogen 160 (s/sco 1.33) and roche (s/sco 3.46) assays in parallel ( figure 2 ). as well, nine of the twelve pretested 161 routine samples were analyzed by the blot and pre-known results were largely confirmed (figure 3, 162 supplementary material). all measured sars-cov-2 iggs were of low avidity (supplementary material). 163 eighteen samples, including eight sera from confirmed covid-19 cases were retested in a lateral flow 164 assay. of the eight covid-19 sera, two were clearly positive for igg, while three were tested weakly 165 positive and three negative (figure 1, supplementary) . five archived samples that were valued reactive in 166 at least one of the six sars-cov-2 igg/total antibody tests were also re-evaluated by this lateral flow assay. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint sample #7 was taken nine days before pcr and the corresponding sample #8 19 days after the pcr. serum 180 #9 was taken on the day of the pcr and serum #10 29 days thereafter. results of the rapid lateral flow 181 test, the plaque reduction neutralization assay (prnt50) and the line assay (blot) are shown in the table. p, 182 positive; wp, weakly positive; n, negative; nd, not determined. 183 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint in a rather short period, various assays for detection of sars-cov-2 antibodies were introduced to the 203 market. a meta-analysis of 38 studies on the performance of different format sars-cov-2 antibody tests 204 mainly manufactured from chinese companies was reported recently [9] . furthermore, studies on the 205 sensitivity and specificity of sars-cov-2 igg tests, including the elisas from epitope, euroimmun, and 206 mikrogen, were published previously [6,10,8,7]. here, we compare six different commercially available 207 tests, all of which have been released within the past few months. three of them (abbott, diasorin, roche) 208 were applied in a random access manner which reduces the needed hands-on-time markedly. a subset of 209 samples were retested in a laboratory-developed prnt50, in an immunoblot including the determination 210 of sars-cov-2 igg avidities and in a rapid lateral flow assay. to our knowledge, such a comprehensive 211 study has not yet been carried out. 212 the sensitivities between the six tests differ from 76.9% to 97.1% (table 1) , which is in accordance to other 213 studies [6,8,10] and to the meta-analysis [9] . our data indicate that assays based on the more abundant 214 viral nucleocapsid protein as an epitope are slightly more sensitive compared to tests using domains of 215 the spike protein as an antigen. furthermore, a lack of reactivity in the latter igg tests does not necessarily 216 mean that sars-cov-2 neutralizing antibodies are missing (compare figure 1) . however, three sera from 217 two covid-19 patients, which were only recognized as igg positive in the abbott test, could not be 218 the specificities of sars-cov-2 igg/total antibody tests are at a comparatively high level between 96.0 226 and 100.0%, as has also been reported by others [6,8,10]. interestingly, one sample collected in winter 227 2018/2019 was found to be reactive in the mikrogen and roche assays as well as in the blot but could not 228 be confirmed by the prnt50 (figure 2, supplementary material) . for all other 99 archived samples, a 229 random pattern of rare, isolated reactivity was demonstrated and none was confirmed as possessing sars-230 cov-2 neutralizing antibodies by prnt50 (figure 2, supplementary material) . in addition, two sera obtained 231 from patients with acute ebv infection were tested negative for sars-cov-2 igg/total antibodies. thus, 232 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint china novel coronavirus i, research t (2020) a novel coronavirus from 279 patients with pneumonia in china the species severe 281 acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 a novel 284 coronavirus genome identified in a cluster of pneumonia cases -wuhan interpreting diagnostic tests for sars-cov-2 detection of 2019 291 novel coronavirus (2019-ncov) by real-time rt-pcr comparison of four new 294 commercial serologic assays for determination of sars-cov-2 igg haagmans bl (2020) severe acute respiratory syndrome coronavirus 2-299 specific antibody responses in coronavirus disease clinical performance of sars-cov-2 igg 302 antibody tests and potential protective immunity neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their 309 implications cross-reactivity to epitopes of endemic human coronaviruses or triggered by active ebv infection may not 233 represent a major problem. nevertheless, it should be noted that due to the currently estimated low 234 prevalence of sars-cov-2 antibodies in the overall german population, even a rather high specificity of 235 99% would produce a relevant number of false positive results. thus, these assays -that all show a 236 comparable high accuracy of 92.5%-98.5% (table 1 ) -should be preferentially used for testing of patients 237 with a history of a probable infection. in case of doubt, the implementation of the labor-intensive and 238 time-consuming prnt50 should be considered. 239the majority of sars-cov-2 iggs in sera from covid-19 patients were confirmed by an immunoblot but for sars-cov-2 igg/total antibodies by at least one of the six assays were all negative by this lateral flow 256 assay. thus, this rapid antibody test is believed to have a good specificity but sensitivity is reduced. 257particularly the occurrence of faint igg bands is a diagnostic challenge. the interpretation of results 258 obtained from this assay should be done with caution. 259taken together, all six tested sars-cov-2 igg/total antibody assays demonstrate a good performance. 260their slightly reduced specificities, however, may produce a relevant number of false positive results in 261 low prevalence countries. future studies should address the long-term course of sars-cov-2 antibodies 262 as well as the virus-specific cellular immune response. for the latter, the development of routine test 263 all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint 14 procedures is urgently required. the prnt results indicate that a high proportion of antibody-positive sera 264 are actually able to neutralize the virus in culture and, thus, may be relevant for immunity. 265 all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all authors declare no conflict of interest. 276 all rights reserved. no reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint this version posted june 17, 2020. . https://doi.org/10.1101/2020.06.15.20131672 doi: medrxiv preprint key: cord-296426-upwsdgso authors: virmani, sarthak; gleeson, shana e.; girone, gianna f.; malhotra, divyanshu; cohen, elizabeth a.; klarman, sharon e.; asch, william s. title: identifying a kidney transplant recipient covid phenotype to aid test utilization in the setting of limited testing availability does one exist? date: 2020-06-20 journal: transplant proc doi: 10.1016/j.transproceed.2020.05.033 sha: doc_id: 296426 cord_uid: upwsdgso abstract: the high morbidity and mortality of covid-19 in immunocompetent patients raises significant concern for immunosuppressed kidney transplant recipients (ktrs). this level of concern, both on the part of the ktrs and transplant professionals, is heightened by a lack of prior knowledge on how sars-cov-2 may manifest differently in immunosuppressed patients. characterizing how ktrs may present differently than the general population would allow for more targeted and timely evaluation and treatment of ktrs with covid-19 infection. methods without prior knowledge of how this virus would affect our transplant center’s delivery of care to ktrs who are sars-cov-2 positive or patients under investigation (puis), and in the setting of limited testing availability, we initiated a quality assurance and improvement project (qapi) to track ktrs followed at our transplant center through the sars-cov-2 testing process. results of the 53 symptomatic patients, 20 (38%) tested positive for sars-cov-2 either on presentation to the emergency department, or referral to a designated outpatient testing center. in addition, 16 (80%) of the 20 patients who tested positive required inpatient treatment. intriguingly, patients with a history of polyoma bk viremia (bkv) had a higher incidence of testing positive for sars-cov-2 compared to patients without history of bkv (80% and 28%, respectively; p= 0.002). the positive predictive value and likelihood ratio was 80% and 6.6 for this association, respectively. among our ktrs tested, those receiving belatacept had a lower likelihood of testing positive for sars-cov-2. this finding approached, but did not achieve, statistical significance (p=0.06). the high morbidity and mortality of covid-19 in immunocompetent patients raises significant concern for immunosuppressed kidney transplant recipients (ktrs). this level of concern, both on the part of the ktrs and transplant professionals, is heightened by a lack of prior knowledge on how sars-cov-2 may manifest differently in immunosuppressed patients. characterizing how ktrs may present differently than the general population would allow for more targeted and timely evaluation and treatment of ktrs with covid-19 infection. methods: without prior knowledge of how this virus would affect our transplant center's delivery of care to ktrs who are sars-cov-2 positive or patients under investigation (puis), and in the setting of limited testing availability, we initiated a quality assurance and improvement project (qapi) to track ktrs followed at our transplant center through the sars-cov-2 testing process. results: of the 53 symptomatic patients, 20 (38%) tested positive for sars-cov-2 either on presentation to the emergency department, or referral to a designated outpatient testing center. in addition, 16 (80%) of the 20 patients who tested positive required inpatient treatment. intriguingly, patients with a history of polyoma bk viremia (bkv) had a higher incidence of testing positive for sars-cov-2 compared to patients without history of bkv (80% and 28%, respectively; p= 0.002). the positive predictive value and likelihood ratio was 80% and 6.6 for this association, respectively. among our ktrs tested, those receiving belatacept had a lower likelihood of testing positive for sars-cov-2. this finding approached, but did not achieve, statistical significance (p=0.06). the landscape of severe acute respiratory syndrome 2 virus (sars-cov2) pandemic is constantly changing. limited resources and swiftly evolving information are influencing real time changes in our approach to triaging, evaluation, and management of patients suspected to be infected by this virus. it is unclear if immunocompromised patients are at a higher risk for contracting this viral infection as compared to the general population. while it is true that other non-novel viruses tend to cause more severe disease in immunocompromised patients [1] , no conclusive data is available to suggest an increased susceptibility or severity of sars-cov-2 infection in immunosuppressed kidney transplant recipients (ktrs). at the time of writing, isolated case reports from early days of the pandemic in the wuhan district of china are yet to be comprehensively analyzed and validated. emerging claims of co-morbid associations and effective therapeutics in the mainstream and social media lack consistent and conclusive evidence, raise uncertainty among healthcare professionals, and cause confusion among the general public. the initial knowledge of a syndromic presentation with fever and flu-like symptoms has now expanded to include other systemic symptoms such as vague malaise, body aches, and gastrointestinal symptoms, mimicking many common infectious diseases. [2] in order to utilize our transplant center's resources most efficiently during a pandemic with potentially scarce testing capability, we recognized that it might be necessary to limit the use of tests to those patients most likely to benefit from diagnostic testing. we started a qapi project to identify a patient phenotype that is associated with confirmed covid-19 disease, so that we may effectively prioritize patients for testing. here we report our initial observations on the first 53 patients from our center tested for sars-cov-2. this was a single center, retrospective chart review performed as a qapi project to assess similarities in kidney transplant recipients who tested positive for sars-cov-2 as compared to those who tested negative, and guide testing recommendations in the setting of limited testing availability during the covid-19 pandemic. data was collected for kidney or kidney-pancreas transplant recipients from march 10, 2020 through april 9, 2020. patients were included if they were over the age of 18, were receiving transplant care at our center, and were tested for sars-cov-2 during this time period. asymptomatic patients screened for sars-cov-2 as a prerequisite for placement in a skilled nursing facility were excluded. all variables were collected from the electronic medical record. chi-squared test was used to compare categorical data and a student's t-test was used to compare continuous data. a pvalue of <0.05 was considered to be statistically significant. all of the 53 patients included in this cohort were tested for sars-cov-2. individuals who remained puis were excluded from the analysis. overall, the average age was 59.5 years for sars-cov-2 positive patients and 56.7 years for patients who tested negative, slightly older than the average (55.4 years old) ktr followed at our center (table 1) . we did not observe any significant association between patient gender, level of education, or history of diabetes on the sars-cov-2 test result. cough was the most common symptom, followed by fever and shortness of breath ( table 2) . none of these symptoms, individually, had a statistically significant association with a positive test result. patients presenting with only one symptom (53% of our total cohort) were more likely to test negative for sars-cov-2. however, presenting with more than 1 symptom (fever, cough, and shortness of breath) did associate with a positive test result x 2 (1, n = 53) =1.63, p = 0.047. the majority (75%) of the sars-cov-2 positive patients required hospital admission. there is emerging data from china that patients in the abo-a blood group may be more susceptible to sars-cov-2 infection. we observe a similar trend (table 1) . compared with the total ktr population followed at our center, a larger percentage of sars-cov-2 positive patients are abo-a (40%, compared with 34.11%). however, this trend failed to achieve statistical significance (p=0.22). similarly, among patients who presented for sars-cov-2 testing, patients with abo-a were slightly more likely to test positive compared to the non-abo-a patients tested (42% and 38%, respectively). however, this trend also failed to achieve statistical significance, (p=0.74). our cohort of ktrs showed no significant difference in alc between patients who tested positive and negative for sars-cov-2 (table 3 ). in this cohort, both the average and median alc were less than 1 in all patients who were tested for sars-cov-2 infection, both those with and without covid-19. however, hemoglobin and hematocrit were both significantly higher in patients with covid-19 when compared to patients who tested negative for sars-cov-2, possibly reflecting hemoconcentration in the former. no significant associations were identified among the other laboratory data points reviewed (table 3) . we also tested the hypothesis that patients with a history of a transplant related virus might be more prone to covid-19 (table 4 ). interestingly, we found a high association between a sars-cov-2 positive test result and a known history of polyoma bkv (specifically, serum quantitative pcr greater than log 3 viremia, p = 0.002). though our cohort is relatively small and larger cohorts are necessary to confirm our findings, history of bk infection had a positive predictive value of 80% at predicting a sars-cov-2 positive test result and a specificity of 94%. furthermore, a history of bk infection had a positive likelihood ratio of 6.6. we considered the possibility that continuous variables associated with a bk infection history might also predict sars-cov-2 infection. in particular, receiver operator curves (roc) were constructed using bk log titer at peak level of viremia and duration of viremia exceeding log 3. however, this data exploration was limited by having only two patients with a positive sars-cov-2 test and a history of bk viremia ( figure 1 ). we did not find an association with a history of cytomegalovirus infection. our transplant center has a large cohort of patients receiving belatacept as their primary immunosuppressant agent (table 4 ). intriguingly, we observed a trend toward a lower rate of sars-cov-2 positive testing among the patients on belatacept maintenance therapy in this cohort. in the patients who resulted sars-cov-2 positive, only 2 (10%) were on belatacept based immunosuppression. this was in contrast to cnis, where 85% of sars-cov-2 positive patients were on tacrolimus. belatacept based immunosuppression regimens were utilized for 13 patients within our total cohort tested. we observed a lower rate of sars-cov-2 positive testing in the belatacept patients compared to the patients receiving other regimens (15% and 45%, respectively). this lower rate nearly achieved significance (p = 0.056) in this small cohort. we did not find an association between either a biopsy proven rejection history, or active therapy with an ace inhibitor or arb in our cohort (table 4) . medical record archiving prevented ascertainment of transplant induction medication and abo blood group for 2 patients transplanted prior to 1998. these two patients were not included in the analysis of these two categories. the covid19 pandemic represents the most significant health crisis to face our society in over a century. many changes to how we assess, test, and monitor ktrs during this unique time in medical history are necessary. the initially limited availability of viral testing, coupled with the recommendation to utilize telehealth as a way to reduce patient exposure to the medical center, prompted a qapi to search for a ktr phenotype that indicated a higher probability of having covid19 disease. in our cohort, patients with a history of bkv were at increased risk to test positive for covid-19 compared to patients without a history of bkv. the reason for this association is unclear. one hypothesis is that despite a reduction in immunosuppression to manage bkv, these ktrs remain too immunosuppressed to control and clear the sars-cov-2 infection. early reports indicate that the severe acute respiratory syndrome associated with covid-19 can be attributed to excessive pro-inflammatory host immune responses. [3] therefore, an alternative hypothesis is that patients with a history of bkv may be at risk for more clinically apparent disease compared to ktrs without a history of bkv because of their reduced immunosuppression. the lack of a reliable laboratory test to quantitatively measure the responsiveness of the immune system in immunosuppressed patients complicates the search for a mechanistic link between bkv and covid-19. though statistically significant in our small patient cohort, larger studies of ktrs with covid-19 disease and a history of bkv will be required to confirm and better understand this association. as a finding from this qapi, we recommend that until additional data becomes available, ktrs with a history of bkv should be prioritized for sars-cov-2 testing. an interesting finding in our cohort was the signal towards a potential protective effect of belatacept immunosuppression in patients who were tested, but not sars-cov-2 positive. this difference was significantly different from the sars-cov-2 positive cohort, who were more likely to be on tacrolimus-based immunosuppression. over 250 kidney transplant recipients at our center are maintained on belatacept as part of their immunosuppressive regimen, and the majority are late conversions (>3 months post-transplant) from cni therapy. recently, concerns about belatacept and an increased risk of opportunistic infection have been emphasized. [4] bertrand and colleagues found that belatacept was associated with an incidence of 9.8 opportunistic infections/100 -person years, including pneumocystis jirovecii pneumonia and cytomegalovirus disease. [4] this was more commonly seen in the group who converted to belatacept early, <6 months post-transplant. this observation is in contrast to our qapi data which showed a lower rate of sars-cov-2 positive testing in belatacept treated patients. notably, bertrand and colleagues did not propose an explanation for the increase in opportunistic infections seen with belatacept. based on what is known about belatacept's impact on the immune system, it is unclear why belatacept was associated with a protective effect in our cohort. not surprisingly, we identified an association between the number of classic symptoms (fever, cough, and shortness of breath) reported and the likelihood of testing positive for sars-cov-2. nonetheless, 24% of screened patients presenting with only one symptom were confirmed as sars-cov-2 positive on testing. therefore, similar to the general population, a high level of suspicion for covid19 is warranted in ktrs with limited symptomatology. patients presenting with 2 or 3 symptoms were more likely to have a positive test. but, this finding may be skewed by the relatively high false negative rate of the sars-cov-2 test, which is reported to be as high as 40% in the general population. given concerns regarding testing availability and the need to limit the ktr's contact with medical facilities during this pandemic, we sought to identify symptoms more frequently associated with covid-19 positivity. we reviewed multiple symptom types, including fever, dyspnea, cough, gastrointestinal symptoms, neurologic symptoms, anosmia, and dysgeusia. as a cohort, ktrs with covid-19 demonstrated all of these symptoms in varying combinations, with the exception of anosmia (none of our sars-cov-2 positive ktrs had documented anosmia). the most common presenting symptoms were cough (70%), dyspnea (60%) and fever (55%). while not as prevalent, these symptoms were also frequently present among those who tested negative for sars-cov-2 (42% of such patients had cough, 39% had dyspnea, and 58% had fever). when only one of these symptoms was present, there was no statistically significant difference between patients with or without covid-19. however, the presence of more than one of these common presenting symptoms did correlate with sars-cov-2 positivity. if patients had 2 or 3 of these symptoms (cough, dyspnea, and/or fever), they were more likely to test positive for sars-cov-2, and this difference was statistically significant. given this, we advocate for expediting testing of our ktrs who present with at least 2 of these 3 symptoms during the covid-19 pandemic. as knowledge and experience with covid-19 increased, anosmia and dysgeusia became increasingly recognized as symptoms of this syndrome. [5, 6] some of these patients remain minimally symptomatic (and so can easily spread the infection), while others were reported to progress and require hospitalization. the exact prevalence of these symptoms is not entirely clear, though one italian study that surveyed hospitalized patients with covid-19 found that 33.9% of patients had alteration in either taste or smell, the vast majority of whom developed these symptoms prior to requiring hospitalization. [7] in our cohort of patients, we did not identify anosmia or dysgeusia as common presenting symptoms in ktrs. even if one speculated that there could have been false negative test results, the overall prevalence of these symptoms was quite low in the entire cohort of patients, much lower than reported in other studies [7] . many of the reports of anosmia and dysgeusia in covid-19 were not published until late march and early april 2020, and so our ktrs were not routinely queried about them in our early experience with this infection. given this, our low numbers may reflect under-reporting of taste and smell alterations. based on our currently available data, we cannot state that anosmia or dysgeusia are common presenting symptoms in ktrs. however, given the potential for underreporting, and reports in the literature that these symptoms present early in the disease process it seems prudent to screen ktrs for anosmia and dysgeusia when evaluating for possible covid-19 [8] . recent media reports have highlighted the ways in which race and socioeconomic status are playing a role in the covid-19 pandemic [9] . little peer reviewed data has been published on this topic, particularly regarding how education level may affect incidence and outcomes of sars-cov-2 infection in different populations. one group reported that those with higher education level (i.e. doctoral degree) reported more adherence to social distancing and other preventative measures; this could have implications on infection risk, but this data has yet to be published in a peer reviewed journal [10] . among our ktrs who were tested for covid -19, 44 patients had data available regarding educational history. of these 44 patients, 14 patients (31.8%) had obtained education beyond the high school level. although a minority of our ktrs had been educated beyond high school, there was no significant difference between those who had covid-19 and those who did not. it is unknown whether this is because educational history does not correlate with infection rates, or if this reflects the fact that the majority of our cohort have not completed education beyond high school. there was no statistically significant difference in gender between sars-cov-2 positive and negative patients in our ktr cohort. some published data have indicated there may be a slight male predominance in this infection; one such paper reported 54.3% of patients hospitalized with covid-19 were male [11] . other data from china has also raised the question of a gender difference in infection, though it is unclear if this data can be extrapolated to the us, as there is a larger proportion of males in china as compared to the us population [12] . though some authors have reported a slight male predominance among patients, other cohorts show similar rates of infection among males and females [13] . our data also shows similar rates of covid-19 between the genders. some data suggests that even if men and women are infected at similar rates, mortality may be higher in males, though this has yet to be definitively proven. one analysis from china showed a statistically significant case fatality rate between the genders (case fatality of 2.8% among males and 1.7% among females) [13] . since the focus of this work was on presenting symptoms and not final outcomes, we cannot comment on any potential gender difference in disease severity or mortality risk in our ktrs. this may be worth exploring in future work, but gender should not be a factor in deciding which ktrs to test for sars-cov-2 infection. it is common knowledge that individuals with diabetes are at higher risk of infectious diseases, including respiratory pathogens. moreover, diabetics are known to have a more severe disease when infected with respiratory viruses such as influenza, respiratory syncytial virus, etc. thus far, there have been conflicting reports identifying diabetes as an isolated risk factor for the sars-cov-2 infection. several small studies of patients hospitalized with covid19 [14, 15] showed that a comorbid diagnosis of diabetes or overt hyperglycemia was not an identifiable risk factor for sars-cov-2 infection. contrasting these findings, a report by the chinese center for disease control and prevention, reviewing more than 72,000 cases showed a 3 fold higher mortality risk of patients with diabetes as compared to the general population ( 7.3% vs 2.3%) [16] . among the 20 patients that tested positive in our cohort, more than half, 55% were known to have diabetes with a mean hba1c of 7.9. a lower proportion (36%) of patients that tested negative for sars-cov-2, were known to have diabetes. we speculate that ktrs with diabetes are also more likely to have other cardiopulmonary comorbidities placing them at a higher risk of contracting covid-19 and perhaps having worse outcomes in comparison to the general population. many viral illnesses can cause bone marrow suppression, and it is not unusual for a viral infection to present with cytopenias. early experience with covid-19 indicates lymphopenia to be a common feature, with one study showing a significant lymphopenia in as many as 72% of patients with covid-19 [11] . while lymphopenia may be a diagnostic clue to covid-19 upon initial evaluation of a patient, the literature also suggests that severity of lymphopenia may correlate with the overall severity of disease. multiple retrospective studies have found that patients with more severe disease had lower absolute lymphocyte counts (alc) than patients with more mild disease, and one group noted that lymphopenia was more common in patients who ultimately died from their infection [14, 15] . while this data may be significant in risk stratification and prognostication of patients with covid-19 in general, we believe it will be less helpful in the evaluation of ktrs with suspected or confirmed covid-19. our cohort of ktrs showed no significant difference in the alc between patients with and without covid-19. lymphopenia is common in our ktrs; in this cohort, both the average and median alc were less than 1 in all patients who were tested for sars-cov-2 infection, both those with and without covid-19. we therefore conclude that lymphopenia is not particularly suggestive of covid-19 in ktrs, and should not be used in deciding whom to test for the infection. although the goal of our work was to describe a phenotype amongst our ktrs (and thus improve our testing process), it would seem that the baseline prevalence of lymphopenia among ktrs limits the overall utility of using this parameter in prognostication. based on knowledge of abo blood groups and susceptibility risk during the previous sars coronavirus outbreak in 2003 [17] , there have been attempts to identify an association between abo blood type frequencies and the novel coronavirus. early pre-print reports from china found a higher risk of sars-cov-2 infection among patients with abo blood group a as compared to non-a blood groups [18] . blood type o was found to have a protective effect towards the infection. differential inhibition of adhesion of sars-cov-2 to ace2 expressing cell lines by natural anti-blood group antibodies is hypothesized as a possible mechanism of these findings. interestingly, the relative proportion of sars-cov-2 positive patients in our cohort with abo blood type a was significantly higher (40%) than the rest of the non-a blood types. although our observation did not reach statistical significance ( p-value 0.7) due to the limited sample size, further studies into this association with larger cohorts is certainly warranted. recognizing that our early cohort of ktrs tested for sars-cov-2 is relatively small, and additional factors which may have confounded our results or their interpretation are unknown at this time, this data seeking to identify a ktr covid-19 phenotype to aid test utilization in the setting of limited testing availability is valuable to the transplant community. as transplant centers across the world race to identify which of their ktrs are at a higher risk for covid-19, data from our qapi should influence decisions about whom to prioritize for testing while test availability remains a limiting factor. we initiated a qapi at our transplant center to track ktrs through the sars-cov-2 testing process. recognizing that limited testing availability forces healthcare professionals to restrict testing to ktrs most likely to benefit from testing, we collected data to determine if patient demographics and reported symptoms could aid in predicting disease. interestingly, we identified a strong association between the history of bkv and testing positive for sars-cov-2 indicating that this group of ktrs should be prioritized for sars-cov-2 testing. we also identified a signal suggesting that belatacept maintenance immunosuppression was protective against presenting with symptomatic covid-19 infection. additional data from larger and longer term cohorts will be necessary to validate these associations. • with limited testing availability, and to most efficiently utilize our transplant center resources, we started a qapi project to identify a patient phenotype to prioritize for sars-cov2 testing. • patients with history of polyoma bk viremia had a higher incidence of testing positive for sars-cov2 infection as compared to those without a history of polyoma bkv viremia. • patients receiving belatacept had a lower likelihood of testing positive for sars-cov2 infection as compared to those on calcineurin based immunosuppression. infection in renal transplant recipients covid-19 in solid organ transplant recipients: initial report from the us epicenter the pathogenesis and treatment of the opportunistic infections after conversion to belatacept in kidney transplantation anosmia and dysgeusia in the absence of other respiratory diseases: should covid-19 infection be considered? sudden and complete olfactory loss function as a possible symptom of covid-19 self-reported olfactory and taste disorders in sars-cov-2 patients: a cross-sectional study smell and taste dysfunction in patients with covid-19 black americans face alarming rates of coronavirus infection in some states. . the new york times individuals with low incomes, less education report higher perceived financial, health threats from covid-19. the evidence base characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia covid-19-new insights on a rapidly changing epidemic the novel coronavirus pneumonia emergency response epidemiology team. vital surveillances: the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) -china clinical characteristics of 140 patients infected with sars-cov-2 in wuhan clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china. intensive care characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention abo blood group and susceptibility to severe acute respiratory syndrome relationship between the abo blood group and the covid-19 susceptibility key: cord-302584-fwdpzv85 authors: zhu, ying; liu, mo; zhao, weiguang; zhang, jianlin; zhang, xue; wang, ke; gu, chunfang; wu, kailang; li, yan; zheng, congyi; xiao, gengfu; yan, huimin; zhang, jiamin; guo, deyin; tien, po; wu, jianguo title: isolation of virus from a sars patient and genome-wide analysis of genetic mutations related to pathogenesis and epidemiology from 47 sars-cov isolates date: 2005-01-01 journal: virus genes doi: 10.1007/s11262-004-4586-9 sha: doc_id: 302584 cord_uid: fwdpzv85 severe acute respiratory syndrome (sars) caused by sars-associated coronavirus (sars-cov) is a fatal disease. prevention of future outbreaks is essential and requires understanding pathogenesis and evolution of the virus. we have isolated a sars-cov in china and analyzed 47 sars-cov genomes with the aims to reveal the evolution trends of the virus and provide insights into understanding pathogenesis and sars epidemic. specimen from a sars patient was inoculated into cell culture. the presence of sars-cov was determined by rt-pcr and confirmed by electron microscopy. virus was isolated followed by the determination of its genome sequences, which were then analyzed by comparing with other 46 sars-cov genomes. genetic mutations with potential implications to pathogenesis and the epidemic were characterized. this viral genome consists of 29,728 nucleotides with overall organization in agreement with that of published isolates. a total of 348 positions were mutated on 47 viral genomes. among them 22 had mutations in more than three genomes. hot spots of nucleotide variations and unique trends of mutations were identified on the viral genomes. mutation rates were different from gene to gene and were correlated well with periodical or geographic characteristics of the epidemic. in november 2002, first case of a novel infectious disease named severe acute respiratory syndrome (sars) suddenly appeared in southern china [1] . this illness emerged and rapidly spread to different areas of asia and then other countries around the world with a high morbidity (about 25% required intensive care) and 9.6% fatality [2] . in march 2003, the world health organization (who) made an unprecedented international effort by organizing world-leading laboratories to find the causative agent. this effort resulted in the declaration made simultaneously by three research groups that a new sars-associated coronavirus (sars-cov) was the pathogen of this disease [3] [4] [5] . when the outbreak of sars came to an end in july 2003, it had caused a cumulative total of 8437 cases and 813 deaths worldwide [6] . since the discovery of sars-cov, progresses regarding the studies of this virus have been swift dramatically as the complete viral genome was sequenced [7] . although the definition of sars case still largely relied on clinical and epidemiological criteria, diagnostic tests based on the detection of viral rna and proteins have been developed [8] , along with the development of vaccines [9] . results from both phylogenetic analysis and epidemiological studies suggested the origin of sars-cov was animal-oriented, most likely from himalayan palm civets, ferrets and raccoon dogs [10] [11] [12] [13] . as a member of the coronoviridae family, sars-cov is enveloped and positive-stranded rna virus. it harbors 23 coding sequences, including 4 primary structural proteins (nucleocapsid protein n, spike protein s, membrane protein m, and small envelope protein e); 5 non-structural proteins (x1, x2, x3, x4, x5); and 1 polyprotein that compose two orfs (orf1a and orf1b). polyprotein catalytically auto-processes to produce a group of proteins including proteases (plppro and 3clpro), rnadependent polymerase (pol), rna helicase (hel), and function unknown proteins [4, 5, 7] . like other rna viruses, whose most striking characteristic is the high rate of genetic mutation [11, [14] [15] [16] [17] [18] . despite the fact that the sars-cov can cause an atypical and fatal form of pneumonia, the genome structure, gene expression pattern, and protein profiles of the virus are similar to those of other conventional coronaviruses [17] , which are only responsible for mild respiratory tract infections in a wide range of animals including humans, pigs, cows, mice, cats, and birds [10, 19] . it is possible that distinct patterns of several genes and unique variations in the sars-cov genome may contribute to its severe virulence or pathogenesis. the mechanism of sars-cov pathogenesis may involve both direct viral cytocidal effects on the target cells and immunemediated mechanisms. potential mutability of the viral genome may pose problems in the control of future sars epidemics. in this report, we described the isolation of a new sars-cov strain (whu) from a patient in hubei province, china during the late period of sars outbreak. complete genome sequence of whu isolate was determined and compared with that of 46 other sars-cov strains whose complete genomic sequences were available at the time analyzed. comparative study of genetic characterization and nucleotide variation of all known sars-cov offers insights into understanding functions of the viral genes and revealing the evolution trends of the virus. it would also provide basis for clinical diagnosis, future developing potential drugs and vaccines against sars-cov infections. the sars patient was an 18-year-old male from jiayu county, hubei province, china. he worked in beijing during that time when sars outbreak was occurring. he came back to hubei province and became ill on april 29th, 2003 with fever and atypical pneumonia, and was admitted to hospital for isolation and treatment on may 3rd 2003. veroe6 cells were inoculated with specimen obtained from the sars patient. the presence of the sars-associated coronavirus in infected cell cultures was determined by the appearance of cytopathic effects (cpe) as well as by rt-pcr amplification using primers (primer-1/primer-2 and primer-3/primer-4; table 1 ) specific to the sars-cov. viral particles were examined under electron microscope. viral rna was extracted from infected veroe6 cells based on the procedures described by the manufacture (invitrogen, carlsbad, ca). the first strand of the viral cdna was synthesized from extracted viral rna by reverse transcription pcr using random primers provided by the manufacture (promega, madison, wi). double-stranded dna fragments were produced by pcr amplification of the viral cdna using 10 pairs of specific primers (primer 5 to primer 24; table 1 ) designed to cover entire viral genome based on the sequences of sars-cov strain hku-39849 (accession number ay278491). each of the pcr products was cloned into vector pgem-t, respectively. random clones were selected for dna sequencing analysis. sequences representing the entire viral genome was fully assembled and edited by dnasis software programs. nucleotide sequences of complete genome of the sars-cov isolate (whu) were deposited to genbank (accession number ay394850). the complete genome sequences of all 47 sars-associated coronaviruses were downloaded from genbank (table 2 ). homology searches for the dna sequences were conducted and their deduced amino acid sequences were analyzed through the public database with the blast search program provided by the national center for biotechnology information (ncbi). sequence alignment was performed using software clustalw and further analyzed using software bioedit. nucleotide sequences of the entire genome of newly identified whu strain along with that of other 46 sars-cov isolates released in the genbank were aligned with the clustalw software program. phylogenetic trees were created for all nucleotide sequences by neighbor-joining and parsimony methods. sequences were analyzed with reference to the trees to reveal character states relevant to phylogenetic branching. during late period of the sars outbreak in 2003, three patients were identified as probable sars cases in hubei province, a less sars representative area in china. in order to study the sars-cov caused disease, we obtained specimen from one of the patients. seven days after inoculation of veroe6 cells with patient specimens, cpe was appeared on the infected cells ( fig. 1) indicating the presence of an infectious agent. two specific amplicons were detected by rt-pcr amplifications using extracted viral rna as templates when two pairs of sars-cov specific primers were used, respectively (data not shown). these results implicated that exist of a sars-cov in the specimen was highly possible. coronavirustable 2 . accession numbers of genomic sequences of 47 sarsassociated coronaviruses released in the genbank accession number accession number urbani ay278741 twy ap008581 tws ap006560 twk ap006559 twj ap006558 twh ap006557 cuhk-w1 ay278554 taiwan tc3 ay348314 taiwan tc2 ay338175 taiwan tc1 ay338174 twc ay321118 frankfurt ay291315 bj04 ay279354 bj03 ay278490 bj02 ay278487 zj01 ay297028 tor2 ay274119 tw1 ay291451 bjo1 ay278488 shangai qxc1 ay463059 shangai qxc2 ay463060 like particles were observed when we further examined infected cells under electron microscope (data not shown). in addition, sars-cov antibodies were detected from the patient's serum. all together, these results provided substantial evidence to suggest that this patient was infected by sars-cov, named whu strain. after identification of the whu strain, we isolated the virus and determined complete nucleotide sequences of its genome (accession numberay394850). since this virus was the only sars-cov that has ever been isolated and sequenced from hubei province, we carried out detailed sequence analysis of its entire genome. results from sequence analysis indicated that the genome of whu strain consisted of 29,728 nucleotides with a two-nucleotide deletion at residuals 27,825 and 27,826. phylogenetic analysis was conducted with the genome sequences of the whu strain and that of all 46 sars-cov isolates, whose genomic sequence information was fully available in the public databases (table 2 ). both phylogenetic study and sequence analysis indicated that the overall genome organization and predicted proteins of whu isolate were in agreement with published studies on other sars-cov isolates (fig. 2) . like all sars-cov isolates, the whu strain belongs to a new group of coronavirus [3] . however, the whu isolate with a two-nucleotide deletion was genetically diverse from most of the published sars-cov isolates, but closely related to twc strain (fig. 3) . to investigate the variations of nucleotide sequences among sars coronaviruses, we performed a genome-wide analysis of genetic mutations on all 47 sars-cov genomes. results indicated that a total of 348 positions on the 47 viral genomes had alterative nucleotides. among them, 22 positions with mutations occurred on more than three viral genomes ( table 3 , fig. 4 (table 3 and fig. 4) . our next step was to determine whether the high mutability had any implications linked to the viral genes or their functions ( fig. 4 and table 3 ). after further comparison and analysis of the viral sequences, we realized that polyprotein gene (orf1 a and orf1 b) had the highest variation rate among all genes. this region not only carried 11 mutations, but also had the second highest variable positions (residual 3852 and 11,493). orf1b gene contains additional two residuals (17,564 and 19 ,084) at which 7 viruses were mutated. we also noticed that the s gene had a high mutability with residual 22222 mutated in 7 viruses, residual 21721 in 6, and residual 24933 in 3. two positions with high mutation rate were identified within the m gene. one was located at the most variable residual 26477, at which 20 viruses were mutated. the other one was residual 26600, at which 6 viral genomes were changed. e gene and n gene had one mutation spot at residual 26203 and 28276, respectively. among five nonstructural genes, x4 had one mutation site at residual 27243 with mutation rate of 5, while x5 gene had two mutation spots at residual 27813 and 27827 with mutation rate of 7 ( fig. 4 and table 3 ). based on the recommendations from who [6] , all sars cases can be divided periodically into early-period case, mid-period case, and late-period case (table 4 ). in this study, we proposed all 47 known viral isolates into two groups, early-mid period and mid-late period group (table 5) . based on results from sequence analysis, we realized that there were some correlations between genetic mutations of the virus and periodical or geographic characteristics of the outbreak. several residuals (9404, 9854, 17564, 19838 (tables 3 5) . in addition, some genetic mutations were linked to certain geographic regions where the viruses isolated. for instance, high genetic mutation rate at position 3852 was mainly found in viruses isolated from taiwan. mutations at residual 26203 occurred in most taiwan isolates (60%), but not found in any isolates identified from other regions around the world. moreover, all three viral strains (fra, sod and frankfurt) isolated from europe had mutations at the same residuals, 2557, 11448 and 24933, while the rest isolates showed no changes in these positions (tables 3 and 5 ). although the sars epidemic ended after 6 months spreading, many important questions remain unclear. what is the natural reservoir of sars-cov; where and how the virus crossed the barriers between its reservoir and human to initiate reservoir-human transmission, and subsequent human-to-human infection. it was proposed that the natural reservoir of sars-cov was animal originated [10, 11, 13] , most likely himalayan palm civets [12] . this was not a surprise, since many fatal human viruses including hiv and influenza virus were originated by transmission from animals. hiv pandemic had happened as a consequence of the combination of transmission of sivcpz from chimpanzee and common practice of ''hunting and field-dressing chimpanzee'' in west central africa [20] . similarly in southern china, where sars-cov initially emerged, people used to consume wild animal meat and some of the animals are now confirmed to carry sars-like coronavirus [12] . another question is whether sars outbreak will come back. at the beginning of 2004, three sars cases were reported indicating sars do come back. however, the situation of this year seems quite different from last year, since transmission, infection and severity of sars-cov were clearly weakened. one possible explanation is that it might be just a preface of sars epidemics. like last year, in the early period of sars pandemics, the virus did not show strong toxicity. another possibility is that sars-cov might be truly weakened due to many reasons including genetic mutations, like the influenza flua virus which has caused a disaster outbreak in 1918 and was weakened after the pandemic that took 20 million lives [21] . influenza epidemics throughout the world occurred periodically between the first pandemic and present time due to the viral antigenic drift and shift. these processes also resulted in the appearance of influenza b and c virus with significant differences in genetic characterizations [22] . it would be important to find out if sars-cov has similar epidemic rules as influenza virus dose, whether sars-cov is weakening or will sars breakout periodically. while these questions remain to be addressed, it is for sure that the sars-cov certainly has a high mutation rate on table 3 . summary of genetic mutations within genes of 47 sars-associated coronaviruses orf 1a position 2557 3852 9404 9854 11448 11493 mutation rate 3 14 7 6 3 14 its genome, which could in turn play significant roles in its pathogenecity and epidemics of the disease. molecular epidemiology and genome-wide analysis of mutations among sars-cov have provided insights into our understanding some of the questions [11, [14] [15] [16] [17] [18] . for instance, except the geographic distribution of potential animal reservoirs, the high homologies among sars-cov of human and sars-like coronavirus of animals strongly supported the hypothesis of animal origin of sars-cov [12] . it is possible that some mutations on the viral genome were responsible for the transmission of sars-cov from animals to human. in an effort to study the sars-cov, we identified and genetically sequenced a new sars-cov isolated from a patient with sars in hubei province. hubei was a less sars representative area in china, because there were only a total of three patients confirmed as probable sars cases and only one viral strain was isolated from this region. these facts prompted us to study this virus further. our sequence analysis indicated that although the overall genome organization of whu (fig. 2 ) is in agreement with published studies on other isolates, whu carried a two-nucleotide deletion at residuals 27825 and 27826 was genetically diverse from most sars-cov isolates. these results implicated that mutations occurred during the viral transmission from beijing to hubei, although we do not know at this point whether these mutations have any biological significance. it is interesting to notice that although the sars-cov virus evaded human population only for 6 months, its genetic information already altered in many ways during its short journey of human transmission. individual viral genes displayed distinct patterns of genetic mutations at different time during the sars outbreak. for instance, mutability of the s gene was high during early-mid period, but low during mid-late period of the epidemic, which suggested that mutability of s gene decreased as viral transmission increased. one possible explanation for this observation is that during early-mid period of the epidemic, as the gene encoding protein for the recognition of receptors of the host and for the mediation of viral entry into host cells, s gene had to change at a high frequency in order to quickly fulfill its biological roles. once the viral adaptation to human cells completed or reached its equilibrium, genetic changes were less important or no longer needed. thus, genetic information of s gene became relatively stable during mid-late period of the outbreak [23] . another example is orf lab that encodes the polyprotein of sars-cov. like s gene, orf lab was also actively involved in genetic mutations. however, in contrast to s gene, mutability of orf lab was low at the beginning, but high during midlate period of the epidemics. this observation can be explained well by the fact that the toxicity of sars-cov was weakened in mid-late period. other structural genes including e, m, and n genes were more conserved at beginning of the outbreak, but underwent genetic changes at the end of transmission. this pattern of genetic mutation obviously reflects biological roles of these structural genes in viral particles assembly, which in turn crucial for the virus to fight with increasing immune pressures from the hosts. genetic analysis of non-structural genes showed that they intended to keep genetic information conserved throughout the entire process of transmission. therefore, these genes may prove to be ideal targets for the diagnosis of sars-co.v, screening antiviral drugs, and perhaps developing antiviral vaccines. patterns of genetic mutations of certain viral genes were linked to geographic locations from where the virus isolated. mutations at residuals 3825 and 26203 within the x5 and e genes could clearly set the taiwan isolates apart from others. thus, these two positions may be used as molecular signatures in the identification of taiwan isolates. similar phenomena were also found in three viral strains (sod, fra, and frankfurt) isolated from europe during mid-late period of the outbreak. these viral strains had mutations at the same residuals (2557, 11,448 and 24,933), while all isolates from other regions did not show any changes at these positions. this kind of specific mutation pattern may reflect relatively independent geographical locations of taiwan and europe. we speculated that population in these regions perhaps developed unique immunity due to their unique locations, for which the virus had to make specific genetic mutations in order to invade these populations. in addition, based on genome-wide mutation analysis, some viral strains isolated from beijing had a close relationship to isolates identified from southern china during early-mid period of the outbreak. it could be translated to that at least these sars-cov isolates found in beijing were originally from southern china. much have to be done in order to understand thoroughly the evolution, transmission, origin, and infection of sars-associated coronavirus. it is interesting to recognize that genome-wide mutation analysis could provide new insights into our understanding the route of viral transmission and predication or perhaps prevention of future sars epidemics. our study would provide a rational and hypothesis-driven approach to study these questions, develop rapid diagnostic tests, and design measurement to prevent this fatal disease. in addition, fully understand molecular mechanism of genetic mutations would provide insights into understanding plausible transmission route of sars-cov from animal to humans as well as from human to human, and trends of changing in pathogenecity of sars-cov during its rout of transmission and path of evolution. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) department of communicable disease surveillance and response. who consensus document on the epidemiology of severe acute respiratory syndrome (sars) this research was supported by the sars special grant of wuhan university. key: cord-293988-f5gvwjyh authors: musso, nicolò; costantino, angelita; la spina, sebastiano; finocchiaro, alessandra; andronico, francesca; stracquadanio, stefano; liotta, luigi; visalli, rosanna; emmanuele, giovanni title: new sars-cov-2 infection detected in an italian pet cat by rt-qpcr from deep pharyngeal swab date: 2020-09-11 journal: pathogens doi: 10.3390/pathogens9090746 sha: doc_id: 293988 cord_uid: f5gvwjyh the pandemic respiratory disease covid-19, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), emerged in wuhan in december 2019 and then spread throughout the world; italy was the most affected european country. despite close pet–human contact, little is known about the predisposition of pets to sars-cov-2. among these, felines are the most susceptible. in this study, a domestic cat with clear clinical signs of pneumonia, confirmed by rx imaging, was found to be infected by sars-cov-2 using quantitative rt–qpcr from a nasal swab. this is the first italian study responding to the request of the scientific community to focus attention on the possible role of pets as a viral reservoir. an important question remains unanswered: did the cat actually die due to sars-cov-2 infection? the world health organization (who) declared covid-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), as a worldwide pandemic [1] . the first epidemic cluster occurred in china, namely in wuhan city [2, 3] , as early as 1 december 2019. as of mid-july 2020, there have been over 5,525,245 confirmed covid-19 cases worldwide, with more than 30% cases in the eu and uk and more than 347,108 deaths globally [2] , (https://www.worldometers.info/coronavirus, accessed on 10 may 2020). italy was severely affected [4] , and it was one of the first and hardest hit countries in europe, with over 219,000 cases and 30,500 deaths reported [2] , for which reason on 9 march 2020 a lockdown was declared for the entire country and progressively stricter restrictions adopted [4, 5] . sars-cov-2 is structurally and functionally closely related to the already known coronaviruses responsible for the middle east respiratory syndrome (mers-cov) and the severe acute respiratory syndrome (sars-cov). in detail, the mechanism of host cell infection is the same for sars-cov and sars-cov2, i.e., it depends on the external protein of the virus, the glycoprotein (s) spike, which is able to bind and recognize the human receptor angiotensin converting enzyme 2 (ace2), primed by the transmembrane protease, serine 2 (tmprss2) and/or extracellular matrix metalloproteinase inducer cd147 [2, [6] [7] [8] . because of the close contact between people and pets, such as dogs and cats, the scientific community started to investigate the possibility of animal-human virus transmission [9] . furthermore, the animal ace2 receptor has high human ace2 aminoacidic sequence identity [10] [11] [12] . it has been shown that some animal species, particularly felines, can occasionally become infected with sars-cov-2 [10, [13] [14] [15] [16] . so far, some cases of domestic animal infection have been reported in belgium [17] , hong kong [14] , and france [18] , but no cases had yet been investigated in italy. in particular, viral rna and infectious viral particles were found in the upper respiratory tract of domestic cats after introduction of sars-cov-2 virus samples through their nasal cavities; nevertheless, none of the infected cats showed clinical signs of the disease. in addition, viral rna was detected in 1:3 healthy cats exposed to infected felines, suggesting that they had contracted the virus from the droplets exhaled by infected cats [13, 19] . in this study, we identified the natural infection of a cat with sars-cov-2 for the first time in italy. the rna obtained from the nasal swab was processed by rt-qpcr using two different chemistries, revealing the presence of two sars-cov-2 genes. finally, part of one gene was further sequenced to evaluate its nature. a sterilized, three-year-old, male european shorthair cat was presented to a veterinary clinic with the owner reporting serious respiratory distress of about three days. the mandatory vaccinations required by the current italian legislation, including viral rhinotracheitis, calicivirus and panleukopenia, had been completed. on physical examination, the cat had severe dyspnea and sialorrhea, kussmaul breathing, and asynchronous chest and abdomen. chest auscultation revealed increased vesicular murmur. due to the presence of pathological infiltration and ground-glass opacity of the lungs, the differential diagnosis suggested an idiopathic interstitial pneumonia of bacterial, viral or mycotic origin. despite amoxicillin and clavulanic acid administration in combination with aerosol, the cat died shortly after admission and the corpse was cremated according to the current health protocol. according to good veterinary practice, a blood sample was collected from the jugular vein and 1 ml was transferred to a sterile tube containing ethylenediamine tetra-acetic acid (edta) for hematological analysis, whereas another aliquot of about 3 ml was collected in a sterile glass tube to obtain serum sample for biochemical analyses. moreover, clinical imaging evaluation was performed using ultrasound and x-ray techniques. given the similar clinical picture and the current situation caused by sars-cov-2, a nasal swab was collected to test for sars-cov-2. a nasal swab was collected using the virus test kit diagnostics sterile pack swabs universal viral transport system (cod. ryco-vart10b03, jiangsu rongye technology co., ltd., touqiao town, yangzhou city, china). to enhance rna uptake, a variation to the standard protocol was made: 700 µl of swab buffer was processed in the same column, instead of the original 140 µl. ave buffer and ethanol were added in the same proportions in order to reach a final extraction volume of 6.3 ml. then, the total volume was eluted in the same column ten times. for the rest, the extraction process followed the protocol provided by the manufacturers. viral rna from the original swab was purified using the qiaamp ® viral rna mini-kit (qiagen, hilden, germany; cod. 52904). the extracted rna was quantified by fluorometric technique using the qubit™ rna hs assay kit (thermo fisher, waltham, ma, usa; cod. q32852) according to the standard procedure. nasal swab rna reverse transcription was carried out using the quantitect ® reverse transcription kit (qiagen, hilden, germany; cod. 205311). rt-qpcrs targeting sars-cov-2 were performed using taqman and sybr chemistries on a rotor-gene q thermocycler (qiagen) to amplify two different sars-cov-2 genes: the n1 gene, as indicated by the centers for disease control and prevention (cdc), and the spike gene, respectively. the primers used, their concentrations and the qpcr thermal profiles are listed in table 1 . rt-qpcr targeting sars-cov-2 was performed with taqman chemistry: the taqman ® probe (qiagen, hilden, germany) was labeled at the 5 -end with the reporter molecule 6-carboxyfluorescein (fam) and at the 3 -end with the black hole quencher 1 (bhq-1) (eurofins genomics, luxembourg); the reaction was performed using the quantinova probe pcr (qiagen, hilden, germany; cod. 208252) according to the manufacturer's recommendations. rt-qpcr targeting the sars-cov-2 spike was performed using sybr rt-qpcr (quantitech primers, qiagen, hilden, germany; cod. qt00016786) giving an amplicon of 88 bp. finally, amplicons were verified by running the rt-qpcr products on 1.8% agarose gel stained with (canvax, córdoba, spain) greensafe dna gel stain (cod. e0206) and fastruler ladder (thermo fisher, waltham, ma, usa; cod. sm1103). to exclude human mrna cross-contamination, human beta-actin primers at a final concentration of 1 µm were used as a control at the sybr rt-qpcr (quantitech primers, qiagen, hilden, germany; cod. qt00016786) giving an amplicon of 88 bp. the amplicons obtained by pcr from the amplification with the n1 portion gene primers (without probe) were purified using the qiaquick pcr purification kit (qiagen, hilden, germany; cod. 28106) and quantified using the fluorimeter qubit dsdna br assay kit (invitrogen, carlsbad, ca, usa; cod. 32850), then 5 ng of the product were sequenced in a seqstudio genetic analyzer (thermo fisher scientific, waltham, ma, usa) using the applied biosystems bigdye terminator cycle sequencing 3.1v (thermo fisher scientific, waltham, ma, usa; cod. 4337455) as previously described [21] . amplicons were then sequenced in a seqstudio genetic analyzer (thermo fisher scientific, waltham, ma, usa) using the applied biosystems bigdye terminator cycle sequencing 3.1v (cod. 4337455, thermo fisher scientific, waltham, ma, usa; cod. 4337455) as previously described [22] and compared with the reference sequence "mt077125 severe acute respiratory syndrome coronavirus 2 isolated sars-cov-2/human/ita/inmi1/2020 (complete genome sequence release date: 11-apr-2020)" using the blast tool (https://blast.ncbi.nlm.nih.gov/blast.cgi). ethical approval was not necessary as per institutional and national guidelines and regulations. the main biochemical and hematological parameters revealed lower alkaline phosphatase and higher glycemia values, while the blood count test showed relative and absolute neutrophilia (figure 1a) . the radiographic analysis revealed an unstructured interstitial pattern caused by the widespread presence of pathological infiltrate throughout the lung interstitium. the increased radiodensity of the lung parenchyma, defined as "ground glass", with little or no evidence of the intrathoracic vessels, clearly confirmed this condition (figure 1b) . furthermore, x-ray imaging revealed interstitial pneumonia with an area of pulmonary opacity (figure 1b) leading to the suspicion of effusion, excluded by the ultrasound examination (data not shown). the rna extracted from the nasal swab was quantified at 6.56 ng/µl and tested in duplicate by taqman probes for the detection of the sars-cov-2 n1 portion gene. sars-cov-2 synthetic rna and nasal swab rna amplified within the 28th and 34th cycle threshold (ct), respectively, while no amplification for the no template control (ntc) was detected. the amplification curves and ct, average and sd data are reported in figure s1a (supplementary materials). furthermore, the real-time qpcr products were run on a 1.8% agarose gel to confirm the correct size of the amplicon ( figure s1a, right) . further evidence of sars-cov-2 was given by the detection of the spike gene within the 40th ct during the real-time qpcr assay, while no amplification for sars-cov-2 synthetic rna and ntc was found ( figure s1b ). to ensure that two different real-time activities were conducted on the same rna during the sample collection and processing phases, and to exclude any possible contamination with human rna, a feline housekeeping gene (available in the techne-fcov kit) was used to verify the presence of correct feline rna. the nasal swab sample amplified within the 39th ct, while there was no amplification for the ntc (figure s1c ). at the same time, the feline sample tested negative for the human beta-actin that was used as control. finally, the amplified fragment obtained by endpoint pcr using the same rt-qpcr primers ( figure s1a, right) was sequenced using the sanger method to verify the accuracy of the amplification, resulting in a perfect match with the sars-cov-2 n1 gene according to the blast tool (https: //blast.ncbi.nlm.nih.gov/blast.cgi) ( figure s2 ). in late december 2019, the severe acute respiratory syndrome-coronavirus 2 (sars-cov-2), identified as a novel coronavirus [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] , first caused uncommon pneumonia in humans in wuhan (china) and then rapidly spread internationally. thus, the world health organization (who) named the disease caused by this virus "coronavirus disease 2019" (covid-19) and officially declared covid-19 as a pandemic [34] . there is evidence that sars-cov-2 shares 96.2% of its nucleotide identity with the ratg13 coronavirus detected in horseshow bats in china [35] . indeed, first evidence indicates that the infection took place through the consumption of meat derived from infected bats (bat meat is commonly eaten in china). the fact that pets are in close contact is given, but it became especially relevant once it was shown that pets were also susceptible to infection. knowing their susceptibility to sars-cov-2 is therefore very important as several cases of infected pets have been reported. unlike several studies carried out on dogs, which have shown little, if any, susceptibility to the virus, felines-especially cats-seem to have greater predisposition to the infection [13, 16] . in the same way as in other coronaviruses such as sars-cov, the feline coronaviruses described until now seem to be able to change their tissue tropism [36] . in this study, we report-for the first time in italy-the case of a male european cat positive for sars-cov-2. unfortunately, we were not able to confirm the presence of the virus in the lungs of the cat because it was cremated before any tissues could be collected. as such, no association can be made between detection of sars-cov-2 in the nasal swab of the cat and its clinical condition. sars-cov-2 may well have been the cause of the cat's death, but it may have equally been an accidental finding [18] . the cat presented clinical signs of pulmonary disease and the blood test and subsequent x-ray and ultrasound investigation confirmed the diagnosis of severe pneumonia. as the cat's pathology evolved rapidly and harmfully (the animal died in as little as three days), with clinical signs and rate of disease progression similar to human covid-19 patients, and because previously published papers reported different cases of feline infection [10, [13] [14] [15] [16] , a nasal swab was collected in order to verify a possible infection with sars-cov-2. the identification of sars-cov-2 virus was supported not only by the qpcr amplification of the commonly used n1 gene within the 34th ct, by means of the taqman probe, but also by the amplification, using a more economic sybr green chemistry, of another portion of the sars-cov-2 virus spike gene-missing in the synthetic rna commonly used as positive control-which may represent a new molecular identification target. at the same time, the feline sample was free from human cross-contamination, as confirmed by positive amplification for a feline housekeeping gene and by the absence of amplification of the human housekeeping gene beta-actin. finally, to verify the presence of sars-cov-2, an endpoint pcr was performed with cdc primers targeting n1 and the products were subsequently sequenced using the sanger method. although the clinical history and the investigations performed clearly show positivity with clinical manifestations for sars-cov-2 in the lung, the infection source could not be clarified. as a resident of a ground floor apartment, the cat was used to going outside. moreover, it was the only pet living with the owner. probably, the contamination was due to the habit of cats of licking potentially contaminated surfaces or through contact, not detected by the owners, with other unidentified positive cats or positive asymptomatic visitors. at the same time, the presence of sars-cov-2 in the nasal swab is not enough to assert that it caused the pathological event [37] . as per health protocols and due to the time required for the swab transport and analysis, the cat corpse was cremated before knowing the test results. for this reason, unfortunately it was not possible to perform histological analysis to confirm that the pet died due to sars-cov2, although the concomitant presence of severe pneumonia and virus rna was ascertained. therefore, the link between sars-cov-2 infection and the cat death remains an open question. to date, the crisis that has affected several world regions seems to be over, but the natural reservoirs of the virus and its high contagion rate, as well as environmental and social conditions may pave the way for a second epidemic wave. particularly, as cats and dogs are more common and in closer contact with humans than bats, they should be checked for sars-cov-2 when affected by severe pneumoniae, as several al studies have demonstrated the propensity of coronaviruses for interspecies transmission [38, 39] . at this point, there is no evidence that cats can spread covid-19 and owners should not abandon their pets nor compromise their wellbeing [18] . supplementary materials: the following are available online at http://www.mdpi.com/2076-0817/9/9/746/s1, figure s1 : amplification profiles for the detection of sars-cov-2. figure s2 : sequencing of amplified end-point pcr derived fragment compared with sars-cov-2 genome. author contributions: n.m., a.c. and g.e. conceived and designed the study. n.m., a.c. and f.a. performed the experiments. s.s., r.v., l.l., s.l.s. and a.f. contributed to data analysis. n.m. and a.c. wrote the draft manuscript, s.s. reviewed and edited the manuscript. all authors have read and agreed to the published version of the manuscript. the authors received no financial support for the research, authorship, and/or publication of this article. world health organization. who director-general's opening remarks at the media briefing on covid-19 covid-19 infection: the china and italy perspectives clinical features of patients infected with 2019 novel coronavirus in modelling the covid-19 epidemic and implementation of population-wide interventions in italy chronology of main steps and legal acts taken by the italian government for the containment of the covid-19 epidemiological emergency 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novel coronavirus: implications for virus origins and receptor binding early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of coronavirus disease 2019 in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study world health organization (who) a pneumonia outbreak associated with a new coronavirus of probable bat origin spike protein fusion peptide and feline coronavirus virulence causation and causal inference in epidemiology cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: we wish to thank stefania stefani and massimo gulisano for their suggestions. the authors declare no conflict of interest.pathogens 2020, 9, 746 pathogens 2020, 9, 746 key: cord-298989-qk0k2lmz authors: , umesh; kundu, debanjan; selvaraj, chandrabose; singh, sanjeev kumar; dubey, vikash kumar title: identification of new anti-ncov drug chemical compounds from indian spices exploiting sars-cov-2 main protease as target date: 2020-05-13 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1763202 sha: doc_id: 298989 cord_uid: qk0k2lmz the 2019-novel coronavirus (ncov) has caused a global health crisis by causing coronavirus disease-19 (covid-19) pandemic in the human population. the unavailability of specific vaccines and anti-viral drug for ncov, science demands sincere efforts in the field of drug design and discovery for covid-19. the novel coronavirus main protease (sars-cov-2 mpro) play a crucial role during the disease propagation, and hence sars-cov-2 mpro represents as a drug target for the drug discovery. herein, we have applied bioinformatics approach for screening of chemical compounds from indian spices as potent inhibitors of sars-cov-2 main protease (pdbid: 6y84). the structure files of indian spices chemical compounds were taken from pubchem database or zinc database and screened by molecular docking, by using autodock-4.2, mgltools-1.5.6, raccoon virtual screening tools. top 04 hits based on their highest binding affinity were analyzed. carnosol exhibited highest binding affinity -8.2 kcal/mol and strong and stable interactions with the amino acid residues present on the active site of sars-cov-2 mpro. arjunglucoside-i (-7.88 kcal/mol) and rosmanol (-7.99 kcal/mol) also showed a strong and stable binding affinity with favourable adme properties. these compounds on md simulations for 50 ns shows strong hydrogen-bonding interactions with the protein active site and remains stable inside the active site. our virtual screening results suggest that these small chemical molecules can be used as potential inhibitors against sars-cov-2 mpro and may have an anti-viral effect on ncov. however, further validation and investigation of these inhibitors against sars-cov-2 main protease are needed to claim their candidacy for clinical trials. communicated by ramaswamy h. sarma since december 2019, a outbreak of covid-19 with massive global impact has started in hubei and wuhan city in china caused by a novel coronavirus, sars-cov-2 (bhoopathi et al., 2020; kumar & rathi, 2020; wang et al., 2020) . on january 2020, world health organization emergency committee declared a global health emergency based on the high rate of spreading of the infection with high fatality rate (chhikara et al., 2020; kumar & rathi, 2020) . as of now on 4th april 2020, europe and usa are new epicentres of pandemic. even in the past, different coronaviruses have caused multiple human diseases that resulted in global epidemics such as the middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars) and coronavirus disease 2019 . while the coronaviruses have a significant impact on human health, the general public has an inferior awareness of coronavirus pathogenesis and infection. covid-19 was declared a pandemic in march 2020 as the worldwide human population is facing high risk from contracting the ncov infection. many countries, including india, has announced lockdown in the country and to maintain social distancing to avoid further spread as no drug or vaccine is available against sars-cov-2. coronavirus has been classified into four subfamilies based on their shape and host. these subfamilies are alpha-coronavirus, beta-coronavirus, gamma-coronavirus and delta coronavirus (paules et al., 2020) . alpha-coronavirus and beta-coronavirus are considered to have been originated from bats, while the gamma and delta coronaviruses are considered to be derived from birds and pigs (banerjee et al., 2019) . coronaviruses contain a positive sense, single-strand rna genome coding for viral polymerase, rna synthesis materials, and large nonstructural polypeptide. coronavirus genome contains transcriptional modification, including 5'methylated cap and a 3'polyadenylated tail. coronaviruses have very high rates of error in rna replication due to constant errors by rna dependent rna polymerase (banerjee et al., 2019; lau et al., 2018) . only a few protein crystal structures of sars-cov-2 are available on protein databank. sars-cov-2 main protease, a potential drug target, crystal structure (pdb-id: 6y84) was available and used for docking simulation and identification of potential drug molecule form indian spices. sars-cov-2 main protease has a vital role in the processing of polyprotein that is translated from viral rna, and the protease is considered as key for viral survival and growth . medicinal plants yielding biologically active compounds have always been of great interest to scientists as they play an essential role in preventing human diseases. in the entire world, india is recognized where spices have been traditionally used as a source of medicine. many active pharmaceutical gradients have been identified and extracted having a wide range of physiological and pharmacological properties (sachan et al., 2018) . apart from the regular uses of spices in culinary activities, they are widely used as indigenous medicines, nutraceuticals, in aromatherapy, as natural colouring agents, perfumes, cosmetics etc. in recent years there has been experimental evidence on physiological benefits that could be drawn in the context of various diseases like diabetes, cardiovascular issues and inflammatory disorders like arthritis and cancer. they have also been identified as preventive agents in certain conditions. spices like red pepper, garlic, and fenugreek have been reported to have hypercholesterolemic activity, whereas fenugreek and garlic are also known to reduce and control blood sugar levels (bhagya & raveendra, 2017) . the main active ingredient of turmeric, curcumin has been widely studied to have a broad spectrum of medicinal value ranging from anti-cancerous, anti-inflammatory and an anti-amyloidogenic activity (bhagya & raveendra, 2017) . several computational studies related to drug or vaccine development against sars-cov-2 is recently published (aanouz et al., 2020; gupta et al., 2020; joshi et al., 2020; muralidharan et al., 2020; sarma et al., 2020) . in the current study, we have utilized the prior knowledge on the medicinal values and potential applications of indian spices, we have tried to explore if they can be used as novel agents for controlling sars-cov-2. the data generated using computational approaches give very encouraging results. in this study, prepared list of 45 chemical compounds from indian spices and compound structure file was downloaded from pubchem database or zinc database. structure of protein sars-cov-2 mpro was downloaded from rcsb protein database. (pdb id: 6y84). the atomic coordinates of active site were defined using report available in the literature and identification of catalytic his41, his164 and cys145 (khaerunnisa et al., 2020) . energy minimization of the protein molecule was done by swiss pdb viewer (guex & peitsch, 1997) . before docking, the assignment of charge, solvation parameters and fragmental volumes to the protein was done using the autodock tool 4 (adt). the protein 6y84 pdb molecule was further optimized by using adt for the molecular docking using established procedure. the structure data files of the all chemical compounds were downloaded from pubchem database and converted into mol2 structures by using open babel. in order to further simplify the analyses, ligands were fist optimized and converted mol2 to pdbqt format by using the graphical user interface version of raccoon. mgltools-1.5.6, raccoon is preparing autodock virtual screening tool-python (forli, scripps research institute). compound screening using raccoon jautodock program molecular screening of the all chemical compound libraries was performed by using raccoon and mgltools-1.5.6 software by autodock as the engine for docking (morris et al., 2009) . during the molecular docking period, the ligands were considered as a flexible molecule and the protein was considered as a rigid structure molecule. the configuration file for the grid parameters file and docking parameters file was generated by using autodock. autodock and autogrid tools integrated with the autodock4 were used to generate grid maps for each atom of the ligand. the grid boxes were made, such as to include one site at a time and perform docking. grid x, y and z coordinates were 9.204, à4.557, and 19.602. we analyzed each ligand by setting default docking parameters except in the number of runs: we ran the lga for 100 runs with each ligand with an initial population size of 150 random starting positions and conformation, 2.5 million number of energy evaluations. the application of grid parameters file was also used to predict the amino acid residue in the active site of the protein that interacts with the ligands. positional root-meansquare deviation (rmsd) values were less than 1.0 å considered ideal and clustered together for finding the favourable binding. the highest negative binding energy was considered as the ligand molecule with maximum binding affinity. visual analysis of the docking site was performed using pymol-2.3.3 and the results were validated by using autodock tools-1.5.6 (morris et al., 1998) . binding interaction analyses of identifying inhibitor and the sars-cov-2 mpro (pdb id: 6y84) was done using an online program by using ligplot analysis (wallace et al., 1995) . the classical molecular dynamics simulation is carried out for the prepared co-crystal structure and selected ligand-binding complex poses through desmond molecular dynamics package incorporated in schrodinger suite (chow et al., 2008) . both the apo and ligand complex is solvated in the tip3p model (specifies a three-site rigid water molecule with charges), using the volume occupancy in an orthorhombic box with periodic boundary conditions. for neutralizing the system, the overall charge of apo and ligand complex is solvated with appropriate cation (naþ) or anion (cl-) along with a salt concentration of 0.15 mol/l. the prepared system is energy minimized for a convergence threshold of 1.0 kcal/ mol/å by using the steepest descent method, and for minimization and relaxation of the system, the npt ensemble is applied (selvaraj et al., 2015) . the standard temperature is kept constant at 300 k and pressure at the level of 1.013 bar for the total simulation, and each simulation is started for the total time scale of 50 ns. for the analysis of md trajectories, the rmsd, rmsf and hydrogen bond interactions are analyzed using the trajectory analysis incorporated in the desmond (selvaraj & singh, 2014) . the absorption, distribution, metabolism, and excretion (adme) properties of the studied top hits compound were calculated by using online swissadme program (guex & peitsch, 1997) . the significant parameters for adme associated properties such as lipinski's rule of five, pharmacokinetic properties the solubilities of drug and drug likeness were considered. we have created a database of 45 indian spices compounds and their structures downloaded from pubchem and zinc databases. four small molecules were selected based on their binding affinity with sars-cov-2 mpro as shown in the table 1 . the list of remaining compounds used of moleculer docking is submitted as supplementary material (table 1s ). the top three compounds include carnosol, rosmanol, and arjunglucoside-i. the further insights into binding interactions of these compounds with sars-cov-2 mpro were analysed using ligplot as shown in figures 1 and 2. sars-cov-2 mpro protein (pdbid-6y84) active site defined but unliganded (owen et al., 2019) was used and the molecules where docked in the region where a-ketoamide was bound to protein (pdbid-6y2f) (sars-cov-2) main protease with bound with a-ketoamide (zhang et al., 2020) involving his41,164 and cys145 in the active site. a-ketoamide was hence used as a positive control for this study and resulting interactions were analytically compared with the other indian spices. a-ketoamide showed interactions with cys145 via hydrogen bonds and table 1 . details of various kinds of interaction shown between the amino acids near the active site of sars-cov-2 main protease along with their respective inhibitor constant (ki) and biological source and binding energy. the active site residues are indicated in bold. hydrophobic interactions his41. it was also seen to form hydrogen bonds with thr26. upon further analysis of the docking results, it was seen that our molecules had comparable binding energy as to a-ketoamide suggesting that these ingredients could indeed interact with same site (amino acids) as a-ketoamide. carnasol (-8.2 kcal/mol) was seen to form hydrogen bonds with leu141, ser144 and cys145. hydrophobic interactions for carnasol include his41, thr25, asn142, phe140, glu166, met165 (figure 1) . rosmanol, isolated from rosemary (rosmarinus officinalis), has been previously reported to have antioxidant activity, shows binding energy of -7.99 kcal/mol and forms hydrogen bonds with leu141, gly 143, ser144 and cys145. further hydrophobic interactions are shown with thr25, 26, his41, phe140, his163, and leu27 (figure 2a ). arjunglucoside-i binds with hydrophobic interaction with his41, 164 and cys145 additional to hydrogen bonding with thr25, 26, his163, and glu166 ( figure 2b ). the results of md simulation for both apo and ligand complex is analyzed for the 50 ns of time scale to understand the dynamic behavior and stability. md simulation is performed for the total of 50 ns and the trajectories are for rmsd plot as shown in the the protein secondary structure is framed as 3 sheets, 7 beta hairpins, 9 beta bulges, 13 strands, 32 beta turns, 3 gamma turns from this 182-304 residues are occupied dominantly by loop regions. these 122 residues in the c-terminal functionally act in the md simulation, and thus the sudden drift happens in the 45 th ns. this values of rmsd for the apo protein, is compared with ligand molecules in the figure 3 , and for understanding the deviations, the 25 th to 50 th ns is focused. the results of ligand complex for the md simulation of 50 ns of timescale shows that the ligand complex alpha ketoamide (red color) and arjun glycoside (blue color) shows stable throughout the simulation. while comparing these two alpha ketoamide and arjun glycoside ligand complex with apo protein, the ligand complex is matched with apo protein for the timescale of 45 th ns. after the 45 th ns the apo protein is drifted upwards, but the alpha ketoamide and arjun glycoside ligand complex remains stable. this may be due to the strong interaction pattern seen in alpha ketoamide and arjun glycoside interactions with the protein. both the alpha ketoamide and arjun glycoside ligand bound complex are stable and positioned in the range of $2å, which is close to the apo protein till 47 th ns. the figures 5a and 5b shows the hydrogen bond interactions for the alpha ketoamide and arjun glycoside, which clearly shows the minimum participation of 2-3 hydrogen bonds seen in between the alpha ketoamide and protein, and minimum participation of 4-6 hydrogen bonds between the arjun glycoside and protein. this active participation of hydrogen bonds between the alpha ketoamide and arjun glycoside with protein makes the ligand complex stable for 50 ns of md simulations. the ligand carnosol shows stable movement in terms of rmsd values by showing a narrow graph, but while comparing with apo protein, the carnosol ligand complex is deviated from the 14 th ns and stays in the range bound of $3.4 å till the end of the simulations. for attaining the stable md simulation for the carnosol ligand bound complex, 2-3 hydrogen bonds are actively contributed and makes the complex stable in the dynamic state. the ligand rosmanol bound complex shows stability till the 26 th ns with the range of $2.5 å, but after that, the drift happens to make the rmsd value deviated in upward direction and fix the positional rmsd with the range of $4 å. this may be due to the loss of hydrogen bonding interactions after the 27 th ns seen in the figure 5d . figure 5d says that the initial 27 ns shows the hydrogen bond interactions in the range of 2-3 between the protein and ligand, but after 27 th ns the ligand losses the hydrogen bonding ability and shows the hydrogen bond interactions in the range of 1-2 between the protein and ligand. overall, the apo protein shows a narrow range of stability and notable fluctuations are also shown, that indicates the participation of loop structures. those fluctuations are arrested through the active interactions of ligand molecules that shows strong binding between the protein and ligand. we have also analysed the molecules we report here for violation of lipinski's rule (table 2) . rosmanol, carnosol fits perfectly as within the defined parameters for non-violation of lipinski's rule. the molecules have log p values ranging from 1.15 to 3.27 which imply that these can effectively have suitable cell membrane permeability. their number of hydrogen bond donors as well acceptors are well within range for carnosol and rosmanol but arjunglucoside-i show high number of hydrogen bond donors and acceptors. arjunglucoside is a large molecule having high molecular weight and total polar solvent area. despite these factors it might prove to be essential in terms of potential drug once preceded with advanced studies. amidst the unforeseeable outbreak of covid-19 there has been a sudden rise in demand of drug development, vaccines and identification of potential bioactive molecules which could prove to be useful fulfilling the purpose of broadening treatment options. in the quest for finding novel treatment regimen for these kinds of viral outbreaks, screening of already known molecules could also prove to be vital. in this context, we report here some active pharmaceutical ingredients which are present in the commonly used spices in india could prove to be useful. preliminary in silico investigations show that indeed some molecules like carnosol and rosmanol have the properties which can further exploited and investigated for drug candidate against sars-cov-2. although it is imperative to understand that development of rigid and highly specific treatment options will require further expeimental studies. no potential conflict of interest was reported by the author(s). moroccan medicinal plants as inhibitors of covid-19: computational investigations bats and coronaviruses mulibenificial uses of spices: a brief review novel 2019 coronavirus structure, mechanism of action, antiviral drug promises 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of pathological features of corona virus disease crystal structure of sars-cov-2 main protease provides a basis for design of improved a-ketoamide inhibitors key: cord-279105-e2zjxjox authors: lee, cheryl yi-pin; lin, raymond t. p.; renia, laurent; ng, lisa f. p. title: serological approaches for covid-19: epidemiologic perspective on surveillance and control date: 2020-04-24 journal: front immunol doi: 10.3389/fimmu.2020.00879 sha: doc_id: 279105 cord_uid: e2zjxjox since december 2019, the novel coronavirus, sars-cov-2, has garnered global attention due to its rapid transmission, which has infected more than two million people worldwide. early detection of sars-cov-2 is one of the crucial interventions to control virus spread and dissemination. molecular assays have been the gold standard to directly detect for the presence of viral genetic material in infected individuals. however, insufficient viral rna at the point of detection may lead to false negative results. as such, it is important to also employ immune-based assays to determine one's exposure to sars-cov-2, as well as to assist in the surveillance of individuals with prior exposure to sars-cov-2. within a span of 4 months, extensive studies have been done to develop serological systems to characterize the antibody profiles, as well as to identify and generate potentially neutralizing antibodies during sars-cov-2 infection. the vast diversity of novel findings has added value to coronavirus research, and a strategic consolidation is crucial to encompass the latest advances and developments. this review aims to provide a concise yet extensive collation of current immunoassays for sars-cov-2, while discussing the strengths, limitations and applications of antibody detection in sars-cov-2 research and control. the ongoing pandemic, which originates from a newly emerged coronavirus, sars-cov-2, was discovered in the city of wuhan in china's hubei province in december 2019 (1) . to date, due to rapid transmission globally, there are more than two million laboratory-confirmed human infection cases, with a few hundred thousand deaths across 210 countries and territories (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/ situation-reports/). this unprecedented crisis led to a worldwide effort to rapidly characterize the immunobiology of sars-cov-2, while mitigating further spread of this deadly pathogen. sars-cov-2 is a single stranded, positive sense rna virus that belongs to the coronaviridae family of the betacoronavirus genus (2) . it has a genome size of ∼30 kilobases that encodes for multiple structural proteins comprising the spike (s), the envelope (e), the membrane (m), and the nucleocapsid (n), as well as non-structural proteins (3) (figure 1) . infection by sars-cov-2 causes an acute respiratory disease termed the coronavirus disease 2019 . the clinical manifestations of covid-19 form a spectrum, from being asymptomatic to fever with mild respiratory illness, to acute respiratory distress syndrome, and death from respiratory failure or associated complications (3) (4) (5) . as the reported incubation period varies among different patient cohorts, it is often difficult to ascertain the actual day of onset, and infected subjects who are asymptomatic or pre-symptomatic may go undetected (5) (6) (7) . early detection of sars-cov-2 infection is one of the crucial interventions to control virus transmission. with the discovery of the genome organization of sars-cov-2 viral rna, which is adapted from genbank accession number: mn908947, is characterized by sequence alignment against two representative members of the betacoronavirus genus. the entire genome sequence is ∼30 kilobases (kb) long. the virus, numerous diagnostic assays using quantitative reverse transcriptase pcr (qrt-pcr) were developed (3) . qrt-pcr is the reference standard for diagnosing infections with high sensitivity and accuracy in the acute phase of illness. sars-cov-2 viral rna has been detected in both throat and nasal swabs of infected individuals by qrt-pcr, which becomes almost undetectable by 14 days post-illness onset (pio) (or symptom onset) (8, 9) (figure 2) . apart from being costly and time consuming to perform, false negative results may arise due to improper handling of nucleic acid samples, inadequate and variable sampling resulting in insufficient viral genetic material at the point of detection (after 14 days pio), or biological variation on when viral rna is detectable by qrt-pcr (10, 15) . with the limitations of qrt-pcr, immunoassays may offer another figure 2 | schematic illustration on the window period of detection for either viral rna or antibodies in sars-cov-2-infected individuals. presence of sars-cov-2 viral rna (boxed in pink) in throat or nasal swab of patients are typically undetectable by 14 day post illness onset (pio) (8, 9) . sars-cov-2-specific antibodies (boxed in blue): igm is detectable as early as 3 days pio, and peaks between 2 and 3 weeks pio (10, 11) . igm response was still detectable after more than 1 month pio (12) . both iga and igg are present as early as 4 days pio, and peaks after 2 weeks pio in serum samples (10, 11, 13, 14) . there are currently no reports on the presence of these sars-cov-2-specific antibodies in the later phase pio, as indicated by dotted lines. this depicts the importance of serological studies to identify individuals with current or prior exposure to sars-cov-2 that went undetected, by testing for either igm, igg, or iga antibodies against sars-cov-2. illustration was created using biorender. avenue to reduce undiagnosed cases, with the advantage that rapid test formats may deliver results in a relatively shorter time and lower cost (10). immunoassays are another diagnostic approach that can provide information on both active viral infections and past exposures (figure 2 ). to date, many commercial companies and research institutes have developed serological assays to detect sars-cov-2 antibodies from patient serum or plasma samples (16, 17) . closely related to another pathogen, severe acute respiratory syndrome coronavirus (sars-cov), these assays mainly target immunogenic coronavirus proteins: s protein, which is the most exposed viral protein, and n protein, which is abundantly expressed during infection (3, 14, 18) . in addition, the receptor-binding domain (rbd), which is located along the s protein, is also a target of interest to detect the presence of sars-cov-2-specific antibodies (19, 20) . in recent pre-prints deposited in medxriv and bioxriv, it was shown that both anti-sars-cov-2-igm and igg levels increase gradually along with infection phases, with igm being detected as early as 3 days pio, which peaks between two to three weeks pio (10, 11) . one study has reported that sars-cov-2-specific igm is still present in the serum after 1 month pio (12) . sars-cov-2-specific igg antibodies, on the other hand, can be present as early as 4 days pio, and peak after 17 days pio (10, 11) (figure 2) . these observations are similar to what was previously reported during a sars-cov infection (21) . however, interestingly, one study demonstrated that longitudinal profiling of both antibodies in a population of 63 covid-19 patients showed no specific chronological order in terms of igm and igg seroconversion (10) , which was also observed in patients infected with sars-cov and another human coronavirus, middle east respiratory syndrome coronavirus (mers-cov) (22, 23) . in addition, there seems to be no correlation between seroconversion rates with age, gender or time of hospitalization (10) . these findings on sars-cov-2-specific antibodies seroconversion against the s viral protein suggest the importance to test for both igm and igg antibodies to confirm a positive infection. expectedly, similar to what was reported for sars-cov and mers-cov, both igm and igg levels seems to be correlated with disease severity, with a higher level of both antibodies present in patients with more severe sars-cov-2 infection (10, 11, 14, (24) (25) (26) . in contrast to other flu-like infections such as influenza, instead of igg1, igg3 appears to be the dominant igg subtype during sars-cov-2 infection (13, 27, 28) . as a majority of the human population has prior exposure to endemic human coronavirus infections including alphacoronaviruses (229e and nl63), and other betacoronaviruses (oc42 and hku1) (29) , it is crucial to validate the specificity and sensitivity of current immunoassays against sars-cov-2 to avoid false positive outcomes. within the s protein antigen, cross-reactivity was observed when samples were tested against sars-cov s and s1 subunit proteins, and to a smaller extent, with mers-cov s protein ( table 1) . interestingly, there was no cross-reactivity with the s1 subunit of mers-cov (14) . the high level of crossreactivity between sars-cov and sars-cov-2 can be attributed to the high degree of genetic homology (3, 14, 19) . furthermore, detailed analysis revealed a highly conserved s2 subunit domain across coronaviruses, which may explain for the cross-reactivity observed with only the s protein of mers-cov, and not with the s1 subunit (14, 19) . these data suggest that using an s1 subunit-based immunoassay may be more specific than the entire s antigen for diagnosing sars-cov-2 infections. another immunogenic target, the rbd, which lies along the s protein is usually the target of many neutralizing antibodies against sars-cov (30) . a substantial level of cross-reactivity by sars-cov rbd-induced antibodies to sars-cov-2 rbd was described ( table 1 ) (20) . of clinical relevance, these antibodies were also able to cross-neutralize sars-cov-2 pseudovirus infection, signifying the potential of an immunotherapy-based treatment (20) . while one non-peer reviewed study has shown that rbd-based serological assays are more sensitive than s1 subunit-based assays in identifying antibodies in mild covid-19 patients (14), other non-peer-reviewed studies have described a lower degree of antibody response to the rbd as compared to full-length s protein, plausibly reflecting the larger number of epitopes present on the larger s antigen (13, 19 ). due to a high level of similarity of 90% between sars-cov and sars-cov-2 n proteins, the n antigen of sars-cov was also used for serological detection of sars-cov-2-specific antibodies ( table 1 ) (14) . these n-based assays were reported to be more sensitive than s1 subunit-based tests (14) . the use of sars-cov antigens to diagnose sars-cov-2 infections may be reliable, given that sars-cov has not circulated in the human population since 2004 (3). in addition, an earlier report has demonstrated waning of sars-cov-specific antibodies, therefore being undetectable in 91% of patient serum samples after 6 years (31) . since respiratory diseases are the hallmark of coronavirus infections, which activate mucosal immunity, several studies have exploited the detection of iga to diagnose sars-cov-2 infection in patients (table 1 ) (13, 14) . although a strong iga response was also detected in covid-19 patients where peak seroconversion was achieved by two weeks pio (figure 2) , igabased immunoassay has been hypothesized to be less specific than igg-based elisa due to cross-reactivity with serum samples from patients infected by other coronaviruses (14) . with the availability of immunoassays utilizing various coronavirus structural proteins, the use of more than one different antigen-based serological approach may be essential to establish a true positive sars-cov-2 infection. in addition, frontiers in immunology | www.frontiersin.org the use of saliva samples and other bodily fluid swabs as a less invasive alternative, which have been done for other viral infections including hiv and measles, should also be explored for serological testing of sars-cov-2 infections (32, 33). apart from using immunoassays for the early detection of sars-cov-2 infected individuals, it is also critical to determine the regions where sars-cov-2-specific antibodies bind to help guide vaccine designs. using sars-cov-derived b-cell epitopes that have been experimentally identified from positive b-cell assays (34), 49 out of 298 linear b-cell epitopes have an identical match with sars-cov-2 protein sequences without any mutations (3) . notably, majority of these matches were located at both the s and n viral antigens, with only 4 from the m protein, and none in the e protein (3). on the other hand, 6 conformational b-cell epitopes identified from the same database were located on the s antigen. however, unlike the linear epitopes, none of these mapped identically to the sars-cov-2 protein (3). further mapping the residues of linear b-cell epitopes onto available sars-cov s protein structure revealed several regions on the s2 subunit that may allow cross-neutralization of both sars-cov and sars-cov-2 (3, 35) . in contrast, conformational b-cell epitopes mapped onto the s1 subunit, resulting in very few identical residues within sars-cov and sars-cov-2 (3). these findings indicate that sars-cov-specific antibodies targeting these discontinuous regions may not be able to cross-react with sars-cov-2 (3, 36) . as these regions are computationally predicted, serological studies using patient samples are necessary to validate the importance of these regions for serology and in controlling sars-cov-2 infection. it also remains imperative to identify other sars-cov antibodies that may recognize the conformational epitopes of sars-cov-2 s protein, which can greatly reduce the amount of time needed to develop novel neutralizing antibodies. the findings derived from serological assays can provide valuable information that would help to support the diagnosis, treatment, and prevention of sars-cov-2 infections. characterization of antibody profiles suggested that any suspected individuals with undetectable antibody levels against sars-cov-2 after 20 days pio may be a true negative case, since both anti-sars-cov-2 igm or igg seroconversion should have already occurred (10, 11) . however, these findings may be limited to the relatively small sample size (<300 patients) and may require further validation with a larger cohort. in order to reinforce diagnosis, it would be advisable to perform multiple assays against different viral antigen. in addition, the information of antibody seroconversion is crucial in determining the optimal timepoints to collect serum or plasma samples for immunoassay screening, as well as obtaining peripheral blood b cells for the generation of therapeutic monoclonal antibodies (37) . currently, in order to rapidly generate neutralizing monoclonal antibodies against sars-cov-2, repurposing of existing sars-cov-specific antibodies was demonstrated. to date, two human sars-cov-specific antibodies, cr3022 and 47d11, have been shown to recognize sars-cov-2 (38, 39) . cr3022 recognizes an epitope along the rbd of sars-cov-2, which differs largely at the c-terminus residues to the rbd of sars-cov (38) . unfortunately, this variation in sequence impacted the ability of cr3022 to crossneutralize sars-cov-2. monoclonal antibody 47d11, on the other hand, targets the rbd along the s1 subunit of both sars-cov and sars-cov-2 with similar affinities, thereby enabling cross-neutralization against sars-cov-2 infection (39) . while combinatory therapy has exhibited a stronger neutralization capability against sars-cov infection (40), a cocktail antibody approach for sars-cov-2 could be explored. surprisingly, reports on antibodies against the coronavirus e protein are scarce, possibly due to it being the smallest protein. however, the e antigen is involved in viral assembly, release of virions, as well as virus pathogenesis (41) . it was demonstrated that recombinant coronaviruses lacking the e protein displayed significantly reduced viral titers, impaired viral maturation and produced avirulent virus progenies, suggesting a similar importance of e protein during sars-cov-2 infection (42, 43) . thus, it would be worthwhile to identify or generate neutralizing antibodies that are specific against the viral e protein. during the course of an epidemic, one of the main challenges is the identification of asymptomatic infection. since these individuals do not present any distinguishable symptoms, they could be the major source of transmission (10) . immunoassays may be able to detect mildly infected cases (14) , which is important to ascertain the extent of community spread. while it is fast, robust and easy to perform, there are several limitations to serological assays. one of the major setbacks of immunoassays is the inability to detect the presence of infection during the early stage of disease, as antibodies take several days to be generated after exposure to foreign material (44) . as such, a recent infection may provide false negative results during serological testing. thus, the use of rt-pcr may be more suitable to diagnose an early acute sars-cov-2 infection. furthermore, due to the unique genetic makeup of each individual, there would be an inherent variability of the antibody response (45) . this could possibly explain the difference in antibody profiles elicited among individuals infected with sars-cov-2 (10). cross-reactivity could potentially be a limitation of immunoassays as it severely impacts the specificity and sensitivity of the test. although the phylogenetically closest coronavirus, sars-cov, has not been reported to be circulating in the human population since 2004 (3), other endemic human coronaviruses may still pose a problem to accurately diagnose patients with true sars-cov-2 infection. while a recent study has demonstrated negligible cross-reactivity from human coronavirus, nl63, to sars-cov-2 (13), validation with other human coronaviruses remains to be investigated. in addition, prior findings on the s protein sequence and neutralization antigenicity of other coronaviruses suggest that antibodies neutralizing clinical human coronavirus isolates may not have the same degree of cross-reactivity with laboratory strains of human coronaviruses, thereby affecting the sensitivity of immunoassays (46) (47) (48) . given the rapid increase in the number of confirmed covid-19 cases coupled with the shortage in test kits to meet rising demands, decentralized point-of-care tests (poct) may be another alternative to facilitate sars-cov-2 diagnosis. such tests include lateral flow assay (lfa), which is a paper-based platform for the detection and quantification of analytes in complex mixtures (49) . to design lfa for sars-cov-2 detection, an antibody specific to the viral antigen, or a viral antigen that is detectable by patient serum or plasma samples can be immobilized on a nitrocellulose membrane. detection of binding between the analyte and capture antibody by a detector antibody will give rise to a colored line, closely resembling home pregnancy kits (50) . poct is advantageous as it is usually designed to be rapid, sensitive, highly accessible, and easily performed, requiring only a small amount of sample (50) . meanwhile, several hundreds of candidate pocts are being evaluated for their applicability toward identifying sars-cov-2-infected individuals (50) . however, pocts can't replace rt-pcr and it is crucial that these developing tests are rigorously assessed prior to use. it is important to note that wrong use and interpretation could lead to disastrous public health consequences (51) . rapid development of diagnostic tools and immune-based assays are important early interventions against the ongoing sars-cov-2 pandemic. the availability of serological assays that target a diverse range of viral antigen has no doubt assisted in the accurate diagnosis of covid-19 patients. essentially, data generated through serological studies can greatly aid in supplementing the results from qrt-pcr, as well as contribute to seroepidemiology, which has been shown to help in the design of virus elimination programs (52) . moving forward, this extensive collation of the current immunoassays against sars-cov-2 will provide insights toward monoclonal antibodies discovery and characterization for the development of a sars-cov-2 vaccine. ln and lr conceived the presented idea. cl wrote the manuscript and prepared the figures. ln, lr, and rl revised the manuscript. all authors approved this manuscript for publication. this work was supported by core research grants provided to singapore immunology network by the biomedical research council (bmrc), and by the a * ccelerate gap-funded project (accl/19-gap064-r20h-h). new sars-like virus in china triggers alarm novel coronavirus 2019-ncov: prevalence, biological and clinical characteristics comparison with sars-cov and mers-cov preliminary identification of potential vaccine targets for the covid-19 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(2020) seroepidemiology: an underused tool for designing and monitoring vaccination programmes in low-and middle-income countries diagrams are created with biorender. the authors wish to thank drs. siew-wai fong and yi-hao chan for critical comments of this manuscript. key: cord-297599-y4lu8m4k authors: luo, hua; zhao, mingming; tan, dechao; liu, chang; yang, lin; qiu, ling; gao, yan; yu, hua title: anti-covid-19 drug screening: frontier concepts and core technologies date: 2020-10-28 journal: chin med doi: 10.1186/s13020-020-00393-z sha: doc_id: 297599 cord_uid: y4lu8m4k the outbreak of covid-19 has recently evolved into a global pandemic. up to july 2020, almost every country has confirmed covid-19 cases reported worldwide. many leading experts have predicted that the epidemic will persist for relatively a long period of time. thus far, there have been no remedies proven effective against the disease. as the nation where covid-19 broke out first, china has adopted a combination of traditional chinese medicine and western medicine to fight against the disease, and has achieved significant clinical result. up to now, the covid-19 pandemic has been effectively controlled in china. however, the rest of the world (except for a limited number of countries and regions) is still in deep water. this paper thoroughly summarizes interdisciplinary notions and techniques, including disease model, biochip, network pharmacology, and molecular docking technology, etc., providing a reference for researchers in the screening of drugs for covid-19 prevention and treatment. these methodologies may facilitate researchers to screen out more potential drugs for treating covid-19 pneumonia and to tackle this global crisis. covid-19 is an acute respiratory infection caused by a novel coronavirus (sars-cov-2). the main symptoms of this disease include fever, dry cough, and fatigue, often accompanied by diarrhea, sore throat, runny nose, muscle pain, and so on. severe cases can rapidly develop into respiratory distress syndrome, septic shock, metabolic acidosis and coagulopathy, and multiple organ failure [1] . sars-cov-2 is a coronavirus with a membranous envelope, in a spherical or oval shape, often polymorphic, with a diameter of 60-140 nm [1] . the viral infection involves entry of sars-cov-2 virus into host cells [2] [3] [4] [5] mainly via a cell receptor angiotensin-converting enzyme ii (ace2). covid-19 can be transmitted through human saliva and physical contact, with an incubation period of 1-14 days. it is also highly contagious during the asymptomatic period, with 44 percent of transmission occurring before the onset of symptoms. the viral load in the patient's saliva peaked during the first week of the onset of new covid-19 symptoms such as fever and cough [3, [6] [7] [8] [9] . no precise vaccine or drug has been approved to prevent covid-19 effectively, leading to the rapid global spike of confirmed cases. until 3rd august in 2020, more than 17 million cases of covid-19 infections and with more than 680,000 deaths have been confirmed in 209 countries and regions worldwide. in china, anti-viral drugs such as interferon-α, ribavirin, lopinavir/ritonavir, chloroquine phosphate and arbidol, as well as a combination with traditional chinese medicine (tcm) are recommended to treat covid-19 [1] . however, lopinavir or ritonavir did not outperform the benefits to those of the standard treatment in hospitalized adults with severe covid-19 [10] . on the other hand, tcm has been observed to play an important role in preventing and controlling the epidemic. according luo et al. chin med (2020) 15:115 to data released by chinese center for disease control and prevention on 23rd march 2020, 74,187 of the newly diagnosed patients of covid-19 in china were treated with chinese medicine, accounting for 91.5 percent of total patient population at that time. in hubei province, 61,449 patients were treated with tcm, accounting for 90.6 percent [11] . clinical observation showed that the total effective rate of chinese medicine reached more than 90% [11] . tcm efficiently relieved the symptoms of the patients and promoted a fast recovery of their bodies, thus effectively reducing the number of patients whom changed from mild to severe conditions, as well as the overall mortality rate of the patients [11] . at present, a large amount of research work has been invested in searching for the treatments of coivd-19 worldwide. in china, promising therapies for coivd19 have been recommended according to the accumulated experiences of tcm in fighting with various epidemics during the long history. in this paper, we mainly focus on summarizing the advantages of tcm in treating covid-19 and several advanced techniques and concepts for drug screening of anti-covid-19 drug candidates. varieties of in vitro and in vivo models for drug screening are also introduced. we expect that this review to provide researchers valuable ideas and approaches in fast finding out the precise therapies and/or effective drugs against covid-19. as a single-stranded rna virus belonging to the c family of coronavirus, sars-cov-2 features four structural proteins, s protein, n protein, m protein, and e protein [12] . the current results suggest that the virus enters cells by binding to the host surface receptor, ace2, via the s protein, and then fuse with the host cell [13] . compared with sars-cov and mers-cov, the pathogenicity of sars-cov-2 is lower but is easier to spread [14] . whether fecaloral route transmission of sars-cov-2 plays an important role in spreading the virus, reminiscent of sars-cov, needs to be further studied for confirmation. of particular concern is whether sars-cov-2 will become as seasonal as community-acquired hcov. the heredity, pathogenicity, and continuous transmission of sars-cov-2 in the human body will influence the developing trend of covid-19 which is currently outbreaking globally [14] . to the origin of sars-cov-2 virus, researchers have thus far put forward two hypotheses: (1) it is a natural selection in animal hosts before zoonosis transfer; (2) it is natural selection after the zoonosis shift [15] . both hypotheses are helpful for further investigate and elucidate how the virus initially transmitted from animals to humans. at this stage, some researchers believed that the virus was originally from a natural host of bat, a report show that the 2019-ncov is 96% identical at the whole-genome level to a bat coronavirus [3] . the rtg13 sequence helped reveal key rbd (receptor-binding domain) mutations and multiple base-cutting sites. the virus may recombine to evolve in wild animals such as pangolins, and then cross the species barrier to humans through other intermediate hosts [16] . as of april 5, sars-cov-2 has been observed and reported to be effectively replicated in ferrets and cats, and transmitted through respiratory droplets in cats [17] . at present, there are two main detection methods for sars-cov-2 virus: nucleic acid detection and immunological detection. nucleic acid detection methods include gene sequencing, fluorescent quantitative pcr, digital micro-drop pcr, gene chip, and loop-mediated isothermal amplification. immunoassays include immunochromatographic strips, elisa, and chemiluminescence immunoassay [12] . since the outbreak of covid-19 pneumonia, there has been no precise drugs for treatment of this disease with regulatory approval. next, we will review and make a comprehensive analysis of drug targets, as well as possible therapeutic paradigms such as vaccine and therapeutic antibody, general antiviral drugs, protease inhibitor, and convalescent plasma therapy. the s protein is the receptor via which the virus binds to the host cell, and participates in cell fusion. it is a key target for the development of antibodies, vaccines (especially polypeptide and mrna vaccines), small molecular compounds against this virus. the receptor for sars-cov-2 infected cells is ace2, which is similar to sars-cov and also relies on transmembrane serine protease, is also a target for drug development [18] . according to the previous research of sars-cov, a rbd region on the correct conformation of s protein may be an ideal immunogen for vaccine design and development [16] . besides, an inhibitor of lipoprotein nucleotide fusion based on hr2 sequence was designed, called ipb02, which is highly active in sars-cov-2 s protein-mediated cell-to-cell fusion and pseudovirus infection [19] . sars-cov-2 major protease (mpro) is a key cov enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target. shanghai institute of materia medica, in collaboration with several institutions, has designed and synthesized two lead compounds against sars-cov-2 mpro, both of which have shown effective anti-infectious activity [19] . besides, activation of nlrp3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domaincontaining-3) inflammatory bodies is lacking in bats, so the use of nlrp3 inhibitor mcc950 may be helpful in the treatment of covid-19 [14] . viral 3-chymotrypsinlike cysteine protease (3clpro), which controls the replication of coronavirus, is a drug target of sars-cov and mers-cov. nine potential anti-sars-cov-2 active compounds have been identified via this molecular pathway [20] . as the most effective medical means of epidemic prevention and control, vaccine can effectively block the spread of the virus [21] . at present, china is focusing on the simultaneous implementation of three technical routes: influenza vector vaccine, recombinant protein vaccine, and nucleic acid vaccine. led by academician wei chen from the medical research institute of the academy of military medical sciences, the team has successfully developed a recombinant new crown vaccine, which is now in phase i clinical trial (registration number of recombinant sars-cov-2 vaccine (adenovirus vector) registration no. chictr200030906). at the same time, some international pharmaceutical companies include gene one life science inc. and inovio pharmaceuticals inc. have conducted relevant animal experiments to develop the covid-19 vaccine. moderna and the vaccine research center of the national institute of allergy and infectious diseases conducted the first phase i clinical trial of a novel lipid nanoparticle (lnp)encapsulated mrna-based vaccine, mrna-1273, which encodes the spike protein (s protein) of sars-cov-2, began in the united states [22, 23] . scholars proposed two very far-sighted global solutions in the event of a new virus that triggers a pandemic in the future. one solution is to establish a global distribution of production capacity, where surveillance in animal reservoirs combined with characterization of the virus can identify members of the virus family that are likely to cause a pandemic, candidate vaccines using these isolates can then be produced, and this capability can be activated if new viruses emerge. this strategy is currently applied to h5 and h7 avian influenza vaccines. another solution is the development of a broad-based protective vaccine covering the entire family or genus of the virus, which is currently underway for influenza viruses and may be applied to coronaviruses in the future [24] . therapeutic antibodies are also specific therapeutic drugs. sars-cov-2 and rbd of sars-cov have high homology. the strong neutralizing antibody cr3022, which targets sars-cov rbd, can be effectively combined with sars-cov-2 rbd. thus it is a potential anti-sars-cov-2 antibody [18] . mode rna therapeutics, wuxi biologics and vir biotechnology are developing monoclonal antibody for sars-cov-2 and several pre-clinical studies have been completed and clinical trials have also begun. vaccines, in the form of monoclonal antibody, oligopeptides, and peptide molecules, take months to years to develop. considering approved antiviral drugs against a variety of viruses such as hiv, hbv, hcv, sars, and mers, guangdi li et al. recommended some of these current antiviral drugs for treating covid-19, such as nucleoside inhibitors favipiravir, ribavirin or ridgisivir [25] ; common antiviral drugs chloroquine phosphate, favipiravir, and arbidol; protease inhibitors disulfiram or lopinavir/ritonavir and immunosuppressants may also work against covid-19 [25] . in the absence of specific therapeutic drugs, convalescent plasma therapy (cpt), a passive immunotherapy, has been one of the potential treatment options for covid-19 [26] . the strategy has been used to treat flu, viral infections, sars, and other infectious diseases. since the plasma who meets the medical requirement is rather limited, and the quality of blood products is demanding, the use of cpt has failed to popularize, in spite of demonstrated efficacy [26] . therefore, researches on exploring and developing effective drugs for preventing and treating covid-19 are necessary and urgent. as very limited modern medicine has shown clinically approven efficacy for covid-19, lessons can be drawn from the effective treatment experience from the use of the combination of tcm and western medicines. during the screening of effective drugs and potential formulations or medicinal combinations, a variety of in vitro and in vivo screening models has been employed. therefore, disease models and some core technologies would be introduced in the following paragraphs for facilitating researchers to screen drugs for treating covid-19. using in vitro models (e.g. certain cell lines), the antiviral effect of a drug candidate could be quickly predicted preliminarily. at present, the most commonly used cell line in antiviral test of chinese medicine is mdck (madin-darby canine kidney) cells. in addition, hep-2, a549, mbck, and mouse macrophage raw264.7 are also frequently applied for screening of antiviral drugs. mdck cells are susceptible to multiple influenza virus strains, supporting numerous passages of virus replication (capable of 35 consecutive rounds), and are widely used in antiviral activity assessment, mechanism research, and influenza vaccine development. mdck cells are the most widely used cell lines for antiviral research in tcm [27] [28] [29] . single herb such as scutellariae radix (黄芩) [30] , isatidis radix (板蓝根) [31] , lonicerae japonicae flos (金银花) [32] , houttuyniae herba (鱼腥草) [32] , etc. and tcm formulations such as fufangyizhihao granule (复方一枝蒿颗粒) [33] and lianhua qingwen capsule (连花清瘟胶囊) [33] have shown potentials on attenuating the proliferation of a/ pr/8/34(h1n1), a/aichi/2/1968(h3n2) and other influenza viruses in mdck cells. a549 human lung adenocarcinoma epithelial cell line is a commonly used tool to investigate the interactions between virus and host, the biochemistry of viral proteins, and to analysis genes that are overexpressed to suppress viral infections, to perform large-scale drug screening, for example, screening for crispr activation, [34] . it was previously demonstrated that tcm reseda odorata (木犀草) decreased the caspase-9, caspase-8 and caspase-3 expression in a549 cells infected with h1n1 virus [35] and chuanxiong rhizoma (川芎) [36] . in addition, the inhibitory effects of scutellariae radix on a 1/fm/166/85(h1n1) virus and influenza a and b viruses have been demonstrated in a549 cell line [37, 38] . researchers have used vero e6 cells to understand covid-19. for instance, vero e6 cells were used to test the antiviral activity of lianhua qingwen capsule against sars-cov-2 [39] . more interestingly, recent studies on vero e6 cells demonstrated that tmprss2 could enhance sars-cov-2 infection, which was in common with middle east respiratory syndrome and sars-cov. given the various variants containing point mutations or 15-30-bp deletions (del-mut) found accordingly at the s1/s2 junction via plaque purification of vero-e6 cells cultured with sars-cov-2 and the fact that adaptive function could disappear due to replication of permissive vero-e6 cells, it was suggested that strong selective pressure might be responsible for sars-cov-2 infection in humans promoted by the unique cleavage motif [40] . additionally, a tmprss2-expressing veroe6 cell line was considered helpful for propagating and isolating sars-cov-2, because it was easily accessible to infection of sars-cov-2 [41] . yi zhi hao granule has been reported to present obvious anti-h1n1 activity on human laryngeal carcinoma epithelial cell hep-2, and can alleviate lung injury and reduce the mortality of infected mice [42] . xiaoqinglong decoction (小青龙汤) can effectively inhibit the infection of hep-2 cells by the human respiratory syncytial virus [43] . in addition, mouse macrophages, raw264.7 are increasingly favored for screening antiviral drugs. for instance, yinhua ping gan granule (银花平感颗粒) was observed to efficiently inhibit the proliferation of a/pr/8/34(h1n1) virus in raw264.7 cells, which might be related to the following elements: regulates type i interferon and pattern recognition receptor signaling pathway; up-regulates the expression of ifn-γ and anti-myxvirus protein 1; downregulates the expression of il-6, tnf-α and phosphorylated tank binding kinase 1, and signal transduction and transcription activator 1 [33] . as in vitro screening can only offer potential drugs against covid-19, proper animal models have to be established to further understand covid-19 disease evolvement and to screen drugs for effective treatment, before possible clinical trials. researchers are testing mice, rats, ferrets, and even monkeys to answer key questions about the diseases and to fast-track potential drugs and vaccines for clinical trials. after outbreak of covid-19, china institute of laboratory animal sciences (cnilas) has taken the lead in establishing the disease models in transgenic mice and rhesus monkeys with covid-19, enriching the understanding of the etiology and pathology of covid-19. with the model, 5 patent medicines have been thoroughly evaluated in vivo for potential treatment of covid-19, and 6 vaccines and 4 more patent medicines are currently evaluated [44] . it was reported that academician dr. zhong nanshan's team has created the world's first non-genetically modified mouse model infected with sars-cov-2. compared with the traditional receptor transgenic mouse model, this model has a shorter construction time and does not need to reproduce; therefore, it is suitable for large-scale popularization in a short time. this model is useful for in vivo validation of antiviral drug and protective neutralizing antibodies, and vaccines. besides, the model can be used to study the immune response and pathogenesis of sars-cov-2 in vivo. in addition to mice and rhesus monkeys, other animals similar to human viral infections have been used in previous studies to screen for an antiviral drug. mouse plays an important role in researches on the pathogenesis of respiratory diseases associated with human virus infection [45] [46] [47] [48] [49] . some researchers are currently using mice as an animal model to test drugs and vaccines and to investigate the nature of the infection of sars-cov-2 [49] [50] [51] . however, mice often shrug off infection with sars-cov-2 and only exhibited a relatively mild clinical disease [24, 52] , because mouse ace2 receptor features several differences from that of humans [53] . for instance, out of 29 key amino acids in this important domain,11 in mouse were different from that in human [54] . luckily, engineering mice that could express both the human and mouse version of the receptor's gene, ace2, has been suggested as an effective way to remove the roadblock. in fact, in a study led by qin chuan on sars, engineered mice that could express human ace2 protein was successfully established, leading this chinese team pioneered the establishment of a sars-cov-2 infected hace2 transgenic mouse model [54] . during investigation of the pathogenicity of the virus in this mouse model, weight loss and virus replication in the lung was observed. therefore, this model still needs to be optimized since the weight loss and signs of pneumonia were mild and very different from that of humans [54] . currently, researchers are accelerating animal model research for sars-cov-2, and the transgenic mice developed for sars-cov are in short supply. for example, an effective and convenient novel mouse model in evaluating in vivo protective capacity of the sars-cov-2 vaccines was developed through stitching the human gene for ace2 into an adenovirus by perlman et al. in his unpublished work, in which mice was infected with it then receptor was created by some of their lung cells [55] . surprisingly, 20% of weight loss, which was greater than twice in qin's study, ruffled fur, other illness sign with no death were occurred in those mice infected with sars-cov-2. in addition, pacific inflammatory responses and pneumonia were found in the most recent study, in which both young and aged wild-type balb/c mice were effectively infected with mouse-adapted sars-cov-2 at passage 6 (macsp6) [55] . furtherly, a receptor-binding domain (rbd)-based sars-cov-2 subunit vaccine was found to conferred full protection against sars-cov-2 macsp6 challenge and elicited highly potent neutralizing antibodies, and the protective activity of it was determined in vivo. of the 29 amino acids in the key region of the human ace2 receptor that binds to the virus, 13 amino acids differ from that of the rat [54] . therefore, rats might not be a suitable model for studying sars-cov-2 infection. in terms of susceptibility to sars-cov-2, rats also had no advantage. however, the large size of rats has some advantages in many experiments that require repeated blood collection. syrian hamsters are gaining increasing attention in the fight against covid-19. in hamster, only 4 of 29 amino acids of ace2 receptor are different from those of human [56] . despite of the subtle symptoms, coronavirus was found to easily infect hamsters and cause serious acute respiratory syndrome (sars) as early as fifteen years ago, nevertheless, hamster did not catch much attention as an effective animal model for the disease at that time [57] . now, however, the tables have turned in the case of covid-19 since this model comes under spotlight again caused by a related virus, sars-cov-2. the first new hamster model was published in january by a physicianscientist of the university of hong kong (hku) jasper fuk-woo chan, whose work become one of the earliest studies in which asymptomatic infection and humanto-human transmission of sars-cov-2 were reported. eight hamsters were infected with sars-cov-2 by chan and his co-workers in their study, afterwards, a body weight loss, ruffled fur, lethargy, a hunched posture and rapid breath occurred with high levels of sars-cov-2 found in the lungs and intestines, tissues studded with the virus' target, ace2 of the animals [58] . interestingly, another team also at hku. led by hui-ling yen suggested comparable results closely behind of this study [58] . what's more inspiring, those hamster studies may contribute to understand the spreading way of the virus. in both above studies, transmission of sars-cov-2 happened whenever an uninfected hamster was put together with an infected one in a cage, apart from the possible transmission through respiratory droplets, chan and his colleagues also noticed the fact that hamsters ate feces, and therefore, a fecal-oral spread way could not be ruled out [58] . ferret shares remarkably similar lung physiology with humans, unsurprisingly, it is widely used in researches of respiratory viruses that infected humans and as one of the best animal models for another respiratory disease and influenza [59] . they are susceptible to human influenza viruses and various of the clinical symptoms of influenza infection in humans can be found on them [59] . a team of researchers in china recently found that sars-cov-2 could replicate in the upper respiratory tract of ferrets for up to eight days, without causing severe disease or death. the authors believed that the ferret could be a potential candidate animal model for assessing vaccine candidates and antiviral drugs against covid-19 [60] . by contrast, in the same study, livestock including chickens, pigs and ducks were demonstrated to not susceptible to sars-cov-2, while dogs had low susceptibility and cats were highly susceptible to the virus. besides, kim et al. [61] reported similar results. they found that ferrets were highly susceptible to sars-cov-2 infection, and exhibited the elevated body temperature and virus replication. although fatalities were not observed, ferrets effectively transmitted the virus by direct or indirect contact, recapitulating human infection and transmission. more importantly, kim also suggested that older ferrets might serve as a better animal model than younger ones. regardless of the unclear reasons, it is widely accepted that sars-cov-2 strikes the elderly much harder than the younger in humans, and similar phenomenon happened in the ferrets. kim had observed more severe symptoms causing loss of white blood cells and platelets in older ferrets infected with the virus, besides, 93% of older ferrets died of viral infection while no symptoms showed in younger ones infected with the same virus [62] , which indicated again that ferrets might be an ideal animal model for understanding and defeating against covid-19 since it presents similar features. non-human primates, especially rhesus monkeys, have similar immune responses to human viral infections due to their genetic and physiological similarities [63] . nonhuman primates are the closest species to human being who is subject to the infection of viruses [64] . different species of monkey have been infected with sars-cov-2 by intense efforts that closely followed by the isolation of the virus from human. a team from wuhan institute of virology, chinese academy of sciences, published the first study of sars-cov-2 infected rhesus monkey model [65] . they found that sars-cov-2 caused acute localized-to-wide to spread pneumonia as proved by pathological studies in rhesus macaques, although without obvious clinical symptoms of respiratory disease. another research team observed more serious interstitial pneumonia in old monkeys infected by sars-cov-2 than that in young monkeys, which provided insights into the pathogenic mechanism and may facilitate the development of vaccines and therapeutics against sars-cov-2 infection. in their study, two 15 years old and three 3-5 years old rhesus macaques were infected with sars-cov-2, then viral replication of anal swabs, nasopharyngeal swabs and the lung in old monkeys was found to be more active than that in young monkeys 14 days after the virus infection [44] . typical interstitial pneumonia characterized by thickened alveolar septum along with edema and inflammation was developed in monkeys, and diffuse severe interstitial pneumonia was observed in old monkeys. the research team led by qin chuan has obtained similar conclusions. they further found that sars-cov-2 had a conjunctival infection transmission route in the rhesus monkey model, which suggested conjunctival infection could cause mild covid-19 symptoms [66] . another critical finding was that the reinfection by sars-cov-2 of rhesus macaques might be decreased by a remarkably enhanced neutralizing antibody response, suggesting that primary sars-cov-2 infection might contribute to protection of monkeys from subsequent reinfection [67] . besides, some scientists inoculated cynomolgus macaques with sars-cov-2 or mers-cov and compared with historical sars-cov infections and found that the severity of sars-cov-2 infection was intermediate between that of sars-cov and mers-cov [68] . lu et.al recommended that macaca mulatta could be used to investigate viral pathogenesis and evaluate vaccines and drugs of covid-19. two different families of non-human primates, including new world monkeys (6 callithrix jacchus) and old world monkeys (6 macaca fascicularis, 12 macaca mulatta) were involved in their study, and they reported that macaca mulatta was most susceptible to sars-cov2 infection, followed by macaca fascicularis and callithrix jacchus [69] . a mers-cov primate model was successfully established in a latest study, which firstly reported the dosedependent effects of highly pathogenic coronavirus infection of primates and used a route of infection (small particle aerosol) with potential relevance to mers-cov transmission in humans with twelve african green monkeys [70] . african green monkeys may serve as primate model for covid-19, with considerably potential value in viral pathogenesis and therapeutic development researches [70] . taken together, monkeys might be used as suitable animal models to evaluate vaccines and drugs for covid-19. through data mining to screen the clinical use of the tcm formula for covid-19, high frequency/core drugs in tcm formula could be selected. also, the induction and analysis of four properties and five tastes (四气五 味) and meridian tropism (归经) of many chinese medicines from tcm formula could be made. effective guidance for the prevention as well as clinical treatment of covid-19 could be provided through investigations of the characteristics of commonly used medicine. through data mining, the key chinese medicine to prevent new coronary pneumonia is saposhnikoviae radix [72] . from the meridian tropism point of view, the lung meridian, stomach meridian are dominant. according to the classification of efficacy, the top four herbs used most frequently were heat-clearing herbs, antipyretic herbs, qi-invigorating herbs, and yin-tonifying herbs. it was found that in the prescriptions of tcm for the prevention and treatment of covid-19 in various regions, the main methods were to clear away heat and reinforce deficiency, and to strengthen the spleen and stomach. in the later period, most of tcm used were tonifying qi and yin. molecular docking is a model based on applied mathematics, biology, and computers to predict the binding affinity of small molecules to specific receptors, which can be used to predict drug affinity at target binding sites and drug-specific metabolic enzyme interactions to better understand the complexity of living systems. studies have shown that sars-cov virus infections are through the expression of s-protein and human ace-2 binding, which leads the virus to enter the cells [65, 73] . through molecular docking technology, 46 active components from tcm formula with high binding capacity could act on the binding region of sars-cov-2 s-protein of human ace2 protein. the screened components mainly belong to lonicerae japonicae flos, mori folium and other seven herbs [74] . papain-like protease (plp) plays an important role in the replication of coronavirus. in the process of virus replication, the small rna virus first encodes a large polymer precursor protein, and then the polymer protein is hydrolyzed to produce a functional protein, the hydrolysis process is mainly completed by 3clpro. therefore, it is important to search for inhibitors of 3cl hydrolase of sars-cov-2 coronavirus for the prevention and treatment of covid-19. through molecular docking technology, trichosanthis fructus (瓜蒌) and fritillariae cirrhosae bulbus (川 贝母), obtained by plp inhibitors screening and ace2 inhibitors screening respectively, were parts of the sangbei zhisou powder (桑贝止嗽散) and xiaoxianxiong decoction (小陷胸汤), which had the effect of clearing away heat and phlegm [75] . they can be applied to the syndrome of cough with slight dyspnea caused by external pathogenic factors in the early stage of viral action. radix et rhizoma rhei (大黄) and trichosanthis fructus, screened by mpro and plp respectively, are in maxing shigan decoction (麻杏石甘汤) and xuanbai chengqi decoction (宣白承气汤). they can be applied to syndrome types of the lung obstructed by pathogenic heat or blocked by toxin, and viscera-solid knot in the severe stage of the disease. mori folium, lonicerae japonicae flos and forsythiae fructus obtained by ace2 inhibitors screening have been included in the sangju beverage (桑 菊饮) and yinqiao powder (银翘散), which can be used in the early stage of the disease, such as the syndromes of warming evil invading the lung and slight cough and asthma. network pharmacology, which constructs network models centering on medicinal materials and disease targets, can provide a reasonable experimental basis and a brandnew research idea for discovery of new drugs and excavation of potential drugs. combining the study of the entire network with the holistic characteristics of tcm, network pharmacology can be used to study the basic theory of tcm from the perspectives of multi-approach, multi-component, and multi-target. screening the active substances, predicting the target protein, finding the signal pathway, constructing the network and analyzing it by network pharmacology, so the potential mechanism between the active components, target proteins and the network of pathways related to the occurrence and development of diseases may get understood, consistent with the overall concept of tcm. subsequently, the function of the key active components and covid-19 related proteins can be studied by molecular docking technique, and the potential material basis of treating covid-19 with prescription can be therefore explored, which could provide theoretical reference for preventing and treating covid-19 with prescriptions. using jean-baptiste de lamarck genetic algorithm and virtual docking technology, small molecular components of chinese herbal medicine were screened from the component library of chinese herbal medicine based on s-protein and ace2. using s-protein as the target, 12 [76] . through the analysis of weixuening mixture by network pharmacology, 326 components and 555 related targets were obtained, of which 35 targets corresponding to 101 components were closely related to covid-19. more specifically, herbs-active compounds-targets network and gene ontology function enrichment analysis as well as kyoto encyclopedia of genes and genomes pathway enrichment analysis were performed by cytoscape software and database for annotation, visualization and integrated discovery. the enrichment analysis of the target pathway showed that weixuening mixture mainly acted on infectious disease, inflammatory reaction, and immune regulation. it is suggested that weixuening mixture may play a potential role in the prevention and treatment of covid-19 by regulating the immune and inflammatory responses [77] . besides, with network pharmacology, it was found that the core active compounds in yupingfeng powder (玉屏风散) regulates multiple signal pathways by binding with ace2 to esr1, ar, ptgs2, and other targets to prevent and treat covid-19 [78] . jinhua qing gan granule (金花 清感颗粒) can regulate the signal pathway of ptgs2, hsp90ab1, hsp90aa1, ptgs1, and ncoa2 by combining 2019-ncov3cl hydrolase and ace2 to prevent and treat covid-19 [79] . taking advantage of the nascent computer technology and professional software, researchers could quickly screen out the regular pattern of anti-covid-19 drugs according to the existing pharmacological knowledge. in combination with clinical experience, it can further provide effective suggestions to clinical medication. this method can also screen out the active substances, target proteins, and possible signal pathways of related drugs. the application of computer virtual screening technology can greatly reduce the blindness in the screening of prescriptions and medicines, and save manpower, material, and financial resources. however, this method is limited by the information of drug structures and targets. biochip technology can detect numerous information simultaneously, which is helpful to explore the molecular mechanism of compounds from chinese medicine, optimize the dosage and prescription. gene chip can connect the characteristics of multicomponent, multi-target, multi-pathway action, and gene expression together; compare their difference in expression profiles; and determine the corresponding gene expression targets. according to the expression of organ specificity and the level of expression and the compound theory of monarch, minister, assistant and guide, and correlates with medication dose. at the same time, according to the interaction of the corresponding gene targets of different compatibility, the close relationship between each compound medicine can be analyzed, and the material basis of drug action and internal compatibility law would be elucidated. through gene chip technology observing the changes of gene transcription in an apoptotic signal pathway, a study found that luteolin could down-regulate the differentially expressed genes of casp3, casp8, and myd88, and interfere with cell apoptosis in a549 cell infected by h1n1 [80] . therefore, biochip technology presents great potential being used for screening tcm formulas in treating covid-19. the response of clinical studies of tcm in the prevention and treatment of covid-19 have been very rapid, and the current registration scheme covers the whole process of disease prevention, treatment, and rehabilitation. tcm formula has the characteristics of multi-component, multitarget, and multi-way synergy, which are the advantages of clinical treatment of tcm. however, perspective from the modern medicine, clarification of the relationship between chemical basis and biological mechanisms for tcm is still a challenge to be concerned. the substance basis, curative effect, and mechanism of tcm formula need to be further investigated and demonstrated. besides, many means of tcm diagnosis and treatment rely on personal experience. to guide the clinical medication, it is important to establish an objective and standardized diagnosis and treatment standards. traditional methods for screening antiviral chinese medicines are limited by the experimental cycle; since the high infectivity of sars-cov-2, the high requirements of laboratory conditions greatly lag the progress of drug screening. the common techniques of computer-aided drug design (cadd) include molecular docking, reverse docking, qsar, pharmacophore, and prediction of drug absorption, distribution, and metabolic transport. discovery and optimization of drug targets and the application of lead compound in the absorption, distribution, metabolism, excretion, and toxicity prediction of compounds have greatly accelerated the process of drug screening. cadd is also a tool for studying tcm, expounding the mechanism of action of tcm, and optimizing the development of new tcm prescriptions. among them, the technologies of molecular docking (dock), inverse molecular docking (invdock), similarity search, substructure search, pharmacophore search, pharmacophore screening, and chemical component toxicity prediction are of great significance to the study of tcm and its prescription. it is an important tool to explain the mechanism of tcm scientifically and reasonably [80] . in order to find out the potential chinese medicines or active ingredients against covid-19, scholars used data mining and network to mine the highfrequency chinese medicines and formulas from ancient prescriptions, then explored binding rates between the main ingredients in high frequency chinese medicines and the key targets of sars-cov-2 via using molecular docking approach. finally, a network pharmacology was used to uncover the potential molecular mechanism [81] . researchers can quickly screen out potential medicines by cadd, but further animal experiments and clinical studies should be performed to confirm the safety and effectiveness of these medicines. the tcm prescription is composed of different compounds, and its function is to target multiple target genes. it can directly inhibit viruses by target protein, and indirectly inhibit virus by human target protein. therefore, predicting the gene-target interactions of active components could help to decipher the mechanism of multitarget action for tcm. in the search for new antiviral drugs, studies have been conducted on molecular docking and molecular dynamics simulations to identify components or derivatives of chinese medicines or tcm formulations that can block channel activity or drugs at sialic acid binding sites. a study examined six viral proteins that could be targeted by antiviral drugs, including neuraminidase, hemagglutinin, matrix protein 1, m2 proton channel, nucleoprotein, and nonstructural protein 1 [82] . molecular docking techniques were used to identify potential inhibitors of 13,144 tcm compounds. the results showed that 56 compounds could inhibit more than two drug targets simultaneously. the interactions of these compounds with host targets were studied by molecular reverse docking. finally, it was found that 22 compounds inhibitors could stably bind to host targets with high binding free energy [82] . significant research works have provided indispensable insights into the technology and methodologies in screening drugs treated coivd-19 that likely contribute to the development and progression of interdisciplinary strategies in the understanding to prevent and treat coivd-19. as stated prior, in vivo and in vitro models have been developed to verify the effectiveness of anti-covid-19 drugs. but there is still wanting in the most commonly used model that provides an inexpensive, simple and reproducible model to study coivd-19. it is our hope that our further understanding of the role of frontier concepts and core technologies in screening anti-covid-19 drugs that may lead to new therapeutic targets for preventing and treating covid-19. national administration of traditional chinese medicine. diagnosis and treatment protocol for novel coronavirus pneumonia structure, function, and evolution of coronavirus spike proteins a pneumonia outbreak associated with a new coronavirus of probable bat origin the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission temporal dynamics in viral shedding and transmissibility of covid-19 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical features of patients infected with 2019 novel coronavirus in wuhan a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 three chinese patent medicines and three tcm prescriptions" have obvious curative effect, and traditional chinese medicine played an important role in fighting against novel coronavirus disease research progress on detection methods of sars-cov-2 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding zoonotic origins of human coronaviruses the proximal origin of sars-cov-2 research progress in covid-19 susceptibility of ferrets, cats, dogs, and different domestic animals to sars-coronavirus-2. biorxiv advances in therapeutic drugs and vaccine of corona virus disease design of potent membrane fusion inhibitors against sars-cov-2, an emerging coronavirus with high fusogenic activity. biorxiv structural basis of sars-cov-2 3cl(pro) and anti-covid-19 drug discovery from medicinal plants immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic moderna doses first patient with mrna-1273 in coronavirus vaccine trial safety and immunogenicity study of 2019-ncov vaccine (mrna-1273) for prophylaxis of sars-cov-2 infection (covid-19) sars-cov-2 vaccines: status report therapeutic options for the 2019 novel coronavirus (2019-ncov) convalescent plasma therapy for covid-19: state of the art evaluation of potential herb-drug interactions between oseltamivir and commonly used anti-influenza chinese medicinal herbs screening of neuraminidase inhibitory activities of some medicinal plants traditionally used in lingnan chinese medicines sheng jiang san, a traditional multi-herb formulation, exerts anti-influenza effects in vitro and in vivo via neuraminidase inhibition and immune regulation anti-influenza virus effect of radix scutellariae-rhizoma zingiberis in vitro research progress on anti-influenza effective components of isatidis radix the research on inhibitory effect of honeysuckle and houttuynia on influenza a virus replication in vitro new progress on anti-influenza activity and mechanism of chinese materia medica alternative experimental models for studying influenza proteins, host-virus interactions and anti-influenza drugs luteolin's intervention effect and its mechanism of apoptosis induced by h1n1 in vitro effects and mechanism of ligustrazine on apoptosis of a549 cells induced by h1n1 virus in vitro wogonin, a flavonoid isolated from scutellaria baicalensis, has anti-viral activities against influenza infection via modulation of ampk pathways inhibitory effect of baicalin on influenza virus a h1n1 in vitro lianhuaqingwen exerts anti-viral and antiinflammatory activity against novel coronavirus (sars-cov-2) attenuated sars-cov-2 variants with deletions at the s1/s2 junction enhanced isolation of sars-cov-2 by tmprss2-expressing cells antivirus effect of compound yizhihao pellets on influenza virus h1n1 infection in mice xiao-qing-long-tang (sho-seiryu-to) inhibited cytopathic effect of human respiratory syncytial virus in cell lines of human respiratory tract age-related rhesus macaque models of covid-19 evaluation of the potential effects of as03-adjuvanted a(h1n1)pdm09 vaccine administration on the central nervous system of non-primed and a(h1n1)pdm09-primed cotton rats genetic contributions to influenza virus attenuation in the rat brain viromimetic sting agonist-loaded hollow polymeric nanoparticles for safe and effective vaccination against middle east respiratory syndrome coronavirus single-dose, intranasal immunization with recombinant parainfluenza virus 5 expressing middle east respiratory syndrome coronavirus (mers-cov) spike protein protects mice from fatal mers-cov infection an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year ready to submit your research ? choose bmc and benefit from an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 and multiple endemic, epidemic and bat coronavirus does sars-cov-2 invade the brain? translational lessons from animal models pathogenicity of severe acute respiratory coronavirus deletion mutants in hace-2 transgenic mice the pathogenicity of sars-cov-2 in hace2 transgenic mice rapid adaptation of sars-cov-2 in balb/c mice: novel mouse model for vaccine efficacy spike protein recognition of mammalian ace2 predicts the host range and an optimized ace2 for sars-cov-2 infection design, synthesis and biological evaluation of novel cholesteryl ester transfer protein inhibitors bearing a cycloalkene scaffold simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility the ferret as a model organism to study influenza a virus infection susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 infection and rapid transmission of sars-cov-2 in ferrets ferret animal model of severe fever with thrombocytopenia syndrome phlebovirus for human lethal infection and pathogenesis nonhuman primate models of human viral infections influenza a virus challenge models in cynomolgus macaques using the authentic inhaled aerosol and intranasal routes of infection infection with novel coronavirus (sars-cov-2) causes pneumonia in the rhesus macaques ocular conjunctival inoculation of sars-cov-2 can cause mild covid-19 in rhesus macaques lack of reinfection in rhesus macaques infected with sars-cov-2. biorxiv comparative pathogenesis of covid-19 comparison of sars-cov-2 infections among 3 species of non-human primates small particle aerosol exposure of african green monkeys to mers-cov as a model for highly pathogenic coronavirus infection. res square study on pneumonia prescription of traditional chinese medicine to prevent novel coronavirus coronavirus infection in different regions based on data mining study on the medication regularity of traditional chinese medicine in the prevention and treatment of covid-19 based on data minin structure of sars coronavirus spike receptor-binding domain complexed with receptor rapid establishment of traditional chinese medicine prevention and treatment of 2019-ncov based on clinical experience and molecular docking study on screening potential traditional chinese medicines against 2019-ncov based on mpro and plp screening of active ingredients from traditional chinese medicine against the novel coronavirus based on molecular docking prediction of active components and potential targets of weixuening mixture for the treatment of novel coronavirus pneumonia based on network pharmacology study on active compounds of yupingfeng san in preventing and treating covid-19 based on network pharmacology and molecular docking technology exploring active compounds of jinhua qinggan granules for prevention of covid-19 based on network pharmacology and molecular docking research advances in application of biochip in traditional chinese medicine identifying potential treatments of covid-19 from traditional chinese medicine (tcm) by using a data-driven approach study on the mechanisms of active compounds in traditional chinese medicine for the treatment of influenza virus by virtual screening publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to acknowledge and thank prof. yitao wang for his wonderful help with organizing and designing this study; we would like to thank prof. xiuping chen for participating in the revision. authors' contributions hy organized, conceived, and supervised the study. hl, mz, dt, cl, ly, and ql drafted the manuscript. yg and hl revised the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests.received: 25 may 2020 accepted: 15 october 2020 key: cord-281281-knelqmzx authors: villas-boas, gustavo r.; rescia, vanessa c.; paes, marina m.; lavorato, stefânia n.; de magalhães-filho, manoel f.; cunha, mila s.; simões, rafael da c.; de lacerda, roseli b.; de freitas-júnior, renilson s.; ramos, bruno h. da s.; mapeli, ana m.; henriques, matheus da s. t.; de freitas, william r.; lopes, luiz a. f.; oliveira, luiz g. r.; da silva, jonatas g.; silva-filho, saulo e.; da silveira, ana p. s.; leão, katyuscya v.; matos, maria m. de s.; fernandes, jamille s.; cuman, roberto k. n.; silva-comar, francielli m. de s.; comar, jurandir f.; brasileiro, luana do a.; dos santos, jussileide n.; oesterreich, silvia a. title: the new coronavirus (sars-cov-2): a comprehensive review on immunity and the application of bioinformatics and molecular modeling to the discovery of potential anti-sars-cov-2 agents date: 2020-09-07 journal: molecules doi: 10.3390/molecules25184086 sha: doc_id: 281281 cord_uid: knelqmzx on march 11, 2020, the world health organization (who) officially declared the outbreak caused by the new coronavirus (sars-cov-2) a pandemic. the rapid spread of the disease surprised the scientific and medical community. based on the latest reports, news, and scientific articles published, there is no doubt that the coronavirus has overloaded health systems globally. practical actions against the recent emergence and rapid expansion of the sars-cov-2 require the development and use of tools for discovering new molecular anti-sars-cov-2 targets. thus, this review presents bioinformatics and molecular modeling strategies that aim to assist in the discovery of potential anti-sars-cov-2 agents. besides, we reviewed the relationship between sars-cov-2 and innate immunity, since understanding the structures involved in this infection can contribute to the development of new therapeutic targets. bioinformatics is a technology that assists researchers in coping with diseases by investigating genetic sequencing and seeking structural models of potential molecular targets present in sars-cov2. the details provided in this review provide future points of consideration in the field of virology and medical sciences that will contribute to clarifying potential therapeutic targets for anti-sars-cov-2 and for understanding the molecular mechanisms responsible for the pathogenesis and virulence of sars-cov-2. . continental map of sars-cov-2 infection cases. data were collected on 28.08.2020 [15] . designed by freepik. currently, in the absence of any efficient therapy known for the treatment of covid-2019 infections and, also, to the process of developing new drugs is time-consuming and cumbersome, the use of bioinformatics as a tool can redirect old drugs against covid-19, helping to identify treatments with known pharmacokinetic, pharmacodynamic and toxicity profiles [5] . some recent studies have provided critical insights using bioinformatics and molecular modeling to help the rapid development of treatments that can be tested in clinical trials [16, 17] . for example, using xml-like web effort (q-uel) systems to access relevant and emerging literature and interact with standard publicly available bioinformatics tools on the internet helped quickly identify sequences of amino acids that are well conserved across many coronaviruses, including sars-cov-2. the theory behind q-uel has been described and developed in several essentially mathematical papers [17] . q-uel is a biomedical and pharmaceutical data mining tool that comprises knowledge bearing tags as "probabilistic statements" from relatively structured data sources [18] and specialist text [19] , as well as "common sense" and general wisdom from thesauruses and encyclopedias, and automatic surfing of the internet [20] . research using this type of tool can contribute to the proposition of specific synthetic vaccine epitope and peptidomimetic agents (see [17] ). the role of bioinformatics in conjunction with molecular modeling in the search for methods of diagnosis, treatment and prevention of covid-19 is unquestionable. processes such as screening of bioactive compounds, modeling of biomacromolecule structures, primer selection and genetic sequencing compounds can be faster, more accurate and less expensive when aided by computer 28 .08.2020 [15] . designed by freepik. currently, in the absence of any efficient therapy known for the treatment of covid-2019 infections and, also, to the process of developing new drugs is time-consuming and cumbersome, the use of bioinformatics as a tool can redirect old drugs against covid-19, helping to identify treatments with known pharmacokinetic, pharmacodynamic and toxicity profiles [5] . some recent studies have provided critical insights using bioinformatics and molecular modeling to help the rapid development of treatments that can be tested in clinical trials [16, 17] . for example, using xml-like web effort (q-uel) systems to access relevant and emerging literature and interact with standard publicly available bioinformatics tools on the internet helped quickly identify sequences of amino acids that are well conserved across many coronaviruses, including sars-cov-2. the theory behind q-uel has been described and developed in several essentially mathematical papers [17] . q-uel is a biomedical and pharmaceutical data mining tool that comprises knowledge bearing tags as "probabilistic statements" from relatively structured data sources [18] and specialist text [19] , as well as "common sense" and general wisdom from thesauruses and encyclopedias, and automatic surfing of the internet [20] . research using this type of tool can contribute to the proposition of specific synthetic vaccine epitope and peptidomimetic agents (see [17] ). the role of bioinformatics in conjunction with molecular modeling in the search for methods of diagnosis, treatment and prevention of covid-19 is unquestionable. processes such as screening of bioactive compounds, modeling of biomacromolecule structures, primer selection and genetic sequencing compounds can be faster, more accurate and less expensive when aided by computer tools. experts from all over the world believe in the potential of bioinformatics in combating the covid-19 pandemic. bioinformatics contributes to understanding the variations in sars-cov-2 proteins and molecules 2020, 25, 4086 4 of 46 how the virulence of this pathogen can increase. based on this, this tool can also clarify how the virus subverts the immune system. our know-how about the molecular elements involved in triggering this type of response can bring to light essential points that may otherwise be neglected. in addition to issues related to bioinformatics and molecular modeling, understanding the participation of innate immunity in the infectious process is crucial for the screening of new target molecules for the treatment and diagnosis of covid-19. it is well established in the current literature that innate immunity plays a central role in determining the outcome of viral infections in general [21] . therefore, the present review explicitly focuses on this aspect of the immune system, demonstrating its role in the formation of the adaptive immune response downstream and its importance as a therapeutic target for the development of new drugs for the treatment of patients with severe covid-19. researchers put much effort to understand the origin and pathophysiology of this novel coronavirus and have been testing multiple drugs to screen for therapeutic effective substances [22] . despite little understanding about the pathophysiology and high pathogenicity of sars-cov-2 infection, early studies have shown that increased amounts of proinflammatory cytokines in serum (e.g., (interleukin il) il-1β, il-6, il-12, interferon-γ (ifnγ), interferon-inducible protein 10 (ip10), and monocytic chemotactic protein 1 (mcp1)) were associated with pulmonary inflammation and extensive lung damage in patients with severe acute respiratory syndrome (sars) [23] . besides, research has shown that infection by mers-cov leads to a significant increase in serum levels of pro-inflammatory cytokines (ifnγ, tnf-α, il15, and il17) [24] . patients who required intensive care unit (icu) admission had higher concentrations of gcsf, ip10, mcp1, mip1a, and tnf-α than those who did not require icu admission, suggesting that massive cytokine synthesis and secretion are associated with the severity of the disease [6] . given the above, the present study aimed to develop a comprehensive review of aspects of bioinformatics and molecular modeling as auxiliary tools to pharmacology and immunology in the discovery of potential anti-sars-cov-2 agents. understanding these aspects streamlines the biomedical research process, without any operational costs. besides, we researched and gathered evidence of the interactions between sars-cov-2 and innate immunity. discovering new therapeutic targets and understanding pathophysiological processes are the key to the development of effective treatments and efficient management of patients who progress to severe sars-cov-2 infection, which can save lives worldwide. most coronavirus (cov) infections in humans are caused by low-pathogenicity species, causing common cold symptoms; however, they can eventually lead to serious infections in higher risk groups such as the elderly, children, patients with comorbidities (hypertension, diabetes mellitus, asthma, among others) and/or those suffering immunosuppression. prior to 2019, two highly pathogenic and animal-derived coronavirus species (sars and mers) were responsible for outbreaks of severe acute respiratory syndromes. regarding human infection by sars-cov-2, the clinical spectrum is not fully described, and the lethality, mortality, infectivity, and transmissibility pattern is not yet fully elucidated. in addition, there is no specific treatment with antivirals that are effective against sars-cov-2 or available vaccines and, currently, treatment is supportive and nonspecific for covid-19 in hospitalized patients with fever, accompanied by cough or sore throat and with dyspnea or o 2 saturation below 95% or respiratory discomfort [25] . cov, of the order nidovirales, coronavirin subfamily and coronaviridae family, is a single-stranded rna virus with diameter of 80-120 nm [26] with appearance of crown under electron microscope ("coronam" is the latin term for "crown") due to the presence of glycoproteins in the viral envelope [27] . it is a virus capable of infecting humans and a wide variety of other mammalian hosts (e.g., mice, swine, rats, dogs, cats, rabbits, horses, cattle, cetaceans and bats) and birds (chickens, pheasants and molecules 2020, 25, 4086 5 of 46 turkeys) and develop respiratory, enteric, liver and central nervous system (cns) diseases. based on its genotypic and serological characteristics, cov is classified into 3 subfamilies, previously called groups 1, 2 and 3. group 1 and 2 were composed of cov that has mammals as hosts and group 3 was composed, until recently, only of avian cov [28] [29] [30] [31] [32] . currently, the study group of the international committee for viral taxonomy (icvt) has proposed replacing the 3 traditional groups by subfamilies alfacoronavirus (α-cov) (group 1), betacoronavirus (β-cov) (group 2) and gamacoronavirus (γ-cov) (group 3). after that, the presence of a fourth cov subfamily was detected in birds and pigs and called deltacoronavirus (δ-cov) [33, 34] . the most common human covs (hcov) are hcov-oc43, hcov-hku1, both β-covs of strain a, and hcov-229e and hcov-nl63, both α-covs. generally, they cause common colds and self-limited upper respiratory infections in immunocompetent individuals, that is, they are eliminated in a short period of time by the immune system without the need for intervention through specific pharmacotherapy. in immunocompromised and elderly individuals, lower respiratory tract infections may also occur. other hcov include sars-cov, sars-cov-2 (or sars-cov-2) and mers-cov (β-covs of lineage b and c, respectively). these cov categories can cause epidemics of varying clinical severity, with respiratory and extra-respiratory manifestations. regarding sars-cov, mers-cov, mortality rates are up to 10% and 35%, respectively [27] and sars-cov-2 belongs to β-cov subfamily. an important feature of the sars-cov epidemic between 2002 and 2003 was the virus efficiency in transmitting from species such as masked palm civet (paguma larvata), raccoon dog (nyctereutes procyonoides) and the chinese ferret-badger (melogale moschata) and infect human populations (fig 1a) [35] . it is postulated that sars-cov is the result of the recombination of covs transmitted from these animals to humans. however, research has shown that sars-cov has not been detected in domestic or wild masked palm civet [36] , suggesting that these, and other animals marketed in wholesale seafood markets in china, were not the main reservoirs of the virus [37] . cov similar to sars-cov was isolated from the chinese horseshoe bat (rhinolophus spp.) [38, 39] , which were also marketed in live-animal markets, strongly suggesting that the virus may have recently been transmitted from bats to other mammals, such as masked palm civets, and later to humans (figure 2a ) [37] . in addition to sars-cov, there are other situations of cross-transmission of covs among different animal species. bovine coronavirus (bcov) and hcov-oc43 have broad similarities. bcov is believed to have been transmitted from bovine hosts to humans a hundred years ago [40] . previous studies have demonstrated the isolation of bcov from alpaca that showed signs of enteritis and in captive wild ruminants [41, 42] , strongly suggesting that this cov has been transmitted to others species (figure 2b) . previous studies have shown that canine (ccov), feline (fcov) and swine coronaviruses exchanged genetic material at random, showing that they were infecting the same host. recombination processes among the first ccov and fcov (ccov-i and fcov-i) strains and an unknown cov resulted in two new groups of viruses, ccov-ii and fcov-ii. in addition, studies have shown that the sequence of the transmissible gastroenteritis virus (tgev) genetic material shows that this cov was originated through cross-transmission of ccov-ii species from an infected dog (figure 2c ) [43] . molecules 2020, 25, x for peer review 5 of 48 swine, rats, dogs, cats, rabbits, horses, cattle, cetaceans and bats) and birds (chickens, pheasants and turkeys) and develop respiratory, enteric, liver and central nervous system (cns) diseases. based on its genotypic and serological characteristics, cov is classified into 3 subfamilies, previously called groups 1, 2 and 3. group 1 and 2 were composed of cov that has mammals as hosts and group 3 was composed, until recently, only of avian cov [28] [29] [30] [31] [32] . currently, the study group of the international committee for viral taxonomy (icvt) has proposed replacing the 3 traditional groups by subfamilies alfacoronavirus (α-cov) (group 1), betacoronavirus (β-cov) (group 2) and gamacoronavirus (γ-cov) (group 3). after that, the presence of a fourth cov subfamily was detected in birds and pigs and called deltacoronavirus (δ-cov) [33, 34] . the most common human covs (hcov) are hcov-oc43, hcov-hku1, both β-covs of strain a, and hcov-229e and hcov-nl63, both α-covs. generally, they cause common colds and selflimited upper respiratory infections in immunocompetent individuals, that is, they are eliminated in a short period of time by the immune system without the need for intervention through specific pharmacotherapy. in immunocompromised and elderly individuals, lower respiratory tract infections may also occur. other hcov include sars-cov, sars-cov-2 (or sars-cov-2) and mers-cov (β-covs of lineage b and c, respectively). these cov categories can cause epidemics of varying clinical severity, with respiratory and extra-respiratory manifestations. regarding sars-cov, mers-cov, mortality rates are up to 10% and 35%, respectively [27] and sars-cov-2 belongs to β-cov subfamily. an important feature of the sars-cov epidemic between 2002 and 2003 was the virus efficiency in transmitting from species such as masked palm civet (paguma larvata), raccoon dog (nyctereutes procyonoides) and the chinese ferret-badger (melogale moschata) and infect human populations (fig 1a) [35] . it is postulated that sars-cov is the result of the recombination of covs transmitted from these animals to humans. however, research has shown that sars-cov has not been detected in domestic or wild masked palm civet [36] , suggesting that these, and other animals marketed in wholesale seafood markets in china, were not the main reservoirs of the virus [37] . cov similar to sars-cov was isolated from the chinese horseshoe bat (rhinolophus spp.) [38, 39] , which were also marketed in live-animal markets, strongly suggesting that the virus may have recently been transmitted from bats to other mammals, such as masked palm civets, and later to humans (figure 2a ) [37] . . this virus has spread and adapted to wild animals, for example, masked palm civet, which is marketed for human consumption in wholesale seafood markets in china. the employees of these markets that manipulate these wild animals have been infected; however, they did not present important clinical signs, and symptoms were minimal. the process of adapting the virus to new hosts resulted in strains with efficient replication capacity in human hosts, which cause diseases with clinical conditions ranging from mild to severe and with great ability to spread from person to person; (b) oc43 coronavirus, whose natural reservoir are humans (hcov-oc43) and bovine coronavirus (bcov) are closely related. it is postulated that these coronaviruses originated in another animal species and subsequently have crossed their species. bcov has effectively spread among other animal species, for example, alpaca (south american mammal of the camelid family) and wild ruminants (such as deer); (c) currently, some canine viruses are believed to have common ancestors with feline species. this occurs with coronaviruses that infect these species. currently, feline coronavirus i (fcov-i) and canine coronavirus i (ccov-i) are believed to share a common ancestor. a recombination process (random exchange of genetic material) of ccov-i with an unknown coronavirus gave rise to a second type of canine coronavirus (ccov-ii). the recombination of ccov-ii with fcov-i in an unknown host gave rise to a second type of feline coronavirus (fcov-ii). there is evidence that ccov-ii was transmitted to pigs, originating the transmissible gastroenteritis virus (tgev) [36] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). in addition to sars-cov, there are other situations of cross-transmission of covs among different animal species. bovine coronavirus (bcov) and hcov-oc43 have broad similarities. bcov is believed to have been transmitted from bovine hosts to humans a hundred years ago [40] . previous this virus has spread and adapted to wild animals, for example, masked palm civet, which is marketed for human consumption in wholesale seafood markets in china. the employees of these markets that manipulate these wild animals have been infected; however, they did not present important clinical signs, and symptoms were minimal. the process of adapting the virus to new hosts resulted in strains with efficient replication capacity in human hosts, which cause diseases with clinical conditions ranging from mild to severe and with great ability to spread from person to person; (b) oc43 coronavirus, whose natural reservoir are humans (hcov-oc43) and bovine coronavirus (bcov) are closely related. it is postulated that these coronaviruses originated in another animal species and subsequently have crossed their species. bcov has effectively spread among other animal species, for example, alpaca (south american mammal of the camelid family) and wild ruminants (such as deer); (c) currently, some canine viruses are believed to have common ancestors with feline species. this occurs with coronaviruses that infect these species. currently, feline coronavirus i (fcov-i) and canine coronavirus i (ccov-i) are believed to share a common ancestor. a recombination process (random exchange of genetic material) of ccov-i with an unknown coronavirus gave rise to a second type of canine coronavirus (ccov-ii). the recombination of ccov-ii with fcov-i in an unknown host gave rise to a second type of feline coronavirus (fcov-ii). there is evidence that ccov-ii was transmitted to pigs, originating the transmissible gastroenteritis virus (tgev) [36] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). in general, statistical data suggest that 2% of the population are asymptomatic cov carriers and that these viruses are responsible for about 5% to 10% of acute respiratory infections [44] . as for transmission and infection, rarely animal covs infect people and spread among them, as occurred with sars-cov and mers-cov. at first, many of the patients with outbreaks of respiratory diseases caused by sars-cov-2 in wuhan, china, had some connection with a large seafood and live-animal market, strongly suggesting that the spread occurred from animal to people. however, after the outbreak began, an increasing number of patients supposedly did not have exposure to the animal market, also indicating the occurrence of spread from person to person [25] . the possibility of sars-cov-2 transmission from seafood to humans is unlikely. as the wuhan seafood wholesale market also sells other animals, the sars-cov-2 natural host remains unknown [44] . the sustained spread from people to people is occurring in the globe. this culminated in the statement that the now-pandemic sars-cov-2 outbreak constitutes a public health emergency of international concern (pheic) by who on january 30, 2020 in geneva, switzerland. person-to-person transmission cases have already been reported worldwide. transmission in health institutions, such as hospitals, has frequently occurred, having already been reported in almost all countries where sars-cov-2 is present. regarding the person-to-person spread that occurred with sars-cov or mers-cov, the main means is believed to have been through respiratory droplets produced when an infected person coughs or sneezes, similar to the way influenza and other respiratory pathogens are disseminated. in addition, aerosol transmission has been identified in patients undergoing airway procedures, such as endotracheal intubation or airway aspiration. in the population, sars-cov and mers-cov dissemination among people usually occurs after close contacts, and health professionals involved in assisting these patients are particularly vulnerable [25] . a recent study conducted with women who were in the third trimester of pregnancy and who were confirmed with sars-cov-2 infection showed no evidence of vertical transmission, that is, from mother to child. despite that, all pregnant women underwent cesarean section and it remains unclear whether transmission can occur during normal delivery [45] . further studies with pregnant women in other gestation periods and even in the third trimester should be carried out, since the sample used was small. studies like this are crucial for the care of pregnant women, since they are relatively more susceptible to respiratory pathogen infection and severe pneumonia. recent studies have shown that interpersonal sars-cov-2 transmission has occurred since mid-december 2019 in wuhan (china) and from there, considerable efforts to reduce transmission and control the current pandemic will be necessary, since the transmission dynamics is very similar in all places where the number of infected and dead people is high. furthermore, these results suggest that measures to prevent or reduce transmission should be urgently implemented in populations at risk [1] . for this reason, all government spheres worldwide, together with regulatory agencies from the health area, recommended complete social distancing as an emergency measure to contain the spread of sars-cov-2 through interpersonal contact. it is important to highlight that, through the experience of chinese and italians, the possibility of sars-cov-2 transmission before the onset of symptoms cannot be excluded and although it seems uncommon, there is evidence that individuals who remain asymptomatic can transmit the virus. these data suggest that the use of isolation is the best way to contain the sars-cov-2-associated pandemic [27] . china's cdc and local cdcs used the chinese experience in the sars-cov-2 outbreak to conduct investigations that determined the viral incubation time. they demonstrated that this period can vary from 3 to 7 days to 2 weeks, since the longest time from infection to symptoms was 12.5 days (95% ci, 9.2-18) [1] . in addition, these results showed that the new pandemic doubled every seven days, while the basic reproduction number (r0-r zero) is 2.2, that is, each patient transmits the infection to an additional 2.2 individuals, on average. it is important to note that the r0 estimates of the sars-cov epidemic in 2002-2003 were approximately 3 [46] . for viral infection to occur, the connection of the virus to a receptor expressed by host cells is the first step, followed by fusion with the cell membrane. specifically for sars-cov-2, it is possible to deduce that the epithelial cells of lungs are its main target, since in part of infected patients, covid-19 progresses as an acute respiratory infection [47] . previous studies have shown that human-to-human sars-cov transmission occurs by the association between the binding domain to the spike receptor located on the viral envelope and the cell receptor. recently, it was identified that cell receptor for sars-cov is the angiotensin-converting enzyme 2 (ace2) [48, 49] . it is important to note that the sequence of the binding domain to the spike receptor of the sars-cov-2 viral envelope is similar to that of sars-cov. these data strongly suggest that the entry into host cells occurs through the ace2 receptor ( figure 3 ) [48] . infection to an additional 2.2 individuals, on average. it is important to note that the r0 estimates of the sars-cov epidemic in 2002-2003 were approximately 3 [46] . for viral infection to occur, the connection of the virus to a receptor expressed by host cells is the first step, followed by fusion with the cell membrane. specifically for sars-cov-2, it is possible to deduce that the epithelial cells of lungs are its main target, since in part of infected patients, covid-19 progresses as an acute respiratory infection [47] . previous studies have shown that human-to-human sars-cov transmission occurs by the association between the binding domain to the spike receptor located on the viral envelope and the cell receptor. recently, it was identified that cell receptor for sars-cov is the angiotensin-converting enzyme 2 (ace2) [48, 49] . it is important to note that the sequence of the binding domain to the spike receptor of the sars-cov-2 viral envelope is similar to that of sars-cov. these data strongly suggest that the entry into host cells occurs through the ace2 receptor ( figure 3 ) [48] . it is assumed that covs that cause covid-19 in humans are related to bats (upper left corner). this selected subset of viruses has the necessary resources to infect the human respiratory tract (lower left corner), with a certain tropism for this system. infection (right panel) requires the interaction of spike proteins present in the sars-cov-2 viral envelope (s proteins) with host sites for type 2 angiotensin-converting enzyme (ace2) present in the lung. subsequently, proteases present on the surface of pneumocytes cleave the s2 region of the s protein, the subunit responsible for the fusion of the s protein with the cell membrane. after cleavage, a series of conformational changes are triggered, resulting in the fusion between viral envelope and the target cell membrane. the structural features of sars-cov-2 that can facilitate infection in humans include: (1) presence of reasons for binding to the s1b receptor (rbms) (in purple) that bind to ace2 orthologous receptors. ace2 is believed to be orthologous because it exhibits homology to s1b rbms (since they complement each other to the point of binding) and were probably duplicated from a common ancestor, shared by the two underlying sister species, where in the course of evolution, both receptors gradually differentiate but continue to have affinity for each other; (2) an s1a domain that provides additional interactions with the host and; (3) a cleavage substrate for a furin protease (represented by the green starry shape bound to the protease at the bottom right of the figure 3 . infection of pneumococytes during covid-19. it is assumed that covs that cause covid-19 in humans are related to bats (upper left corner). this selected subset of viruses has the necessary resources to infect the human respiratory tract (lower left corner), with a certain tropism for this system. infection (right panel) requires the interaction of spike proteins present in the sars-cov-2 viral envelope (s proteins) with host sites for type 2 angiotensin-converting enzyme (ace2) present in the lung. subsequently, proteases present on the surface of pneumocytes cleave the s2 region of the s protein, the subunit responsible for the fusion of the s protein with the cell membrane. after cleavage, a series of conformational changes are triggered, resulting in the fusion between viral envelope and the target cell membrane. the structural features of sars-cov-2 that can facilitate infection in humans include: (1) presence of reasons for binding to the s1b receptor (rbms) (in purple) that bind to ace2 orthologous receptors. ace2 is believed to be orthologous because it exhibits homology to s1b rbms (since they complement each other to the point of binding) and were probably duplicated from a common ancestor, shared by the two underlying sister species, where in the course of evolution, both receptors gradually differentiate but continue to have affinity for each other; (2) an s1a domain that provides additional interactions with the host and; (3) a cleavage substrate for a furin protease (represented by the green starry shape bound to the protease at the bottom right of the figure), which can provide greater sensitivity to cleavages by host proteases. anti-cov antibodies (shown at the bottom right of the figure) can prevent infection through the following mechanisms: (a) binding to s1brbms of the virus, blocking access to ace2 receptor and consequently preventing the continuation of the process of virus fusion with the target cell; (b) distal connection in relation to rbms, generating steric impairment and, consequently, blocking the connection between the virus rbms and the ace2 receptor of the host cell; (c) binding in the s1a region of the viral spike, blocking alternative connections to different receptors; and (d) binding to s2, the region responsible for the fusion of the virus with the membrane of the target cell, consequently preventing fusion. as future perspectives, future research should aim at the development of protease inhibitor antiviral compounds, which play a crucial role in the fusion of the virus to the host cell membrane, suppressing the entry of the virus [50] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). in a recent study, [7] demonstrated the interactions of sars-cov-2 with the host organism, specifically interactions between spike proteins ("coronan") located in the viral envelope (s proteins), projected from the envelope, and the host ace2 receptors. covs derived from the most virulent bats are those in which s proteins have tropism by distinctly human receptors, as is the case with sars-cov-2. after the first contact with humans, the virus migrates to lungs and s proteins interact with various proteins and receptors, which are factors of host susceptibility. such interactions cause substantial changes in the conformation of proteins involved in the infection process of host cells, triggering the fusion between virus and human cells and, consequently, the infection. it is noteworthy that specific antibodies against s proteins and antiviral agents that block the link between virus and human susceptibility factors can prevent sars-cov-2 infection [51] (figure 2 ). studies like these have shown that there is a wide spread of covs in their natural reservoirs and by demonstrating interactions between virus and human hosts and, consequently, how the infectious process occurs, they drive the development of vaccines and antiviral drugs. in addition, further studies should be aimed at the active surveillance of these viruses in geographic regions that go beyond the beginning of the sars-cov-2 pandemic outbreak. in the long term, broad-spectrum antiviral drugs and vaccines should be developed with the aim of controlling emerging infectious diseases caused by covs. in addition, regulatory agencies in the health sector should develop specific regulations to control domestication and consumption of wild animals. the emergence of the highly pathogenic sars-cov-2 led to the need for deeper knowledge on the biology and pathogenesis of cov. an emerging theme in the cov pathogenesis and covid-19 pathophysiology is the interaction between specific viral genes and the host's immune system, which acts as a key determinant in the regulation of disease virulence and development results. viral interactions with innate immune system play a key role in determining the course of the infection. the initial control of viral replication by type i interferons (ifn), complement system proteins and other innate immune mediators limit viral spread in the host during the early stages of the disease [52] . in addition, the initial innate response also plays a key role in the development of the subsequent adaptive immune response [53] . despite that, it is well established in current literature that the hyperactive innate immune response can also result in pathology and subsequent tissue damage. regarding innate immunity, there are two main pathways by which cells detect viruses that invade the organism and activate the ifn pathway [21] . toll-like receptors (tlrs), which include tlr3, tlr7, tlr8 and tlr9, are responsible for detecting viruses in endosomal compartments as they penetrate host cells. the cytoplasmic domain of tlrs (called card) contains two specific rna-helicases, rig-i and mda5, which detect viral rna in the cell cytoplasm. for activation of both pathways, interactions between sensors with pathogen-associated molecular patterns (pamps) are required, such as, for example, single-stranded rnas associated with viral genomes or double-stranded rna, which is a by-product of viral replication, being common targets. different adapter proteins participate in signal transduction in the induction pathways of tlrs and cytoplasmic ifn. the tlr-dependent pathway uses tir-domain-containing adapter-inducing interferon-beta adapter proteins (trif) and/or the myeloid differentiation factor 88 (myd88) and the ifn cytoplasmic induction pathway uses the mavs/ips-1/visa/cardif mitochondrial adapter protein [54] (figure 4 ). the tlr-dependent pathway uses tir-domain-containing adapter-inducing interferon-beta adapter proteins (trif) and/or the myeloid differentiation factor 88 (myd88) and the ifn cytoplasmic induction pathway uses the mavs/ips-1/visa/cardif mitochondrial adapter protein [54] ( figure 4 ). after internalized, the virus exposes the genomic rna to the dsrna detection mechanism in the cell, that is, tlr3, rigi and mda5. these proteins are responsible for the irf-3 cascade signaling, leading to ifnb induction and, consequently, the production of ifnβ protein. the newly synthesized ifnβ can bind to ifn receptors on the surface of the same cell or surrounding cells and induce the synthesis of more ifn molecules. binding to ifn receptors activates the signal transducer and activator of transcription 1 (stat1) signaling pathway to activate several distinct antiviral genes located in isre promoter elements [21, 55] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). the complement system plays a crucial role in the innate immune response to viruses. it represents one of the main factors responsible for the development of pro-inflammatory reactions during these diseases [56, 57] . research has shown that, just as in human infection, intranasal sars-cov infection in c57bl/6j mice, results in virus replication with high rates in the lung, in addition to induction of inflammatory cytokines and chemokines and infiltration of immune cells in the pneumocytes [58] . using c3-deficient mice (c3 -/-), these researchers demonstrated that animals c3 -/-infected with sars-cov lost less weight and obtained a significant reduction in respiratory dysfunction when compared to control groups, despite viral loads equivalent in the lung. besides, a significantly lower rate of neutrophils and inflammatory monocytes were present in the lungs of c3 -/-mice than in the c56bl/6j controls, and subsequent studies showed that lung injury was reduced, as were cytokine (il-6, mainly) and chemokine levels in lungs and serum of c3 -/-mice than when purchased from animals in the control group [58] . this suggests that c3 inhibition may significantly reduce the pulmonary inflammatory complications of sars-cov-2 infection. the decrease in neutrophilic infiltration in the lungs and the reduced levels of intrapulmonary and plasma il-6 that have been observed in mice with c3 -/-infected with sars-cov suggest a potential treatment that combines c3 inhibitors and anti-il-6 drugs [59] . besides, as c3 is one of the first proteins synthesized by the complement system in the innate immune cascade, the anti-inflammatory potential for c3 blockage is broader. drugs like amy-101 are being investigated for this [60] , which is currently being tested in patients with covid-19 [59] . c3 blockade can simultaneously inhibit the synthesis of c3a and c5a. furthermore, this block reduces the intrapulmonary activation of c3 and the release of ilafter internalized, the virus exposes the genomic rna to the dsrna detection mechanism in the cell, that is, tlr3, rigi and mda5. these proteins are responsible for the irf-3 cascade signaling, leading to ifnb induction and, consequently, the production of ifnβ protein. the newly synthesized ifnβ can bind to ifn receptors on the surface of the same cell or surrounding cells and induce the synthesis of more ifn molecules. binding to ifn receptors activates the signal transducer and activator of transcription 1 (stat1) signaling pathway to activate several distinct antiviral genes located in isre promoter elements [21, 55] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). the complement system plays a crucial role in the innate immune response to viruses. it represents one of the main factors responsible for the development of pro-inflammatory reactions during these diseases [56, 57] . research has shown that, just as in human infection, intranasal sars-cov infection in c57bl/6j mice, results in virus replication with high rates in the lung, in addition to induction of inflammatory cytokines and chemokines and infiltration of immune cells in the pneumocytes [58] . using c3-deficient mice (c3 -/-), these researchers demonstrated that animals c3 -/infected with sars-cov lost less weight and obtained a significant reduction in respiratory dysfunction when compared to control groups, despite viral loads equivalent in the lung. besides, a significantly lower rate of neutrophils and inflammatory monocytes were present in the lungs of c3 -/mice than in the c56bl/6j controls, and subsequent studies showed that lung injury was reduced, as were cytokine (il-6, mainly) and chemokine levels in lungs and serum of c3 -/mice than when purchased from animals in the control group [58] . this suggests that c3 inhibition may significantly reduce the pulmonary inflammatory complications of sars-cov-2 infection. the decrease in neutrophilic infiltration in the lungs and the reduced levels of intrapulmonary and plasma il-6 that have been observed in mice with c3 -/infected with sars-cov suggest a potential treatment that combines c3 inhibitors and anti-il-6 drugs [59] . besides, as c3 is one of the first proteins synthesized by the complement system in the innate immune cascade, the anti-inflammatory potential for c3 blockage is broader. drugs like amy-101 are being investigated for this [60] , which is currently being tested in patients with covid-19 [59] . c3 blockade can simultaneously inhibit the synthesis of c3a and c5a. furthermore, this block reduces the intrapulmonary activation of c3 and the release of il-6 from alveolar macrophages or other cells that express c3a receptors (c3ars) and/or c5a receptors (c5ars), causing the beneficial evolution and cure of lung injury [60] . the role of complement system activation in the development of severe acute respiratory syndrome associated with sars-cov-2 infection is not yet fully understood, and clinical data are scarce. recent research has shown that the sars-cov, mers-cov, and sars-cov-2 "n" proteins bind to masp-2, the essential serine protease in the complement activation lectin pathway, leading to over complement activation and lung injury severe inflammatory disease. thus, it is evident that the blockade between the interaction of protein n with masp-2 can significantly reduce the hyperactivation of complement induced by protein n and lung injury in vitro and in vivo. complement overactivation is present in patients with covid-19, and a promising suppressive effect has been seen in patients with severe lung injury treated with an anti-c5a monoclonal antibody [61] . due to the cascade organization of the complement system, inhibitors that target c3 or its upstream activators may be more effective and, potentially, may prevent the initial stages of the infectious process that lead to severe lung inflammation. despite this, no substance that acts with these mechanisms of action has been approved for use in humans, although phase ii clinical studies are already underway [59] . figure 5 outlines the moment when complement system inhibitors can be used in lung injury associated with sars-cov-2. molecules 2020, 25, x for peer review 12 of 48 figure 5 . a possible scheme for using complement system inhibitors in lung injury associated with sars-cov-2: (a) sars-cov-2 penetrates the host's pneumocytes and uses the cellular machinery for protein synthesis and replication of the genetic material and causing activation of the complement system through different pathways; (b) complement activation contributes to the massive inflammatory response of pneumocytes observed in some patients with severe covid-19. the inhibition of c3 or c5 can have significant therapeutic potential [59] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). the mechanism of natural sars-cov-2 infection occurs very similar to previously studied sars-cov [62] and produces adaptive immune responses against the structural antigens of these viruses in humans and animals [63] . spike glycoproteins located in the viral envelope (s proteins) are figure 5 . a possible scheme for using complement system inhibitors in lung injury associated with sars-cov-2: (a) sars-cov-2 penetrates the host's pneumocytes and uses the cellular machinery for protein synthesis and replication of the genetic material and causing activation of the complement system through different pathways; (b) complement activation contributes to the massive inflammatory response of pneumocytes observed in some patients with severe covid-19. the inhibition of c3 or c5 can have significant therapeutic potential [59] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). the mechanism of natural sars-cov-2 infection occurs very similar to previously studied sars-cov [62] and produces adaptive immune responses against the structural antigens of these viruses in humans and animals [63] . spike glycoproteins located in the viral envelope (s proteins) are responsible for binding to the cell receptor and fusing the virus membrane with the host's cell membrane [64] . these glycoproteins also act as antigens responsible for the activation of t cells and development of humoral and cellular immunity [65] . literature data have demonstrated that the majority of antigenic peptides are located in structural proteins (mainly s protein) [66, 67] . it is believed that hypervirulent cov variants, such as sars-cov-2, have the capacity to develop viremia, producing systemic disease, being often fatal [6, [68] [69] [70] [71] [72] [73] . previous studies have shown that sars-cov infection of macrophages and dendritic cells leads to an aberrant cytokine/chemokine expression pattern [21] , so that the ability to infect and replicate in such phagocytes appears to be a determinant factor for establishing the course of viral infection. in addition, it has recently been reported that sars-cov-2 causes lymphocyte depletion, resulting in high viral titers [6, 37, 74, 75] . previous studies demonstrate the spread of sars-cov to lymphoid organs such as spleen, thymus, peyer plaques and mesenteric lymph nodes, developing intense lymphoid depletion [69, 73, 76] . it is already well established in literature that lymphocytes and their subtypes are essential to maintain the immune system function in basal activity or during infectious processes. viral infections, immunodeficiency syndromes and other infectious diseases, such as tuberculosis, are known to determine abnormal plasma counts in lymphocyte levels and their subtypes [77] [78] [79] ; and for such infections to evolve into more serious events, lymphopenia is crucial, since the reduction of this cell type is necessary for the survival and persistence of many microorganisms. in wuhan, where sars-cov-2 infection started, 63% of patients with covid-19 had significant lymphopenia induction [6] . although the mechanisms that cause lymphopenia in cov infections are not fully understood, it is already known that lymphocytes can undergo lysis by direct or indirect viral action [70] . previous studies have shown that indirect events, secondary to viral infection, are the main responsible for cell lysis through the synthesis of soluble factors such as ifn, cytokines and chemokines by host cells [70, 73] . as already mentioned, very recent studies have identified that the sars-cov cell receptor is ace2 and that the sequence of the binding domain to the spike receptor of the sars-cov-2 viral envelope is similar to that of sars-cov, suggesting that the entry into host cells occurs through the ace2 receptor [48] . however, the presence of ace2 receptors has been identified in the oral mucosa, in type ii pneumocytes, along the intestine and in the renal and cardiac endotheliums [80] , with no evidence of the presence of these receptors in circulating mature lymphocytes. for this reason, it is believed that the cytolytic effect of sars-cov-2 that led infected patients to develop lymphopenia is not direct, that is, caused by the virus itself, since the receptor determines the tropism of the pathogen by the tissue. in addition, previous studies have demonstrated the presence of cov rna in lymphoid tissues, such as the thymus, in infected patients. this suggests that one of the lymphopenia mechanisms in these patients may be related to the reduction in the production of mature lymphocytes by the thymus, which is the target organ in several infectious diseases, including coronavirus [81] . since these viruses infect the thymus and cause tissue damage, there is an important interference in the lymphocyte differentiation process, which can cause lymphopenia [82] . it is well established in literature that t cells are crucial to produce immunoglobulins and, consequently, for the development of humoral and memory immunity. therefore, pharmacological treatments or the use of integrative and complementary practices that prevent the reduction or increase the levels of cells of the immune system can be essential tools in combination with treatment and/or prevention of sars-cov-2 infection. the use of bioinformatics and other computational tools in addition to molecular modeling has helped researchers from different areas in the search for strategies for diagnosing viral infection, in the development of vaccines for its prevention, as well as in the discovery of new anti-sars-cov-2 agents. the knowledge of the genome of a species based on the genetic sequencing technique is the starting point for the structure and function of its genes to be understood. in the context of diseases transmitted by microorganisms, such as sars-cov-2, the mapping of the genome of microorganisms collected from infected patients in different regions of the world also allows tracing a transmission profile, including its dissemination in different regions and countries, contributing to the search for strategies to combat the disease and monitor mutations [16, 83] . data from the ncbi genbank ® gene sequence database https://www.ncbi.nlm.nih.gov/genbank) accessed on 08/04/2020 indicated more than 14,000 nucleotide sequences inserted since december 2019 for sars-cov-2, most of them coming from cities in china and the usa. in february 2020, researchers from the university of são paulo and instituto adolf lutz, in brazil, together with researchers from the university of oxford, in the united kingdom, managed to elucidate and publish the complete gene sequence of the virus (genbank accession number mt126808) obtained from of the first confirmed cases of the disease in brazil within just two days from the confirmation of the diagnosis [84] . the speed in obtaining this information is only possible through the application of bioinformatics techniques. bioinformatics showed great advance in the 1990s, mainly in view of the development of the genomics area, which generated large amount of biological data incompatible to be quickly analyzed in a manual way. the rapid and adequate manipulation of these data has been possible through the application of data comparison and analysis software and easy access to previously available data from the sharing and storage of information in virtual databases [85] . in view of experimentally obtained sequences, it is possible to perform the alignment of these sequences to other sequences available in virtual databases, leading to the knowledge of the family to which the microorganism is related [83, 86, 87] . in one of the first studies on the new virus that caused respiratory infections in china, the researchers were able to determine from the sequencing of rna obtained from bronchoalveolar fluid samples that the etiologic agent was rna virus of the coronaviridae family. in addition, using bioinformatics tools, it was possible to perform a phylogenetic analysis, revealing 89.1% similarity of the nucleotide sequence of this virus with a group of coronaviruses of the genus betacoronavirus already identified in bats in china, which gave evidence of the virus origin [88] (figure 6 ). one of the algorithms most widely used for comparing biological sequences, whether from nucleotides to nucleic acids or from amino acids to proteins, is the blast (basic local alignment search tool) [89] . the diversity of information on biological sequences from organisms of different degrees of complexity allows blast to be a starting point to understand the genetic relationship with other species, also tracing a possible origin. the use of such tool allowed [90] to identify high similarity between sars-cov-2 genomic sequence extracted from ncbi genbank ® (genbank accession number mn908947) and viral metagenomas found in the pangolin mammal present in the database. these studies, in turn, guided analyses that led to findings of high amino acid identity of s, e, m and n genes of pangolin coronavirus with those isolated from humans, suggesting that the virus capable of infecting humans may have emerged from a recombination of coronaviruses isolated from bat and pangolin. the large capacity for storing information in virtual databases made possible by advances in the field of information technology allows these databases to be continuously updated with new genomic sequences to be universally available. in the context of covid-19, this characteristic was important for a better understanding of the origin of sars-cov-2 from the comparative analysis of genomic data of the new virus with others from the same family, suggesting its origin from natural selection, with modifications in its spike protein, more specifically in the host receptor binding domain, which may have enhanced its interaction and recognition by the human cell [83, 91] . related [83, 86, 87] . in one of the first studies on the new virus that caused respiratory infections in china, the researchers were able to determine from the sequencing of rna obtained from bronchoalveolar fluid samples that the etiologic agent was rna virus of the coronaviridae family. in addition, using bioinformatics tools, it was possible to perform a phylogenetic analysis, revealing 89.1% similarity of the nucleotide sequence of this virus with a group of coronaviruses of the genus betacoronavirus already identified in bats in china, which gave evidence of the virus origin [88] ( figure 6 ). figure 6 . bioinformatics as technologies applied to health as allies to coping with the disease: bioinformatics is a technology that assists researchers in coping with diseases by investigating genetic sequencing and seeking structural models of potential molecular targets present in sars-cov-2. note: this image was developed using the coreldraw software (2017 corel corporation id 410003). one of the algorithms most widely used for comparing biological sequences, whether from nucleotides to nucleic acids or from amino acids to proteins, is the blast (basic local alignment figure 6 . bioinformatics as technologies applied to health as allies to coping with the disease: bioinformatics is a technology that assists researchers in coping with diseases by investigating genetic sequencing and seeking structural models of potential molecular targets present in sars-cov-2. note: this image was developed using the coreldraw software (2017 corel corporation id 410003). the rt-pcr test is considered the gold standard for diagnosing covid-19 [92] . by this technique, the reverse transcriptase enzyme initially participates in transforming the viral rna into complementary dna (cdna). then, the genetic material is amplified. in this process, the regions of the genetic material to be amplified are recognized by polymerase by means of oligonucleotides complementary to the region of interest called primers [93] . the step of primer selection is essential to ensure the quality of the analysis and requires a thorough analysis of the reference genetic sequence [94] . it is also important to consider that recognition is specific, and the method can be applied worldwide, that is, genetic material conserved regions should be prioritized over regions of low similarity. in this context, bioinformatics can contribute by allowing the design of primers based on the comparison of different genetic sequences from different geographic regions, available in virtual databases, identifying regions conserved in the genome, which would enhance the method's specificity [95] . in the work of [92] , which is also recommended by who as a reference in primers to be used in rt-pcr tests, researchers used complete and partial genetic sequences of sars-related viruses present in the ncbi genbank ® database for the design of primers and, in view of the first publications of sars-cov-2 sequence on virological.org [84] and gisaid [96] , it was possible to align the primers proposed for these sequences and to select those with greater correspondence. several online servers and software have been used in order to optimize this process [95, 97, 98] . one of the main strategies to control viral diseases has been vaccination prevention. the development of a vaccine involves the identification of either synthetic or natural antigens that can prevent or even treat a disease. for viral diseases, the most explored strategies involve the use of attenuated viruses in their composition, use of viral structures that can be neutralized by the immune system, or application of part of the viral genetic material related to the expression of proteins that can behave as antigens for the development of antibodies against the pathogen of origin [99] . regardless of strategy to be used, knowledge about the viral genome and the structure and function of virus proteins is essential for a vaccine to be successfully developed. in this context, bioinformatics tools have also contributed to greater quality and speed in the process. the proposal of vaccines based on b and t-cell epitopes has been a possible strategy for obtaining vaccines for covid-19 prevention. such epitopes can be predicted from the analysis of amino acid sequences of proteins of the sars-cov-2 pathogen and comparison with virtual epitope databases [100, 101] . even without studies on specific immune responses against sars-cov-2, the genetic similarity between this virus and sars-cov obtained from sequential alignment and phylogenetic analysis of genomes indicates that possible common epitopes related to structural s and n proteins could be incorporated into vaccines to be developed, in view of the immunological response already observed against sars-cov structures [102] . the search for regions conserved in genetic sequences of different coronavirus species aided by bioinformatics tools has also been a way to identify sequences of amino acids that can be models for the design of specific synthetic epitopes to compose vaccines for covid-19 prevention [17] . the discovery and planning of new drugs that can be used in the treatment of covid-19 benefited from the several advances observed over the last decades in the field of bioinformatics. a potential antiviral agent should target processes or macromolecules essential for maintaining the cycle of viral replication and infection of new host cells [103] . to do so, knowing the genetic sequence of the virus is not enough, but it is also important to determine the functions related to each of the genes that compose it. the sequential identity between genetic materials of different organisms may indicate equivalent genes in terms of function [104] , which encode common proteins that can be exploited as molecular targets for the action of drugs aimed at the treatment of viral infections, for example [105] . prior knowledge of potential molecular targets in other coronaviruses combined with the similarity of genetic sequences among viruses of this family opens several doors for the discovery of new bioactive compounds aided by computational tools [11] . to modulate the action of a molecular target, it is important to know its three-dimensional structure. the determination of this structure is essential for the understanding of how its connection site is organized, how it interferes with its mechanism of action and, given this information, how agents that interfere with its functioning can be proposed. information on the structural organization of a protein is obtained by various techniques such as x-ray crystallography [106] , cryogenic electron microscopy [107] and nuclear magnetic resonance (nmr) [108] . however, bioinformatics tools are those that enable the detailed analysis of data obtained by these techniques and, consequently, the proposition and refinement of three-dimensional models, also exploring comparative analyses with models already proposed [109] . in the same way that there are virtual databases powered by genetic sequences, advances in bioinformatics have also enabled the creation of virtual databases of models of three-dimensional structures of biomacromolecules, which data are freely accessible and can be downloaded and viewed using visualization and molecular modeling software. the most popular three-dimensional structure database is the protein data bank (pdb-www.rcsb.org), with more than 160,000 structures inserted so far [110] . so far, more than 300 structural models of sars-cov-2 macromolecules were deposited in the pdb. most refer to structures proposed for the main protease complexed or not with possible inhibitors [11, 111] , but structural models can also be found for the nucleocapsid protein, non-structural proteins nsp3, nsp9, nsp11, nsp15 [112] and nsp16 and spike glycoprotein [113, 114] . the determination of structural models of the main sars-cov-2 protease, an important enzyme in the processing of polyproteins translated from viral rna in free forms and complexed with the α-ketoamide 13b inhibitor [11] started from obtaining crystallographic data via x-ray diffraction. structural models were then determined by the molecular substitution method, using as reference a structural model from sars-cov already available in pdb, with 96% sequential identity. previously determined sars-cov spike glycoprotein models were also used as a reference in the construction of models of the same sars-cov-2 protein, whose structural data had been obtained from cryogenic electron microscopy [114] . the construction of structural models using crystallographic data as a reference allows the knowledge of the real arrangement of the structure under given experimental condition. when determining the structure of the main protease complexed with 13b inhibitor, [11] were able to "visualize" at molecular level, aided by computational tools, the main points of interaction between inhibitor and enzyme, which may contribute to direct structural modifications that lead to the optimization of the compound's inhibitory activity. likewise, the knowledge of interactions involved in the protein-protein complex between the rbd domain of the sars-cov-2 spike glycoprotein and the human cell ace2 receptor helps to better understand how viral recognition occurs and the consequent search for epitopes conserved in this viral protein that can be targets of neutralizing antibodies to enable the production of vaccines [113] . obtaining crystals from biomolecules; however, can be a laborious or even unfeasible process [115] . in these situations, we choose to build structural models using homology modeling. by this technique, a macromolecule of known three-dimensional structure with considerable degree of sequential identity to that to be determined is used as reference for the construction of this structural model [116] . comparative studies between models determined with the aid of crystallography and models obtained by homology modeling indicate that they have high prediction quality and can be widely applied in studies of interaction between ligand and molecular target [117] [118] [119] . considering the urgency of obtaining structural models of potential molecular targets present in sars-cov-2 and the availability of models of three-dimensional protein structure, mainly of other viruses of the coronaviridae family, with sequential similarity to those of the virus that causes covid-19, homology modeling is the fastest, cheapest and most accessible way for structural determination. thus, many research groups have used this strategy to obtain structural models of different sars-cov-2 proteins, such as the nucleocapsid protein [120] , envelope protein, orf7a protein [88, 120] , non-structural proteins (nsp) [88, 120, 121] , 3clpro [121, 122] and spike glycoprotein [88, 120, 123] , which have been used for the discovery of antiviral agents and the development of vaccines against covid-19. the use of molecular modeling at the early stages of searching for new bioactive compounds has contributed for the faster development of new drugs at reduced costs. in view of the urgency for effective treatments of covid-19, virtual screening methods for bioactive compounds end up by optimizing this discovery pathway by allowing the initial selection of those that present steric and electronic similarities with compounds of known activity (drug planning based on the ligand) or that have the potential to strongly bind to the molecular target of interest (structure-based drug planning) [124] . in view of the low number of compounds with known anti-sars-cov-2 activity and considering the availability of structural models of potential molecular targets of the virus, the strategy based on the structure of the molecular target has been further explored, in which molecular docking techniques play a key role in this planning method. molecular docking consists of anchoring a molecule at the binding site of a molecular target, evaluating its assumed conformations, always searching for the one that generates the lowest-energy complex or the most stable with the target [125] . the binding energy of this complex can be related to its ability to interact and thus modulate the target's action. based on this information, it is possible to select, from virtual databases with a multitude of compounds, potential inhibitors, agonists, or antagonists, against a specific target, which will be hit compounds for more targeted structural changes. in this process of identifying new hit compounds via virtual screening, virtual libraries of chemical compounds, such as zinc databases (https://zinc.docking.org/) [126] and pubchem (https: //pubchem.ncbi.nlm.nih.gov/) [127] , offer a great diversity regarding structural patterns and origin (natural or synthetic), which can contribute to higher quality in the search process. these two databases were explored by [128] to identify possible inhibitors of the sars-cov-2 main protease. from these chemical libraries, researchers selected compounds that already had the ability to inhibit proteases found in other organisms, totaling more than 300 tested compounds, which were submitted to molecular docking studies against the main protease (pdb id 6lu7), thus determining the interaction energy of the target-ligand complex. in this study, the pubchem cid 444,745 compound (figure 7a) showed the greatest potential for inhibiting the enzyme, forming the most stable complex with the main protease. there are published works that have focused on the screening of compounds of natural origin, which have great historical importance in the process of discovering new drugs [129] . libraries of phytochemical compounds with antiviral action from traditional chinese medicine plants have already been virtually screened against different sars-cov-2 molecular targets such as the 3clpro protein [122, 130] , papain-like protease (plpro) and spike glycoprotein [130] . among selected compounds, pubchem cid 11,610,052 [122] and quercetin [130] showed strong interaction with 3clpro; dihydrotanshinone i with the spike glycoprotein; and cryptotanshinone, with both plpro there are published works that have focused on the screening of compounds of natural origin, which have great historical importance in the process of discovering new drugs [129] . libraries of phytochemical compounds with antiviral action from traditional chinese medicine plants have already been virtually screened against different sars-cov-2 molecular targets such as the 3clpro protein [122, 130] , papain-like protease (plpro) and spike glycoprotein [130] . among selected compounds, pubchem cid 11,610,052 [122] and quercetin [130] showed strong interaction with 3clpro; dihydrotanshinone i with the spike glycoprotein; and cryptotanshinone, with both plpro and 3clpro proteins [130] (figure 7a ). in addition to phytochemicals, compounds of marine origin have also been in silico evaluated against the sars-cov-2 main protease [131] . the virtual screening strategy adopted by the authors involved the initial construction of a pharmacophoric model based on the structure, which was created from information on the structure of the enzyme cocrystallized with n3 inhibitor (pdb id 6lu7). in this work, the virtual screening of structures from libraries of marine compounds was then carried out using the pharmacophoric model created as a molecular filter to select those that presented structural characteristics corresponding to the created model. subsequently, the selected compounds were submitted to anchoring at the active site of the enzyme as a way of assessing complementarity with the target. different compounds were identified as potential main protease inhibitors in this analysis, highlighting the compound with the greatest potential heptafuhalol a (figure 7a ). in addition to the virtual screening strategies adopted, another that has stood out among works searching for new anti-sars-cov-2 agents is the one that uses drug groups already approved, preferably those with antiviral action, such as search libraries, characterizing a drug repositioning strategy. the interest in studying compounds already in pharmaceutical use for other purposes has the advantages of reducing time and costs mainly related to the stage of drug discovery, but above all the fact of knowing the toxicological profile of the compound, previously attested in clinical trials [132] . in two studies found in literature, the use of a set of drugs obtained from the zinc database [126] was observed as a library of screening compounds. in the work of [133] , the interaction between this set of drugs and the sars-cov-2 spike glycoprotein was evaluated, highlighting as a result the high potential for interaction between digitoxin and zorubicin (figure 7b ). in the work of [88] , the set of drugs obtained from zinc was virtually evaluated regarding their interaction with different probable molecular targets of the virus, highlighting the potential of ribavirin against plpro; limecycline against 3clpro and valganciclovir against rna-dependent rna polymerase (rdrp) (figure 7b ). in the study by [134] , the drugs analyzed were obtained from the sweetlead database [135] and screened against the spike glycoprotein, with drug pemirolast (figure 2b ) being indicated as a promising inhibitor of the action of this glycoprotein. [136] conducted in silico evaluation against sars-cov-2 rdrp only the potential inhibitor of drugs or compounds in clinical trials with anti-hcv action. among drugs tested, the potential of ribavirin is highlighted, which had also demonstrated potential antiviral action in the work of [88] , but acting against the plpro target ( figure 7b ). currently, a comprehensive and standardized repository of knowledge of the interaction mechanisms between sars-cov-2 and the host has been created. the repository is called covid-19 disease map and was built with the contribution of experts using the results of published research, including bioinformatics data. this technology is a platform that contributes to the computational analysis of molecular processes involved in the 2019-ncoc input and replication interactions in the host. besides, information about the immune system's performance, recovery of host cells, and repair mechanisms can be obtained through the covid-19 disease map [137] . figure 8 demonstrates the objective and the initial layout of the map and its operating cycle. a recent study involving the phylogenetic analysis of 160 complete genomes of covid-19, researchers revealed three central variants distinguished by changes in amino acids, which were called variants a, b, and c, with a being the ancestral type according to the bat outgroup coronavirus. viral strains a and c have been identified in many countries outside east asia, that is, in europe and the united states of america. in contrast, variant b is the most common type in east asia, and there is no evidence that its ancestral genome spread outside east asia without first undergoing mutations [138] . this update is carried out according to the materials available in databases on the subject to support visual and computational exploration, as well as efforts to model diseases [137] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). a recent study involving the phylogenetic analysis of 160 complete genomes of covid-19, researchers revealed three central variants distinguished by changes in amino acids, which were called variants a, b, and c, with a being the ancestral type according to the bat outgroup coronavirus. viral strains a and c have been identified in many countries outside east asia, that is, in europe and the united states of america. in contrast, variant b is the most common type in east asia, and there is no evidence that its ancestral genome spread outside east asia without first undergoing mutations [138] . a phylogenetic analysis carried out in turkey, with the first 30 genomes isolated from sars-cov-2 in this region, showed that the introduction of the virus in the country occurred before the first reported case of infection. the study demonstrated that the virus circulated in turkey from several independent international introductions and revealed a hub for inland transmission [139] . another phylogenetic analysis carried out recently sequenced nine viral genomes of patients with covid-19 previously reported in connecticut, united states. the research placed most of these genomes with viruses sequenced from washington state. interestingly, when the researchers combined the genomic data obtained in the study with national and international travel patterns, they found that domestic introductions probably drove the initial transmission of sars-cov-2 in connecticut. this study provides evidence for the sustained and widespread transmission of sars-cov-2 in the usa and highlights the critical need for local surveillance [140] . important research on the evolutionary and epidemiological dynamics of the current outbreak of covid-19 analyzed 12 genomes of sars-cov-2 strains collected from china and 12 other countries with sampling dates between 24 december 2019 and 9 february 2020. the phylogenetic study results showed that the time to the most recent common ancestor was 2 november 2019, and the evolutionary rate of sars-cov-2 was 9.90 × 10 −4 substitutions per site per year. these results highlight that the use of phylodynamic analyzes is crucial to provide insights into the possibilities of interventions to limit the spread of sars-cov-2 worldwide [141] . disease map is selected and reviewed from databases and knowledge continuously. this update is carried out according to the materials available in databases on the subject to support visual and computational exploration, as well as efforts to model diseases [137] . note: this image was developed using the coreldraw software (2017 corel corporation id 410003). a phylogenetic analysis carried out in turkey, with the first 30 genomes isolated from sars-cov-2 in this region, showed that the introduction of the virus in the country occurred before the first reported case of infection. the study demonstrated that the virus circulated in turkey from several independent international introductions and revealed a hub for inland transmission [139] . another phylogenetic analysis carried out recently sequenced nine viral genomes of patients with covid-19 previously reported in connecticut, united states. the research placed most of these genomes with viruses sequenced from washington state. interestingly, when the researchers combined the genomic data obtained in the study with national and international travel patterns, they found that domestic introductions probably drove the initial transmission of sars-cov-2 in connecticut. this study provides evidence for the sustained and widespread transmission of sars-cov-2 in the usa and highlights the critical need for local surveillance [140] . important research on the evolutionary and epidemiological dynamics of the current outbreak of covid-19 analyzed 12 genomes of sars-cov-2 strains collected from china and 12 other countries with sampling dates between 24 december 2019 and 9 february 2020. the phylogenetic study results showed that the time to the most recent common ancestor was 2 november 2019, and the evolutionary rate of sars-cov-2 was 9.90 × 10 −4 substitutions per site per year. these results highlight that the use of phylodynamic analyzes is crucial to provide insights into the possibilities of interventions to limit the spread of sars-cov-2 worldwide [141] . from studies like these, it is possible to suggest that phylogenetic networks developed with bioinformatics aid can also be used successfully to trace undocumented sources of covid-19 infection. after that, cases can be reported and quarantined to prevent the disease's recurrent spread worldwide [138] . the contributions of bioinformatics and molecular modeling in elucidating essential targets for the planning and development of new drugs, and the analysis of already known compounds, support the search for safer and more effective treatments against sars-cov-2 infection. identifying targets, planning interactions, understanding the structure-activity relationship, coupling, and molecular dynamics allow a projection of the mechanism of antiviral action in silico. an example of this is the prospect of using molecules of natural origin tested in vitro or in vivo and which showed significant antiviral activity. therefore, the use of software to identify viral targets and the prediction of their interaction with the molecule under analysis is necessary so that that research can proceed safely and with a higher probability of success. the verification of the performance of these compounds through computational analysis can predict physical-chemical properties, which are related to pharmacodynamic and pharmacokinetic parameters, whose knowledge is crucial when it comes to the planning of new drugs [142] . table 1 presents studies where the researchers tested natural compounds from plants and fungi against sars-cov-2. the table shows the results of these analyzes on viral replication. it provides an essential overview of the antiviral action of phytochemicals that can be studied in bioinformatics and molecular modeling for the design of new anti-sars-cov-2 therapies. in an attempt to mitigate the symptoms of covid-19, accelerate recovery, and reduce the mortality rate, several known drugs are being tested against sars-cov-2, aiming at an antiviral action, the development of effective treatments, and prevention of infections by opportunistic microorganisms. the interaction of these drugs with proteins and viral receptors can be clarified through bioinformatics, which can identify the target and, with the support of molecular modeling, test the structural overlap, chemical interactions, and molecular coupling. studies like these are fundamental to elucidate the antiviral action mechanism of the compounds, favoring the projection of molecular modifications that modulate the affinity and specificity of the drug and, consequently, its aspects of pharmacological potency and safety. proposals for the repurposing of drugs known are a more reliable option for the development of an efficient and developed therapy with the urgency that the moment requires, to reduce the number of deaths [143] . table 2 presents studies on the effects of using drugs already known, and therapies with combinations of drugs, and the results of these research on the clinical condition of that infected and viral replication. these studies serve as a basis for more accurate analyzes of the drug's mechanism of action and the test outcome. in the context of the covid-19 pandemic, some studies and clinical reports describe the clinical worsening of some patients. the condition became known as cytokine storm or hypercytokinemia and is associated with increased mortality in infected patients. among the proposed pharmacological strategies, is the use of anti-cytokine molecules, such as anti-interleukin drugs and inhibitors of ifn-γ and tnf-α (see figure 4) , capable of reducing the inflammatory process. the elucidation of the mechanisms associated with the action of immunomodulatory drugs on hypercytokinaemia is essential for the knowledge of safety regarding the use of these drugs, the best pharmacotherapeutic management, and the effectiveness to reduce the risk of death. bioinformatics, when applied to the analysis of inflammatory mediators, is a tool capable of evaluating the virus's performance and its ability to trigger an intense inflammatory response and how anti-cytokine molecules can normalize this condition. in this sense, bioinformatics aims to plan therapies that prevent the disease from worsening without causing immunodeficiency [144, 145] . table 3 shows the research results that used anti-cytokine compounds on respiratory function and inflammatory indexes in patients with covid-19. the table reveals the clinical outcomes of the use of these substances, which can be investigated with the aid of bioinformatics and molecular modeling as a potential therapy against hypercytokinaemia resulting from sars-cov-2 infection, significantly reducing patient mortality. humans 500 mg of aztm once daily on day 1, followed by 250 mg once daily for the next 4 days no benefit was seen [160] 8 mg/kg intravenously 6 days improvement in respiratory and laboratory parameters [177] 8 mg/kg once daily 2 days the treatment associated with hemoadsorption, improved gas exchange and reduced levels of inflammatory mediators [178] sarilumab humans 400 mg intravenously 10 days treatment was associated with faster recovery [179] 5 days reduction of inflammation and rapid recovery [180] 200 mg _ clinical improvement and lower mortality [174] [185] it is crucial to mention that several of the drugs mentioned in this review have already received temporary regulatory approval. for example, remdesivir, hydroxychloroquine, chloroquine, and azithromycin are being used with consent in treatment protocols, depending on the country. the combination of these substances has also been used. despite this, studies on its effectiveness in the different stages of sars-cov-2 infection are controversial, and additional research is needed to elucidate the efficacy of these substances in infected patients. the use of molecular modeling at the early stages of searching for new bioactive compounds contributes to the faster development of new drugs at significantly reduced costs. given the low number of compounds with known anti-sars-cov-2 activity and considering the availability of structural models of potential molecular targets of the virus, the strategy based on the structure of the molecular target has been further explored, in which molecular docking techniques play a crucial role in this planning method. the compound pubchem cid 444,745 showed the highest potential to inhibit sars-cov-2 proteases during studies in search of possible molecular targets against the virus. about phytochemicals, the virtual libraries that store information about these compounds have already been extensively explored. among the selected compounds, pubchem cid 11,610,052 and quercetin showed a strong interaction with 3clpro from sars-cov-2. also, dihydrotansinone i interact strongly with spike glycoprotein and cryptotanshinone, with proteins plpro and 3clpro. marine compounds were also evaluated in silico against the main sars-cov-2 protease. the results identified different compounds as potential inhibitors of this protease, highlighting heptafuhalol a as the most promising. another virtual screening strategy that stands out among the research is the one that uses groups of drugs with antiviral action already approved for use in humans, characterizing a drug repositioning strategy. it was shown that digitoxin and zorubicin have a high potential for interaction with the sars-cov-2 spike glycoprotein. another highlight was the potential for ribavirin to interact with the plpro, lymecycline against 3clpro, and valganciclovir against rna-dependent rna polymerase, essential proteins for the survival of sars-cov-2. besides, the present review showed that the pemirolast is promising as an inhibitor of the action of spikes glycoproteins. in addition to issues related to bioinformatics and molecular modeling, the details provided in the present review envision future points of consideration in the field of virology and medical sciences that will contribute to understanding the molecular mechanisms responsible for the pathogenesis and virulence of sars-cov-2 well as the development of the human acute severe respiratory syndrome. it is well established that the pathophysiology and lung injury caused by sars-cov-2 is related to innate immunity and factors such as the release of pro-inflammatory cytokines and the complement system action. besides, we strongly emphasize that: (1) the complement system is an essential element of protective immunity against pathogens, but its excessive or unregulated activation can result in critical tissue damage and; (2) the viral s2 subunit can be an important target for future antiviral compounds. thus, anti-s2 antiviral compounds may be potential treatments for sars-cov-2 infection. during this unprecedented period, we encourage scientists to actively contribute to understanding the role of the complement system in the development of covid-19. in addition to what was previously mentioned, all authors made due contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; or have drafted the work or substantively revised it; has approved the submitted version and; agrees to be personally accountable for the author's own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and documented in the literature. all authors have read and agreed to the published version of the manuscript. funding: this research 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in severe covid-19: a case report favorable anakinra responses in severe covid-19 patients with secondary hemophagocytic lymphohistiocytosis interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study pediatric crohn disease and multisystem inflammatory syndrome in children (mis-c) and covid-19 treated with infliximab eculizumab treatment in patients with covid-19: preliminary results from real life asl napoli 2 nord experience this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we especially thank the research groups: research group on development of pharmaceutical products (p&dprofar); research group on biomolecules and catalyze; research group on biodiversity and health (biosa). the map for figure 2 was "designed by freepik". we appreciate the opportunity to enrich our work with your art. we are grateful for the participation of everyone who contributed to the writing and revision of this manuscript. the authors declare no conflict of interest. key: cord-281684-m3m4mhye authors: fagre, anna c.; manhard, john; adams, rachel; eckley, miles; zhan, shijun; lewis, juliette; rocha, savannah m.; woods, catherine; kuo, karina; liao, wuxiang; li, lin; corper, adam; challa, dilip; mount, emily; tumanut, christine; tjalkens, ronald b.; aboelleil, tawfik; fan, xiaomin; schountz, tony title: a potent sars-cov-2 neutralizing human monoclonal antibody that reduces viral burden and disease severity in syrian hamsters date: 2020-09-28 journal: biorxiv doi: 10.1101/2020.09.25.313601 sha: doc_id: 281684 cord_uid: m3m4mhye the emergence of covid-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of covid-19 are needed. we identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the sars-cov-2 spike protein receptor binding domain that neutralized the virus in vitro. administration of the lead antibody clone to syrian hamsters challenged with sars-cov-2 significantly reduced viral load and histopathology score in the lungs. moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. the use of this antibody could provide an important therapy for treatment of covid-19 patients. the emergence of a novel coronavirus disease , caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first described in december 2019 in wuhan, china 1 . symptoms include cough, dyspnea, chest tightness and fever, plus occasional adverse gastrointestinal (gi) disturbances. in a vulnerable subset of patients, the disease often progresses to an atypical pneumonia with high morbidity and mortality rates [2] [3] [4] [5] [6] . mortality based on case fatality rates have ranged from early estimates of 3.9% in hubei province, china, to 0.9% in populations from countries with widespread testing. recent estimates of infection fatality rates (ifrs) are between 0.6% to 1% and overall indicate that covid-19 has a 10-fold greater mortality than seasonal influenza [6] [7] [8] [9] [10] . alarmingly, the ifr is considerably higher (5.6%) in individuals ≥65 years of age where the prognosis for those hospitalized patients ranges from guarded to poor; they frequently require long periods of breathing support, with mortality rates of 20-30% 6, 11 . consequently, the effect on healthcare resources is of major concern and there is an increasingly unmet medical need for effective therapies, particularly for older patients with comorbidities, including atherosclerosis, hypertension and diabetes mellitus, to prevent lethal pneumonia. cellular infection by coronaviruses is mediated by the viral homotrimeric spike glycoprotein binding to specific host cell receptors. each protomer consists of an s1 domain, which mediates receptor binding, and an s2 domain that mediates membrane fusion and cell infection following conformational changes induced by host cell receptor binding [12] [13] [14] . the receptor for sars-cov, sars-cov-2 and hcov-nl63 is angiotensin converting enzyme 2 (ace2), which is expressed on mucosal epithelia of the upper respiratory tract, bronchioles, lungs and the gi tract [15] [16] [17] [18] [19] . convalescent serum and purified antibodies from recovering patients have yielded promising results in past viral outbreaks 20-26 , and this approach may also be promising for sars-cov-2. one key challenge in the development of these antibodies is how to prevent viral escape whereby variant mutations nullify antibody binding to the rbd epitope. multiple variants of sars-cov-2 have emerged, some of which have mutations in the rbd of the s protein and are predicted to have increased binding to ace2 [27] [28] [29] [30] . the best neutralizing antibodies are likely to recognize epitopes with high sequence conservation due to structural or functional constraints. the rbm binding site to ace2 is such an example because sequence divergence within this region is constrained to the prerequisite that binding to ace2 must be maintained. an analysis of the crystal structures of the ace2 -rbd sars-cov complex 31 and the ace2-sars-cov-2 rbd complex 29, 32 confirms that eight of the key rbm contact residues are conserved between these respiratory coronaviruses, although the sars-cov-2 rbm forms a more compact interface with ace2 than that formed by sars-cov rbd and has a higher affinity for ace2 29, 32 . many of the previously identified neutralizing antibody clones against sars-cov were shown to block s1 binding to its ace2 receptor by binding to epitopes located within the rbm [21] [22] [23] [24] 33, 34 . however, not all monoclonal antibodies isolated previously against the sars-cov rbd recognize the sars-cov-2 spike protein in the context of the full-length spike homotrimer 35, 36 . similarly, recent studies evaluating anti-sars-cov-2 antibodies isolated from convalescent patients indicate only a minor subset of the clones have the requisite viral neutralizing activity 35, 36, 46 . together, these studies indicate that there is an urgent need for sars-cov-2-specific human neutralizing antibody therapeutics that have proven ability to both neutralize sars-cov-2 viral host cell infection in vitro as well as the appropriate activity in vivo. the syrian hamster has been shown to be a suitable model for sars-cov-2 infection given the similarity of clinical signs and lung pathology to human disease in the first few days of infection [37] [38] [39] . however, to date, there has been only a gross histological analysis of the lung pathological changes following infection and the impact of sars-cov-2 neutralizing antibody clones on lung immune infiltrates has yet to be fully assessed. in this study, we describe an anti-sars-cov-2 spike rbd clone isolated from a rationally designed, fully human antibody library that bound to native spike protein. this potent antibody could block the interaction of the spike protein with ace2 and could also block sars-cov-2 infection of vero e6 cells and the resultant cytopathic effect. this clone was then tested in a pilot therapeutic study in syrian golden hamsters (mesocricetus auratus) and was shown to reduce viral load and ameliorated the severity of bronchointerstitial pneumonia. detailed histological and image analysis suggests that macrophages play a key role in the lung response to sars-cov-2 infection in hamsters. production and purification of antibody clones. the dna inserts encoding the light and heavy chains of clone avgn-b and its variants were separately cloned into different expression vectors carrying the constant regions of human igg1 heavy chain and the kappa chain. the heavy and light chain plasmids were co-transfected into expi293 suspension cells. after 5-7 days, secreted antibody was then purified from the culture supernatants by protein a chromatography, bufferexchanged into pbs ph 7.4 and its concentration determined by nanodrop a280 assay. the quality of the igg was assessed by sds-page and by hplc. endotoxin levels were tested using a limulus amoebocyte lysate (lal) assay (thermofisher scientific, cat # a39552). variants. the wells of immulon 2 hb elisa 96-well plates were coated with avgn-b or its variant iggs at 4° c overnight. the wells were blocked, washed, then serial dilutions of the monomeric biotinylated rbd-antigen added to the wells and incubated for 1 h at room temperature. bound antigen was then detected with hrp-labeled streptavidin. the od readings were plotted against concentration using prism software (graphpad ca) for curve fitting and determination of the apparent kd value. the ic50 values for inhibition of rbd binding to the ace2 receptor were determined by pre-mixing a serial dilution of antibody with 2 nm of biotinylated sars-cov-2-rbd and incubating for 30 min before adding to wells coated with recombinant ace2-fc fusion protein (residue gln18-ser760 fused to human fc [kactus biosystems, cat# ace-hm401]). after 1 h, bound rbd was detected using hrp-labeled streptavidin and the od readings plotted against antibody concentration using prism software. real time kinetics were measured by bilayer interferometry (bli) at a temperature of 30° c using vero e6 cells were plated overnight in 96-well plates at 20,000 cells per well. antibodies were diluted in complete dmem and serially diluted 1:3 resulting in a 12-point dose response dilution series run in 4 or 8 replicates. the dilutions of antibody were incubated with 100 tcid50 per 50 µl of sars-cov-2 (strain 2019-ncov/usa-wa1/2020) for 1 h and added to the assay plates. the plates were incubated for 3 days at 37° c, 5% co2 and 95% relative humidity and the inhibitory effects of the antibodies were assessed. approval of the study protocol was obtained from the colorado state university institutional animal care and use committee (protocol 993). male syrian hamsters (n=20, 10 weeks of age, obtained from charles river laboratory). the animals were held in the csu animal facility and provided access to standard pelleted feed and water ad libitum prior to being moved into the biosafety level 3 facility for experimental challenge. of the 20 hamsters, 18 were intranasally infected with 2.5x10 4 tcid50/ml equivalents of sars-cov-2 (strain 2019-ncov/usa-wa1/2020) and divided into treatment groups as follows: "avgn-b high" (2.5 mg avgn-b) (n=5), "avgn-b low" (1 mg avgn-b) (n=5), "untreated" (no antibody) (n=6), and "ab control" (2.5 mg isotype control igg) (n=2). two uninfected hamsters received 2.5 mg avgn-b (termed "uninfected"). each animal was dosed intraperitoneally with corresponding treatment at 24 and 72 hours post dosing (hpi). at 24, 48, 72, 96, and 120 hpi, each hamster was weighed and assessed for presence of clinical signs (lethargy, ruffled fur, hunched back posture, nasolacrimal discharge, and rapid breathing). at 120 hpi (5 dpi), each hamster was anesthetized with isoflurane and then euthanized via cardiac exsanguination and blood was collected. weight loss (calculated as percentage decrease from 0 dpi weight) and viral rna load in lung were compared between treatment groups in prism using multiple t-tests and mann-whitney u tests, respectively. p<0.05 was considered significant. swabs in viral transport medium were vortexed thoroughly and centrifuged to pellet cellular debris. lungs, tracheobronchial and hilar lymph nodes, thymus, esophagus, heart and liver from 20 hamsters labeled with ear tags 26-45 were extirpated en bloc and fixed whole in 10% neutral buffered formalin for at least 3 days to ensure virus inactivation prior to transfer to the csu veterinary diagnostic laboratory for trimming. four transverse whole-lung sections were stained with h&e or processed for ihc. sections, 5 µm thick, were subjected to heat-induced epitope retrieval performed online on a leica bond-iii ihc automated stainer using bond-epitope retrieval solution. antibodies to sars-cov-2 nucleocapsid protein (mouse, 1:500), pancytokeratin, factor-viii and ionized calcium binding adaptor molecule (iba-1) (leica biosystems) or negative control slides primary antibody was replaced by a rabbit non-specific igg isotype negative control antibody for 20 minutes. labeling was performed on an automated staining platform. fast red was used a chromogen and slides were counterstained with hematoxylin. immunoreactions were visualized and blindly scored by a single pathologist. in all treated categories, reactive lung sections incubated with primary antibodies were used as positive immunohistochemical control. negative control sections were incubated in diluent composed of tris-buffered saline with a carrier protein and homologous nonimmune serum. all sequential steps of the immunostaining procedure were performed on negative controls following incubation. paraffin embedded tissue sections were stained for sars-cov-2 nucleocapsid protein (1:500) and ionized calcium binding adaptor molecule 1 (abcam; ab5076; 1:50) using a leica bond rx m automated staining instrument following permeabilization using 0.01% triton x diluted in tristable 1 . the precise digital quantification of the total affected pulmonary parenchyma as well as counting of inflammatory cells per area (roi, 1mm 2 ) in histological section was determined. a digital montage was compiled at 100´ magnification to include the four tissue classes that were differentially characterized (table 1) to establish algorithm classifier. affected rois were subsequently automatically identified using olympus cellsens software by quantifying whole-lung mounts scanned from each hamster for total number of nuclei or nucleated cells (to exclude erythrocytes) stained with hematoxylin. sections labeled by immunofluorescence for iba-1 and sars-cov-2 were analyzed using the count and measure module of cellsens. the algorithm predominantly extracted multispectral information from images with additional dia processing using spatial, logical and threshold separators after manual annotations. all four classes were accurately identified as expected by a board-certified pathologist who was blinded to experimental groups of hamsters. a rationally designed fully human antibody library displayed on yeast was screened by magnetic individual antibody clones were tested for their abilities to block sars-cov-2-rbd binding to ace2 using a competition elisa and tested for their ability to bind to native sars-cov-2 spike protein expressed as a gfp fusion protein by transfected 293 cells compared to binding activity to non-transfected 293 cell controls. those antibody clones that blocked the interaction of the rbd with ace2 and bound to native spike protein were then tested for neutralization of sars-cov-2 in a cytopathic effect (cpe) assay with vero e6 cells. clone avgn-b was identified as the most potent in these assays. it exhibited an apparent affinity by elisa for sars-cov-2 rbd of 0.17 nm (fig. 1a) , an apparent ic50 value for blocking rbd binding to ace2-fc of 2.2 nm (fig. 1b) , and an apparent kd for binding to native spike protein expressed by 293 cells of 1.02 nm compared to 10.9 nm for ace2-fc (fig. 1d) . real time binding kinetics of avgn-b to the isolated rbd recombinant protein measured by bli indicated a kd of 0.37 nm (fig. 1c) . when tested for its ability to neutralize sars-cov-2 infectivity, clone avgn-b exhibited 100% protection from sars-cov-2 induced cell death down to 0.017 µg/ml. using a colorimetric assay for quantitation of cell death, avgn-b exhibited an ic50 value of that ranged from 0.008 µg/ml (experiment with 4 replicates) to 0.0054 µg/ml (experiment with 8 replicates) in the cpe assay (fig. 1e) . at 2 dpi, infected hamsters appeared quiet and began to progressively lose weight over the course of the study (up to ~13% reduction in untreated animals). there was no significant reduction in weight loss associated with avgn-b treatment (p>0.05) ( fig. 2a) . none of the hamsters in the study died or met euthanasia criteria prior to study termination at 5 dpi. there was a significant reduction in viral rna in the lungs of hamsters treated with avgn-b (both 2.5 mg and 1 mg doses) compared to those that were untreated (p = 0.0173 and 0.0303, respectively) (fig. 2b ). lungs from the 2 uninfected and the 2 ab control hamsters (treated with 2.5 mg control isotype lungs were examined for the extent of macrophage infiltration and sars-cov-2 viral load at 5 days post infection by histopathological examination and by immunofluorescence imaging (fig. 5 ). whole mount sections of paraffin-embedded lung tissue were stained with hematoxylin and eosin and brightfield grayscale images were collected using a microscope equipped with a scanning motorized stage (fig. 5a-e) . hematoxylin-positive macrophage soma were rendered as focal points within the regions of interest to calculate the percent hypercellularity of tissue following infection with sars-cov-2. by 5 dpi, lung tissue showed extensive infiltration of macrophages (fig. 5a ) that was decreased in dose-dependent fashion by treatment with avgn-b (fig. 5b, 5c) . treatment of uninfected hamsters with avgn-b alone did not increase macrophage infiltration, whereas co-treatment with igg control ab during infection with sars-cov-2 (ab control group) still resulted in marked infiltration of macrophages (fig. 5e ). the percent of total lung area displaying macrophage hypercellularity was quantified in fig. 5n , and consequently, in parallel to the in vivo studies described above, antibody engineering of clone avgn-b has been performed and a panel of more potent variants has now been isolated. as shown in table 2 future studies will explore the efficacy of the avgn-b and/or its variants in non-human primate models, several of which have been described for use in sars-cov-2 pathogenesis and countermeasure development studies 40, 60, 61 . larger studies will also be conducted to examine whether avgn-b and/or its variants by reducing viral load and accumulation of macrophages within the lung will prevent downstream inflammatory and coagulation sequalae of sars-cov-2 infection within other parenchymatous organs, particularly heart, kidneys and liver. should avgn-b and/or its variants advance to clinical trials in human patients, its effect on kawasaki-like disease (kd) in sars-cov-2 infected children merits clinical investigation. there is accumulating evidence that the monocyte/macrophage system releases cytokines that directly lead to vascular endothelial damage during acute kd 62, 63 . investigating the role of avgn-b and/or its variants in suppressing the cytokine storm by suppressing a pivotal player, monocyte-macrophage system will be important. affected lung area "blue" and "red" for cellular density intra-bronchiolar, intra-alveolar, peribronchiolar and perivascular inflammation or alveolar septal thickening, edema and hemorrhage 2 unaffected lung tissue "light blue" normal lung parenchyma 3 background "grey" intravascular erythrocytes, blood vessel walls, bronchiolar mucosa 4 glass "white" glass a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical characteristics of coronavirus disease 2019 in china risk factors of fatal outcome in hospitalized subjects with coronavirus disease 2019 from a nationwide analysis in china clinical features of patients infected with 2019 novel coronavirus in wuhan clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical, laboratory and 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cells cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody human neutralizing antibodies elicited by sars-cov-2 infection a human monoclonal antibody blocking sars-cov-2 infection a human neutralizing antibody targets the receptor binding site of sars-cov-2 isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model a sars-cov-2 infection model in mice demonstrates protection by neutralizing antibodies massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by sars-cov-2 in golden syrian hamsters identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury sars-cov-2 and myocardial injury: a role for nox2? prospects for the use of regulators of oxidative stress in the comprehensive treatment of the novel coronavirus disease 2019 (covid-19) and its complications is macrophages heterogeneity important in determining covid-19 lethality? trained immunity: a program of innate immune memory in health and disease monocyte and macrophage immunometabolism in atherosclerosis covid-19: immunopathology and its implications for therapy role of oxidized ldl-induced "trained macrophages" in the pathogenesis of covid-19 and benefits of pioglitazone: a hypothesis coagulopathy and antiphospholipid antibodies in patients with covid-19 metabolic modulation of inflammation-induced activation of coagulation infection with novel coronavirus (sars-cov-2) causes pneumonia in rhesus macaques chadox1 ncov-19 vaccine prevents sars-cov-2 pneumonia in rhesus macaques peripheral blood monocyte/macrophages and serum tumor necrosis factor in kawasaki disease nf-κb activation in peripheral blood monocytes/macrophages and t cells during acute kawasaki disease key: cord-294527-fct2y5vn authors: guadarrama-ortiz, parménides; choreño-parra, josé alberto; sánchez-martínez, claudia marisol; pacheco-sánchez, francisco javier; rodríguez-nava, alberto iván; garcía-quintero, gabriela title: neurological aspects of sars-cov-2 infection: mechanisms and manifestations date: 2020-09-04 journal: front neurol doi: 10.3389/fneur.2020.01039 sha: doc_id: 294527 cord_uid: fct2y5vn the human infection of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a public health emergency of international concern that has caused more than 16.8 million new cases and 662,000 deaths as of july 30, 2020. although coronavirus disease 2019 (covid-19), which is associated with this virus, mainly affects the lungs, recent evidence from clinical and pathological studies indicates that this pathogen has a broad infective ability to spread to extrapulmonary tissues, causing multiorgan failure in severely ill patients. in this regard, there is increasing preoccupation with the neuroinvasive potential of sars-cov-2 due to the observation of neurological manifestations in covid-19 patients. this concern is also supported by the neurotropism previously documented in other human coronaviruses, including the 2002–2003 sars-cov-1 outbreak. hence, in the current review article, we aimed to summarize the spectrum of neurological findings associated with covid-19, which include signs of peripheral neuropathy, myopathy, olfactory dysfunction, meningoencephalitis, guillain-barré syndrome, and neuropsychiatric disorders. furthermore, we analyze the mechanisms underlying such neurological sequela and discuss possible therapeutics for patients with neurological findings associated with covid-19. finally, we describe the hostand pathogen-specific factors that determine the tissue tropism of sars-cov-2 and possible routes employed by the virus to invade the nervous system from a pathophysiological and molecular perspective. in this manner, the current manuscript contributes to increasing the current understanding of the neurological aspects of covid-19 and the impact of the current pandemic on the neurology field. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) originated in wuhan, china (1), in december 2019, and it has engulfed the world in an unprecedented global pandemic. the coronavirus disease 2019 (covid-19) associated with this virus has caused more than 16.8 million new cases and 662,000 deaths as of july 30, 2020 (2) , generating a high burden of disease that has exceeded the assistance capacities of several healthcare systems around the world. the clinical characteristics of patients with covid-19 are similar to those observed during the outbreak of sars-cov-1, which emerged in 2002-2003, causing more than 8,000 confirmed cases and ∼800 deaths (3) . as such, the human infection with sars-cov-2 mainly affects the lower respiratory tract, causing mild to moderate respiratory symptoms in about 85% of patients with covid-19 (4) (5) (6) , including fever, headache, fatigue, myalgia, dry cough, and diarrhea. most symptomatic individuals are men in their fifth and sixth decades of life that attend medical centers after an incubation period of about 4 to 5 days (4) (5) (6) (7) . another 15% of patients present moderately severe forms of the disease manifested as pneumonia of atypical features in radiological studies of the lung, such as bilateral multi-lobe consolidations and ground-glass opacities (8) . finally, in 5 to 30% of covid-19 patients, the virus causes severe acute respiratory syndrome (sars), which is characterized by profound respiratory distress that obligates the establishment of intensive life support interventions, such as intubation and mechanical ventilation (4) (5) (6) . initial reports estimated mortality rates of about 2 to 4%. however, new analyses indicate a higher lethality of covid-19 (9) , especially among individuals older than 65 years, and patients with comorbidities (4) (5) (6) . unfortunately, the risk of a fatal outcome is disproportionally higher among patients requiring mechanical ventilation, with a mortality rate close to 80% (7) . the rapid transmission of sars-cov-2 and the increasing number of positive cases reported around the world have overcome the resources of the healthcare systems in different regions. this has significantly impacted all areas of medicine, especially those directly related to the management of severe respiratory infections, such as pneumology and critical care medicine. nonetheless, sars-cov-2 has the potential to spread to different extrapulmonary tissues, and, in some of the most severe cases, the infection can progress to multiorgan failure (5) . therefore, all healthcare providers from any area of medicine must acquire adequate knowledge of the principal characteristics of covid-19. currently, there is increasing preoccupation about the potential capacity of sars-cov-2 to invade the central nervous system (cns) and the peripheral nervous system (pns). these concerns are based on recent observations in individuals infected with sars-cov-2 that present neurological findings (10) . a better understanding of the mechanisms underlying the neurologic sequela of patients with covid-19 is urgent to discover novel targets for therapeutics development. in the current review article, we therefore provide an overview of the spectrum of neurological manifestations of covid-19. additionally, we analyze host-and pathogen-specific factors that determine the tissue tropism of sars-cov-2 and discuss the possible routes employed by the virus to invade the nervous system. furthermore, we propose possible therapeutics for the neurologic complications of covid-19. virology sars-cov-2 is a novel member of the group of human coronaviruses (hcovs), which is constituted of hcov-229e, hcov-nl63, hcov-hku1, hcov-oc43, sars-cov, and mers-cov (11) . these are rna single-stranded viruses belonging to the coronaviridae family. some of these pathogens have caused a variety of respiratory diseases in the past. for instance, sars-cov-1 infected more than 8,000 individuals around the world (3) . also, the human coronavirus related to the respiratory syndrome of the middle east (mers-cov), which emerged in saudi arabia in 2012 and caused high mortality rates among infected people (12, 13) . based on sequence comparisons of viral genomes, hcovs are grouped into four genera: alpha, beta, gamma, and delta coronaviruses. sars-cov-2 is a beta coronavirus genetically related to another bat coronavirus named batcov ratg13 as well as sars-cov-1 (14, 15) . furthermore, sars-cov-2 also shares its genetic identity with coronaviruses isolated from pangolins (16, 17) . hence, it is believed that covid-19 is a zoonotic disease that originated from bats or pangolins. the genome of sars-cov-2 consists of a single rna strand of 29.903 bp that codifies for the replicase-transcriptase, as well as for the structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n) (15) . the initial step of sars-cov-2 infection is the recognition of its receptors on the surface of host cells. this step is mediated by the viral spike (s) protein, which recognizes the human receptor angiotensin i-converting enzyme 2 (ace2), the same receptor for the s protein of sars-cov-1 (18) (19) (20) . this protein owns two functional domains: the s1 domain contains the receptorbinding domain (rbd), which attaches to ace2, whereas the s2 domain mediates the fusion of the viral and host cell membranes (20) . therefore, the organ distribution of the ace2 receptor is a crucial determinant of the virus infectivity and tropism. a second step determinant in the infection process of sars-cov-2 is the activation of the s protein. this process is mediated by different host proteases, which execute the cleavage of the molecule at the s1/s2 and s'2 sites. this protein processing allows the complete activity of the s2 domain and the fusion of the viral and cellular membranes. for this purpose, and as in the case of sars-cov-1, sars-cov-2 uses the transmembrane serine protease 2 (tmprss2) (19, 21, 22) . interestingly, the proteases tmprss4 and cathepsin l also promote sars-cov-2 infection of human small intestinal enterocytes and 293/hace2 cells (23, 24) . hence, the tissue patterns of expression of tmprss2, tmprss4, and cathepsin l is another decisive factor that determines the tropism of the virus, and, indeed, some drugs that inhibit the activity of these proteases are now proposed as potential therapeutic agents to prevent and treat covid-19 (19, 23) . other factors implicated in the process of sars-cov-2 infection include the phosphatidylinositol 3-phosphate 5kinase (pikfyve) (23) . this enzyme mediates the production of phosphatidylinositol-3,5-bisphosphate [pi (3, 5) p2], a phosphoinositide that participates in the maturation process of endosomes. treatment with apilimod, a potent inhibitor for pikfyve, reduced the infectivity of sars-cov-2 and could be a novel candidate for therapeutic applications. after the entry into host cells, viral replication begins with the translation of the replicase-polymerase gene and the assembly of the replication-transcription complex. this complex subsequently transcribes the genomic regions that codify for structural proteins. new virions are assembled in the endoplasmic reticulum and golgi apparatus to finally egress from the cell (11) . a particular feature of sars-cov-2 is that it possesses a polybasic furin cleavage sequence (prra) in the s1/s2 site, which is absent in other close related coronaviruses (14, 20) . this inserted furin cleavage sequence is processed at the golgi apparatus during the biosynthesis of the s protein of novel virions inside the host infected cells (20) . the novel virions of sars-cov-2 may thus contain an s protein primed and ready to infect any other cells expressing the ace2 receptor, with no further requirement of tmprss2 activity. as virtually all cells express furin under normal conditions, the inserted furin cleavage sequence may expand the transmissibility and tissue tropism of sars-cov-2. figure 1 illustrates the process of sars-cov-2 infection. the immune response against sars-cov-2 sars-cov-2 elicits an exuberant immune response characterized by a dysregulated production of soluble immune mediators. this phenomenon has been called a "cytokine storm" and is responsible for mediating tissue damage in patients with covid-19 that progress to severe illness (25) (26) (27) (28) . the immune receptors that recognize the viral infection and initiate the immune responses against sars-cov-2 are unknown. as this virus is genetically related to sars-cov-1, it is presumed that both viruses share mechanisms of infection. in this sense, sars-cov-1 is recognized by the toll-like receptors (tlr) tlr3 and tlr4, which induce an immune reaction via myd88 and trif pathways (29, 30) . furthermore, sars-cov-1 triggers the production of il-1β through the activation of the inflammasome (31) . in this regard, the activation of the inflammasome is also possible to occur during sars-cov-2 infection, as high levels of il-1β have been observed in covid-19 patients (32) . other immune mediators that are exaggeratedly produced in response to sars-cov-2 include il2, il-6, il7, il10, g-scf, cxcl10, mcp-1, mip-1a, and tnfα (5, 28, 33) . from these, il-1β, il-6, cxcl10, and tnfα are the cytokines with a higher capacity to generate tissue damage in several organs, including the cns, due to their pro-inflammatory properties. for instance, il-1β and il-6 have been implicated in neurotoxicity associated with chimeric antigen receptor (car) t cell therapy in patients with hematological malignancies (34, 35) . these cytokines possess detrimental effects on endothelial function at several vascular niches, which may be implicated in the pathophysiology of the neurological complications of covid-19, as discussed later. notably, despite the dysregulated production of immune mediators, an ample range of immune cell subtypes are depleted from the circulation of patients with severe sars-cov-2 infection. these cells include monocytes, dendritic cells, cd4+ and cd8+ t cells, and nk cells (36) . furthermore, the few adaptive lymphocytes that remain in the blood express markers of functional exhaustion (37) . these data suggest that severe covid-19 is a state of immunosuppression similar to the known sepsis-induced immunosuppression (38) . however, it is also possible that the robust recruitment of functional immune cells to the sites of sars-cov-2 infection may explain the leukopenia observed during covid-19. several hcovs, including sars-cov-1, have the potential to infect different human tissues and organs (39, 40) . similarly, sars-cov-2 may also spread to several extrapulmonary tissues, including the cns. although recent analyses of autopsy specimens from patients with covid-19 have not explored the presence of this virus in the brain (41), the neurological manifestations observed among individuals with covid-19 (10) and the isolation of other human coronaviruses from neurological specimens support the notion of a possible neurotropism of sars-cov-2 (42) (43) (44) (45) (46) (47) . the neurotropism of other hcovs has been extensively revised elsewhere (48) . as mentioned before, the patterns of expression of ace2, tmprss2, tmprss4, furin, cathepsin l, and other entry factors used by sars-cov-2 for infection determine the tropism of the virus. the ace2 receptor is highly expressed in cells of the alveolar epithelium (49, 50) , which explains the vulnerability of the lungs to infection. the expression of ace2 has also been found in other tissues, including the oral mucosa, endothelium, heart, kidney, lymphoid organs, testis, gut, and urinary tract (49) (50) (51) (52) . nonetheless, distribution of tmprss2 has been assessed in a limited variety of human tissues, showing high expression in prostate cells, respiratory epithelial cells, salivary gland, kidney, liver, stomach, small intestine, and colon (53) (54) (55) . tmprss2 is regulated by androgens (53) , which may explain the higher susceptibility of men to suffer from severe forms of covid-19. few studies have analyzed the expression of tmprss4 and cathepsin l in healthy human organs. according to the human protein atlas (https://www.proteinatlas.org/) dataset, tmprss4 is present in the cerebral cortex, hippocampus, caudate, thyroid gland, adrenal gland, nasopharynx, bronchi, lung, stomach, duodenum, colon, rectum, gallbladder, pancreas, and genitourinary tract. meanwhile, cathepsin l shows medium expression in lung and liver, and low expression at bronchi, salivary gland, liver, kidney, pancreas, and genitourinary tract. the expression in the human body of the known genes mediating the entry of sars-cov-2 into human cells coincides with the multiorgan pattern of covid-19 manifestations. nonetheless, the presence of such factors is low in the cns under normal conditions. this may be in part because previous studies have only analyzed the bulk organ gene expression patterns of ace2 and tmprss2. more recently, a comprehensive analysis of several single-cell rna-seq databases showed that there are dual-positive ace2+tmprss2+ cells in tissues beyond the respiratory system, including oligodendrocytes in the brain, and inhibitory enteric neurons (56) . furthermore, ace2+ctsl+ cells were enriched in the olfactory epithelium. these data support some of the possible routes of sars-cov-2 entry into the cns discussed below. the mechanisms employed by sars-cov-2 to infect the nervous system are unknown. currently, the hypotheses about the routes of viral entry into the cns rely on previous observations made in experimental studies of sars-cov-1 infection. one hypothesis proposes that sars-cov-2 invades the brain by breaching the blood-brain barrier (bbb). evidence in favor of this infection mechanism includes the high expression of the ace2 receptor in endothelial cells of blood vessels (49, 50) . the virus may therefore infect endothelial cells of the brain vasculature in the first term, and it may then spread to the surrounding ace2+tmprss2+ oligodendrocytes and, finally, to the neurons. this would explain why sars-cov-1 has been observed inside neurons even when they have a mild expression of ace2 under normal conditions (39, 40, 57) . despite this, sars-cov-1 has not been isolated from or observed inside endothelial cells (58) . conversely, the high concentrations of pro-inflammatory cytokines in the systemic circulation of patients with severe forms of covid-19 might induce structural and functional alterations of the bbb (5, 28, 59) . in this sense, it is well-known that different inflammatory mediators have detrimental effects on bbb integrity, increasing its permeability to neurotoxic molecules and immune cells (60) . sars-cov-2 could thus gain access to the cns directly through a paracellular route or within the immune cells, a mechanism that has been called a "trojan horse" (61) . it might be feasible for this mechanism to occur during sars-cov-2 infection since the previous sars-cov-1 has also been observed inside different leukocyte subsets (40) . secondly, the virus could enter the cns through the olfactory epithelium, crossing the cribriform plate of the ethmoid bone and reaching the olfactory bulb from which it could spread to different areas of the brain (62) . this route was demonstrated in mice intranasally inoculated with sars-cov-1, among which a rapid viral spread from the olfactory bulb to the brain stem was observed. this exposure to the virus caused high lethality among infected animals due to the occurrence of neuronal death in the respiratory centers of the brain stem (63) . similar results were observed in another animal model of cns infection with the hcov-oc43 coronavirus (64) . in humans, some studies demonstrated olfactory neuropathy in patients with sars-cov-1 infection (65) . likewise, recent investigations have shown that the olfactory epithelium is enriched with ace2+ctsl+ cells (56) . moreover, hyposmia and anosmia are frequent manifestations of covid-19 (10, 66) , suggesting that sars-cov-2 might also infect the olfactory bulb in covid-19 patients. in light of these findings, some researchers have proposed that the neuroinvasive potential of sars-cov-2 could contribute to the respiratory failure observed in patients with severe covid-19 (67) . finally, as in the case of other respiratory viruses with neurotropic potential, including the influenza a virus (68), sars-cov-2 could gain access to the cns through the vagus nerve. the terminals of this nerve are located along the respiratory and gastrointestinal tracts, sites with high expression of ace2 (49, 50) and enriched with ace2+tmprss2+ enteric neurons (56) . from these organs, the virus could gain access to the brain stem, taking advantage of the polarization of neurons and the machinery responsible for retrograde neuronal communication, or through endocytosis and clathrin-mediated exocytosis, as observed in the case of the transsynaptic transmission of the porcine coronavirus hev 67n (69). the possible cns invasion routes used by sars-cov-2 are illustrated in figure 2 . some of the initial descriptions of the clinical phenotype of patients infected with sars-cov-2 showed that up to 10% of individuals with covid-19 manifested non-specific neurological symptoms such as headache and dizziness (5, 6, 70) . in a more recent report by mao et al., a third of patients with covid-19 presented non-specific neurological manifestations, including dizziness (16.8%), headache (13.1%), loss of consciousness (7.5%), and seizures (0.5%) (10) . two recent systematic reviews and meta-analyses showed that headache and dizziness are among the most frequent neurological symptoms affecting covid-19 patients (71, 72) . interestingly, headache can occur even in the absence of fever and can be manifested as a migraine, tension-type, or cluster headache (73) . these non-specific neurological symptoms may reflect the neurotoxic effect of hypoxemia and the cytokine storm observed in patients with severe covid-19. however, some of these manifestations must be differentiated from delirium, and other secondary causes, such as metabolic, gastrointestinal, renal, and hematological complications, must be ruled out, particularly in aged patients with underlying comorbidities. interestingly, non-specific neurological manifestations are more frequent in patients who progress to respiratory failure, which supports the hypothesis about a possible contribution of the cns infection to the respiratory failure caused by sars-cov-2 (67) . furthermore, patients with unspecific neurological symptoms have shown higher degrees of leukopenia, thrombocytopenia, and elevated blood urea nitrogen levels (bun) (10) . these data suggest that some of these findings might have certain prognostic value to predict the occurrence of more severe neurological complications. however, the predictive potential of non-specific neurological symptoms needs to be further evaluated in prospective studies. this would be of great help for the timely detection of patients at risk of neurologic sequela. acute meningitis and encephalitis are dramatic complications of various viral infections that often result in high morbidity and mortality rates due to their severity. coronaviruses have also been associated with these neurological complications in humans. for instance, the hcov-oc43 coronavirus was isolated from the brain of a patient who died of viral encephalitis (74) . likewise, both sars-cov-1 (46) and mers-cov have been reported to cause encephalitis (75) . in this context, sars-cov-2 can also cause viral meningitis and encephalitis, as demonstrated by a recent report of a 64-yearold patient with laboratory-confirmed covid-19 who presented neurologic manifestations during the infection, including lethargy, clonus, and pyramidal signs in the lower limbs as well as stiff neck and brudzinski sign (76) . the cerebrospinal fluid (csf) study revealed high protein levels and hypoglycorrhachia, although the virus could not be isolated from csf in this case. the patient received antiviral drugs and symptomatic measures, progressing favorably without neurological complications. similarly, in another report, a 24-year-old man with a 3-day history of headache developed fever, loss of consciousness, and seizures (77) . upon hospital admission, he presented signs of meningeal irritation and a low glasgow coma scale (gcs) score, requiring mechanical ventilation. during his medical follow-up, he continued presenting seizures resistant to pharmacological treatment. his laboratory studies revealed high protein levels and pleocytosis in the csf, and the mri showed hyperintensities in the mesial temporal lobe. the patient tested negative for covid-19 when his nasopharyngeal swab specimen was analyzed. nonetheless, the result of the test was positive in the csf sample. this finding was the first demonstration of the presence of sars-cov-2 in the cns. the occurrence of encephalitis as the initial and even only manifestation of covid-19 has latter been reported in three additional cases from china and the united states (78) (79) (80) . finally, another case report showed that sars-cov-2 infection could cause acute necrotizing encephalopathy (81) . this entity is a severe neurological complication resulting from the disruption of the bbb associated with the cytokine storm observed in individuals with a critical illness. together, these cases illustrate the development of severe acute neurological complications in patients with covid-19, confirming the neurotropism of sars-cov-2. these findings justify the intentional search for sars-cov-2 infection in patients attending with acute neurological manifestations and the antecedent of a recent respiratory infection, such as influenza, or influenza-like illness, has been related to acute cardiovascular complications, including acute myocardial infarction and stroke (82) . among infectious causes of vascular events, the infection with the varicella-zoster virus (vsv) is the most frequent viral cause of stroke (83) . the study by mao et al. revealed that 3% of patients with covid-19 also present stroke as their only neurological manifestation of the infection. from these, the majority were affected by ischemic strokes (six ischemic vs. one hemorrhagic from a total of 214 patients included in the study) (10) . stroke in individuals with covid-19 occurs late during the disease and is more frequently observed among patients with severe respiratory failure. interestingly, some covid-19 patients were admitted to medical centers with hemiplegia and no history of respiratory symptoms (10) . other studies have also reported stroke in patients with severe covid-19, even among young individuals (84, 85) . this might indicate that stroke could be a result of vascular alterations directly driven by the virus, although the presence of cardiovascular risk factors can increase the incidence of this complication. a recent meta-analysis found that stroke, apart from being a frequent complication of covid-19, has a considerable prognostic value to predict the risk of mortality. as such, patients with stroke have a 3-fold increase in the risk of death due to covid-19 (86) . the association between stroke and covid-19 might result from the critical condition and the pro-inflammatory state that prevails in most severely ill patients. the increased levels of pro-inflammatory cytokines in these individuals could alter the normal function of the cerebral vessels. these factors, along with the high prevalence of cardiovascular risk factors among patients with covid-19, can trigger cerebrovascular complications. the endothelial infection of the cerebral vasculature due to its high expression of the ace2 receptor might further contribute to these complications (49) . in fact, the incidence of stroke in patients with vsv infection is a consequence of the invasion of the cerebral arteries by the virus (83) . other mechanisms by which sars-cov-2 could cause stroke include coagulation disorders. the abnormalities in the coagulation of severe covid-19 patients have unique characteristics that partially resemble disseminated intravascular coagulation (dic) or thrombotic microangiopathy. these alterations increase the risk of thrombotic complications and death due to covid-19 (87) . acute myocarditis has been reported in patients with sars-cov-2 infection (88, 89) . this cardiac complication could trigger events of brain embolization and stroke, which might explain the incidence of cerebral infarctions in young patients with covid-19 in the absence of cardiovascular risk factors. during the outbreak caused by sars-cov-1, some studies described the occurrence of peripheral motor neuropathy and myopathy among infected individuals, mostly as part of the spectrum of manifestations of the critical illness polyneuropathy and myopathy disorders (90) . similarly, 2.3% of patients with covid-19 have presented neuropathic pain, probably associated with peripheral neuropathy, whereas 10.7% of the cases showed data of skeletal muscle injury with elevated serum levels of creatine phosphokinase (cpk) (10) . as in the case of other neurological manifestations, neuropathic pain and myopathy have been more frequently observed in patients with severe forms of covid-19. notably, these symptoms occur earlier during the disease in individuals with sars-cov-2 infection as compared to patients affected by sars-cov-1 (10) . this suggests that the neuropathy and myopathy of covid-19 are directly associated with the injury of peripheral nerves and striated muscles driven by the virus. covid-19 patients with neuropathy and myopathy, however, exhibit higher levels of neutrophils and acute phase reactants than individuals without neurological manifestations (10) . the nerve and muscle disorders observed during sars-cov-2 infection might thus be associated with a critical illness polyneuropathy of rapid development due to the more deteriorated clinical condition of patients with covid-19 as compared to cases of sars-cov-1 infection. vascular, thrombotic, ischemic, and direct nerve and muscle alterations driven by sars-cov-2 can contribute to neuropathy and myopathy of covid-19 patients. a wide range of infectious diseases can cause complicate within cranial neuropathies like facial palsy and ophthalmoplegia. among the pathogens that cause these manifestations with more frequency are hiv and vsv (91) . cranial nerves might also be susceptible to a direct or indirect injury caused by sars-cov-2. in fact, according to a recent study, about 85% and 88% of patients with covid-19 develop olfactory and gustatory dysfunction (66) . similarly, in another investigation conducted in italy, about 33% of patients with covid-19 reported taste and/or olfactory disorders (92) . these findings might be specific for sars-cov-2 infection and could predict the causative pathogen in patients with acute respiratory illness. indeed, smell and/or taste disorders were more frequently observed among covid-19 patients as compared to individuals with laboratory-confirmed influenza infection in a case-control study conducted in spain (93) . these observations might be of particular relevance for the upcoming flu season, which is predicted to be historically unique due to the convergence of influenza and covid-19. during such an envisioned scenario, the differentiation of these diseases by clinical manifestations could be complicated. nonetheless, the correct identification of the causative pathogen have therapeutic implications, such as the selection of adequate antiviral treatment. the presence of smell and taste dysfunction could thus be useful to distinguish covid-19 from influenza. furthermore, these symptoms can precede the onset of respiratory symptoms (94, 95) , and their presence may predict a milder clinical course of the disease (96) . notably, covid-19-related olfactory dysfunction has not been associated with rhinorrhea or nasal congestion, and more than half of the affected patients did not recover the function of the olfactory nerve (66) . this suggests direct damage to the olfactory epithelium, which further reaffirms the possibility of an entry route of sars-cov-2 through the cribriform plate, reaching the olfactory bulb from where it spreads to other parts of the cns (62). guillain-barré syndrome the association between viral infections and guillain-barré syndrome has been largely described in the past (97) . infection with campylobacter jejuni, cytomegalovirus, or mycoplasma pneumoniae precedes guillain-barré syndrome in up to twothirds of affected individuals (98) . more recently, several viral emerging diseases have shown this syndrome as one of its more common and severe complications, as is the case of the infection with the zika virus (99) . although this association was not described in patients infected with sars-cov-1, cases of guillain-barré syndrome were observed during the outbreak of mers-cov (100) . in this context, the infection with sars-cov-2 could also complicate with or manifests as guillain-barré syndrome. in a recent report, zhao et al. described the case of a woman that developed asymmetric and progressive muscle weakness in the lower limbs, associated with leukopenia and high protein levels in the csf without pleocytosis, accompanied by data of demyelinating neuropathy in studies of nerve conduction (101) . the patient had a history of a recent trip to the city of wuhan, and she showed no evidence of respiratory system involvement at the time of symptom onset. a total of 8days later, the appearance of fever, respiratory symptoms, and radiological data compatible with pneumonia was documented, confirming the sars-cov-2 infection by laboratory testing in a nasopharyngeal swab sample. the patient received intravenous immunoglobulin treatment recovering the neurological function without sequel. similarly, in another case from italy, a 71-yearold male also developed guillain-barré syndrome before the onset of any respiratory symptom (102) . these reports suggest that guillain-barré syndrome may be the first manifestation of covid-19, presenting as a parainfectious phenomenon. this is due to the parallel occurrence of neurological and respiratory symptoms, instead of the postinfectious pattern observed in other infections, including the mers-cov (100). nonetheless, two new case reports of six covid-19 patients demonstrated that guillain-barré syndrome could occur a few days after the onset of respiratory symptoms (103, 104) . collectively, these findings obligate physicians to continuously monitor the neurological condition of patients with suspected or confirmed sars-cov-2 infection to identify any sign of demyelinating neuropathy. furthermore, the occurrence of guillain-barré syndrome associated with covid-19 must be carefully distinguished from the myopathy and neuropathy disorders of the critically ill patient. miller fisher syndrome is a rare neurological disease that is considered a variant of the guillain-barré syndrome. this disorder is characterized by the triad of abnormal muscle coordination, paralysis of the eye muscles, and the absence of tendon reflexes. miller fisher syndrome can be associated with a history of recent viral illness; however, no evidence exists of its association with human coronavirus infections. interestingly, a recent paper has reported the occurrence of the triad of ataxia, areflexia, and ophthalmoplegia in a 50-yearold man that presented cough, fever, anosmia, and hypogeusia some days before the onset of the neurological symptoms. the infection with the sars-cov-2 infection was confirmed in an oropharyngeal swab sample, and blood tests showed lymphopenia, elevated c-reactive protein levels, and anti-gd1b-igg antibodies. he received treatment with intravenous immunoglobulin, which caused significant improvement in his neurological functions. collectively, the literature summarized here indicates that neurological syndromes caused by aberrant immune responses, such as guillain-barré and miller fisher syndromes, can occur as part of the clinical spectrum of sars-cov-2 infection. despite this, the immune mechanisms implicated in these phenomena, and the possibility of a direct neuropathic effect driven by the virus are unknown. molecular mimicry has been proposed as the primary immune alteration underlying the development of guillain-barré syndrome during infections (105) . this phenomenon is related to the presence of carbohydrates in infectious agents of similar structural characteristics as compared to carbohydrates expressed on neuronal membrane ganglioside and galactocerebrosides. for instance, the lipopolysaccharides of campylobacter jejuni share ganglioside-like epitopes with peripheral nerves (106), whereas galactocerebroside-like structures are present in glycolipids of mycoplasma pneumoniae (107). these molecular similarities trigger the production of anti-glycolipid antibodies, which mediate autoimmune damage to peripheral nerves. gangliosideor galactocerebroside-like epitopes have not been described in sars-cov-2. however, the s protein of this virus possesses 22 nlinked glycan sequons per promoter (20) , that could potentially share certain similitude with carbohydrates localized on the surface of the host nerve cells. future studies are required to evaluate the serologic features of anti-glycolipid antibodies in patients with covid-19 to elucidate possible mechanisms underlying the association between sars-cov-2 infection and guillain-barré syndrome. several viruses affecting humans have been implicated in the development of psychiatric symptoms due to their neurotropism. the immediate antecedent for the global transmission of a respiratory pathogen was the 2009 pandemic associated with the emergence of a novel influenza a (h1n1) virus. several neuropsychiatric manifestations during the outbreak of influenza were observed among infected patients, including fear and behavioral changes (108) . similarly, during the sars-cov-1, a range of psychiatric disorders was identified, including anxiety, depression, suicidal ideation, and hallucinations (109, 110) . a recent systematic review found a high incidence of confusion, depression, anxiety, memory impairment, insomnia, and steroidinduced psychosis among patients with sars-cov-1 or mers-cov infection (111) . the mechanisms underlying psychiatric disorders in patients with viral infections are not precise, but they might be related to the structural and functional disruption of the bbb mediated by circulating inflammatory cytokines produced in response to viruses. these mediators might also alter neuronal networks implicated in cognitive functions. indeed, several psychiatric illnesses, including schizophrenia, have been proposed to result from immune-mediated pathogenic mechanisms (112) . in this sense, it is clear that the current pandemic can cause indirect effects on the mental health of infected and non-infected people due to quarantine and social distancing measures, which constitute sources of distress additional to the daily life problems (113) . at this moment, however, there is little literature about neuropsychiatric disorders directly associated with the infection with sars-cov-2. in a case series of three patients with laboratory-confirmed covid-19 and no evidence of respiratory symptoms, it was found that such individuals presented anxiety, agitation, paranoid behavior, disorganized thinking, and auditory hallucinations (114) . other authors have also reported that delirium can be present in a high percentage of covid-19 patients (111) . thus, as mentioned before, delirium must also be considered in the differential diagnosis of individuals with acute neuropsychiatric manifestations associated with sars-cov-2 infection. finally, the virus could lead to long-term neuropsychiatric and cognitive sequels. in fact, survivors of sars-cov-1 and mers-cov have been found to present depression, anxiety, and posttraumatic distress syndrome several months after the diagnosis (111) . therefore, it is essential to conduct long prospective observational studies to estimate the incidence of psychiatric disorders in the post-illness stage of covid-19. future studies must address possible links between the antecedent of sars-cov-2 infection and the incidence of chronic neurodegenerative disorders. the studies about the spectrum of neurological and psychiatric manifestations of covid-19 are summarized in table 1 . supportive measures, along with strict control and prevention of fever, high blood pressure, elevated glucose, and seizures, may ameliorate the neurotoxic potential of sars-cov-2 infection. currently, there are no mechanism-based therapeutics specific for neurological complications of covid-19. the evidence curated in this review suggests possible pathologic processes underlying the involvement of the cns/pns during sars-cov-2 infection. a better understanding of these mechanisms may reveal targets for therapeutic interventions. direct effects driven by the virus, such as the infection of brain blood vessels and nerve cells, are proposed to play a role in neurologic manifestations of covid-19. although studies addressing the other neurological manifestations reported in covid-19 patients include ataxia, acute hemorrhagic necrotizing encephalopathy, polyneuritis cranialis, and neuralgia (10, 81) . relationship between viral loads in the cns and the severity of such manifestations are required, reducing the number of copies of sars-cov-2 in the circulation and tissues might be a useful strategy. remdesivir is the only antiviral drug with demonstrated capacity to blocking sars-cov-2 replication in pre-clinical trials approved for usage in humans. this antiviral drug reduces the time of clinical recovery in patients with covid-19 (116) . the benefits of remdesivir for patients with neurological complications have not been evaluated. other candidate agents that antagonize the activity of the host proteases implicated in the infection process of sars-cov-2 may be useful to limit the infective capacity of this virus. these include the camostat mesylate and nafamostat mesylate, two compounds that block the activity of tmprss2 (19, 117) . the effects of these drugs on the severity and recovery of neurologic sequela associated with covid-19 must be evaluated in future clinical trials. passive immunization by transfer of convalescent human plasma may also contribute to reduce the viral loads and prevent or reverse the development of neurological symptoms. this assumption is supported by recent systematic reviews that found that convalescent plasma therapy improves symptoms, reduce viral loads, and diminishes mortality in covid-19 patients (118) . the time at which these proposed interventions would be more useful to counteract neurologic sequela of covid-19 is unknown. immune-mediated neurotoxicity is an obvious therapeutic target for individuals with sars-cov-2 infection (59). the immune profile of patients with severe covid-19 resembles the cytokine release syndrome (crs) observed after car-tcell therapy (34, 35) . as aforementioned, individuals receiving car-t cells who develop crs are at risk of injury to the nervous system. therefore, lessons from the treatment of patients with car-t-cell-therapy-associated neurotoxicity might be applicable to covid-19 patients. tocilizumab, an anti-il-6 monoclonal antibody, is the primary treatment for crs (119) , and is currently being used to ameliorate inflammatory manifestations caused by sars-cov-2 infection (120). in patients with severe covid-19, tocilizumab declined cytokine production and reduced the risk of intubation requirement (120) . however, the utility of tocilizumab for management of crsassociated neurotoxicity is controversial (121) . interestingly, mouse models have revealed that the antagonist of the receptor of il-1β anakinra might be a better option than tocilizumab for the treatment of crs and neurotoxicity after car-t-cell therapy (122) . as a strong induction of il-1β has been observed in severely ill covid-19 patients (32), the potential use of anakinra deserves further investigation. monocytes and macrophages also contribute to the development of crs and neurotoxicity after car-t-cell therapy. as such, inhibition of gm-csf with monoclonal antibodies has shown to reduce neuroinflammation in phase i studies of patients receiving car-t-cell therapy (123) . interestingly, in a recent study conducted in patients with covid-19, the gm-csf blockade with mavrilimumab improved clinical symptoms, survival, and reduced intubation requirement (124) . inhibition of gm-csf is thus a potential therapeutic for patients with covid-19 that develop neurological complications. short courses of steroids are safe and provide some benefits for the treatment of immune-mediated neurotoxicity. specifically, dexamethasone may constitute a good candidate due to its excellent cns penetration and beneficial effect on the integrity of the bbb. dexamethasone has been safely used in patients with acute respiratory distress syndrome, reducing the requirement of mechanical ventilation and mortality (125) . these data may support the use of dexamethasone for the management of neurological manifestations of sars-cov-2 infection, although possible consequences of the dexamethasoneinduced immunosuppression need to be evaluated. the breaching of the bbb is an important event for the development of neurotoxicity in covid-19 patients and individuals under car-t-cell therapy. pharmacological agents targeting the bbb may thus be useful to treat severe neurological manifestations of covid-19. the sonic hedgehog (shh) signaling pathway is essential for the maintenance of integrity and immune homeostasis of the bbb (126) . agonists of shh, such as purmorphamine, a compound that activates the shh by promoting smoothened protein (127) , reduce bbb damage in animal models of infection and ischemic stroke (128) (129) (130) . although little evidence exists of the safety of purmorphamine in humans, this agent warrants further exploration for the management of neurotoxicity associated with severe infections, including covid-19. the magnitude of the current pandemic has generated concerns among professionals from various areas of medicine. the evidence summarized in this review shows that neurology is an active area of medicine at the frontline of the current pandemic. actually, due to the increasing number of confirmed cases around the world and the neurotropic potential of sars-cov-2, it is highly likely that neurologists would have to provide medical care to patients with covid-19, some of which might present neurological manifestations. the first and most important precautionary measure that must be taken by neurological centers around the world is to improve the knowledge of the disease among health care professionals. obviously, measures of personal protection and social distancing should be extreme in neurology and neurosurgery clinics since it is possible that some covid-19 patients attending with neurological symptoms with a not yet confirmed sars-cov-2 infection may constitute potential foci of inadvertent contagion for doctors, especially if they do not manifest respiratory symptoms initially. to prevent contagions, it is important to investigate previous contact with people with laboratoryconfirmed covid-19 in patients attending to neurological centers. similarly, it is crucial to maintain a high degree of clinical suspicion about possible sars-cov-2 infection in patients presenting an acute neurological condition. furthermore, the continuous surveillance and intentional search for neurological complications in patients with confirmed sars-cov-2 infection are necessary and even mandatory because these measures could allow the timely establishment of therapeutic strategies for limiting neurologic sequelae. finally, the current pandemic may obligate some changes in normal care to patients with neurologic disorders at medical centers receiving many covid-19 cases. of particular importance is the possible impact of this pandemic in the triage and management of patients with stroke and other acute neurological emergencies due to resource re-allocation. also, the interruption of activities at many neurology outpatient clinics may affect the management of chronic neurological conditions, requiring increased usage of tools such as telemedicine and e-care. the clinical phenotype of covid-19 encompasses a spectrum of neurological manifestations of varying severity that, besides respiratory symptoms, can cause high morbidity and mortality rates in individuals 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simbaña-rivera, katherine; gómez-barreno, lenin; rubio-neira, mario; guaman, linda p.; kyriakidis, nikolaos c; muslin, claire; jaramillo, ana maría gómez; barba-ostria, carlos; cevallos-robalino, doménica; sanches-sanmiguel, hugo; unigarro, luis; zalakeviciute, rasa; gadian, naomi; lópez-cortés, andrés title: clinical, molecular and epidemiological characterization of the sars-cov2 virus and the coronavirus disease 2019 (covid-19), a comprehensive literature review date: 2020-05-30 journal: diagn microbiol infect dis doi: 10.1016/j.diagmicrobio.2020.115094 sha: doc_id: 286683 cord_uid: mettlmhz abstract coronaviruses are an extensive family of viruses that can cause disease in both animals and humans. the current classification of coronaviruses recognizes 39 species in 27 subgenera that belong to the family coronaviridae. from those, at least seven coronaviruses are known to cause respiratory infections in humans. four of these viruses can cause common cold-like symptoms. those that infect animals can evolve and become infectious to humans. three recent examples of these viral jumps include sars cov, mers-cov and sars cov-2 virus. they are responsible for causing severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers) and the most recently discovered coronavirus disease during 2019 (covid-19). covid-19, a respiratory disease caused by the sars-cov-2 virus, was declared a pandemic by the world health organization (who) on 11 march 2020. the rapid spread of the disease has taken the scientific and medical community by surprise. latest figures from 20th may 2020 show more than 5 million people had been infected with the virus, causing more than 330,000 deaths in over 210 countries worldwide. the large amount of information received daily relating to covid-19 is so abundant and dynamic that medical staff, health authorities, academics and the media are not able to keep up with this new pandemic. in order to offer a clear insight of the extensive literature available, we have conducted a comprehensive literature review of the sars cov-2 virus and the coronavirus diseases 2019 (covid-19). the viral membrane contains the spike (s) glycoprotein that forms the peplomers on the virion surface, giving the virus its 'corona'or crown-like morphology in the electron microscope. the membrane (m) glycoprotein and the envelope (e) protein provide the ring structure. within the virion interior lies a helical nucleocapsid comprised of the nucleocapsid (n) protein complexed with a single positive-strand rna genome of about 30 kb in length [16] . the first genome of sars-cov-2 named wuhan-hu-1 (ncbi reference sequence nc_045512) was isolated and sequenced in china in january 2020 [12, 16] . the sars-cov-2 genome has similarities to other viruses: approximately 96% similarity to the bat coronavirus batcov rath13; an estimated 80% similarity with sars-cov [16] , and an estimated 50% identity with mers-cov [17, 18] . sars-cov-2 has a positive-sense single-stranded rna genome. it is approximately 30,000 bases in length and comprises of a 5′ terminal cap structure and a 3′ poly a tail. according to wu et al. [19] , this novel coronavirus (ivdc-hb-01/2019 strain) has 14 open reading frames (orfs) encoding 29 proteins. the 5' terminus of the genome contains the orf1ab and orf1a genes. orf1ab is the largest gene and encodes the pp1ab protein that contains 15 non-structural proteins named nsps (nsp1-nsp10 and nsp12-nsp16). orf1a encodes the pp1a protein and also has 10 nsps (nsp1-nsp10) [19] . the 3' terminus of the genome contains four structural proteins: spike (s) glycoprotein; envelope (e) protein; membrane (m) glycoprotein and nucleocapsid (n) phosphoprotein. it also contains 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14) [20] (figure 3b ). the global scientific community from 58 countries have united to study this novel coronavirus by sequencing and submitting 12,059 sars-cov-2 genomes to the global initiative on sharing all influenza data (gisaid) (https://www.gisaid.org/) between december 2019 and april 2020 [21, 22] . sars-cov-2 has accumulated mutations in its rna genome as the outbreak progresses. as an intracellular obligate microorganism, the coronavirus exploits the host cell machinery for its own replication and spread. since virus-host interactions form the basis of diseases, knowledge about their interplay is of great importance, particularly when identifying key targets for antivirals. j o u r n a l p r e -p r o o f ace2 is a type i membrane protein that participates in the maturation of angiotensin, a peptide hormone that controls vasoconstriction and blood pressure [30] . in the respiratory tract, ace2 is widely expressed on the epithelial cells of alveoli, trachea, bronchi, bronchial serous glands [31] , and alveolar monocytes and macrophages [32] . xu et al. reported the [33] rna-seq profiling data of 13 organs with para-carcinoma normal tissues from the cancer genome atlas (tcga; https://www.cancer.gov/tcga) and 14 organs with normal tissue from fantom5 cage (https://fantom.gsc.riken.jp/). these were used to validate the expression of the human cell receptor ace2 in the virus and may indicate the potential infection routes of sars-cov-2 [34] . interestingly, the ace2 receptor is expressed more in oral cavity than lung. this potentially could indicate that susceptibility and infectivity of sars-cov-2 is greater from oral mucosa surfaces [33] . following the binding of the rbd in the s1 subunit to the receptor ace2, sars-cov-2 s protein is cleaved by the cell surface-associated transmembrane protease serine 2 tmprss2, which activates s2 domain for membrane fusion between the viral and cell membrane [35] . a functional polybasic (furin) cleavage site was found at the s1-s2 boundary through the insertion of 12 nucleotides [13, 29, 36] . the s673, t678 and s686 residues of o-linked glycans flank the cleavage site and are unique in sars-cov-2 [29] . in addition to the s glycoprotein -ace2 receptor complex, wang following the release and uncoating of viral rna to the cytoplasm, coronavirus replication starts with the translation of orf1a and orf1b into polyproteins pp1a and pp1ab via a frameshifting mechanism (figure 4 ) [38] . subsequently, polyproteins pp1a and pp1ab are processed by internal viral proteases, including the main protease m pro , a potential drug target whose crystal structure was recently determined for sars-cov-2 [15] . polyprotein cleavage yields 15 mature replicase proteins, which assemble into a replicationtranscription complex that engages in negative-strand rna synthesis. both full-length and multiple subgenomic negative-strand rnas are produced. the former serves as template for new full-length genomic rnas and the latter template the synthesis of the subgenomic mrnas required to express the structural and accessory protein genes residing in the 3′proximal quarter of the genome [37] . coronavirus rna replication occurs on a virusinduced reticulovesicular network of modified endoplasmic reticulum (er) membranes [39] . the assembly of virions is quickly ensued with the accumulation of new genomic rna and structural components. the n protein complexes with genome rna, forming helical structures. then, the transmembrane m protein, localized to the intracellular membranes of the er -golgi intermediate compartment (ergic) , interacts with the other viral structural proteins (s, e and n proteins) to allow the budding of virions [40, 41] . following assembly and budding, virions are transported in vesicles and eventually released by exocytosis. normal immune responses against the majority of viruses involve a rapid containment phase mediated by innate immunity components and -if necessary-a delayed, yet more sophisticated adaptive immunity phase that should be able to eradicate the pathogen andhopefully-generate long-lasting immunological memory. the former includes antiviral type i interferons (ifns), macrophage and neutrophil activation that leads to proinflammatory cytokine production and nk cells on the other hand, anti-viral adaptive immune responses involve a virus-tailored coordinated attack by antigen specific cd8+ cytotoxic t cells (ctls), the th1 subset of cd4+ t helper cells that orchestrates the j o u r n a l p r e -p r o o f immune response against viruses and other intracellular pathogens, specific antibody producing plasma cells, and finally the production of memory t and b cell subsets. immune responses following sars-cov-2 infection can be a double-edged sword. a rapid and robust type i ifn orchestrated response can lead to virus clearance and -given that antiviral lymphocytes are activated and expanded-immune memory. conversely, a late activation of innate immunity, possibly owing to is usually associated with severe pathology that can lead to pneumonia, ards, septic shock, multi-organ failure and, eventually, death. in this line, a delayed type i ifn response and inefficient sars-cov-2 clearance by alveolar macrophages can promote excessive viral replication that can lead to severe pathology accompanied by increased viral shedding and, thus, viral transmissibility. accordingly, in patients whose immune system is weakened or otherwise dysregulated, such as older men with comorbidities, severe covid-19 is clearly more likely to occur [42] [43] [44] . a recent study has demonstrated that the average duration of sars-cov-2 viral shedding was 20 days after covid-19 onset, raising a debate as to the optimal time of patient isolation [45] . however, in terms of transmission viral shedding seems to be more relevant in the early phases of the infection as it can precede covid-19 symptoms by 2-3 days whilst up to 50% of infections are associated with viral shedding by asymptomatic cases [46] [47] [48] therefore, individuals that mount efficient containment-phase immune responses accompanied by decreased inflammatory responses will not experience infection-or immune response-mediated overt manifestations, but may be important silent spreaders of sars-cov-2. type i ifns are mainly produced by plasmacytoid dendritic cells (pdcs) and have a plethora of antiviral effects such as blocking cell entry and trafficking of viral particles, inducing rnase and dnase expression to degrade virus genetic material, enhancing presentation of viral antigens by mhc-i, inhibiting protein synthesis, inducing apoptosis of j o u r n a l p r e -p r o o f infected cells and activating anti-viral subsets such as macrophages and cytotoxic nk cells and t lymphocytes [49] . pathogen recognition receptors like cytosolic rig-i and mda-5 [50, 51] or endosomal toll like receptors (tlrs) 7 and 8 that recognize viral rna [52] are responsible for the activation of signaling cascades that activate the transcription factors nf-kb, interferon regulatory factor (irf) 3 and irf7 that translocate to the nucleus and induce proinflammatory cytokines and type i interferon (ifn) production. in turn, type i ifns activate the downstream jak-stat signal pathway resulting in expression of ifnstimulated genes (isgs) [53, 54] . our experience from sars-cov and mers-cov infection has shown that delayed type i ifn production and excessive recruitment and activation of infiltrating proinflammatory cells (neutrophils and monocytes-macrophages) are possible mediators of lung dysfunction and bad prognostic factors for the outcome of the infection. delayed type i ifn production allows for highly efficient viral replication that, in turn, results in recruitment of hyperinflammatory neutrophils and monocytes. therefore, the pathogen recognition receptors (prrs) of these proinflammatory cells recognize high numbers of their ligands and subsequently secrete excessive amounts of proinflammatory cytokines that lead to septic shock, lung pathology, pneumonia or acute respiratory distress syndrome. it has been shown that in severe cases both sars-cov and mers-cov fruitfully employ an immune evasion mechanism whereby early type i ifn responses to viral infection are dampened [54] . this can be achieved by blocking signaling both upstream, as well as downstream of type i ifn expression. sars-cov can inhibit irf3 nuclear translocation, whereas mers-cov can impede histone modification [56] . additionally, both viruses can inhibit ifn signaling by decreasing stat1 phosphorylation [57] . due to the many sequence similarities of sars-cov-2 with sars-cov and mers-cov it would be enticing to speculate that similar mechanisms are also present, however further studies are needed to shed light to this hypothesis. hyperactivated neutrophils and monocytes-macrophages are the usual source of the cytokine storm. in this aspect, absolute neutrophil counts and neutrophil to lymphocyte j o u r n a l p r e -p r o o f ratio (nlr) were strongly associated with disease severity in a large cohort of covid-19 patients and were proposed as markers of adverse disease prognosis [58] . interestingly, the increased amounts of proinflammatory cytokines in serum associated with pulmonary inflammation and extensive lung damage described both in sars [59] and mers diseases [60] were also reported in the early study of 41 patients with covid-19 in wuhan [41] . evidence shows that the leading cause of covid-19 mortality is respiratory failure caused by acute respiratory distress syndrome (ards). there is an association with a cytokine storm mediated by high-levels of proinflammatory cytokines including il-2, il-7, il-10, g-csf, ip-10, mcp-1, mip-1a and tnf-α. ards was associated with increased fatality and subsequent studies confirmed il-6 and c-reactive protein are significantly upregulated in patients that died compared to convalescent patients [56] moreover, a recent study of 452 patients in wuhan identified that severe cases showed significantly higher cytokines and chemokines such as tumor necrosis factor-α (tnf-α), il-2, il-6, il-8 and il-10 expressed [58] . in accordance with these findings, therapeutic strategies are being tested. a phase 3 randomized controlled trial of il-1 blockade (anakinra) in sepsis has shown significant survival benefit in patients with hyperinflammation, without apparent increased adverse events [61] . currently, a multicenter, randomized controlled trial of tocilizumab (il-6 receptor blockade, licensed for cytokine release syndrome), is being trialed in patients with covid-19 pneumonia presenting with high levels of il-6 in china (chictr2000029765) [62] . moreover, several clinical trials are exploring if the well-established antiviral [63] and anti-inflammatory effects of hydroxychloroquine will be effective in treating patients with covid-19 as has previously been suggested for sars-cov infection [64] . this has also been demonstrated in vitro for sars-cov-2 [65] . in contrast, janus kinase (jak) inhibition has been proposed as a potential treatment in order to reduce both inflammation and cellular viral entry in covid-19 [66] . thus, it comes as no surprise that in a recent correspondence, lancet authors have identified the following potential therapeutic options for cytokine storm syndrome including ards the use of corticosteroids, selective cytokine blockade (eg, anakinra or tocilizumab) and jak inhibition [67] . j o u r n a l p r e -p r o o f virus presentation to the different t cell subsets stands on the crossroads between innate and adaptive immune responses. studies on sars-cov and mers-cov [72] presentation have identified several susceptibility and protection conferring hla alleles. the dearth of similar data regarding sars-cov-2 antigen presentation to t cells and possible virus evasion mechanisms of this process suggests it is a virgin investigation field to be explored. apart from the sustained inflammation and cytokine storm, lymphopenia has been implicated as a major risk factor for ards and mortality in the context of covid-19 [73] . similar findings were described for sars-cov infected patients who had considerable decreases of cd4+ t and cd8+ t cells [69] . however, in convalescent patients specific tcell memory responses to sars-cov were still found six years post infection [74] . showed no reactivity with viral antigens. however, the small number of sera used (n=4) implies that further investigation is needed to corroborate these results [78] . nonetheless, since we are currently in early stages of sars-cov-2 pandemic more studies need to be carried out to shed light on antibody persistence (both igm and igg) and protective effects. recently, macaques re-challenged with sars-cov-2 after a primary infection did not show signs of re-infection, suggesting that protective immunity and memory responses were fruitfully mounted. this finding can also impact vaccine production strategies [79] . importantly, covid-19 convalescent sera was shown to hold promise as a passive immune therapy alternative to facilitate disease containment [80] . to the best of our knowledge, at j o u r n a l p r e -p r o o f least one pharmaceutical company, takeda, is preparing to purify antibody preparations from covid-19 convalescent sera against sars-cov-2 [81] . a recently published case report of a patient with mild-to-moderate covid-19 revealed the presence of an increased activated cd4+ t cells and cd8+ t cells, antibody-secreting cells (ascs), follicular helper t cells (tfh cells), and anti-sars-cov-2 igm and igg antibodies, suggesting that both cellular and humoral responses are important in containing the virus and inhibiting severe pathology [82] . antibody dependent enhancement (ade) is a mechanism whereby non-protective antibodies produced during an infection with an agent either mediate increased uptake of this agent into target cells or cross-recognize a different pathogen and facilitate its entrance to target cells [83] . evidence emerging over the past two decades suggests that antibodies against different coronavirus can cross-react to some extent and mediate ade [84] . ade in the context of sars-cov was thought to be mediated by antibodies produced against 229e-cov [85] and was contemplated as contributing to high mortality rates in china [86] . the described mechanism suggests that low affinity or low title anti-spike protein antibodies rather than neutralizing the virus result in fc receptor mediated infection of immune cells, further aggravating the dysregulation of anti-sars-cov immune responses [87] . indeed, in vitro as well as in vivo experimental models have shown that ade hinders the ability to manage inflammation in the lung and elsewhere. this may lead to ards and other hyperinflammation-induced clinical manifestations also observed in several of the documented cases of severe covid-19 [88, 89] . while the molecular and immunological host response to sars-cov-2 infection has not yet been fully elucidated to confirm ade is occurring, anti-sars-cov-2 antibodies have been shown to partially cross-react with sars-cov, suggesting enhancement is a possibility. with this in mind, ade in populations previously exposed to other coronavirus can partially explain the geographic discrepancies observed in covid-19 pathogenesis and severity. finally, ade can have several implications in vaccine development as low-affinity or low-titer antibody producing vaccines can increment susceptibility rather than confer protection against the pathogen as has previously been described for a dengue vaccine [90] [91] [92] . j o u r n a l p r e -p r o o f detection methods based on nucleic amplification are often used in the case of sars-cov, mers-cov and other viruses, because have high sensitivity and specificity, particularly in the acute phase of infection [93] . case identification and surveillance of covid-19 spread although rt-qpcr assay is considered the gold-standard method to detect viruses such as sars-cov and mers-cov [94, 95] , currently available rt-qpcr assays targeting sars-cov-2 have important considerations. firstly, due to the genome similarity of j o u r n a l p r e -p r o o f sars-cov-2 to sars-cov (82% of nucleotide identity [96] ), some of the primer-probe sets described by different groups and listed in the who coronavirus disease (covid19) technical guidance [97] , have cross-reaction with sars-cov and other bat-associated sars-related viruses, therefore, it is important to run confirmatory tests. loop-mediated isothermal amplification (lamp) is a one-step isothermal amplification reaction that couples amplification of a target sequence with four to six primers, to ensure high sensitivity and specificity, under isothermal conditions (63-65°c), using a polymerase with high strand displacement activity [129] . in the case of an rna sample, lamp, is preceded by the reverse transcription of the sample rna. rt-lamp has been used before for the detection of various pathogens [130] . including sars-cov-2 and other respiratory viruses [131, 132] . recently, it received emergency use authorization (eua) from the u.s. j o u r n a l p r e -p r o o f serological tests also, called immunoassays, are rapid and simple alternatives for screening of individuals that have been exposed to sars-cov-2 based on the qualitative or quantitative detection of sars-cov-2 antigens and/or anti-sars-cov-2 antibodies. there are several types of serological tests available, including elisa (enzyme-linked immunosorbent assay), iift (indirect immunofluorescence test), lateral flow immunoassays and neutralization tests. immunoassays assays are very useful because they allow to study the immune response(s) to sars-cov-2 in a qualitative and quantitative manner. in addition, help to determine the precise rate of the infection [78, 133] , and to determine more precisely the fatality rate of the infection [78] . several sars-cov-2 targeted serological tests are commercially available or in development [134] . a recently developed kit, reported a sensitivity of 88.66% and specificity of 90.63% [135] using sars-cov-2 igg-igm combined antibody rapid (within 15 minutes) test [135] . despite their simple and fast readout and their potential for being used outside laboratory environments (bedside, small clinics, airports, train stations, etc.), serological tests have a critical disadvantage; given the fact that antibodies specifically targeting the virus would normally appear after 6 days or longer [136] after the illness onset [137] , tests based on this principle have a lag period of approximately 4 to 7 days post-infection. during this lag period, infected and non-infected individuals will both result in a negative output. in addition, it is important to highlight that because serological tests depend on the ability to produce antibodies, intrinsic immunological differences and/or responses between individuals, can significantly affect the outcome of these tests. recently, some commercially available immunoassays received ce mark for professional use [138, 139] , and therefore are registered as in vitro diagnostic devices. currently, there are a plethora of antibody tests for covid-19 with variable performance (sensitivity varying from 45 to 100%, specificity from 96 to 100%, reviewed in (foundation for innovative new diagnostics) [134] . different manufacturers of serological assays declare that their assays have no cross reactivity to other human coronaviruses and other respiratory viruses. however, despite the data provided by manufacturers, recent studies highlighted that several of the commercially available tests have sensitivity and/or specificity issues that should be considered for using and analyzing results of many of these tests [140] [141] [142] [143] [144] . j o u r n a l p r e -p r o o f as mentioned before, immunoassays -particularly tests detecting anti-sars-cov-2 igm and/or igg-indicate that the person has been exposed to the virus. in the case of other viral infections, having antibodies targeting a pathogen has often been considered an indication of having immunity against that pathogen [145] . based on this idea, some governments have suggested using serological tests, to determine who has developed immunity against sars-cov-2 and provide positive individuals a "risk-free certificate" or "immunity passports", which would enable them to travel or to return to work, assuming that they are protected against re-infection [146]. however, based on the limited knowledge of how immunity to this virus works [147] , there is not enough evidence to declare a person immune, or to confirm that people who have anti-sars-cov-2 antibodies are protected from a re-infection. even though covid-19 can be diagnosed using qpcr as the gold standard, inadequate access to reagents and equipment has slowed disease detection even in developed countries such as the us. several low cost and rapid tests using different approaches have been described. unlocking) technique for the detection of covid-19 and the detectr (developed by mammoth biosciences) prototype rapid detection diagnosis kit using crispr to detect the sars-cov-2 in human samples have been described [148] . the use of rna aptamers, have recently emerged as a powerful background-free technology for live-cell rna imaging due to their fluorogenic properties upon ligand binding, a technology that could be of use to diagnose sars-cov-2 infection [149] . finally the use of next generation sequence (explify®) might be used to detect and identify bacterial, viral, fungal, and parasitic pathogens by their unique genome sequences [107] . in covid-19 symptomatic infection, the clinical presentation can range from mild to ventilation assistance [151] . the spectrum of symptoms of covid19 infection are characteristic of a mild disease in most of the cases, however, it is important to point that the progression could lead to a severe respiratory distress. asymptomatic infection (while incubation occurs) was described both in the first cases in wuhan and in other cohorts. a group of isolated patients were screened for sars-cov-2, where 17% (629 cases) were positive for the test, and half of these cases had no symptoms. on the other hand, there are reports of cases without overt symptoms in which there were ground glass images in the chest tomography in up to 50% of patients [152] . of the asymptomatic cases studied in wuhan city, the 2.5% of people exposed developed specific symptoms in 2.2 days, and the remaining 97.5% were symptomatic in the following 11.5 days (ci, 8.2 to 15.6 days). the median estimated incubation period was 5.1 days (95% ci, 4.5 to 5.8 days) [153] . some patients with initially mild symptoms had symptom progression over the course of one week [154] . the descriptive studies available so far have concluded that the majority of cases are mild infections (more than 80% of cases); with up to 15% of patients being sever j o u r n a l p r e -p r o o f in most cohorts, and less than 5% have been considered as critical cases with high vital risk [155] . in a study describing 138 patients with covid-19 pneumonia in wuhan, the most common clinical characteristics at the onset of the disease were described. this is consistent with other international cohorts (table 3 ) [150] . it is important to note that fever is not always present and up to 20% of patients could had a low grade temperature between 37.5 to 38 degrees celsius or normal temperature [156] . if these patients required hospitalization, 89% developed a fever during the course of the illness. rarer accompanying symptoms included headache without warning signs, odynophagia and rhinorrhea. gastrointestinal symptoms such as nausea and watery diarrhea were relatively rare [151] . dyspnea develops after a median of 5 to 8 days from the onset of symptoms. it is important to notice that, if dyspnea is an important clinical finding, not all the patients with this j o u r n a l p r e -p r o o f symptom will develop severe respiratory distress or require oxygen supplementation [150] . according to world health organization (who) guidelines, covid-19 infection can present as pneumonia without signs of severity, and could be managed in the outpatient setting. this is applicable to those patients who do not need supplemental oxygen [157] . as previously mentioned, the most serious manifestation of covid 19 infection is pneumonia, characterized by cough, dyspnea, and infiltrates on chest images; the latter is indistinguishable from other viral lung infections. acute respiratory distress syndrome (ards) is a major complication of covid pneumonia in patients with severe disease. this develops in 20% after a median of eight days. mechanical ventilation is implemented in 12.3% of cases [158] . in different case reports, the need for supplemental oxygen via the nasal cannula was required in approximately 50% of hospitalized patients. 30% required non-invasive mechanical ventilation, and less than 3% required invasive mechanical ventilation with or without extracorporeal membrane oxygenation (ecmo) [159] . it is important to mention that the proportion of severe cases is highly dependent on the study population and may be related to the epidemiological behavior of the infection in each country. additionally, the number of people tested will influence the denominator. in italy, the average age of people infected with covid-19 is between 60 and 65 years, and 16% of those hospitalized require admission to the intensive care unit (icu) [160]. the who recommendations had established that severe covid-19 disease could be defined by the following parameters in table 4 [157] . [162] . among the established risk factors for the development of ards is age greater than 65 years, diabetes mellitus and hypertension, in at least 40% of patients [151] . it should be clarified that, although advanced age is identified as a risk factor for a severe infection, those of any age may suffer from severe illness from covid-19. the descriptions made so far of the patients from china have determined that almost 90% of the patients were between the ages of 30 and 79 years (cohort of 44,500 cases) [155] . in other population settings, such as in the united states, more than 60% of confirmed the clinical characteristics of symptomatic cases and their severity has been described. in addition to the symptoms reported by the patients, the findings on physical examination may be absent during mild coivd-19 infection. those with moderate to severe covid-19 infection have various signs during pulmonary auscultation, however the most common findings include: wet rales; global decrease in respiratory sounds and increased thrill [164] . early recognition is essential to classify cases as potential cases and initiate one of the most important measures to contain the pandemic, isolation. 2. anyone who has resided or been traveling in areas where widespread community transmission has been reported. 3. any patient who has had potential exposure through attending events or has spent time in specific settings where cases of covid-19 have been reported. the scenarios described respond to the context of a high suspicion of covid-19 infection. the world health authorities (cdc, who) continually update these contexts. there have been multiple case definitions and clarifications regarding when to perform a covid-19 test: • they have pointed out the importance of fever, cough and dyspnea as sentinel symptoms, since these should form part of the clinical judgment that guides doctors. this allows to expand the group of suspicious patients. • in cases of severe respiratory distress of undetermined etiology and that do not meet the previously indicated criteria, a screening for covid-19 would be indicated. • in areas of limited resources, the suggestion is to prioritize cases that require hospital care, and in this way guide the epidemiological fence to order isolation and protect the most vulnerable people (chronically ill and over 65 years of age), as well as test those with the greatest possibility of exposure (travelers and health personnel). currently, there is no laboratory data profile that is framed in covid 19 infection. from a cohort of 43 patients confirmed with covid 19, these findings were classified as mild, moderate and severe disease [165] . high levels of d-dimer and more severe lymphopenia have been associated with mortality due to a prothrombotic state that determines multi-organ failure. in general, leukopenia and / or leukocytosis can be found in the interpretation of blood biometry, however, the most widely described finding is lymphopenia [166] . it should be considered that in the context of viral pneumonia biomarkers such as procalcitonin and pcr are not useful, as often these biomarkers are in the normal range for patients with covid-19. among other findings, descriptive studies have reported considerable elevations of lactate dehydrogenase and ferritin as well as alteration in aminotransferases; although elevation ranges for these parameters have not been established [167] . about the imaging findings, covid 19 viral pneumonia has similar features on imaging to other viral infections. although computed tomography (ct) is the test of choice, it is not useful for a definitive diagnosis due to the wide variety of images that can be found in patients with covid 19 infection. this statement is derived from a large cohort of more than 1000 wuhan patients, where rt-pcr confirmation of covid 19 and chest ct images of these patients were correspondingly analyzed. ct images were determined to have a sensitivity of 98%; however, the specificity was only 25% [168] . in general, the majority of descriptive studies concur that the finding of ground glass opacifications is most common. it is typically basal and bilateral, and rarely associated with underlying consolidation. a multicenter chinese study that retrospectively reviewed the ct scans of 101 patients found that 87% had typical ground-glass images and up to 53% had this finding along with consolidations. these findings were more frequent in the most j o u r n a l p r e -p r o o f severe and older age groups of patients [169] . pleural effusion (4%), and lymphadenopathy (2.7%) [170] . the emergence and outbreak of sars-cov-2, the causative agent of covid-19, has rapidly become a global concern that highlights the need for fast, sensitive, and specific tools to monitor the spread of this infectious agent. diagnostic protocols to detect sars-cov-2 using real-time quantitative polymerase chain reaction (rt-qpcr) were listed on the world health organization (who) website as guidance, however, various institutions and governments have chosen to establish their own protocols that might not be publicly available or listed by who. there are important challenges associated with close surveillance of the current sars-cov-2 outbreak. firstly, the rapid increase of cases has overwhelmed diagnostic testing capacity in many countries, highlighting the need for a high-throughput, scalable pipelines for sample processing [171, 172] . secondly, given that sars-cov-2 is closely related to other coronaviruses [96] , some of the currently available nucleic acid detection assays can result in false positives [173] . thirdly, critical concern for molecular detection is the low sensitivity reported for rt-qpcr assays [168] and serological tests [135] , particularly in the early stages of infection. additionally, most of the available rt-qpcr assays require sample processing and equipment only available in diagnostic and/or research laboratories. it is important to mention that coinfection is a possibility, as some reports from italy and in spain, less than 1% of cases in a cohort correspond to plwh, which have had a satisfactory evolution and less than half required an intensive care unit [177] . in the us, of the 5,700 hospitalized patients in the new york area, only 47 patients had hiv, while in san francisco, data was published on 1,233 people who had diagnosed with sars-cov-2 infection, of which less than 3% had hiv and none of them developed severe covid-19 [178] . despite the existence of in vitro studies on the efficacy of the use of lopinavir / ritonavir, it is currently known that its effect in cases of moderate and severe covid-19 is null, and therefore at the moment no recommendation can be given nor how treatment, much less as prophylaxis [179] . this clarification is important given that there is a belief that plwhs could be protected if they take antiretroviral therapy. therefore, current recommendations for plwhs are to maintain antiretroviral therapy with the goal of controlling hiv as well as following the same standards of care as the general population to avoid acquiring a sars-cov-2 infection [180] . regarding sars-ncov infection in pregnant women, there is currently limited evidence about the effect of the virus on the mother or fetus. however, due to the physiological changes typical of pregnancy, especially on the immune system (immunosuppression) and the cardiopulmonary system, pregnant women are thought to be more susceptible to developing severe symptoms when they acquire the viral respiratory disease. in 2009, when influenza a h1n1 infection occurred, only 1% of the infected population were pregnant, yet they accounted for 5% of infection-related deaths [181] . pregnancy (25%) [182] . in another study of 11 pregnant patients infected with mers-cov, 9 presented adverse results (91%), 6 neonates were admitted to the neonatal intensive care unit (55%) and three of them died (27%) [183] . however, it is important to note the small sample size which could increase the risk of bias and low power of the study. with information obtained so far from the wuhan sars-cov-2 outbreak, the infection appears to be less severe for pregnant women, compared to previous sars-cov and mers-cov outbreaks [181] . however, it is important to take into account that the data from covid-19 infection should be monitored with a doppler ultrasound every two weeks, due to the risk of developing intrauterine growth restriction [187, 188] . the time of termination of the pregnancy, as well as the method, also depend on several factors, including gestational age, maternal condition in relation to sars-cov-2 infection, presence of maternal comorbidities, and fetal condition. decisions must be made collaboratively during multidisciplinary team discussions, with individualized management plans established for each patient [189] . a diagnosis of covid-19 alone is not an indication for the termination of pregnancy, rather it should be made in combination with consideration of morbidity and mortality of both the fetus and mother. after delivery, the use of corticosteroids is recommended for antenatal fetal lung maturation, with betamethasone or dexamethasone [190] ; taking special care in critically nursing patients, as this may worsen their condition, and may delay delivery, which is necessary for the management of these patients [187, 191] . the symptoms children present with are similar to adults, as is the incubation period ranging from 1 to 14 days (mean of 5.2). a cough is the most frequent presenting symptom (65%) followed by fever (60%). there is a higher occurrence of gastrointestinal symptoms including diarrhea (15%), nausea, vomiting (10 %) and abdominal pain. these gastrointestinal symptoms are usually more variable in children than adults and are sometimes the only clinical manifestation in associations with fevers. [192, 193] . the clinical progression and disease severity in pediatric patients is markedly different from that of adults. over 90% of affected children are asymptomatic or have mild to moderate disease [192] . the majority of serious cases in children are related to those with significant comorbidities such as heart disease, immunosuppression, etc. to date of this review, only a few cases of children without underlying comorbidities have died as a result of covid 19 have been reported. this difference of severity of illness between adults and j o u r n a l p r e -p r o o f children has not been clarified, however, several theories have been postulated. these include that children express more ace2 receptors in their lungs which confer some protection to severe injuries such as those caused by rsv and which would decrease dramatically with age [194, 195] . immunological factors may also influence outcomes, as in childhood we are most exposed to frequent challenges including recent seasonal viruses such as rsv in the winter months. most likely, it is multifactorial and depends on factors from both the host and the virus itself [195] . abnormal radiological (ct) findings are found in asymptomatic children and consist of bilateral lung lesions (50%). elevated crp (creactive protein), procalcitonin pct (80%), and liver enzymes are present in most affected children, unlike adults in whom pct is not a reliable marker. virus elimination via the stool even after the negativity in the nasopharyngeal mucosa and the disappearance of symptoms makes them a potential source of contagion through the fecal-oral route [196] . patients with cancer are generally more susceptible to infections than healthy people, because they have a state of systemic immunosuppression that is exacerbated during chemotherapy or radiotherapy [197] . in china, according to national surveillance data, coronavirus infection occurs in 1.3% of patients with malignant tumors. this is a much higher proportion than the general incidence of 0.3% [198] . p<0.0001) even after adjusting for age [197] . further research, completed in a tertiary hospital in wuhuan -china similarly found that 25% of patients with cancer and sars-cov-2 infection died, most of them over 60 years of age [199] . due to these findings, it has been proposed by many international entities that during the pandemic, for prevention, an individualized plan based on the patient's specific conditions is required, with the aim to minimize the number of visits to health institutions.  for early-stage patients with need of post-surgical adjuvant chemotherapy, especially those whose clinical, pathologic, and molecular biologic staging suggest a better prognosis, the start time of adjuvant chemotherapy may be delayed up to 90 days after surgery without affecting the overall effect of treatment [200] .  for patients with advanced cancer, the main approach should be to minimize hospitalization in covid-19 positive installations. replacing the existing intravenous treatment regimen with oral chemotherapy during this special period may be considered, to ensure that treatment is not interrupted for a long time during the pandemic [201] . however, if there is a suspicion of covid-19 infection in this population group, the same updated diagnostic guidelines and the corresponding management should be followed depending on their severity of illness. moreover, an individualized follow-up plan should be outlined due to higher likelihood of complications in this group of population [202] . it should be noted that patients attending out-patient appointments for cancer have higher levels of anxiety, depression and other mental health problems than the general population. studies have shown that approximately 50% of malignant tumor survivors have a moderate to severe fear of tumor recurrence [203] . for this reason, psychologist surveillance of outpatients in quarantine or during hospitalization should be considered. reported complications derived from covid-19 describe a severe disease that requires management in an intensive care unit (icu) in approximately 5% of proven infections. j o u r n a l p r e -p r o o f appear to be most susceptible to the life-threatening complications. the risk of patient-topatient transmission in the icu is currently unknown, therefore adherence to infection control precautions is paramount [204, 205] . progressive deterioration of respiratory function is undoubtedly the most common and lifethreatening complication of the infection. the prevalence of hypoxic respiratory failure in covid-19 patients is 19%, and it can progress to acute respiratory distress syndrome (ards), with the need of mechanical ventilation support at 10.5 days on average. one study found that between 10 and 32% of hospitalized patients require admission to the icu due to respiratory deterioration [205] . as respiratory complications are the most common cause of severe deterioration, early identification of them will undoubtedly help in timely support. support provided should be adapted to take into account risk factors such as advanced age, neutrophilia and organic dysfunction for the development of ards. the diagnostic support of pulmonary tomography is undoubtedly a valid tool; images in patients with different clinical types of covid-19 have characteristic manifestations, but it can become an operational problem due to the difficulty in performing imaging on critically ill patients. on the contrary, lung ultrasound at the bed-side could provide an alternative to radiographs and tomography during the diagnosis of covid-19 [206, 207] . since more than 70% of hospitalized patients will require supplemental oxygen, it is recommended that oxygen should be started when pulse oximetry values fall below 90%. an upper-limit of 96% saturation should be established, since higher values have been shown to be harmful [208, 209] . hemodynamic deterioration has a variability of presentation, this depends on the study population and the definition [223] , the presence of shock in the intensive care unit may be present between 25 to 35% [204, 224] . cardiomyopathy related to viral infection is one of j o u r n a l p r e -p r o o f the main causes of hemodynamic detriment, occurring in up to 23% of patients with covid-19 [43] . hemodynamic failure is one of the main causes of death in these patients, with percentages of up to 40%, inconclusive risk factors are associated to date such as diabetes, hypertension, lymphopenia, and elevation of d-dimer [225] . acute kidney injury (aki) is present in up to 12% of critically ill patients, podocytes and proximal tubule cells are potential host cells for sars-cov-2, caused by the virus induced cytopathic effect. the diagnosis is based on markers of early kidney injury and urinary output [210] . initial management of shock is based on fluid resuscitation, based on the application of dynamic parameters to predict response to fluids, such as variation in stroke volume (svv), variation in pulse pressure volume (ppv) and change in stroke volume with passive leg elevation or fluid challenge above static parameters [225] . variables such as skin temperature, capillary refill time and/or serum lactate measurement are currently valid tools to assess shock. the volume of liquids used in resuscitation should be restricted and administered in relation to dynamic assessment. a liberal water resuscitation strategy is not recommended, rather a balance of crystalloids over colloids as resuscitation liquids should be encouraged and avoiding the use of hydroxyethyl starches, albumin, dextrans or gelatins [226, 227] . indirect evidence suggests that the target mean arterial pressure (tam) for patients with septic shock is 65 mmhg using vasoactive support [228] . the recommendation of norepinephrine use as the first agent is maintained. if norepinephrine is unavailable, vasopressin or epinephrine could be used, avoiding the use of dopamine as the initial vasopressor due to the potential development of arrhythmias [229, 230] . in patients with covid-19 and shock with evidence of cardiac dysfunction and persistent hypoperfusion despite fluid resuscitation and norepinephrine use, dobutamine as inotropic is recommended. given the development of refractory septic shock, the suggestion of the use of hydrocortisone in continuous infusion is maintained, as indirect evidence, this in favor of reducing the length of stay in the icu and the resolution time of the shock [229] . according to the investigative mission of the who in china, the case-fatality rate ranged other reports from china have coincided with this clinical risk profile, for example, a study that included 41 confirmed cases, 12 patients who had ards had as main underlying diseases: diabetes and high blood pressure. of these cases, 6 patients died [156] . according to who, the recovery time is estimated to be two weeks for patients with mild infections and three to six weeks for those with serious illnesses. on the other hand, cdc established that people who had symptoms in the mild to moderate spectrum and maintained home isolation have a resolution of 3 days after the fever decrease, and there was a substantial improvement in respiratory symptoms, even without use of medications. isolation may be limited to 7 days from resolution of symptoms, however, it must be adapted to the population circumstances of the epidemic [158] . the evolution of the epidemiological curve in covid-19 outbreak makes consider containment strategies in china primarily, and other countries based on nonpharmaceutical interventions (npis). according to the who, the most effective measure is hands washing [231] . combination as public health measures reduced contact rates in the population and therefore reduce virus transmission (table 6 ) [232] . table 6 non -pharmacological measures. increasing the level of hand cleanliness to 60% in places with a high concentration of people, like all airports in the world would have a reduction of 69% in the impact of a potential disease spreading [233] . the epidemiological evolution of the covid-19 pandemic through phases has required the application of specific measures according to the time or phase in which the virus is found in each country. the evolution in the non-pharmacological measures has been as variable as the pharmacological ones, in such a way, since january to march, it was ensured that the use of face masks was limited only to people who had contact with epidemiological foci, not to healthy people [234] . this concept was also reinforced by cdc, in order to optimize the use of masks for health workforce. definitely the course of the pandemic was changing rapidly, which also demanded the change from containment measures to mitigation. the recommendations in the current context remain regarding the use of a facial mask in the community, but its optimization is important for health professionals. the use of the mask is not a substitute for handwashing and social distancing measures, as these ones together allow avoiding viral particles in aerosols or drops, as a low cost and accessible measure for general population [235, 236] . there is still non-specific information for the recommendation of masks, in general, having in several studies claims that surgical or cotton cloth masks do not prevent the spread of the sars-cov-2 virus [237] . the evidence about the transmission of the virus in the asymptomatic period also changed the containment measures, suggesting the community use of masks. it is from this that the recommendations for the rational use of masks arise since in some j o u r n a l p r e -p r o o f countries the massive use of n95 masks was reported, masks indicated for the use of medical personnel [238] . regarding to this non pharmaceutical recommendations, the studies suggest to priories the resources on vulnerable population, in endemic areas, older people, adult with comorbidities and health workforce. studies are still needed on the duration of the protective effect of the masks and above all the possibility of their reuse for resource optimization. meanwhile the most important recommendation continues to be its use in addition to hand hygiene and social distancing [238] . therapeutic j o u r n a l p r e -p r o o f this effect is reinforced by azithromycin. there were the best results in terms of viral load reduction, even though is mentioned some limitations in the study like small sample size, a short long-term outcome follow-up, and dropout of six patients from the study [246] . concerning to mortality rates, a study was conducted in new york with 1376 patients (0.63-1.85). thus, it concludes that the use of hcq is not associated with either a decreased or increased clinical impairment, intubation or death [247] results reinforced by other study in 1438 hospitalized patients with covid-19 diagnosis in new york city, whom received treatment with hydroxychloroquine, azithromycin, or both drugs was not associated with significantly reduction in mortality [248] . relating to safety of this drug, in a study carried out in 200 patients in which the theoretical complications of the use of hcq and its combination with macrolides (azithromycin) were assessed by serial electrocardiograms, the following results were obtained. in 5% of patients (10) received chloroquine, 95% (191) received hcq, and 59% (119) [252] . supportive therapies in immune regulation, together with the use of antivirals, are important to take into account, especially in those patients in a serious and critical state, in which they could improve the clinical response and perhaps avoid residual lung injuries. the convalescent plasma is extracted from recovered individuals from an infection, being an antibody transfer medium to provide passive immunity (neutralizing antibodies and globulin). the goal is to provide a rapid immune response until the patient can develop their own active immune response in the hope that there will be clinical improvement [253] . improvement in most individuals, as well as viral suppression 7 days after treatment [255] . in the same country, at the shenzhen hospital, 5 cases of patients with severe covid-19 were reported who met criteria for acute respiratory distress syndrome (ards), who were administered convalescent plasma (titration greater than 1: 1000 and neutralizing antibodies greater than 40). it was found that clinical recovery occurred approximately 12 days after the transfusion (4 patients) and 3 of the 5 patients were discharged 55 days after admission. it is important to mention that this group of patients also received antivirals, methylprednisolone and all the necessary support measures in intensive care [256] . other drugs like ivermectin, nitazoxanide, and others have been studied in the context of covid-19 treatment, but the results are inconsistent. all of the clinical trials evidence, supporting or against the use of mentioned drugs are detailed in (supplementary table 2 ). this review summarized some drug repurposing agents previously known to has efficacy against other virus like sars-cov, mers-cov, influenza. actually, exist some new drugs with high potential action on targets for covid-19 therapeutics. it is important to notice that there is no specific treatment for the coronavirus approach. in context of the scientific evidence exposed and the particular clinical features of each patient, the reader will be able to make the best clinical and therapeutic decisions. when it comes to vaccine design and manufacturing, the main objectives are to ensure its safety, its efficacy in activating specific adaptive immune responses and the production ofideally-long term memory. thus, eliciting protective immune responses including neutralization antibodies and/or ctl generation is of paramount importance. huge challenges need to be tackled in order to minimize the long and cumbersome process of vaccine generation. among them, candidate antigen targets need to be identified, immunization routes and delivery systems investigated, animal models set, adjuvants optimized, scalability and production facility considered, target population selected, and vaccine safety and long-term efficiency evaluated. currently there are no approved vaccines against any human coronavirus, suggesting that their generation is quite novel. several candidate vaccines against sars-cov had shown promise reaching phase i or phase ii clinical trials [258, 259] , but the rapid containment of sars-cov expansion rendered them redundant, did not allow for a test population for phase iii trials and, therefore, put their further assessment to a halt. ctl memory could last up to 11 years after infection [260] . these data suggest that vaccine strategies employing viral structural proteins that can elicit effective, long-term memory t cell responses could yield fruitful results. on the other hand, the s1 spike protein region containing the ace receptor binding domain (rdb) is the obvious option when neutralizing antibody responses are considered [261] [262] [263] . indeed, a candidate sars vaccine antigen consisting of the rbd of sars-cov spike protein was created and found it could elicit robust neutralizing antibody responses and long-term protection in vaccinated animals [264] . the fact that covid-19 convalescent sera shows potential as a therapeutic approach [80] aligns with the theory that efficient b cell responses are mounted and lead to production of protective antibodies. two different groups, using an immunoinformatic approach mapped several ctl and b cell epitopes on different proteins of the virus [265, 266] . moreover, various ctl epitopes were found to be binding mhc class i peptide-binding grooves via multiple contacts, illustrating their probable capacity to elicit immune responses [265, 266] . consequently, these identified b and t cell epitopes could be potential targets for therapeutic vaccines. however, important safety considerations should be taken into account before releasing a new vaccine in the market. previous studies on macaque models have shown that a vaccineinduced anti-spike protein antibody at the acute stage of sars-cov infection can provoke severe acute lung injury [267] . similar observations of sars-cov vaccine-induced pulmonary injury have also been described in multiple several murine and monkey animal models [268] . an additional factor that needs to be checked in phase ii and iii trials is that the vaccine does not cause ade of the pathogen, as has previously been described. such concerns have risen in the context of a dengue vaccine [269] . the pharmaceutical companies that are currently on a race to produce a vaccine for covid-19 along with the vaccine developing strategies they are using are summarized in table 8 and figure 7 . as can be easily deduced from table 8 hospitalization and admission to already heavily charged icus due to these pathologies that could prove critical for weaker health systems that would struggle to carry the burden of combined outbreaks. moreover, vaccinating health care workers is crucial for reducing the risk of absence due to disease, thereby strengthening the healthcare workforce and minimizing the risk to infect covid-19 hospitalized patients with additional pneumoniacausing pathogens. lastly, covid-19 patients vaccinated for influenza and streptococcus pneumoniae allow their immune system to focus on one pathogen and, therefore, give it a better fighting chance against sars-cov-2 infection [276] . high risk groups prioritized for vaccination for these two pathogens include pregnant women, persons with immunocompromised immune systems (either due to congenital or acquired immunodeficiencies), children, adults ≥ 65 years and health care professionals. j o u r n a l p r e -p r o o f heat or chemical treatment inactivation. f) attenuated live pathogen vaccine strategies consist in administering a live pathogen that due to cell culture passaging has lost its virulence. they usually elicit robust and long-term memory immune responses without the need to administer an adjuvant. g) in dna vaccines the dna codifying a highly immunogenic antigen is administered and captured by professional antigen presenting cells (apcs) leading to antigen production and presentation by these cells. h) moderna's vaccine candidate already in phase i clinical trials uses an mrna vaccine approach whereby the genetic information codifying for the s protein of sars-cov-2 is delivered in lnps to enhance absorption by apcs. once uptaken by apcs the mrna induces the expression of s antigen that is subsequently mounted on and presented by mhc molecules to elicit adaptive immune response. numerous studies confirm that climate has an impact on virus (i.e., influenza, coronavirus, etc.) spread through manipulating the 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fang; wu, yan; ye, yang; xu, yechun title: discovery of baicalin and baicalein as novel, natural product inhibitors of sars-cov-2 3cl protease in vitro date: 2020-04-14 journal: biorxiv doi: 10.1101/2020.04.13.038687 sha: doc_id: 291710 cord_uid: ixun0c8g human infections with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) cause coronavirus disease 19 (covid-19) and there is currently no cure. the 3c-like protease (3clpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. to advance the speed of drug discovery and development, we investigated the inhibition of sars-cov-2 3clpro by natural products derived from chinese traditional medicines. baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of sars-cov-2 3clpro and exhibited potent antiviral activities in a cell-based system. remarkably, the binding mode of baicalein with sars-cov-2 3clpro determined by x-ray protein crystallography is distinctly different from those of known inhibitors. baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial s1/s2 subsites and the oxyanion loop, acting as a “shield” in front of the catalytic dyad to prevent the peptide substrate approaching the active site. the simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs. traditional chinese medicines (tcms) have evolved over thousands of years and are an invaluable source for drug discovery and development. as a notable example, the discovery of artemisinin (qinghaosu), which was originally isolated from the tcm artemisia annua l. (qinghao), is a milestone in the treatment of malaria. tcms as well as purified natural products also provide a rich resource for novel antiviral drug development. several herbal medicines and natural products have shown antiviral activities against viral pathogens (8) (9) (10) (11) . among these, the roots of scutellaria baicalensis georgi (huangqin in chinese) are frequently used in tcm for the prophylaxis and treatment of hepatitis and respiratory disorders (12) (13) (14) . in the present study, an enzymatic assay was performed to test if the ingredients isolated from s. baicalensis are inhibitors of sars-cov-2 3clpro. as a result, baicalin and baicalein, two bioactive components from s. baicalensis, are identified as novel inhibitors of sars-cov-2 3clpro with an antiviral activity in the sars-cov-2 infected cells. a crystal structure of sars-cov-2 3clpro in complex with baicalein, the first non-covalent, non-peptidomimetic small-molecule inhibitor, was also determined, revealing a unique binding mode of this natural product with the protease. the pivotal role of the 3cl protease in processing polyproteins into individual functional proteins for viral replication and a highly conserved substrate specificity of the enzyme among various covs make it a promising target for screening of inhibitors. a fluorescence resonance energy transfer (fret) protease assay was applied to measure the proteolytic activity of the recombinant sars-cov-2 3clpro on a fluorogenic substrate. the detail of the assay is described in the experimental section. this fret-based protease assay was utilized to screen natural products as novel inhibitors of sars-cov-2 3clpro. it was first used to determine the inhibitory activities of the total aqueous extract, fractionations, and purified compounds from s. baicalensis against sars-cov-2 3clpro (see supplementary materials, fig. s1 ). as the result, two fractions from s. baicalensis showed significant inhibition on sars-cov-2 3clpro at 10.0 g/ml (table s1) . surprisingly, baicalin, the major component in fraction 8, shows an ic50 of 6.41 m against the protease, while baicalein, the major component in fraction 12, has an ic50 of 0.94 m (fig. s2 ; table 1 ). accordingly, baicalin and baicalein are identified as novel non-peptidomimetic inhibitors of sars-cov-2 3clpro with single-digit micromolar potency. to validate the binding of baicalin and baicalein with sars-cov-2 3clpro and exclude the suspicion of being the pan-assay interference compounds (pains) (15) , their binding affinities with the protease were measured by isothermal titration calorimetry (itc), widely known as an invaluable tool used to determine thermodynamic parameters of protein-ligand interactions such as kd (fig. 1, a and b ; table 1 ). the resulting kd of baicalin and baicalein binding with sars-cov-2 3clpro is 11.50 and 4.03 m, respectively, which has a good correlation with the ic50s mentioned above, demonstrating that specific binding of the compounds with the enzyme is responsible for their bioactivities. moreover, the itc profiles in combination with their chemical structures suggest that baicalin and baicalein act as noncovalent inhibitors of sars-cov-2 3clpro with a high ligand binding efficiency. native state electrospray ionization mass spectrometry (esi-ms) has been used extensively to directly observe native state proteins and protein complexes, allowing direct detection of protein-ligand non-covalent complexes with kds as weak as 1 mm (16) . the determination of m/z between [protein + ligand] m/z and [unbound protein] m/z is able to identify a ligand as a binder with the correct molecular weight, while the ratio of the intensity of the [protein + ligand] peaks relative to [unbound protein] peaks provides a qualitative indication of the ligand-binding affinity. herein, an esi-ms analysis using high-resolution magnetic resonance mass spectrometry (mrms) was carried out to detect the binding of baicalin and baicalein with sars-cov-2 3clpro. for the free protease performance optimization, the mass range around the change stated 18+ was isolated with a center mass of the quadrupole of m/z 3750 (fig. s3 ). for the ligand-binding screening studies, two charge states (18+ and 19+) have been used for calculation of the free protease and protein-ligand complex intensities. the representative spectra of samples containing sars-cov-2 3clpro (1 m) and baicalin (3.13 m) or baicalein (0.31 m) acquired under both optimized and screening conditions are shown in fig. 2c and d, demonstrating a specific binding of baicalin or baicalein with the protease. moreover, the plot of the fraction of the bound protease versus the total concentration of baicalin or baicalein obtained kds of 12.73 and 1.40 µm for baicalin and baicalein, respectively (fig. 1e and f), in keeping with the results from the itc measurements. the mode of action of baicalein and the structural determinants associated with its binding with sars-cov-2 3clpro were further explored using x-ray protein crystallography. the crystal structure of sars-cov-2 3clpro in complex with baicalein was determined at a resolution of 2.2 å ( fig. 2 ; table s2 ). the protease has a catalytic cys145-his41 dyad and an extended binding site, features shared by sars-cov 3clpro and mers-cov 3clpro ( hydrophobic interactions. consequently, baicalein is perfectly ensconced in the core of the substrate-binding pocket and interacts with two catalytic residues, the oxyanion loop (residues 138-145), glu166, and the s1/s2 subsites, which are the key elements for recognition of substrates as well as peptidomimetic inhibitors (17) . although baicalein did not move deeply into the s1 sub-pocket, its phenolic hydroxyl groups did form contacts with the crucial residue of this sub-pocket, his163, via the water molecule. by the aid of an array of direct and indirect hydrogen bonds with leu141/gly141/ser144, baicalein fixed the conformation of the flexible oxyanion loop, which serves to stabilize the tetrahedral transition state of the proteolytic reaction. these results together provide the molecular details of baicalein recognition by sars-cov-2 3clpro and an explanation for the observed potent activity of such a small molecule against the protease. the amino sequence of sars-cov-2 3clpro displays 96% sequence identity to sars-cov 3clpro. there are 12 residues differed in two proteases and none of them participates in the direct contacts with baicalein. the high level of sequence identified between two proteases allows one to assume that baicalein will bind to the sars-cov 3clpro in the same way as it does to sars-cov-2 3clpro. the inhibition assay shows that baicalein can also inhibit sars-cov 3clpro, with an ic50 of 1.18 ± 0.37 µm. thus, a three-dimensional model of sars-cov 3clpro in complex with baicalein was constructed by superimposing the crystal structure of sars-cov-2 3clpro/baicalein with that of sars-cov 3clpro/tg-0204998 (pdb code 2zu4) (fig. s4a ). the binding mode of baicalein with sars-cov 3clpro is distinctively different from those of known inhibitors. all of the crystal structures of the inhibitor-bound sars-cov 3clpro were collected for a comparison analysis (fig. s4b ). if those peptidomimetic inhibitors are delineated like "swords" to compete with the binding of substrate, baicalein works as a "shield" in front of two catalytic dyads to prevent the approach of the substrate to the active site (fig. s4b ). such a unique binding mode in combination with its high ligand-binding efficiency and small molecular weight renders baicalein valuable for drug development. we substrate-binding sites, particularly for the crucial s1/s2 subsites (7, 19, 20) . accordingly, substrate analogs or mimetics attached with a chemical warhead targeting the catalytic cysteine were designed as peptidomimetic inhibitors of 3clpros with a covalent mechanism of action (6). a series of diamide acetamides acting as non-covalent sars-cov 3clpro inhibitors and their binding modes examined by crystal structures have been reported, but they are more or less peptidomimetic inhibitors and a continuous development of these compounds is absent (21) . although several other small molecules have been declared as 3clpro inhibitors, a solid validation of their binding with 3clpros by itc or complex structure determination is lacking. as none of the known inhibitors has been moved to clinical trials, considerable efforts to discover novel small molecule inhibitors of 3clpros are urgently needed. in the authors sincerely thank prof. zihe rao and prof. haitao yang for kindly providing the protein as well as the substrate for the enzymatic assay. we also thank the staff from beamlines bl17u1 and bl18u1 at shanghai synchrotron radiation facility. we thank letpub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. the authors declare no competing interests. the sars-cov-2 3clpro-baicalein complex structure was deposited with the protein data bank with accession code 6m2n. all other data are available from the corresponding author upon reasonable request. figures s1-s4 tables s1 and s2 references (24-29) chemical shifts were reported in ppm (δ) coupling constants (j) in hertz. chemical shifts are reported in ppm units with me4si as a reference standard. the cdna of full length sars-cov-2 3clpro or sars-cov 3clpro was cloned into the for ligand binding screening and dissociation constant (kd) determination, the magnitude size was set to 128 k and 1000 single scans were added. (24). the purified sars-cov-2-3clpro protein was concentrated to 7 mg/ml for crystallization. one hour incubation of the protein with 10 mm baicalein was carried out before crystallization condition screening. crystals of the complex were obtained at 20 °c by mixing equal volumes of protein-baicalein and a reservoir (16 % peg6000, 100 mm mes, ph 5.8, 3% dmso) with a handing-drop vapor diffusion method. crystals were flash frozen in liquid nitrogen in the presence of the reservoir solution supplemented with 20% glycerol. x-ray diffraction data were collected at beamline bl18u1 at the shanghai synchrotron radiation facility (25) . the data were processed with hkl3000 software packages (26) . the complex structure was solved by molecular replacement using the program phaser (27) with a search model of pdb code 6lu7. the model was built using coot (28) and refined with a simulated-annealing protocol implemented in the program phenix (29) . the refined structure was deposited to protein data bank with an accession code listed in table s1 . the complete statistics as well as the quality of the solved structure are also shown in table s1 . the vero e6 cell line was obtained from american type culture collection (atcc, manassas, usa) and maintained in minimum eagle's medium (mem; gibco invitrogen) supplemented with 10% fetal bovine serum (fbs; invitrogen, uk) in a humid incubator with baicalein is shown as green spheres and other inhibitors together with two catalytic residues are shown as sticks. coronaviruses -drug discovery and therapeutic options sars and mers: recent insights into emerging coronaviruses early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster a pneumonia outbreak associated with a new coronavirus of probable bat origin an overview of severe acute respiratory syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy design of wide-spectrum inhibitors targeting coronavirus main proteases antiviral effect of forsythoside a from forsythia suspensa (thunb.) vahl fruit against influenza a virus through reduction of viral m1 protein antiviral activity of chlorogenic acid against influenza a (h1n1/h3n2) virus and its inhibition of neuraminidase chemistry and pharmacology of the herb pair flos lonicerae japonicae-forsythiae fructus antiviral natural products and herbal medicines baicalin and its aglycone: a novel approach for treatment of metabolic disorders the comparative study of the therapeutic effects and mechanism of baicalin, baicalein, and their combination on ulcerative colitis rat therapeutic potentials of baicalin and its aglycone, baicalein against inflammatory disorders the ecstasy and agony of assay interference compounds native state mass spectrometry, surface plasmon resonance, and x-ray crystallography correlate strongly as a fragment screening combination ph-dependent conformational flexibility of the sars-cov main proteinase (m(pro)) dimer: molecular dynamics simulations and multiple x-ray structure analyses remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs sars-cov 3cl protease cleaves its c-terminal autoprocessing site by novel subsite cooperativity discovery, synthesis, and structure-based optimization of a series of n-(tert-butyl)-2-(n-arylamido)-2-(pyridin-3-yl) acetamides (ml188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (sars-cov) 3cl protease combination of western medicine and chinese traditional patent medicine in treating a family case of covid-19 in wuhan safety, tolerability, and pharmacokinetics of a single ascending dose of baicalein chewable tablets in healthy subjects optimization of electrospray ionization by statistical design of experiments and response surface methodology: protein-ligand equilibrium dissociation constant determinations upgrade of macromolecular crystallography beamline bl17u1 at ssrf hkl-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes phaser crystallographic software coot: model-building tools for molecular graphics phenix: building new software for automated crystallographic structure determination a clinical isolate sars-cov-2 (5) was propagated in the vero e6 cells all the infection experiments were performed at biosafety level-3 (bls-3) after that, the virus-compound mixture was removed and cells were further cultured with a fresh compound containing medium. at 48 h p.i., the cell supernatant was collected and the viral rna in supernatant was subjected to qrt-pcr analysis as described previously (18). dmso was used in the controls. the experiments were performed in triplicates and three hplc-ms profiling of the active fraction 8 and 12 hplc-ms profiling of fration 8. (b) hplc-ms profiling of fration 12 key: cord-271648-m2c5bvuj authors: ashour, hossam m.; elkhatib, walid f.; rahman, md. masudur; elshabrawy, hatem a. title: insights into the recent 2019 novel coronavirus (sars-cov-2) in light of past human coronavirus outbreaks date: 2020-03-04 journal: pathogens doi: 10.3390/pathogens9030186 sha: doc_id: 271648 cord_uid: m2c5bvuj coronaviruses (covs) are rna viruses that have become a major public health concern since the severe acute respiratory syndrome-cov (sars-cov) outbreak in 2002. the continuous evolution of coronaviruses was further highlighted with the emergence of the middle east respiratory syndrome-cov (mers-cov) outbreak in 2012. currently, the world is concerned about the 2019 novel cov (sars-cov-2) that was initially identified in the city of wuhan, china in december 2019. patients presented with severe viral pneumonia and respiratory illness. the number of cases has been mounting since then. as of late february 2020, tens of thousands of cases and several thousand deaths have been reported in china alone, in addition to thousands of cases in other countries. although the fatality rate of sars-cov-2 is currently lower than sars-cov, the virus seems to be highly contagious based on the number of infected cases to date. in this review, we discuss structure, genome organization, entry of covs into target cells, and provide insights into past and present outbreaks. the future of human cov outbreaks will not only depend on how the viruses will evolve, but will also depend on how we develop efficient prevention and treatment strategies to deal with this continuous threat. coronaviruses (covs) were discovered in the 1960s and they were classified under family coronaviridae, which is the largest family within the order nidovirales ( figure 1 ) [1] . family coronaviridae encompasses two subfamilies: subfamily orthocoronavirinae and subfamily torovirinae ( figure 1 ) [1] . subfamily orthocoronavirinae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus ( figure 1 ) [1] . covs are typically harbored in mammals and birds and are common in camels, cattle, cats, bats, and other animals [2] . alpha and betacoronaviruses circulate in mammals, including bats ( figure 1 ) [2] . gammacoronaviruses mostly infect avian species the first discovered covs were ibv that causes respiratory disease in chickens and the human covs, human cov-229e (hcov-229e) and human cov-oc43 (hcov-oc43), which cause the common cold in humans [8, 9] . since the emergence of hcov-229e and hcov-oc43, several other hcovs were discovered, such as severe acute respiratory syndrome-cov (sars-cov) in 2002, hcov-nl63 in 2004, hcov-hku1 in 2005, middle east respiratory syndrome-cov (mers-cov) in 2012 [10] . starting december 2019, there were reports of patients presenting with severe viral pneumonia in the city of wuhan, china [11] . sequencing of the virus from these patients has identified a novel cov as the causative agent of this respiratory disease [11] . the 2019 novel cov virus (2019-ncov) was recently named sars-cov-2 by the world health organization (who). the disease caused by sars-cov-2 has been named covid-19. prior to 2002, covs were treated as nuisances but never as serious viruses. things changed after the emergence of sars-cov, which caused serious illnesses and deaths in 2002-2003 [12] . unlike all human covs that cause mild respiratory symptoms, sars-cov, mers-cov, and sars-cov-2 are associated with serious respiratory diseases [12, 13] . since its emergence, the sars-cov-2 has drawn well-deserved attention from the world. efforts are underway in an attempt to control this new cov outbreak. covs, including the newly discovered sars-cov-2, are spherical positive single-stranded rna viruses that are characterized by spike proteins projecting from the virion surface [14, 15] . the spherical morphology of the viral particle together with the spike projections led to the name coronavirus from the latin word corona meaning crown, due to the appearance of the virus as a royal crown under the electron microscope [14, 15] . covs are enveloped viruses (envelope is a lipid bilayer derived from the host cell membrane) with the viral structure formed primarily of structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, and hemagglutininfigure 1 . classification of different types of coronaviruses within the family coronaviridae, subfamily orthocoronavirinae, and the respective genera: alpha-, beta-, gamma-, and deltacoronaviruses. the sars-cov-2 is classified as a betacoronavirus. the first discovered covs were ibv that causes respiratory disease in chickens and the human covs, human cov-229e (hcov-229e) and human cov-oc43 (hcov-oc43), which cause the common cold in humans [8, 9] . since the emergence of hcov-229e and hcov-oc43, several other hcovs were discovered, such as severe acute respiratory syndrome-cov (sars-cov) in 2002, hcov-nl63 in 2004, hcov-hku1 in 2005, middle east respiratory syndrome-cov (mers-cov) in 2012 [10] . starting december 2019, there were reports of patients presenting with severe viral pneumonia in the city of wuhan, china [11] . sequencing of the virus from these patients has identified a novel cov as the causative agent of this respiratory disease [11] . the 2019 novel cov virus (2019-ncov) was recently named sars-cov-2 by the world health organization (who). the disease caused by sars-cov-2 has been named covid-19. prior to 2002, covs were treated as nuisances but never as serious viruses. things changed after the emergence of sars-cov, which caused serious illnesses and deaths in 2002-2003 [12] . unlike all human covs that cause mild respiratory symptoms, sars-cov, mers-cov, and sars-cov-2 are associated with serious respiratory diseases [12, 13] . since its emergence, the sars-cov-2 has drawn well-deserved attention from the world. efforts are underway in an attempt to control this new cov outbreak. covs, including the newly discovered sars-cov-2, are spherical positive single-stranded rna viruses that are characterized by spike proteins projecting from the virion surface [14, 15] . the spherical morphology of the viral particle together with the spike projections led to the name coronavirus from the latin word corona meaning crown, due to the appearance of the virus as a royal crown under the electron microscope [14, 15] . covs are enveloped viruses (envelope is a lipid bilayer derived from the host cell membrane) with the viral structure formed primarily of structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, and hemagglutinin-esterase (he) protein in some betacoronaviruses [16] . the s, m, and e proteins are all embedded in the viral envelope; pathogens 2020, 9, 186 3 of 15 however, n protein interacts with the viral rna and is located in the core of the viral particle, forming the nucleocapsid [16] . the s protein is a heavily glycosylated protein that forms homotrimeric spikes on the surface of the viral particle and mediates viral entry into host cells [17] . in some covs, each monomer of the homotimeric s protein exists as two subunits (s1 and s2) on the viral particle due to cleavage of s protein by host furin-like proteases during viral replication [17, 18] . however, in other covs including sars-cov, s protein forms s1 and s2 domains, remains intact on viral particles, and only gets cleaved inside endocytic vesicles during viral entry [19, 20] . the m protein is one of the most important proteins in the virion structure. it exists in higher quantities than any other protein in the viral particle, in contrast to the e protein which is found in small quantities within the virion [21] . the difference in abundancy may due to the fact that m protein gives the virus its shape and is critical together with e protein in orchestrating the assembly of the virus and in forming mature viral envelopes [22] . the e protein also functions in release of viral particles from host cells, in addition to other functions [22] . the n protein binds the viral rna and is required for packaging of viral rna into the viral particle during viral assembly [23, 24] . as mentioned previously, he is present on the surface of some betacoronaviruses. it is a hemagglutinin similar to influenza virus hemagglutinin (binds sialic acid on host cell-surface glycoproteins) and possesses acetyl-esterase activity [25] . he characteristics may enhance entry and pathogenesis of coronaviruses that contain such protein in their viral structure. the rna genome of covs is the second largest of all rna viruses, ranging from 26 to 32 kilobases (kb) in size [26] . the largest genome of all rna viruses is that of the recently described planarian secretory cell nidovirus, pscnv (41.1 kb genome size) [27] . viral rna codes for structural and nonstructural proteins [28] . the structural proteins together with a few nonstructural proteins, with different functions, are coded within the 3 end of the viral genome [28] . however, the 5' two-thirds of the genome codes for nonstructural proteins that are important in viral replication, including the rna-dependent rna polymerase (rdrp) [28] . once the viral genome is inside the host cell cytoplasm following viral entry, translation of the 5 end of viral rna produces the rdrp, which uses viral rna as a template to generate virus-specific mrnas (subgenomic mrnas) from subgenomic negative strand intermediates [29, 30] . subgenomic mrnas share the same 3 ends and the same leader sequence of 70-90 nucleotides at their 5 ends [28, 31] . translation of subgenomic mrnas leads to production of structural and nonstructural viral proteins [28] . once sufficient structural proteins and genomic viral rna are formed, viral rna is then assembled with viral structural proteins into virions. viral assembly and budding occur in smooth-walled vesicles in the endoplasmic reticulum-golgi intermediate compartment (ergic) [28] . s protein is the viral protein that mediates the entry of covs into host cells [32] . receptor-binding domain (rbd) within the s1 domain mediates binding to the cognate host cell receptor; however, the s2 domain mediates the fusion events, between viral membrane and host cell membrane, that are required for entry of covs into host cells [33, 34] . the rbd is located at n-terminus of the s1 subunit as in mouse hepatitis virus (mhv) or at the c-terminus as in sars-cov and mers-cov [34, 35] . the tissue tropism of covs is determined by the s protein interaction with the receptors on host cells. several cellular receptors were described as receptors for covs. for example, aminopeptidase n (apn) was identified as the receptor for several alphacoronaviruses [36] , angiotensin-converting enzyme 2 (ace2) as the receptor for sars-cov [37] , hcov-nl63 [38] and possibly for the newly discovered sars-cov-2 [39] , ceacam1 as the receptor for mhv [40] , and dipeptidyl-peptidase 4 (dpp4 which is also known as cd26) as the receptor for mers-cov [41] . a study on the rbd of sars-cov-2 s protein showed its similarity in structure to that of sars-cov with some key amino acid differences [42] . the previous finding suggests that sars-cov-2 could employ human ace2 as its cellular receptor. a recent study published in science showed that sars-cov-2 s protein has higher affinity to ace2 than sars-cov s protein [43] . however, the receptor usage and the cellular tropism of sars-cov-2 need further investigation. the trimeric cov s protein is cleaved by host cell proteases during infection to expose the fusion peptide of the s2 domain, which induces the fusion of viral and cellular membranes [18, 20, 44, 45] . fusion of viral envelope with host cell membrane results in the release of the viral genome into the cytoplasm [33, 34] . cleavage of s protein occurs at different sites that were identified, in different coronaviruses, to be between the s1 and s2 domains (s1/s2 site) and within the s2 domain proximal to the fusion peptide (s2 site) [18, 20, 44, 45] . it is believed that cleavage at both sites is required for viral entry [46] . different proteases have been identified to cleave the s1/s2 site depending on its amino acid sequence in different viruses. for example, the s1/s2 site of mers-cov s protein (rsvr↓sv) is cleaved by furin after its biosynthesis during viral replication [47] . sars-cov-2 has an s1/s2 site (ayt↓m) that is identical to the one in sars-cov [48] . this sars-cov s1/s2 site has been shown to be cleaved by cathepsin l following receptor binding and during viral entry in late endosomes [20] . we believe that the sars-cov-2 s1/s2 site may be cleaved by cathepsin l similar to sars-cov. other proteases such as trypsin, elastase, and tmprss2 have been shown to cleave sars-cov s protein at other sites between s1 and s2 domains [44, 46, 49] . unlike sars-cov, sars-cov-2 has an additional furin-like protease cleavage site (rrar↓sv) that is n-terminus to the s1/s2 site (ayt↓m), and is absent in sars-cov [48] . the presence of this furin-like cleavage site in sars-cov-2 suggests its cleavage by furin during viral egress. in addition to the previous s1/s2 sites, sars-cov-2 has a furin-like protease cleavage s2 site (kr↓sf) that is identical to that in sars-cov [48] . however, there is no evidence that sars-cov s protein is cleaved by furin-like proteases at the s2 site during viral egress [20] . similar to s1/s2 site, we believe that sars-covs protein is cleaved at the s2 site by cathepsin l or tmprss2 in different cellular locations during viral entry. the previous notion is supported by several studies which showed that cathepsin l and tmprss2 promote sars-cov entry while their inhibition suppressed infection of permissive cells [20, 44, 50] . given that sars-cov-2 has the same s2 site as sars-cov, we believe that processing of sars-cov-2 s protein at s2 site is similar to sars-cov. similarly, mers-cov has an s2 site (rxxr↓sa) that is less efficiently cleaved by furin and most probably cleaved by tmprss2 or cathepsin l during viral entry [51] . despite the identification of putative protease cleavage sites in sars-cov-2 s protein, their relative importance for sars-cov-2 s protein activation, viral pathogenesis, and cellular tropism needs further investigation. antiviral small molecules that inhibited cathepsin l were able to inhibit sars-cov infections in vitro and that of other viruses that depend on cathepsin l for entry, such as ebola, hendra, and nipah viruses [50] . the presence of a cathepsin l cleavage site in sars-cov-2 s protein (s1/s2 site) suggests that cathepsin l inhibitors may be valuable in inhibiting sars-cov-2 infections [48] . antibodies against rbd and s2 domain of sars-cov and mers-cov s proteins have been found effective in neutralizing infections of permissive cell lines in vitro [52] [53] [54] [55] . in addition, neutralizing antibodies were capable of treating infections in experimental animals and in infected patients during these major outbreaks [56] [57] [58] [59] . in one study, several sars-cov rbd-specific monoclonal antibodies did not bind to sars-cov-2 s protein [43] . another study showed that the sars-cov-specific monoclonal antibody, cr3022, bound with high affinity to sars-cov-2 rbd [60] . the previous studies suggest that there are differences between the two rbds which impact the cross reactivity of many neutralizing antibodies. however, neutralizing antibodies against sars-cov-2, once developed, could be promising in controlling the current sars-cov-2 outbreak. around 8098 cases that were infected over nine months (around 10% fatality) ( table 1 ) [12] . sars-cov was found to infect unciliated bronchial epithelial cells and type ii pneumocytes and cause fever, cough, shortness of breath, and severe complications such as pneumonia and kidney failure [12, 37] . the incubation period for sars-cov was estimated to range from 2 to 10 days, and up to 14 days (table 1 ) [61] . studies have shown that bats harbor covs that are ancestral to sars-cov (table 1 ) [62] . civets and raccoon dogs, of chinese local markets, were shown to harbor sars-like covs (table 1 ) [63] . the detection of sars-related covs (sarsr-covs) in bats and small animals in retail markets may indicate an interspecies transmission from bats to small animals and finally to humans (figure 2 ). studies in bats from different regions of china have identified several sarsr-covs [64] . the previous finding indicates that sars-cov has been circulating in bats for a long time before genetically changing and jumping to humans. since ace2 was identified as the receptor for sars-cov, it is not surprising that sars-cov has adapted itself to bind human ace2 and efficiently infect human cells [65] . that sort of adaptation required a set of amino acid changes in the rbd of s protein of sars viruses that were circulating in bats [65] . therefore, we conclude that the human-to-human transmission that was seen during the sars-cov outbreak is attributed to the ability of sars-cov to adapt its s protein (particularly rbd) to efficiently bind to human ace2 and infect airway epithelia ( figure 2 ). [62] . civets and raccoon dogs, of chinese local markets, were shown to harbor sarslike covs (table 1 ) [63] . the detection of sars-related covs (sarsr-covs) in bats and small animals in retail markets may indicate an interspecies transmission from bats to small animals and finally to humans (figure 2 ). studies in bats from different regions of china have identified several sarsr-covs [64] . the previous finding indicates that sars-cov has been circulating in bats for a long time before genetically changing and jumping to humans. since ace2 was identified as the receptor for sars-cov, it is not surprising that sars-cov has adapted itself to bind human ace2 and efficiently infect human cells [65] . that sort of adaptation required a set of amino acid changes in the rbd of s protein of sars viruses that were circulating in bats [65] . therefore, we conclude that the human-tohuman transmission that was seen during the sars-cov outbreak is attributed to the ability of sars-cov to adapt its s protein (particularly rbd) to efficiently bind to human ace2 and infect airway epithelia ( figure 2 ). mers-cov was first described in 2012 as a new cov that causes a severe respiratory disease in saudi arabia [13] . similar to sars-cov, mers-cov infects unciliated bronchial epithelial cells and mers-cov was first described in 2012 as a new cov that causes a severe respiratory disease in saudi arabia [13] . similar to sars-cov, mers-cov infects unciliated bronchial epithelial cells and type ii pneumocytes and causes severe illness of the respiratory tract, which is characterized by fever, cough, shortness of breath, and severe complications such as pneumonia and kidney failure [13] . the incubation period of mers-cov is quite similar to sars-cov and ranges from 2 to 14 days (table 1 ) [66] . as of january 2020 and since 2012, 862 of 2506 infected cases in 27 countries have died (≈35% fatality), which is more than three times the fatality seen in sars-cov infections (table 1 ) [67] . however, unlike sars-cov, human-to-human transmission of mers-cov is not easy and has not been confirmed except in cases of very close contact with infected patients in health care settings [67] . mers-related covs (mersr-covs) were detected in bats, suggesting a potential bat origin (table 1) [68, 69] . mers-cov was transmitted to humans from dromedary camels ( table 1 ) [70] . studies have also shown that camel mers-cov strains are almost identical to human mers-cov strains [71] . it was postulated that mers-cov existed in camels at least 30 years ago since antibodies to mers-cov were detected in samples that were collected from camels in 1983 [72] . sequence analyses have shown that mersr-covs' rbds share only 60-70% sequence identity with that of human and camel mers-covs [73] . similar to the adaptation of sars-cov to human host, mersr-covs that are circulating in bats had to undergo several amino acid changes in rbd of s protein to become capable of infecting camels and humans ( figure 2 ) [74] . we believe that the amino acid changes in mersr-covs' rbd led to the emergence of mers-cov strains that are capable of binding to human dpp4 with high affinity, infecting humans, and causing the 2012 outbreak ( figure 2) . as mentioned previously, the sars-cov-2 was isolated and sequenced from patients that showed symptoms of respiratory illness and pneumonia in wuhan, china during december 2019. sars-cov-2 is the third identified human cov that causes severe respiratory illness with symptoms and incubation period resembling that of sars-cov and mers-cov infections (table 1) [11, 75] . since december 2019, sars-cov-2 infection rates have been rising in china and worldwide [42] . similar to sars-cov and unlike mers-cov, human-to-human transmission has been confirmed [42] . initial cases of sars-cov-2 infections were somehow connected to the huanan seafood market in wuhan, in the hubei province of china [11] . in this market, a number of nonaquatic animals were on sale, such as birds, snakes, marmots, bats, and rabbits [11] . genetic analyses of viral samples from patients with sars-cov-2 infections revealed that the sars-cov-2 is a betacoronavirus that has 88% sequence identity to two bat sarsr-cov: 79% identity to sars-cov and only 50% identity to mers-cov [42] . the previous findings suggest that sars-cov-2 is a new virus that is distinct from sars-cov and mers-cov but most probably originated in bats, similar to sars-cov and mers-cov [42] . another recent study confirmed that sars-cov-2 significantly clustered with a sequence from the bat sars-like cov that was isolated in 2015 [76] . however, the existence of an intermediate host for sars-cov-2 is still not verified (figure 2 ). to date, the fatality of sars-cov-2 appears to be less than that observed in sars-cov and mers-cov infections. however, since new cases are confirmed everyday as we write this review, the fatality of this virus may keep changing and will not be accurately calculated until after the end of this outbreak. the virus appears to be more fatal in elderly patients or patients with comorbidities [77] . however, it is important to note that there could be cases that went undetected, which makes it hard to accurately calculate the fatality of this new virus. our lessons learned from sars-cov and mers-cov outbreaks include the high mutation rates that characterize all rna viruses [78] , the evolving nature of covs [6, 7] , and the ease of transmission from one species to another [6, 7] . as mentioned previously, it appears that sars-and mers-covs arose at sometime from ancestral covs harbored by bats (figure 2 ). whereas animals served as intermediate hosts, humans served as terminal hosts (figure 2 ). sars-cov was transmitted to civets and raccoon dogs, and to camels in the case of mers-cov (figure 2 ). they were then transmitted pathogens 2020, 9, 186 7 of 15 from these intermediate animal hosts to humans (figure 2 ). the practice of eating raw meat and the close contact between humans and animals are both risk factors for the initiation of a new human cov outbreak. this is due to the constant exposure of humans in these cultures to the ever-changing mutant covs. the first cases of sars-cov-2 infections were reported in the chinese city of wuhan during december 2019 [11] . although other options have not been completely ruled out yet, it is believed that the sars-cov-2 stemmed from a large seafood and animal market in wuhan, the capital of hubei province, china [11] . as for other wet markets in china, live animals are sold, mostly for food or medicine. the attributed medicinal and/or magical uses of wildlife and rare animal parts (such as pangolin scales and tiger paws) are mainly based on traditional chinese medicine (tcm), which has been widely promoted by the current chinese government. however, it is important to note that most of these folk remedies are never prescribed in reputable tcm hospitals. the wuhan market is known to have a lot of exotic animals and exotic animal parts [42] . thus, sars-cov-2 disease can be considered a zoonotic disease (like sars) that has initially spread from animals to humans. however, human-to-human transmission has also been confirmed [79] . it is not well understood why outbreaks of cov infections are mostly occurring in china. we speculate that those viruses may be predominantly circulating in animals in china rather than other animals in different parts of the world. one of the reasons for these sudden outbreaks could be the close interactions with live and wild animals that are consumed as food in wholesale food markets in china. in a very recent study, the genomes of covs isolated from nine patients having viral pneumonia in wuhan were analyzed [42] . the study showed that the genomes of these viruses differed by less than 0.1 percent (more than 99.98% of sequence identity), which indicates that the virus has only recently emerged in humans and has been detected rapidly after its emergence [42] . as the virus keeps spreading to more individuals, more mutations may arise which can potentially make the virus more virulent and thus constant surveillance will be necessary. the u.s. center for disease control and prevention (cdc) reported that sars-cov-2 causes a respiratory illness that is characterized by fever, cough, and shortness of breath. radiographs of some sars-cov-2 patients demonstrated invasive lesions in both lungs [80] . the cdc also reports that the elderly, individuals with underlying health problems, and people with compromised immune systems are at a particularly higher risk of developing severe pneumonia from the virus [77] . we believe that it is too early to assess the impact of the new virus on children. sars-cov infections were significantly less common among children than adults, and kids younger than 12 reported much less severe symptoms than patients who were more than 12 [81] . this may have to do with children being exposed to more cov in school and the outdoors than adults or because of the better overall health status of children as compared to adults. also, children tend to be more up-to-date with vaccinations, which may protect them from secondary infections that are often triggered by the main infections. during the sars-cov outbreak, most schools and factories in china remained open. following the sars-cov-2 outbreak and confirmation of many infected cases, china responded by locking down residents of wuhan city, banning wildlife trade until the epidemic is over, and attempting to build two new hospitals in the city of wuhan to specifically handle the new outbreak. it is currently unclear if these two hospitals will be able to handle a major outbreak in a city of 11 million residents. there is no definite information about the exact time of the start of the outbreak. that is why it is hard to assess how contagious the virus is (the rate of sustained spread) at the present time. however, it seems likely and plausible that it is highly contagious, based on the mounting data about human-to-human transmission outside china [42] . in order to be able to precisely assess the rate of sustained spread, information about numbers of cases and deaths (the overall number of patients) will need to be divided by the overall number of people at risk of acquiring the disease (the number of individuals who have been in contact with the patients). once this is determined, the overall risk can be assessed. underreporting or misdiagnosis of cases can negatively impact the calculations and thus delay the ability of public health officials to truly assess the situation. in an outbreak of this magnitude, the most important number that public health experts will be looking for is the basic reproduction number, also known as the r0 [82] . this number measures the disease's potential and represents the average number of people who will catch the disease from one infected person in a population that has never had the disease in the past [82] . in one study, the mean estimate of r0 was calculated to be between 2.24 and 3.58 [83] . another study estimated the r0 to have a high average value of 2.5 [84] , which is consistent with other groups that reported values from 2 to 3 [83, [85] [86] [87] [88] . these r0 estimates for the sars-cov-2 are consistent with r0 estimates for sars-and mers-covs (from 2 to 5) (table 1 ) [89, 90] . the virus is less deadly than sars, which killed about 10% of the infected patients. because r0 represents an average, outliers (carriers who infect so many people or carriers who infect nobody) can have a huge impact on the final value of the r0. in other words, a bigger r0 does not necessarily mean more infections. for example, the r0 for the seasonal flu typically ranges from 1.2 to 1.4 [91] , but it still infected many more people than the number of people who got infected with sars-cov. at any rate, any r0 above 1 should be taken seriously. the goal will be to reduce the r0 to a value that is below 1. finally, r0 estimates can be higher than the "true" r0 values because of two main reasons: infected people who did not show symptoms and infected people who did not report their symptoms. since r0 is not an intrinsic property of the virus itself, r0 estimates tend to be lower in places where there are sound infection control methods and vice versa. typically, r0 estimates are highest at the beginning of an outbreak and then subside gradually once countries become aware of the outbreak and manage to put in effective control measures to prevent the spread of the virus. the lockdown strategy that china is implementing works best during the early stages of an infection, which is not the case in wuhan, as there are several million people who already left the city before the restrictions were imposed. if we are beyond the early stages, then there can be disadvantages associated with such lockdown on wuhan. among the disadvantages are people evading care to avoid any restrictions on their life. given that the incubation period can be up to 11 days or more, a two-week federal quarantine was ordered for 195 u.s. citizens who were flown back from china [92] . the action was described by the cdc as precautionary and preventive. for comparison, there were no quarantines ordered in the u.s. for the recent sars-cov or mers-cov outbreaks. the last federal quarantine in the u.s. was ordered back in the 1963 to prevent the spread of smallpox from sweden to the u.s. during a smallpox outbreak in sweden [93] . as shown in table 1 , the number of people infected by the virus has exceeded the global total infected with sars-cov (8098 individuals) in a nine-month period that extended from 2002 until 2003. it will be difficult to control the disease without a level of disruption of air travel. we believe that limiting travel to and from china will be a necessary measure. respiratory viruses spread through respiratory droplets that are produced when an infected person coughs or sneezes. the exact modes of transmission of sars-cov-2 are not entirely clear at this early stage, but reports of healthcare professionals in china who contracted the disease suggest a highly contagious virus [77] . prevention of the sars-cov-2 infections entail precautions that are common to other respiratory viruses. the most obvious measure is to avoid contact with people who are sick. this is especially important due to the contagious nature of the virus. if someone is sick, they should stay home. once they recover, they may consider using disposable face masks (while frequently changing them) and avoiding close contact with coworkers. however, the value of wearing face masks is controversial, to say the least [94] . surgical masks do not fully protect against airborne viruses as they do not fully seal the nose and the mouth. thus, small droplets, which can travel farther than large droplets and in more unpredictable patterns, can be inhaled around the sides of the masks. the n95 masks offer a better protection as long as they fit properly. it is worth noting that n95 masks are not suitable for people with facial hair [95] . being an enveloped virus, washing hands with water and soap for at least 30 s would be beneficial in deactivating the sars-cov-2 [96] . hand sanitizers can also be used if water and soap are not readily available, while touching eyes, nose, and mouth should be prevented [97] . disinfection of different environmental surfaces, tools, and objects is crucial in limiting the spread of the virus [97] . the more you use one of these objects or touch one of these surfaces, the more pressing cleaning and disinfection become. public health officials, local health departments, hospitals, doctors, and cdc personnel should work closely with universities and other workplaces to educate and provide needed supplies to contain the spread of the virus. the world health organization (who) declared sars-cov-2 as a public health emergency of international concern (pheic) on 30 january 2020 [98] . a pheic is an atypical event that constitutes a public health risk and potential for the disease to spread to other countries, thus requiring a coordinated international response [99] . this designation could help mobilize more resources to the impacted areas. the current outbreak represents the sixth time the who has declared a global emergency since it gained the power to declare an international emergency in 2005 [100] . the previous five times were the 2009 h1n1 swine flu, the 2013 ebola outbreak in west africa, the 2014 polio outbreak, the 2016 zika outbreak, and 2019 ebola outbreak in the democratic republic of congo [100] . none of these previous emergencies led a worldwide pandemic. however, we predict that the current outbreak is more likely to become a pandemic. the current diagnostic test for the virus is pcr-based [101] . since this test typically takes 48 h, a new quicker diagnostic test needs to be developed. it will not be practical to isolate (quarantine) a large number of individuals until results of the pcr-based diagnostic test become available. this can also overwhelm healthcare facilities, already overwhelmed by the increasing number of cases that are discovered every day in china and elsewhere in the world. containing the outbreak before it can spread is the best way to prevent pandemics. border closures and screening at airports and checkpoints are classical measures that were previously implemented in the 2009 h1n1 flu pandemic [102] . this can reduce the spread of the virus but will not be a fool-proof strategy. the reason is that the incubation period of the virus is believed to be as long as 14 days, as was the case with mers-cov [103, 104] . this means that carriers of the virus can show up at the border with no apparent symptoms and readily pass through security without raising any red flags. compared to the sars-cov outbreak in 2003, the current increase in air traffic in china and worldwide has likely contributed to the more rapid spread of sars-cov-2 in 2020. without a quick diagnostic test, there are not many good options that can completely stop the transmission of the virus. once a test is available, cases can be identified and isolated. based on previous genetic experience with sars-cov, scientists will need to quickly develop a vaccine for sars-cov-2. the world is hoping to succeed in containing this virus as soon as possible. even after success, there needs to be follow-up with patients who are cured and declared virus-free. this is a lesson we learned from the ebola outbreak, in which some patients who walked out from the hospital "virus-free" were found later to harbor the ebola virus that was carefully hiding itself in other parts of the body, such as the immune-privileged eye [105] [106] [107] . although this hidden ebola virus was no longer transmissible to other humans, it rendered the label "virus-free" 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transmission and control retinal pigment epithelial cells are a potential reservoir for ebola virus in the human eye immune tolerance elicited via unique ocular and oral routes key: cord-301974-4wn40ivq authors: berry, jody d; jones, steven; drebot, michael a; andonov, anton; sabara, marta; yuan, xin y; weingartl, hana; fernando, lisa; marszal, peter; gren, jason; nicolas, brigitte; andonova, maya; ranada, francesca; gubbins, michael j; ball, t.blake; kitching, paul; li, yan; kabani, amin; plummer, frank title: development and characterisation of neutralising monoclonal antibody to the sars-coronavirus date: 2004-09-01 journal: j virol methods doi: 10.1016/j.jviromet.2004.04.009 sha: doc_id: 301974 cord_uid: 4wn40ivq there is a global need to elucidate protective antigens expressed by the sars-coronavirus (sars-cov). monoclonal antibody reagents that recognise specific antigens on sars-cov are needed urgently. in this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mabs) against the sars-cov is presented, based upon their specificity, binding requirements, and biological activity. initial screening by elisa, using highly purified virus as the coating antigen, resulted in the selection of 103 mabs to the sars virus. subsequent screening steps reduced this panel to seventeen igg mabs. a single mab, f26g15, is specific for the nucleoprotein as seen in western immunoblot while five other mabs react with the spike protein. two of these spike-specific mabs demonstrate the ability to neutralise sars-cov in vitro while another four western immunoblot-negative mabs also neutralise the virus. the utility of these mabs for diagnostic development is demonstrated. antibody from convalescent sars patients, but not normal human serum, is also shown to specifically compete off binding of mabs to whole sars-cov. these studies highlight the importance of using standardised assays and reagents. these mabs will be useful for the development of diagnostic tests, studies of sars-cov pathogenesis and vaccine development. the sars-coronavirus (sars-cov) is recognised as the causal agent of severe acute respiratory syndrome (sars) in humans. this virus caused nearly 800 deaths and infected more than 8000 people in various affected countries throughout the world (stadler et al., 2003) . the sars-coronavirus spike protein has only 20-27% pairwise identity at the amino acid level to the spike proteins of other previously characterised coronaviruses. recently, the genomes of sars-cov isolates, implicated in the 2003 toronto outbreak, were sequenced in their entirety (marra et al., 2003; rota et al., 2003) . the production of mabs to the sars-cov virus is critical for diagnostic development, vaccine research and studies of viral pathogenesis. assays that detect the presence of virally encoded proteins or nucleic acids may be preferable for diagnosis of sars infections as the development of serum antibodies in infected individuals is quite protracted (li et al., 2003a) . coronaviruses are enveloped, single-stranded rna viruses that replicate in the host cell cytoplasm (fields et al., 2001) . the coronaviruses form a single genus of the family coronaviridae and the virions are large (80-160 nm in diameter) pleomorphic, but generally spherical, particles. virions of most coronaviruses contain three major proteins: the phosphorylated nucleocapsid protein (n); a small membrane-embedded glycoprotein (m); and a large club-shaped peplomer glycoprotein (s) which appears in em micrographs as protruding spikes 20 nm in length. the m protein is synthesised on ribosomes bound to the endoplasmic reticulum and accumulates in the golgi apparatus. the subcellular localisation of m protein to the golgi is believed to influence the site of virus budding in the infected cell. the s-protein mediates many of the biological properties of the virus, including attachment to cell receptors, penetration, and cell-fusion, and it is the major target for virus-neutralising antibodies (collins et al., 1982; talbot et al., 1984; wege et al., 1984; jimenez et al., 1986; laude et al., 1986; godet et al., 1994) . a portion of the s glycoprotein that is not incorporated into budding virions is transported to the plasma membrane of the cell where it remains bound to the cell surface (gerna et al., 1982) . coronaviruses infect a wide range of mammalian hosts to produce a variety of disease outcomes including respiratory disease, enteritis and encephalitis. antigenic similarities between various coronaviruses have been demonstrated to reside in the s-protein and have been used to study the evolution of this virus family (brian et al., 1983) . for most coronaviruses causing enteric and respiratory diseases the pathophysiological events leading to clinical symptoms are due to the acute cytocidal infection of the target cells. these infections can be limited by the local immune response resulting in the production of secretory antibodies specific for the s-protein (enjuanes et al., 1995) . in contrast, many coronaviruses are maintained and spread in the population as inapparent and subclinical infections. the sequence of events leading to chronic versus acute disease is unknown but likely depends on the expression of viral genes, the functional impairment of host cells, and the interaction with the host immune response. there is a critical need to elucidate the immunologic basis for protection against sars-cov infection. recently the sars s-protein was shown to be a functional fusogen and is about 180-200 kda in size (xiao et al., 2003) . a host cell receptor, angiotensin-converting enzyme 2 (ace-2) was recently identified as a functional receptor for the sars-cov, and mediated infection of 293t cells in vitro (li et al., 2003b) . therefore, antibody responses to the s-protein may neutralise the infectivity of the sars-cov. the immunogenetics of antibody responses to protective epitopes is of particular importance and will lead to a clearer understanding of the nature of protective antibody responses to sars. lastly, the production of protective monoclonal antibodies may lead to the development of new recombinant therapeutic antibodies in order to provide rapid protection in sars patients. in the present work, a description of the development of murine mabs against the sars-cov involved in the toronto sars outbreak is presented. the mabs were analysed for pertinent immunochemical properties and for their ability to neutralise sars-cov in vitro. for preparation of partially purified whole-virus antigen, sars-cov was expanded after plaque purification in vero-6 cell monolayers and partially purified through a sucrose cushion. highly purified sars-cov (tor-3 strain, isolated from a patient infected in the toronto sars outbreak; krokhin et al., 2003) was prepared in the same way, except the viral particles were further purified using gradient centrifugation. briefly, 500 ml of supernatant from sars-cov infected vero-6 cells was concentrated first on top of a cushion of iodixanol in a beckman sw32 rotor (mississauga, on). the virus was subsequently mixed to form a suspension of 20% iodixanol and subjected to centrifugation in a beckman nvt 90 rotor (mississauga, on) for 3.5 h at 400,000 × g. fractions were collected from the bottom of the self-generated gradient, tested by western immunoblot with sars-cov-infected, convalescent human patient serum, and the sars-cov positive fractions were pooled and dialysed against phosphate buffered saline (pbs). the dialysed virus preparation was further concentrated by ultracentifugation for 1.5 h at 150,000 × g. immunisation of mice was performed according to ncfad standard operating procedures under iso17025. five-to six-week-old female balb/c mice (charles river, wilmington, ma) were injected subcutaneously (sc) with 50 g of beta-propiolactone-inactivated, partially purified sars-cov (tor-3 strain) with an equal part of complete freund's adjuvant (h37-ra, cfa) from difco (bd, oakville, on) on day 1. on day 30 the mice received 50 g of partially purified sars-cov s.c. in incomplete freund's adjuvant (ifa) in a total volume of 100 l. on days 48 and 63, the mice received 5 g of the same antigen in a total volume of 100 l sc with ifa. the mice received a final booster injection with 5 g of highly purified sars-cov in 200 l pbs to the intra-peritoneal cavity 3 days prior to hybridoma fusion. mice were euthanised by anaesthesia overdose and exsanguinated by cardiac puncture. the spleens were subsequently excised under aseptic conditions. infected vero cells were scraped off of 162 cm 2 corning tissue culture flasks (corning, ny) and clarified by centrifugation. a borate saline mixture (0.05 m boric acid, 0.12 m, nacl, 0.024 m naoh) was used to wash the cell pellet twice and the pellet was resuspended in 2 ml borate saline +1% triton x-100 for each t162 flask. the pellet was kept at 4 • c using a water bath and sonicated for ten minutes at 50% power. the debris was pelleted via centrifugation at 10,000 × g for 10 min and the supernatant was collected and stored at −20 • c in aliquots for later use. removal of mouse spleens, preparation of spleen and myeloma cells, and the fusion for hybridoma production were performed according to ncfad standard operating procedures under iso17025. ampoules of the myeloma cell line p3x63ag8.653 (atcc, rockville, md) were thawed 1 week prior to fusion and grown in bd cell mab quantum yield medium in the presence of 8-azaguanine (sigma, oakville, on). cells were in log-phase growth at the time of fusion. hybridoma fusion was performed essentially as originally described (kohler and milstein, 1975) with the following modifications. briefly, spleens were harvested 3 days after a final boost with a given antigen and the splenocytes were prepared by splenic perfusion as follows. under aseptic conditions, the spleens were perforated with a 10 cm 3 syringe with a 21 gauge sterile disposable needle. the spleen cells were perfused out of the spleen with injections of serum free bd cell mab quantum yield medium (bd-pharmingen, oakville, on). two identically immunised mouse spleens were used to produce these hybridoma clones. the fusion was performed using the p3x63ag8.653 myeloma line in log-phase growth. peg1500 (1 ml; roche, basel, sw) was added drop-wise over 1 min while gently tapping the tube containing the thoroughly washed myeloma-splenocyte pellet. the peg 1500 was slowly diluted out over three minutes with serum free bd-cell mab quantum yield medium. the cells were resuspended and mixed into 90 ml of stemcell clonacell medium d (hat) (vancouver, bc) containing 5 ml origen hybridoma cloning factor (hcf) (igen, gaithersburg, md) and plated out according to the manufacturer's instructions. the plates were incubated at 37 • c under a 5% co 2 overlay for 10-18 days in humidified chambers. visible colonies were picked from the plates after approximately 2 weeks growth and placed into 96-well plates containing 150-200 l of complete hybridoma medium supplemented with 1× hypoxanthine thymidine (sigma, oakville, on), 4% hcf and 10% fbs (wisent). supernatants were screened 4 days later via elisa using purified virus as antigen. isotyping was performed using a commercial murine isotyping dipstick test (roche, basel, sw) according to the manufacturer's instructions. hybridoma culture supernatants were concentrated 5-10 fold using amicon stirred cell nitrogen concentrators with 30 kda cutoff millipore (ym-30) membranes (both from millipore, billerica, ma). hybridoma culture supernatants were assayed for binding to highly purified sars-cov in an elisa assay when the cultured cells were confluent in the culture plates. the costar 3690 96-well 1/2 well elisa plates (corning, ny) were coated with either bovine serum albumin (bsa) or highly purified sars-cov (18-37 ng/well) in pbs overnight at 4 • c and then blocked with 0.4% bsa in pbs, for 2 h at 37 • c. the supernatant (30 l/well) was incubated neat for 1 h at 37 • c. the elisa plates were washed 10 times with distilled water and patted dry on a paper towel. a pan-goat anti-mouse igg-hrp antibody (southern biotechnology associates, birmingham, alabama) was diluted to 1:2000 in 0.2% bsa in pbs, applied to the elisa plates for 45 min at 37 • c, and then washed as described above. positive binding was detected with commercial abts used according to the manufacturer's instructions (roche, basel, sw). the od was read at 405 nm at 15 and 60 min intervals after addition of the developing reagent. mouse immune and preimmune sera were diluted 1:2000 with 2%-bsa in pbs for use as positive and negative controls, respectively, and for the establishment of the hybridoma screening assay. competition elisa (c-elisa) measured the binding of murine mabs to highly purified sars-cov in the presence of human serum. infected serum was from confirmed infected patients with well-established sars. these samples were from the initial set of patients from which the virus was isolated. plates were coated with highly purified sars-cov at 18 ng per well, and normal human serum or serum from convalescent sars patient s3, serially diluted in 2% bsa-pbs, was allowed to react on the pre-blocked plates for 30 min. mab f26g6 (spike specific) or f26g15 (nucleoprotein specific) were pre-diluted to a concentration that gives approximately half-maximum optical density after 1 h development in the presence of no competing serum. the diluted mab preparations were then applied to the wells, and the incubation and development of the c-elisa was performed as described above. whole virions and sars-cov-infected vero cell lysates, at a final total protein concentration of 1 g per lane, were boiled in sds-loading buffer for 10 min. the samples were loaded in criterion pre-cast gels (biorad, mississauga, on) and electrophoresed at 200 v for 30 min. the proteins were transferred to immobilon nylon membranes (millipore, billerica, ma) for 2 h at room temperature at 100 v, or overnight at 27 v at 4 • c. blots were blocked with 3% bsa in tbs, rinsed three times with tbs, and reacted with monoclonal antibody overnight at 4 • c. the antibody supernatants were reacted neat and the concentrated supernatants were diluted 1:50 in 0.2% bsa in pbs. blots were washed three times with tbs-tween-20 (0.05%) for 5 min before being incubated with secondary antibody (same as above) at 1:1000 in tbs, 0.2% bsa for 1 h. the blots were washed as above and developed using dab insoluble substrate (pierce, rockford, il). monolayers of sars-infected vero cells were stained as follows. glass slides were coated with infected vero cell monolayers and fixed with acetone. the slides were irradiated with 20 kilogreys from a cobalt gamma irradiator, removed from biocontainment, and then stored at −80 • c. dilutions of antibodies and test sera were made initially in 96-well plates (bd-falcon, oakville, on) in sterile phosphate-buffered saline (ph 7.3). samples were allowed to incubate for 45 min in a 37 • c incubator, and were washed with distilled water. fluorescein labelled goat anti-mouse secondary antibodies (sigma, oakville, on) diluted in pbs were added to the slides and incubated for 45 min at 37 • c, washed as above, and air dried. slides were coated with mounting medium and stored at 4 • c until examined. immuno-dotblot analysis was performed using immobilon nylon membranes (millipore, billerica, ma). a total of 15 l of sars-cov antigen (infected vero cell lysate) or 5 g of highly purified virus is coated (per spot) for 1 h at 37 • c. in an attempt to further characterise the immunochemical properties of the individual mabs, antigen was pre-treated in several different conditions as follows: untreated native antigen; denatured by heat treatment at 95 • c for 5 min; denatured by sodium dodecyl sulphate treatment (sds) 2% prior to coating on the membrane; denatured by both heat treatment and sds as above together; reduced with beta-mercaptoethanol (5%) prior to coating on the membrane; denatured by temperature as above and reduced as above, together; denatured by both heat and sds in the presence of beta-mercaptoethanol. antigen-coated blots were blocked with 3% bsa in tbs for 2 h at 37 • c, and rinsed three times with tbs, prior to incubation with mabs. the concentrated mab supernatants were diluted 1:50 in 0.2% bsa in tbs (tris-buffered saline, ph 7.2) and reacted overnight at 4 • c on a rotary platform shaker with gentle agitation. blots were washed three times with tbs-tween-20 (0.05%) for 5 min before being incubated with secondary antibody (goat anti-mouse igg-hrp, southern biotech, same as above) at 1:2000 in tbs, 0.2% bsa for 1 h. the blots were washed as above and developed using dab insoluble substrate (pierce, rockford, il). the elisa positive monoclonal antibodies were screened for neutralisation of sars-cov using two independent formats. the first was a standard plaque reduction assay and the second measured reduction of cytopathic effect (cpe) in a microtiter format. a standard plaque reduction neutralisation test was performed as previously described (beaty et al., 1989) using highly purified sars-cov. briefly, mixtures of pre-titred (100 pfus) sars-cov and serial two-fold dilutions of hybridoma supernatant were incubated at 37 • c for 1 h and added to six well plates containing vero cell monolayers. after incubation at 37 • c for 1 h, a nutrient-agar overlay was added and the plates were placed in a co 2 incubator at 37 • c for approximately 3 days. a second overlay was then added which contained neutral red as a vital stain. plates were then checked periodically over the next few days for plaque formation. the highest dilution tested that produced a plaque reduction of at least 90% was defined as the titration end point. a microtiter format cpe reduction assay was used to test the sars-cov reactive mabs for neutralisation of sars-cov and transmissible gastroenteritis virus diamond strain (kindly provided by dr. susy carman, lsd, university of guelph). briefly, concentrated hybridoma supernatants were diluted 1:10 in cell culture medium and incubated with 100 tcid50 of either highly purified sars-cov (tor-3 strain), or tgev, for 1 h at 37 • c, for a final dilution of 1:20. the virus-antibody mix was then transferred onto cell monolayers in 96-well plates (costar, corning, ny). vero v-76 cells were used for the sars-cov, st cells for the tgev. the plates were incubated until cpe developed in virus back titration controls. elisa screening on highly purified sars-cov identified a panel of 103 mabs reactive to the sars-cov antigen preparation. negative screening on bsa reduced this number to a panel of 17 igg/k type mabs ( fig. 1; table 1 ). in general, binding reactivity of these mabs is greatly affected by both the conformation and purity of the antigen as illustrated by the decreased binding of these mabs, when tested in elisa, to heat denatured pure sars-cov as antigen compared to native virus (fig. 1) . this clearly shows the importance of selecting suitable antigen for screening assays. western immunoblot analysis identified mabs to the sars-cov spike and nucleoprotein. five mabs (f26g1, g6, g8, g18 and g19) react specifically with the sars-cov spike protein in western immunoblot. these mabs recognise spike in western immunoblot on both highly purified virus and infected cell lysates (fig. 2) , but do not react with mock-infected cell lysates (data not shown). this result suggests that these mabs target linear epitopes within the spike protein. another mab, f26g15, bound fig. 1 . elisa reactivity of mabs with whole, inactivated sars-cov. hybridoma supernatants were tested at a 1:4 dilution in pbs + 0.2% bsa on pre-blocked plates, coated with 18 ng per well of inactivated, highly purified sars-cov. positive clones were identified as having positive binding (color) in wells that were at least four-fold higher than the background level reactivity on bsa. antigen legend: black bars-native, highly purified sars-cov; white bars-heat denatured, highly purified sars-cov; grey bars-bsa control. a virus neutralisation tests were performed independently in separate containment laboratories (nml, national microbiology laboratory; ncfad, national centre for foreign animal disease). the last six rows denote neutralising clones. b only a single dilution of 1/20 was tested in microwell format. c protein specificity tests shown here were determined by western immunoblot with purified virus and infected cell lysate under denaturing conditions (fig. 1) . u, unknown target antigen. d immuno-dotblot was performed using antigen treated under various conditions described in section 2. n, native; h, heat denatured, 95 • c for 5 min; d, sds treated (2%); h+d, heated in the presence of sds (2%); r, treated with reducing agent, beta-mercaptoethanol (5%); h + r, heated in the presence of reducing agent, beta-mercaptoethanol (5%); a, treated with heat, sds (2%) and reducing agent beta-mercaptoethanol (5%). e immune-fluorescence on whole cell slides infected with sars-cov (see fig. 2 ); ++, strong positive reaction; +, positive reaction; ±, weak positive reaction; −, negative reaction. f epitope properties described as follows: l, linear or continuous; e, surface exposed; c, conformational; p, protective in vitro; nd, not determined. to the nucleoprotein in western immunoblot assays. interestingly, the majority of the identified mabs bound to the spike protein and not the nucleoprotein, despite the strong sero-reactivity of the polyclonal serum of the immunised mice for the nucleoprotein (fig. 2) . the identity of the target antigen of eleven other mabs could not be determined by western immunoblot analysis. this observation suggests that these mabs likely target conformational epitopes that are sensitive to the conditions employed in such analyses. work is planned to identify the specific targets of these mabs. immuno-dotblot analysis reveals a spectrum of conformational requirements for binding (summarised in table 1 ). the effects of different denaturing treatments on the binding activity of a subset of neutralising and some non-neutralising mabs were examined using immuno-dotblot assays on infected lysates compared to uninfected lysates. the series of conditions tested include exposure to heat, detergent, a reducing agent, and combinations thereof. interestingly, mab f26g15, which binds to the nucleoprotein in western immunoblot, does not bind to the sars-cov antigen in immuno-dotblot under any of the conditions tested. the inherent charge of the highly phosphorylated nucleoprotein, or of the immobilon membrane used in the assays, may provide an explanation for these results. immuno-dot blots were not performed with mabs f26g2, f26g4, f26g12, f26g14, or f26g17, however the binding of these mabs is considered conformational as they do not bind to sars-cov proteins in western immunoblot (fig. 2) . the binding of spike protein specific mabs f26g1, f26g18, and f26g19 is inhibited in immuno-dotblot assays when antigens are treated with heat plus detergent, or heat plus detergent plus reducing agent. when the immuno-dotblot assay was instead performed using purified virus particles, the result was identical for nucleoprotein specific mab f26g15, and spike specific mabs f26g1, f26g18, and f26g19 (data not shown). antigen pre-treatment for western immunoblot, which depends on the application of an electrical current, differs when compared to passive adsorption in elisa and immuno-dotblot assays, and clearly do not always correlate with one another. these studies illustrate the need to use multiple assays for epitope characterisation and reveal limitations of current epitope classification schemes. the spike protein is an immunodominant antigen when inactivated sars-cov is used as an antigen. murine antibody responses to inactivated whole sars-cov are focused upon the sars spike and np protein as shown by the specificity of the recovered mabs. while multiple sars-cov proteins appear to be the target of mouse serum antibody as shown in western blot (fig. 2, immune sera) , the majority of the mabs recognise the spike protein in western immunoblot. in a sars-cov infection, the host immune system is exposed to a large load of nucleoprotein antigen, due to the presence of replicating virus in infected tissues. this would suggest that a competitive elisa format based upon the np as the target antigen might provide fig. 3 . competition elisa measuring the binding of murine mabs to highly purified sars-cov in the presence of human patient serum. dilutions (as indicated) of a normal human serum control (white bar), or serum from convalescent sars patient s3 were applied to wells coated with highly purified whole sars-cov. mab f26g6 (spike specific; black bars) or f26g15 (nucleoprotein specific; grey bars) were then added to the reactions. the results depicted for the pooled normal human serum (nhs) represent the mean of three replicate tests performed in the presence of mab f26g6 combined with three replicate tests performed in the presence of mab f26g15. results are representative of identical assays performed in duplicate with gamma-irradiated patient sera (2 mrad) (*p = 0.01, **p = 0.004, student's t-test). increased sensitivity for early detection of infection. towards this goal, the ability of sars convalescent serum to compete for binding of mabs to the nucleoprotein or to spike on whole sars-cov was tested via elisa. the serum samples were gamma-irrradiated prior to use. antibodies in the serum of patient s3 clearly inhibit binding of both the nucleoprotein mab f26g15 and the spike protein specific mab f26g6 (fig. 3) . in contrast, antibody in pooled normal human sera does not compete for binding by either mab. while not a statistical analysis these data show that these mabs are useful for the development of serological competition assays which can be subjected to validation tests. the sera from several other sars-cov infected patients demonstrate a similar ability to inhibit binding by the f26g15 (nucleoprotein specific mab) and f26g6 (spike specific mab). however, in some samples there is inhibition of only the binding to nucleoprotein without inhibition of the mab to the spike protein suggesting that spike antibody responses take longer to develop in infected humans (data not shown). the predominance of mabs to the spike protein in mice immunised with intact viral particles led us to test for biological activity in virus neutralisation assays. the sars-cov spike protein is the target of neutralising antibodies. neutralisation positive mabs bind both to linear epitopes in the spike protein and to unknown conformational epitope(s) either in the spike protein or in other proteins. a total of six mabs were identified that could neutralise sars-cov infectivity: f26g3, g7, g9, g10, g18, and g19 (table 1) . significantly, two of these mabs, f26 g18 and 19, were positively identified as being specific for spike, as determined by western immunoblotting. the specific targets of the remaining neutralising mabs remain to be elucidated. no cross-neutralisation was observed for the animal coronavirus tgev. this shows that these mabs are specific for sars-cov epitopes and do not cross-neutralise via tgev epitopes. the remaining mabs were unable to prevent viral growth when they were applied to the neutralisation assays. these data suggest that vaccines capable of engendering neutralising antibody responses to the spike protein may be effective in blocking infection with sars-cov. the four western immunoblot negative, virus-neutralising mabs were tested for their ability to bind native sars-cov in infected cells by immunofluorescence assay. nonneutralising mab f26g6 was used as a positive control, since this mab recognises spike protein in immunohistochemical staining of infected vero cells (data not shown). immunofluorescence analysis reveals that the neutralising mabs f26g3, g7, g9, and g10 specifically recognise sars-cov infected but not uninfected vero cells (fig. 4) . irrelevant, isotype matched mabs, produced in an identical fashion, do not react with sars-cov infected vero cells. the sars-neutralising mabs bind epitopes with higher conformational requirements than the non-neutralising mabs as they are less tolerant to denaturation of the epitopes. the method of antigen preparation and quality of the antigen greatly affect the interpretation of mab binding results obtained from immuno-dotblot assays. importantly, none of the mabs react with mock-infected lysates as assayed in immuno-dotblots (data not shown). this observation suggests that the majority of the neutralising mabs likely target surface exposed, protein epitopes on the native viral particle. one such putative protective antigen has been identified as the spike protein by western immunoblot analysis with neutralising mabs f26g18 and f26g19. antigen quality and conformation affects the binding of the anti-sars-cov mabs in elisa. while purified virus is clearly the optimal antigen tested in this series of experiments, the lower quality sars-cov-infected vero cell lysates are, however, much easier to prepare for diagnostic assays. therefore, the limits of mab binding to sars-cov infected vero cell lysates were further examined via elisa. the majority of these mabs exhibit decreased binding when the antigen is heat denatured ( table 2) . heat denaturation has very little effect on the binding of non-neutralising mab f26g16, which also maintains a high level of binding in elisa using infected vero cell lysates. f26g16 does, however, show higher background on the irrelevant antigen, bsa, and has inconsistent reactivity in western immunoblots with heat denatured viral lysate (fig. 1, table 1 ). the combination of lower quality antigen in infected vero cell lysates, along with heat denaturation of the antigens, has a stronger negative effect on binding by the neutralising mabs compared to non-neutralising mabs (table 2) . indeed, regardless of western immunoblot reactivity, the non-neutralising clones retain a greater ability to bind heat denatured antigen compared to the neutralising mabs (lower mean percent reduction in od per group p < 0.001, student's t-test, table 2 ). this supports the earlier observation in elisa on purified sars-cov (fig. 1) that shows that the neutralising mabs have higher requirements for native epitope conformation compared to non-neutralising mabs. the higher conformational requirement by neutralising mabs may help to explain some discrepancies observed in the immuno-dotblot methods. the immuno-dotblot is, overall, a less sensitive method, and the results are more difficult to quantify, compared to elisa. for example, mab f26g18 binds to the sars-cov spike protein in western immunoblot and neutralises sars-cov infection in vitro. while binding in western immunoblot generally suggests the epitope is linear in nature, nonetheless, heat treatments clearly affect binding of some mabs to the whole virion. the immuno-dotblot data reveal that with the lower quality antigen of the infected vero cell lysate, under the conditions of heat-plus detergent, or heat-plus detergent and reducing agent, mab f26g18 cannot bind (table 1) . this paper describes the development of murine mabs which recognise sars-cov antigens in elisa, immuno-dotblot, western immunoblot, on the surface of infected cells, and in neutralisation assays. these data are consistent with the appearance of coronavirus antigens on the surface of the infected cell during replication (talbot et al., 1984) , although the fixation process may allow for reactivity of these mabs with internal antigens as well. the conformational sensitivity of most of the sars-cov neutralising mabs is consistent with properties of neutralising mabs raised against other enveloped viruses, which generally require more native conformation for binding (wilson et al., 2000; zwick et al., 2001) . the immuno-dotblot assays contradict the classification of several putative epitopes as being linear as is suggested by positive western immunoblot reaction (for example with neutralising mab f26g18). indeed, it appears that the strict traditional classification of epitopes as being linear or conformational must account for a broader spectrum of conformational requirements, especially when dealing with antigens of variable quality in different immunological tests. however, in general the neutralising mabs can be considered to have a higher requirement for correct epitope conformation compared to non-neutralising mabs in elisa. it will be important to verify, under optimised conditions (opstelten et al., 1995) the use of viral lysates designed for maximal recovery of coronavirus proteins, and to this end the production of high quality recombinant protein antigens will provide useful insights. unfortunately, preparation of highly purified viral antigen requires enormous efforts under bio-containment conditions, which emphasises the need for a quality recombinant antigen assay. collectively, these data demonstrate that development of mab-based diagnostic tools for the detection of sars-cov infection is well within reach. the spike protein of the sars-cov is a target of neutralising mabs. epitopes on the spike protein provide neutralising targets on sars-cov in vitro, and this is consistent with the spike being the target of neutralising antibodies for other coronavirus strains (godet et al., 1994) . moreover, these mabs may be useful for the identification of protective epitopes for vaccine formulations (enjuanes et al., 1995) . studies are underway to determine the identity of the antigen(s) recognised by the neutralising, western immunnoblot negative mabs. preliminary analysis of the nucleotide and predicted amino acid sequences of the cloned v h and v l coding regions of these mabs suggests that the mabs are distinct. this observation implies that the hybridoma clones expressing the anti-sars-cov neutralising mabs were derived from individually rearranged and clonally selected b cells in vivo. it also reveals that there is no apparent consensus sequence within the complementarity determining regions that is required for the mabs to exhibit virus neutralisation activity. this finding makes it feasible to engineer multiple mabs with high specificity and avidity for various sars-cov epitopes for preparations of defined cocktails of therapeutic mabs. a detailed description of the immunogenetic properties of these mabs is in preparation (berry et al., manuscript in preparation) . the np specific mab, f26g15, is useful in competitive elisa with patient sera. this is important as early detection of sars infections is key to risk management of this disease. this is the first description of neutralising mabs from a host immunised with whole sars-cov and these antibodies should prove useful for the development of new diagnostic tests, studies of antigenic variation, and vaccine development in the global fight against sars. arboviruses proceedings of the fourth international symposium on neonatal diarrhea. s.d. acres monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion tropism and immunoprotection in transmissible gastroenteritis coronaviruses fields virology reactivity of human coronavirus oc43 and neonatal calf diarrhoea coronavirus membrane-associated antigens major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein critical epitopes in transmissible gastroenteritis virus neutralization continuous cultures of fused cells secreting antibody of predefined specificity mass spectrometric characterization of proteins from the sars virus: a preliminary report antigenic structure of transmissible gastroenteritis virus. i. properties of monoclonal antibodies directed against virion proteins profile of specific antibodies to the sars-associated coronavirus angiotensin converting enzyme 2 is a functional receptor for the sars coronavirus envelope glycoprotein interactions in coronavirus assembly sars-beginning to understand a new virus topographical mapping of epitopes on the glycoproteins of murine hepatitis virus-4 (strain jhm): correlation with biological activities hybridoma antibodies to the murine coronavirus jhm: characterization of epitopes on the peplomer protein (e2) epitopes involved in antibody-mediated protection from ebola-virus the sars-cov s glycoprotein: expression and functional characterization identification and characterization of a peptide that specifically binds the human broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12 the authors would like to thank ms. nicole beausoleil, mr. daryl dick, mr. darrell johnstone, ms. kathy frost, ms. hilary holland, and mr. richard nickel for expert technical assistance. mg is supported by a health canada ocs postdoctoral fellowship. thanks to dr. john copps (nc-fad) for expert veterinarian services and for assisting in the design of the emergency animal use document. thanks also to dr. susy carman (university of guelph, canada) and dr. lorne babiuk (vido, canada) for providing animal coronavirus strains. funding for this work was provided by health canada and the canadian food inspection agency. this work is dedicated to the memory of lloyd d. berry. key: cord-296237-i9cti2ok authors: díez, josé-maría; romero, carolina; vergara-alert, júlia; belló-perez, melissa; rodon, jordi; honrubia, josé manuel; segalés, joaquim; sola, isabel; enjuanes, luis; gajardo, rodrigo title: cross-neutralization activity against sars-cov-2 is present in currently available intravenous immunoglobulins date: 2020-06-19 journal: biorxiv doi: 10.1101/2020.06.19.160879 sha: doc_id: 296237 cord_uid: i9cti2ok background there is a crucial need for effective therapies that are immediately available to counteract covid-19 disease. recently, elisa binding cross-reactivity against components of human epidemic coronaviruses with currently available intravenous immunoglobulins (ivig) gamunex-c and flebogamma dif (5% and 10%) have been reported. in this study, the same products were tested for neutralization activity against sars-cov-2, sars-cov and mers-cov and their potential as an antiviral therapy. methods the neutralization capacity of six selected lots of ivig was assessed against sars-cov-2 (two different isolates), sars-cov and mers-cov in cell cultures. infectivity neutralization was measured by determining the percent reduction in plaque-forming units (pfu) and by cytopathic effects for two ivig lots in one of the sars-cov-2 isolates. neutralization was quantified using the plaque reduction neutralization test 50 (prnt50) in the pfu assay and the half maximal inhibitory concentration (ic50) in the cytopathic/cytotoxic method (calculated as the minus log10 dilution which reduced the viral titer by 50%). results all ivig preparations showed neutralization of both sars-cov-2 isolates, ranging from 79 to 89.5% with prnt50 titers from 4.5 to >5 for the pfu method and ranging from 47.0%-64.7% with an ic50 ~1 for the cytopathic method. all ivig lots produced neutralization of sars-cov ranging from 39.5 to 55.1 % and prnt50 values ranging from 2.0 to 3.3. no ivig preparation showed significant neutralizing activity against mers-cov. conclusion in cell culture neutralization assays, the tested ivig products contain antibodies with significant cross-neutralization capacity against sars-cov-2 and sars-cov. however, no neutralization capacity was demonstrated against mers-cov. these preparations are currently available and may be immediately useful for covid-19 management. the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which causes the respiratory disease covid-19 was declared a pandemic by the who in march 2020. most infected patients (80%) have mild symptoms. however, about 20% of covid-19 patients can progress to severe pneumonia and to acute respiratory distress syndrome (ards) which is associated with multiorgan failure and death 1 . the current critical situation demands an effective and reliable therapy that is immediately available to control the progression of the disease. convalescent plasma or plasma-derived immunoglobulin (ig) (either polyvalent ig prepared from healthy donors or hyperimmune ig prepared from donors with high antibody titers against a specific antigen) have been historically used as a readily available therapeutic option in outbreaks of emerging or re-emerging infections 2 . to date, seven human coronaviruses (hcov) have been identified. four of them are endemic and globally distributed (hcov-229e, hcov-nl63, hcov-oc43 and hcov-hku1) 3 . these viruses typically cause mild symptoms and are associated with about 15% of common colds 4 [5] [6] [7] . more recently (december 2019), the novel coronavirus sars-cov-2 emerged in china and because of its extraordinary human-tohuman transmissibility is currently causing an unprecedented pandemic 8 . although several therapeutic approaches against sars-cov-2 are under investigation, therapeutic agents of proven efficacy are still lacking. interestingly, coronaviruses share some morphological and functional properties that may be associated with cross-reactive immune responses. this cross-reactivity may have important therapeutic implications 9 . sars-cov, sars-cov-2, and mers-cov are classified within the family coronaviridae, genus betacoronavirus, subgenera sarbecovirus (sars-cov, sars-cov-2) and merbecovirus (mers-cov). the spike protein (s), which is exposed on the virion surface, is the main determinant of the coronavirus entry into the host cell and is also the major target of neutralizing antibodies 10 . spikes are formed by trimers of protein s, which is in turn formed by subunit (s1) that mediates the binding to the cell receptor and a membraneanchored subunit (s2) that mediates the fusion of the virus with cell membranes 11 . potent neutralizing antibodies often target the receptor interaction site on s1. however, the s1 subunit shows a higher variability than s2. antibodies targeting s1 are often virus-specific making s2 a better target for cross-neutralizing antibodies 12, 13 . the amino-acid sequence identity among the s proteins of human betacoronaviruses causing mild (hcov-oc43 and hcov-hku1) and severe (sars-cov, sars-cov-2, and mers-cov) respiratory infections varies between 22% and 33% 10 . however, the s proteins of sars-cov and sars-cov-2 share 77% amino-acid identity 14 and more than 90% rna sequence homology 15 . cross-reactivity in antigenic responses has been described among human coronaviruses of the same genus, particularly betacoronaviruses. crossreactivity between sars-cov, mers-cov and other endemic human coronaviruses has been reported in some neutralization assays 16-18 . recently, cross-reactivity in elisa binding assays against antigens of sars-cov, sars-cov-2, and mers-cov has been reported with currently available intravenous immunoglobulins (ivig) such as gamunex-c and flebogamma dif 19 . in this study, the neutralization capacity of the ivig products gamunex-c and flebogamma dif against these epidemic human coronaviruses -sars-cov, sars-cov-2, and mers-cov-was evaluated. ivig products used in this study were flebogamma ® dif 5% and 10% (instituto grifols s.a., barcelona, spain) and gamunex ® -c 10% (grifols therapeutics inc., raleigh nc, usa), two highly purified (≥ 98%-99%igg), unmodified human immunoglobulins. each product is manufactured from plasma collected from thousands of donors in the us and/or several european countries. igg concentrations in flebogamma dif products were 50 mg/ml and 100 mg/ml (5% and 10%) and in gamunex-c, the concentration was 100 mg/ml (10%). to ensure a virus-free product, both ivig manufacturing processes contain dedicated steps with high pathogen clearance capacity, such as solvent/detergent treatment, heat treatment, caprylate treatment and planova™ nanofiltration down to 20 nm pore size. the plasma used to manufacture the ivig lots tested was collected from march 2018 to october 2019. six different lots of flebogamma dif and gamunex-c were tested at several dilutions for cross-reactivity against sars-cov, sars-cov-2, and mers-cov by: i) elisa techniques; and ii) well-stablished neutralization assays in cell cultures. lots were identified as f1 and f2 for flebogamma 5% dif, f3 and f4 for flebogamma 10% dif and g1 and g2 for gamunex-c. each experiment was performed in duplicate. handling of viruses and cell cultures was carried out at the recombinant sars-cov was generated from urbani strain using a previously described reverse genetic technique 20 . two different sars-cov-2 isolates were tested: a) sars-cov-2 mad6 isolated from a covid-19 patient in spain; and b) sars-cov-2 (accession id epi_isl_418268 at gisaid repository: http://gisaid.org) isolated from a covid-19 patient in spain. both stock viruses (a and b) were prepared by collecting the supernatant from vero e6 cells, as previously described 21 . recombinant mers-cov was generated using a previously described reverse genetic system 22 from the reference sequence of mers-cov isolated from the index patient emc/2012 (genebank jx869059) 23 . huh7 is a well differentiated human hepatocyte-derived carcinoma cell line, kindly provided by dr. luis carrasco vero e6 is a cell line isolated from kidney epithelial cells extracted from an african green monkey. vero e6 is composed of epithelial-like cells susceptible to infection by sars-cov and sars-cov-2 25 . at cnb-csic, vero e6 cell lines were kindly provided by dr. eric snjider (university of leiden medical center, the netherlands). both huh7 and vero e6 cell lines were cultured in dulbecco-modified eagle medium (dmem) supplemented with 25 mm hepes buffer, 2 mm l-glutamine (sigma-aldrich, st. louis, mi, usa), 1% nonessential amino-acids (sigma-aldrich), 10% fetal bovine serum (fbs; biowhittaker, inc., walkersville, md, usa). in the postinfection semisolid medium, the percentage of fbs was reduced to 2%, and deae-dextran was added to a final concentration of 0.08 mg/ml. at irta-cresa, vero e6 cells were obtained from the atcc (atcc crl-1586) and cultured in dmem (lonza, basel, switzerland) supplemented with 5% (fbs (euroclone, pero, italy), 100 u/ml penicillin, 100 µg/ml streptomycin, and 2 mm glutamine 8 (all thermofisher scientific, waltham, ma, usa). in the post-infection medium, the percentage of fbs was reduced to 2%. qualitative determination of igg class antibodies crossreactivity against antigens of the tested coronaviruses was performed using elisa techniques. ivig samples were serially diluted using the buffer solutions provided in each igg elisa kit. the following kits were used for the qualitative determination of igg class antibodies in the experimental ivig lots: sars coronavirus igg elisa kit (creative diagnostics, shirley, ny, usa), against virus lysate; human anti-sars-cov-2 virus spike 1 [s1] igg elisa kit (alpha diagnostic intl. inc., san antonio, tx, usa), against s1 subunit spike protein; rv-402100-1, human anti-mers-np igg elisa kit (alpha diagnostic intl. inc.), against n protein; rv-402400-1, human anti-mers-rbd igg elisa kit (alpha diagnostic intl. inc.), against receptor-binding domain (rbd) of s1 subunit spike protein (s1/rbd); rv-402300-1, human anti-mers-s2 igg elisa kit (alpha diagnostic intl. inc.), against s2 subunit spike protein; rv-405200 (formerly rv-404100-1). in all cases the determinations were carried out following the manufacturer's instructions. reactivity was rated as negative if no reaction was observed with neat ivig or positive if the lowest ivig dilution demonstrated reactivity. aliquots of 50 µl of each ivig dilution-virus complex were added in duplicate to confluent monolayers of vero e6 cells (for sars-cov and sars-cov-2) or huh7 (for mers-cov), seeded in 12-well plates and incubated for 1 h (37°c; 5% co2). after this adsorption time, the igg-virus complex inoculum was removed, and a semi-solid overlay was added (dmem 2% fbs + 0.6% agarose). cells were incubated for 72 h at 37ºc. the semi-solid medium was removed, and the cells were fixed with 10% neutral buffered formaldehyde (sigma-aldrich) for 1 hour at room temperature and stained with 0.2% aqueous gentian violet for 10 min, followed by plaque counting. the sensitivity threshold of the technique was 20 pfu per ml. the neutralization potency of the ivig products was expressed in two ways: 1) percent reduction in pfu calculated from the pfu count after neutralization by ivig relative to initial pfu count inoculated onto the cells; and 2) plaque reduction neutralization test (prnt50) value, calculated as the -log10 of the reciprocal of the highest ivig dilution to reduce the number of plaques by 50% compared to the number of plaques without ivig. a fixed concentration of a sars-cov-2 stock (10 1.8 tcid 50/ml, a concentration that achieves 50% cytopathic effect) was mixed with decreasing concentrations of the ivig samples (range 1:10 to 1:5120), each mixture was incubated for 1 h at 37º c and added to vero e6 cells. to assess potential plasma-induced cytotoxicity, vero e6 cells were also cultured with the same decreasing concentrations of plasma in the absence of sars-cov-2. uninfected cells and untreated virus infected cells were used as negative and positive infection controls, respectively. plasma from a covid-19 positive patient with a high half-maximal inhibitory concentration (ic50) was included as an active positive control (expressed as the -log10 of the reciprocal of the dilution). all the cultures were incubated at 37ºc and 5% co2 for 3 days. cytopathic or cytotoxic effects of the virus or plasma samples were measured at 3 days post infection, using the cell titer-glo luminescent cell viability assay (promega, madison, wi, usa). luminescence was measured in a fluoroskan ascent fl luminometer (thermofisher scientific). neutralization curves are shown as nonlinear regressions. ic50 values were determined from the fitted curves as the plasma dilutions that produced 50% neutralization. details of the technique are available elsewhere 21 . ivig products showed consistent reactivity to antigens of sars-cov (culture lysate) at 10-100 mg/ml igg, sars-cov-2 (s1 subunit protein) at 100 µg/ml igg, and mers-cov (n protein, s1 subunit/rhd protein and s2 subunit protein) at 50 µg/ml igg (table 1) . all the assayed ivig preparations had neutralizing activity against sars-cov ranging from 39% to 61% (figure 1 ). all 10% igg ivig preparations (f3, f4, g1, and g2) showed prnt50 neutralization titers between 2.0 and 3.3, corresponding to 50-61% pfu reduction ( figures 1b, 1c) . the highest pfu reductions, 59.3% and 61.9% (prnt50 neutralization titers of 3.2 and 3.3), were observed with lots f4 and g1, respectively, at 1 and 0.1 mg/ml igg (dilution factors 2 and 3). the f1 and f2 lots, (5% igg) showed a lower neutralization capacity with pfu reductions of 39.5% and 43.3%, respectively ( figure 1a ). for sars-cov-2 mad6 isolate, all ivig lots, except f1 (inconclusive results) showed a significant neutralizing activity and reached prnt50 titers ranging from 4.5 to >5 (figure 2 ). pfu reductions ranging from 78.2% to 82.5% were observed with lots f2, f3 and f4 at a dilution factor of 1. even at the highest dilution factor (5 = 0.5 and 1 µg/ml), the pfu reduction ranged from 38.5% to 50.9% corresponding to prnt50 titers of 4.5-5.0 (figures 2a, 2b) . for lots g1 and g2, the pfu reduction was even higher, ranging from 88.5% -89.5% at a dilution factor of 1 to 61.7% -62.5% at a dilution factor of 5 with prnt50 titers greater than 5 ( figure 2c ). for the sars-cov-2 epi_isl_418268 isolate, f4 and g1 lots neutralized 58.4% and 64.7%, respectively, tcid50 counts at a dilution factor of 1 ( figure 3 ). as shown in figure 3 , one replicate of f4 product failed to demonstrate neutralization. no ivig lot showed any significant pfu reduction (i.e., >10%) on mers-cov even at the lowest dilution factor (10 mg/ml igg). the results presented here demonstrate, for the first time, significant cross-neutralization activity against sars-cov and especially sars-cov-2 in therapeutic ivig concentrates (flebogamma dif and gamunex-c). this neutralizing activity correlates with the cross-reactivity to different coronavirus antigens observed in elisa binding assays with ivig, as shown in a previous study 19 . the plasma used to manufacture the tested ivig lots was collected prior the detection of sars-cov-2 in europe and the usa. therefore, these results should be ascribed to cross-reactivity against sars-cov-2 by antibodies against endemic human coronaviruses in the human population at large. similar results have been reported for sars-cov and mers-cov. [16] [17] [18] these neutralization studies showed that ivig products contain antibodies with cross-neutralizing capacity against sars-cov (40-60%) and sars-cov-2 (80%-90%), but not against mers-cov (<10%). these results suggest that the cross-neutralizing antibodies target antigenic regions more conserved in sars-cov and sars-cov-2 than in mers-cov. no significant differences in the neutralizing capacity were observed among ivig lots regardless the country of origin for the plasma. this reinforces the broad applicability of these results. two different neutralization techniques were used for sars-cov-2 and both techniques showed not only the ivig neutralization capacity, but also the reliability of the results. in addition, results obtained with two different sars-cov-2 isolates confirm that the neutralization capacity is not dependent on the isolate. this was not unexpected since no significant sequence differences have been observed among sars-cov-2 isolates currently circulating throughout the world. the percentage of sars-cov-2 cross-neutralization was higher in the pfu reduction technique than in the cytopathic effect/cytotoxic technique with very low or negative values in some few cases (inconclusive for lot f1 by the pfu study, and cytopathic effect in one replicate of lot f4). this suggests that the technique used and/or slight variations in methodology may significantly influence the nature or magnitude of the results. therefore, further evaluation this cross-neutralizing activity should be conducted. cross-neutralization is gaining attention as a protective mechanism against viral infection in the context of the covid-19 health emergency. the results of this study are in agreement with recent studies that describe crossneutralization of sars-cov-2 by monoclonal antibodies from memory b cells of an individual who was infected with sars-cov 26 . furthermore, sars-cov-2-reactive cd4+ t cells have been detected in around half of unexposed individuals, suggesting that there is cross-reactive t cell recognition between circulating common cold coronaviruses and sars-cov-2 27 . however, the levels of crossneutralizing antibodies against sars-cov-2 in the sera of sars-cov patients can be highly variable 28 . ivig products are prepared using plasma from thousands of different donors, hence containing a broad representation of the state of immunity in the population at that time. this is consistent with the low rate of variability found among the different lots of ivig products tested. nevertheless, greater variability is expected among individuals with respect to infection by a given endemic human coronavirus. therefore, it has been hypothesized that the diversity of symptoms observed in sars-cov-2-infected individuals and even the potential for getting infected may depend on pre-existing cross-immunity due to previous exposure to other endemic human coronaviruses. in this regard, a detailed study of the state of immunity in the general population distinguishing those affected and not affected by the sars-cov-2 may be warranted. the higher cross-neutralizing capacity of the tested ivig preparations against sars-cov and sars-cov-2 than mers-cov may be explained by higher sequence identity of the s proteins of circulating mild human coronaviruses (hcov-oc43 and hcov-hku1) and sars-cov and sars-cov-2 compared to mers-cov (32%-33% vs. 23%-25) 14, 15 . additionally, differences in specific domains of the s protein between sars-cov and sars-cov-2 might explain higher cross-reactivity of the tested ivig against sars-cov-2 compared to sars-cov (80%-90% vs. 40%-60%). the absence of cross-neutralization against mers-cov despite the cross-reactivity observed in elisa assays suggest that these antibodies are not neutralizing. this does not necessarily indicate that the antibodies are not functional by another mechanism. for example, these non-neutralizing antibodies could be labelling the virion for identification by immune cells and subsequent destruction 29 . despite the limitations of the in vitro nature of this study, the clinical implications of the findings are encouraging. although ivig are considered a therapeutic option for hyperinflammation in patients with severe covid-19 30 , the results of this study may support the use of high dose ivig as a therapy for covid-19. positive results have already been reported for ivig in case studies 31, 32 . ivig is being tested in an ongoing clinical trial 33 . further studies looking at the functionality of these antibodies could improve our understanding the human coronavirus acquired immunity. this could pave the way for ivig (and other igg products such as intramuscular or subcutaneous preparations) as a potential therapeutic/prophylactic approach to fight future epidemics by emerging human coronaviruses. in conclusion, under the experimental conditions of this study, ivig (flebogamma dif and gamunex-c) contained antibodies with significant neutralization capacity against sars-cov and sars-cov-2, but not against mers-cov. additional research is warranted to advance ivig towards clinical use for covid-19. strategies for the prevention and management of coronavirus disease 2019 use of human immunoglobulins as an anti-infective treatment: the experience so far and their possible re-emerging role genetic recombination, and pathogenesis of coronaviruses update on human rhinovirus and coronavirus infections sars: prognosis, outcome and sequelae sars-cov-2/covid-19: viral genomics, epidemiology, vaccines, and therapeutic interventions middle east respiratory syndrome european centre for disease prevention and control. covid-19. situation update worldwide global patterns in coronavirus diversity. virus evolution identification of the receptor-binding domain of the spike glycoprotein of human betacoronavirus hku1 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-cov-2, sars-cov, and mers-cov: a comparative overview phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop profiling early humoral response to diagnose novel coronavirus disease (covid-19) an outbreak of human coronavirus oc43 infection and serological cross-reactivity with sars coronavirus cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests antigenic crossreactivity between severe acute respiratory syndromeassociated coronavirus and human coronaviruses 229e and oc43 currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus 2 antigens construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis search for sars-cov-2 inhibitors in currently approved drugs to tackle covid-19 pandemia engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans comparative host gene transcription by microarray analysis early after infection of the huh7 cell line by sars coronavirus and human coronavirus 229e the life cycle of sars coronavirus in vero e6 cells crossneutralization of sars-cov-2 by a human monoclonal sars-cov antibody targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals lack of crossneutralization by sars patient sera towards sars-cov-2 protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts covid-19: consider cytokine storm syndromes and immunosuppression high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with covid-19 the efficacy of intravenous immunoglobulin therapy for severe 2019-ncov infected pneumonia jordi bozzo phd, cmpp and michael k. james phd (grifols) are acknowledged for medical writing and editorial support in the preparation of this manuscript. contribution from antonio páez md (grifols) who provided his expert opinion is acknowledged. the authors acknowledge the expert technical assistance from daniel casals, eduard sala, judith luque, laura gómez, and gonzalo mercado (grifols, viral and cell culture laboratory). neutralization experiments were funded by grifols, the manufacturer of flebogamma ® dif and gamunex ® -c. jva, jr, mbp, jmh, is, and le declare having no other conflict of interest. jmd, cr and rg are full-time employees of grifols. key: cord-292256-jp80u828 authors: moriguchi, takeshi; harii, norikazu; goto, junko; harada, daiki; sugawara, hisanori; takamino, junichi; ueno, masateru; sakata, hiroki; kondo, kengo; myose, natsuhiko; nakao, atsuhito; takeda, masayuki; haro, hirotaka; inoue, osamu; suzuki-inoue, katsue; kubokawa, kayo; ogihara, shinji; sasaki, tomoyuki; kinouchi, hiroyuki; kojin, hiroyuki; ito, masami; onishi, hiroshi; shimizu, tatsuya; sasaki, yu; enomoto, nobuyuki; ishihara, hiroshi; furuya, shiomi; yamamoto, tomoko; shimada, shinji title: a first case of meningitis/encephalitis associated with sars-coronavirus-2 date: 2020-04-03 journal: int j infect dis doi: 10.1016/j.ijid.2020.03.062 sha: doc_id: 292256 cord_uid: jp80u828 novel coronavirus (sars-coronavirus-2:sars-cov-2) which emerged in wuhan, china, has spread to multiple countries rapidly. we report the first case of meningitis associated with sars-cov-2 who was brought in by ambulance due to a convulsion accompanied by unconsciousness. he had never been to any foreign countries. he felt generalized fatigue and fever (day 1). he saw doctors nearby twice (day 2 and 5) and was prescribed laninamivir and antipyretic agents, his family visited his home and found that he was unconsciousness and lying on the floor in his vomit. he was immediately transported to this hospital by ambulance (day 9). under emergency transport, he had transient generalized seizures that lasted about a minute. he had obvious neck stiffness. the specific sars-cov-2 rna was not detected in the nasopharyngeal swab but was detected in a csf. antihsv 1 and varicella-zoster igm antibodies were not detected in serum samples. a brain mri showed hyperintensity along the wall of right lateral ventricle and hyperintense signal changes in the right mesial temporal lobe and hippocampus, suggesting the possibility of sars-cov-2 meningitis. this case warns the physicians of patients who have cns symptoms. novel coronavirus (sars-coronavirus-2:sars-cov-2) emerged in december 2019 in wuhan, china, and has become a global health emergency. (wang et al., 2020a,b) a preliminary report warned that sars-cov-2 could have neuroinvasive potential because some patients showed neurologic symptoms such as headache, nausea, and vomiting . in order to end the pandemic of sars-coronavirus-2 diseases, the diagnosis of the disease must be prompt and not overlook any findings. this brief report describes the first case of the patient, which brought in by the ambulance due to a convulsion accompanied by unconsciousness, was diagnosed with aseptic encephalitis with sars-cov-2 rna in cerebrospinal fluid. a 24-year-old man he has never been to foreign countries. he felt headache, generalized fatigue and fever in late february 2020 (day 1). on day 2, he consulted a doctor nearby. there, he was prescribed laninamivir and antipyretic agents under the diagnosis of influenza due to his clinical symptoms in spite of the negative result of the diagnostic test. three days later (day 5), he visited another clinic because of the worsening of his previous symptoms, headache, and sore throat. he underwent chest x-ray examination and blood test resulted in negative findings. on day 9, he was found lying on the floor with consciousness disturbance. he was immediately transferred to our hospital by ambulance. during emergency transport, he presented with transient generalized seizures for about a minute. upon arrival at our hospital, he had a glasgow coma scale (gcs) of 6 (e4 v1 m1) with hemodynamically stability. he had obvious neck stiffness. blood investigation showed an increased white cell count, neutrophil dominant, relatively decreased lymphocytes, increased c-reactive protein. subsequent investigations included systemic ct demonstrating no evidence of brain edema. the chest ct showed that there was small ground glass opacity on the right superior lobe and both sides of the inferior lobe. on a further lumbar puncture examination, his cerebrospinal fluid was clear and colorless, and the initial pressure was greater than 320 mmh 2 o. the csf cell count was 12/ml-10 mononuclear and 2 polymorphonuclear cells without red blood cells. anti-hsv 1 and varicella-zoster igm antibodies were not detected in serum samples. the rt-pcr test for sars-cov-2 was performed using a nasopharyngeal swab and csf because we assumed that a sars-cov-2 was involved in the outbreak. although the specific sars-cov-2 rna was not detected in the nasopharyngeal swab, it was detected in csf (supplementary table 1) . during emergency treatment, the endotracheal intubation and mechanical ventilation were required because of multiple epileptic seizures. he was transferred to the icu with the clinical diagnosis of meningitis and viral pneumonia. after the icu admission, he was empirically started on intravenous (iv) ceftriaxone, vancomycin, aciclovir and steroids. he also underwent intravenous administration of levetiraceta for seizure. favipiravir had been administered via nasogastric tube for 10 days since day 2. brain mri was performed 20 h after admission to the icu (figure 1 ). diffusion weighted images (dwi) showed hyperintensity along the wall of inferior horn of right lateral ventricle. fluid-attenuated inversion recovery (flair) images showed hyperintense signal changes in the right mesial temporal lobe and hippocampus with slight hippocampal atrophy. contrast-enhanced imaging showed no definite dural enhancement. these findings indicated right lateral ventriculitis and encephalitis mainly on right mesial lobe and hippocampus. a differential diagnosis was considered to be hippocampal sclerosis accompanying post convulsive encephalopathy. besides, t2-weighted image showed pan-paranasal sinusitis. at day 15, we are continuing treatment for bacterial pneumonia and impaired consciousness due to encephalitis associated with sars-cov-2 in intensive care unit. we declare no competing interests. patient relative's written consent was obtained for publication. clinical specimens for sars-cov-2 diagnostic testing were obtained in accordance with guidelines of national institute of infectious diseases in japan. nasopharyngeal swab specimens were collected with synthetic fiber swabs; each swab was inserted into a separate sterile tube containing 1 ml of phosphate-buffered saline (pbs) supplemented with 0.5% bsa. spinal fluid was collected in sterile specimen containers. specimens were immediately examined at the yamanashi university hospital laboratory department or stored at 4 c until ready for examination. viral rna was extracted from clinical specimen using maglead 6gc (precision system science co., ltd.). the sars-cov-2 rna was detected using agpath-id tm one-step rt-pcr reagents (am1005) (applied biosystems) on cobasz480 (roche). the diagnostic assay for sars-cov-2 has three nucleocapsid gene targets (supplementary materials). the nasopharyngeal swabs obtained from this patient on day 1 (66 minutes after admission) were negative for n and n2 (supplementary table 1 ). as for spinal fluid, however, 1 sample out of 2 (1/2) on day 1 (84 min after admission) was positive for n, but not for n2. therefore, we re-examined the same specimen again and found that 2/2 samples were positive for n, but not for n2. again, the nasopharyngeal swabs were negative for both n and n2. our report described the first case of meningitis/encephalitis associated with sars-cov-2. this case shows the neuroinvasive potential of the virus and that we cannot exclude sars-cov-2 infections even if the rt-pcr test for sars-cov-2 using the patient's nasopharyngeal specimen is negative. in 2002-2003, severe acute respiratory syndrome (sars) pandemic appeared and sars-cov was isolated as the pathogen and as the new family of the human coronaviruses (drosten et al., 2003; ksiazek et al., 2003) . over a number of years, human coronaviruses including sars-cov was identified as possible pathogens for pathologies outside the respiratory systems. (gu et al., 2005; raj et al., 2014) . a report shows that sars-cov genome sequences were detected in the brain of all sars autopsies with real-time rt-pcr (gu et al., 2005) . importantly, the signals were strong in the hippocampus where we found inflammation in the patient's brain. recent study claims that the genomic sequence is similar between sars-cov and sars-cov-2 (yu et al., 2020) , especially the receptor-binding domains of sars-cov is structurally similar to that of sars-cov-2 (lu et al., 2020) . this may lead that sars-cov and sars-cov-2 shares the ace2 as a receptor. that might be the reason why sars-cov and sars-cov-2 might invade the same place in human brains. in the present case, mri demonstrated the abnormal findings of medial temporal lobe including hippocampus suggesting encephalitis, hippocampal sclerosis or post convulsive encephalitis. hippocampal sclerosis would be unlikely because he had no episodes of mesial temporal epilepsy in his past history. in addition, this case was presented with significant paranasal sinusitis. although the relation between sinusitis and retrograde trans-synaptic transfer is obscure, we should pay attention to nasal and paranasal condition in the diagnosis and treatment for sars-cov-2 infection. we claim that this case is important because this case shows that the unconscious patients are potentially infected by sars-cov-2 and might cause the horizontal infection. in order to end the pandemic of sars-cov-2 diseases, the diagnosis of the disease must be prompt and not overlook any findings. finding the suspected patient is the first step of a preventive measure against the pandemic. it should be kept in mind that the symptoms of the encephalitis or cerebropathia may be the first indication, as well as respiratory symptoms, to find the hidden sars-cov-2 patients. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors declare no conflicts of interest. proofreading the manuscript, prof. kohji moriishi for detailed knowledge about coronavirus, prof. zentaro yamagata for ethical considerations, and dr. kenichi matsuda for helpful advice for everything about the medical treatment for the critically ills and discussions over the case. identification of a novel coronavirus in patients with severe acute respiratory syndrome multiple organ infection and the pathogenesis of sars a novel coronavirus associated with severe acute respiratory syndrome the neuroinvasive potential of sars-cov2 may be at least partially responsible for the respiratory failure of covid-19 patients genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding mers: emergence of a novel human coronavirus clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in wuhan the authors would like to thank yamanashi university hospital sars-cov-2 nursing team and respiratory medical team that are fighting against the illness, mr. charles r. allala for carefully supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ijid.2020.03.062. key: cord-306373-61snvddh authors: xu, xiao-wei; wu, xiao-xin; jiang, xian-gao; xu, kai-jin; ying, ling-jun; ma, chun-lian; li, shi-bo; wang, hua-ying; zhang, sheng; gao, hai-nv; sheng, ji-fang; cai, hong-liu; qiu, yun-qing; li, lan-juan title: clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series date: 2020-02-19 journal: bmj doi: 10.1136/bmj.m606 sha: doc_id: 306373 cord_uid: 61snvddh objective: to study the clinical characteristics of patients in zhejiang province, china, infected with the 2019 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) responsible for coronavirus disease 2019 (covid-2019). design: retrospective case series. setting: seven hospitals in zhejiang province, china. participants: 62 patients admitted to hospital with laboratory confirmed sars-cov-2 infection. data were collected from 10 january 2020 to 26 january 2020. main outcome measures: clinical data, collected using a standardised case report form, such as temperature, history of exposure, incubation period. if information was not clear, the working group in hangzhou contacted the doctor responsible for treating the patient for clarification. results: of the 62 patients studied (median age 41 years), only one was admitted to an intensive care unit, and no patients died during the study. according to research, none of the infected patients in zhejiang province were ever exposed to the huanan seafood market, the original source of the virus; all studied cases were infected by human to human transmission. the most common symptoms at onset of illness were fever in 48 (77%) patients, cough in 50 (81%), expectoration in 35 (56%), headache in 21 (34%), myalgia or fatigue in 32 (52%), diarrhoea in 3 (8%), and haemoptysis in 2 (3%). only two patients (3%) developed shortness of breath on admission. the median time from exposure to onset of illness was 4 days (interquartile range 3-5 days), and from onset of symptoms to first hospital admission was 2 (1-4) days. conclusion: as of early february 2020, compared with patients initially infected with sars-cov-2 in wuhan, the symptoms of patients in zhejiang province are relatively mild. in december 2019 a group of patients with pneumonia of unknown cause were confirmed to be infected with a novel coronavirus, known as 2019-ncov, in wuhan, hubei province, china, which had previously not been detected in humans or animals. 1 epidemiological evidence suggested that most of these patients had visited a local seafood market in wuhan 2 and that the gene sequence of the virus obtained from these patients was highly similar to that identified in bats. 3 the virus was subsequently renamed sars-cov-2 as it is similar to the coronavirus responsible for severe acute respiratory syndrome (sars-cov), a member of the subgenus sarbecovirus (beta-cov lineage b), with which it shares more than 79% of its sequence, but it is more distant to the coronavirus responsible for middle east respiratory syndrome (mers-cov), a member of the merbecovirus subgenus (only 50% homology with sars-cov-2). all these viruses are categorised within the same genus of the subfamily orthocoronavirinae within the family coronaviridae. [4] [5] [6] [7] some researchers have found that sars-cov-2 has strong affinity to human respiratory receptors, 8 suggesting a potential threat to global public health. initially, the first confirmed cases were nearly all related to the huanan seafood market (closed on 1 january 2020) and were concentrated in wuhan. 9 coronavirus disease 2019 (covid-19) soon drew global attention because of the rapidly increasing numbers of new cases. 2 the new type of coronavirus infection was believed to have been transmitted from animals, and by january 2020 it was suspected that the initially doi: 10 .1136/bmj.m606 | bmj 2020;368:m606 | the bmj affected patients had been infected with the virus through human to human transmission. 10 since january 2020 the spread of covid-19 has escalated and the virus has extended rapidly to most parts of china as well as to other countries. as of 8 february 2020 a confirmed 37 589 people have been infected with sars-cov-2 globally, including 302 people across 24 other countries. 11 these figures are updated daily and are expected to increase further. despite the increasing number of confirmed cases, the clinical investigation of patients was insufficient. a previous study reported the clinical characteristics of the first 41 infected patients in the greater wuhan area, contributing to an understanding of the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of those patients. 9 a second study found a familial cluster of sars-cov-2, clearly suggesting human to human transmission in family homes and hospitals and showing that spread of the virus between cities is possible. 10 in late january 2020 large numbers of people across china were returning to their home towns after visiting wuhan for the chinese lunar new year. this weeklong holiday accounts for the largest mass movement of people worldwide each year. consequently, over time more patients are expected to emerge across china and perhaps the world. we found that the characteristics of patients outside of wuhan differed from those initially reported in patients in wuhan. 10 we describe the clinical characteristics and laboratory findings of patients in zhejiang province infected with sars-cov-2 to provide an insight into the prevention and treatment of covid-19 across china and elsewhere. we conducted a retrospective study focusing on the clinical characteristics of confirmed cases of covid-19 in zhejiang province from 10 january 2020 to 26 january 2020. since the outbreak of covid-19, strict precautionary measures have been implemented in zhejiang province, including the creation of fever clinics that exclusively receive patients with suspected sars-cov-2 infection, defined as presenting with a fever or any respiratory symptoms, including dry cough, and especially in those with a history of travel to wuhan or exposure to infected people within two weeks before the onset of illness since january 2020. case definitions of confirmed human infection with sars-cov-2 are in accordance with the interim guidance from the world health organization. 1 only patients with a laboratory confirmed infection were enrolled in this study. we collected data on 62 patients admitted to hospital with laboratory confirmed sars-cov-2 infection in seven designated tertiary hospitals in zhejiang province (see supplementary file for further details). information was collected on dates of illness onset, visits to clinical facilities, and hospital admissions. epidemiological data were collected through brief interviews with each patient. several investigators interviewed each patient to collect exposure histories during the two weeks before illness onset, including the dates and times of close contact (gathering, living, or working together) with individuals from wuhan with confirmed or suspected sars-cov-2 infection. the incubation period was defined as the time from exposure to the onset of illness, which was estimated among patients who could provide the exact date of close contact with individuals from wuhan with confirmed or suspected sars-cov-2 infection. we also investigated the possibility of familial clusters-that is, index patients who travelled to wuhan and then infected others in their families. we extracted the medical records of patients and sent these to the data collection centre in hangzhou. a team of doctors who had been treating patients with covid-19 collected and reviewed the data. because of the urgent need to collect data on this emerging pathogen, the requirement for informed consent was waived. we used a standardised case report form to collect clinical data. if information was not clear, the working group in hangzhou contacted the doctor responsible for the treatment of the patient for clarification. sputum and throat swab specimens collected from all patients at admission were tested by real time polymerase chain reaction for sars-cov-2 rna within three hours. laboratory confirmation of the virus was performed using real time reverse transcription polymerase chain reaction. 9 virus detection was repeated twice every 24 hours. laboratory tests were conducted at admission, including a complete blood count, serum biochemistry, and identification of other respiratory pathogens such as influenza a virus (h1n1, h3n2, h7n9), influenza b virus, respiratory syncytial virus, parainfluenza virus, and adenovirus. most patients received antiviral treatment with interferon alpha inhalation (50 μg twice daily), lopinavir and ritonavir (400 mg twice daily and 100 mg twice daily, respectively), and arbidol (200 mg three times daily). patients received treatment with corticosteroid (40-80 mg/day) and gamma globulin (15-20 g/day) for 3-5 days when their resting respiratory rate was more than 30 per minute, or oxygen saturation was below 93% without oxygen, or multiple pulmonary lobes showed more than 50% progression of disease in 48 hours on imaging. patients also received treatment with probiotics in most cases. quinolones and second generation beta lactams (oral and intravenous) were administered if fever lasted for more than seven days or c reactive protein levels were 30 mg/l or more (normal range 0-8 mg/l). patients suspected of being infected with sars-cov-2 were discharged from hospital once the results of two real time reverse transcription polymerase chain reaction tests taken 24 hours apart were negative for sars-cov-2 antigens. as a previous study 9 has shown that patients' condition worsens on the 10th day after illness onset, we divided the cohort into patients with symptoms for more than 10 days and those with symptoms for less than 10 days. we summarised continuous variables as either means and standard deviations or medians with interquartile ranges. for categorical variables, we calculated the percentages of patients in each category. all analyses were done with spss software, version 22.0. this was a retrospective case series study and no patients were involved in the study design, setting the research questions, or the outcome measures directly. no patients were asked to advise on interpretation or writing up of results. by 26 january 2020, clinical data were collected on 62 patients in zhejiang province with laboratory confirmed sars-cov-2 infection. twenty five (40%) of the patients were aged 19-40 years, 33 (53%) were aged 41-65 years, 2 (3%) were aged 10 and 11 years, and 2 (3%) were aged 65 years and older. the median age was 41 years (interquartile range 32-52 years; table 1). as of 26 january 2020, more than half of the 62 patients (35, 56%) were men. no patients had a history of exposure to the huanan seafood market and all 62 patients had been exposed to individuals with confirmed sars-cov-2 infection. among the 62 patients, 23 (37%) resided in wuhan and the remaining 39 (63%) had made short term trips to wuhan before illness onset. fifty six (90%) patients could provide the exact date of close contact with someone with confirmed or suspected sars-cov-2 infection. of the 33 patients with symptoms for more than 10 days after illness onset, 10 (30%) were aged 19-40 years, 22 (67%) were aged 41-65 years, and 1 (3%) was older than 65 years. the median age of patients was 45 years (interquartile range 37-54 years; table 1). twenty of the 62 patients (32%) had underlying diseases-seven (11%) had liver disease, five (8%) of the 33 patients with symptoms for more than 10 days after illness onset, 13 (39%) had underlying diseases: four (12%) patients had liver disease, four (12%) had hypertension, and one each had chronic obstructive pulmonary disease (3%), diabetes (3%), and cerebrovascular disease (3%). among 56 patients who could provide the exact date of close contact with someone with confirmed or suspected sars-cov-2 infection, the median incubation period from exposure to symptoms was 4 days (interquartile range 3-5 days). the median time from onset of symptoms to first hospital admission was 2.0 (1.0-4.3) days. the most common symptoms at illness onset were fever (48, 77%), cough (50, 81%), expectoration (35, 56%), headache (21, 34%), myalgia or fatigue (32, 52%), diarrhoea (3, 8%), and haemoptysis (2, 3%). only two (3%) patients developed shortness of breath. among the 33 patients who had symptoms for more than 10 days after illness onset, the median incubation period from exposure to symptoms was 3 days (interquartile range 3-4 days). the median time from onset of symptoms to first hospital admission was 6.5 (5.0-9.0) days. the most common symptoms at onset of illness were cough (27, 82%), fever (24, 73%), expectoration (19, 58%), myalgia or fatigue (19, 58%), headache (15, 45%), diarrhoea (3, 9%), and haemoptysis (2, 6%). only one (3%) patient developed shortness of breath. on admission, the blood counts of 19 of the 62 (31%) patients showed leucopenia (white blood cell count <4×10 9 /l) and 26 (42%) showed lymphopenia (lymphocyte count <1.0×10 9 /l; table 2). the d-dimer levels were within normal range (median 0.2 mg/l (interquartile range 0.2-0.5 mg/l). levels of aspartate aminotransferase increased in 10 (16%) patients. 1) . only one patient did not have pneumonia. of the 62 patients, only one was transferred to an intensive care unit for acute respiratory distress syndrome and received mechanical ventilation (table 3) . fifty five (89%) patients received antiviral treatment, 28 (45%) were given empirical antibiotic treatment, and 16 (26%) were given systematic corticosteroid and gamma globulin treatment. at this point, one (2%) patient had been discharged and no patients had died. fitness for discharge was based on abatement of fever for at least three days, with improved evidence on chest radiography and viral clearance in samples from the lower respiratory tract. as of 8 february 2020, more than 30 000 laboratory confirmed cases of infection with the novel coronavirus (sars-cov-19) were reported in china. 12 the number of infections is increasing rapidly. it is possible that an even greater number of infected patients exist without a diagnosis because their symptoms were less severe and because of the incubation period. thousands of patients with suspected sars-cov-2 infection could eventually receive a diagnosis. the clinical features of early cases of covid-19 in wuhan were not the same as those in other areas of china. according to our data, none of the infected patients in zhejiang province had been exposed to the huanan seafood market, and as the number of familial clusters in infected patients in our study is large, this might suggest human to human transmission. this finding is also consistent with a published article. 10 further detailed investigations should aim to ascertain the exact mode of transmission. most of the infected individuals in zhejiang province were male patients, but the age range of patients is large as sars-cov-2 also infected children and those older than 65 years. 9 13 no major differences were found between the initial clinical symptoms of patients in zhejiang province and those in wuhan. most of the patients in zhejiang province, however, had mild to moderate symptoms, and only a small portion of them had dyspnoea. only one patient developed acute respiratory distress syndrome and was admitted to an intensive care unit. the laboratory test results showed that the patients also experienced mild illness. there were fewer patients with abnormal renal function and lactate dehydrogenase and procalcitonin levels. through media and national advocacy, patients with fever, cough, expectoration, and other upper respiratory tract symptoms were asked to go to hospital at an early stage. even those who had contact with other patients, or patients with suspected infection were asked to go to hospital. we also analysed patients with symptoms for more than 10 days after illness onset. we found that the clinical features of patients with symptoms for longer than 10 days in zhejiang province were less severe than those of the primary infected patients from wuhan. 9 13 this phenomenon was also apparent during the transmission of mers-cov. the global case mortality of mers-cov was about 40%, whereas the mortality from second generation mers-cov was about 20%. 14 15 patients in the two cohorts received antiviral treatment, but the types of drugs used varied between patients. treatment with lopinavir and ritonavir were reported to have the potential to treat sars infections, 16 and we suppose this treatment might be a beneficial part of the treatment for covid-19. the rate of antibiotic and corticosteroid use was different. less than half the patients in zhejiang province received antibiotics. whether the use of antivirals, antibiotics, or steroids affects the prognosis of patients remains unknown. given that most infections in zhejiang province were in patients who had no direct contact with the original site of outbreak, our findings provide valuable information in the understanding of the clinical features of covid-19, as the number of people with confirmed disease continues to increase rapidly. our study population might represent most of the clinical characteristics of infected patients since january 2020. further containment proposals should be implemented by the chinese government, such as preventing people from wuhan having contact with those elsewhere, banning gatherings of more than 100 people, conducting daily public-wide educational campaigns on precautionary measures against exposure to sars-cov-2, encouraging people to cancel traditional family gatherings such as during the chinese lunar new year, and extending the chinese lunar new year holiday to prevent large scale spread. our study has several limitations. firstly, only 62 patients were included. a large number of patients were continually being admitted to hospital as data were being collected, and thus we obtained data on most but not all of the patients with laboratory confirmed infection in zhejiang province during the study period. secondly, as the patients were only from zhejiang province, it might be that more clinical features related to covid-19 will be identified. thirdly, at the time of study submission, most patients had not been discharged, so we are unable to estimate either the case fatality rate or the predictors of fatality. moreover, the time since illness onset in some of our patients might be shorter than the observation period of 10 days, which could result in biases of clinical observation characteristics. compared with the symptoms of the initial patients with sars-cov-2 infection in wuhan, those of patients from zhejiang province in our study were relatively mild. currently, no effective drug treatment or vaccine exists. it is necessary for monitoring of the virus to be strengthened and drugs and vaccines to be developed against sars-cov-2 infection as soon as possible. 1 we thank song-jia tang for assisting with english translation. contributors: x-wx and x-xw contributed equally to this article. xwx and ljl conceptualised the paper. xxw analysed the data, with input from jfs, kjx, xgj, ljy, clm, sbl, hyw, sz, hng, hlc, and yqq. xwx and xxw wrote the initial draft with all authors providing critical feedback and edits to subsequent revisions. all authors approved the final draft of the manuscript. l-lj is the guarantor. the corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. funding: no funding. competing interests: all authors have completed the icmje uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work. ethical approval: this study was approved by the ethics committee of the first affiliated hospital, zhejiang university school of medicine (2020iit a0001). patient consent: obtained. data sharing: no additional data available. transparency: the lead authors and manuscript's guarantor affirm that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained. dissemination to participants and related patient and public communities: no study participants were involved in the preparation of this article. the results of the article will be summarised in media see rights and reprints this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is noncommercial clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding who. middle east respiratory syndrome coronavirus evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission clinical features of patients infected with 2019 novel coronavirus in wuhan, china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster novel coronavirus (2019-ncov) epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings supplementary information: description of clinical centres and cases enrolled in this study, and case report form key: cord-284589-j1609xlu authors: sedova, mayya; jaroszewski, lukasz; alisoltani, arghavan; godzik, adam title: coronavirus3d: 3d structural visualization of covid-19 genomic divergence date: 2020-05-29 journal: bioinformatics doi: 10.1093/bioinformatics/btaa550 sha: doc_id: 284589 cord_uid: j1609xlu motivation: as the covid-19 pandemics is spreading around the world, the sars-cov-2 virus is evolving with mutations that potentially change and fine-tune functions of the proteins coded in its genome. results: coronavirus3d website integrates data on the sars-cov-2 virus mutations with information about 3d structures of its proteins, allowing users to visually analyze the mutations in their 3d context. availability: coronavirus3d server is freely available at https://coronavirus3d.org. the main challenge in the rapidly developing covid-19 outbreak is the management of the current pandemic, but predicting its future course is quickly becoming a major focus. differences in the societal responses, such as various levels of social distancing and screening/quarantine implementation are probably the main reason behind the different courses that covid-19 takes in different countries and regions. but at the same time, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus is mutating, which might result in virus escape from diagnostic tests or virus resistance to therapeutic interventions. over twenty-seven thousand sars-cov-2 genomes have been sequenced as of may 15 th , 2020 and their phylogenetic analysis identified the emergence of three major viral clades (gisaid as of may 15 th , 2020). some of the widespread mutations observed in these clades result in amino-acid substitutions. inspection of the corresponding protein structures strongly suggests that they may have an impact on the conformation and functions of the proteins they are found in and, possibly, on the covid-19 outcomes. while there are no confirmed clinical differences between sars-cov-2 from different clades, the ongoing growth of the number of mutations create a high demand for the systematic analysis of nonsynonymous mutations and their possible influence on the covid-19 pandemics. this provided motivation for the development of the coronavirus3d server that provides a unique platform for exploring the distribution of the mutations in the context of the 3d structure of the proteins they are found in. information on the growing genetic diversity of sars-cov-2 is being studied intensively and continuously updated data can be obtained from resources such as gisaid (https://www.gisaid.org) or nextstrain (https://nextstrain.org). at the same time, with the exception of the spike protein mutations, there are no publicly available resources that provide analysis for all the other structurally characterized regions of the sars-cov-2 proteins. coronavirus3d server integrates information about the threedimensional structures of sars-cov-2 virus proteins from the pdb (http://rcsb.org) (berman, et al., 2000) , with the data on sars-cov-2 genomic variations retrieved from china national center for bioinformation (cncb) (https://bigd.big.ac.cn/ncov?lang=en). the server is updated automatically as new data becomes available, the date and details of the last update are listed on the top of the genome viewer panel. the coronaviusr3d website was developed with the protael package (sedova, et al., 2016) and 3d visualizations use the 3dmol.js library (rego and koes, 2015) . the structural models of sars-cov-2 proteins without experimental structures were built using modeller (webb and sali, 2016) based on ffas (xu, et al., 2014) alignments. the central page of the coronavirus server (see figure 1a ,) provides an interactive view of the sars-cov-2 genome (genbank id: © the author(s) 2020. published by oxford university press. this is an open access article distributed under the terms of the creative commons attribution non-commercial license (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. for commercial re-use, please contact journals.permissions@oup.com mn908947.3), with information on boundaries of the predicted proteins, currently available sars-cov-2 structures and a histogram of the aminoacid mutation frequency. if no sars-cov-2 structure is available, links are provided to the models based on the sars-cov structures. in the future we plan to incorporate ab initio models. currently, we provide references to the resources for such predictions on the help page. using buttons on the top of the viewer or selecting specific regions with a mouse, users can zoom in to the display of the selected regions at higher resolution. users can also select individual structures or models, which automatically displays information on the selected structure in the lower panels. detailed information about the functions of the user interface is provided in the help pages available via the link located at the top of the page. the first of the lower level panels (figure 1b) provides interactive visualization of the selected structure or model, with an option for coloring the chain according to the mutation frequency. the example in figure 1b shows the sars-cov-2 structure of the complex between nsp10 and nsp16 (pdb id: 6w4h (rosas-lemus, et al., 2020) ). because chain a was selected for viewing, this chain is shown in color, with the second chain shown with lower intensity. as seen in the figure, some mutations fall on the nsp10/nsp16 interface, possibly changing the stability of the complex. the second of the lower panels ( figure 1d ) provides a list of mutations in the selected protein (or in the selected genomic regions) that can be downloaded for further analysis. the coronavirus3d server was designed to provide users with information and tools to carry out their own analysis of how mutations in the sars-cov-2 proteins may affect their 3d-structures and their functions. we show here two examples of such analyses. the first example is the most common mutation in rna-directed rna polymerase (rdrp or nsp12). this mutation, p323>l (genomic position 14408), is located at the interface between nsp12 and nsp8 proteins in the rdrp complex, as shown on the experimental structure of the nsp7/nsp8/nsp12 complex (pdb id:6yyt (hillen, et al., 2020) ) (figure 1c, top) . mutations at this interface may change the strength of the interactions in the complex and its activation profile. interestingly, genomes with this mutation were demonstrated to have significantly (~3 times) higher mutation frequency as compared to genomes without this mutation (pachetti, et al., 2020) . which could be related to the overactivation of the rna polymerase complex. the second example shows the most widespread mutation in spike glycoprotein -d614>g (genomic position 23403), visualized here on the experimental structure of the spike protein and the human ace2 receptor (pdb id:6vxx (laha, et al., 2020) ) (figure 1c, bottom) . this is the defining mutation of the g clade of the sars-cov-2. the corresponding position in sars-cov is part of an immunodominant epitope (wang, et al., 2016) . interestingly, the mutations mentioned in these two examples are observed in practically the same set of genomes corresponding to the g clade. it is still unclear which of these mutations (if any) contributes to the apparent recent expansion of the g clade. the protein data bank structure of replicating sars-cov-2 polymerase characterizations of sars-cov-2 mutational profile, spike protein stability and viral transmission emerging sars-cov-2 mutation hot spots include a novel rnadependent-rna polymerase variant js: molecular visualization with webgl the crystal structure of nsp10-nsp16 heterodimer from sars-cov-2 in complex with s-adenosylmethionine protael: protein data visualization library for the web immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates comparative protein structure modeling using modeller ffas-3d: improving fold recognition by including optimized structural features and template re-ranking we acknowledge efforts of the teams at pdb, cncb, gisaid and nextstrain for maintaining and distributing information on covid-19 and all the individual laboratories that make their results public and available through these depositories. this work is supported in whole or in part by nih institutes: niaid under contract no. hhsn272201700060c and nigms by a grant gm118187.conflict of interest: none declared. key: cord-304073-f3iwclkm authors: mullick, jhinuk basu; simmons, chelsey s.; gaire, janak title: animal models to study emerging technologies against sars-cov-2 date: 2020-07-27 journal: cell mol bioeng doi: 10.1007/s12195-020-00638-9 sha: doc_id: 304073 cord_uid: f3iwclkm new technologies are being developed toward the novel coronavirus sars-cov-2 to understand its pathogenesis and transmission, to develop therapeutics and vaccines, and to formulate preventive strategies. animal models are indispensable to understand these processes and develop and test emerging technologies; however, the mechanism of infection for sars-cov-2 requires certain similarities to humans that do not exist in common laboratory rodents. here, we review important elements of viral infection, transmission, and clinical presentation reflected by various animal models readily available or being developed and studied for sars-cov-2 to help bioengineers evaluate appropriate preclinical models for their emerging technologies. importantly, applications of traditional mice and rat models are limited for studying sars-cov-2 and development of covid-19. non-human primates, syrian hamsters, ferrets, cats, and engineered chimeras mimic the human infection more closely and hold strong potential as animal models of sars-cov-2 infection and progression of resulting human disease. the novel coronavirus sars-cov-2, which emerged in december 2019 and causes the disease ''coronavirus disease , has galvanized biomedical research to understand, prevent, and treat this life-threatening condition. 43 as with many diseases, animal models are being leveraged to study cellular pathogenesis and transmission of infection, as well as to examine the efficacy of viral vaccines and analyzing herd immunity post-vaccination. however, unique features of sars-cov-2 limit the utility of traditional laboratory animals. sars-cov-2 has a solar-corona-like appearance with spike surface proteins, the characteristic namesake of coronaviruses. these spike proteins have two subunits: the surface unit s1 binds with high affinity to human angiotensin-converting enzyme 2 (ace2) receptors and the transmembrane unit s2, which is then cleaved by human transmembrane serine proteases tmprss1 and tmptss2. 24 ace2 and tmprsss co-expression are essential for sars-cov-2 infection, but mice, rabbits, rats, and guinea pigs do not have ace2 receptors susceptible to sars-cov-2 binding. 49 furthermore, due to their anatomical difference and small size, intranasal viral inoculation of typical small rodent models can result in inhalation and ingestion, thus making it difficult to discriminate between intranasal, oral, and intrapulmonary vaccination 31 and limiting their utility as a model of vaccination against sars-cov-2. here, we discuss alternative rodent models, large animals, and chimeras that may be useful to the bioengineering community working on innovative treatments and vaccinations against sars-cov-2. to investigate sars-cov-2 as with most diseases, no single animal model perfectly matches the clinical profile of sars-cov-2, as observed in humans. a necessary condition for animal models to model the cellular pathogenesis of sars-cov-2 infection is ace2 receptor and serine proteases similar to humans, as discussed above. several larger mammals such as palm civets, pigs, ferrets, cats, and nonhuman primates have ace2 receptors which the virus can recognize. 54 however, many animals known to be susceptible to similar coronaviruses, e.g., chickens, pigs, and dogs, 19 have not been reported to be susceptible to sars-cov-2. given the nuances of each animal model, the choice of animal model for studying covid-19 should vary based on the application. to investigate treatments, for example, models should be considered that show characteristic symptoms similar to humans such as shortness of breath, fever, weight loss, loss of appetite, and pneumonia. 20 since older humans have exhibited increased susceptibility to sars-cov-2 33 models utilizing aged adult animals should be favored. for genomic and pathological studies, viral rna or histopathological tissues need to be obtained, for which animal models should be preferred in whom similar organs as those affected during human pathogenesis are involved. for covid-19, these organs include the respiratory tract, lungs, and gastrointestinal tract. mode of transmission must be similar for projects working to develop personal protective equipment such as respiratory masks or eye protection. in such urgent times, large animal and non-traditional rodent models can be readily utilized without the time delay associated with developing transgenic and chimeric small animal models. large mammals also have the advantage of physiological, anatomical and immunological proximity to humans. 16 their mucosal surfaces can be easily accessed for intranasal and oral vaccine administration and, often being outbred species, may yield more clinically relevant results. a brief overview of readily available animal models of cov-id-19 is provided in table 1 . hamsters are adept at amplifying many viruses and often studied for viral persistence and shedding. 4 the golden syrian hamster (mesocricetus auratus) had been used previously to study other respiratory viruses such as severe acute respiratory syndrome coronavirus (sars), adenovirus, and influenza virus. the structure of the ace2 receptor of hamsters is similar to humans, allowing the spike protein of sars-cov-2 to bind with high affinity and generating preference for the syrian hamster as a model in both sars and sars-cov-2 studies. to mimic human transmission, recent studies challenged male and female syrian hamsters intranasally. 10, 45 the viral loads were found to be highest in the lungs at 2 days post-inoculation (dpi) and were reduced below the detection limits by 7 dpi, when the animals started to recover. the animals showed passive immunization whereby hamsters developed neutralizing antibodies (nabs) by 7 dpi 10 or by 14 dpi. 45 high transmission rate, as shown by transmission to all the naı¨ve co-housed hamsters, did mimic the pathogenesis in humans and was likely through respiratory droplets or oral-fecal contamination. despite high viral loads observed throughout many organs, the hamsters showed only mild symptoms such as weight loss, lethargy, ruffled fur, hunched back posture, and rapid breathing. 10 the main disadvantage of the syrian hamster model was the lack of mortality which did not match the human clinical profile. these studies also used younger hamsters between 4 and 10 weeks of age, likely following previous sars-cov experiments. 39 however, in light of known complications in elderly patients, 33 it may have resembled human disease more closely if aged hamsters were used. overall, the studies show that the syrian hamster is a useful animal model for sars-cov-2 infection especially to study viral replication, shedding, and transmission through the respiratory tract. having anatomical and physiological similarity with the human respiratory system, ferrets (mustula putorius furo) have previously been used for studying sars infection. 56 recent reports suggest affinity of sars-cov-2 for binding to ferret ace2, and the mechanism of transmission correlated with human transmission, which required direct physical contact with potential airborne transmission as viral rna was animal models of sars-cov-2 detected in nasal washes, saliva, blood, fecal, and urine samples. 30, 42 both in humans and ferrets, the virus affected similar organs such as the nasal turbinate, trachea, lung, soft palate, tonsils, intestine, and kidney. 6, 30, 42 clinical signs were mild compared to human patients and included fever and loss of appetite. nabs were detected starting 13 dpi. 42 blanco-melo et al. analyzed the transcriptional regulation of immune modulators to sars-cov-2 in ferret model and inferred that severity of covid-19 in aged population was linked with a severe inflammatory response. 6 they suggest that controlling the sars-cov-2-associated cytokine storm 59 should be the primary focus of treatment. the ferret thus can serve as a useful model for studying the pathogenesis of sars-cov-2 infection in the respiratory tract. ferrets have been considered to be outbred species and hence also used widely in vaccination studies. however recent findings suggest that due to inbreeding, ferrets can be clustered based on geographical locations, where north american and australian clusters have been found to be very low in genetic diversity. 21 researchers using ferrets as a model should consider these issues of the genetic background when interpreting their findings. sars and sars-cov-2 viruses cannot bind to mouse ace2 (mace2) due to differences in key amino acid residues. 49 due to this low sensitivity of mice to sars viruses, several transgenic mice lines were created during the sars epidemic to study its pathogenesis. human ace2 (hace2) genes were expressed under a hybrid chicken beta-actin promoter with cytomegalovirus enhancer 51 or cytokeratin 18 (k18) promoter. 35 the jackson laboratory (bar harbor, me) has recently started re-producing the k18-hace2 strain used to study sars infection, 55 although systemic damage and neuroinflammation still did not represent the human clinical profile. 49 yang et al. developed an efficient model where the endogenous mouse ace2 promoter was used and had tissue distribution similar to the natural hace2 distribution; however, this model still could not mimic the high mortality observed in humans. 58 since both the viruses have a similar mechanism of entry through the ace2 receptor, the developed models are still relevant for sars-cov-2 study. bao et al. used the mace2 promoter line to study the pathogenesis of sars-cov-2 infection. 2 viral rna was detected in the lungs, bronchi, and alveoli, while the wild-type mice failed to be infected by sars-cov-2. the study thus found that the ace2 receptor was essential for sars-cov-2 infection, as expected. soldatov et al. have been working on producing a transgenic line where hace2 and htmprss2 are coexpressed with the mice tmprss2 promoter through gene editing using crispr/cas9. 49 the group hypothesizes that the co-expression will make the model mimic human pathogenesis more closely, particularly in the lungs. other transgenic mice models studied during sars infection can also be helpful for sars-cov-2 studies. one such model is the ace2 knockout mouse, which lacks the ace2 receptor and upon sars infection has been seen to suffer from acute lung failure. 27 tmprss2 knockout mice have shown lower levels of inflammatory cytokines and infiltration by immune cells, leading to less severe tissue damage in the lungs after sars and middle east respiratory syndrome coronavirus (mers) infections, 28 which has pointed researchers to tmprss2 as a potential therapeutic target for serine protease inhibitors. additional strains such as stat1 knockout, balb/c, c57bl/6, and 129s6 mice that were studied for sars may prove to be useful as well. 50 the immune system of feral mice or ''dirty mice'', mice exposed to a diverse group of microbes, have broad t cell distribution that could rapidly control pathogens 18 and render them potential candidates to gain insights into vaccine development for sars-cov-2. as close evolutionary relatives to humans, non-human primates have very similar physiology and immunology to humans 31 and hence greater translational potential. issues with non-human primate models, however, include additional ethical justifications, scarcity of biosafety level-3 facilities, and lack of trained personnel to carry out work. 10 nonetheless, many primate models have yielded helpful insights into sars-cov-2. the rhesus macaque (macaca mulatta) is broadly popular for studying re-infection potentials and designing vaccines. when infected with sars-cov-2, specifically, the rhesus macaque model manifests mild to moderate symptoms of decreased appetite, weight loss, fever, and increased or irregular respiration patterns along with hunched posture. 3, 11, 34, 37 viral rna can be detected throughout the respiratory and gastrointestinal tract, 3, 11, 34, 37, 41 mimicking humans, and also from atypical organs such as spinal cord, heart, skeletal muscles, and bladder 3 ; liver and kidney 11, 14 ; and the spleen. 34 the hallmarks of human sars-cov-2 infection of pulmonary edema and diffused interstitial pneumonia were observed on radiographs throughout these studies. in age-related comparative studies, yu et al. studied the effects of sars-cov-2 infection with three ''young'' (3-5 years) and two ''geriatric'' (15 years) animals. 62 they found geriatric macaques suffered from more severe interstitial pneumonia with more viral load in lungs and anus. the older macaques had viral replication in the entire lung while the younger ones had only in the upper lobes of the lung. another age-related study found that older animals had higher levels of virus-specific antibodies which were detectable early, by 4 dpi. 34 rhesus macaques were also used to investigate uncommon transmission routes; deng et al. inoculated sars-cov-2 through conjunctival and gastric routes. 14 though sars-cov-2 failed to replicate via the gastric route, mild interstitial pneumonia and alimentary canal infection were observed in the conjunctival route challenge. rhesus macaques also have been used to observe if primary infection with sars-cov-2 can protect against re-infections. in these studies, viral loads measured by bronchoalveolar lavage, nasopharyngeal swabs, or anal swabs were found to be reduced manyfold upon reinfection. 3, 11 chandrashekar et al., upon rechallenging the animals, found viral loads were reduced > 100,000-fold. 11 similar results were observed by bao et al., where no clinical symptoms were observed upon reinfection. 3 in a vaccine screening study, yu et al. developed dna vaccine candidates against sars-cov-2 spike protein, which were tested on 35 adult rhesus macaques. 61 after vaccination and upon a second exposure, viral loads were reduced > 1,000-fold. in all studies, animals developed nabs. overall, the rhesus macaque model has been similar in many aspects to the human covid-19 pathogenesis. nonetheless, though studies utilized adult animals up to 15 years old, they could not mimic the high fatality rate of humans. 3, 11, 37 cynomolgus macaques the crab-eating macaque (macaca fascicularis), originally found in southeast asia, had been studied previously for sars. 17 similar to rhesus macaques, when infected with sars-cov-2, geriatric animals showed prolonged viral shedding from the upper respiratory tract in comparison to younger animals through 21 dpi. 40 however, animals infected with sars-cov-2 showed no obvious clinical signs or mortality, even in the geriatric group, 40 which is in contrast to severe systemic responses seen in older macaques for sars. 48 when comparing male and female macaques, no difference in levels of antibodies was observed, though nabs could be detected as early as 4 dpi. 34 lu et al. did report fever and weight loss, and both studies reported diffuse interstitial pneumonia, a common complication in humans. 34, 40 early and prolonged virus shedding of sars-cov-2 compounded with the lack of symptoms highlights an area of concern for community transmission among humans as well. while macaques do not mimic the severity of human symptoms, they may still be helpful to understand disease progression in older and potentially asymptomatic subjects and thus to formulate strategies for containment of covid-19. common marmosets (callithrix jacchus), a new world monkey, were widely used to understand the pathogenesis of and immunization against the deadly mers as their pathophysiology mimicked lethal pneumonia seen in humans. 9 its utility as a model to study sars-cov-2 has not been explored much, though. a single comparative study by lu et al. was found in which six marmosets were intranasally infected with the virus for comparison with other nonhuman primates. 34 viral rna was observed till 14 dpi in blood and nasal, throat, and anal swabs; however, viral-specific antibodies could not be detected in serum, which could be due to lack of marmoset specific antibody detection kit. only one-third of the marmosets had elevated body temperature, lung tissue showed no indication of pneumonia, and no other organs showed the presence of viral rna. overall, the common marmosets were found to be relatively resistant to sars-cov-2 as compared to rhesus macaques and cynomolgus macaques. there has been evidence of infection with sars-cov-2 in tigers at the bronx zoo and isolated reports of transmission from humans to domestic cats. 25 it is helpful therefore, to look into the infectivity of sars-cov-2 on cats to prevent chain of transmission between cats and humans. cats previously had been found susceptible to sars infection and to have the ace2 receptor important in the development of covid-like symptoms. 52 more recently, shi et al. inoculated seven 6-9 months-old ''sub-adult'' cats intranasally with a sars-cov-2 viral strain. 42 viral rna was detected in the nasal turbinates, soft palates, tonsils, tracheas, and the small intestine on 3-6 dpi. the group also found transmission through droplets to the exposed cats. in 70-100 days-old ''juvenile'' cats, by 3 dpi, massive viral lesions were found in the nasal cavity, trachea and lungs, indicating that juvenile cats allow for better replication of the sars-cov-2 virus. nabs were detected in the sub-adult cats between 11 and 12 dpi and in the juvenile cats between 10 and 20 dpi. in a similar study by halfmann et al., inoculated cats showed viral shedding in nasal swab by 1-3 dpi. 22 transmission was also observed in cats that were cohoused. all tested cats were asymptomatic with no clinical symptoms of fever or weight loss. all inoculated and transmission-infected cats produced anti-sars-cov-2 igg antibodies at 24 dpi. 22 in another study, it was reported that 14.7% of cats who were exposed to infected humans harbored antibodies against sars-cov-2 spike protein. 63 the above studies indicate that cats are susceptible to the virus and may harbor it without showing any evident clinical symptoms or mortality. since humans are in close contact with domesticated cats, there is therefore a risk of transmission to humans from asymptomatic feline companion animals. canine ace2 is similar to hace2, 19 and though dogs are known to be infected by some coronaviruses, sars-cov-2 does not seem to be a concern in canines. the first suspected case of human-to-animal transmission of sars-cov-2 was in a pomeranian dog belonging to an infected person in hong kong. 47 generally, isolated cases of sars-cov-2 infection in companion canines have found dogs to remain asymptomatic though viral rna has been found in nasal, oral and rectal swabs. 47 shi et al. inoculated five 3-month-old beagles with sars-cov-2 intranasally, and two un-inoculated beagles were co-housed for transmission studies. 42 while viral rna was observed in rectal swabs of three virus-inoculated dogs by 2-6 dpi, viral rna was not observed in any other organ. by the 14 dpi, two virus-inoculated dogs produced antibodies while the other dogs were found to be seronegative. overall, results from these studies and broad testing of pets 26 indicate low susceptibility for sars-cov-2 among dogs, suggesting a minimal risk of asymptomatic transmission from canine companion animals to humans. the first coronavirus infections were identified in chickens in the 1930s, thirty years before the discovery of the disease in humans. 8 however, the current pandemic-causing coronavirus sars-cov-2 has been reported to be non-infective towards chickens, ducks, and pigs. 42 in both inoculated animals and co-housed uninoculated animals, no viral rna could be isolated from swabs. all animals were also found to be seronegative by 14 dpi. in horses, the enteric equine coronavirus has surged over the last few years, but there is no evidence horses are susceptible to infection with sars-cov-2. 13 while farm animals are unlikely to transmit sars-cov-2, extensive outbreaks have been documented at meat processing plants because of the close working conditions among human workers, suggesting that food production will likely remain a concern throughout the pandemic. 15 despite highly conserved genes broadly, therapeutic and immunization strategies involving adaptive immunity are hard to translate from traditional animal models like mice, rats, and non-human primates into humans. to address these limitations, various strains of immunodeficient mice have been leveraged to create chimeras by transplanting cells and tissues of interest from humans. 12, 38, 44 in particular, engineered chimeras incorporating human immune cells tend to better recapitulate the human immune system, enabling human-specific immune responses to deadly viruses. for example, humanized mice created by injecting human peripheral mononuclear cells (pbmcs) or human cd34+ cells into nod scid gamma (nsg) mice, commonly known as hupbmc-nsg or cd34+ humanized mice, respectively, have been a valuable tool for studying viral infections including hiv, 29 dengue, 36 and hepatitis. 5 as an example, kim et al. created hupbmc-nsg mice and infected with hiv-1 to test the effectiveness of antiviral drugs and the usefulness of nabs. 29 following infection, the authors observed an increase in plasma viral load and a decrease in cd4+ t cells. after the use of an antiviral drug and a nab in hiv-infected hupbmc-nsg mice, they observed a decrease in plasma viral loads and no decline in cd4+ t cells, demonstrating the effectiveness of intervention strategy. although we have not identified reports using humanized mice to study covid-19, success from the aforementioned viral studies highlight the relevance and utility of humanized mice for covid-19 research. cd34+ humanized mice and pbmc humanized mice are available from commercial vendors such as the jackson laboratory, and humanized mice can be further tailored according to research applications. recently, wahl et al. created mice with human lung tissue by subcutaneous transplantation of human fetal lung in nsg mice and later combined these mice with bone marrow-thymic-liver mice, a mouse line with a very robust human immune system. 53 thus, they created a humanized mouse containing both the human immune system and human ''lungs'' that could potentially be used for studying pathogenesis of respiratory viruses like sars-cov-2 and screening for antiviral drugs and therapeutic vaccines. humanized mice, though being the closest replication of the human immune system in a small animal model, come with some limitations. while mice with a degree of ''humanized'' immune function are available commercially, it will take additional effort to develop enhanced variants of humanized mice with a more functional human immune system, e.g. with human leukocyte antigens. 7 unlike readily available small animal models with a competent immune system, it takes a longer time to develop humanized mice and requires additional aseptic handling practices and pathogen-free facilities. nonetheless, humanized mice offer a promising test-bed for rapid development and testing of antiviral drugs, vaccines, and their delivery related to covid 19 research. inspired by the success of using humanized mice to study species-specific response in small animal models, researchers have leveraged the utility of immunodeficient mice to develop chimeras using immune cells from complex animals such as bats. bats are thought to harbor a number of viruses dangerous to humans and other animals without effect, but how viruses and the bat immune system coexist asymptomatically is largely unknown. moreover, sars-cov-2 has~90% similarities to beta-coronaviruses isolated from bats, suggesting bat-to-human transmission was at the nexus of the current pandemic. in vivo studies of bats are limited, though, by challenging breeding conditions and long gestation periods, 23 concerns with capturing large numbers of conserved wild bats, 46 and other challenges of outbred species. most studies on bats are limited to specialized bat cell lines, 1,64 and limits to the availability of these bat-specific cell lines and batspecific antibodies make even in vitro research more challenging than anticipated. to mitigate some of these challenges, a chimera model has been developed by yong et al. that stably expresses the immune system of the bat (eonycteris spelaea) on a mouse background. 60 these ''bat-mice'' have been developed by transplanting bat bone marrow cells into immunodeficient nsg mice. this study showed that~80 to 100 bat-mice can be generated from one bat, thus reducing problems associated with animal numbers. the bat-mice model reconstituted all major immune cells including monocytes, t and b lymphocytes, natural killer cells, and dendritic cells. the model has been found to generate bat specific antibodies in response to antigens as well as resistance to graft rejection when transplanted with bat cells even after 40 weeks. the development of bat-mice has opened up new avenues for research that were previously deemed challenging due to the limited availability of bats for immunological research. a better understanding of how the bat's immune system handles various pathogens, including deadly viruses such as sars-cov-2, will provide valuable insights to strategize the development of effective therapeutic and preventative approaches and additional understanding of bat-tohuman transmission of a variety of viruses. an efficient covid-19 animal model which closely resembles the human clinical picture can accelerate the path for vaccine development and therapeutics as well as shine light on viral pathogenesis and formulation of preventive strategies. for most diseases, mice and other small rodents have been modified to cater to various demands of the research community. an abundant range of reagents and assays are available, too, for such analysis. the problem specific to sars-cov-2, however, has been the dominant role of the ace2 receptor in covid-19 pathogenesis. the virus so far has not been found to bind to the ace2 receptor of any of the commonly used small animals unless genetically modified. genetic modification is a timeconsuming process, and the rapid spread of sars-cov-2 infection and its compounding socioeconomic effects require more rapid solutions. thus, researchers are shifting interest toward readily available larger animal models. many large animals are closer anatomically and physiologically to humans, can be outbred, show many clinical similarities to the human infection, and very importantly have ace2 receptors recognized by the sars-cov-2 virus. there are several drawbacks, however. no animal model has manifested clinical symptoms as severe as observed in humans, especially regarding the mortality rate. only a few studies could be found which involved an aged animal, 34, 40 where even then no fatality was observed. we conjecture that environmental factors could be at play as animals are typically kept in well-controlled indoor environments, whereas patient morbidity correlates with regions of increased air pollution. 57 the results of therapeutic studies on animals housed in sterile conditions should therefore be interpreted cautiously. also, the animal models discussed above have been tested with sars-cov-2 strains which were clinically available from local human subjects. recent reports show as many as fourteen mutated variants of the spike proteins from different geographical locations, some of which are more virulent than the others. 32 studies on one or two specific local strains may be hard to extrapolate to a global scale. as with many large animal studies, most done for sars-cov-2 were limited in the number of animals tested compared to gold-standard preclinical rodent studies, except a large study of dna vaccine candidates on 35 adult rhesus macaques. 61 these limited numbers, coupled with a lack of investigation into the duration of protection afforded by neutralizing antibodies, leave much work in the development and testing of anti-viral treatments and vaccination strategies. drawbacks notwithstanding, a wide range of animal models with ace2 receptors and serine proteases susceptible to sars-cov-2 are available to study emerging 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through grant r35gm128831 to css. nothing to disclose. key: cord-257958-yehnlabq authors: barh, debmalya; tiwari, sandeep; weener, marianna e.; azevedo, vasco; góes-neto, aristóteles; gromiha, m. michael; ghosh, preetam title: multi-omics-based identification of sars-cov-2 infection biology and candidate drugs against covid-19 date: 2020-10-10 journal: comput biol med doi: 10.1016/j.compbiomed.2020.104051 sha: doc_id: 257958 cord_uid: yehnlabq sars-cov-2 has ushered a global pandemic with no effective drug being available at present. although several fda-approved drugs are currently under clinical trials for drug repositioning, there is an on-going global effort for new drug identification. in this paper, using multi-omics (interactome, proteome, transcriptome, and bibliome) data and subsequent integrated analysis, we present the biological events associated with sars-cov-2 infection and identify several candidate drugs against this viral disease. we found that: (i) interactome-based infection pathways differ from the other three omics-based profiles. (ii) viral process, mrna splicing, cytokine and interferon signaling, and ubiquitin mediated proteolysis are important pathways in sars-cov-2 infection. (iii) sars-cov-2 infection also shares pathways with influenza a, epstein-barr virus, htlv-i, measles, and hepatitis virus. (iv) further, bacterial, parasitic, and protozoan infection pathways such as tuberculosis, malaria, and leishmaniasis are also shared by this virus. (v) a total of 50 candidate drugs including the prophylaxis agents and pathway specific inhibitors are identified against covid-19. (vi) betamethasone, estrogen, simvastatin, hydrocortisone, tositumomab, cyclosporin a etc. are among the important drugs. (vii) ozone, nitric oxide, and photosensitizer drugs are also identified as possible therapeutic candidates. (viii) curcumin, retinoic acids, vitamin d, arsenic, copper, and zinc may be the candidate prophylaxis agents. nearly 70% of our identified agents are previously suggested to have anti-covid-19 effects or under clinical trials. among our identified drugs, the ones that are not yet tested, need validation with caution while an appropriate drug combination from these candidate drugs along with a sars-cov-2 specific antiviral agent is needed for effective covid-19 management. the first case of covid-19 caused by sars-cov-2 infection was reported between december 7, 2019 to december 12, 2019 from huanan, hubei province, china [1] [2] [3] and as of september 28, 2020, the virus has infected 32,730,945 people with 9,91,224 deaths globally [4] . currently, the usa is the most affected country by the covid-19 pandemic with an estimated 6,960,152 infections and 2,02,478 deaths, followed by india (5, 992, 532 infected, 94,503 deaths), and brazil (4,689,613 infected, 1,40,537 deaths) [4] . although, the "person-to-person transmission" of sars-cov-2 was reported on january 24, 2020 [5] and several quarantine and management strategies were adopted in various countries, the infection could still not be controlled and neither the morbidity. one of the major causes for this uncontrolled number of infection and deaths is the unavailability of sars-cov-2 specific vaccines and antiviral drugs. to overcome the crisis and to identify effective anti-sars-cov-2 drugs, repurposing of the existing fda approved antiviral drugs are given high priority [6] . although, individual host omics such as interactome [7] , proteome [8] , and transcriptome [9, 10] based pathways of sars-cov-2 infected host cell or subject have been described and potential drugs targeting such pathways were reported, so far, no integrative omics based approaches have been employed to study the sars-cov-2 infected host biology and pathways in order to predict the potential drugs. in this paper, we have used an integrative omics approach considering the sars-cov-2 infected host interactome, proteome, transcriptome, and bibliome datasets and analysed the covid-19 associated host genetic information to identify common host pathways that are deregulated during sars-cov-2 infection and potential drugs targeting those pathways. we have also reported the sars-cov-2 infected host biology/ pathways and potential drugs from each omics-based approach separately. in this work, we analysed five different peer-reviewed published omics data sets to identify the infection biology and candidate drugs against sars-cov-2. the overall approach applied in our work is presented in fig-1 . j o u r n a l p r e -p r o o f we have used five omics data sets in this analysis. the first data set, designated as the interactome, outlines sars-cov-2 interaction with 332 human (hek293t/17 cell line) proteins as described by gordon et al., 2020 [7] . the second data set used in this study is the transcriptome (designated as transcriptome-1) of lung epithelial cells upon viral infection. we have considered 88 up-regulated genes from this transcriptome profile [9] . the third data set comprise of 1067 up-regulated genes from the peripheral blood of covid-19 patients (designated as transcriptome-2) as described by xiong for identification of potential drugs from various gene sets, we used two additional tools enrichr (https://amp.pharm.mssm.edu/enrichr/) [13] and web-based gene set analysis toolkit (webgestalt, www.webgestalt.org) [13] apart from the toppfun module of toppgene suite [12] . enrichr uses dsigdb (a drug signatures database) [14] and drugmatrix (a comprehensive toxicogenomic database) for gene set analysis based candidate drug enrichment. all default parameters were used in enrichr and toppfun. in webgestalt [15] , the over-representation analysis was performed using human genome as reference set and drugbank [16] as the functional database. default parameters for all these tools were used and a p-value <0.05 was considered significant and used to generate the results. to understand the network-based biological processes, pathways, and protein-drug interactions for each data set and also the combined data set, networkanalyst 3.0 (www.networkanalyst.ca) [17] was used. since sars-cov-2 predominantly infects the lung cells, in our network analysis the human species was used, and to generate the lung tissuespecific protein-protein interaction (ppi) network, from the tissue-specific ppi module of networkanalyst, the lung tissue type was selected. for generic ppi, the imex interactome database (innatedb) [18] was used. the top ten genes, based on their degree and j o u r n a l p r e -p r o o f betweenness, were considered. for network-based pathways, the kyoto encyclopedia of genes and genomes (kegg) database [19] and for biological processes, the panther:bp [20] modules that were integrated with networkanalyst was used. the common pathways and bps under top ten hits were selected for further analysis. the genes associated with the selected common bps and pathways were collected and analysed using toppfun [12] to identify drugs for these gene sets. additionally, the protein-drug interactions module of networkanalyst 3.0 [17] was also used to identify drugs for each omics sample-based network using the drugbank database [16] and filtered with minimum-network. in this approach, we combined the top ten pathways from each of our analysis (individual and combined omics analysis). based on their cumulative number of appearances, the common pathways were ranked. pathways enriched with at least 3 omics-based analyses are presented in the results. we followed a similar approach for selecting bps also. to identify the most frequent drugs enriched across all our analysis, we also adopted a similar strategy. additionally, in this case, we classified all the drugs following the drug categories as suggested by the drug information portal of the u.s. national library of medicine (https://druginfo.nlm.nih.gov/drugportal/) and listed the most common drug categories across all the analysis. further, we also presented the most frequent drugs under each category for better identification of the important drugs. this drug based enrichment was performed for both omics-based and pathway-based drugs that were identified in our analysis. in our analysis, several common and unique biological processes and drugs are enriched depending on the omic data sets or the analysis tools used. here, we report the important pathways, biological process, and drugs. the detailed outcomes of each analysis can be found in the supplementary materials. first, we used the toppgene suite [12] for candidate gene prioritization. as the bibliome based gene set is a combination of all kinds of sars-cov-2 infection associated human genetic data, we used the bibliome based gene set collected through disgenet [11] as the training set for this toppgene based analysis. (table s1 ). however, while we combined the top 10 genes from all the candidate gene-based analysis and table s1) . gordon et al., 2020 [7] in their ppi study showed that 40% of the interactome are related to endomembrane and er/golgi/ mitochondrial trafficking. sars-cov-2 predominantly interacts with innate immune pathways, host translation machinery, and cullin ubiquitin ligase complex. in our interactome set enrichment, we also observed that, the interactome is mostly associated with organelle membranes and involved in intracellular protein targeting or transport. additionally, we observed that the sars-cov-2 interacting human proteins are mostly involved in cell cycle, nuclear pore complex (npc) disassembly, mitochondrial protein import, and regulation of glucokinase (table s2 (i)). in our lung-specific interactome analysis, this interactome was also found to be involved in viral carcinogenesis, mrna splicing, negative regulation of apoptosis, ubiquitin mediated proteolysis, protein processing in endoplasmic reticulum, and spliceosome pathways (table s3 (i)) . in their analysis, gordon et al., 2020 [7] suggested that protein biogenesis inhibitors, sigma1 and sigma2 receptor inhibitors, eif4a inhibitors, sec61 inhibitors, anti-cancer compounds, and antipsychotic antihistamines could be potential drugs against sars-cov-2. in our analysis, we also observed enrichment of anticancer or antipsychotic drugs. our identified potential drugs are antineoplastic antibiotics (idarubicin, romidepsin, daunorubicin, epirubicin), antineoplastic agent (irinotecan), anti-depressive and anti-psychotic agents (fluoxetine, fluphenazine), estrogens (coumestrol, conjugated estrogen), anti-bacterial agents (tunicamycin, latamoxef), enzyme inhibitor (thapsigargin), central nervous system agent (chlorzoxazone), dermatologic agent (verteporfin), adrenergic agent (dobutamine), and metal/growth substance (polaprezinc). apart from these, flavin adenine dinucleotide, guanosine-5'-diphosphate, myristic acid, and kappadione are also enriched in our analysis (table s2 (i), table s3 (i)). anticoagulants (citric acid) are also found to be good candidates (table s2 (p), table s3 (p)). blanco-melo et al., 2020 [9] in their in vitro transcriptome (transcriptome-1) study using normal human bronchial epithelial (nhbe) cells have shown that sars-cov-2 induces a low ifn-i and -iii and high chemokine signalling. this unique and inflammatory response was not observed in sars-cov-1, mers-cov, iav, and rsv [9] . in our analysis, we also found that this up-regulated transcriptome is associated with interferon and cytokine signaling pathways. further, the transcriptome-1 is also enriched for influenza a, herpes, and hepatitis c infections (table s2 (t-1)). the network-based analysis enriched negative regulation of apoptotic process, mrna splicing, viral carcinogenesis, hepatitis b, epstein-barr virus infection, nod-like receptor signaling pathway, ubiquitin mediated proteolysis, il-17 signaling pathway, measles, hepatitis c, and influenza a (table s3 (t-1)). the results are mostly common to the cellular proteome-based analysis, except the ubiquitin mediated proteolysis, which is not found in case of the proteome data set (table s3 (p)). for the up-regulated cellular transcriptome (transcriptome-1), we identified several potential drugs. these drugs belongs to several drug categories such as-adrenal cortex hormones (table s2 (t-1), table s3 (t-1)) xiong et al., 2020 [10] reported that, upon infection with sars-cov-2, the patient's blood transcriptome (transcriptome-2) profile shows increased complement activation, humoral immune response, immunoglobulin mediated response, acute inflammatory response, and lymphocyte apoptosis. we also observed a similar profile for transcriptome-2, based on our gene set enrichment analysis. however, in addition to these biological processes and pathways, we found that the malaria pathway is also shared by sars-cov-2 (table s2 (t-2) ). in the network-based lung-specific ppi analysis, we found a profile that is similar to the bronchial epithelial cell specific transcriptome-based (transcriptome-1) findings by blanco-melo et al., 2020 [9] . we found viral carcinogenesis, negative regulation of apoptotic process, endocytosis, mrna splicing, epstein-barr virus infection, hepatitis b, htlv-i infection, measles, and ubiquitin mediated proteolysis pathways in our analysis (table s3 (t-2)). the candidate drugs for this up-regulated patient's blood transcriptome are identified as: antihypertensive agent (hydrochlorothiazide), antirheumatic agent (leflunomide), antiasthmatic agent (theophylline), and anesthetic (procaine). we also found that anthelmintic drug lucanthone is enriched in our analysis (table s2 (t-2), table s3 (t-2)). the bibliome-based gene set analysis showed mostly similar biological events that we had (table s2 (b), table s3 (b)). our combined omics associated gene set enrichment analysis (proteome and transcriptomes) revealed a similar biology and pathways of sars-cov-2 as we found in each individual transcriptome analysis. the key bps and pathways involved are complement activation, humoral immune response, and cytokine and interferon signaling ( (table s1 (p+t), table s3 (p+t)). for the combination of all the five omics data sets, we identified antineoplastic agents (table s1 (p+t+i+b), table s3 (p+t+i+b)). in our analysis, we observed sars-cov-2 infection shares other viral pathways such as to identify pathway specific drugs, we used the genes involved in the five most important common pathways (viral processes including all the individual virus pathways, mrna splicing, ubiquitin mediated proteolysis, cytokine signaling in immune system, and protein processing in endoplasmic reticulum). for each pathway, genes from all the five omics data sets (identified from our network based analysis and toppfun) were combined and toppfun [12] , webgestalt [15] , and enrichr [13] were used to identify gene set specific drugs. (table s4) . additionally, several candidate drugs are also identified for each pathway. glibenclamide (table s4) . since we identified several pathways, biological process, and drugs in our various analyses, to identify the most important ones, we combined all the results and then ranked them according to their number of appearances. we selected the pathways, biological process, and drugs that are commonly enriched in at least 3 of our omics-based analysis. while we combined biological processes from all our analysis and performed the ranking, we found that rna splicing, viral process, mrna processing, mrna splicing via spliceosome, negative regulation of apoptotic process, regulation of cell cycle, rhythmic process, cell proliferation, defense response, immune system process, protein phosphorylation, and complement activation are the top 15 pathways involved in the sars-cov-2 infection process ( fig-2a , table s5 ). similarly, epstein-barr virus infection, htlv-i infection, pathways in cancer, cell cycle, cytokine signaling in immune system, focal adhesion, hepatitis b, influenza a, leishmaniasis, ubiquitin mediated proteolysis, viral carcinogenesis, and mrna splicing/spliceosome are found as top pathways (fig-2b , table s5 ). apart from these pathways, measles, tuberculosis, toxoplasmosis, and malaria pathways are also found to be common in at least three of our analyses (fig-2b , table s5 ). (fig-4) . purvalanol, resveratrol, sorafenib, tanespimycin, and vorinostat (fig-5a , table s7 ). the identified top drug categories are antineoplastic agents, analgesics, anti-infective agents, antibacterial agents, enzyme inhibitors, metal/ growth substances, alkylating agents, antihemophilic factors, and vitamin d that have at least 3 drugs in each category (fig-5b , (fig-6) . among the anticancer drugs identified in our analysis, a combination of topoisomerase inhibitors (irinotecan and etoposide) is reported to be an effective treatment strategy to counter cytokine storms in critically ill covid-19 patients [21] . our identified doxorubicin was previously suggested for repurposing to treat covid-19 patients [22] . another identified anticancer drug dasatinib is found to be safer to treat chronic myeloid leukemia (cml) patients infected with sars-cov-2 [23] . binimetinib, brigatinib , seliciclib, sorafenib, and vorinostat were also previously predicted to be effective against sars-cov-2 infection [24] [25] [26] [27] . therefore, the additional anticancer drugs identified by our analyses, such as phenethyl isothiocyanate, abciximab, lucanthone, cisplatin, tositumomab, procarbazine, and noscapine may also be tested against sars-cov-2. our identified antidepressant drug fluoxetine (prozac) was previously reported to inhibit the replication of sars-cov-2 [29] and therefore, may be tested for potential covid-19 therapy. the antipsychotic medicine fluphenazine is also enriched in our analysis that has previously been tested on sars-cov-2 [30] . similarly, sertraline (a serotonin (5-ht) inhibitor), that is identified in our study, has recently been found to block sars cov 2 endocytosis and suggested for drug repurposing against covid-19 [31] . thus, the additional j o u r n a l p r e -p r o o f antidepressant/ antipsychotic drugs identified by our analyses, such as droperidol (antidopaminergic), may also be tested to understand their effectiveness against sars-cov-2. in our analysis, several cyclooxygenase (cox) inhibitors and anti-histamine compounds found potential against covid-19. among these drugs, indomethacin (cox inhibitor) is shown to exert its anti-sars-cov-2 activity in canine coronavirus model [32] . further, indomethacin in combination with resveratrol (also identified in our analysis) has been proposed to be used for treating covid 19 [33] . two other cox inhibitors (loxoprofen and acetaminophen) identified in our analysis were also previously tested in silico for their potential antiviral activity against sars-cov-2 [34] . niflumic acid which is also a cox inhibitor as well as a ca2+-activated clchannel blocker is also enriched in our analysis and also been previously identified as a potential drug against sars-cov-2 [35] . other antiinflammatory drugs such as andrographolide, apremilast (phosphodiesterase type 4 inhibitor) that are identified in our analysis were also predicted by previous studies as potential covid-19 therapeutics [36, 37] . in our analysis, we also found the rheumatoid arthritis drug tofacitinib as a good candidate for covid-19 management. tofacitinib is a jak inhibitor and reduces immune system inflammation and is currently under clinical trials for covid-19 treatment (nct04415151, nct04412252), therefore, the additional anti-allergic and antiinflammatory drugs identified by our analyses including sulindac (cox inhibitor) and terfenadine (h1-receptor binding anti-histamine drug) need attention to be tested against sars-cov-2. similarly, other channel inhibitors enriched in our analysis such as acetohexamide (atp-dependent k + channel inhibitor), suloctidil (calcium antagonist), glibenclamide (atp-sensitive potassium channels inhibitor), and thapsigargin (a sarco/endoplasmic reticulum ca²⁺ atpase inhibitor) may also be tested. tissue plasminogen activators (tpa), antihemophilic factors, immune globulin, and antithymocyte globulin are found to be good candidates in covid-19 treatment as per our various omics-based analyses. reports suggest that, tpa with targeted anti-inflammatory treatment may be a potential therapy against covid-19 [38] . tenecteplase, a tpa, that is identified in our analysis, is considered in managing acute coronary syndromes associated with covid-19 [39] and is currently under clinical trial to manage covid-19 patients (nct04505592). lanoteplase, a third generation recombinant plasminogen derived from alteplase, is also identified in our analysis. alteplase is currently under clinical trial to j o u r n a l p r e -p r o o f evaluate its efficacy against covid-19 (nct04357730). we also found antihemophilic factors could be also effective against covid-19. the factor viii which is an antihemophilic factor is elevated in covid-19 patients [40] . therefore, factor viii inhibitors may be a potential therapeutic strategy. immune globulin is also enriched in our analysis. immune globulin is present in plasma and convalescent plasma therapy is effective in treating covid-19 patients [41] . our identified human serum albumin may be a good delivery vehicle to maximize the cellular internalization and enhancement of other drugs used in covid-19 treatment [42] . since most of our identified blood based components are suggested to be effective in covid-19, the anti-thymocyte globulin which is also identified in our analysis may also be tested for its efficacy in the management of sars-cov-2 infection. estrogen or estrogen analogues are enriched in almost all of our omics data set-based analyses. dimorphism of covid-19 infection has been recently reported. men are found to be more prone to sars-cov-2 infection or covid-19 associated deaths as compared to women [43] . the disease severity of covid-19 is also found to be dependent on erα:erβ expression ratio and e2 level [43] . reports also suggest that the expression of human ace2, which is the receptor for sars-cov-2, is regulated by estrogen [44] . therefore, estradiol agonist may have a protective role against covid-19 [45] . recently, estrogen patch is under clinical trial to evaluate its efficacy against covid-19 (nct04359329). in our analysis, we observed that phytoestrogens including coumestrol could also be a potential therapeutic against covid-19. we observed that, steroids could play an important role in fighting against covid-19. the corticosteroid dexamethasone and methylprednisolone which we identified in our analysis, were previously found to significantly reduce mortality rates from covid-19 [46, 47] and currently under clinical trials (nct04327401, nct04323592). the other corticosteroid that is predominantly enriched in our integrative omics approach is betamethasone. betamethasone is found to be safe for pregnant subjects infected with sars-cov-2 [48] and is currently also under clinical trial at sites where dexamethasone is not easily available (nct04509973). therefore, other corticosteroids identified in our analysis such as fludrocortisone acetate may also be tested against covid-19. among our identified glucocorticoids, hydrocortisone has been tested on covid-19 patients [49] and prednisone and procaine may be tested in the coming days. j o u r n a l p r e -p r o o f no known targeted antiviral drug is enriched in our analysis. however, we identified several antibiotics. among these antibiotics, clindamycin was previously suggested by a computational approach [50] and is recommended to treat sars-cov-2 infection [51] . we identified two additional antitumor antibiotics, geldanamycin and tanespimycin, which are also heat shock protein 90 (hsp90) inhibitors. while geldanamycin is suggested for covid-19 treatment [52] , tanespimycin was previously identified by an in silico method [52] . apart from sharing various viral pathways, we also found that sars-cov-2 shares pathways associated with tuberculosis, leishmaniasis, and malaria. the antimalarial drug hydroxychloroquine alone or in combination with remdesivir effectively inhibits sars-cov-2 infection [54, 55] . hydroxychloroquine is under clinical trial (nct04345692). in our analysis, we found that artenimol or artesunate, an alternative of chloroquine could also be effective in treating covid-19. moreover, artesunate was previously predicted to be a good candidate against sars-cov-2 [56, 57] and is currently under clinical trial (nct04387240). in our analysis, we found immunosuppressant drugs may also be effective against covid-19. among the identified immunosuppressants, cyclosporin a is enriched in our multiple analyses. a recent in vitro study indicates that cyclosporin a is effective against sars-cov-2 [58] and therefore, can be a potential drug to be further tested in covid-19 patients [59] . cyclosporin a is currently under clinical trials (nct04412785, nct04451239) . similarly, the other immunosuppressant leflunomide identified in our analyses is also under clinical trial (nct04361214). in our integrative omics approach, we found that ozone could be a potential therapy against ozone-based management for early control of covid-19 are under various stages of clinical trials (nct04366089, nct04370223). nitric oxide, which is known to provide innate antiviral protection [61] , is also enriched in our analysis. nitric oxide inhalation is recently reported to be beneficial to manage the acute respiratory distress syndrome due to sars-cov-2 infection [62] and currently nitric oxide inhalation therapy is under clinical trials to understand its efficacy in managing covid-19 patients (nct04383002, nct04421508, nct04306393). verteporfin, a photosensitizer drug for photodynamic therapy is found to be an effective drug in our analysis. verteporfin is also previously reported to be a potential antiviral therapy against sars-cov-2 [63] . in our multi-omics-based analysis, acetylcysteine is enriched. acetylcysteine is a mucolytic agent that can prevent the severity of sars). therefore, our results suggest that our analytic approach in this study is highly accurate and curcumin, retinoic acids, and ergocalciferol could be good prophylaxis candidates against covid-19. arsenenous acid and arsenic trioxide are identified both in our candidate gene-based integrative omics analysis as well as viral pathway-based analysis. recent in silico analysis suggests that arsenic based drug darinaparsin may inhibit sars-cov-2 rna polymerase and proteases, and therefore, may inhibit viral replication [72] . in homeopathy medicine arsenicum album 30, prepared from arsenic trioxide, is recommended as a prophylactic medicine against sars-cov-2 infection [73] . in our analysis, copper is found to inhibit all three important pathways: mrna splicing, ubiquitin mediated proteolysis, and protein processing in endoplasmic reticulum pathways. copper irreversibly disintegrates coronavirus genomes, envelope, and spike [74] . report also suggests that enrichment of copper levels in j o u r n a l p r e -p r o o f plasma could boost both the innate and adaptive immunity [75] and therefore, copper could also be a good prophylactic agent against sars-cov-2 infection. similarly, polaprezinc is identified as a potential molecule to block protein processing in the endoplasmic reticulum pathway. zinc supplementation is recommended as a good prophylaxis and therapeutic strategy against covid-19 [76] . currently, several clinical trials are on-going to evaluate the anti-covid-19 efficacy of zinc in combination with other drugs (nct04351490, nct04447534). in this work, we have used four different host specific omics data sets that were recently published along with a bibliome based gene set to identify the sars-cov-2 infection biology, potential drugs, and prophylaxis agents against this virus. based on our individual omics-based or their combination-based analysis, it is evident that the sars-cov-2 infection shares various virus, bacteria, and protozoon infection pathways. although, we identified several candidate drugs and prophylaxis candidates, we were not able to identify any sars-cov-2 specific antiviral agent. nearly ~70% of our identified agents were previously reported to have anti-covid-19 activity, and a number of these agents are currently under various stages of clinical trials. since the most observed effective drugs/agents are among those that were used (or introduced) previously, our used integrative omics-based methodology is highly credible and warrants its wide-spread adoption and application. this method would also be highly applicable for drug repositioning research in future waves of covid-19 infection and also other possible pandemics. however, as sars-cov-2 shares multiple pathogens' infection pathways, individual drugs targeting a single pathway may not be effective and therefore, a combination of drugs needs to be formulated to block the multiple infection pathways of this virus. further, the prophylaxis agents identified here also need an effective combination. all these identified drugs need multiple validations, large scale clinical trials, and caution before their use in covid-19 management. db: conceived and designed the experiment, data collection and analysis, result interpretation, and wrote the paper; st: preformed re-analysis; pg: data interpretation and edited the article, mei, agn, mmg and va: provided technical inputs. authors declare no competing interest. j o u r n a l p r e -p r o o f supplementary table legends table s1 : candidate gene based analysis of multi-omics data. table s2 : gene set enrichment based analysis of multi-omics data. table s3 : network analysis based lung-specific multi-omics data. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a new coronavirus associated with human respiratory disease in china a novel coronavirus from patients with pneumonia in china coronavirus disease 2019 (covid-19) a familial cluster of pneumonia associated with the 2019 novel 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covid-19 patients with acute respiratory failure? potential cytoprotective activity of ozone therapy in sars-cov-2/covid-19 the nitric oxide pathway provides innate antiviral protection in conjunction with the type i interferon pathway in fibroblasts nitric oxide inhalation as an interventional rescue therapy for covid-19-induced acute respiratory distress syndrome potent antiviral effect of protoporphyrin ix and verteporfin on sars-cov-2 infection, biorxiv catechin and curcumin interact with corona (2019-ncov/sars-cov2) viral s protein and ace2 of human cell membrane: insights from computational study and implication for intervention a novel combination of vitamin c, curcumin and glycyrrhizic acid potentially regulates immune and inflammatory response associated with coronavirus infections: a perspective from system biology analysis inhibitory effects of resveratrol on the epstein-barr virus lytic cycle resveratrol as a novel anti-herpes simplex virus nutraceutical agent: an overview effective inhibition of mers-cov infection by resveratrol natural compounds as potential inhibitors of novel coronavirus (covid-19) main protease: an in silico study vitamin d deficiency and co-morbidities in covid-19 patients -a fatal relationship? letter: covid-19, and vitamin d in silico identification of a potent arsenic based approved drug darinaparsin against sars-cov-2: inhibitor of rna dependent rna polymerase (rdrp) and necessary proteases advisory for corona virus homoeopathy for prevention of corona virus infections unani medicines useful in symptomatic management of corona virus infection is copper beneficial for covid-19 patients? potential role of zinc supplementation in prophylaxis and treatment of covid-19 htlv-i, measles, and hepatitis virus infection pathways. • sars-cov-2 also shares tuberculosis, malaria, and leishmaniasis infection pathways. • mrna splicing, cytokine and ifn signaling, and ubiquitin are important pathways. • anticancer, antipsychotic, anti-inflammatory, antibiotic, immunosuppressant, corticosteroid cyclosporin a are top candidate drugs nitric oxide, and photosensitizer drugs are also important against covid-19 retinoic acids, vit-d, arsenic, copper, and zinc are candidate prophylaxis agents • ~70% of our identified drugs are previously suggested or under clinical trial for covid-19. • our newly identified candidate drugs need validation with caution none key: cord-293082-fw7deem8 authors: zhang, guangzhi; li, bin; yoo, dongwan; qin, tong; zhang, xiaodon; jia, yaxiong; cui, shangjin title: animal coronaviruses and sars‐cov‐2 date: 2020-08-16 journal: transbound emerg dis doi: 10.1111/tbed.13791 sha: doc_id: 293082 cord_uid: fw7deem8 covid‐19 is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2). it has rapidly spread to 216 countries and territories since first outbreak in december of 2019, posing a substantial economic losses and extraordinary threats to the public health worldwide. although bats have been suggested as the natural host of sars‐cov‐2, transmission chains of this virus, role of animals during cross‐species transmission, and future concerns remain unclear. diverse animal coronaviruses have extensively been studied since the discovery of avian coronavirus in 1930s. the current article comprehensively reviews and discusses the current understanding about animal coronaviruses and sars‐cov‐2 for their emergence, transmission, zoonotic potential, alteration of tissue/host tropism, evolution, status of vaccines, and surveillance. this study aims at providing guidance for control of covid‐19 and preventative strategies for possible future outbreaks of zoonotic coronavirus via cross‐species transmission. the coronavirus disease 2019 broke out in wuhan, china in december 2019, and 46 spread rapidly across the world. on march 11th of 2020, world health organization (who) 47 announced covid-19 a pandemic. as of april 7, just four months since its first outbreak, more 48 than 3.4 million confirmed cases and 238,000 deaths have been recorded in 215 countries, areas, 49 and territories, and moreover it seems that severe acute respiratory syndrome coronavirus 2 50 (sars-cov-2) that causes covid-19 will probably continue to circulate around the globe 51 (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). the health 52 authorities and governments of affected countries have paid attention to current pandemic and 53 have taken immediate measures to block covid-19 transmission, including utilization of personal 54 protective equipment, quarantine, epidemiological investigation, isolation, clinical data analysis 55 and sharing, public health education, maintaining social distance, the creation of diagnostics, 56 therapeutics, and vaccines, etc (xiao and torok 2020) . the new information is rapidly 57 accumulating and uncovers the nature of sars-cov-2, but many questions remain to be 58 answered. (table 1) . these six covs usually cause infections in pigs, but pdcov seems to have the ability 141 to infect other species such as badgers, calves, and cats (li et al. 2018) . moreover, it was reported our phylogenetic analysis with s protein shows that pedv and tgev share only 42.8%-43.5% of 154 genome similarity with sars-cov-2, and phev and pdcov share 49.2%-49.3% and 155 40.3%-40.4% genome similarity with sars-cov-2, respectively (table 2) . obviously, porcine covs are unlikely the origin of sars-cov-2. however, the rbd of sars-cov-2 is likely to 157 recognize porcine ace2 based on the high similarity of critical virus-binding residues between 158 porcine ace2 and human ace2 (wan, shang, graham, et al. 2020 this article is protected by copyright. all rights reserved the investigation needs to be explored. ibv normally binds to cell receptors via sialic acid for its 185 attachment and entry (shahwan et al. 2013; toro, van santen, and jackwood 2012) (table 1) . live-attenuated vaccines for ibv are usually adopted by the farms, which are developed by serial 187 passages of virulent strains in embryonated chicken eggs (laconi et al. 2020; masoudi, pishraft 188 sabet, and shahsavadi 2020; baron, iqbal, and nair 2018 kong. an oie report shows that the antibody in a pet dog living with a receptor for both fecv and fipv (hohdatsu et al. 1998 ) ( table 1 ). as shown in table 2 showed that the receptor-binding motif of sars-cov-2 rbd can engage with ace2 of humans 260 and cats at similar efficiencies (wan, shang, graham, et al. 2020 for neonates through colostrum (nemoto et al. 2017; kanno et al. 2013) . as for the receptors utilized by sars-cov-2, biophysical and structural data showed that the s 374 protein engages with ace2 with at least 10 times higher affinity compared to that of sars-cov 375 (wrapp et al. 2020 ). in addition to ace2, sars-cov-2 may also invade the cell via another 376 receptor: cd147 ibrahim et al. 2020 accepted article ibrahim et al. 2020; hao et al. 2020) . it was reported that furin is involved in the cleavage of 381 sars-cov-2 s protein and further facilitate viral entry . in principle, tissues 382 with consistently high expression of ace2 or cd147 are likely susceptible to sars-cov-2, 383 including small intestine, kidney (renal tubular cells), testis (leydig cells and cells in seminiferous 384 ducts), gall bladder, heart, stomach, and liver (fagerberg et al. 2014 ). according to a recent report shen et al. 2020 phylogenetic analysis by researchers from institut pasteur in paris showed that sars-cov-2 409 might already circulated in france prior to first local case (gámbaro et al. 2020 ). all of these data 410 undoubtedly showed an intricate situation of this virus. thus, more surveillance needs to be taken 411 to explore the origin and diversity of this virus worldwide. importantly, the careful analyses demonstrate that some of the animal covs have evolved to all data generated or analyzed in this study are included in this manuscript and in the 526 supplementary material. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved strength of infectivity. survey of sars-cov-2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection isolation and sequence analysis of canine respiratory coronavirus detection of a group 2 coronavirus in dogs with canine infectious respiratory disease analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics diagnosis of feline infectious peritonitis: a review of the current literature phylogenetic network analysis of sars-cov-2 genomes evolution of infectious bronchitis virus in the field after homologous vaccination introduction', vet this article is protected by copyright. all rights reserved j. saif feline coronavirus type ii strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type i and canine coronavirus differences in virus receptor for type i and type ii feline infectious peritonitis virus engineering a live attenuated porcine epidemic diarrhea virus vaccine candidate via inactivation of the viral 2'-o-methyltransferase and the endocytosis signal of the spike protein coronavirus spike protein and tropism changes' human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a covid-19 spike-host cell receptor grp78 binding site prediction experimental bovine coronavirus in turkey poults and young chickens tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus triple-reassortant influenza a virus with h3 of human seasonal origin, na of swine origin, and internal a(h1n1) pandemic 2009 genes is established in danish pigs attenuated live infectious bronchitis virus qx vaccine disseminates slowly to target organs distant from the site of inoculation identifying sars-cov-2-related coronaviruses in malayan pangolins structure, function, and evolution of coronavirus spike proteins broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility cats under the shadow of the sars-cov-2 pandemic emergence of sars-cov-2 through recombination and strong purifying selection emerging lethal infectious bronchitis coronavirus variants with multiorgan tropism', transbound emerg this article is protected by copyright. all rights reserved ferrets immunogenicity and efficacy of live infectious bronchitis 793/b.08ir vaccine in spf chickens origins of the 2009 h1n1 influenza pandemic in swine in mexico contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection antibody response to equine coronavirus in horses inoculated with a bovine coronavirus vaccine emergence and pandemic potential of swine-origin h1n1 influenza virus swine enteric coronavirus disease: a review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the united states and canada mutation of the s and 3c genes in genomes of feline coronaviruses prevalence of feline coronavirus (fcov) and feline leukemia virus (felv) in turkish cats this article is protected by copyright. all rights reserved antibody-binding epitopes in the spike 2 domain and the nucleocapsid protein of feline infectious peritonitis virus sialic acid binding properties of soluble coronavirus spike (s1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus cell entry mechanisms of sars-cov-2 pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus genomic diversity of sars-cov-2 sars-cov-2 and sars-cov spike-rbd structure and receptor binding comparison and potential implications on neutralizing antibody and vaccine development canine respiratory coronavirus, bovine coronavirus, and human coronavirus oc43: receptors and attachment factors', viruses prevalence of feline coronavirus antibodies in japanese domestic cats during the past decade on the origin and continuing evolution accepted article this article is protected by copyright. all rights reserved of sars-cov-2 genetic diversity and selection regulates evolution of infectious bronchitis virus structural basis for human coronavirus attachment to sialic acid receptors mutational analysis of aminopeptidase n, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses pathogenic characteristics of persistent feline enteric coronavirus infection in cats receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus molecular mechanism for antibody-dependent enhancement of coronavirus entry development of an inactivated vaccine candidate sars-cov-2 invades host cells via a novel route: cd147-spike protein importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on the middle east respiratory syndrome coronavirus spike glycoprotein to avoid neutralization escape emerging and re-emerging coronaviruses in pigs an outbreak of feline infectious peritonitis in a taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type ii feline coronavirus evidence of recombination in coronaviruses implicating pangolin origins of ncov-2019 discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus cryo-em structure of the 2019-ncov spike in the prefusion conformation wenjie tan, and genhong cheng. 2020. 'mutations evidence for gastrointestinal infection of sars-cov-2 isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins taking the right measures to control covid-19 systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective broad cross-species infection of cultured cells by bat hku2-related swine acute diarrhea syndrome coronavirus and identification of its replication in murine dendritic cells in vivo highlight its potential for diverse interspecies transmission 2019 novel coronavirus is undergoing active recombination covid-19 and veterinarians for one health, zoonotic-and reverse-zoonotic transmissions and team singapore novel coronavirus outbreak research. 2020. 'epidemiologic features and clinical course of patients infected with sars-cov-2 in sars-cov-2 spike protein variant d614g increases infectivity and retains sensitivity to antibodies that target the receptor binding domain the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity sars-cov-2 neutralizing serum antibodies in cats: a serological investigation probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child tissue tropisms opt for transmissible reassortants during avian and swine influenza a virus co-infection in swine a genomic perspective on the origin and emergence of sars-cov-2 evolution of infectious bronchitis virus in china over the past two decades fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva', medrxiv: accepted article this article is protected by copyright immunogenicity and safety of a recombinant adenovirus type-5-vectored covid-19 vaccine in healthy adults aged 18 years or older: a randomised contribution of porcine aminopeptidase n to porcine deltacoronavirus infection virulence factors in porcine coronaviruses and vaccine design this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-304295-3mpymd8a authors: khan, muhammad muzamil; noor, amna; madni*, asadullah; shafiq, mudassir title: emergence of novel coronavirus and progress toward treatment and vaccine date: 2020-06-04 journal: rev med virol doi: 10.1002/rmv.2116 sha: doc_id: 304295 cord_uid: 3mpymd8a in late december 2019, a group of patients was observed with pneumonia‐like symptoms that were linked with a wet market in wuhan, china. the patients were found to have a novel coronavirus genetically related to a bat coronavirus that was termed sars‐cov‐2. the virus gradually spread worldwide and was declared a pandemic by who. scientists have started trials on potential preventive and treatment options. currently, there is no specific approved treatment for sars‐cov‐2, and various clinical trials are underway to explore better treatments. some previously approved antiviral and other drugs have shown some in vitro activity. here we summarize the fight against this novel coronavirus with particular focus on the different treatment options and clinical trials exploring treatment as well as work done toward development of vaccines. coronaviruses are a form of positive-strand non-segmented rna viruses distributed among birds, mammals and humans that cause respiratory and neurological illnesses. 1 there are six different types that can cause disease among humans. four of these (hku1, 229e, nl63 and oc43) cause only the common cold in patients, 2 5 due to the broad genetic variation and diversity of coronaviruses and higher chances of animal to human spread they are likely to emerge periodically in future. 6 during december 2019, an outbreak of pneumonia-like symptoms occurred in patients that were linked to the seafood market in wuhan china. 7 investigation identified a new strain of coronavirus called sars-cov-2. 8 the detection of this novel coronavirus is key to confirm the cases and proceed to treatment. in an early method for detecting sars-cov-2, the samples from bronchoalveolar-lavage fluids were collected, centrifuged to remove debris and inoculated onto human epithelial cells of airway origin. 9 about 20 000 sequences from each sample were obtained and genome matches showed more than 85% identity with sars-like betacornavirus. results were also obtained from real-time pcr, and isolated viruses were named sars-cov-2. 8 to further characterize sars-cov-2, the de-novo sequence was obtained by using nanopore sequencing and illumina methods. the airway epithelial cell cultures were suitable for visualization and identification of the novel coronavirus. 9 check efficacy against this virus. the possible options include nucleoside analogs, interferon that can act as immune modulators and approved antimalarials having antiviral activities such as chloroquine and hydroxychloroquine. the nucleoside analogs such as ribavirin, favipiravir, galidesivir and remdesivir target rna polymerase and block the synthesis of viral rna. 10 favipiravir was effectively used against influenza and has the potential to inhibit viral rna synthesis and a new study also supports its activity against sars-cov-2. 11 different clinical trials are underway where patients are being recruited to evaluate the efficacy of a combination of favipiravir and interferon-α 12 and a combination of favipiravir and baloxavir marboxil is being evaluated. 13 ribavirin is a guanine derivative antiviral drug approved for the treatment of hcv and rsv, but it causes anemia at higher doses that limit its use and its efficacy against coronavirus is uncertain. 14 remdesivir is an adenine derivative antiviral drug. it has activity against a variety of viral strains such as sars and mers and a recent study also supports its activity against sars-cov-2. 11 a recent patient in us with sars-cov-2 has shown recovery with intravenous administration of remdesivir. 15 recently, phase-iii clinical trials have been started in usa to evaluate the efficacy of iv remdesivir as 200 mg od or 100 mg od for 9 days. 16 a recent rct apparently reported no significant efficacy, but we await the published findings. small molecular weight drugs such as chloroquine have shown inhibitory effects against sars-cov-2 (ec50 = 1.14 μm in vero e6 cells) and is under evaluation in open-label trials.. 15 chloroquine (cq) is a recognized anti-malarial drug but also has antiviral activity. the antiviral activity of chloroquine was first noticed in the late 1960s. 21 chloroquine and its analog hydroxychloroquine both have inhibitory effects against various viruses including sars, 22 enterovirus 23 and zika virus. 24 chloroquine inhibits the virus by increasing endosomal ph and so reducing viral cell fusion and also interferes with cellular receptor glycosylation. 25 the ec90 value of chloroquine against sars-cov-2 is 6.90 μm that can be achieved with administration of 500 mg dose. 26 remdesivir and chloroquine have shown activity in in vitro studies and can easily be tested in patients with sars-cov-2. 15 in another recent study, gao et al found that chloroquine phosphate reduced the symptoms of pneumonia in sars-cov-2 patients and shortening the duration of disease. 27 the guidelines for the treatment of sars-cov-2 were revised six times since its issuance on 15 january 2020. the recent guidelines also include ifn-α, remdesivir, ribavirin, ritonavir and chloroquine. the mode of administration of ifn-α is through inhalation at a dose of 5 million units diluted with water for injection. the dose of ritonavir is 100 mg bd for adults. ribavirin may be given in combination with ifn-α or ritonavir at a dose of 500 mg bd or tds. chloroquine phosphate should be administered at a dose of 500 mg bd. arbidol can be given three times a day at 200 mg. 28 hydroxychloroquine (hcq) is an approved disease-modifying antirheumatic drug that also has immunomodulatory effects and prevents organ damage. 29 hcq alters endosomal ph and interrupts the biding between rna/dna and toll-like receptors that leads to suppression of tlr signaling. [30] [31] [32] inside the cytoplasm, hcq also interferes with the interaction between nucleic acid sensor cyclic gmp and cytosolic dna. 33 these two mechanisms lead to increase production of il-1, tnf and type-1 interferon. such mechanism supports the idea that hcq suppress the cytokine release storm (crs) which is due to sars-cov-2 triggered overreaction of immune system. 34 in a recent study, hcq was found to be more effective than chloroquine; a loading dose of 400 mg twice daily and maintenance dose of 200 mg twice a day is recommended for sars-cov2 infection. 35 the mechanism involves in antiviral role of hcq and cq is inhibition of receptor binding and fusion of cell membrane. these two are crucial steps that are required for cell entry of sars-cov-2. chloroquine interferes with the glycosylation of ace-2 (angiotensinconverting enzyme receptors) receptors that are considered as cellular receptors for sars-cov-2 and block the fusion of sars-cov-2 with host cell. thus, the binding of virus is blocked and infection is prevented. the hcq and cq after entry into the cell tend to concentrate in lysosomes and endosomes. the sars-cov-2 use endosome as a tool for cellular entry. hcq and cq increase the ph of endosomes and create a negative influence on the binding of sars-c0v-2 with endosomes. 25 lysosomal protease helps in viral fusion with cell membrane. increase lysosomal ph prevents the action of protease and fusion and replication of virus is blocked 36 . the mechanism of action of chloroquine and hydroxychloroquine is represented as (figure 1 ). cinanserin is an antagonist of serotonin receptors. it can inhibit chymotrypsin-like (3c-like) protease and has shown promising results against sars coronavirus. 37 the 3c-protease was also investigated to be encoded in sars-cov-2. 38 flavonoids such as chalcones, flavones and isoflavones produce antioxidant effects but they also have antiviral effects. 39 a study has found that flavonoids can inhibit the entry of hepatitis-c virus. 40 some flavonoids have activity against mers coronavirus, presumably due to inhibition of 3c-like protease. 41 nitric oxide (no) is a biological gas produced from arginine. no after reaction with superoxide forms peroxynitrite which has cytotoxic bactericidal action. 43 nitric oxide is also known to regulate airway function and reduce inflammation of airways. 44 the beneficial effect of no in sars patients was observed in some studies. 45 no can also inhibit the synthesis of rna and viral protein. 46 a study has found that organic nitric oxide could inhibit the replication cycle of sars-cov-2. 47 αlpha-lipoic acid (ala) is used in hepatic disorders and polyneuropathies. 48 ala is an antioxidant that protects cells against oxidative stress by increasing the glutathione level. 49 some studies support the role of angiotensin-converting enzyme (ace) inhibitors. this is based on the hypothesis that ace-2 serves as receptor for sars-cov-2. 54,55 so, ace inhibitor could potentially compete for site binding and reduce the mortality and morbidity associated with sars-cov-2. 56 the various ongoing clinical trials are summarized in table 1 . vaccine provides immunity against a particular pathogen before exposure of that infectious agent. several types of vaccines exist that can be nucleic acid based, live attenuated vaccine, subunit proteins or nanoparticle vaccines. 67 different technologies are being utilized to f i g u r e 1 mechanism of action of hcq and cq by blocking binding of virus with ace-2 receptors and increasing endosomal ph and preventing fusion with the cell develop potential vaccine for sars-cov-2 including dna and mrnabased nanoparticles. phase-i trials of potential vaccines focus on safety and immunogenicity, for example, against mers. 68 two of the clinical trials on mers vaccine are expected to be complete by the end of 2020 69, 70 in russia and one in germany by december 2021. 71 the inovo pharmaceutical company has tested its vaccine against mers coronavirus funded by coalition for epidemic preparedness innovation (cepi) using dnabased technology and named as ino-4700. 72 the university of oxford recombinant chimpanzee adenovirus has also begun phase 1 randomized multicenter trials for intramuscular injection of vaccine chadox1 against sars-cov-2. the 1100 participants have been divided into four groups and they will be observed for any serious adverse event for 6 months. the ongoing clinical trials for development of vaccines have been summarized in table 2 and completed vaccine trials for sars and mers viruses have been summarized in table 3 . the fusion of coronavirus with a cell occurs after biding of s protein to a target receptor delivers viral nucleocapsid and initiates replication. the s protein causes syncytial formation at the receptor site. 85 a neutralizing antibody that can target the s protein on the surface of clinical trials for various treatment options for sars-cov-2 clinical trial registration number trial information date type references the efficacy of newgen beta-gluten probiotic powder in patient with 2019-ncov. 16 march 2020 interventional 57 the efficacy of combination of favipiravir + tocilizumab in patient with pneumonia (covid-19 the immune response was dose-independent and further trials needed to establish efficacy 84 coronavirus pathogenesis epidemiology, genetic recombination, and pathogenesis of coronaviruses origin and evolution of pathogenic coronaviruses epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china isolation of a novel coronavirus from a man with pneumonia in saudi arabia the role of super-spreaders in infectious disease wuhan municipal health commission. report of clustering pneumonia of unknown etiology in wuhan city china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china coronaviruses and the human airway: a universal system for virus-host interaction studies new nucleoside analogues for the treatment of hemorrhagic fever virus infections washington state 2019-ncov case investigation team. first case of 2019 novel coronavirus in the united states clinical study for safety and efficacy of favipiravir in the treatment of novel coronavirus pneumonia (covid-19) a randomized controlled trial for the efficacy and safety of baloxavir marboxil, favipiravir tablets in 2019-ncov pneumonia (novel coronavirus pneumonia, ncp) patients who are still positive on virus detection under the current antiviral therapy coronavirusesdrug discovery and therapeutic options remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro bin cao yw. mild/moderate 2019-ncov remdesivir rct role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset approved antiviral drugs over the past 50 years clinical management of respiratory syndrome in patients hospitalized for suspected middle east respiratory syndrome coronavirus infection in the paris area from effect of chloroquine on the growth of animal viruses in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine an evaluation of chloroquine as a broad-acting antiviral against hand, foot and mouth disease fda-approved drug, prevents zika virus infection and its associated congenital microcephaly in mice chloroquine is a potent inhibitor of sars coronavirus infection and spread dose refinements in long-term therapy of rheumatoid arthritis with antimalarials breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies discovering drugs to treat coronavirus disease 2019 (covid-19) clinical efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic review mechanism of endosomal tlr inhibition by antimalarial drugs and imidazoquinolines the ectodomain of toll-like receptor 9 is cleaved to generate a functional receptor immune stimulation mediated by autoantigen binding sites within small nuclear rnas involves toll-like receptors 7 and 8 cutting edge: antimalarial drugs inhibit ifn-β production through blockade of cyclic gmp-amp synthase-dna interaction chloroquine and hydroxychloroquine equally affect tumor necrosis factor-alpha, interleukin 6, and interferongamma production by peripheral blood mononuclear cells in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases cinanserin is an inhibitor of the 3c-like proteinase of severe acute respiratory syndrome coronavirus and 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research and preparation development of qingfei detoxification decoction (mixture) for prevention and treatment of novel coronavirus pneumonia clinical study for bronchoscopic alveolar lavage in the treatment of critically trachea intubation patients with new coronavirus pneumonia (covid-19) evaluation of the protective effect of dexmedetomidine on patients with severe novel coronavirus pneumonia (covid-19) coronoavirus clinical trial evaluating the efficacy and safety of bromhexine hydrochloride tablets combined with standard treatment/ standard treatment in patients with suspected and mild novel coronavirus pneumonia (covid-19) fingolimod in covid-19 eculizumab (soliris) in covid-19 infected patients (solid-c19) 2019-ncov (wuhan virus), a novel coronavirus: human-to-human transmission, travel-related cases, and vaccine readiness potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review study of safety and immunogenicity of bvrs-gamvac study of safety and immunogenicity of bvrs-gamvac-combi randomized, double-blind, placebo-controlled, phase ib study to assess the safety and immunogenicity of mva-mers-s_df-1 inovio selected by cepi to develop vaccine against new coronavirus an obscure biotech stock skyrockets 38% after saying it's testing a coronavirus antibody (vir) a study of a candidate covid-19 vaccine (cov001) race to develop coronavirus vaccine j&j scientific officer 'pretty confident' they can create coronavirus vaccine as outbreak widens saskatchewan lab joins global effort to develop coronavirus vaccine china) to collaborate on development of coronavirus vaccine a phase i clinical trial for recombinant novel coronavirus (2019-cov) vaccine (adenoviral vector safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine vrc 301 study team. a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase 1, open-label, single-arm, dose-escalation trial structural insights into coronavirus entry the ebola epidemic crystallizes the potential of passive antibody therapy for infectious diseases isolation of hcv neutralizing antibodies by yeast display emergence of novel coronavirus and progress toward treatment and vaccine key: cord-298850-tgxfki7n authors: figuero-pérez, luis; olivares-hernández, alejandro; escala-cornejo, roberto a.; terán-brage, eduardo; lópez-gutiérrez, álvaro; cruz-hernández, juan j. title: anakinra as a potential alternative in the treatment of severe acute respiratory infection associated with sars-cov-2 refractory to tocilizumab date: 2020-10-15 journal: nan doi: 10.1016/j.reumae.2020.06.008 sha: doc_id: 298850 cord_uid: tgxfki7n sars-cov-2 is a new rna virus which causes coronavirus disease 2019 (covid-19), declared a pandemic by the world health organization (who). it triggers an atypical pneumonia that can progress to multiorgan failure. covid-19 can cause dysregulation of the immune system, triggering an inflammatory response, and simulate haemophagocytic lymphohistiocytosis. several studies have proposed that anti-il-6 receptor antibodies, such as tocilizumab, play an important role in the treatment of severe acute respiratory infection associated with sars-cov-2. however, the role of anti-il-1 receptor antibodies, such as anakinra, in the treatment of covid-19 has not been established. we present a case report of a 51-year-old man diagnosed with severe respiratory infection associated with sars-cov-2 that was refractory to antiviral and anti-il-6 treatment, with a favourable clinical outcome and analytical improvement after treatment with anti-il-1 (anakinra). el virus sars-cov-2 es un nuevo virus rna causante de la enfermedad covid-19, declarada como pandemia por la organización mundial de la salud (oms). produce un cuadro de neumonía atípica que puede desembocar en un fallo multiorgánico. la desregulación del sistema inmune secundaria a la infección produce un cuadro similar al síndrome de linfohistiocitosis hemofagocítica (slhh). varios estudios han definido la importancia que los inhibidores de la il-6 (tocilizumab) tienen en el tratamiento de la infección por sars-cov-2, sin embargo, la indicación de tratamiento con inhibidores de il-1 (anakinra) no se encuentra establecida de forma clara. presentamos el caso de un paciente de 51 años con neumonía bilateral secundaria a infección por sars-cov-2 refractaria al tratamiento antiviral y anti-il-6 que presentó mejoría clínica y analítica tras el tratamiento con anti-il-1 (anakinra). the sars-cov-2 virus is a new rna virus that was first identified in december 2019 in the city of wuhan, china. 1 sars-cov-2 causes a picture of atypical pneumonia that can lead to multiorgan failure. 2 the deregulation of the immune system secondary to the infection produces a picture similar to haemophagocytic lymphohistiocytosis syndrome (hlhs) syndrome). 3 the different pathways of immune activation culminate in cytotoxic dysfunction whose main trigger is a "cytokine storm". several studies have defined the importance that il-6 inhibitors (tocilizumab) have in the treatment of sars-cov-2 infection. 4 however, the indication for treatment with il-1 inhibitors (anakinra) has not been clearly established. we present the case of a 51-year-old patient with bilateral pneumonia secondary to sars-cov-2 infection refractory to treatment with tocilizumab who showed improvement after treatment with anakinra. a 51-year-old male who attended the emergency department with a fever (>38c) and dyspnoea of one week's duration. the patient had a history of copd, liver cirrhosis of unrelated origin and adenocarcinoma of the rectum (pt4n1m0). the presence of generalized hypoventilation with fine bibasal crackles was noteworthy in the physical examination. chest xray ( fig. 1) showed bilateral infiltrations with a ground glass pattern. blood analysis showed the values described in table 1 . blood and urine cultures were negative. sars-cov-2 virus was detected by polymerase chain reaction in pharyngeal exudate, which was positive. on diagnosis of bilateral pneumonia secondary to sars-cov-2 infection, the patient was admitted to hospital and treatment was initiated with broad-spectrum antibiotics (ceftriaxone, azithromycin and later escalated to piperacillin-tazobactam), hydroxychloroquine (hcq) and lopinavir/ritonavir (lpv/r). in view of the need for respiratory support, treatment with tocilizumab was initiated (8mg/kg every 12h, 2 subcutaneous doses). given the absence of respiratory and analytical improvement (table 1 ) 48h after administration of tocilizumab, it was decided to administer anakinra (100mg single total dose, subcutaneous). subsequently, the patient made good clinical progress, ventilatory support was discontinued and he was discharged from the hospital 14 days after admission. the "cytokine storm" secondary to sars-cov-2 infection determines severe covid-19 disease. the excessive activation of the immune system produces a picture similar to shlh. 3 the use of anti-il-6 antibodies in the treatment of sars-cov-2 infection is currently under study, being one of the current pillars of covid-19 disease treatment. in the study by le et al., 5 tocilizumab demonstrated clinical response after one or two doses of the drug in 69% of patients with cytokine activation syndrome. the use of inhibitory molecules of other interleukins is currently under study, anakinra being the most studied anti-interleukin molecule after tocilizumab in the treatment of covid-19. 6 in the case of our patient, treatment with tocilizumab did not provide clinical or analytical improvement, while the use of anakinra allowed clear improvement at 48h. although a possible benefit due to the late effect of tocilizumab cannot be ruled out. although a single dose of anakinra was administered to our patient, there are studies such as that of monteagudo et al., which show the efficacy of this drug in continuous infusion at doses greater than 2400mg/day for the treatment of macrophage activation syndrome (mas). 7 in conclusion, specific il-1 blockade could be an effective alternative in the management of patients with sars-cov-2 infection who have not benefited from other treatments. j o u r n a l p r e -p r o o f severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the callengues cancer patients in sars-cov-2 infection: a nationwide analysis in china iv anakinra for macrophage activation syndrome may hold lessons for treatment of cytokine storm in the setting of covid19. acr open rheumatol why tocilizumab could be an effective treatment for severe covid-19? fda approval summary: tocilizumab for treatment of chimeric antigen receptor t cell-induced severe of life-threatening cytokine release syndrome the rheumatologist`s role in covid-19 continuous intravenous anakinra infusion to calm the cytokine storm in macrophage activation syndrome figure 1 chest x-ray on admission bilateral alveolar infiltrations with a ground glass pattern key: cord-300041-1d9xu4ts authors: chen, sharon c-a; rawlinson, william d. title: focus on sars-cov-2 and covid-19 date: 2020-10-08 journal: pathology doi: 10.1016/j.pathol.2020.09.010 sha: doc_id: 300041 cord_uid: 1d9xu4ts nan emerging infections, particularly those that have occurred in pandemic proportions, have caused concern for governments and communities for thousands of years. pandemic 'plague' (possibly typhus) in 430 bc in athens during the peloponnesian war likely killed two-thirds of the local population, whilst bubonic plague in the 14th century killed one-third of the world's population. as with many of their predecessors, these were caused by bacteria, and in true contagion-style, able to take flight in the setting of severe crowding, poor public health control, limited (if any) antimicrobial intervention, and in association with major events such as wars. the word 'quarantine', or a period or place of isolation, comes from the italian word 'quaranta' which means 40. in the mid-1600s the italians stopped people from disembarking from ships for 40 days if it was believed there was a disease outbreak onboard, a legacy we still embrace today. in contrast, more recent pandemics have been dominated by viruses such as influenza h1n1 and h3n2, localised epidemics by ebola virus, severe acute respiratory syndrome coronavirus-1 (sars-cov-1), mers-cov, and now, sars-cov-2, the causative agent of covid-19. these can often be directly transferred from animals as occurs with other zoonoses, and have occurred in settings of modern public health systems, where antimicrobial agents of varying efficacy cannot be readily deployed, and in the absence of any identifiable major global event. we welcome this collection of papers from colleagues on sars-cov-2 and covid-19 from both the diagnostic laboratory and clinical viewpoints. our aim is to provide up-to-date perspectives at a national level on this significant pandemic, and lessons learned during the greater part of the current pandemic. the exponential growth in manuscripts globally on sars-cov-2 means that no single cross section of papers will provide all the important information. however, by having a single edition, with broad focus on human pathology of sars-cov-2 infection, we aim to provide the readers of pathology with insights from different areas of covid-19 diagnosis. these include an overview of the variety of diagnostic tools and their application as summarised by gulholm et al., 1 together with technique-focussed articles on molecular diagnostics (williams et al. 2 substantive progress continues to be made in the arena of diagnostic tests for sars-cov-2 infection with improvements in molecular diagnostics, rapid antigen detection tests and serological assays. as for all diagnostic tests, the clinical utility will depend on clinical context and, importantly, on disease prevalence which should inform their use. delineation of case clusters using next generation sequencing methods are critical for timely infection control and public health measures. these aspects have not been included in this mini collection but are described elsewhere. 16, 17 we conclude with two thoughts. given the rise in cases in some regions of the asia pacific and their likely adverse social impact, australia is a good position to deploy assistance if required. branley et al. 18 have provided one example of such deployment to a remote australian quarantine setting. we continue to be faced by the risk of pandemics and we must learn from our observations at present with sars-cov-2 infection, and resulting covid-19 disease. p.g. wodehouse wrote in 1926: 'to the thinking person nothing is more remarkable in this life than the way in which humanity adjusts itself to conditions which at their outset might well have appeared intolerable'. our ability to tolerate, as well as learn and share in a collaborative manner new knowledge about sars-cov-2, and to harness this knowledge to reduce human suffering, is something of which we can be proud. hand-in-hand, however, we must also be cognisant that science changes constantly and hence (as noted by the honourable mr. justice archie campbell in the sars commission executive summary) 'the point is not about who is right and who is wrong… we should not be driven by the scientific dogma of yesterday or even the scientific dogma of today. we should be driven by the … principle' 18 to take every reasonable step to reduce risk of covid-19 infection. during the coming months and years, this, together with use of knowledge in designing diagnostic tests, research into antivirals, vaccines and novel interventions will be important in how we improve the outcomes of this pandemic. the authors state that there are no conflicts of interest to disclose. laboratory diagnosis of severe acute respiratory syndrome coronavirus 2 implementation and evaluation of a novel real-time multiplex assay for sars-cov-2: in-field learnings from a clinical microbiology laboratory virus isolation of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) for diagnostic and research purposes comparative analysis of three laboratory based serological assays for sars-cov-2 in an australian cohort head-to-head evaluation on diagnostic accuracies of six sars-cov-2 serological assays clinical evaluation of four commercial immunoassays for the detection of antibodies against established sars-cov-2 infection clinical evaluation of sars-cov-2 point-of-care tests laboratory biosafety measures involving sars-cov-2 and the classification as a risk group 3 biological agent sample pooling is a viable strategy for sars-cov-2 detection in low-prevalence settings sars-cov-2 in children: spectrum of disease, transmission and immunopathological underpinnings accuracy amidst ambiguity: false positive sars-cov-2 nucleic acid tests when covid-19 prevalence is low the impact of viral transport media on pcr assay results for the detection of nucleic acid from sars-cov-2 contamination of sars-cov-2 rt-pcr probes at the oligonucleotide manufacturer histopathology of cutaneous covid-19 lesion: possible sars-cov-2 cytopathogenic effect is prostate infarction and acute urinary retention a possible complication of severe covid-19 infection? isolation and rapid sharing of the 2019 novel coronavirus (sars-cov-2) from the first patient diagnosed with covid-19 in australia revealing covid-19 transmission in australia by sars-cov-2 genome sequencing and agent-based modeling rapid deployment of pathology services to a remote australian quarantine setting during the covid-19 pandemic toronto: the sars commission key: cord-293852-r72c6584 authors: greco, s.; madè, a.; gaetano, c.; devaux, y.; emanueli, c.; martelli, f. title: noncoding rnas implication in cardiovascular diseases in the covid-19 era date: 2020-10-31 journal: j transl med doi: 10.1186/s12967-020-02582-8 sha: doc_id: 293852 cord_uid: r72c6584 coronavirus disease 19 (covid-19) is caused by the infection of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). although the main clinical manifestations of covid-19 are respiratory, many patients also display acute myocardial injury and chronic damage to the cardiovascular system. understanding both direct and indirect damage caused to the heart and the vascular system by sars-cov-2 infection is necessary to identify optimal clinical care strategies. the homeostasis of the cardiovascular system requires a tight regulation of the gene expression, which is controlled by multiple types of rna molecules, including rna encoding proteins (messenger rnas) (mrnas) and those lacking protein-coding potential, the noncoding-rnas. in the last few years, dysregulation of noncoding-rnas has emerged as a crucial component in the pathophysiology of virtually all cardiovascular diseases. here we will discuss the potential role of noncoding rnas in covid-19 disease mechanisms and their possible use as biomarkers of clinical use. the novel human severe acute respiratory syndrome coronavirus 2 (sars-cov-2), isolated on 7th january 2020, has been identified as the cause of acute respiratory distress syndrome (ards) cases of unknown etiology detected in wuhan city, hubei province of china and then indicated as coronavirus disease 2019 (covid-19) [1] . because of the rapid global spread of the covid-19, on 11th march 2020, the director-general of the world health organization (who) defined the disease as a pandemic [2] and up to october 27th 2020, there have been more than 43 million confirmed cases and more than 1 million deaths worldwide [3] . the main clinical manifestations of covid-19 are respiratory. however, many patients also display a severe involvement of the cardiovascular system [4] [5] [6] [7] [8] [9] . thus, it is of paramount importance to understand the direct and indirect damage caused to the cardiovascular system by sars-cov-2 infection, as well as the underpinning pathogenetic mechanisms. here we will review the importance of transcriptomics techniques in our understanding of human coronavirus disease mechanisms in the cardiovascular system and for the identification of biomarkers of potential clinical use. specifically, we will focus on noncoding rnas (ncrnas), an emerging class of regulatory rnas [10] . given their fundamental role in gene expression regulation, we propose ncrnas as promising candidates for understanding the consequences of sars-cov-2 infection on the cardiovascular system. threshold: small noncoding rnas (small ncrnas) and long noncoding rnas (lncrnas). the most studied small ncrnas are micrornas (mirnas) and long ncr-nas, which include also circular rnas (circrnas) [11, 13] . micrornas (mirnas) are short noncoding rnas (18-25 nucleotides) that modulate gene expression by sequence-specific recognition of their target transcripts. most mirna genes are transcribed by rna polymerase ii from intergenic, intronic or polycistronic loci forming an intermediate hairpin of 60-70 nucleotide, named ''precursor mirna'' (pre-mirna) [14] . the pre-mirna is then transported out of the nucleus and cleaved by the cytoplasmic rnase iii dicer into a short mirna duplex [15, 16] . one strand of this duplex binds to the argonaute (ago) protein forming the rna-induced silencing complex (risc), which can recognize the target mrna by sequence complementarity [17, 18] . this binding leads to the degradation and/or translational inhibition of the target mrnas. each mirna can have hundreds of rna targets and also a single mrna may have several mirna recognition sequences, generating complex regulatory networks [19] . lncrnas are ncrnas regulating the transcription and translation of protein-coding gene expression [20] [21] [22] [23] . they have some similarities with coding genes, such as the presence of epigenetic marks, introns and the existence of splice variants, and the transcription driven by promoter elements. moreover, they can be either polyadenylated or non-polyadenylated [24] . they may originate from either the sense or antisense dna strand, can overlap coding genes entirely or partly. they can be divided in sense, antisense, intronic, intergenic, and bidirectional lncrnas, enhancer-associated rnas (ernas), and promoter associated long rnas (palrs) [25] . finally, there are circrnas, that are circularized rnas generated by back-splicing events of pre-mrnas [26] . lncrnas can function as epigenetic regulators, can regulate the transcription rate by assembling transcriptional activators and repressors [27] . some nuclear lncrnas have been implicated in maintaining nuclear structures, including interchromatin granules, nuclear speckles and paraspeckles [22] . lncrnas can also regulate the gene expression by binding to mrnas and modulating their translation and/or stability [28, 29] . despite their generally low-abundance, some lncrnas can accumulate because they high stability and, by sequestering mirnas, can function as competing endogenous rnas (cernas) [30, 31] . sars-cov-2 belongs to the beta-coronavirus genus of the coronaviridae family; like the other members of the family, sars-cov-2 is enveloped and its genome is a single-stranded positive-sense rna of around 30 kb [32] . coronaviruses genome encodes for nonstructural proteins and for four structural proteins: spike (s), envelope (e), membrane (m) and nucleocapsid (n) proteins ( fig. 1 ) [33] . the s protein can recognize the receptor of the host cell and is responsible for cell membrane fusion [34] , the n protein interacts with the viral rna to assemble the ribonucleoprotein [35] , the e protein is necessary for virion assembly [36] and the m protein has a pivotal role in virus assembly [37] . all the members of the coronaviridae family seem to have the same replication mechanism: the genomic rna represents the template used to translate two partly overlapping open reading frames in two polyproteins, which encode the nonstructural proteins necessary to the replication-transcription complexes assembly in association with cytoplasmic membranes [32, 38] . coronaviruses can infect many animal species, causing different symptoms, such as respiratory and intestinal diseases [39, 40] . among human coronaviruses, severe acute respiratory syndrome-coronavirus (sars-cov) and middle east respiratory syndrome-coronavirus (mers-cov) are the best studied, due to the epidemics that originated in 2002 and 2012, respectively [39] . virology and genetic studies indicate that human coronaviruses have a reservoir in nature not corresponding with the intermediary host species, which is responsible for dissemination to humans [39] [40] [41] . since sars-cov-2 genome is more than 96% identical to a bat coronavirus, it is very likely that bats may have been the initial zoonotic host [42] . however, the intermediary species is unknown. the most common human-human transmission is through respiratory droplets generated by sneezing and coughing [43] . genetic sequence analysis revealed that sars-cov-2 has 79.0% nucleotide identity to sars-cov and 51.8% identity to mers-cov [44, 45] . comparing sars-cov-2 and sars-cov genomes, six regions of difference have been recognized (fig. 1 ). based on proteomic comparison, sars-cov-2 proteins are highly homologous (about 95%-100%) to the sars-cov virus proteins [45] . the similarity of sars-cov and sars-cov-2 viruses is also confirmed by the fact that they both use angiotensinconverting enzyme 2 (ace2) as cellular receptor, via the receptor-binding domain of surface spike glycoprotein s [46] [47] [48] . for their similar structural and pathogenicity features, the better studied sars-cov and mers-cov constitute important models when developing hypothesis for sars-cov-2 disease mechanisms. patients with pre-existing cardiovascular diseases (cvds) have a higher risk of severe disease and death upon sars-cov-2 infection [4] [5] [6] 8] . it is plausible that covid-19 can affect the cardiovascular system at various levels. indeed, ace2 is expressed by a multitude of cell types, including cardiac and vascular cells [49] , which may represent direct targets. accordingly, sars-cov-2 infection has been associated with multiple direct and indirect cardiovascular complications including arrhythmias as well as myocardial injury due to hypoxia, microvascular thrombosis and systemic cytokine release syndrome ("cytokine storm") [8, 50] . indeed, the immune response is crucial for infection resolution, but this response can also result in immunopathogenesis. during the disease course, sars-cov viral loads were observed to decrease while disease severity increased, suggesting that immunopathogenesis may contribute to ards [51] and may be an important cause of cardiovascular damage (fig. 2) . moreover, antiviral therapies under investigation for covid-19 may damage the heart and have other cardiovascular side effects, such as arrhythmias and repolarization abnormalities [52] . ace2 is an integral membrane carboxymonopeptidase that has been identified as a functional receptor for coronaviruses, including sars-cov and sars-cov-2 [48, [53] [54] [55] . in this process, it is crucial the s-protein priming by the transmembrane serine protease 2 tmprss2 [54] . ace2 is expressed in alveolar epithelial cells, in line with the respiratory symptoms of covid-19, but also in other epithelial and non-epithelial cells, in the kidneys and the gut. with relevance to the potential cardiac impact of covid-19, ace2 is also expressed by cardiomyocytes, fibroblasts, pericytes, macrophages and the epicardial fat [49, [56] [57] [58] . ace2 is an important member of the renin angiotensin system [54] . as shown in fig. 2 , ace2 activity is necessary to generate angiotensin 1-7, which, via the activation of the g protein-coupled mas receptor, leads to vasodilatory, anti-hypertrophic, and anti-fibrotic effects. this represents an important mechanism to compensate the negative cardiovascular action of an excessive angiotensin ii stimulation on the angiotensin ii type 1 receptor (at1r) (fig. 3) . in keeping with an important role of ace2 in the heart, ace2 knockout mice display impaired cardiac contractility and the upregulation of the hypoxia-associated gene program [58] . in both the mouse model of acute myocardial infarction (ami) and in idiopathic and ischemic heart failure (hf) patients, increased expression of ace2 protein and mrna has been observed [59, 60] . moreover, ace2 is a transmembrane protein, whose catalytic domain is located at the extracellular side of the cell and it can be released into the blood after cleavage by adam17 (also named tace). previous studies have reported increases in circulating ace2, possibly due to augmented ace2 shedding, to be associated with cardiovascular risk factors, such as advanced age and diabetes mellitus [61] , which are also risk factors for covid-19 [8, 50] . given ace2 expression in the heart, it is conceivable that the sars-covs virus can have a direct effect the renin-angiotensin system. ace2 can cleave both angiotensin i and angiotensin ii to generate ang 1-9 and ang 1-7, respectively. ang 1-9 and ang 1-7, in turn, bind angiotensin ii type 2 receptor (at2r) and mas and have vasodilatory, pro-apoptotic, anti-fibrotic and anti-inflammatory effects. ace2 converts angiotensin i to angiotensin ii, which through the binding to at1r has opposite effects, i.e. vasoconstriction, anti-apoptotic, pro-fibrotic and pro-inflammatory effects on cardiac function. accordingly, in several sars-cov patients, viral rna was detected not only in the lungs, but also in the myocardium [62, 63] . increased ace2 expression in cardiovascular patients [59, 60] may increase their susceptibility to sars-covs infection. however, in the heart of both sars-cov infected mice and of sars patients, decreased ace2-levels were observed [64] , suggesting a complex virus/receptor dynamic that needs to be elucidated. further complicating the scenario, ace2 levels can also be increased by inhibitors of the renin angiotensin system [65] . however, while the impact of these drugs on covid-19 is still largely unknown, a retrospective study did not identify inhibitors of the renin angiotensin system as independent predictors of poor outcome [66, 67] . different studies found that the values of cardiac troponins were increased in covid-19 patients with more severe disease [4, 5, [68] [69] [70] , indicating an association of sars-cov-2 with myocardial damage. indeed, in a study surveying 187 covid-19 hospitalized patients [9] , myocardial injury identified by increased troponin t and n-terminal pro brain natriuretic peptide (nt-pro-bnp) levels was significantly associated with death, while the prognosis of patients with underlying cvds, but without myocardial injury, was less severe. moreover, cardiac injury was associated with cardiac dysfunction and arrhythmias. accordingly, there are several studies reporting a direct effect on heart function of coronaviruses with a pathogenicity similar to that of sars-cov-2. in a rabbit model of coronavirus infection, viral-cardiomyopathy and progression into dilated cardiomyopathy (dcm) have been described [71] . moreover, mers-cov infection has been associated with acute myocarditis and hf [72] . interestingly, in the hearts of both sars-cov infected mice and sars patients, macrophage infiltration with evidence of myocardial damage was observed [64] . a similar pattern with low-grade myocardial inflammation and viral particles in the heart has been reported in one covid-19 case [73] . cardiomyocytes showed non-specific features such as focal myofibrillar lysis, and lipid droplets, but no viral particles were observed in myocytes and endothelia, suggesting infected macrophage migration from the lung or during a viraemic phase. other myocarditis single cases in which endomyocardial biopsy was performed scored negative for sars-cov-2 genome presence [74, 75] . thus, further studies with higher numerosity are definitely needed to ascertain the nature of covid-19 myocarditis. li et al. [76] reported in sars patients an impairment in left ventricular performance during the acute phase, but reversible on clinical recovery. this impairment was more severe in critically ill patients and elevated lactate dehydrogenase levels, reflecting the severity of tissue damage, were associated with decreased ejection fraction [76] . while the expression of ace2 is well known in heart, it is controversial the expression of tmprss2 [77] . these data draw the attention on the mechanism by which the virus could enter in the cardiomyocytes, and on the existence of secondary effects related to hypoxia and systemic inflammation during complicated covid-19 courses. indeed, the finding of a more prominent immune reaction in patients with more critical illness, and the association of cytokines as pro-inflammatory mediators in hf, support the hypothesis that the heart function impairment may be due to the cytokine storm in response to sars infection [76, 78] . likewise, also in covid-19 patients, a cytokine storm triggered by the sars-cov2 infection may result in damage to myocardial cells. this strong inflammatory response may also confer risk for atherosclerotic plaque rupture in coronary artery disease patients, increasing the risk of acute coronary syndrome in more severely affected covid-19 patients, akin to what has been observed in influenza viral illness [79] . also important in elevating the risk of cardiac injury is hypoxemia caused by respiratory dysfunction, higher risk of capillary embolism [80] and the increased metabolic demands. moreover, many antiviral drugs can cause cardiac damage [81] , further complicating the clinical situation. in hospitalized covid-19 patients, cardiac arrhythmia was present in more than 15% of the patients and was far more common in those presenting serious symptoms and requiring intensive care [5, 9] . while myocarditis should be considered, high prevalence of arrhythmia might be also attributable to hypoxia, cytokine storm syndrome and metabolic disorder in the setting of viral infection, possibly in the presence of prior cvds. indeed, there are several possible pathophysiological causes of arrhythmias such as: 1) the direct injury to cardiomyocytes that alters the membrane electrical conduction, 2) massive edema, 3) ischemia originating from microvascular disease following the infection, 4) re-entrant arrhythmias caused by myocardial fibrosis or scars and 5) proinflammatory cytokines storm. the first three events could occur in the acute setting, while the fourth and fifth are associated with chronic or healed myocarditis. moreover, the proinflammatory cytokines storm (e.g., il-6) might cause alteration in the desmosomal proteins of the cardiomyocyte membrane that could be arrhythmogenic [75, 82] . there is increasing evidence that patients with cvds and/or diabetes mellitus are more likely to develop severe symptoms when affected by covid-19 [4-6, 83, 84] . among covid-19 patients with severe symptoms, many had hypertension, heart disease and arrhythmia. acute coronary syndrome seems to have a particularly poor prognosis in covid-19 patients. indeed, upon sars-cov-2 infection, cardiac insufficiency is more likely to occur in these patients where cardiac function is already compromised by myocardial ischemia [4-6, 83, 84] . moreover, sars-cov infection leads to long term disorders of lipid and glucose metabolism [85] . considering the similarities between sars-cov and sars-cov-2, this novel virus might also inflict a similar chronic damage. transcriptomics has been extensively used to understand cvd pathogenesis, to identify coding and ncrnas with a potential as therapeutic targets and clinically useful biomarkers. these studies have been extensively reviewed elsewhere [10, [86] [87] [88] [89] . here we will only give few examples that may be paradigmatic also in the investigation of the cardiovascular implications of covid-19. transcriptomics first focused on mrnas expressed in the heart, identifying transcripts encoding mostly contractile sarcomeric proteins, cytoskeletal proteins, ion channels, intracellular signal transducers, including apoptosis and cell proliferation genes, and proteins maintaining the redox state of the myocardium [90, 91] . transcriptomic profiling identified several mirnas deregulated in left ventricles (lv) of both dilated (dcm) and ischemic cardiomyopathy (icm) patients [92] . additional studies allowed identification also of signatures specific to particular cardiomyopathies, such as dcm in patients with reduced catecholamine sensitivity [93] , dcm in patients with a reverse-remodeling response following β-blocker treatment [94] and icm in patients affected by type 2 diabetes mellitus [95] . mirna expression is also dysregulated in viral myocarditis [96] [97] [98] . in particular, mir-155-5p is one of the mir-nas consistently modulated in both human and mouse viral myocarditis, contributing to myocardial damage through the modulation of monocyte-macrophages cardiac infiltration and t lymphocyte activation [98] . genome-wide analyses of lv samples of hf patients identified a subset of lncrnas significantly deregulated compared with healthy controls; despite the limited evolutionary conservation of lncrnas, many of these findings were also confirmed in relevant mouse models of disease [86, [99] [100] [101] [102] [103] . particularly relevant are circrnas originating from the multi-exon gene titin, that are dysregulated in both dilated and hypertrophic cardiomyopathies, and are regulated by the splicing factor rna binding motif protein 20 (rbm20) [104] . of great translational relevance is the fact that ncrnas are released into the blood where they are sufficiently stable to be readily measured with common laboratory techniques, such as qpcr, and that their concentration levels can differentiate diseased patients from healthy subjects. in addition, their measurement requires a minimally invasive procedure, thus representing an enormous reservoir for biomarkers discovery, for both diagnostic and prognostic applications. many genome-wide profiling studies of circulating ncrnas have been performed. among mirnas, heart and muscle-enriched mirnas, also named myomirs (mir-1-3p, mir-133a-3p, mir-133b-3p, mir-208a/b-3p, and mir-499-5p) seem to be particularly relevant [105] [106] [107] [108] [109] [110] . indeed, in patients with ami, myomirs have been reported to be elevated in plasma, likely due to cardiomyocyte necrosis and thus released into the circulating system [111, 112] . accordingly, mir-208a-3p and mir-499-5p were also elevated in the plasma of viral-cardiomyopathy patients and their levels correlated positively with myocardial damage assessed by measuring troponin t levels [113] . among myomirs, mir-208 is of particular interest, since its expression is highly cardiacspecific. moreover, voellenkle et al., analyzing the pattern of expression of peripheral blood mononuclear cells (pbmcs) identified a mirna signature characterizing dcm patients [109] . also lncrnas can be found in the peripheral blood and hold a promising biomarker potential. the expression of lipcar (long intergenic noncoding rna predicting cardiac remodeling) in plasma has been found to be correlated to cardiac remodeling progression after ami [114, 115] . other potential biomarkers are miat (myocardial infarction associated transcript), discriminating st-elevation from non st-elevation ami [116] , sencr (smooth muscle and endothelial cell-enriched migration/ differentiation-associated long noncoding rna), associated to lv remodeling [117] , as well as nron (noncoding repressor of nfat) and mhrt (myosin heavy chain associated rna transcripts), lncrnas elevated in hf patients and independent predictors of hf events [117, 118] . greco et al. [102] , analyzing icm patients found that anril, hotair, and loc285194/tusc7 had similar modulation in pbmc and heart tissue, suggesting a potential role as functional biomarkers. among circrnas, micra (myocardial infarctionassociated circular rna) levels in the blood are associated with ischemic hf [119] , while hsa_circ_0124644 and hsa-circ-0005870 are potential diagnostic biomarkers of coronary artery disease [120] and hypertension [121] respectively (fig. 4b) . the sars-cov-2 host-response transcriptomics changes have just started to be investigated, but reports on them are increasing over time. the gene expression profile of blood samples derived from covid-19 patients not only allows the definition of the host responses to the infection and, thus, a better understanding of the disease pathogenesis, but also the identification of potential biomarkers that could help monitoring patient responses to the disease. specifically, a distinct pattern of inflammatory cytokines was identified in bronchoalveolar lavage fluid and in the peripheral blood mononuclear cells, including ccl-2, -3 and -4, and cxcl-10, as well as the activation of the p53 signaling-pathway in lymphocytes that may be related to covid-19 lymphopenia [122] . in addition, several differential expressed transcripts were identified in pbmcs of covid-19 patients with severe or mild symptoms [123] compared to controls. cytokinemediated signaling, the natural killer cell mediated toxicity and t cell activation pathways were enriched terms in common between the two disease stages, while interleukins-and tnf-signaling pathways were enriched terms the release of ncrnas by diseased cardiac tissues in the peripheral blood and the immunomodulation associated to cvd allowed the identification of dysregulated ncrnas in whole blood, plasma/serum or in pbmcs, to be used as potential biomarkers [123] . moreover, the single-cell rna-sequencing (scrnaseq) approach has been used to profile the sars-cov-2 host-response in the pbmcs of covid-19 patients, and to comprehensively characterize the immunological changes [124] [125] [126] [127] [128] [129] [130] . in eight covid-19 patients, scrna-seq of pbmc indicated the depletion of the innate immune subsets compared to healthy pbmc [125] . in addition, a new population, which was annotated as 'developing neutrophils' , has been found increased only in patients with ards; this population expressed several genes in common with the neutrophil progenitors, but not canonical neutrophil markers, indicating that they could be neutrophils at various developmental stages [125] . the monocyte subset was enriched in cd14, but without a substantial expression of pro-inflammatory cytokine genes (tnf, il6, il1b, ccl3, ccl4 or cxcl2), which is in contrast with other reports [124, 126] . additionally, cd14 + monocytes showed the upregulation of a signature of interferon (ifn)-stimulated genes that correlated with clinically relevant parameters [125] . this strong activation of interferon-stimulated genes was also confirmed by arunachalam et al. [127] , primarily in patients with mild or moderate disease, that is associated to reduced expression of genes encoding proinflammatory cytokines [127] . similarly, a more pronounced expression of type i ifn in mild covid-19 patients and lack of il1b mrna levels increase were also confirmed in a recent study that analyzed by scrna-seq two independent large cohorts of covid-19 patients [128] . conversely, an increased tnf/il-1β-driven inflammatory response was observed in severe covid-19 patients as compared to severe influenza, and to mild covid-19 patients [129] . the scrna-seq approach has also been used to study the immune cell landscape of two severe-stage covid-19 patients prior to and following tocilizumab-induced remission [126] . in this study, a monocyte subpopulation that contributes to the inflammatory cytokine storms was identified. bioinformatics analysis predicted a severe stage-specific interaction-networks formed by monocyte receptors and cytokines [126] . in addition, tocilizumab treatment in severe-stage covid-19 patients led to an increased number of antibody-secreting plasma b cells, while cd8 + t cell-related cytotoxicity and cytokine production did not change [126] . these data provide insights into the understanding of the inflammatory storms observed in covid-19 patients and identify also potential drug targets. zhang et al. analyzed by scrna-seq the pbmcs of thirteen covid-19 patients with clinical conditions ranging from moderate to severe and to convalescent [130] . differentially expressed genes of cd14 + and cd16 + covid-19 monocytes, especially in severe covid-19 state, were associated with ifn responses, myeloid leukocyte activation, cytokine production and nuclear factor (nf)-κb signaling pathway [130] . interestingly, in patients at the early phase of convalescence, despite of the recovery of most of the clinical parameters to a normal range, the immune system was still fully activated, as demonstrated by a still high naive t and t reg subsets ratios [130] . early and late stages of recovery have been analyzed in detail by wen et al., confirming that covid-19 patients are still vulnerable after hospital discharge [124] . covid-19 early recovery stage was characterized by cd14 ++ monocytes with high inflammatory gene expression as well as by the abundance of cd14 ++il1β + cells, while t cells decreased remarkably, compared with both late recovery stage and healthy control subjects. due to the limited cardiac tissue availability, data on sars-cov-2-mediated heart transcriptome changes have not been reported yet. however, sars-cov-2 infection of human induced pluripotent stem cell-derived cardiomyocytes (ipsc-cms) induced cytotoxic effects and rna-seq findings highlighted significant transcriptional changes in gene pathways related to cellular metabolism and immune response [131] [132] [133] . indeed, sharma et al. [131] observed in infected ipsc-cms a downregulation of transcriptional pathways related to mitochondrial function, oxidative phosphorylation, and cardiac function, whereas upregulated pathways included immune cytokines, immunomodulators, antiviral response, and apoptosis [131] . accordingly, bojkova and collaborators showed that sars-cov-2 infection of ipsc-cms induced cytotoxic and proapoptotic effects and abolished cardiomyocyte beating [132] . virus infection produced a transcriptional response including the up-regulation of genes associated to viral response and interferon signaling, apoptosis and reactive oxygen stress [132] . on the same line, pérez-bermejo et al. [133] , found that human ipsc-cms exposed to sars-cov-2 demonstrated a productive infection and morphological signatures of damage, which included a distinct pattern of myofibrillar fragmentation and numerous ipsc-cms lacking nuclear dna. these morphological changes were also confirmed in human autopsy specimens from covid-19 patients [133] . rnaseq transcriptomic data obtained in infected ipsc-cms suggested a compensatory overexpression of myosin heavy chain genes in response to targeted degradation and also a significant depression of the ubiquitin-proteasome system upon infection [133] . as for the heart, a limited number of reports on lung transcriptomic changes mediated by sars-cov-2 infections has been published so far. one of the first studies on lung cells was the analysis of the transcriptional footprint in post-mortem lung samples of covid-19 patients [134] . in this study a reduced ifn-i and -iii response and a consistent chemokine signature compared to other respiratory viruses were observed [134] . the reanalysis of a previously reported dataset identified, as expected, upregulated expression of chemokines and neutrophils in the lung tissue and bronchoalveolar lavage fluid of covid-19 patients and, in addition, the upregulation of genes coordinating heme biosynthesis [135] . this effect, which has been shown in sepsis secondary to pneumonia to have a protective role against oxidative stress [136] , could be responsible of pro-inflammatory cytokine production amplification [137] or cause intravascular coagulation [138] . the possible involvement of altered coagulation following sars-cov-2 infection has been also proposed by a study analyzing three publicly available rna sequencing datasets obtained from clinically isolated samples of bronchoalveolar lavage fluid, pbmcs and from in vitro sars-cov-2 infected primary normal human bronchial epithelial cells compared to the respective controls [139] . gene expression analysis of both bronchoalveolar lavage and bronchial epithelial cells, but not of pbmcs, highlighted the activation of the extrinsic blood coagulation cascade and the suppression of the plasminogen activation system [139] . moreover, a study performed in autopsy lung specimens from patients who succumbed to sars-cov-2 infection supported two phases of disease evolution in patients with severe covid-19 pneumonia [140] . in the first phase, high expression of ifn pathway genes and of endothelial genes related to tissue damage and fibrosis were observed [140] . this first phase was morphologically characterized by high viral rna, a histological pattern of exudative diffuse alveolar damage, a shorter disease duration, while the second phase showed a low (or undetectable) viral rna, and an organizing form of diffuse alveolar damage [140] . of note, the aforementioned pro-inflammatory cytokines, such as tnf-α, ifn-γ, il-1β, il-6, il-17, and il-18, are also elevated in hf and in viral myocarditis, and their sustained elevation correlates to hf progression. they are responsible for both compensatory cardiac hypertrophy and fibrosis in the setting of cardiac injury and induce further inflammation [141] . there are contrasting effects attributed to ifn-γ in the heart. upon adverse stimuli, such as myocarditis or hypertension, the release of ifn-γ by recruited inflammatory cells to the heart results in cardiac fibrosis and hypertrophy. however, other studies have found that ifn-γ has also protective effects, limiting cardiac hypertrophy [142] . the signaling of the pro-inflammatory cytokines is counterbalanced by the release of anti-inflammatory cytokines and tgf-β, which mitigate hypertrophic cardiac remodeling [141] . these data indicate that, along with the direct effect mediated by the virus, such as in myocarditis, the cardiac sequelae may be due to the cytokines storm following the infection, which reinforces the cytokines release, observed in dilated cardiomyopathies. noncoding rnas regulating inflammation and the cardiovascular system: are they playing a role in covid-19? an over-activation of the inflammatory response and the ensuing cytokine storm seem to be a crucial pathogenetic mechanism in covid-19 patients and, as illustrated in sect. "transcriptomics studies of sars-covs infection: focus on inflammation", the innate immune response pathway has emerged as profoundly dysregulated in sars-covs infections. the immune system contributes to heart development, composition and function and, in specific circumstances, immune cells can also cause damage, participating to cardiac disease [143] . for instance, in patients with hf, a chronic activation of the innate immune system is often observed, with t-cells and macrophages myocardial infiltration and increased pro-inflammatory cytokine levels (i.e. tnf-a, il-1b, and il-6) [144] . ncrnas are important regulators of these processes and we propose that ncrnas may play a fundamental role also in the cardiovascular dysfunctions of covid-19 patients. as mentioned above, the expression of ace2 is well known in adult cardiomyocytes [48, [53] [54] [55] 77] , as well as in neonatal rat cardiomyocytes and in human ipsc-cms [145] . bioinformatics analysis predicted mir-200b, mir-200c and mir-429 among the mirnas targeting ace2, and in vitro experiments demonstrated that mir-200c modulation regulated the expression of ace2 [145] . mir-200c is upregulated by oxidative stress [146] and is also involved in cvds [146] , suggesting that mir-200c-mediated regulation of ace2 may be important for sars-cov-2 entry. computational analyses have predicted that sars-cov-2 can divert the cellular mirnas from their transcriptional regulating activity, contributing to the abnormal immunity activation in patients with covid-19 [147] , or synthesizes its own viral mirnas to reduce the host cell apoptosis preventing host defense [147] [148] [149] . on the other side, host mirnas were predicted to target sars-cov-2 spike proteins, suggesting a potential role as therapeutic molecules [149, 150] . direct or indirect effects of sars-cov-2 infection can alter host response and produce noncoding rna differential expression. in particular, in bronchial epithelial cells infected with sars-cov-2, a complex bioinformatics and computational pipeline, revealed several activated networks, including those involved in immunoglobulin g and interferon lambda [151] . in addition, acute inflammatory response and activation of tnf were also observed. this analysis also revealed several host-derived lncrnas differentially expressed in covid-19 patient-derived lung tissue, and in sars-cov-2 infected epithelial cells, including malat1 (metastasis-associated lung adenocarcinoma transcript 1) and neat1 (nuclear-enriched autosomal transcript 1) [151] (fig. 5) . the computational reanalysis of rna-seq dataset of sars-cov-2 infected bronchial epithelial cells [134] identified several protein-coding rnas and lncrnas differentially modulated [152] . the interaction of lncrnas with the differentially expressed protein-coding genes was analyzed by network enrichment analysis indicating significant interactions involved in cellular signaling, metabolism, immune response and rna homeostasis [152] . the rna-seq of rna extracted from peripheral blood samples from 10 covid-19 patients compared to 4 controls demonstrated that 35 mirnas were upregulated and 38 mirnas were downregulated in the human patients with covid-19 [153] . enrichment analysis of differentially expressed mirna target genes revealed that peptidases, protein kinases, and the ubiquitin system were shown to be the highest enrichment categories [153] (fig. 5) . there are also some hints provided by transcriptomics studies on sars-cov. when this infection had a multicountry outbreak in 2002 to 2003, coding rnas were still the focus of attention for the scientific community and microarrays were the preferential profiling tool. nevertheless, some studies performed in the following years on lung samples or on bronchoalveolar stem cells collected from mouse infected with adapted sars-cov are available [154] [155] [156] [157] . these studies identified lncrnas and mirnas involved in innate immune response in cvds. in particular, the lncrnas neat1 (nuclear paraspeckle assembly transcript 1) and malat1 (metastasis-associated lung adenocarcinoma transcript 1) [154] were up-and down-regulated after infection, respectively, and mir-155-5p [156] , mir-21-5p and mir-223-3p were among the most deregulated mirnas upon sars-cov infection (fig. 5) . mir-155-5p, mir-223-3p and mir-21-5p are expressed in immune cells and involved in both innate immune response [158, 159] and in myocardial infarction or hf [10, [86] [87] [88] [158] [159] [160] [161] . these mirnas control the production and secretion of pro-inflammatory cytokines in the heart by toll-like (trl) receptors and their downstream signaling pathway, which involves the transcription nuclear factor-kappa b (nf-κb). indeed, mir-155-5p, along with mir-146a-5p, represents a unique regulatory network for the fine-tuning of the macrophage inflammatory response via regulation of nf-κb activity [162] . in particular, the activation of nf-κb by a stressor stimulates mir-155-5p expression which, amplifying nf-κb activity, enables a strong macrophage activation. as the inflammatory response develops, mir-146a-5p transcription increases, inhibiting its targets irak1 and traf6, whereby dampening nf-κb activation (fig. 5) . in addition, also mir-21-5p targets a component of the nf-κb pathway, pdcd4 (programmed cell death 4), thus stimulating the release of pro-inflammatory cytokines and inhibiting the release of anti-inflammatory cytokines [163, 164] . finally, mir-223-3p seems to have cardio-protective and anti-inflammatory roles by enhancing glucose metabolism and inhibiting granulocyte activation. accordingly, a decrease of mir-223-3p levels in tissue and plasma has been observed in diabetes and cvds [95, [165] [166] [167] . another ncrna of relevance in cardio-immunology is the lncrna neat1, which was reported to induce intima thickening in vascular smooth muscle cells [168] . neat1 is also part of the innate immune response by promoting the activation of the inflammasome in macrophages and the release of il-1β [169] . together, these data indicate that some of the ncrnas reported to be dysregulated by transcriptomic profiling in cvds, are also involved in viral innate immune responses, and that they may be identified as candidate transcripts for our query in understanding the pathogenesis of covid-19. investigating the interplay between sars-cov-2, the host antiviral defenses and the cardiovascular system, is fundamental to understand the viral pathogenesis and the infection outcome. indications from sars-cov and other coronaviruses are very helpful; however, sars-cov-2 is a novel human pathogen and many aspects of its interaction with the host could be unique. it is now clear that pre-existing cvds increase both the severity of the primary respiratory syndrome and the risk of adverse outcomes. sars-cov-2 infection consequences on the cardiovascular system should be investigated both for their acute and prolonged 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cardiovascular diseases call to action for the cardiovascular side of covid-19 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we are thankful to jeremy c. hill (imperial college london) for editorial assistance. figures were partly generated by using biorender app and https :// www.somer sault 1824.com/scien ce-illus trati ons/. all authors contributed to the conception, design and revision of the article. sg, am and fm wrote most of the text and prepared the figures. cg, yd and key: cord-292742-mio4przi authors: mcaloose, denise; laverack, melissa; wang, leyi; killian, mary lea; caserta, leonardo c.; yuan, fangfeng; mitchell, patrick k.; queen, krista; mauldin, matthew r.; cronk, brittany d.; bartlett, susan l.; sykes, john m.; zec, stephanie; stokol, tracy; ingerman, karen; delaney, martha a.; fredrickson, richard; ivančić, marina; jenkins-moore, melinda; mozingo, katie; franzen, kerrie; bergeson, nichole hines; goodman, laura; wang, haibin; fang, ying; olmstead, colleen; mccann, colleen; thomas, patrick; goodrich, erin; elvinger, françois; smith, david c.; tong, suxiang; slavinski, sally; calle, paul p.; terio, karen; torchetti, mia kim; diel, diego g. title: from people to panthera: natural sars-cov-2 infection in tigers and lions at the bronx zoo date: 2020-10-13 journal: mbio doi: 10.1128/mbio.02220-20 sha: doc_id: 292742 cord_uid: mio4przi despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. among these, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and ebolaviruses have killed thousands; the human immunodeficiency virus (hiv) has killed millions. zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. the current sars-cov-2 pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. in march 2020, new york city was a global epicenter for sars-cov-2 infections. during this time, four tigers and three lions at the bronx zoo, ny, developed mild, abnormal respiratory signs. we detected sars-cov-2 rna in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral rna with cellular damage in one. we produced nine whole sars-cov-2 genomes from the animals and keepers and identified different sars-cov-2 genotypes in the tigers and lions. epidemiologic and genomic data indicated human-to-tiger transmission. these were the first confirmed cases of natural sars-cov-2 animal infections in the united states and the first in nondomestic species in the world. we highlight disease transmission at a nontraditional interface and provide information that contributes to understanding sars-cov-2 transmission across species. and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. our data show that tigers and lions were infected with different genotypes of sars-cov-2, indicating two independent transmission events to the animals. importantly, infected animals shed infectious virus in respiratory secretions and feces. a better understanding of the susceptibility of animal species to sars-cov-2 may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health. keywords one health, panthera leo, panthera tigris, sars-cov-2, in situ hybridization, lion, rrt-pcr, tiger, virus isolation, whole-genome sequencing, zoo, zoonotic infection c oronaviruses are a recognized cause of disease in humans and animals (1) . among them are the viruses that cause colds in humans, multisystemic disease in domestic cats, and gastrointestinal and respiratory diseases in pigs and poultry. coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome-related coronavirus (sars-cov-2) (2) was first reported in wuhan, hubei province, china at the end of december 2019 (3) . within weeks the virus spread globally, and by july 2020, over 10 million people were infected and more than 500,000 had died (https://www.who.int/ emergencies/diseases/novel-coronavirus-2019; accessed 1 july 2020). genome sequence analysis has shown sars-cov-2 to be most closely related to a bat coronavirus (ratg13-2013), and horseshoe bats are currently considered the likely source of the ancestral virus from which the currently circulating sars-cov-2 virus was derived (4, 5) . subsequent genetic adaptation of the currently circulating virus in an intermediate animal host(s) or after human transmission has been proposed (5, 6) . the exact details of how long the virus had been circulating in animals prior to transmission to people is unknown. however, a recent study suggests the virus may have been circulating in bats for several decades (7) , and an early cluster of human covid-19 cases had an epidemiological link to the huanan seafood wholesale market in wuhan where a variety of live wild animals were sold (4) . the current sars-cov-2 pandemic and outbreaks of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) before it raise awareness and concerns about zoonotic (animal-tohuman) diseases and cross-species transmission of coronaviruses (8) (9) (10) (11) . given the suspected zoonotic origin of sars-cov-2, identifying susceptible animal species, reservoirs, and transmission routes between species is a topic of global scientific and public interest. natural sars-cov-2 infections in animals have been reported in dogs, cats, and farmed mink in hong kong, europe, china, and the united states (12) (13) (14) . infection in most of these cases has been linked to households or settings in which human owners or caretakers have tested positive for sars-cov-2 and infection from humans to animals has been presumed. experimental inoculation studies have shown that sars-cov-2 infects and replicates with high efficiency in domestic cats, ferrets, and fruit bats and poorly in dogs; pigs, chickens, and ducks do not seem to support productive sars-cov-2 infection (15, 16) . importantly, virus shedding and horizontal transmission have been shown in cats and ferrets (15) (16) (17) following experimental inoculation. in this study, we report natural infection of tigers (panthera tigris) and lions (panthera leo) with sars-cov-2 at the wildlife conservation society's (wcs's) bronx zoo, new york, ny, and provide a detailed genomic characterization of viruses obtained from infected animals and keepers who had close contact with the sars-cov-2-positive animals. these were the first confirmed animal infections in the united states and occurred in march 2020, when, due to widespread community transmission (18) , new york was a global sars-cov-2 epicenter. audible wheezing despite remaining eupneic. by 3 april, an additional malayan tiger (tiger 2) and two amur tigers (panthera tigris altaica) (tigers 3 and 4) housed in the same building as tiger 1 but in different enclosures and three african lions (panthera leo krugeri) (lions 1, 2, and 3) housed in a separate building developed similar respiratory signs. all animals otherwise exhibited normal behavior and activity. clinical respiratory signs resolved in less than 5 days (3 to 5 april 2020) in all animals except tiger 1, whose clinical signs lasted 16 days (resolution of clinical signs on 12 april 2020). an additional amur tiger (tiger 5) in the same building as tigers 1 to 4 did not develop clinical respiratory disease. detection of sars-cov-2 in affected animals. a broad diagnostic investigation was performed in tiger 1 on 2 april, 6 days after the onset of clinical signs. this included physical examination, thoracic and abdominal radiography and ultrasonography, and collection of respiratory (nasal swab, oropharyngeal swab, and tracheal wash) and blood samples. thoracic radiography and ultrasonography revealed small, multifocal regions of peribronchial consolidation. cytologic examination of tracheal wash fluid identified necrotic epithelial and inflammatory cells consistent with tracheitis ( fig. 1a fig 1 tracheal wash cytology (a and b) and in situ hybridization (ish) (c and d). tiger 1. (a) flocculent material from the trachea consists of stringy mucus with enmeshed degenerate cells characterized by condensed nuclei and loss of distinct cellular features (arrows). (b) few intact cells (short arrow) and degenerate epithelial cells (long arrow) are admixed with abundant round to amorphous cellular debris and granular degenerate mucus (arrowheads). inset: degenerate epithelial cell (upper right) with nuclear fragmentation (karyolysis) and an adjacent intact neutrophil (lower left). modified wright's stain. (c and d) incubation with sars-cov-2-specific probe is positive (red puncta) throughout the mucinous material, in the cytoplasm of intact and degenerate epithelial and inflammatory cells, and in cellular debris. red chromogenic assay; hematoxylin counterstain. (note: a software or equipment malfunction produced a faint horizontal line that may be visible in panels a and b.) and b). in situ hybridization (ish) colocalized sars-cov-2 rna within necrotic epithelial and inflammatory cells in the fluid ( fig. 1c and d and see also fig. s1). all respiratory samples (nasal, oropharyngeal, and tracheal wash) were negative on virus isolation for feline herpesvirus and feline calicivirus and on targeted pcr testing and metagenomic analysis for common feline pathogens (table s1 , bioproject accession no. prjna627354); all were positive for sars-cov-2 by real-time reverse transcription-pcr (rrt-pcr) using primers and probes targeting portions of the nucleocapsid (n1, n2, and n3) and envelope (e) genes (table s2 ). minion and sanger-based sequencing of the full sars-cov-2 spike (s) gene, an internal region of the n gene, and the rna-dependent rna polymerase (rdrp) confirmed the virus in the respiratory samples (table s3 and data set 1 [see "data availability" in materials and methods for definitions and locations of all data sets]). fecal samples collected opportunistically from each animal (symptomatic tigers 1 to 4, asymptomatic tiger 5, and symptomatic lions 1 to 3) tested positive for sars-cov-2 by rrt-pcr (table s4 ). results were confirmed by amplicon sequencing (table s3 and data set 2). infectious sars-cov-2 detected in respiratory and fecal samples from affected animals. virus isolation was performed on all respiratory and fecal samples. cytopathic effect (cpe) was observed in vero cells inoculated with tracheal wash fluid from tiger 1 ( fig. 2a and b ) and fecal samples from tiger 3 and lion 2 (table s5 ). results were confirmed by rrt-pcr (cdc n1 assay) and/or ish and immunofluorescence assays ( fig. 2c and d) . additionally, a neutralizing antibody titer of 64 detected in tiger 1 confirmed active sars-cov-2 infection in this animal (table s6) . epidemiologic and diagnostic investigation of zoo staff. subsequent to confirmation of sars-cov-2 infection in the animals, an epidemiologic investigation of zoo staff identified 10 zoo keepers and two managers who provided care for and had close (յ1.8-m) but not direct contact with the tigers or lions between 16 march 2020 (the date on which the zoo was closed to the public due to the pandemic) and 27 march to 3 april 2020 (timeline of disease onset in the animals). four staff (2 tiger and 2 lion keepers) reported mild respiratory symptoms (including fever, cough, chills, myalgia, and fatigue) between 20 and 28 march 2020. nasopharyngeal samples and blood were collected from these staff members on 6 april 2020, and rrt-pcr and a microsphere immunoassay (mia; to detect igg antibodies) were performed; staff who did not report symptoms were not tested. all tested keepers had evidence of current or prior sars-cov comparative genomics and phylogenetic and haplotype network analysis. nine complete sars-cov-2 genome sequences (from four tigers, three lions, and two keepers) and eight full-length s gene sequences (from seven symptomatic animals and one asymptomatic animal) were generated directly from respiratory and/or fecal samples (data sets 3 and 4). compared to the wuhan-hu-1 sequence (genbank accession number nc_045512), all tiger and keeper sequences contained six single-nucleotide polymorphisms (snps) with nine additional ambiguous sites ( fig. s2 and table s7 ). a total of 20 sites differed between the three lion sequences and wuhan-hu-1 ( fig. s2 and table s7 ). viral sequences in the tigers, lions, and keepers clustered into common sars-cov-2 clades (fig. 3a) . those from tigers and tiger keepers clustered with clade g (defined by the d614g substitution in the spike protein); the lion sequences clustered with clade v (defined by the g251v substitution in orf3a) (fig. 3a ). median-joining haplotype network analysis of the viral sequences corroborated results of phylogenetic analyses ( fig. 3b and data set 4). nucleotide sequence and amino acid analysis of the spike protein of sars-cov-2 in tigers and lions was performed. compared with the wuhan-hu-1 strain, the tiger and lion sars-cov-2 s gene sequences had 1 to 4 nucleotide differences that resulted in several nonsynonymous substitutions (fig. 4 ). of five substitutions in the tiger strains, only one (g496d) was found in available human sars-cov-2 strains. these changes were not observed in the viral sequences from the lions (fig. 4 ). our results document susceptibility and natural sars-cov-2 infection in tigers and lions. these were the first confirmed animal infections in the united states and the first to be described in a nondomestic species in the world. genomic and epidemiological data support a close evolutionary relationship between the viral strains in the tigers and those in the tiger keepers. notably, the genetic differences and the distant phylogenetic relationship between sequences recovered from the tigers/tiger keepers and lions and the relationship of these strains in the context of global sequences indicate that tigers and lions were infected by two different sars-cov-2 genotypes. these data suggest that at least two independent sars-cov-2 introductions occurred, one in tigers and another in lions. importantly, the sars-cov-2 genome sequence from tiger 1 was identical to the viral sequence from keeper 1 (a tiger keeper) and to other human sars-cov-2 strains detected in new york (ny-cdc-2929 [mt304486] and ny-qdx-00000001 [mt452574.1]). these observations, temporal overlap in animal and human infections, and a lack of new animal introductions to the collection support the conclusion of transmission from an infected keeper(s) to the tigers. whether this was direct or indirect (e.g., fomite, food handling/preparation, or shared enrichment items) and whether subsequent tiger-to-tiger transmission (aerosol, respiratory droplet, etc.) occurred were not determined. a clear association and transmission source were not identified for the lions. the lion sars-cov-2 sequences were more divergent than those in the tigers and keepers. interestingly, nine of the 12 snps (relative to the wuhan-hu-1 reference strain) shared by all three lion viruses were also found in a human strain (closest strain, gisaid accession no. epi_isl_427161) detected in connecticut, usa. two lion keepers were serologically positive for sars-cov-2, but viral rna was not detected, and sars-cov-2 could not be confirmed in their respiratory samples. however, given close contact between keepers and animals and the serological evidence indicating infection of two of the lion keepers, it is possible that while asymptomatic, they or other asymptomatic staff may have transmitted virus to the lions. the host range of sars-cov-2 and other coronaviruses is determined primarily by the interaction of the virus s glycoprotein, specifically the spike 1 subunit (s1), and the cellular receptor, angiotensin-converting enzyme ii (ace2) (19) . in silico predictions have shown high binding potential between the s receptor binding domain (rbd) and domestic cat ace2 receptor and conservation of three of five amino acid residues that are critical for interaction with the sars-cov-2 s glycoprotein in human and domestic cat ace2 (19) . these observations are supported by reports describing natural and experimental infection of domestic cats with sars-cov-2 (15, 20) and the data here that show a high degree of conservation between ace2 in humans and domestic and wild felids. further work is needed to determine if these changes affect sars-cov-2 receptor binding and pathogenicity in felids and humans. infections in the tigers and lions occurred at a time before sars-cov-2 testing was widely available in the united states and when there was limited evidence of pre-or asymptomatic viral shedding (21) . additionally, at that time, keepers caring for the tigers and lions did not generally wear personal protective equipment (ppe) given the (historical) low risk of infectious respiratory disease transmission between humans and domestic or nondomestic felid species. results of this investigation prompted the immediate development of new protocols for ppe use in the enclosures of nondomestic felids and other known or susceptible species including mustelids, viverrids, and chiroptera (ppe was already in place for work with nonhuman primates) at the bronx zoo. they also contributed to the development of similar recommendations by other zoo and wildlife organizations (https://www.aza.org/aza-news-releases/posts/aza-and-aazv-statement-on -covid-19-positive-tiger-in-new-york). the role of domestic and wild animal species in the epidemiology of sars-cov-2 is not completely understood. to date, the reported number of cases of sars-cov-2 infection in domestic and wild animal species is low, and to our knowledge, no other zoos worldwide have confirmed cases in their animals. this is notable when considered in the context of the large number of human cases and close interactions between people, their pets, and wild animals in their care. however, the fact that companion animals, farmed mink, and zoo animals are susceptible to sars-cov-2 infection and shed infectious virus in respiratory secretions and/or feces (13-15) makes the humananimal-environment interface an important area for further one health-based studies (22) . in general, a better understanding of sars-cov-2 susceptibility across a wide range of animal species will help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection important in both animal and human health. in the last 2 decades, at least three major coronavirus epidemics (sars, mers, and covid-19) have occurred. a feature shared by these and other novel viruses of humans including ebolaviruses and human immunodeficiency virus (hiv) is an origin in a wild sars-cov-2 in tigers, lions, and keepers at a zoo ® animal host. despite a traditionally held perception of low risk, scientists and conservationists around the world have long recognized and shared concerns related to human activities that increase human-wildlife interactions and zoonotic disease transmission risk (22) (23) (24) . as long as anthropogenic development and population growth bring humans and wildlife into increasing proximity, legal and illegal harvesting persists, and consumption of wildlife and wildlife products exists, there will be continued and significant risk of pandemic viral emergence with devastating global impact on human and animal health, economies, food security, and biodiversity. personal protective equipment (ppe), including n95 or surgical masks, face shields or goggles, and disposable gloves, were not generally worn when working with the tigers and lions prior to the sars-cov-2 pandemic but were worn during all animal handling and sample collection subsequent to the development of clinical signs in tiger 1. diagnostic specimens were submitted to the university of illinois veterinary diagnostic laboratory (uiuc-vdl) and animal health diagnostic center at cornell university (cornell ahdc) (both part of the national animal health laboratory network) for broad diagnostic investigation (respiratory samples-tiger 1) and specific sars-cov-2 testing (fecal samplesall animals). epidemiologic investigation. subsequent to the development of clinical signs and positive test results in the tigers and lions, an epidemiologic investigation into possible human infections in staff working with these animals was conducted by the new york city (nyc) and new york state (nys) public health laboratories in conjunction with the u.s. centers for disease control and prevention (cdc). the investigation focused on the time period between 16 march 2020 (date on which the zoo was closed to the public due to the pandemic) and 27 march to 3 april 2020 (the timeline of disease onset in the animals). twelve staff members (10 keepers and 2 managers) were identified who had responsibilities that offered opportunities for close (յ1.8-m) but not direct contact with the animals during this time period. this included moving animals between enclosures and exhibits, feeding, training sessions, enrichment activities, greetings, and social interactions (e.g., chuffing, a form of vocalization that tigers performed that involves air exhalation and which keepers also use to greet tigers). additionally, lion keepers worked at a desk that was located less than 1.8 m from metal-mesh-fronted lion enclosures. four keepers reported being mildly symptomatic (including fever, cough, chills, myalgia, and fatigue) with signs beginning in each prior to or concurrently with illness in animals on 20, 22, 27, and 28 march 2020. nasopharyngeal swab and blood samples were collected on 6 april 2020 from the symptomatic keepers, and sars-cov-2-specific rrt-pcr and a microsphere immunoassay (to detect igg antibodies) were performed; sampling and testing were not performed in the eight additional staff who did not report symptoms. all four of the tested keepers had evidence of sars-cov-2 infection (one rrt-pcr-positive tiger keeper [keeper 1], one rrt-pcr-and serology-positive tiger keeper [tiger 2], and two serology-positive lion keepers [keeper 3 and keeper 4]). none of the keepers reported being sick at work. all stayed home for at least 7 days from the onset of illness, and none returned to work prior to a minimum of 7 symptom-free days and 72 fever-free hours in compliance with organizational covid-19 policies and cdc and ny department of health (doh) guidelines. rrt-pcr-positive specimens were forwarded to cdc for whole-genome sequencing (wgs) and haplotype network analysis to characterize the human samples and further compare the human and animal viral genome sequences. interviews with the tiger and lion keepers suggested that up to two additional keepers may have had signs or symptoms suggestive of mild and transient covid-19; however, they did not self-report being sick, may not have recognized their symptoms as being consistent with covid-19, and were not tested. non-sars-cov-2 respiratory pathogen testing. nucleic acid extracted from the respiratory specimens (nasal and oropharyngeal swabs and tracheal wash fluid) from tiger 1 was tested by real-time pcr (rpcr) or rrt-pcr for several common feline respiratory pathogens (cornell ahdc) including bordetella bronchiseptica (25) , influenza a virus (cdc universal assay) (26) , mycoplasma cynos (27) , mycoplasma felis (28) , pneumovirus (29) with probe modification (6-carboxyfluorescein [fam]-cttcatcacttttggcctgg cccag-bhq1), and streptococcus equi subsp. zooepidemicus (30) . additionally, samples were tested for chlamydia psittaci, chlamydia felis, and chlamydia abortus using a conventional pcr assay (31) , and virus isolation was performed using inoculated feline pulmonary cells to test for feline herpesvirus and feline calicivirus. all assays have been adapted and optimized and are used routinely in feline infectious respiratory disease diagnostic testing. tracheal wash fluid cytology. direct smears of buoyant, flocculent material in the tracheal wash fluid and cytocentrifuge smears of the remaining fluid were prepared and stained with a romanoski stain (modified wright's stain) using an automated stainer (hema-tek 1000; siemens). the stained slides were examined using standard bright-field microscopy by a board-certified veterinary clinical pathologist (cornell ahdc). in situ hybridization for sars-cov-2 rna. unstained cytologic smears of tracheal wash fluid from tiger 1 were fixed in ice-cold 100% methanol for 20 min and stored at ϫ80°c until shipped to uiuc college of veterinary medicine zoological pathology program (zpp) on cold packs. upon arrival, the slides were submerged for an additional 30 min in 10% neutral buffered formalin at 4°c, air dried, and then placed in 100% ethyl alcohol for 5 min at room temperature. an in situ hybridization (ish) chromogenic manual assay was performed using the rnascope 2.5 hd detection kit red and a 20-pair oligonucleotide probe targeting the sars-cov-2 s gene of the wuhan hu-1 complete genome (nc_045512.2; advanced cell diagnostics catalog no. 848561) according to the manufacturer's directions (advanced cell diagnostics, inc., newark, ca). positive-control slides consisted of vero cells infected with sars-cov-2 isolated from tiger 1. a control probe targeting the dapb gene from the bacillus subtilis strain smy (advanced cell diagnostics catalog no. 310043) was used as a negative control on all cytology sections in parallel with the sars-cov-2 target probe (see fig. s1 ). additional negative controls to rule out cross-reactivity with tiger rna and the felid alphacoronavirus included a cytocentrifuge preparation of cell cultures infected with the alphacoronavirus feline enteric coronavirus (fecov) (kindly provided by gary whittaker, cornell university); formalin-fixed, paraffin-embedded (ffpe) unstained sections of normal malayan tiger trachea, lung, and oropharyngeal tissue; and lung, lymph node, and intestine from a fecov-positive domestic cat (fig. s1) . samples from the control tiger and domestic cat were collected opportunistically in 2014 and 2013, respectively, and archived as part of routine necropsy procedures (wildlife conservation society's [wcs's] bronx zoo and uiuc-zpp, respectively). virus isolation. virus isolation on respiratory and fecal samples was performed in vero (atcc ccl-81), vero e6, and vero 76 cells under biosafety level 3 conditions at the cornell ahdc and the national veterinary services laboratory (nvsl). cells were cultured in minimum essential medium eagle (mem-e; gibco, gaithersburg, md) supplemented with 2.5 to 10% fetal bovine serum (fbs; gibco), 100 iu/ml penicillin, and 100 g/ml streptomycin (growth medium). cells were seeded in 12-well culture plates or t25 flasks and cultured at 37°c with 5% co 2 for 24 to 48h. before inoculation, respiratory swabs and tracheal wash fluid samples were diluted at 1:10, 1:100, and 1:1,000 in serum-free mem-e containing 200 ui/ml penicillin, 200 g/ml streptomycin, and 2.5 g/ml amphotericin b (all from gibco); swabs from fecal samples were placed in 1 ml of sterile pbs supplemented with 2.5% bovine serum albumin (bsa) (sigma-aldrich, st. louis, mo) containing 200 ui/ml penicillin, 200 g/ml streptomycin, and 2.5 g/ml amphotericin b (all from gibco). cells were rinsed with mem-e and inoculated with 300 l of each respiratory sample dilution in individual wells of a 12-well plate or 1.5 ml of the diluted fecal sample in a t25 flask and adsorbed for 1 h at 37°c with 5% co 2 for 1 h. mock-inoculated cells were used as negative controls. after adsorption, replacement medium was added, and cells were incubated at 37°c with 5% co 2 and monitored daily for cytopathic effect (cpe) for 5 days. cell cultures with no cpe were frozen, thawed, and subjected to three blind passages with inoculation of fresh vero cell cultures with the lysates as described above. sars-cov-2 infection in cpe-positive cultures was confirmed with sars-cov-2specific rrt-pcr using the cdc n1 primer and probe set (sequences available upon request), an immunofluorescence assay using a mouse monoclonal antibody against the sars-cov n protein (32, 33) , and rnascope in situ hybridization as described above. virus neutralization assay. seroconversion of tiger 1 to sars-cov-2 was assessed by a virus neutralization assay (vn; cornell ahdc). twofold serial dilutions (1:8 to 1:4,096) of a serum sample collected on 2 april 2020 (6 days after the onset of clinical signs) were incubated with 100 50% tissue culture infective doses (tcid 50 ) of sars-cov-2 on vero cells for 1 h at 37°c. following incubation of serum and virus, 50 l of a cell suspension of vero ccl-81 cells was added to each well of a 96-well plate and incubated for 72 h at 37°c with 5% co 2 . virus cytopathic effect (cpe) was used as an indicator of virus infection/replication. neutralizing antibody titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited cpe. archived frozen sera from another tiger (archived frozen at cornell ahdc) and positive human control sera (deidentified convalescent human sera provided by cayuga medical center, irb protocol 0420ep) were included, and all samples were tested in triplicate with results averaged. a cell culture control was included in the assays, and the virus working dilution was back-titrated. sars-cov-2 rrt-pcr. nucleic acid was extracted from nasal and oropharyngeal swabs and tracheal wash fluid (tiger 1) using the magmax isolation kit (thermo fisher scientific, waltham, ma) and from fecal samples (tigers 1 to 5 and lions 1 to 3) using the magmax core nucleic acid purification kit (thermo fisher scientific, waltham, ma) and an automated nucleic acid extractor (king fisher flex purification system; thermo fisher scientific, waltham, ma) according to the manufacturer's instructions. analysis for sars-cov-2 rna was performed with real-time reverse transcriptase (rrt) pcr and either the 2019-ncov cdc qpcr probe assay targeting three regions of the nucleocapsid (n) gene (n1, n2, and/or n3; integrated dna technologies [idt], inc., coralville, ia) (uiuc-vdl, cornell ahdc) or the cdc qpcr probe assay, and in-house primers for the envelope (e) gene with the agpath-id one-step rt-pcr kit (applied biosystems, foster city, ca) (uiuc-vdl) (primer and probe sequences are available upon request). thermal cycler conditions consisted of reverse transcription and enzyme activation at 45 to 48°c for 10 min and 95°c for 10 min, respectively, followed by 40 to 45 cycles of 95°c for 3 to 15 s and 55 to 60°c for 45 s. positive (2019-ncov_n_positive control; idt, coralville, ia, and a synthesized plasmid [genscript] e gene control) and negative (distilled or nuclease-free water) controls, plus internal amplification controls (xeno or beta-actin; thermo fisher scientific, waltham, ma), were included as separate reaction mixtures. following initial rrt-pcr testing at both institutions, samples were submitted to the nvsl for confirmatory testing, using the 2019-ncov cdc qpcr probe assay and n1 and n2 primers (sequences available upon request). amplicon sequencing. minion and sanger amplicon sequencing was used to confirm rrt-pcr results at cornell ahdc and nvsl, respectively (primer sequences are available upon request). for minion-based sequencing, targets were amplified directly from tracheal wash or fecal samples using the superscript iv one-step rt-pcr system (thermo fisher scientific, waltham, ma). primers targeted the complete spike (s) gene (4,023 bp) and an internal region of the n gene (634 bp). universal oxford nanopore-compatible adapter sequences were added to the 5= end of each primer sequence to allow pcr-based barcoding. amplicons were purified (ampure xp beads [beckman coulter, brea, ca]; 1.6:1 volumetric bead-to-dna ratio), and dna quantification was performed on a qubit fluorometer 3.0 (double-stranded dna [dsdna] high-sensitivity assay kit; thermo fisher scientific, waltham, ma). samples were subsequently diluted to 0.5 nm in a total of 24 l and used as the input for the library preparation following the 1d pcr barcoding (96) genomic dna (sqk-lsk109) protocol (oxford nanopore technologies, oxford, uk). final dna libraries were loaded in a flo-min106 r9.4 flow cell to start the sequencing runs. sanger sequencing was performed using primers targeting partial regions of s, n, and rnadependent rna polymerase (rdrp) genes (primer sequences available upon request). amplicons were generated directly from nasal and oropharyngeal swabs and tracheal wash fluid using the superscript iii one-step rt-pcr system (thermo fisher scientific, waltham, ma). reaction mixtures were purified using the qiagen pcr purification kit (qiagen, germantown, md). dna was amplified for sanger sequencing using the bigdye terminator v 3.1 cycle sequencing kit (thermo fisher scientific, waltham, ma) and sequenced on the applied biosystems 3500xl genetic analyzer (thermo fisher scientific, waltham, ma). whole-genome sequencing and phylogenetic analysis. whole-genome sequencing (wgs) was performed on tracheal wash fluid and fecal specimens from all individual tigers and lions as previously described (34) . individual fecal samples from tigers 1 to 5 and lions 1 to 3 and cell culture viral isolates were subjected to sequencing with either minion-based amplicon sequencing using overlapping primers covering the full viral genome (amplicons with an average size of ϳ1,500 bp; primer sequences are available upon request) or the ion ampliseq kit for chef dl8 and ion ampliseq sars-cov-2 research panel (thermo fisher scientific, waltham, ma) (data sets 2 and 3). minion libraries were prepared as previously described (35) using the native barcode kit, exp-nbd104, ligation sequencing kit, sqk-sqk109 (oxford nanopore technologies), and sequenced on an r9.4 flow cell for 6 h. ion targeted libraries were sequenced using an ion 530 chip on the ion s5 system using the ion 510-ion 520-ion 530 kit (thermo fisher scientific, waltham, ma). viral isolates were sequenced with the minion or ion ampliseq approach as described above. whole-genome sequencing on the rrt-pcr-positive specimens from the two sars-cov-2-positive keepers was performed as previously described using an amplicon sequencing approach and sanger sequencing (36) (data set 4). all genomes for each animal that were assembled using data generated from different sequencing platforms (illumina, minion, and/or ion torrent) were combined into a single consensus sequence for each animal (data set 2). the assemblies for a given animal were aligned with the wuhan-hu-1 reference sequence (nc_045512.2) using mafft v. 7.453 (37) . the reference sequence was then removed from the alignment, and a consensus sequence for the virus sequence recovered from each animal was generated using the consambig program in emboss v. 6.6.0.0 (38) . when a single assembly shifted alignment due to a single base insertion or repeat nucleotide, the alignment was rerun after removal of the offending nucleotides. to compare the outbreak genomes to others isolated from humans in the same geographic region, all available sars-cov-2 genomes from new york were downloaded from ncbi on 23 april 2020. these were clustered at 99.99% identity using vsearch v. 2.14.2 (39) , and the consensus sequences from each cluster were aligned along with wuhan-hu-1 reference sequence, using mafft v. 7.453 (37) . a phylogenetic tree was constructed using the consensus sequence from each animal, keepers, sequences from new york, and the wuhan-hu-1 reference sequence (nc_045512.2) using the gtr-gamma model in raxml v. 8.2.12 (40) . haplotype network analysis. haplotype network analyses were conducted with two overlapping data sets using popart software (41) using the median joining algorithm (42) . data set 1 contained nine genomes generated from the bronx zoo cases: four tigers, three lions, and two tiger keepers. data set 2 contained the nine genomes from data set 1, as well as 500 additional genomes (including the sars-cov-2 reference wuhan-hu genome) generated from a total of 53 countries to better understand genetic relatedness of bronx zoo cases in the context of the global pandemic. the top 10 blast results for the lion sequences were included in data set 2. for both data sets, the entire genome alignment was examined visually for accuracy and evidence of large-scale rearrangements to rule out the likelihood of multiple single-nucleotide polymorphisms (snps) being the result of a single evolutionary event. subsequently, whole-genome alignments were converted into an snp matrix by removing columns containing identical bases, gaps, and ambiguous bases. the lengths of final snp matrices were 25 nucleotides (nt) (data set 1) and 567 nt (data set 2). data availability. primer and probe sequence information for rrt-pcr and amplicon and wholegenome sequencing is available upon request. all remaining data are available in the main text or the supplemental material or as follows: data set 1, metagenomics data obtained from respiratory specimens from tiger 1 have been deposited in srr11587605 under prjna627354; data set 2, whole-genome consensus sequences obtained from sars-cov-2 detected in fecal samples from tigers 2 to 4 and lions 1 to 3 have been deposited in genbank under accession numbers mt704313, mt704315, mt704316, mt704312, mt704310, and mt704311, respectively; data set 3, whole-genome sequences obtained from sars-cov-2 tiger and lion isolates (tgr1/ny/20, tgr1/ny/20, and ln2/ny/20) have been deposited in genbank under accession numbers mt704317, mt704314, and mt747978, respectively; data set 4, whole-genome sequences obtained from sars-cov-2 strains in keepers 1 and 2 have been deposited in genbank under accession numbers mt703883 and mt703884, respectively. supplemental material is available online only. the emergence of sars, mers and novel sars-2 coronaviruses in the 21st century the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 china novel coronavirus investigating and research team. 2020. a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov-2 full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic pathways to zoonotic spillover host range and emerging and reemerging pathogens risk factors for human disease emergence reverse zoonotic disease transmission (zooanthroponosis): a systematic review of seldomdocumented human biological threats to animals infection of dogs with sars-cov-2 first reported cases of sars-cov-2 infection in companion animals sars-cov-2 infection in farmed minks, the netherlands susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 infection and rapid transmission of sars-cov-2 in ferrets sars-cov-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study introductions and early spread of sars-cov-2 in spike protein recognition of mammalian ace2 predicts the host range and an optimized ace2 for sars-cov-2 infection sars-cov-2 neutralizing serum antibodies in cats: a serological investigation presymptomatic transmission of sars-cov-2 -singapore one health proof of concept: bringing a transdisciplinary approach to surveillance for zoonotic viruses at the human-wild animal interface sustainable development must account for pandemic risk host and viral traits predict zoonotic spillover from mammals factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, chlamydophila felis and bordetella bronchiseptica in cats: experience from 218 european catteries design and performance of the cdc real-time reverse transcriptase pcr swine flu panel for detection of 2009 a (h1n1) pandemic influenza virus characterization of a novel mycoplasma cynos real-time pcr assay development and evaluation of a real-time polymerase chain reaction method for the detection of mycoplasma felis detection of canine pneumovirus in dogs with canine infectious respiratory disease real-time pcr for detection and differentiation of streptococcus equi subsp. equi and streptococcus equi subsp. zooepidemicus detection of chlamydia psittaci dna in avian clinical samples by polymerase chain reaction intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells production and characterization of monoclonal antibodies against the nucleocapsid protein of sars-cov complete genome sequence of sars-cov-2 in a tiger from a u ncov-2019 sequencing protocol rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome virus coronavirus 2 mafft multiple sequence alignment software version 7: improvements in performance and usability emboss: the european molecular biology open software suite vsearch: a versatile open source tool for metagenomics raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies popart: full-feature software for haplotype network construction median-joining networks for inferring intraspecific phylogenies we declare no competing interests. this manuscript represents the opinions of the authors and does not necessarily reflect the position of the u.s. centers for disease control and prevention. key: cord-275313-mfyff9ne authors: modjarrad, kayvon title: treatment strategies for middle east respiratory syndrome coronavirus date: 2016-01-01 journal: journal of virus eradication doi: nan sha: doc_id: 275313 cord_uid: mfyff9ne middle east respiratory syndrome coronavirus (mers-cov), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than 1600 people, resulting in almost 600 deaths. the high case fatality rate, growing geographic distribution and vaguely defined epidemiology of this novel pathogen have created an urgent need for effective public health countermeasures, including safe and effective treatment strategies. despite the relatively few numbers of cases to date, research and development of mers-cov therapeutic candidates is advancing quickly. this review surveys the landscape of these efforts and assesses their potential for use in affected populations. respiratory tract infections are the leading cause of mortality in resource-limited settings, accounting for more than 4 million deaths each year globally [1] . epidemic-and pandemic-prone respiratory viruses are the aetiological pathogens in many cases, and have caused several of the most prominent infectious disease outbreaks of the past two decades: these include h5n1 influenza in 1997, severe acute respiratory syndrome (sars) in 2003 and pandemic h1n1 influenza in 2009. most recently, middle east respiratory syndrome coronavirus (mers-cov) has emerged as a novel cause of severe acute respiratory illness after first being identified in a saudi arabian patient in 2012 [2] . although initially restricted to the arabian peninsula, this emerging pathogen has respectively infected and killed more than 1600 and 580 people on four continents across 26 countries [3, 4] . phylogenetically related to sars-cov [5] , mers-cov has a similar clinical presentation [6] [7] [8] [9] , albeit with a higher case fatality rate (~40% versus 10%) [3] [4] [5] . dromedary camels serve as the principal animal reservoir for this virus; and zoonotic spillover from dromedaries to humans has, thus far, driven the course of the epidemic [10] [11] [12] [13] [14] [15] [16] [17] [18] . although humanto-human transmission has been documented -particularly in the context of nosocomial outbreaks [19] [20] [21] [22] [23] [24] -the spread of mers-cov is inefficient and unsustained, as reflected in an estimated reproduction rate of no higher than 0.7 [25, 26] . mers-cov is an enveloped, single-stranded, positive-sense rna virus that comprises a 30-kilobase genome that codes for four structural proteins and an rna polymerase [27] , typical of the coronaviridae family ( figure 1 ). the most immunogenic of these proteins is the virus' only surface glycoprotein, spike (s) [28] [29] [30] that mediates viral attachment and fusion via the host cognate receptor, dipeptidyl peptidase 4 (dpp4) [31] . although the broad principles of the virus' life cycle and its mechanisms of pathogenesis are beginning to be understood, this knowledge has not yet translated to a licensed therapy or vaccine. much of the work to develop safe and effective mers-cov countermeasures has centred on vaccines, but the relatively low prevalence of the disease, the sporadic nature of the case clusters and the dearth of detailed knowledge on chains of transmission highlight the need for greater investments into the discovery of effective therapeutic and secondary prophylactic regimens for infected and exposed individuals. efforts to research and develop treatment strategies for mers-cov are accelerating but remain limited in their scope and stage of advancement. there are few novel compounds being studied that are specific for mers-cov molecular targets, as most treatment options, investigational and licensed, are being repurposed from their use for other rna viruses or other non-infectious diseases. the current landscape of mers-cov therapies, therefore, is dominated by an armamentarium of repositioned drugs with in vitro activity against mers-cov replication, but is also speckled with agents that are directed towards and derived from host immunity. the current review surveys the landscape of therapeutic products in each category and assesses their potential for advanced testing and development. respiratory and circulatory support, preservation of renal, hepatic and neurological function, and prevention of secondary infections. beyond implementing basic principles of critical care medicine, immune-based therapies have been used most commonly during both the sars-cov pandemic of 2003 and the current mers-cov epidemic, each time yielding equivocal results. there have been some promising animal data where combination treatment with ribavirin and interferon (ifn)-α2b improved clinical outcomes in mers-cov-infected non-human primates (nhps). however, treatment was initiated very soon after viral challenge (~8 hours), a window that is unlikely to be replicated in a real-world clinical setting [32] . various ifn regimens, in combination with ribavirin, have been intermittently administered to severely ill patients, although typically in an ad hoc manner and in the absence of systematic evaluation [33] [34] [35] [36] [37] . individual case reports and uncontrolled case series not only limit determination of whether an intervention works but if it is safe as well. ribavirin, for example, is a potent nucleoside analogue that has been used with varying measures of success against a range of rna viruses [38] . however, patients can experience significant toxicities when given the drug alone or in combination with an interferon, including but not limited to haemolytic anaemia and metabolic abnormalities. interferons also can elicit systemic adverse effects, psychiatric disturbances and neutropenia [39] . thus, without the benefit of randomised controlled trial data, it becomes difficult to assess whether the treatment is worse than the disease. certain strategies, however, have been shown to worsen clinical outcomes in the setting of a coronavirus infection. for example, studies during the sars pandemic showed that corticosteroids, when used early on sars-cov infected patients, significantly increased viral load, icu admission and mortality [40, 41] . the role for interferon therapies has been less clear in the current mers-cov epidemic, as some data show a positive impact on proximate outcomes, such as oxygenation and inflammation, but no effect on more significant outcomes like hospital stay and long-term survival [35, 36, 42] . rapidly scaled treatments based on naturally occurring neutralising antibodies such as convalescent plasma or hyperimmune globulin, on the other hand, have been shown to be relatively safe and potentially effective for reducing mortality from several infections such as sars-cov and influenza [43] [44] [45] , and may hold promise for mers-cov as well. this strategy, however, relies on the rapid identification of cases and contacts and immediate deployment of products to have maximal impact. one study found that convalescent plasma decreased mortality in sars-cov patients only if administered within 14 days of illness [44] . a network for the use of convalescent plasma for case clusters of mers-cov is currently being assembled [43] to test its safety, efficacy and feasibility. however, actualisation of this plan is limited by logistical challenges, local technical capacity and donor supply. unfortunately, no host-derived experimental interventions have yet demonstrated appreciable benefit in acutely ill, mers-covinfected patients in a consistent or controlled manner. this reality, although, has not slowed down the discovery and advancement of passive prophylactic products derived from vaccinated and infected animals and humans. despite intensive efforts to develop a mers-cov vaccine, the prevalence and transmissibility of this emerging pathogen are both relatively low [3, 26] , making it difficult to define a target population for vaccination. mabs, on the other hand, can be administered in the setting of an outbreak without the need to discriminate who might be at greatest risk for infection. they can be used to treat cases early in their natural history and for post-exposure prophylaxis of case contacts. mabs also carry the benefits of higher potency, greater specificity, more extensive pre-licensing evaluation and consequently a more vetted safety profile. additionally, mabs can help define immunogenic epitopes through crystallographic analysis, thereby providing atomic-level detail for the design of better immunogens. they also have been proven as effective therapies in the areas of cancer treatment and autoimmune disease management. although there is only one pathogen, respiratory syncytial virus, for which a mab is licensed for use, there are a number of other infectious disease indications-such as ebola virus disease treatment and human immunodeficiency virus primary and secondary prevention-for which mabs are being tested in advanced phase clinical trials (www.clinicaltrials.gov). despite all of these advantages, the timelines and costs of mab research and development (r&d) are respectively longer and higher than that for polyclonal antibody preparations. in spite of the requirements for greater upfront investments and a more rigorous testing and approval process, several groups have identified highly potent mers-cov mabs and are advancing them through preclinical stages of development (table 1) . some have been isolated from immunised animals (mice/humanised mice/nhps) [46] [47] [48] [49] [50] [51] [52] [53] [54] , while others have been identified from either an antibody human phage library [55] or memory b cells of infected and recovered human survivors [56] . almost all of the published mabs and all of those in development target the s receptor-binding domain (rbd), which contains the most immunogenic epitopes on the virus. many bind to the rbd, expressed both on a recombinant s and on the surface of live virus, with picomolar affinity and neutralise mers-cov at a half maximal inhibitory concentration (ic 50) of 10 ng/μl or less. additionally, several groups have demonstrated new york blood center mersmab1 s1 imunised mouse rbd in vitro [46] nih national cancer institute m336, m337, m338 human antibody library rbd in vitro [52] nih niaid d12, f11, g2, g4 s/s1 immunised mouse rbd, s1, s2 nhp efficacy [51] regeneron regn3048/regn3051 humanised mouse rbd mouse/nhp efficacy [49] tsinghua university mers-4, mers-27 human antibody library rbd in vitro [47] rbd: receptor binding domain; s: spike glycoprotein; s1: spike domain containing rbd; s2: spike domain containing fusion machinery. journal of virus eradication 2016; 2: 1-4 protective efficacy in pre-and post-exposure prophylaxis animal models. because most of the antibodies target the rbd, there is a potential for viral escape from any one mab. thus, there may be a need to develop antibodies against other vulnerable sites on s or to investigate the use of combination mabs to overcome the potential emergence of therapeutic resistance. it is likely that mabs directed at other sites on the s glycoprotein have already been recovered but are not as potent neutralisers, as is the case in one report [51] . a more efficient search for potent neutralising antibodies that target epitopes outside the rbd could be facilitated by a more detailed understanding of the atomic-level structure of the entire s glycoprotein, as has already been resolved for the rbd. the successes thus far in isolating potent and protective mabs, although significant, are likely to be tempered by the challenges of advancing these products to licensing and full-scale production at affordable costs for as of yet undefined populations in a relatively short timeframe. thus, mabs should be advanced along a development pipeline in parallel with a program of rational drug design and discovery. although intensive, supportive care still serves as the primary treatment option for mers-cov and mabs are the focus of the most advanced r&d efforts, antiviral therapies are being actively investigated for use in severely ill patients. there are two main pathways for drug discovery: de novo development and repurposing licensed medications. there are few new antivirals for mers-cov; however, the ebola epidemic has had an unanticipated consequence of facilitating their development. one in particular, gs-5734 developed by gilead sciences, is an adenine analogue that is incorporated into viral rna to disrupt replication [57] . it has shown survival benefit in nhps inoculated with ebola virus and is now advancing through a phase i dose escalation trial. it has been claimed to have in vitro activity against mers-cov as well, but publication of these data is pending. similarly, bcx4430 is a nucleoside analogue that is being developed by biocryst pharmaceuticals for potential treatment of filoviruses, coronaviruses and other rna viruses [58] . additionally, small interfering rna molecules and peptide inhibitors are being investigated for their ability to disrupt mers-cov replication, although these products are still in very early phases of investigation [59, 60] . as the life cycle and genetic sequence of this new coronavirus has become better elucidated, the rational design and development of novel and approved agents with potent antiviral activity have become possible. the advent of high-throughput screens of licensed compounds and small molecules has also allowed researchers to efficiently evaluate large libraries of drugs for their in vitro antiviral activity against novel targets [61] [62] [63] [64] [65] [66] . to date, several dozen licensed drugs have been reported to inhibit mers-cov replication. using slightly different screening technologies, different groups have converged on some common classes of compounds, including nucleoside analogues, antibacterial protein synthesis inhibitors, kinase signalling modifiers, antimetabolites and antiprotozoal agents. mycophenolic acid, an inhibitor of both t an b lymphocytes, has also been found to have strong activity against mers-cov, as it does against other rna viruses such as west nile, hepatitis c and dengue [63] . only one of the drugs to show in vitro activity against mers-cov, lopinavir, however, has been tested in an animal model. mers-cov-challenged marmosets that were treated with this protease inhibitor had better clinical, pathological, virological and radiological outcomes than controls or those treated with mycophenolate mofetil [67] . additionally, two peptides, hr1p and hr2p are being developed as potential fusion inhibitors [59] . by acting on the six-helix bundle core of the mers-cov s protein to prevent protein-mediated cell-to-cell fusion, this class of compounds may hold potential beyond mers-cov towards a long-term objective of a pan-coronavirus antiviral. given some of the common life cycles and pathways of pathogenesis for rna viruses and homologies in protein structures across different coronaviruses, there may be economies of effort and investment in developing antivirals that have activity against more than one virus or family of viruses. irrespective of the breadth of these novel or repurposed compounds, treatment studies should be carried out prospectively according to protocols that plan for the collection of quality controlled data and serial biological sampling to assess viral evolution and biomarkers of favourable clinical outcomes. recent infectious disease outbreaks such as the 2009 h1n1 influenza pandemic, the h7n9 influenza epidemic in china, the ebola crisis in west africa and now the mers-cov outbreak have highlighted the need for better r&d preparedness and improved coordination of clinical testing in the face of the accelerating number of emerging and re-emerging infectious diseases. the ability to have an armamentarium of countermeasures and clinical trial infrastructure in the early phases of an outbreak is critical for mounting an effective public health campaign. for example, the sars-cov pandemic caused more than 8000 cases of severe acute respiratory illness and nearly 900 deaths but few prospective, controlled studies were undertaken to determine the optimal management of the disease. consequently, treatment options for sars-cov were never defined clearly and thus difficult to adapt for mers-cov. although global coordination has resulted in the advancement of some urgently needed, novel countermeasures for mers-cov, they will have to be developed along faster timelines than before, with greater investments earlier in the preclinical development pipeline that can generate products for more timely efficacy testing in affected populations. as the global community takes lessons from the most recent outbreak and prepares for the potential of another regional epidemic or broader pandemic, stakeholders in mers-cov r&d must 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broad-spectrum filovirus inhibitor that providescomplete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates id week biocryst announces nature publication demonstrating efficacy of bcx4430 in a non-human primate model of filovirus infection structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus design of potential rnai (mirna and sirna) molecules for middle east respiratory syndrome coronavirus (mers-cov) gene silencing by computational method antiviral drugs specific for coronaviruses in preclinical development a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors alternative screening approaches for discovery of middle east respiratory syndrome coronavirus inhibitors treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset the opinions expressed herein are those of the authors and should not be construed as official or representing the views of the us department of defense or the department of the army. key: cord-301693-3hsu2u1k authors: he, yuwen; luo, jiangyan; yang, jie; song, jinlong; wei, li; ma, weifeng title: value of viral nucleic acid in sputum and feces and specific igm/igg in serum for the diagnosis of coronavirus disease 2019 date: 2020-08-06 journal: front cell infect microbiol doi: 10.3389/fcimb.2020.00445 sha: doc_id: 301693 cord_uid: 3hsu2u1k a new type of coronavirus-induced pneumonia eventually termed “coronavirus disease 2019” (covid-19) was diagnosed in patients in wuhan (hubei province, china) in december 2019, and soon spread worldwide. to improve the detection rate of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), we analyzed the results of viral nucleic acid and serum-specific antibody tests on clinical samples from 20 patients with sars-cov-2 infection diagnosed at the first affiliated hospital of guangzhou medical university in china. by comparing various sample types collected from covid-19 patients, we revealed multiple pathways for sars-cov-2 shedding, and a prolonged detectable period for viral nucleic acid test in sputum specimens, demonstrating that the timeline of the viral shedding is of great value in determining the time of release from quarantine or discharge from hospital. we also recommend for the application of serological test to assist in confirming sars-cov-2 infection judged by viral nucleic acid test, especially when covid-19-related symptoms have appeared and the viral nucleic acid test was negative. our findings are critical for the diagnosis of sars-cov-2 infection and for determining deadline of restriction measures to prevent transmission caused by convalescent patients with covid-19. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), identified in wuhan, china at the end of 2019, spread rapidly worldwide. in order to diagnose a large number of patients, samples of lower respiratory tract such as sputum with high positive rate are generally collected for viral nucleic acid detection (han et al., 2020; qu et al., 2020) . in addition, some studies have reported the presence of viruses in feces wu et al., 2020) , implying the risk of fecal-oral transmission, and indicating that specimen collection should not be limited to respiratory samples. common symptoms at onset of illness were fever (98%), cough (76%), myalgia, or fatigue (44%) . however, it is worth noting that carriers who are subclinical can also infect people (rothe et al., 2020) . therefore, it is necessary to deliver viral nucleic acid and serological tests for people with a history of exposure to sars-cov-2. we undertook a study on the viral nucleic acids of sars-cov-2 in swabs (nasal, pharyngeal), sputum and feces, as well as antibodies in the serum of covid-19 patients admitted to the first affiliated hospital of guangzhou medical university, china. we aimed to clarify the importance of the test results of different specimen types for the diagnosis and deisolation of patients with coronavirus disease 2019 . the study protocol was approved by the medical ethics committee of the first affiliated hospital of guangzhou medical university (guangzhou, china). written informed consent was obtained from all study participants. from 29 january to 6 june 2020, samples (swabs, feces, sputum, and blood) of 20 covid-19 patients were collected from the first affiliated hospital of guangzhou medical university, a designated hospital for the treatment of critically ill patients with covid-19 in guangzhou, a first-tier city in china. by the way, there were only 20 patients in this hospital who were almost critically ill or severe, other mild or asymptomatic patients were not admitted. the subjects of our study were all covid-19 patients admitted to our hospital, and there were no additional inclusion or exclusion criteria. after admission, the patient was tested for viral nucleic acid continuously for a week and then every 2-3 days. antibodies have been tested every 2-3 days since 15 february. to improve the efficiency of clinical work, collection of long-lasting negative specimens was suspended, while positive specimens were continuously collected until discharge. in the first affiliated hospital of guangzhou medical university, extraction of nucleic acids from the respiratory and fecal samples was performed with the commercialized nucleic acid extraction kits (magnetic bead method, daan gene co., ltd. of sun yat-sen university, guangzhou, china). the operations were completely following the instruction of the kit. the blood specimen was centrifuged with 3,000 rpm for 15 min to separate the serum within 24 h after collection, and then inactivated at 56 • c for 30 min and stored at 4 • c until use. rna was detected using the new coronavirus 2019-ncov nucleic acid detection kit (daan gene co., ltd. of sun yatsen university, guangzhou, china). the analytical sensitivity of our kit was 500 copies/ml. this kit had no cross-reaction with other pathogens similar to sars-cov-2 or causing similar symptoms. exogenous substances such as covid-19 therapeutic drugs and endogenous substances such as blood and mucus in specimens did not interfere with the detection results of the kit. the primer and probe sequences designed for the open reading frame (orf1ab), nucleoprotein (n) gene regions of sars-cov-2 are shown in table 1 . nc (orf1ab/n) pcr reaction solution a and solution b were oscillated thoroughly after melting at room temperature followed by 8,000 rpm centrifugation for several seconds. next, 17 µl of nc (orf1ab/n) pcr reaction solution a and 3 µl of nc (orf1ab/n) pcr reaction solution b were fully mixed and centrifuged briefly to bring down all the liquid to the bottom of the tube. each pcr reaction tube was added with 20 µl of the above amplification system followed by adding 5 µl of the negative quality control (qc) substance, the viral nucleic acid of the sample to be tested, or positive qc substance. it was a one-step rt-pcr. the total pcr reaction volume was 25 µl. the volume of the template was 5 µl. the concentration of primers and probes was 1 µm and 10 µm in the final pcr reaction volume respectively. these pcr reaction tubes were centrifuged (8,000 rpm) for a few seconds at room temperature and placed in an rt-pcr instrument (abi prism 7500; applied biosystems, foster city, ca, usa) for amplification and detection. the amplification process was as follows: 50 • c for 15 min, and 95 • c for 15 min, then 45 cycles were performed, including 94 • c for 15 s, 55 • c for 45 s, and the default melting curve steps of the rt-pcr instrument. if the ct value of the tested sample was < 40 in the fam and vic channels (fam and vic are fluorescent dyes), and there was an obvious amplification curve, the sample was judged to be positive for sars-cov-2. our serological test used for sars-cov-2 specific igm/igg antibodies was a rapid detection method. the detection principle of the igm/igg antibody detection kit (livzon diagnostics co., ltd, zhuhai, china) for sars-cov-2 is based on colloidal gold immunochromatography. the antibody detection kit was proved that there was no cross-reaction to the kit in the detection of igm/igg-positive samples of similar or other viruses. it has been experimentally verified that a variety of exogenous and endogenous substances have not interfered with our antibody detection kit, and the presence of the sars-cov-2 igg antibody does not affect the detection of sars-cov-2 igm antibody and vice versa. the clinical test results of this kit showed that the detection sensitivity of igm was 79.0% and the specificity was 99.7%; the detection sensitivity of igg was 84.3% and the specificity was 99.4%; the combined detection sensitivity of igm and igg was 90.6% and the specificity was 99.2%. serum (10 µl) was added to the sample well of the igm and igg detection cards. then, two drops (∼100 µl) of sample diluent were added vertically. if the detection line and qc line appeared within 15 min, then the sample was judged to be positive. data processing was carried out using spss v22.0 (ibm, armonk, ny, usa). the differences between samples or individuals were analyzed by anova of randomized block design data and bonferroni multiple comparison. data are presented as mean ± sd. time from onset to admission and hospital stay are presented as median (iqr). anova of randomized block design data with 2-sided p < 0.05 was considered significant. multiple comparison with 2-sided p < 0.0083 was considered significant. a total of 22 patients were diagnosed as covid-19 in our hospital, of which two (patients #19, #20) were transferred the next day after hospital admission. the median time from onset to admission to our hospital for 20 patients was 9.5 (7.5-14.0), some patients had been treated elsewhere during this period. primer sequences probe sequences forward reverse and the median hospital stay was 63.5 (28.0-93.8) days. in fact, a longer time from onset to admission may result in more serious symptoms or organic damages, thereby increasing the difficulty of clinical cure (qi et al., 2020) . of 20 patients with covid-19, the mean age were 57.35 years old. the ratio of males: females was 14:6. according to the clinical diagnosis, the ratio of patients with critical: severe: mild disease was 15:2:3. the clinical characteristics were summarized in ). in addition, 17 (85%) patients had comorbidities, including 14 (70%) acute respiratory distress syndrome, 9 (45%) myocardial damage, and 8 (40%) hypertension, etc. specimens were collected from these 20 patients admitted to the hospital since january 29. as of may 10, all 20 patients had turned negative for viral nucleic acid. the discharge criteria include: (i) body temperature returned to normal for more than 3 days; (ii) respiratory symptoms were significantly improved; (iii) chest images showed that acute exudative lesions were significantly improved; (iv) viral nucleic acid tests were negative in sputum, nasopharyngeal swabs, and feces samples for two consecutive times (at intervals of more than 24 h). seventeen patients had been discharged from the hospital on june 7, while three patients (patients #8, #13, #22) had not been discharged so far because of other underlying diseases. among the 20 tested patients, the time-dependent diagnostic results of sars-cov-2 rna in throat swabs, nasal swabs, sputum, and feces were summarized in figure 1 , as well as serum specific igm/igg antibodies. when all types of specimens turned negative, the symptoms of most patients had completely disappeared or were alleviated. sputum samples were positive in 19 of the 20 (95%) patients with covid-19. in other words, of all the sample types tested, the detection rate of the viral nucleic acids of sars-cov-2 was highest in sputum samples. interestingly, sars-cov-2 nucleic acid tests in nasal swabs, throat swabs and feces for a small number of patients (patients #10, #14, #17, and #21) were invariably negative from diagnosis to discharge but lasted for 94, 29, 41, and 4 days in sputum, respectively. moreover, sputum samples remained positive for an average of 42.8 ± 4.2 (mean ± sd) days since diagnosis. the comparison of the viruscarrying duration showed that the persistence of sars-cov-2 rna in sputum stayed significantly longer than that in throat swabs, nasal swabs, and feces (figure 2) , which prolonged by 32.0, 24.0, and 20.6 days, respectively. among them, patient #5 even continued to be positive for 99 days. in short, sputum has a high detection rate of viral nucleic acid and a long-term continuous positive. the feces samples remained sars-cov-2 rna positive for 22.3 ± 29.8 (mean ± sd) days since diagnosis, of which 11 patients (55%) were positive. although the existence time of virus in feces was not significantly different from that in nasal or throat swabs (figure 2) , it is worth noting that patients #2 and #8 presented consecutively positive in feces samples for 24.0 and 29.0 days, respectively after the nasal-swab, throat-swab, and sputum samples consistently became negative. moreover, patients #5 with the longest positive duration in the sputum had a longer feces duration, which took 103 days before it turned negative. although the detection of virus nucleic acid based on rt-pcr has high sensitivity, it is inevitable to produce false-negative results according to our experience and other references . the potential problem of a high false-negative rate caused by the nucleic-acid test prompted us to increase the detection of igm and igg antibodies against sars-cov-2 from 15 february 2020. these specific antibodies against sars-cov-2 were successfully detected in all of 20 patients (100%) (figure 1) , with the exception of two patients (patients #19, #20) who were not tested. otherwise, a few cases such as patient #16 would likely be considered to be sars-cov-2-negative through routine rna testing and, thus, pose a threat to other people. initially, we observed that patient #16 presented fever and diarrhea, and chest x-ray/ct showed unilateral pulmonary infection. epidemiological investigation showed a history of passing through the epidemic area and similar symptoms appear on her spouse. for this patient, sars-cov-2 rnas in all kinds of samples were negative while sars-cov-2 igg and igm remained positive. in view of the above circumstances, we finally took igm/igg positive as the diagnostic criteria to include this patient. in a word, we found that the combined detection of viral nucleic figure 2 | the number of days it took for sars-cov-2 rna to shed in four types of sample (throat swab, nasal swab, sputum, and feces). *p < 0.00833, **p < 0.00167, ***p <0.00017. acid and specific antibody successfully improved the detection rate of sars-cov-2. in this study, we examined the clinical biological samples of all covid-19 patients in the first affiliated hospital of guangzhou medical university, most of them were critical or severe patients. compared with mild patients (wölfel et al., 2020) , our specimens usually have a longer time for virus shedding, and critically ill patients have a longer period of treatment and observation, which provides a reference for the discharge and isolation time of critically ill patients. secondly, since covid-19 was first prevalent in china, we successfully demonstrated the whole process from diagnosis to discharge, while other areas may have not detected until the endpoints of some critically ill patients from the beginning of the epidemic to the present. one limitation is that the sample size is relatively small, and more data on critically ill patients need to be collected in the future to further confirm our results. we also call for a follow-up of discharged patients to observe the prognosis or re-positive conditions, although we failed to collect these useful data in time. our results show that viral nucleic acid has a high detection rate and a significantly long existence time in the sputum from covid-19 patients. research by qu et al. (2020) also showed that sars-cov-2 rna is still detectable in sputum obtained by atomization from cured patients, although the pharynx swab test is negative. in addition, the viral load in the sputum is the highest compared to other specimens in the later stage of covid-19 (yoon et al., 2020) . therefore, it is necessary to collect lower respiratory tract specimens for sars-cov-2 rna testing before covid-19 patients can be discharged. taken together, these results indicate that the sputum test was a more reliable criterion for discharge from hospital or quarantine release testing from a specimen taken from the upper airways. moreover, since some patients (10%, such as patients #2 and #8) have positive feces longer than sputum, we recommend that the combined detection of feces and sputum is more reliable. our data suggest that the presence of sars-cov-2 rna in feces may be prolonged for more than a month in exceptional cases after a negative result in nasal-swab, throat-swab, and sputum samples. this observation is similar to that of wu et al. (2020) , who found that the positivity of sars-cov-2 rna in fecal samples lagged behind that in respiratory samples. currently, although no cases of sars-cov-2 transmission through fecal-oral route have been reported, the potential risk of fecal-oral transmission may be increased in closed residential spaces and areas with poor sanitary conditions. for themselves, sars-cov-2 can actively replicate in human intestinal organs, and infectious viruses can be isolated from fecal samples of covid-19 patients . our results suggested that infected patients could potentially shed sars-cov-2 through respiratory and fecal-oral routes. therefore, we suggest that rt-pcr should be employed to routine diagnosis of fecal samples after removal of sars-cov-2 rna from the respiratory tract. a negative fecal result for sars-cov-2 nucleic acids in feces could be included in the rules for discharge from the hospital or lifting of quarantine measures as a supplement to sputum detection for patients recovering from covid-19. however, a negative result of viral nucleic acid cannot rule out the possibility of sars-cov-2 infection. we have further confirmed that combined detection of serum sars-cov-2 igm and igg was a practical and sensitive indicator, and also an effective complement to viral nucleic acid testing considering false-negative results upon it . in addition, the duration of igm/igg-positivity from serum samples was much longer than that of sars-cov-2 nucleic acids from clinical samples. serological test appears to be more meaningful for patients with an exposure history but who are negative for sars-cov-2 nucleic acids, regardless of whether the patients present symptoms or not. we speculate that serological test can effectively make up for the omission risk of viral nucleic acid detection, thus possessing important value in the timely diagnosis and prevention of covid-19. although nucleic acid test is difficult to avoid false negative, it can directly detect whether there is sars-cov-2 virus in human body, and its positive result is of great significance and is the gold standard for covid-19 diagnosis (shen et al., 2020) . compared with serological detection, nucleic acid detection can be applied in the early stage of sars-cov-2 infection, and indicate that it is now undergoing an infected state. however, in view of the high sensitivity of specific antibody detection, we suggest that it can be used as a supplementary indicator for nucleic acid detection, or even as a targeted remedy for leakage. in summary, sars-cov-2 has multiple shedding ways and a more prolonged survival period in sputum specimens from covid-19 patients. a comprehensive understanding of the viral shedding period in human body is extremely helpful to determine the time of release from quarantine or discharge from the hospital. we also recommend the application of a serological test to assist in identifying sars-cov-2 infection judged by viral nucleic acid test, especially when covid-19related symptoms have presented. since covid-19 has progress to one of the latest severe infectious diseases threatening human and restricting social activities worldwide, our findings are critical for the diagnosis of sars-cov-2 infection and the prevention of virus transmission in convalescent covid-19 patients. all datasets generated for this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by the first affiliated hospital of guangzhou medical university (guangzhou, china). the patients/participants provided their written informed consent to participate in this study. written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. sars-cov-2 rna more readily detected in induced sputum than in throat swabs of convalescent covid-19 patients clinical features of patients infected with 2019 novel coronavirus in wuhan false-negative results of real-time reverse-transcriptase polymerase chain reaction for severe acute respiratory syndrome coronavirus 2: role of deep-learning-based ct diagnosis and insights from two cases multicenter analysis of clinical characteristics and outcomes in patients with covid-19 who develop liver injury positive result of sars-cov-2 in sputum from a cured patient with covid-19 transmission of 2019-ncov infection from an asymptomatic contact in germany recent advances and perspectives of nucleic acid detection for coronavirus detection of novel coronavirus by rt-pcr in stool specimen from asymptomatic child virological assessment of hospitalized patients with covid-2019 prolonged presence of sars-cov-2 viral rna in faecal samples clinical significance of a high sars-cov-2 viral load in the saliva infection of bat and human intestinal organoids by sars-cov-2 wm and lw conceived and designed the study. js conducted the experiments. jl analyzed the data. yh and jy wrote the paper. all authors reviewed and edited the manuscript. all authors contributed to the article and approved the submitted version. this work was supported by the china national science foundation (grant no. 81503103). we thank the patients involved in the study. we acknowledge all healthcare workers involved in the diagnosis and treatment of patients with 2019 novel coronavirus disease in the first affiliated hospital of guangzhou medical university. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 he, luo, yang, song, wei and ma. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-290290-wyx9ib7s authors: sinegubova, maria v.; orlova, nadezhda a.; kovnir, sergey v.; dayanova, lutsia k.; vorobiev, ivan i title: high-level expression of the monomeric sars-cov-2 s protein rbd 320-537 in stably transfected cho cells by the eef1a1-based plasmid vector date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.04.368092 sha: doc_id: 290290 cord_uid: wyx9ib7s the spike (s) protein is one of the three proteins forming the coronaviruses’ viral envelope. the s protein of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has a spatial structure similar to the s proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (rbd), which is a significant inducer of host immune response. recombinant sars-cov-2 rbd is widely used as a highly specific minimal antigen for serological tests. correct exposure of antigenic determinants has a significant impact on the accuracy of such tests – the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native s protein. based on the previously developed p1.1 vector, containing the regulatory sequences of the eukaryotic translation elongation factor 1 alpha gene (eef1a1) from chinese hamster, we created two expression constructs encoding sars-cov-2 rbd with c-terminal c-myc and polyhistidine tags. rbdv1 contained a native viral signal peptide, rbdv2 – human tpa signal peptide. we transfected a cho dg44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. for the rbdv2 variant, a high-yield clonal producer cell line was obtained. we developed a simple purification scheme that consistently yielded up to 30 mg of rbd protein per liter of the simple shake flask cell culture. purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for rbdv2 protein, the monomeric form content exceeded 90% for several series. deglycosylation with pngase f and mass spectrometry confirmed the presence of n-glycosylation. the antigen produced by the described technique is suitable for serological tests and similar applications. humanity is faced with an unprecedented challenge -the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which causes a severe respiratory illness -coronavirus disease 2019 (covid19) pandemic. countries were sent to lockdown; people could not make informed decisions about the possibility of social contacts; the need for diagnostic tests is very high. existing tests for sars-cov2 are reviewed in [1] . at the beginning of the pandemic, pcr testing methods dominated since such test systems can be developed urgently, soon after the emergence of a new virus in the population. among the disadvantages of pcr-tests is a high sensitivity to contamination and dependence on sampling's correctness, a high proportion of falsepositive signals. unlike pcr diagnostics, serological testing gives positive results long after the event of infection, at least for several months. this testing method makes it possible to reliably determine whether a person is infected with the sars-cov-2, even in the absence of disease symptoms. we need serological tests, both in express format and screening tests based on elisa. serologic tests are also needed to detect convalescent plasma of therapeutic interest and assess emerging vaccines' effectiveness. in order for serological testing to have a more significant predictive value, mapping of the epitopes to which neutralizing antibodies appear should be carried out, as was done for sars-cov1 [2] , аnd convalescent or postvaccinal sera should be massively tested for the presence of neutralizing antibodies, for example, with a surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction [3] or another that can be carried out on a relatively large scale. the use of highly specific and high-affinity viral antigens is already a big step towards improving diagnostic accuracy. the immunodominant antigen of sars-cov2 is the rbd domain of the spike protein [4] . another antigen widely used for diagnostics -the nucleocapsid (n) protein -combines high sensitivity and low specificity; therefore, it needs accurate antigen mutagenesis to remove highly conserved areas without compromising affinity. cases are described for sars-cov when the results of testing with n-protein were clarified using two subunits of spike protein [5] . the coronaviruses' spike (s) protein forms large coronal-like protrusions on the virions surface, hence the name of the family coronaviridae. the s protein plays a crucial role in receptor recognition, cell membrane fusion, internalization of viruses, and their exit from the endosomes. it is described in detail in the review [6] . it consists of s1 and s2 subunits and, in the case of the sars-cov-2 virus, has 1260 amino acids [7] . the s protein is co-translationally incorporated into the rough endoplasmic reticulum (er) and is glycosylated by n-linked glycans. glycosylation is essential for proper folding and transport of the s protein. the s protein trimer is transported from the er. interacting with the m and e proteins s protein trimer is transported to the virus's assembly site. s protein is required for cell entry but not necessary for virus assembly [8] . during their intracellular processing, s proteins of many types of coronaviruses, including sars-cov-2 and mers-cov, but not sars-cov, undergo partial proteolytic degradation at the furin signal protease recognition site with the formation of two subunits s1 and s2. apparently, most of the s protein copies on the membrane of sars-cov-2 viral particles are trimers of s2 subunits that are incapable of interacting with the receptor. the full-length s protein trimer on the viral particle's surface also undergoes complex conformational rearrangements during the formation of the rbd-receptor complex and the virus's penetration into the cell. the s protein homotrimer binds to the ace2 dimer, detailed study of this interaction is available here [9] . as part of the trimer, the spike protein's monomer "moves its head" -the s1 subunit can form the open or closed conformation; that is, it can have a raised fragment or a lowered rbd domain, this can influence the affinity of antibodies targeted to it. the s protein of sars-cov-2 amino acid sequence is variable, with more than 200 and 18 relatively frequent s protein amino acid variations. a glycan shield is formed by n-linked glycans on the s protein surface, which is likely to help viral immune escape. in a comparative study of genome-wide sequencing data of natural isolates of sars-cov-2 [10] for the detected 228 variants of the s protein, all 22 potential nglycosylation sites within the s protein's ectodomain were completely conserved, which confirms the importance of each of these sites for maintaining the integrity of the s protein oligosaccharide envelope. it should be noted that not all of the 22 potential n-glycosylation sites are occupied, for s1 and s2 subunits, obtained from transiently transfected hek293 cells [11] n-glycosylation events were experimentally confirmed only for 17 out of 22 sites, also at least one o-glycosylation site was experimentally found inside the rbd-domain area of the s1 subunit with the mucin-like structures. non-vertebrate cells may be used to produce the s protein or its fragments; in this case, n-glycans are present mostly in the form of bulky high mannose or paucimannose structures, possibly blocking the interaction of antibodies with the folded s protein [12] . computational modeling of the glycan shield, performed for the hek293-derived s protein, revealed that in the case of human cells, around 40% of the protein's surface is effectively shielded from igg antibodies [13] . the use of full-length s protein for practical serological testing is nearly impossible due to its insolubility, caused by the presence of transmembrane domain. an artificial trimer of its ectodomain has been successfully used as an antigen in serological tests; however, such complex protein cannot be obtained in large quantities in mammalian cells, apparently due to the limitation on the folding of the trimerized abundantly glycosylated protein and subsequent difficulties in its isolation and purification. it is generally believed that the sars-cov-2 s protein receptor-binding domain is a minimal proteinaceous antigen, adequately resembling the immunogenicity of the whole spike protein. this domain contains only two occupied n-glycosylation sites [11] and 1-2 occupied o-glycosylation sites. it does not contribute to the trimer formation, and its surface is mostly unshielded. isolated rbd's of the s proteins of beta-coronaviruses were produced in various expression systems. bacterial expression of the rbd from mers-cov produced no soluble target protein, refolding attempts also were unsuccessful [14] . budding yeasts pichia pastoris were the suitable host for the secretion of mers-cov rbd with at least two (from three) n-linked glycosylation sites present. similar data were obtained for the rbd from sars-cov virus -removal of all n-glycosylation sites resulted in the sharp drop of protein secretion rate in the p. pastoris yeast, in the case of full rbd domain (residues 318-536), secretion of the unglycosylated target protein was stopped completely [15] . it may be proposed that the addition of n-glycans in these sites is needed for correct folding of the rbd in the er of eukaryotic cells. the sars-cov-2 s protein rbd, expressed in e.coli, also was detected only as inclusion bodies and was found to be unreactive even on blotting [16] . hyperglycosylated yeast-derived sars-cov-2 rbd was obtained in reasonable quantities (50 mg/l in bioreactor culture) by the p. pastoris expression system and successfully used for mice immunization [17] . unfortunately, yeast-derived glycosylated proteins contain immunogenic glycans and cannot be used for immune assays with human antibodies. similarly, sars-cov-2 rbd may be produced in the nicotiana benthamiana plant, resulting in non-vertebrate n-glycans addition, potentially reactive with human antibodies [18] . most early preprints and peer-reviewed articles describing the sars-cov-2 s protein and its rbd domain production methods were focused on transient transfection of hek293 cells [11] [19] and purification of small protein lots in a very short time. for example, d. stadlbauer [20] reports more than 20 mg/l target protein titer in transiently transfected hek-293 cells. simultaneously, the scalability of transiently transfected cell lines cultivation is still questionable, and gram quantities of rbd, needed for large scale in vitro diagnostic activity, may be produced only by stably transfected cell lines. previously we have developed the plasmid vector p1.1, containing large fragments of non-coding dna from the eef1a1 gene of the chinese hamster and fragment of the epstein-barr virus long terminal repeat concatemer [21] and employed it for unusually high-level expression of various proteins in cho cells, including blood clotting factors viii [22] , ix [23] , and heterodimeric follicle-stimulating hormone [24] . cho cells were successfully used for transient sars-cov rbd expression at 10 mg/l secretion level [25] . we have proposed that sars-cov-2 rbd, suitable for in vitro diagnostics use, may be expressed in large quantities by stably transfected cho cells, bearing the eef1a1-based plasmid. p1.1-tr2-rbdv1 construction. the rbd coding sequence was synthesized according to [26] . the dna fragment encoding the rbdv1 orf with kozak consensus sequence and c-terminal c-myc and 6xhis tags were obtained by pcr using primers ad-cov-absf and ad-rbd-myc6hnher (listed in table 1 the resulting ptm vector was sequenced as described above, available from addgene, plasmid # 162783. ptm-rbdv2 construction. rbd orf was amplified using adaptor primers ad-sfr2-nhef and ad-sfr2-xmar restricted by nhei and xmai (sibenzyme, novosibirsk, russia) and cloned into ptm vector, restricted by nhei and asigi (sibenzyme, novosibirsk, russia). the resulting construct was sequenced using sq-5ch6-f and sq-mych-r primers. ptm-rbdv2 is available from addgene, plasmid # 162785 plasmids for cell transfections were purified by the plasmid midiprep kit (evrogen, moscow, russia) and concentrated by ethanol precipitation in sterile conditions. the transgene copy number in the cho genome was determined by the quantitative real-time-pcr (qpcr) as described in [22, 24] . serial dilutions of p1.1-egfp [21] or pgem-rab1 plasmids were used for calibration curves generation. the weight of one cho haploid genome was taken as 3 pg, according to [27] . genomic chinese hamster ovary dg-44 cells (thermo fischer scientific) were cultured in the procho 5 medium (lonza, switzerland), supplemented by 4 mm glutamine, 4 mm alanyl-glutamine and hypoxanthinethymidine supplement (ht) (paneco, moscow, russia). cells were grown as a suspension culture in sterile 125 ml erlenmeyer flasks with vented caps, routinely passaged 3 to 4 days with centrifugation (300 g, 5 min) and seeding density 3-4*10 5 cells/ml. the 50-80 µg of each plasmid were precipitated by the addition of 96% ethanol and 3m sodium acetate, washed with 70% ethanol, dried, and resuspended in 100 µl of sterile r-buffer, neon transfection kit (thermo seeding cell culture was grown in 125 ml erlenmeyer shake flasks with 30 ml of lonza procho 5 medium, supplemented with 4 mm glutamine, 4 mm alanyl-glutamine and 2-8 µm mtx until cell concentration exceeds 1-1.5 mln cells/ml. cell suspension was transferred to four 250 ml erlenmeyer flasks, each containing 60 ml of culture medium, and grown to the same cell density. the entire cell suspension was transferred to a single 2 l erlenmeyer flask with 1 l culture medium, final seeding density 3-4*10 5 cells/ml. cells were cultured for three days, on the fourth day of culture, daily glucose measurements were started. glucose concentration in the cell supernatant was measured by the accutrend plus system (roche, switzerland); if glucose level was below 20 mm, it was added up to 50 mm as the sterile 45% solution. the culture in 2 l flask was grown for 6 to 8 days until the cell viability, measured by trypan blue exclusion, dropped below 50%. the clonal cell line was obtained by the limiting dilution method from the cell population, cultured in 8 µm mtx. methotrexate was omitted in the culture medium for two 3 d passage before cloning. cells were additionally split by 1:1 dilution 24 hours before the cloning procedure. cells were diluted in excell-cho (merck, germany) culture medium supplemented with 4 mm glutamine, 4 mm alanyl-glutamine, ht and 10% of untransfected cho dg 44 conditioned medium resulting in seeding density 0.5 cell/well, and the suspension was seeded into 96-well plates (200 μl/well). plates were left undisturbed for 14 days at 37°c, 5% co2 atmosphere. wells with single colonies were screened by microscopy; well grown colonies were detached by pipetting and transferred to the wells of 12-well plate, containing 4 ml of the excell-cho, supplemented as described above and grown for 7 days undisturbed. product titer was measured by elisa, as described below, 6 wells with highest rbdv2 titer were used for further cultivation. best-producing clonal cell lines were transferred to 125 ml erlenmeyer flasks with the procho 5 culture medium supplemented with 4 mm glutamine, 4 mm alanyl-glutamine and 8 µm mtx and after 5 days in suspension culture, the best producing clone was determined by measuring the product titer and cell concentration. sds-page was performed with the 12.5% acrylamide in the separating gel, in reducing conditions, if not stated otherwise, with the pageruler prestained marker, 5 µl/lane (thermofisher scientific). gels were stained by the colloidal coomassie blue according to [28] , scanned by the conventional flatbed scanner in the transparent mode as 16-bit grayscale images and analyzed by the totallab tl120 gel densitometry software (nonlinear dynamics, uk). sds-page was performed as described above, protein transfer, blocking, hybridization and color development were done according to [29] using nitrocellulose transfer membrane (gvs group, bologna, italy) and towbin buffer with methanol. primary anti-c-myc antibody (sci store, moscow, russia #psm103-100) was used at the 1:2000 dilution, anti-mouse-hrp conjugate (abcam, cambridge, uk, ab6789) was used at 1:2000 dilution; membrane was developed by the dab-metal substrate and scanned by the flatbed scanner in the reflection mode. multimeric forms of the rbd were quantified by size exclusion chromatography, utilizing waters extracts were vacuum-dried and redissolved in the 0.5% trifluoroacetic acid (tfa), 3% acn solution. prepared solutions were mixed at 3:1 ratio with 20% α-cyano-4-hydroxycinnamic acid (merck) solution in 20% acn, 0.5% tfa on the target plate. solutions of intact and deglycosylated proteins were passed through the ziptip c18 microcolumns (millipore), washed and eluted according to manufacturer protocol. one and a half µl of protein solutions were mixed on the target plate with 0.5 µl of the 20% 2,5-dihydroxybenzoic acid (merck) solution in 20% acn, 0.5% tfa. mass spectra were obtained by the maldi-tof mass spectrometer ultraflextreme peptides identification was performed by the gpmaw 4.0 software (lighthouse data, denmark) and by the mascot server (matrix science, boston, usa). glycopeptides mass assignment was performed by the glycomod online software tool [30] . sandwich elisa with anti-s protein antibodies was performed using a prototype of the sars-cov-2 antigen detection kit (xema co., ltd., moscow, russia, a generous gift of dr. yuri lebedin). pre-covid-19 normal human plasma sample (renam, moscow, russia) was used for preparation of the sars-cov-2 negative serum sample. serum samples of five patients with the pcr-confirmed sars-cov-2 infection were pooled for testing and one serum sample with the borderline igg titer level was tested separately. the blood sampling protocol conformed to the local hospital human ethics committee guidelines. antibody capture elisa with human serum samples was performed according to [29] at the 100 ng per well antigens load. antigens were applied on elisa 96-well plates (corning, usa) overnight at + 4oc, in pbs, the t-test was performed using the graphpad quickcalcs web site: https://www.graphpad.com/quickcalcs/ttest1.cfm (accessed november 2020). the native n-terminal signal peptide of sars-cov-2 s protein (amino acid sequence mfvflvllplvssq) was fused to the rbd sequence (319 -541, according to yp_009724390.1) and joined with a c-terminal c-myc epitope (eqkliseedl), short linker sequence, and hexahistidine tag. n-terminal part of the rbdv1 gene was constructed according to [26] , utilizing the optimized codon usage gene structure. c-terminal tags were not optimized for codon usage frequencies. the resulting synthetic gene was cloned into the p1.1-tr2 vector plasmid, a shortened derivative of the p1.1 plasmid [21] , and used for transfection of dhfr-deficient cho dg44 cells. the resulting expression plasmid p1.1-tr2-rbdv1 [genbank: mw187858] is shown on fig 1a. the stably transfected cell population was obtained by selection in the presence of 200 nm of dhfr inhibitor methotrexate, rbd titer 0.33 mg/l was detected for 3-days culture (fig. s1 ). one-step target gene amplification was performed by increasing the mtx concentration tenfold and maintaining the cell culture for 17 days until cell viability restored to more than 85%; the resulting polyclonal cell population could secrete up to 3,0 mg/l rbd in the 3-days culture. the target protein was purified by a single imac chromatography step, utilizing the ida-based resin chelating sepharose fast flow (cytiva), ni2+ ions, and step elution by increasing imidazole concentrations (fig 1b, fig 1c) . the resulting protein production method was found to be sub-optimal due to unexpectedly low secretion rate, signs of cellular toxicity of the target gene -33 h cell duplication time, maximal cell density in shake flask of 2.3 mln сells/ml (fig s2) , and unacceptable level of contaminant proteins co-eluting with the rbdv1. at the same time, the rbdv1 protein was stable in the culture medium during the extended batch cultivation of cells for at least 7 days (fig 1d) , making the long-term feed batch cultivations a viable option for its production in large quantities. we proposed that target protein secretion rate and its purity after one-step purification could be significantly improved by a simultaneous shift of the rbd domain boundaries, exchange of the sars-cov-2 s protein native signal peptide to the signal peptide of more abundantly expressed protein, two-step genome amplification and switch from ida-based resin to the nta-based one (fig 1e) . human tissue plasminogen activator signal peptide (htpa sp, amino acid sequence mdamkrglccvlllcgavfvsas) is commonly used for heterologous protein expression in mammalian cells. it was successfully used for the expression of sars-cov s protein in the form of dna vaccine [31] and envelope viral protein gp120 [32] . in the case of mers-cov s protein rbd -fc fusion protein, various heterologous signal peptides modulate target protein secretion rate by the factor of two [14] . corrected boundaries of the sars-cov-2 rbd were determined according to the cryo-em data [pdb id: 6vxx] [33] obtained for the trimeric sars-cov-2 s protein ectodomain. initially used 319 -541 coordinates, described in the [26] include one unpaired cys residue originated from the n-terminal part of the next domain sd1 (structural domain 1), so we excluded lys319 from the n-terminus of the mature rbd protein, aiming at the maximization of signal peptide processing, and removed c-terminal aminoacids c 538 vnf 541 , which form the structure of the sd1 domain. both linker areas surrounding the folded rbd domain core remain present in the rbdv2 protein (320 -537, according to yp_009724390.1). additionally, we redesigned c-terminal tags by introducing the pro residue immediately upstream of the c-myc tag, adding the short linker sequence sagg between the c-myc tag and polyhistidine tag, and extending the polyhistidine tag up to 10 residues. we expected this structure to expose the c-myc tag properly on the protein globule's surface and move the decahistidine tag away from possible masking negatively charged protein surface areas. we constructed an expression vector ptm [genbank: mw187855], where consensus kozak sequence, htpa sp and c-myc and 10-histidine tags are coded in the polylinker. rbd coding fragment was cloned in-frame, resulting ptm-rbdv2 expression plasmid [genbank: mw187856] is shown on fig.2a . cho dg44 cells were transfected by the ptm-rbdv2 plasmid, stably transfected cell population was established at the 200 nm mtx selection pressure. target protein titer was similar to the previous plasmid design -0.9 mg/l for 3-days culture, but after one step of the mtx-driven genome amplification, it increased eleven-fold to 9.7 mg/l at 2 µm mtx ( fig 2b) and then increased by a factor of 2.5 after second amplification step at 8 µm mtx, resulting titer was 24.6 mg/l for 3-days culture (fig 3a) . a steady increase of the target protein titer was detected for the extended batch cultivation of polyclonal cell population obtained at 8 µm mtx, peaking at 50 mg/l at 8 days of cultivation in the 2 l shake flask (fig 3d, 3e) . a similar ratio of product titer increase after multi-step mtx-driven genome amplification was described for the mers-cov rbd -40-fold increase after 9 steps of consecutive increments of mtx concentration, overall amplification period length was 60 days [14] . vector plasmid ptm, used in this study, allowed a much more rapid amplification course -a 27-fold titer increase in two steps, 33 days total. this all cell populations, secreting rbd proteins, were analyzed by the quantitative pcr and it was found, that increased productivity of populations, adapted to higher concentrations of mtx corresponds to higher copy numbers of target gene (fig 3c) . higher cell productivity in the case of rbdv2 protein was not due to higher target gene copy numbers, then in the case of rbdv1. cell culture medium pro cho5 (lonza), utilized in this study, contains unknown components, blocking histagged rbd protein's interaction with the ni-nta chromatography resin. clarified conditioned medium, used for protein purification, was concentrated approximately tenfold by tangential flow ultrafiltration on the 5 kda mwco cassettes and completely desalted by diafiltration, 20 diafiltration volumes of the 10 mm imidazole-hcl, ph 8.0 solution. rbdv1 and rbdv2 proteins were purified by imac utilizing ni-nta agarose (thermo fischer scientific, usa) in the same conditions. desalted conditioned medium was applied onto the column in the presence of 10 mm imidazole; the column was washed by the solution containing elution was performed by the 300 mm imidazole solution; further column strip by the 50 mm edta-na solution revealed no detectable target protein rbdv2 in the eluate (fig 2c) . purified proteins were desalted by another round of ultrafiltration/diafiltration on the centrifugal concentrators with 5 kda mwco membranes; diafiltration solution was pbs; final concentration 3-7 mg/ml. purified proteins were flashfrozen in liquid nitrogen and stored frozen in aliquots. overall protein yield for rbdv2 was 64%, 32 mg of purified rbdv2 were obtained from 1 l shake flask culture. the apparent molecular weight of intact rbdv1 was determined as 35.3 kda, deglycosylated rbdv1 -26.1 kda, theoretical molecular weight -27647 da. rbdv2 molecular weight was determined as 35.7 kda for the intact protein, deglycosylated protein -28.5 kda, theoretical molecular mass -27459 da (fig 4a) . both protein variants possess two distinct forms of intramolecular disulfide bonds sets, visible as two closely adjacent bands in non-reducing conditions and complete absence of such band pattern in reducing conditions. previously it was reported that sars-cov-2 rbd 319-541, expressed transiently in hek-293 cells, tends to form a covalent dimer, around 30% from the total, visible as the 60 kda band on the denaturing gel in nonreducing conditions [19] . we confirmed this observation; in the case of stably transfected cho cells, covalent dimerization was also 31% according to gel densitometry data. at the same time, it should be noted that the rbdv2 protein, redesigned explicitly for mitigation of this unwanted dimerization and containing an even number of cys residues, still forms 6 % of the covalent dimer. purified rbdv2 was tested by size exclusion chromatography. the major monomer form's apparent molecular weight was determined as 32.4 kda (fig s3) , admixtures peaks apparent molecular masses corresponded well to rbd dimer, tetramer, and two high molecular mass oligomers accounting for 6% of all peak areas (fig 4b) . mass-spectrometry analysis of rbdv1 and rbdv2 revealed that both proteins' molecular masses diminished ( fig s5, s6 ). this long peptide was completely absent in both spectra of de-glycosylated proteins (table s1 -s4) . a more detailed analysis of this area of the rbd protein may be of some interest for the s protein structure-function investigation but is out of scope for the present study. purified rbd variants were used as antigens for microplates coating and subsequent direct elisa with pooled sera obtained from patients with the rt-pcr-confirmed covid-19 diagnosis, 1 weakly positive serum sample from the rt-pcr-confirmed covid-19 patient, and serum sample obtained from a healthy volunteer before december 2019 (fig 4e) . both rbd variants perform equally -all serum samples produce highly similar od readings for all dilutions tested with both antigens. here we describe a method of generating stably transfected cho cell lines, secreting large quantities of monomeric sars-cov-2 rbd, suitable for serological assays. at present, serological assays for detection of seroconversion upon sars-cov-2 infection are mostly based on two viral antigens -nucleoprotein (np) and s protein or fragments of the s protein, including the rbd. there are various reports on the specificity and sensitivity of assays based on these two antigens. in some cases, the sensitivity of clinically approved npbased assays was challenged by direct re-testing of np-negative serum samples by the rbd-based assays [34] . other studies question the specificity of np-based elisa tests, demonstrating a significant level of false-positive results for the full-length sars-cov-2 np [35] . it may be proposed that testing of serum samples with both sars-cov-2 antigens will produce the most accurate results, as was done, for example, in the south-east england population study [36] ; this conclusion was made in the microarray study of a limited number of patients serum samples [37] . it is unclear yet, which part of the s protein is the optimal antigen for serological assays; microarray analysis revealed that s2 fragment generates more false-positive results than s1 or rbd antigen variants [37] in the case of igg detection, at the same time the rbd protein generated much lower signals on covid-19 patients serum samples then s1 or s1+s2 antigens. in another microarray study it was found that igg response toward the rbd domain in the convalescent plasma samples correlates well with the response toward full-length soluble s protein [38] . in the conventional elisa test format, rbd demonstrated nearly 100% specificity and sensitivity on a limited number of sars-cov-2 patients and control serum samples [4] . as of 26.10.20, at least 104 various immunoassays for sars-cov-2 antibodies were authorized for in vitro diagnostic use in the eu [39], many of them use rbd as the antigen. a simple elisa screening test with the 96-well microplate will consume around 10 µg of the rbd antigen for 40 test samples, so even one million tests will require 250 mg of the purified rbd protein, making the antigen supply a critical step in the production of such tests. method of the generation of highly productive stably transfected cho cell line, secreting the rbd protein, may be important for ivd test manufacturers in securing the sources of rbd antigen with highly predictable properties. although the rbd fragment of the s protein from sars-cov-2 is not the most popular antigen variant in the current efforts of anti-sars-cov-2 vaccine development [40] , it may be considered as the viable candidate for a simple subunit vaccine. it demonstrated the significant protective immune response development in rodents, without signs of ade effect [41] and some rbd-based protein subunit vaccine have advanced to phase ii clinical trials. cultured cho cells are the reliable source of rbd protein for this kind of vaccines; at the productivity level achieved in our study, only 30 m 3 of cell culture supernatant will provide enough antigen material for 100 mln of typical 10 µg/vial vaccine doses. с, d -protein sequence coverage by tryptic peptides, maldi-tof analysis. glycosylated peptides found are not pictured, signal peptides are yellow, detected tryptic peptides -violet, experimentally obtained masses, [m+h]+, are stated in the boxes. e -immunoreactivity of rbdv1 and rbdv2 by elisa with pooled serum samples from pcr-positive patients -(+)pooled, single serum sample from pcr-positive patient (+) and pre-covid-19 pooled sera (-). all sera samples were analyzed in duplicates, data are mean. supporting figure s1 . cell growth and viability dynamics of initial selection and mtx-driven target gene amplification. supporting figure s2 . cell growth curve for the extended batch cultivation of rbdv1 and rbdv2producing cell populations, 2 um mtx selection pressure. supporting figure s3 . size exclusion chromatography trace of molecular mass calibrators and molecular mass calibration curve. supporting figure s4 . maldi-tof spectra traces of intact proteins in glycosylated and deglycosylated forms. supporting figure s5 . maldi-tof spectra traces of tryptic peptides mxtures from intact and deglycosylated rbdv1. supporting figure s6 . maldi-tof spectra traces of tryptic peptides mxtures from intact and deglycosylated rbdv2. supporting table s1 . peptides mass list of the rbdv1 intact protein, in-gel digestion, reduced protein. supporting table s2 . peptides mass list of the rbdv2 intact protein, in-gel digestion, reduced protein. supporting molecular and immunological diagnostic tests of covid-19: current status and challenges. iscience antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design a sars-cov-2 surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients false-positive results in a recombinant severe acute respiratory syndromeassociated coronavirus (sars-cov) nucleocapsid-based western blot assay were rectified by the use of two subunits (s1 and s2) of spike for detection of antibody to sars-cov structure, function, and evolution of coronavirus spike proteins structural and functional properties of sars-cov-2 spike protein: potential 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population sample from se england analysis of sars-cov-2 antibodies in covid-19 convalescent blood using a ): p. 3581. 39. database. foundation for innovative new diagnostics. sars-cov-2 diagnostic pipeline a systematic review of sars-cov-2 vaccine candidates the sars-cov-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement we thank mr. arthur isaev (genetico, moscow, russia) and dr. alexander ivanov (institute of molecular biology russian academy of sciences, moscow, russia) for valuable comments and early access to the sars-cov-2 s protein sequence data, dr. yuri lebedin, eugenia kostrikina and xema co., ltd., for providing anti-rbd mabs and conjugates.the measurements were carried out on the equipment of the shared-access equipment centre "industrial biotechnology" the research center of biotechnology of the russian academy of sciences. dna sequencing was carried out in the inter-institutional center for collective use "genome" imb ras, organized with the support of the russian foundation of basic research.the authors would like to acknowledge all the doctors who diagnose and treat patients during the covid-19 pandemic. primers for rbdv1 cloning, restriction sites are underlined ad-cov-absf aacctcgaggccgccaccatgttcatgccttctt ad-rbd-myc6hnher gctagcctaatggtgatggtgatgatgaccggtatgcatat tcagatcctcttctgagatgagtttttgttcgaagttcacgc atttgtt primers for ptm construction, sticky ends of annealed pairs are underlinedctagtgatggtgatggtgatggtgatggtgatgaccgcctg cagacagatcctcttcgctgatcagtttttgttcaccggta primers for rbdv2 cloning, restriction sites are underlined ad-sfr2-nhef gctagcgtgcagcccaccgaatcc ad-sfr2-xmar cccgggtttgttcttcacgagattggt sequencing primers sq-5ch6-f gccgctgcttcctgtgac iresa rev aggtttccgggccctcacattg sq-mych-r gatgaccgcctgcagac key: cord-298067-awo3smgp authors: li, huanjie; wang, yangyang; ji, mingyu; pei, fengyan; zhao, qianqian; zhou, yunying; hong, yatian; han, shuyi; wang, jun; wang, qingxi; li, qiang; wang, yunshan title: transmission routes analysis of sars-cov-2: a systematic review and case report date: 2020-07-10 journal: front cell dev biol doi: 10.3389/fcell.2020.00618 sha: doc_id: 298067 cord_uid: awo3smgp the global outbreak of sars-cov-2 spread rapidly throughout the world which transmitted among humans through various routes. asymptomatic (carriers) and possible fecal-oral transmission, resulted into a large-scale spread. these issues pose great challenges to disease diagnosis and epidemic control. we obtained data on 29 cases of covid-19 patients in jinan, china, and reported the clinical data of asymptomatic patients confirmed with stool samples positive. some patients with gastrointestinal infections are secondary to pulmonary infections, and during the patients' recovery period, the virus may still existin the patient's gastrointestinal tract over 7 days. we combined with epidemiological and clinical data of asymptomatic patients to analyze the possible routes of viral transmission and infection, including eyes-nose, hands-eyes, fecal-oral, and eyes-oral, et al., thus first presented the two-way transmission through eyes-oral. through associating infection symptoms with the transmission routes of virus and the patient course of the disease, we expect to provide guidelines for clinical diagnosis and the basis for suppressing the spread of the virus and antiviral treatment. in late december 2019, the global outbreak of a novel coronavirus (2019-ncov) spread rapidly throughout the world resulting in more than 5,404,512 cases and 343,514 deaths till may 26th, 2020 (who, 2020) . the numbers of confirmed cases are still rising, posing higher challenges for disease control and patient treatment. the new coronavirus was isolated from human airway epithelial cells and sequenced through the unbiased, high-throughput sequencing to identify microbial sequences . different from both mers-cov and sars-cov virus, 2019-ncov was identified as the seventh member of the family of coronavirus that infects humans and has been formally named as the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the international committee on taxonomy of viruses. sars-cov-2 has strong transmission power, and it is easy to cause acute respiratory distress syndrome, septic shock, coagulation dysfunction, intestinal dysfunction, and other clinical symptoms post infection . at the onset of the illness, several patients were observed with extra-pulmonary manifestations, such as conjunctivitis, or even presented with asymptomatic infections (rothe et al., 2020) . but the potential routes of viral spread from patients or asymptomatic carriers to a healthy person has poorly understood . little was known about why and how the sars-cov-2 induced enteric symptoms, ocular diseases, or cardiac diseases. scientific literature on sars-cov-2 infection is growing rapidly, and further research to determine the infectivity and viability of sars-cov-2 is essential to control its spread especially in asymptomatic carriers. the entire genome of sars-cov-2 has been sequenced, and it encodes rna polymerase, spike (s) glycoprotein, membrane (m) glycoprotein, envelope (e) glycoprotein, and nucleocapsid (n) proteins. sars-cov-2 possessed a typical genome structure of the coronavirus and belonged to the cluster of beta-coronaviruses (lu r. et al., 2020) . it has a close evolutionary association with sars-cov and mers-cov and is more than 82% nucleotide identity with sars-cov (chan et al., 2020a) . although sars-cov-2 shares the same receptor ace2 with sars-cov, there are some difference between the viruses in structure and function (wan et al., 2020; zheng and song, 2020) . the cryo-em structure of the sars-cov-2 s trimer in the profusion conformation was reported recently that ace2 bound to sars-cov-2 s ectodomain with a higher affinity than binding to sars-cov. the high affinity may contribute to the apparent ease with which sars-cov-2 can spread from human to human (wrapp et al., 2020) . the estimated mean r0 for sars-cov-2 is around 3.28, with a median of 2.79 and iqr of 1.16, which is considerably higher than sars-cov . thus, ace2 plays a vital role in the sars-cov-2 infection . sars-cov-2 has evolved the stronger capability to infect and transmit among humans. to this day, there is no specific medicine to prevent or treat covid-19. studying on the viral transmission will provide significant information for understanding the pathogenesis of sars-cov-2 infection and preventing the massive spread of infectious diseases. sars-cov-2 has been detected in multiple organs, for example, eyes, nasopharynx, saliva, alveolar lavage fluid, blood, intestine, feces after infection , which brings a great difficulty to reach the diagnosis. on february 1, 2020, respiratory samples of four patients were confirmed sars-cov-2 infections by real-time pcr in jinan central hospital, shandong province, china. clinical characteristics and blood biochemical indexes of patients were recorded and examined at the hospital. on february 2, we collected samples of four patients' conjunctival secretions, feces, cell phone surfaces, hands surface, an inner surface of the mask, ground, door handles surface, the head surface of bed, and other samples in the isolation ward immediately. the sars-cov-2 nucleic acid for the above samples of patients was also detected immediately. besides, we also collected the medical records of 29 confirmed patients during hospitalization from the isolation ward of jinan infectious disease hospital. in this study, we systematically investigated the clinical and laboratory characteristics of confirmed 29 cases provided by jinan infectious disease hospital, shandong university, and the environmental samples collected from jinan central hospital, shandong university. epidemiological and clinical characteristics of asymptomatic patients in jinan are reported. summarizing the published articles, including sars-cov and sars-cov-2, we combined with epidemiological and clinical data to analyze the possible routes of asymptomatic patients with virus infection in order to provide the basis for suppressing the spread of the virus, and antiviral treatment and advice for the protection of medical staff. four patients were diagnosed with covid-19 in jinan central hospital, shandong province. we collected throat swabs and blood samples and other environmental samples, including the four patients' conjunctival secretions, feces, cell phone surfaces, hands surfaces, an inner surface of the mask, ground, door handles surface, the head surface of bed, et al. in the isolation ward before they were transferred to jinan infectious disease hospital. in 2 weeks, there were 29 cases of covid-19 patients transferred to jinan infectious disease hospital for hospitalization, including the above-mentioned four patients. all the cases were confirmed by rt-pcr and were analyzed for epidemiological, clinical, radiological features, and laboratory data. this study was approved by the ethics commission of jinan central hospital affiliated to shandong university and jinan infectious disease hospital affiliated to shandong university. the patients waived the right to informed consent. we recorded and analyzed the contact history, hematological, biochemical, radiological, and microbiological investigation results. the respiratory tract, blood, and stool samples of 29 patients were collected. rna of biological and environmental samples were extracted using liferiver (shanghai liferiver biotechnology co., ltd.) nucleic acid extraction kit, which had been registered for detecting orf1ab gene, e gene, and n gene of sars-cov-2. samples were tested by rt-pcr following the steps of the kit in a tertiary protection laboratory. respiratory samples of the patients were also tested for influenza a and b viruses and respiratory syncytial virus using the xpert xpress flu/rsv assay (genexpert system) according to the instructions. by searching pubmed web, we analyzed and compared the reports of sars-cov, mers-cov, and sars-cov-2 in different countries in the early outbreak stage. local case analysis in this paper adopts the method of descriptive statistics, tables, and graphs. compared with wuhan, the 29 cases of covid-19 in jinan exhibited mild or moderate symptoms, which are mainly imported cases from wuhan's contact history (tables 1, 2) . of the 29 cases, 44.8% had a history of contact with figure 1 | symptoms according to day of illness and hospitalization among the cases confirmed sars-cov-2. representative cases were combined the timeline of positive throat and anal swabs. case07-10 were treated in jinan infectious disease hospital and the remaining cases were reported in other hospitals. the patients are some particular cases of being discharged. after the throat swab turned to negative, the stool samples of patients became positive again appeared 5-7 days after discharged, or even longer. 2) . we sorted out 10 representative cases and combed the timeline of the positive throat and stool samples (figure 1) . four cases are enrolled in jinan, and the rest of the cases are reported by other agencies. in addition to patients whose initial symptoms of infection are diarrhea, there are some particular cases. after the throat swab turned to negative, their stool samples became positive again, and most of the clinical manifestations appeared 5-7 days after the pulmonary symptoms. some patients with gastrointestinal infections were secondary to pulmonary infections, and during the patients' recovery period, the virus may still be released from the patient's gastrointestinal tract for 7 days, or even longer. we inferred virus being excreted through the intestine may be more beneficial to the recovery of patients. the replication and duration of sars-cov-2 detoxification is directly related to the prognosis of patients. patients with different clinical symptoms may be closely related to the route of the virus during transmission. the infection sites of virus causing different clinical symptoms and directly affects the course of disease. we speculated several different clinical symptoms may reflect various transmission pathways of sars-cov-2 shown in figure 2 . the symptomless sars-cov-2 carriers were divided into two groups, i.e., the throat swab positive or the stool sample positive, which reflected the different target sites of a susceptible patient. study on the first clinical symptoms and the progression of the disease, lungs-intestine transmission route showed that there are three kinds of routes: from the lungs to intestine, the self-infection from the intestine to lungs, co-infection of lungs, and intestine. combing the time points of patients' hospitalization may be helpful to provide a guideline for covid-19 diagnosis and treatment. whether the excretion of feces in vitro during the recovery period is infectious remains to be further studied. the criteria of patients discharged should be reassessed, and the nucleic acid testing of anal swabs or stool samples should be added. environmental samples detection of four patients with early diagnosis, including eye and conjunctival secretions, telephone surfaces, hands surface, masks, the surface of door handles and bed at home, and other samples in the isolation ward, were all negative except their masks. the virus nucleic acid testing of the blood and stool samples were all negative on admission. but the nucleic acid test of their stool samples became positive after the patients were hospitalized for 8-10 days, while nucleic acid test of throat swab turned to negative. sars-cov-2 is mainly transmitted through the respiratory tract in the early stage and can be survival longer on the mask. the epidemiological survey showed the four patients had no recurrence of human transmission before isolated by the hospital. cases in jinan are mainly mild symptoms, which indicated that timely diagnosis, early detection, early isolation are very effective approaches before the virus becomes highly contagious. there are no new cases reported for the 26 successive days in jinan. the fundamental measure for controlling infectious diseases is to cut the source of infection. it is suggested that study on the transmission routes of the virus is a feasible pathway in controlling the disease effectively and we summarize the transmission routes of sars-cov-2 systematically. the transmission of respiratory droplets and mucous membranes infections are the main route of sars-cov-2, but not the only one just like sars-cov, sars-cov-2 is a typical respiratory virus causing highly contagious potentially lethal disease. the mucous membranes infection is still the main transmission. many clinical cases developed influenza-like symptoms, with a 2-4 days history of cough and subjective fever wu f. et al., 2020; zhu et al., 2020) . coronavirus gains entry into host cells through recognizing and binding to the host receptor ace2 distributed from the conjunctiva (wan et al., 2020 ). once exposed directly or indirectly to the virus (infectious droplets, body fluids, virus-carrying hands), the mucosal cells in the conjunctiva, mouth, nasal cavity, or throat were susceptibility infected by the virus for replicating (gao et al., 2016; zhou p. et al., 2020) . a larger amount of virus was assembled and released into the human lungs through the respiratory tract, resulting in various types of fever, cough, or groundglass opacity of lung on ct examination results and even respiratory failure. the sars-cov-2 mainly spread from person to another through small respiratory droplets from the nose or mouth when a person confirmed covid-19 coughs or exhales (figures 3, 4) . these droplets land on objects and surfaces around the person. other people may catch the virus by breathing in droplets or touching these objects or surfaces, then touching their eyes, nose, or mouth. the risk of catching sars-cov-2 from someone with no symptoms is very low. however, many people with sars-cov-2 experience only mild symptoms, particularly true in the early stages of the disease. some cases reported that conjunctivitis was the first symptom and they were infected while had a history of close contact with a patient with covid-19 lu c. w. et al., 2020) . it speculates that there may be a risk of tears and conjunctival transmission. growing evidence shows that the virus attacks multiple organs in the body. during the outbreak of sars-cov-2, some patients developed symptoms of conjunctivitis. some patients even suffered the ocular diseases in clinical diagnosis before fever and cough . there have been case reports in which many ophthalmologists were found to be infected through routine diagnosis and treatment with only his eyes unprotected (chan et al., 2020a; xia et al., 2020) . therefore, if conjunctivitis as the initial symptom of confirmed covid-19 patients was neglected and contacted without comprehensive measures, the infectious tears and body fluids containing the virus could infect other persons (belser et al., 2013; lu c. w. et al., 2020) . those results suggested that the eyes route of transmission existed (chan et al., 2020b; huang et al., 2020) . deng et al. (2020) demonstrated that macaques can be infected with sars-cov-2 via the conjunctival route. we proposed that the sars-cov-2 transmitted and infected through eyes including two routes (figure 3) . one is direct contact and the other is indirect contact transmission. the direct route is that droplets with virus enter through the eyes. for example, in a fever clinic, a relatively closed environment, there is a high risk of eye infections. when medical personnel performs close-up operations, virus droplets (aerosols pollution) will spray out which may splash into the eyes and cause infections, so medical staff are strongly recommended wearing goggles or a mask. a virus presented in these body fluids may affect our precautionary practices and sites of sampling for diagnostic tests. recently, jiang et al. found that the virus was present both on surfaces and in the air (jiang et al., 2020) . the two positive areas were the surfaces of the nurse station in the isolation area with suspected patients and the air of the isolation ward with an intensive care patient. so high contraction of nucleic acid may exist in the aerosol and influence operator, even the test result. another route is through indirect contact infection, that is, accidentally touching the virus droplets with your hands and rubbing your eyes or noses may cause conjunctival infection. therefore, once sars-cov-2 infected and replicated in eyes, it will be transmitted through two ways (figure 3) , one is outward transmission, eye secretions, or tears with virus contaminating the hands, and then there is a risk of viral transmission through hands. another route is an inward transmission. if the virus infects person through eyes, conjunctival secretions, and tears can flow into the mouth through the nasopharyngeal tube which will ultimately reach the lungs or gastrointestinal tract and more infections may occur. routes of transmission of sars-cov-2 other than respiratory droplets and stool are still enigmatic. the proportion of patients confirmed sars-cov-2 with conjunctivitis is much smaller than respiratory symptoms, which reflect that the eyes are not the most important organ for propagating the virus. for instance, the eyes cannot generate infectious aerosol unless an eye ezamination has performed. but we still insist that the eyes are important portals of entry for virus. moreover, increasing reports each day suggesting that sars-cov-2 cases began with eye redness and tingling as the leading symptoms, and the literature suggests that viruses can infect the human body through conjunctiva (lu c. w. et al., 2020; wang w. et al., 2020) . these results showed that a few new cases of covid-19 began with conjunctivitis as the first symptom, and the sars-cov-2 containing in the eye surface may enter into the nasal cavity and throat through drain tears. sars-cov-2 can be transmitted through the nose-eye, possibly through the way of increased oral pressure caused by coughing or sneezing, and reverse transmission of the virus through the nasolacrimal duct to the dacryocyst and then infect the conjunctival cornea (sun et al., 2020; zhou y. et al., 2020) . thus, this route is a two-way transmission route (figure 4) . the lacrimal route, via drainage of tear fluid including virus from punctum in the upper and lower eyelid through canaliculi to the lacrimal sac, and further through the nasolacrimal duct to the nasal cavity, would be another pathway available for sars-cov-2 infection. during replication in the ocular tract there will be a continuous influx of virions to the nasal cavity, and respiratory infection may be established. the possibility of subclinical and/or prolonged virus replication in the eye, followed by continuous transfer to the respiratory tract cannot be excluded. generally, many respiratory pathogens, such as influenza, sars-cov and mers-cov, cause enteric symptoms, so is sars-cov-2 (holshue et al., 2020) . diarrhea was observed in a considerable number of patients. in early reports from wuhan, 2-10% of patients with covid-19 had gastrointestinal symptoms such as diarrhea or vomiting (chen n. et al., 2020; wang d. et al., 2020) . abdominal pain was reported more frequently in patients admitted to the intensive care unit than in individuals who did not require intensive care unit care, and 10% of patients presented with diarrhea and nausea 1-2 days before the development of fever and respiratory symptoms (yeo et al., 2020) . the study found that the detection of sars-cov-2 nucleic acid positive in a few feces of patients with confirmed covid-19 cases indicated the presence of a live virus. nanshan zhong and lanjuan li teams have isolated sars-cov-2 from the fecal swab specimens of the pneumonia patient with covid-19 separately. these findings demonstrated the presence of live viruses in the feces of patients. the recent occurrence of two covid-19 patients in the same building in hongkong also provide the evidence of fecal transmission. indeed, the sars-cov-2 receptor ace2 is highly expressed on differentiated enterocytes. sars-cov-2 can infect enterocyte lineage cells in a human intestinal organoid model (lamers et al., 2020) . fecal transmission mode accounts for a small proportion of respiratory virus transmission. most of the virus transmitted through the feces are enteroviruses, and respiratory viruses are mainly transmitted through droplets and contact. however, more than 15% of cases showed that the anal test of several patients had become positive at the later stage, while the chest radiographic evidence and viral clearance in respiratory samples from the upper respiratory tract showed significant improvement, so fecal formation route cannot be ignored. pan et al. (2020) reported that the viral loads of stool samples were less than those of respiratory samples, so whether the excretion of feces in vitro during the recovery period is infectious remains to be further studied. considering the evidence of fecal excretion for sars-cov-2, the virus can also be transmitted via the fecal-oral transmission route or re-transmitted through the formation of aerosols in virus-containing feces. the transmission route of the tract may not be a single transmission. it may be a medium channel for other routes (figure 4) . therefore, the standard procedure of stool sample collection and examination in patients with sars-cov-2 is important to protect medical staff and reduce the risk of infection. the blood-borne spread of sars-cov-2 may be caused by cytokine storms (css) cytokines (ck) are key factors regulating the immune response caused by many infectious pathogens that can significantly damage host organs and tissues. in the early stages of sars-cov infection, cytokine levels in the blood, such as il-6, il-8, and tnf-î±, are rapidly elevated, the elevation of which is associated with the progression of lung invasion and injury (wong et al., 2004) . mers-cov infection was also reported inducing increased concentrations of proinflammatory cytokines (ifnî³, tnfî±, il15, and il17) (mahallawi et al., 2018) . huang et al. reported 41 patients with laboratory confirmed sars-cov-2 infection and those high concentrations of cytokines recorded in plasma of critically ill patients . the concentrations of various cytokines (il-6) in the blood of severe patients with sars-cov-2 infection were significantly higher than those of non-serious patients. the concentration of cytokines can indicate the severity of the disease. liu et al. analyzed the blood immunological indexes of 33 patients with new coronary pneumonia and found that after sars-cov-2 infection, the pathogenic t cells were quickly activated to produce granulocyte-macrophage colony stimulating factor (gm-csf) and il-6 . gm-csf further activates cd14, cd16 inflammatory monocytes, producing more il-6 and other cytokines, resulting in a cytokine storm that leads to severe immune damage to the lungs and other organs. more and more cases reported that the brain, kidney, and heart impairment induced by the virus infection, may contribute to multi-organ failure and death eventually wu c. et al., 2020) . those cases with sars-cov-2 indicate that there is a dissemination way (lymphatic, hematogenous, direct invasion of adjacent tissues, and pathogenic implantation) in blood vessels of viral infection, which usually occurs in critical patients. this potential way for the viral spread is that sars-cov-2 enters into the lung from mouth and throat and then infects cells. the virus replicates in the cell and releases more new viruses. massive accumulation of sars-cov-2 leads to a surge of immune cells and more and more pro-inflammatory cytokines, resulting in a rapid increase in ck levels in the blood, or releasing more virus particles into the blood circulation. the virus and cytokines positively induce high expression of ace2 in the intestinal epithelium and other organs which accelerated over-expression of ace2 and viral binding, causing systemic infections with the virus (figure 5) . the model might explain why the sars-cov-2 nucleic acid testing in the stool of some patients turns to positive occurs in the later days of treatment. the ongoing outbreak of sars-cov-2 causes an epidemic of acute respiratory syndrome in humans in wuhan, china. it has rapidly spread to national regions and other countries, thus, pose a serious threat to public health. our research reported the characteristics of sars-cov-2 epidemiology in jinan. compared with wuhan, 29 cases of covid-19 in jinan exhibited mild or moderate symptoms, which are mainly imported cases from wuhan's contact history. the infection and transmission capacity of the virus is greatly weakened, which may be due to the fact that the cases in jinan are mostly second or third generation communicators. the incubation period of sars-cov-2 is 1-14 days, which is infectious and the incubation period is equally contagious. the main routes of viral transmission are respiratory tract, including the spatter (e.g., blood spatter, spatter during intubation, etc.) and droplet transmission (sneezing or cough), contact transmission (e.g., hand wiping eyes), fecal-eye transmission, nasal-eye transmission, mouth-eye transmission (through contaminated hands or objects) and the transmission of eye secretions and tears (figure 6 ). if the virus first contacts the conjunctiva of the patients' eyes, or the hand touches the virus and rubs the eye, the virus will invade the patient's eye conjunctiva, infect and reproduce, causing eye swelling which can even lead to conjunctivitis. the replicated virus may pass through the tear fluid to patient's nasolacrimal duct and enter the respiratory infection pathway when coughing or enter the digestive tract infection pathway when swallowing food. it insisted that the eyes are important entrance and replication sites of sars-cov-2. although direct droplet transmission is the main route of transmission, fecal excretion, environmental contamination, and fomites might contribute to the viral transmission. considering the evidence of fecal excretion, sars-cov-2 can also be transmitted via the fecal-oral transmission route. the virus can stay active in the digestive tract for more than 7 days, even longer time than in the lungs. after the throat swab turns negative, sars-cov-2 can also be detected in the feces of patients. investigating on the infection and transmission of the virus, we reported the asymptomatic person with sars-cov-2 positive. it is not clear whether the virus discharged from the feces is infectious or not, the body excrete the virus through the feces is a beneficial way for human self-regulation. the replication and duration of virus detoxification may be directly related to the prognosis of patients. besides, when pulmonary infection secondary to intestinal infection, patients should also avoid the occurrence of self-infection. in high prevalence season of influenza, it may be necessary to detect the influenza virus if diarrhea appeared, to avoid the occurrence of fecaloral transmission and lung infection. moreover, asymptomatic carriers could acquire and transmit the sars-cov-2, which makes the prevention of covid-19 infection great challenge in the world. all datasets generated for this study are included in the article/supplementary material. this study was approved by the medical ethics committee of jinan central hospital affiliated to shandong university. the committee waived the need for specific consent. hl and yaw conceived and wrote the manuscript. hl and yaw drew the pictures. yh and mj collected patient's clinical information. ql provided the cases. qz, yz, fp, and sh did the nucleic acid testing of sars-cov-2. yuw and qw managed the project. all other authors contributed to the analysis, reviewed results, and reviewed the manuscript. ocular tropism of respiratory viruses. microbiol genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster ocular manifestations of a hospitalized patient with confirmed 2019 novel coronavirus disease epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study ocular conjunctival inoculation of sars-cov-2 can cause mild covid-19 in rhesus macaques from sars to mers: evidence and speculation first case of 2019 novel coronavirus in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan clinical data on hospital 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bioinformatics analysis based on single-cell transcriptomes. biorxiv novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov a pneumonia outbreak associated with a new coronavirus of probable bat origin ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva. medrxiv a novel coronavirus from patients with pneumonia in china key: cord-280821-kc0ut4oy authors: venturini, elisabetta; montagnani, carlotta; garazzino, silvia; donà, daniele; pierantoni, luca; lo vecchio, andrea; nicolini, giangiacomo; bianchini, sonia; krzysztofiak, andrzej; galli, luisa; villani, alberto; castelli-gattinara, guido title: treatment of children with covid-19: position paper of the italian society of pediatric infectious disease date: 2020-09-24 journal: ital j pediatr doi: 10.1186/s13052-020-00900-w sha: doc_id: 280821 cord_uid: kc0ut4oy a statement of consensus was formulated after reviewing available literature on pediatric treatment strategies for covid-19 by the steering and scientific committee of the italian society of infectious pediatric diseases in connection with the italian society of paediatrics. since december 2019, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection has been reported in hubei province, china, and from there it spread worldwide. starting from the end of february 2020, the number of cases of coronavirus disease 2019 (covid19) outside china rapidly increased, urging the world health organization (who) to declare covid-19 as a pandemic on march 11 [1] . most children with sars-cov-2 infection develop none or mild symptoms, needing only supportive treatment [2, 3] . however, few cases of severe covid-19 in children have been reported [3] [4] [5] , including multisystem inflammatory syndromes [6, 7] . as no evidence of at least good quality is available regarding therapy of covid-19, treatment regimens of covid-19 are not standardized. at present, few clinical trials for covid-19 treatment involving children are ongoing [8] . moreover, evidences are rapidly evolving and the therapeutic indications can change very quickly and even this work has changed quite a bit in the course of its writing. to date, many national and international guidelines have been issued for the adult population [9] [10] [11] . however, there is a need of guidance on covid-19 management in children from scientific societies and expert groups. recently, an expert opinion has been released by a group of pediatric infectious disease specialists from north america, suggesting that supportive care alone is indicated for the overwhelming majority of cases [12] . the italian society of pediatric infectious diseases steering and scientific committee developed a position paper on treatment of children with covid-19, reviewing the current literature on this topic and providing indications based on the available literature data. since new evidences will be available in the next weeks/months, the italian society of pediatric infectious diseases will guarantee to maintain treatment indication updated on the website www.sitip.org (updated english and italian versions of the present document). the consensus statement was formulated by the steering and scientific committee of the italian society of pediatric infectious diseases in connection with the italian society of pediatrics. decision was made after reviewing available literature on pediatric treatment strategies for covid-19 at 16 june 2020 on pubmed with the following search strategies: (sars-cov-19 or covid-19 or coronavirus) and treatment. moreover, national and international recommendations of who and scientific societies available online were evaluated. ongoing clinical trials were searched on https://clinicaltrials.gov/ and https://www. aifa.gov.it/sperimentazioni-cliniche-covid-19. clinical syndromes associated with covid-19 in children were defined adapting the who classification as follows [13] [14] [15] . asymptomatic case: infection identified during screening or contact tracing without symptoms. mild case: fever and/or fatigue and/or upper airways symptoms without radiological/ultrasound findings (if performed). moderate case: fever and/or fatigue and/or upper airways symptoms (cough or mild respiratory distress) and/ or poor feeding and/or pneumonia identified with chest x-ray or ultrasound. severe case: [16] . sepsis-associated organ dysfunction and septic shock were defined according to surviving sepsis campaign definition [17] . regardless of the early stage of the disease, the following indicators should be assessed as related to an increased risk of rapid progression to the severe/critical stage. clinical early warning indicators: increased tachypnoea, despite 2 h of intravenous rehydration and low flow nasal cannula oxygen therapy impaired consciousness progressive increasing of lactate values bilateral lung infiltration or multiple lobes involvement, pleural effusion or rapid progression of the lesions in a short period of time age < 3 months underlying diseases (congenital heart disease, bronchopulmonary dysplasia, anomalies of respiratory tract, abnormal hemoglobin, anemia, severe malnutrition, congenital or acquired immunodeficiency) children and adolescents 0-19 years of age with fever > 3 days and two of the following: skin rash or bilateral non-purulent conjunctivitis or muco-cutaneous inflammation signs (oral, hands or feet) hypotension or shock signs of myocardial dysfunction, pericarditis, valvulitis, or coronary abnormalities (including echocardiography findings or elevated troponin/ nt-probnp) evidence of coagulopathy acute gastrointestinal problems (diarrhea, vomiting, or abdominal pain) elevated markers of inflammation such as erythrocyte sedimentation rate, c-reactive protein, or procalcitonin. and no other obvious microbial cause of inflammation, including bacterial sepsis, staphylococcal or streptococcal shock syndromes. and evidence of covid-19 (positive real-time polymerase chain reaction, antigen test or serology), or a likely contact with patients with covid-19. asymptomatic cases: no treatment. moderate and mild cases: only antipyretic therapy. remdesivir if not available hydroxychloroquine or lopinavir/ritonavir all these drugs should be preferably administered within a clinical trial. warning: hydroxychloroquine can cause qtc prolongation. every 2 days perform electrocardiogram (ecg) and qtc assessment for qtc prolongation increased risk. for hydroxychloroquine, carry out glucose 6 phosphate dehydrogenase (g6pdh) dosage in case of risk factors for deficiency. remdesivir should be administered in patients with normal renal function. liver function assessment should be performed in all patients prior to starting remdesivir and every other day while receiving remdesivir. duration of therapy: 5-7 days, extendable according to clinical course. immunomodulating therapy: this therapy must be considered in case of: -ards or progressive deterioration of respiratory function. -multisystem inflammatory syndrome. -marked alteration or increasing trend of il-6 and/or d-dimer and/or ferritin and/or c-reactive protein. -interval of at least 7 days from the beginning of the symptoms. methylprednisolone or dexamethasone or anakinra (or tocilizumab) the available dosages and formulations of the various drugs are indicated in the text. covid-19 management and treatment in children, according to disease severity, are reported in table 1 . antipyretic therapy: prefer paracetamol (10-15 mg/kg every 4-6 h) in case of fever > 38.5°c. avoid ibuprofen in case of dehydration, vomiting and diarrhea, as it is associated with an increased risk of kidney failure. some authors have suggested a correlation between the use of ibuprofen and an unfavorable course of sars-cov-2 infection [19] . however, these data are not currently confirmed and the european medicines agency does not contraindicate the use of non-steroidal antiinflammatory drugs [20] . inhalation therapy: if topic steroids and/or bronchodilators are needed (e.g. patient with recurrent wheezing undergoing exacerbation and suggestive symptoms or confirmed sars-cov-2 infection) the use of pressurized suspensions with spacer chamber is suggested. conversely, the use of nebulisers is not recommended in order to avoid particles aerosolization and increased contagiousness. ongoing steroid treatment should not be stopped [21] . venous thromboembolism prophylaxis: severe covid-19 seems to be associated in adults with an increased risk of disseminated intravascular coagulation and venous thromboembolism. a study on 449 adult patients with severe infection showed a lower mortality rate in those receiving anticoagulant therapy [22] . therefore, recently adults protocols suggest strategies for prevention and management of coagulative disorders secondary to covid19 infection with low molecularweight heparin, especially in case of ards [13, 23] . however, children have a much lower incidence of thrombotic complications than adults, even under higher risk conditions such as major surgery or polytrauma [24] . therefore, such prophylaxis is not routinely suggested in children. an exception can be made for neonatal age and adolescents, where constitutionally the incidence of thrombotic complications is higher [25] . preventive anticoagulant therapy can therefore be considered for these age groups, in cases where severe inflammatory conditions occur and therefore hyperactivation of the clotting process could lead to the appearance of important thrombotic complications. the suggested treatment is with subcutaneous enoxaparin 100-200 u/kg/day, that can be increased to 150-300 u/kg/day in neonates. lopinavir/ritonavir lopinavir/ritonavir is a boosted protease inhibitor, used, in association with other drugs, in the therapy of human immunodeficiency virus (hiv) infection, starting from the age of 14 days of live [26] . its security profile is well known, as it has been largely used in hiv infections in the pediatric age, it is available both in oral suspension and tablets. on the base of some clinical trials, the chinese guideline on sars-cov-2 pneumoniae recommend the early use of lopinavir/ritonavir [27] . on 18 march 2020, the results of a double blinded, randomized, open-label trial, which compared lopinavir/ ritonavir associated to standard of care and standard of care alone, on 199 sars-cov-2 pneumonia hospitalized patients, have been published on new england journal of medicine [28] . the study did not evidence any statistically significant differences in the clinical improvement. mortality at 28 days showed a 5.8% difference in favor of lopinavir/ritonavir use, although not statistically significant (95% confidence interval, ci95%: − 17.3-5.7). patients in which lopinavir/ritonavir therapy has been started before the 12 days of symptoms had a significantly reduction of symptoms duration, hazard ratio: 1.25 (95%ci, 1.77-2.05). data about mortality, stay in intensive care unit and duration of hospitalization were not stratified for the start of therapy before and after 12 days. the results of this study cannot be transferred to pediatric population, to patients with mildmoderate symptoms, and to patients who underwent an early therapy. currently, american guidelines on covid-19 treatment published in may 2020, recommend both in children and adults to use lopinavir/ritonavir only in the context of clinical trials, given the lack of effectiveness reported now in literature [9, 12] . the latest chinese guidelines on sars-cov-2 pneumoniae do not recommend the use of a specific antiviral for the treatment of covid-19, and nevertheless include lopinavir/ritonavir among the available therapeutic options for hospitalized patients [29] . less than 40 clinical trials are currently registered to evaluate the effectiveness of lopinavir/ritonavir against covid-19. lopinavir/ritonavir is not indicated in premature neonates before the 42 weeks of corrected age and in all cases before 14 days of live [30] . remdesivir remdesivir is a nucleotide analogue, which is incorporated in the viral rna chain, determining its premature termination. it has been developed from gilead in 2017 for ebola therapy. in vitro studies, its high spectrum efficacy against different coronavirus has been demonstrated [31, 32] . interestingly, remdesivir seems to have a high genetic barrier to viral resistance development (demonstrated in sars studies). it has a short plasmatic half-life, but it is rapidly converted into its active form (in 2 h from the infusion) and it has a 14 h intracellular half-life [33] . in may 2020, following an assessment of the emergency use authorization criteria and available scientific evidence, the fda issued an emergency use authorization allowing for the administration of remdesivir intravenously by health care providers for the treatment of covid-19 suspected or laboratoryconfirmed in adults and pediatric patients hospitalized with severe disease [34] . in a guidance approved by the american pediatric infectious diseases society for covid-19 treatment in children, if an antiviral is used, panel suggested remdesivir as the preferred agent [12] . antivirals should preferably be used as part of a clinical trial, if available. however, a warning has been issued on the coadministration of remdesivir and chloroquine phosphate or hydroxychloroquine sulfate as it may result in reduced antiviral activity of remdesivir [34] . as at 26 june 2020, there are 39 ongoing trials evaluating clinical efficacy of this drug in the therapy of moderate and severe sars-cov-2 infections [35] . four studies (gs-us-540-5774, gs-us-540-5773, gs-us-540-5821 and gs-us-540-5823) are currently ongoing in italy. all these studies are sponsored by gilead and only one study involve children from birth to 18 years whereas the other three enroll children older than 12 years and adults [36] . dosage: adults: 1st day 200 mg iv in 30 min, followed by 100 mg iv /day for other 9 days children (< 40 kg): 1st day 5 mg/kg iv (in 30 min), followed by 2.5 mg/kg iv (in 30 min)/day for other 9 days. at present, the dosage has not been established for the first 2 weeks of life and weight < 2.5 kg. hydroxychloroquine (and chloroquine) could have an antiviral activity [37] . the mechanism is currently not fully clarified. however, in vitro studies suggest that they could act by increasing the endosomal ph required for virus / host cell fusion and interfering with the glycosylation of the sars-cov-2 cell receptor [38] . in particular, hydroxychloroquine appears to have better in vitro activity towards sars-cov-2. the anti-inflammatory activity of these molecules, through the inhibition of the production of interleukin (il) -6 and tumor necrosis factor (tnf)-α, could contribute to their effectiveness. in addition, these molecules have been in use for decades, showing a good safety profile. compared to chloroquine, hydroxychloroquine is a drug more readily available and with a higher safety profile [39] . in february 2020, a panel of experts in china summarized the results of the use of chloroquine (500 mg every 12 h for 10 days) in adults with covid-19, suggesting that its use would be associated with an improvement in the clinical success rate, reducing the hospitalization and improving the outcome [40] . since then, hydroxychloroquine has been widely used in adult patients [41, 42] . to date, there are more than 200 registered clinical trials for pre-exposure or postexposure prophylaxis of sars-cov-2 infection, and treatment of patients with mild, moderate, and severe covid-19 [35] . however, it is not yet clear if the benefits outweigh the risks, especially in children. in fact, on 24 april 2020, the fda warned against the use of hydroxychloroquine or chloroquine for covid-19 outside hospital or clinical trial setting due to the risk of qt interval prolongation and ventricular tachycardia [43] . this risk could be also increased when combined with some antibiotics such as azithromycin. moreover, since the majority of children manifest only mild symptoms, a widespread use of hydroxychloroquine may confer only minimal benefit. furthermore, pharmacokinetic modeling and simulation used to identify pediatric-specific dosages for hydroxychloroquine have raised concerns on plasma exposures lower than those needed to mediate an antiviral effect [44] . however, these findings do not exclude a potential utility of hydroxychloroquine for covid-19 treatment based on different mechanisms of action, such as immunomodulation. conclusive data are needed from ongoing trial to understand the possible role of this drug in children with covid-19. available formulations: hydroxychloroquine tablets 200 mg. dosage: adults: 400 mg twice a day the first day, followed by 200 mg twice a day for overall 5-7 days. children: 6 mg/kg (maximum: 400 mg/dose) twice a day on day 1, followed by 3 mg/kg (maximum: 200 mg/ dose) twice a day for up to 5 days. *. * hydroxychloroquine solution preparation is recommended for children. precautions for use: perform ecg before administering the drug to rule out long qt. in case of risk factors, dose g6pdh before use. other antiviral therapy favipiravir is an antiviral drug authorized in japan for the treatment of flu. it works by inhibiting rna polymerase-rna-dependent. the medicine is not authorized in europe or in the usa. preliminary results on 80 patients with non-serious sars-cov-2 infection, published only in chinese, seems to show a better efficacy of this drug compared to lopinavir/ritonavir [37] . few clinical trials are currently undergoing to evaluate the efficacy of favipiravir in sars-cov-2 infection and the aifa scientific technical commission approved a clinical trial program for this drug [36, 45] . ivermectin, an fda-approved anti-parasitic, seems to have broad-spectrum anti-viral activity in vitro, is an inhibitor of the causative virus (sars-cov-2), 2 h post infection with sars-cov-2 able to effect 5000-fold reduction in viral rna at 48 h [46] . a single center, double-blind, randomized, placebo-controlled trial is ongoing on ivermectin efficacy in reducing sars-cov-2 viral load in adult patients with low risk, non-severe covid-19 [47] . steroids at present, there are no clear evidences to support the use of systemic steroids during sars-cov-2 infection unless specific needs (e.g. asthmatic patient who does not respond to 3 doses of bronchodilator, severe allergic reaction). in particular, in patients on chronic systemic or inhaled steroid therapy, treatment stopping is not necessary. some data suggest that methylprednisolone could have an immunomodulating activity in case of ards, decreasing the risk of death [48] . its use is also indicated in case of a worsening of pulmonary function after at least 7 days from the beginning of symptoms, in association with marked alteration or tendency to increasing of il-6 and/or d-dimer and/or ferritin and/or c-reactive protein. in these cases, can be used: -methylprednisolone 1-2 mg / kg (max 80 mg) once a day. a short course is indicated (2-5 days). in severe cases high doses (methylprednisolone 30 mg/ kg) can be considered. recently some good results were reported by the interim analysis of the recovery trial [49], a study where 2104 adult patients were randomised to receive dexamethasone 6 mg once per day (either orally or intravenously) for 10 days and 4321 patients were randomised to usual care alone. dexamethasone significantly reduced deaths by one-third in ventilated patients (relative risk, rr 0.65) and by one fifth in those receiving only oxygen (rr 0.80), but there was no benefit among patients who did not require respiratory support. based on these results, a treatment with dexamethasone (0.2-0.4 mg/kg, maximum 6 mg) in patients requiring oxygen should be considered. anakinra the multisystem inflammatory syndrome reported in patients with covid-19 shares considerable biochemical overlapping with the cytokine storm complicating macrophage activation syndrome associated with rheumatic disease. anakinra is licensed for treating people with rheumatoid arthritis, aged at least 8 months. this is a 17 kd recombinant, non-glycosylated human il-1 receptor antagonist who inhibits the proinflammatory cytokines interleukin (il)-1α and il-1β, with a short half-life of about 3-4 h and good safety profile. anakinra has also been used with some success to treat macrophage activation syndrome caused by various inflammatory conditions. several case series report that anakinra reduced both need for invasive mechanical ventilation in the intensive care unit and mortality among patients with severe forms of covid-19, without serious side-effects [50, 51] . currently, there are 10 ongoing clinical trials on anakinra use in covid-19, all in adults [52] . however, there are few pediatric reports of children treated with anakinra showing that such treatment in serious cases can be safe and beneficial [53, 54] . indeed, anakinra has been proposed as the best option if such treatment is considered necessary, because it has a relatively short half-life and may be discontinued rapidly in case of adverse effect or concern of worsening the infection [55] . anakinra can be administered intravenously (off label) or subcutaneously and has a wide therapeutic window; when anakinra is effective for cytokine storm syndromes, it works within 2 to 3 days [56] . therapeutic scheme: -anakinra vial 100 mg/0.67 ml intravenously: 8-10 mg/kg/day in 2 or 4 administrations depending on the total dose (up to a maximum of 100 mg 4 times a day) after 48-72 h, repeat the plasma dosage of il-6 and/ or d-dimer. tocilizumab tocilizumab is a recombinant humanized monoclonal antibody belonging to the g1 immunoglobulin subclass and directed against both soluble and membrane il-6 receptors [57] . this drug is indicated for the treatment of moderate and severe rheumatoid arthritis, systemic juvenile idiopathic arthritis (from the age of 1 year), juvenile idiopathic polyarthritis (from the age of 2 years) and severe release of cytokines induced by car-t lymphocytes (chimeric antigen receptor t cell) (from the age of 2 years). studies suggested that the alveolar damage in covid-19 is caused by a cytokine storm (including il-6) and that symptoms improve with the use of tocilizumab [58, 59] . based on these results, different clinical trials have been started. the italian multicenter study tocivid-19 has been promoted by the national cancer institute, irccs, g. pascale foundation, naples. the recruitment of the prospective phase has been completed, while the observational study continues. the protocol includes the enrollment of patients of any age. two other studies started at the end of march in italy, but not enrolling pediatric patients. one of them, aimed at assessing the efficacy of the early tocilizumab administered early in patients suffering from recently emerging covid-19 pneumonia and requiring hospital care but not invasive or semi-invasive ventilation, was concluded early because it was not effective [36] . therapeutic scheme: -tocilizumab vial 20 mg/ml -first infusion at a dosage of 10-12 mg/kg < 30 kg and 8 mg/kg > 30 kg, (maximum dosage 800 mg, duration of infusion at least 60 min) -second infusion 12 h after the first (at the discretion of the doctor, in case of no response) -a possible third infusion after 24 h may be considered. after 24 h from the last administration, repeat the plasma dosage of il-6 and / or d-dimer. when to use biologic drugs: • serious or critical cases. • end of the initial phase of high viral load of covid-19 (afebrile> 72 h and/or at least 7 days after the onset of symptoms). • high levels of il-6 (> 40 pg/ml); alternatively, high levels of d-dimer and/or pcr and/or ferritin and/or fibrinogen increasing progressively. when not to use biologic drugs: • ast / alt value above 5 times normal levels. • neutrophils value lower than 500 cells/ml. • platelets value lower than 50,000 cells /ml. • documented sepsis from other pathogens other than covid-19. • presence of comorbidities related, according to clinical judgment, to an unfavorable outcome. • complicated diverticulitis or intestinal perforation. • immunomodulating and anti-rejection therapy. • known hypersensitivity to the drug. it is also recommended to avoid administration of biologics if anti-mrpv vaccination has been carried out in the past 30 days. if possible, before starting therapy: -quantiferon test -hbv and hcv markers other immunomodulant therapies in view of the fact that the alveolar damage of severe forms of sars-cov-2 infection is caused by a cytokine storm [60] , two other clinical trials have been started in italy on the use of emapalumab (anti-interferon gamma monoclonal antibody) and associated anakinra (il-1 receptor antagonist) and the use of sarilumab (monoclonal antibody that binds to the il-6 receptor). a belgian study comparing tocilizumab, anakinra and siltuximab is going to start soon. information and forms relating to the studies are available on aifa website, but there is no prevision for enrollment of pediatric patients [36] . among the new therapies planned for the future is the infusion of hyperimmune plasma from cured patients. this approach, already used in china and previously for ebola and sars, seems to give good results at least in the most serious cases. in the usa 3 studies have been launched that will evaluate this approach [61] . the choice to add empirical antibiotic therapy should only be made if there is reasonable evidence of bacterial superinfection. an increased procalcitonin, c-reactive protein and the presence of neutrophilic leukocytosis represent laboratory parameters suggestive of bacterial infection. from a clinical point of view, the persistence of fever for more than 3 days can be suggestive of bacterial infection [62] . tests for atypical bacteria (i.e. mycoplasma) should be performed in case of clinical suspect, in order to start targeted therapy. the start of an empirical antibiotic therapy is also recommended in the presence of comorbidities, such as immunodeficiency, cystic fibrosis, other chronic diseases of the respiratory tract, severe neuromotor disability. in case of productive cough, the collection of a sputum sample for culture examination before the start of antibiotic therapy would be indicated. in patients without risk factors, we recommend: -amoxicillin 90 mg /kg/day in 3 doses, in case of possible oral intake -ceftriaxone 80-100 mg/kg/day, in case of impossibility of oral intake. this drug is also recommended for the possibility of administering once a day and therefore reducing the risks for healthcare professionals. azithromycin preliminary data have suggested a possible efficacy of azithromycin in combination with hydroxychloroquine as a background therapy for sars-cov-2 infection. the exact role of this drug in covid-19 is unknown, as no in vitro data are available at present. it has been supposed a dual role, antiviral and anti-inflammatory. the anti-inflammatory action of azithromycin has been already demonstrated in many conditions. regarding the theoretical antiviral activity, the first published study was on a small sample size (36 patients, of whom only 6 receiving the combination of azithromycin/hydroxychloroquine) [41] . in this study, by gautret and colleagues, 100% of the patients taking hydroxychloroquine and azithromycin had negative nasopharyngeal swab 6 days from the start of therapy, compared to 57.1% of the hydroxychloroquine alone and 12.5% of the control group. the same results have been replicated in a larger population [63] . however, a more recent study on virological and clinical results of 11 adult patients with a serious disease raises doubts about the antiviral efficacy of this combination, as it reports results in contrast with those of gautret et al. [64] . this combination have been also evaluated in a larger observational study including 1061 adults, resulting to be safe and associate with a very low fatality rate. however, its efficacy has been doubted in a study on a large adult population in new york among hospitalized patients with covid-19, where treatment with hydroxychloroquine, azithromycin, or both were not associated with significantly lower in-hospital mortality [65] . considering the lack of a strong rationale and the absence of evidence of certain effectiveness in the treatment of covid-19 patients, the use of azithromycin should be considered carefully and the qtc interval strongly monitored. in fact, on april 2020, european medicines agency (ema) warned about risk of serious side effects particularly if associated with chloroquine and hydroxychloroquine [66] . dosage: adults: 500 mg the first day, then 250 mg/day for other 4 days children: 15 mg/kg the first day, then 7.5 mg/kg once a day for other 4 days the drugs used for the therapy of sars-cov-2 infection, especially lopinavir/ritonavir and azithromycin/ hydroxychloroquine, can have interactions with other drugs. before administering those treatment, evaluate possible interactions on drugs [67] . for all drugs, consent for off-label use must be requested and the procedures provided by the reference healthcare company must be followed. as for remdesivir, since it is an experimental drug, the procedures for compassionate use must be followed. in conclusion this position paper summarizes the suggested treatments in covid-19 infected children based on a review of the current literature carried out by the scientific committee of the italian society of infectious pediatric diseases. since most sars-cov-2 infections in children have a benign course, pharmacological treatment, other than supportive therapy, should be reserved to those with more severe cases. as new evidences are expected to be available in the next weeks/months, the italian society of pediatric infectious diseases will guarantee to maintain treatment indication updated on the website www.sitip.org world health organization. who director-general's opening remarks at the media briefing on covid-19 -11 systematic review of covid-19 in children shows milder cases and a better prognosis than adults the italian sitip-sip pediatric infection study group, et al. multicentre italian study of sars-cov-2 infection in children and adolescents screening and severity of coronavirus disease 2019 (covid-19) in children in madrid clinical features of severe pediatric patients with coronavirus disease 2019 in wuhan: a single center's observational study kawasaki-like multisystem inflammatory syndrome in children during the covid-19 pandemic in paris, france: prospective observational study an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study covid-19) treatment guidelines società italiana di malattie infettive tropicali. sezione regione lazio. raccomandazioni per la gestione clinica e terapeutica della covid-19 maggio 2020 multicenter initial guidance on use of antivirals for children with covid-19/ sars-cov-2 clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance multisystem inflammatory syndrome in children and adolescents with covid-19 pediatric acute respiratory distress syndrome: consensus recommendations from the pediatric acute lung injury consensus conference surviving sepsis campaign international guidelines for the management of septic shock and sepsis-associated organ dysfunction in children multicentre validation of the bedside paediatric early warning system score: a severity of illness score to detect evolving critical illness in hospitalised children covid-19: ibuprofen should not be used for managing symptoms, say doctors and scientists ema gives advice on the use of non-steroidal anti-inflammatories for covid-19 recommendations for inhaled asthma controller medications anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy isth interim guidance on recognition and management of coagulopathy in covid-19 hospital-associated venous thromboembolism in pediatrics: a systematic review and meta-analysis of risk factors and risk-assessment models developmental hemostasis: clinical implications from the fetus to the adolescent paediatric european network for treatment of aids (penta) guidelines for treatment of paediatric hiv-1 infection 2015: optimizing health in preparation for adult life national health commission & state administration of traditional chinese medicine. diagnosis and treatment protocol for novel coronavirus pneumonia a trial of lopinavirritonavir in adults hospitalized with severe covid-19 national health commission & national administration of traditional chinese medicine. diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7) european public assessment report (epar) for kaletra arguments in favour of remdesivir for treating sars-cov-2 infections remdesivir as a possible therapeutic option for the covid-19 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro remdesivir by gilead sciences: fda warns of newly discovered potential drug interaction that may reduce effectiveness of treatment sperimentazioni cliniche -covid-19 discovering drugs to treat coronavirus disease 2019 (covid-19) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) za zhi for the multicenter collaboration group of department of science and technology of guangdong province and health commission of guangdong province for chloroquine in the treatment of novel coronavirus pneumonia. expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia (article in chinese hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial information for clinicians on therapeutic options for covid-19 patients fda cautions against use of hydroxychloroquine or chloroquine for covid-19 outside of the hospital setting or a clinical trial due to risk of heart rhythm problems best pharmaceuticals for children act-pediatric trials network steering committee, et al. simulated assessment of pharmacokinetically guided dosing for investigational treatments of pediatric patients with coronavirus disease favipiravir: aggiornamento della valutazione della cts the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro the sars-cov-2 ivermectin navarra-isglobal trial (saint) to evaluate the potential of ivermectin to reduce transmission in low risk, non-severe covid-19 patients in the first 48 hours after symptoms onset: a structured summary of a study protocol for a randomized control pilot trial risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china anakinra for severe forms of covid-19: a cohort study interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study anakinra in covid-19: important considerations for clinical trials the lancet reumatology clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 novel paediatric presentation of covid-19 with ards and cytokine storm syndrome without respiratory symptoms intravenous anakinra for macrophage activation syndrome may hold lessons for treatment of cytokine storm in the setting of coronavirus disease 2019 the rheumatologist's role in covid-19 european public assessment report (epar) for roactemra (tocilizumab ema gives advice on the use of non-steroidal anti-inflammatories for covid-19 effective treatment of severe covid-19 patients with tocilizumab covid-19: consider cytokine storm syndromes and immunosuppression how blood from coronavirus survivors might save lives diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxy-chloroquine and azithromycin in patients with severe covid-19 infection association of treatment with hydroxychloroquine or azithromycin with inhospital mortality in patients with covid-19 in new york state covid-19: reminder of risk of serious side effects with chloroquine and hydroxychloroquine covid-19 drug interactions publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. all authors contribute to prepare the manuscript read and approved the final version. key: cord-310807-p5cb6idp authors: kanwar, anubhav; selvaraju, suresh; esper, frank title: human coronavirus-hku1 infection among adults in cleveland, ohio date: 2017-03-25 journal: open forum infect dis doi: 10.1093/ofid/ofx052 sha: doc_id: 310807 cord_uid: p5cb6idp background. human coronaviruses (cov) have been long recognized as a common cause of respiratory tract disease including severe respiratory tract illness. coronavirus-hku1 has been described predominantly among children less than 5 years of age in the united states with few studies characterizing the disease spectrum among adults. methods. nasopharyngeal specimens of patients with respiratory symptoms were analyzed for cov-hku1 by nxtag respiratory pathogen panel multiplex assay from february 7, 2016 to april 30, 2016. epidemiologic, clinical, and laboratory data were collected on adults (patients >18 years) whose samples screened positive. results. of 832 adult respiratory specimens screened, 13 (1.6%) cases of cov-hku1 were identified. adults age ranged between 23 and 75 years and 6 (46%) were males. all of whom had 1 or more respiratory symptoms, and 5 (38%) also reported 1 or more gastrointestinal symptoms. eleven (85%) reported history of smoking and 5 (38%) used inhaled steroids. seven (54%) required hospitalization, 5 (71%) of these needed supplemental oxygen, and 2 (29%) were admitted to intensive care. median length of hospitalization was 5 days. eight (62%) received antibiotics despite identification of cov-hku1. infectious work-up in 1 patient who died did not reveal any other pathogen. in 2 (15%) cov-hku1-positive adults, the only viral coinfection detected was influenza a. conclusions. coronavirus-hku1 accounted for 1.6% of adult respiratory infections and should be considered in differential diagnosis of severe respiratory illnesses among adults. coronavirus-hku1 has been predominantly reported in children in united states [5, 6] but less commonly among adults [7] [8] [9] . silva et al [9] found 1 case (0.5%) among 200 adults presenting with flu-like symptoms over a 5-month period, and gorse et al [7] reported 1 patient with cov-hku1 among 585 adults (0.17%) with history of copd. in this study, we report one of the largest case series of cov-hku1 infections among adults presenting with respiratory tract illness and describe their clinical characteristics. wards, and emergency department (ed) at the discretion of the on-service physicians. samples were placed in m4-rt viral transport media (remel, lenexa, ks) and transported to the laboratory where they were frozen until testing. testing was performed using the nxtag respiratory pathogen panel ce-ivd assay kits (luminex, austin, tx) per the manufacturer's guidelines (package insert, nxtag respiratory pathogen panel assay; luminex). in brief, 35 µl of extracted nucleic acid were added directly to nxtag respiratory pathogen panel preplated, lyophilized reagents. multiplexed reverse-transcription polymerase chain reaction and bead hybridizations were performed in each plate well under a single cycling program per manufacturer's instructions. the sealed plates were placed directly on the magpix instrument (luminex) for data acquisition. raw signals generated by the magpix instrument were subsequently analyzed by the assay-specific software accessory package using synct software to establish the presence or absence of each pathogen in each sample. for detection of covs, the nucleocapsid protein gene (n) was used to identify covs 229e, oc43, and nl63. the open reading frame 1aboratory region of the ribonucleic acid polymerase was used to identify cov-hku1. the limit of detection titer for cov-hku1 was 1.57e+04 genome copies/ml (package insert, nxtag respiratory pathogen panel assay). sample collection and data analysis were approved by the metrohealth medical center institutional review board (irb16-00162). clinical data analysis for adults (patients >18 years of age) included the following: age, gender, hospitalization status, length of hospitalization, clinical features, discharge diagnosis, outcome (death or survived), comorbidities (smoking, lung disease), laboratory and radiology results, intensive care unit (icu) stay, oxygen use and duration, treatment provided, and exposure history. clinical data were collected using redcap software (redcap 6.17.0, vanderbilt university). coronaviruses detected from all respiratory specimens during the study period is summarized in table 1 . of these, 832 originated from adult patients (>18 years) with 35 (4.2%) screening positive for covs. thirteen (1.6%) samples tested positive for cov-hku1 and represented 37% of all cov species detected among adults. median age of adult patients was 52 years (range, 23 to 79 years). eleven cov-hku1-positive adults (85%) had an underlying respiratory comorbidity, predominantly history of smoking, with 6 (46%) being current smokers (table 2) . five patients (38%) reported use of inhaled corticosteroids. one patient was on systemic immunosuppression (prednisone, azathioprine). in addition, 2 patients had underlying gastrointestinal comorbid conditions. all adults with cov-hku1 infection had respiratory symptoms ( table 3 ). shortness of breath was the most common respiratory complaint (77%) followed by cough (62%) and rhinorrhea (54%). however, fever, wheezing, and chest pain were less common (38%, 23%, and 15%, respectively). upper or lower gastrointestinal symptoms were reported in 5 (38%) adults. other symptoms included myalgias (31%) and sore throat (23%). seven patients (54%) required hospital admission, 5 (71%) of whom were diagnosed with pneumonia by the on-service team. among hospitalized adults, 2 (29%) were admitted to the icu. both patients were male. three patients (23%) were diagnosed with sepsis, 1 of whom developed septic shock and died. this individual had a history of smoking (64-pack years) and copd. another individual admitted to the icu developed pancreatitis, pericarditis, peritonitis, and altered mental status during hospitalization and recovered. all blood, urine, and sputum cultures from these individuals were negative for other bacterial, fungal, and viral infections. laboratory findings (table 3) were obtained in 9 (69%) of the cov-hku1 adults. of these, leukocytosis (white blood cell count [wbc] >11 000 cells/µl) was observed in 5 (56%), with an average of 29 600 cells/µl (range, 13 600-49 700 cells/µl). of these, neutrophilic predominance (neutrophil count >8000 cells/µl) was seen in 4 (80%) and bandemia (>10% bands) was seen in 2 (40%). one (8%) adult had leukopenia (wbc of 3700 cells/µl). twelve (92%) adults had chest radiography performed with 6 (50%) showing infiltrative disease. the chest imaging findings included prominence of interstitial markings with a reticulonodular pattern, ground-glass opacities, patchy consolidation, interlobular and intralobular septal thickening, basilar atelectasis, small pleural effusions, and hyperinflated lungs ( figure 2 ). eight (62%) received antibiotics, and 2 (15%) received oseltamivir, despite identification of cov-hku1 as the table 1 eighty-three percent of all cov-hku1-positive samples occurred within a 4-week period between february 7 and march 5 ( figure 1 ). this period included 10 (67%) of the adult cases. coronaviruses oc43 and nl63 had a more prolonged circulation but also predominated during these same 4 weeks. our report describes the largest number of cov-hku1 infections among the general adult population in the united states to date. we find that 1.6% of adults screened for respiratory viruses have evidence of cov-hku1. previous studies in north america have reported lower rates during a typical viral respiratory season [7-9]. on the other hand, cov-hku1 infection among adults outside the united states have been reported with rates ranging between 0.04% and 2.1% [11] [12] [13] [14] [15] . our rate of 1.6% is similar to these findings. we hypothesize that the overall rate might change if surveillance occurred throughout the year. it is likely that the study period represents peak cov-hku1 circulation in our community. we identified smoking as the predominant comorbid condition (85% of all cov-hku1-positive adults). smoking has been found to be an independent predictor of mers-cov acquisition in saudi arabia [16] and among 50% of smokers with cov-hku1 pneumonia in hong kong, china [11] . increased respiratory infections are hypothesized to be secondary to continuing parenchymal trauma and suppression of host resistance over time [17] . in our study, smoking was identified commonly among all age groups of adults with cov-hku1 disease irrespective of the age of onset of smoking. we found a high rate of cov-hku1 infections (38%) in patients using inhaled corticosteroids. although inhaled steroids have been accepted as safe agents with no or minimal systemic absorption, the effects on local cell-mediated immunity is unclear. they have been shown to be a risk factor for bacterial pneumonia in patients with asthma and copd, as well as influenza in copd patients [18, 19] . in a recent meta-analysis by dong et al [20] , there was an increased but nonsignificant trend towards influenza infection among patients taking inhaled fluticasone. further studies are needed to confirm any relationship between respiratory illness due to cov-hku1 and other cov with inhaled corticosteroids use. all patients we identified with cov-hku1 infection had respiratory symptoms with dyspnea, cough, and rhinorrhea being the most common. this is not unexpected due to our study design. similar studies describing hku1 in pediatric and adult patients have also found an association with upper and lower respiratory tract illness [7, 9, 12, 15, 21] . however, cov-hku1 has also been reported in patients presenting with nonrespiratory symptoms [8] . we identified gastrointestinal symptoms in 38% of cov-hku1-positive adults, consisting of diarrhea and nausea and/or vomiting. the mechanism for gastrointestinal symptoms seen in cov-hku1 disease remains unclear. vabret et al [22] and esper et al [23] have isolated cov-hku1 from stool samples in patients with diarrhea and nausea and/or vomiting. non-cov, such as influenza [24] , and other covs, such as sars-cov [25] and mers-cov [26] , have also been detected in stool specimens among patients with and without gastrointestinal symptoms. further studies are needed to determine whether cov-hku1 detection in stools is part of gastrointestinal disease versus asymptomatic viral shedding and whether it plays any role in disease transmission. it is interesting to note that adults identified with cov-hku1 had higher than expected severity of disease in our study, with increased occurrence of hospital admissions, oxygen use, and icu admission. this is in contrast to other studies in which at-risk patient populations had lower morbidity and hospitalization with cov-hku1 infections [7, 8, 27] . looking at other covs, the rates of hospitalization and morbidity were relatively similar in combined pediatric and adult studies [12] . our finding of 54% hospitalization and 15% icu admission could be accounted for by selection bias of the sample population (screening those presenting to the ed or hospitalized with respiratory tract illness). studies focusing on outpatient and asymptomatic adults will provide a better understanding of disease severity. however, this study does demonstrate cov-hku1's potential for severe illness. a 75-year-old with no history of immunosuppression died due to shock while meeting clinical criteria for sepsis and negative infectious work-up. it remains unclear what, if any, role cov-hku1 played in this patient's demise. most studies of cov-hku1 have demonstrated lower mortality [11] . in the 2 deaths reported by woo et al [11] , both patients were immunocompromised with history of cancer, diabetes mellitus, advanced age, and lymphopenia. another death linked to cov-hku1 was reported in a severely immunocompromised patient lymphodepleted by conditioning for autologous hematopoietic stem cell transplantation [28] . in this case, cov-hku1 was isolated from his lung tissue and bronchoalveolar lavage with autopsy revealing bronchopneumonia, organizing pneumonitis, and diffuse alveolar damage. in our study, hku1-positive adults who did not require hospitalization had a normal chest x-ray, whereas 86% among inpatients with cov-hku1 had chest infiltrates. garbino et al [29] reported interstitial and alveolar infiltrates in 52% of hospitalized patients with respiratory samples positive for cov-oc43, cov-nl63, cov-229e, and cov-hku1. given paucity of chest imaging data, complete characterization of radiographic findings in cov-hku1 infections is incomplete. approximately 62% of all patients with cov-hku1 infection in our study received antibiotics. this is similar to other studies in which up to 76% of patients with cov-positive bronchoalveolar lavage received antibiotics [29] . antibiotic use after a positive respiratory pathogen panel for viral pathogen was observed for median duration of 7 days in our study. this is consistent with other studies suggesting that mere disease diagnosis with respiratory pathogen panel might not alter antibiotic prescribing practices among healthcare providers for viral respiratory illnesses [30] . such practices are concerning for unnecessary antibiotic exposure and the potential to aid the development of multidrug-resistant organisms [31] . this highlights the need for education of providers to consider cov-hku1 as a pathogen associated with upper and lower respiratory tract infections and de-escalate antibiotics with negative bacterial cultures and antigen [32] . among all cov-positive adults, 17% had coinfections, the most common being influenza a and rhinoviruses. these findings are comparable to other studies [21] . higher coinfections with cov and other viruses could suggest common reservoirs, overlap in seasonality, and persistence of virus or viral genome in respiratory secretions [33] . among our 2 adults with cov-hku1 and influenza a coinfection, it was unclear whether symptoms were due to cov-hku1 or influenza or both. the clinical relevance of respiratory viral coinfections is not fully understood. higher severity of respiratory disease has been reported in certain pathogen coinfection combinations, including influenza a with covs [34] . in other studies, there was no increased severity of illness in cov-associated coinfections [15, 27, 35] . gaunt et al [27] showed that a quantitative viral load for cov was unchanged in cases of coinfection with rsv and monoinfection. further investigation on the role viral coinfections play in alteration of respiratory disease severity should be undertaken. our study has several limitations. by evaluating only symptomatic patients, we could have missed asymptomatic or subclinical infections. this could lead to an underestimation of the disease prevalence in the adult population and an overestimation of disease severity. the retrospective aspect of our study is insufficient to establish causality. larger study periods will also be required to establish a complete understanding regarding seasonal distribution patterns of this virus in our community. however, the majority of cov-hku studies in the literature are single-center experiences occurring over 1 season [12, 21] . our study provides needed insight into clinical characteristics and severity associated with cov-hku1 infection in adults in the northeast region of the united states. we opine that cov-hku1 infection should be considered in differential diagnosis of community-acquired pneumonia in adults, including those that require hospitalization. more studies are needed to fully understand the seasonality, risk factors, incidence, and epidemiology of cov-hku1. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. history and recent advances in coronavirus discovery identification of a new human coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus hku1 infection in the united states coronavirus infection and hospitalizations for acute respiratory illness in 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humans, saudi arabia smoking, alcohol consumption, and susceptibility to the common cold inhaled corticosteroids and the increased risk of pneumonia: what's new? a 2015 updated review salmeterol and fluticasone propionate and survival in chronic obstructive pulmonary disease use of inhaled corticosteroids in patients with copd and the risk of tb and influenza: a systematic review and meta-analysis of randomized controlled trials. a systematic review and meta-analysis of randomized controlled trials etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing detection of the new human coronavirus hku1: a report of 6 cases human coronaviruses are uncommon in patients with gastrointestinal illness prevalence of gastrointestinal symptoms in patients with influenza, clinical significance, and pathophysiology of human influenza viruses in faecal samples: what do we know enteric involvement of severe acute respiratory syndrome-associated coronavirus infection viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method use of a novel virus detection assay to identify coronavirus hku1 in the lungs of a hematopoietic stem cell transplant recipient with fatal pneumonia a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection antibiotic discontinuation rates associated with positive respiratory viral panel and low procalcitonin results in proven or suspected respiratory infections impact of antibacterials on subsequent resistance and clinical outcomes in adult patients with viral pneumonia: an opportunity for stewardship access to a polymerase chain reaction assay method targeting 13 respiratory viruses can reduce antibiotics: a randomised, controlled trial the role of infections and coinfections with newly identified and emerging respiratory viruses in children rate and influence of respiratory virus co-infection on pandemic (h1n1) influenza disease detection of four human coronaviruses in respiratory infections in children: a one-year study in colorado key: cord-293890-thfros7x authors: carbo, ellen c.; sidorov, igor a.; zevenhoven-dobbe, jessica c.; snijder, eric j.; claas, eric c.; laros, jeroen f.j.; kroes, aloys c.m.; de vries, jutte j.c. title: coronavirus discovery by metagenomic sequencing: a tool for pandemic preparedness date: 2020-08-21 journal: j clin virol doi: 10.1016/j.jcv.2020.104594 sha: doc_id: 293890 cord_uid: thfros7x introduction: the sars-cov-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. a protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness. aim: the aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness. methods: the performance of a viral metagenomic protocol in a clinical setting for the identification of novel coronaviruses was tested using clinical samples containing sars-cov-2, sars-cov, and mers-cov, in combination with databases generated to contain only viruses of before the discovery dates of these coronaviruses, to mimic virus discovery. results: classification of ngs reads using centrifuge and genome detective resulted in assignment of the reads to the closest relatives of the emerging coronaviruses. low nucleotide and amino acid identity (81% and 84%, respectively, for sars-cov-2) in combination with up to 98% genome coverage were indicative for a related, novel coronavirus. capture probes targeting vertebrate viruses, designed in 2015, enhanced both sequencing depth and coverage of the sars-cov-2 genome, the latter increasing from 71 to 98%. conclusion: the model used for simulation of virus discovery enabled validation of the metagenomic sequencing protocol. the metagenomic protocol with virus probes designed before the pandemic, can assist the detection and identification of novel coronaviruses directly in clinical samples. the severe acute respiratory syndrome coronavirus type 2 (sars-cov-2) pandemic of 2020 demonstrates the devastating effect an emerging virus can have. although previous pandemics such as the spanish flu (1918) and asian flu (1957) resulted in a multitude of fatal cases, the sars-cov-2 pandemic exhibits an unprecedented impact on public health, the economy and society as a whole. metagenomic next-generation sequencing (mngs) enables hypothesis-free sequencing of all nucleic acids in a given sample, including genomes of pathogens. all sequences are amplified, followed by classification of sequences based on a reference database. while research applications are more common, mngs is being introduced in clinical diagnostic laboratories as indicated by recently diagnosed cases of encephalitis [6] . implementation of mngs in clinical diagnostics requires validation of metagenomic protocols. metagenomic protocols and pipelines have been successfully used for detection of known pathogens [6] [7] [8] . however, detection and identification of novel, j o u r n a l p r e -p r o o f previously unknown emerging viruses presents a challenge due to the absence of their genome sequences in reference databases. in this study, we validated the identification of emerging coronaviruses by a viral metagenomic protocol, using clinical samples with sars-cov-2, and samples spiked with cultivated isolates sars-cov frankfurt-1 (sars-cov) and mers-cov emc/2012 (mers-cov). the validation included analysis of the performance of both an in-house and a commercially available data analysis pipeline, genome detective [9] . identification of coronaviruses was tested using modified databases lacking sars-cov-2, sars-cov, and mers-cov, mimicking the situation at the time of virus discovery. additionally, the efficacy of detection of novel coronaviruses using capture probes targeting vertebrate viruses [10] [11] known before the current pandemic was analyzed using a sars-cov-2 clinical sample. nasopharyngeal swabs were obtained from two patients who tested positive for sars-cov-2 by realtime pcr targeting the sars-cov-2 e-gene [12] with cq values of 20 and 30, respectively. these pcrs library preparation and sequencing were performed using a previously validated protocol [15] [16]. briefly, 200μl of patient samples were spiked with equine arteritis virus (eav) and phocid herpesvirus-1 (phhv-1) prior to na extraction using the magnapure 96 dna and viral na small volume extraction kit on the magnapure 96 system (roche, basel, switzerland) resulting in 100μl nucleic acid-containing eluate. of this eluate, 50μl per sample was used as input for the library prep, utilizing the nebnext ultra ii directional rna library prep kit for illumina (new england biolabs, ipswich, ma, usa), dual indexed nebnext multiplex oligos for illumina (1.5µm), and a protocol optimized for processing rna and dna simultaneously in a single tube [15] . library preps of the samples where processed both with and without enrichment for viruses using sequence capture probes (see below). subsequent sequence analysis was performed using a after quality pre-processing using an in-house qc pipeline, biopet version 0.9.0 [17] and removal of human reads after mapping them to human reference genome grch38 [18] with bowtie2 version 2.3.4 [19] , the remaining sequencing reads were taxonomically classified using centrifuge 1.0.2-beta [20] pre-processed short reads were de novo assembled into contigs using spades version 3.10.1 [22] . all contigs were analyzed using the ncbi basic local alignment search tool (blast 2.8.1) [23] using the blast ncbi's nucleotide (nt) database (accessed april 2018). only viral hits for contigs with a length of ≥500bp were selected to identify the best shared homology to viruses. a length of 500bp was taken to ensure coverage of the built contigs by at least 3 reads, to rule out any possible contamination. only hits dated prior to 2020 genomes were considered to mimic the virus discovery setting for sars-cov-2. after extraction of human reads, fastq files generated for sars-cov-2 samples (with and without viral enrichment) were uploaded for classification and de novo assembly by the commercial webbased tool genome detective v1.120 (www.genomedetective.com, accessed 2020-05-11) [9] , using a reference database (generated 2019-09-21). in brief, after removal of low-quality reads and trimming by trimmomatic [24] , candidate viral reads were identified using the protein-based alignment method diamond [25] in combination with the swissprot uniref90 protein database followed by de novo assembly using metaspades [26] . blastx and blastn [23] were used to search for candidate reference sequences using the ncbi refseq virus database (accessed 2019-09-21). consensus sequences were produced by joining de novo contigs using advanced genome aligner [27] . classification results of viral reads are shown in figure 1 and results of de novo assembly of all samples for contigs longer than 500bp are shown in table 2 . blastn was used to search for hits with sequence homology. only viral hits with the lowest e-value of all matches identified that were submitted before the publication of sars-cov-2 genomes were considered. blastn search results of the contigs with coronaviridae hits are listed in table 2 including the length of the longest contig for each sample. identity data of the hits with the lowest evalue are listed in supplementary table 1 . additional blast alignment figures of the longest contigs of both the sars-cov and mers-cov samples can be found in supplementary figure 1 and 2, respectively. genomedetective results of identification of sars-cov-2 sequences using a database created before the emergence of sars-cov-2 are shown in figure 2 . sars-cov-2 sequences were identified as sars-cov, with nucleotide and amino acid identity of 80-81% and 83-85% respectively in combination with up to 98% genome coverage, being indicative for a novel finding. the efficacy of a metagenomic sequencing protocol using capture probes targeting vertebrate virus sequences designed before the emergence of sars-cov-2, was studied in the context of virus discovery. we analyzed metagenomic data from the two sars-cov-2 positive samples prepared both with and without viral enrichment. the total amount of contigs and the number of contigs matching genomes of viruses form coronaviridae are shown in table 2 and reads mapping to the sars-cov-2 reference genome were used to visualize the difference in using capture probes as depicted in figure 3 , where the sars-cov-2 genome is almost completely covered. the two largest contigs built by spades that had a hit with the lowest e-value when blasted against genomes from coronaviridae, were 4866bp and 5811bp in length for the two sars-cov-2 samples enriched using probes. in this study, we evaluated the performance of a metagenomic sequencing protocol for the identification of emerging viruses using clinical samples in combination with a simulated reference database. high and low loads of sars-cov-2, sars-cov, and mers-cov in clinical samples could be detected as 'novel' viruses, using only reference sequences created before these viruses emerged. sequence reads were assigned to the closest relatives of these viruses available at that time and assembled with heterologous sequences to 'novel' consensus genomes. low identity of these consensus genomes with genomes of closely related ones indicated a novel virus. additionally, j o u r n a l p r e -p r o o f probes targeting sequences of vertebrate viruses, available prior to the coronavirus pandemic of 2020, succeeded in the capture of nearly the full genome of sars-cov-2. it must be noted that the validation was performed using emerging viruses with nucleotide identity of over 76% to their closest known relatives and conclusions cannot be extended to novel viruses which are less closely related. nucleotide (and amino acid) identities reported in literature with regard to novel human pathogenic viruses vary, for example 50% for older viruses like sars-cov [1] , 80% for mers-cov [14] , 88% for parts of the human metapneumovirus [28] and up to 97.2% for parts of sars-cov-2 [29] . several reports have shown an increase of 100-10.000 fold in sensitivity for detection of known viruses when using capture probes [10] , [30] and here we report the potential of using capture probes in the detection of novel viruses. sequence variation was addressed in the probe design by retaining mutant or variant sequences if sequences diverged by more than 90% [10] . lipkin and colleagues describe the capture of conserved regions of a rodent hepacivirus isolate with 75% identity using virseqcap vert, and even 40% for detection rather than whole genome sequencing is suggested [10] . the capture probes used in this study targeted sequences of several isolates of alpha-, beta-, gamma-, and deltacoronaviruses. in this study the whole genome of sars-cov-2, with 76-100% overall nucleotide identity to the probe targets, was detected using these probes. metagenomic sequencing is increasingly being used in diagnostic laboratories as a hypothesis-free approach for suspected infectious diseases in undiagnosed cases. metagenomic sequencing in diagnostic laboratories has resulted in the detection of pathogens present in the reference database but either not tested for by routine methods due to rare or unknown associations with a specific disease, or for which routine testing failed (e.g., due to primer mismatches). additionally, mngs enables the detection of novel pathogens not (yet) present in the databases. common bioinformatic classifiers are usually not designed for discovery purposes, so additional algorithms including a separate validation to assess the performance in a discovery setting are needed. reports on specific j o u r n a l p r e -p r o o f bioinformatic discovery tools typically describe the algorithm and an in silico analysis and here we present validation studies on the performance of virus discovery tools using clinical samples. implementation of virus discovery protocols in diagnostic laboratories may contribute to increased vigilance for emerging viruses and therefore aids in surveillance and pandemic preparedness. the authors have no conflicts of interest. j o u r n a l p r e -p r o o f table showing the total number of built contigs with a length >=500bp, the number of these contigs where the hit with the lowest e-value would be a hit to viruses, the number of contigs where the hit with the lowest e-value would be a hit to coronaviridae and of this last group the length of the longest contig, the alignment length, identity match, taxonomic name of blast result and the release years of sequences belonging to the species and subjects found by blast. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid vip: an integrated pipeline for metagenomics of virus identification and discovery metagenomics for pathogen detection in public health genome detective: an automated system for virus identification from highthroughput sequencing data virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis', mbio enhanced virome sequencing using targeted sequence capture detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr mechanisms and enzymes involved in sars coronavirus genome expression isolation of a novel coronavirus from a man with pneumonia in saudi arabia retrospective validation of a metagenomic sequencing protocol for combined detection of rna and dna viruses using respiratory samples from pediatric patients the respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease fast gapped-read alignment with bowtie 2 centrifuge: rapid and sensitive classification of metagenomic sequences interactive metagenomic visualization in a web browser spades: a new genome assembly algorithm and its applications to single-cell sequencing basic local alignment search tool trimmomatic: a flexible trimmer for illumina sequence data fast and sensitive protein alignment using diamond metaspades: a new versatile metagenomic assembler an alignment method for nucleic acid sequences against annotated genomes analysis of the genomic sequence of a human metapneumovirus a novel bat coronavirus closely related to sars-cov-2 contains natural insertions at the s1/s2 cleavage site of the spike protein improved diagnosis of viral encephalitis in adult and pediatric hematological patients using viral metagenomics we would like to thank joost van harinxma thoe slooten and alhena reyes for the library preparations and viral probe enrichments. additionally, we would like to thank lopje höcker, margriet kraakman and tom vreeswijk for all their technical assistance in the lab. key: cord-271505-eot38721 authors: wang, hongliang; rao, shuan; jiang, chengyu title: molecular pathogenesis of severe acute respiratory syndrome date: 2006-09-28 journal: microbes infect doi: 10.1016/j.micinf.2006.06.012 sha: doc_id: 271505 cord_uid: eot38721 the global outbreak in 2002–2003 of severe acute respiratory syndrome (sars) posed a serious threat to public health and had a significant impact on socioeconomic stability. although the global outbreak of sars has been contained, there are serious concerns over its re-emergence and bioterrorism potential, and up to date, no specific treatment exists for this disease. here we review the progress of studies on the pathogenesis of the disease, in particular, studies on the molecular level. during the winter of 2002e2003, a new 'plague' emerged in guangdong province, china, and quickly spread to other countries. patients were characterized by fever, dry cough, dyspnea, headache, and hypoxemia; and death could be a result of progressive respiratory failure due to alveolar damage. the syndrome was designated 'severe acute respiratory syndrome' (sars). the identification of the etiologic agent, a novel coronavirus, as sars-associated coronavirus (sars-cov) was quickly made by international collaboration [1, 2] . by 31 july 2003, when the pandemic terminated, 8096 people in 26 countries had been diagnosed with probable sars, 774 of whom died [http://www.who.int/csr/sars/country/ table2004_04_21/en/index.html]. in the winter of 2003e 2004, sporadic cases were reported, including four cases in guangdong province, and laboratory acquired sars from singapore, taiwan and beijing [http://www.who.int/csr/don/ archive/disease/severe_acute_respiratory_syndrome/en/index. html]. although the death rate is low in comparison with fatalities during previous pandemics, the rapidity of spread due to air travel, the coverage in the media and the enormous economic and social impacts, the fear of renewed outbreaks as well as the potential misuse of the virus as a biological weapon all contribute to the far more pronounced impact of sars-cov. since the identification of the etiological agent, rapid progress has been made towards understanding this newly emerged pathogen. most previously published reviews focused on the epidemiology, clinical presentation and potential treatment of sars-cov infection; this review focuses on the molecular mechanisms of sars pathogenesis. the newly identified virus is a new member of the family coronaviridae, genus coronavirus. it is a family of large, enveloped, positive-sense single-stranded rna viruses that replicate in the cytoplasm of animal host cells. the genome of sars-cov is about 29 kb with 13e15 open reading frames (orfs). downstream of orf1a and 1b is orfs that encode the four main structural proteins, namely, spike (s), envelope (e), membrane (m), and nucleocapsid (n) protein [3, 4] . the spike protein of sars-cov, which is involved in virus binding, fusion, and entry, is a typical class i viral fusion protein, similar to the transmembrane glycoproteins of many enveloped viruses [5] . the amino-terminal s1 and carboxylterminal s2 subunits of the sars-cov s protein can be identified through their homology with the s1 and s2 subunits of other coronaviruses. coronaviruses are usually subdivided into three phylogenetic groups. sars-cov is different from the known coronaviruses; depending on the method of sequence analysis used, sars-cov either constitutes a new phylogenetic group [1e4] or a subgroup of group 2 [6] . studies using pseudotyped lentiviruses carrying the s, m and e glycoproteins of sars-cov demonstrated that the spike protein is both necessary and sufficient for virus attachment to susceptible cells [5] . the cellular receptors were detected by taking advantage of this tag. in an in vitro study, li et al. demonstrated that the angiotensin-converting enzyme 2 (ace2) is a functional cellular receptor of sars-cov, by using coimmunoprecipitation of the virus glycoprotein (s1) with lysates from cells that are susceptible to virus infection (vero e6 cells) followed by mass spectrometry analysis [7] . later, our group proved that ace2 is crucial for sars-cov infection in vivo employing an ace2 knockout mouse [8] . later, the structure of sars coronavirus spike receptor-binding domain (rbd) complexed with ace2 was determined [9] . all these findings will influence the ongoing vaccine development and biological analysis. immunostaining techniques identified ace2 on the surface of type 1 and type 2 alveolar epithelial cells, enterocytes of the small intestine and the brush border of the proximal tubular cells of the kidney [10] . these localizations explain the documented tissue tropism of sars-cov for the lung and gastrointestinal tract. however, it should be noted that colonic enterocytes as well as the liver tissue were largely negative for ace2 protein expression, while sars-cov replication does occur therein. in contrast, whereas ace2 is strongly expressed on the endothelial cells of arteries and veins of all tissues and the smooth muscle cells of the intestinal tract, there is no evidence of virus infection at any of these sites [10] . these observations suggest that another cellular factor is required for successful virus infection. pseudotyped virus containing the spike protein has also been shown to bind to dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign) [5] . dc-sign is a type ii transmembrane adhesion molecule found on dendritic cells. it consists of tandem repeats of a highly conserved 23-amino acid sequence and a c-type lectin domain that recognizes carbohydrate residues on a variety of pathogens. unlike the ace2 receptor on pneumocytes and enterocytes, dc-sign does not permit sars-cov infection of the dendritic cells but can transfer the virus to susceptible target cells through a synapse-like structure (trans infection), and this cell-mediated transfer can be blocked by the mouse anti-sars-cov spike protein antiserum [5] . besides dc-sign, there is also a report showing that cells expressing a molecule which is 77% identical to dc-signe l-sign (cd209l) can enhance sars-cov infection [11] . another study proved that l-sign is a receptor for sars-cov with the expression cloning method [12] . this study also indicated that cells expressing l-sign became susceptible to sars-cov infection, but l-sign was a less efficient receptor when compared with ace2 [12] . l-sign also is a type ii transmembrane glycoprotein in the c-type lectin family. it has a structure similar to that of dc-sign, except that l-sign has considerable polymorphism in the tandem repeat domain. since l-sign has much higher polymorphism, there must exist different combinations in the population. chan et al. found that homozygous expression of polymorphic variants of l-sign plays a protective role in sars-cov infection, because cells homozygous for l-sign show higher binding capacity for sars-cov, higher proteasomedependent viral degradation and a low capacity for trans infection [13] . all these findings indicate that lectins may play a role in the pathogenehost interaction. whether there are other molecules involved in the sars-covehost interaction remains open. after the virus attaches to the host cells, the virus enters the cell either through ph-dependent receptor-mediated endocytosis or through direct membrane fusion. currently, there is no consensus about this question in the sars-cov case. some experiments demonstrated that sars-cov might gain cell entry via ph-dependent endocytosis, but there are also studies showing that the sars coronavirus entered the cells through direct membrane fusion. the n-terminal half of the s protein (s1) contains the receptor-binding domain, whereas the c-terminal half (s2) is the membrane-anchored membrane-fusion subunit, which contains two heptad repeat regions (hr1 and hr2). in the native state, spike proteins on the virus surface could be in oligomeric form and form the stems of the spikes on sars-cov; hr1 regions may be in random coil conformation covered by the s1 domain. after binding to ace2 on the target cells, the s2 domain changes conformation by forming a-helices, extending and inserting its inert fusion peptide into the target cell membrane, and exposing hr1 and hr2 regions. then the hr1 and hr2 regions form a six-helix oligomeric complex, with the hr2 trimer as a core. this fusion-active core structure brings the viral and target cell membranes into close proximity, resulting in fusion between the membranes and formation of fusion pores, which allows the virus genome to enter the target cell [14] . apart from direct membrane fusion at the target cell surface, sars-cov might gain cell entry via ph-dependent endocytosis, which is also mediated by the s protein. the spike glycoproteins of most of the enveloped rna viruses can mediate the attachment, fusion and entry of the virus. however, an activating trigger, which can result in a conformational change, is usually required to make the spike glycoproteins fulfill their functions. for example, binding to specific receptors/coreceptors is the activating trigger for mlv and hiv, while the influenza virus requires only an acidic milieu. however, this is different in the case of sars-cov, as some groups have found that a special endosomal protease, cathepsin l, was crucial in the activation of viral infectivity; inhibitors of cathepsin l can prevent sars-cov entry [15] . so sars-cov may first bind to ace2 on the cell surface and be taken up into a vesicle (endocytosis). later, cleavage of spike protein or ace2 by cathepsin l facilitates fusion of the viral membrane and the vesicle membrane. these results are consistent with previous findings showing that the inhibitors of vacuolar acidification could block infection by s-bearing pseudotypes in a dose-dependent manner [5] , as cathepsin l is a ph-dependent cysteine protease with its maximal activity in an acidic milieu and may lose its activity with increasing ph. therefore, the spike protein mediated entry of sars-cov might be through direct membrane fusion or in a ph-dependent endocytosis fashion, and certain factors might influence this process. the mean incubation period for this disease is estimated to be 6.4 days (95% ci 5.2e7.7). the mean time from onset of clinical symptoms to hospital admission varied from 3 to 5 days [16] . the most common symptoms included fever, chills, rigors, and myalgia. cough and headache were also reported in more than 50% of the patients. other common findings were lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase and creatine kinase levels [17] watery diarrhea has also been reported [18] . results from autopsied organs showed that the predominant damage occurred in lungs. the pathological characteristics of the lung include acute pulmonary exudative and hemorrhagic inflammation in lung tissue, greatly increased permeability of capillaries leading to a leakage of proteins such as cellulose, or even erythrocytes, into the alveolar wall and space, and accumulation of exudates and edema fluids in most of the alveoli spaces, and even hyaline membrane formation in some alveoli spaces [19] . airspace opacity distributed peripherally in the lower lung zone is the most commonly encountered radiographic image in patients with sars at presentation. radiographic progression to multifocal or bilateral lung involvement followed by radiographic improvement occurs during treatment in about 70% of patients with sars. no cavitation, lymphadenopathy, or pleural effusion was demonstrated [20] . typically, sars follows a three-phase clinical course [18] . phase 1 is a viral replication phase that involves an initial presentation of high fever and myalgia of a few days' duration, which generally improve after a few days. the increasing viral load during this phase suggests that the symptoms are largely related to the effect of viral replication and cytolysis; however, this may also be due to antiviral and immunomodulatory therapies. in phase 2, which begins about 8 days after onset of fever, patients frequently had recurrence of fever, onset of diarrhea, and oxygen desaturation. the timing of igg seroconversion, which starts on day 10, seems to correlate with falls in viral load, which occurs between days 10 and 15. severe clinical worsening also occurs at this time. therefore, the lung damage at this phase is most likely related to immunopathological damage as a result of host response, rather than uncontrolled viral replication itself [18] . most cases improve after steroid treatment and enter a third phase of rehabilitation, while about 20% deteriorate with evidence of severe lung injury characterized by acute respiratory distress syndrome (ards) necessitating ventilation [18] . epidemiological analysis of the patient population shows that some factors may influence the final outcome of the disease. firstly, age: in patients older than 65 years, the mortality rate exceeds 50% [21] , while sars in children, especially those under 12 years, is generally associated with an uneventful course and a good outcome. secondly, coexisting illnesses, especially diabetes mellitus and heart disease, are consistently found to be independent prognostic factors for poor outcome, which is defined as death, need for mechanical ventilation, or admission to an intensive care unit [18, 21] . thirdly, in some studies, an increased lactate dehydrogenase level and elevated neutrophil count at the time of admission, as well as low cd4 and cd8 lymphocyte counts, were associated with a poor prognosis [22] . although much has been learned about sars in the three years since its discovery, aspects of the pathogenesis of the disease are still not fully understood. but it needs to be emphasized that since there is no specific drug or vaccine available, research on molecular mechanisms is crucial to identify potential treatment targets. and those with immunosuppressive conditions such as hiv infection or treatment with immunosuppressors is less severe, also indicating that immunopathology plays an important role in phase 2. the use of steroids for sars in this phase seems to be beneficial, which is also evidence that the inflammation and immune response play a role in this phase. in general, during a viral infection, most cell types in the body respond by secreting high levels of type 1 interferons. this is not the case in the sars-cov infection. the immune-related genes that were overexpressed after the onset of sars are usually associated with the innate immune response against bacterial infection and not against a viral infection. for example, the expression of lactoferrin is upregulated [23] . in addition, other innate immune defenses, such as the collectins, which can bind the glycosylated sars-cov s protein, may play an important role in host defense. this suggests that the response of sars affected patients is mainly an innate inflammatory response rather than a specific immune response against a viral infection [23] . besides the function of limiting virus spread, the immunological response against viral infection can also cause pathological damage to the host tissues. this is especially a concern in the case of proinflammatory cytokines. nicholls and colleagues suggested that pro-inflammatory cytokines released by activated macrophages in alveoli could have a prominent role in the pathogenesis of sars [24] . t cells are essential for adaptive immunity against viral infections in vivo. antiviral cd4 þ helper t cells help in the production of virus-specific antibodies by b cells, while cd8 þ ctls can kill virus-infected host cells. in the case of sars-cov infection, there is no conclusion as to the degree that t cells can influence the progress of the disease, since lymphopenia with a rapid decrease in both cd4 and cd8 t cells is common during the acute phase of sars [22] . neutralizing antibodies (igg) have been detected in sars patients, suggesting that humoral immunity plays an important role in the elimination of the virus. this is further evidenced by the activity of an anti-s1 human monoclonal antibody, 80r, in neutralizing sars-cov infection and inhibiting syncytia formation [25] . virus-induced apoptosis is a common phenomenon in viral infection, especially rna virus infections, including some coronaviruses. apoptosis can be used by the host cells to clear off the virus in the infected cells and also can assist virus dissemination by the release of viral particles, thereby facilitating its survival in vivo. therefore, apoptosis can have two opposite roles on the pathogenicity of viral infection, enhancing or suppressing the viral infection. in the case of sars, apoptosis was observed in patients' lung epithelial cells; thus, sars-cov induced apoptosis would certainly have a deleterious pathogenic role, leading to severe tissue damage [26] . this might explain the severe respiratory system damage in sars patients. apoptosis may also play an important role in the hematological changes besides the direct injury in the lung. as mentioned above, hematological changes in patients with sars are common and include lymphopenia, thrombocytopenia and occasionally leukopenia. the mechanism underlying this phenomenon remains unclear, but there are studies indicating that apoptosis as well as the immune response are involved [27] . several studies have focused on the mechanism of sars-cov induced apoptosis, for example, proapoptotic components of the virus genes and apoptosis pathways. sars-cov infection in vero e6 cells can lead to apoptosis. further studies showed that a variety of signaling pathways were phosphorylated or dephosphorylated when the cells were infected with sars-cov. specifically, p38 mitogen-activated protein kinase (mapk) is thought to be involved in induction of apoptosis, since mizutani et al. found that the p38 mapk and its downstream targets, mapkapk-2 and hsp-27 were activated during viral replication [28] . a p38 inhibitor was able to partially prevent cytopathic effects induced by sars-cov infection. the signal transducer and activator of transcription (stat)-3, which is usually constitutively phosphorylated at a tyrosine residue (705), is dephosphorylated by sars-cov-induced activation of p38 [28] . although akt, an inhibitor of apoptosis, was also partially activated, this weak activation cannot prevent sars-cov infection-induced apoptosis in vero e6 cells. and recently, research on the 90 kda ribosomal s6 kinases (p90rsks), an important substrate of the erk, showed that the specific serine residue (380) of p90rsks, that has been reported to be involved in autophosphorylation by activation of the c-terminal kinase domain, was phosphorylated in confluent sars-cov infected cells, and this phosphorylation can be inhibited by an inhibitor of p38 mapk [29] . all these results suggest that sars-cov induced apoptosis in vero e6 cells is related to p38 mapk. besides the whole virus, certain components of the virus can also induce apoptosis. it has been reported that sars-cov proteins 3a, 3b and 7a can induce apoptosis; the sars coronavirus nucleocapsid protein can induce apoptosis in cos-1 cells in the absence of growth factors; the c-terminal domain of sars-cov spike protein is sufficient to induce apoptosis in vero e6 cells. a recent study showed that the induction of apoptosis by the 7a protein also is related to its ability to activate p38 mapk [30] . in adult sars patients, respiratory distress is the principal cause of mortality. the histological change associated with ards is called diffuse alveolar damage (dad), which is characterized by structureless non-cellular exudates filling the bronchioles. dad is associated with a high mortality rate, and apart from supportive clinical care, there are few specific therapeutic options of proven benefit. recent studies have shown that the renin-angiotensin system plays an important role in the sars-cov caused acute lung failure. acid aspiration or sepsis in wild-type mice, which mimics human acute lung injury, resulted in impairment of lung function as assessed by lung elasticity, blood oxygenation and pulmonary edema. the mice developed edema, alveolar wall thickening, bleeding, inflammatory cell infiltration and hyaline membrane formation. but in the ace2 knockout mice, lung injury was much more severe than in the wildtype mice when assessed similarly. a rescue experiment using the recombinant human ace2 protein showed a decreased degree of acute lung injury in both the knockout and wildtype mice. all these results demonstrated that loss of ace2 is essential for lung injury [31] . both ace and ace2 are key enzymes in the renin-angiotensin system. ace cleaves the decapeptide angiotensini (angi) into octapeptide angiotensinii (angii). ace2 cleaves a single residue from angi to generate ang1e9, and a residue from angii to generate ang1e7. in this way, ace2 counterbalances the function of ace and negatively regulates the angii level. thus loss of ace2 expression will lead to a high level of angii, which will then, through receptor at1a, have a causative role in acute lung failure (fig. 1 ). we speculated that the acute lung failure caused by sars-cov was mainly due to the function of ace2. first we found that experimental infections of sars-cov in wild-type mice resulted in considerably reduced ace2 expression in the lungs, while ace expression was not changed. to further test our hypothesis, we established a defined model system using recombinant sars-cov spike protein. this model system allowed us to avoid possible secondary effects resulting from viral replication or infections in vivo and to directly test whether sars-cov spike protein might adversely affect acute lung injury through modulation of ace2. with this system we found that binding of spike to endogenous ace2 in vero e6 cells also resulted in downregulation of ace2 surface expression. further studies showed that treatment with spike protein worsened the lung function in wild-type mice. moreover, spike treatment of acid-challenged wildtype mice augmented the pathological changes in the lung parenchyma and increased lung edema, while in vivo spike protein administration did not affect the severity of lung failure in ace2 knockout mice, indicating that the effect of spike protein on acute lung injury is ace2 specific. thus we hypothesize that infection with sars-cov can result in ace2 downregulation through binding of sars-cov spike protein to ace2. given that ace2 is a key negative regulatory factor for severity of lung edema and acute lung failure, sars-cov spike protein-mediated ace2 downregulation then contributes to the severity of lung pathologies [8] (fig. 1) . about one million people suffer from ards due to different disposing factors, and the death rate can reach 50%. as the recombinant ace2 and angiotensin ii receptor inhibitors are already in clinical use for control of blood pressure, this finding points to a possible therapy for the otherwise incurable disease (fig. 1) . besides ards that contributes significantly to sars-cov caused death, other sars-cov associated diseases, such as hypertension, glucose increase, and heart dysfunctions cannot be ignored [18, 21] . the mechanisms underlying the pathogenesis are still unclear and need to be understood. currently, there is no antiviral therapy of proven value for sars. clinically, treatment of sars includes anti-sars-cov therapy and anti-inflammatory treatment to limit viral pneumonitis and subsequent pulmonary fibrosis. with insights into the field of sars pathogenesis and sars-cov genome structure, novel potential therapeutic targets for antiviral therapy were evaluated. for example, inhibitors of each step of the virus life cycle, like binding inhibitors, fusion inhibitors, rna transcription (replication) inhibitors as well as protease inhibitors, were designed and evaluated. only a few potential anti-sars agents have been tested in animal models, and their efficacy in humans is still unknown. inconsistent results between different groups investigating the same compound may be related to testing methodology, in particular, differences between in vitro and in vivo antiviral mechanisms. new technologies such as sirna may be important. however, this new technology is still riddled with practical difficulties, like efficacious delivery systems and safety concerns, limiting its application to patient treatment. using sera from people convalescing from sars to treat sars patients was proved to be effective, implying that neutralizing antibody can serve as a therapeutic strategy. a retrospective study in a limited number of patients using human sars convalescent plasma suggested that passive immunization had no obvious adverse effects [32] . however, the use of convalescent sera is not a practical therapy, especially when there is an outbreak worldwide. there are still no effective antiviral therapies that can be used immediately on patients. the most promising method to prevent the disease is a protective vaccine. immediately after the sars outbreak, researchers began to investigate whether inactivated vaccine could be used to prevent sars. in china, a collaborative group from the chinese academy of medical sciences, china's cdc, and the sinovac biotech company passed the inactivated sars-cov strain pumc001 vaccine clinical trial phase 1 from the sfda in the summer of 2005. although the inactivated sars vaccine seems useful and reasonable in sars prevention, the safety of the inactivated sars-cov vaccine is a serious concern. vaccines targeting structural genes, especially the recombinant spike protein, seem the safest strategy. a dna vaccine encoding s protein can elicit neutralizing antibodies and induce t cell response, which can protect mice from sars infection [33] . varieties of vectors were used to express the spike protein and then used to immunize mice, african green monkeys, and hamsters. neutralizing antibodies were detected which protected them from infection. the dna sars vaccine invented by scientists at the national institute of allergy and infectious diseases and produced by vical inc. of san diego entered clinical trial phase 1 under us fda guidelines at the end of 2004. recently, a group proved that rbd could elicit much higher titers of neutralizing antibodies than did the full-length s protein [34] , suggesting that rbd may be a potential vaccine candidate. however, the discovery that spike rbd alone could worsen acute lung injury in mice indicated the potential safety issue of spike as protein vaccine. the harm of spike rbd to the lung was thought to be caused by its binding to sars receptor ace2 [8] . it is reported that s proteins from the 2002e2003 sars outbreak and from the much less severe 2003e2004 outbreak have different binding affinities to the receptor ace2. the latter has lower affinity to human ace2, mainly due to an alteration of amino acid residues at sites 479 and 487 [35] . therefore, in order to obtain safer recombinant spike protein vaccine, site-directed mutagenesis at these sites might be necessary to eliminate the binding affinity of spike to ace2. in summary, the global outbreak of sars has led to the formation of a successful network of laboratories, and much has been learned about sars in the three years since its discovery. however, some aspects of the molecular pathogenesis of the disease are still not fully understood. further investigations of various aspects, including the life cycle of the virus, the molecular mechanisms of the disease, and the factors that can influence the progress of the disease, will result in a more thorough understanding of this new pathogen. results from further research will certainly suggest more promising treatment strategies and may lead to prevention of this disease. fig. 1 . schematic representation of the role of ras in the sars-cov induced acute lung injury and the potential therapeutic drugs. ace can cleave angiotensin i to produce angiotensin ii, which can then either bind to at1ar leading to lung injury, or bind to at2r reducing the severity of lung injury. ace2, on the contrary, can counteract ace by converting the angiotensin ii to a less damaging molecule. sars-cov infection or spike protein treatment can down-regulate the expression of ace2, and thus aggravate lung injury. based on these findings, ace inhibitor (such as lisinopri, captopri), at1ar inhibitor (such as losartan, valsartan) as well as recombinant human ace2 (rhuace2), are all potential drugs for this kind of acute lung injury. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome nabel, ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars coronavirus-induced lung injury structure of sars coronavirus spike receptor-binding domain complexed with receptor tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong a cluster of cases of severe acute respiratory syndrome in hong kong clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study pathological study on severe acute respiratory syndrome severe acute respiratory syndrome: radiographic appearances and pattern of progression in 138 patients clinical features and short-term outcomes of 144 patients with sars in the greater toronto area haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis expression profile of immune response genes in patients with severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome evaluation of human monoclonal antibody 80r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants sars coronavirus induces apoptosis in vero e6 cells hematological findings in sars patients and possible mechanisms tyrosine dephosphorylation of stat3 in sars coronavirus-infected vero e6 cells regulation of p90rsk phosphorylation by sars-cov infection in vero e6 cells palese, 7a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen-activated protein kinase angiotensinconverting enzyme 2 protects from severe acute lung failure retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone reatment in sars patients a dna vaccine induces sars coronavirus neutralization and protective immunity in mice severe acute respiratory syndrome: vaccine on the way receptor and viral determinants of sars-coronavirus adaptation to human ace2 the authors thank dr. wenhui li of harvard medical school for reading this manuscript and for his useful comments and suggestions. key: cord-287758-da11ypiy authors: mônica vitalino de almeida, sinara; cleberson santos soares, josé; lima dos santos, keriolaine; emanuel ferreira alves, josival; galdino ribeiro, amélia; trindade tenório jacob, íris; juliane da silva ferreira, cindy; celerino dos santos, jéssica; ferreira de oliveira, jamerson; bezerra de carvalho junior, luiz; do carmo alves de lima, maria title: covid-19 therapy: what weapons do we bring into battle? date: 2020-09-10 journal: bioorg med chem doi: 10.1016/j.bmc.2020.115757 sha: doc_id: 287758 cord_uid: da11ypiy urgent treatments, in any modality, to fight sars-cov-2 infections are desired by society in general, by health professionals, by estate-leaders and, mainly, by the scientific community, because one thing is certain amidst the numerous uncertainties regarding covid-19: knowledge is the means to discover or to produce an effective treatment against this global disease. scientists from several areas in the world are still committed to this mission, as shown by the accelerated scientific production in the first half of 2020 with over 25,000 published articles related to the new coronavirus. three great lines of publications related to covid-19 were identified for building this article: the first refers to knowledge production concerning the virus and pathophysiology of covid-19; the second regards efforts to produce vaccines against sars-cov-2 at a speed without precedent in the history of science; the third comprehends the attempts to find a marketed drug that can be used to treat covid-19 by drug repurposing. in this review, the drugs that have been repurposed so far are grouped according to their chemical class. their structures will be presented to provide better understanding of their structural similarities and possible correlations with mechanisms of actions. this can help identifying anti-sars-cov-2 promising therapeutic agents. the world is facing a huge challenge in the coronavirus disease (covid-19) pandemic: how to fight an enemy without weapons in terms of therapy? unfortunately, even before the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) worldwide spread, there were no clinical treatments nor prevention strategies available for any human coronavirus. 1 it is understandable that both society and researchers urge the discovery of new compounds or even of a drug that is commercially available that can be employed by physicians mainly for patients with the extreme presentation of covid-19. there is also urgency in the discovery of medicine with prophylactic action to prevent the entry of the virus in host cells after exposure. vaccine research experts already indicate that rescue from sars-cov-2 will come from a long but effective journey to produce a vaccine. 2 while this is not a reality, the scientific community, including medicinal chemists and doctors who accompany patients, are trying to identify therapeutic alternatives. this is a meritorious attitude: the commitment with the protection of humanity. nevertheless, the rigorous feature of science in the discovery of a new drug cannot be disregarded, even during a pandemic and in the face of the urgent demand for a treatment, to avoid eventual mistakes and spurious hope. the increase in studies related to sars-cov-2 during the first semester in 2020 has allowed the rather speedy identification of promising therapeutic targets for both developing immunotherapies and producing/identifying antiviral drugs. it is noteworthy the increase in outbreaks of sars-cov (2002) and mers-cov (2013), with accelerated production of knowledge on these hcovs, which has been very useful for ongoing investigations on sars-cov-2. one example is the availability of technological devices that allowed the fast sequencing of sars-cov-2 genome and the elucidation of a promising antigen target, the s glycoprotein. nonetheless, the development of a human vaccine can take years, especially because employing emergent technologies requires extensive safety tests and expansion to large scale production in order to assist the world population, as demanded in the case of the covid-19 pandemic. 3 the development of new medicine also demands many years of research that involve stages of reasonable planning, synthesis, structural characterization, formulation of prototypes, preclinical and clinical trials. therefore, the literature highlights, as alternative treatments for covid-19, the repurposing of drugs, which is fast and useful in emergencies such as the one experienced today. the repurposing of drugs means the use of broad-spectrum medicine for a new disease, once its metabolic characteristics, doses, potential efficacy and adverse effects are pre-established due to drug studies extracellular liquid volume and arterial pressure of the human body. it is largely expressed in fifteen human tissues, including ciliated bronchial epithelial cells and type ii pneumocytes form pulmonary alveoli, the main location of lesions caused by sars-cov-2. 36, 37 after ace2 receptor-binding, a conformational alteration occurs in protein s allowing the fusion between the viral envelope and the host cell membrane via endosomal. then, sars-cov-2 releases the rna into the cytoplasm to be translated into viral replicase polyproteins pp1a and pp1ab, which are processed by 3cl pro and pl pro proteases, respectively. the cleavage products are 16 nsps that form the transcription and replication complex. 38 next, the positive rna strand is translated into a template of negative strand that allows the synthesis of new genomics and sub genomics mrnas. these mrnas are translated and transcribed producing structural and accessory proteins. viral proteins and rna genomic are put together in virions at endoplasmic reticulum and golgi complex, finally transported through vesicles and released from the cell host for infecting new cells. 38, 39 the covid-19 symptomatology starts after the virus is installed in host cells. in general, the symptoms include lasting and high fever, dry cough, shortness of breath, muscles aches or tiredness, sputum production, headaches, and a small percentage of individuals presented gastrointestinal symptoms such as diarrhoea and vomit. 40 the incubation period of sars-cov-2, from exposure to first symptoms, lasts 2 to 14 days. the pre-symptomatic stage lasts from 1-3 days (possibly more) before the beginning of symptoms. the post-symptomatic stage lasts at least 7 days after the beginning of symptoms and 3 days after lowering of fever and improvement of respiratory symptoms. 41 there are many unanswered questions such as the duration of potential immunity of both symptomatic and asymptomatic individuals when infected with sars-cov-2. 31 it is noteworthy that efficient strategies to fight the disease should not depend on the symptoms of patients, once asymptomatic or pre-symptomatic subjects can play an important role in the direct and indirect transmission to others, as demonstrated by arons et al. 42 this investigation reports that half the residents in a nursing facility, who tested positive, were asymptomatic when tested and probably contributed to the transmission to other residents. thus, control strategies focused on symptomatic residents were not sufficient to prevent transmission once sars-cov-2 had been introduced in the facility. laboratory exams of infected patients showed alterations in haematology and biochemistry. it was verified the increase of leukocytes and the reduction of lymphocytes; increased d-dimer and erythrocyte sedimentation rate (esr), prolongation in prothrombin times (pt), followed by increase in bilirubin levels, aspartate transaminase (ast), alanine transaminase (alt), creatinine, lactate dehydrogenase (ldh), protein c reactive (pcr), hypoalbuminemia (low albumin), microcytosis and thrombocytopenia. 43 in addition, inflammatory factors that indicate the immune condition of patients, such as interleukins (il) il-2, il-6, il-7, and il-10 and the tumoral necrosis factor-α (tnf-α) become elevated. plasma levels of granulocyte-colony stimulating factor (gcsf), protein induced by interferon gamma, monocyte chemoattractant protein-1 (mcp-1), macrophages inflammatory protein 1α and tnf-α also display significant increase. 44 potential risk factors or comorbidities that can lead to complications of covid-19 include elderly individuals (specially above 65 years of age), cardiovascular issues, cerebrovascular, chronic pulmonary diseases, immunocompromising, renal problems, hepatic disease, hypertension, diabetes and obesity. [44] [45] [46] [47] [48] [49] there is a notorious concern regard the medicine administered to fight these comorbidities because some of them can lead to greater expression of ace2, such as treatments for diabetes 48 or hypertension. 50 this may favour or even aggravate covid-19 infection. these facts justify the urgency of research that contemplate alternative therapeutic targets such as calcium channels blockers for hypertensive individuals as suggested by fang et al. 48 however, there is little clinical evidence on the risk of treating covid-19 patients with therapies that induce greater expression of ace2. further investigation is necessary to explore whether these medicines inhibit or trigger the viral entry into the cells of an infected host. 51 a frequent report in epidemiological data regarding the mortality of covid-19 concerns the sex of individuals, as men are the predominant fatal victims of the disease. therefore, being of the male sex is considered a bad prognostic factor for infection. 52 ,53 a possible explanation lies in the relation between gonadal hormones and the expression of ace2 enzymes or even an alleged vitamin d deficiency, according to vignera et al. 54 the latter suggest monitoring of serum levels of testosterone and vitamin d in infected patients for a better understanding of the different fatality rates between sexes, including the hypothesis that women's hygiene justify a lesser rate of infection. understanding the pathogenic effects of sars-cov-2 for the different organs affected by the disease has also been object of investigation, such as gut-lung crosstalk. 55 data from research conducted thus far indicate that the infection caused by sars-cov-2 is not only capable of causing pneumonia, but it can also damage other organs such as the heart, the liver, the kidneys and organic systems such as the blood and the immune system. 44, 56, 57 patients with the extreme form of the disease frequently manifest lymphopenia, 30, 57 hepatic insufficiency 58 and viral sepsis diagnostic, 51 whose complications can be related to the severity of the cases and the mortality of patients. 56, 57 there are reports that the eventual death of such patients is due to multiple organ insufficiency, acute respiratory distress syndrome (ards), cardiac insufficiency, arrhythmia and renal insufficiency. 56, 59 therefore, great attention is necessary to the disease's potential damage to multiple organs and to therapeutic alternatives to fight covid-19, 60 given that some of these alternatives can have side effects on organs initially unrelated to the respiratory system, but that may be susceptible to a systemic compromise prompted by the virus once the treatment has begun. hence, it is possible to observe the existence of different forms of aggravating the disease. 41 in this regard, wang et al. 60 recommend the creation of a system to categorize patients with the severe form of covid-19. several investigations report that all hcovs, sars-cov, mers-cov and sars-cov-2 induce exaggerated immune responses in the host, which are associated to the severity of pulmonary pathology and might lead to the development of acute respiratory distress syndrome (ards) or death. 57 the incidence of the extreme form of the infection is associated with cytokine storm syndrome, characterized by high plasma concentration of several interleukin, inflammatory cytokine, inflammatory chemokines, among other factors that cause infiltrated inflammatory in the organs. 44, 61, 62 survivors of this excessive response by the immune system can develop long-term fibrosis and pulmonary damages that might culminate in functional injuries to these organs, thus reducing the patient's quality of life. 63 during the development of drugs to fight microorganisms, the adoption of strategies that allow the design of molecules to act against specific biological targets of bacteria, parasites or viruses is preferred. therapies for covs can be divided into several categories, based on specific paths: (1) covs proteins or functional enzymes that are essential for viral replication; (2) structural proteins of the virus that prevent its binding to the respective receptors in human cells or its assembly process; (3) some viral factor that restores the host's inherent immunity and; (4) host-specific enzymes or receptors, that prevent the entry of the virus in the host cells. 5, 64 so far, structural proteins and enzymes that participate actively in the process of viral replication are the most investigated targets for the development of molecules for anti-covs therapies (fig. 1) . investigations by wu et al. 5 through bioinformatics, analysed possible sars-cov-2 therapeutic targets. the proteins coded by this virus were verified and compared to proteins coded by other covs. the results enabled the detection of structural similarities to sars-cov, from which it was possible to conduct homology modelling to build 19 proteins for sars-cov-2. among the targets were spike (s) glycoprotein, nsp (rna-dependent rna polymerase -rdrp), enzyme helicase (3cl pro and pl pro ), tmprss, orf7a factor and ace2 presents in the host cells. 5 these targets have pivotal roles in the development of the virus and have great influence on its pathogenicity, hence, some details are provided next. molecular modelling showed that spike (s) glycoprotein is a transmembrane protein of approximately 180 to 200 kda type i whose n-terminal turns to the exterior of the virus and its cterminal segment turns to the interior of the virus. the typical structure of covs is given by the assemble of a bulbous projection of a corolla as trimers of protein s and it is cleaved into two important subunits from the pathogenic perspective: s1 and s2. sars-cov and sars-cov-2 (s) glycoprotein share about 76% of amino acid identity and enable the entry of the virus in the host cells. therefore, s glycoprotein present in covs has been considered a promising biological target for antiviral mechanisms. 35, 65 the moment when the virus approximates the target cell prompts the recognition by the receptor-binding domain (rbd) in the s glycoprotein of its receptor, which leads to the binding to subunit s1. next, the subunit s2 allows fusion of viral and cellular membranes, which enables entry in the cell and the release of viral rna genome. 35, 66, 67 some investigations suggest that the strong binding affinity between s protein and ace2 is essential for viral entry, hence, ace2 is also relevant for the development of drugs. 5, 68 molecules that bind to the surface of the virus can destabilize the formation of s glycoproteins and interfere both with the trimerization of the protein and with the continuity of the life cycle of covs. 69 several studies have been conducted on s protein to clarify its sars-cov-2 structure and its binding process as well as to evaluate its relevance as target for in-silico and in-vitro assays on molecules for anti-sars-cov-2 therapies. one study conducted by hoffman et al. 35 investigated how the sars-cov-2 s protein facilitates viral entry in the target cells and how this process could be blocked. results showed that ace2 is used as receptor for the entry of sars-cov-2 in host cells and that the spread of this cov in the infected host depends on the activity of tmprss2 (a cellular serine protease responsible for initiating the binding process between s protein and ace2). this process can be blocked with clinically approved tmprss2 inhibitor. prior to this, the relevance of tmprss2 was highlighted in the dissemination of several types of viruses such as influenza a and other covs, which also makes it a relevant target for covid-19 therapeutic intervention. [70] [71] [72] [73] [74] [75] [76] binding between s proteins and ace2 receptors was corroborated through x-ray crystallography conducted by lan et al. 67 to elucidate the interaction between the sars-cov-2 rbd and ace2 at a higher resolution. in spite of different interactions with ace2, the sars-cov-2 rbd /ace2 and sars-cov rbd /ace2 interfaces share a substantial similarity regarding the surface area, the number of interacting residues and the networks of hydrophilic interactions. such similarity strongly points to a convergent evolution of both sars-cov-2 and sars-cov rbd structure which improves the binding affinity for the same receptor, the ace2. the non-conserved rbd regions in s protein, such as subunit s2, could be potential targets for cross-reactive antibodies. considering rbd as a critical region for receptor binding, antibodies that target the conserved epitopes in the rbd are also good candidates for the development of highly potent cross-reactive therapeutic agents against several species of covs, including sars-cov-2. 67 investigations on ligands obtained from drugbank 5.1 used molecular docking to identify target regions in the pockets of the quaternary structure of sars-cov-2 s glycoprotein (from protein data bank-pdb). six pockets present in s glycoprotein deserve further investigation in medicinal chemistry due to suitable features for small molecule binding. among the six pockets, the eight best ligand candidates from drugbank were all binding pocket #1, which contained residues of amino acids proline, leucine, lysine, asparagine, phenylalanine, glycine, threonine, glutamine, alanine, methionine and tyrosine. one of the best ligands was the drug saquinavir, an antiviral from the class of protease inhibitors, used in anti-hiv therapy. 69 nsps are involved in the rna transcription, translation, protein synthesis, processing and modification, viral replication and infection of the host. significant functional proteins, 3cl pro , pl pro , helicases and rdrp are important targets for the development of small-molecule inhibitors, due to their biological function and vital enzyme active site. 77 factors nsp1, nsp3c and orf7a are related to assistance to the immune evasion of sars-cov-2. interaction between nsp 1 and the host ribosomal subunit induce the degradation of mrna, allowing the virus to develop resistance to the host innate immunity. binding between orf7a and bone marrow matrix antigen 2 (bst-2) inhibits activity and blocks bst-2 glycosylation. these results suggest that all three structures are potential targets for antiviral medicine. 5 proteases pl pro and 3cl pro mediate the proteolytic cleavage of polypeptides produced by βcoronavirus sars after genome transcription, thus generating other proteins. the 3cl pro , known as nsp5, cleavages several non-structural proteins of importance for viral replication and the maturation of nsps, which is essential in the life cycle of the virus. therefore, it is an attractive biological target for that has been inhibited in-silico by several antiviral, anti-inflammatory and anti-hypertensive drugs from the database zinc (fda). 5 in addition, docking and molecular dynamic studies conducted by qamar et al. 78 showed that non-toxic natural products formed strong bonds with sars-cov-2 catalytic dyad cis145-his41 of 3cl pro . moreover, the proteinase pl pro is responsible for cleavages of n-terminus in the replicase polyprotein to release nsp1, nsp2 and nsp3, which are essential for correcting virus replication significant to antagonize the host's innate immunity. analysis of the docking model showed that ribavirin formed hydrogen bonds with residues gly164, gln270, tyr274, asp303 as well as hydrophobic interactions between tyr265 and the pl pro residue. these results indicate ribavirin as a powerful pl pro enzyme inhibitor, which means it has promising features for anti-covid-19 therapy given the inhibition of a likely pl pro therapeutic target. 5 helicase (nsp13) has been identified as a promising target for antiviral drug discovery, particularly against sars-cov-2. it is a multifunctional protein necessary for a wide range of biological processes, such as genome replication, recombination and dislocation of proteins related to chromatin and nucleic acid remodelling. for covs, helicase is indispensable for viral replication. in studies on molecular modelling, several antibacterial, antifungal and antiviral drugs were analysed and presented elevated affinity to helicase, suggesting it as a good target for sars-cov-2 therapy. 5 rna-dependent rna polymerase (rdrp -also nominated nsp12) catalyses the viral rna, which performs a key role in the replication/transcription complex of sars-cov-2, possibly aided by nsp7 and nsp8 complex as cofactor. 5, 79 nsp12 has been studied as potential target for several sars-cov and mers-cov inhibitors, due to its importance for viral control. satisfactory results of rdrp inhibition by several ligands were presented in the modelling studies by gao et al. 79 yin et al. 80 those ligands included antiviral analogous to nucleotides, such as remdesivir, which already shows great potential in the treatment of covid-19 infections. in addition, some nonstructural proteins, including nsp3b, nsp3e, nsp7, nsp8, nsp9, nsp10, nsp14, nsp15 and nsp16, also stood out as useful targets due to their significant role in the synthesis and replication of viral rna. 5 3cl pro is key enzyme for covs, also called main protease (m pro ), that plays a pivotal role in mediating viral replication and transcription, making it an attractive target for anti-sars-cov-2 drugs. such claim is reinforced by studies by jin et al. 81 after the virtual screening of n3 inhibitor. results show that n3 (1) is a time-dependent irreversible inhibitor of this enzyme and that a stable covalent bond is formed between n3 and 3cl pro . high-throughput screening (hts) was applied to 10,000 drugs and drug candidates, demonstrating that ebselen (2), px-12 (3) and carmofur (4) are all able to covalently bind to 3cl pro do sars-cov-2, with ic 50 that varied from 0.67 to 21.4 µm (fig. 2) . it is likely that a part of the hits identified by hts are bonded to the catalytic cysteine of 3cl pro through their sulfhydryl groups. in-vitro studies on antiviral activity were performed to corroborate the results. real time quantitative pcr (qrt-pcr) demonstrated that ebselen and n3 had the strongest antiviral effects at a concentration of 10 μm treatment in sars-cov-2 infected vero cells. after plaque-reduction assay, the dose-response curves suggested that both could penetrate cellular membrane to access their targets. this result strongly supports the hypothesis that developing a single antiviral agent targeting 3cl pro or in combination with other therapies could provide an effective first line of defence against all covs related diseases. in relation to sars-cov-2 therapy, some of the aforementioned targets have been explored for both new drug proposition as well as for sars-cov-2 drug repurposing. our focus is on this last type, and for each medicine, the putative mechanism of action and viral target will be described trying to find an understandable rational therapy even for an immediate illness situation like covid-19 pandemic. carmofur (4). as previously mentioned, sars-cov-2 is an enveloped virus, whose nucleocapsid consists of a positive rna genome surrounded by multiple copies of nucleocapsid protein. this virus, after entry in the host cell, replicates fast the viral genome with new virion production. the rna replication into the cell host depends on enzymes and substrates for rna synthesis, such as ribonucleotides (adenine, guanine, cytosine or uracil) that have nitrogenous bases in the purine or pyrimidine classes. compounds can mimic these chemical structures and interfere with the formation or use of one of these essential normal organism metabolites. the interference is generally prompted by enzyme inhibition in the biosynthetic pathway of the metabolite or by incorporation, as a false building block, into vital macromolecules such as proteins and polynucleotides. so, this class of therapeutic agents is called antimetabolites. 82, 83 diverse antimetabolites have been indicated as promising anti-sars-cov-2. they are described next. pyrimidine derivatives are aromatic organic compounds necessary for all life forms. examples of pyrimidine derivatives are nitrogenous bases cytosine (5), uracil (6) and thymine (7) (fig. 3) . they are found in dna and rna and participate in the metabolic process that involves carbohydrate and lipids. 84, 85 these heterocyclic rings share two nitrogen atoms at 1 and 3 positions, but display variations between themselves, such as an amine group at 4-position in the cytosine and a methyl at 5-position in the thymine. from the pharmacologic perspective, nitrogenous bases are investigated as pharmacophores and are found in the structure of many drugs and experimental substances with various activities, 86 such as antitumoral, 87 antibacterial, 88 antiparasitic, 89 and antiviral. 90, 91 regarding antiviral activity, there are several approved drugs that are classified as pyrimidine nucleotide biosynthesis inhibitors (pnbi) because, after phosphorylation, they are incorporated either into the dna or into the rna and inhibit hosts or pathogenic enzymes, such as polymerases. 85 therefore, the likely mechanism of action of some pyrimidine derivative drugs has been considered for repurposing. some pyrimidine derivatives with antiviral activity are often formulated as prodrugs. this format solves issues of high polarity in its final structure prompted by the phosphonic acid, which interferes with pharmacological properties and causes low cellular permeability and low oral bioavailability. 91 one compound appointed as potential anti-sars-cov-2 is the 5-fluorouracil (8) (5-fu) (fig. 3) , a heterocyclic aromatic amine similar to uracil (u) that presents a fluorine-carbon bond at 5-position. this compound is used in the treatment of oesophageal cancer, 83 stomach cancer, 92 breast and colon cancer. 93 the similarity between 5-fu and uracil allows the direct action on nuclei acid as it is incorporated into the genetic material and inhibits replication. 83 tests with 5-fu as monotherapy confirmed its failure against any coronaviruses. the reason proposed to such failure relied on the fact that coronaviruses rna proofreading activities involve a 3' → 5' exoribonuclease in the nsp14, which removes 5-fu during replication and metabolism. hence, the combination between 5-fu and deoxyribonucleoside and deoxyribose was suggested so that, after its insertion in the rna, it escapes rna proofreading and prompt lethality and/or lethal mutagenesis in the virus. despite the proposition of using a widely marketed drug to treat several types of cancer, which means it has well-established efficiency and safety, no other type of test has been made to confirm its efficacy against sars-cov-2. therefore, further experiments are necessary to explore 5-fu potentialities. 94 another antitumoral drug considered for its anti-sars-cov-2 potential is gemcitabine (gct) (9) (fig. 3) , an analogue of deoxycytidine whose pharmacological action is triggered after the intracellular transformation into triphosphate gemcitabine. the latter competes with endogenous nucleoside triphosphates by incorporation into the genetic material, thus inhibiting dna synthesis. 95 initially, gct was developed for antiviral activity, however, initial results caused it to be redirected for anticancer therapy. it became, then, widely used against non-small cell lung cancer, pancreas, bladder and breast cancers as well. [96] [97] [98] in-vitro analyses of gemcitabine hydrochloride inhibited mers-cov and sars-cov, with a ce 50 of 1.2 μm and 4.9 μm, respectively, in addition to low cell toxicity for vero e6 cells. 99, 100 these data are indicative of a possible activity against sars-cov-2, but complementary preclinical investigations are necessary before clinical trials. albeit considered a safe drug under predetermined doses, gct adverse effects are noteworthy and include myelosuppression and disruption of liver functions. in february 2017, the european union (eu) approved baricitinib (10) as second-line oral treatment for mild to severe active rheumatoid arthritis in adults. 9 a differential feature of baricitinib structure is the azetidine ring bearing an ethylsulfonyl, beyond an acetonitrile group at 3-position. the same ring binds to the n atom at 1-position in the pyrazole, which, in its turn, binds to the pyrimidine conjugated to a pyrrole ring. 101 this medicine can modulate human innate and adaptive immune system. based on this property, presumably, one of the important mechanisms of action of baricitinib in the treatment of rheumatoid arthritis is the inhibition of the il-6 / jak1 / jak2 pathway. 102 the promising nature of baricitinib and other small molecule inhibitors against sars-cov-2 was pointed by richardson et al. 9 through in-silico tests using benevolent ai. the authors evaluated 378 compounds to show that sunitinib (11) and erlotinib (12) inhibit ap2-associated protein kinase 1 (aak1) interrupting the virus entry to the cells and the intracellular assembly of new viral particles (fig. 3) . regarding these two antitumor drugs, it is known that sunitinib is an oral oxindole multitargeted kinase inhibitor that inhibits certain tyrosine kinases including vascular endothelial growth factor receptors (vegfr types 1 and 2), platelet-derived growth factor receptors (pdgfr-α and pdgfr-β), stem cell factor receptor (kit), fms-like tyrosine kinase-3 (flt3), glial cell-line derived neurotrophic factor receptor (ret) and the receptor of macrophage-colony stimulating factor (csf1r). 103 concerning erlotinib, it was developed as reversible and highly specific small-molecule tyrosine kinase inhibitor that competitively blocks the binding of adenosine triphosphate to its binding site in the tyrosine kinase domain of epidermal growth factor receptor (egfr), thereby inhibiting autophosphorylation and blocking downstream signalling. 104 however, these oncological drugs have serious adverse effects such as diarrhoea, loss of appetite and skin rashes. in addition, high doses of these medications can aggravate those effects. in relation to baricitinib, its anti-sars-cov-2 potential was explained in three ways: aak1 inhibition like sunitinib and erlotinib; the kinase associated to cyclin g, which is another endocytosis regulator; and the janus kinase, that inhibits the action of cytosines that triggers the inflammatory process. because baricitinib can inhibit aak1 at the therapeutic dose (2 or 4 mg/day), the drug is indicated for clinical trials. it is highlighted that baricitinib is not indicated for patients with neutropenia or lymphopenia, once it lowers rates of neutrocytes and lymphocytes, which can lead the disease to progress and increase anaemia. furthermore, treatment with baricitinib can reactivate varicella-zoster, herpes simplex and epstein-barr viruses. this implicates in a conflict between the potential effect and the adverse effects of baricitinib against covid-19 to prevent aggravating the disease and the mortality of patients. 105 an analogue of adenosine, galidesivir (gsv) (13) is a broad-spectrum antiviral drug that blocks viral rna polymerase by replacing a natural nucleotide with galidesivir triphosphate. this alteration prompts changes in electrostatic interactions and prevents the formation of the rna elongated strand. 106, 107 adenosine and gsv differ in that galidesivir has one carbon at 7-position in the pyrimidine ring and nitrogen in the ribose ring, whereas adenosine has one nitrogen in the former and oxygen in the latter (fig. 2) . 106 it is noteworthy that gsv has not been approved for clinical trial and is an experimental drug in advanced stages of development. 108 gsv was first developed against hepatitis c (hcv) but first clinical trials were conducted to ensure its safety (in healthy individuals) and efficacy against yellow fever. furthermore, gsv displayed in-vitro and in-vivo antiviral activity against filoviridae, alphavirus, bunyavirus, arenavirus, paramyxovirus, flavivirus, orthomyxovirus, picornavirus and sars and mers coronaviruses. 11, 106, 109 recent in-silico studies have shown the existence of a strong bond between gsv and sars-cov-rdrp to demonstrate the capacity of alterations in rna polymerase, which can eradicate the virus. although, preclinical and clinical trials are necessary to either confirm or deny this hypothesis. 7 it is noteworthy that investigations have pointed the inactivity of gsv against sars-cov-2 at concentrations lower than 100 mμ. 110 the existence of antiviral activity against other coronaviruses indicates that more investigations on gsv against sars-cov-2 are required to elucidate its potential activity in advanced testing. next, sofosbuvir (sbv) (14) is an example of successful nucleotide prodrug, approved by the food drug administration (fda) since 2013, against chronic hepatitis c infections. sbv is also combined with other antiviral drugs, such as ledipasvir, velpatasvir and voxilaprevir. 111, 112 the structural similarity between sbv (fig. 2) and uridine allows that drug to act on hcv rdrp, incorporate itself into the viral rna and terminate the synthesis of the nucleotide sequence. 82 structural analysis of sbv revealed that its elevated potential is partly due to the presence of the 5'phosphate, which terminates the primary enzyme transformation monophosphate inhibitor. 113 the antiviral activity has been explored against other viruses through in-vitro and in-silico studies and shown potential for inhibiting the dengue virus, 114 yellow fever, 115 telbivudine (tbv) (15) (fig. 3) is a thymidine nucleoside analogue used with specific activity against the hepatitis b virus (hbv). it starts acting after phosphorylation by cellular kinases, which results in the active metabolite, telbivudine 5'-triphosphate, enabling dna polymerase and inhibiting viral replication. the hydroxyl at 3-position in the sugar β-l-2'-desoxirribose provides specificity to hbv polymerase. 120 suggesting repurposing tbv to fight covid-19 was prompted by virtual screening to find drugs that act on viral m pro . among other results were ribavirin, tbv and two vitamins, cyanocobalamin (b12) and nicotinamide (b3). researchers suggest that these four drugs can be combined and used against covid-19, once they are safe, marketed and approved by the authorities. 8 notwithstanding, the suggestion of repurposing these drugs requires more information, including on drug interaction parameters. in spite of well-tolerated and safe for monotherapy, associating tbv and ribavirin, another antiviral drug, can increase hepatotoxic activities of tbv. 121, 122 it is also important to consider the elevated risk of resistance to tbv, which and pyrimidine derivatives drugs: 5-fluorouracil (8); gemcitabine (9); baricitinib (10); sunitinib (11); erlotinib (12); galidesivir (13); sofosbuvir (14) ; telbivudine (15). purine is a 5 and 6-membered bicyclic ring. similar to pyrimidines, purine derivatives are essential to life. they are basic constituents of nitrogenous bases adenine (a) (16) and guanine (g) (17) (fig. 4) . 84 the safety of rdv for humans infected with ebov was evaluated in the democratic republic of congo. results confirmed its safety but did not point rdv as the best therapeutic option, once its mortality rate reached 53% of treated group. 133 it has been proved that gs-5734 inhibits epidemic and zoonotic hcov. 134 of viral loads and weight loss in murine. 135 therefore, rdv is a potential drug to treat mers-cov infections. regarding covid-19, rdv was used to treat the first us case. the patient was 35 years old, had slight cough, low fever and no evidence of pneumonia at day 4 of the disease. when the clinical symptoms became worse, the patient was given vancomycin and cefepime. as the symptoms worsened, intravenous treatment with rdv was administered at day 7, and vancomycin and cefepime were no longer administered. at day 8, the patient displayed clinical improvement, unfortunately details on the doses and duration of treatment were not provided. 136 after this first case, a clinical trial with a larger number of covid-19 patients was conducted. 137 this study of efficacy involved 53 patients infected with sars-cov-2 who displayed saturation equal or inferior to 94% while they were breathing ambient air or receiving oxygen support. the treatment lasted 10 days, patients were given 200 mg intravenously on day 1, followed by 100 mg daily for the remaining 9 days of treatment. follow up of patients treated for 18 days indicated that, after the first dose of rdv, 68% improved oxygen support whereas 15% of patients got sicker, 47% were discharged and mortality rate was 13%. the most common adverse events (60% of patients) were increased hepatic enzymes, diarrhoea, rash, renal impairment, and hypotension. some limitations were noted in the study, such as the small size of the cohort, the short duration of follow-up and the lack of information on the patients. hence, the efficacy of rdv requires validation by the ongoing randomized, placebo-controlled trials. one advantage of repurposing rdv is the availability of data on safety and pharmacokinetics, which were obtained previously at phase 1 clinical trial. in addition to the promising results shown by rdv, other purine analogues have been investigated for sars-cov2. ganciclovir (gcv) (19) also named, according to its chemical structure, 9-(1,3-dihydroxy-2-propoxymethyl) guanine (fig. 4) , is a guanine analogue, similar to acyclovir, except for the bond between the methyl group and one hydroxyl. gcv inhibits the human herpesvirus and is also indicated in the treatment of cytomegalovirus infections related to acquired immunodeficiency syndrome (aids). 139 gcv is converted into ganciclovir triphosphate by cellular kinase, which inhibits dgtp and disrupts viral dna synthesis due to substitution of various adenosine bases in the dna chain. 140 recently, gcv was used to treat covid-19 patients in china. 141 the drug was administered with other antivirals, such as oseltamivir and kaletra. as a descriptive study, the relation between gcv as key factor in clinical outcome of the 99 patients (31% of which were discharges) was not possible. 141 therefore, gcv efficacy as monotherapy or part of combined therapy is yet necessary for more robust investigations. valganciclovir (20) (fig. 4) is the antiviral prodrug of gcv taken by mouth. it is indicated for the same treatments as gcv (cytomegalovirus in people who have acquired immunodeficiency syndrome, gastrointestinal disorders related to aids). the drug has great bioavailability and is converted by hydrolysis into ganciclovir. using valganciclovir in its oral form enables clinical treatment and makes patients more comfortable. [142] [143] [144] the mechanism of action is the same of gcv. 145 valganciclovir was computationally evaluated for covid-19. 5 the assay with the main proteins coded for sars-cov-2 allowed the determination of 21 possible binding targets, of which 19 were proteins and 2 host targets. valganciclovir, one of the drugs used in the study, was presented as a possible anti-sars-cov-2 therapeutic drug due to its high binding affinity to two wellestablished viral targets. the first target was pl pro , indispensable enzyme in viral replication; the second, rdrp, conserved nsp12 in coronavirus, which is vital for its replication/transcription. therefore, valganciclovir could be a significant antiviral drug to treat sars-cov-2. but there are no clinical reports on valganciclovir used to treat covid-19 in addition to what has been reported about gcv. 141 hence, its efficacy is yet to be confirmed as anti-sars-cov-2 therapeutic. tenofovir (tfv) (19) is another adenine analogue pointed as promising covid-19 therapeutic (fig. 4) , it is also called tenofovir disoproxil fumarate or alafenamide tenofovir (taf). approved by the fda in 2001, tfv is a prodrug used to treat hiv and cases of nucleoside resistance. 146 tfv is an analogue reverse-transcriptase inhibitor (ntrti). inside cells, tfv is phosphorylated and competes with deoxyadenosine 5'-monophosphate (d-amp), thus preventing the formation of dna. once incorporated into a growing dna strand, it causes premature termination of dna transcription and prevents viral replication. 146, 147 modelling and docking studies evaluated the antiviral effects of tfv and verified a strong bond to sars-cov-rdrp, which can disrupt this polymerase and terminate the viral infection. 7 however, in-vitro tests showed that tfv lacks apparent antiviral effect at concentrations inferior to 100 μm for sars-cov-2. 110 in spite of lukewarm in-vitro and in-silico outcomes, an ongoing clinical study on tfv (chictr2000029468), expected to end in june 2020, aims at assessing the effect of the combination tenofovir + emtricitabine (cytidine analogue) related to lpv/r in covid-19 patients. 131 in addition to the efficacy of treatment, clinical trials can validate the prevalence of adverse effects related to the toxicity of tfv in patients. tfv is also a powerful nephrotoxic drug causing damage to proximal tubular cells. in spite of that, interrupting the treatment is sufficient to improve adverse effects, which makes monitoring of patients essential. 145 heterocyclic compounds with different heteroatoms such as nitrogen, sulphur and oxygen can present different pharmacological properties. one such property is to serve as analogue of nitrogenous bases of nucleic acids, such as triazoles, which have a five-membered ring of two carbon atoms and three nitrogen atoms. this aromatic ring can assume two isometric forms, 1,2,3-triazole and 1,2,4-triazole. the former is stable under acid and basic conditions and becomes more reactive when binding to electronegative elements. 148, 149 triazoles are important and stand out for their various biological activities, such as anticancer, 150 antituberculosis, 151 anti-inflammatory, 152 antimicrobial, 153 and antiviral. 154 specifically for the latter action, triazole-based derivatives have shown promising invitro activity against coronavirus, probably by 3cl pro inhibition. 155 ribavirin (22) (fig. 4) is a powerful triazole-based antiviral analogue to guanosine. it presents a wide range of pharmacological activities related to several viruses, for instance: herpes simplex virus, human immunodeficiency (hvi-1), influenza, respiratory syncytial (rsv) and hepatitis c. 149, 156 the drug was initially used in 1980 to treat syncytial virus in children, generally combined with interferon (inf). however, ribavirin treatment presents undesirable adverse effects, like lowering of haemoglobin, which limit clinical use. 157 its action mechanism relies on the inhibition of enzyme inosine monophosphate dehydrogenase, necessary in the synthesis of guanosine triphosphate, which prevents viral dna and, mainly, rna replication. the necessary concentration for in-vitro inhibition of rsv and influenza ranges from 3-10 μg/ml. 158 site similar to other sars-pl pro inhibitors. the formation of hydrogen bonds and π-π stacking were also predicted. these findings suggest that ribavirin as a powerful pl pro inhibitor. nonetheless, investigation on triazole derivatives for anti-sars-cov-2 therapy are still preliminary. on the other hand, favipiravir (fpv) (23) (fig. 4) is a prodrug, approved in 2014 in japan, to treat cases of influenza a and b that displayed resistance to first line drugs. it has provided results that indicate a promising character and is currently undergoing clinical trials against covid-19. its antiviral efficacy has also been investigated in different countries to fight ebola and lassa, for example. the molecular structure of the drug consists of a pyrazine heterocyclic ring with fluorine at 5-position, carboxamide at 3-position and a double bond between oxygen and carbon 2, which renders its analogue to guanine (17). 162, 163 the metabolization of the prodrug into its active form, favipiravir-ribofuranosyl-5'-triphosphate, requires intracellular ribosylation and phosphorylation. 163 fpv therapeutic targets are rdrp enzymes, necessary in viral transcription and replication, and its inhibition blocks synthesis of viral rna for a spectrum of viruses, including human coronavirus. a different investigation compared patients treated with fpv plus interferon inhalation to lpv/rtv. patients under fpv therapy responded better to the progression of the disease with accentuated viral depuration. also, the incidence of nausea and vomit was higher for lpv/rtv. 166 in addition to these clinical trials, the antiviral activity of fpv against sars-cov-2 was also evaluated but no clear antiviral effect was noted for doses lower than 100 μm. 110 an in-vitro study using molecular docking focused on the binding properties of sars-cov-2 protein structures to 61 antiviral agents, including oseltamivir. the study showed that 37 molecules form bonds to sars-cov-2 crystal proteins. however, data did not show oseltamivir as the best structure because lopinavir, asunaprevir and remdesivir interacted with more than two protein structures in the virus. hence, they are likely more promising than oseltamivir. 178 notwithstanding, we suggest further look into oseltamivir against other enzyme targets since different studies achieved positive results regarding its use as anti-sars-cov-2. nelfinavir (27) (fig. 5) is a safe anti-retroviral drug largely used for hiv-1 protease inhibition with strong in-vivo activity. 179 generally, nelfinavir is combined with other anti-retroviral medication as part of a highly active antiretroviral therapy (haart) that reduces significantly the viral load by increasing cell number to 200 mm -3 cd4(+) lymphocytes. the drug is prescribed for children, young individuals, adults and pregnant women. 180, 181 nelfinavir and its active metabolite m8 strongly bind to serum proteins, displaying optimal tissue distribution. a frequent adverse effect is light to moderate diarrhoea, reported for 15 to 20% of patients. 180 the sars-cov outbreak in several countries triggered the search for antiviral drugs active against the disease. among the 24 drugs likely to inhibit sars-cov, nelfinavir stands out in all assays. 181 the mechanism of action suggested for nelfinavir involves preventing sars-cov replication after its entry in the host cell and disrupting virion production. based on results from previous studies as well, nelfinavir was considered a likely therapy for covid-19 after its indication for clinical trials as a promising anti-sars drug. recently, 1,903 drugs were evaluated for their binding affinity to sars-cov-2 m pro . 182 among the compounds, 15 drugs were selected based on the docking score and three-dimensional atazanavir (28) (fig. 5) is an antiretroviral drug protease inhibitor used to treat hiv infections with in-vitro inhibitory concentration of 2,6-5,3 nmol. compared to other protease inhibitors, atazanavir has the advantage of allowing a daily posology regimen with a favourable metabolic profile and low frequency of adverse effects. 183, 184 several hiv-1 resistant to protease inhibitors are still sensitive to in-vitro atazanavir, which is considered safe and well tolerated. 185 the atazanavir acts to inhibit hiv-1 protease, which is indispensable in the processing of polyproteins precursors of viral structures and prevents the formation of infectious and mature viral particles. 183 the good activities reported for this drug as well as the search for safe and fast therapy for captopril (29) (fig. 5) is an angiotensin-converting enzyme inhibitor (acei). it is a zinc metallopeptidases inhibitor that converts angiotensin-i into angiotensin-ii, an essential function that regulates arterial pressure. it is predominantly indicated as vasodilator in patients with cardiac insufficiency. this drug was suggested as potential antibiotic capable of inhibiting zinc succinyls/dipeptidase by blocking its zinc catalytic center. 189, 190 tolerance to captopril has been largely investigated; its single dose by mouth is well-established and confirms the pharmacological activity in the short term (10-30 minutes) at the cellular level. this capacity is related to captopril transport mainly through plasma proteins such as albumins with absorption rate between 70-75%. reported adverse effects are neutropenia, proteinuria, dysgeusia and cough, but less frequent for low doses. 189, 190 some investigations have suggested captopril as possible covid-19 treatment. serafin et al. 100 indicated captopril as potential for inhibiting the bond between human sars-cov-2 and ace-2 and reduce severe pneumonia symptoms. in-silico studies using molecular docking were conducted with fda-approved drugs capable of binding to the main active site in proteinase 3cl pro . 189 two drugs were identified as ligands for the enzyme active site: captopril and disulfiram. the former binds to the active site at the same position of n3 inhibitor (a standard inhibitor that reacts irreversibly in the same site with 3cl pro cys145). it is, thus, suggested that captopril binds to the same site of n3, obstructing the function of cys145-his41 catalytic dyad. captopril probably inhibits the enzyme in two stages. initially, it establishes non-covalent bonds to sites in the enzyme targets, then, a reaction takes place between the critic groups, which results in a more stable inhibitor complex. the hypothesis is that captopril can bind covalently to 3cl pro cys145. although the potential of captopril on the enzyme has been demonstrated, therapeutic use against covid-19 is controversial, once the drug induces overexpression of ace-2 -the main receptor used by sars-cov-2 to entry the cells. therefore, combination with other drugs, such as angiotensin-ii receptor blockers, needs analysis to clarify the effects of captopril in covid-19 treatment. the cyclosporin a (csa) (30) (fig. 5) is isolated from the fungus beauveria nivea and was approved for use by the fda in 1983. this drug has been used for decades to prevent organ rejection and to treat t cell-associated autoimmune diseases such as behcet's disease, psoriatic arthritis, lupus nephritis, rheumatoid arthritis, systemic lupus erythematosus or interstitial lung disease. such drug exerts its immunosuppressive function and anti-inflammatory effects by inhibiting the transcription of genes required for t cell proliferation, notably interleukin-2. [191] [192] [193] due to the severity of covid-19, csa can be potential to prevent hyperinflammation-induced lung injury. 194 in this regard, it is known sars-cov nps1 induces the expression of interleukin-2 via nuclear factor of activated t cell (nf-at) activation, 195 which can trigger the cytokine storm seen in patients with severe covid-19 status. 138 another advantage presented by csa in relation to other antiinflammatory drugs is its already known anti-cov action against all genus, including sars-cov, [195] [196] [197] at low and non-cytotoxic micromolar concentrations verified in cell culture assays. this antiviral property is thought to be mediated by the inhibition of cyclophilin-a-dependent viral assembly as well as inhibition of the nf-at pathway or even by genetic or pharmacological specific inhibition of cyclophilin-d, hindering the viral replication. 195, 198 as already reported, sars-cov and sars-cov-2 are very similar (79.5% sequence identity). 17 teicoplanin (31) (fig. 6) is an antibiotic used against gram-positive bacteria with 5 major compounds at different side chains. it prevents polymerization of peptidoglycans and inhibits the development of the cell-wall, thus prompting cell death. 203 it is a big molecule that has displayed antiviral activity on an early stage of the viral life cycle by inhibiting the low-ph cleavage of the viral spike protein by cathepsin l in the late endosomes thereby preventing release of viral rna and replication. this compound has already shown inhibitory activity against ebola virus, mers-cov and sars-cov. 203 recent investigations have suggested teicoplanin as alternative treatment for covid-19 after an in-vitro assay achieved ic 50 value of 1.66 µm, thus proving its efficacy against sars-cov-2. these results need to be confirmed through randomized clinical trials, which are still to be conducted. 100, 204, 205 using antibiotics to fight viruses, albeit completely ignored, can become useful to treat covid-19. 206, 207 some studies report the possibility of repurposing drugs like terconazole, which displayed good in-vitro results against mers-cov and sars-cov, 100 dasabuvir (32) (fig. 6) is a drug from the naphthalene class and phenyl-naphthalene subclass due to the bond between its naphthalene ring and a phenyl group. dasabuvir is a first line drug used as combined therapy for chronic hepatitis c. 208 dasabuvir is a non-nucleoside inhibitor that binds to nps5b (non-structural protein 5b -rdrp) and induces conformational change that makes rdrp incapable of elongating the viral rna. 209 repurposing of this drug can be useful as sars-cov-2 therapy due to its antiviral activity. 210 dasabuvir was subjected to docking studies against sars briefly, dasabuvir forms π-cation interactions with lys31a (present in the ace2), π-π interactions with phe170b (s protein residue) and hydrogen bonds to ace2 residues glu35a and asp38a and gly176b and ser174b (s protein residue). the authors highlight the importance of repurposing drugs as new therapeutic alternatives not only for the new coronavirus but for the next viral outbreaks. darunavir (33) (fig. 6) is a benzene derivative that has been evaluated for repurposing against covid-19. this drug is an antiviral used in the treatment of hiv-1 infections. it provides a great genetic barrier to resistance and is highly active against resistant strains of hiv-1 that are not susceptible to other protease inhibitors. 211 darunavir is administered orally as pills or suspension and is often used with low doses of ritonavir as part of a combined art protocol. 212 its mechanism of actions works by protease inhibition. darunavir establishes high affinity bonds to hiv-1 protease forming a stable complex, thereby selectively inhibiting polyprotein gag-pol coded by the virus. this prevents the formation of mature viral particles. 211 fda-approved drugs against 3cl pro , rdrp, helicase, exonuclease 3′ a 5′, endornase e 2′-o-ribose methyltransferase. among the best drugs in the assay, darunavir was a surprise because, despite inhibiting viral proteinase, the study showed that it binds to the replication complex components of sars-cov-2 with inhibitory potency kd < 1000 nm. one example is rdrp, whose kd value was 148.74 nm and exonuclease 3′ to 5′ with k d value of 195.73 nm. a docking study was conducted by sang et al. 215 as in-silico evaluation of anti-hiv drugs in their interaction capacity to proteinase 3cl pro . results suggest that all drugs have higher binding affinity to sars-cov-2 3cl pro than to the homolog sars-cov proteinase. among the evaluated drugs, indinavir and darunavir displayed the highest docking scores, therefore, they were subjected to molecular dynamic (md) simulations, free binding energy calculations and molecular mechanics poisson-boltzmann surface area (mm-pbsa) to detail molecular interactions between inhibitors and proteinase. the data suggest darunavir had better binding affinity to sars-cov-2 3cl pro with binding affinity of -10,24 kj / mol. in addition, darunavir bind to sars-cov-2 3cl pro via 19 contact residues and to sars-cov 3cl pro via 17 residues. this difference explains the lower binding energy values between darunavir and sars-cov 3cl pro . it was also noted 5 hydrogen bonds between darunavir and sars-cov-2 3cl pro but none for indinavir and this proteinase. because hydrogen bonds are important in the stability of the inhibitor-enzyme complex, darunavir is probably more promising against covid-19. finally, a different in-silico assay was conducted by pant at al. 216 to assess a large variety of compounds (300) from several data banks and 66 potential compounds from fda-approved drugs. the compounds were tested against sars-cov-2 3cl pro . darunavir was among the 20 best fda-approved drugs, with a score of -7.208. all data collected from in-silico studies still require experimental studies to validate the anti-sars-cov-2 activity of darunavir. a research conducted by dyall et al. 99 performed a robust in-vitro assay and showed that the drug dasatinib (34) (fig. 6) , a kinase signalling inhibitor developed to treat human cancers, inhibited mers-cov and sars-cov, exhibiting ec 50 values 5.4 and 2.1, respectively. this study also revealed that kinase signalling may also be important for replication of this hcovs. nevertheless, the authors reported that dasatinib may be valuable against coronaviruses infections if a dosing regimen that minimizes immunotoxicity while still blocking viral replication can be defined. results indicated this drug as a likely therapeutic alternative against sars-cov-2 infection. an in-silico study carried out by qiao et al. 217 showed that dasatinib, among others, is one of the most promising drugs for the inhibition of sars-cov-2 3cl pro . more preclinical and clinical studies are required to prove whether dasatinib is really promising for covid-19 patient treatment. imatinib (35) (fig. 6) is an oral anticancer agent that inhibits the activity of some tyrosine kinases, most prominently the bcr-abl fusion oncoprotein (whose overactivation can lead to chronic myeloid leukaemia, cml), c-kit (involved in gastrointestinal stromal tumours development), platelet-derived growth factor receptor (pdgfr), and the native abl kinase, which has a ubiquitous expression and plays important roles in several biological processes. 218 in addition to this well-known antitumor effect, imatinib has also shown in-vitro antiviral properties against several virus, such as infectious bronchitis virus (a viral model for studying the role of tyrosine kinase activity during cov infection), by interfering with virus-cell fusion, 219 and other rna viruses including coxsackie virus, 220 hepatitis c virus, 221 ebola, 222 among others, mainly by blocking viral entry or egress from the host cell. besides, this drug showed activity against sars-cov and mers-cov, 223 both phylogenetically related to sars-cov-2. 24 in this regard, it is reported that imatinib has anti-cov activity in two points of the virus life cycle. in the early phases of infection, it inhibits virion fusion with the endosome and subsequent release into the cytoplasm, thus preventing viral entry and viral replication via abl-mediated cytoskeletal rearrangement. in a later phase of the infection, abl2 protein expression, which is inhibited by this drug, enables sars-cov and mers-cov replication, which suggests that abl2 is a new host cell protein required for viral growth. 223 furthermore, evidences suggest that imatinib can modulate the immune response by sundry mechanisms, [224] [225] [226] for several diseases, such as rheumatoid arthritis, 227 asthma, 228 and crohn's disease. 229 this information insinuates that such drug might perform its potentially beneficial immunomodulatory role as a treatment alternative for covid-19 pneumonia. in addition, the use of imatinib as treatment appears to be reasonable from an economic point of view and its high availability in hospitals, 230 since this drug is well tolerated and the risk of severe adverse effects is relatively low, especially in short-term administration. 231 it is also recognized that adverse effects, mostly mild to moderate in intensity, will be easily controlled by dose reduction or discontinuation. 232 in light of this information, tatar and turhan 233 used the docking methodology to better understand the mechanism of inhibition of the sars-cov-2 n protein with 34 antiviral compounds. based on this study results, imatinib was one of the highly binding affinities performed against the aforementioned target, with the lowest micromolar ki values among the compounds evaluated. in line with this study, an in-vitro research carried out by weston et al. 234 found 17 fda approved drugs that inhibited sars-cov-2 at non-cytotoxic concentrations. the authors indicate imatinib as one of the hits, since it exhibited ic 50 value of 3.24 μm. they subsequently determined the mechanism of action, demonstrating this drug inhibits fusion of covs with cellular membranes, precluding their entry. this result indicates imatinib use against sars-cov-2. however, its efficacy and safety need to be better confirmed in further preclinical and clinical trials in order of elect him as candidate drug in the treatment of covid-19. synthesized for the first time by jean francois rossignol in the beginning of the 1970s, the 2acetyloxy-n-(5-nitro-2-thiazolyl) benzamide, sold under the name nitazoxanide (ntz) (36) (fig. 6) , is the result of a structural modification in the antiparasitic niclosamide when a benzene ring is replaced with a nitrothiazole. 235, 236 developed and sold as antiparasitic, ntz is also a first line broad spectrum antiviral with good results against parainfluenza, coronavirus, rotavirus, hepatitis and other respiratory infections. [236] [237] [238] [239] following oral administration, ntz is absorbed in the intestine, where it is rapidly hydrolysed by plasma esterase into the active metabolite tizoxanide. 236 the mechanism of action of ntz varies according to the pathogen. in relation to antiviral activity, ntz blocks the maturation of the viral hemagglutinin at the post-translation stage in treatments against influenza. in treatments against hcv (hepatitis c), it activates protein kinase r (pkr), which leads to phosphorylation of the eukaryotic initiation factor-2α thereby preventing translation. 240 cao et al. 241 conducted an in-vitro evaluation of ntz against recombinant murine coronavirus expressing the firefly luciferase (mhv-2afls). the strand was pivotal to triage the 727 drugs with likely anti-cov activity. the first assay resulted in 84 molecules among which was ntz. the antiviral effect of ntz verified for mouse astrocytoma (dbt) and fibroblasts (17cl-1). 241 the dbt cells infected with mhv-2afls were treated with ntz at 5 µm for 12 hours, after which the viral titer (tcid 50 ) was determined and viral n protein was subjected to western blot. results show the strong inhibitory effect of ntz on the viral titer. 241 in-vitro studies on ntz or its metabolite tizoxanide were also conducted to verify efficacy against different coronaviruses. the replication of canine coronavirus (strain k378) in a72 cells, for instance, was blocked by the tizoxanide with ic 50 of 1 µg/ml. on the other hand, ntz inhibited the viral n protein in bovine coronavirus l9 (βcov-l9) and human enteric coronavirus 4408 (hecov-4408) with approximate values of 0.3 µg/ml. 239, 241 ntz is also responsible for inhibiting pro-inflammatory cytokines, such as tnf-α, il-2, ilnitazoxanide (36) drugs. more recently, molecules with a quinoline group have been widely investigated as treatment for the new coronavirus (sars-cov-2), such as chloroquine (cq) (37) and hydroxychloroquine (hcq) (38) (fig. 7) that belong to the quinoline class and aminoquinoline subclass. both are quickabsorption synthetic drugs approved to treat malaria (plasmodium falciparum) by several regulating agencies in the world. cq and hcq are water soluble; the latter is more soluble due to presence of hydroxyl group. they are currently used to treat autoimmune diseases such as lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis as they have immunomodulatory and antithrombosis properties. [243] [244] [245] therefore, these drugs could be useful against covid-19 due to the elevated levels of cytokine caused by cov infections in humans. 246 the mechanism of action of cq for anti-malarial treatment is not entirely clear, but the interference with the digestion of haemoglobin by the parasite has been suggested. 245 hcq has a similar mechanism, however, in regard to sars-cov-2, clinical trials showed it to be safer. [246] [247] [248] therefore, hcq, rather than cq, is used against sars-cov-2. recent studies report antiviral activity of cq and hcq as they impair viral entry and release in different in-vitro and in-vivo models. 244, 249 a factor that can also justify viral mechanisms is the aminoquinoline bioaccumulation in the tissues, as defended by patil, singhal & masand. 243 a factor that facilitates viral replication is the acidic ph of endosomes, lysosomes and golgi complex of the host. thus, cq is promising because it increases the ph of intracellular vacuoles, binds to the cellular receptors, changes the glycosylation and because of its selective and reversible immunomodulator effect on human cd4+ t cells. hcq exerts similar mechanism of action: a) increases the ph; b) modulation of activated immune cells; c) reduces the number of proinflammatory cytokine and other mediators to control inflammation. 243 it has also been suggested by roldan et al. 244 a likely involvement of hcq in iron homeostasis during sars-cov-2 infection, which is a similar mechanism to other viral infections in humans. [250] [251] [252] the little difference between the therapeutic and the toxic dose of cq is also known, and poisoning is related to cardiovascular complications that can be fatal. using either cq or hcq, then, requires strict prescription and self-medication is not advised. 249 based on the recovery data, it was concluded that hcq is not effective in patients admitted with covid-19. this highlights the importance of randomized clinical trials and the collection of clear data on the efficacy and safety of medications. ivermectin (39) (fig. 7) is an fda-approved broad-spectrum antiparasitic agent used in the treatment of tropical diseases, such as onchocerciasis, lymphatic filariasis, strongyloidiasis and lice. there is also evidence of its effectiveness in the management of myiasis, trichinosis, malaria, leishmaniasis, trypanosomiasis, chagas disease and schistosomiasis as well as bed bugs, inflammatory skin lesions, epilepsy, neurological diseases, tuberculosis and some cancers. 266 it is known that ivermectin is capable of inhibiting the bond between a virus and the nuclear transport mediated by the superfamily of importin proteins (imp α/β1) 267 based on the fact that sars-cov-2 is a rna virus deeply related to sars-cov, studies on sars-cov proteins have revealed a potential role for imp α/β1 during infection in signal-dependent nucleocytoplasmic shutting of the sars-cov nucleocapsid protein. 273 furthermore, the sars-cov accessory protein orf6 has been shown to antagonize the antiviral activity of the stat1 transcription factor by sequestering imp α/β1 on the rough er/golgi membrane. 274 considering the ivermectin nuclear transport inhibitory activity, such drug is widely believed as a promising therapeutic approach against sars-cov-2. recently, caly et al. 275 a different mechanism of action that consolidates the use of ivermectin against covid-19 is its immunomodulatory property. the inflammatory response (proinflammatory cytokine) is exaggerated in patients with the extreme case of the disease, which is likely explained by the hypoxiainducible factor (hif-1α) that is activated by the virus when no inhibitory medication is administered. this understanding can be explained by the study conducted by kosyna et al. 276 , whose lab tests with and without ivermectin aimed to examine whether the properties of the bond between hif-1α and imp α/β1 and between hif-1 and nuclear localization signals were affected by the hypoxia mechanism on the cellular level. the authors concluded that ivermectin inhibited both imp α/β1 and hif-1α. regarding this hinders its indication and clinical decisions. thus far, data indicate ivermectin is useful at the early stages of the disease, even the epidemiologic profile of covid-19 shows significant differences regarding the age of patients for the affected countries and patients with comorbidity. 279 therefore, large scale randomized clinical trials are necessary to standardize clinical, laboratory and image evaluations, as well as combined drug therapy with vitamins and zinc, for example. 280 financially, ivermectin is inexpensive and its doses and protocols are well established for different purposes. in addition, this drug has little side effects. 281 indole, also named benzo [b] pyrrole, is a planar bicyclic heteroaromatic, whose ten π electrons move across its structure making this chemical group behave as a weak base. 282, the indole ring is the most abundant heterocyclic in nature and is commonly found in biologically active natural products, such as vegetables and seafood. it is also present in the structure of the essential amino acid tryptophan, which interferes with protein synthesis and with the regulation of physiological mechanisms such as precursors for serotonin and vitamin b3. 283 288 and adding the indole ring to spirothiazolidinones conducted to better influenza a/h3n2 inhibition. 289 now, the antiviral activity of indole and its derivatives for covid-19 therapy is supposed. arbidol (40) is also a promising drug to fight covid-19 (fig. 7) . this drug is classified as antiviral and has been used for 25 there is high expression of eca2, identified as human receptor for virus entry. 290 notwithstanding, a different clinic trial concluded that arbidol monotherapy is best for the patient than lpv/rtv. 291 despite the results, both investigations recognize their own limitations due to small cohort size and lack of placebo-control group. according to the authors, these limitations are inherent to pandemic times when placebo-control groups are difficult to conduct due to life-threatening conditions. most studies with arbidol use 200 mg/3*day 164, 290, 291 following the chinese guidelines. previous investigations on the pharmacokinetics of arbidol in healthy chinese patients showed that a single 800 mg oral dose is sufficient for cmax de ~4,1μm. 293 this value was obtained through invitro assays that pointed the drug as effective and promising against sars-cov-2. hence, clinical trials are still necessary to confirm the efficacy of arbidol at elevated doses to treat covid-19. 293 rizatriptan (rzt) (41) (fig. 7) is used to treat migraine and is a selective receptor of serotonin (5-ht) type 1b and 1d, structurally and pharmacologically related to other selective antagonists at these receptors. 294 its structure is based on an indole ring replaced with methyltriazole at 5-position and unsubstituted ethanamine replaced with methyl at 3-position, the substitution sites are the same of melatonin (mlt). after virtual triage through molecular dock at spike-ace2 interface, ligations π-cation, interactions π-π and hydrogen bonds were identified between rzt and the sars-cov-2 protein complex. as one of the outstanding compounds in the analysis, in-vitro tests are still necessary. 187 it is noteworthy that overdosing of the drug can trigger dizziness, fainting, cardiac issues, hypertension, bradycardia and vomiting. despite the safety of the drug in regular doses, there are no reports on either in-vitro or in-vivo tests to support the theoretical data and the antiviral action thus far. one last indole derivative that could be repurposed to treat covid-19 is melatonin (mlt) (42) (fig. 7) . mlt is classified as a hormone and nutraceutical, as it is naturally produced by the pineal gland and released into the bloodstream. it regulates the sleep-wake cycle as well as our mood, learning and memory, fertility, reproduction and the immune system. from a chemical perspective, it is an indole derivative with a methoxy group at 5-position and one ethylacetamide at 3-position. 295 regarding its antiviral potential, mlt acts indirectly through anti-inflammatory, antioxidant and immune modulating activities. 296 an investigation with murine infected with semliki forest virus (sfv) and west nile virus (wnv) showed the efficiency of mlt in reducing mortality rates for these viruses as well as in reducing the levels of pro-inflammatory cytokines. 297 the anti-inflammatory and antioxidant properties of mlt point to its antiviral effects in humans. 298 based on computational data, zhou et al. 299 suggested using combined medication to fight sars-cov-2. one such combination involves mlt plus mercaptopurine in synergic action against the following targets: pl pro , ace2, c-jun signal and anti-inflammatory vias. therefore, experimental studies on modifications of ace2 pathways caused by mlt are useful to understand this drug. 299 on the other hand, it has been suggested that using this neuro-hormone can mitigate the extreme form of the disease, the acute respiratory syndrome that has caused most deaths by sars-cov-2 cases. despite its safety for humans, the lack of data on the relevance of its use for covid-19 patients emetine (43) is an approved anti-protozoal drug used against amebae with reported inhibitory activity for enterovirus infections, 301 zika virus and ebola by interfering with the process of viral replication and entry in host cells. 302 emetine is an isoquinoline alkaloid that presents 4 methoxy groups in its structure (fig. 7) . studies also confirm emetine has in-vitro activity against coronaviruses, including sars-cov and mers-cov. 99 assays to clarify the activity of these drugs and the mechanism involved in the antiviral action of emetine both in isolation and combined to other drugs. another alkaloid candidate to repurpose against covid-19 is homoharringtonine (hht) (44) (fig. 8) . hht is an fda-approved drug in semi-synthetic form known as omacetaxine. this drug displays antitumoral activity in the treatment of myeloid chronic leukaemia. the mechanism of action implicates the ribosomal bond to prevent protein translation. in addition to antitumor activity, there are data in the literature that describe antiviral activity of hht against several types of viruses including covs. 304, 305 a recent in-vitro evaluation of anti-sars-cov-2 activity displayed ec 50 of 2.10 µm. however, the mechanism of action is not yet clear, which demands further investigation on ideal doses of hht to achieve the clinical results expected of a covid-19 therapeutic drug. 110 the first reports on tetraethylthiuram disulfide, disulfiram (dsf) (45) (fig. 8) , date back to 1881. however, only in the 1940s that dsf would become popular when it was discovered that it could form copper chelates which favoured the death of micro-organisms and enabled treatment of intestinal parasites. [306] [307] [308] in 1945, dsf alcohol sensitivity was discovered accidentally and it was soon used in the clinical treatment of alcohol dependence. 309, 310 dsf is used to treat alcohol dependence because it irreversibly inhibits the acetaldehyde dehydrogenase enzyme and modifies cysteine residues in its active site. this change prompts the formation of a disulphide bond between two cysteine residues in the active site. 311 dsf effectiveness is based on its similarly to several proteins yielding a range of biological activities, such as antitumoral, 312, 313 antimicrobial, 310 and anti-sars and mers-cov. 314 adding to the list of drugs to be repurposed against sars-cov-2, recent studies indicate that dsf is able to inhibit other enzymes, such as methyltransferase, urease and kinase, all by reacting with important cysteine residues that suppress the natural cycle of the enzymes, suggesting broad-spectrum characteristics. 312, 314 covs have two viral enzymes, m pro and pl pro , that are cysteine protease involved in the formation of structural and non-structural proteins that constitute the viruses and favour control of host cells. 315 different assays, such as proteolytic and binding synergy assays, were also conducted and described. 314 although outcomes indicate dsf for anti-mers-cov and anti-sars-cov therapy, to the present date (15 th june 2020), no other article was published claiming the availability of the compound as promising anti-sars-cov-2. recently, an announcement was published on the oxford university website on the results of one randomized evaluation of covid-19 therapy. 317 more specifically, the study focused on dexamethasone (46), a corticosteroid with fluorine at 9-position (fig. 8) . the drug is mostly used as anti-inflammatory, which works by inhibiting vasodilation, reducing leukocyte migration to the inflammation site and increasing vascular permeability. 318 immunotherapy is an effective intervention in viral infections. most attempts at immunotherapy were successful in fighting viruses similar to sars-cov-2. the principal methods include vaccine, neutralizing antibodies (nabs) candidates and convalescent plasma. 35, 61, 67, 68, 156, 319, 320 in addition, according to the evidences from viral infections (ebola, influenza, sars and mers), immunotherapeutic interventions can reduce viral load and mortality rate of patients. 321, 322 development of either monoclonal (mabs) or polyclonal (pabs) neutralizing antibodies is a commonly adopted immunotherapeutic alternative due to its specificity, purity, low contamination by blood-transmitted pathogens and relative safety. however, there are limitations to the use of nabs once its development and large-scale production for clinical use are a complex, expensive and slow process. 321 promising scientific investigations have suggested using mabs or pabs as prophylactic and therapeutic measures against influenza 323 and hcovs, such as mers-cov 324 and sars-cov. 325 targets reported as promising for hcovs immunotherapy were cytokine, 326 s1-receptor-binding domain (s1-rbd), s1 n-terminal domain (s1-ntd) and some other region of subunit s2 in order to block the rbds bonds to their respective receptors and to interfere either with s2-mediated membrane fusion or with the entry in the host cells, thus inhibiting infection. 327 these researchers have encouraged the development of nabs with cross reactivity potential and/or cross neutralization effect on sars-cov-2 infections, as shown by tian et al. 328 data suggest that mab cr3022 can be developed as therapeutic candidate, either isolated or combined with neutralizing antibodies to prevent and treat covid-19, given that it could potently form bonds with sars-cov-2 rbd (kd of 6.3 nm). a different study by wang et al. 329 reported the discovery of a human mab (47d11) that promoted cross neutralization of sars-cov and sars-cov-2 in a culture of cells through an independent receptor-binding inhibition mechanism that targets a conserved epitope on the spike hcovs rbd mentioned above. it is also reported the on-going investigation of convalescent plasma or immunoglobulin as last resource to improve the survival rate of patients with several viral infections such as h 5 n 1 avian influenza, 330 334 a possible explanation for the efficacy of convalescent plasma is that immunoglobulin antibodies in the plasma of recovered patients can suppress viremia. 156 shen et al. 335 reported that five patients with extreme symptoms of covid-19 received blood transfusion containing convalescent plasma with specific sars-cov-2 antibodies. after a series of blood transfusions, the improvement in clinical status of patients was observed. viral loads also decreased and became negative within 12 days after the transfusion, and sars-cov-2-specific elisa and nabs titers increased following the transfusion. in spite of the limited sample, the authors concluded that convalescent plasma transfusion benefited patients infected with sars-cov-2. therefore, testing safety and efficacy of transfusing convalescent plasma in patients infected with sars-cov-2 can be of value. 319, 336 among the modalities of immunotherapy, vaccines are expected to be more promising, hence the global engagement in their production. over the last decade, the scientific community and the vaccine industry had to answer urgently to the epidemics of h1n1, ebola, zika and, more recently, sars-cov-2. vaccine development is an expensive and slow process with high risks of failure, which often motivate developers to follow a linear sequence of steps with several breaks for data analysis and fabrication processes. therefore, it is fundamental that vaccines be developed through faithful methods even if it takes longer to move them onto clinical trials or to make a large number of doses available, a challenge during a pandemic. 337 developing efficient vaccines for sars-cov-2 will be essential to reduce the severity of the disease, viral shedding and transmission to control future outbreaks. prior to the covid-19 pandemic, multiple strategies were used to generate vaccines for the first hcovs (sars-cov and mers-cov). 338 several studies related to sars-cov vaccine production targeting the protein s, due to its function in the receptor binding and fusion to the host membrane, were successful in animal tests against that coronavirus. 339-341 these vaccines employed live-attenuated virus vaccines, killed virus, dna vaccines and viral vector vaccines. theoretically, these techniques could be applied to develop sars-cov-2 vaccines given their similarities from both the genomic perspective and the mechanisms employed in the invasion and infection of host cells. 326 gao et al. 342 promoted the pilotscale production of a purified inactivated sars-cov-2 virus vaccine candidate (picovacc), which induced sars-cov-2-specific nabs in mice, rats and non-human primates. in addition, three immunizations using two different doses (3 μg or 6 μg per dose) in macaques provided partial or complete protection against the sars-cov-2 challenge, respectively, without observable antibodydependent enhancement of infection. these data reinforce the use of picovacc in the next steps of clinical trials targeting sars-cov-2 still for the present year. given the magnitude of the covid-19 pandemic, it has become indispensable to work as fast as possible to develop vaccines for global distribution. however, protocols are necessary to safekeep the population's health. hence, before allowing human testing of covid-19 vaccines, regulatory organizations must evaluate their safety against a series of virus strains and more than one animal model. they also must demand preclinical evidences that experimental vaccines prevent infectioneven if it means waiting weeks or months for models to become available. this is time well-spent, once testing vaccines without investing the due amount of time to completely understand the risks can lead to setbacks for current and future pandemics. 343 repurposing potential relies on the mechanism for other viruses, such as hepatitis c, by inducing conformational changes that compromise rdrp activity. it was also possible to identify benzene derivatives used to treat cancer (dasatinib and imatinib) and one antiviral (darunavir), but predominant in-silico and some in-vitro outcomes demand more conclusive studies. representative of benzoic derivatives, ntz displayed significant inhibitory activity against pro-inflammatory cytokines, which can benefit control of ards, in spite of an unclear mechanism against sars-cov-2. quinoline derivatives, hcq and cq, were some of the first drugs investigated. after several in-silico, in-vitro and in-vivo assays, based on results presented by recovery to the present date, unfortunately, these drugs were proven ineffective in hospitalized covid-19 patients. ivermectin represents macrolide derivatives and is suggested as promising due to both immunostimulatory activity and inhibitory activity on nuclear transport when administered at the early stages of the disease. among indole derivatives, arbidol was pointed out as useful for reducing viral binding and releasing of intracellular vacuoles that contain the virus. nonetheless, clinical trials are still necessary after dose adjustment for better outcomes. both indole derivatives (emetine and hht), in spite of promising results, were only submitted to in-silico and in-vitro tests, thus demanding further investigation on their toxicity and mechanism of control. hence, the currently available data on the classes of drugs investigated here revealed that drugs were considered promising mainly after in-silico tests only. in fact, the inefficacy of some of these drugs became evident after in-vitro or in-vitro tests, as cq and hcq, whose clinical trials failed to confirm the so-expected anti-sars-cov-2 activity. it is necessary to highlight the importance of clinical trials with 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p.; prostko, john; rodgers, mary; coller, kelly; pearce, sandra; franz, sergej; du, li; stone, mars; pillai, satish k.; sotomayor-gonzalez, alicia; servellita, venice; martin, claudia sanchez san; granados, andrea; glasner, dustin r.; han, lucy m.; truong, kent; akagi, naomi; nguyen, david n.; neumann, neil m.; qazi, daniel; hsu, elaine; gu, wei; santos, yale a.; custer, brian; green, valerie; williamson, phillip; hills, nancy k.; lu, chuanyi m.; whitman, jeffrey d.; stramer, susan l.; wang, candace; reyes, kevin; hakim, jill m. c.; sujishi, kirk; alazzeh, fariba; pham, lori; thornborrow, edward; oon, ching-ying; miller, steve; kurtz, theodore; simmons, graham; hackett, john; busch, michael p.; chiu, charles y. title: sars-cov-2 seroprevalence and neutralizing activity in donor and patient blood date: 2020-09-17 journal: nat commun doi: 10.1038/s41467-020-18468-8 sha: doc_id: 302707 cord_uid: cap2rgf7 given the limited availability of serological testing to date, the seroprevalence of sars-cov-2-specific antibodies in different populations has remained unclear. here, we report very low sars-cov-2 seroprevalence in two san francisco bay area populations. seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early april 2020. we additionally describe the longitudinal dynamics of immunoglobulin-g (igg), immunoglobulin-m (igm), and in vitro neutralizing antibody titers in covid-19 patients. the median time to seroconversion ranged from 10.3–11.0 days for these 3 assays. neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of igg and neutralizing titers was >93%. these findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using sars-cov-2 anti-nucleocapsid protein igg and anti-spike igm assays are generally predictive of in vitro neutralizing capacity. c oronavirus disease 2019 (covid-19) is a novel respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 1 . the symptoms of covid-19 range from asymptomatic infection to acute respiratory distress syndrome and death, and the covid-19 pandemic has resulted in substantial burdens on healthcare systems worldwide 2, 3 . accurate and large-scale serologic testing that includes detection of neutralizing antibodies is essential in evaluating spread of infection in the community, informing public health containment efforts, and identifying donors for convalescent plasma therapy trials. given the current state of diagnostic testing which largely relies on molecular techniques, the seroprevalence of sars-cov-2-specific antibodies-a proxy for prior infection-in different populations has remained unclear. here, we present data validating the use of the eua authorized abbott architect sars-cov-2 igg test for antibody detection in two populations in march 2020, a hospitalized covid-19 patient cohort at a tertiary care hospital in san francisco and a cohort of blood donors from the san francisco bay area. we also investigate the longitudinal dynamics of igg, igm, and in vitro neutralizing antibody titers in hospitalized covid-19 patients over time. these studies demonstrate that sars-cov-2 seroprevalence in the san francisco bay area was very low, suggesting limited circulation of the virus in the community as of early march, and that igg and igm titers are predictive of neutralizing activity, with high positive percent agreement. performance characteristics of the abbott architect sars-cov-2 igg and igm assays. prior to assessing seroprevalence of sars-cov-2 antibodies in san francisco bay area populations, we verified the performance of the abbott architect sars-cov-2 igg (fda emergency use authorization (eua)) and igm (prototype) assays. these assays are chemiluminescent microparticle immunoassays that target the nucleocapsid and spike proteins, respectively. the nucleocapsid protein was targeted in the architect igg assay as it was found to have increased sensitivity compared to the spike protein [4] [5] [6] . to evaluate assay sensitivity, we assembled a cohort of 38 hospitalized patients and 5 outpatients at university of california, san francisco (ucsf) medical center and the san francisco veterans affairs (sfva) health care system, all of whom received care at adult inpatient units or clinics and were real-time polymerase chain reaction (rt-pcr) positive for sars-cov-2 from nasopharyngeal and/or oropharyngeal swab testing ( fig. 1a and supplementary table 1 ). the percentage of patients seroconverting for igg at weekly time intervals following reported symptom onset reached 94.4% at ≥22 days (fig. 1b, left) . correspondingly, igg assay sensitivity from analysis of all 423 samples increased weekly to reach 96.9% at ≥22 days, and was 99% when samples from seven immunocompromised patients (see below) were excluded (fig. 1b , right, and table 1 ). the percentage of patients seroconverting for igm was also 94.4% at ≥22 days (fig. 1c, left) and igm assay sensitivity from analysis of 346 samples was 97.9% (98.9% with immunocompromised patients excluded) (fig. 1c , right, and table 1 ). to evaluate assay specificity, serum and plasma samples collected by abbott laboratories from us blood donors from miami, florida prior to the covid-19 pandemic ("pre-covid-19") were tested for igg (n = 1013) and igm (n = 1492) antibody seroreactivity. two samples out of 1013 were positive by igg testing, yielding an igg specificity of 99.8% (95% ci: 99.3-100%) (fig. 1d, top) , concordant with the 99.9% specificity reported in an independent study by the university of washington 7, 8 . six samples out of 1492 from us blood donors were positive by igm testing, yielding a igm specificity of 99.6% (95% ci: 99.2-99.9%) (fig. 1d, bottom) . thus, the architect sars-cov-2 igg and igm assays demonstrated high sensitivity (96.9% at ≥22 days in a primarily hospitalized patient cohort) and specificity (99.6-99.8% % in pre-covid blood donors). longitudinal dynamics of igg and igm titers in covid-19 patients. given the availability of longitudinal samples from our cohort of 43 sars-cov-2 pcr positive patients, we next analyzed the longitudinal dynamics of plasma igg (286 samples) and igm (249 samples) titers during the course of hospitalization (fig. 1e, f) . anti-nucleocapsid igg and anti-spike igm antibody titers were observed to rise approximately in tandem, with similar median times to seroconversion of 11.0 and 10.8 days, respectively. these results are consistent with a previous study reporting concomitant detection of igg and igm antibodies to spike protein, or, in some instances, earlier detection of igg antibodies than igm in covid-19 patients 9-11 . thus, it is possible that sequential igm followed by igg production may not be a general feature of the immune response to sars-cov-2, as is the case for many viral infections. in addition, the differences in time to seroconversion may be related to biologic variability among patients. it is also possible that some of observed variability and early seroconversion may be a result of initially mild disease symptoms leading patients to self-report delayed symptom onset dates. of the four patients in week 3 who had not yet seroconverted for igg (fig. 1b , left, days 15-21), two were kidney transplant recipients on tacrolimus and mycophenolate mofetil (mmf) immunosuppressive therapy; one was >90 years old; and one was an asymptomatic patient receiving acute psychiatric care who provided an unreliable history. both renal transplant recipients were observed to ultimately seroconvert for igg and igm. notably, delayed seroconversion for igg and igm was not universal among immunosuppressed patients. three additional solid organ transplant (sot) recipients on tacrolimus and mmf, as well as one patient with rheumatoid arthritis on methotrexate and infliximab, all seroconverted within 2 weeks (fig. 1e, f) , while another sot recipient was positive for igg and igm in the earliest available serum sample from day 17 post symptom onset. we did not have samples beyond day 18 for the remaining two patients who were not immunosuppressed; however, as seroconversion was observed as late as three weeks after symptom onset (fig. 1e, f) , it is possible that analysis of later samples would have demonstrated detectable antibodies in their serum. the one patient who was still igg negative in the 22+ day time frame (fig. 1b, left) from a day 29 plasma sample had only mild symptoms and was positive by igm and neutralizing antibody testing (described below). conversely, the only igm negative case in the 22+ day time frame (fig. 1c, left) was igm negative from a day 50 plasma sample but igg positive, likely due to waning of igm antibody titers to undetectable levels by this later time point. to further evaluate assay specificity, we tested 235 remnant plasma samples from 163 sars-cov-2 pcr-negative ucsf patients collected from late march to early april 2020. the testing resulted in detection of only one reactive sample, yielding a specificity of 99.6% (95% ci: 97.7-100%) (fig. 2a, 2nd column) . the igg reactive sample was from a patient admitted for syncope but who reported a cough of 1-month duration, suggesting potential prior infection from either sars-cov-2 or another human coronavirus that may have elicited a cross-reactive antibody response. we also tested 39 sars-cov-2 pcr negative ucsf patients for igm antibody, none of whom were positive (fig. 2b, 2nd column) . these results of 99.6-100% specificity from sars-cov-2 pcr-negative ucsf patients are thus comparable to the 99.6-99.8% specificity of the architect cov-2 igg and igm assays calculated from pre-covid-19 samples (fig. 1d) . seroprevalence of sars-cov-2 in blood donors and patients from the san francisco bay area. to investigate sars-cov-2 seroprevalence in the san francisco bay area, we performed antinucleocapsid igg testing on plasma and serum samples from two cohorts of individuals with low suspicion of infection from covid-19. one cohort consisted of 1000 individuals who donated blood in march 2020 at blood bank centers throughout the bay area ( fig. 1a and supplementary table 2 ). routine blood donor screening was performed to exclude those with selfreported symptoms of acute illness and abnormal vital signs. we detected four igg positive samples in this cohort, yielding a seroreactivity rate of 0.40% (fig. 2a, 4th column) . this cohort was not tested for igm antibody. we then analyzed the four igg positive samples using two orthogonal tests, the vitros anti-sars-cov-2 total antibody assay (ortho clinical diagnostics eua) and a sars-cov-2 pseudovirus neutralization assay (described below). of the four samples, three (circled in fig. 2a) were negative by both the vitros and neutralization assays, and thus were designated likely false reactives by the architect igg assay. thus, the calculated seroprevalence after confirmatory orthogonal testing for bay area blood donors in march 2020 was 0.1% (95% ci: 0.00-0.56%). the false reactive rate in this population of 0.3% is consistent with the reported specificity of the architect sars-cov-igg test of 99.6% (fig. 1d) 7 . we additionally evaluated seroprevalence in a cross-section of patients who received care at adult inpatient units or clinics at the ucsf medical center for indications other than covid-19 respiratory disease (non-covid-19, never tested for sars-cov-2 by rt-pcr) from late march to early april 2020 (supplementary table 3 ). remnant samples from 387 patients were obtained from ucsf clinical laboratories (fig. 2a, 3rd column). only one patient had igg seroreactivity; this patient had presented with respiratory failure and ground-glass opacities on chest imaging but was never tested for sars-2-cov by rt-pcr. igg seroprevalence in this population was thus low at 0.26% (95% ci: 0-0.76%), comparable to the 0.1% seroprevalence in the 1000 tested bay area blood donors. although only 23 of the remnant samples were able to be subsequently tested for igm antibodies, importantly and as expected, none were positive (fig. 2b, column 3) . next, we sought to directly compare igg and igm antibody titers with sars-cov-2 in vitro neutralizing activity in 54 available plasma samples from 22 of the 43 sars-cov-2 pcr positive patients for whom residual longitudinal samples were available (fig. 2c) . neutralizing antibody activity measured using sars-cov-2 pseudoviruses has previously been shown to correlate well with that measured using cultured sars-cov-2 isolates 12 . plasma titers that achieved 80% neutralization of infectivity (nt80) using a sars-cov-2 pseudovirus, a vesicular stomatitis virus (vsv) pseudotype expressing the sars-cov-2 spike protein, were measured by luciferase assay (see "methods"). the positive percent agreement (ppa) and negative percent agreement (npa) were 92.5% and 93.7%, respectively, between igg and igm positivity; 93.8% and 75.0%, respectively, between nt80 and igg positivity; and 84.8% and 78.6%, respectively, between nt80 and igm positivity (fig. 2c, left to right) . all pairwise comparisons were linearly related with rho correlations (fig. 2d-g) . there was no significant correlation observed between median igg, igm, and neutralizing antibody titers and severity of disease (supplementary table 4 ). in this study, we provide evidence that seropositive results using the architect sars-cov-2 anti-nucleocapsid protein igg and anti-spike igm assays are generally predictive of in vitro neutralizing capacity. given that anti-nucleocapsid antibodies are not thought to be directly neutralizing, these results suggest that detection of anti-nucleocapsid igg antibodies may be indicative of a productive and polyclonal humoral immune response that includes neutralizing (likely anti-spike) activity. indeed, we also found high positive and npa of >92% and good correlation (rho = 0.65) for detection of anti-nucleocapsid igg and anti-spike igm antibodies. these findings are also of relevance for serologic testing, as 44% (7 of 16) of the fda eua authorized serological assays as of june 2020 target the nucleocapsid protein, and also suggest that igg and igm titers may be predictive of neutralizing activity when identifying potential candidate donors for experimental convalescent plasma therapy. however, in vitro neutralization activity may not necessarily confer protective immunity and the efficacy of convalescent plasma therapy for treatment of covid-19 disease remains to be determined. importantly, our results also show that the seroprevalence of igg antibodies against sars-cov-2 in blood donors and non-covid-19 patients seen at a tertiary care hospital in the san francisco bay area from march to april 2020 is very low at 0.10% (95% ci: 0.00-0.56%) and 0.26% (0.00-0.76%), respectively. these seroprevalence rates in two distinct populations in the san francisco bay area are near the specificity limit of the architect assay, and are far lower than the specificity limits for many lateral flow immunoassays 13 . these findings contrast with those from other community-based studies that reported higher rates of seropositivity in california 14, 15 , and underscore the importance of using a highly accurate test for surveillance studies in low-prevalence populations. they also indicate a very low likelihood of widespread cryptic circulation of sars-cov-2 in the bay area prior to march 2020, consistent with the low detection rate by direct viral testing of respiratory samples collected during that early time period 16 . study design and ethics. the study population consisted of patients with available remnant serum and plasma specimens from the clinical laboratories at ucsf. samples from patients who were positive or negative by sars-cov-2 rt-pcr testing of nasopharyngeal, oropharyngeal, and/or pooled nasopharyngealoropharyngeal swabs were collected in march-april 2020. additional samples were collected from randomly selected cohorts of outpatients and hospitalized patients at ucsf during the same time period seen for indications other than covid-19 respiratory disease (non-covid). serum samples from blood donors in the san francisco bay area were collected by vitalant research institute in march 2020. clinical data for ucsf patients were extracted from electronic health records and entered in a hipaa (health insurance portability and accountability act)-secure redcap research database. collected data included demographics, major comorbidities, patient-reported symptom onset date, clinical symptoms and indicators of covid-19 severity such as admission to the intensive care unit and requirement for mechanical ventilation. this study was approved by the institutional review board (irb) at ucsf (ucsf irb #10-02598) as a no-subject contact study with waiver of consent. serologic testing. the abbott architect sars-cov-2 igg assay (fda eua) and sars-cov-2 igm (prototype) testing was performed using either serum or plasma samples on the architect instrument according to the manufacturer instructions 7 . these tests are chemiluminescent microparticle immunoassay reactions that target the nucleocapsid protein (igg assay) or the spike protein (igm assay) and measure relative light units that are then used to calculate an index value. at a predefined index value threshold of 0.6 signal-to-cutoff (s/c) ratio for igm seropositivity and 1.4 s/c for igg for seropositivity, these assays were found to have specificities of 99.6-99.8%. the linear range is 1.4-4.0 s/c for the igg assay, and 0.6-21 s/c for the igm assay. the vitros anti-sars-cov-2 total antibody assay approved under fda eua was performed using either serum or plasma samples at vitalant research institute according to the manufacturer instructions 15 . the test is a chemiluminescent immunoassay that targets the spike protein and measures relative light units that are then used to calculate an index value. at a predefined index value threshold of 1.0 signal-to-cutoff (s/c) ratio for igg seropositivity, this assay was found to have a sensitivity of 100% (92.7-100%) and specificity of 100% (95% ci = 99.1-100.0%). production of pseudoviruses for the sars-cov-2 neutralization assay. vsvδg-luciferase-based viruses, in which the glycoprotein (g) gene has been replaced with luciferase, were produced by transient transfection of viral glycoprotein expression plasmids (pcg sars-cov-2 spike, provided courtesy of stefan pölhmann 17 , as well as pcaggs vsv-g or pcaggs ebogp as controls 18 ) or no glycoprotein controls into hek293t cells by transit-2020. briefly, cells were seeded into 15-cm culture dishes and allowed to attach for 24 h before transfection with 30 μg expression plasmid per plate. the transfection medium was changed at 16 h post-transfection. the expression-enhancing reagent valproic acid was added to a final concentration of 3.75 mm, and the cells were incubated for 3-4 h. the medium was changed again, and the cells were inoculated with vsvδg-luc virus at a multiplicity of infection of 0.3 for 4 h before the medium was changed again. at about 24 h post-infection, the supernatants were collected and cleared of debris by filtration through a 0.45-μm syringe filter. antibody neutralization. hek293t cells were transfected with human ace2 and tmprss2 by transit-2020 19 . after 24 h cells were plated into black 96-well tissue culture treated plates. plasma, collected in lithium heparin tubes, was diluted to 1:20 followed by four subsequent 1:4 dilutions. per well, 50 µl of pseudovirus harboring either sars-cov-2 s, vsv-g, or ebogp (adjusted to result in~10,000 rlu in target cells) was mixed with 50 µl of the respective serum or plasma dilution to give a final series of longitudinal serum or plasma dilutions starting at 1:40 and incubated for 1 h at 37°c. controls included wells with vsvδg (no envelope), without added serum/plasma, and with serum predetermined to possess or lack neutralizing activity. subsequently, the 100 µl mix was added to the target cells (performed in duplicate) and cells were incubated for 24 h at 37°c. supernatants were then removed, cells were lysed, and luciferase activity was read as per manufacturer instructions. results were calculated as a percentage of no serum control. each plate was qualified by lack of infection with the no envelope control, and performance of positive and negative controls. nonlinear regression curves and 80% neutralization titers (nt80) were calculated in graphpad prism v8. statistical analysis. we calculated ppa, npa, and overall percent agreement (opa) between the neutralizing antibody result and igg, assuming igg to be the gold standard. we then calculated ppa, npa, and opa between the neutralizing antibody result and igm, assuming igm to be the gold standard. we calculated 95% exact binomial (clopper-pearson) confidence intervals for each proportion. igg, igm, and nt80 titers were non-normally distributed and were summarized using medians and interquartile ranges. we compared antibody titers to dichotomously defined clinical characteristics at various time points using two-sided wilcoxon fig. 2 longitudinal dynamics and in vitro neutralizing activity of antibodies against sars-cov-2. a igg s/c ratios were determined for hospitalized patients and outpatients and blood donors on whom sars-cov-2 pcr testing was positive or negative or was not performed. numbers of seroreactive and total individuals tested are shown in tables below the graphs. the circled data points were additionally tested by the vitros and neutralization assays and were negative by both assays. for patients with multiple samples, the single highest s/c value is plotted. b igm s/c ratios, as in a. c (left) igg and igm titers for sars-cov-2 pcr positive matched patient samples. percent of data points in each quadrant and positive percent agreement (ppa), negative percent agreement (npa), and overall percent agreement (opa) between igg and igm are shown. (middle) 80% neutralization titers (nt80) plotted against igg s/c values. (right) 80% nt80 plotted against igm s/c values. the cutoff for nt80 is a titer level of >40; negative results (<40) are nonnumeric and are plotted at 35 for visualization purposes. d nt80 titers for sars-cov-2 pcr-positive patients were plotted against day post symptom onset. immunocompromised patients are shown in blue. e for the 6 sars-cov-2 pcr-positive patients whose igm, igg, and nt80 seroconversion events were captured during serial sampling, the days post-symptom onset seroconversion events are compared. f nt80 activity was evaluated per patient for the indicated time frames post onset of symptoms. the percent of patients with detectable nt80 activity measured within each time frame is indicated below the graphs. if multiple samples per patient were collected, the sample with the highest nt80 value within each time frame was used. g the average nt80 activity (right axis) and igg and igm (left axis) titers are plotted by day post-symptom onset (left); corresponding graphs for individual patients are shown in a 3 × 3 grid (right). if multiple samples per patient were collected, the sample with the highest s/c or nt80 value per time frame was used. for f, the box outlines denote the iqr, the solid line in the box denotes median neutralizing antibody titer, and the whiskers outside of the box extend to the minimum and maximum neutralizing antibody titers. rank sum tests. the correlations between age and igg, igm, and nt titers were calculated using spearman nonparametric correlation coefficients. statistical calculations were performed using python 3.7.7 using scipy.stats, sklearn.metrics.auc and statsmodels.stats libraries as well as stata v15.1 (college station, tx). all data generated or analyzed during this study are included in this published article and associated supplementary information and source data files. source data are provided with this paper. received: 19 june 2020; accepted: 18 august 2020; a novel coronavirus from patients with pneumonia in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study sensitivity in detection of antibodies to nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 in patients with coronavirus disease 2019 severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients evaluation of nucleocapsid and spike protein-based enzymelinked immunosorbent assays for detecting antibodies against sars-cov-2 cov-2 igg instructions for use. h14806r03. en. 6r86-20 performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in antibody responses to sars-cov-2 in patients with covid-19 antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 kinetics of sars-cov-2 specific igm and igg responses in covid-19 patients safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial evaluation of sars-cov-2 serology assays reveals a range of test performance covid-19 antibody seroprevalence in seroprevalence of sars-cov-2-specific antibodies among adults in sample pooling as a strategy to detect community transmission of sars-cov-2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor inhibitors of sars-cov entry-identification using an internally-controlled dual envelope pseudovirion assay protease inhibitors targeting coronavirus and filovirus entry we thank the patients and their families at ucsf without whom collecting and providing this aggregate data would not have been possible. this work was funded by nih grants r01-hl105704 (cyc) and r38hl143581 (g.m.g., s.p.b., and j.w.) from the national heart, lung, and blood institute, r33-ai129077 (cyc) the national institute of allergy and infectious diseases, the charles and helen schwab foundation (cyc). these funders had no role in study design, data collection and analysis, writing the paper, or decision to publish. this work was also funded in part by abbott laboratories. employees of abbott laboratories (j.p., m.r., k.c., s.p., j.h.) contributed to sample collection, igg and igm testing, and data analysis but had no role in the study design, writing the paper, or decision to publish. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-18468-8.correspondence and requests for materials should be addressed to c.y.c.peer review information nature communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. peer review reports are available. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-289588-n61gz7pi authors: samudrala, pavan kumar; kumar, pramod; choudhary, kamlesh; thakur, nagender; wadekar, gaurav suresh; dayaramani, richa; agrawal, mukta; alexander, amit title: virology, pathogenesis, diagnosis and in-line treatment of covid-19 date: 2020-07-17 journal: eur j pharmacol doi: 10.1016/j.ejphar.2020.173375 sha: doc_id: 289588 cord_uid: n61gz7pi sars-cov-2, a newly emerged pathogen in december 2019, marked as one of the highly pathogenic coronavirus, and altogether this is the third coronavirus attack that crossed the species barrier. as of 1(st) july 2020, it is spreading around 216 countries, areas or territories, and a total of 10,185,374 and 503,862 confirmed cases and death reports, respectively. the sars-cov-2 virus entered into the target cells by binding with the hace2 receptors. spike glycoprotein promotes the entry of the virus into host target cells. literature reported a significant mutation in receptor binding sites and membrane proteins of the previous sars-cov to turned as sars-cov-2 virus, responsible for most dreadful pandemic covid-19. these modifications may be the probable reason for the extreme transmission and pathogenicity of the virus. a hasty spread of covid-19 throughout the world is highly threatening, but still, scientists do not have a proper therapeutic measure to fight with it. scientists are endeavoring across the world to find effective therapy to combat covid 19. several drugs such as remdesivir, hydroxychloroquine, chloroquine, ribavirin, ritonavir, lopinavir, favipiravir, interferons, bevacizumab, azithromycin, etc. are currently under clinical trials. vaccine development from various pharmaceutical companies and research institutes is under progress, and more than ten vaccine candidates are in the various phases of clinical trials. this review work highlighted the origin, emergence, structural features, pathogenesis, and clinical features of covid-19. we have also discussed the in-line treatment strategies, preventive measures, and vaccines to combat the emergence of covid-19. the coronavirus (cov) as believed to be originated from bats, was known for 800 years, 79 confirmed by many works of literature. these are positive-sense single-strand rna viruses 80 with around 24 similar species from the family of coronaviridae. this family of 81 coronaviridae is further categorized as α, β, λ, and δ based on its distinct genetic features. 82 however, among these, only alpha (α) and beta (β) coronavirus genera are pathogenic to 83 mammalian and humans (chen et africa is listed as a minimally affected region (297,290 cases and 6,010 deaths) till date 118 (who, 2020a) . during this outbreak of covid-19, the world is frightened with an 119 unpredictable and hasty impact of the infection, and the data is changing day by day. 120 coronaviruses are crown shape peplomers, positive-sense ssrna (single strand rna) virus, 124 which was reported in the pleomorphic form with 80-160 nm size . it is a 125 nonsegmented and rna virus ranging from 26 to 32 kb. coronavirus comes under the order-126 nidovirus, family-coronaviridae, subfamily-coronavineae which were further divided into α, 127 β, ϒ and δ genus. among these, α and β genus of coronavirus mainly affect the human 128 population. the α genus contains hcov-229e (human coronavirus) and nl63, while the β 129 genus consists of hku1, 229e, oc43, mers-cov, sars-cov and latest outbreak sars-130 cov-2. coronavirus has been mainly reported with single-strand rna, nucleocapsid protein, 131 envelop protein, membrane protein, spike glycoprotein (s), etc., as shown in figure 2 (lei et 132 al., 2018) . 133 spike (s) glycoprotein is responsible for the characteristic feature of the coronavirus because 134 it forms the crown-like structure on the outer surface of the virus. the s-protein divides into 135 two subunits, namely, s1 and s2. the s1 subunit further classified into three domains, 136 particularly a, b, and c (angeletti et al.) . generally, domain a of the subunit s1 present on 137 cov-oc43 and cov-hku1 is binding with the host receptors (hulswit et al., 2019) . interestingly, the structure of the s protein in both sars-cov and novel sars-cov-2 143 viruses is almost similar to a few differences. 144 recent literature reveals that rbd of the s1 subunit of the " s " protein is responsible for 154 binding with ace2 (angiotensin-converting enzyme) receptors (letko et al., 2020) . this 155 rbd in coronaviruses is highly variable. based on studies, six rbd amino acids that remain 156 present on the s1 subunit are very critical for binding to the receptors. interestingly, different 157 structural and functional studies stated that five of these six residual proteins are distinct in 158 sars-cov-2 compared to sars-cov. accordingly, the coordinates based on the covid-159 19 (l455, f486, q493, s494, n501, and y505) compared to those found in sars-cov 160 (y442, l472, n479, d480, t487, and y4911) differ in only five distinct residues except 161 y4911 (andersen et al., 2020). these differences may be the probable reason for the high 162 affinity of sars-cov-2 with the receptors and therefore are optimal for receptor binding 163 a polybasic cleavage site (rrar) is present at the union site of s1 and s2 subunit of spike 166 protein. in addition, proline residue is also introduced prior to this cleavage site, and the 167 sequence in sars-cov-2 becomes prra (unique protein sequencing in sars-cov-2). insertion of the cleavage site has been seen but also the conversion of low pathogenicity virus 177 into highly pathogenic forms is well observed (alexander and brown, 2009; ito et al., 2001) . 178 however, the function of the predicated o-linked glycans is still not clear, but based on the 179 literature, they may create a mucin-like domain that helps in immunoevasion ( are assembled into the nucleocapsid and viral envelope at the er or ergic, followed by 250 release of virus by exocytosis or by fusing with the plasma membrane. 251 although the pathogenesis behind covid-19 is not well understood, the pathogenesis of 253 mers-cov and sars-cov still can be the best source of information regarding covid-19 254 (li et al., 2020b) . 255 recent literature suggests that the modified residues of rbd of s1 subunit, presence of 256 rrar and partially opened state of 's' trimer may be a reason for high pathogenicity and 257 transmission capacity of covid-19. these rbd of s1 subunit on spike proteins binds to the 258 hace2 receptor which are mainly present in the lungs, particularly type-2 pneumocytes. this the clinical symptoms of covid-19 infection can be seen after 5 to 6 days of incubation 272 which mainly differs from age and immune system of the person. males are more susceptible 273 to sars-cov-2 as compare to females. people with the age of more than 60 are more 274 sensitive to sars-cov-2 (li et al., 2020a; the, 2020). 275 the most common clinical features of sars-cov-2 infection are fever (more than 80% 276 cases), cough (more than 60% cases), fatigue (more than 35% cases), sputum production 277 (more than 30% cases), and shortness of breath (more than 15% cases). however, less 278 common features include headache, muscle weakness, breathlessness, sore throat, and 279 pleuritic pain (10-15%). apart from this nausea, vomiting, chest tightness is the rare features pro-inflammatory cytokines are secreted by alveolar macrophages results in the recruitment 320 of neutrophil and monocyte or macrophage along with activation of t-cells of epithelial cells, 321 which further starts inflammation and tissue injury (ritchie and singanayagam, 2020). in 322 medical term, ard is called "diffuse alveolar damage," or epithelial-cell hyperplasia. the 323 diagnostic parameter for ard is hypoxia, which mainly decides based on the ratio between 324 the blood pressure of oxygen and the percentage of oxygen supply, formulated as pao2/fio2 325 mmhg. in severe cases, pao2/fio2 found to be ≤100 mmhg while in moderate cases, found 326 to be ≤ 200. this ratio falls from 200 to 300 in mild cases of ard due to sars-cov-2 327 . other parameters that can distinguish ard are bilateral opacities 328 followed by the collapse of lungs, which can be diagnosed through ct scan or ultrasound, 329 where lung infiltration found to be >50% (lai et al., 2020a) . 330 it is a reliable technique to isolate the affected population from the large population. it is also 350 essential for initial screening for auxiliary diagnosis. a thermal camera was first introduced at 351 hospital entrances and in the emergency department to recognize any persons with 352 incremental body temperature. the scanning distance required for such cameras is 10 meters. however, numerous potential pre-analytical and analytical risks are associated with rt-373 pcr. such risks are false (-) identification, sample preparation, specificity, sensitivity, 374 stability issues, and also, it is a time-consuming process. although calibration of the 375 instruments is also a matter of great concern, as it may provide false or misleading results, 376 therefore it is highly recommended to calibrate the rt-pcr regularly to avoid any 377 misleading readings. besides, the diagnostic kit should also be pre-validated in terms of the 378 accuracy and reliability of the data (lippi et al., 2020) . 379 during the outbreak, remdesivir was tested on three covid-19 infected patients in the u.s., 422 which showed improvement of symptoms with no significant side effects, and also fda has 423 given permission about 250 patients to use this product. as far as safety is a major concern, 424 gilead sciences announced phase iii clinical trial of remdesivir to prove its safety and 425 efficacy in covid-19 infection (keown, 16 .03.2020). after a preliminary investigation, 426 gilead announced the results of the initial phase 3 simple trial. the drug significantly 427 reduced the mortality rate upon 14 days of treatment and showed improvement in 64 % of 428 cases (gilead, 2020) . based on these results, the fda issued emergency use authorizations 429 for the use of remdesivir against covid-19; however, full approval is still not provided 430 anywhere in the world (fda, 2020). 431 the fda already approves the drug chloroquine for treating malaria, arthritis and lupus 433 hydroxychloroquine and azithromycin. they reviewed the data of 84 patients, they treated 499 with hydroxychloroquine on the first day at a dose of 400 mg twice a day, followed by 200 500 mg twice daily. azithromycin was given at a dose of 500 mg per day. they observed 501 prolongation of qt interval from day 4 of therapy. very importantly, severely prolonged qt 502 interval was observed in a subset of nine patients with more than 500 mg against the baseline 503 average of 435±24 mg (mean ± sd). qt interval prolongation is a known marker for the 504 high risk of sudden cardiac death (chorin et al., 2020) 505 in the latest news, on 18 th march 2020, reported that favipiravir marketed as avigan found 507 effective against covid-19. xinmin zhang from the ministry of science and technology, 508 china, said that favipiravir gives positive results in a clinical trial on 340 patients in 509 shenzhen and wuhan city. the drug was developed by toyama chemicals and prescribed for 510 influenza infection. in february 2020, it got approval for the experimental treatment of novel 511 coronavirus infection. in the wuhan trial, the treatment with favipiravir improved the 512 symptoms of infection and reduced the duration of fever. according to the guardian report, 513 some patients with early covid-19 symptoms showed recovery; however, the patients with 514 severe or advanced symptoms do not show a similar response (bryner, 2020) . 515 bevacizumab, a recombinant humanized monoclonal antibody against vegf, was first 517 approved by usfda on 26 th february 2004 for the first-line treatment for metastatic 518 colorectal cancer (cowling and leung, 2020) . subsequently, the fda approved this product 519 along with chemotherapy to treat many cancers like lung cancer, renal cancer, cervical 520 cancer, ovarian cancer, etc. (zhang and zhou, 2018 ). in addition, recent studies suggest that 521 higher levels of blood vegf in covid-19 patients compared with normal and also 522 pulmonary edema, dyspnea, acute respiratory distress and acute lung injury are the most 523 detrimental symptoms of covid-19. numerous studies reported that vegf was a critical 524 factor in pulmonary edema, acute respiratory distress and acute lung injury (schoeman and 525 fielding, 2019). based on this evidences, qilu hospital of shandong university has also 526 initiated clinical trials on this product for treating the covid-19 (clinical trials.gov). 527 protease inhibitors are approved by usfda to treat hiv, and also, these drugs previously 529 immunodeficiency virus) (pronker et al., 2013) . usually, successful development of a 572 vaccine takes around ten years. in this pandemic crisis of covid-19, the world is sorely 573 needed a potential vaccine to combat this threatful situation. as per the available data, around 574 ten vaccines are presently under clinical trial against sars-cov-2 from which the scientists 575 from oxford university and astrazeneca expected to have the data of phase iii trial by this 576 summer (as shown in table 1). however, many experts around the globe suspect the 577 credibility of this work as it takes a minimum of 18 months to develop the first vaccine even 578 after tremendous efforts. while some presume that a huge dose of vaccines (around hundreds 579 of million) will be ready to launch by the end of this year (iserson, 2020) . 580 ppes are being used by health care professionals to protect from the infection of sar-cov-2 623 to avoid secondary transmission in hospitals. ppe should cover the whole body to prevent the 624 chances of infection through the patient. health care professionals should also wear respirator 625 masks such as n95 (filters >95% of airborne particles), ffp2 (filters > 94% of airborne 626 particles), and ffp3 (filters > 99% of airborne particles) to avoid infection. adequate training 627 of ppes handling should be given to health care workers to avoid careless handling. also, 628 proper care should be taken for the disposal of used ppes (kraemer et al., 2020) . 629 many health advisory bodies have compelled the use of masks across the world as a 631 preventive measure to avoid the spread of covid-19. mask control the virus spread through 632 the droplets of an infected person by acting as a shield/barrier to prevent the exposure of the 633 critical body parts responsible for the spreading of the covid-19. however, in the absence 634 of a mask, it is advisable to cover the face with bending elbow over the face or to cover with 635 a handkerchief/tissue paper while sneezing or coughing (adhikari et al., 2020; who, 2020b). 636 cleaning of hands from time to time with alcohol-based sanitizers is advisable to protect 638 individuals against coronavirus disease. washing hands with soap and water before and after 639 meals must be practiced as a preventive measure. as suggested, the content of alcohol should 640 be more than 70% in hand sanitizes, and therefore children should be kept away from 641 alcohol-based sanitizers (who, 2020b). although, there is a great need to produce sufficient evidence by successful clinical studies to 667 assure the safety and efficacy of these drug substances. the development of a vaccine for the 668 prevention and cure of covid-19 and a promising diagnostic tool is also essential. 669 hopefully, the constant scientific efforts and thorough understanding of sars-cov-2 670 pathogenesis and life cycle will soon find out the solution for this. gaurav suresh wadekar: writing-original draft epidemiology, causes, clinical manifestation and 687 diagnosis, prevention and control of coronavirus disease (covid-19) during the early 688 outbreak period: a scoping review 690 correlation of chest ct and rt-pcr testing in coronavirus disease permeation enhancers: specialized components of mucoadhesives history of highly pathogenic avian influenza. revue 696 scientifique et technique membrane binding proteins of coronaviruses the proximal 700 origin of sars-cov-2 how will 702 country-based mitigation measures influence the course of the covid-19 epidemic? the role of the nsp2 and nsp3 in its pathogenesis global aspects of viral glycosylation 710 covid-19: towards controlling of a pandemic activation of the sars coronavirus spike 712 protein via sequential proteolytic cleavage at two distinct sites identification of new respiratory viruses in 715 the new millennium. viruses coronavirus infections reported by promed flu drug used in japan shows promise in treating covid-19 human 722 immunopathogenesis of severe acute respiratory syndrome (sars) zhejiang da xue xue bao. yi xue ban = journal of 728 zhejiang university emerging coronaviruses: genome structure, replication, 730 and pathogenesis the 733 qt interval in patients with covid-19 treated with hydroxychloroquine and azithromycin. 734 nature medicine role of lopinavir/ritonavir in the 737 treatment of sars: initial virological and clinical findings epidemiological research priorities for public health 739 control of the ongoing global novel coronavirus (2019-ncov) outbreak. euro surveillance : 740 bulletin europeen sur les maladies transmissibles origin and evolution of pathogenic coronaviruses ct imaging and differential diagnosis of covid-19. canadian 746 association of radiologists journal = journal l'association canadienne des radiologistes screening of an 750 fda-approved compound library identifies four small-molecule inhibitors of middle east 751 respiratory syndrome coronavirus replication in cell culture roche coronavirus test receives emergency use authorisation from 754 fda coronaviruses: an overview of their replication and 760 pathogenesis breakthrough: chloroquine phosphate has shown apparent 762 efficacy in treatment of covid-19 associated pneumonia in clinical studies hydroxychloroquine and 767 azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical 768 trial hydroxychloroquine and 772 azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical 773 trial observational study of 776 hydroxychloroquine in hospitalized patients with covid-19. the new england journal of 777 medicine first known person-to-784 person transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in 785 the usa gilead announces results from phase 3 trial of investigational antiviral 787 remdesivir in patients with severe covid-19 clinical characteristics of coronavirus disease coronavirus disease 2019 (covid-19): what we know? 795 covid-19: what is next for public health? azithromycin-induced torsade de 799 pointes clinical 803 features of patients infected with 2019 novel coronavirus in wuhan use 806 of chest ct in combination with negative rt-pcr assay for the 2019 novel coronavirus 807 but high clinical suspicion human 810 coronaviruses oc43 and hku1 bind to 9-<em>o</em>-acetylated sialic acids via a 811 conserved receptor-binding site in spike protein domain a angiotensin-converting enzyme 2 protects from severe 815 acute lung failure sars-cov-2 (covid-19) vaccine development and production: an 817 cambridge quarterly of healthcare ethics : cq : the international 818 journal of healthcare ethics committees generation of a highly pathogenic avian influenza a virus from an avirulent 821 field isolate by passaging in chickens a paper raises some safety concerns for gilead's covid-19 823 treatment azithromycin as a 825 cause of qt-interval prolongation and torsade de pointes in the absence of other known 826 precipitating factors sars-coronavirus replication is supported by a 830 reticulovesicular network of modified endoplasmic reticulum the effect of human mobility and control 834 measures on the covid-19 epidemic in china a 838 crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung 839 injury asymptomatic carrier state, acute respiratory disease, and pneumonia due to 842 severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths severe acute respiratory 845 syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the 846 epidemic and the challenges effective 848 strategies to prevent coronavirus disease-2019 (covid-19) outbreak in hospital. the journal 849 of hospital infection nsp3 of coronaviruses: structures and functions of a 851 large multi-domain protein functional assessment of cell entry and receptor 853 usage for sars-cov-2 and other lineage b betacoronaviruses structure of sars coronavirus spike 856 receptor-binding domain complexed with receptor molecular immune pathogenesis and 862 diagnosis of covid-19 development and clinical application of 866 a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis potential preanalytical and analytical 869 vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (covid-19) receptor usage and cell entry of porcine epidemic diarrhea coronavirus hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting 876 sars-cov-2 infection in vitro overlapping and discrete aspects of the pathology and pathogenesis of the 879 emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov covid-19 vaccines: knowing the unknown neurological manifestations of hospitalized patients with china: a retrospective case series study coronavirus (covid-19): managing stress and anxiety trypsin treatment unlocks barrier for zoonotic bat coronavirus 891 infection clinical management of severe acute respiratory infection when 893 novel coronavirus (ncov) infection is suspected structures of mers-cov spike glycoprotein in complex with 896 sialoside attachment receptors coronavirus infections-more than just the 898 common cold findings of lung ultrasonography of novel 900 corona virus pneumonia during the 2019-2020 epidemic coronaviruses post-sars: update on replication and 902 pathogenesis risk 904 in vaccine research and development quantified immunosuppression for hyperinflammation in 906 covid-19: a double-edged sword? the lancet roche's cobas sars-cov-2 test to detect novel coronavirus receives fda 908 emergency use authorization and is available in markets accepting the ce mark the epidemiology and pathogenesis of coronavirus 910 disease (covid-19) outbreak coronavirus envelope protein: current knowledge structure of mouse coronavirus spike protein complexed with receptor 915 reveals mechanism for viral entry a review of coronavirus disease-2019 (covid-19) regulatory updates and 919 analytical methodologies for nitrosamine impurities detection in sartans, ranitidine, nizatidine 920 and metformin along with sample preparation techniques animal models and novel therapeutic 924 interventions for middle east respiratory syndrome coronavirus infections. frontiers in 925 microbiology 10 chloroquine and its analogs: a new promise of an old drug for 927 effective and safe cancer therapies genetic recombination, and pathogenesis of coronaviruses. trends in 930 microbiology the covid-19 vaccine development landscape covid-19: fighting panic with information of chloroquine and covid-19 function, and antigenicity of the sars-cov-2 spike glycoprotein function, and antigenicity of the sars-cov-2 spike glycoprotein receptor recognition by the novel 943 coronavirus from wuhan: an analysis based on decade-long structural studies of sars 944 coronavirus xiao, 946 g., 2020a. remdesivir and chloroquine effectively inhibit the recently emerged novel 947 coronavirus (2019-ncov) in vitro unique epidemiological and clinical features 949 of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control 950 measures who, 2020a. coronavirus disease 2019 (covid-19): situation report -144 coronavirus disease (covid-19) advice for the public, advice for public. 954 world health organization cryo-em structure of the 2019-ncov spike in the prefusion 957 conformation overview of the 2019 novel coronavirus (2019-959 ncov): the pathogen of severe specific contagious pneumonia (sscp) pathological 963 findings of covid-19 associated with acute respiratory distress syndrome. the lancet. 964 respiratory medicine the 966 deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in 967 china the 969 deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in 970 china 974 clinical characteristics of 82 death cases with covid-19 bevacizumab with dose-dense paclitaxel/carboplatin as first-line 976 chemotherapy for advanced ovarian cancer a pneumonia outbreak associated 981 with a new coronavirus of probable bat origin covid-19): a perspective from china ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests:none key: cord-277076-yvsyo4l9 authors: berger, a.; preiser, w. title: sars date: 2019-09-12 journal: encyclopedia of environmental health doi: 10.1016/b978-0-444-63951-6.00624-0 sha: doc_id: 277076 cord_uid: yvsyo4l9 severe acute respiratory syndrome (sars) emerged in southern china in late 2002. it first spread within guangdong province and then to other parts of china. via air travelers, it quickly reached various countries around the globe, causing several major hospital outbreaks. within weeks, the causative agent, a previously unknown coronavirus (sars-cov), was identified, thanks to an unprecedented international effort led by the world health organization (who). its origin was quickly traced to wild animals traded locally for culinary purposes. masked palm civet and some other species seem to have acted as intermediate hosts. since then, sars-like coronaviruses were found in different bat species in china and elsewhere, and bats are now regarded as the wildlife reservoir for sars-cov. fortunately, the sars outbreak could be contained within months. until july 2003, it had caused 8096 cases, with 774 deaths. once adequate measures such as isolating patients and quarantining their contacts were strictly adhered to, further transmission between human beings could be interrupted. sars is an example of how rapidly an infectious agent can spread in the modern world. at the same time, it should serve as a showcase of how international cooperation and modern science can help to combat the spread of infectious diseases. in an unprecedented move, who initiated a collaborative multicenter research project to identify the causative agent of sars. avian influenza was quickly ruled out. several laboratories diagnosed active infections with chlamydia or paramyxoviruses in somebut far from allpatients suffering from sars. however, there was good evidence that neither of these were plausible etiological agents for the novel infectious disease. the network comprised laboratories with access to sars patient samples and those with particular expertise on emerging or respiratory viruses. for several weeks, a spirit of cooperation rather than competition prevailed. samples were exchanged and results and findings shared continuously via daily telephone conferences and a password-protected website. within weeks, groups in hong kong, germany, canada, and the united states of america found a hitherto unknown member of the coronavirus family in sars patients. this was achieved through independent studies but certainly benefited greatly from the real-time sharing of interim results. although different approaches were employed, including cell culture, electron microscopy, and molecular techniques, sequencing data showed all laboratories had identified the same coronavirus, previously unknown to human and veterinary virology ( figure 1) . it was shown that sars patients seroconverted against this virus in the course of their illness; healthy, unexposed control individuals lacked antibody reactivity. however, it remained to be proven that this novel coronavirus was indeed the etiological agent for sars rather than an 'innocent bystander' newly discovered by thorough studies. after experimental infection of macaques with the newly isolated agent was shown to cause a sars-like illness, and subsequent reisolation of the agent, all of koch's postulates had been fulfilled. on 16 april 2003, who officially announced that the provisionally termed sars-associated coronavirus (sars-cov) was the causative agent of sars. based on this breakthrough, tests for the detection of viral sequences and specific antibodies were quickly developed and made available to affected countries. in addition, numerous scientists embarked on programmes to develop vaccines and drugs or antibodies for prophylactic or therapeutic use. within a few months, the sars outbreak was brought under control. on 5 july 2003, who declared that the last chain of person-toperson transmission had been interrupted. measures including source isolation of patientswho only became infectious after onset of clinical symptomsstrict infection control in health care facilities, timely identification and quarantining of exposed contacts, and perhaps also measures to increase social distance, such as travel warnings and screening of travelers, had led to this remarkable and remarkably rapid success. thorough and consistent implementation of these measures eventually brought an end to the sars outbreak even in the worst affected areas. in the meantime, however, several areasdifferent chinese provinces other than guangdong, most prominently the capital, beijing, but also toronto in canada, and taiwanpaid a high price for not implementing adequate countermeasures in a timely fashion. typically, a so-called 'superspreader,' that is, a highly contagious sars patient, would seek treatment at a poorly prepared facility, and by the time the danger was realized, scores of staff and patients had become infected and themselves become sources of spread. interestingly, despite the rapid identification of the agent and laboratory tests becoming available almost immediately, these formidable achievements did not contribute much to the containment of the outbreak. instead, it was the prudent and thorough use of 'old-fashioned' measures such as isolation and quarantine that proved to be the key to success. identification of suspected cases was based on clinical and epidemiological criteria: high fever (>38 c) plus symptoms of respiratory tract infection plus an exposure history, the details of which depended on each location's sars status at the time. an additional positive sars-cov test result or radiological or pathological evidence of pneumonia or respiratory distress syndrome would make it a probable case. the final case count from 1 november 2002 until 31 july 2003 is 8096, with 774 deaths. since mid-2003, sars has reappeared on four occasions. three involved laboratory-acquired infections, which demonstrates the dangers of breaching biosafety procedures and the risks of subsequent further spread in the community by secondary transmission outside of the laboratory. the fourth sars outbreak was due to reintroduction from the reservoir. to minimize the risk of reemergence, who has issued guidelines for the surveillance of possible sars cases. risk categories to guide adequate national surveillance strategies to guard against the possible (re-)emergence of sars are emergence from wildlife or other animal reservoirs, emergence or introduction from laboratories or via international travel, or low risk of sars-cov emergence or introduction. who also urges all countries to conduct an inventory of all laboratories working with or storing sars-cov and to ensure strict enforcement of biosafety procedures. before sars, only two coronaviruses (hcov-229 e and hcov-oc43) were known to infect humans. because they both cause only relatively minor disease and are difficult to propagate in the laboratory, they received relatively little attention by diagnostic and research laboratories. in contrast, several coronaviruses were recognized as animal pathogens, infecting pigs, dogs, rabbits, cats, mice, rats, turkeys and chicken. some of these had for some time played important roles in veterinary medicine. it was obvious that the majority of human sars cases were acquired through transmission from other sars sufferers (which proved key to its eventual control). however, it seemed highly unlikely that this was a previously existing disease entity that was only then being recognized; its propensity to cause nosocomial outbreaks, often in hospital settings, would not have been overlooked for long. the very first sars cases were identified retrospectively in various localities in guangdong province. the first of these early cases occurred in foshan in november 2002, and several more over the following weeks in nearby places. interest soon focused on these early index cases: was there any characteristic these patients had in common that could point to a source of their sars-cov infection? there was indeed; many of these early index patients either worked in kitchens or at markets or lived nearby where they were constantly exposed to a multitude of species of domestic and wild animals that were being traded, kept in captivity, and finally slaughtered and prepared for consumption. this provided the impetus for studying animals being sold at guangdong markets. infection with coronaviruses closely related to sars-cov was identified in different species, most commonly in the masked palm civet (paguma larvata) but also in the chinese ferret badger (melogale moschata) and the raccoon dog (nyctereutes procyonoides). a further, small sars outbreak occurred again in guangdong in late 2003/early 2004; molecular analysis of virus isolates from human cases and animals sampled at the same place and time confirmed that this was zoonotically acquired from paguma larvata. however, further investigations revealed that this widely consumed species is most likely not the animal reservoir, as most populations studied were uninfected. later research identified a multitude of coronaviruses in different species of bats in asia and elsewhere, among them what is probably the progenitor of sars-cov. studies by different groups demonstrate that sars was the result of spillover from a wildlife reservoirmost likely batsinto an intermediate host (or hosts; most importantly paguma larvata) and from there to the human population. rapid viral evolution was demonstrated and most likely was the pivotal factor that allowed sars-cov to rapidly adapt to nonreservoir species. table 1 shows the distribution of several different coronaviruses in humans and animals and their classification into different groups. disease caused by sars-cov may present with rather nonspecific clinical signs and symptoms. the differential diagnosis is therefore wide and may include various common respiratory pathogens, including influenza, parainfluenza, and respiratory syncytial viruses (rsv) the laboratory diagnosis of sars remains a challenge; in fact, despite the rapid identification of sars-cov as the etiological agent, testing contributed little to the successful control of the 2003 outbreak. insufficient test specificity on occasions caused false-positive results, leading to considerable confusion. in many viral diseases, virus shedding is greatest during the early symptomatic phase, that is, around, and immediately following the onset of symptoms. unfortunately, virus excretion is comparatively low during the initial phase of sars. it peaks in respiratory specimens and in stools at around day 10 after the onset of the clinical illness. in addition, there are currently no laboratory tests available to reliably diagnose sars in the first few days of illness. the highest test sensitivity is achieved with bronchoalveolar lavage fluid or lung biopsy tissue at the onset of illness. because of the invasiveness of such procedures and the associated risk of transmission, nasopharyngeal aspirates and throat washings, taken with respiratory precautions and preserved in viral transport medium, remain the most important diagnostic specimens. most commonly, viral genome detection, usually by reverse transcriptase-polymerase chain reaction (rt-pcr), is used diagnostically. nucleic acid amplification tests have been designed targeting the orf1b or nucleoprotein genes; it has not been clearly proven in clinical studies that they are superior. real-time rt-pcr of nasopharyngeal aspirates is the most sensitive and rapid method. furthermore, the determination of viral load in nasopharyngeal specimens or serum has been shown to be of clinical value, as it is an important prognostic factor. to avoid false-positive results, who stipulates that the following should be tested: at least two different clinical specimen types (e.g., nasopharyngeal aspirate and stool), or the same type of clinical specimen but collected on at least two occasions during the course of the illness (e.g., sequential nasopharyngeal aspirates), or the same original clinical sample but two different assays or by repeating the same assay but using a new rna extract for each test. sars-cov is cultivable on vero and caco2 cells not only from respiratory materials but also from fecal samples ( figure 2 ). once isolated, the virus must be identified as sars-cov using further tests. cell culture is a very demanding test, but currently (with the exception of animal trials) the only means to show the existence of a live virus. antigen detection in respiratory and fecal specimens using enzyme immunoassay (eia) is generally less sensitive than rt-pcr. however, antigen detection in serum specimens with monoclonal antibodies or monospecific polyclonal antibody against the viral n protein was found to be a sensitive and specific test for the diagnosis of sars. as serum antibody levels start to rise from day 7 after onset of illness, the sensitivity of the serum antigen assay progressively decreased to 0% at day 21. antibody testing allows the indirect diagnosis of sars-cov infection and is unsuitable during the acute illness. positive antibody test results indicate previous infection with sars-cov. seroconversion from negative to positive or a fourfold rise in the antibody titer from acute to convalescent serum indicates a recent infection. a negative antibody test result later than 21 days after the onset of illness is likely to indicate that no infection with sars-cov has taken place. virus-specific serum igg, igm, and iga antibodies against sars-cov appear at around the same time, between days 5 and 17 after the onset of symptoms. antibody testing is therefore generally not useful during the first week of illness. for antibody testing, the indirect immunofluorescent antibody test is more commonly performed than the neutralizing antibody test in cell cultures that again requires a biosafety level 3 laboratory (figure 3) . a recombinant nucleocapsid eia may be used as a rapid screening test and possesses a higher sensitivity, with detection as early as day 5 after onset of illness. single low-titer positive antibody results can be due to cross-reactivity with other human coronaviruses and require confirmation by more specific western blotting or neutralization assays. who regards seroconversion or an at least fourfold rise in antibody titer between an acute and a convalescent serum as proof of infection. the updated who guidelines for the global surveillance of sars of october 2004 replace all previous who guidance on sars surveillance and response. one key recommendation is that independent of the test used, who strongly recommends that during the interepidemic period all countries seek verification of laboratory-confirmed cases of sars ('preliminary positive' cases), preferably by an external laboratory that is part of the who sars international reference and verification laboratory network (figure 4) . human sars-cov are no longer circulating in the human population, nevertheless the virus is still present in the animal reservoir and virological laboratories and can reemerge anytime. there is no single test that can be used to diagnose sars with a reasonable degree of accuracy. diagnosis, therefore, continues to rely on the clinical examination, supported by case definitions that include the risk assessment according to the 2004 updated who guidelines (tables 2 and 3 ). the initial symptoms of sars are nonspecific, complicating the differential diagnosis. the mean incubation period is 5 days with the range of 2-10 days although there are isolated reports of longer incubation periods. unlike influenza virus, where the patients are most infectious in the first 2 days of illness, transmission from symptomatic sars patients usually occurred on or after the fifth day of onset of disease, which is in line with the rising viral load in nasopharyngeal secretions that peaked at around day 10. there have been no reports of transmission occurring before the onset of symptoms. the typical clinical presentation of sars is that of viral pneumonia with rapid respiratory deterioration. patients initially develop influenza-like prodromal symptoms. fever, malaise, myalgia, headache, rigors, and nonproductive cough are the major presenting symptoms, whereas rhinorrhea and sore throat are less frequently seen. clinical deterioration, often accompanied by watery diarrhea, commonly occurs 1 week after the onset of illness. although history of fever is the most frequently reported symptom, it may be absent on initial measurement. severe cases develop rapidly progressing respiratory distress and oxygen desaturation with approximately 20% requiring intensive care. chest radiographs typically show ground-glass opacities and focal consolidations, especially in the periphery and subpleural regions of the lower zones. progressive involvement of both lungs is not uncommon. features during the later stages have sometimes included spontaneous pneumothorax, pneumomediastinum, subpleural fibrosis and cystic changes. nosocomial transmission of sars-cov has been a striking feature of the sars outbreak. the majority of the cases have occurred in adults. children are less commonly affected than adults and usually have a milder illness. the overall mortality rate was approximately 10%. age and the presence of comorbidities are poor prognostic indicators. although the sars outbreak was still ongoing during the first half of 2003, significant funding was made available for sars-related research. immunomodulators (i.e., corticosteroids, intravenous immunoglobulins, thymosin, and anti-tnf) were empirically used for the treatment of sars during the initial epidemic. the correlation between viral load and clinical outcome suggests that suppression of viral replication by effective antiviral drugs should be the key to preventing morbidity and mortality. numerous potential antiviral agents have been identified using different approaches. in vitro susceptibility test results demonstrate that ifn-alpha and ifnbeta have some potential activity. ribavirin has good activity when tested in human caco-2 cells despite its lack of activity in vero cells. the viral proteases are important targets for the development of antiviral drugs. protease inhibitors like nelfinavir, glycyrrhizin, chloroquine, and many others as well as many herbal formulations have been found to possess some antiviral activity against sars-cov in vitro. in addition, the use of nitric oxide (s-nitro-n-acetylpenicillamine) inhalation as an experimental form of rescue therapy for sars appeared to have inhibitory activity against sars-cov. screening of chemical libraries has identified several inhibitors of the viral protease and helicase. identification of angiotensin-converting enzyme 2 (ace2) as an obligatory cellular receptor for table 2 risk categories for the emergence of sars emergence of sars-cov-like viruses from wildlife or other animal reservoirs -countries/areas identified as source(s) of the epidemic in 2002-03 in southern china or areas with an increased likelihood of animal-to-human transmission of sars-cov-like viruses from wildlife or other animal reservoirs. emergence or introduction of sars-cov from laboratories or international travel -countries/areas at potentially higher risk of sars-cov emergence or introduction due to the presence of laboratories in which sars-cov or sars-covlike viruses are being studied or in which clinical specimens infected with sars-cov are being processed or stored. or -countries/areas with entry of large numbers of persons from areas in which wildlife or other animal reservoirs of sars-cov-like viruses are found. low risk of sars-cov emergence or introduction -countries/areas that never reported cases or reported only imported cases during the 2002-03 epidemic, and that do not conduct research using live sars-cov-like viruses or store clinical samples from sars cases. sars-cov contributed to understanding of the sars-cov entry process, and helped to characterize two targets of antiviral therapeutics: the sars-cov spike protein and ace2. however, most of the chemicals or approaches have not been evaluated in human or animal models. various approaches toward producing a vaccine against sars have been pursued, including the use of inactivated sars-cov, plasmid dna, and adenovirus vectors. one obvious problem any vaccine would face is whom it should be given to, in the absence of sars-cov transmission. however, waiting until a renewed outbreak occurs before commencing vaccination means that precious weeks would be lost until individuals at risk become immune. the administration of preformed antibodies (obtained from human or animal donors or, more recently, produced in vitro) is effective in preventing a number of different infections, such as hepatitis b, varicella, and rsv. similarly, it could be shown that neutralizing antibodies against sars-cov can protect experimental animals from infection. using screening of a large naive antibody library by antibody phage display technology, neutralizing antibodies were identified and produced that were protective in vitro. there was no evidence of enhancement of sars-cov infection by subneutralizing concentrations of these antibodies, and immune escape mutants were not generated. in theory, this could be an ideal tool: if sufficient stocks of such human monoclonal antibodies could be procured, they would be ready for use when and wherever sars reemerges. one could then passively immunize anyone in contact with the source or patients, affording immediate protection. in summary, sars was a novel severe infectious disease that presumably originated in guangdong in southern china. large-scale wildlife trade and consumption favored the emergence of this zoonosis from a hitherto unrecognized animal reservoir. after its zoonotic origins, the new agent quickly spread within the human population, being transmitted mostly via the respiratory route through close human-to-human contact. sars was rapidly disseminated via the metropolis hong kong through international air travel. its important potential for nosocomial transmission in the community and in hospitals was soon recognized and allowed for appropriate measures to be instituted in most places. there was an unprecedented rapid gain of knowledge through global networking and international collaboration, led by who. unfortunately it took some major outbreaks, some of them affecting industrialized countries with modern health care systems, until the first sars epidemic was controlled. thus in the end rapid success was achieved through 'traditional' sanitary measures, mainly rapid identification of suspect cases and isolation of the diseased. fortunately, sars-cov hasat least so farnot established itself permanently in the human population. its relatively low transmissibility (with the exception of 'superspreaders') led to a low basic reproduction number r 0 ; together with the fact that infected individuals only become infectious themselves once they have developed clinical disease, this made 'traditional' public health measures very effective. is the fact that the first pandemic of the twenty-first century was quickly contained reason to take comfort in the knowledge that the history of humankind has reached a phase in which biomedical sciences and information technology are able to deal with such threats? probably not. first, the rapid success was remarkable. unfortunately, though, this does not reflect preparedness. in 1998, donald burke proposed the following criteria for identifying virus families with a high pandemic risk: (1) those that had caused pandemics in human populations in the recent past; (2) those with the proven ability to cause larger epizootics; and (3) those with an intrinsic propensity to rapidly undergo evolution on the basis of high mutation rate or genome organization favoring recombination ('intrinsic evolvability'). interestingly, even before the sars outbreak (which obviously fulfills the first criterion), the coronavirus family should clearly have been regarded as high risk: starting in the late 1970s, the porcine epidemic diarrhea coronavirus (pedv) caused a severe swine epizootic in europe and asia (second criterion). the high evolvability of the coronavirus family had also been demonstrated: the porcine respiratory coronavirus (prcv) evolved through a deletion mutation in the s gene from the transmissible gastroenteritis virus (tgev); the mutant has a different tissue tropism and is less virulent. second, infectious disease emergence from zoonotic sources was a well-recognized threat long before sars. nevertheless, it took anotherfortunately minor -sars outbreak before decisive steps were taken to control at least the trade in the directly implicated paguma larvata in southern china. starting shortly after sars had been controlled, another agent with pandemic potential has caused an ongoing epizootic of unprecedented proportions: highly pathogenic influenza a (h5n1) virus. transmission to humans occurs almost exclusively through close contact with infectedand sickpoultry. despite the possibility that this avian influenza virus will become fully 'humanized' and trigger the next influenza pandemic, even activities recognized as high risk, such as 'wet markets,' have mostly not been curtailed. finally, the lessons from sarsparticularly the experiences of beijing, toronto, and taiwan that suffered devastating sars outbreaks during the later phase of the outbreakemphasize over and over again the enormous importance of open and timely communication. at times, this may mean admitting to problems that (at least initially) reflect negatively on the country or territory concerned. in the medium to long term, however, such openness is the only way to prevent consequences that would be much worse. although this was widely accepted in the wake of the sars experience and has had a strong influence on the new international health regulations that entered into force in june 2007, the information policies of some countries during the h5n1 epizootic unfortunately do not attest to this at all. notwithstanding its grave consequences in humanitarian, political, and economic terms, sars should serve as an example what can be achieved through international cooperation, modern science, and rigorous use of 'traditional' approaches. even if prudent precautions were suddenly adopted, sars will certainly not have been the last highly pathogenic novel infectious agent that crosses the species barrier into the human population. although bats must be very high on the list of 'culprits' for emerging viral diseases, having been identified as reservoir hosts for a number of emerging viruses, there is no reason why other groups of animals should not figure as prominently in future. the mechanisms behind emergence have been studied. these may be linked to the agent, the new host species (i.e., human beings and in certain cases also the intermediate hosts) or to the connection between those. many of the factors and determinants of disease emergence are related to human activities. although this has accompanied humankind throughout history, and in fact may have helped shape human history, the speed and the extent of human-induced changes has accelerated markedly in recent times. although in the case of sars people were lucky in the end, this may be quite different next time round. it will be important to improve understanding of emergence to minimize the risks of what is a natural phenomenon but much aggravated by human behavior. severe acute respiratory syndrome (sars)ã�paradigm of an emerging viral infection the evolvability of emerging viruses development of antiviral therapy for severe acute respiratory syndrome severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome: identification of the etiological agent evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses viral zoonosesã�a threat under control? human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets bats, civets and the emergence of sars wet marketsã�a continuing source of severe acute respiratory syndrome and influenza? relevant websites www.cdc.gov/eid. emerging infectious diseases: (search for sars) health protection agency (uk) information and guidance on sars released by the world health organization (who) international office of epizootics key: cord-309147-c3ikb81g authors: nadeem, muhammad shahid; zamzami, mazin a.; choudhry, hani; murtaza, bibi nazia; kazmi, imran; ahmad, habib; shakoori, abdul rauf title: origin, potential therapeutic targets and treatment for coronavirus disease (covid-19) date: 2020-04-22 journal: pathogens doi: 10.3390/pathogens9040307 sha: doc_id: 309147 cord_uid: c3ikb81g the ongoing episode of coronavirus disease 19 (covid-19) has imposed a serious threat to global health and the world economy. the disease has rapidly acquired a pandemic status affecting almost all populated areas of the planet. the causative agent of covid-19 is a novel coronavirus known as sars-cov-2. the virus has an approximate 30 kb single-stranded positive-sense rna genome, which is 74.5% to 99% identical to that of sars-cov, cov-pangolin, and the coronavirus the from horseshoe bat. according to available information, sars-cov-2 is inferred to be a recombinant virus that originated from bats and was transmitted to humans, possibly using the pangolin as the intermediate host. the interaction of the sars-cov-2 spike protein with the human ace2 (angiotensin-converting enzyme 2) receptor, and its subsequent cleavage by serine protease and fusion, are the main events in the pathophysiology. the serine protease inhibitors, spike protein-based vaccines, or ace2 blockers may have therapeutic potential in the near future. at present, no vaccine is available against covid-19. the disease is being treated with antiviral, antimalarial, anti-inflammatory, herbal medicines, and active plasma antibodies. in this context, the present review article provides a cumulative account of the recent information regarding the viral characteristics, potential therapeutic targets, treatment options, and prospective research questions. the outbreak of a novel respiratory syndrome, referred to as coronavirus disease 2019 (covid19) , was first recognized in wuhan, china, in december 2019. the causative agent for this deadly condition is a coronavirus known as sars-cov-2. covid-19 is demonstrated by fever, dry cough, persistent pressure in the chest, and shortness of breath [1, 2] . sneezing, runny nose, and symptoms similar to the common cold are observed in only 5% of patients. about 2% to 10% of patients have shown diarrhea like symptoms [3, 4] . the mortality rate of covid-19 is 4.5% to 6%, which is less than that of sars (severe acute respiratory syndrome), which has a mortality rate of 9.6%, and less than that of mers (middle east respiratory syndrome), up to 34.4% deaths. individuals already suffering from cardiovascular disease, hypertension, respiratory disease, or diabetes are at a high risk of mortality. age and gender-specific variations in the death rate have also been reported [5] . the disease became pandemic within a few months of its emergence, indicating a high transmission ability as coronaviruses, first discovered in the 1960s, are found in birds and mammals, especially in bats, cats, camels, and rats [19] . the causative agent of covid-19 (sars-cov-2) belongs to the genus β-coronavirus, family coronaviridae, and order nidovirales. a similar human coronavirus was found to be responsible for sars in 2002 and 2003. the virus responsible for covid-19 has a single-stranded positive-sense rna genome of about 30 kb, which has 74% to 99% identity with that of the coronavirus from the pangolin (manis javanica) and horseshoe bat (rhinolophus sinicus) (bat-covratg13), respectively [2, [20] [21] [22] [23] . bats have been reported as being the rich source of coronaviruses [24, 25] , although only a few of these coronaviruses can infect humans [26, 27] . according to the literature, the sars and mers viruses have zoonotic transmission, originating from bats using palm civets and camels, respectively, as the intermediate hosts [28] [29] [30] [31] [32] . the recent reports have suggested that sars-cov-2 is a modified coronavirus of bat origin [22, 32] , which came to humans as a result of zoonotic transmission [33, 34] . a coronavirus identified from the malayan pangolin has been shown to have a 99% similarity with sars-cov-2. the receptor-binding domain (rbd) of pangolin-cov has only a one amino acid difference with that of sars-cov-2; the infected pangolins exhibit pathological symptoms similar to humans suffering from covid-19, and their blood circulating antibodies can react with the spike protein of sars-cov-2 [35, 36] . although the ratg13 coronavirus isolated from bat has about 96% identity with sars-cov-2, its rbd is different from that of the later, exhibiting a low binding ability to the human ace2 [37] . however, the rbd of the s-protein from pangolin-cov is highly similar to that of sars-cov-2, six residues critical for receptor binding being identical in both [38] . a comparative analysis of genetic data available to date has suggested that sars-cov-2 originated by the recombination of pangolin-cov and the bat-cov-ratg13-like virus [35, [39] [40] [41] . based on this information, the pangolin is considered to be one of the possible intermediate hosts between bat and human. snakes, minks, and turtles are also being investigated as the potential intermediate hosts [42, 43] (figure 1 ). five out of the six critical amino acid residues comprising the rbd of the s-protein from sars-cov and sars-cov-2 are different, contradicting the theories about the laboratory origin of sars-cov-2 by the manipulation of sars or mers like viruses [43] . studies based on the analysis of n, s, and orf1a/1b genes have shown conserved sequences suggesting that sars-cov-2 is an animal virus, which was transmitted to humans by undergoing evolutionary adaptations [22, 44] . the sars infection from 2003, also involved zoonotic transmission of the virus to humans. hence, further studies are required to confirm the intermediate hosts of coronaviruses to control zoonotic transmission and avoid the outbreak of such viral infections in the future [28] . on march 2, 2020, who published a pcr based detection method for sars-cov-2. the procedure could detect the virus in the blood, sputum, and nasopharyngeal swab [45, 46] . noncontrast chest ct (computed tomography) can also be used for the diagnosis of viral pneumonia. however, ct scans can be negative in the case of covid-19 [47] . on the other hand, patients with negative rt-pcr test results can show pneumonia-like symptoms on a ct scan [48] . in a comparative study, the sensitivity of a chest ct was found to be 98%, whereas the sensitivity of the pcr test was only 71% [49, 50] . rt-pcr based diagnosis also gave false-positive results [51] . low viral load, inefficient sampling, poor sample storage or processing conditions, along with a lack of specific primers due to the high rate of mutations in rna viruses, are some of the apparent factors for the poor sensitivity of pcr based diagnoses. recently, some parallel procedures have also been reported for the diagnosis of covid-19. one of these procedures is loop-mediated isothermal amplification (lamp), which is a faster single-step procedure, having > 95% sensitivity [52, 53] . further modifications of lamp-based procedures have been reported, which can reduce the testing time with minimum equipment requirements [54, 55] . to develop serological procedures, iga and igm have been evaluated against sars-cov-2 by immunofluorescence assays [56] [57] [58] . however, further refining of rt-pcr and the serological procedures are required to improve the sensitivity and specificity. on 2 march 2020, who published a pcr based detection method for sars-cov-2. the procedure could detect the virus in the blood, sputum, and nasopharyngeal swab [45, 46] . noncontrast chest ct (computed tomography) can also be used for the diagnosis of viral pneumonia. however, ct scans can be negative in the case of covid-19 [47] . on the other hand, patients with negative rt-pcr test results can show pneumonia-like symptoms on a ct scan [48] . in a comparative study, the sensitivity of a chest ct was found to be 98%, whereas the sensitivity of the pcr test was only 71% [49, 50] . rt-pcr based diagnosis also gave false-positive results [51] . low viral load, inefficient sampling, poor sample storage or processing conditions, along with a lack of specific primers due to the high rate of mutations in rna viruses, are some of the apparent factors for the poor sensitivity of pcr based diagnoses. recently, some parallel procedures have also been reported for the diagnosis of covid-19. one of these procedures is loop-mediated isothermal amplification (lamp), which is a faster single-step procedure, having >95% sensitivity [52, 53] . further modifications of lamp-based procedures have been reported, which can reduce the testing time with minimum equipment requirements [54, 55] . to develop serological procedures, iga and igm have been evaluated against sars-cov-2 by immunofluorescence assays [56] [57] [58] . however, further refining of rt-pcr and the serological procedures are required to improve the sensitivity and specificity. sars became epidemic in many countries around the world in 2002 and 2003. the disease had many symptoms similar to those of covid-19. however, sars-cov-2 and sars-cov have shown differences, as well as similarities, in their genomic composition, incubation time, and infection mechanisms. a set of affinities has been tabulated that can help us to establish the correlation between the two viruses (table 1) . to date, the mortality rate of covid-19 is 4.5% to 5.5%. there are more than 1 million reported infections and 50,000 deaths (as recorded on 3 april 2020). the mortality rate was between 9.6% to 21%. it was restricted to 8437 individuals and 813 deaths. [6, 7, 62] 3 the virus needs a longer incubation time (average 14 days) to represent covid-19 symptoms. the virus needed a relatively short incubation time (1-4 days) to exhibit symptoms. [11, 63] 4 in covid-19, the infection ratio between males and females is 2.7:1, indicating that the disease is more prevalent among males. old aged people also have a high mortality rate. the male to female ratio was 1:1.25; more prevalent in females. there was a higher death rate in old aged patients. the intra-or inter-species transmission of β-coronaviruses (covs) requires a viral interaction with the host cell receptors, and the subsequent invasion of the host cells [85] . the genome of the coronavirus codes for a surface glycoprotein, known as a "spike" protein (s-protein), that specifically binds to the host cellular receptors to initiate the infection process. in fact, the spike protein performs a "key" like function to "unlock" the door and facilitate the cellular entry of a coronavirus. studies based on 3d models of spike proteins from the sars-cov and sars-cov-2 viruses, have shown a considerable overall similarity [34, 86] . the cryo-em structure of the sars-cov-2 s-protein has also been reported [87] (figure 2) . the overall structure of the s-protein consists of several functional domains. the rbd, fusion domain (fd), and the s2 cleavage site could be critical for future studies to develop therapeutic strategies [87] . the protein exhibits a high binding affinity with ace2, as represented by a low dissociation constant value (kd~15 nm). the receptor binding affinity of the sars-cov-2 s-protein is 10 times higher than that of the sars-cov s-protein [37, [87] [88] [89] [90] . furthermore, studies using human, pig, and civet cell lines have allowed sars-cov-2 infection and replication, indicating that the virus makes use of the ace2 receptor for infection [22, [91] [92] [93] . ace2 is cleaved by a protease (tmprss2) in order to activate virus entry. this can be inhibited by protease inhibitors such as camostat mesylate [92] . ace2 is highly expressed in the lungs; a vast surface area makes the lung tissue highly susceptible to sars-cov-2 infection [94] . in addition to the lungs, the ace2 receptor is also expressed in the endothelial cells of intestine, kidney, and heart cells [95] . pathogens 2020, 9, x for peer review 5 of 13 of six amino acids are different to that of sars-cov-2. the intra-or inter-species transmission of β-coronaviruses (covs) requires a viral interaction with the host cell receptors, and the subsequent invasion of the host cells [85] . the genome of the coronavirus codes for a surface glycoprotein, known as a "spike" protein (s-protein), that specifically binds to the host cellular receptors to initiate the infection process. in fact, the spike protein performs a "key" like function to "unlock" the door and facilitate the cellular entry of a coronavirus. studies based on 3d models of spike proteins from the sars-cov and sars-cov-2 viruses, have shown a considerable overall similarity [34, 86] . the cryo-em structure of the sars-cov-2 s-protein has also been reported [87] (figure 2) . the overall structure of the s-protein consists of several functional domains. the rbd, fusion domain (fd), and the s2 cleavage site could be critical for future studies to develop therapeutic strategies [87] . the protein exhibits a high binding affinity with ace2, as represented by a low dissociation constant value (kd ⁓ 15 nm). the receptor binding affinity of the sars-cov-2 s-protein is 10 times higher than that of the sars-cov s-protein [37, [87] [88] [89] [90] . furthermore, studies using human, pig, and civet cell lines have allowed sars-cov-2 infection and replication, indicating that the virus makes use of the ace2 receptor for infection [22, [91] [92] [93] . ace2 is cleaved by a protease (tmprss2) in order to activate virus entry. this can be inhibited by protease inhibitors such as camostat mesylate [92] . ace2 is highly expressed in the lungs; a vast surface area makes the lung tissue highly susceptible to sars-cov-2 infection [94] . in addition to the lungs, the ace2 receptor is also expressed in the endothelial cells of intestine, kidney, and heart cells [95] . the interaction of the viral s-protein with ace2, its subsequent activation by protease (tmprss2), and its viral entry into the cell. the schematic primary structure of the sars-cov-2 spike protein is elaborated indicating the major domains. ss-signal sequence, rbd-receptor binding domain, rbd subdomains 1 and 2-sd1 and sd2, s1/s2-the protease cleavage site, s2′-the protease restriction site indicated by the arrows, fp-fusion peptide, hr1 and hr2-heptad repeats 1 and 2, figure 2 . the interaction of the viral s-protein with ace2, its subsequent activation by protease (tmprss2), and its viral entry into the cell. the schematic primary structure of the sars-cov-2 spike protein is elaborated indicating the major domains. ss-signal sequence, rbd-receptor binding domain, rbd subdomains 1 and 2-sd1 and sd2, s1/s2-the protease cleavage site, s2 -the protease restriction site indicated by the arrows, fp-fusion peptide, hr1 and hr2-heptad repeats 1 and 2, ch-central helix, cd-connector domain, tm-transmembrane domain, and ct-cytoplasmic tail. (the schematic was adopted and modified from [87, 88] .) according to recent information, the glutamine at the amino acid position 394 in the receptor-binding protein of sars-cov-2 that corresponds to the residue 479 in sars-cov, is recognized by lysine 31 residue in the human ace2 receptor [90] . an interaction of polar residues in the ectodomain of ace2 with the receptor binding domain of sars-cov-2 spike protein has been reported [96] . downstream interaction and the invasion process include the cleavage of the spike by a serine protease at the "s2" domain ( figure 2 ) [97] , followed by the interaction of the virus s-protein fusion domain with the host cell plasma membrane. virus entry takes place either by fusion with the plasma membrane or by endocytosis, and the subsequent fusion of membranes in endosomes [98] . in addition to virus-plasma membrane fusion, sars-cov can adopt clathrin-dependent endocytosis [27] . once inside the host cell, the viral rna is translated in the cytoplasm producing polyproteins and structural proteins. after translation, the replication of the genome occurs [99] . new virus particles are formed in the membranes of the golgi apparatus and the endoplasmic reticulum, after which the vesicles harboring the viral particles are fused with plasma membranes for the release of the virus [100, 101] . because sars-cov-2, in binding with ace2, is dependent on an interaction and the cleavage of the spike protein by a serine protease, a spike protein-based vaccine or serine protease specific inhibitors could be potential therapies against sars-cov-2 infection [102] . the comparative homology studies of sars-cov-2 proteins (s, n, m, and e proteins) with those from sars and mers viruses, have suggested some targets for vaccine development [103] . polyclonal antibodies raised against sars-cov are found to prevent a spike-mediated host cell invasion of sars-cov-2 [104] . recently, 1.3 billion potential protease blockers have been investigated by molecular docking studies. many of these molecules can be evaluated in wet labs in the near future [105] . ace2 blockers can be another option to avoid the infection [106] . similarly, there are some molecules including gsk1838705a, kt203, kt185, and bms195614 that have strong binding affinities with rbd of the viral s-protein [107] . these molecules can help to control rapid infections by engaging the virus at entry points [107] . currently, a tremendous amount of research is in progress to develop a vaccine against covid-19. however, vaccine development is time consuming process, and the newly introduced vaccine will require several safety evaluations [4] . according to estimates, a vaccine against covid-19 may take more than a year to become available [108] . even after the preparation of an effective vaccine, under the present pandemic situation, human trials will be a big challenge for researchers. at present covid-19 is being treated with some broad-spectrum antiviral drugs including remdesivir, favipiravir, and chinese herbal medicine [101, 109] . in vitro studies have shown that chloroquine and remdesivir are effective against sars-cov-2 [73] [74] [75] . chloroquine phosphate has shown treatment efficacy and safety against sars-cov-2 associated pneumonia; these findings are based on multiple trials in hospitals in china [76] . application of an anti-inflammatory drug such as baricitinib, together with an antiviral drug, has also been recommended to treat covid-19 [110] . high doses of ascorbic acid (vitamin c) are suggested for the prevention of the covid-19 disease [111] . type i interferon can inhibit viral replication. according to studies, interferon β could inhibit the replication of sars-cov [95, 112] . however, its efficacy against sars-cov-2 needs further investigation. the use of convalescent plasma for the treatment of covid-19 has been suggested. however, the absence of multiple trials on a large scale, and the concern that antibodies may demonstrate donor dependent titters and specificities, are the main deficits of convalescent plasma therapy [113] . there are several challenges in the management and control of coronaviruses. a wide range of coronaviruses with a highly mutable single-stranded rna genome are found [20, 21] in many mammalian and avian sources [19] that closely interact with each other, as well as with humans. the long-term viability of coronaviruses in airborne aerosols, and on daily utensils composed of plastics, stainless steel, and cardboard, enhance the chances of transmission of infection between individuals and species [8, 81, 114] . the recurrence of sars-cov-2 has been reported in convalescence times [115] , which can make the treatment more difficult and increase the chance of complications. the interventions into the ace2 binding abilities of sars-cov-2 and similar viruses, by the inhibition of the corresponding serine protease [106] , can be an area of investigation. easy, highly reliable, and early-stage diagnosis procedures are still required as a challenge for biomedical researchers [52, 53] . the presence of sars-cov-2 in stool samples of infected individuals raises the question about the fecal-oral transmission of the disease [10] . recently, the viability of sars-cov and sars-cov-2 has been described (115); however, the genetic factors behind the long-term survival of these coronaviruses need further investigation [81] . sars-cov has shown low stability at higher temperatures and at specific air humidity levels [9, 116] ; the effect of temperature and other environmental factors on the viability of sars-cov-2 are unclear. inactivation of coronaviruses by disinfectants, such as 60% to 70% ethanol or 0.1% sodium hypochlorite, is well established [117] . the efficacy and specificity of antiviral and antimalarial drugs being used in the treatment of covid-19 need further clinical trials. the ongoing covid-19 outbreak that emerged from wuhan, china, has acquired 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approach who says vaccines against novel coronavirus 18 months away, pushes global research can chinese medicine be used for prevention of corona virus disease 2019 (covid-19)? a review of historical classics, research evidence and current prevention programs covid-19: combining antiviral and anti-inflammatory treatments treatment for severe acute respiratory distress syndrome from covid-19 ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines convalescent plasma to treat covid-19: possibilities and challenges the authors are grateful to the doctors, medical staff, and volunteers contributing to overcome the coronavirus disease (covid-19) . the authors have no conflict of interest. key: cord-294363-bv6xa8v8 authors: zhou, hong; fang, yan; xu, tao; ni, wei‐jian; shen, ai‐zong; meng, xiao‐ming title: potential therapeutic targets and promising drugs for combating sars‐cov‐2 date: 2020-05-05 journal: br j pharmacol doi: 10.1111/bph.15092 sha: doc_id: 294363 cord_uid: bv6xa8v8 as of april 9, 2020, a novel coronavirus (sars‐cov‐2) had caused 89,931 deaths and 1,503,900 confirmed cases worldwide, which indicates an increasingly severe and uncontrollable situation. initially, little was known about the virus. as research continues, we have learned the genome structure, epidemiological and clinical characteristics and pathogenic mechanisms of sars‐cov‐2. based on these discoveries, identifying potential targets involved in the processes of virus pathogenesis is urgently needed, and discovering or developing promising drugs based on potential targets is the most pressing need. therefore, we summarize the potential therapeutic targets involved in virus pathogenesis and discuss the advancements, possibilities and significance of promising drugs based on these targets for treating sars‐cov‐2. this review will facilitate the identification of potential targets and provide promising clues for drug development that can be translated into clinical applications for combating sars‐cov‐2. in december 2019, a new coronavirus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2) caused an epidemic in wuhan, a central city in central china. this novel coronavirus has never been reported before and was found to have the ability to infect humans for the first time after nearly one month of spread. subsequently, it spread rapidly throughout china and caused outbreaks in many other countries, such as japan, south korea, italy and the united states . on january 31, 2020, the world health organization (who) declared that sars-cov-2 caused coronavirus disease 2019 , a public health emergency of international concern, and then designated it a pandemic on march 11, 2020. according to the latest data as of april 9, 2020 from the who, there are 89,931 deaths and 1,503,900 confirmed cases worldwide, with the mortality reaching approximately 5.980%, and the epidemic has followed a rapid growth model. a series of initiatives and statistical data hinted at the unusually strong destructive effect of sars-cov-2, which should merit adequate attention and active prevention from all over the worldwide. although we are confident that a resolution will eventually come, no specific vaccines or ideal drugs for sars-cov-2 have been formally utilized clinically at present, so it is critical to understand the exact pathogenic mechanism and develop effective drugs and vaccines to respond to sars-cov-2 outbreaks (cabrini et al., 2020) . several studies demonstrated angiotensin converting enzyme 2 (ace2) as an important therapeutic target of sars-cov-2 entry and infection, and many potential targets were subsequently proposed, such as the spike (s) protein and transmembrane serine protease 2 (tmprss2). additionally, multiple potential drugs have been suggested and verification work for virus treatment is underway. excitingly, several vaccines that have proven safety, efficacy and quality were also proven in early clinical trials. however, information on these works is distributed and diverse, which makes it difficult to form a comprehensive understanding for reference. therefore, in this review, we comprehensively summarize the potential therapeutic targets involved in the processes of virus transmission, infection and pathogenesis, based on recent studies. additionally, we discuss in depth the advancements, possibilities and significance of promising drugs based on these targets in the treatment of sars-cov-2. this review will this article is protected by copyright. all rights reserved. facilitate the identification of potential targets and provide promising clues for promising drug discovery and development that can be translated into clinical applications for combating sars-cov-2. coronaviruses (covs) are nonsegmented, positive sense, single-stranded rna viruses that belong to the subspecies coronaviridae. the length of cov genomes ranges from 26 to 32 kilobases, which makes it the largest viral rna known (livingston et al., 2020a) . currently, six kinds of covs are known to cause human diseases, which can be classified into two groups: slightly pathogenic and highly pathogenic covs. among the highly pathogenic covs, both severe acute respiratory syndrome coronavirus (sars-cov, 2002, southern china) and middle east respiratory syndrome coronavirus (mers-cov, 2012, saudi arabia) , which mainly infect the lower airways and cause serious fatal pneumonia, are known and can affect humans (cui et al., 2019) . the third known highly pathogenic cov has been identified as the pathogen causing outbreaks of sars-like clinical symptoms in wuhan city of china and was officially designated sars-cov-2 by the who. sars-cov-2 belongs to lineage b of the β-covs, a subgroup of sarbecovirus, which contains a positive-sense single-stranded rna as the hereditary substance and is wrapped by nucleocapsid protein in the core region and a peripheral envelope consisting of the spike protein, envelope protein, and membrane protein ( figure 1a ) (chan et al., 2020) . after conducting in-depth research, researchers found that the genomic organization of sars-cov-2 was consistent with a single-stranded positive-sense rna, which contains 5'-methylated caps and 3'-polyadenylated tails and is arranged in the following order: 5' end; replicase orf1a/b; spike (s); envelope (e); membrane (m); nucleoprotein (n); accessory proteins such as orf 3, 6, 7a, 7b, 8 and 9b; and the 3´ end ( figure 1b) . phylogenetic analysis studies found that sars-cov-2 has the closest relationship with bat-derived sars-cov but is not completely identical to sars-cov (approximately 79% identity) or mers-cov (approaching 50% identity) (paraskevis et al., 2020) . to date, studies have shown that the transmission of sars-cov-2 is spread mainly between people by means of the respiratory system and droplets (phan et al., 2020) . at present, several studies have put forward that sars-cov-2 can be transmitted not only by droplets but also this article is protected by copyright. all rights reserved. potentially via the oral faecal route (huang et al., 2020a) , but the latter requires formal proof. approximately 2 to 14 days after infection, with a median period of 4 days (interquartile range, 2 to 7 days) (livingston et al., 2020b) . the lungs and immune organs are the two main targets attacked by sars-cov-2. in addition, the virus also attacks other organs, such as the heart, kidney, oesophagus and multiple specific cell types (including alveolar cells, myocardial cells, renal proximal tubule cells, oesophageal epithelial cells, etc.), and the most likely reason is related to infections and ace2 distribution zou et al., 2020) . epidemiological surveys have found that the clinical features of sars-cov-2 infection are similar to those of sars-cov and are characterized by fever (>37.3℃), dry cough, dyspnoea or shortness of breath in most patients, whereas nonrespiratory clinical symptoms such as diarrhoea, sore throat, muscle ache, headache, and vomiting have also been reported in a minority of patients (meo et al., 2020) . confirmed patients have also developed acute respiratory distress syndrome, while critical illness may present with respiratory and lung function failure, even multiple organ dysfunction and septic shock, which require extracorporeal membrane oxygenation (ecmo) and intensive care support . the major pathological characteristics of the lung include pulmonary alveolitis and bronchiolitis with epithelial cell proliferation, desquamation, squamous metaplasia and the production of mucus and oedema fluid. meanwhile, massive diffuse pulmonary interstitial fibrosis accompanied by excessive inflammation, a certain amount of hyaline degeneration, and variable levels of pulmonary haemorrhagic infarction have been observed at the lesion site (luo et al., 2020) . moreover, several kinds of immune cells, including focal monocytes, lymphocytes, plasma cells and several multinucleate giant cells along with intracytoplasmic viral inclusion bodies, infiltrate into the pulmonary interstitium . in addition, immunohistochemistry results indicated positive results for immune cells, and pathological staining also showed extensive pulmonary interstitial fibrosis. eventually, these abnormal pathological changes result in lung function failure or multiple organ failure. although there has been some progress in the investigation and research of sars-cov-2, knowledge about this virus is still insufficient (chen et al., 2020a) . at present, the number of new confirmed cases and mortality are rapidly increasing daily under symptomatic treatment, this article is protected by copyright. all rights reserved. which requires research to confirm potential therapeutic targets and discover promising drugs as soon as possible, as the current priorities for the response to the sars-cov-2 outbreak. we know that the outbreaks of sars-cov-2 will be controlled with ideal drugs or effective vaccines to eventually end the pandemic. however, what worries us most is that many existing traditional models of drug and vaccine development are inadequate for this outbreak; a long-term process (maybe a few months or years) cannot rescue those patients who are dying as well as the slumping economy in a timely manner. vaccine/drug research and development should be given great attention because there is currently no ideal solution to clear sars-cov-2 infection, despite a pressing need to identify symptomatic strategies to ease suffering and preclude potential death (morse et al., 2020a) . therefore, accelerating research for potential therapeutic target confirmation, promising drug discovery, and clinical verification development will speed up efforts to combat sars-cov-2. based on the current studies, we summarize the potential therapeutic targets involved in virus transmission, infection and pathogenesis processes. additionally, we discuss the advancements, possibilities and significance of promising drugs based on these targets for combating sars-cov-2 (figure 2 ). in the initial stage of infection, the spike (s) protein of sars-cov-2 first combines with a cell surface receptor (wrapp et al., 2020) . the s protein consists of two subunits, the s1 (bulb) and s2 (stalk) subunits. s1 is responsible for receptor binding, while s2 is responsible for membrane fusion . more specifically, s1 combines with the cognate receptor to induce a strong conformational change in s2, thereby resulting in the fusion of the virus envelope and host cellular membrane and then releasing the nucleocapsid into the cytoplasm (sekimukai et al., 2020) . in view of this mechanism, targeting the s protein is likely to cut off sars-cov-2 infection, which can be regarded as the primary factor that needs to be studied in depth. based on past experience, sars-cov-2 s protein-neutralizing antibodies (nabs) are most likely to become the preferred research and development strategy that should be modelled and considered by researchers and drug r&d institutions to provide passive immunization against the infection . according to the functional this article is protected by copyright. all rights reserved. analysis of the s2 subunit, the interaction between heptad repeat 1 (hr1) and hr2 of s2 can form a six-helical bundle (6-hb), hence facilitating the fusion process of the viral and host cell membranes. drawing on experience with sars-cov, xia et al designed hr1-and hr2-derived peptides, designated sars-cov-2-hr1p (aa 924-965) and sars-cov-2-hr2p (aa 1168-1203), respectively . since sars-cov-2 and sars-cov s-hr2 sequences are 100% identical, sars-cov-2-hr2p most likely acts as a membrane fusion inhibitor in a way similar to sars-hr2p, the reported sars-cov fusion inhibitor . a fusion experiment of sars-cov-2-hr2p showed a potent fusion inhibitory effect with a half maximal inhibitory concentration (ic50) of 0.18 µm, while sars-cov-2-hr1p exhibited no obvious fusion inhibitory activity up to 40 µm, indicating that sars-cov-2-hr2p may be a promising therapeutic agent for sars-cov-2; however, the safety and actual effect await verification . research did not end there, so in a subsequent experiment, a pancoronavirus fusion inhibitor denoted as ek1 was designed based on the properties of the hr1 region (xia et al., 2019) . the results showed that ek1 also exhibited a potent fusion inhibitory effect with an ic50 of 0.19 µm, suggesting that ek1 is also a promising treatment as a sars-cov-2 drug pending verification and development. according to other research results, we know that sars-cov-2 enters the host cell by binding to membrane angiotensin-converting enzyme 2 (ace2) via the s protein receptor-binding domain (rbd) . based on this situation, zhang et al synthesized a first-in-class peptide binder, named 23-mer peptide binder (sbp1), by using automated fast-flow peptide synthesis technology. after careful analysis, they found that sbp1 can potentially restrain the entry process of sars-cov-2 into human cells through binding to the sars-cov-2-rbd with extremely low affinity (kd value = 47 nm) . moreover, the human protein-derived sequence of sbp1 prevented the binder from being immunogenic, which further promoted the peptide binder sbp1 as a promising preclinical drug lead for anti-sars-cov-2 drug discovery and development. receptor binding is one of the major determinants of tissue tropism and host range for covs. a study showed that covs adopt cell surface enzymes as their binding receptors, such as the ace2 receptor for sars-cov and the dipeptidyl peptidase 4 (dpp4) receptor for this article is protected by copyright. all rights reserved. (fung et al., 2019) . recently, a study found that sars-cov-2 can enter into the identical set of cell lines as sars-cov, suggesting that they have a similar receptor, ace2. the results of sequence analysis showed that some but not all sars-cov-2 clusters can use ace2 for host cell entry . according to receptor-binding motif (rbm) analysis, the majority of amino acid residues indispensable to ace2 binding were retained in the s protein of sars-cov-2, which was consistent a previous conclusion that the virus used ace2 for host cell entry . in agreement with these findings, sars-cov-2 uses ace2 for cellular entry, similar to sars-cov. therefore, targeting ace2 can prevent the replication of sars-cov-2, which can be regarded as a potential therapeutic strategy that needs to be studied in depth. a recent hypothesis proposed that ace inhibitors (aceis), such as captopril and enalapril, or angiotensin receptor 1 (at 1r) inhibitors, including losartan and valsartan, might be beneficial for those patients who experienced pneumonia induced by sars-cov-2 . however, unfortunately, this is just a plausible speculation without basic or clinical research verification. at present, researchers envision administering a kind of antibody that could bind to the host cell membrane ace2 protein, thus preventing the entry and infection of sars-cov-2. this promising intervention strategy was shown to significantly block viral entry and replication in experiments, but additional tests are needed to identify any anti-sars-cov-2 infection effects (li et al., 2003) . alternatively, people could simply develop a single chain variable fragment (scfv) that binds to the ace2 receptor to inhibit its complexation with sars-cov-2. in addition, vhh domains or nanobodies from camelids are two possible choices for consideration as well (desmyter et al., 2013) . a potentially more promising strategy was proposed by a researcher, who claimed that an antibody-like molecule could be created that would bind to the virus itself rather than protecting host cells against infection. based on this strategy, researchers proposed using clinical-grade soluble human ace2 (hrsace2) to bind the s protein of sars-cov-2, thereby neutralizing the virus. research on sars-cov indicated that this strategy is quite promising. thus, the hrsace2 is feasible to inhibit sars-cov-2 infection of cells, including vero-e6 cells, human capillary organoids and human kidney organoids, in a dose-dependent manner (vanessa, 2020) . since ace2 is a valuable therapeutic target and hrsace2 shows good therapeutic properties in vitro, it may be another good choice to fuse hrsace2 to an this article is protected by copyright. all rights reserved. immunoadhesin to form an immunoglobulin-fc domain to prolong the availability of the circulating molecule and boost immune system functions to fight sars-cov-2 infection. this research offers solid evidence that hrsace2-immunoglobulin-fc may similarly suppress sars-cov-2 both in vitro and potentially in patients (kruse, 2020) . overall, the aforementioned therapeutic strategies indicated that ace2 must be a potential therapeutic target in the treatment of sars-cov-2. meanwhile, an ace2 antibody, ace2-scfv, ace2 nanobody and ace2-fc may be promising anti-sars-cov-2 drugs after animal testing and clinical trials. in addition to the s protein and ace2, another study suggested that proteases may help activate the s protein by priming it to promote sars-cov-2 cellular entry. the endosomal cysteine proteases cathepsin b and l (catb/l) and the serine protease tmprss2 are two critical proteases contributing to the pathogenicity of coronavirus infections(iwata-yoshikawa et al., 2019). research has found that sars-cov-2 can use tmprss2 rather than catb/l for s protein priming, and the spread of sars-cov-2 might also be intimately associated with tmprss2 activity. in addition, an in vitro study found that the serine protease inhibitor camostat mesylate could significantly block the activity of tmprss2, which can efficiently prevent the virus from entering caco-2 (tmprss2 + ) cells rather than 293t (tmprss2 -) cells (hoffmann et al., 2020) . this result indicates that blocking tmprss2 should be considered a potential therapeutic target for the treatment of sars-cov-2-infected patients. more remarkably, camostat mesylate has already been approved for safety for the treatment of pancreatic inflammation disease in japan, which reminds us that camostat mesylate should be given sufficient consideration as a promising therapeutic drug for combating sars-cov-2 without safety concerns (gibo et al., 2005) . according to a new finding from the university of tokyo, a comparable drug named nafamostat mesylate can prevent tmprss2-triggered sars-cov-2 membrane fusion at a concentration less than one-tenth that of camostat mesylate, which indicated that nafamostat mesylate may also be a promising inhibitor of sars-cov-2 infection by targeting tmprss2 (inoue, 2020) . as the two drugs have been prescribed in japan for many years and have adequate clinical data with regard to safety, we expect that they can enter clinical trials this article is protected by copyright. all rights reserved. for treating sars-cov-2 as soon as possible. more importantly, the research process of the two tmprss2 inhibitors for treating sars-cov-2 reminds us that searching for therapeutics from marketed drugs that have been confirmed to be safe (drug repurposing) appears to be a good strategy and extremely worthwhile. previous studies have focused on ace2 in host cells and its partner molecule tmprss2. although progress has been made in determining the structures and binding domains and in drug screening, it is unclear whether there are other receptors on the host cell membrane for s protein binding, which reminds us of the challenges of identifying drug targets for sars-cov-2. therefore, as a result of in-depth study, another receptor was discovered recently to be involved in sars-cov-2 infection. cd147, usually referred to as extracellular matrix metalloproteinase inducer (emmprin) or basic immunoglobulin (basigin), is a kind of transmembrane glycoprotein belonging to the immunoglobulin family, which is closely associated with many disease processes, such as tumour progression, plasmodium invasion and virus infection (lu et al., 2018; nguyen et al., 2018; zhang et al., 2018) . previous studies showed that cd147 can promote the entry of sars-cov into host cells, while the cd147-antagonistic peptide (ap)-9 has high binding activity to effectively prevent sars-cov infection of hek293 cells (chen et al., 2005) (chen et al., 2005; . additionally, multiple studies showed the tissue distribution specificity of cd147 in tumour tissues, inflamed tissues and pathogen-infected cells, which indicates relatively low cross-reactivity with normal cells (kosugi et al., 2015; su et al., 2018) . therefore, the route of viral entry involving cd147 suggests a novel potential target with promising druggability for specific anti-sars-cov-2 drug (such as metuximab, metuzumab and meplazumab, etc.) verification and exploration. in a review of past research, we found that coronavirus genomic rna acts as the transcript to induce orf1a translation in a cap-dependent manner to form the polyprotein ppan after entering and uncoiling in the host cell. near the end of orf1a, there are two important structures, i.e., a slippery sequence and an rna pseudoknot, which were reported to be important for inducing 25-30% of ribosomes to undergo frame shifting, thereby promoting orf1b translation to provide the longer polyproteins pp1a and pp1ab (masters, 2006) . after autoproteolytic separation, the polyproteins pp1a and pp1ab produce several nonstructural proteins, i.e., the well-known nsps. through the functional study of nsps, researchers found that nsp12 encodes rna-dependent rna polymerase (rdrp) (xu et al., 2003) , while nsp5 this article is protected by copyright. all rights reserved. and nsp3 encode the main protease (mpro) and papain-like protease (plpro) (ziebuhr et al., 2000) , respectively. during the replication process of rna viruses, rdrp determines the fidelity and the rates of replication and mutation of the virus to condition its adaptation to the environment and even to a new host, thus influencing the evolution of the virus(hukowska-szematowicz, 2020). targeting for sars-cov-2. aurintricarboxylic acid (ata), a known anionic polymer, is known to combine with several protein targets, such as gp120 and e-selectin, and to prevent the replication of sars-cov through binding to rdrp with an ic50 of 0.2 mg/ml (cushman et al., 1991; he et al., 2004; yap et al., 2005) , which indicates its potential for anti-sars-cov-2 drug development. ribavirin (rbv), a broad-spectrum antiviral drug, has been studied in mers-and sars-infected patients to combat mers/sars-cov, but its efficacy against sars-cov-2 is unclear. however, whether to choose rbv as a candidate drug is unclear, as some studies have shown a worsening of patient outcomes by rbv administration, indicating that rbv (at least itself) is not a good choice for anti-sars-cov-2 drug development (stockman et al., 2006; zhang et al., 2020) . favipiravir is a new type of rdrp inhibitor. research shows that favipiravir is activated through phosphoribosylation in cells to form favipiravir-rtp, which is considered a substrate for viral rna polymerase, thereby binding with rdrp to inhibit its activity (furuta et al., 2017) . a favipiravir clinical trial (chictr2000029600) for treating sars-cov-2 infection achieved the expected results. in the clinical trial, a total of 80 patients were divided into two groups, i.e., a control group and a favipiravir intervention group. the results showed that favipiravir has a better anti-sars-cov-2 effect than similar drugs (lopinavir/ritonavir). in addition, another this article is protected by copyright. all rights reserved. prospective, multicentre, open-label, randomized superiority trial (chictr200030254) has come to the similar conclusion that favipiravir (1600 mg/time/bid on the first day; 600 mg/time/bid from the second day to the end of treatment) should be regarded as one of the most promising candidate drugs for combating sars-cov-2 due to its higher rate of 7-day clinical recovery and more effectively decreasing the incidence of clinical symptoms, such as fever and cough, than the currently recommended drug, arbidol . moreover, no significant adverse reactions were observed, and relatively high patient compliance was noted in the favipiravir treatment group, indicating that favipiravir is a potential anti-sars-cov-2 drug that requires the greatest attention . remdesivir is also a nucleotide analogue inhibitor of rdrp (brown et al., 2019) . recently, wang et al. found that remdesivir can effectively prevent the infection of sars-cov-2 at a very low concentration with a relatively high efficiency of selection (ec50 = 0.77 μm and a half-cytotoxic concentration (cc50)>100 μm (si>129.87)), suggesting that remdesivir is very likely to be a potential drug against sars-cov-2 infection . furthermore, a report that remdesivir achieved the expected treatment effect in a patient infected with sars-cov-2 in the united states attracted much attention (holshue et al., 2020) . currently, remdesivir has become the most promising drug that has entered the phase iii clinical trial stage, shedding new light on the treatment of sars-cov-2-induced disease. based on the above research, rdrp is one of the most promising therapeutic targets, and rdrp inhibitors (such as aurintricarboxylic acid, favipiravir and remdesivir) will be the most promising drugs for sars-cov-2. plpro is a kind of protease responsible for processing the polypeptide translated from the independently (cheng et al., 2015) . to date, no inhibitor targeting plpro has been confirmed to be effective against sars-cov-2. however, the successful experience of designing inhibitors targeting sars-cov and mers-cov plpro enzymes with antiviral activity and selectivity reminds us that it is a good choice to design specific inhibitors targeting sars-cov-2 plpro. the main protease is also known as 3c-like main protease (3clpro) due to its similar cleavage site specificity to that of picornavirus 3c protease (3cpro) (zhao et al., 2013) . previous studies demonstrated that 3clpro is one of the most vital proteases for rdrp generation, virus replication and infection (zhou et al., 2019) . similar to the rdrp protein, sars-cov-2 and sars-cov share approximately 96 percent 3clpro sequence identity at the protein level (morse et al., 2020a) . research has discovered that 3clpro easily forms a dimer under natural conditions through hydrogen bonds. in-depth structure analysis found that each monomer contains two main regions, the n-terminal catalytic region and the c-terminal region, and that the difference is dependent mainly on the catalytic region located on the protein surface (lee et al., 2005) . because sars-cov-2 may share with sars-cov similar possible interactions of substrates or inhibitors towards its active sites, imperceptible this article is protected by copyright. all rights reserved. structural alterations from a to s residues are not expected to significantly affect the binding characteristics of substrates and inhibitors to those active sites. therefore, 3clpro is expected to become another potential enzyme target for the treatment of sars-cov-2. lopinavir and ritonavir, two inhibitors of 3clpro, were originally designed to treat hiv (pillaiyar et al., 2020) . as the two major 3clpro inhibitors, lopinavir and ritonavir are used mainly for clinically treating hiv in combination with other drugs, that is, the commonly discussed antiviral cocktails. at present, several studies have shown that the two 3clpro inhibitors have anti-cov properties both in vitro and in mers-cov-infected primates (not humans), and they have also shown activity in nonrandomized trials of sars-cov patients (wu et al., 2004 indicating that this combination most likely failed . although hopes are fading for treatment with lopinavir and ritonavir, targeting 3clpro is still a potential therapeutic strategy for combating sars-cov-2. as research has continued, a growing number of small molecule inhibitors have been found to have high potential therapeutic effects against sars-cov-2 by targeting 3clpro. chen et al found nine promising small molecule inhibitors for the treatment of sars-cov-2 after optimization based on targeting 3clpro by 32,297 potential antiviral medicinal plant compounds (sun et 11b can likely prevent sars-cov-2 replication with an ic50 of 0.67 μμ . based on the above research, we believe that small molecule inhibitors that potently inhibit 3clpro are promising drugs for sars-cov-2. in addition to rdrp, plpro and 3clpro, helicases and glycogen synthase kinase 3 (gsk3) should also be of interest because they are essential components during the coronavirus replication process in host cells and could act as viable targets for anti-sars/mers chemical therapies, according to studies that have already been confirmed by researchers (adedeji et al., 2012; adedeji et al., 2014; mizutani et al., 2006; wu et al., 2009) . although there have been no related approved inhibitor-based anti-sars-cov-2 therapies so far, those candidate compounds, especially remdesivir, could serve as potential leads and clinical medicines for developing effective anti-sars-cov-2 drugs with interpretation of the crystal structure of replicases. with the publication of the rna genome sequence of sars-cov-2 (genbank: mn908947), one strategy could aim to target the viral rna genome itself for degradation, in addition to targeting the surface proteins and viral replicases. therefore, using small interfering rnas (sirnas), rna aptamers or antisense oligonucleotides (asos) against the sars-cov rna genome may provide important insights and promising therapeutic targets for sars-cov-2 treatment (ahn et al., 2009; asha et al., 2018; qureshi et al., 2018; shum et al., 2008) . recent studies have shown that two double-stranded rnas (dsrnas) specifically bind to sars-cov m protein gene were greater than 70% . in addition, the patent application cn1569233 describes promising sirnas targeting sars-cov genes that encode main components, including rdrp, the nucleoprotein n, and proteolytic enzymes. these sirnas are reported to have a significant inhibitory or killing effect of approximately 50-90% of the sars-cov virus bj01 strain, in which the most effective are proteolytic enzyme-targeted sirnas. based on these findings, sirna may be a potential biological agent for consideration as a great treatment strategy to kill sars-cov-2. two patents in korea present the usage of rna aptamers for inhibiting sars-cov. one asos have also been designed to detect sars-cov infections and to prevent or cure sars-cov-related disease (lim et al., 2006) . before the occurrence of sars-cov-2, a patent application submitted by ionis pharmaceuticals (wo2005023083) showed hybrid dna/rna asos designed for disrupting the pseudoknot in the site of the sars-cov rna frame shift. in addition to inhibiting the virus directly, asos are also expected to target the disease-related proteins involved in the inflammatory cytokine storm process, which could be considered a promising therapeutic strategy for combating sars-cov-2 . therefore, asos may be a kind of potential biological agent useful for the treatment of sars-cov-2, similar to the situation for sars-cov. in addition, nucleoside analogues (such as eidd-1931 and eidd-2801) should be considered a class of biologic resources with potential antiviral effects. although the viral genome may be a potential target of sirnas, rna aptamers, asos and nucleoside analogues for sars-cov-2, as was observed for sars-cov, this approach presents several challenges. one of the most important challenges is the delivery of oligonucleotides into the lungs (youngren-ortiz et al., 2016) . in addition, even if sirnas, rna aptamers, asos and other biologic resources are effective clinically, how to scale up the mass production of these potential biological agents to cover the large infected population will be a problem worthy of this article is protected by copyright. all rights reserved. study . therefore, we have a long way to go to reach the goals for the production of these potential biological agents (sirnas, rna aptamers and asos). although many strategies have been used to block the attachment, entry, replication and release processes to inhibit sars-cov-2 infection, how to prevent viral evasion from host immune responses and virus-induced cytopathic effects is considered one of the most urgent problems that need to be solved in sars-cov-2-induced pneumonia-associated respiratory syndrome (pars) patients. studies from sars-cov-induced deaths and animal models have shown that an aberrant and excessive host inflammatory cytokine storm results in a strong immunopathological response and lethal disease (hui et al., 2019; rockx et al., 2009; smits et al., 2010) , which was also confirmed in sars-cov-2-induced pars patients based on pathological features and autopsies. inflammatory cytokine storm refers to the dysfunction of the immune system and an excessive inflammatory response that becomes uncontrollable , and it is closely associated with multiple infectious and non-infection conditions and diseases including graft-versus-host disease, autoimmune disease, severe virus infection, multiple organ dysfunction syndrome and chimeric antigen receptor (car)-t cell therapy (channappanavar et al., 2017; giavridis et al., 2018; riddell, 2018) . during infection, cd4-positive t cells (cd4 + t cells) are rapidly activated to form pathogenic t helper (th) 1 cells and to produce granulocyte-macrophage colony-stimulating factor (gm-csf). the cytokine condition contributes to cd14 + cd16 + monocyte recruitment and il-6 secretion, which further aggravate the inflammatory response after sars-cov-2 infection . recently, several clinical reports revealed that most patients infected with sars-cov-2 have increased plasma concentrations of inflammatory cytokines, such as interleukins (il-2/7/10), granulocyte/-macrophage-csf, monocyte chemoattractant protein-1 (mcp-1), and tumour necrosis factor  (tnf-), especially the critically ill patients (chen et al., 2020b; huang et al., 2020b; peeri et al., 2020) . these clinical indicators reveal the occurrence of severe pulmonary inflammation and the production of inflammatory cytokine storms during sars-cov-2 infection. therefore, symptomatic treatments, especially strategies to eliminate inflammation and inflammatory cytokine storms, combined with organ support, in these critically ill patients are this article is protected by copyright. all rights reserved. the most critical part of clinical management (zumla et al., 2020) . therefore, the inflammatory cytokine storm is a promising research and therapeutic target that may not only identify the immunopathological mechanism but also benefit the discovery of potential drugs. as it takes a long time to evaluate and develop specific new drugs targeting sars-cov-2, several currently marketed drugs that target inflammatory cytokine storms and reduce immunopathology could be considered at this critical moment. tocilizumab, which can specifically bind to both the soluble il-6 receptor and membrane-bound il-6 receptor to inhibit related signal transduction, is the first il-6-blocking antibody approved for marketing and shows proven safety and effectiveness in therapy for rheumatoid arthritis (safy-khan et al., 2020; verhoeven et al., 2019) . to date, a treatment programme utilizing tocilizumab based on conventional therapy has been administered to 20 patients (including 18 severe cases and 2 critical cases). the elevated body temperature was reduced to normal within 24 hours, which was accompanied by varying degrees of improvement in the oxygenation index of respiratory function. after two weeks of careful treatment by the scheme, 19 patients recovered, and only one patient became severely ill due to critical illness . this treatment programme utilizing tocilizumab based on conventional therapy has been carried out in many hospitals in wuhan, china, and has achieved good results, which indicates that tocilizumab is a drug with great potential for targeting the inflammatory cytokine storm on the basis of conventional treatment(xinhua, 2020). ge et al reported a poly-adp-ribose polymerase 1 (parp1) inhibitor named cvl218, which was identified by their data-driven drug repurposing framework. their study showed that cvl218 could effectively inhibit sars-cov-2 replication with an ec50 of 5.12 µm. additionally, it significantly suppressed cpg-induced il-6 production by 50% and 72.65% in peripheral blood mononuclear cells at 1 µm and 3 µm concentrations after 12 hours, respectively. the aforementioned finding suggests that cvl218 has a significant anti-inflammatory cytokine storm effect, which is closely associated with sars-cov-2-induced immunopathology prevention, especially for intensive care unit (icu) patients (ge et al., 2020) . further in vivo pharmacokinetic and toxicokinetic studies showed that cvl218 is distributed mainly in lung tissue without apparent toxicity, which makes it an appealing and promising drug candidate for the treatment of sars-cov-2 induced inflammatory cytokine storms. this article is protected by copyright. all rights reserved. above all, the basic research/results of clinical treatment with tocilizumab and cvl218 based on conventional therapy reminds researchers that exploring novel uses of marketed drugs may also be an effective strategy for treating sars-cov-2-induced pars. vaccination is the most ideal strategy to prevent infections and diseases by exposure to specific pathogens, especially in vulnerable populations (ralph et al., 2020) . therefore, it is absolutely critical for us to develop safe and efficient vaccines to control the spread of the pandemic and prevent the future recurrence of sars-cov-2 outbreaks. in the early stages of the outbreak, the chinese government said that at least five vaccine technologies would be explored in china, including an inactivated vaccine, a subunit protein vaccine, a nucleic acid vaccine, an adenoviral vector vaccine and a recombinant vaccine (zhang, 2020a) . on january 23, 2020, the coalition for epidemic preparedness innovations (cepi) declared that they would fund three vaccine technology platform to develop effective vaccines against sars-cov-2 in the shortest period including dna, mrna, and "molecular clamp" platforms (lu, 2020) . it was encouraging that on march 16, 2020, the nih announced that the first vaccine against sars-cov-2, named mrna-1273, entered the phase i human study nct04283461, which represented a total of 63 days from sequence selection to first human dosing by using the mrna platform (moderna, 2020) . subsequently, on the same day, another exciting development of a subunit vaccine (adenovirus type 5 vector) against sars-cov-2 was created by experts of the academy of military medical sciences of china, and was approved for clinical trials (zhang, 2020b) . the subunit vaccine, which has been approved in terms of safety, efficacy and quality by a third party, is a type of vaccine containing only a fragment of the sars-cov-2 pathogen to induce a protective immune response, according to the who. since the start of the sars-cov-2 outbreak, the world's major academic institutions and biopharmaceutical companies have joined the race to develop a safe and effective prophylactic vaccine through multiple platforms such as mrna, dna, adenoviral vector and recombinant protein platforms (pang et al., 2020 sars-cov-and mers-cov-related vaccine experience may promote the design and development of anti-sars-cov-2 vaccines due to the remarkable sequence homology among sars-cov-2, sars-cov and mers-cov . we believe that there will be good news regarding sars-cov-2 vaccines soon as a result of joint efforts in scientific research. in addition to vaccines, a simple but potentially very effective strategy that can be used for combating sars-cov-2 is using convalescent patient sera, which can be obtained from patients who have recovered from virus infection. this passive strategy has been proven applicable according to experiences in the treatment of other viral diseases (mire et al., 2016) . based on experience, patients with resolved infection will develop viral antibodies at a high titre in response to different antigens of sars-cov-2 . one or more passive antibodies derived from convalescent patient sera will likely neutralize sars-cov-2 and prevent new rounds of infection. during the outbreak of ebola in 2014-2015, this rationale was used to treat ebola patients with convalescent serum and achieved very good results (kraft et al., 2015) . at present, in china, many patients with resolved cases of sars-cov-2 said they are willing to donate plasma if necessary. if possible, this plasma can be transfused into other infected patients to help them overcome viral infections. since plasma donation is a mature technology, and plasma transfusion is also a part of routine medical care, this proposal is the simplest and most feasible under consideration (thorpe et al., 2020) . at the same time, we also need to consider that this proposal is not a long-term solution because the growing number of cases is far outpacing the speed of the recovery. as sars-cov-2 infection has become more rampant worldwide, all researchers are trying to search for potential therapeutic targets and promising drugs. in china, many researchers have been trying to find clues from traditional chinese medicine (tcm), and now, significant progress has been achieved in the treatment of sars-cov-2. since january 25, 2020, the early intervention of tcm has played an important role in this epidemic situation. according to statistics, a total of 60,107 patients infected with sars-cov-2 in china were treated with tcm by february 17th, 2020. this article is protected by copyright. all rights reserved. a recent study screened eighty-three chemical structures from tcm compounds, and found that theaflavin (zinc3978446) has a low idock score (-9.11 kcal/mol) and binding energy (-8.8 kcal/mol) in the catalytic pocket of sars-cov-2 rdrp. the results from the protein-ligand interaction profiler (plip) showed that theaflavin can form extra π-cation interactions and hydrogen bonds with the catalytic pocket (at the site of asp452, lys545, arg555, thr556, tyr619, lys621, cys622, asp623, arg624, and asp760) of sars-cov-2 rdrp (lung et al., 2020) , which indicated that thealfavin should be recognized as a lead compound for developing promising anti-sars-cov-2 drugs. moreover, an in silico integrative model of absorption, distribution, metabolism and excretion (adme) was used to screen promising ingredients or compounds from tcm for directly inhibiting sars-cov-2 . of the screened compounds, 13 (such as kaempferol, moupinamide and dihydrotanshinone i, etc.) that exist in tcms were found to have potential anti-sars-cov-2 activity related to similar possible targets, including plpro, 3clpro and s protein. furthermore, 125 tcms were detected to contain 2 or more of these 13 compounds, and 26 (such as forsythiae fructus, coptidis rhizome and mori cortex, etc.) of them are classically catalogued as treating viral respiratory infections. in addition, network pharmacology analysis predicted that these 26 tcms have general roles, including regulating viral infection, immune/inflammation reactions and the hypoxia response in vivo (zhang et al., 2020b) . the aforementioned results of this research showed that many ingredients or compounds of tcms could be considered lead compounds for developing promising anti-sars-cov-2 drugs. therefore, researchers should also concentrate a certain effort on screening, discovery and development of promising tcm compounds/extracts for the treatment of sars-cov-2. although the outbreak of sars-cov-2 in china was contained by the joint efforts of the government, society, and medical staff, outbreaks in other countries and regions outside of china have become increasingly severe and uncontrollable. by april 9, 2020, the number of cumulative confirmed cases is estimated to be close to 1,503,900 leaving approximately 89,931 people dead worldwide. as the epidemic spreads, scientists around the world are attempting to investigate the virus to understand its pathogenesis and explore potential targets this article is protected by copyright. all rights reserved. and promising drugs that would be effective in combating sars-cov-2. although there have been some clues regarding viral pathogenesis and potential targets, there are no verified antiviral drugs with specific effects against sars-cov-2. the efficacy and safety of these promising candidate drugs in the treatment of sars-cov-2 need to be confirmed in further preclinical and clinical trials. with unremitting efforts to block the outbreak of sars-cov-2 worldwide, we hope that the infection and transmission of this virus will recede in a few months, as was observed for sars-cov and mers-cov. although it will be a long and difficult road, the outbreak of sars-cov-2 worldwide has underlined the urgent need for renewed efforts to develop potential broad-spectrum and targeted antiviral drugs to overcome this virus. key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the iuphar/bps guide to pharmacology (harding, sharman et al., 2018) , and are permanently archived in the concise guide to 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article is protected by copyright. all rights reserved. zhang, my, zhang, y, wu, xd, zhang, k, lin, p, bian, hj, qin, mm, huang, w, wei, d, zhang, z, wu, j, chen, r, feng, f, wang, b, nan, g, zhu, p, chen, zn (2018) this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. these replicases synthesize the full-length negative-sense antigenome template to produce new genomic rna and further form the assembled virion, which is then released into the extracellular space by exocytosis. uncontrolled replication promotes sars-cov-2 infection, leading to immune disturbances and inflammatory cytokine storms and eventually resulting in multiorgan functional damage, particularly in the lung. key: cord-289535-srrfr1es authors: tregoning, j. s.; brown, e. s.; cheeseman, h. m.; flight, k. e.; higham, s. l.; lemm, n.‐m.; pierce, b. f.; stirling, d. c.; wang, z.; pollock, k. m. title: vaccines for covid‐19 date: 2020-10-18 journal: clin exp immunol doi: 10.1111/cei.13517 sha: doc_id: 289535 cord_uid: srrfr1es since the emergence of covid‐19, caused by the sars‐cov‐2 virus at the end of 2019, there has been an explosion of vaccine development. by 24 september 2020, a staggering number of vaccines (more than 200) had started preclinical development, of which 43 had entered clinical trials, including some approaches that have not previously been licensed for human vaccines. vaccines have been widely considered as part of the exit strategy to enable the return to previous patterns of working, schooling and socializing. importantly, to effectively control the covid‐19 pandemic, production needs to be scaled‐up from a small number of preclinical doses to enough filled vials to immunize the world’s population, which requires close engagement with manufacturers and regulators. it will require a global effort to control the virus, necessitating equitable access for all countries to effective vaccines. this review explores the immune responses required to protect against sars‐cov‐2 and the potential for vaccine‐induced immunopathology. we describe the profile of the different platforms and the advantages and disadvantages of each approach. the review also addresses the critical steps between promising preclinical leads and manufacturing at scale. the issues faced during this pandemic and the platforms being developed to address it will be invaluable for future outbreak control. nine months after the outbreak began we are at a point where preclinical and early clinical data are being generated for the vaccines; an overview of this important area will help our understanding of the next phases. in november 2019, a cluster of pneumonia cases was detected in wuhan, china [1] . these were the first cases of covid-19 caused by the novel beta-coronavirus sars-cov-2. the genetic information was made publicly available on 10 january 2020, 54 days after the first declared case. sixty-three days after the sars-cov-2 sequence was published, on 13 march 2020, the first doses of the first human vaccine were being tested. by 24 september 2020, the sars-cov-2 vaccine landscape included 43 candidates being tested in clinical trials and more than 200 candidates. as the results from the phase i trials and earliest phase ii/iii trials emerge, this review will cover the platforms under development, the type of immune response required and the path to a clinical product. coronaviruses are unusually large enveloped rna viruses, with a large positive-sense, single-stranded rna genome. the integrity of this lengthy genome is maintained by a proof-reading replicase. the sars-cov-2 genome encodes 11 open reading frames (orf), many of which have unknown functions (fig. 1 ). orf1a and orf1b both encode polyproteins, which are cleaved into multiple non-structural proteins. orf4 encodes the envelope protein, a viroporin [2] , and orf5 encodes the membrane protein; together, they coordinate viral assembly and release. orf9 encodes the nucleocapsid (n) protein. orf2 encodes the spike (s) surface glycoprotein, the viral entry protein and key antigenic determinant, which binds the angiotensin converting enzyme 2 (ace2) 163 receptor on the host cells. ace2 is commonly found on type ii pneumocyte cells in the airways. sars-cov-2 has a 10-20 times higher affinity for ace2 than the related coronavirus sars-cov-1 [3] , which was responsible for the 2002-04 sars outbreak. sars-cov-2 is able to bind ace2 from a wide range of mammalian species [4] . having bound ace2, spike protein is cleaved by a host cell surface bound proteinase, either furin or tmprss2, enabling entry of the viral capsid. there may be a relationship between the mechanism of viral entry via ace2 and the pathogenesis of disease. human coronaviruses can cause both mild (oc43, hku1, 229e and nl63) and severe (sars-cov-1, sars-cov-2 and mers) disease. for most patients (approximately 80%), sars-cov-2 causes an asymptomatic infection or mild symptoms [5] . the following signs are associated with a virus positive polymerase chain reaction (pcr) test: fatigue, fever, chills, loss of appetite and persistent cough [6] . a striking feature of infection with sars-cov-2 is anosmia, a loss of smell and taste, reported in approximately 64% of cases in one study [7] . whether viral spread to the lower respiratory tract is a precursor for severe disease is unclear; pneumonia with characteristic pulmonary ground glass opacity changes on chest ct scans is common, even in asymptomatic individuals [8] . blood clotting, respiratory compromise, renal damage and cardiovascular collapse are all features of severe disease. the greatest risk factor for severe covid-19 disease is age: the remarkable relationship with age is consistently observed, despite geographic variability in reported case fatality rates [9] . while sars-cov-2 is a new virus and, therefore, the exact correlates of protection are not completely defined, there are precedents from other respiratory infections in general and coronaviruses in particular [10] . there has been discussion that natural immunity to sars-cov-2 declines quickly; whether this is the case is still unclear. it is our speculation that because vaccines aim to evoke an immune response they could be more immunogenic than the virus itself, which might have mechanisms to dampen immune response: whether this speculation is correct or not is yet to be determined. the t cell response is important in the control of other respiratory infections, and therefore likely to be the spike (s) protein is the major antigenic determinant, coat is made of spike (s), membrane (m) and envelope (e) proteins. the rna in encapsulated with the nucleocapsid (n) protein). created with biorender.com important in covid-19 [11] . models of sars-cov-1 indicate that t cells can be protective. cd4 + t cell depletion in mouse models delayed viral clearance and enhanced disease [12] ; similarly, t cell transfer resulted in rapid viral clearance and disease amelioration [13, 14] . sars-cov-1-specific cd8 + t resident memory were protective in a mouse model in the absence of antibody [15] . t cell memory can be long-lived; sars-cov-1 t cells were detected 4 years after infection [15, 16] . for sars-cov-2, t cell responses have been observed to a range of antigens, including s, m, n and other orfs [17] . sars-cov-2-specific t cells have been detected in individuals who had asymptomatic or mild covid-19 [18] and sars-cov-2-specific t cells have been observed in contacts of infected individuals [19] . patients suffering from covid-19 had fewer t cells than healthy controls [20] . t cells, especially cd4 + t cells, can influence the immune response through the production of cytokines, and elevated cytokines have been associated with exacerbated disease [20] . the skewing of the cd4 + t cell response is likely to be important. t helper type 1 (th1) responses are central to the successful control of sars-cov-1 and mers-cov [21] . th17 responses have been speculated to be deleterious [22] , and increased th2 cytokines were seen in severe disease [23] . regulatory t cells are important in the resolution of infection, and were observed to be elevated in covid-19 patients [20] . circulating follicular t helper cells, important in defining recall antibody response to infection, have been observed in a small number of individuals with covid-19 [24] . it is not clear whether the 'cytokine storm' is a cause or effect of disease; understanding this relationship is critical in monitoring vaccine safety. antibody response. the humoral response is pivotal in later stages of infection and helps to inhibit subsequent reinfection. virus-specific antibodies were detectable in 80-100% of sars-cov-1 and mers-cov patients 2 weeks after onset of symptoms [25] [26] [27] [28] [29] [30] [31] , with delayed antibody responses associated with more severe disease. a number of studies have been performed to try to more clearly understand the antibody response to sars-cov-2; a systematic review of studies on antibody to coronaviruses [32] observed that antibody was rarely seen in the first 7 days of infection, but rose in the second and third weeks postinfection. it is unclear whether antibodies correlate with covid-19 severity. antibodies are likely to be an important part of vaccine-induced protection. in sars-cov-1, the antibody response is short-lived [immunoglobulin (ig)m and iga responses last less than 6 months and igg lasts approximately 1 year]; this is possibly the same for sars-cov-2 [33] . human challenge studies using non-covid-19 coronavirus strains suggest that higher antibody levels correlate with protection [32] . these challenge studies have also suggested that reinfection is possible [34] , but the dose in challenge studies may be higher than experienced during natural infection. two recent studies have observed natural reinfections with sars-cov-2, one asymptomatic [35] and one symptomatic [36] , although this is in the context of more than 25 million recorded cases globally, suggesting that it is a rare event. because of the overlap between sars-cov-1 and sars-cov-2 spike proteins, antibodies could be cross-neutralizing [37] . however, the most potent specific, neutralizing monoclonal antibodies against the receptor binding domain (rbd) of sars-cov-1 did not bind to the spike protein of sars-cov-2 [38] . one promising observation is that isolated neutralizing antibodies have minimally mutated vdj genes, which make inducing them possible with fewer rounds of vaccination [39] . most attention has focused upon neutralizing igg antibodies in the serum, but other antibody-mediated mechanisms may be important in disease pathogenesis. fragment crystallizable (fc) and fc receptor (fcr) interactions can regulate the inflammatory response [40] and the sars-cov-2 virus-antibody complex could potentially trigger such fcr-mediated inflammatory responses, causing acute lung injury [41] . the iga response may be important in determining disease severity of covid-19 patients, but remains relatively unexplored so far [42] . one concern with vaccine development for sars-cov-2 is that the immune response can cause disease, often in the act of clearing the infection. understanding vaccineinduced immunopathology is critically important for all emerging infectious diseases. vaccines for emerging infections will, by necessity, require a shorter turn-around from discovery to deployment, and therefore predicting safety early in the process is critical. vaccine-induced immunopathology can either present as an acute response to the vaccine itself or as disease enhancement after viral infection. vaccines can occasionally induce an acute autoimmune disease. this was observed during the 1976 h1n1 swine flu outbreak, where vaccination in the united states led to an increased risk of guillain-barré syndrome (gbs) [43] . the mechanism has not been fully determined, but one suggestion is off-target antibodies against ganglioside gm1. off-target autoimmune effects were also observed during the 2009 h1n1 swine flu pandemic, with narcolepsy observed in a subset of children immunized with a vaccine adjuvanted with as03 [pandemrix; glaxosmithkline (gsk)]. there was a very tight association with hla-dqb1*06:02 [44] . the proposed mechanism is inhibition of the hypocretin signalling pathway. curiously, another swine flu vaccine made by gsk (arepanrix; gsk) using the same adjuvant was not associated with narcolepsy [45] , suggesting that the side effect was not caused by the adjuvant. the level of viral proteins, specifically nucleoprotein, may have been the problem [46] ; anti-nucleoprotein antibodies have been seen to cross-react with hypocretin [47] . these acute events are relatively rare; the rate of gbs was 8 per million individuals vaccinated and narcolepsy at approximately 30 per million individuals vaccinated (all in individuals aged less than 20 years) [48] . the delayed effects of vaccines are difficult to predict; post-licensure monitoring will be critical, especially as the vaccines will potentially have been tested in fewer people during the prelicensure phases than other licensed products. disease enhancement following infection of vaccinated individuals has been seen in other viral diseases; for example, measles, respiratory syncytial virus (rsv) and dengue virus. of children who received formalin-inactivated measles vaccine and were then subsequently exposed to the wild-type measles virus, 15-60% developed a severe form of the disease [49] , causing the vaccine to be withdrawn in 1967. a similar situation was observed with formalin-inactivated rsv vaccination (fi-rsv) in a clinical trial in 1966. the fi-rsv vaccine induced mainly non-protective antibodies, and children who were seronegative to the virus prevaccination had enhanced disease and hospitalization compared to the control groups [50] . vaccine-enhanced disease has also been observed with the live attenuated tetravalent dengue vaccine (dengvaxia; sanofi pasteur inc., swiftwater, pa, usa), specifically in seronegative children [51] . disease enhancement following vaccination can occur by two main mechanisms: priming for a detrimental t cell response and priming for antibodies that can increase the risk of infection or severe disease. the cellular response to vaccination, particularly t cells and eosinophils, and the inflammatory mediators these cells release has been suggested to promote vaccineenhanced disease [52] [53] [54] . whether sars-cov-2 vaccine platforms will have negative outcomes on infection is currently speculative, and draws upon experience with other respiratory viruses. one important factor determining the t cell response is antigen selection. specific epitopes can affect t cell polarization and activation, therefore antigen selection for vaccine applications requires careful consideration [55] . both the s and n proteins of sars-cov-1 have epitopes that are recognized by cd4 + and cd8 + t cells. some vaccines which used the n protein induced an eosinophilic response associated with vaccine-enhanced disease [13] , and post-vaccination challenge of animals immunized with sars-cov-1 n protein induced severe pneumonia [56] . mismatch of epitopes between vaccine and challenge strain can also lead to t cell enhanced disease due to original antigenic sin, as seen in dengue [57] . the vaccine platform may be critical in determining disease outcome on infection. immunopathology in animal models has most commonly been linked to inactivated, alum-adjuvanted vaccines. for example, double inactivation [ultraviolet (uv) and formalin] of sars-cov-1 enhanced the eosinophilic response from the vaccine, eliciting a proinflammatory pulmonary response and failing to provide complete protection [56] . enhanced disease was also observed following immunization with a gamma-irradiated mers-cov vaccine [56] . the mode of inactivation can influence both the quality of antibodies and the polarization of the t cell response to the vaccine. formalin inactivation in particular has been associated with deleterious th2 skewing by the addition of carbonyl groups [58] , and th2 skewing has been seen for a formalin-inactivated vaccine for sars-cov-1 [59] . other methods of inactivation have been explored; for example, beta-propiolactone, uv or gamma radiation, which could prove to be a promising avenue forward for eliciting the correct t cell response [60] . immunopathology is not restricted to inactivated vaccines. it can occur following immunization with a range of vaccine platforms; for example, it has been seen in animal models of rsv vaccination with both viral vectors and dna vaccines [61] . similarly, a range of vaccines against sars-cov-1 induced th2-directed pulmonary immunopathology in mouse models [56] . age at vaccination may also be an important consideration in immunopathology: the fi-rsv vaccine was given to infants. infants have a different immune response to adults, and this may predispose towards a qualitatively different immune memory [62] . the humoral arm of the adaptive immune response can also contribute to disease, called 'antibody-dependent enhancement' (ade). ade has been observed with flaviviruses, coronaviruses and some viruses of the paramyxoviridae family [63] . ade can occur in two ways, either by causing immune complexes or by enhancing infection. antibodies are bispecific molecules; as such, they can form antigen-antibody complexes. these complexes can cause direct damage when complex deposition in the vasculature leads to complement deposition and vessel damage, as seen after the feline coronavirus infection [64] . immune complexes can trigger macrophage activation leading to the release of proinflammatory cytokines. immune complexes have been proposed to have a role in the enhanced disease seen after fi-rsv immunization [65] , and may have a role in sars-cov-2 [41] . antibodies can also increase viral disease by enhancing infection; some viruses utilize antibodies to enter target cells. in the case of dengue virus, pre-existing antibodies for one serotype of the virus can cause enhancement of infection upon subsequent exposure to a new serotype [63] . a number of mechanisms have been proposed: antibody bound to virus could facilitate entry into macrophages through their fcrs [66] and antibody might stabilize viral surface antigen into a mature form [67] . the avidity of the antibody has been suggested as an important factor, with low antibody avidity a risk factor [68] . ade has been reported in sars-cov-1 after viral challenge in mice [69] , ferrets [70] and macaques [71] using a range of different vaccine strategies. in mers-cov, a neutralizing monoclonal antibody targeting the spike protein promoted viral entry via the fc receptor [72] . it is not yet known whether antibodies to sars-cov-2 will enhance disease, but it is something that is being closely monitored [73] . animal models. as coronaviruses have previously been associated with immunopathogenesis, vaccine-enhanced disease is a potential concern for efficient vaccine design for sars-cov-2. the use of models can improve understanding [74] , potentially predicting correlates of protection or disease. the ideal animal model is permissive to infection with the virus and reproduces the pathology and clinical course observed in humans. since the sars-cov-1 outbreak in 2002-04 a range of species, including hamsters, cats, ferrets and non-human primates, have all been used to study pathogenesis of coronaviruses [75, 76] . despite productive infection in a wide range of laboratory species, few displayed overt clinical disease. several inbred mouse strains have been investigated to model sars-cov-1, including balb/c, c57bl/6, rag1 −/− and 129svev mice. although young adult mice infected with varying doses of sars-cov-1 showed evidence of infection, the inbred strains do not accurately reflect the alveolar damage seen in humans [74] . however, aged mice show signs of clinical disease despite, in many cases, the absence of the lung lesions seen in humans [77] , and therefore have been used more extensively than younger mice. transgenic mice expressing human ace2 (hace2) have also been generated; disease severity in transgenic mice largely correlated with the level of hace2 expression, and when challenged with sars-cov-1 they developed severe infection and 100% mortality was reached by day 7 [78] . mers-cov appears to be even more challenging to model, with most species resistant to infection, except for some primate species [79] and camelids [80, 81] . the same models are being used for sars-cov-2. infection of human ace2 transgenic mice with sars-cov-2 led to weight loss and viral rna was detectable in the lungs, as well as lung pathology [82] . symptomatic infection and transmission of sars-cov-2 between animals has been observed in hamsters [83] , and asymptomatic infection and transmission of sars-cov-2 has been observed in ferrets [83] . sars-cov-2 is also infectious in experimental settings using cats, but not dogs, pigs, chickens or ducks [84] . as with sars-cov-1, non-human primates, e.g. rhesus or cynomologous macaques, have been helpful for evaluating immune protection [85] . human challenge. as animal models do not fully recapitulate human disease, alternative strategies may be required. controlled human infection models (chim) are studies in which participants (either vaccinated or not) are intentionally challenged with an infectious organism [86] . chim trials of sars-cov-2 vaccine candidates could be particularly beneficial in vaccine and drug efficacy studies, especially if the community infection rate has declined due to epidemiological interventions [87] . the deliberate exposure of healthy individuals to sars-cov-2 requires a tight ethical and regulatory framework [88] . the major concerns are that we do not have complete understanding of the long-term sequelae of sars-cov-2 infection and there is a lack of rescue therapy to enable the resolution of severe infection, although recent findings suggest that dexamethasone may reduce mortality in severe disease [89] and remdesivir (gilead sciences, foster city, ca, usa) may improve clinical status [90] . the lack of rescue therapy is not unique to a sars-cov-2 chim. rhinovirus and rsv chim do not have a specific anti-viral treatment but are self-resolving, which may also be true for sars-cov-2 in healthy young adults. there are also challenges associated with the manufacture of a challenge virus stock, which requires a high-containment [biosafety level iii (bsliii)] laboratory. at the time of writing, no study had been established, although the world health organization (who) has published guidance [91] and several academic and contract research organizations are investigating the approach [92] . an alternative use of deliberate human infection has been proposed: to infect young, lowrisk individuals to build herd-immunity, and therefore safeguard the unvaccinated, immunocompromised and 167 immunologically naive [93, 94] . however, this strategy is unattractive because the risk factors for severe disease are not fully understood: ethically there are also questions about infecting groups of individuals for the greater benefit, especially if there is a financial incentive. a huge range of vaccine approaches against sars-cov-2 have been proposed (table 1) . these include traditional approaches -inactivated, live attenuated and protein/adjuvant approaches and more novel, as yet, unlicensed approaches -viral vectors and nucleic acids. this has been a rapidly evolving field and some of the vaccines are more advanced than others. we are focusing upon those that are in clinical trials at the time of writing ( table 2) . several factors need to be considered before any vaccine progresses to widespread usage. first and foremost is vaccine safety and efficacy. closely linked is the scope for global scale-up manufacture to produce enough doses to achieve herd immunity. the spike (s) protein. before looking at the platforms being developed, the antigen needs to be considered. based on experience with sars-cov-1, most vaccines target the sars-cov-2 spike protein. within the spike, the receptor binding domain (rbd) responsible for binding to and entering host cells is the primary target of neutralizing antibodies [95] , and some vaccines only include this region. however, a recent study that isolated monoclonal antibodies found that most of them targeted areas outside the rbd [39] . an important consideration is the correct folding of the protein, both during production and when the vaccine is in storage prior to deployment. the coronavirus spike is a type 1 fusion protein and is metastable, undergoing an irreversible conformational change to enable membrane fusion [3, 96, 97] . this may affect the ability of the antigen to induce neutralizing antibodies. a similar effect has been seen with the rsv fusion (f) glycoprotein. antibodies specific to prefusion f (pre-f) have better neutralizing capacity than post-fusion f-specific antibodies [98] [99] [100] [101] : stabilization of the pre-f form can lead to better responses. based on this, prefusion sars-cov-2 spike protein could elicit a more potent immune response and stabilized sars-cov-2 spike proteins have been generated with stabilizing proline mutations in the s2 domain [3, 102, 103] . coronavirus nucleocapsid (n) is also immunogenic: antibodies against the sars-cov-1 n protein are abundant and longer-lived than those against the s protein in recovered patients [104] . interestingly, in model systems of sars-cov-1, immunization with the n protein is associated with vaccine-enhanced disease [105, 106] . it is not known whether the n protein is a potential protective immunogen for sars-cov-2, although vaccine approaches that use whole virus -either inactivated virus or live attenuated approaches -will potentially include n protein. the n protein can be a useful diagnostic for infection during phase iii trials of s protein-based vaccines. while the emphasis has been on the generation of neutralizing antibodies, targeting t cell epitopes may provide additional protection [11] . in other respiratory viruses, for example rsv, t cell only strategies can enhance disease [61] and t cells can be deleterious in dengue [57] , although less evidence of this has been seen with influenza vaccines [107] . it is not yet clear whether sars-cov-2 behaves more like rsv or influenza. drawing on information about sars-cov-1 and mers-cov and using bioinformatics, potential immunogenic epitopes in the sars-cov-2 proteome have been predicted. a total of 781 human leucocyte antigen (hla) class i and 418 hla class ii epitopes common between sars-cov-1 and sars-cov-2 were found [108]. t cell responses against the structural proteins of sars-cov-1 were found to be more immunogenic than non-structural proteins [13] . a wide range of different platforms have been developed, which can be loosely grouped as proteins, inactivated virus, vectored vaccines, live attenuated and nucleic acid (fig. 2) . this is clearly a fast-moving space and the following is based on data accessed in september 2020; an updated website is available at https://vac-lshtm.shiny apps. io/ncov_vacci ne_lands cape/ and the who has a vaccine tracker [109] . as many of the vaccines under development are produced by commercial organizations, peer-reviewed publications concerning their development and efficacy are limited, as such some information has been taken from press releases which may be less robust in their scrutiny. published results from clinical trials are summarized in table 3 . as with other pathogens, recombinantly produced viral surface proteins can safely be used as vaccines for covid-19. although protein vaccines have a good safety profile they can have low levels of immunogenicity, which means that many require adjuvants to improve their efficacy. while bacterial protein vaccines can be made through the purification of whole pathogen preparations, viral subunit vaccines necessitate recombinant genetic 168 engineering. the genes encoding the chosen antigens are cloned or synthesized, expressed and purified using a variety of expression systems, including insect, bacterial, yeast and mammalian cells [110] . bacterial expression systems are often used because they have high levels of expression and are easy to scale-up, with fermenter repurposing relatively easy. however, for viral antigens, where post-translational modification can be important, the use of insect cells or mammalian cells may be preferential [111, 112] . several protein subunit vaccination approaches were under development for sars-cov-1 and mers-cov [113] [114] [115] . a subunit vaccine made up of sars-cov-1 spike protein fragments, expressed in escherichia coli, induced neutralizing pre-clinical (mice) [197] phase i trials [198, 199, 200] curevac pre-clinical (mice) [195] phase i trial [196] people's liberation army/ walvax antibodies in rabbits [116] . neutralizing antibodies were induced in mice after immunization with transgenic plants [117] or mammalian expressed [118] several sars-cov-2 protein vaccines are in development; eight candidates are in clinical trials, but no data are yet available from these trials. two of the earliest to be announced are from clover biopharmaceuticals and the university of queensland. clover biopharmaceuticals has used 'trimer-tag' technology to make a mammalian cellexpressed, spike protein subunit trimer vaccine [120] . this antigen can be recognized by antibodies in the sera of people who have recovered from sars-cov-2 [121] . the vaccine will be given in conjunction with gsk's adjuvant as03 or cytosine-phosphate-guanosine (cpg)/alum during the phase i trial (nct04405908). the university of queensland, funded by the coalition for epidemic preparedness innovations (cepi) has developed a recombinant subunit vaccine using spike protein that has been 'locked' in prefusion conformation using the molecular clamp technique [122] . this is currently being tested with mf59 (nct04495933). sanofi are developing a protein subunit vaccine against sars-cov-2, expressed using a baculovirus platform, funded by the us biomedical advanced research and development authority (barda). this has been reported to be delivered in conjunction with as03 from gsk [122, 123] or potentially one other adjuvant which has not been revealed. phase i clinical trials were initiated on 3 september 2020 (nct04537208), with an aim to make the vaccine available in early 2021 [122] . other protein candidates in clinical trials (table 2 ) are from adimmune (baculovirus-derived, alum adjuvanted), anhui zhifei (rbd only), instituto finlay de vacunas (rbd), kentucky bioprocessing (tobacco-derived protein), medigen (alum/cpg adjuvanted) and vaxxine (advax adjuvanted). differences in cost of manufacturing, location of the manufacturer and impact of the adjuvant will determine which candidates progress beyond clinical trials. virus-like particles (vlps) are a subset of protein vaccines which are artificially produced nanoparticles that resemble viruses. rather than an individual protein, vlps are made up of some or all of the proteins that form the viral capsid [124] . they have some similarities to live attenuated or inactivated vaccines, and can produce strong cellular and humoral immune responses with no risk of reversion, because they contain none of the genetic material of the virus. they are used for a wide range of viruses, including hpv, and a preclinical sars-cov-1 vlp has been tested [125] . vlp nanoparticles are self-assembling protein particles, not necessarily derived from the virus capsid proteins. novavax, funded by cepi and us operation warp speed, have developed a recombinant nanoparticle vaccine (nvx-cov2373) that displays the sars-cov-2 spike protein [122, 126] . this is produced using engineered baculovirus to infect sf9 insect cells [127] . for the clinical trial with nvx-cov2373 novavax are using their own saponin-based matrix-m adjuvant (nct04368988), the data from which have recently been published [128] . the vaccine was immunogenic, but required the addition of adjuvant to achieve 100% seroconversion; two doses were required for neutralising antibody in all individuals. immunized animal models develop spike proteinspecific antibodies that prevent the attachment of the spike protein to host cell ace-2 receptors and also neutralize the wild-type virus [129] . another company (medicago) are using a plant-based system, nicotiana benthamiana, to produce a vlp [130] which is currently in clinical trial in combination with cpg or as03 adjuvant (nct04450004). other groups at the preclinical stage include saiba ag, based in switzerland, who are using a cucumber mosaic virus vlp that is bound to sars-cov-2 rbd, which induced neutralizing antibody in mice [131] . peptide vaccination is based upon the concept that, as induction of t cell responses can be achieved using a fraction of the entire protein [132, 133] , only the minimal immunogenic peptide sequence needs to be included. by selecting conserved epitopes, peptide vaccines can potentially induce broad-spectrum immunity against multiple strains of a given pathogen [134, 135] . peptides are easier to produce than whole protein antigens, as they can be produced synthetically and do not require folding into a tertiary structure. however, peptide vaccines are often weakly immunogenic. this is due to several factors, including the relatively small size of the peptide and differences in mhc processing; they therefore may require carrier proteins or adjuvants [136, 137] . several groups are exploring the use of multi-epitope peptide vaccines against sars-cov-2; following bioinformatic and immune-informatic-based predictions of immunogenic epitopes [138] [139] [140] [141] , the studies are focusing upon t rather than b cell epitopes. ose immunotherapeutics have used a multiepitope peptide approach to induce t cell responses in mice [142] . covaxx and the university of nebraska medical center have recently registered a phase i clinical trial for a multi-epitope peptide vaccine (nct04545749) as has the vector institute (nct04527575) currently in clinical trial. artificial antigen-presenting cells (aapc) are immunotherapeutic agents that can stimulate antigen-specific t cell responses [143] they have been widely explored for cancer vaccines and have also been proposed for infectious disease vaccines. in the case of sars-cov-2, aapcs are transfected with a lentivirus encoding the structural and protease proteins. the cells are then administered via subcutaneous injection [144] . the shenzhen geno-immune medical institute in china are undertaking an ongoing phase i clinical trial with an aapc approach (nct04299724) and a modified dendritic cell platform (nct04276896). aivita biomedical inc. are following a similar platform (nct04386252). due to the need to isolate and purify cells and maintain them at gmp quality, this approach seems impractical for mass vaccination campaigns. isolating and then inactivating a virus, historically with formaldehyde, is one of the oldest methods of viral vaccination. inactivation of viruses has been effective for a range of different viruses. however, there have been major safety concerns relating to sars-cov-1 and mers-cov-inactivated vaccines, reminiscent of fi-rsv, and these concerns are also valid for sars-cov-2. lung pathology of vaccinated animals on virus challenge has been seen for both a gamma-irradiated mers-cov vaccine [56] and a uv irradiation-inactivated sars-cov-1 vaccine [145] . the choice of both the adjuvant and the inactivating agent is important in shaping the immune response. for example, a formaldehyde inactivated mers-cov vaccine adjuvanted with alum and cpg demonstrated enhanced protection without inducing eosinophil-mediated vaccine-related pathology [146] . there are four inactivated vaccine candidates in clinical trials. sinovac biotech are using a platform previously developed for sars-cov-1 [147] . the virus is grown in vero cells and inactivated with beta-propiolactone. the inactivated vaccine was safe and immunogenic in rhesus macaques and offered complete protection against sars-cov-2 challenge, where no virus was detected in the pharynx or lungs [148] . two different versions of this inactivated vaccine have been developed, adjuvanted with either alum or cpg108. this vaccine has completed a phase ii human trial in 600 healthy adults aged 18-59 years (nct04352608), with 90% seroconversion observed after the second dose of vaccine and some neutralizing antibody detected [149] . it is interesting to note that the production method for the virus was changed between phases i and ii trials, and this may have increased immunogenicity. the vaccine has entered phase iii clinical trials in brazil (nct04456595) and indonesia (nct04508075). sinopharm, working with both the beijing institute of biological products and the wuhan institute of biological products, have also developed an inactivated vaccine. this vaccine has now been tested in a phases i/ii clinical trial (chictr2000031809). no serious adverse effects were observed, and more than 95% of individuals seroconverted with detectable neutralizing antibody in the two different trials [150] . the antibody was mainly observed after the second dose. two other organizations, bharat biotech (india) and the institute of medical biology/chinese academy of medical sciences, are running clinical trials of inactivated vaccines, but these are ongoing with no published data as yet. valneva, based in scotland, have just expanded their bsl3 manufacturing capacity and have signed a deal with the uk government for 100 million doses of a formaldehyde-inactivated vaccine adjuvanted with cpg, based on their japanese encephalitis virus vaccine [151] . the use of a live virus to prevent infection is the oldest vaccine approach. the original vaccine, cowpox, used exactly this approach to prevent smallpox. we are grouping two approaches under live viral vaccine platforms: attenuation of the virus or the use of a viral vector to deliver transgenes. live attenuated vaccines closely resemble natural infection. as a result, they are often immunogenic with a single administration without an adjuvant [152] . one consideration is balancing attenuation and replication -over-attenuated vaccines may not replicate enough to be immunogenic, and this balance can vary between different individuals, especially the very young or immunocompromised. historically, serial passage for attenuating mutations has been used; for example, live attenuated influenza vaccine (laiv) is cold-adapted, restricting it to the upper airway. this method requires time and extensive testing: the yellow fever vaccine yf17d was passaged more than 200 times. alternatively, attenuated viruses can be generated by reverse genetics [153] , introducing site-directed mutations into genes associated with virulence. the e protein has been targeted for both sars-cov-1 and mers-cov [154, 155] . however, this method requires the identification of genes that would attenuate viral replication and the mutation(s) inserted to be phenotypically stable [153] . a novel method of codon-pair de-optimization has been developed. the codon de-optimized virus is chemically synthesized to retain 100% amino acid sequence identical to the parent virus, but to contain an increased number of cpg and upa rna dinucleotides to up-regulate host responses. codon-pair de-optimization has been used for attenuating rsv [156] . codagenix and the serum institute of india are developing a live attenuated sars-cov-2 vaccine, using codon de-optimization technology, building on previous experience with rsv and influenza [157] . in vectored vaccines, the antigenic gene of interest is expressed from another micro-organism, either virus or bacteria. adenovirus, vsv and modified vaccinia virus ankara (mva) are some of the common viral vectors used [158] . the vectors can either be replication-deficient, delivering a gene cargo but not growing themselves, or replication-competent, reproducing in the immunization site. the different platforms may alter the reactogenicity and immunogenicity of the vaccine. a recombinant mva expressing the sars-cov-1 s protein delivered via intranasal or intramuscular routes induced protective immunity in mice [159] . an adenovirus vaccine against mers-cov offered complete protection against challenge in mice [152] . as pre-existing immunity against human adenovirus is widespread and can hamper its clinical application as a vector [158] , a chimpanzee adenovirus can be used. a recombinant chimpanzee adenovirus (chadox1) encoding the s protein, known as mers001, was immunogenic in mice and safe in phase i clinical trials in humans [160] . five non-replicating viral vectored vaccines are currently in clinical trials all based around adenoviral vectors. replication-deficient adenoviral vectors lack the e1a and e1b genes; these are the early genes which are essential for reproduction of the virus [161] , and deliver the antigen gene without replicating in the vaccinated individual. building on experience with mers-cov, the university of oxford are developing a chimpanzee adenovirus vaccine vector expressing the wild-type s protein (chadox1 ncov-19, also known as azd1222). the azd1222 vaccine was immunogenic in mice and pigs [162] . in rhesus macaques it reduced viral load and pneumonia after challenge with sars-cov-2 [163, 164] . the azd1222 vaccine entered phase i clinical trial on april 23 2020 in 543 volunteers aged 18-55 years (nct04324606). in this study, there were local and systemic reactions to the vaccine, controlled by paracetamol, but no severe adverse effects. the vaccine was immunogenic, with 91% participants having neutralizing antibody after one dose and 100% after two doses. interferon (ifn)-γ-producing t cells were also detectable [165] . in partnership with astrazeneca, this vaccine received a further $1.2 billion from barda towards its global development, manufacturing and distribution. the vaccine has now progressed into phases ii/iii trials in the united kingdom (nct04444674), brazil (isrctn89951424) and the united states (nct04516746). cansino biologics (china) are developing a human ad5vectored vaccine. in phase i trials (chictr2000030906), the ad5 vectored covid-19 vaccine was tolerable and immunogenic at 28 days post-vaccination [166] . both humoral responses and specific t cell responses were observed in healthy individuals 28 days after vaccination. transient and self-limiting adverse events such as severe fever, fatigue and muscle pain were reported in the high vaccine dose group. similar results were reported after the phase ii trial [167] . the vaccine is now in a phases i/ii trial in canada (nct04398147). [198, 199, 200] cansino biological subset had t cell responses analysed. [128] janssen (part of j and j) are using an experimental, replication incompetent adenovirus vector (advac ® ) in their per.c6 ® cell line technology [168] . this platform has been used for zika, rsv and hiv vaccine candidates. an ebola vaccine (ad26.zebov) using the same platform has been proven safe and immunogenic, and has been used as part of efforts to contain democratic republic of the congo (drc) ebola outbreaks [169] . the vaccine has been seen to be protective against sars-cov-2 challenge in rhesus macaques [170] and is in phase i trials in the united states, belgium (nct04436276) and japan (nct04509947). one other vectored vaccine that has received a great deal of press attention is from the gamaleya research institute, which has been given the tradename sputnik v. this vaccine uses two different adenovirus vectors, ad26 and ad5. to date, two clinical trials have been registered giving individually or as a prime-boost, either as a solution (nct04436471) or lyophilized formulation (nct04437875) in a total of 75 people. the trial recorded mild-moderate systemic effects and mild local effects, including injection site pain; 100% seroconversion rate by binding enzyme-linked immunosorbent assay (elisa) was observed. interestingly, there was also some anti-vector antibody detected after immunization [171] . the registration of this vaccine is presumably subject to larger efficacy trials, with a phase iii trial registered in september 2020 (nct04530396). an alternative to replication deficient vectors is to use a live attenuated vector. merck has recently acquired themis, who have developed an attenuated measles vector vaccine approach using an attenuated strain of measles derived from the original 1954 vaccine strain. themis have previously used this approach to develop a chikungunya vaccine, which was safe and immunogenic [172] . in collaboration with institut pasteur and the university of pittsburgh they are now running clinical trials with these vaccines (nct04497298 and nct04498247). live and vectored vaccines may lend themselves to mucosal delivery which may achieve better local immunity and has been used for other vaccines; for example, intranasal live attenuated influenza vaccine (laiv). however, the enthusiasm for mucosal vaccines based on preclinical data has not always translated into clinical success. symvivo is using oral delivery of a probiotic bacteria, bifidobacterium longum, to deliver the spike transgene (nct04334980). the migal galilee research institute have adopted an existing vaccine against infectious bronchitis virus (ibv), which has been used in a preclinical veterinary trial inducing humoral, cellular and mucosal immunity [173] to be delivered orally, but this is not yet in clinical trials. beijing wantai biological pharmacy and xiamen university have recently registered a phase i clinical trial using an influenza viral vector (chictr2000037782). nucleic acid vaccines have been highlighted for their potential in pandemic situations due to their low cost and potential rapid development, although this potential has yet to be translated into a real-world vaccine [174] . they utilize either plasmid dna or rna, encoding a target antigen. following delivery of the vaccine, the nucleic acid is taken up by the cells and the encoded antigen is expressed. conceptually, one facility can produce any required nucleic acid vaccine and production can be theoretically scaled-up to meet pandemic level demands. the covid-19 pandemic will serve as an important test case for nucleic acid vaccines, with six rna platforms and four dna platforms currently in clinical trial. most dna vaccines are constructed from plasmids that contain prokaryotic sequences that support the plasmids' propagation in e. coli, and a mammalian expression cassette that controls the expression of the target transgene in the vaccinated organism. the expression cassette contains an upstream promoter to drive transgene expression, a kozak sequence, the inserted transgene and a 3′ polyadenylation (polya) tail. following delivery, the dna vaccine is taken up by host cells local at the immunization site or by migrating apcs [175] . to induce an adaptive immune response the dna must enter the cell nucleus. in transiting to the nucleus the dna passes through the cytosol which is inflammatory, being sensed by intracellular pattern recognition receptors, for example sting1 [175] or tbk1 [176] , inducing an innate immune response. the triggering of innate immunity is essential for promoting adaptive immunity to dna vaccines. if apcs are transfected directly with a dna vaccine, they will load vaccine-encoded peptides onto both mhci and mhcii molecules and activate t cells [177] . transfected stromal cells will generate antigen, which will be encountered by apcs and b cells following antigen release from cell exosomes or apoptotic bodies. transit of injected naked dna to the nucleus is highly inefficient, with a large majority of the dna failing to cross the cell membrane or nuclear envelope [178, 179] . to mitigate this loss, dna vaccine programmes employ delivery platforms such as electroporation and bio-injection. preclinical animal studies have demonstrated that dna vaccines encoding the m, n, 3a or s proteins of the sars-cov-1 virus could elicit immune responses [180] [181] [182] . a multivalent dna vaccine encoding s and m protein epitopes could protect from sars-cov-1 cytopathic effects. the s protein is the target of the only sars-cov-1 dna vaccine to progress to phase i clinical trial, delivered by bio-injector, and it was safe and induced neutralizing antibody responses [183] . the leading dna vaccine against mers-cov (ino-4700) was developed by inovio. phase i clinical trials were completed in 2019, with the vaccine showing a good safety profile and inducing humoral immunity and polyfunctional cd8 + t cell responses [184] . the inovio mers ino4700 (gls-5300) vaccine that was due to be taken to phase ii clinical trials (nct03721718) has now been redeployed as ino-4800 (nct04336410) to begin clinical trials for protection against sars-cov-2. in preclinical studies of the ino-4800 vaccine, neutralizing antibody and t cell responses were observed in mice and blocking antibody responses in vaccinated guinea pigs [185] and macaques [186] . the phase i trial (nct04336410) is ongoing, but the data have not yet been published. genexine, in south korea (nct04445389), zydus cadila in india (ctri/2020/07/026352) and osaka university in japan (nct04463472) have initiated phase i trials of dna vaccines. rna vaccines are based on the same premise as dna vaccines of expressing a vaccine antigen transgene in the host cell, but they are one step further along the expression pathway, skipping the transcription step. unlike dna vaccines, expression of rna vaccines begins once they enter the cell cytosol, which can increase the efficiency of expression. as with dna vaccines, the presence of 'foreign' rna is sensed in both the endosome and cytosol [187] , giving rna vaccines a self-adjuvanting effect [188] . however, the early triggering of type i ifn responses can downregulate protein expression [189] . modified nucleosides can be incorporated into the mrna product to create a 'silenced' rna vaccine that avoids detection by tlrs and does not trigger a type i ifn response [190, 191] , but there is a balance between antigen expression from the vaccine construct and triggering enough inflammation to activate the immune response. this balance may be altered by the formulation to deliver the vaccine and can be different between different animal species, making predictions from preclinical studies difficult. there are two primary types of rna vaccine mrna and self-amplifying mrna (sarna). non-replicating mrna vaccines are constructs engineered to encode the gene of interest, and typically have a 5′ cap, utrs flanking the gene of interest and poly a tail. the 5′ cap is essential for mrna to associate with the eukaryotic translation complex. utrs are selected to optimize rna protein expression, avoiding the inclusion of sequences that would hamper translation [192, 193] . mrna vaccine constructs are made using bacteriophagederived rna polymerases and ntps to transcribe linearized dna in vitro. self-amplifying rna vaccines are alphavirus-derived rna replicons modified to encode the antigen of interest in place of rna structural proteins. the viral replicon also contains an open reading frame (orf) that encodes four alphavirus non-structural proteins (nsp1-4) and a subgenomic promoter. the non-structural proteins form an rna-dependent rna polymerase (rdrp). the rdrp complex transcribes more copies of the vaccine in the transfected cell. as a result, sarna vaccines express protein at higher levels and persist for longer than non-replicating rna [194] . as it is a newer technology, rna vaccines were not developed against sars-cov-1. six rna vaccines are in clinical trials for sar-cov-2. moderna fast-tracked their candidate vaccine mrna-1273 and were first to begin clinical trials on 17 march 2020 with the national institute of health's national institute of allergy and infectious diseases (niaid) (nct04283461). this phase i study involved 45 patient volunteers, divided into three group cohorts, as a dose escalation: low (25 µg), middle (100 µg) and high (250 µg) in a prime boost. a preclinical study using the same vaccines was protective in mice against viral challenge [195] . the vaccine was immunogenic, with increasing antibody titres with increasing dose administered; of note, three individuals (of 15, 21%) in the 250-µg group reported severe adverse events, with severity increasing after the second vaccination [196] . the vaccine is now in phase ii (nct04405076) and phase iii (nct04470427) trials, focusing on the 100-µg dose. biontech is collaborating with pfizer to develop four s protein vaccine candidates. they are using a nucleoside modified mrna. phases i/ii clinical trials are running in germany (nct04380701) and the united states (nct04368728), with a multi-site phase iii study planned. the vaccine induced both cellular and humoral responses in mice [197] and induced neutralizing antibody in the clinical study [198] . they have performed a further two clinical studies, observing similar responses in a second study with their initial construct (bnt162b1) which encodes a rbd trimer [199] . in a comparator study they observed similar levels of immunogenicity to a stabilised membrane anchored spike protein (bnt162b2), but with lower levels of reactogenicity [200] . both curevac and the people's liberation army have also developed mrna vaccines that are in clinical trials, but no results have been published as yet. imperial college london and its spin-out social enterprise, vacequity global health, are developing an sarna vaccine encoding the s protein. intramuscular injection with lnp formulation induced high neutralizing antibody titres in mice [103] . tested doses of the preclinical vaccine ranged from 0·01 µg to 10 µg, with a boost of the same dose at week 4 post-vaccination. human trials of the vaccine with 420 participants started in june 2020 (isrctn17072692). arcturus, based in singapore, are also developing an sarna vaccine encoding a prefusion spike, which is in phase i clinical trial (nct04480957). protein vaccines can have low levels of immunogenicity. this can be boosted by adjuvants [201] . adjuvants enhance the immune response through multiple mechanisms, causing a depot effect; up-regulating the production of chemokines and cytokines; enhancing the cellular recruitment to site of injection; increasing antigen uptake and presentation by apcs and increasing inflammasome activation [202] . adjuvants can also tailor the immune response, guiding it towards producing the most effective form of immunity against the specific pathogen being vaccinated against [203] [204] [205] . a range of adjuvants have been proposed for use with sars-cov-2 protein vaccines. these include advax, alum, as03 (gsk), matrix-m (novavax), cpg (dynavax) and mf59 (csl). additional components are also included with nucleic acid vaccines to enhance uptake and immunogenicity. nucleic acids are combined with a range of formulations, including lipid nanoparticles (lnps), liposomes and polyplexes. such formulations are essential for rna vaccines, as 'naked' rna is susceptible to being degraded by extracellular rnases which will prevent efficient cell uptake. lnps have previously been used for other rna therapeutics and moderna, imperial college london, arcturus, curevac and biontech vaccines all utilize this technology. the stability of these formulations can be a concern and may necessitate vaccine storage at a lower temperature which might, in turn, impact access to the vaccine. for dna vaccines, delivery devices are often used to increase uptake. inovio uses an electroporation device (cellectra ® 2000), which delivers an electric current to the site of injection: in a study on acceptability, acute pain (six of 10 on the vas score) was recorded for the first 5 min after immunization, but this receded [206] . a similar effect was observed in a study using a different electroporation device [207] . genexine are also using electroporation in their trial, but they are also comparing with a needle-free biojector. biojector devices have been shown to increase the antibody response to dna vaccines [208, 209] . one of the more experimental approaches proposed to reduce the impact of covid-19 has been the use of other live vaccines as non-specific vaccines [210] . this has been proposed for bacille calmette-guérin (bcg), oral polio vaccine [211] and measles, mumps and rubella (mmr) [212] . the proposed mechanism is described as trained immunity, where exposure to one agent alters the epigenetic profile of innate immune cells, potentially increasing the production of cytokines. in preclinical models, bcg pretreatment has been shown to reduce influenza viral titres [213] . early ecological data (in april 2020) suggested that countries with mandatory bcg vaccination had reduced mortality from covid-19, but this analysis has a number of issues, mainly associated with demographics and the timing of when the virus reached different countries [214] . a more recent study has supported this protective effect [215] . remarkably, a number of randomized clinical trials have been set up to directly test whether bcg can reduce the burden of covid-19. the covid-19 pandemic has led to a surge of different vaccines being rapidly moved to clinical trials. a number of these vaccines have been around for several years as promising preclinical platforms, but not necessarily been attractive enough to generate funding to support human trials. each of the approaches has advantages and disadvantages (table 4 ): which aspects are the most important will only be identified following efficacy studies. live attenuated vaccines have a long track record of safety and efficacy, but they may not be feasible in the current pandemic due to the length of time it takes to generate a candidate and test for attenuation. inactivated vaccines also have a long track record of protective efficacy, and they have the advantage that they are fast to generate; however, they require highcontainment facilities to generate the virus stock. there is also a concern about vaccine-induced immunopathology with an inactivated vaccine, which has been seen for some other respiratory viruses and in preclinical models of sars-cov-1; whether this is the case for inactivated sars-cov-2 vaccines will only be seen after larger and longer phases ii/iii trials. recombinant protein vaccines have been in use since the 1980s; they are a more targeted approach than using a whole virus, which may focus the immune response on a key antigen, but this may lose some breadth of protection. protein candidates were somewhat slower to enter clinical trials but may have a faster route to licensure, being a more known product than newer vaccines. one challenge is to use the correct conformation of the protein, spike is metastable and may be less protective if used in a post-fusion form. one peptide vaccine has registered a clinical trial (nct04527575) from the vector institute in koltsovo, russia; it is adjuvanted with alum. the cellular-based approaches, using aapc, do not seem to be practical for wide-scale rollout. nucleic acid vaccines, both dna and rna, have much potential in terms of speed of response and scale-up: this outbreak will be an important test for whether they can deliver on their promise. dna vaccines have historically been less immunogenic than other platforms, although with alternate delivery devices that may be overcome. rna vaccines have not been widely tested for infectious diseases; this is the first time an sarna vaccine has been trialled. rna may have a slight issue concerning heatstability, necessitating -80˚c storage. viral vectored vaccines are the furthest ahead in clinical trials, with three candidates in later phase clinical trials. they are known to be safe, but may be reactogenic at higher doses. historically, these approaches have had mixed results for efficacy and one concern is pre-existing immunity against the vector, especially when a human viral-derived vector such as ad5 is used. it will be of great interest to see which platforms and candidates are protective in efficacy trials. as of september 2020, the furthest advanced candidates have completed phase i trials (table 3) , although so far not all the organizations involved have published data from completed trials. all the data published so far indicate that the vaccines are safe, but there are more adverse events at higher doses: both the moderna [196] and biontech [198] vaccines had severe adverse effects at the highest doses, leading to a lower dose in later studies; some severe adverse effects were also recorded in the cansino [166, 167] and university of oxford [165] vectored vaccine trials. the vaccines all appear to be immunogenic, although it is hard to compare directly, as different groups will have used subtly different elisa and neutralization assays. a further complication for comparison is when data have been published as press releases rather than peer-reviewed papers. ultimately, vaccine efficacy in a randomized trial is the most important issue, but here again, different primary end-points are being explored. some studies are looking at reduction of disease, while others are looking at reduction of confirmed infections. the covid-19 crisis represents an opportunity for several experimental vaccine platforms to progress to clinical trials. however, there are considerations between a promising preclinical candidate and a global vaccination campaign, including trials, regulation and manufacture. clinical vaccine trials conventionally undergo four broad phases, from early safety in small numbers of volunteers (phase i) to wide-scale post-licensure monitoring (phase iv). usually, each of the phases take months or even years to complete before moving on. in a pandemic setting, there is need to speed up the transition between phases; this has been achieved with co-operation of regulatory agencies and research ethics committees. additionally, phases can be merged, with planning of phases ii/iii trials initiated before the phase i trial has even begun. there is the potential that data obtained from phase i will mean that planned later-phase trials are cancelled due to safety concerns or futility. ultimately, pushing a vaccine through the different stages of a clinical trial does not negate the need for complete safety data sets to be collected, but close co-operation with the researchers and regulators can accelerate a progress that could reduce 10 years to 18 months. one consideration is that due to the accelerated time-scale, postlicensure monitoring will be extremely important. another issue concerns the sample size required for efficacy studies; as cases fall, larger studies will be necessary or an alternate trial design. a ring trial was used in ebola [216] , which allowed efficacy to be assessed in a few individuals. one of the major hurdles to a vaccine relieving the covid-19 pandemic is manufacturing enough doses to achieve global herd immunity. the number of doses needed to achieve global coverage depends upon the regime used, but is potentially as many as 16 billion (assuming a prime boost regime with some contingency). to ensure licensure and prequalification status, good manufacturing process (gmp) standards must be upheld during up-scaled manufacturing and clinical studies. manufacturing a vaccine at a global scale in the timeframe required is a unique challenge. vaccines that are not only safe and effective but also highly scalable, to produce millions or even billions of doses, would be the most desirable tool for curbing the pandemic. logistics are a key consideration, including access to components to manufacture the vaccines -for example, nucleic acid vaccines require nucleotide triphosphates (ntps), which are also in high demand for the diagnostic tests. the vaccines also require plant and materials to fill and finish the final product; one bottleneck is exactly that: glass bottles. in parallel with the accelerated clinical trials, accelerated manufacturing scale-up is required. this telescoped manufacturing process means that investment in the next step is being made before the results of the previous step are known. this has considerable financial risk, especially in terms of setting up the necessary manufacturing plant if it cannot be repurposed, either for other pandemic vaccines or other biologicals. funding is a critical part of the vaccine development process. it remains to be seen whether the total costs of research, development and licensure of any novel vaccine platforms for sars-cov-2 is comparable to traditional vaccines, such as dengvaxia, which costed approximately $1.5 billion until licensure [217] . a range of funding mechanisms have supported vaccine development. one of the major bodies co-ordinating the funding is cepi, which has received funding from multi-national sources, including governments and charities. other vaccine candidate teams are being supported by their governments 'fast-tracking' their candidates through clinical trials and streamlining their manufacturing. for example, operation warp speed in the united states, is supporting six candidates from astrazeneca (azd1222), moderna (rna), pfizer (rna with biontech), merck (vectored vaccine with themis), johnson and johnson (vectored vaccine) and novavax (recombinant protein). a uk vaccine task force was announced on 20 april 2020 [218] to support uk-based candidates and reviewing government regulations to facilitate rapid and safe vaccine trials. several vaccine candidates have never progressed past phase i before, therefore manufacturing gmp material at scale poses new challenges. many of the vaccines are being developed by either academic groups or small-to mediumsized biotech companies, neither of which necessarily have capacity to manufacture at a large scale. one approach is outsourcing to contract manufacturing organizations (cmos), which can have licensing complications. some companies have invested in manufacturing capacity; for example in july 2018, moderna opened a manufacturing facility in norwood (usa) [219] , which produce rna up to the gram scale. however, they still need to work with external companies to complete formulation protocols. new manufacturing facilities are also being constructed elsewhere, including the vaccines manufacture and innovation centre (vmic) in the united kingdom, which has been accelerated and is planned to open in mid-2021, and valneva opened an expanded bsliii facility in scotland in august 2020. larger companies may have more experience and capacity of manufacturing; for example, janssen and astrazeneca are making adenovirus vectors and sanofi is making a protein vaccine. in the initial stages of the outbreak there was relatively little publicized activity from the larger pharmaceutical companies, with most of the attention on smaller biotechs and academic groups. it is not clear why this was the case. one possible reason could have been exemption from liability, although vaccine manufacturers are exempt in the united states under the 2005 public readiness and emergency preparedness, or prep act. integrating national funding programmes with equitable global access to vaccines is vital. there is a concern that wealthy countries will monopolize initial production runs of vaccines, with preorders outstripping manufacturing capacity. alternative models of licensure, social enterprise and spoke and hub manufacture may be necessary. for example, the astrazeneca/oxford vaccine has been licensed to the serum institute of india, the largest global manufacturer of vaccines by doses produced, and imperial college london have established a social enterprise company to enable equitable access. vaccines require regulation to be introduced as a licensed product. there are a range of national and international bodies which cover this process; for example, any product trialled in the united kingdom will be considered for approval by the medicines and healthcare products regulatory agency (mhra). the product is then considered for prequalification by the who for multinational distribution, which has supported the regulation and distribution of vaccines for 33 years [220] . this process can normally take a substantial amount of time; however, during the covid-19 pandemic manufacturers and regulators are striving towards the delivery of a vaccine that is safe and effective within 18 months from february 2020. there is a precedent for accelerated licensure in the context of a pandemic, as seen with the approval of rvsv-zebov [221] as a vaccine for ebola. the who supported the accelerated regulatory approval for rvsv-zebov-gp using an expedited prequalification review following its receiving conditional marketing authorization from the european medicines agency (ema) [222, 223] . the who has issued guidance and recommendations for the regulation of sars-cov-2 vaccines [220] . the issues faced during this pandemic and the vaccine platforms being developed to address it will be invaluable for future outbreak control. while, at this stage, it is not possible to say which platform is best, and what works best for one infection may not be best for all infections or for all populations. one of the biggest considerations of all the platforms is speed into trials versus ability to deploy the vaccine. while some of the high-speed platforms, for example rna, have entered into phase i trials faster than other approaches, their lack of track record means that approaches for global scale-up are potentially slower. older approaches may leapfrog the newer platforms in the scale-up and manufacturing stage. one observation of interest is the speed with which one of the oldest technologies for viral vaccines, inactivation, has been able to move forwards. prior to covid-19, much of the attention for pandemic vaccine preparedness was on newer technologies; however, of the candidates in phase iii in august 2020, two of four are inactivated vaccines. safety and efficacy data from these large studies will be critical. another consideration is that while distributed global manufacture is effective and appropriate for routine scheduled vaccination, local surge manufacturing capacity for new vaccines is important. this capacity may have to be maintained at a loss for large amounts of time, unless alternative commercial contracts can be found for the same facility that can then be replaced at short notice. this reflects a broader consideration that investment in public health, which may appear expensive to begin with, can save a considerable amount in the long term; one study estimated that every £1 spent on public health saves £14 in return [224] . another consideration is for greater standardization of assays and end-points. comparisons of the different trials has been made significantly harder by the use of different methodologies. this is part of the broader global context of a true pandemic. collaborative worldwide action is required to control the virus, which necessitates leadership and a willingness to share by countries with more developed scientific research programmes. ultimately, while lessons will be learned from this pandemic, the next one will be different and sticking rigidly to a plan that controlled this coronavirus will not necessarily work for a different virus, as the german strategist helmuth von moltke 'sort of ' said: 'no plan survives contact with the enemy' . it is still far too early to know what the best approach will be to control covid-19 with vaccines. speculating as to which vaccine platform is 'best' , while academically enjoyable, is not of value here. that so many platforms, both new and old, are moving into efficacy study makes this an extremely exciting time for vaccinology. the outbreak will certainly be a test case for the novel vaccine platforms, particularly nucleic acid vaccines, which have promised much to date but not been licensed for human use. one issue is whether vaccines will play a role in reducing the burden of the pandemic. even at maximum speed, the first efficacy trials will start 9 months after the start of the pandemic and the first licensed doses are unlikely to be ready for 18 months, by which time the virus will have caused a large wave of mortality and a larger wave of global disruption. important questions remain (box 1) regarding what is a successful vaccine, how should it be deployed and who should be prioritized. these 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vaccines prevent covid-19? could an unrelated live attenuated vaccine serve as a preventive measure to dampen septic inflammation associated with covid-19 infection? nonspecific protection of mice against influenza virus infection by local or systemic immunization with bacille calmette-guerin is global bcg vaccination coverage relevant to the progression of sars-cov-2 pandemic? efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola ça suffit!) a review of dengvaxia®: development to deployment government launches vaccine taskforce to combat coronavirus 2020 moderna manufacturing: why norwood? world health organization. standardization of vaccines for coronavirus disease (covid-19) the authors. clinical and experimental immunology published by john wiley & sons ltd on behalf of british society for immunology successful ebola vaccine provides 100% protection in trial roadmap for introduction and roll-out of merck rvsv-zebov world health organization. major milestone for whosupported ebola vaccine return on investment of public health interventions: a systematic review legal agreements: barriers and enablers to global equitable covid-19 vaccine access the authors thank dr ed parker, professor beate kampmann and the team at the vaccine centre at the london school of hygiene and tropical medicine for developing and maintaining the covid-19 vaccine tracker tool, which was invaluable for this review. b.f.p. is funded by the key: cord-301730-flv5lnv8 authors: pandey, anamika; khan, mohd kamran; hamurcu, mehmet; gezgin, sait title: natural plant products: a less focused aspect for the covid-19 viral outbreak date: 2020-10-15 journal: front plant sci doi: 10.3389/fpls.2020.568890 sha: doc_id: 301730 cord_uid: flv5lnv8 the sudden emergence of covid-19 caused by a novel coronavirus (ncov) led the entire world to search for relevant solutions to fight the pandemic. although continuous trials are being conducted to develop precise vaccines and therapeutic antibodies, a potential remedy is yet to be developed. plants have largely contributed to the treatment of several human diseases and different phytoconstituents have been previously described to impede the replication of numerous viruses. despite the previous positive reports of plant-based medications, no successful clinical trials of phyto-anti-covid drugs could be conducted to date. in this article, we discuss varying perspectives on why phyto-anti-viral drug clinical trials were not successful in the case of covid-19. the issue has been discussed in light of the usage of plant-based therapeutics in previous coronavirus outbreaks. through this article, we aim to identify the disadvantages in this research area and suggest some measures to ensure that phytoconstituents can efficiently contribute to future random viral outbreaks. it is emphasized that if used strategically phyto-inhibitors with pre-established clinical data for other diseases can save the time required for long clinical trials. the scientific community should competently tap into phytoconstituents and take their research up to the final stage of clinical trials so that potential phyto-anti-covid drugs can be developed. nobody knew that the coronavirus disease of 2019 was going to be a greater disaster when compared to the former coronavirus outbreaks, severe acute respiratory syndrome (sars), and middle east respiratory syndrome (mers). the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), that caused covid-19 is alarming to the scientific world as it has resulted in the unexpected viral pandemic. however, was covid-19 an unexpected viral pandemic? the answer is no. the previous two corona outbreaks, sars-cov, and mers-cov, were first reported in guangdong, china in november 2002 (zhong et al., 2003) and saudi arabia in april 2012, respectively (de wit et al., 2016; cui et al., 2019) . after the identification of sars-cov (ksiazek et al., 2003) , several strategies were adopted to eradicate the disease, however, infection control was proven to be more effective than medical intervention leading to the termination of the sars pandemic. although the end of the sars pandemic was announced in july 2003, different types of sars re-occurred in different years either by zoonotic or human-to-human transmission in several countries including canada, vietnam, the middle east, hong kong, south korea, and jordan zaki et al., 2012; hijawi et al., 2013) . later, evidence proved that intermediate hosts may not be required for straight human infection as chinese horseshoe bats are the main reservoirs of sars-cov (ge et al., 2013) . besides, it was suggested that there was a higher risk of recurrence of sars-cov from circulating viruses in bat populations (menachery et al., 2015) . the emergence of sars-cov-2 is just a confirmation of the previous predictions. after previous outbreaks, several studies determined the poor efficacy of therapeutics and both monoclonal antibodies and vaccines against cov infection (menachery et al., 2015) . in this scenario, new measures should have been created to fight the problem. the fact that more than 619,150 deaths around the world have been reported, as of july 23 2020, has proven that we, as a scientific world, were less prepared for such an abrupt viral outbreak (who, 2020, siuation report, 185) . though plants and their extracts have been recognized as effective anti-viral agents for several decades (chantrill et al., 1952) , plant-based medications have been largely ignored. despite the vast research conducted in several directions after the previous cov pandemics, plant-based therapeutics could not achieve a satisfactory level in clinical trials against the disease. medicinal plant extracts have been reported to impede the replication of several viruses including human immunodeficiency virus (hiv), hepatitis b virus (hbv), poxvirus, severe acute respiratory syndrome (sars) virus, and herpes simplex virus type 2 (hsv-2) (vermani and garg, 2002; kotwal et al., 2005; huang et al., 2006) . despite that, there are no reports of plant-based medicines that have been successful in preventing the spread of covid-19 or curing covid-19. thus, further study is required to affirm why plant-based medications could not work in the case of covid-19. in this article, we discuss different aspects of the utilization of plant-based therapeutics in controlling viral outbreaks like covid-19. future research for such viral pandemics should be designed in light of the success stories of previous plant-based medications. the disadvantages in this field of research have been deliberated so that lessons can be learned and the scientific community can prepare for future outbreaks. medicinal plants can be used as anti-viral agents either as firstgeneration drugs where plant crudes are used in their natural forms or as second-generation drugs where the active metabolites of plants which are responsible for the anti-viral activity are employed (jassim and naji, 2003) . however, to be successful, plant-based herbal medicines have to address the issue of genetic variability of viruses, competent replication of dna and rna viruses within the host cells, and their capability to survive in the host cells (wagner and hewlett, 1999; jassim and naji, 2003) . in contrast to synthetic drugs, some of the plantbased metabolites hinder the replication of viruses without disturbing the host metabolism; which consequently have restricted side effects as drugs (hussain et al., 2017) . the interest in anti-viral plant research development started with the suppression of the amplification of the influenza a virus by 12 plant extracts (chantrill et al., 1952) . afterward, continuous efforts have been made to screen different plant sources via in silico, in vitro, and in vivo assays for anti-viral activity towards several viruses such as parainfluenza virus type 3, respiratory syncytial virus, poliovirus type 1, herpes simplex virus (hsv), enteric coronavirus, and rotavirus (rv). (ahmad et al., 1996; rajbhandari et al., 2001; jassim and naji, 2003) . plant crudes contain several metabolites and it is extremely crucial to identify which component makes it a potential candidate for an effective anti-viral drug. different anti-viral compounds of plants including peptides, lignans, terpenoids, polysaccharides, flavonoids, polyacetylenes, and alkaloids are effective against different targets of viruses such as dna, rna genomes, membranes, the replication process, and ribosomal activity (jassim and naji, 2003; ireland et al., 2008; sencanski et al., 2015; vilas boas et al., 2019) . as an example, the strong in vitro activity of extracts of macaranga barteri against e7 and e19 echoviruses suggests that it can be an effective therapeutic agent for enteroviral infections such as encephalitis (ogbole et al., 2018) . however, among all the components, three stilbenoids (especially vedelianin) isolated from m. barteri extracts are largely responsible for the anti-viral activity against echoviruses (segun et al., 2019) . similarly, other stilbenes such as resveratrol and trans-arachidin isolated from grapes and the hairy root culture of peanut are capable of reducing the replication rate of the african swine fever virus (asfv) and rvs, respectively (abba et al., 2015; ball et al., 2015) . coronaviruses, which have the largest genomes among the rna viruses, consist of a long positive-sense rna that behaves like mrna encoding the synthesis of two replicase polyproteins (pp), i.e., pp1a and pp1ab. these polyproteins are processed by a main protease, i.e., chymotrypsin-like (3clpro) protease and papain like protease (plp). while the main protease cleaves at 11 sites, plp cleaves at two or more than two sites on the polyproteins. due to the essential role of these proteases in proteolytic processing during viral replication, these proteases are considered as the main targets for the development of therapeutic drugs (deng et al., 2014) . covs are grouped into four categories: alpha, beta, gamma, and delta types; among which only alpha and beta types are known to infect humans. coronaviruses are difficult to handle because of the higher mutation rates of their nucleotides as compared to other singlestranded rna viruses. before the present sars-cov-2, six different types of human coronaviruses (hcov) had been discovered including hcov-hku1, hcov-oc43, hcov-nl63, hcov-229e, mers-cov, and sars-cov (friedman et al., 2018) . the enveloped hcov viruses fit in to coronaviridae family and are known to develop respiratory diseases (geller et al., 2012) . sars-cov and mers-cov were reported as the most deadly viral corona outbreaks causing an epidemic in different countries with a fatality rate of 9% (during 2002 (during and 2003 (during ) and 35.4% (up to december 2016 , respectively (de wit et al., 2016) . the first outbreak of sars-cov in china led to a sprint of screening of chinese medicinal herbs against the disease and some of them came out as potential anti-viral agents. during the sars outbreak in the absence of effective therapies, ribavirin, a licensed drug for the respiratory syncytial virus (rsv) was commonly suggested as a treatment (van vonderen et al., 2003) . however, it was later found to cause the death of sars patients by inducing anemia and hemolysis (booth et al., 2003; chiou et al., 2005) . glycyrrhizin (triterpene glycoside glycyrrhizic acid), a phytoconstituent extracted from liquorice roots (glycyrrhiza radix) proved to be a more efficient anti-viral agent for sars-cov when compared to ribavirin (cinatl et al., 2003) . glycyrrhizin was tried against the isolates of sars-cov replicated in vero cells (kidney epithelial cells isolated from african green monkeys) and was proven to be the most compelling interceptor of replication of sars-cov when compared to other anti-viral agents such as mycophenolic acid, pyrazofurin, 6-azauridine, and ribavirin (cinatl et al., 2003) . besides it also hinders the entry of the virus which is a main step in the replication cycle. the concentration of glycyrrhizin required to impede the cytopathic effect of a virus in a vero cell culture to 50% of the control value, i.e., ec50, was 300 mg/l. ec50 stands for the effective concentration of a drug that gives a half-maximal response. although the complete effect of glycyrrhizin activity towards sars-cov is not clear, it increases the production of nitrous oxide by the overexpression of nitrous oxide synthase that inhibits the viral replication (jeong and kim, 2002; cinatl et al., 2003) . a concentration of 1000 mg/l of glycyrrhizin was found to be effective in lowering the expression of sars-cov antigens (extracted from patient's serum) in a vero cell culture. further analysis of glycyrrhizin derivatives against sars-cov showed that the addition of different compounds to functional groups may lead to a 10-70 fold increase in the anti-sars activity; however, they may also increase the cytotoxic effects (hoever et al., 2005) . although cytotoxic effects need to be discussed, such studies open the platform for the modification of glycyrrhizin for the production of novel anti-sars-cov drugs with enhanced activity. several plants and their extracts have been reported to have a remedial approach against sars-cov and mers-cov by modulating the immune response. based on a virus-induced cytopathic effect (cpe) assay and an mts [(5-(3carboxymethoxyphenyl)-2-(4,5-dimethyl-thiazoly)-3-(4sulfophenyl) tetrazolium] cell proliferation assay, extracts of linder aggregata, pyrrosia lingua, artemisia annua, and lycoris radiata with ec50 values ranging from 2.4 ± 0.2 to 88.2 ± 7.7 mg/l showed much better anti-sars-cov activity in a vero cell culture when compared to glycyrrhizin (li et al., 2005) . however, among these four extracts, lycoris radiata with an ec50 value of 2.4 ± 0.2 mg/l was found to be the most effective candidate for anti-viral medicine against sars-cov. lycorine, one of the phytoconstituents fractionated from the extract of lycoris radiata is mainly responsible for its anti-sars-cov activity showing a lower ec50 value than its original extract ( ± 0.0012 µm). lycorine is also recognized for its potential inhibition activity against herpes simplex virus (type i) and the poliomyelitis virus. thus, it is crucial to explore the broad antiviral feature of lycorine in detail using real-time pcr to assess its capacity to inhibit viral rna replication and its interface with viral antigens. aescin, an extensively utilized drug in europe, which is extracted from horse chestnut trees and reserpine which is extracted from the rauwolfia species have shown ec50 values of 3.4 µm and 6.0 µm against sars-cov in a vero cell culture, respectively (wu et al., 2004) . in addition, radix ginseng, eucalyptus, and lonicera japonica extracts have shown antiviral activity towards sars-cov at 100 µm. later, ginsenoside-rb1 isolated from the traditional chinese herb, radix ginseng, was reported to lessen acute lung injury in rats by inhibiting the inflammatory signaling pathway (yuan et al., 2014) . many research experiments have been conducted to determine the inhibition capacity of phytoconstituents against the main protease (3clpro) and papain-like protease (plpro) of sars and mers coronaviruses. strangely, even the constituents in tea can be potentially effective against a deadly virus like sars-cov. it is one of the positive points that can be counted on while considering the negative effects of tannic acid in the tea. the 3-isotheaflavin-3-gallate (tf2b), theaflavin-3,3'-digallate (tf3), and tannic acid compounds that are abundant in black tea extracts are potent inhibitors of the main protease of sars-cov, 3clpro at ic50 < 10 µm as determined by an hplc proteolytic assay . ic50 stands for the concentration of an inhibitor where the response is reduced by half. quercetin, a plant flavonoid, and its derivative quercetin-3b-galactoside show potent inhibition of viral replication in sars-cov 3clpro where sugar moiety is crucial for inhibitory action (yi et al., 2004; lin et al., 2005; chen et al., 2006 ). an extract of houttuynia cordata, a traditional chinese medicine, at 200µg/ml had been reported to have an inhibitory effect on the rna-dependent rna polymerase (rdrp) and 3c-like protease (3clpro) of sars-cov which was non-toxic to mice at an oral dosage of 16 g/kg (lau et al., 2008) . scutellarein extracted from scutettaria baicalensis can be a potential sars-cov inhibitor as it hinders the atpase activity of the helicase protein of sars-cov in vitro (yu et al., 2012) . few compounds such as dihydrotanshinone isolated from the root of salvia miltiorrhiza showed anti-viral activity against both sars and mers cov by the inhibition of proteases and hindering of the viral entry, respectively (park et al., 2012; kim et al., 2018) . a phlorotannin, dieckol, extracted from the edible brown algae ecklonia cava showed potential inhibitory effects on the 3clpro of sars-cov at ic50 = 2.7 µm. it is more repressive on the cell-based 3clpro cis-cleavage when compared to the other natural cov protease inhibitors such as quinone-methide triterpene extracted from tripterygium regelii (ryu et al., 2010; park et al., 2013) . not only are these extracts potent against 3clpro but also several natural compounds have been reported to have a greater inhibitory action against plpro. a polyphenol compound, papyriflavonol a, derived from broussonetia papyrifera which hinders the sars-cov plpro with an ic50 value of 3.7 µm can be utilized for the development of anti-cov agents (park et al., 2017) . a cinnamic amide with an infrequent carbinolamide motif derived from the methanol extract of t. terrestris fruit showed effective inhibitory action towards sars-cov plpro with ic50 = 15.8 µm (song et al., 2014) . resveratrol (trans-3, 5, 4′-trihydroxystilbene), a natural stilbene derivative, can be extracted from different plants including cranberry (vaccinium macrocarpon), grape (vitis vinifera), and huzhang (polygonum cuspidatum) is efficient in inhibiting mers-cov replication in vitro by reducing cell death and alleviating the expression of nucleocapsid protein that is required for viral replication (lin et al., 2017) . the inhibition of the nf-kb pathway by resveratrol in signal transduction shows its potential capacity to be an effective broad-spectrum anti-viral agent and thus, its functioning against cov should be investigated in vivo. the anti-viral activity of several medicinal herbal extracts such as sophora subprostrata radix, phellodendron cortex, coptidis rhizoma, meliae cortex, and cimicifuga rhizome was identified against mouse hepatitis virus (mhv) which is widely considered as a prototype of coronavirus. the ec50 values of these compounds is in the range of 2.0 to 27.5 µg/ml suggesting that these can be potent candidates for developing anti-viral therapeutics (kim et al., 2008) . the accidental outbreaks of sars and mers coronaviruses pointed towards the chances of the emergence of novel covs in the future. until 2020, there was a dearth of approved drugs for sars-cov and mers-cov and it emphasized the significance of broad-spectrum viral inhibitors. the extent of conservation in crucial active domains of different human coronaviruses such as rna helicase and 3clpro can be utilized as a target when developing potential broad-spectrum anti-cov drugs. silvestrol, extracted from aglaia sp., inhibits the cap-dependent mrna translation of hcov-229e and mers-cov in human embryonic lung fibroblast (mrc-5) cells with ec50 values of less than 0.003 µm and 0.0013 µm, respectively (muller et al., 2018) . several phytocompounds including mycophenolate mofetil, emetine, and lycorine were identified as the potential broad-spectrum inhibitors that hindered the in vitro replication of four covs; mers-cov, mhv-a59, hcov-nl63, and hcov-oc43-wt with ec50 values less than 5 µm (shen et al., 2019) . among these, the inhibition capacity of lycorine against hcov-oc43 by reducing the viral lethality in the central nervous system of mice was reported in vivo via bioluminescence imaging (shen et al., 2019) . the effectiveness of most of these phytocompounds as broad-spectrum inhibitors has been confirmed in in vitro infection models and was tested in a particular cell line that may be influenced by specific host cell types. the identification of broad-spectrum inhibitors and determining their inhibition capacity in an in vivo system may lead to the production of potential drugs. the emergence of the covid-19 outbreak and its spread around the world led to an urgent search for a solution against the disease. while expected methods such as the combined medication of systematic corticosteroids and anti-viral treatment along with interferons are being tried for the speedy recovery of patients, plant-based treatment regimes are also potentially being explored in the race. the release of the gene sequence of sars-cov-2, the crystallization of its main protease, and its availability in the protein data bank (pdb) showed that the main proteins of sars-cov-2 shares great similarities with those of sars-cov and mers-cov zhou et al., 2020) . it should be considered that despite the reported mutations in the novel coronavirus as compared to sars-cov and mers-cov, the effectiveness of potential plants and their phytoconstituents that were operative against sars-cov and mers-cov could have been employed for sars-cov-2. sars-cov and sars-cov-2 belong to beta coronaviruses with high homology in the genomic sequence at the nucleotide level; however, there are six regions of differences in their genome sequence. these regions can be supportive to developing new drugs for sars-cov-2 (xu et al., 2020) . in addition, proteins of sars-cov-2 share 95% -100% homology with sars-cov with only two non-homologous proteins, orf8 and orf10. the amino acid sequence of orf8 is different in both the viruses (chan et al., 2020) . a blastp comparison of sars-cov and sars-cov-2 showed a more than 95% similarity in helicase, nsp7,8,9,10, 3clike proteinase, 3'-to-5' exonuclease, and rna-dependent rna polymerase (xu et al., 2020) . the antibodies active towards the n protein of sars-cov may have increased the probability of binding to the n protein of sars-cov-2 due to approximately 90% identity in the n protein amino acids of both viruses (gralinski and menachery, 2020) . these similarities and dissimilarities between the two viruses should be considered while utilizing the phyto-inhibitors active against sars-cov for the novel coronavirus (ncov), sars-cov-2. however, not taking potential plants or their phytoconstituents to an effective therapeutic anti-viral drug stage during previous corona outbreaks is one of the major disadvantages of the present scenario. the availability of potent plant-based drugs against sars-cov and mers-cov could have opened new treatment pathways for sudden outbreaks such as covid-19. given this disadvantage, most of the plant-based investigations directed towards covid-19 either focus on bioinformatic tools such as in silico processing, molecular docking, or concentrate on molecular farming such as the production of recombinant proteins involving vaccines and antibodies (islam et al., 2020; rosales-mendoza et al., 2020) . several molecules of known herbal medicines, when docked with the proteins of sars-cov-2, have been reported to inhibit 3clpro, plpro, spike proteins, and viral replication by binding in different domains (islam et al., 2020) . this binding hinders the substrate from going to the enzyme's active sites, prevents dimer formation, or averts viral entry (park et al., 2013; zhang d. h. et al., 2020) . the traditional herbs which contain these potential anti-viral compounds and are regularly used in handling viral respiratory infections might be employed to provide immediate support in the treatment of covid-19. however, clinical manifestations are required for the routine implementation of these phytoconstituents as drugs. additionally, it should also be kept in mind that molecular docking is a crucial process in the identification of potential antiviral compounds and is based on the available genome information of the novel coronavirus. in the case of mutation in the existing sars-cov-2, the suggested compounds may not be effective and new investigations will be required (figure 1) . ul qamar et al. (2020) created a 3d homology model of the 3clpro sequence of sars-cov-2 and highlighted its conserved nature comparable with the main protease sequence of sars-cov which shared a 99.02% sequence similarity. however, 12 point-mutations have been reported in sars-cov-2 which dislocate crucial hydrogen bonds, change the receptor binding site of its main protease, and thus, sars-cov-2 may behave differently towards some phyto-inhibitors that were effective towards sars-cov and need to be tested. a detailed molecular docking-based screening of more than 32,000 phytoconstituents resulted in nine potential compounds (including myricitrin, licoleafol, and amaranthin) that may hinder the activity of the sars-cov-2 3clpro (ul qamar et al., 2020) . another molecular docking-based study using lopinavir and nelfinavir as standards revealed that epicatechingallate, catechin, curcumin, oleuropein, apigenin-7-glucoside, figure 1 | this picture shows the current status of phytoconstituents in the development process of anti-covid-19 drugs. due to the identified mutations in sars-cov-2 as compared to sars-cov, the detection of potent anti-viral compounds is necessary. molecular docking can largely contribute to this process. the phytocompounds (a) that are identified as potential inhibitors of sars-cov-2 should be immediately forwarded to pre-clinical and clinical trials (aanouz et al., 2020; khaerunnisa et al., 2020; ul qamar et al., 2020) . the phytocompounds (b) that were effective against sars-cov and are potent for sars-cov-2 based on in vitro experiments saved the time of target compound selection and extraction assays (nemunaitis et al., 2013; choy et al., 2020) . as their efficacy and safety has already been proven in previous the sars-cov outbreak, these can be directly taken to advanced stages in covid-19 clinical trials. the phytocompounds (c) that are approved for clinical trials or are currently in the process of clinical trials (d) should be taken forward to phase iv as soon as possible so that the wide effect of the developed drugs can be observed before the covid-19 pandemic ends (borba et al., 2020; gautret et al., 2020; qiu et al., 2020) . this is extremely critical for future random viral corona outbreaks. naringenin, demethoxycurcumin, luteolin-7-glucoside, quercetin, and kaempferol compounds extracted from medicinal plants may act as potential inhibitors of the main protease of covid-19 (khaerunnisa et al., 2020) . three phytocompounds, b-eudesmol, digitoxigenin, and ccrocin isolated from lauris nobilis l, nerium oleander, and crocus sativus l, respectively, have been proposed as potential inhibitors of the spike protein of sars-cov-2 based on the molecular docking study (aanouz et al., 2020) (figure 1) . another important aspect while dealing with phyto-inhibitors against covs is that the results obtained from in vitro experiments may be different from the clinical efficacy in in vivo experiments. this may be due to the fact that the oral intake of these phyto-drugs may not reach the expected blood serum concentration as observed in in vitro experiments. emetine, an alkaloid extracted from the root of the plant psychotria ipecacuanha (ipecac root), that was determined to be a broad-spectrum inhibitor regulating different covs in vitro was found to inhibit sars-cov-2 replication at 0.5 mm. however, as a therapeutic its plasma concentration stretches up to 0.156 mm that is much lower than its toxic plasma concentration of 1.04 mm and its ec50 value towards sars-cov-2 (regenthal et al., 1999; choy et al., 2020) . homoharringtonine, isolated from cephalotoxus fortunei, is a broad spectrum anti-viral drug effective against murine hepatitis and porcine epidemic diarrhea coronaviruses and inhibits sars-cov-2 at ec50 = 2.10 mm. however, a semi-synthetic type of homoharringtonine, omacetaxine, is reported to have a therapeutic plasma concentration of 0.066 mm after 11 days of treatment that was much lower than its ec50 value against sars-cov-2 in vitro (nemunaitis et al., 2013; choy et al., 2020) . these results of emetine and homoharringtonine confirmed that plantbased therapeutics which are potentially effective against sars-cov-2 should be immediately considered for dose optimization. isolated from liquorice roots, diammonium glycyrrhizinate combined with vitamin c tablets had been prescribed for common covid-19 symptoms and had been approved for randomized clinical trials qiu et al., 2020) . these results also give us hope that broad-spectrum phyto-antivirals can be effective for sudden viral outbreaks in the future. different researchers are investigating diverse plant forms based on ethnopharmacological data to find effective anti-cov drugs with novel action mechanisms especially targeting viral replication. in this crucial situation, it is required to discuss why phyto-inhibitors could not reach an effective drug level in previous corona outbreaks so that proper strategies could be developed for future viral epidemics. the development of drugs is a costly and long-term process. as the screening of plant-based anti-virals is very similar to the testing procedure of synthetic drugs, a better correlation of their in vitro and in vivo (ivivc) results may hasten their approval process (babar, 2013; bose et al., 2020) (figure 1) . the clinical trials of plants-based anti-virals pass through five phases including the preclinical phase (unrestricted dose on animals or in vitro experiments), phase i, ii, iii, and iv (figure 1) . phase i includes the testing of the drug and its doses on healthy people, while phase ii and phase iii are comprised of screening in patients to check the efficacy, side-effects, and safety issues associated with the drug. the number of participants increases in each phase with approximately 300-3000 patients in phase iii. phase iv is one of the most crucial steps including post-marketing surveillance to observe the safety and long-term effects of the drug when used in public. several potential plant-based anti-virals against different viral diseases have entered into the market of licensed products as they are effective against particular cellular responses without an added destruction of the cell. echinacea purpurea has reached phase iv of nonrandomized clinical trials in spain to illustrate the interaction between the anti-retroviral drug, darunavir, and echinacea purpurea in hiv-1 infected patients (mólto et al., 2010; kurapati et al., 2016) . triptolide woldifiion in china made it up to phase iii in randomized clinical trials and its impact on the hiv-1 reservoir was estimated (li, 2014) . (+)-calanolide a extracted from calophyllum lanigerum hinders hiv-1 reverse transcriptase and was one of the few initial anti-hiv agents that went into a clinical trial. it successfully passed the phase i clinical trial which was performed on healthy people (creagh et al., 2001) ; however, it was not further evaluated for efficacy and safety (usach et al., 2013) . phyllanthus urinaria and phyllanthus niruri were found to block endogenous dna polymerase enzyme necessary for hepatitis b virus (hbv) replication and made it up to clinical trial (jassim and naji, 2003) . however, a randomized controlled trial on 47 patients suffering from chronic hbv showed that 12 months of the intervention of 250 mg capsule of phyllanthus niruri twice in a day did not reduce the virus load and could not clear the hepatitis b antigens. accordingly, phyllanthus niruri was not recommended as a standard drug for chronic hepatitis b patients as its efficiency was found to be wide-ranging according to the variations in the treated populations (baiguera et al., 2018) . similarly, in the case of covid-19, two forms of cinchona bark-based antimalarials, chloroquine diphosphate and hydroxychloroquine have also been the center of interest due to the reported inhibitory effects of these compounds on sars-cov. positive reports of recovery in sars-cov-2 affected patients after hydroxychloroquine treatment are available where a daily dose of 600mg of hydroxychloroquine along with azithromycin significantly reduced virus load after six days of inclusion (gautret et al., 2020) . however, the main drawback of this study was the small sample size and limited time for a long-term follow-up of the patients. another study on chloroquine diphosphate reported a lethality rate of 15% and 39% in lowdosage (450 mg twice a day on the 1st day and once a day for 4 days) and high-dosage (600 mg twice a day for 10 days) groups in critical sars-cov-2 patients after 13 days of treatment (borba et al., 2020) . thus, a higher dosage of chloroquine diphosphate along with azithromycin in critically ill patients especially suffering from cardio disorders has been reported to be unsafe. thus, evaluation of chloroquine as a drug through randomized clinical trials is required. the chances of the effective use of similar phytocompounds in the covid-19 outbreak would have been higher if experiments focusing on these phyto-inhibitors had been performed for and after sars-cov. one of the possible reasons for the failure of such natural products could be the differences in the prepared drug due to the ecological and seasonal variations in the plant growth, genotypic variation, timing of harvest, variations in the storage, and manufacturing conditions (islam et al., 2020) . changes in these factors may influence the production of the main component and contaminants (shimanovskii, 2020) . studies such as that of borba et al. (2020) and gautret et al. (2020) reported a daily requirement of a minimum 600 mg of chloroquine compounds per sars-cov-2 patient. though plants produce enough of these natural products for their own use, it is not sufficient to fulfill the commercial manufacturing needs of pharmaceutical companies. thus, sustainable and reproducible large-scale production of these natural products is another challenge in their successful utilization for the treatment of sars-cov-2. plant cell and tissue culture approaches can be effective for the extraction and multiplication of many of these natural constituents (hussain et al., 2012; shimanovskii, 2020) . extraction of phytocompounds from the tissue culture is quick and efficient when compared to the isolation from whole plants. moreover, plant tissue culture techniques facilitate the production of phytocompounds in completely controlled conditions following the regulations of good manufacturing practices (gmp). phytocompounds extracted from tissue cultures can be free of microbes and other compounds found in soil-grown plants and are protected from climatic changes (hussain et al., 2012) . other than plant tissue culture methods, vertical farming units (vfus) can be promising for the controlled and monitored exponential production of the target crop that prevents the cross-pollination of the target crop with genetically compatible species (ma et al., 2005; buyel et al., 2017; buyel, 2018) . not only for growing and harvesting the plants, gmps must be followed for the extraction and purification of the pure and homogenous phytocompound from the harvested biomass. this downstream processing of phytocompounds may account for approximately 80% of the total production cost depending on the removal of the contaminants and purity of the extracted compound (ma et al., 2005; fischer et al., 2012) . moreover, the chances of success of natural anti-viral products may largely increase if they are prepared in line with ethnopharmacological guidelines. the proper application of in silico and in vitro methods followed by in vivo experiments may smooth the way for clinical trials of phytocompounds. more than a hundred phytocompounds have been found to inhibit different types of coronaviruses either by inhibiting the interaction of the sars-cov (s) protein and the ace2 receptor or by inhibiting the viral replication, cell division, 3cl protease, papain-like protease (pl pro) or by hindering the viral entry (islam et al., 2020) . it should be noted that for sars-cov that emerged in 2003, it was not until 2017 when in vitro studies confirming the action mechanism of natural products were conducted schwarz et al., 2014; park et al., 2017) ; however, the natural products with the same mechanism against sars-cov and even more effective mechanisms were already identified in the initial years of the sars-cov epidemic. if more efforts had been given to those phyto-resources from the start of the sars-cov epidemic, they could have reached successful clinical trials. moreover, the inability of sars-cov phyto-inhibitors-based research to reach an extensive level of in vivo studies reduced the chances of phyto-anti-cov drugs being developed and passing successfully through clinical trials. the emergency sars-cov-2 outbreak led to the utilization of several phytocompounds for the treatment of patients. the direct benefit of most of these compounds is that their effects on the human body and their safety are already established through clinical trials for other diseases. however, utmost care is required while prescribing the dose of these phytocompounds as their uncontrolled use can have long term side-effects on the patient's body. though numerous phytoconstituents were found to be effective against sars and mers covs, they fell behind in the drug development process as most of them could not reach the clinical trial stage. one of the possible reasons for incomplete clinical trials could be the intermittent nature of the sars and mers epidemics. the absence of patients for the advanced phase trials (phase ii, iii, iv) of phytoconstituents may have contributed to their failure as licensed drugs. thus, one of the potential suggestions for covid-19 recovery could be to identify the potential phytoconstituents based on in vitro results and with minimum side effects in phase i trials and take them up to advanced phase trials (phase ii, iii, iv) as soon as possible (figure 1 ). so that, in case the covid-19 pandemic ends abruptly by chance like sars-cov, then we can have at least a few efficient phyto-anti-covid drugs that would have completed randomized clinical trials. such drugs may not only be effective on re-emergence of sars-cov-2 in the coming years but can also be potent against similar viral respiratory outbreaks. moreover, creating an effective phyto-anti-covid drug during this pandemic may provide an idea on the duration and the strategy required for the development of potent plant-based therapeutics in case of such random viral outbreaks (figure 1) . we as plant biologists need to be more concerned and vigilant about the status of the plants, their extracts, and phytoconstituents in developing phyto-anti-viral drugs and controlling pandemics like covid-19. it is as crucial as the production of important cereals in the world. ap and mkk conceived, wrote, and edited the manuscript. mh and sg made intellectual contributions to the manuscript. all authors contributed to the article and approved the submitted version. moroccan medicinal plants as inhibitors of covid-19: computational investigations antiviral activity of resveratrol against human and animal viruses antiviral properties of extract of opuntia streptacantha phyllanthus niruri versus placebo for chronic hepatitis b virus infection: a randomized controlled trial investigation of stilbenoids as potential therapeutic agents for rotavirus gastroenteritis clinical features and short-term outcomes of 144 patients with sars in the greater 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molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 attenuating effect of ginsenoside rb1 on lps-induced lung injury in rats isolation of a novel coronavirus from a man with pneumonia in saudi arabia in silico screening of chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus crystal structure of sars-cov-2 main protease provides a basis for design of improved a-ketoamide inhibitors epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china a pneumonia outbreak associated with a new coronavirus of probable bat origin key: cord-296250-7ln7p715 authors: wang, sheng-fan; chen, kuan-hsuan; wang, szu-yu; yarmishyn, aliaksandr a.; lai, wei-yi; lin, yi-ying; wang, mong-lien; chou, shih-jie; yang, yi-ping; chang, yuh-lih title: the pharmacological development of direct acting agents for emerging needed therapy against severe acute respiratory syndrome coronavirus-2 date: 2020-05-20 journal: j chin med assoc doi: 10.1097/jcma.0000000000000353 sha: doc_id: 296250 cord_uid: 7ln7p715 recently, the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) was quickly identified as the causal pathogen leading to the outbreak of sars-like illness all over the world. as the sars-cov-2 infection pandemic proceeds, many efforts are being dedicated to the development of diverse treatment strategies. increasing evidence showed potential therapeutic agents directly acting against sars-cov-2 virus, such as interferon, rna-dependent rna polymerase inhibitors, protease inhibitors, viral entry blockers, neuraminidase inhibitor, vaccine, antibody agent targeting the sars-cov-2 rna genome, natural killer cells, and nucleocytoplasmic trafficking inhibitor. to date, several direct anti-sars-cov-2 agents have demonstrated promising in vitro and clinical efficacy. this article reviews the current and future development of direct acting agents against sars-cov-2. the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) was quickly confirmed as the causal pathogen leading to the outbreak of sars-like illness (later named coronavirus disease 2019 ) in china. 1 as for now, covid-19 has become a pandemic that led to healthcare crisis all over the world. sars-cov-2 belongs to betacoronavirus genus of coronaviruses (covs) that are known to cause multiple respiratory infections in humans. the history of covs can trace back to the middle of nineteenth century. 2 the cov particles are enveloped and contain a single-stranded positive-sense rna genome. the cov genome encodes both structural (such as envelope [e] protein, transmembrane [m] glycoprotein, spike [s] glycoprotein and nucleocapsid [n] protein) proteins responsible for virus replication and virus entry and nonstructural proteins that are involved in genome replication and transcription. the viral membrane harbors the s protein that plays an important role in virus entry and is responsible for inducing host immune response. 3 the cov infection begins with the interaction of the receptor binding domain (rbd) of the viral s protein and the receptor on the cell surface such as dipeptidyl peptidase-4 for mers-cov and angiotensin-converting enzyme 2 (ace2) for sars-cov. 4 according to the recent results, sars-cov-2 utilizes a similar host receptor, ace2 for its attachment and entry. therefore, the treatment strategy designed for sars-cov can be potentially applied to use against sars-cov-2. 5 after the entry, the virus gets uncoated and translates its open reading frames 1a and 1b (orf1 and orf1b) into polyproteins pp1a and pp1ab. subsequently, these polyproteins are cleaved into several nonstructural proteins by several proteases, which further assemble and form the transcription-replication complex. the rna-dependent rna polymerase (rdrp) transcribes the positive strand of rna to negative strand, which is further transcribed into positive-strand subgenomic mrnas. these subgenomic mrnas are translated to make new accessory and structural proteins. interfering with any specific step of the virus replication cycle would be a potential therapeutic target to combat sars-cov-2. increasing evidence reveals potential therapeutic agents acting directly against sars-cov-2, such as interferon (ifn), rdrp inhibitors, protease inhibitors, coronaviral protease inhibitor, viral entry blocker, neuraminidase inhibitor, vaccine, antibody, agent targeting the sars-cov-2 rna genome, natural killer cells, and nucleocytoplasmic trafficking inhibitors. some of them have already demonstrated both in vitro and clinical efficacy. the drug discovery process to develop new antiviral agents and obtaining the clinical approval usually takes a long period of time. until now, no significantly effective antiviral drugs are clinically approved for treating coronavirus infections. to enhance the progress of potential treatment for coronaviruses especially for sars-cov and mers-cov, repurposing of broadly acting antiviral drugs such as ifns and ribavirin that have been used for other viral infections or other indications is usually employed. these drugs have the significant superiority for their well-known characteristics of pharmacokinetic and pharmacodynamics. another approach for discovery of anti-cov drugs includes the de novo development of ideal, novel agents according to the biochemical understanding of the specific coronavirus. the novel specific anti-sars-cov-2 agents might comprise inhibitors interfering with the viral replication cycle, antibody targeting the host receptor and virus s protein, and inhibitors of host cellular proteases involved in the virus endocytosis pathway. ifns are a group of cytokine mediators that are induced in response to virus infection. ifns are classified into subtypes i and ii, both of which are involved in innate immunity and adaptive immune response. several effector antiviral mechanisms of ifn are known, such as mrna translation inhibition, enhancement of rna degradation, rna editing, targeting viral nucleocapsids, and inhibiting rna synthesis. 6 among the type i ifn, ifnα is quickly stimulated in innate immune response to initial virus infection. ifn-α and β inhibit the replication of sars-cov. 7, 8 however, ifn-γ was identified to lack antiviral activity against sars-cov. 9 previous study demonstrated that sars-cov inhibited ifn transcription in infected cells and the additional ifn could partly resume innate immunity against sars-cov. 10 pegylated ifn-α2b, a licensed drug for chronic hepatitis b and c, showed the anti-sars-cov activity by decreasing viral replication and lung damage. 11,12 ifn-α is a candidate medication for sars and mers treatment. 13, 14 therefore, the current clinical evidence showed that ifns might be used for the development of novel anti-sars-cov-2 therapy. ribavirin is a broad-spectrum antiviral agent that has previously been used for hepatitis c patients treatment. it is a guanosine derivative and can target rdrp enzyme to inhibit the synthesis of viral rna and capping of mrna. during the previous sars epidemic, ribavirin was widely used for patients in china. 15 however, evidence showed ribavirin might have no significant antiviral activity against sars-cov in vitro and had significant toxicity concern in clinical practice. 16, 17 hence, ribavirin usage for sars-cov is questionable and has been criticized. 18 earlier evidence demonstrated that the combination of ribavirin and ifn-β had a synergistic effect for inhibiting the replication of sars coronavirus in animal and human cell lines. 7 regarding the adverse reactions and absence of the in vitro efficacy for sars-cov, the usage of ribavirin against sars-cov-2 should be carefully considered. remdesivir, by far, is the most promising drug among all ongoing clinical trials. due to its structural similarity to adenosine, it can inhibit the rdrp by incorporating into viral rna, thus halting viral genome replication. recently, the first experience of remdesivir usage in the united states for the covid-19-infected patient was reported. 19 the in vitro/in vivo antiviral activity of remdesivir and ifn-β was found to be superior to the combination of lopinavir/ritonavir/lpv/rtv/ifn-β for mers-cov. 20 the most updated cohort study showed clinical improvement observed in 36 of 53 patients (68%). 21 therefore, remdesivir alone or together with ifn-β might be the potential regimen for the therapy of covid-19. favipiravir (fpv) is a guanine analog, also selectively inhibiting rdrp (like remdesivir) that has been approved for the treatment of influenza. 22 the clinical evaluation for its efficacy and safety in the therapy of covid-19 is ongoing (chictr2000029600). a co-formulation of lopinavir and ritonavir, which was reported to have in vitro activity against the sars-cov and was revealed to have some activity against mers-cov in vivo, has been tested for the therapy of covid-19. [23] [24] [25] the component of lopinavir can bind to hiv-1 protease and block the cleavage of viral gag-pol precursor polyprotein into specific proteins essential for hiv infection, resulting in dysfunctional viral particles. another component, ritonavir, can increase the plasma level of lopinavir by inhibiting the cyp3a-mediated metabolism of the latter. it has been used for the treatment of covid-19 patients. [26] [27] [28] however, lopinavir-ritonavir treatment might not have benefit in the severe covid-19 hospitalized patients. 29 nelfinavir, another hiv protease inhibitor, has previously been shown to inhibit the replication of sars-cov. 30 in addition, nelfinavir has revealed rational binding conformation with the viral main protease of sars-cov-2. (biorxiv doi:10.1101/2020.01.28.922922.) therefore, nelfinavir might be a potential therapeutic option for covid-19. coronaviruses encode two proteases, chymotrypsin-like and papain-like proteases, which are responsible for the viral replication and interfering with the host innate immunity. hence, targeting specific coronaviral proteases is a potential treatment option against coronaviruses. 31 cinanserin, an old serotonin receptor antagonist, inhibits the chymotrypsin-like protease and might be a potential inhibitor of replication of sars-cov. 32 in addition, some flavonoids, which have inhibitory activity on chymotrypsin-like protease might be used against sars-cov. 33, 34 moreover, evidence showed that diarylheptanoids, the natural products derived from japanese alder (alnus japonica), can inhibit the papain-like protease and restore the host innate immunity response against sars-cov through maintaining the function of ifns. 31 therefore, specific coronaviral proteases might be good candidate targets for developing new drugs to fight covid-19. hydroxychloroquine and chloroquine are aminoquinolines, which have been used to treat autoimmune diseases and malaria. they are weak diprotic bases that can elevate the ph of the endosomes, which in turn prevent viral entry fusion. 35 chloroquine can also interfere with ace2 glycosylation of the cellular receptor of sars-cov and sars-cov-2. in vitro tests revealed its capability to reduce viral copy number. its in vivo antiviral capability is now under clinical trial (open-label trial chictr2000029609). the preliminary study in france evaluated the efficacy of hydroxychloroquine in sars-cov-2 patients. the results showed that the virologic cure rate was significantly higher in hydroxychloroquine/azithromycin-treated group (p = 0.001). 36 however, the comprehensive evaluation of safety and clinical efficacy in chloroquine/hydroxychloroquine therapy for covid-19 patients is still under investigation. hence, the benefits and risk of chloroquine or hydroxychloroquine therapy for covid-19 remains to be carefully elucidated. in a recent study, it was shown that sars-cov-2 recognizes ace2 more efficiently than sars-cov. 5 therefore, interfering with the interaction between s protein of sars-cov-2 and ace2 might be a potential approach for antiviral therapy. recently, the covid-19 high-performance computing (hpc) consortium has been established for the development of anti-sars-cov-2 therapy and prediction of the covid-19 pandemic. using the world's most powerful supercomputer, summit, the high-throughput screening of the possible docking models and identification of novel small molecules which can bind to the viral s protein or s protein-human ace2 interface has been proposed. at present, the initial 77 novel compounds were identified for the subsequent clinical efficacy tests against sars-cov-2 (chemrxiv. preprint. https://doi.org/10.26434/ chemrxiv.11871402.v4). in addition to the ace2, it has been revealed that s proteinderived cell entry also depends on transmembrane protease serine 2 (tmprss2). 37 hence, camostat mesylate, a tmprss2 inhibitor, can eliminate sars-cov-2 infection in vitro assay. on the other hand, the heptad repeat (hr) of sars-cov-2 s protein is also involved in the process of viral entry. 38 moreover, anti-hr peptides possess anti-sars-cov-2 activity through inhibition of viral fusion. 38 the ph-and receptordependent endocytosis are also involved in coronavirus entry mechanism. 39, 40 adaptor protein complex 2-associated protein kinase 1 is a host kinase responsible for regulation of clathrinmediated endocytosis. 41 recent evidence suggested that baricitinib, a janus kinase inhibitor and also an adaptor protein complex 2-associated protein kinase 1-binding drug identified by artificial intelligence technology research, might be a novel candidate drug for the future covid-19 treatment. 42 targeting novel coronavirus entry mechanisms might provide an alternative potential strategy for future anti-sar-cov-2 drug development. oseltamivir, an antiviral medication used for influenza a/b through neuraminidase inhibition, has gained considerable attention and has been proposed for evaluation of its efficacy for covid-19 treatment. there is no evidence that oseltamivir has significant anti-sars-cov activity in vitro. 16 the efficacy of oseltamivir against coronavirus is still unknown in the clinic. 43 coronaviruses do not utilize neuraminidase and thus there is no specific enzyme to be targeted by oseltamivir or any other neuraminidase inhibitor. hence, the in vitro data have suggested that neuraminidase inhibitors such as baloxavir would exert activity against sars cov-2 or other coronaviruses should be carefully interpreted. 44 amantadine, indicated for influenza and parkinson disease, has showed the antiviral activity against human coronavirus oc43 and sars-cov. 9,45 sars-cov-2 entry into host cells might be dependent on interacting with the s protein and host cellular receptor. subsequently, cathepsin l and cathepsin b (ctsl/ctsb), the host cell proteases responsible for cleavage of the spike protein, play an important role in the deenveloping process. it provides potential target for covid-19 therapy development. recently, amantadine was screened for the possibility to downregulate the expression of ctsl/ ctsb through high-throughput drug screen gene expression analysis (doi: https://doi.org/10.1101/2020.04.05.026187). however, other evidence indicated its poor activity against sars-cov. 16 at present, the amantadine and its analog are not recommended for influenza due to influenza a resistance and side effect on lactation due to decrease of serum prolactin. 46 although amantadine and its analog are not commonly discussed, this aspect whatever used for adjuvant or single treatment might be worthy to reassess in the future development for anti-sars-cov-2 therapy. effective vaccines are important to prevent and control viral pandemics such as influenza vaccines. unfortunately, even after numerous studies on coronaviruses, the licensed vaccines are still lacking. theoretically, the s protein of sars-cov-2 is a promising target for vaccine development. recently, wrapp et al. 47 found that the cryogenic electron microscopy structure of sars-cov-2 s trimer provides additional protein engineering information and thus can speed the progress of vaccine development. in addition, discovering the unique pentapeptides of sars-cov-2 s protein provides several candidate epitopes for vaccine development. 48 due to high similarity between sars-cov-2 and sars-cov, a researcher identified the sars-cov-derived b-and t-cell epitopes were identically mapped to sars-cov-2 proteins which might be helpful for the initial phase of vaccine development. 49 inactive vaccines derived from sars, might be tested for sars-cov-2. besides, rhesus θ-defensin and protein cage nanoparticles, which are innate immunomodulators, exhibit high anti-sars-cov potency. 50, 51 protein cage nanoparticles designed vaccine for sars-cov might be further evaluated for their antiviral activity against sars-cov-2. to date, despite the fact that the sequence information of sars-cov-2 has been revealed, moderna's mrna-1273 is the pioneer vaccine candidate on the market that has stepped its foot into clinical trials. 52 if the synthetic strand of mrna could prove its safety in humans and pass through the phase i trial, evaluation of its clinical benefits would be subsequently executed. it is highly expected for its antiviral response against sars-cov-2 through targeting s protein (nct04283461). numerous potential sars-cov-2 vaccines are being developed, including viral-like particles, nucleic acid-based vaccine, adenoviral vector-based vaccine, recombinant subunits vaccine, recombinant influenza viral vector vaccine, inactivated vaccine, and so on. 53 upon current development progress, further work is needed to develop safe and effective vaccines for the control of the sars-cov-2 ongoing epidemics. passive immunity by antibody therapy might be another strategy to control the sars-cov-2 pandemic. passive immunization with the antibody, which can neutralize sars-cov-2 virus, might inhibit the virus replication and mitigate the disease severity. according to the prior experience in treating mers, sars, ebola, and influenza viral infections with convalescent plasma from recovered patients that contains high antibody titers against virus, convalescent plasma could ameliorate the viral level and mortality. [54] [55] [56] [57] however, the challenges such as unknown viral kinetics of sars-cov-2 and insufficient donors with suitable clinical status still exist. the usage of monoclonal antibodies for infectious disease is a new area, which addresses many concerns related to serum therapy and intravenous immunoglobulin medication in terms of better specificity, purity, safety, and reducing risk of contamination. like the sars-cov, sars-cov-2 also targetd ace2 for its attachment and entry into host cells. hence, several neutralizing monoclonal antibodies targeting sars-cov by recognized s1 and s2 subunits, and amino acid residues might be applied to sars-cov-2. 58 a recent study showed that cr3022, a neutralizing antibody from a recovered sars patient, might have the cross-reactive activity between sars-cov-2 and sars-cov through structural modeling with highly conserved distal epitope from the rbd. 59 nevertheless, evidence showed that even sars-cov-2 spike protein displayed high (about 75%) homology with sars-cov, the novel epitopes only contributed to about 85% of the rbd antibody epitopes, implied concerned alterations in their antigenicity. 60 61 on the other hand, the rationale of ace2 receptor as a specific target against sars-cov-2 has been proposed. 62 it is reasonable to use the ace2 receptor as a target for neutralizing the virus. currently, recombinant human ace2 (rhace2; gsk2586881) is being investigated for evaluation of its clinical effects in covid-19 patients (nct04287686). moreover, in order to target ace2 against sars-cov-2, it would be advisable to convert the soluble ace2 receptor to the immunoadhesin form, which can be achieved by binding with the immunoglobulin fc domain (ace2-fc). this modification will extend its lifespan and recruit immune response against the virus in the future antibody development. nevertheless, the sars-cov-2 s protein-derived cell entry not only depends on ace2 but also on the host cellular serine protease tmprss2. 37 moreover, the hr loops including hr1 and hr2 domains on the s2 fragment of sars-cov-2 are involved in viral entry mechanism. 38 furthermore, ultimately, the cocktail antibodies strategy might be required to pursue full sars-cov-2 protection coverage for specific population, which would elevate the complexity for formulation and manufacturing. hence, antibodies targeting the fusion/entry mechanism of sars-cov-2 are still ongoing investigated and be worthy to expected in the future. beyond targeting the s protein of sars-cov-2, directly targeting the viral rna genome through inducing its degradation might be a reasonable strategy. sars-cov-2 rna genome sequence has been recently identified (gen-bank: mn908947.3), and using rna interference by small interfering rna (sirna) or antisense oligonucleotides against the virus might be another potential treatment strategy. 63 however, several challenges still exist, first, the conserved rna sequence domains of sars-cov-2 are still unclear; second, it is unclear whether the oligonucleotides can be efficiently delivered into the lungs. by using such tools as liposomes, it is possible to deliver oligonucleotides to some extent; however, it is still unclear whether sufficient amount of oligonucleotides could be effectively delivered into the lung lesions and could eliminate the virus in clinical settings. 64 therefore, there still exists an uncrossed gap for immediate and quick development of anti-sars-cov-2 therapy using oligonucleotides. targeting the rna genome of sars-cov-2 by a novel crispr rna knockdown system might be another option. recent evidence revealed that the crispr/cas13 rna knockdown system might be used against sars-cov-2 rna genome. 65 the crispr/cas13d system encompasses cas13d protein and guides rna specifically targeting the sars-cov-2 rna genome. it is believed that the crispr/cas13 system might be delivered to the sars-cov-2-infected lung tissue by adeno-associated virus vector. therefore, the crispr system against the sars-cov-2 might be a potential developmental strategy in the future. until now, no well-established evidence has been revealed for the details of the immunological response in sars-cov-2 infected patients. however, we can initially glean some information from previous cov studies such as sars-cov and mers-cov. after virus entering the host cell, it activates several receptors such as rig-i-like receptor, nod-like receptor, toll-like receptor, and c-type lectin-like receptors. subsequently, it stimulates the expression of several inflammatory factors, dendritic cell maturation, and synthesis of type i ifns which can control the viral spread and eliminate virus via phagocytosis by macrophages. 66 learning to recognize the virus and raising a defense immune system may take several days to weeks. during this period, natural killer (nk) cells might initially be an important gate keeper for exogenous virus. recent evidence suggested that nk cells might be helpful against particular viruses in vivo, but there were not any clearly successful clinical results against virus infection. 67 hence, celularity, a new jersey-based therapeutics company declared that, "cynk-001, a cancer treatment is under clinical trial against covid-19 awaiting for fda permission." however, several challenges exist such as novel viral evading mechanisms of immune system including n protein of sars-cov. 68 another important challenge is cytokine storm, a characteristic developed by some covid-19 patients. therefore, the clinical testing should proceed with extreme caution and carefully monitor the clinical status to see whether it over-activates immune system and further harms healthy lung cells. ivermectin, an approved antiparasitic medication have been recognized as an antiviral agent against several viruses such as influenza, hiv, dengue virus, west nile virus, and venezuelan equine encephalitis virus in vitro. [69] [70] [71] [72] initially, the inhibitory activity of ivermectin on interaction of virus integrase protein (in) and the importin (imp) α/β1 heterodimer was proposed as the antiviral mechanism because various rna viruses are dependent on impα/β1 for infection. [73] [74] [75] previous studies demonstrated that impα/β1 was involved in a signaldependent sars-cov nucleocapsid protein nucleocytoplasmic trafficking during sars-cov infection and could affect host cell division. [76] [77] [78] [79] [80] besides, the sars-cov accessory protein can evade innate immune antiviral response through sequestering impα/β1 on the rough er/golgi membrane. 81 the evidence suggests that ivermectin might be used against sars-cov-2 through nuclear transport inhibitory activity. recently, an in vitro experiment showed that a single treatment with ivermectin resulted in about 5000-fold reduction in sars-cov-2 virus. 82 the next step of drug development is to clarify the dosing regimen. based on its in vitro efficacy and a well-known safety information, ivermectin might be worthy to further evaluate as a potential anti-sars-cov-2 drug. in conclusion, several direct anti-sars-cov-2 agents have demonstrated some promising in vitro and clinical efficacy (fig. 1) . however, most of them are case reports or preliminary data from clinical trials with small sample sizes; therefore, further clinical trials are required. according to the current knowledge about the pandemic of sars-cov-2, the pharmacological development of direct-acting agents against covid-19 is an urgent issue and a critical challenge for all of the pharmaceutical scientists and healthcare professionals. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study recent discovery and development of inhibitors targeting coronaviruses mers-cov spike protein: a key target for antivirals dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus antiviral actions of interferons ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines prevention of experimental coronavirus colds with intranasal alpha-2b interferon in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds interferon priming enables cells to partially overturn the sars coronavirus-induced block in innate immune activation pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques proposal for vaccination against sars coronavirus using avian infectious bronchitis virus strain h from the netherlands interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review managing sars amidst uncertainty inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs clinical features and short-term outcomes of 144 patients with sars in the greater toronto area critics slam treatment for sars as ineffective and perhaps dangerous washington state 2019-ncov case investigation team. first case of 2019 novel coronavirus in the united states comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov compassionate use of remdesivir for patients with severe covid-19 mechanism of action of t-705 against influenza virus treatment and vaccines for severe acute respiratory syndrome treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset coronavirus 2019-ncov: a brief perspective from the front line the author's response: case of the index patient who caused tertiary transmission of coronavirus disease 2019 in korea: the application of lopinavir/ritonavir for the treatment of covid-19 pneumonia monitored by quantitative rt-pcr epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment a trial of lopinavirritonavir in adults hospitalized with severe covid-19 hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus diarylheptanoids from alnus japonica inhibit papain-like protease of severe acute respiratory syndrome coronavirus cinanserin is an inhibitor of the 3c-like proteinase of severe acute respiratory review article. 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convalescent plasma study group. the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease-19 (covid-19) a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody angiotensinconverting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target a review on current status of antiviral sirna aerosol delivery of sirna to the lungs. part 1: rationale for gene delivery systems virus against virus: a potential treatment for 2019-ncov (sars-cov-2) and other rna viruses modulation of murine dendritic cell function by adenine nucleotides and adenosine: involvement of the a(2b) receptor continuous engagement of a self-specific activation receptor induces nk cell tolerance sars-cov nucleocapsid protein antagonizes ifn-β response by targeting initial step of ifn-β induction pathway, and its c-terminal region is critical for the antagonism influenza a viruses escape from mxa restriction at the expense of efficient nuclear vrnp import nuclear import and export inhibitors alter capsid protein distribution in mammalian cells and reduce venezuelan equine encephalitis virus replication nuclear localization of dengue virus (denv) 1-4 non-structural protein 5; protection against all 4 denv serotypes by the inhibitor ivermectin ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of hiv-1 and dengue virus an alphascreen®-based assay for high-throughput screening for specific inhibitors of nuclear import nuclear trafficking of proteins from rna viruses: potential target for antivirals? inhibitors of nuclear transport intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus nucleocytoplasmic transport of nucleocapsid proteins of enveloped rna viruses localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane the fdaapproved drug ivermectin inhibits the replication of sars-cov-2 in vitro key: cord-286298-pn9nwl64 authors: helmy, yosra a.; fawzy, mohamed; elaswad, ahmed; sobieh, ahmed; kenney, scott p.; shehata, awad a. title: the covid-19 pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control date: 2020-04-24 journal: j clin med doi: 10.3390/jcm9041225 sha: doc_id: 286298 cord_uid: pn9nwl64 a pneumonia outbreak with unknown etiology was reported in wuhan, hubei province, china, in december 2019, associated with the huanan seafood wholesale market. the causative agent of the outbreak was identified by the who as the severe acute respiratory syndrome coronavirus-2 (sars-cov-2), producing the disease named coronavirus disease-2019 (covid-19). the virus is closely related (96.3%) to bat coronavirus ratg13, based on phylogenetic analysis. human-to-human transmission has been confirmed even from asymptomatic carriers. the virus has spread to at least 200 countries, and more than 1,700,000 confirmed cases and 111,600 deaths have been recorded, with massive global increases in the number of cases daily. therefore, the who has declared covid-19 a pandemic. the disease is characterized by fever, dry cough, and chest pain with pneumonia in severe cases. in the beginning, the world public health authorities tried to eradicate the disease in china through quarantine but are now transitioning to prevention strategies worldwide to delay its spread. to date, there are no available vaccines or specific therapeutic drugs to treat the virus. there are many knowledge gaps about the newly emerged sars-cov-2, leading to misinformation. therefore, in this review, we provide recent information about the covid-19 pandemic. this review also provides insights for the control of pathogenic infections in humans such as sars-cov-2 infection and future spillovers. coronaviruses are enveloped, single-strand rna viruses that can infect a wide range of hosts including avian, wild, domestic mammalian species, and humans. coronaviruses are well known for their ability to mutate rapidly, alter tissue tropism, cross the species barrier, and adapt to different coronaviruses are enveloped, icosahedral symmetric particles, approximately 80-220 nm in diameter containing a non-segmented, single-strand, positive-sense rna genome of about 26-32 kb in size [34] . coronaviruses (covs) are one of the largest groups of viruses that belong to the order nidovirales, suborder cornidovirineae, and family coronaviridae. coronaviridae is classified into two subfamilies, namely, letovirinae and orthocoronavirinae. letovirinae includes the alphaletovirus genus, while orthocoronaviridae is further classified on the basis of phylogenetic analysis and genome structure into four genera: alphacoronavirus (αcov), betacoronavirus (βcov), gammacoronavirus (γcov), and deltacoronavirus (δcov), which contain 17, 12, 2, and 7 unique species, respectively (ictv 2018). the most recent classification of the coronaviridae is shown in table 1 . corona in latin means crown, and this name was attributed to the virus due to the presence of spike projections from the virus envelope that give it the shape of a crown under the electron microscope; nido means nest and refers to the ability of the viruses of this order to make a nested set of subgenomic mrna [25, 35] . coronaviruses infect a wide range of wild and domestic animals; αand βcovs infect mammals, while γand δcovs primarily infect birds (table 1) . a human coronavirus (hcov) was first isolated in 1960 from hospitalized patients who suffered from common cold symptoms and was named b814 [36] . so far, the seven different hcovs that infect humans are 229e, nl63, which belong to α covs, and hku1, oc43, sars, mers, sars-cov-2, which belong to βcovs. in 2002-2003, a pandemic caused by sars-cov (lineage b βcov) originated in china [37] . in the middle east, mers-cov (lineage c βcov) emerged in 2012 [27] . in 2019, a newly emerged sars-cov-2, closely related to bat sars-related covs, was clustered with lineage b βcov. chan et al. [13] demonstrated that sars-cov-2 represents a distinct lineage in the subgenus sarbecovirus (previously, lineage 2b of βcov) [38] . additionally, other coronaviruses have caused pandemic diseases in domestic and wild mammals and birds, leading to high mortality rates and severe economic losses. these viruses include ibv in chickens [40] , beluga whale coronavirus sw1 (bwcov-sw1) [41] , bat coronaviruses cdphe15 and hku10 (ictv 2018), porcine epidemic diarrhea virus (pedv), tgev, and sudden acute diarrhea syndrome (sads-cov) [42] . since the emergence of sars-cov-2 in wuhan city, china, in december 2019, many laboratories have been working on sequencing the genome of the causative agent. as of 14 april 2020, there are a total of 7655 complete genomes from 67 countries in the global initiative on sharing all influenza data (gisaid) database [43] (table 2) . a reference genome is now available in the ncbi genome database (29,903 nucleotide, reference sequence: nc_045512.3) [44] . to date, there are a total of 875 sequences including one refseq sequence and 768 complete genomes at ncbi. sars-cov-2 is a monopartite, single-stranded, and positive-sense rna virus with a genome size of 29,903 nucleotides, making it the second-largest known rna genome. the virus genome consists of two untranslated regions (utrs) at the 5 and 3 ends and 11 open reading frames (orfs) that encode 27 proteins (table 3) . the first orf (orf1/ab) constitutes about two-thirds of the virus genome, encoding 16 non-structural proteins (nsps), while the remaining third of the genome encodes 4 structural proteins and at least 6 accessory proteins. the structural proteins are spike glycoprotein (s), matrix protein (m), envelope protein (e), and nucleocapsid protein (n), while the accessory proteins are orf3a, orf6, orf7a, orf7b, orf8, and orf10, as shown in figure 1 [2, 13, 38, 45] . the 5′utr and 3′utr of sars cov-2 are comprised of 265 and 229 nucleotides, respectively. orf1ab is 21,290 nucleotides and encodes either replicase proteins pp1a of 4405 amino acids (aa) (nsp1-nsp11) or pp1ab of 7096 aa (nsp1-nsp16), according to ribosomal frameshift. of these proteins, (1) nsp1 suppresses the antiviral host response, (2) nsp3 is a papain-like protease, (3) nsp5 is a 3clpro (3c-like protease domain), (4) nsp7 makes a complex with nsp8 to form a primase, (5) nsp9 is responsible for rna/dna binding activity, (6) nsp12 is an rna-dependent rna polymerase (rdrp), (7) nsp13 is confirmed as a helicase, (8) nsp14 is a 3′-5′ exonuclease (exon), 9) nsp15 is a poly(u)-specific endoribonuclease (xendou). the remaining nsps are involved in transcription and replication of the viral genome [13, 46, 47] . single-stranded rna viruses exhibit a faster biological mutation rate due to the lack of proofreading activity of viral rna polymerases [48] ; however, unlike other mutation-prone rna viruses, with the exception of the arenaviridae family, covs do have limited proofreading capabilities, with the nsp14 protein allowing for the enhanced genome size of cov family members [49] . recombination is another mechanism of evolution in coronaviruses [50] . a high recombination frequency was demonstrated in murine hepatitis virus during mixed infection, where the majority of viruses recovered after three passages were recombinants [51] . recombination was also reported for mers-cov and sars-cov. seven putative recombination regions were detected in orf1ab and s protein between sars-cov and six other coronaviruses by in silico analysis of their genomes [52] . similarly, bioinformatic analysis of mers-cov genomic data revealed 28 recombinant sequences from humans and camels [53] . recombination in sars-cov-2 is not yet clearly understood. initial studies suggested that it may have occurred in the course of sars-cov-2 evolution [9] , while other researchers excluded the possibility of recombination based on a full genome evolutionary analysis investigating putative recombination events [11] . to better understand the evolution of sars-cov-2, we performed a phylogenetic analysis of 45 representative coronaviruses from 18 countries including sars-cov, sars-cov-2, hcov, bat sars . the other third of sars cov-2 includes four genes (in green) that encode four structural proteins (s, m, e, n), and six accessory genes (in blue) that encode six accessory proteins (orf3a, orf6, orf7a, orf7b, orf8, and orf10). the 5 utr and 3 utr of sars cov-2 are comprised of 265 and 229 nucleotides, respectively. orf1ab is 21,290 nucleotides and encodes either replicase proteins pp1a of 4405 amino acids (aa) (nsp1-nsp11) or pp1ab of 7096 aa (nsp1-nsp16), according to ribosomal frameshift. of these proteins, (1) nsp1 suppresses the antiviral host response, (2) nsp3 is a papain-like protease, (3) nsp5 is a 3clpro (3c-like protease domain), (4) nsp7 makes a complex with nsp8 to form a primase, (5) nsp9 is responsible for rna/dna binding activity, (6) nsp12 is an rna-dependent rna polymerase (rdrp), (7) nsp13 is confirmed as a helicase, (8) nsp14 is a 3 -5 exonuclease (exon), 9) nsp15 is a poly(u)-specific endoribonuclease (xendou). the remaining nsps are involved in transcription and replication of the viral genome [13, 46, 47] . single-stranded rna viruses exhibit a faster biological mutation rate due to the lack of proofreading activity of viral rna polymerases [48] ; however, unlike other mutation-prone rna viruses, with the exception of the arenaviridae family, covs do have limited proofreading capabilities, with the nsp14 protein allowing for the enhanced genome size of cov family members [49] . recombination is another mechanism of evolution in coronaviruses [50] . a high recombination frequency was demonstrated in murine hepatitis virus during mixed infection, where the majority of viruses recovered after three passages were recombinants [51] . recombination was also reported for mers-cov and sars-cov. seven putative recombination regions were detected in orf1ab and s protein between sars-cov and six other coronaviruses by in silico analysis of their genomes [52] . similarly, bioinformatic analysis of mers-cov genomic data revealed 28 recombinant sequences from humans and camels [53] . recombination in sars-cov-2 is not yet clearly understood. initial studies suggested that it may have occurred in the course of sars-cov-2 evolution [9] , while other researchers excluded the possibility of recombination based on a full genome evolutionary analysis investigating putative recombination events [11] . to better understand the evolution of sars-cov-2, we performed a phylogenetic analysis of 45 representative coronaviruses from 18 countries including sars-cov, sars-cov-2, hcov, bat sars cov, bat sars-like cov, and mers-cov. the viral genomes were obtained from the gisaid and ncbi databases. multiple sequence alignment was performed using kalign 3 [54] . a phylogenetic tree was constructed based on whole-genome sequences (coding sequences of all genes) in iq-tree, using the maximum likelihood method, ultrafast bootstrap approximation, and modelfinder [55, 56] . the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. the bootstrap values were determined by 1,000 replicates. the tree was visualized in mega x [57] ( figure 2 ). cov, bat sars-like cov, and mers-cov. the viral genomes were obtained from the gisaid and ncbi databases. multiple sequence alignment was performed using kalign 3 [54] . a phylogenetic tree was constructed based on whole-genome sequences (coding sequences of all genes) in iq-tree, using the maximum likelihood method, ultrafast bootstrap approximation, and modelfinder [55, 56] . the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. the bootstrap values were determined by 1,000 replicates. the tree was visualized in mega x [57] ( figure 2 ). in the analysis we performed, all sars-cov-2 samples from the 18 countries clustered together and were close to bat sars or sars-like coronaviruses, with wuhan bat cov ratg13 being the closest virus. in addition, mers-cov and human cov hku1 were very distant from sars-cov-2 ( figure 2 ). within mers-cov samples, the south china mers-nl13892 clustered separately from other mers-covs. the two bat sars-like covs (bat-sl-covzc45 and bat-sl-covzxc21) were the second closest viruses from bats to sars-cov-2 ( figure 2 ). all sars-covs from china, canada, england, and the us were in a single cluster. the tree was constructed in iq-tree using the maximum likelihood method, modelfinder, and ultrafast bootstrap approximation (1000 replicates). the tree is drawn to scale, with branch lengths (numbers below the branches) measured in the number of substitutions per site. branch lengths less than 0.3 are not shown. numbers above the branches represent the percentage of replicate trees in which the associated viruses clustered together in the bootstrap test. the tree is rooted with two human coronavirus species from the genus alphacoronavirus as an outgroup (hcov-229e and hcov-nl63). in the analysis we performed, all sars-cov-2 samples from the 18 countries clustered together and were close to bat sars or sars-like coronaviruses, with wuhan bat cov ratg13 being the closest virus. in addition, mers-cov and human cov hku1 were very distant from sars-cov-2 ( figure 2 ). within mers-cov samples, the south china mers-nl13892 clustered separately from other mers-covs. the two bat sars-like covs (bat-sl-covzc45 and bat-sl-covzxc21) were the second closest viruses from bats to sars-cov-2 ( figure 2 ). all sars-covs from china, canada, england, and the us were in a single cluster. zhou et al. [9] conducted a phylogenetic analysis of sars-cov-2 against previously identified coronaviruses based on their whole-genome sequences, main structural protein genes, and non-structural protein genes. sars-cov-2 clustering was different depending on whether the whole genome or specific genes were used in the analysis. for example, sars-cov-2 clustered with the members of the subgenus sarbecovirus including the sars-cov (79.5% identical) that caused the global pandemic in 2003 and other bat sars-like viruses (96% identical at the whole-genome level), but the topological position within the sarbecoviruses changed when individual genes (orf1ab, s, e, m, and n) were used for clustering [2, 9] . based on the whole-genome sequence alignment, sars-cov-2 shares 89% identity with bat sars-like covzxc21, 82% with sars-cov, and 96.3% with bat cov ratg13 [11, 13] . alignment of the predicted protein sequences of sars-cov-2 to those of sars-cov or sars-like coronaviruses revealed a total of 380 amino acid substitutions between these viruses [2] . these amino acid substitutions were distributed as follows: 348 mutations in nonstructural proteins (orf1ab, 3a, 3b, 7a, 7b, 9b, and orf14), 27 in s protein, and 5 in n protein. no amino acid substitutions were detected in e or m proteins, indicating that e and m proteins are highly conserved among these viruses. it has been reported that sars-cov-2 uses the same cellular receptor, hace2, as sars-cov to gain entry into the cell [9, 58, 59] . the analysis of the receptor-binding domains (rbd) of sars-cov and sars-cov-2 s protein revealed similar binding affinities [60] . wu et al. [2] found a total of 27 amino acid substitutions in the s protein but not in the receptor-binding motif (rbm) that directly interacts with hace2, which may affect host tropism. these 27 substituted residues were distributed as follows: 17 in the s1 subunit [6 in the rbd and 6 in the subdomain (sd)] and 10 in the s2 subunit. wan et al. [58] reported similarity in the spike protein rbd, including rbm, of both sars-cov and sars-cov-2, in addition to the presence of several residues in sars-cov-2 rbm that favor the interaction with human ace2. these results agree with the genomic analysis of sars-cov-2, according to which the s2 subunit of the spike protein shares 99% identity with those of two bat sars-like covs (sl-covzxc21 and zc45) and of human sars-cov [13] . while the sars-cov-2 s2 subunit was conserved, the s1 subunit shares an overall 70% identity with those of bat and human sars-cov. the rbd core domain of s1 is highly conserved, with most of the amino acid differences located in the external subdomain that is responsible for the direct interaction with host receptors [13] . investigators have also reported the presence of a polybasic cleavage site and predicted o-linked glycans that are unique to sars-cov-2 s protein. differences in sars-cov-2 s protein and the high contagious nature of this virus suggest that sars-cov-2 has evolved via natural selection for binding to human ace2 receptor [61] . orf3b also differs in sars-cov-2. orf3b deletion mutations in sars-cov do not affect viral replication in vitro [62] . orf3b may play a role in viral pathogenicity in addition to its inhibitory effects on interferon (ifn) expression and signaling [63, 64] . recently, a novel short putative protein was identified in orf3b of sars-cov-2 [13] ; however, the function of this novel protein is still not known. sars-cov-2 orf8 is closer to those of bat sars cov zxc21 and zc45 and distant from that of human sars-cov [13] . the assessment of genetic diversity among 86 complete or semi-complete genomes of sars-cov-2 viruses revealed three deletions in the genome of isolates from japan, usa, and australia in addition to many other substitution mutations. the deletion mutations were in the orf1ab gene (3-nucleotide and 24-nucleotide deletion) and at the 3 end of the genome (10-nucleotide deletion). of the 93 substitution mutations, 42 changed the amino acid sequence of structural and non-structural proteins [65] . the 3and 24-nucleotide deletions in orf1ab are expected to reduce the protein sequence by 1 and 8 amino acid residues, respectively, without changing the reading frame, but the functional effects have yet to be investigated. the alignment of sars-cov-2 reference s protein gene against all sars-cov-2 sequenced genomes from china, usa, japan, australia, and taiwan revealed 99.97-100% identity, with 100% query coverage (also confirmed by our phylogenetic analysis, figure 2 ), while the identity and coverage for sars-cov s protein gene were 74.5% and 91%, respectively. also, the s protein gene from bat sars and sars-like coronavirus isolates shared 76.5-83% identity with that of sars-cov-2. this agrees with previous conclusions regarding the evolutionary analysis of sars-cov-2 [11, 44] . in the phylogenetic analysis we performed, sars-cov-2 viruses were in the same cluster regardless of the geographic region ( figure 2 ). these results strongly suggest the possibility of a recent common ancestor for all sars-cov-2 or the transmission of the same virus strain across countries. the outbreak of covid-19 originated from wuhan city, hubei province, in china. fifty-five percent of the infected cases before 1 january 2020 were linked to the huanan seafood wholesale market. however, the first human-to-human case of sars-cov-2 infection reported on 1 december 2019 did not have any exposure to this market [66, 67] . in mid-january 2020, sars-cov-2 spread to other provinces of china due to the spring festival travel season. sars-cov-2 was transmitted from china to other countries via international travelers. on 13 january 2020, the first case of sars-cov-2 infection was confirmed outside china in thailand, and on 16 january 2020 the first infected case was confirmed in japan. these cases were also linked to the huanan seafood wholesale market. by 25 january 2020, the number of confirmed cases had risen to 2062, including 2,016 in china, thailand, hong kong, macau, australia, malaysia, singapore, france, japan, south korea, taiwan, the us, vietnam, nepal, and sweden. on 30 january 2020, china reported a sharp rise in the number of infected cases, with the presence of infection in more than 18 countries. therefore, who declared the sars-cov-2 outbreak to be a public health emergency of international concern [68] . as of 16 march 2020, more than 150 countries and territories have been affected, with major outbreaks in central china, south korea, italy, iran, france, and germany [69] . there were 167,511 confirmed cases of sars-cov-2 infections, with 6606 deaths and about 8% estimated mortality rate. more than 73% of these cases have been reported in mainland china [69] . at this time, the number of global cases has shown a drastic increase within a short time, confirmed cases and deaths in china have not increased too much, while confirmed cases and deaths in other countries have drastically increased ( table 4 ). the number of confirmed cases increased from 2798 to 17,391 in one week (between 27 january and 3 february), and the number of infected countries doubled (from 12 to 24). due to the rapid increase of the number of infected cases and infected countries, the who declared sars-cov-2 a pandemic on 11 march 2020 and on 13 march 2020, the who declared europe to be the new center of the pandemic due to the massive increase of confirmed cases there [70] . on 15 ,889, were in italy). one week later (13 april 2020), the number of confirmed cases of sars-cov-2 increased 1.7 times (up to 524,514 confirmed cases), and the number of deaths increased 2.5 times (up to 20,444 deaths) in the usa alone. the number of confirmed cases, deaths, and infected countries are shown in table 4 . the origins of more than 75% of coronavirus infections are considered zoonotic, i.e., animals are the main source of the outbreaks. for example, sars-cov was transmitted from palm civets to humans, and mers-cov from dromedary camels to humans. bats are currently considered a reservoir for all human coronaviruses, as mentioned above [5, 71] . many coronaviruses are circulating in animals but have not yet infected humans. the type of animal that sars-cov-2 originated from is still unclear. at the beginning of the outbreak in wuhan, china, many patients were linked to the huanan seafood wholesale market, suggesting animal-to-person spread. after retrospectively studying case reports, the number of patients that did not have exposure to animal markets has risen, indicating person-to-person spread was also occurring at that time [66] . sars-cov-2 is closely related to bat coronaviruses and sars-cov [8] . a group of researchers reported early in the outbreak that the novel sars-cov-2 has the highest similarity of codon usage bias with snakes [72, 73] ; however, this method to determine initial host origins is dubious. interestingly, researchers also reported one amino acid difference in the receptor-binding domain of the s protein of pangolin-cov compared to that of sars-cov-2, suggesting that pangolins might play a role as an intermediate host (xiao et al., data currently under review). another group of researchers reported that the virus originated from bats based on the genome sequence of sars-cov-2, which is 96% identical to bat coronavirus ratg13. there were speculations that sars-cov-2 is a laboratory-engineered cov and leaked directly from a laboratory in wuhan where a bat cov (ratg13) was recently reported. however, there is no evidence to support this allegation [74] . recently, a group of researchers found that sars-cov-2 replicates poorly in dogs, pigs, chickens, and ducks but efficiently in ferrets and cats [75] . scientists are still trying to find the main source of the disease outbreak and identify the definitive intermediate hosts. both established (sars-cov, mers-cov) and novel (sars-cov-2) coronaviruses were reported to spread from an infected person to a non-infected person through direct or indirect contact. sars-cov-2 infection was reported to be transmitted directly from person to person like most respiratory viruses via close contact with an infected person or through respiratory droplets (aerosol) produced when an infected person coughs or sneezes. these droplets can be inhaled to reach the lung. the virus can be indirectly transmitted via touching a surface or an object that was previously contaminated with the virus and then touching the face, eyes, or mouth [76] and possibly via the fecal-oral route [77, 78] . asymptomatic carriers (during the incubation period of the virus) and patients after recovery from the acute form of the disease are also considered a potential source of virus transmission to healthy persons [10, 12] . interestingly, human coronaviruses are able to survive on steel, metal, wood, aluminum, paper, glass, plastic, ceramic, disposable gowns, and surgical gloves for 2-9 days. high temperature (≥30 • c) can reduce the persistence period, while low temperature (4 • c) increases the persistence time up to 28 days [79] . transmission of the virus vertically from mother to fetus or via breast milk has not been confirmed yet [80] . the transmission cycle of coronavirus among animals and humans is shown in figure 3 . both established (sars-cov, mers-cov) and novel (sars-cov-2) coronaviruses were reported to spread from an infected person to a non-infected person through direct or indirect contact. sars-cov-2 infection was reported to be transmitted directly from person to person like most respiratory viruses via close contact with an infected person or through respiratory droplets (aerosol) produced when an infected person coughs or sneezes. these droplets can be inhaled to reach the lung. the virus can be indirectly transmitted via touching a surface or an object that was previously contaminated with the virus and then touching the face, eyes, or mouth [76] and possibly via the fecal-oral route [77, 78] . asymptomatic carriers (during the incubation period of the virus) and patients after recovery from the acute form of the disease are also considered a potential source of virus transmission to healthy persons [10, 12] . interestingly, human coronaviruses are able to survive on steel, metal, wood, aluminum, paper, glass, plastic, ceramic, disposable gowns, and surgical gloves for 2-9 days. high temperature (≥30 °c) can reduce the persistence period, while low temperature (4 °c) increases the persistence time up to 28 days [79] . transmission of the virus vertically from mother to fetus or via breast milk has not been confirmed yet [80] . the transmission cycle of coronavirus among animals and humans is shown in figure 3 . there are many factors that affect sars-cov-2 transmission and spread. these factors include, but are not limited to: (1) travel to or contact with individuals who have recently visited wuhan, china, or other places experiencing an outbreak; (2) close contact with persons who are diagnosed positive for the disease, such as healthcare workers caring for patients with sars-cov-2; (3) contact with droplets and secretions (produced by sneezing or coughing) from an infected person and eating or handling wild animals native to china such as bats. additionally, the risk of infection is higher for the elderly and for patients suffering from pre-existing illnesses such as cardiovascular disease, hypertension, diabetes, and chronic respiratory disease [66] . the reported fatality rate based on age is 14.8% for people ˃80 years of age, 8% for people between 70 and 79 years, 3.6% for people between 60 and 69 years, 1.3% for people between 50 and 59 years, 0.4% for people between 40 and 49 years, there are many factors that affect sars-cov-2 transmission and spread. these factors include, but are not limited to: (1) travel to or contact with individuals who have recently visited wuhan, china, or other places experiencing an outbreak; (2) close contact with persons who are diagnosed positive for the disease, such as healthcare workers caring for patients with sars-cov-2; (3) contact with droplets and secretions (produced by sneezing or coughing) from an infected person and eating or handling wild animals native to china such as bats. additionally, the risk of infection is higher for the elderly and for patients suffering from pre-existing illnesses such as cardiovascular disease, hypertension, diabetes, and chronic respiratory disease [66] . the reported fatality rate based on age is 14.8% for people >80 years of age, 8% for people between 70 and 79 years, 3.6% for people between 60 and 69 years, 1.3% for people between 50 and 59 years, 0.4% for people between 40 and 49 years, 0.2% for people between 10 and 39 years; no fatalities have been reported for children under 10 years of age. notably, the fatality rate is higher in males (2.8%) than in females (1.7%) [81, 82] . the estimated incubation period of the novel coronavirus ranges from 2 to 14 days. however, some cases had an incubation period of 21, 24, or 27 days [83] . the complete clinical picture of sars-cov-2 is still unclear. the disease begins with flu-like symptoms that include fever, fatigue, dry cough, sore throat, shortness of breath, headache, chest tightness, chest pain, and muscle pain. some of sars-cov-2 patients have runny nose, nausea, vomiting, and diarrhea [84] . people can be infected without showing symptoms, which allows the virus to spread more effectively from person to person. complications can occur due to covid-19 leading to severe infections, such as pneumonia (infection of the lungs), kidney failure, and death [8] . the mild phase of the disease can last up to 2 weeks, while severe or critical disease lasts approximately 3 to 6 weeks (this analysis was conducted on 55,924 confirmed cases). additionally, the time from the disease onset to the development of severe disease is one week, while the time from the onset of symptoms to death ranges from 2 to 8 weeks [82] . based on the data analysis of 72,314 confirmed cases of sars-cov-2 in wuhan city, china, by 11 february, 80.9% of the cases were mild with flu-like symptoms, and patients recovered at home, 13.8% were severe with pneumonia and shortness of breath, 4.7% were critical with respiratory failure and septic shock resulting in organs failure, and approximately 2% of the cases were fatal [85] . another study was conducted on 99 hospitalized patients, and symptoms were classified as follow: fever (83%), cough (82%), shortness of breath (31%), muscle ache (11%), confusion (9%), headache (8%), sore throat (5%), runny nose (4%), chest pain (2%), diarrhea (2%), and nausea and vomiting (1%) [8] . the rapid diagnosis of sars-cov-2 infection is the cornerstone of disease control. it depends on several criteria including case history, clinical symptoms, serology, molecular diagnosis, and computed tomography (ct) imaging. on 2 march 2020, who published interim guidance for laboratory testing of suspected human cases, with precautions for specimen collection, packing, shipment, and amplification of nucleic acid to detect viral genes (n, e, s, and rdrp) [82] . sars-cov-2 uses the same cell entry receptor, hace2, as sars-cov. therefore, oral swabs, bronchoalveolar lavage fluid (balf), blood, as well as anal swabs are the best samples used for virus diagnosis [86] . a proper diagnosis depends primarily on the factors described below. the strict monitoring of case history in clinically suspicious patients is considered the first step in the early diagnosis of sars-cov-2 infection. clinically suspicious patients are those who suffer from fever and lower respiratory tract infection symptoms (for details, see the clinical characteristics section) and reside within or have traveled to endemic regions or had close contact with a confirmed or suspected case. additionally, sars-cov-2 can be transmitted by symptomatic and asymptomatic patients especially to the high-risk group mentioned above (for details, see the risk assessment section) [13] . the blood profiles of patients suffering from sars-cov-2 infection revealed the following: (1) increased c-reactive protein and erythrocytes, (2) increased myohemoglobin, liver enzymes, and muscle enzymes, with a high level of d-dimer in severe cases, and (3) normal or decreased white blood cell counts and lymphocytes in the early stage of the disease, with advanced lymphocytopenia in severe cases [13] . in icu patients, high levels of plasma granulocyte colony-stimulating factor (gcsf), ip10, il2, il7, il10, tnf-α, and mip1a were reported [38] . electron microscope examination of sars-cov-2 revealed the typical coronavirus morphology. further, sars-cov-2 was successfully isolated from human respiratory epithelial cells or balf samples of infected patients using huh7 cells and vero e6 cells. the isolated strain was confirmed by immunofluorescent antibody techniques using the cross-reactive nucleoprotein (np) antibody. serum neutralization tests (snt) using vero e6 cells were conducted to confirm the neutralization activity in igg-positive viral samples [87] . igm and igg elisa detection kits using bat sarsr-cov rp3 np were developed with no cross-reaction against human coronaviruses except sarsr-cov [80] . using these serological tools, viral antibody titers were increased in sars-cov-2-infected patients [38] . the procedures of elisa for the determination of sars-cov-2 igg were described before [86] . nucleic acid detection is the main, fastest, and most sensitive test for the diagnosis of sars-cov-2 infection. recently, two nested rt-pcr and two real-time rt-pcr assays have been developed with successful detection of the first 25 positive cases of infection in japan [88] . three real-time rt-pcr techniques have been designed based on the e, rdrp, and n genes [89] . also, scientists established molecular detection tools for sars-cov-2 based on the s gene [86] . chest x-ray examination in the early stage of the disease shows interstitial changes and multiple small plaque shadows. chest ct scans play an important role in the diagnosis of acute respiratory disease syndrome (ards) and pneumonia as well as in the early detection of lung parenchymal abnormalities in patients at risk and provide an impression of secondary infection (figure 4 ). electron microscope examination of sars-cov-2 revealed the typical coronavirus morphology. further, sars-cov-2 was successfully isolated from human respiratory epithelial cells or balf samples of infected patients using huh7 cells and vero e6 cells. the isolated strain was confirmed by immunofluorescent antibody techniques using the cross-reactive nucleoprotein (np) antibody. serum neutralization tests (snt) using vero e6 cells were conducted to confirm the neutralization activity in igg-positive viral samples [87] . igm and igg elisa detection kits using bat sarsr-cov rp3 np were developed with no cross-reaction against human coronaviruses except sarsr-cov [80] . using these serological tools, viral antibody titers were increased in sars-cov-2-infected patients [38] . the procedures of elisa for the determination of sars-cov-2 igg were described before [86] . nucleic acid detection is the main, fastest, and most sensitive test for the diagnosis of sars-cov-2 infection. recently, two nested rt-pcr and two real-time rt-pcr assays have been developed with successful detection of the first 25 positive cases of infection in japan [88] . three real-time rt-pcr techniques have been designed based on the e, rdrp, and n genes [89] . also, scientists established molecular detection tools for sars-cov-2 based on the s gene [86] . chest x-ray examination in the early stage of the disease shows interstitial changes and multiple small plaque shadows. chest ct scans play an important role in the diagnosis of acute respiratory disease syndrome (ards) and pneumonia as well as in the early detection of lung parenchymal abnormalities in patients at risk and provide an impression of secondary infection ( figure 4 ). assessing these lungs parenchymal abnormalities conveys disease severity to clinicians. using artificial intelligence models in the future may be useful in mass screening, to allow risk prioritization and help to minimize turnaround time [90] . pan et al. [91] conducted a retrospective study to elaborate the time course of lung changes during recovery from infection. they described assessing these lungs parenchymal abnormalities conveys disease severity to clinicians. using artificial intelligence models in the future may be useful in mass screening, to allow risk prioritization and help to minimize turnaround time [90] . pan et al. [91] conducted a retrospective study to elaborate the time course of lung changes during recovery from infection. they described findings using international standard nomenclatures such as ground-glass opacity (ggo), consolidation, and crazy paving patterns. they established a semi-quantitative scoring system of 5 grades to quantify the degree of involvement based on an area ranging from 0% to >75%. the total score ranged from 0 to 25 (max), and involvement was subpleural, random, or diffuse. they found that in early stages (0-4 days after the onset of symptoms), ggo was the main finding in lower lung lobes; in progressive stages (5-8 days), the progression of lung disease involved three patterns of ground-glass, consolidation, and crazy paving, while in peak stages (9-13 days) , dense consolidation became the prevalent feature; in absorption stages (>14 days), ggo was detected with no crazy paving and resolution of consolidations [91] . more than 75% of sars-cov-2-affected patients suffered from bilateral lung involvement, and 71% have multilobe involvement. ct examinations of 21 patients showed 29% consolidation and 86% ggo in the chest [8, 9, 92, 93] . another study examined 51 cases by ct and reported that 77% showed pure ggo, 75% exhibited ggo with reticular and/or interlobular septal thickness, 59% had ggo with consolidation, while 55% revealed pure consolidation. bilateral lung involvement was reported in 86% of cases; in 80% of the cases the posterior part of the lung was involved, while in 86%, the periphery was involved [94] . in january 2020, the who issued guidance for the clinical management of sars when sars-cov-2 infection was suspected. in this guidance, the start of emergency treatments, immediate implementation of prevention and control strategies, early supportive therapy and prevention of sars-cov-2 complications were described in detail [15] . so far, there are no approved specific antiviral drugs for sars-cov-2 infection. therefore, preventive measures and inactivation of the virus are essential to stop and control the spread of the disease. human coronaviruses can be inactivated using 0.5% hydrogen peroxide, 62-71% ethanol, 0.1% sodium hypochlorite, 0.7-1% formaldehyde, 2% glutaraldehyde, or 0.23% povidone iodine within 1 minute. other disinfectants such as 0.02% chlorhexidine digluconate, 0.55% orthophtalaldehyde, or 0.05-0.2% benzalkonium chloride are less effective [79] . in light of the urgent clinical demand, many drugs are approved to be used for clinical trials against sars-cov-2 infection, such as lopinavir/ritonavir, arbidol, interferon-alpha, favipiravir, chloroquine phosphate, darunavir/cobicistat, oseltamivir, and methylprednisolone. the most used antiviral drugs [95] are summarized in table 5 . generally, coronaviruses are not sensitive to current antiviral drugs, and high concentrations of drugs effective on these viruses cannot be used in vivo. therefore, combinations of different therapies have been used for the treatment of coronavirus infections [96] . some drug combinations that could be successful for the treatment of sars-cov-2 patients are lopinavir and ritonavir [97, 98] , lopinavir/ritonavir plus arbidol [99] , and ribavirin and interferon [100, 101] . the use of anti-inflammatory drugs such as glucocorticoids, il-6 antagonist, janus kinase inhibitors (jak), and choloroquine/hydrocholoroquine in sars-cov-2 patients is a dilemma, especially in patients suffering from an impaired immune system. balancing the risk-benefit ratio is a critical issue. corticosteroids may delay the elimination of the virus and increase the risk of secondary infection. in addition, drugs targeting pro-inflammatory cytokines can only inhibit specific inflammatory factors and thus may not be very effective in curbing the cytokine storm (excessive and uncontrolled release of pro-inflammatory cytokines). moreover, some anti-inflammatory drugs such as jak block inf-α production, which is important in fighting the virus [102] . additionally, fecal transplantation was approved for clinical trials as a therapeutic option for sars-cov-2-related pneumonia based on the promising results obtained from fecal microbiota transplantation in patients suffering from antibiotic-associated diarrhea, active ulcerative colitis, and other viral infections [103] [104] [105] [106] . recently, it was found that intestinal microbiota-derived ifn in lung stroma confers protection against viral diseases such as avian influenza and respiratory syncytial virus [103] . moreover, based on historical records of the effect of antiviral herbs on sars and influenza h1n1, chinese herbal formulas could be an alternative approach for the prevention of sars-cov-2 in a high-risk population [107] , if no scientifically based therapeutics are available. it was found that sambucus formasana nakai exhibited a strong antiviral effect against human coronavirus nl63 [108]. increases ph in host cell lysosomes and negatively influences virus-receptor binding, as well as interferes with the glycosylation of cellular receptors of sars-cov -exhibited a promising antiviral effect against sars-cov-2 in vitro -improved covid-19-pneumonia patients and shortened the course of the disease [114] remdesivir a monophosphoramidate of adenosine prodrug that incorporates into nascent viral rna chains causing pre-mature termination -used against a wide range of rna viruses such as filoviridae, paramyxoviridae, pneumoviridae, and coronaviridae; used successfully in covid-19 treatment in the united states and showed no adverse events [115] darunavir and cobicistat inhibit 3 c-like protease (3clpro). -used for the treatment of mers-cov in experimental animals -used for the treatment of hiv-1 patients [116] to date, there is no vaccine to prevent sars-cov-2 infection, and trials for vaccine development are in the preliminary stages of research. several vaccine candidates such as live attenuated, adenovirus-vectored, recombinant protein, and nucleic acid (dna and mrna) vaccines are in the pipeline [123] . the epidemiology of sars-cov-2 is still unclear, and data availability is limited. therefore, it is imperative to follow preventive measures and safety precautions issued by health authorities to limit exposure to the virus and to reduce further spread. general hygienic measures should be implemented, such as (1) washing hands often with soap and water or an alcohol-based hand sanitizer, (2) cough or sneeze etiquette, recommending covering of the mouth, (3) avoiding touching eyes, nose, and mouth if the hands are not clean, (4) avoiding close contact with sick persons, (5) avoiding sharing dishes, glasses, bedding, and other household items with sick people, (6) cleaning and disinfection of surfaces that are often touched, and (7) staying home from work, school, and public areas when feeling sick. the transmission route of sars-cov-2 is probably not only through cough, respiratory droplets, and/or contaminated surfaces [13, 124] , but also through fecal-oral transmission [78] . therefore, strict hygienic measures should be followed, especially in dense cities or agricultural spaces [125] . since the sars-cov-2 spread is primarily driven by travel, screening of travelers who arrive at airports from pandemic areas for possible sars-cov-2 infection and entry-screening procedures are necessary. also, general hygienic precautions during travel are highly recommended. travelers who suffered from acute respiratory infection should be tested and reported to the respective public health authorities [73] . in addition, people should be motivated to notify and report about travel history and close contacts in case of sars-cov-2 infection. asymptomatic carriers (during the incubation period) and patients after recovery from the acute form are also considered potential sources of the virus [12, 13] . strict hygienic measures should be implemented to avoid virus transmission to healthcare workers and other contacts, i.e., placement of sars-cov-2 suspected or confirmed patients in single-person rooms and wearing personal protection equipment (ppe) such as masks, goggles, and protective gowns. because early diagnosis and detection of asymptomatic carriers of sars-cov-2 are successful factors for the treatment and prevention of transmission, health authorities should designate laboratories to implement tests for a rapid and accurate diagnosis [126] . the control of coronaviruses is based on biosecurity regarding animals as well as on shifts in food habits, including discouraging the consumption of bushmeat and of animal products without appropriate cooking [127] . ban of wet marketplaces where live or dead animals are handled should be implemented. surveillance among people who have contact with wildlife and improvement of biosecurity regarding wildlife trade are urgently needed to prevent the next pandemic outbreak [128] . the epidemiology of sars-cov-2 is still unclear. many unresolved questions related to sars-cov-2 epidemiology and pathogenicity pose great challenges for researchers. these unresolved questions include: what is the origin of sars-cov-2? what is the intermediate host that transmitted the virus from bats? why does the virus cause severe disease and mortality in the elderly or those with co-morbidities, while it is milder in children? are aerosol, saliva, feces, urine, and foodborne the only routes of transmission? what are the other unknown routes of transmission? control of the sars-cov-2 outbreak and future epidemics requires global efforts among medical and veterinary clinicians, diagnosticians, epidemiologists, public health experts, vaccinologists, pharmaceutical industries, economists, and governments to implement a one-health approach [128, 129] . these measures must include: (1) writing policies and supporting funds required for the implementation of one health, prevention, and control measures, (2) hiring well-trained and professional personnel, (3) performing rapid and accurate diagnosis and treatment of infected persons, (4) developing and providing vaccines for virus control in humans, (5) conducting surveillance among wildlife for the identification and characterization of possible reservoirs and surveillance among people who are in contact with wildlife to identify risk factors in human behaviors and living environment, (6) improving hygienic measures, (7) assessing the social and economic impacts of covid-19 on the population, (8) utilizing veterinary experience in the disinfection of premises and gatherings under the supervision of health authorities to decrease outbreaks in humans, (9) providing antiviral drugs for the treatment of the disease in humans, and (10) increasing public health awareness about the virus and its transmission. the sars-cov-2 outbreak started in wuhan city, china, in december 2019. it is now a global pandemic, with 1,773,084 confirmed cases, 111,652 deaths, and 467,074 recoveries (as of 13 april 2020). the virus has the potential for rapid and extensive spread between people and countries. there are a lot of misleading information and knowledge gaps on the newly emerged sars-cov-2. therefore, we reviewed the latest updates about different aspects including epidemiology, source of infection, transmission dynamics, zoonotic potential, virus characteristics, and discovery of novel strategies for disease control to avoid spillover of infection in the future. bats play an important role in the transmission of the infection to humans. coronaviruses are genetically diverse and have a high tendency towards frequent genetic mutations and gene recombination, which increases the risk of interspecies transmission. information about the incubation period can help in establishing an effective quarantine for asymptomatic carriers, thus preventing the virus spread. from our perspectives and based on the currently available information about the virus and its epidemiology, the control of the sars-cov-2 requires an effective and global disease coordination effort including multidisciplinary research efforts (one-health approach) through collaboration between governments, epidemiologists, virologists, health authorities, veterinarians, and physicians. at this stage of the disease outbreak, developing vaccines is crucial to limit the spread of the infection. genome composition and divergence of the novel coronavirus (2019-ncov) originating in china isolation and characterization of viruses related to the sars coronavirus from animals in southern china supplement to: transmission of mers-coronavirus in household contacts origin and evolution of pathogenic coronaviruses summary of probable sars cases with onset of illness from 1 situation update clinical features of patients infected with 2019 novel coronavirus in a pneumonia outbreak associated with a new coronavirus of probable bat origin a familial cluster of pneumonia 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future pandemics similar to the 2019-ncov outbreak the one health approach is necessary for the control of rift valley fever infections in egypt: a comprehensive review this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we gratefully acknowledge the authors, originating, and submitting laboratories of the sequences from the gisaid's epicov™ database, on which part of the phylogenetic tree construction is based. the authors declare no conflict of interest. key: cord-294136-e69ao8j0 authors: han, dongsheng; li, rui; han, yanxi; zhang, rui; li, jinming title: covid-19: insight into the asymptomatic sars-cov-2 infection and transmission date: 2020-08-27 journal: int j biol sci doi: 10.7150/ijbs.48991 sha: doc_id: 294136 cord_uid: e69ao8j0 the existence of a substantial but unclear number of asymptomatic sars-cov-2 patients worldwide has raised concerns among global public health authorities. in this review, according to the published literature, we provided the evidence that asymptomatic infections can result in person-to-person transmission. four studies suggested that the virus can be transmitted by asymptomatic patients for at least two consecutive generations, indicating its strong infectivity. asymptomatic infection tends to be, but is not only, identified among young people (<20 years old). the majority of asymptomatic patients appear to have a milder clinical course during hospitalization, but the severity of the symptoms of the secondary patients infected by sars-cov-2 from asymptomatic patients varies with their physical constitution. the proportion of asymptomatic individuals among all confirmed cases widely differed (from 1.95% to 87.9%) according to the study setting and the populations studied. the increasing large-scale tests are expected to give more information about the true number of asymptomatic infections in the population. in china and other countries, various guidelines for management of asymptomatic cases have been issued. importantly, early detection, early reporting, early isolation and early treatment of asymptomatic patients require the joint efforts of policy makers, clinicians, technicians, epidemiologists, virologists and patients. the covid-19 pandemic caused by the sars-cov-2 virus is now straining or overwhelming health care systems worldwide. the clinical manifestations of covid-19 are protean, including asymptomatic carrier, acute respiratory disease, and pneumonia of varying degrees of severity [1] . through the joint efforts of government administrations, academic institutions, medical workers, technology enterprises and ordinary community residents, the epidemic in china has been basically brought under control. however, in many countries, new infections and deaths are still increasing. as of july 4, 2020, over 11 million confirmed cases and more than 525,000 fatalities at a global scale have been attributed to covid-19 infection. as the situation is rapidly evolving, it is unclear what the final scope and impact of this pandemic will be. in addition to the potent transmissibility of the virus itself, there is increasing evidence that unnoticed, asymptomatic cases in the infected population may greatly accelerate the spread of sars-cov-2 from person to person [2] [3] [4] . therefore, strict screening and management of patients with asymptomatic infection may be a key breakthrough to control the spread of the pandemic [5] . asymptomatic covid-19 patients are those who carry the virus but do not show any symptoms (e.g., fever, gastrointestinal or respiratory symptoms), and no ivyspring international publisher significant abnormalities on chest radiograph at time of laboratory confirmation [2, 6, 7] . however, asymptomatic cases are difficult to identify because they seem to be normal except for the presence of viruses in their bodies, making the transmission caused by these silent patients difficult to prevent. moreover, in the early stage of the outbreak, all medical resources tended to be used for the identification and management of critically ill patients, and asymptomatic patients have not attracted attention. these factors have led to a lack of systematic awareness of the prevalence and potential role of asymptomatic disease. due to heightened concerns of the risks posed by these stealth virus carriers, china has published the numbers and conditions of asymptomatic people every day since april 1 and has further promulgated guidance for the management of asymptomatic covid-19 cases nationwide [8] . in this review, based on the findings of existing studies and reports, we summarize the clinical and epidemiological characteristics and discuss current screening and management strategies for asymptomatic patients. this knowledge is critical to design and implement efficient and globally coordinated interventions. according to recently published studies, we reviewed the clinical, virological and epidemiological characteristics of asymptomatic infections, which were usually determined by reverse transcriptionpolymerase chain reaction (rt-pcr) ( table 1) . we found that (1) some asymptomatic cases at the time of the earliest test remained asymptomatic throughout the whole duration of laboratory and clinical monitoring, while others, which may account for approximately 80%, experienced clinical symptoms at a later stage of infection (presymptomatic patients) [9] [10] [11] [12] ; (2) the viral load in respiratory specimens of asymptomatic patients was similar to that in symptomatic patients, ranging from 1×10 4 to 1×10 7 copies per milliliter [10, 13] ; (3) the period of positive nucleic acid tests of asymptomatic patients (the interval from the first day of positive nucleic acid tests to the first day of continuous negative tests) could be up to 3 weeks (ranging from 1-24 days) [14, 15] ; (4) hoehl et al. successfully isolated sars-cov-2 from throat swabs of two asymptomatic patients in a cell culture of caco-2 cells, suggesting the potential for presymptomatic transmission [16] ; (5) increasing studies show clear epidemiological evidence of human-to-human asymptomatic spread of covid-19 (described in the following section); (6) asymptomatic infection tends to be, but is not only, identified among young people (<20 years old) [14, 15, [17] [18] [19] ; and (7) the majority (>90%) of asymptomatic patients appears to have a milder clinical course during hospitalization [15] , but the severity of the symptoms of the secondary patients infected by sars-cov-2 from asymptomatic patients varies based on their physical constitution [2, 20] . these findings may be adjusted as the pandemic continues to unfold. with the accumulation of more research data, researchers will thoroughly clarify the occurrence, development and outcome of asymptomatic infections, providing reliable evidence for finalizing prevention, diagnosis and treatment strategies. asymptomatic transmission could be defined as the transmission of sars-cov-2 from an asymptomatic person (source or index patient) to a secondary patient, as ascertained by exposure and symptom onset dates, with no evidence that the secondary patient had been exposed to anyone else with covid-19 [4] . asymptomatic transmission is driven by two groups of patients, one with no self-perceived symptoms or clinically detectable signs throughout the 14 days of quarantine and the other in the incubation period (i.e., the time period between getting infected and showing symptoms) (presymptomatic transmission). as early as january 2020, chan et al. reported evidence of human-to-human transmission of sars-cov-2 in family settings, suggesting that asymptomatic patients might serve as a possible source to propagate the outbreak [17] . by estimating the epidemiological data of distinct outbreak clusters from singapore and tianjin, china, tindale et al. identified that the serial interval (i.e., the number of days between symptom onset in a primary case and a secondary case) was shorter than the incubation period of sars-cov-2 by 2-4 days, supporting the likelihood that sars-cov-2 viral shedding can occur in the absence of symptoms and before symptom onset [21] . similar findings were reported by other researchers [22] [23] [24] . to date, epidemiologic studies have clearly documented sars-cov-2 transmission during the asymptomatic period. the evidence was mostly observed in cluster outbreaks, especially in family cluster outbreaks that occurred in china (figure 1 ). in general, in these clusters, there is a clear history of contact between the source patients and the secondary patients; there is no other explanation for the infection; and according to the epidemiological survey, the source patients were asymptomatic when transmitting the virus to the secondary patients. for example, a study reported in january 2020 identified five patients with covid-19 pneumonia infected by a 20-year-old asymptomatic woman (the source patient) who was thought to have acquired sars-cov-2 infection from the epidemic center of wuhan [2] . this source patient showed no symptoms during the entire period of monitoring and isolation ( figure 1a) . tong et al. identified 2 covid-19 patients (patients a and d) after their exposure to an asymptomatic person from wuhan who was later confirmed to be positive for sars-cov-2 [3] . patients a and d later transmitted sars-cov-2 to 3 family members (patients b, d and e) ( figure 1b) . li et al. confirmed asymptomatic and human-to-human transmission through close contacts in familial and hospital settings by analyzing the epidemiologic, laboratory, and clinical data of 7 covid-19 patients in a 2-family cluster [20] . the epidemiological survey showed that the source patient (56-year-old man) had stayed at hankou station in wuhan, china for 6 hours on his way from guangzhou to xuzhou ( figure 1c ). qian and colleagues reported a family cluster of asymptomatic covid-19 transmission in zhejiang china [9] . two source patients became infected with sars-cov-2 after a visit to a temple and then transmitted the virus to four other family members before experiencing symptoms. the four secondary patients then unknowingly (because they were asymptomatic) infected three other relatives through close contact ( figure 1d) . importantly, the latter three reports provided by tong et al., li et al. and qian et al. together with another report from luo et al. [11] , showed that sars-cov-2 can be spread through sustained transmission (at least two generations of spread) through asymptomatic/presymptomatic individuals ( figure 1b-e) , suggesting that the virus has a strong infectivity. in another study in mainland china, the source patient (case 13), who had a travel history to the city of huanggang, hubei province on jan 19-20, 2020 and was diagnosed as an asymptomatic covid-19 carrier, transmitted the virus to his cohabiting family members [14] ( figure 1f ). in addition, the transmission of sars-cov-2 infection from asymptomatic contacts has also been reported in other countries outside china, such as germany [25] , the usa [26] and singapore [4] . . the label (letter or number) on each person represents the person's id in the original literature. the 6 cluster outbreaks are as follows: a is from reference [2] , b is from reference [3] , c is from reference [20] , d is from reference [9] , e is from reference [11] , and f is from reference [14] . in all the above reports, the source patients remained without any symptoms of infection during the clinical course ( figure 1a, d and f) , or developed varying degrees of pneumonia ( figure 1b, c and e) . through comprehensive analysis of 24 asymptomatic covid-19 patients screened among close contacts, the authors reported that asymptomatic carriers among the close contacts were prone to mild illness during admission. however, the communicable period could last as long as 21 days, and the communicated patients could develop severe illness [14] . in four clusters of covid-19 in singapore, asymptomatic transmission occurred approximately 1-3 days before the source patient developed symptoms [4] . since asymptomatic transmission is an established fact, how many individuals in the infected population are attributed to asymptomatic transmission? several studies have made preliminary assessments. for example, using available data of 468 confirmed covid-19 cases compiled from online reports from 18 provincial centers for disease control and prevention (cdc) in china, du et al. identified that presymptomatic transmission might be responsible for 12.6% (59) of confirmed cases [23] . in the singapore outbreak, 6.4% (10/157) of locally acquired cases were attributed to presymptomatic transmission [4] . another study published by scientists in belgium and the netherlands showed that up to 48% (95% ci: 32-67%) of the 91 included covid-19 cases in singapore and 62% (95% ci: 50-76%) of the 135 included covid-19 cases in tianjin, china contracted the infection from someone who was presymptomatic [27] . now that the transmission of sars-cov-2 through asymptomatic patients via person-to-person contact has been confirmed, reliable estimations of the proportion of asymptomatic infections among all covid-19 patients are crucially needed to guide public health policy, especially to clarify the intensity and range of social distancing strategies to be implemented to prevent the spread of covid-19 [12] . robert redfield, director of the us cdc, warned that up to a quarter of covid-19 patients may not display any symptoms [28] . we summarized the data of currently published literature. overall, the detected asymptomatic proportion widely differed (from 1.95% to 87.9%) in the available studies with more than 10 confirmed individuals (table 1) . this situation is similar to the reported asymptomatic proportion of mers-cov (another coronavirus that infects humans) infections, which is varied from 12.5% to 91.7% based on the populations studied [29] . although reliable comparison may not be possible due to the differences in the study setting and the included populations, it can be seen, in general, that the asymptomatic proportions of sars-cov-2 infections reported in china (<16%) and singapore (<5%) are lower than those reported in japan (>30%), iceland (>40%) and the united states (>50%) ( table 1) . according to the national health commission (nhc) update, as of april 14, a total of 6,764 asymptomatic infections were reported nationwide in china, close to 8% of all confirmed sars-cov-2 cases (approximately 83,000 cases) [31] . in pediatric patients (<16 years of age) with covid-19, a nationwide epidemiologic survey in china showed that 12.9% (94/731) of cases were asymptomatic [7] . in wuhan city, 15.8% (27/117) of diagnosed children did not exhibit any symptoms of infection or radiologic features of pneumonia during the course of hospitalization [33] . the latest implemented universal sars-cov-2 testing in all pregnant patients presenting for delivery in new york city revealed that at this point in the pandemic, 87.9% of the patients who were positive for sars-cov-2 at delivery were asymptomatic, underscoring the risk of covid-19 among asymptomatic obstetrical patients [34] . several studies suggested that the vast majority of cases (approximately 80%) that were asymptomatic at the time of testing would eventually develop into symptomatic cases [10] [11] [12] , highlighting the importance of timely detection, treatment and management of asymptomatic patients. however, most of the above results were not obtained by random sampling or testing all individuals in the population, but mainly through the analysis of screening data from high-risk groups (i.e., individuals who have symptoms, have traveled to areas infected with the virus or have been in contact with infected patients). thus, these early results might not accurately reflect the real situation of the entire population. the large-scale population tests launched in increasing countries/regions are expected to reveal the true number of sars-cov-2 infections in the population and the contribution of asymptomatic individuals. recently, iceland reported the results of its population screening based on nucleic acid testing, and found that 43% of the participants who tested positive had no symptoms [76] . importantly, before large-scale testing begins, full consideration should be given to the rationality of the research design, the standardization of the experimental operation, and the reliability of the quality of the reagents/kits. this is the premise to ensure the reliability of the final results. the current first-line laboratory diagnostic tests for covid-19 worldwide include nucleic acid testing (mainly rt-pcr) and rapid serological antibody (mainly igm and igg) testing. as the transmission of sars-cov-2 may occur in the early course of infection and a high viral load in respiratory samples could be detected [13] , rt-pcr testing for this virus is more suitable for screening at earlier stages of infection in key populations, such as patients with obvious symptoms and close contacts of asymptomatic patients [35] . however, due to various factors (such as nonstandard sampling, inadequately trained personnel, unqualified reagents, etc.), false negative results in nucleic acid testing are almost inevitable [36] . it has been reported that the detection rate of currently available rt-pcr methods is between 30% and 60% [37] [38] . the issues contributing to false negative results in nucleic acid assays include insufficient viral load in infected persons, nonstandard sampling and handling, imperfect sensitivity of in vitro diagnostic reagents, and inadequate clinical laboratory testing capabilities [39] . for some cases, multiple samples and repeated testing may be required to obtain a final diagnosis [40] . in addition, in the early stages of outbreaks, many countries did not have sufficient reagents to carry out large-scale testing, which led to nucleic acid testing being more inclined to be performed on persons with obvious symptoms and reduced the detection for asymptomatic infections [41] . in theory, nucleic acid testing will miss individuals who have been asymptomatic for past infections (convalescent cases). these reasons make the true number of infected individuals, including asymptomatic individuals, in the population is currently unclear. detecting serum antibodies is an effective supplement to nucleic acid assays. data show that the positive rate can be increased from 51.9%, for a single rt-pcr test, to 98.6% when combining an igm antibody assay with an rt-pcr test [42] . serological assays are rapidly being developed and have proven to be useful in confirming covid-19 infection [35, 43] . in view of its simple operation and low cost, serological detection is suitable for large-scale screening [41] . a study showed that the median time of igm antibody response was 5 days (interquartile range [iqr], 3-6), while the appearance of igg antibody occurred on day 14 (iqr, 10-18) after symptom onset [42] . thus, serological testing is unlikely to play an important diagnostic role in the early stage of sars-cov-2 infection, but is more suitable in retrospective investigations for accurately determining the burden of infection, the contribution of asymptomatic infection, the basic reproduction number and total mortality, which have important implications for public health [44, 45] . eran et al. carried out serologic testing for sars-cov-2 antibodies in 3,330 people in santa clara county, northern california, suggesting that the actual number of infected people may be 50-to 85-fold the reported number of confirmed cases [41] . however, the reliability of antibody kits they used was been questioned by some scientists because of the lack of rigorously assessment. on april 10-14, a random seroprevalence survey of 865 residents in los angeles county, california suggested that only 4.06% (35/865) of the tested people have sars-cov-2 antibodies, suggesting that most people are still susceptible to contracting covid-19 [46] . at present, large-scale serological screening projects are running in many countries and regions [47] [48] [49] , if the study design is rigorous (full consideration is given to variation factors such as sampling, research population, reagent quality, etc.), the results of which will have important guiding significance for understanding the true number of asymptomatic infections and controlling the current disease outbreak worldwide. asymptomatic cases are easily neglected in disease screening and epidemic prevention. the asymptomatic covid-19 patients who have been identified include close contacts of covid-19 cases during their medical observation period, individuals involved in cluster outbreaks, people who were unknowingly exposed to infected patients and were identified when the infection source was being traced, and individuals who recently traveled to areas/ countries/regions with high infection rates (i.e., imported cases) [14, 18] . however, in reality, there may be more undetected asymptomatic patients. these cases may not be aware that they have been infected and therefore will not actively self-isolate or seek medical attention [50] , or they may be misdiagnosed during the screening and thus transmit the virus to others unknowingly. this situation complicates fighting the pandemic, forcing healthcare workers around the world to shift their focus from containing the infection to mitigation [51] . as the large-scale domestic epidemic has passed, one of china's current core tasks is to strictly screen, report and manage patients with asymptomatic infections to prevent possible future outbreaks. the actions china takes next will provide experience for the rest of the world to respond to the subsequent sars-cov-2 outbreak. on april 08, 2020, china's state council issued guidance for the management of asymptomatic covid-19 cases [8] . briefly, the guidelines call for standardized reporting of asymptomatic cases, which means that all healthcare facilities should report asymptomatic cases detected by them within 2 hours through direct online reporting. county-level cdcs are required to complete a case investigation within 24 hours after receiving the report of an asymptomatic case and report all the close contacts of the case. then, the asymptomatic case and all the close contacts need to undergo a 14-day centralized quarantine for medical observation. similarly, 14 days of active monitoring or quarantine for contacts of asymptomatic cases are also recommended by other countries [12, 52, 53] . once the individuals complete the 14-day quarantine and test negative twice in a row with one test every 24 hours, they are released from medical observation. those who show no symptoms and still test positive will be kept under centralized medical observation. if asymptomatic cases display clinical manifestations during the period of centralized medical observation, they shall be immediately transferred to designated medical institutions for standardized treatment in accordance with the confirmed cases [54] . furthermore, the guidance also pointed out that those who are released from centralized quarantine still need to be under medical observation for another fortnight. this strategy is useful in monitoring the risk of possible recurrence of the virus in recovered covid-19 patients [55, 56] . these series of management measures for asymptomatic patients in china are very strict compared to those of other countries. in the united states and the united kingdom, asymptomatic patients might discontinue isolation if they still have no symptoms within one week of the first positive covid-19 diagnostic test [57, 58] . there are currently no proven pharmaceutical treatments (vaccines or antivirals). rigorous implementation of traditional public health measures such as isolation and quarantine, social distancing and community containment is now the preferred option to reduce transmission of the virus [59, 60] . since the sars-cov-2 outbreak began, china has implemented all these nonpharmaceutical measures, such as case isolation, contact tracing and quarantine, physical distancing and community containment, with unprecedented efforts and successfully curbed the spread of the virus across the country in a relatively short period of time [24, 61] . other countries are also aware of the necessity of implementing such measures. once community transmission is detected, implementing the combined intervention of isolating infected individuals and their family members (including voluntary isolation at home); closing schools, factories or office buildings; suspending public markets; cancelling gatherings; and even locking-down affected cities are recommended [51, 53, 59, [62] [63] [64] . related studies have also suggested the importance of these measures in reducing sars-cov-2 spread, including the spread from asymptomatic individuals [32, [65] [66] [67] . furthermore, for personal protection, wearing masks is another important public health intervention for limiting the spread of respiratory pathogens. as transmission from asymptomatic infected individuals has been documented for covid-19, wearing masks in public places would help to intercept the transmission and prevent the spread by these apparently healthy infectious sources (asymptomatic patients) [68] [69] [70] . other suggestions should be accepted as well, such as restricting movement in affected cities, avoiding close contact with patients who are symptomatic, avoiding unnecessary gatherings, and washing hands frequently (with soap and water for at least 20 s) [70] [71] [72] . when coming into contact with confirmed/suspicious patients, people should proactively inform health authorities and seek testing to determine whether they are infected. addressing covid-19 is now an urgent health and social concern. however, controlling the pandemic only by detecting, isolating and treating symptomatic or critically ill patients is not sufficient because accumulating evidence has confirmed the asymptomatic transmission of sars-cov-2. several studies have even suggested that the virus can be transmitted by asymptomatic patients for at least two consecutive generations, indicating its strong infectivity. therefore, it is of great public health significance to screen and manage asymptomatic patients and their close contacts via multiple strategies to contain potential outbreaks. several scattered studies have evaluated the contribution of asymptomatic patients in the population. however, there is no comparability between their results due to the differences in the distribution and size of the studied populations, so the true scale of asymptomatic infections remains unclear. ongoing large-scale population screening is expected to answer this question. studies have provided a cautionary warning that sars-cov-2 virus may be transmitted through multiple routes in addition to the most common respiratory droplet spread. it is necessary to fully consider the sample type and sampling time to improve the detection rate. in addition, a combination of molecular and serological tests is needed to identify virus carriers when necessary. asymptomatic patients themselves should complete the standard isolation and treatment process as required by governments and doctors. additionally, when considering that the spread of sars-cov-2 may occur through respiratory droplets, contact with contaminated objects or fecal viral shedding, which is currently being investigated [4, 73] , individuals should also pay attention to personal hygiene and reduce close contact with healthy people to avoid causing new infections. in short, early detection, early reporting, early isolation and early treatment of asymptomatic patients require the joint efforts of policy makers, clinicians, technicians, epidemiologists, virologists and patients. cdc: centers for disease control and prevention; iqr: interquartile range; rt-pcr: reverse transcription-polymerase chain reaction. asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths presumed asymptomatic carrier transmission of covid-19 potential 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population prevalence of sars-cov-2 infection in residents of a large homeless shelter in boston this work was supported by the "aids and hepatitis, and other major infectious disease control and prevention" program of china under grant (no. 2018zx10102001) and the national natural science foundation of china under grant (no. 81703276). the funder had no role in study design, data collection, analysis, interpretation, or writing of the paper. the authors have declared that no competing interest exists. key: cord-305856-xt3zxajf authors: shanmugam, chandrakumar; mohammed, abdul rafi; ravuri, swarupa; luthra, vishwas; rajagopal, narasimhamurthy; karre, saritha title: covid-2019 – a comprehensive pathology insight date: 2020-09-18 journal: pathol res pract doi: 10.1016/j.prp.2020.153222 sha: doc_id: 305856 cord_uid: xt3zxajf corona virus disease-2019 (covid-19) caused by severe acute respiratory syndrome corona virus-2 (sars cov-2), a highly contagious single stranded rna virus genetically related to sars cov. the lungs are the main organs affected leading to pneumonia and respiratory failure in severe cases that may need mechanical ventilation. occasionally patient may present with gastro-intestinal, cardiac and neurologic symptoms with or without lung involvement. pathologically, the lungs show either mild congestion and alveolar exudation or acute respiratory distress syndrome (ards) with hyaline membrane or histopathology of acute fibrinous organizing pneumonia (afop) that parallels disease severity. other organs like liver and kidneys may be involved secondarily. currently the treatment is principally symptomatic and prevention by proper use of personal protective equipment and other measures is crucial to limit the spread. in the midst of pandemic there is paucity of literature on pathological features including pathogenesis, hence in this review we provide the current pathology centered understanding of covid-19. furthermore, the pathogenetic pathway is pivotal in the development of therapeutic targets. the current pandemic of corona virus disease-2019 (covid-19) caused by severe acute respiratory syndrome corona virus-2 (sars cov-2) led to complete lockdown in many countries contributing to major socio-economic crisis and irreparable recession, globally. sars cov-2, a novel β cov was first identified in adults presenting with acute lower respiratory tract infection of unexplained etiology in china. [1] though no age group is spared, severe forms occur in patients older than 60 years specifically with co-morbidities. the majority of the infected individuals are asymptomatic or with mild form of disease and are potential transmitters. this disease is highly contagious and mainly spread through respiratory droplets, close contact with infected cases or materials (fomites) and nosocomially to other patients and health care workers in the hospitals. [2, 3] covid-19 has a much lower case fatality ratio and significantly greater transmission rate than 2003 sars pandemic. [4, 5] currently rt-pcr of upper and lower respiratory swabs or samples is the gold standard diagnostic test. serological tests based on antibody detection, though not helpful during the early phases of disease, can be used to confirm infection in later phase. a thorough literature search (pubmed, preprint servers and google scholar) using terms covid-19 and pathology/pathogenesis, sars cov-2 and pathology/pathogenesis and 2019-ncov and pathology/pathogenesis was done to maximize the yield of literature, which ended on 24 may 2020. in this review, we have comprehensively discussed all aspects of covid-19 with special emphasis on the pathology including pathogenesis and therapeutic targets. it forms a ready resource for clinicians, pathologists, and researchers including epidemiologists aiding them in the diagnosis and treatment of these patients, and may also pave way to further research. the earliest case of sars cov-2 infection currently known was reported on 31st december 2019 in wuhan, hubei province of china. [1] after this it spread rapidly to other parts of china as well as internationally affecting over 185 countries as of 23 april 2020, leading to the current global pandemic. [2] the world health organization declared covid-19 to be a public health emergency of international concern on 30 january 2020, and recognized it as a pandemic on 11 march 2020.[6,7] as of 24 may 2020, globally 5.48 million cases of covid19 have been reported, resulting in 346,071 deaths and 2,290,776 people have recovered. [8] j o u r n a l p r e -p r o o f the basic reproduction number (r0) of the sars cov-2 is estimated to be between 1.4 and 3.9, indicating its highly contagious nature. [9, 10] the r0 may be even higher in places of public gatherings like in cruise ships, religious/political/academic/business congregations as well as in hospitals non-compliant with personal protective measures. [9, 10, 11] the incubation period and serial interval is estimated at 5-6 days and 8 days, respectively, which is similar to that for sars cov and mers cov. [9, 12, 13, 14] early in the pandemic, the case-fatality rate (cfr) was estimated to be between 0.9% and 3%, [15, 16] lower than other hcovs (sars cov (6%-17%) and mers cov (20%-40%)). [17, 18, 19] however, by the 24 th of may 2020 many countries exhibited exponential rise in cfr. [8] (table:i) unlike sars cov, the high percentage of sars cov-2 infected individuals manifest as asymptomatic or pauci-symptomatic infection who escape detection and become potential transmitters. [20, 21] it is important to note that, not all close contacts are infected suggesting a role for individual genetic susceptibility. [22, 23, 24] in humans, the virus usually gains entry through upper aero-digestive tract. more recently sars cov-2 was isolated from the feces of patients, indicating the possibility of fecal-oral spread. [24, 25] furthermore, sars cov-2 infection in pregnant women raised a possibility of vertical transmission. [26] however, the vertical transmission was ruled out based on negative testing for the virus on the swabs collected from the amniotic fluid, cord blood, neonatal throat and breast milk of the six infected pregnant women. [26] the long range airborne transmission is also speculated which depends on flow dynamics of the virus from the infected person and also on ventilation status of the area. [27] moreover, the expansion and spread of covid-19 can be visualized by mapping techniques like cartograms. [28] the understanding of modes of transmission of sars cov-2 will enable application of appropriate containment measures. though there is generalized susceptibility to sars cov-2 infection for all age groups, body defense against infection as well as their underlying age related organ system compromise. [22, 31, 32, 33] similar to sars cov, a recent study reported non-o blood group specifically group a had higher infection and death rates due to covid-19 owing to absence of protective anti-a igm antibodies. [34, 35] many uncertainities still persist in the sars cov-2 epidemiology especially virus-host interaction including host susceptibility and the evolution of epidemic. the corona viruses (covs) are classified into α and β (seen in mammals including humans); γ and δ (seen in avian species). [ to pangolin cov with a difference of only one amino acid. [46] recently another study suggested pangolin involvement in sars cov-2 origin due to evidence of re-assortment in covs. [47] sars cov-2 differs from other β covs by the presence of unique polybasic cleavage site that contributes to increased pathogenicity and transmissibility. [48] each virion is a enveloped, non-segmented, positive sense single stranded rna virus the sars cov-2 because of its similarity with sars cov is presumed to infect human cells through its densely glycosylated spike (s) proteins s1 fraction with receptor binding domain (rbd) which binds to the angiotensin-converting enzyme 2 receptor (ace-2 r) with 10 to 20 fold higher affinity than sars cov. [ [56, 57] after the virus gets attached to this receptor, the sars cov-2 with its unique polybasic s1/s2 protease cleavage site with sprr insertion on the spike protein which is recognized and cleaved by transmembrane protease serine (tmprsss) expressed on host cells to expose the fusion protein (s2 fraction) that enables the fusion of both viral and the host cell j o u r n a l p r e -p r o o f membrane. [56] it has been demonstrated that ace-2 r and tmprsss are highly co-expressed in alveolar type 2 pneumocytes, epithelium of upper esophagus and absorptive enterocytes, forming the basis of speculation that the sars cov-2 can gain access into host through esophageal and intestinal epithelium apart from alveolar epithelium. hence, the potential target tissues for sars cov-2 should co-express ace-2 r and tmprsss. the current understanding of pathology stems from few case reports and autopsy case studies. the gross features include heavy and boggy lungs, patchy consolidation along with pleural fibrinous exudate and /or fibrosis, sometimes with purulent inflammation due to secondary bacterial infection with/without evidence of pericarditis. [62] the microscopic features depend on stage and severity of the disease. early stages (asymptomatic/mildly symptomatic patients) show non-specific changes including pulmonary j o u r n a l p r e -p r o o f edema, focal pneumocyte hyperplasia, focal chronic inflammatory infiltrate and multinucleated giant cells with absence of prominent hyaline membrane formation. [63] as, the disease progress there is diffuse alveolar damage with transparent hyaline membrane formation and severe pulmonary edema. however, in sars cov-2, there is firbomyxoid exudates with visible fibrinous cords along with mucous plugging of bronchioles which has a bearing with respect to oxygen therapy. there is also widespread interstitial inflammatory infiltrates with severe epithelial damage,diffuse type ii pneumocyte hyperplasia consistent with ards. [64, 65, 66, 67, 68] one study reported massive pulmonary interstitial fibrosis with variable degree of hemorrhagic necrosis, chronic inflammation with multinucleate giant cells and intracytoplasmic viral inclusion bodies in severe cases. [69] interestingly, another study showed features of lymphocytic viral pneumonia in a patient who died early in the disease (5th day after development of symptoms), whereas five other patients who succumbed later (20th day after development of symptoms) exhibited acute fibrinous and organizing pneumonia (afop) showing extensive fibrinous deposits forming balls/mounds but not hyaline membrane in their alveoli. these patients also showed prominent vascular injury evidenced by endothelial cell detachment and prominent intracytoplasmic vacuolization in small and medium-sized pulmonary blood vessels. [70] also, severe covid-19 infection has been associated with a novel pulmonary-specific vasculopathy known as pulmonary intravascular coagulopathy (pic), that parallels disease severity. [71] these findings may be considered as important indicators of disease severity and prognosis. the liver shows mild lobular lymphocytic infiltration and moderate micro-vesicular steatosis along with mild lobular activity, possibly related to the viral infection itself and ischemia. there were no obvious histological changes in heart tissue except for mild interstitial chronic mononuclear infiltrate. [57, 64, 65] hence the changes in the liver and heart are more likely secondary or related to the underlying diseases. [64] the pathology in other organs have not been elucidated. it is too early to determine the specificity and consistency of these histopathological findings with respect to the stage and severity of the covid-19 owing to the paucity of information obtained from few biopsy/autopsy case reports. in addition, the histopathological features may be modified or altered by patients' immunity, presence of co-morbidities, secondary infections and therapy given to these patients especially steroids. only few patients present with gastrointestinal symptoms like diarrhea (5.7%) and nausea/vomiting (6.1%). [72] in 26.7% of covid-19 patients there was at least one underlying co-morbidity(hypertension, diabetes, chronic cardiovascular/ pulmonary/ renal disease and cancer). [72] the severe form is characterized by ards that necessitates mechanical ventilatory support in an intensive care unit (icu), and also leads to multiorgan involvement resulting in shock, septicemia, and mods with high mortality. [73, 74] a substantial proportion of patients developed diarrhea during hospitalization, potentially aggravated by various drugs including antibiotics. [75] these patients may also present with cardiac sounding chest pain due to myocarditis and myocardial infarction. children are either asymptomatic or pauci-symptomatic (fever (50%), cough (38%), fatigue, rhinorrhoea or nasal congestion) and are less likely to have severe infections. [82, 83] gastrointestinal symptoms like diarrhoea, abdominal cramps and vomiting, common in children, covid-19 positive patients frequently exhibit hematologic abnormalities in the form of lymphopenia, leukopenia, and thrombocytopenia, along with elevated levels of liver enzymes, lactate dehydrogenase, prothrombin time and d-dimers. [72] lymphopenia is associated with disease severity and mortality. [1] acute phase reactants such as crp, ferritin and procalcitonin and pro-inflammatory cytokine levels were higher in covid-19 than healthy adults. [86] covid-19 patients needing icu management when compared to non-icu patients had higher plasma levels of pro-inflammatory cytokines (il2, il7, il10, gscf, ip10, mcp1, mip1a, and tnfα), increased total wbc and neutrophil counts, higher levels of d-dimer, creatine kinase, and creatinine. [31, 74] similar laboratory findings were seen in children with covid-19. [29] findings on chest imaging in sars cov-2 pneumonia seems to be similar to ordinary viral pneumonia, with some peculiarities. chest x-ray and ct changes may be seen even before the detection of the virus from swab. in contrast, the chest x-ray may be normal in 31% of laboratory confirmed covid-19 cases. [87] the commonest feature on chest x-ray is presence of bilaterally symmetrical ground glass opacities with or without associated consolidation in the posterior and peripheral lung fields. [32] however, the ct findings vary with the duration of symptoms. [88] in the initial phase (days 2-4) basal multifocal peripheral ground-glass opacities are noted. with disease progression (mid phase (days 4-7) there is linear opacities developing on a background of ground-glass opacities (crazy pavement pattern). in the late phase (days 8-14) the central ground-glass opacities become surrounded by denser crescentic shaped consolidation (forming more than three-fourths of a circle) or form complete ring of at least 2 mm in thicknesscalled as 'reversed halo sign' or 'atoll sign' [88] children also exhibit similar radiologic features. [29] chest ct suggesting covid-19 had 97% sensitivity in concordance with positive these tests are complex, time consuming, expensive and need expertise to perform as well as to interpret. [91, 95, 96] the molecular test based on point of care testing using cartridges are rapid and needs less expertise. high throughput technologies including ngs can be used for simultaneous screening of large number of samples but its application is limited to research only due to high expenditure. [97] the serological tests detects either the viral antigens (spike protein and nucleo-capsid being target antigens), or the antibody response to the virus by immunochromatography and elisa methods. specifically, the antibody testing is not helpful in the early phases of infection. though simple, cost effective, easy to perform and interpret, there are chances of false positives especially due to cross reactive antibodies against other hcovs. additionally, a negative antibody test does not exclude sars cov-2 infection. [95, 98] the role of virus isolation and culture as well as detection of the virus by its cytopathic effects on cell lines is highly limited due to requirements of bio-safety level-3 facility. [99, 100] hence it is not j o u r n a l p r e -p r o o f recommended by who for diagnostic purpose [101] currently the diagnostic tests for detecting sars cov-2 infection is variable and non-uniform owing to the use of different probes, kits and reagents. though there are numerous reports claiming efficacy of various drugs and vaccine against covid-19, none are effective and safe to receive approval by regulatory authorities. the management of covid-19 mainly relies on effective implementation of infection preventive and control measures and delivery of timely supportive care including oxygen therapy and mechanical ventilation as and when indicated. as the r0 value is >1 (range 1.4 to 3.9), [9, 10] efficiency of intervention strategies such as screening of incoming people, wearing masks, quarantine for travellers has already been proved. [102] specifically, reducing travel volume to and from china has had a positive impact on transmission dynamics of covid-19. [103] . though preventive vaccines against sars cov-2 can be developed targeting the spike (s) glycoprotein or its receptor-binding domain (rbd), these are made ineffective due to generation of altered immunogens in the target proteins owing to rapid mutations and recombinations. [104, 105] in sars cov, live attenuated vaccine with the deleted structural e gene mutant was effective in producing neutralizing antibodies which lowered viral loads and reduced disease severity. [106] the development of inactivated vaccines against sars cov was hindered due to occurrence of harmful immune and/or inflammatory responses post challenge. j o u r n a l p r e -p r o o f 13 [107, 108, 109] sub-unit vaccines (purified proteins combined with adjuvants) and viral vector (adeno virus) vaccines against s glycoprotein or its rbd and n protein of sars cov and mers cov elicited higher humoral response as well as enhanced mucosal immunity with intranasal administration. [110, 111, 112] furthermore, dna based vaccine against s glycoprotein of mers cov also showed robust neutralizing antibody response and is currently under clinical trial. [113] based on these reports vaccines for sars cov-2 is likely possible. however, its efficacy and safety has to be proved before approval. in the absence of specific anti-viral therapy, treatment is mainly symptomatic and supportive that includes oxygen therapy, conservative fluid management, hemodynamic support and / or mechanical ventilation. mechanical ventilatory support with low tidal volume and low inspiratory pressure is indicated when the respiratory distress is refractory to conventional oxygen therapy or niv. [2] extracorporeal membrane oxygenation (ecmo) is indicated in patients with refractory hypoxemia despite prone position mechanical ventilation. [2] a recent retrospective study identified older age, high sequential organ failure assessment score (sofa) score, and d-dimer greater than 1 µg/ml as poor prognostic factors which aid the clinician early in instituting aggressive treatment and monitoring for such patients. [114] steroids, and injudicious antibiotic use should be discouraged. some studies report effective use of rna polymerase inhibitors remdesivir and immucillin-a as prophylactic and therapeutic agents against hcovs including sars cov-2. [115, 116] anti-malarial drug chloroquine and its analogue may show protective effect against virus by decreasing intracellular ph but may cause cardiac arrythmias owing to prolonged qtc interval in some patients. [117] monoclonal antibodies against interleukin-6 (il-6) like sarilumab, siltuximab, tocilizumab and interleukin-1 (il-1) inhibitor like anakinra may be useful in severe cases and may control the effects of sirs which is the main culprit in the pathogenesis of severe cases. [118] currently, there are 1,673 studies registered in clinical trials involving various investigational drugs and vaccine apart from those mentioned above and are still at phase-i level. [119] theoretically, molecules involved at each step of sars cov-2 pathogenesis may become potential therapeutic targets. (figure: to conclude, sars cov-2 is highly infective and its control depends on strict implementation of preventive measures. though rt-pcr is the gold standard for sars cov-2 diagnosis, the results are variable and there is scope of false negatives owing to either sampling errors or due to usage of different primers and reagents by different vendors. serologic estimations of antibody titres though not helpful for diagnosis, may be useful for prognostication and follow-up. though currently available pathologic data is limited, it is of prime importance to unveil the pathogenesis which will enable the development of therapeutic options. however, studies on larger cohorts are needed to validate the findings obtained for generalized application. importantly, due to limited availability of time and resources for research during the current emergency situation there is a huge lag and gap in understanding covid-19; hence the current review removes the lag and bridges the gap and provides pathology centered understanding of the disease. furthermore, the currently approved clinical trials that are in various stages of development on all aspects of covid-19 including vaccines and potential therapeutic targets will shed light in future. there are no conflicts of interest to disclose. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster evaluation and treatment of corona virus (covid-19) statpearls ncbi-bookshelf likelihood of survival of corona virus disease 2019. lancet coronavirus: covid-19 has killed more people than sars and mers combined, despite lower case fatality rate early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia pattern of early human-to-human transmission of wuhan covid-19 outbreak on the diamond princess cruise ship: estimating the epidemic potential and j o u r n a l p r e -p r o o f effectiveness of public health countermeasures 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coronavirus that lacks the e gene is attenuated in vitro and in vivo safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine immunization with inactivated middle east respiratory syndrome coronavirus vaccineleads to lung immunopathology on challenge with live virus inactivated sars-cov vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry sars cov subunit vaccine: bodymediated neutralisation and enhancement intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immuniza-tion: implication for designing novel mucosal mers vaccines comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study the antiviralcompound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 sars-cov-2 and the use of chloroquine as an antiviral treatment we thank dr. vaseemuddin mohammad (consultant nephrologist, tables: table i key: cord-313344-rqvi2ksc authors: farcas, gabriella a.; poutanen, susan m.; mazzulli, tony; willey, barbara m.; butany, jagdish; asa, sylvia l.; faure, peter; akhavan, poolak; low, donald e.; kain, kevin c. title: fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus date: 2005-01-15 journal: j infect dis doi: 10.1086/426870 sha: doc_id: 313344 cord_uid: rqvi2ksc severe acute respiratory syndrome (sars) is characterized by pulmonary compromise; however, patients often have evidence of other organ dysfunction that may reflect extrapulmonary dissemination of sars coronavirus (sars-cov). we report on the distribution and viral load of sars-cov in multiple organ samples from patients who died of sars during the toronto outbreak. sars-cov was detected in lung (100%), bowel (73%), liver (41%), and kidney (38%) in 19 patients who died of sars, with the highest viral loads observed in lung (1.0 × 10(10) copies/g) and bowel (2.7 × 10(9) copies/g). fatal sars was associated with multiorgan viral dissemination in a distribution that has implications for disease manifestation, viral shedding, and transmission. epidemic resulted in 251 probable cases and 44 deaths, making toronto the most affected center outside of asia [4] . coronaviruses are a diverse family of enveloped, positivestrand rna viruses that cause a wide spectrum of intestinal and respiratory diseases. for animal coronaviruses, data suggest that changes in the spike glycoprotein may contribute to differences in tissue tropism and virulence [5] . unlike the enteric and pneumonic illnesses associated with the animal coronaviruses, illnesses associated with previously recognized human coronaviruses (hcov-229e, hcov-oc43, and hcov-nl63) had been largely confined to the upper respiratory tract [6] . however, sars-cov appears to have arisen as a result of the zoonotic transmission of an animal coronavirus to humans, and little is known about its potential tissue tropism in humans [6] . although sars is primarily characterized by the presence of lower respiratory tract infection and subsequent pulmonary compromise, patients often have evidence of other organ dysfunction, including gastrointestinal symptoms and abnormal liver function [2, 7] , as well as splenic atrophy and lymphadenopathy [8] . this may reflect widespread immunopathology or the presence of extrapulmonary sars-cov dissemination and replication, as has been observed in other species infected with animal coronavirus [9] . the purpose of the present study was to investigate the presence of sars-cov, the degree of viral dissemination, and the viral loads in multiple organ samples from all patients who died of sars during the toronto outbreak (march to september 2003) and underwent a postmortem examination and compare the results with those found in patients who died of other causes during the outbreak. we demonstrate that, in fatal cases of sars, sars-cov is often disseminated to multiple organs, in patterns that have implications for clinical manifestations, viral shedding, and disease transmission. subjects and methods. all patients whose condition met the centers for disease control and prevention (cdc) and world health organization (who) case definition of probable sars in toronto, canada, and who underwent a postmortem examination during the period from the beginning of the outbreak in march to september 2003 were eligible for inclusion in this study. autopsies were performed on 21 of the 44 patients with deaths attributed to sars. fifteen autopsies were performed using a modified protocol for highly infectious cases, and 6 autopsies were restricted to biopsies of specified tissues. fifty-one patients who died of causes other than sars during the outbreak and who underwent postmortem examination were included as controls. causes of death in these individuals included congestive heart failure, cerebrovascular accidents, ath-erosclerotic heart disease, chronic obstructive pulmonary disease, invasive group a streptococcal infection, amiodorone pulmonary toxicity, and pulmonary fibrosis. clinical details were extracted, by use of hospital records, into standardized data extraction forms. results of antemortem microbiologic examination for routine bacterial and viral respiratory pathogens from the 21 individuals with probable sars were negative. however, on the basis of postmortem examination, 3 of these patients had evidence of coinfection with other organisms: 2 women had evidence of coinfection with aspergillus species, and 1 patient had evidence of cytomegalovirus infection. this study was reviewed and approved by the research ethics boards of the mount sinai hospital and the university health network, toronto, canada, and by the chief coroner's office of ontario, canada. a total of 212 discrete postmortem organ samples, including lung, liver, spleen, kidney, small bowel, large bowel, lymph nodes, heart, and skeletal muscle, were prospectively collected from the 21 patients who died of sars and underwent autopsies. two of these patients died 1100 days after disease onset, and all organ samples tested were determined by real-time reverse-transcriptase polymerase chain reaction (rt-pcr) to be negative for sars-cov. these patients have been excluded from all subsequent analyses. as controls, 228 postmortem organ samples from the same tissues were prospectively obtained from 51 subjects who died of causes other than sars. all samples collected at the time of autopsy were snap frozen in a mixture of absolute ethanol and dry ice and were subsequently stored at ϫ70њc until analyzed. all samples were coded and were independently processed and examined. specimen analysis and interpretation were performed blinded to other diagnostic investigations. for rt-pcr, organ tissue samples were homogenized, in rlt buffer (qiagen), by use of disposable tissue grinders (kendall precision), and rna was isolated by use of the rneasy mini kit (qiagen). the rt-pcr was performed using the commercially available realart hpa-coronavirus lc-rt assay (artus) on a lightcycler real-time pcr platform, as described elsewhere [10] . viral load was calculated from a standard curve based on 4 quantification standards. a standard preparation of sars-cov isolated from cell culture supernatants of veroe6 cells was used as a calibrator in each run. in addition, a second heterologous amplification system (i.e., an internal control) was included to ensure the quality of rna isolation and to identify pcr inhibition. the sensitivity and specificity of this standardized realtime rt-pcr assay for the detection of sars-cov in postmortem lung tissues were previously assessed at 100% [10] . specificity of amplicons was verified by nucleic acid sequencing. results. the 19 patients who died within 51 days after infection and underwent a postmortem examination had a mean age of 68 years (range, 43-99 years). ten of the patients were female. the mean duration of illness was 22.5 days (range, 5-51 days). sars-cov was found in 100% (19/19 patients, each with specimens collected from multiple sites, totaling 66 discrete samples) of lung samples, 73% (11/15) of bowel samples, 69% (9/13) of lymph node samples, 41% (7/17) of liver samples, 40% (7/18) of heart samples, 38% (6/16) of kidney samples, and 12% (2/17) of skeletal muscle samples. the viral loads for each organ are presented in tables 1 and 2. of interest, particularly high viral loads were observed in a recent lung-transplant recipient. all 206 postmortem samples from the 51 non-sars fatalities that occurred during the sars outbreak were negative for sars-cov. the corresponding sensitivity and specificity of the real-time rt-pcr assay were 100% (95% confidence interval [ci], 94.6%-100%) and 100% (95% ci, 98.2%-100%), respectively, for the detection of sars-cov in lung tissue within 51 days of disease onset. pathologic findings in sars-cov-infected lung specimens showed diffuse alveolar damage; however, histopathologic changes in bowel were minimal, despite the presence of sars-cov in 170% of these cases, often at high viral loads (data not shown). there were no changes on gross examination, and only minimal inflammatory changes were observed on microscopic examination. similarly, despite evidence of elevated antemortem levels of transaminases and the presence of sars-cov in the liver at autopsy in 41% of these cases, there were only minor inflammatory changes observed in the liver on microscopic examination. of note, 100% (7/7) of patients who had sars-cov detected in the liver had abnormal antemortem liverfunction test results, whereas abnormal results were found in 40% (4/10) who did not have sars-cov detected. discussion. there are limited data available detailing viral loads and extrapulmonary dissemination of sars-cov in humans. in this study, we used a standardized and validated realtime rt-pcr assay to detect and determine the viral load of sars-cov in multiple organs from a large number of individuals who died of sars during the toronto outbreak, compared with those who died of other causes. we demonstrate that, even 51 days after disease onset, sars-cov was consistently found in multiple lung lobes, suggesting that there is widespread dissemination of the virus throughout the lung at time of death. further, we observed extrapulmonary dissemination of the virus into all major organs, especially the bowel and lymph nodes. these data have implications for the clinical manifestation, disease course and outcome, and transmission of sars-cov. sars-cov viral dissemination has previously been examined in a simian model [11] . necropsy results indicated the widespread presence of sars-cov in lung but only sporadic presence of the virus in the duodenum, kidney, and spleen. although our findings that sars-cov is detected in postmortem these data, combined with evidence of viral shedding in stool and urine, viral replication in the gut, and reports of transient viremia and relatively low viral loads in the blood, suggest that viral overspill is perhaps a less likely explanation for the extrapulmonary dissemination of sars-cov that we observed in humans [12] . of interest, angiotensin-converting enzyme 2 (ace2), the putative functional receptor of sars-cov [13] , is expressed in many of the organs in which we observed sars-cov dissemination, including the gastrointestinal tract, heart, kidney, lung, lymph nodes, skeletal muscle, liver, and spleen [14] . gastrointestinal complaints-and watery diarrhea, in particular-are frequent symptoms of sars, reported at presentation by у20% of infected individuals [12] and developing in as many as 70% of individuals during the course of illness [15] . in this study, we demonstrated that the majority of patients had evidence of sars-cov in both large and small bowel, often at high viral loads. these data suggest that sars-cov displays tissue tropism for the bowel and provide a putative mechanism for the frequent occurrence of gastrointestinal symptoms in this population. this hypothesis is supported by observations that sars-cov is frequently and persistently identified in fecal specimens [1, 2, 11] and by recent electronic microscopic evidence indicating viral replication and recovery of sars-cov from postmortem small-bowel specimens [12] . collectively, these observations indicate that enteric involvement is common in sars and have implications for infection-control measures and the potential for fecal-oral transmission in community outbreaks. evidence of hepatic dysfunction is common in sars, and elevated serum alanine aminotransferase levels are observed before death in у40% of patients [6] . in this study, we observed a relationship between the presence of sars-cov in the liver and antemortem abnormal liver function tests, supporting a role for sars-cov in mediating hepatic dysfunction. furthermore, splenic atrophy and lymphadenopathy with tissue necrosis have also been reported in sars [2, 7, 8] . these findings can potentially be explained by our observations of elevated viral loads at these sites. ding et al. hypothesized that the degenerative changes observed in these organs are most likely due to disturbed cell metabolism caused by rapid viral replication [8] , but the authors lacked the viral-load evidence we present here to support these contentions. although organ damage in patients with sars may be the immunopathologic consequence of an exuberant host response [2] , evidence of elevated viral loads and of extrapulmonary viral dissemination suggests that regional viral replication could also potentially contribute to organ dysfunction and exacerbate the host response at these sites. although sars-cov has previously been detected in urine and stool [1, 2, 11] from patients with sars, to our knowledge there is only 1 report of sars-cov detection in kidney [3] . in addition, kuiken et al. found pcr evidence of sars-cov in kidney, duodenum, and stomach in 1 of 4 sars-cov-infected macaques, but no viral loads were given [11] . our findings of high viral loads in the gut and liver and moderate viral loads in the kidney are consistent with previous reports of elevated viral shedding in the stool and moderate to low shedding in urine [1, 2] . the current findings provide insight into multiorgan sars-cov dissemination and viral load at the time of death, on the basis of a prospective and systematic examination of a large number of fatal sars cases. sars-cov was consistently identified in the lungs of patients who died of sars and was not found in control individuals, supporting a direct role for sars-cov in contributing to fatal outcomes. sars-cov disseminates to other organs, which may explain, at least in part, the clinical manifestations and pattern of viral shedding observed in sars-cov-infected patients and may provide insight into the selection of appropriate clinical samples in the event of a future outbreak. identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence the severe acute respiratory syndrome outbreak of severe acute respiratory syndrome in hong kong special administrative region: case report the clinical pathology of severe acute respiratory syndrome (sars): a report from china sars coronavirus: a new challenge for prevention and therapy absolute association of coronavirus in lung tissue from fatal cases of severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study we thank the health care providers of the greater toronto region for their dedication and devotion to patient care during the severe acute respiratory syndrome (sars) outbreak. in addition, we thank nate kreiswirth, olga imas, george moussa, and the office of the chief coroner of ontario, for expert technical assistance with this study. we also thank hans-wilhelm doerr (university of frankfurt, frankfurt, germany) and matthias niedrig (robert koch-institute, berlin, germany) for generously providing the standard preparation of sars coronavirus cell culture isolate. key: cord-308342-ycdok8fc authors: shutler, j.; zaraska, k.; holding, t. m.; machnik, m.; uppuluri, k.; ashton, i.; migdal, l.; dahiya, r. title: risk of sars-cov-2 infection from contaminated water systems date: 2020-06-20 journal: nan doi: 10.1101/2020.06.17.20133504 sha: doc_id: 308342 cord_uid: ycdok8fc following the outbreak of severe acute respiratory syndrome coronavirus (sars-cov-2) in china, airborne water droplets (aerosols) have been identified as the main transmission route, although other transmission routes are likely to exist. we quantify sars-cov-2 virus survivability within water and the risk of infection posed by faecal contaminated water within 39 countries. we identify that the virus can remain stable within water for up to 25 days, and country specific relative risk of infection posed by faecal contaminated water is related to the environment. faecal contaminated rivers, waterways and water systems within countries with high infection rates can provide infectious doses >100 copies within 100 ml of water. the implications for freshwater systems, the coastal marine environment and virus resurgence are discussed. the outbreak of the severe acute respiratory syndrome coronavirus (sars-cov-2) began 28 in wuhan province, china in december 2019 and has now spread throughout the world 29 with about 6 million cases confirmed globally within 214 countries and territories. water 30 aerosols originating from individuals infected by sars-cov-2 are considered a major 31 pathway for infection 1 , and the virus has been shown to remain stable in saline solution 2 32 and under varying environmental conditions 3 . viral shedding in faeces of viable sars-33 cov-2 virus is documented (eg 4 ) and sars-cov-2 ribonucleic acid (rna) has been 34 detected in the shed faeces of both symptomatic and asymptomatic children and adults 35 (eg 5 ); with potentially 43% of infections being asymptomatic and unreported 6 . 36 human viral pathogens that can be transmitted by water that pose moderate to high health 38 significance as defined by the who include adenovirus, astrovirus, hepatitis a and e, 39 rotavirus, norovirus and other enteroviruses. the survival of the large family of 40 coronavirus in water systems has been highlighted 7 , and viral loads within untreated 41 wastewater, consistent with population infection rates, have been identified 8 . while 42 evidence for sars cov-2 is limited, other human coronaviruses are documented to 43 survive in wastewater effluent 9 , with colder water temperature likely to increase survival 44 considerably 3 . collectively this evidence suggests that sars-cov-2 virus can survive 45 within water and the viral loads within untreated sewage effluent are likely high in countries 46 of high infection rates, a portion of which is viable virus, and therefore water contaminated 47 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 20, 2020. . and c) countries where relative risk has been calculated with relative risk as a linear scale; grey signifies a country not included. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 20, 2020. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 20, 2020. virus copies, the proportion of infectious viruses in sewage must be known. the presence 117 of infectious virus in stool samples has been demonstrated 4 , but there is a lack of 118 quantitative data on this ratio for sars-cov-2 in stool. we instead used literature on the 119 number of infectious adenovirus copies in sewage (eg 16 ) and wastewater discharge into 120 rivers 17 to select high (10 -1 ) medium (10 -2 ) and low (10 -3 ) estimates for the ratio of 121 infectious virus to genome copies to infectious viruses. we note that adenoviruses are 122 known to be particularly resilient, and therefore likely to represent an upper estimate, but 123 also that our selected range is consistent with the 10 -3 value used elsewhere for assessing 124 viral risk in water systems (eg 14 ), including one assessment for sars cov-2 transmission 125 risk to wastewater workers 18 . 126 the temperature dependent survivability means that it is likely that the risk posed by 128 wastewater will increase during winter months as the sewage temperature will be lower 129 enabling longer viral survival, but temperature history and age of the sewage will be 130 needed to fully understand any detected viral loads. sars-cov-2 infection to, and spread 131 between, domestic cats has occurred due to similarities between human and some animal 132 angiotensin converting enzyme 2 (ace2) gene 20 . increased animal foraging can occur 133 downstream from water treatment facilities, relative to upstream, highlighting possible risk 134 of some riparian wildlife infection if feeding occurs after a spill. 135 136 it is possible that sars-cov-2 survivability and transport within rivers could impact 138 drinking water supplies in countries where rivers or reservoirs are the primary drinking 139 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 20, 2020. . https://doi.org/10.1101/2020.06.17.20133504 doi: medrxiv preprint water sources and where large populations, with little or no sewage treatment, exist close 140 to the water source, such as within refugee camps or shanty towns. riverine enteric virus 141 transport and catchment accumulation can occur for common viruses (eg 21 ) and under 142 stratified conditions it would be possible for a river plume to enter a reservoir and 143 subsequently exit through the reservoir outlet without mixing with the main body of water. 144 filtering of water, followed by ultraviolet disinfection or chlorination are the recommended 145 approaches for virus removal from drinking water sources 22 . filtering is normally used to 146 remove large particulates. the effective ultraviolet dose for sars-cov-2 disinfection 147 appears highly variable and dependent upon the surface to which the virus is attached 23 . 148 the upper dosage value of 1 joule (j) cm -2 to ensure effective ultraviolet disinfection of 149 sars-cov-2 23 is an order of magnitude larger than that typically used (~40 to 90 mj cm -150 2 ) for low volume domestic drinking water treatment. the world health organization 151 (who) guidelines state that effective chlorination disinfection occurs at residual chlorine 152 concentrations of ≥0.5 mg l -1 22 , which matches the minimum needed to deactivate 153 sars-cov-1 24 . however, the actual chlorine dosage used for water treatment can vary, 154 based on country, region, water origin and infrastructure (eg uk guidelines are 155 concentrations of 0.2 to 0.5 mg l -1 ). collectively this means that if a drinking water source 156 was to become infected with sars-cov-2 the standard virus removal and disinfection 157 approaches of ultraviolet exposure and chlorination may not reduce the virus below 158 detectable limits. reviewing of regional or countrywide drinking water processing 159 approaches is recommended to reduce the potential for sars-cov-2 surviving through 160 drinking water processing systems. boiling of drinking water will result in the virus being 161 orca and pilot whales 20 . of particular concern are whales whose throats are exposed to 177 large volumes of water during feeding and who visit coastlines for prey that are known to 178 accumulate around sewage outfalls, such as minke whales feeding on mackerel or orca 179 feeding on chinook salmon. in these instances, the animal could be exposed to a large 180 viral dose, even if the virus is only present within the water in low concentrations. for 181 example, if the riverine viral concentration is low at 1 copie ml -1 , which is undetectable by 182 pcr (detection limit is >100 copies ml -1 ), then a medium sized whale filtering water during 183 feeding could receive repeated doses of 5.65 million copies every second (see methods 184 for calculation). a seafood market is among the suspected sources for the origin of the 185 sars-cov-2 virus, so any viral transmission from land to sea may be a circular process. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 20, 2020. . https://doi.org/10.1101/2020.06.17.20133504 doi: medrxiv preprint the relative risk of sars cov-2 from waste water systems is calculated by using a 217 modified version of equations 1 and 2 from 13 , given as 218 where v ww,c is the per capital daily volume of domestic water usage for country c, and df c 220 is the dilution factor downloaded from 13 supplemental table 1 and supplemental table 2 we note that measured wastewater viral counts in paris on the 9 th april were 3.1 × 10 6 235 genome copies l -1 with 82,000 active cases 19 , whereas using our (albeit country specific) 236 method gives the estimate of 1.3 × 10 6 genome copies l -1 , which is within the correct order 237 of magnitude (this calculation used the same number of active cases). 238 239 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 20, 2020. . https://doi.org/10.1101/2020.06.17.20133504 doi: medrxiv preprint to calculate c faeces we assumed a log-normal distribution and calculated the expected 240 value using the mean and standard deviation from 12 using the standard equation: 241 where µ is the sample mean and σ is the sample standard deviation of the log normal 243 distribution. 12 state that µ of the distribution is 5.22 log 10 copies ml -1 and σ = 1.86 log 10 244 copies ml -1 which results in an expected c faeces concentration within the sewage effluent of 245 1595.9 million copies ml -1 . v faeces is the mean daily volume of faeces generated per person 246 (0.149 kg, from table 3 of 26 and assuming faeces has a density approximately equal to 247 water 27 . note we used the 'rich country' value from 26 because the rt-pcr data 12 that 248 we use to estimate c faeces was measured from samples collected in germany. the four orders of magnitude, and as such we selected high (10 -1 ), medium (10 -2 ) and low (10 -257 3 ) estimates (which equate to 10%, 1% and 0.1% proportion of viable versus within the 258 total viral genome counts). 259 the expected number of copies of infectious virus resulting from a sewage spill into a river, 261 lake or coastal region for a given country can therefore be calculated as 262 c spill,c was estimated for may 3 2020 21 countries that contain large inland water bodies 264 and were known to rely upon reservoirs for drinking water 28 . long-term statistical mean 265 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 20, 2020. . https://doi.org/10.1101/2020.06.17.20133504 doi: medrxiv preprint water temperature, needed to calculate virus survivability, was calculated from a climate 266 quality global lake temperature dataset (see below). temperature values for each country 267 were the countrywide mean lake temperature within a rectangular box matching a 268 simplified country outline. the dilution factors reported in 13 can vary by several orders of 269 magnitude and were deemed to provide the major source of uncertainty in the calculation. the concentration of sars-cov-2 virus needed for infection is not known. 30 provides 10 3 284 copies for influenza. the infectious dose for sars-cov-2 is likely significantly lower 285 because 31 ranks influenza as "very high infective dose" and sars-cov-2 as "low". we 286 therefore use a value of 100 copies as a concentration that could result in infection. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 20, 2020. . https://doi.org/10.1101/2020.06.17.20133504 doi: medrxiv preprint number of active cases (±20%), mass of faeces generated per capita per day (0.095 kg, 292 see table 3 of 26 , mean number of viral genome copies in faeces (3.54×10 12 ) and density of 293 faeces was not included in the uncertainty analysis. this resulted in a combined 294 uncertainty budget of ±68% copies ml -1 . it is important to note that this value does not 295 include uncertainty in the dilution factors or the ratio of viral genome copies to infectious 296 virus. instead, the c inf calculation was repeated for high, medium and low values of these 297 parameters. 298 299 as reported in 3 , the virus concentration in water follows an exponential decay, with its 301 half-life decreasing with decreasing temperature and the ph control of half life is very small 302 over the ph range of 3-10 (which encompasses the range found in natural freshwater and 303 marine systems). based on the in vitro data presented in 3 , the following empirical model 304 was derived to describe virus concentration reduction factor due to the temperature-305 dependent die-off: 306 = 10 !.05!°! !!.32 (5) 307 = ! 10 !!" (6) 308 where c 0 is initial virus concentration (copies ml -1 ), n(t) is virus concentration after time t 309 (days) and r is 24 hour survival factor due to temperature t driven die off. this model fit to 310 the in vitro data gives a root mean square difference (rmsd) of ±1% for water at 4°c 311 which increases to ±7.5% at 22°c. when considering temperature controlled survival in 312 the waste water system, c ww,c becomes the value used for the initial viral load c 0 following 313 a sewage effluent spill. as noted in 5,12 , the viral load follows a heavy-tailed distribution 314 with the majority of patients shedding around 10 5 copies ml -1 ) but some having viral loads 315 as high as 10 12 copies ml -1 . this results in the super-spreader problem where a tiny 316 proportion of the infected population can become responsible for contributing a majority of 317 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 20, 2020. . https://doi.org/10.1101/2020.06.17.20133504 doi: medrxiv preprint viral load in the wastewater. for a large infected population, this approach allows robust 318 statistical modeling of viral load. however, in case of smaller communities with low number 319 of infections, the actual viral load could be severely underestimated if a super-spreader is 320 present within the population. 321 322 the example volume flow rate through the mouth of a medium sized bowhead whale 324 whilst feeding was provided by 32 ) a flow rate of 5.65 m 3 s -1 is given for a 15 m whale 325 (mouth pressure of -1768 pa at a 4 km h -1 foraging speed, assuming an oral opening of 326 5.09 m 2 with an opening radius = 1.27 m). assuming a low viral concentration of 1 copies 327 per ml -1 , which equates to 1000 copies l -1 . 5.65 m 3 s -1 equates to 5650 l s -1 . the dosage 328 per second as the whale swims during feeding is given by 1000 (copies l -1 ) × 5650 (l s -1 ) 329 = 5.65 million copies s -1 . 330 severe acute 333 respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 334 (covid-19): the epidemic and the challenges evaluation of saline, phosphate buffered saline and minimum 337 essential medium as potential alternatives to viral transport media for sars-cov-2 338 testing stability of sars-cov-2 in different environmental conditions infectious sars-cov-2 in feces of patient with severe covid-19 virological assessment of hospitalized patients with covid-2019. 344 suppression of covid-19 outbreak in the municipality of first detection of sars-cov-2 in untreated wastewaters in italy first confirmed detection of sars-cov-2 in untreated wastewater 350 in australia: a proof of concept for the wastewater surveillance of covid-19 in the 351 community potential fecal transmission of sars-cov-2: current evidence and 356 implications for public health high levels of sewage contamination released from urban areas 359 after storm events: a quantitative survey with sewage specific bacterial indicators an analysis of sars-cov-2 viral load by patient age worldwide estimation 364 of river concentrations of any chemical originating from sewage-treatment plants 365 using dilution factors discharge-based 368 qmra for estimation of public health risks from exposure to stormwater-borne 369 pathogens in recreational waters in the united states coronavirus 372 in water environments: occurrence, persistence and concentration methods -a 373 scoping review an improved 375 infectivity assay combining cell culture with real-time pcr for rapid quantification of 376 human adenoviruses 41 and semi-quantification of human adenovirus in sewage quantification of human adenovirus and norovirus in river water in 379 the north-east of france qmra of sars-cov-2 for workers in wastewater treatment 382 plants evaluation of lockdown impact on sars-cov-2 dynamics through 384 viral genome quantification in paris wastewaters broad host range of sars-cov-2 predicted by comparative and 387 structural analysis of ace2 in vertebrates evidence of viral dissemination and seasonality in a mediterranean 390 river catchment: implications for water pollution management water, sanitation, hygiene, and waste water management for the covid-19 393 virus: interim guidance rapid 395 evidence summary on sars-cov-2 survivorship and disinfection, and a reusable 396 ppe protocol using a double-hit process. medrxiv (2020) study on the resistance of severe acute respiratory syndrome-399 associated coronavirus freshwater clams as bioconcentrators of avian influenza 402 virus in water. vector-borne zoonotic dis the characterization of feces and 405 urine: a review of the literature to inform advanced treatment technology review of synthetic human faeces 409 and faecal sludge for sanitation and wastewater research world water: resources, usage and the role of man made reservoirs lake surface water temperature (lswt) 414 v4.0 (1995-2016) influenza virus aerosols in the 417 air and their infectiousness emerging threats from zoonotic coronaviruses-from sars 419 and mers to 2019-ncov models of hydrodynamic flow in the bowhead whale filter feeding 422 apparatus key: cord-309474-9h9w46eq authors: schiaffini, riccardo; barbetti, fabrizio; rapini, novella; inzaghi, elena; deodati, annalisa; patera, ippolita p; matteoli, maria c; ciampalini, paolo; carducci, chiara; lorubbio, antonella; schiaffini, gabriele; cianfarani, stefano title: school and pre-school children with type 1 diabetes during covid-19 quarantine: the synergic effect of parental care and technology date: 2020-07-03 journal: diabetes res clin pract doi: 10.1016/j.diabres.2020.108302 sha: doc_id: 309474 cord_uid: 9h9w46eq abstract introduction management of type 1 diabetes (t1d) poses numerous challenges, especially for young children and their families. parental care positively influencesthe outcomesofchildren with t1d, while there are often criticisms in school environment. the covid-19 pandemic has forced children and parents to spend many hours at home and diabetes care has returned mainly in the hands of parents. aim of the study to evaluate the effectiveness of exclusive return to parental care in pre-school and school children with t1d treated with tandem basal iq system during the covid-19 pandemic. patients and methods 22 children (m:f = 14:8) with t1d have been evaluated. we compared insulin and cgm data (tir, tbr and tar) of two periods: pre-cov and in-cov, in which children have transitioned from normal school attendance to the exclusive care of their parents. results during the in-cov period a significantly (p < 0.001) higher median value of tir (66,41%) was observed as compared to pre-cov period (61,45%). patients also showed a statistically significant difference (p < 0.002) between the in-cov period and the pre-cov period as concerning the tar metric: respectively 29,86 ± 10,6 % vs 34,73 ± 12,8 %. the difference between the bolus insulin doses was statistically significant (pre-cov 5,3 iu/day, in-cov 7,9 iu/day – p<0.05). conclusion our observational real-life study confirms the positive effect of parental care in t1d very young children and demonstrates that during the covid-19 pandemic it was possible to obtain a good glycometabolic compensation despite the significant change in lifestyle. type 1 diabetes (t1d) is one of the most common chronic diseases in infancy 1 and the most frequent endocrinopathy in childhood. it is estimated that about 20000 children are affected by t1d in italy 2. correct management of t1d involves frequent blood glucose monitoring, insulin therapy, dietary indications and structured physical activity, representing a high burden on young children and their families. because of these daily challenges, effective diabetes treatment requires -in principlecomplete parental dedication and involvement 3. moreover, parents and teachers of kindergarteners and of children in primary school usually experience the additional challenges of a critical management in school hours, due in particular to the fear of hypoglycemia, the extreme glycemic variability of this age group and the difficulties in correcting the hyperglycemic peaks. parents of kids diagnosed with t1d early in life tend to be proactive in the care of diabetes of their children during school and pre-school periods with a strong parental involvement in disease management and a positive influence on the metabolic and psychosocial outcomes 4-5. conversely, parents of patients diagnosed in late childhood or in adolescence are less involved in care, and usually this is associated with less than optimal glycemic control 6. nevertheless, parental care remains important throughout childhood into young adulthood and a progressive sharing of responsibilities is considered an important step towards a therapeutic approach well balanced between self-monitoring and quality of life 7. the gap between family and school care capacities can be bridged by the use of technologies in the treatment of diabetes in children. in the last two decades, new tools for the management of children with t1d have been proven to be safe and useful. continuous subcutaneous insulin infusion (csii), continuous glucose monitoring (cgm) and remote monitoring in case of multiple daily injections (mdi) seem to be valid options to manage children with diabetes. in particular, cgm systems and remote control access have improved the treatment and management of diabetes during school hours. parents of kindergartner (pre-school) and school children reported that the use of remote monitoring and cgm was effective in control glucose excursions 8. in particular, the use of technologies capable of reducing hypoglycemic risk, such as the tandem basal iq system, have proven to be helpful in reducing parental burden. this system is able to prevent/reduce hypoglycemia thanks to cgm real-time data and was introduced in italy about 6 months ago. covid-19 pandemic has forced children and parents to spend many hours together at home, reducing structured physical activity while bringing back diabetes care in the hands of parents. we aimed at investigating whether this unusual situation lead to an improvement or a worsening of the glucose control. this is a real-life, retrospective, observational study aimed at evaluating how constant parental care compared to spending time outside home affected glycemic control in pre-school and school children with t1d utilizing tandem basal iq system before and during the quarantine period due to pandemic covid-19 infection. the diabetes unit of the bambino gesù children's hospital -rome, italy, regularly follows 980 type 1 diabetes pediatric patients (age 0-18 years); of these patients, 90 use the tandem basal iq technology (considered as an inclusion criteria) and 29 are in the school-preschool age range; finally 22 pre-school and school children (m:f = 14:8) with t1d have been retrospectively evaluated (7 patients/parents resulted not reachable during the lockdown period). the mean age was 8,7 ± 1,9 years (range 3,5-10,5 years) and the diabetes duration was at least of 1 year. enrolled patients were all being intensively insulin treated with the tandem basal iq technology for at least six months. tandem basal iq technology consists of an insulin pump integrated with a dexcom g6 glucose sensor capable of previously suspending insulin delivery in case of hypoglycemia prediction. a multidisciplinary team (diabetologist, nurse, dietitian and psychologist) dispensed a standardized protocol of education to all patients enrolled and their parents at the time of diabetes diagnosis and at 6 months intervals. all the patients and their families were educated in carbo-counting procedures and were instructed to follow a balanced nutritional program consisting in 55% of carbohydrates, 20% of proteins and 25% of lipids. the study was conducted according to the declaration of helsinki. participants and their parents provided informed consent to have their cgm data downloaded at regular intervals, as part of routine clinical control and the study was ethically approved. potential conflict of interest do not exist. beginning march 9, 2020, the start of the lockdown in italy, all patients and families were asked to stay at home due to the covid-19 pandemic emergency. during the pandemic, the children maintained the same diet with a similar distribution between the different macronutrients and they were unable to carry out any structured physical activity, as often happens also in routine life, considering the young age of the group. we compared cgm data of the last two weeks of "normal life" (normal school attendance) (pre-cov period) with the first two weeks of confinement at home (in-cov period) . the following cgm metrics were evaluated: time in range (tir -percent of time in the ideal range of glucose between 70 and 180 mg/dl), time above range (tar -percent of time above 180 mg/dl), time below range (tbr -percent of time below 70 mg/dl). we also compared the insulin requirement in the 2 different periods, in terms of total insulin (iu/day), basal insulin delivery and insulin administered as boluses. all data were extracted from the dexcom clarity and diasend platforms. results are reported as the mean ± sd. normal distribution was assessed by shapiro-wilk test. mean variations of distributions were evaluated using the student's t test for paired data. the entire analyses were performed using spss 25.0 (spss inc., chicago, ill, united states) with p < 0.05 considered significant. data from 22 children (mean age 8,7 ± 1,9 years) (m:f 14:8) with t1d and disease duration longer than 1 year were analyzed. the percentage of time of cgm wearing was 98%. no significant differences between the two evaluated periods (in-cov vs pre-cov) were found in tbr (hypoglycemia): respectively 3,73 ± 3,04 % vs 3,95 ± 4,4 % (tab. 1). in contrast, during the in-cov period a significantly (p <0.001) higher median value of tir (66,41%) was observed as compared to pre-cov period (61,45%) (tab 1 and fig.1 ). conversely, patients showed a lower tar during in-cov period than pre-cov period 29,86 ± 10,6 % vs 34,73 ± 12,8 %, p < 0.002). interestingly, no differences between in-cov and pre-cov periods were observed regarding the total insulin dose (20,7 vs 18,2 iu/day) and the basal insulin delivery (13,3 vs 11,9 iu/day), while a statistically significant difference (p<0.05) was found between the mean bolus doses (7,9 vs 5,3 iu/day) and the daily number of correction boluses (3,1 vs 1,9 iu/day). in this group of pre-school and school t1d children using cgm and semi-automated insulin delivery systems (tandem basal iq), the forced and exclusive return to parental care due to the "stay at home" rule decided by the italian government was associated with a better metabolic performance with higher percentage values of tir and lower mean values of tar. this result seems to be due to a greater use of correction boluses; in fact, our evaluation shows a significant increase in insulin boluses during the in-cov period as compared to pre-cov one. we speculate that similar results will be hardly seen in adolescents and middle school teens, because the management of the disease in these age groups remains in the hands of the patients even during confinement the and physical activity has no significant impact in younger children. nonetheless, we think that it would be worthwhile to investigate older age groups in countries where the lockdown is still in force, though we recognize that other variables -say good or bad interaction with parents-are probably at work in adolescents with t1d. we predict that a better outcome in terms of tbr could be likely in this group because of increased regularity of meals' content and stringent control on alcohol consumption 9. the study has some limitations: first, patients selection limited only to preschool/school children who used tandem basal iq technology can represent a selection bias; secondly, the small size of the sample may not make the results obtained generalizable. our observational real-life study confirms the positive effect of parental care in t1d very young children and that, though new technologies can potentially improve diabetes outcomes also in this sub-population, maintenance of a good glucose control remains largely dependent on family competence and education 10. usually the majority of young people with t1d spend many hours at school. trained school-staff is therefore essential to provide a safe environment for children with diabetes. teachers, along with the school's auxiliary staff, play a key role in reducing strong glycemic oscillations typical of younger children 11. our study shows also a no significant differences in tbr, despite the shift to predominant parental care. this can be explained by the fact that all the evaluated children were already using a technology (tandem basal iq system) that effectively reduces the risk of hypoglycemia and therefore for this specific metric it can be assumed that parental intervention does not change the outcomes. in the past 2 decades, technological innovations have revolutionized the treatment of t1d and the real-world data highlighted that patients using insulin pump therapy have a better short and long-term glycemic control relative to the matched injection therapy groups 12. however, despite the use of technology, parental intervention still seems to be more effective and probably the difference in the near future could be made by the spread of hybrid closed loop (hcl) systems. in the real-world experiences hcl use is associated with improved glycemic control and no change in psychosocial outcomes 13. why children with t1d attending school are often not adequately managed? recent studies showed that a limited availability of glucagon kits, the shortage of trained personnel able to manage daily diabetes-specific emergencies and a reduced ability to perform an adequate carbohydrate counting, considered an effective means to provide good glycemic control, are the main causes 14-15. consequently, family-based interventions for youth with t1d are believed to be effective at improving diabetes outcomes 16. since young patients with t1d spend most of daytime at school, teachers and assisting personnel should receive appropriate training in order to provide a safe environment. however, it is likely that teachers of kindergartens, preschools and primary schools have not enough knowledge to appropriately assist children with t1d 17-18. this may negatively reverberate on trust in school personnel of parents of very young children with t1d, whose perception of the burden of care is very strong. thus, health care providers, parents, teachers, and school assistants of youngsters with t1d should team up to improve, by specific educational programs 19-20, the skills to handle the disease and guarantee a safe attendance at school. type 1 diabetes evaluating parents' self-efficacy for diabetes management in pediatric type 1 diabetes collaborative involvement of primary and secondary caregivers: associations with youths' diabetes outcomes the impact of managing school-aged children's diabetes: the role of child behavior problems and parental discipline strategies caregiving for children with type 1 diabetes and clinical outcomes in central india: the idream study shared responsibility for type 1 diabetes care is associated with glycemic variability and risk of glycemic excursions in youth use of remote monitoring with continuous glucose monitoring in young children with type 1 diabetes: the parents' perspective social consumption of alcohol in adolescents with type 1 diabetes is associated with increased glucose lability, but not hypoglycaemia the impact of technology on current diabetes management diabetes care in the school setting: a position statement of the american diabetes association real-world outcomes of insulin pump compared to injection therapy in a population-based sample of children with type 1 diabetes six months of hybrid closed loop in the real-world: an evaluation of children and young adults using the 670g system are children and adolescents with type 1 diabetes in saudi arabia safe at school? effects of carbohydrate counting method on metabolic control in children with type 1 diabetes mellitus family-based interventions targeting improvements in health and family outcomes of children and adolescents with type 1 diabetes: a systematic review level of knowledge and evaluation of perceptions regarding pediatric diabetes among greek teachers special needs of children with type 1 diabetes at primary school: perceptions from parents, children, and teachers i'm essentially his pancreas": parent perceptions of diabetes burden and opportunities to reduce burden in the care of children <8 years old with type 1 diabetes ispad clinical practice consensus guidelines 2018: management and support of children and adolescents with type 1 diabetes in school the authors declare no conflict of interest. the authors received no funding from an external source. key: cord-310239-mmvuij3k authors: arentz, susan; yang, guoyan; goldenberg, joshua; beardsley, jennifer; myers, stephen p.; mertz, dominik; leeder, stephen; hunter, jennifer title: clinical significance summary: preliminary results of a rapid review of zinc for the prevention and treatment of sars-cov-2 and other acute viral respiratory infections date: 2020-08-01 journal: adv integr med doi: 10.1016/j.aimed.2020.07.009 sha: doc_id: 310239 cord_uid: mmvuij3k nan zinc may potentially reduce the risk of sars-cov-2 infections and shorten the duration and severity of illness, including recovery from stroke, through several mechanisms. indirect evidence from systematic reviews have found zinc supplementation is effective for the prevention of acute respiratory infections in young children and zinc lozenges may reduce the duration of the common cold in adults. safety concerns associated with high doses or prolonged intake of zinc include anosmia (loss of smell) and copper deficiency. as of the 9 june 2020, the preliminary findings of a rapid review of zinc for the prevention or treatment pending any definitive evidence, clinicians might consider assessing the zinc status of people with chronic disease co-morbidities and older adults as part of a sars-cov-2 clinical work-up, as both groups have a higher risk of zinc deficiency/insufficiency and poorer outcomes from sars-cov-2. supplementation might be indicated for those with low or borderline low results, low dietary intake and/or increased needs. the global covid-19 pandemic has prompted an urgent search for pharmaceutical and traditional, complementary and integrative medicine (tcim) interventions. data from all countries indicate that the case fatality and morbidity rates from sars-cov-2 increases with age and for those with noncommunicable chronic disease co-morbidities. [1] [2] [3] [4] notably, zinc deficiency/insufficiency is prevalent in populations aged over 71 years, [5] [6] [7] [8] [9] , in people with chronic diseases [10] [11] [12] including diabetes, [10, 12, 13] and cardiovascular diseases [10, 12] and hospitalised patients following stroke [14] -see box 1. [ insert box 1] j o u r n a l p r e -p r o o f south-east asia, sub-saharan and central and south american regions, however, marginal deficiencies are also prevalent in developed regions. [33, 34] assessment of zinc status is notoriously difficult due to absence of sensitive and precise biochemical indicators. the most reliable methods involve combining a clinical assessment with laboratory tests assessing tissue concentrations of zinc in plasma or hair. [35] clinical manifestations of mildmoderate zinc deficiency include recurrent infections, slow tissue repair, rough skin, mental lethargy, irritability, headaches and reduced lean body mass. [36] assessment of dietary zinc with validated food frequency instruments may help identify dietary insufficiency [37] however zinc status is still likely to be underestimated due to individual physiological characteristics. [31] for instance, whilst zinc insufficiency/deficiency is known to diminish antibody and cell-mediated immunity in humans that in turn increases the risk of infections, this may only become apparent upon immune system provocation. [38, 39] through several mechanisms, zinc has the potential to reduce the risk of viral respiratory tract infections, including sars-cov-2, and shorten the duration and severity of illness. the authors of a recent non-systematic narrative review of the underlying mechanisms postulate that along with its direct antiviral properties, zinc has the potential to reduce inflammation, improve mucocillary clearance, prevent of ventilator-induced lung injury, and modulate antiviral immunity. in vitro studies have demonstrated that zinc can inhibit the enzymatic activity and replication of sars-cov rna polymerase and may inhibit angiotensin-converting enzyme 2 (ace2) activity. [40, 42, 43] the antiviral effects of zinc are also hypothesised to potentiate the therapeutic effects of chloroquine, [44] , as chloroquine acts as a zinc ionophore increasing zn 2+ influx into the cell. [40] zinc may also modify the host's response to an infection as it is an essential co-factor element with a broad range of functions in the body. zinc has an essential role in immune and airways function, wound healing and tissue repair that in turn, may delay or prevent recovery from viral respiratory illnesses. [45] [46] [47] [48] [49] [50] [51] other consequences of zinc deficiency include an increased risk of vitamin a deficiency that is also critical for immune function, due to carrier proteins and activation enzymes being dependant on sufficient zinc status. [52] the potential role of zinc as an adjuvant therapy for sars-cov-2 may be broader than just antiviral and/or immunological support. zinc also plays a complex role in haemostatic modulation acting as j o u r n a l p r e -p r o o f an effector of coagulation, anticoagulation and fibrinolysis . [53, 54] zinc is also essential for neurological function and normalisation of zinc intake has been shown to improve neurological recovery following stroke. [14] the effectiveness of zinc in preventing or treating sars-cov-2 infections is yet to be systematically evaluated and, along with other nutritional supplements, was not mentioned in a recent narrative review of tcim for the treatment of coronavirus disease 2019 . [55] the findings of systematic reviews of related populations are promising; however, the reviews are limited by population, intervention, or are out of date. [56] [57] [58] a 2016 cochrane review of six rcts concluded zinc supplementation was effective for the prevention of pneumonia in children aged two to 59 months. [57] unlike an earlier review in 2000 of seven rcts with adult participants and one rct with children, [59] an updated 2011 systematic review of 13 rcts found a dose-dependent effect of zinc lozenges compared to placebo controls for reduced duration of common colds in adults. [60] daily dosages less than 75mg of zinc had no significant effect on duration of colds, however, daily dosage over 75mg reduced the duration of colds by 42% (95% ci: 35% to 48%). in a subsequent 2017 systematic review of seven rcts of zinc lozenges with a daily dose >75mg, a smaller reduction of 33% (95% ci 21% to 45%) in the duration of common colds was found. [61] no differences in duration were found for daily doses of 192-207mg compared to doses of 80-92mg. other formats of zinc for preventing or treating upper respiratory infections were examined in three cochrane systematic reviews, however, all were withdrawn. [56, 62, 63] a protocol for the systematic review of zinc for prevention and treatment of common colds was withdrawn in 2019 due to noncompletion within the editorial time-frame. [64] search strategy: the primary objective of this rapid review was to assess the effects of zinc on the incidence, duration and severity of acute upper or lower respiratory tract infections caused by sars-cov-2 infection in people of any age and of any zinc status when used as a preventive supplement or as a therapy. the secondary objectives are to assess the effects of zinc on the incidence, duration and severity of assessed rob and extracted data for each study. primary studies included were randomized controlled trials (rcts) and quasi-randomised controlled trials. there were no date nor language restrictions, however, studies published in languages other than english or chinese are yet to be translated. included were any zinc conjugates, such as salts or amino-chelates as a single ingredient, in any form (e.g. tablet, syrup, lozenge, gel, spray, liquid), dose and duration, administered via oral, intranasal, sublingual, transdermal, intramuscular or intravenous routes. excluded excluded were systematic reviews, non-randomised studies of interventions and studies without a concurrent control, such as case series and case reports. excluded were people with respiratory tract infections or other upper/lower respiratory illnesses when the cause was confirmed not to be a viral infection, or a non-viral cause is common. excluded were co-interventions and zinc administered alongside other nutraceuticals, herbs or pharmaceuticals unless both the intervention and control groups received the co-intervention. the exception were co-ingredients with the primary purpose to facilitate absorption (e.g. vitamin b12) or cellular retention (e.g. vitamin b6 or magnesium) of zinc. the a total of 1,625 records were retrieved from the database searches, of which 1,182 records remained after duplicates were removed. a further 981 records were excluded at title and abstract screening, and 80 following full-text screening (due to ineligible study design n=29, population n=11 or intervention n=32; full-text not available n=7; or awaiting translation n=1), leaving 121 records reporting 122 primary studies (86 published in english and 35 in chinese). one study published in spanish is pending translation. four trials specific to sars-cov-2 were included, all of which are currently ongoing, and the investigators have been contacted are yet to report their results. a further 15 ongoing trials were excluded as the interventions used zinc in combination with other nutraceuticals (most commonly vitamin c and d) and/or as an agonist (additive) to hydroxychloroquine. as such, the independent effects of zinc cannot be determined. of the remaining 118 published studies, none investigated zinc for prevention or treatment of acute respiratory infections caused only by a coronavirus infection. most of the studies (79%) evaluated zinc for treating or preventing upper and/or lower acute respiratory infections in children. (table 1 ). all the studies of adult participants were for acute upper respiratory infections i.e. the common cold (table 1) , of which 21 were naturally occurring infections and six inoculated the participants with human rhinovirus species. the prevention effect of zinc was assessed in a variety of ways, mostly as the incidence or recurrence of respiratory infections as reported by study clinicians, the participants' physician or other healthcare workers, parents or self-reports, hospitalisation and/or laboratory tests. treatment effects for severity and duration included time to symptom resolution, fever or respiratory distress, time in hospital, viral shedding, and self or clinician reported clinical severity. a wide range of zinc formulations and dosages were used, including lozenges, nasal gels and sprays, and oral zinc delivered in syrup, tablet or capsule formats. only one study evaluated intravenous zinc. is a four-arm pragmatic rct comparing zinc gluconate only, zinc gluconate and vitamin c, vitamin c only, and usual care (standard prescribed medication/supplements). the dose of zinc gluconate is 50mg daily, taken at bedtime. the primary outcome is the number of days required to reach a 50% reduction in symptom severity score (derived from a composite self-rating score of fever, cough, shortness of breath and fatigue rated on a 0-3 scale). secondary outcomes are time to symptom resolution for each symptom, total symptom composite score at day 5, proportion requiring hospitalisation, use of prescribed adjunctive medicines, and adverse events. methodological limitations include subjective primary outcome measures from unblinded participants, potential uncertainty around the quality and quantity of the ingredients in the supplements, [68] a potentially insufficient dose of elemental zinc and that the usual care group may use any combination of readily available prescribed medications / supplements, including zinc or vitamin c. strengths of the pragmatic design include a capacity to inform 'real-world' decisions about any benefits and risks of additional zinc supplementation using products that are readily available compared to usual care alone. the second study, "high-dose intravenous zinc (hdivzn) as adjunctive therapy in covid-19 positive critically ill patients: a pilot randomized controlled trial" (actrn12620000454976), is being conducted in a hospital setting in australia. hdivzn is a two-arm, double-blind rct comparing intravenous zinc chloride (0.5mg/kg/d) or placebo in 250ml saline bags infused daily over 3-6 hours for seven days. hdivzn aims to recruit 160 patients who are hospitalised with sars-cov-2 infection. the primary outcome is oxygenation. secondary outcomes are concerned with feasibility, including adequacy of blinding, availability/delivery/storage of the zinc infusions and per-patient costs. methodological strengths include blinding and the use of an objective primary outcome measure. limitations include not assessing any other clinical outcomes listed in the core outcome set (cos) for clinical trials on covid-19. [4] the dose of zinc, approximately 50% more than the minimum daily requirement and without an intracellular transporter co-factor, may be insufficient to effect change of the outcome measurements. [69] given this is a single-centre trial located in australia with a low incidence of sars-cov-2, as of the 14 th june 2020, no eligible participants had been recruited to the study; and, according to the investigator a/professor ischia, "due to the low numbers of covid-19 infections, the trial is unlikely to reach full recruitment to achieve its desired statistical power." [70] prevention of sars-cov-2 is being evaluated in a multicentre trial of 660 military health professionals exposed to sars-cov-2 and located in tunisia (nct04377646: a study of hydroxychloroquine and zinc in the prevention of covid-19 infection in military healthcare workers (covid-milit)). participants will be randomized to one of three study arms; either hydroxychloroquine and zinc, hydroxychloroquine and placebo, or two placebo controls. in covid-milit, hydroxychloroquine 400 mg will be administered at day 1 and day 2, then as a weekly dose for up to 2 months. zinc will consist of 15mg per day for up to two months. the primary outcome is the frequency of infection at two months, secondary outcomes are frequency of ten symptoms and adverse events. the low dose of zinc will provide minimum intake required for health. the treatment of sars-cov-2 with either hydroxychloroquine plus zinc compared to hydroxychloroquine alone will be evaluated in 80 hospitalised adults with confirmed sars-cov-2. this study, registered on the iranian clinical registry (irct20180425039414n2; the effect of zinc on the treatment and clinical course of patients with sars-cov2 (covid-19)), is being conducted at the amin hospital in isfahan. participants will be randomised to either hydroxychloroquine 200 mg every 12 hours plus zinc 220mg twice daily, or to hydroxychloroquine alone during their hospital stay. outcomes include mortality rates, length of hospital stay and the clinical course of sars-cov-2 (fever, shortness of breath, cough, blood oxygenation (sao2) and hemodynamic parameters). the treatment j o u r n a l p r e -p r o o f study was designed to ensure that all study participants diagnosed with sars-cov-2 received treatment. preliminary findings of this rapid systematic review found limited direct evidence evaluating zinc for the prevention or treatment of sars-cov-2, as the results of the four registered rcts that were identified are pending. once available, the findings from the covidatoz trial that is evaluating the comparative effectiveness of zinc supplements against vitamin c and usual care for treatment of mild to moderate symptoms of community-based sars-cov2-19 infections, will be relevant to the general population who can self-prescribe, along with a wide range of health practitioners who provide tcim advice. the findings from the hdivzn trial that is evaluating the efficacy and safety of intravenous zinc infusions for hospitalised patients may provide safer and less expensive therapeutic options compared to pharmaceuticals currently being evaluated. delivery of the intervention, however, requires medical oversight that will restrict its application to hospital settings and perhaps a few primary care settings. the two comparative effectiveness studies will not explain the preventative or treatment effects of zinc as a stand-alone therapy, however they will explain the potential benefits of zinc adjunct to hydroxychloroquine in populations at high risk of zinc deficiency, [34] for the prevention of sars-cov-range of functions in the body that modulate immunity, respiratory tract inflammation, coagulation and neurological function to name a few. [14, [38] [39] [40] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] pending any definitive evidence, it might be reasonable for clinicians to consider assessing the zinc status of people with chronic disease co-morbidities and older adults as part of a sars-cov-2 clinical work-up, as both groups have a higher risk of zinc deficiency/insufficiency and poorer outcomes from sars-cov-2. zinc status can be assessed by taking a diet and clinical history (see box 1), clinical examination and laboratory tests. plasma zinc may be more reliable than serum zinc and whilst hair mineral analysis is another option a timely result may not be available. [35] for prevention of sars-cov-2 and most importantly for general health, given that zinc supplements are readily available, they may be indicated for people with low or borderline low results, low dietary intake and/or increased needs. to optimise safety, a daily dose lower than the tolerable upper limits (<7mg for children aged 1-3 years up to 22mg for those aged 15-17 years) should be used along with dietary modifications whenever possible. in adults, doses up to the no observed adverse effect level (noael) of 50 mg/day should be considered. [28] at this stage, it is unclear if there is any additional benefit from supplementing zinc for the prevention of sars-cov-2 or other viral respiratory infections in low risk populations nor for people with normal zinc status. it is also unclear if there are any benefits from supplementing with zinc for the treatment of sars-cov-2. there is limited indirect evidence from viral upper respiratory infections that zinc lozenges with a daily dose of >75mg of zinc may shorten the duration of the common cold. however, there are risks with higher doses above the noael including permanent loss of smell. [28] therefore, a daily dose higher than 100mg of elemental zinc in a lozenge is probably not advisable, as it is questionable whether there are any additional therapeutic effects. [61] disclaimer: this article has not been peer-reviewed; it should not replace individual clinical judgement. the views expressed in this rapid review are the views of the authors and not necessarily from the host institutions. the views are not a substitute for professional medical advice. publications [41] j o u r n a l p r e -p r o o f di napoli r: features, evaluation and treatment coronavirus (covid-19). in: statpearls the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak diabetes is a risk factor for the progression and prognosis of covid-19 a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) low zinc status: a new risk factor for pneumonia in the elderly? mineral intakes of elderly adult supplement and nonsupplement users in the third national health and nutrition examination survey rink l: correlation between zinc status and immune function in the elderly prevalence of zinc deficiency and its clinical relevance among hospitalised elderly. archives of gerontology and geriatrics zinc intake of the us population: findings from the third national health and nutrition examination survey discovery of human zinc deficiency: its impact on human health and disease zinc fluxes and zinc transporter genes in chronic diseases. mutation research/fundamental and molecular mechanisms of mutagenesis serum zinc level and coronary heart disease events in patients with type 2 diabetes copper, chromium, manganese, iron, nickel, and zinc levels in biological samples of diabetes mellitus patients normalization of zinc intake enhances neurological retrieval of patients suffering from ischemic strokes the nutrition crsp: what is marginal malnutrition, and does it affect human function? plant breeding: a long-term strategy for the control of zinc deficiency in vulnerable populations. the american journal of clinical nutrition dietary calcium, phytate, and zinc intakes and the calcium, phytate, and zinc molar ratios of the diets of a selected group of east african children. the american journal of clinical nutrition effect of vegetarian diets on zinc status: a systematic review and meta-analysis of studies in humans effect of gestational zinc deficiency on pregnancy outcomes: summary of observation studies and zinc supplementation trials effects of maternal zinc supplementation on pregnancy and lactation outcomes. food and nutrition bulletin zinc deficiency in pregnancy and fetal-neonatal outcomes and impact of the supplements on pregnancy outcomes the dynamic link between the integrity of the immune system and zinc status zinc in health and chronic disease. the journal of nutrition, health & aging alcoholism causes alveolar macrophage zinc deficiency and immune dysfunction. american journal of respiratory and critical care medicine practice patterns of naturopathic physicians: results from a random survey of licensed practitioners in two us states recent aspects of the effects of zinc on human health ineffectiveness of zinc gluconate nasal spray and zinc orotate lozenges in common-cold treatment: a double-blind, placebo-controlled clinical trial. alternative therapies in health and medicine european commission health & consumer protection directorate-general: scientific committee on food estimating the global prevalence of zinc deficiency: results based on zinc availability in national food supplies and the prevalence of stunting national risk of zinc deficiency as estimated by national surveys use of national food balance data to estimate the adequacy of zinc in national food supplies: methodology and regional estimates indicators of zinc status at the population level: a review of the evidence zinc: the missing link in combating micronutrient malnutrition in developing countries assessing zinc in humans. current opinion in clinical nutrition and metabolic care as: discovery of zinc for human health and biomarkers of zinc deficiency. in: molecular, genetic, and nutritional aspects of major and trace minerals a short 18 items food frequency questionnaire biochemically validated to estimate zinc status in humans reprogramming of the immune system during zinc deficiency zinc supplementation of young men alters metallothionein, zinc transporter, and cytokine gene expression in leukocyte populations zinc and respiratory tract infections: perspectives for covid-19 (review) zinc and respiratory tract infections: perspectives for covid-19. permission to reprint figure 1 papain-like protease 2 (plp2) from severe acute respiratory syndrome coronavirus (sars-cov): expression, purification, characterization, and inhibition zn(2+) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture does zinc supplementation enhance the clinical efficacy of chloroquine/hydroxychloroquine to win todays battle against covid-19? zinc modulates cytokine-induced lung epithelial cell barrier permeability zinc modulates airway epithelium susceptibility to death receptormediated apoptosis apoptosis in the normal and inflamed airway epithelium: role of zinc in epithelial protection and procaspase-3 regulation new insights into the role of zinc in the respiratory epithelium zinc metabolism in airway epithelium and airway inflammation: basic mechanisms and clinical targets. a review impact of zinc metabolism on innate immune function in the setting of sepsis. international journal for vitamin and nutrition research zinc: mechanisms of host defense. the journal of nutrition effect of zinc deficiency on hepatic enzymes regulating vitamin a status zinc homeostasis in platelet-related diseases zinc: an important cofactor in haemostasis and thrombosis complementary, alternative and integrative medicine (cam) for the treatment of coronavirus disease 2019 (covid-19): an overview: licensed under a creative commons attribution 4.0 international license zinc for the common cold zinc supplementation for the prevention of pneumonia in children aged 2 months to 59 months efficacy of zinc against common cold viruses: an overview zinc and the common cold: a meta-analysis revisited zinc lozenges may shorten the duration of colds: a systematic review. the open respiratory medicine journal zinc lozenges and the common cold: a meta-analysis comparing zinc acetate and zinc gluconate, and the role of zinc dosage zinc for the common cold withdrawn: zinc for the common cold zinc for preventing and treating the common cold protocol for a rapid review of zinc for the prevention or treatment of covid-19 and other coronavirusrelated respiratory tract infections in humans rob 2: a revised tool for assessing risk of bias in randomised trials a revised tool for assessing risk of bias in randomized trials the contents of herbal and dietary supplements implicated in liver injury in the united states are frequently mislabeled hdivzn trial progress update on human rhinovirus and coronavirus infections key: cord-297842-hkr1wm3k authors: tilley, kimberly; ayvazyan, vladimir; martinez, lauren; nanda, neha; kawaguchi, eric s.; o’gorman, maurice; conti, david; gauderman, w. james; van orman, sarah title: a cross-sectional study examining the seroprevalence of severe acute respiratory syndrome coronavirus 2 antibodies in a university student population date: 2020-10-15 journal: j adolesc health doi: 10.1016/j.jadohealth.2020.09.001 sha: doc_id: 297842 cord_uid: hkr1wm3k purpose: the aim of the study was to determine the prevalence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) antibodies in a university student population. methods: this was a cross-sectional survey study based on the world health organization population-based seroepidemiological investigational protocol for sars-cov-2 conducted between april 29, 2020, and may 8, 2020, examining sars-cov-2 antibody prevalence among 790 university students in los angeles, ca. participants completed a questionnaire on potential risk factors before blood sampling. samples were analyzed using the euroimmun anti-sars-cov-2 elisa (igg) for the qualitative detection of igg class antibodies to sars-cov-2 in human serum or plasma. results: the estimated prevalence of sars-cov-2 antibody was 4.0% (3.0%, 5.1%). factors associated with having a positive test included history of anosmia and/or loss of taste (95% ci: 1.4–9.6). a history of respiratory symptoms, with or without fever, was not associated with a positive antibody test. conclusions: prevalence of sars-cov-2 antibodies in the undergraduate and graduate student university population was similar to community prevalence. this study demonstrates that the seroprevalence of sars-cov-2 antibodies in a representative sample of a large urban university population is similar to that of the surrounding community. symptoms of prior sars-cov-2 infection in the college-aged student include loss of taste or smell but not a history of respiratory symptoms. at the end of december 2019, a novel coronavirus was identified in patients with pneumonia of unclear etiology in wuhan, china [1, 2] . the disease was termed covid-19 by the world health organization, and the underlying viral agent was subsequently termed severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [3] . over the ensuing months, sars-cov-2 spread across six continents, infecting millions of persons. in attempts to control the rapid spread of the virus, governments around the world have taken unprecedented steps to contain the virusdissuing shelter-in-place orders and closing of all nonessential businesses, including institutes of higher education (ihe). because of widespread testing shortages and the varied range of clinical presentations for covid-19, including asymptomatic infection, the true disease incidence in the u.s. since january 2020 is unknown. given public health concerns with reopening ihe, serologic evaluation to determine the student population prevalence of antibodies to the virus is of utmost importance. in the fall of 2019, approximately 19.9 million students were attending ihe in the u.s., representing more than 5% of the u.s. population [4, 5] . there is limited data on the number of infections with sars-cov-2 associated with ihe. surveys done in the spring of 2020 indicate that <1% of college students report having a confirmed case of covid-19 [6, 7] . early cases of covid-19 infection were associated with international travel, yet by mid-march 2020, community spread was evident in many locations across the u.s. for many ihe, with diverse, interconnected, often residential populations, it was unclear what proportion of the population had been infected with sars-cov-2 before the closure of many campuses in march 2020. a national survey of college students conducted between march and may 2020 reported that 14% of college students indicated that they may be or probably had covid-19 based on health care provider assessment or symptoms but not confirmed by a test [6] . given that the severity of the symptoms increases with increasing age and existence of medical comorbidities, many infections among undergraduate and graduate university students are expected to be asymptomatic or minimally symptomatic [6, 8] . thus, the presence of antibodies to sars-cov-2 is important in assessing the current prevalence of infection on college campuses. the assessment of risk factors in students both with and without the presence of antibodies to sars-cov-2 will help guide ihe as they plan for reopening their physical campuses. given the close proximity in which many university students live in dormitories or other off-campus high-density housing [9] , the potential for rapid spread of sars-cov-2 is a relevant concern. university students are also expected to have different social connectedness compared with the general population, such as participation in athletics or social clubs [9, 10] . in addition, the academic course structure of both undergraduate and graduate student education has been shown to have a high degree of connected networks, thus fostering the social conditions for the spread of an infectious disease such as sars-cov-2 [11] . a recent survey on the behaviors of college students who experienced symptoms consistent with covid-19 found that 30.1% continued to attend classes [7] . previously published seroprevalence studies have focused on community-level spread in cities or countries [12e14]. this study aimed to provide an estimate of infection in students attending a los angeles university with a diverse population, including a large number of international students. it also aimed to explore the risk factors for infection in this population. this information will offer evidence-based strategies for control measures, as ihe plan to reopen campuses. the study was a cross-sectional study examining the prevalence of covid-19 antibodies from blood samples obtained from college students. the study design was based on the world health organization population-based seroepidemiological investigational protocol for covid-19 virus infection [15] . data were collected from april 29, 2020, to may 8, 2020, and prevalence estimates reflect this specific snapshot in time. it is not known how long antibodies persist after infection. there is also potential for some lag in ability to detect antibodies after infection, so prevalence estimates could reflect cumulative infection up to approximately late april 2020. the study was conducted at a large urban university in los angeles, ca. approval was obtained from the institutional review board at this institution, and all participants provided electronic informed consent and health insurance portability and accountability act authorization. on april 29, 2020, the county of los angeles, ca, reported 27,641 cumulative cases of sars-cov-2 infection and had 1,156 recorded deaths related to covid-19 [16] . participants were invited to participate in this study via an email invitation, which was sent to 9,135 students (25% of eligible students) enrolled in the spring 2020 semester. inclusion criteria were (1) participants were eligible to access services at the student health center (i.e., students must be enrolled in six or more credit hours for on-campus programs, not in online degree programs) and (2) students' primary campus was the main campus for this institution (this was a practical consideration). exclusion criteria included (1) students aged <18 years and (2) students who were not in the randomly selected pool of potential participants (i.e., students who heard of the study via word of mouth or other similar means were excluded from participating). although not a criterion for receiving a study invitation, only students still living in the region were truly eligible to participate in this study, given that they had to come to the student health center for a blood draw. a stratified random sampling approach was used with the following subgroups: female undergraduates, male undergraduates, female graduate students, and male graduate students. with the goal of having the sample distributions match distributions in the population, these strata were selected based on internal university data, indicating that females are more likely to participate in health-related research projects compared with males, and fewer undergraduates (36.4%) spent the end of the spring 2020 semester in los angeles relative to graduate students (76.5%). within each stratum, a random selection of students was invited to participate. sign-ups were monitored, and demographic distributions of invitations were adjusted as needed. during recruitment, it was also determined that white domestic students were overrepresented in sign-ups, and students in this group were downweighted for random selection in later waves of invitations so that sufficient data could be collected from nonwhite domestic and international students. five waves of invitations were sent. the invitation email was sent to the student's university email address through the student health electronic health record. each email had a unique study code to ensure that only invited students participated in the study. students accepted the invitation by scheduling an appointment at the student health center. the number of appointments available for students to participate in this study was capped at 800. the euroimmun anti-sars-cov-2 elisa (igg) is an enzymelinked immunosorbent assay intended for the qualitative detection of igg class antibodies to sars-cov-2 in human serum or plasma. euroimmun is licensed for use under the food and drug administration's emergency use authorization. testing requires .5 ml of serum or 2 ml of whole blood. specimens were collected at the student health center by the laboratory staff and sent via courier within 24 hours of collection for testing. the validation program at the frederick national laboratory for cancer research determined that sensitivity of this test is 90% (27/30; 95% confidence interval [ci]: 74.4%e96.5%) and specificity is 100% (80/80; 95% ci: 95.4%e100%) [17] . this test classifies individuals into negative (ratio <.8 is considered negativedthis is a normal result for patients who have no or early sars-cov-2 exposure), borderline (ratio .8 to <1.1), and positive (ratio 1.1) for anti-sars-cov-2. in the independent clinical agreement validation study from the manufacturer borderline, results were counted as negative [17] . individuals who were classified as borderline were asked to return for a follow-up test 14 days later. all participants completed a study questionnaire with questions on the following items: prior exposure to a person with suspected or confirmed covid-19 infection; prior covid-19 diagnosis; prior illness history and symptoms since january 1, 2020; prior loss of smell and/or taste since january 1, 2020; receipt of the influenza vaccine during this academic year; travel history since december 1, 2019, including international and domestic; living situation in early march 2020; and current living situation. those who reported coughing, wheezing, shortness of breath or "other" respiratory symptoms were classified as having experienced respiratory symptoms. demographic data on students were obtained from electronic health records, and demographic data in these records are provided by the university. available data included sex (two categories available only: male and female), race/ethnicity/ international status (the university combines these into one variable with levels including asian/pacific islander, black/african american, hispanic/latino, international from any non-u.s. country, multiracial/multiethnic, white/caucasian, and other/ unknown; at this school, the majority of international students are from china), and degree program category (collapsed into two categories: undergraduate and graduate). to reduce potential bias, weights were created for each participant using iterative proportional fitting to match demographics available on the overall university population. the following variables were included: sex by degree program category and race/ethnicity/international status by degree program category. we used a bayesian approach to estimate the antibody prevalence of covid-19 in our student population by incorporating the sensitivity and specificity of the diagnostic test into the prevalence estimate [18] . to be consistent with the euroimmun anti-sars-cov-2 elisa (igg) manual [17] , we classified a borderline test result as negative. prior values for the sensitivity and specificity were taken from the validation program at the frederick national laboratory for cancer research [17] . the prior on the true prevalence was taken from a recent study on antibody prevalence in la county [13] and reflects the community from which our sample was derived from. of the 863 adults in la county that consented to the study and were tested, 35 individuals tested positive. more information on the prior and model specifications are found in the appendix. odds ratios (ors; unadjusted for covariates in the model) were used to explore potential factors associated with a positive antibody test. for these analyses, positive cases were compared with borderline and negative cases combined. given the exploratory nature of this work, adjustments were not made for multiple comparisons, so results should be interpreted within this context. reference groups for demographic factors were female for sex, white/caucasian for race/ethnicity/international status, and undergraduate for degree program category. for all other variables, the reference was not having the given characteristic. there were 790 students who participated in this study. the sample was 48.2% female, 30% international, and 52.2% undergraduate (table 1 ). at the time of data collection, most students lived in off-campus housing, alone or with roommates/friends (61.3%); in their family home (26.5%); and in university-owned housing (9.1%). initially, 23 individuals tested as borderline. all but four of these people returned for follow-up testing, and five people had a borderline test the second time, with one person having a positive test. estimates for the prevalence and its 95% credible intervals are reported in table 2 . the estimated prevalence of covid-19 antibody positivity at the time of our study was 4.0% (3.0%, 5.1%). posterior estimates for the sensitivity and specificity can be found in the appendix. furthermore, when stratified by academic status, the estimated prevalence was 4.0% (3.0%, 5.2%) and 4.0% (2.9%, 5.2%) for undergraduate and graduate students, respectively, suggesting that the two student groups may not have differing lifestyles that will make them more (or less) susceptible to infection. our analysis treated borderline subjects (n ¼ 10) as test negative. to investigate the influence of potentially misclassifying borderline subjects, we provide a supplementary analysis where we treat borderline subjects as test positive. this increased the prevalence slightly to 4.3% (3.2%, 5.7%). these results can be found in the appendix. to assess the influence of prior specifications to the prevalence of covid-19 antibody positivity, we ran the model, classifying borderline results as test negative, by placing a noninformative prior to the prevalence. with a noninformative prior, the estimated prevalence of antibody positivity was 3.6% (.7%, 6.0%). there were three participants (reflecting 2.8 weighting observations) who reported that they had a previous covid-19 diagnosis via a nasal or throat swab; all these participants also had positive antibody tests (reflects 8.8% of positive antibody cases). more than half (61.4%, weighted n ¼ 19.4) of the positive cases did not report any prior illness with respiratory symptoms, and more than one third (37.0%) did not report any prior illness at all (others reported symptoms such as fatigue, headache, sore throat, and runny nose). we report the unadjusted ors and their 95% cis in table 3 . in this sample of university students, the following factors were associated with having a positive test for sars-cov-2 antibodies: students with a history of loss of taste and/or smell were 4.0 times as likely to have a positive test, compared with those without this history (95% ci: 1.4e9.6); students with confirmed or suspected exposure to a positive covid-19 case were 3.3 times as likely to have a positive test, relative to those without confirmed or suspected exposure (95% ci: 1.4e7.0). we observed increased test positivity for those reporting international travel (or ¼ 1.6, 95% ci: .8e3.3) and domestic travel (or ¼ 2.1, 95% ci: .9e5.2) since december 1, 2019, although neither was statistically significant. of the 26 positive cases who reported domestic travel, only 10 reported regional travel within southern california. sex, race/ethnicity, academic level, current and prior living in university-owned housing, and history of flu shot during the 2019e2020 academic year were not significantly associated with having a positive antibody test (data not shown for all variables). a history of respiratory symptoms, with or without fever, was also not associated with a positive antibody test. we perform a supplementary analysis by creating weights for each participant using iterative proportional fitting to match demographics available on the overall university population to reduce potential bias. the following variables were included in calculating these: sex by degree program category and race/ethnicity/international status by degree program category. we present the weighted unadjusted or in the appendix and note that the overall conclusions are consistent with what we previously observed. seroprevalence of sars-cov-2 antibodies in a los angeles university student population as of may 8, 2020, was estimated to be 4.0%. this does not substantially differ from what has been reported in prior population studies from santa clara county and los angeles county. in the los angeles county study, done in april 2020, prevalence was estimated at 4.65% (95% ci: 2.8%e 5.6%) [13] . likewise, an analysis of the blood samples from approximately 3,300 people living in santa clara county in early april estimated prevalence at 2.8% (95% ci: 1.3%e4.7%) [14] . this study demonstrates that the prevalence of infection at this institute did not differ from the larger community. the low prevalence indicates that sars-cov-2 was not widely circulating in the student population before the closure of the physical campus in mid-march 2020. this institute started the transition to virtual learning on march 11, 2020, with spring break the following week. for the remainder of the spring semester, the students completed all coursework virtually, and the physical campus was closed except for a small percentage of students who remained in universityowned housing. on march 19, 2020, governor gavin newsom issued a stay-at-home order for the state of california. it is likely that these events played a part in the low seroprevalence of sars-cov-2 in this study. with the low seroprevalence to sars-cov-2 in our population at the time of this study, there is not a strong argument for widespread antibody testing to inform decisions on reopening the college campuses. the reopening of ihe will rest on the ability to mitigate spread through continued physical distancing measures, environmental measures, promotion of behaviors that reduce spread, contact tracing, and access to testing. the centers for disease control is collaborating with public health departments and private laboratories to use seroprevalence surveys in different locations and populations to help estimate the number of persons who may have been infected with sars-cov-2 and not included in official case counts [19] . although widespread antibody testing at this time has limited usefulness, additional seroprevalence surveys from ihe will be important to estimate the number of infections associated with ihe and to further our understanding of risk factors for infection as physical college campuses reopen. approximately 1% of the samples in our study fell in the borderline or indeterminate range. this may represent early infection with a rising antibody titer, prior infection with waning antibody production, or cross-reactivity with another virus. loss of smell and/or taste could be a relevant indicator in this population, which has also been reported by others [20] . this may be a screening tool relevant to ihes, although more information on duration and intensity of lost smell/taste would be beneficial. notably, 39.6% of negative cases reported respiratory symptoms, suggesting that their answers to this question likely reflected respiratory effects from seasonal conditions unrelated to sars-cov-2. the presence of sars-cov-2 antibodies indicates prior infection, but it is still unclear whether this indicates immunity. we did not perform additional neutralization antibody assays to determine further characteristics of the antibodies. it is also unknown what percentage of students with asymptomatic or mild infections develop detectable antibody response. prior studies with the middle east respiratory syndrome (mers-cov) demonstrated that the severity of the disease correlated with the antibody response [21] . this consideration is important for ihe; if large numbers of the population have asymptomatic or mildly symptomatic infections, seroprevalence studies may underrepresent the prior disease incidence. further longitudinal serological studies on the college population are needed to determine ongoing disease incidence as well as the extent and duration of immunity to sars-cov-2. there are several other potential limitations of this study. there is limited data available on the validity of the assay used in this study, although this uncertainty was accounted for in statistical analyses. limitations in our understanding of borderline findings also make it challenging to draw conclusions. in analyses of potential risk factors, adjustments were not made for multiple comparisons, and estimated associations of each factor with prevalence were not adjusted for other risk factors, and so these findings should be interpreted with caution. only students currently residing in the region were eligible to participate in this study, so true prevalence in this university population is unknown. demographic data are limited in their availability, so there may be other biases present because of unmeasured or insufficiently measured variables. it is also possible that there are factors that drove interest in participating in this study, such as prior symptoms, which may affect prevalence estimates. furthermore, it is not known how generalizable these findings may be to other university populations. although it is still unknown the frequency in which positive sars-cov-2 antibodies develop or are sustained in asymptomatic or subclinical infection or if antibodies confer protective immunity and how long that immunity will last, antibody testing remains a powerful tool to examine prevalence of a disease in a population after the initial infection. over time, this information can be used to assess risk factors, monitor spread, and help administrators and public health officials plan for easing current mitigation and future health care needs. our results reflect antibody positivity in this university population because of infection with sars-cov-2 in the early days of the pandemic and during the statewide shutdown. in addition to showing comparability with general population prevalence, our estimates serve as a useful comparison for future studies that may be conducted in this population as this university campus returns to normal operations. a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 digest of education statistics united states census bureau. us and world population clock the impact of covid-19 on college student well-being. american college health association a descriptive study of coronavirus disease 2019 e related experiences and perspective of a national sample of college students in spring 2020 evidence supporting transmission of severe acute respiratory syndrome coronavirus 2 while presymptomatic or asymptomatic american college health association-national college health assessment ii: reference group executive summary spring 2019. silver spring, md: american college health association club and intermural sports participation and college student academic success the small-world network of college classes: implications for epidemic spread on a university campus spread of sars-cov-2 in the icelandic population seroprevalence of sars-cov-2especific antibodies among adults in covid-19 antibody seroprevalence population-based age-stratified seroepidemiological investigation protocol for covid-19 virus infection county of los angeles public health. covid-19 data dashboard anti-sars-cov-2 elisa (igg) instruction for use a tutorial in estimating the prevalence of disease in humans and animals in the absence of a gold standard diagnostic large-scale geographic seroprevalence survey real-time tracking of self-reported symptoms to predict potential covid-19 mers-cov antibody responses 1 year after symptom onset, south korea the authors thank the students who participated in this study for completing the risk factor questionnaire and providing a sample for antibody testing in a timely manner. supplementary data related to this article can be found at https://doi.org/10.1016/j.jadohealth.2020.09.001. key: cord-293274-ysr1l557 authors: perisé-barrios, ana judith; tomeo-martín, beatriz davinia; gómez-ochoa, pablo; delgado-bonet, pablo; plaza, pedro; palau-concejo, paula; gonzález, jorge; ortiz-diez, gustavo; meléndez-lazo, antonio; gentil, michaela; garcía-castro, javier; barbero-fernández, alicia title: humoral response to sars-cov-2 by healthy and sick dogs during covid-19 pandemic in spain date: 2020-09-22 journal: biorxiv doi: 10.1101/2020.09.22.308023 sha: doc_id: 293274 cord_uid: ysr1l557 covid-19 is a zoonotic disease originated by sars-cov-2. infection of animals with sars-cov-2 are being reported during last months, and also an increase of severe lung pathologies in domestic dogs has been detected by veterinarians in spain. therefore it is necessary to describe the pathological processes in those animals that show symptoms similar to those described in humans affected by covid-19. the potential for companion animals contributing to the continued human-to-human disease, infectivity, and community spread is an urgent issue to be considered. forty animals with pulmonary pathologies were studied by chest x-ray, ultrasound study, and computed tomography. nasopharyngeal and rectal swab were analyzed to detect canine pathogens, including sars-cov-2. twenty healthy dogs living in sars-cov-2 positive households were included. immunoglobulin detection by different immunoassays was performed. our findings show that sick dogs presented severe alveolar or interstitial pattern, with pulmonary opacity, parenchymal abnormalities, and bilateral lesions. forty dogs were negative for sars-cov-2 but mycoplasma spp. was detected in 26 of 33 dogs. five healthy and one pathological dog presented igg against sars-cov-2. here we report that despite detecting dogs with igg α-sars-cov-2, we never obtained a positive rt-qpcr, not even in dogs with severe pulmonary disease; suggesting that even in the case of a canine infection transmission would be unlikely. moreover, dogs living in covid-19 positive households could have been more exposed to be infected during outbreaks. covid-19 is a zoonotic disease originated by sars-cov-2. infection of animals with sars-cov-2 are being reported during last months, and also an increase of severe lung pathologies in domestic dogs has been detected by veterinarians in spain. therefore it is necessary to describe the pathological processes in those animals that show symptoms similar to those described in humans affected by covid-19. the potential for companion animals contributing to the continued human-to-human disease, infectivity, and community spread is an urgent issue to be considered. forty animals with pulmonary pathologies were studied by chest x-ray, ultrasound study, and computed tomography. nasopharyngeal and rectal swab were analyzed to detect canine pathogens, including sars-cov-2. twenty healthy dogs living in sars-cov-2 positive households were included. immunoglobulin detection by different immunoassays was performed. our findings show that sick dogs presented severe alveolar or interstitial pattern, with pulmonary opacity, parenchymal abnormalities, and bilateral lesions. forty dogs were negative for sars-cov-2 but mycoplasma spp. was detected in 26 of 33 dogs. five healthy and one pathological dog presented igg against sars-cov-2. here we report that despite detecting dogs with igg α-sars-cov-2, we never obtained a positive rt-qpcr, not even in dogs with severe pulmonary disease; suggesting that even in the case of a canine infection transmission would be unlikely. moreover, dogs living in covid-19 positive households could have been more exposed to be infected during outbreaks. we are currently in an international health emergency generated by the emerging zoonotic coronavirus sars-cov-2 that began its expansion by the end of the year 2019 in wuhan (china) and has caused a pandemic in a few months. 1 covid-19 is a pathology with various clinical manifestations caused by sars-cov-2, and the severity of its infection is mainly associated with lung injury, with findings similar to macrophage activation syndrome that causes hyperinflammation and lung damage by an uncontrolled activation and proliferation of t lymphocytes and macrophages. 2, 3 four genera of coronavirus have been described: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (α-cov, β-cov, γ-cov, and δ-cov) according to their genetic structure. with the detection of sars-cov-2 in humans, seven coronaviruses had been isolated from people, but only two of them (sars-cov and mers-cov) predominantly infect the lower airways and cause fatal pneumonia. severe pneumonia is associated with rapid viral replication, massive inflammatory cell infiltration, and proinflammatory cytokine responses. 4 in humans, the other coronaviruses cause mild upper respiratory tract infections in immunocompetent adults and serious symptoms in children and elderly people. α-cov and β-cov infect mammals and have been also described in dogs and cats; mostly they are responsible for respiratory infections in humans and gastroenteritis in animals. in dogs, canine enteric coronavirus (ccov), an α-cov, causes an enteritis of variable severity (rarely fatal) and develop immunity; however some of the recovered dogs become carriers with the ability to infect other dogs. however, the described fecal-oral transmission pattern for ccov includes only the canine species, and is not currently postulated as a possible zoonotic agent. however, canine respiratory coronavirus (crcov), that belong to the β-cov (like sars-cov-2), cause respiratory symptoms in dogs, in general with mild clinical signs 5 and occasionally as a coinfection with other respiratory pathogens. recently, the first cases of asymptomatic dogs infected with sars-cov-2 have been described. 6 due to the zoonotic origin of sars-cov-2 and the described transmission between species, the hypothesis of the spread between animals becomes more plausible. 7 cases of infected cats, dogs, tigers, lions, minks, and ferrets have been reported during sars-cov-2 outbreaks, and all of them had close contact with infected people. 6, 8 experimental infections using different animal species report an increased susceptibility to infection by ferrets and cats, and indicate a low susceptibility to sars-cov-2 infection by golden hamsters, macaques, fruit bats, and dogs. 9 furthermore, under natural conditions, infections in more than 20 mink farms have been reported. the transmission pattern postulated is that virus was transmitted to the animals by infected people and then the virus spread between minks. 10 later, in may 2020, two workers of the mink farms that could have acquired the infection from the minks have been reported. these would be the first described transmissions of sars cov-2 from animal to human (apart the first one that originated the pandemic). 11 some data suggests that also transmission from minks to cats and dogs occurred in the farms. the world organization for animal health-oie stated that some animals can become infected by being in permanent contact with infected people, although they note that there are no evidences defining the role of infected pets in the spread of sars-cov-2. to date, no cases of transmission from domestic or captive wild animals to humans have been described (excluding, if confirmed, mink farm workers). information regarding the possibility of companion animals becoming infected is confusing and controversial. some authors describe that dogs whose owners were positive for sars-cov-2, showed serological negative to sars-cov-2, postulating that pets are not virus carriers. 12 by contrast, some cases of companion dogs have been reported that were positive by rt-qpcr detection 6 and others that have developed neutralizing antibodies against sars-cov-2. 13 currently, around ten rt-qpcr-positive dogs have been detected worldwide (hong kong, denmark, and usa, but none in spain), all of them in close contact with covid-19 positive humans. 14 less than half of them were asymptomatic, one presented mild respiratory illness, and only one present also neutralizing antibodies coursing with hemolytic anemia. 15 on the other hand, two negative-pcr dogs have developed neutralizing antibodies, one was asymptomatic and the other had breathing problems, but it is not clear if those were related to the infection. 15 further, molecular testing of 3500 dogs, cat and horse companion animals were done by idexx company in usa and korea and no positive cases were found. no positive cases were reported in dogs exposed to sars-cov-2 in france. 16 another recent study in italy carried out with pets has shown that none of the 817 animals studied was positive to sars-cov-2 by rt-qpcr test but 13 dogs and 6 cats had neutralizing antibodies. 13 in this manuscript we report that, during the months of the pandemic, an increase of aggressive lung pathologies in dogs was detected by veterinarians in spain. therefore, it is important to determine the infectious agent and a potential role of a sars-cov-2 infection. considering the information that is currently available, it is therefore necessary to better describe the pathological processes that could occur in those animals that could be infected by sars-cov-2 and also showing symptoms similar to those described in humans affected by covid-19. it is also highly relevant to determine if dogs could become infected in a home environment where close humanpet relationships occur. therefore, the potential for companion animals contributing to the continued human-to-human disease, infectivity, and community spread is an urgent issue to be considered. here, we describe the study of sick and healthy dogs regarding a potential infection with sars-cov-2. a prospective study with forty dogs presenting pneumonia was performed between april and june 2020 in spain. a clinical follow-up of all patients was performed and mortality was also recorded. twenty healthy dogs living with people affected by covid-19 were included as animals exposed to the virus. inclusion/exclusion criteria and more information are available in suppl. methods. the study was approved by the ethical committee of the faculty of health sciences, alfonso x el sabio university and all dog owners gave written informed consent. chest x-ray (cxr), thoracic radiographic and a study ultrasound were performed in sick dogs. the pattern type, distribution and intensity were analyzed. the pathological lung was recognized when the ultrasound lung rockets also called b lines were observed ( figure 1 ) or by the presence of other pulmonary ultrasound findings for consolidation (crushing, tissue, nodule sign) ( figure 1 ). computed tomographic (ct) was perform to assess the lesions distribution, classified as generalized, focal, and uni or bilateral. more information is available in suppl. methods. blood samples from either sick or healthy-exposed dogs were analyzed to determine immunoglobulins (igg) against sars-cov-2. a high-sensitive sars-cov-2 spike s1 protein elisa kit was used. values >2·5od of the negative control were considered as positives. to determine antibodies (igm and igg) against ccov, dog plasma samples were analyzed by eia assays. to determine neutralizing antibodies (igg) against canine adenovirus (cav), canine parvovirus (cpv) and canine distemper virus (cdv), plasma samples were analyzed by an elisa in solid phase. more information is available in suppl. methods. nasopharyngeal and rectal swabs were collected from sick dogs and analyzed by laboklin gmbh & co.kg using conventional pcr or real-time pcr (qpcr and rt-qpcr). all samples were tested for canine adenovirus type 2 (cav-2), bordetella bronchiseptica, cdv, canine parainfluenza virus (cpiv), canine influenza a virus (civ), canine herpesvirus-1 (canid alphaherpesvirus-1: cahv-1), and sars-cov-2 by taqman real-time pcr, and for mycoplasma spp. by conventional pcr. more information is available in suppl. methods. lungs of two dogs (ser209 and ser222) were histologically evaluated after necropsy. the macroscopic exam evaluated congestion, oedema and the lung injury pattern. lung samples were fixed in formalin 4% for 24 hours, paraffin-embedded and 3µm thick sections stained with hematoxylin-eosin. more information is available in suppl. categorical variables were presented as percentages. for continuous variables, data distribution normality was evaluated with the kolmogorov-smirnov test. continuous data were presented as mean with standard deviation (sd) or median with interquartile range (iqr). forty pathologic dogs met the inclusion criteria with the mean age of 8 years (range: 2 months to 13 years). fifteen breeds were recorded being the most common cross-breed 22·5% (9/40), yorkshire terrier 12·5% (5/40), and german shepherd 10% (4/40). there were 22 females and 18 males. the most common clinical signs were crackles on lung auscultation, followed by cough, tachypnoea, fatigue, fever, tachycardia, vomiting, and diarrhoea ( table 1 ). the radiographic findings in all analyzed dogs were consistent with mild to severe alveolar or interstitial pattern, with pulmonary opacity accentuated in the caudodorsal lung field. in 32·5% (13/40) in order to assess the overall status of dogs, and considering that the number of white blood cells is frequently altered in an infection, we consider it relevant to perform a hematologic evaluation on twenty-four pathologic patients. the count of white blood cells was out of range in 58·3% of dogs being the number of neutrophils abnormal in 75%, lymphocytes in 37·5%, and monocytes in 45·8% (table 2) . considering the altered number of immune cells observed in peripheral blood, together with the clinical course, we proceeded to evaluate possible pulmonary pathogens. in order to determine whether the observed pathologies could be related to a sars-cov-2 infection or to other pathogens, pcr analysis was performed. all forty dogs were negative for sars-cov-2 (table 3) . furthermore, thirty-three dogs were analyzed for a complete profile including the most common canine infectious agents. all of them were negative for cpiv, civ, and cahv-1. mycoplasma spp. and cdv were detected as a single agent with 57·6% (19/33) and 3% (1/33), respectively ( table 3 ). the pathologies in our patients were very aggressive and 42·5% (17/40) of dogs died of pneumonia during follow-up ( table 1) scant and disperse inflammatory cells, mainly macrophages with intracytoplasmic brown granular pigment were observed in alveolar septa. interestingly, some of these findings, mainly the scattered syncytia, are usually present in some viral infections. 17 after analyzing the possible pathogens in the diagnosed dogs and the findings in the evaluated tissues, we set out to study the immune response against some of these infectious agents in 17 dogs. further, we also decided to study 20 dogs that lived with people diagnosed with covid-19, as a group of dogs exposed to the virus but which did not present symptoms at the time of sampling. first of all, information about vaccination was gathered to determine the immune status of dogs. ten sick dogs had been vaccinated routinely as it is recommended by veterinarians but eight sick dogs did not received any vaccine (suppl. table s1). we have not detected any association between the vaccination patterns of pathological animals compared to healthy dogs. immunoglobulins g (igg) against cav, cpv, and cdv were analyzed in peripheral blood samples from these sick and healthy dogs (table 4 ). further, antibodies (igm and igg isotypes) against canine coronavirus that affects enteric tract, and also igg against sars-cov-2 were studied in both groups ( table 4 ). the number of dogs that presented igg antibodies against sars-cov-2 was higher in the group of healthy dogs (25%; 5/20), compared to the pathological ones (5·88%; 1/17). interestingly, the sick dog that presented antibodies against sars-cov-2 was negative for the detection of the virus in swabs studied by rt-qpcr, however mycoplasma spp. and cdv were detected in this patient ( table 3 ). all of the five igg α-sars-cov-2 positives healthy dogs showed the same pattern of antibodies against the other studied pathogens, being positives for igg α-cav, igg α-cpv, and igg α-cdv (table 4 ). nevertheless, two of them presented igg α-ccov while the remaining three were not protected against canine coronavirus. twelve healthy dogs presented igg α-ccov and two of them were positive for igg α-sars-cov-2 (table 4 ). seven pathological dogs presented igg α-ccov but in this group all of them were negative to α-sars-cov-2 (table 4 ). professionals and pet owners demand more information about a possible infection of their animals with sars-cov-2. a survey of us veterinarians reported that 60% of them seeing owners concerned that their pets had covid-19, 18 so many owners are restless and demand a more in-depth clinical study from their animals. pets are often in close contact with humans, and thus, it is important to determine their susceptibility to sars-cov-2 as well as the risk that infected pets are as a source of infection for humans. dogs are currently not considered to be susceptible hosts for sars-cov-2, despite few positive rt-qpcr test results in dogs were reported. 6 surveillance data from idexx laboratories, a multinational veterinary diagnostics company, showing that there were no positive results for sars-cov-2 in any of more than 1500 dog´s specimens submitted for respiratory pcr panels, according with the idea of transmission from human to pet is very rare. however, veterinarians, in spain, have observed an increase in aggressive lung pathologies in dogs in the months of the human pandemic, which did not respond to conventional antibiotic treatments. moreover, in veterinary medicine the respiratory disease is rarely lethal in the pet dog population. a mortality rate of 1·2% due to respiratory disease (only 0·3% due to pneumonia) has been reported, 19, 20 nevertheless in our study we found a mortality rate of 42·5% during follow-up without a clarified etiology. these dogs, with very aggressive lung diseases, showed a very similar appearance to those described for covid-19 pneumonia in human medicine. 21 historically, the most common pathogens associated with canine infectious respiratory disease complex have been cpiv, cav-2, bordetella bronchiseptica, streptococcus equi subsp. zooepidemicus, mycoplasma cynos, chv-1, cdv, civ, and crcov. 20 in our study, we detect eight of 33 analyzed dogs presenting classical primary respiratory pathogens, and we also detect igm for ccov in four dogs (3/17 pathological dogs and 1/20 healthy dog). in this regard, the presence of crcov is detected more frequently in dogs with mild clinical signs than in dogs with moderate or severe clinical signs, 5 therefore, we would rule out that it was the agent responsible for the severe respiratory pathologies in these three dogs. moreover, mycoplasma cynos is the only mycoplasma spp. significantly associated with pneumonia in dogs but it is still also unclear if m. cynos is a primary or secondary pathogen in dogs, because it can be cultured from the lungs of dogs, both with and without other identifiable infectious agents. 22 in a european study of dogs with canine infectious respiratory disease seroprevalence of mycoplasma spp. levels ranging from 20·7% to 61·9%, 23 but in other study with healthy dogs mycoplasma were isolated from 78% to 93% of throat swabs. 20 moreover, mycoplasma infections are usually associated with other infections. it is interesting to note that mycoplasma coinfections are very common in covid-19 human patients 24 and it also has been suggested that a co-infection or activation of latent mycoplasma infections in covid-19 disease may be important in determining a fatal disease course. 25, 26 normally, the therapy response when treating respiratory tract diseases with drugs (antibiotics, bronchodilators, anti-inflammatories, antitussives, decongestants, mucolytics, mucokinetics or expectorants) is adequate or complete, nevertheless our patients did not respond adequately to the therapeutic protocol. a major pathogen has not been detected in our patients, so at the moment the causative agent of the pathologies is unknown. further, the number of deaths was more than 30 times higher than expected without clarified etiology and curiously during the peak of the covid-19 pandemic in spain. when analyzing deceased dogs, interstitial pneumonia that usually courses with nonspecific lesions was detected, that has been described also in canine pathologies, such as canine distemper, herbicide poisoning or systemic processes (septicemia or uremia principally). 17 however, it should be noted that showed lesions are similar to described for covid-19 affected humans. 1 specially, striking lesions observed in vessels, both lymphocytic vasculitis, and the hyalinosis of the arteriolar wall. 27 however, all of them were negative in rt-qpcr tests for sars-cov-2 using nasopharyngeal and rectal samples. these results agree with a large-scale study that recently has been shown to assess sars-cov-2 infection in 817 companion animals living in northern italy and neither animals tested positive using rt-qpcr were found. 13 likewise, it should be considered that viral particles have been detected in the skin endothelium of human patients despite they were negative when tested by rt-qpcr. therefore, it would be useful analyze sars-cov-2 by ihc in necropsy samples from our patients. 28 regarding the presence of immunoglobulins against sars-cov-2 in peripheral blood of pets, in a previous study 487 dogs were tested in china. they were serological negative for anti-sars-cov-2 iggs. among them, 15 pet dog and 99 street dog sera were collected from wuhan city but it should be noted that only one pet dog living with a confirmed covid-19 human patient presented antibodies against sars-cov-2. 12 however, in the italian study, 3·4% of 188 dogs (and 3·9% of 63 cats) had measurable sars-cov-2 neutralizing antibody titers. none of these animals with neutralizing antibodies displayed respiratory symptoms at the time of sampling. interestingly, dogs from covid-19 positive households seem to be significantly more likely to test igg positive than those from covid-19 negative households. 13 finally, it has been determined that only half of the dogs artificially inoculated with sars-cov-2 seroconverted. 8 here we detected specific anti-sars-cov-2 canine immunoglobulins in one sick dog (1/17) infection; nevertheless, they were not tested and confirmed. other owners had not exhibited symptoms during the pandemic. therefore, it was decided to exclude this data from the statistical study due to a lack of reliable information. most people infected with sars-cov-2 display an antibody response between day 10 and day 21 after infection, and several studies have suggested that previous antibodies and t cells against endemic human coronavirus may provide some degree of crossprotection to sars-cov-2 infection. 29 further, it has been reported pre-existing memory cd4+ t cells that are cross-reactive with sars-cov-2 and the common human cold coronaviruses hcov-oc43, hcov-229e, hcov-nl63, or hcov-hku1. 30 in our data we did not find any correlation between ccov and sars-cov-2 igg-positive dogs, although the low number of cases makes it difficult to reach a valid conclusion. in sum, we analyzed dogs affected by severe pulmonary disease, all of them being negative for sars-cov-2 by rt-qpcr, however some of them present igg α-sars-cov-2, as well as the healthy dogs; suggesting that even in the case of a canine infection it would be little transmissible. moreover, dogs with owners positive for sars-cov-2 could have been more exposed to be infected during outbreaks. a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical features of patients infected with 2019 novel coronavirus in wuhan the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex infection of dogs with sars-cov-2 the proximal origin of sars-cov-2 susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 transmission and response to re-exposure of sars-cov-2 in domestic cats sars-cov2 infection in farmed mink sars-cov-2 infection in farmed minks, the netherlands serological survey of sars-cov-2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals evidence of exposure to sars-cov-2 in cats and dogs from households in italy in: associate administrator u-a, united states department of agriculture absence of sars-cov-2 infection in cats and dogs in close contact with a cluster of covid-19 patients in a veterinary campus s pathology of domestic animals owner concerns that pets have covid-19 methods and mortality results of a health survey of purebred dogs in the uk aetiology of canine infectious respiratory disease complex and prevalence of its pathogens in europe point-of-care lung ultrasound in patients with covid-19 -a narrative review canine infectious respiratory disease european surveillance of emerging pathogens associated with canine infectious respiratory disease precautions are needed for covid-19 patients with coinfection of common respiratory pathogens covid-19 coronavirus: is infection along with mycoplasma or other bacteria linked to progression to a lethal outcome? characterization of microbial co-infections in the respiratory tract of hospitalized covid-19 patients the pathological autopsy of coronavirus disease 2019 (covid-2019) in china: a review sars-cov-2 endothelial infection causes covid-19 chilblains: histopathological, immunohistochemical and ultrastructural study of seven paediatric cases targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals selective and cross-reactive sars-cov-2 t cell epitopes in unexposed humans the authors would like to thank the owners of the dogs for their participation, as well as the veterinary clinics and hospitals that have collaborated in this study (veterinary hospital vetcare, veterinary hospital madrid norte, among others). the authors would like to thank the "centro de transfusión veterinario" for their donation of samples of virus not-exposed dogs. key: cord-302608-fw4pmaoc authors: huang, jiao-mei; jan, syed sajid; wei, xiaobin; wan, yi; ouyang, songying title: evidence of the recombinant origin and ongoing mutations in severe acute respiratory syndrome coronavirus 2 (sars-cov-2) date: 2020-03-19 journal: biorxiv doi: 10.1101/2020.03.16.993816 sha: doc_id: 302608 cord_uid: fw4pmaoc the recent global outbreak of viral pneumonia designated as coronavirus disease 2019 (covid-19) by coronavirus (sars-cov-2) has threatened global public health and urged to investigate its source. whole genome analysis of sars-cov-2 revealed ~96% genomic similarity with bat cov (ratg13) and clustered together in phylogenetic tree. furthermore, ratgl3 also showed 97.43% spike protein similarity with sars-cov-2 suggesting that ratgl3 is the closest strain. however, rbd and key amino acid residues supposed to be crucial for human-to-human and cross-species transmission are homologues between sars-cov-2 and pangolin covs. these results from our analysis suggest that sars-cov-2 is a recombinant virus of bat and pangolin covs. moreover, this study also reports mutations in coding regions of 125 sars-cov-2 genomes signifying its aptitude for evolution. in short, our findings propose that homologous recombination has been occurred between bat and pangolin covs that triggered cross-species transmission and emergence of sars-cov-2, and, during the ongoing outbreak, sars-cov-2 is still evolving for its adaptability. the family coronaviridae is comprised of large, enveloped, single stranded, and positivesense rna viruses that can infect a wide range of animals including humans guan et al., 2003) . the viruses are further classified into four genera: alpha, beta, gamma, and delta coronavirus (king et al., 2012) . so far, all coronaviruses (covs) identified in human belong to the genera alpha and beta. among them betacovs are of particular importance. different novel strains of highly infectious betacovs have been emerged in human populations in the past two decades that have caused severe health concern all over the world. severe acute respiratory syndrome coronavirus (sars-cov) was first recognized in 2003, causing a global outbreak (zhong, 2004; peiris et al., 2004; cherry, 2004) . it was followed by another pandemic event in 2012 by a novel strain of coronavirus designated as middle east respiratory syndrome coronavirus (mers-cov) (lu et al., 2013) . both covs were zoonotic pathogens and evolved in animals. bats in the genus rhinolophus are natural reservoir of coronaviruses worldwide, and it is presumed that both sars-cov and mers-cov have been transmitted to human through some intermediate mammalian hosts (li et al., 2005a; bolles et al., 2011; al-tawfiq and memish, 2014) . recently, emergence of another pandemic termed as coronavirus disease 2019 (covid-19) by world health organization (who) caused by a novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been reported (zhu et al., 2020) . to date, more than 174,000 people are infected and over 6,600 death tolls, having transmission clusters worldwide including china, italy, south korea, iran, japan, usa, france, spain, germany and several other countries causing alarming global health concern. the large trimeric spike glycoprotein (s) located on the surface of covs is crucial for viral infection and pathogenesis, which is further subdivided into n-terminal s1 subunit and c-terminal s2 domain. the s1 subunit is specialized in recognizing receptors on host cell, comprising of two separate domains located at n-and c-terminal which can fold independently and facilitate receptor engagement (masters, 2006) . receptor-binding domains (rbds) of most covs are located on s1 c-terminus and enable attachment to its host receptor (li et al., 2005b) . the host specificity of virus particle is determined by amino acid sequence of rbd and is usually dissimilar among different covs. therefore, rbd is a core determinant for tissue tropism and host range of covs. this article presents sars-cov-2 phylogenetic trees, comparison and analysis of genome, spike protein, and rbd amino acid sequences of different covs, deducing source and etiology of covid-19 and evolutionary relationship among sars-cov-2 in human. to determine the evolutionary relationship of the sars-cov-2, phylogenetic analysis was performed on whole genomic sequences of different covs from various hosts. the maximum-likelihood (ml) phylogenetic tree is shown in figure 1 , which illustrates four main groups representing four genera of covs, alpha, beta, gamma, and delta. in the phylogenetic tree, strains of sars-cov-2 (red colored) are cluster together and belong to the genera betacoronavirus. among beta-covs, sars-cov, civet sars cov, bat sars-like covs, bat/ratg13 cov, and sars-covs-2 clustered together forming a discrete clade from mers-covs. the clade is further divided into two branches and one of the branches comprises all sars-cov-2 strains clustered together with bat/yunnan/ratg13 cov forming a monophyletic group. bat/yunnan/ratg13 exhibited ~96% genomic similarity with sars-cov-2. this specifies that sars-cov-2 is closely related to bat/yunnan/ratg13 cov. the ml phylogenetic tree demonstrates that covs from bat source are found in the inner joint or neighboring clade of sars-cov-2. this indicates that bats covs particularly bat/yunnan/ratg13 are the source of sars-cov-2, and they are emerged and transmitted from bats to humans through some recombination and transformation events in intermediate host. to explore the emergence of sars-cov-2 in humans, we investigated covs s-protein and its rbd as they are responsible for determining the host range ( table 1) . the s-protein amino acid sequence identity between sars-cov-2 and related beta-covs showed that bat/yunnan/ratg13 shares highest similarity of 97.43%. however, the amino acid sequence identity of rbd of sars-cov-2 with bat/yunnan/ratg13 is 89.57%. on the other hand, beta-covs from pangolin sources (pangolin/guandong/1/2019 and pangolin/guangdong/lung08) revealed highest rbd amino acid sequence identity of 96.68% and 96.08% respectively with sars-cov-2. these indication shows the existence of homologous recombination events within the s-protein gene between bat and pangolin covs. similarity plot analysis of covs genome sequences from bat, pangolin and human also indicated a possible recombination within s-protein of sars-covs-19 ( figure s1 ). the amino acid residues change in s-protein of sars-cov-2 was further analyzed with sars-cov, pangolin and bat covs including pangolin/guandong/1/2019, pangolin/guangdong/lung08, and bat/yunnan/ratg13 (figure 2) . regardless of low homology between sars-cov-2 (wuhan-hu-1_mn908947) and sars-cov (sars_aar07630), they had many homologues areas in s-protein. the five key amino acid residues of s-protein at positions 442,472, 479,480, and 487 of sars-covs are described to be at the angiotensin-converting enzyme-2 (ace2) receptor complex interface and supposed to be crucial for human to human and cross-species transmission (li et al., 2005b; wu et al., 2012) . figure 2b and table s1 describe that all key amino acid residues of rbd (except two positions) are completely homologues between sars-cov-2 (wuhan-hu-1_mn908947) and pangolin covs (pangolin/guandong/1/2019 and pangolin/guangdong/lung08), supporting our postulation of recombination event in s-protein gene. even though, all five crucial amino acid residues of sars-cov-2 for binding to ace2 are different from sars-cov, their hydrophobicity and polarity are similar, having same s-protein structural confirmation and identical rbd 3-d structure (xu et al., 2020) . in addition, six critical key residues in mers-cov rbd binding to its receptor dipeptidyl peptidase 4 (dpp4) are all different in sars-cov and sars-cov-2 related coronavirus (figure 2a) . we also investigated some of the important evolutionary and phylogenetic aspects of sarstable 2 . among different orfs of sars-cov-2, orf1a was most variable segment with total number of 44 dissimilar amino acid substitutions. it was followed by spike segment s orf with 13 amino acid residue substitutions. however, orf6 and orf7b are the most conserved regions without amino acid changes. in addition, orf10, e, m and orf7a have tended to be more conserved, with only one or two amino acid substitutions. with the global spread of sars-cov-2, its amino acid sequence is also significantly varied (figure 3) . usually, rna viruses have high rate of genetic mutations, which leads to evolution and provide them with increased adaptability (lin et al., 2019) . to further explore sars-cov-2 evolution in human, we have performed phylogenetic analysis based on the aforementioned sars-cov-2 in correspondence with their amino acid substitution. one hundred and twenty-five newly sequenced sars-cov-2 complete genomes were obtained from global initiative on sharing all influenza data epiflutm database (gisaid epiflu tm ) and genbank. closely related beta-covs genomes sequences from different hosts were also collected and analyzed together with sars-cov-2. open reading frames (orfs) of covs genomes were predicted using orffinder (v0.4.3) with default parameters ignoring nested orfs. raw pair-end reads of pangolin dataset sample (srr10168377) obtained from ncbi were filtered with bbmap.sh (v38.79) by removing adaptors, trimming low quality reads from both sides (quality value < 20), and reads length less than 50 nt were ignored. host reference genome (pangolin manjav1.0, gcf_001685135.1) contaminant reads were removed by bowtie2 (v2.3.5.1) [13] . pangolin cov genome fragments were assembled via megahit (v1.2.9) (li et al., 2015) . the sequences of covs were aligned using multiple sequence alignment mafft (v7.450) (katoh et al., 2019) . aligned sequences were visualized with jalview (v2.10.3) (waterhouse et al., 2009) . poorly aligned regions and gaps were removed by trimal (v1.4.rev22) (capellagutiérrez et al., 2009) . maximum likelihood (ml) phylogenetic trees of whole genome sequences were constructed in iq-tree (v1.6.12) (nguyen et al., 2015) . support for inferred relationships in the phylogenetic tree was assessed by bootstrap analysis with 1000 replicates and the best-fit substitution model was determined by iq-tree model test. to be involved in recombination. similarity scores between genomic sequences were generated by simplot (v3.5.1) (lole et al., 1999) . red color highlights interaction positions of sars-cov-2 and pangolin covs with different amino acids residues. middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution sars-cov and emergent coronaviruses: viral determinants of interspecies transmission trimal: a tool for automated alignment trimming in large-scale phylogenetic analyses the chronology of the 2002-2003 sars mini pandemic isolation and characterization of viruses related to the sars coronavirus from animals in southern china mafft online service: multiple sequence alignment, interactive sequence choice and visualization virus taxonomy. ninth report of the international committee on taxonomy of viruses fast gapped-read alignment with bowtie 2 megahit: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de bruijn graph structure of sars coronavirus spike receptor-binding domain complexed with receptor bats are natural reservoirs of sars-like coronaviruses naturally occurring mutations in pb1 affect influenza a virus replication fidelity, virulence, and adaptability full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread the molecular biology of coronaviruses iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies severe acute respiratory syndrome from sars coronavirus to novel animal and human coronaviruses jalview version 2-a multiple sequence alignment editor and analysis workbench mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission management and prevention of sars in china a novel coronavirus from patients with pneumonia in china the authors have declared that no competing interests exist. key: cord-294108-uvnh0s9r authors: dube, taru; ghosh, amrito; mishra, jibanananda; kompella, uday b.; panda, jiban jyoti title: repurposed drugs, molecular vaccines, immune‐modulators, and nanotherapeutics to treat and prevent covid‐19 associated with sars‐cov‐2, a deadly nanovector date: 2020-10-25 journal: adv ther (weinh) doi: 10.1002/adtp.202000172 sha: doc_id: 294108 cord_uid: uvnh0s9r the deadly pandemic, coronavirus disease 2019 (covid‐19), caused due to the severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), has paralyzed the world. although significant methodological advances have been made in the field of viral detection/diagnosis with 251 in vitro diagnostic tests receiving emergency use approval by the us‐fda, little progress has been made in identifying curative or preventive therapies. this review discusses the current trends and potential future approaches for developing covid‐19 therapeutics, including repurposed drugs, vaccine candidates, immune‐modulators, convalescent plasma therapy, and antiviral nanoparticles/nanovaccines/combinatorial nanotherapeutics to surmount the pandemic viral strain. many potent therapeutic candidates emerging via drug‐repurposing could significantly reduce the cost and duration of anti‐covid‐19 drug development. gene/protein‐based vaccine candidates that could elicit both humoral and cell‐based immunity would be on the frontlines to prevent the disease. many emerging nanotechnology‐based interventions will be critical in the fight against the deadly virus by facilitating early detection and enabling target oriented multidrug therapeutics. the therapeutic candidates discussed in this article include remdesivir, dexamethasone, hydroxychloroquine, favilavir, lopinavir/ritonavir, antibody therapeutics like gimsilumab and tjm2, anti‐viral nanoparticles, and nanoparticle‐based dna and mrna vaccines. the entire world is facing the pandemic coronavirus disease of 2019 (covid19) due to the outbreak of 2019 novel coronavirus (2019-ncov; sars-cov-2) and an international health emergency has been declared. the disease has touched nearly every corner of the world. 2019-ncov infection is highly contagious and containment efforts mostly deal with identification and quarantine of exposed and asymptomatic suspects, contact tracing, detection, and strict isolation of infected patients to limit its spread. [1] [2] [3] [4] world health organization (who), on 30th january 2020, declared the disease caused by 2019-ncov as the sixth international public health emergency. due to its rapid escalation across the globe, the who declared the 2019-ncov outbreak a global pandemic on the 11th of march 2020. so far, i.e., from 31st december 2019 to 21st september 2020, more than 30 949 804 individuals were afflicted by 2019-ncov, and 959 116 deaths have occurred worldwide. [3] on the 11th of february 2020, the who officially declared a name for the ongoing 2019-ncov related disease pandemic as coronavirus disease 2019 whereas, the international committee on taxonomy of viruses (ictv) has renamed 2019-ncov as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) based on its close genetic resemblance with another coronavirus sars-cov. [2, 5] sars-cov-2 can transfer from an infected person with or without symptoms to another individual, with a high basic reproduction number (r 0 ) value. although the initial r 0 was estimated to be between 2.2 and 2.7 for sars-cov-2, one estimate published in february 2020 indicated that the r 0 value is as high as 4.7 to 6.6, with the infected individual doubling time of 2.4 d. [6, 7] the r 0 value indicates that each individual can spread the infection to 4.7-6.6 individuals on average. more recent studies published in august 2020 indicated that covid-19 has a higher transmission rate than severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) with a basic reproduction number of 3.32 and an incubation period of 5.24 d. [7] globalization and the convenience of country to country travel had additionally fueled the worldwide spread of the disease. [2, [8] [9] [10] this article discusses sars-cov-2 nanostructure, the virus biology in connection to its epidemiology, clinical manifestations, and potential and future therapeutic options including repurposed drugs, vaccine/protein therapies, immune therapies, and nanotherapeutics. additionally, the recommended preventive and protective measures are presented (figure 1 ). coronaviruses (covs) are a large family of viruses and are known causative agents for respiratory, hepatic, intestinal, and neurological diseases of altering severity in several animal species. tyrell and bynoe in 1966 were the first to cultivate and describe the covs from patients having common colds. [11] covs are single-stranded ribonucleic acid (rna) viruses. morphologically, they are spherical virions, having a core of rna that is being surrounded by a membrane decorated with protein projections (called spike proteins) like a ring/crown (latin: corona = crown). nanomaterials with very complex structures. they are efficient nanovectors capable of transferring their genetic materials to the infected host cells, followed by hijacking host-cell machinery to express their proteins. [28] many viruses have proven their worth in the field of drug, gene, and vaccine delivery. [29, 30] each sars-cov-2 virion has an average diameter of 50-150 nm (figure 2) . [14, 31, 32] similar to other covs, sars-cov-2 carries four major structural genes encoding major structural proteins, known as the spike protein (s), an envelope protein (e), the membrane protein (m), and the nucleocapsid protein (n); n protein embraces the genome consisting of ssrna and the proteins s, e, and m constitute the envelope of the virus (figure 3) . the s protein enables the virus to bind to and amalgamate with the host cell membrane. it is the key immunogenic antigen and has a decisive role in defining virulence, tissue tropism, host range, and protective immunity. [25] the trimeric s protein belongs to a class i fusion protein consisting of s1 (receptor binding) and s2 (membrane fusion) subunits. the subunit s1 consists of a c domain carrying the rbd, and an n domain. s2 contains fusion peptide, the heptad repeat (hr-1 and hr-2), transmembrane, and cytoplasmic domains. the locking of the rbd to the ace2 receptor of the host cell initiates conformational modifications in the s2 subunit that further facilitates fusion among viral and host cell membranes. the s1/s2 juncture is the cleavage site for proteases and is also required to trigger membrane fusion, viral entry, and in the formation of syncytium. [16, 27] other structural proteins, like e, m, and n, are involved in the viral assembly. alternatively, nonstructural proteins constituting viral cysteine proteases such as papain-like protease (pl pro), 3chymotrypsin like protease (3cl pro), rna-dependent rna polymerase (rdrp), helicase, and other accessory proteins, participate in the viral transcription followed by replication. to counter the host immune response, the m protein and pl pro, antagonize the host interferon (ifn) response and help the virus to control in vivo replication efficacy and pathogenesis. [16] alike sars-cov, sars-cov-2 ingress into target cells is also aided by the s protein. viral entry depends on i) the locking of s1 unit of s protein to host cell ace2 receptors; ii) the s protein priming by host cellular protease tmprss2, which facilitates s protein cleavage right at the s1/s2 juncture and the s2' site, facilitating the merger between viral and cellular membranes. following cellular entry, covs disassemble to release their genome (nucleocapsid and viral rna) into the cytoplasm of the infected host cell for initiating the translation of viral polyproteins and subsequent replication of genomic rna (figure 4) . [33] replicase polyproteins of sars-cov-2 are processed by pl pro and 3cl pro to form nonstructural proteins (helicase/rdrp). [16] various 3cl pro inhibitors can be expected to repress viral replication by obstructing the cleavage function of the protein. rdrp is a fundamental enzyme of the rna synthesizing machinery of rna viruses, and it is highly conserved in all hcovs. therefore, it served as a prime target in various viral infections to halt genome replication and can be inhibited by various polymerase inhibitors (favilavir, remdesivir). [16, [33] [34] sars-cov-2 utilizes the ace2 receptors to gain entry into the host cells. [35] ace2 is a counteractive component of the renin angiotensin system (ras) that is responsible for maintaining a balance between fluid volume and pressure by utilizing the cleavage products of angiotensin (agt) and their receptors. [36] [37] [38] among these peptides, angiotensin ii (ang-ii) causes vasoconstriction and helps in sodium retention by means of agtr1 receptor and results in vasodilation and natriuresis by binding to the agtr2 receptor. the enzyme ace is responsible for producing ang-ii. after infecting host cells, the virus also uses its protease 3cl pro to suppress nfkappab by degrading the activating factor ikkgamma. [39] since, nfkappab is involved in the induction of ace by attaching to its promoter and enhancing transcription. [40] the virus thus reduces the expression of ace. ace further downregulates the expression of ace2 in-part by ang-ii, which is its catalytic product. [41] thus, the viral infection results in upregulation of ace2 and downregulation of ace. it has been shown that, bradykinin, another important molecule that forms an important component of vasosuppressor system involved in inducing hypotension and vasodilation, is being regulated by aces. bradykinin is degraded by ace and its concentration is enhanced in presence of angiotensin1-9 a peptide produced by ace2. thus, the enhanced ace2 expression caused by the virus leads to increased levels of bradykinin causing "bradykinin storm." this is because sars-cov-2 increases the amount of bradykinin in the infected tissues. bradykinin causes vasodilation leading to swelling and inflammation of the tissue and induces pain. it also increases the production of hyaluronic acid. the bradykinin storm induced leakage of fluid and hyaluronic acid induced formation of hydrogel into the lungs may be the reason behind low oxygen uptake in severe covid-19 patients and generation of severe symptoms like hypokalemia associated with arrhythmia and sudden cardiac death. [42] [43] [44] [45] hence, the drugs which can take care of the bradykinin storm can also be considered as potential therapeutics for covid-19. sars-cov-2 phylogenetic analysis showed that it is meticulously related to two bat-derived sars-like covs (bat-sl-covzc45 and bat-sl-covzxc21) found in horseshoe bats (rhinolophus) with ≈89% nucleotide sequence resemblance with bat-sl-covzc45. it exhibits ≈79% nucleotide sequence resemblance with sars-cov and ≈50% with mers-cov. further, a 98.7% nucleotide sequence resemblance to the partial rdrp gene of the bat-cov strain btcov/4991 was also observed. [2, 10] gaining in-depth insight into the sars-cov-2 virus epidemiology, and characterizing its possible impact, is a pressing need of the current time in order to expand health care measures to tackle the pandemic. the overall impact of a pandemic is governed by the total number of infected persons, transmissibility, and its medical severity (asymptomatic, mild-to-severe symptomatic, requiring hospitalization, or fatal). [46] covid-19 pandemic is continuously evolving, with massive number of cases and deaths each day. based on data from the initial outbreak and considering worldwide incidence, the trend of an increasing incidence of covid-19 principally follows exponential growth in the number of reporting cases. [2, 47] the present mean incubation time for covid-19 is 5.5 d, and the median incubation time is 5.1 d, with the potential asymptomatic transmission. following sars-cov-2 infection, most patients (≈97.5%) develop symptoms within 11.5 d and almost every patient shows symptoms within 14 d. very few (≈2.5%) sars-cov-2 infected patients develop symptoms within 2.2 d. [1, 2, 31, 48] as of 21st september 2020, 216 countries/areas/territories/ regions have reported confirmed cases of the disease. usa has reported the largest number of confirmed covid-19 patients (6 703 698), followed by india (5 487 580), brazil (4 528 240), (136 532). [49] sars-cov-2 can affect all age groups especially the higher risk groups which include: i) children <59 months, ii) pregnant women, iii) elderly, iv) people with chronic medical ailments (cardiac, pulmonary, hepatic, renal, metabolic, hematologic, neurodevelopmental diseases, or weak im-mune system), v) people with immunosuppressive conditions (hiv/aids, under chemotherapy or steroids), and vi) health care professionals. [4, 47, 50] lower risk groups should self-isolate themselves at home, drink plenty of fluids, and follow general good-health guidelines to keep their immune system strong and healthy. sars-cov-2 infection is highly contagious, and containment efforts mostly emphasize on quarantine of exposed and asymptomatic suspects and strict isolation of infected patients during the incubation period to limit the disease spread. preliminary clinical feature of the sars-cov-2 infection in humans is pneumonia, which formed the basis of identification, detection as well as isolation of patients. sars-cov-2 infection manifests varied clinical features, extending from asymptomatic to a condition with acute respiratory distress syndrome (ards). [51] in symptomatic patients so far, the clinical manifestations consist of fever >39.1°c (most common symptom), dry cough, nasal congestion, sore throat, dyspnea, headache, myalgia, fatigue, upper respiratory tract infections, smell/taste dysfunctions, and diarrhea. very few patients are reported with rhinorrhea. pneumonia mostly occurs by 2-3 weeks following the initial infection with prominent signs of hypoxemia and deviations in blood gas. [1, 2, 47, 50, 52] visible changes observed in chest x-rays and computed tomography demonstrating deterioration in lung tissue are observed to be the ground glass irregularities of the disease. pulmonary consolidation, alveolar exudates, and interlobular involvements have also been observed. patients also show low white blood cell count (leukopenia), and elevation in inflammatory markers (c-reactive proteins, proinflammatory cytokines, etc.) and formation of blood clots in some cases. covid-19 patients seeking intensive care unit (icu) are particularly older and more likely to carry pre-existing comorbid conditions like hypertension and related heart diseases followed by diabetes. although, certain epidemiological features were identified, additional studies are still warranted. [1, 2, 46] recent reports have also revealed the existence of asymptomatic infections and gastrointestinal symptoms, specifically among youngsters. [53] the occurrence of asymptomatic individuals with the ability to spread the infection may be a great hurdle in the control and containment of the disease and are posing significant public health threats. however, detailed studies on the extent of asymptomatic persons, their viral loads, and share in transmission need further evaluation. this allows a better understanding of the viral pathogenesis and will aid policy makers to develop scientifically sound guidelines. [8] currently, there is no specific approved therapeutic agent available to treat sars-cov-2. treatment is supportive and based on the patient's medical condition to alleviate symptoms. proper public health measures to check the viral infections are of an urgent need to tackle the growing pandemic. additionally, stringent measures must be taken to check the human-to-human transmission to minimize secondary infections and prevent community transmission among near contacts, and health care professionals, and to prevent further spread. based on earlier knowledge of the mers and sars outbreak, the who recommends adaptation of various infection control measures to lessen the overall risk of the disease transmission and prevent overall spread. this includes avoiding close proximity with people showing symptoms of acute respiratory infections, washing hands with soap frequently (minimum for a span of 20 s), using 60-95% alcohol-based hand sanitizers after contacting infected people, and avoiding contact with infected inanimate surfaces and pet/wild animals without having proper protection. [54] the practice of cough etiquettes by people suffering from acute respiratory tract infection, maintaining distance from these patients, proper covering of cough or sneezes, and washing hands frequently are few other disease preventive and control measures being recommended. the who further recommends proper and consistent adoption of environmental disinfection and cleaning methods like cleaning of supposedly infected surfaces with water, soaps, disinfectants, and detergents. people are recommended to stay at home, avoiding social contact (stay home stay safe). while the governments have imposed lockdown measures, travel bans, and ban on people gathering in parks and public places. people should leave their homes putting facemasks and only for essential/basic commodities or medical needs. people should avoid unnecessary journeys and travel to or from work only when they cannot work from home. people going outside should maintain a social distancing of more than 2 m apart from anyone other than members of their own family. a person showing sars-cov-2 symptoms (fever of ≥37.8°c, a persistent cough or breathing problems) should take extra precautions and self-isolate (quarantine) himself/herself for 10-14 d including his family members. [55] although, significant methodological advances have been made in the field of viral detection of covid-19 with many companies receiving emergency use approvals for their in vitro diagnostic tests, molecular-based high complexity laboratory tests, or antibody tests as mentioned by the us-fda website (a total of 251 as of september 21, 2020), little progress has been made in identifying curative or preventive therapies. [56] thus, there is a definite need to find therapeutic agents directly targeting sars-cov-2, and several scientists around the world are focusing on the development of fruitful prevention/treatment strategies and finding new drugs/antivirals/vaccines against sars-cov-2, to halt the ongoing outbreak. the us-fda website lists dozens (a total of 100 companies as of september 21, 2020) of companies involved in developing antiviral or vaccine therapies for covid-19. [57] antivirals can be broadly categorized into two classes, i.e., 1) virus targeting antivirals, which target the viral life cycle, machinery and pathways or directly inactivate viral structural proteins; and 2) host targeting antivirals, which either target the host cellular machinery important for viral infection or target the host's immune response pathways and cascades elicited toward the viral infection. [12, 58] in the course of viral infection, various events in the viral life cycle and virus-host protein-protein interactions have been identified as the potential targets for antivirals. [59] cellular events such as virion adsorption, intracellular transport, uncoating, genome and protein synthesis, and assembly inside infected host cells play a decisive role in the viral pathogenesis and targeting these can be a good strategy in the development of current therapies for tackling viral infections. the existence of a potentially small number of viral targets and fast mutating genes sometimes make the business of finding and developing novel and potent antivirals a challenging task. viruses offer limited intrinsic targets for engineering antivirals. [60] as viruses are greatly dependent on the host cellular machinery for replication, [61] targeting various host factor(s) [62] and molecular pathways hijacked by the virus opens a pandora of opportunities to construct novel antivirals. [12, 62, 63] potential therapeutic targets in sars-cov-2: mostly various genes and their encoded proteins of sars-cov-2 can act as favorable diagnostic, therapeutic, or vaccine targets for the virus (figures 4 and 5) . mechanisms such as inhibition of viral enzymes (dna and rna polymerases, 3cl pro, tmprss2, reverse transcriptase, neuraminidase, endonucleases, and other proteases) or processes such as ace2 cellular receptor inhibitors and endosomal acidification mediators prohibiting viral fusion; molecules interfering with glycosylation of the viral protein, viral assembly, new viral particle transport, and release, and immunomodulation of cytokine release can be potential targets in developing various antiviral drugs for the sars-cov-2. [64] [65] [66] tmprss2 inhibitors (camostat, nafamostat) and furin inhibitors can abrogate the s1/s2 proteolytic cleavage, thereby blocking the viral entry. [35] in addition, the viral entry can be curbed by using carbohydrate-binding protein inhibitors such as griffithsin, which bind to the spike glycoprotein. [67] endosomal cell entry and s protein activation inside endosomes depend on the ph-dependent endosomal protease cathepsins and might be clogged using lysosomotropic agents like ammonium chloride, chloroquine, and cathepsin inhibitors. [68] antibodies against the rbd and other viral proteins can effectively inhibit virus entry into the host cell. the enzyme 3cl pro, one of the essential proteases of the sars-cov life cycle, plays a crucial role in the proteolysis of various viral polyproteins, controlling viral replication. the 3cl pro enzyme is considered as a key drug target in the case of sars-cov and mers. [69] recent studies revealed that sars-cov-2 3cl pro is conserved and carries 99% nucleotide sequence identity with sars-cov 3cl pro. hence, 3cl pro inhibitors that impede the cleaving ability of 3cl pro might repress virus replication, rendering this enzyme an attractive therapeutic target against covid-19. [69, 70] however, most of the potential drugs/vaccines in the spotlight as potential covid-19 therapeutics are only experimental candidates, and vigorous clinical studies are warranted to verify their efficacy and safety. the antivirals being investigated may be toxic and many of them can merely alleviate certain conditions. to date, apart from the emergency use approval of the antiviral drug favilavir in china, india, russia, and parts of the middle east and the emergency use approval of remdesivir by the us-fda and japan in covid-19 patients, there are no approved therapeutic molecules to treat the covid-19 pandemic. [56, 71] thus far, therapeutic modalities including various western, [72] natural product-based (nct04382040), and traditional chinese medicines [73, 74] with some potential activity against covid-19 have been briskly tested clinically, and they exhibited preliminary efficacy against covid-19. [65] drug repurposing (repositioning, or retasking) expedites drug product development by identifying new uses for existing approved or experimental drugs in the current global crisis. this www.advancedsciencenews.com www.advtherap.com strategy could significantly reduce the cost and duration of drug development compared to discovering and developing entirely new therapeutics. [75] the following are the lists of various drugs/therapeutics under consideration and testing to be repositioned against covid-19. favilavir (also known as favipiravir, t-705) a guanosine nucleotide analogue, is an antiviral with broad-spectrum activity. it is a pyrazine carboxamide derivative and is currently being mar-keted in china and japan as avigan for treating influenza. it became the first antiviral drug to gain approval from the national medical products administration of china for clinical trials in treating sars-cov-2. favilavir is highly effective in treating rna virus infections by inhibiting the rdrp. favilavir, originally was formulated to battle catarrhal (inflammation in nose and throat), has shown efficacy in clinical trials carried out in covid-19 patients. chictr2000029600 and chictr2000029544 are ongoing clinical studies evaluating the safety profile and efficacy of favilavir. it has been approved in italy by italian medicines agency (aifa) for experimental use against covid-19 and clinical trials are underway (nct04336904). it is being administered to www.advancedsciencenews.com www.advtherap.com moderate covid-19 patients at 1800 mg twice a day on the first day, and thereafter 600 mg thrice a day up to 14 d; however, the dose may vary based on indications. favilavir is one of the potential treatment options currently being tried for possible use in the treatment of patients suffering from covid-19. despite its potential effectiveness and mass production in china, favilavir is not yet approved as a drug product for covid-19. nct04310228 is another ongoing clinical trial assessing the usefulness and safety of favilavir in combination with tocilizumab. vero e6 cells infected with ncov2019betacov/wuhan/wiv04/2019 exhibited a half-maximal effective concentration value (ec 50 ) of 61.9 × 10 −6 m against favilavir. [72] remdesivir has been labeled as the most promising antiviral by the who for the ongoing sars-cov-2 caused covid-19 pandemic. [76] remdesivir (development code gs-5734), an adenosine nucleotide analogue, is a broad-spectrum antiviral drug. a study reported the ec 50 value of remdesivir against vero e6 cells infected with ncov2019betacov/wuhan/wiv04/2019 to be around 0.77 × 10 −6 m and also showed its antiviral effect against 2019-ncov infected human liver cancer huh-7 cell line. [72] it is a monophosphoramidate prodrug and is metabolized to the active form, gs-441524, which disguises and gets incorporated in new rna strand by viral rna polymerase, thereby escapes proofreading by viral exonuclease, halting genome replication. [77] [78] [79] originally, remdesivir was developed and synthesized to battle ebola and was reported to have treated an american covid-19 patient, who has now fully recovered after receiving the drug. [80] however, more clinical data are required before the drug can be approved and is considered as an effective and official drug to treat either sars-cov-2 or ebola virus. warren and group reported the ec 50 of remdesivir against ebola virus infected macrophages, human endothelial and liver cells to be around 0.01 × 10 −6 to 0.20 × 10 −6 m, and demonstrated its therapeutic efficacy in an ebola virus infected monkey model of the disease. [81] initially china planned phase iii clinical studies to estimate the safety profile and efficacy of remdesivir in covid-19 patients based on its promising preclinical data in sars-cov and mers-cov infections, [82, 83] evaluating remdesivir in severe covid-19 (nct04257656) and in mild/moderate covid-19 patients (nct04252664). however, these trials were either terminated or suspended due to the unavailabilty of eligible covid-19 patients. similarly, in february 2020, the u.s. national institute of allergy and infectious diseases (niaid) carried out a phase iii adaptive covid-19 treatment trial (actt) globally to assess the efficacy of various investigational molecules compared to the control arm (nct04280705). preliminary results from the trial indicated that remdesivir was superior as compared to placebo in shortening the recovery time in hospitalized covid-19 patients. [84] another multicentric trial for remdesivir compared the drug with the standard of care treatment, which includes supplementary oxygen and ventilator support when indicated, in severe covid-19 (nct04292899) patients and patients with moderate covid-19 (nct04292730). in the trial, along with the standard care, a 200 mg loading dose of remdesivir was administered on day 1, followed by 100 mg dose administered as intravenous (iv) infusions every 24 h for 2-5 or 2-10 d. [85] results of the trials indicated no significant difference in clinical status of covid-19 patients compared to the standard of care. [86, 87] other drugs like chloroquine or hydroxychloroquine are also under consideration in the global hunt to discover an effective covid-19 therapy [88] after their approval for limited and emergency use for covid-19 by the us-fda. chloroquine a "4-aminoquinoline" is an old drug used in the prevention and therapy of malaria. it is further prescribed in systemic lupus erythematosus (sle) and rheumatoid arthritis (ra) due to its anti-inflammatory activity. in preliminary studies, the drug has shown efficacy and tolerable safety profile against covid-19 associated pneumonia. [89] wang and group reported effective in vitro inhibition of the virus, using chloroquine. vero e6 cells infected with ncov2019betacov/wuhan/wiv04/2019 and treated with chloroquine exhibited an ec 50 of 1.13 × 10 −6 m. [72] chloroquine also acts as zinc ionophore, and allows passage of extracellular zinc to the cytoplasm and inhibits viral rdrp. [90] [91] [92] it also interferes with viral entry by inhibiting host receptor glycosylation. administered to a patient orally at a dose of 500 mg in 12 to 24 h for 5 to 10 d. [85] hydroxychloroquine, a chloroquine derivative carrying a similar mechanism of action as well as therapeutic activity as chloroquine [93] but with minimal adverse effects, has also been evaluated as an anti-covid-19 therapeutic. [94] in a study by yao and group, hydroxychloroquine inhibited sars-cov-2 infected vero cells in vitro after 48 h of growth with a lower ec 50 value (0.72 × 10 −6 m) as compared to chloroquine (5.47 × 10 −6 m). [93] multicentric clinical trials in china have also revealed that the drug shows a potent broad-spectrum antiviral effect. the mechanism of action may be increased endosomal ph, thereby interfering with the fusion process of the virus with the cell membrane. thus, the virus is incapable of releasing its genetic payload inside the cell and replicate further. unlike the us-fda, european regulators restricted the general use of chloroquine for covid-19 without significant data and limited their use to clinical trials only. therefore, clinical research at a large-scale is still looked-for to elucidate its mode of action and potential prophylactic/therapeutic efficacy against covid-19. a study (chictr2000029559) in china has tested the drug in 62 covid-19 patients showing mild to moderate symptoms. all the patients received treatments like antiviral and antibacterial drugs, oxygen therapy, immunoglobulin as standard care, whereas only half of them received hydroxychloroquine (400 mg per day) together with standard care up to 5 d. clinical symptoms like the rate of recovery of the body temperature, the time required for cough remission were significantly shortened in patients receiving hydroxychloroquine. larger percentage of patients (80.6%) with improved pneumonia symptoms in the treatment group compared to the control was reported (54.8%). [95] another phase ii clinical study (nct04335084) testing the efficacy of hydroxychloroquine in combination with vitamin c, d, www.advancedsciencenews.com www.advtherap.com and zinc toward the prevention of covid-19 infection is underway. a report by dr. raoult from france further demonstrated significant improvements in covid-19 patients administered with hydroxychloroquine at a dose of 600 mg kg -1 for 6 d along with azithromycin. [88] however, who has pulled out the drugs from their solidarity trial due to lack of efficacy in treating covid-19. [96] 6.1.4. np-120 np-120 (ifenprodil-brand name cerocal), a potential therapeutic option for idiopathic pulmonary fibrosis (ipf), acute lung injury (ali), and persistent coughs may be repurposed for covid-19. np-120 is n-methyl-d-aspartate (ndma) receptor glutamate receptor antagonist, which targets the nmda-type subunit 2b (glu2nb). [97] ifenprodil is a vasodilator, originally developed by sanofi as an oral medication to treat blood circulation disorders in french and japanese markets. it is no longer sold in france but is still marketed in japan. ifenprodil is an approved drug in countries like japan and south korea to treat certain neurological conditions. an independent animal study showing a considerable reduction in ali and enhanced survivability in avian h5n1 infected mice encourages researchers to expand clinical programs to ali and ards associated with covid-19 infection. [97] an adaptive phase iib/iii study assessing the safety and efficacy of ifenprodil (20/40 mg three times a day) together with standard of care in comparison to standard of care alone in the treatment of hospitalized covid-19 patients is underway (nct04382924). ifenprodil is already being tested in clinical trials in patients suffering from ipf and its associated cough (nct04318704). a repurposed drug combination treatment against sars-cov-2, lopinavir and ritonavir (protease inhibitor combination; kaletra), is currently used as both first-and second-line antiretroviral medication for hiv. lopinavir is given in conjunction with ritonavir to increase its half-life. [98, 99] several clinical trials of lopinavir/ritonavir, either alone or with various combinations, are underway. china launched a controlled trial (chictr2000029308) to test the efficacy of lopinavir/ritonavir and ifn -2b combination in patients hospitalized with covid-19. the most commonly studied dosing regimen of lopinavir/ritonavir for covid-19 infection is 200 mg/50 mg orally twice daily for 14 d. [98] some other antiretrovirals were also screened for anti-sars-cov-2 activity. [85] a randomized controlled phase iii trial (nct04252274) assessing the efficacy and safety of darunavir/cobicistat combination (prezcobix) for treatment of covid-19 is underway. however, due to the lack of efficacy in hospitalized covid-19 patients, who withdrew lopinavir/ritonavir from their solidarity trial. [96] 6.1. 6 hoffmann and co-workers showed that sars-cov-2 infection relies on the host cellular factors, such as ace2 and tmprss2, and could be successfully blocked by using protease inhibitors. [35] ace2 enzyme is a protein recognized by various covs (sars-cov and sars-cov-2) to gain cell entry. [27, 100] ace2 receptor is expressed by the epithelial cells covering organs like lung, intestine, and blood vessels. [101] in addition to ace2 as the entry receptor, the virus also requires cellular proteases for the priming of s protein to enter the host cells. tmprss2 is a serine protease employed by covs for s protein priming. [35] therefore, it is anticipated that the viral entry inside host cells can be obstructed by serine protease tmprss2 inhibitors. the marketed tmprss2 inhibitors, nafamostat and camostat have been demonstrated to be effective in blocking the sars-cov-2 cellular entry. [102] a phase iia trial studying the impact of camostat mesilate (foipan) on 180 covid-19 patients was initiated during march 2020 (nct04321096). nafamostat exhibits inhibitory effect against ncov2019betacov/wuhan/wiv04/2019 infected vero e6 cells with an ec 50 value of 22.5 × 10 −6 m. [72] leronlimab (pro 140) is used to treat breast cancer and hiv. leronlimab is a humanized monoclonal antibody (mab) capable of blocking ccr5 (ccr5 antagonist). it is in clinical trials for mild to moderate covid-19 patients (nct04343651) and it is expected to benefit patients showing respiratory complications by diminishing the cytokine storm. currently, leronlimab has received fast track approval by the us-fda as a combination therapy with antiretroviral therapy for hiv and triple-negative metastatic breast cancer and has completed nine clinical trials, including a phase iii trial in hivinfected patients. [103] 6.1. 8 another promising repurposed antiviral drug is umifenovir, also known as arbidol which inhibits the virion membrane fusion to host cell by targeting the interaction between s protein/ace2 receptors. arbidol is currently approved in china and russia as prophylactic and treatment drug against influenza. in vitro cellular models showed antiviral activity against sars and therefore it is anticipated to have promising results against sars-cov-2. the most studied dosing regimen of umifenovir for covid-19 infection is 200 mg orally every 8 h. ongoing clinical trials are further evaluating the efficacy of umifenovir. [85] a randomized, placebo-controlled, phase iv clinical trial assessing the safety and efficacy of umifenovir as an adjuvant therapy to the combined therapeutic regimen of ifn 1a, lopinavir/ritonavir and hydroxychloroquine in moderate to severe covid-19 patients (nct04350684) is underway. our immune system relies on two vital pillars: 1) the innate/general immunity; and 2) the adaptive/specialized immunity. recent literature suggests that innate immunity can also influence the nature of adaptive responses apart from adv. therap. 2020, 2000172 figure 6 . the immune responses generated against sars-cov-2. mainly two types of immune responses are generated in the host against the virus. one is the normal or positive immune response, which leads to virus neutralization and ceasing of the disease progression and the other one is the abnormal or aggressive immune response that gives rise to disease associated complications like the ards during a severe covid-19 infection. protecting the host against viruses during early infection when the initial adaptive immune responses take effect. [104] the synchronized activities of innate and adaptive immunity are vital for gaining overall protection against viruses. innate immunity gives early reactions by enabling the detection and destruction of pathogens quickly within hours and consists of the skin and mucous membranes in the body openings forming external barriers, phagocytic cells, natural killer (nk) cells, various substances in the blood and body fluids. [105] adaptive immunity takes more time in the detection and destruction of pathogens, but it targets the pathogen more precisely and can patrol the body against antigens for months/years by producing memory cells. adaptive immunity consists of t cells, b cells, antibodies, and cytokines as soluble proteins in the blood and tissue. [105] upon virus entry inside host cells, here most specifically the airway epithelial cells for sars-cov-2, two types of immune responses are being observed (figure 6) . one is the positive and healthy immune response and the other is the negative and defective immune response. in the case of rna viruses, after the virus entry and replication and further release inside the host cells, the infection is detected by a set of pattern/pathogen recognition receptors (prrs) of innate immune system comprising first-line of defense against viral infection. inside the cell, prrs like toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) sense the viral ss/dsrna genome and its replication intermediates. upon cellular entry, the virus is recognized by the endosomal ssrna sensor (tlr7/8), the cytosolic dsrna sensor (rig-i, mda-5), and the cytosolic inflammasome sensor (nlrp3). subsequently, these different sensors recruit adaptor proteins including myeloid differentiation primary response 88 (myd88) and mitochondrial antiviral signaling protein (mavs) respectively to further activate downstream signaling pathways. this causes activation of the transcription factors like nuclear factor kappa b (nf-b) and ifn regulatory factors (i.e., irf3 and irf7), and subsequently triggers the production of type i/iii interferons (ifn-and ifn-) and proinflammatory cytokines like interleukin 1 beta (il-1 ), il-6, and tumor necrosis factor alpha (tnf-), respectively. [106] the antiviral activity of ifn-and ifn-is further amplified by the expression of ifn stimulated genes (isgs) such as ribonuclease l (rnase l) and the proinflammatory chemokine (cxcl10) and is vital in limiting the spread and replication of the virus and modulating the innate/adaptive immune responses. cytokines released by infected cells further modulate the adaptive immune response by recruiting and activating immune cells in eliminating the virus. this comprises the positive immune response, wherein the infection attracts t cells specific to the virus, and destroys the virus checking the infection. neutralizing antibodies are also produced against the viruses, which block the viral infection, and alveolar macrophages further clear the viruses by phagocytosis. [107] the virus additionally activates the inflammasome sensor, nlrp3, that lead to the secretion of highly inflammatory cytokine il-1 and the initiation of pyroptosis (highly reproduced with permission. [107] copyright 2020, invivogen. inflammatory form of programmed cell death). this comprises the negative immune response, wherein the host cells undergo pyroptosis, elicited against cytopathic viruses, in response to the viral release. then pathogen-associated molecular patterns (pamps) like viral m-rna and damage-associated molecular patterns (damps) like atp, nucleic acids, and asc oligomers are released from the host cells in reaction to the virus. these molecular patterns are then recognized by other neighboring cells, which include macrophages, epithelial and endothelial cells causing them to release various chemokines and proinflammatory cytokines like ifn gamma-induced protein 10 (ip-10), macrophage inflammatory protein 1 (mip-1 ), mip-1 , and mip-1 , and il-10. these chemokines and cytokines attract immune cells like macrophages, monocytes, and t-cells to the infection site leading to further inflammation and additional production of ifn by t cells. in a defective response, higher accumulation of immune cells causes overproduction of the proinflammatory cytokines causing a cytokine storm that damages the organ being infected, and then it circulates to other organs causing multiorgan damage. it has also been observed that non-neutralizing antibodies produced against the virus by the activated b cells further enhance the damage by sars-cov-2 infection by a phenomenon called antibody-dependent enhancement. [108] downregulation of the host ifn response can cause an unbalanced immune response producing high levels of proinflammatory cytokines and infiltration of inflammatory cells leading to hyperinflammation causing more severe clinical symptoms of covid-19. covs have evolved many mechanisms to stop type i ifn induction and signaling. severe covid-19 patients demonstrated unusually impaired type i ifn signatures in comparison to mild or moderate cases. [107] therefore the category of host immune response generated against the virus (figure 7) defines the disease severity in the sars-cov-2 infection, and aggressive immune response against the virus causes significant damage to the lung airways. [109] so, the adoption of immune therapeutic strategy against the virus could be either based on fortifying the positive immune response or minimizing the dysfunction immune response by means of immune-suppressive therapies. the process of vaccine development against the virus should be dealt with caution. following are some of the list of therapeutic molecules/drug candidates that are meant to target the immune response generated against the virus and under consideration in the present scenario. one promising vaccine candidate for covid-19 is the fusogenix dna vaccine (covigenix). fusogenix is a proteo-lipid vehicle (plv), formulated with well-tolerated neutral lipids and entos proprietary fusion-associated small trans-membrane www.advancedsciencenews.com www.advtherap.com proteins (fast proteins), which have a novel fusion mechanism to deliver therapeutic nucleic acid payloads directly into target cells, intact and unmodified. fusogenix dna vaccine utilizes plasmid dna, which encodes several protein epitopes (antigen) from key immunogenic sars-cov-2 proteins to develop an advanced therapeutic payload with maximum protection. these protein epitopes will help activate the body's inherent antibody production and elicitation of a protective immune response against covid-19. further, in comparison to traditional vaccines, the dna-based vaccine avoids the use of the infectious agent, shows enhanced stability, easy large-scale production, and is expected to stimulate both b-and t-cell responses. in preclinical in vivo studies, covigenix showed high immunogenicity, efficacy, and safety. in phase i/ii human clinical trials, covigenix will be further evaluated for its safety, tolerability, immunogenicity, and efficacy. [103] another vaccine candidate developed against sars-cov-2 is mrna-1273. it is a lipid nanoparticle (lnp) loaded with an mrna that encodes for a complete, prefusion stabilized form of the s protein of the sars-cov-2. mrna-1273 is in phase i human clinical trial to examine the safety and immunogenicity of its five dose levels (10, 25, 50, 100, and 250 µg) on a double-dose vaccination schedule (nct04283461). on 12th may 2020, mrna-1273 was granted fast track designation by us-fda. data from the phase 1 study (first clinical batch), with 25 µg and 100 µg dose levels, have shown the presence of well-tolerated neutralizing antibodies titers at or above convalescent sera. safety data obtained from the phase 1 study led to the discontinuation of the 250 µg dose level in the subsequent phase ii studies. [110] phase iii trial was next launched to assess the efficacy, safety, as well as immunogenicity of the vaccine at a dose of 100 µg in adult participants (nct04470427). another vaccine for covid-19 is based on an adenovirus and is named as ad5-ncov. this vaccine is being developed by the chinese company cansino biologics in partnership with china's institute of biology at the country's academy of military medical sciences. results from a phase i safety (nct04313127) and phase ii (nct04341389) trials conducted for the vaccine, demonstrated strong immune response against sars-cov-2. therefore, in an unprecedented move, the chinese military approved the vaccine as a "specially needed drug." [111, 112] its phase iii trial in saudi arabia is underway (nct04526990). another rna based vaccine candidate developed against sars-cov-2 is bnt162b2. it is based on lnps loaded with nucleosidemodified messenger rna (modrna) that encodes for an optimized sars-cov-2 full length s protein and rbd. based on preclinical and phase i/ii studies (nct04380701) data and in consultation with the us-fda's center for biologics evaluation and research (cber), bnt162b2 has received fasttrack designation by the us-fda and will be tested at a 30 µg dose level (in a two dose regimen) in a global phase ii/iii study (nct04368728). [113] another vaccine candidate against covid-19 is a purified inactivated sars-cov-2 virus vaccine called coronavac (formerly called as picovacc). preclinical studies for the vaccine showed the induction of specific neutralizing antibodies against sars-cov-2 in rats, mice, and macaques (nonhuman primates) those were able to neutralize 10 representative sars-cov-2 strains. three immunizations with the vaccine at two different doses (3/6 µg per dose) provided partial or complete protection in non-human primates (macaques) against sars-cov-2 infection. [114] preliminary results of phase i/ii clinical trials (nct04383574 and nct04352608), which was tested at three/two different doses in a two doses regimen either scheduled on day 0/28 or on day 0/14 or both. (antigen content of 300, 600, 1200 su/0.5 ml) showed promising immunogenicity and no severe adverse events. coronavac got an emergency approval for limited use by the chinese government. phase iii clinical trial (nct04456595) is underway to assess its safety and tolerability. the immunization schedule is two doses im (600 su/0.5ml) with a 14 d interval. ino-4800 dna vaccine is designed in such a way to directly deliver optimized dna plasmids into cells using inovio's trademarked device called cellectra. cellectra utilizes a transitory electric pulse to reversibly open tiny cellular pores, enabling the plasmids to enter. once inside the cell, the plasmids begin replicating along with the cell's dna and are translated into proteins (mabs), producing a specific immune response. this methodology has the potential to generate therapeutic mabs in vivo. clinical trials are underway to assess its safety, tolerability, and immunogenicity (nct04336410). smith and co-workers in a preclinical study testing humoral immunogenicity in both mice and guinea pigs have shown the successful generation of neutralizing antibodies and t cell immune responses against sars-cov-2. these encouraging preclinical results further support its translation to large randomized clinical trials. [115] an adenoviral vaccine candidate azd1222 (provisionally named chadox1 ncov-19) based on the sars-cov-2 s protein that was originally developed to target mers is also a potential vaccine candidate for covid-19. it is a weakened and safer form of a common cold adenovirus (chadox1) that cannot reproduce within the body but can produce cov s protein after vaccination resulting in the formation of antibodies against the s proteins. the vaccine's seed stock was in production at oxford university's biomanufacturing facility, uk, and phase i/ii clinical trials (nct04324606/isrctn15281137 and nct04444674) assessing its safety, efficacy, and immunogenicity against sars-cov-2 are underway. chadox1 ncov-19 is being administered intramuscularly (im) either at a single dose of 5 × 10 10 viral particles (vp) or a single dose of 5 × 10 10 vp followed by a booster dose. the preliminary results from phase i/ii trial (nct04324606) showed an acceptable safety profile, and a second vaccine dose (boosting dose) further enhanced the antibody responses inducing both humoral and cellular immune responses. [116] doremalen and co-workers in a study successfully demonstrated a robust humoral and cell-mediated response from a single dose of an investigational vaccine, azd1222. a single vaccination has prevented pneumonia caused by sars-cov-2 in six rhesus macaques by halting sars-cov-2 replication. [117] the vaccine has progressed to phase ii/iii (nct04400838) as well as phase iii trials (isrctn89951424). on september 6, 2020 astrazeneca stopped global trials of the vaccine after finding a volunteer, who developed a type of inflammation called transverse myelitis. the british trial resumed on september 12, 2020, but trials in other countries are still on hold. another vaccine in progress is based on virus-like particles (vlp). benthamiana. vlp mimic viruses without genetic material and enables the body's immune system to generate an immune response. vlps cannot replicate and are noninfectious. in this plant-based approach, the genetic sequence of a virus is inserted into agrobacterium, which then enters into the plant tissues. the plant begins to produce the protein that is used as a vaccine. in case the virus begins to mutate, the production may be updated in new plants. using plants and genetically engineered agrobacteria, the mass-production of vaccines is easy, faster, and inexpensive. the phase i trial (nct04450004) evaluating safety, tolerability, and immunogenicinity at dosages of 3.75, 7.5, or 15 µg of the vaccine candidate alone or followed by a booster dose in healthy human volunteers is underway. medicago is also planning to initiate its phase ii/iii trials. [118] a genetically modified infectious bronchitis virus (ibv) vaccine is also in the pipeline to treat covid-19. ibv was originally developed to treat avian coronavirus. it is available in oral form and has shown efficacy in preclinical trials. the new vaccine is anticipated to turn sars-cov-2 infection into a very mild cold. [119] another potential protein-based vaccine candidate in the pipeline is covid-19 s-trimer (scb-2019). it is a recombinant subunit vaccine developed using clover's patented trimer-tag technology via a prompt mammalian cell expression system. s-trimer vaccine is based on the s protein of the sars-cov-2. the company also identified the antibodies against the trimeric s protein in the serum of patients fully recovered from covid-19. a randomized, placebo-controlled phase i trial of scb-2019 has been launched and will be evaluating the safety, reactogenicity, as well as immunogenicity at multiple dose levels (3, 9, and 30 µg) with and without the adjuvant (nct04405908). [103, 118] vaast, oral recombinant vaccine tablets based on the genome covid-19 causative virus using vaxart's proprietary oral vaccine platform, are in preclinical trials. each vaccine constructs is a nonreplicating viral vector based on a different sars-cov-2 antigen combination. [103, 118] adcovid is another potent single dose, intranasal vaccine candidate in preclinical trials to protect against covid-19. it is an adenovirus-based vaccine expressing sars-cov-2 s protein. [103] 6.2.13. sputnik v sputnik v a covid-19 vaccine was registered on 11th august 2020 and approved for early use by the russian ministry of health under the adopted emergency rules during the pandemic. it is a dual adenovirus vectors (rad26 and rad5) based vaccine loaded with a fragment of gene coding s protein of the sars-cov-2. the use of two dissimilar forms of adenovirus vectors, for the first and second vaccination doses is a unique technology of the gamaleya national center for ensuring long lasting immunity. on 1st august 2020, phase i/ii clinical trials of sputnik v (nct04437875 and nct04436471) have been completed and results suggested the induction of strong antibody and cellular immune responses. postregistration phase iii clinical trial comprising ≥ 40000 people in russia was expected to get started 31st august, 2020 onwards (nct04530396). other countries (uae, saudi arabia, philippines, india, and brazil) may also join the phase iii clinical trial locally. [118] the key focus of developing therapeutics against the virus has been revolving around finding antivirals and vaccines. however, many reports point toward various patients associated complications such as cytokine storm syndrome (crs) and macrophage activation syndrome (mas), which lead to ards during severe covid-19 infection. covid-19 patients developing crs secondary to covid-19 pneumonia show increased production of various proinflammatory cytokines, chemokines, and other growth factors. therefore, treatment of hyperinflammation or the aggressive immune dysfunctions raised in response to the viral infection using immunosuppression mechanism can be advantageous and aid in reducing the mortality. [52, 120] www.advancedsciencenews.com although immunomodulatory therapy is not recommended in covid-19 pneumonia in general, the compassionate use of immunomodulators might be advantageous in covid-19 patients exhibiting crs complications, decreasing the high levels of proinflammatory cytokines, and thereby preventing multiorgan failure. [121] the following are some of the immunomodulators under consideration as anti-covid-19 therapeutics. gimsilumab (kin 1901) , a human mab, working by targeting granulocyte macrophage colony stimulating factor (gm-csf), is under clinical trials to prevent and treat ards in covid-19 patients. as gm-csf elevated levels in blood further augment the expression of other proinflammatory cytokines causing the progression of ards and serum from covid-19 patients have shown high levels of gm-csf; therefore, gimsilumab is anticipated to reduce the mortality rate by reducing lung damage. a multicenter, phase ii trial (nct04351243) is underway to assess gimsilumab's efficacy and safety profile in patients with ards/lung injury secondary to covid-19. [103] tjm2 (tj003234) is a neutralizing antibody against human gm-csf. tjm2 shows a high affinity towards human gm-csf, thereby blocking gm-csf binding to its receptor. this will further prevent downstream signaling cascades resulting in response to gm-csf binding, halt target cell activation, resulting in inhibition of inflammatory responses causing reduced tissue inflammation and death. tjm2 is in clinical trials to study its efficacy in reducing the severity of complications associated with covid-19. [103] a phase i/ii, randomized, multicenter trial (nct04341116) will assess the efficacy of tjm2 under supportive care when administered as an iv infusion (3 mg kg -1 or 6 mg kg -1 ) in subjects with severe covid-19. lenzilumab, another gm-csf neutralizing immunotherapy, is being studied in minimizing and treating the cytokine storm and associated lung dysfunction/ards in covid-19 patients. lenzilumab is humanigen's proprietary humaneered mab targeting gm-csf and is being approved by us-fda for sympathetic use in covid-19 patients. [103] currently, it is being studied in a multicentric, phase iii trial in comparison to the current standard of care for taking care of respiratory failure and prevention of death in hospitalized covid-19 patients (nct04351152). namilumab (izn-101, amg203) is another fully human immunoglobulin g1 mab targeting gm-csf, which has received compassionate use approval from us-fda in the treatment of critical as well as hospitalized covid-19 patients before their admission to icu. 103 the double center compassionate use study of the drug will be conducted in bergamo and milan cities in italy. it is currently in later stages of clinical development programs against ra and ankylosing spondylitis. [122] tzls-501 is another antibody in progress as a potential treatment for covid-19. tzls-501 is a humanized mab targeting interleukin-6 receptor (anti-il-6r). early clinical studies channeled in china suggest that anti-il-6r mabs might be used clinically for treating covid-19 patients. tzls-501 binds to both forms of il-6r (membrane-bound or soluble), thereby speedily depleting the levels of il-6 in blood. overproduction of il-6 leads to the elicitation of long-lasting inflammation, causing lung damage following sars-cov-2 infection. [103] 6.3.6. at-100 another immunotherapeutic candidate explored for covid-19 is at-100. at-100 is a human recombinant surfactant protein d (rhsp-d). initially, at-100 was developed for broncho-pulmonary dysplasia (bpd) in premature infants who requisite mechanical ventilation and oxygen therapy. [103] tocilizumab (actemra) is a recombinant version of a humanized mab targeting the il-6 receptor, which binds to il-6r, thereby inhibiting signal transduction. us-fda has approved it for the treatment of crs, rheumatoid arthritis, giant cell arthritis, polyarticular juvenile idiopathic arthritis, and systematic juvenile idiopathic arthritis. [121, 123] xu and group from china in their pilot study used tocilizumab (single dose of 400 mg iv) to treat a total of 21 covid-19 patients. tocilizumab treated patients showed a significant reduction in fever and il-6 levels. lymphocyte counts in the patients returned to normal after treatment and they demonstrated an improvement in lung function. [123] a multicentered randomized phase iii trial of tocilizumab is underway in china to assess its efficacy and safety in covid-19 patients with pneumonia and elevated il-6 (chictr2000029765). another proof of concept study (tosca) in italy is underway for its efficacy and safety profile assessment in the treatment of patients with ards and crs secondary to covid-19 (nct04332913). another multicentric phase iii placebo-controlled clinical trial, called covacta (nct04320615), is underway to evaluate tocilizumab (maximum single dose of 800 mg iv and one more dose only if clinical symptoms deteriorate/or exhibits no improvements) along with standard of care versus placebo along with standard of care in severe covid-19 patients globally. sarilumab (kevzara) is another il-6 receptor blocker under clinical trials for its ability to lessen the effect of the inflammatory adv. therap. 2020, 2000172 www.advancedsciencenews.com www.advtherap.com immune response against sars-cov-2 infection. it is a fully human mab against the il-6 receptor. it has been approved by us-fda toward the treatment of rheumatoid arthritis, but its use to treat covid-19 patients is investigational. sarilumab was developed using proprietary velocimmune technology (regeneron's) using a genetically engineered mouse equipped with a genetically humanized immune system to produce fully human antibodies. a multicenter, double blind, phase ii/iii trial (nct04327388) is underway to assess the efficacy and safety of a single iv dose of sarilumab compared to supportive care plus placebo in severe/critical covid-19 patients. in phase ii, patients were distributed into three groups at random, with two doses of sarilumab (200 and 400 mg) and placebo. preliminary data from the trial led to its immediate amendment to enroll only critically ill covid-19 patients (either on icu or requiring mechanical ventilation/high flow oxygenation) receiving a higher dose of the drug (400 mg) or placebo. [103] cytosorb is an extracorporeal blood purification technology based on hemo-adsorbent polymer to treat uncontrolled inflammation in patients those have undergone cardiac surgery. it consists of polymer beads with high porosity those can successfully adsorb and remove toxic substances and inflammatory mediators from blood and other bodily fluids. cytosorb has received us-fda's emergency use authorization in the us for use in severely ill covid-19 patients approaching or confirmed respiratory/multiple organ failure and are admitted to icu. it is approved in the european regions as an adjuvant therapy designed to lessen the complications associated with crs by absorbing a wide range of cytokines, pamps and damps, thereby reducing their levels in circulation. [124] the use of convalescent sera is a promising treatment strategy against sars-cov-2. it consists of patient derived serum enriched with passive neutralizing antibodies against the virus, obtained from covid-19 recovered patients. [125] many reports from china suggest the use of convalescent serum as therapy for patients with covid-19. china has offered convalescent plasma therapy to 200 covid-19 patients (chictr2000029757). convalescent plasma therapy is promising, but its safety and efficacy as a treatment modality for covid-19 are yet to be established. therefore, it is important to study and establish the safety as well as efficacy of convalescent plasma therapy in covid-19 patients in clinical trials. the us-fda based on encouraging results from early trials has given it the emergency use authorization against covid-19. glucocorticoids may modulate inflammation-mediated lung injury in covid-19 patients and thereby reduce progression to respiratory failure and the resulting death. however, there is uncertainty about the effectiveness of glucocorticoids in covid-19. dexamethasone is a corticosteroid used in a wide range of conditions for its anti-inflammatory along with immunosuppressant effects. preliminary findings of the phase ii/iii recovery trial (nct04381936), a randomized, controlled, open-label study conducted in the uk, noted a survival benefit with the use of dexamethasone (oral or iv at a dose of 6 mg per day for up to 10 d) as compared to standard care alone (invasive mechanical ventilation or oxygen) in hospitalized covid-19 patients. patients on ventilators showed one third and patients requiring only oxygen showed one fifth reductions in the 28 day mortality. dexamethasone can also be used in both children and elderly, but in pregnancy/breast-feeding cases, the recovery trial used prednisolone (40 mg orally) or hydrocortisone (80 mg twice daily iv) instead of dexamethasone. [126] among several drugs employed for the treatment of covid-19, n-acetylcysteine (nac) is a potential therapeutic/preventive/adjuvant against sars-cov-2. nac is a precursor of reduced glutathione (gsh). it has antiinflammatory, antioxidant, antiviral, and anticoagulant/plateletinhibiting properties. it is approved as an over-the-counter dietary supplement and is in the who list of essential medications. the us-fda has approved the usage of nac to treat acetaminophen overdose associated liver side effects, and also as mucolytic agent (due to its free sulfhydryl group) in cystic fibrosis or chronic obstructive pulmonary disease (copd). it has been also used as an antioxidant in a variety of disorders involving gsh depletion and oxidative stress. thiols can block ace2 receptors thereby blocking penetration of sars-cov-2 into cells. nac can act as a potential therapeutic agent in the treatment and/or attenuating the risk associated with covid-19 through a variety of potential mechanisms, including increasing glutathione level, improving t cell response, and modulating inflammation thereby preventing covid-19 patients from moving to icus. [127] an ongoing phase ii clinical trial (nct04374461) is assessing the number of successful covid-19 patients discharged/transferred from critical care units after receiving nac (6 g per day iv) in addition to supportive or other treatments as prescribed against covid-19. other phase iii (nct04455243) and phase iv (nct04419025) clinical trials of nac are assessing nac's anti-inflammatory effect and its efficacy in minimizing the severity associated with covid-19, respectively. [128] cyclosporine a (csa) is a calcineurin inhibitor and is used to avert organ rejection and for treating t-cell associated autoimmune diseases. anti-inflammatory and immunosuppressive effects of csa are exerted due to its bonding to cyclophilin-a (cyp-a), thereby preventing the activation of nuclear factor of activated t-cell (nfat) and production of il-2 (a cytokine crucial for the proliferation of t-cells). csa has shown potent antiviral activity at noncytotoxic micromolar concentrations against many covs including sars-cov in cell cultures. csa may be successful in covid-19 due to the close resemblance between sars-cov-2 and sars-cov. this antiviral property might have resulted due to the inhibition of cyp-a-dependent viral assembly and inhibition of the nfat pathway. [129] [130] [131] ongoing phase i safety study of csa (oral or iv) is assessing the tolerability and clinical effects in covid-19 patients requiring oxygen supplementation (nct04412785). a phase iia randomized clinical trial (nct04492891) is underway for the treatment of non-icu hospitalized covid-19 patients. patients will receive csa orally for up to 7 d at 2.5 mg kg -1 body weight twice a day along with standard of care. another ongoing phase iv clinical trial (nct04392531) is assessing the efficacy and safety of csa plus standard treatment in comparison to standard treatment in hospitalized covid-19 patients. another corticosteroid in pipeline is inhaled budesonide. it may directly inhibit the viral replication or may exert the inflammatory effect. steroids reduce the effect of cytokines and thereby modulate immune response. budesonide may prevent the ards associated with covid-19. inhaled corticosteroid's therapeutic effect against covid-19 is limited and still need to be explored. various clinical trials assessing their efficacy in covid-19 are ongoing (phase iv: nct04355637). vitamin d is a steroid hormone. vitamin d is being evaluated as an adjuvant therapy for covid-19 based on its immunomodulatory, anti-inflammatory, antifibrotic, and antioxidant effects. vitamin d exerts direct effect on immune cell proliferation and activity, and ace2 expression. [132] the aim of one of its ongoing randomized phase iii trial (nct04385940) in covid-19 patients is to assess its efficacy at daily low dose (1000 iu) versus weekly high dose (50000 iu) for 3 weeks and to determine the correlation between vitamin d deficiency and clinical characteristics. another phase iii trial (nct04344041) assessing the efficacy of vitamin d3 at high dose (400000 iu) in comparison to standard dose (50000 iu) is underway. apart from the currently available drugs, immunomodulators, and vaccine candidates, there are many other potent molecules like novel protein or peptide therapeutics and even nanoparticulate based strategies yet to be tested against the virus. these new therapeutics, once proven, can be the game changers in tackling the pandemic. below we list some of the potential future therapeutics for treating covid-19 patients. in the last decades, peptides as therapeutics and diagnostic molecules have gained massive interest in drug development due to the dynamic research conducted by pharmaceutical and biotech companies. [133] currently, a large number of both natural as well as synthetic therapeutic peptides are in clinical development for various diseases ranging from cancer, neurodegenerative, cardiovascular, metabolic disorders, autoimmune disorders, hormonal deficiencies, to infectious diseases. [133, 134] peptide based therapeutics offer numerous benefits over other molecules such as high selectivity and specificity, high efficacy and safety, tolerability, less immunogenicity, biocompatibility, low toxicity, easy, and cost-effective production. according to their source and method of production, peptide based therapeutics can be classified as: 1) natural peptides (derived from peptide hormones, plant proteins, animals, and human-derived peptides); 2) recombinant peptides; and 3) synthetic peptides. [133] [134] [135] peptides are relatively easy to modify structurally. improved therapeutic performances can be achieved by utilization of modified amino acids, cyclization, etc. many antiviral peptides are being explored for viral diseases like hepatitis, influenza, and hiv. [136] peptides exhibit a reduced possibility of developing resistance during the treatment. antiviral activities of the peptides can be attributed to three major mechanisms: 1) inhibition of viral attachment and penetration into host cells; 2) inhibition of replication by interacting with viral polymerases, and 3) disruption of the viral envelope. they can be presented as a new generation of antiviral agents with broad-spectrum activities. [136, 137] potential peptide-based therapeutics which are in the pipeline against covid-19 include flowvax peptide vaccine from flow pharma, [103] corvax signal peptide vaccine from vaxil bio, (vax-hit platform), [103] epitope-based peptide vaccine from onco-gen (vaccinomics strategy), [103] ii-key peptide vaccine from generex biotechnology (epivax's computational tools and li-key technology), [118, 138] etc. peptide-based therapeutics can be effective alternatives for covid-19 prophylaxis. nanotechnology, as one of the ground-breaking approaches, can be really promising in offering remedial solutions toward fighting the on-going covid-19 outbreak and future pandemics. nanotechnology allows a number of tactics to fabricate highly advanced therapeutic options to cope with sars-cov-2 both outside and inside the host. the scope of nanotechnology against covid-19 includes conventional as well as advanced biomimetic approaches and engineered nanomaterials with versatile chemical functionalities. therefore, nano intervention will be extremely relevant in advancing covid-19 prophylaxis, diagnosis, and treatment (figure 8) . in view of what we know so far about the sars-cov-2 structure, its pathophysiology, life cycle, and related immunological response, we envisage key steps where nanotechnologist and nanotechnology could play a pivotal part. herein, nanotechnology is discussed in terms of fabricating engineered nanocarriers to overcome the limitations associated with conventional antiviral and other biological therapeutics, nanomaterials as novel therapeutics, risk free immunization techniques, engineered nanomaterials in the development of point-of-care diagnostics, and alternative advanced disinfection methodologies. nanotechnology aims toward safe, effective, and targeted delivery of existing as well as future therapeutics, fabricating risk-free vaccines for safe and effective immunization, curbing the interactions between viruses and host cells, and permanent disruption of viral particles. [139] [140] [141] in the recent past, many nanoparticulate systems have been tested for their efficacy and made their way to preclinical studies against several pathogenic viruses such as hiv, herpes simplex, human papilloma virus, and respiratory viruses. [140] [141] [142] in the dearth of a specified/broad spectrum antiviral agent against sars-cov-2, current therapy focusses on repurposing existing molecules. therefore, to make the repurposed therapeutics more effective and safer, an appropriate nanocarrier delivery system might be useful. successful delivery of therapeutics especially peptide/protein, dna/rna, and other biologicals to the target site is most often hampered due to their poor aqueous solubility, degradation, fast-clearance, low-bioavailability leading to subtherapeutic concentrations. nanomaterials with large surface area to volume ratios, versatile functionalities and surface modification, unique physicochemical properties, and suitable for various routes of administration can easily address the challenges associated with conventional therapeutics. biocompatible organic/inorganic nanoparticles can be potentially tuned to encapsulate therapeutics with large payloads, control release, minimize drug resistance, and improve pharmacokinetic/pharmacodynamic properties and patient compliance. further actively targeted nanocarriers can cross the biological barriers thereby reaching the protected target sites with higher specificity excluding the unwanted exposure to nontargeted areas resulting in dosage reduction, reduced systemic toxicity, improved bioavailability, and enhanced efficacy. nanotechnology further makes it possible to tailor the therapy according to individual needs. [140] itani and group have demonstrated the potential role of different nanoparticles as efficient delivery agents for potential therapeutics or immunomodulators against covid-19. [142] nanoparticle based delivery of therapeutics (drugs, vaccines, rna/dna, and peptides) ensures targeted and optimized concentration in the desired site while minimizing adverse effects and unwanted degradation enhancing efficacy. donalisio et al. demonstrated nanotechnological approach for the tropical treatment of herpes simplex virus (hsv-1 and hsv-2) by encapsulating acyclovir in chitosan nanoparticles. the obtained gel formulation showed enhanced penetration across the porcine skin as compared to the commercial cream product, and revealed increased efficacy as compared to its free form. [143] labauve and group used a lipid-coated mesoporous silica nanocarrier system to deliver antiviral molecule ml336 for inhibiting venezuelan equine encephalitis virus (veev), in order to improve its solubility and stability. the designed ml336 loaded nanosystem showed enhanced circulation time, biocompatibility, and viral inhibition in vivo in a murine model of veev infection. [144] kumar et al. demonstrated the fabrication of zidovudine loaded lactoferrin nanoparticles. zidovudine is a highly bioavailable antiretroviral drug, which shows serious side effects and toxicity. results revealed higher efficacy and reduced toxicity of nanocarrier based formulation as compared to their free soluble counterparts. [145] liu et al. demonstrated the fabrication of liposomes encapsulating cholesterol modified hydroxychloroquine for the treatment of bleomycin induced pulmonary fibrosis. the engineered nanosystem inhibited the proliferation of rat lung fibroblasts and reduced any toxicity associated with hydroxychloroquine thereby, providing evidence as a possible therapeutic agent for pulmonary fibrosis. [146] encapsulation in a nanosystem enhances nucleic acid-based vaccine stability and offers a newer fusion mechanism to transport the genetic material loaded inside them directly to the cells. similarly, the efficacy of other mrna/sirna vaccines can be significantly enhanced via complexing them with lipid/polymerbased nanoparticles. these lipid/polymer-based nanoparticles are based on ionizable monomers, which complex negatively charged rna/dna and thus facilitate escape of the entrapped rna/dna from endosomes into the cytosol where the genetic material can be translated. [30] the successful inhibition of viruses achieved with nanoplatforms could have extensive advantage in combatting various viral infections including sars-cov-2. combinatorial therapy aims in suppressing more than one pathway simultaneously, thereby, producing synergistic effect. therefore, combinatorial therapy may represent another therapeutic approach against sars-cov-2. it displays numerous advantages such as dose reduction of the individual therapeutics, leading to fewer adverse events. it may also overcome drug resistance. nanocarriers are very promising systems for the co-delivery of multiple drugs with different physicochemical properties. they enable encapsulation and sequential release of both hydrophobic and hydrophilic drugs in a single platform. nanoparticles can also be instrumental in attaining combinatorial multidrug therapeutics for controlling uncontrolled inflammation and manage disease severity. a very recent work by dormont et al. developed targeted multidrug nanoparticles composed of an en-dogenous lipid squalene, an immunomodulator adenosine and an antioxidant -tocopherol for treating acute viral inflammation (figure 9 ). [147] freeling et al. fabricated a lipid-drug nanoparticle system comprising of three antiretroviral drugs (lopinavir, ritonavir, and tenofovir) to overcome lymphatic drug insufficiency and reported 50-fold higher and sustained intracellular concentrations in lymph nodes compared to current oral therapy of free drugs. this combinatorial nanotherapy can be used to target sars-cov-2. [148] bearing in mind the risk of random mutations in sars-cov-2 genome, which may result in variation in antigenic protein, nanoparticle functionalization with a varied number of antigenic moieties/proteins concurrently will enhance the efficacy of vaccines against viral infections. these next-generation nanocarrier-based vaccines will not only help in guarding the antigenic proteins against metabolism/degradation but will also help in greater exposure of antigenic proteins to antigen-presenting cells, thereby improving their delivery and efficacy. nanomaterials have emerged as a favorable tool both as immune stimulators and immune suppressors for immune modulation. nanomaterial based nonviral vectored vaccines can successfully overcome two major limitations associated with conventional vaccines of weak immunogenicity and reversion risk associated with live/attenuated viral vectors, and can be a reliable alternative to viral vaccines. nanosystems can imitate the genome transfer ability of viral vaccines which show high pathogenicity and can also deliver molecular adjuvants simultaneously. [139] recombinant protein and inactivated vaccines in general necessitate the use of adjuvants to enhance their immunogenicity. many nanomaterials depending on their functionalization possess an intrinsic adjuvant property and can amplify the immune response in a host. therefore, fabricating nanomaterials with intrinsic ability to amplify host's immune response will be very helpful in enhancing the efficacy of vaccines especially in patients with impaired immune system. nanosystems can also enhance the targeted delivery of immunosuppressants to both adaptive and innate immune systems. [139] various nanoparticles like graphene oxide, silver, gold, copper, and zinc nanoparticles show intrinsic antiviral/antibacterial properties and can act as potential therapeutics against the disease. [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] they may offer alternative methods to traditional disinfection protocols used in healthcare sites. nanoparticles can potentially sustain release, enhance cell entry, and reduce efflux of toxic metal ions. the use of metal nanoparticles would be particularly advantageous as antiviral agents because metals may attack a wide range of viral targets. also, there is a lower risk of developing drug resistance in the case of metals as compared to conventional antivirals. [149] silver nanoparticles (agnps) have been investigated for their antimicrobial potency against bacteria, but many reports [147] copyright 2020, the authors. point toward their antiviral activities against viruses, which includes hiv, herpes simplex virus, respiratory syncytial virus, hepatitis b virus, etc. [150] [151] [152] li and group have demonstrated efficient in vitro inhibition of h1n1 infection utilizing agnps based codelivery of oseltamivir (neuraminidase inhibitor) by inhibiting viral attachment and release. [153] the fact that agnps are less toxic and are nontoxic at lower concentrations to humans, vouch for their possible usage for therapeutic purposes. given the fact that silver ion itself attacks a broad range of targets in the microbial entry, microorganisms generally do not develop resistance against silver. [152] nanocolloid agnps have also been shown to be effective in controlling viral proliferation in the respiratory tract after inhalation delivery, thus controlling the disease progression. [154] another study by levina and group demonstrated in vitro antiviral activity of dna decorated titanium dioxide (tio 2 ) nanosystem against different influenza a virus subtypes infected mdck cells. the nanosystem was found to be highly efficient in delivering nucleic acids directly inside the cells without the aid of any transfection agent with an ic 50 value of 1.5 µg ml -1 (30 × 10 −9 m for dna). [155] another nanostructured material, graphene, has shown promising antimicrobial/antiviral properties. [156] [157] [158] recently, the efficacy of cu to inactivate hucov-229e was demonstrated. it was found that the hucov-229e (10 3 plaque forming units) was inactivated irreversibly in less than 60 min on brasses/alloys containing at least 70-90% cu, whereas the virus was shown to persist on surfaces like glass, rubber, and stainless steel in an infectious state for at least 5 d. [159] dhanasezhian et al. fabricated biogenic gold and agnps using the seaweed sargassum wightii. their cytotoxic and antiviral activity against hsv-1 and hsv-2 strains on vero cells showed that functionalized metallic nanoparticles act as antiviral agents by hindering virion attachment and cellular entry, depending on their particle size. [160] figure 10 . schematic illustration for the selective naked-eye detection of sars-cov-2 rna facilitated by the suitably designed aso-capped au nanoparticles. reproduced with permission. [163] copyright 2020, american chemical society (acs). https://pubs.acs.org/doi/10.1021/acsnano.0c03822 technologies will accelerate patient management and identification and containment of infection hotspots. nanoparticle based disease monitoring mediated by various metallic nanoparticles carrying magnetic, fluorescence, and surface-enhanced raman scattering features would aid in tracking treatment response. [161, 162] moitra and group reported the development of gold nanoparticles (aunps) based colorimetric assay for rapid, selective, and visual diagnosis of positive covid-19 patients within 10 min after the isolation of viral rna. in the presence of targeted rna from the virus, thiol-modified antisense oligonucleotides against n protein of sars-cov-2 capped aunps exhibited selective agglomeration leading to altered surface plasmon resonance. subsequent addition of rnaseh further cleaved the rna from the rna-antisense oligonucleotides complex leading to the formation of a precipitate (figure 10) . [163] graphene sheets conjugated to antibodies can rapidly detect targeted viral proteins and viral targeting antibody functionalized graphenes can further be useful in the development of biosensors against covid-19. [164] similarly, utilizing theranostic nanoparticles, both therapy and diagnosis can be combined in order to detect and neutralize the virus in a single go, halting the virus to survive and reproduce within the host. the theranostic nanoparticles can efficiently complex with sars-cov-2, detecting and disrupting their structure. [142] potential nanoparticle based therapeutics which are in the pipeline against the current covid-19 outbreak include proteo-lipid nanovehicle based fusogenix dna vaccine, lipid nanoparticle-loaded mrna vaccine (mrna-1273), plantbased virus-like particles (vlp), recombinant protein nanoparticle vaccine (nvx-cov2373), [103, 165] and nanomicelles based antiviral. [138] analogous to cancer nanotherapeutics, covid-19 can also be managed successfully by exercising 3d nanotechnologybased approaches that include efficient detection, diagnosis, and delivery. the tissue and cell targeting principle achieved by targeting antibodies and peptides in handling other dreaded diseases like cancer and neurodegeneration can be adopted in the case of the antiviral nanoparticles for achieving targeted and effective therapy of infected patients. furthermore, theranostic nanoparticles can be developed to detect and kill the virus simultaneously. nanoparticle based kits can be developed to detect the virus at an early stage in asymptomatic patients with lesser viral loads. nanoparticle based antiviral material can be developed for fabricating superfine filters for face masks, personal protective equipment, and surface coatings. further, nanotechnology may assist in large-scale production of therapeutics and equipment addressing covid-19 and similar pandemics. thus, there are varieties of therapeutic molecules against covid-19. the list of these potential therapeutic molecules under consideration for covid-19 is summarized in table 1 . the exceptional speed, with which the sars-cov-2 pandemic is escalating across the world, is causing global anxiety to attain new heights. covid-19 has become the reason for the globally prevalent fear, stress, and concern. all these are natural and common reactions to the fast-changing and undefined situation that all of us find ourselves in. further, there is a noticeable degree of fear, stress, and apprehension in certain groups with the higher risk factor, such as children, older people, health care professionals, frontline workers, and people with pre-existing maladies (such as asthma, diabetes, heart diseases, and hypertension). the impact phase iii nct04530396 [ 118] linear dna vaccine dna based on sars-cov-2 spike (s) protein applied dna sciences/ takis biotech preclinical [ 118] zycov-d dna plasmid dna vaccine zydus cadila phase i/ii ctri/2020/07/026352 [ 103, 118] bacillus calmette-guerin (bcg) vaccine phase iii chictr2000034780 [ 118] (continued) adv. therap. 2020, 2000172 phase iii [ 103, 118] covaxin inactivated coronavirus whole-virion inactivated sars-cov-2 vaccine (bbv152) phase i/ii nct04471519 [ 118] nvx-cov2373 protein recombinant sars-cov-2 nanoparticle vaccine consisting of trimeric full-length sars-cov-2 spike glycoproteins and saponin based matrix-m1 adjuvant novavax phase i/ii nct04368988 [ 103, 165] flowvax peptide vaccine adjuvanted microsphere peptide vaccine targeting sars-cov-2 nucleocapsid adjuvanted microsphere peptide vaccine targeting sars-cov-2 nucleocapsid flow pharma preclinical ebola, marburg, hiv, zika, influenza, hpv [ 103] signal peptide vaccine (vxl-301, 302, 303) signal peptide vaccine using vaxhit bioinformatics platform vaxil bio preclinical [ 103] epitope-based peptide vaccine peptide synthetic long peptide vaccine candidate for s and m proteins (vaccinomics strategy) oncogen preclinical [ 103] ii-key peptide peptide synthetic peptides that mimic the epitopes of the sars-cov-2 using epivax's computational tools and li-key technology generex/ epivax preclinical influenza, hiv, sars-cov [ 138] monoclonal antibodies gimsilumab (kin 1901) human mab targets gm-csf roivant sciences phase ii nct04351243 [ 103] tjm2 (tj003234) neutralizing antibody neutralizing antibody against human gm-csf phase i/ii nct04341116 [ 103] lenzilumab neutralizing antibody neutralizing antibody against human gm-csf humanigen phase iii nct04351152 compassionate use [ 103] namilumab (izn-101, amg203) human mab targets gm-csf izana bioscience compassionate use ra and ankylosing spondylitis [ 103, 122] tzls-501 human monoclonal antibody mab against il-6 receptor tiziana life sciences preclinical autoimmune diseases [ 103] at-100 protein human recombinant surfactant protein d (rhsp-d) airway therapeutics preclinical bronchopulmonary dysplasia (bpd) in premature infants [ 103] tocilizumab (actemra) human mab mab against il-6 receptor roche phase iii nct04320615 chictr2000029765 ra, giant cell arthritis [ 121, 123] sarilumab (kevzara) human mab il-6 receptor blocker sanofi and regeneron phase ii/iii nct04327388 ra [ 103] convalescent sera passive neutralizing antibody passive neutralizing antibody developed in covid-19 recovered patients -p h a s e 0 chictr2000029757 phase ii nct04343755 [ 125] anti-inflammatory molecules glucocorticoid anti-inflammatory and immunosuppressant -phase ii/iii nct04381936 [ 126] (continued) adv. therap. 2020, 2000172 phase iv nct04419025 [ 127, 128] cyclosporine a calcineurin inhibitor anti-inflammatory and immunosuppressant phase iia nct04492891 phase iv nct04392531 [129] [130] [131] budesonide corticosteroid anti-inflammatory -phase iv nct04355637 vitamin d steroid anti-inflammatory, antioxidant, immunomodulatory, -phase iii nct04385940 nct04344041 [ 132] of the crisis on people's mental health is also of major concern. as the new measures to prevent the infection are being introduced, such as quarantine (social isolation), cases of depression and loneliness are expected to rise. the disruptive effects of the covid-19 outbreak are dominating our daily lives; therefore, it is of prime importance that we should manage and react properly to this challenging situation unfolding fast in our lives. the world is in total chaos, lives have frozen, and everything around us has come to a standstill situation. nature has forced us to stay indoors. let us hope, together; we will be able to find a cure and curb the grave epidemic as soon as possible. many drug candidates are under investigation for the disease. initial reports suggest some of them are promising at this juncture. however, whether these drugs will act following virus dynamics and host response is a big question. how they will behave in a clinical trial is yet another long-term debate. besides, their affordability in developing countries is a significant concern. antiviral therapy shows a wide margin of ineffectiveness as many viruses possess extraordinary genetic adaptability, which endows them with the ability to escape antiviral repression. in some instances, the viruses reclaim advantage over the host by online mutagenesis, which creates novel strains with additional acquired resistance to the existing antivirals. therefore, finding new and multifaceted antiviral agents is the unmet need of the hour. an approach analogous to cancer nanotechnology, including early diagnosis, targeted delivery, and combinatorial approaches can be followed to tackle this deadly viral nanovector. us-fda's extraordinary speed in reviewing emergency use authorization (euas) and investigational new drug (ind) applications and their prompt approval to expedite anti-covid-19 therapeutics into the clinical development is highly appreciable. who and partners had launched an international clinical trial named solidarity, for comparing potential options against standard of care to find an effective treatment for covid-19. the trial's aim is to expedite the discovery of any potential drugs that can slow down the disease progression or improve patient's survival rate. on 4th july 2020, who has withdrawn hydroxychloroquine and lopinavir/ritonavir from their solidarity trial. the solidarity trial's committee found that these drugs were ineffective in treating the infection. similar findings were also reported from the uk's recovery trial. further, us-fda's approval for the emergency use of remdesivir and other drugs, and other medical countermeasures (mcms) and facilitating their availability is another noteworthy move in combating covid-19 and is an example of using science and technology at its best in protecting and rescuing the world from emerging chemical, biological, radiological and nuclear (cbrn) threats. with the current focused, rapid, worldwide discoveries in basic science, translational research, and clinical studies, and the expedited regulatory scrutiny, it is anticipated that preventive and curative therapies for covid-19 will be a reality in the near future. coronavirus disease (covid-19) weekly epidemiological update and weekly operational update world health organization naming the coronavirus disease (covid-19) and the virus that causes it proc. natl. acad. sci proc. natl. acad. sci summary of probable sars cases with onset of illness from 1 epidemic and pandemicprone diseases (mers situation update sars-cov-2 life cycle: stages and inhibition targets who coronavirus disease (covid-19) dashboard infection control guidance for healthcare professionals about coronavirus (covid-19 coronavirus disease (covid-19) advice for the public emergency use authorization (eua) information, and list of all current euas covid-19 company directory clinical drug information drug discovery today favilavir: first approved drug to possibly treat coronavirus outline of trial designs for experimental therapeutics solidarity" clinical trial for covid-19 treatments biopharma products in development for covid-19 immunobiology: the immune system in health and disease predicted immune responses to sars-cov-2 coronavirus vaccine tracker israeli-made oral vaccine for coronavirus on track, but testing will take months proc. natl. acad. sci. usa 2020 catching up to coronavirus: top 60 treatments in development therapeutic advances in infectious disease 2017 theranostics 2020 ieee 15th int. conf. on nanotechnology antibacterial and antiviral graphene-based coating for public settings key: cord-289407-8fje16z1 authors: moore, g.; rickard, h.; stevenson, d.; aranega bou, p.; pitman, j.; crook, a.; davies, k.; spencer, a.; burton, c.; easterbrook, l.; love, h. e.; summers, s.; welch, s. r.; wand, n.; thompson, k.-a.; pottage, t.; richards, k. s.; dunning, j.; bennett, a. title: detection of sars-cov-2 within the healthcare environment: a multicentre study conducted during the first wave of the covid-19 outbreak in england date: 2020-09-25 journal: nan doi: 10.1101/2020.09.24.20191411 sha: doc_id: 289407 cord_uid: 8fje16z1 understanding how severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is spread within the hospital setting is essential if staff are to be adequately protected, effective infection control measures are to be implemented and nosocomial transmission is to be prevented. the presence of sars-cov-2 in the air and on environmental surfaces around hospitalised patients, with and without respiratory symptoms, was investigated. environmental sampling was carried out within eight hospitals in england during the first wave of the covid-19 outbreak. samples were analysed using reverse transcription polymerase chain reaction (rt-pcr) and virus isolation assays. sars-cov-2 rna was detected on 30 (8.9%) of 336 environmental surfaces. ct values ranged from 28.8 to 39.1 equating to 2.2 x 105 to 59 genomic copies/swab. concomitant bacterial counts were low, suggesting the cleaning performed by nursing and domestic staff across all eight hospitals was effective. sars-cov-2 rna was detected in four of 55 air samples taken < 1 m from four different patients. in all cases, the concentration of viral rna was low and ranged from < 10 to 460 genomic copies per m3 of air. infectious virus was not recovered from any of the pcr positive samples analysed. effective cleaning can reduce the risk of fomite (contact) transmission but some surface types may facilitate the survival, persistence and/or dispersal of sars-cov-2. the presence of low or undetectable concentrations of viral rna in the air supports current guidance on the use of specific ppe ensembles for aerosol and non-aerosol generating procedures. over the course of 2020, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of coronavirus disease-19 has spread rapidly across the globe and, as of 15 august 2020, had infected 21 million people and caused over 750,000 deaths. 1 transmission of respiratory viruses can occur through inhalation of respiratory droplets (particles >5µm in diameter) and infectious aerosols (< 5µm in diameter) and/or contact with respiratory droplets either directly or indirectly via contaminated surfaces. the rapid spread of covid-19 has led many to conclude that airborne transmission must be involved. 2 this, though, is widely debated and, according to current evidence, sars-cov-2 is primarily transmitted via droplet and contact routes, although it is acknowledged, that airborne transmission could occur in specific circumstances and settings. 3 healthcare workers (hcws) and others on the front line are at increased risk of acquiring infection. 4 medical aerosol generating procedures (agps), such as intubation, non-invasive ventilation and airway suctioning can produce droplets < 5µm in diameter and have been associated with increased transmission of sars-cov-1 from patients to hcws. 5 it is argued, however, that there is limited evidence to link agps with transmission of respiratory infections, including covid-19. 6 air samples taken during tracheostomy procedures, high flow nasal oxygen treatment, non-invasive ventilation and nebulisation have not contained sars-cov-2 rna 7 and hcws exposed to unrecognised covid-19 patients undergoing similar high-risk agps have not become infected. 8 nonetheless, occupational exposure has resulted in infection 9 and it has been estimated that in england, patient to hcw transmissions could be responsible for 57% of hcw infections. 10 nosocomial transmission may also account for 20% of infections in inpatients 10 and so understanding how sars-cov-2 is spread within the hospital setting is essential to ensure staff are adequately protected and effective infection control measures are implemented. . cc-by-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint several studies, utilising a range of air and surface sampling methods, have been carried out to determine the presence and prevalence of sars-cov-2 in the healthcare environment. [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] the detection of viral rna in air samples differs with study with some reporting widespread airborne contamination 14, 18, 21 but many reporting low or non-detectable concentrations 13, 15, 16, 19 even in samples collected 10 cm from the face of positive patients. 12 surfaces frequently touched by staff and/or patients are often contaminated with bacterial pathogens. likewise, sars-cov-2 rna has been detected on high-contact surfaces such as computers, bed rails and door handles. again, the extent of this surface contamination differs with study. reported positivity rates range from 0·8% to over 70% with those studies reporting a comparatively higher level of airborne contamination also detecting widespread surface contamination. 18, 21 in many cases, sampling was performed before routine cleaning but the efficacy of cleaning was not assessed. 11, 13, 15 when comparative samples were taken, sars-cov-2 rna was detected on 61% of surfaces sampled prior to cleaning but was not detected on any surface after it had been cleaned. 17 the proportion of surfaces contaminated with viral rna can also differ with ward type. some studies have detected little to no surface contamination in intensive care units (icus) but have detected widespread contamination within general wards. 11, 21 by contrast, others report comparatively higher positivity rates within the icu setting. 14, 20 environmental sampling can provide important information about the spread of healthcareassociated infections. it is though, resource-intensive and time-consuming and, thus, many studies investigating sars-cov-2 and its contamination of the healthcare environment have focused on a single hospital and, in the context of the covid-19 pandemic, a single point in time. sampling frequency is also, in general, low, meaning that results often represent a snapshot in time and place. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint in a rapid evolving outbreak, there is a need to gain quick understanding of certain trends and whilst snapshot samples by themselves cannot be considered representative, they can, when taken together, provide useful data relating to type, level and location of environmental contamination. to date, however, differences in study setting, protocol and methodology have led to inconsistency in the results obtained making it difficult to draw any firm conclusions relating to sars-cov-2 and its presence within the healthcare environment. as part of the public health england (phe) national incident response, the presence of sars-cov-2 in the air and on environmental surfaces around hospitalised patients, with and without respiratory symptoms, was investigated. environmental sampling, utilising standard methods, was carried out within 8 acute hospital trusts in england. trends, in terms of type and level of surface contamination and the potential for agps to disperse sars-cov-2 have been identified and these provide evidence to support current infection prevention and control guidance including the use of personal protective equipment. between 03/03/20 and 12/05/20, the study team visited eight hospitals (three on more than one occasion; figure 1 ) and carried out environmental sampling in areas where patients infected with sars-cov-2 were receiving care. these included 11 negative pressure isolation rooms, 11 neutral pressure side rooms, six icu/hdu open cohorts and 12 non-icu cohort bays. whilst sampling primarily focused on 44 individual bed spaces (table 1) , samples were also taken from the wider ward environment (e.g. nursing stations; patient toilet areas) and from non-covid wards. medical procedures being performed and obvious symptoms such as coughing were observed and recorded. patient details (hospital number; date of admission; date of diagnosis) were collected for future correlation with clinical virology results. details regarding routine and terminal (discharge) cleaning were also collected. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint surfaces deemed to be high-contact sites were sampled using nylon flocked swabs (copan, brescia, italy) wetted with universal transport medium and from 27/03/20, to provide an indication of general surface cleanliness, tryptone soya agar contact plates (oxoid ltd, basingstoke, uk). air samples were taken using two types of active air sampler: a coriolis µ air sampler (bertin instruments, montigny-le-bretonneux, france), operating at 300 l/min and collecting into 15 ml rnase-free phosphate buffered saline (pbs) and an md8 air sampler (sartorius, göttingen, germany), operating at 50 l/min and collecting onto a gelatine membrane filter. both samplers were positioned close to patients (< 1 m) with and without respiratory symptoms and operated for 10 minutes. the type and duration of agp, if any, was noted. ambient temperature and relative humidity were monitored. all samples were returned to phe porton down. agar contact plates were incubated at 37 o c for 48h whilst the air and swab samples (for virus detection) were frozen at -80 o c prior to processing. laboratory-based validation experiments confirmed that neither the transport or storage conditions adversely affected subsequent rt-pcr analysis. rna was extracted from aliquots (140 μl) of each swab and coriolis air sample using the qiaamp viral rna mini kit (qiagen ltd, manchester, uk). the remaining coriolis sample was concentrated to < 1 ml using a vivaspin® 20 centrifugal concentrator. each gelatine membrane was dissolved in 10 ml minimum essential medium (mem). aliquots (140 μl) of both were extracted. in total 425 samples (surface swabs (n=336) and air (n=89)) were analysed for sars-cov-2 using rt-pcr. all samples were screened in duplicate using one of the following targets: rna dependent rna polymerase (rdrp) with probe 2, envelope (e) or nucleocapsid (n) and orf1ab (viasure, certest biotec, zaragoza). a sample was considered positive when amplification was detected in both replicates or 'suspect' when it was detected in only one. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint both replicates. all positive samples were quantified using the n target on the viasure platform. amplification in one replicate was considered sufficient for quantification. samples that could not be quantified were re-extracted and quantification reattempted. virus isolation was carried out on all positive samples with a ct value < 34. vero e6 cells (vero c1008; atcc crl-1586) in culture medium (mem supplemented with glutamax™cell monolayers that did not display cpe were sub-cultured a maximum of three times, providing continuous cultures of ~30 days. environmental sampling was carried out in and around the bed space of 44 different patients, 35 (80%) of whom were male (table 1) . twenty-three patients had been admitted to intensive care (n=15) or a respiratory high dependency unit (n=8) whilst 21 patients occupied beds in a non-icu setting. these included 10 patients who, after being diagnosed early in the outbreak, were admitted to infectious diseases units. at the time of sampling, 21 patients were receiving mechanical ventilation either invasively (n=8) or non-invasively (n=13), six patients were receiving oxygen via a venturi mask and three patients required drugs or saline to be administered by nebulisation. all patients had tested positive for sars-cov-2 and the median time since diagnosis was 5 days (range 1 -44 days). time since symptom onset ranged from 3 to 45 days. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint in total, 336 surfaces were sampled for bacteria and/or sars-cov-2. the mean aerobic colony count was 1 cfu/cm 2 . of those surfaces with more extensive bacterial contamination (>2·5 cfu/cm 2 ), 18 (70%) were associated with a patient's bed (bed rail, bed control and/or nurse call button) or mobile phone. sars-cov-2 rna was detected on 30 (8·9%) of the 336 surfaces sampled (table 2) . of the 44 individual bed spaces, 10 were contaminated with viral rna and accounted for 19 (63%) of all positive sites. in addition to nurse call buttons (n = 4), bed control panels (n = 3) and mobile phones (n = 3), viral rna was also detected on bedside equipment (e.g. monitor screens, syringe drivers and computer keyboards) particularly in the icu/hdu setting. however, in the non-icu setting, 27% of surfaces contaminated with sars-cov-2 rna were located outside the patient bed area. these included toilet door handles and portable vital signs monitors which together accounted for 26% of all positive sites. rt-pcr ct (cycle threshold) values ranged from 28·8 to 39·1 which when quantified equated to 2·2 x 10 5 to 59 genomic copies per swab. samples with a ct value < 34 were incubated on vero e6 cells. no cpe or a decrease in ct values across the course of three serial passages were observed suggesting the samples did not contain infectious virus. ambient temperature and relative humidity differed with ward and ranged from 21 o c to 25 o c and from 21% to 41% respectively. air samples were collected using two types of highvolume air sampler but sars-cov-2 rna was only detected in four (7·3%) of the 55 samples taken using the coriolis μ sampler. two of these samples were taken in two different single rooms (neutral pressure). in both cases, the sampler was positioned close (< 1 m) to a patient being treated with continuous positive airway pressure (cpap) via a mask that covered the nose and mouth. time since diagnosis was 4 and 7 days with both patients reporting symptoms at least 8 days prior to sampling. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint viral rna was also detected in two air samples taken in two 4-bed cohort bays. on one of these occasions, the air sampler was positioned close to a patient who was receiving oxygen via a venturi mask. this patient had tested positive for sars-cov-2 the previous day with a ct value of 21·35. the second patient, diagnosed 6 days earlier (ct value of 17·68), was not receiving any notable treatment. however, approximately 30-40 minutes before sampling was carried out, there was a 'crash call' elsewhere within the bay. there was no intubation or cpr but a significant increase in staff activity was observed and may have facilitated dispersal of airborne particles. the total volume of each air sample was 3 m 3 and the associated ct values ranged from 37 to 39 which, when quantified, equated to 460 to < 10 genomic copies per m 3 of air. when sampling the healthcare environment, many variables can impact the results obtained. this can make interpretation of the data difficult, particularly if a frame of reference is lacking. in this study and to provide context, agar contact plates were used to provide an aerobic bacterial colony count and an indication of surface cleanliness. whilst no microbiological standards exist for healthcare surfaces, a benchmark of < 2·5 cfu/cm 2 has been suggested. 22 sars-cov-2 was detected on 30 (8·9%) of the 336 surfaces sampled ( table 2 ). the proportion of surfaces positive for viral rna differed with hospital and ranged from 0-27%. this likely reflects the fact that sampling was carried out on different types of ward occupied by different types of patient requiring different types of care and/or treatment (table 1) . overall, however, the results are similar to those of other studies 13, 15, 20 and suggest that, whilst sars-cov-2 can contaminate healthcare surfaces, widespread contamination is unlikely. 17 the bacterial load on the majority (89%) of surfaces sampled was < 2·5 cfu/cm 2 suggesting that in general is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint and despite increased pressure on beds and workload, the routine cleaning performed by the nursing and domestic staff across all eight hospitals was effective. nonetheless, contamination of the healthcare environment can occur and sars-cov-2 rna was detected on the same type of surface across multiple hospitals (table 2) implying that, despite the effectiveness of the cleaning protocols employed, some types of surface could facilitate the survival, persistence and/or dispersal of sars-cov-2. patients consider the nurse call button a direct conduit to care and many patients were observed to hold the button close even whilst dozing. intensity and frequency of contact can increase microbial transfer from hands to surface 23 and sars-cov-2 rna was detected on 4 (17%) of the nurse call buttons sampled. ct values ranged from 30·8 to 36·2 equating to 2·9 x 10 4 to 1·2 x 10 3 genomic copies/swab (table 2) . to reduce the risk of transmission of sars-cov-2 in the hospital setting, it is recommended that surfaces such as over-bed tables, bed rails and nurse call buttons are cleaned at least twice daily. 24 the median number of bacteria recovered from nurse call buttons was 50 cfu/25cm 2 . comparatively fewer bacteria (< 1 cfu/cm 2 ) were recovered from tables and bed rails suggesting these surfaces are (and can be) effectively cleaned. heavy contamination of the nurse call button has been described previously 25 and staff should be reminded that routine cleaning should include all aspects of the patient bed. future consideration should be given to design modification and/or improving the ability to clean nurse call buttons. patient mobility can contribute greatly to the spread of bacteria within a ward. 25 similarly, sars-cov-2 rna has been detected on patient-contact sites outside the immediate bed space 13, 17 and, in the current study, outside of cohort bays; specifically, toilet door handles. the presence of sars-cov-2 rna on door handles has been reported previously 13, 14, 20 and the contact area between the hand and handle and the pressure of grip likely facilitates transfer to and from the hands. in this study, the amount of sars-cov-2 rna detected on is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint one door handle was 2·2 x 10 5 genomic copies/swab implying significant transfer from a contaminated hand. despite this, we were unable to culture viable virus. the lowest genomic copy number (n gene) required to isolate virus from clinical samples is reportedly 5 x 10 5 genomic copies/ml 26 ; higher than the copy number in any of the environmental samples collected during this study. subjecting the samples to multiple freeze-thaw cycles may also have impacted infectivity by disrupting virion and genome integrity 26 . regardless, there is potential for viable virus to contaminate a single door handle and to be transferred to the hands of numerous successive contacts and as a consequence, to other inanimate surfaces. 27 sars-cov-2 rna was detected on 4·9% (7/143) and 13·8% (22/159) of the surfaces sampled in the icu/hdu and non-icu wards respectively. in contrast to patients admitted to cohort wards, patients in icus/hdus are more likely to be bed bound and be receiving mechanical ventilation. reduced patient mobility likely contributed to the less frequent detection of sars-cov-2. however, viral rna was still detected on staff contact sites (e.g. monitor screens, syringe drivers; table 2 ). disposable gloves are an important element of ppe and can prevent the hands of hcws from acquiring pathogens. however, during routine patient care, the glove surface itself can become contaminated. if gloves are not regularly and appropriately changed then contamination of surfaces via gloved hands can occur. 19 when caring for covid-19 patients, particularly in the icu/hdu setting, the requirement to don full ppe presents additional challenges in terms of preserving ppe and ensuring staff know how to implement appropriate hand hygiene within an outbreak setting. 14, 19 non-critical medical devices (e.g. blood pressure cuffs and temperature probes) have been implicated in nosocomial infection. 28 sars-cov-2 was detected on four (31%) of 13 portable vital signs monitors ( table 2 ). the highest level of viral rna (1·6 x 10 3 genomic copies/swab) was detected on a fingertip pulse-oximeter associated with a machine that had been removed is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint from a single room occupied by a covid-positive patient. the other three machines were located on cohort bays. when (or on whom) these machines were last used or when they were last cleaned is not known and results demonstrate the presence and/or persistence of viral rna and not infectious virus. nonetheless, contact pressure has been shown to significantly affect viral transfer to and from fingerpads 29 and in the absence of cleaning, fingertip pulse-oximeters could facilitate transmission of sars-cov-2, particularly between asymptomatic and noninfected patients. sars-cov-2 rna was detected in four (7·3%) of the high volume (3 m 3 ) air samples taken using the coriolis sampler. it is not known what may have contributed to this airborne contamination but two of these samples were taken < 1 m from two patients receiving cpap therapy (table 1) . cpap is considered an agp. however, air samples were taken close to 11 other patients receiving non-invasive ventilation (niv), 7 of whom had also tested positive < 7 days earlier. no viral rna was detected. the make/model of cpap machine used to treat these two patients was not used elsewhere and it is possible that the equipment used to deliver niv to patients may promote the generation and/or release of aerosols. 30 how the apparatus is used or tolerated may also have an effect. during sampling, one of the two patients was observed to turn in bed -multiple times and on one occasion disconnect the cpap machine to aid movement. the dispersal distance of exhaled air from a jet nebulizer and venturi-type oxygen mask is estimated to be 0.8 m and 0.4 m respectively. 30 in this study, sars-cov-2 was not detected in any air sample collected during drug nebulisation. viral rna was detected < 1 m from one (of six) patients receiving oxygen. time since diagnosis and symptom onset was 1 and 6 days respectively; comparatively earlier than many of the other patients (table 1) . others hypothesise that the concentration of sars-cov-2 in the air and/or on high-touch surfaces is highest during the first week of illness, 11 suggesting that new admissions to hospital may have is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint greater potential to transmit the virus to others. it has been suggested that placing suspected covid-19 patients in single rooms or bays that are fully disinfected between admissions could reduce nosocomial infection rates by 80%. 10 in all four cases where sars-cov-2 was detected in air samples, the concentration of viral rna was low and ranged from 460 to < 10 genomic copies per m 3 of air. as discussed, samples containing this level of viral nucleic acid are unlikely to contain viable (infectious) virus 27 and this finding, together with the inability to detect sars-cov-2 rna in all other air samples, supports current guidance on the use of specific ppe ensembles for aerosol-and non-aerosol generating procedures. it is acknowledged, however, that many of the procedures believed to generate aerosols and droplets were not captured during this study and that samples were only collected over a 10-minute period. unprotected, prolonged exposure to an infected patient has been linked to transmission. 9 in a rapidly evolving outbreak situation, there is a need to gain quick understanding of certain trends; in this case, the contamination of the healthcare environment. despite its limitations, this multi-centre study supports the findings of others 13, 15, [19] [20] and should provide assurance to hcws. sars-cov-2 may be present on frequently touched surfaces but effective cleaning should reduce the risk of fomite-transmission 21 is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 25, 2020. . https://doi.org/10.1101/2020.09.24.20191411 doi: medrxiv preprint it is time to address airborne transmission of covid-19. clin room/commentaries/detail/transmission-of-sars-cov-2-implications-for-infection-preventionprecautions estimating the burden of united states workers 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disease 2019 (covid-19) due to sars-cov-2 in hong kong toilets dominate environmental detection of sars-cov-15 aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards clinical data on hospital environmental hygiene monitoring and medical staff protection during the coronavirus disease 2019 outbreak aerodynamic analysis of sars-cov-2 in two wuhan hospitals air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient environmental contamination by sars cov-2 in a designated hospital for coronavirus disease 2019 environmental contamination of sars-cov-2 in healthcare premises investigating sars-cov-2 surface and air contamination in an acute healthcare setting during the peak of the covid-19 pandemic in london are hygiene standards useful in assessing infection risk? transfer of pathogens to and from patients, healthcare providers and medical devices during care activity -a systematic review and metaanalysis we thank the staff and patients at the hospitals that participated in this study. the views expressed in this article are those of the author(s) and are not necessarily those of public health england or the department of health and social care. all authors declare no competing interests key: cord-284068-sbon3aes authors: mok, chee keng; ng, yan ling; ahidjo, bintou ahmadou; hua lee, regina ching; choy loe, marcus wing; liu, jing; tan, kai sen; kaur, parveen; chng, wee joo; wong, john eu-li; wang, de yun; hao, erwei; hou, xiaotao; tan, yong wah; mak, tze minn; lin, cui; lin, raymond; tambyah, paul; deng, jiagang; hann chu, justin jang title: calcitriol, the active form of vitamin d, is a promising candidate for covid-19 prophylaxis date: 2020-06-22 journal: biorxiv doi: 10.1101/2020.06.21.162396 sha: doc_id: 284068 cord_uid: sbon3aes covid-19, the disease caused by sars-cov-2 (1), was declared a pandemic by the world health organization (who) in march 2020 (2). while awaiting a vaccine, several antivirals are being used to manage the disease with limited success (3, 4). to expand this arsenal, we screened 4 compound libraries: a united states food and drug administration (fda) approved drug library, an angiotensin converting enzyme-2 (ace2) targeted compound library, a flavonoid compound library as well as a natural product library. of the 121 compounds identified with activity against sars-cov-2, 7 were shortlisted for validation. we show for the first time that the active form of vitamin d, calcitriol, exhibits significant potent activity against sars-cov-2. this finding paves the way for consideration of host-directed therapies for ring prophylaxis of contacts of sars-cov-2 patients. abstract: covid-19, the disease caused by sars-cov-2 (1), was declared a pandemic by the world health organization (who) in march 2020 (2) . while awaiting a vaccine, several antivirals are being used to manage the disease with limited success (3, 4) . to expand this arsenal, we screened 4 compound libraries: a united states food and drug administration (fda) approved drug library, an angiotensin converting enzyme-2 (ace2) targeted compound library, a flavonoid compound library as well as a natural product library. of the 121 compounds identified with activity against sars-cov-2, 7 were shortlisted for validation. we show for the first time that the active form of vitamin d, calcitriol, exhibits significant potent activity against sars-cov-2. this finding paves the way for consideration of host-directed therapies for ring prophylaxis of contacts of sars-cov-2 patients. despite implementation of physical distancing, mask wearing, quarantine and the tireless efforts expended for contact tracing, the rapid transmissibility of sars-cov-2 even during the asymptomatic phase has made containment of this virus extremely difficult. the main proposed strategy to curb this pandemic is the implementation of mass vaccination programs. once a suitable vaccine is discovered, the significant challenges associated with vaccination programs e.g. limitations in manufacturing capabilities and associated costs, are anticipated to significantly affect uptake of vaccinations globally. we therefore propose that ring prophylaxis, which had been previously proposed for influenza pandemics and involves treating close contacts of a confirmed case with an antiviral prophylaxis to further curb community spread (5) , be considered as a viable strategy to reduce transmission of sars-cov-2. in an effort to identify potential candidates for sars-cov-2 chemoprophylaxis, we performed a virusinduced cytopathic effect (cpe) based screen of several small molecule libraries in sars-cov-2infected vero e6 cells (fig. 1a) . the african green monkey kidney epithelial vero e6 cells were used for the screen as these cells are highly susceptible to coronaviruses and exhibit obvious cpe upon infection. a 57-compound natural product library and a library of 462 ace2 targeted inhibitors (the ace2 receptor was identified to be necessary for sars-cov-2 infection (6)) were used in a preinfection treatment screen to identify potential viral entry inhibitors, while a post-infection treatment screen was performed using both a 500 compound flavonoid library and a 1172 fda-approved compound libraries in order to identify potential inhibitors targeting post-entry steps of the sars-cov-2 replication cycle. for the pre-infection treatment screen, vero e6 cells were treated with compounds for two hours prior to infection with sars-cov-2. the post-infection treatment screen on the other hand was performed by adding compounds to the vero e6 cells 1 hour post-infection with sars-cov-2. compounds which showed less than 50% cpe compared to the 0.1%dmso vehicle control with sars-cov-2 infection were identified as hits (tables s1-s3) . using this method, we identified 31 compounds from the pre-infection treatment screen and 90 compounds from the post-infection treatment screen with activity against sars-cov-2 (table s4) . as expected, our hit list included the tyrosine kinase inhibitors masitinib and imatinib mesylate (7), the antiretroviral drug lopinavir(8) and the calpain inhibitor calpeptin (9) -all compounds reported to inhibit sars-cov, sars-cov-2 or mers-cov. this provides the robustness and confidence of our primary screen for potential antivirals against sars-cov-2. of these primary hits, 7 compounds were selected for downstream validation (table 1 ). these included 3 compounds from the pre-infection treatment screen (citicoline, pravastatin sodium and tenofovir alafenamide) and four compounds from the post-infection treatment screen (imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate). these compounds were selected based on level of cpe inhibition in the primary screens (fig. 1b) , known mechanism of action and existing fda approval or generally recognized as safe (gras) status. fda approval was considered an important factor as pre-existing data on safety and dosage would allow expedited decisions to be made regarding the potential use of these compounds in vulnerable populations to stymie the current pandemic. validation assays to determine changes in infectious virus titres upon treatment was carried out by testing selected hit compounds in dose-dependent assays in vero e6 to confirm the primary screen observation and also in the human hepatocarcinoma huh7 cell line as the latter cell line expresses high levels of the ace2 receptor (10) and supports replication of coronaviruses (11) . cell viability assays were also carried out to ensure that reduction of sars-cov-2 titres was not due to cytotoxic effects of the compounds on host cells. cc50, ic50 were obtained for each of the 7 compounds in vero e6 cells (table s5 ) and huh7 cells (table s6) (table s6 ). given that the huh7 cell line is a hepatocarcinoma cell line and therefore not the first point of entry for sars-cov-2 in humans, we decided to test the three most promising compounds (imatinib mesylate, citicoline and calcitriol) against sars-cov-2 in the primary human nasal epithelial cell line (hnec) that is a known in vivo target of sars-cov-2 (12) (fig. 1a) . despite its significant activity in the continuous cell lines (vero e6 and huh7), in hnecs, imatinib mesylate only displayed a 0.2 log 10 reduction in viral titre (fig. 3) . interestingly, out of the three compounds only calcitriol proved effective against sars-cov-2 with a reduction of 0.69 log 10 in viral titre (fig. 3 ). while recent data has shown that vitamin d levels are negatively associated with morbidity and mortality of covid-19 cases (13, 14) , this is the first report of a direct inhibitory effect of calcitriol on sars-cov-2. vitamin d is well known to modulate host immune responses through the production of the antimicrobial peptides such as cathelicidin to promote autophagy (15) . it has proven essential for host defenses against many intracellular pathogens including respiratory pathogens such as mycobacterium tuberculosis, and has been shown to also possess anti-inflammatory properties (15) . a recent study by smith and colleagues (16) showed an association between vitamin d deficiency and sars-cov-2 infection and covid-19 associated mortality. the authors speculated that vitamin d supplementation could protect against sars-cov-2 infection and improve patient disease outcomes (16) , and our finding certainly provides credence to this hypothesis. given that calcitriolmediated inhibition occurred upon post-treatment of vero e6 cells and hnecs, it is likely that its mechanism of antiviral action targets the post-entry phase of viral replication. use of host-directed therapies (hdts) for prevention of infections is certainly not a new idea. most of these therapies however rely mainly on the use of vaccines, convalescent plasma and monoclonal antibodies (17, 18) . small molecule hdts have been used adjunctively for diseases such as tuberculosis (19) and have been proposed for viral pandemics (5) . this strategy would overcome some of the costs and challenges associated with antiviral production, including the emergence of drug resistance (20) . vitamin d is a dietary supplement that is cheap and widely available even in low and middle income countries and is converted by the liver and kidneys into the active compound calcitriol (21, 22) . it is however important that our findings be confirmed in vivo as well as in clinical trials in order to assess efficacy, optimal dosage, treatment duration, toxicity and safety of calcitriol. given the high transmissibility of sars-cov-2 globally (23), if these findings can be replicated in clinical trials, calcitriol may certainly prove to be an effective tool in the effort to control the pandemic while waiting for an effective vaccine to be rolled out globally. at the very least, these findings certainly pave the way for consideration of host-directed therapies for ring prophylaxis of contacts of sars-cov-2 patients. coronaviridae study group of the international committee on taxonomy of v. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 who. rolling updates on coronavirus disease accessed 8 treatments administered to the first 9152 reported cases of covid-19: a systematic review current perspective of antiviral strategies against covid-19 tackling the next influenza pandemic a pneumonia outbreak associated with a new coronavirus of probable bat origin repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection factors associated with prolonged viral shedding and impact of lopinavir/ritonavir treatment in hospitalised noncritically ill patients with sars-cov-2 infection inhibition of severe acute respiratory syndrome-associated coronavirus (sarscov) by calpain inhibitors and beta-d-n4-hydroxycytidine highly infectious sars-cov pseudotyped virus reveals the cell tropism and its correlation with receptor expression replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in huh7 hepatocarcinoma cell line sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes the role of vitamin d in the prevention of coronavirus disease 2019 infection and mortality evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths the vitamin d-antimicrobial peptide pathway and its role in protection against infection the role of vitamin d in the prevention of coronavirus disease 2019 infection and mortality accessed 21 a review of sars-cov-2 and the ongoing clinical trials pandemic influenza and healthcare demand in the netherlands: scenario analysis vitamin d deficiency and liver disease therapeutic use of calcitriol. current vascular pharmacology presymptomatic transmission of sars-cov-2 -singapore human nasal epithelial cells derived from multiple subjects exhibit differential responses to h3n2 influenza virus infection in vitro the use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro key: cord-307489-2liu4anc authors: elavia, nasha; sharma, nishant; li, si; wang, yichen; milekic, bojana title: an atypical presentation of acute pulmonary embolism with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pneumonia date: 2020-05-23 journal: cureus doi: 10.7759/cureus.8249 sha: doc_id: 307489 cord_uid: 2liu4anc clinical presentation and severity of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) varies greatly amongst patients, as supported by recent literature. this poses an ongoing challenge in the diagnostic and therapeutic approach for managing these patients. here, we would like to describe a case of acute bilateral pulmonary embolism (pe) presenting with atypical gastrointestinal symptoms in a patient with sars-cov-2 infection. this atypical presentation of pe is unique to our case and highlights the significance of a high index of clinical suspicion for sars-cov-2 and its associated thrombogenic effect, even in patients with atypical symptoms. although knowledge about the effects of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been emerging, so far, the most commonly documented reason for hospitalization was respiratory distress [1] . proinflammatory states, such as acute infections, were known to be associated with an increased risk of thromboembolic events. venous thromboembolism is an uncharacteristic complication of the sars-cov-2, and only few descriptions of potential correlation exist in the literature [2] . newly discovered atypical features of the sars-cov-2 infection continue to be frequently reported, while increasing clinician's awareness and mindfulness for uncommon presentations. here, we would like to describe a case of acute bilateral pulmonary embolism (pe) in a patient with sars-cov-2 pneumonia who mainly presented with gastrointestinal symptoms. a 64-year-old male with a past medical history notable for diabetes mellitus type 2, obstructive sleep apnea treated with nocturnal continuous positive airway pressure ventilation (cpap) and glaucoma presented to the emergency department with diarrhea and loss of appetite. the patient reported having four to five episodes of watery bowel movements per day, which started about seven days prior to admission. he denied any nausea, vomiting, abdominal pain, blood in stool, fever or any respiratory complaints of chest pain, shortness of breath or cough. social history was negative for smoking, recent travel, sick contact exposure, immobilization, hospitalization or recent antibiotic use. on presentation, he was tachypneic at 20 breaths per 1, 2 1 1 1 1 minute and hypoxic with an oxygen saturation of 81% while breathing ambient air. his physical exam revealed bilateral basilar rhonchi, but otherwise he did not appear in any acute respiratory distress. laboratory studies were notable for lymphopenia (absolute lymphocyte count 0.85 k/ul) along with significantly elevated lactate dehydrogenase (624 u/l), ferritin (2,237 ng/ml) and c-reactive protein (66 mg/l) levels. chest x-ray revealed bilateral airspace opacification as seen with sars-cov-2. a nasopharyngeal swab for sars-cov-2 was positive. the pretest probability of pe calculated using the wells score was less than 4, which was unlikely for pe. bilateral lower extremity duplex was negative for deep venous thrombosis (dvt). d-dimer level was 1.52 ng/ml feu. in the light of significant discrepancy between severe hypoxia and the absence of respiratory symptoms or a respiratory viral syndrome, a pulmonary ct angiogram (cta) was performed, which confirmed acute bilateral pe extending from the distal right main pulmonary artery into all right lobes along with patchy ground-glass opacities consistent with sars-cov-2 pneumonia (figure 1 ). axial image (a) and coronal image (b) demonstrating bilateral filling defects of the pulmonary artery (red arrows). the patient was admitted to a negative pressure room and started on anticoagulation with heparin. he was eventually discharged home on an oral novel anticoagulant and continuous oxygen at 4 liters/minute via nasal cannula. the sars-cov-2 pandemic is challenging and, in many cases, an overwhelming situation for the medical community. research on the characteristics of this novel coronavirus is evolving every day as cases of sars-cov-2 continue to emerge with a multitude of clinical presentations. so far, fever is the most commonly reported symptom (approximately 88% of cases), followed by cough (68%), vomiting (5%) and diarrhea (3.8%) [3] . up to 15% of patients develop complications of severe interstitial pneumonia leading to acute respiratory distress syndrome (ards), multiorgan failure, disseminated intravascular coagulation and death [3] . [4, 5] . in contrast to our case, patients with suspected pe have been reported to have overlapping symptoms of sars-cov-2 [5] . our patient however presented mainly with gastrointestinal symptoms, which have been reported with sars-cov-2; however, with significant hypoxia in the absence of a respiratory viral syndrome although with a low pretest probability for pe, we decided to further evaluate the patient for hypoxia. the presence of gastrointestinal symptoms and cardiopulmonary complications are reported in patients with sars-cov-2; however, the presence of gastrointestinal symptoms in sars-cov-2 complicated with pe in the absence of an overlapping respiratory viral syndrome makes our case unique. risk assessment scores and laboratory data are widely used to assist in clinical decision making; nevertheless, the importance of clinicians' degree of suspicion and mindfulness for atypical presentations and complications cannot be overstated, especially in a currently evolving disease like sars-cov-2 infection. at many centers across the united states, inflammatory marker levels are now routinely checked in sars-cov-2 patients. studies have shown that elevated d-dimer levels correlate with the severity of sars-cov-2 illness, poor prognosis, higher likelihood of admission to the intensive care unit (icu) and even death [3, 6] . although our patient did not have symptoms of a viral respiratory syndrome, an elevated d-dimer level prompted us to do a chest cta. the decision was supported by the literature (algorithm by zuckier et al. published in april 2020 suggests obtaining a chest cta in sars-cov-2 patients with elevated d-dimer levels if there are no contraindications) [7] . additionally, patients with severe sars-cov-2 disease, including those requiring invasive mechanical ventilation and intensive care, are known to have an increased risk of venous thrombosis [8] . whether this thrombogenic phenomena is due to the virus itself or the acute inflammation in critically ill patients is not clear. as such, algorithms have been developed but the decision for therapeutic anticoagulation in sars-cov-2 patients at this time remains largely individualized, accounting for patient and physician factors. further studies are required to aid in developing evidence-based guidelines for therapeutic anticoagulation in patients with sars-cov-2 depending on patient characteristics and severity of the infection. cases of sars-cov-2 continue to emerge with atypical presentations such as the case highlighted here. therefore, it is important for all physicians (and particularly, the emergency department) to have a high clinical suspicion for both sars-cov-2 and venous thrombosis and include these in the differential diagnoses and early identification of the disease. human subjects: consent was obtained by all participants in this study. in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. acute pulmonary embolism and covid-19 pneumonia: a random association covid-19 complicated by acute pulmonary embolism clinical characteristics of coronavirus disease 2019 in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy diagnostic evaluation of pulmonary embolism during the covid-19 pandemic covid-19 complicated by acute pulmonary embolism and right-sided heart failure key: cord-305931-0pgu2gvh authors: janus, scott e; hajjari, jamal; cunningham, michael j; hoit, brian d title: covid19: a case report of thrombus in transit date: 2020-06-17 journal: eur heart j case rep doi: 10.1093/ehjcr/ytaa189 sha: doc_id: 305931 cord_uid: 0pgu2gvh background: the global pandemic of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused significant morbidity and mortality, not only through devastating lung injury, but also due to multiple malfunctions in the cardiovascular system. the primary aetiology is believed to be mediated through lung alveolar injury; however, a few published reports have linked sars-cov-2 to significant organ dysfunction, venous thrombo-embolism, and coagulopathy. in view of the fact that the utility of tissue plasminogen activator in this population is not well studied, we present this case of rapid improvement in oxygenation after successful lytic therapy for thrombus in transit in this patient with sars-cov-2. case summary: we discuss a patient admitted with sars-cov-2 pneumonia. due to the development of dramatic hypoxia, he underwent echocardiography which demonstrated extensive thrombus in transit. he received successful thrombolytic therapy with tissue plasminogen activator, with subsequent improvement in oxygenation. the patient was successfully discharged home on 2 l of oxygen via nasal cannula, and continues to improve at follow-up with his cardiologist and primary care physician. conclusion: this case not only highlights embolic causes of hypoxia in sars-cov-2, but demonstrates the important utility of an echocardiogram and tissue plasminogen activator in this population. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was declared a pandemic by the world health organization on 11 march 2020. 1,2 while the majority of infected patients do not require hospitalization, those admitted to the hospital typically present with shortness of breath. 3 a significant proportion of hospitalized patients develop hypoxia (27.4%), with 14.2 % requiring intensive care, and a substantial number progress to acute respiratory distress syndrome (ards). 3 preliminary postmortem autopsies have demonstrated deposition of fibrin in the airspaces of lung parenchyma with fibrin-platelet microthrombi in the pulmonary vasculature which may be contributing to the hypoxia. 4 although the utility of tissue plasminogen activator (tpa) for thrombus in transit in other clinical settings has previously been reported, 5 the literature regarding cardiovascular events in sars-cov-2 remains scarce; we therefore describe the case of a 64-year-old male who presented with learning points sars-cov-2 pneumonia, who was found to have extensive clot in transit on echocardiography and underwent successful lytic therapy. a 64-year-old male with a history of well-controlled diabetes mellitus and hypertension presented with 10 days of cough and congestion after attending a regional conference. despite driving to the meeting, he had multiple interactions with individuals from italy. he visited his primary care physician, who diagnosed community-acquired pneumonia, and prescribed a 5-day course of levofloxacin 750 mg daily and prednisone 50 mg. over the next week his symptoms did not improve, and he presented to the emergency room after battling symptoms for 17 days. his vitals on presentation demonstrated a temperature of 36.6 c, heart rate of 102 b.p.m., a respiratory rate of 20 breaths/min, and blood pressure of 126/77 mmhg. cardiovascular exam was reported as normal; however, his lung exam demonstrated scattered rhonchi. his medications on admission were aspirin 81 mg daily, atorvastatin 20 mg daily, metformin 1000 mg b.i.d., linagliptin 5 mg daily, and isinopril 20 mg daily. differential diagnosis included sars-cov-2 pneumonia, community-acquired pneumonia, pulmonary embolism, acute heart failure exacerbation, acute coronary syndrome, myocarditis, and pericardial disease. his chest radiogram ( figure 1 ) demonstrated bilateral opacities, left worse than right. he was given ceftriaxone 1 g every 24 h and azithromycin 500 mg once, and was admitted to the general medical ward. he was continued on his home aspirin 81 mg, atorvastatin 20 mg, and lisinopril 20 mg, in addition to being placed on an insulin lispro sliding algorithm and enoxaparin 40 mg subcutaneously daily for deep vein thrombosis prophylaxis. on hospital day 1, an infectious disease consultant recommended sars-cov-2 nasal swab pcr testing. the following day, his sars-cov-2 test returned positive and 2.5 l of o 2 by nasal cannula was required for an oxygen saturation of 80% with ambulation. azithromycin and ceftriaxone were discontinued, and he was managed with an albuterol metered dose inhaler 2.5 mg every 6 h. on hospital day 3, a persistent sinus tachycardia (110 b.p.m.) was unfortunately only treated with metoprolol tartrate 12.5 mg b.i.d. on hospital day 5, tachycardia worsened with heart rates in the 130s, hypoxia requiring 15 l by non-rebreather mask, and acute kidney injury with creatinine elevation to 1.7 from 1. figure 3a and b; supplementarty material online, movie 1) in the right atrium. d-dimer returned at 14 000 ng/ml (normal range <500 ng/ml). worsening hypoxia and acute kidney injury precluded a ct pulmonary embolism scan. thrombolytic therapy with tissue plasminogen activator (tpa) followed by a heparin drip was recommended by the heart team (cardiology and cardiothoracic surgery consultation teams). oxygenation improved and required only a 2 l nasal cannula within 24 h. on hospital day 6, an echocardiogram demonstrated resolution of the right atrial thrombus (figure 4 , supplementarty material online, movie 2) with improvement in pulmonary artery pressures. on hospital day 9, he was transitioned to apixaban with a planned duration for 3-6 months. the patient was discharged home on 2 l of oxygen on hospital day 11. he followed up with his cardiologist via telephone encounter 5 days after discharge and continues to improve. his last encounter was 1 month after hospitalization with his primary care physician where his dyspnoea has improved and no longer requires oxygen by nasal cannula. he is scheduled to see vascular medicine to discuss the risk/benefit of prolonged anticoagulation. despite a global pandemic of >2 million cases 6 of sars-cov-2, there remains a paucity of literature on the mechanisms underpinning the high associated morbidity and mortality. although the majority of patients suffer from hypoxia related to sars-cov-2 pneumonia, consideration must be given to other cardiovascular complications which may contribute to hypoxia. 7 limited reports have cited increased risk of coagulopathy and thrombo-embolism; 8 therefore, obstructive embolism/microthrombi may exacerbate the severe ventilation-perfusion mismatch. 9 this patient had severe right ventricular enlargement and dysfunction, severe pulmonary hypertension, and a large thrombus in transit demonstrated on echocardiography. it remains unclear whether the aetiology of the thrombus was secondary to coronavirus hypercoagulability, endothelial injury, or stasis from his previous travel, or a combination of these factors. evolving literature has demonstrated the importance of echocardiography in determining right heart dysfunction and pulmonary hypertension in the sars-cov-2 population, 1 which may not be from ards alone. therefore, heightened awareness of thrombo-embolism in the sars-cov-2 population is critical. the acute and dramatic resolution of hypoxia in our patient after tpa supports the conclusion of hypoxia from embolism rather than progressive sars-cov-2 pneumonia. in view of the fact that the utility of tpa in this population is not well studied, 10 we present this case of rapid improvement in oxygenation after successful lytic therapy for thrombus in transit in this patient with sars-cov-2. we present this remarkable case of a 64-year-old male admitted with sars-cov-2 pneumonia, who was found to have extensive clot in transit on echocardiography, and underwent successful thrombolytic therapy. this case not only highlights embolic causes of hypoxia in sars-cov-2, but demonstrates the important diagnostic utility of an echocardiogram and the potential therapeutic role of tpa in this population. supplementary material is available at european heart journal -cardiovascular imaging online. the authors confirm that written consent for submission and publication of this case report including images and associated text has been obtained from the patient in line with cope guidance. cardiovascular considerations for patients, health care workers, and health systems during the coronavirus disease 2019 (covid-19) andemic world health organization. who director-general's opening remarks at the media briefing on covid-19 -11 presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the pulmonary and cardiac pathology in covid-19: the first autopsy series from new orleans to lyse or not to lyse: the dangerous right venricular thrombus world health organization covid-19 complicated by acute pulmonary embolism prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ards): a case series key: cord-294069-7zr77r71 authors: hu, xiaowen; ni, wei; wang, zhaoguo; ma, guangren; pan, bei; dong, liyan; gao, ruqin; jiang, fachun title: the distribution of sars-cov-2 contamination on the environmental surfaces during incubation period of covid-19 patients date: 2020-09-30 journal: ecotoxicol environ saf doi: 10.1016/j.ecoenv.2020.111438 sha: doc_id: 294069 cord_uid: 7zr77r71 roles of environmental factors in transmission of covid-19 have been highlighted. in this study, we sampled the high-touch environmental surfaces in the quarantine room, aiming to detect the distribution of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) on the environmental surfaces during the incubation period of coronavirus disease 2019 (covid-19) patients. fifteen sites were sampled from the quarantine room, distributing in the functional areas such as bedroom, bathroom and living room. all environmental surface samples were collected with sterile polyester-tipped applicator pre-moistened in viral transport medium and tested for sars-cov-2. overall, 34.1% of samples were detected positively for sars-cov-2. the positive rates of patient a, b and c, were 46.2%, 0 and 61.5%, respectively. sars-cov-2 was detected positively in bedroom and bathroom, with the positive rate of 50.0% and 46.7%, respectively. in contrast, living room had no positive sample detected. environmental contamination of sars-cov-2 distributes widely during the incubation period of ccovid-19, and the positive rates of sars-cov-2 on environmental surfaces are relatively high in bathroom and bedroom. since a novel human coronavirus, named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first detected in wuhan, china, in late 2019, the coronavirus disease 2019 caused by this virus has been reported drastically all over the world. as of may 25, 2020, about 5,408,301 confirmed covid-19 cases including 345,064 deaths have been reported in 188 countries (jhu, 2020) . so far, compared with severe acute respiratory syndrome coronavirus 1 (sars-cov-1) and middle east respiratory syndrome coronavirus (mers-cov), a relatively low fatality (about 7%) has been summarized from sars-cov-2 infected cases, however, it is obvious that sars-cov-2 is much more contagious as evidenced by its spread to 188 countries across the globe within a very short time. roles of environmental factors in transmission of covid-19 have been highlighted before. several impressive published works on environmental roles revealed the potential risk of environmental factors on the transmission and prevalence of covid-19 pandemics, such as climate change, fomites, water transfer, air and food (eslami & jalili, 2020; qu et al., 2020; wigginton & boehm, 2020) . kampf et al. emphasized that inanimate surface contact is an important way of transmitting the sars-cov-2 (kampf et al., 2020) . sars-cov-2 can survive on surfaces for hours to days, which can live on inanimate surfaces such as metals, glass and plastic at room temperature increasing the opportunity for transmission via touch (kampf et al., 2020; van doremalen et al., 2020) . eslami and jalili concluded that reducing the frequency of touching surfaces by hands and disinfecting surfaces can j o u r n a l p r e -p r o o f reduce the amount of coronavirus load on surfaces and the rate of transmission (eslami & jalili, 2020) . although transmission from contaminated surfaces and fomites are emphasized as an indirect transmission route of sars-cov-2, detection of the virus on the environmental surfaces is largely unknown. from march 18 to 24, 2020, three chinese oversea students returned to qingdao, china. after tested negative nucleic acid without any symptoms at the entry quarantine, they were then transferred to the hotel for 14 days quarantine. during quarantine period in hotel, they presented initial symptoms or were tested positive sars-cov-2 as a presymptomatic person. meanwhile, they were transferred to the local hospital for further diagnosis and treatment. in addition, we synchronously sampled the high-touch environmental surfaces in the quarantine room, aiming to detect the sars-cov-2 distribution on the environmental surfaces during the incubation period of covid-19 patients. our findings would extremely address the importance of touching the contaminated environment transmission, and help extend an effective protocol to interrupt the indirect environmental transmission of sars-cov-2, limit its spread, and mitigate its risks. three covid-19 patients, as chinese oversea students in america and england, returned to qingdao from march 18 to 24, 2020. they had no fever and other symptoms at the entry quarantine, and transferred to a hotel for 14 days quarantine. during the quarantine, their nasopharyngeal swabs were collected every one to three j o u r n a l p r e -p r o o f days. if the swab was tested negatively for sars-cov-2, the student would be still in quarantine in hotel, whereas if the swab was tested positively for sars-cov-2, or presenting initial symptoms of covid-19, such as fever, cough, myalgia and fatigue, etc., they would be immediately transferred to the hospital for further diagnosis and treatment. personal, clinical, and radiological characteristics onset of illness were obtained with standardized data collection forms from the interview, field reports as well as electronic medical records. additionally, their frequency of face washing, hands washing, tooth brushing, bathing and excrement during the quarantine were interviewed by telephone after discharged from hospital. this study was approved by the ethics commission of municipal centre of disease control and prevention of qingdao and written informed consent was waived considering the emergency of infectious disease. environmental surface samples were collected with sterile polyester-tipped applicator pre-moistened in viral transport medium. fifteen sites were sampled from the quarantine room, including light switch, bathroom door knob, inner wall of toilet, towel, inner surface of washbowl, sewer inlet, floor (0.5-1.5meters from the bed), bedside table surface, pillow, sheet, duvet cover, television (tv), tv remote controller, telephone, and bay window. all sampling sites were marked in fig. 1 . the surface of the entire item was swabbed except for pillow, sheet, and duvet cover by swabbing 10 times vigorously from 2 directions (horizontally and vertically) in an about 100cm 2 area that they contacted. all sites were sampled within 4 hours after the j o u r n a l p r e -p r o o f 6 positive nucleic acid test of the patients. all samples after sampling were transferred to the qingdao municipal center for disease control and prevention for sars-cov-2 detection. tests were carried out in biosafety level 2 facilities, using a commercial novel coronavirus nucleic acid detection kit (shanghai biogerm medical technology company) in a total reaction volume of 25μl, targeting sars-cov-2 virus frame1ab (orfab1). viral rna was extracted from sample material and collected in elution buffer, and then underwent real-time reverse-transcription-polymerase-chain-reaction (rt-pcr) with sars-cov-2-specific primers and probes. reverse transcription was performed at 50°c for 10 min, 95°c for 5 min, followed by 40 cycles of rt-pcr analysis at 95 °c for 10 s, and annealing/elongation/fluorescence detection at 55°c for 40 s. a cycle threshold (ct) value were used to approximately reflect the viral loads (inversely related to ct-value) in the respiratory tract, and lower ct values indicated higher viral loads and vice versa (xu et al., 2020; zhu et al., 2020) . according to the technical commission the people's republic of china (chinese cdc, 2020), a ct value less than 37 was defined as a positive test, and a ct value of 40 or more was considered as a negative test. an equivocal result, defined as a ct value between 37 to 40, required confirmation by retesting. if the repeated ct value was less than 40 and an obvious peak was observed, or if the repeated ct value was less than 37, the result was deemed positive. quality controls in sars-cov-2 detection was conducted as follows: environmental surface samples were at the room temperature for no more than 2 hours before laboratory detection; viral rna was isolated from sample material by automatic nucleic acid isolation instruments (magna pure 96, roche diagnostics gmbh, germany) to minimize the possibility of laboratory contamination; novel coronavirus nucleic acid detection kit recommended by national health commission of china was adopted, and laboratory steps were performed according to the instruction of the manufacturer. in addition, sars-cov-2 medium quality control and negative quality control (tmnqc, randox, uk) were used for rna isolation to conduct quality controls. after comparing with stability and sensitivity of products from six manufacturers, we purchased the most precise primers from shanghai biogerm medical technology company. according to the stability of weak positive quality control results (synthetic sars-cov-2-rna, twist bioscience, #102024), the effectiveness of primers was monitored in real time. three patients returned to qingdao, china, during march 18 and 24, 2020, and their characteristics were shown in table 1 . patient a and c were onset of illness (fever, cough or fatigue) during the quarantine time. when presenting initial symptoms, they immediately reported to professional medical staffs stayed in hotel. then, medical staffs stayed in hotel immediately sampled their nasopharyngeal swabs and environmental surfaces, and transferred them to the hospital for further diagnosis additionally, the frequency of washing behaviors of patients at the quarantine room, including face washing, hands washing, tooth brushing, bathing and excrement, were shown in table 2 . all sampling sites were marked in fig. 1 . in total, forty-one samples were collected from the quarantine rooms of the three covid-19 patients. as shown in fig. 3 as shown in fig. 1 and table 3 , the quarantine room was divided into three functional areas. bedroom and bathroom were tested positively for sars-cov-2, with the positivity rate of 50.0% (7/14) and 46.7% (7/15), respectively. in the bathroom, all the six sites were positive for rt-pcr tests, including light switch, bathroom door knob, inner wall of toilet, towel, inner surface of washbowl and sewer inlet, and the lowest ct value was detected from inner wall of toilet. in the bedroom, all the five sites were positive for rt-pcr tests, including floor, beside table surface, pillow, sheet and duvet cover, and the lowest ct value was detected from pillow. in contrast, living room was negative for virus, and all sites, such as tv, tv remote controller, telephone and bay window, were negative for rt-pcr test. in addition, all environmental sites came from five types of material. as shown in table 3 , the positive rate was highest in cotton sites (60.0%, 6/10), followed by ceramic sites (40.0%, 2/5), metal sites (40.0%, 2/5), wood sites (33.3%, 2/6) and plastic sites (16.7%, 2/12) although researchers enhanced the importance of environmental contamination on the route of sars-cov-2 transmission (faridi et al., 2020; guo et al., 2020; ong et al., 2020; yung et al., 2020; ) , there has been limit data revealing the contribution of temperature, humidity and surface material types may be the important environmental factors for the survivability of sars-cov-2 on surfaces (aboubakr et al., 2020; biryukov et al., 2020; ren et al., 2020) . in our study, we found that environmental samples from bedroom and bathroom were detected positively for sars-cov-2 with close positive rates (50.0% and 46.7%, respectively), and the average ct value in bathroom (32) was lower than that in bedroom (35) several limitations should be known in our study. firstly, we did not sample any indoor air, which should be strengthened in future study. compared with other sampling sites in the room, the frequency of touching main door knob may be relatively low. however, main door knob should be still as necessary sampling site. unfortunately, we did not sample it in all rooms, which should be paid for attention in our future study. although we used central heating to describe the situation of room environment, other environmental factors, such as room temperature and humidity, were not detected accurately in the quarantine rooms. in addition, our study only analyzed the environmental contamination in the quarantine room based on a small size of covid-19 patients, and further research on a larger size should be performed to evaluate the contribution of environmental contamination to the transmissibility of covid-19. environmental contamination of sars-cov-2 distributes widely during the incubation period of cvoid-19, and the positive rates of sars-cov-2 on environmental surfaces are relatively high in bathroom and bedroom. our findings would extremely address the importance of touching the contaminated environment transmission, and help extend an effective protocol to interrupt the indirect environmental transmission of sars-cov-2, limit its spread, and mitigate its risks. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. conceptualization, writing-original draft. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. stability of sars-cov-2 and other coronaviruses in the environment and on common touch surfaces and the influence of climatic conditions: a review public health-seattle and king county and cdc public health-seattle and king county and cdc covid-19 investigation team. presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility increasing temperature and relative humidity accelerates inactivation of sars-cov-2 on technical guidelines for laboratory testing of covid-19 the role of environmental factors to transmission of sars-cov-2 (covid-19) a field j o u r n a l p r e -p r o o f indoor air measurement of sars-cov-2 in the patient rooms of the largest hospital in iran aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards test and analysis of the central heating residential indoor thermal environment in qingdao city coronavirus resource center persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents viral rna load as determined by cell culture as a management tool for discharge of sars-cov-2 patients from infectious disease wards air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient sars-cov-2 virus culture and subgenomic rna for respiratory specimens from patients with mild coronavirus disease an imperative need for research on the role of environmental factors in transmission of novel coronavirus (covid-19) stability and infectivity of coronaviruses in inanimate environments duration of infectiousness and correlation with rt-pcr cycle threshold values in cases of covid-19 aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 2020. coronavirus disease 2019 (covid-19)-who environmental engineers and scientists have important roles to play in stemming outbreaks and pandemics caused by enveloped viruses virological assessment of hospitalized patients with covid-2019 characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding environment and personal protective equipment tests for sars-cov-2 in the isolation room of an infant with infection a novel coronavirus from patients with pneumonia in china wei ni: formal analysis, writing-original draft we are deeply thankful to all health-care workers involved in the diagnosis and treatment of patients in qingdao. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord-300968-dtaasxk1 authors: kliger, yossef; levanon, erez y.; gerber, doron title: from genome to antivirals: sars as a test tube date: 2005-03-01 journal: drug discovery today doi: 10.1016/s1359-6446(04)03320-3 sha: doc_id: 300968 cord_uid: dtaasxk1 abstract the severe acute respiratory syndrome (sars) epidemic brought into the spotlight the need for rapid development of effective anti-viral drugs against newly emerging viruses. researchers have leveraged the 20-year battle against aids into a variety of possible treatments for sars. most prominently, based solely on viral genome information, silencers of viral genes, viral-enzyme blockers and viral-entry inhibitors were suggested as potential therapeutic agents for sars. in particular, inhibitors of viral entry, comprising therapeutic peptides, were based on the recently launched anti-hiv drug enfuvirtide. this could represent one of the most direct routes from genome sequencing to the discovery of antiviral drugs. severe acute respiratory syndrome (sars) is a pulmonary infection that has been identified in multiple outbreaks around the world, emerging initially in guangdong province, china, in november 2002. the number of reported cases increased exponentially and reached 8422, which resulted in 916 deaths by august 2003 (who website). the syndrome is caused by a previously unknown virus -sars-associated coronavirus (sars-cov) [1] [2] [3] . the global sars outbreak has been contained, mainly owing to strict patient isolation and aggressive containment of infected regions, but the virus itself has potential to reappear. this concern is supported by studies reporting a cyclic pattern for other human-infective coronaviruses that attack mainly in the winter, sometimes skipping years, for example, the related human coronavirus, hcov-oc43, which breaks out every two to four years [4] . to date, there are ~36 antiviral drugs, half of which were developed in the past 15 years to treat a single virus, hiv-1. development of these antiviral drugs gained from the advances in molecular and structural biology coupled with advances in medicinal chemistry and in the industrialization of the drug discovery process. the sars epidemic has emphasized the need to develop drugs against emerging viral infections quickly, and demonstrates the usage of genomic technologies in antiviral research. in the past two decades, biology has become an information-driven science as a result of the emergence of genomic technologies and the expansion of the internet that allows analysis of genomic databases at every researcher's desktop. these advanced genomic technologies led to rapid sequencing of sars-cov [5, 6] and, for the first time in history, the sequencing of a genome of an infective agent preceded the understanding of its basic biology and etiology. armed with this genomic information, research groups around the world suggested multidisciplinary approaches to attain anti-sars drugs. in general, physicians tried to relieve the symptoms mainly by modulating parts of the immune system, while vaccinologists began the long process of developing a vaccine against the virus. molecular and structural biologists suggested ways to interfere with the viral life cycle, and these are the focus of this review (figure 1 ). the strategy starts from virus identification, goes through genome sequencing and, hopefully, ends with an antiviral drug. drug development remains a challenge, but the acceleration in drug discovery offered by genome technologies will hopefully enable significant timetable cuts in achieving antiviral medicine. other, more classical anti-viral strategies used to treat sars patients, like the use of interferon, will not be discussed here. the interested reader is referred to [7, 8] . this review includes a retrospective summary of the development of anti-hiv drugs, followed by an appraisal of anti-sars strategies and their applicability for rapid development of antivirals against sars-cov, if it does resurface, and against the next, probably inevitable, viral threat. in the early 1980s, a sudden increase in life-threatening opportunistic infections and kaposi's sarcoma, considered rare until then, was observed among homosexuals and injecting drug users. these infections were attributed to an acquired immunodeficiency syndrome (aids). about two years of extensive studies passed until the human immunodeficiency virus (hiv) was identified as the etiological agent [9, 10] . an additional two years passed before completing the sequencing of its genome [11] . the huge efforts invested in developing the first drug to treat aids patients bore fruit in record speed when the fda approved azidothymidine (azt) in 1987. azt, a nucleoside analog, inhibits the viral reverse transcriptase -an enzyme that is essential for hiv replication. unfortunately, isolates from these patients showed decreased sensitivity after six months of azt administration. the finding that some of these isolates also exhibited cross-resistance to other nucleoside analogs [12] raised further concern. azt and other nucleoside reverse transcriptase inhibitors were still the sole treatment available for almost a decade. the next important milestones were the development of inhibitors against the protease, another essential viral enzyme, and nonnucleoside reverse transcriptase inhibitors, first approved in 1995 and 1997, respectively. while the nucleoside reverse transcriptase inhibitors were discovered by cell culture screening directed towards a specific class of chemical agent, the non-nucleoside reverse transcriptase inhibitors were discovered by hts of large compound libraries. the discovery of hiv protease inhibitors represents one of the best examples of the application of protein structural knowledge to rational drug design. since then, aids patients have been treated with a cocktail of inhibitors against hiv reverse transcriptase and protease. however, despite the unprecedented successes in the therapy of hiv infection, hiv continues to spread, causing more than 14,000 new infections every day, 95% of these in the developing world (who website). a major problem with current aids treatment is the high frequency of hiv mutations, resulting in drug resistance. thus, efforts are being made to develop agents addressing yet untargeted steps in the hiv life cycle. viral-induced fusion between viral and host cell membranes was acknowledged as a the sars-cov life cycle is vulnerable to therapeutic intervention in several places. (1) virus binding to cellular receptors. outside the cell, blocking the interaction of sars-cov with the cellular receptor will prevent the virus from attaching to host cells. co-receptor antagonists will prevent the initiation of the next step. www.drugdiscoverytoday.com useful drug target with the approval of the hiv fusion inhibitor, enfuvirtide (fuzeon), in 2003 [13] . unlike other hiv drugs that are small molecules developed against viral enzymes, enfuvirtide is a peptide that corresponds to a specific segment of the viral envelope protein. importantly, this segment can be directly pinpointed by computational sequence analysis [14, 15] . this strategy seems promising in developing anti-viral therapeutic peptides to other viruses that possess type 1 viral fusion proteins [e.g. measles virus and respiratory syncytial virus (rsv)], which share some structural motifs with hiv. little is known about viral-induced membrane fusion of other viruses that do not share these motifs. viruses can be divided into two groups based on the composition of their outer surface: (a) non-enveloped viruses are enclosed by a protein shell called a capsid; (b) enveloped viruses are surrounded by a membrane 'stolen' from their last host. in order to infect host cells, that is, to inject their genetic material into the cell, enveloped viruses need to overcome both viral and cellular membrane barriers. viral entry of many enveloped viruses, including sars-cov, involves two major steps. first, the virion binds to receptor(s) localized on the surface of its host cell, and second, the viral membrane fuses with the host cell membrane. sars-cov spike glycoprotein, responsible for these two steps, is translated as a large polypeptide that is subsequently cleaved to produce two functional subunits, s1 and s2. s1 is the peripheral protein, which binds to cellular receptor(s), whereas s2 is a type i transmembrane protein that catalyzes the membrane fusion reaction. both steps are crucial for viral infection, and therefore were suggested as targets for antivirals. since the identification of cd4 as the cellular receptor for hiv in 1984 [16, 17] , several therapeutic agents aiming to inhibit the binding of hiv to cd4 were suggested. unfortunately, these efforts have yet to bear fruit. major difficulties that slow down the development of inhibitors for the binding of cd4 to gp120 include: (i) the gp120binding site for cd4 consists largely of a recessed pocket; (ii) antibodies that bind to cd4 antigen are likely to block virus attachment but can be immunosuppressive because they will lead to depletion of cd4 cells. currently, both a recombinant cd4-igg2 fusion protein (pro-542) and a small-molecule (bms-488043) aiming to prevent hiv from attaching to cd4, are in clinical trials. these efforts reflect the motivation of inhibiting the first step in the viral life cycle, that is, the binding of the virus to its host cell. this approach was strengthened when cxcr4 and ccr5 were identified as additional essential cellular receptors for hiv [18, 19] , and with the discovery that ccr5deficient people are resistant to infection by hiv [20] . similar to hiv, binding of the viral spike glycoprotein to some receptor(s) on host cells is the first step in sars-cov infection. blocking the interaction between these receptors and the virus could prevent infection, thus inspiring the search for sars-cov cellular receptors. recently, human angiotensin converting enzyme-related carboxypeptidase (ace2), a type i integral membrane metalloprotease, was identified as a receptor for sars-cov [21] . a soluble form of ace2 and an antibody recognizing sars-cov s1 efficiently neutralized sars-cov in vitro, supporting the speculation that the ace2-binding site of the spike glycoprotein is an attractive target for vaccine and drug development [21, 22] . this is further supported by an ace2 inhibitor, which also inhibits sars-cov infection in vitro [23] . notably, a 193-amino acid fragment of sars-cov s1, which efficiently bound ace2, blocked spike glycoprotein-mediated infection with an ic 50 of less than 10 nm [24] . more recently, a human lung cdna library was screened to identify receptors for sars-cov, revealing that human cd209l can also mediate infection by sars-cov, although it is a much less efficient receptor than ace2. interestingly, cd209l is expressed in human lung in type ii alveolar cells, which are an important target for sars-cov infection [25] . it is still not known whether interactions between ace2 and cd209l play a role in sars-cov infection and pathogenesis. hiv entry involves the binding of the viral envelope glycoproteins (comprising gp120 and gp41, which are the homologous of sars-cov s1 and s2, respectively) to cd4 on the host cell plasma membrane. this induces conformational changes, enabling the n-terminal heptad repeat region (n-hr) of gp41 to be exposed. at this stage, enfuvirtide binds to the n-hr of gp41, hence blocking further conformational changes required for membrane fusion. enfuvirtide is a synthetic peptide inhibitor corresponding to a segment of gp41, known as the c-terminal heptad repeat (c-hr). following the cd4-induced conformational change of gp41, plasma membrane ccr5 (or cxcr4) molecules are recruited to the binding site, and bind to the cd4-envelope complexes. this triggers a highly stable interaction between the c-hr and the n-hr regions of gp41, which drives the membrane fusion reaction to completion. thus, enfuvirtide can no longer inhibit the fusion process [26] . slower engagement of the co-receptor with the cd4-envelope complexes, results in a stronger inhibition by c-hr-derived peptides [27, 28] . furthermore, reduction in ccr5 binding efficiency resulted in slower fusion kinetics and increased sensitivity to enfuvirtide [28, 29] . further support for this model is provided by the finding that ccr5 and cxcr4 antagonists showed strong anti-hiv synergy with enfuvirtide against ccr5-dependent and cxcr4-dependent hiv isolates, respectively [30, 31] . in addition, pro-542 acts in concert with enfuvirtide in virus-cell and cell-cell fusion assay, by triggering formation there are no peptide fusion inhibitors for influenza virus. it is noteworthy that influenza virus uses a different mechanism to enter its host cells: it is first endocytosed into the cell, followed by a ph-dependent fusion between the viral and the endosome membranes. strikingly, it takes only few milliseconds from the time the ph drops in the endosomes until the fusion process is completed [33] [34] [35] [36] . in contrast, the time scale of hiv infection is about 20 minutes, allowing ample time for binding of entry inhibitors [26, 27, 37] . sars-cov entry kinetics resembles that of hiv. at 5 minutes after exposure, the sars-cov lined the plasma membrane of vero cells [38] . fusion and entry of the viral load into the cytoplasm was observed mainly between 15 and 20 minutes [38] . the timescale similarity between hiv and sars-cov fusion process, as opposed to the fast membrane fusion of influenza virus, indicates that entry inhibitors could be successful with sars-cov. despite the lack of sequence similarity between the fusion proteins of hiv-1 and sars-cov. schematic illustration of (a) hiv-1 gp41 and (b) the equivalent s2 protein from the sars-cov. a leucine/isoleucine heptad repeat adjacent to the n-terminus of both proteins appears in red.the c-hr is in green. cysteine residues (purple) confining a loop structure are located between the two heptad repeats. an aromatic residues-rich motif is marked blue, and the transmembrane segment is in orange. a peptide corresponding to the c-hr, which acts as potent inhibitor of hiv-1 entry into the cell, appears in yellow.the helical wheel is a top view of a single strand of a coiled coil. in the wheel projection of the n-hr (c) and c-hr (d) of sars-cov s2 protein, each of the seven positions (a-g) corresponds to the location of an amino acid residue that makes up the coiled coil.the arrows between the seven positions indicate the relative locations of adjacent residues in an amino acid subsequence.the helical wheel demonstrates how a potential antiviral drug can be discovered based solely on sequence information. homology and the difference in length between sars-cov s2 and hiv gp41, homologous regions of the n-hr and c-hr in sars-cov s2 were identified immediately after the sars-cov genome sequence was published ( figure 2) . thus, a similar strategy might be applied to inhibit the entry of sars-cov [39] , (http://www.virology.net/articles/ sars/s2model.html). indeed, preliminary reports revealed anti-sars activity for peptides corresponding to the c-hr of sars-cov s2 protein [40] [41] [42] , and indicated a mode of action similar to that of enfuvirtide [43] [44] [45] [46] . the kinetic similarity of sars-cov and hiv entries suggests a synergism between sars-cov spike glycoprotein inhibitors and agents that block some of its receptors. the role of different cellular receptors in sars-cov entry should be characterized to discover the receptor(s) that trigger conformational changes and transform the spike protein into the stable 'fusogenic' form. antagonists for these receptor(s) could synergize with fusion inhibitors. the synergy between sars fusion inhibitors and ace2 or cd209l antagonists has not yet been investigated. the first step is to determine optimal fusion inhibitors. intriguingly, whereas polar residues disrupt the heptad repeat in the c-hr of hiv-1 gp41, the c-hr of sars-cov s2 has a perfect leucine/isoleucine heptad repeat ( figure 2d ). this could explain why the exact sequence boundaries of the c-hr-derived peptides are crucial for efficient inhibition [41, 42, 44, 47] , as aggregation of the peptides in solution could abolish anti-viral activity. interestingly, two reports demonstrate that n-hr-derived peptides are also active [40, 41] , while others found that only c-hrderived peptides have anti-sars activity [42, 47] . it is noteworthy that the reason for the poor inhibitory activities of n-hr-derived peptides in other viruses is contributed to their tendency to aggregate in solution, suggesting that, similar to the c-hr-derived peptides, the exact sequence boundaries of the n-hr-derived peptides are important. the main advantage of fusion inhibitors is their immediate discovery as they are simply the corresponding fragments of a known protein. however, their drawbacks as therapeutic peptides are lack of oral bioavailability and high production costs. auspiciously, sars is a respiratory syndrome, thus, peptidic fusion inhibitors could be given by inhalation. this approach was applied successfully in rsv-infected mice [48] . to serve as drug targets, viral proteins should fulfill two criteria: (i) they should be essential for the viral life cycle; and (ii) they should exhibit low similarity to host proteins. sars-cov genome analysis was performed to predict its proteome [49] , and three viral enzymes were suggested as targets for drug discovery: the helicase, the rna-dependent rna polymerase and the main protease. these enzymes are crucial for replication, transcription, translation and posttranslational polyprotein processing (box 1). assay development based on these three sars-cov target enzymes was initiated [50] [51] [52] , thus paving the way for highthroughput in vitro screening approaches to identify candidate inhibitors in compound libraries. the traditional and, in many cases, the most cost-efficient way of dealing with viruses has been through vaccines. the logic of vaccine development against sars-cov emerges from the combination of several findings: (i) re-infection with sars-cov causes only mild illness; (ii) sars is fatal mainly to old people who have difficulty in producing good humoral and cellular immune responses; and (iii) the case fatality ratio of sars ranges from 0-50% depending on the age group affected, with an overall reviews box 1 currently, inhibitors target three enzymes, crucial for sars-cov life cycle: helicase: protein-fold recognition methods followed by a biochemical study suggested a dual use of sars-cov helicase in both rna synthesis and cap formation, suggesting new avenues to treat the virus [65, 66] . screening of a compound library by plaque reduction assay resulted in a helicase inhibitor at the low microm range. in vitro assay confirmed sars-cov helicase as a validated drug target [67] . rna-dependent rna polymerase: molecular modeling revealed that sars-cov rna-dependent rna polymerase does not contain a hydrophobic pocket for non-nucleoside inhibitors.this is in contrast with the non-nucleoside inhibitors activity against hiv-1 reverse transcriptase [68] . of the many nucleoside analogues screened, sars-cov most selective nucleoside analogue inhibitor is beta-d-n4hydroxycytidine, albeit at low efficacy (ec90 of 6 microm by virus yield reduction assay) [69] . ribavirin, a broad-spectrum nucleoside analogue efficacious in the treatment of several viral infections, was used in various countries against sars-cov.while ribavirin was promising in vitro [70] , recent reports revealed that ribavirin did not appear to confer any benefit for patients with sars [71, 72] . main protease: sequence similarity was found between the substrate-binding sites of sars-cov main protease and the main protease of related viruses. remarkably, the sars-cov main protease cleaved the porcine coronavirus transmissible gastroenteritis coronavirus (tgev) main protease substrate [73] .this rationalizes screening of known protease inhibitors for anti-sars activity.this approach was reinforced by the crystallization of the sars-cov main protease together with a tgev inhibitor [74] . the findings revealed that homology modeling is often inadequate for the prediction of the mutual orientation of domains in multidomain proteins. however, the tgev-based homology model also shows that a reasonable model of a substrate-binding site can serve to develop useful ideas for inhibitor design that can inspire medicinal chemists to start a synthesis program long before the 3d structure of the target enzyme is experimentally determined (reviewed in [75] ). in parallel, hts of compound libraries identified inhibitors of the sars-cov main protease in the low âµm range [67, 76, 77] .there are a few studies demonstrating that inhibitors of the hiv protease can also inhibit sars-cov, albeit with much lower efficiency [52, 78] . www.drugdiscoverytoday.com estimate of case fatality of 14-15% (who website). thus, most infected individuals recover from sars. furthermore, the success of a vaccine against other mammal-infective coronaviruses is encouraging [53, 54] . modern antiviral vaccine development depends heavily on the viral genome. the availability of the human genome, together with the recent sequencing of the sars-cov genome, largely increases the probability of success of vaccine development. the full-length spike glycoprotein of sars-cov, expressed by vaccinia virus, induces binding and neutralizing antibody and protectively immunizes mice against a subsequent infection with sars-cov [55] . in addition, dna vaccine encoding the spike glycoprotein of the sars-cov induces t-cell and neutralizing antibody responses, as well as protective immunity, in a mouse model [56] . the discovery of rnai raises many hopes regarding antiviral strategies and carries the promise of a shortcut in the drug discovery process. usually, target discovery is followed by exhaustive hts and/or structure-based screening of many thousands of compounds in the hope that some of them will efficiently bind to the target. theoretically, with small-interfering rna (sirna) as a drug, the course from target to drug is much shorter. encouraging results in mice were obtained using an rnaibased therapy against hepatitis b virus (hbv): transfection with plasmids expressing short hairpin rnas (shrnas) homologous to hbv mrnas effectively inhibited replication initiation in cultured cells and mice liver, showing that such an approach could be useful in the treatment of viral diseases [57] . currently, there are attempts to use sirna as anti-sars drugs, but they are still in preliminary in vitro stages [58] [59] [60] . the application of this relatively new technology to therapeutics faces several safety and technical issues, including delivery of the rna molecule into the virus-infected cells and the activation of interferon system [61, 62] . sars-cov reminds us that viral infections are a global threat. it is vital that the scientific community acquire the ability to develop anti-viral therapy promptly. we can be encouraged by the remarkable speed with which the global community acted in a coordinated research effort to investigate sars-cov. immediately after the last nucleotide of the sars-cov genome was verified, the sequence was distributed through the internet to the worldwide scientific community. among the genomicbased approaches that followed, inhibitors of the viralinduced membrane fusion seem the most promising. the mutation rate of sars-cov is much slower than that of hiv-1 and is among the lowest of rna viruses [63, 64] . however, viral resistance will be an obstacle. the solution could lie in the use of a drug cocktail, combining antiviral drugs with different modes of action (e.g. protease and polymerase inhibitors), to lower the chances for drug-resistant viruses to arise. in addition, drug cocktails are beneficial when the optimal dose of a drug, given as a mono-therapy, is toxic -then, combining drugs with distinct modes of action, in sub-optimal doses, might alleviate toxicity issues. moreover, the recent advancement in the understanding of hiv entry into its host cell revealed an opportunity for synergism, based on the molecular mechanism of viral entry. drugs that inhibit the interaction between ccr5 and the cd4-envelope complexes enhance the efficiency of hiv fusion inhibitors by elongating the exposure time of their target site. the timescale similarity of the sars-cov fusion process to that of hiv is encouraging. hopefully, future identification and characterization of sars-cov receptors will open a way for an efficient antiviral strategy, by synergistically combining viral entry inhibitors. within a few months, scientists have managed to leverage the technological advances of the past 20 years of anti-aids research into an unprecedented antiviral campaign against sars (figure 3) . sars served as a test tube for novel approaches developed following the aids epidemic. the current sars epidemic was finally contained, yet quick development of antivirals is still high priority. today, we are closer than ever to achieving therapeutic solutions for a viral epidemic shortly after viral outbreak. a time line comparing key achievements in aids and sars research. effective international 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vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds investigational use of ribavirin in the treatment of severe acute respiratory syndrome temporal relationship of viral load, ribavirin, interleukin (il)-6, il-8, and clinical progression in patients with severe acute respiratory syndrome coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor utility of homology models in the drug discovery process characterization of sars-cov main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay high-throughput screening identifies inhibitors of the sars coronavirus main proteinase hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus pages 1020-1028) an important literature citation was omitted from the published reference list.the details of this article we thank l. rychlewski, a. wool, e. eisenberg, n. rabbie, a. toporik, m. olshansky and s.g. peisajovich for critical reading of the manuscript. we are also grateful to artist r. lieber for capturing the central idea of the article with her illustration. key: cord-307860-iqk1yiw4 authors: ionescu, mihaela ileana title: an overview of the crystallized structures of the sars-cov-2 date: 2020-10-24 journal: protein j doi: 10.1007/s10930-020-09933-w sha: doc_id: 307860 cord_uid: iqk1yiw4 many research teams all over the world focus their research on the sars-cov-2, the new coronavirus that causes the so-called covid-19 disease. most of the studies identify the main protease or 3c-like protease (m(pro)/3cl(pro)) as a valid target for large-spectrum inhibitors. also, the interaction of the human receptor angiotensin-converting enzyme 2 (ace2) with the viral surface glycoprotein (s) is studied in depth. structural studies tried to identify the residues responsible for enhancement/weaken virus-ace2 interactions or the cross-reactivity of the neutralizing antibodies. although the understanding of the immune system and the hyper-inflammatory process in covid-19 are crucial for managing the immediate and the long-term consequences of the disease, not many x-ray/nmr/cryo-em crystals are available. in addition to 3cl(pro), the crystal structures of other nonstructural proteins offer valuable information for elucidating some aspects of the sars-cov-2 infection. thus, the structural analysis of the sars-cov-2 is currently mainly focused on three directions—finding m(pro)/3cl(pro) inhibitors, the virus-host cell invasion, and the virus-neutralizing antibody interaction. electronic supplementary material: the online version of this article (10.1007/s10930-020-09933-w) contains supplementary material, which is available to authorized users. latest works [6, 7] . much of the knowledge about the transmission of sars-cov-2 has gleaned from the sars-cov and mers-cov studies [5, 8] . there are some notable differences between tissue tropism of the hcovs. analysis of structural similarities and structural differences between hcovs species could advance the understanding of sars-cov-2 pathogenesis [8] [9] [10] [11] [12] . the review aims to collect and synthesize the x-ray/ nmr/cryo-em structures of the sars-cov-2 deposited in the public database protein data bank (pdb) (https ://www. rcsb.org/pdb). structures retrieved from pdb (august 12, 2020) were analyzed for relevant information on covid-19 infection, synthesis of new inhibitors, sars-cov-2 interaction with host receptors, and the neutralizing antibodies interactions with spike glycoprotein. in this review, the sars-cov-2 related structures published in peer-reviewed papers are analyzed in depth. very often the authors add minor/major revisions of the coordinate files after the pdb structure was submitted in the pdb. an association of scientists has created a public database in which covs structures are systematically validated (https ://covid -19.biore produ cibil ity.org/) [13] . the aim of this review is to provide an analysis of the sars-cov-2 structures deposited in the pdb. every wednesday, the covid-19/sars-cov-2 resources announce the new pdb structures (https ://www.rcsb.org/). the rapid accumulation of the x-ray/nmr/cryo-em structures of the sars-cov-2 in the protein data base (pdb) needs an objective selection analysis of these crystal structures for further research. the covid-19 is an ongoing pandemic and the virus undergoes mutations reflected in differences of the crystal structures. there are many crystal structures of the sars-cov-2 spike (s) co-crystallized with antibodies that further advance the understanding of the immunogenicity of the s [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . also, there is an intense work on designing effective inhibitors. there are many compounds mainly co-crystallized with the viral main protease (3cl-protease). the subgroup analysis of structural and non-structural protein (nsp) of the sars-cov-2 and other covs includes the multiple sequence alignment. the x-ray/nmr/cryo-em structures of the sars-cov-2 were retrieved by searching "sars-cov-2" in pdb (https ://www.rcsb.org/pdb). inclusion criteria: the present review narrows the analysis of the sars-cov-2 crystal structures deposited in the pdb only for the crystal structures published in peerreviewed journals (august 12, 2020). the further analysis of some important characteristics of the sars-cov-2 needs the inclusions of other five crystal structures (fig. 1) . the structures were checked in the validated sars-cov-2 related structural models of potential drug targets site (https ://covid -19.biore produ cibil ity.org/) [13] . exclusion criteria: the pdb sars-cov-2 related crystal structures non-published in peer-reviewed journals. the structures were visualize and analyzed on dassault systèmes biova program-discovery studio modeling environment, release 2017, san diego: dassault systèmes, 2016 (https ://accel rys.com). [26] [27] [28] . the search of "sars-cov-2" in pdb retrieved 337 entries (august 12, 2020) (table s1 ). among them, only 65 of 2 x-ray/nmr/cryo-em structures are published until now-pdb id(s) 6lxt, 6vyb, 6vxx, 7btf, 6m71, 6m0j, 6m3m, 6w01, 6vww, 6vsb, 6lzg, 7bv1, 7bv2, 6yyt, 6m17, 6vw1,6xr8, 6xra, 6lu7, 7bqy, 6lze, 6m0k, 7buy, 6y2e, 6y2g, 6y2f, 6w41, 6yor, 7bz5, 7c01, 7bwj, 7byr, 6xcm, 6xcn, 6xca, 7c2l, 7bzf, 7c2k, 6z97, 6zlw, 6zm7, 6zn5, 6zon, 6zp4, 6xdg, 6wqf, 7bw4, 7cah, 6xey, 6zox, 6zoz, 6zoy, 6zp0, 6zp1, 6zp2, 6yva, 6zcz, 6zdh, 6zdg, 6zer, 6zfo, 6zmi, 6zmo, 6zmt, and 6zme [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . the first x-ray structure found (pdb id 6lu7) belongs to the nonstructural protein 5 (3c-like protease) of the sars-cov-2 in complex with the michael acceptor-based inhibitor n3 (prd_002214). the structure was deposited in pdb in 2020-01-26 and released in 2020-02-05 by the liu x. et al. [41] . two pdb entries (6m1d and 6m18) of the human ace2 co-crystallized with the sodium-dependent neutral amino acid transporter b(0)at1were added because these structures are part of the same work with the spike-receptor binding domain (rbd)/ace2/ b(0)at1 em-structure (pdb id 6m17) [39] . three nsp9 structures non-published in the peer-reviewed papers were included in the study (pdb id(s) 6w9q, 6w4b, and 6wxd). their sequences were analyzed by multiple sequence alignment and compared with previous works. the selection flow of the 70 pdb structures included in the present review is shown in fig. 1 . one of the most urgent aims of controlling the sars-cov-2 pandemic is to find out an efficient therapy. healthcare professionals have established different clinical practice guidelines. the update of the clinical results helps the medical teams to monitor and refine their therapy, but a conclusion could be drawn only at the end of the sars-cov-2 pandemic. there is no specific cure for covid-19, which explains the race to discover effective inhibitors and a universal vaccine. despite numerous sars-cov-2 pdb structures co-crystallized with inhibitors, only a few of them have been publishing in peer-reviewed papers. many research teams focus on designing inhibitors of viral proteases due to their role in viral replication [51] . there are two viral proteases: main protease/3c-like protease (m pro /3cl pro ) and papainlike protease (pl pro ). these proteases cleave the polyprotein 1ab (uniprotkb p0c6x7) to yield the viral proteins [44] . there are seven pdb structures of sars-cov-2 3cl pro cocrystallized with antiviral drug candidates. two structures are co-crystallized with n3 (prd_002214) (pdb id(s) 6lu7 and 7bqy) [41] . the 6lze and 6m0k structures are co-crystallized with compounds 11a and 11b, respectively [42] . two structures are co-crystallized with alphaketoamide 13b (pdb id(s) 6y2f and 6y2g) [44] . the 7buy structure is co-crystallized with the antineoplastic drug carmofur [43] . there is a cryo-em crystal structure of the sars-cov-2 rna-dependent rna polymerase (rdrp) complex (nsp12/nsp8/nsp7) with the antiviral drug remdesivir (pdb id 7bv2) [37] . however, these compounds are not optimal inhibitors of sars-cov-2. the comparison of the antiviral drug candidates against sars-cov-2 and other covs is shown in table 1 . the 3cl pro (uniprotkb p0dtd1) is extensively studied for designing of new inhibitors due to its unique characteristics. it is also known as non-structural protein 5 (nsp5). it is a cysteine protease involved in the cleavage of the viral polyproteins 1a and 1ab. moreover, there are no human counterparts for 3cl pro [52] [53] [54] [55] [56] [57] . the 3cl pro is one of the targets for the control of zoonotic reservoir of covs. the peptidomimetics with 3-thiophene and 1-methylbenzotriazole backbones inhibit the bat covs hku4-cov and hku5-cov at sub-micromolar concentrations [58] . the need for specific treatment of covid-19 had led to testing the inhibitory activity of other antiviral drugs. at the beginning of the sars-cov-2 pandemic, wang et al. published the comparison of inhibitory activity of five antiviral drugs (ribavirin, penciclovir, favipiravir, nefamostat, and remdesivir) and two antiprotozoal drugs (nitazoxanide, and cloroquine). the ec50 of remdesivir and cloroquine against sars-cov-2 are 0.72 μm and 1.13 μm, respectively [59] . dai et al. published the compounds 11b that showed ec50 of 0.72 μm, identical to the ec50 of remdesivir (table 1 ) [42] . compounds 11a and 11b (pdb id(s) 6lze and 6m0k) show good inhibitory activity at a concentration of 1 μm-100% and 96%, respectively [42] . zhang et al. published three sars-cov-2 3cl pro -one free enzyme (pdb id 6y2e) and two crystal structures with a new inhibitor candidate-an alpha-ketoamide compound 13b (pdb id(s) 6y2g and 6y2f) [44] . compound 13b is a non-polymer compound derived from a peptidomimetic inhibitor against enterovirus proteases [60] . the x-ray crystal structures of the sars-cov-2 3cl pro co-crystallized with 13b demonstrated that the interactions from the dimer interface are essential for shaping the binding pocket. also, the authors greatly improved the pharmacokinetic characteristics of the compound 13b. the compound 13b can be administered as an inhaler [44] . two x-ray crystal structures of the sars-cov-2 3cl pro co-crystallized with michael acceptor-based inhibitor (prd_002214) were recently published: pdb id(s) 6lu7 and 7bqy. the inhibitor prd_002214, the inhibitor n3, is the result of the high-throughput screening of more 10,000 inhibitory compounds against sars-cov-2 3cl pro . the kinetic experiments showed that n3 is a time-dependent irreversible peptide-like inhibitor of the sars-cov-2 3cl pro . molecular docking analysis demonstrated that n3 fit inside the substrate-binding pocket (cys145-his41 catalytic dyad) highly conserved in covs [41, 61] . a recent structural study hypothesized that a cluster of residues outside the catalytic site are possibly conformationally relevant when bound to the n3 [62] . the inhibition of hydrolytic activity of the n3 was previously determined for other covs (hcov-22e, fipv, and mhv-a59) ( table 1 ). the overall analysis of the available data suggests that n3 is a wide-spectrum anti-cov lead compound [63] . another structural study demonstrated that the antineoplastic drug carmofur inhibits the 3cl pro sars-cov-2 (pdb id 7buy). carmofur (pubchem cid 2577) (c 11 h 16 fn 3 o 3 ) is an antimetabolite (pyrimidine analog) derivative of 5′-fluorouracil. carmofur is used to treat colorectal cancer and breast cancer (drugbank db09010). the jin et al. study shows that carmofur inhibits viral replication by forming a covalent bond with the cysteine residue from the catalytic site of the 3cl pro sars-cov-2 (table 1 ) [64] . the 3cl pro is a particular cysteine protease that comprises three domains. the 3cl pro proteolytic activity requires the formation of the cys(−)/his(+) zwitterionic table 1 the comparison of the antiviral drug candidates against sars-cov-2 and other covs grl-0617 is naphthalene-based inhibitor, cc 50 is the cytotoxic concentration of the extracts to cause death to 50% of viable cells in the host; ec 50 is half maximal effective concentration; ic 50 is half maximal inhibitory concentration, na not available; sd is standard deviation, ci95 confidence interval 95% state onto the catalytic dyad [65, 66] . although the catalytic mechanism of cov 3cl pro is not fully understood, extensive studies that connected the structural, computational, and biochemical approaches of different wildtype and mutated 3cl pro decipher important aspects of catalytic efficiency [67] . thus, wang et al. identify that, besides the cysteine from the catalytic dyad, the 3cl pro mers-cov has another cysteine nearby. the role of the second cysteine in catalysis is not fully understood. a conserved motif gscgs forms consecutive three turns when starting the catalysis. two structural characteristics are essential for catalysis. the first characteristic is a partial negative cluster formed by arg-tyr-asp. secondly, there is a conserved water molecule that mediates remote control between the partial negative charged cluster and the cys-his dyad. also, a conserved pair (glu-his) very well conserved in 3cl pro covs forms a stable hydrogen-bond. the glutamine substrate recognizes the glu-his pair by a steric effect. three more residues have an essential role in the glutamine substrate interactions-the his166 and the nearby tyr164 and phe143. the tyrosine residue forms a strong hydrogen bond with his and the phenylalanine residue employs a steric effect to restrain the rotation of his [67] . the multiple sequence alignment of the 3cl pro sequences from different covs allows an overall analysis. thus seven 3cl pro from the following covs were aligned using clustal omega program: feline infectious peritonitis virus fipv (pdb id 5eu8), porcine epidemic diarrhea virus pedv (pdb id 5gwz), human hcov-nl63 (pdb id 5gwy), sars-cov-2 (pdb id 6lu7), sars-cov (pdb id 3iwm), human hcov-hku1 (pdb id 3d23), mers-cov (pdb id 4rsp), and murine coronavirus (strain a59) mhv-a59 (pdb id 6jij). the residues essential for catalysis-the cys145-his41 dyad and the gscgs motif highly conserved in covs-are shown in fig. 2 . the phylogenetic tree (cladogram) of the 3cl pro from different covs sequences analyzed confirms that sars-cov-2 is closer to the sars-cov (fig. 3 ). the multiple sequence alignment of 3cl pro of covs of different origin (using the clustal omega program). the cys145 of sars-cov-2 3cl pro that interact with the compounds 11a and 11b is shown in black background along with the cys145-his41 catalytic dyad highly conserved in 3cl pro from covs [42, 44, 59, 65, 66] ; in green are marked the cluster of ser with high affinity for small molecule inhibitors [44, 55] ; in green it is shown the gscgs motif essential for starting the catalysis; in light blue are marked the glu-his residues critical for substrate binding by means of steric effect; in yellow background is marked the triad arg-tyr-asp that forms a partial neg-ative charge cluster that, by a conserved water molecule, mediates the interaction with cys-his catalytic dyad; in brown are shown the residues involved in the glutamine substrate recognition-the conserved his and the conserved tyr and phe that interacts with by the phenolic hydroxyl group with his and employs a steric effect to restrain the rotation of his, respectively; in bold-underline are marked the glu and ser residues that are demonstrated to be essential in the dimer interactions in sars-cov-2 [60] ; with dot "." are marked the semiconservative replacements; with colon ":" are marked the conservative replacements; with "*" are marked the identities of the residues the rna-dependent rna polymerase (rdrp) (uniprotkb p0dtd1), also known as nsp12, is responsible for the replication and transcription of the viral genome. the rdrp complex is composed of the nsp12, nsp7, and nsp8. the nsp12 is the catalytic subunit and needs the nsp7 and nsp8 (eight subunits of each) to fulfill the replication process. the rdrp is considered an important target for new/already known drugs because of its evolutionary stability compared with the s glycoproteins that are more prone to mutations under selections of host immunity [48, 68] . a recent study investigated the feasibility of the rdrp to be targeted by novel nucleoside inhibitors or small molecules [69] . one previous work has shown that nsp7 and nsp8 of feline cov form a 2:1 heterodimer (pdb id 3ub0). the nsp7 and nsp8 from the sars-cov form an 8:8 hexadecamer (pdb id 6nur) and act as primase during viral replication [70] [71] [72] . remdesivir (gs-5734) (pubchem cid 121304016) (drugbank db14761) is a prodrug of adenosine triphosphate (atp) analog. remdesivir is metabolized into the active form remdesivir-triphosphate (remdesivir-tp) (nci thesaurus gs-441524). the antiviral activity of the remdesivir-tp consists of the competition with atp for rna incorporation thus inhibiting viral rdrp. remdesivir is a valuable therapeutic agent against ebola virus infections [73] . many studies investigated the potential therapeutic use of remdesivir against other viral infections including covid-19 [74] [75] [76] [77] . the therapeutic potency of the remdesivir in covid-19 needs much more well-conducted studies and a thorough analysis of the clinical results [78] . the sars-cov-2 rdrp structure provides important structural details about rdrp -rna interactions (pdb id(s) 6yyt, 7btf, 6m71, and 7bv1) [31, 37, 38] . the structures co-crystallized with remdesivir further advances the understanding of the mechanisms of viral rna replication (pdb id 7bv2) [37] . yin et al. demonstrates that rdrp inhibition depends on the low remdesivir-triphosphate (rtp) concentration and low rtp/atp ratio. only rtp inhibits the rdrp polymerization activity. the structural studies have shown that the asn691, ser682, and asp623 in the rdrp complex explain the binding of the rtp to the ntps site [38] . however, the structural studies should be interpreted according to biochemical studies to further elucidate the incorporation of rtp into the growing rna chain [38, 77, 79, 80] . the recent structural works provide insight into the nucleotide analog inhibitors' ability to hamper sars-cov-2 rna replication. the pre-translocated and post-translocated rdrp complex structures (pdb id(s) 7c2k and 7bzf) show that an efficient inhibitory strategy could be the blocking of the interaction between nsp8 and nsp12 [45] . the biochemical and structural works of peng et al. (pdb id 7bw4) demonstrate that sars-cov-2 rdrp enzymatic activity is lower, about 35% for rna synthesis than its sars-cov counterpart. the reason is the residue substitution in nsp12 and nsp8. also, the thermal stability of the sars-cov-2 rdrp is lower compared to that of sars-cov rdrp the thermal stability of rdrp could advance the understanding of the adaptive evolution of sars-cov-2 in the human host that have a lower body temperature than bats [3, 48, 81 ]. the papain-like protease (pl pro ) is a cysteine protease. pl pro , rna binding domain, and membrane-anchoring domain form the non-structural protein 3 (nsp3). pl pro from different hcovs has been screened for a large panel of chemicals because it is involved in cov replication [82, 83] . the biochemical, structural, and functional studies advance the development of new inhibitors against sars-cov-2 pl pro . the naphthalene-based inhibitor grl-0617 (pubchem cid 44828571) is an effective inhibitor of the sars-cov pl pro (table 1 ) [84] . shin et al. advances the hypothesis that the grl-0617 has two effects against sars-cov-2 pl pro . first, the grl-0617 binding to the amino acid y268 was confirmed by the reduction of the inhibitory effect in mutated pl pro y268t and y268g. second, the grl-0617 promotes the antiviral interferon pathway and reduces viral replication (pdb id 6yva) [50] . the cov nucleocapsid (n) is a highly immunogenic multifunctional protein. the cov n binds the viral rna strand into a long helical structure attached to the membrane (m) protein [85] . there are nine sars-cov-2 x-ray structures of n protein, but only one crystal structure is published until now (pdb id 6m3m) [33] . similar to the n proteins of other covs, the sars-cov-2n protein consists of the n-terminal rna-binding domain (n-ntd), the c-terminal dimerization domain (n-ctd), and a ser/arg rich linker [86] . the ribonucleotide binding site of the cov n-ntd domain was subject to structural studies to develop inhibitors that specifically reduce the rna-binding affinity, thus altering the viral replication. the sars-cov-2n structures available are not co-crystallized with any inhibitor, but the kang et al. structural study demonstrated that the sars-cov-2n employs a unique pattern for ribonucleotide binding, the residues arg89 being one reason for the weak guanosine base recognition (k d for guanosine monophosphate gmp is 8 mmol/l) [33] . the kang et al. claimed that these structural characteristics could be exploited for further investigation of inhibitor compounds, mainly the inhibitor pj34 of the hcov-oc43 n-ntd (pdb id 4kxj) which reduces the n protein's rna-binding affinity by 10% [86] . herein, a further molecular docking of the inhibitor pj34 with the sars-cov-2n-ntd (pdb id 6m3m) was made. pj34 interactions with sars-cov-2n proteins' n-terminal domain (ntd) were compared with those of the hcov-oc43 co-crystallized with the pj34 inhibitor (pdb id 4kxj). the molecular docking of the compound pj34 with the pdb id 6m3m shows the following parameters: ∆g = − 5.66 and ki = 71.34 μm. the interactions involved the residues that bind the pj34 are different from the residues previously described at its counterpart from hcov-oc43 (fig. 4) [86] . the molecular docking offers only a quick view about ligand-protein interaction, deeper theoretical analysis, and experimental validation should be made. sequence comparison of the n proteins' n-terminal domain (ntd) of the hcov-oc43 and sars-cov-2 reveals some particularities that are worth further investigation. the sequence positions 48 to 51 (48-nnta-51) allows easier access of the nucleotides. the threonine t55 and alanine a56 increase the steric clash with ribonucleotide phosphate moiety. the arginine r89 increases the polar features in the nitrogenous base binding site. (fig. 5) [33]. there are many works about the cell entry of covs because, on the one hand, this step is crucial for tissue and cell tropism, and on the other hand, it gains insight into the ability for interspecies transmission. the viral entry is crucial for viral replication. therefore its deep understanding explains the sars-cov-2 pathogenesis and speeds up the finding of a specific treatment. the cell entry is a different multi-step process for each cov. the first step of any viral infection is the presence of at least one suitable host receptor. the spike (s), a heavily glycosylated type i transmembrane glycoprotein, is the same to all hcovs, but the host receptor is different [87] . similar to the sars-cov and human coronavirus nl63 (hcov-nl63), the sars-cov-2 attaches to host receptor ace2 by s glycoprotein [3, 88, 89] . in contrast, the mers-cov uses cellular receptor dipeptidyl peptidase-4 (dpp4), known as cd2 receptor [90] [91] [92] . the s glycoprotein (uniprotkb p59594) is a homotrimer and each monomer contains two distinct functional subunits-s1 and s2-that are subject to proteolytic cleavage by cellular proteases. the host factors involved in priming the s glycoprotein are the type ii transmembrane serine protease 2 (tmprss2) and furin [30, [93] [94] [95] . the tmprss2 is predominantly expressed in the prostate. thus, the demonstration that its expression is androgen-dependent could explain the gender differences in the covid-19 outcome. also, the tmprss2 has a crucial role in other viral infections explaining the pneumotropism of h7n9 and some subtypes of influenza viruses [96] . the furin (e.c. 3.4.21.75) (pdb id(s) 4omc, 4omd, etc.) (uniprotkb p09958) is a ubiquitous endoprotease that belongs to the family of proprotein convertases. the furin mediates the priming of various proteins-some bacterial toxins, h7n1and h5n1 influenza virus haemagglutinin [97, 98] . the proprotein convertases (ppc) cleave at single or paired basic residues within the motif r/k-(x) 0,2,4,6 -r/k (where x means any residue) [93, 99] . there is one cleavage at the s1/s2 boundary and the second one s2′ is within s2 upstream of the putative fusion protein [94, 96] . some researchers claim that there are three cleavage sites (s1, s2, and s2′) [100] . the s1 subunit harbors the receptor binding domain (rbd) which contains the core and receptor-binding motif (rbm) [101] . the s1 subunit binds the peptidase domain of the human ace2. then, the internalization of the virus particle into the endosomes causes conformational changes of the s1 [35, 102] . the proteolysis by the cathepsin ctsl (e.c. 3.4.22.15; uniprotkb p07711) reveals the s2 subunit which mediates the virus-cell membrane fusion [2, [103] [104] [105] . herein, a comparison of the s1/s2 and s2′ cleavage sites of the covs and sars-cov-2 was made [93] . the multiple sequence alignment of the different sars-cov-2 s glycoprotein with their counterparts of covs described by (figs. 6, 7) . the first striking difference between the analyzed s' sars-cov-2 concerns the s1/s2 cleavage site. thus, only some pdb entries (6xr8/6xra, 7c2l, and 6xcn/6xcm) show the ppc or furin cleavage motif rrar↓ [106] . moreover, the furin cleavage motif from the s1/s2 sequence of the sars-cov-2 is more similar to its hcov-oc43 counterpart than to furin cleavage motif of the sars-cov and mers-cov. in contrast, the s2′cleavage site sequence of all sars-cov-2 pdb entries analyzed has the furin cleavage motif similar to its sars-cov counterpart. the phylogenetic tree (cladogram) of the s sequences analyzed shows that the sars-cov-2 s is closer to sars-cov s, but the differences observed between the nine sars-cov-2 s sequences are reflected in the evolutionary relationship (fig. 8) . there are 22 published structures of sars-cov-2 s glycoprotein retrieved from the pdb. among them, seven are s glycoprotein structures (pdd id(s) 6vxx, 6vyb, 6vsb, 6lxt, 6z97, 6xr8, and 6ra) and four structures are co-crystallized with the human ace2 receptor (pdb id(s) 6m0j, 6lzg, 6m17, and 6vw1). out of 32 sars-cov-2 s glycoprotein structures co-crystallized with neutralizing antibodies, eleven has been published (pdb id(s) 6w41, 7bz5, 7c01, 7bwj, 7byr, 6xcm, 6xcn, 6xca, 7c2l, 6xdg, and 6yor) (fig. 9) . cai et al. (pdb id(s) 6xr8 and 6xra) assume a protective role of the s2 post-fusion structure that could elicit the non-neutralizing antibodies to evade the host immune system [40] . also, the authors identify a fusion peptide proximal region (fprp) with a critical role in rearrangements of s protein and membrane fusion, demonstrated by a mutation d614g that lead to more efficient cell entry. the aspartic acid (d614) forms a salt bridge with the lysine (k854) belonging to fprp [40, [107] [108] [109] . barnes et al. the multiple sequence alignment of sars-cov-2 nucleocapsid (n) (pdb id 6m3m) and hcov-oc43 (pdb id 4kxj) sequences (using the clustal omega program). the residues that interact with the compound pj34 are shown in gray background, according to molecular docking results for 6m3m and according to lin et al. findings 4kxj; are considered all types of interactions-van der waals, hydrogen bonds, carbon hydrogen bonds, and hydrophobic interactions; the striking differences between the two n sequences observed by the kang et al. are marked in yellow background; the amp-binding residues are underlined; in red is marked the tyr residue whose mutation y110a leads to a significant decrease of kd for rna binding [33] ; with dot "." are marked the semi-conservative replacements; with colon ":" are marked the conservative replacements; with "*" are marked the identities of the residues study demonstrates that the mutation d614g, a mutation that enhances the sars-cov-2 transmissibility, is unlikely to affect antibodies from recovering covid-19 patients included in the study. [24] . the ace2 (e.c.3.4.17.23; uniprotkb q9byf1) is a homodimer that catalyzes the reaction: angiotensin ii + h 2 o = angiotensin-(1-7) + l-phenylalanine [39, 110] . the human ace2 gene is expressed in a large panel of organs-lungs (type ii pneumocytes), heart, kidney, intestine, cerebral neurons-or immune cells-alveolar monocytes/macrophages. the ace2 expression is up-regulated by interferon and influenza a virus in human nasal epithelia and lung cells [111, 112] . shang et al. recently published the structure of the complex sars-cov-2 rbd/human ace2 (pdb id 6vw1). a comparison with the sars-cov highlighted that sars-cov-2 rbm forms larger binding interface and more contacts with human ace2. the authors focused on the receptor-binding motif (rbm) of the viral rbd (s1 subunit) and the two virus-binding hotspots-31 and 353-of the human ace2 that are stabilized by the viral residues q493 and l455 and n501, respectively. the study successfully determined that the tight sars-cov-2 binding onto the human ace2 due to a four residue motif 482 gveg 485 and to the mutation l486f in the rbm sequence [101] . yan et al. have recently published the cryo-electron microscopy structures of the full-length human ace2-b(0)at1 complex and rbd-ace2-b(0)at1 (pdb id 6m17, 6m1d, 6m18) [39] . the authors have shown that two s glycoprotein trimers simultaneously bind to an ace2 homodimer. moreover, the authors highlighted some residues that could explain the difference between the cell entry of sars-cov-2 and sars-cov. two mutations (val404 to lys317 and leu472 to phe486) are responsible for an enhancement interaction of sars-cov-2-rbd and ace2. in contrast, the mutation arg436 to asn439 weakens the sars-cov-2-rbd and hace2 interaction [39] . the sodium-dependent neutral amino acid transporter b(0)at1 fig. 6 the multiple sequence alignment of spike (s) s1/s2 cleavage site sequences using the clustal omega program. the s1/s2 sequences are bolded and the basic arginine and lysine residues are marked in red [93] (slc6a19) (uniprotkb q695t7) mediates absorption of neutral amino acids across the membrane of the renal and intestinal epithelial cells and mutations in b(0)at1 (such as a69t and r240q) may cause harnup disorder [113] [114] [115] [116] . the b(0)at1 expression onto the small intestinal cells depends on the co-expression of the ace2 and is enhanced by aminopeptidase n (cd13) [117] . the aminopeptidase n (e.c. 3.4.11.2; uniprot uk p15114) mediates other viral infections-transmissible gastroenteritis virus responsible for diarrheal disease in piglets, cytomegalovirus or human cov-229e [118] [119] [120] . lan j. et al. compared the sars-cov and sars-cov-2 rbd bound to hace2 and suggested a convergent evolution between the two coronaviruses. they further suggest that a unique "rrar" cleavage site at the s1/s2 boundary of the sars-cov-2 has a crucial role in rapid inter-human transmission (pdb id 6m0j). the authors emphasize the importance of neutralizing antibodies in the evolution of sars-cov-2 infection. their structural study suggests that the lack of cross-reactivity by m396 and 80r antibodies that neutralizes sars-cov resides in several residue changes in the sars-cov-2 rbd [121] . wang et al. firstly identify that s1 c-terminal domain (sars-cov-2 s-ctd) is the key region that interacts with the human ace2. a comprehensive comparison between sars-cov-2 (pdb id 6lzg) with similar sars-cov, reveals that the former has a higher affinity for the receptor hace2. moreover, the authors advance the hypothesis of the "hotspot" region in s for receptor binding. further experiments with the polyclonal antibodies, confirm the differences between sars-cov-2 and sars-cov' s glycoprotein [36] . the covid-19 is the disease due to the interaction of the pathogen sars-cov-2 with the human host. consequently, all aspects of this interaction draw the picture of fig. 7 the multiple sequence alignment of spike (s) s2′ cleavage site sequences using the clustal omega program. the s1/s2 sequences are bolded and the basic arginine and lysine residues are marked in red [93] ; in yellow background are marked the cysteine residues that form an internal disulfide bond (c840 and c851) and the residues that form a salt bridge that reinforces the previous disulfide bond (k835-d848); in light blue is shown the lysine (k854) that form a salt bridge with the aspartic acid (d614) (not shown) [40, [107] [108] [109] the covid-19. the interaction of sars-cov-2 with the human host is dynamic and has new features compared to other pandemics in human history. first, immunity developed after the sars-cov-2 infection is not yet well understood [6, 122, 123] . then, the mechanisms of tissue damage in covid-19 are not fully understood. the most striking characteristic of covid-19 is the storm of proinflammatory cytokines that ultimately is responsible for the vast majority of the death [124] . the biggest challenge is the best protocol to treat critically ill patients. methylprednisolone has already been reported to reduce the worst outcomes (https ://advai tabio .com/news/covid 19-analy sis/) [125] . the managing of the hyper-inflammatory process in covid-19 proved to be crucial for the evolution of the disease. in sars-cov infections, some pro-inflammatory cytokines (ip-10, il-8, and mcp-1) are elevated. meanwhile, there is no antiviral (ifn) response [126] . in many diseases associated with systemic inflammatory response syndrome, the control of damage-associated molecular patterns (damps) by their counteracting molecules suppressing/inhibiting damps (samps) is crucial [127] . previous studies on the crystal structures of sars-cov s glycoprotein mutants neutralized by 80r-specific antibodies have been considered a hope for the immunotherapeutic fig. 8 the phylogenetic tree (cladogram) of the covs spike (s) sequences of covs with different origin. feline infectious peritonitis virus (fipv), porcine epidemic diarrhea virus (pedv), hcov-nl63, sars-cov-2, sars-cov, hcov-hku1, and coronavirus (strain a59) mhv-a59; performed by clustal omega program fig. 9 the crystal structures co-crystallized with neutralizing antibodies. the epitopes of the sars-cov-2 spike are ntd (n terminal domain), rbd (receptor binding domain, quaternary epitopes, and ectodomain; there are indicated the pdb entries and in parenthesis the neutralizing antibody; * the most potent neutralizing antibodies from convalescent patients according to liu et al. work [16] strategy for the future outbreak of sars (pdb id 2ghv) [128] . there are 32 structures of sars-cov-2 about the immune system published in peer-reviewed papers (pdb id9s) 6w41, 7bz5, 7c01, 7bwj, 7byr, 6xcm, 6xcn, 7c2l, 6xdg, 6yor, 7cah, 6xey, 6zcz, 6zdh, 6zdg , 6zer, and 6zfo). more than eight months after the onset of the pandemic, health care professionals and researchers are able to analyze the clinical evolution of many patients. the host immune response to sars-cov-2 infection is perhaps the most controversial issue. most of the last pdb entries are about the sars-cov-2 neutralizing antibodies. the patients recovered with covid-19 rapidly advanced studies with plasma samples from convalescents. numerous research teams already identified numerous potent neutralizing antibodies against multiple epitopes on the sars-cov-2 spike (fig. 9 ). yuan et al. published the cryo-em crystal structure of the sars-cov-2 s1 with the cr3022 antibody as an effort to understand the antigenicity of the sars-cov-2 (pdb id 6w41). their results show that cr3022 fab binds to sars-cov rbd with higher affinity ( table 2 ). in the selection process of the selection of the neutralizing antibodies against sars-cov-2, ju et al. provide evidence that binding affinity does not predict ace2 competing capacity [20] . the cr3022 antibodies fail to neutralize sars-cov-2 in vitro. the cr3022 epitope does not overlap with the ace2 binding site in sars -cov-2 s-rbd. however, sars-cov and sars-cov-2 have a conserved, but cryptic epitope that could be worth considering for a vaccine [14] . moreover, the huo et al. suggest that cr3022 binding facilitates conversion to the fusion-incompetent post-fusion state (pdb id 6yor) [15] . these findings offer promising perspectives on covid-19 therapy with neutralizing antibodies. the challenge of the therapy with neutralizing antibodies is to overcome the virus mutants. the therapeutic cocktail of neutralizing antibodies against sars-cov-2 is an efficient approach. the hansen et al. team discovered a pair of non-competitive neutralizing antibodies that simultaneously bind to s-rbd sars-cov-2 (namely regn10933 and regn10987) (pdb id 6xdg) [23] . the mapping of the sars-cov-2 epitopes targeted by potent neutralizing mabs demonstrated the diversity of the neutralizing antibodies directed against the s-rbd, s-ntd, and the epitopes that do not map to the s-rbd or s-ntd. even though the authors liu et al. do not establish the role of s-ntd in [16] blocking sars-cov-2 infection, these findings are crucial for vaccine development [16] . the studies about the host immune system are, on the one hand, about the production of neutralizing antibodies, and on the other hand, about the virus's ability to hamper defense immunity. the covs have an impressive ability to suppress the ifn response. an antagonist of the ifn response is the nsp15 [129] . the viral nsp3 has an active role in suppressing innate immunity by blocking the interferon regulatory factor (irf3) (gene id 3661) and altering the nf-kappab signaling, which controls the expression of some inflammatory cytokine genes [130, 131] . the nsp3 has a crucial role in damaging the host's first line of defense against sars-cov-2. nsp3 probably has a role in producing the unusual inflammation described in patients with severe covid-19 [132] . the sars-cov-2 nsp3 has no crystal structures published until now. the nsp3 (~ 200 kda) is a large multidomain protein (e.c. 3.4.19.12) (uniprotuk p0dtd1) responsible for the cleavage of the replicase polyprotein 1ab at the n-terminus [133] . the papain-like protease (pl pro ) domain of the viral nsp3 exhibits deubiquitinating activity [134] . a recent study has shown that deubiquitinating/deis-gylating (dub/deisg) nsp3 deficient mutants (h1652r, v1691k, and v1691r) result in attenuating the mers virus [135] . a recent study identified specific mutations in nsp3 corroborated with nsp2 that suggest potential mechanisms that explain higher contagiousness of sars-cov-2 compared to sars-cov [136] . thoms the organization of the non-structural (nsp) proteins in cov is not fully understood. the cov has two overlapping open reading frames (orfs) orf 1a and orf 1b, and by 1 ribosomal frameshifting-a translational regulation mechanism described in retroviruses-there is a regulation of relative ratio of structural to enzymatic proteins [137, 138] . the cov synthesizes two polyproteins that are further cleaved by the viral proteases. the replicase polyprotein 1ab (uni-protkb p0dtd1) also contains the proteases responsible for the cleavage [139] . the viral protease 3cl pro is extensively studied for the designing of new drugs to control the spread of human and zoonotic covs [140] . the nsp9 is an rna-replicase which binds the viral rna and has a regulatory effect in viral replication [141] . three unpublished crystal structures of nsp9 from sars-cov-2 were retrieved from the pdb (pdb id(s) 6w9q, 6w4b, and 6wxd) (litter et al., and tan et al.). previous studies focused on nsp9 from other covs, demonstrated that the residues from the dimer interface greatly influence their nucleic acid binding affinity [142] . according to zeng et al. findings , the dimerization greatly depends on the n-finger and the two glycine residues from a conserved region gxxxg [142] . (fig. 10) . the nsp15 is uridylate-specific endoribonulcease (nen-dou) (e.c. 3.1.-.-) highly conserved among vertebrates coronaviruses. the nsp15 plays an important role in the life cycle of covs [143, 144] . the recent sars-cov-2 nsp15 structures show that it is a hexamer and the catalytic site belong to the c-terminal domain (pdb id(s) 6w01 and 6vww). the study concludes that the sars-cov-2 nsp15 differs by mers-cov nsp15 by residues that coordinate the manganese ion [145] . the sars-cov-2 emerged at the late of the year 2019. the epidemiology of covid-19 pandemic is beyond the subject of this paper. however, we can learn how other epidemics have been handled throughout history. there are many historical records, but an episode about the plaque epidemic, brilliantly described by irving stone in the agony and the ecstasy, could be a school-case. in the florence transformed in morgue and deserted by people, michelangelo-michelangelo di lodovico buonarroti simoni (1475-1564)-chose to stay in the city and take care of his ill younger brother until the last moment and doing the best for a decent funeral. strikingly, in the xvi century, michelangelo applied the recommendation for covid-19 -hygiene and social distance. he thoroughly brushed himself into a bathtub with hot water and sent away his sister-in-law and his nephews. even he was asymptomatic, in our days terms, he refused granacci's wine. unlike the plague described by irving stone, covid-19 pandemic is considerably longer and for the time being there is no hope for its end soon. until the emergence of sars-cov in 2003 and mers in 2012, the covs infections do not pay much attention. now, facing the sars-cov-2, the spectrum of re-emergence of the sars-cov-2 or the animal-human transmission of a new cov strain is more real than ever. so, the understanding of all aspects of the sars-cov-2 is of great interest. the rapid involvement of the scientific communities greatly advances the elucidation of some aspects of covid-19 pandemic to be prepared in the event of a new contagious and fatal cov disease. the structural studies focused on designing specific inhibitors to gain knowledge about the covid-19 treatment, and further studies involving biophysical methods will speed up the sars-cov-2 cure [146, 147] . likewise, many research teams advance their studies on understanding the most subtle details of the virus-host cell interaction, the emergence of the neutralizing antibodies, or the designing of new inhibitors. the virus and host factors are both relevant for the initiation and evolution of a viral infection. the sars-cov-2 shares epidemiological patterns with sars-cov and mers, but there are some structural differences. thus, even though sars-cov and sars-cov-2 use the same host receptor, sars-cov-2 binds to the ace2 with higher affinity [101] . two main features are associated with the worst outcome in sars-cov-2 infection-co-morbidities and elderly. the traits of immune system at different ages could be a strong explanation about the accompanying symptoms of various viruses when they present with an underlying disease. for example, episodes of wheezing in young children have been observed in respiratory syncytial virus (rsv) infections. in contrast, in adults and older children, the episodes of wheezing have been observed in human rhinoviruses (hrv) and hcovs (hcov-nl63 and hcov-hku1) infections [148] . recent studies identify ace-2 maturation stages, and the hypertension treatment with ace inhibitors could be an explanation for mild disease in children [149, 150] . thus, structural studies about spike -ace-2 and ace2-neutralizing antibodies are of great interest. the crystal structures of sars-cov-2 reveal important details about the viral infection. first, the full-length human ace2 structure reveals that viral cell entry involves the simultaneous binding of two s glycoproteins to ace2. also, the use of b(0)at1 in stabilization of the full-length human ace2 structure advances the hypothesis that b(0)at1 may play a role in enteric infections of some covs [39] . certain virus components trigger the immune system and limit the interspecies transmission of different covs. the structural studies greatly advance the selection of the best immunogens for sars-cov-2 prophylaxis. a comparison of the hcov suggests that the spike's sars-cov-2-ctd domain could be a valid immunogen for a future vaccine [36] . the viral immune evasion depends on more than one factor and structural work of many research teams greatly advances the understanding of immune response. the sars-cov-2 nsp1 is one of the major immune factors that impedes the host immune clearance. a recent structural work demonstrated that sars-cov-2 nsp1 blocks rig-idependent innate immune response [46] . the hypothesis of the bats being at the origin of the sars-cov-2 is, until now, not either demonstrated or rejected. however, it is about 96% identity between the genome of sars-cov-2 and the genome of batcov ratg13 [3] . even the genome identity between sars-cov-2 and sars-cov is about 80%-recent studies claim fig. 10 the multiple sequence alignment of nsp9 sequences using the clustal omega program. the n-finger (in red) and gxxxg motif (grey background) are important for dimerization. the conserved residues of n-finger are underlined a close evolutionary relationship between the two viruses [3, 101] . the results of the structural studies could bring valuable perspective of the host specificity. thus, ziegler et al. work about the human and mouse ace2 demonstrated that different to mouse ace2, the ace2 is an interferonstimulated gene (isg) in human primary upper airway epithelial basal cells [112] . these results are useful not only for using an appropriate animal/cellular model in experimental research but for screening for the potential animal host of the sars-cov-2, which could elucidate the chain of the virus passing from the animal host to human. also, the differences of an essential step in covs replication, the priming of s glycoprotein by host cell proteases, could be exploited to elucidate the zoonotic potential of the sars-cov-2. the findings of hoffmann et al. highlighted that the s1/s2 cleavage site sequence of sars-cov-2 s glycoprotein harbors several arginine residues (multibasic) on the contrary to its closely related the bat cov ratg13 [94] . current theory holds that most of the deaths are due to an excessive level of pro-inflammatory cytokines in circulation. the humoral immune response is most studied and structural studies add important results that further explain biochemical findings [121] . the most optimistic outcome is to obtain a vaccine that elicits strong and long-term immunity. until then, understanding the evolutions of phenotypic changes of the sars-cov-2 and elucidation of epidemiological aspects of the covid-19 pandemic greatly depends on the analysis of all available research results. there is a huge effort in deciphering the sars-cov-2 pandemic, leading to many studies conducted around the world. the scientific work relies on observation, experiments, and structural studies to provide a deep view of sars-cov-2. the current structural analysis of the sars-cov-2 is mainly focused on three major lines-finding new hydrolase inhibitors, the virus-host cell invasion, and the virus-neutralizing antibody interaction. there is an intense work on structural studies of sars-cov-2 proteins, mainly for s glycoprotein that is crucial for the pathogenesis of all covs, but totum est majus sua parte (the whole is bigger than the part). the convalescent patients' b cells demonstrated that not a single spike epitope elicits the neutralizing antibodies. the host-virus interaction is a dynamic process, and the host 's defense mechanisms are crucial not only to cure the covid-19 infection but to prevent further re-infection. in this sense, structural studies involving neutralizing antibodies gain a perspective in deciphering the immunity of cured persons. also, the works about the viral nsp(s) aid the understanding of the viral immune evasion. despite the limits of x-ray/cryo-em crystal structure studies -mainly the lack of information about the cell-mediated immunity and time-consuming experiments for clinical validation of the new inhibitors -the thorough analysis of the pdb entries is a powerful tool for further understanding of the human covs infections. emergence of novel coronavirus and covid-19: whether to stay or die out? the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak: an update on the status a pneumonia outbreak associated with a new coronavirus of probable bat origin keep up with the latest coronavirus research origin and evolution of pathogenic coronaviruses composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 evolutionary history, potential intermediate animal host, 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jurisdictional claims in published maps and institutional affiliations acknowledgements many thanks to the editorial of the protein journal for the flexible approach during the covid-19 pandemic. i would like to thank professor lawrence berliner and the referees for their comments that much improved the quality of the manuscript. many thanks to tudor constantin badea for the fruitful discussions during the mobility project. the author declares no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-291436-cu5o8ipw authors: martínez-hernández, fernando; isaak-delgado, ana belem; alfonso-toledo, jorge alberto; muñoz-garcía, claudia irais; villalobos, guiehdani; aréchiga-ceballos, nidia; rendón-franco, emilio title: assessing the sars-cov-2 threat to wildlife: potential risk to a broad range of mammals date: 2020-10-05 journal: perspect ecol conserv doi: 10.1016/j.pecon.2020.09.008 sha: doc_id: 291436 cord_uid: cu5o8ipw severe acute respiratory syndrome coronavirus 2 (sars-cov-2) can infect animals, however, the whole range of potential hosts is still unknown. this work makes an assessment of wildlife susceptibility to sars-cov-2 by analyzing the similarities of angiotensin converting enzyme 2 (ace2) and transmembrane protease, serine 2 (tmprss2) —both recognized as receptors and protease for coronavirus spike protein— and the genetic variation of the viral protein spike in the recognition sites. the sequences from different mammals, birds, reptiles, and amphibians, and the sequence from sars-cov-2 s protein were obtained from the genbank. comparisons of aligned sequences were made by selecting amino acids residues of ace2, tmprss2 and s protein; phylogenetic trees were reconstructed using the same sequences. the species susceptibility was ranked by substituting the values of amino acid residues for both proteins. our results ranked primates at the top, but surprisingly, just below are carnivores, cetaceans and wild rodents, showing a relatively high potential risk, as opposed to lab rodents that are typically mammals at lower risk. most of the sequences from birds, reptiles and amphibians occupied the lowest ranges in the analyses. models and phylogenetic trees outputs showed the species that are more prone to getting infected with sars-cov-2. interestingly, during this short pandemic period, a high haplotypic variation was observed in the rbd of the viral s protein, suggesting new risks for other hosts. our findings are consistent with other published results reporting laboratory and natural infections in different species. finally, urgent measures of wildlife monitoring are needed regarding sars-cov-2, as well as measures for avoiding or limiting human contact with wildlife, and precautionary measures to protect wildlife workers and researchers; monitoring disposal of waste and sewage than can potentially affect the environment, and designing protocols for dealing with the outbreak. severe acute respiratory syndrome coronavirus 2(sars-cov-2) is causing the biggest pandemic 52 of this century, and could potentially infect between 30 to 40% of the world´s populations (de 53 soto et al., 2020) . due to its high transmission rate and mortality, researches around the world 54 are trying to get some insight about its origin through the analysis of related-virus genes 55 sequences in humans and animals (lu r et al., 2020) , and looking for angiotensin-converting 56 enzyme 2 (ace2) sequence similarities in animals, the putative receptor of this virus (andersen 57 probabilistic models were constructed based on the amino acid residue sequence for ace2 and 186 tmprss2. two models were created for the ace2 receptor and the third model includes the 187 tmprss2. the significant residue amino acids for ace2 that interact with the viral s protein, so the assignation of the pcsv was as follow: four amino acid properties were used (size, 205 hydrophobicity, charge and polarity), each one with three categories: 1) size (tiny, small and 206 large); 2) hydrophobicity (hydrophilic, moderate, hydrophobic); 3) charge (positive, neutral, 207 negative); and 4) polarity (polar, amphipathic, non-polar). for polarity, some amino acids are 208 reported in two categories (i.e., amphipathic and polar or non-polar), for these cases the higher 209 value was assigned. when human and animal showed the same amino acid residue the pcsv was 210 assigned as 1. we assumed that an amino acid residue substitution by a different amino acid 211 residue even sharing all the physical properties will give a 0.9 pcsv tops. values for each physical 212 property were assigned as follows: for each similar physical property a 0.225 was assigned; if the 213 property was not the same but in the closest category 0.112 was assigned; and if the property 214 was neither of those it was assigned a 0. the pcsv for each amino acid residue position was the 215 sum of the values of the four categories. the minimum assigned value was 0.225 and the 216 maximum was 0.9 (s3 table) . to find the total pcsv (tpcsv) for each animal species, the product 217 of all pcsv was calculated (s4 table) . protein sequences of the three genes described above were subjected to multiple alignments in 8 226 different data matrices: 1) ace2 gene matrix; 2) tmprss2 gene matrix; 3) ace2 pbd sites; 4) 227 tmprss2 proteolytic site; 5) gene matrix ace2 and tmprss2; 6) matrix genes ace2 pbd site 228 and tmprss2 proteolytic site; 7) viral protein s pbd site; and 8) viral protein s proteolytic site 229 j o u r n a l p r e -p r o o f 11 by tmprss2. all alignments were established using the clustal w and muscle algorithms 230 included in mega software version 7.0.26 (tamura et al., 2011) . phylogenetic reconstructions 231 were performed using the eight matrices by bayesian approximations with mr. bayes software 232 version 3.2 (huelsenbeck et al., 2001) . the analysis was performed for 2 million generations with 233 sampling trees every 100 generations. trees with scores lower than those at the stationary phase 234 (burn-in) were discarded, and the trees that reached the stationary phase were collected and 235 used to build majority consensus trees. 236 237 the three models were consistent among them for most of the species, with some variation in the 240 rank of certain particular species (fig 1) . model 1 gave the most different results with respect to 241 the others. most of the primates ranked in the first places, but interestingly, after that, carnivores, 242 cetaceans, and wild rodents showed a relatively high potential risk. lab rat and mouse ranked in 243 the lowest places along with bats. 244 245 reptiles, amphibians and birds ranked below mammals, with some exception. birds such as 246 empidonax traillii (models 2 and 3), and nothoprocta perdicaria (model 1) classified to a level 247 similar to that of lab rodents and bats. reptiles with the highest rank were chelonia mydas 248 (model 1), and protobothrops mucrosquamatus (models 2 and 3) even around some mammals 249 such as mus musculus and myotis lucifugus but below rattus norvegicus. for models 2 and 3 most 250 of the reptiles were at the bottom of the rank. amphibians were at similar rank than reptiles in 251 model 1, but for models 2 and 3 were around reptiles. in particular, the highest rank was xenopus 252 tropicalis (model 2), and rhinatrema bivittatum (model 3; s5 table) . the analyses with the surface glycoprotein s showed little genetic variation, so the trees were 271 observed with polytomies (data not show). however, both domains showed genetic variation for 272 the rbd region, 12 haplotypes identified (fig 4a) , with h1 being the most frequent (98.5%), and 273 for the tmprss2 cut region 6 haplotypes were identified (fig 4b) , being the most frequent h1 274 (99.6%). the other haplotypes presented unique frequencies. actually, experimental evidence found between one species of nwp and two of owp infected 312 with sars-cov-2, showed that the former presents higher frequency of viremia than the latter, 313 particularly 100% of six callithrix jacchus individuals versus 44% of eighteen macaca spp; but 314 they also found that viral loads and pathological lesions at microscopic level were greater in 315 macaca spp. than in c. jacchus (lu s., et al., 2020) ; also proved that nwp, at least the species c. (fip), since feral cats were found prior to and during the outbreak and diagnostic test cross-344 reactivity with fip (bossart and schwartz, 1990). however, the lack of molecular evidence in that 345 case and the existence of a short coronavirus sequence from another infected harbor seal left 346 more questions than answers, because there was no case-information but just the submitted 347 genbank sequence (schütze, 2016) . interestingly, in our phylogenetic trees, these marine 348 mammalian species share the same clade with the feline species, which also supports this 349 hypothesis. people that inhabit or travel through the seacoast, or wastewater released by urban other in silico studies using the docking approach showed that some carnivores, as panthera 359 pardus, p. tigris, puma concolor, lyxn pardinus, and crocuta crocuta, have also less free energy, 360 (which means more stable union, because less energy is available to make a structural change) 361 than humans, which means more affinity for the sars-cov-2 s-protein . the another element of dissent, other than the host, is the virus per se. for example, the high 397 variation observed in the sars-cov-2 rbd region (fig 4a) , during the five months that the 398 pandemic has lasted, made feasible the generation of new variants. these variations are non-399 synonymous mutations, which suggests a low pressure of natural selection that, in turn, can 400 impact over an elevated sequence diversity in this genomic region and, consequently, may 401 increase its hosts-species affinity. and in the case of the viral tmprss2 recognition region, a less 402 genetic variation was detected, since it is represented by 6 different haplotypes that have been 403 observed with numerous non-synonymous substitutions (fig 4b) . however, for the viral 404 the sars-cov-2 impact on new wild hosts is uncertain. to date, this virus is considered by many 436 authors as a deadly pathogen for humans, but with varying degrees of severity . 437 however, its effects on animal health is almost unknown. in humans, the severity of the disease is 438 once the virus enters in wild environments, social cohesion in animals could be a decisive factor 447 for an outbreak. then, taking individuals proximity into consideration, primate order are at the 448 greatest risk, not only because its receptor and protease homology, as we see in our models and 449 phylogenetic trees, but also for its social organization and troops´ fusion-fission dynamic (sueur 450 et al., 2011) . however, high social cohesion also exists amongst some carnivora and many 451 cetacean species, even at interspecies level (brakes, 2017; gittleman, 1989) . in this sense, we 452 must emphasize that some scientist claim that airborne transmission is feasible through small 453 droplets exhaled by humans, being able to move freely tens of meters from its origin (morawska 454 and cao, 2020). the risk increases when social wild species are gathering in great numbers at 455 places where people go, like national parks, and sometimes even face crowded situations (bath 456 and enck, 2003) . 457 in order to prevent person to person disease spread, during this sars-cov-2 global health crisis 459 a quarantine in most of the world was imposed (wilder-smith and freedman, 2020). the human 460 containment has resulted in the appearance of wild animals in some cities around world, and 461 their presence indicates that humans and wildlife are closer than ever (lewis, 2020) . this 462 proximity exists even in natural areas, where ecotourism has become one of the major economic 463 activities, owing to wildlife observations . although at the moment 464 ecotourism has collapsed by sars-cov-2 quarantine, it will be reactivated in the near future 465 aerosol exposure, and environmental exposure), and some mitigation strategies (minimize, 507 assess, and protect). at the same time this document provides some additional resource for 508 specific groups such as great apes, bats, felid, and small carnivores (oie, 2020). we encourage to 509 review this document as a first step in the strategy to limit sars-cov-2 spread among wildlife. 510 however, we should keep in mind that transmission could occur, and we still need protocols for 511 monitoring and mitigation strategies once sars-cov-2 is detected. sars-594 associated coronavirus transmitted from human to pig broad host range of sars-cov-2 600 predicted by comparative and structural analysis of ace2 in vertebrates the pathophysiology of virulence of the covid-19 las áreas protegidas de américa latina: situación actual y perspectivas 610 para el futuro ferret models of viral pathogenesis. virology 479-480 the significant but understudied impact of pathogen 616 transmission from humans to animals zoönotisch risico van het sars-cov2 virus (covid-19) bij gezelschapsdieren: 624 infectie van dier naar mens en van mens naar dier protecting 630 healthcare workers from sars-cov-2 infection: practical indications 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discovery of a 899 novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal 900 coronavirus in gammacoronavirus silico analysis of intermediate hosts and susceptible animals of sars-cov-2. chemrxiv characteristics of pediatric sars-cov-2 909 infection and potential evidence for persistent fecal viral shedding the deadly 913 coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in 87. yu, p., qi, f., xu, y., li, f., liu, p., liu, j., bao, l., deng, w., gao, h., xiang, z., xiao, c., lv, q., 917gong, s., liu, j., song, z., qu, y., xue, j., wei, q., liu, m., wang, g., wang, s., yu, h., liu, x., 918 huang, b., wang, w., zhao, l., wang, h., ye, f., zhou, w., zhen, w., han, j., wu, g., jin, q., 919wang, j., tan, w., qin, c., 2020. age-related rhesus macaque models of covid-19. animal. key: cord-303297-fiievwy7 authors: oberemok, volodymyr v.; laikova, kateryna v.; yurchenko, kseniya a.; fomochkina, irina i.; kubyshkin, anatolii v. title: sars-cov-2 will continue to circulate in the human population: an opinion from the point of view of the virus-host relationship date: 2020-04-30 journal: inflamm res doi: 10.1007/s00011-020-01352-y sha: doc_id: 303297 cord_uid: fiievwy7 at the population level, the virus-host relationship is not set up to end with the complete elimination of either or both. pathogen-resistant individuals will always remain in the host population. in turn, the virus can never completely eliminate the host population, because evolutionarily such an event is a dead end for the virus as an obligate intracellular parasite. a certain existential balance exists in the virus-host relationship. against this backdrop, viral epidemics and pandemics only become manifest and egregious to human beings when tens and hundreds of thousands of people die and the question emerges what caused the high mortality peaks on the death chart. the answer seems clear; the emerging strain of the virus is new to the host population, and new mutations of the virus and natural selection will lead to a survival of only genetically resistant individuals in a host population. the dangers inherent to a novel virus are due to new features generally inthe molecular structure of proteins, which enable the virus to infect the cells of the host organism more intensively, dramatically challenging host immunity, and thus be transmitted more readily in the host population. in this article, we will concentrate on the facts currently available about severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which has caused covid-19 (coronavirus disease 2019) pandemic and try to predict its development and consequences based on the virus-host relationship. in fact, only two scenarios will occur simultaneously in the very near future: people who are genetically resistant to the virus will get sick, recover, and develop immunity, while people who are sensitive to the virus will need drugs and vaccines, which will have to be researched and developed if they are to recover. if the pandemic does not stop, in a few decades it is anticipated that sars-cov-2 will become as safe as the four non-severe acute respiratory syndrome human coronaviruses (hcov-nl63, hcov-hku1, hcov-oc43, and hcov-229e) currently circulating but causing low mortality in the human population. genomic alignments suggest that the covid-19 virus (sars-cov-2) from the genus betacoronavirus may be the result of recombination of genetic material from two different viruses, one similar to the chinese horseshoe bat virus [1] and the other closer to the pangolin virus (two divergent viruses could have infected the same organism simultaneously). to date, aside from bat coronavirus ratg13 (96.2% similarity), the pangolin-cov (91.02% similarity) is the coronavirus most closely related to sars-cov-2 [2] . even though the exact origin of the virus remains unclear, it is possible to infect a chinese horseshoe bat or a pangolin with the sars-cov-2 and evaluate the success of the infection to determine the origin of the new virus responsible for the pandemic: did it come from animals or from a laboratory? while the absence of disease symptoms in these animals may be due to the effectiveness of their immune systems, the intensity of viral proliferation can be easily detected using the real-time pcr technique. however, the genie has already escaped from the bottle, and now we need to deal with the result, not the cause. moreover, a 'hand-made' virus is likely to be effective against both animals and humans. investigations suggest that sars-cov-2 may bind human ace2 (angiotensin-converting enzyme 2) receptor with high responsible editor: john di battista. * anatolii v. kubyshkin kubyshkin_av@mail.ru 1 v.i. vernadsky crimean federal university, simferopol, russia affinity but computational analyses predict that the interaction is not ideal and that the rbd (receptor-binding domain) of the spike protein of sars-cov-2 is optimized for binding to human ace2 receptor with an efficient solution different from those previously predicted [3, 4] . this evidence is strongly arguing against culture-based scenarios. thus, the high-affinity binding of the sars-cov-2 spike protein to human ace2 receptor is most likely the result of natural selection on a human or human-like ace2 receptor that permits another optimal binding solution to arise. however, more scientific facts are needed to answer the question about the origin of the virus. obtaining related viral sequences from animal sources would be the most definitive way of revealing viral origins [1] . the possibility of the existence of a potential intermediate host of sars-cov-2, a missing link between a bat and human or a pangolin and human can also shed light on the current pandemic. to date, however, there is no credible evidence to support the claim that sars-cov-2 originated from a laboratory-engineered coronavirus [5] . in early december 2019, the first cases of pneumonia of unknown origin were identified in wuhan, the capital city of hubei province in china [6] . the pathogen has since been identified as a novel enveloped positive-sense, single-stranded rna betacoronavirus [7] . this virus, which has a phylogenetic similarity to sars-cov [8] , has been given the name severe acute respiratory syndrome coronavirus 2 (sars-cov-2; also, covid-19 virus). to date, among the total number of patients with covid-19 globally, the case-fatality rate (ratio between the dead and total number of infected) ranges from 5.1 to 5.2% and is constantly increasing. this is ten times more than from the usual seasonal flu. also, sars-cov-2 is supposed to be more contagious and environmentally stable than seasonal flu viruses, since seasonal flu usually ends by april in northern hemisphere. in addition, it seems that the virus is also more likely to affect the heart than any other similar viruses, so although pneumonia is often the main cause of death, cardiologists and infectionists, for example in russia, are seeing infected patients whose worst symptoms are not respiratory, but cardiac and many people infected with covid-19 are dying from heart attacks, as a possible complication of sars-cov-2 infection. high expression of the ace2 receptor, via which covid-19 virus enters cells using its spike glycoprotein, was identified in type ii alveolar cells (at2) of the lung [9] [10] [11] , esophagus upper and stratified epithelial cells, absorptive enterocytes from the ileum and colon [11] , cholangiocytes [12] , myocardial cells, kidney proximal tubule cells, and bladder urothelial cells. these findings indicate that those organs with high ace2 expressing cells should be considered as a potential high risk for sars-cov-2 infection [9] . investigations suggest that the spike glycoprotein of sars-cov-2 is the result of the combination of a bat sars-cov with an unknown beta-cov [13] , probably a coronavirus of pangolin origin. the s1 subunit containing the receptor-binding domain region of the spike protein of pangolin-cov is much more closely related to that of sars-cov-2 than to that of the bat coronavirus ratg13. moreover, while five key amino acid residues involved in the interaction with human angiotensin-converting enzyme 2 (ace2), mainly expressed in a small subset of cells in the lung called type 2 alveolar cells [8] , are completely consistent between pangolin cov and sars-cov-2, four amino acid mutations are present in ratg13 [2] . the spike protein of sars-cov-2 contains a 3-d structure in the receptorbinding domain region to maintain the van der waals forces [14] . the 394 glutamine residue in the receptor-binding domain region of sars-cov-2 is recognized by the critical lysine 31 residue on the human angiotensin-converting enzyme 2 as a key receptor for virus penetration. it is also believed that the single n501t mutation in the sars-cov-2 spike protein (corresponding to the s487t mutation in sars-cov) may have significantly enhanced its binding affinity for human ace2 and subsequent penetration [3] . for the human population, this is the third significant coronavirus infection to occur in the twenty-first century, following severe acute respiratory syndrome (sars) in 2002-2003 and middle east respiratory syndrome (mers) in 2011. both sars-cov and mers-cov were newly identified coronaviruses of zoonotic origin in the genus betacoronavirus, with a much lower incidence than sars-cov-2: 8,096 cases for sars since 2002 (mortality rate ~ 10%) and 2,494 cases for mers since 2012 (mortality rate ~ 35%). oddly enough, sars-cov-2 is not very similar to the genomes of sars-cov (about 79%) or mers-cov (about 50%) [7, 15] . despite the initial reports stating that most of the laboratory-confirmed infected patients (27 of 41 cases) had links to the wuhan seafood market where different animals, including bats, snakes, birds, pangolins, and other small mammals are normally traded within the market [6] , it is now obvious that the newly identified coronavirus sars-cov-2 is transmitted with enormous efficacy from human to human via respiratory droplets or close contact. the highly contagious nature of sars-cov-2 is probably due in large part to the virus spreading via asymptomatic infected individuals [16] , which is exacerbated by an unusually long incubation period of up to 11-14 days. based on data from hospitalized patients, the majority of covid-19 cases (about 80%) presented as asymptomatic or with mild symptoms while the remainder were severe or critical [6, 17] . most covid-19 patients developed lymphopenia and pneumonia, with characteristic pulmonary ground-glass opacity changes seen on chest computed tomography [6, 17, 18] . sars-cov-2 is believed to dampen anti-viral interferon responses resulting in uncontrolled viral replication (for sars-cov and mers-cov, the response to viral infection by type i ifn is suppressed). the influx of neutrophils and monocytes/macrophages results in hyperproduction of pro-inflammatory cytokines [19] . the study of 41 hospitalized patients revealed high-levels of pro-inflammatory cytokines, including il-2, il-7, g-csf, ip-10, mcp-1, mip-1a, and tnfα, in the most severe cases of covid-19 [6] . this so-called 'cytokine storm' can initiate viral sepsis and inflammatory-induced lung injury, which lead to other complications including pneumonitis, acute respiratory distress syndrome (ards), respiratory failure, shock, organ failure, and potentially death [19] . individuals particularly susceptible to covid-19 are elderly people with underlying diseases, including diabetes, hypertension, and cardiovascular disease [6] . since sars-cov-2 mutates constantly and more frequently than other rna viruses (coronaviruses possess the longest genomes of all known rna viruses, so more errors are made when they are copied; also viral rnadependent rna polymerases do not have a proofreading nuclease activity), it is likely that strains of the virus will appear whose host 'preference' will change, for instance, by reducing the average age of patients with a severe course of the disease. as global infection has progressed, we are now more frequently seeing young patients dying from covid-19. tang et al. found single nucleotide polymorphisms (snps) in sars-cov-2 at location 8,782 (orf1ab polyprotein: t8517c, synonymous) and 28,144 (orf8 protein: c251t, s84l, non-synonymous). among the 103 sars-cov-2 virus strains in genbank, 101 of them exhibited complete linkage between the two snps: 72 strains (~ 71%) exhibited a 'ct' haplotype (defined as l type because t28144 is in the codon of leucine) and 29 strains (~ 29%) exhibited a 'tc' haplotype (defined as s type because c28144 is in the codon of serine) at these two sites. although the l type (~ 71%) was more prevalent than the s type (~ 29%) in the sars-cov-2 viruses examined, the s type is actually the ancestral version of sars-cov-2 (nucleotides of the s type at sites 8782 and 28,144 were identical to the orthologous sites in the most closely related viruses). the ratio observed, 71:29, suggests that the l type has a higher transmission rate than the s type or was more prevalent at the start of the pandemic. interestingly, while the l type was more prevalent in the early stages of the outbreak in wuhan (96.3%l:3.7%s), the frequency of the l type decreased after early january 2020, possibly because human intervention efforts may have caused severe selective pressure against the l type [20] . what about reinfection? earlier, a pivotal role for virusspecific memory t-cells in broad and long-term protection against sars-cov infection was elucidated [21, 22] . indeed, the crucial protective role of t-cell immune responses in coronavirus infections has been clearly documented in several animal models, e.g., feline infectious peritonitis virus (fipv), mouse hepatitis virus (mhv), and avian infectious bronchitis virus (ibv) [23] [24] [25] . moreover, reinfection did not occur in sars-cov-2 infected rhesus macaques with the same strain [26] . what about reinfection with other possible strains of people who recovered from initial infection with one strain? at present, it remains unclear whether convalescing patients are at risk for relapse or reinfection. it is also worth noting the theory that coronavirus infections might play a part in multiple sclerosis. it is known that mouse hepatitis virus, a murine coronavirus, is a close relative of common human coronavirus hcov-oc43, which causes a multiple sclerosis-like demyelinating disease in the central nervous system of rodents [27] . some studies have suggested that human coronaviruses, in particular hcov-oc43, may be involved in gastrointestinal disease. coronaviruses can be detected in stool samples, and antibodies directed to hcov-oc43 have been observed more frequently in children with gastroenteritis [28] . thus, the future health of people who have recovered from covid-19 may be accompanied by complications. it should be noted that in nature the number of host organisms is regulated by parasites, including viruses. outbreaks are eventually replaced by a population decline (lotka-volterra system of equations, as an example of a kolmogorov model). it is necessary to consider the r-strategy of virus survival in which a large number of descendants as well as mutants are formed. based on the lotka-volterra equations, when the human population declines as the result of the action of a virus, following a delay, the overall population of the virus also decreases. thus, in the human population, during the decline phase, the majority of individuals have genotypes adapted for the virus strains prevailing at the moment. under these circumstances, the human population will start to grow until the virus genotypes capable of suppressing the human population significantly appear again. the same line of reasoning can be applied to a 'medical drug-virus' system, except that a drug cannot change by itself. therefore, it is necessary to control viruses constantly with new drugs developed for use against both new strains of viruses and new species of viruses to save the lives of virus-sensitive people. taking into consideration the natural genetic mechanisms of mutations and recombination, it is impossible to imagine how to deprive a virus of the opportunity to generate new strains and time to time threaten our world with new pandemics. it must be understood that the short-term relationship between the virus and the host is always the result of darwin's microevolution. individuals who do not die in the fight against the virus will form a generation that is more resistant to the virus over time. the virus will continue to reproduce in these individuals, but will not pose a great danger to them. on the other hand, humanity is capable of inventing drugs against viruses and slowing down the progress of darwin's microevolution, saving the lives of those who otherwise might die without treatment. would covid-19 patients after successful treatment or recovery again become severely infected with other strains of this virus? there are no guarantees at the moment, but it is clear that the virus will continue to circulate and kill the people who are most susceptible to it. thus, based on the facts accumulated so far, it is clear that while the new coronavirus is more aggressive, it does not have mortality rates as high as those seen with sars-cov and mers-cov. humanity will definitely survive. drugs and vaccines will save the lives of some virussensitive people. deaths among people of reproductive age will gradually lead to a human population in which the next generations will be more resistant to this virus (fig. 1 ). this is one of the primary reasons why pandemics of spanish flu (h1n1 subtype) did not last for many years because the overwhelming majority of people who died were aged 18-45. individuals genetically resistant to the virus survived it and formed the next virus-resistant generation. published studies indicate that 20-40% of populations in some areas have been infected by the h1n1 virus and thus have some level of protective immunity [29] . the origin of sars-cov-2 is not completely understood. if this virus is man-made, then it is necessary to go back 45 years and rethink the results of the asilomar conference on recombinant dna held in 1975, when scientists met to discuss the potential biohazards and regulation of biotechnology and banned some potentially dangerous experiments, including cloning of recombinant dnas derived from highly pathogenic organisms. if the virus is of natural origin, then it is necessary to understand how new coronaviruses are transferred from animal reservoirs to humans and reduce the likelihood of a person coming into contact with them. in addition, it is imperative to develop new vaccines and drugs against coronavirus infection in case of emergency. is it important today to stop the spread of infection as quickly as possible? yes, if possible, it must be stopped. however, is it in our power to do so when the official number of diagnosed cases has already exceeded 1 million people worldwide? probably not. in some cases, the human population has been able to completely obliterate a viral disease by enacting successful anti-epidemiological measures, as happened with the smallpox virus [30] , but smallpox virus had no animal reservoir. apparently, covid-19 is not one of these cases. in our opinion, the high contagiousness of sars-cov-2 and the ability of coronaviruses to persist for a long time in host organisms [31, 32] will allow the covid-19 virus to continue to circulate in the human population until the fall of 2020 when a new pandemic wave seems to occur again. the most likely outcome is that this new coronavirus (as occurred with the four other well-known non-severe acute respiratory syndrome (nonsars)-related human coronaviruses (hcovs), including hcov-229e, -oc43, -hku1, and -nl63) will take its place next to the seasonal flu viruses, where the infection rate is high and the mortality rate is very low. in the near future, your doctor will routinely prescribe you something like 'covidol' and say that in the coming days you are likely to recover. interestingly, after the discovery of non-sars hcov-nl63 and hcov-hku1, several groups reported infections by these viruses in different countries, illustrating that these viruses have spread worldwide [33] [34] [35] [36] [37] [38] [39] . the viruses can be detected in 1-10% of patients with acute respiratory tract infections, and double infections with other respiratory viruses are common [37] . of note, for sars-cov, a close relative of sars-cov-2, there is evidence of at least seven potential regions of recombination in the sars-cov genome in the replicase-and spike-coding regions, with possible recombination partners that include porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), bovine coronavirus (bcov), hcov-229e, mhv, and ibv [40] . a similar study involving the human coronavirus hcov-nl63 likewise demonstrated that hcov-nl63 exhibited signs of having arisen from multiple recombination events from its nearest relative over the course of hundreds of years [41] . thus, it is possible that sars-cov-2, through multiple recombination events, can trigger an outbreak of respiratory diseases caused by new strains of nonsars-related human hcovs. now, as a century ago during the spanish flu pandemic, the emphasis on washing hands frequently, avoiding crowds, and wearing masks are recurrent in newspaper records and on websites, as is disdain for people flouting the rules. in the meantime, when we wash our hands with soap, we should thank providence that the sars-cov-2 does not have the same mortality rate as mers-cov. as the situation stands right now, it is better to become infected with covid-19 as late as possible to allow vaccines and drugs to be developed and tested, as there are no guarantees yet concerning who is at risk of a severe course of the disease and who is not. apparently, the bitter and unpopular truth is that most of the world's inhabitants need to make contact with the sars-cov-2 before we can all return to normal life. quarantine slows down this process, gaining time for doctors and scientists who, through their professional activities, can save virus-sensitive individuals. so often, we have to choose: quickly but painfully, or gently but slowly. today we find ourselves in a zugzwang situation: a greater number of deaths, or the collapse of the global economy. we are on the second path so far, but soon we will need to reach a compromise because sars-cov-2 will continue to circulate in the human population. this coronavirus is with us forever. perhaps we can learn from the seasonal flu viruses that kill hundreds of thousands of people every year; since we are used to these viruses, they do not scare us anymore, so we have reached the compromise that lets us all move forward. the proximal origin of sars-cov-2 probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak receptor recognition by novel coronavirus from wuhan: an analysis based on decadelong structural studies of sars mechanism of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium no credible evidence supporting claims of the laboratory engineering of sars-cov-2 clinical features of patients infected with 2019 novel coronavirus in wuhan genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a novel coronavirus from patients with pneumonia in china the single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to wuhan 2019-ncov infection single-cell rna expression profiling of ace2, the putative receptor of wuhan the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection discovery of bat coronaviruses through surveillance and probe capture-based next-generation sequencing evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission return of the coronavirus: 2019-ncov. viruses. 2020 transmission of 2019-ncov infection from an asymptomatic contact in germany a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster a new coronavirus associated with human respiratory disease in china immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic on the origin and continuing evolution of sars-cov-2 t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection recombinant duck enteritis viruses expressing major structural proteins of the infectious bronchitis virus provide protection against infectious bronchitis in chickens evaluation of protective efficacy of the synthetic peptide vaccine containing the t-helper 1 epitope with cpg oligodeoxynucleotide against feline infectious peritonitis virus infection in cats structural and functional correlates of enhanced antiviral immunity generated by heteroclitic cd8 t cell epitopes reinfection could not occur in sars-cov-2 infected rhesus macaques pathogenesis of virus-induced demyelination and gastroenteritis: evidence of antigenic relatedness between human enteric coronavirus strains and human coronavirus oc43 the global eradication of smallpox recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission host factors in coronavirus replication new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia human coronavirus nl63 infection in canada human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong the association of newly identified respiratory viruses with lower respiratory tract infections in korean children detection of human coronavirus nl63 in young children with bronchiolitis coronavirus hku1 infection in the united states human coronavirus nl63, a new respiratory virus testing the hypothesis of a recombinant origin of the sars-associated coronavirus mosaic structure of human coronavirus nl63, one thousand years of evolution key: cord-318204-t024w7h6 authors: fang, ferric c; naccache, samia n; greninger, alexander l title: the laboratory diagnosis of covid-19-frequently-asked questions date: 2020-06-08 journal: clin infect dis doi: 10.1093/cid/ciaa742 sha: doc_id: 318204 cord_uid: t024w7h6 diagnostic testing has played and will continue to play a major role in the covid-19 pandemic. the ability to detect the sars-cov-2 coronavirus in respiratory secretions is essential to determine when an individual is infected and potentially infectious to others. viral detection is used for the identification, management and isolation of individual patients. viral detection is also used to determine when the virus has entered a community and how rapidly it is spreading. as communities attempt to re-open following periods of shutdown, the detection of both sars-cov-2 and specific antibodies recognizing the virus will become increasingly important as a means to assess infection and immunity in individuals and communities. here we discuss questions commonly asked by clinicians about covid-19 diagnostic testing. limitations in the availability of ppe and testing supplies, in addition to the operational difficulty of scaling np swab collections for asymptomatic screening, have led to the evaluation of alternative samples including nasal swabs, mid-turbinate swabs, oropharyngeal swabs, and saliva. saliva is particularly attractive as it requires neither swabs nor transport media, although collection and processing of saliva presents other challenges (20) . the comparability of these specimen types for a qualitative test depends on the viral load present at the time of infection. oropharyngeal swabs have shown less sensitivity compared to nasopharyngeal and nasal swabs (21) . nasal swabs and nasopharyngeal swabs may be comparable in sensitivity, but more studies are needed to compare these specimens across different patient populations. new specimen types for an eua test must receive a specific amendment for that specimen type before they can be reported. some studies have indicated that self-collected samples are comparable in sensitivity to those collected by health care providers, which can obviate the need to use ppe for testing (22) (23) (24) (25) . although sputum and bronchoalveolar lavage samples may have higher viral loads and can therefore provide greater test sensitivity than upper respiratory samples (26, 27) , particularly at later stages of illness, they entail a higher risk of aerosol generation or require an invasive procedure, so these sample types are obtained more selectively. a c c e p t e d m a n u s c r i p t (28, 29) . this may be a consequence of the variable distribution of the ace2 viral receptor protein in the respiratory tract (30) . interestingly, viral tropism for the lower respiratory tract was also seen during the h1n1 influenza epidemic (31) . is it worth repeating a test after a patient has tested negative? in view of the less than ideal sensitivity of an np swab to detect sars-cov-2 infection, it may be useful to repeat testing in a patient in whom the clinical suspicion is high (32) . in our experience, the yield of repeat testing from the same source is low (33) . however, the yield from repeat testing may be substantial in higher prevalence settings (34) . shedding culturable virus after about one week from symptom onset, but can continue to have detectable viral rna in their respiratory tracts for longer periods of time (35, 36) . more severely ill patients will remain pcr-positive for longer, sometimes extending for weeks-to-months (37) . sars-cov-2 rna can also be found in stool samples and typically continues to be detectable in the feces for weeks (38) . evidence indicates that human intestinal epithelial cells can support replication of a c c e p t e d m a n u s c r i p t 7 the virus (39) , and sars-cov-2 has been cultured from stool samples (40) . fecal-oral transmission of sars-cov-2 has not been demonstrated, but there is concern that fecal shedding might contribute to the spread of infection. does a positive specimen mean that a patient is infectious? not necessarily. although viral nucleic acids can be detected during convalescence (35) , culturable virus is believed to represent a better correlate of infectivity (41) . however, sars-cov-2 culture requires a bsl-3 facility and there are no authorized clinical assays utilizing sars-cov-2 viral culture at this time, so clinical monitoring is dependent on rna detection. negative? two negative pcr assays at least 24 hours apart are commonly used as a criterion to discontinue isolation (42) . however, a number of patients will revert to pcr-positivity after two negative samples (43) . this may reflect fluctuations in the quantity of viral rna shedding during recovery. when monitoring for viral load as inferred by real-time pcr cycle threshold (ct), cases that have reverted to positivity consistently exhibit high ct values indicative of a low viral load (44) . there is presently little evidence of true virological and clinical relapse, and the prognosis for these patients appears to be good. a c c e p t e d m a n u s c r i p t 8 is quantitative pcr (viral load) useful? there is some correlation between illness severity and viral load on presentation, as inferred by the real-time pcr cycle threshold (ct) (37, 45) . however, an isolated viral load estimate is of limited prognostic value, and even asymptomatic individuals may have high viral loads (46) . viral load trends may have greater value and might help to inform decisions to initiate a trial of antiviral therapy, but viral loads typically decline regardless of the clinical course (3, 6, 47) . older patients tend to have higher viral loads (6), just as they are at greater risk for more severe or critical illness (48, 49) . imply that only one of two pcr targets was detected, and confirmation with a different assay that detects alternative targets is recommended. given the high specificity of the pcr assays, most inconclusive results will ultimately be confirmed as positive. inconclusive or indeterminate results could also indicate that the internal controls failed and may indicate a technical issue, such as the presence of a pcr-inhibitory substance in the sample. different labs and tests may use different terminology, so it is worth contacting a given clinical laboratory to determine how they report low positive results. diagnostic platforms used for the detection of specific antibodies to sars-cov-2 proteins include rapid diagnostic tests (rdt) such as lateral flow assays (lfa), enzyme-linked immunosorbent assays (elisa), neutralization assays and chemiluminescent immunoassays (50) . only neutralization assays can provide information regarding the ability of antibodies to inhibit viral growth. the performance of various serologic tests is more variable than the rt-pcr assays for sars-cov-2 (8, (51) (52) (53) . this is particularly important because the positive or negative predictive value of a test is dependent not only on the intrinsic test accuracy but also on the prevalence of disease in the population. an insensitive test will have a poor ability to exclude the presence of disease when prevalence is high, but more germane to covid-19, a test with low specificity will have a poor ability to indicate the presence of disease when the prevalence is low. when only a few percent of the population have immunity to sars-cov-2, as is presently the case in most regions, a positive result from a serologic test with low specificity will be more likely to represent a false-positive. there are four kinds of human coronavirus that cause mild-to-moderate seasonal respiratory tract infections: 229e, nl63, oc43 and hku1. cross-reactivity with antibodies to seasonal coronaviruses is a theoretical concern for a sars-cov-2 serologic test, but for most of the commercial assays evaluated thus far, this does not seem to be the case (54, 55) . to rule-out cross reactivity, a new assay should be tested against a panel of serum samples that pre-date the emergence of sars-cov-2, ideally more than 500, to a c c e p t e d m a n u s c r i p t 10 accurately ascertain specificity. cross-reactivity between sars-cov-1 or mers-cov and sars-cov-2 is more likely (56) , but should be a limited concern. are serologic tests useful to diagnose acute covid-19? although the primary use of serologic tests is to determine prior exposure to sars-cov-2, the detection of specific antibodies may support the diagnosis of covid-19 in a patient with a high clinical suspicion but negative pcr tests (57-59). igm and igg directed against sars-cov-2 may appear as early as 3-6 days after the onset of symptoms. by three weeks, nearly all patients have seroconverted, and the antibodies persist for at least two months, with igg showing greater persistence (4-8, 59). the duration of antibody responses to sars-cov2 is unknown. antibody responses to the common respiratory coronaviruses decay after a few years (56) , and it is suspected that immunity to sars-cov-2 will be similar. antibody responses have been observed in nearly all patients with covid-19, although it is possible that some very mild or asymptomatic infections, or infections in immunocompromised patients, may not result in seroconversion (5, 60) . a c c e p t e d m a n u s c r i p t 11 does a positive antibody test mean that a patient is immune? it is likely that the detection of specific antibodies in a patient with a history of covid-19-like illness will be indicative of at least some degree of immunity (61) . experimental animals re-challenged with pathogenic coronaviruses exhibit resistance to re-infection (62) . however, a quantitative cutoff of antibody titer that correlates with protective immunity is undefined. as with other viruses, it is possible that a low titer of antibodies is not protective. also, it is not presently known whether neutralizing antibodies are the primary mechanism of immune protection. in fact, higher antibody titers are observed in patients with more severe illness (5, 6, 51) . as patients with mild covid-19 may recover despite low antibody titers, and patients with severe covid-19 have persistent illness despite the development of high antibody titers, one may question whether neutralizing antibodies are in fact protective. the reported therapeutic benefits of convalescent plasma might be due to constituents other than neutralizing antibodies (63) . moreover, the development of neutralizing antibodies is accompanied by t cell responses (64, 65) , which may contribute to protection. are quantitative serologies helpful? because sars-cov-2 is a new human pathogen, pre-existing adaptive immunity is non-existent, so acute and convalescent titers are not required to establish the diagnosis of covid-19. until specific cutoffs are identified as a correlate of protective immunity, qualitative serologic results are sufficient to provide clinical guidance. however, reporting of quantitative serological read-outs could offer clinicians more information about the potential for false positives and false negatives for values near the positivity threshold. a c c e p t e d m a n u s c r i p t 12 is there a correlation between age and antibody titer? older age correlates with a higher likelihood of severe illness from covid-19 and with the development of higher antibody titers (59), perhaps due to a higher antigen load. is point-of-care testing available? affordable point-of-care (poc) diagnostics for sars-cov-2 could facilitate the widespread testing and contact tracing strategies proposed for post-pandemic wave containment (66) . however, performance characteristics and usability are critical parameters for these tests, as they are typically deployed without the quality assurance apparatus of a high complexity laboratory. a poc antigen detection assay was recently authorized by the fda, but is known to be less sensitive than pcr, so its clinical role has yet to be defined. negative results using this assay should be confirmed by a more sensitive method in most instances. other assay technologies, including a crispr-based nucleic acid detection system (67), may be utilized in poc formats in the future if appropriate sensitivity can be achieved. a variety of biomarkers, including lymphocyte count, neutrophil-tolymphocyte ratio, crp, troponin t, d-dimer, ldh, procalcitonin, il-6 and ferritin are predictive of disease progression and mortality in covid-19 (table 1) (68, 69) . these laboratory tests play a vital role in identifying patients at risk for complications and to guide treatment interventions. early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application temporal dynamics in viral shedding and transmissibility of covid-19 diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 antibody responses to sars-cov-2 in patients with covid-19 temporal profiles of viral load in posterior 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sars-cov-2 infection protects against rechallenge in rhesus macaques convalescent plasma in covid-19: possible mechanisms of action detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals laboratory diagnosis of emerging human coronavirus infections -the state of the art point-of-care testing for covid-19 using sherlock diagnostics hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19): a meta-analysis elevated levels of il-6 and crp predict the need for mechanical ventilation in covid-19 interpreting diagnostic tests for sars-cov-2 a c c e p t e d m a n u s c r i p t 15 a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-308857-otsrexqu authors: goel, saurav; hawi, sara; goel, gaurav; thakur, vijay kumar; pearce, oliver; hoskins, clare; hussain, tanvir; agrawal, anupam; upadhyaya, hari m.; cross, graham; barber, asa h. title: resilient and agile engineering solutions to address societal challenges such as coronavirus pandemic date: 2020-05-28 journal: mater today chem doi: 10.1016/j.mtchem.2020.100300 sha: doc_id: 308857 cord_uid: otsrexqu the world is witnessing tumultuous times as major economic powers including the us, uk, russia, india, and most of europe continue to be in a state of lockdown. the worst-hit sectors due to this lockdown are sales, production (manufacturing), transport (aerospace and automotive) and tourism. lockdowns became necessary as a preventive measure to avoid the spread of the contagious and infectious “coronavirus disease 2019” (covid-19). this newly identified disease is caused by a new strain of the virus being referred to as severe acute respiratory syndrome coronavirus 2 (sars cov-2; formerly called 2019-ncov). we review the current medical and manufacturing response to covid-19, including advances in instrumentation, sensing, use of lasers, fumigation chambers and development of novel tools such as lab-on-the-chip using combinatorial additive and subtractive manufacturing techniques and use of molecular modelling and molecular docking in drug and vaccine discovery. we also offer perspectives on future considerations on climate change, outsourced versus indigenous manufacturing, automation, and antimicrobial resistance. overall, this paper attempts to identify key areas where manufacturing can be employed to address societal challenges such as covid-19. coronaviruses (covs) belong to the family coronaviridae which includes four genera: α, β, γ and δ as well as several subgenera and species [1] . sars cov-2 is a β-coronavirus with a single-stranded rna genome of ~30 kb [2] . recent topical research has revealed several new covs (three α-coronaviruses, three new βcoronaviruses, and one previously described α-coronavirus) from bats captured from myanmar and future emergence of new diseases caused by these covs due to change of land use has been speculated [3] . furthermore, newer mutations of the virus that originally spread from wuhan were confirmed as deadlier in some countries compared to others, which has led to added confusion and concern [4] . sars cov-2 was first identified from the outbreak of respiratory illness cases in wuhan city in the hubei province of china. initial reports of the virus were made to the world health organisation (who) on december 31, 2019. this was followed by the who declaring covid-19 as a global health emergency on january 30, 2020 due to rapid spreading, and a later pandemic declaration on march 11, 2020. the disease has quickly engulfed most of the world and has caused severe infection to populations across numerous countries as shown in figure 1 . covid-19 is shorthand for "coronavirus disease 2019" which is caused by the sars cov-2 virus, also called coronavirus in typical usage. viruses cause disease by binding to receptors on cells in the human body and then replicating at a rapid rate which triggers a variety of pathogenic processes. in the case of covid-19, the structures on the surface of the virus bind to receptors in the airway or the lungs of human beings. the lungs may become inflamed, making breathing more difficult. for some people, the infection becomes severe and leads to critical care requirements. the anatomy of sars cov-2, including its internal biological structure of spike protein legs, envelope protein, and membrane protein, surrounding the genomic rna is shown in figure 2 (a). the virus shown in figure 2(a) highlights a large diameter of about 0.1 µm with variations in sizes reported by different researchers. the spike-shaped protein legs that make up a portion of the outer capsule of the virus create the crown-like or corona-like appearance. thus, the name coronavirus. the mechanism of infection due to the surface interactions between the spike protein and the lung cells is depicted in figure 2 (b). this understanding was gathered from the genetic analysis which revealed that the mutations located in the spike surface glycoprotein might induce conformal changes and play an essential role in binding to receptors on the host cell (lungs) and determine host tropisms by leading to possible changing antigenicity [7] . viruses are fundamentally different to bacteria so the classically developed work on nano-structuring and biomimicking surfaces, such as the cicada wing, primarily targeting bacterial killing, should not be confused as a readily available knowledge to kill sars cov-2. physically, a bacterium is larger than a virus (the biggest size of a virus is smaller than the smallest known bacteria). bacteria are living cells which are 100±60 nm capable of prolific reproduction independently, whereas viruses are non-living particles, requiring a host cell for replication. also, bacteria possess a cell-wall engulfing a chromosome, whereas viruses consist of genetic material, either dna or rna, covered by a protein coating. bacterial infections can therefore be treated with high success using antibiotic drugs. although some antiviral medicines are now available, the susceptibility of viruses to react to the treatment rate of medicines is significantly reduced as they are non-living. hence, the only long-term option to eradicate viruses is the development of vaccines that stimulate the natural production of antibodies. studies suggest that the source of sars cov-2 could either be ratg13 from horseshoe bats (rhinolophus affinis), pangolins (manis javanica), or a mix of both. their transmission has occurred by zoonotic mechanisms [6, 7] . however, the coronavirus isolated from pangolins is 99% similar in a specific region of the spike protein, which corresponds to the 74 amino acids involved in the angiotensin-converting enzyme 2 (ace 2) receptor binding domain, which allows the virus to enter human cells to infect them as shown in figure 2 (b). the virus ratg13 isolated from rhinolophus affinis bats is highly divergent in this specific region (only 77 % similarity). this observation indicates that the coronavirus isolated from pangolins can enter human cells, whereas coronavirus isolated from rhinolophus affinis bats is unable to enter human cells. the sars cov-2 has a high transmissible efficiency and covid-19 has high morbidity and mortality. a popular but possibly flawed measure for assessing fatality of disease is the use of deaths/case counts. this measure would yield a fatality rate of 6.6% for covid-19 as of 17th may 2020 [8] . the problem with this measure is that case counts reflect the number of tests that were done rather than infections, and the deaths lag the cases because fatality (if observed) may happen several days after the case is identified. a lag in reporting case numbers and incorrect tests may also occur. an alternative measure is the case fatality rate (cfr), which is the ratio of deaths / (deaths + recovered cases). this measure would yield a fatality rate of 14.6% [9] . the consensus is that the covid-19 disease has high fatality and can exceed the fatality ratio of the century-old "spanish flu", which had a 10% cfr [11] . however, the data analysis of callaway et al. [10] shown in figure 3 suggests that covid-19's cfr is lower than that of mers and ebola and that its infection rate (r0 -the expected number of cases directly generated by one case in a population where all individuals are susceptible to infection) suggests that the infection can spread more easily than other diseases, including seasonal influenza. a further comparison of three different episodes of epidemics and pandemics caused by the family of coronavirus, namely, sars cov-1, mers and sars cov-2, is depicted in table 1 . the exact mechanism of these drugs towards the virus is yet to be completely understood. however, the mechanism where a drug affects the replication of viruses in-vivo is a broadly accepted concept. in general, viruses replicate via protein processing using endosomes within golgi bodies. a drug such as hcq increases the ph of the golgi bodies making them more basic, thus disrupting the integrity of the internal nucleocapsid protein (see figure 2a ). this denatures the protein of the coronavirus rendering it dysfunctional. therefore, the rationale for using hcq is built on the premise that the change in ph brought about by the drug inhibits the endosomal transport necessary to spread the infection, hence the patient recovers. another short-term treatment being implemented by some hospitals across the world is to infuse patients with the antibody-rich blood plasma of people who have recovered from covid19 [13] . this approach has been used during disease outbreaks for over a century. however, the approach carries significant risk in terms of the already immunocompromised patients' immune response after administration, and results may vary between patients. as an early effort of investigation of the problem from scratch, researchers at toho university in japan used high-sensitivity cameras and laser beam guidance experiments to deduce that saliva spray during a sneeze (potentially containing thousands of viruses) can be classified into large vs small droplets or droplets vs aerosols. the droplets, due to their heavier weight, fall off, whereas aerosols remain airborne for a few hours due to their relatively small size. prima facie evidence suggests that the coronavirus has escalated to a pandemic due to the high contagion occurring via this 'airborne' spread model. recently, the possibility of asymptomatic or oligosymptomatic infection has also been highlighted [14] . the aerosols may circulate near an infected patient in an airborne condition depending on the local conditions (airflow rate, humidity, dryness) for up to a few hours, even after the infected person has left the location. hence, the chances of contracting coronavirus are relatively high by merely occupying the same vicinity where an infected person has been or passed through. researchers have experimentally evaluated the stability of sars cov-2 recently, and these comparisons are shown in figure 4 [15, 16] . it is noteworthy that sars cov-2 was stable in the aerosol (airborne) form for up to 3 hours. as opposed to this, the virus appears to be stable on surgical masks or stainless-steel surfaces for up to 7 days and for up to 4 days on smooth surfaces such as glass or currency notes. the same research also shows that the application of hand soap does not immediately deactivate the virus but rather takes up to 5 minutes. therefore, a 5-minute waiting period after a handwash is required before bringing hands in contact with face, mouth or nose. covid-19 is not only present in the airway secretions of infected patients when they sneeze, but also when they simply breathe out or speak. studies have shown that covid-19 is also present in other body fluids of infected people, such as faeces, blood [17], oral fluids, anal secretions [18] , tears [19] , and urine [20] . the virus primarily attacks the respiratory system, however, new evidence shows the virus is not filtered by the kidneys, as traces of virus can be seen in sewage systems [8] . therefore, as a mitigation strategy, testing of these fluids, which can be available and abundant in the wider population can be done for identifying the most effective areas. overall, when people are in close contact with one another, then transmission is more likely. most sources for infection worldwide have happened in an enclosed space, including homes, offices, public transport and restaurants. a second spreading pathway for the virus is through touching a surface or object that has the virus on it and then touching one's own mouth, nose, or eyes. if one is in a wellventilated space with fewer people, even for a longer period, the risk of infection is low. sustained contact with an infected person, even for a short period, even without a cough or sneeze, can spread the infection. by the virtue of disturbing the cell programming, covid-19 possesses the capability to cause a surprise attack on almost any part of the body ranging from lungs, heart, blood vessels, brain, eyes, nose, liver, kidneys and intestine. a schematic representation of this adverse impact is shown in figure 5(a) [21] . recent literature suggests that apart from the airway, the human brain may also be targeted by the virus -see figure 5 (b) and figure 6 [22] . patients have reported having mild (anosmia and ageusia) to severe (encephalopathy) neurological manifestations, and if true, it makes sars cov-2 more lethal. [21] (b) tissue distribution of ace2 receptors in humans [23] (c) possible neuro disorders due to sars cov-2 [22] and (d) life cycle of sars cov-2 in host cells [24] . (figures reprinted with permission) our nasal lining tissue contains a rich number of cell receptors called angiotensinconverting enzyme 2 (ace2), which are favourable sites for the sars cov-2 to attach its spiked protein to, thus paving way for the entrance of the virus inside the body. once attached to the cell, sars cov-2 can change cell programming, replicate itself, and attack new cells at a very rapid pace. within a week of entrance into the body, the virus exhibits effects. during this time, the person may show early symptoms such as fever, dry cough, sore throat, loss of smell and taste, or head and body aches. at this point, the failure of the immunity in defeating the virus means giving the virus a way forward to enter into the respiratory system, where the lungs are attacked. the lungs also have a cell lining rich in ace2 [22] . as the immune system continues to fight the sars cov-2, the supply of healthy oxygen is disrupted. as the disease progresses inside the body, the white blood cells release inflammatory molecules or chemokines, which attack and kill the virusinfected immune cells, leaving the dead cells and pus behind. this affects healthy oxygen transfer in the lungs and is the stage where the patient starts to complain of pneumonia, coughing, fever, and rapid, shallow respiration. acute respiratory distress syndrome may develop. at this stage, the lungs start to be filled with opaque black spots signifying closure of the air pores, and such patients require ventilator support. the survivors of this severity of infection can end up having long-term complications. a few hospitals have successfully applied artificial intelligence to monitor the recovery rate by monitoring the coronascorea general term used to quantify the extent of the blocked pores (in cm 3 ) [25] long-term research measures, advanced manufacturing and metrology have pivotal roles to play in containing a pandemic. specifically, the development of engineering innovations is timely for the control of the covid-19 pandemic. for diagnostic testing to be useful for informing doctors and governments about the true incidence at any one time of coronavirus in the population, it needs to be inexpensive, high in sensitivity and specificity (sensitivity refers to the ability of a test to correctly identify those with the disease or true positive rate, whereas specificity refers to the ability of a test to correctly identify those without the disease or true negative rate) and easy to use for non-experts. as doctors are treating infected patients in this emergent time in an unprepared state, emergent manufacturing measures can help design better testing equipment and thus help in both short and long term to tackle this problem. the most significant challenge now lies in probing early-stage symptoms of covid-19. for a relatively late stage detection in severe cases, chest computer tomography (ct) using x-ray probes (providing >90% sensitivity) has proved to be a more reliable test assay in comparison to the reverse-transcription polymerasechain-reaction (rt-pcr) test and other sensor-based detection methods currently being pursued [27] . since the nervous system and respiratory dysregulation are both likely to co-exist, covid-19 testing should be done by combining brain mri and rt-pcr tests as shown in figure 7(a) [28] . however, caution is required in the analysis of results, such that covid-19 should not be confused with drug-resistant tuberculosis (tb) as both would show symptoms of damages in the lungs. in an attempt to boost the testing in a populous country such as india, the indian council of medical research (icmr) has approved the use of diagnostic machines used for testing drug-resistant tuberculosis under the guidance that the throat/nasal swabs are collected in the viral transport medium [29] . interestingly, the pcr test cannot identify asymptomatic infections or those people who were exposed to or infected with covid-19 in the past and did not suffer from the disease or have recovered from covid-19 and may still be spreading the virus (carriers). therefore, different tests may be required to collate vital information required by government bodies to carry out a risk-benefit analysis to relax lockdown measures to protect their economies. immunodiagnostic kits such as a lab-on-a-chip are being developed for asymptomatic detection of covid-19 [30] . a lab-on-chip (figure 7 (b) works on the principle of detecting the presence of patient-generated antibodies against the virus that causes a specific disease, in this case, covid-19. the test uses a lateral flow immunoassay to assess the presence of an analyte from a patient sample or specimen and detects two types of antibody isotypes: igm and igg. igm antibodies are the first antibodies to appear in response to a novel antigen and imply a more recently initiated infection while igg antibodies are generated later in the course of infection and possess a higher affinity for the target antigen, meaning they are more specifically able to bind the substance which caused the immune response. a test is declared positive if either one or both antibodies are detected during the test, similar to widely used pregnancy tests. the test consists of an anti-human igg coating in the g test line region and an igm coating the m test line region (see figure 7 (b). during testing, the sample (blood, urine etc) reacts with sars-cov-2 antigen-coated gold nanoparticles (aunp) in the conjugation pad of the test cassette. any antibody in the patient sample that recognises the sars-cov-2 antigen binds to the antigen-aunp complex. the mixture then migrates laterally across the membrane by capillary action/lateral flow. as these human antibody/antigen/aunp complexes move across the test lines, they are captured at the anti-human igm 'm' line, the anti-human igg 'g' line, or both, depending on the antibody contents of the specimen. the sample first reaches the anti-human igm antibodies which coat the m line. if the specimen contains igm antibodies to sars-cov-2, a coloured line will appear in the m test line region. next, the sample reaches the anti-human igg antibodies which coat the g line. if a specimen contains igg antibodies to sars-cov-2, the conjugate-specimen complex reacts with anti-human igg. a coloured line appears in the g test line region as a result. the rabbit igg-aunp complexes are captured by the control line (which contains anti-rabbit-igg). this visible line indicates that there has been successful lateral flow across the detection strip. the last check ensures that the sample had enough volume to move across the entirety of the test cassette. only human antibody/sars-cov-2 antigen/aunp complexes will produce a visible red or pink line at the m or g line. other antibodies produce no colour. the accuracy of these lab-on-chip tests is still being debated as it depends on two key parameters, sensitivity and specificity. to date, igg related sensitivity and specificity has been found to be higher than that of igm. a schematic diagram in figure 7(c) and figure 7(d) shows that unlike currently available diagnostic methods, field-effect transistor (fet)-based biosensing devices may have several advantages, including the ability to make highly sensitive and instantaneous measurements using small amounts of analytes [31] . the fet sensor shown in figure 7(d) makes use of a graphene sheet since graphene-based fet biosensors can detect surrounding changes on their surface and provide an optimal sensing environment for ultrasensitive and low-noise detection. from this standpoint, graphene-based fet technology is attractive for applications related to the sensitive immunological diagnosis. graphene as a sensing material is selected, and sars-cov-2 spike antibody is conjugated onto the graphene sheet via 1-pyrenebutyric acid n-hydroxysuccinimide ester [31] (figures reprinted with permission) a group of researchers at mit and harvard are developing coronavirus-identifying sensor embedded face masks. this ongoing work is an extension to their previous work where they developed a low-cost method to detect a zika virus [33] . in this technique, the sensor is made by using genetic material such as the rna or the dna which binds to the viruses. the researchers used a lyophilizer to freeze-dry the genetic material onto the fabric of the mask. the material deposited onto the fabric remains stable for many months (at room temperature) and the detection process starts merely by the presence of moisture (such as saliva). the detection signal is small (in terms of voltage) and can be detected by an additional fluorimeter device that can quantify this signal and emit in form of a fluorescent light. thus, one can expect to see light glowing masks to detect coronavirus in the future. fumigation chambers allow quick disinfection of a person while visiting areas such as hospitals, universities or airports. the aim is to use tubes releasing hydrogen peroxide in a chamber designed to be five-feet wide and seven-feet tall. the fumigation chamber usually seals automatically after the person's entry to avoid any leakage outside of the chamber and has designated sensors facilitating the chamber entry. the disinfection lasts for five seconds. it is to be noted here that hydrogen peroxide has also been recommended by the food and drug administration (fda) to be used as a sterilisation material to decontaminate n95 or n95-equivalent respirators for reuse by health care workers in hospital settings [34] . a populist view is that sunlight and high temperature during peak summers kills the coronavirus, which would help containment of its spread. it is reported that sars cov-2 does not survive beyond 5 min after being exposed to 70ºc [16] . however, it has yet to be ascertained as to how long does sunlight take to deactivate sars cov-2 and at what intensity. a more specific question is whether ultra-violet (uv) light can kill coronavirus? sunlight usually contains three types of uv: uva (320-400 nm), uvb (280-320 nm), and uvc (200-280 nm). uva and uvb can both cause sunburn, however, uvc is shorter and a more energetic wavelength of light. while uvc is effective at destroying genetic material, whether in humans or viral particles, it is filtered by the ozone layer and does not reach the earth's surface. preliminary findings from the national biodefense analysis and countermeasures centre of the usa (which houses a bsl-4 level lab) indicate that "sunlight seems to be very detrimental to the virus… within minutes, the majority of the virus is inactivated on surfaces and in the air in direct sunlight." [35] . research on the use of uv as a treatment is still evolving and the behaviour of sars cov-2 under uv light is unknown. results were relatively favourable for sars cov-1 treatment with uvc [36, 37] . sars cov-1 was efficiently inactivated after 40 minutes of uvc exposure (at about a wavelength of 254 nm), whereas addition of psoralen (a light-sensitive drug) to uva enhances inactivation of the sars cov-1. popular press reports suggest that uv light booths are capable of deactivating coronavirus without any human contact and are proposed to be deployed ( figure 8 ). one must be cautious, since uvc can be dangerous to the skin, causing burns within seconds and harmful to the eyes if observed directly. therefore, risk assessment and associated precautions would need to be deployed that may render this strategy difficult in the short-term. 3.5.1. antimicrobial coatings chouirfa et al. [39] summarised nanomaterials based coatings for antibacterial applications and xing et al. [40] have summarised the potential of natural polymer chitosan as an antimicrobial agent ( figure 9 ). their research on antimicrobial properties of a nanocomposite coating formed by polysaccharide 1-deoxylactit-1-yl chitosan (chitlac) and silver nanoparticles (nag) on methacrylate thermosets showed satisfactory results. figure 9 : antimicrobial mechanism of chitosan-based coating with antimicrobial agents. reprinted with permission from [40] . antimicrobial surfaces are based on three main mechanisms (see figure 10 ): the anti-biofouling mechanism which repels microbes and prevents them from adhering to the surface, the release-killing mechanism where microbes are killed in the nearsurface environment with a release of antimicrobial agents and the contact-killing mechanism where microbes adhere and are killed on the surface [41, 42] . researchers have also experimented with developing long-lasting antimicrobial surfaces to stop spreading pathogenic microbes through commonly touched surfaces or at-risk surfaces and have focused on the use of antimicrobial/antiviral materials such as copper. experimental trials for copper oxide impregnated respiratory protective facemasks have yielded 100% efficiency in eradicating the infectivity of human and avian influenza a virus in simulated breathing conditions [43] . warnes et al. [44] have shown that the surfaces of dry copper alloys are lethal to viruses such as mnv-1 at room temperature, with higher copper content being more time-efficient. generally, the antimicrobial activity of copper is attributed to oxidative behaviour of copper and the solubility properties of copper oxides [45] . more recently, surface texturing of copper via cold spray methods has shown enhanced promise as an antiviral agent [46] . combining the aforementioned antimicrobial contact-killing properties of copper, the anti-adhesion properties of polymeric micelles and the release-kill abilities of chlorine dioxide (clo2), li et al. [47] developed a multifunctional coating viricidal for influenza virus h1n1. this multifunctional coating is composed of clo2 encapsulated in polymeric micelles (with slow on-demand release) on which copper nanoparticles were covalently tethered. other studies have investigated hydrophobic polycationic coatings as antimicrobial coatings [48] [49] [50] . hsu et al. [51] investigated the mechanism by which the n,n-dodecyl,methyl-polyethelenimine (pei) coated surfaces killed the influenza a virus. it was concluded that upon contact with the coated surface, the virus adheres irreversibly and through hydrophobic and electrostatic interactions, the virus' disintegration is then initiated resulting in rna leakage into the solution. the incorporation of pei into protective mask textiles has also been investigated with nearly a 1000-fold improvement in the capture of the t4d bacteriophage virus of escherichia coli b [52] . finally, the direct mechanical action of sharp nanostructures such as darts, blades and spikes as a kind "mechano-cide" has been shown to have some success for bacteria, but remains largely unexplored for viruses [53] . from a contact mechanics perspective, the combined effect of sharp ( somewhat surprisingly, while researchers have investigated bactericidal properties of nanoparticles of silver, yet the current understanding of the interaction of these nanoparticles with viruses is limited. however, some positive results are reported and, based on these reports, this direction of manufacturing research holds a good promise to tackle sars cov-2. previous studies showed that the size of nanoparticle is critical to manifest a viricidal effect. for example, a nanoparticle size of less than 25 nm was found to be effective against tacaribe virus [56] , while a particle size of between 7 to 20 nm worked well against herpes simplex virus (hsv) type 1/2 and human parainfluenza virus type-3 [57] . nakamura et al. [58] reported that materials with immobilised silver nanoparticles possess enhanced microbicidal activities against the virus. these researchers developed a material using silver nanoparticle absorbed on a chitin sheet having a nanoscale fibre-like surface structure shown in figure 11 and obtained favourable results against h1n1influenza a virus. figure 11 : the mechanism of viricidal activity of silver (ag) nanoparticles (np) chitin nanofiber sheets showing strong antimicrobial activity via reactive oxygen species (ros) and silver ions on the substrate. reprinted with permission from [58] . moreover, the unique characteristics of metallic nanoparticles such as high surface to volume ratio, surface-enhanced raman scattering and localized surface plasmon resonance can be utilised for virus detection and therapy via destruction through laser-induced localised hypothermia as well. some of the candidate nanoparticles are metallic and high entropy alloy nanoparticles (agauptpdcu high entropy nanoparticles, feo nps, ag nps, tio2 nps, au nps, ag-au core-shell nps) [59] . for this purpose, uv-visible spectroscopy can be deployed to study the surface plasmon resonance, refractive index, and fluorescence change when nanoparticles interact with virus particles. nanoparticle-based investigation can help detect and inactivate viruses (figure 12 ). specific to coronavirus, graphene oxide sheets with silver nanoparticles (go-ag nps) were reported to inhibit the growth of feline coronavirus (fcov) by up to 25%, in comparison to pure go that inhibited it only up to 16% [60] . additionally, chiral biosensor with self-assembled chiral gold nanohybrids (cau nps) on account of multiple plasmonic scattering showed better detection performance on coronavirus [61] . figure 12 : nanoparticle based therapy for sars cov-2. reprinted with permission from [59] as with all fundamental and translation nanomaterial research, progress is not immediate. caution is required with nanomaterial technologies, as the long-term impact on human health and the environment of free nanoparticles has not been ascertained, and this may ultimately pose greater risk, particularly in the liver and respiratory tract of human beings and in ecosystems. lack of governmental regulation strategies for nanomaterials can also hinder the speed of approval and ultimately the availability in the marketplace. nevertheless, a call has gone out to nanomedicine researchers to utilise their existing knowledge base and translate their technologies towards covid-19, should the outbreak last more than 12 months [62] . filtration efficiency is a strong measure of penetration prevention of aerosols through mask filters and is usually required to be above 98% for surgical face masks [63] . ultrasonic welding is usually deployed to produce filters with sufficient filtration efficiency. both polymer and textile-based masks are popular and can benefit from the adoption of sequential micromachining techniques [64] . a surgical mask (procedure mask) is worn by health professionals during surgery to avoid exposure to aerosols. such masks are not designed to protect the wearer from inhaling airborne bacteria or virus particles and are less effective than respirators, such as n95 or niosh masks which provide better protection due to their material, shape and tight seal. the who laboratory biosafety manual necessitates biosafety level 2 (bsl-2) requirements for non-propagative diagnostic laboratories and bsl-3 for laboratories handling high concentrations of live sars-cov-2. according to the manual and the who biosafety guidance for sars-cov-2, the exhaust air from such laboratories should be discharged through high-efficiency particulate air (hepa) filters. it is worth mentioning that particle collection efficiency of such mechanical filtering methods decreases to about 50% at particle sizes of 0.5 µm due to diffusion and diffusioninterception regimes of particles with sizes in the range from 0.05 up to 1 µm [21] . therefore, hepa filters are not ideal in screening the viruses like sars cov-2 which has a typical diameter from 60 nm to 160 nm [22] . mass production of appropriate filters to screen the virus entry is a micromanufacturing challenge. this could potentially involve technologies such as direct laser micromachining or punch-based microstamping methods. for this purpose, a plasma spraying, or laser micromachining method may be used to obtain a well-suited punch which can be used to stamp (pierce) polymer sheets (e.g., pet), to achieve appropriate filter sizes. another candidate process of subtractive manufacturing is metal anisotropic reactive ion etching with oxidation (mario) [65] . an illustration of how the proposed micromachining strategies can be deployed for scalable fabrication of filters to screen virus scale particles is shown in figure 13 . while self-assembly techniques such as block copolymers have shown promise filtering small viral particles such as human rhinovirus [66] , direct printing methods allow greater engineering control over pore size and spacing, critical to tuning membrane efficiency, fluid resistance and mechanical strength. protection of surrounding or nearby people is important and an infected patient can help to control the spread by wearing a mask, leading to the concept of social distancing suggested by various governments. the safe distance guideline is an important concept especially in populous countries where an assembly of people on the streets can cause the spread to grow exponentially. keeping this in mind, the who urged people to maintain a safe social physical distance of about 3 ft (~1 m), while the centre of disease control and prevention recommends this to be 6 ft (~2 m). these limits are now challenged by recent research that suggests this safe distance should be 23 to 27 ft (7 to 8 m) [69] . the same study also suggested that the currently available commercial masks need to be redesigned. it was suggested that the masks available at present are not suitable for containment of sprayed aerosols (during a sneeze) travelling at the velocity of 108 kmph [70] , as this increases the possibility of escape of viruses through contaminated droplets from edges of the mask making nearby people more vulnerable to contracting the coronavirus. also, chin et al. [16] tested the strength of the virus and suggested that the virus is highly stable at 4ºc as well as in the ph range of 3 to 10. the virus was found to be detectable on a mask surface even after 7 days. these findings suggest a product design strategy to develop more appropriate masks for handling exhalations travelling at high speeds by considering human ergonomics, material aspects, antimicrobial nature and structural aspect. while the design and strategies for developing these new types of masks are underway, a possible technique for deploying the extant fabrication methods would be to merge the two approaches, namely, additive and subtractive manufacturing methods described above. this development would mean that even in the current landscape, a new mask-making strategy would provide more protection than the currently available masks. astm f2100-07 is the relevant international standard that details specifications of materials to be used in medical face masks. however, the current standards do not capture the techniques required to make the mask reusable especially as the current materials are yet to be tested against sars cov-2. as of now, curad antiviral isolation masks making use of a cocktail coating (citric acid, zinc and copper) are available as an option to disinfecting the outer surface of the mask. these are useful for medical professionals as they can come in contact with the virus infected aerosols more frequently. another concept is that of a 'germ trap' surface [71] , which is akin to fooling a virus. the team at the university of manchester, uk identified specific glycoproteins that have carbohydrates attached to their surface, similar to those seen on the surface of the cell of the nasal passages. this study suggests that a glycoprotein biocoating on a snood surface [72] denatures a virus by causing the same interfacial chemical reaction between the spike proteins and carbohydrate cell surface as the one when the coronavirus attaches to the ace2 receptors, but due to unavailability of any physical cells to invade on the snood surface, the virus decomposes and becomes deactivated immediately after landing on the textile surface (see figure 14 ). the efficiency of germ trap coating in achieving its goal is indicated to be 96%. based upon the existing literature, an immediate measure to functionalise the personal protective equipment's (ppes) could be to introduce the use of antimicrobial coatings comprising of proven materials (e.g., zinc and sodium [73, 74] based compounds) on the surface of ppes, especially the shields and visors. such coatings can be deposited by additive methods such as spraying (in 3d) or via roll to roll (r2r) manufacturing. in terms of processing techniques, one limitation of thermal spraying is that it requires heat resistant feedstock materials, limiting the use of most of the chemical species traditionally used to functionalise surfaces, such as polyethylene glycol (peg) [75] . in view of this limitation, a possible research avenue is to develop functional coatings with viricidal properties such that the coating ingredients do not degrade at high temperatures, thus providing a controlled release over time. with this goal in mind, a suitable processing and material matrix availability is crucial to obtain a coating to possess desirable advantages in the medical field. on the other hand, the use of thermal sprayed hydroxyapatite (ha) coatings on orthopaedic implants has been widely adopted in the medical field since the late 1980s [76] . as reported in the early 2000s [77] , the use of thermal sprayed ha coatings on metal implants presents several advantages and a positive potential for its use in the medical field [78] . taking into consideration the status of ha as the standard in thermal sprayed coatings and the decades of research on its behaviour and response to living tissue [79] , the choice of ha as a base material for the development of anti-microbial functional coatings has been most popular to date. at a personal level, deploying smart ppe incorporating viral detection capability into masks, gloves or wearing "viral dosimeter" badges might help monitor the strength of direct transmission vectors as well as environmental exposure rates and accumulation. conversely, masks could collect exhaled viral particles for early detection of infection [70] . detection could be implemented as microfluidic channels functionalized with bioelectronic miniaturized detection schemes [80, 81] with specific virus sensitivity [82, 83] . local area smart sensing filter textiles incorporated into mobile or in-place ventilation systems designed to both filter and detect could also help provide early alert alarms and protection in buildings and other areas of restricted air replacement. the importance of wearing a mask by the infected person and by a healthy person coming in the vicinity of the infected person is ranked on the priority order shown by the cartoon model in figure 15 . safe disposal of materials used in the treatment of infected patients such as gloves, masks, test samples, clothes or the human waste, especially in icu patients who may be closer to death is a challenging task. in addition to the possibility of an infection, this waste could lead to other secondary issues including emergence of a new category of virus/bacteria. hence, safe disposal of biomedical waste is very important. additionally, gaining support from members of the public to adequately dispose of their used personal masks etc. is essential. recently, an absorbent gel with embedded disinfecting material has been developed at sree chitra tirunal institute for medical sciences and technology, india for liquid respiratory and other body fluid solidification and disinfection for the safe management of infected respiratory secretions. this material helps to solidify the liquid respiratory secretions from icu patients or those with copious secretions treated in the wards. the material so collected becomes fit for disposal through the usual incineration system for biomedical wastes. newer guidelines are required to refine the existing risk assessments procedures [84] . big data analytics and extant research utilizes accelerated development of techniques for data mining, machine learning and use of artificial intelligence (ai) that shows promise in the treatment of sars cov-2. as a direct benefit to this approach, a digitalised shadow of the data to study plausible scenarios of certainty of an event becomes easier. as for sars cov-2, genome data now exist offering scope for soft-computing application researchers as well as those involved in ai based research to offer new insights into the area. as an example, the global initiative on sharing all influenza data (gisaid) database (https://www.gisaid.org), made available in march 2020, contains a compilation of over 24,000 sars-cov-2 complete and partial genomes from all over the globe since december 2019 [85] . recently, researchers from cambridge used phylogenetic network on 160 complete genomes of sars cov-2 [85] to predict probable mutation and migration paths of the coronavirus. atomistic modelling and molecular docking [86] can hold the key to a scientific breakthrough to address questions surrounding sars cov-2. the development of vaccines, antibodies and diagnostics is dependent in a large measure on our understanding of the interfacial science between the spike (s) glycoprotein of sars cov-2 and ace2 receptor in human lung cell (as shown earlier in figure 2 ). wrapp et al. [87] obtained a cryo-electron microscopy structure of the sars cov-2 s trimer in the metastable prefusion conformation, showing it undergoes a transient (hide and active states) structural rearrangement. their results relying on surface plasmonic resonance elucidated a stronger affinity of sars cov-2 with ace2 compared to sars cov-1, and shed light on two states, namely, a "down state" or receptorinaccessible and an "up state" or receptor-accessible state shown in figure 16 in yet another interesting study [89] , computer-aided drug screening was used to run a test assay over 10,000 compounds as inhibitors to a key cov enzyme, m pro . ebselen was found to exhibit antiviral activity and illustrate a way forward use of this potentially promising modelling strategy to discover targeted drug and vaccine down: transient hidden state of rbd up: active binding state of rbd development. a molecular docking example shown in figure 16 (b) is yet another effective example of accelerating the drug discovery [88] . recently, liu et al. [90] carried out an aerodynamic analysis by measurement of the rna of sars cov-2. they used pre-sterilized gelatin filters (pore size of 3 μm) to collect the aerosols from different areas of two wuhan hospitals. this study classified the sampling location in three areas: ( surprisingly, the researchers detected maximum stains of sars cov-2 in patients' toilet areas, while the levels were low in the isolation wards and ventilated patient rooms. also, the medical staff areas showed peaks of high concentrations with aerosol size ranging from 0.01 micrometres to 2.5 micrometres and they were neutralised after rigorous sanitisation procedures. the study suggests that there is a likelihood of resuspension of virus-laden aerosols from the surface of medical staff's protective equipment including surgical masks during their removal while having lunch, visiting toilets or breaks etc. the source of these virus suspensions was speculated to be from the direct deposition of patient's respiratory droplets or airborne sars-cov-2 onto the protective apparel. the researchers also found that the sars-cov-2 in aerosol forms had relatively longer residence time, implying the infection remains for a relatively longer time causing further transmissions. this study serves as a practice guide on the requirements and specification after the lockdown procedures are eased to avoid recurrent infection. the exit-strategy after the lockdown must consider carefully the ventilation of offices/classrooms, maximum use of open space (preferable sunlight), regular sanitisation of clothes, hairs and hands, and proper use and disinfection of toilet areas. the data obtained by toho university (figure 17) suggests that laser technology can be employed efficiently to monitor the streamlines and microdroplets movements from a sneeze. this monitoring could be a highly effective lab-scale activity to not just guide the futuristic cfd models but also to efficiently design new masks effective for preventing diseases caused by high velocity spray caused by a sneeze. while engineering and manufacturing will undoubtedly play their part over the coming years, the interventions proposed herein can help avoid the recurrent spread and emergence of new infections. the patients who have survived while battling against covid-19 showed that they have developed antibodies to the virus. antibodies are virus-specific proteins, which have a memory of the exposure to the virus and will recognize the virus on a second exposure. antibodies help prevent future infections by detecting the virus and binding to their surfaces signalling the body's immune system to destroy such viruses or virus-infected cells. while reasons are not clear, common wisdom is that the human body does not have an adequate immune response to hiv. for covid-19, an adequate immune response does exist. therefore, a vaccine is more likely to be created successfully for sars cov-2. additionally, the observation that a large majority of people recover, either without any symptoms or with minimal symptoms such as fever and body ache, also bodes well for the development of a vaccine. three major scenarios are currently being pursued or envisaged in tackling the disease. (i) research and trials on vaccines: clinical trials are being conducted across the globe into novel vaccines and fast-tracking approval processes are being deployed. however, scientists fear that the virus itself, like the flu, can mutate and form different strains. therefore, immunity after immunisation or contracting and surviving covid-19 may only last as short as two years. (ii) therapeutic trials using available drugs: attention is being focused to drug repurposing and advanced formulations, including antimalarial, hiv, and other antiviral and immune-modulating drugs and biomolecules. (iii) nanotechnology: study of molecules that show promise but exhibit poor physiochemical properties or toxicity is currently being formulated across the globe into advanced nanomedicine platforms, translating the knowledge gained in drug targeting and efficacy enhancement from conditions such as cancer and cardiovascular disease. such technologies aim to provide better treatment prognosis for those patients who fall ill with coronavirus related diseases. attention to immunemodulating molecules is growing, as patients who contracted severe forms of covid-19 were reported to experience notorious cytokine storms which ultimately 2 m 1 m lead to their death. ability to suppress such immunological responses could be one key to controlling the progression of the virus from mild to severe infection. lessons learned during this pandemic will also impact the outlook for other viruses. these include governmental pandemic funding allocation, stockpiling of medical protective equipment and emergency aid strategies within and between countries across the globe. as the coronavirus problem increased across the globe in 2020, many questions have been raised regarding what more governments could have done, why they did not act more quickly, and what were the fundamental failures within their policy. over time, addressing these issues will pave the way for a more streamlined and effective future response. in addition to this, contact tracing technology is being heavily invested in across the globe, with the intention that citizens would be able to download mobile applications, which could inform them (and the authorities) if they had been exposed to another person who tested positive to covid-19. whilst early trials seem positive, the longer-term question remains over privacy and ownership of personal data collected by such applications. access to death records inside countries and across continents will allow identification of high-risk population categories which will aid risk prevention. the positive news is that this pandemic has cemented the importance of scientific integrity, the inclusion of scientific advisors into government, and the role of science in society. while this may offer little solace at such an upsetting time, yet, with the power of science, this disease will be managed unlike many other viral threats over the years. currently, vaccine trials are ongoing at various phases of progress in different countries (see table 3 ) and good news may be expected soon. [95] . the world has seen a surge in the use of digital tools being growingly used for teaching, research, consultation, banking and day to day activities. digital health innovation has been a prime example. these approaches culminated in the development of telemedicine consultation. the current situation has also driven many changes in work-life routines such as less travel and digital technology-based remote work. the coronavirus pandemic has brought significant disruption and caused severe emotional, sentimental and financial losses. there is even a chance that life on the planet will never be the same again and many stringent safety measures will continue to be followed in the time to come while using public transport, air travel, or while visiting touristic places. the continued extension of lockdowns (e.g., lockdown 1.0 to lockdown 4.0 in india) has resulted in making people restless when staying indoors. countries who were not very resilient in adapting to the situation even saw disrupted supply chains, disturbed migrant workers, lack of food and other supplies and unavailability of essential items -a situation comparable to a natural disaster. however, every disaster brings forth a new cycle of life and the episode of covid-19 did bring some good things. countries facing severe pollution in air, water, and land saw a life not seen in the last many decades. social websites and digital tools were used heavily to share the beauty of nature. rivers got cleaner, the air became more breathable, and the sky got clearer [96] . as a result of this transition, even climate change and transition in weather were observed in many places and it will be befitting to say that we witnessed how "nature self-heals and mends itself". a prime example of climate change as a result of lockdown can be seen in china, europe and in india (see figure 18 ). india has faced two major challenges for many years in the wake of rapid urbanisation: alarming levels of air pollution and a rapid decrease in the availability of clean water, especially from rivers like the ganga. the government of india had earlier set up a mega project called "namami gange" [97] for freeing the rivers from industrial wastes. the project is of such importance that when addressing the indian community at madison square garden in new york in 2014, the prime minister of india mr narendra modi had said, "if we can clean the ganga, it will be a huge help for 40% population of the country." covid-19 has rejuvenated the ganga and many other rivers around the world, making them cleaner to a point that their water was reported to be potable [98] . the learning from the episode of covid-19 has been that resources offered by mother nature are meant to be used and not to be "exploited". if this simple rule is forgotten, then the world may continue to witness a regular self-healing cycle. on this occasion it was covid-19 but the next time, it could be readjustments in the ecosystem caused by a surge in the carbon emissions. researchers are reporting a decrease in solar activity leading to an increased flux of energetic particles in our galaxy [101] . before it is too late to realize, and the entire human race wipes-off the face of the earth due to the climate change, we must take this pandemic as a wake-up call towards being considerate to the environment. what has apparently come clearer though is that problems like biohazards primarily emerge due to our lifestyle (e.g., consumption patterns including eating habits) or mishandling of biowastes (see figure 19 ). there is an absolute necessity to pay utmost attention to biosafety and implementation of effective iso standards worldwide. health of people is also closely connected to the health of animals and our shared environment. this is because people, animals, plants, and our environment are interdependent [102] and this direction of research considers the concept of "one health". in the wake of the covid-19 pandemic, the world witnessed an all-time-high demand of ventilators, ppe's, masks, bed liners, other essential health supplies and medicines. the pandemic struck everywhere, including the largest manufacturing economiesand pretty much all at once. as a result, most nations experienced manufacturing shocks. the two shocks that have impacted manufacturing worldwide are: • supply shock: the containment measures (such as lockdowns and social distancing) kept the workers away and resulted in reduced productivity and output. • demand shock: customer consumption patterns started changing (e.g., movement away from "non-essentials") and this affected the demand for a large variety of manufactured goods and associated services. as the pandemic spread, a few countries were resilient enough to cope with the pace of transformed supply chain requirements while many others who had earlier (in pre-covid-19 era) aligned themselves to outsourced or cloud-based manufacturing, primarily driven by low-cost, became hugely dependent for their essential supplies on other countries (primarily china the manufacturing related lesson learned from the covid-19 episode is that pandemics can affect manufacturing in several geographical locations simultaneously and therefore can affect an individual nation's surge capacity to deliver essential supplies such as diagnostics, drugs and vaccines to its population. going forward, this lesson will critically affect thoughts on manufacturing strategy and low-cost based outsourcing, and we may see a sustained push towards development of resilient indigenous manufacturing capabilities and a decreasing reliance on low-cost based outsourced manufacturing. a second trend that we perceive is an increasing focus on digitised automation and use of automated production systems including the use of robotics in day to day life as well as deployment of embedded robotics in manufacturing operations. an example of a futuristic automation capability was displayed at the indian institute of technology guwahati who developed remote controlled food delivery robot for covid-19 isolation wards (see figure 20) . the robot was designed for 6 hours run when fully charged. antimicrobial resistance (amr) refers to the resistance of microbes to antimicrobial drugs or "microbial immunity". the who as well as several national health organizations have identified antibiotic resistance as one of the greatest threats to global public health, economic growth, agriculture, economic security and national security. around the globe, people are being admitted to hospitals with infections that do not respond to antibiotic treatment. the problem of amr is rooted in the fact that when microbes are repeatedly exposed to antibiotics, they mutate to produce strains that are resistant to the antibiotic and are therefore able to resist the effects of medication that could successfully treat the microbe. in 2014, lord jim o'neill and his team published a review commissioned by the united kingdom government entitled, "antimicrobial resistance: tackling a crisis for the health and wealth of nations" (the amr review) which is in line to the question that de kraker et al. [104] asked "will 10 million people die a year due to amr by 2050?" the problem of amr is connected to that of covid-19 with respect to the emergence of zoonotic diseases. the trend of overuse and/or misuse of antimicrobials, excess use of certain antibiotics in animals, and pharmaceutical industry pollution can lead to continuous emergence of zoonotic diseases. excessive use of antimicrobials stresses the naturally occurring microbiome and allows for resistant bacteria to become dominant. indeed, research has suggested that amr might spread to humans through food products of animal origin, the environment, and by direct contact in the case of agricultural workers. addressing the issue of amr as well as others shown in figure 21 is thus a global priority as its impact on claiming lives is no less than that of covid-19 [105]. the rapid emergence of the covid-19 pandemic provided minimal time for welldirected resource mobilization, and almost every country was tested for its resilience in its medical and manufacturing abilities during the first half of 2020. the number of tragic health cases pushed organizations such as the fda to allow emergency use authorisation of various drugs without a systematic testing protocol, and among the 70 drugs tried, favipiravir and tocilizumab were rated most effective. amidst the chaotic situation, the growing use of digital tools for professional communications and artificial intelligence to monitor the recovery of covid-19 patients showed some positive outcomes. a major question remains open to date, "how can the symptoms of covid-19 be detected in their early stages?" while more research is needed on the instrumentation and manufacturing sides, there are clear opportunities identified from the available scientific knowledgebase. in relatively advanced stages of the symptoms, rapid measurements by combining chest computer tomography (ct) and reverse-transcription polymerase-chain-reaction (rt-pcr) tests seem to provide a high confidence level in the screening. more work is needed on the sensing side for detection of the virus in the primitive stages. definitive promise has been exhibited using laser technology to monitor the aerosol spread and to detect the rna of the virus and gain a deeper understanding of the aerodynamic properties of emergent viruses. further development of fumigation chambers and ultra-violet chambers seem to hold promise as precautionary measures that can be implemented in public places like malls, airports, theatres and train stations etc. in this paper, we have also highlighted the possibilities of developing novel tools such as lab-on-the-chip, in addition to developing advanced personal protective equipment (ppe) using combinatorial additive and subtractive manufacturing techniques such as roll-to-roll manufacturing and thermal spray. these technologies allow the capability to filter the coronavirus, which has a diameter in the range of 100 ± 60 nm. it is envisioned that in future, wearing masks and social distancing would be mandated to avoid the recurrent spread of the virus. in the long term, micro-manufacturers, medical professionals, metrology engineers, material scientists and virologists will need to work in a more interdisciplinary way to develop modelling informed fabrication strategies. accelerated use of molecular modelling approaches -like molecular dynamics and molecular dockingalso seems to hold promise in drug and vaccine discovery to end this pandemic from its root. as society progresses to meet challenges such as covid-19, many overarching issues will become important. strong measures will need to be implemented to ensure careful handling of biowastes, indigenous capability-based manufacturing focus will become stronger and issues such as antimicrobial resistance will demand increased attention. our hope is that the post-pandemic society will better heed the warning signs from nature and work on making manufacturing systems resilient to 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antimicrobial resistance by 2050? plos medicine authors are very much thankful to the research support provided by the ukri via grants no.: (ep/k503241/1, ep/l016567/1, ep/s013652/1, ep/t001100/1, ep/s036180/1 and ep/t024607/1) as well as the gcrf/ epsrc supported sunrise program (ep/p032591/1). additionally, we acknowledge the support received from h2020 (cost actions (ca18125, ca18224, ca17136 and ca16235) and euramet empir a185 (2018)), royal academy of engineering grant no. iapp18-19\295 (indo-uk partnership), royal academy of engineering grant no. tsp1332 (south africa-uk partnership) and newton fellowship award from the royal society (nif\r1\191571). also, numerical calculations performed on the isambard bristol, uk and archer hpc were made available by the epsrc resource allocation panel (rap). all data in the manuscript will be available through cranfield university open repository. key: cord-303517-8971aq02 authors: cajamarca-baron, jairo; guavita-navarro, diana; buitrago-bohorquez, jhon; gallego-cardona, laura; navas, angela; cubides, hector; arredondo, ana maría; escobar, alejandro; rojas-villarraga, adriana title: sars-cov-2 (covid-19) in patients with some degree of immunosuppression date: 2020-10-16 journal: nan doi: 10.1016/j.reumae.2020.08.001 sha: doc_id: 303517 cord_uid: 8971aq02 background it is not clear whether patients with some degree of immunosuppression have worse outcomes in sars-cov-2 infection, compared to healthy people. objective to carry out a narrative review of the information available on infection by sars-cov-2 in immunosuppressed patients, especially patients with cancer, transplanted, neurological diseases, primary and secondary immunodeficiencies. results patients with cancer and recent cancer treatment (chemotherapy or surgery) and sars-cov-2 infection have a higher risk of worse outcomes. in transplant patients (renal, cardiac and hepatic), with neurological pathologies (multiple sclerosis (ms), neuromyelitis optica (nmods), myasthenia gravis (mg)), primary immunodeficiencies and infection with human immunodeficiency virus (hiv) in association with immunosuppressants, studies have shown no tendency for worse outcomes. conclusion given the little evidence we have so far, the behaviour of sars-cov-2 infection in immunosuppressed patients is unclear, but current studies have not shown worse outcomes, except for patients with cancer. in december 2019, a group of five patients with severe pneumonia of unknown origin were reported to have had contact with a seafood market in the city of wuhan, hubei province, china, as an epidemiological link. the chinese centre for disease control and prevention (china cdc), deployed a rapid response for the epidemiological and aetiological investigation of cases and identified a new coronavirus with the ability to cause severe lung disease that can rapidly progress to death in affected patients. 1, 2 given its rapid progression and the poor knowledge of the infection, how it behaves in patients with multiple comorbidities is not clear, especially patients with some degree of immunosuppression, due either to their underlying disease or to the use of immunosuppressants to manage it. in this review, we will focus on describing the literature on sars-cov-2 infection in patients with some degree of immunosuppression, other than rheumatological diseases, among these, cancer patients, transplant recipients, primary immunodeficiency, and hiv patients. initially the virus was termed the new coronavirus (2019-ncov) and variations of same. sars-cov-2 is the name currently used for the virus, which shares genetic similarities with the sars-cov virus. covid-19 (coronavirus disease 2019) is the name of the disease generated by sars-cov-2 infection. 3 j o u r n a l p r e -p r o o f epidemiology from december 18th to 29th, samples of bronchoalveolar lavage fluid were collected from patients hospitalized for severe pneumonia in the city of wuhan, the epicentre of the pandemic, and the new coronavirus was isolated. results for viruses such as severe acute respiratory syndrome (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), influenza, avian influenza, and other common respiratory pathogens were negative. 1, 3 on january 24, a case series of 41 confirmed sars-cov-2 patients treated at a wuhan hospital was released. most were male (73%), with a median age of 49 years, and less than half had comorbidities (32%) such as diabetes mellitus (20%), hypertension (15%), and cardiovascular disease (15%). of these patients, 66% had a history of exposure to the seafood market. 4 in another case series of 138 hospitalized patients in wuhan, china, 41% of the infected patients were presumed to have been infected by nosocomial transmission, 26% of the patients required icu hospitalization, and mortality was about 4.3%. 5 the infection rapidly escalated and was declared a public health emergency by the world health organization (who) on january 30, 2020. as of today, june 27, 214 affected countries have been reported, and the number of confirmed cases worldwide is close to ten million (9, 770, 954) , with a total of 493,898 deaths and 5,391,416 cases that have recovered. the country with the most confirmed cases is currently the united states, followed by brazil and russia. china, the first country affected, has a declining case curve and is now in twentieth place worldwide. 6 in the americas, there are 4,933,972 confirmed cases with 241,931 deaths; the united states and brazil together account for 75% of all cases and 75% of all deaths currently reported in the region. 7 infection in children is less frequent and most reported cases are among family members and, to a lesser extent, from close contact with infected patients. 8 j o u r n a l p r e -p r o o f the coronaviruses (cov) are a group of viruses discovered in 1960 that have a single strand of rna (~26-32 kb in length) that codes for structural, envelope, membrane and nucleotide proteins, as well as for non-structural proteins. 9 they belong to the coronaviridae family which in turn is part of a larger family, the nidovirals. the coronaviridae family is divided into two subfamilies: orthocoronavirinae and torovirinae. the former is classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. they are zoonotic viruses, bats have been acknowledged as natural hosts, but six types have been recognized as having the ability to infect humans: two alpha-coronaviruses (229e and nl63) and four betacoronaviruses (oc43, hku1, sars-cov and mers-cov). 1, 9, 10 in 2003, there was an outbreak of sars-cov that caused 794 deaths worldwide. in 2012, mers-cov was discovered in middle eastern countries with a fatality rate of 35.5%. 1 sars-cov-2 is a betacoronavirus, subgenus sarbecovirus and from the subfamily orthocoronavirinae with an envelope composed of a lipid bilayer derived from the host membrane. the genome encodes for spike glycoprotein (s), small envelope protein (e), membrane protein (m), and nucleocapsid protein (n). it also encodes accessory proteins that interfere with the host's immune response. 11 its name is due to its similarity to a crown, given the spherical morphology of the virus and the projections on its surface that correspond to the s protein, which is glycosylated and mediates the viral entry into the host cells. the m protein gives the shape to the viral particle and together with the e protein directs the assembly of the virus and its maturation. the n protein participates in the packaging of the viral rna during assembly. haemagglutinin is one of the accessory proteins, which binds to sialic acid in host glycoproteins, improving entry into the cell. 10 like sars-cov, sars-cov-2 uses the receptor for the angiotensin 2 converting enzyme (ace2) as a means of entry into the cell where it binds by means of the s protein, however, unlike the other viruses, sars-cov-2 binding is much stronger since this protein undergoes a residue substitution in its c-terminal domain that increases affinity for the receptor. 11, 12 the s protein has two subunits, s1 which determines the cell tropism and s2 which mediates the fusion of the virion to the membrane so that it can enter the cell where it rapidly translates two polyproteins that form the replication/transcription complex into a double-membrane vesicle; the virion contained in these vesicles fuses with the plasma membrane to be released later. 11 the viral genome found in cytoplasm acts as pathogen-associated molecular patterns (pamps) and are recognized by the molecular pattern recognition receptors (prrs) that are toll-like receptors (tlr 3, tlr7, tlr8 and tlr9). the rig-i receptor (retinoic-acid inducible gene-i), the cytosolic receptor mda-5 (melanoma differentiation-associated gene 5) and cgas (nucleotidyltransferase cyclic gmp-amp synthase) recognize viral rna and recruit adaptive molecules that trigger a response cascade leading to the activation of the nuclear transcription factor- and interferon regulatory factor 3 (irf3), producing interferon  and  and pro-inflammatory cytokines. 11 different elevated cytokines have been found in patients with covid-19: il-1, il2, il-4, il-7, il-10, il-12, il-13, il-17, macrophage colony-stimulating factor (mcsf), mcp-1, hepatocyte growth factor (hgf), ifn- and tnf-. this supports the fact that lung damage is secondary to a cytokine storm induced by the inflammatory response, resulting in the person entering a critical condition. 11, 13 the transmission dynamics are not yet fully known. the intermediate host between the natural reservoir and humans is unknown. however, it has been possible to confirm person-to-person transmission, which contributes to the rapid spread of the disease, and this is confirmed by the data found in the case series, which show that as of january 1, 55% of cases were linked to the seafood market in wuhan (china); however, of the cases reported after this date, only 8.6% had this link. 14 to date, person-to-person transmission has been considered to occur via respiratory droplets produced by coughing or sneezing. however, the presence of the virus has been detected in other fluids such as blood, faeces, and saliva. 15, 16 initially, it was believed that spread was by people with clinical manifestations. however, it has been shown that asymptomatic carriers also transmit, and some people have even been recognized as "super spreaders", infecting many people, including health workers. 3 vertical transmission of the virus in pregnancy has not been proven, however it is not known whether there is a risk during delivery through the vaginal canal. 17 clinical manifestations can range from being asymptomatic to acute respiratory distress syndrome and multiorgan dysfunction. the incubation period is estimated at 5.2 days; however, this can vary. 18 in the first case series of zhu et al. and ren et al. of patients treated at a hospital in hubei, the predominant symptoms were dry cough, dyspnoea, and fever. lung opacities consistent with a pneumonic process, worsening rapidly (two to four days) and requiring invasive mechanical ventilation, are recorded. 1, 2 in general, the most common clinical manifestations are fever (although not present in all cases), cough, odynophagia, fatigue, and myalgia. other less frequent symptoms are sputum production, headache, haemoptysis, and diarrhoea. cases of keratoconjunctivitis and fulminant myocarditis have also been described. 4, 5, 14 in early stages of the disease, chest x-ray can be normal, but as the disease progresses, bilateral ground glass opacity or consolidation can be found in more than 89% of patients. chest ct is much more sensitive than radiography. these findings can be found in asymptomatic patients. 13, 19 it has been reported that up to 17% of patients will have no radiological changes. 20 as for laboratory findings, lymphopenia was common, found in more than 80% of patients, with less frequent evidence of thrombocytopenia and leukopenia. a large proportion presented with elevated c-reactive protein, and in fewer cases, elevated alanine aminotransferase, aspartate aminotransferase, creatine kinase, and d-dimer were found. disturbances are pronounced in patients with severe disease. 20 in the different case series and epidemiological reports published since the appearance of sars-cov-2, comorbidities have been highlighted as risk factors associated with severity. it has also been established that patients admitted to the intensive care unit have more comorbidities than those in general hospitalization. the most prevalent underlying diseases are high blood pressure, diabetes mellitus, cardiovascular and cerebrovascular disease. 21, 22 the results of nine metaanalyses in patients with covid-19 published to date are summarised in tables 1 and 2. table 1 presents the prevalence of comorbidities in patients with covid-19, most of them hospitalized, and analysed through seven meta-analyses. in turn, table 2 shows the risk associated with severity, death, or fatality due to the presence of comorbidities, through seven meta-analyses. the meta-analysis by wang et al. is noteworthy, which identifies arterial hypertension, diabetes mellitus, chronic obstructive pulmonary disease (copd), cardiovascular and cerebrovascular disease as factors that negatively impact mortality. specifically, copd increases by 5.9 times 23 the risk of progression and deterioration of patients with covid-19. these data are supported by the results of another meta-analysis by zhao et al. where the severity of covid-19 is four times higher in copd patients; they also assessed the impact of active smoking, which increases the risk of severe covid-19 two-fold. 24 it has been widely demonstrated that patients suffering from diabetes mellitus are admitted more frequently to the intensive care unit and have a higher mortality, as shown in table 2 . the results of a primary study by roncon et al. are highlighted (or 3.21, 95% ci 1.82-5.64; p < .0001, i 2 = 16%). 25 these patients tend to present a more severe pneumonic process, with greater inflammatory response and tissue damage, which makes them more prone to cytokine storm leading to rapid deterioration, which is why patients with this history should be strictly monitored. 26 cardiovascular disease is a risk factor per se increased by covid-19 infection that generates or aggravates myocardial damage, but when associated with myocardial injury the results are usually fatal for patients. 27, 28 among other comorbidities, chronic kidney disease is associated with in-hospital mortality, as are cancer and cerebrovascular disease, demonstrated through two meta-analyses that included over fifteen thousand patients ( table 2) ; studies suggest that superficial fungal infections and psoriasis confer vulnerability to covid-19; a body mass index (bmi) > 40 kg/m2 is an independent risk factor for complications from the infection; and there are discouraging results regarding underlying neurological disease and sars-cov-2. [29] [30] [31] [32] in general, the presence of comorbidities should imply strict follow-up of patients to detect early complications; however, more attention should be paid to certain comorbidities where strong associations have been found with covid-19 infection and its severe outcomes. overall mortality is .5% to 4%, but it changes to 5% to 15% among patients requiring hospitalization and increases to 62% in critically ill patients. studies have shown two peaks, at 14 and 22 days. among the causes of death, respiratory failure prevails, followed by shock due to myocardial dysfunction and finally, the combination of the two. 33 it is important to maintain a high degree of diagnostic suspicion in patients with fever or respiratory symptoms who have travelled to affected areas or have had close contact with j o u r n a l p r e -p r o o f suspected or confirmed cases 14 days before the onset of symptoms. in this scenario, confirmation by molecular testing is required. real-time reverse transcription polymerase chain reaction is performed on specimens collected from the lower or upper respiratory tract if the former cannot be obtained. 34 there are several assays that are performed on serum or plasma for the detection of both viral proteins and antibodies. the most widely used are to detect immunoglobulin g (igg) and immunoglobulin m (igm) antibodies, which are produced in the second week of infection. 35 there are several therapeutic goals and they are directed at different levels: inhibition of virus entry into the cell, inhibition of fusion of the viral envelope to the membrane, transcription inhibition, inhibition of viral proteins and blockade of il-6 signalling to prevent the cytokine storm. 35 at first, chloroquine (clc) and hydroxychloroquine (hcq) were shown to block sars-cov-2 in vitro, with better results for the latter and therefore its use was indicated for the management of covid-19 infection. 36 promising results have been shown in case series; however, the studies have limitations such as the sample size used. however, preliminary results were recently published from the recovery study, 37 a randomized clinical trial to evaluate potential drugs for management of the infection in the united kingdom, in which they concluded no beneficial effect of the use of antimalarials in hospitalized patients, and therefore they stopped including patients for this treatment arm and the recommendation that it should not be used has been extended worldwide. additional results have recently been published in several articles on the efficacy and safety of the antimalarials chloroquine and hydroxychloroquine for the treatment of different phases of sars-cov-2 infection. however, the data are controversial, some not demonstrating efficacy or reporting a high number of adverse events, primarily associated with cardiac arrhythmias. 38 it is important to note that a number of criticisms and concerns have been raised regarding the accuracy of the data from these studies and they have therefore been withdrawn. 39 to date there are more than 200 clinical trials underway with hcq, and 80 with clq, 35 several of which are in prophylactic use in healthcare workers and others post-exposure. 36, 40 lopinavir/ritonavir has studies in sars and mers, the data published for covid-19 are reports and retrospective studies with which the effect cannot be established with certainty. there is a clinical j o u r n a l p r e -p r o o f trial of 199 patients with covid-19 with no difference in mortality, hospital discharge or recovery. however, despite this, in some centres it is still being used at doses of 400 mg/100 mg twice a day for 14 days. 35 ribavirin, like lopinavir/ritonavir, has activity against other coronaviruses and was considered to be a possible treatment for sars-cov-2; however, studies carried out for sars show limited, highdose activity leading to a high rate of haematological and hepatic adverse events, therefore its use is now limited. 35, 41 other antivirals such as oseltamivir were used in the first cases in hubei, china, because it was suspected to be seasonal influenza; they are now not indicated for use in sars-cov-2. 35 remdesivir is a nucleoside analogue that showed in vitro activity against sars-cov-2, used later in a 53-patient cohort in canada, the united states, europe, and japan, achieving a satisfactory response in 36 patients. 42 based on preliminary studies, some drug regulatory agencies (united states and japan), 43 conducted emergency approvals for use in hospitalized patients. results from ongoing studies are expected to evaluate efficacy and safety. currently umifenovir or arbidol is under study, an antiviral that aims to inhibit the interaction between protein s and ace2. 35 the use of corticosteroids is limited in sars-cov-2 infection to scenarios of chronic obstructive pulmonary disease exacerbation and refractory shock, taking into account previous studies in influenza pneumonia where they were associated with increased mortality. 35 recently, preliminary results from the recovery study 44 showed that the use of dexamethasone reduced mortality in one-third of critically ill patients who were on mechanical ventilation, while reduction was onefifth in those receiving non-invasive oxygen. definitive results are expected from this and the more than 10 clinical trials currently underway to define the particular subgroups that would benefit from this treatment. monoclonal antibodies against il-6 are another therapy studied, in phases of adult respiratory distress syndrome (ards), with promising results in small case series. 35 convalescent plasma is another therapy used as salvage in sars and mers. at the beginning of the pandemic, a case series of five critical patients from china, who were given convalescent plasma, showed improvement in their clinical status. more recently, several case series and preliminary trial results have demonstrated clinical benefits and decreased mortality with its use, 45 particularly in hospitalized patients with moderate to severe involvement; however, results from more than j o u r n a l p r e -p r o o f 200 clinical trials 46 are awaited to clarify the characteristics of the plasma, the donors, and the specific individuals who could benefit. 47 despite the abovementioned therapies, there is still no specific treatment and therefore the recommendations are symptomatic management in mild cases, supportive therapy in cases of critical illness and management of ventilation in cases of ards. 35 immunosuppression and sars-cov-2 given the suddenness of the pandemic, and its rapid spread, little is known about sars-cov-2 infection and certain types of condition or disease, this is the case for people with some type of immunosuppression (either primary, associated to underlying or pharmacological diseases), which given the physiopathogenesis of sars-cov-2 infection known so far, would raise two hypotheses: it could be a possible benefit, since this state of immunosuppression could avoid that uncontrolled immune response or "cytokine storm", but on the other hand, it is equally clear from previous studies that immunosuppressant use or status is associated with increased risk of infection. in epidemics such as the abovementioned sars-cov, immunosuppressed patients, especially transplant recipients, did not have worse outcomes than the general population. 48, 49 similar findings were presented in the mers epidemic, being male, advanced age and comorbidities such as diabetes mellitus, obesity, pulmonary pathology and renal disease being found as risk factors, and immunosuppression status not being associated as a factor of poor prognosis. 50 to date, the centres for disease control and prevention (cdc) and other international agencies 51 have included as poor prognostic factors patients with some degree of immunosuppression, including people with a history of cancer treatment, smokers, transplant recipients, people with immunodeficiencies, poorly controlled hiv or aids and people with prolonged use of steroids or immunosuppressive drugs, all based on previous studies that associate such diseases with respiratory infections, especially of viral aetiology. 52 current evidence of conditions associated with immunosuppression and sars-cov-2 infection j o u r n a l p r e -p r o o f c a n c e r on march 21, liang et al., 53 published a study collecting data from 575 hospitals in china, up until january 31, 2020, on patients with sars-cov-2, comparing those with a history of cancer and those without. they collected 1,519 patients, 18 (1%) with a history of cancer. the most frequent neoplasm was lung (five cases [28%]), and of the total cancer patients, four (25%) had undergone chemotherapy or surgery within the previous month, and the rest were cancer survivors, with strict follow-up. in terms of sociodemographic characteristics, the cancer patients were older, had a greater history of exposure to cigarettes, presented more polypnoea and had more severe pulmonary tomographic manifestations. in the analysis of outcomes, they showed that patients with a history of cancer and sar s-cov-2 infection were at greater risk of serious events (defined as the percentage of patients admitted to the intensive care unit requiring invasive ventilation or death) compared to patients without cancer (seven (39%) of 18 patients vs. 124 (8%) of 1,572 patients; p = .0003). in addition, patients who underwent chemotherapy or surgery in the last month had a higher risk (three (75%) out of four patients) of clinically severe events than those who had not undergone chemotherapy or surgery (six (43%) out of 14 patients). these data were confirmed by logistic regression (odds ratio (or) 5.34; 95% ci 1.80-16.18; p = .0026) after adjusting for other risk factors such as age and smoking history. in addition, the patients with cancer deteriorated more rapidly than those without cancer (median time to severe events 13 days (ci 6-15 vs. 43 days, 20-unreached; p <.0001). furthermore, desai et al., 54 recently published a meta-analysis, in which they included 11 studies, finding a prevalence of cancer in patients with covid-19 of 2% (ci 2.0%-3.0%; i2 = 83.2%). we should clarify that some authors consider that the current evidence is insufficient in this field, however, the number of research results has been increasing, showing similar results. 55, 56 taking into account the results mentioned, it can be stated that cancer and its recent treatment are bad prognostic factors for sars-cov-2 infection. therefore, special recommendations should be considered for these patients, such as postponing adjuvant chemotherapy or elective surgery in people with "stable" cancer, especially in endemic areas, adopting stricter personal protection measures for cancer patients or cancer survivors, and considering stricter surveillance or treatment when cancer patients are infected with sars-cov-2. 57 in general, decisions should be made on a "patient-to-patient" basis. 55, 58 t r a n s p l an t we highlight the study of a case series, 59 two heart and kidney transplants and one liver (paediatric population). the heart transplant patients were confirmed by pcr to be infected, one of them was 51 years old, came with immunosuppression with tacrolimus 2 mg per day and mycophenolate 1 g per day, and attended consultation for fever, fatigue and liquid stools, with characteristic findings of sars-cov-2 infection on chest tomography. he presented criteria of severe pneumonia, immunosuppression was discontinued, and he was managed with immunoglobulin (ivig) 10g/day and methylprednisolone 80 mg/day and made adequate medical progress. the second patient, 43 years old, in immunosuppression with tacrolimus 3.5 mg a day and mycophenolate 1 g a day, attended with fever and fatigue, had lymphopenia, did not require hospitalization, nor discontinuation of immunosuppression, and was managed with ceftriaxone and ganciclovir, with an adequate outcome. their adequate clinical course suggests that in patients with this type of transplant the disease has a similar presentation to non-transplanted patients. 59 it should be noted that in a series of seven cases 60 (two liver, three kidney, one lung and one heart) an initial attenuated inflammatory response was evident, suggesting that although patients with transplant immunosuppression may have higher susceptibility to sars-cov-2 infection, their clinical course could be similar to that of immunocompetent patients. with regard to the renal transplantation patients, until the time of the report, a 50-year-old patient remained in icu managed with lopinavir/ritonavir, requiring suspension of immunosuppression who came under management with tracrolimus, everolimus and prednisolone at intermediate doses. he was admitted for fever and vomiting that progressed to respiratory symptoms, and had thrombocytopenia, lymphopenia, and elevated d-dimer as factors of poor prognosis. the second, a 52-year-old, under immunosuppression with tacrolimus, mycophenolate, and prednisolone, consulted for fatigue, abdominal pain, dyspnoea, fever, and dry cough, presented lymphopenia and imaging findings typical of sars-cov-2 infection. he was managed with ivig (5 g, then 10 g/day x 11 days), methylprednisolone 40 mg/day and interferon α (5 million/u day), in addition to suspension of immunosuppression, and responded adequately to treatment. with regard to this type of transplant, the atypical presentation of the first case is noteworthy, and the adequate response of the second that could be associated with the use of multiple therapies, without it being possible conclude whether renal transplantation is associated or not with a worse prognosis. 61 gandolfini et al., 62 publish two cases of renal transplant and covid-19, a 75-year-old male and a 52-year-old female patient under management with tacrolimus, corticoids and mycophenolate, who developed severe pneumonia; in addition to suspending immunosuppressants, management with hydroxychloroquine, lopinavir/ritonavir and colchicine was started, due to the unavailability of tocilizumab. the administration of colchicine achieved an impact in decreasing il-6 serum levels, thanks to its interfering with inflammasome assembly which leads to the production of il-1b and other interleukins such as il-6. in italy, the paediatric liver transplant group of hospital papa giovanni xxiii bergamo followed up 700 liver transplants (two in the last three months), associated with autoimmune liver diseases (100 patients), three additionally in chemotherapy (for hepatoblastoma). of the total number of transplant recipients, three were confirmed to be infected with sars-cov2, and all remained asymptomatic without requiring hospitalization or suspension of immunosuppression. additionally, qin et al. 63 , report the case of a patient with hepatocellular carcinoma who underwent liver transplantation and suffered an undetected sars-cov-2 infection in the perioperative period; immunosuppression was initiated with tacrolimus and glucocorticoids; however, persistence of fever led to confirmation of sars-cov-2 infection; management with oseltamivir and immunoglobulin was initiated, and despite a prolonged convalescence, they did not present multiorgan failure, thus immunosuppression was maintained. the importance of sars cov-2 detection is highlighted for organ receptors and donors to reduce the transmission and risk of severe infection or rejection due to adjustments in immunosuppression. given the above, it is not clear whether transplantation and use of immunosuppressants in this context is a risk or severity factor for sars-cov-2 infection. likewise, in the event of sars-cov-2 infection, the adjustment or suspension of immunosuppressors should be assessed, and we should always seek to protect graft function with the administration of glucocorticoid doses and support measures, among others. 64 neurology is a continuously growing specialty. many diseases have a component that compromises autoimmune aggression to a greater or lesser extent and therefore go on to require immunosuppressant or immunomodulatory management. within the multiple entities, two diseases have become relevant in recent times, multiple sclerosis and optical neuromyelitis, due on the one hand to their physiopathological mechanism that involves neurodegeneration and inflammation by excessive activity of the immune system derived from antigenic epitopes and proinflammatory molecules, and on the other hand the use of therapies that trigger regulation of immune cells, affecting in some cases innate and adaptive immunity in most cases. if we consider that the response mechanisms to viral infections are based on inhibition of the infection by type i interferons and the death of the infected cells by nk lymphocytes (innate immunity), the generation of antibodies that block the union and entry of the virus into the cells, and the elimination of cells infected by cytotoxic t cells (adaptive immunity), the different drugs currently used could to a greater or lesser extent alter the immune response to sars-cov-2 infection, and this is why there are now different considerations when initiating or continuing therapies. 65 people with ms are at higher risk of admission to the intensive care unit due to infections, and higher mortality at one year after admission than the general population. the use of diseasemodifying therapies implies a higher risk of infections, however, to date there is no data to indicate that patients with ms are at higher risk of sars-cov-2 infection, or more severe infection. it is even possible that such disease-modifying therapies and their immunosuppressive effect may play a protective role during 19-covid infection by preventing or dampening hyperimmune activity that, in some cases, could lead to clinical deterioration; there is even a report of a patient with primary progressive multiple sclerosis receiving treatment with ocrelizumab and becoming infected with sars-cov-2, in the context of lymphopenia and hypogammaglobulinema expected for this type of treatment, without generating major clinical complications, this hypothesis is obviously limited for now only to academic deductions and limited information. 66, 67 in recent results of the multicentre registry covisep, 68 which includes information from 347 patients with ms and covid-19, it was demonstrated that age, obesity and highest score in the expanded disability status scale, were independent risk factors for severity of covid-19. it is suggested that people with ms and related disorders receiving immunotherapy continue to receive the therapy during mild viral infections. in those with documented mild sars-cov-2 infection, it may be reasonable to continue treatment. neurologists should have a lower threshold for suspending treatment in people taking therapies with greater immunosuppressant effects. 69 consideration should be given to suspending treatment in those who are hospitalized with severe or complicated sars-cov-2 infection. treatment may be restarted after four weeks or when symptoms have completely resolved, considering the risk of rebound of ms activity with s1p modulators and natalizumab. neurologists should alert intensive care physicians to the importance of fever management in people with ms. 70 in people with ms and disease-modifying treatment, the decision to start, continue, temporarily suspend, or defer doses should be individualized, 71 taking into account factors such as disease activity and the possibility of disease progression, as well as considerations of the mechanism of action of the drugs and their ability to deplete lymphocytes. recommendations from experts suggest not suspending first-line drugs (interferons, glatiramer acetate, teriflunomide, or dimethyl fumarate) and considering deferring therapies such as cladribidine and alemtuzumab based on their ability to deplete lymphocyte counts rapidly and aggressively. 72, 73 a survey of patients with nmosd or ms from china, from 10 centres, did not find an increased risk of infection by covid-19, suggesting as a possibility the role of self-care and protective measures taken by patients and their healthcare team, regardless of their condition and immunosuppression drug. 74 relapses in patients with nmosd can be devastating and patients should be encouraged to continue therapies for the prevention of attacks, including corticosteroids, azathioprine, mycophenolate mofetil, rituximab, tocilizumab and eculizumab. if there is a clinical need to discontinue or delay treatment in patients with nmosd, corticosteroids may be used in moderate doses to prevent relapses in the short-term, it is important to consider individualized therapy and comorbidities when deciding on management of this condition during the covid-19 pandemic. 69 my a s t h e n i a g r a v i s / l a mb e r t -e a to n my a s t h e n i c s y n d r o me ( mg / l e ms ) because most patients with myasthenia gravis (mg) are on immunosuppressant or immunomodulatory therapies and may also have muscle weakness and ventilatory failure, there is a theoretical concern that they may be at increased risk for infection or experience severe manifestations of sars-cov-2 infection. in a series of five cases 75 with mg hospitalized for covid-19 infection, a variable clinical course was demonstrated, with three requiring mechanical ventilation and one presenting mg crises, and although it is difficult to assess the latter due to intubation and sedation in two of the cases, none had a fatal outcome. two additional cases have been reported, one developing crises due to myasthenia 76 and the other with chronic refractory mg, 77 with good outcome, without complications or worsening of their baseline condition. there are numerous recommendations circulating that attempt to provide clarity and guidance. however, the differences between the recommendations have created confusion, because decision-making varies in different countries, and due to the lack of databases with an adequate number of patients. patients with mg/lems should continue their treatment and are advised not to discontinue any existing medications; there is no scientific evidence to suggest that symptomatic therapies such as pyridostigmine increase the risk of infection and they should not be discontinued unless there are other clinical reasons to do so, given the risk of increased disease activity and/or mg exacerbation or crisis. 1 with regard to certain therapies (immunoglobulins, plasmapheresis) there is no information pointing to increased risk of infection, however, the use of immunoglobulin should be based on the individual need of the patient and indiscriminate use should be avoided. in general, these therapies should be reserved for patients with acute exacerbation and if required as maintenance therapy on an exceptional basis, additional precautions should be taken. in patients with severe sars-cov-2 infection, temporary suspension of immunosuppression may need to be considered. it is important to note that decisions to intensify or change treatment should be individualized based on the relative severity of the sars-cov-2 infection. 78 there is very little data regarding the impact of sars-cov-2 infection on primary immunodeficiencies (pi), which is why several international organizations such as the european society for immunodeficiency (esid), 79 the reference centre for hereditary immunodeficiencies (le centre de référence déficits immunitaires héréditaires, ceredih) 80 organization for primary immunodeficiencies (ipop) 81 are collecting data through a survey of physicians in order to gather information and provide them better care. both the idf (immune deficiency foundation) 82 and the ascia (australasian society of clinical immunology and allergy) 83 and other agencies 84 have considered their patients' increased risk of severe respiratory infections or of experiencing a more severe disease course, however, they recognize that it cannot be said whether people with primary immunodeficiencies are at higher or lower risk of severe sars-cov-2 infection. ascia and ipopi promote measures to prevent the spread of the virus, social isolation, and call for early consultation with medical services when infection is suspected, and recommend maintaining continuity of medication, especially in those receiving immunoglobulin. 81, 83 virtual resources with patient and medical community education are available in the idf. these experts on the subject have theorized the possible effects of sars-cov-2 in different populations, 82 for example, in the group of t lymphocyte (tl) immunodeficiency (combined immunodeficiency, digeorge syndrome, among others) measures of isolation and protection must be maximised, since the action of these defence cells is necessary for the control of the virus; in b-lymphocyte deficiency (agammaglobulinaemia, common variable immunodeficiency) the risk of infection is not thought to be higher than in the community, except in patients with structural involvement at the lung level, and in the phagocytosis deficiency immunodeficiency group (neutropenia and chronic granulomatous disease) although neutrophils are not as important in controlling the virus, the possibility of co-infection needs to be considered, while the chronic granulomatous disease group is not thought to be at increased risk of infection or severe manifestation. 85 other recommendations according to the ipopi joint statement on the current coronavirus pandemic, are the use of pcr tests for diagnosis, since for some forms of pi there is no production of antibodies, and therefore tests based on immunoglobulins are not effective. in this same publication, as of april 5, 2020, 15 sars-cov-2 cases in different types of pi, exhibiting typical symptoms (fever, cough, and upper respiratory symptoms), 13 of which were under 45 years of age; seven required hospitalization (two developed adult respiratory distress syndrome) and all were under 45 years of age. in the most recent update, based on collected (unpublished) data, there does not appear to be an increased risk of sars-cov-2 infection, especially in its severe form, however, given the still limited information and risk for these patients, isolation and infection prevention measures should be maintained as much as possible. 81 another important aspect for these patients is the impact during the sars-cov-2 pandemic on their health-related quality of life (hrqol), requiring strict isolation and a remote care programme. in an italian cohort of 158 patients with pi due to a bl defect, two scales were evaluated, one specific to health-related quality of life, cvidqol (common variable immune deficiency quality of life), and another generic scale to assess anxiety and depression, ghq-12 (12-item general health questionnaire); finding that the remote care programme does not affect hrqol, however, in the group of patients at risk of anxiety/depression there is impaired quality of life, emphasizing the importance of individualizing each patient and psychosocial support. 86 hu during the stay at this institution infection was documented by nasopharyngeal sample associated with amplification of the betacoronavirus e gene and the specific sars-cov-2 rdrp gene by pcr, comorbidities such as hypothyroidism and asthma were also identified in these patients. all came under antiretroviral treatment, with cd4 cell count (> 400 cell/mm3 in four of the patients). prior classification according to the patient's clinical status as mild, moderate or severe, under the precept that protease inhibitors could have activity against coronavirus protease, cobicistat + darunavir was considered appropriate in two of them, starting ritonavir + lopinavir in the rest combined with hydroxychloroquine, azithromycin, corticosteroids, interferon β-1b and even tocilizumab, according to duration and progression. the survival of all is documented up to the time of publication and the conclusion is that patients in advanced stages of the disease should be guaranteed differential diagnosis by opportunistic pulmonary agents, inferring that they may have a poorer outcome, as well as an ominous prognosis. 89, 90 in a recent study of incidence and severity 91 of covid-19 involving 60 hiv clinics in spain (77,590 patients), 236 patients were diagnosed with covid-19, 151 were hospitalized, 15 required intensive therapy and 20 died. it was found that patients receiving tenofovir disoproxil fumarate (tdf)-emtricitabine (ftc) reverse transcriptase inhibitors had a lower risk of developing covid-19 and of hospitalization compared to groups receiving other treatments. there are isolated cases 92 in which patients coinfected with hiv and covid-19 in management with lopinavir and ritonavir had a favourable clinical response, considering that this reaction can have two effects: inhibition of sars-cov-2 replication, as well as inhibition of hiv replication, allowing a slight activation of the immune system capable of responding to sars-cov-2 without the progression of the patient to hyperinflammatory status, even highlighting that lymphopenia would not be considered a marker of poor prognosis but a protective immune effect, being considered an disturbance of the overactive response of the immune system, avoiding serious clinical manifestations. 92 other hypotheses raised as a favourable clinical response dependent on the patient's immune status, coinfection or history of opportunistic infections, mainly at the pulmonary level, the risk or benefit in relation to the use of glucocorticoids and even the benefit of introducing tocilizumab early were not omitted. 93 in patients with hiv and sars-cov-2 infection it can be concluded that the immune response, prognosis and outcome are highly variable and / or subjective according to the antiretroviral treatment in place, duration in relation to diagnosis and viral suppression, because hiv patients without treatment, newly diagnosed or with no viral suppression may have a compromised immune system (mediated by a low cd4 count), even being vulnerable not only to the worst outcomes due to sarv-cov-2, but alleged coinfection by other agents in pulmonary opportunists. patients with adherence to antiretroviral treatment, who have achieved viral suppression and do not have low cd4 count, will be affected by sars-cov-2 with the same chances as immunocompetent patients who develop mild manifestations, regardless of change in antiretroviral treatment or adjustment with ritonavir and lopinavir, however, it is important to emphasize the divergences in approach, management and choice versus antiretroviral treatment documented to date. for this type of patients, as well as those mentioned previously, recommendations have already been published on their management with covid-19. 94 the degree of pharmacological immunosuppression is assessed according to the patient's immunological risk, the type of protocol used, the type of target at which the drug or group of drugs is directed, and the type of disease for which it is indicated (e.g., neoplasms, transplants, immunological, etc.). although in all cases it is not easy to directly assess the degree of immunosuppression, some biomarkers have been developed that reflect the individual's response to immunosuppressants, which can range from general tests such as liver function enzymes, to the use of specific genotypes, including cell counts of specific lymphocyte populations, cytokines, leukocyte markers and target enzymes, among others. 95 in other cases, the degree is assessed according to the number of immunosuppressant drugs, dose and time employed, being low when a drug is used in low or moderate doses for a short time, and increasing to a higher degree when two or more immunosuppressants are used in combination, regardless of the dose. 96 there are many drugs used in different medical specialties that are associated with a certain degree of immunosuppression and that render patients vulnerable to one or another infectious process. however, in the field of sars-cov-2 infection, the data are scarce and unknown. as mentioned throughout the review, comorbidities are the most important risk factors compared to drugs used. the literature search predominantly reveals information about possible drug interactions that can occur with the treatment of covid-19, which can clearly also generate damage and worsen the clinical picture. to date there are no data on specific drugs that are associated to a greater or lesser degree with infection by the new coronavirus. 97 there is a warning about possible drug interactions between immunosuppressive drugs and those under investigation for the treatment of covid-19, generating alerts and guidelines for the development of this complex task by clinicians. remdesivir, an antiviral with promising results in the treatment of covid-19 as mentioned, has no data so far on possible drug interactions with immunosuppressive drugs, unlike chloroquine/hydroxychloroquine and lopinavir/ritonavir, which although their use is declining, were initially an active part of treatment, suggesting major interactions with calcineurin inhibitors, mtor (mammalian target of rapamycin inhibitors) and corticosteroids, especially lopinavir/ritonavir. 97 there are recommendations about discontinuing mycophenolate in critically ill transplant recipients, 98 which would also apply to covid-19, bearing in mind that during the h1n1 pandemic it was documented that this drug decreased the serological response in transplant recipients. 97 however, given the scarcity of data, this decision must be tailored to each patient in this special subgroup. in reference to calcineurin inhibitors (tacrolimus, cyclosporine), a dose minimization scheme is proposed, with the possibility of increasing the interval for its administration, suggesting safety in these regimens, particularly in individuals with kidney transplantation and covid-19. 99 multiple recommendations and guidelines have been generated around the use of immunosuppressors or cytostatics in the oncology field, 100 such as cyclophosphamide, doxorubicin, cytarabine, vinblastine, as well as immunotherapy and the use of biological drugs in the context of cancer, according to the type of neoplasm in the context of risk or presence of covid-19 infection, suggesting in general a decrease in dose, but always balancing individual cases according to the type of neoplasm, stage and immunosuppressive scheme proposed. 58 the recommendation is to avoid high doses of corticosteroids since they could, as observed in patients with mers-cov, prolong viral replication in patients with covid-19. 97 as mentioned above, their use would be reserved for specific subgroups of critically ill patients other drugs, not immunosuppressants, but associated with the physiopathogenesis of the disease, such as angiotensin-converting enzyme inhibitors or renin-angiotensin-aldosterone system j o u r n a l p r e -p r o o f inhibitors, have not been shown to increase the risk of sars-cov-2 infection, and conversely, their withdrawal could be harmful. 101 considering the evidence available to date on sars-cov-2 infection outcomes in patients with immunosuppression (either due to their disease or the use of immunosuppressants) its behaviour is not clear in this type of individuals. we can highlight that patients with cancer and recent treatment of cancer (chemotherapy or surgery) have a higher risk of worse outcomes, with faster deterioration than those without cancer, an increased risk of severity and mortality having been shown through two meta-analyses. with regard to transplant patients (kidney, heart and liver), patients with neurological disease associated with the use of immunosuppression (ms, nmods, mg), primary immunodeficiencies and hiv, studies have not shown a tendency to poorer outcomes than patients without these diseases or drugs and have sars-cov-2 infection, similar to that found in rheumatological diseases. 102 this could perhaps be explained in that the severity of sars-cov-2 infection has been associated with an aberrant inflammatory response (cytokine storm). for the time being and as more information is obtained, and based on the aforementioned literature and recommendations of societies, it is suggested that immunosuppressant therapy be continued, starting new therapies should be avoided as much as is possible (especially in endemic areas) and in case of infection, depending on its severity, the risk/benefit should be evaluated of suspending it during the period of infection. it should be noted that the presence of comorbidities, such as high blood pressure, diabetes mellitus and copd, increases the risk of severity, intensive care 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treated with immunosuppressive targeted therapies key: cord-314445-4cb4a9r5 authors: mcnamara, ryan p.; caro-vegas, carolina; landis, justin t.; moorad, razia; pluta, linda j.; eason, anthony b.; thompson, cecilia; bailey, aubrey; villamor, femi cleola s.; lange, philip t.; wong, jason p.; seltzer, tischan; seltzer, jedediah; zhou, yijun; vahrson, wolfgang; juarez, angelica; meyo, james o.; calabre, tiphaine; broussard, grant; rivera-soto, ricardo; chappell, danielle l.; baric, ralph s.; damania, blossom; miller, melissa b.; dittmer, dirk p. title: high-density amplicon sequencing identifies community spread and ongoing evolution of sars-cov-2 in the southern united states date: 2020-06-19 journal: biorxiv doi: 10.1101/2020.06.19.161141 sha: doc_id: 314445 cord_uid: 4cb4a9r5 sars-cov-2 is constantly evolving. prior studies have focused on high case-density locations, such as the northern and western metropolitan areas in the u.s. this study demonstrates continued sars-cov-2 evolution in a suburban southern u.s. region by high-density amplicon sequencing of symptomatic cases. 57% of strains carried the spike d614g variant. the presence of d614g was associated with a higher genome copy number and its prevalence expanded with time. four strains carried a deletion in a predicted stem loop of the 3’ untranslated region. the data are consistent with community spread within the local population and the larger continental u.s. no strain had mutations in the target sites used in common diagnostic assays. the data instill confidence in the sensitivity of current tests and validate “testing by sequencing” as a new option to uncover cases, particularly those not conforming to the standard clinical presentation of covid-19. this study contributes to the understanding of covid-19 by providing an extensive set of genomes from a non-urban setting and further informs vaccine design by defining d614g as a dominant and emergent sars-cov-2 isolate in the u.s. the current covid-19 pandemic is an urgent public health emergency with over 112,000 deaths in the united states (u.s.) alone. covid-19 is caused by infection with the severe acute 48 respiratory syndrome coronavirus-2 (sars-cov-2). the typical symptoms for covid-19 may include 49 the following: fever, cough, shortness of breath, fatigue, myalgias, headache, sore throat, abdominal to provide finer granularity about biological changes during sars-cov-2 transmission, we 106 employed next generation sequencing (ngs) as an independent screening modality. this allowed us 107 to reconstruct the mutational landscape of cases seen at a tertiary clinical care center in the 108 southeastern u.s. from the start of the u.s. epidemic on march 3, 2020, until past the peak of the first distribution of 10x coverage for all samples is presented in figure 1a . as expected, more mapped 220 reads yielded higher coverage. of the 33 negative controls, none had >10 2 total reads aligned. of the 221 positive samples, greater than 5*10 3 total mapped reads were needed to obtain 1x coverage of the 222 whole genome, a minimum of 3.1x10 4 reads were needed to obtain >90% coverage at 10x. the 223 number of reads aligned varied depending on the viral load, as determined by real-time qpcr using 224 cdc primer n1, but not total rna, as determined using rnase p, of the samples ( figure 1b) . in this 225 assay, any cp <35 for sars-cov-2 qpcr yielded reliable coverage, which increased linearly with 226 viral load. at a cp ≥35 most positive samples still yielded reads that mapped to the target genome 227 and thus allowed detection of sars-cov-2 sequences; however, the results were less consistent, 228 and coverage was more variable. as expected, total rna (measured by rnase p) was not 229 associated with sequencing coverage and varied considerably across samples, even though each 230 sample used the same amount of virus transport medium (vtm). the coverage level distribution is shown in figure 1c independently derived consensus genomes from the sars-cov-2/human/usa-wa1/2020 295 isolates showed evidence of divergence between the original isolate, the seed stock, and 296 commercially distributed standard (figure 2b) . similar culture-associated changes were recently 297 reported for a second, culture-amplified reference isolate: hong kong/vm20001061/2020. this is not 298 surprising, given that any large-scale virus amplification in culture is accompanied by virus evolution, 299 but it raises concerns about the utility of using a natural isolate, rather than a molecular clone 300 (graham et al., 2018; thao et al., 2020) as standard for sequencing. the phylogeny based on whole genome nucleotide sequences revealed several interesting 302 facets. predictably, all unc isolates of sars-cov-2 were significantly different from sars-cov and 303 rattg13 (figure 2b, purple color) . rattg13 was used as an outgroup for clustering. the first nc 304 case (nc_6999, (figure 2b , arrow labeled "wa")) was a person returning from washington (wa) and one large deletion was identified in four independent samples: 14 nucleotides were deleted 318 beginning at position 29745 (indicated in figure 2c by a delta symbol) . this region is within the 319 previously recognized "coronavirus 3' stem-loop ii-like motif (s2m)". this was confirmed in multiple 320 isolates, supported by multiple, independent junction-spanning reads (figure 3a, b) . junctions were 321 mapped to single nucleotide resolution directly from individual reads. the variant 3' end does not 322 destroy overall folding but introduces a shorter stable hairpin (figure 3c, d) . how this mutation 323 affects viral fitness remains to be established. in sum, this study generated exhaustive snv information representing the introduction and 325 spread of sars-cov-2 across a suburban low-density area in the southern u.s. all samples were 326 from symptomatic cases and the majority of genomes clustered with variants that predominate the 327 outbreak in the u.s., rather than europe or china. this supports the notion that the majority of u.s. there seems to be partial overlap between the bulged stem-loop and the pseudoknot, suggesting that 360 these two structures are mutually exclusive and may serve as a switch to regulate the ratio of full 361 length rna and defective rna (goebel et al., 2004) . these two structures are also present in sarscov. these isolates represent full-length genomes from symptomatic patients rather than disjointed 363 rna fragments recovered after clinical disease had subsided, thus we speculate that these deletion about half of the specimen not clinically tested for sars-cov-2 tested positive by sequencing. this was not surprising, as to this day testing capabilities are limited, and probable cases are triaged 419 based on clinical and public health indications. these unknown cases were not asymptomatic but 420 represent patients with a clinically indicated need for upper respiratory sampling. finding additional 421 sars-cov-2 cases in this population suggests that case counts based on nat represent a lower 422 estimate of sars-cov-2 prevalence. it may also suggest that the current triage criteria for sarscov-2 testing are too limited to understand spread of this virus. in sum, this study underscores the 424 sensitivity and accuracy of current nat assays and demonstrates the utility of testing by sequencing. it contributes to the worldwide effort to understand and combat the covid-19 pandemic by providing 426 the first set of full-length sars-cov-2 genomes from a non-urban setting. coronavirus susceptibility to the antiviral remdesivir (gs-5734) is 496 mediated by the viral polymerase and the proofreading exoribonuclease the proximal origin of 499 sars-cov-2 presymptomatic sars-cov-2 infections and transmission in 502 a skilled nursing facility sars-cov-2 viral spike g614 mutation exhibits higher case 504 fatality rate covid-19 in critically ill patients in the seattle 507 region 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sars-cov-2 using a synthetic 619 genomics platform aerosol and surface stability of sars-cov-622 2 as compared with sars-cov-1 emergence of genomic diversity and recurrent mutations in sars-626 an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-629 cov-2 epidemic: an observational cohort study antigenicity of the sars-cov-2 spike glycoprotein receptor recognition by the novel 635 coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus receptor recognition by the novel 638 coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus. 639 a phylogenetically conserved hairpin-type 3' 641 untranslated region pseudoknot functions in coronavirus rna replication virological assessment of hospitalized patients with covid-645 2019 cryo-em structure of the 2019-ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china factors 653 associated with prolonged viral rna shedding in patients with covid-19 characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral 657 shedding quantitative detection and viral load analysis of sars-cov-2 in infected patients viral and 662 host factors related to the clinical outcome of covid-19 clinical 665 course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a 666 retrospective cohort study a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china sars-cov-2 viral load in upper respiratory specimens of infected patients genetic interactions between an 678 essential 3' cis-acting rna pseudoknot, replicase gene products, and the extreme 3' end of the mouse 679 coronavirus genome this work was funded by public health service grants ca016086, ca019014, and ca239583 key: cord-297941-7yut9vt4 authors: haq, m.; rehman, a.; noor, m.; ahmed, j.; ahmad, j.; irfan, m.; anwar, s.; ahmad, s.; amin, s.; rahim, f.; haq, n. u. title: seroprevalence and risk factors of sars cov-2 in health care workers of tertiary-care hospitals in the province of khyber pakhtunkhwa, pakistan date: 2020-09-30 journal: nan doi: 10.1101/2020.09.29.20203125 sha: doc_id: 297941 cord_uid: 7yut9vt4 background: high number of sars cov2 infected patients has overburdened healthcare delivery system, particularly in low-income countries. in the recent past many studies from the developed countries have been published on the prevalence of sars cov2 antibodies and the risk factors of covid 19 in healthcare-workers but little is known from developing countries. methods: this cross-sectional study was conducted on prevalence of sars cov2 antibody and risk factors for seropositivity in hcws in tertiary care hospitals of peshawar city, khyber pakhtunkhwa province pakistan. findings: the overall seroprevalence of sars cov2 antibodies was 30.7% (ci, 27.8 to 33.6) in 1011 hcws. laboratory technicians had the highest seropositivity (50.0%, ci, 31.8 to 68.1). risk analysis revealed that wearing face-mask and observing social-distancing within a family could reduce the risk (or:0.67. p<0.05) and (or:0.73. p<0.05) while the odds of seropositivity were higher among those attending funeral and visiting local-markets (or:1.83. p<0.05) and (or:1.66. p<0.01). in univariable analysis, being a nursing staff and a paramedical staff led to higher risk of seropositivity (or:1.58. p< 0.05), (or:1.79. p< 0.05). fever (or:2.36, ci, 1.52 to 3.68) and loss of smell (or:2.95, ci: 1.46 to 5.98) were significantly associated with increased risk of seropositivity (p<0.01). among the seropositive hcws, 165 (53.2%) had no symptoms at all while 145 (46.8%) had one or more symptoms. interpretation: the high prevalence of sars cov2 antibodies in hcws warrants for better training and use of protective measure to reduce their risk. early detection of asymptomatic hcws may be of special importance because they are likely to be potential threat to others during the active phase of viremia. funding: prime foundation pakistan. the first case of corona virus was reported in bmj in 1965 1 many corona viruses have been recovered from animals or humans, however, only two of them have gained attention in the past two decades. 2, 3, 4 the transmission of virus to others is typically like that of the "common cold". healthcare workers are exposed to and at higher risk of acquiring infection while dealing with patients suffering from highly infectious diseases like covid-19. pcr may be negative even in acute phase in certain cases. 5 antibodies tests (anti sars-cov-2 antibodies) may be useful in diagnosing pcr negative cases and also provide information about past infection .5,6 a cochrane review of 54 studies on antibody testing reported that 94% patients may be positive after the third week of onset of symptoms 7 and hence may be a better index of past exposure to sars cov-2. the role of antibodies in preventing further infection from covic-19 is still not clear 8 however it is assumed that antibodies may provide some protection. 9 many studies have been published on the prevalence of sars cov-2 antibodies in healthcare workers from developed countries in recent past, however little is known from developing countries. to our knowledge this is the first study of assessing sars-cov-2 antibodies of hcws form both public and private tertiary care hospitals in peshawar, pakistan. the present study aims to estimate the seroprevalence of sars-cov-2 in hcwes and explores the possible risk factors of exposure to sars-cov-2. this is a cross-sectional study, following the strobe (strengthening the reporting of observational studies in epidemiology) reporting guidelines, conducted from june 15 to 29, 2020 using purposive sampling technique. the number of hcws included in the study was 1011. to participate, were included in the study. the hcws included doctors, paramedics, nurses, medical technicians, laboratory and other staff of the hospitals. the study was approved by institutional review board of prime foundation pakistan. data about detailed history of risk factors, co-morbid factors, demographic information and symptoms was collected on a semi-structured proforma. five ml peripheral venous blood was collected in li . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 30, 2020. . https://doi.org/10.1101/2020.09.29.20203125 doi: medrxiv preprint heparinised tube, after informed, serum separated using 2500 rpm centrifuge and stored in labelled serum cup for analysis using 20 micro litre serum volume while remaining serum was stored at -80 c 0 temperature. cobas e411 system was used for immunoassay. the fda approved kit was used for detection of anti-sars-cov-2 antibodies which has high specificity (100% and sensitivity (more than 98·8%) according to the manufacturers. 10 , however public health england estimated its specificity to be 100% but a sensitivity of 87%. 11 results were interpreted against a cut off value of 1 au/ml and less than 1 au/ml was considered negative and more than or equal to 1au/ml as positive. statistical analyses were performed using spss v.24·0. the means and standard deviations were used to present the continuous variables and the categorical variables were described as the counts and the percentages. variables with p values < 0·01 in the univariate analysis were further used for a multivariate logistic regression analysis and p value ≤0·05 was considered significant. socio-demographic characteristics: the demographic characteristics of healthcare workers are summarized in table 1 below. the fcws included 688 (68·1%) males and 323 (31·9%) female. the mean age was 33.6 years (sd ±10·5) while 454 (45·0%) were in the age group 20-29 years and 312 (31.0%) 30-39 years. and only 34 (3·40%) in age group 60 years and above. the professional categories of hcws were, nursing staff (26·1%), paramedical staff (21·3%), trainee doctors / medical officers (11·6%), ward staffs (11·3%), consultants (9%), house officers (6.8%), lab technicians 5·2% and 8·7% were ward support staff members. the overall seroprevalence of sars-cov-2 antibodies was 30·7% (ci 95%: 27·8 -33·6). the seroprevalence was not significantly different (p>0·02) in males 31·8% (ci 95%: 28·3 -35·4) than female 28·2% (ci 95%: 23·3 -33·4) female subjects [table: 1]. the age wise seroprevalence of sars-cov-2 antibodies was 29·5% (95% ci 25·7-33·5) in age group 20-29 years, 33·3%(95% ci, 28·1 -39·8) and it increasing with older age until plateauing . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint in different professional category, the highest seroprevalence were identified in lab technicians (50·0%, 95% ci 31·8-68·1) followed by paramedical staff (42.0%, 95% ci 34.2 -50.1), ward staff (39·8%, 95% ci 29·4 -50·7) and nursing staff (38·8%, 95% ci 32·1 -45·7). while consultant, trainee doctors and house officer had seroprevalence of (18·2%, 95% ci 12·4 -25·1), (19·9%, 95% ci 14·3 -26·4) and (18·4%, 95% ci 11·7 -26·7) respectively [ among the seropositive hcws, 165 (53·2%) were completely asymptomatic while 145 (46·8%) had one or more symptoms. the mean antibody level was 26·12 (sd ± 26·79) au/ml in seropositive participants (males 24·63 sd ±25·68, females 29·72 sd ±29·14). the mean antibody level in seropositive asymptomatic participants was 30·20 (sd ± 29·63) while in symptomatic it was 21·48 (sd ± 22·35). gender was not an independent risk factor and the odds of being seropositive were similar between males and females (or: 1·02, 95% ci, 0.89-1·41. p> 0·05). the use of face masks and observing social distancing within a family had lesser odds of being seropositive with a statistical significant association (or: 0·67, 95% ci, 0·49 -0·92. p<0·05), (or: 0·73, 95% ci, 0·55 -1·98. p<0·05) in multivariable regression models (mlm) [ table: 2]. in mlm, the odds of seropositivity were higher among those attending funeral and visiting local markets for shopping (or: 1·83, 95% ci, 1·05 -3·16. p<0·05) and (or: 1·66, 95% ci, 1·16 -2·37. p<0·01). however the risk of seropositivity did not increase with attending congregational prayers in mosques (or: 0·52, 95% ci, 0·34 -0·79. p<0·05) [ the risk of being seropositivity was strongly (p< 0.01) associated with fever (or: 2·36, 95% ci: 1·52-3·68) and loss of smell (or: 2·95, 95% ci: 1·46-5·98) while loss of taste was strongly associated with seropositivity (or: 2·4, 95%ci, 1·44-4.00, p<0·001) in univariable analysis but . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 30, 2020. . https://doi.org/10.1101/2020.09.29.20203125 doi: medrxiv preprint multivariable logistic regression did not show any significant association. [table: 2] co-morbidities were present in 17% in seropositive subjects and included diabetes (30%), hypertension (36·4%), cardiac disease (15·4%), asthma (18·2%) and recent surgery (40%). to our knowledge this is the first study on prevalence of sars cov-2 antibodies in hcw of tertiary care hospitals in pakistan. studies form other counties observed lower seroprevalence in hcws. the seroprevalence of sars-cov-2 antibodies in healthcare workers were 30.7% (ci 95%: 27·8 -33·6). it varied from 18.2% among doctors to 50% in laboratory technicians. the highest seroprevalence were reported in lab technicians (50%) and paramedical staff (42%) compared to the rest of hcws. in a study from china, the seroprevalence was 17.14% while 24% and 9·3% have been reported from uk and spain. 12, 13, 14 much lower weighted prevalence (1·07%) was reported in a greek study in 1952 hcws. 15 the higher seroprevalence of antibodies in our study may indicate higher exposure of hcws to covid-19 positive subjects or patients. it may also be due to inadequate use of ppe and education/awareness levels of hcws. a recent meta-analysis published in the lancet journal concluded that physical distancing, use of mask and goggles significantly decrease the risk of infection. 16 the risk of increased seropositivity was also not associated with attending congregational prayers in mosques (or:0·52, 95% ci, 0·34-0·79). this could be possibly due to two main reasons. first, the overall personal and environmental hygienic practices observed as religious obligation in . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this this version posted september 30, 2020. . https://doi.org/10.1101/2020.09.29.20203125 doi: medrxiv preprint mosques that includes washing hands and face at least five times a day before prayers and keeping the prayer area clean. second, voluntary implementation of preventive measures after the consensus decrees on the same by religious scholars. 18 this also highlights the need of involvement of clergy for effective implementation of public health strategies in conservative societies like pakistan. the risk of becoming positive for sars-cov-2 antibodies did not increase with history of direct contact with covid patients within or outside the hospital. this could be due to more careful approach of hcws when coming in contact with known covid patients. the same has been reported in other studies that frontline hcws dealing with covid patients do not show higher risk of acquiring the infection when compared to non frontline hcws. 19 in our study most of the subjects were asymptomatic. the mean antibodies level in seropositive asymptomatic participants were significantly higher compared to symptomatic subjects (p<0·001). in contrast other studies reported lower antibodies level in asymptomatic patients. 20 it is also suggested that asymptomatic patients may have lower seroconversion levels but the duration of virus shedding is longer in them when compared to symptomatic patients 21 the risk of becoming seropositive was not different significantly in males and females but the mean antibodies titres were significantly high in females (p<0·03). increasing age was a significant risk for sars-cov 2 antibodies levels. the highest mean antibody level (38·95 ± 34·88) was seen in the age group (50-59) while the lowest (23·73 ± 25·44) was in the age group (20-29) (p = 0·05). in a mathematical model to epidemic data from six countries a positive correlation was found with increasing age and susceptibility of young was almost half to that of adults. 22 profession of hcws was a significant risk and seropositivity with higher prevalence in nursing and paramedical staff compared to consultants and trainee doctors (hos and mos/tmos) in univariate analysis. this is consistent with sars-cov study epidemic in 2003. 23 and could be due to longer duration of contact (more than 30 minutes) 14 of specific hcws. however multivariate analysis did not show any significant difference. the three commonest reported co-morbidities in other studies are hypertension, diabetes and cardiovascular diseases. 24 in our study the overall co-morbidities were 17% in seropositive subjects and these were recent history of surgery 40%, hypertension 36·4%, diabetes 30%, asthma 18·2% and cardiac disease 15·4%. the . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 30, 2020. . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 30, 2020. . https://doi.org/10.1101/2020.09.29.20203125 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 30, 2020. . https://doi.org/10.1101/2020.09.29.20203125 doi: medrxiv preprint upper ci . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint the copyright holder for this this version posted september 30, 2020. . https://doi.org/10.1101/2020.09.29.20203125 doi: medrxiv preprint covid-19: first coronavirus was described in the bmj in 1965 a cluster of cases of severe acute respiratory syndrome in hong kong middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation. the lancet infectious diseases corona viruses: an overview of their replication and pathogenesis, in corona viruses advice on the use of point-of-care immunodiagnostic tests for covid-19 antibody responses to sars-cov-2 in patients of novel coronavirus disease antibody tests for identification of current and past infection with sars-cov-2 antibody responses to sars-cov-2 in patients with covid-19 testing for covid-19. the lancet. respiratory medicine roche's covid-19 antibody test receives fda emergency use authorization and is available in markets accepting the ce mark covid-19 antibody tests: a briefing high sars-cov-2 antibody prevalence among healthcare workers exposed to covid-19 patients pandemic peak sars-cov-2 infection and seroconversion rates in london frontline health care workers. the lancet seroprevalence of antibodies against sars-cov-2 among health care workers in a large spanish reference hospital antibodies against sars-cov-2 among health care workers in a country with low burden of covid-19 physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis. the lancet sars-cov-2) within new york city during exponential phase of covid-19pandemic: report of the new york city residency program directors covid-19 research group president alvi outlines plan agreed with ulema on congregational prayers during ramzan hospital-wide sars-cov-2 antibody screening in 3056 staff in a tertiary center clinical and immunological assessment of asymptomatic sars-cov-2 infections seroprevalence of sars-cov-2 among frontline healthcare personnel during the first month of caring for patients with covid-19 age-dependent effects in the transmission and control of covid-19 epidemics seroprevalence of antibody to severe acute respiratory syndrome (sars)-associated coronavirus among health care workers in sars and non-sars medical wards key: cord-274802-7ioiwsd8 authors: varghese, praveen mathews; tsolaki, anthony g.; yasmin, hadida; shastri, abhishek; ferluga, janez; vatish, manu; madan, taruna; kishore, uday title: host-pathogen interaction in covid-19: pathogenesis, potential therapeutics and vaccination strategies date: 2020-08-19 journal: immunobiology doi: 10.1016/j.imbio.2020.152008 sha: doc_id: 274802 cord_uid: 7ioiwsd8 abstract the current coronavirus pandemic, covid-19, is the third outbreak of disease caused by the coronavirus family, after severe acute respiratory syndrome and middle east respiratory syndrome. it is an acute infectious disease caused by severe acute respiratory syndrome coronavirus 2 virus (sars-cov-2). the severe disease is characterised by acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation dysfunction, and multiple organ dysfunction syndromes. currently, no drugs or vaccine exist against the disease and the only course of treatment is symptom management involving mechanical ventilation, immune suppressants, and repurposed drugs. as such the severe form of the disease has a relatively high mortality rate. last 6 months have seen an explosion of information related to the host receptors, virus transmission, virus structure-function relationships, pathophysiology, co-morbidities, immune response, treatment and most promising vaccines. this review takes a critically comprehensive look at various aspects of host-pathogen interaction in covid-19. we examine genomic aspects of sars-cov-2, modulation of innate and adaptive immunity, complement-triggered microangiopathy, and host transmission modalities. we also examine its pathophysiological impact during pregnancy, in addition to various gaps in our knowledge. the lessons learnt from various clinical trials involving repurposed drugs have been summarised. we also highlight the rationale and likely success of the most promising vaccine candidates. receptor on enterocytes in the small intestine and is consistent with clinical reports of gastrointestinal symptoms and viral shedding in faeces (34, 35) . this has been further resolved with the comprehensive identification of host cells/tissues expressing both ace2 and tmprss2 ( figure 2 ). thus, likely targets for sars-cov-2 primarily include secretory goblets of the nasal mucosa, lung type ii pneumocytes and absorptive erythrocytes of the small intestine (36) . of note, this study also showed that the ace2 receptor is an interferonstimulated gene in sars-cov-2 infection in the cells of the human upper nasal epithelium and lung, predominatly mediated by ifn-α2 and ifn-γ (36) . moreover, bystander cells are subject to interferon-mediated effects (upregulation of ace2 receptor) rather than sars-cov-2 infected cells, suggesting a mechanism of enhanced viral targeting and entry during pathogenesis and a possible avenue for therapeutic intervention (36) . analysis of genetic variation in the ace2 gene has identified single nucleotide polymorphisms (snps) that differ in frequency globally among the human population, particularly between males and females (37). characterising these snps more fully with epidemiological and clinical data on covid-19 will in time shed light on the precise molecular mechanisms of transmission and disease. furthermore, in the sars-cov-2 viral s protein, 27 amino acid substitutions have been described, although these occurred outside the rbd that directly interacts with ace2 (14) . of paramount importance is characterising the genetic variation and its consequences in the s protein and its rbd, as this will determine whether the sars-cov-2 virus is evolving and is likely to be a seasonal infection with new variants for the human population. undoubtedly, variation in the s protein and ace2, the central interface of hostpathogen interaction in covid-19 will have evolved from natural selection contributing to the pathogenesis of this disease. proteins and transported into the endoplasmic reticulum (er). these proteins are processed via the secretory pathway and are transported into the er-golgi intermediate compartment (69, 70) , where the full-length viral genomes are packaged with the nucleocapsid n protein, budding from the membrane, and thus forming the enveloped mature virion (71) . the n protein has two domains that can bind the rna genome, with the aid of nsp3 protein, and attaching it to the rtc, facilitating the packaging of the virus (72) (73) (74) . the viral m protein has three transmembrane domains and is responsible for the majority of protein-protein interactions needed for virus assembly, including membrane curvature and binding the nucleocapsid (75, 76) . pseudo-virus particles can also only be formed when there is a co-expression of m protein and e protein, indicating the requirement of both these two proteins to form the coronavirus envelope (77) . the viral e protein is also involved in structural shaping of the viral membrane envelope and in inhibiting m protein aggregation, as well as a role in pathogenesis (78) (79) (80) (81) . after the assembly of the mature virions, they are transported in vesicles, where they are released from the infected cell via exocytosis (82) . unlike sars, covid-19 patients had the highest viral load near presentation, which could account for the fast-spreading nature of this epidemic. in a study involving covid-19 patients in hong kong recorded high viral load on presentation with the onset of symptoms and also when the symptoms are mild (83) . sars cov-2 viral rna load was detected in the deep throat (posterior oropharyngeal) saliva samples for 20 days or even longer. the peak of the viral load correlated positively with age. viral load in posterior oropharyngeal saliva samples was higher during the first week of symptom onset, which gradually declined. thus, the location of sample collection and the timing for the onset of symptoms both are important factors to be considered for the detection of sars cov-2 positive cases. in the same study, most of the patients showed rising antibody titres 10 days after symptom onset, though the serum antibody levels did not show correlation with clinical severity (83) . the patient's antibody to sars-cov-2 viral nucleocapsid protein using infected cell lysates was identified on the 10th day after symptom onset by western blot (84) . in another study involving 285 patients with covid-19, all were tested positive for antiviral igg within 19 days after symptom onset. both igg and igm titres reached a plateau within 6 days after seroconversion (85) . in wuhan tongji hospital, around 60 convalescent patients tested positive for the igg against the virus, while 13 patients tested negative for igm, where igg titre was higher comparatively. both the antibody titres showed a decrease when tested weeks apart (86) . thus, titres of sars-cov-2 antibodies can reflect the progress of viral infection and can be a vital component to understand the development and prognosis of the disease and similarly timing of antibody seroconversion is also crucial for determining the optimum duration for collecting serum specimens for antibody diagnosis. as previously mentioned, several other studies also confirmed the presence of sars-cov-2 nucleic acids in the faecal, urine samples and rectal swabs of covid-19 patients and thus it becomes essential to ascertain viral load dynamics in such samples too (87) (88) (89) . transmission sars-cov-2 is transmitted through "respiratory droplets", which are large droplets of virusladen mucus or through close contact with infected individuals (90) (91) (92) (93) . at the same time virus has also been reported to spread via asymptomatic but infected individuals in several countries, including china, germany, usa, and india (91, (94) (95) (96) (97) . a systematic review and meta-analysis of 172 observational studies with no randomised controlled trials and 44 relevant comparative studies in health-care and non-health-care settings revealed transmission of virus decreased as physical distancing increased to 1 metre or more (98) . eye protection, n95 or similar respirators in health-care settings and 12-16-layer cotton or surgical masks in the community were found to greatly control the transmission (98) . studies have also established that the median half-life of the aerosolised virus is ~1.2 hours under lab conditions, similar to the sars-cov. however currently, no evidence supports real-world airborne transmission of the virus through aerosols (99) . sars-cov-2 was found to remain viable for up to 4 hours on copper surfaces, up to 24 hours on cardboard surfaces, and up to 72 hours on plastic and stainless-steel surfaces. thus, there exists a possibility of contact transmission to occur, although no confirmed cases of contact transmission have been reported (99) . the virus was also found in the faeces of infected patients showing that the virus can survive and replicate in the digestive system (100) . this suggests that there may be a possibility of an oral-faecal route of transmission, though again no confirmed cases have been reported (101) . the royal college of obstetricians and gynaecologist uk have reported that transmission from mother to baby antenatally or intrapartum is possible although this requires further study for confirmation; there appears to be no evidence supporting vertical transmission to the foetus (102) (103) (104) . additionally, as reported by who and cdc, the virus has not been found to be transmitted by breastfeeding and has not been found in breastmilk of covid-19 mothers (105, 106) . covid-19 was found to have low severity and mortality than sars, but it is highly contagious and affecting comparatively more men than women (94, 107, 108) . the difference in fatality rate between males and females may probably be explained by the fact that as ace2 is located on the x chromosome. there may be alleles that confer resistance to covid-19, at the same time, oestrogen and testosterone sex hormones have different immunoregulatory functions that may contribute to protection or severity of the disease (109, 110) . the disease has also been found to disproportionately affect older aged persons and people suffering from social deprivation, diabetes, severe asthma, cardiovascular disease, obesity, haematological malignancy, recent cancer, kidney, liver, neurological or autoimmune conditions (111) . studies have also reported that members of minority communities such as the black and south asian populations, are at a higher risk of the disease (111) . the incubation period of the disease ranges between 2 to 14 days and the median incubation period is approximately 4-5 days before symptom onset (87, 92, 112, 113) . during the onset of the illness, the common symptoms that most patients exhibited were fever and cough. other symptoms include conjunctivitis, myalgia (muscle pain) or fatigue, headache, dyspnoea (short of breath), chest pain, diarrhoea, nausea, rhinorrhoea (runny nose), vomiting, loss of appetite, abdominal pain, gastrointestinal bleeding, autoimmune haemolytic anaemia, and sometimes haemoptysis (coughing of blood) (108, (114) (115) (116) (117) (118) . patients have also reported anosmia (loss of smell), dysgeusia (distortion of the sense of taste) (119) (120) (121) (122) . for sars-cov-2 asymptomatic patients, anosmia, hyposmia, or dysgeusia are symptoms that were suggested for screening (123) . in addition to these, neurological manifestations such as dizziness, headache, impaired consciousness, acute cerebrovascular disease, ataxia, seizure, nerve pain, skeletal muscular injury manifestations, intracerebral haemorrhage, central nervous system vasculitis, encephalopathy, encephalitis, cranial neuropathies and psychosis were reported predominantly in older people (124) (125) (126) . in paediatric patients, an autoimmune and autoinflammatory disease, paediatric inflammatory multisystem syndrome (pims), also known as multisystem inflammatory syndrome in children (mis-c), has been reported to occur after sars-cov-2 infection (127) (128) (129) (130) (131) (132) (133) (134) . cutaneous manifestations of covid-19 have also been reported (135) (136) (137) . a case report from strasbourg, france reported purpuric lesions in the lower extremity (137) . an italian study reported patients presenting with an erythematous rash, urticaria and chickenpox-like vesicles mainly in the trunk with little or no itching that did not correspond to disease severity (138) . the prolonged use of personal protective equipment and repeated washing have also led to an increase in dermal conditions such as pressure injury, contact dermatitis, itch, pressure urticaria, and exacerbation of pre-existing skin diseases (124) . the first step of infection is the inhalation of viral particles present in respiratory droplets from an infected host. once inhaled, the virion enters the nasal cavity of a healthy host and likely binds to goblet and ciliated cells in the nose that express ace2 (32) . at this time, a limited innate immune response may occur, and the virus replicates and moves further down the respiratory tract via the conducting airways. as the virions proliferate and spread towards the upper respiratory tract, usually a robust innate immune response is triggered by the detection of the virions by pattern recognition receptors (prrs) like toll-like receptors, rig-1, and mda-5. this may present several symptoms starting from dysphonia (hoarseness), ulceration of the epiglottis and subglottis, and profound oedema and granulations in the subglottis and also in the upper trachea (139) . in a few patients, mild tachypnoea and coarse breath sounds were also observed while the virus is in the upper airway (91) . furthermore, the detection by prr leads to the expression of type 1 interferons (ifn) in the early stages of infection, which helps establish an anti-viral state in the cells by producing inflammatory cytokines and chemokines. the sars-cov produces an enzyme that adds 2' o-methyl group to viral rna, which helps it evade detection by mda-5, thereby delaying the induction of ifn. studies have established that unlike an early ifn response, a delayed ifn response causes an inability to control viral replication, leading to cellular damage of airway epithelia and the lung parenchyma and an eventual lethal inflammatory cytokine storm (140) (141) (142) . the sars-cov-2 papain-like protease, which is essential to generate the rtc, has been shown to preferentially cleave the ubiquitin-like protein isg15 from interferon responsive factor 3 (irf3), attenuating type i interferon responses (143) . the c-terminus of the sars-cov-2 non-structural protein 1 was reported to bind to the 40s ribosomal subunit and block the mrna entry tunnel (144) . this obstruction effectively inhibits the rig-i-dependent innate immune response (144) . accordingly, no significant expression of ifn was detected up to 48 hours post-infection with sars-cov-2. only il-6, which correlates with respiratory failure, acute respiratory distress syndrome (ards), and adverse clinical outcomes were upregulated. monocyte chemoattractant protein-1 (mcp1), c-x-c motif chemokine (cxcl) 1, cxcl5, and cxlc10, were also upregulated 48 h post-infection with sars-cov-2 (145) . the suppression of innate immune activation and annihilation of t cells can help explain the mild or even the lack of symptoms in many infected patients. the increased viral replication efficiency in the respiratory tract early on leads to the highly efficient person-to-person transmission of the virus in the community (145) . the virions further migrate towards the lower respiratory tract and reaches the alveoli where it binds to the type 2 pneumocytes and begins replication. as the type 2 pneumocytes undergo apoptosis after viral release, they secrete inflammatory mediators like cxlc proteins that attract macrophages and neutrophils ( figure 5 ) (146) . the stimulated macrophages further secrete cytokines such as il-1β, il-6 and tumor necrotic factor α (tnf-α). the released cytokines trigger a "cytokine storm", which stimulates the release of vascular endothelial growth factor (vegf), monocyte chemoattractant protein-1 (mcp-1), il-8, and additional il-6, as well as reduced e-cadherin expression on endothelial cells causing vasodilation and increase capillary permeability (147) . this causes the plasma to leak into the interstitial spaces and alveoli, increasing interstitial and alveolar oedema. the increased alveolar oedema decreases the level of surfactant in the alveoli. this causes an increase in the surface tension in the alveoli, which leads to alveolar collapse. oedema and alveolar collapse may present as multiple peripheral ground-glass opacities in subpleural regions of both lungs, which is observed in many patients (148) . chest ct scan of patients also revealed bilateral multifocal infiltrates and mediastinal and hilar lymphadenopathy in some patients (91) . these decrease the gas exchange efficiency causing hypoxemia and increased work of breathing presenting as dyspnoea (shortness of breath), culminating in ards (149) . abnormal coagulation parameters, mainly elevated d-dimers seem to be associated with a higher risk of development of ards in covid-19 patients (150) . the aberrant wound healing may even lead to fibrosis than other forms of ards (151) . stimulated neutrophils secrete reactive oxygen species (ros) and proteases which destroy both infected and uninfected type 1 and type 2 pneumocytes, leading to further reduced gas exchange and alveolar collapse, respectively (152) . furthermore, the dead pneumocytes slough off into alveoli filling them up with fluid, protein deposits, cell debris, macrophages, and neutrophils. this causes pulmonary consolidation, which leads to altered gas exchange and causes hypoxemia (153, 154) . the consolidation also leads to productive cough. the hypoxemia can further trigger chemoreceptors that stimulate the sympathetic nervous system (sns) that causes tachycardia (increased heart rate) and tachypnoea (increased respiratory rate) (155, 156) . the central nervous system (cns) is also affected by the high concentrations of il-1β, il-6 and tnf-α in the blood, as these cytokines stimulate the hypothalamus to release prostaglandins such as pge2, which causes an increased body temperature leading to fever (157) . studies have also reported elevated levels of myeloperoxidase (mpo)-dna and citrullinated histone h3 (cit-h3), which are markers used to detect neutrophil extracellular traps (nets), in the serum of covid-19 patients (158) . furthermore, control neutrophils treated with covid-19 patient serum exhibited netosis (158) . nets, while protecting the host from invasive pathogens, have been attributed to play a role in many autoimmune and vascular diseases. for example, nets are known to contribute to ards, pathogen-induced acute lung injury, thrombosis and can contribute to further cytokine release lading to the inflammation (158) . an increased frequency of neutrophils, eosinophils and monocytes was reported in severe covid-19 positive patients; severe patients showed further increase in neutrophils though their activation status had not altered. there was no significant change in the immature granulocyte frequencies. however, there was an inverse correlation between frequency of immature granulocytes in moderate and severe patients with the duration since the appearance of symptoms. severe patients exhibited lower percentages of both conventional and plasmacytoid dendritic cells (dc) (159) . response syndrome (sirs). the spread of the inflammation from the lungs into the circulatory system causes increased capillary permeability within the systemic circulation. this leads to a decrease in blood volume along with increased vasodilation of systemic arteries, leading to decreased peripheral resistance. the decreased blood volume, along with peripheral resistance, causes hypotension (decreased blood pressure), which decreases perfusion to other organs leading to multisystemic organ failure (mof) (160-162). the cytokine storm has also been shown to trigger autoimmune haemolytic anaemias (aiha) (with warm or cold antibodies) (117, 163, 164) . most of the studies report manifestation of aiha early, during the active phase of covid-19 (within 4 to 13 days), a timeframe matching that of the cytokine storm (117, 127, 163, 164) . as a result of ards, sirs and mof, patients suffering from severe sars-cov-2 infection exhibit significantly elevated levels of, il-2, il-8, il-17, g-csf, gm-csf, mip-1α, crp, and ddimer, in addition to il-6, il-1β and tnf-α (145). there are reports suggesting that in addition to the lungs, sars-cov-2 infection may induce the multiorgan injury in patients involving brain, heart, liver, kidney, intestine and eyes (165, 166) . covid-19 associated neurological complications the neurological pathologies observed in covid-19 are similar to those observed in previous coronavirus epidemics (167) . myoclonus and demyelination are reported in a few cases (126, 168, 169) . a study conducted in wuhan, china involving 214 covid-19 patients, reported that 78 patients developed neurological manifestations (125) . in another study from strasbourg, france where effectively 58 patients were recruited for an observational study, reported agitation in 69% of the patients, confusion in 65%, and 67% of the patients had corticospinal tract signs (170) . a systemic review and meta-analysis of literature databases for psychiatric and neuropsychiatric presentations in coronavirus infections reported transient encephalopathies with features of delirium and psychosis (171) . the study also reported cognitive dysexecutive syndrome and delirium with agitation in a few cases (171) . there is also a reported case of autoimmune encephalitis with the typical clinical features of opsoclonus and myoclonus, and another case of autoimmune encephalitis with a radiological imagery showing typical limbic encephalitis (167) . the exact mechanism for encephalopathy may be multifactorial (effect of sepsis, hypoxia, and/or cytokine storm) (172) . a few cases of guillain-barré syndrome (gbs) associated with sars-cov-2 have been reported from italy (173) . however, further epidemiological and mechanistic study is required to confirm the incidents of gbs in covid-19. the binding of the virus to the ace-2 receptors on endothelial cells causes extravasation of red blood cells leading to cerebral microbleeds (137, 167) . there have also been reports of severe strokes in covid-19 patients, but further study is required to determine its association with covid-19 (167) . magnetic resonance imaging (mri) revealed abnormalities such as meningeal enhancement, ischaemic stroke, perfusion changes, microhaemorrhages, medial temporal lobe signal abnormalities similar to that seen in viral or autoimmune encephalitis (170, 174) . very few cases have been reported where sars-cov-2 was detected in csf and its supportive histopathological features; no reports of the virus in the brain exist yet (167, 175, 176) . thus, it is important to establish whether the above-described syndromes may be caused due to either direct viral injury, hyperinflammation, vasculopathy and/or coagulopathy, autoantibody production to neuronal antigens, sepsis and hypoxia, or a combination of these (172) . out of the first 41 patients diagnosed with covid-19 in wuhan, 5 of them had myocardial injury associated with the sars-cov-2, which mainly manifested as an increase in highsensitivity cardiac troponin i (115) . the hemogas analysis showed hypoxia; laboratory tests showed elevation of c-reactive protein, transaminases and lactate dehydrogenase, and lymphopenia (177) . several patients showed abnormal myocardial zymogram, showing high levels of creatine kinase (112) . because of an excessive inflammation, hypoxia, immobilisation and diffuse intravascular coagulation (dic), covid-19 patients may predispose to both venous and arterial thromboembolic disease (87, 115, 178) . it has also been observed that concomitant acute thrombosis of the abdominal aorta and pulmonary embolism induces cardiovascular complications in covid-19 patients, suggesting an association of hypercoagulable condition with the disease (179) . covid-19 patients with abnormal liver function were also documented, where patients had alanine aminotransferase (alt) or aspartate aminotransferase (ast), bilirubin, acute phase recants (apr) like crp, fibrinogen and il-6 above the normal range (112, 180) . sepsis, hypovolaemia, and nephrotoxins were found to be important contributors to kidney damage in covid-19 patients. cardiorenal syndrome, particularly right ventricular failure, might lead to kidney congestion and acute kidney injury in covid-19 patients (112) . symptoms such as olfactory and gustatory dysfunctions were also found to be related to covid-19 (181) . sars-cov-2, facilitated by tmprss2 and tmprss4, was found to infect and reproduce in ace-2 + mature enterocytes (100) . however, the virions released into the intestinal lumen were inactivated by stimulated human colonic fluid and no infectious virions were recovered in stool samples, in spite of the presence of viral rna in stools. this study thus established the intestine as a site of viral replication and its effect on local and systemic illness and overall covid-19 progression (100). as in the case respiratory infections by respiratory syncytial virus and sars-cov, the eyes have been shown to act as a portal of entry for the virus. while there have been no reports of sars-cov-2 transmission in humans via ocular tissues, further studies are required to exclude the eyes as a source of infection and as a portal of entry. moderate conjunctivitis could be the first sign of severe respiratory distress in covid-19 patients (182) . studies from china on patients with covid-19 reported conjunctivitis and other ocular manifestations, such as epiphora, conjunctival congestion, or chemosis in patients with severe covid-19 (183) (184) (185) (186) . the studies also reported a few patients with positive conjunctival swab for covid-19 determined by rt-pcr (183, 184, 186) . similar results were also reported in a study conducted by the national institute for infectious diseases in rome, italy (187) . in addition to the conjunctivitis, the ocular swabs were positive for sars-cov-2 even when nasopharyngeal swabs tested negative for the virus. this suggests that the conjunctiva may sustain viral replication for an extended period of time (118) . there are reports from france, italy, united kingdom and the united states of america, suggesting the presentation of autoimmune and auto inflammatory diseases in children, especially in children of african descent, such as paediatric inflammatory multisystemic syndrome (pims), also known as, multisystemic inflammatory syndrome in children (mis-c) (127) (128) (129) (130) (131) (132) (133) (134) . this syndrome includes kawasaki-like disease, kawasaki disease shock syndrome, toxic shock syndrome, myocarditis and macrophage activation syndrome (127) (128) (129) (130) (131) (132) (133) (134) . the exact cause for kawasaki disease remains unknown; however, it is believed that it is caused by an apparent atypical immune response to pathogens in genetically predisposed individuals (188) (189) (190) . previous studies have implicated the pathogenesis of kawasaki disease with the infection of certain members of the coronavirus family (191, 192) . the temporal association between the beginning of covid-19, sars-cov-2 infection and the onset of pims suggest a causal link (129) . this is further supported by the fact that in most cases, the patients exhibiting pims tested positive for igm or igg sars-cov-2 antibodies (127) (128) (129) (130) (131) (132) (133) (134) . the presence of igg antibodies clearly indicates a delayed onset of pims following sars-cov-2 infection (127) (128) (129) (130) (131) (132) (133) (134) . the onset of pims occurred 4-5 weeks after acute covid-19 (193) . the patients presented with fever, diffused skin rashes, rash/oedema of hands and feet, conjunctivitis, dry cracked lips, cervical lymphadenopathy and arthritis. the kawasaki-like disease caused by covid-19 exhibited a few differences in both clinical and biochemical features from patients suffering from kawasaki disease without sars-cov-2 infection. clinically, the patients suffering from covid-19 associated kawasaki-like disease were older and the disease occurred in both sexes, unlike the classical kawasaki disease that occurs in younger male children (193) . the covid-19 associated kawasaki-like disease also had a higher incidence of abdominal pain and/or more frequent diarrhoea, meningeal and respiratory involvement, and a strikingly different myocarditis severity and frequency when compared to classical kawasaki disease (127) (128) (129) (130) (131) (132) (133) (134) . biochemically the patients exhibited leukopenia with thrombocytopenia, increased ferritin, elevated myocarditis markers and high levels of procalcitonin, crp and cytokines were observed when compared to classical kawasaki disease (127) (128) (129) (130) (131) (132) (133) (134) . nearly 62% patients also showed resistance to the initial treatment with intravenous ivig infusion, and required a second infusion for successful treatment (128, 132) . while the children exhibited the devastating effects of the cytokine storm associated with covid-19, such as heart failure, pneumonia, gastrointestinal, neurological and renal manifestations, the paediatric patients in the french study rarely had respiratory manifestations (128) . this suggests a different host immune response in children compared to adults. treatment for pims involves the administration of il-1 receptor antagonist, il-6 receptor blockers such as tocilizumab or sarilumab, ivig, and steroids or biologics to control inflammation (128, 132) . covid-19 is known to affect older members of the population disproportionately, with adults over the age of 65 years making up to 80% of hospitalization and having a 23-fold greater risk of death (111, 194) . one possible explanation for this could be the increased baseline inflammation, called inflammaging, commonly observed in individuals over the age of 60 (195) . studies have shown increased baseline serum concentrations of crp and cytokines such as il-6 and il-8 (196) . inflammaging could be the result of the accumulation of mis-folded proteins, compromised gut barrier, obesity and impaired clearance of dead or dying cells (196, 197) . senescent nonlymphoid cells have been known to secrete inflammatory cytokines, chemokines, growth factors, and matrix metalloproteinases (195, 198) . this increased baseline inflammation inhibits antigen-specific immunity affecting the efficacy of many vaccines (199) . studies have shown that treatment with rapamycin, mapk inhibitor or steroids reduces this excessive inflammation and enhances vaccine efficacy (195, 200, 201) . in case of covid-19, this baseline inflammation may itself not be detrimental but contributes to the initiation of an inflammatory cascade that ends in the deadly cytokine storm (195) . furthermore the accumulation of senescent cells in the lungs of older patients could inhibit t cell response, induce nkr ligand expression, which marks the cells for elimination by infiltrating t cells expressing nkrs (195) . as observed in the case of vaccines against other respiratory viruses, inflammaging may reduce the efficacy of covid-19 vaccinations in this already disproportionately affected group. as with any infection, both the innate and adaptive arms of the immune system are required to mount a successful defence against a viral incursion. in case of covid-19, a decrease in the circulating t helper cells (cd4 + cells), cytotoxic t cells (cd8 + cells), b cells, natural killers cells, lymphocytes, monocytes, eosinophils and basophils has been reported (108, 149, 159, 202) . a retrospective, single-centre study involving 452 patients revealed a significant decrease in the total number of regulatory t cells, memory t cells and suppressor t cells (203) . the study also reported an increase in the percentage of naïve t cells (203) . as naïve t cells help respond to novel pathogens that the immune system has not yet encountered by managing release of cytokines, this may help explain the hyperinflammation (204) . the lower levels of memory t cells reported may also explain the relapses reported in covid-19 convalescent individuals (204) . direct infection of thp-1 cells, human peripheral blood monocyte-derived macrophages and dendritic cells by mers-cov and infection of t cells and macrophages by sars-cov has been reported (6, 205) . hence, it can be speculated that sars-cov-2 may also infect monocytes and macrophages by a mechanism that is yet to be elucidated (204) . receptors such as cd147 on the surface of t cells and other immune cells may mediate viral entry (206) . the clinical trial with anti-cd147 monoclonal antibody, meplazumab, showed promising efficacy and safety in covid-19 patients (207) . however, cd147 did not show a direct interaction with the s protein of sars-cov-2 (208) . similarly, lymphopenia can be attributed to sars-cov-2 direct infection and lymphocyte death, destruction of the lymphatic organs, and/or high levels of the programmed cell death protein 1 (pd-1) on cd8 + t cells (which is known to trigger t cell exhaustion) (204, 209, 210) . lymphocytopenia, neutrophilia and neutrophil-to-lymphocyte ratio are being used as a predictor for the severity of the illness during early stages of infection and a poor outcome in covid-19 (159, 202, 211, 212) . this further alludes to the hyper-inflammatory nature of covid-19. furthermore, covid-19 patients were reported to have elevated serum levels of highsensitivity c-reactive protein and procalcitonin, whose levels have been associated with high risks of mortality and organ injury (213) . lower percentage and count of cd3 + , cd4 + , and cd8 + lymphocytes populations serve as a prognostic marker for mortality, organ injury, and severe pneumonia (213) . sars-cov exposed as well as a subset of non-exposed people exhibit a cross-reactive t cell repertoire (214) . studies have also reported the presence of sars-cov-2 spike glycoprotein-reactive cd4 + t cells in peripheral blood of a subset of donor who were not infected with sars-cov-2 (215, 216) . these s reactive cd4 + t cells were found to primarily react with the c-terminal of the s epitope (216) . this binding preference could be attributed to the presence of overlapping human coronavirus mhc-ii epitopes in the c-terminal domain. hence, these cd4 + t cells are cross reactive clones generated during previous infections with endemic human coronavirus (216) . a long-term information and knowledge for ageing -camden' (linkage) sub-study is currently underway to study if pre-existing antibodies and specific t cells contribute to the devastating effect observed in old people (217) . the b cell response occurs alongside the t helper cell response (~1 week post infection) in covid-19 patients and helps mount a humoral response via antibodies that would help neutralise the virus (109) . characterisation of the transcriptome during the recovery stage of the disease revealed significantly lower levels of naive b cells, while plasma b cell levels had increased in peripheral blood mononuclear cells (159, 218) . it was found that a certain subset of patients who contract the disease may not develop long-lasting antibodies against the pathogen; it is possible that these patients may be susceptible to the re-infection (109) . immune cell profiling of covid-19 patients in the recovery stage by single-cell sequencing has identified several new b cell-receptor changes such as ighv3-23 and ighv3-7, and isotypes used earlier for vaccine development including ighv3-15, ighv3-30, and igkv3-11 (218) . the strongest pairing frequencies, ighv3-23-ighj4, has been suggested to indicate a monoclonal state associated with sars-cov-2 specificity (218) . antibodies analysed from the serum of covid-19 patients revealed no cross-reactivity with the s1 subunit of the sars cov spike antigen, while some reactivity was observed between the nucleocapsid antigens of sars-cov and sars-cov-2 (85) . the rbd-specific igm and igg antibodies were significantly elevated in the severe and recovered patients (159) . investigations conducted on covid-19 recuperating rhesus macaque models, re-infected with sars-cov-2, reported no measurable viral spreading, clinical manifestations, or histopathological changes associated with covid-19 (219) . the study found lower viral loads in nasopharyngeal or anal swabs 5 or 7 days after reinfection, compared to the recorded viral loads 5 or 7 days after the initial infection with sars-cov-2 at similar sites. similarly, increased levels of leukocytes and neutrophils were recorded 14 days after reinfection, compared to the levels measured during the initial infection. significantly higher specific antibody titres were recorded 14 day post reinfection. there were also increased activation of cd8 + t cells, changes in cd4 + tcm cells and memory b cells. thus, increased production of neutralising antibodies protected the primates against covid-19 re-infection (220, 221) . a study on 149 covid-19 convalescent individuals revealed that plasma collected after 39 days of symptom manifestation had a variable half-maximal pseudovirus neutralizing titres of less than 1:50 in 33%, below 1:1,000 in 79%, and only 1% showed titres above 1:5,000 (222) . interestingly, in spite of the low titres reported, antibodies specific to three distinct epitopes on the rbd of the sars-cov-2 s protein neutralized at half-maximal inhibitory concentrations as low as single digit ng/ml (222) . hence, a vaccine that can elicit the production of such highly potent antibodies, or monoclonal antibodies raised against the rbd of the sars-cov-2 s protein, may be highly protective. however, studies on sars-cov and mers-cov revealed that neutralizing antibodies to s protein can potentially augment severe lung injury by exacerbating inflammatory responses (109, (223) (224) (225) . hence, therapeutic antibodies should be carefully studied to minimise any unwanted pro-inflammatory activity while retaining maximum virus neutralizing capacity. additional specific insights on the intracellular life cycle have also been gained from nextgeneration sequencing (ngs) studies on the transcriptome and epi-transcriptome profile of sars-cov-2 virus and infected host cell. this fundamental approach has given an insight into the specific molecular dialogue between the pathogen and the host cell. this dialogue is complex. the sars-cov-2 transcriptome has been studied in high resolution. it has revealed its highly complex nature, mainly as a result of numerous discontinuous transcription events, revealing canonical and non-canonical rna transcripts with rna modifications (16) . in addition to the canonical full-length genome and other 9 sgrnas, this study also found numerous non-canonical rna transcripts of unknown orfs that contained rna modifications. 41 putative rna medications were identified at an aagaa motif. these previously unknown orfs represent the epi-transcriptome of sars-cov-2 and has revealed numerous viral transcripts that may be involved in pathogenesis (16) . another study looked at transcriptome profiling in the primary human lung epithelium and compared differences between sars-cov-2 and sars-cov infection and identified several pathways potentially involved in pathogenesis and gender-specific differences in clinical presentation (226) . among the genes that were upregulated were a cluster involved in the cytokine-mediated signalling pathways, and in particular, the il-17 signalling pathway (226) . specifically, cytokine pathways driven by nuclear factor kappa-light-chain-enhancer of activated b cell (nf-κb), toll-like receptors (tlrs), mitogen-activated protein kinase (mapk), bone marrow stromal cell antigen 2 (bst2), il-32, tnf alpha induced protein 3 (tnfaip3), tnfaip3 interacting protein 1 (tn1p1), intercellular adhesion molecule 1 (icam-1), intercellular adhesion molecule 2 (icam-2), matrix metallopeptidase 9 (mmp9), baculoviral iap repeat containing 3 (birc3), and rho family gtpase 1 (rnd1), were significantly upregulated during sars-cov-2 infection, suggesting a significant role in pathogenesis (226) . moreover, rela (nf-κb p65 subunit) seems to be significantly upregulated in sars-cov-2 infection, leading to il-8 involvement (226) . of note is the expression of oestrogen receptor 1 (esr1), which was also enhanced under sars-cov-2 infection, suggesting sex hormones may be involved in differential expression during viral infection and may have implications for the differences in clinical severity seen between genders (226) . additionally, over 24 and 48 hour post-infection, cxcl-2 was significantly upregulated in sars-cov-2 infection compared to sars-cov (226) . a recent study using single-cell rna-seq in human, non-human primate and mouse tissues/cells was able to resolve further the host cellular targets for sars-cov-2 and their abundance in specific tissue/cell types (36) . the study identified ace2 and tmprss2 co-expressing cells (lung type ii pneumocytes, ileal absorptive enterocytes and nasal goblet secretory cells) and also determined that that ace2 is induced by interferon-stimulated gene, suggesting a possible mechanism for enhanced viral infection (36) . the clinical pathways of covid-19 disease severity may also depend on host-specific factors that may contribute to the 'cytokine storm', or cytokines release syndrome (crs), which is the massive release of pro-inflammatory cytokines including cytokines (il-1β, il-2, il-6, il-7, il-8, and tnf-α) and chemokines such as cxcl10 and ccl2 in the lungs (172, 227) . these genomic approaches also shed light on the specific genetic host factors that predispose individuals to this severe clinical presentation. proteomic and transcriptomic studies on bronchoalveolar lavage (bal) samples from covid-19 patients have also revealed considerable insights into the expression of sars-cov-2 receptors, co-receptors, immune responses, as well as risk factors for severe disease e.g. age and co-morbidities. asthma, chronic obstructive pulmonary disease (copd), hypertension, smoking, obesity, and male gender status were all associated with higher expression of ace2 and cd147 in bal, as well as bronchial biopsy and blood from covid-19 patients (206) . furthermore, there was a positive correlation between the expression of cd147-related genes in bal and the age and body mass index (bmi) of covid-19 patients (206) . in another study on bal from covid-19 patients, an association was observed between covid-19 severity and enhanced levels of certain cytokines, e.g. ccl2/mcp-1, cxcl10/ip-10, ccl3/mip-1a, and ccl4/mip1b (228) . this study also found that sars-cov-2 triggered apoptosis and the p53 signalling pathway in lymphocytes, probably causing additional lymphopenia in these patients (228) . a comparison of transcriptome profiles between patients with covid-19 and influenza a virus infection revealed an absence of significant type i interferon/antiviral responses with sars-cov-2 infection, with enhanced expression of genes involved in metabolic pathways e.g. haem biosynthesis, oxidative phosphorylation and tryptophan metabolism, suggesting an important role for mitochondria during sars-cov-2 infection (229) . furthermore, a meta-analysis on bal data from covid-19 patients also revealed an excess for neutrophils and chemokines (229) . in meta-transcriptomic sequencing of bal from 8 covid-19 patients, the expression of proinflammatory genes, especially chemokines, was significantly elevated in these patients compared to community-acquired pneumonia patients and healthy controls, suggesting hypercytokinemia (230) . it also revealed enhanced dendritic cell and neutrophil activity (230) . in contrast to sars-cov, which induces an ineffective interferon response, sars-cov-2 was found to strongly initiate expression of numerous interferon stimulated genes, which are thought to significantly contribute to immunopathogenesis (230) . similarly, an analysis of rna-seq data sets of bal from covid-19 patients identified upregulation of neutrophil, inflammatory genes and chemokines, which may be involved in immunopathology, e.g. tnfr, il-8, cxcr1, cxcr2, adam10, gpr84, mme, anpep, and lap3 (231) . chronic co-morbidities for covid-19 patients include cardiovascular disease, hypertension, diabetes, stroke and malignant tumour (112) . it was also found that parameters such as older age, underlying hypertension, high cytokine levels (il-6, il-10, and tnf-α), and high lactate dehydrogenase level were significantly associated with severe covid-19 during hospital admission (166) . in a study involving 184 icu patients with covid-19 pneumonia, all of them showed an incidence of thrombotic complications such as symptomatic acute pulmonary embolism (pe), deep vein thrombosis, ischemic stroke, myocardial infarction or systemic arterial embolism (165) . approximately, one-third of patients experienced gastrointestinal symptoms. during hospitalization, a substantial proportion of patients presented cardiac injury, liver, and kidney dysfunction, and hyperglycaemia. icu covid-19 patients had higher plasma levels of il-2, il-7, il-10, gscf, ip10, mcp-1, mip-1, and tnf-α, compared to non-icu patients. majority of icu patients diagnosed with covid-19 were found to be at highest thrombotic risk (165) . patients with severe covid-19 likely developed ards and died of respiratory failure. biopsy samples at autopsy from a patient who died from severe covid-19 showed bilateral diffuse alveolar damage with cellular fibromyxoid exudates, and mononuclear inflammatory lymphocytes in both lungs (149, 177) . diffuse alveolar damage with fibrin rich hyaline membranes are pathological results of covid-19. in a study, 12 covid-19-infected cancer patients were found to have underlying diseases, such as hypertension, diabetes and chronic obstructive pulmonary disease (232) . cancer patients with accompanying covid-19 infection showed deteriorating conditions and poor outcomes, and thus it was recommended to avoid treatments causing immunosuppression (233) . the complement system is an integral part of the innate immune response. it consists of a group of plasma proteins produced mainly by the liver or membrane proteins expressed on cell surface. these proteins interact in a cascade that leads to the opsonization of pathogens and the induction of inflammatory responses. the complement system comprises of three distinct activation pathways, i.e. classical, alternative or lectin (mbl). the activation of these pathways is based on different molecules present on the pathogen surfaces. the classical pathway is initiated by the binding of c1q to the pathogen surface or antibody complex. the initiation of the alternative pathway is triggered by the binding of a spontaneously activated complement component to pathogen surface. the binding of the mbl to mannose-containing carbohydrates on pathogens triggers the initiation of the lectin pathway. the early events of three pathways eventually converge to generate a protease called, c3 convertase, which is covalently bound to the pathogen. the c3 convertase then cleaves c3, present in plasma, into c3a and c3b. the c3b binds to the pathogen and targets it for destruction by phagocytes. furthermore, c3b binds with the c3 convertase to form c5 convertase, which produces c5a and c5b. c5b triggers the late events of the complement cascade, which are a series of polymerization reactions where c6, c7, c8 and c9 interact with each other to form the membrane attack complex (mac). the mac can damage the membrane of certain pathogens by creating a pore in it. the c5a and c3a produced are important small peptide mediators of inflammation [reviewed in (234) ]. studies in c3 -/-(gene-deficient) mice infected with sars-cov revealed the presence of c3 activation products such as c3a, c3b, ic3b, c3c, and c3dg 1 day post infection (235) . the c3 deficient mice showed significantly less respiratory dysfunction and lower weight loss as compared to control. the mice also showed significantly lower levels of neutrophils and monocytes compared to the control. lower il-6, tnf-α and il-1 levels were reported in the lungs of the c3 deficient mice (235) . the study also reported lower weight loss in mice deficient in factor b or c4. in view of the critical role of the complement system in sars-cov infection since the first day of infection, it raised possibility for complement involvement in sars-cov-2. levels of the terminal component of the complement system (mac) and c5a are increased in patients with ards (236, 237) . mac is known to damage endothelial cells, and thus, regulation or inhibition of mac by its known regulators such as cd59 or clusterin could be a potential treatment for endothelial dysfunction/damage in ards or covid-19 (238) (239) (240) . considering the lectin pathway of the complement system, mbl was shown to bind sars-cov in vitro and inhibit its infectivity (241) . the n-protein of sars-cov and sars-cov-2 has been shown to interact with mbl-associated serine proteases-2 (masp2), which is known to initiate the lectin pathway (242) , leading to over-activation of the complement system. this same study also highlighted excess complement proteins found in post-mortem covid-19 patient lungs (242) . furthermore, deletion of masp2 or perturbance of the masp-2-n protein interaction was found to reduce lung injury. these studies, along with human proteomic studies, demonstrate the activation of multiple complement pathways during a coronavirus infection. in case of covid-19, the alternative and lectin pathways of the complement system seem to be preferentially activated (243) . increased levels of plasma c5a and mac were recorded in patients with moderate and severe covid-19 (244) . a post-mortem study of lung and skin vasculature in 5 covid-19 patients showed significant deposits of mac and c4d that colocalized with the sars-cov-2 s-protein, and masp2 in the micro-vasculature. this study did not find prominent classical features of ards such as hyaline membranes and inflammation in the histopathological examination (245) . a recent study reported an increase in levels of c5a, which correlated with increased covid-19 disease severity, as well as high levels of expression of c5ar1 in blood and pulmonary myeloid cells of covid-19 patients (246) . furthermore, use of anti-c5ar1 monoclonal antibodies in human c5ar1 knock-in mice was found to successfully prevent c5a-mediated myeloid cell recruitment and activation, thereby inhibiting acute lung injury (246) . a recent genetic study in covid-19 patients as reported that gene variants associated with complement regulatory protein, cd55 (decayaccelerating factor, which accelerates the decay of complement proteins, and thus inhibits complement activation) is associated with increased risk in clinical outcome (odds ratio 2.34-2.4); gene variants that map to c3 showed some protective effect (odds ratio 0.66-0.68) (247) . neutrophils along with the complement system are another important component in the defence of the host against invading pathogens. neutrophil infiltration in pulmonary capillaries, acute capillaritis with fibrin deposition, extravasation of neutrophils into the alveolar space, and neutrophilic mucositis have been reported in the case of sars-cov-2 infection (158) . the neutrophilic extracellular traps (nets) and the neutrophils activated by sars-cov-2 infection contain c3, factor b and properdin, triggering the alternative pathway of the complement system (243) . while this is usually beneficial, the sustained activation and nets formation leads to a hyper-inflammatory immune response that damages and destroy surrounding tissue. this aberrant behaviour, in concert with the abnormal complement activation, leads to the well recorded clinical manifestations observed in the case of covid-19 such as ards and pulmonary inflammation (248) . additionally, nets have been reported to induce the production of excessive thrombin and subsequently generate c5a (248) . hence, it has been speculated that feedback loop that begins with complement activation leading to netosis causing an increases in thrombin production, that further stimulates the complement activation causing enhanced net formation (243) . microangiopathy refers to a disease of the small blood vessels. the term is used when small blood vessels pathologically thicken or weaken, which leads to impaired flow of blood as well as leaking of cells and proteins. sustained inflammation in the vascular system due to the sars-cov-2 infection leads to thrombosis and microangiopathy (109, 249) . this is supported by reports of increased lactate dehydrogenase, bilirubin, activation of platelets, elevated d-dimer levels and hyper-fibrinolysis (250) . a post-mortem case series of 4 patients with covid-19 found thrombotic microangiopathy, which was restricted to the lungs, along with diffuse alveolar damage could have contributed to causing death (251) . another such study of 21 cases found similar diffuse alveolar damage with significant capillary congestion and microthrombi despite anti-coagulation therapy (252) . due to the presence of severe pulmonary vascular dysfunction in ards, it has been argued that ards is a type of vascular microthrombotic disease with lung phenotype involvement. this argument is supported by the association of mortality in ards with thrombocytopenia and mof as a result of disseminated intravascular coagulation (253) (254) (255) . in recent times, a couple of theories on the pathogenesis of ards in sepsis have evolved: the 'two-path unifying theory' in which certain homeostasis mechanisms lead to microthrombogenesis, and the 'twoactivation theory of the endothelium' in which the complement mac leads to inflammation via cytokines and microthrombogenesis via platelet activation (256, 257 ). the complement system plays a key role in the pathogenesis of thrombotic microangiopathy. this is a syndrome characterised by thrombocytopenia (low platelet count), microangiopathic haemolytic anaemia and systemic organ damage. atypical haemolytic uremic syndrome (ahus) is an example of such a disorder that typically leads to kidney damage. it is caused by excessive activation of the alternative pathway due to mutations in complement regulators factor h (common), factor i, membrane-cofactor protein, or c3. analysis of renal tissue morphology from autopsies of covid-19 patients revealed strong c5b-9 staining (via the alternative pathway) on the apical brush border of tubular epithelial cells with minimal deposition on glomeruli and capillaries of the kidney (258) . treatment with eculizumab (c5 inhibitor) dramatically improved outcomes of survival in ahus. features similar to complement-mediated thrombotic angiopathy such as kidney and cardiac injury increased lactate dehydrogenase and d-dimer, and decreased platelets were observed in covid-19 (137, 259) . eculizumab was used successfully as part of management of four covid-19 patients with severe pneumonia or ards in the intensive care unit, and this preliminary data is being used to conduct further full-fledged clinical trials with eculizumab (260) . considering the overlap with complement-mediated thrombotic angiopathy in covid-19, few studies are underway to test the effectiveness of complement regulators. a recent case study demonstrated a favourable outcome for the compstatin-based c3 inhibitor amy-101. the study, which involved a 71-year-old caucasian male with severe pneumonia and systemic inflammation, found that amy-101 was safe and had a favourable outcome in improving the clinical presentation of the patient significantly (261) . furthermore, treatment with a recombinant c5a antibody on 2 male covid-19 patients aged 54 and 67 years showed significant benefit in suppressing complement hyperactivation, which contributes to the excessive immune response causing aggravated inflammatory lung injury, a hallmark of sars-cov-2 pathogenesis and lethality (242) . one of the many challenges includes determining patients who have a dysregulated complement activation. c3 bound to erythrocytes has been detected in patients with covid-19 (262) , which may prove to be a useful blood marker as well as in identifying patients who potentially merit intervention with complement regulators (263) . in covid-19, endothelial injury has been found to be a key pathophysiological feature. a case series found direct evidence of viral infection of endothelial cells and endothelial inflammation, leading to endothelial cell death (264) . in covid-19 patients, endothelial cell abnormalities were recorded in the kidney, lung, heart, small bowel, and liver. 5 of 26 deceased covid-19 patients were found to have suffered endothelial cell swelling with variable foamy degeneration in the glomeruli and an additional 3 patients were found to have severe injury to the endothelium due to segmental fibrin thrombi in glomerular capillary loops (264) (265) (266) . mac deposition has been observed in the endothelium of covid-19 patients (245) . such studies have led to notion that in covid-19, there are strong vascular and inflammatory components as well, which play a significant role in the pathophysiology of the illness (267) . consistent with endothelial injury, the significantly elevated levels of von willebrand factor found in the patient with severe covid-19 has led to the idea that the infection of the ace2 expressing endothelium by sars-cov-2 induces injury and activates the complement , which sets up a feedback loop that maintains a state of inflammation (243, (268) (269) (270) . it is worth noting that ards may occur in covid-19 despite well-preserved lung gas volume, which could indicate a key role for inflammatory processes, leading to vascular constriction and subsequent low oxygen levels in the blood (271) . furthermore, d-dimer (a fibrin degradation product) levels are also found to be elevated in covid-19 and are associated with poorer prognosis (268, 272) . these factors add to the importance of understanding vascular changes in this disease, including microangiopathic processes and coagulopathies in patients with covid-19. pregnancy is associated with several maternal adaptations in both immune function (immunosuppression) and cardiovascular physiology (increased cardiac output, physiological anaemia, cardiac hypertrophy) that would likely alter susceptibility to viral respiratory infections including sars-cov-2. maternal death occurred in 15% of patients admitted to the icu for covid-19 and in 25% of those who required invasive mechanical ventilation (273) . to date, the literature consists of case reports, case series and retrospective studies. the most common presenting symptoms of maternal covid-19 are fever, cough, dyspnoea, and gastrointestinal symptoms (274) . clinical findings of respiratory manifestations were similar to those seen in the non-pregnant populations, with similar ct findings together with elevations in c-reactive protein with decreased white blood cell counts (275) . although the portal of entry is inhalational, there are widespread systemic effects. the immobility, hypoxia and acute inflammation lead to a prothrombotic hypercoagulable state, and indeed, elevated ddimers are correlated with disease severity (276) . covid-19 is thus associated with venous or arterial thromboembolism (277) . the mechanism by which this occurs is currently thought to be as a result of inflammatory cytokines (203) inducing production of tissue factor with subsequent thrombin activation. the elevations of d-dimer (often seen in acute phases of infection) may be related to this increased thrombin generation. while serious maternal morbidity has been seen, the vast majority of pregnant women with sars-cov-2 infection remained asymptomatic for respiratory symptoms (278) (279) (280) . pregnancy is coupled to physiological changes in cardiorespiratory status (281) which might be expected to alter susceptibility to a respiratory upset. nevertheless, the evidence suggests that this is less prevalent than thought. however, the changes in immune function and coagulation in pregnancy appear to increase some complications. similarly, the coagulopathies seen in covid-19 in the non-pregnant population might be expected to have deleterious effects in pregnancy, which is already a prothrombotic state. covid-19 has been seen to be associated with preeclampsia (274, 282) with one non-peerreviewed report suggesting a causal link (283) . the placenta has also been reported as having vascular malperfusion and thrombosis (284, 285) , which may provide an underlying explanation for the preeclampsia, a disease, associated with poor placental perfusion and altered vascular function (286) . evidence of increased clotting at the placental surface suggests that this mechanism may be responsible (in part) for the increased incidence of preeclampsia. sars-cov-2 virions have been seen in the syncytiotrophoblast, the part of the placenta responsible for the angiogenic imbalance seen in preeclampsia and effects on the release of known factors associated with the disease (sflt-1 -soluble fms like tyrosine kinase 1 and plgf -placental growth factor) are unknown. the disease is also linked to preterm premature rupture of membrane (pprom) (104, 282) , and preterm labour (287) , both of which are linked to inflammation. the underlying mechanism by which pprom occurs is not entirely elucidated. however, reports have suggested that activation of the coagulation system and thrombin causes fetal membrane weakening and subsequent rupture of membranes (288, 289) . the alterations in clotting and thrombin seen in covid-19 may well provide a mechanism for this. similarly, thrombin has been related to the induction of preterm labour and weakened fetal membranes by induction of decidual colony-stimulating factor (csf)-2 (290). at present, there are no drugs, therapeutics, or vaccines approved for curing, preventing, or treating sars-cov-2 specifically. as of june 2020, a total of 239 (147 in human trails, 92 in preclinical development) therapeutic drugs are under development against covid-19. the current treatment for sars-cov-2 patients involves the management of clinical symptoms and providing supportive care. while research into developing new drugs and treatments against sars-cov-2 are ongoing, much of the effort currently focuses on the repurposing existing medicines used against viruses, multiple sclerosis, arthritis, blood plasma derivatives and malaria. moreover, although immunosuppressive treatments, e.g. corticosteroids have shown promise for covid-19, there is considerable concern about possible side effects. other immunotherapeutic approaches given as adjunct therapy and based on neutralizing inflammatory cytokines and other immunomodulators, passive viral neutralization to reduce lung pathology and viral load, could be a promising approach (291) . a number of these approaches are discussed below. the antiviral drug remdesivir, developed by gilead sciences, is an adenosine analogue, which incorporates into nascent viral rna chains and results in premature termination, effectively inhibiting viral rna synthesis (292) . it was developed for the treatment of ebola and marburg virus infections (293) , and animal studies have shown that it is effective against the other coronavirus (294) . in vitro studies have established its efficacy against sars-cov-2 (295) . an open-label trial across the united states, europe, canada and japan showed clinical improvement of 36 of the 53 covid-19 patients who were treated with a 10-day course of remdesivir on a compassionate basis (296) . however, a follow-up multi-centre, randomized, double-blinded, placebo-controlled trial of 237 patients showed that the drug was not associated with a difference in time to clinical improvement. compared to the placebo, the drug was found to have a non-significant but, numerically faster time to clinical improvement in patients with a symptom duration of 10 days or lower (297) . currently, japan and the usa have allowed the use of the drug under emergency use authorization for the treatment of covid-19. in a randomized, open-label, multi-centre phase 3 clinical trials, a 5-day course remdesivir brought about a significant clinical improvement compared to standard alone in patients with moderate covid-19. this clinical study assessed the effect of 5-day (n=191) and 10-day (n=193) investigational remdesivir courses plus standard of care, versus standard of care alone (n=200) on clinical improvement on day 11 (298) . in case of patients with severe disease, both 5 day and 10 courses of the drug have been found to have similar clinical outcomes, but as the study lacked placebo control, the magnitude of benefit cannot be determined (299) . umifenovir, marketed as arbidol, is a derivative of indole carboxylic acids used for the treatment of influenza a and b virus infection, and other arboviruses (300) . it functions by incorporating into cell membranes and interfering with the hydrogen bonding network of phospholipids, blocking both the fusion of the virus to the cell membrane and the virusendosome fusion (301) . in vitro studies have established anti-viral efficacy against ebola virus, human herpesvirus 8, hepatitis c virus, tacaribe arena virus, sars-cov and sars-cov-2 (302) (303) (304) . a retrospective study on 81 sars-cov-2 patients treated with umifenovir did not reveal any improvement in clinical prognosis or accelerated viral clearance (304) . currently, two randomized and open-label trials to determine the safety and efficiency of the drug are ongoing in china. favipiravir, another anti-viral drug, developed by fujifilm toyama chemical (as avigan) and zhejiang hisun pharmaceutical, is a pyrazinecarboxamide derivative. it is converted into an active phosphoribosylated form (favipiravir-rtp) in cells and is recognized as a substrate by viral rna polymerase, thereby blocking the activity of rna-dependent rna polymerase. it was developed as a treatment against influenza. the drug is currently approved for the treatment of sars-cov-2 in china and italy. a study with 80 sars-cov-2 patients treated using the drug has reported that better efficacy was observed in anti-viral activity and lower adverse reactions compared to the control group that was treated with lopinavir/ritonavir (303) . another prospective, multi-centre, open-label, randomized superiority study with 240 sars-cov-2 infected patients was conducted at three hospitals. they showed faster recovery from clinical symptoms when compared to the controls that were treated with umifenovir, even though similar numbers required the use of ventilators and oxygen (305) . there are currently six trials ongoing in china evaluating the efficiency of this drug against other antivirals for the treatment of covid-19 and a phase 3 clinal trial to assess its effectiveness and safety is scheduled in japan and usa. anti-malaria drugs, chloroquine and hydroxychloroquine, are lysosomotropic agents that function by increasing late endosomal and lysosomal ph, which results in impaired viral release from the endosome or lysosome (306) (307) (308) . in vitro studies have shown antiviral activity against sars-cov-2 with hydroxychloroquine, a weak diprotic base, to have higher potency against the virus (295, 309) . in sars-cov-2, chloroquine, along with its lysosomotropic activity, is believed to reduce glycosylation of ace2 affecting the binding of the virus to the cells (310) . furthermore, chloroquine is also shown to block the production of proinflammatory cytokines such as il-6 preventing ards (311); hydroxychloroquine was found to possess an anti-inflammatory effect on th17-related cytokines (il-6, il-17 and il-22) (312) . initial clinical studies in china involving 100 sars-cov-2 infected patients, who were treated with chloroquine, showed amelioration of pneumonia, shortened disease progression, increased resolution of lung lesions on ct, and a better virus-negative conversion (313, 314) . hydroxychloroquine and combination therapy with azithromycin was found to reduce viral load in a french open-label non-randomised clinical trial and in an observational pilot study (315, 316) . nevertheless, these studies were plagued with several limitations, such as small sample size, very short observation period, no randomisation, lack of reports on clinical progression, poorly described inclusion and exclusion criteria, and low national early warning score (87, 315, 316) . another trial with 30 sars-cov-2 infected patients treated with hydroxychloroquine for seven days in china and a study with effectively 10 sars-cov-2 patients, revealed no significant difference in the nasopharyngeal viral carriage when compared to the controls that were provided with the local standard care (317, 318) . a third randomized clinical trial conducted in china with 62 patients exhibiting mild sars-cov-2 when treated with hydroxychloroquine were found to have recovered faster from cough and fever when compared to the placebo. however, the result of this study cannot be extrapolated to patients with severe sars-cov-2 (319). a retrospective cohort study of 1438 random sample of inpatients with laboratory-confirmed sars-cov-2 admitted to hospitals in the new york city was conducted. it did not find any significant differences in in-hospital mortality associated with the treatment with hydroxychloroquine, azithromycin, or both, compared to the controls where the patients were given neither of the drugs (320). the us fda and european medicines agency (ema) and many other countries like india and poland have authorized emergency use of hydroxychloroquine to treat sars-cov-2 infected patients. however, the fda and ema have issued warnings against the reported side effects of the drugs. these include abnormal electrical activity that affects the heart rhythm (qt interval prolongation, ventricular tachycardia, and ventricular fibrillation), particularly when taken at high doses or in combination with the antibiotic azithromycin. other side effects reported are liver and kidney problems, nerve cell damage that can lead to seizures and hypoglycaemia (321, 322) . around 30 clinical trials have been registered to study the effects of hydroxychloroquine and chloroquine independently or in combination with each other on sars-cov-2 have been registered in the usa and china (323) . another anti-parasitic drug, ivermectin, has been shown to be effective against sars-cov-2 in vitro (324) . a clinical trial to assess the efficiency of ivermectin against sars-cov-2 has been planned to take place in japan soon. the corticosteroid, dexamethasone, functions as an immunosuppressant. the drug is believed to modulate the lung injury caused by a dysregulated immune system, thereby reducing the progression to respiratory failure and death (325) . in a randomized, controlled, open-label, adaptive, platform trial, 2,104 patients treated with 6 mg of dexamethasone (orally or intravenously) for 10 days were found to have a significantly reduced 28 day mortality rate among those receiving mechanical ventilation by 33.33%, and by 20% among those receiving oxygen without mechanical ventilation, compared to 4,321 patients treated with standard care (325) . treatment with the drug did not provide any benefit to patients who did not require oxygen or mechanical ventilation, hinting at possible harm. the use of corticosteroid in the case of severe respiratory infections requires the use of "the right dose, at the right time, in the right patient" (325) . this is because a high or an early dose may help the virus proliferate by suppressing the immune system, instead of reducing inflammation. in case of covid-19, the peak of viral shedding is higher early in the disease. the benefit of dexamethasone when patients require respiratory support or after the first week of the disease suggest that this stage is dominated by an irrepressible immune response versus active viral replication (325) . dexamethasone is the first drug found to reduce mortality in covid-19 (326). lopinavir/ritonavir is a drug combination. lopinavir is a protease inhibitor, developed by abbott laboratories against hiv-1 that functions by blocking essential viral proteases (327) . due to poor pharmacokinetics, it is administered exclusively in combination with ritonavir which increases lopinavir's plasma half-life through inhibition of cyp3a-mediated metabolism of lopinavir, thereby increasing its exposure and improving the anti-viral activity of the drug (327) . in vitro studies have revealed that lopinavir inhibited the replication of the sars-cov-2 virus in vero e6 cell (328) . in a randomized, controlled, open-label trial with 199 patients with laboratory-confirmed sars-cov-2 infection, no benefit was observed with lopinavir-ritonavir treatment beyond standard care (329) . another single-blind randomised controlled trial in china treated 44 patients with mild/moderate covid-19 for 14 days, or umifenovir or standard care with no antiviral (219) . in the study, no differences were found in the time taken for viral clearance, as assessed by pcr of nasopharyngeal swabs, fever, cough, or lung ct findings. clinical status deterioration to severe/critical from mild or moderate clinical status and gastrointestinal side effects was seen highest in patients treated with lopinavir/ritonavir when compared to umifenovir treated or those treated with standard care and no antivirals (219) . both these randomised clinical trials suffer from small sample sizes and lack of blinding. a multi-centre, prospective, open-label, randomised, phase 2 trial in hong kong with 127 sars-cov-2 infected patients involved treatment for 14 days with only lopinavir-ritonavir (control), or with a combination of lopinavir-ritonavir, ribavirin, an oral hepatitis c virus drug, and ifn-. it found that the combination treatment was effective in reducing symptoms and viral shedding faster, as well as duration of hospital stay (330) . currently, about a dozen trials are studying the effect of the drug against sars-cov-2. one such study is a phase 4 randomized controlled trial in china in which the effectiveness of lopinavir-ritonavir against influenza drugs, umifenovir and oseltamivir, is to be studied. another south korean trial is looking to compare the efficacy of lopinavir-ritonavir against hydroxychloroquine. the who solidarity trial and uk-based recovery trial is looking to study the effectiveness of lopinavir-ritonavir independently; the who solidarity trial also looks to the explore the drug in combination with interferon-. another second-generation protease inhibitor against hiv-1, darunavir, has shown significant inhibition of sars-cov-2 replication (in vitro). according to a press release by johnson & johnson, an unpublished single-centre, open-label, randomized, and controlled trial in china in which 30 sars-cov-2 patients were treated with darunavir and cobicistat was not effective in treating sars-cov-2 (331). however, a further three clinical studies in china are scheduled. other drugs currently being tested against sars-cov-2 include tocilizumab, a monoclonal antibody against il-6 developed by roche, which is used for the treatment of moderate to severe rheumatoid arthritis by blocking il-6 activity. the drug was found to have helped cure 19 of 20 covid-19 patients in a trial conducted in china (332) . another open multi-centre randomized controlled trial french study awaits publication, in which 129 patients were split into two groups, i.e. routine treatment with and without tocilizumab: in the group treated with tocilizumab, the combination of ventilation requirement (mechanical or non-invasive) or death was achieved in a significantly lower proportion of patients (333) . a phase 3 trial to test its efficacy in treating patients with severe covid-19 has been authorised by the fda. moreover, an italian multi-centre, retrospective study of 544 patients with severe covid-19 pneumonia, revealed that the use of tocilizumab given either intravenously or subcutaneously was associated with reduced risk of mechanical ventilation and death (334) . anakinra is a recombinant il-1 receptor antagonist that has shown promise in treating severe covid-19 disease. in a retrospective cohort study of patients with covid-19 and ards that were managed with non-invasive ventilation (outside the icu), their treatment with high-dose anakinra was observed to be safe and associated with clinical improvement in 72% of patients (335) . another study has also described the early use of anakinra in covid-19 patients with cytokine storm syndrome (css) and acute hypoxic respiratory failure (ahrf) which may be beneficial in preventing the need for mechanical ventilation (336) . these results have encouraged further clinical trials to validate its safety and efficacy (337) . approaches targeting inhibition of bruton tyrosine kinase (btk) has also shown promise. btk plays a significant role in human innate immune responses. tlrs recognize ssrna of viruses like sars-cov-2 and induce signalling via btk-dependent activation of nf-κb, initiating a pro-inflammatory response (338) (339) (340) (341) . btk also plays a key role in the activation of the nlrp3 inflammasome, resulting in maturation and secretion of il-1β, a key pro-inflammatory cytokine (342) (343) (344) . thus, btk seems a favourable target against the cytokine storm in covid-19. in one study, acalabrutinib (a selective inhibitor of btk) was given to 19 patients with severe covid-19 and clinical improvements were observed over a 2-week treatment period, with reduced biomarkers of inflammation (c-reactive protein and il-6) to normal levels (345) . other dual inhibitors e.g. ibrutinib which target btk/il-2-inducible t-cell kinase (itk) signalling have also shown promise (346) . in one study of patients given ibutinib for treatment of b-cell malignancies and chronic graft-versus-host disease (cgvhd), there was evidence that ibutinib may also protect against pulmonary injury in covid-19, which these patients subsequently had, suggesting ibutinib as a possible prophylactic for vulnerable patient groups (347) . similar findings demonstrating a possible protective role of btk inhibitors in cancer with covid-19 have also been subsequently described (348) (349) (350) . these promising findings now merit a controlled randomised trial to demonstrate efficacy and drug safety of these btk inhibitors. intravenous immunoglobulin (ivig) is a pooled preparation of normal igg obtained from several thousand healthy donors. it is generally used in the immunotherapy of several autoimmune and inflammatory diseases, (351) , and thus has been investigated for treating covid-19 to mitigate the css. ivig therapy has shown promise through several studies, although careful selection of covid-19 patients and timing of ivig administration appear to be the key for good clinical outcome. preliminary findings from one multi-centre study showed that early administration of high dose ivig improved the prognosis of critical patients with covid-19 (352) . similarly, 3 patients with severe covid-19 who received high-dose ivig made a satisfactory recovery (353) . in another study, the use of ivig as an adjuvant treatment for covid-19 pneumonia within 48 hours of admission to the icu reduced the use of mechanical ventilation, icu and hospital time, and the 28-day mortality rate of patients with severe covid-19 pneumonia (354) . in a case study of a covid-19 patient with respiratory failure and shock, treatment with plasma exchange before ivig treatment resulted in prompt recovery without the need for mechanical ventilation and may be an additional early treatment step to treat critically ill covid-19 patients (355) . ivig treatment of severely-ill covid-19 patients on mechanical ventilation has also shown promise. in one study of 5 patients, treatment with ivig improved clinical and respiratory outcome, particularly saturation o2 levels, resulting in earlier extubation of the patients (356) . furthermore, ct graphs obtained after ivig therapy also revealed improvements in pulmonary lesions of these patients (356) . convalescent plasma therapy (cpt) is another classical adaptive immunotherapy used for the treatment of infectious disease for over a century. it has currently been approved for covid-19 by the fda under compassionate use rules. the treatment involves the transfusion of high neutralizing antibody titre containing blood plasma from sars-cov-2 recovered patients. this provides immediate short-term immunity. this is accomplished by binding of the pathogen to the antibody, which results in the activation of the immune system causing cellular cytotoxicity, phagocytosis, or direct pathogen neutralisation. five clinical studies, conducted involving 27 covid-19 patients who were treated with cpt, revealed significantly lower viral titres, increased levels of neutralizing antibody, improved clinical symptoms such as apyrexia, resolved ards and unassisted breathing (357) (358) (359) (360) (361) . among the 27 cpt-treated patients, no fatalities were recorded, and no severe adverse reactions or treatment complications associated with cpt were reported (357) (358) (359) (360) (361) . while providing with valuable initial data, these studies suffer from several limitations such as lack of proper control groups, non-randomized evaluations, concomitant drug treatments, poor participant selection, lack of proper cpt dosage, and duration of therapy (362) . three clinical trials are currently being evaluated by the fda to test the safety and efficiency of cpt in patients who have been exposed to the virus and are at high risk of developing severe covid-19, patients who are admitted in hospital with acute respiratory symptoms, and for covid 19 patients under mechanical ventilation (363) . further trials are also planned or ongoing in china, columbia, iran, mexico and the netherlands (363) . early safety indicators of covid-19 cpt were evaluated in a study of 5,000 patients and showed that the mortality rate was not unduly high and concluded that transfusion of convalescent plasma appears safe in hospitalized patients with covid-19 (364) . while the repertoire of antivirals and repurposed drugs tested against sars-cov-2 are expected to help manage the disease, the development of a safe and effective vaccine would help cut down the overall number of deaths and prevent the population from getting the disease in the first place. a recent study suggested that mandatory bcg vaccination can possibly be associated in flattening the curve in the spread of covid-19. it analysed the rate of day-wise increase in positive cases in 135 countries and deaths in 134 countries for the first 30-day period (365) . while arguments for the potentially beneficial effects of pre-existing vaccines have been sporadically made, including giving mmr (mumps, measles and rubella) vaccines to elderly population, generating a sars-cov-2 specific vaccine seems a logical and obligatory choice. as of 31 july 2020, the who landscape document reports 139 candidate vaccines developed on various platforms ( figure 6 ) in preclinical stages of development: only 26 are under clinical evaluation. mrna-1273 vaccine is a sequence optimized mrna/lnp expressing a perfusion stabilized form of sars-cov-2 s-2p a transmembrane anchored protein with the native furin cleavage site, developed by moderna in collaboration with the national institute of allergy and infectious diseases vaccine research center (366, 367) . the vaccine is undergoing an openlabel phase 1 clinical trial that started in march, 2020 with 45 healthy adult (18-55-year-old) volunteers for six weeks in three dose cohorts (25µg, 100µg and 250µg) as two doses approximately 28 days apart via intramuscular injection in the upper arm. three cohorts of 56-70-year-old volunteers and three cohorts of healthy volunteers aged 71 and above are being enrolled in addition to the initial volunteers. the volunteers will be followed through 12 months after the second vaccination to assess safety data, common vaccination symptoms, review trial data and advise niaid (367) . a phase ii trial with 600 healthy participants in two cohorts (18-55 years old adults and adults aged 55 years and above) treated with a placebo, a 50μg or a 250μg dose has begun from may, 29 th , 2020. the in vivo studies in murine models suggested the vaccine to be immunogenic and could elicit igg2a and igg1 subclass s-binding antibodies. mrna-1273 immunized mice splenocytes showed higher secretion of ifn- than il-4, il-5 or il-3 upon re-stimulation with peptide pools (s1 and s2). a dose of 1μg of mrna-127 was found to induce robust cd8 + t cell response to the s1 peptide pool with balanced th1/th2 ab isotype response in mice. thus, a 100 μg dose of vaccine has been decided for human trial in phase 3 study, which is equivalent to 1μg dose induced in mice (368) . the fda has granted fast track designation for the vaccine. the pfizer licensed biontech's bnt162 vaccine development programme has developed four coronavirus vaccine candidates (366) . two of the vaccines contain mrna coding for the spike protein of sars-cov-2, while the other two contain only the rbd of the spike protein (369) . furthermore, the four vaccine candidates are made of three different mrna formats. two of the vaccine are based on nucleoside modified mrna (modrna), which incorporates modified nucleosides in the mrna (370). this suppresses intrinsic immune activation and the production of anti-drug antibodies against the mrna itself (370). the suppressed immune activity against the therapeutic mrna helps produce the antigenic protein for longer periods (370). the next vaccine candidate is based on the optimised unmodified mrna (urna) format (370). urna uses uridine in the mrna, making it more immunogenic (370). finally, the last vaccine candidate uses self-amplifying mrna (sarna) (370). it is based on the principle of viral replication. the sarna, in addition to encoding a protein of interest, also encodes, replicase (370). this enables the self-amplification of the mrna inside the cell (370). the dsrna intermediate created during the replication of the rna triggers an immune response making sarna a potent activator of the immune system (370). a phase 1/2, randomized, placebo-controlled, observer-blind, dose-finding, and vaccine candidate-selection study to evaluate the safety, tolerability, immunogenicity, and potential efficacy of the candidate in 200 healthy adult volunteers is ongoing (100). another frontrunner among the candidates is cansino bio's ad5-ncov (366) . it is a genetically engineered vaccine candidate with the replication-defective adenovirus type 5 (live virus) as the vector to express sars-cov-2 spike protein. this would help the body to produce neutralizing antibodies against sars-cov-2. it has been shown to induce a strong anti-viral activity against sars-cov-2 in animal and in vitro studies. a single-centre, non-random, open, and dose-escalation phase i clinical trial for recombinant novel coronavirus vaccine (adenoviral vector) in 108 healthy adults aged between 18 and 60 years were conducted. the vaccine has been administered as a liquid formulation intramuscularly in the deltoid muscle (371) . three different doses were chosen: (a) low dose of 5x10 10 viral particles/0.5ml; (b) intermediate dose of 1.5x10 11 viral particles/ml; and (c) high dose combines both low and intermediate dose (one in each arm). the volunteers are assessed for a period for 6 months to study any adverse reactions or other relevant outcomes (371) . most common systematic adverse reaction observed were fever, fatigue, headache and muscular pain but with no serious adverse effect were noted within 28 days. participants showed four-fold increase in anti-rbd antibodies in all the groups; neutralizing antibodies increased gradually being highest at 28 days post vaccination. ad5 neutralizing antibody titres were boosted significantly postvaccination. il-2 and tnf- were detected and polyfunctional memory cd4 + t cell phenotypes were higher than cd8 + t cells. this suggested ad5 vectored covid-19 vaccine to be immunogenic and capable of stimulating both b and t cell response. for phase 2 clinical trial, intermediate dose was chosen and is expected to be completed by 31 january 2021 (372) . the vaccine may have some negative effects in older age people thus in the 2 nd clinical trial participants above 60 years will be included. t cell response peaked earlier from 14 th day after the 1 st shot of vaccine whereas the antibodies production level peaked at 28 th day post vaccination. the study also highlighted the possibility of negative effect on vaccine elicited immune response due to pre-existing ad5 immunity (372) . chadox1-ncov19 is being developed by oxford university, uk (366) . it is a replication deficient simian adenovirus vector chadox1, containing full length s-protein of sars cov-2 along with a tissue plasminogen activator leader sequence. the vaccine is reported to be effective in inducing an antiviral response in animal models (373) . chadox1-ncov19 was found to be immunogenic in mice mounting robust anti-viral response. single dose of this vaccine was capable of inducing humoral and cellular immune response in rhesus macaques (373) . a phase i/ii single-blinded, randomised, multi-centre study to determine efficacy, safety and immunogenicity of the vaccine in about 1090 healthy adult volunteers aged 18-55 years was initiated on april 23 rd , 2020 (374). the volunteers have been subjected to either one dose of 5x10 10 vp of chadox1 ncov-19, an additional booster dose of 2.5x10 10 vp of chadox1 ncov-19, or a control of menacwy vaccine delivered intramuscularly (374). the volunteers were assessed for a period for 6 months to study any adverse reactions or other relevant outcomes (375) . the results showed increase in s-specific antibodies with a single dose by 28 th day and increase in neutralizing antibodies with booster dose in all participants. chadox1-ncov19 was also capable of inducing heightened effector t-cell response quite earlier than antibody response. t cell response peaked on day 14 th and sustained up to 56 days. the results showed chadox1 ncov-19 vaccine to be safe, tolerant and immunogenic, which further supported phase 3 trial which is now underway (375) . picovacc is a purified inactivated sars-cov-2 vaccine candidate which is capable of inducing neutralizing antibodies in mice, rats, and nonhuman primates specific to sars-cov-2. cn2 strain of sars cov-2 virus was chosen to develop picovacc which was inactivated with β-propiolactone. this inactivated vaccine candidate was able to produce about 10-fold higher s-specific antibody titres in murine model when compared to covid-19 recovered patients. efficacy of picovacc was also tested in rhesus macaques with an intramuscular low (1.5µg), medium (3 µg) and high (6 µg) dose administered three times (0, 7 th and 14 th day) and on day 22 nd sars cov-2 cn1 strain was inoculated through intratracheal (lungs) route. all vaccinated macaques showed protection towards sars cov-2 infection and their viral loads declined significantly. no notable haemato-and histopathological changes were observed; human clinical trials are awaited (376) . a group of us scientists have come up with a series of prototype dna vaccines expressing variants of the sars-cov-2 spike protein. the efficacy of the dna vaccine candidates was evaluated in 35 rhesus macaques (6-12-year-old). intramuscular dose (5mg) of dna vaccine was administered, followed by booster dose on 3 rd week and antigenic challenge (1.2 x 10 8 viral particles) on 6 th week (both intranasal and intratracheal route). dna vaccine was found to be protective with dramatic reduction of viral replication and enhanced production of sspecific binding as well as neutralizing antibodies compared to controls. the study has not yet addressed the possibility of mutations that may emerge in escaping neutralizing antibodies, though it seems to be protective in primates against sars-cov-2 (377). j o u r n a l p r e -p r o o f ino-4800, developed by inovio, is a dna vaccine candidate (366) . the optimized spike protein of sars-cov-2 virus dna plasmids are introduced into cells by the use of a proprietary platform, cellectra®, via electroporation (378) . once inserted, the plasmids are expected to strengthen the body's own natural response. a phase i open-label study to evaluate the safety, tolerability and immunogenicity of ino-4800 as a prophylactic vaccine against sars-cov-2 in 40 healthy volunteers aged 18-50 years is ongoing (378) . the volunteers will be treated with either one or two doses of 1 mg of vaccine administered intradermally followed by electroporation the following day (378) . the volunteers will be assessed for a period for 1 year to study any adverse reactions or other relevant outcomes (378) . once the initial safety and immunogenicity of the vaccine are satisfied, phase ii efficacy studies are planned. qualitative and quantitative properties of cd4 + and cd8 + t cell responses in covid-19 and prophylactic vaccine development necessitate identifying viral regions and potential epitopes. thus, a total of 423 peptides (15-to 18-mer), which span the full proteome of the sars-cov-2 excluding orf-1, were designed and used to assess the memory t cell responses upon challenge on 42 patients following recovery from covid-19. 39 peptides were identified containing cd4 + and/or cd8 + epitopes. the memory of t cell responses from convalescent individuals with covid-19 was found to be greater in severe covid-19 cases compared to mild ones. immunodominant epitope clusters and peptides were most markedly observed to belong to spike, m, and orf3 proteins. in about 35% of study groups, strong cd8 + t cell responses specific to the np protein were observed, suggesting the possibility of inclusion of non-spike proteins in future covid-19 vaccine design (379) . in another study, a comprehensive immunogenicity map of the sars-cov-2 virus was carried out; 65 peptide sequences (33-mers) were generated based on computational algorithms. a single 33-mer peptide containing multiple epitopes that can possibly present on hla class i and class ii across majority of population and provide long-term immunity in most people acting as b and t cell epitopes had been identified. this in silico analysis needs further evaluation for safety and efficacy as a vaccine (380) . in an unprecedented short span of time and speed since the beginning of the covid-19 pandemic, significant progress has been made in our understanding of the pathogenesis of sars-cov-2 infection. however, there are endless unanswered questions; hopefully and most likely, they will be answered in near future. why there are a huge population that are asymptomatic carriers? what are the genetic contributors to susceptibility and resistance to developing covid-19? how pregnant women are so resilient to developing covidsymptoms; for that matter young children as well! what happens during the period of latency, i.e. between being infected and showing symptoms? how far the lung surfactant system gets affected during severe symptoms? what triggers thrombotic microangiopathy in addition to complement activation. on the adaptive immune aspects, what variations exist within population in terms of the proportion of neutralising antibodies? persistence of neutralising antibodies and recall memory magnitude following second infection (on vaccination trials) will yield serious information about how to finetune the dose, duration and vaccination strategies. in this acute crisis, a number of existing drugs have been repurposed empirically; clinical trials have yielded variable results. it is becoming clear that combination therapies are more likely to be successful. deciphering, at high resolution, the mechanisms and consequences of hostpathogen interactions in covid-19 will lead to novel therapies and preventative vaccination strategies. primary cellular host and co-receptor for sar-cov-2. 1) attachment and entry of sar2-cov-2 requires priming by transmembrane serine protease 2 (tmprss2) which cleaves the s protein into s1 and s2 portions, facilitating, 2), s1 targeting and binding of the receptor angiotensin-converting enzyme 2 (ace2), followed by receptor-mediated endocytosis of the virion into the host cell. j o u r n a l p r e -p r o o f tmprss2 is the key protease involved in priming sars-cov-2, which forms a receptorprotease complex with ace2 on the host cell surface, thus facilitating viral targeting and entry to the host cell. co-expression of aec2 and tmprss2 has been found in proximal as well in distal airways. the nasal cavity has the highest expression of both the receptors in ciliated and secretory (goblet) cells compared to lung bronchi (ciliated and secretory cells) and lung parenchyma (alveolar type 2 progenitor cells, at2). structural conformation of receptor-binding domain (rbd) present in s1 region of sars-cov-2 spike protein is capable of influencing the ace2-binding affinity. in case of sars-cov-2, the rbd contains a four-residue motif glycine-valine/glutamine-glutamate/threonineglycine which enables the binding loop to take a different conformation. it can undergo two possible conformational changes, a "lying down state" which has low affinity towards aec2 and a "standing up state" with high binding affinity. sars-cov-2 rbd is found mostly in lying down state, and thus being less accessible to aec2. this hidden conformation of rbd in the spike protein can possibly be a masking strategy for immune evasion by sars-cov-2. 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(bcg) vaccination predicts flattened curves for the spread of covid-19 world health organization. draft landscape of covid-19 candidate vaccines -15 phase i, open-label, dose-ranging study of the safety and immunogenicity of 2019-ncov vaccine (mrna-1273) in healthy adults sars-cov-2 mrna vaccine development enabled by prototype pathogen preparedness a close look at the frontrunning coronavirus vaccines as of may 1 (updated) chinese clinical trial registry. a randomized, double-blinded, placebo-controlled phase ii clinical trial for recombinant novel coronavirus (2019-ncov) vaccine (adenovirus vector) safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques a study of a candidate covid-19 vaccine (cov001) -full text view -clinicaltrials.gov safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial development of an inactivated vaccine candidate for sars-cov-2 dna vaccine protection against sars-cov-2 in rhesus macaques tolerability and immunogenicity of ino-4800 for covid-19 in healthy volunteers broad and strong memory cd4 + and cd8 + t cells induced by sars-cov-2 in uk convalescent covid-19 patients identification of sars-cov-2 vaccine epitopes predicted to induce long-term population-scale immunity key: cord-297132-lhfa9fl5 authors: aghagoli, ghazal; gallo marin, benjamin; katchur, nicole j.; chaves-sell, franz; asaad, wael f.; murphy, sarah a. title: neurological involvement in covid-19 and potential mechanisms: a review date: 2020-07-13 journal: neurocrit care doi: 10.1007/s12028-020-01049-4 sha: doc_id: 297132 cord_uid: lhfa9fl5 as the current understanding of covid-19 continues to evolve, a synthesis of the literature on the neurological impact of this novel virus may help inform clinical management and highlight potentially important avenues of investigation. additionally, understanding the potential mechanisms of neurologic injury may guide efforts to better detect and ameliorate these complications. in this review, we synthesize a range of clinical observations and initial case series describing potential neurologic manifestations of covid-19 and place these observations in the context of coronavirus neuro-pathophysiology as it may relate to sars-cov-2 infection. reported nervous system manifestations range from anosmia and ageusia, to cerebral hemorrhage and infarction. while the volume of covid-19-related case studies continues to grow, previous work examining related viruses suggests potential mechanisms through which the novel coronavirus may impact the cns and result in neurological complications. namely, animal studies examining the sars-cov have implicated the angiotensin-converting-enzyme-2 receptor as a mediator of coronavirus-related neuronal damage and have shown that sars-cov can infect cerebrovascular endothelium and brain parenchyma, the latter predominantly in the medial temporal lobe, resulting in apoptosis and necrosis. human postmortem brain studies indicate that human coronavirus variants and sars-cov can infect neurons and glia, implying sars-cov-2 may have similar neurovirulence. additionally, studies have demonstrated an increase in cytokine serum levels as a result of sars-cov infection, consistent with the notion that cytokine overproduction and toxicity may be a relevant potential mechanism of neurologic injury, paralleling a known pathway of pulmonary injury. we also discuss evidence that suggests that sars-cov-2 may be a vasculotropic and neurotropic virus. early reports suggest covid-19 may be associated with severe neurologic complications, and several plausible mechanisms exist to account for these observations. a heightened awareness of the potential for neurologic involvement and further investigation into the relevant pathophysiology will be necessary to understand and ultimately mitigate sars-cov-2-associated neurologic injury. the novel 2019 coronavirus disease (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) results in a variety of symptoms including fever, cough, and fatigue [1] . as more is learned, it has become apparent that neurologic involvement in covid-19 may be important in some patients. a subset of patients presents with neurologic symptoms such as headache, dizziness, or a cerebrovascular event [2] . reports have also implicated isolated, sudden onset of anosmia and ageusia as early indicators of sars-cov-2 infection, suggesting that early neurological involvement may be relevant [3] . of great concern are the potential long-term neurologic complications from covid-19 infection. here, we synthesize the literature to highlight clinical observations that suggest important associations between sars-cov-2 infection and the nervous system and discuss potential mechanisms of neural injury. awareness of the possible neurological manifestations in covid-19 patients is of utmost importance to assist providers in the recognition, treatment, and management of potentially life-threatening neurologic complications. while sars-cov-2 presents primarily as a respiratory disease, injury to other organ systems, including the nervous system, is well documented [4] . these observations shed light on the broad physiological impact of covid-19, and awareness of these extrapulmonary features may help inform the overall prognosis in patients affected. the reported neurologic effects of covid-19 infection are myriad and may include complications related to viral infection, immune response, critical illness, related therapies and recovery. a retrospective study of 214 covid-19 patients from wuhan, china, found that 36.4% of patients had neurologic manifestations of the disease, including symptoms relating to the central nervous system (24.8%), peripheral nervous system (8.9%) and skeletal muscle injury (10.7%). the most common neurologic manifestations were dizziness (16.8%) and headache (13.1%). severely ill patients were more likely than less severely afflicted patients to exhibit neurologic symptoms (45.5% vs. 30 .2%, respectively) including cerebrovascular disease (seen in 5.7% and 0.8%, respectively), impaired consciousness (14.8% vs. 2.4%), and skeletal muscle injury (19.3% vs. 4.8%) [4] . a recently published report of 58 patients admitted to two intensive care units in strasbourg, france, with covid-19-associated-ards found neurological features associated with the illness in 14% of the patients on admission to the icu, in 67% when sedation was lifted, and overall in 84% when considering all neurological complications through hospital discharge. neurologic symptoms cataloged in this study included agitation (69%), confusion (65%), and corticospinal tract signs (67%). notably, 33% of the patients discharged from the hospital were found to have an executive dysfunction syndrome such as inattention, disorientation or poorly organized movements. mri was performed in 13 of 64 patients for encephalopathic features. among these, bilateral frontotemporal hypoperfusion abnormalities were seen in 11/11 (100%), acute or subacute stroke in 3/13 (23%) and enhancement of leptomeningeal spaces in 8/13 (62%). eight patients underwent electroencephalography. one demonstrated a pattern of diffuse bilateral slowing while others had non-specific findings. csf samples were obtained from 7 patients; two patients had oligoclonal bands, one had elevated protein and igg levels and no patients had pcr assays positive for sars-cov-2 [5] . in another multi-center, retrospective cohort, 50 of 235 icu patients were found to have neurologic symptoms (21%) [6] . twenty-seven of these underwent mri, and acute imaging abnormalities were found in 44% (12/27) including, most commonly, cortical fluid-attenuated inversion recovery (flair) signal abnormalities in non-specific distributions affecting the frontal, temporal, parietal, occipital, cingulate or insular cortex, as might be seen with infectious or autoimmune encephalitis, a post-ictal state, hypoxia or hypoglycemia. subcortical and deep white matter signal changes accompanied these findings (6/10), and leptomeningeal enhancement (5/8 patients) was also seen. in addition, one patient presented with a large-vessel ais and another with cvst. csf testing demonstrated elevated protein in 6 out of 7 patients tested; all had normal cell counts and 2 had negative pcr assays for sars-cov-2 [6] . though case reports of possible encephalitis associated with covid-19 are reported [7, 8] , we found one isolated report of sars-cov-2 being detected in the csf of symptomatic patients. sars-cov-2 rna was detected in the csf by rt-pcr, but not the nasal swab, of a 24-year-old man in japan [9] . this patient had clinical findings of encephalitis, including seizure, headache, stiff neck, elevated csf pressure, and a wbc count of 12/μl [9] . magnetic resonance imaging (mri) with diffusion weighted imaging (dwi) demonstrated hyperintensity along the inferior horn of the right lateral ventricle, flair sequences showed hyperintense signal changes in the right mesial temporal lobe with hippocampal atrophy, and t2-weighted imaging revealed paranasal sinusitis [9] . this patient was diagnosed with encephalitis associated with sars-cov-2 [9] . in contrast, doung et al. describe a patient with headache and fever who presented with new seizure in the absence of respiratory symptoms. csf was not tested for sars-cov-2, but additional tests did suggest an aseptic meningitis with an elevated wbc count and lymphocytic predominance. a nasopharyngeal swab confirmed covid-19 infection [7] . in another case, a patient admitted with covid-19 developed signs of meningeal irritation and alteration in consciousness. a head ct and lumbar puncture were normal. bacterial and viral csf studies were negative including sars-cov-2 pcr testing, but the patient was diagnosed with covid-19-associated meningoencephalitis with the authors postulating transient dissemination of the virus in the csf with a robust inflammatory response [10] . interestingly, postmortem examination of a patient infected with sars-cov-2, who presented with confusion and mental status changes, detected virus in frontal lobe neurons by electron microscopy despite negative csf pcr testing. viral particles were also identified in brain capillary endothelial cell and seen actively budding from endothelial cells, both providing the first direct evidence of sars-cov-2 in human brain tissue and implicating a potential direct hematogenous route for cns seeding [11] . this report has been followed by a second, identifying and quantifying sars-cov-2 virus in brain tissue samples from 8 of 22 patients (36%) who died from covid-19 infection [12] , more firmly establishing cov-id-19's neurotropic potential. another case describes a covid-19 patient with cough, fever, and altered mental status who was diagnosed with acute necrotizing encephalopathy (ane) [8] . a non-contrast ct demonstrated symmetric hypoattenuation within the bilateral medial thalami with normal ct angiogram and venogram. mri showed characteristic hemorrhagic rim enhancing lesions within the thalami, medial temporal lobes, and subinsular regions [8] . ane is a rare but well-recognized complication of viral illnesses, particularly in children [13, 14] and was reported in adults during the novel influenza a h1n1 pandemic [15] . although the pathogenesis of ane is not known, it is hypothesized to be immune-mediated, with more than 90% of cases preceded by fever and upper respiratory infection. elevated csf and serum cytokines have been linked to the disease presentation in both adults and children, and some authors have suggested hypercytokinesis may play a role in driving endothelial damage and blood-brain barrier disruption [16] [17] [18] . whether sars-cov-2 may cause acute viral meningitis-encephalitis or viral associated encephalopathy syndromes, as have been associated with other viral illnesses, remains to be elucidated. acute demyelinating polyneuropathy associated with sars-cov-2 infection has also been described [19] . a case series from italy reports 5 patients who developed guillain-barre syndrome (gbs) between 5 and 10 days after the onset of typically described covid-19 symptoms, including fever, cough, anosmia and ageusia [20] , a similar time-interval to the development of gbs observed in other viral illnesses. sars-cov-2 rt-pcr of the csf was negative in all 5 of these patients [21] . a case of a patient who developed the typical progressive weakness of the distal lower extremities evolving to quadriplegia approximately 2 weeks after acute covid-19 infection is reported separately [22] , as is a series of two patients who developed miller fisher variant and polyneuritis cranialis, respectively, notably in the time-course of acute covid-19 infection [23] . in china, a 61-year-old otherwise asymptomatic woman presented with acute symmetric weakness and areflexia in the lower extremities. this patient was also diagnosed with gbs; 8 days later she developed typical covid-19 symptoms and sars-cov-2 infection was confirmed. at the time of hospital discharge, both respiratory and neurological symptoms had resolved [19] . because relatively little is yet known about sars-cov-2, profound muscle weakness and difficulty weaning off the ventilator are well described, and there is significant clinical overlap between critical illness neuropathy-myopathy and the symptoms of gbs, it may be important to consider this atypical complication of viral infection in critically ill patients. there is now a well-documented association of anosmia and ageusia with covid-19. although olfactory dysfunction can commonly occur with viral illness second to mucosal inflammation, what appears to be unique to covid-19 may be the development of anosmia and hyposmia in the absence of nasal obstruction or rhinorrhea. a study that included 417 patients with mild-to-moderate covid-19 infection admitted to 12 european hospitals found that 85.6% and 88.0% of patients reported anosmia and ageusia, respectively [3] . the concurrent appearance of both symptoms was statistically significant (p < 0.001), though either anosmia or ageusia can also occur alone. olfactory dysfunction manifested before any classical upper respiratory or pulmonary symptoms in 11.8% of cases [3] . while 18.2% of patients denied rhinorrhea symptoms, nearly 80% of these patients reported reduced olfaction or complete anosmia [3] . a cross-sectional study that included 59 patients with severe covid-19 hospitalized in italy found 33.9% reported anosmia or ageusia [24] . loss of smell was threefold higher in subjects who tested positive for sars-cov-2 (59%) than in subjects who tested negative (18%) in a population-based study in the uk [25] , and ten-fold higher among ambulatory clinic patients in the usa who tested positive for covid-19 [26] . the etiology of this phenomenon, and whether it is limited to injury or inflammation in epithelial tissue or represents a possible route of retrograde axonal transport to the cns is an area of investigation. for example, yet-to-be peerreviewed reports of bulk sequencing gene expression studies have suggested that, as with the respiratory epithelium, the human olfactory epithelium expresses both ace2 and tmprss2, key genes thought to be involved in sars-cov-2 infection [27, 28] . however, by using single cell rna-sequencing analysis and immunostaining techniques, expression of ace2 was isolated, not to olfactory sensory neurons or neurons of the olfactory bulb, but to the non-neuronal support cells of the olfactory epithelium, and sustentacular cells in particular [28] . this suggests that non-neuronal epithelial tissue may be the infectious target. a clinical report describes a sars-cov-2 patient with mild respiratory symptoms and acute anosmia in which bilateral hyperintensity of the olfactory bulbs and of the right gyrus rectus on flair sequence was seen on an mri performed on day 4 of symptoms [29] . followup imaging 28 days later showed resolution of cortical hyperintensities with slight reduction in thickness of the olfactory bulbs, though clinically the patient had recovered. notably, two other clinically similar patients had mri with flair performed on days of illness 12 and 25 that were completely unremarkable. this case, therefore, represents an intriguing and unique report-possibly an in vivo snapshot of viral involvement in an area of the brain associated with olfaction, and it points to what may be one pathway of viral infection from olfactory mucosa to the brain. recent studies have shed light on vascular-thrombotic complications associated with covid-19 infection [1, 30, 31] . coagulation abnormalities are common in severe illness and appear to be an important indicator of poor prognosis [31] . sepsis-induced coagulopathy (sic) has been described in 21.6% of patients classified with severe covid-19 and associated with mortality. heparin treatment in those with sic or with a markedly elevated d-dimer was also associated with improved survival [31, 32] . the incidence of arterial or venous thromboembolism has been reported to be between 8 and 31% in patients hospitalized with covid-19, despite treatment with prophylactic anticoagulation, including deep venous thromboembolism (vte), pulmonary embolism, acute ischemic stroke (ais), and cerebral venous sinus thrombosis [33] [34] [35] . one center noted that the rate of vte in covid-19 infected patients was more than five times greater than that of comparable historical control groups such as patients admitted with non-covid ards or influenza [36] . the american society of hematology has recommended that hospitalized patients with covid-19 be treated with standard thromboprophylaxis [37] , but there remains intense debate about the possible benefit of intensified anticoagulation regimens [38] . ais has been reported in 2.3-5% of patients hospitalized with covid-19 [4, 5, 39] , though patient series and case reports of ais continue to accumulate [40] [41] [42] . three covid-19 patients with multi-vessel ais and positive antiphospholipid antibodies are described in a recent case series. a 69-year-old patient with no significant past medical history was found to have multiple bilateral cerebral infarcts and bilateral jugular venous thrombi. in the same series, another 69-year-old patient developed ischemia in both lower limbs and two digits of his left hand and had bilateral cerebral infarcts in multiple vascular territories as revealed by ct imaging [40] . in these patients, anti-cardiolipin and anti-b2-glycoprotein antibodies were positive and lupus anticoagulant (lac) was negative. in a prospective french series of covid-19 patients, anti-cardiolipin antibodies were found in 10% and lac in 45% of a hospitalized cohort [43] . a retrospective study of 221 hospitalized covid-19 patients found cerebrovascular disease in a significant minority with 5% of the patients developing acute ischemic stroke, 0.5% cerebral venous sinus thrombosis, and 0.5% cerebral hemorrhage [39] . patients with cerebrovascular disease had a heightened inflammatory response and abnormal coagulation with elevated c-reactive protein (crp) and d-dimer levels. in this series, older age was a risk factor (mean age 71.6 years in the group with cerebrovascular disease versus 52.1 years in those without; p < 0.05) [39] . however, ais has also been described in young patients [42] and even as a presenting feature of covid-19. a case series from new york described five sars-cov-2-positive patients under the age of 50 who presented to medical attention with large-vessel strokes. only one of the patients in this notable series had a previous history of stroke [41] . there is mounting evidence to suggest that the vascular endothelium may be a key organ in covid-19 infection. clinically, it has been noted that the most frequent comorbidities of patients hospitalized with sars-cov-2 infection are hypertension, diabetes, and cardiovascular disease, which share endothelial dysfunction as a common feature [44] . the endothelium is also a principal regulator of thrombosis and hemostasis, and endothelial cell dysfunction, induced by covid-19 infection, may be an important driver of coagulopathy and increased thrombotic burden. endothelial cell activation induced by infection and resultant disruption of the antithrombotic endothelial surface, excess thrombin generation, and early termination of fibrinolysis are possible contributors to the thrombotic state [45] . because vascular endothelial cells express ace2 receptors in abundance, in addition to many other of the cell surface receptors used by sars-cov-2 for cell entry, direct viral infection of vascular endothelial cells has also been posited [38, 44, 45] . varga et al. demonstrated endothelial viral invasion by sars-cov-2 on pathology specimens affecting blood vessels in the heart, kidneys, lungs, and small intestine [46] . virus in the endothelium was accompanied by inflammatory cells and evidence of endothelial cell death, suggestive of an endotheliitis, which might explain microcirculatory injury or failure exacerbating critical illness and organ injury. an interesting clinical case report describes a severely ill patient with covid-19, ards, acute renal failure, and altered mental status in whom von willebrand factor, a marker of endothelial stimulation and damage, was massively elevated at 500% of normal [47] . the kawasaki-like syndrome that is now described in young patients following covid-19 infection and associated with a hyper-inflammatory state is further suggestive of a vascular inflammatory potential of sars-cov-2 [48, 49] . the longer-term complications of covid-19 infection remain unknown. as the virus proliferates in lung tissue, inducing inflammation and edema, alveolar gas exchange is disrupted, with the potential to cause hypoxia, anaerobic metabolism, and acid accumulation [50] . acidosis risks increasing cerebral vasodilation and promoting cerebral edema [50] . some concern may be raised by autopsy findings of patients infected with other coronaviruses. autopsies of 8 confirmed sars-cov cases in china revealed cerebral edema and marked neuronal injury [51] . recently, brain autopsies of 18 patients who tested positive for sars-cov-2 revealed hypoxic changes in the cerebellum and cerebrum, with neuronal loss in the cerebral cortex, hippocampus, and cerebellar purkinje cell layer [52] . these studies suggest that cns injury secondary to severe pulmonary dysfunction may occur. the degree and extent of cns injury in earlier phases of the disease process, and to what extent this may be clinically relevant to sars-cov-2, are still unclear. the clinical observations that have been reported in covid-19 shed light on the many potential adverse effects of sars-cov-2 infection on the nervous system and, in particular, how any specific effects of the virus might overlay on commonly observed consequences of critical illness such as critical illness encephalopathy, critical illness neuropathy, and critical illness myopathy. subsequent studies must address both the short and important longterm neurological consequences of covid-19 infection and how we can best mitigate disability and avoid morbidities. the known pathophysiology of sars-cov-2 and the other human coronaviruses offer clues regarding possible mechanisms of neurological damage. sars-cov-2 has now been shown to be capable of invading the cns, as have other human coronaviruses (hcov), the viral group of which sars-cov-2 is a member. sars-cov-2 invasion is thought to require both a cell surface receptor for the viral spike (s) protein to bind to as well as priming of the s protein by cell proteases. more specifically, sars-cov-2 utilizes ace2 as its entry receptor and tmprss2 cell protease for s protein priming [53] . cross human tissue surveys of ace2 and tmprss2 positive cells found co-expression of these proteins in nasal goblet and ciliated epithelial cells as well as oligodendrocytes [54] . ace2/tmprss2 co-expression in oligodendrocytes could be one means of cns infiltration or proliferation. cases of acute encephalitis were reported during the sars-cov epidemic with virus detected in patient csf [55, 56] . further insight also comes from pathology studies wherein both viral rna and infectious virus have been detected in brain tissue. a postmortem study examining four individuals with sars-cov-related deaths and four control individuals found sars-cov antigen and rna in the cerebrum of the sars-cov infected individuals [57] . other human coronaviruses have previously been found in the brain by autopsy studies: hcov strains 229e and oc43 were found in 44 out of 90 brain donors as determined by rt-pcr [58] . interestingly, the prevalence of oc43 was significantly higher in patients with multiple sclerosis (ms) than in controls. additionally, another study demonstrated an increase in mcp-1 chemokine mrna in astrocyte cells lines following hcov-oc43 infection [59] . elevation in mcp-1 has been linked to increased permeability of the blood-brain barrier [60] . thus, these results suggest that hcov infection may exacerbate a predisposition for ms neuropathology and highlight the possibility that coronavirus infection may interact with preexisting or coexisting neuropathology to yield additive or chronic neurologic complications. coronaviruses may invade the cns by either a transneuronal or hematogenous route. one unique feature of sars-cov-2, early anosmia, may signify early neuroinvasion through the olfactory bulb as retrograde transport of hcov from the nasal epithelium to the olfactory nerve and the cns has been demonstrated in mice models. three days after intranasal inoculation with hcov-oc4, transgenic mice were found to have cells containing viral-specific antigens in their olfactory bulbs, but not in the perivascular spaces. by 7 days post-inoculation, there was propagation of the virus throughout the whole brain, coincident with a fatal clinical encephalitis. like hcov-oc43, sars-cov has also been found in the cns of mice following experimental nasal inoculation. an approximately eightfold increase in the density of sars-cov-positive cells in the cns was observed over 1-2 weeks after infection, principally clustered in the hippocampus [61] . sars-cov has been clinically associated with cases of encephalitis, ischemic changes in neurons, and viral particles, and genome sequences have been detected in the brain upon human autopsy [51] . though sars-cov and sars-cov-2 share 82% of their genomic identity, sars-cov-2 has unique genetic characteristics, notably encoding proteins that may affect both viral replication and pathogenicity [62] . the implications and significance of these genetic differences are not yet known. coronaviruses may alternatively cross into the cns through a blood-brain barrier compromised by endothelial injury or endotheliitis, inflammatory mediators, transmigration of macrophages carrying the virus, or direct infection of the endothelial cells themselves [11, 53, 54, 58] . once established in the cns, sars-cov, the virus responsible for severe acute respiratory syndrome (sars), has been shown to be capable of inducing rapid transneuronal spread and death of infected neurons in transgenic mice models expressing human ace2 receptors [63] . on the other hand, some mice infected with hcov-oc43, a human coronavirus that causes the common cold, develop an acute encephalitis with neuronal infection, or may survive acute infection and develop chronic encephalitis characterized by behavioral changes and persistence of the oc43 virus in affected neurons [64] . infection of hippocampal and cortical neural cells with hcov-oc43 in tissue culture has indicated that cell death may occur by apoptosis of both infected and neighboring, non-infected cells [64] . tnf-⍺, a known trigger for apoptosis, was found to be released by the infected cells and may have contributed to apoptosis in uninfected cells and in infiltration and activation of microglia, a finding consistent with previous studies [65] . both sars-cov and sars-cov-2 enter host cells through ace2 receptors, but phylogenetic data and atomic-level resolution virus-receptor complex analyses suggest that the novel coronavirus may recognize human ace2 more efficiently [66, 67] . in a study that introduced clinical-grade soluble human ace2 (hrsace2) and sars-cov-2 in engineered human tissue, hrsace2 was able to effectively scavenge the virus inhibiting its attachment to cells [68] . ace2, which is expressed at high levels in various tissues including alveolar type-2 cells, brain endothelial cells, neurons, and glial cells [51, 69, 70] , regulates the renin-angiotensin system by opposing angiotensin-converting-enzyme (ace) signaling through the production of the vasodilator peptide angiotensin [1] [2] [3] [4] [5] [6] [7] 71] . sars-cov has been shown to reduce ace2 levels in the mouse lung without a detectable alteration in ace expression [72] . in a study of sars-cov transgenic mice that expressed human ace2 receptors, the transgenic mice showed susceptibility to the virus, more efficient replication of the virus as compared to wild-type mice, more severe pulmonary lesions, detectable viral antigen in the brain, and cerebral vasculitis and hemorrhages [73] . by downregulating ace2 expression, sars-cov-2 may upset the delicate balance of ace/ace2 cerebrovascular control which may result in unopposed ace signal, excessive vasoconstriction, or disrupted cerebral autoregulation. infection with sars-cov has previously been shown to be associated with high levels of cytokines, including tumor necrosis factor-alpha (tnfα), interleukin (il)-1β, il-6, il-12, and interferon gamma (infγ), a phenomenon known as "cytokine storm" [1, 74, 75] , and high levels of these "pro-inflammatory" cytokines have been linked to poor outcomes. sars-cov-2 shares such pathogenicity, as covid-19 severity has now been associated with increased levels of il-1β, il-2, il-6, il-7, il-8, il-10, il-17, infγ, infγ-inducible protein-10, mcp1, g-csf, tnfα, and macrophage inflammatory protein 1α [1, [76] [77] [78] [79] [80] [81] . elevated ferritin and il-6, markers of hyper-inflammation, have already been linked to mortality in covid-19 [1, 82] . cytokine storms may contribute to both acute lung injury and neurotoxicity; mice infected with influenza a virus demonstrated significant increases in cytokines il-6, il-1β, and tnf-α with vascular hyperpermeability seen in lungs and also the brain within 6 days of inoculation [83] . the integrity of the blood-brain barrier may be disrupted by cytokine-driven injury and immune-mediated toxicity in the absence of direct viral spread or invasion (fig. 1) . observations suggest that acute necrotizing encephalopathy (ane), as an example, may be mediated by cytokine toxicity [84] . cytokines may also be directly neurotoxic, mediate or even inhibit injury to cells of the cns either alone or acting in synergy [85] . the ways in which the highly activated cytokine signaling seen in sars-cov-2 infection may impact neurologic outcome via alteration of neuroinflammatory pathways are not understood. the effects of covid-19 on the nervous system and neurological outcomes after successful treatment have not been well studied. there is an urgent need for clinical and laboratory research to characterize the relationship between sars-cov-2 and neurologic injury. in particular, the broad variety of neurologic complications reported in association with covid-19, such as ischemic or hemorrhagic stroke, encephalopathy, and seizures, suggests direct effects of viral tropism for the cns, indirect effects through injury to other organ systems, or sporadic synergy between infectious mechanisms and underlying conditions. mounting evidence suggests that the novel coronavirus is both vasculotropic and neurotropic. to elucidate these pathogenic pathways, larger and more systematic studies will be required, and relevant animal and tissue models must be developed and refined. current data on neuropathology associated with covid-19 are severely limited. this likely reflects under-reporting of neurologic manifestations in the setting of massive coexisting pulmonary injury and sedation, which makes it difficult if not impossible to conduct thorough neurological clinical examinations, particularly in severe cases where such complications may be more common. furthermore, even when there may be clinical suspicion of neurological involvement, patients with covid-19 are maintained under strict isolation precautions, and the ability to obtain neuroimaging is limited, further restricting opportunities to observe and study complications and sequelae. however, efforts must be made to circumvent these challenges in order to better characterize sars-cov-2 and its neuropathologic potential. these findings will ultimately help clinicians detect neuropathological signs earlier, attempt therapeutic intervention prior to irreversible injury, and identify compelling neurobiological targets for more optimal treatment and prevention of neurologic injury. fig. 1 infection with the sars-cov-2 virus may lead to brain pathology through potential direct and indirect mechanisms. sars-cov-2 infection leads to cytokine storms. recent evidence suggests that interleukin (il)-1ra, il-6, monocyte chemoattractant protein (mcp)-3, and serum interferon gammainduced protein (ip)-10 are implicated in the fatal outcomes of covid-19 infection [81, 86] . cytokine storms can damage an intact bloodbrain barrier and disrupt normal functioning in the cns without the virus crossing the blood-brain barrier from the systemic circulation. in addition, covid-19 has been associated with a pro-thrombotic state, which may lead to occlusion of cerebral vessels and lead to brain injury [81] . finally, ace2, a functional receptor of sars-cov-2, may facilitate direct invasion of neurons and cerebrovascular endothelial cells, leading to apoptosis and necrosis of neurons and neighboring cells [64] . original figure created by nicole j. katchur clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study neurologic manifestations of hospitalized patients with coronavirus disease neurologic features in severe 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critically ill covid-19 cov-2: a storm is raging influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza novel human reovirus isolated from children with acute necrotizing encephalopathy cytokines and acute neurodegeneration exuberant elevation of ip-10, mcp-3 and il-1ra during sars-cov-2 infection is associated with disease severity and fatal outcome key: cord-298669-g2up0cfi authors: pollock, david d; castoe, todd a; perry, blair w; lytras, spyros; wade, kristen j; robertson, david l; holmes, edward c; boni, maciej f; kosakovsky pond, sergei l; parry, rhys; carlton, elizabeth j; wood, james l n; pennings, pleuni s; goldstein, richard a title: viral cpg deficiency provides no evidence that dogs were intermediate hosts for sars-cov-2 date: 2020-07-13 journal: mol biol evol doi: 10.1093/molbev/msaa178 sha: doc_id: 298669 cord_uid: g2up0cfi due to the scope and impact of the covid-19 pandemic there exists a strong desire to understand where the sars-cov-2 virus came from and how it jumped species boundaries to humans. molecular evolutionary analyses can trace viral origins by establishing relatedness and divergence times of viruses and identifying past selective pressures. however, we must uphold rigorous standards of inference and interpretation on this topic because of the ramifications of being wrong. here, we dispute the conclusions of xia (2020) that dogs are a likely intermediate host of a sars-cov-2 ancestor. we highlight major flaws in xia’s inference process and his analysis of cpg deficiencies, and conclude that there is no direct evidence for the role of dogs as intermediate hosts. bats and pangolins currently have the greatest support as ancestral hosts of sars-cov-2, with the strong caveat that sampling of wildlife species for coronaviruses has been limited. the covid-19 pandemic began following a cross-species transmission event of the causative virus, sars-cov-2, sometime in late 2019 lu et al., 2020; gorbalenya et al., 2020; . as the scientific community works to understand the origins, biology, impacts, and treatment strategies for this virus, it is key that we avoid over interpretation of findings and speculation not well supported by available evidence. otherwise, we risk diversion of time and resources from following more plausible and scientifically justified leads. accordingly, there is a heightened urgency for the scientific community to diligently survey and critically evaluate new research findings before they are accepted as sound or actionable knowledge. understanding the pre-human origins of sars-cov-2 is important because it may provide insight into how and why it was able to jump into human populations, in turn better defining the risks of future pandemics. molecular evolutionary studies have an important role to play in inferring the origins of the virus because they can confirm the relatedness of viruses, shed light on evolutionary time-scales, and potentially identify past selective pressures that allowed the virus to successfully infect and replicate in human hosts. a recent study by xia (2020) used patterns of cpg deficiency in sars-cov-2 and related coronaviruses, and a series of compounding assumptions, to promote "the importance of monitoring sars-like coronaviruses in feral dogs". his conclusions rest upon the observation that values of cpg deficiency in sars-cov-2 (genus betacoronavirus) resemble those observed in distantly related canine alphacoronaviruses that constitute a separate genus within the coronaviridae. here, we conduct a critical re-evaluation of the conclusions of xia (2020), highlight key flaws in his underlying logic, and illustrate why his conclusion that dogs are likely intermediate hosts of sars-cov-2 is unjustified based on available data. we re-analyze viral cpg deficiency data to incorporate key pangolin viral genomes that were available but omitted from xia's study. these data further undermine the key inferences and conclusions of xia (2020) . to date, the closest known relative of sars-cov-2 across its genome as a whole is the ratg13 virus that was isolated from a horseshoe bat, the established reservoir of the earlier sars coronaviruses that emerged in 2002 -2003 (zhou h. et al., 2020 . interestingly, rmyn02, isolated from another horseshoe bat, is more closely related to sars-cov-2 in the long replicase 1a reading frame (orf1ab; zhou p. et al., 2020) . the next closest relative of sars-cov-2, pangolin-2020, was isolated from pangolins illegally smuggled into guangdong province, china xiao et al., 2020) . thus, until a closer relative is identified, bats, followed by pangolins, are the most likely source of the originating or reservoir host species for sars-cov-2. however, all these viruses are divergent enough from sars-cov-2 on an evolutionary time-scale that their role is uncertain (boni et al., 2020) . a potentially informative feature of the cluster of bat and pangolin coronaviruses similar to sars-cov-2 is a region of the spike protein. this is a key viral feature that binds to the ace2 receptor in sars-cov-2 to enter host cells, and shows strong signs of multiple past recombination events. the spike binding regions of the pangolin-2020 coronavirus, and that of the 2017 pangolin coronavirus sequence, are more similar to sars-cov-2 than that of ratg13. this suggests that there were multiple recombination events between ancestral viruses related to the bat ratg13, rmyn02, pangolin-2020, and sars-cov-2 lineages (boni et al., 2020) . these findings suggest that such inter-viral recombination events occur commonly among coronaviruses in nature (zhou h. et al., 2020) . further, there was likely a recombination event in the past involving the variable loop region of the bat ratg13 virus, although current sampling is insufficient to determine what the parental and offspring sequences were in this recombination event (boni et al., 2020) . for these recombination events to have occurred, divergent viruses must have co-infected the same host. while bats are the only group known to host both ancestral forms of sars-cov-2, the two recent host-jumping events indicate that other organisms are also possible candidate hosts. the timing of these events is informed by the extent of divergence among these sequences and the viral mutation rate. estimated divergence dates between sars-cov-2 and ratg13, suggest that the coronavirus lineage that gave rise to sars-cov-2 circulated unnoticed for decades in bats or other intermediate hosts prior to infecting humans (boni et al. 2020; nielsen et al., 2020) . a well-known feature of most rna viruses is that they tend to have lower levels of cpg dinucleotides than expected based on the relative frequencies of c and g nucleotides independently (cheng, 2013; jenkins et al., 2001; karlin et al., 1994; rima and mcferran, 1997) . the sars-cov-2 viral genome is more depleted in cpgs than many related coronaviruses (fig. 1) , a trait shared with distantly related alphacoronaviruses in dogs. based primarily on this observation, xia (2020) concluded that canines are a likely intermediate (pre-human) host for sars-cov-2. the idea is founded on the assumption that cpg levels in sars-cov-2 and dog alphacoronavirus are notably low, requiring an unusual environment to evolve, and that the gastro-intestinal tract of dogs is the singular prime candidate to provide that environment. however, the basis of this argument is undermined by the observation that the most closely related sequences from bats and pangolins, several of which were omitted from xia's (2020) analysis, are also highly depleted in cpgs ( fig. 1 and supplementary table s1 ). in addition, many other rna viruses are far more depleted in cpgs than is sars-cov-2, including pestiviruses that also happen to be found in the pangolin (gao et al., 2020; fig. 1) . hence, cpg depletion is not a unique feature of dog viruses or sars-cov-2. many factors can influence the genomic composition of viruses, including random genetic drift, recombination, and underlying stochastic mutational bias, as well as natural selection (dunham et al. 2009; jenkins et al., 2001; theys et al., 2018) . normally in molecular evolutionary analyses, we assume mutation and drift as the null model, and inference of natural selection, adaptation, and recombination need to be demonstrated by obtaining strong evidence in their favor. xia (2020) , however, provided no compelling evidence for natural selection. it is reasonable to think that natural selection can play a role in viral cpg levels because viral cpg is a target for mammalian defense systems and viruses are likely to evolve to evade such host defense mechanisms. nevertheless, the evolutionary reasons for low gc content are still debated in even exceptionally well-studied systems with unquestioned animal origins (2020) points out, the mammalian zinc finger antiviral protein (zap) binds to cpg dinucleotides in viral rna genomes and inhibits viral replication and mediates viral degradation (ficarelli et al., 2020; ficarelli et al., 2019; meagher et al., 2019; takata et al., 2017) . additionally, mammalian apobec3g is known to modify viral rna, deaminating c to u (sharma et al., 2016; sharma et al., 2015; sharma et al., 2019) . notably, bats show unusual and extensive adaptation of apobec3g, potentially driving their anti-viral response and perhaps correlating with low cpg content in sars-like coronaviruses in bats (jebb et al., 2020) . at any point in time, natural selection affecting cpg content may be in a rough balance with mutation and drift, but differences in cpg content among species could be caused by strengthening or weakening of any of these factors. an altered host environment could induce more extensive targeting of cpgs and positive selection for their removal, or an altered viral life history could lead to stronger selection on viral protein function, including cpgs, and stronger selection for their retention. we can speculate that sequence context-dependency, such as that shown for gatc motifs (henaut et al., 1996) , may also play a role. likewise, relaxed selection could influence cpg levels in either direction. further, it has been shown that the genomic dinucleotide composition of rna viruses is a poor-predictor of host species, suggesting that there is minimal host-specific impact on cpg suppression (di giallonardo et al., 2017) . for these reasons, gross similarities in cpg depletion characteristics are unreliable for inferring their shared causative nature. in summary, cpg depletion levels are known to be diverse among rna viruses broadly, cpg levels are also depleted in non-canine viruses closely related to sars-cov-2, evidence that natural selection drove the cpg depletion in sars-cov-2 ancestors is lacking, and there are a variety of competing mechanisms for genomes to become relatively depleted in cpg over evolutionary time. despite this, xia (2020) speculated that low viral genomic cpg levels in sars-cov-2 required evolutionary time in a previous host species and tissue that more actively selected for cpg depletion than do bats. because low cpg levels, similar to those in sars-cov-2, were observed in alphacoronaviruses that infect dog digestive tracts, he then concluded: "… canine tissue infected by the canine coronavirus may provide a cellular environment selecting against cpg", and "this suggests the importance of monitoring sars-like coronaviruses in feral dogs in the fight against sars-cov-2." however, there is no evidence for the logical premise of xia's argument, considering that all mammals have digestive tracts. additionally, a recent inoculation study found that while other domesticated mammalian hosts are highly susceptible to sars-cov-2, canines exhibited low susceptibility, and no traces of viral rna were detectable in any dog organs (shi et al., 2020) . further, it is notable that based on a study modeling ace2 binding affinity with the spike protein from sars-cov-2, it seems highly unlikely that dogs played an important role in the recent evolution of sars-cov-2 (damas et al., 2020) . these findings cast further doubt on the relevance of dogs as hosts of viruses related to sars-cov-2. hence, there is no reason to conclude that dogs or dog digestive tracts are special in this respect. we reanalyzed the "sars-related" subset of the data shown in fig. 1 from xia (2020) , but also including seven betacoronaviruses from pangolins and a bat (rmyn02), four additional dog alphacoronaviruses, and two additional non-coronaviruses (pestiviruses) from pangolins, using the same indices (icpg -a measure of genomic cpg deficiency, and genomic gc content; fig. 1 ). the names of all viruses used in our analysis, along with estimated gc content and icpg estimates, are provided in supplementary table s1 ). multiple bat and pangolin betacoronaviruses have low icpg comparable to sars-cov-2, and the other pangolin viruses have even lower icpg. this non-exhaustive sample is sufficient to refute the claim by xia (2020) that "no betacoronaviruses from their natural hosts have the genomic icpg and gc% combination close to sars-cov-2 and batcov ratg13". notably, dog alphacoronaviruses are also not exceptional in terms of cpg deficiency. furthermore, while humans and dogs have zap, which xia (2020) hypothesizes targets and selects for cpg depletion, our analyses suggest zap is highly conserved in mammalian genomes. in particular, bat and pangolin genomes also appear to contain functional zap (supplementary table s2 ). apobec3g may also be conserved across mammals, but the results are less clear, as similarity to human apobec3g is low in other mammals; however, human apobec3g is more similar to genes in bats and the pangolin than in dogs (supplementary material table s3 ). these results are relevant because they mean that bats and pangolins, the most likely pre-human hosts at present, have equal mechanistic potential to select against viral cpg content as dogs. while there is no evidence that sars-cov-2 has a low cpg content due to the action or evasion of these mechanisms (or if such a process is responsible for any cpg patterns in any organisms), the distribution of these proteins provides no prior mechanistic basis to exclude bats and pangolins as either reservoirs or intermediate hosts, and provides no evidence to specifically implicate dogs. in addition to being unsupported by positive evidence, xia's (2020) hypothesis for dogs as intermediate hosts of ancestral viruses giving rise to sars-cov-2 requires an unlikely history of cross-species viral transmission (see fig. 2 for potential hypotheses) for which there is no evidence. specifically, this hypothesis minimally requires: 1) an ancestral sars virus in bats (the main reservoir for sars-lineage viruses) was passed to dogs, which drove depletion of viral cpgs, 2) dogs passed this virus back to an unknown host or hosts that passed it to bats and pangolins (which gave rise to pangolin 2020, bat rmyn02, and bat ratg13 observed coronaviruses), 3) and descendant lineages of this virus were passed to humans via an unknown host (fig. 2) . in addition to this primary hypothesis, xia's manuscript and subsequent online comments further imply dogs were a more recent host of sars-cov-2, and thus the need for monitoring "in feral dogs" (fig. 2) . a simpler alternative to this improbable transmission hypothesis is that bats transferred this virus directly to humans or through a yet undetermined host (fig. 2) . in our view, it is a problem that potential wild animal hosts have not yet been well sampled. while it may be worthwhile to test dog samples as part of broader efforts to sample diverse potential hosts, a narrow focus on dogs is unjustified by existing evidence. in summary, the proposition of xia (2020) that dogs are a likely pre-human host for sars-cov-2 is not 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epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding cpg dinucleotides inhibit hiv-1 replication through zinc finger antiviral protein (zap)-dependent and -independent mechanisms khnyn is essential for the zinc finger antiviral protein (zap) to restrict hiv-1 containing clustered cpg dinucleotides uneven distribution of gatc motifs in the escherichia coli chromosome, its plasmids and its phages evolution of base composition and codon usage bias in the genus flavivirus why is cpg suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? identifying sars-cov-2 related coronaviruses in malayan pangolins structure of the zinc-finger antiviral protein in complex with rna reveals a mechanism for selective targeting of cg-rich viral sequences a novel coronavirus emerging in china -key questions for impact assessment synonymous mutations and the molecular evolution of sars-cov-2 origins dinucleotide and stop codon frequencies in single-stranded rna viruses the double-domain cytidine deaminase apobec3g is a cellular site-specific rna editing enzyme apobec3a cytidine deaminase induces rna editing in monocytes and macrophages mitochondrial hypoxic stress induces widespread rna editing by apobec3g in natural killer cells susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 cg dinucleotide suppression enables antiviral defense targeting non-self rna within-patient mutation frequencies reveal fitness costs of cpg dinucleotides and drastic amino acid changes in hiv the cpg dinucleotide content of the hiv-1 envelope gene may predict disease progression extreme genomic cpg deficiency in sars-cov-2 and evasion of host antiviral defense isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins commentary: a genomic perspective on the origin and emergence of sars-cov-2 a novel bat coronavirus reveals natural insertions at the s1/s2 cleavage site of the spike protein and a possible recombinant origin of hcov-19 a pneumonia outbreak associated with a new coronavirus of probable bat origin longer timespan, and there could plausibly have been multiple hosts from divergent species during this time. dogs are represented by grey outlines because no viruses closely related to sars-cov-2 have been discovered in dogs supplementary table s1. viral sequences used in figure 1, along with icpg and gc content blast results comparing human zap proteins to homologous annotated genes in bat, dog, and pangolin genomes blast results comparing human apobec3g proteins to homologous annotated genes in bat, dog, and pangolin genomes ddp, tac, and elc are funded by nih (1r01ai134673); ddp is also funded by nih gm083127; tac is also funded by national science foundation (deb-1655571, ios-1655735); ech is funded by an arc australian laureate fellowship (fl170100022); sl and dlr are funded by the mrc (mc_uu_12014/12); jw is supported by the alborada trust. supplementary data are available at molecular biology and evolution online. d.d.p. and t.a.c wrote the manuscript; b.w.p. and s.l. analyzed data; all authors contributed concerns about the dog cpg paper and to editing the final manuscript. the authors declare no competing interests. key: cord-311762-f6muhf3d authors: chen, yu wai; yiu, chin-pang bennu; wong, kwok-yin title: prediction of the sars-cov-2 (2019-ncov) 3c-like protease (3cl (pro)) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates date: 2020-02-21 journal: f1000res doi: 10.12688/f1000research.22457.1 sha: doc_id: 311762 cord_uid: f6muhf3d we prepared the three-dimensional model of the sars-cov-2 (aka 2019-ncov) 3c-like protease (3cl (pro)) using the crystal structure of the highly similar (96% identity) ortholog from the sars-cov. all residues involved in the catalysis, substrate binding and dimerisation are 100% conserved. comparison of the polyprotein pp1ab sequences showed 86% identity. the 3c-like cleavage sites on the coronaviral polyproteins are highly conserved. based on the near-identical substrate specificities and high sequence identities, we are of the opinion that some of the previous progress of specific inhibitors development for the sars-cov enzyme can be conferred on its sars-cov-2 counterpart. with the 3cl (pro) molecular model, we performed virtual screening for purchasable drugs and proposed 16 candidates for consideration. among these, the antivirals ledipasvir or velpatasvir are particularly attractive as therapeutics to combat the new coronavirus with minimal side effects, commonly fatigue and headache. the drugs epclusa (velpatasvir/sofosbuvir) and harvoni (ledipasvir/sofosbuvir) could be very effective owing to their dual inhibitory actions on two viral enzymes. on 7 january 2020, a new coronavirus, 2019-ncov (now officially named sars-cov-2) was implicated in an alarming outbreak of a pneumonia-like illness covid-19, originating from wuhan city, hubei, china. human-to-human transmission was first confirmed in guangdong, china 1 . the world health organisation has declared this a global public health emergency -on 15 february 2020, there are more than 65,000 confirmed cases reported, and the death toll is over 1500. in the height of the crisis, this virus is spreading at a rate and scale far worse than previous coronaviral epidemics. it was immediately evident from its genome that the coronavirus is evolutionarily related (80% identity) to the beta-coronavirus implicated in the severe acute respiratory syndrome (sars), which originated in bats and was causative of a global outbreak in 2003. the momentum of research on developing antiviral agents against the sars-cov carried on after the epidemic subsided. despite this, no sars treatment has yet come to fruition; however, knowledge acquired from the extensive research and development efforts may be of use to inform the current therapeutic options. the viral genome encodes more than 20 proteins, among which are two proteases (pl pro and 3cl pro ) that are vital to virus replication; they cleave the two translated polyproteins (pp1a and pp1ab) into individual functional components. the 3-chymotrypsin-like protease (3cl pro , aka main protease, m pro ) is considered to be a promising drug target. tremendous effort has been spent on studying this protein in order to identify therapeutics against the sars-cov in particular and other pathogenic coronaviruses (e.g. mers-cov, the middle east respiratory syndrome coronavirus) in general because they share similar active sites and enzymatic mechanisms. the purpose of this study is to build a molecular model of the 3cl pro of the sars-cov-2 and to carry out virtual screening to identify readily usable therapeutics. it was not our intention, however, to comment on other structure-based drug design research as these will not be timely for the current epidemic. the translated polyprotein (pp1ab) sequence was obtained from the annotation of the genbank entry of the sars-cov-2 genome (accession number mn908947). by comparing this sequence with the sars-cov pp1ab sequence (accession number abi96956), the protease cleavage sites and all mature protein sequences were obtained. sequence comparison and alignment were performed with blastp. the high-resolution apo-enzyme structure of sars-cov 3cl pro (pdbid: 2duc) 2 was employed as the template. the variant residues were "mutated" in silico by scwrl4 3 , followed by manual adjustment to ensure that the best side-chain rotamer was employed ( table 2 ). the rebuilt model was subjected to steepest descent energy minimisation by gromacs 2018.4 using the gromos 54a7 forcefield, with a restraint force constant of 1000 kj mol -1 nm -2 applied on all backbone atoms and all atoms of the vital residues (table 1) . accessible surface area of residues were calculated with areaimol of the ccp4 suite v7.0. mtiopenscreen web service 4 was used for screening against its library of 7173 purchasable drugs (drugs-lib), with the binding site grid specified by the active-site residues. the active sites on chain a and chain b were screened independently with autodock vina 5 . when the crystal structure was released, it was stripped of its inhibitor and subjected to a screening. a list of 4,500 target:ligand docking combinations ranked by binding energies was produced for each screen. the top 10 or 11 (ranked using a binding energy cut-off) hits for chains a and b were examined visually in pymol (version 1.7.x) 6 . an earlier version of this article can be found on chemrxiv (doi: 10.26434/chemrxiv.11831103.v2). the first available genome was genbank mn908947, now ncbi reference sequence nc_045512. from it, the pp1ab sequence of sars-cov-2 was extracted and aligned with that of sars-cov. the overall amino-acid sequence identity is very high (86%). the conservation is noticeable at the polyprotein cleavage sites. all 11 3cl pro sites 2 are highly conserved or identical (extended data 7 , table s1), inferring that their respective proteases have very similar specificities. the 3cl pro sequence of sars-cov-2 has only 12 out of 306 residues different from that of sars-cov (identity = 96%). we compared the polyprotein pp1ab and the 3cl pro sequences among all 11 sars-cov-2 genomes (genbank mn908947, 3d model of the sars-cov-2 3cl pro the amino acids that are known to be important for the enzyme's functions are listed in table 1 . not unexpectedly, none of the 12 variant positions are involved in major roles. therefore, we are confident to prepare a structural model of the sars-cov-2 3cl pro by molecular modelling (extended data 7 , figure s1 ), which will be immediately useful for in silico development of targeted treatment. after we submitted the first draft of this study, the crystal structure of sars-cov-2 3cl pro was solved and released (pdb id 6lu7), which confirms that the predicted model is good within experimental errors (extended data 7 , figure s2 ). when examined in molecular graphics 6 , all solutions were found to fit into their respective active sites convincingly. the binding energies of chain a complexes were generally higher than those of chain b by approximately 1.4 kcal mol -1 (table 3) . this presumably demonstrates the intrinsic conformational variability between the a-and b-chain active sites in the crystal structure (the average root-mean-square deviation (rmsd) in cα atomic positions of active-site residues is 0.83 å). in each screen, the differences in binding energies are small, suggesting that the ranking is not discriminatory, and all top scorers should be examined. we combined the two screens and found 16 candidates which give promising binding models (etoposide and its phosphate counted as one) ( table 3) . we checked the actions, targets and side effects of the 16 candidates. among these, we first noticed velpatasvir ( figure 1a , d) and ledipasvir, which are inhibitors of the ns5a protein of the hepatitis c virus (hcv). both are marketed as approved drugs in combination with sofosbuvir, which is a prodrug nucleotide analogue inhibitor of rna-dependent rna polymerase (rdrp, or ns5b). interestingly, sofosbuvir has recently been proposed as an antiviral for the sars-cov-2 based on the similarity between the replication mechanisms of the hcv and the coronaviruses 14 . our results further strengthen that these dual-component hcv drugs, epclusa (velpatasvir/sofosbuvir) and harvoni (ledipasvir/sofosbuvir), may be attractive candidates to repurpose because they may inhibit two coronaviral enzymes. a drug that can target two viral proteins substantially reduces the ability of the virus to develop resistance. these direct-acting antiviral drugs are also associated with very minimal side effects and are conveniently orally administered (table 4 ). the flavonoid glycosides diosmin ( figure 1b ) and hesperidin ( figure 1e ), obtained from citrus fruits, fit very well into and block the substrate binding site. yet, these compounds table 2 . in silico mutagenesis of the sars-cov-2 3cl pro . the 12 variant residues with reference to the sars-cov enzyme are shown with the respective treatment of rotamer. "a" and "b" refers to the individual chains of the dimeric model. both chains are in the crystal asymmetric unit and are not identical. the rotamer symbol (bracketed) is defined according to the conventions of richardson 15 , followed by its respective rank of popularity. asa: accessible surface area (average of a and b chains) of the residue in the sars-cov 3cl pro structure, in å 2 and in % relative to the asa of a residue x in the gly-x-gly conformation. residue rotamer asa, å 2 (%) remarks on replacement (table 4) . hesperidin hits showed up multiple times, suggesting it has many modes of binding ( figure 1a ). teniposide and etoposide (and its phosphate) are chemically related and turned up in multiple hits with good binding models ( figure 1f ). however, these chemotherapy drugs have a lot of strong side effects and need intravenous administration (table 4 ). the approved drug venetoclax ( figure 1c ) and investigational drugs mk-3207 and r428 scored well in both screens. venetoclax is another chemotherapy drug that is burdened by side effects including upper respiratory tract infection (table 4) . not much has been disclosed about mk-3207 and r428. we subjected the crystal structure to the same virtual screening procedures. a very similar list of candidates showed up consistently (extended data 7 , table s2 ) with high scores although ledipasvir was not found. we noticed that most of the compounds on the list have molecular weights (mw) over 500, except lumacaftor (mw=452). the largest one is ledipasvir (mw=889). this is because the size of the peptide substrate and the deeply buried protease active site demand a large molecule that has many rotatable dynamics to fit into it. we identified five trials on clinicaltrials.gov involving antiviral and immunomodulatory drug treatments for sars ( one record which receives a lot of attention amid the current outbreak is the lopinavir/ritonavir combination 18 . they are protease inhibitors originally developed against hiv. during the 2003 sars outbreak, despite lacking a clinical trial, they were tried as an emergency measure and found to offer improved clinical outcome 18 . however, some scientists did express scepticism 19 . by analogy, these compounds were speculated to act on sars-cov 3cl pro specifically, but there is as yet no crystal structure to support that, although docking studies were carried out to propose various binding modes 20-23 . the ic 50 value of lopinavir is 50 μm (k i = 14 μm) and that for ritonavir cannot be established 24 . although this is far from a cure, based on our results that the two cov 3cl pro enzymes are identical as far as protein sequences and substrate specificities are concerned, we are of the opinion that this is still one of the recommended routes for immediate treatment at the time of writing (early february 2020). if we look beyond the 3cl pro , an earlier screen produced 27 candidates that could be repurposed against both sars-cov and mers-cov 25 . in addition, the other coronaviral proteins could be targeted for screening. treatment of the covid-19 with remdesivir (a repurposed drug in development targeting the rdrp) showing improved clinical outcome has just been reported and clinical trial is now underway 26 . we consider this work part of the global efforts responding in a timely fashion to fight this deadly communicable disease. we are aware that there are similar modelling, screening and repurposing exercises targeting 3cl pro reported or announced 20,27-33 . our methods did not overlap, and we share no common results with these studies. the "extended results" folder contains the following extended data: • tab s1.docx (sequence homology of the 3cl pro cleavage junctions of pp1ab between sars-cov-2 and sars-cov). • tab s2.docx (the results of virtual screening of drugs on the active site of sars-cov-2 3cl pro crystal structure). • fig s1. pptx (the structural model of the sars-cov-2 3cl pro protease). • compare crystal.docx (a comparison, with figure s2 , of the active sites of model chains a, b and the crystal structure). data are available under the terms of the creative commons zero "no rights reserved" data waiver (cc0 1.0 public domain dedication). if applicable, is the statistical analysis and its interpretation appropriate? yes 1. 3. more details of the docking should be provided. what's the binding energy cutoff used? how is the hits (reported in table 3 ) used? 3clpro is catalytically active as a dimer. how is this considered in the virtual screening? what does the "(b top scorers)" mean? in the extended data of virtual screening, one compound could have multiple entries with different zinc numbers. for example hesperidin corresponds to at least 20 different compounds. what are the difference? and how are different results assembled? table 1 is not clear. please do a column-by-column comparison between different sites of sars-cov and sars-cov-2. also please add one-letter amino acid codes for the residues. the constructed protein structure is very similar to the recently solved crystal structure (6lu7), as "... confirms that the predicted model is good within experimental errors", but the docking results seem to differ significantly. could the authors explain? are all the source data underlying the results available to ensure full reproducibility? yes no competing interests were disclosed. competing interests: we confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster reported the computational modelling and virtual screening results of the 3c-like protease et. al.(3clpro) of sars-cov-2. this study is timely in view of the recent outbreak of covid-19. the rationale of repurposing existing drugs to tackle the global viral outbreak is sound. the manuscript is also well-written and structured. it should be noted that:the authors compared their model with the recently published crystal structure of 3clpro and found a high similarity between the two structures. they also obtained a similar list of top-ranked drug candidates when the crystal structure was subjected to the same screening protocol.several studies using similar modeling and virtual screening approaches have also been published recently. some suggestions for improving the manuscript:the authors proposed that the hcv drugs velpatasvir and ledipasvir, and thus epclusa and harvoni, could be attractive drug candidates for treating sars-cov-2 infection. however, there is no direct evidence to support this claim. to support this claim, the authors should connect the computational results with experimental data. to test their hypothesis, the authors should at least prove (or disprove) that the two hcv drugs could inhibit the biochemical activity of 3clpro of sars-cov-2.to further test the hypothesis, the two ns5a inhibitors should be tested using in vitro assays such as viral rna pcr assay.if there are no such experimental data to support the claim, the authors may consider revising their conclusion to "the computational results provide a rationale for further experimental validation of treating sars-cov-2 with velpatasvir and ledipasvir". no competing interests were disclosed. reviewer expertise: medicinal chemistry, drug discovery, chemical biology yu wai chen and co-workers presented a molecular modeling and docking study of the 3cl protease in the sars-cov-2 virus. the manuscript started with comparing polyprotein pp1ab sequences of sars-cov-2 and sars-cov, based on which the 3d structure of sars-cov-2 3clpro protein was constructed. the authors then performed virtual screening against sars-cov-2 3clpro using a library of 7173 purchasable drugs. considering both binding affinities and known side effects, the authors recommend velpatasvir and ledipasvir, and further suggest combining them with another hcv rdrp inhibitor sofosbuvir, aka repurposing the epclusa and harvoni for treating the coronavirus. this is a concise and timely report, and has proposed new therapeutic possibilities for the sars-cov-2 virus. the manuscript could be further improved by addressing the following comments. more details of the docking should be provided. what's the binding energy cutoff used? how is the the benefits of publishing with f1000research:your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and morethe peer review process is transparent and collaborative your article is indexed in pubmed after passing peer review dedicated customer support at every stage for pre-submission enquiries, contact research@f1000.com key: cord-296268-kb7fgfaq authors: mendonça, luiza; howe, andrew; gilchrist, james b.; sun, dapeng; knight, michael l.; zanetti-domingues, laura c.; bateman, benji; krebs, anna-sophia; chen, long; radecke, julika; sheng, yuewen; li, vivian d.; ni, tao; kounatidis, ilias; koronfel, mohamed a.; szynkiewicz, marta; harkiolaki, maria; martin-fernandez, marisa l.; james, william; zhang, peijun title: sars-cov-2 assembly and egress pathway revealed by correlative multi-modal multi-scale cryo-imaging date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.05.370239 sha: doc_id: 296268 cord_uid: kb7fgfaq since the outbreak of the sars-cov-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. however, investigation of the sars-cov-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on sars-cov-2 replicative cycle. understanding the genome replication, assembly and egress of sars-cov-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. here, we investigated sars-cov-2 replication in vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft x-ray cryo-tomography and serial cryofib/sem volume imaging of the entire sars-cov-2 infected cell with cryo-electron tomography (cryoet) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. our results reveal at the whole cell level profound cytopathic effects of sars-cov-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for rna synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. furthermore, cryoet of cell lamellae reveals how viral rnas are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and rnps assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of sars-cov-2 genome replication, virus assembly and egress pathways. since the outbreak of the sars-cov-2 pandemic, there have been intense structural studies 24 on purified recombinant viral components and inactivated viruses. however, investigation of 25 the sars-cov-2 infection in the native cellular context is scarce, and there is a lack of 26 comprehensive knowledge on sars-cov-2 replicative cycle. understanding the genome 27 replication, assembly and egress of sars-cov-2, a multistage process that involves different 28 cellular compartments and the activity of many viral and cellular proteins, is critically krios to identify each individual infected cell (39.2 % for moi of 0.1 and 45.4% for moi 124 0.5) where cryoet tilt series were collected at the cell periphery. the grids were then 125 transferred to a fib/sem dualbeam instrument and the same infected cells were subjected to 126 either serial cryofib/sem volume imaging (zhu et al., 2021) or cryofib milling of cellular 127 lamellae where additional cryoet tilt series were collected (sutton et al., 2020) . 128 alternatively, we imaged infected cells on cryoem grids by soft x-ray cryo-tomography 129 figure 1d , movie 5). we noticed that in a recent study, such 160 cytoplasm invagination was also seen in one of the conventional em images of stained 161 plastic sections of sars-cov-2 infected cells, although no description was given (lamers et 162 al., 2020) . 163 164 independently, we investigated cytopathic effect of sars-cov-2 infection using soft x-ray 165 cryo-tomography. consistent with the cryofib/sem volume imaging results, the overview 166 images of soft x-ray display profound changes in mitochondria morphology, as they appear 167 fragmented in the sars-cov-2 infected cell ( figure s3b&d on the cell surface are clearly distinguishable in soft x-ray cryo-tomogram ( figure s3e , 174 black arrow). also clearly observed are extensive vesiculation ( figure s3f ) and a partial 175 nucleus invagination in the infected cell ( figure 3g) . cov-2 (total 9 portals from 24 dmvs) compared to those of mhv (8 portals per dmv), 207 signifying the difference between coronaviruses. we also observed vaultosomes near the 208 dmv outer membranes ( figure 2f , black arrow). it is still unclear what is the physiological 209 role of vaults is, but they have been associated with rna nucleocytoplasmic transport, innate suggests that proteolytic processing has taken place in these compartments resulting in s1 262 shedding. therefore, we suggest that assembly at the ergic smvs is the only pathway 263 which will lead to infectious viral progeny. 264 there has not been much detailed studies on how sars-cov-2 viruses are released from 267 cell. we investigated sars-cov-2 egress using both serial cryofib/sem volume imaging 268 and cryoet. cryofib/sem images clearly reveal virus exiting tunnels in 3d at the cell 269 periphery connecting to cell membrane ( figure 5a -b, movie 2). this likely resulted from the 270 fusion of very large multi-virus containing vesicles with cell membrane, i.e. egress through exocytosis. consistent with cryofib/sem analysis, we also observed virus exiting tunnels in 272 cryo-tomograms ( figure 5c ). the fact that these compartments often contained many viral 273 particles suggests that this is a snapshot of viral exit, rather than cellular entry. 274 however, in addition to exocytosis, we also frequently found plasma membrane 276 discontinuities next to viral particles outside the cell ( figure 5e we used a multi-modal, multi-scale and correlative approach to investigate sars-cov-2 305 infection process in native cells, from the whole cell to subcellular and to the molecular level. 306 the integration of multi-scale imaging data, achieved through this advanced workflow 307 ( figure s1 ), has led us to propose a pathway for sars-cov-2 replication, in particular virus 308 genome replication, assembly and egress. the replication of sars-cov-2 appears spatially 309 well-organized and highly efficient. from genome replication, to protein synthesis and 310 transport, to virus assembly and budding, these processes take place in close-knit cytoplasmic 311 compartments. as illustrated in figure the cells were incubated at room temperature for 15 minutes after which a further 1.5 ml of 446 media was added to each well. the plate was then incubated at 37 °c for 24 hours following 447 which supernatants were discarded and cells washed with 2 ml of pbs. the cells were then 448 fixed by addition of 3 ml of 4% paraformaldehyde in pbs for 1 hour at room temperature. 449 after fixation, grids were plunge-frozen on a leica grid plunger 2. 1 ul of concentrated 10 450 nm gold fiducials was applied to the gold-side of the em grid and blotted from the gold-side. 451 the grid was quickly immersed in liquid ethane after blotting. frozen grids were stored in 452 liquid nitrogen until data collection. milling of sars-cov-2 infected cells was carried out using a scios dualbeam cryofib 477 (thermofisher scientific) equipped with a pp3010t transfer system and stage (quorum 478 technologies). grids were sputter coated within the pp3010t transfer chamber maintained at 479 -175 °c. after loading onto the scios stage at -168 °c, the grids were inspected using the 480 sem (operated at 5 kv and 13 pa) and cells, identified as infected from tem, were found. 481 the grid surface was coated using the gas injection system 482 (trimethyl(methylcyclopentadienyl)platinum(iv), thermofisher scientific) for 3 s, yielding 483 a thickness of ~3 um. milling was performed using the ion beam operated at 30 kv and 484 currents decreasing from 300 pa to 30 pa. at 30 pa lamella thickness was less than 300 nm. 485 during the final stage of milling, sem inspection of the lamellae was conducted at 2 kv and 486 13 pa. 487 488 samples were imaged on a zeis crossbeam 550xl fitted with a quorum transfer station and 490 cryo-stage. they were mounted on a quorum-compatible custom sample holder and coated 491 with platinum for 60 sec at 10 ma on the quorum transfer stage, prior to loading on the cryo-492 stage. stage temperature was set at -165°c, while the anticontaminator was held at -185°c. 493 samples were imaged at 45° tilt after being coated again with pt for 2x 30sec using the fib-494 sem's internal gis system, with the pt reservoir held at 25°c. initial trapezoid trenches were 495 milled at 30kv 7na over 15 μm to reach a final depth of 10 μm, with a polish step over a 496 rectangular box with a depth of 10 μm performed at 30kv 1.5 na. serial sectioning and 497 imaging acquisition was performed as follows: fib milling was set up using the 30kv 700 498 pa probe, a z-slice step of 20 nm and a depth of 10 μm over the entire milling box; sem 499 imaging was performed at a pixel depth of 3024x2304 pixels, which resulted in a pixel size of 500 6.5 nm, with the beam set at 2kv 35pa, dwell time 100 nsec and scan speed 1, averaging the 501 signal over 100 line scans as a noise-reduction strategy. 502 cryoet image processing 504 the frames in each tilt angle in a tilt series were processed to correct drift using motioncor2 505 (zheng et al., 2017) . for the intact cells dataset, all tilt series were aligned using the default 506 parameters in imod version 4.10.22 with the etomo interface, using gold-fiducial markers 507 (kremer et al., 1996) . for lamella dataset, the tilt series were aligned in the framework of 508 appion-protomo fiducial-less tilt-series alignment suite (noble and stagg, 2015) . after tilt series alignment, the tilt-series stacks together with the files describing the projection 510 transformation and fitted tilt angles were transferred to emclarity for the subsequent sub-511 tomogram averaging analysis (himes and zhang, 2018) . 512 513 all sub-tomogram averaging analysis steps were performed using emclarity, mostly 515 following previously published protocols described workflow (himes and zhang, 2018) . the 516 ctf estimation for each tilt was performed by using emclarity version 1.4.3 , and the 517 subvolumes were selected by using automatic template matching function within emclarity 518 using reference derived from emdb-21452 (walls et al., 2020) that was low-pass filtered to 519 30-å resolution in emclarity. the template matching results were cleaned manually by 520 comparison of the binned tomograms overlaid with the emclarity-generated imod model 521 showing the x,y,z coordinates of each cross-correlation peak detected. after manually 522 template cleaning, a total of 450 subvolumes from the lamella dataset and a total of 7090 523 subvolumes from the extracellular viruses dataset were retained, deriving from 3 tilt-series 524 and 50 tilt-series respectively, for the following averaging and alignment steps in emclarity. 525 for the extracellular viruses dataset, the 3d iterative averaging and alignment procedures 526 were carried out gradually with binning of 4x, 3x, 2x , each with 2-3 iterations with 527 increasingly restrictive search angles and translational shifts. 3-fold symmetry was applied 528 during all the steps. final converged average map was generated using bin2 tomograms with 529 pixel size of 3.26 å/pixel and a box size of 123×123×123 voxels. resolution indicated by 530 0.143 fsc cut-off was 8.7 å. the same process was carried out for lamella dataset, except 531 for the final average map was generated with pixel size of 4.26 å/pixel 532 and a box size of 90×90×90 voxels and a final resolution at 11 å (gold standard fsc at 533 0.143 cut-off). 534 cell structures were manually segmented from stacks of images using imagej (koppensteiner 537 et al., 2012) (kounatidis et al., 2020) . grids were loaded on the x-ray microscope at 553 b24 and were first mapped using visible light with a 20x objective. the resulting coordinate-554 map was used to locate areas of interest where 2d x-ray mosaics were collected (x-ray light 555 used was at 500ev) and used to identify areas of interest within. tilt series of 100-120 º were 556 collected for each field of view area of interest at 0.2 or 0.5º steps with constant exposure of 557 0.5 sec keeping average pixel intensity to between 5-30k counts. all tilt series were background subtracted, saved as raw tiff stacks and reconstructed using either imod 559 (kremer et al., 1996) or batchruntomo (mastronarde, 2005) . the membrane rearrangements mediated 614 by coronavirus nonstructural proteins 3 and 4. virology 458-459 cryo-soft x-ray tomography: using soft x-rays to explore the 617 ultrastructure of whole cells emclarity: software for high-resolution 619 cryo-electron tomography and subtomogram averaging sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by 624 a clinically proven protease inhibitor 5'-triphosphate 627 rna is the ligand for rig-i structures and 630 distributions of sars-cov-2 spike proteins on intact virions sars-633 coronavirus replication is supported by a reticulovesicular network of 634 modified endoplasmic reticulum macrophage internal hiv-1 is protected from 637 neutralizing antibodies 3d correlative cryo-structured illumination 641 fluorescence and soft x-ray microscopy elucidates reovirus intracellular 642 computer 644 visualization of three-dimensional image data using imod sars-cov-2 productively infects human gut enterocytes structure of the sars-cov-2 spike 652 receptor-binding domain bound to the ace2 receptor the sars-cov-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with rna and the membrane-associated m protein. biorxiv : the 656 preprint server for biology automated electron microscope tomography using 662 robust prediction of specimen movements the hepatitis c virus-665 induced membranous web and associated nuclear transport machinery limit 666 access of pattern recognition receptors to viral replication sites a 670 structural analysis of m protein in coronavirus assembly and morphology automated batch fiducial-less tilt-673 series alignment in appion using protomo sars-coronavirus-2 replication in vero e6 678 cells: replication kinetics, rapid adaptation and cytopathology expression and cleavage 682 of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce 683 the formation of double-membrane vesicles that mimic those associated with 684 coronaviral rna replication morphological and biochemical characterization of the membranous 687 hepatitis c virus replication compartment ucsf chimera--a visualization system 691 for exploratory research and analysis er-derived vesicles 695 exporting short-lived erad regulators, for replication structural basis of receptor recognition 699 by sars-cov-2 cardiomyocytes are susceptible to sars-cov-2 infection characterization of severe acute respiratory syndrome-706 associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry a unifying structural and functional model of the 712 coronavirus replication organelle: tracking down rna synthesis cryo-em structure of the 715 sars coronavirus spike glycoprotein in complex with its host cell receptor the intracellular sites 719 of early replication and budding of sars-coronavirus structural model of the sars 722 coronavirus e channel in lmpg micelles assembly intermediates of 726 orthoreovirus captured in the cell free fatty acid binding pocket in the locked structure of sars cov-2 spike protein. science in situ 733 structural analysis of sars-cov-2 spike reveals flexibility mediated by 734 three hinges nucleocapsid-independent 737 assembly of coronavirus-like particles by co-expression of viral envelope 738 protein genes structure, function, and antigenicity of the sars-cov structural and functional basis of 744 sars-cov-2 entry by using human ace2 a molecular pore spans the double membrane of the 748 coronavirus replication organelle membrane vesicles as platforms for viral replication. trends in 751 microbiology direct 753 visualization of vaults within intact cells by electron cryo-tomography structural basis for the recognition of the sars-cov-2 by full-length human 757 ace2 molecular architecture of the sars-cov-2 760 virus motioncor2: anisotropic correction of beam-induced 763 motion for improved cryo-electron microscopy short 770 article serial cryofib / sem reveals cytoarchitectural disruptions in leigh 771 syndrome patient cells serial cryofib / sem reveals cytoarchitectural 772 disruptions in leigh syndrome patient cells key: cord-302382-eifh95zm authors: owji, hajar; negahdaripour, manica; hajighahramani, nasim title: immunotherapeutic approaches to curtail covid-19 date: 2020-08-21 journal: int immunopharmacol doi: 10.1016/j.intimp.2020.106924 sha: doc_id: 302382 cord_uid: eifh95zm covid-19, the disease induced by the recently emerged severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has imposed an unpredictable burden on the world. drug repurposing has been employed to rapidly find a cure; but despite great efforts, no drug or vaccine is presently available for treating or prevention of covid-19. apart from antivirals, immunotherapeutic strategies are suggested considering the role of the immune response as the host defense again the virus, and the fact that sars-cov-2 suppresses interferon induction as an immune evasion strategy. active immunization through vaccines, interferon administration, passive immunotherapy by convalescent plasma or synthesized monoclonal and polyclonal antibodies, as well as immunomodulatory drugs, are different immunotherapeutic approaches that will be mentioned in this review. the focus would be on passive immunotherapeutic interventions. interferons might be helpful in some stages. vaccine development has been followed with unprecedented speed. some of these vaccines have been advanced to human clinical trials. convalescent plasma therapy is already practiced in many countries to help save the lives of severely ill patients. different antibodies that target various steps of sars-cov-2 pathogenesis or the associated immune responses are also proposed. for treating the cytokine storm induced at a late stage of the disease in some patients, immune modulation through jak inhibitors, corticosteroids, and some other cognate classes are evaluated. given the changing pattern of cytokine induction and immune responses throughout the covid-19 disease course, different adapted approaches are needed to help patients. gaining more knowledge about the detailed pathogenesis of sars-cov-2, its interplay with the immune system, and viral-mediated responses are crucial to identify efficient preventive and therapeutic approaches. a systemic approach seems essential in this regard. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a newly emerged betacoronavirus, is responsible for coronavirus disease 2019 (covid19) , which was first reported in wuhan, china in december 2019. sars-cov-2, the third fatal virus of its group, is an enveloped positive-sense, singlestranded rna virus [1] . while the other viruses of this family induce only mild cold symptoms, severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov), the two other virulent betacoronaviruses, have been presented with higher fatality rates than sars-cov-2. on the other hand, sars-cov-2 has shown a higher transmission rate and therefore a wider spread around the globe [2] . the burden of coronavirus disease 2019 (covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has been very huge on health, economy, and many other aspects of life. it has already claimed more than 550750,000 lives (as of 12 15 julyaugust 2020 according to who covid-19 dashboard at https://covid19.who.int/). the urgency of this threat has prompted scientists in many countries to seek solutions through drug repurposing and repositioning of the previously approved drugs, while the fast-tracking of vaccine and drug development are seriously followed. however, some of the repurposed candidate drugs have already failed in some clinical trials [3] . some antiviral drugs, developed for other similar viruses, are suggested, which may inhibit the cell entry or replication of the virus [2] . on the other hand, supporting the immune system's potential to function properly and fight with the virus is another viable strategy. normalization of the dysregulated immune responses or even their suppression at the final stages of the disease may also be required [4] . generally, following the entry of an invading pathogen into the body and its recognition by the first-line immune cells, the innate immune system would be stimulated, which may be later supported by adaptive immune responses as well. the interaction of each pathogen with the immune cells could define the defense the body against it, thus understanding such interplays is fundamental. although the role of the the immune system function in the control or overcoming covid-19 is indisputable, and many therapeutic and preventive approaches implicate modulation of the immune system activity, there are still many questions to be answered in this regard. for instance, the pattern of cytokine secretion during the disease course has been the subject of vast investigations. the complexity of the immune system responses as well as the variations in the level of cytokines that happens during the disease period and may lead to cytokine storm, highlights the need for gaining a deep overview on such events as well as the interventional possibilities to be able to cure or prevent the disease. in this review, the pathogenesis of sars-cov-2 and its interplay with the immune system , which are discussed in detail in other publications [5] [6] [7] , are briefly stated. a classification of possible immune-based approaches to combat covid-19 is presented with a focus on convalescent plasma therapy, manufactured antibodies (monoclonal and polyclonal antibodies), and immunomodulators that could potentially modify the immune system activity. vaccines, as a potential immune-based approach to curtail covid-19, were discussed previously [8] [9] [10], so will not be discussed in detail here. despite the large volume of ongoing studies on sars-cov-2, parts of our current knowledge on its pathogenesis is based on the studies of sars-cov and mers-cov, since the clinical manifestations of sars-cov-2 highly resemble the ones observed in the two latter viral infections [11] . sars-cov-2 may enters the lungs from through the nasopharyngeal mucosal membrane and infect. alveolar alveolar macrophages and type i and ii epithelial cells in the lungs are shown to be infected by sars-cov-2 [5] . the most prominent way of the viral entry was shown to be through the attachment of s protein and angiotensin-converting enzyme 2 (ace2) receptors, which. some studies clarified that viral entry may be enhanced through some proteases such as a serine protease called tmprss2 (transmembrane protease, serine 2) or cathepsin l/b (ctsl/b) [6] . tmprss2 may, in fact, proteolytically activates sars-cov-2 by cleaving the s protein and promote replication and syncytium formation in the virus. hence, tmprss2-expressing cells were shown to be highly susceptible to sars-cov-2 infection [13] . cathepsin, which is expressed in endosomes, was shown to facilitate the fusion of the virus and endosome membrane [14] . recent studies revealed that a combinational effect between tmprss2 and cathepsin is needed for an effective viral entry [15] . another serine exopeptidase receptor, a serine exopeptidase, called dipeptidyl peptidase 4 (dpp4) or cluster of differentiation 26 (cd26), also was also shown to provide additional interactions with sars-cov-2 spike beside the ace2 receptor [7] . despite the noteworthy pieces of evidence supporting the role of ace2 and the associated proteases in the viral entry, iit was shown revealed that the virus can also enter the cell through clathrin-dependent and -independent endocytosis pathways. for instance, sars-cov-2 may attack lymphocytes through the jak-stat pathway [8] . covid-19 patients manifest mild to severe symptoms, including fever, non-productive cough, dyspnea, malaise, fatigue, lymphopenia, and pneumonia 2-14 days after the viral attack. moreover, laboratory results including leucopenia, elevated c-reactive protein (crp), and higher erythrocyte sedimentation rate (esr) are detected. a wide range of other clinical manifestations has been observed in covid-19 involving different organs, namely heart, eyes, nose, brain, pancreas, kidney, and bladder. as reported, 7-14 days upon the manifestation of the initial symptoms of the disease, the virus may cause a second attack and an aggravation of the disease symptoms in which severe pneumonia, ground-glass opacity, acute cardiac injury, and rnaaemia are observed [9] . in this phase of the disease, the level of lymphocytes drops dramatically, and cytokine storm occurs. cytokine storm is an uncontrolled release of inflammatory cytokines, including ifn-α, ifn-γ, gm-csf, g-csf, il-1ß, il-6, il-12, il-18, il-33, tnf-α, tgf-ß and chemokines, including ccl2, ccl3, ccl5, cxcl8, cxcl9, cxcl10. the cytokines level reduces as the patient recovers. cytokine storm has been recognized as the main cause of acute respiratory distress syndrome (ards), which causes lung injury, and multiple organ failure. moreover, patients who succumbed to covid-19 represented a higher level of neutrophils, d-dimer, blood urea nitrogen (bun), and creatinine than the survivors [10, 11] . as the virus enters the cell, the viral rna is released and proceeds to the translation step. the viral proteins and rnas, produced by the host cell, are inserted into the endoplasmic reticulum and golgi, where the viral nucleocapsid is formed, and the viral particles are prepared to be released out of the cell membrane. the virus may enter the peripheral blood from the lungs and attack other organs that express the ace2 receptor, including heart, kidneys, and gastrointestinal tract. the viral antigen is presented to t cells and b cells via major histocompatibility complex (mhc) on antigen-presenting cells (apcs), thus innate and humoral adaptive immunities are activated. innate immunity is initiated by interferon secretion from the infected cells in viral infections for signaling to other cells and making them ready for the battle [4] . sars-cov-2 is found to antagonize the induction of type i interferons (interferon-alpha and -beta) [21, 22] thereby evading from the innate immune system defense [23] . macrophage and dendritic cells were shown to play important roles in viral destruction and mucosal immunity. the level of cd4+ and cd8+ cells also increases, which leads to long-term immunity. it was shown that cd4+ and cd8+ cells are responsible for promoting neutralizing antibody proliferation and destruction of viral-infected cells, respectively [24] . different immunoglobulins against viral s (spike), m (membrane), n (nucleoprotein) proteins, nsp (nonstructural proteins), and orf (open reading frame) are produced. however, the prevalent types of immunoglobulins are against s protein and among them igm and igg induce short-term and long-term immunities, respectively. moreover, it was shown that the immune system decision between th1 response (cellular response) or th2 response (humoral response), which is affected by the cytokine pattern, determines the viral infection control. in fact, it was reported that some infections are well controlled by th1 response and this response was observed in 20 recovered covid-19 patients [25] . sars-cov-2 was found to suppress antigen presentation through downregulation of mhc class i and ii molecules, which may lead to the impediment of t cell-mediated immune defense [26] . what mentioned above are known as the most prevalent process of sars-cov-2 pathogenesis and clinical manifestations; however, a closer scrutiny recently revealed a wider range of clinical manifestation for covid-19. in fact, a direct association has been observed between the expression of viral receptors, including ace2 and tmprss2 and the affected organ. herein, organs including heart, eyes, nose, brain, pancreas, kidney, and bladderfor instance, the abundance of ace2 receptors on endothelial cells explain why cardiovascular complications are considered as the second threat caused by sars-cov-2 after respiratory symptoms [27] . moreover, sars-cov-2 may target the central nervous system and infect neurons in the nasal passage, which can be the cause of smell and taste disruption in some covid -19 patients. however, the loss of taste and smell is of the most initial inflammatory responses to sars-cov-2 and can be reversible as the body defeats the virus [28] . were also shown to be affected by sars-cov-2 [12] . in addition to the role of nasal cells in sars-cov-2 infection, the nasal secretory cells may be exploited by the virus for transmission to the other persons. viral antigens are presented to t cells and b cells via major histocompatibility complex (mhc) on antigenpresenting cells (apcs), thus innate and adaptive immunities are activated. innate immunity response is initiated by interferon secretion from the infected cells in viral infections for signaling to other cells and making them ready for the battle [4] . sars-cov-2 is found to antagonize the induction of type i interferons (interferon-alpha and -beta) [12, 13] thereby evading the innate immune system defense [14] . moreover, it was shown that the immune system decision between th1 response (cellular response) or th2 response (humoral response), which is affected by the cytokine pattern, determines the viral infection control. in fact, it was reported that some infections were well controlled by a th1 response, and this response was observed in 20 recovered covid-19 patients [15] . sars-cov-2 was found to suppress antigen presentation through the downregulation of mhc class i and ii molecules, which may lead to the impediment of t cell-mediated immune defense [16] . in the second attack phase of the disease (usually one or two weeks after the presentation of the first symptoms), the level of lymphocytes drops dramatically, and the cytokine storm occurs. cytokine storm is an uncontrolled release of inflammatory cytokines, including ifn-α, ifn-γ, gm-csf, g-csf, il-1ß, il-6, il-12, il-18, il-33, tnf-α, tgf-ß, and chemokines, particularly ccl2, ccl3, ccl5, cxcl8, cxcl9, cxcl10. the cytokine level reduces as the patient recovers. cytokine storm has been recognized as the main cause of acute respiratory distress syndrome (ards), which causes lung injury, and multiple organ failure. taken together, the immune system is highly impaired in critically ill covid-19 patients. given the role of the immune system in host defense, immunomodulation could be regarded as an important strategy to curtail covid-19 considering the patient's immune system condition at various phases of the disease. such immunomodulatory interventions can be achieved using vaccines, interferons, convalescent plasma, anti-inflammatory agents, antibodies, and other classes of immunomodulators, which are described in the following. innate immunity, as the first immediate and general defense response, plays a key role in the protection against invading pathogens, which is initiated by interferon secretion from the infected cells in viral infections for signaling to other cells and making them ready for the battle [4] . sars-cov-2 is found to antagonize the induction of type i interferons (interferon-alpha and -beta) [21, 22] thereby evading from the innate immune system defense, as a mechanism of its pathogenesis [23] . besides the yet questions in the initial responses to sars-cov-2 (e.g. types of receptors that are recruited for viral entry or cells that are affected), secondary responses (e.g. immune system responses) have been also under debate. for example, data concerning the beneficial or detrimental role of inflammatory mediators are controversial [4] . cytokine storm, which is the result of increase in the levels of some cytokines as explained before, may also happen as a pathological situation of the innate immune system as well. another disruptive activity of the virus involves the functional exhaustion of lymphocytes, which would affect the adaptive immunity. the immunopathological conditions aroused in covid-19, are mentioned in brief here, as they were discussed in detail elsewhere . there are still unknowns regarding the exact interactions of the immune and adaptive immunities with the virus as well as the methods of virus for confounding the host immune system. the in-depth understanding of viral-mediated responses and interplay of the virus and the host immune system might not be feasible without a systemic approach . nevertheless, the similarity of severe respiratory failure induced by sars-cov-2 to acute respiratory distress syndrome (ards) and the deterioration of patients' conditions in around a week following the first symptoms implicate the role of immunity dysregulation in covid-19 profile [6] . therefore, immunotherapy and modulation of the immune system, which could modify these disorders, are potentially effective strategies to fight with sars-cov-2. such interventions may be done through vaccines, which actively stimulates the immune responses, or administration of interferons i, which are discussed in previous publications. passive immunotherapy and the modulation of the immune system through anti-inflammatory or immunostimulatory agents are other alternatives, which will be examined in detail in the following. of note, these immunotherapeutic approaches should be carefully selected based on the stage of the disease and the state of the patient's immune system. the suppression of interferon i-mediated immune responses by sars-cov-2 is already confirmed [12, 13] thereby evading from the innate immune system defense, as a mechanism of its pathogenesis [23] . although interferon has beenwas shown to fight against the virus and are is suggested for to treatment of the disease [17] , some contradictory data demonstrated that interferon may enhance ace2 expression and thus viral entry [18] . on the other hand, positive results were found by using interferons type i, including interferon-beta-1a in several clinical trials [19] . the difference in the route of administration, either subcutaneous (s.c.) and intravenous (i.v.), was proposed as a reason for the diverse effects reported about interferon beta-1a in some studies [20] . interferon-beta is already being examined in a combination protocol in the international clinical trial launched by who in the partner countries, called as "solidarity" [3] . the outcomes of the investigations on interferon therapy in covid-19 were presented in some other publications [17, [19] [20] and as a systematic review [21] . interferon-beta is already being examined in a combination protocol in the international clinical trial launched by who, called the "solidarity" trial, in the partner countries [3] . vaccines are believed as the ultimate protection for saving the public from the novel virus. the lack of previous exposure of the human immune system to sars-cov-2 [22] is regarded as the major contributor to its high risk. hence, active immunization through vaccines could prepare the body to resist against this infection. very soon after finding the virus genetic sequence, vaccine development was started and followed with unprecedented speed by several research groups and pharmaceutical companies. huge investments are dedicated by several public and private bodies to advance the this project [2] . various platforms are employed in these investigational vaccines, including inactivated, killed or weakened pathogen, non-replicating viral vector, rna, and dna, vlp (virus-like particle), and protein subunit structures virus. there also some inactivated as well as replicating viral vector-based vaccines under development at preclinical stage. each of these platforms has its own advantage and limitations. while dna and rna vaccines are intensively studied mainly because of their rapid development, easy production, and safety, there are novel types not commercially available previously. thus, their largescale production might take need more time to be set up, due to novelty and lack of previous experience in their commercial production. additionally, more than one dose of these vaccine types is required for the immunization because of their short half-life [23] . as tthis topic is reviewed in details elsewhere [23] [24] [25] . all in all, vaccine development is a time-consuming process. moreover, the induction of memory in the immune system is still under question. therefore, despite preliminary promising results, the efficiency of the investigational vaccines should be confirmed in large clinical trials. considering the lack of an approved drug or vaccine against sars-cov-2, taking advantage of a helpful alternative intervention is an urgent need [26] [27] [28] . passive immunotherapy via antibodies has been considered as a possible strategy for defeating covid-19 apart from anti-viral therapeutics and vaccines ( fig. 1 ). antibodies can be either isolated from a convalescent patient or produced in a lab [29] . these two approaches would be discussed in more detail in the following. active immunization is provided through vaccines, which are still under development for covid-19. passive immunization can be performed via natural antibodies using convalescent plasma therapy (cpt) or antibodies that are manufactured. in cpt, natural neutralizing antibodies derived from a hyperimmune patient would be administered to a covid-19 patient through plasma transfusion. this approach is already being used and investigated in many countries with acceptable levels of success. on the other hands, different polyclonal or monoclonal antibodies could be produced via using hybridoma cell-lines, animals, or cell-free protein synthesis. neutralizing antibodies derived from a hyperimmune patient through plasma transfusion is the most prevalent and accessible empirical approach used to treat several viral infections previously. this method, so-called convalescent plasma therapy (cpt), is also considered as a viable therapy for covid-19 [30] [31] [32] . it has been used to reduce the hospital stay and the mortality rate of critically ill patients with severe acute respiratory syndromes [32, 33] and shown was found beneficial in some previous epidemics of infectious diseases [34] . the use of this approach had been suggested for the first time during the outbreak of spanish influenza (pandemic of 1918-1920) [33] . subsequently, plasma transfusion was recommended as a safe and effective way for the prevention or treatment of the ebola virus in 2014 and also several other severe viral infections, including mers, sars-cov, and avian influenza a [35, 36] . in fact, neutralizing antibodies in the convalescent plasma (cp) could suppress the viremia through binding to the external antigens of the viruses and blocking their entry into the host cells [34, 37] . the effectiveness of cpt could vary according to the type of microorganism, it's pathogenesis, and treatment protocols, such as timing, dosing, and volume of administration [33] . according to the previous evidence for plasma therapy of other coronaviruses, such as sars-cov and mers, the early transfusion of cp can probably be more effective and improves the survival rate of critical covd-19 patients at the early disease stage. it could be explained by the fact that in most viral infections, viremia rises in the first week of the disease [37, 38] . it should be noted that cpt may not be useful for mild or end-stage patients. indeed, cpt is not able to significantly reduce the mortality rate well among end-stage patients because of their disease severity. on the other hand, mild patients can be self-recovered and cpt would not be required [39] . the titer of sars-cov-2 neutralizing antibodies in the cp could be another important factor to increase treatment efficacy. although, the antibodies level in the donor plasma before transfusion is not determined, some studies indicated that the specific igg increases about three weeks after symptom onset and peaks at week 12. therefore, the cp from donors who are at week 12 after the initiation of the symptoms is predicted to be more efficient [27, 40] . generally, patients with primary and secondary antibody deficiencies, need intravenous immunoglobulin (ivig) treatment as the standard replacement therapy [41] ; and historically, administration of ivig has been one of the important treatments in immunodeficient patients [42] . these patients are considered as a high-risk group, which can encounter several severe complications if infected with sars-cov-2 virus [43] . therefore, an effective treatment is required to help such patients survive. cp extracted from the sars-cov-2 survivors may be a promising approach for the protection of covid-19 patients with antibody deficiency before the development of an effective vaccine [44] . however, the data about the potency of cpt in covid-19 patients with primary and secondary humoral immunodeficiency is limited and has not been fully established, there are some case studies that have reported its proper effectiveness. for example, clark et al. reported a 76-year-old covid-19 case with lymphoma who was treated with a combination of bendamustine and rituximab that could lead to the impairment of humoral and cellular responses. after the failure of different treatment protocols against sars-cov-2, the administration of hyperimmune plasma resulted in a rapid recovery in this patient [45] . in another report, an immunosuppressed covid-19 patient with myeloid malignancy, disseminated tuberculosis, and kidney disease, was successfully treated after transfusion of cp and tocilizumab [46] . male patient with covid-19, whose health condition was improved following cp administration [47] . presently, several clinical trials investigating the usage of cpt in covid-19 are ongoing (as recorded in https://clinicaltrials.gov/), a number of which are summarized in table 1 . the trials are selected according to the recruitment status of the study (recruitment or completed state), age (18 years and older), and severity of symptoms in participants (admitted to the hospital or icu with severe symptoms). several other publications have discussed the usage of cpt in covid-19 [32, 33, 35] , thus no more details would not be addressed here. all in all, cpt has demonstrated acceptable safety and in the current situation and seems a promising choice for treating severe covid-19 patients besides other therapeutic strategies [48] . as mentioned before,the previous experiences and efficacy in other similar diseases, as well as its feasibility, are the important advantages of cpt. moreover, according to different reports, cpt is welltolerated by receivers [35] , but as all treatment approaches, this method may have some minor adverse effects as well. generally, the most common adverse effects of cpt is related to transfusion events, such as chills, fever, rash, allergic reactions, circulatory overload, and hemolysis [40, 49] . additionally, several other problems are attributed to the mentioned approach, including lack of plasma donors, risk of cross-contamination, inconsistency from batch to batch, non-scalability, and the possibility of host reaction [50] . these pitfalls behoove pharmaceutical companies to manufacture polyclonal or monoclonal antibodies. the first and most common targets are spike (s) proteins, which are located on the virus surface, generating its specific 'crown' shape [53] . sars-cov-2 starts its pathogenesis through the attachment of receptorbinding domain (rbd), located in the s1 subunit of the s protein, with angiotensin-converting enzyme 2 (ace2). thus, s proteins are considered as the most antigenic part of the virus with the main responsibility for the host immune responses [54] . it has been widely suggested that the previously-experimented sars-cov rbd neutralizing antibodies can be repurposed for sars-cov-2 [1, 55, 56] . the cross-neutralization capacity of antibodies relies on the conservation of the particular residues that are essential for the formation of specific bonds between rbd and the antibody, between the two types of viruses. for instance, an analysis was performed to determine which of the previously-proposed monoclonal antibodies against sars-cov can also cross-neutralize sars-cov-2. in the mentioned study, the residues for the formation of a salt bridge and electrostatic interaction between the m396 antibody and rbd were conserved between sars-cov and sars-cov-2. by contrast, antibodies, including r80 and f26g19 failed to interact with sars-cov-2 rbd in a similar way they did with sars-cov rbd due to the differences in their residues [57] . surprisingly, it was shown that f26g19 neutralized sars-cov-2 more potently than sars-cov through other interactions [51] . studies including cryoelectron microscopy of sars-cov-2 spike and analysis of the protein-protein interactions of sars-cov-2 and ace2 receptor with energy-based methods revealed the amino acids 319-591 of sars-cov-2 rbd as important residues; the latter study also introduced the linear and conformational epitopes within this region as antibody targets [54, 58] . though the rbd has been considered as the main target of interest, some neutralizing or blocking antibodies have been shown to recognize other epitopes, including domains in s1 subunit, s-ectodomain, hr1 and hr2 domains in the s2 subunit, nucleoprotein (np), or envelope (e) protein [59] [60] [61] . for example, cr3022 cross-neutralized sars-cov-2 more strongly than other neutralizing antibodies against sars-cov, while it did not compete with ace2 for binding to sars-cov-2. this observation indicates that cr3022 neutralizes sars-cov-2 through binding epitopes other than rbd [57] . another study showed that the hr2 domain with an identity of 93% is highly conserved between sars-cov and sars-cov-2, and thus neutralizing antibodies that target the hr2 domain, including 2b2, 1a9, 4b12, and 1g10 potently cross-neutralized sars-cov-2 [62] . similarly, the s309 monoclonal antibody, retrieved from convalescent sars-cov patients, potently neutralized sars-cov-2 through a highly conserved domain distinct from rbd and did not interfere with the binding of s protein with the ace2 receptor [63] . moreover, in contrast to the above-mentioned importance of rbd in designing monoclonal antibodies, some studies have revealed the antibody-dependent enhancement of viral entry when the monoclonal antibody targets the rbd. it was shown that the binding of monoclonal antibody to the rbd triggers conformational changes that are similar to the alterations made following the binding of viral receptors to the viral rbd; hence, the binding mediates the virus entry to the cell via viral receptor-dependent pathways. however, as stated in the mentioned study, this mechanism depends on the monoclonal antibody dosage, expression of particular viral receptors (e.g. fc receptors), and particular features of the monoclonal antibody [64] . although ace2 has been introduced as the main receptor of sars-cov, it may not be sufficient for the interaction between the cell and the virus. other cellular factors, such as vimentin, a cytoskeleton protein, were revealed to be important in the formation of the ace2-sars-cov complex [65] . therefore, surface vimentin was recognized as a potential target for sars-cov. ds-sign/cd209 is also a transmembrane adhesion molecule, which is mainly expressed on interstitial dendritic cells and lung alveolar macrophages. it was shown that ds-sign also mediates the entry of sars-cov. a humanized monoclonal antibody was produced to interfere with the interaction of ds-sign and intercellular adhesion molecule 3 (icam-3), and thus inhibit sars-cov entry to the cell [66] . similar proteins may be identified in regards to sars-cov-2. in addition to inhibiting the virus entry, antibodies can intrude into the biological activities of the virus thereby preventing its replication. fully human antibodies, which are capable of traversing across the cell membrane of infected cells and preventing virus replication, were made against several kinds of viruses, including influenza, hepatitis c virus, and ebola [67] . papain-like proteases (plpro), cysteine-like protease (3clpro), and other non-structural proteins (nsps) can be suggested as targets of interests that hinder sars-cov-2 replication [1, 68] . besides structural parts of the virus, various steps associated with the innate and the adaptive immune responses have been proposed as the most important targets of interest. for instance, the significant rise in the level of chemokines and cytokines, including il-1β, ifn-γ, ip-10, and mcp-1, which is called the cytokine storm, can be inhibited by antibodies [69] . the preliminary studies of critically-ill covid-19 patients have shown that il-6 may cause severe inflammatory responses that lead to acute respiratory distress syndrome [70] . herein, tocilizumab, an il-6 inhibitor monoclonal antibody, has gained significant attention. in a 21-patient clinical study recruited in china, tocilizumab resulted in the reduction of oxygen need in 75% of patients, lung lesion opacity absorption in 90.5% of patients, and correction of lymphocyte and c-reactive protein levels [70] . the significance of tocilizumab can be better explained since a noticeable number of 46 clinical trials regarding its use against sars-cov-2 related pneumonia and respiratory tract infections were recorded in nih until may 27, 2020. an increase in the level of some of these proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor (gm-csf) results in a positive feedback in the number of other inflammatory mediators, including il6, il-23, and tnf. gm-csf along with il6 and il23 also induce th1/th17 differentiation and the polarization of macrophages to m1 phenotypes, which in turn boost the inflammatory responses [71] . th17 immune response also can aggravate the cytokine storm by raising the level of il17, gm-csf, il21, and il22 [72] . high levels of gm-csf and th17 cells were observed in the plasma of severe-to critically-ill covid-19 patients [73, 74] . herein, harnessing the upregulation of gm-csf can prevent a cascade of inflammatory responses, which result in acute respiratory syndrome. since monoclonal antibodies targeting one inflammatory mediator, may fail to control the whole cytokine storm and prevent the lung injury induced by acute respiratory distress syndrome, a newer approach to prevent lung injury was proposed. it was shown that physical stress such as excessive mechanical stress caused by ventilators upregulates the expression of a gene, called nicotinamide phosphoribosyltransferase (nampt). as the bioavailability of nampt increases, toll-like receptor 4 (tlr4), which is responsible for lung inflammation, gets activated [75, 76] . herein, neutralization of circulating nampt by monoclonal antibodies can be another viable approach in preventing the lung injury caused by sars-cov-2. in addition to the role of inflammatory mediators, understanding the adaptive immune responses helps us to repurpose or invent immunomodulatory antibodies to defeat sars-cov-2. for example, cd4+ and cd8+ t-cells are expected to promote the proliferation of neutralizing antibodies and the destruction of infected cells, respectively. however, lymphocytopenia is identified to play a role in the pathogenesis of severely-ill covid-19 patients [77] , which can be prevented or restored by regulating lymphocyte proliferation and apoptosis [78] . herein, some studies have shown that sepsis may occur secondary to inflammatory responses. the immune imbalance, which occurs in sepsis, maybe as a result of t-cell depletion. pd-1 and ctla-4 receptors are immune checkpoints that are expressed on the surface of t-cells and play a role as the negative regulator of t-cell function. therefore, inhibiting the immune checkpoints by monoclonal antibodies is also an intriguing approach in defeating sars-cov-2 [79] . furthermore, cd4+ cells express a receptor called c-chemokine receptor 5 (ccr5), which was established as a way of hiv entry to the cell [80] . this receptor could also be a potential target for sars-cov-2. the level of different immunoglobulins is raised in response to sars-cov-2. even though an increase in the level of immunoglobulins is attributed to pose neutralizing effect on sars-cov-2, a rise in anti-s igg is associated with lung failure [81, 82] . complement systems also play a role in conferring both humoral and natural immunity; however, they have been also attributed to the refractory inflammatory diseases. herein, c5a and c5a receptor, the members of the complement system, have been successfully targeted in different clinical trials of inflammatory diseases and thus can be recognized as possible targets in new diseases such as covid-19 [83] . additionally, tlr3, cd16, immunoreceptor tyrosin-based activation motif (itam), g-csf, monocyte chemoattractant protein 1 (mcp1), tnfα, il4, and il10 are the other members of the immune system for which anti-sars-cov monoclonal antibodies were patented. these monoclonal antibodies could be reevaluated for sars-cov-2 [84] . besides the main strategies described above for designing monoclonal antibodies against sars-cov-2, some more novel approaches with a completely different mechanism were suggested. one of these strategies is using dewetting monoclonal antibodies. dewetting transition is a process in which hydrophobic pores of the ion channels inhibit water transmission and thus impair the cellular performance. this phenomenon can be deployed to block viruses, bacteria, and autoimmune activities. dewetting monoclonal antibodies are antibodies with a lipophilic fragment that target the transmembrane receptors and hinder the physiological water flow inside the channel. such antibodies were produced against the influenza virus [85, 86] . recently, viroporins were identified in sars-cov-2, the ion channel proteins that are generated by the virus e protein and are responsible for different parts of the virus life cycle, including virus entry, assembly, release, and the whole pathogenesis cycle [87] . as suggested, dewetting monoclonal antibodies could be developed against sars-cov-2 viroporins and administered through the nose. these antibodies can deactivate the virus by targeting viroporins even before the virus will be able to bind to the host cells [88] . generally speaking, tthe antibodies used for treatment could be usually produced at large-scale, either as monoclonal or polyclonal antibodies, using hybridoma cell-lines, animals, or cell-free protein synthesis either, [89] . monoclonal antibodies can be produced via several technologies, including the production of high-affinity human antibodies in immunized transgenic mice (e.g. xenomouse® or humab® mice), various phagedisplay systems such as generating antibodies from immunoglobulin cdna libraries in bacteria or mammalian cells, and obtaining memory b cells of convalescent patients that are immortalized by ebv transformation. all of these techniques were previously recruited to produce monoclonal antibodies against sars-cov [90] . production the production of monoclonal antibodies against sars-cov-2 is in its incipient phase. currently, our data about sars-cov-2 antibodies greatly comes from the studies in which the antibodies derived from the plasma of convalescent patients were analyzed. a recent study of the convalescent patients' antibodies demonstrated that anti-sars-cov-2 antibodies were versatile among the convalescent patients and each patient represented a unique pattern of antibody biodistribution. these results explain why it may be difficult to design specific anti-sars-cov-2 antibodies [91] . therefore, the introduced antibodies in this study were mainly tested against sars-cov or approved for other immune inflammatory diseases such as rheumatoid arthritis, cancers, and other viral infections. an extra method for designing antibodies against sars-cov-2 can be based on the antibody-antigen computational simulation [51] . for instance, an online docking server using the codockpp engine is constructed to predict the docking modes between antibodies or other peptides. this server is freely available at http://ncov.schanglab.org.cn. identified structural parts of sars-cov-2 and the homologous parts of other coronaviruses were gathered to produce the mentioned docking server [92] . table 2 consists of tthe antibodies retrieved from the previous studies on sars-cov or computational studies concerning sars-cov-2, which mainly target the virus structure or host receptors are shown in table 2 . despite a great homology between sars-cov and sars-cov-2, the cross-reactivity of sars-cov antibodies against sars-cov-2 is still under debate. for instance, some highly-conserved regions are found in sars-cov-2, which are absent in sars-cov. the c-terminal of sars-cov-2 rbd highly differs from that of sars-cov. moreover, an extra furin cleavage site was found between s1 and s2 subunits in sars-cov-2, which is absent in sars-cov. these differences may not affect the ability of both viruses in interacting with the ace2 receptor but explain the different levels of affinity among neutralizing antibodies with the two viruses [51, 57, 93, 94] . moreover, a recent study revealed that the antibodies targeting rbd of the coronavirus family are virus species-specific, while those that target viral parts outside rbd are capable of cross recognition [91] . in an antibody epitope computational analysis, it was revealed that 85.3% of antibody epitopes in the sars-cov-2 spike were novel in comparison with those in sars-cov [95] . therefore, antibodies suggested in table 2 ought to be reevaluated to be used against sars-cov-2. taken together, among the introduced monoclonal antibodies in these study f26g19, cr3022, and 47d11 were shown to cross-neutralize sars-cov and sars-cov-2. antibodies, including s101.1, s102.1, s103.3, s104.1, s105.2, s106.1, s107.4, s108.1, s109.2, neutralize spike by binding to residues 318-510 analysis of immune sars-cov patients' serum and in vivo study in mice [60] s132.9, s128.5, s127.6, s124.4, s159.1, s160. [111] further information in this study concerning monoclonal antibody data against sars-cov-2 was retrieved from clinical trial data recorded in clinicaltrials.gov or biotechnology and pharmaceutical companies' websites, which are summarized in tables 3 and 4 and harbour biomed, are designed to neutralize sars-cov-2 structure. however, the mechanisms and specificities of the latter antibodies have not been elaborated to date and thus should be followed in companies' websites. not mentioned binds to highlyconserved epitopes within sars-cov and sars-cov-2 vir biotechnology with wuxi biologics and biogen enters clinical trial within 3-5 months aimed to confer short-and long-term immunity and use as prophylaxis [116] not for effective passive immunotherapy, several epitopes ought to be targeted rather than one epitope. moreover, the design of monoclonal antibodies and their testing at a clinical stage is a long pathway followed by the fact that the massive production of monoclonal antibodies might be costly, time-consuming, (table 4 ). polyclonal antibodies produced by immunized animals or a particular cell line technology are expected to mimic convalescent plasma therapy with a higher potency than their plasma-derived equivalents as well as better clinical outcomes. furthermore, the risk of contamination and host reactions will be reduced compared with their plasma equivalents; dosing and kinetics also would be more predictable and scalable. moreover, targeting more than one epitope can cause synergistic effects in neutralization and would limit the formation of escape-mutants. for instance, a recent study revealed that a cocktail of antibody noticeably enhanced sars-cov-2 neutralization compared with the use of one monoclonal antibody [63] . no doubt the immune system is highly impaired in critically ill covid-19 patients, which denotes the possibility of using immunomodulators in this disease. monoclonal or polyclonal antibodies, interferons, and hydroxychloroquine are immunomodulators, which were widely discussed in previous sections. in this section, other several classes of immunomodulators including tyrosine kinase inhibitors, mtor inhibitors, calcineurin inhibitors, antimetabolites, tnf blockers, metal-based agents, and other anti-inflammatory agents are might be of value in the treatment of covid-19.discussed janus-associated kinase (jak) inhibitors are of high interest among the mentioned immunomodulators. although ace2 receptors have been recognized as the main receptor for the entry of sars-cov-2, it was shown that sars-cov-2 also attacks the cells that do not have ace2 receptors, including lymphocytes. it inhibitors have a marginal effect on il21, which is responsible for b cell function, and do not disrupt innate immune response, since the inhibition caused by jak2 inhibitors is transient and reversible [135] . another member of this family, baricitinib, is of special interest due to its advantages over other jak inhibitors and has been highly studied. baricitinib inhibits another regulator of endocytosis, called cyclin g associated kinase, through which it can defeat the viral infection [136] . baricitinib has been also suggested as the best choice among other jak inhibitors due to its acceptable profile of side effects, the possibility of once-daily dosing, higher potency, and advantageous pharmacokinetics. the inhibitory doses of baricitinib were welltolerated by patients with inflammatory diseases in comparison with other kinase inhibitors [137, 138] . moreover, baricitinib represents low protein binding and minimal interaction with drug transporters or metabolic enzymes; thus, it is preferred over other jak inhibitors for administration along with an antiviral regimen [138] . in contrast to the above-mentioned benefits of baricitinib, some clinical studies suggested that baricitinib may not be an ideal option for the treatment of covid-19 due to the possibility of causing lymphocytopenia, neutropenia, viral reactivation, and enhancement of coinfection [139] . other jak inhibitors, including upadacitinib and filgotinib, were also shown to impair interferon-mediated antiviral responses and perpetuate sars-cov-2 infection. herein, the incidence of secondary viral infections, including herpes zoster was observed, which was shown to be more prevalent in immunocompromised patients. hence, jak inhibitors, particularly baricitinib, should be administered with meticulous consideration, especially in susceptible and immunocompromised patients [140] . next, ruxolitinib was listed as one of the top hit compounds in an advanced bioinformatics analysis of available medications for sars-cov-2. the mentioned study aimed to identify compounds that counteract the expression of sars-cov-2-related genes [141] . to date, only three jak inhibitors have entered clinical studies on covid-19 patients, including baricitinib, ruxolitinib, and tofacitinib, among which many of the studies are related to baricitinib and ruxolitinib (table 5) . bruton tyrosine kinase (btk) inhibitors are another group of tyrosine kinase inhibitors, which has been repurposed to modulate the cytokine storm ensuing covid-19 infection. btk signaling leads to b cell proliferation and activation of cytokine pathway. btk inhibitors are mainly approved for the treatment of an aggressive form of b cell lymphoma, called mantle cell lymphoma. this group includes acalabrutinib, zanubrutinib, tirabrutinib, and ibrutinib, among which ibrutinib was shown to have less efficacy and more toxicity [142] . astrazeneca ® incorporation has designed a clinical trial to assess the efficacy of one of these btk inhibitors, called acalabrutinib, to alleviate the cytokine storm of sars-cov-2 infection (table 5 ). in addition to btk inhibitors, other kinase inhibitors, including erlotinib and sunitinib were also shown to interfere in viral entry through inhibiting jak and aak1. however, they are not classified as jak inhibitors and are not preferred over jak inhibitors due to less efficacy and higher toxicity [137, 138] . sorafenib was also hypothesized to be repurposed in covid-19 based on a drug-gene interaction analysis [143] . another mechanism of immunosuppression, which has been proposed, is related to the use of mtor inhibitors. the cytokine storm in covid-19 patients is attributed to a mechanism, called antibodydependent enhancement, in some systematic reviews [144] . this phenomenon happens when the virus triggers the production of cross-reactive antibodies by memory b cells. these cross-reactive antibodies enhance virus delivery to the macrophages and thus contribute to the massive replication of the virus without being captured by the immune system. mtor inhibitors were found to inhibit the activation of memory b cell and prevent the antibody-dependent enhancement mechanism. mtor inhibitors also were shown to inhibit the replication of mers-cov in the in vitro studies [144] . some mtor inhibitors, including rapamycin or sirolimus, were hypothesized to be repurposed in covid-19 clinical studies. sirolimus was shown to inhibit viral replication and release in patients with severe pneumonia and acute respiratory failure [145] . the computational analysis of protein-protein interactions and gene-enrichment network also suggested that sirolimus can be repurposed for sasr-cov-2 [146] . moreover, a clinical study of sirolimus on covid-19 patients has been recently started (table 5 ). papain-like protease (plpro) is a viral protease responsible for coronavirus genome replication with deubiquitinating activity [147] . plpro has been mainly the target of viral inhibitor class of medications. however, antimetabolites were also shown to be effective on plpro due to their pharmacological action. for instance, 6-mercaptopurine (6mp) and 6-thioguanine (6tg) were shown to be the specific inhibitors of sars-cov plpro [148] . mycophenolate mofetil is another immunosuppressant that was shown to target plpro in sars-cov and mers-cov in both in vitro and in vivo studies. however, further clinical studies are needed to assess its efficacy against sars-cov-2 [149] . there is no definitive evidence about the efficacy of other antimetabolites including methotrexate in covid-19 patients [149] . calcineurin inhibitors such as tacrolimus might be effective against sars-cov-2, since they inhibit calcineurin thereby blocking t cell activation. tacrolimus, which is mainly used in organ transplant, was shown to be effective against mers-cov in a renal transplant patient compared with a similar patient who did not receive tacrolimus as a part of the transplant regimen [150] . tacrolimus was also found to be effective against sars-cov in a study on cell lines. however, further studies undoubtedly are required to assess its efficacy against sars-cov-2 [151] . there is no definitive evidence about the efficacy of other calcineurin inhibitors including cyclosporine [149] . metal-based agents with different metal centers, including gold, ruthenium, and bismuth were suggested to be used in covid-19 patients [152] . the gold compound, called auranofin (ridaura®) is an fda approved compound, which was initially proposed for rheumatoid arthritis. the exact mechanism of auranofin is still unclear; however, it is classified as an immunomodulatory and anti-inflammatory agent. in recent years, auranofin has gained attention in viral infections, including hiv. in the case of hiv, it was revealed to be more effective than hydroxychloroquine in control of viral production, latency, and viral reactivation [153] . it was also hypothesized that auranofin can interfere with il-6 signaling by inhibiting jak1 and stat3 pathways [154] . it was shown that a low micromolar concentration of auranofin strongly inhibited sars-cov-2 viral replication and reduced the viral-induced cytokine expression in human cells [155] . tnf-α was shown to be associated with sars-cov pulmonary injury; therefore, tnf-α blockers, which are mainly used in the treatment of autoimmune and inflammatory diseases, such as rheumatoid arthritis, ankylosing spondylitis, and psoriasis, can be suggested as a potential target for sars-cov-2 [156] . besides the monoclonal antibodies that modulate the tnf-α responses, etanercept was suggested as another immunomodulator for covid-19 patients [149] . lenalidomide and thalidomide, which are not specifically classified as tnf-α blockers, were hypothesized to be repurposed based on a drug-gene interaction analysis [143] . thalidomide has antiinflammatory, anti-fibrotic, and immunoregulatory effects, which proved to be safe and effective in the treatment of lung injuries with different etiologies, including h1ni-induced lung injury [157] . it has also entered a clinical trial of covid-19, as shown in table 5 . cd24fc, which is a fusion protein constituted of human cd24 attached to the human igg fc region, is another biological immunomodulator. cd24fc was demonstrated to successfully ameliorate cytokine responses in viral infections and reduce the graft versus host disease [158, 159] . hence, cd24fc could be effective against the sars-cov-2 cytokine storm and the associated pneumonia. cd24fc has entered a clinical trial by oncimmune ® incorporation (table 5 ). the rationale for usinge of corticosteroids in covid-19 patients relies on their inhibitory effects on the inflammatory factors. corticosteroids inhibit a massive proportion of cytokines, chemokines, inflammatory enzymes, and receptors that are overexpressed in response to the viral infection [160] . results regarding the beneficial effects of corticosteroids against coronavirus are controversial. a metaanalysis including 5270 patients from 15 studies revealed that the use of corticosteroids resulted in higher mortality rate, the longer length of stay, a higher rate of bacterial infection, hypokalemia, and hypercalcemia [161] . furthermore, russell and colleagues in a comment published in the lancet did not advocate the use of corticosteroids in covid-19-induced lung injury or shock, except in the clinical trial settings [162] . by contrast, a retrospective analysis of 401 patients with severe sars revealed that corticosteroids led to reduced mortality rate and shortened hospital stay [163] . taken together, corticosteroids are indicated for critically-ill critically ill covid-19 patients and its use in mild to moderate patients is highly disregarded. for instance, the use of corticosteroids is recommended in mechanically-ventilated covid-19 patients with respiratory failure (with ards), while is inappropriate for covid-19 patients without ards [164] . in fact, administering corticosteroids in the onset of disease was shown to deter the immune system and increase the viral load; thereby inducing additional complications, such as diabetes and vascular necrosis [165, 166] . the timing and dosage of corticosteroids are of special importance in the outcomes of treatment in critically-ill critically ill patients [167] . for instance, in patients with high inflammatory responses including severe deterioration of oxygenation indicators and rapid imaging progress, a short term (3-5 days) of corticosteroids doses equivalent to 1-2 mg/kg/day methylprednisolone is recommended [168] . moreover, a recent study revealed that low doses of corticosteroids (e.g. 25-150 mg/day methylprednisolone) did not delay viral clearance and did not increase the mortality rate compared with the control group [169] . recently dexamethasone was introduced as the first medicine presented with a survival benefit in treating critically-ill critically ill covid-19 patients, and who welcomes preliminary results regarding its use [170, 171] . according to a large, multi-center and randomized clinical trial called recovery (randomized evaluation of covid-19 therapy) trial, the use of 6 mg/day dexamethasone in covid-19 patients on mechanical ventilation or oxygen supplementation was associated with reduced mortality rate compared with those who received standard treatment. in the patients who did not need respiratory support, no improvement was observed [172, 173] . this well-known steroid is believed to support the suppression of hyperactive inflammatory responses, so-called cytokine storm [174] . interestingly, an extra mechanism in defeating sars-cov-2 by dexamethasone was suggested. a computational study revealed that dexamethasone tightly binds to 3c-like protease (the main protease) in sars-cov-2 as remdesivir does; however, dexamethasone surprisingly forms a better contact with the enzyme active site than remdesivir [175] . in addition to the importance of dose and timing, other considerations concerning the use of corticosteroids ought to be made according to a recent correspondence consensually made by experts in the lancet, including: (1) the pros and cons of corticosteroids should be carefully evaluated prior to administration; (2) prudent consideration should be made for the further use of corticosteroids in patients who are on the regular use of corticosteroids for chronic diseases [176] . modulation of the immune system is obviously a major strategy in the control and treatment of covid-19, which should be adjusted according to different stages of the disease. it can be done through vaccines, interferons, antibodies (either as convalescent plasma therapy or monoclonal and polyclonal antibodies), or potential therapeutic immunomodulators. at the pre-disease or disease prevention stage, active immunization by vaccines could be an optimal approach. however, vaccine development is a time-consuming and complicated process. despite enormous efforts on finding vaccines against sars-cov-2, there is still a fairly long way to the market availability of a proper vaccine. following covid-19 disease onset, regulating the activities of the aberrant immune system may be a valuable treatment goal. at the early stages of the 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cti, a leading contract research organization, for planned phase iii study for lenzilumab for coronavirus treatment vir biotechnology proceeding with two clinical development candidates for covid-19 distributed bio optimizes anti-sars protective antibodies to block the novel coronavirus tiziana life sciences plc to expedite development of its fully human anti-interleukin-6-receptor monoclonal antibody, a potential treatment of certain patients infected with coronavirus aqualung therapeutics advances its investigational monoclonal antibody into ind-enabling studies of acute respiratory distress syndrome (ards) and ventilator-induced lung injury (vili) advances-its-investigational-monoclonal-antibody-into-ind-enabling-studies-of-acute-respiratory-distress-syndrome-ards-and-ventilator-induced-lung-injury-vili mount sinai and harbour biomed collaborate to advance novel biotherapies for the treatment of cancer and coronavirus covid-19 nascent biotech investigation drug product, pritumumab, to be studied as a potential therapeutic agent against (coronavirus) covid-19 inflarx doses first patient in multicenter randomized clinical trial in severe progressed covid-19 pneumonia in europe upon receipt of initial positive human data with inflarx's anti-c5a technology amgen and adaptive biotechnologies announce strategic partnership to develop a therapeutic to prevent or treat covid-19 identification and characterization of a potential therapeutic covid-19 antibody by vir biotechnology published in nature immunoprecise provides updates on b cell select™ and deep display™ programs for sars-cov-2 antibody discovery emergent biosolutions initiates development of plasma-derived product candidates for the treatment and prevention of coronavirus disease initiates-development-of-recombinant-polyclonal-antibody-therapy-for-covid-19.html. 131. nugenerex. ii-key-ncov peptide vaccine anticancer kinase inhibitors impair intracellular viral trafficking and exert broadspectrum antiviral effects the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the experience of clinical immunologists from china immunosuppression for hyperinflammation in covid-19: a doubleedged sword? the lancet th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib family-wide structural analysis of human numb-associated protein kinases feasibility and biological rationale of repurposing sunitinib and erlotinib for dengue treatment covid-19: combining antiviral and anti-inflammatory treatments baricitinib-a januase kinase inhibitor-not an ideal option for management of covid 19 baricitinib for covid-19: a suitable treatment advanced bioinformatics rapidly identifies existing therapeutics for patients with coronavirus disease-2019 (covid-19) review of bruton tyrosine kinase inhibitors for the treatment of relapsed or refractory mantle cell lymphoma covid-19: a drug repurposing and biomarker identification by using comprehensive genedisease associations through protein-protein interaction network analysis prevent covid-19 severity by repurposing mtor inhibitors. 2020.preprints adjuvant treatment with a mammalian target of rapamycin inhibitor, sirolimus, and steroids improves outcomes in patients with severe h1n1 pneumonia and acute respiratory failure network-based drug repurposing for human coronavirus the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deisgylating enzyme associations between immune-suppressive and stimulating drugs and novel covid-19-a systematic review of current evidence. ecancermedicalscience mers cov infection in two renal transplant recipients: case report replication of human coronaviruses sars-cov, hcov-nl63 and hcov-229e is inhibited by the drug fk506. virus research a role for metal-based drugs in fighting covid-19 infection? the case of auranofin chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids auranofin blocks interleukin-6 signalling by inhibiting phosphorylation of jak1 and stat3. immunology the fda-approved gold drug auranofin inhibits novel coronavirus (sars-cov-2) replication and attenuates inflammation in human cells tnf-[alpha] inhibition for potential therapeutic modulation of sars coronavirus infection anti-inflammatory effect of thalidomide on h1n1 influenza virus-induced pulmonary injury in mice cd24 and siglec-10 selectively repress tissue damage-induced immune responses cd24 and fc fusion protein protects sivmac239-infected chinese rhesus macaque against progression to aids one hormone, two actions: anti-and pro-inflammatory effects of glucocorticoids the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury. the lancet treatment of severe acute respiratory syndrome with glucosteroids: the guangzhou experience surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) glucocorticoid-induced diabetes in severe acute respiratory syndrome: the impact of high dosage and duration of methylprednisolone therapy factors of avascular necrosis of femoral head and osteoporosis in sars patients' convalescence. zhonghua yi xue za zhi the pathogenesis and treatment of thecytokine storm'in covid-19 effectiveness of glucocorticoid therapy in patients with severe coronavirus disease 2019: protocol of a randomized controlled trial low-dose corticosteroid therapy does not delay viral clearance in patients with covid-1 582: 469 171. who welcomes preliminary results about dexamethasone use in treating critically ill covid-19 patients covid-19) treatment guidelines. national institutes of health effect of dexamethasone in hospitalized patients with covid-19: preliminary report. medrxiv coronavirus breakthrough: dexamethasone is first drug shown to save lives deciphering the binding mechanism of dexamethasone against sars-cov-2 main protease: computational molecular modelling approach on the use of corticosteroids for 2019-ncov pneumonia key: cord-304418-k9owyolj authors: le maréchal, m.; morand, p.; epaulard, o.; némoz, b. title: covid-19 in clinical practice: a narrative synthesis date: 2020-09-29 journal: med mal infect doi: 10.1016/j.medmal.2020.09.012 sha: doc_id: 304418 cord_uid: k9owyolj the coronavirus disease 2019 (covid-19) was first reported in the city of wuhan, china. the disease rapidly spread to the rest of china, to southern-east asia, then to europe, america, and on to the rest of the world. covid-19 is associated with a betacoronavirus named sars-cov-2. the virus penetrates the organism through the respiratory tract, conveyed by contaminated droplets. the main cell receptor targeted is the surface-bound ace-2. as of the 26th july 2020, 15,200,000 covid-19 cases and 650,000 deaths were reported worldwide. the mortality rate is estimated between 1.3 and 18.3%. the reproductive rate without any public health intervention is estimated around 4-5.1 in france. most hospitalized patients for covid-19 present respiratory symptoms, which in some cases is associated with fever. up to 86% of admissions to icu are related to acute respiratory failure. to date, no anti-viral therapy has proven its efficacy considering randomized trials. only immunomodulatory treatments such as corticosteroids have shown to cause significant improvement in patient outcome. a study estimated the rt in the city of wuhan, china, between the 1 st january 2020 and the 8 t march 2020 [22] , during five successive time periods (corresponding to different public health interventions). rt peaked at 3.82 on the 24 th january 2020 (with no major public health intervention), and subsequently declined, to below 1.0 on the 6 th february 2020 (after one week of city lockdown, traffic suspension, and home quarantine) and was below 0.3 on the 1 st march 2020 (after 15 days of quarantine) [22] . in south korea, reproduction number rt was estimated at 1.5 (95% confidence interval (ci) 1.4-1.6) [18] . in france, flaxman et al. estimated rt before lockdown between 4-5.1 (95%ci), and around 0.5 during lockdown [23] . the virus penetrates the organism through the respiratory tract, conveyed by contaminated droplets. while the exact viral progression remains elusive, the virus seems to have a favorable tropism for epithelial cells within the airways, leading to viral replication both in the nasal cavities and in distal bronchioles [24] . the main cell receptor targeted by the viral surface glycoprotein (s) is the surfacebound angiotensin-converter enzyme 2 (ace2) [8] , yet it is still unclear if the virus can benefit from a second receptor to achieve gain cell entry. tmprss2 is a cellular serine proteinase whose role has been pointed out to prime sars-cov-2 cellular entry [25] . the viral s protein, also known as spike, is responsible for both cell attachment and membrane fusion. it is formed by three dimers of two non-covalently bound domains s1 and s2. both subunits are synthetized as a protomer resulting from the expression of the s gene. s1 and s2 are efficiently cleaved by a cellular furin [26] thanks to a polybasic motif (prra) specific of sars-cov-2 [27] . because of its specificity and key role in viral cycle, the s protein constitutes the main target for vaccine candidates [28] . diagnosis of covid-19 requires laboratory confirmation, usually performed by detection of viral rna by a reverse-transcription polymerase chain reaction (rt-pcr). while the first laboratory confirmed cases in wuhan early in january 2020 relied on deep sequencing techniques [29] , highly scalable assays have since been developed after the viral sequence was published. the first rt-pcr protocol was published by drosten and colleagues [30] from charité university in berlin, and since then adopted by the who. this approach relies on the amplification and detection of the following sequences among the sars-cov-2 genetic sequence: the e gene encoding the envelope protein is used as a screening assay targeting all members of the betacoronavirus genera, and both the rdrp gene or the n gene are used as confirmatory assays due to their specific sequence in the sars-cov-2 species. this protocol has been widely adopted in commercially available test kits and is now used in most facilities. the paris institut pasteur has developed its own assay [31] , based on the detection of two sequences in the rdrp gene spanning nucleotides 12621-12727 and 14010-14116. this protocol has been widely used in french laboratories to manage early covid-19 suspicions and cases [32] . such assays can be performed on several samples, and the choice of sampling site has been a debated issue considering the consequences of false negative results. nasal swabs, oropharyngeal swabs, saliva, sputum and bronchoalveolar lavage fluid (bal) were mostly reported. nasal swabs have proven to be effective and provided adequate sensitivity (80%) [33, 34] , if performed properly. oropharyngeal swabs tend to be less sensitive (32%) [35] . saliva collection has been proposed as a viable alternative yielding higher sensitivity (91.7%) [36] , also addressing the availability of swabs issue as worldwide demand surge and laboratories face shortages in stocks. sputum samples, while being more sensitive than upper respiratory tract samples (95.7%) [37, 38] , raise security concerns for the safety of healthcare workers regarding the droplet dissemination nature of the covid-19. bal and other lower respiratory tract samples are widely used in intensive care units and yield very good results (93% sensitivity) [35] . the viral load seemed higher in early stages of the disease [39] , with symptomatic patients exhibiting higher viral titers as assessed by the lower ct numbers [40] . when analyzed by rt-pcr, stools are frequently positive (41%), and high viral loads have been pointed out in such samples in both asymptomatic and symptomatic patients [41] . no relation has been established as of today between enteric viral excretion and clinical outcome and disease severity. in children, the average duration of viral rna shedding in stools are 29 days (+/-12 days). the duration of viral shredding seemed to decrease with age [42] and infectivity of stools is low to non-existent. due to feces positivity, the sampling of wastewater has been proposed to assess viral circulation [43] . plasmatic detection of sars-cov-2 has been reported but only with low viral titers, and mainly in clinically severe cases [44] ; bloodstream infectivity has yet to be demonstrated. urine has remained virus-free except in one study [45] . most genomic testing in asymptomatic patients [46] returned negative after 2-3 weeks, with exceptional long shedding up to 45 days after symptom onset [47, 48] , raising the question of the infectivity of such viral shedding. excretion of infectious virions is thought to span two days before onset of symptoms up to 8 days after onset [49] , viral excretion peaking at day 4 post-infection [50] , supporting the effectiveness of a 14-day quarantine period for case isolation. however, results do not fully correlate with infectivity: viral culture from clinical samples is usually infeasible after 8 days after onset of symptoms [51] . nucleic acid testing (nat) is key to patient management and surveillance of disease propagation. according to several works, incubation period has been estimated between a mean of 4.8 and 6.4 days [52] [53] [54] [55] . lauer et al. estimated that symptom onset will occur within 11.5 days for 97.5% of patients, and that a 14 days quarantine would be sufficient [53] . a large work from zhou et al. described clinical timeline for an 813 patient cohort [56] . the mean time from illness onset to intensive care unit (icu) admission was 9-12 days [21, 56] ; it was 7 days (4-9) to dyspnea [56] , 11-12.5 days to hospital admission [56, 57] , 9 days (7-13) to sepsis, 12 days (8-15) to respiratory failure, and 21-44 days to death, depending on studies [56, 58] . time between onset of symptoms and dyspnea is 5-7 days, to ards 8-12 days [56, 59] . one initially described symptom was fever; however, up to 60% of patients were described as nonfebrile, and up to 52% of patients admitted in icu were non-febrile [52] . coughing was reported in 60-82% of cases, asthenia in 38-70% of cases, myalgia in 11-44% of cases, dyspnea/shortness of breath in 19-55% of cases, and diarrhea in 2-10% of cases [55, [59] [60] [61] . no symptoms are specific of covid-19, but surprisingly, anosmia and ageusia appeared to be strongly linked with covid-19 infection. mechanism for these symptoms is still to be unveiled, as well as for digestive forms in elderly patients presenting with only diarrhea. a study described imaging data among 37 asymptomatic patients [48] . chest ct evidenced in 11 of them (30%) focal ground glass opacities, and in 10/37 (27%) stripe shadows and/or diffuse consolidation; 16/37 (43%) had a normal ct scan. in a 63 asymptomatic chinese cohort, 29/63 patients had abnormal ct scans; few patients (13%) had comorbidities [62] . the most frequent presentation of hospitalized covid-19 is pneumonia (91-100%) with dyspnea and a rarely productive cough [52, 55, 56, 59, 61] . in case of patients diagnosed with clinical pneumonia, chest x-ray and ct-scanner found bilateral ground-glass opacity in 25-100% of cases [56, 59] . when considering all hospitalized covid-19 patients, ct scans evidenced ground-glass opacities in 56-71% of patients and consolidation in 59% of patients [52, 56] . pulmonary injury was bilateral in 52-75% of patients [52, 56] . thromboembolic complications several works have described the high incidence in covid-19 patients of both venous and arterial thromboembolic diseases. in klok's study of 184 patients with proven covid-19 pneumonia admitted to the icu in the netherlands, and who all received at least standard dose thromboprophylaxis, the incidence of thrombotic complication was 31% (95%ci 20-41%) [63] . in an italian study of 362 cases (in icu and on general wards), 28 presented a thromboembolic event (7.7%). the authors estimated that those events were highly underestimated due to the low number of specific imaging tests performed [64] . known risk factors for thromboembolic events that are reported in covid-19 are excessive inflammation and immobilization [65] . however, in a large multicenter international work published by freund et al among 3,253 patients who underwent a computed tomography pulmonary angiogram for a suspected pulmonary embolism, a positive covid-19 status was not associated with pulmonary embolism in multivariate analysis (p=0.40) [66] . cardiac injuries have been described in covid-19 patients. viruses are a common cause of myocarditis [67] ; myocardial injury can be related to direct cell injury caused by the virus, to t lymphocyte mediated cytotoxicity, to hemodynamic damage induced by hypoxia or shock, or related to cytokine storm [67] . arrhythmia has been described as a cause of transfer in icu in 44% of covid-19 patients [59] ; in an covid-19 acute setting, it can result from direct cardiomyocyte injury, to an infection of the pericardium causing massive edema, or to an ischemia due to microcirculation lesions [68] . in a 416 cohort of patients in wuhan, china, 82 patients with elevated levels of cardiac troponin had a higher risk of hospital death [69] . several neurological manifestations of covid-19 have been reported. manifestations are very diverse: -olfactory dysfunction: generally, post-viral olfactory loss account for 11% of acute olfactory dysfunction [70, 71] . several studies reported olfactory dysfunction among covid-19 patients (5-86%) [72] [73] [74] . this may be related to a localized olfactory cleft edema (local inflammation), or a direct neuroinvasion of the olfactory nerve [70, 73] . the loss of flavor perception is also frequently reported; it is considered to be mainly due to a loss of retronasal olfaction rather than a loss of sense of taste itself [70] , -central nervous system manifestations: confusion [72, 75, 76] , acute cognitive disorder, acute myelitis, encephalopathy, encephalitis [77] , intracranial hemorrhage, strokes [72, 75, 76] , seizures, -peripheral nervous system manifestations: guillain-barré syndrome [78] [79] [80] [81] , skeletal muscle damage (hyperckemia, rhabdomyolysis, myopathy [72, 76] ), dysautonomia), -neuropsychiatric symptoms [82] : anxiety, depression, insomnia, and psychosis suggested mechanisms are the hypoxic brain injury on severe pneumonia with peripheral vasodilatation, hypoxia, hypercapnia, and anaerobic metabolism, immune mediated injury related to the cytokine storm, and sars-cov-2 direct neurovirulence, since it has already been described for other coronaviruses [76, 83] . prevalence of reported comorbidities among patients with covid-19 has largely varied according to countries. the largest published cohorts are among chinese and american patients, and main comorbidities are hypertension (17-57%) [19, 84, 85] , obesity (42%) [84] , diabetes (8-34%) [84] , and cardiovascular disease (4%) [19, 85] . being a man was described as the main risk factor for covid-19 [61, 85] . most icu admissions were related with a respiratory failure (54-86%) [56, 86, 87] , that is also the leading cause of mortality (93-100%) [56, 87] . patients with respiratory failure are described to present an acute respiratory distress syndrome (ards), defined as a respiratory failure not fully explained by cardiac failure or fluid overload, bilateral opacities in chest imaging, and oxygenation pao2/fio2 < 300 mmhg [88] . report from critically-ill-patients series suggested that those patients presented a "cytokine storm". biological data showed a higher level of il-6 in critically-ill and non-surviving patients, a higher level of crp and a higher level of ferritin [56, 59, 86, 89] . among severe patients, the lymphocytes count was lower than mid patients or healthy controls (respectively 1,132mol/l, 1,256mol/l, and 2,215mol/l). cd4+ and cd8+ lymphocytes were also lower in the severe-patients group [89] . necropsy of patients who died from covid-19 allowed histological analysis of lung tissue samples. reported patients spent between 1 and 23 days in icu before death. macroscopic examination found lungs which were heavy, congested and edematous with patchy involvement. histological examination found features corresponding to the exudative and early or intermediate proliferative phases of diffuse alveolar damage (capillary congestion, interstitial and intra-alveolar edema, dilated alveolar ducts, collapsed alveoli and loss of pneumocytes). authors also reported interstitial pneumonia (inflammatory lymphomonocytic infiltrate along the slightly thickened interalveolar septa), organizing pneumonia, and acute fibrinous organizing pneumonia [90] . a review article on children presenting covid-19 was published by cui et al. reporting clinical, biological and imaging features on 2.597 children [91] . among all cases 7.6% were asymptomatic, 45 .5% were mild, 41.5% were moderate, 4.4% were severe, 0.9% were critical, and 0.1% (3) led to death. regarding clinical characteristics, authors collected data from 23 articles (452 children): 43% presented with fever, 43% with cough, 20% with sore throat, 17% with tachycardia, 16% with rhinorrhea, 15% with nasal congestion, and 13% with shortness of breath. among 23 critical cases, six had an underlying disease. pulmonary imaging in 294 cases reported 30% of ground glass opacities, 20% of local patchy shadow, 15% of bilateral patchy shadow, and 1% of interstitial lesions. in an international study among 582 children presenting covid-19 infection, reported risk factors for admission to icu or requiring mechanical ventilation were being less than 1-month-old (p<0.001) and having an underlying disease (p<0.001) [92] . observations described a higher risk of kawasaki-like inflammatory syndrome in children infected by sars-cov-2, also called by who the covid-19 associated pediatric multisystem inflammatory syndrome [93] ; e.g., a 497% increase of children admitted for a kawasaki-like syndrome during covid-19 epidemic was described in a small french cohort [94] ; igg antibodies against sars-cov-2 infection were detected among 19/21 of children presenting with a kawasaki-like syndrome during the epidemic in another french cohort [95] . kawasaki disease is described as an acute febrile systemic vasculitis that affects medium and small-sized blood vessels. one suspected mechanism is a post-viral immunological reaction to several viruses (influenza [96] , enterovirus [97] , adenovirus [98] , parvovirus [99] , vzv [100] , ebv [100] , measles [101] , or dengue [102] ). upon infection, humoral antiviral immunity is triggered, owing to the development of specific antibodies. a vast majority of infected patients will generate anti-sars-cov-2 antibodies [103] . antibody titers peak around day 30 post-infection [104] , but from this point forward only decrease.. igm and igg kinetics do not differ significantly [105] , thus making differential isolation of these markers void. seroconversion is witnessed at a median of 14 days after symptom onset [106] . high antibody titers are associated with severe respiratory symptoms, asymptomatic patients having lower titers [105] . this raises the so far unresolved question of covid-19 immune mechanisms and protection. it is not clear whether high antibody titers could promote severe clinical presentations by a mechanism similar to antibody dependent enhancement [107, 108] . on the other hand, pauci-symptomatic forms of covid-19 could trigger a reduced humoral response only that could correlate with a shortened duration of protection [109] . anti-sars-cov-2 antibodies are directed towards both the spike (s) protein and the nucleocapsid (np) [110] . neutralising antibodies are observed in most patients and recognise specifically the spike (s) protein [111] . neutralising activity appears to be correlated with the presence of antibodies binding the receptor-binding domain (rbd) in its closed conformation within the spike protein [112] , and the detection of such antibodies could be a surrogate marker of protection. no cross-reactivity with other human coronavirus (hcov-oc43, hcov-nl63, hcov-229e and hcov-hku1) has been evidenced to date (ref). however, cross-reactivity with sars-cov-1 has been shown at least in vitro [26] . while serological assays allow large epidemiological studies and enable better evaluations of epidemic parameters (rt, for instance), individual benefit is scarce, if existent. accordingly, we believe patient management should focus on molecular based assays. to date, no anti-viral therapy has proven its efficacy and the current management of covid-19 remains supportive care and ventilatory support when needed. remdesivir remdesivir (gs-5734) is a nucleotide analog that targets viral rna polymerases. it has an established in vitro (culture cells) and in vivo (mouse and primate models) efficiency on multiple genetically distinct coronaviruses, and on ebola virus [113, 114] . its in vitro effect on sars-cov-2 has been reported in wang et al.'s work, with a high 90% effective concentration value against infection of vero e6 cells [115] . williamson explored remdesivir's efficiency in 12 rhesus macaques infected with sars-cov-2 infection [116] . one over six macaques in the remdesivir group developed a respiratory disease vs 6/6 in the control group. after euthanasia and lung analysis, no virus was detected in lung tissue samples in the remdesivir group; remdesivir was detected in all six lungs of the treated animals. only 3/36 lobe lungs in the control group were virus-free. there was no difference in viral load in bal between both groups. gilead restricted access to remdesivir since the beginning of covid-19 pandemic to compassionate use and to clinical trials [117] . the first clinical study on remdesivir was published by grein et al. on 53 patients, without any control group. treatment was started 12 days after symptom onset; 68% of treated patients showed an improvement regarding oxygen support, and 13% of patients who completed treatment died [118] . a large international clinical trial compared 541 patients receiving remdesivir with 522 patients treated with placebo. a shorter time to recovery (11 days vs 15 days) was reported in the remdesivir group (p<0.001). adverse events were reported (21% in the remdesivir group and 27% in the placebo group [119] ). goldman et al. compared 5 vs 10 days of remdesivir treatment in a multicentric, international clinical trial among 397 patients and did not observe any difference between the 2 groups at 14 days after treatment onset on primary outcome (clinical efficacy on a 7-points scale) [120] . lopinavir is an antiviral agent developed to target hiv protease; it is generally used in association with ritonavir, a pharmacokinetic "booster" increasing lopinavir plasma concentration. it is widely used in adults living with hiv/aids [121] . lpv/rtv is regarded as a potential anti-sars-cov-2 agent since several trials on sars-cov-1 showed a favorable effect. indeed, in a 2003 study of 1,521 patients with sars, chan et al. reported a 2% death rate in the lpv/rtv group vs 16% in the soc group (p<0.05) if lpv/rtv was used as initial treatment, but with no difference as a rescue treatment [122] . in a 2004 study exploring the efficacy of lpv/rtv in patients with sars regarding a composite primary outcome (severe hypoxemia or death at day 21), chu et al observed that 2% in the lpv/rtv group met the primary outcome, vs 29% in a historic control group (p<0.001) [123] . the first large clinical trial published on lpv/rtv on sars-cov-2 compared 99 patients receiving the antiviral vs 100 receiving soc alone [124] ; there was no difference between the 2 groups regarding the primary end point (time to improvement) (15 vs 16 days, p=0.09). noteworthily, the median time between symptom onset and treatment was 13 days. 48% of patients in the lpv/rtv group vs 50% in the soc group who underwent an adverse event. hydroxychloroquine (hcq) is a widely used molecule in limited forms of lupus, with a low price, and it has an established clinical safety profile [125] in vitro studies showed the effect of chloroquine [115] and hcq in inhibiting sars-cov2 infection [126] . in vitro activity of hcq and chloroquine against sars-cov-2 were not different in liu et. al's work [126] , and hcq was found, in vitro, to reach three times the potent antiviral activity of chloroquine in yao et. al's work [127] . noteworthily, hcq was four times less toxic than chloroquine in animal study [128] . however, a recent in vitro study showed that chloroquine did not block sars-cov-2 infection of the tmprss2-positive lung cell [129] . another in vitro study also found that hcq did not show any antiviral activity in a model of reconstituted human airway epithelium [130] . due to such in vitro results, several trials assessed the efficiency of hcq on viral load in respiratory samples (without clinical considerations [131] [132] [133] [135] . association of hcq and azithromycin was associated with a higher risk of ventricular tachycardia and prolonged qt. hcq alone was responsible for more conduction disorder. however, in patients receiving azithromycin alone compared with hcq alone, there was more prolonged qt and ventricular tachycardia. unchanged before and after hemodialysis [137] . moreover, several observations reported the onset of severe covid-19 in patients who were already receiving hcq as a long-term treatment for an inflammatory disease [138] . one randomized trial evaluated the outcome of hcq whether or not in association with azithromycine on mild to moderate covid-19 [139] . primary endpoint (escalation in icu, mechanical ventilation or death) was reached by 35% of patients in the early corticosteroid group vs 54% in the control group (p=0.005) [141] . recovery was a randomized, controlled trial of the use of 6 mg of dexamethasone vs soc in hospitalized patients with covid-19. at 28 days, 454/2,104 (21.6%) patients had died in the dexamethasone group vs 1,065/4,321 (24.6%) in the soc group (p<0.001) [142] . tocilizumab is a monoclonal antibody against il-6 receptor. it is mainly prescribed in rheumatoid arthritis. an italian team reported the use of tocilizumab to the first 100 patients presenting to the brescia university hospital with a covid-19 ards requiring ventilatory support. at 72h, 58% of patients showed an improvement, and at 10 days after treatment inset, 77% of patients had improved and/or stabilized [143] . anakinra is an antagonist of il-1 receptor. an italian team reported the use of anakinra in 29 patients with moderate to severe covid-19 ards before mechanical ventilation, compared with 16 patients not receiving anakinra. survival rate at 21 days was 90% in the anakinra group vs 56% in the control group (p =0.009). however, there was no difference regarding the mechanical ventilation-free survival (72% in the anakinra group vs 50% in the control group, p=0.150) [144] . a chinese team reported the use in 51 covid-19 patients of convalescent plasma compared with a control group of 50 patients. their primary endpoint was clinical improvement within a 28 day-period (reduction of 2 points on a 6-points disease severity scale). there was no difference between both groups (p = 0.260). in sub-group analysis among patients presenting a severe disease, 91% of patients met the primary endpoint in the plasma therapy group vs 68% in the control group (p = 0.003) [145] . in another study, the outcomes of 1,430 severe or critical patients treated with soc and 138 patients treated with convalescent plasma therapy were compared; 2.2% of patients died in the plasma group vs 4.1% in the soc group (no comparison). 2.4% of patients were admitted to icu in the plasma group vs 5.1% in the soc group (p=0.2) [146] . the effect of ace inhibitor and arb treatment on covid-19 severity and/or mortality has been reported in several studies but seems variable. in zhang et al's work, on 1,128 patients with hypertension and covid-19, multivariate analysis found a lower all-cause mortality in the ace inhibitor/arb group, than in the other group (p = 0.03) [147] . however, li et al did not found any difference in covid-19 severity (p=0.645) or mortality (p=0.340) among 362 patients admitted for hypertension and covid-19 [148] . in chu et al.'s meta-analysis, social distancing was considered as efficient with a -10.2% risk difference (95%ci [-11.5 to -7.5%] of infection in short distance vs further distance [149] . wearing respirators or face masks was associated with a large reduction in risk of infection (risk difference -14.3%, 95%ci [-15.9 to -10.7%]) [149] . eye protection also seemed efficient in infection reduction with a risk difference of -10.6% 95%ci [-12.5 to 7.7%] [149] . however, lockdown had the larger impact on transmission with an 81% [75%-87%] reduction [23] . in france, the number of daily cases of covid-19 was growing by the end of summer 2020, suggesting the debut of a second epidemic wave, as many other countries are facing. the first wave allowed us to develop and strengthen our laboratory tests. since then, french health authorities have implemented a systematic contact tracing (contact-covid) around each patient presenting a positive sars-cov-2 rt-pcr. this highlighted the high number of asymptomatic and mild cases, and made french people massively adapt their daily habits with a systematic face mask protection in all public areas, and restrictions in social events. however, none of the evaluated pharmacological treatments have showna clear efficacy on sars-cov-2. one of the main limitations seem to be the long period between symptom onset and initiation of treatment. an effective vaccine against sars-cov-2 appears to be the only way to end this pandemic. at the end of august 2020, 203 trials were registered on clinicaltrial for a vaccine against covid-19, thus raising hope to end this pandemic in a reasonable amount of time. promed post -promed-mail n diseases is for i. promed post -promed-mail china is consistent with substantial human-to-human transmission who coronavirus disease (covid-19) dashboard | who coronavirus disease who director-general's opening remarks at the media briefing on covid-19 -11 the species severe acute respiratory syndrome-related 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against sars-cov-2 infection in non-human primates hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial preliminary evidence from a multicenter prospective observational study of the safety and efficacy of chloroquine for the treatment of covid-19 observational study of hydroxychloroquine in hospitalized patients with covid-19 cardiovascular toxicities associated with hydroxychloroquine and azithromycin: an analysis of the world health organization pharmacovigilance database hydroxychloroquine pharmacokinetic in covid-19 critically ill patients: an observational cohort study covid-19 infection also page 28 of 29 patients taking hydroxychloroquine hydroxychloroquine with or without azithromycin in mild-to-moderate covid-19 successful use of methylprednisolone for treating severe covid-19 early short course corticosteroids in hospitalized patients with covid-19 effect of dexamethasone in hospitalized patients with covid-19: preliminary tocilizumab for the treatment of severe covid-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in brescia interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study treatment of 5 critically ill patients with covid-19 with convalescent plasma improved clinical symptoms and mortality on severe/critical covid-19 patients utilizing convalescent plasma transfusion association of inpatient use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease 2019 (covid-19) infection in wuhan, china physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis key: cord-293688-g6kag5ij authors: nora, holtmann; philippos, edimiris; marcel, andree; cornelius, doehmen; dunja, baston-buest; ortwin, adams; jan-steffen, kruessel; petra, bielfeld alexandra title: assessment of sars-cov-2 in human semen a cohort study date: 2020-05-29 journal: fertil steril doi: 10.1016/j.fertnstert.2020.05.028 sha: doc_id: 293688 cord_uid: g6kag5ij objective: to investigate the presence of viral rna in human semen of severe-acute-respiratory syndrome coronavirus 2 (sars-cov-2) recovered and positive patients and to evaluate its presence and relevance on semen parameters. design: pilot cohort study setting: university hospital patients: 34 adult males were distributed into a) patients in convalescence (patients with confirmed sars-cov-2 infection in pharyngeal swab by rt-pcr and/or antibodies), b) negative control group (no antibodies) and c) patients with an acute infection (detection of sars-cov-2 in pharyngeal swab). intervention: semen and a blood sample were collected from each individual. main outcome measures: analysis of semen quality according to the who standards. detection of sars-cov-2 by rt-pcr in the native semen sample and after density gradient preparation. confirmation of immunoglobulin (ig)-a und ig-g antibodies in the blood. results: 18 semen samples from recovered males were obtained 8 to 54 days after absence of symptoms, 14 samples from controls and 2 samples from patients with an active covid-19 infection. no rna was detected by rt-pcr in the semen including semen samples from two patients with an acute covid-19 infection. subjects with a moderate infection showed an impairment of sperm quality. conclusion: a mild covid-19 infection is not likely to affect testis and epididymis function, whereas semen parameters did seem impaired after a moderate infection . sars-cov-2 rna could not be detected in semen of recovered and acute covid-19 positive males. this suggests no viral transmission during sexual contact and assisted reproductive techniques (art), however, further data need to be obtained. in december 2019 clusters of a novel type of pneumonia were reported in wuhan city, hubai province, china (1) and defined by the world health organisation (who) as coronavirus disease 2019 in february 2020 (2). the severe acute respiratory syndrome 108 coronavirus 2 (sars-cov-2) was identified as the causing viral pathogen for the pandemic (3) . in order to constrain the world-wide outbreak of covid-19, viral transmission pathways are intensively studied. so far, it is known that the coronavirus is predominantly transmitted 111 through respiratory droplets (4) . in addition, viral rna has been detected in various biological samples, such as faeces, urine and blood (5). sars-cov-2 seems to have a high affinity binding capability to the angiotensin converting enzyme-2 (ace-2) in human cells, 114 which is expressed in multiple organ systems, including the testis (6) . although the testis are immunologically privileged in case of viremia, some viruses can cross the blood-testis barrier causing local inflammation of the testis (7) . the virus may persist after an acute infection as 117 for example human immunodeficiency virus (hiv) and can theoretically replicate within the male reproductive tract (8). hence, viral rna of primarily non-sexual transmitted diseases can be found in semen (9). 120 the presence of sars-cov-2 in the male reproductive tract may reduce male fertility through orchitis or spermatogonial stem cell infection and may have implications for sexual transmission and consequently for embryonic infection, miscarriage and congenital disease 123 (10) . in order to thoroughly advise couples with the acute desire for a child, information about the impact of covid-19 on male reproductive function and viral seeding are needed. the aim of this study is to a) determine any possible implications of covid-19 on male 126 semen parameters and b) analyse the semen for any presence of sars-cov-2 rna in recovered men and males with an active covid-19 infection. defined patients requiring hospitalisation with oxygen supply up to 6 litres to achieve more than 92% of peripheral oxygenation. the control group consisted of healthy volunteers with 153 no reported andrological pathology. a semen sample of each participant was obtained by masturbation and ejaculation directly into non-cytotoxic sterile containers. freshly collected semen was liquefied for 30-60min at room temperature and processed within one hour of ejaculation for analysis of sperm 159 characteristics according to the criteria published by the who. sperm morphology was not assessed due to safety concerns. samples were homogenized, and 500µl were transferred to the tube for viral testing of the native sample. 162 to prepare the semen sample for the viral testing, the remaining semen was prepared in a 2step washing process modified according to the center's standards procedure for hiv or hep c infected males. first, the semen was counted and filtered through a 30°c pre-warmed 165 90%/45% colloidal silica density gradient with 1.800rpm for 20min (spermfilter®, gynotec b.v., gc malden, netherlands) prepared with gm 501 spermair (spa) sperm processing media (gynemed, lensahn, germany). second, the pellet was washed in 3ml pre-warmed 168 spa with 2.300rpm for 10min and the resulting pellet resuspended in 500µl spa, counted and transferred to the viral testing tube of the processed sperm. detection of sars-cov-2 in semen centrifugation of the native and processed sperm sample for 1min at 3.500rpm. rna extraction was performed from 200µl supernatant using the ez1 virus mini kit v2. 174 (qiagen®, hilden, germany) following the manufacturer's instructions. 60µl were eluted from 200µl starting material. 5µl of the eluate were tested in rt-pcr using the taqman®technique. a 113-base pair amplicon in the e-gene of sars-cov-2 was amplified and 177 detected, as described with minor modifications (11) . rt-pcr was performed with an abi 7500 fast sequence detector system (pe applied biosystems, weiterstadt, germany). the thermal protocol described was shortened to 40 cycles of 95°c. we used the lightmix®, we used a commercial anti-sars-cov-2 s1 igg and iga elisa (igg cat. no. ei 2606following sensitivity and specificity of the commercial anti-sars-cov-2 s1 igg and iga elisa are indicated: igg sensitivity increases from <10 days (30.3%) after start of symptoms to >21 days after start to ~94%. igg specificity is high with ~99%. iga sensitivity increases 195 from 51.5% <10 days after start of symptoms to 100% >21 days. iga specificity is also high ~88%. statistical analysis was performed using spss 23 and mann-whitney u test. two-sided p values <0.5 were considered statistically significant. table 1 ). 17 out of 18 recovered participants described symptoms, mainly fever (10 out of 18), cough, headache, ague, muscle pain, body ache, dyspnea and fatigue. 2 participants had anosmia and loss of taste. one participant reported testicular discomfort. 237 4 participants with a moderate course of disease were hospitalized due to high fever and dyspnea. none of them needed endotracheal intubation. however, 2 subjects received an antiretroviral therapy with lopinavir/ritonavir for 1 day in one case and for 3 days in the other 240 case. the third subject was given hydroxychloroquine and moxifloxacin. other medication taken by some participants was paracetamol. the control group did not suffer from any symptoms related to covid-19 in the past 8 243 table 1) . 252 sars-cov-2 rna could neither be detected in semen samples from recovered nor from acute infected subjects. the results of the sperm analysis are summarized in table 2 . patients with a moderate 258 infection have a statistical significant impairment of sperm quality (sperm concentration, total number of sperm per ejaculate, total number of progressive motility, total number of complete motility) in comparison with men recovered from a mild infection and the control group. we 261 divided the individuals in fever positive vs. fever negative regardless of their classification into mild and moderate and analyzed the semen accordingly as depicted in table 3 . although there were statistical significant differences regarding the volume, the complete motility and 264 the number of immotile sperms, the values were all still in the normal range (table 3 ). ace2 is the cell entry receptor for sars-cov-2 which is not only found in the respiratory system but also in the testis. this finding led to the hypothesis that the human testis and 270 therewith semen is a target for a sars-cov-2 infection which might increase the understanding of this rapidly spreading disease (12) . furthermore, the investigation of semen samples regarding the presence of sars-cov-2 rna is highly important since it was shown 273 before for several different viruses, that viremic patients can shed viruses into their semen (9). in the investigated semen samples (14) . the fact, that only very low titers of sars-cov-2 have been detected so far in non-respiratory sites like feces and urine specimens (5,15) additionally supports the hypothesis that sars-cov-2 shows only a minor risk of virus 285 shedding into the semen. nevertheless, even a minor risk is not acceptable in the light of treating otherwise healthy couples for infertility reasons. therefore, it is of tremendous importance to investigate particularly non-treated males' semen since many individuals 288 suffering from a mild form of covid-19 might not even have associated their symptoms to an actual infection with sars-cov-2. here, our study differs from the report of song et al. showed though that 6 out of 38 males with a positive nasopharyngeal swap who still had 303 symptoms or stopped having symptoms 2-3 days before semen analysis presented with sars cov-2 in the semen (17) . on another note, it is of interest, that although it was described before in the literature that viral infections have a negative impact on semen parameters like 306 volume, number of spermatozoa and motility we could not detect a negative influence of the sars-cov-2 infection in respect of the aforementioned sperm count parameters in recovered subjects with mild symptoms. however, patients facing a moderate course of disease and 309 being in need of hospital care had a reduced sperm quality (table 2) . on one hand this could be an effect of the infection with sars-cov-2 in association with the severity of the illness or due to a transitory higher viral load. on the other hand, the impaired male fertility in this 312 subgroup could be pre-existing. as these subjects were treated with lopinavir/ritonavir and hydroxychloroquin, an impact of this medication on sperm parameters is possible, however unlikely, since it was only applied for a few days. moreover, there exists no evidence that 315 lopinavir/ritonavir or hydroxychloroquin have an impact on male fertility (18, 19) . additionally it is noteworthy, that in general modifications of the sperm count due to trauma, injury or infection might be seen only after 3 months of time. subsequently, another semen 318 analysis after the aforementioned time would be desirable. our study has certain limitations. first, we investigated a relative small sample size. second, sperm analysis of tested individuals performed before the outbreak of the pandemic was not obtained, limiting the 321 diagnosis of pre-existing male infertility. third, we only analyzed 2 patients with an active covid-19 infection, it will be necessary to ascertain our findings in a lager sample size. finally, our preliminary results lack any data about long-term effects of sars-cov-2 on 324 male reproductive function. in summary, sars-cov-2 does not seem to have a short-term impact on male fertility in 327 patients with mild symptoms regarding sperm characteristics according to the who criteria. we found no evidence of sars-cov-2 shedding in semen of recovered males or patients with an acute covid-19 infection after a recovery time of 32,7 days on average. statistical analysis according to mann-whitney u test for non parametric distribution dedicated by asterisk (* or **; *mild vs. moderate; **moderate vs. control) with p< .05. no statistically significant differences could be detected between mild and control. § : 2 patients with cryptozoospermia were excluded of the analysis according to who statistical analysis according to mann-whitney u test for non parametric distribution dedicated by asterisk (*) with p< .05 considered significant. *** different by trend 468 360 2. who director-general's remarks at the media briefing on 2019-ncov on 11 a pneumonia outbreak associated with a new coronavirus of probable bat origin severe acute respiratory syndrome: historical, epidemiologic, and clinical features first case of 2019 novel coronavirus in the united states structure analysis of the receptor binding of 2019-372 ncov structural, cellular and molecular aspects of immune privilege in the testis zika virus in semen and spermatozoa the breadth of viruses in human semen viruses in the mammalian male genital tract and their effects on 381 the reproductive system detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr scrna-seq profiling of human testes reveals the presence of the ace2 receptor, a target for sars-cov-2 infection in spermatogonia persistence and clinical relevance of zika virus in the male genital 390 tract no evidence of sars-cov-2 in semen of males recovering from covid-19 absence of 2019 novel coronavirus in semen and testes of covid-19 patients in press detectable hiv-1 in semen in individuals with very low blood viral loads clinical characteristics and results of semen 399 tests among men with coronavirus disease antivirals and male reproduction paternal safety of anti-rheumatic medications 333 we would like to thank all individuals for their participation in our study. additionally, we would like to thank stephanie engels and stefanie van den boom for technical assistance. key: cord-311848-8n9ee57a authors: giesen, nicola; sprute, rosanne; rüthrich, maria; khodamoradi, yascha; mellinghoff, sibylle c.; beutel, gernot; lueck, catherina; koldehoff, michael; hentrich, marcus; sandherr, michael; bergwelt-baildon, michael von; wolf, hans-heinrich; hirsch, hans h.; wörmann, bernhard; cornely, oliver a.; köhler, philipp; schalk, enrico; lilienfeld-toal, marie von title: evidence-based management of covid-19 in cancer patients – guideline by the infectious diseases working party (agiho) of the german society for haematology and medical oncology (dgho) date: 2020-09-21 journal: eur j cancer doi: 10.1016/j.ejca.2020.09.009 sha: doc_id: 311848 cord_uid: 8n9ee57a since its first detection in china in late 2019 the novel coronavirus sars-cov-2 and the associated infectious disease covid-19 continue to have a major impact on global health care and clinical practice. cancer patients, in particular those with haematological malignancies, seem to be at an increased risk for a severe course of infection. deliberations to avoid or defer potentially immunosuppressive therapies in these patients need to be balanced against the overarching goal of providing optimal antineoplastic treatment. this poses a unique challenge to treating physicians. this guideline provides evidence-based recommendations regarding prevention, diagnostics and treatment of sars-cov-2 infection and covid-19 as well as strategies towards safe antineoplastic care during the covid-19 pandemic. it was prepared by the infectious diseases working party (agiho) of the german society for haematology and medical oncology (dgho) by critically reviewing the currently available data on sars-cov-2 and covid-19 in cancer patients applying evidence-based medicine criteria. specialists certified in haematology, medical oncology, infectious diseases, critical care, emergency 119 medicine, and virology. 120 after definition of topics and formation of subgroups, a systematic search of medline for 122 publications in english language was performed using one of the following search terms: 123 "coronavirus", "sars-cov-2", or "covid-19". given the current dynamic of research into 124 publications on the pre-print server www.medrxiv.org were also evaluated, however, the lack of 125 formal peer-review in these cases was taken into consideration with regard to grading of quality of 126 evidence. publications were evaluated that appeared online until august 19 th 2020. 127 j o u r n a l p r e -p r o o f guideline process 128 relevant literature was thoroughly reviewed, the data extracted and rated. based on the results of 129 data assessment, preliminary recommendations were first discussed within subgroups and then 130 discussed and revised in a step-by-step process by the specialist panel. strength of recommendation 131 and quality of evidence were graded applying the scale proposed by the european society of clinical 132 microbiology and infectious diseases (escmid) ( table 1) . 23 in short, recommendations were graded 133 as follows: a -agiho strongly supports a recommendation for use, b -agiho moderately supports 134 a recommendation for use, c -agiho marginally supports a recommendation for use, and d -135 agiho supports a recommendation against use. the final recommendations presented in this 136 guideline were discussed and agreed upon by the agiho general assembly in a web meeting on june 137 23 rd 2020 and again on july 9 th 2020. 138 139 cancer patients are generally assumed to be at an increased risk of severe illness by respiratory virus 140 infections when compared to healthy individuals, amongst others as they tend to be older and more 141 frequently suffer from comorbidities than the general population. 24 however, in case of sars-cov-2, 142 both healthy and immunocompromised individuals are immunologically naïve to this infection. data 143 on sars-cov-2 infection rates vary among patients with malignant diseases. 1,13,25-28 144 overrepresentation of cancer patients among hospitalized patient populations may contribute to a 145 higher reported prevalence of sars-cov-2 infections among cancer patients compared to the general 146 population, which is supported by a study showing similar infection rates in hospitalized patients 147 with haematological malignancies and a comparator group of health care workers (hcws). 29 148 in cancer patients, uncontrolled malignancy seems to confer a higher risk of severe or even fatal 149 outcome of 30 with regard to specific cancer types, both haematological malignancies 150 and lung cancer were repeatedly identified as factors for poor prognosis compared to other (solid) 151 cancers. 12, [29] [30] [31] [32] [33] [34] [35] interestingly, myeloid or lymphoid malignancies as underlying disease do not appear 152 j o u r n a l p r e -p r o o f to differ in their impact on covid-19 mortality. 36 among cancer patients, advanced stage 11, 12 and 153 recent antineoplastic therapy within the last 2-4 weeks were reported as risk factors. [37] [38] [39] however, 154 data on the impact of different cancer treatment modalities (immunotherapy, endocrine therapy, 155 targeted therapy, radiotherapy, chemotherapy, or surgery) on the outcome of 20, 39, 40 157 of note, patients with lymphopenia 11,41-43 and granulocytosis 33,44 were reported to be at an increased 158 risk for severe or fatal covid-19. further factors with possible impact on covid-19 course and 159 outcome are listed in table 2 . 160 prevention 161 given the current lack of herd immunity, an effective vaccine, or antiviral prophylaxis, hygiene 163 measures and contact precautions are the cornerstones in preventing sars-cov-2 infection and 164 transmission (table 3) . community-wide face masks and physical distancing measures were effective 165 in several population-based studies and are thus strongly recommended (aii u ). [45] [46] [47] [48] [49] a distance of at 166 least 1.5m (6ft) is usually considered appropriate, however, depending on environmental conditions 167 a wider distance may be considered. 50 168 hand hygiene is crucial for infection control and regular washing of hands with water and soap is 169 strongly recommended for any population (aii t ). 51,52 alcohol-based hand-rubs were shown to be 170 virucidal to sars-cov-2 if applied for at least 30 seconds at a concentration of ethanol or 2-propanol 171 ≥ 30%. 53 we strongly recommend hand disinfection for hcws and cancer patients in health care 172 settings (aii u ). 173 sars-cov-2 can remain viable on surfaces for up to 3 days. 54, 55 we strongly recommend disinfection 174 of frequently touched surfaces such as doorknobs, elevator buttons or hand rails for cancer patients 175 in health care settings (aii r,u ) and moderatly outside of health care settings (bii r,u ) . 54-56 176 j o u r n a l p r e -p r o o f surgical masks covering nose and mouths of an infected person reduce coronavirus rna in expiration 177 air. 57, 58 particulate-filtering facepieces such as the us regulated n95 respirators and the functionally 178 equivalent e.u. regulated ffp2 masks are characterized by a tighter fit and a finer mesh. several 179 randomized trials in hcws provide evidence of the protective effect of surgical masks against 180 respiratory virus infections with a potential additional benefit of ffp2/n95 respirators. [59] [60] [61] if worn to 181 prevent infection, ffp2/n95 masks may be equipped with an exhalation valve for greater comfort, 182 whereas in order to prevent transmission they must not have an exhalation valve. 22 183 we strongly recommend that cancer patients and hcws wear a surgical mask to prevent sars-cov-2 184 transmission and infection (aii t ). 57-60 if caring for covid-19 patients, we strongly recommend that 185 hcws wear ffp2/n95 respirators (aii t ) and personal protective equipment including gloves, gowns 186 and eye protection such as goggles or face shields (aii r ). 22, [61] [62] [63] [64] patients with covid-19 are strongly 187 recommended to wear a surgical mask or ffp2/n95 respirator without exhalation valve (aii t ) taking 188 into account the protective equipment of their surroundings. 22,57,58 189 cancer patients diagnosed with sars-cov-2 infection should undergo either self-quarantine, single 190 room or cohort isolation (aii t,u ). 65 while infectiousness of sars-cov-2 seems to decline significantly 191 within 7-8 days after onset of symptoms, 66,67 prolonged shedding of viral rna for many weeks was 192 observed, especially in immunocompromised patients and in severe we strongly 193 recommend requirement of a negative sars-cov-2 pcr test result prior to discontinuation of 194 isolation (aii t,u ), which should be considered no earlier than 14 days after onset of symptoms and 2 195 days after cessation of symptoms. the possibility of false-negative test results must be kept in mind. 196 a positive test after one negative test was reported in up to 30% of which 197 declined to 5% after three consecutive negative tests. 68, 71 requirement of more than one consecutive 198 negative test prior to discontinuation of isolation should therefore be considered, especially in 199 patients with risk factors for prolonged viral shedding. 200 with regard to participation in activities of daily life of cancer patients not in quarantine/isolation 201 due to suspected or confirmed sars-cov-2 infection, special consideration should be given to 202 j o u r n a l p r e -p r o o f current local epidemiology and requirements of local and national health authorities. as a general 203 recommendation, restriction of activities to places that have adequate hygiene concepts 204 implemented seems to be reasonable as well as a preference of outdoor versus indoor activities, 205 where possible. 206 several large trials could not establish an association between renin-angiotensin-aldosterone-system 208 (raas) blockers and risk of sars-cov-2 infection or severe covid-19 disease. 72-74 discontinuation of 209 raas blockers is therefore not recommended (dii u selenium or vitamin c no conclusive data support supplementation with regard to administration of intravenous immunoglobulin (ivig) may be considered in cancer patients with 215 hypogammaglobulinaemia and covid-19 (bii t,u ). 77 as specific antibodies against sars-cov-2 are most 216 likely absent in current products due to low herd immunity at the moment, ivig will primarily act 217 against possible co-infections with other pathogens. however, with an increase of sars-cov-2 218 infections in populations over the course of the covid-19 pandemic future ivig preparations may 219 contain specific antibodies against sars-cov-2 possibly allowing for a broader use of ivig in covid-220 19 patients than according to present recommendations. 221 prophylaxis with g-csf might help in reducing vulnerability to infections due to shortened 222 neutropenia. however, g-csf has also been associated with a risk of hyperinflammation during 223 neutrophil regeneration and cases of severe covid-19 have been reported after g-csf 224 administration. 44 we therefore do not recommend additional g-csf prophylaxis on top of current 225 recommendations (diii). 78 226 j o u r n a l p r e -p r o o f several preclinical and early clinical trials on vaccine candidates against sars-cov-2 have shown 227 promising results. [79] [80] [81] as cancer patients are usually not included in these trials, it is too early to 228 make any specific deliberations on sars-cov-2 vaccine strategies in these patients. however, based 229 on experiences from other vaccines, depending on the type of vaccine, efficacy and/or safety might 230 be an issue in immunocompromised cancer patients, rendering vaccinations of hcws, caregivers and 231 relatives especially important. 82 232 organizational aspects the prevention of nosocomial sars-cov-2 transmission is of major importance during treatment of 234 cancer patients. this relates to both inpatient and outpatient management. therefore, we strongly 235 recommend the implementation of specific organizational pathways in hospitals and outpatient 236 clinics (aiii , table 4 ). 21, 83, 84 this includes precise scheduling of in-person appointments to reduce 237 waiting times, increasing telemedical approaches including phone or video consultations when 238 clinically possible as well as special routing and zoning for cancer patients. particularly with regard to 239 patient care in outpatient clinics we recommend to reduce the seating capacity in waiting areas and 240 treatment rooms to ensure a distancing of at least 1.5m. in order to reduce the number of visitors, 241 relatives and non-essential other attendants should be advised to stay outside the clinic during the 242 patient visit. in high-volume contact areas such as front desks, installation of transparent shields may 243 offer additional protection for hcws . 244 in order to provide best care for cancer patients with covid-19, icu and respirator capacity should 245 be increased (biii). if possible, dedicated treatment teams should be implemented to ensure 246 continued cancer care in case of infected medical personnel (aiii). 19 early detection of infected staff 247 is crucial. surveillance screening related to local epidemiology should be considered, especially in 248 inpatients, to prevent pre-symptomatic transmission of sars-cov2 (aii t,u ). 85 249 the risk of transmission strongly correlates with the number of consultation and treatment 250 appointments. 21, 29, 86, 87 however, optimal control of the underlying malignancy is considered 251 j o u r n a l p r e -p r o o f favorable as patients with active cancer appear to have an increased risk of severe in 252 order to ensure high-quality cancer care, visits should by no means be avoided or unnecessarily 253 delayed, but reduced if possible without interfering with treatment goals. we strongly recommend 254 considering therapeutic strategies with the fewest and shortest clinic visits adapted to curative or 255 palliative intent taking the patient's individual situation and risk into account (aiii). this might e.g. 256 include substitution of intravenous by oral regimens (e.g. 5-fluorouracil/capecitabine) or 257 hypofractionated radiotherapy. given the immunocompromising effect of many cancer therapies and the fact that most cancer 264 patients belong to high-risk groups regarding adverse outcome of covid-19 it seems reasonable to 265 debate whether it may be the safer course of action to delay or discontinue certain antineoplastic 266 therapies. however, uncontrolled malignancy was identified as an independent risk factor for severe 267 covid-19. 20 we therefore strongly recommend performing antineoplastic therapy to reach the best 268 possible remission (aii u , table 5 ). 269 routine delay or discontinuation of antineoplastic therapy in patients without suspected/confirmed 270 sars-cov-2 infection is not recommended even in times of pandemic (dii u ). 18, 20, 31, 33, 40, [92] [93] [94] in case of 271 suspected sars-cov-2 infection, e.g. due to contact with a confirmed case or a high incidence in the 272 area, we strongly recommend to quarantine the patient and delay antineoplastic therapy for up to 14 273 days, if not detrimental for cancer prognosis (aiii). given the average incubation time of 3-5 days, a 274 delay for a shorter time period and (re-)start of antineoplastic therapy under quarantine conditions 275 may be considered especially in patients with significant prognostic impact of per-protocol 276 j o u r n a l p r e -p r o o f administration of treatment. obviously, these patients should be tested for sars-cov-2 (aiii) but the 277 possibility of false-negative test results should be kept in mind. 278 cytotoxic chemotherapy was reported as a risk factor for severe covid-19 by some, 11, 13, 37, 39 although 279 not consistently across all studies. 20,40 we therefore moderately recommend to consider to 280 delay/discontinue chemotherapy in areas with high sars-cov-2 infection rates in patients with 281 controlled malignancy if no significant detrimental impact on cancer prognosis is to be expected 282 (bii u ). this might be especially relevant in the palliative setting, if the benefit of chemotherapy is 283 marginal, and if other risk factors for severe covid-19 are present. furthermore, dose reductions 284 might be a reasonable strategy in the palliative setting in order to reduce neutropenia. we do not 285 recommend to delay/discontinue radiotherapy, targeted therapy, endocrine therapy or surgery in 286 cancer patients without suspected/confirmed sars-cov-2 infection (dii u ) as no impact on mortality 287 of such prior treatments was seen in several large cohort studies of 20, 31, 40, 94 288 in patients with covid-19, it is strongly recommended to delay/discontinue chemotherapy, if 289 possible, as chemotherapy within two weeks of admission was a major risk factor for severe covid-290 19 in a large chinese cohort study (aii u ). 11 similarly, we strongly recommend delaying surgery in 291 covid-19 patients (aii u ), as perioperative sars-cov-2 infection was associated with a high rate of 292 pulmonary complications and increased mortality. 95 we recommend to delay/discontinue 293 radiotherapy in patients with covid-19 with moderate strength (bii u ) taking into account field size, 294 location and dosage. 11,12,31,40 295 a small cohort study in breast cancer patients with covid-19 reported very favorable outcomes in 296 several patients who did not discontinue their endocrine therapy despite diagnosis of infection. 18 297 given that endocrine therapy is usually not associated with significant immunosuppression, we do 298 not recommend to discontinue endocrine therapy in patients with covid-19 (diii). it is important to 299 note that this does not apply to cdk4/6 inhibitors jointly administered with endocrine therapy which 300 can induce significant neutropenia. 96 301 targeted therapy was reported as a risk factor for severe covid-19 in one study, although patient 302 numbers for this subgroup were small. 11 given that many targeted agents adversely affect immune 303 function we marginally support discontinuation of targeted therapy in covid-19 patients (ciii). in 304 support, a recent small german study on multiple myeloma (mm) patients with covid-19 reported 305 favorable outcomes after discontinuation of various types of targeted anti-mm therapies until 306 resolution of symptoms. 17 however, the heterogeneity of drugs summarized as targeted therapy has 307 to be acknowledged and depending on the available data, substance specific recommendations 308 should be applied. 309 in the following we summarize current knowledge regarding specific cancer treatments. this 311 summary is in no way complete and subject to change as knowledge accumulates. 312 while corticosteroid therapy can be beneficial to treat severe covid-19, 97 long term systemic 313 steroids were identified as a risk factor to develop severe covid-19 in a large registry study of 314 patients with inflammatory bowel disease. 98 we marginally recommend considering to delay, 315 discontinue or reduce treatment with systemic steroids in cancer patients during the covid-19 316 pandemic (cii t,u ). any potential impact of steroid reduction on treatment success needs to be 317 carefully evaluated, most importantly in curative settings. 318 immune checkpoint inhibitors were initially suspected to increase the risk of severe covid-19. 12 319 however, later studies did not find a significant association after adjustment for smoking. 99,100 we 320 therefore do not recommend to delay/discontinue immune checkpoint inhibitors (dii u ). 321 prior treatment with tyrosine kinase inhibitors (tki) was not associated with adverse outcome in a 322 cohort study of lung cancer patients with covid-19, although patient numbers in this subgroup were 323 small and no further details on the type of tki were provided. 100 routine delay/discontinuation of tki 324 in lung cancer patients is thus not recommended (dii u the jak inhibitor ruxolitinib was evaluated in a small randomized controlled trial (rct) against 330 placebo for the treatment of severe covid-19 given its anti-inflammatory properties. while no 331 statistically significant difference in outcome was observed, time to clinical improvement of patients 332 receiving ruxolitinib was numerically shorter. 103 discontinuation of ruxolitinib in patients with covid-333 19 is therefore not recommended (dii t ). 334 further data on the risks of specific antineoplastic drugs with regard to covid-19 is scarce at this 335 time. in a small case series of five patients with cml and covid-19, tki treatment could safely be 336 continued. 104 regarding the impact of rituximab on covid-19, several cases are published reporting 337 outcomes ranging from very mild to fatal. 105,106 b-cell depletion seems to be associated with 338 prolonged shedding of sars-cov-2. 68,105 several case reports on lenalidomide-based therapies 339 describe severe to fatal outcomes of covid-19. 107,108 however, it remains unclear whether this is 340 mainly due to the drug or the underlying malignancy. as hypersensitivity pneumonitis has been 341 reported as a rare side-effect of lenalidomide, an adverse impact on the course of covid-19 is, 342 however, conceivable. 109 in contrast, a recent german study on mm patients on active therapy at 343 time of covid-19 diagnosis including lenalidomide-, proteasome inhibitor-, and daratumumab-based 344 therapies reported favorable outcomes after therapy was discontinued in all patients until resolution 345 of symptoms with no fatalities. 17 346 diagnostics 347 for diagnosis of sars-cov-2 infection, the agiho guideline panel categorized the following clinical 348 situations: a) asymptomatic cancer patients scheduled for antineoplastic treatment in whom delay is 349 likely to increase risk of death, b) asymptomatic cancer patients scheduled for antineoplastic 350 treatment in whom delay is unlikely to increase risk of death, and c) cancer patients presenting with 351 respiratory symptoms compatible with covid-19. with regard to diagnosing sars-cov-2 infection 352 and covid-19 there should be no differences between these groups. a comprehensive approach 353 should be applied to all cancer patients (table 6) . 354 upper respiratory samples obtained by nasopharyngeal or posterior oropharyngeal swab at the time 356 of symptom onset are standard to diagnose acute sars-cov-2 infection. 110,111 sampling bias may be 357 decreased by combining a nasopharyngeal swab and an oropharyngeal swab in one universal 358 transport medium. 112 if a nasopharyngeal swab is contraindicated, expectorated sputum can be used, 359 in particular during thrombocytopenia or if nasopharynx tumors increase bleeding risks. 111 new 360 evidence has become available indicating that morning saliva may be a viable alternative, but data is 361 currently only available preprint. 113 in case of mechanically ventilated patients, lower respiratory 362 samples by tracheal aspirate or bronchoalveolar lavage are standard in the icu population. 111 363 tracheal aspirate is often preferred to limit droplet and aerosol exposure of hcws. generally, it has 364 to be emphasized that diagnostic material should be sampled from the focus of symptoms -that is, 365 samples from the upper respiratory tract for those with symptoms of urtid only and samples from 366 the lower respiratory tract for those with lrtid. 367 currently, antigen assays for diagnosing sars-cov-2 infection are being developed and should only 368 be used in clinical studies. antibody assays should not be used to diagnose active/ongoing sars-cov-369 2 infection, but depending on sensitivity and specificity may be helpful to identify patients with 370 previous sars-cov-2 infection. 114-116 a major caveat is the uncertainty associated with undetectable 371 or low antibody levels in individuals with a-/oligosymptomatic course of sars-cov-2 infection, the 372 level and duration of detectable antibodies in immunocompromised cancer patients, and the 373 protection from re-infection or severe disease. 374 we strongly recommend that all cancer patients prior to antineoplastic therapy receive upper 375 respiratory sampling to diagnose sars-cov-2 infection by pcr (aii u ), taking into account local 376 epidemiology, individual patient risk and potential for nosocomial transmission. in intubated 377 patients, we strongly recommend additional testing of tracheal aspirate (aii u ). if the above 378 techniques are contraindicated in individual patients, testing of saliva or expectorated sputum is 379 recommended with moderate strength (bii a,u ). 380 imaging studies show characteristic findings and are highly sensitive to identify patients with covid-382 19 lrtid in a timely manner. 117-119 they complement molecular testing strategies. chest ct imaging 383 abnormalities can evolve rapidly from focal unilateral to diffuse bilateral ground-glass opacities, even 384 in asymptomatic patients. ground-glass opacities may be accompanied by consolidations which 385 evolve during the course of disease. 119 386 we strongly recommend low-dose chest ct in all cancer patients with suspected covid-19 to 387 diagnose lrtid due to sars-cov-2 (aii u ). 117-119 388 treatment 389 since the treatment of covid-19 is a rapidly changing field, it is strongly recommended to include 390 patients into clinical trials if at all possible (aiii). to evaluate treatment indications and outcomes in 391 covid-19, the who ordinal scale for clinical improvement should be applied (suppl. table 1 ). 120 it 392 summarizes disease severity during the course of covid-19 from uninfected to ambulatory, 393 hospitalized with mild disease or severe disease or dead and allocates scores from 0-8. the who 394 ordinal scale is foundation to most clinical trials on covid-19 and allows to measure endpoints and 395 to facilitate interpretation of results across studies. 396 prophylaxis 397 to date, there is no agent that has shown convincing efficacy as post-exposure prophylaxis. a double-398 blind rct (n=821) compared post-exposure prophylaxis with hydroxychloroquine or placebo. the 399 incidence of either laboratory confirmed infection or illness compatible with covid-19 within 14 days 400 did not differ between the groups. participants receiving hydroxychloroquine experienced a higher 401 rate of adverse events, in particular gastrointestinal or neurological. 121 a retrospective study 402 evaluated umifenovir, an antiviral agent targeting haemagglutinin, as prophylaxis and reported an 403 effect in hcws, albeit with small sample size. 122 other compounds have not been tested so far. 404 therefore, post-exposure prophylaxis with any drug with presumed antiviral activity outside of 405 controlled clinical trials is not recommended (diii, for hydroxychloroquine di, table 7 ). 406 remdesivir 408 a double-blind rct (n=1063) compared intravenous remdesivir with placebo in adults hospitalized 409 with covid-19 and evidence of lrtid. preliminary results were published after the data and safety 410 monitoring board recommended to unblind. remdesivir was superior to placebo in shortening the 411 time to recovery (11 days versus 15 days). this effect was most pronounced in patients requiring 412 oxygen but not mechanical ventilation (corresponding to who scale 5) at presentation. the meier estimates of mortality by 14 days were 7·1% with remdesivir and 11·9% with placebo which 414 was not statistically significant. 123 a second, open-label rct compared remdesivir for 5 days with 415 remdesivir for 10 days. in patients with who scale 3-5 the trial did not show a difference between 416 the short or longer course of remdesivir. however, as the study did not include placebo control the 417 degree of benefit cannot be determined. 124 418 we strongly support a recommendation for use of remdesivir for 10 days in patients with covid-19 419 lrtid (who scale 3-7) to shorten time to recovery in covid-19 (aii t ). 420 hydroxychloroquine 421 an open-label rct (n=150) compared hydroxychloroquine with standard of care in mild to moderate 422 disease. conversion rates to sars-cov-2 negative did not differ between the groups, patients on 423 active drug experienced a higher rate of gastrointestinal adverse events. 125 a cohort study (n=928) 424 found a 3-fold risk of death in cancer patients treated with hydroxychloroquine/azithromycin 425 combination for covid-19. 20 426 we therefore recommend against hydroxychloroquine treatment of covid-19 (dii u the effect of corticosteroids was studied in an open-label rct with hospitalized covid-19 patients 441 receiving standard of care (n=4321) compared to additional low-dose dexamethasone (n=2104). in 442 the entire cohort, application of dexamethasone showed a significant reduction in 28-day mortality 443 (rate ratio 0.83) and a shorter time to hospital discharge (12 days versus 13 days). the impact was 444 most pronounced in patients requiring mechanical ventilation with 28-day mortality reduced by one 445 third compared to a reduction of one fifth in patients only requiring non-invasive oxygen 446 supplementation. in contrast, patients not requiring oxygen had a numerically higher mortality when 447 treated with dexamethasone, however, without reaching statistical significance. 97 448 we strongly recommend dexamethasone in covid-19 patients requiring oxygen or mechanical 449 ventilation (who scale 4-7, ai). in contrast, all asymptomatic patients or those well enough to be on 450 ambient air should not receive low-dose dexamethasone for treatment of covid-19 (di). clinicians 451 need to be aware of potential adverse effects of corticosteroid treatment. 452 tocilizumab, a humanized monoclonal antibody against interleukin-6 showed mixed results in severe 454 we marginally recommend tocilizumab in patients with a severe course of covid-19 likely due to 463 hyperinflammation (who scale ≥ 5, cii u ). further randomized trials are needed to confirm whether 464 tocilizumab is effective and, if so, identify subsets of patients most likely to benefit from the drug. 465 in a prospective cohort study (n=52) covid-19 who scale ≥4 patients received the human 466 interleukin-1 receptor antagonist anakinra and were compared to a historical control (n=44). rates 467 of progression to mechanical ventilation or death were 25% vs 73%. 136 468 we therefore marginally recommend anakinra in covid-19 patients with who scale ≥4 (cii h,t ). 469 baricitinib, an inhibitor of the jak/stat pathway commonly used in rheumatology patients, was 471 evaluated in a retrospective cohort study in patients with covid-19 who scale 3 (n=113). patients 472 treated with baricitinib experienced a significantly lower case fatality rate and rate of icu admission 473 as well as a higher rate of hospital discharge after two weeks. 137 in all hospitalized covid-19 cancer patients (who scale 3-6), we strongly recommend thrombosis 498 prophylaxis with lmwh (aii t ) to prevent thromboembolic complications. despite prophylactic 499 anticoagulation, an increased incidence of thromboembolic disease associated with covid-19 in icu 500 patients was reported. 7,143 in a single-center cohort study, the use of therapeutic anticoagulation in 501 covid-19 patients requiring ventilation (n=234) significantly reduced hospital mortality (29·1% 502 versus 62·7 %) compared to ventilated patients without anticoagulation (n=161). 144 we therefore 503 moderately recommend therapeutic anticoagulation in ventilated covid-19 patients (who scale 6-504 7) with cancer to reduce mortality while carefully weighing the risk-benefit ratio of bleeding (bii t ). 505 health, personal fees from jazz pharmaceuticals, personal fees from msd, personal fees from 538 newconceptoncology, outside the submitted work. cl reports non-financial support from jazz 539 pharmaceuticals, non-financial support from neovii, outside the submitted work. mh reports 540 personal fees from amgen, personal fees from janssen, personal fees from celgene, personal fees 541 from sanofi, personal fees from takeda, outside the submitted work. mbb 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incidence of 335 thrombotic complications in critically ill icu patients with covid-19 dose anticoagulation with in-hospital survival among hospitalized patients with covid-19 extracorporeal life support 340 organization covid-19 interim guidelines patients with covid-19. j med virol. 2020. 77 18. tang lv, hu y. poor clinical outcomes for patients with cancer during the covid-19 78 pandemic. lancet oncol. 2020. 79 all authors actively participated in the guideline panel. ng coordinated the guideline panel and wrote the final version of the manuscript. all authors agreed upon guideline topics, performed a systematic literature search, extracted and rated the data, discussed and agreed upon the final recommendations, helped in writing and critically revised the first draft of the manuscript, and approved the final version of the manuscript. ☐ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☒the authors declare the following financial interests/personal relationships which may be key: cord-311847-2czqs84q authors: pennisi, manuela; lanza, giuseppe; falzone, luca; fisicaro, francesco; ferri, raffaele; bella, rita title: sars-cov-2 and the nervous system: from clinical features to molecular mechanisms date: 2020-07-31 journal: int j mol sci doi: 10.3390/ijms21155475 sha: doc_id: 311847 cord_uid: 2czqs84q increasing evidence suggests that severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) can also invade the central nervous system (cns). however, findings available on its neurological manifestations and their pathogenic mechanisms have not yet been systematically addressed. a literature search on neurological complications reported in patients with covid-19 until june 2020 produced a total of 23 studies. overall, these papers report that patients may exhibit a wide range of neurological manifestations, including encephalopathy, encephalitis, seizures, cerebrovascular events, acute polyneuropathy, headache, hypogeusia, and hyposmia, as well as some non-specific symptoms. whether these features can be an indirect and unspecific consequence of the pulmonary disease or a generalized inflammatory state on the cns remains to be determined; also, they may rather reflect direct sars-cov-2-related neuronal damage. hematogenous versus transsynaptic propagation, the role of the angiotensin ii converting enzyme receptor-2, the spread across the blood-brain barrier, the impact of the hyperimmune response (the so-called “cytokine storm”), and the possibility of virus persistence within some cns resident cells are still debated. the different levels and severity of neurotropism and neurovirulence in patients with covid-19 might be explained by a combination of viral and host factors and by their interaction. coronaviruses (covs) are a group of large enveloped non-segmented positive-sense rna viruses, causing respiratory and enteric diseases in animals and humans [1] . a highly pathogenic cov, named severe acute respiratory syndrome (sars)-cov-2 (formerly known as 2019-ncov), emerged in december 2019 in the hubei region of china, and in the city of wuhan in particular. the initial cases, presenting with a dry cough, sore throat, fever, dyspnea, and bilateral lung infiltrates on chest imaging, were all linked to the wuhan's huanan seafood wholesale market, which trades fish and a variety of live animals, including bats, poultry, marmots, and snakes [2] . this novel cov has caused an outbreak viral antigen and more rapid antibody detection systems (such as the enzyme-linked immunosorbent assay) are currently being developed, although their accuracy is still limited by the relatively high rate of false-negative cases and, therefore, needs to be improved [25] . more recently, other studies are trying to propose droplet digital pcr-based methods as more effective diagnostic strategies for the identification of sars-cov-2 positive patients with low viral load [26] . to date, there is no effective treatment for patients with covid-19. adenosine analogs (e.g., remdesivir, favipiravir, ribavirin, and galidesivir) acting on the rna-dependent polymerase and blocking the viral rna synthesis are promising. chloroquine (cq) and hydroxychloroquine (hcq) can effectively inhibit sars-cov-2 in vitro, but their efficacy in vivo is under evaluation, as well as the effect of serum rich in anti-sars-cov-2 antibodies obtained from convalescent subjects [23] . very recently, a systematic review assessed the efficacy and safety of cq/hcq for treatment or prophylaxis of adult patients with covid-19 [27] . thirty-two studies were included, of which 6 randomized clinical trials (rcts) and 26 non-randomized, with a total of 29, 192 participants. overall, studies suggest that the treatment of hospitalized patients with cq/hcq may not reduce the risk of death compared to standard care. high dose regimens or combination with macrolides may be associated with harm, particularly qtc prolongation and cardiac arrhythmias [28] . post-exposure prophylaxis may not reduce the rate of infection, although the quality of the evidence is low. the authors concluded that patients should be treated with cq/hcq only if monitored and in the context of high-quality rcts [27] . therefore, rationalization of the use of these drugs is also advised [29] . other non-specific immune modulators include human immunoglobulin and corticosteroids, such as dexamethasone, a glucocorticoid that has proved to be the first life-saving drug in these patients. in particular, dexamethasone 6 mg once daily (either per os or by intravenous injection) for 10 days may result in a reduction in mortality by 1/3 in patients on ventilators and by 1/5 in those receiving oxygen [30] . other treatment options include specific monoclonal antibodies that bind the receptor-receptor domain of sars-cov-2 and antibodies that block inflammatory interleukins (il), such as tocilizumab. finally, several vaccines are under analysis and include live attenuated viruses, inactivated viruses, use of recombinant dna, and vaccines based on sars-cov-2 specific proteins and subunits [31] . until these therapeutic options are confirmed, the main measures are prevention, isolation, social distancing, frequent hand washing, and the use of personal protective equipment. some experimental and clinical studies previously performed on other covs and preclinical models seem to converge on the evidence that these viruses may have a tropism into the central nervous system (cns). seven types of covs are currently known that can infect humans and cause neurological damage [23] . in some animal and human covs (including those causing sars and mers), a neuroinvasive potential has been demonstrated [32] . although there are limitations in the epidemiological studies carried on covid-19, as well as limited case records for determining the actual incidence of these complications, some patients reported neurological symptoms, but clinical findings and pathogenic features have not yet systematically addressed. the penetration of several respiratory viruses into the cns has already been shown, a mechanism called "neuroinvasion," affecting both glia and neurons [31] . in some cases and under certain conditions, the neurotropism can cause neurovirulence, which refers to the development of neurological manifestations [33] . in the case of sars-cov-2, the existence of these phenomena is supported by previous evidence showing human cns infection from other respiratory viruses, the cns of other species infected by covs, animal and in vitro models of cns infection by human covs, and the occurrence of neurological complications in the course of other human covs infections. the aims of this review are i) to summarize the available information on the relationship between covs and the nervous system, ii) to identify the potential targets and routes of entry of sars-cov-2 into the nervous system, and iii) to describe the range of the neurological features reported to date in patients with covid-19 and the proposed pathogenic mechanisms. all the common respiratory viruses affecting humans, such as influenza, covs, and respiratory syncytial virus (rsv), can be associated with various neurological manifestations, particularly in subjects experiencing severe pulmonary symptoms [34] . for instance, the effects that rsv may cause include seizures, encephalitis, ataxia, and cerebellitis, and the virus has been detected in the cerebrospinal fluid (csf). influenza is known to cause neurological complications, such as encephalitis, myelitis, meningitis, and guillain-barré syndrome (gbs) [34] . respiratory viruses, including the cov family, affect the cns of other species, such as birds, felines, and livestock [35] . meningitis and spinal cord inflammation have been reported in cats affected by a pathogenic feline cov [36] . a 91% homology resemblance has been assessed between the human oc43 cov and the swine hemagglutinating encephalomyelitis virus (hev), which can invade the porcine brain by retrograde neuronal propagation through the peripheral nerves [37] . a subspecies of murine covs, called mouse hepatitis virus, induces a demyelinating disease resembling multiple sclerosis (ms) [35] . human covs can induce acute or persistent infections in neuronal cell lineages, neuroglia, and oligodendrocytes [38] [39] [40] . moreover, flaccid paralysis and demyelination in animal models can be caused by the human oc43 cov [41] . in particular, it has been shown that the spread of the oc43 in susceptible mice runs from the olfactory bulb to the brainstem and spinal cord, and uses the axonal transport system as the avenue for the neuron-to-neuron spread [33] . further, neuron-to-neuron propagation strategies observed in cell cultures include both passive viral particle diffusion and axonal transport [42] . sars-cov, which may enter the cns through the olfactory bulb and transneuronally spread to other brain regions, can cause neuronal death in the human ace2 receptor transgenic mouse [43] . at least four types of human covs have shown neuroinvasive capacity based on the detection of viral rna or other nucleic acids in the human brain [35] . in a 12-month-old infant with severe immunodeficiency, a case of fatal oc43 cov-related encephalitis was confirmed through rna sequencing techniques and rt-pcr in samples of brain biopsy [44] . immunohistochemical study of the brain showed a microglial reaction, t lymphocyte infiltrates, and presence of the oc43 cov nucleocapsid in neurons. in a 15-year-old adolescent with disseminated acute encephalomyelitis associated with oc43 cov infection, magnetic resonance imaging (mri) disclosed demyelination in the subcortical white matter, cerebellum, and spinal cord [45] . the oc43 cov was detected in the csf and nasopharynx secretions by using the pcr. there has also been a report of gbs associated with 229e and oc43 cov co-infection in a pediatric patient [46] . encephalitis, ischemic stroke, and polyneuropathy can result from sars-cov exposure, with viral rna detectable in the csf [47, 48] . in a necropsy study carried out on 8 victims of sars-cov, infected neurons were found in the cortex and hypothalamus, and genomic sequences of sars-cov were detected in all cases by using the rt-pcr [49] . encephalomyelitis and vasculitis may result also from mers-cov infection. a series of three patients showed that they all suffered from an altered level of consciousness, ranging from confusion to coma, along with ataxia and motor deficit [50] . at brain mri, bilateral lesions were evident in the white matter of the frontal, parietal, and temporal lobes, as well as in the basal ganglia and corpus callosum. two of these patients showed an increased protein level in the csf, while all had lymphocytopenia and severe multiple organ involvement, including kidney, liver, and the cardiovascular system [50] . during the mers-cov infection, other neurological complications were reported: brainstem encephalitis, gbs [51] , and cerebral hemorrhage in the context of thrombocytopenia and disseminated intravascular coagulation [52] . a retrospective study involving 70 mers patients reported that 8.6% had seizures, while four had gbs in a series of 23 cases. the latency of the neurological symptoms ranged from 7 and 26 days after the onset of the pulmonary disease [53] . a pubmed-based literature search was performed to find all relevant reports published until june 2020. the search queries were "covid-19 and nervous system", "brain", "neurology", "neurological", "encephalopathy", "encephalitis", "stroke", "seizures", "neuropathy". the search was also repeated by using the above-mentioned keywords and the term "sars-cov-2" instead of "covid-19". the authors selected all the articles based on the abstract and full-text examination and without a priori appraisal of inclusion/exclusion criteria. indeed, the number of studies reporting neurological complications of covid-19 is still limited and the majority include case reports/case series or retrospective samples, without a systematic specific assessment of the neurological complications. the references of the articles retrieved were also examined in search of more data. after this process, a total of 23 studies were included. figure 1 summarizes the main neurological manifestations of covid-19 and proposed mechanisms. reported: brainstem encephalitis, gbs [51] , and cerebral hemorrhage in the context of thrombocytopenia and disseminated intravascular coagulation [52] . a retrospective study involving 70 mers patients reported that 8.6% had seizures, while four had gbs in a series of 23 cases. the latency of the neurological symptoms ranged from 7 and 26 days after the onset of the pulmonary disease [53] . a pubmed-based literature search was performed to find all relevant reports published until june 2020. the search queries were "covid-19 and nervous system", "brain", "neurology", "neurological", "encephalopathy", "encephalitis", "stroke", "seizures", "neuropathy". the search was also repeated by using the above-mentioned keywords and the term "sars-cov-2" instead of "covid-19". the authors selected all the articles based on the abstract and full-text examination and without a priori appraisal of inclusion/exclusion criteria. indeed, the number of studies reporting neurological complications of covid-19 is still limited and the majority include case reports/case series or retrospective samples, without a systematic specific assessment of the neurological complications. the references of the articles retrieved were also examined in search of more data. after this process, a total of 23 studies were included. figure 1 summarizes the main neurological manifestations of covid-19 and proposed mechanisms. the available research on the neurological involvement in sars-cov-2 makes it hard to causally link a specific neurological manifestation to the viral infection. as a general rule, severe forms of covid-19 are more likely to produce neurological complications when compared to the mild forms (45.5% versus 30%). an autopsy study on deceased patients with covid-19 due to respiratory failure indicated the presence of cerebral edema and neuronal degeneration in these subjects [77] . a study in wuhan (china), reported neurological findings in 214 hospitalized patients with covid-19 [68] . another systematic study in france [60] noted neurological symptoms in 49 of 58 patients, including confusion, encephalopathy, and cortico-spinal tract signs at clinical examination, along with leptomeningeal enhancement and perfusion abnormalities on brain mri. overall, the most common neurological symptoms reported in some patients with covid-19 were headache, anosmia, ageusia, asthenia, and myalgia, followed by encephalopathy, seizures, stroke, and encephalitis [78] . elderly patients and those with previous cognitive decline, multiple comorbidities, other infections, severe medical illness, poor premorbid functional status, malnutrition, and vascular risk factors (especially hypertension) have a higher risk to show an altered level of consciousness related to covid-19 [55, 68, 79] . moreover, metabolic or endocrine derangements, including hypoor hypernatremia, hypo-or hypercalcemia, hypo-or hyperglycemia, renal and/or liver dysfunction, among others, put patients at further risk for encephalopathy. sepsis and the subsequent inflammatory and the so-called "cytokine storm" may also contribute to encephalopathy with il-6, il-8, il-10, and tumor necrosis factor α (tnfα) being implicated in confusional states [80] . finally, patients with previous neurological disorders and acute respiratory symptoms seem to be at increased risk for encephalopathy as the initial symptom of covid-19. coherently, in the study by mao et al. [68] , 15% of patients with a severe form of the disease presented an altered level of consciousness. toxic and metabolic causes, as well as the effects of drugs or hypoxia, may result in covid-19-associated encephalopathy [57] . interestingly, an electroencephalography (eeg) report on a patient with altered mental status who was unable to follow verbal orders as the presenting symptom of covid-19 showed diffuse slow waves, particularly in the left temporal region, whereas pathological findings demonstrated cerebral edema without inflammatory signs [55] . in these cases, treatment is symptomatic and includes fever control, treatment of hypoxia, and antiepileptic medications [77] . a case of covid-19-associated (confirmed by rt-pcr in a nasopharyngeal sample) acute hemorrhagic necrotizing encephalopathy has also been described [71] . brain computed tomography (ct) detected a symmetrical bilateral hypodense area in the medial thalamic nucleus, whereas mri showed contrast-enhanced hemorrhagic lesions, with multifocal and symmetrical disposition, in both thalami, insula, and the mesial region of temporal lobes [71] . although relatively rare, acute necrotizing encephalopathy can be a severe complication of some viral infections, including the influenza virus. the authors postulated that the pathogenesis might be related to the "cytokine storm" induced by covid-19 [81] . a posterior reversible encephalopathy-like syndrome, associated with transient cortical blindness, was also reported [64] . based on the available evidence, sars-cov-2 should be included in the differential diagnosis algorithm of viral encephalitis. typical symptoms are fever, headache, seizures, behavioral disorders, and altered level of consciousness. in these patients, an early diagnosis is of crucial importance to increase the survival rate, especially in those with severe pneumonia and hypoxia [23] . in a report of a 56-year-old woman from wuhan with covid-19, the brain ct remained normal but the diagnosis of encephalitis was confirmed through the isolation of sars-cov-2 in the csf using genomic sequencing techniques [75] . the case of a 24-year-old japanese man presenting with multiple generalized epileptic seizures and decreased level of consciousness led to a diagnosis of meningoencephalitis [69] . brain mri showed hyperintense areas in the right mesial region of the temporal lobe and hippocampus. while the sars-cov-2 rna was not detected in the nasopharynx, it was identified in the csf by using rt-pcr, although it was unclear if some of the patient features were present in the context of seizures due to other causes [69] . anyhow, high levels of proinflammatory cytokines in the csf can cause breakdown and increased permeability of the blood-brain barrier (bbb) which may, in turn, lead to viral invasion and clinical manifestation [71] . seizures have already been reported in cov infections, and there has been a high proportion of breakthrough seizures in patients with epilepsy who developed covid-19 [78] . nevertheless, a recent analysis of 304 patients with covid-19 [67] reported two seizure-like events only, with no confirmed cases of new-onset seizures. however, the study was limited by a lack of instrumental investigation (e.g., eeg, neuroimaging) and by its retrospective approach. a case report of a patient with no history of epilepsy who had multiple apparent tonic-clonic seizures in the context of covid-19 might be interpreted as an unmasked seizure disorder or a direct effect of covid-19 on the brain, although further confirmations are needed [63] . when compared to younger subjects without comorbidities, elderly patients with covid-19 and vascular risk factors appear to be at greater risk for developing cerebrovascular complications [23] . in a retrospective study of 221 patients [66] , 11 (5%) presented with ischemic stroke, one (0.5%) with cerebral venous thrombosis, and one (0.5%) with a cerebral hemorrhage. the risk factors for stroke in this population were: advanced age (mean age: 71.6 years), severe pulmonary disease, hypertension, diabetes, marked inflammatory or procoagulant response (e.g., increased c-reactive protein and d-dimer), and previous cerebrovascular events [66] . other researchers described five patients with stroke (80% ischemic), who had severe forms of covid-19, increased d-dimer, thrombocytopenia, and multiple organ failure [68] . notably, a study in the usa demonstrated that young patients (aged < 50 years) more likely developed large-vessel strokes in the context of covid-19, suggesting that all ages are vulnerable [70] . regarding pathomechanisms, the increased risk of cerebrovascular disease during the covid-19 infection is likely multifactorial. it has been shown that sars-cov-2 can bind to the ace2 receptor on endothelial cells, which might result in increased blood pressure. both ischemic and hemorrhagic strokes can be secondary to the increase in blood pressure, together with the presence of thrombocytopenia and coagulation disorders. the "cytokine storm" may act as another pathogenic mechanism [23] . the levels of c-reactive protein, ferritin, d-dimer, lactate dehydrogenase, and the leukocyte count have often been found to be elevated in these patients [82] . moreover, increased inflammatory markers and hypercoagulability state seem to characterize severe cases, along with a substantially enhanced risk of stroke [68] . the likelihood of ischemic or hemorrhagic stroke may be also increased by some viral-related mechanisms, including vascular endothelial cell infection and consequent vessel damage. on the other hand, it is well known that infection-associated systemic inflammation, thrombosis, or vasculitis increase the risk of stroke [83] . finally, systemic vasculitis and cns vasculitis have been demonstrated at autopsy in patients with sars-cov [84] . most patients with covid-19 complain of headaches. guan et al. [57] found that 13.6% of a series of more than 1000 patients reported headache, and in 15% of those with severe forms. the intensity of headache was generally referred to be mild, although the study did not mention whether a prior history of headache or any meningeal sign was present. in a recent case series [58] , headaches were a predominant complaint, along with fever, cough, sore throat, and breathlessness. the prevalence varies in different reports, but headache may affect up to 1/3 of patients [54, 62] . headache is a well-known clinical feature of meningitis, encephalitis, intracranial hypertension, cerebrovascular diseases, and vasculitis, whereas scarce pathophysiological data link it to covid-19. in some cases, cytokines and chemokines released by macrophages may activate nociceptive sensory neurons [85] , with a neuroinflammatory mechanism similar to that involved in pain [86] . in this scenario, screening patients for secondary causes of headache, including covid-19, is mandatory, especially for patients in whom frequency or severity of headache change, or present with systemic symptoms, or do not respond to first-line or habitual treatments. anosmia and secondarily taste disorders are commonly reported in patients with covid-19 and may appear suddenly [56] . in italy, 19.4% of patients had some form of chemosensory dysfunction [74] , whereas in a case register of twelve european hospitals, the prevalence of olfactory and gustatory dysfunction in 417 patients with covid-19 with mild-to-moderate symptoms [65] was 85.6% and 88%, respectively. notably, 12% of them declared the olfactory dysfunction as the initial symptom and 18% had no runny nose or nasal obstruction [65] . indeed, although anosmia is noted in many other respiratory infections, such as cold and influenza, in covid-19 it is typically not accompanied by nasal swelling or rhinitis [65, 74, 87] . given the reports of anosmia presenting as an early symptom of covid-19, dedicated testing may offer the potential for early detection of sars-cov-2 infection. nevertheless, the chemosensory deficit in covid19 has not yet been systematically investigated, although it is a current research "hot topic", both at the clinical-epidemiological and cellular level. initial observations and early studies suggested as possible mechanisms for anosmia in covid-19 the cleft syndrome, nasal obstruction and rhinorrhea, "cytokine storm", direct damage to olfactory receptor neurons (orns), and impairment of the brain olfactory centers. the most obvious cause would be a direct damage to orns, since other human covs (e.g., oc43) have shown to directly bind to orns. however, although anosmia is linked with human viruses causing the common cold, such as influenza and other covs (respiratory or not), the exact mechanism has not yet been clearly established. a new model of olfactory dysfunction in covid-19 has been recently drawn from the observation that sustentacular cells (suss) are the primary target of the virus and that suss infection triggers a cascade of events leading to anosmia [88] . suss express ace2 and would be infected first. impairment of sus would negatively affect orns, leading to the inhibition of the odor perception. simultaneously, rapid immune response would be induced in a subset of orns and microvillar cells, which would lead to activation of lymphocytes and macrophages and their infiltration into the olfactory epithelium, as well as secretion of proinflammatory cytokines. stem cell infection may potentially explain why a small fraction of patients with covid-19 experience long-term dysosmia [88] . of note, such a model does not imply that sars-cov-2 travels from the olfactory epithelium to the brain along the olfactory axons. indeed, no axonal transport of sars-cov-2 to the brain has been demonstrated in the hamster model during the first two weeks after infection [89] , and no viral accumulation or persistence has been reported in cerebral olfactory regions of autopsy material from patients with covid-19 [90] . on the other hand, rapid sars-cov-2 accumulation in the brain after intranasal injection was recently shown using the new humanized ace2 knock-in mouse [91] . yet, this is not synonymous with transport along olfactory axons, as other routes are also possible. if sars-cov-2 travels within the olfactory axons, this would require an ace2-independent passage of the virus from suss to orns within the olfactory epithelium. in addition, it would be relevant to examine progenitor or stem cell infection, as these olfactory epithelium cells also express significant levels of ace2. probably, also host genetic factors play a role in individual susceptibility to anosmia in covid-19 and the characterization of these factors is of particular interest [92] . regarding gustatory dysfunction in covid-19, it is known that the ability to separate flavors depends on the retronasal stimulation pathway. therefore, in patients with ageusia, retronasal olfactory dysfunction is commonly suggested, although some studies reported high ace2 expression on the oral cavity mucosa and epithelial cells of the tongue [93] . another possibility is that sars-cov-2 may have a direct effect on the taste buds or receptors [94] . gbs (i.e., acute inflammatory demyelinating polyneuropathy) can follow a gastrointestinal or respiratory infection. a molecular mimicry mechanism, in which infecting viruses likely share epitopes similar to some peripheral nerve components, is believed to occur and to stimulate autoreactive t or b lymphocytes. antibodies against the virus cross-react and bind to peripheral nerve components, thus causing neuronal dysfunction and clinical manifestations. after sars-and mers-cov infections, both gbs and acute motor axonal neuropathy have been described [32, 51] . reports of gbs in patients with covid-19 are emerging. a case series [73] reported five cases of gbs in italy, and four of them presented with lower-extremity weakness and paresthesia. patients developed symptoms five to 10 days after the onset of the viral infection. at electromyography, two patients had gbs and three acute motor axonal neuropathy [73] . another patient in iran [72] and an italian patient with the miller-fisher gbs variant [59] were also reported. in a 62-year-old patient presenting with motor weakness of the lower extremities and covid-19 symptoms, gbs associated with sars-cov-2 infection was observed a week later [76] . an increase in proteins (124 mg/dl) but not in cells was found in the csf, while at neurophysiological examination increased distal latencies and f-waves absence was detected, suggesting a severe and diffuse peripheral nerve demyelination. although the authors reported that the patient was infected by sars-cov-2 at the onset of gbs symptoms (because the patient had lymphopenia and thrombocytopenia), it cannot be excluded that covid-19 and gbs presented coincidentally [76] , and therefore further evidence is needed. some of the most frequently described non-specific symptoms are myalgia, unsteadiness, and fatigue. of note, 36.4% of 214 patients with covid-19 admitted in a wuhan hospital exhibited neurological manifestations [68] , which were categorized as "cns involvement" (24.8%), "peripheral involvement" (10.7%), and "muscular-skeletal involvement" (10.7%). among the latter, 15% of the non-severe patients reported myalgia, while 13.7% had elevated levels of creatine kinase. two cases of rhabdomyolysis (0.2%) were also described [61] . apart from a general unspecific reaction to the awareness of infection, some psychiatric illnesses might directly result from exposure to human covs. in patients with psychotic symptoms compared to non-psychiatric controls, a higher prevalence of immune reactivity for hku1 and nl63 covs was found [95] , suggesting that viral exposure may represent a comorbid risk factor in neuropsychiatric disease. however, the role that sars-cov-2 may play in the etiopathogenesis of psychiatric diseases needs to be explored. sars-cov entry into the human host cell seems to be mediated primarily by cellular receptors ace2, which are expressed in the lung, kidney, vascular endothelia, small intestine, and human airway epithelia [96] [97] [98] . conversely, mers-cov may enter human host cells primarily through the dipeptidyl peptidase-4 (dpp4) protein located in the membrane of cells of the immune system, liver, small intestine, and lower respiratory tract [99, 100] . however, ace2 or dpp4 alone are not enough to make the host cell susceptible to infections. this is particularly true when considering that sars or mers infections were also reported in the cns, where ace2 or ddp4 expression level is low under normal conditions [101] . the exact route through which sars and mers covs enter the cns is still not clear, although the glymphatic or a pure hematogenous path seems to be unlikely, particularly in the initial infection stage, during which no virus particle is detected in the brain [49, 102, 103] . however, some evidence indicates that covs might initially invade peripheral nerve terminals, and later the cns through a synapse-connected route [104] [105] [106] [107] . coherently, the sars infection seems to be able to cause significant neuronal damage without substantial inflammatory infiltration [43] . earlier studies have shown the presence of viral particles in the brain of patients with sars, located exclusively in the neurons [102, 103] . in vivo experiments using transgenic mice showed that, when sars or mers covs are given intranasally, they can enter the brain via the olfactory nerve, and quickly spread to specific brain regions, such as the brainstem and the thalamus [43, 108] . in these cases, the brain expresses ace2 receptors, that have been detected in neurons and glial cells, making them a potential target for covid-19. another important observation is that in mice infected with mers-cov with low inoculum doses, virus particles are not detected in the lung but only in the brain, which indicates that the cns infection is relevant for the high mortality of the disease [108] . however, although murine models develop cns infection, mers-cov has never been detected in the human cns, thus suggesting a different disease model [109] . viruses are present in the brain of patients with sars-covs [49] . baig et al. [24] have recently suggested a putative transcribral sars-cov-2 route to the brain and the presence of its rna in the csf would be the conclusive evidence to support the covid-19 neurovirulence. however, the pathomechanisms underlying the cns invasion seem to be more complex. a cns invasion can occur in both the initial and late phases of sars-cov-2 infection [24] . however, research is yet to determine the exact route for the entrance of the virus into the brain. a direct entry along the olfactory nerve can be considered among the potential mechanisms. in particular, the nasal olfactory epithelium is the probable site of enhanced binding of sars-cov-2. multiple non-neuronal cell types within the olfactory epithelium express two host receptors, ace2 and transmembrane protease serine 2 (tmprss2), that facilitate sars-cov-2 binding, replication, and accumulation. moreover, a subsequent brain infection beginning from the olfactory neurons might be considered, as well as the possibility that orns may initiate a rapid immune response at the early stages of the disease [110] . using a mouse model, bilinska et al. [111] determined whether cells in the olfactory epithelium expressed the receptors allowing the entry of the sars-cov-2 virus. they showed that ace2 and tmprss2 were expressed in the suss of the olfactory epithelium but not, or much less, in most olfactory receptor neurons, suggesting that suss are involved in sars-cov-2 virus entry and smell impairment. moreover, the expression of the entry proteins increased in older animals, thus possibly explaining, if verified also in humans, why older individuals are more susceptible to sars-cov-2 infection [111] . translationally, these preliminary findings suggest that damage to the olfactory epithelium may not only underlie clinical anosmia but also represent a preferential gate to the brain. namely, sars-cov-2 might spread via the transcribral route from the olfactory epithelium along the olfactory nerve to the olfactory bulb within the cns or spread retrogradely via transsynaptic transfer using an endocytosis or exocytosis mechanism and a fast axonal transport mechanism of vesicle transport moving the virus along microtubules back to neuronal cell bodies [78] . additionally, another possible transsynaptic route from the nasal respiratory epithelium to the brain via the trigeminal nerve branch has recently been hypothesized, although replication of the findings is needed [112] . the biological plausibility of the retrograde transsynaptic pathway from the peripheral nerve endings is based on the evidence that some covs appear to be capable of penetrating the cns through the cribriform plate of the ethmoid bone, even if the olfactory bulb is efficient enough to control viral invasion [113] . according to li et al. [113] , mechanoreceptors and chemoreceptors in the lung and respiratory tract can act as a possible retrograde pathway for sars-cov-2, as the nucleus of the solitary tract receives sensory information from these anatomical structures. indeed, a dysfunction of the cardiac-respiratory control centers in the medulla oblongata would aggravate the symptoms till death [113] . however, the neurogenic hypothesis of respiratory failure is not supported by other researchers [114] , as they argue that patients with covid-19 pneumonia do develop hypoxia and low co 2 levels accompanied by increased respiratory rate. while these patients can breathe spontaneously, they do it with great effort; thus, a respiratory failure resulting from a neurological origin would be characterized by a reduced respiratory rate, low oxygen levels, and high co 2 levels [114] . further virologic, histopathological, and immunohistochemical studies are necessary to demonstrate a specific neurotropism of sars-cov-2 for the brain respiratory control centers. the hypotheses behind sars-cov-2 transsynaptic propagation are further corroborated by other studies demonstrating that the virus may use a transsynaptic route for infecting the cns. one of the first pieces of evidence was provided in 1986 by gosztonyi [115] , who described the axonal transport of viral nucleic acid of some neurotropic viruses. in particular, he described that the rabies virus (rv) could be transmitted to other neurons by transsynaptic passage, without involving the complete virus replication, thus reaching various brain areas. li et al. [105] demonstrated that hev was able to propagate into cns via transsynaptic routes. namely, the peripheral inoculation of hev in both piglets and rodents results in encephalomyelitis via the primary motor cortex, where membranous-coating-mediated endo-/exocytosis events favor the hev transsynaptic transfer. in a recent review, taylor and enquist [116] deeply described the axonal route of propagation of several neuroinvasive viruses, including herpes simplex, varicella-zoster, pseudorabies, rhabdoviridae (including rv), flaviviridae, vesicular stomatitis (vsv), and theiler's murine encephalitis virus (belonging to the picornaviridae). they also described the mechanisms by which viruses were able to move in and out axons, both anterogradely or retrogradely, thanks to coupled or separate transports (mediated by vesicles or not, respectively) [116] . the anterograde and retrograde transsynaptic propagation lead researchers to adopt this viral feature for mapping the axon transports of neuronal impulses, viruses, and other factors. in this context, the transsynaptic transport of vsv has been used for tracing and mapping neuronal circuits by using specific virus-labeling techniques [117, 118] . of note, transsynaptic propagation is not a prerogative of cns viruses. for instance, the measles virus (mv) can reach the cns and cause subacute sclerosing panencephalitis, which is often fatal. the cns complications of mv infection are known to be the results of transsynaptic viral propagation thanks to the binding between mv envelope f protein and several host proteins, including hemagglutinin and neurokinin-1 [119] . taken together, these observations support the concept that some viruses, including respiratory viruses like the sars-cov-2, may propagate through the cns via a transsynaptic transport. the cell invasion of sars-cov-2 and its rapid replication seem to be supported by the ace2 receptor [120] . the damaging effects of angiotensin ii may be enhanced because of the depletion of the ace2 receptor on the cell membrane, which leads to an acute deterioration in lung function. therefore, the down-regulation of the ace2 receptor could put the hypertensive and diabetic population at higher risk for covid-19 due to the increase in angiotensin ii. a hypothesis related to this issue is that ace inhibitors, when used in patients with covid-19, can lead to an increased expression of ace2, thus probably making the cells more vulnerable to sars-cov-2 infection [120] . a study examining the risk factors for mortality in patients with covid-19 found that 40% of the deceased people presented single or multiple comorbidities, with high blood pressure being the most common (30%) [121] . the neurovirulence of sars-cov-2 could be related to the degree of expression of the ace receptor in the cns, although this receptor is expressed in endothelial cells, so it is necessary to further investigate its role in the etiopathogenesis of some neurological complications, such as stroke [23] . the viral s protein might allow the virus interaction in brain microcirculation with ace2 receptors expressed in the capillary endothelium, possibly leading to endothelial cells infection and subsequent spreading to the neurons after that the endothelial damage has occurred [24] . damage of the epithelial barrier by covs may occur, thus allowing the virus to reach the bloodstream or the lymphatic system and to spread to other tissues, including the brain. in this scenario, however, it is important to distinguish between the nasal olfactory epithelium and the nasal respiratory epithelium. while the former has been indicated as the main route for the trans-synaptic propagation of covs, the latter seems to be involved in the hematogenous propagation [78] . nevertheless, it is not well clearly understood how this could take place, although the bbb seems to be involved. two hypotheses have been proposed for the crossing of the bbb by sars-cov-2. the first mechanisms would involve the infection and transport across vascular endothelial cells, which express ace2 and, as such, are at risk for sars-cov-2 infection [78] . sars-cov-2 particles have been found in capillary endothelia and neurons of a frontal lobe specimen from an autopsy case study [122] . in particular, viral particles were packaged in intraneuronal dilated vesicles, and endocytosis or exocytosis of viral particles across endothelial cells were detected by electron microscopic imaging [78] . as soon as the virus enters vascular and neuronal cells, it might interact with ace2 on neurons, glia, and vessels, and then begin a cycle of viral budding, thus further damaging both vascular and neuronal tissue [24] . the second hypothesis is based on the so-called "trojan horse mechanism," through the infection of leukocytes that pass the bbb [35] . as lymphocytes, granulocytes, and monocytes all express ace2, the sars-cov might able to infect them [49, [123] [124] [125] , and it is likely that sars-cov-2 too may act in the same manner. moreover, the covid-19-related systemic inflammation would increase the bbb permeability, thus facilitating the invasion of the cns by the infected immune cells [126] . covid-19-related hypoxia may be responsible for indirect neuronal damage as it induces anaerobic metabolism in the cns cells, ischemia, interstitial edema, and vasodilatation in the cerebral circulation, which eventually causes stroke, syncope, and anoxic crisis [127] . the fact that covs can infect macrophages, astroglia, and microglia makes it possible for the host's immune-mediated response to playing a role. in some patients who died because of covid-19, a multiple organ failure and a hyperinflammatory syndrome (the "cytokine storm") were hypothesized as possible underlying causes [81] . in this context, a previous study in mice showed t-lymphocyte infiltration into the cns and significantly increased levels of the proinflammatory cytokine il-6 and the chemokine monocyte chemoattractant protein-1 after cov exposure [128] . finally, in genetically predisposed individuals, the persistence of covs in some cns resident cells cannot be excluded, where they would act as a cofactor of clinical exacerbations. serological techniques have identified covs in various neurological diseases, such as parkinson's disease, ms, and optic neuritis [129] [130] [131] [132] . therefore, it was proposed that a persistent cov infection might be a pathogenic factor in the development and course of some neurological diseases. for instance, infectious agents may play a triggering role in ms, with viruses being the most likely culprit in genetically predisposed individuals [133] . taken together, all the mechanisms discussed here might, at least in part, explain why and how sars-cov-2 could be moved in or within the cns despite the low expression of ace2 receptor in the brain. further studies are needed to better understand these pivotal aspects of neuroinfection. the research on the neurological manifestations of covid-19 has recently made significant progress, although the exact neuropathogenic mechanisms of sars-cov-2 are not yet completely clear. essential questions are emerging with the identification of people with covid-19 and cns involvement. although these patients may exhibit a wide range of neurological complications, it remains to be determined whether these can be an indirect and unspecific consequence of the pulmonary disease, hypoxia, or generalized inflammatory state on the cns, or if they may rather reflect a direct viral-related neuronal damage. some symptoms, such as headache and unsteadiness, are non-specific manifestations of several viral infections but, in some cases, they might accompany more severe diseases, such as meningitis, encephalitis, and stroke. a hematogenous versus a transsynaptic propagation is still debated, as well as the role of the ace2 receptor, the impact of hyperimmune response, and the viral persistence within some cns cells. the different levels and severity of human neurotropism and neurovirulence in patients with covid-19 might be explained by a combination of viral and host factors and their interaction. although researchers are yet to elucidate the real degree of neurovirulence of sars-cov-2, there is a demonstration of its presence in the csf or tissue samples at autopsy. however, in the current epidemic, some difficulties may occur in performing mri or a lumbar puncture, especially in severely affected patients and in those admitted in intensive care units. nevertheless, it remains of pivotal importance for all patients with altered consciousness or any unexplained neurological manifestation to receive an accurate neurological exam and appropriate instrumental investigations (i.e., neuroimaging, eeg, evoked potentials, csf), when necessary [134] . another warning related to this topic is that lymphopenia in immunosuppressed patients with covid-19 can be a serious risk factor; these are not only patients with cancer or with systemic autoimmune diseases but also patients with neurological disorders. indeed, taking high-dose corticosteroids or immunosuppressive/biological treatments is relevant for diseases like cerebral vasculitis, neuromyelitis optica, neurosarcoidosis, polymyositis, myasthenia gravis, or ms, and the scientific community should rapidly develop ad hoc guidelines for the management (especially in terms of reevaluation of dosages and treatment cycles) of these diseases during the covid-19 era. another relevant consideration concerns the possibility of developing anti-covid-19 drugs to cross the bbb and to selectively target the sars-cov-2 inside the brain. the role of the bbb needs to be further explored in patients with covid-19. hence, the possible neuroinvasion may be a significant mechanism to take into account for treating and preventing covid-19. in this context, the design of safe and effective brain penetrating drugs would be very helpful in preventing and possibly treating the neurological complications of covid-19, although the studies on this "cutting-edge topic" are still at their beginning [135] . the differences in the sequence of spike proteins between sars-cov and sars-cov-2 will enable scientists to identify epitopes in covid-19 patients for the development of monoclonal antibodies against this virus. basic research studies on sars-cov-2 and host interactions are the key to several unanswered questions in the prevention and control of the disease, including the challenging question of why not all patients with covid-19 show neuroinvasion and why, among those experiencing neuroinvasion, not all show neurotropism or neurovirulence [136] . moreover, the difference in terms of neurological involvement and pathomechanisms between the current pandemic and the sars and mers infections needs to be further studied. lastly, longitudinal neurological assessments of patients after their recovery will be crucial in the 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involvement after infection with covid-19 and other coronaviruses mutations in the spike glycoprotein of human coronavirus oc43 modulate disease in balb/c mice from encephalitis to flaccid paralysis and demyelination cerebrospinal fluid antibodies to coronavirus in patients with parkinson's disease two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients antibodies to coronaviruses oc43 and 229e in multiple sclerosis patients coronaviruses in spinal fluid of patients with acute monosymptomatic optic neuritis epidemiologic evidence for multiple sclerosis as an infection height, and sex on motor evoked potentials: translational data from a large italian cohort in a clinical environment cns penetration of potential anti-covid-19 drugs the emergence of a novel coronavirus (sars-cov-2) disease and their neuroinvasive propensity may affect in covid-19 patients key: cord-309418-dx6e0lri authors: segalés, joaquim; puig, mariona; rodon, jordi; avila-nieto, carlos; carrillo, jorge; cantero, guillermo; terrón, maria teresa; cruz, sílvia; parera, mariona; noguera-julián, marc; izquierdo-useros, nuria; guallar, víctor; vidal, enric; valencia, alfonso; blanco, ignacio; blanco, julià; clotet, bonaventura; vergara-alert, júlia title: detection of sars-cov-2 in a cat owned by a covid-19−affected patient in spain date: 2020-10-06 journal: proc natl acad sci u s a doi: 10.1073/pnas.2010817117 sha: doc_id: 309418 cord_uid: dx6e0lri severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the etiological agent of covid-19, is considered a zoonotic pathogen mainly transmitted human to human. few reports indicate that pets may be exposed to the virus. the present report describes a cat suffering from severe respiratory distress and thrombocytopenia living with a family with several members affected by covid-19. clinical signs of the cat prompted humanitarian euthanasia and a detailed postmortem investigation to assess whether a covid-19−like disease was causing the condition. necropsy results showed the animal suffered from feline hypertrophic cardiomyopathy and severe pulmonary edema and thrombosis. sars-cov-2 rna was only detected in nasal swab, nasal turbinates, and mesenteric lymph node, but no evidence of histopathological lesions compatible with a viral infection were detected. the cat seroconverted against sars-cov-2, further evidencing a productive infection in this animal. we conclude that the animal had a subclinical sars-cov-2 infection concomitant to an unrelated cardiomyopathy that led to euthanasia. edited by tak w. mak, university of toronto, toronto, on, canada, and approved august 22, 2020 (received for review may 27, 2020) severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the etiological agent of covid-19, is considered a zoonotic pathogen mainly transmitted human to human. few reports indicate that pets may be exposed to the virus. the present report describes a cat suffering from severe respiratory distress and thrombocytopenia living with a family with several members affected by . clinical signs of the cat prompted humanitarian euthanasia and a detailed postmortem investigation to assess whether a covid-19−like disease was causing the condition. necropsy results showed the animal suffered from feline hypertrophic cardiomyopathy and severe pulmonary edema and thrombosis. sars-cov-2 rna was only detected in nasal swab, nasal turbinates, and mesenteric lymph node, but no evidence of histopathological lesions compatible with a viral infection were detected. the cat seroconverted against sars-cov-2, further evidencing a productive infection in this animal. we conclude that the animal had a subclinical sars-cov-2 infection concomitant to an unrelated cardiomyopathy that led to euthanasia. sars-cov-2 | covid-19 | cat | transmission t he severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the etiological agent of covid-19, declared as pandemic on march 11, 2020 . since its initial report at the end of 2019, the world health organization has reported more than 17.3 million human cases of covid-19 causing over 674,000 deaths (accessed on august 1, 2020, https://covid19.who. int/). sars-cov-2 is believed to have originated in bats (1). however, the species barrier jump from bats to humans is considered unlikely, and the most probable hypothesis includes the existence of an intermediate host (2) . such a scenario points out the importance of animals in the emergence of covid-19 in china, which further emphasizes the need of a one health approach to tackle emerging diseases. the role of animals in the context of covid-19 is not only circumscribed to its origin and spillover events, but also at other key levels. first is the need to develop animal infection models for sars-cov-2 to accelerate the preclinical phase for developing vaccines and antiviral drugs against this novel agent. several models for sars-cov-2 infection have been so far developed in animals, including egyptian fruit bat, ferret, golden syrian hamster, cat, humanized angiotensin-converting enzyme 2 (ace2) transgenic mice (hace2 mice), and some nonhuman primate species (3) (4) (5) (6) (7) (8) . second, livestock and zoo animals as well as pets can be exposed to sars-cov-2 by contacts with covid-19 patients or sars-cov-2 subclinically infected humans, resulting, eventually, in reverse zoonosis (human to animal transmission of a pathogen originated in animals). such reverse zoonosis has been reported so far in at least four dogs (two in hong kong and two in the united states) (9), six cats (two in the united states, and one each in belgium, hong kong, france, and germany), farmed minks (in at least four farms in the netherlands) (10) , and eight big felines in the bronx zoo in new york (five tigers and three african lions). considering the number of infected people all over the world and the very few cases reported in different animal species, it is thought that animals play no or a negligible role in the epidemiology of sars-cov-2. just very recently, there has been speculation on the possibility of a mink farm worker being exposed from an infected animal (10) , which might be the first potential documented zoonosis event within the pandemic. the present case report aims to describe evidence in spain of sars-cov-2 infection in a 4-y-old domestic cat (european × persian crossbreed). the owner of the animal died from covid-19 (rt-pcr confirmed), and the animal was then taken by some relatives who were also diagnosed with a mild-to-moderate form of the disease, although sars-cov-2 infection was not confirmed at the laboratory. the animal (c1) had another cat mate (c2) at home that never showed any clinical sign. a relative of the deceased owner referred the cat (c1) to a veterinary hospital due to severe dyspnoea. the clinical examination of the pet was performed with personal protective significance covid-19 is the most devastating pandemic in recent history. as with many emerging infectious diseases, it is of zoonotic origin, meaning that animals played a major role in the initial transmission events. despite sars-cov-2 being highly adapted to jump from human to human, several animal species are naturally susceptible to sars-cov-2, including pets such as cats. in the present report, a cat from a family with several relatives affected by covid-19 developed severe respiratory clinical signs, leading to humanitarian euthanasia. due to the suspicion of a potential covid-19 infection in the cat, different antemortem and postmortem tests were assayed. the clinical condition was finally attributed to a feline hypertrophic cardiomyopathy, but the animal was also infected by sars-cov-2. 1 to whom correspondence may be addressed. email: joaquim.segales@irta.cat. first published september 18, 2020. equipment (ppe) consisting of laboratory coat, face shield, mask, and a double pair of gloves. the animal was normothermic, and blood analyses revealed mild anemia and severe thrombocytopenia. radiographically, a moderate bronchointerstitial pattern was observed bilaterally, but with more intensity in craneoventral areas and right side of the lung. the probnp (brain natriuretic peptide) test (idexx snap feline probnp test) resulted positive; this test measures the b-type natriuretic peptide in blood, which is increased in response to excessive stretching of heart muscle cells. in consequence, cardiac insufficiency was suspected, and feline hypertrophic cardiomyopathy was subsequently diagnosed after echocardiography. tests to detect antigen against feline immunodeficiency virus and feline leukemia virus resulted negative. the animal was kept at the veterinary hospital overnight, but the course worsened during the following afternoon, with lateral decubitus, shortness of breath, agonic violent respiratory efforts, and apnoea. the current owner, following the advice of the veterinary clinician, decided to humanely euthanize the cat. due to the nature of the case, the necropsy was performed at the biosafety level 3 facilities at the centre de recerca en sanitat animal (institut de recerca i tecnologia agroalimentàries [irta-cresa]). for such necropsy, the ppe used consisted of double overall, triple glove pairs, ffp3 mask, and a beltmounted powered air-purifying respirator. externally, the animal had abundant fat resources, remnants of blood in the nostrils and mouth, and evidence of tearing at the medial eye edges. at necropsy, the most significant gross findings included moderate to severe hypertrophy of the left ventricular and interventricular wall with no cardiomegaly; lack of pulmonary collapse with marked redness of lung lobes; 15 ml of thoracic serosanguineous fluid; liver paleness; presence of blood in mouth, trachea, stomach, and intestines; and marked redness of the nasal turbinates. samples from lung, trachea, turbinates, lymph nodes (mediastinal, submandibular, and mesenteric), tonsil, spleen, bone marrow, heart (ventricles and atria), skeletal muscle, pancreas, stomach, small and large intestines, adrenal gland, parotid gland, brain, and skin were taken and fixed by immersion in 10% buffered formalin for subsequent histopathological and immunohistochemistry studies. a second set of similar samples were taken within dulbecco's modified eagle's medium to perform sars-cov-2 real-time rt-pcr (11) . in addition, nasal swabs, rectal swabs, and lung swabs were taken for virological analyses. the most significant histological features consisted of severe pulmonary edema, congestion and hemorrhages, pulmonary thrombosis of capillaries and small/medium-sized blood vessels, nasal turbinate hemorrhages, interstitial fibrosis of left ventricular and interventricular walls, mild hepatic lipidosis, splenic hematopoiesis, mild membranoproliferative glomerulonephritis, and focal nodular adrenal hyperplasia. in addition, blood, plasma, and rectal and nasal swabs from c2 were obtained, and plasma from c1 taken by the veterinarian the day before the euthanasia was also available. rt-qpcr and/ or antibody detection was also performed in these extra samples. results of rt-qpcr to detect three sars-cov-2 genes are shown in table 1 . viral genome was found in nasal swabs and turbinates as well as mesenteric lymph node of c1, although with high ct values. the rest of the analyzed tissue results and c2 swabs were negative for the rt-qpcr methods. immunohistochemistry to detect sars-cov-2 antigen in formalin-fixed, paraffin-embedded tissues also gave negative results. the serum from both c1 and c2 yielded positive elisa titers of ≥1:4,000 for spike, s2, and receptor binding domain (rbd), but was negative for n protein (fig. 1) . also, both animals had sars-cov-2 neutralizing antibodies (reciprocal dilution of the half maximal inhibitory concentration, ic 50, of 191 and 205 for c1 and c2, respectively). viral genome could be partially obtained from the lymph node sample of c1 (global initiative on sharing avian influenza data, gisaid acc. epi_isl_482820) showing high identity (99.997%) with the genomic sequence obtained from the initial animal owner (gisaid acc. epi_isl_483059). considering the clinical signs and both gross and microscopic lesions, it was concluded that the cat developed cardiorespiratory failure due to hypertrophic cardiomyopathy and secondary thromboembolism. the severity of the condition during the visit and the poor prognosis prompted the suggestion to euthanize the animal. the detection of sars-cov-2 rna in several samples of c1, all of them with ct values over 30 (low viral load), and presence of antibodies (neutralizing and nonneutralizing) in both c1 and c2, indicated both animals suffered from a productive viral infection, probably linked to the exposure of the cats to covid-19−affected owners. experimental studies have demonstrated that cats are susceptible to sars-cov-2 infection and able to transmit it to direct contact mates (4, 7). those authors did not find clinical signs in any of the inoculated cats. however, a certain degree of interstitial pneumonia, and loss of cilia and epithelial necrosis and inflammation in nasal turbinates and trachea, were observed in one of these studies (7) . importantly, virus antigen was found in epithelial cells of the nasal turbinates, necrotic debris in the tonsil, submucosal glands of the trachea, and enterocytes of the small intestine. these experimental results, together with the few reports on sars-cov-2 detection in domestic cats and wild felids, indicate that felines are susceptible to infection by the novel coronavirus. moreover, the presence of lesions in the respiratory tract and detection of viral antigen in both respiratory and digestive tracts are compatible with the mild to moderate clinical course with dyspnoea and diarrhea already seen in some of these naturally exposed felines. since the cat referred to in the present report showed severe respiratory clinical signs and came from a covid-19 environment, a serious concern regarding the cause of the signs was established. however, the clinical and pathological diagnostic efforts indicated that the animal had significant previous comorbidities such as feline hypertrophic cardiomyopathy (12) and severe thrombocytopenia. therefore, the presence of pulmonary edema, congestion and hemorrhage, and pulmonary thrombosis and hydrothorax, all of them attributable to the preexisting condition, accounted for the severity of the clinical signs of the studied cat, and was the major reason to recommend euthanasia. in fact, no evidence of viral pneumonia was found, and no sars-cov-2 was retrieved from the lungs, findings that have been observed in experimentally infected cats (7) . the detection of sars-cov-2 rna in the mesenteric lymph node may be explained by the tropism of the virus to the digestive tract as well (7), since the virus seems to replicate in enterocytes, and it would be feasible that remnants of virus or viral rna may reach the mesenteric lymph node by draining the small intestine. in the cat of the present report, however, no lesions were observed in the digestive tract. therefore, no evidence of clinical signs or lesions potentially caused by sars-cov-2 was attributed to c1. the second animal (c2) never showed any clinical sign compatible to sars-cov-2 infection, but this is not surprising, since such infection can remain completely asymptomatic (4) . to date, all described cases in cats have been related to covid-19−affected owners or other people that came into contact with them. for the wild felids in new york, it has been speculated that they were infected by an asymptomatic animal caretaker or, alternatively, by one in a preclinical phase of the covid-19. this is also the situation of the present report's cats. since the owner (who finally died because of the disease) and several relatives suffered from covid-19, it is very likely that the pet was exposed, in a relatively continuous manner, to sars-cov-2. although the exact moment of exposure or infection was not possible to determine based on the reconstruction of the chronological events around the present case (fig. 2) , the low amount of sars-cov-2 (ct values of >32) found in positive samples of c1, evidence of sars-cov-2 seroconversion, the lack of compatible lesions with a viral disease, and the preexisting morbid conditions suggest that sars-cov-2 infection in c1 was an incidental epidemiological finding. therefore, it was concluded that the cause of death of the studied cat was unrelated to the novel coronavirus. however, since the susceptibility of domestic cats has now been established, extensive studies on domestic cat sars-cov-2 prevalence are needed to precisely ascertain the role of this sympatric species in the covid19 pandemic. in addition, although highly speculative at this stage, it would be important to ascertain whether sars-cov-2 infection may be able to worsen already existing disorders in cats and other animal species. immunohistochemistry. a previously described immunohistochemistry technique to detect sars-cov-2 antigen (6), using the monoclonal antibody 40143-r019 (sino biological) at dilution 1:1,000, was applied on nasal turbinates, trachea, lung, mesenteric lymph nodes, and intestine. in-house elisa and virus neutralizing tests. in-house elisa techniques to detect antibodies against sars-cov-2 spike, s2, and nucleocapsid protein (n) as well as rbd were assayed on the plasma samples of c1 and c2. briefly, nunc maxisorp elisa plates were coated with 50 ng per well of spike, s2, rbd, or nucleocapsid protein (sinobiologicals) in phosphate-buffered saline (pbs) overnight at 4°c. after washing with pbs + 0.05% of tween-20 (sigma-aldrich), plates were blocked using pbs/1% of bovine serum albumin (miltenyi biotech) for 2 h at room temperature. four serial dilutions of serum samples were added in duplicate starting at 1/500 dilution in blocking buffer for 1 h at room temperature. to calculate the specific signal, the background obtained using antigen-free wells was subtracted. serum samples from four cats collected before 2019 were used as negative controls. all samples were assayed in parallel in the same plate. the horseradish peroxidaseconjugated goat anti-cat igg (h+l) (1/20,000) (jackson immunoresearch) was used as detection antibodies. plates were revealed with o-phenylenediamine dihydrochloride (sigma aldrich) and stopped using 2 n of h 2 so 4 . the signal was analyzed as the optical density (od) at 492 to 620. a virus neutralization assay in plasma of both c1 and c2 was performed following a previously published protocol (13) . sars-cov-2 genome sequencing. viral rna was extracted directly from c1 samples using the indimag pathogen kit (indical bioscience) and transcribed to complementary dna with the primescript rt reagent kit, using a combination of oligo-dt and random hexamers, according to the manufacturer's protocol. for human nasopharyngeal swab samples, rna was extracted using magmax pathogen rna/dna kit (thermofisher scientific inc.). dna library preparation was performed using swift amplicon sars-cov-2 panel (swift biosciences). sequencing-ready libraries where then loaded onto illumina miseq platform and a 300-base pair paired-end sequencing kit. sequence reads were quality filtered, and primer sequences were trimmed off using cutadapt (14) . paired-end reads were matched using pear (15) and mapped against coronavirus reference (nc_045512.2) using bowtie2 tool (16) . consensus genomic sequence was called using samtools from the resulting alignment at a 20× coverage for frontal nasal turbinate and mesenteric lymph node samples of c1 (gisaid accid epi_isl_482820) and human sample (gisaid accid epi_isl_483059). only genomic positions with at least 5× q25 coverage were used. the nasal swab and other nasal turbinate samples (medial and caudal) from c1 failed to provide sufficient data to perform the analysis. data availability. all study data are included in the article. a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov-2 the pathogenicity of sars-cov-2 in hace2 transgenic mice transmission of sars-cov-2 in domestic cats infection and rapid transmission of sars-cov-2 in ferrets comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 age-related rhesus macaque models of covid-19 infection of dogs with sars-cov-2 sars-cov2 infection in farmed mink detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr feline hypertrophic cardiomyopathy: an update search for sars-cov-2 inhibitors in currently approved drugs to tackle covid-19 pandemia cutadapt removes adapter sequences from high-throughput sequencing reads pear: a fast and accurate illumina paired-end read merger fast gapped-read alignment with bowtie 2 acknowledgments. we appreciate the collaboration of esther zamora, carla zamora, and their relatives, the owners of negrito (c1) and whisky (c2). we also thank mónica pérez from irta-cresa for the histopathology laboratory work, and m. pilar armengol and the translational genomics platform team at the institut de recerca germans trias i pujol for the sequencing studies. we also acknowledge the crowdfunding initiative of irsicaixa, https://www.yomecorono.com. key: cord-311114-ggcpsjk8 authors: radhakrishnan, chandni; divakar, mohit kumar; jain, abhinav; viswanathan, prasanth; bhoyar, rahul c.; jolly, bani; imran, mohamed; sharma, disha; rophina, mercy; ranjan, gyan; jose, beena philomina; raman, rajendran vadukkoot; kesavan, thulaseedharan nallaveettil; george, kalpana; mathew, sheela; poovullathil, jayesh kumar; govindan, sajeeth kumar keeriyatt; nair, priyanka raveendranadhan; vadekkandiyil, shameer; gladson, vineeth; mohan, midhun; parambath, fairoz cheriyalingal; mangla, mohit; shamnath, afra; sivasubbu, sridhar; scaria, vinod title: initial insights into the genetic epidemiology of sars-cov-2 isolates from kerala suggest local spread from limited introductions date: 2020-09-09 journal: biorxiv doi: 10.1101/2020.09.09.289892 sha: doc_id: 311114 cord_uid: ggcpsjk8 coronavirus disease 2019 (covid-19) rapidly spread from a city in china to almost every country in the world, affecting millions of individuals. genomic approaches have been extensively used to understand the evolution and epidemiology of sars-cov-2 across the world. kerala is a unique state in india well connected with the rest of the world through a large number of expatriates, trade, and tourism. the first case of covid-19 in india was reported in kerala in january 2020, during the initial days of the pandemic. the rapid increase in the covid-19 cases in the state of kerala has necessitated the understanding of the genetic epidemiology of circulating virus, evolution, and mutations in sars-cov-2. we sequenced a total of 200 samples from patients at a tertiary hospital in kerala using covidseq protocol at a mean coverage of 7,755x. the analysis identified 166 unique high-quality variants encompassing 4 novel variants and 89 new variants identified for the first time in sars-cov-2 samples isolated from india. phylogenetic and haplotype analysis revealed that the circulating population of the virus was dominated (94.6% of genomes) by three distinct introductions followed by local spread, apart from identifying polytomies suggesting recent outbreaks. the genomes formed a monophyletic distribution exclusively mapping to the a2a clade. further analysis of the functional variants revealed two variants in the s gene of the virus reportedly associated with increased infectivity and 5 variants that mapped to five primer/probe binding sites that could potentially compromise the efficacy of rt-pcr detection. to the best of our knowledge, this is the first and most comprehensive report of genetic epidemiology and evolution of sars-cov-2 isolates from kerala. the covid-19 pandemic has seen a widespread application of genomic approaches to understand the epidemiology and evolution of sars-cov-2. the accelerated efforts to sequence genomes of clinical isolates of sars-cov-2 from across the world picked up pace following the initial genome sequencing of the virus from a patient in wuhan, the epicenter for the pandemic [1] ). as the virus evolves through the accumulation of mutations, it has split into major lineages with strong geographical affinities [2] . the availability of the genome sequences in the public domain has provided a unique view of the introduction, evolution, and dynamics of sars-cov-2 in different parts of the world [3] . a number of approaches have emerged for rapid and scalable sequencing of sars-cov-2 from clinical isolates. this includes direct shotgun approaches as well as targeted amplicon-based and targeted capture-based approaches [4] [5] [6] . sequencing based approaches provide a unique opportunity for high fidelity of detection and for understanding the genetic epidemiology of sars-cov-2 [7] . additionally, the genetic variants could offer insights into the mutational spectrum, evolution, infectivity, and attenuation of the virus [8, 9] . additional analyses on genomic variants have also provided useful insights into the efficacy of primer/probe-based diagnostic assays as well as immune epitopes and resistance to antisera [10, 11] . an approach for high-throughput multiplex amplicon sequencing of sars-cov-2 has been previously reported from our group [7] . kerala is a unique state in india with a population of 35 million people and extensively connected with the global populations through over 1.6 million expatriates, apart from being a traditional trade post and a global tourist destination. the state is therefore in a distinct position, affected by local as well as global epidemics. in fact, the first identified case of covid-19 in india was from kerala, early in the epidemic. the patient had traveled from wuhan, china [12, 13] . the initial genomic identity of the virus was also established which mapped to the b superclade of sars-cov-2 [14] . further introductions into the state during the later days of the pandemic through international and regional travel could have contributed to the spread of the epidemic in the state and the pool of circulating genetic lineages or clades. while a number of studies on the genetic epidemiology of sars-cov-2 from different states in india have emerged [14] [15] [16] [17] , there has been a paucity of information on the genetic architecture and epidemiology of sars-cov-2 isolates in the state of kerala. we intended to fulfill the gap in knowledge on the identity of the circulating genetic lineages/clades contributing to the epidemic in the state of kerala. to this end, we employed a high-throughput sequencing-based approach for the genetic epidemiology of sars-cov-2. to the best of our knowledge, this is the first comprehensive overview of the genetic architecture of sars-cov-2 isolates from the state of kerala. the institutional human ethics committee approved the project (gmc kkd/rp2020/iec438). rna samples were isolated from nasopharyngeal/oropharyngeal swabs of patients presenting to government medical college, kozhikode, a major tertiary care center in kerala. rna extraction was done using magmax viral/pathogen nucleic acid isolation kit in thermo scientific kingfisher flex automated extraction system according to the manufacturer's instructions. all the rna samples were transferred within 72 hours of collection at a cold temperature (2-8°c) and were stored at -80°c until further processing. sequencing was performed using the covidseq protocol as reported previously [7] . briefly, this protocol involved multiplex amplicon sequencing on the illumina novaseq platform. the base calls generated in the binary base call (bcl) format were demultiplexed to fastq reads using bcl2fastq (v2.20). for reference-based assembly, we followed a previously defined protocol from poojary et al. [18] . as per the protocol, the quality control of fastq reads was performed using trimmomatic (v0.39) at a phred score of q30 [19] with adapter trimming. these reads were further aligned to the severe acute respiratory syndrome 2 (sars-cov-2) wuhan-hu-1 reference genome (nc_045512.2) using hisat2-2.1 [20] . the human reads were removed using samtools (v1.10) [21] . the samples with coverage >99% and <5% unassigned nucleotides underwent variant calling and consensus sequences generation using varscan (v2.4.4) [22] and samtools (v1.10) [21] , bcftools (v1.10.2), and seqtk (v 1.3-r114) [23] respectively. variants were annotated using annovar [24] employing a range of custom annotation datasets and tables. all the variants identified were systematically compared with a compendium of other indian and global variants. a total of 93,995 complete sars-cov-2 genomes deposited in the global initiative on sharing all influenza data (gisaid) database till september 1, 2020 were used for comparative analysis. summary of the sample details along with their originating and submitting laboratories are provided in supplementary table 1 . viral genomes with a pairwise alignment ≥ 99% and gaps < 1% with the reference genome (nc_045512.2) were considered for further variant calling using snp-sites [25] . genetic variants compiled from a total of 1,855 high-quality genomes from india and 32,286 global genomes were considered for analysis. phylogenetic analysis was performed according to the pipeline provided by nextstrain [26] . the dataset of 2,476 complete sars-cov-2 genomes deposited in the gisaid database from india was used for the analysis supplementary table 2 , along with 113 genomes from the current study which have 99% coverage and at least 98% pairwise alignment with the reference genome (nc_045512.2). genomes having more than 5% ns or missing dates of sample collection were excluded from the analysis. the phylogenetic tree was constructed and refined to a molecular clock phylogeny using the augur framework provided by nextstrain and was visualized using auspice. the phylogenetic assignment of named global outbreak lineages (pangolin) package was used to assign lineages to the genomes from this study [27] . the lineages were visualized and annotated on the phylogenetic tree using itol [28] . for haplotype analysis, the genomes were aligned to the wuhan-hu-1 (nc_045512.2) reference genome using mafft [29] and problematic genomic loci (low coverage, high sequencing error rate, hypermutable and homoplasic sites) were masked from the alignment [30] . the aligned sequences were imported into the dna sequence polymorphism tool (dnasp v6.12.03) [31] to generate haplotypes. a tcs haplotype network [32] for the genomes was constructed using the population analysis with reticulate trees software (popart v 1.7) [33] . times to the most recent common ancestor (tmrca) for the haplogroups were computed following the bayesian markov chain monte carlo (mcmc) method using beast v1.10.4 [34] . the analysis was performed using a coalescent growth rate model along with a strict molecular clock and the hky+γ substitution model with gamma-distributed rate variation (gamma categories=4). mcmc was run for 50 million steps. the output was analyzed in tracer v1.7.1 [35] and burn-in was adjusted to attain an appropriate effective sample size (ess). further, we have evaluated the sars-cov-2 variants based on their functional relevance. we curated a comprehensive compendium of sars-cov-2 variants of functional relevance as well as variants that are associated with increased infectivity and attenuation of sars-cov-2 from literature and preprint servers. the variants were systematically annotated and mapped to the reference genome coordinates and their respective amino acid changes. this variant compendium encompassed about 337 variants curated from 35 publications. the variants in this study were compared with the genomic variants generated using bespoke scripts. we were also interested to evaluate the effect of sars-cov-2 variants on the efficacy of rt-pcr detection. we took a compiled list of 132 primer/probe sequences widely used in the molecular detection of sars-cov-2 around the globe [11] . in our analysis, we mapped the sars-cov-2 genetic variants obtained from kerala genomes to the 132 primer or probes sequence and calculated the melting temperature (tm) of the mutant with the wild type sequence. the length of primers in the curated list is greater than 13 nucleotides. the formula applied for calculating melting temperature is tm=64.9 +41*(yg+zc-16.4)/(wa+xt+yg+zc) where w, x, y, and z are the number of a, t, g, and c nucleotides respectively [36] . figure 1 summarises the schematic for the overall data analysis. a total of 200 isolates of sars-cov-2 from kerala were processed for genome sequencing. the genomes were sequenced using covidseq protocol [7] and generated approximately 8.1 million raw reads per sample. the reads were subjected to quality control and resulted in approximately 7.5 million reads per sample, of which around 6.4 million reads per sample aligned to the sars-cov-2 reference genome (nc_045512.2). the reads had a mapping percentage of 84.93% and mean 7,755x coverage. the data has been summarized in supplementary table 3 and the mean coverage of the sample across the amplicons has been represented in figure 2 . of the 200 isolates of sars-cov-2 sequenced, a total of 179 samples had > 99% coverage and <5% unassigned nucleotides across the genome. these samples were further processed for variant calling and consensus generation. our analysis identified a total of 195 unique variants, with a median variant count of 12 per sample. variant quality has been ensured with the average variation percentage across genomes ≥ 50. of the total 195 unique variants, 166 were categorized as high-quality variants. the detailed information on variant quality is provided in supplementary table 4 . the distribution of variants across the sars-cov-2 genomes used in the study was analyzed. also, the proportional distribution of variants for every 100 bps across the genome was calculated and compared among various datasets. variant distribution across genomes and comparison of variant proportions across genome datasets are represented in figure 3 . out of the 166 high-quality unique variants, 4 variants were found to be reported for the first time in the global compilation of variants table 1 . we have also added 89 new variants (2.61%) to the indian repertoire of genetic variants. details of these variants are systematically compiled in supplementary table 5 . the overlap in the variants between the present study of kerala, other indian, and global datasets is summarized in supplementary figure 1 . out of the 4 novel variants, 1 variant in the s gene, 25281g>a, was a personal variant and was not shared by any other isolate. the remaining three novel variants were shared variants and were present in different genes (orf1b, orf7a and s). of the total 166 high-quality unique variants, 162 variants were located in the protein-coding regions while 4 variants mapped to either downstream or upstream regions. of the total variants in protein-coding regions, 93 variants were non-synonymous, 67 were synonymous, and 2 variants resulted in stopgain mutation. these two stopgain variants were found in orf3a (26113:g>t) and orf8 (28028:g>a) genes and were present in one individual each. the annotation of the variants based on the location and consequence is represented in figure 3 . the phylogenetic tree was constructed using the genome wuhan/wh01 (epi_isl_406798) as root and 2366 genomes from india which met the inclusion criteria (ns < 5%, no missing/ambiguous date of sample collection) including 113 genomes sequenced in this study. all 113 genomes from this study were found to cluster under the globally predominant clade a2a (gisaid clade g and gh). in contrast, one of the previous genomes available from kerala (epi_isl_413523, submitted by national institute of virology, pune, india), which is also one of the first sars-cov-2 genomes sequenced in india, belongs to the clade b [13] . haplotype analysis was done using a dataset of 850 sars-cov-2 genomes from india (including 113 genomes from kerala) that fell under clade a2a in the phylogenetic tree and clustered close to the 113 genomes from kerala. among the 850 genomes, there were 592 variable sites and 400 unique haplotypes supplementary figure 5 summarizes the haplotype network of the a2a clade genomes. may) for the three major haplogroups k1, k2, and k3 respectively. taken together, the analysis suggests that the majority of the sars-cov-2 isolates are outcomes of limited introductions early in the epidemic followed by local circulation. annotating the variants for their functional consequences using custom annotation datasets, revealed a total of 42 genetic variants that were predicted as deleterious by sift [37] . the filtered variants were found to span 13 unique protein domains as per uniprot [38] annotations. we found 15 and 120 genetic variants that mapped back to potential b and t cell epitopes from the immune epitope database (iedb) [39] respectively. in addition, 5 variants were found to span predicted error-prone sites including sequencing error sites, homoplasic positions, and hypermutable sites. functional annotation details of all the filtered variants are summarised in supplementary table 7 . we also explored whether the variants mapped to the rt-pcr primers and probes sites. on mapping the genetic variants with the curated primers and probes, we found 5 unique variants at 5 unique primer or probes binding sites. a total of four unique variants had allele frequency > 1% at 4 unique primer binding sites. summary of novel variants and diagnostic primer/probe spanning variants are compiled in table 1 and table 2 respectively. details on the read count and depth of coverage of these variants are systematically documented in supplementary table 8.a and 8. b . with the view of identifying potential functionally relevant variants, we overlapped the variants obtained from the present study with a manually curated compilation of functionally relevant sars-cov-2 variants. our analysis identified 2 variants in the s gene which were reported to be associated with increased infectivity. l5f, a variation co-occurring with d614g was earlier demonstrated to possess increased infectivity [8, 40] using cell line studies. in our study, 23403a>g (d614g) and 21575c>t (l5f) mutations were observed at frequencies of 99.44% and 15.64% respectively in the genomes. the combination of these variations was found to occur at a higher frequency in genomes from kerala. [2] and therefore leaves the mutational fingerprint which is widely used for tracing the spread of the virus [41] . the availability of high-throughput sequencing approaches has enabled researchers to sequence genomes as the pandemic progressed in their respective countries. a number of methods have been adopted for rapid high throughput sequencing of sars-cov-2 including shotgun sequencing [4] , pcr amplicon, and hybridization/capture-based enrichment and sequencing [5] [6] [7] . genome sequencing of sars-cov-2 in various countries [42] has led to to insights into the temporal and geographical spread of the virus [43] , introductions, and spread of the virus through travelers [44, 45] , local transmission, and dynamics [46] , investigating the origin of outbreaks [47] , just to name a few. by virtue of its connectivity to major cities through its expatriate population, trade and tourism is uniquely poised in this pandemic. it is not surprising therefore that the first case of covid-19 in india, early in the pandemic, was reported from the state [12, 13] . the genome of the isolate suggested it originated from china [13] . the following months have seen the number of cases increase to over 80 thousand in the state with a paucity of information on the origin, spread, and dynamics of the virus [ https://dashboard.kerala.gov.in/ ]. in this present study, we performed sequencing and analysis of sars-cov-2 isolates from kerala which revealed unique patterns of the transmission. these genomes are clustered into a monophyletic group mapping to the a2a clade. the a2a clade is also marked by the d614g variant, which is suggested to confer higher infectivity to the virus in experimental in vitro settings [48, 49] and is therefore thought to have emerged globally as the predominant clade [8] , though the cause-effect relationship still remains speculative. haplotype analysis suggests that three major haplogroups with distinct ancestry groups encompass the majority of the isolates. the haplotype analysis in the context of other genomes from india suggests the introductions were from inter-state travel. the prevalent haplotypes were not found in any of the global genomes, supporting this observation. this also suggests that focussed testing, tracing and quarantine of expatriates and international travelers implemented during the epidemic would have been effective in curbing the spread from international travelers. the genome clusters also suggested polytomies, suggesting a recent outbreak [17] . close follow-up of the cluster members confirmed the potential source of the outbreak, suggesting genetic epidemiology could be used in conjunction with case follow-ups to uncover potential outbreaks and possibly connect outbreaks which are apparently not related. this study uncovered a total of 4 novel genetic variants and 89 variants which were identified only in kerala and not in the rest of india. the genome sequences could also uncover insights into the variants of functional relevance. one of the variants of significance is a stopgain variant (28028:g>a) in the orf8 gene. variants including deletions in orf8 have been suggested to attenuate the virus [50, 51] . similar variants have also been identified in other related viruses like the sars-cov and mers-cov [9, 52] . a variant 21575c>t (l5f) in the s gene associated with increased infectivity of the virus [40] was present in 15.64% of the genomes sequenced. following recent reports which suggest variants in the primer/probe binding sites could impact the efficiency of rt-pcr assays [11, 53, 54] , we explored whether any of the variants in the present study mapped to the primer/probe binding sites. we identified 5 unique variants in 5 unique binding sites. the maximum number of variants were the primer set published by won et al. [55] spanning multiple genes, apart from the 2019-ncov-nfp ggggaacttctcctgctagaat binding sites in the n gene [ https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf?sfvrsn=de3a7 6aa_2 ]. the latter is part of the china centers for disease control and prevention (cdc) protocol with variants in 1.117% in genomes from kerala. we have earlier reported variants in this primer site in 39.5% of the genomes from india [11] and 18.8% [56] of global genomes. this information would be potentially valuable for laboratories in selecting reagents for screening and diagnosis. the study has two caveats; first is that the samples were collected from a single major tertiary care center in north kerala. however, the center caters to a large population and region and has close proximity to an international airport. secondly, the sampling was limited to a short period of time, thus enabling only a cross-sectional view of the epidemic and precluding an accurate and temporal view of the dynamics of the epidemic in the state. nevertheless, this provides a unique opportunity to create a snapshot of the epidemic in time and space. notwithstanding the limitations, this is the first and most comprehensive overview of the genetic epidemiology of sars-cov-2 in the state of kerala. while providing insights into the epidemiology of the epidemic, the study also enabled tracing outbreaks thereby highlighting the utility of genome sequencing as an adjunct to high-throughput screening and testing. it has not escaped our mind that scalable technologies that can combine both the approaches [7] could potentially find a place in understanding epidemics better. a new coronavirus associated with human respiratory disease in china evolutionary history, potential intermediate animal host, and cross-species analyses of sars-cov-2 global initiative on sharing all influenza data -from vision to reality rapid implementation of sars-cov-2 sequencing to investigate cases of health-care 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nomenclature proposal for sars-cov-2 lineages to assist genomic epidemiology interactive tree of life (itol) v4: recent updates and new developments recent developments in the mafft multiple sequence alignment program issues with sars-cov-2 sequencing data 2020 dnasp 6: dna sequence polymorphism analysis of large data sets tcs: a computer program to estimate gene genealogies popart : full-feature software for haplotype network construction bayesian phylogenetic and phylodynamic data integration using beast 1.10 posterior summarization in bayesian phylogenetics using tracer 1.7 hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 dna: the effect of single base pair mismatch sift: predicting amino acid changes that affect protein function uniprot: the universal protein knowledgebase the immune epitope database (iedb): 2018 update the impact of mutations in sars-cov-2 spike on viral infectivity and antigenicity genome-wide analysis of sars-cov-2 virus strains circulating worldwide implicates heterogeneity. sci rep cog-uk) consortiumcontact@cogconsortium.uk. an integrated national scale sars-cov-2 genomic surveillance network geographical and temporal distribution of sars-cov-2 clades in the who european region rapid sars-cov-2 whole-genome sequencing and analysis for informed public health decision-making in the netherlands whole genome and phylogenetic analysis of two sars-cov-2 strains isolated in italy in genomic epidemiology of sars-cov-2 in guangdong province clinical features of patients infected with 2019 novel coronavirus in wuhan the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity d614g mutation of sars-cov-2 spike protein enhances viral infectivity. microbiology. biorxiv; 2020 identification of unique mutations in sars-cov-2 strains isolated from india suggests its attenuated pathotype biorxiv effects of a major deletion in the sars-cov-2 genome on the severity of infection and the inflammatory response: an observational cohort study deletion variants of middle east respiratory syndrome coronavirus from humans newly emerging mutations in the matrix genes of the human influenza a(h1n1)pdm09 and a(h3n2) viruses reduce the detection sensitivity of real-time reverse transcription-pcr the impact of primer and probe-template mismatches on the sensitivity of pandemic influenza a/h1n1/2009 virus detection by real-time rt-pcr development of a laboratory-safe and low-cost detection protocol for sars-cov-2 of the coronavirus disease 2019 (covid-19) presence of mismatches between diagnostic pcr assays and coronavirus sars-cov-2 genome authors thank anjali bajaj for editorial assistance. aj, md, bj acknowledge research fellowships from csir. ds acknowledges a research fellowship from intel. authors acknowledge funding for the work from the council of scientific and industrial research (csir), india through grants codest and mlp2005. the funders had no role in the design of experiment, analysis, or decision to publish . supplementary table 1: gisaid acknowledgment table for global genomes used in the study supplementary table 2: gisaid acknowledgment table for genomes from india considered for phylogenetic analysis supplementary table 3 : data summary of the samples sequenced by covidseq protocol and processed using a custom pipeline. table 4 . summary of quality details of the variants identified in the study key: cord-304617-5ozf18lg authors: al-khafaji, khattab; al-duhaidahawil, dunya; taskin tok, tugba title: using integrated computational approaches to identify safe and rapid treatment for sars-cov-2 date: 2020-05-15 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1764392 sha: doc_id: 304617 cord_uid: 5ozf18lg sars-cov-2 is a new generation of coronavirus, which was first determined in wuhan, china, in december 2019. so far, however, there no effective treatment has been found to stop this new generation of coronavirus but discovering of the crystal structure of sars-cov-2 main protease (sars-cov-2 mpro) may facilitate searching for new therapies for sars-cov-2. the aim was to assess the effectiveness of available fda approved drugs which can construct a covalent bond with cys145 inside binding site sars-cov-2 main protease by using covalent docking screening. we conducted the covdock module mmgbsa module in the schrodinger suite 2020-1, to examine the covalent bonding utilizing. besides, we submitted the top three drugs to molecular dynamics simulations via gromacs 2018.1. the covalent docking showed that saquinavir, ritonavir, remdesivir, delavirdine, cefuroxime axetil, oseltamivir and prevacid have the highest binding energies mmgbsa of –72.17, −72.02, −65.19, −57.65, −54.25, −51.8, and −51.14 kcal/mol, respectively. the 50 ns molecular dynamics simulation was conducted for saquinavir, ritonavir and remdesivir to evaluate the stability of these drugs inside the binding pocket of sars-cov-2 main protease. the current study provides a powerful in silico results, means for rapid screening of drugs as anti-protease medications and recommend that the above-mentioned drugs can be used in the treatment of sars-cov-2 in combined or sole therapy. communicated by ramaswamy h. sarma sars-cov-2 also called 'severe acute respiratory syndrome coronavirus 2' abbreviated sars-cov-2 was recognized to be the causative of atypical pneumonia (joshi, 2020; pant et al., 2020) outbreak in wuhan, china (hasan, 2020; s. a. khan et al., 2020) . the virus belongs to the family known as 'coronaviruses' because of the crown-like appearance of spikes glycoproteins on the envelope under an electron microscope . world health organization (who) recently announced that the virus transforms from epidemic to pandemic, which requires urgent intervention to prevent the growing spread of the virus across the globe (chan et al., 2020) . the total confirmed cases 2,347,884 with 738,923 cases in the united state of america (usa) alone and the total death of 161,138 (as of april 19), with mortality estimated within 2% and about 3.4%, according to estimates of approved cases and death worldwide (n. . the most familiar is a virus that arose from the rhinolophus bat which is > 96% homologous with the modern sars-cov-2 virus and it is just 79% homologous with the initial sars-cov (fisher & heymann, 2020) . the fast-growing number of infected cases globally urged the world health organization to announce a state of global health emergency to correlate scientific and medical disciplines to develop rapidly an effective treatment for patients, (morse et al., 2020; sarma et al., 2020) elderly patients and people with severe underlying health diseases like heart diseases, lung illness, and diabetic patients, for instance, appear to be at greater risk of revealing severe sars-cov-2 requires immediate intervention rather than waiting virus vaccine which may require 1 year to be available (enayatkhani, 2020) . while drug repurposing could be a short-term and fast resolution to handle sars-cov-2 patients (elfiky, 2020 ; r. j. kumar et al., 2019) , repurposing existing drugs can offer a good choice to overcome the virus and offer better risk-versus trade-off as compared with discovering new drug and can help overcome time waiting for new therapy rather than use the available resources (elmezayen, 2020; muralidharan, 2020) one successful repurposing drug story includes duloxetine which originally developed for depression and fda approved as the first-inclass choice for stress urinary incontinence (sweeney & chancellor, 2005) , duloxetine initially created as antidepressant also is now passed to phase iii clinical trials as a first-in-class treatment for premature ejaculation (mcmahon, 2012) and thalidomide, which had a tragic start as an over-the-counter sedative for morning sickness in pregnancy is now being applied to manage leprosy and multiple myeloma (hideshima & anderson, 2002) . as a result of, a newly issued x-ray crystal of sars-cov-2 main protein (mpro), we planning to use computational approaches (cameron et al., 2013) to contribute to find an effective treatment for sars-cov-2.thereby, computational analyses speed up these approaches since they allow to handle millions of data simultaneously (gupta et al., 2020) . molecular docking includes a set of computational methods and algorithms that aimed to identify novel relationships between chemical ligands and targets through using the modelling of their direct physical interaction (aanouz, 2020; ekins et al., 2007) . in present study, we attend to evaluate some of approved drugs to be as covalent binders, irreversible interactions, which can provide a powerful strategy to fight against epidemic viruses. and molecular dynamics simulations can give a more detail for the image which got from molecular covalent docking. the crystal structure of fda-available covalent drugs which available in table 1 were selected based on the review of kumalo et al. (2015) and some of the antiviral drugs that can form a covalent bond to the target protein. and we aim also here to redirect them for other indications specially to see their possibility to fight against sars-cov-2. thence, we searched about the chosen drugs in pubchem (https://pubchem.ncbi.nlm.nih.gov/) to identify the possibility of the selected drugs to be as covalent binders toward sars-cov-2 mpro. where, pubchem provides detailed information about the selected drugs rather than other repositories, especially, the property of drug to form covalent bond. before starting covalent docking, we downloaded the selected drugs one by one and optimize them by using the ligprep (lim et al., 2020) based on the opls_2005 force field and generated possible state employing epik in the schrodinger 2020 (elfiky 2020; s. a. khan et al.,2020) . in the next step, the structure of sars-cov-2 mpro (6lu7) was downloaded from protein data bank (http://www.rcsb.org/pdb) (jin et al. , 2020) . the protease structure was optimized by adding hydrogens, removing water molecules and optimizing charge using the protein preparation wizard module (kumalo et al., 2015) in schrodinger suite 2020-1. the covalent docking protocol was preferred since the cysteine 145 residue which is available in the binding site of sars-cov-2 mpro considered as a vital residue that can form a covalent bond with the drug if it can interact covalently. the different mechanisms for the cys145-fda drug were mentioned in table 1 based on the nature of the drug so that the reactive functional group on the ligand and receptor residue are identified and the bond is formed between the correct atoms. these covalently docked complexes were created using covdock in schrodinger suite 2020-1. finally, we selected the lowest the mmgbsa value for each drug as a propriate conformation of the drug inside the binding pocket. the chosen drugs binding energies were calculated using prime mm-gbsa modules (vijayakumar et al., 2014) in the schr€ odinger (2020). the best poses of selected drugs-sars-cov-2 mpro-were chosen to obtain the binding free energy calculation. prime mmgbsa is a method that combines optimized potential for liquid simulations-all atoms (oplsaa) force field, molecular mechanics energies (emm), an sgb solvation model for polar solvation (gsgb), and a non-polar solvation term (gnp) composed of the non-polar solvent accessible surface area and van der waals interactions. the total binding free energy: molecular dynamics simulations are a decision-making process for inspections of protein-drug complexes' stabilities (al-khafaji & taskin tok, 2020b) . it is used to clarify the dynamic behavior at an atomic level of biological systems, which is hard to handle in labs (shukla et al., 2019) . in the current study, we conducted molecular dynamics simulations for the top three drugs based on mmgbsa values. the got protein-drug complex structures from covalent docking were submitted to md simulations (saquinavir, ritonavir, and remdesivir with sars-cov-2 mpro). we hired gromacs 2018.1 to run 50 ns md simulations (abraham et al., 2015) . charmm 27 force field for all atoms were chosen to run md simulation (bjelkmar et al., 2010) . we used swiss param to produce the topologies of drugs (zoete et al., 2011) . all protein-drug systems were solvated with three-point transferable intermolecular potential (tip3p) and their charges were neutralized via adding na or cl ions. in the following step, the protein-drugs systems were energetically minimized through the steepest descent algorithm at a tolerance value of 1000 kj/mol.nm. then the equilibration with position restraint on the protein molecules for 0.1 ns using nvt and npt ensembles were done. electrostatic interactions were evaluated by particle mesh ewald summation (darden et al., 1993) . we performed the molecular dynamics simulation with no restraint on the protein molecules or ligand to determine the stability in the final step (time step of 0.015 ns). rmsd, rmsf, rg, and number of hydrogen bonds were chosen to analyze md trajectories by using gromacs utilities. the principal component analysis (pca) approach was employed to calculate eigenvectors and eigenvalues and their projection along with the first two principal components (al-khafaji & taskin tok, 2020a). this approach is based on the protocol of gromacs 2018.1 (abraham et al., 2015) . it is used to simplify the effect of drugs on the dynamic motion of the targeted protein where it extracts the dynamic motions in simulations that are required for their biological function (amadei et al., 1993) . we got the principal component analysis from the md trajectories. a series of eigenvectors and eigenvalues were generated by diagonalizing the matrix. we chose trajectories of the protein backbone of the complexes to get 2 d-projection of motion of trajectory. to assess the possibility of selected fda drugs to work as treatment of sars-cov-2, the covalent docking was utilized to screen the selected library and rank them according to their binding affinities. the calculated binding free energies of some available drugs using docking score, glide gscore, and ensemble-average mm/gbsa are shown in table 1 . to validate our covalent docking results, the correlation between mmgbsa and docking score was constructed ( figure 1 ). the binding energy mmgbsa-docking score correlation shows a good correlation (r 2 ¼ 0.6299). based on this correlation, we chose the top three ranked-mmgbsa and docking score values of fda drugs for dissection their binding modes inside the binding site. covalent docking showed that saquinavir, ritonavir, remdesivir, delavirdine, cefuroxime axetil, oseltamivir, and prevacid have à72.17, à72.02, à65.19, à57.65, à54.25, à51.8, and à51.14, respectively. where all the topeight fda drugs show a higher affinity to form a covalent, irreversible bond with cys145 of sars-cov-2 mpro. here we investigated the role of molecular weight upon the affinity of selected drugs to bind covalently to cys145, whereas 57% of selected drugs which have molecular weight over than 600 g/ mol ( figure 2 ). and have higher free binding energy mmgbsa than à50 kcal/mol. while the ratio of selected drugs decreased to be 16% of drugs which can form covalent bonding with over than à50 kcal/mol. surprisingly, none of the selected drugs which have a molecular weight below than 300 g/mol can form a good affinity of binding energy. this indicates that higher molecular weight covalent warheads can form stable and efficient binding energy. what stands out in the table 1 is saquinavir has the highest binding affinity (lowest binding energy mmgbsa of 72.17 kcal/mol). therefore, the deep examination of saquinavir is needed. figure 3 shows that saquinavir not only formed covalent bond of 1.81 å with cys145 but also formed five hydrogen bonds inside the pocket of sars-cov-2 mpro. in the second rank ritonavir as presented in table 1 has à72.02 kcal/mol. this high affinity resulted from covalent bond between ritonavir and cys145 of 1.82 å (figure 4 ) through nucleophilic addition to double bond reaction besides it interacted within binding site by forming three hydrogen bonds. despite remdesivir formed covalent bond with cys145 of 1.82 å and three hydrogen bonds ( figure 5 ) and this is similar to ritonavir, but the mmgbsa value is lower than that of ritonavir this may due to nature of reaction which in remdesivir nucleophilic addition to triple bond. the effect of drug-protein interactions upon dynamics of biological system is a fundamental in drug discovery thereby we used rmsd to investigate the influence of saquinavir, ritonavir, and remdesivir upon the stability of sars-cov-2 mpro. we utilized gromacs to execute the md simulations of 50 ns for three drug-protein systems besides of apo protein. rmsd fluctuations for both apo and hollo forms were measured and presented. the rmsd was calculated to assess the overall dynamics, stability, and convergence of the various systems and the results are presented in figure 6 (a). figure 6 (a) shows there is a significant decrease in the rmsd value when sars-cov-2 main protease whether it bound to saquinavir, ritonavir, or remedisivir. further analysis revealed that the rmsd average of apo sars-cov-2 main protease was 0.294 nm but when it bound to saquinavir, ritonavir, and remedisivir the rmsd averages were.01865, 0.2130, and 0.2053 nm, respectively. another significant aspect of md simulation is the flexibility of protein's backbone which can be assessed through measuring rmsf value. the results of the comparative analysis between these drugs and their effects upon sars-cov-2 main protease are illustrated in figure 6 (b). closer scrutiny of figure 6 (b) exhibits the binding of saquinavir diminished the fluctuations of the protein's backbone. and behavior is also can be seen from figure 6 (b) where the binding of both ritonavir and remedisivir led to reducing the flexibility of the protein. the radius of gyration (rg) is a definition of system's density, and substantially influences the folding rate and stability of proteins. rg was employed to assess the compactness of all complexes. in this work, rg values are in agreement with rmsf values where there are no significant differences between apo form and hollo forms as presented in figure 6 (c). this reveals that protein remained stable and compact all through the 50 ns time. the number of intermolecular hydrogen bonds is a vital aspect to give an impression about the stability of drug and protein. also, the number of hydrogen bonds is relevant to the binding scores of molecular docking process. we calculated the hydrogen bonds over time to validate the stable interactions between top three drugs and their corresponding target. as seen in the figure 6 (d) the ritonavir has highest order of hydrogen bonding (the average ¼3.35), while saquinavir has an average of 1.68. the essential dynamic method is a tool to explore the dynamical behavior in the space of sars-cov-2 main protease combined with saquinavir, ritonavir, and remedisivir. basically, the comparison of the drug-bound sars-cov-2 mpro and drug-unbound was made as reference. in order to further understand the configurational space, we selected the first two principal components (pc1, pc2) to analyze their projection of trajectories during the simulations of ligand free and ligand bound sars-cov-2 mpro of the phase space (shown in figure 7) . during the four system simulations, the results clearly show that the unbound ligand protein covered a wider region of phase space, while all three drug-protein system occupied a smaller region of phase space. especially, saquinavir reduced the essential dynamics to lowest degree of functional motions as compared with another drugs. moreover, the pca results suggest that the drug-bound sars-cov-2 mpro is more stable than ligandunbound sars-cov-2 mpro form of sars-cov-2 mpro. in short, the pca results are also in agreement with the rmsd and rmsf results, which enhance the validity of the performed analysis. sars-cov-2 causes major pandemic health issue since its spread across the world and can infect humans mainly respiratory system causing severe pneumonia with no vaccine and drug treatment available. prior study that have referred to the significance of molecular docking to determine effective treatment in short time (wu, et al., 2020) . in reviewing the literature, we took the advantage of possibility of fda available drugs that can be as a covalent warhead to inhibit the sars-cov-2 mpro with cys145. an initial objective of the project was to identify effective and applicable treatment. the present study focused on the main protease (mpro), especially pdb id (6lu7) as a potential target for several marketed drugs as possible therapeutic option to combat the virus to see the capability of these drugs to bind with the cysteine 145 residue which is available in the binding site of sars-cov-2 main protease. the mpro in sars-cov-2 is necessary for the proteolytic maturation of the virus targeting this protein to limit the expansion of infection by hindering the cleavage of the viral polyprotein. the most interesting finding was that the both saquinavir and ritonavir have the same affinity (binding energy mmgbsa % à72 kcal/mol) to block binding site of sars-cov-2 with irreversible interactions. this study supports evidence from previous observations that lopinavir/ritonavir can inhibit sars-cov-2 (lim et al., 2020) . another important outcome was that the remdesivir comes in second rank with binding affinity (mmgbsa ¼ à65 kcal/mol ). whereas previous research has established that remdesivir can inhibit sars-cov-2 m proenzyme through docking results. another significant finding, delavirdine computationally showed the ability to form irreversible covalent bond of à57.65 kcal/mol mmgbsa binding energy. moreover, indinavir and cefuroxime axetil exhibited the possibility to halt the pocket of sars-cov-2 mpro by forming stable interaction through covalent bonding and hydrogen bonding (mmgbsa binding energies % à54 kcal/ mol). furthermore, oseltamivir and prevacid exhibited same affinity to bind with sars-cov-2 protease through one covalent bond and one hydrogen bond (% à51 kcal/mol), which is a good explanation for the activity of oseltamivir (peeri et al., 2020) . in this study, results obtained show the binding affinity of drugs to an active site depends on several factors mainly the ability of a compound to form a covalent bond with amino acid residues of the mpro (cys145) and length of a covalent bond, the number of h-bonds that can form with a pocket of the active site and type of nucleophilic addition of unsaturated bonds. in this concept, the structural features in saquinavir and ritonavir like free amine group (-nh2), hydroxyl groups (-oh), carbonyl groups (c¼o) in addition to ether group play key structural feature to form h-bond. results show the promising activities for antiretroviral drugs saquinavir that used for hiv/aids more than ritonavir and remdesivir followed by lopinavir as the best drugs to bind covalently toward sars-cov-2 mpro with lowest energy of binding. as discussed above, saquinavir, ritonavir, remdesivir, delavirdine, cefuroxime axetil, oseltamivir, and prevacid showed the ability to block the binding site of sars-cov-2 mpro by forming covalent bond and stabilized with hydrogen bonding. these outcomes are contrary to that of kandeel and al-nazawi (2020), they found that ribavirin, telbivudine, vitamin b12, and nicotinamide can be can form non-bonding interactions. whilst our results showed that, saquinavir, ritonavir, and remdesivir can form irreversible interactions, which are considered an effective way for viral infections. in the present work, comparative molecular dynamics simulations were conducted to evaluate the effects of saquinavir, ritonavir, and remdesivir on the conformational and dynamical demeanor in either apo or hollo forms to understand the inhibitory possibility at atomic level. an initial mission of the project was to identify rapid, effective, safe, and available for large proportions of people, so we run covalent docking and mmgbsa to sift the possibility of these drug to form irreversible interactions. therefore, we had chosen these drugs because they carry covalent warheads that can bind covalently to the target. but the docking results are not adequate, so we run md simulations examine how much these drugs are able to form stable interactions with targeted protein. one interesting finding is the rmsd of protein backbone in apo status has higher average value, whereas the binding with top three drugs diminished these rmsd average. another important finding was that the rmsf of protein's backbone be less flexible when it compared to apo form of sars-cov-2 main protease. it is somewhat surprising that the binding of three drugs have not noted in rg values. it is not yet clear whether the top three drugs can show an evidence to inhibit the targeted protein, thereby we lean on the principal component analysis to analyze the md trajectories to judge without dispute. the most obvious finding to emerge from 2d pca analysis is that the binding of under investigated drugs caused stately impact on essential dynamics of protein by reducing its essential dynamics to its least possible motions. these results seem to be consistent with rmsd results. a possible explanation for this might be that binding of the drugs make the binding site much narrower due to hydrogen bindings this makes the 3d structure of targeted protein more rigid so it will lose its biological functions. it is possible, therefore, that using these available drugs in two ways: either alone or in combined way. these findings suggest the possible use of nominated drugs against sars-cov-2 in short time as approved by covalent docking screening. also, the present results are significant in at least directing the clinicians to use these safe drugs to stop development of corona virus and second giving hope that available drugs that can be efficient treatment. coronavirus today emphasizes as a potential threat to all people worldwide. although extensive researches have been directed to stop sars-cov-2, but till now there is no medication. meanwhile, the spreading with complicated crisis requires immediate therapy to overcome the spread and minimize mortality of sars-cov-2. the aim of the present study was to discover effective treatment through repurposing some of available fda-approved drugs against sars-cov-2 mpro. where, they can provide covalent warheads in virtual screening. the most prominent finding to emerge from this study is that the affinity of covalent binder toward sars-cov-2 mpro is ranked: saquinavir > ritonavir > remdesivir > delavirdine > cefuroxime axetil > oseltamivir ¼ prevacid. one of the more noteworthy findings in this study is that md simulation analysis that saquinavir, ritonavir, and remdesivir can form stable interaction inside the binding site of sars-cov-2 mpro. also, they restricted the essential motions of protein. overall, the results of screening toward mpret encourage for further clinical evaluations. they can be easy to reach and exploit as persuasive treatments for sars-cov-2. moroccan medicinal plants as inhibitors of covid-19: computational investigations gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers amygdalin as multi-target anticancer drug against targets of cell division cycle: double docking and molecular dynamics simulation understanding the mechanism of amygdalin's multifunctional anti-cancer action using computational approach essential dynamics of proteins implementation of the charmm force field in gromacs: analysis of protein stability effects from correction maps, virtual interaction sites, and water models a graph-based recovery and decomposition of swanson's hypothesis using semantic predications a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study emerging coronaviruses: genome structure, replication, and pathogenesis particle mesh ewald: an nálog(n) method for ewald sums in large systems in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling anti-hcv, nucleotide inhibitors, repurposing against covid-19 drug repurposing for coronavirus (covid-19): in silico screening of known drugs against coronavirus 3cl hydrolase and protease enzymes reverse vaccinology approach to design a novel multi-epitope vaccine candidate against covid-19: an in silico study two-dimensional projection of motion of trajectory of sars-cov-2 mpro bound with drugs over the pc1 and pc2 q&a: the novel coronavirus outbreak causing covid-19 in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin molecular mechanisms of novel therapeutic approaches for multiple myeloma structure of mpro from covid-19 virus and discovery of its inhibitors discovery of potential multi-target-directed ligands by targeting host-specific sars-cov-2 structurally conserved main protease virtual screening and repurposing of fda approved drugs against covid-19 main protease identification of chymotrypsin-like protease inhibitors of sars-cov-2 via integrated computational approach targeting sars-cov-2: a systematic drug repurposing approach to identify promising inhibitors against 3c-like proteinase and 2 0 -o-ribose methyltransferase theory and applications of covalent docking in drug discovery: merits and pitfalls extrapolation of phenolic compounds as multi-target agents against cancer and inflammation case of the index patient who caused tertiary transmission of coronavirus disease 2019 in korea: the application of lopinavir/ritonavir for the treatment of covid-19 pneumonia monitored by quantitative rt-pcr dapoxetine: a new option in the medical management of premature ejaculation learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 peptide-like and small-molecule inhibitors against covid-19 the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? in-silico homology assisted identification of inhibitor of rna binding against 2019-ncov n-protein (n terminal domain) identification of novel small molecules against gsk3b for alzheimer's disease using chemoinformatics approach treatment of stress urinary incontinence with duloxetine hydrochloride identification of natural inhibitors against angiotensin i converting enzyme for cardiac safety using induced fit docking and mm-gbsa studies analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods swissparam: a fast force field generation tool for small organic molecules authors thankful for staff membered of gaziantep university institute of health sciences, department of bioinformatics and computational biology. the authors declare no conflict of interest. key: cord-304263-5kddk5fa authors: c., selvaa kumar; kumar, senthil arun; wei, haiyan title: comparative docking studies to understand the binding affinity of nicotine with soluble ace2 (sace2)-sars-cov-2 complex over sace2 date: 2020-10-08 journal: toxicol rep doi: 10.1016/j.toxrep.2020.10.002 sha: doc_id: 304263 cord_uid: 5kddk5fa the study aimed to validate the proficiency of nicotine binding with the soluble angiotensin-converting enzyme ii receptor (sace2) with or without sars-cov-2 in the context of its binding affinity. modelled human sace2 and the spike (s1) protein of indian sars-cov-2 (ins1) docked with each other. on the other hand, nicotine docked with sace2 in the presence or absence of sars-cov-2. nicotine established a stable interaction with negatively charged asp368 of sace2, which in turn binds with amino acids like thr362, lys363, thr365, thr371, and ala372. in the presence of nicotine, ins1 and sace2 showed a reduced binding affinity score of -12.6 kcal/mol (vs -15.7 kcal/mol without nicotine), and a lowered interface area of 1933.6 å(2) (vs 2057.3å(2) without nicotine). the neuronal nicotinic acetylcholine receptor and angiotensin-converting enzyme 2 (ace2) receptor showed 19.85 % sequence identity among themselves. following these receptors possessed conserved trp302 and cys344 amino acids between them for nicotine binding. however, nicotine showed a higher binding affinity score of -6.33 kcal/mol for the sace2-ins1 complex than the sace2 alone with -5.24 kcal/mol. a lowered inhibitory constant value of 22.95µm recorded while nicotine interacted with the sace2-ins1 complex over the sace2 alone with 151.69 µm. in summary, nicotine showed a profound binding affinity for the sace2-ins1 complex than the sace2 alone paving for the clinical trials to validate its therapeutic efficacy as a bitter compound against the sars-cov-2 virulence. the sars-cov-2 pandemic that originated from wuhan, china has become a dreadful human threat with its unprecedented outbreak (1) . clinicians and researchers across global countries have abided various clinical strategies to attenuate the sars-cov-2 transmission by targeting its virulence characteristics using diverse anti-viral agents, including, the indigenous bioactives; sars-cov-2-protein specific monoclonal antibodies; and convalescent plasma transfusion therapy (2) (3) (4) . angiotensin-converting enzyme ii (ace2) has been the principal target of attenuating the sars-cov-2 virulence that acts as the gateway of sars-cov-2 (as a receptor agonist) amongst the humans (5) (6) (7) . the proliferation of sars-cov-2 in the upper respiratory tract has been mediated by its stable interaction with the ace2 (8) . ace2 predominantly expressed on the alveolar epithelial cells of lungs; arterial-venous endothelial cells; enterocytes of the small intestine; and arterial smooth muscle cells initiates the conversion of angiotensin-converting enzyme ii to i (8, 9) . with the increased angiotensin-ii levels, sars-cov-2 binding with ace2 perturbs its structural characteristics and biological function preceding the acute lung injury among severely ill patients (9, 10) . acute-lung injury is the outcome of the dysregulated-ace2 function via sars-cov-2 invasion thus triggering j o u r n a l p r e -p r o o f the innate-immune response overwhelmingly with the increased pro-inflammatory cytokines production, especially il-6, il-8 and il-1β (11, 12) . in compliance with these adverse clinical outcomes associated with the disrupted ace2 function via sars-cov-2 pathogenesis (9, 13) , the smokers must be the worst victims of this virus attack with the increased mortality. surprisingly, the smokers showed mild adverse symptoms compared with the non-smokers whereby the mortality rate remains unchanged between these two groups (14) (15) (16) (17) . indeed, this has gathered a wide attention on the medicinal nicotine, a bitter compound exposure among the smokers could intervene the sars-cov-2 virulence and therefore its adverse clinical symptoms (18, 19) . we hypothesize that nicotine by interrupting the sars-cov-2 binding with the ace2 could attenuate its binding, irrespective of its other likely effects in reversing the cytokine storm regulated by the α4/α7 nicotinic acetylcholine (cholinergic) receptor expressed on the neuronal, muscles, and immune-macrophage cells (18) . research studies unveiled the interaction between the structural spike 1 (s1) protein of sars-cov-2 with the nicotinic acetylcholine receptors (nachrs) that are likely to intervene with its biological function (20, 21) . amino acid residues of 381 to 386 of the s1 protein of sars-cov-2 actively indulged in establishing a stable interaction with α9 subunit of nachrs (20, 21) . with this interaction, the sars-cov-2 could effectively degrade the immune responses of the invaded host. nicotine by stably interacting with its receptor agonist nachrs could profoundly overrule the sars-cov-2 binding with nachrs. indeed, this could replenish the functioning of the nicotinic cholinergic system disrupted by the sars-cov-2 invasion (20, 22) . thus, the insilico study performed to unveil the nicotine's urge for binding with the soluble ace2 with or without sars-cov-2 in compliance with its interaction with the known human neuronal alpha4-beta2 nicotine-acetylcholine receptor (nn-achr). as a result, this would unravel the nicotine's efficacy in hindering the stable-interaction between sars-cov-2 and its receptor agonist ace2. also, the study outcome will confer a better understanding of the effect of nicotine as a bitter bio-active to tackle the sars-cov-2 binding affirmative with ace2. the human soluble angiotensin-converting enzyme 2 (sace2) protein sequence was downloaded from the uniprot database (accession number: q9byf1) (23) . and a cytoplasmic domain (aa 792-805). precisely, the amino acids of ace2 (aa 30-41; aa 82-84; aa 353-357) would be targeted by the spike 1 (s1) protein of sars-cov-2 during the hostinvasion via ace2 binding. we found the reported structural protein template of ace2-sars-cov-2 complex with the enlisted pdb id: 6vw1 (24) incomplete with numerous missing amino acid residues. the reported ace2-sars-cov-2 complex possessed 614 residues of ace2 and 527 residues of sars-cov-2. for this reason, we performed the homology modelling of each of ace2 and s1 protein of sars-cov-2 study proteins of the ace2-sars-cov-2 complex over utilizing this available crystal ace2-sars-cov-2 complex structure for the study. from the protein data bank (25) , the crystal structure of ace2 with pdb id: 6m18 (26) with 814 amino acids downloaded with reported missing residues (25, 26) . in compliance with the obtained ace2 template, the three-dimensional (3d) structure of ace2 developed using swiss-model (27) . furthermore, sace2 region extracted from the modelled ace2 protein using chimera, which later considered for detailed-structural analysis. protein sequence (mt012098) (28) of the spike 1 (s1) of sars-cov-2 of indian origin (ins1) was obtained from ncbi and utilized for its 3d-homology modelling using swiss-model online server. the ins1 protein sequence modelled using pdb id: 6vsb as a template. this 3d structure showed the sequence identity of 99.17% and a query coverage of 95% (29) . the 3d-modelled protein structures of sace2 and ins1 were subjected to energy minimization using chimera (30) . collectively, the structural evaluation of the modelled sace2 and ins1 performed using procheck-ramachandran plot server (31) . the in-silico modelled protein structures of the study proteins: ace2 and s1 protein of sars-cov-2 were superimposed with each of its existing crystal structures of ace2-sars-cov-2 complex to elucidate its structural variations (supplementary figure 1a ,b). structurally evaluated sace2 and ins1 protein models considered for docking using haddock server (32) . based on the uniprot report, the chosen interface regions of the 3d modelled protein for protein-protein docking lies within the amino acid ranges: 30-41; 82-84; and 353-357. details regarding "active site" residues of ins1 obtained from the literature review (33, 34) which form the whole receptor-binding domain (rbd) with the amino acid j o u r n a l p r e -p r o o f residues length of 319-541. passive amino acids were selected automatically using the checkbox option for sace2 and ins1. amino acids proximal to the active site regions chosen as passive residues. wholly, ten clusters of four poses each generated by the haddock server. out of ten clusters, the least haddock scored poses were subjected to binding energy evaluation using pdbepisa (35) . discovery studio visualizer standalone software was used to study the docked poses and the interactions (36). the 3d structure of nicotine downloaded from pdb id: 1uw6, which later docked with modelled sace2 protein (37) using autodock tools (version 1.5.6) (38) . based on the previous studies; we found arg273, his345, pro346, glu375, his505, and tyr515 amino acids as the active-interacting residues of sars-cov-2 with sace2 (34). kollman and gasteiger charges were added to both protein and ligands, respectively. grid box confined to the active site amino acids. prior to docking the grid box was generated with a size of 44 å, 116 å, and 48å for x, y, and z, respectively. additionally, grid centre values were maintained at -0.861, -6.417, and 3.778 were customized for x, y, and z, respectively. aside, grid maps developed using auto dock 4.0 and auto grid 4.0 program. based on the lamarckian genetic algorithm ten conformers were generated. furthermore, for protein ligand docking-sace2-sars-cov-2 complex was considered wherein the grid box developed within the sace2 site with the measured size values of 72å, 44 å, and 82å for x, y, and z, respectively. the customized grid centre was at 42.694, -9.11, and 1.389 for x, y, and z, respectively. concomitantly, the associated grid maps developed using auto grid 4.0 and autodock 4.0. the region of interaction and the poses visualized using discovery studio visualizer (29) . ace2 protein modelled using swiss-model online server wherein protein data bank id: 6m18 listed as a potential structural template with 100% amino acid identity and 99% query coverage. structurally modelled ace2 possessed both extracellular and helical domains with the amino acid length of 21 to 768. structural analysis by ramachandra plot confirms that 91.4% amino acids are in the favoured region; 8.2% in the additional allowed region; 0.4% in the generously allowed region and 0% in the unfavourable region. thus, sace2 model with the total size of 21-708 residues extracted from the complete modelled structure. fusion peptide (amino acid 788-806); heptad repeat region (amino acid 912-984); and the partial heptad repeat region 2 (amino acid 1163-1213). as per ramachandran plot report, the core region comprised of 86.1% residues; extended allowed region with 11.9% residues; generously allowed region with 1.7% and the outlier was 0.3%. nicotine established a stable interaction within the active site pocket of sace2. alternatively, ins1 interacted at the s1 protein binding residue site of sace2. collectively, nicotine showed a higher binding affinity with the lowered inhibition constant for the sace2-ins1 complex over sace2 alone. this inference unveils the keenness of nicotine binding with the ins1 bound sace2 complex than sace2. moreover, with this profound interaction, it is more likely for nicotine to interrupt the sars-cov-2 interaction with ace2. crystal structure of the human neuronal alpha4-beta2 nicotinic receptor bound with the nicotine (ligand) procured from the protein data bank (pdb id: 5kxi) (40) . based on the pdbsum report, nicotine established a profound interaction with the nn-achr through amino acids such as tyr100, trp156, cys199, cys200, and tyr204 ( figure 3a ) with a binding affinity of 8.3nm from an experimental perspective. these crucial interacting residues were mapped on the modelled human sace2 by the clustal omega-pairwise alignment (41) . as per the alignment report, the overall amino acid identity between sace2 and nn-achr was 19.85%. their global alignment analysis has unravelled the whole 109 identical residues accompanied by the 102 semiconservative residues, and 74 weakly conservative residues (supplementary figure 3 a). two dimensional (2d) structural comparison between the conserved motifs in nn-achr and sace2 revealed that amino acids like "ccaeiy" from nn-achr and "chptaw" from sace2 are a sheet with a short loop region. conversely, wt and wd of nn-achr and sace2, respectively are short loop region (supplementary figure 3b) surprisingly, the vital nicotine interacting residues of nn-achr: trp156 and cys199 found conserved in sace2, but with the varied residue numbers of trp302 and cys344, respectively. a noticeable conserved residue substitution of try204 of nn-achr with trp349 spotted in sace2 ( figure 4 ). all crucial nicotine interacting and flanking residues of sace2 such as his401, asp382, gly405, his378 and tyr385 complies well with the nicotine interacting residues: trp349, cys344, and his345 of nn-achr (figure 5a) . nevertheless, compared with nn-achr, the residues: asn210, asp303, and trp302 were spotted distinctly away from the j o u r n a l p r e -p r o o f nicotine interacting residues in sace2. while the residue asn210 was undetectable in sace2 ( figure 4) . mapping of nicotine binding residues of nn-achr on ins1-sace2 complex showed the nicotine-interacting residues flanked by the asp368, thr362, lys363, thr365, thr371 and ala372 residues located proximally to cys344 ( figure 5 ). cys344 (as cys199 in nn-achr) plays a significant role in affirming the nicotine interaction in both sace2 and nn-achr (figure 5a and 5b) . the represented study images generated using the discovery studio. blocking the entry of sars-cov-2 via the host based ace2 receptor has received more attention among the research communities (42) . to gain access into the host system, ace2 acts as the entry point of sars-cov-2 infiltration in all essential organs of the human physiology, in particular the respiratory system (43, 44) . a study report has shown the effective tackling of sars-cov-2 pathogenesis using monoclonal ig-antibodies developed in specific to the human-ace2 agonist of sars-cov-2 (45, 46) . in addition to recruiting ace-2 as the gateway (47), sars-cov-2 by establishing a strong interaction with the host-ace2 destabilize its structural conformation, and interfere its biological role in converting the angiotensin ii to angiotensin i (6, 48) . the increased angiotensin ii levels with the dysregulated ace2 receptor functions could inflict the acute lung injury among the sars-cov-2 patients (49, 50) . smokers being highly vulnerable to sars-cov-2 virulence associated with the increased airway-ace2 expression than the non-smokers (51), incredibly, they showed moderate adverse-clinical symptoms of sars-cov-2 than the non-smokers (14, 16, 18) . this noticeable clinical outcome witnessed among the smokers has compelled us (14, 16) to seek for the significant therapeutic action of bitter-bioactive nicotine compound to attenuate the sars-cov-2 virulence in the context of its interaction (as an agonist) with the ace2 receptor (15, 52) . the protein-protein docking was performed between nicotine and the 3d-modelled sace2 protein to understand the crucial nicotine-interacting residues of sace2. in this study, nicotine's preference for the sace2 binding resembles its binding affinity characteristics for the known nn-achr (53-55) whereby the conserved trp302 and cys344 residues flanked by the other crucial his378, asp382, his401,gly405, and tyr385 residues have predominantly determined its interaction with the sace2 (figure 2a and 4) . overall, the interaction of nicotine was wrapped by the crucial positively charged his401 through which the other noticeable bindings with his378, asp382, tyr385 and gly405 residues of sace2 were to summarize, nicotine's better binding preference for the ins1 bound sace2 complex than the naïve sace2 alone prone to interlude the binding of sace2 with sars-cov-2. undeniably, this could be the likely mechanism of nicotine exposure to attenuate the adverse clinical symptoms of sars-cov-2 patients who habituated to smoking over the non-smokers. we are very cautious about this study, wherein we don't encourage nicotine exposure via injurious cigarette smoking among the population as it is detrimental to lungs and health. the study outcomes merely emphasize the likely-benefits of nicotine as a bitter compound to counter the adverse clinical symptoms of sars-cov-2 pathogenesis by intervening the profound interaction of sars-cov-2 with ace2; that further demands an in-detail clinical validation among the sars-cov-2 patients. medicinal nicotine referred for the treatment of various neurological disturbances (56, 57) , could be utilized to treat the sars-cov-2 patients after being clinically evaluated of its therapeutic effects of dose/usage under the recommended safety-guidelines (15, 18, 19) . (b) nicotine bound more firmly into the active pocket site of sace2-sars-cov-2 complex (mimicking the diseased condition) by establishing the stable interactions with the ala372, thr362, lys363, thr365, and thr371 residues through asp368 in proximal to the spike (s1) protein binding site of sars-cov-2. j o u r n a l p r e -p r o o f figure 3 : nicotine binding (in black coloured structure) with the human neuronal alpha4-beta2 nicotine-acetylcholine receptor (in green coloured structure) with the key amino acid residues engaged in its interaction shown in the surface view. figure 4 : pairwise sequence alignment profile of the soluble angiotensin-converting enzyme 2 (sace2) with the neuronal nicotinic acetylcholine receptor (nn-achr). paired residues that are exclusively engaged in nicotine binding with the nn-achr has been marked using the downward arrows on the profile template. the trp302 and cys344 of sace2 residues are highly conserved in compliance to the trp 156 and cys 199 residues of nn-achr. also, the trp349 residue of sace2 and its alternative tyr204 residue of nn-achr shared similar characteristics in nature. . conserved residues such as trp349, cys344, and his345 of nn-achr (enlightened in maroon colour) shares proximity with the sace2 (enlightened in green colour). the conserved residues underlie the stable interaction of nicotine with the sace2 residues through his401. asp303 and trp302 residues of nn-achr are located far away from the nicotine binding site (highlighted using the maroon colour). structural binding characteristics of nicotine with the soluble angiotensinconverting enzyme 2 (sace2)-sars-cov-2 complex in the context of its interaction with the neuronal nicotinic acetylcholine receptor (nn-achr). nicotine bound proximal to the conserved cys344 residue (marked in maroon colour) of the sace2-sars-cov-2 complex which in-turn facilitates its further interaction with the other flanking residues of sace2 through asp368 (marked in green colour). brief review on covid-19: the 2020 pandemic caused by sars-cov-2 current and future therapeutical approaches for covid-19. drug discovery today clinical trials during the sars-cov-2 pandemic novel coronavirus 2019-ncov: prevalence, biological and clinical characteristics comparison with sars-cov and mers-cov. european review for medical and pharmacological sciences physiological and pathological regulation of ace2, the sars-cov-2 receptor sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2 angiotensin receptor blockers as tentative sars-cov-2 therapeutics. drug development research severe acute respiratory syndrome coronavirus 2 (sars-cov-2): an overview of viral structure and host response. diabetes & metabolic syndrome much more than just a receptor for sars-cov-2 angiotensin-converting enzyme 2 protects from severe acute lung failure early upregulation of acute respiratory distress syndrome-associated 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prevent and treat covid-19 nicotinic cholinergic system and covid-19: in silico identification of an interaction between sars-cov-2 and nicotinic receptors with potential therapeutic targeting implications simulations support the interaction of the sars-cov-2 spike protein with nicotinic acetylcholine receptors and suggest subtype specificity. biorxiv : the preprint server for biology editorial: nicotine and sars-cov-2: covid-19 may be a disease of the nicotinic cholinergic system the swiss-prot protein sequence data bank and its new supplement trembl structural basis of receptor recognition by sars-cov-2 the protein data bank structural basis for the recognition of sars-cov-2 by full-length human ace2 swiss-model: homology modelling of protein structures and complexes fullgenome sequences of the first two sars-cov-2 viruses from india cryo-em structure of the 2019-ncov spike in the prefusion conformation ucsf chimera--a visualization system for exploratory research and analysis 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expressions and significances of the angiotensin-converting enzyme 2 gene, the receptor of sars-cov-2 for covid-19 expression of the sars-cov-2 cell receptor gene ace2 in a wide variety of human tissues. infectious diseases of poverty a review of sars-cov-2 and the ongoing clinical trials neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig cell entry mechanisms of sars-cov-2 assessing ace2 expression patterns in lung tissues in the pathogenesis of covid-19 a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome clinical implications of sars-cov interaction with renin angiotensin system: jacc review topic of the week ace-2 expression in the small airway epithelia of smokers and copd patients: implications for covid-19. the european respiratory journal editorial: nicotine and sars-cov-2: covid-19 may be a disease of the nicotinic cholinergic system understanding of nicotinic acetylcholine receptors nicotinic receptor contributions to smoking: insights from human studies and animal models covid-19 and smoking. is nicotine the hidden link? the european respiratory journal chronic high dose transdermal nicotine in parkinson's disease: an open trial nicotine treatment of mild cognitive impairment: a 6-month double-blind pilot clinical trial the overall technical support was provided by school of biotechnology and bioinformatics, d.y. patil deemed to be university, cbd belapur. all the authors would like to acknowledge the same. no external funding was provided for this in-silico based study. key: cord-306177-5wefp31y authors: iheagwam, franklyn nonso; rotimi, solomon oladapo title: computer-aided analysis of multiple sars-cov-2 therapeutic targets: identification of potent molecules from african medicinal plants date: 2020-09-12 journal: scientifica (cairo) doi: 10.1155/2020/1878410 sha: doc_id: 306177 cord_uid: 5wefp31y the covid-19 pandemic, which started in wuhan, china, has spread rapidly over the world with no known antiviral therapy or vaccine. interestingly, traditional chinese medicine helped in flattening the pandemic curve in china. in this study, molecules from african medicinal plants were analysed as potential candidates against multiple sars-cov-2 therapeutic targets. sixty-five molecules from the zinc database subset (afrodb natural products) were virtually screened with some reported repurposed therapeutics against six sars-cov-2 and two human targets. molecular docking, druglikeness, absorption, distribution, metabolism, excretion, and toxicity (admet) of the best hits were further simulated. of the 65 compounds, only three, namely, 3-galloylcatechin, proanthocyanidin b1, and luteolin 7-galactoside found in almond (terminalia catappa), grape (vitis vinifera), and common verbena (verbena officinalis), were able to bind to all eight targets better than the reported repurposed drugs. the findings suggest these molecules may play a role as therapeutic leads in tackling this pandemic due to their multitarget activity. coronaviruses (covs) are members of coronaviridae family belonging to the order nidovirales. ey are enveloped viruses of about 60 to 140 nm in diameter with positive-sense single-stranded rna genome (+ssrna) classified into four genera, namely, alpha (α), beta (β), gamma (c), and delta (δ) [1] . ese viruses have spikes protruding from their surface, giving them a crown-like structure (hence, the name corona), which makes them bind to the human lower respiratory system [2] . since the turn of the 21 st century, covs have caused several pandemics that are not only of significant public health concerns but also distort socioeconomic activities in infected regions [3] . in 2003, severe acute respiratory syndrome (sars) was identified in guangdong, china, with sars-cov as the causative pathogen, while the middle east respiratory syndrome (mers) caused by mers-cov resurfaced a decade later in jeddah, saudi arabia [4] . ese zoonotic pathogens belong to the β-cov genus, with pneumonia and acute respiratory distress syndrome (ards) as prominent symptoms [5] . in wuhan, china, a novel pandemic initially known as 2019 novel coronavirus (2019-ncov) was reported in december 2019. it was later called coronavirus disease (covid-19) by the world health organization (who) on 11 february 2020 [6] . e disease exhibits similar symptoms with sars, consequently making the international committee on taxonomy of viruses (ictv) name the viral pathogen severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [7] . irrespective of the numerous ways and policies to contain this pandemic, it has continued to spread worldwide with an increase in its related mortality. e who situation report as of 8 june 2020 shows 6,931,000 confirmed cases with 400,857 reported deaths worldwide. in africa, 135,412 confirmed cases with 3,236 deaths were reported with the transmission classified majorly as community-based [8] . e novelty of this disease means there is no antiviral therapy or vaccine to combat it. nonetheless, the severity of this disease has led to its prioritisation and increased research on the disease and the virus by researchers worldwide, leading to a better understanding of its aetiology, pathogenesis, management, and treatment [1] . recently, lopinavir, ritonavir, remdesivir, chloroquine, hydroxychloroquine, camostat, and nafamostat, to mention a few, have been proposed as potential drug candidates that could be repurposed to combat this pandemic [9] [10] [11] [12] . traditional chinese medicine (tcm) and ayurveda system of medicine have been attributed to play a part in the flattening of the pandemic curve in china with about 60,000 people treated with tcm [13, 14] . eoretically, the secondary active metabolites in these natural products may have been responsible for the attributed success of tcm against covid-19 in china. computational method approaches are important techniques which efficiently filter, screen, select, and identify potential leads for drug development from diverse chemical databases [15] . numerous computational screening analyses have been carried out to screen and identify drug candidates with therapeutic and prophylactic potential against various proteins of sars-cov-2 from the zinc database [16] [17] [18] [19] [20] . network analysis leaning algorithm (machine learning, deep learning, and artificial intelligence (ai)) approach based on a fully connected neural network in combination with virtual screening methods as well as network-based meta-analysis has been utilised in investigating potential anti-sars-cov-2 leads from zinc database [21] [22] [23] [24] [25] . e unites states food and drug administration-(usfda-) approved drugs [26] , drugbank [27, 28] , traditional ayurvedic, chinese and natural medicine [20, [28] [29] [30] [31] , dark chemical matter, and foodb [25] are some of the zinc database subsets that have been rigourously screened for molecules to combat sars-cov-2 with main protease, rna-dependent rna polymerase, and angiotensin-converting enzyme-2 as the major therapeutic targets. despite the volume of research on computational screening analyses from different databases, there is a paucity of information on small molecules from african medicinal plants and other therapeutic targets that can help combat sars-cov-2. hence, this study analysed a plethora of natural products (nps) from african medicinal plants with known bioactivities in human as therapeutic candidates targeting and inhibiting sars-cov-2 rna synthesis, replication, structural protein function, and host-specific receptors/enzymes. a total of 65 compounds from african medicinal plants with known bioactivities in human were downloaded as a subset of afrodb natural products (http://zinc15.docking.org/catalogs/afronp/substances/ subsets/in-man/) as shown in table s1 . at the same time, lopinavir, ritonavir, remdesivir, chloroquine, hydroxychloroquine, camostat, and nafamostat which are proposed drug candidates in treating covid-19 were downloaded from pubchem (http://www.pubchem.ncbi.nlm.nih.gov) in .sdf format and used as standards. conversion to their threedimensional (3d) structures, force field generation, and the addition of nonpolar hydrogen and gasteiger charges were carried out using open babel v2.3.2 [32] , ligpargen [33] , and autodock4.2 [34, 35] , respectively. prediction. e protein structures of sars-cov-2 papainlike protease (plpro), main/3-chymotrypsin-like protease (3clpro), rna-dependent rna polymerase (rdrp), helicase, 2-o-methyltransferase (2omt), spike receptor-binding domain (s-rbd), human angiotensin-converting enzyme 2 (ace2), and human type-ii transmembrane serine protease (tmprss2) with pdb codes 6w9c, 6lu7, 6m71, 6w4h, 6m17.e, 6m17.b, and 5ce1, respectively, were retrieved from protein data bank (http://www.rcsb.org). experimental sars-cov-2 helicase structure is yet to be deposited in the protein data bank; hence, a homology structure was modelled as described by iheagwam and ogunlana [36] . briefly, sars-cov-2 helicase sequence was acquired from uniprot (http:// www.uniprot.org) via the uniprot identifier (p0dtd1) with a feature identifier (pro_0000449630). e fasta sequence was inputted in swiss-model [37] to generate a theoretical model which was evaluated using procheck, errat [38] , and verify 3d [39] . 3drefine was used to minimise the energy levels of all protein crystal structures [40] . all therapeutic targets were run on the dogsitescorer server for prediction of their molecular druggable pocket [41] . docking. igemdock v2.1 was used to screen the ligands in the binding pockets of the proteins with population size 300, 80 generations, and 10 solutions as the set screening parameters [42] . top ranking ligands present across all eight proteins [8] were further subjected to molecular docking using autodock vina [43] . gasteiger charges were computed, polar hydrogen was assigned, and grid map was set at 1å space for plpro, 3clpro, rdrp, helicase, 2omt, s-rbd, ace2, and tmprss2 protein crystal structures using autodock 4.2 as shown in table 1 before molecular docking was carried out [34, 35] . evaluation. physicochemical, pharmacokinetic, and toxicity parameters of the identified hit therapeutic molecules were predicted using swissadme [44] and admetlab [45] . e sars-cov-2 helicase fasta sequence (601 amino acid residues) which was inputted into swiss-model generated five helicase homology models from 5 templates, namely, 6jyt, 5wwp, 4non, 5ftf, and 6sje. however, the model built using 5wwp as template was selected based on its global model quality estimate, qmean, template resolution, sequence identity, similarity, and coverage over other templates (table s2, figure s1 ). in swiss-model, structural information of templates is extracted based on openstructure comparative modelling engine to provide complete stoichiometry and generate a 3d homology model structure [37] . according to xiang [46] , if the sequence identity similarity of 30% upward is shared between target and template, the homology model is considered dependable and successful. with a 72.2% similarity and qmean of −1.72, the selected model was not only successful but might have structural similarity and behaviour with experimental models [47] . upon energy minimisation of the modelled helicase, a root-mean-square deviation (rmsd) score of 0.387 and 0.588å was observed when superimposed with the modelled helicase and 5wwp, respectively ( figure s2 ). is observation suggests the homology model has a high similarity and structural orderliness with the template as corroborated by studies on tmprss2 and dipeptidyl peptidase 4 [17, 36] . e model also had good stereochemical quality as conferred by the ramachandran plot with reduced noise as shown in table s3 with a 3d verification score of 94.7% and quality factor of 88.14% further corroborating the reliability of the model ( figures s3 and s4 , respectively). docking. in the course of drug discovery, structure-based virtual screening is a computational approach utilised to identify promising novel small chemical ligands from curated chemical compound databases with potential activity against drug targets [48] . also known as 3-galloylcatechin, proanthocyanidin b1, and luteolin 7-galactoside, respectively, appeared across all therapeutic targets as the 3 rd , 10 th , and 5 th respective ranking molecule for plpro; 1 st , 3 rd , and 9 th respective ranking molecule for 3clpro; 2 nd , 4 th , and 9 th respective ranking molecule for rdrp; 4 th , 1 st , and 3 rd respective ranking molecule for helicase; 3 rd , 4 th , and 1 st respective ranking molecule for 2omt; 8 th , 2 nd , and 12 th respective ranking molecule for s-rbd; 10 th , 1 st , and 7 th respective ranking molecule for ace2; and 4 th , 5 th , and 10 th respective ranking molecule for tmprss2 (table s4) . ese three compounds were further docked using autodock vina in the binding pocket of the therapeutic targets predicted by dogsitescorer, as shown in figure s5 . pocket and subpocket detection and analysis tools are useful for assessing the druggability of therapeutic targets. dogsitescorer detects druggable pockets using a support vector machine based on the integration of grid-based method and gaussian filter difference [41] . pocket detection is usually done prior to molecular docking simulations. e selected hit ligands exhibited better autodock binding fitness than the proposed covid-19 treatment drug candidates in the binding pocket of plpro (−5.7, −4.1, and −4.6 kcal/mol, respectively), 3clpro (−6.3, −5.1, and −5.6 kcal/mol, respectively), rdrp (−8.7, −9.8, and −8.5 kcal/mol, respectively), helicase (−9.8, −10.3, and −9.2 kcal/mol, respectively), 2omt (−8.6, −10.5, and −9.6 kcal/mol, respectively), s-rbd (−5.2, −6.4, and −6.1 kcal/ mol, respectively), ace2 (−8.5, −9.7, and −8.9 kcal/mol, respectively), and tmprss2 (−8.4, −8.9, and −8.9 kcal/mol, respectively) ( table 2) . interestingly, none of them were able to target the s-rbd-ace2 interface (result not shown). in autodock vina, ligands with more electronegative binding energy are ranked higher and believed to have better binding fitness [43] . 3-galloylcatechin, proanthocyanidin b1, and luteolin 7-galactoside all had lower autodock vina scores than the proposed standards in most of the targets, suggesting better binding fitness, lower inhibition constant, and better experimental activity values [49] . 3-galloylcatechin is an extremely weak base flavonoid with almond (terminalia catappa) and grapes (vitis vinifera) as rich sources [50] . proanthocyanidin b1-like 3-galloylcatechin is also a flavonoid made up of (−)-epicatechin and (+)-catechin units, joined by a β-interflavanyl bond. it is found in cocoa powder and grapes at high concentration [51] . luteolin 7galactoside is a flavonoid glycoside found in fruits, herbs, and spices such as common verbena (verbena officinalis) [52] . e antiviral activity of 3-galloylcatechin and proanthocyanidin b1 has previously been reported [53, 54] . ghosh and chakraborty [55] and nguyen and woo [56] in their respective studies have identified gallocatechin gallate, a closely related molecule to 3-galloylcatechin, as a candidate molecule against sars-cov 3clpro. competitive inhibition of this enzyme was the proposed mode of gallocatechin gallate action. e inhibitory activity of procyanidin b1 on sars-cov infection has been reported by zhuang et al. [57] . ough procyanidin b1 mode of action was not elucidated, transferrin receptor and ace2 expression were found not to play a role in the inhibitory property. luteolin 7-galactoside, on the other hand, does not have any reported antiviral activity, but rather its effectiveness in treating sore throats, respiratory tract diseases, and its anti-inflammatory activity is well established [58, 59] . e carbohydrate moiety stereoisomer, luteolin 7-glucoside, on the other hand, has been reported to possess antiviral activity [60, 61] . luteolin was recently reported to possess possible strong antiviral activity against sars-cov-2 as a result of its low binding energy in the active site of some therapeutic targets [62] . molecular docking simulation predicts the binding properties and interactions between a ligand and its target [63] . looking at the binding mode and molecular interaction of the hit ligands in the binding pocket of sars-cov-2 therapeutic targets (plpro, 3clpro, rdrp, helicase, 2omt, s-rbd, ace2, and tmprss2) as simulated by autodock vina, conventional hydrogen bonds, carbon-hydrogen bond, van der waal forces, and various pi (π) interactions were responsible for stabilising the ligand interactions (figures 1-16 ). his1017 and cys1015 were noteworthy common amino acid residues that interacted via hydrogen bond and van der waal interaction, respectively, with all the hit ligands and proposed drugs in the binding site of plpro (figure 1 ). in the binding pocket of 2omt as depicted in figures 9-10 , asn101 (hydrogen bond), gly71, asp133 (van der waal forces), leu100, and met131 (π interactions) were the common amino acid residues that interacted with the hit molecules and proposed standards to stabilise them in the binding site while asp618 (van der waal interaction) was synonymous in the binding site of rdrp (figures 7-8) . ese amino acid residues could be targeted as possible therapeutic targets in the inhibition of these proteins due to their noncovalent stabilising activity in the druggable pocket [64] . his contains an imidazole side chain with acid-base properties which is essential in the catalytic mechanism of enzymes where it is found [65] . for 3clpro, helicase, s-rbd, ace2, and tmprss2, the amino acid residues responsible for stabilising the molecules in the binding site were not consistent for the ligands and proposed standards (figures 3-6 and 11, 12-15, and 16, respectively) . all ligands and proposed standards were able to bind properly in the binding pocket of the therapeutic targets as predicted by dogsitescorer after the docking simulation by autodock vina (figure s5 ). on the other hand, luteolin 7galactoside and ritonavir bound to the allosteric site of plpro with ritonavir extending into the active site while lopinavir as well as ritonavir bound to the allosteric site of 3clpro. is binding pose could be attributed to their chemical structures requiring a more substantial binding pocket. chloroquine (s-rbd and ace2) and hydroxychloroquine (tmprss2) were also found to bind to allosteric sites after docking ( figures s6 and s7, respectively) . e binding of hydroxychloroquine in the positively charged allosteric site of tmprss2 was similar to earlier findings of remdesivir binding with tmprss2 where hydrogen bonds with asn and arg were also formed [20] . it is of interest that the high binding energies of 3-galloylcatechin, proanthocyanidin b1, and luteolin 7-galactoside to ace2 and tmprss2 could have clinical implication in the prevention of sars-cov-2 transmission [66] . various studies have reported other phytomolecules present in african medicinal plants that have exhibited potential activity as antagonists against sars-cov-2 by interfering with different therapeutic targets [67, 68] . ese findings further buttress the role of nps and their identified bioactives in tackling the covid-19 pandemic. e physicochemical properties of the hit ligands are presented in table 3 . interestingly, none of the hit molecules were able to pass lipinski's druglikeness test with 3-galloylcatechin, proanthocyanidin b1, and luteolin 7galactoside violating 1, 3, and 2 rules, respectively. ey also violated ghose, veber, egan, muegge, and leadlikeness [36] . e druglikeness failure exhibited by the compounds is not surprising as nps have been reported to fail lipinski's druglikeness test, which is attributed to their mechanism of absorption [73] . nps have been reported to be bioavailable by exploiting complex mechanisms such as active transport unlike synthetic drugs that utilise passive diffusion [74] . is is because nps look a lot like biosynthetic intermediates and endogenous metabolites than synthetic compounds [75] . ey also differ in terms of elemental composition and stereochemical complexity [76] . interestingly, many nps that violate ro5 have been reported to remain bioavailable by lipinski [77] . advances in synthetic biology and organic synthesis methodology such as biosynthetic gene cluster manipulation, total synthesis, semisynthesis, or a combination of these methods have identified a new generation of natural product scaffolds that can be systematically targeted, to increase the activity, decrease the toxicity, and/or improve the physicochemical and pharmacokinetic properties [78] . is can also be applied as done in other natural leads during synthesis and lead optimization to improve the druglikeness of the compounds [79, 80] . e synthetic accessibility score of the compounds ranged from 4.16 to 5.32. e score which ranges from 1 to 10 is based primarily on the assumption that molecular fragment frequency in easily obtainable molecules actually correlate with the ease of synthesis [44] . e observed values for the hit ligands suggest their fragmental contribution and chemical moieties should be moderately favourable for synthetic synthesis in the pharmaceutical industry leading to potential drug discovery outcome [81] [82] [83] . in the course of drug discovery, compounds with predicted favourable pharmacokinetic and toxicity properties have the potential to pass standard clinical trial criteria's making them drug candidates [84] . looking at the absorption properties presented in table 4 , none of the hit compounds were permeable to caco-2 and human intestine. ey were also not p-glycoprotein substrates, inhibitors, and bioavailable at 30%. however, 3-galloylcatechin was predicted to inhibit the p-glycoprotein, permeate the blood-brain barrier, and become bioavailable at 20%. in contrast, proanthocyanidin b1 and luteolin 7-galactoside could permeate the blood-brain barrier and become bioavailable at 20%, respectively. ese compounds are not p-glycoprotein substrates, ensuring their bioavailability and intracellular concentration are not affected [85] . a look at the distribution properties showed 87.287, 76.369, and 78.270% as the plasma protein binding and −1.129, −0.720, and −1.028 l/kg as the volume distribution for 3-galloylcatechin, proanthocyanidin b1, and luteolin 7-galactoside, respectively. ese hit compounds were predicted to be neither inhibitors nor substrate of the various cytochrome p 450 (cyp 450 ) isoforms. 3-galloylcatechin was nonetheless the only exception inhibiting cyp3a4 and cyp2c19 isoforms (table 4) . is inhibition could lead to the accumulation of drugs metabolised by these cyp isoforms and, hence, drug toxicity may occur [86] . a half-life of 1.534, 2.11, and 1.483 hours as well as a clearance rate of 1.204, 1.015, and 1.232 ml/min/kg were the respective elimination properties of 3-galloylcatechin, proanthocyanidin b1, and luteolin 7-galactoside (table 4) . toxicity property presented in table 4 showed these compounds met the maximum recommended daily dose by the u.s. food and drug administration without being hepatotoxic, skin sensitisers, and mutagenic. ey were found to be human ether-a-go-go-related (herg) gene blocker with the ability to induce drug liver injury. on the other hand, 3-galloylcatechin was found to be mutagenic, while luteolin 7-galactoside was not a predicted hergrelated gene blocker. compounds identified as inhibitors of cyp isoforms, herg blockers, and ames mutagen can be optimised by the addition of analogues to their cores during optimization process to avoid the development of long qt syndrome and mutagenicity and overcome the few lapses in the pharmacokinetic properties [78] . ese identified hits, however, possess good admet properties while meeting the maximum recommended daily dose of usfda. in this study, computer-aided analysis was utilised to identify 3-galloylcatechin, proanthocyanidin b1, and luteolin 7-galactoside (zinc 3978503, zinc 5085289, and zinc 40422816, respectively) found in some medicinal plants (almond (terminalia catappa), grape (vitis vinifera), and common verbena (verbena officinalis)) of african flora as hit compounds against multiple sars-cov-2 targets. ese compounds could be possible leads and nutraceuticals involved in the treatment or as prophylaxis of covid-19. e scaffolds of these compounds can be optimised to improve the few lapses in its metabolism and toxicity. e results further suggest these compounds will help to overcome in some degree the old paradigm "one gene, one drug, one disease" of drug discovery. nonetheless, further in vitro, in vivo, and clinical research is required to validate the pharmacotherapeutic significance of these hit compounds as anti-sars-cov-2 therapy. e data used to support the findings of this study are included within the article and supplementary files. e authors declare no conflicts of interest regarding the publication of this paper. table s1: list of molecules downloaded from zinc database subset (afrodb natural products). table s2 : homology modelling result for sars-cov-2 helicase. table s3 : verification of stereochemical quality of sars-cov-2 helicase template and modelled and minimised modelled structure. table s4 : virtual screening result of molecules against multiple sars-cov-2 targets using igemdock. figure s1 : helicase homology model-template sequence alignment. figure s2 : 3d crystal structure of (a) homology modelled sars-cov-2 and (b) structural superimposition of 5wwp (blue), modelled helicase (white), and energy minimised modelled helicase (green). figure s3 : 3d verification plot of the minimised modelled sars-cov-2 helicase structure. figure s4 : quality factor plot of the minimised modelled sars-cov-2 helicase structure. figure s5 : predicted binding pockets of (a) plpro, (b) 3clpro, (c) helicase, (d) rdrp, (e) 2omt, (f ) s-rbd, and (g) ace2 and tmprss2 by dogsitescorer. figure s6 : 3d representation of zinc 3978503, zinc 5085289, zinc 40422816, chloroquine, hydroxychloroquine, lopinavir, remdesivir, and ritonavir colour coded as red, blue, green, yellow, purple, black, orange, and magenta, respectively, in the binding pocket of (a) plpro, (b) 3clpro, (c) helicase, (d) rdrp, (e) 2omt, (f ) s-rbd, and (g) ace2. figure s7 : 3d representation of zinc 3978503, zinc 5085289, zinc 40422816, camostat, chloroquine, hydroxychloroquine, and nafamostat in the binding pocket of tmprss2 colour coded as red, blue, green, yellow, black, orange, and pink, respectively. 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prevention and management of the novel coronavirus (sars-cov-2) using molecular docking approach identification of new anti-ncov drug chemical compounds from indian spices exploiting sars-cov-2 main protease as target open babel: an open chemical toolbox ligpargen web server: an automatic opls-aa parameter generator for organic ligands a semiempirical free energy force field with charge-based desolvation automated docking using a lamarckian genetic algorithm and an empirical binding free energy function model optimization and in silico analysis of potential dipeptidyl peptidase iv antagonists from gc-ms identified compounds in nauclea latifolia leaf extracts swiss-model: homology modelling of protein structures and complexes verification of protein structures: patterns of nonbonded atomic interactions assessment of protein models with three-dimensional profiles 3d refine: an interactive web server for efficient protein structure refinement combining global and local measures for 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lacking c-4 (c-ring) deoxy flavonoid nucleophiles e flavonoid chemosystematics of egyptian verbena species bioactivity guided fractionation of phyllanthus orbicularis and identification of the principal anti hsv-2 compounds procyanidin b1 purified from cinnamomi cortex suppresses hepatitis c virus replication identification of polyphenols from broussonetia papyrifera as sars cov-2 main protease inhibitors using in silico docking and molecular dynamics simulation approaches flavonoidmediated inhibition of sars coronavirus 3c-like protease expressed in pichia pastoris procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection e role of flavonoids in the antiinflammatory activity of chamomilla recutita e chemistry and biological activities of natural products from northern african plant families: from taccaceae to zygophyllaceae luteolin-7-o-glucoside present in lettuce extracts inhibits hepatitis b surface antigen production and viral replication by human hepatoma cells in vitro anti-hbv active flavone glucosides from euphorbia humifusa willd computational screening of antagonists against the sars-cov-2 (covid-19) coronavirus by molecular docking in silico computational screening of kabasura kudineer-official siddha formulation and jacom against sars-cov-2 spike protein amino acid transporters as disease modifiers and drug targets histidine biosynthesis sars-cov-2 prophylactic and treatment; a counter argument against the sole use of chloroquine molecular docking analyses of activity against therapeutic targets of sars-cov-2 potential inhibitors of coronavirus 3-chymotrypsin-like protease (3clpro): an in silico screening of alkaloids and terpenoids from african medicinal plants a "rule of three" for fragment-based lead discovery? a knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. a qualitative and quantitative characterization of known drug databases lead-and drug-like compounds: the rule-offive revolution molecular properties that influence the oral bioavailability of drug candidates gene expression profiling analysis reveals putative phytochemotherapeutic target for castration-resistant prostate cancer structural diversity of biologically interesting datasets: a scaffold analysis approach e impact of natural products upon modern drug discovery properties and architecture of drugs and natural products revisited chris lipinski discusses life and chemistry after the rule of five natural product synthesis in the age of scalability case history: discovery of eribulin (halaven ™ ), a halichondrin b analogue that prolongs overall survival in patients with metastatic breast cancer fixing the unfixable: the art of optimizing natural products for human medicine twenty years on: the impact of fragments on drug discovery unraveling plant natural chemical diversity for drug discovery purposes fragment-to-lead medicinal chemistry publications in 2017: miniperspective a molecular modeling approach to identify effective antiviral phytochemicals against the main protease of sars-cov-2 on the mechanism of substrate/non-substrate recognition by p-glycoprotein covid-19: cadd to the rescue acknowledgments e authors are grateful to the frontline workers who have remained persistent during this pandemic. key: cord-259229-e8m8m4ut authors: samidurai, arun; das, anindita title: cardiovascular complications associated with covid-19 and potential therapeutic strategies date: 2020-09-16 journal: int j mol sci doi: 10.3390/ijms21186790 sha: doc_id: 259229 cord_uid: e8m8m4ut the outbreak of coronavirus disease 2019 (covid-19), an infectious disease with severe acute respiratory syndrome, has now become a worldwide pandemic. despite the respiratory complication, covid-19 is also associated with significant multiple organ dysfunction, including severe cardiac impairment. emerging evidence reveals a direct interplay between covid-19 and dire cardiovascular complications, including myocardial injury, heart failure, heart attack, myocarditis, arrhythmias as well as blood clots, which are accompanied with elevated risk and adverse outcome among infected patients, even sudden death. the proposed pathophysiological mechanisms of myocardial impairment include invasion of sars-cov-2 virus via angiotensin-converting enzyme 2 to cardiovascular cells/tissue, which leads to endothelial inflammation and dysfunction, de-stabilization of vulnerable atherosclerotic plaques, stent thrombosis, cardiac stress due to diminish oxygen supply and cardiac muscle damage, and myocardial infarction. several promising therapeutics are under investigation to the overall prognosis of covid-19 patients with high risk of cardiovascular impairment, nevertheless to date, none have shown proven clinical efficacy. in this comprehensive review, we aimed to highlight the current integrated therapeutic approaches for covid-19 and we summarized the potential therapeutic options, currently under clinical trials, with their mechanisms of action and associated adverse cardiac events in highly infectious covid-19 patients. coronavirus-19 is an emerging infectious disease caused by the novel single-stranded rna enveloped severe acute respiratory syndrome-coronavirus-2 (sars-cov-2). the first case of covid-19 was reported on 8 december 2019 in hubei province of china [1] and within a short span of time, the disease quickly spread to other parts of the world [2] and has rapidly evolved as a global pandemic situation. the first confirmed case of covid-19 in the united states of america (usa) was reported on 20 january 2020 in the state of washington when a 35-year-old man showed symptoms of sars-cov-2 infection after returning from wuhan, china [3] . the first person-to-person transmission of a confirmed covid-19 case in usa was reported in illinois on 30 january 2020 after an initial positive diagnosis of covid-19 on the patient's wife, who returned from wuhan, china in mid-january 2020, and unfortunately, covid-19 is now widespread in all 50 states across the usa [4, 5] . according to johns hopkins coronavirus resource center, as of 9 september 2020, there are 27,699,974 confirmed covid-19 cases and 900,239 confirmed deaths worldwide [6] . usa is now the epicenter of the disease; the death toll has reached 190,763 with 6,358,983 confirmed covid-19 cases [7] . figure 1 is the graphical representation of most affected regions of confirmed cases with reported deaths across the world. countries with more than 100,000 confirmed cases with reported deaths are presented in figure 1a ,b; and countries with more than 50,000 cases (but fewer than 100,000) and corresponding reported deaths are shown in figure 1c ,d. understanding the structure and the genetic makeup of sars-cov-2 is important to appreciate the ongoing efforts to address this disease and for the discovery of drugs and vaccines. sars-cov-2 is spherical in shape and consists of multiple components which are essential for their replication and transcription: (1) several club shaped projections on the surface of the envelope, called spike glycoprotein (s), which helps in anchoring to the host cell and acts as an inducer to neutralize antibodies, (2) a small membrane envelope protein (e), (3) structural membrane protein (m), which spans the lipid bilayer, (4) hemagglutinin-esterase glycoprotein (he), which destroys the sialic acid present on the host cell and helps the virus to inject its genetic material, (5) nucleoprotein (n) and (6) the key component, the positive-sense single-stranded genomic rna [8] [9] [10] . the typical structure of covid-19 virus depicting the above-mentioned components is shown in figure 2a . the genome size of covid-19 is about 26.4-31.7 kb [11] and is the largest among all known rna viruses in this category (sars-cov and middle east respiratory syndrome, mers). sars-cov-2 encompasses several open reading frames (orf) along with its 5 utr and 3 utr regions. orf1a/b (frame shift) is the full-length gene (29.8 kb size) that encodes replicase polyprotein named pp1a protein and 16 accessary (non-structural proteins (nsps) and orf1b codes for pp1b and 10 nsps [11] [12] [13] [14] . the structural proteins including spike (s), envelope (e), membrane protein (m) and nucleoprotein (n) are coded by orfs 10 and 11 present on the 3' utr region. several other essential accessary proteins are coded by orf3, orf7a, orf7b, and orf8 [15] . the domain structure of sars-cov-2 is presented in figure 2b . the genomic sequencing data obtained from covid-19-infected patients in china revealed the distinct features of sars-cov-2 [16] . sequence comparison showed the novel sars-cov-2 was more distantly correlated with sars-cov (about 79%) and mers-cov (about 50%). some of the salient features of sars-cov-2 make it unique and virulent compared to previously known coronaviruses. reports suggest that sars-cov-2 lacks the orf8a protein present in sars-cov and has variation in the 3c and 8b proteins [12] . in addition, a single mutation at n501t in the spike protein strengthened the binding efficiency of sars-cov-2 to angiotensin-converting enzyme 2 (ace2), the primary mode of entry into host cell [17] . structural details of sars-cov binding to the host cell through spike protein give us the clue about the importance of the mutation in this region [18] . studies show that this mutation in the spike protein of sars-cov-2 increases its binding affinity to ace2 in human by 10-20 times higher than sars-cov [19] . this mutation in the spike protein may be one of the key attributes of sars-cov-2 that led to its rapid spreading around the world in a very short period. there are six different strains of sars-cov-2 identified so far, namely, (1) l strain (originated in wuhan, china and the parent orf8-l84s strain), (2) s strain (mutation of orf8, l84s), (3) v strain (variant of orf3a coding protein ns3, g251v), (4) g (mutation in spike protein, d614g), (5) gh strain (mutations in spike protein, d614g and orf3a, q57h) and (6) gr (mutation in nucleocapsid gene, rg203kr) [20, 21] . among these variants, g strain is the most widespread, has undergone several mutations since january 2020, and branched into subtypes gr and gh. the g and gr strains are prevalent in europe, including in italy, whereas the gh strain is widespread in germany and france. the most predominant variant found in north america is the gh strain, and this subtype along with the gr clades accounts for 74% of all global sequences of sars-cov-2 genome [22] . as of now, strains g, gh and gr are constantly increasing, globally, and it is yet to be determined whether the unique nature of these strains is associated with the disease intensity. covid-19 virus is predominantly transmitted from human to human through respiratory droplets or aerosols (>5-10 µm in diameter) and contact routes. inhalation of respiratory droplets and aerosols from covid-19-infected persons is the most likely potential mode of transmission of the disease [23] . once in the host system, sars-cov-2 follows the traditional steps similar to any other virus for its mode of entry into the host cells [24] . the spike protein anchors the virus to the surface of the host cell by binding to the ace2 receptor [25] . the virus undergoes conformational changes to fuse to the cell membrane of the host cells and engulfs into the cytoplasm of the cell by endosomal pathway. once inside the cell, the virus releases its genomic rna and multiplies using the host's molecular machinery. experimental evidence using hela cells demonstrate that sars-cov-2 entry into host cell is activated by cell surface proteases and lysosomal proteases such as transmembrane serine protease 2 (tmprss2) and lysosomal cathepsin [26] . ace2 is expressed in type ii alveolar cells, the predominant portal of entry in the lungs; it is also expressed in the heart, intestine and kidney as well as on the epithelial cells of oral mucosa and the tongue [27, 28] . sars-cov-2 primarily affects the respiratory tract, and infected patients suffer from pneumonia and flu-like symptoms ( figure 3 ). the patients might need intensive care and artificial ventilation after developing acute respiratory syndrome (ards) or multiple organ dysfunction syndrome (mods). the abundance of sars-cov-2 compromises the normal function and leads to complications in lungs (inflammation, hypoxia, cytokine storm, pulmonary edema, acute respiratory distress syndrome) and in heart (myocardial infarction, heart failure, myocarditis and arrhythmia). ace1, angiotensin i-converting enzyme; ace2, angiotensin-converting enzyme 2; acei, ace inhibitor; at1r, angiotensin type 1 receptor; at2r, angiotensin type 2 receptor; arbs, angiotensin ii type-i receptor blockers; ctni, cardiac troponin i; mas, mitochondrial assembly receptor; mras, mineralocorticoid receptor antagonists; tmprss2, transmembrane serine protease 2. the lungs, being a major organ targeted by sars-cov-2 infection, are severely compromised in delivering their function. one of the common clinical manifestations of covid-19 at the late stage of the disease is shortness of breath, pneumonia-like symptoms and hypoxia, which ultimately is fatal to the patients. in the pulmonary vasculature, sars-cov-2 enters through endocytosis and activates adam metallopeptidase domain 17 (adam17), which in turn cleaves ace2, which indicates the loss of protection against the renin-angiotensin-aldosterone system (raas), which is mediated by cleaved ace2 [29, 30] . the activation of adam17 also triggers acute pulmonary inflammation and infiltration of cytokines and leukocytes in the alveolar space and results in pulmonary edema [9, 31] . overactive systemic inflammation as a response to covid-19 infection results in cytokine storm and leads to respiratory difficulties and accounts for majority of the deaths during end stage of the treatment [32] . pulmonary complications associated with covid-19 infection include diseases such as acute respiratory distress syndrome (ards), vascular endothelialitis, sepsis, pulmonary edema and pulmonary embolism [33, 34] . a multicenter cohort study involving 235 hospitals from 24 countries, which includes 1128 patients and 294 cases of confirmed covid-19, suggests that up to 51.2% suffer severe pulmonary complications post-surgery and the majority of the deaths are largely due to pulmonary embolism [33] . lung autopsy reports obtained from covid-19 patients who died due to ards showed severe alveolar damage and infiltration of perivascular t-cells. histology analysis also demonstrated increased thrombus formation, intussusceptive angiogenesis and microangiopathy in covid-19 patients compared to influenza [32] . gene expression analysis using rna isolated from covid-19 patients showed several inflammatory markers and angiogenesis-related genes were differently regulated compared to healthy lungs. most importantly, there was a significantly increased positive count for ace2 in covid-19 tissues compared to control. covid-19 patients with ards were also characterized by an increased deposition of fibrin and high expression of d-dimers and fibrinogen, suggesting fibrinolysis as a determining factor of mortality [35] . although pulmonary complication is the dominant clinical manifestation of covid-19, underlying cardiovascular complication as well as developed acute cardiac injury enhances the vulnerability of the patient. acute respiratory complication/failure and cytokine storm may cause reduced oxygen supply, which could lead to acute myocardial injury in covid-19 patients [36] . patients with cardiovascular disease (cvd) have an increased risk for severity and mortality with covid-19 infection, mainly because of the abundance of ace2 receptor in the cardiovascular system, which serves as a gateway for the entry of virus in lungs and heart [37] . respiratory illness and acute cardiac injury are major clinical manifestations observed in patients infected with sars-cov-2 during the late stage complications of the disease [38] . reasonably, patients with coronary artery disease or heart failure are vulnerable to developing major cardiac injury, and once such patients are infected with sars-cov-2, they are at greatest risk of serious myocardial impairment or cardiac dysfunction, requiring hospitalization due to unexpected deterioration, and eventual mortality is greater among these patients. a brief view of cardiovascular complications associated with covid-19 is presented in figure 3 . voluminous available clinical evidence confirms that the severity of covid-19 is pronounced in patients with a prevalence of underlying cardiovascular diseases, and in many of these patients, the virus causes severe myocardial injury [39] , including myocardial dysfunction, cardiomyopathy, arrhythmias and heart failure during the course of critical illness [40] [41] [42] [43] [44] [45] [46] . according to the death data of the cdc (centers for disease control and prevention), different health conditions contribute to the deaths of covid-19 patients in united states, which are summarized in figure 4 [47] . deaths that are associated with more than one underlying condition, e.g., deaths involving both diabetes and respiratory arrest or cardiovascular complications and respiratory arrest, etc. hypertension, diabetes, cardiac arrest, ischemic heart disease, and heart failure are the major risk factors and have contributed to the fatalities in covid-19 cases. the renin-angiotensin-aldosterone system (raas) consists of an enzymatic cascade that controls blood pressure by regulating circulatory homeostasis, body fluid and systemic vascular resistance, all of which are involved in the regulation of a myriad of cardiovascular system [48] . ace1 (angiotensin i-converting enzyme) cleaves angiotensin i (ang-i) to angiotensin ii (ang-ii), which binds to and activates angiotensin type 1 receptor (at1r), which leads to vasoconstriction, inflammation, fibrosis and proliferation [28] (figure 3 ). ace2 converts ang-ii into angiotensin 1-7 (ang 1-7), which has vasodilating and anti-inflammatory effects by binding to mas receptor (mas-r). ace2 also cleaves ang-i into angiotensin-1-9, which is further converted into ang 1-7 by ace. therefore, ace2 regulates abnormal activation of the raas, which can prevent the development of hypertension, cardiac hypertrophy, and heart failure [49] . an increase in ace2/ace1 ratio protects against endothelial dysfunctions and vascular constriction, and exogenous ace2 activation attenuates thrombus formation and reduces platelet attachment to vessels [50, 51] . the etiology of ace2-dependent cardiovascular complications with covid-19 infection is rather complex. sars-cov-2 enters cardiovascular cells/tissue by binding to the membrane-bound form of ace2 (ace2 receptor). elevated levels of ace2 and its activity are the biomarkers of cardiovascular disease including patients with heart failure [52] , which indicate that these patients may be more susceptible to covid-19 infection [53] , with worsened prognosis of cardiovascular disease treatment [54] . measuring plasma angiotensin peptides and plasma ace2 levels can provide a direct evaluation on the progress of treatment and the state of the raas in covid-19 patients [55] . nevertheless, earlier clinical studies conveyed that treatment with soluble form of recombinant human ace2 (rhace2; apn01 (0.4 mg/kg, iv, bid for 7 days), gsk2586881: 0.4 mg/kg, iv, bid for 3 days) neutralized the excessive sars-cov virus in the system and preserved the protective cellular effects of ace2 in ards patients [56, 57] . consequently, scientists propose the therapeutic potential of soluble recombinant ace2, which can overwhelm sars-cov-2 to prevent its binding to cellular ace2 [58] . in addition, ace inhibitors (acei), which upregulate ace2 expression on the cell surface, have been proven to be successful, and improved the survival rate in patients undergoing covid-19 treatment [49] . abundant expression of ace2 on the cell surface following virus infection may maintain ang-ii degradation, which could reduce at1r activation and the risk of deleterious outcomes of covid-19. while the ace2 gene is located on the x-chromosome, gender has an impact among covid-19 patients, where men are at increased risk of susceptibility to covid-19 infection and cvd complications due to their hemizygous allele for ace2 compared to heterozygous allele in female [28] . interestingly, clinical data from 1485 european men and 537 women with heart failure showed elevated level of circulating plasma ace2 in men than in female [59] . this data complements with an observation of increased prevalence and susceptibility of covid-19 in males and demonstrates that abundance of ace2 receptor in cardiovascular cells can lead to severe clinical complications [60] [61] [62] . considering the importance of ace2 in the development of hypertension and diabetes mellitus, patients with covid-19 exhibit severe comorbidities including hypertension and diabetes with poor prognosis. initial evidence from 44,672 confirmed cases in china showed 4.2% with cvd and 12.8% with hypertension. however, among the death rate, 6% had hypertension, 7.3% had diabetes and 6.3% suffered from chronic respiratory disease [62, 63] . in another study involving a small population of 99 patients, 40% had underlying cvd or cerebrovascular disease [64] . interestingly, data from a small registry of 41 admitted patients showed an alarming 73% were men, with a median age of 49 [9] . however, data involving 5700 patients (admitted during 1 march to 4 april 2020) with a median age of 63 from new york city, the epicenter of covid-19 spread in the usa, showed a slightly different picture [38] . the most common underlying comorbidities were hypertension (57%), obesity (42%), and diabetes (34%). figure 4 depicts the statistics on distribution of underlying conditions among covid-19 patients based on the data from center for disease control department (cdc), usa. the data reveals that hypertension is a major comorbid factor for covid-19 fatalities. an increased risk of covid-19 death was associated with an age greater than 65 years (mortality of 10%), cvds (coronary artery disease: 10.2%; heart failure: 15.3%; cardiac arrhythmia: 11.5%), chronic obstructive pulmonary disease (14.2%), and current smoking (9.4%). another detailed observational meta-analysis (49,076 confirmed covid-19 case) of data available from public domains including databases from medline, embase and web of science showed patients with preexisting condition of cvd, diabetes and hypertension are significantly associated with a higher risk of developing severe complication with covid-19 disease. precisely, the analysis comparing the complications between severe vs non-severe (mild to moderate) covid-19 cases concluded that cvd was significantly associated with increased illness severity and adverse outcomes among covid-19 patients [65] . recently, cdc suggests that children with certain medical conditions, like neurological, genetic, and metabolic conditions, or congenital heart disease might be at increased risk of severe illness from covid-19 compared to other children. additional study comprising 191 patients from two hospitals in wuhan, china reported 48% of patients had underlying comorbidity factors: 30% with hypertension, 19% with diabetes and 8% with coronary heart disease [40] . in another cohort with 1591 confirmed covid-19 patients (during 20 february and 18 march 2020) with an average median age of 63 from lombardy, italy, 68% had at least one underlying comorbidity, hypertension (49%), cvd (21%), hypercholesterolemia (18%), or diabetes (17%) [66] . moreover, a staggering 82% of the patients were male and the mortality rate was higher in elderly patients aged ≥64 years compared to younger patients (36% vs. 15%). due to the high prevalence of hypertension in the older population, elderly male individuals may be at the highest risk of infection with worse outcomes, and an increased mortality rate with respect to younger patients. patients with hypertension are mostly treated with ace inhibitors (acei) and angiotensin ii type-i receptor blockers (arbs), which substantially increases the expression of ace2, due to negative feedback activation caused by low level of ang-i in the system [67] . considering ace2 as a preferential receptor of sars-cov-2, the patient with antihypertensive therapy with acei/arbs may be at higher risk of developing severe covid-19 with poor prognosis [67] . remarkably, clinical studies do not support this hypothesis and found no evidence to demonstrate use of acei or arb as a risk factor in covid-19 patients [49] . multiple investigators have demonstrated the beneficial therapeutic effect of acei or arb in prevention of covid-19 infection [68, 69] . independent studies conducted among hypertensive patients found no association between the use of acei or arb and increased risk of mortality in covid-19-positive cases [70, 71] . a population-based case-control study in the lombardy region of italy with a total of 6272 patients with covid-19 (21 february and 11 march 2020) reported that 22.2% patients were receiving arb and 23.9% patients were receiving acei [70] . other antihypertensive drugs were also used more in covid-19 patients than in controls and they had a more frequent history of hospitalization due to cardiovascular complications. however, this study showed no evidence of association of use of anti-hypertrophic drugs including acei or arbs and susceptibility of covid-19. another study with 12,594 patients in the new york university (nyu) langone health, in which 5894 patients were positive for covid-19 (46.8%), reported 4357 patients had a history of hypertension (34.6%) [72] . among these hypertensive patients, 2573 (59.1%) patients were positive for covid-19 (59.1%). this study also identified no substantial adverse effect with the use of antihypertensive drugs including acei or arbs in the covid-19 positive patients. therefore, prospective research is warranted to clarify the accuracy of existing contradictory hypotheses regarding the use of acei or arbs to control of blood pressure in hypertensive patients during viral infections. fundamentally, after entering into the cells via ace2 receptors and excessive binding of the sars-cov-2 result in the downregulation of ace2 by intracellular degradation and shedding, which could reduce the ang-ii degradation and activation of at1r with induction of myocardial hyper-inflammatory reaction in response to covid-19 [49, 73] . due to acei or arb treatment, more ace2 may be localized in the cell surface after virus binding, which could facilitate ang-ii degradation with reduction of at1r activation [49] . apart from hypertension and age, acute cardiac injury, chronic heart damage and heart failure have all been observed in patients treated for covid-19 infection [44, 74] . due to acute inflammation, procoagulant stimulus and endothelial cell dysfunction, various influenza rna viruses are involved in the development of human atherosclerotic plaques and progression of atherosclerosis. de-stabilization of vulnerable atherosclerotic plaques triggers acute myocardial infarction (mi) or cardiovascular death [75, 76] . myocardial infarction, commonly known as heart attack, is a clinical condition, where oxygen supply to the heart is restricted and results in the irreversible loss of cardiomyocytes due to activation of cardiac apoptosis [77] [78] [79] . a large population of patients diagnosed with covid-19 has died due to mi [80] . data obtained from a laboratory in lombardy, italy suggest that 60.7% (17 out of 28 cases) of patients with confirmed covid-19 and an existing condition of st-elevation myocardial infarction (stemi) had to undergo a repeated coronary angiogram and coronary lesion was identified as a major cause of the complication [81] . myocardial injury was also identified as a major contributor of mortality in covid-19 patients, as derived using data from hospitals in wuhan, china [39] . strikingly, the cardiac troponin i (ctni) level, a distinct marker of myocardial injury, was noticeably elevated in 52 patients out of 187 hospitalized patients with covid-19 (27.8%) and the mortality was nearly 70% in these patients with elevated ctni. progressive increased levels of c-reactive protein and n-terminal pro-b-type natriuretic peptide (nt-probnp) coexisted with elevated ctni levels in these covid-19 patients, which enhance the severity of inflammation and ventricular dysfunction. among 138 patients treated for covid-19 (admitted to zhongnan hospital of wuhan university during january 2020), 33 patients had either acute myocardial injury or cardiac arrhythmia, as suggested by their elevated ctni level of 0.011 ng/ml versus 0.0051 ng/ml for those who were treated in non-icu [82] . several other retrospective multi-center cohort studies from china have also confirmed the significant elevation of biomarkers of myocardial injury over the course of covid-19 infection that were strongly associated with rapid surge of irreversible clinical deterioration and increased mortality [40, 41, 44, 83] . although limited data are available on the incidence of heart failure in patients with covid-19, the study with 191 hospitalized patients with confirmed covid-19 (ranging in age from 18 to 87 years) in wuhan, china (until 31 january 2020) reported that heart failure was identified in 44 patients (23%), among them, 28 (52%) patients died and 16 (12%) patients recovered [40] . cardiac injury, as a common complication (19.7%), was associated with an unexpected high risk of mortality during hospitalization of elderly patients with covid-19 in wuhan, china [41] . evidence indicated in another retrospective study that apart from ards and sepsis, acute cardiac injury (77%) and heart failure (49%) were the most common critical complications of death in 113 deceased patients with covid-19 in wuhan, china [84] . several other case reports also established that acute or end-stage heart failure was the main pathophysiological manifestation of covid-19 [61, 85, 86] , which might be associated with hyperinflammation and oxidative stress [53, 87, 88] . interestingly, one recent study indicated a decline in emergency department visits for heart failure during the covid-19 pandemic, partly due to effective remote clinician-patient interactions [89] . since patients with cvd are considered to be more vulnerable to sars-cov-2 infection, with higher risk of negative consequences, these patients avoid frequent hospital visits and prefer alternative remote management. however, analyzing the clinical records during the covid-19 pandemic (between 20 february and 20 april 2020) of emergency department of san filippo neri hospital in rome, italy, a study revealed patients with acute heart failure often reported to the emergency department after significant clinical deterioration with high mortality due to failure of routine clinical assessment [90] . emerging studies indicate that severe covid-19-related death is associated with coagulopathy, venous thromboembolism ((vte) and disseminated intravascular coagulation (dic) [91] . data obtained from the covid-19 patient population in wuhan, china indicate an abnormal coagulation pattern with prolonged prothrombin time [91] . there were 183 patients registered in this study and parameters such as (dic), antithrombin activity, prothrombin time (pt) and d-dimer, a fibrin degradation product, were measured and compared between survivors and non-survivors. the results showed elevated levels of dic and d-dimers and prolonged pt in non-survivors and suggest thrombus formation may have contributed to the mortality in these patients. this notion is strongly supported by the observation that treatment of covid-19 patients with anti-coagulation drug heparin resulted in reduced mortality rate [92] . the 28-day mortality study between heparin users and nonusers indicated that only selected covid-19 patients with markedly higher sepsis-induced coagulopathy (sic) score or elevated d-dimer were benefited from the anticoagulant therapy. notably, anticoagulant treatment may endanger those patients without significant coagulopathy, because the activation of coagulation with local thrombosis/fibrin deposition could limit the survival and dissemination of microbial pathogens and reduce their invasion [93] . myocarditis is a disease marked by the inflammation of the heart muscle, most often due to viral infection. this inflammation interferes with the electrical system and compromises the pumping capacity of the heart and results in arrhythmia and cardiac arrest [94] . common diagnosis procedures include electrocardiogram (ecg), mri (magnetic resonance imaging) and a manifestation of increased cardiac troponin i (ctni) level. covid-19 patients with severe stage of illness manifest systemic hyperinflammation syndrome [95] . this data suggests an effect of adverse inflammatory reaction or cytokine storm in response to covid-19 treatment and defines a strong role for ace2 signaling in covid-19 disease [95] . several reports have shown that patients with covid-19 infection are diagnosed with myocarditis [41, 44, 61, 66] . in a case report of a 69-year-old man admitted in lombardy, italy with respiratory difficulties and required mechanical ventilator, with worsening heart condition. transthoracic echocardiography showed mild left ventricle hypertrophy (lvh) with preserved left ventricular ejection fraction and normal wall motion and elevated plasma troponin level (at 9.0 ng/ml) [83, 96] . cardiovascular mri was suggestive of myocarditis and the patient tested positive for covid-19 infection demonstrating sars-cov-2 infection was the most likely cause for the incidence of myocarditis [96] . similarly, a 53-year-old healthy woman was diagnosed with acute myopericarditis upon covid-19 infection. cardiac mri showed a severe left ventricular dysfunction (ejection fraction-35%). the patient also had myocyte necrosis with high-sensitivity cardiac troponin t (hstnt) level concentration of 0.24 ng/ml [61] . these reports suggest that patient with covid-19 infection are prone to myocarditis, and physicians would suspect such conditions along with underlying morbidity factors like hypertension and other cvd. emerging clinical and epidemiological evidence suggests that metabolic disarray, hypoxia and accentuated myocardial inflammation due to sars-cov-2 infection plays a critical role in the pathophysiology of myocardial injury and prevalence of arrhythmic complications [97] . in a clinical cohort with 138 patients with covid-19 in wuhan, china, cardiac arrhythmias were considered a major complication in 23 patients (16.7%) who were transferred to the intensive care unit (icu) [44] . specifically, cardiac arrhythmia was more common in icu patients than in non-icu patients (44.4% vs. 6.9%). a recent study from new york-presbyterian/columbia university irving medical center highlighted the spectrum of life-threatening arrhythmias observed in four patients with covid-19 infection [98] . fulminant myocarditis with cardiogenic shock could also coexist with atrial and ventricular arrhythmias, which could increase the severity of covid-19 patients, including death [99, 100] . therefore, the expected cardiac arrhythmogenic effect of covid-19 may be an important underlying risk of disease complication, which needs additional precautions and specialized management. based on the available clinical data, potential myocardial injury is a relevant challenge among hospitalized patients with covid-19 with increased risk of mortality; therefore, it is essential for multidisciplinary assessment, including blood pressure control in hypertensive patients as well as cardiovascular evaluation and therapy to reduce the morality for covid-19 infection. strikingly, a recent study in germany involving 100 patients with an average age of 49 years who recently recovered from covid-19 infection recognized the cardiovascular sequelae, irrespective of preexisting cardiac conditions [101] . cardiovascular magnetic resonance imaging (cmr) revealed that 78 patients had abnormal cardiac structural changes, 76 had detectable levels of biomarker of cardiac injury, e.g., elevated level of high-sensitivity cardiac troponin t (hstnt), lower left ventricular ejection fraction, higher left ventricle volumes, higher left ventricle mass, and raised native t1 and t2 (quantitative assessments of the myocardium composition), commonly found after a heart attack, and 60 had signs of inflammation. the exact molecular mechanism by which sars-cov-2 virus leads to cardiomyocyte injury is not completely understood. however, the abundant expression of ace-2 receptors in the heart plays an important role in the accumulation of sars-cov-2 virus in the cardiac tissue, which eventually results in hyperactivation of inflammation and cardiac tissue injury in patients. recently, autopsy results of 39 patients, who died at early stage of covid-19 infection in germany, revealed the most likely localization of sars-cov-2 not to be in the cardiomyocytes, but in interstitial cells or macrophages invading the myocardial tissue [102] . however, another emerging study using human induced pluripotent stem cell-derived cardiomyocytes (hipsc-cms) shows sars-cov-2 can directly enter and replicate in hipsc-cms and induce apoptosis, which results in cessation of cardiomyocyte beating after 72 h of infection [103] . the majority of the covid-19 patients suffering from cardiovascular complications show a significant elevation of ctni, nt-probnp and interleukin-6 (il-6) or other cytokines [il1b, il1ra, il7, il8, il9, il10, c-x-c motif chemokine 10 (cxcl10), chemokine (c-c motif) ligand 2 (ccl2), granulocyte-macrophage colony-stimulating factor (gm-csf), and tumor necrosis factor-α (tnf-α)] in their blood stream [9, 40, 104] . severe hyperinflammation or cytokine storm due to immunological dysregulation may be the primary contributor to cardiomyocyte injury [105] . epidemiological studies with other viral rnas indicated that after entering into the cytoplasm of cardiomyocytes, viral rna is further transcribed and translated into the viral structural proteins to form the complete infectious virion [106] . ultimately, infected cardiomyocytes would be lysed, which could lead to activation of the innate immune response with induction of pro-inflammatory cytokines, inflammation-induced destabilization of coronary artery plaques and development of left ventricular dysfunction [107] . collectively, uncontrolled hyperactivated t-lymphocytes with systemic inflammation appears to be the most common mechanisms of the cardiomyocyte injury in covid-19 patients with profound cardiovascular consequences. in addition to binding to ace2 of the host cell, the priming of the transmembrane spike (s) glycoprotein of sars-cov-2 by host proteases (furin, a signature protease of highly pathogenic viruses) through cleavage at the s1/s2 and the s2 sites could enhance its transmissibility and pathogenicity [108] . multiple evidences have revealed that the notch signaling plays a major role in maintaining the homeostasis of the cardiovascular system, including atherosclerosis progression and ventricular remodeling after myocardial infarction [109] [110] [111] . furin is transcriptionally induced by notch signaling, but notch is cleaved at the cell membrane by adam10/adam17 to enable final cleavage by γ-secretase to form active notch intracellular domain, which regulates the transcription of target genes in nucleus. therefore, targeting notch activation using inhibitor γ-secretase (gsi) could be a promising therapeutic strategy to block the virus entry into the cardiac cells by reducing furin and increase adam17 shedding. the notch signaling also modulates the activity of innate and adaptive immune responses by inducing macrophage polarization [112] . in microphages, it directly binds to il-6 promoter in response to interferon (ifn)-γ and promotes il-6 production, which may cause severe myocardial injury due to triggered "cytokine storm" [113] . our current understanding on the molecular mechanisms of cardiomyocyte injury for sars-cov-2 infection is limited and future in depth rigorous studies are warranted. early diagnosis of covid-19 infection in patients is crucial for the recommendation of appropriate treatment strategy and to address associated cvd complications. initial symptoms of sars-cov-2 infection include high fever or chills, cough, shortness of breath, headache, sore throat, new loss of taste or smell, diarrhea and fatigue that appears during 2-14 days after the exposure to the virus. these early indications, though similar to regular viral infection, should be taken seriously during this pandemic time and diagnosed further for the presence of covid-19 infection. currently, the established diagnostic test for the identification of sars-cov-2 infection is based on nucleic acid amplification testing (naat) or commonly called real-time reverse transcription-polymerase chain reaction (rt-pcr) assay, nucleic acid-based meta-genomic next-generation sequencing (mngs), reverse transcription loop-mediated isothermal amplification (rt-lamp) and antigen testing performed with nasopharyngeal swab specimen [114, 115] . in the absence of any pharmaceutical interventions, traditional public health measures are considered to be the mainstay of management tools to curb this worldwide covid-19 epidemic. most widely accepted practices are hygienic precautions, isolation and quarantine, social distancing and community containment [44, 116] . to minimize cardiovascular complications in highly infectious covid-19 patients, the patients with covid-19 infection require routine monitoring of cardiac parameters with echocardiography, telemetry to assess qt interval and electrocardiograph (ecg) to identify the development of cardiomyopathy, arrhythmia, ischemic heart disease and heart failure. potential therapeutic options to impede the propagation of covid-19 and its associated cardiovascular complication are desperately needed during this ongoing severe pandemic. researchers and clinicians are focusing on developing new drugs against coronavirus as well as repurposing already approved drugs for the treatment of covid-19 patients. unapproved antiviral drugs for sars-cov-1 and/or middle east respiratory syndrome coronavirus (mers-cov) diseases are also currently being reevaluated as treatment options for covid-19. however, covid-19 poses unique problems that were not encountered with the previous known viruses. the major issue was to address the cvd complications, systemic and vascular inflammation, and to deal with comorbid risk like hypertension, diabetes and heart failure. initial approaches were to emphasis on obstructing the viral replication and inflammation by using antiviral drugs, such as, remdesivir, liponovir/ritonavir, hydroxy chloroquine (hcq), corticosteroids and broad-spectrum antibiotics like azithromycin, clarithromycin to address inflammation [117, 118] . table 1 summarizes the mechanisms of action and beneficial as well as adverse effects of drug treatments used for covid-19. the antiviral drug, remdesivir (veklury, gs-5734), initially developed for ebola, inhibits rna-dependent rna polymerase and prematurely terminates the viral rna transcription and shows broad-spectrum antiviral activity against rna viruses, including sars-cov-2 in vitro, and inhibits mers-cov, sars-cov-1, and sars-cov-2 replication in animal models [119] . remdesivir is a substrate for the drug metabolizing enzymes cyp2c8, cyp2d6, and cyp3a4, as well as a substrate for organic anion transporting polypeptides 1b1 (oatp1b1) and p-glycoprotein (p-gp) transporters. remdesivir (100-200 mg/day for 10 days) either treated alone or in combination with anti-inflammatory drugs was effective in curbing the virus and shortening the recovery time of patients undergoing treatment for covid-19 [120] . a multicenter randomized, double-blind, clinical trial, involving 237 patients with severe covid-19, conducted in ten hospitals in wuhan, china, reported that seriously ill patients, receiving remdesivir (200 mg on day 1 followed by 100 mg on days 2-10 in single daily infusions) within 10 days of symptom onset, showed a numerically faster time to clinical improvement than those receiving placebo, without any antiviral effect [121] . the study also reported early termination of the treatment due to multiple adverse events (including gastrointestinal symptoms, aminotransferase or bilirubin increases, and worsened cardiopulmonary status) in the remdesivir-treated patients (66%) ( table 1) . in a small pilot study of four critically ill covid-19 patients with remdesivir, three patients tested negative for sars-cov-2 rna (swap test) after 3 days of therapy. however, these reports also indicated some adverse side effects including liver injury [122, 123] . lopinavir-ritonavir antiviral drugs such as lopinavir-ritonavir (mylan or kaletra; 400 mg and 100 mg, respectively, twice a day for 14 days), hiv protease inhibitors, used in the clinical trial provided only a moderate benefit of reducing the recovery time by 1 day [124] . although in vivo animal study shows that a combination of remdesivir with lopinavir-ritonavir yields better outcome for coronavirus infection [125] . however, the treatment with these protease inhibitors (lopinavir-ritonavir) develop cardio-metabolic complications including development of dyslipidemia with an adverse cholesterol profile, which could elicit inflammation with elevated reactive oxygen species (ros) production, altered myocardial ubiquitin proteasome and calcium-handling pathways together with decreased contractile function [126, 127] (table 1) . lopinavir-ritonavir treatment inhibits the myocardial ups (ubiquitin proteasome system) and leads to elevated calcineurin and connexin 43 expression that may contribute to cardiac contractile dysfunction [127] . without any benefit, lopinavir-ritonavir may also cause bradycardia, qt and pr interval prolongation due to the interaction with cytochrome p450 enzymes [124, 128, 129] . baricitinib (olumiant®), an inhibitor of janus kinase (jak1 and jak2) molecule and a drug for the treatment of rheumatoid arthritis was tested (2 mg or 4 mg once daily) in covid-19 patients [130] . this drug was repurposed in covid-19 treatment to curb the occurrence of inflammation process due to the use of ace inhibitors, which moderately reduced the lung inflammation and cytokine [131] . the management of hyperinflammation or cytokine storm has been challenging and accounts for the majority of the mortality associated with adverse cases of covid-19 patients. clinical practices to address this complication involves treatment with monoclonal antibody against interleukin-6 receptor (il-6r) such as tocilizumab (actrema®), siltuximab (sylvant®) and sarilumab (kevzara®) to control the infiltration of macrophages and cytokines in the respiratory system and suppression t-cell activation [132, 133] . tocilizumab specifically binds membrane-bound (mil-6r) and soluble interleukin-6 receptor (sil-6r) and inhibits signal transduction. covid-19 patients treated with tocilizumab (4 to 8 mg/kg with recommended dose of 400 mg with a maximum dose of 800 mg) in addition to routine therapy showed significant improvement of the clinical outcomes, effectively controlled body temperature with improvement of peripheral oxygen saturation and reduction of inflammatory storm [134] . considering the emergency to identify a drug that is effective in reducing the complications associated with covid-19, efforts are also underway to repurpose old drugs that are proven to be clinically safe. data from recovery trial indicates that dexamethasone, a steroid drug generally used as an anti-inflammatory agent, is effective in reducing the mortality rate by one-third in covid-19 patients subjected to mechanical ventilation or who were on ventilators compared to patients receiving standard therapy [135] . among 6400 registered covid-19 patients, 2100 of them who received 6 mg of dexamethasone for 10 days, had reduced mortality by 20% compared to 4300 patients who were on standard treatment. more importantly, patients on ventilator support during the critical stage of treatment responded better to dexamethasone compared to patients just receiving oxygen therapy. the outcome of this study is considered a breakthrough in the fight against covid-19 because dexamethasone is a commonly available drug and cost effective. however, further evidence is required to use dexamethasone in covid-19 patients. another drug that gained much attention for the treatment of covid-19 is hydroxychloroquine (hcq, plaquenil), an anti-malarial compound, which is also widely used for attenuation of systemic lupus erythematosus (sle), rheumatoid arthritis (ra), juvenile idiopathic arthritis (jia) and sjogren's syndrome [136] . several clinical studies, including trials from nih (nct04358068), are testing this drug for covid-19 treatment, either alone or in combination with azithromycin [137] . the treatments with hydroxychloroquine alone (400 mg by mouth twice daily for 1 day followed by 200 mg by mouth twice daily for 4 days) or in combination with azithromycin (500 mg by mouth or intravenous daily for 5 days) lead to a prolongation of the qt interval, possibly increasing the risk of sudden cardiac death [137] (table 1) . another retrospective multicenter cohort study was conducted involving 1438 patients admitted across various hospitals in the city of new york who were diagnosed with covid-19 (between 15-28 march 2020), those receiving either hcq alone (dose ranges: 200-600 mg; once or twice a day) or in combination with azithromycin (dose ranges: 200 mg to 500 mg; once or twice a day) or azithromycin alone. the results from the study showed that the probability of death for patients receiving hcq + azithromycin was 25.7% (189 out of 735), while patients receiving hcq alone was 19.9% (54 out of 271) and 10.0% (21 out of 211) in azithromycin alone group. cardiac arrest was significantly high in patients receiving hcq + azithromycin combination than treatment with placebo or hcq alone [138] . another cohort study performed at an academic tertiary care center in boston, massachusetts, showed similar high risk of qt prolongation with subsequently developed other ventricular arrhythmias in the hcq alone (400 mg, twice on day 1, then 400 mg daily on days 2 through 5) or with azithromycin-treated patients with covid-19 [139] . an observational study of 1446 admitted patients to the hospital with covid-19 (between 7 march and 8 april 2020) in new york, revealed that hcq administration alone was not associated with either a greatly lowered or an increased risk of the composite end point of intubation or death [140] . the treatment regimen of hydroxychloroquine was a loading dose of 600 mg twice on day 1, followed by 400 mg daily for 4 additional days. however, recently, the u.s. food and drug administration (fda) revoked its approval to use hcq for covid-19 treatment due to disappointing results [141] . data from randomized clinical trials suggest that hcq had no beneficial effects compared to placebo and was not successful in decreasing the mortality rate or in hospital stay (based on fda report, updated on 1 july 2020) [142] . therefore, rigorous, and large-scale studies with careful risk assessment of hcq should be conducted prior to initiating covid-19 therapeutics, with close monitoring cardiac manifestations including evaluation of cardiac biomarkers, routine electrocardiograms and electrolyte monitoring. there is an urgency for the development of a safe and effective vaccine for covid-19; however, no specific vaccines against sar-cov-2 are currently available [40] . multiple inactivated vaccine candidates for sars-cov-2, such as dna-, rna-based formulations, recombinant-subunits containing viral epitopes, adenovirus-based vectors and purified inactivated virus are under development [143, 144] . several candidate vaccines are still in the preliminary stage of phase i clinical trial. the mrna-based vaccine prepared by the usa national institute of allergy and infectious diseases against sars-cov-2 is under phase 1 trial [145] . ino-4800, a dna-based vaccine, is also in pipeline and will soon be available for human trial. preliminary results from pilot studies and clinical trials on new vaccine are encouraging and gives hope for a successful availability of an effective vaccine by end of 2020. several pharmaceutical companies, including pfizer, novartis and astrazeneca and moderna, are testing their candidate vaccine. university of oxford in collaboration with astrazeneca are in the development of covid-19 vaccine and expect to produce 30 million doses in uk by september 2020. jenner institute, oxford, uk is a leader in this effort and launched a phase iii clinical trial of more than 6000 people in may. however, due to the suspected adverse event in a person receiving the vaccine in the united kingdom, the clinical trials have been temporarily paused. moderna, a usa-based company in collaboration with switzerland's lonza, released positive outcomes from its phase i clinical trial of their mrna1273 vaccine for sars-cov-2 [146] . preliminary results are very promising, showing good immune response, and due to effectiveness and safety profiles, this vaccine is approved by the u.s. food and drug administration (fda) for phase ii and phase iii studies [147, 148] . novartis announced its plans to initiate a phase iii clinical trial to study effects of canakinumab, an interleukin (il)-1β blocker, in covid-19 patients with pneumonia [149] . they aim to rapidly enroll 450 patients at multiple medical centers across france, germany, italy, spain, uk and the usa and randomize them to receive either canakinumab or placebo on top of standard of care (soc) [150] . pfizer, in partnership with biontech (bnt), has initiated its phase i/ii clinical trial in the usa for its mrna-based vaccine, the bnt162 prevent covid-19 [151] . sinopharm, a wuhan, china-based pharmaceutical company received approval from the national medical products administration (china) and conducting phase ii clinical trials for its inactivated vaccine bbibp-corv. the company already tested 2000 doses of this vaccine and expect to release in the marker by the end of the year 2020. sinovac is planning to enter its phase iii clinical trial in collaboration with instituto butantan in brazil after observing positive results in its preclinical trail with the vaccine coronavac [144] . ad5-ncov, an adenovirus type 5 vector-based vaccine developed by cansiobiologics, china is also in phase iii clinical trial and demonstrated promising effects in the early phase of testing on 108 participants [152] . inovio pharmaceuticals in collaboration with university of pennsylvania and center for pharmaceutical research, kansas city, missouri, is testing its dna-based vaccine ino-4800 [153] . preclinical experiments conducted in guinea pigs showed antibody titer against ace2 receptor/sars-cov2 binding protein. when countries all over the world are racing to develop their own vaccine against covid-19, russia has already approved a vaccine candidate for public use named sputnik v, that was developed in collaboration with gamaleya research institute of epidemiology and microbiology in moscow [154] . the vaccines comprise either recombinant adenovirus type 26 (rad26) or recombinant adenovirus type 5 (rad5) vectors, which contain the gene for sars-cov-2 spike glycoprotein (rad26-s and rad5-s). initial results from the ongoing phase i and ii clinical trials are promising, which include total population size of 76 healthy adult volunteers [155] . among them 38 volunteers were intramuscularly vaccinated with gam-covid-vac lyo (lyophilized vaccine formulation) and other 38 participants were subjected to gam-covid-vac (frozen vaccine formulation) [155] . both heterologous recombinant adenoviral (rad26 and rad5) vector-based covid-19 vaccines induced a strong humoral and cellular immune responses with reported safety profiles in participants. however, further investigations with larger scale population (including different underlying medical complications) are needed to demonstrate the effectiveness of this vaccine for prevention of covid-19. nevertheless, scientists globally have serious concerns about unforeseen adverse effects of this vaccine without the outcomes of the phase iii trial. even though, for the development of an efficient vaccine for covid-19, extensive preclinical studies and clinical trials are essential to carefully evaluate the adverse effect of vaccine, the aforementioned fast-paced preclinical data are encouraging for advancing the preventive strategies against covid-19. several other treatment options such as convalescent plasma therapy (cpt) and monoclonal antibody therapy have been evaluated with some moderate success. cpt is a traditional method where plasma containing the antibody from recovered patients infected with covid-19 was transfused to the severely ill covid-19 patients [25, [156] [157] [158] . studies showed that cp therapy was effective, and the level of neutralizing increased as high as 1:640 times in patients infected with sars-cov-2 [25] . transfusing antibodies from covid-19 survivors into high-risk patients to neutralize sars-cov-2 could provide a quick treatment option until an optimistic vaccine will arrive to prevent this viral infection. efforts are also underway to design a monoclonal antibody that can target the specific epitope on the spike protein of sars-cov-2 and block the virus entry in to the host cell [108, 159, 160] . such efforts are still in their preliminary stage [161] and are time consuming; however, they could provide a long-lasting solution for dealing with sars viruses in general. recently, stem cell therapies with secreted extracellular vesicles (evs) offer a potential therapeutic benefit in covid-19 patients by attenuating inflammation with regeneration of the damaged lung. mesenchymal stem cells (mscs)-derived evs-based therapy could be the most promising reparative strategy in people with covid-19, because of its high proliferation rate, low invasive nature, and the immunomodulatory, antioxidant and anti-inflammatory properties of mscs [162] . there are several promising clinical trials with msc-derived evs underway, which could reveal convincing evidence in the encouraging prospect of msc-based therapies for respiratory complications of covid-19 patients [163, 164] . despite the above-mentioned beneficial effects of different therapeutics, the safety profiles of these therapies have not been proficiently identified. specifically, the potential adverse cardiovascular effects of these drugs in covid-19 patients need urgent attention before rushing the approval of any new drug into clinical application. for most effective treatments for covid-19, it is important to pay attention to emerging evidence about potential harmful risk of drug interactions. due to the highly transmissible novel coronavirus, sars-cov-2, the covid-19 outbreak has become an unprecedented worldwide pandemic with a record number of infected individuals and an excess of mortality. the desperate need for effective therapeutics for covid-19 during this pandemic integrated scientist around the world across multiple research fields while sharing their research findings and knowledge to fast-track the process of drug discovery. considering the high mortality of covid-19 patients with cardiovascular comorbidities, it is important to understand whether it is attributable to underlying cardiovascular disease (cvd) or if cvd is the consequence of inflammatory response to sar-cov-2 infection or severe respiratory symptoms. the precise mechanisms linking cvds and worsened prognosis or higher mortality rate in covid-19 patients remain unknown. recent therapies under investigation for severe multi-organ failure in covid-19 patients may have adverse cardiovascular effects, while their clinical efficacy for combating covid-19 is yet to be established. new advanced technological tools, like information technology based on smart phone apps, social media, artificial intelligence (ai), machine learning, etc., accelerate the diagnosis/screening of patients with virus, analysis of available literature, and identification of potential therapeutic targets and other specific clinical features to tackle covid-19 pandemic. moreover, ai, particularly, plays an important role in predicting the harmful interaction between cardiovascular consequences with the drugs used for covid-19, by automated interpretation of collected meta-data from various sources. in the context of disease progression with cardiovascular complications, the researchers are focusing on developing new drugs in parallel to repurposing already clinically approved drugs to avoid a massive surge of covid-19 patients with a prevalence of cvd. therefore, urgent understanding of molecular mechanism as well as retrospective and prospective studies with robust diagnosis of cardiovascular impairments will be crucial for development of advanced therapies for the treatment of sars-cov-2 virus, which could mitigate the adverse cardiovascular events among covid-19 patients and save humankind around the globe from this deadly pandemic. the authors declare no conflict 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terms and conditions of the creative commons attribution (cc by) license key: cord-297786-jz1d1m2e authors: hasan, md. mahbub; das, rasel; rasheduzzaman, md.; hussain, md hamed; muzahid, nazmul hasan; salauddin, asma; rumi, meheadi hasan; rashid, s m mahbubur; siddiki, amam zonaed; mannan, adnan title: global and local mutations in bangladeshi sars-cov-2 genomes date: 2020-08-26 journal: biorxiv doi: 10.1101/2020.08.25.267658 sha: doc_id: 297786 cord_uid: jz1d1m2e corona virus disease-2019 (covid-19) warrants comprehensive investigations of publicly available severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) genomes to gain new insight about their epidemiology, mutations and pathogenesis. nearly 0.4 million mutations were identified so far in ∼60,000 sars-cov-2 genomic sequences. in this study, we compared 207 of sars-cov-2 genomes reported from different parts of bangladesh and their comparison with 467 globally reported sequences to understand the origin of viruses, possible patterns of mutations, availability of unique mutations, and their apparent impact on pathogenicity of the virus in victims of bangladeshi population. phylogenetic analyses indicates that in bangladesh, sars-cov-2 viruses might arrived through infected travelers from european countries, and the gr clade was found as predominant in this region. we found 2602 mutations including 1602 missense mutations, 612 synonymous mutations, 36 insertions and deletions with 352 other mutations types. in line with the global trend, d614g mutation in spike glycoprotein was predominantly high (95.6%) in bangladeshi isolates. interestingly, we found the average number of mutations in orf1ab, s, orf3a, m and n of genomes, having nucleotide shift at g614 (n=459), were significantly higher (p≤0.001) than those having mutation at d614 (n=215). previously reported frequent mutations such as p4715l, d614g, r203k, g204r and i300f were also prevalent in bangladeshi isolates. additionally, 87 unique amino acid changes were revealed and were categorized as originating from different cities of bangladesh. the analyses would increase our understanding of variations in virus genomes circulating in bangladesh and elsewhere and help develop novel therapeutic targets against sars-cov-2. severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) has become an etiological agent of the disease called coronavirus disease-2019 (covid19) . as of 21 august 2020, globally there have been 22,492,312 confirmed cases of covid-19, including 788,503 deaths, reported to who. to explore the viral pathogenesis, modern genomics tools are highly crucial and has been employed by researchers around the world. hundreds of virus whole genomes are now submitted in publicly accessible databases from different parts of the globe everyday. it is hightime to analyze the variations among those sequences which will help future strategic efforts for its preventive measures such as vaccine design and therapeutics. sars-cov-2 consists of positive-sense single-stranded rna with a genome size ranging from ~27 to 34 kb. it contains a variable number (6) (7) (8) (9) (10) (11) of open-reading frames (orfs). the first orf is almost two-third of the whole genome and encodes four structural, 16 non-structural and eight accessory proteins [1, 2] . according to a recent study on 48,635 of sars cov-2 genome sequences, 353,341 mutation events have been observed globally in comparison to the reference genome of wuhan. among these sequences, india, congo, bangladesh and kazakhstan have significantly high numbers of mutations per sample compared to the global average [3] . out of these mutations, d614g mutation (causing aspartate to glycine in s protein position 614) is reported to be the most prevalent mutations reported from europe, oceania, south america, africa [4] . zhang et al. (2020) reported that the level of angiotensin-converting enzyme 2 (ace2) expression was distinctly higher by the retroviruses pseudotyped with g614 compared to that of d614 [4, 5] . the functional properties of the (d614) and (g614) were compared in this study and g614 was found to be more stable than d614 with more transmission efficiency, supporting the previous epidemiological data [5] . another reported mutation of orf1ab is p4715l linked with d614g, that has been reported to have a strong relationship with higher fatality rates in 28 countries and 17 states of the united states [3, 6] . three other mutations namely c14408t (orf1b), c241t (5' utr), c3037t (orf1a) is reported to be common and co-occurring in the same genome while g11083t has been found mostly in asian countries [6] . hassan et al. (2020) investigated the accumulation of orf3a mutations of sars-cov-2 from india where they revealed four types of mutations (q>h, d>y and s>l) near traf, ion channel and caveolin binding domain, respectively [7] . notable that all these mutations might have implications in maintaining the virulence of the virus and nlrp3 inflammasome activation. in bangladesh, as of 21 august, 2020, nearly 287,959 people are infected and 3822 people have died due to covid-19 (https://iedcr.gov.bd/). among them, 329 of sars-cov-2 genome sequences were deposited in gisaid database (https://www.gisaid.org). analysis of these sequences is sparsely reported in the literature. analyzed 64 sequences and identified the presence of 180 mutations in the coding regions of the viruses and mutations at nsp2 was the most prevalent [11] . due to the small numbers of genome sequence analyzed, most of these findings were not conclusive and representative. further comprehensive analyses is therefore necessary to better understand the circulating virus in the country. in this study, we first compared 207 genome sequences isolated from bangladesh with time-resolved phylogenetic analysis and investigated the origin of imported covid-19 cases to bangladesh. then, we studied the variants present in different isolates of bangladesh to investigate the pattern of mutations, identify ums, and discuss the pseudo-effect of these mutations on the structure and function of encoded proteins, with their role in pathogenicity. most interestingly, we found 87 ums with a total count of 113 in bangladeshi isolates which will increase our understanding of distribution of sars-cov-2 virus in different regions and associated pathogenicity. as of 30 june, 2020, a total of 35,723 whole genomic rna sequences of sars-cov-2 had been submitted to gisaid. from these downloaded sequences, a custom python script was used to retrieve unique sequences. the same script also removed any sequence containing "n" and other ambiguous iupac codes [12] . this resulted in a total of 8723 complete genomic sequences. to select representative sequences from curated 8723 sequences and make a comparison against sequences from bangladesh, priorities were given to those countries that had a higher number of infections in each continent (source: https://www.worldometers.info/coronavirus/). we selected these sequences in such a way that each continent must contain at least one sequence from each gisaid clade. the number of sequences selected from a country was based on the total number of unique sequences retrieved. this resulted in a total of 467 unique representative sequences from 42 countries (see supporting file table s1 ). from bangladesh, 215 whole-genome rna sequences of sars-cov-2 were uploaded to gisaid as of 15 july, 2020. only high coverage complete sequences (n=207) were kept for analysis. all these 207 sequences were aligned with the previously selected 467 representative sequences along with that of wuhan-1 (accession id mn908947) as a reference sequence [13] . to ensure comparability, the flanks of all the sequences were truncated to the consensus range from 56 to 29,797 [14] , with nucleotide position numbering according to the wuhan 1 reference sequence, prior to alignment. multiple sequence alignment (msa) and phylogenetic tree construction were carried out using molecular evolutionary genetic analysis (mega x) software [15] . all selected sequences were aligned using muscle software tool [16] . later, an nj (neighbor-joining) phylogenetic tree [17] was constructed using the tamura-nei method. tree topology was assessed using a fast bootstrapping function with 1000 replicates. tree visualization and annotations were performed in the interactive tree of life (itol) v5 [18] . the genome detective coronavirus typing tool version 1.13 was used for variant analyses of sars-cov-2 genome which is specially designed for this virus analysis (https://www.genomedetective.com/app/typingtool/cov/) [19] . for analysis of (um) among 207 genomic sequences from bangladesh, we used a cov server hosted in gisaid server (https://www.gisaid.org/epiflu-applications/covsurver-app/). the server analyzed our dataset against all available genomic sequences of sars-cov-2 including the wuhan reference sequence deposited on gisaid until july 16, 2020. descriptive and inferential statistics were used to analyze different mutations and their correlation with different categorical variables. for correlation, we used one-way analysis of variance using spss statistics 25 (ibm, armonk, new york) licensed to king's college london. to understand the sars-cov-2 viral transmission in bangladesh, we performed phylogenetic analysis on the selected 207 viral genomes reported from different districts of bangladesh along with selected 467 globally submitted sequences as reported from 42 countries and 6 continents ( figure 1 ). this apparently represents the overall clade distribution of all global sequences along with sequences from bangladeshi isolates. gr clade was found predominant in bangladesh as about 84% of the sequences were grouped to this clade followed by gh and g with ~6 and ~5%, respectively. similar clade distribution has been found in isolates submitted from european countries. we also attempted to compare the sequence data among different districts of bangladesh from where patient samples were collected for sequencing, looking at the districtwise distribution of clade (figure 2) , it was found that the sequences from three districts, namely dhaka, narayanganj and rangpur primarily belong to gr clade. conversely, only sequences from chattogram district were from s and gh clades. on another note, phylogenetic analyses of the clade distribution of isolates from countries like saudi arabia, where gh clade was predominant [3] it is highly likely that the introduction of gh and s clades in bangladesh could be of middle-eastern origin. based on the datsets, we hypothesized that bangladeshi sars-cov-2 isolates belonging to different clades might have critical implications concerning viral transmission rate, virulence, severity and other aspects of disease pathogenesis. in addition, the presence of different clades of sars-cov-2 strains in different districts could also have implications in the accuracy of diagnostic tests that are underway. our analyses revealed a total of 2602 mutations observed among 207 bangladeshi genomic sequences that constitutes a number of 1602 missense, 612 synonymous, 36 insertion/deletions and 352 other mutations (table 1) . we identified mutations like i300f (nsp2), p4715l (nsp12), d614g (s glycoprotein), r203k and g204r (n protein) are the most frequently occurring common mutations found in bangladesh with a frequency of 138, 199, 198, 177 and 177, respectively ( figure 3 ). notable that, no particular mutations occurred at any specific time period rather they have been observed over the whole period of disease incidence. firstly, 1163a>t (i300f), can be a destabilizing factor for nsp2 and thus modulating the strategy of host cell survival [11, 20] . this mutation can lead to a reduction of conformational entropy due to the presence of the side chains that can result in charge neutralization of the phosphorylated serine residues [21] . secondly, rna dependent rna polymerase (nsp12) is significant for replication and transcription of the viral rna genome. therefore, p > l at 4715 in nsp12 may have some effects on rna transcription. this mutation was also observed in most of the usa states (28 out of 31). the same mutation was prevalent in european countries like spain, france etc. this alteration could affect the pathogenesis triggered by antibody escape variants with the epitope loss [22, 23] . thirdly, korber et al. (2020) stated that the g614 type might have originated either in europe or china [24] . they also reported that the original wuhan d614 form was also predominant in asian samples. meanwhile, the g614 form had clearly established and started expanding in countries outside of china. we also noticed 95.6% of genomes from bangladesh have d614g mutation, which is also dominant in the world. however, the average number of mutations per orf is varied among d614 and g614 containing genomes that we have studied (n=674) as revealed by table 2 . the average number of mutations in orf1ab, s, orf3a, m and n of genomes having mutated g614 (n=459) are significantly higher (p≤0.001) than those having wild d614 (n=215) in s glycoprotein (table 2; figure 1 ). interestingly, the average mutation number is declined in orf8 of genomes having g614 mutation (p<0.001). this correlation indicates that the genomes containing d614g mutation are more prone to bear other mutations which may facilitate the notion that the link of this mutation with the transmission and pathogenesis of sars-cov-2 [4, 12, 25] . finally, r203k and g204r mutations in n protein were previously reported in indian, spanish, italian, and french samples [21, 26] . these mutations are located in the site of the sr-rich region which has been reported to be intrinsically disordered [27] . this region further incorporates a few phosphorylation sites [28] , including the gsk3 phosphorylation at ser202 and a cdk phosphorylation site at ser206 which are located close to the position of this mutation. the 'srgts' (202-206) and 'spar' (206-209) sequence motifs are dependent on gsk3 and cdk phosphorylation motifs, respectively. other variations (28881g>a and 28882g>a) together convert polar to non-polar amino acid (r203k) and 28883g>c variation converts nonpolar to polar amino acid (g204r). we observed 87 unique shifts from the different proteins of sars-cov-2 genomes found in bangladesh (table s2 ). details of their pseudo-effects on viral replication, assembly, transmissivity and pathogenicity are corroborated in table s2 . surprisingly, most of these um were localized except a few exceptions. for example, the ums e8d (e), s180t (n), l139j and is involved in the transcription and replication of cov rnas. we observed 4 ums in nsp1, but the location of these amino acids was not in the kh domain (k164 and h165 of nsp1), which binds with ribosome 40s subunit [30] . however, nsp1 acts as a primary virulence factor in sars-cov-2 infection, and mutation in this protein could affect the structure and functional properties, thereby altering its virulence properties. nine ums were seen in nsp2, but their effects on host cells were merely reported in the literature. since nsp2 interacts with the host proteins and disrupts the host cell survival signaling pathway [31] , any mutation in nsp2 may play a crucial role in sars-cov infections. in a recent study, it has been found that, compared to bat sars-cov, sars-cov-2 has a stabilizing mutation at amino acid position 501, t501q, which alters the viral pathogenicity and makes the virus more contagious [32] . we did not observe this mutation in our study. the mutations of nsp3 are responsible for affecting the virus assembly and hence their replication. it is due to the disruption of replicase polyprotein processing into nsps. these nsps assemble with cellular membranes and facilitate virus replication. the ums found in nsp3 might have some probable effects. firstly, the ums (a889v, r883g, s1038f and v843f) were found in the main domain of nsp3 that is important for processing endopeptidases from coronaviruses. secondly, many um (e.g. a1803v, a1819s, g1691c, i1672s, a602s, d782b, k462r, l373m, n1337s, n51d, r883g, t1363is, y246c and y272h etc) are found in topological (cytoplasmic) domain rather than transmembrane domain which could interfere less on cytoplasmic double-membrane vesicle formation, necessary for viral replication. thirdly, we observed three ums (l373m, y246c and y272h) in adp-ribose-1′-phosphatase (adrp) or (macro) domain. it has been shown that mutations of the adrp domains does not diminish virus replication in mice, but reduces the production of the cytokine il-6, which is an important pro-inflammatory molecule [33] . however, we did not observe any mutation in the active sites and zinc finger motif, attributing normal catalytic activity of nsp3. the general opinion is that sars-cov pl-pro domain is important for the development of antiviral drugs and of the actual role of this enzyme in the biogenesis of the covid-19 replicase complex is yet not explored. it is proposed that the proteins nsp3, nsp4 and nsp6, through their transmembrane domains, are involved in the replicative and transcription complex [34] . in our study, we observed only 3 ums in nsp4 and nsp6, respectively. meanwhile, nsp5 encodes 3c-like proteinase which cleaves the c-terminus of replicase polyprotein at 11 sites. the five ums that we found in nsp5 did not fall in its active sites (3304 and 3408 in orf1ab) requiring further investigation with large number of sequence datasets. the present global outbreak of covid-19, caused by sars-cov-2, has already taken ~0.8 million lives. to combat this deadly disease, we need a greater understanding of the pathobiology of the virus. hence, it is essential to minimize the translational gap between viral genomic information and its clinical consequences for developing effective therapeutic strategies. in this study, we have attempted to explore genomic variations of bangladeshi sars-cov-2 viral isolates while comparing with a large cohort of global isolates. our analyses will facilitate the understanding of the origin, mutation patterns and their possible effect on viral pathogenicity. this study tries to address the importance of the variations in the viral genomes and their necessity for therapeutic interventions. the unique insights from this study will undoubtedly be 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pathogenesis sars-cov-2: virus mutations in specific european populations key: cord-291523-4dtk1kyh authors: nguyen, thanh thi; abdelrazek, mohamed; nguyen, dung tien; aryal, sunil; nguyen, duc thanh; khatami, amin title: origin of novel coronavirus (covid-19): a computational biology study using artificial intelligence date: 2020-07-01 journal: biorxiv doi: 10.1101/2020.05.12.091397 sha: doc_id: 291523 cord_uid: 4dtk1kyh origin of the covid-19 virus has been intensely debated in the scientific community since the first infected cases were detected in december 2019. the disease has caused a global pandemic, leading to deaths of thousands of people across the world and thus finding origin of this novel coronavirus is important in responding and controlling the pandemic. recent research results suggest that bats or pangolins might be the original hosts for the virus based on comparative studies using its genomic sequences. this paper investigates the covid-19 virus origin by using artificial intelligence (ai) and raw genomic sequences of the virus. more than 300 genome sequences of covid-19 infected cases collected from different countries are explored and analysed using unsupervised clustering methods. the results obtained from various ai-enabled experiments using clustering algorithms demonstrate that all examined covid-19 virus genomes belong to a cluster that also contains bat and pangolin coronavirus genomes. this provides evidences strongly supporting scientific hypotheses that bats and pangolins are probable hosts for the covid-19 virus. at the whole genome analysis level, our findings also indicate that bats are more likely the hosts for the covid-19 virus than pangolins. the covid-19 pandemic has rapidly spread across many countries and disturbed lives of millions of people around the globe. there have been approximately 10 million confirmed cases of covid-19 globally, including nearly 500,000 deaths, reported to the world health organization at the end of june 2020 [1] . studies on understanding the virus, which was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), are important to propose appropriate intervention strategies and contribute to the development of therapeutics and vaccines. finding origin of the covid-19 virus is crucial as it helps to understand where the virus comes from via its evolutionary relationships with other biological organisms and species. this will facilitate the process of identifying and isolating the source and preventing further transmissions of the pathogen to the human population. this will also help to understand the outbreak dynamics, leading to the creation of informed plans for public health responses [2] . a study by wu et al. [3] using a complete genome obtained from a patient who was a worker at a seafood market in wuhan city, hubei province, china shows that the virus is closely related to a group of sars-like covs that were previously found present in bats in china. it is believed that bats are the most likely reservoir hosts for the covid-19 virus as it is very similar to a bat coronavirus. these results are supported by a separate study by lu et al. [4] using genome sequences acquired from nine covid-19 patients who were among early cases in wuhan, china. outcomes of a phylogenetic analysis suggest that the virus belongs to the genus betacoronavirus, sub-genus sarbecovirus, which includes many bat sars-like covs and sars covs. another study in [5] confirms this finding by analysing genomes obtained from three adult patients admitted to a hospital in wuhan on december 27, 2019. likewise, zhou et al. [6] advocate a probable bat origin of sars-cov-2 by using complete genome sequences of five patients at the beginning of the outbreak in wuhan, china. one of these sequences shows 96.2% similarity to a genome sequence of a coronavirus, denoted ratg13, which was previously obtained from a rhinolophus affinis bat found in yunnan province of china. zhang and holmes [7] also highlight a similarity of approximately 85% between sars-cov-2 and ratg13 in their receptor binding domain, which is an important region of the viral genomes for binding the viruses to the human angiotensin-converting enzyme 2 receptor. in another study, lam et al. [8] found two related lineages of covs in pangolin genome sequences sampled in guangxi and guangdong provinces in china, which have similar genomic organizations to sars-cov-2. that study suggests that pangolins could be possible hosts for sars-cov-2 although they are solitary animals in an endangered status with relatively small population sizes. these findings are corroborated by zhang et al. [9] who assembled a pangolin cov draft genome using a reference-guided scaffolding approach based on contigs taxonomically annotated to sars-cov-2, sars-cov, and bat sars-like cov. xiao et al. [10] furthermore suggest that sars-cov-2 may have been formed by a recombination of a pangolin cov-like virus with one similar to ratg13, and pangolins are potentially the intermediate hosts for sars-cov-2. on the other hand, by analysing genomic features of sars-cov-2, i.e. mutations in the receptor binding domain portion of the spike protein and distinct backbone of the virus, andersen et al. [11] determined that this novel coronavirus originated through natural processes rather than through a laboratory manipulation. this study presents a step further to suggesting the likely origin of the covid-19 virus by using artificial intelligence (ai) methods to explore genome sequences obtained from more than 300 covid-19 patients across the world. we use ai-enabled unsupervised clustering methods to demonstrate and emphasize the relationships between covid-19 virus, bat covs and pangolin covs. through analysing the results of clustering methods, we are able to suggest the sub-genus sarbecovirus of the genus betacoronavirus of sars-cov-2 and the more likely bat origin of the virus rather than a pangolin origin. we downloaded 334 complete genome sequences of sars-cov-2 available from the genbank database, which is maintained by the national center for biotechnology information (ncbi), in early april 2020. among these sequences, 258 were reported from usa, 49 were from china and the rest were distributed through various countries from asia to europe and south america. accession numbers and detailed distribution of these genome sequences across different countries are presented in tables 4 and 5 in appendix 1. most of reference sequences, e.g. ones within the alphacoronavirus and betacoronavirus genera, are also downloaded from the ncbi genbank and virus-host db (https://www.genome.jp/virushostdb/) that covers ncbi reference sequences (refseq, release 99, march 2, 2020). genome sequences of guangxi pangolin covs [8] are downloaded from the gisaid database (https://www.gisaid.org) with accession numbers epi_isl_410538 -epi_isl_410543. a guangdong pangolin cov genome [10] is also downloaded from gisaid with accession number epi_isl_410721. we employ three sets of reference sequences in this study with details presented in tables 1-3. the selection of reference genomes at different taxonomic levels is based on a study in [12] that uses the ai-based supervised decision tree method to classify novel pathogens, which include sars-cov-2 sequences. we aim to traverse from high to low taxonomic levels to search for the covid-19 virus origin through discovering its genus and sub-genus taxonomy and its closest genome sequences. unsupervised clustering methods are employed to cluster datasets comprising both query sequences (sars-cov-2) and reference sequences into clusters. in this paper, we propose the use of hierarchical clustering algorithm [13] and densitybased spatial clustering of applications with noise (dbscan) method [14] for this purpose. with these two methods, we perform two steps to observe the clustering results that lead to interpretations about the taxonomy and origin of sars-cov-2. in the first step, we apply clustering algorithms to cluster the set of reference sequences only, and then use the same settings (i.e. values of parameters) of clustering algorithms to cluster a dataset that merges reference sequences and sars-cov-2 sequences. through this step, we can find out reference sequences by which sars-cov-2 sequences form a group with. in the second step, we vary the settings of the clustering algorithms and observe changes in the clustering outcomes. with the second step, we are able to discover the closest reference sequences to the sars-cov-2 sequences and compare the similarities between genomes. in the hierarchical clustering method, the cut-off parameter c plays as a threshold in defining clusters and thus c is allowed to change during our experiments. with regard to the dbscan method, the neighbourhood search radius parameter ε and the minimum number of neighbours parameter, which is required to identify a core point, are crucial in partitioning observations into clusters. in our experiments, we set the minimum number of neighbours to 3 and allow only the search radius parameter ε to vary. outputs of the dbscan method may also include outliers, which are normally labelled as cluster "-1". to facilitate the execution of the clustering methods, we propose the use of pairwise distances between sequences based on the jukes-cantor method [15] and the maximum composite likelihood method [16] . the jukes-cantor method estimates evolutionary distances by the maximum likelihood approach using the number of substitutions between two sequences. with nucleotide sequences, the distance is defined as d = −3/4 * ln(1−p * 4/3) where p is the ratio between the number of positions where the substitution is to a different nucleotide and the number of positions in the sequences. on the other hand, the maximum composite likelihood method considers the sum of loglikelihoods of all pairwise distances in a distance matrix as a composite likelihood because these distances are correlated owing to their phylogenetic relationships. tamura et al. [16] showed that estimates of pairwise distances and their related substitution parameters such as those of the tamura-nei model [17] can be obtained accurately and efficiently by maximizing this composite likelihood. the unweighted pair group method with arithmetic mean (upgma) method is applied to create hierarchical cluster trees, which are used to construct dendrogram plots for the hierarchical clustering method. the upgma method is also employed to generate phylogenetic trees in order to show results of the dbscan algorithm. we start the experiments to search for taxonomy and origin of sars-cov-2 with the first set of reference genome sequences (set 1 in table 1 ). this set consists of much more diversified viruses than the other two sets (sets 2 and 3 in tables 2 and 3 ) as it includes representatives from major virus classes at the highest available virus taxonomic level. with a large coverage of various types of viruses, the use of this reference set minimizes the probability of missing out any known virus types. outcomes of the hierarchical clustering and dbscan methods are presented in figs. 1 and 2, respectively. in these experiments, we use 16 sars-cov-2 sequences representing 16 countries in table 5 (appendix 1) for the demonstration purpose. the first released sars-cov-2 genome of each country is selected for these experiments. clustering outcomes on all 334 sequences are presented in fig. 9 in appendix 2, which shows results similar to those reported here. both clustering methods consistently demonstrate that sars-cov-2 sequences form a cluster with a representative virus of riboviria among 12 major virus classes (adenoviridae, anelloviridae, caudovirales, geminiviridae, genomoviridae, microviridae, ortervirales, papillomaviridae, parvoviridae, polydnaviridae, polyomaviridae, and riboviria). the middle east respiratory syndrome (mers) cov, which caused the mers outbreak in 2012, is chosen as a representative of the riboviria realm. in hierarchical clustering ( fig. 1 ), when combined with reference genomes, sars-cov-2 genomes do not create a new cluster on their own but form a cluster with the mers cov, i.e. cluster "8". with the dbscan method ( fig. 2) , sars-cov-2 genomes also do not create their own cluster but form the cluster "1" with the mers cov. these clustering results suggest that sars-cov-2 belongs to the riboviria realm. (table 1) with the cut-off parameter c equal to 5 * 10 −4 (left), and using a set that merges 16 representative sars-cov-2 sequences and reference sequences with c also set to 5 * 10 −4 (right). a number at the beginning of each virus name indicates the cluster that virus belongs to after clustering. once we have been able to identify sars-cov-2 as belonging to the riboviria realm, we move to the next lower taxonomic level that consists of 12 virus families within riboviria. these families are presented in set 2 ( , and using a set that merges sars-cov-2 sequences and reference sequences with ε also set to 0.7 (right). as set 1 includes representatives of major virus classes and the minimum number of neighbours is set to 3 while ε is set to 0.7, dbscan considers individual viruses as outliers (left). when the dataset is expanded to include sars-cov-2 sequences, dbscan forms cluster "1" that includes all sars-cov-2 sequences and the mers cov, which represents the riboviria realm (right). (table 2) with the cut-off parameter c equal to 0.001 (left), and using a set that merges sars-cov-2 sequences and reference sequences with c also set to 0.001 (right). covs and bat sars-like covs. notably, we also include in this set 6 sequences of guangxi pangolin covs deposited to the gisaid database by lam et al. [8] and a sequence of guangdong pangolin cov by xiao et al. [10] . evolutionary distances between each of the reference genomes in set 3 (table 3) to the 334 sars-cov-2 genomes based on the jukes-cantor method are presented in fig. 5 . we can observe that these distances are almost constant across 334 sars-cov-2 sequences, which are collected in 16 countries (table 5) table 2) with the search radius parameter ε equal to 0.6 (left), and using a set that merges sars-cov-2 sequences and reference sequences with ε also set to 0.6 (right). group contains genomes of alphacov viruses (refer to the taxonomy in table 3 ) that are much evolutionarily divergent from sars-cov-2 sequences. the middle group of lines comprises most of the betacov viruses, especially those in the sarbecovirus sub-genus. the bottom lines identify reference viruses that are closest to sars-cov-2, which include bat cov ratg13, guangdong pangolin cov, bat sars cov zc45 and bat sars cov zxc21. the bat cov ratg13 line at the bottom is notably distinguished from other lines while the guangdong pangolin cov line is the second closest to sars-cov-2. the similarities between bat cov ratg13, guangdong pangolin cov and guangxi pangolin cov gx/p4l with sars-cov-2/australia/vic01/2020, produced by the simplot software [18] , are displayed in fig. 6 . consistent with the results presented in fig. 5 , bat cov ratg13 is shown closer to sars-cov-2 than pangolin covs. fig. 7 shows outcomes of the hierarchical clustering method using set 3 of reference sequences in table 3 . with the cut-off parameter c is set equal to 0.7, the hierarchical clustering algorithm separates the reference sequences into 6 clusters in which cluster "5" comprises all examined viruses of the sarbecovirus sub-genus, including many sars covs, bat sars-like covs and pangolin covs (fig. 7a) . it is observed that the algorithm reasonably groups viruses into clusters, for example, the genus alphacov is represented by cluster "4" while the sub-genera embecovirus, nobecovirus, merbecovirus, and hibecovirus are labelled as clusters "3", "6", "2", and "1", respectively. using the same cut-off value of 0.7, we next perform clustering on a dataset that merges reference sequences and 16 representative sars-cov-2 sequences (see fig. 7b ). results on all 334 sars-cov-2 sequences, which are similar to those on the 16 representative sequences, are provided in fig. 10 in appendix 2. the outcome presented in fig. 7b shows that the 16 representative sars-cov-2 sequences fall into cluster "5", which comprises the sarbecovirus sub-genus. the number of clusters is still 6 and the membership structure of the clusters is the same as in the case of clustering reference sequences only (fig. 7a) , except that the sarbecovirus cluster now has been expanded to also contain sars-cov-2 sequences. by comparing figs. 7a and 7b, we believe that sars-cov-2 is naturally part of the sarbecovirus sub-genus. this realization is substantiated by moving to fig. 7c that shows a clustering outcome when the cut-off parameter c is decreased to 0.1. in fig. 7c , while 3 members of the merbecovirus sub-genus (i.e. pipistrellus bat cov hku5, tylonycteris bat cov hku4 and mers cov) are divided into 3 clusters ("12", "2" and "4") or members of the sarbecovirus cluster are separated themselves into 2 clusters "'1" and "11", sequences of sars-cov-2 still join the cluster "11' with other members of sarbecovirus such as 3 bat viruses (bat sars cov zc45, bat sars cov zxc21, bat cov ratg13) and 7 pangolin covs. as the cut-off parameter c decreases, the number of clusters increases. this is an expected outcome because the cut-off threshold line moves closer to the leaves of the dendrogram. when the cut-off c is reduced to 0.03 (fig. 7d) , there are only 2 viruses (bat cov ratg13 and guangdong pangolin cov) that can form a cluster with sars-cov-2 (labelled as cluster "15"). these are 2 viruses closest to sars-cov-2 based on the whole genome analysis. results in figs. 7c and 7d therefore provide evidence that bats or pangolins could be possible hosts for sars-cov-2. we next reduce the cut-off c to 0.01 as in fig. 7e . at this stage, only bat cov ratg13 is within the same cluster with sars-cov-2 (cluster "17"). we thus believe that bats are the more probable hosts for sars-cov-2 than pangolins. the inference of our ai-enabled analysis is in line with a result in [19] that investigates the polyprotein 1ab of sars-cov-2 and suggests that this novel coronavirus has more likely been arisen from viruses infecting bats rather than pangolins. when the cut-off c is reduced to 0.001 as in fig. 7f , we observe that the total number of clusters now increases to 29 and more importantly, sars-cov-2 sequences do not combine with any other reference viruses but form its own cluster "19". could we use this clustering result (fig. 7f) to infer that sars-cov-2 might not originate in bats or pangolins? this is a debatable question because the answer depends on the level of details we use to differentiate between the species or organisms. the cut-off parameter in hierarchical clustering can be considered as the level of details. with the results obtained in fig. 7d (and also in the experiments with the dbscan method presented next), we support a hypothesis that bats or pangolins are the probable origin of sars-cov-2. this is because we observe that the similarity between sars-cov-2 and bat cov ratg13 (or guangdong pangolin cov) is considerably large compared to the similarity between viruses that originated in the same host. for example, bat sars-like covs such as bat sars cov rf1, bat sars cov longquan-140, bat sars cov hku3-1, bat sars cov rp3, bat sars cov rs672/2006, bat sars cov rsshc014, bat sars cov wiv1, bat sars cov zc45 and bat sars cov zxc21 had the same bat origin. in fig. 7d , these viruses however are separated into 2 different clusters ("3" and "2") while all 16 sars-cov-2 representatives are grouped together with bat cov ratg13 and guangdong pangolin cov in cluster "15". this demonstrates that the difference between the same origin viruses (e.g. bat sars cov wiv1 and bat sars cov zc45) is larger than the difference between sars-cov-2 and bat cov ratg13 (or guangdong pangolin cov). therefore, sars-cov-2 is deemed very likely originated in the same host with bat cov ratg13 or guangdong pangolin cov, which is bat or pangolin, respectively. clustering outcomes of the dbscan method via phylogenetic trees using set 3 of reference sequences (table 3) are presented in fig. 8 . we first apply dbscan to reference sequences only, which results in 3 clusters and several outliers (fig. 8a) . the search radius parameter ε is set equal to 0.55. as we set the minimum number of neighbours parameter to 3, it is expected that viruses of the sub-genera embecovirus, nobecovirus and hibecovirus are detected as outliers "-1" because there are only 1 or 2 viruses in these sub-genera. three viruses of the merbecovirus sub-genus (i.e. tylonycteris bat cov hku4, pipistrellus bat cov hku5 and mers cov) are grouped into the cluster "2". all examined viruses of the sarbecovirus sub-genus are joined in cluster "1" while the alphacov viruses are combined into cluster "3". fig. 8b shows an outcome of dbscan with the same ε value of 0.55 and the dataset has been expanded to include 16 representative sars-cov-2 sequences. we observe that genomes of sars-cov-2 fall into the cluster "1", which includes all the examined sarbecovirus viruses. when ε is decreased to 0.3 in fig. 8c , all members of the merbecovirus cluster or the alphacov cluster become outliers while 16 sars-cov-2 genomes still stick with the sarbecovirus cluster. in line with the results obtained by using hierarchical clustering in fig. 7 , those obtained in fig. 8b and 8c using the dbscan method give us the confidence to confirm that sars-cov-2 is part of the sarbecovirus sub-genus. fig. 8d shows that bat cov ratg13 and guangdong pangolin cov are closest to sars-cov-2 as they join with 16 sars-cov-2 representatives in cluster "2". this again substantiates the probable bat or pangolin origin of sars-cov-2. by reducing ε to 0.1 as in fig. 8e , the guangdong pangolin cov becomes an outlier whilst sars-cov-2 sequences form a cluster ("3") with only bat cov ratg13. this further confirms our findings when using the hierarchical clustering in fig. 7 that bats are more likely the reservoir hosts for the sars-cov-2 than pangolins. when ε is decreased to 0.01 as in fig. 8f , sars-cov-2 genomes form its own cluster "3", which is separated with any bat or pangolin genomes. as with the result in fig. 7f by the hierarchical clustering, this result also raises a question whether sars-cov-2 really originated in bats or pangolins. in fig. 8d , it is again observed that the similarity between sars-cov-2 and bat cov ratg13 (or guangdong pangolin cov) is larger than the similarity between bat sars covs, which have the same bat origin. specifically, sars-cov-2, bat cov ratg13 and guangdong pangolin cov are grouped together in cluster "2" while bat sars covs are divided into 2 clusters, i.e. bat sars cov zxc21 and bat sars cov zc45 are in cluster "2" whereas other bat sars covs are in cluster "3". we thus suggest that sars-cov-2 probably has the same origin with bat cov ratg13 or guangdong pangolin cov. in other words, bats or pangolins are the probable origin of sars-cov-2. all results presented above are obtained using the pairwise distances estimated by the jukes-cantor method. results based on distances calculated by the maximum composite likelihood method are reported in appendix 3, which are similar to those obtained by using the jukes-cantor method. these ai-based quantitative results using the unsupervised hierarchical clustering and dbscan methods provide more evidences to suggest that 1) sars-cov-2 belongs to the sarbecovirus sub-genus of the betacoronavirus genus, 2) bats and pangolins may have served as the hosts for sars-cov-2, and 3) bats are the more probable origin of sars-cov-2 than pangolins. the severity of covid-19 pandemic has initiated a race in finding origin of the covid-19 virus. studies on genome sequences obtained from early patients in wuhan city in china suggest the probable bat origin of the virus based on similarities between these sequences and those obtained from bat covs previously reported in china. other studies afterwards found that sars-cov-2 genome sequences are also similar to pangolin cov sequences and accordingly raised a hypothesis on the pangolin origin of the covid-19 virus. this paper has investigated origin of the covid-19 virus using unsupervised clustering methods and more than 300 raw genome sequences of sars-cov-2 collected from various countries around the world. outcomes of these ai-enabled methods are analysed, leading to a confirmation on the coronaviridae family of the covid-19 virus. more specifically, the sars-cov-2 belongs to the sub-genus sarbecovirus within the genus betacoronavirus that includes sars-cov, which caused the global sars pandemic in 2002-2003 [20; 21] . the results of various clustering experiments show that sars-cov-2 genomes are more likely to form a cluster with the bat cov ratg13 genome than pangolin cov genomes, which were constructed from samples collected in guangxi and guangdong provinces in china. this indicates that bats are more likely the reservoir host for the covid-19 virus than pangolins. this study among many ai studies in the fight against the covid-19 pandemic [22] has shown the power and capabilities of ai in this challenging battle, especially from the computational biology and medicine perspective. the findings of this research on the large dataset of 334 sars-cov-2 genomic sequences provide more insights about the covid-19 virus and thus facilitate the progress on discovering medicines and vaccines to mitigate its impacts and prevent a similar pandemic in future. the race to produce treatment drugs and vaccines is still ongoing and no effective results have been reported yet. a further research in this direction is strongly encouraged by a recent success of ai in identifying powerful new kinds of antibiotic from a pool of more than 100 million molecules as published in [23] . as ai is capable of analysing large datasets and discovering knowledge from them in an intelligent and efficient manner, finding a covid-19 vaccine using ai is a realistic hope [24] . in this appendix, we first present results of the hierarchical clustering method applied to the dataset that combines set 1 of reference sequences (table 1 ) with all 334 sars-cov-2 sequences (see fig. 9 ). we then show results of the hierarchical clustering (fig. 10) and dbscan (fig. 11 ) on a dataset that combines all 334 sars-cov-2 sequences and reference sequences in set 3 (table 3) . fig. 9 . results shown via a dendrogram plot (left) of the hierarchical clustering method applied to the dataset that combines reference sequences in set 1 (table 1 ) and all 334 sars-cov-2 sequences. the middle figure shows in detail (zoom in) the top part of the dendrogram plot while the right figure shows the bottom part of the plot. all 334 sars-cov-2 sequences are grouped in cluster "8", which also includes the middle east respiratory syndrome cov of the riboviria realm. this means that sars-cov-2 belongs to the riboviria realm. these results are consistent with those shown in fig. 1b that, for the demonstration purpose, employed only 16 sars-cov-2 genomes, which are representatives of 16 countries in table 5 . this appendix presents results of two clustering methods, i.e. hierarchical clustering and dbscan, using the sequence distances computed by the maximum composite likelihood method [16] , which was conducted in the mega x software [25] . these results are greatly similar to those obtained by using the jukes-cantor distance method shown throughout the paper. in these experiments, the clustering methods are applied to a dataset that combines reference sequences in set 3 (table 3 ) and 16 representative genomes of 16 countries in table 5 . when a country has more than one collected genome, the first released genome of that country is selected for this experiment. fig. 12 demonstrates the distances estimated by the maximum composite likelihood method between each of the reference sequences and 16 representative sars-cov-2 genomes. the lines are almost parallel indicating that sars-cov-2 genome is not altered much across countries, which is in line with the results obtained using the jukes-cantor distance estimates in fig. 5 . the bat cov ratg13 is again shown much closer to sars-cov-2 than pangolin covs and other reference viruses although the distance range in fig. 12 is larger than that in fig. 5 in fig. 13a , when the hierarchical clustering cut-off parameter is set equal to 0.1, all 16 representative sars-cov-2 genomes are grouped into cluster "12", which also includes other viruses of the sarbecovirus sub-genus of the betacov genus. when moving from fig. 13a to fig. 13b , even though members of the sarbecovirus cluster ("12" in fig. 13a ) are split into 2 clusters "1" and "2" in fig. 13b , the sars-cov-2 sequences are still grouped into cluster "14" with other members of the sarbecovirus sub-genus such as bat cov ratg13, guangdong pangolin cov, bat sars cov zxc21 and bat sars cov zc45. these results provide us with a confidence on confirming the sarbecovirus sub-genus of the sars-cov-2. this is consistent with the result based on the jukes-cantor distances shown in fig. 7 . fig. 13c shows that sars-cov-2 genomes are combined only with that of bat cov ratg13 when the cut-off parameter is decreased to 0.001. this again indicates that bats are the more likely origin of sars-cov-2 than pangolins. when we reduce the cut-off parameter to 0.0001, the sars-cov-2 sequences create their own cluster "22" and this questions the probable bat or pangolin origin of sars-cov-2. however, in fig. 13b , we also find that the similarity between sars-cov-2 and bat cov ratg13 (or guangdong pangolin cov) is larger than the similarity between viruses having the same origin. for example, bat sars cov wiv1 and bat sars cov zc45 have the same bat origin but they are divided into 2 clusters ("1" and "14") while all 16 sars-cov-2 representatives are grouped into cluster "14" with bat cov ratg13 and guangdong pangolin cov. this implies that sars-cov-2 may have originated in bats or pangolins. results of dbscan using distances estimated by the maximum composite likelihood method are presented in fig. 14 , which are also consistent with those obtained by the jukes-cantor distance method in fig. 8 , leading to the same suggestions on the sub-genus sarbecovirus membership of sars-cov-2, its likely bat or pangolin origin, and the more probable bat origin than the pangolin origin of the virus at the whole genome analysis level. who coronavirus disease (covid-19) dashboard. available at origin of sars-cov-2 a new coronavirus associated with human respiratory disease in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin a genomic perspective on the origin and emergence of sars-cov-2 identifying sars-cov-2 related coronaviruses in malayan pangolins probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak isolation of sars-cov-2-related coronavirus from malayan pangolins the proximal origin of sars-cov-2 machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: covid-19 case study clustering methods a density-based algorithm for discovering clusters in large spatial databases with noise evolution of protein molecules prospects for inferring very large phylogenies by using the neighbor-joining method estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination an exclusive 42 amino acid signature in pp1ab protein provides insights into the evolutive history of the 2019 novel human-pathogenic coronavirus (sars-cov-2) identification of a novel coronavirus in patients with severe acute respiratory syndrome origins of major human infectious diseases artificial intelligence in the battle against coronavirus (covid-19): a survey and future research directions a deep learning approach to antibiotic discovery ai can help scientists find a covid-19 vaccine mega x: molecular evolutionary genetics analysis across computing platforms table 4 . accession numbers of 334 sars-cov-2 genome sequences obtained from ncbi genbank in early april 2020, sorted by date released mn908947, mn985325, mn975262, mn938384, mn988713, mn997409, mn994468, mn994467, mn988669, mn988668, mn996531, mn996530, mn996529, mn996528, mn996527, mt007544, mt019533, mt019532, mt019531, mt019530, mt019529, mt020881, mt020880, mt027064, mt027063, mt027062, mt039890, mt039888, mt039887, mt039873, mt049951, mt044258, mt044257, mt066176, mt066175, mt072688, mt093631, mt093571, mt106054, mt106053, mt106052, mt118835, mt123293, mt123292, mt123291 tables 4 and 5 in this appendix present accession numbers and detailed distribution of 334 sars-cov-2 complete genomes across different countries obtained from the ncbi genbank database. key: cord-309138-44qpk2vf authors: khanna, kanika; kohli, sukhmeen kaur; kaur, ravdeep; bhardwaj, abhay; bhardwaj, vinay; ohri, puja; sharma, anket; ahmad, ajaz; bhardwaj, renu; ahmad, parvaiz title: herbal immune-boosters: substantial warriors of pandemic covid-19 battle date: 2020-10-03 journal: phytomedicine doi: 10.1016/j.phymed.2020.153361 sha: doc_id: 309138 cord_uid: 44qpk2vf current scenario depicts that world has been clenched by covid-19 pandemic. inevitably, public health and safety measures could be undertaken in order to dwindle the infection threat and mortality. moreover, to overcome the global menace and drawing out world from moribund stage, there is an exigency for social distancing and quarantines. since december, 2019, coronavirus, sars-cov-2 (covid-19) have came into existence and up till now world is still in the state of shock.at this point of time, covid-19 has entered perilous phase, creating havoc among individuals, and this has been directly implied due to enhanced globalisation and ability of the virus to acclimatize at all conditions. the unabated transmission is due to lack of drugs, vaccines and therapeutics against this viral outbreak. but research is still underway to formulate the vaccines or drugs by this means, as scientific communities are continuously working to unravel the pharmacologically active compounds that might offer a new insight for curbing infections and pandemics. therefore, the topical covid-19 situation highlights an immediate need for effective therapeutics against sars-cov-2. towards this effort, the present review discusses the vital concepts related to covid-19, in terms of its origin, transmission, clinical aspects and diagnosis. however, here, we have formulated the novel concept hitherto, ancient means of traditional medicines or herbal plants to beat this pandemic. pandemic diseases are of global concern in the present era, to cause gigantic morbidity and transience, regardless of, extensive medical facilities. particularly, anti-viral therapies have been fraught because of surfacing of mutants competent enough to subdue the drugs targeting disease that has created panic all over. by this review, we suggest that herbal or medicinal plant formulations could be essential alternative strategy, a step ahead to battle these awful viruses. zhang and holmes, 2020). moreover, sars-cov-2 just like sars-cov utilizes the same ace2 receptors to infect its hosts (guo et al., 2020) . thus, the sites where ace2 protein is mainly expressed are the potential target sites for sars-cov-2 respectively. these regions are belong to type ii alveolar cells of the lungs and enterocytes of the small intestine (hamming et al., 2004; zheng, 2020) . nevertheless, there are some remarkable biological differences between the sars-cov-2 and the other beta-covs, which probably make it more infectious. consequently, the epidemiological dynamics of sars-cov-2 is different from previous human-cov outbreaks having striking local and global spread (zhang and holmes, 2020; zheng, 2020). although, sars-cov-2 shows greater human-to-human transmission efficiency, its crude fatality rate (0.25% to 5%) is comparatively far less than that of sars-cov which is approx. 10%. furthermore, sars-cov-2 has r 0 (basic reproduction number) of 4.7 to 6.6. this highly contagious nature of sars-cov-2 is supported by the fact that its spike (s) protein possess 10 to 20 time's greater affinity for ace2 receptors than sars-cov (zheng, 2020) . s-protein is the surface glycoprotein that assists the virus in the attachment to the host cells through its receptor-binding domain (rbd). s-protein has several domains, one of the sections termed as ectodomain has two subunits, s1 and s2, which form a crown-like structure around the virus (vellingiri et al., 2020) . besides, s-protein of sars-cov-2 contains a furin-like cleavage site at the s1-s2 junction, missing in other members of its sister clade. this additional cleavage site might also be responsible for greater pathogenicity of sars-cov-2 as it also occurs in highly infectious form of influenza virus but lacking in less pathogenic ones (coutard et al., 2020; zhang and holmes, 2020).3. origin and the natural reservoir for both the sars-cov and mers-cov were bats; however, these viruses infect humans through an intermediate host. palm civets are supposed to be the intermediate host of sars-cov while dromedary camels are of mers-cov (yin and wunderink, 2018) .unfortunately, the exact zoonotic origin of sars-cov-2 is still elusive. since sars-cov-2 shares 88% nucleotide homology with two sars-like cov found in bats (bat-sl-covzc45 and bat-sl-covzxc21) and 96% with ratg13 virus found in horseshoe bat (rhinolophus affinis), bats are considered to be its natural host (lu et al., 2020b; zhou et al., 2020b) . despite the 96% nucleotide similarity, the rbd of both the viruses varies significantly (mackenzie and smith, 2020) . however, due to the ecological separation of the bats from the humans and the requirement of some necessary mutations in the virus genome to cross the species barrier, sars-cov-2 likely has one or more mammalian intermediate hosts for efficient animal-to-human transmission (zhang and holmes, 2020) . analyses of interactions between rbd in sars-cov-2 and ace2 receptors in different hosts indicate that pangolins, snakes, and turtles may be the immediate host of sars-cov-2 (liu et al., 2020) (fig. 1) . recent phylogenetic studies on the genome of pangolin-cov found from dead malayan pangolins, which are illegally imported in china revealed that its genome is about 91% identical to sars-cov-2 and around 90% to ratg13 (bat-cov). besides, five of the six amino acids (contact residues) on rbd, vital for binding to ace2 receptors on hosts are consistent between sars-cov-2 and pangolin-cov, but only four between pangolin-cov and ratg13 and only one out of six between sars-cov and sars-cov-2 (zhang et al., person-to-person transmission is primarily reported in families, communities, and hospitals (guo et al., 2020) . droplet transmission is considered as the main route of person-to-person transmission (han et al., 2020) . the infection also spreads through direct contact and via fomite exposure i.e., direct contact to eyes, nose, and mouth after touching surfaces and objects contaminated by an infected individual morawskaa and cao, 2020) . in general, human-covs can remain infectious on some material surfaces for even 9 days (kampf et al., 2020) . besides, sars-cov-2 also spreads through contact with asymptomatic carriers . since sars-cov-2 infection is spreading at an unprecedented pace, airborne transmission too warrants meticulous determination (morawskaa and cao, 2020). in addition, sars-cov-2 was found in a stool sample, gastrointestinal tissue even urine and saliva of an infected person, suggests the possibility of intestinal and fecal-oral transmission (xiao et al., 2020; guan et al., 2020) . although no case of mother to child transmission has been reported yet, infection in two newborns of wuhan, china raises the question for vertical transmission (han et al., 2020) . apart from this, there are recent studies that reveal that there is a possibility of sexual transmission of covid-19 infection. there are an array of evidences for this transmission such as through faecal-oral route via gastrointestinal infection . this is most probably due to ace2 that enable virus entry and ace2 mrna is overexpressed in gastro-intestinal system as revealed through immunofluorescent analysis of rectal epithelial cells (hindson, 2020). moreover, rna analysis of sars-cov-2 also revealed that the viral particles can infect these cells therefore, it is quite predictable that sexual intercourse could be the possible way of contagion. there have been cases when person shows negative results on nasopharyngeal swabs, while, positive on rectal swabs, depicting that sexual transmission might be the possibility . henceforth, the physicians as well as doctors in particular recommend a strong message to discourage sexual practices if in any case of covid-19 infection. hence, comprehensive possible means of sars-cov-2 transmission warrants further study. suggests this novel sars-cov-2 affects elderly people with coexisting other medical complications more seriously. these results are consistent with previous studies conducted on severely ill patients which also advocates high mortality rate in elderly people with acute respiratory distress syndrome (ards) and comorbidities such as hypertension, diabetes, chronic obstructive lung disease and coronary heart disease zhou et al., 2020a) . the most common complications associated with sars-cov-2 infection are sepsis, respiratory and heart failure, ards, and septic shock (zhou et al., 2020a) . even though children of all ages face risk of sars-cov infection, the serious progression of infection and morbidity is rare in children and adolescents relative to the adults (lu et al., 2020a). sars-cov-2 infection is often categorized into three stages: first, asymptomatic phase; second, non-severe symptomatic phase; and third, severe respiratory symptomatic phase (shi et al., 2020) . usually, a small number of patient's progress to the severe stage and develop ards and/or multiorgan failure (cao et al., 2020). host's immune responses initiate as soon as sars-cov-2 binds to ace2 receptors and releases viral rna for replication. both the innate and adaptive immune response could be triggered in response to the sars-cov-2 infection (cao et al., 2020). however, immune responses are different between severely and moderately infected persons. in a blood sample of symptomatic hospitalised patients with mild to moderate sars-cov-2 infection before resolution of symptoms, immunological changes such as increase in the number of activated cd4+ helper t cells and cd8+ killer t cells, follicular helper t (tfh) cells, antibodysecreting cells (ascs) and antibodies particularly igg (immunoglobulin g)and igm (immunoglobulin m) were detected (thevarajan, 2020) . on the other hand, in severely infected patients, lymphocytopenia is a common denominator with substantial fall in numbers of natural killer cells, b cells, cd3+ t cells, cd4+ helper t cells, cd8+ killer t cells along with the increase in neutrophil-to-lymphocyte ratio (nlr) and c-reactive protein levels. additionally, in comparison to the non-severe patients, pro-inflammatory cytokines and chemokines such as tumor necrosis factor (tnf)-α, interleukin (il)-2, il-6, il-7, il-8, il-10, granulocyte-colony stimulating factor (gcsf), monocytechemoattractant protein 1 (mcp1) and macrophage inflammatory protein 1-alpha (mip1-alpha) are often reported to be elevated in serum levels of critically ill patients (huang et al., 2020; qin et al., 2020; wang et al., 2020a) . the elevated ratio of nlr, which is a biomarker of systemic inflammatory response syndrome, points to the devastated inflammatory state of icu patients (salciccioli et al., 2015) . moreover, uncontrolled levels of cytokines and chemokines cause over-active inflammatory responses or cytokine storm. this hyperactive immune response along with impaired adaptive immune response may trigger pulmonary injury, ards, viral sepsis and organ failure like complications, and eventually death in some cases (prompetchara et al., 2020) . the traditional chinese medicines and ayurveda since the vedic period (1500-500 bce) provides globally with potential remedies to lessen the severity of the illnesses caused by the traditional medicines have been in general disregarded in the novel research and expansion of contemporary drugs due to the fact that their translational ability is commonly i.e. curumin, is identified to block cytokine release, specifically interleukin-1, interleukin-6, pro-inflammatory cytokines and tumor necrosis factor-α and is directed to be consumed with milk (omara et al., 2010) . inhibition of the cytokine discharge is one of the prime clinical development associated with experimental modules of flu and other infectious diseases and have also been compared to covid-19 where similar cytokine storm play an imperative role in transience (sordillo et al., 2015) . moreover, ayush has recommended certain preventive and medicinal plants for prevention and prophylactic of covid-19 including warm extracts of tinospora cordifolia (advised for chronic fever), andrograhis paniculata (advised for fever and cold), cydonia oblonga, zizyphus jujube and cordia myxa (enhancing antioxidant, immune-modulatory, anti-allergic, smooth muscle relaxant, anti-influenza activity) and arsenicum album 30 (found effective against sars-cov-2, immune-modulator). the symptomatic management of covid-19 was suggested to be acquired from agastya haritaki the polysaccharides are a structurally multifaceted class of biomolecules that have diverse therefore, these biocompatible compounds were suggested to be employed in coating materials of various sanitary items for covid-19 deterrence. the naturally occurring products and phytomedicines are coming to the fore all around the (sengupta, 2020 ). according to their study, 'aurantiamide', an active metabolite from piper aurantiacum has chymotrypsin like-proteases like activities that is a ratified agent for inhibiting coronavirus. in addition to this, it also encompassespiperol, eugenol, catechol, caryophyllene, etragol, chavibetol, betlol, quercetin etc. that also grows body's self-defense mechanism either through reception of viruses or in waning off the viral load within the infected hosts (sengupta, 2020) .alongside, studies have been depicted the role of herbal drug gene-eden-vir/novirin against many noxious viruses and this drug is mainly comprised of quercetin, green tea, cinnamon, licorice and selenium (polansky and lori, 2020). they found that it disrupts the viral entry, infection, replication, viral proteases, viral quasi-speciesand triggers the immunity, thereby, can be evidently utilised against sars-cov-2 respectively (polansky and lori, 2020). interestingly, the role of tulsi for scientific evidence against covid-19 has also been elucidated (goothy et al., 2020). as, it is well known herbal plant for antiviral effects in inhibiting many deadly viruses like vaccinia, dengue, hepatitis, encephalitis etc. by enhancing their survival and defense ability. moreover, it also reinstate the physiological functions of body through its phenolic and antioxidative property that in turns shields the body from toxic substances (shivananjappa and joshi, 2012) . the most possible mechanism underlying its immunity boosters lies in triggering humoral and cellular immunity responses (vaghasiya et al., 2010) . apart from this, modulatory actions of gaba pathway also encompass their multi-modal therapeutic properties, therefore, we can conjecture that it could be efficient in cure of covid-19. nevertheless, the role of persian herbs have also been untangled against coronavirus covid-19 pandemic is a huge catastrophe that has caused devastating effects however, the biological activities can also be improved by ingesting high levels of naturally existing polyphenols that not only elicit immunity against viruses but they are also comprised of receptors that identifies and permit their cellular uptake for activating signaling cascade are also hindered by smoke.therefore, these steps can reboot the immune system and reinforce the inner forces to battle against severe viral infections covid-19 respectively. the most momentous weapon against any kind of viral infection is a strong immune system. vitamin a is an essential fat-soluble vitamin which has a major involvement in regulating vision, growth and maturity as well as protection of the mucosal and epithelium had the highest inhibitory potential against sars-cov-2, followed by galangal, sappan wood and curcuma species and it was further suggested that they might have antiviral potency against covid-19. additionally, different plant based sources of vitamins include, mushrooms, carrots, broccoli, almonds, citrus, guava, amla, avocados etc. fig. 3 describes the role of vitamins (a, c, e and d) in combating covid-19. zinc is one of the indispensible elements which has significant role in modulation of growth and development and the regulation of the immunomodulatory responses in addition to this, another trace element that has been reported to have wide range of pleiotropic effects ranges from anti-inflammatory to antioxidative is selenium (se) (rayman, 2012) . comparatively, the lower concentrations of se are related to enhancement in the jeopardy of mortality, meager immune responses and cognitive reduction, whereas the higher doses are affirmed to show antiviral activity (rayman, 2012) . supplementation of se results in elevation in concentration of se in the plasma, up-regulation of lymphocyte phospholipid levels and activity of gpox in the cell, subsequently leading elevation in cellular immune response. the humoral response was shown to be un-affected (broome et al., 2004) . similar reports of hasty clearance of poliovirus were also reported by se supplementation (ivory et al., 2017). various plants namely, beans, nuts, peanuts, grains, mushrooms, cereals, lentils, garlic, grains, etc. have been wide sources of minerals and must be included in diet. nutraceuticals are the products which are argued to provide physiological assistance and protection against varied persistent diseases. wide range of nutraceutical products have been isolated herbal products, dietary supplements, isolated nutrients, genetically engineered foods and processed cereals, beverages and soups (kalra, 2003) . certain nutraceuticals have been shown earlier to enhance the immune function. mccarty and dinicolantonio (2020) elucidated that, a few nutraceuticals were able to lower the degree of symptoms and provide respite to the patients infected from coronavirus and influenza. another significant product is probiotic, they are defined as live micro-organisms which bestow varied health benefits such as improvement in gastrointestinal activity (sanders, 2008) . they also induce certain specific immune responses by augmenting antibody synthesis (kanauchi et al., 2018) . a report by paks belong to mammalian kinases family also known as rac/cdc42-activated kinases that have been came into existence since decades. amid, these classes pak1 is most prominent category of "pathogenic kinases" that can cause a plethora of diseases like cancer, inflammation, malaria, dengue, immuno-suppression and many antiviral diseases, if behave abnormally (maruta, 2020) . strikingly, pak1-blockers (naturally existing) such as caffeic acid, its esters, bee-product propolis have been observed to inhibit rac that directly activates pak1 (xu et al., 2005) . however, previous studies reported that chloroquine a drug active against malaria also suppressed the incidence of sars/coronavirus infection, but its mode of action still remains unknown (vinecent, 2005) . later, it was reported that this drug induced cdk inhibitor (p21) to inhibit pak1 activity (maruta, 2014) . in line of forging argument, a recent study depicted that tumor-repressed phosphatase pten, mainly involved in impeding pak1 also inhibited the coronavirus-curbed llc2-linked fibrosis (lu, 2020). moreover, llc2 expression has been observed to correlate with ace2, a coronavirus receptor and ck2/ras-pak1-ap1 signaling cascade (chen et al., 2010) . henceforth, through all these assumptions we can deduce the pak1-reliance of corona pathogenesis and a suggestion of using pak1-blockers for treating pandemic covid-19 outbreak (fig. 4) inevitably, the conventional and recently accepted product propolis, obtained from bees have been known since ancient times. it is formerly obtained from honey-bee extract via honey-bees feeding the buds of poplar and willow trees, thereby, mixing their saliva into it to form hexagonal bee-hives in order to protect their larval species against many pathogenic organisms. this product synthesised is called as propolis and is regarded as herbal medicine with anti-viral properties. in contemporary world, propolis is identified in possessing anticancer characteristics due to the presence of an ingredient cape (caffeic acid phenyl ester) involved in down-regulation of rac for pak1 inhibition respectively (xu et al., 2005) . although, the anticancer constituents in propolis varies from product to product according to harvesting property of bee extracts. for instance, the anti-cancer element in brazilian propolis is artepillin c (arc), while in sub-tropical regions of taiwan and okinawa, polyphenols namely, nymphaeols act as direct inhibitors of pak1 .in view of the fact that, familiar thing among all categories is that they are comprised of pak1-blockers. ever since, has been elucidated that, pak1 tends to cause cancers, viral diseases like hiv, hepatitis, pappiloma, influenza, ebola, sars and corona virus along with immune system suppression of hosts, henceforth, propolis would be quintessential in blocking covid/coronavirus curbed fibrosis in respiratory tract and boosting the immunity of an individual (maruta, 2014) . the effectiveness of propolis also depends on the product, its chemical nature and action. to expedite, cape-containing propolis named 'bio 30', commercialised by new zealand is found to be highly active (maruta, 2014) . the optimal doses are 1 ml/10 kg of the body weight. regardless of this pre-formed formulation in the market, it could not be used against covid-19 eruption, due to paucity of its stock in market place. moreover, the available stocks are being predominantly used for treating patients with deadly cancers and prolonged treatment of genetic brain disorder, nf tumers (neuro fibromatosis type 1, 2) (maruta and he, 2020). in addition to this, the cell membrane permeability of caffeic acid and arc is relatively weak, owing to its -cooh moiety. nonetheless, this feature could be surmounted by triggering the cell membrane permeability via conversion their esters into 1, 2, 3-triazolyl esters that are thousand times more powerful than arc or caffeine esters (maruta and ahn, 2017) . along with this, melatonin hormone (pineal gland hormone) is a serotonin and possess anti-melanogenic activity that is a primitive factor as melanogenesis is directly correlated to pak1 . further, it also owns similarity with other anti-pak1 actions like anti-cancer, antiviral, antiinflammatory, immuno-boosters etc. therefore, it is pertinent to mention here that melatonin, a commonly available sleeping pill could be utilized against coronaviral infections. currently, many investigations are being carried out to high-light the significance of melatonin as another therapeutic resort or covid-19 adjuvant (zhang et al., 2020b) . furthermore, glucocorticoid hormone 'ciclesonide' is another choice to cure different inflammatory disorders and it has been widely commercialised under the brand name of alvesco. it has been officially approved since 1990 and been widely associated with treating adults as well as children. the molecular mechanism forming the basis for anti-inflammatory action is based on the potential of pak1-blockers that lays the foundation for its pathway. majorly, it works according to two basic reasons, firstly, inflammation is not possible without pak1 and apart from this, a herbal formulation mainly triterpenes or steroid named 'triptolide' derived from thunder god vine also inhibited rac followed by blocking pak1 route (maruta and ahn, 2017) . this medicine was previously treating the patients infected with dengue virus in restricting the proliferation of virus in lungs by blocking pak1 signaling cascade (liou et al., 2008) . conversely, due to its lower water solubility, few years back, its -oh group was phosphorylated to enhance its solubility (patil et al., 2015) . resultantly, phosphotase-receptive pro-drug of triptolide 'minnelide' is currently under clinical trials to cure covid-19 infections as well. another compound ivermectin has been screened from bacteria but with some lethal effects. interestingly, these ill-effects were removed later by chemically re-designing the compound. (hirokawa et al., 2005) . therefore, possessing all these features it was approved to cure rare cancer disease cutaneous lymphoma and commercialised as 'isodax' in market. however, research is being conducted with the belief that it could be most probably utilized for covid-19 therapy. it is need of an hour to clinically test various pak1-blockers against blocking virus replication to develop anti-covid-19 therapy for patients suffering worldwide. strata of mankind has been marred with the onset of the unexpected pandemics, leading to the atrocious effects on their entire communities. considering themselves invincible to conquer the world, they have now become the cocoons of their houses against covid-19 storm. unfortunately, sars-cov-2 has led the global population on their toes due to its highly infectious nature and susceptibility towards humans in causing higher mortalities among them. we can also introspect that about a lot many cases of covid-19 have been undetected and by looking into the ongoing trends, the cases are doing to inflate in the coming future. this would ultimately affect the world economy, specifically within developing and under-developing nations. besides, who has advised the world to take preventive measures, owing to which government has facilitated nationwide shutdowns, selfquarantine, physical distancing to ensure public safety. thus far, the prevailing drugs and vaccines for antiviral diseases have been used as clinical trials to fight against coronavirus. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influencethe work reported in this paper. vitamin d supplementation improves sustained virologic response in chronic hepatitis c (genotype 1)-naïve patients zinc supplementation promotes a a review on antiviral activity of the 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extracts as antiviral agents water extract of licorice had anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines mers, sars and other coronaviruses as causes of pneumonia identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 the traditional medicine and modern medicine from natural products covid-19: melatonin as a potential adjuvant treatment probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak a genomic perspective on the origin and emergence of sars-cov-2 sars-cov-2: an emerging coronavirus that causes a global threat clinical course and risk factors for mortality of adult in patients with covid-19 in wuhan, china: a retrospective cohort study a pneumonia outbreak associated with a new coronavirus of probable bat origin none list of tables: table 1 . herbal formulations as possible therapeutics against covid-19 infection mechanism of action therapeutic property references anti-inflammatory action by via modulation of gene expression of ace2 enzyme, serine protease tmprss2, protein pre-requisite for sars-cov2 invasion into host cells.adjunct therapy and utilised as mouthwash and throat gargle products clinically and home use owing to their potential to decrease viral entry via the oral mucosa. 15. highest ace2 enzyme inhibition, anti-inflammatory activity, modulated -gene expression for tnf-production in macrophages.bioactive compounds could be used for drug formulations. 17. ginkgolic acids impeded dna and protein synthesis by binding towards host cell receptors to activate cellsignaling pathways for arresting cell cycle as an inhibitory action. key: cord-291397-look6ddt authors: roberto, palumbo; francesco, londrino; emanuela, cordova; giorgia, gambardella; pasquale, niscola; sara, dominijanni title: current treatment of covid-19 in renal patients: hope or hype? date: 2020-09-28 journal: intern emerg med doi: 10.1007/s11739-020-02510-0 sha: doc_id: 291397 cord_uid: look6ddt to date the severe acute respiratory syndrome coronavirus 2 (sarscov-2), known as covid-19, is for clinicians the most difficult global therapeutic problem. in this landscape, the management of patients with chronic kidney disease, acute kidney injury or patients undergoing immunosuppressant therapies for kidney transplant or glomerular diseases, represent a clinical challenge for nephrologists, especially in patients with severe acute lung involvement. therefore in this setting, due to the lack of anti-covid treatment schedules, tailored management is mandatory to reduce the side effects, as consequence of impaired renal function and drugs interactions. we report the main treatment actually used against sars-cov-2, underlining its possible use in the nephropatic patients and the central role of nephrologists to improve the clinical outcome. despite the drastic containment measures implemented in many countries across the world, sars-cov-2 outbreak (also known as , started in the city of wuhan (hubei province of china) in december 2019, is spreading all over the world [1, 2] . in this landscape, due to the sudden and rapid explosion of the infection and to the lack of new effective therapies, clinicians are investigating the effect of drugs used for other viral diseases. recently, alberici et al. reported the therapeutic management of the sars-cov-2 infection in a population of renal patients in the city of brescia, in italy (brescia covid task force) based on the disease activity [3, 4] . currently, nephrologists are using the available drugs with particular attention to the renal impairment degree, interactions with other medications and, regarding dialysis patients, to the dialytic clearance. the novel sars-cov-2 coronavirus is a rna obligate intracellular virus and its replication depends on synthetic processes of the host cell. to date, none of the drugs currently used have shown therapeutics effects. as a rule, to be effective, selective antiviral agents should block viral entry into the cell and should be active inside the host cell, whereas nonselective viral inhibitors may interfere with human cell function and promote the side effects. the clinical onset of sars-cov-2 infection is variable from mild self-limited influenza-like symptoms to severe acute respiratory syndrome with possible association of multi-organ failure (mof) secondary to cytokine storm and sometimes of haemophagocitic syndrome [3, 4] . it is noteworthy that in renal patients, the latter conditions represent a real challenge in terms of care and survival. the diagnosis of covid-19 has to be confirmed by the reverse transcription polymerase chain reaction (rt-pcr) or gene sequencing for respiratory tract samples or by serologic test. moreover, chest computed tomography (ct) represents an indispensable tool for screening and diagnosing covid-19 patients. ct chest imaging in the early stages, from mild to severe cases, shows interstitial changes that developed into multiple ground glass opacification with or without consolidative abnormalities. pleural effusions and mediastinal lymphnode enlargement should be detected in severe cases. findings are often peripheral, bilateral and involving the lower lobes. also chest x-ray can be considered a reliable diagnostic tool, especially in the emergency setting [2, [5] [6] [7] . actually, the therapeutic management of sars-cov-2 infection has many fields of action. on one side to block the viral replication, on the other side to treat the interstitial pneumonia and mof which cause the most severe clinical setting. currently, the antiviral drugs available are: hydroxychloroquine (hcl), chloroquine (cl), azithromycin, lopinavir/ritonavir, darunavir/cobicistat, remdesivir, and favipavir. the other drugs, in particular those necessary to contrast the mof and haemophagocytic syndrome are corticosteroids and monoclonal antibodies; also dialysis therapy with special filters should be used. given the lack of specific therapy about the ongoing sars-cov-2 infection, we conducted a brief review to summarize the mechanism of action and the potentially side effects of the treatment currently available, focusing on the effects of the drugs on renal disease at different stages in terms of therapeutic management and survival. hydroxychloroquine (hcl) and chloroquine (cl) are the cornerstone therapies of sensitive falciparum malaria and the second-line treatment of many rheumatologic diseases. both drugs are currently under investigation for use in the sars-cov-2 outbreaks. cl and hcl were described as potent in vitro inhibitors for other coronaviruses, including sars-cov-1, so they are used in the treatment of sars-cov-2 [8, 9] . it has been demonstrated that sars-cov-2 links the angiotensin-converting enzyme 2 expressed in the lung. cl/hcl could also reduce the expression of sialic acids that may be required for binding sars-cov-2. moreover, cl/hcl increase endosomal ph required for virus/cell fusion, as well as interfering with the glycosylation of cellular receptors of sars-cov. finally, cl/hcl interfere with the virion assembly through the blockage of cytokine storm reducing the pro-inflammatory mediators production and, consequently, the systemic inflammatory response syndrome (sirs). in a chinese study [9] , patients diagnosed as mild, moderate, and severe cases of pneumonia received chloroquine 500 mg twice per day for 10 days, improving clinical outcomes. moreover, hcl plus azithromycin (azt) are significantly associated with sars-cov-2 viral load reduction [8, [10] [11] [12] [13] [14] [15] [16] [17] although the association leads to the more common side effect of qtc interval prolongation. recently, concerns have been raised about the toxicity of cl and hcl in patients with sars-cov-2 infection. in a multinational study, 96,032 covid patients were enrolled. a group of 14,888 treated patients (1868 received chloroquine, 3783 received cl plus a macrolide, 3016 received hcl, and 6221 received hcl plus macrolide) was compared with 81,144 patients in the control group. a multivariate analysis showed that the treatments with hcl and cl with or without a macrolide, were independently associated with an increased risk of in-hospital mortality. furthermore, the hcl regimens were independently associated with an increased risk of ventricular arrhythmia if compared with the control group [18] . in addition, borba et al. have compared two dosage regimens of cl (450 mg two times per day on day one then 450 mg once daily for 5 days and 600 mg two times per day for 10 days) and the study was finished early due to ventricular tachycardia and qtc prolongation especially in the high dose arm [19] . based on the above studies, it is crucial to emphasize the importance of performing a tailor-made dosage of drugs through a continual monitoring of renal function which often worsens during sars-cov-2 infection, with risk of accumulation and significant increase in toxicity. ideal dose and duration of sars-cov-2 treatment are unknown. more data are available for hcl sulfate dose recommended in general population than cl: 200 mg, two times per day (bid) or three times per day (tid) for 10 days. the adjusted doses of hcl required for renal patients are based on the glomerular filtration rate (gfr). for gfr between 15 and 30 ml/min the suggested dose is 200 mg/die once a day (od). for gfr < 15 ml/min the suggested dose is 200 mg every other day (alternate days). cl is administered 500 mg bid for 10 days. the adjusted doses of cl required for renal patients are: if gfr < 10 ml/min, in hemodialysis (hd) and peritoneal dialysis (pd) 50% of dose should be administered. in continuous renal replacement therapy (crrt), no dosage adjustment is required. standard dose, renal dose, and side effects of hcl, cl and of all the other drugs described below are summarized in table 1 . side effects are reported in table 2 . azt is a bacteriostatic antibiotic of macrolides class with a broad spectrum of activity against gram-positive and atypical bacteria. the immune-modulatory and antiinflammatory effects of azt take place by the downregulation of the inflammatory response, the reduction in cytokine production, and the induction of immunoglobulin antibodies production. moreover, azt is active in vitro against zika and ebola viruses [20, 21] . given these properties, azt and other macrolides have been studied for their potential use as target therapy for viral respiratory infections including mers (middle east respiratory syndrome) and sars (severe acute respiratory syndrome). in a recent publication, the authors suggested that macrolide therapy had no significant association with mers cov rna clearance and was not associated with a significant change in 90-days patient mortality [22] . furthermore, another study conducted in a small population suggested that the use of hcl in combination with azt was associated with significant viral load reduction in sars-cov-2 infected patients, although the risk of qt prolongation induced by the association of the two drugs should be considered. as for each treatment, the cost benefits of the risk should be individually evaluated [10] . in april 2020, two clinical trials began to verify the efficacy of azt therapy in sars-cov-2. currently there is no clear evidence of the efficacy of azt treatment in sars-cov-2 pneumonia [23, 24] . the who guidelines and surviving sepsis campaign guidelines (march 2020) recommended empiric antimicrobial therapy only for prevention or treatment of superinfections [25] . the dose recommended for patients with mild to severe symptoms is 500 mg od for three days. in patient with severe ckd (gfr < 10 ml/min), dose adjustment is required (50% of dose). the co-formulation lopinavir/ritonavir is one of the main treatment for hiv infections. lopinavir is a hiv protease inhibitor, whereas ritonavir inhibits the cyp3a-mediated metabolism of lopinavir providing to increase plasma level of lopinavir. furthermore, lopinavir is mainly metabolized and eliminated by the liver [26] . many studies, carried out during the past epidemic respiratory syndromes (sars and mers), suggested a reduction in viral load, steroid usage and, consequently, in nosocomial infections, in patients treated with lopinavir/ritonavir, showing a clinical improvement [27, 28] . a randomized clinical trial, handled by a chinese group, suggested that in hospitalized adult patients with severe infection, no benefit was observed with lopinavir/ritonavir beyond standard care in terms of time to clinical improvement, reduction of mortality and safety (side effects and discontinuation of treatment) [29, 30] . the recommended doses of lopinavir/ritonavir are 400/100 mg bid for 5-10 days [31] [32] [33] . this therapeutic schemes has been suggested in symptomatic elderly patients with underlying diseases or patient with acute respiratory distress syndrome (ards) when remdesivir is not available (mild/high-risk groups) starting from the early stage. lopinavir pharmacokinetics have not been studied in patients with renal disease; however, since the renal clearance of drug is negligible, a decrease in total body clearance is not expected in patients with renal disease. in hd, pd and crrt, no dosage adjustment is required. darunavir (drv) is a hiv protease inhibitor that blocks the cut of gag-pol proteins encoded by hiv in the cells infected by the virus preventing the development of mature infective viral particles. it was initially used in combination with ritonavir [34] and then boosted with cobicistat [35] . cobicistat is a booster for the improvement of pharmacokinetics and pharmacodynamics of drv by cytochrome p450 (cyp3a) inhibition [36] . darunavir/cobicistat (drv/c) is approved by the united states food and drug administration (fda) only for use with a boosting agent, and in combination with other anti-retrovirals, for the treatment of hiv-1 infection. janssen pharmaceutical companies, a subsidiary of johnson&johnson, yielded its brand drv/c used for hiv treatment for the use in activities research for sars-cov-2 treatment. anecdotal reports suggested that darunavir has potential antiviral activity against sars-cov-2 virus. janssen have found low interactions between drv and the active site of sars-cov-2 protease. actually, the use of drv as treatment for sars-cov-2 is not supported by in vitro or clinical studies [37, 38] . drv/c is metabolized by the liver. caution, without any dose adaptation, is suggested in case of slight and mild liver dysfunction, whereas the use of drv/c is not recommended in case of severe liver dysfunction. actually, the therapeutic scheme for the treatment of sars-cov-2 infection is off-label. the drug doses suggested is 800/150 mg od for 5-7 days. drv/c has the same mechanism of action of lopinavir/ritonavir; therefore, it is reasonable to consider the use of drv/c as an alternative treatment for sars-cov-2 infection disease when lopinavir/ritonavir is not available [39] . drv is poorly eliminated by the kidney [40]; changing in dosage is not requested in patient with renal disease. instead, considering that cobicistat should reduce the renal clearance, its use is contraindicated in patient with gfr < 70 ml/min. finally, no evidence or clinical study is available regarding the dialysis clearance [35] . remdesivir (rdv) is a novel antiviral drug, recently used for the ebola treatment. rdv is an adenosine nucleotide analogue with a broad spectrum of antiviral activity. it works as rnadependent rna polymerase (rprd) inhibitor. rdv creates a tightly bind with rprd, showing a high level of binding affinity comparable to these of native nucleotides. rdv inhibits viral replication through premature termination of rna, blocking the transcription and consequently the viral replication [41] . rdv has been studied in clinical trials and it is still under investigation for the ebola treatment in humans, mers, and sars-cov viruses [42, 43] . it is currently under investigation for sars-cov-2 treatment. indeed, clinical trials are currently in progress to verify the efficacy and safety of rdv use in hospitalized adult patients with mild or severe sars-cov-2 disease [44, 45] . successful results have been reached in one case of sars-cov-2-infected patients treated with intravenous rdv [46] . rdv is a prodrug, predominantly metabolized by hepatic enzymes with hydrolase activity. based on rapid distribution, metabolism and clearance, the drug has scarce clinically significant interactions and does not prolong the qtc interval [47] . there are three different therapeutic schemes proposed by the ongoing trials; all of them share the same dosage but differ in the duration of the treatment based on the clinical stage of the disease (mild or severe). the recommended dose of rdv is 200 mg intravenous (iv) on day 1, 100 mg iv daily for 5 up to 10 days. the use of rdv has not been approved by fda (food and drug administration) and ema (european medicines agency) even if its compassionate use is recommended in patients with severe sars-cov-2 infection who are not eligible for inclusion criteria in clinical trials and in absence of alternative therapeutic options [48] . in a recent randomized, double-blinded, placebo-controlled, multicenter trial, the efficacy of the rdv in adults hospitalized sars-cov-2 patients with pulmonary involvement has been demonstrated. rdv has received the authorization for the treatment of suspected or laboratory-confirmed sars-cov-2 adults and children hospitalized patients with severe disease (low blood oxygen levels, needing oxygen therapy, needing mechanical ventilation), due to shortening the time to recovery [49] . the proposed dosage is: 200 mg as a single dose on day 1, followed by 100 mg once daily. instead, the renal recommended doses are: if egfr ≥ 30 ml/min no dosage adjustment necessary, if egfr ≤ 30 ml/min avoid use, in renal replacement therapies avoid use. the antiviral 6-fluoro-3-hydroxy-2-pyrazinecarboxamide (t-705, favipiravir) favipavir (fp) is a rna-dependent rna polymerase (rdrp) inhibitor. its use was approved only in japan for the treatment of influenza infection and it is distributed only upon request by the minister of health, labor and welfare of japan to avoid irrational prescriptions [50] in addition to its anti-influenza activity, fp blocks the replication of other rna viruses [51] . fp has been used for the treatment of human infection with life-threatening ebola virus and severe fever with thrombocytopenia syndrome [52] . moreover, the fp activity against multiple rna viruses has been demonstrated [53] . due to its inhibition of rna polymerase, fp may have potential antiviral activity on sars-cov-2 infection [54] . the preliminary results of a clinical chinese trial suggested that fp should have stronger antiviral action than lopinavir/ritonavir [55] . fp was approved in china for sars-cov-2 treatment since february 15, 2020 [56] . dosage and treatment duration for sars-cov-2 infection are unavailable. the approved fp dose in japan is 1600 mg tid on day 1 followed by 600 mg tid for 4 days. the viral replication period is 6 days or longer for seasonal influenza. when drug administration is stopped during the virus replication period or when resistant strains appear, virus replication and fever relapse. thus, 10 days of administration should be required for severe influenza or novel influenza [57] . however, no published clinical studies are available about the efficacy and safety of the drug in the treatment of sars-cov-2 disease. corticosteroids (cst) are immunomodulatory agents that modulate the host response by the secretion of various interleukins (il-1, il-6, il-8, il-11, il-12, ifn-γ e tnfα) and the expression of their receptors. some corticosteroids attenuate the cd28 co-stimulatory pathway through the inhibition of naïve t-cell proliferation and differentiation, a key mechanism in the pathogenesis of ards in sars-cov-2-associated pneumoniae. cts have been largely used in the treatment of past coronavirus infection (sars, mers). no evidence was found on the antiviral effect of corticosteroids alone in resisting sars-cov-2 in vivo and in vitro [58] [59] [60] . the dose of methylprednisolone varied, depending on disease severity. moreover, administration of cst in the early phases of sars-cov-2 infection, is not recommended due to the increasing risk of superinfection and prolonged viral replication [61] . however, it has been demonstrated that methylprednisolone may be beneficial for patients with sars-cov-2 pneumonia who have developed ards on progression [62] . the use of systemic cst in adults with sars-cov-2 and ards mechanically ventilated has been suggested by guidelines on the management of critically ill adults with coronavirus disease 2019 (covid19) of surviving sepsis campaign (weak recommendation, low-quality evidence). systemic cst are recommended for a short-term use (1 ~ 2 mg·kg × 3 ~ 5 days) [25, 63] . in patients on chronic dialysis or with renal transplant and rapid progression of sars-cov-2 disease the guidelines of the "brescia renal covid task force" indicate an initial dose of desametasone 20 mg/die for 5 days followed by a dose of 10 mg die for 5 days [3, 4] . tociluzumab. tociluzumab (tc) is a humanized monoclonal antibody (mab) anti-human il-6 receptor of the igg1 subclass. tc, interrupting il-6 pathway, seems to be highly effective in reducing the inflammatory response through its pro-inflammatory effects and it has been successfully used for the treatment of rheumatologic diseases. the use of tc for the treatment of sars-cov-2 infection has shown encouraging preliminary clinical results. nevertheless, the right time of therapy initiation, the duration and the recommended dose are not fully clear. a multicenter, randomized controlled trial is ongoing in the investigation of the efficacy and safety of tc in the treatment of new coronavirus pneumonia [64] [65] [66] [67] . moreover, a screening for tuberculosis and hepatitis b infection should be done before initiating tc. the brescia renal covid task force [3, 4] suggested a dose of 8 mg/kg iv, repeatable after 12 h. eculizumab a multicenter trial (nct04288713) is underway on eculizumab (ez), a humanized mab that binds the complement component c5, which is required for formation of the membrane attack complex (mac); mac can cause organs' damage in other types of coronaviruses infection; therefore, modulation of this part of the immune response during sars-cov-2 infection can be helpful. in unvaccinated patients against neisseria meningitides daily antimicrobial prophylaxis is mandatory (ceftriaxone iv) to avoid life-threatening infections. ez dose recommended is 900 mg iv every 7 days [65] . sarilumab sarilumab (sa) is a specific mab for the interleukin-6 (il-6) receptor; it may potentially contrast cytokine release syndrome in severely ill patients of covid-19 disease [68, 69] . anakinra. anakinra (ank). ank is a recombinant human interleukin-1 (il-1) receptor antagonist. nct04324021 is a phase 3 randomized, open-label, multicenter trial on ank. dose is 100 mg every 6 h (total of 400 mg daily) for 15 days [70] . since the elimination through the kidney of mab is considered not significant, in ckd patients no dosage adjustment is required. treatment of acute severe pneumonia with immunoglobulin from healed patients could accelerate clinical recovery. currently several trials are ongoing in china [71, 72] . in some patients with sars-cov-2 infection, the systemic involvement should induce acute kidney injury (aki) and mof. the cause of mof in patients with sars-cov-2 pneumonia, similar to septic shock, is an uncontrolled inflammatory state or a subsequent immune-paralysis. when pharmacological treatment is not effective, extracorporeal therapies offer a possibility to support different organs in a multiple organ dysfunction [73] . the indications for extracorporeal therapy in patients with sars-cov-2 infection and mof include renal replacement therapy and sepsis. aki, in patients affected by sars-cov-2-related pneumonia, is uncommon, but it might result from a sirs involving combined myocardial and kidney function. in sepsis, ronco et al. observed the reduction of circulating levels of cytokines using cartridges containing highly biocompatible sorbents and microporous resins (ex. cytosorb). the suggested schemes of application is 1 unit every 12 h in the first 24 h and 1 unit per day in the following 2 days [74] . in dialysis patients with chronic kidney disease (ckd), alberici et al. suggested expanded intermittent hemodialysis (hdx) whit medium cut-off (mco) membrane to increase removal of inflammatory cytokines [3, 4] . high rate of pulmonary thromboembolism due to thrombophilic and inflammatory state has been reported. therefore, the prophylactic use of low molecular weight heparin (lmwh) is indicated [75, 76] . since the sars-cov-2 outbreak began, last december 2019 in china, millions of people have been infected; sars-cov-2 should cause a large spectrum of clinical signs and symptoms ranging from mild to critical [1, 2] . currently, there are no vaccines or drugs approved for prevention and treatment of the infection. moreover, the kidney involvement of the sars-cov-2 infection should be associated with three different clinical settings. the first includes patients with ckd, the second includes patients with aki, the third includes immunosuppressed patients such as renal graft recipients or patients with glomerular diseases. moreover, it is known that kidney injury is associated with an increased risk of death in patients with influenza a virus subtype h1n1 and sars [77, 78] . in addition, a recent report suggested that the kidney involvement, in patients infected by sars-cov-2 at admission, is associated with a higher risk of in-hospital death [79] . aki sars-cov-2 related is probably multifactorial, including a major role of inflammatory cytokines. in severe cases, cytokine storm causes a multiple organ failure syndrome, including kidney's disease [80] . kidney histology was examined in an autopsy series of 26 patients with evidence of acute tubular damage. nine biopsies were tested for intracellular virus and coronaviruses were identified in seven [81] . to date, two cases were reported with collapsing glomerulopathy presenting with severe aki and heavy proteinuria [82, 83] . since the clinical presentation of sars-cov-2 infection should be misleading until the severe stages, with rapid clinical deterioration, clinicians should pay more attention to renal patients, carrying out early treatment and intensive monitoring to improve the clinical outcome. alberici et al. [3, 4] describe the experience of the nephrology ward of the university hospital of brescia in the northern of italy, place of a dramatic covid-19 outbreak, focusing the attention on dialysis and transplant patients. the authors suggested a better outcome especially in the setting of the transplanted group managed by a dedicated nephrology unit. preliminary results on general population with mild symptoms suggested a synergic effect of hcl in combination with azt, with a possible preventive effect of severe respiratory tract infections, in order to confirm the antiviral activity showed against sars-cov-1, zika and ebola viruses [45] [46] [47] [48] . on the basis of the latest toxicity warnings, when using cl and hct, it is crucial to adapt the dosage to the renal function to avoid the overload and to carry out a close monitoring of the side effects, particularly cardiological ones. during this phase, nephrologist should consider the possibility to combine antiviral therapy and standard drugs, although in a recent trial, no benefit was observed with the association of lopinavir/ritonavir treatment [29] . in severe cases of mof and haemophagocitic syndrome, immunosuppressive strategies must be considered. the massive lung involvement and interstitial infiltrates with respiratory failure (pao2/ fio < 100) is usually considered the main life-threatening process of sars-cov-2 infection. moreover, the administration of high dose of steroids in association with monoclonal antibody as rescue procedures was strongly suggested [3, 4] . unfortunately, to date, a monoclonal antibody against sars-cov-2 has not been developed and tc is the most used in ongoing studies. when sars-cov-2 infection causes aki unresponsive to pharmacological treatment, renal replacement therapy is necessary. moreover, in severely ill patients with sepsis/mof or haemophagocitic syndrome, extracorporeal therapies with highly biocompatible resins are also used and seem to have a remarkable benefit in terms of hemodynamic support and organ function rescue [73, 74] . convalescent plasma could be a promising approach in selected cases with severe disease [71, 72] . in renal transplant recipients and glomerulonephritis patients affected by sars-cov-2 infection, it is mandatory to verify the interactions of the drugs with immunosuppressive therapies to reduce and/or avoid harmful side effects. notably, it has been suggested to discontinue calcineurin inhibitors and antimetabolite in renal graft recipients, using only low doses of cst as anti-rejection therapies [3, 4] . pulmonary thromboembolism prophylaxis is useful in all patients with covid-19 infection [75, 76] . in covid-19 pediatric kidney patient, rdv is preferred to other agents. it is described a multisystem inflammatory syndrome (mic-s) that shares clinical features with kawasaki disease. children who present with clinical features of mis-c should be treated with steroid and if refractory, with monoclonal antibody (anakinra, tocilizumab) [84] . finally, we would underline the nephrologists' role and its importance in the global clinical evaluation in kidney patients suffering from covid-19 disease. waiting for the availability of new drugs, nephrologists should contribute to choose the best therapy for renal patients in order to reserve the kidney function, to consider possible interactions between drugs and to avoid nephrotoxicity of combined therapies. furthermore, in unclear cases of renal function impairment, a kidney biopsy would be mandatory. in conclusion, the sars-cov-2 outbreaks therapy represents for clinicians one of the most difficult challenge. within this setting, renal patients represent a population at high risk of morbidity and mortality, requiring a tailored therapeutic approach. moreover, it is well known that the therapeutic target to treat patients with ckd, aki, and kidney transplant is complicated by pharmacodynamics and pharmacokinetics aspects, drug interactions, and extracorporeal therapies. thus, there are no doubts that in renal patients, within the sars-cov-2 infection management, the role of nephrologists is useful in order to draw a balance between beneficial and potentially harmful effects of current treatment available. author contributions pr, lf, and ds conceived of the study and participated in its organization and coordination. lf, gg, ce, and np contributed to the acquisition of scientific materials. lf, gg, ce, and ds drafted the manuscript. all authors contributed to the interpretation of data, manuscript revisions, and read and approved the final manuscript. the author(s) declare that they have no conflict of interest. ethical approval not required for this paper. informed consent for this type of study, formal consent is not required. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention li x et al 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-globa l-kevza ra-r-saril umab-clini cal-trial -progr am-in-patie nts-withsever e-covid -19 sobi to initiate a clinical study to evaluate whether anakinra and emapalumab may relieve complications associated with severe covid-19 disease convalescent plasma for the treatment of severe and critical novel coronavirus pneumonia (covid-19): a prospective randomized controlled trial (chictr2000029757) latest version coronavirus epidemic: preparing for extracorporeal organ support in intensive care coronavirus epidemic and extracorporeal therapies in intensive care: si vis pace para bellum diagnostic evaluation of pulmonary embolism during the covid-19 pandemic acute pulmonary embolism and covid-19 pneumonia: a random association? acute renal impairment in coronavirus-associated severe acute respiratory syndrome acute kidney injury in critically ill patients with pandemic influenza a pneumonia 2009 in korea: a multicenter study kidney disease is associated with in-hospital death of patients with covid-19 covid-19 nephropathy; an emerging condition caused by novel coronavirus infection renal histopathological analysis of 26 postmortem findings of patients with covid-19 in china collapsing glomerulopathy in a patient with coronavirus disease collapsing glomerulopathy in a covid-19 patient hyperinflammatory shock in children during covid-19 pandemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-310221-car394ou authors: chandrashekar, abishek; liu, jinyan; martinot, amanda j.; mcmahan, katherine; mercado, noe b.; peter, lauren; tostanoski, lisa h.; yu, jingyou; maliga, zoltan; nekorchuk, michael; busman-sahay, kathleen; terry, margaret; wrijil, linda m.; ducat, sarah; martinez, david r.; atyeo, caroline; fischinger, stephanie; burke, john s.; slein, matthew d.; pessaint, laurent; van ry, alex; greenhouse, jack; taylor, tammy; blade, kelvin; cook, anthony; finneyfrock, brad; brown, renita; teow, elyse; velasco, jason; zahn, roland; wegmann, frank; abbink, peter; bondzie, esther a.; dagotto, gabriel; gebre, makda s.; he, xuan; jacob-dolan, catherine; kordana, nicole; li, zhenfeng; lifton, michelle a.; mahrokhian, shant h.; maxfield, lori f.; nityanandam, ramya; nkolola, joseph p.; schmidt, aaron g.; miller, andrew d.; baric, ralph s.; alter, galit; sorger, peter k.; estes, jacob d.; andersen, hanne; lewis, mark g.; barouch, dan h. title: sars-cov-2 infection protects against rechallenge in rhesus macaques date: 2020-05-20 journal: science doi: 10.1126/science.abc4776 sha: doc_id: 310221 cord_uid: car394ou an understanding of protective immunity to sars-cov-2 is critical for vaccine and public health strategies aimed at ending the global covid-19 pandemic. a key unanswered question is whether infection with sars-cov-2 results in protective immunity against re-exposure. we developed a rhesus macaque model of sars-cov-2 infection and observed that macaques had high viral loads in the upper and lower respiratory tract, humoral and cellular immune responses, and pathologic evidence of viral pneumonia. following initial viral clearance, animals were rechallenged with sars-cov-2 and showed 5 log(10) reductions in median viral loads in bronchoalveolar lavage and nasal mucosa compared with primary infection. anamnestic immune responses following rechallenge suggested that protection was mediated by immunologic control. these data show that sars-cov-2 infection induced protective immunity against re-exposure in nonhuman primates. *these authors contributed equally to this work. †corresponding author. email: dbarouch@bidmc.harvard.edu an understanding of protective immunity to sars-cov-2 is critical for vaccine and public health strategies aimed at ending the global covid-19 pandemic. a key unanswered question is whether infection with sars-cov-2 results in protective immunity against re-exposure. we developed a rhesus macaque model of sars-cov-2 infection and observed that macaques had high viral loads in the upper and lower respiratory tract, humoral and cellular immune responses, and pathologic evidence of viral pneumonia. following initial viral clearance, animals were rechallenged with sars-cov-2 and showed 5 log 10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa compared with primary infection. anamnestic immune responses following rechallenge suggested that protection was mediated by immunologic control. these data show that sars-cov-2 infection induced protective immunity against reexposure in nonhuman primates. . s3 ), but fever, weight loss, respiratory distress, and mortality were not observed. to help differentiate input challenge virus from newly replicating virus, we developed an rt-pcr assay to assess e gene subgenomic mrna (sgmrna). e gene sgmrna reflects viral replication cellular intermediates that are not packaged into virions and thus represent putative replicating virus in cells (9) . compared with total viral rna ( fig. 1b) , sgmrna levels were lower in ns on day 1 with a median of 5.11 (range <1.70-5.94) log10 sgmrna copies/swab, but then increased by day 2 to a median of 6.50 (range 4.16-7.81) log 10 sgmrna copies/swab (fig. 1c) . we next evaluated sars-cov-2-specific humoral and cellular immune responses in these animals. all 9 macaques developed binding antibody responses to the sars-cov-2 spike (s) protein by elisa ( fig. 2a) and neutralizing antibody (nab) responses using both a pseudovirus neutralization assay (10) (fig. 2b ) and a live virus neutralization assay (11, 12) (fig. 2c ). nab titers of approximately 100 were observed in all animals on day 35 regardless of dose group (range 83-197 by the pseudovirus neutralization assay and 35-326 by the live virus neutralization assay). antibody responses of multiple subclasses were observed against the receptor binding domain (rbd), the prefusion s ectodomain (s), and the nucleocapsid (n), and antibodies exhibited diverse effector functions, including antibody-dependent complement deposition (adcd), antibody-dependent cellular phagocytosis (adcp), antibody-dependent neutrophil phagocytosis (adnp), and antibody-dependent nk cell degranulation (nk cd107a) and cytokine secretion (nk mip1β, nk ifnγ) (13) (fig. 2d ). cellular immune responses to pooled s peptides were observed in the majority of animals by ifn-γ elispot assays on day 35, with a trend toward lower responses in the lower dose groups (fig. 2e) . intracellular cytokine staining assays demonstrated induction of both s-specific cd8+ and cd4+ t cell responses (fig. 2f ). only limited pathology data from sars-cov-2 infected humans are currently available. to assess the pathologic char-acteristics of sars-cov-2 infection in rhesus macaques, we inoculated 4 animals with 1.1 × 10 5 pfu virus by the in and it routes as above and necropsied them on day 2 (n = 2) and day 4 (n = 2) following challenge. multiple regions of the upper respiratory tract, lower respiratory tract, gastrointestinal tract, lymph nodes, and other organs were harvested for virologic and pathologic analyses. high levels of viral rna were observed in all nasal mucosa, pharynx, trachea, and lung tissues, and lower levels of virus were found in the gastrointestinal tract, liver, and kidney ( fig. s4 ). viral rna was readily detected in paratracheal lymph nodes but was only sporadically found in distal lymph nodes and spleen ( fig. s4 ). upper airway mucosae, trachea, and lungs were paraformaldehyde fixed, paraffin embeded, and evaluated by histopathology. on day 2 following challenge, both necropsied animals demonstrated multifocal regions of inflammation and evidence of viral pneumonia, including expansion of alveolar septae with mononuclear cell infiltrates, consolidation, and edema (fig. 3, a and b) . regions with edema also contained numerous polymorphonuclear cells, predominantly neutrophils. terminal bronchiolar epithelium was necrotic and sloughed with clumps of epithelial cells detected within airways and distally within alveolar spaces (fig. 3 , c and d) with formation of occasional bronchiolar epithelial syncytial cells (fig. 3e ). hyaline membranes were occasionally observed within alveolar septa, consistent with damage to type i and type ii pneumocytes (fig. 3f ). diffusely reactive alveolar macrophages filled alveoli, and some were multinucleated and labeled positive for nucleocapsid by immunohistochemistry (fig. 3g ). alveolar lining cells (pneumocytes) also prominently labeled positive for nucleocapsid (fig. 3h ). multifocal clusters of virus infected cells were present throughout the lung parenchyma, as detected by immunohistochemistry and in situ rna hybridization (rnascope) (14, 15) to further characterize infected tissues, we performed cyclic immunofluorescence (cycif) imaging, a method for multiplex immunophenotyping of paraformaldehyde fixed tissue specimens (16) . tissues were stained for nucleocapsid (sars-n), pan-cytokeratin (to identify epithelial cells), iba-1 (ionized calcium binding adaptor as a pan-macrophage marker), cd68 (monocyte/macrophage marker), and cd206 (macrophage marker), in addition to a panel of markers to identify other immune cells and anatomical structures (table s1), and counterstaining for dna to label all nuclei. foci of virus infected cells were randomly dispersed throughout the lung and were variably associated with inflammatory infiltrates (fig. 4 , a to d). some areas of parenchymal consolidation and inflammation contained little to no virus ( fig. 4a, arrows, and fig. s8 ). virus infected cells frequently co-stained with pan-cytokeratin (fig. 4 , e to h), suggesting that they were alveolar epithelial cells (pneumocytes). uninfected iba-1+ cd68+ cd206+ activated macrophages were also frequently detected adjacent to virally infected epithelial cells (fig. 4 , e and i to k). these data demonstrate that sars-cov-2 induced multifocal areas of acute inflammation and viral pneumonia involving infected pneumocytes, ciliated bronchial epithelial cells, and likely other cell types. on day 35 following initial viral infection (figs. 1 and 2), we rechallenged all 9 rhesus macaques with the same doses of sars-cov-2 that were utilized for the primary infection, namely 1.1 × 10 6 pfu (group 1; n = 3), 1.1 × 10 5 pfu (group 2; n = 3), or 1.1 × 10 4 pfu (group 3; n = 3). we included 3 naïve animals as positive controls in the rechallenge experiment. very limited viral rna was observed in bal on day 1 following rechallenge in two group 1 animals and in one group 2 animal, with no viral rna detected at subsequent timepoints (fig. 5a ). in contrast, high levels of viral rna were observed in the concurrently challenged naïve animals (fig. 5a) , as expected. median peak viral loads in bal were >5.1 log10 lower following rechallenge as compared with the primary challenge (p < 0.0001, two-sided mann-whitney test; fig. 5b ). viral rna following rechallenge was higher in ns compared with bal, but exhibited dose dependence and rapid decline (fig. 5c) , and median peak viral loads in ns were still >1.7 log 10 lower following rechallenge as compared with the primary challenge (p = 0.0011, two-sided mann-whitney test; fig. 5d ). we speculated that the majority of virus detected in ns following rechallenge was input challenge virus, and we therefore assessed sgmrna levels in ns following rechallenge. low but detectable levels of sgmrna were still observed in 4 of 9 animals in ns on day 1 following rechallenge, but sgmrna levels declined quickly (fig. 5e) , and median peak sgmrna levels in ns were >4.8 log10 lower following rechallenge as compared with the primary challenge (p = 0.0003, two-sided mann-whitney test; fig. 5f ). consistent with these data, plaque assays in bal and ns samples following rechallenge showed no recoverable virus and were lower than following primary infection (p = 0.009 and 0.002, respectively, two-sided mann-whitney tests; fig. s9 ). moreover, little or no clinical disease was observed in the animals following rechallenge (fig. s10) . following sars-cov-2 rechallenge, animals exhibited rapid anamnestic immune responses, including increased virus-specific elisa titers (p = 0.0034, two-sided mann-whitney test), pseudovirus nab titers (p = 0.0003), and live virus nab titers (p = 0.0003) as well as a trend toward increased ifn-γ elispot responses (p = 0.1837) by day 7 after rechallenge ( fig. 6 ). in particular, nab titers were markedly higher on day 14 following rechallenge compared with day 14 following primary challenge (p < 0.0001, two-sided mann-whitney test) (fig. s11). all animals developed anamnestic antibody responses following rechallenge, regardless of the presence or absence of residual viral rna or sgmrna in bal or ns, and thus we speculate that the protective efficacy against rechallenge was mediated by rapid immunologic control. individuals who recover from certain viral infections typically develop virus-specific antibody responses that provide robust protective immunity against re-exposure, but some viruses do not generate protective natural immunity, such as hiv-1 (17) . human challenge studies for the common cold coronavirus 229e have suggested that there may be partial natural immunity (18) . however, there is currently no data whether humans who have recovered from sars-cov-2 infection are protected from re-exposure (world health organization, scientific brief, april 24, 2020; https:// www.who.int/news-room/commentaries/detail/immunitypassports-in-the-context-of-covid-19). this is a critical issue with profound implications for vaccine development, public health strategies, antibody-based therapeutics, and epidemiologic modeling of herd immunity. in this study, we demonstrate that sars-cov-2 infection in rhesus macaques provided protective efficacy against sars-cov-2 rechallenge. we developed a rhesus macaque model of sars-cov-2 infection that recapitulates many aspects of human sars-cov-2 infection, including high levels of viral replication in the upper and lower respiratory tract (fig. 1) (19) . however, neither nonhuman primate model led to respiratory failure or mortality, and thus further research will be required to develop a nonhuman primate model of severe covid-19 disease. sars-cov-2 infection in rhesus macaques led to humoral and cellular immune responses (fig. 2) and provided protection against rechallenge (fig. 5) . residual low levels of subgenomic mrna in nasal swabs in a subset of animals (fig. 5 ) and anamnestic immune responses in all animals (fig. 6) following sars-cov-2 rechallenge suggest that protection was mediated by immunologic control and likely was not sterilizing. given the near-complete protection in all animals following sars-cov-2 rechallenge, we were unable to determine immune correlates of protection in this study. sars-cov-2 infection in rhesus monkeys resulted in the induction of neutralizing antibody titers of approximately 100 by both a pseudovirus neutralization assay and a live virus neutralization assay, but the relative importance of neutralizing antibodies, other functional antibodies, cellular immunity, and innate immunity to protective efficacy against sars-cov-2 remains to be determined. moreover, additional research will be required to define the durability of natural immunity. in summary, sars-cov-2 infection in rhesus macaques induced humoral and cellular immune responses and provided protective efficacy against sars-cov-2 rechallenge. these data raise the possibility that immunologic approaches to the prevention and treatment of sars-cov-2 infection may in fact be possible. however, it is critical to emphasize that there are important differences between sars-cov-2 infection in macaques and humans, with many parameters still yet to be defined in both species, and thus our data should be interpreted cautiously. rigorous clinical studies will be required to determine whether sars-cov-2 infection effectively protects against sars-cov-2 re-exposure in humans. international (cc by 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. to view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. this license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material. science.sciencemag.org/cgi/content/full/science.abc4776/dc1 materials and methods table s1 figs. s1 to s11 a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin washington state 2019-ncov case investigation team, first case of 2019 novel coronavirus in the united states china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster virological assessment of hospitalized patients with covid-2019 a dna vaccine induces sars coronavirus neutralization and protective immunity in mice reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus dissecting polyclonal vaccine-induced humoral immunity against hiv using systems serology impact of early cart in the gut during acute hiv infection defining hiv and siv reservoirs in lymphoid tissues highly multiplexed immunofluorescence imaging of human tissues and tumors using t-cycif and conventional optical microscopes hiv-1 superinfection despite broad cd8 + t-cell responses containing replication of the primary virus the time course of the immune response to experimental coronavirus infection of man cwiak for generous advice, assistance, and reagents. funding: we acknowledge support from the ragon institute of mgh, mit, and harvard, mark and lisa schwartz foundation led the clinical care of the animals and performed the virologic assays. r.z. and f.w. participated in study design and interpretation of data is an inventor on patent application wo 2017/184733 a1 submitted by massachusetts general hospital that covers systems serology key: cord-314135-udce22id authors: geisslinger, franz; vollmar, angelika m.; bartel, karin title: cancer patients have a higher risk regarding covid-19–and vice versa? date: 2020-07-06 journal: pharmaceuticals (basel) doi: 10.3390/ph13070143 sha: doc_id: 314135 cord_uid: udce22id the world is currently suffering from a pandemic which has claimed the lives of over 230,000 people to date. the responsible virus is called severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and causes the coronavirus disease 2019 (covid-19), which is mainly characterized by fever, cough and shortness of breath. in severe cases, the disease can lead to respiratory distress syndrome and septic shock, which are mostly fatal for the patient. the severity of disease progression was hypothesized to be related to an overshooting immune response and was correlated with age and comorbidities, including cancer. a lot of research has lately been focused on the pathogenesis and acute consequences of covid-19. however, the possibility of long-term consequences caused by viral infections which has been shown for other viruses are not to be neglected. in this regard, this opinion discusses the interplay of sars-cov-2 infection and cancer with special focus on the inflammatory immune response and tissue damage caused by infection. we summarize the available literature on covid-19 suggesting an increased risk for severe disease progression in cancer patients, and we discuss the possibility that sars-cov-2 could contribute to cancer development. we offer lines of thought to provide ideas for urgently needed studies on the potential long-term effects of sars-cov-2 infection. over the past few months, the novel coronavirus, called severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has become a worldwide concern, globally threatening public health. originating in wuhan, china, it dramatically spread to countries all over the world, causing a pandemic [1] . as of the 3rd of may 2020, there are more than 3.3 million people infected with the novel coronavirus sars-cov-2 and more than 230,000 people have died from the associated disease, called coronavirus disease 2019 (covid-19) [2] . sars-cov-2 belongs to the subfamily of betacoronaviruses and is an enveloped ssrna virus. like most other coronaviruses, sars-cov-2 is a zoonotic virus, suggested to have its origin in bats, from which it was transmitted to humans in december 2019 [3, 4] . in comparison to earlier identified sars-related coronaviruses, namely sars-cov-1 and mers-cov, sars-cov-2 is more contagious and, like sars-cov-1, sars-cov-2 is also transmitted from human to human [3] . subsequently, there are more infections and fatalities documented for sars-cov-2 as compared to sars-cov-1 [2, 5] , despite a supposedly lower lethality [6] . as it is a novel identified virus, there is no vaccine available to prevent sars-cov-2 infection and moreover there are no licensed drugs available for therapy [7, 8] . the main symptoms of covid-19, the lung disease following sars-cov-2 infection are fever, cough, shortness of breath and respiratory distress syndrome with risk for septic shock. furthermore, the main symptoms of covid-19, the lung disease following sars-cov-2 infection are fever, cough, shortness of breath and respiratory distress syndrome with risk for septic shock. furthermore, lymphopenia, myalgia, nausea and vomiting frequently occur [9, 10] . interestingly, about 80% of infected people have no or only mild symptoms, while only approximately 20% have severe events, making hospital stays and icu support necessary [11] . the reason behind this phenomenon remains predominantly unknown, but it is hypothesized that the outcome is linked to overshooting immune responses [12] . furthermore, severity seems to correlate with age and comorbidities, as older people as well as patients with comorbidities, including cancer, have a higher risk for severe events and a poor outcome [13, 14] . recent studies have mainly focused on acute complications upon sars-cov-2 infection. however, viral infections may also have long-term consequences in terms of risk for cancer development, mainly mediated by inflammatory tissue damage [15] . in this context, this opinion focuses on the interplay between cancer and sars-cov-2, summarizing the risk of cancer patients for sars-cov-2 infection and covid-19 fatality. furthermore, the immune system and inflammation are put into focus as significant factors of fatality. additionally, potential capability and mechanisms of sars-cov-2 to contribute to cancer development are discussed, opening questions to be addressed in future research ( figure 1 ). as already mentioned above, age and comorbidities are the main factors for severe covid-19 progress and fatality, putting patients at increased risk [16] [17] [18] . these comorbidities are very versatile, including hypertension, cardiovascular diseases, diabetes, chronic obstructive pulmonary disease (copd), and diseases [13, 14] . initial studies suggested that all cancer patients, regardless of the affected organ or tissue, are more at risk regarding a severe covid-19 progression and, subsequently, fatality. there is evidence that cancer patients are around 5 times more at risk of dying from covid-19 than patients without comorbidities [17, 18] . in contrast, dai et al. and kong et al. recently pointed out that only patients suffering from hematological, lung or metastatic cancer have an increased risk for severity and fatality as compared to healthy people [19, 20] . due to the recentness of the sars-cov-2 pandemic, these studies were only based on small patient cohorts. therefore, further investigations are necessary to assess the susceptibility of cancer patients regarding covid-19 fatality for certainty. however, increased risk for fatalities of cancer patients is not restricted to sars-cov-2 infections, but occurs upon various infectious diseases, such as influenza, varicella-zoster, tuberculosis and infections with toxoplasma gondii [21] [22] [23] [24] . the supposedly higher fatality rates of cancer patients might be caused by their susceptible immune system. anti-cancer therapy, including chemotherapy and radiotherapy, but also malignancy itself, contribute to immune suppression. in as already mentioned above, age and comorbidities are the main factors for severe covid-19 progress and fatality, putting patients at increased risk [16] [17] [18] . these comorbidities are very versatile, including hypertension, cardiovascular diseases, diabetes, chronic obstructive pulmonary disease (copd), and diseases [13, 14] . initial studies suggested that all cancer patients, regardless of the affected organ or tissue, are more at risk regarding a severe covid-19 progression and, subsequently, fatality. there is evidence that cancer patients are around 5 times more at risk of dying from covid-19 than patients without comorbidities [17, 18] . in contrast, dai et al. and kong et al. recently pointed out that only patients suffering from hematological, lung or metastatic cancer have an increased risk for severity and fatality as compared to healthy people [19, 20] . due to the recentness of the sars-cov-2 pandemic, these studies were only based on small patient cohorts. therefore, further investigations are necessary to assess the susceptibility of cancer patients regarding covid-19 fatality for certainty. however, increased risk for fatalities of cancer patients is not restricted to sars-cov-2 infections, but occurs upon various infectious diseases, such as influenza, varicella-zoster, tuberculosis and infections with toxoplasma gondii [21] [22] [23] [24] . the supposedly higher fatality rates of cancer patients might be caused by their susceptible immune system. anti-cancer therapy, including chemotherapy and radiotherapy, but also malignancy itself, contribute to immune suppression. in line with that, patients who received chemotherapy up to 14 days prior to infection have a much higher risk for a severe outcome than other cancer patients [17, 18, 25] therefore, oncologists recommend isolating cancer patients and testing them for sars-cov-2 infection regularly to protect this vulnerable group to the best ability from infection and thus severe covid-19 progression and fatal events [26, 27] . given the high risk for fatalities, especially for cancer patients receiving chemotherapy, the resulting altered risk benefit ratio during the sars-cov-2 pandemic should be taken into consideration. for a proper evaluation of cancer and covid-19 relation and vice versa, a detailed understanding of the underlying pathogenesis is mandatory. while sars-cov-2 infection predominantly affects the respiratory tract, developing covid-19, resulting in fever, cough, shortness of breath and respiratory distress syndrome [9, 10] , this might not be the major reason for causing severe events. interestingly, an overshooting immune response resulting in a cytokine storm and the modulation of angiotensin converting enzyme 2 (ace2) have been connected to severity. a cytokine storm or cytokine releasing syndrome is described as a quick and massive cytokine release which may lead to multiple organ failure. high amounts of pro-inflammatory cytokines are secreted after the activation of immune cells, initiating an overshooting immune response [28] . the subsequent, severe inflammation can cause acute, life-threatening disorders by inducing septic shock, impairing proper oxygen absorption, inducing lung failure and multi-organ failure [29] [30] [31] [32] . cytokine storms frequently occur as complications after infection with pathogens, such as sars-cov-1, epstein-barr virus and influenza virus, for example, as well as a side effect of cancer therapy [33] . preliminary evidence suggests that such a cytokine storm in response to infection with sars-cov-2 is a major factor, promoting severe covid-19 progress and subsequently disease fatality [8, 12] . in detail, it has been reported by wan et al. that in a study with patients that suffered from severe covid-19 pneumonia (21 patients included), levels of cd8 + t-cells and b-cells were reduced, while the cytokines il-6 and il-10 were elevated as compared to patients with a mild pneumonia (102 patients included) [34] . these findings were confirmed by other studies, showing elevated levels of pro-inflammatory cytokines of covid-19 patients. in this context, a study of qin et al. on 452 covid-19 patients, of which 286 were classified as severe cases, revealed significantly elevated levels of il-2, il-6, il-8, il-10 and tnfα associated with the disease [35] . il-2, il-6, and il-10 were furthermore found to be remarkably upregulated in patients with severe covid-19 in a study by shi et al. [36] . along this line, huang et al. reported increased plasma levels of pro-inflammatory mediators il-2, il-7, il-10, gscf, ip10, mcp-1, mip-1a, and tnf-α in covid-19 patients that were hospitalized in an icu. however, this study only included 49 patients [6] . consequently, mild and severe cases seem to show a different cytokine secretion profile, providing an option as biomarkers to monitor disease progression. on the other hand, targeting excessive cytokine release with immunosuppressive agents might serve as a therapeutic option for severe sars-cov-2 infection [12] . evidence suggests that patients suffering from rheumatism which are typically treated with cytokine blockers, such as infliximab, adalimumab and ustekinumab, do not have a higher risk for severe covid-19 progress, despite their comorbidity [37] . subsequently, cytokine blockers, especially the il-6 antagonist tocilizumab, are considered as potential drugs to prevent cytokine releasing syndrome [38] . however, a major problem of an immunosuppressive therapy approach is optimal timing, as, especially in the initial phase, a properly working immune system is of great importance [39] . in this context, monitoring of cytokine levels in bronchoalveolar lavage fluid and peripheral blood mononuclear cells as well as virus load could be a suitable approach. [40] 3. in addition to cytokine release syndrome, fatality has recently been connected to the modulation of ace2. ace2 is physiologically responsible for the conversion of angiotensin ii to angiotensin 1-7 and is therefore part of the renin-angiotensin system, playing an important role in the homeostasis of the cardiovascular and renal system (reviewed in [41, 42] ). in addition, angiotensin ii and angiotensin 1-7 also play an important role in inflammation. angiotensin ii promotes inflammation and vasoconstriction by activating at1 and at2 receptors, while angiotensin 1-7 has anti-inflammatory properties and, moreover, causes vasodilatation [43] [44] [45] . furthermore, it is widely known that ace2 is important for lung function, and accumulation of angiotensin ii is a marker for pulmonary arterial hypertension disease progression [41] . subsequently, ace2 dysregulation could promote overshooting immune responses. intriguingly, sars-cov-2 invades cells via ace2, as it was shown that its spike proteins bind to ace2 and that ace2 is necessary for virus entry [46, 47] . after virus entry, ace2 is probably downregulated, causing accumulation of antiogensin ii and thereby influencing immune response [48, 49] . as ace2 is also expressed in the cardiovascular system, it is likely that sars-cov-2 might also cause cardiovascular complications. indeed, it has been reported that a proportion of covid-19 patients suffer from myocardial damage and heart failure [50, 51] . interestingly, these patients complain only about cardiovascular symptoms like heart palpitations and chest tightness, but not about common respiratory symptoms connected to covid-19 [51] . of note, studies showed that sars-cov-1, whose spike protein has a high homology to sars-cov-2 [48] , binds to ace2 and modulates its expression during sars disease [52, 53] . interestingly, coronaviruses causing common colds, such as nl63, which also enters its host cells via ace2, does not influence ace2 expression [52] . therefore, ace2 modulation is considered as a major factor for severity, probably mediated by dysregulation of the immune response. inflammation, in principle, is an essential physiological process, which involves the activation, recruitment, and action of cells belonging to the innate and adaptive immunity and protects the body from harmful pathogens. yet, when deregulated or persisting, inflammation can cause severe diseases, such as diabetes, atherosclerosis and rheumatoid arthritis [54, 55] . usually, acute inflammation is resolved quickly with the removal of the pathogen from the body; however, if the pathogen cannot be removed completely, the inflammatory response becomes chronic [56] . an estimated 15-20% of cancers are preceded by inflammation within the same tissue, that is initiated long before tumor formation. causes of these chronic inflammations can be pathogenic infections, such as hepatitis virus or helicobacter pylori infections, autoimmunity, or environmental factors, like alcohol or cigarette smoke [55] . the major problem with unresolved, persistent inflammation is the generation of a pro-tumorigenic environment by immune cells and their secreted cytokine mediators. this microenvironment is rich in reactive oxygen species (ros), such as superoxide, nitric oxide, hydrogen peroxide, or radicals. continuous exposition to ros kills infected cells, yet also poses a threat to healthy host cells, as it drives dna mutations [57] . furthermore, immune cells infiltrate the site of inflammation and secrete cytokines like tnf-α, tgf-β, il-1β and il-6, which increase vascular permeability and favor a mesenchymal phenotype of cells that is important for migration. along this line, matrix metalloproteinases are also secreted to digest the extracellular matrix proteins and enable migration (reviewed in [56] ). as described above, there are some studies that investigated cytokine levels in patients suffering from covid-19 and revealed an elevation in il-2, il-6, il-8, il-7, il-10, g-csf, ip10, mcp-1, mip1a, and tnf-α [6, [34] [35] [36] . these data indicate that sars-cov-2 infection might bear the potential to cause carcinogenic inflammations. of note, elevated levels of mcp-1, tgf-β1, tnf-α, il-1β, and il-6 were found in patients that died from the related sars-cov-1 infection [58] . however, whether sars-cov-2-induced inflammation can become chronic is unknown to date and should be a part of further research. if chronic, the damage to healthy tissue caused by inflammatory processes is often followed by excessive tissue remodeling, leading to fibrosis. inflammation-induced fibrosis is accompanied by a loss of organ function and tumorigenesis, for instance in the development of hepatocellular carcinoma and lung cancer [59, 60] . in hepatocellular carcinoma, chronic inflammation, especially that mediated by ros, induces the proliferation of hepatocytes, and activates hepatic stellate cells and myofibroblasts, which create extracellular matrix proteins. these are key steps that lead to fibrosis, accompanied by the genomic instability of constantly proliferating hepatocytes and alterations in blood flow. subsequent production of vegf drives neoangiogenesis and promotes tumor survival [61] . similar mechanisms are described in the lung. lung fibrosis can be driven by tgf-β, which induces myofibroblasts and the synthesis of ecm proteins and promotes tumorigenesis [62] . again, specific data on sars-cov-2 infection in that regard are scarce, yet there is a study which reports pathological changes to the lung. zhang et al. reported a case study of a 72-year-old man who suffered from covid-19, had a rapid progression of the disease which required icu support, and later died from the disease. the postmortem transthoracic needle biopsies showed pathologic changes in the lung displayed by diffuse alveolar damage with loose fibrous plugs [63] . related to that, a study of seven patients that died from sars-cov-1 infection revealed diffuse alveolar damage, with the presence of multinucleated pneumocytes. additionally, they discovered mild to moderate fibrosis in the lung [64] . besides these first hints, which connect sars-cov infections with fibrosis, the fact that mechanical injury to the lung by intubation and high pressure ventilatory assistance in icu support are also capable of inducing fibrosis should not be neglected [65] . hence, infections that cause excessive and persisting inflammation and pathogenic conditions favoring fibrotic lesions bear the risk of developing tumors over time. while it is not clear to date whether infection with sars-cov-2 leads to persisting pathogen presence or continuous low-level inflammatory processes, there are few in vitro studies on the related sars-cov-1 viral infection and cellular persistence. chan et al. analyzed seven different cell lines for their permissiveness to sars-cov-1 infection. they found that the cell line permissive for infection also showed a persistent chronic infection, which could also be maintained during passaging [66] . their findings are supported by those of palacios et al., who reported the presence of viral particles in infected cells after multiple passaging [67] . furthermore, pacciarini et al. reported that sars cov-1 infection persisted in proximal tubular epithelial cells but not in glomerular mesangial cells. these findings indicate that the virus may cause persistent infection and that this might be cell type-dependent and yet not restricted to the lung [68] . in summary, these studies point to the possibility that sars-cov-2 might also be able to form persisting infection sites. however, caution is necessary when interpreting these studies, as of course transferring in vitro findings to in vivo is difficult. interestingly, a study by ling et al. analyzed the clearance of viral rna in oropharyngeal swabs and feces and reports that the median time from onset of the symptoms to clearance is 9.5 and 11 days, respectively. they further found that the time until tests turned negative was delayed by glucocorticoid therapy [69] . as the time until clearance from oropharyngeal swabs and feces differs, it is not clear how the virus is transported in the body and whether negative samples are proof for the eradication of the virus. hence, presently it cannot be estimated if sars-cov-2 is able to form persistent infection and studies addressing this question are urgently needed. despite the drawbacks of existing studies on sars-cov-2 and the limited information on related sars-cov-1 infection, these studies nevertheless indicate a pro-inflammatory phenotype, that might favor carcinogenesis, especially if it turns out to be persistent. in this regard, we suggest that follow-up studies on a broad number of covid-19 survivors would be beneficial to assess the risk of possible long-term consequences of the disease. the idea that viral infection might be the reason for the development of cancer is not new. in the 1980s, researchers hypothesized that viral infection with the human papilloma virus (hpv) could lead to the development of genital cancer [70] . this hypothesis was later proven and even rewarded with a nobel prize for physiology and medicine in 2008 [71] . of course, since then a lot of research has been done in the field. viruses that are capable of causing tumorigenesis are termed oncoviruses and are currently estimated to be responsible for about 15-20% of cancerous diseases [59, 72] . to date, several viruses have been shown to cause cancer by influencing a various number of cancer cell hallmarks that have been described by hanahan and weinberg [73] , among them human papilloma virus (hpv), hepatitis virus b and c (hbv, hcv) or the epstein-barr virus (ebv) [59, 72, 74] . in addition, several other viruses have been implicated in carcinogenesis [75] (table 1) . adult t-cell leukemia [82] [83] [84] kshv kaposi's sarcoma [85] mcpyv merkel cell carcinoma [86] [87] [88] hcmv mucoepidermoid carcinoma [89, 90] ebv however, the necessary prerequisites and mechanisms that lead to cancer development upon oncovirus infection are still not entirely clear. nevertheless, several characteristics are common among oncoviruses. they are widespread among the population, and also in individuals that do not develop cancer, they seem to cause persistent infection rather than leading to host cell lysis, and they are usually not sufficient to cause cancer on their own, but need an additional risk factor (e.g., inflammation, co-infection, immune suppression or host mutations) [59, 91] . in general, oncoviruses can be classified in direct oncoviruses like ebv, hpv, htlv-1, kshv or mcpyv, which activate oncoproteins, either encoded by the virus or the host cell, or indirect oncoviruses. indirect oncoviruses mainly cause chronic inflammation processes, that will eventually lead to tumor development, a mechanism typical for hbv and hcv [59] . usually, virus-driven cancers take about 15-40 years post-infection to develop. during this period, virus replication is either strongly diminished or even absent and the virus only exists in form of its nucleic acid, present as episome, plasmid or integrated into the host genome [72, 92] . infection with the hcv virus, for example, causes carcinogenesis over a period of 20-40 years by direct and indirect oncogenic events. direct events include interactions with host factors leading to alterations in transcriptional regulation, metabolism and apoptosis [93] . indirect events are oxidative stress and inflammation, the driving factors of hcv oncogenesis [94] . even though oncoviruses are roughly categorized in direct and indirect oncoviruses, this does not mean that the tumorigenic mechanisms are exclusive. as a matter of fact, many oncoviruses are associated with inflammation. of course, infection with the indirect oncovirus hcv causes the release of pro-fibrinogenic factors, such as tgfβ, and a chronic low-level inflammation caused by the ifn-γand il-2-dominated immune response. the cytokine milieu and related production of reactive oxygen species is a strong driving factor for genetic mutations and hence cancer development [94, 95] . nevertheless, direct oncoviruses, such as ebv, hpv or kshv, have also been linked with inflammatory immune responses. in the case of ebv, the viral protein lmp-1 activates signaling via the pi3k/akt, the jak/stat and the nfκb pathway induces cancer cell hallmarks and, in that regard, tumor-promoting inflammation [96] [97] [98] . furthermore, ebers, viral rna transcripts, activate the il6/stat3 pathway and induce il6, il9, il10 and igf-1, which promote inflammation and growth [99, 100] . the release of inflammatory cytokines il6, il8, tnfα, mip-1α and mip-1β has also been reported following kshv infections and is linked to the viral protein kaposin b [101] [102] [103] . along this line, the expression of hpv viral protein e6 is linked to alterations in cytokine levels [104, 105] . e6 facilitates the activation of nfκb, il6 production and subsequent autocrine and paracrine activation of stat3 [106] . promoting inflammation is therefore a common feature of oncoviruses, and the il6/stat3 axis is frequently activated. il6 is a pleiotropic, inflammatory mediator, which can activate mapk or pi3k signaling and stat3. this blocks apoptosis and favors cell proliferation, even in a harsh, inflammatory environment, thereby essentially contributing to malignant transformation [107] . the recent out-break of sars-cov-2 with a rising number of infected individuals brings about the question about possible long-term consequences, such as a potential risk of developing cancer after sars-cov-2 infection. since the virus has only recently emerged as a human infection, there are no studies available so far. however, there are some hints that might indicate pro-tumorigenic activity of sars-cov-2 which we want to point out in the following paragraph to provide ideas for future research in this area. as described above, increased cytokine levels, including those of il-6, have also been reported in covid-19 patients. whether the elevation of il-6, which is typical for oncoviruses, is a feature of sars-cov-2 infection or is an unspecific consequence of the cytokine storm in these patients remains unknown to date. however, we suggest that the potential of sars-cov-2 to elevate cytokines, especially il-6, and the subsequent activation of pro-inflammatory signaling pathways should be addressed in future studies, helping to estimate an oncogenic potential of sars-cov-2 as mentioned, there are no studies on sars-cov-2 in that regard; however, there are a few publications that investigated the relation between sars-cov, the cause of the sars disease and now known as sars-cov-1, and cancer incidence. these studies might be able to guide future research on the relation of cancer and sars-cov-2. for instance, li et al. reported a study on a connection between the sars disease and childhood acute lymphatic leukemia (all) by the hong kong paediatric haematology and oncology study group. they analyzed the development of novel childhood all cases prior to, during and after 2003, the year in which hong kong suffered from a sars pandemic. the study shows a decline in standard-risk all incidence during the time of social isolation measures that were taken to prevent sars-cov-1 from spreading. while the study links this decrease to a less likely infection with sars-cov-1, the authors could not rule out other infections as a cause and only concluded that the reduced number of infections in general is responsible [108] . another study reported an interaction of the endoribonuclease nsp15, which is commonly expressed in coronaviruses, with the tumor suppressor protein retinoblastoma (prb). the authors showed that nsp15 could bind to prb, leading to a reduction in prb levels and alterations in its regulation of cell growth and gene expression [109] . of note, established oncoviruses like hcv, ebv, kshv, mcpyv, hpv, hbv, htlv, are also known to interact with prb [72] . ebv protein ebna-3, kshv protein lana1, mcpyv or hcv protein ns5b, lead to the inactivation and degradation of rb, which leads to an enhancement in cell cycle progression [95, 102, [110] [111] [112] [113] [114] [115] . therefore, the ability to modulate rb function seems to be a hint for oncogenic potential. studies which investigate the ability of sars-cov-2 to modulate tumor suppressor function, in particular rb function, would shed interesting light on this matter. along this line, sars-cov-1 was also shown to interfere with a number of signaling pathways, that are associated with the malignant transformation of cells, such as p53, egfr, jak/stat, or mapk signaling [116] . for instance, the virus directly interacts with the protein rchy1, an e3 ubiquitin-ligase, which leads to enhanced degradation of the tumor suppressor p53 [117] . furthermore, sars-cov-1 activates p38 mapk and subsequently elevates pro-inflammatory cytokines, including il6 [118, 119] . interestingly, egfr signaling is overactivated after viral infection and linked to the development of fibrosis [120, 121] . as discussed above, fibrosis is highly linked to cancer development, giving further hints that sars-cov-1 might favor tumorigenesis. if, however, this is the case, and to what extent these pathology mechanisms also apply to sars-cov-2, is unknown as of yet. nevertheless, we suggest that future research should investigate the discussed pathways after sars-cov-2 infection to shed light on the matter. interestingly, the angiotensin-converting enzyme 2/angiotensin-(1-7)/mitochondrial assembly receptor (ace2/ang-(1-7)/masr) axis is debated for its role in cancer. there have been studies suggesting the pro-and anti-tumorigenic potential of its activation (reviewed in [4] ). as mentioned above, the sars-cov-2 virus uses ace2 to invade cells, probably followed by the downregulation of the receptor [48, 52, 53] . this deregulation in ace2 could therefore favor carcinogenesis. these studies suggest that infection with coronaviruses such as sars cov-1 and sars-cov-2 might influence carcinogenic events. it would therefore be important in our opinion to dedicate future research to address this question. the novel coronavirus sars-cov-2, which causes covid-19, has recently emerged as major worldwide health problem, driving health care systems to their limits and causing many deaths, especially in high-risk patient cohorts. high risk factors for a severe progression are high age and comorbidities, especially cancer. chemotherapy-and radiation therapy-induced immunosuppression is a major risk factor for cancer patients to acquire a severe and probably fatal sars-cov-2 infection. hence, sars-cov-2 and cancer form a dangerous couple. however, this connection might be of a bidirectional nature, as there are some initial hints, suggesting that an infection with sars-cov-2 might lead to long-term pathological consequences, such as cancer development. of course, since the virus has just recently emerged, there are no specific data available so far; however, the pathogenesis of covid-19 shows parallels to other oncovirus infections. the excessive inflammatory immune response during severe covid-19 cases might drive cancer progression. furthermore, inflammatory processes, as well as mechanical lung injury potentially caused by ventilatory assistance during icu stay, can lead to fibrosis. inflammatory and fibrotic changes caused by viral infection are known to be cancer drivers. therefore, studies specifically designed to investigate a possible tumorigenic potential of sars-cov-2 are urgently needed. currently, a lot of strategies are being discussed concerning management of sars-cov-2 infections. while the world is waiting for the first vaccines to be approved, it is also hypothesized to induce herd immunity by gradually infecting most of the population with the virus. however, in this regard, caution is necessary, as the long-term consequences of infections, also with an initial mild or symptom-free progression, are not clear. overall, further research is greatly needed to understand sars-cov-2 pathogenesis and to develop vaccines and therapies. it is essential to keep in mind that the currently available studies on sars-cov-2 include only small numbers of patients and they therefore have to be interpreted with caution and the statistical resilience has to be proven by studies including large cohorts. in this regard, we think that follow-up studies on a broad number of covid-19 survivors are inevitable to assess the risk of possible long-term consequences of the disease, especially regarding oncogenic potential. author contributions: f.g. and k.b. conception; f.g. wrote the manuscript; k.b. wrote sections of the manuscript. a.m.v. and k.b. critically revised the manuscript. all authors contributed to manuscript 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is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-296602-19noki6p authors: law, helen kw; cheung, chung yan; sia, sin fun; chan, yuk on; peiris, js malik; lau, yu lung title: toll-like receptors, chemokine receptors and death receptor ligands responses in sars coronavirus infected human monocyte derived dendritic cells date: 2009-06-08 journal: bmc immunol doi: 10.1186/1471-2172-10-35 sha: doc_id: 296602 cord_uid: 19noki6p background: the sars outbreak in 2003 provides a unique opportunity for the study of human responses to a novel virus. we have previously reported that dendritic cells (dcs) might be involved in the immune escape mechanisms for sars-cov. in this study, we focussed on the gene expression of toll-like receptors (tlrs), chemokine receptors (ccrs) and death receptor ligands in sars-cov infected dcs. we also compared adult and cord blood (cb) dcs to find a possible explanation for the age-dependent severity of sars. results: our results demonstrates that sars-cov did not modulate tlr-1 to tlr-10 gene expression but significantly induced the expression of ccr-1, ccr-3, and ccr-5. there was also strong induction of tnf-related apoptosis-inducing ligand (trail), but not fas ligand gene expression in sars-cov infected dcs. interestingly, the expressions of most genes studied were higher in cb dcs than adult dcs. conclusion: the upregulation of chemokines and ccrs may facilitate dc migration from the infection site to the lymph nodes, whereas the increase of trail may induce lymphocyte apoptosis. these findings may explain the increased lung infiltrations and lymphoid depletion in sars patients. further explorations of the biological significance of these findings are warranted. the sars outbreak in 2003 provides a unique opportunity for the study of human response to a novel virus. the etiological agent was identified to be a coronavirus (cov) originating from an animal reservoir [1, 2] . clinically, patients presented with an atypical pneumonia followed by diffuse alveolar damage, with mortality up to 52% in patients over 65 years old. interestingly, the disease pres-entations of sars were less severe in children than adults, and none of the infected children (< 12 years old) died of sars [3, 4] . evidences in the literature, including ours, support the role of immune evasion in the severity and immunopathology of sars (reviewed by [5] [6] [7] ), and we further suggested that the developmental status of the host immune system may be responsible for the agedependence of disease severity in sars. in our previous report [8] , we have demonstrated that dendritic cells (dcs), the key antigen presenting cells with crucial role in anti-viral immune responses, might also be involved in the immune escape mechanisms for sars-cov. there was entry and incomplete replication of sars-cov in dcs but there was no production of infectious virus released into the culture medium [8] . sars-cov infection did not lead to dcs apoptosis or dc maturation. interestingly, the sars-cov infected dcs showed low expression of antiviral cytokines (ifn-α, ifn-β, ifn-γ and il-12p40), moderate upregulation of proinflammatory cytokines (tnf-α and il-6) but significant upregulation of inflammatory chemokines (macrophage inflammatory protein (mip)-1α/ccl3, regulated upon activation, normal t cell expressed and secreted (rantes)/ccl-5, interferon-inducible protein of 10 kd (ip-10)/cxcl10 and monocyte chemotactic protein (mcp)-1/ccl2. we postulated that this lack of antiviral cytokine response against a background of intense chemokine upregulation could represent a mechanism of immune evasion by sars-cov. in the current study, we focused on the receptor responses in sars-cov infected dcs and compared adult and cord blood (cb) dcs to establish possible explanation for the age dependent severity of sars. dcs express a wide range of receptors for the recognition of conserved pathogen patterns as well as the induction of subsequent immune responses. some toll like receptors (tlrs) are expressed on the cell surface (tlr-1, tlr-2, tlr-4, tlr-5, tlr-6, tlr-10) while some are located within intracellular compartments (tlr-3, tlr-7, tlr-8, tlr-9) [9] . they are differentially expressed in different dc subsets and are modulated in response to a variety of stimuli [10, 11] . viral proteins may bind to tlr-2 or tlr-4, single stranded rna binds to tlr-7 and tlr-8, and double stranded rna binds tlr-3 while viral dna binds to tlr-9. the binding of ligands to tlrs may trigger downstream signaling pathways that are involved in both the cytokine release during the primary induction of inflammation and secondary activation of anti-inflammatory mechanisms [12] . cross talks between tlrs are common and the formation of tlr heterodimers allows a higher level of complexity in ligand-receptor binding and subsequent signaling. the migration of dcs from peripheral tissues to lymph nodes is essential for antigen presentation and triggering of adaptive immune responses. the trafficking of dcs is regulated by chemokines in their microenvironment and their expression of chemokine receptors (ccrs). differential expressions of ccrs are observed during dc maturation [13, 14] and some viruses, such as herpes simplex virus (hsv), can block ccr expressions on dcs to alter their migratory properties [15] . there are redundancies in the chemokines and ccrs interactions as many different ligands bind the same receptor and many receptors bind the same ligand. for example, rantes binds to ccr-1, ccr-3 and ccr-5, while mip-1α also binds to ccr-1 and ccr-5 [16] . we determined if the expression of these receptors on dcs are altered by sars-cov and contribute to autocrine regulation of dc migration. death receptors (drs) and their ligands also play important roles in innate and adaptive immune responses by regulating cell death and survival [17] . well-characterized death receptor ligands (drls) include tumor necrosis factor (tnf)-α, fasl and tnf-related apoptosis-inducing ligand (trail/apo2l). recently, several viruses, including measles virus [18] , human immunodeficiency virus (hiv) [19] , cytomegalovirus (cmv) [20] , and herpes simplex virus (hsv) [21] , were shown to induce trail expression on dcs. these "killer dcs" may be involved in the killing of virus-infected cells or bystander lymphocytes and natural killer cells. in view of the lymphopenia observed in sars patient, we determine if drls expression on dcs is modulated by sars-cov. this study provides evidence that sars-cov does not alter the tlrs, but modulates ccrs and dlrs expression in dcs; and further suggests possible mechanisms of immune escape and amplification of immunopathology in sars. gene expression of extracellular tlrs (tlr-1, tlr-2, tlr-4, tlr-5, tlr-6) in dcs are summarised in fig. 1 . there was a low (<50 copies/per 10 4 β-actin), but significant upregulation of tlr-1 and tlr-2 in sars-cov infected adult dcs at 3 h post infection. a similar trend was observed in cb dcs but the difference did not reach statistical significance. in cb dcs, the basal gene expressions of tlr-1, tlr-2, and tlr-5 and the sars-cov induced tlr-2 and tlr-4 expression were significantly higher than that in adult dcs (table 1) . no expression of tlr-10 was detected in either adult or cb dcs (data not shown). gene expression of intracellular tlrs (tlr-3, tlr-7, tlr-8, tlr-9) in dcs are summarised in fig. 2 . in adult dcs, the expression was low (average in range of 0 -20 copies per 10 4 β-actin). the expression of tlr-3, tlr-7 and tlr-9 were also low in cb dcs (average in range of 0 -20 copies per 10 4 β-actin). the basal gene expression of tlr-8 was high in cb dcs and the expression was nearly doubled after sars-cov infection at both 3 h and 9 h post infection. however, due to sample variations the difference between mock and sars-cov infected cb dcs was not statistically significant. comparing the mock and sars-cov infected adult dcs, there was moderate induction of ccr-1, ccr-3, ccr-5 and ccr-7 at both 3 h and 9 h post infection ( fig. 3 ) but only that observed for ccr-1 and ccr-5 reached statistical significance. it is important to note that at 3 h post infection, the ccr-3 expression in mock infected adult dcs was very low (average <1 copies per 10 4 β-actin) but it increased to 183.91 ± 128.35 copies per 10 4 β-actin after sars-cov infection. in sars-cov infected cb dcs, a strong and significant upregulation was observed for ccr-3 at both 3 h and 9 h post infection. in general, the expressions of these ccrs in cb dcs were significantly higher than in adult dcs (table 1) . we also assayed the expression of drls in sars-cov infected dcs as they may have an impact on the killing of bystanding immune cells. the gene expressions of fasl and trail in dcs were summarised in fig. 4 . in both adult and cb dcs, fasl gene expression was low (~0-40 copies per 10 4 β-actin) and there was a slight and insignificant induction by sars-cov. in the contrary, a strong induction of trail gene expression was observed in sars-cov infected dcs at both 3 h and 9 h post infection. the upregulated trail expression in cb dcs were significantly higher than that in adult dcs (table 1 ). dendritic cells are professional antigen presenting cells that play an important role as sentinels at the infection site by interacting directly with pathogens. some pathogens hijack different parts of the immune pathways involving dcs to enhance their survival. in the present study, we demonstrate that sars-cov does not have significant effect on the expression of tlrs on adult or cb dcs (figs. 1 &2) . however, there was significant upregulation of ccr-1 and ccr5 in sars-cov infected adult dcs and ccr-3 in sars-cov infected cb dcs which may be related to the autocrine regulation of dc migration (fig. 3) . moreover, we demonstrate a significant upregulation of trail on both adult and cb dcs (fig. 4) . we hypothesize that the upregulation of chemokine receptors may facilitate the autocrine mobilisation of dcs away from the infection sites and the upregulation of death receptor liganads may result in apoptosis of other immune cells, leading to immunosuppression and lymphopenia. tlrs are expressed on many cell types and their expression on different dc subsets have been characterised. visintin et al. [22] measured tlr expression on immature human dcs by monoclonal antibodies binding, but the expression was very low, corresponding to a few hundred molecules or less in immature dcs. subsequent studies of tlrs expressions were based on comparing rna expression quantitatively and some reports for immature dcs have been controversial. our detection of tlr-1 to tlr-9 expressions in mock infected adult dcs were consistent with that reported by jarrossay et al. [10] and the low level of tlr-10 was similar to that reported by kadowaki et al. [11] . viruses may subvert the tlr response to evade the host defense by: (a) producting specific viral particles that block tlr function, (b) blocking tlr recognition and (c) stimulating viral replication through tlrs [23] . viral infections, by rsv or influenza a, have been shown to upregulate tlr-3 and tlr-4 expression in host airway epithelial cells, leading to increased signalling activity and proinflammatory cytokine production [23] . more recently, tlr-4 was shown to be upregulated in mice with acid-induced or inactivated avian influenza-induced lung injuries; and tlr-4 deficient mice showed less severe acute lung injuries to these challenges [24] . we, therefore, determined if sars-cov may modulate the tlrs expression on dcs. in this study, there was no induction nor suppression of tlrs in sars-cov infected dcs (figs. 1 &2) . the variations between samples were high, but the expression levels of some extracellular tlrs (tlr-1, tlr-2, tlr-4, tlr-5) and some intracellular tlrs (tlr-7, tlr-8) in cb dcs were higher than that in adult dcs (table 1 ). further investigation is needed to determine if the expression is responsible for the slightly higher type i inferferon expression reported in cb dcs [8] and to identify any correlation with less severe sars in children. interestingly, impaired immune responses to tlr-3 and tlr-4 ligands have been reported in cb dcs [25] . the majority of tlr research is focussed on the downstream signalling pathways triggered by the binding of specific ligands to tlrs. activation of tlrs induce signalling cascades which activate transcription factors and gene expression for anti-inflammatory responses [9] . the identification of such ligands may have therapeutic applications for the treatment of sars. in a recent study on the control of coronavirus infection by plasmocytoid dcs (pdcs), it was demonstrated that mouse hepatitis virus (mhv) induces type i ifn response and the recognition of mhv is mediated by tlr7 [26] . the authors demonstrated that sars-cov also induced a strong type i interferon response in human pdcs and proposed that tlr7 may play a similar role in sars-cov infection. another group developed a recombinant mouse-adapted sars-cov (rma15) that was lethal in balb/c mice and demonstrated that myd88 played an important role in sars-cov pathogenesis [27] . both groups suggested the involvement of tlr7/myd88/ifnα dependent signaling and further exploration is needed to determine if tlr7 agonist may elicit potent antiviral effects against sars-cov infection. to date, approximately 50 chemokines and 20 ccrs have been identified in humans [16] and they play important roles in inflammation and infectious diseases [28] . in our previous study, there was significant increase in the inflammatory chemokines (mip 1α, rantes, ip-10 and mcp-1) in sars-infected dcs [8] . in addition, we have shown that sars patients who have inherited the highproduction gene allele of rantes have more deaths [29] . in this study, we determined whether the expressions of representative ccrs (ccr1, ccr-3, ccr-5 and ccr-7) are also upregulated by sars. indeed, there was signifi-cant induction of ccr-1, ccr-3 and ccr-5 mrna expression in sars-cov infected dcs (fig. 3) suggesting the possibility of a autocrine loop in facilitating the trafficking of dcs. therefore, further investigation into therapeutic strategies which aim at reducing chemokine and ccrs expresssion are warranted. among the ccrs studied, the upregulation of ccr-3 is the strongest (fig. 3) . it has been reported that the expression of ccr-3, unlike ccr-5 and ccr-7, are independent of the maturation status of dcs [13, 14] . further investigation is needed to determine which mechanism is involved in ccr-3 upregulation. in addition to cell trafficking, ccr-3 has also been reported to function as a death receptor on b cells [30] . whether ccr-3 expression is related to the characteristic antibody responses in sars and the alternative function of ccrs on sars-cov infected dcs will need to be explored further. ccr-7 has been identified as a key molecule for the establishment of central and peripheral tolerance. it is strongly upregulated in herpesvirus infected t cells [31] , ebv infected b cells [32] and mature dcs [14] . in addition, microarray analysis of pbmc infected with sars-cov have shown an upregulation of ccr7 [33] . in this study, we only detected a slight but insignificant change in ccr-7 expression in adult sars-cov infected dcs (fig. 3) . this observation is consistent with previous findings that sars-cov did not stimulate dc maturation [8, 34] . however, this is in contrary to observations made by others [35, 36] . we speculate as these immature dcs migrate to the lymph nodes, they may act as regulatory dcs which are less effective for the activation of allogeneic cd4+ t cells than normal dcs [37] or they may drive the development of regulatory t cells [38] . these sars-cov-driven regulatory immature dcs may facilitate the immune evasion of sars-cov [39] . interestingly, we detected significantly higher levels of chemokines and ccrs genes in cb dcs than in adults dcs. based on the function of chemokines on cell trafficking, more severe infiltration of cells to the lungs would be expected in children. on the contrary, sars was less severe in children than adults [3, 4] . the age-dependency of disease severity in sars merits further studies to elucidate the underlying mechanisms. the development of animal models for sars has been difficult because relevant signs of clinical illness and death cannot be reproducible in most animal models [40] . recently, aged balb/ c mice have shown to develop more severe disease after sars infection [41] and have lower vaccine efficacy than young mice [42] . the comparison of sars-cov infected senescent mice with adult mice can be extended to younger mice and knock out models for the study of agedependency in the pathogenesis of sars. a recent study has shown that mice deficient in either ccr1, ccr2 or ccr5 exhibited more prominent airway epithelial cell apoptosis and more severe lung pathology. this suggests that ccrs may be playing different roles at the site of infection and in the trafficking of immune cells [27] . using real time quantitative assay, we have shown significant upregulation of trail gene expression in avian influenza (h5n1) infected macrophages [43] . similarly, sars-cov infected dc also showed a "killer dc' phenotype with high expression of trail (fig. 4) . comparing the two studies, the level of trail expression in sars-cov infected adult dcs (~300 copies/10 4 β actin genes) was lower than that in h5n1 infected adult macrophages (~1800 copies/10 4 β actin genes). trail expression in sars-cov infected cb dcs was the highest (~5000 copies/10 4 β actin genes). further investigation is needed to confirm the cytotoxic function of sars-cov infected dcs on immune cells. based on the possible dc migration induced by chemokines and the role of trail in inducing lymphocyte apoptosis, our findings may explain the white pulp atrophy in the spleen and lymphoid depletion in lymph nodes of sars patients. the mechanism involved in children may be more complicated and it is important to determine if the cb dcs are functionally immature in spite of the high expression of chemokines, ccrs and trail. more recently, trail expression is also reported to be upregulated after the binding of hiv to tlr-7 [44] or activation of tlr-8 [45] . hence the high trail expression in sars-infected cb dcs may also be related to their tlr-8 expression. this study has characterized important receptor responses of dcs to sars-cov. the suggested mechanisms need to be substantiated by further research involving a larger sample size for different pathways as pathogens may have multiple ways to modify the physiology of dcs to their advantage. adult blood samples were from the white cell fraction of blood donated to the hong kong red cross by normal healthy volunteers. human umbilical cord blood (cb) samples were collected from the placenta of normal fullterm uncomplicated pregnancies. informed consent was obtained from the mothers prior to delivery. the protocol was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster [ec1473-00]. blood mononuclear cells were isolated from whole blood by centrifugation, using ficoll-hypaque gradients (phar-macia biotech, uppsala, sweden), washed, and labeled with immunomagnetic antibodies. positive selection was performed according to manufacturer's specification (miltenyi biotec, bergisch gladbach, germany) as in previous experiments [8] . isolated cd14+ monocytes from the positive fraction were resuspended in rpmi 1640, supplemented with 50 iu/ml penicillin and 50 μg/ml streptomycin and 10% fetal bovine serum (invitrogen, grand island, usa). cell viability, as measured by trypan blue exclusion, was more than 95%. the purity of the isolated cells as measured by flow cytometry was consistantly between 90% to 95%. cd14+ monocytes were cultured in the presence of il-4 (10 ng/ml; r&d, minneapolis, usa) and gm-csf (50 ng/ ml; r&d, minneapolis, usa) for 7 days at 37°c in a humidified atmosphere containing 5% co 2 as in our previous study [8] . the cultures were fed with fresh medium and cytokines on day 3 and cell differentiation was monitored by light microscopy. on day 5, dcs were harvested, centrifuged, washed and adjusted to 1 × 10 6 cells/ ml before virus infection. laboratory procedures involving live viruses was performed in biosafety level-3 containment. sars-cov, strain hku-39849 [1] was cultured in fetal rhesus kidney-4 (frhk-4) cells. the cell culture supernatant was harvested, centrifuged to remove cell fragments, aliquoted and kept frozen at -70°c. sars-cov titre of the stock virus was determined by infection of frhk-4 cells. cytopathic changes on frhk-4 cells was monitored every day up to 4 days and virus titre expressed as tissue culture infective dose (tcid 50 ). cells were inoculated by sars-cov at a multiplicity of infection (moi) of 1. the virus was allowed to be adsorbed for 1 hour at 37°c and unbound virus was washed off by excess volume of pbs (time = 0 h post infection). mock infected cells were treated in parallel, except that virus was not added. total rna was extracted from ~1.5 × 10 5 cells harvested at 3 h and 9 h post infection by trizol reagent (invitrogen life technologies, usa). in later experiments, qiashredder columns (qiagen, hilden, germany) were used to ensure adequate homogenisation and rna was extracted by the rneasy mini kit (qiagen, hilden, germany). reverse transcription was performed on the dnasetreated total rna using oligo (dt) primers and superscript ii reverse transcriptase (invitrogen life technologies, usa) according to the manufacturer's recommendation. the cdna synthesised were diluted (1:50) and quantified by real-time pcr using taqman technology (applied biosystems, ca, usa). specific primers ( table 2) were used and non-specific reactions and primer-dimer artifacts have been minimised (as evaluated by gel electrophoresis). detection of pcr product was based on taqman fluorescence signal. standard curves were generated using serial dilutions of plasmids (~10 -10 10 copies) containing cloned sequences involved. results were calculated as the number of targeted molecules/μl cdna. to standardise results for variability in rna and cdna quantity and quality, we express the results as the number of target copies per 10 4 copies of β actin gene, which was determined previously [8] . all data were expressed as mean ± sem. all samples were paired and differences between groups were analyzed by paired student t test or the non-parametric equivalents using the instat software (graphpad software, inc. san diego, ca, usa). coronavirus as a possible cause of severe acute respiratory syndrome isolation and characterization of 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ifn-alpha tumoricidal activity of tlr7/8-activated inflammatory dendritic cells the authors thank the staff of department of microbiology, the university of hong kong for their technical support; and the staff of labor ward, queen mary hospital, in facilitating the collection of cord blood. the work described in this paper is supported partially by the special sars research fund and the outstanding researcher award (yll, jsmp) from the university of hong kong; the public health research grant (ai 95357) from the national institute of allergy and infectious diseases, usa and the research fund for the control of infectious diseases (rfcid 03040772) from the hksar government. we also thank dr. matthew buckwalter, insitut pasteur, france for proofreading the manuscript. hkwl prepared the dendritic cells. sfs and yoc performed the experiments involving the virus in the biosafety level 3 laboratories. hkwl and sfs carried out the molecular studies. hkwl, cyc, jsmp and yll participated in the design of the study and analyzed data. hkwl and yll drafted the manuscript. all authors read and approved the final manuscript. key: cord-314171-431buxxr authors: dariya, begum; nagaraju, ganji purnchandra title: understanding novel covid-19: its impact on organ failure and risk assessment for diabetic and cancer patients date: 2020-05-06 journal: cytokine growth factor rev doi: 10.1016/j.cytogfr.2020.05.001 sha: doc_id: 314171 cord_uid: 431buxxr the current pandemic outbreak of covid-19 originated from wuhan, china. it is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) with significant mortality and morbidity rate. the severe risk factors are commonly detected in patients of older age and with medical comorbidities like cancer and diabetes. scientists and doctors have scrambled to gain knowledge about the novel virus and its pathophysiology in order to discover possible therapeutic regimens and vaccines for covid-19. the therapeutic strategies like targeting the viral genome emphasize the promising approach to target covid-19. additionally, blocking the receptor, ace2 via the neutralizing antibodies for viral escape that prevents it from entering into the cells provides another therapeutic regimen. in this review article, we have presented the effect of sars-cov-2 infection in comorbid patients and discussed organ failure caused by this virus. based on the data available from the scientific literature and ongoing clinical trials, we have focused on therapeutic strategies. we hope that we would fill the gaps that puzzled the researchers and clinicians with the best of our knowledge collected for the betterment of the patients for the coming future. the sars-cov-2 is the frightful pathogen responsible for the outbreak of covid-19 [1] . the disease emerged in wuhan city, china, in late december 2019 [2] and is highly contagious with rapid human-to-human transmission [3] . the who declared the covid-19 outbreak a global emergency [1] . the confirmed cases rose to 2,101,164 with 140,773 deaths and approximately 532,830 recoveries in almost 210 countries (reported by csse). organizations around the world debated potential therapeutic strategies for treating covid-19 patients and public health strategies. this pandemic also threatened the economy of the entire world. the complete role and severity of sars-cov-2 remain undefined. in this review article, we have focused on determining the relationship between sars-cov-2 infection and organ failure. additionally, we focused on the comorbid population that includes patients with diabetes, and/or cardiovascular disease, who are covid-19. it is well established that morphologically, a virus is composed of genetic material surrounded by a protein capsid, which in some animal viruses is then surrounded by a lipid j o u r n a l p r e -p r o o f bilayer [4] . this small pathogen can cause a pandemic, raising global alarms. looking back through history, many viruses have triggered pandemics similar to covid-19, including smallpox, tuberculosis, plague, spanish flu, hiv/aids, and h1n1 flu. table 1 shows a list of viruses that have infected humans and had non-human host species. sars-cov-2 belongs to the family coronaviridae, which includes viruses responsible for diseases from the cold to mers and sars [1] . sars coronavirus, mers coronavirus, 229e, and oc43 primarily infect humans. sars-cov-2 shares many similarities with sars: they both have a crown or halo-like appearance and a glycoprotein-studded envelope. the sars-cov-2 virus varies structurally from other viruses that have transmembrane crown-like spiked glycoproteins [5] . it has 4 structural proteins, including envelope, spike, nucleocapsid and membrane. they are comprised of the functional subunits s1 and s2, which are responsible for detecting ace2 receptors present on the host cells. ace2 receptors are utilized by the virus to enter the host cells [6] [7] [8] . ace2 expression is detected on type i and type ii alveolar epithelium, upper respiratory system, heart, kidney tubular epithelium, pancreas, endothelial cells and enterocytes. thus, the external spike protein determines the infectious nature and host specificity of sars-cov-2. the host cells allow the entry of the virus through the process called endocytosis. moreover, the transmembrane proximal serine protease 2 (tmprss2) is the host protein that facilitates the entry of the virus through the s protein [5, 9] . additionally, it is involved in priming the s protein and potentiates the cleavage of the spike (fig.1 ). later, in the cytoplasm, the endosome exposes single-stranded rna, the virus' genetic material. the genome of the virus encodes various non-structural proteins like papain-like protease (plpro), rna-dependent rna polymerase (rdrp) and the coronavirus main protease, 3c-like protease (3clpro) [10, 11] . the virus then hijacks the machinery of the cell to synthesize the viral polypeptides that encode for the replicase transcriptase complex. the active virus produces rna through rdrp. moreover, plpro actively deubiquitinases certain immune regulator cells like if3 and nf-κb to suppress the immune response [11, 12] . it uses the endoplasmic reticulum to synthesize m and s proteins, which are essential for its outer capsule. the viral proteinases 3clpro and plpro more effectively cleave the viral polyproteins with the help of the host translation machinery [10, 11] . they produce new spikes and glycoproteins that are assembled into numerous copies of the virus. after replication of the genetic material, the golgi bodies exocytose the viruses, which then attack other cells. the created stress on the endoplasmic reticulum by the virus also induces apoptosis of the healthy host cells after releasing millions of viral copies. the viruses continue to attack other cells or end up as droplets and enter the lungs [13] . as an immune response, a fever is generated as the host's immune system fights to clear the virus out of the body. pro-inflammatory chemokines are activated to produce inflammatory cells. cd4+ t helper cells develop immunity against sars-cov-2 by producing ifn-γ and il-17 [14] . sars-cov-2 also targets these circulating immune cells and induces apoptosis of cd3, cd8 and cd4 cells, causing lymphocytopenia [15] [16] [17] [18] . this results in the overproduction of cytokines, causing a cytokine storm as it is released from the inhibition of innate immunity. the cytokine storm results in hyper inflammation, ultimately causing failure of multiple organs [19] [20] [21] . for instance, under severe conditions, a patient's immune system can attack the lung cells. this results in fluid filling the lungs and cell apoptosis, causing difficulty in breathing. in some cases, this leads to death. j o u r n a l p r e -p r o o f the typical signs of covid-19 infection are fatigue, cough, fever, and myalgia, and some patients have also developed dyspnoea. respiratory symptoms like cough, shortness of breath, acute respiratory syndrome and organ injury are also detected as serious complications [2, 13] . the patients also experience lung alterations and reduced circulating lymphocytes and platelet counts. human-to-human transmission usually occurs during the incubation phase or when the patient exhibits symptoms [5] . however, sometimes, a person in the asymptomatic phase is also found to be contagious. such individuals are referred to as super-spreaders. for these cases, the transmission route was skin to skin or touching inanimate objects mediated via the nose, mouth or eyes [5] . exhaled respiratory aerosols droplets are airborne and can remain in the air for long periods. thus, direct inhalation of these aerosol droplets or touching the surface of infected objects like latex, steel, aluminium, or surgical gloves are possible modes of transmission [5] . thus viruses like sars-cov-2 virus remain active outside the host body [22] . furthermore, faecal and sewage transmission are also possible modes of transmission [22] . similarly, the virus in sewage was found to be active for a few days to a week [23] . as of now, there are 400 sars-cov-2 genomes available from the ncbi database. the sequence analysis of these available genomes could form the platform for vaccine development. sardar et al [24] compared genomes of sars-cov-2 from diverse geographical origins including wuhan (china), italy, india, the usa, and nepal by performing an integrated phylogenetic analysis using bioinformatics tools. additionally, they also predicted that unique antiviral host mirnas may target sars-cov-2 genes. they related past research studies about the inhibitory nature of has-mir-27b-3p in the ace2 cascade [15] . moreover, they also determined that has-mir-27b is complimentary to the spike of the indian strain of sars-cov-2. previous research data also suggested that mir-27b inhibits the replication of j o u r n a l p r e -p r o o f hiv-1 [25] . however, they could not provide any experimental evidence regarding this related to sars-cov-2, and there are other no research data available in this regard. the life-threatening pneumonia outbreak started as an epidemic within wuhan city, china. on december 31, 2019, the wuhan municipal health commission reported critically ill cases of viral pneumonia. later, the international committee on taxonomy of viruses (ictv) identified the virus as sars-cov-2 [26] . the chinese government then mandated a quarantine to control the epidemic stage and to determine the effectiveness of a quarantine against the virus [27] . however, sars-cov-2 was transmitted rapidly, quickly crossing borders of various countries. thus, the number of established cases climbed quickly, causing the who to announce covid-19 a global pandemic disease on march 11, 2020. initially, china had the most confirmed cases, followed by north america and europe. italy and the us, however, surpassed china in terms of the number of established cases. thus, at this stage of vigorous spread, it is essential to determine the mechanism of viral infection to control and treat the disease. until now, it was believed that bat, especially rhinolophus affinis, was the natural host of the virus [7] . it is still uncertain how this virus was transmitted from bat to human, although some research has suggested that pangolin (manis javanica) could be the intermediate host [14, 28, 29] . the aggressive intervention steps were taken by governments, such as isolating and quarantining latent patients, have reduced the contact rate and effectively decreased the peak for covid-19 cases. in addition to the symptoms discussed previously, there are other clinical manifestations observed with covid-19 infection, including the failure of multiple organs. sars-cov and j o u r n a l p r e -p r o o f 2019-ncov/ sars-cov-2 were found to share a common progenitor with hku9-1, the bat coronavirus. the similarity in the spike structure proteins of sars-cov-2 showed a higher affinity with ace2 as discussed earlier [5] . ace2 receptors are present on the target cells, making the cells highly susceptible to the entry of sars-cov-2 and subsequent pathogenesis. for instance, type ii alveolar cells (at2) of the lungs are believed to have higher ace2 expression and are the primary targets for sars-cov-2 [7] . in this study, also examined different cell types from different organs that showed ace2 expression using single-cell-rna seq (scrna-seq) data to determine the effect of covid-19 for the first time. we tabulated their results for different organs showing the type of cells tested, the proportion of cells expressing ace2, and the risk of organ failure in table 2 [6, [30] [31] [32] [33] . additionally, myocardial infarctions increase ace2 expression, increasing the likelihood of cardiac injury [34] . this contributes to the understanding of the effect of covid-19 on various organs. cardiac injury was the most common complication that was related to an elevated risk of disease severity in covid-19 patients. about 23% of covid-19 ill patients had cardiac injury [17] , and 13% showed an elevation in creatinine kinase [35] . the mrna and protein ace2 expression levels are higher in these patients with cardiac disease, creating an increased risk for severe covid-19 complications, including heart failure. moreover, it was also suggested by liang chen et al. that the human heart infected with sars-cov-2 attacks pericytes, resulting in microvascular disorders [36] . this microvascular disorder causes j o u r n a l p r e -p r o o f dysfunction in capillary endothelial cells, low microvascular reactivity and high vascular permeability. the modelling and docking results also showed that sars-cov-2 has a receptor-binding domain that fits well with human ace2 receptors. moreover, the amino acid residues including 491tyr, 441leu, 479gln, 487asn, 472phe and 480ser of the sars-cov-2 mature spike protein are involved in binding with the ace2 receptor [36] . thus, these amino acid residues in the binding domain are important to consider in the design of potential drug treatments. taken together, these findings show that covid-19 in patients with cardiovascular disease often results in a critical condition and death. the organ failure common in covid-19 patients results in alveolar and acute respiratory failure [3] . because there is a substantial comparison between the pathogenesis of sars-cov-2 and sars, researchers and clinicians should explore the infection mechanisms of sars-cov-2 based on the effect of sars. earlier research showed that about 6.7% of sars patients possessed acute kidney injury (aki), leading to a mortality of almost 91.7% [37] . thus, physicians need to consider the effect of sars-cov-2 on the kidney. cheng et al [38] explored the occurrence of aki in covid-19 patients to determine their association. they reported that 14.4% of the covid-19 patients in the study showed elevated levels of serum creatinine upon admission. most of these patients were male, older, or had other illnesses. additionally, 13.1% had elevated blood urea nitrogen (bun). after hospitalization (10 days), the serum creatinine peak elevated to 91±67 μmol/l, and about 43.9% of the patients showed proteinuria. these patients also showed a higher incidence of aki (11.9%) after hospitalization; only 5.1% had aki during admission. the in-hospital mortality recorded was 16.1%; however, those with raised baseline serum creatinine had a mortality rate of 33.7%. furthermore, the univariate cox regression analysis also showed that covid-19 patients who were male and/or older than 65 were highly prone to in-hospital death. therefore, j o u r n a l p r e -p r o o f clinicians must be aware of kidney diseases in covid-19 patients after hospitalization as early diagnosis and effective treatment would reduce the death rate in patients. recent clinical data has shown that cancer patients receiving antineoplastic treatment are highly vulnerable to the effects of covid-19, similar to immune-suppressed and older (>60 years) patients [39] . patients with haematological malignancy, neutropenia or lymphopenia as well as those receiving multiple doses of chemotherapy are at higher risk for hospitalization (4 times) higher and death (10 times) higher compared with the healthy population of covid-19 patients [40] . moreover, cancer patients diagnosed with covid-19 who received antitumor therapy within 14 days before diagnosis are at elevated risk for severe complications. zhang et al. [41] also concluded that malignant patients with covid-19 had poor prognosis. they suggested that vital covid-19 screening should be carried out for the cancer patients receiving antitumor therapy, and cancer patients with covid-19 should avoid using immunosuppression drugs or use a decreased dose. the demographic data from italy showed that among 3000 reported covid-19 cases, 20% of the patients who died had a medical history of malignancy in the previous 5 years [42] . among 1,524 patients joined to the zhongnan hospital of wuhan university, 12 patients had covid-19 [39] . this same study showed that the patients with nsclc (7 patients) who were >60 years of age had the highest incidence rate for covid-19 disease [39] , followed by oesophageal (4 patients) and breast cancer (3 patients) [41] . the report also showed that these patients were more likely to suffer life-threatening complications and require icu admission or mechanical ventilation [39] . these deaths were due to acute myocardial infarction, acute respiratory syndrome, septic shock and pulmonary embolism [41] . for these 28 cancer patients in wuhan who also had covid-19, the lab findings at the time of admission included low blood count (anaemic), leucopenia and lymphopenia [41] . additionally, low levels of serum albumin, high levels of serum globulin and high lactate dehydrogenase with sensitive c-reactive protein in higher levels and erythrocyte sedimentation rate were also detected [41] . the radiological findings included ground-glass opacity, patchy consolidation [43] , interlobular septal thickening, reticular appearances and fibrous strips. the patients with patchy consolidations were at greater risk for developing severe problems than those without [43] . moreover, lung cancer patients were reported with decreased lung volume along with pneumonia [41] . it was also shown that among these malignant patients with covid-19, 70% of the patients had stage iv cancer and are severe complications [41] . the research and clinical data available now contribute to the proper understanding of the risk of covid-19 occurrence in malignant patients. thus, this helps oncologists tailor the covid-19 clinical management for the benefit of such patients. additionally, establishing essential guidelines and recommendations for the care of cancer patients based on a patient's age, affected organ and stage of cancer during the covid-19 outbreak is very crucial [44] . patients with diabetes mellitus (dm), obesity and/or hypertension and covid-19 have increased mortality and morbidity rates [17, 18, 35, 42, [45] [46] [47] . however, the association of dm with hypertension and cardiovascular diseases as risk factors for covid-19 is still unknown and not clear. in one study that included 52 icu-admitted covid-19 patients, about 22% of the patients were diabetic. of the 52 admitted patients, 32 did not survive [17, 44] . this suggests that dm is the predominant comorbidity with covid-19. a few mechanisms have been suggested by researchers to explain the high susceptible of dm patients to covid-j o u r n a l p r e -p r o o f 19 pathogenesis. these include having an efficient cellular binding and easy entry of the virus, low chance for viral clearance, weakened t-cell function, highly prone to cytokine storm and hyperinflammation. additionally, ace2 expression weakens with the administration of insulin [48, 49] , whereas ace2 expression is upregulated by hypoglycemic agents like thiazolidinediones, glucagon-like peptides-1, ace inhibitors, angiotensin-receptor blockers, antihypertensives and statins [50] [51] [52] [53] [54] . the rodent dm model showed higher expression ace2 in heart, lung, kidney and pancreas compared with normal controls [48, 49] . furthermore, rao et al [55] . from their mendelian randomization study determined from the diseases and traits that the lungs of dm patient showed increased expression of ace2. the protein levels of proteases like furin that facilitate the cleaving of the spike protein for virus entry were also found to be overexpressed in dm patients [56] . in order to understand the association between ace2 expression in dm patients and covid-19 infection, we must understand the mechanism of ace2 action. ace2 catalyses the conversion of angiotensin ii to angiotensin 1-7 and angiotensin 1-9 (fig. 2) . these act as antioxidants and anti-inflammatory enzymes and are found to be protective against lung ards [57] . after sars-cov-2 binds with ace2, the virus degrades it, and thus the free angiotensin ii induces acute lung injury [58] . additionally, increased loss of potassium levels through urine and increased secretion of aldosterone also result after the intrusion of the virus [35] . thus, ace2 is unable to protect against lung injury after the entry of sars-cov-2 because ace2 is degraded by the virus. the aetiology is still confusing, but it is clear that dm patients are at higher risk for severe complications with covid-19. thus, we have an urgent need for research studies focused on determining how hyperinsulinemia and hyperglycaemic conditions affect sars-cov-2 infection and how dm might affect vaccine efficacy. j o u r n a l p r e -p r o o f unfortunately, therapeutic treatments are not yet available for this covid-19 outbreak as it takes years for a drug to come to market. however, at present, the clinical trials and sympathetic use are being expedited. thus currently, social distancing, patient isolation and supportive medical care and monitoring are the only available approaches. the potential pharmaceutical approaches to blocking the ace2 receptor or a viral protein to mediate the inhibition of covid-19 are tabulated in table 3 . these include the following potential options: developing safe and efficient vaccines during the covid-19 pandemic stage is very crucial. the antibodies developed against viral spike proteins by the host immune system are utilized in developing vaccines. the process includes purifying the plasma that contains antibodies from a recovered covid-19 patient. the next step is targeting these antibodies to the spike protein of the virus to neutralize it and likely to develop passive immunity against disease [68] . moreover, structural and sequence homology of sars-cov-2 with the other lethal cov viruses such as sars and mers would help determine the best epitope to use in the design of an anti-sars-cov-2 vaccine. this would essentially neutralize the viral s protein, although creating a vaccine is a slow process. the sars-cov and sars-cov-2 spike proteins are 76.5% homologous at the amino acid level [5] . recently, wan et al. [69] also determined that sars-cov with residue 479 (lysine) and sars-cov-2 with glutamine 394 residue recognize and bind to lysine 31 of the ace2 receptor in humans [70] . an alternate approach is to inject neutralizing antibodies into covid-19-infected animals to obtain purified polyclonal antibodies from these animals [71] . this process can be expedited, but the outcome is not guaranteed as the animals might not produce the expected neutralizing antisera [72] . j o u r n a l p r e -p r o o f tmprss2 is a proximal serine protease present in the host that plays a crucial role in proteolytic processing called priming. it is actively involved in priming of the s protein of sars-cov after its binding with the ace2 receptor [73] . thus, it is crucial for the entry and spread of sars-cov. a couple of reports recently showed that sars-cov-2 also enters through this mechanism, and thus the entry of sars-cov-2 into cells may be blocked by using inhibitors of tmprss2, such as camostat mesylate (fig. 1) [7, 67 ]. ace2 is a mono-carboxy peptidase that hydrolyses angiotensin ii. as discussed earlier, ace2 binds with high affinity to sars-cov-2, thereby allowing the virus to enter the host cells. thus, targeting the binding site of the ace2 receptor and sars-cov-2 with antibodies or therapeutic drugs might provide a successful treatment strategy. moreover, ace2 shows similar homology with ace, which hydrolyses angiotensin i to angiotensin ii [74] . this led to a lot of confusion between ace inhibitors and ace2 inhibitors. increased ace activity and reduced ace2 activity promote lung injury. furthermore, there have been several reports that ace inhibitors (acei) inhibit the expression of ace2 in kidney and heart [50] . as determined histologically, ace2 is primarily membrane-bound but is also present as a soluble form in body fluids at a very low level [75, 76] . the cleavage in the membrane adam17 anchor by a disintegrin and metalloprotease 17 present at the membrane-bound ace2 receptor increases the occurrence of more soluble ace2 in the body fluids (fig. 1) . thus, the membrane-bound ace2 is no longer available for sars-cov-2 to bind with it ( fig. 1 ). angiotensin ii via its type 1 receptor (at1r) induces the upregulation of adam17, which potentiates the cleavage and increases the soluble ace2 concentration in the body fluids. thus, at1r upregulates adam17 and increases soluble ace2 [77] , whereas the j o u r n a l p r e -p r o o f administration of at1r blockers (arb) prevents the process and maintains the membranebound ace2 receptors (fig. 1) . additionally, arb is also found to consistently alter the expression of ace2 at the protein and mrna levels. ace2 expression is upregulated in the renal vasculature and cardiac tissue [78] [79] [80] . therefore, using arb to treat for pathological conditions like lung injury without any complaint of covid-19 infection is beneficial, but it may be critical during covid-19 infection [81, 82] . lung injury without sars-cov-2 infection is caused by downregulated alveolar ace2 levels and decreased angiotensin ii metabolism [83, 84] . however, several researchers have proposed the use of arb to protect against lung injury during covid-19 infection. to summarize, comorbidities including cardiovascular, hypertension diseases and diabetes 2, are generally treated with blockers such as ras blockers, arbs, ace inhibitors and ar blockers. the current clinical trials performed to determine the efficacy of drugs against covid-19 are tabulated in table 4 . moreover, in the most recent studies, liu et al. [85] determined that the serum levels of angiotensin ii are increased in covid-19 patients who had pneumonia and lung injury. this indicates that sars-cov-2 disrupts the balance of the ratio of ace/ace2, resulting in increased levels of angiotensin ii. this further induces inflammation, pulmonary vasoconstriction and oxidative organ damage, causing acute lung injury. thus, the modulation of ras by arbs or recombinant ace2 increases the amount of ace2 receptors and controls the levels of angiotensin ii [86] . moreover, this also increases the level of soluble ace2 that competitively binds with sars-cov-2, causing delayed entry of the virus into cells and protecting against lung injury. additionally, rapid sequencing of the sars-cov-2 genome has enabled epidemiological tracking and diagnosis and promotes the development of therapeutic [87] . one approach is to prevent the reproduction of sars-cov-2 by targeting the viral rna polymerase, which is used to synthesize the viral rna genome and is not produced naturally in the host body [88] . if the viral polymerase could be selectively targeted or blocked, the virus could no longer produce rna copies and the viral infection could be stopped. for instance, remdesivir is a drug developed against the ebola virus specifically, but it showed promising results when used to treat mers-cov infection [63] . remdesivir, conceals the viral rna polymerase and prevents the proofreading to occur via the viral exonucleases. this way it decreases the viral rna production in the host cells (fig. 1) . remdesivir was also suggested to give positive results when used against sars-cov-2, and a clinical trial sponsored by the nih began in february 2020. however, the clinical impact of this drug against covid-19 is still unclear and researchers are waiting for the outcome of the patient's ongoing clinical trials. targeting viral processing is another possible treatment strategy, which takes advantage of the fact that viral proteases have a specific site where protein cutting occurs [89] . therefore, drugs that fit into this specific site can block the functionality of a viral protease, thereby inhibiting viral protein production. viral proteases are promising therapeutic targets since they are available in limited numbers in the host cells and have a specific site for catalytic j o u r n a l p r e -p r o o f activity [89] . this type of therapy has been successfully used against hiv, and now researchers are seeking to use protease inhibitors against sars-cov-2. targeted disruption of the packaging of a virus is another possible approach since, after packaging, the virus leaves the host cell through exocytosis and attacks other cells [90] . however, in coronaviruses there are no proteins specifically designed for packaging; all of the viral proteins are actively involved in viral packaging. thus, this type of approach does not seem promising for the treatment of covid-19. other potential treatments target the virulent shell. the fully matured virus particles have a shell made up of spike and membrane proteins. the membrane proteins are buried within the membrane, but the spike proteins are external. thus, a virus can be targeted exteriorly by attacking the spike proteins with drugs. since it is on the exterior, the spike protein is also the main target of antibodies produced by the host's immune system [91] . this led to the development of vaccines against sars-cov-2, which we have already discussed earlier in this article. chloroquine and its derivative hydroxychloroquine have long been used for the prevention and treatment of malaria and chronic inflammatory diseases like rheumatoid arthritis [92] . both of these drugs are found to be very effective in treating dysregulations caused by a coronavirus in china [63] . it was notably reported that chloroquine inhibits the replication of hcov-229e and sars-cov-1. it was also found to prevent the entry of virus by inhibiting the glycosylation of ace2 receptors, block proteases and regulate acidification in the endosome (fig. 1) . chloroquine and hydroxychloroquine also promote an immunomodulatory j o u r n a l p r e -p r o o f effect via the production of cytokines and suppress autophagy and lysosomal functionality in host cells [90, 93] . chloroquine was identified to suppress sars-cov-2 growth in vitro, showing more than a half-maximal effective concentration (ec50) with a low micromolar range [94] . there are no adverse side effects known for the proposed usage of chloroquine against covid-19 [95] . there was a sharp increase in the prescription of these drugs for covid-19 patients with the reference of usa president mr donald trump in late march in the us (https://www.bbc.com/news/51980731). furthermore, it was also granted by the food and drugs administration (fda) for the use of hydroxychloroquine and chloroquine drug as an emergency as authorized for covid-19 therapy. there are more than 20 clinical trials carried in the us, uk, china and spain on these drugs. the later reports from the clinical trials also suggested that chloroquine drug would minimise the disease duration [95] . bur, later on, april 24, the fda also licenced the usage of the drug issued under the warning regarding the dangerous threat for the serious heart rhythm adverse side effects in covid-19 patients when used. however, to get a complete clearance of virus, chloroquine is combined with other drugs. for instance, hydroxychloroquine combined with azithromycin resulted in greater clearance of virus than hydroxychloroquine alone [96] . the outbreak of sars-cov-2 is an ongoing global public health crisis. the expedition of clinical trials for various potential therapeutic drugs is essential to evaluate their efficacy at this midpoint of the pandemic. the pressure on researchers to develop promising therapeutic drugs for covid-19 is very high. these drugs include cytokines and bioengineered and vector-based antibodies to block the gene expression of the virus and to develop a vaccine. however, there are currently no therapeutic drugs available to treat covid-19. clinical symptoms caused by sars-cov-2 infection should be closely monitored to determine j o u r n a l p r e -p r o o f whether the virus has infected internal organs so that appropriate therapy can be performed. a patient's demographic data and past medical history are crucial to forming a good therapeutic plan. moreover, together, antiviral research and clinical trials will improve the effectiveness of therapy and facilitate the production of a drug or vaccine in record time. in this article we focused on the effect of covid-19 with other diseases (comorbidities) and associated organ injury to provide researchers with a better understanding of the clinical implications of covid-19. we also focused on the ongoing research and clinical trials conducted for the benefit of covid-19 patients to determine the efficacy of drugs for treatment. gpn involved in literature search, figures, and. study design, bd involved in literature search, and figures. bd and gpn drafted and finalized the paper. both authors read, and approved this review. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. none to declared his phd, both in biotechnology, from sri venkateswara university in tirupati, andhra pradesh, india. dr. nagaraju received his dsc from berhampur university in berhampur, odisha, india. dr. nagaraju's research focuses on translational projects related to gastrointestinal malignancies. he has published over 90 research papers in highly reputed international journals and has presented more than 50 abstracts at various national and international conferences. dr, nagaraju is author and editor of several published books. he serves as editorial board member of several internationally recognized academic journals. dr. nagaraju is an associate member of the discovery and developmental therapeutics research program at winship cancer institute. dr. nagaraju has received several international awards including faacc. he also holds memberships with the asioa, the sicb, the science advisory board, the rna society, the aacc and the aacr. injury. 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which is important for the sars-cov-2 infection key: cord-306581-g3d0lqxp authors: khattab, mohamed h.; sherry, alexander d.; jessop, aaron c.; ciombor, kristen k.; chakravarthy, bapsi title: early detection of sars-cov-2 from staging pet-ct date: 2020-09-29 journal: j radiat oncol doi: 10.1007/s13566-020-00436-w sha: doc_id: 306581 cord_uid: g3d0lqxp objective: sars-cov-2 infection may manifest with minimal or no clinical symptoms. however, signs of infection may appear on routine imaging obtained in the care of patients with cancer. the management of patients planned for chemoradiation with asymptomatic or mildly symptomatic sars-cov-2 infection is uncertain. methods: here, we present a case study of a mildly symptomatic patient with anal cancer diagnosed with sars-cov-2 from a staging pet-ct scan. results: pet-ct scan for anal cancer staging demonstrated pulmonary avidity suspicious for an infectious, rather than malignant, process. in the setting of these imaging findings and new-onset anosmia, viral polymerase chain reaction was ordered and found to be positive for sars-cov-2. to avoid myelosuppression in the setting of active infection, planned chemoradiation was delayed until cessation of viral shedding. conclusion: in the covid-19 era, oncologists obtaining routine staging imaging should have high diagnostic suspicion for subclinical sars-cov-2 infection. to avoid precipitating severe pneumonia and hospitalization, multidisciplinary discussion with risk-benefit analysis is recommended before initiating immunosuppressive therapies such as chemoradiation. a 61-year-old female with biopsy-proven anal squamous cell carcinoma was referred for staging positron emission tomography-computed tomography (pet-ct) to rule out metastatic disease, prior to planned definitive chemotherapy and radiation. her anal cancer had been detected on routine screening colonoscopy. she denied having any cough, fever, shortness of breath, or any gastrointestinal symptoms. she did recall a peculiar, subjective anosmia during the prior week, although she was without congestion or nasal obstruction. at presentation, she was largely asymptomatic and was unconcerned regarding her reduced ability to smell. p e t -c t f o r o n c o l o g i c s t a g i n g d e m o n s t r a t e d fluorodeoxyglucose-avid multifocal lower lung, rounded, peripheral ground-glass nodules within the right lower, right middle, and left lower lobes with several opacities demonstrating the reversed halo sign (fig. 1) . given the presence of the reversed halo sign, the differential diagnosis strongly suggested viral infection, although this is not diagnostic for covid-19 pneumonia and includes other non-specific viral infections as well as organizing pneumonia and eosinophilic pneumonia. in this patient with anal cancer, metastatic disease was also considered, even though ground-glass metastases are unusual for squamous cell carcinoma and these findings were not present on a previous ct obtained 6 weeks prior; moreover, ct correlate did not reveal any distant masses or concern for metastatic disease. the findings can be also present in drug pneumonitis, although no therapy had been initiated in this patient. given the ongoing covid-19 pandemic, a nasopharyngeal swab with polymerase chain reaction (pcr) was obtained and was confirmed positive for the potentially lethal sars-cov-2 viral infection. as the treatment for early-stage anal cancer includes fluorouracil and mitomycin-c chemotherapy with concurrent radiotherapy, a multidisciplinary discussion was held and recommended delaying oncologic treatment in order to prevent myelosuppression. the patient was quarantined with an uneventful recovery, although repeat nasopharyngeal swab with pcr demonstrated persistent sars-cov-2 positivity at 14 days. her anosmia resolved after a total of 2 weeks. after repeat pcr was negative, suggesting a lack of active detectable viral shedding, she initiated standard of care chemoradiation. sars-cov-2 is a potentially lethal viral infection, although many patients will be largely asymptomatic or have a vague sense of microsmia or anosmia as their only symptom [1] [2] [3] . oncologists should have a low threshold for diagnostic testing for sars-cov-2, especially in patients planned to undergo chemotherapy or radiation. subtle signs, such as decreased olfaction, should raise oncologists' suspicion for sars-cov-2 infection. furthermore, routine scans ordered by oncologists, such as staging pet-ct or even chest ct image-guidance for radiation, can suggest early-onset infection, and oncologists should remain vigilant when reviewing these images [4] . this raises the question of how oncologists should manage common incidental lung findings during a respiratory viral pandemic. ground-glass nodules are not specific for covid-19 pneumonia, although certain radiologic findings, such as the reversed halo sign, may raise the pre-test probability for viral pneumonia. caution should be taken prior to initiating cytotoxic or immunosuppressive treatments in sars-cov-2 confirmed cases, even in asymptomatic or mildly symptomatic patients, as oncologic treatments may adversely impact patient ability to combat the viral infection and even potentiate an acceleration and/or worsening of the infectious course [5] . therefore, in certain vulnerable patient populations, pcr testing prompted by non-specific pulmonary radiologic findings is likely indicated, especially when taken together with a thorough clinical review for signs and symptoms of covid-19 such as anosmia. even in the absence of clinical symptomatology, incidental radiologic findings alone should prompt pcr testing to verify the safety of anti-neoplastic treatments. in geographic regions with a fig. 1 screening positron emission tomography fused with computed tomography demonstrating fluorodeoxyglucose-avid multifocal, rounded, peripheral ground-glass nodules, some demonstrating the reversed halo sign, within the right lower, right middle, and left lower lung lobes concerning for an infectious process significant and increasing covid-19 case burden, routine pcr testing in the absence of clinical or radiologic findings may be indicated in patients undergoing chemoradiation or radiation, and it is our institutional practice to test all patients receiving any chemotherapy or greater than 10 days of radiation. in the setting of asymptomatic or mildly symptomatic patients with confirmed sars-cov-2 infection, multidisciplinary discussion with oncology and infectious disease teams is important to ascertain the risks and benefits of delaying cancer therapy. author contributions all authors contributed to the study conception and design, data collection, and interpretation. the first draft of the manuscript was written by mohamed h. khattab and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. data availability data from this study are maintained in an institutional repository and are available from the corresponding author upon reasonable request. conflict of interest the authors declare that they have no conflict of interest. ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. this study was exempt from irb review per vanderbilt university irb policy i.b.1. all identifiable patient information has been omitted, and the information contained in the manuscript is anonymized. consent to participation/publish the participant has consented to the submission of the case report to the journal. self-reported olfactory and taste disorders in sars-cov-2 patients: a cross-sectional study alterations in smell or taste in mildly symptomatic outpatients with sars-cov-2 infection temporal dynamics in viral shedding and transmissibility of covid-19 rapid detection of asymptomatic covid-19 by ct image-guidance for stereotactic ablative radiotherapy cancer patients in sars-cov-2 infection: a nationwide analysis in china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-289598-t8upoq9a authors: yoon, jane c; montgomery, martha p; buff, ann m; boyd, andrew t; jamison, calla; hernandez, alfonso; schmit, kristine; shah, sarita; ajoku, sophia; holland, david p; prieto, juliana; smith, sasha; swancutt, mark a; turner, kim; andrews, tom; flowers, kevin; wells, alyssa; marchman, cathryn; laney, emaline; bixler, danae; cavanaugh, sean; flowers, nicole; gaffga, nicholas; ko, jean y; paulin, heather n; weng, mark k; mosites, emily; morris, sapna bamrah title: covid-19 prevalence among people experiencing homelessness and homelessness service staff during early community transmission in atlanta, georgia, april–may 2020 date: 2020-09-08 journal: clin infect dis doi: 10.1093/cid/ciaa1340 sha: doc_id: 289598 cord_uid: t8upoq9a background: in response to reported covid-19 outbreaks among people experiencing homelessness (peh) in other u.s. cities, we conducted multiple, proactive, facility-wide testing events for peh living sheltered and unsheltered and homelessness service staff in atlanta, georgia. we describe sars-cov-2 prevalence and associated symptoms and review shelter infection prevention and control (ipc) policies methods: peh and staff were tested for sars-cov-2 by reverse transcription polymerase chain reaction (rt-pcr) during april 7–may 6, 2020. a subset of peh and staff was screened for symptoms. shelter assessments were conducted concurrently at a convenience sample of shelters using a standardized questionnaire results: overall, 2,875 individuals at 24 shelters and nine unsheltered outreach events underwent sars-cov-2 testing and 2,860 (99.5%) had conclusive test results. sars-cov-2 prevalence was 2.1% (36/1,684) among peh living sheltered, 0.5% (3/628) among peh living unsheltered, and 1.3% (7/548) among staff. reporting fever, cough, or shortness of breath in the last week during symptom screening was 14% sensitive and 89% specific for identifying covid-19 cases compared with rt-pcr. prevalence by shelter ranged 0%–27.6%. repeat testing 3–4 weeks later at four shelters documented decreased sars-cov-2 prevalence (0%–3.9%). nine of 24 shelters completed shelter assessments and implemented ipc measures as part of the covid-19 response conclusions: peh living in shelters experienced higher sars-cov-2 prevalence compared with peh living unsheltered. facility-wide testing in congregate settings allowed for identification and isolation of covid-19 cases and is an important strategy to interrupt sars-cov-2 transmission in 2019, approximately 570,000 people experienced homelessness on any given night in the united states (u.s.), and 63% used congregate shelters [1] . in atlanta, georgia, an estimated 3, 200 people experienced homelessness on any given night in 2019, and approximately one-quarter were living unsheltered (i.e., living in a place not meant for human habitation) [2] . risk of sars-cov-2 infection, the virus that causes covid-19, may be higher among people experiencing homelessness (peh) because of challenges in preventing respiratory disease transmission in congregate shelter settings. peh might also be at increased risk of severe covid-19 if infected due to a high prevalence of untreated, chronic medical conditions and obstacles to accessing healthcare [3] [4] [5] [6] [7] [8] . fulton county, the largest county in georgia, which includes 90% of the city of atlanta, reported the first covid-19 case on march 2, 2020. a sharp increase in cases was recorded in mid-april 2020. a door-to-door household survey conducted in fulton and neighboring dekalb counties during april 28-may 3, 2020, found an estimated 2.5% seroprevalence of sars-cov-2 antibodies [9] . reports of high sars-cov-2 infection rates and outbreaks within shelters in other metropolitan areas, in parallel with increasing local case-rates, led to concerns for widespread transmission in atlanta shelters [10] [11] [12] . to understand sars-cov-2 prevalence and prevent transmission among peh in atlanta, homeless service agencies partnered with local and federal government agencies to: (1) determine sars-cov-2 prevalence among clients living sheltered and unsheltered and homelessness service staff through viral testing; (2) a c c e p t e d m a n u s c r i p t 6 methods participants included clients living in shelters, clients living unsheltered, and staff in atlanta, georgia, during april 7-may 6, 2020. testing at homeless shelters was offered facility-wide to all clients and staff. testing was offered to clients living unsheltered during homeless outreach service events (e.g., meal services). participation was voluntary but encouraged by service agencies. written consent was obtained from each adult (≥16 years of age) or parent or guardian (for children <16 years) for administration of a brief, standardized screening questionnaire and testing for sars-cov-2 (see supplemental material #1). all screening interviews and specimen collections were conducted on-site at shelters or community events serving peh. at shelters with more than five people with positive sars-cov-2 results upon initial testing, clients and staff were re-screened and re-tested 3-4 weeks later, and testing was also offered to any new clients or staff. all participants (including parents or guardians of children <16 years) self-reported their sex, race, and ethnicity based on fixed-response categories. race and ethnicity were combined into mutually exclusive categories and were considered missing when race was missing and ethnicity was either non-hispanic or missing. participants from a convenience sample of testing events were interviewed using the screening questionnaire to collect information on symptoms, underlying medical conditions, pregnancy status, and tobacco use. persons interviewed were asked if they had any medical conditions in the following categories: diabetes, cardiovascular disease, chronic lung, kidney, and liver disease, immunocompromising conditions (e.g., hiv, chronic steroid use), and neurological conditions (e.g., seizure disorder). nasopharyngeal, oropharyngeal, or nasal mid-turbinate specimens were collected by clinical providers or supervised self-collection and tested by contracted commercial laboratories for sars-cov-2 by reverse transcription polymerase chain reaction (rt-pcr). positive sars-cov-2 test results were provided directly to the person (or parent or guardian for children <16 years) or to the shelter via the county public health department's standard notification procedure. clients living in shelters who had positive sars-cov-2 were isolated immediately in a a c c e p t e d m a n u s c r i p t 7 separate housing unit at the shelter or isolated until transported to an isolation hotel. for clients living unsheltered, results were provided via a clinic hotline number or clinical outreach teams, which located the clients with positive sars-cov-2 and arranged transport to the isolation hotel. staff with positive sars-cov-2 isolated at home. clients were not allowed to return to shared spaces in shelters, and isolated staff did not return to work until they met symptom-or time-based criteria in accordance with the centers for disease control and prevention (cdc) guidelines at the time [13] . shelter assessments were conducted in conjunction with screening and testing at a convenience sample of shelters. these shelters were selected based on availability of testing staff on the day of testing. using a standardized assessment questionnaire (see supplemental materials #2), shelter management was interviewed to collect quantitative and qualitative data on shelter characteristics and services offered. shelter characteristics included number of clients and staff, type (e.g., daytime only, 24 hours per day, transitional housing), services provided, and sleeping space configurations. measures and policies implemented by the shelter to mitigate sars-cov-2 transmission, including standardized client and staff screening, isolation and quarantine protocols, and ipc, were also collected. shelter staff were counseled on best practices to prevent transmission in the shelter. descriptive statistics were used to characterize the population tested and the proportions with current and recent symptoms, underlying medical conditions, and positive sars-cov-2. continuous variables were compared using student's t-test, and categorical variables were compared using the chi-square test. shelter characteristics were described in aggregate but were not analyzed in relation to sars-cov-2 prevalence due to small numbers. cdc determined this project to be non-research as demographic characteristics for tested participants are included in table 1 a c c e p t e d m a n u s c r i p t 9 the same subset of 1,997 people was screened for symptoms; 12 with inconclusive results were subsequently excluded (table 3) in one shelter housing adult men, testing revealed a widespread, undetected outbreak. this shelter represented 1% (29/2,050) of people tested on the initial round but 20% (8/40) of those with positive sars-cov-2. client census had been decreased by approximately half to facilitate physical distancing, but the shelter did not enforce city or state shelter-in-place orders nor restrict client movement. the shelter only had congregate sleeping spaces; sleeping mats and beds were placed at least six feet apart, but head-to-toe alignment was inconsistently used. showers upon entry were encouraged, but face coverings and hand sanitizer were not provided due to insufficient supply. household cleaning solutions and environmental protection agency-registered disinfectants appropriate for cleaning high-touch surfaces or items were also not available [15, 16] . 70% of all peh in atlanta based on the 2019 point-in-time count in atlanta [2] . overall sars-cov-2 prevalence among peh and staff tested in atlanta from april to may 2020 was low compared with reports among peh in other large, urban settings [10] [11] [12] . although sars-cov-2 prevalence among clients living in shelters was only 2.1%, it was four times higher than the 0.5% prevalence among a c c e p t e d m a n u s c r i p t 11 peh living unsheltered at the time. to our knowledge, this is the first report of sars-cov-2 prevalence in a population of peh living unsheltered. although the risk of sars-cov-2 was lower for peh living unsheltered, unsheltered living situations pose an increased risk of morbidity and mortality compared with living sheltered, so linkages to permanent supportive housing should remain a priority [17] . testing at shelters in other large, urban settings in the united states has primarily occurred in response to covid-19 clusters or outbreaks [10] [11] [12] . in early april 2020, baggett et al. investigated an outbreak in a large homeless shelter in boston with a sars-cov-2 detection rate of 36% [10] . during an outbreak across three affiliated shelters in seattle from march-april 2020, 18% of clients and 21% of staff had positive sars-cov-2 [11] . in atlanta, universal screening and testing of peh and staff was a proactive strategy, rather than in response to known covid-19 cases. a recent study of long-term care facilities in fulton county, georgia, found 1.5% of residents had positive sars-cov-2 when testing was proactive, compared to 47.2% after a case was already diagnosed [18] . only one shelter we tested was found to have a widespread, undetected outbreak. on march 23, 2020, 3 weeks after the first covid-19 case was identified in atlanta, the city implemented a 14-day shelterin-place order, which was followed by a georgia state-wide shelter-in-place order during april 3-30, 2020. over half of shelters assessed stopped taking clients during these shelter-in-place orders, and all reported a lower-than-average number of clients, which helped facilitate physical distancing. the low sars-cov-2 prevalence among peh and staff in atlanta compared with other cities may reflect the impact of shelter-in-place orders coupled with low community prevalence at the time of testing and the proactive testing strategy. [19, 20] . although the specificity was higher (81%-89%), screening would produce a high proportion of false positive results in low prevalence settings. most shelters assessed were conducting standardized symptom screening for clients, but only one-third were screening staff. despite the limitations of symptom screening, cdc recommends that homeless service providers regularly assess both clients and staff for symptoms using a standardized protocol [21] . people with covid-19 symptoms should immediately be provided with a face covering, isolated, and tested for sars-cov-2. shelters, in coordination with local public health authorities and community coalitions, should have plans to isolate people with suspected or confirmed sars-cov-2 infection to prevent spread [21] . however, because an estimated 40-45% of people with sars-cov-2 are asymptomatic, shelters should reduce the number of people served or expand to alternative housing sites, increase physical distancing (i.e., >6 feet), and mandate use of face coverings by all staff and clients inside shelters except when in bed or individual rooms [21, 22] . our finding of decreased prevalence in four shelters during repeat testing is consistent with reports from skilled nursing facilities and correctional facilities, supporting the use of universal (facility-wide) testing for early identification and isolation of those with positive sars-cov-2 as a strategy to interrupt transmission in congregate settings [23] [24] [25] . a potentially important factor in the observed decreased prevalence in these shelters was the ability to move all peh identified with sars-cov-2 infections to separate housing units at the shelter or to offsite locations. in homeless shelters and encampments located in areas where sars-cov-2 community transmission is substantial, cdc recommends that initial (baseline) and regular testing be considered, regardless of whether an initial covid-19 case has been identified [26] . in areas where community transmission is minimal to moderate, cdc provides examples of testing strategies that can be used to identify asymptomatic cases among both clients and staff, such as sentinel surveillance, positive symptom screening thresholds, or random testing (e.g., every third person) on a regular basis [26] . if a covid-19 case is identified, cdc recommends repeat testing of all previously negative or untested individuals until testing identifies no new covid-19 cases for at least 14 days [26] . a c c e p t e d m a n u s c r i p t 13 our findings are subject to several limitations. sars-cov-2 transmission dynamics are complex, and these results represent a single point in time early in the covid-19 pandemic. we did not screen or test all peh or shelters in atlanta; therefore, the results might not be representative of all peh or shelters; they are not generalizable to other large metropolitan areas. we cannot compare this prevalence to the general population of fulton county during this time period because representative testing had not been conducted among the general population. additionally, misclassification of sheltered and unsheltered housing status might have resulted from movement between settings and the difficulty of verifying unsheltered status. due to shortages of nasopharyngeal swabs, three specimen collection methods were used. specimen collection methods have different sars-cov-2 detection sensitivities; however, nasal and nasal midturbinate specimens were shown to have >90% sensitivity compared with nasopharyngeal samples [27] . symptom and medical condition screenings were only conducted at a subset of testing events, which oversampled the larger shelters and may not be generalizable. shelter clients might not have disclosed symptoms due to fears that they would be removed from shelters. thus, the proportion of people who reported symptoms might be underestimated. only nine shelters of 24 underwent facility assessments, including two shelters at which assessments were conducted after initial testing. these findings may reflect improved or modified ipc policies as a result of knowledge of positive cases at these two shelters. the findings provide an early view into the effects of the covid-19 pandemic on peh and homelessness service staff in atlanta. as evidence grows and guidance evolves during the covid-19 pandemic, shelters should prioritize mitigation strategies and best practices for congregate and highrisk settings to prevent sars-cov-2 transmission while continuing to provide essential services for a c c e p t e d m a n u s c r i p t 14 m a n u s c r i p t m a n u s c r i p t 19 a c c e p t e d m a n u s c r i p t 21 a c c e p t e d m a n u s c r i p t 22 the 2019 annual homeless assessment report (ahar) to congress. part 1: point-in-time estimates of homelessness state of homelessness: atlanta continuum of care america/homelessness-statistics/state-of-homelessness-dashboards/?state=georgia. accessed 29 preventing and controlling emerging and reemerging transmissible diseases in the homeless morbidity and mortality in homeless individuals, prisoners, sex workers, and individuals with substance use disorders in high-income countries: a systematic review and meta-analysis chronic disease burden of the homeless: a descriptive study of student-run free clinics in tampa, florida cardiovascular disease and homelessness out-of-hospital and emergency department utilization by adult homeless patients barriers to homeless persons acquiring health insurance through the affordable care act estimated community seroprevalence of sars-cov-2 antibodies -two georgia counties prevalence of sars-cov-2 infection in residents of a large homeless shelter in boston covid-19 outbreak among three affiliated homeless service sites assessment of sars-cov-2 infection prevalence in homeless shelters -four u.s. cities discontinuation of isolation for persons with covid -19 not in healthcare settings outbreak of drug-resistant mycobacterium tuberculosis among homeless people in list n: disinfectants for use against sars-cov-2 (covid-19) unsheltered status, and risk factors for mortality mass screening for sars-cov-2 infection among residents and staff in twenty-eight long-term care facilities in fulton county, georgia. medrxiv symptom screening at illness onset of health care personnel with sars-cov-2 infection in king county estimated effectiveness of symptom and risk screening to prevent the spread of covid-19 interim guidance for homeless service providers to plan and respond to coronavirus disease 2019 (covid-19) universal and serial laboratory testing for sars-cov-2 at a long-term care skilled nursing facility for veterans initial and repeated point prevalence surveys to inform sars-cov-2 infection prevention in 26 skilled nursing facilities -detroit serial laboratory testing for sars-cov-2 infection among incarcerated and detained persons in a correctional and detention facility -louisiana interim considerations for health departments for sars-cov-2 testing in homeless shelters and encampments swabs collected by patients or health care workers for sars-cov-2 testing we would like to thank anitra walker (mercy care), catherine christie (mercy key: cord-294831-pem059zk authors: zhang, ling-pu; wang, meixian; wang, yanping; zhu, jun; zhang, nannan title: focus on a 2019-novel coronavirus (sars-cov-2) date: 2020-06-11 journal: future microbiology doi: 10.2217/fmb-2020-0063 sha: doc_id: 294831 cord_uid: pem059zk a new coronavirus, severe acute respiratory syndrome coronavirus 2, was first discovered in wuhan, china, in december 2019. as of april 7, 2020, the new coronavirus has spread quickly to 184 countries and aroused the attention of the entire world. no targeted drugs have yet been available for intervention and treatment of this virus. the sharing of academic information is crucial to risk assessment and control activities in outbreak countries. in this review, we summarize the epidemiological, genetic and clinical characteristics of the virus as well as laboratory testing and treatments to understand the nature of the virus. we hope this review will be helpful to prevent viral infections in outbreak countries and regions. clinical symptoms fever (98%), sough (77%), dyspnea (63.5%), myalgia (11.5%), malaise (35%) and so on fever (ͼ99%), cough (62%-100%),chills or rigor (15%-73%), diarrhea 20%, dyspnea (40%) fever (77%), cough (90%), dyspnea (68%), sputum production (40%), odynophagia (39%), digestive system /signs (20%), hemoptysis (4.3%), myalgia (43%) and headache (20%) [53, 69, 85] radiology critically ill patients with bilateral multiple lobular and subsegmental areas of consolidation; mild patients with bilateral ground-glass opacity and subsegmental areas of consolidation almost 100% patients with abnormal ct unilateral/bilateral ground-glass opacities or focal unilateral/bilateral consolidation. chest radiography or ct abnormal rate was ͼ94% unilateral/bilateral patchy densities or infiltrates, bilateral hilar infiltration, segmented/lobar opacities, ground-glass opacities and possible small pleural effusions. chest radiography or ct abnormal rate was between 90% to 100% was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the international committee on taxonomy of viruses on 11 february 2020 [1] . the disease caused by sars-cov-2 was named covid-19 by the world health organization (who). on 30 january 2020, the who declared the outbreak of covid-19 to be a global health emergency and further labeled it a pandemic on 11 march 2020. the virus can be transmitted not only from animals to humans but also from humans to humans [23] . a lancet report demonstrated that the virus could have recently acquired the ability to transmit between humans [6] . a report of five patients in a family cluster who traveled to wuhan and were infected with sars-cov-2 was the first report directly illustrating that the virus is capable of person-to-person transmission in hospital and family settings [23] . sars-cov-2 can spread via direct contact and respiratory droplets. respiratory particles are spread while breathing, speaking, coughing or sneezing [24] . in addition, aerosol and fomite transfer may promote transmission of the virus according to a study in the new england journal of medicine [25] . aerosolized virus may be generated by respiratory and surgical procedures. the study showed that the half-life of the virus is about 1.1-1.2 h, and it remained viable for 3 h in aerosols. meanwhile, the virus on plastic, stainless steel, copper and cardboard remained stable for 4-72 h [25] . these results indicate that aerosol or fomites may be able to spread sars-cov-2 [25] . a fluid-resistant (type-r) surgical face mask is used to protect against droplets. fecal-oral transmission may also play an important role in sars-cov-2 spread [26] . xiao and colleagues showed that 53.42% of 73 hospitalized covid-19 patients had sars-cov-2 rna in stool specimens, and the duration time of positive stool results ranged from 1 to 12 days [27] . in addition, viral nucleic acid in 64.29% patients remained positive in the feces after sars-cov-2 rna in pharyngeal swabs turned negative. furthermore, positive sars-cov-2 rna in stool specimens was not associated with gastrointestinal symptoms [28] . together, these findings indicate the possibility of sars-cov-2 via the fecal-oral transmission. the basic reproduction number (r0) of this virus reflects the dynamics of transmission during this coronavirus outbreak. the who has estimated that sars-cov-2 has a reproduction number of 1.4-2.5. however, a recent study shows the average r0 to be 3.28 (median: 2.79; interquartile range [iqr]: 1.16), an r0 considerably higher than the who estimate at 1.95 [29] . in an earlier phase of the outbreak, li et al. reported that the mean incubation period of the virus is 5.2 days (95% ci: 4.1-7.0), the epidemic doubled in size every 7.4 days, and the r0 was estimated to be 2.2 (95% ci: 1.4-3.9) [30] . zhao and collaborators estimated an r0 ranged from 2.24 (95% ci: 2.49-2.63) to 3.58 (95% ci: 2.89-4.39) [31] . in another study, r0 was computed to oscillate between 3.30 (95% ci: 2.73-3.96) and 5.47 (95% ci: 4.16-7.10) [32] . in addition, the study reported a transmission rate within wuhan of 1.94 days (95% ci: 1.25-6.71), an infectious period of 1.61 days (95% ci: 0.35-3.23) and an r0 value of 3.11 (95% ci: 2.39-4.31) [32] . based on the nowcasting and forecasting approach, wu et al. showed an estimated reproduction number of 2.68 (95% ci: 2.47-2.86), and an epidemic doubling time of 6.4 days [33] . recently, tang et al. calculated the r0 as 6.47 (95% ci: 5.71-7.23), using mathematical seir-type epidemiological model [34] . nevertheless, sars-cov-2 has demonstrated a higher transmission rate than that of sars-cov and mers-cov. variation in viral transmissibility should be considered, and estimates of the reproduction number may change in the future. sars-cov-2 is similar to sars-cov. both single-stranded rna viruses share 82% nucleotide identity, and sars-cov-2 shares 89% identity with sars-like covzxc21 [35] . the genome of sars-cov-2 has 29,891 nucleotides encoding 9,860 amino acids. genetically, sars-cov-2 is similar to sars-cov (about 79%) and mers-cov (about 50%) [36] . the virus contains a replicase, spike (s) protein, envelope (e) protein, membrane (m) protein and nucleocapsid [35] (figure 1a & b) . however, sars-cov-2 lacks the hemagglutinin-esterase gene, which is found in lineage a β-covs. sars-cov-2 has 12 open reading frames (orfs) encoded by nine subgenomic mrnas that carry nine transcription regulatory sequences, two terminal untranslated regions (utr) and a conserved leader sequence [37] . the large replicase polyprotein pp1a contains ten nonstructural proteins (nsp1-nsp10), and pp1ab contains 15 nonstructural proteins (nsp1-nsp10, nsp12-nsp16), which are all encoded by orf1a and orf1ab [38] (figure 1a ). with the exception of nsp3 and nsp5, which are cysteine proteases, most nonstructural proteins play an important role in the transcription and replication of sars-cov-2 [39] . pp1ab has different lengths in covid-19, sars-cov and mers-cov of 29,844 bp (7096 aa), 29,751 bp (7073 aa) and 30,119 bp (7078 aa), respectively ( figure 1a) . furthermore, there is no obvious difference between sars-cov-2and sars-cov nonstructural proteins and orfs. the spike glycoprotein plays an important role in binding to receptors on host cells and, therefore, is involved in host tropism [40] . sars-cov-2, sars-cov and mers-cov have s proteins containing 1,273, 1,255 and 1,270 aa, respectively ( figure 1a ) [36] . the s protein mediates entrance into human respiratory epithelial cells by interacting with the cell-surface receptor angiotensin-converting enzyme 2 (ace2) [35, 41] . it is comprised of s1 and s2 subunits ( figure 1c ), and the s1 subunit shares approximately 70% identity with that of human sars-cov and bat sars-like covs (sl-covzxc21 and zc45). the s1 subunit has an n-terminal domain and a receptorbinding domain (rbd) that are both responsible for the binding of virions to host cells [16] . both sars-cov-2 and sars-cov bind to ace2 through the c-terminal domains (ctd) of their s1 subunits, and mers-cov utilizes the ctd to bind proteinaceous dipeptidyl peptidase 4 (dpp4) [42] . the rbd of sars-cov-2 has 73% identity to that of sars-cov [43] . the transmissibility of the sars-cov-2 virus is greater than that of sars-cov, which may be because the rbd of sars-cov-2 is slightly different from that of sars-cov. the s2 subunit shares 99% identity with two bat sars-like covs and human sars-covs [38] . the s2 subunit contains a fusion peptide (fp) and heptad repeats (hrs) 1 and 2 ( figure 1c ). after the s1 rbd binds to the ace receptor on the host cell, the fp of s2 is inserted into the host cell membrane, and then hr1 and hr2 form a six-helix bundle (6-hb), which helps the virus fuse with host cell membranes [16, 44] . sars-cov-2 envelope (e) protein, matrix protein, accessory proteins p6 and p8, nonstructural protein 7 (nsp7), and nsp13 are homologous with those of sars virus [39] . therefore, the sars-cov-2 virus has a high level of identity with sars-cov. this suggests that an anti-sars-cov antibody, which could cross-react with the sars-cov-2 s protein, may be useful to treat patients with the virus. sars-cov-2 infection has caused clusters of severe respiratory illness similar to that of sars-cov. the virus causes symptoms such as fever, cough, shortness of breath, leukopenia and pneumonia in both lungs [6] . the symptoms are observed approximately 5.2 days after the sars-cov-2 infection [5] . in a study published in the lancet, 41 of 41 patients who were identified as positive for sars-cov-2 infection presented with pneumonia and abnormal chest computed tomography (ct) [6] . covid-19 symptoms included fever (98%), cough (76%) and myalgia or fatigue (44%). less common symptoms such as sputum production (28%), headache (8%), hemoptysis (5%) and diarrhea (3%), were also observed [6] . another clinical study containing 138 patients showed that common symptoms were fever, fatigue, dry cough, lymphopenia, prolonged prothrombin time and an increased lactate dehydrogenase level (table 1 & figure 2 ) [5] . common complications included shock, acute respiratory distress syndrome, acute renal injury, acute liver failure, arrhythmia, rnaaemia and acute cardiac injury [45] . in addition, it is now understood that sars-cov-2 can infect children as well as adults [46] . one study showed that during the early infection period, most patients had normal white blood cell counts; however, 56.8% of patients had leukopenia in cases of serious infection [46] . patients with dyspnea were more frequently admitted to the intensive care unit (icu) [46] . most chest computed tomography (ct) showed bilateral patchy shadows or ground-glass opacity (ggo) in the lungs [5, 6] . in another study, 86% of patients showed ggo by chest ct, and 29% of patients had consolidation [47] . following the appearance of ggo, 75% of patient lung cts showed reticular or interlobular septal thickening [48] . however, no direct cavitation, pleural effusion, lymphadenopathy or nodules were observed in the lungs of covid-19 patients [49] (table 1 ). in view of the large amounts of cytokines produced during sars-cov infection, infection with sars-cov-2 similarly induces the production of proinflammatory cytokines such as, interleukin 1 beta (il-1β), interferon gamma (ifn-γ), ip10 and monocyte chemoattractant protein 1 (mcp). moreover, the levels of granulocyte colony stimulating factor (gcsf), ip10, mcp1, mip1α and tumor necrosis factor-alpha (tnf-α) were found to be higher in intensive care unit (icu) than non-icu patients [50] . however, secretion of immunosuppressive cytokines (e.g.,interleukin 4 [il-4] and interleukin 10 [il-10] by t-helper type 2 [th2] cells was also increased during sars-cov-2 infection [6] ) ( table 1) . the mortality rate of sars-cov-2-infected patients varies in different studies. a study of 41 patients showed a mortality rate of 15% [6] , and the fatality of 138 patients in a clinical study was 4.3% [5] . however, chen showed that the mortality was 3%, a number closer to the official national statistics of china (2.01%) [51] . these differences in mortality rates are possibly due to the difference in sample size. the reported mortality rate of sars-cov was 10%, and that of mers-cov was about 36% [11] . the currently estimated mortality rate of sars-cov-2 is therefore lower than that of sars-cov and mers-cov [51, 52] (table 1 ). in summary, sars-cov-2 spreads more rapidly but has a relatively lower fatality rate as compared with two other related coronaviruses. the number of total leukocytes, lymphocytes and monocytes has been detected from hospitalized patients with covid-19 [53] . approximately 25% of cases formed leucopenia [6] . moreover, lymphopenia was observed in 63-70.3% of patients [5, 6] . cd4 + or cd8 + t-cell numbers decreased as the disease severity increased [6, 54] . patients with severe cases had more prominent abnormalities than those with non-severe cases. patients infected with covid-19 had some unique clinical features including rhinorrhoea, sneezing, sore throat. most patients had haemoptysis dyspnea, fever, headache, fatigue, sputum production, pneumonia and ground-glass opacities. however, only a low percentage of patients developed intestinal symptoms such as diarrhea and vomiting. leucopenia, lymphopenia, pro-inflammation cytokines increasing, acute respiratory distress syndrome and acute organs damages (such as cardiac, liver or kidney) were common features in some intensive care unit patients. virus from throat swabs, blood, urine, stool or respiratory tracts have been assessed by fluorescent reverse transcription-polymerase chain reaction (rt-pcr) methods [55] . primers and probes targeting rdrp/helicase (hel), e, s, n and replicase orf 1a/b genes were designed and tested for sars-cov-2 [55] . rt-pcr showed that primers of the e and rdrp genes demonstrated better sensitivity than that of the n gene, and the limit of detection (lod) of rdrp/hel and n gene was lower than that of s gene and rdrp-p2 [18] . chan et al. showed that the covid-19-rdrp/hel assay (rdrp/hel probe, fam-ttaagatgtggtgcttgcatacgtagac-labkfq) was significantly more sensitive than the rdrp-p2 assay for the detection of sars-cov-2 rna, and detection targeted orf1a/b, orf1b-nsp14, rdrp, s, e or n genes was with less specificity for sars-cov-2 [18] . moreover, a previous study showed that the specific probe rdrp sarsr-p2 (fam-caggtggaacctcatcaggagatgc-bbq) detected only the sars-cov-2 rna transcript but not the sars-cov rna [55] . however, a novel rt-pcr assay showed that targeting rdrp2 was a non-specific assay for sars-cov-2, because this detected other betacoronaviruses such as sars-cov [18] . the covid-19-rdrp/hel assay had the lowest lod in vitro and higher sensitivity and specificity, which helped to reduce the false-negative rate and improve the laboratory diagnosis of covid-19 [18] . sars-cov-2 viral load is detectable from throat-and lung-derived samples; however, blood and urine have not yet yielded virus [56] . patients with covid-19 produced the highest viral load near symptom presentation, which may be the reason for the fast spread of the virus [57] . an observational cohort showed that viral load in saliva was highest following symptom onset in the first week, then gradually declined with time [56] . endotracheal aspirate viral load was available from day 8 after symptom onset and did not significantly decline thereafter [57] . following symptom onset, virus load in 33% patients could be detected for 20 days or longer in that study. viral load in respiratory tract specimens was about sixfold higher than that in the nonrespiratory tract specimens [57] . in addition, elder patients reportedly had a greater virus load than that of younger patients. studies showed that higher initial viral load was related to the severity of covid-19 symptoms [57] . although the positive test ratio was only 47.4% in previous study, it was improved by novel assay methods [58] . the sample quality, collection time, detection kits and technical abilities of clinical doctors may affect the accuracy of detection. therefore, precise diagnosis of covid-19 should be combined with ct scans and nucleic acid testing. bronchoalveolar lavage fluid or throat swabs from patients have been sequenced, and viral genomes were searched via blast with the sars-cov-2 sequence [38, 39] . genome sequencing has also been used to identify patients with suspected infection. in addition to hematological detection and nucleic acid testing, serological diagnosis is important for patients who present late with a very low viral load, below the detection limit of rt-pcr assays [59] . igm and igg titers were relatively increased on day 5 and rapidly raised 10 days after symptom onset in most patients [59, 60] . the igm-positive rate increased from 50 to 94%, whereas the igg-positive rate increased from 81 to 100% [60] . in addition, the igm-and igg-positive rates were not significantly different before and after patients were found to be virally negative [61] . therefore, virus-specific igm and igg serological testing may be used to confirm current or previous infection with sars-cov-2. an ideal animal model for covid-19 would reflect the clinical signs, viral replication and pathology displayed in humans. non-human primate models (rhesus, cynomolgus macaques, african green monkeys, common marmoset, squirrel monkeys and mustached tamarins) have all been evaluated as models of sars-cov infection [62] . all non-human primates had pneumonia, cough and respiratory distress after virus infection [15] . mice (balb/c, c57bl6 and 129s strains) supported sars-cov replication and showed clinical signs of sars [15] . rag1 −/− mice, cd1 −/− mice, stat1 −/− mice and beige mice have been used to determine the role of immune effectors of the virus [63] . ac70 and ac63 transgene-positive mice also showed clinical manifestation after sars-cov infection, demonstrating their usefulness to study the pathogenesis and evaluation of vaccines and other therapeutics [63] . hamsters also have been used to study immuno-prophylaxis and drug research as they harbored high viral titers and pulmonary histopathology upon virus infection [63] . ferrets are another appropriate animal model to study this respiratory virus because the clinical symptoms, viral titers and histologic changes were similar to those of patients with virus infection [63] . ace2, the receptor of sars-cov, was also identified as the functional receptor for sars-cov-2, therefore, mice, hamsters and ferrets may be animal models for studying the sars-cov-2 [63] . an article reported in science shows that sars-cov-2 can replicate in the upper respiratory tract of ferrets, indicating that ferrets represent an ideal animal model for evaluating antiviral drugs or vaccine candidates against covid-19 [64] . in addition, the domestic cat has shown multifocal pulmonary consolidation with infection of sars-cov, and sars-cov-2 can replicate efficiently in cats and transmit between cats via the airborne route [64] . by contrast, dogs, pigs, chickens and ducks are poorly susceptible to sars-cov-2 [64] . no specific therapeutic medicine has been approved for the treatment of sars-cov-2 infection. all patients are given empirical antibiotic or antiviral drugs. moxifloxacin or levofloxacin are empirically used to treat early coinfections with bacteria [65] . linezolid is effective against streptococcus pneumoniae and staphylococcus aureus and is combined with nemonoxacin in cases of severe infection [65] . a combination of lopinavir and ritonavir has been used to treat covid-19 because the lopinavir/ritonavir (lpv/r) combination has been confirmed to be effective against sars-cov and mers-cov [6, 66, 67] . another antiviral drug, remdesivir (rdv), was predicted to be efficacious against covid-19 by target-based virtual ligand screening [68] . rdv is a novel nucleotide analog prodrug that is in development and has demonstrated effective pan-cov therapy. the first case of sars-cov-2 infection in the usa was successfully treated with rda. moreover, a randomized, double-blind, parallel-controlled phase iii clinical trial was conducted to recruit sars-cov-2-infected patients [4, 69] . favipiravir is a nucleoside analog that can lead to lethal viral mutagenesis, chain termination or the inhibition of nucleotide biosynthesis [70] . favipiravir in combination with oseltamivir, which was given to patients infected with sars-cov-2, has been used to treat severe influenza [45] . oseltamivir, a neuraminidase inhibitor, is recommended as an antiviral treatment for influenza and has been widely used to inhibit covid-19 in china [45, 70] . other neuraminidase inhibitors, zanamivir and peramivir, are also effective treatments for mers-cov [71] . at this time, these are all empirical therapies for covid-19. however, whether oseltamivir or zanamivir are effective treatments for covid-19 also needs further study. arbidol (arb) was licensed for the treatment of influenza and other respiratory viral infections in russia and china [72, 73] . blaising and coauthors consider arb to be a broad spectrum antiviral drug [72] . in addition, arb has been reported to inhibit sars-cov in vitro. therefore, a clinical trial of arb-treated covid-19-positive patients has been registered [65] . glucocorticoids have been commonly used in patients with sars-cov or mers-cov infection [74, 75] . glucocorticoids prolonged the survival time of sars cases [75] . patients with covid-19 were given glucocorticoids in some hospitals in china. however, the mortality rate did not decrease with corticosteroid treatment in patients infected with sars-cov-2, and viral clearance was not delayed [6, 74] . therefore, it is still controversial whether corticosteroids should be used to treat sars-cov-2 infections [69] . chloroquine/hydroxychloroquine was used as an antimalarial, broad spectrum antiviral drug, and has been broadly used in autoimmune diseases including lupus and rheumatoid arthritis [76] . a recent study indicates that chloroquine and the antiviral drug rdv-inhibited sars-cov-2 in vitro [77] . clinical symptoms of patients treated with chloroquine were obviously relieved: for example, more rapid decline in fever and improvement of lung ct [77] . chloroquine was suggested to treat covid-19 in the sars-cov-2 treatment guidelines by the chinese medical advisory. chloroquine has been highly effective in reducing sars-cov-2 viral replication by increasing endosomal ph and interfering with the glycosylation of cellular receptors [76] . moreover, chloroquine was probably the first molecule to be used to treat covid-19. another antiviral drug, nelfinavir, an hiv protease inhibitor, was predicted to be a potential inhibitor of covid-19 [26] . in addition, cytokine immunotherapy with ifn-α, a broad spectrum antiviral drug, has been used to inhibit hbv. ifn-α was used to treat patients infected with sars-cov-2 according to established guidelines [70] . ifn-α combined with lpv/r was shown to be beneficial for treatment of covid-19 [78] . tocilizumab is a humanized anti-il-6-receptor (il-6r) monoclonal antibody that inhibits il-6 signaling and is used as a treatment in rheumatoid arthritis [79] . il-6 is one of the most important cytokines involved in covid-19-induced cytokine storms. tocilizumab (tcz) has been used to treat covid-19 in china and italy. tcz was recommended for covid-19 patients to prevent or treat cytokine storms and could reduce the mortality of covid-19 [80] . other drug types such as rna synthesis inhibitors (tdf and 3tc) and an fp (ek1), have also effectively inhibited sars-cov-2 in vitro. in addition, traditional chinese medicines (lian hua qing wen capsules, shu feng jie du capsules) have also been used to treat covid-19 in the latest version of the diagnosis and treatment of pneumonia induced by covid-19 [81] . in addition, human seroalbumin and γ-immunoglobulin were given to some patients with severe infections [82] . in conclusion, there are specific vaccines or antiviral drugs for covid-19. all of the drugs described above have shown some usefulness in treating sars-cov-2 infections, and their efficacy merits further study. covid-19 is a serious human infectious disease of global concern. as of 7 april 2020, sars-cov-2 has infected a total of 134,784 patients globally at least ( figure 3d & g) . the covid-19 outbreak poses a serious challenge to china and the whole world; it has profoundly affected public health. although the overall mortality rate of sars-cov-2 appears to be lower than that of sars-cov and mers-cov (table 1) , the transmissibility of covid-19 is more rapid. moreover, the fatality rate of elderly patients with reduced immunity or chronic diseases is as high as 15% [6] . in addition, asymptomatic carriers may be a potential source of infection, sustaining a local 10 epidemic and global spread [83] . therefore, covid-19 may cause disruptions to the global public health system for an extended period of time. the outbreak of covid-19 has shown that there are still shortcomings in the prevention of public health diseases such as the lack of awareness of the frontline doctors and the early vigilance and attention from the government. meanwhile, the awareness of the general public to health concerns also must be improved. individuals should develop good living habits: for example, keeping away from wild animals, not consuming wild animals, maintaining hand hygiene -among others. in addition, the development of targeted antiviral drugs should be anticipated and accelerated in the future. certainly, vigorous measures to prevent contagion have been taken in china and other countries. to prevent the spread of infection, the chinese government imposed a full lockdown and canceled public events such as the new year festival; contact with wild animals was also restricted, and travel was reduced with screening at airports, railway stations and subway stations [84] . moreover, two emergency hospitals in wuhan were constructed, and army medical units and medical staff from other areas were deployed to help prevent infections in wuhan [84] . the who and various governments advised people to reduce public activity, maintain social distance, wear masks, wash their hands frequently, and practice respiratory hygiene. as of 7 april 2020, the number of fully recovered patients was 277,420 worldwide ( figure 3e & h) , and the number of recovered patients was 77,468 in china ( figure 3b ). therefore, we are confident that the outbreak of covid-19 will be effectively curbed. most countries have isolated any suspected cases as rapidly as possible to contain infection and prevent local outbreaks. the ability to rapidly test patients suspected of having a sars-cov-2 infection is the cornerstone of case isolation. the experience gained from sars-cov and mers-cov by the health community over the last 20 years could also help in dealing with sars-cov-2 infections throughout the world. to date, no sars-cov-2-specific antiviral drugs or vaccines have been described for covid-19. therefore, a safe and stable vaccine for covid-19 is urgently needed, and it is expected to be ready within 18 months [81] . vaccines specific for sars-cov-2 will immunize people worldwide in the future. it is hoped that drugs specifically targeted for covid-19 also will be widely developed. meanwhile, the origin, intermediate host, structure and pathogenesis of sars-cov-2 will be the focus of future research. moreover, prediction of whether the similar coronaviruses can infect humans will be import for all the world. we hope that vaccines specific for the similar coronaviruses will be developed in advance to prevent epidemics in the world. • coronavirus disease 2019 (covid-19) was first reported in china and currently poses a serious challenge worldwide. the pneumonia was caused by a novel coronavirus named sars-cov-2. • more than 1,347,804 cases of covid-19 and 74,596 deaths have been reported as of april 7, 2020 according to data from johns hopkins resource center. • sars-cov-2 belongs to betacoronavirus, a large genus of viruses prevalent in nature. sars-cov-2 has 82% nucleotide identity with human sars-cov and 96% nucleotide identity with bat sars coronavirus (sarsr-cov-ratg13). • the fatality rate of covid-19 has been approximately 3.4% and the r0 has ranged from 1.4 to 6.4. compared with previous coronavirus outbreaks, has been reported to have a lower mortality rate and more rapid transmissibility and caused severe acute respiratory syndrome similarly to sars. • the main symptoms are fever, cough, shortness of breath, leukopenia and pneumonia. • diagnostic laboratory testing of covid-19 includes hematology testing, nucleic acid testing, viral genome sequencing and serology testing. to date, no specific antiviral drugs or vaccines for covid-19 have been developed. therefore, empirical antiviral drugs (lopinavir/ritonavir, favipiravir, oseltamivir, zanamivir and peramivir, arbidol), antibiotic drugs (moxifloxacin, levofloxacin, linezolid), chloroquine/hydroxychloroquine, glucocorticoids, monoclonal anti-inflammatory antibody (tocilizumab), have been used to treat sars-cov-2 infection. traditional chinese medicines are also used for therapy during infection with sars-cov-2. • this review is presented in the hope of helping the public effectively recognize and combat covid-19 and to provide a reference for future studies or outbreaks. the authors gratefully acknowledge all doctors who participate in the fight against covid-19 on the frontline. financial & competing interests disclosure no writing assistance was utilized in the production of this manuscript. 10.2217/fmb-2020-0063 future microbiol. (epub ahead of print) future science group 52279 ireland:5364 norway:5865 netherlands:18926 germany:28700 belgium:3986 turkey:1326 iran:24236 china:77468 us 5373 france:8911 belgium:1632 germany:1810 ltaly:16523 china 2019 novel coronavirus-important information for clinicians estimating the unreported number of novel coronavirus (2019-ncov) cases in china in the first half of january 2020: a data-driven modelling analysis of the early outbreak lessons learned from the 2019-ncov epidemic on prevention of future infectious diseases coronavirus 2019-ncov: a brief perspective from the front line clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan • provides the first evidence for the diagnosis and treatment of covid-19 patients. the common symptoms among 41 patients were fever, cough, myalgia, fatigue and pneumonia clinical and epidemiological features of 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cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality tocilizumab treatment in covid-19: a single center experience review of the 2019 novel coronavirus (sars-cov-2) based on current evidence a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) a novel coronavirus (covid-19) outbreak: a call for action novel coronavirus pneumonia emergency in zhuhai: impact and challenges focus on middle east respiratory syndrome coronavirus (mers-cov) key: cord-318934-dxipu00r authors: matsuyama, shutoku; ujike, makoto; ishii, koji; fukushi, shuetsu; morikawa, shigeru; tashiro, masato; taguchi, fumihiro title: enhancement of sars-cov infection by proteases date: 2006 journal: the nidoviruses doi: 10.1007/978-0-387-33012-9_42 sha: doc_id: 318934 cord_uid: dxipu00r nan the severe acute respiratory syndrome (sars) is caused by a newly emergent coronavirus (sars-cov). 1, 2 this virus grows in a variety of tissues that express its receptor, but the mechanism of the severe respiratory illness is not well understood. sars-cov is supposed to enter cells via endosome, and its spike (s) protein, which is responsible for cell entry of this virus, is activated by a certain protease active only in acidic conditions in the endosome. 3 to see whether this is correct or not, we began to study the sars-cov entry mechanism. in the course of this study, we found that various proteases facilitated sars-cov entry from cell surface. this indicated that sars-cov has a potential to enter cells via two different pathways, endosomal and cell-surface pathways, depending upon the presence of proteases. moreover, sars-cov entry from the cell surface mediated by proteases was a 100-fold more efficient infection than entry through endosomes. these results suggest that severe illness in the lung and intestine can be attributed to the proteases produced in these organs in inflammatory responses or physiological conditions. the sars-cov frankfurt 1 strain, kindly provided by dr. j. ziebuhr, 1 was propagated and titered using vero e6 cells. recombinant vaccinia virus harboring sars-cov s gene (dis-s) was used to express s protein. this recombinant virus was made from highly attenuated vaccinia virus dis. 4 vesicular stomatitis virus (vsv) pseudotype bearing sars-cov s protein was produced as reported previously. 5 various proteases were dissolved in phosphate-buffered saline, ph 7.2 (pbs), and used at the indicated concentration in dmem containing 5% fcs. the proteases used in this study were trypsin (sigma, st louis, mo, t-8802), thermolysin (sigma, p1512), chymotrypsin (sigma, c-3142), dispase (roche, 1 276 921, branchburg, nj), papain (worthington biochemicals, 53j6521, freehold, nj), proteinase k (wako, tokyo, japan), collagenase (sigma, c-5183) and elastase (sigma, e-0258). s protein expressed in vero e6 cells was analyzed by western blotting. preparation of cell lysates, electrophoresis in sds-polyacrylamide gel, and electrical transfer of the protein onto a transfer membrane were described previously. 6 s protein was detected with anti-s antibody, img-557 (imgenex, san diego, ca, usa). sars-cov entry or replication in veroe6 cells was examined by real-time pcr to detect the copy number of mrna9. the primers for amplification were complementary to the leader sequence (forward) and n gene (reverse) of sars-cov. the reaction was performed using a lightcycler instrument (roche). veroe6 cells were infected with the frankfurt-1 strain of sars-cov at a multiplicity of infection (moi) of 0.5, and infected cells were treated with trypsin (200 g/ml) at room temperature (rt) for 5 min after 20 h incubation. cell fusion was detected from approximately 2 h after trypsin treatment. fusion was also found after treatment with thermolysin or dispase. little or no fusion occurred following treatment with papain, chymotrypsin, proteinase k, or collagenase. s proteins in cells treated with proteases that induce fusion were cleaved approximately in the middle, and a fragment corresponding to s2 of ca. 100 kda protein was detected. however, no s2 band was detected in sars-cov infected cells treated with proteases that failed to induce fusion. veroe6 cells were infected with dis-s that harbors sars-cov s gene at moi of 1, and these cells were also treated with various proteases as described above. trypsin, thermolysin, and dispase induced fusion of s protein expressing cells, while other proteases failed to induce substantial cell fusion (fig. 1) . the results obtained using veroe6 cells expressing s protein by recombinant vaccinia virus were very similar to those observed in veroe6 cells infected with sars-cov. these results showed that various proteases activate the fusion activity of the sars-cov s protein by inducing its cleavage. it was also revealed that sars-cov infection was extensively inhibited by treatment of cells with bafilomycin (1 mm), which perturbs endosomal ph (fig. 2) . collectively, these results suggest that sars-cov takes an endosomal pathway for its entry and that s protein cleavage is important for fusion activity, which is in good agreement with the observations of a previous report. the hypothesis proposed by simmons et al. 3 stated that sars-cov is able to enter cells directly from their surface, if receptor-bound virus is treated with trypsin and other proteases that induce fusion. treatment of veroe6 cells with bafilomycin was shown to suppress sars-cov infection via the endosomal pathway to less than 1/100 (fig. 2) . bafilomycin-treated cells were inoculated with sars-cov at an moi of 1 and incubated on ice for 30 min. this allows virus binding to its receptor, but does not allow virus entry into cells. cells were then treated with trypsin for 5 min at rt and incubated at 37 o c for 6 h in the presence of bafilomycin. virus entry was estimated by the newly synthesized mrna9 measured quantitatively by real-time pcr. it was shown that trypsin with fusion-inducing activity extensively facilitated viral entry (fig. 2) . thermolysin and dispase also facilitated entry into veroe6 cells treated with bafilomycin. in contrast, two proteases that did not induce fusion, papain and collagenase, failed to do so. pseudotype vsv bearing sars-cov s protein infection was also facilitated in bafilomycin-treated veroe6 cells after treatment with proteases that induce fusion of sars-cov infected cells. treatment of cells with trypsin before virus infection did not enhance viral entry (fig. 2) , indicating that the effects of trypsin on cells are not involved in this infection. trypsin treatment of sars-cov prior to infection did not enhance infectivity, but reduced it by 10-to 100-fold (fig. 3) . these results demonstrate that sars-cov, when adsorbed onto the cell surface, fuses with the plasma membrane via the s protein after cleavage, suggesting a non-endosomal, direct entry of sars-cov into cells in the presence of proteases. those findings also support the hypothesis drawn by simmons et al. 3 that trypsin-like protease plays an important role in facilitating membrane fusion. treatment with a high concentration of trypsin augmented virus entry or replication by approximately 10-fold during an early phase of the infection, from 3 to 6 h postinfection, compared with the standard infection. this implies that infection through the cell surface is approximately 10-fold more efficient than infection via the endosomal pathway. these data also imply that viral replication after entry via the cell surface proceeds approximately 1 h ahead of that via the endosomal pathway. because sars-cov replication was enhanced by trypsin treatment, we next assessed the efficiency of virus spread in the presence or absence of trypsin in a low moi that mimics natural infection in humans. ten pfu of virus were inoculated onto 10 5 confluent veroe6 cells (moi = 0.0001), and the cells were incubated at 37 o c for 20 h in the media with or without various concentration of trypsin. the level of mrna9 showed that virus replication was 100-to 1000-fold higher when cells were cultured in the presence of trypsin, compared to replication in the absence of trypsin. viral infectivity also indicated that trypsin treatment enhanced viral growth by ca. 100-fold. this enhancement of viral replication observed in the presence of trypsin was also observed when infected veroe6 cells were cultured in the presence of proteases, such as thermolysin and dispase, which induce fusion, but no enhancement was encountered when cultured in the presence of papain or collagenase, which fail to induce fusion. these observations suggest that proteases that facilitate sars-cov entry from the cell surface support efficient sars-cov infection. thus, protease is likely to be responsible for the high multiplication of sars-cov in the target organs of sars, such as the lungs, where various proteases are produced (e.g., by inflammatory cells), as well as in the intestines, where a number of proteases are physiologically secreted. elastase is reported to be one of major proteases produced in inflammatory lungs. 7 thus we examined whether elastase enhances infection in cultured veroe6 cells at low multiplicities of infection. this finding strongly suggests that sars-cov replication can be enhanced in the lungs by elastase. thus, elastase is possibly a protease that is responsible for an acute severe illness caused by sars-cov. sars-cov infection was evident in a number of organs, such as the liver, cerebrum, and kidneys, as well as in major target organs such as the lungs and intestines. 8, 9 in the latter organs, drastic tissue damage by sars-cov infection was observed, while the other organs were not so severely affected. although the pathogenic mechanism of sars has not been elucidated, the present study suggests that proteases secreted in major target organs play an important role in the high multiplication of virus in those organs, which could result in severe tissue damage. sars-cov may initially infect pneumocytes via an endosomal pathway. this would induce inflammation that generates a variety of proteases such as elastase. once those proteases are present in the lungs, they may mediate a robust infection, which may result in enhanced replication. although lung damage is reportedly mediated by cytokine storm, 8 another target organ, the small intestines, could also contribute to the high viral titers detected in these tissues, which, in turn, may result in diarrhea. 10 the present studies suggest that co-infection of sars-cov with nonpathogenic respiratory agents, such as chlamydia or mycoplasma, could result in severe lung disease as a consequence of protease production or induction by the non-sars-cov agents, as has been shown by the enhancement of disease caused by influenza virus co-infected with nonpathogenic bacteria. 11, 12 studies are in progress to examine whether co-infection exacerbates pneumonia in mice infected with sars-cov. a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry structural analysis of vaccinia virus dis strain: application as a new replication-deficient viral vector vesicular stomatitis virus pseudotyped with severe acute respiratory syndrome coronavirus spike protein impaired entry of soluble receptor-resistant mutants of mouse hepatitits virus into cells expressing mhvr2 receptor the role of neutrophil elastase in acute lung injury lung pathology of fatal severe acute respiratory syndrome digestive system manifestations in patients with severe acute respiratory syndrome role of staphylococcus protease in the development of influenza pneumonia haemophilus paragallinarum exacerbates h9n2 influenza a virus infection in chickens co-infection of staphylococcus aureus or pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) we thank miyuki kawase for her excellent technical assistance throughout the experiments. this work was financially supported by a grant from the ministry of education, culture, sports, science and technology (16017308) and grant from the ministry of health, labor and welfare (h16-shinkoh-9). key: cord-317786-iv1br2oj authors: waterfield, t.; watson, c.; moore, r.; ferris, k.; tonry, c.; watt, a. p.; mcginn, c.; foster, s.; evans, j.; lyttle, m. d.; ahmad, s.; ladhani, s.; corr, m.; mcfetridge, l.; mitchell, h.; brown, k.; amirthalingam, g.; maney, j.-a.; christie, s. title: seroprevalence of sars-cov-2 antibodies in children a prospective multicentre cohort study. date: 2020-09-02 journal: nan doi: 10.1101/2020.08.31.20183095 sha: doc_id: 317786 cord_uid: iv1br2oj background studies based on molecular testing of oral/nasal swabs underestimate sars-cov-2 infection due to issues with test sensitivity and timing of testing. the objective of this study was to report the presence of sars-cov-2 antibodies, consistent with previous infection, and to report the symptomatology of infection in children. design this multicentre observational cohort study, conducted between 16th april 3rd july 2020 at 5 uk sites, aimed to recruit 900 children aged 2 to 15 years of age. participants provided blood samples for sars-cov-2 antibody testing and data were gathered regarding unwell contacts and symptoms. results 1007 participants were enrolled, and 992 were included in the final analysis. the median age of participants was 10.1 years. there were 68 (6.9%) participants with positive sars-cov-2 antibody tests indicative of previous sars-cov-2 infection. of these, 34/68 (50%) reported no symptoms. the presence of antibodies and the mean antibody titre was not influenced by age. following multivariate analysis 4 independent variables were identified as significantly associated with sars-cov-2 infection. these were: known infected household contact; fatigue; gastrointestinal symptoms; and changes in sense of smell or taste. discussion in this study children demonstrated similar antibody titres in response to sars-cov-2 irrespective of age. the symptoms of sars-cov-2 infection in children were subtle but of those reported, fatigue, gastrointestinal symptoms and changes in sense of smell or taste were most strongly associated with antibody positivity. registration this study was registered at https://www.clinicaltrials.gov (trial registration: nct04347408) on the 15/04/2020. during the first wave of the sars-cov-2 pandemic in england, children accounted for just 1% of confirmed infections,(1) had a milder clinical course, and had a much lower mortality than adults (1) (2) (3) (4) , a pattern similar to other international settings (3, 4) . the reasons for this are unknown, but various hypothesises exist. public health measures, such as school closures, may have minimised children's exposure to sars-cov-2. it is also possible that children have a different immune response to the virus for example the reduced expression of the ace2 gene, the host receptor for sar-cov-2 virus in airway cells (5) (6) (7) . despite existing data, it is impossible to state accurately what proportion of children were infected with sars-cov-2 in the uk. studies based on molecular testing of oral/nasal swabs with real-time reverse transcription polymerase chain reaction (rt-qpcr) underestimate infection due to issues with test sensitivity, timing of testing and non-testing of asymptomatic individuals (8) . a potentially more reliable method is to test for specific antibodies. existing antibody tests typically detect immunoglobulin g (igg or total antibody) to either the nucleocapsid or spike proteins of the virus (9) . antibody testing has greater potential than rt-qpcr to detect previous asymptomatic/mildly symptomatic infection, and is not dependent on coinciding with active infection. current best seroprevalence estimates from adults in the uk indicate that approximately 6.2% have antibodies consistent with previous sars-cov-2 infection (10) . these findings are similar to other international seroprevalence studies (11) (12) (13) . currently there are no published data reporting the current seroprevalence of sars-cov-2 antibodies in uk children. it is unclear what proportion of children are asymptomatic and which symptoms are most associated with paediatric sars-cov-2 infection. estimates based on rt-qpcr testing of oral/nasal swabs suggest that cough or fever are the most common symptoms (14) (15) (16) (17) (18) (19) . however, these studies focus on symptomatic cohorts, introducing selection bias (14) (15) (16) (17) (18) (19) , which leads to underestimation of the asymptomatic proportion. the objective of this study was to report the presence, and titres, of sars-cov-2 antibodies in healthy children of healthcare workers across the uk and to report the symptomatology of infection including the asymptomatic rate. . cc-by-nc 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint this multicentre observational prospective cohort study was designed to determine the seroprevalence of sars-cov-2 antibodies in healthy children, and report the symptomatology of infection. this study has been written in conjunction with the strengthening the reporting of observational studies in epidemiology (strobe) guidelines (20) . the study protocol has undergone external peer review and is available as an open access publication (21) . participants were recruited from 5 uk centres, in the 4 regions of the uk, between 16 th april 2020 and 3 rd july 2020. the sites included tertiary nhs hospitals (belfast, cardiff, manchester, and glasgow) and a public health england site (london). children of healthcare workers, aged between 2 and 15 years at the time of recruitment, were eligible to participate. a "healthcare worker" was defined as a national health service (nhs) employee. healthcare workers were categorised according to role, including whether that role involved patient facing activities. approximately 150 non-patient facing staff were included to provide a comparison group, and to improve the generalisability of the results. participants were identified at each participating nhs organisation using internal intranet advertisements and email circulars. children were excluded if they were receiving antibiotics, had been admitted to hospital within the last 7 days, were receiving oral immunosuppressive treatment, or if ever diagnosed with a malignancy. informed consent was obtained, and assent given by children where possible. participants were free to decline/withdraw consent at any time without providing a reason and without being subject to any resulting detriment. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint all children underwent phlebotomy performed by experienced paediatric medical and nursing professionals. serum and/or plasma were tested for antibodies to sars-cov-2, in ukas accredited laboratories using the following assays, which have been validated for use (22) (23) (24) : • nucleocapsid assays -(abbott architect® sars-cov-2 igg and roche elecsys® anti-sars-cov-2 total antibody) • spike protein assays -(diasorin liaison® sars cov-2 s1/s2 igg assay) the abbott, roche and diasorin assays are highly specific for sars-cov-2 antibodies, using the manufacturer's suggested cut-offs, with specificities of 1.00 (95% ci 0.98 to 1. (22) (23) (24) . a summary of the tests used is provided in table 1 . study data were collected on a case report form (crf) using redcap (research electronic data capture) electronic data capture tools (25) . participants and their parents provided information at enrollment relating to age, sex, previous health and potential predictors of sars-cov-2 infection including; known contact with individuals with covid-19, contact with individuals who have been symptomatic and/or self-isolating and results of any diagnostic testing such as rt-qpcr testing/antibody testing. to minimise recall bias, data relating to exposures and illness episodes were collected blinded to antibody testing results. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint  presence of antibodies (igg/total antibody) to sars-cov-2 in serum or plasma reported as titres.  previous sars-cov-2 infection defined as a positive antibody test using the manufacturer's advised positivity cut-off. the study was powered to detect a change in seroprevalence of sars-cov-2 antibodies at 3 time-points (enrollment, and 2 and 6 months following enrollment). to achieve this, 675 participants were required (assuming alpha of, 0.05 and beta of 0.2). allowing for 30% dropout rate, we aimed to recruit 900 participants from 5 sites. variables including sex, age, parent role, symptomatology, household contacts, and sars-cov-2 antibody prevalence were analysed using descriptive statistics (number and proportion for discrete variables, median and interquartile range for continuous variables). seroprevalence rates between sites were compared using fisher's exact test and antibody titres were correlated with age using the kendall's rank correlation test and mean titres were compared between symptomatic and asymptomatic participants using the wilcoxon rank sum test. variables associated with sars-cov-2 infection were analysed in a stepwise approach. initially all possible variables were assessed using univariate analysis with fisher's exact testing of categorical data, and the mann-whitney u test for continuous data (continuous data is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint from univariate and multivariate analysis. analysis was conducted in r (r core team, 2014). a ppi group comprising parents and children was convened. the ppi group met virtually and via socially distanced meetings. the group have contributed to the design of the study through online surveys and video discussions. they have also contributed to media interviews on national television and the lead young person has co-authored a manuscript outlining their experience of taking part in the study (26) . this study was registered at https://www.clinicaltrials.gov (trial registration: nct04347408) on the 15/04/2020 (last updated 27/05/20). at the time of registration no patients had been recruited to the study which opened on the 16/04/20. the end of the study will be the last study visit. . cc-by-nc 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint in total, 1042 potential participants were screened for inclusion, of whom 35 were excluded; 18 were outside the specified age range, 1 met specific exclusion criteria, and 16 declined consent. the remaining 1007 children were enrolled, of which 15 were excluded from analysis due to unsuccessful phlebotomy; 992 were included in the final analysis ( figure 1 ). the recruitment by site can be visualised in table 2 table 3 . seroprevalence of sars-cov-2 antibodies varied between sites. belfast had significantly lower seroprevalence than all other sites at 0.9% (95% ci 0.2 to 3.3, n=215); p<0.0001, and in london seroprevalence was significantly higher than all other sites at 11.6% (95% ci 7.8 to 16.8 n=199); p=0.0069. the remaining 3 sites reported seroprevalence rates between 5.6% and 8.9%. the difference between these 3 sites were not significant ( table 2) .. the mean antibody titres, for those testing positive, were; is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. there was no correlation between age and antibody titres ( figure 2 ). the results from the abbott architect® sars-cov-2 igg assay indicated a small but significant difference in mean antibody titres between asymptomatic 4.3 s/c (95% ci 3.4 to 5.2) and symptomatic participants 5.5 s/c (95% ci 4.7 to 6.2); p=0.04. there was no significant difference in mean antibody titres for the roche elecsys® or diasorin liaison® assays when comparing symptomatic and asymptomatic participants (p =0.23 and 0.58 respectively) (figure2). the univariate analysis of individual variables associated with sars-cov-2 infection is shown in table 3 . in addition to clinical features, variables such as age, gender, the role of the parent (patient facing or not), and known household contacts were included. age and gender were not associated with sars-cov-2 infection (table 3) . parental role showed significant association in the univariate analysis, but this was no longer significant once corrected for site and other variables in the multivariate analysis. contact with a household member with confirmed sars-cov-2 infection was significantly associated with sars-cov-2 infection in the participant in both the univariate and multivariate analyses ( table 3) is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint this observational study is one of the largest uk studies of paediatric sars-cov-2 antibody seroprevalence, and the only study to recruit from all regions of the uk. following the first pandemic wave in the uk, 68/992 (6.9%) children of healthcare workers had evidence of prior infection with sars-cov-2. whilst this is likely to be higher than the general population it is surprisingly similar to the seroprevalence reported by the ons study of adults from england and wales (6.2%) (10) , and similar to international estimates (11) (12) (13) . as expected there was marked geographical variation, with london reporting the highest infection rates (11.6%) and belfast the lowest (0.9%) p<0.0001. these regional variations are consistent with published adult estimates of seroprevalence from the same time period (10) . in this study there was a near equal number of children under 10 years of age 32/68 (47%) and children over 10 years of age 36/68 (53%) developing antibodies consistent with previous sars-cov-2 infection. age, as a categorical or continuous variable, was not a statistically significant factor in predicting the presence of antibodies, or the overall titres in children irrespective of the assay used ( figure 2 ). this is in contrast to several studies that have reported a lower seroprevalence in young children (under 10 years of age) and in elderly adults (over 65 years of age) following the first wave of the pandemic (11) (12) (13) . this has led some authors to suggest that children are less susceptible to sars-cov-2 infection (27) (28) (29) (30) . the studies on which these assumptions are based have typically reported a binary antibody outcome (positive or negative) rather than absolute titres (11) (12) (13) . it is possible that the lower seroprevalence reported thus far in younger children merely reflects the effect of social distancing measures on this group. this may go some way to explain why the over 65s also demonstrated lower seroprevalence in the same studies (27) (28) (29) (30) . in our cohort, children were more likely to be exposed to sars-cov-2 in the home due to fact that their parent(s) worked in healthcare. the findings from this study may therefore provide a greater insight into how younger children react when exposed to sars-cov-2. further research is required to understand if younger children are really less susceptible to sars-cov-2. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. there is evidence from adult serological studies that those with severe illness develop a significantly greater antibody response than those with mild or asymptomatic disease (31) (32) (33) . this has raised concerns that children, who typically have mild disease, may fail to develop a meaningful antibody response to sars-cov-2 infection. more recently, emerging adult data suggest that even asymptomatic adults are capable of mounting a potentially lasting and protective immune response (34) (35) . in our study antibody titres, measured using the abbott architect® sars-cov-2 igg assay, were significantly higher in symptomatic children compared with asymptomatic children p=0.04. these findings were not replicated with either the roche elecsys® anti-sars-cov-2 or diasorin liaison® sars cov-2 s1/s2 igg assays. it therefore remains unclear to what extent the severity of symptoms in children influences the antibody response. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint this study demonstrates that approximately half of children are asymptomatic when infected with sars-cov-2 and that current uk testing strategies will fail to diagnose the majority of paediatric infections. this study also demonstrates that younger children were just as likely to become infected with sars-cov-2 as older children and that they are capable of mounting a similar antibody response. the strengths of this study are that is a large multicentre study including children from across the four nations of the uk. the findings are based on serological antibody testing rather than rt-qpcr testing of swabs and are therefore more likely to report the true asymptomatic rate and the true symptomatology of paediatric infection with sars-cov-2. the limitations of this study are that all of the children in this study had only mild disease making comparison between severe and mild disease impossible. the children were also recruited from healthcare workers and the prevalence of antibodies is likely to be lower in the general population. the children of healthcare workers were chosen for a number of reasons. firstly, the study was conducted during the lock-down phase of the pandemic response thereby making face-to-face discussions challenging due to a need to conform with social distancing rules. healthcare workers were felt to be more likely to be able to understand the study and consent without the need for face-to-face discussions with members of the research team. secondly, healthcare workers were at higher risk of exposure to sars-cov-2 and their children were more likely to be infected making a study of symptomatology more practical. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. .  data sharing: all of the individual participant data collected during this study will be available (including data dictionaries) on the queen's university belfast database within 3 months of completion of the study. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint diasorin liaison® reported in au/ml. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint . cc-by-nc 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 2, 2020. . https://doi.org/10.1101/2020.08.31.20183095 doi: medrxiv preprint et alcovid-19 in children: analysis of the first pandemic peak in englandarchives of disease in childhood published online first: 12 defining the epidemiology of covid-19 -studies needed coronavirus disease 2019 in children-united states amsterdam: national institute for public health and the environment (rivm) nasal gene expression of angiotensin-converting enzyme 2 in children and adults physiological and pathological regulation of ace2, the sars142 cov-2 receptor angiotensin-converting enzyme-2 (ace2),144 sars-cov-2 and pathophysiology of coronavirus disease 2019 (covid-19). the journal of 145 pathology a cautionary tale of false-negative nasopharyngeal covid-19 covid-19: phe laboratory assessments of molecular tests latest data and analysis on coronavirus (covid-19) in the uk and its effect on the economy and society prevalence of sars-cov-2 in spain (ene-covid): a nationwide, populationbased seroepidemiological study seroprevalence of anti-sars-cov-2 igg antibodies in geneva, switzerland (serocov-pop): a population-based study seroprevalence of sars-cov-2 igg significantly varies with age: results from a mass population screening (sars-2-screen-cda) epidemiology of covid-19 among children in china detection of covid-19 in children in early sars-cov-2 infection in children children with covid-19 in pediatric emergency departments in italy clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study screening and severity of coronavirus disease 2019 (covid-19) in children in madrid the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies seroprevalence of sars-cov-2 antibodies in children of healthcare workers-a prospective multicentre cohort study protocol -accepted for publication evaluation of the abbott sars-cov-2 igg for the detection of anti-sarscov-2 antibodies evaluation of roche elecsys antisars-cov-2 serology assay for the detection of anti-sars-cov-2 antibodies evaluation of diasorin liaison sarscov-2 s1/s2 igg serology assay for the detection of anti-sars-cov-2 antibodies research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support listening to the voices of children and young people involved in medical research spread of sars-cov-2 in the icelandic population characteristics of household transmission of covid-19 covid-19 in schools -the experience in nsw 2020 early release -antibody responses to sars-cov-2 at 8 weeks 552 postinfection in asymptomatic patients antibody responses to sars-cov-2 in patients with covid-19 profile of 623 immunoglobulin g and igm antibodies against severe acute respiratory syndrome 624 coronavirus 2 (sars-cov-2) etection, prevalence, and duration of humoral responses to sars-cov-2 under conditions of limited population exposure doi key: cord-309289-vm0k7hfx authors: rothan, hussin a.; stone, shannon; natekar, janhavi; kumari, pratima; arora, komal; kumar, mukesh title: the fdaapproved gold drug auranofin inhibits novel coronavirus (sars-cov-2) replication and attenuates inflammation in human cells date: 2020-04-14 journal: biorxiv doi: 10.1101/2020.04.14.041228 sha: doc_id: 309289 cord_uid: vm0k7hfx sars-cov-2 has recently emerged as a new public health threat. herein, we report that the fda-approved gold drug, auranofin, inhibits sars-cov-2 replication in human cells at low micro molar concentration. treatment of cells with auranofin resulted in a 95% reduction in the viral rna at 48 hours after infection. auranofin treatment dramatically reduced the expression of sars-cov-2-induced cytokines in human cells. these data indicate that auranofin could be a useful drug to limit sars-cov-2 infection and associated lung injury due to its anti-viral, anti-inflammatory and anti-ros properties. auranofin has a well-known toxicity profile and is considered safe for human use. sars-cov-2 has recently emerged as a new public health threat. herein, we report that the fda-approved gold drug, auranofin, inhibits sars-cov-2 replication in human cells at low micro molar concentration. treatment of cells with auranofin resulted in a 95% reduction in the viral rna at 48 hours after infection. auranofin treatment dramatically reduced the expression of sars-cov-2-induced cytokines in human cells. these data indicate that auranofin could be a useful drug to limit sars-cov-2 infection and associated lung injury due to its anti-viral, antiinflammatory and anti-ros properties. auranofin has a well-known toxicity profile and is considered safe for human use. gold-based compounds have shown promising activity against a wide range of clinical conditions and microorganism infections. auranofin, a gold-containing triethyl phosphine, is an fda-approved drug for the treatment of rheumatoid arthritis since 1985 (1). it has been investigated for potential therapeutic application in a number of other diseases including cancer, neurodegenerative disorders, hiv/aids, parasitic infections and bacterial infections (1, 2) . recently, auranofin was approved by fda for phase ii clinical trials for cancer therapy. the mechanism of action of auranofin involves the inhibition of redox enzymes such as thioredoxin reductase, induction of endoplasmic reticulum (er) stress and subsequent activation of the unfolded protein response (upr) (2) (3) (4) (5) . inhibition of these redox enzymes leads to cellular oxidative stress and intrinsic apoptosis (6, 7) . in addition, auranofin is an anti-inflammatory drug that reduces cytokines production and stimulate cell-mediated immunity (8) . the dual inhibition of inflammatory pathways and thiol redox enzymes by auranofin makes it an attractive candidate for cancer therapy and treating microbial infections. coronaviruses are a family of enveloped viruses with positive sense, single-stranded rna genomes (9) . sars-cov-2, the causative agent of covid-19, is closely related to severe acute respiratory syndrome coronavirus (sars-cov-1) (9, 10). it is known that er stress and upr activation contribute significantly to the viral replication and pathogenesis during a coronavirus infection (11) . infection with sars-cov-1 increases the expression of the er protein folding chaperons grp78, grp94 and other er stress related genes to maintain protein folding (12) . cells overexpressing the sars-cov spike protein and other viral proteins exhibit high levels of upr activation (13, 14) . thus, inhibition of redox enzymes such as thioredoxin reductase and induction of er stress by auranofin could significantly affect sars-cov-2 protein synthesis (15) . in addition, sars-cov-2 infection causes acute inflammation and neutrophilia that leads to a cytokine storm with over expression of tnf-alpha, monocyte chemoattractant protein (mcp-1) and reactive oxygen species (ros) (10). the severe covid-19 illness represents a devastating inflammatory lung disorder due to cytokines storm that is associated with multiple organ dysfunction leading to high mortality (10, 16) . taken together, these studies suggest that auranofin could mitigate sars-cov-2 infection and associated lung damage due to its anti-viral, anti-inflammatory and anti-ros properties. auranofin has a well-known toxicity profile and is considered safe for human use. we investigated the anti-viral activity of auranofin against sars-cov-2 and its effect on virus-induced inflammation in human cells. we infected huh7 cells with sars-cov-2 (usa-wa1/2020) at a multiplicity of infection (moi) of 1 for 2 hours, followed by the addition of 4 µm of auranofin (17, 18) . dmso (0.1%) was used as control (the solvent was used to prepare drug stock). cell culture supernatants and cell lysates were collected at 24 and 48 hours after infection. virus rna copies were measured by rt-pcr using two separate primers specific for the viral n1 gene and n2 gene (19, 20) . as depicted in figure 1 taken together these results demonstrate that auranofin inhibits replication of sars-cov-2 in human cells at low micro molar concentration. we also demonstrate that auranofin treatment resulted in significant reduction in virus-induced inflammation. these data indicate that auranofin could be a useful drug to limit sars-cov-2 infection and associated lung injury. further animal studies are warranted to evaluate the efficacy of auranofin for the management of sars-cov-2 associated disease. in this study, we used a novel sars-cov-2 (usa-wa1/2020) isolated from an oropharyngeal swab from a patient in washington, usa (bei nr-52281). virus strain was amplified once in vero e6 cells and had titers of 5 x 10 6 plaque-forming units (pfu)/ml. huh7 cells (human liver cell line) were grown in dmem (gibco) supplemented with 5% heat-inactivated fetal bovine serum. cells were infected with sars-cov-2 or pbs (mock) at a multiplicity of infection (moi) of 1 for 2 hours (17, 18, 21, 22) . cell were washed twice with pbs and media containing different concentrations of auranofin (sigma) or dmso (sigma) was added to cells. supernatants and cell lysates were harvested at 24 and 48 hours after infection. virus rna levels were analyzed in the supernatant and cell lysates by quantitative reverse transcription-polymerase chain reaction (qrt-pcr). rna from cell culture supernatants was extracted using a viral rna mini kit (qiagen) and rna from cell lysates was extracted using a rneasy mini kit (qiagen) as described previously (20, 21, 23) . qrt-pcr was used to measure viral rna levels using previously published primers and probes specific for the sars-cov-2. forward (5′-gaccccaaaatc agcgaaat-3′), reverse (5′-tctggttactgccagttgaatctg-3′) for mrna analysis of il-6, il-1β, tnfα and nf-kb, cdna was prepared from rna isolated from the cell lysates using a iscript™ cdna synthesis kit (bio-rad, hercules, ca, usa), and qrt-pcr was conducted as described previously (21, 23, 24) . the primer sequences used for qrt-pcr are listed in table 1 . auranofin: repurposing an old drug for a golden new age auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis repurposing auranofin, ebselen, and px-12 as antimicrobial agents targeting the thioredoxin system repurposing auranofin as an antifungal: in vitro activity against a variety of medically important fungi repurposing auranofin for the treatment of cutaneous staphylococcal infections the combination of alcohol and cigarette smoke induces endoplasmic reticulum stress and cell death in pancreatic acinar cells the unfolded protein response: controlling cell fate decisions under er stress and beyond biologic actions and pharmacokinetic studies of auranofin. the american journal of medicine the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak hlh across speciality collaboration uk. covid-19: consider cytokine storm syndromes and immunosuppression coronavirus infection, er stress, apoptosis and innate immunity comparative host gene transcription by microarray analysis early after infection of the huh7 cell line by severe acute respiratory syndrome coronavirus and human coronavirus 229e comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus hku1 the 8ab protein of sars-cov is a luminal er membrane-associated protein and induces the activation of atf6 role of endoplasmic reticulum-associated proteins in flavivirus replication and assembly complexes covid-19, cytokines and immunosuppression: what can we learn from severe acute respiratory syndrome? clinical and experimental rheumatology integrated microrna and mrna profiling in zika virus-infected neurons favipiravir and ribavirin inhibit replication of asian and african strains of zika virus in different cell models z-dna-binding protein 1 is critical for controlling virus replication and survival in west nile virus encephalitis a guinea pig model of zika virus infection cellular microrna-155 regulates virus-induced inflammatory response and protects against lethal west nile virus infection deletion of pregnancy zone protein and murinoglobulin-1 restricts the pathogenesis of west nile virus infection in mice identification of host genes leading to west nile virus encephalitis in mice brain using rna-seq analysis integrated analysis of micrornas and their disease related targets in the brain of mice infected with west nile virus national institute of neurological disorders and stroke, grant (r21od024896) from the office of the director, national institutes of health, and institutional funds. key: cord-294212-nlekz39f authors: wang, dongliang; mai, jinhui; zhou, wenfeng; yu, wanting; zhan, yang; wang, naidong; epstein, neal d.; yang, yi title: immunoinformatic analysis of tand b-cell epitopes for sars-cov-2 vaccine design date: 2020-07-03 journal: vaccines (basel) doi: 10.3390/vaccines8030355 sha: doc_id: 294212 cord_uid: nlekz39f currently, there is limited knowledge about the immunological profiles of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). we used computer-based immunoinformatic analysis and the newly resolved 3-dimensional (3d) structures of the sars-cov-2 s trimeric protein, together with analyses of the immunogenic profiles of sars-cov, to anticipate potential b-cell and t-cell epitopes of the sars-cov-2 s protein for vaccine design, particularly for peptide-driven vaccine design and serological diagnosis. nine conserved linear b-cell epitopes and multiple discontinuous b-cell epitopes composed of 69 residues on the surface of the sars-cov-2 trimeric s protein were predicted to be highly antigenic. we found that the sars-cov-2 s protein has a different antigenic profile than that of the sars-cov s protein due to the variations in their primary and 3d structures. importantly, sars-cov-2 may exploit an immune evasion mechanism through two point mutations in the critical and conserved linear neutralization epitope (overlap with fusion peptide) around a sparsely glycosylated area. these mutations lead to a significant decrease in the antigenicity of this epitope in the sars-cov-2 s protein. in addition, 62 t-cell epitopes in the sars-cov-2 s protein were predicted in our study. the structure-based immunoinformatic analysis for the sars-cov-2 s protein in this study may improve vaccine design, diagnosis, and immunotherapy against the pandemic of covid-19. the outbreak of the coronavirus disease 2019 (covid-19) is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] . by 16 june 2020, sars-cov-2 has been reported in 216 nations and has resulted in 7,941,791 confirmed cases (https: //www.who.int/emergencies/diseases/novel-coronavirus-2019). coronavirus (cov) belongs to the family of coronaviridae, and it is an enveloped, positive-sense single-stranded rna virus. both sars-cov-2 and sars-cov fit into the subgenus of sarbecovirus within the genus of betacoronavirus (beta-cov), based on phylogenetic tree analysis [1] [2] [3] . the viral genome, approximately 30 kb in size, encodes four structural proteins including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. the s protein is composed of an ectodomain, sars-cov infection triggers a series of humoral and cellular immune responses, including the production of high titers of specific neutralizing antibodies and specific cytotoxic t lymphocyte responses to sars-cov [12, 13] . the s protein is the major structural antigenic component through which effective protective immunity is raised against virus infection. a vaccine based on the s protein could elicit antibodies to neutralize virus infection by blocking virus fusion and entry. the sars-cov-2 s protein shares a high degree of similarity to the sars-cov s protein [14, 15] , and it also binds in similar fashion to the human ace2 receptor and thus is likely to employ a similar cell entry mechanism [4, 16] . as such, the s protein is an effective antigenic component for sars-cov-2 vaccine design and development. however, currently, there is little or limited information about the immunogenic profiles of sars-cov-2 and the immune responses against sars-cov-2. despite this, computer-based immunoinformatics [17] , together with the recent progress on the 3-dimensional (3d) sars-cov-2 s protein [14, 15, [18] [19] [20] [21] , offers a powerful strategy providing rational and rapid guidelines for the design and development of effective vaccines against this emerging infectious disease. in this study, the close genetic relationship of sars-cov-2 with other members of the genus of beta-cov, especially with sars-cov, prompted us to explore the potential immunogenic profiles of sars-cov-2 for vaccine design and development. we used computer-based immunoinformatic analysis, together with analyses of the immunogenic profiles of sars-cov, to anticipate potential b-cell and t-cell epitopes of the s protein of sars-cov-2 for vaccine design, particularly peptide-driven vaccine design, immunotherapy, and serological diagnosis. linear b-cell epitopes of the sars-cov-2 s protein were predicted by bepipred 2.0 in iedb (bepipred 2.0., immune epitope database and analysis resource, national institute of allergy and infectious diseases, bethesda, md, usa) with a threshold of 0.55 (corresponding specificity > 0.817 and sensitivity < 0.292), and only the epitopes with more than 8 residues were considered for subsequent antigenicity analysis. antigenicity was evaluated via the vaxijen v2.0 server online tool (vaxijen v2.0., the jenner institute, oxford, uk) [22] . discontinuous b-cell epitopes were predicted via the discotope 2.0 server tool in iedb with a default threshold of −3.7 (corresponding specificity > 0.75 and sensitivity < 0.47), based on the 3-dimensional (3d) structures of the sars-cov-2 s protein (pdb id: 6vyb, b chain) and the sars-cov-2 s protein rbd (pdb id: 6m0j, b chain). cd8 t-cell epitopes were predicted based on the net mhc pan 4.0 algorithm in iedb with a peptide size of 9 residues, and the 8 most frequent hla class i alleles (hla-a*01:01, hla-a*02:01, hla-a*03:01, hla-a*11:01, hla-a*24:02, hla-b*07:02, hla-b*08:01, and hla-b*40:01) in the worldwide population (phenotypic frequency > 10%) were selected [23] . the top 1% of peptides with high scores were chosen for subsequent immunogenicity evaluation, which was analyzed by the vaxijen v2.0 server. for cd4 t-cell epitope prediction, an iedb-recommended 2.22 algorithm based on 7 alleles (drb1*0301, drb1*0701, drb1*0501, drb3*0101, drb3*0202, drb4*0101, and drb5*0101) [24] at a default 15-aa peptide was used with a median consensus percentile of prediction threshold ≤ 20, as recommended. b-cell and t-cell epitopes of the sars-cov s protein were searched in iedb by using iedb's immunobrowser tool. to identify b-and t-cell epitopes tested by experiments, only the epitopes with the response frequency (rf) values more than 0.5 were considered as positive. 3d structures of all peptides were modelled via the pep-fold3 online server [25] . all the peptides were docked to the mhc i molecules hla-b7 (pdb id: 3vcl) and hla-a*01:01 (pdb id: 4nqv) via the patchdock rigid-body docking server based on the defined threshold [26] . the docking transformation with good molecular shape complementarity was selected based on the geometry docking algorithm in patchdock, and then scoring and refining of the docked complexes were performed using the firedock server [27, 28] . the docking complexes with high global energy, attractive van der waals (vdw) energy, and hydrogen-bonding energy were used for subsequent analysis. protein-peptide connection was examined via ligplot+ v.2.2, and pymol (version 1.8.4.0, schrödinger, inc, new york, nj, usa) was used to analyze docked complexes. in all, 17 potential linear b-cell epitopes were predicted by the bepipred 2.0 program (table s1 , supplementary materials), and nine linear b-cell epitopes were chosen for further analysis after their antigenicity was evaluated via the vaxijen v2.0 program, based on the scores (table 1) . all the predicted b-cell epitopes were localized to a strictly conserved region and shared 100% identity throughout the 138 sars-cov-2 isolates. structure simulations demonstrated that all the nine epitopes were located on the surfaces of either the monomer or the trimer of the s protein ( figure 2a , top panel). of note, of the nine epitopes, epitope 5 ( 405 devrqiapgqtgki 418 ) was localized to the rbd and (table 1) . we also reviewed seven dominant linear b-cell epitopes of the sars-cov s proteins based on previous experimental tests and response frequency (see methods). of the seven epitopes, two epitopes were identical throughout all the 87 sars-cov isolates and four were highly conserved (≥93.1% sars-cov isolates had identical epitopes) (table s2 , supplementary materials). these results suggested that the majority of linear b-cell epitopes of the s protein were highly conserved in sars-cov and sars-cov-2 isolates, respectively ( table 1 and table s2 ). it is worth noting that one epitope ( 786 qilpdplkptkrsfiedllfnkvtla 811 ) located in the s2 subunit of the sars-cov s protein is an important linear b-cell epitope capable of eliciting the production of a neutralizing antibody (nab) identified in patients who recovered from sars-cov infection (table s2 ) [13] . in addition, we also predicted linear b-cell epitopes of the sars-cov s protein, and six of the seven dominant linear b-cell epitopes were predicted by bepipred 2.0, since the six dominant b-cell epitopes had overlapping sequences with their counterparts in the predicted epitope pool, thereby supporting bepipred 2.0 as a reliable and powerful tool for predicting linear b-cell epitopes. finally, the comparison of the epitope sequences revealed that there were no overlapping sequences between the nine potential linear b-cell epitopes of sars-cov-2 and the seven dominant linear b-cell epitopes of sars-cov (table 1 and table s2 ), suggesting that the immunogenetic profile of the sars-cov-2 s protein may be different from that of sars-cov. besides the linear b-cell epitopes, 69 residues on the surface of the s protein of the sars-cov-2 were predicted to form the multiple discontinuous b-cell epitopes ( table 2) . furthermore, based on the primary structure and 3d structure of the trimeric s protein, these residues were mainly distributed within eight regions ( table 2 and figure 2b ). notably, region s1-2 containing 35 residues accounted for more than half of the residues (35/69) comprising the discontinuous b-cell epitopes, and these 35 residues of region s1-2 were all located in the rbd. furthermore, 31 of the 35 residues were in the rbm (region s1-2 in table 2 ). this result suggested that the rbd, particularly the rbm, was highly antigenic. in addition, among the discontinuous epitope(s) of region s1-2, 10 residues (g417, g446, y449, q493, g496, q498, t500, n501, g502, and y505) were identified as the key residues contributing to the binding to the host receptor ace2 [19] (table 2 ). in region s2-2, two residues (p793 and i794) were located in the fusion peptide (fp) and exposed on the surface of the s2 subunit (table 2 and figure 2b , top panel). therefore, antibodies targeting these two regions may block the virus binding to the host cell receptor and the subsequent membrane fusion between virus and host cell. in addition, sequence alignments revealed that these 69 residues were strictly conserved among the s proteins of the 138 sars-cov-2 isolates, except that a point mutation (p1143l, region s2-5, table 2 ) occurred in the australia/qld02/2020 strain. although this mutation did not change the secondary structure ( figure s1a , supplementary materials), it caused a slight increase in the antigenicity (the antigenic scores increasing from 0.558 to 0.565). indeed, as shown in figure s1b , the longer side chain of 1143 l caused an apparent alteration of the surface structure. note: residues in the epitopes that are present in the crystal structure of the sars-cov-2 trimeric s protein are underlined; otherwise, they were absent in the crystal structure. table 2 . predicted discontinuous b-cell epitopes of the sars-cov-2 s protein. sequences domain/motif s1-1 k97, s98, k187, p209, n211, e281, n282 ntd s1-2 t415, k417, d420, y421, n439, n440, s443, k444, v445, g446, g447, n448, * y449, * n450, l452, r454, k458, s459, n460, k462, s477, p491, * l492, q493, s494, * g496, f497, q498, p499, * t500, n501, * g502, v503, g504, * y505 rbd between fp and hr1 s2-4 y917, e918 hr1 s2-5 q1071, n1074, t1100, l1141, q1142, p1143, e1144, l1145, d1146, s1147 between hr1 and hr2 residues in the epitopes that are involved in binding of the sars-cov-2 rbd to hace2 are underlined. * indicates the residues that are present at the identical positions of both sars-cov and sars-cov-2 s proteins. cell. in addition, sequence alignments revealed that these 69 residues were strictly conserved among the s proteins of the 138 sars-cov-2 isolates, except that a point mutation (p1143l, region s2-5, table 2 ) occurred in the australia/qld02/2020 strain. although this mutation did not change the secondary structure ( figure s1a , supplementary materials), it caused a slight increase in the antigenicity (the antigenic scores increasing from 0.558 to 0.565). indeed, as shown in figure s1b , the longer side chain of 1143 l caused an apparent alteration of the surface structure. next, we examined all the discontinuous b-cell epitopes of the sars-cov s protein deposited in the iedb database, and three main epitopes (epitope id: 77442, 77444, and 910052) were obtained from the database (table s3 , supplementary materials). furthermore, these conformational epitopes could be recognized by a variety of neutralizing mabs (80r, m396 and s230) in previous studies [29, 30] . 3d structure analyses revealed that the residues among the three discontinuous b-cell epitopes were exclusively mapped onto the rbd surface of the s1 subunit, suggesting that the rbd of the sars-cov s protein is also highly antigenic. we also compared the common residues comprising the discontinuous epitopes within the rbds of both the rbds of sars-cov and sars-cov-2 s proteins. only seven residues (y449, n450, l492, g496, t500, g502, and y505 of the sars-cov-2 s protein) were present in the identical positions of both s proteins (table 2) . therefore, the three mabs (80r, m396, and s230) recognizing the rbd of the sars-cov s protein hardly bound the sars-cov-2 rbd, although the rbds of both sars-cov and sars-cov-2 exhibit a high degree of 3d structural homology [14] . altogether, compared to the sars-cov s protein, the sars-cov-2 s protein may have a distinct antigenic profile, although both viruses are closely related by phylogenetic analysis (figures s2 and s3 , supplementary materials). in all, 40 peptides were predicted as the potential cd8 t-cell epitopes following analysis of peptide-mhc-i binding of the sars-cov-2 s protein using the net mhc pan 4.0 server and their subsequent evaluation of antigenicity using vaxijen v2.0 ( 22 potential cd4 t-cell epitopes were predicted to be present in the sars-cov-2 s protein ( table 3) . three of the 62 predicted t-cell epitopes (cd8 and cd4 t-cell epitopes above) of the sars-cov-2 s protein have been reported as t-cell epitopes of the sars-cov s protein in previous studies [31] [32] [33] [34] (table s5 , supplementary materials). the first and second were cd8 t-cell epitopes ( 493 pyrvvvlsf 501 , epitope id: 50166; 1174 nlneslidl 1182 , epitope id: 44814), while the first one was located in the rbd of the sars-cov s protein, which is known to be important for receptor binding and virus entry [35] . the third one was encompassed in one of the cd4 t-cell epitopes ( 993 qliraaeirasanlaatk 1010 , epitope id: 100428) of the sars-cov s protein (the epitope highlighted with underline showed the predicted cd4 t-cell epitope derived from the sars-cov-2 s protein). the underlined epitope is also identified as a t-cell epitope of the sars-cov s protein. before molecular docking with hla molecules, the 3d structures of the 40 potential cd8 t-cell epitopes were modelled via pep-fold3. only the best 3d model of each epitope was chosen for the subsequent molecular docking with hla molecules. among the 40 epitopes, four were docked into hla-b7 and nine were docked into hla-a*01:01. for the four peptide-hla-b7 molecular docking, the binding efficiency of each epitope was evaluated by the global and vdw energies, which were computed ranging from −11.55 to −26.14 kcal/mol and −18.15 to −25.38 kcal/mol, respectively (table 4 ). all the four peptides were predicted to be well docked into the groove of the hla-b7 molecule and formed stable hydrogen bonds with the residues in the groove of the hla ( figure 3a) . notably, both t73 and e152 in the hla-b7 groove frequently interacted with the epitopes via hydrogen bonding within 3.1å ( figure s4a, supplementary materials) . furthermore, the global and vdw energies of the nine peptide-hla-a*01:01 dockings ranged from −12.90 to −46.66 kcal/mol and −12.10 to −25.71 kcal/mol, respectively (table 4 ). of these nine peptides, five peptides showed high binding affinities and the other four peptides showed even higher binding affinities with hla-a*01:01. hydrogen bonds less than 3.1å were frequently observed in docking complexes. t73, n77, t143, and r156 within the groove were the major residues interacting with these peptides and formed stable complexes ( figure 3b and figure s4b ). the underlined epitopes are also identified as t-cell epitopes of the sars-cov s protein. (table 4 ). of these nine peptides, five peptides showed high binding affinities and the other four peptides showed even higher binding affinities with hla-a*01:01. hydrogen bonds less than 3.1å were frequently observed in docking complexes. t73, n77, t143, and r156 within the groove were the major residues interacting with these peptides and formed stable complexes ( figure 3b and figure s4b ). table 4 . table 4 . vaccination is the most effective medical strategy against a variety of infectious diseases. unfortunately, to date, no vaccine against coronavirus-associated diseases has been approved by the fda for use in humans. therefore, a vaccine against covid-19 is urgently needed to control the pandemic caused by the highly contagious sars-cov-2. the lack of knowledge about sars-cov-2 immunogenic profiles and immune responses is a challenge to vaccine design and development. the s protein is a leading potential target for vaccine design for either sars-cov or sars-cov-2 infection because of its strong immunogenicity and its roles in virus attachment and cell entry [16, 36] . importantly, the s protein of sars-cov is capable of inducing the production of neutralizing antibodies (nabs), which have been found in convalescent plasma samples from sars patients [13] and in animal models [37, 38] . therefore, antibodies targeting the s protein, particularly the rbd/rbm or the s2 fusion machinery, may exhibit neutralizing activity against sars-cov-2 infections. sars-cov-2 and sars-cov belong to the same genus of the betacoronavirus and both of them, together with the three coronaviruses from bat, show a very close genetic relationship in the evolution of the virus ( figure s2 , supplementary materials). however, the similarity of the immunogenic properties of these viruses remains to be determined. several immunological questions are critical: "are the immunogenic profiles of sars-cov-2 and sars-cov as similar as the genetic relationship shown in the phylogenetic trees?"; "do the immunogenic properties of both viruses differ significantly from each other?"; and "can the nabs raised against sars-cov provide effective protection against the infection of sars-cov-2?". we sought to identify the potential b-cell and t-cell epitopes of the sars-cov-2 s protein by using various state-of-the-art tools. the results improve our understanding of s protein immunogenesis and vaccine design. nine linear b-cell epitopes were predicted and localized to the surface of the sars-cov-2 s protein (table 1) , while seven linear b-cell epitopes of the sars-cov s protein have been confirmed by previous investigations [13, [39] [40] [41] [42] . the two groups of epitopes do not share any similarities, even though the s proteins from both viruses are close to each other in their primary structure. one critical linear b-cell epitope ( 786 qilpdplkptkrsfiedllfnkvtla 811 ) of the sars-cov s protein was reported to be recognized by nabs obtained from convalescent sars patients in a previous report [13] . another group also reported that an epitope ( 803 llfnkvtladagfmkqygeclgdina 828 ) was able to induce the production of nabs in animal models [41] . both epitopes localize to the s2 subunit and have a nine-residue overlap from position 803 to 811. furthermore, two of our predicted linear b-cell epitopes (predicted via bepipred 2.0 in iedb) were also found to map to the region between 786q-828a (data not shown), suggesting that the region (786q-828a) is an epitope-rich region of the sars-cov s protein. the function of this region in the s2 subunit is still unknown, but we note that the region has a three-residue overlap with the fusion peptide (fp) from 770 to 788 (figure 1) , and a proteolytic cleavage site (s2 ) upstream of the fusion peptide is conserved in all known coronaviruses [43] . therefore, antibodies targeting this epitope-rich site could potentially block fp function in membrane fusion during virus cell entry. comparison of the sars-cov and sars-cov-2 s proteins reveals that the s2 subunit is structurally conserved and shares higher aa identity (~90%), than does the s1 subunit (~68%). likewise, we also identified a homologous peptide ( 804 qilpdpskpskrsfiedllfnkvtla 829 ) in the s2 subunit of the sars-cov-2 s protein, which differs by only two residues from the corresponding region in sars-cov (see the residues indicated by the underlines). however, this homologous peptide in the sars-cov-2 s protein does not qualify as a predicted linear b-cell epitope. we noticed that the two residues in sars-cov (792l and 795t) are replaced by residues with less bulky side chains in sars-cov-2 (810s and 813s), which may decrease the antigenicity of the peptides and support sars-cov-2 countering host immune surveillance and clearance. indeed, we evaluated the antigenicity of both peptides using vaxijen v2.0, and found that the antigenicity score of the linear b-cell epitope in the sars-cov s protein was 0.4121, almost double the score of the peptide in the sars-cov-2 s protein (0.2114). although the antigenicity of this peptide in sars-cov-2 remains to be experimentally determined, and currently, most vaccine designs are focusing on the rbd of the s1 subunit, we rspeculate that a vaccine based on this epitope-rich region (786q-828a) in the s2 subunit of the sars-cov s protein may also elicit broad nabs that can cross-react with other virus members among this coronavirus family to provide broader protections (i.e., against simultaneous infections of both sars-cov and sars-cov-2). very recently, poh et al. used sera from covid-19 convalescent patients to identify peptides eliciting nabs from a pool constructed from overlapping sequences of the sars-cov-2 s protein [44] . one of the identified peptides ( 809 pskpskrsfiedllfnkv 826 ) is also from the s2 subunit, located near the fusion peptide of the sars-cov-2 s protein. however, as discussed above, the antigenicity of this peptide and its effect across human populations of different ages, genetic backgrounds, and immune status requires further evaluation before being used for vaccine design, due to its lower antigenicity score compared to the concomitant sars-cov s protein. in addition, s proteins of coronaviruses are decorated with an extensive glycan shield, which blocks neutralizing antibody recognition and presents a challenge for vaccine development. walls et al. recently characterized the s glycan shield of the sars-cov s protein [43] . according to their result, this epitope-rich region (786q-828a) located in a glycan hole that is sparsely glycosylated provides access to host protease for further proteolysis and subsequent induction of membrane fusion. taken together, this epitope-rich region is an ideal target for sars-cov-2 vaccine design. besides these critical linear b-cell epitopes, multiple discontinuous conformational b-cell epitopes distributed throughout eight regions were located on the surface of the trimeric s protein (table 2 and figure 2b ). region s1-2 is one of the critical sites that could be targeted by nabs since it is located in the rbd, and specifically on the surface of the rbm. ten of the 35 residues among the conformational epitope(s)/regions are potentially involved in binding of the sars-cov-2 rbd to hace2: (g417, g446, y449, q493, g496, q498, t500, n501, g502, and y505) ( table 2 ) [19] . it is possible that antibodies compete with hace2 to bind the sars-cov-2 rbd, and thereafter block the interaction of the virus with the receptor and the subsequent virus cell entry. in addition, seven of the 35 residues (y449, n450, l492, g496, t500, g502, and y505) of the sars-cov-2 rbd are also identified in the sars-cov rbd. these are the key residues forming a conformational epitope recognized by nabs in previous studies [45, 46] . cell-mediated immunity plays crucial roles in the response to virus infection as well as cancer therapy. cd8 cytotoxic t cells kill cells via t-cell receptor (tcr) recognition of the cognate peptide presented by mhc class i. two critical cd8 t-cell epitopes ( table 4 , a5 and a9 in figure 3 ), previously reported in sars-cov studies, were also predicted as cd8 t-cell epitopes of the sars-cov-2 s protein in our study. importantly, both the epitopes can be docked onto the hla-a*01:01 allele in an energetically favorable manner (table 4 ). these results strongly suggest that both of the cd8 t-cell epitopes are authentic epitopes of the sars-cov-2 s protein and are possibly involved in cell-mediated immune responses against sars-cov-2 infection. currently, most vaccine designs of the virus focus on the nab production elicited by the s protein, but t cell-mediated immunity (both cd8+ and cd4+ helper cells) against the viral infection deserves more attention. furthermore, "human-like" t-cell epitopes in sars-cov-2 should be removed from the vaccine since these epitopes are able to activate treg cells and suppress the immune response [47] . epitope prediction via immunoinformatics has accelerated the identification of antigens capable of eliciting a strong immune protective response against pathogen infections. likewise, it can remove deleterious epitopes from the antigen pool, which may cause antibody-dependent enhancement (ade), cytokine storm, autoimmune responses, and pathological lesions. the authenticity and effectiveness of these predicted epitopes may be improved through the use of threshold scoring and further confirmed by in vitro experiments and animal models. since epitope mapping of a new pathogen is time-consuming and laborious work, epitope prediction by immunoinformatics improves the efficiency of vaccine design and development. for instance, gutiérrez et al. predicted cross-conserved t-cell epitopes of seven representative strains of influenza a virus (iav) in us swine herds [48] . following the prediction, researchers in tanja opriessnig's group designed and tested a dna vaccine containing these predicted cross-conserved t-cell epitopes followed by an inactivated vaccine for boost. the new designed vaccine (prime-boosting regimen) exhibits an additive increase in cell-mediated immunity and an excellent clinical protection [49] . a pool of epitopes may be chosen as the core immunogen to develop various peptide-driven vaccines, such as a peptide vaccine, a dna/rna vaccine encoding these tandem epitopes, or a subunit vaccine via grafting these epitopes onto a defined nanoparticle, i.e., virus-like particles (vlps). in contrast to the whole pathogen-based vaccine, the injurious side effects (i.e., ade, cytokine storm, autoimmune responses, and pathological lesions) of these isolated epitopes can be evaluated in vitro or in animal models. thereafter, the peptide-driven vaccine with the optimized epitope combination will be safer and more effective as a result of its more precise design guided by a variety of versatile immunoinformatic tools. cytokine storm is one of the most dangerous and potentially lethal sequelae of covid-19 infection but the details of its onset and why it affects one patient rather than another remain unknown. several possible pathways may be responsible for sars-cov-2-associated cytokine storm, but it is likely that a failure to initially suppress viral replication leads to severe tissue damage from an overwhelming infection and a subsequent uncontrolled immune response. the initial cytokine wave following sars-cov-2 infection comes from the innate immune response. pattern recognition receptors (prrs), such as toll-like receptors (tlr-3, -7, and -8), rig-i-like receptors (rlrs), and nod-like receptors (nlrs) recognize the viral rna genome or its intermediates during replication. this recognition causes substantial releases of proinflammatory cytokines, such as tnf-α, il-1β, and il-6. second, the proliferation of sars-cov-2 in host cells leads to a large amount of cell death, and the content of the dead cells release damage signals, which further amplify cytokine release leading to cytokine storm [50] . lastly, cytokine storm may be exacerbated through a complement cascade after infection. the large number and combinatorial diversity of n-linked glycans on the surface of the sars-cov-2 s protein can be recognized by mannose-binding lectin (mbl), which is able to initiate the complement cascade and activate macrophages. subsequently, these activated macrophages also release a large amount of cytokine, i.e., il-1, il-6, and tnf-α. thus, a peptide-driven vaccine in the absence of viral genome and various glycan-conjugated antigens, theoretically, promises to limit inappropriate cytokine release. an autoimmune response induced by foreign antigens presents another safety issue for vaccine design. generally, pathogens may evade host immunosurveillance through producing "host-like" b-cell or t-cell epitopes and activating self-reactive t-regulatory cells that suppress the immune response or induce tolerance to the pathogens. however, in the context of a vaccine formula (combinations of antigens and adjuvants), these "host-like" epitopes may reversibly activate to induce an autoimmune response. recently, epivax, inc., has launched a new immunoinformatic tool called janusmatrix that helps to identify "human-like" epitopes from pathogens [47] . removing these "human-like" epitopes from a vaccine formula further enhances the safety and efficacy of such vaccines. compared to the use of the whole virus proteome, the epitopes predicted in our study are more easily evaluated by these newly developed tools such as janusmatrix, which removes the "human-like" epitopes from the vaccine. finally, ade is a critical safety concern for vaccine design and development. in general, ade is mediated by non-neutralizing antibodies binding to virus and then promoting virus cell entry via fcγ receptors (fcγrs). actually, ade was discovered in the course of vaccinations (i.e., rsv and dengue virus) and is difficult to predict. ade has been reported in animal experiments during vaccine trials of sars-cov and mers-cov [51, 52] . however, ade during vaccination for sars-cov or mers-cov, let alone sars-cov-2, remains to be determined in human. epitope prediction and in vitro immunological assays can aid scientists in the identification and removal of potential ade-promoting b-cell epitopes. the refined pool of candidate vaccine antigens can then be more exhaustively tested in animal models for evidence of ade. the results presented in this study highlight sars-cov-2 evolution and the structure-relevant immune profiles of both s proteins (sars-cov-2 and sars-cov). this perspective improves vaccine design and immunotherapy and works to minimize the side effects of vaccination for sars-cov-2. supplementary materials: the following are available online at http://www.mdpi.com/2076-393x/8/3/355/s1, figure s1 : the effect of p1143l mutation on the secondary structure and predicted 3d structure, figure s2 : neighbor-joining tree analysis of sars-cov-2, figure s3 : maximum-likelihood tree analysis of sars-cov-2, figure s4 : (a) 2d graphical representation of interaction analyses between human hla-b7 protein and mhc-i binding peptides, (b) 2d graphical representation of interaction analyses between human hla-a*01:01 protein and mhc-i binding peptides, figure s5 : (a) sequence variability of the s protein between ratg13 and sars-cov-2, (b) sequence variability of the s protein among 138 sars-cov-2 clinical isolates, table s1 : 17 predicted linear b-cell epitopes of the sars-cov-2 s protein using bepipred 2.0, table s2 : linear b-cell epitopes of the sars-cov s protein, table s3 : discontinuous b-cell epitopes of the sars-cov s protein, table s4 : predicted mhc class-i epitopes in the sars-cov-2 s protein, table s5 : mhc class i and class ii epitopes identified in the sars-cov s protein, table s6 : detailed information of 138 sars-cov-2 sequences, table s7 : detailed information of reference coronavirus strains in this study, table s8 : percentage of nucleotide sequence and amino acid sequence identity of sars-cov-2 and reference cov strains, table s9 : sequence variability among structural proteins of sars-cov-2 clinical isolates. a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus structure analysis of the receptor binding of 2019-ncov dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc receptor usage and cell entry of porcine epidemic diarrhea coronavirus a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 conformational states 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server for prediction of protective antigens, tumour antigens and subunit vaccines comprehensive analysis of dengue virus-specific responses supports an hla-linked protective role for cd8(+) t cells development and validation of a broad scheme for prediction of hla class ii restricted t cell epitopes pep-fold3: faster de novo structure prediction for linear peptides in solution and in complex patchdock and symmdock: servers for rigid and symmetric docking fast interaction refinement in molecular docking a web server for fast interaction refinement in molecular docking potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies identification of a novel conserved hla-a*0201-restricted epitope from the spike protein of sars-cov hla-a*0201 t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins identification of an hla-a*0201-restricted cd8+ t-cell epitope ssp-1 of sars-cov spike protein searching immunodominant epitopes prior to epidemic: hla class ii-restricted sars-cov spike protein epitopes in unexposed individuals structure of sars coronavirus spike receptor-binding domain complexed with receptor the spike protein of sars-cov-a target for vaccine and therapeutic development severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice evaluation of modified vaccinia virus ankara based recombinant sars vaccine in ferrets identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines a study on antigenicity and receptor-binding ability of fragment 450-650 of the spike protein of sars coronavirus identification of an antigenic determinant on the s2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies screening and identification of linear b-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (sars)-associated coronavirus using synthetic overlapping peptide library unexpected receptor functional mimicry elucidates activation of coronavirus fusion two linear epitopes on the sars-cov-2 spike protein that elicit neutralising antibodies in covid-19 patients structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80r structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody better epitope discovery, precision immune engineering, and accelerated vaccine design using immunoinformatics tools. front in vivo validation of predicted and conserved t cell epitopes in a swine influenza model a prime-boost concept using a t-cell epitope-driven dna vaccine followed by a whole virus vaccine effectively protected pigs in the pandemic h1n1 pig challenge model cytokine storm induced by sars-cov-2 a decade after sars: strategies for controlling emerging coronaviruses recent advances in the vaccine development against middle east respiratory syndrome-coronavirus we thank all the researchers who submitted their sequencing data from gisaid on which this study is based. the authors declare no conflict of interest. key: cord-314051-dr27bsvt authors: lother, sylvain a. title: preoperative sars-cov-2 screening: can it really rule out covid-19? date: 2020-06-23 journal: can j anaesth doi: 10.1007/s12630-020-01746-w sha: doc_id: 314051 cord_uid: dr27bsvt nan expanded indications for testing the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) have been recommended as health systems emerge from pandemic-related slowdowns. routine preoperative screening prior to elective surgery has been broadly discussed. the putative benefits are intuitive: identify sars-cov-2 carriers before surgery to prevent adverse patient events, prevent further transmission, reduce consumption of resources and personal protective equipment, and improve hospital system efficiency. the harm associated with routine testing are less frequently considered. this editorial will address the deficiencies of real-time reverse transcriptase polymerase chain reaction (rt-pcr) as a method for detecting sars-cov-2 and provide information required to appropriately interpret test results. the purpose of sars-cov-2 screening with rt-pcr is to detect viral genetic material (rna) in the presymptomatic phase of infection. this incubation period for sars-cov-2 is protracted with initial low levels of viral rna until replication increases in the hours or days leading up to symptom onset. 1 on average, logarithmic viral replication and subsequent symptoms only start five to six days after exposure, but in some instances, this can be delayed up to 14 days. 2 before viral rna reaches detectable thresholds, patients may appear well prior to elective surgery despite being exposed to sars-cov-2 in the preceding 14 days. if viral carriage is not detected by testing, patients may proceed with elective surgery whereby signs and symptoms of coronavirus disease (covid-19) may arise in the postoperative period, leading to adverse outcomes. 3 while screening with rt-pcr may detect some presymptomatic preoperative patients, the window of diagnostic utility is small, and careful interpretation of negative and positive test results must be considered prior to altering the course of therapy. when evaluating any screening test, one must consider the sensitivity, specificity, and population prevalence of the target condition. without a reference standard, measuring rt-pcr sensitivity for sars-cov-2 in asymptomatic patients remains an unresolved problem. 4 as of 7 june 2020, clinical sensitivity has not been reported for any commercial tests in asymptomatic people. in the symptomatic cohort, considerable concern exists over false negative results, ranging from 11% to 40%. 5 -7 the probability of detecting sars-cov-2 also varies based on time from the exposure, being as low as 0% in the immediate days following exposure, 33% one day before symptom onset, 62% on the day of symptom onset, and peaking at 80% on day 3 of symptoms. 8 prevalence varies widely depending on the characteristics of the population of interest. testing in the united kingdom between 27 april and 10 may 2020 indicated that sars-cov-2 was present in 0.27% of the community population (95% confidence interval, 0.17 to 0.41). 9 thirty-three samples from 215 labouring women (15%) in new york taken between 22 march and 4 april 2020 were positive, but only four of these women were symptomatic. 10 highly variable testing accuracy and population characteristics will inevitably lead to false positives and false negatives. to illustrate, consider an imaginary population of 1,000 patients awaiting elective surgery in whom the true population prevalence of sars-cov-2 is 2% (table) . assume that all patients with sars-cov-2 are tested very soon before their symptoms emerge. the evidence cited above suggests a sensitivity of 70% and specificity of 95% are probably reasonable estimates. when the population prevalence is low the number of patients falsely diagnosed will be substantially greater than those correctly diagnosed. some will be incorrectly cleared for surgery. how will health systems deal with these results? interpreting a negative rt-pcr screening result for sars-cov-2 our imaginary population will largely test negative, so let's begin there. while the observed negative predictive value of 99% sounds attractive, health systems are given no guidance on how to manage these negative test results. should these patients proceed to surgery? can providers safely discontinue infection prevention and control measures? how should patients be counselled about their test results? what is their risk of postoperative covid-19? the answers to these questions are critically important prior to implementing widespread screening. while reports of rt-pcr sensitivity have varied widely, sensitivity is consistently low in the early or presymptomatic period. in our example, six patients with negative rt-pcr will be harbouring sars-cov-2 immediately before surgery. these patients are at high risk of morbidity and mortality. in a recent analysis of 278 patients undergoing elective surgery, the 30-day mortality was 18.9% when sars-cov-2 infection was detected between seven days preoperatively and 30 days postoperatively. 11 without detection, patients may also propagate sars-cov-2 transmission. to prevent transmission to care providers and others, appropriate infection control measures should be maintained for any patients with possible exposures in the preceding 14 days, regardless of test results. 12 this is supported by the american society of anesthesiologists/ anesthesia patient safety foundation joint statement, stating: ''because false negatives may occur with testing, droplet precautions should be used by or [operating room] staff for operative cases. before performing agmps [aerosol-generating medical procedures], healthcare providers within the room should wear an n95 mask, eye protection, gown and gloves''. 13 in this context, it is unclear if testing will effectively minimize risk while reducing resource utilization and improving efficiency. additionally, appropriate counselling on the benefits of proceeding with surgery weighted against the uncertain risks (based on false negative test results) in the postoperative period require careful discussion. interpretation of negative rt-pcr screening must be considered in the context of its many limitations. first, the probability of detecting viral rna is extremely low in the first few days following exposure. 8 if potential for a recent exposure exists, defer testing in favour of an observation period while limiting further exposure. 14 second, high-risk populations (e.g., close contact to a known covid-19 case, recent travel, high-risk occupation) will increase the pre-test probability and the chance of false negative results. these individuals should isolate and limit exposures prior to proceeding to surgery. lastly, rapid point of care tests are not yet validated in asymptomatic patients, so delays from sample collection to results (up to three days in some institutions) can lead to false reassurance. patients may screen negative despite being in the early incubation phase. viral load may increase in the time waiting for results. proceeding to surgery without considering testing limitations could lead to devastating outcomes. a positive rt-pcr result identifies a group of patients who may be infected with sars-cov-2 and should have elective surgeries delayed. this is clearly the most prudent path to take, and would allow for a period of observation, treatment, and recovery prior to pursuing elective surgery. importantly, providers will need to be comfortable discussing the risks, benefits, and alternatives of delaying surgery further, and how their underlying comorbidities could affect covid-19 outcomes. the impact of false positive testing is unknown. mass screening of patients in low prevalence settings will lead to considerable false positives, regardless of test specificity. using our imaginary population, this test would yield a positive predictive value of only 22%, leading to 49 false positive results for every 1,000 patients tested. these individuals are likely to experience further delays in elective procedures, unnecessary self-quarantine, and other psychosocial harms associated with receiving an erroneous covid-19 diagnosis. alternative strategy to routine sars-cov-2 screening before elective surgery screening with rt-pcr is insensitive at detecting sars-cov-2 early in its incubation phase. alternate strategies are required to minimize harm to patients and providers. perhaps the single best strategy to ensure patients arrive for surgery without subclinical sars-cov-2 infection is to mandate a period of strict self-quarantine for 14 days prior to elective surgery. any prior exposures would have sufficient time to dissipate or enter the symptomatic phase. patients who are asymptomatic after quarantine can safely proceed with surgery, without preoperative testing. nevertheless, not all patients will have the means to self-quarantine and this extreme strategy may increase social and economic stressors before elective surgery. few health systems will have the resources to ensure patients self-isolate for this length of time. thus, preoperative rt-pcr testing should be considered one of a suite of tools in the multipronged approach to minimizing patient/provider risk as elective surgeries ramp up. the downsides of testing must be considered and incorporated into patient/provider discussions before proceeding to surgery and altering the clinical course. in conclusion, sars-cov-2 preoperative screening programs are being considered to improve patient and provider safety, outcomes, and resource management. nevertheless, the putative benefits of screening with rt-pcr may not be realized because of low disease prevalence and poor test accuracy. further evidence and guidance is needed to manage both false positive and false negative results. in addition to testing, alternative strategies that include self-isolation and other distancing measures are needed to enhance outcomes as we emerge from pandemic-related slowdowns. dépistage préopératoire du sars-cov-2 : est-il véritablement possible d'exclure la présence de covid-19? l'élargissement des indications pour les tests de dépistage du syndrome respiratoire aigu sévère du coronavirus 2 (sars-cov-2) a été recommandé alors que les systèmes de santé émergent des ralentissements liés à la pandémie. le dépistage préopératoire de routine avant une chirurgie non urgente a été discuté dans les grandes lignes. les avantages supposés d'un tel dépistage sont intuitifs : identifier les porteurs du sars-cov-2 avant leur chirurgie afin de prévenir les complications pour le patient, prévenir une transmission supplémentaire, réduire l'utilisation des ressources et des équipements de protection individuelle, et améliorer l'efficience du système hospitalier. les effets nocifs d'un dépistage systématique sont moins souvent considérés. cet éditorial abordera les écueils du test d'amplification par la polymérase avec transcription inverse en temps réel (rt-pcr) comme méthode de dépistage du sars-cov-2 et fournira les informations nécessaires à interpréter adéquatement les résultats de ce test. l'objectif du dépistage du sars-cov-2 par rt-pcr est de détecter le matériel génétique viral (arn) pendant la phase présymptomatique de l'infection. cette période d'incubation du sars-cov-2 commence par de faibles taux initiaux d'arn viral jusqu'à ce que la réplication augmente dans les heures ou jours précédant l'apparition des symptômes. 1 en moyenne, la réplication virale logarithmique et les symptômes subséquents ne s'amorcent que cinq à six jours après l'exposition, et dans certains cas, jusqu'à 14 jours. 2 avant que l'arn viral n'atteigne des seuils détectables, les patients pourraient apparaître en bonne santé avant une chirurgie non urgente malgré avoir été exposés au sars-cov-2 au cours des 14 jours précédents. si la présence virale n'est pas dépistée par un test, les patients peuvent aller de l'avant avec leur chirurgie non urgente, à la suite de laquelle les signes et symptômes d'une atteinte au coronavirus (covid-19) pourraient survenir en période postopératoire, entraînant des devenirs défavorables. 3 bien que le dépistage par rt-pcr puisse identifier certains patients préopératoires présymptomatiques, sa fenêtre d'utilité diagnostique est étroite, et une interprétation prudente des résultats négatifs et positifs du test doit être envisagée avant de modifier le déroulement du traitement. lors de l'évaluation de tout test de dépistage, il faut tenir compte de sa sensibilité, de sa spécificité et de la prévalence dans la population de la condition ciblée. sans norme de référence, la mesure de la sensibilité du rt-pcr pour dépister le sars-cov-2 chez les patients asymptomatiques demeure un problème non résolu. 4 en date du 7 juin 2020, aucune sensibilité clinique n'a été rapportée pour un test commercial réalisé chez des personnes asymptomatiques. dans la cohorte symptomatique, il existe d'importantes inquiétudes quant aux résultats faux négatifs, allant de 11 % à 40 %. 5 -7 la probabilité de dépistage du sars-cov-2 varie également en fonction du temps écoulé depuis l'exposition, étant aussi faible que 0 % dans les jours suivant immédiatement l'exposition, 33 % un jour avant l'apparition des symptômes, 62 % le jour de l'apparition des symptômes, et atteignant un pic à 80 % au troisième jour de présence de symptômes. 8 à titre d'exemple, imaginons une population fictive de 1000 patients devant subir une chirurgie non urgente et dans laquelle la véritable prévalence du sars-cov-2 est de 2 % (voir tableau). partons du principe que tous les patients atteints par le sars-cov-2 sont testés peu après l'apparition de leurs symptômes. selon les données probantes citées ci-dessus, une sensibilité de 70 % et une spécificité de 95 % seraient probablement des estimations raisonnables. si la prévalence dans la population est faible, le nombre de patients diagnostiqués à tort sera considérablement plus élevé que le nombre de patients correctement diagnostiqués. certains patients recevront à tort l'autorisation de subir leur chirurgie. comment les systèmes de santé gèreront-ils ces résultats? prenons comme point de départ les résultats de test de notre population fictive, qui seront en grande partie négatifs. alors que la valeur prédictive négative observée de 99 % semble séduisante, les systèmes de santé n'ont reçu aucune directive sur la façon de gérer ces résultats de test négatifs. ces patients devraient-ils subir leur chirurgie? le personnel médical peut-il en toute sécurité ne prendre aucune mesure de prévention et de contrôle des infections? comment faut-il conseiller les patients concernant les résultats de leur test? quel est leur risque de covid-19 postopératoire? avant d'instaurer un dépistage généralisé, il est crucial de répondre à ces questions. alors que les comptes rendus portant sur la sensibilité du rt-pcr varient considérablement, la sensibilité est invariablement basse au début ou durant la période présymptomatique. dans notre cohorte fictive, six patients dont le test rt-pcr était négatif seront porteurs du sars-cov-2 immédiatement avant leur chirurgie. ces patients courent un risque élevé de morbidité et de mortalité. dans une analyse récente de 278 patients subissant une chirurgie non urgente, la mortalité à 30 jours était de 18,9 % lorsqu'une infection au sars-cov-2 était décelée entre sept jours préopératoires et 30 jours postopératoires. 11 sans détection, ces patients pourraient également devenir des vecteurs de transmission du sars-cov-2. afin de prévenir la transmission au personnel soignant et aux autres, il est important de maintenir des mesures de contrôle des infections adaptées pour tout patient ayant été possiblement exposé au virus dans les 14 jours précédents sa chirurgie, indépendamment des résultats de leur test. 12 la déclaration conjointe de l'american society of anesthesiologists et de l'anesthesia patient safety foundation soutient cette approche : « parce qu'il peut y avoir des faux négatifs dans les tests, les précautions contre les gouttelettes devraient être employées par le personnel de sop [salle d'opération] pour les cas opératoires. avant de réaliser une imga [intervention médicale générant des aérosols], les fournisseurs de soins de santé dans la salle devraient porter un masque n95, une protection oculaire, une blouse et des gants ». 13 dans ce contexte, il est impossible de déterminer si le dépistage minimisera effectivement les risques tout en réduisant l'utilisation des ressources et en améliorant l'efficacité. en outre, une consultation adaptée soupesant les avantages de la réalisation de la chirurgie et les risques incertains (si l'on se fonde sur les résultats de test faux négatifs) en période postopératoire justifie une discussion prudente. dans l'interprétation d'un dépistage négatif par rt-pcr, il faut tenir compte des nombreuses limitations une stratégie alternative au dépistage systématique du sars-cov-2 avant une chirurgie non urgente le dépistage par rt-pcr n'est pas suffisamment sensible pour détecter le sars-cov-2 au début de la phase d'incubation. il nous faut donc d'autres stratégies pour minimiser les effets néfastes pour les patients et les fournisseurs de soins de santé. la meilleure stratégie pour garantir que les patients se présentent pour leur chirurgie sans infection subclinique au sars-cov-2 serait peut-être d'exiger une période d'isolement volontaire stricte de 14 jours avant toute chirurgie non urgente. toute exposition passée aurait ainsi le temps nécessaire pour montrer une non-infection ou entrer en phase symptomatique. les patients asymptomatiques après cette période de quarantaine pourraient alors subir leur chirurgie en toute sécurité, sans dépistage préopératoire. il faut toutefois garder à l'esprit que tous les patients n'auront pas les moyens de se mettre en isolement volontaire, et cette stratégie extrême pourrait augmenter les facteurs de stress sociaux et économiques avant une chirurgie non urgente. peu nombreux sont les systèmes de santé qui disposeront des ressources nécessaires pour tableau précision diagnostique du dépistage préopératoire du sars-cov-2 dans une population avec une prévalence de 2 % de la maladie en conclusion, des programmes de dépistage préopératoire du sars-cov-2 sont envisagés pour améliorer la sécurité des patients et des professionnels de la santé, les pronostics, et la gestion des ressources. toutefois, les avantages supposés d'un dépistage par rt-pcr pourraient ne pas se concrétiser en raison d'une faible prévalence de la maladie et du manque de précision du test. des données probantes et des directives supplémentaires sont nécessaires pour gérer tant les résultats faux positifs que faux négatifs. outre le dépistage, des stratégies alternatives comprenant l'isolement volontaire ainsi que d'autres mesures de distanciation sont nécessaires pour améliorer les devenirs alors que nous émergeons du ralentissement lié à la pandémie. temporal dynamics in viral shedding and transmissibility of covid-19 incubation period and other epidemiological characteristics of, 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection false negative tests for sars-cov-2 infection -challenges and implications antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 false-negative results of initial rt-pcr assays for covid-19: a systematic review evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure coronavirus (covid-19) infection survey pilot: england universal screening for sars-cov-2 in women admitted for delivery mortality and pulmonary complications in patients undergoing surgery with perioperative sars-cov-2 infection: an international cohort study surgery in a time of uncertainty: a need for universal respiratory precautions in the operating room american society of anesthesiologists; anesthesia patient safety foundation. the asa and apsf joint statement on perioperative testing for the covid-19 virus ramping up delivery of cardiac surgery during the covid-19 pandemic: a guidance statement from the society of thoracic surgeons disclosures none. editorial responsibility this submission was handled by dr. gregory l. bryson, deputy editor-in-chief, canadian journal of anesthesia.déclarations aucune.déclaration de financement aucune.responsabilité éditoriale cet article a été traité par dr gregory l. bryson, rédacteur en chef adjoint, journal canadien d'anesthésie. key: cord-311125-v9ddes3c authors: cooper, keiland w.; brann, david h.; farruggia, michael c.; bhutani, surabhi; pellegrino, robert; tsukahara, tatsuya; weinreb, caleb; joseph, paule v.; larson, eric d.; parma, valentina; albers, mark w.; barlow, linda a.; datta, sandeep robert; di pizio, antonella title: covid-19 and the chemical senses: supporting players take center stage date: 2020-07-01 journal: neuron doi: 10.1016/j.neuron.2020.06.032 sha: doc_id: 311125 cord_uid: v9ddes3c abstract the main neurological manifestation of covid-19 is loss of smell or taste. the high incidence of smell loss without significant rhinorrhea or nasal congestion suggests that sars-cov-2 targets the chemical senses through mechanisms distinct from those used by endemic coronaviruses or other common cold-causing agents. here we review recently developed hypotheses about how sars-cov-2 might alter the cells and circuits involved in chemosensory processing and thereby change perception. given our limited understanding of sars-cov-2 pathogenesis, we propose future experiments to elucidate disease mechanisms and highlight the relevance of this ongoing work to understanding how the virus might alter brain function more broadly. disturbances in smell and taste have emerged as the predominant neurological symptom of the coronavirus disease 2019 , which is caused by sars-cov-2. perhaps as many as 80 percent or more of patients infected with sars-cov-2 report anosmia, hyposmia, ageusia, dysgeusia, or changes in chemesthesis (the ability to sense chemical irritants) (giacomelli et al., 2020; kaye et al., 2020; lechien et al., 2020a; parma et al., 2020; spinato et al., 2020; yan et al., 2020) . self-reported changes in chemical perception can predict whether a subject will test positive for sars-cov-2 (bénézit et al., 2020; fontanet et al., 2020; haehner et al., 2020; moein et al., 2020; shweta et al., 2020) ; one recent observational study that included more than two million participants revealed that the loss of smell and taste is more predictive than all other symptoms, including fatigue, fever or cough (menni et al., 2020) . most of these studies have lacked objective chemosensory assessment, raising the possibility that chemosensory disturbances are even more prevalent than currently appreciated; indeed, smell testing reveals increased odor detection thresholds in a subset of covid-19 patients who subjectively report a normal sense of smell (hornuss et al., 2020; iravani et al., 2020; moein et al., 2020; qiu et al., 2020) . these findings have prompted researchers to develop accessible smell tests (in which individuals rate the quality and intensity of scents originating from e.g., scratch-and-sniff cards or common kitchen items) for potential use as screening tools for covid-19 (iravani et al., 2020; rodriguez et al., 2020) . the close relationship between covid-19 and changes in chemical sensation raises questions about how sars-cov-2 might alter the cells and circuits charged with detecting stimuli and creating perception. identifying these pathophysiological mechanisms has important implications for the development of possible treatments, as well as for the design of clinical chemosensory assessments to detect sars-cov-2 infection. further, given that the covid-19 syndrome is associated with neurological symptoms (including dizziness, headache, and altered consciousness) and stroke, characterizing these mechanisms may shed light on how sars-cov-2 disrupts neural systems more broadly (docherty et al., 2020; helms et al., 2020; mao et al., 2020) . here we largely focus on interactions between sars-cov-2 and the olfactory system, which have been explored in some detail as the pandemic has progressed; as recent data suggests that sars-cov-2 may independently target taste and chemesthesis , we also briefly speculate on possible pathophysiological mechanisms in those systems. cov-2 belongs to the coronavirus family, which includes the pandemic mers-cov and sars-cov, and the lesser known but more common endemic coronaviruses hcov-oc43, hcov-hku1, hcov-229e and hcov-nl63. the endemic coronaviruses can infect the upper airway and frequently cause the common cold, which in turn is associated with both acute and chronic changes in smell and taste (dalton, 2004; mäkelä et al., 1998; pellegrino et al., 2020; rowan et al., 2015; suzuki et al., 2007; wood et al., 2011) . the main proposed mechanisms for acute viral-mediated changes in smell include conductive deficits caused by loss of patency due to swelling of the mucosa and increased mucus production, changes in mucus composition, and secondary changes in olfactory signaling caused by local release of inflammatory intermediates like cytokines (åkerlund et al., 1995; chen et al., 2019; damm et al., 2002; schlosser et al., 2016; trotier et al., 2007; victores et al., 2018; zhao et al., 2004) . while cold-causing viruses likely act through multiple mechanisms to influence smell, recovery from virus-associated olfactory deficits tend to resolve with a time course similar to that of other cold-related symptoms like nasal congestion (hummel et al., 1998a; hummel et al., 1998b; zhao et al., 2014) . in a subset of patients viral infections lead to long-lasting (i.e., months) post-viral anosmia, which is thought to result from direct damage to the olfactory sensory neurons (osns) responsible for odor detection in the olfactory epithelium (oe) (cavazzana et al., 2018; duncan and seiden, 1995; welge-lüssen, 2005; welge-lussen and wolfensberger, 2006) . partial or full recovery of olfactory function in these patients is likely due to the recruitment of stem cells in the olfactory epithelium, which can replace damaged osns over long timescales. the recovery process is often accompanied by parosmias -distortions of smell perception -associated with wiring errors between newborn osns and their post-synaptic targets in the olfactory bulb (ob) (figure 1 ) (leopold, 2002; rombaux et al., 2009) . some cases of post-viral anosmia have been hypothesized to be the consequence of viral damage to central nervous system structures; in these cases, coronaviruses and other viruses are thought to gain access to the ob either directly via osn axons, or indirectly by passing through perforations in the cribriform plate ( figure 1 ) (barnett and perlman, 1993; schwob et al., 2001; van riel et al., 2015) . however, the natural history of covid-19-associated anosmia argues that sars-cov-2 attacks the olfactory system through mechanisms distinct from those used by the more benign endemic coronaviruses. many patients report anosmia as their first symptom, or in the absence of rhinorrhea or nasal congestion, suggesting that if inflammation is a key component of pathogenesis it is local rather than generalized (giacomelli et al., 2020; kaye et al., 2020; lechien et al., 2020b; parma et al., 2020; spinato et al., 2020; vaira et al., 2020) ; indeed the sensitivity of anosmia as a predictor of covid-19 increases in patients without other nasal symptoms shoer et al., 2020) . consistent with this observation, imaging studies of the olfactory system in covid-19 patients are either normal or reveal focal inflammation (eliezer et al., 2020; galougahi et al., 2020) . in addition, resolution of anosmia seems more rapid than observed in post-viral olfactory loss, in many cases occurring in weeks (rather than months) after initial symptoms develop kaye et al., 2020; lechien et al., 2020a; yan et al., 2020) . finally, the limited data currently available suggest that parosmias are infrequent during or after covid-19 recovery (although this may change as we learn more) (lechien et al., 2020a; parma et al., 2020) . coronaviruses are so named because of the halo of spike (s) proteins that decorate their surface (du et al., 2009; perlman and netland, 2009 ). these s proteins interact with specific cellular receptors to bind host cells; binding is followed by protease-mediated s protein cleavage, which exposes fusion-promoting domains that enable viral entry. sars-cov-2 infects cells through interactions between its s protein and the angiotensin i converting enzyme 2 (ace2) receptor on target cells; ace2 plays a crucial modulatory role in the renin-angiotensin system, which regulates blood pressure and salt water balance (bader, 2010; shang et al., 2020b; walls et al., 2020; zhou et al., 2020) . infection requires s protein cleavage, likely by the host cell serine protease tmprss2, although other proteases may also be involved (hoffmann et al., 2020a; hoffmann et al., 2020b; zang et al., 2020) . with the exception of hcov-nl63, the endemic coronaviruses do not use ace2 as their primary cellular receptor (belouzard et al., 2012; zumla et al., 2016) , a molecular distinction that likely underlies key differences in pathophysiology. sars-cov also uses ace2 as its main receptor, and in one case study sars-cov infection was associated with anosmia (hwang, 2006) , although (unlike covid-19) chemosensory disturbances are not a hallmark of sars. differences between sars-cov and sars-cov-2 in terms of their impacts on chemosensory systems may relate to biophysical differences, as the receptor-binding domain of the sars-cov-2 spike protein binds ace2 with higher affinity and with a different binding mode than that of sars-cov (li et al., 2003; shang et al., 2020a; walls et al., 2020) . species variation in the protein sequence of ace2 significantly affects its affinity for the sars-cov-2 s protein, which in turn renders distinct model organisms differentially susceptible to infection (wan et al., 2020; zhao et al., 2020) . given data suggesting that ace2 is necessary for sars-cov2 to infect host cells, researchers have used a variety of approaches to discern the pattern of expression of ace2 and other viral entry proteins across the tissue landscape, with the goal of inferring possible target cells and disease mechanisms. for example, two studies have reported that cells in the nasal respiratory epithelium (re) have higher expression of sars-cov-2 entry genes than cells in the re that line the trachea or lungs (hou et al., 2020; sungnak et al., 2020) . consistent with this finding, recent work in macaques, ferrets and cats identifies the nasal epithelium as a major source of viral rna after sars-cov-2 infection (munster et al., 2020; shi et al., 2020) . these data suggest that the nasal epithelium may act as a major reservoir for the virus (figures 1 and 2 ). although much of the human nose is lined with re, sensory detection occurs in the olfactory epithelium, which houses osns and is located in the superior-most regions of the nasal epithelium (figures 1 and 2) . the oe is a complex chemosensory tissue composed of multiple cell types, including immature and mature osns, non-neuronal cell types, such as the sustentacular, bowman's gland and microvillar cells, and stem cells including globose and horizontal basal cells. sustentacular cells are particularly intimately associated with osns, and "enwrap" the sensory dendritic cilia that project into the airspace and enable odor detection (liang, 2020) . four recently published studies -using species ranging from mouse to human -have explored the cell types in the oe that express ace2 and other viral entry genes chen et al., 2020b; fodoulian et al., 2020; ziegler et al., 2020) ; all four conclude that osns do not express ace2 ( figure 2 ). instead, co-expression of ace2 and tmprss2 was observed in key support cells (including sustentacular, bowman's gland and microvillar cells) and stem cells that repopulate the epithelium after damage. although ace2 mrna was identified using single cell rna sequencing (scseq) techniques in only a small subset of these cells, both brann et al. and fodoulian et al. have demonstrated high level expression of ace2 protein in a large population of sustentacular cells concentrated in the dorso-medial aspect of the mouse oe, which corresponds to the dorsal "zone" traditionally identified via molecular markers (gussing and bohm, 2004; miyamichi et al., 2005) . consistent with this observation, mouse and human sustentacular cells identified as ace2 positive by scseq largely derive from the dorsal zone . the co-expression of ace2 and tmprss2 suggests that oe support cells may be the initial targets of sars-cov-2 infection. these inference-based conclusions are increasingly being pressure-tested by experiments in which model organisms are directly subject to sars-cov-2 infection. one recent paper in the golden hamster reports that sars-cov-2 infects sustentacular cells but not osns (bryche et al., 2020) . intriguingly, this phenotype is accompanied by damage to the osn ciliary layer and an increase in the number of microglia present in the oe, both of which are partially reverted to normal at 14 days after infection. a similar study in the hamster identified a large number of cells in the oe that were infected by sars-cov-2, although oe cell types were not definitively identified; inspection of the photomicrographs in that paper reveals that most sars-cov-2-positive cells traverse the thickness of the oe, suggesting that sustentacular cells are primary targets for infection (sia et al., 2020) . human oe samples obtained from covid-19 patients have similarly been queried for infection by sars-cov-2, which has revealed coronaviral antigens present in oe cells; infected cell types were not unambiguously identified, but their shape and position is consistent with the virus targeting sustentacular cells rather than osns (cantuti-castelvetri et al., 2020; meinhardt et al., 2020) . smell reported in covid-19 ( figure 3 )? localized inflammation in the epithelium might block the olfactory clefts, a pair of narrow passages located in the superior regions of the nasal epithelium through which air must flow to reach the oe, which comprises only five percent of the total nasal epithelium in humans (besser et al., 2020; trotier et al., 2007) . consistent with this possibility, a recent ct study of a covid-19 patient who presented with anosmia revealed blocked olfactory clefts (eliezer et al., 2020) . alternatively, infection of support cells and the attendant inflammation may cause local increases in inflammatory intermediates such as cytokines, which have been shown to influence osn function in a non-cell autonomous manner . indeed, a recent study demonstrates elevated levels of inflammatory cytokines in the oe of infected patients (torabi et al., 2020) . inflammatory intermediates have been suggested to indirectly lower the expression of odorant receptor (or) genes by osns, which could cause significant changes in odor perception; this work also shows that or expression levels return to normal after cessation of the inflammatory insult . finally, sars-cov-2 infection of support cells might alter the oe microenvironment in a manner deleterious to function. bowman's glands, for example, secrete mucus, which is essential to normal odor detection (dear et al., 1991; kern, 2000; solbu and holen, 2011) . sustentacular and microvillar cells remain somewhat mysterious, but seem to structurally support sensory neurons, phagocytose and/or detoxify potentially damaging agents, generate inflammatory cytokines, manage energetics through delivery of glucose to osns, and maintain local salt and water balance (baxter et al., 2020; lemons et al., 2017; o'leary et al., 2019; suzuki et al., 1996; ualiyeva et al., 2020; vogalis et al., 2005) . damage to support cells thus has the potential to change ion gradients and fuel availability, which in turn could acutely influence osn firing rates. furthermore, the important role of sustentacular cells in anatomically supporting osn sensory cilia (which are surrounded by sustentacular cell processes) is highlighted by the observation that infection of sustentacular cells by sars-cov-2 in the golden hamster is linked to the denuding of osn cilia, even in the absence of osn infection (bryche et al., 2020) . at least a subset of patients with covid-19 appear to have longer lasting anosmia, which may reflect more widespread damage to osns and the oe (as is seen in typical post-viral syndromes) (jafek et al., 1990; lechien et al., 2020b; welge-lussen and wolfensberger, 2006) . such damage could be a consequence of high levels of local inflammation, or a secondary consequence of the death of support cells, which in mouse models can lead to cascading changes in the structure and function of the oe (bergström et al., 2003; chen et al., 2019; herrick et al., 2017) . of note, the horizontal basal cells that are normally quiescent but play a particularly important role in regenerating damaged oe also express ace2 schwob et al., 2017) ; infection of these cells may therefore slow functional recovery over long timescales. recent mri studies have revealed transient changes in the ob that accompany covid-related anosmia, consistent with some degree of central involvement in at least a subset of patients (laurendon et al., 2020; politi et al., 2020) . certain coronaviruses can pass from the oe through the cribriform plate to infect the ob (dubé et al., 2018; durrant et al., 2016) . it remains unclear whether sars-cov-2 (given that it likely does not directly infect osns, and thus cannot pass directly through the olfactory nerve, see however, scseq and immunostaining of the mouse ob has revealed -as in the nose -that bulb neurons do not express detectable levels of ace2 ( figure 2 ) . consistent with this finding, targeted deep sequencing of dopaminergic juxtaglomerular cells, which receive direct inputs from nose osns, failed to reveal ace2 mrna . furthermore, ob cell targeting was not observed in studies of hamsters infected with sars-cov-2 (bryche et al., 2020) . in contrast, vascular pericytes in the ob expressed high levels of ace2 protein , consistent with the reported expression of ace2 in perivascular cells in the brain and throughout the body (chen et al., 2020a; he et al., 2020) . pericytes are critical for maintaining the blood-brain barrier, for defining local blood pressure, and for mediating neuroimmune responses (armulik et al., 2011) ; infection of these cells thus has the potential to alter perfusion or recruit inflammation, both of which can indirectly influence the function of neural circuits. thus, while vascular effects and inflammation could influence central brain structures involved in odor perception, current data suggest that it is unlikely that sars-cov-2 directly infects ob neurons responsible for processing odor information from the nose and conveying it to the cortex. consistent with observations in the ob, there is an increasing amount of data suggesting that ace2 is not appreciably expressed in neurons in the brain at either the rna or protein level. meta-analysis of 10 separate mouse deep sequencing datasets drawn from across the central and peripheral nervous system did not reveal significant expression of either ace2 or tmprss2 in any neural cell type ; scseq analysis of human prefrontal cortex and hippocampus failed to identify any ace2-expressing cells (chen et al., 2020c) ; multiple immunostaining studies have demonstrated ace2 expression in brain vasculature but not in neurons or glia (brann et al., 2020; hamming et al., 2004; kehoe et al., 2016) . these expression-based studies are now being complemented by data from a variety of animal models that can be infected with sars-cov-2. infection of macaques, cats and ferrets with sars-cov-2 (which in the monkey and cat resulted in pulmonary infection) did not reveal any virus present in the brain (munster et al., 2020; shi et al., 2020) . in addition, recent human autopsy studies reveal that the brain contains the least amount of sars-cov-2 of any sampled tissue in the body (puelles et al., 2020) . in contrast to these observations, several reports have suggested that sars-cov-2 may be "neuroinvasive," which we take here to mean the direct infection of neurons or glia in the central nervous system (baig et al., 2020; li et al., 2020; meinhardt et al., 2020; morris and zohrabian, 2020; wu et al., 2020) . arguments supporting this position often refer to data demonstrating that coronaviruses can hop from the nose to the bulb in experimental mouse models (dubé et al., 2018; durrant et al., 2016; perlman et al., 1989; van riel et al., 2015) ; however, without exception, the coronaviruses that have this property either do not use ace2 as a receptor, were explicitly selected during passage for their neurotropic potential, or both (butler et al., 2006; cowley and weiss, 2010) . many lines of evidence for neuro-invasiveness are drawn from prior work on sars-cov, and its ability to infect ace2-expressing cells. for example, it has been observed that nasal instillation of sars-cov in mouse models expressing human ace2 (which has much higher affinity for the cov and sars-cov-2 spike protein than does mouse ace2) can result in widespread infection of brain tissues (netland et al., 2008; tseng et al., 2007; yang et al., 2007) ; however, these models transgenically express human ace2 in many cells in which it is normally absent, making it an inappropriate tool to probe viral tropism. notably, a recent experiment assessed the consequences of sars-cov-2 exposure in mice in which human ace2 is transgenically expressed under control of the mouse ace2 promoter (which should largely, although not perfectly, recapitulate endogenous patterns of ace2 expression); while nasal infection and subsequent spread was sufficient to ultimately kill these mice, post-mortem analysis failed to reveal sars-cov-2 in the brain (bao et al., 2020) . although there is a case report describing detection of sars-cov rna in the csf of a patient, as this rna could have originated from many cell types (including vascular cells), it does not clearly demonstrate the neuroinvasive potential of either sars-cov or sars-cov-2 (hung et al., 2003) . furthermore, two studies have failed to identify sars-cov-2 rna in the csf of patients (farhadian et al., 2020; schaller et al., 2020) . however, other lines of evidence more directly support the possibility that sars-cov-2 can directly infect neurons or glia. in one patient autopsy sample, sars-cov was identified via antigen staining to be distributed across the brain parenchyma . two papers have reported ace2 staining in astrocytes in the rat cerebellum and brainstem (gallagher et al., 2006; gowrisankar and clark, 2016) , a paper from the human cell atlas has reported ace2 expression in oligodendrocytes (muus et al., 2020) , and a meta-analysis of a single scseq dataset revealed low-level expression of ace2 in human middle temporal gyrus and posterior cingulate cortex (although that same dataset included data for only a handful of vascular cells, making it difficult to assess relative expression levels) (chen et al., 2020c) . two recent papers have observed anti-ace2 staining in the mouse olfactory bulb, the first in a subset of mitral cells (bilinska et al., 2020) , the second in all mitral cells (ueha et al., 2020) ; the latter paper also reports the presence of ace2 protein in all osn nuclei (but not osn cilia, raising the possibility of non-specific staining). cortical neurons in human brain organoids (which express ace2) can be infected by sars-cov-2 (gopalakrishnan et al., 2020) . perhaps most convincingly, nasal instillation of sars-cov-2 in a mouse with the human ace2 gene knocked into the mouse ace2 locus caused infection of brain neurons, although the number, distribution and identity of these cells was not characterized . we are only now beginning to understand sars-cov-2 pathogenesis and so it is important to recognize key caveats in interpreting data suggesting sars-cov-2 does or does not infect neurons and glia directly. conclusions drawn based upon the presence or absence of ace2 message or protein depend upon the specific methods being used: for example, scseq techniques have low sensitivity but an identifiable noise floor, whereas immunohistochemistry depends upon the vagaries of each anti-ace2 antibody. similarly, conclusions based upon sars-cov-2 infection of model organisms (transgenic or otherwise) depend upon the expression pattern and s protein affinity of the ace2 protein in each model, as well as a number of other species-specific factors relating to viral tropism and immune responses. it is clear that there are significant inconsistencies amongst the datasets that require reconciliation; definitively addressing the question of whether sars-cov-2 can infect human neurons (in the bulb or elsewhere) will ultimately require detailed analysis of autopsy tissue. it is also important to note that sars-cov-2 in principle has access to the brain through routes independent of the nasal epithelium, including via vasculature and nerves innervating infected tissues; these modes of infection have been observed for other viruses (dahm et al., 2016; koyuncu et al., 2013) . in light of this controversy, it is worth noting that many of the observed neurological consequences of covid-19 (like altered consciousness and stroke) can in principle be explained by a primary vasculopathy and hypercoagulability (chen et al., 2020d; helms et al., 2020; zhang et al., 2020) , or by a secondary deficit in brain perfusion caused by elevated cytokines (poyiadji et al., 2020) . in contrast, clinical signs of encephalitis (i.e., infection of neurons), such as increased risk of seizures and inflammatory csf, have not been commonly observed (lu et al., 2020) . this perspective is consistent with recent mri findings in covid-19 patients (beyrouti et al., 2020; coolen et al., 2020) , and two separate autopsy series that failed to identify any signs of encephalitis (schaller et al., 2020; solomon et al., 2020) . at this point, therefore, the balance of the clinical evidence points to sars-cov-2 influencing the brain indirectly through effects on vasculature, although this view may change as we learn more about viral pathogenesis and we gain access to more detailed clinical information. smell, taste and chemesthesis are readily distinguishable but synergize to create the perception of the flavor in the mouth. a vast spectrum of volatile food odors is detected retronasally by osns, while the taste repertoire is restricted to non-volatile sour, salt, sweet, bitter and umami stimuli that directly activate taste buds on the tongue. the spiciness of chili peppers and the cool of mint are neither odors nor tastes, but rather activate sensory neurons of chemesthesis that innervate oral epithelia. most recent reports of smell loss in covid-19 patients (see above) have generally considered smell and taste loss as unitary, and excluded consideration of chemesthesis altogether. importantly, recent data suggest that taste and chemesthesis may be disturbed independently of smell in covid-19 patients (adamczyk et al., 2020; lechien et al., 2020a; parma et al., 2020; vaira et al., 2020) . for example, parma et al. showed that ~60% of participants link taste loss (which could be attributed to flavor) to the deficits in at least one specific taste quality (e.g., salty taste), suggesting that at least some participants distinguish changes in the taste component of flavor . however, the basic taste modalities were not predictably affected, with some taste qualities being differentially impacted across individuals. what mechanistic hypotheses can explain the specific impact of sars-cov-2 on taste function, and which cell populations may be targeted by the virus such that their loss would lead to distorted taste? taste perception is supported by taste buds, which have a characteristic distribution on the tongue and which house taste receptor cells (trcs) that are categorized into 3 morphological types ( figure 4 ) (type i -support, type ii -sweet, bitter, or umami, and type iii -sour) (chaudhari and roper, 2010) . like osns, individual trcs are constantly being renewed by stem cells, and thus taste function and perception also depend on rapid and reliable production of the proper proportions of each of the different trcs (barlow and klein, 2015) . although formal analyses are only now emerging, a cursory mining of publicly available datasets of type ii and iii trcs in mice reveals that ace2 is expressed by sour-sensing type iii trcs, and to a lesser extent by bitter and sweet/umami-sensing type ii trcs (han et al., 2020; lee et al., 2017; qin et al., 2018; shigemura et al., 2019; zhang et al., 2019) . while type ii and type iii trcs express little to no tmprss2, cathepsins (ctsb, ctsl) are abundant, and may function as proteases to cleave sars-cov-2 spike protein at type ii and iii trcs. available datasets do not include transcriptional profiling for the type i trcs, which -much like olfactory sustentacular cells -extend cellular processes that wrap type ii and iii trcs (liang, 2020; yang et al., 2020) . in addition, type i cells degrade atp, which is used as a neurotransmitter by type ii cells to convey taste information to the brain via the gustatory nerves (bartel et al., 2006; finger et al., 2005; vandenbeuch et al., 2013) . if type i cells are indeed ace2 positive and targeted by sars-cov-2, the loss of support cells could lead to taste bud collapse via cell death and/or reduced efficacy of taste signals to gustatory nerves (figure 3 ). while human taste tissue has yet to be profiled, microarray data comparing macaque taste and non-taste lingual epithelia reveal low levels of ace2, tmprss2, ctsl and ctsb in both tissue compartments (hevezi et al., 2009) , suggesting that the machinery necessary for sars-cov-2 infection of taste relevant cells may be present in humans. adult taste stem cells in the posterior tongue (where they are most abundant) express the marker lgr5 and can give rise to all trc lineages (yee et al., 2013) . despite a limited sample size, scseq profiling revealed that murine lgr5-positive stem cells express ace2 and trmpss2, and thus may be competent for infection by sars-cov-2 ( figure 4 ) (qin et al., 2018) . these data raise the possibility that taste dysfunction in covid-19 patients may be caused or exacerbated by insufficient trc renewal due to sars-cov-2 stem cell damage. in addition, it has been shown that experimentally-induced systemic inflammation in mice can reduce taste stem cell output, which in turn leads to depopulated taste buds and perturbed taste function (cohn et al., 2010; feng et al., 2014; kaufman et al., 2018; kim et al., 2012; wang et al., , 2009 ). if sars-cov-2 directly infects the tongue, local inflammatory processes could therefore alter stem cell properties and ultimately influence taste perception (figure 3 ). consistent with this possibility, scseq analysis in mice and humans suggests that ace2 is highly expressed in subsets of tongue epithelial cells, as are other proteases linked to coronavirus entry, e.g., tmprss11d, tmprss4, ctsb (pisco et al., 2020; schaum et al., 2018; venkatakrishnan et al., 2020; xu et al., 2020) . it is important to note that -unlike osns -trcs are not neurons; thus, all of the cell types identified as ace2 positive in the tongue to date are either stem or epithelial cells. transcriptional profiling of sensory neurons in the geniculate ganglion, whose fibers innervate a subset of taste buds, indicate that ace2 and tmprss2 are not expressed (figure 4) (dvoryanchikov et al., 2017; zhang et al., 2019) . similarly, the vagal sensory neurons that innervate taste buds in the larynx do not express ace2 or tmprss2 (prescott et al., 2020) . obtaining more extensive data regarding sars-cov-2 relevant gene expression in taste epithelial populations (which have been woefully undersampled compared to similar populations in the olfactory system) will provide a better basis from which to propose mechanistic hypotheses. in addition to taste and smell perturbations, subsets of covid-19 patients experience a disruption of chemesthesis. chemesthesic stimuli are detected by a variety of epithelial sensors and relayed to the brainstem via trigeminal ganglion sensory neurons, distinct branches of which innervate the nasal respiratory re and the non-taste epithelium of the tongue, among other targets (frasnelli and manescu, 2017; viana, 2011) . these trigeminal somatosensory afferents are enriched for trp channel receptors that detect chemical irritants (roper, 2014) . in the nasal cavity several irritants are detected by solitary chemosensory cells (sccs), which express many of the molecules required for bitter taste detection and signal transduction that characterize type ii trcs and are innervated by trigeminal fibers (finger et al., 2003; gulbransen et al., 2008; tizzano et al., 2010) . sccs are capable of driving systemic physiological responses to aversive substances (like reduced respiration) and can recruit a local neurogenic inflammatory response (saunders et al., 2014; tizzano et al., 2010) . as with the type i trcs, it is not yet clear from sequencing data whether sccs express sars-cov-2 cell entry genes. thus how sars-cov-2 influences chemesthesis remains uncertain, although these effects are unlikely to be mediated by trigeminal neurons, which do not express ace2 or trmpss2 (nguyen et al., 2019; nguyen et al., 2017) . research into possible mechanisms underlying changes in chemical perception due to covid-19 has only just begun, and much remains to be learned about the pathophysiology of sars-cov-2. that said, current evidence favors a model in which sars-cov-2 cell entry genes in the olfactory, gustatory and chemesthetic systems are not expressed in primary or secondary neurons, but rather are expressed in epithelial, support and stem cells responsible for maintaining perception. this model suggests that neural function is altered indirectly due to sequelae of sars-cov-2 infection of peripheral support cells, including (but not limited to) local inflammation and changes in osn gene expression and ciliary structure. observed disease-associated changes in ob mri intensity suggest that in a subset of patients there is central involvement as well; given the transient nature of these changes, it is likely that they reflect either local inflammatory processes (that are e.g., a consequence of vascular infection) or more speculatively are a consequence of inflammatory cytokines diffusing from the dorsal epithelium across the perforations in the cribriform plate. although less likely given current evidence, it also remains possible that sars-cov-2 directly infects osns or bulb neurons. the notion that chemical sensory deficits in covid-19 result from infection of support cells highlights an important lacuna in our understanding: while an enormous amount of effort has been devoted to understanding the molecular mechanisms underlying chemosensation -and the neural pathways that convey information about chemical cues to the brain -we still know little about the non-neuronal cells and structures that support sensory transduction. in this sense, sars-cov-2 provides an important opportunity to gain insight into how the complex peripheral tissues that support taste, smell and chemesthesis enable meaningful interactions with the world. much of our current understanding of pathophysiological mechanisms has been inferred from patterns of ace2 and tmprss2 expression. however, these inferences are constrained by our current understanding of the virus, and the predicted relationship between ace2 mrna and protein. although the evidence that ace2 is obligate for sars-cov-2 entry is strong, it has been suggested that other molecules such as bsg, neuropilin-1, or pikfyve may participate in sars-cov-2 entry (cantuti-castelvetri et al., 2020; chen et al., 2005; kang et al., 2020; ou et al., 2020; wang et al., 2020) . furthermore, it has recently been reported that low level expression of ace2 may be sufficient to support sars-cov-2 infection (lamers et al., 2020) , suggesting that sars-cov-2 may infect apparently ace2-negative cell types. it is also important to note that many of the conclusions drawn about cell entry gene expression (in e.g., the olfactory bulb) are based largely upon mouse data. although the mouse and human ace2 and tmprss2 expression data align nearly perfectly in the olfactory epithelium , there may be clinically relevant divergences between species in the olfactory bulb and brain (hodge et al., 2019; maresh et al., 2008) . finally, it is important to note that the ace2 gene is regulated by inflammation in human cells, and other sars-cov-2 entry genes may be similarly modulated by primary infection and inflammation (ansari et al., 2020; ziegler et al., 2020) . this observation raises the possibility that a broader spectrum of cells expresses ace2 during sars-cov-2 infection than is currently appreciated. addressing mechanistic hypotheses will be facilitated by further experiments in mouse models in which human ace2 is expressed under the control of the mouse ace2 promoter , and by more extensive use of non-mouse model organisms, such as hamsters, ferrets, cats and monkeys, all of which are susceptible to infection by sars-cov-2 (bryche et al., 2020; munster et al., 2020; shi et al., 2020; sia et al., 2020) . similarly, the development and use of benign viruses pseudotyped with the sars-cov-2 spike protein can be used to identify targeted cell types without the burden of a bsl-3 containment system. developing tools to characterize the pattern of infection of sars-cov-2 in the olfactory system should be complemented by physiological studies in the oe, ob and cortex -and parallel behavioral workexploring how the function of the olfactory system evolves after sars-cov-2 infection. importantly, the olfactory epithelium and tongue can be sampled in live human subjects without dissection, which holds the promise for their use as models for characterizing sars-cov-2 interactions with the human nervous system. all of this sars-cov-2specific work will be usefully complemented by additional work exploring the physiological function of support cells in the nose and tongue. finally, ongoing basic science efforts will benefit from more and better clinical data (whitcroft and hummel, 2020) . given the diversity of symptoms reported by patients, it remains unclear whether covid-19 attacks chemosensation through one or many pathophysiological mechanisms, or whether specific smell or taste qualities are particularly affected. similarly, we lack an understanding of how smell, taste and chemesthesis evolve over the long term in the subset of patients that do not exhibit a quick recovery. revealing the mechanisms through which sars-cov-2 influences chemical sensing will have important implications for our understanding of how viruses can functionally alter sensory systems in specific, and neural circuits more generally. chemosensation occurs in sensory epithelia in the nose and mouth. multiple cranial nerves relay the senses of smell, taste, and chemesthesis to the brain. airborne odors are detected by olfactory sensory neurons that reside in the olfactory epithelium; their axons pierce the cribriform plate to enter the olfactory bulb in the brain. taste buds on the tongue are innervated by sensory afferents from the facial nerve (vii) and glossopharyngeal nerve (ix). the vagus nerve (x) also innervates taste buds residing in the pharynx. the detection of pungent chemicals, also known as chemesthesis, is mediated by both oral and nasal afferents of the and olfactory epithelium, as well as the olfactory bulb in the brain are colored (left). for each tissue, a schematic of the anatomy and known major cell types are shown (middle). the nose contains both respiratory and olfactory epithelia. the olfactory epithelium is restricted to medial portion of the superior turbinate and the superior portion of the nasal septum, whereas the respiratory epithelium is continuous with the upper airway (mygind and dahl, 1998) . olfactory sensory neurons within the olfactory epithelium are responsible for detecting odors, and in mice and humans they are continuously regenerated from globose progenitors throughout life (durante et al., 2020; schwob et al., 2017) . the olfactory epithelium also contains other support and non-neuronal cell types, as well as reserve horizontal basal stem cells that respond to injury and can reconstitute olfactory epithelial cell types (choi and goldstein, 2018) . in the respiratory epithelium, basal progenitor cells generate all epithelial cell types, including ciliated and secretory cells. in the olfactory bulb in the brain (tan) the axons from olfactory sensory neurons coalesce into glomeruli, and mitral/tufted cells innervate these glomeruli and send olfactory projections to downstream olfactory areas. ace2 positive cell types are indicated in gray (right). four recent reports have all concluded that ace2 is not expressed in olfactory sensory neurons sustentacular cells, bowman's gland cells, and microvillar cells in the olfactory epithelium express both ace2 and tmprss2 and may be directly infected by sars-cov-2. support cell infection may cause changes in the olfactory mucus or ion imbalances that can inhibit olfactory signaling; such changes may be rapidly reversible. the loss of these support cells in animal models can also result in the death of olfactory sensory neurons. (c) inflammatory cytokines may also directly or indirectly inhibit olfactory sensory neuron function. (d) although current data suggests that sustentacular and other support cells are the primary targets of sars-cov-2, a remaining possibility is that sars-cov-2 directly infects osns. (e) immunostaining for ace2 protein in the mouse olfactory bulb suggests that ace2 expression is restricted to vascular pericytes , which may directly (via perfusion changes) or indirectly (via inflammation) affect chemosensory perception in the brain. (f) taste and chemesthetic disturbances may result from the direct infection of cells in the tongue, the secondary consequences of obstruction due to inflammation, or damage following the release of inflammatory cytokines. (barlow, 2015) . (b) each bud is a collection of ~100 taste receptor cells (trcs) categorized into 3 morphological types (i -support or gliallike, ii -sweet, bitter, umami, and iii -sour). all trcs are continuously renewed from basal stem cells adjacent to taste buds ("basal cells") (gaillard et al., 2015; liu et al., 2013; okubo et al., 2009) . taste bud-fated daughters generated by basal cells exit the cell cycle and enter buds as postmitotic trc precursors, which differentiate into each of the trc types (miura et al., 2006; miura et al., 2014) . basal cells also give rise to keratinocytes of the non-taste lingual epithelium including surrounding taste buds. individual trcs have a brief lifespan ranging from 1-6 weeks (beidler and smallman 1965; hamamichi et al., 2006; perea-martinez et al., 2013) . type iii trcs make conventional presynaptic contacts, while type ii trcs have specialized contacts with sensory afferents to transmit taste information to the cns (finger et al., 2005; romanov et al., 2018; roper and chaudhari, 2017; taruno et al., 2013) . 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associated with a new coronavirus of probable bat origin receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues coronaviruses -drug discovery and therapeutic options pericyte ace2 expression in the olfactory bulb is inferred from the mouse data; there are currently no available human olfactory bulb sequencing datasets the authors would like to thank the global consortium for chemosensory research (gccr) for facilitating the collaborative environment in which this manuscript was conceived. srd is supported by grants r011dc016222 and u19ns112953 from the national institutes of health, and by the simons collaboration on the global brain. lab is supported by grants r01 dc012383 and r21 ca236480. kwc gratefully acknowledges support from uci inp. edl is supported by nidcd k23 dc014747 to v. ramakrishnan, r01s dc012555 and dc017679 to s. kinnamon, and r01 dc014253 to d. restrepo. mcf is supported by the yale medical school fellowship. pvj is supported by the national institute of nursing research under award number 1zianr000035-01. pvj is also supported by the office of workforce diversity, national institutes of health and the rockefeller university heilbrunn nurse scholar award. dhb is supported by an nsf graduate research fellowship. the authors have no conflicts to declare. key: cord-293691-ewerquin authors: sauerhering, lucie; kupke, alexandra; meier, lars; dietzel, erik; hoppe, judith; gruber, achim d.; gattenloehner, stefan; witte, biruta; fink, ludger; hofmann, nina; zimmermann, tobias; goesmann, alexander; nist, andrea; stiewe, thorsten; becker, stephan; herold, susanne; peteranderl, christin title: cyclophilin inhibitors restrict middle east respiratory syndrome coronavirus via interferon λ in vitro and in mice date: 2020-07-02 journal: eur respir j doi: 10.1183/13993003.01826-2019 sha: doc_id: 293691 cord_uid: ewerquin rationale: while severe coronavirus infections, including middle east respiratory syndrome coronavirus (mers-cov) cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use. objectives: we elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin a (csa) and alisporivir (alv) restrict mers-cov to validate their suitability as readily-available therapy in mers-cov infection. methods: calu-3 cells and primary human alveolar epithelial cells (haec) were infected with mers-cov and treated with csa or alv or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nfat, or map kinases. novel csa-induced pathways were identified by rna sequencing and manipulated by gene knockdown or neutralising antibodies. viral replication was quantified by qrt-pcr and tcid(50). data were validated in a murine mers-cov infection model. results: csa and alv both reduced mers-cov titers and viral rna replication in calu-3 and haec improving epithelial integrity. while neither calcineurin nor nfat inhibition reduced mers-cov propagation, blockade of c-jun n-terminal kinase diminished infectious viral particle release but not rna accumulation. importantly, csa induced interferon regulatory factor 1 (irf1), a pronounced type-iii-interferon (ifnλ) response and expression of antiviral genes. down-regulation of irf1 or ifnλ increased mers-cov propagation in presence of csa. importantly, oral application of csa reduced mers-cov replication in vivo, correlating with elevated lung ifnλ levels and improved outcome. conclusions: we provide evidence that cyclophilin inhibitors efficiently decrease mers-cov replication in vitro and in vivo via upregulation of inflammatory, antiviral cell responses, in particular ifnλ. csa might therefore represent a promising candidate to treat mers-cov infection. saudi arabia [1] and led to recurring human infections with more than 2,500 laboratory-confirmed cases and high case fatality rates of about 35% [2] . in ex vivo infection of human lung tissue, mers-cov targets bronchial and alveolar epithelial cells (aec) and leads to a detachment and apoptosis of aec [3] . recent reports analyzing autopsy material of deceased mers-cov-infected patients showed mers-cov antigen in aec and epithelial multinucleated syncytial cell conglomerates in vivo [4, 5] . accordingly, severe human infection presents as pneumonia with progression to acute respiratory distress syndrome [4, 5] . to date, no vaccine or specific treatment for mers-cov -or the recently ongoing pandemic caused by the novel severe acute respiratory syndrome cov 2 (sars-cov-2) -is approved and therapy relies on supportive measures only [2, 6] . while in vitro studies and experiments in non-human primates demonstrated benefits of a combination of type-i-interferon and antiviral compounds including ribavirin against mers-cov [7] [8] [9] , results from retrospective patient cohorts applying similar treatment regimens remain controversial [10] [11] [12] . cyclosporin a (csa) has been found to inhibit several human-pathogenic cov in cell lines originating from kidney or liver epithelia [13] [14] [15] [16] . however, the molecular mechanisms by which csa affects cov, including mers-cov, particularly in its primary target cells, the pulmonary epithelium, remain elusive. moreover, preclinical studies addressing the effect of csa or related compounds on mers-cov replication in vivo have been lacking to date. csa is known to block the peptidyl-prolyl cis-trans isomerase (ppi) activity of cyclophilins that is involved in diverse cellular processes (e.g. protein folding, 17). additionally, csa forms together with cyclophilin a (cypa) and calcineurin (cna) a ternary complex which blocks the cna-dependent activation of nfat (nuclear factor of activated t-cells), a process which accounts for the immunosuppressive effect of csa [18] . in addition, csa has also been shown to inhibit the map kinases jnk (c-jun n-terminal kinase) and p38 [19, 20] . here, we aimed to elucidate the distinct signaling pathways by which csa affects mers-cov in clinically relevant models such as primary human aec and a murine mers-cov infection model [21, 22] . we demonstrate that csa blocks mers-cov infectious particle egress, which is dependent on jnk. moreover, we for the first time provide evidence that csa triggered the activation of an antiviral defense state in lung epithelial cells. we show that csa is a potent inducer of interferon regulatory factor 1 (irf1), type-iii-ifn (ifnλ) and multiple interferon-stimulated genes (isgs). additionally, we demonstrate that oral application of csa induced a robust ifnλ response in vivo and, importantly, significantly reduced mers-cov replication and improved disease progression in infected mice. experiments with mers-cov were performed under biosafety level 4 conditions at the institute of virology, philipps-university of marburg, germany. human alveolar epithelial cells (haec) were isolated and cultured as previously described [23] . human lung tissue was obtained from patients who underwent lobectomy after informed written consent (departments of pathology and surgery, university of giessen, approved by the university of giessen ethics committee; az.58/15). calu-3 or haec were infected at a multiplicity of infection (moi) of 0.1 diluted in dmem/f12 without fcs at 37°c for 1 h. cells were washed with dmem/f12 with 10% fcs and supplemented with stimulatory/ inhibitory reagents as indicated. 24 h after infection (pi) cells were processed for quantitative pcr (maxima-sybr/rox qpcr-mastermix, thermo fisher) and supernatant was harvested for virus titration as described previously [24] . all animal experiments were performed in accordance with the german animal protection laws and were authorized by the regional authorities (g73/2017). c57bl/6 mice were purchased from charles river laboratories and housed under pathogenfree conditions. mice were intratracheally inoculated with adenovirus-hdpp4-mcherry (cloned at viraquest inc.) as described [21, 25] . five days post transduction, mice were infected intranasally with 1.5x10 5 tcid 50 /ml mers-cov (emc/2012). 50 mg/kg/day csa diluted in dmso or dmso alone were mixed with a nut/chocolate-creme, and offered to the mice for voluntary uptake. uptake was controlled daily. csa feeding started 2 days before mers-cov challenge. mice were sacrificed 4 or 7 days post mers-cov infection. all data are given as mean ± sem. statistical significance was analyzed by unpaired two-tailed student's t-test or by 1-way anova and post-hoc multicomparison tests as indicated in the respective figures. a p value of less than 0.05 was considered significant. *p < 0.05; **p < 0.01; ***p < 0.005. further experimental details can be found in the online supplement. to address the previously proposed antiviral activity of csa in clinically relevant cells, we infected the human bronchial epithelial cell line calu-3 and primary human alveolar epithelial cells (haec) with mers-cov and analyzed intracellular viral rna and infectious particle release in presence of dmso or csa ( figure 1 ). in both calu-3 and haec, csa treatment led to a >95% decrease of viral rna ( figure 1a ) and a reduction of viral titers in the supernatant by 2.6-2.8 log 10 , respectively ( figure 1b) . interestingly, and in accordance with reports from autopsy material of mers-cov patients [4] , mers-cov-infected calu-3 and primary haec both showed apoptotic cell loss and formation of multinucleated cell foci ( figure 1c ). addition of csa reduced cell foci formation and significantly reduced apoptosis induction ( figure 1c , d). in line, both cftr (cystic fibrosis transmembrane conductance regulator; figure 1e ) and enacβ (epithelial sodium channel beta; supplement fig.e1) protein expression was improved after addition of csa to mers-cov-infected calu-3. moreover, epithelial structural integrity and ability for vectorial water transport were reduced in mers-cov-infected control cells and significantly improved to normal levels in mers-cov-infected, csa-treated cells ( figure 1f , g). csa is known to act via multiple signaling pathways including cyclophilin ppiase activity, the cna-nfat axis as well as map kinase signaling [17] [18] [19] [20] . using specific inhibitors, we aimed to interfere with csa-affected pathways to identify relevant molecular signaling events involved in the csa-mediated reduction of mers-cov infection. inhibition of cna by its specific inhibitor calcineurin inhibitory peptide (cip), as well as inhibition of the downstream transcription factor nfat resulted in minor, statistically non-significant changes in mers-cov viral titers in both calu-3 and haec (figure 2a , b). the non-immunosuppressive derivate of csa, alisporivir (alv), that binds the ppiase but does not induce ternary complex formation of cypa with cna, reduced viral titers to a similar extent as csa, suggesting that the cypa-ppiase activity elicits the restrictive effect on mers-cov replication rather than ternary complex-mediated signaling events. moreover, alv reduced cell foci formation and loss of epithelial integrity to a similar extent as csa (supplement fig. e2 ). applying specific mapk inhibitors against jnk and p38, we revealed that inhibition of the map kinase jnk, but not of p38 reduced mers-cov titers in both calu-3 and haec ( figure 2a our data suggest that, as opposed to its well-known cna/nfatfigure 4c ). these data indicate that csa treatment mounts a distinct interferon-driven antiviral response in lung epithelial cells. to better understand the transcriptional programs leading to ifnλ induction in csa-treated cells, we analyzed the regulation of interferon regulatory factors (irfs). our data reveal significant upregulation of irf1 mrna levels upon csa treatment, but not of irf3, irf7 or irf9 ( figure 5a ). irf1 is known to be a specific activator of ifnl gene expression [26] . accordingly, we identified a significantly increased number of irf1-expressing cells in csa-stimulated calu-3 cells by immunofluorescence ( figure 5b ). in line, irf1 sirna knockdown significantly reduced ifnl mrna levels in csa treated calu-3 cells ( figure 5c ). accordingly, irf1 knockdown inhibited ifnλ release by more than 75 % as compared to control ( figure 5d ). to understand the extent to which the inhibition of mers-cov propagation in csa treated cells was mediated by irf1-mediated production of ifnλ, we performed knockdown of irf1 or neutralized cell-free ifnλ, respectively. our data demonstrated that silencing of irf1 but not treatment by control sirna lead to a significant increase in mers-cov released viral particles in csa-treated cells ( figure 6a , b). moreover, neutralizing antibodies directed against ifnλ1, ifnλ2 and ifnλ3 or against the less induced ifnβ were applied ( figure 6b ). neutralization of ifnβ had no significant impact on mers-cov replication after csa treatment, whereas application of anti-ifnλ1/2/3 treatment significantly increased mers-cov viral titers by 1.05 log 10 level ( figure 6b ). these data indicate that the antiviral effects of csa were at least partially mediated by an irf1-ifnλ signaling axis, and independent of type-i-ifn. as no specific treatment is approved for mers-cov or sars-cov(-2), current treatment strategies are supportive [29, 30] . treatments including recombinant type-i-ifn and antivirals (e.g. lopinavir/ritonavir) have been applied off-label to treat mers-cov and yielded only moderate efficacy with controversial results in retrospective studies, and data from prospective studies or randomized controlled trials are lacking [29, [31] [32] [33] . due to its receptor specificity to the human dpp4, only few animal models to study mers-cov pathogenesis and mers-cov-directed antiviral compounds have been accessible to date. for this study, mers-cov infection in the mouse was facilitated via intratracheal delivery of a human dpp4encoding adenovirus, that might cause low-level inflammation itself and inhomogeneous receptor distribution within the lung, present for a limited time frame. however, even if this model might not fully recapitulate the native cellular distribution or density of the receptor as seen in the human lung, high transduction efficiencies (≥ 95%, data not shown) allow efficient viral spread in the upper and lower respiratory airways with quick progression to severe lung injury [22] and with moderate changes in morbidity [34] . thus, model-specific neurotropism as seen in some of the transgenic hdpp4 mice [35] or the necessity to adapt virus isolates via multiple passages, which might potentially affect its susceptibility to interventional strategies, are circumvented. while prior exposure to adenovirus evokes moderate histological changes including perivascular and bronchiolar lymphocytic infiltration (data not shown), mers-cov infection led to a clearly distinguishable granulocytic, necrotizing interstitial pneumonia with alveolar edema formation as described previously [22] . moreover, we demonstrate that inhibition of cypa via csa or alv, which both potently block the cypa ppiase activity at the used concentrations [42] , results in a pronounced upregulation of type-iii-ifn on both mrna and protein level, which was mediated via irf1 and was accompanied by expression of antiviral isgs. among those, especially ifit1 (interferon-induced protein with tetratricopeptide repeats 1), has been reported to influence the pathogenesis of mers-cov, highlighting the relevance of our findings [43] . of note, type-iii-ifns have recently emerged as key antiviral players in the innate immune response to viral infections at mucosal and epithelial surfaces [44] [45] [46] [47] . they efficiently restrict different respiratory viruses, and act e.g. by limiting spread from the upper to the lower airways [44, [46] [47] [48] . as opposed to type-i-ifn, type-iii-ifn do not trigger detrimental immune responses that contribute to immunopathology in influenza infection [23, 25, 44, 49] . this might prove to be pivotal in the context of csa-dependent stimulation of ifnλ during cov, as severe human cov infections, like mers-cov and-while data are still limitedalso sars-cov-2, are characterized by an immunopathology with a strong cytokine induction [5, 50, 51] . in addition to defining a novel pro-inflammatory, antiviral expression profile induced by csa on lung epithelial cells, this study also demonstrated for the first time gen. virol. 2017. statistical significance was calculated using unpaired two-way student's t-test (a, b, c, d, g) with *p < 0.05. onestep rt-pcr system (invitrogen life technologies) as described previously [2, 3] . intracellular localization of endogenous irf1 protein was analyzed 3 and 4 hour post csa stimulation. dmso-treated cells were used as negative control. stimulated cells were fixed with 4% pfa and permeabilized with methanol/acetone for 10 min. cells were incubated with a rabbit monoclonal-anti-irf1 (1:100; cell signaling) and an alexafluor 594-conjugated secondary antibody (1:400; dianova). cell nuclei were counterstained with dapi. the samples were mounted in fluoprep (biomérieux) and images were recorded with a confocal laser scanning microscope (leica sp5). calu3 cells were seeded in 0.4µm pore size transwell cell culture dishes (corning) and cultured until achieving electrochemical resistances (ter) of ≥800ω /cm 2 as measured by millicell-ers2 device. cells were infected apically with mers-cov at ; as reported previously [4] . for quantification of mers-cov induced apoptosis a caspase 3/7 gloassay® sds-page and western blot was analyzed as described previously [5] . calu-3 cells were infected with mers-cov using a moi of 0.1 and stimulated with csa 1 hour after virus adsorption. 24 h pi cells were scratched off with 500 µl pbs supplemented with protease-inhibitor mix (calbiochem) and centrifuged for 5 min at 5,000 rpm. cell pellets were resuspended in sample buffer [6] containing 4% sds and boiled at 100°c for 10 min. after discharge of the probes out of the bsl4 laboratory another 10 min boiling step was performed before the samples were separated using an 7,5 % sds-gel. after blotting on a nitrocellulose membrane and blocking using pbsdef with 5% milk powder first antibodies (anti-cftr antibody, clone mm13-4 and mouse monoclonal anti-vinculin antibody both sigma-aldrich; enacβ antibody (e-10), sc-48428; santa cruz biotechnology) diluted in pbsdef with 1% milk powder were incubated overnight followed by secondary antibody-incubation for 1 h (goat anti-mouse/hrp and swine anti-rabbit/hrp; both dako). for visualization of the signals image lab software was used. all animal experiments were performed in accordance with the regulations of german animal protection laws and as authorized by the regional authorities for histopathological analyses of formalin-fixed, paraffin-embedded murine lung tissues, sections of 2 µm thickness were cut from four to six evenly distributed planes throughout the entire lungs and mounted on adhesive glass slides. the slides were stained with hematoxylin and eosin and coverslipped. histopathological evaluation was performed using an established four grade scoring scheme [7] including the following parameters: affected area, severity and distribution of interstitial inflammation, infiltration of macrophages, lymphocytes and granulocytes, necrosis, alveolar hemorrhage and edema as well as formation of bronchus-associated lymphoid tissue (balt) and perivascular, lymphocytic cuffing. figure onestep rt-pcr kit as described previously [2, 3] . quantification was carried out using a standard curve based on 10-fold serial dilutions of appropriately cloned rna ranging from 10 2 to 10 5 copies. bar graphs in represent mean ± sem of n = 4 isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus 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histopathologies in mouse models of acute pneumonia work with live mers-cov was performed in the bsl-4 facility of the philipps university, marburg, germany. we thank julia spengler, larissa hamann, stefanie jarmer, dirk becker and marc ringel for excellent technical and experimental assistance. we thank ralf bartenschlager for providing alisporivir. key: cord-315085-rucfowvv authors: sekulic, miroslav; harper, holly; nezami, behtash g; shen, daniel l; sekulic, simona pichler; koeth, aaron t; harding, clifford v; gilmore, hannah; sadri, navid title: molecular detection of sars-cov-2 infection in ffpe samples and histopathologic findings in fatal sars-cov-2 cases date: 2020-05-26 journal: am j clin pathol doi: 10.1093/ajcp/aqaa091 sha: doc_id: 315085 cord_uid: rucfowvv objectives: to report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) positive individuals. methods: postmortem examination was completed following centers for disease control and prevention public guidelines. numerous formalin-fixed paraffin-embedded (ffpe) tissue types from each case were surveyed for sars-cov-2 rna by quantitative reverse transcription polymerase chain reaction (qrt-pcr). sars-cov-2 viral genome was sequenced by next-generation sequencing (ngs) from ffpe lung tissue blocks. results: postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. viral rna was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qrt-pcr method. rna sequencing using ngs in case 1 revealed mutations most consistent with western european clade a2a with orf1a l3606f mutation. conclusions: sars-cov-2 testing and viral sequencing can be performed from ffpe tissue. detection and sequencing of sars-cov-2 in combination with morphological findings from postmortem tissue examination can aid in gaining a better understanding of the virus’s pathophysiologic effects on human health. used viral culture, electron microscopy, in situ hybridization, or immunohistochemical staining to detect viral involvement in different organs. in this study we report postmortem findings and detection and sequencing of sars-cov-2 viral rna from formalin-fixed paraffinembedded (ffpe) samples of multiple organs collected in 2 patients with antemortem detection of sars-cov-2. the decedents underwent postmortem examination following centers for disease control and prevention (cdc) public guidelines for the collection of specimens and were performed without the examination of brain or spinal cord. the lungs from case 1 were infused with formalin under gravity via the primary/main bronchi and allowed to fix for 24 hours in a container of formalin before dissection and sampling of sections. the lungs of case 2 were dissected fresh, and sampling of sections was done at the same time. for both autopsies the following organ tissues were sampled: lungs (all lung lobes), main bronchi, trachea, heart (left ventricle anterior/lateral/posterior, right ventricle, and interventricular septum), hilar and peribronchial lymph nodes, spleen, esophagus, stomach, small intestine, large intestine, liver (left and right aspects), pancreas, bilateral adrenal glands, bilateral kidneys, urinary bladder, skin, and skeletal muscle (psoas). representative tissue samples were submitted and in standard manner formalin-fixed and paraffin-embedded. paraffin tissue sections were stained with h&e and examined under light microscopy. rna was extracted from 3 unstained slides (0.5 μm thick) using maxwell rsc rna ffpe kit (promega) in elution volume of 30 μl. the cdc 2019-novel coronavirus real-time rt-pcr diagnostic panel assay was adapted as previously described 5 using taqpath 1-step rt-qpcr mastermix (thermo fisher), 2019-ncov n1 and n2 primer/probe sets, and control rnase p primer /probe set (idt). cycle threshold values less than 40 were considered positive. lung tissue samples from 2 non-sars-cov-2 patients with dad were run as controls and were negative. leftover extracted rna used for pcr was used for sequencing with an ampliseq sars-cov-2 panel. seven μl of rna was converted to cdna using superscript vilo iii (thermo fisher). library preparation was performed using ampliseq sars-cov-2 panel set and ampliseq plus library kit (thermo fisher). templating was performed with ion chef kit and sequencing was performed on ion s5 prime with io 530 chip kit (thermo fisher). alignment to sars-cov-2 reference (genbank id: mn908947) and variant calling was performed with ion torrent suite 5.10. variants calls were further confirmed by manual inspection using integrative genomics browser (broad institute). only variants with allele frequency greater than 90% were called. genomic epidemiology classification was performed using nextstrain.org on april 27, 2020. 6 the decedent was an 81-year-old man admitted from an assisted living facility with acute respiratory failure, fever, and positive nasopharyngeal swab sars-cov-2 test. eight days prior to admission the patient was noted to have developed an afebrile cough, initially thought to be associated with congestive heart failure. six days later, the patient was found to be febrile (38.7°c), and the following day he required 2 l/ min of oxygen. empirical management for concern of respiratory tract infection was initiated, and an outpatient nasopharyngeal swab collection for sars-cov-2 testing was performed. once the patient's sars-cov-2 testing returned positive he was immediately admitted the same day. the patient's medical history was otherwise notable for dementia, radiologic evidence of a left lung mass (managed with hospice care), coronary artery disease (status post coronary artery bypass grafting), atrial fibrillation (biventricular pacemaker implanted), congestive heart failure, peripheral artery disease (status post iliac stenting), diabetes mellitus, hypertension, dyslipidemia, chronic kidney disease, gout, smoking, cerebrovascular accidents, and urinary tract infections. surgical history was further notable for carotid endarterectomy, left inguinal hernia repair, and cataract surgery. laboratory testing around the time of admission was notable for cbc with pancytopenia, serum creatinine of 1.55 mg/dl, blood urea nitrogen of 37 mg/dl, serum glucose of 104 mg/dl, and brain natriuretic peptide of 1,156 pg/ml. blood and urine bacterial cultures showed no growth. infectious disease testing was negative for legionella, streptococcus pneumoniae, and hiv. body temperature was 37.8°c. pitting edema around the ankles was noted on examination. chest roentgenogram on the day of admission demonstrated diffuse patchy opacities in the right lung and subtle patchy opacities in the left lower lung ❚image 1❚. chest computed tomography (ct) on the same date revealed multifocal peripheral and central ground-glass opacities throughout the bilateral lungs, a pulmonary mass in the medial aspect of the left lower lobe, small left-sided pleural effusion, moderate cardiomegaly, and calcifications of the coronary arteries and thoracic aorta. after admission, the patient was initially given 2 l/ min of oxygen via nasal cannula but began to require increasing oxygen support up to 6 l/min. the patient was also noted to have evidence of encephalopathy compounding baseline dementia. based upon the patient's overall poor functional/physiologic status, underlying malignancy, comorbidities, and superimposed infection, it was determined that the patient's prognosis would be poor even with further management and as such, the patient was provided comfort measures. the patient was declared dead 5 days after admission, and an autopsy sine brain and spinal cord was permitted by the family. the autopsy was performed 29 hours after death. the postmortem gross examination of the lungs after 24 hours of inflation and submersion fixation with formalin revealed congested parenchyma superimposed on emphysematous changes, and the left lower lobe contained a mass lesion (4.5 cm in greatest dimension) ❚image 2❚. before fixation, the right lung weighed 1,200 g, and the left lung weighed 1,040 g. microscopically, the lungs revealed involvement of all lung lobes by acute/exudative phase of dad characterized by hyaline membrane formation, scattered squamous metaplasia of distal airways (alveoli, alveolar ducts, and respiratory bronchioles), and background emphysematous changes (image 2). bronchial and tracheal sampling showed only minimal submucosal chronic inflammation with unremarkable overlying respiratory epithelium. sars-cov-2 rna was detected in section of lung parenchyma, bronchi, and lymph node ❚table 1❚. the left lower lobe mass lesion was morphologically and immunophenotypically most consistent with large cell carcinoma, and there was evidence of metastatic involvement of ipsilateral hilar and peribronchial lymph nodes. other organs that revealed significant findings that contributed to the patient's overall physiologic status and terminal decline included the kidneys and heart. the kidneys showed evidence of acute tubular injury on a background of moderately advanced chronic changes of the renal parenchyma. the heart was enlarged (620 g) and showed changes associated with chronic ischemic heart disease: severe stenosis of native coronary arteries (left anterior descending, left circumflex, and right main coronary arteries), patent graft vessels, and moderately extensive replacement-type interstitial fibrosis. aside from the aforementioned findings, the gross and microscopic examinations of remaining organs/tissue did not reveal significant findings. lower levels of sars-cov-2 rna were detected in sections of spleen, heart, intestine, liver, and skeletal muscle. sars-cov-2 rna was not detected in samples of esophagus, stomach, kidney, adrenal, and skin samples ( table 1) . sequencing of the extracted viral rna from the lung showed that the virus derived from claude a2a with g11083t (orf1a l3606f) mutation ❚table 2❚. in summary, the cause of death was sars-cov-2 infection occurring in the setting of metastatic carcinoma, diabetes, and chronic ischemic cardiomyopathy, leading to respiratory failure. ❚image 1❚ (case 1) chest roentgenogram performed on the day of admission revealed diffuse patchy opacities in the right lung and subtle patchy opacities in the left lower lung (a), and computed tomography showed multifocal peripheral and central ground-glass opacities throughout the bilateral lungs, a pulmonary mass in the medial aspect of the left lower lobe, small left-sided pleural effusion, moderate cardiomegaly, and calcifications of the coronary arteries and thoracic aorta (b; the pulmonary mass and coronary artery calcifications are not present in the illustrated coronal slice). postmortem gross examination of the right lung showed congested parenchyma superimposed on emphysematous changes (c; bar scale equals 1 cm; pink areas represent incomplete formalin fixation). the second decedent was a 54-year-old man with a history of hypertension and type 2 diabetes mellitus. he presented to a community hospital with an acute episode of shortness of breath, which worsened with exertion, and a generally dry cough of 2 days' duration. the patient denied having fever, headache, malaise, and gastrointestinal symptoms. he reported having never smoked. in the emergency department, his vital signs were notable for tachycardia (heart rate of 118 beats/min) and oxygen saturation of 76%. he was afebrile. physical examination was notable for unlabored breathing with diminished, yet clear, breath sounds bilaterally, tachycardia, and good peripheral pulses. a chest roentgenogram showed diffuse bilateral airspace opacities with some areas of consolidation of the lower lungs, concerning for pneumonia ❚image 3a❚. the patient was admitted to the intensive care unit (icu) with acute hypoxic respiratory failure, requiring 15 l/min of oxygen via nonrebreather mask. initial labs showed an elevated d-dimer, and although there was some concern for pulmonary embolism, a ct scan was not pursued due to underlying renal function issues (elevated serum creatinine). instead, empiric anticoagulation was initiated (heparin bridged to enoxaparin sodium). a nucleic amplification test performed on a nasopharyngeal swab sample came back positive for sars-cov-2 later the same night. tests for influenza and respiratory syncytial virus were negative. on day 1 after admission, ❚image 2❚ (case 1) postmortem microscopic examination of the lungs showed diffuse alveolar damage characterized by hyaline membrane formation (a, ×100) and scattered squamous metaplasia of distal airways (b, ×100) on a background of emphysematous changes. bronchial sampling showed only minimal submucosal chronic inflammation with unremarkable overlying respiratory epithelium (c, ×100), and the left lower lobe lung mass lesion was consistent with large cell carcinoma (d, ×100). the patient was consented and enrolled in a trial to study the utility of the antiviral medication remdesivir in treating sars-cov-2-infected patients. he received his first dose of the 10-day course the same day. over the next few days, the patient's renal function showed improvement, but he still remained hypoxic despite escalation in oxygen therapy. a repeated chest roentgenogram performed 4 days after admission showed increased opacities bilaterally with a more consolidative appearance at the right lung base. blood and urine cultures grew coagulase-negative staphylococcus and enterococcus faecalis, respectively, and the patient was started on antibiotics (vancomycin and piperacillin/tazobactam). the patient's wbc count also increased significantly over time, with a shift to increased absolute neutrophil counts and immature precursors in the peripheral blood. lymphopenia was present beginning on day 2 of admission. a respiratory culture had no growth of bacteria or fungi. serial chest roentgenograms showed initial worsening of his lung disease to more severe diffuse interstitial infiltrates ❚image 3b❚ with subsequent stabilization of his imaging. he had increasing oxygen requirements beginning on day 9 after admission, and, despite appropriate therapy, continued to have low oxygen saturation. ❚image 2❚ (cont) the kidneys were notable for acute tubular injury on a background of moderately extensive chronic changes of the renal parenchyma (e, ×100). the heart showed sequelae of coronary artery atherosclerosis and chronic ischemic injury with replacement-type interstitial fibrosis (f, ×20). laboratory assessments around this time were notable for rapidly rising serum creatinine and liver enzymes. as a result of continued oxygen requirements, he was intubated on day 10 and also started on propofol with subsequent drop in blood pressure. intravenous fluid resuscitation and pressor medications were started. the patient's oxygen saturation still showed no improvement, so a cisatracurium besylate drip was started and the patient was transferred to our institution for further management. while a central line was being placed, the patient became bradycardic with a heart rate of 30 beats/min. an arterial blood gas test at the time showed respiratory and metabolic acidemia. despite further medication, the patient's heart rate continued to decrease to approximately 20 beats/min without a measurable blood pressure. the patient entered pulseless electrical activity arrest and resuscitative measures were initiated, with return of spontaneous circulation in approximately 2 min. the patient was evaluated for possible extracorporeal membrane oxygenation; however, he was deemed to be a poor candidate by the shock team. ventilator settings at the time were as follows: positive end-expiratory pressure, 15 cm h 2 o; fraction of expired o 2 , 100%; and respiratory rate, 20 breaths/min. despite continued icu support, the patient died 10 days after admission to the hospital and 12 days after first reported onset of symptoms. the autopsy was limited to examination of the chest and abdomen only, per the family's request. the autopsy was performed 39 hours after death. at postmortem examination, external findings were primarily limited to those of medical interventions such as intubation and central line placement. the decedent was overweight, bordering on obese, with a body mass index of 29.9 kg/m 2 . in situ examination was significant for bilateral serosanguineous pleural effusions of 300 ml within each hemithorax. grossly, bilateral lungs were strikingly heavy (right lung, 2,050 g; left lung, 1,100 g) with a congested appearance. sectioning revealed a diffuse and relatively uniform firmness to the parenchyma ❚image 3c❚. there were no focal lesions. histologically, sections from all lobes of the lungs showed varying stages of dad with some areas demonstrating prominent hyaline membrane formation and significant pneumocyte hyperplasia (acute/exudative stage) ❚image 4a❚ and others exhibiting patchy to diffuse intra-alveolar fibroblastic proliferation and interstitial edema (organizing stage) ❚image 4b❚. areas of marked intra-alveolar acute inflammation were also present focally involving all lobes except the left lower lobe, diagnostic of acute bronchopneumonia ❚image 4d❚. varying degrees of pulmonary edema, clusters of multinucleated giant cells ❚image 4c❚, and foci of squamous metaplasia were also noted scattered throughout the lung parenchyma. of note, despite extensive sampling and microscopic review, microthrombi were not identified. ❚image 3❚ (case 2) chest roentgenogram performed on the day of admission revealed diffuse bilateral airspace opacities with some areas of consolidation in the lower lungs, consistent with pneumonia (a). chest roentgenogram performed on day 10 of hospital admission (1 day prior to death) showed extensive multifocal patchy infiltrates throughout the lungs bilaterally (b). postmortem gross examination of bilateral lung showed congested and firm parenchyma (c). submitted sections of the trachea ❚image 4e❚ and proximal bronchi showed no significant abnormalities. additional significant findings noted at autopsy included evidence of diabetic glomerulosclerosis and acute tubular necrosis within bilateral kidneys ❚image 4f❚, an enlarged heart (560 g) with left ventricular hypertrophy, mild calcified atherosclerotic coronary artery disease, liver (2,300 g) and spleen (270 g) were enlarged secondary to acute congestion, and necrotizing granulomata within a right peribronchial lymph node and the spleen. a grocott methenamine silver stain performed on the section of lymph node revealed yeast organisms most consistent with incidental histoplasma capsulatum. sars-cov-2 rna was detected in sections of lung parenchyma, bronchi, lymph node, and spleen ( table 1) . the presence of sars-cov-2 rna was not detected in the heart, esophagus, stomach, intestines, liver, skeletal muscle, kidney, adrenal gland, or skin in case 2 (table 1) . attempts at viral genome sequencing did not produce adequate coverage for successful genomic evaluation in this case, possibly due to low viral titers resulting from remdesivir administration. in summary of these findings, the cause of death was sars-cov-2 infection occurring in the setting of diabetes and underlying cardiovascular disease leading to respiratory and subsequent multiorgan system failure. ❚image 4❚ (case 2) postmortem microscopic examination of the lungs revealed varying stages of diffuse alveolar damage characterized by extensive hyaline membrane formation (acute/exudative stage) (a, ×100) and intra-alveolar fibrin deposition (organizing stage) (b, ×100). scattered multinucleated giant cells were also seen (c, ×400). the patient also had significant superimposed bronchopneumonia (d, ×200). here we report the detection of sars-cov-2 rna in a wide range of organs similar to that reported by an investigation of cases from washington state. 7 this is the first study to our knowledge to demonstrate sars-cov-2 rna detection in several major organs and to sequence sars-cov-2 genome from postmortem ffpe sections. sars-cov-2 and sars-cov use spike (s) proteins to bind to the host cell via angiotensin converting enzyme 2 (ace2) as an entry receptor leading to the internalization of the complex by the host cell. [8] [9] [10] [11] ace2, a counterregulatory component of the renin angiotensin aldosterone system, is mainly localized in the heart, kidney, endothelium, and testis, but is also expressed at low levels in many other tissues such as the brain, intestines, and lung. 11 in this study, we detected sars-cov-2 rna in a number of ace2 expressing tissues including lungs, heart, and intestines. we were also able to detect viral rna from ffpe samples from upper airways, lymph node, spleen, and liver, which mirrors the finding of prior reports with analysis from fresh tissue. 7, 12 although virus rna was detected in extrapulmonary tissues, there was no histomorphologic evidence of acute pathologic changes attributable to the virus itself or an inflammatory response. although relatively few reports documenting postmortem pathologic findings in patients with sars-cov-2 have been published to date, specific patterns of injury have emerged. as is expected in the clinical setting of pneumonia and ards, the lungs have been found to be the most consistently and severely affected. several reports have demonstrated features of dad, primarily in the acute or exudative phase in the majority of autopsies performed. 2, 3, 8, 13 these have been characterized by intra-alveolar fibrin with associated hyaline membrane formation, markedly reactive pneumocytes, and variable squamous metaplasia. fewer cases showed more advanced changes associated with dad, including intra-alveolar fibroblastic proliferation and interstitial edema. 8, 13 additional findings that have been frequently noted include multinucleated giant cells, 3, 8, 13 acute inflammation in the form of bronchopneumonia and/or acute bronchiolitis, and relative paucity of chronic inflammatory cells. 2, 8, 11 both of our cases exhibited features of the acute phase of dad, while case 2 showed more prominent organization. in case 1, these features were superimposed on a background of chronic emphysematous changes and in the setting of concomitant large cell carcinoma of the left lung with associated ipsilateral nodal metastasis. additional pulmonary findings in case 2 included significant superimposed acute bronchopneumonia and several clusters of multinucleated giant cells. both patients also demonstrated histopathologic features of acute renal injury superimposed on a background of moderate chronic changes to the renal parenchyma, in keeping with their histories of diabetes mellitus and hypertension. case 2 exhibited classic features of nodular diabetic glomerulosclerosis. both patients also demonstrated congestive splenomegaly and ❚image 4❚ (cont) tracheal sampling showed only minimal submucosal chronic inflammation with unremarkable overlying respiratory epithelium (e, ×100). the kidneys were notable for acute tubular necrosis on a background of diffuse and nodular diabetic glomerulosclerosis (f, ×200). sinusoidal congestion of the liver. while these features have also been mentioned in other reports, 2,13 they may simply be related to terminal changes in these acutely ill patients rather than a direct result of the viral infection. also in keeping with the reported literature, the additional pathologic changes identified in our cases were associated with the patients' underlying chronic medical conditions and did not seem to be specifically related to sars-cov-2 infection. for example, case 1 showed advanced cardiovascular disease as evidenced by cardiomegaly, severe atherosclerotic coronary artery disease, and remote ischemic changes within the myocardium. cardiomegaly without significant coronary artery disease or evidence of ischemic injury was seen in case 2. neither heart showed features of lymphocytic myocarditis, which has been reported as a possible consequence of sars-cov-2 infection. 8 additionally, although several anecdotal reports regarding hypercoagulability have also begun circulating, histologic documentation of microthrombi has only rarely been reported in the published literature. 2 we were unable to identify any microthrombi in our 2 patients. gross and histopathologic examination of the upper respiratory tract (main bronchi and trachea), pancreas, adrenal glands, gastrointestinal tract tissues, urinary bladder, skin, and skeletal muscle in our cases showed no significant changes. other postmortem ancillary studies have been performed on sars-cov-2 patients. localization studies of sars-cov-2 by immunofluorescence revealed prominent expression on alveolar epithelial cells, including damaged, desquamated cells within the alveolar space. 4 ultrastructural examination by electron microscopy was able to locate viral particles in type ii pneumocytes, upper airway and intestinal mucosal epithelial cells, and proximal tubular epithelial cells. 7 tian et al 12 performed reverse transcriptase polymerase chain reaction (rt-pcr) for sars-cov-2 on postmortem ffpe core biopsies collected from 4 patients and reported detection of sars-cov-2 rna in heart, lung, and liver in at least 1 patient in their cohort. in our study we were able to consistently detect the virus in lung and lymph node samples, while bronchus samples also contained viral rna, but at lower relative concentration compared to lung tissue. it has been postulated that moderate levels of rna in lymph nodes reflect viral presence in leukocytes that may serve as a route for the virus to disseminate from airways or lung parenchyma to other organs. 7 similarly, in an autopsy study performed on macaques on day 4 post infection, the highest sars-cov-2 rna levels were detected in lungs, using quantitative rt-pcr. viral rna was also detected in the lymph nodes in 3 out of 4 animals. 13 monitoring the spread of virus in a pandemic is critical in disease control. shared sars-cov-2 genotyping efforts and resources like the global initiative on sharing all influenza data (gisaid, https://www.gisaid.org), nextstrain (https://nextstrain.org), and johns hopkins university dashboard (https://coronavirus.jhu.edu/map. html) have enabled dynamic tracking of the evolution of the pandemic, enhanced monitoring of infection patterns, and have allowed better estimations of the affected population size in communities. 14 like other rna viruses, the rna-dependent rna polymerase of sars-cov-2 lacks proofreading capability, leading to an accumulation of mutations in its genome over time. mutations in the critical proteins, including the s protein, rna polymerase, rna primase, and nucleoprotein, have been discovered. 15, 16 these mutations are not equally distributed across the genome and generate hotspots that can be linked to specific geographic location of the outbreak. knowledge and monitoring of these mutation sites is critical in designing a vaccine for preventing the infection as well as therapies for treatment of sars-cov-2. 14, 17 further studies showing the effect of these mutations on pathogenicity and pathobiology of the virus are needed. sequencing of viral rna from ffpe lung tissue from the case 1 autopsy showed mutations most consistent with a subset of the western european clade a2a (c3037t, c14408t, a23403g), 16 with mutations enriched in new york state a2a cases (c1059t and g25563t). 15 the viral rna from our cases also had g11083t mutation that to date has been described in only 2% (13/648) of the a2a clade that contain c1059t mutation. it has been suggested that the g11083t mutation is more common in regions (italy and brazil) with higher fatality rates. 18 the g11083 mutation was described in the first case of sars-cov-2 in italy. 19 a study examining the transmission and evolution of sars-cov-2 in cruise quarantine suggests that the g11083t mutation can be transmitted via rna recombination. 20 linkage disequilibrium analysis suggests that rna recombination with a g11083t mutation may contribute to the increase of mutations among the viral progeny. 20 in this case we did observe other mutations not extensively described in previous genomic sequencing efforts, including point mutations c9515t, c23378t, and in-frame deletion 12137_12142del (table 2 ). more studies are needed to determine if g11083t, or other mutations detected in this case, may increase the fitness of the carrier virus as a benefit allele in the future. the challenge that pathologists and the medical community at large face is determining if deaths in individuals who present with the clinical signs and symptoms of sars-cov-2 infection are truly infected with the virus. it is not standard for all autopsies to take swabs or put aside fresh tissue for potential china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china covid-19 autopsies pathological findings of covid-19 associated with acute respiratory distress syndrome histopathologic changes and sars-cov-2 immunostaining in the lung of a patient with covid-19 comparison of abbott id now, diasorin simplexa, and cdc fda eua methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from individuals diagnosed with covid-19 nextstrain: real-time tracking of pathogen evolution histopathology and ultrastructural findings of fatal covid-19 infections structure of sars coronavirus spike receptor-binding domain complexed with receptor angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor tissue distribution of ace2 protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model phylogenetic network analysis of sars-cov-2 genomes host, viral, and environmental transcriptome profiles of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) ajcp / original article the role of phylogenetic analysis in clarifying the infection source of a covid-19 patient evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission decoding the lethal effect of sars-cov-2 (novel coronavirus) strains from global perspective: molecular pathogenesis and evolutionary divergence molecular characterization of sars-cov-2 from the first case of covid-19 in italy faster de novo mutation of sars-cov-2 in shipboard quarantine molecular testing for sars-cov-2 detection, whereas production of ffpe tissue blocks is standard for most autopsies that require histologic examination. the ability to detect sars-cov-2 rna from ffpe tissue may assist in cases in which there are clinical, or even histologic, findings that overlap findings found in sars-cov-2 infection. ffpe blocks are stored on many autopsies in many places from the period preceding the recognized onset of community-acquired sars-cov-2 infection. recent reports suggest that sars-cov-2 infection has been detected in cases that preceded the previously recognized onset of community transmission in the united states. our results suggest that sequencing of ffpe materials could contribute to a greater understanding of the early onset and spread of sars-cov-2 infection in the united states or other countries.possible limitations for molecular testing on autopsy samples include sampling bias if virus involvement is focal. this can be addressed by obtaining multiple samples from each organ. our results showed widespread positivity of samples throughout sites in the lung and airways. this study supports sampling of upper and lower airways, immune organs (lymph node and spleen), and to a lesser degree the intestine, heart, and liver. another concern is postmortem tissue autolysis and rna degradation. further studies are required to determine the stability of viral rna in different organs after death. finally, since the virus may affect each organ differently, individual organs may be expected to have different viral loads at any given time in the course of the disease. however, the clinical correlation of such findings is yet to be determined.in conclusion, the findings of our postmortem examinations support major histopathologic features previously reported in the literature, the most striking of which is dad. changes in other organs, like the kidneys and heart, are likely secondary or related to underlying diseases. as might be expected given this pattern of injury, sars-cov-2 rna is more easily detected in lung and upper airway tissue when compared to other organs. interestingly, our study also documents detection in hematopoietic and lymphoid organs (lymph node and spleen) and may lend credence to the theory that leukocytes could serve as a route for dissemination of the virus from airways to other organs. 8 we propose that detection of sars-cov-2 rna from postmortem ffpe tissues from multiple sites can aid in tracking the spread of the novel coronavirus and in gaining a better understanding of its pathophysiologic effects on human health. additionally, the retrospective sequencing of archived ffpe material may allow for more definitive determination of the rate of prevalence of sars-cov-2 infection and therefore providing more accurate epidemiologic data on the virus's course and entry into a given population. key: cord-316702-dj2fo8sn authors: vignesh, ramachandran; shankar, esaki m.; velu, vijayakumar; thyagarajan, sadras panchatcharam title: is herd immunity against sars-cov-2 a silver lining? date: 2020-09-30 journal: front immunol doi: 10.3389/fimmu.2020.586781 sha: doc_id: 316702 cord_uid: dj2fo8sn nan the emergence of a novel coronavirus severe acute respiratory syndrome coronavirus-2 (sars-cov-2) in late 2019 and its wide global spread has led to millions of infections and substantial morbidity and mortality (1) . coronavirus disease 2019 (covid-19) caused by sars-cov-2 infection can range from mild self-limiting disease to acute respiratory distress syndrome and death (2) . with the who having reported 31,174,627 confirmed cases and 962,613 deaths globally as of 22nd september 2020, the covid-19 pandemic seems to show almost poor indication of abating (3) . while the global scientific community is racing against time to strategize combating possibilities, with several vaccine trials, drug discoveries and validations underway, we still need a practical and sustainable solution to face the ongoing threat to global public health. the terminology "herd immunity", coined by topley and wilson in 1923, later formed the basis for vaccines, applications and vaccination programs, especially against certain viral infections (4). the concept of herd immunity refers to the indirect protection from infection conferred on susceptible individuals when a sufficiently large proportion of individuals immune to the infection exist in a population. herd immunity concept is generally applicable to those infections that are transmitted directly from person to person and for those humans serving as the reservoir of infection. history has shown that a significant drop in the number of cases and even eradication is rendered by vaccines, and herd immunity is achieved against infectious diseases like small pox, polio, measles, rubella, diphtheria, pertussis and mumps (4-7). the concept of herd immunity appears to be highly critical in the context of disease elimination programs because the said eradication of an infectious agent becomes possible in spite of the absence of an effectual vaccine. interestingly, natural herd immunity is a classical concept that has been successfully accomplished in instances like the 1918 h1n1 influenza pandemic wherein no vaccine was available (8) . going by this old school modus operandi, in the absence of a vaccine, building natural herd immunity against sars-cov-2 is theoretically considered feasible. however, letting an existing super infectious condition to run amok in the pretext of building up effective herd immunity might not be a smart strategy to end the pandemic. it requires judicious analyses to avoid the colossal burden it could inflict on the healthcare system and the surge in mortalities and associated complications. in a detailed classical analysis, fox et al. has listed down the following four conditions for effective herd immunity to ensue (i) the infectious agent must exist and be restricted to a single host, (ii) the transmission must occur primarily through direct contact, (iii) the infection must induce a robust and life-long immunity, and (iv) the cumulative or herd immunity is amplified if the population possesses a random mixing pattern (9) . applying the aforementioned conditions to herd immunity against sars-cov-2, though the infectious agent has been identified and believed to be zoonotic, we are yet to place a finger on the intermediate host (10) . secondly, the transmission, of course, occurs through direct person-to-person contact (11) . however, regarding the third condition, we have a paucity of data on the immune response elicited by sars-cov-2 in humans, till date (12) and the questions remains about the long-lasting immunity for the exposed individuals. finally, though the entire human population is susceptible to covid-19, the mixing of the pattern varies that is dependent on several societal factors and practices such as universal lockdown, mass quarantine, isolation, social distancing and public health preventive measures, particularly for those at risk (12) . table 1 listed the various infectious agents and their corresponding r 0 values and herd immunity thresholds. while earlier studies have estimated the basic reproductive number (r 0 ) of sars-cov-2 to be in the range of 2 to 3, recent estimates place the r 0 at 5.7 (13, 15) . this indicates the highly infective nature of the virus, meaning that on an average each infected individual can give rise to about 5.7 newer infections and subsequently spread the agent on an exponential scale. assuming an r 0 estimate of 5.7, using the mathematical formula 1-1/r 0 , the herd immunity threshold for covid-19 would be~82.5%, meaning that the incidence of infection will begin to decline once the proportion of individuals with acquired immunity to sars-cov-2 in the population exceeds 82.5%. however, it remains to be noted that the estimate of the proposed threshold is only theoretical with the assumptions of a homogenous population and presence of uniform sterilizing immunity in the recovered patients. mathematical modeling studies have shown that disease-induced herd immunity threshold would be substantially lower than the values calculated by conventional formula due to the population heterogeneity (16, 17) . furthermore, there are several instances of "super spreading events" reported from various countries, wherein a single patient goes on to infect far more number of people than an average patient does, based on estimated r 0 value (18) . several factors such as increased viral load, asymptomatic individuals, immune suppression and extensive social interactions have been implicated in these "super-spreading events" (19) . studies point towards these "super-spreaders" as the reason for the heterogeneous propagation of sars-cov-2 across geographical locations and since these present with a relatively higher r 0 value, they could potentially impact the dynamics of spread and lower the herd immunity threshold (20, 21) . the whole concept of herd immunity against sars-cov-2 hinges on the assumption that an infection would generate sufficient, effective and protective long-lasting immunity. however, data are scarce if the acquired immunity developed by humans is sterilizing enough and whether it would stay long enough. earlier studies on survivors of sars-cov-1 and mers-cov infections have shown that the antibodies against sars-cov-1 persisted for nearly two years and antibodies to mers-cov lasted for almost 3 years (22, 23) . besides, studies have also demonstrated evidence of seroconversion within 14 to 19 days of disease onset among sars-cov-2 patients with covid-19 (24, 25) . a study has reported the interesting finding of t cell reactivity to sars-cov-2 in about 50% of specimens collected between 2015 and 2018 before the viral emergence (26) . this could reflect the circulating t cell memory to the seasonal coronaviruses-229e, oc43, nl63, and hku1. a study also suggests that sars-cov-2-reactive antibody responses have been detected in unexposed individuals who are igg seropositive for oc43 and nl63 and this cross-immune reactivity mainly targets viral 1ab polyprotein and s proteins, these viral antigens have high sequence similarity with low pathogenic human coronaviruses and sars-cov-2 (27, 28) . in addition, a study has also shown that these antibodies were particularly prevalent in children and adolescents (29) . it is also important to note that sera from uninfected individuals exhibited variability in their reactivity, they are reactive with sars-cov-2 s and nucleoprotein but not with the s1 subunit or the receptor binding domain (rbd) of spike protein. since many studies from different geographical locations are documenting preexisting immunity to sars-cov-2, it will be important to define specificities of these t and b cell immune response carefully to assess their association with covid-19 disease severity. this preexisting cross-reactive t and b cell immunity to sars-cov-2 may have wide implications as this could explain differential clinical outcomes in covid-19 patients, disease severity, vaccine development, and important in accessing herd immunity for sars-cov-2 viral infection/covid-19 disease. studies characterizing the sars-cov-2-specific t cell responses suggest that there is marked activation of t cells in acute covid-19 patients (30) (31) (32) . several studies have provided strong evidence for the importance of sars-cov-2 specific ctls, and t helper cells in mild and moderate patients compared to severe covid-19 disease (27, 28, (31) (32) (33) . the effector memory cd8 t cell population is decreased in the severe patients compared to the recovered patients (34) (35) (36) . similarly, the t cells in severe patients compared to convalescent patients express higher levels of exhaustion markers pd-1 and cd39 on the memory cd8 t cells with the signature pertaining to hyperimmune activation (hladr + cd38 + cd8 + ) with lower cd8 t cell response. in line with cd8 t cells, the effector memory cd4 t cell frequencies are also elevated in the covid-19 patients (34) (35) (36) . interestingly, the recovered patients seem to have higher levels of activated circulating t follicular helper (tfh) population, indicative of recent antigen encounter and emigration from germinal center. similar to the pd-1 levels of cd8 t cells, the pd-1 levels were increased in the cd4 t cells. as a result of the decrease in the t cells, non-t cell frequencies that includes, macrophages, monocytes, neutrophils were observed to be elevated in severe covid-19 patients (30, 34) . similar to t cells the b cell subpopulations are altered in the covid-19 disease and it has been shown that the frequency of plasmablast is highly elevated (30% of the circulating b cells) in the covid-19 patients (34) . these blood plasmablast frequencies are shown to correlate with activated circulating tfh cells (34, 37) . the proliferating b cells are markedly elevated in the covid-19 patients with activated phenotype, suggestive of altered b cell response during covid-19 disease. several studies have provided strong evidence for the importance of sars-cov-2specific neutralizing antibodies in association with less disease severity in covid-19 patients (38, 39) . sars-cov-2 specific antibodies are found in convalescent plasma and most recovered individuals have rbd-specific antibodies with potent antiviral activity (40, 41) , importantly severe and critical patients are currently being treated with convalescent plasma therapy in many parts of the world (42, 43) . overall, these data suggest that vaccines or therapeutic strategies that induce functional anti-viral sars-cov-2 specific t and b cell responses are important to curtail sars-cov-2 infection in vivo. there remain many unknowns and several other research questions unanswered. the degree of protection afforded by the antibodies against sars-cov-2 among the infected is still ambiguous, and strong evidence-based findings are awaited to know if the host responses generated would be of strong sterilizing or weak and waning immune responses. earlier studies on the coronavirus 229e, responsible for common cold have shown that the titers of antibodies produced were not sufficient to prevent reinfection in all the individuals studied (44) . interestingly, similar pattern exists in sars-cov-2 infection and where there is a rapid decay of sars-cov-2 of antibodies in persons with mild symptoms and recovered patients (45, 46) . similarly, as a recent study demonstrated the evidence of reinfection of sars-cov-2 by phylogenetically distinct sars-cov-2 re-infection, this may also suggest that the immunity generated during primary sars-cov-2 infection may be short lived and may not protect if reinfection happens by the second distinct virus (47) . these results also suggest that sars-cov-2 may continue to circulate among human population despite herd immunity with natural infection or with vaccination. however, a recent study on monkeys has indicated the development of protective immunity against reexposure to sars-cov-2 (homologous) (48) . hence, massive longitudinal monitoring of sars-cov-2 seroprevalence remains the need of the hour to determine the percentage of the population already infected and if reaching a herd immunity threshold is even feasible. it also remains logical to consider the viral factors such as the possibility of mutations and emergence of new strains of a virus, which can go on to make herd immunity futile (49) . given the unavailability of a vaccine against sars-cov-2, it is prudent to foresee the consequences of achieving the herd immunity threshold in epidemiological and immunological viewpoints. a recent population-based sero-epidemiological study from spain involving 61075 participants has revealed 5% seroprevalence of sars-cov-2, thereby highlighting that the majority of the spanish population remains seronegative (50) . figure 1 represents this scenario of a population with only 5% seroprevalence of sars-cov-2 antibodies and the importance of achieving herd immunity threshold. similarly, seroprevalence among the community in los angeles county has been reported as 4.34%, about 3.3% in japan, and 2.73% in hong kong (51) (52) (53) . based on a serological survey conducted by the indian council for medical research (icmr) in may -june, 2020 across 83 districts in india involving over 26,400 participants, the seroprevalence was found to be only 0.73% (54) . the lower rates of seroprevalence of sars-cov-2 antibodies reported from various geographical locations point in the direction that we are still a long way ahead of achieving herd immunity. according to a modeling study, assuming a scenario of achieving uniform herd immunity threshold of 67% and with an infection fatality rate of 0.6% for sars-cov-2, the estimate of the absolute number of deaths worldwide would easily exceed a whopping 30 million (14) . since these estimates are based on various assumptions and the real-life scenario could throw us curve balls of multiple factors influencing the outcomes, the fatality rate could even be higher. a recent modelling study has estimated that about one in five individuals worldwide would be at increased risk of severe covid-19, upon infection with sars-cov-2, owing to the underlying conditions. the study projects an alarming figure of about 349 million people requiring hospital admission and substantial mortality rates (55) . furthermore, in the case of highly populated and resourcestrapped countries, the increase in the number of infected cases could overwhelm the healthcare facilities and could lead to a shortage of essential medical services exacerbating further complications and deaths. thus, weighing on the immunological and epidemiological consequences, achieving natural herd immunity at the cost of severe morbidities and mortalities worldwide, in the absence of an effective and safe vaccine appears farfetched and seemingly an impracticable solution. with so many vaccines being tested in various phases of clinical trials, the availability of an effective vaccine seems to be the only way forward in this war against covid-19. echoing dr. anthony fauci's statements, even when a vaccine with a modest efficacy is made available in near future, anti-science and anti-vaccine campaigns need to be counteracted and cooperation of the general public is needed to achieve an efficient and successful herd immunity against covid-19 (56). rv, es, vv, and st led the writing of this opinion article. all authors contributed to the article and approved the submitted version. the views, opinions, assumptions, or any other information set out in this article are solely those of the authors and should not be attributed to anyone. the authors salute all the health care workers who are at the front lines of the covid-19 pandemic, helping patients and their families. frontiers in immunology | www.frontiersin.org september 2020 | volume 11 | article 586781 an interactive web-based dashboard to track covid-19 in real time clinical features of patients infected with 2019 novel coronavirus in wuhan who. coronavirus disease (covid-19) dashboard. available at: https://covid19. who.int/?gclid=cjwkcajw_qb3braveiwavwq6vvsmd-becu3ak qlye88rcmzf-muzkht9fj70phh5wl5hb3fcqzsmhrocvoiqavd_bwe the spread of 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preserved serum at an outpatient setting in kobe, japan: a cross-sectional study seroprevalence of sars-cov-2 in hong kong and in residents evacuated from hubei province, china: a multicohort study prevalence of sars-cov-2 infection in india: findings from the national serosurvey global, regional, and national estimates of the population at increased risk of severe covid-19 due to underlying health conditions in 2020: a modelling study fauci says covid-19 vaccine may not get us to herd immunity if too many people refuse to get it the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright â© 2020 vignesh, shankar, velu and thyagarajan. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-309633-1cd74xdl authors: rogers, julia h.; link, amy c.; mcculloch, denise; brandstetter, elisabeth; newman, kira l.; jackson, michael l.; hughes, james p.; englund, janet a.; boeckh, michael; sugg, nancy; ilcisin, misja; sibley, thomas r.; fay, kairsten; lee, jover; han, peter; truong, melissa; richardson, matthew; nickerson, deborah a.; starita, lea m.; bedford, trevor; chu, helen y. title: characteristics of covid-19 in homeless shelters: a community-based surveillance study date: 2020-09-15 journal: ann intern med doi: 10.7326/m20-3799 sha: doc_id: 309633 cord_uid: 1cd74xdl background: homeless shelters are a high-risk setting for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) transmission because of crowding and shared hygiene facilities. objective: to investigate sars-cov-2 case counts across several adult and family homeless shelters in a major metropolitan area. design: cross-sectional, community-based surveillance study. (clinicaltrials.gov: nct04141917) setting: 14 homeless shelters in king county, washington. participants: a total of 1434 study encounters were done in shelter residents and staff, regardless of symptoms. intervention: two strategies were used for sars-cov-2 testing: routine surveillance and contact tracing (“surge testing”) events. measurements: the primary outcome measure was test positivity rate of sars-cov-2 infection at shelters, determined by dividing the number of positive cases by the total number of participant encounters, regardless of symptoms. sociodemographic, clinical, and virologic variables were assessed as correlates of viral positivity. results: among 1434 encounters, 29 (2% [95% ci, 1.4% to 2.9%]) cases of sars-cov-2 infection were detected across 5 shelters. most (n = 21 [72.4%]) were detected during surge testing events rather than routine surveillance, and most (n = 21 [72.4% {ci, 52.8% to 87.3%}]) were asymptomatic at the time of sample collection. persons who were positive for sars-cov-2 were more frequently aged 60 years or older than those without sars-cov-2 (44.8% vs. 15.9%). eighty-six percent of persons with positive test results slept in a communal space rather than in a private or shared room. limitation: selection bias due to voluntary participation and a relatively small case count. conclusion: active surveillance and surge testing were used to detect multiple cases of asymptomatic and symptomatic sars-cov-2 infection in homeless shelters. the findings suggest an unmet need for routine viral testing outside of clinical settings for homeless populations. primary funding source: gates ventures. shelters, and self-reported new or worsening cough alone or 2 or more new or worsening ari symptoms with onset in the past 7 days. eligible ari symptoms included subjective fever, cough, sore throat, shortness of breath, myalgia, headache, and rhinorrhea. data on chills, sweats, ear pain or discharge, nausea or vomiting, diarrhea, and rash were also collected, although these alone were not sufficient to meet ari criteria. once a month, study eligibility was extended to shelter residents aged 3 months or older regardless of symptoms. study staff recruited participants 6 days a week during this period ( figure 1) . in response to sars-cov-2 in washington state, onsite testing and treatment of influenza (that is, the trial intervention) were discontinued on 1 april 2020. we reduced study staff to 3 onsite days per week at each shelter and recruited persons regardless of symptoms. shelter staff were also eligible for study participation at this time. individual participants were not followed longitudinally, but eligible persons could have multiple encounters throughout the study period. study participation was limited to once weekly unless new or worsening ari symptoms developed, in which case a person was permitted to reenroll within 7 days. this study was approved by the human subjects division of the university of washington institutional review board (study00007800). participants were recruited in person using 2 mechanisms: routine surveillance and surge testing events. routine surveillance, as detailed earlier, involved selfselected participation at staffed kiosks in shelters during standardized days and times. surge testing was initiated on 30 march 2020 (and continued through 24 april) in collaboration with public health-seattle & king county's communicable disease epidemiology team to conduct contact tracing at 6 shelters where cases of sars-cov-2 were previously detected ( figure 2 ). during these 1-day events, we offered sars-cov-2 testing to all residents and staff. in addition to 3 shelters participating in routine surveillance, we did surge testing at 3 other shelters where a case of sars-cov-2 was detected. these 3 additional shelters had residents or staff members that had sought services from or worked at 1 of the routine surveillance sites in the prior month. sampling strategies for asymptomatic versus symptomatic study participants were the same at these sites. the 9 original participating shelters included those serving women (shelter a), mixed-sex adults (shelters b and c), mixed-sex adults aged 18 to 25 years (shelter d), families (shelters e, f, and g), men aged 50 years or older (shelter h), and men aged 18 years or older (shelter i). private or shared rooms were available as sleeping accommodations at shelters e, f, and g. shelters b symptomatic encounters include those with ≥1 self-reported symptom. characteristics of covid-19 in homeless shelters and g were closed in early april, and to reduce crowding, residents were moved to shelters j and k, which had private or shared rooms. we did routine surveillance at these new sites. altogether, 11 shelters (shelters a through k) were sites for routine surveillance, and 3 additional shelters (shelters l, m, and n) were sites for surge testing alone. maximum nightly capacity ranged from 45 to 275 persons. supplement table 1 (available at annals.org) shows shelter site characteristics and participant encounter metrics. all questionnaire data were collected electronically in research electronic data capture (redcap) on a tablet (supplement, available at annals.org). participants chose to complete the questionnaire themselves or with the assistance of study staff. telephonic interpretation services were available for non-english-speaking participants. mid-nasal samples were obtained using a sterile nylon flocked nasal swab (copan diagnostics). until 6 march, study staff collected these swabs. thereafter, because of heightened infection control precautions, participants were instructed to self-collect a mid-nasal swab while observed by study staff. visual guides were shared with participants before sample collection to demonstrate self-swabbing. questionnaire data included participant age, race, sex, smoking status, underlying conditions, flu vaccine status, sleeping arrangements, and symptom profiles and duration. smoking status was determined by asking participants if they used tobacco products, e-cigarettes, or vape pens. underlying conditions included asthma, blood disorders, cancer, chronic obstructive pulmonary disease or emphysema, chronic bronchitis, immunosuppression, liver disease, heart disease, diabetes, neurologic conditions, or aspirin therapy. flu vaccine status was determined by self-reported receipt of influenza vaccine since 1 july 2019. sleeping arrangements were reported only by shelter residents and categorized as communal or private room/shared family room. communal included sleeping in a congregate space with bunk beds, bed mats, or rooms shared with more than 1 family. participant encounters with 1 or more new or worsening symptoms with onset in the past 7 days were defined as symptomatic, and those without any new or worsening symptoms in the past 7 days were defined as asymptomatic. participants with ari symptoms also had symptom duration data collected in response to the question, "when did the symptoms you mentioned in the beginning of this survey become new or worsening?" viral co-infection was defined as the presence of 2 or more viral pathogens (supplement table 2 , available at annals.org). influenza-like illness was defined as having a fever and cough or sore throat. coronavirus disease 2019 -like illness was defined as fever and cough or increased difficulty breathing. samples were transported to the university of washington laboratory in universal viral transport medium (becton dickinson) in ice-packed coolers and stored at 4°c before testing. testing was done at the brotman baty institute for precision medicine. total nucleic acids were extracted (magna pure [roche]) and tested for the presence of 27 respiratory pathogens using taqman reverse transcriptase polymerase chain reaction (rt-pcr) on the openarray platform (thermo fisher scientific) as well as sars-cov-2 using a laboratory-developed test or research assay (supplement table 2 ). for the laboratorydeveloped test, sars-cov-2 detection was done using real-time rt-pcr with probe sets targeting orf1b and s with fam fluor (life technologies 4332079 assays #apgzjkf and #apxgvc4apx) multiplexed with a ribonuclease p (rnase p) probe set with vic or hex fluor (life technologies a30064 or idt custom), each in duplicate on a quantstudio 6 instrument (applied biosystems). the research assay uses only the orf1b and rnase p multiplexed rt-pcr in duplicate. shelter specimens collected between 25 february and 18 march 2020 were tested for sars-cov-2 using the research assay in real time. specimens collected after 19 march were tested for sars-cov-2 using the laboratory-developed test under an emergency use authorization issued by washington state. specimens collected before 25 february were tested retrospecasymptomatic symptomatic symptomatic encounters include those with ≥1 self-reported symptom. annals.org annals of internal medicine tively using a single replicate orf1b and rnase p multiplexed rt-pcr research assay to detect sars-cov-2 orf1b. we used cycle threshold (ct) values as a semiquantitative measure of viral load in a sample. cycle threshold values are inversely related to the viral load. three or 4 replicates for rnase p and sars-cov-2 were required to have a ct value less than 39 for a sample to be considered positive for the laboratory-developed test, and both replicates had to be positive for the research assay. the primary outcome of this study was sars-cov-2 infection, defined as detection of sars-cov-2 from a nasal swab, regardless of symptoms. we calculated the test positivity rate of sars-cov-2 infection at shelters by dividing the number of positive cases by the total number of participant encounters in the study period. all data in this analysis are presented by participant encounter, defined as each time an eligible person, either with or without symptoms, completed a nasal swab and survey with an onsite study staff member. we used participant encounters as the primary unit of analysis in this study rather than unique participants because of difficulties in matching names at different encounters in a transient population. (we estimated that there were 925 unique participants identified in this study population, but this number is uncertain.) we used descriptive statistics to evaluate the sociodemographic and clinical characteristics, virologic factors, and symptom profiles of all participant encounters. the 95% cis for study measures of disease occurrence are provided in the results section. responses of "do not know" and "prefer not to say" were coded as missing observations and dropped from the analysis. descriptive statistics comparing demographic characteristics of unique participants versus participant encounters were similar overall. most persons had only 1 encounter during the study period (n = 696 [75.2%]), and all sars-cov-2 cases included in this study involved unique participants. this study was funded by gates ventures. the funder was not involved in the design of the study and does not have any ownership over the management and conduct of the study, the data, or the rights to publish. a total of 1434 participant encounters occurred between 1 january and 24 april 2020 at 14 shelters. of these encounters, 601 (41.9%) involved asymptomatic persons, and 833 (58.1%) involved symptomatic persons. the median age of participants was 46 years (range, 0 to 82 years) ( table 1) . most encounters involved males (67.9%). the predominant racial groups were white (40.9%) and black or african american (30.5%). more than half of the encounters involved smokers (57.7%), and 39.4% involved participants with at least 1 underlying condition. among the 833 symptomatic participant encounters, the mean number of symptoms was 2 (sd, 2.2) ( table 2) . rhinorrhea (43.0%), cough (37.3%), and myalgia (23.7%) were the most common symptoms. of the 725 participant encounters with symptom duration data available, 40.3% had ari symptoms for less than 2 days at the time of testing. the proportion of encounters that met the case definition for influenza-like illness was 13.2%, and the proportion for covid-19 -like illness was 12.7%. samples from 28 (2.1%) participant encounters were positive for 2 or more of 17 respiratory pathogens (plus sars-cov-2) (supplement table 2 ). samples from 201 (15%) encounters were positive for streptococcus pneumoniae. we identified 29 (2.0% [95% ci, 1.4% to 2.9%]) participant encounters with sars-cov-2 infection involving 29 unique persons. four (13.8%) of these persons were shelter staff. the positivity rate among encounters with shelter staff compared with shelter residents was similar (2.5% vs. 2.0%, respectively). approximately half of encounters with sars-cov-2 detected involved persons aged 60 years or older (44.8%), and only 3 involved persons younger than 35 years (10.3%) ( table 1) table 2) . one positive encounter met both the influenza-like illness (3.4%) and covid-19 -like illness (3.4%) case definition. of the 6 positive encounters with symptom duration data available, 5 (83.3%) reported symptoms developing less than 48 hours before study participation. among encounters that were negative for sars-cov-2, 1.9% of persons tested positive for at least 1 other respiratory virus, compared with 10.3% among encounters with positive sars-cov-2 results. mean sars-cov-2 ct values among samples collected from symptomatic (n = 8) and asymptomatic (n = 21) persons were 27.9 (sd, 5.0) and 29.6 (sd, 6.1), respectively. in total, participating shelters had 1482 beds, of which 1183 (80.0%) were at routine surveillance sites. a total of 1119 (78%) participant encounters occurred at routine surveillance sites ( table 1) . shelter h, which served older men, represented the greatest number of participant encounters (18%) from a single site, whereas 21.7% of encounters were in family shelters (supplement table 1 ). between 30 march and 24 april, we held 8 surge testing events at 6 sites, resulting in 315 participant encounters, ranging from 12 to 97 during each event. cases of sars-cov-2 were detected at 5 shelters. the first case was detected on 11 march at shelter h, with a subsequent case on 30 march at shelter i ( figure 2 ). most positive cases were detected during surge testing events (n = 21 [72.4%]) compared with routine surveillance (n = 8 [27.6%]). site-specific positivity rates original research characteristics of covid-19 in homeless shelters ranged between 1% and 20%. overall, 85.7% of positive cases were in participants who had slept in a communal space in the past week, compared with 78.2% of negative encounters ( table 1) . of sars-cov-2 cases, 5 were detected at shelter f (4.6% of total site encounters), which had both private and communal sleeping spaces. three sars-cov-2 cases from this site were among persons sharing the same private room. the remaining 24 sars-cov-2 cases were at shelters serving adult men with only communal sleeping spaces available. most sars-cov-2 cases (79.3%) were detected at shelters serving older male residents, with shared day center services, showering facilities, and a rotating staff (figure 3 ). our findings show detection of sars-cov-2 in homeless shelters during 4 months of active surveillance and surge testing. overall, 2% of participant encounters involved positive sars-cov-2 results, with most cases detected through surge testing events. encounters with positive results were more frequent in older persons and nonsmokers. most sars-cov-2 in-fections were asymptomatic, with similar mean ct values in cases with and without symptoms. in our study, most positive cases reported no or mild symptoms. this may in part be from early detection of presymptomatic cases or identification of persons with mild illness episodes who would not have sought care or testing services. an outbreak investigation at a boston-based shelter serving only men reported a substantially higher positivity rate (36%) among all residents tested at a single time point. however, testing at this shelter was done at a time when the community incidence of sars-cov-2 in massachusetts was higher than that in washington state (14, 15) . similar to our study, the boston group noted that a large proportion of persons with sars-cov-2 were asymptomatic, with only 7.5% reporting cough and 1.4% reporting shortness of breath (13) . although the exact role of presymptomatic and asymptomatic sars-cov-2 transmission remains unclear, recent publications have linked outbreaks to asymptomatic index cases (16 -18) . recent studies have shown that ct values from positive rt-qpcr results may relate to viral transmissibility and may inform clinical decision making about isolation annals.org annals of internal medicine precautions (19) . cycle threshold values were similar in persons with and without symptoms, suggesting that viral load may not be associated with symptoms. prior studies have implicated asymptomatic and presymptomatic persons as a source of infection, but the duration of sars-cov-2 infectivity is unknown (16 -18) . this has major implications for public health and shelter service providers developing guidelines for isolation of residents who are positive for sars-cov-2 and reintroduction into a general shelter population. further research is needed to understand the effect of temporal dynamics in viral shedding on transmissibility of sars-cov-2 in communal settings and the role of asymptomatic cases. shelter characteristics, particularly resident density and sleeping arrangements, may play a role in sars-cov-2 transmission. the outbreak seen in shelters h, l, and m may have been related to the use of floor mats in a communal sleeping space without temporary dividers and less than 6 feet apart (12) . we observed only 1 positive sars-cov-2 result in shelters with bunk beds rather than floor mats in congregate sleeping areas. the family shelters adhered to the centers for disease control and prevention recommendations of using curtains as a temporary barrier between familial bed clusters in congregate sleeping areas (20) . these shelters also implemented social distancing and handwashing protocols in late march, with daily temperature checks and symptom assessments by staff, which were indepen-dent from voluntary participation in this study. these measures may have curtailed further transmission within shelter f. shelters h, l, and m, where more cases were detected, had limited staff-conducted screening and a shortage of hygiene resources. we sampled both staff and residents and found sars-cov-2 test positivity rates to be similar between the groups. future analyses will focus on transmission dynamics within shelters, with sampling from both groups. our positivity rate was lower than the 8.8% rate seen in the university of washington clinical laboratory during that same period (21) . this may be because most clinical samples were obtained from persons seeking medical care. public health and other groups did additional testing at 2 shelters in this study during an outbreak investigation between 31 march and 8 april. interestingly, only 2 of 41 confirmed cases (4.9%) from this investigation were identified through routine symptom-based screening, and only 3 (7.3%) were identified after health care was sought (12) . in addition, our study identified nearly a third of sars-cov-2 cases through routine surveillance, which may have resulted from study eligibility expansion to asymptomatic persons. we speculate that with earlier asymptomatic testing, additional outbreaks may have been detected at study sites. this study's findings may be subject to selection bias because all participation was voluntary. high levels characteristics of covid-19 in homeless shelters of distrust of health care providers and low rates of health care use in homeless populations have been documented (9, 22, 23) . this may account for more asymptomatic cases of sars-cov-2 having been detected through surge testing events when shelter management actively encouraged all residents and staff to participate. in addition, reducing onsite testing from 6 to 3 days per week may have decreased our ability to detect additional positive cases at participating sites. another limitation is the lack of robust follow-up data on participants. we had very low response rates to a follow-up survey sent via text message or e-mail to asymptomatic participants 7 days after onsite study participation to evaluate for new or worsening symptoms; thus, it was excluded from our analysis. therefore, it is unclear what proportion of the asymptomatic sars-cov-2 cases detected in this study were presymptomatic. in addition, the small numbers of sars-cov-2 cases and unmeasured shelter-level covariates limit the extent to which we can draw conclusions about how sleeping arrangements may mitigate transmission. finally, this study was not able to track unique participants and could not reliably identify encounters in the same participant. the sensitivity of self-sampling for sars-cov-2 detection may also be a problem. however, a recent study shelter l was a temporary homeless service site opened on 14 march when half of the residents at shelter h were moved to reduce crowding. residents at shelter h shared day center services, showering facilities, and a rotating staff with shelters g and m during this period. residents were men aged ≥50 y that slept on communal floor mats in 2 separate rooms. participant recruitment was done through surge testing only at shelter l; routine surveillance was never available as a sampling mechanism. characteristics of covid-19 in homeless shelters annals.org annals of internal medicine of self-collected mid-turbinate nasal swabs for influenza detection found rnase p in 100% of nasal swab specimens, but with higher mean ct values among positive results in self-collected swabs compared with cliniciancollected nasopharyngeal swabs (24) . additional studies have found that self-swabbing results in viral positivity rates similar to those of sentinel physician networks and has excellent diagnostic yield (25-27). in conclusion, this study provides key insights into detection strategies for sars-cov-2 in a vulnerable, hard-to-reach population. passive sentinel surveillance for respiratory viruses may only detect symptomatic cases severe enough to prompt health-seeking behavior and may miss milder ones, delaying the recognition of outbreaks and further viral spread (28, 29) . results of this study's combined active surveillance and surge testing strategy suggest an unmet need for routine viral testing outside of clinical settings in homeless shelters and other congregate living facilities. secretary carson certifies annual data: homelessness ticked up in 2019, driven by major increases in california utilization of mental health and substance abuse services among homeless adults in los angeles influenzalike illness among homeless persons respiratory syncytial virus infection in homeless populations homelessness and the response to emerging infectious disease outbreaks: lessons from sars covid-19: a potential public health problem for homeless populations utilization of health care services among subgroups of urban homeless and housed poor high utilizers of emergency health services in a population-based cohort of homeless adults factors associated with the health care utilization of homeless persons infections in the homeless respiratory viruses within homeless shelters in marseille, france covid-19 outbreak among three affiliated homeless service sites-king county, washington institute for health metrics and evaluation. covid-19 projections: massachusetts. accessed at https://covid19.healthdata.org /united-states-of-america/massachusetts on 19 may 2020. 15. institute for health metrics and evaluation. covid-19 projections: washington screening of healthcare workers for sars-cov-2 highlights the role of asymptomatic carriage in covid-19 transmission presumed asymptomatic carrier transmission of covid-19 evidence supporting transmission of severe acute respiratory syndrome coronavirus 2 while presymptomatic or asymptomatic to interpret the sars-cov-2 test, consider the cycle threshold value interim guidance for homeless service providers to plan and respond to coronavirus disease 2019 (covid-19) uw virology covid-19 dashboard a comprehensive assessment of health care utilization among homeless adults under a system of universal health insurance health care for the homeless: what we have learned in the past 30 years and what's next results of a pilot study using self-collected mid-turbinate nasal swabs for detection of influenza virus infection among pregnant women. influenza other respir viruses feasibility study for the use of self-collected nasal swabs to identify pathogens among participants of a population-based surveillance system for acute respiratory infections (grippeweb-plus)-germany population-based active surveillance cohort studies for influenza: lessons from peru characteristics of covid-19 in homeless shelters original research annals.org annals of internal medicine current author addresses: ms department of biostatistics 35-7232 critical revision of the article for important intellectual content appendix: members of the seattle flu study investigators members of the seattle flu study investigators who authored this work: principal investigators washington), louise e. kimball, phd (vaccine and infectious disease division characteristics of covid-19 in homeless shelters key: cord-029112-u507i0t0 authors: smith, keisha; pace, amy; ortiz, stephan; kazani, shamsah; rottinghaus, scott title: a phase 3 open-label, randomized, controlled study to evaluate the efficacy and safety of intravenously administered ravulizumab compared with best supportive care in patients with covid-19 severe pneumonia, acute lung injury, or acute respiratory distress syndrome: a structured summary of a study protocol for a randomised controlled trial date: 2020-07-13 journal: trials doi: 10.1186/s13063-020-04548-z sha: doc_id: 29112 cord_uid: u507i0t0 objectives: primary objective • to evaluate the effect of ravulizumab, a long-acting complement (c5) inhibitor plus best supportive care (bsc) compared with bsc alone on the survival of patients with covid-19. secondary objectives • number of days free of mechanical ventilation at day 29 • duration of intensive care unit stay at day 29 • change from baseline in sequential organ failure assessment (sofa) score at day 29 • change from baseline in peripheral capillary oxygen saturation/ fraction of inspired oxygen (spo2 /fio2) at day 29 • duration of hospitalization at day 29 • survival (based on all-cause mortality) at day 60 and day 90 safety • incidence of treatment-emergent adverse events and treatment-emergent serious adverse events. pk/pd/immunogenicity • change in serum ravulizumab concentrations over time • change in serum free and total c5 concentrations over time • incidence and titer of anti-alxn1210 antibodies biomarkers • change in absolute level of soluble biomarkers in blood associated with complement activation, inflammatory processes, and hypercoagulable states over time exploratory • incidence of progression to renal failure requiring dialysis at day 29 • time to clinical improvement (based on a modified 6-point ordinal scale) over 29 days • sf-12 physical component summary (pcs) and mental component summary (mcs) scores at day 29 (or discharge), day 60, and day 90 • euroqol 5-dimension 5-level (eq-5d-5l) scores at day 29 (or discharge), day 60, and day 90 trial design: this is a multicenter phase 3, open-label, randomized, controlled, study. the study is being conducted in acute care hospital settings in the united states, united kingdom, spain, france, germany, and japan. participants: male or female patients at least 18 years of age, weighing ≥ 40 kg, admitted to a designated hospital facility for treatment will be screened for eligibility in this study. key inclusion criteria • confirmed diagnosis of sars-cov-2 infection (eg, via polymerase chain reaction [pcr] and/or antibody test) presenting as severe covid-19 requiring hospitalization • severe pneumonia, acute lung injury, or ards confirmed by computed tomography (ct) or x-ray at screening or within the 3 days prior to screening, as part of the patient’s routine clinical care • respiratory distress requiring mechanical ventilation, which can be either invasive (requiring endotracheal intubation) or non-invasive (with continuous positive airway pressure [cpap] or bilevel positive airway pressure [bipap]) key exclusion criteria • patient is not expected to survive for more than 24 hours • patient is on invasive mechanical ventilation with intubation for more than 48 hours prior to screening • severe pre-existing cardiac disease (ie, nyha class 3 or class 4, acute coronary syndrome, or persistent ventricular tachyarrhythmias) • patient has an unresolved neisseria meningitidis infection excluded medications and therapies • current treatment with a complement inhibitor • intravenous immunoglobulin (ivig) within 4 weeks prior to randomization on day 1 excluded prior/concurrent clinical study experience • treatment with investigational therapy in a clinical study within 30 days before randomization, or within 5 half-lives of that investigational therapy, whichever is greater • exceptions a. investigational therapies will be allowed if received as part of best supportive care through an expanded access protocol or emergency approval for the treatment of covid-19. b. investigational antiviral therapies (such as remdesivir) will be allowed even if received as part of a clinical study. intervention and comparator: the study consists of a screening period of up to 3 days, a primary evaluation period of 4 weeks, a final assessment at day 29, and a follow-up period of 8 weeks. for patients randomized to ravulizumab plus bsc, a weight-based dose of ravulizumab (≥40 to < 60 kg/2400 mg, 60 to < 100 kg/2700 mg, ≥ 100 kg/3000 mg) will be administered on day 1. on day 5 and day 10, additional doses of 600 mg (≥40 to <60 kg) or 900 mg (>60 kg) ravulizumab will be administered and on day 15 patients will receive 900 mg ravulizumab. there is no active or placebo comparator in this open-label clinical trial. the total duration of each patient’s participation is anticipated to be approximately 3 months. main outcomes: the primary efficacy outcome of this study is survival (based on all-cause mortality) at day 29. randomisation: patients will be randomized in a 2:1 ratio (ravulizumab plus bsc:bsc alone). randomization will be stratified by intubated or not intubated on day 1. computer-generated randomization lists will be prepared by a third party under the direction of the sponsor. investigators, or designees, will enrol patients and then obtain randomization codes using an interactive voice/web response system. the block size will be kept concealed so that investigators cannot select patients for a particular treatment assignment. blinding (masking): this is an open-label study. numbers to be randomised (sample size): approximately 270 patients will be randomly assigned in a 2:1 ratio to ravulizumab plus bsc (n=180) or bsc alone (n=90). trial status: protocol number: alxn1210-cov-305 original protocol: 09 apr 2020 protocol amendment 1 (global): 13 apr 2020 protocol amendment 2 (global): 17 apr 2020 protocol amendment 3 (global): 09 jun 2020 recruitment is currently ongoing. recruitment was initiated on 11 may 2020. we expect recruitment to be completed by 30 nov 2020. trial registration: clinicaltrials.gov: protocol registry number: nct04369469; first posted; 30 apr 2020 eu clinical trials register: eudract number: https://www.clinicaltrialsregister.eu/ctr-search/search?query=alxn1210-cov-305, start date: 07 may 2020 full protocol: the full redacted protocol is attached as an additional file, accessible from the trials website (additional file 1). in the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol. overall rationale for the amendment: this global amendment was initiated to update the inclusion and exclusion criteria, study endpoints and objectives, and the schedule of activities. patient-reported outcomes (sf-12 and eq-5d-5l) were added and changes were also implemented to align content in section 9 (statistical considerations) with version 1and version 2 of the statistical analysis plan. this amendment is considered to be substantial based on the criteria set forth in article 10(a) of directive 2001/20/ec of the european parliament and the council of the european union. substantive changes are documented in the table below. alexion confidential description of change brief rationale 1.1 -synopsis, 3 -objectives and endpoints 8.3. 2 • reordered secondary endpoints the ordering of testing for the secondary endpoints has been changed to align with the order of interest from a clinical and patient perspective. this reordering reduces the risk of a type ii error if a more clinically important endpoint is set below another endpoint in the hierarchy for which the null hypothesis is not rejected. the secondary endpoint of survival at day 60 and day 90 has been removed from the testing procedure when all patients have completed the primary evaluation period, which is day 29, as sufficient information for this endpoint may not be available. table s1 and table 5 correction of a typographical error given the half-life of the molecule, these expanded windows allow the investigational site staff to collect protocol-specified laboratory samples within a reasonable timeframe. footnote #16: revised to indicate that biomarker samples will be collected for all patients; however, for patients randomized to the ravulizumab + bsc treatment group biomarker samples will be collected any time before the start of infusion for clarity 1.3 -schedule of activities footnote #19: added statement to indicate that medical history should include the date of first onset of signs and symptoms of sars-cov-2 infection. for clarity added a clarifying statement that the 3-day screening period is allowed to evaluate patients for eligibility. if the screening and day 1 visits are combined, the patient has to meet all inclusion/no exclusion criteria prior to randomization. to clarify that patients may be evaluated for eligibility at any time during the 3-day screening period. revised criterion #2 -confirmed diagnosis of sars-cov-2 infection can be via pcr and/or antibody test removed rituximab and mitoxantrone as prohibited medications (criteria 5b and 5c) these medications have been historically disallowed in the sponsor's neurology studies due to a potential drug-drug interaction that could lower rituximab levels, and due to a potential confounding effect for efficacy with mitoxantrone. however, there is a very low likelihood that patients with severe covid-19 will be receiving these therapies in an acute setting, and the potential interactions are less important in this acute setting. therefore, the sponsor has elected to remove these medications from the list of excluded concomitant medications. 5.2 -exclusion criteria 6.5.1 -disallowed medicine and therapy revised washout period for ivig (criterion 5d) for clarity revised criterion to indicate that 1) patients who receive medications as part of bsc at the hospital due to emergency authorization under a compassionate use or expanded access program and 2) patients who receive antivirals (such as remdesivir) as part of a clinical study are eligible for participation in the study (criterion 6) to allow for enrollment of patients who may receive antivirals as part of a clinical study and receive treatment with investigational therapies under a compassionate use (emergency approval) or expanded access program removed verbiage indicating female patients will be evaluated for breastfeeding status or pregnancy at day 1 (criterion 7). correction of a typographical error added criterion #9 -allows enrollment of patients not currently vaccinated against neisseria meningitidis but who will receive prophylactic antibiotic treatment for at least 8 months after last infusion of study drug or until at least 2 weeks after they receive vaccination against n. meningitidis • added statement that the assessment of pao2 is optional and that the spo2 will serve as the surrogate for respiratory status • spo2/fio2 and the associated cutoff values were added to table 7 • added footnote to table 7 to allow for an alternate assessment of the respiratory system to help inform sofa scoring and for clarity revised to indicate that body weight has to be measured, but should be estimated using best judgement if it cannot be measured. for clarity revised to indicate that non-protocol-specified laboratory results that are considered clinically significant are not required to be entered in the crf/ecrf; however, the investigator is still required to report (and record in the ae crf/ecrf) the laboratory abnormality if deemed clinically significant the study is being implemented in an acute inpatient setting and non-protocol-specified laboratory results may not be readily accessible to sponsor staff for verification. to assess the effect of c5 inhibition on systemic activation of complement, inflammation, and prothrombic activity in patients with covid-19 • change in absolute levels of soluble biomarkers in blood associated with complement activation, inflammatory processes, and hypercoagulable states over time exploratory to evaluate the effect of ravulizumab + bsc compared with bsc alone on progression to renal failure requiring dialysis in patients with covid-19 • incidence of progression to renal failure requiring dialysis at day 29 to evaluate the effect of ravulizumab + bsc compared with bsc alone on clinical improvement in patients with covid-19 • time to clinical improvement (based on a modified 6-category ordinal scale) over 29 days to evaluate the effect of ravulizumab + bsc compared with bsc alone on the health-related quality of life of patients with covid-19 • sf-12 pcs and mcs scores at day 29 (or discharge), day 60, and day 90 • eq-5d-5l scores at day 29 (or discharge), day 60, and day 90 baseline is defined as the last available assessment on or before day 1 for all patients. day 1 will be defined as the date of the first infusion of ravulizumab for patients randomized and dosed with ravulizumab and as the date of randomization for patients randomized but not dosed with ravulizumab. abbreviations: bsc = best supportive care; c5 = complement component 5; covid-19 = coronavirus disease 2019; eq-5d-5l = euroqol 5-dimension 5-level; fio2 = fraction of inspired oxygen; mcs = mental component summary; pcs = physical component summary; pd = pharmacodynamic; pk = pharmacokinetic; sf-12 = 12-item short form; sofa = sequential organ failure assessment; spo2 = peripheral capillary oxygen saturation; teae = treatment-emergent adverse event; tesae = treatment-emergent serious adverse event. study alxn1210-cov-305 is a multicenter phase 3, open-label, randomized, controlled study designed to evaluate the safety and efficacy of intravenous (iv) ravulizumab + best supportive care (bsc), compared with bsc alone in patients with a confirmed diagnosis of sars-cov-2 infection, and a clinical presentation consistent with covid-19 severe pneumonia, acute lung injury, or ards. patients at least 18 years of age, weighing ≥ 40 kg, and admitted to a designated hospital facility for treatment will be screened for eligibility in this study. accounting for a 10% nonevaluable rate, approximately 270 patients will be randomized in a 2:1 ratio (180 patients to receive ravulizumab + bsc, 90 patients to bsc alone). patients randomized to ravulizumab + bsc will receive a weight-based dose of ravulizumab on day 1 (table s1 ). on day 5 and day 10, doses of 600 mg or 900 mg ravulizumab will be administered (according to weight category) and on day 15 patients will receive 900 mg ravulizumab. patients in both treatment groups will continue to receive medications, therapies, and interventions per standard hospital treatment protocols for the duration of the study. screening and the day 1 visits can occur on the same day if the patient has met all inclusion and no exclusion criteria. protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 this is an open-label parallel-treatment study. approximately 270 patients (180 ravulizumab + bsc, 90 bsc alone) will be randomly assigned to 1 of 2 treatment groups. the study consists of a screening period of up to 3 days, a primary evaluation period of 4 weeks, a final assessment at day 29, and a follow-up period of 8 weeks. the 2 follow-up visits will be conducted 4 weeks apart as a telephone call if the patient is discharged from the hospital or an in-person visit if the patient is still hospitalized. the total duration of each patient's participation is anticipated to be approximately 3 months. the dosage regimen to be administered during this study is provided in table s1 . no additional doses are allowed during the primary evaluation period (ie, from day 1 to day 29). a weight-based dose of ravulizumab will be administered on day 1 as follows: patients weighing ≥ 40 to < 60 kg: 2400 mg; ≥ 60 to < 100 kg: 2700 mg; or ≥ 100 kg: 3000 mg. a weight-based dose of ravulizumab will be administered on day 5 and day 10 as follows: patients weighing ≥ 40 to < 60 kg: 600 mg; ≥ 60 to < 100 kg: 900 mg; or ≥ 100 kg: 900 mg. on day 15, patients will receive 900 mg ravulizumab. **day 29 represents the end of the primary evaluation period. abbreviations: bsc = best supportive care; d = day; eos = end-of-study; n = number of patients. protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 page 22 of 86 alexion confidential 11 x x x x x x x x adverse event review and evaluation x <<monitor continuously>> x review safety card 12 x 12 x 12 x 12 x 12 safety laboratory tests (predose) 13 clinical chemistry x x x x x x x x hematology x x x x x x x x coagulation panel and d-dimer x x x x x x x x urinalysis x x x x x x x x direct coombs test x pk/pd/immunogenicity tests pk 14 x x x x x x x total and free c5 15 x x x x x x x immunogenicity (predose) 14 x x x biomarker tests serum and plasma biomarkers (predose) 16 x x x x x x x x other concomitant medication 17 x <<monitor continuously>> x nonpharmacologic treatments and therapies 18 x <<monitor continuously>> x patient-reported outcomes sf-12 x x eq-5d-5l x x protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 page 24 of 86 alexion confidential 6. confirmation of meningococcal vaccination within the past 5 years prior to dosing for patients randomized to ravulizumab + bsc. if vaccination cannot be confirmed, the patient should receive prophylactic antibiotics prior to initiating ravulizumab treatment and for at least 8 months from the last infusion of ravulizumab. when patients are vaccinated less than 2 weeks prior to treatment with ravulizumab or after initiation of ravulizumab, they should continue antibiotic prophylaxis for at least 2 weeks after meningococcal vaccination. 7. can be performed within the 3 days prior to screening or at screening. imaging performed as part of the patient's routine clinical care is expected and acceptable for inclusion in this study. 8. urine or serum pregnancy tests (beta human chorionic gonadotropin) to be performed in all female patients. a negative pregnancy test result is required before administration of ravulizumab. 9. spo2 to be measured by pulse oximetry. pao2 to be measured by arterial blood gas, if available. fio2 to be measured by supplemental oxygen. for patients treated with ravulizumab, spo2, pao2 (if available), and fio2 should be measured predose on day 1. the highest daily measurement of oxygen pressure or saturation on the lowest inspired supplemental oxygen level will be recorded in the crf/ecrf. complete or abbreviated physical examination is to be performed at the timepoints indicated in the soa. a complete physical examination will include, at a minimum, assessments of the following organs/body systems: skin, head, ears, eyes, nose, throat, neck, lymph nodes, chest, heart, abdomen, extremities, and musculoskeletal. an abbreviated physical examination consists of at least an evaluation of the respiratory and cardiovascular systems. clinically significant abnormalities or findings will be recorded in the ae crf/ecrf. vital sign measurements should include systolic and diastolic bp (millimeters of mercury [mm hg]), heart rate (beats/minute), respiratory rate (breaths/minute), and temperature (degrees celsius [°c] or degrees fahrenheit [°f]). these measurements will be taken predose on dosing days. when the patient is responsive and capable of understanding, review the patient safety information card (including discussion of the risks of meningococcal infections) during the hospitalization and at discharge. upon discharge, patients who received ravulizumab, must carry the patient safety information card at all times and for at least 8 months after the last infusion of ravulizumab. clinical safety laboratory measurements will be collected predose on dosing days. 14. serum samples for pk and immunogenicity analyses will be collected at the timepoints indicated in the soa for patients randomized to ravulizumab + bsc. on day 1/dosing days, immunogenicity and pk samples will be collected within 4 hours before the administration of ravulizumab (predose) and pk samples will be collected within 4 hours after the end-of-infusion (postdose). postdose pk samples must be collected from a separate line or needle stick to the noninfused arm, not from the infusion line. pharmacokinetic and immunogenicity samples can be collected at any time on nondosing days during the primary evaluation period. serum samples for total and free c5 analyses will be collected at the timepoints indicated in the soa for all patients. for patients randomized to ravulizumab + bsc, samples will be collected within 4 hours before the administration of ravulizumab (predose) and within 4 hours after the end-of-infusion (postdose) on dosing days. postdose samples must be collected from a separate line or needle stick to the noninfused arm, not from the infusion line. samples can be collected at any time on nondosing days during the primary evaluation period. serum and plasma biomarker samples for biomarker analyses will be collected for all patients at the timepoints indicated in the soa and stored at the investigational site prior to analysis by alexion or designee. samples will be collected predose (any time before infusion start) for patients who are randomized to the ravulizumab + bsc treatment group. concomitant medications and nonpharmacologic therapies considered relevant to the treatment of covid-19 (bsc) or ravulizumab treatment (eg, antimicrobials, antimalarials, antivirals, steroids, and vasopressors) that the patient is receiving, at the time of screening and for treating teaes/tesaes, will be recorded in the ae crf/ecrf. will be assessed via a telephone call at day 29 for all patients who are discharged before the end of the primary evaluation period (day 29). page 25 of 86 alexion confidential fio2 = fraction of inspired oxygen; na = not applicable; pao2 = partial pressure of oxygen; pd = pharmacodynamics; pk = pharmacokinetics; sars-cov-2 = severe acute respiratory syndrome coronavirus-2; sf-12 = 12-item short form; soa = schedule of activities; spo2 = peripheral capillary oxygen saturation; tesae = treatment emergent serious adverse event. protocol amendment 3 (global) the novel sars-cov-2 is a beta-coronavirus identified as the causative agent in covid-19 (cdc, covid 19 situation summary). clinical manifestations of covid-19 range from mild flu-like symptoms (eg, low grade fever, cough, fatigue) to ards, respiratory failure, multiple organ failure, and eventual death. an accelerating incidence of sars-cov-2 infections have been reported for patients who present with severe pneumonia, acute lung injury, or ards. emerging epidemiologic data indicate that approximately 30 -50% of patients with covid-19 may require hospitalization, approximately 10% may be admitted to critical care units, and 2.5% or more may die of multiorgan failure, especially those individuals who are older or have other comorbidities. for hospitalized patients, the world health organization (who) has issued recommendations on disease management and therapeutic regimens (who, 2020). aside from supportive care, no therapeutic regimens have been proven effective in reducing either the human-to-human transmission of the infection or its associated fatalities. mortality in those with critical illness has been reported as > 50%; therefore, implementation of proven critical care interventions such as lung protective ventilation is recommended by who. acute respiratory distress syndrome is a constellation of immune-mediated pathologies that are observed in severe cases of coronavirus infection (hammerschmidt, 1980) . this pattern was observed in 2002 with the emergence of severe acute respiratory syndrome-coronavirus (sars-cov), and in 2012 when a related coronavirus, middle east respiratory syndrome coronavirus (mers-cov) was identified (rota, 2003 , zaki, 2012 . complement activation and complement component 5a (c5a; the proinflammatory anaphylatoxin) are involved in multiple mechanisms in the development of acute lung disease induced by pathogenic viruses (wang, 2015) . emerging evidence suggests that activation of the complement system is involved in the pathogenesis of coronavirus (cov)-related ards, and that a c5 inhibitor may be an effective therapeutic in cov-mediated disease (gralinski, 2018 ). the complement system is a part of the immune system that enhances the ability of antibodies and phagocytic cells to clear pathogens and damaged cells. it is made up of more than 30 plasma protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 proteins that opsonize pathogens and induce a series of inflammatory responses to help fight infection. the complement system has key roles in innate and adaptive immune responses, but when hyperactivated can lead to tissue injury. within the complement system there are 3 pathways (classical, lectin, and alternative) that lead to cleavage of c5 and formation of the membrane attack complex, or terminal complement pathway. preclinical data have demonstrated a role for complement activation in cov-mediated disease. gralinski evaluated activation of the complement system in a mouse model of cov. the c57bl/6j mice were infected with mouse-adapted sars-cov which resulted in high-titer virus replication within the lung, induction of inflammatory cytokines and chemokines, and immune cell infiltration within the lung. complement activation was measured by detection of complement pathway component cleavage products. complement protein 3 activation products were detected in sars-cov ma15-infected mice, but not in control mice, as early as 1 day post-infection. complement protein 3 deposition was observed in the lungs of infected wildtype mice on day 2 and day 4 post-infection. transgenic animals lacking complement component 3 (c3) were protected from sars-cov-induced weight loss, had reduced pathology (inflammatory cells in the large airway and parenchyma, perivascular cuffing, thickening of the interstitial membrane, and low levels of intra-alveolar edema), had improved respiratory function, and exhibited lower levels of inflammatory cytokines or chemokines in the lung and periphery. notably, the kinetics of viral replication were unaltered in the c3-deficient mice relative to wildtype controls, suggesting that the observed effects were due to control of complement-mediated inflammatory processes and not reduction of viral titer. complement protein 3 is a central hub in the complement cascade and acts as a relay for activation from the alternative pathway. however transgenic mice lacking alternative pathway proteins factor b or c4 did not have the same protection from cov-mediated weight loss as compared with c3-deficient mice, suggesting that inhibition of the complement alternative pathway alone is insufficient. this implies that inhibition of a key relay point such as c3 or potentially c5, may be required. a second model of viral-mediated lung infection also points to a role for complement (jiang, 2018) . a mers-cov infection in mice causes severe acute respiratory failure and high mortality accompanied by elevated secretion of cytokines and chemokines. in these infected mice, excessive complement activation was detected. increased concentrations of c5a and terminal complement complex (c5b-9), activation products resulting from cleavage of c5, were detected in sera and lung tissue, respectively. blocking c5a with a specific antibody to the c5a receptor (c5ar) reduced alveolar macrophage infiltration and interferon (ifn)-gamma receptor expression in lung, resulting in less tissue damage. decreased spleen tissue damage was also observed. interestingly, anti-c5ar treatment led to decreased viral replication in lung tissues. patients infected by avian influenza virus h5n1 can also present with severe pneumonia, acute lung injury, or ards. the histopathological changes in the lungs are like those observed in severe acute respiratory syndrome (sars) (ng, 2006) . in a mouse model of h5n1, complement activates immune effector cells and drives lung inflammation. complement proteins c3a and c5a can increase vascular permeability, recruit and activate leukocytes, activate endothelial cells, upregulate adhesion molecule and cytokine expression, and induce goblet cell secretion of mucus, exacerbating disease through multiple mechanisms. in these mice, deposition of c3, c5b-9, and mannose-binding lectin (mbl)-c was observed in lung tissue. upregulation of mbl-associated serine protease-2 (masp-2) and complement receptors c3ar and c5ar was also detected. specific inhibition of either c3ar or c5a in the infected mice was effective in reducing lung damage, attenuating inflammation and neutrophil infiltration in the lung, and improving survival (sun, 2013 ). clinical evidence suggests that complement is activated during sars infection and that the progression of severe pneumonia, acute lung injury, or ards in these patients is strongly associated with complement activation. the c3c fragment is present in the sera of patients with sars and is a strong indicator of disease severity (pang, 2006) . consistent with this finding, the complement activation product c5a is associated with the inflammatory response and severe lung damage that occurs in patients infected with the 2009 h1n1 influenza virus (ohta, 2011) . it has also been shown that sars-cov can directly activate complement via the lectin pathway (ip, 2005) . patients with sars develop autoantibodies against human epithelial cells and endothelial cells that mediate complement-dependent cytotoxicity (yang, 2005) . multiple lines of evidence support the hypothesis that complement is a key mediator of virally-induced lung damage and that acute lung injury associated with cov infection is partially mediated by complement (wong, 2004) . therefore, it is plausible to hypothesize that covid-19-related injuries and multiorgan failures are mediated, at least in part, by complement activation. the existing data point to the role of c3 and the terminal complement complex, but not the alternative pathway alone. inhibition of complement, specifically at the terminal complement node through inhibition of c5, may control the inflammatory processes which drive ards. at present, there are no therapies that have received global approval by regulatory authorities for the prevention and/or treatment of covid-19. china's national health commission recently updated treatment guidelines for covid-19 recommending the use of tocilizumab (anti-interleukin [il] 6r monoclonal antibody [mab]) to treat chinese patients infected with sars-cov-2 who have developed serious lung damage and have elevated levels of il-6 in the blood. a variety of supportive therapies are being used in an attempt to improve prognosis in critically ill patients with confirmed covid-19 presenting with severe pneumonia, acute lung injury, or potentially life-threatening ards. despite the use of these supportive agents, patients have continued to experience deterioration of respiratory function, a critical contributor to fatal outcomes. ravulizumab is a mab that specifically binds to the complement protein c5 with high affinity, thereby inhibiting its cleavage to c5a and c5b (the initiating subunit of c5b-9) and preventing the generation of the terminal complement complex c5b-9. this mechanism of action provides a therapeutic rationale for the use of ravulizumab in diseases in which complement activation is involved. in addition, the selective blockade of complement cascade at c5 by ravulizumab preserves the activity of upstream components of the complement cascade that are known to be essential for opsonization of microorganisms and prevention of immune complex disorders (prodinger, 1999) . importantly, c5 blockade preserves the immunoprotective and immunoregulatory functions of early complement components. complement inhibition has been shown to be an effective therapeutic target in hematological and neuroinflammatory diseases. ravulizumab is proposed for the treatment of patients with confirmed sars-cov-2 infection with a clinical presentation consistent with covid-19 severe protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 pneumonia, acute lung injury, or ards. treatment with ravulizumab produces complete and sustained inhibition of c5-mediated terminal complement activity. treatment with ravulizumab could improve outcomes in patients with covid-19 severe pneumonia, acute lung injury, or ards. benefit/risk assessment 2.3.1. potential risks associated with participation in this study and risk mitigation measures are enumerated in table 2 . if vaccination cannot be confirmed, the patient should receive prophylactic antibiotics against meningococcal infection prior to initiating ravulizumab treatment and for at least 8 months from the final infusion of ravulizumab (only applicable to patients exposed to study drug). when patients are vaccinated less than 2 weeks prior to treatment with ravulizumab or after initiation of ravulizumab, they should continue antibiotic prophylaxis for at least 2 weeks after meningococcal vaccination. ravulizumab could increase the risk of infection in this patient population. this potential risk is based on the mode of action of ravulizumab and experience with the use of eculizumab. since the relevance of serious infection with ravulizumab therapy has not been confirmed in clinical studies, this remains a potential risk. training healthcare professionals and patients about the potential risk of additional serious infection. monitoring for signs and symptoms of serious infections will be conducted as part of routine safety assessments for this study. in addition to appropriate antibiotic coverage versus infection and opportunistic infections, guidelines for immune reconstitution and revaccination will be followed, as applicable. immunogenicity treatment with any therapeutic protein has the potential to induce an immune response. potential clinical consequences may include severe hypersensitivity type reactions, across 350 patients enrolled in ravulizumab, phase 3 clinical studies, 2 patients were reported with treatment-emergent adas. protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 page 30 of 86 alexion confidential decrease in efficacy and induction of autoimmunity, including antibodies to the endogenous form of the protein (li, 2002; casadevall, 2002) . presence of anti-alxn1210 antibodies will be assessed. protein therapies administered intravenously have the potential risk of causing local (infusion-site) reactions and systemic reactions (infusion-associated reactions). monitoring for infusion reactions will be conducted as part of routine safety assessments for this study. management of potential infusion reactions is detailed in section 10.6. no studies of ravulizumab have been conducted in pregnant women. there are no data available on excretion of ravulizumab in breast milk. pregnant or nursing female patients will be excluded from participating in this clinical study. patients and their spouses/partners must use a highly effective or acceptable method of contraception for a period of 8 months following the final infusion of ravulizumab. breastfeeding should be discontinued during treatment and up to 8 months after the final infusion of ravulizumab. abbreviations: ada = antidrug antibody. potential benefits of study participation include: • improve survival rate of patients with sars-cov-2 infection who are receiving ravulizumab + best supportive care (bsc) compared with bsc alone • decrease lung injury in patients with sars-cov-2 infection while on supportive medical care • improve clinical outcomes in patients with sars-cov-2 infection while on supportive medical care although the efficacy of ravulizumab has not been previously studied in patients with severe pneumonia, acute lung injury, or ards, the emerging evidence for the scientific rationale, the predicted drug concentrations and pharmacodynamic (pd) effects after administration of the recommended dose (section 4.3), and its established safety profile indicate that ravulizumab is an appropriate candidate for clinical investigation, and that the potential for clinical benefit outweighs the risk of treatment with ravulizumab for patients participating in study alxn1210-cov-305. the safety profile of ravulizumab is well characterized in the current clinical development programs, including approved indications in paroxysmal nocturnal hemoglobinuria (pnh) and protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 page 31 of 86 alexion confidential atypical hemolytic uremic syndrome (ahus). known and potential risks can be effectively managed with the risk mitigation strategies currently in place for ravulizumab. more detailed information about the known and expected benefits and risks and reasonably expected adverse events (aes) of ravulizumab may be found in the investigator's brochure (ib). to characterize the pk/pd and immunogenicity of ravulizumab in patients with covid-19 • change in serum ravulizumab concentrations over time • change in serum free and total c5 concentrations over time • incidence and titer of anti-alxn1210 antibodies biomarkers to assess the effect of c5 inhibition on systemic activation of complement, inflammation, and prothrombic activity in patients with covid-19 • change in absolute levels of soluble biomarkers in blood associated with complement activation, inflammatory processes, and hypercoagulable states over time exploratory to evaluate the effect of ravulizumab + bsc compared with bsc alone on progression to renal failure requiring dialysis in patients with covid-19 • incidence of progression to renal failure requiring dialysis at day 29 to evaluate the effect of ravulizumab + bsc compared with bsc alone on clinical improvement in patients with covid-19 • time to clinical improvement (based on a modified 6-point ordinal scale) over 29 days to evaluate the effect of ravulizumab + bsc compared with bsc alone on the health-related quality of life of patients with covid-19 • sf-12 pcs and mcs scores at day 29 (or discharge), day 60, and day 90 • eq-5d-5l scores at day 29 (or discharge), day 60, and day 90 baseline is defined as the last available assessment on or before day 1 for all patients. day 1 will be defined as the date of the first infusion of ravulizumab for patients randomized and dosed with ravulizumab and as the date of randomization for patients randomized but not dosed with ravulizumab. study alxn1210-cov-305 is a multicenter phase 3, open-label, randomized, controlled study designed to evaluate the safety and efficacy of intravenous (iv) ravulizumab + bsc, compared with bsc alone, in patients with a confirmed diagnosis of sars-cov-2 infection, and a clinical presentation consistent with covid-19 severe pneumonia, acute lung injury, or ards. patients at least 18 years of age, weighing ≥ 40 kg, and admitted to a designated hospital facility for treatment will be screened for eligibility in this study. accounting for a 10% nonevaluable rate, approximately 270 patients will be randomized in a 2:1 ratio (180 patients to receive ravulizumab + bsc, 90 patients to receive bsc alone). patients randomized to ravulizumab + bsc will receive a weight-based dose of ravulizumab on day 1 (table s1 ). on day 5 and day 10, doses of 600 mg or 900 mg ravulizumab will be administered (according to weight category) and on day 15 patients will receive 900 mg ravulizumab. patients in both treatment groups will continue to receive medications, therapies, and interventions per standard hospital treatment protocols for the duration of the study. the study consists of a screening period of up to 3 days, a primary evaluation period of 4 weeks, a final assessment at day 29, and a follow-up period of 8 weeks. the 2 follow-up visits will be conducted 4 weeks apart as a telephone call if the patient is discharged from the hospital or an in-person visit if the patient is still hospitalized. the total duration of each patient's participation is anticipated to be approximately 3 months (figure 1 ). screening and the day 1 visits can occur on the same day if the patient has met all inclusion and no exclusion criteria. this is an open-label, 2:1 randomized, controlled study. o a randomized, controlled study design minimizes bias to selection or treatment allocation. this design will ensure identification of an effective treatment that may improve survival in patients with covid-19 severe pneumonia, acute lung injury, or ards. o the 2:1 randomization will ensure that approximately two-thirds of patients are exposed to treatment and provides more safety information in this patient population for benefit-risk assessment. study alxn1210-cov-305 is being conducted in patients with sars-cov-2 infection with a clinical presentation consistent with covid-19 severe pneumonia, acute lung injury, or ards. o there is no approved treatment for patients with the severe form of covid-19. o clinical evidence suggests that complement is activated during sars infection and that the progression of disease is also associated with complement activation. protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 page 34 of 86 alexion confidential o treatment with ravulizumab could 1) decrease lung injury and improve clinical outcomes in patients with sars-cov-2 infection while on supportive medical care and 2) provide additional time for patients to recover. for patients randomized to ravulizumab + bsc, a weight-based dose of ravulizumab will be administered on day 1. on day 5 and day 10, additional doses of 600 mg or 900 mg ravulizumab will be administered (according to weight category) and on day 15 patients will receive 900 mg ravulizumab. o preliminary pk/pd data suggest that the complement system is amplified in patients with covid-19 severe pneumonia, acute lung injury, or ards beyond what has been observed in patients with ahus and that additional doses of ravulizumab are needed to provide complete and sustained complement inhibition. o due to the rapid activation of complement associated with the severe form of covid-19 (ie, hyperinflammatory response known as cytokine storm syndrome [mehta, 2020] ), this ravulizumab dosage regimen is expected to improve survival and clinically relevant endpoints in these patients. o the chosen primary endpoint is anticipated to reflect a ravulizumab treatment effect over 4 weeks and thus an immediate impact on survival. secondary endpoints o most secondary endpoints were selected based on society of critical care medicine recommendations for standardized endpoints in clinical studies using an intervention to reduce the duration of mechanical ventilation (blackwood, 2019) . o the sofa score is a validated endpoint used in the critical care setting to determine clinical outcomes (eg, multiorgan failure) (lambden, 2019) . this is the basis of the therapeutic strategy to achieve complete complement inhibition in patients with covid-19 infection who present with severe pneumonia, acute lung injury, and/or ards. the end of the primary evaluation period is defined as the date when the last surviving patient completes the day 29/early termination (et) visit. the end of the study is defined as the last patient's last visit, which may be the final safety follow-up telephone call or in-person visit. prospective approval of protocol deviations to recruitment and enrollment criteria, also known as protocol waivers or exemptions, are not permitted. the sponsor allows a 3-day screening period to evaluate patients for eligibility. if the screening and day 1 visits are combined, the patient must meet all inclusion criteria and not meet any exclusion criteria prior to randomization. patients are eligible to be included in the study only if all the following criteria apply: age 1. patient must be ≥ 18 years of age at the time of providing informed consent. 7. female patients of childbearing potential and male patients with female partners of childbearing potential must follow protocol-specified contraception guidance for avoiding pregnancy for 8 months after treatment with the study drug (as described in section 10.4 [appendix 4]). patients are excluded from the study if any of the following criteria apply: 1. patient is not expected to survive for more than 24 hours. 2. patient is on invasive mechanical ventilation with intubation for more than 48 hours prior to screening. a. current treatment with a complement inhibitor or b. intravenous immunoglobulin (ivig) within 4 weeks prior to randomization on day 1. 6. treatment with investigational therapy in a clinical study within 30 days before randomization, or within 5 half-lives of that investigational therapy, whichever is greater exceptions: a. investigational therapies will be allowed if received as part of best supportive care through an expanded access protocol or emergency approval for the treatment of covid-19. b. investigational antiviral therapies (such as remdesivir) will be allowed even if received as part of a clinical study. 7. female patients who are breastfeeding or who have a positive pregnancy test result at screening. 8. history of hypersensitivity to any ingredient contained in the study drug, including hypersensitivity to murine proteins. 9. patient who is not currently vaccinated against n. meningitidis, unless the patient agrees to receive prophylactic treatment with appropriate antibiotics for at least 8 months after the last infusion of study drug or until at least 2 weeks after the patient receives vaccination against n. meningitidis. no lifestyle considerations are applicable for this study. screen failures are defined as patients who provide informed consent, did not meet any inclusion/exclusion criteria, and are not randomly assigned to treatment. a minimal set of screen failure information is required to ensure transparent reporting of screen failure patients to meet the consolidated standards of reporting trials (consort) publishing requirements. minimal information includes demography (if allowable per local regulations), screen failure details (eg, failed eligibility criteria), and any aes, including any serious adverse events (saes) and any relevant concomitant medication use during the screening period. individuals who do not meet the criteria for participation in this study (screen failure) due to a reason that is expected to resolve or has resolved may be rescreened based on discussion and agreement between the investigator and the medical monitor. ravulizumab, a recombinant humanized anti-c5 mab composed of two 448 amino acid heavy chains and two 214 amino acid light chains, is an igg2/4 kappa immunoglobulin consisting of human constant regions, and murine complementarity-determining regions grafted onto human framework light-and heavy-chain variable regions. ravulizumab is produced in chinese hamster ovarian cell lines and was designed through minimal targeted engineering of eculizumab by introducing 4 unique amino acid substitutions to its heavy chain to extend antibody half-life. ravulizumab drug product is supplied for clinical studies as a sterile, preservative-free 10 mg/ml solution in single-use vials and designed for infusion by diluting into commercially available saline (0.9% sodium chloride injection; country-specific pharmacopeia) for administration via iv infusion. the proposed dosage regimen for the treatment of patients with covid-19 who are ≥ 18 years and ≥ 40 kg and are randomized to ravulizumab + bsc is presented in table 5 . the patient's body weight will be recorded on the day of the infusion visit. if the weight at the day of the infusion cannot be obtained, the weight recorded during the previous study visit may be used. abbreviations: covid-19 = coronavirus disease 2019. ravulizumab drug product is formulated at ph 7.0 and each 30 ml vial contains 300 mg of ravulizumab, 0.02% polysorbate 80, 150 mm sodium chloride, 6.63 mm sodium phosphate dibasic, 3.34 mm sodium phosphate monobasic, and water for injection, united states pharmacopeia. the ravulizumab admixture will be administered to the patient using an iv tubing set via an infusion pump followed by an iv flush. use of a 0.2 micron filter is required during the infusion. the iv flush is infused at the same rate of the infusion and end of flush is considered the end of infusion. the iv flush volume is not to be included in the total volume of study drug administered. additional details are provided in the pharmacy manual. ravulizumab will be manufactured and supplied by alexion in single 30 ml vials as a solution concentration of 10 mg/ml (table 6 ). each vial contains 300 mg of ravulizumab for iv administration. protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 stability studies of the diluted admixture of ravulizumab (10 mg/ml) in 0.9% sodium chloride injection support an in-use stability of 6 hours at room temperature at 23°c -27°c (73°f -80°f) and 24 hours when refrigerated at 2°c -8°c (36°f -46°f). ravulizumab vials should not be frozen or shaken. the investigator or designee must confirm appropriate temperature conditions have been maintained during transit for all study intervention received and any discrepancies are reported and resolved before use of the study intervention. only patients enrolled in the study and randomized to the ravulizumab + bsc group may receive the study intervention and only authorized site staff may supply or administer the study intervention. all study intervention must be stored in a secure, environmentally controlled, and monitored (manual or automated) area in accordance with the labeled storage conditions with access limited to the investigator and authorized site staff. the investigator, institution, or the head of the medical institution (where applicable) is responsible for study intervention accountability, reconciliation, and record maintenance (ie, receipt, reconciliation, and final disposition records). this responsibility includes the reporting of any product complaints* to within 1 business day of awareness. *a product complaint is defined as any written, electronic, or oral communication that alleges deficiencies related to the identity, quality, durability, reliability, usability, safety, effectiveness, or performance of a product or clinical study material and/or its packaging components after it is has been released for distribution to an end customer that affects the performance of such product. further guidance and information are provided in the pharmacy manual. alxn1210-cov-305 09 jun 2020 this is an open-label study. eligible patients who meet all inclusion and no exclusion criteria will be randomized in a 2:1 ratio to receive either ravulizumab + bsc or bsc alone. randomization will be stratified by intubated or not intubated on day 1. a randomization schedule will be developed by a centralized third party. when patients are dosed at the investigational site, they will receive ravulizumab directly from the investigator or designee, under medical supervision, thereby minimizing noncompliance. the date and time of the dose administered will be recorded in the source documents and recorded in the case report form (crf)/electronic case report form (ecrf). the dose of ravulizumab and study patient identification will be confirmed at the time of dosing by a member of the investigational site staff. patients may receive appropriate concomitant medications, including antivirals, as part of bsc during this clinical study, unless prohibited per exclusion criterion 5. concomitant medications considered relevant to treatment of covid-19 or ravulizumab treatment (eg, vaccines, antimicrobials, antimalarials, antivirals, steroids, and vasopressors) that the patient is receiving at the time of enrollment or receives during the study must be recorded in the crf/ecrf along with: • reason for use, • dates of administration, including start and end dates, and • dosage information including dose and frequency. the medical monitor should be contacted if there are any questions regarding concomitant therapy. use of the following medications and therapies is prohibited for the specified duration prior to screening and for the duration of the study: • current treatment with a complement inhibitor, and • intravenous immunoglobulin (ivig) within 4 weeks prior to randomization on day 1. additional doses are not allowed during the study. the dosage regimen to be administered during this study is provided in table 5 . patients will continue to be under the care of their treating physician after the study has concluded. this is an open-label study. study drug discontinuation is only applicable for patients who are randomized to ravulizumab + bsc. in rare instances, it may be necessary for a patient to permanently discontinue (definitive discontinuation) the study drug. if the study drug is definitively discontinued, the patient should remain in the study to be evaluated for safety. patients should be considered for discontinuation from study drug if any of the following occur: • serious hypersensitivity reaction; • severe uncontrolled infection; • use of disallowed medication as defined in section 6.5; • pregnancy or planned pregnancy; or • alexion or the investigator deems it is necessary for the participant. data to be collected at the time of discontinuation of study drug, including follow-up for any further evaluations that need to be completed is provided in the schedule of activities (soa , table 1 ). • when applicable, all efforts should be made to ensure patients are willing to comply with study participation prior to conducting the screening procedures. the study staff should notify alexion and their site monitor of all study withdrawals as soon as possible. the reason for patient discontinuation must be recorded in the source documents and crf/ecrf. • a patient may withdraw from the study at any time at his/her own request or may be withdrawn at any time at the discretion of the investigator for safety, behavioral, compliance, or administrative reasons. this is expected to be uncommon. • at the time of discontinuing from the study, if possible, an et visit should be conducted. refer to the soa (table 1) for assessments to be collected at the time of study discontinuation and follow-up. • if the patient withdraws consent for disclosure of future information, alexion may retain and continue to use any data collected before such a withdrawal of consent. • if a patient withdraws from the study, he/she may request destruction of any samples taken and not tested, and the investigator must document this in the site study records. if a patient is unreachable for a scheduled visit within the acceptable visit window (soa , table 1 ), the site study staff must make a reasonable attempt to contact the patient to determine protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 page 45 of 86 alexion confidential the reason for missing the visit. if the patient continues to be unreachable, he/she will be considered as lost to follow-up. discontinuation of specific sites or of the study as a whole is handled as part of section 10.1.8. alxn1210-cov-305 09 jun 2020 page 46 of 86 alexion confidential • study procedures and their timing are summarized in the soa (table 1 ). the list of clinical laboratory tests to be performed is provided in section 10.2. • immediate safety concerns should be discussed with alexion immediately upon occurrence or awareness to determine if the patient should continue or discontinue study intervention. • adherence to the study design requirements, including those specified in the soa (table 1) , is essential and required for study conduct. • all screening evaluations must be completed and reviewed to confirm that potential patients meet all eligibility criteria. the investigator will maintain a screening log to record details of all patients screened and to confirm eligibility or record reasons for screening failure, as applicable. • procedures conducted as part of the patient's routine clinical management (eg, blood count) and obtained before signing of the informed consent form (icf) may be utilized for screening or baseline purposes provided the procedures met the protocol-specified criteria and were performed within the time frame defined in the soa (section 1.3). patients or their legally acceptable representative must be consented per the informed consent process outlined in section 10.1.3. if allowable per local regulations, exceptions may be granted in cases where the patient is unable to provide informed consent. all inclusion (section 5.1) and exclusion (section 5.2) criteria must be reviewed by the investigator or qualified designee to ensure the patient qualifies for study participation. the patient's relevant medical history, including prior and concomitant conditions/disorders, treatment history, and history of medical conditions (ie, cardiovascular and respiratory, including smoking status) will be evaluated by the investigator and documented in the source documents and crf/ecrf. medical history should also include date of first onset of signs and symptoms of sars-cov-2 infection. a review of demographic parameters, including age, gender, race, and ethnicity will be performed, if allowable per local regulations. protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 confirmation of meningococcal vaccination within the past 5 years prior to dosing for patients randomized to ravulizumab + bsc. if vaccination cannot be confirmed, the patient should receive prophylactic antibiotics prior to initiating ravulizumab treatment and for at least 8 months from the last infusion of ravulizumab. when patients are vaccinated less than 2 weeks prior to treatment with ravulizumab or after initiation of ravulizumab, they should continue antibiotic prophylaxis for at least 2 weeks after meningococcal vaccination. additional guidance is provided in section 8.4.9. the sars-cov-2 infection will be evaluated at the designated hospital. a confirmed positive result (eg, via pcr and/or antibody test) is required before randomization. chest ct or x-ray scans will be performed during the screening period to confirm findings consistent with severe pneumonia, acute lung injury, or ards in patients with covid-19. scans performed during the course of the patient's clinical care are accepted and expected to fulfil this diagnostic inclusion criterion for study alxn1210-cov-305. urine or serum pregnancy tests (beta human chorionic gonadotropin) will performed in all female patients. a negative pregnancy test result is required before administration of ravulizumab. survival at day 29 will be determined. the following secondary efficacy parameters will also be measured through day 29: • mechanical ventilation status, • time in the intensive care unit (icu), • sequential organ failure assessment (sofa) score, • oxygen saturation levels (peripheral capillary oxygen saturation [spo2]), • duration of hospitalization. the following secondary efficacy parameter will be measured at day 60 and day 90: protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 • survival (based on all-cause mortality) multiple organ failure is a significant indicator of mortality in patients admitted to the icu. in this study, patients will be evaluated using the sofa score, an assessment tool that includes a review of 6 organ systems: respiratory, renal, hepatic, cardiac, coagulation, and central nervous system (vincent, 1998) . each organ system is scored from 0 to 4 points using the worst value observed within the previous 24 hours (table 7) . arterial blood gas may not be drawn on a protocol-specified visit day; therefore, the assessment of partial pressure of oxygen (pao2) is optional and the highly correlated spo2 will be a surrogate for the respiratory system assessment. 13 -14 10 -12 6 -9 < 6 1. as arterial blood gas may not be drawn on a protocol-specified visit day, the pao2 assessment is optional. abbreviations: cns = central nervous system; gcs = glasgow coma scale; fio2 = fraction of inspired oxygen; pao2 = partial pressure of oxygen; spo2 = peripheral capillary oxygen saturation. source: vincent, 1998; pandharipande, 2006 8.4. safety assessments the following safety-related parameters will be measured through day 29. protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 • complete or abbreviated physical examinations will be assessed by the investigator or designee. a complete physical examination will include, at a minimum, assessments of the skin, head, ears, eyes, nose, throat, neck, lymph nodes, chest, heart, abdomen, extremities, and musculoskeletal. an abbreviated physical examination will include at a minimum, assessment of the respiratory system and cardiovascular systems. • body weight should be measured, but if the site does not have the capacity to measure the patient's body weight it should be estimated using best judgement. investigators or designees should pay special attention to clinical signs related to previous serious illnesses. clinically significant abnormalities or findings will be recorded on the ae crf/ecrf. vital sign measurements will include systolic and diastolic blood pressure (millimeters of mercury [mm hg]), heart rate (hr, beats/minute), respiratory rate (rr, breaths/minute), and temperature (degrees celsius [°c] or degrees fahrenheit [°f]). vital sign measurements will be taken predose on dosing days. • a single 12-lead electrocardiogram (ecg) will be conducted to obtain hr, pulse rate (pr) interval, combination of the q wave, r wave and s wave (qrs) interval, interval between the start of the q wave and the end of the t wave (qt), and the corrected qt (qtc) interval(s). • the investigator must review the laboratory report, document this review, and record any clinically relevant changes occurring during the study in the ae crf/ecrf. the laboratory reports must be filed with the source documents. clinically significant abnormal laboratory findings are those that are not associated with the underlying disease, unless judged by the investigator or designee to be more severe than expected for the patient's condition. • all laboratory tests with values considered clinically significantly abnormal during participation in the study should be repeated until the values return to normal or baseline or are no longer considered clinically significant by the investigator or the medical monitor. − if such values do not return to normal/baseline within a period of time judged reasonable by the investigator, the etiology should be identified, and alexion notified. − all protocol-required laboratory assessments, as defined in section 10.2, must be conducted in accordance with the laboratory manual and the soa (table 1) . − laboratory assessments performed at the institution's local laboratory that require a change in patient management or are considered clinically significant by the protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 investigator (eg, reported as an sae or ae), must be recorded in the ae crf/ecrf. details of immunogenicity assessments are presented in section 8.12. the glasgow coma scale (gcs) is a validated prognostic tool used in the clinical assessment of unconsciousness (eg, patients who are comatose) (sternbach, 2000) .the gcs is comprised of 3 domains -eye response, verbal response, and motor response and within each domain contains a subset of responses that are separately assigned a score ( table 8 ). the gcs has also been used in the critical care setting as an aid in managing respiratory support. a total gcs score of < 8 is indicative of a patient's need for endotracheal intubation. the gcs will be measured to enable calculation of the secondary efficacy endpoint, sofa score. • pregnancy data from all female patients and female spouses/partners of male patients will be collected from the signing of the icf until the conclusion of the study participation. if a pregnancy is reported, the investigator must immediately inform alexion within 24 hours of awareness of the pregnancy and follow the procedures outlined in section 10.4. • for all alexion products, both in development or post-approval, exposure during pregnancy must be recorded and the pregnancy followed, until the outcome of the pregnancy is known (ie, spontaneous miscarriage, elective termination, normal birth, or protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 congenital abnormality), even if the patient discontinues the study intervention or withdraws from the study. the corresponding infant must be followed-up with for 3 months postpartum. • pregnancy is not considered as an ae (section 10.4.3) unless there is a suspicion that ravulizumab may have interfered with the effectiveness of a contraceptive medication. however, complications of pregnancy and abnormal outcomes of pregnancy are aes and may meet the criteria for an sae (eg, ectopic pregnancy, spontaneous abortion, intrauterine fetal demise, neonatal death, or congenital anomaly) (section 8.6). elective abortions without complications should not be reported as aes. when patients randomized to ravulizumab + bsc are able to understand, a patient safety information card will be provided to carry with them at all times. the card is provided to increase patient awareness of the risk of meningococcal infections, and promote quick recognition and disclosure of any potential signs or symptoms of infection experienced during the course of the study and to inform patients on what actions must be taken if they are experiencing signs or symptoms of infection. at each visit throughout the study, the study staff will ensure that the patient has the patient safety information card. patients are required to carry the patient safety information card for 8 months after the last infusion of ravulizumab. it is anticipated that patients randomized to ravulizumab + bsc who have not received a meningococcal vaccination within the past 5 years may be unable to receive meningococcal vaccinations prior to initiating treatment with ravulizumab during this study. if vaccination cannot be confirmed, the patient should receive prophylactic antibiotics against meningococcal infection prior to initiating ravulizumab treatment and for at least 8 months from the last infusion of ravulizumab. when patients can be vaccinated, vaccines against meningococcal serotypes a, c, y, w135, and b, where available, are recommended to prevent common pathogenic meningococcal serotypes. patients must be vaccinated or revaccinated according to the current national vaccination guidelines or local practice for vaccination use with complement inhibitors (eg, ravulizumab). vaccination may not be sufficient to prevent meningococcal infection. consideration should be given per official guidance and local practice on the appropriate use of antibacterial agents. when patients are vaccinated after initiation of ravulizumab, they should continue antibiotic prophylaxis for at least 2 weeks after meningococcal vaccination. if a patient is discharged before the end of the primary evaluation period, the patient will be contacted via telephone on day 29 to assess health status (survival, mechanical ventilation, hospitalization, intensive care unit, and dialysis). alxn1210-cov-305 09 jun 2020 follow-up visits will be conducted as indicated in the soa (table 1) to review patient status; including survival, monitoring for pregnancy, and to obtain information about new or worsening treatment-emergent saes (tesaes). the follow-up visits will be conducted as a telephone call if the patient is discharged from the hospital or an in person visit if the patient is still hospitalized. if a patient is discontinued from the study during the primary evaluation period (ie, from day 1 through day 29), the patient should present for the early termination visit, to be conducted as specified in the soa (table 1) . the patient will be contacted via telephone on day 29 to assess health status (eg, survival, mechanical ventilation, hospitalization, intensive care unit, and dialysis). the following exploratory parameters will also be measured: • progression to renal failure requiring dialysis at day 29 a reduction in the time to clinical improvement, especially when the patient is treated within a short timeframe from symptom onset has been reported in studies comparing antivirals to placebo (wang, 2020) . time to clinical improvement will be evaluated during this study and is defined as a live discharge, a decrease from of least 2 points (ie, #5 to #3) from baseline, or both. a modified 6-category ordinal scale (itemized below) will be used to evaluated clinical improvement. the short-form (sf)-12 is a validated health-related quality of life (hr-qol) instrument that is widely used across a broad spectrum of disease indications. adapted from the 36 -item sf survey that was designed to evaluate physical and mental health status, the sf-12 survey contains only 12 questions but covers the same 8 domains. there is a further stratification into 2 summary measures (physical component summary and mental component summary ) as specified below. (2 items) the pcs-12 and mcs-12 summary measures are scored using a norm-based method (ie, mean = 50, sd = 10) (jenkinson, 1997) . a pcs-12 or mcs-12 score of 50 indicates an average score with respect to a healthy population. scores lower than 50 reflect less than average health and scores greater than 50 reflect better than average health (ware, 1995) . the sf-12 assumes a recall of 1 week before responding to questions. the survey is anticipated to be completed in several minutes and can be completed by the patient or via an interviewer (in-person or over the telephone). the euroqol 5-dimension, 5 severity level (eq-5d-5l) questionnaire is a brief, validated, hr-qol instrument that is intended to assess the patient's health status at the time of administration. the questionnaire contains 5 dimensions (mobility, self-care, usual activities, pain/discomfort, and anxiety/depression), each of which includes 5 response variables (no problems, slight problems, moderate problems, severe problems, and extreme problems) (eq -5d, 2019) there is no summary score generated upon completion, but rather a 5-digit profile (termed "health state") based on each of the dimensions that can be further converted to a single numerical score (index value).value sets (a collection of index values) have been derived for multiple countries/regions. a vertical visual analogue scale (vas) is included for patients to indicate a self-rated estimate of their health. the vas ranges from 100 (best health you can imagine) to 0 (worst health you can imagine). the eq-5d-5l questionnaire and vas are anticipated to be completed in several minutes and can be completed by the patient, via an interviewer (in-person or over the telephone); or via proxy. the definitions of aes and saes can be found in section 10.3. all aes will be reported to the investigator or qualified designee by the patient (or, when appropriate, by a caregiver, surrogate, or the patient's legally acceptable representative). protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 the investigator and any qualified designees are responsible for detecting, documenting, and recording events that meet the definition of an ae or sae and remain responsible for following up aes that are serious, considered related to the study intervention or study procedures, or that caused the patient to discontinue the study intervention (see section 7). procedures for recording, evaluating, follow-up, and reporting aes and saes are outlined in section 10.3 (appendix 3). all aes and saes will be collected from the time of informed consent until through the timepoints specified in the soa (table 1) . all saes will be recorded and reported to alexion or the designee immediately and under no circumstance should this exceed 24 hours, as indicated in section 10.3 (appendix 3). the investigator will submit any updated sae data to alexion within 24 hours of it being available. investigators are not obligated to actively seek ae or sae data after conclusion of the study participation. however, if the investigator learns of any sae, including a death, at any time after a patient has been discharged from the study, and he/she considers the event to be reasonably related to the study intervention or study participation, the investigator must promptly notify alexion. the method of recording, evaluating, and assessing causality of ae and sae and the procedures for completing and transmitting sae reports are provided in section 10.3. care will be taken not to introduce bias when detecting aes and/or saes. open-ended and non-leading verbal questioning of the patient is the preferred method to inquire about ae occurrences. after the initial ae/sae report, the investigator is required to proactively follow-up on each patient at subsequent visits/contacts. all saes will be followed-up until resolution, stabilization, the event is otherwise explained, or the patient is lost to follow-up (as defined in section 7.3). further information on follow-up procedures is provided in section 10.3. • prompt notification of an sae by the investigator to alexion is essential so that legal obligations and ethical responsibilities towards the safety of patients and the safety of a study intervention under clinical investigation are met. • alexion has a legal responsibility to notify both the local regulatory authority and other regulatory agencies about the safety of a study intervention under clinical investigation. alexion will comply with country-specific regulatory requirements relating to safety reporting to the regulatory authority, institutional review board/independent ethics committee (irbs/iec), and investigators. • suspected unexpected serious adverse reactions (susars) must be reported according to local regulatory requirements and alexion policy and forwarded to investigators as necessary. • an investigator who receives an investigator safety report describing an sae or other specific safety information (eg, summary or listing of saes) from alexion will review and then file it along with the ib and will notify the (irb/iec), if appropriate according to local requirements. adverse events of special interest that will be monitored during this study are meningococcal infections. no cases of ravulizumab overdose have been reported during clinical studies. any dose of ravulizumab greater than that specified in the protocol will be considered an overdose. overdoses are medication errors that are not considered teaes unless there is an untoward medical occurrence resulting from the overdose. in the event of an overdose, the investigator or designee should: 1. contact the medical monitor immediately. 2. closely monitor the patient for any sae. 3. obtain a plasma sample for pk analysis if requested by the medical monitor (determined on a case-by-case basis). 4. document the quantity of the excess dose as well as the duration of the overdose in the crf/ecrf. samples will be collected from patients randomized to ravulizumab + bsc as specified in the soa (table 1) to determine serum concentrations of ravulizumab. the actual date and time (24-hour clock time) of each sample will be recorded. samples will be collected from all patients as specified in the soa (table 1) to assess the effect serum and plasma samples will be collected from all patients for biomarker analysis to evaluate complement activation and related pathways and cardiovascular health, and their clinical response to ravulizumab. these biomarkers include complement pathway proteins (eg, total and free c5, soluble c5b-9 [sc5b-9]), cytokines associated with inflammation and disease (eg, il-1, il-2r, il-6, il-8, il-21, tumor necrosis factor [tnf]-b, pentraxin-3, citrullinated histone h3, and monocyte chemoattractant protein [mcp]-1), factor ii, and markers associated with cardiovascular disease (procalcitonin, myoglobin, high sensitivity troponin i [hs-tni] and n-terminal pro-b-type natriuretic peptide [nt-probnp]). antibodies to alxn1210 (ie, antidrug antibody [ada]) will be evaluated in serum samples collected from patients randomized to ravulizumab + bsc according to the soa (table 1) . additionally, serum samples should also be collected at the final visit from patients who discontinued ravulizumab or were withdrawn from the study. these samples will be tested by alexion or alexion's designee. serum samples will be screened for antibodies binding to ravulizumab and the titer of confirmed positive samples will be reported. other analyses may be performed to further characterize the immunogenicity of ravulizumab. the detection and characterization of antibodies to ravulizumab will be performed using a validated assay method. samples collected for detection of antibodies to ravulizumab will also be evaluated for study intervention serum concentration to enable interpretation of the antibody data. confirmed antibody positive samples will be further evaluated for antibody titer and the presence of neutralizing antibodies. data collected during this study that may be used to conduct economic analyses include: • duration of hospitalization (total days or length of stay), • duration of icu stay (including total days), and • patient reported outcomes (eg, sf-12, version 2 and eq-5d-5l). protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 9. statistical considerations the primary null hypothesis is that there is no difference in survival between ravulizumab + bsc and bsc alone as measured by the difference in the proportions surviving at day 29 between the 2 treatment groups. the alternative hypothesis is that ravulizumab + bsc will improve survival at day 29 compared with bsc alone. the null hypotheses associated with the secondary objectives are that ravulizumab + bsc is no different than bsc alone for the respective endpoints; the alternative hypotheses are described below: 1. number of days free of mechanical ventilation: the alternative hypothesis is that treatment with ravulizumab + bsc will increase the number days free of mechanical ventilation at day 29 compared with bsc alone. the alternative hypothesis is that treatment with ravulizumab + bsc will reduce the number days in the icu at day 29 compared with bsc alone. 3. change in sofa score: the alternative hypothesis is that treatment with ravulizumab + bsc will improve changes in sofa score at day 29 compared with bsc alone. 4. change in spo2/fio2: the alternative hypothesis is that treatment with ravulizumab + bsc will improve changes in spo2/fio2 at day 29 compared with bsc alone. the alternative hypothesis is that treatment with ravulizumab + bsc will reduce the number days in the hospital at day 29 compared with bsc alone. 6. survival (based on all-cause mortality) at day 60 and day 90: the alternative hypothesis is that ravulizumab + bsc will improve survival at day 60 and day 90 compared with bsc alone. a sample size of 243 patients (162 ravulizumab + bsc, 81 bsc alone) is required to ensure at least 90% power and detect an improvement in survival from 60% in the bsc alone group to 80% in the ravulizumab + bsc group at day 29. this sample size calculation assumes: • 1-sided z-test of the difference in 2 proportions, • type i error = 0.025, • pooled variance, • 2:1 randomization on the 2 treatment groups, protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 • one interim analysis at 50% information which will be after collecting primary efficacy data on approximately 122 patients. the early stopping boundaries for efficacy and futility (nonbinding) will be constructed using α-spending function as lan-demets spending function with o'brien-fleming flavor and β-spending function as gamma(-4) (lan, 1983; hwang, 1990) . considering a nonevaluable rate of 10%, this study is planned to randomize approximately 270 patients (180 ravulizumab + bsc, 90 bsc alone). the population sets used for analysis sets are defined in the following: the itt consists of all randomized patients and participants will be analyzed as randomized. the itt will be used for the analysis of efficacy data and is considered the primary analysis population. the pps is a subset of the itt without any important protocol deviations that could impact efficacy analyses. determination of applicable important protocol deviations for this purpose will be made prior to database lock. the pps will be used for sensitivity analyses of the primary and secondary efficacy endpoints. safety set (ss) the ss consists of all randomized patients who receive at least 1 dose of ravulizumab for patients randomized to ravulizumab + bsc or who were randomized to bsc alone. the ss will be used for the analysis of safety data. abbreviation: bsc = best supportive care. the primary analysis will be conducted when all patients have completed the primary evaluation period. this analysis will include all efficacy, safety, and available pk/pd/immunogenicity study data for regulatory submission purposes and will be the final analysis of the primary evaluation period. summary statistics will be presented by treatment group and by visit, where applicable. descriptive statistics for continuous variables will minimally include the number of patients, mean, standard deviation, median, minimum, and maximum. for categorical variables, frequencies and percentages will be presented. graphical displays will be provided as appropriate. all statistical analyses will be performed based on a 2-sided type i error of 5%, unless otherwise noted. baseline is defined as the last available assessment on or before day 1 for all patients. day 1 will be defined as the date of the first infusion of ravulizumab for patients randomized and dosed with ravulizumab and as the date of randomization for patients randomized but not dosed with ravulizumab. analyses will be performed using sas ® software version 9.4 or higher. protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 9.4.1. efficacy analyses the primary efficacy endpoint is survival (based on all-cause mortality) at day 29 and will be compared between the 2 treatment groups using a 1-sided mantel-haenszel (mh) test of the difference in 2 proportions stratified by intubated or not intubated on day 1 and a type i error of 0.025. the estimated mh risk difference will be summarized along with the 95% confidence interval using mantel-haenszel stratum weights (mantel, 1959) and the sato variance estimator (sato, 1989) . missing survival data for the primary analysis will be imputed using a multiple imputation approach assuming the data are missing at random (mar) using a logistic regression model with covariates for treatment group, the randomization stratification factor, age, sex, and presence of a pre-existing condition at baseline. sensitivity analyses will include the worst-case, all available, and best-case scenarios. survival will also be analyzed using the method of kaplan and meier (km) and compared using a log-rank test stratified by intubated or not intubated on day 1 as a sensitivity analysis. hazard ratio and risk reduction will be summarized from a cox proportional hazards model stratified by intubated or not intubated on day 1. confidence intervals (95%) will be presented for the survival estimate at day 29 based on the complementary log-log transformation. kaplan-meier curves for both treatment groups will be produced. a sensitivity analysis of the primary endpoint will also be performed using a 3-level categorical outcome of 3) alive and discharged from the icu; 2) alive and in the icu or 1) death. the 2 treatment groups will be compared using an ordinal logistic regression with covariates for treatment group and the randomization stratification factor. additional sensitivity analyses will include statistical models adjusting for age, randomization stratification factor, and other important baseline covariates. subgroup analyses will also be performed by age group, randomization stratification factor, and other important baseline covariates. the statistical analysis plan (sap) will describe the sensitivity and subgroup analyses in greater detail. an interim analysis of the primary endpoint will also be conducted as described in section 9.5. number of days free of mechanical ventilation at day 29 will be compared between treatment groups using an analysis of covariance (ancova), adjusting for age, and randomization stratification factor, among survivors. missing data will be imputed using a multiple imputation approach assuming the data are mar. sensitivity analyses will include the worst-case, all available, and best-case scenarios. duration of icu stay at day 29 will be compared between treatment groups using an ancova, adjusting for age and randomization stratification factor, among survivors. missing data will be imputed using a multiple imputation approach assuming the data are mar. sensitivity analyses will include the worst-case, all available, and best-case scenarios. changes in sofa score from day 1 to day 29 will be summarized by treatment group and study visit for all patients and will be analyzed using a mixed model for repeated measures (mmrm) with baseline sofa score, age, randomization stratification factor, treatment group indicator, protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 study day (days 5, 10, 15, 22, and 29) , and study day by treatment group interaction as covariates. sensitivity analyses will include imputations for missing data. change from baseline in spo2/fio2 at day 29 will be analyzed using a mmrm with baseline spo2/fio2, age, randomization stratification factor, treatment group indicator, study day (days 5, 10, 15, 22, and 29) , and study day by treatment group interaction as covariates. all patients will be included in the model. sensitivity analyses will include imputations for missing data. change from baseline in pao2/fio2 at day 29 will also be analyzed using a mmrm with baseline pao2/fio2, age, randomization stratification factor, treatment group indicator, study day, and study day by treatment group interaction as fixed covariates. all patients will be included in the model. sensitivity analyses will include imputations for missing data. duration of hospitalization at day 29 will be analyzed in a similar manner as duration of icu stay. survival (based on all-cause mortality) at day 60 and day 90 will be estimated using the km method and compared using a log-rank test stratified by intubated or not intubated on day 1. hazard ratio and risk reduction will be summarized from a cox proportional hazards model stratified by intubated or not intubated on day 1. confidence intervals (95%) will be presented for the survival estimates at day 60 and day 90 based on the complementary loglog transformation. kaplan and meier curves for both treatment groups will be produced. a closed testing procedure will be applied to control the type i error for the analyses of the primary and secondary endpoints. if the primary endpoint is statistically significant in favor of ravulizumab, the secondary endpoints will be evaluated according to the following rank order: the hypothesis testing will proceed from highest rank (#1) the number of days free of mechanical ventilation at day 29 to the lowest rank (#5) duration of hospitalization at day 29, and if statistical significance is not achieved at an endpoint (p≥0.05), then endpoints of lower rank will not be considered to be statistically significant. confidence intervals and p-values will be presented for all secondary efficacy endpoints for descriptive purposes, regardless of the outcome of the closed testing procedure. an additional secondary endpoint will be assessed beyond day 29 regardless of the results of the closed testing procedure: survival (based on all-cause mortality) at day 60 and day 90. all safety analyses will be made on the safety set (ss). safety results will be reported by treatment group. the analysis and reporting of aes and saes will be based on teaes and tesaes, defined as aes and saes with onset during or after treatment with ravulizumab. the incidence of teaes and tesaes will be summarized by system organ class and preferred term, with additional summaries showing relationship to ravulizumab, severity, teaes or tesaes leading to ravulizumab discontinuation, and tesaes resulting in death. laboratory measurements as well as their changes from baseline at each visit and shift from baseline, if applicable, will be summarized. vital sign measurements, physical examination findings, and ecg data will also be summarized over time. all patients who have evaluable pk/pd data will be used to summarize pk/pd parameters for ravulizumab. descriptive statistics of ravulizumab concentration data will be presented for patients randomized and treated with ravulizumab for each scheduled sampling timepoint. total and free c5 concentrations will be evaluated by assessing the absolute values and changes and percentage changes from baseline, as appropriate. descriptive statistics will be presented by treatment group and for each scheduled sampling timepoint. serum and plasma biomarkers' actual values, and changes from baseline, and their association with observed clinical responses to ravulizumab will be summarized over time, as appropriate. biomarker data will only be summarized at the final analysis at the end of the study. the incidence and titers for adas to ravulizumab will be summarized in tabular format by treatment group. the proportion of patients ever positive and the proportion of patients always negative may be explored. confirmed ada positive samples will be evaluated for the presence of neutralizing antibodies. incidence of and time to progression to renal failure requiring dialysis at day 29 will be analyzed in a similar manner as the primary endpoint. time to clinical improvement will be analyzed using the km method and compared using a log-rank test stratified by intubated or not intubated on day 1. the sf-12 pcs and mcs scores and eq-5d-5l index and vas scores will be analyzed using an ancova, adjusting for age and the randomization stratification factor. an interim analysis for efficacy and futility will be conducted when approximately 122 patients have completed day 29. if the stopping criteria are met, the study may be terminated early for efficacy or futility depending on which stopping boundary is crossed. the early stopping boundaries for efficacy and futility (nonbinding) will be constructed using α-spending function as lan-demets (o'brien-fleming) spending function and β-spending function as gamma (-4). a 1-sided t-test based on the results from combining all imputed datasets for overall inference will be used with an overall type i error of 0.025. the sap will describe the planned interim analyses in greater detail. provided the study was not stopped early for efficacy or futility, the final primary analysis will be conducted when all patients have completed the primary evaluation period. this analysis will include all efficacy, safety, and available pk/pd/immunogenicity study data for regulatory submission purposes. this analysis will not be considered an interim analysis. an independent data monitoring committee (dmc), comprising experts in relevant fields with no direct relationship to the study, will be appointed by alexion. a minimum of 3 experts (including 1 biostatistician) will be selected. the dmc will review and evaluate cumulative safety data and key efficacy data at prespecified intervals. the dmc's purview will include recommendations to continue or terminate the study. final decisions regarding study conduct will be made by alexion. substantive decisions will be communicated to investigators, irbs/iecs, and appropriate regulatory authorities. the specific responsibilities of the dmc including frequency of meetings will be described in the dmc charter. • the protocol, protocol amendments, icf, ib, and other relevant documents (eg, advertisements) must be submitted to an irb/iec by the investigator and reviewed and approved by the irb/iec before the study is initiated. • any amendments to the protocol will require irb/iec approval before implementation of changes made to the study design, except for changes necessary to eliminate an immediate hazard to study patients. • for studies to be approved by medicines and healthcare products regulatory agency: the investigator will notify the irb/iec of deviations from the study protocol or gcp as defined by uk legislation as a serious breach or as required by irb/iec procedures. • the investigator will be responsible for the following: − providing written summaries of the status of the study to the irb/iec annually or more frequently in accordance with the requirements, policies, and procedures established by the irb/iec investigators or designees and sub-investigators will provide alexion with sufficient, accurate financial information as requested to allow alexion to submit complete and accurate financial certification or disclosure statements to the appropriate regulatory authorities. investigators are responsible for providing information on financial interests during the course of the study and for 1 year after completion of the study. • it is the responsibility of the investigator or designee to obtain informed consent from all patients or their legally acceptable representative as defined per local and country regulations where the study is taking place, and answer all questions regarding the study, prior to any study related procedures including screening assessments. • where applicable by national laws and allowed by local regulations, and following irb/iec approval, patients who are unable to provide informed consent, and whose legally acceptable representative is unavailable, can be enrolled per the judgement of the investigator or designee. • in the exceptional circumstance where informed consent cannot be obtained because of an inability to communicate with, or obtain legally effective consent from the patient and time is not sufficient to obtain consent from the patient's legally acceptable representative, the following procedure should be followed: − written certification from the investigator and a physician who is not involved with the research must be submitted to the irb/iec within 5 working days after administration of the initial dose. − if the patient is enrolled without their consent or that of their legally acceptable representative, all reasonable attempts should be made to inform the patient or their legally acceptable representative and/or family of the patient's enrollment in the study as soon as possible. − document efforts to contact the legally acceptable representative in the study records. • the investigator or designee will explain the nature of the study (including but not limited to the objectives, potential benefits and risks, inconveniences, and the patient's rights and responsibilities) to the patient or his/her legally acceptable representative, defined according to local and country regulations where the study is taking place, and answer all questions regarding the study. • patients must be informed that their participation is voluntary. patients or their legally acceptable representative will be required to sign a statement of informed consent or a certified translation if applicable, that meets the requirements of 21 cfr 50, local regulations, eu general data protection regulation, ich guidelines, health insurance portability and accountability act requirements, where applicable, and the irb/iec or study center. • the patient's medical record must include a statement that informed consent was obtained before the patient was screened in the study (or as soon as feasible in the case of emergency enrollment by the investigator or designee) and date the written consent was obtained. the authorized person obtaining the informed consent must also sign the icf(s). approved protocol and any other study agreements, ich gcp, and all applicable regulatory requirements. − due to the covid-19 pandemic, remote source data verification may be employed where permitted by local regulations. − the scope of the source data verification will be described in detail in the monitoring plan. • records and documents, including signed icfs, pertaining to the conduct of this study must be retained by the investigator for 2 years after the last marketing application approval, or if not approved, 2 years following the discontinuance of the study intervention, unless local regulations or institutional policies require a longer retention period. no records may be destroyed during the retention period without the written approval of alexion. no records may be transferred to another location or party without written notification to alexion. source documents provide evidence for the existence of the patient and substantiate the integrity of the data collected. the investigator or designee will prepare and maintain adequate and accurate source documents (eg, medical records, ecgs, ae and concomitant medication reporting, raw data collection forms) designed to record all observations and other pertinent data for each patient. data reported on the crf/ecrf that are transcribed from source documents must be consistent with the source documents or the discrepancies must be explained. the investigator may need to request previous medical records or transfer records, depending on the study. also, current medical records must be available. source documents are filed at the investigator's site. the study start date is the date on which the first patient is consented. alexion reserves the right to close the study site or terminate the study at any time for any reason at the sole discretion of alexion. study sites will be closed after the study is completed or following the decision to close or terminate the study. a study site is considered closed when all patients have completed the end of study or et visit, all data have been collected and entered into electronic data capture (edc) system, all required documents and study supplies have been collected, and a study-site closure visit has been performed. the investigator may initiate study-site closure at any time, provided there is reasonable cause and sufficient notice is given in advance of the intended termination. reasons for the early closure of a study site by alexion or investigator may include but are not limited to: • failure of the investigator to comply with the protocol, the requirements of the irb/iec or local health authorities, alexion's procedures, or gcp guidelines • inadequate recruitment of patients by the investigator if the study is prematurely terminated or suspended, alexion shall promptly inform the investigators, the irbs/iecs, the regulatory authorities, and any contract research organization(s) used in the study of the reason for termination or suspension, as specified by the applicable regulatory requirements. the investigator shall promptly inform the patient and should assure appropriate patient therapy and/or follow-up. • where possible, primary manuscripts reporting results of the primary efficacy endpoint or the final results will be submitted for publication within 12 to 18 months of the primary evaluation date or end of study, whichever is earlier. • investigators who participate as authors in manuscripts derived from alexion-sponsored studies will agree to the prerequisites as outlined in the alexion author engagement agreement prior to engaging in manuscript development. • the investigator agrees to submit proposals for new manuscripts (whether or not the proposed analyses are derived from protocol-specified endpoints) to alexion for review and consideration. all manuscripts or abstracts emanating from approved proposals are to be submitted to alexion for review before submission to the journal/society. this allows alexion to protect proprietary information and to provide comments. − the proprietary nature of some development work may preclude publication. in some cases, it may be necessary to delay a publication to allow alexion to ensure protection of intellectual property. • in general, primary publications, including congress and journal publications, containing the protocol-specified results of a study should occur prior to the publication of individual study site results or case reports. alexion's policy prohibits duplicate publication, whereby the same results must not be published in multiple peer-reviewed journal manuscripts. − encore congress publications may be appropriate to allow communication of research findings to relevant audience and geographical regions. • alexion will comply with the requirements for publication of study results. in accordance with standard editorial and ethical practice, alexion will generally support publication of multicenter studies only in their entirety and not as individual site data. in this case, a coordinating investigator will be designated by mutual agreement. • authorship will be determined by mutual agreement and in line with international committee of medical journal editors authorship requirements and per the alexion publication policy. • the tests listed in table 9 may be performed by the local or central laboratory, as appropriate for all patients unless otherwise noted. • protocol-specific requirements for inclusion or exclusion of patients are detailed in section 5 of the protocol. • additional tests may be performed at any time during the study as determined necessary by the investigator or required by local regulations. • women of childbearing potential should only be enrolled after a negative serum or urine pregnancy test result at screening. additional serum or urine pregnancy testing will be employed as required by site policies, local regulations, or per the requirements of the irb/iec and should be performed per the timepoints specified in the soa (table 1) . • investigators must document their review of each laboratory safety report. clinically significant findings resulting in an assessment of a tesae should be recorded on the ae crf/ecrf. • an ae is any untoward medical occurrence in a patient, temporally associated with the use of study intervention, whether or not considered related to the study intervention. • note: an ae can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of study intervention. events meeting the ae definition • any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments (eg, ecg, radiological scans, vital signs measurements), including those that worsen from baseline, considered clinically significant in the medical and scientific judgment of the investigator (ie, not related to progression of underlying disease). • exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition. • new conditions detected or diagnosed after study intervention administration even though it may have been present before the start of the study. • signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction. • signs, symptoms, or the clinical sequelae of a suspected overdose of either study intervention or a concomitant medication. overdose per se will not be reported as an ae/sae unless it is an intentional overdose taken with possible suicidal/self-harming intent. such overdoses should be reported regardless of sequelae. • "lack of efficacy" or "failure of expected pharmacological action" per se will not be reported as an ae or sae. such instances will be captured in the efficacy assessments. however, the signs, symptoms, and/or clinical sequelae resulting from lack of efficacy will be reported as ae or sae if they fulfill the definition of an ae or sae. • medical or surgical procedure (eg, endoscopy, appendectomy): the condition that leads to the procedure is the ae. situations in which an untoward medical occurrence did not occur (eg, hospitalization for elective surgery if planned before the signing the icf, admissions for social reasons or for convenience). • anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected at the start of the study that do not worsen. • a medication error (including intentional misuse, abuse, and overdose of the product) or use other than what is defined in the protocol is not considered an ae unless there is an untoward medical occurrence as a result of a medication error. • cases of pregnancy that occur during maternal or paternal exposure to study intervention are to be reported within 24 hours of investigator/site awareness. data on fetal outcome and breastfeeding will be collected for regulatory reporting and safety evaluation. • any clinically significant abnormal laboratory findings or other abnormal safety assessments which are associated with the underlying disease, unless judged by the investigator to be more severe than expected for the patient's condition. • the disease/disorder being studied or expected progression, signs, or symptoms of the disease/disorder being studied, unless more severe than expected for the patient's condition. • situations in which an untoward medical occurrence did not occur (social and/or convenience admission to a hospital). • when further information becomes available, the edc should be updated within 24 hours with the new information and an updated sae report should be submitted to alexion global drug safety (gds). • after the study is completed at a given site, the electronic data collection tool will be taken off-line to prevent the entry of new data or changes to existing data. • if a site receives a report of a new sae from a study patient or receives updated data on a previously reported sae after the electronic data collection tool has been taken off-line, then the site can report this information on a paper sae form (see next section) or to the alexion/medical monitor/sae coordinator by telephone. • all saes will be recorded and reported to alexion or designee immediately and within 24 hours awareness. • saes will be reported using the safety reporting form and submitted to alexion gds. the investigator must complete, sign, and date the sae pages, verify the accuracy of the information recorded on the sae pages with the corresponding source documents, and send a copy via email or facsimile to the contact information provided below: − email: or fax: • additional follow-up information, if required or available, should be entered into the crf/ecrf and sent to alexion gds within 24 hours of the investigator or study site staff becoming aware of this additional information via the reporting process outlined above. • for all saes, the investigator must provide the following: − appropriate and requested follow-up information in the time frame detailed above − causality of the sae(s) − treatment of/intervention for the sae(s) − outcome of the sae(s) − medical records and laboratory/diagnostic information • all paper forms and follow-up information submitted to alexion gds must be accompanied by a cover page signed by the investigator. • paper source documents and/or reports should be kept in the appropriate section of the study file. female patients randomized to ravulizumab + bsc must not donate ova from the day 1 visit until 8 months after treatment with the last infusion of study drug. contraception is the responsibility of the heterosexually active male patients, regardless of his female partner's method of contraception. male patients who have had a vasectomy > 6 months prior must use a condom during heterosexual intercourse. male patients who have had a vasectomy < 6 months prior must use a condom and spermicide during heterosexual intercourse. male patients who have not had a vasectomy must use a condom and spermicide during heterosexual intercourse from the day 1 visit until 8 months after treatment with the last infusion of study drug. sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual intercourse. in this study, abstinence is only acceptable if consistent with the patients' s preferred and usual lifestyle. abstinent male patients who become heterosexually active must use a condom and spermicide during intercourse. periodic abstinence (eg, calendar, symptothermal, or post-ovulation methods for a female partner) is not considered a highly effective method of contraception for male patients. male patients randomized to ravulizumab + bsc must not donate sperm from the day 1 visit until 8 months after treatment with the last infusion of study drug. pregnancy data will be collected during this study for all female patients and female spouses/partners of male patients. exposure during pregnancy (also referred to as exposure in utero) can be the result of either maternal exposure or transmission of study intervention via semen following paternal exposure. if a female patient or a male patient's female partner becomes pregnant during the conduct of this study, the investigator must submit the "pregnancy reporting and outcome/breastfeeding" form to alexion gds via facsimile or email. when the outcome of the pregnancy becomes known, the form should be updated and submitted to alexion gds. if additional follow-up is required, the investigator will be requested to provide the information. exposure of an infant to study intervention during breastfeeding must also be reported (via the "pregnancy reporting and outcome form/breastfeeding") and any aes experienced by the infant must be reported to alexion gds or designee via email or facsimile. pregnancy is not regarded as an ae unless there is a suspicion that the study intervention may have interfered with the effectiveness of a contraceptive medication. however, complications of pregnancy and abnormal outcomes of pregnancy are aes and may meet the criteria for an sae (eg, ectopic pregnancy, spontaneous abortion, intrauterine fetal demise, neonatal death, or congenital anomaly). elective abortions without complications should not be reported as aes. anaphylaxis is highly likely when any 1 of the following 3 criteria is fulfilled: • acute onset of an illness (minutes to several hours) with involvement of the skin, mucosal tissue, or both (eg, generalized hives, pruritus or flushing, swollen lips-tongue-uvula), and at least 1 of the following: o respiratory compromise (eg, dyspnea, wheeze-bronchospasm, stridor, reduced peak expiratory flow, hypoxemia) o reduced blood pressure or associated symptoms of end-organ dysfunction (eg, hypotonia [collapse], syncope, incontinence) • two or more of the following that occur rapidly after exposure to a likely allergen for that participant (minutes to several hours): o involvement of the skin-mucosal tissue (eg, generalized hives, itch-flush, swollen lips/tongue/uvula) o respiratory compromise (eg, dyspnea, wheeze/bronchospasm, stridor, reduced peak expiratory flow, hypoxemia) o reduced blood pressure or associated symptoms (eg, hypotonia [collapse], syncope, incontinence) o persistent gastrointestinal symptoms (eg, crampy abdominal pain, vomiting) • reduced blood pressure after exposure to known allergen for that participant (minutes to several hours): o systolic blood pressure of less than 90 mmhg or greater than 30% decrease from that participant's baseline source: (sampson, 2006) appendix 6: management of potential drug infusion reactions prior to first menses 2. postmenopausal, as documented by amenorrhea for at least 1 year prior to the day 1 visit and fsh serum levels consistent with postmenopausal status of contraception, including at least one of the following: 1. intrauterine device (without copper) in place for at least 6 weeks. 2. progestogen-only hormonal contraception (either oral, injectable combined (estrogen-and progestogen-containing) hormonal contraception (either oral, intravaginal, or transdermal) for at least 6 weeks. estrogen-containing hormonal contraception is acceptable only if it has been used for at least 6 weeks immediately prior to the day 1 visit. estrogen-containing hormonal contraception may not be initiated during the study period surgical sterilization of the male partner (medical assessment of azoospermia is required if vasectomy was performed within the prior 6 months) abstinent female patients who wish to initiate a highly effective method of contraception during the study must refrain from heterosexual intercourse for at least 1 menstrual cycle. b. periodic abstinence (eg, calendar, symptothermal other methods of contraception that are not considered highly effective for female patients: 1. barrier methods, such as male or female condoms, diaphragm, or cervical cap spermicides or spermicidal sponges, used alone or in combination with barrier methods, are not acceptable a core outcome set for critical care ventilation trials pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin covid-19) situation summary eq-5d-5l user guide complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis. mbio association of complement activation and elevated plasma-c5a with adult respiratory distress syndrome. pathophysiological relevance and possible prognostic value group sequential designs using a family of type i error probability spending functions mannose-binding lectin in severe acute respiratory syndrome coronavirus infection a shorter form health survey: can the sf-12 replicate results from the sf-36 longitudinal studies? blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov variable eculizumab clearance requires pharmacodynamic monitoring to optimize therapy for thrombotic microangiopathy after hematopoietic stem cell transplantation the sofa score -development, utility and challenges of accurate assessment in clinical trials discrete sequential boundaries for clinical trials transplant-associated thrombotic microangiopathy is a multifactorial disease unresponsive to immunosuppressant withdrawal statistical aspects of analysis of data from retrospective studies of disease covid-19: consider cytokine storm syndromes and immunosuppression the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h5n1-a review serum concentrations of complement anaphylatoxins and proinflammatory mediators in patients with 2009 h1n1 influenza serum proteomic fingerprints of adult patients with severe acute respiratory syndrome calculating sofa scores when arterial blood gasses are not available: validating spo2/fio2 ratios for imputing pao2/fio2 ratios in the sofa score characterization of a novel coronavirus associated with severe acute respiratory syndrome second symposium on the definition and management of anaphylaxis: summary report--second national institute of allergy and infectious disease/food allergy and anaphylaxis network symposium on the variance estimator of the mantel-haenszel risk difference. letter to the editor the glasgow coma scale inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza h5n1 virus infection use of the sofa score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study the role of c5a in acute lung injury induced by highly pathogenic viral infections remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial sf-12: how to score the sf-12 physical and mental health summary scales clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected. interim guidance plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome autoantibodies against human epithelial cells and endothelial cells after severe acute respiratory syndrome (sars)-associated coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia determine complement activation in patients randomized to bsc alone. the actual date and time (24-hour clock time) of each sample will be recorded. genetics will not be evaluated in this study.protocol amendment 3 (global)alxn1210-cov-305 09 jun 2020• patients must be reconsented to the most current version of the icfs during their participation in the study.• a copy of the icf must be provided to the patient or the patient's legally acceptable representative, as applicable. this document may require translation into the local language. documentation of icfs must remain in each patient's study file and must be available for verification at any time. • patients will be assigned a unique identifier by alexion. any patient records or datasets that are transferred to alexion will contain the identifier only; patient names or any information which would make the patient identifiable will not be transferred.• patients or their legally acceptable representative must be informed that their personal study-related data will be used by alexion in accordance with local data protection law. the level of disclosure must also be explained to the patients who will be required to give consent for their data to be used as described in the informed consent.• patients or their legally acceptable representative must be informed that their medical records may be examined by clinical quality assurance auditors or other authorized personnel appointed by alexion, by appropriate irb/iec members, and by inspectors from regulatory authorities. study-related information and study results may be posted on publicly accessible clinical study databases (eg, the us website www.clinicaltrials.gov or the eu website www.clinicaltrialsregister.eu), as appropriate, and in accordance with national, regional, and local regulations. • all patient data relating to the study will be recorded on printed or ecrf unless transmitted to alexion or designee electronically (eg, laboratory data). the investigator is responsible for verifying that data entries are accurate and correct by physically or electronically signing the ecrf.• the investigator must maintain accurate documentation (source data) that supports the information entered in the crf/ecrf.• the investigator must permit study-related monitoring, audits, irb/iec review, and regulatory agency inspections and provide direct access to source data documents.• alexion or designee is responsible for the data management of this study including quality checking of the data.• study monitors will perform ongoing source data verification to confirm that data entered into the crf/ecrf by authorized site personnel are accurate, complete, and verifiable from source documents; that the safety and rights of patients are being protected; and that the study is being conducted in accordance with the currently protocol amendment 3 (global)alxn1210-cov-305 09 jun 2020 protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 il1, il-2r, il-6, il-8,il-21, pentraxin-3, and citrullinated histone h3 total and free c5 and sc5b-9 procalcitonin myoglobin nt-probnp hs-tni immunogenicity assay (only collect from patients randomized to ravulizumab + bsc) pharmacokinetic assay (only collect from patients randomized to ravulizumab + bsc) factor ii abbreviations: bsc = best supportive care; c = complement protein; hs-tni = high sensitivity troponin i; il = interleukin, mcp = monocyte chemoattractant protein; nt-probnp = n-terminal pro b-type natriuretic peptide; sc5b-9 = soluble c5b-9; tnf = tumor necrosis factor; wbc = white blood cell.protocol amendment 3 (global)alxn1210-cov-305 09 jun 2020 if an event is not an ae per definition above, then it cannot be an sae even if serious conditions are met (eg, hospitalization for signs/symptoms of the disease under study, death due to progression of disease).an sae is defined as any untoward medical occurrence that, at any dose: 1. results in death 2. is life-threatening the term "life-threatening" in the definition of "serious" refers to an event in which the patient was at risk of death at the time of the event. it does not refer to an event, which hypothetically might have caused death, if it was more severe. in general, hospitalization signifies that the patient has been detained (usually involving at least an overnight stay) at the hospital or emergency ward for observation and/or treatment that would not have been appropriate in the physician's office or outpatient setting. complications that occur during hospitalization are aes. if a complication prolongs hospitalization or fulfills any other serious criteria, the event is serious. when in doubt as to whether "hospitalization" occurred or was necessary, the ae should be considered serious. hospitalization for elective treatment of a pre-existing condition that did not worsen from baseline is not considered an ae. • the term disability means a substantial disruption of a person's ability to conduct normal life functions.• this definition is not intended to include experiences of relatively minor medical significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and accidental trauma (eg, sprained ankle) which may interfere with or prevent everyday life functions but do not constitute a substantial disruption. 5. is a congenital anomaly/birth defect 6. other situations:• medical or scientific judgment should be exercised in deciding whether sae reporting is appropriate in other situations such as important medical events that may not be immediately life-threatening or result in death or hospitalization but may jeopardize the patient or may require medical or surgical intervention to prevent one of the other outcomes listed in the above definition. these events should usually be considered serious.• examples of such events include invasive or malignant cancers, intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsions that do not result in hospitalization, or development of drug dependency or drug abuse. • when an ae/sae occurs, it is the responsibility of the investigator to review all documentation (eg, hospital progress notes, laboratory reports, and diagnostics reports) related to the event. • the investigator will then record all relevant ae/sae information in the crf/ecrf. • it is not acceptable for the investigator to send photocopies of the patient's medical records to alexion in lieu of completion of the alexion/ae/sae crf/ecrf page. • there may be instances when copies of medical records for certain cases are requested by alexion. in this case, all patient identifiers, with the exception of the patient number, will be redacted on the copies of the medical records before submission to alexion. • the investigator will attempt to establish a diagnosis of the event based on signs, symptoms, and/or other clinical information. whenever possible, the diagnosis (not the individual signs/symptoms) will be documented as the ae/sae. the investigator will make an assessment of intensity for each ae and sae reported during the study and assign it to one of the following categories from national cancer institute ctcae v5.0, published 27 nov 2017: • grade 1: mild (awareness of sign or symptom, but easily tolerated) • grade 2: moderate (discomfort sufficient to cause interference with normal activities) protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020• grade 3: severe (incapacitating, with inability to perform normal activities) • grade 4: life-threatening • grade 5: fatal • an event is defined as "serious" when it meets at least one of the predefined outcomes as described in the definition of an sae, not when it is rated as severe. • the investigator is obligated to assess the relationship between the study intervention and each occurrence of each ae or sae. an investigator causality assessment must be provided for all aes (both nonserious and serious). this assessment must be recorded in the crf/ecrf and on any additional forms, as appropriate.the definitions for the causality assessments are as follows: − not related: there is no reasonable possibility the study intervention caused the ae.  the ae has a more likely alternative etiology; it may be due to underlying or concurrent illness, complications, concurrent treatments, or effects of another concurrent drug.  the event does not follow a reasonable temporal relationship to administration of the study intervention. − related: there is a reasonable possibility the study intervention caused the ae. the ae has a temporal relationship to the administration of the study intervention.  the event does not have a likely alternative etiology.  the event corresponds with the known pharmaceutical profile of the study intervention.  there is improvement on discontinuation and/or reappearance on rechallenge.• the investigator will use clinical judgment to determine the relationship.• alternative causes, such as underlying disease(s), concomitant therapy, and other risk factors, as well as the temporal relationship of the event to study intervention administration will be considered and investigated. • the investigator will also consult the ib and/or product information, for marketed products, in his/her assessment. • for each ae/sae, the investigator must document in the medical notes that he/she has reviewed the ae/sae and has provided an assessment of causality. • there may be situations in which an sae has occurred, and the investigator has minimal information to include in the initial report to alexion. however, it is very important that the investigator always make an assessment of causality for every event before the initial transmission of the sae data to alexion. • the investigator may change his/her opinion of causality in light of follow-up information and send an sae follow-up report with the updated causality assessment. • the causality assessment is one of the criteria used when determining regulatory reporting requirements. • the investigator is obligated to perform or arrange for the conduct of supplemental measurements and/or evaluations as medically indicated or as requested by alexion to elucidate the nature and/or causality of the ae or sae as fully as possible. this may include additional laboratory tests or investigations, histopathological examinations, or consultation with other health care professionals. • new or updated information will be recorded in the originally completed crf/ecrf. • the investigator will submit any updated sae data to alexion within 24 hours of receipt of the information. • all saes will be recorded and reported to alexion or designee immediately and within 24 hours of awareness. • the primary mechanism for reporting an sae to alexion will be the electronic data collection tool.• if the electronic system is unavailable at the time that the investigator or site becomes aware of an sae, the site will use the paper contingency form for sae reporting via fax or email. facsimile transmission or email may be used in the event of electronic submission failure. − email: or fax: • the site will enter the sae data into the edc system as soon as it becomes available. a woman is considered fertile following menarche and until becoming postmenopausal unless permanently sterile (see below).if fertility is unclear (eg, amenorrhea in adolescents or athletes) and a menstrual cycle cannot be confirmed before administering the dose of study intervention, additional evaluation should be considered.women in the following categories are not considered wocbp 1. premenarchal 2. premenopausal female with one of the following:• documented hysterectomy• documented bilateral salpingectomy• documented bilateral oophorectomy• for individuals with permanent infertility due to an alternate medical cause other than the above, (eg, mullerian agenesis, androgen insensitivity), investigator discretion should be applied to determining study entry.• note: documentation can come from the site personnel's: review of the patient's medical records, medical examination, or medical history interview. • a postmenopausal state is defined as no menses for 12 months without an alternative medical cause.− a high follicle stimulating hormone (fsh) level in the postmenopausal range may be used to confirm a postmenopausal state in women not using hormonal contraception or hormonal replacement therapy (hrt). however, in the absence of 12 months of amenorrhea, confirmation with more than 1 fsh measurement is required.• females on hrt and whose menopausal status is in doubt will be required to use one of the non-estrogen hormonal highly effective contraception methods if they wish to continue their hrt during the study. otherwise, they must discontinue hrt to allow confirmation of postmenopausal status before study enrollment. female patients of non-childbearing potential are exempt from contraception requirements. nonchildbearing potential for female patients is defined as any of the following:protocol amendment 3 (global) alxn1210-cov-305 09 jun 2020 any female patient who becomes pregnant while participating in the study will be discontinued from study intervention. • the investigator will attempt to collect pregnancy information on any male patient's female partner who becomes pregnant while the male patient is in this study. this applies only to male patients who receive ravulizumab.• after obtaining the necessary signed informed consent from the pregnant female partner directly, the investigator will record pregnancy information on the appropriate pregnancy outcome/breastfeeding form and submit it to alexion within 24 hours of learning of the partner's pregnancy. the female partner will also be followed to determine the outcome of the pregnancy. information on the status of the mother and child will be forwarded to alexion. generally, the follow-up will be no longer than 6 to 8 weeks following the estimated delivery date. any termination of the pregnancy will be reported regardless of fetal status (presence or absence of anomalies) or indication for the procedure. • the investigator will collect pregnancy information on any female patient who becomes pregnant while participating in this study. the initial information will be recorded on the appropriate form and submitted to alexion within 24 hours of learning of a patient's pregnancy.• the patient will be followed-up to determine the outcome of the pregnancy. the investigator will collect follow-up information on the patient and the neonate and the information will be forwarded to alexion. generally, follow-up will not be required for longer than 3 months beyond the estimated delivery date. any termination of pregnancy will be reported, regardless of fetal status (presence or absence of anomalies) or indication for the procedure.• while pregnancy itself is not considered to be an ae or sae, any pregnancy complication or elective termination of a pregnancy for medical reasons will be reported as an ae or sae. a spontaneous abortion (occurring at < 22 weeks gestational age) or still birth (occurring at > 22 weeks gestational age) is always considered to be an sae and will be reported as such. any post-study pregnancy related sae considered reasonably related to the study intervention by the investigator will be reported to alexion as described in section 8.6.4. while the investigator is not obligated to actively seek this information in former study patients, he or she may learn of an sae through spontaneous reporting.protocol amendment 3 (global)alxn1210-cov-305 09 jun 2020 appendix 5: biomarkers• blood samples will be collected for biomarker analyses and the data may be used for future exploratory research related to complement activation and inflammatory processes. the samples may also be used to develop tests/assays including diagnostic tests related to c5 inhibitors and covid-19 with clinical presentation of severe pneumonia, acute lung injury, or ards.• the samples may be analyzed as part of a multistudy assessment of biomarkers in the response to ravulizumab to understand covid-19 or related conditions.• the results of biomarker analyses may be reported in a final clinical study report or in a separate summary report.• alexion or designee will store the samples obtained for biomarker analyses in a secure storage space with adequate measures to protect confidentiality.protocol amendment 3 (global) intravenous and infusion-associated reactions are a potential risk with the use of monoclonal antibodies; these reactions can be nonimmune or immune mediated (eg, hypersensitivity reactions). signs and symptoms may include headache, fever, facial flushing, pruritus, myalgia, nausea, chest tightness, dyspnea, vomiting, erythema, abdominal discomfort, diaphoresis, shivers, hypertension, lightheadedness, hypotension, palpitations, and somnolence. signs and symptoms of hypersensitivity or allergic reactions may include hives, swollen face, eyelids, lips, or tongue, or trouble with breathing.all administration-, iv-, and infusion-associated reactions will be reported to the investigator and qualified designee. the investigator and qualified designee are responsible for detecting, documenting, and recording events that meet the definition of ae or sae and remain responsible for following up events that are serious, considered related to the study drug, or study procedures; or that caused the participant to discontinue ravulizumab (section 7).definitions and procedures for recording, evaluating, follow-up, and reporting aes and saes are outlined in section 10.3.before any infusion is started, the treating physician and other appropriate personnel must make certain that medication (ie, adrenaline, inhaled beta agonists, antihistamines, corticosteroids) and other equipment to treat anaphylaxis are readily available. the infusion must be stopped immediately if grade ≥ 2 allergic/hypersensitivity reactions (including drug fever) or grade ≥ 3 cytokine release syndrome/acute infusion reaction occurs. the sponsor must be notified within 24 hours of any infusion reaction requiring interruption or discontinuation of study drug.patients who experience a reaction during the administration of study drug should be treated according to institutional guidelines. for a grade 1 or grade 2 infusion reaction, the infusion should be temporarily stopped and treatment with an antihistamine (eg, diphenhydramine 25 to 50 mg orally or equivalent) and acetaminophen (650 mg orally or equivalent) may be considered. if the patient's signs and symptoms have resolved (with or without administration of the above medication), the infusion may be restarted. however, the patients should be infused at a slower rate and be monitored closely for any signs and symptoms of infusion reactions during the remainder of the infusion. patients experiencing an infusion reaction should be observed in the clinic until resolution of the reaction, or until the investigator determines the patient is no longer at risk. patients who experience a severe reaction during administration of study drug resulting in discontinuation of study drug should undergo all scheduled safety, pk, and pd evaluations required by the protocol.if anaphylaxis occurs according to the criteria listed below, then administration of subcutaneous epinephrine (1/1000, 0.3 ml to 0.5 ml, or equivalent) should be considered. in the case of bronchospasm, treatment with an inhaled beta agonist also should be considered. patients administered an antihistamine for the treatment or prevention of an infusion reaction should be given appropriate warnings about drowsiness and impairment of driving ability before being discharged from the center. key: cord-296219-zzg9hds0 authors: battaglini, denise; brunetti, iole; anania, pasquale; fiaschi, pietro; zona, gianluigi; ball, lorenzo; giacobbe, daniele roberto; vena, antonio; bassetti, matteo; patroniti, nicolò; schenone, angelo; pelosi, paolo; rocco, patricia r. m.; robba, chiara title: neurological manifestations of severe sars-cov-2 infection: potential mechanisms and implications of individualized mechanical ventilation settings date: 2020-08-12 journal: front neurol doi: 10.3389/fneur.2020.00845 sha: doc_id: 296219 cord_uid: zzg9hds0 in december 2019, an outbreak of illness caused by a novel coronavirus (2019-ncov, subsequently renamed sars-cov-2) was reported in wuhan, china. coronavirus disease 2019 (covid-19) quickly spread worldwide to become a pandemic. typical manifestations of covid-19 include fever, dry cough, fatigue, and respiratory distress. in addition, both the central and peripheral nervous system can be affected by sars-cov-2 infection. these neurological changes may be caused by viral neurotropism, by a hyperinflammatory and hypercoagulative state, or even by mechanical ventilation-associated impairment. hypoxia, endothelial cell damage, and the different impacts of different ventilatory strategies may all lead to increased stress and strain, potentially exacerbating the inflammatory response and leading to a complex interaction between the lungs and the brain. to date, no studies have taken into consideration the possible secondary effect of mechanical ventilation on brain recovery and outcomes. the aim of our review is to provide an updated overview of the potential pathogenic mechanisms of neurological manifestations in covid-19, discuss the physiological issues related to brain-lung interactions, and propose strategies for optimization of respiratory support in critically ill patients with sars-cov-2 pneumonia. in december 2019, an outbreak of illness caused by a novel coronavirus (2019-ncov, subsequently renamed sars-cov-2) was reported in wuhan, china. coronavirus disease 2019 (covid-19) quickly spread worldwide to become a pandemic. typical manifestations of covid-19 include fever, dry cough, fatigue, and respiratory distress. in addition, both the central and peripheral nervous system can be affected by sars-cov-2 infection. these neurological changes may be caused by viral neurotropism, by a hyperinflammatory and hypercoagulative state, or even by mechanical ventilation-associated impairment. hypoxia, endothelial cell damage, and the different impacts of different ventilatory strategies may all lead to increased stress and strain, potentially exacerbating the inflammatory response and leading to a complex interaction between the lungs and the brain. to date, no studies have taken into consideration the possible secondary effect of mechanical ventilation on brain recovery and outcomes. the aim of our review is to provide an updated overview of the potential pathogenic mechanisms of neurological manifestations in covid-19, discuss the physiological issues related to brain-lung interactions, and propose strategies for optimization of respiratory support in critically ill patients with sars-cov-2 pneumonia. in december 2019, an outbreak of disease caused by a novel coronavirus (2019 novel coronavirus, 2019-ncov) was reported in wuhan, china (1). on february 11, 2020, the novel virus was renamed the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) by the international committee on taxonomy of viruses, and on the same day, the disease it causes was named coronavirus disease 2019 (covid-19) by the world health organization (who) (2) . the rising number of daily confirmed cases globally led the who to characterize the outbreak as a pandemic on march 11, 2020 (3-8) . the typical manifestations of covid-19 include fever, dry cough, fatigue, and respiratory distress (9) . among patients with symptoms requiring hospitalization, 5-20% require invasive mechanical ventilation and admittance to an intensive care unit (10) . covid-19 is a complex, multisystem disease, perhaps best defined as a multiple organ dysfunction syndrome (mods-cov-2) (11) which includes neurologic manifestations (9) . in a recent meta-analysis (12) , headache was identified as one of the most common neurologic symptoms in the early stages of the disease (occurring in 3.5 to 34% of patients), followed by dizziness. more specific neurological manifestations were also observed, including impairment of smell, taste, or vision; limb weakness; acute cerebrovascular disease; and seizures. the causative mechanisms for neurological involvement in covid-19 are still under-investigated because of a lack of prospective studies (12, 13) . furthermore, mechanical ventilation, commonly used in the management of covid-19 patients, can itself induce an inflammatory response, causing distal organ failure. thus, a complex cross-talk between the lungs and other organs, including the brain (14) , may occur during severe covid-19. despite the paucity of evidence, there are three key hypotheses for the neurological manifestations of covid-19 patients (figure 1) : (1) viral neurotropism; (2) a hyperinflammatory and hypercoagulable state; and (3) brainlung crosstalk. while neuroinvasion may be restricted to most severe cases, other cases may be epiphenomena of systemic disease (11) . the latter hypothesis is particularly interesting because it may be amenable to adjustment of ventilator settings to minimize lung and brain injury. within this abbreviations: ace2, angiotensin-converting enzyme-2; ane, acute necrotizing encephalopathy; ards, acute respiratory distress syndrome; balf, bronchoalveolar lavage fluid; bbb, blood brain-barrier; ca, ammon's horn; cd, cluster of differentiation; ci, confidence interval; cns, central nervous system; cov, coronavirus; covid-19, coronavirus disease 2019; ct, computed tomography; cxcr, chemokine receptor; dic, disseminated intravascular coagulation; do 2 , oxygen delivery; dpp4, dipeptidyl dipeptidase-4; ecmo, extracorporeal membrane oxygenation; fio 2 fraction of inspired oxygen; fox, forkhead box; hlh, hemophagocytic lymphohistiocytosis; icam, intracellular adhesion molecule; ich, intracerebral hemorrhage; icp, intracranial pressure; ifn, interferon; mers, middle east respiratory syndrome; mhv, mouse hepatitis virus; mri, magnetic resonance images; ncov, novel coronavirus; or, odds ratio; paco 2 , partial pressure of carbon dioxide; pao 2 partial pressure of oxygen; pbto 2 brain tissue oxygenation tension; pcr, polymerase chain reaction; peep, positive end-expiratory pressure; pres posterior reversible encephalopathy syndrome; rm, recruitment maneuvers; rna, ribonucleic acid; sars, severe acute respiratory syndrome; tlrs, toll-like receptor; tmprss2 transmembrane serine protease 2; tnf, tumor necrosis factor; who, world health organization. context, the aim of this manuscript is to provide an updated overview of the potential pathogenic mechanisms of neurological manifestations in covid-19, discuss the physiological issues related to brain-lung interactions, and propose strategies for optimization of respiratory support in critically ill patients with sars-cov-2 pneumonia. the coronaviruses are large, enveloped, non-segmented, singlestranded, positive-sense ribonucleic acid (rna) viruses. seven coronaviruses in two genera have been identified as possibly infectious in humans, of which sars-cov-1, middle east respiratory syndrome (mers-cov), and sars-cov-2 can cause life-threatening respiratory failure (15, 16) . genomic and structural analyses have shown that sars-cov-1 binds to angiotensin-converting enzyme-2 (ace2) receptors and transmembrane serine protease-2 (tmprss2) (17) . mers-cov instead binds to dipeptidyl dipeptidase-4 (dpp4) receptors, which are mainly present on the epithelium of the lower respiratory tract, small intestine, liver, kidneys, and immune cells (18) . ace2 receptors are widely distributed in the lung alveolar epithelial cells, nasopharyngeal and oral mucosa, endothelium and vascular smooth muscle cells in the brain, vascular endothelium and smooth muscle cells of the liver, vascular and red pulp sinus endothelium of the spleen, and cytoplasm of distal tubules and collecting ducts in the kidney (17) . however, binding to ace2 and dpp4 receptors alone is not enough to make host cells susceptible to infection. some human epithelial cells which overexpress these receptors are not infected, whereas other cells with lower expression of these receptors, such as central nervous system (cns) cells, have shown sars-cov-1 and mers-cov infection (19) . as with the other coronaviruses, the classical route of sars-cov-2 infection is the passage of infected droplets through the upper airway and binding to ace2 receptors. ocular transmission has also been proposed as a possible alternative route for sars-cov-2 infection, since the aqueous humor contains ace2 receptors (20) . sars-cov-2 enters the host cell by endocytosis. after viral uncoating, the virion is released, followed by translation, replication, virion assembly, and new virion coating, a process which induces programmed cell death (21) . a cascade of cerebral involvement in sars-cov-2 infection has been proposed by many authors (22) (23) (24) . coronaviruses may pass from the systemic to the cerebral circulation by several routes. transsynaptic passage through infected neurons via the olfactory bulb has been demonstrated with other coronaviruses, which are able to invade peripheral nerve terminals and spread in a retrograde fashion through synapses into the cns; neuroimaging evidence from covid-19 patients suggests sars-cov-2 can do so as well. sars-cov-2 can also spread across the blood-brain barrier (bbb) by two distinct mechanisms: (a) leukocyte migration across the bbb (named the trojan horse mechanism); and (b) sluggish movement of blood within the microcirculation, crossing the bbb by binding to endothelial cells (17) . infected leukocytes can bind to ace2 receptors and cross the bbb, migrating into the cns (22) (23) (24) (25) (26) . expression of ace2 receptors has been demonstrated in neurons, astrocytes, oligodendrocytes, the motor cortex, the cytoplasm of neurons, and sympathetic pathways (22) . binding to ace2 produces vasodilatation and counteracts inflammation, while binding to the mas receptor exerts neuroprotective and cardioprotective effects (27) . literature from the previous sars epidemic revealed that the virus primarily infects pneumocytes, but can also enter neuronal cells (28) . trans-synaptic spread has been demonstrated in experimental studies; in sars-cov-1 infected mice, extensive virus replication in brain cells was mediated by cerebral invasion through the olfactory epithelium (29) . this has been also confirmed by another murine study with human coronavirus oc43 (30) . in the clinical setting, sars-cov-1 genome sequences were detected in brain cells of infected patients by electron microscopy, real time-polymerase chain reaction (pcr), and light microscopy. among brain areas, the thalami, cerebellum, white matter, and brainstem were primarily affected, with edema and scattered red degeneration of neurons (31) . sars-cov-1 has been also detected in cerebrospinal fluid, probably reflecting spread through the bbb (29) . coronaviruses can also spread to the medullary cardiorespiratory center, which may at least partially account for the acute respiratory failure of sars (32) . although previous literature on other coronaviruses clearly suggests neuronal involvement, data specific to sars-cov-2 are still limited; magnetic resonance imaging (mri), autopsy findings, and brain biopsies should unravel the mystery. as with other coronaviruses, sars-cov-2 could potentially enter the nervous system through the olfactory bulb and spread to specific brain areas (33) . this trans-synaptic spread theory is corroborated by multiple retrospective reports of anosmia and ageusia in covid-19 patients (9, 29, 34) . most recently, anosmia and hyposmia were identified in 5.6% of 214 hospitalized patients (9), while 33.9% of 20 patients who completed a questionnaire experienced either olfactory or taste disorder and 18.6% experienced both (35 (34) . a single center study on 1,480 patients with influenza-like symptoms revealed that smell and taste loss occurred in the majority of patients who tested positive for sars-cov-2, was significantly associated with covid-19 (p < 0.001), and resolved after illness remission (36) . a multicenter european study of 417 covid-19 patients identified olfactory and gustatory dysfunctions as prevalent, early symptoms, which can indeed be used to identify sars-cov-2 infection (37) . finally, this hypothesis was confirmed in vivo by mri evidence of cortical hyperintensity in the right gyrus rectus and olfactory bulb, suggesting viral invasion of the brain-although not all the patients who develop olfactory dysfunction present with abnormal brain imaging (38)-and in post-mortem brain mri studies, which found olfactory bulb and tract impairment without brainstem involvement (39) . this provides very compelling evidence of sars-cov-2 entry via the olfactory tract and subsequent spread to specific brain areas, although limited to isolated cases (2) . electron microscopy studies have recently demonstrated that sars-cov-2 can cross the bbb by binding to endothelial cells (40) . sars-cov-2 neurotropism was further confirmed in autopsies of infected patients who died of cardiorespiratory failure (>65 years old) and massive intracranial hemorrhage (younger). in both groups, all patients showed lymphocytic panencephalitis and meningitis (41) , confirming the neurotropic hypothesis, perhaps guided by leukocyte invasion. irrespective of mechanism, neurotropism is thus clearly demonstrated. when brain involvement does occur, the presence and persistence of human coronaviruses in the cns, as occurs in mice, can determine long-term neurological sequelae. mice surviving acute coronaviral encephalitis exhibited long-term sequelae associated with decreased activity in an open field test and a reduced hippocampus, with neuronal loss in the ammon's horn (ca)1 and ca3 areas (42) . it has also been hypothesized that human coronaviruses may play a triggering role in long-term neurological conditions, such as multiple sclerosis. although research has not led yet to a direct link to any specific virus, an association of coronaviruses with multiple sclerosis has been suggested (43, 44) . a significantly higher prevalence of human cov-oc43 was observed in the brains of multiple sclerosis patients than in controls (45) . moreover, during infection by human cov-oc43 and cov-229e, an autoreactive t-cell response directed to both viral and myelin antigens was discovered in multiple sclerosis patients, but not in controls (46, 47) . this underlines the possibility that longterm infection of the cns by human coronaviruses may play a role in the onset of multiple sclerosis-like demyelinating lesions, as reported during the covid-19 pandemic (48) . evidence of cns infection by sars-cov-2 has been associated with poor prognosis, worse clinical condition, and sudden death in covid-19 patients (9) . however, there is limited evidence to confirm this hypothesis, since the majority of observed cerebrospinal fluid (csf) samples have been negative for sars-cov-2 infection (49, 50) . this makes it difficult to confirm that neurotropism could be the main mechanism of neurological complications in covid-19. pathogenesis sars-cov-2 may pass across the respiratory epithelium and spread from the alveolar-epithelial barrier to the systemic circulation, enhancing the local inflammatory response (51) and producing a systemic "cytokine storm, " affecting other organs such as the brain (52) . furthermore, inflammation is one of the main mechanisms that trigger the coagulation cascade and promote hypercoagulability. in severe sars-cov-2 infection, recent findings suggest a key role of endothelial cells (ecs) in vascular dysfunction, immunothrombosis, and inflammation (53) . histopathological studies have provided evidence of direct viral infection of ecs, diffuse endotheliitis, and micro-and macrovascular thrombosis, both in the venous and arterial circulations. the pro-inflammatory cytokine storm, with elevated levels of interleukin-6 (il-6), il-2 receptor, and tumor necrosis factor (tnf)-α, could also participate in endothelial dysfunction and leukocyte recruitment in the microvasculature. covid-19-induced endotheliitis may explain the systemic impaired microcirculatory function in different organs observed in covid-19 patients. next, we will discuss the role of hyperinflammation and hypercoagulability as potential mechanisms for secondary brain involvement in covid-19. on the immune side, after antigen binding to the host receptor, monocytes are activated, with the release of proinflammatory cytokines (such as mmp9, which increases bbb permeability, and tnf-α, which that increases expression of intracellular adhesion molecule [icam]-1 on endothelial cells). infected and activated monocytes cross the damaged bbb, inducing the local release of pro-inflammatory cytokines and resulting in oligodendrocyte and neuronal damage. coronavirus primarily infects monocyte-derived macrophages, which produce chemokines and then present cov antigens to t-cells and other pro-inflammatory cells (51) . astrocytes may also release other chemokines that will recruit other leukocytes. this hyperactive neuroinflammatory response could induce immune-mediated neuropathology (2, 51) . on the coagulation side, increased consumption and decreased production of platelets in the damaged lungs are all factors that can contribute to thrombocytopenia (54) . as a consequence, it seems reasonable that infected patients are more prone to developing posttraumatic or spontaneous intracranial hemorrhage (55) , as well as these alterations suggest a trend of sars-cov-2 infection to induce consumption coagulopathy, which, if unchecked, could lead to disseminated intravascular coagulation (dic) and an unfavorable clinical course (56) . in fact, viral infections may lead to sepsis, which represents the most common cause of dic. dic is determined by the release of injury-related cytokines, which activate monocytes and endothelial cells, leading to overexpression of tissue factors and secretion of von willebrand factor. the presence of free thrombin in the circulation can activate platelets, stimulating fibrinolysis (57). inflammation inflammatory involvement was recently confirmed by an experimental murine model of murine coronavirus (mhv-a59), which can enter the brain via intranasal or intracerebral exposure and whose virulence is mediated by cytokine secretion. in one experimental study, injection of mouse hepatitis virus (mhv), a member of the coronaviridae family, into the murine cns demonstrated that coronavirus infection elicits both innate and adaptive immune responses (58) . the genomic rna is then translated, replicated, assembled, and coated for future release and infection of other cells. as replication increases with the aid of macrophages, microglia, astrocytes, and oligodendrocytes, the virus can spread from the ependyma to the brain parenchyma. by this point, inflammation is established, and is followed by bbb damage and enhanced innate and adaptive immune responses (58) . immunofluorescence and immunohistochemistry revealed that microglia and astrocytes are involved in activation of the innate immune system of the brain, releasing cytokines that are involved in the pathogenesis of encephalitis (59) . a study on human autopsy specimens showed that sars-cov-1 was able to infect brain tissue, with necrosis of neuronal cells and gliocyte hyperplasia. these studies suggested that neuronal involvement in sars was characterized by a massive inflammatory process, especially with enhancement of monokine expression in gliocytes induced by interferon (ifn)-γ (60) . an experimental study on bronchoalveolar lavage fluid (balf) of covid-19 patients identified that sars-cov-2 infection of the airway leads to proinflammatory cytokine and chemokine release. this enhances the interaction with receptors expressed on thoracic sensory neurons of the lung, thus causing the release of neuropeptides, followed by vasodilation, immune-cell recruitment, neurogenic inflammation, and potential pain. this mechanism could be theoretically involved in the hyperinflammatory state, which first involves the lung and then extends to the nervous system, with sensory neurons thus potentially acting as drivers of neurogenic pulmonary dysfunction (61) . in a retrospective cohort cited above, severe patients were more likely to exhibit impaired consciousness and acute cerebrovascular disease than non-severe patients (p < 0.001 and p < 0.05, respectively). severe patients also showed a more florid inflammatory response (higher white blood cell and neutrophil counts, lower lymphocyte counts, higher c-reactive protein levels) and higher d-dimer levels than non-severe patients, and developed more extensive multiple organ involvement (9) . acute necrotizing encephalopathy (ane) has been related to a brain cytokine storm, which results in bbb disruption (62) . ane has been previously reported as a rare complication of viral infections such as influenza (62) . radiological findings from computed tomography (ct) scans and mri in covid-19 have been recently published (62) . ane was also identified in a patient with aplastic anemia (63) . non-contrast ct scan demonstrated bilateral symmetric hypoattenuation in the medial thalami with negative ct angiogram and venogram findings, while mri showed bilateral hemorrhagic rims in the thalami, sub-insular regions, and medial temporal lobes. ane usually presents a bilateral distribution, with predominance of lesions in the thalami, brainstem, cerebral white matter and cerebellum, which is consistent with the cerebral insults observed in covid-19 (62) . studies have concluded that men and women might show different responses to covid-19. women seem to be less susceptible to viral infections than men overall. the presence of two x chromosomes influences immune regulatory genes to blunt the inflammatory response and increase levels of antibodies and cluster of differentiation (cd)4 + tcells, and consequently, promoting the expression of cytokines. moreover, the x chromosome acts on other proteins and genes, including forkhead box (fox)p-3, toll like receptor (tlr)-8, cd40l, and chemokine receptor (cxcr)3. nevertheless, the increased susceptibility of women to autoimmune and autoinflammatory disorders has to be taken into account (64) . coronavirus infection of the cns has long provided a model for studying demyelinating diseases such as multiple sclerosis, vaccine design, and novel immunotherapeutic to limit virus spread (58) . hemophagocytic lymphohistiocytosis (hlh) is characterized by a severe dysregulation of t-lymphocytes, natural killer cells, and macrophages within the contest of cytokine storm and multiorgan failure, and represents a clear link between hyperinflammation and hypercoagulability (65) . this condition has been described in patients with sars-cov-2 (1). hlh patients present with pancytopenia, coagulopathy, hepatic dysfunction, hypertriglyceridemia, and high ferritin levels (66) . neurological damage in covid-19 patients may also be associated with coagulopathy. in a recent meta-analysis, lippi et al. showed that low platelet counts are associated with poor prognosis in covid-19 (67) . as reported by yang et al. (54) , hematological changes were common in patients with sars, most notably including lymphopenia and thrombocytopenia, through different potential mechanisms. preliminary data from covid-19 cohorts described a major impairment of blood coagulation and derangement of hemostasis in a large number of patients. han et al. (68) studied alterations in blood coagulation parameters of patients with sars-cov-2 infection, observing lower antithrombin values and higher d-dimer, fibrin/fibrinogen degradation products, and fibrinogen levels. tang et al. (56) observed high levels of d-dimer and fibrin/fibrinogen degradation products in all non-survivors, confirming activation of coagulation cascade and secondary hyperfibrinolysis. within this context, the neurological manifestations associated with sars-cov-2 may be determined by a hypercoagulable state with high d-dimer levels. the association between ischemic stroke and high d-dimer levels has been previously described in the literature (69, 70) . d-dimer elevation reflects ongoing thrombus formation, although it is also an acute-phase reactant that enhances the inflammatory process itself by stimulating monocyte synthesis and release of proinflammatory cytokines (e.g., il-6), thus contributing to stroke occurrence and progression (71) . coagulopathy and antiphospholipid antibodies were found in patients affected by covid-19. these findings were associated with both arterial and venous thrombotic events, including cerebral infarcts and limb ischemia. patients presented with prolonged activated partial thromboplastin and prothrombin times, while two of three patients showed thrombocytopenia (72) . fourteen cases of stroke have been reported out of 214 patients in china (9) . likewise, mri and ct scans revealed a high prevalence of stroke in covid-19 patients (49, (73) (74) (75) , including in patients younger than 50 years (76) . the association between stroke and covid-19 could be explained also by the fact that both diseases share the same risk factors such as hypertension and diabetes (77, 78) , and by the pathological hypercoagulability state that characterize covid-19. an association between high levels of d-dimer and intracerebral hemorrhage (ich) was described in a prospective study carried out by di castelnuovo et al. (79) , although a previous meta-analysis did not show a causal relationship (80) . a recent meta-analysis by zhou et al. (81) , which included 13 studies on 891 patients with ich, concluded that high levels of d-dimer were associated with an elevated risk of ich. in fact, high d-dimer levels stimulate fibrinolysis with subsequent plasmin generation and microvascular lesions, which might cause the inhibition of hemostasis and a hypo-coagulable state, thus triggering cerebral hemorrhage (79) . moreover, an association between elevated d-dimer levels and large hematoma volume, intraventricular and subarachnoid blood extension, and early mortality has been reported in ich (82) . in summary, although literature is inconclusive concerning the relationship between covid-19related hypercoagulability and neurological complications, a possible correlation should be taken into account. possible mechanisms for activation of intrinsic and extrinsic coagulation pathways, followed by inflammation by sars-cov-2 infection are proposed in figure 2 . brain-lung crosstalk and its implications for ventilator management are illustrated in figure 3 . the respiratory management of covid-19 shares some characteristics with that of the acute respiratory distress syndrome (ards) (83) , but different hallmarks must be considered and discussed. covid-19 pneumonia is as a typical "pulmonary" ards (84) . in experimental settings (85) , "pulmonary" as compared to "extrapulmonary" ards is distinguished by increased alveolarepithelial damage, more neutrophil cell infiltration and fibrinous exudate, increased collagen fibers in the alveoli and interstitium. in clinical studies, different radiological patterns have been identified, with different characteristics and responses to alveolar recruitment. in non-covid patients, ards is characterized by interstitial and alveolar edema homogeneously distributed along the vertical gradient (86, 87) , leading to collapse of the most dependent alveoli in the supine position. regional perfusion follows a gravitational gradient (more perfusion in dependent lung regions), and severe hypoxemia is explained mainly by increased "true shunt" in atelectatic, dependent lung regions. application of higher levels of positive end-expiratory pressure (peep) is associated with alveolar recruitment, improving respiratory mechanics and gas exchange. thus, in classical ards patients, therapeutic maneuvers leading to improvement in gas exchange are associated with better lung aeration. conversely, covid-19 pneumonia is characterized by minimal interstitial and alveolar edema, alveolar cellular infiltration and necrosis, with alveolar consolidation and pneumolysis. regional perfusion follows a non-gravitational gradient (more perfusion in non-dependent lung regions), with hyperperfusion of normally aerated and poorly aerated ("ground glass") tissue, leading to major changes in ventilation-perfusion ratio. additionally, perfusion in consolidated, dependent lung regions contributes to "true" shunt. application of higher levels of peep does not recruit alveoli; instead, it leads to deterioration of respiratory mechanics, gas exchange, and hemodynamics. thus, in covid-19 patients, therapeutic maneuvers leading to improvement in gas-exchange are not associated with improved lung aeration, but rather with redistribution of regional perfusion (88) . interestingly, areas of hypoperfusion may occur in poorly aerated ground-glass areas as well as in non-aerated lung regions. this suggests that some hypoperfusion might be protective against further deterioration of ventilation-perfusion ratio as well as "true" shunt. three distinct radiological phenotypes of covid-19 pneumonia have been described (79) . phenotype 1 is characterized by multiple, focal, overperfused ground-glass opacities, normal or high lung compliance, and severe hypoxemia, probably caused by low ventilation/perfusion and regional shunting. in this case, peep should be set according to the lowest driving pressure and/or minimal oxygenation, and inhaled nitric oxide might be useful. phenotype 2 is characterized by an inhomogeneous and/or asymmetrical distribution of atelectasis, partial alveolar derecruitment, and/or consolidation with peribronchial opacities. in these cases, lateral or prone positioning might be helpful. finally, phenotype 3 is characterized by patchy, ards-like diffuse lung infiltration, with a mixed pattern of overperfused, normally aerated and groundglass areas as well as hypoperfused, non-aerated lung regions with low compliance. in this setting, mechanical ventilation should follow standard protective ventilatory strategies used for ards, with minimal peep, prone positioning, and escalation to extracorporeal membrane oxygenation (ecmo) as needed. in all cases, possible microthrombosis and multiorgan failure must be considered. a correlation between acute lung injury and brain hypoxia has been described by oddo et al. (89) . reduced systemic oxygenation may affect brain tissue oxygenation, thus leading to secondary brain damage. measurement of brain tissue oxygenation tension (pbto 2 ) has confirmed that this parameter is strongly correlated with systemic oxygenation and markers of lung function, including partial pressure of carbon dioxide (paco 2 ) and mean arterial pressure. accordingly, impaired partial pressure of oxygen (pao 2 )/fraction of inspired oxygen (fio 2 ) ratio has been associated with lower pbto 2 (89). in another study, patients who underwent an oxygen challenge with 100% fio 2 showed higher pbto 2 (90) . hypoxic-ischemic damage is also associated with impaired outcome (91) . we believe this phenomenon should be considered one of the main mechanisms implicated in neurological dysfunction following sars-cov-2 infection. in fact, given these respiratory characteristics, "silent" hypoxia with normal/hypercapnic respiratory failure can occur due to compromised alveolar gas figure 2 | sars-cov-2-induced hypercoagulability. passage of the virus from the airway to the systemic circulation is facilitated by the sluggish movement of blood within the microcirculation and subsequent binding of ace-2 receptors, expressed on the capillary endothelium, followed by endothelial damage, enhanced inflammation, and hypercoagulability. in this figure, we represent the activation of both intrinsic and extrinsic coagulation pathways as a possible mechanism for hypercoagulability and potential brain damage. intrinsic pathway: activation of factor (f) xiia, followed by activation of fxia and viii. extrinsic pathway: activation of fviia and tissue factor. both pathways converge in the common pathway with activation of fxa, fva, prothrombin into thrombin, fibrinogen into fibrin, and fibrin degradation products (fdp) such as d-dimer. figure 3 | bohr effect. the oxyhemoglobin dissociation curve is shifted to the left in response to respiratory alkalosis (lower paco 2 and higher ph), with increased affinity of oxygen for the hemoglobin. conversely, during respiratory acidosis (higher paco 2 and lower ph), the alveolar oxygen tension and systemic saturation improve, thus reducing alveolar carbon dioxide tension, as explained by the bohr effect: the higher the acidity, the more carbon dioxide is eliminated. exchange (92) . our knowledge concerning hypobaric hypoxia can be derived from aviation medicine (93) . high altitude correlates with severe hypoxemia, which triggers the carotid chemoreceptors, activating the respiratory drive; hypocapnia ensues. the oxyhemoglobin dissociation curve shifts to the left in response to respiratory alkalosis and increased affinity of oxygen for hemoglobin, thereby increasing the alveolar oxygen tension and systemic saturation after reducing alveolar carbon dioxide tension, as explained by the bohr effect-the greater the acidity, the more carbon dioxide is eliminated (94) . pao 2 and oxygen delivery (do 2 ) can be optimized by modulating blood ph and paco 2 , hemoglobin concentration, cardiac output, and arterial content of oxygen. these factors mean close attention is warranted when implementing lung-protective strategies, particularly when using low oxygen targets and permissive hypercapnia. in phenotype 1, characterized by lower potential alveolar recruitability, raising hemoglobin and cardiac output should be considered as a strategy to improve do 2 , as explained in figure 4 . one possible side effect of higher hemoglobin is increased blood viscosity, raising the risk of cerebrovascular events (95) . in phenotype 3 (ards-like covid), prone positioning, higher peep, and rms should be attempted instead to increase pao 2 and control paco 2 levels. at figure 4 | improving oxygen delivery to the brain. raising hemoglobin and cardiac output should be considered for improving oxygen delivery, especially in covid-19 phenotype 1. this figure represents different delivery of oxygen (do 2 ) at a fixed cardiac output, by changing hemoglobin, or at fixed hemoglobin, by changing cardiac output. this point, it is crucial that brain-lung-hemodynamics crosstalk be addressed (figures 5a-c) (96) . current knowledge on the cerebral effects of mechanical ventilation has shifted in favor of moderate-peep strategies instead of low-or zero-peep strategies, due to possible beneficial effects on brain tissue oxygenation (97) (98) (99) . nevertheless, higher peep levels may be considered in covid-19 phenotype 3 to reach acceptable levels of oxygen saturation in the brain (100), thus improving cerebral blood flow and perfusion (101) . in this phenotype (but not in phenotypes 1 or 2), lung recruitment maneuvers might also improve oxygenation by improving gas exchange, although their effects on intracranial pressure (icp) could be detrimental due to impaired jugular venous outflow and venous return (102) . according to the "blast injury theory, " the sympathetic storm, cytokine storm, and hyperinflammatory state caused by infection can induce a transient increase in intravascular pressure, with endothelial damage, raised pulmonary vascular hydrostatic pressure, and increased capillary permeability, thus promoting lung derangement and a secondary brain insult (103) . this could explain, at least in part, why patients with severe covid-19 have worse neurological outcomes (104) . both oxygen and carbon dioxide have been considered important determinants of cerebral homeostasis, due to their effects on cerebral blood flow (105) . low cerebral blood flow due to low paco 2 is associated with cerebral ischemia, while high cerebral blood flow results in cerebral hyperemia and higher icp (105) . a rise in icp may also be achieved by increasing paco 2 if intracranial compliance is reduced. in patients not amenable to alveolar recruitment maneuvers, such as those with covid-19 phenotype 1, overdistension of alveolar areas contributes to a rise in paco 2 due to the increase in dead space, followed by cerebral vasodilatation. conversely, in patients responsive to recruitment maneuvers (covid-19 phenotype 3), shunt is reduced, oxygenation improves, and the paco 2 is decreased, with lower dead space and less changes in icp and cerebral perfusion (83, 106) . pao 2 , paco 2 , ph, hemoglobin, and do 2 might all be considered as clinical targets for bedside monitoring where available, to protect both the brain and the lung. the first report of brain autopsies in covid-19 patients was published on june 12, 2020. impressively, the authors reported that, at histologic analysis, all 18 examined patients (100%) had evidence of acute hypoxic ischemic damage to the cerebrum and cerebellum. neither encephalitis nor any evidence of specific viral invasion was identified (107) . the neuroimaging features of 108 hospitalized covid-19 patients demonstrated a non-specific pattern, with predominance of acute figure 5 | (a-c) brain-lung-heart cross talk. sars-cov-2 lung infection can require mechanical ventilation, which heightens the pro-inflammatory cascade. in this figure, we propose the effect of increased peep on the cardiovascular system and cns in healthy subjects (a), ards (b), and covid-19 (c). in normal lungs (a), high peep and alveolar hyperdistention cause increased plateau pressure (pplat), driving pressure ( p), and pleural pressure (ppl), with consequent reduction of venous return (vr) and cardiac index (ci) and reduced cerebral perfusion pressure (cpp) and increased intracranial pressure (icp). this can be partially offset by the presence of preserved gas exchange. in ards patients (b), the increase in peep with recruitment of collapsed areas does not cause significant changes in hemodynamics or cerebral function, and can increase oxygen delivery (cdo 2 ). conversely, in covid-19 patients (c) who do not respond to recruitment, the concomitance of alveolar hyperdistention after peep increase and hypoxemia can cause serious impairment of cerebral dynamics and cerebral hypoxemia (low pbto 2 ). ischemic infarcts and intracranial hemorrhage. mri findings included the posterior reversible encephalopathy syndrome (pres), hypoxic-ischemic encephalopathy, and exacerbation of preexisting demyelinating disease, corroborating the role of a hyperinflammatory/hypercoagulable state and brain-lung crosstalk as major mechanisms potentially underpinning neurological complications in covid-19 (108) . further evidence of neurological involvement is the higher incidence of icu delirium in covid-19 patients when compared to non-covid patients (26.8 vs. 7.7%, p = 0.003) (109) . this may be explained by the fact that profound hypoxia is known to predispose to long-term cognitive impairment and hypoxic delirium phenotypes, whether caused by bbb dysfunction, inflammation, hypoperfusion, hypoxemia, or a combination thereof (110) (111) (112) . in summary, encephalopathy and cerebrovascular disease are the main neurological features identified in severe covid-19 (73, 113) . despite compelling evidence of viral neurotropism, we believe this is not the primary causative factor of neurological involvement. instead, in most cases it is likely due to impairment of the delicate equilibrium between the brain and the lung and to the hyperinflammatory, pro-coagulative state that is characteristic of sars-cov-2 infection. in covid-19 patients, central and peripheral nervous system changes may be caused by viral neurotropism (such as impairment of olfaction and taste), by a hyperinflammatory and hypercoagulative state, or even by mechanical ventilation-associated impairment. three distinct phenotypes of pulmonary injury have been identified in association with covid-19 pneumonia, each requiring individualized respiratory support strategies to minimize lung injury and optimize oxygen delivery to different organs-including the brain. data from prospective observational studies, randomized clinical trials, and autopsies are urgently needed to confirm the latest 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disease-19 (covid-19) we express our gratitude to mrs. moira elizabeth schottler and mr. filippe vasconcellos for their assistance in editing the manuscript. outside the submitted work dg reports honoraria from stepstone pharma gmbh and unconditional grants from msd italia and correvio italia. outside the submitted work, mb has received funding for scientific advisory boards, travel, and speaker honoraria from angelini, astellas, astrazeneca, basilea, bayer, biomerieux, cidara, correvio, cubist, menarini, molteni, msd, nabriva, paratek, pfizer, roche, shionogi, tetraphase, thermo fisher, and the medicine company.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 battaglini, brunetti, anania, fiaschi, zona, ball, giacobbe, vena, bassetti, patroniti, schenone, pelosi, rocco and robba. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-291916-5yqc3zcx authors: hozhabri, hossein; piceci sparascio, francesca; sohrabi, hamidreza; mousavifar, leila; roy, rené; scribano, daniela; de luca, alessandro; ambrosi, cecilia; sarshar, meysam title: the global emergency of novel coronavirus (sars-cov-2): an update of the current status and forecasting date: 2020-08-05 journal: int j environ res public health doi: 10.3390/ijerph17165648 sha: doc_id: 291916 cord_uid: 5yqc3zcx over the past two decades, there have been two major outbreaks where the crossover of animal betacoronaviruses to humans has resulted in severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). in december 2019, a global public health concern started with the emergence of a new strain of coronavirus (sars-cov-2 or 2019 novel coronavirus, 2019-ncov) which has rapidly spread all over the world from its origin in wuhan, china. sars-cov-2 belongs to the betacoronavirus genus, which includes human sars-cov, mers and two other human coronaviruses (hcovs), hcov-oc43 and hcov-hku1. the fatality rate of sars-cov-2 is lower than the two previous coronavirus epidemics, but it is faster spreading and the large number of infected people with severe viral pneumonia and respiratory illness, showed sars-cov-2 to be highly contagious. based on the current published evidence, herein we summarize the origin, genetics, epidemiology, clinical manifestations, preventions, diagnosis and up to date treatments of sars-cov-2 infections in comparison with those caused by sars-cov and mers-cov. moreover, the possible impact of weather conditions on the transmission of sars-cov-2 is also discussed. therefore, the aim of the present review is to reconsider the two previous pandemics and provide a reference for future studies as well as therapeutic approaches. coronaviruses (covs) are a group of highly enveloped viruses that are diversely found in humans and wildlife. with their high mutation rate and infectivity, covs are important zoonotic pathogens that can infect animals [1, 2] and humans, leading to 5-10% of acute respiratory syndromes [3] . apart from infecting a variety of economically important vertebrates (such as pigs and chickens), six species have been identified to cause disease in humans [4] . they are known to infect respiratory, gastrointestinal, hepatic and neurologic systems with a wide range of clinical features from asymptomatic course to severe disease that require hospitalization in the intensive care unit [4, 5] . the first human coronaviruses (hcovs), hcov-229e and oc43, shown to be significant respiratory pathogens, were identified in the 1960s [6, 7] . however, it is assumed that the first recorded coronavirus-related disease was feline infectious peritonitis (fip) in 1912 [8] . the "corona"-like or crown-like morphology of these viruses leads to choose the name "coronavirus," in 1968 [6] . coronaviruses were not considered as highly pathogenic for humans before the beginning of the 21st century. afterward, two highly pathogenic hcovs, including severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), emerging from animal reservoirs, have led to global epidemics of deadly pneumonia in humans with high morbidity and mortality [9, 10] . in december 2019, seven years after mers outbreak, the third pathogenic hcov emerged in wuhan, the capital city of hubei province in china, causing severe pneumonia [11, 12] . considered as agents that are a great public health threat, an epidemiological alert was placed by the world health organization (who) and this new coronavirus was named sars-cov-2 and the related respiratory disease covid-19 (https://www.who.int). compared with sars-cov, sars-cov-2 appears to be more readily transmitted from human-to-human, spreading to multiple continents and the outbreak of sars-cov-2 was declared on january 30, 2020 [13] (https://www.who.int). in this review, we will introduce the current knowledge on the origin and evolution of sars-cov-2, emphasizing its characteristics and its genetic diversity from previous coronaviruses, with a brief comment on its epidemiology and pathogenesis. we also highlight the environmental factors involved in virus transmission. knowledge about this novel coronavirus is rapidly evolving, and efforts must be implemented in order to protect the populations by reducing transmission and controlling the spread of this fatal disease. according to the international committee on taxonomy of viruses, covs are classified under the order of nidovirales, a family of coronaviridae and subfamily of coronavirinae [14] . based on previous serologic and recent genomic evidences, the family of coronaviridae encompasses two subfamilies: subfamily orthocoronavirinae and subfamily torovirinae ( figure 1) [7, 15] . the subfamily of orthocoronavirinae consists of four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus [7, 16, 17] . covs can be isolated from different animal species, including birds, livestock and mammals such as camels, bats, masked palm civets, mice, dogs and cats [18, 19] . animal covs are known to cause acute diseases in several animals and could be responsible for economic losses in domestic animals or birds [20, 21] . domestic animals may play an important role as intermediate hosts that enable virus transmission from one species to humans [17] . the genera gamma-and deltacoronavirus infect birds, but some of them can also infect mammals [16] . these animal covs include transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), avian infectious bronchitis virus (ibv)-and more recently-swine acute diarrhea syndrome coronavirus (sads-cov). however, animal covs can also infect humans that can spread the infection through human-tohuman transmission [17, 22] . on the other hand, alpha-and betacoronavirus infect only mammals and usually cause respiratory illness in humans; among these, strains 229e, oc43, hku1 and nl63 are the most widespread infecting young children, infants as well as elderly individuals [23] [24] [25] . the high rates of mutation characterizing all rna viruses [23, 26] , the evolving nature of covs and the simplicity of transmission from one species to another are the most relevant features learned from sars-cov and mers-cov previous outbreaks [15, 23, 25] . importantly, most of alpha-and betacoronavirus were found only in bats, and many genetically diverse coronaviruses phylogenetically related to sars-cov and mers-cov have been discovered in diverse bat species worldwide [17] . therefore, hcovs such as sars-and mers-covs seem to have originated in bats by sequential mutations and recombination, including those occurring in the intermediate hosts, civets and raccoon dogs for sars-cov and camels in the case of mers-cov, finally acquiring the ability to infect humans [15, 17] . comparative genome studies published in recent papers strongly support the hypothesis that sars-cov-2 originated in bats and that pangolins (manis javanica) acted as intermediate mammalian hosts [11, 27] (figure 2) . indeed, the genetic sequence of the sars-cov-2 showed more than 79% nucleotide identity with the sequence of sars-cov and 50% with mers-cov [17, 19] . the high degree of homology of the angiotensin-converting enzyme 2 (ace2) receptor in several animal species can be considered as an additional evidence to support that sars-cov-2 originated from bats [28] . based on findings from molecular studies, the ace2 proteins of non-human primates, pigs, cats and ferrets closely resemble the human ace2 receptor. therefore, these species covs can be isolated from different animal species, including birds, livestock and mammals such as camels, bats, masked palm civets, mice, dogs and cats [18, 19] . animal covs are known to cause acute diseases in several animals and could be responsible for economic losses in domestic animals or birds [20, 21] . domestic animals may play an important role as intermediate hosts that enable virus transmission from one species to humans [17] . the genera gammaand deltacoronavirus infect birds, but some of them can also infect mammals [16] . these animal covs include transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), avian infectious bronchitis virus (ibv)-and more recently-swine acute diarrhea syndrome coronavirus (sads-cov). however, animal covs can also infect humans that can spread the infection through human-to-human transmission [17, 22] . on the other hand, alphaand betacoronavirus infect only mammals and usually cause respiratory illness in humans; among these, strains 229e, oc43, hku1 and nl63 are the most widespread infecting young children, infants as well as elderly individuals [23] [24] [25] . the high rates of mutation characterizing all rna viruses [23, 26] , the evolving nature of covs and the simplicity of transmission from one species to another are the most relevant features learned from sars-cov and mers-cov previous outbreaks [15, 23, 25] . importantly, most of alphaand betacoronavirus were found only in bats, and many genetically diverse coronaviruses phylogenetically related to sars-cov and mers-cov have been discovered in diverse bat species worldwide [17] . therefore, hcovs such as sars-and mers-covs seem to have originated in bats by sequential mutations and recombination, including those occurring in the intermediate hosts, civets and raccoon dogs for sars-cov and camels in the case of mers-cov, finally acquiring the ability to infect humans [15, 17] . comparative genome studies published in recent papers strongly support the hypothesis that sars-cov-2 originated in bats and that pangolins (manis javanica) acted as intermediate mammalian hosts [11, 27] (figure 2) . indeed, the genetic sequence of the sars-cov-2 showed more than 79% nucleotide identity with the sequence of sars-cov and 50% with mers-cov [17, 19] . the high degree of homology of the angiotensin-converting enzyme 2 (ace2) receptor in several animal species can be considered as an additional evidence to support that sars-cov-2 originated from bats [28] . based on findings from molecular studies, the ace2 proteins of non-human primates, pigs, cats and ferrets closely resemble the human ace2 receptor. therefore, these species may be susceptible to sars-cov-2 infection, as has been shown for sars-cov. although a recent study showed that neither pigs nor chickens are susceptible to sars-cov-2 by intranasal or oculo-oronasal infections, more evidences are needed to exclude pigs as intermediate host of sars-cov-2 [29] . [15] . based on the genetic sequence identity and the phylogenetic reports, sars-cov-2 is sufficiently different from sars-cov; thus, who has classified it as a new betacoronavirus that infects humans [30] . the genome of hcovs is a single-stranded positive-sense rna (+ssrna) (~26-32 kb) with 5′cap structure and 3′-poly a tail, which is among the largest known rna genomes [31] [32] [33] . the typical hcovs gene order is 5′-replicase-s-e-m-n-3′, with numerous (6 to 11) open reading frames (orfs) encoding accessory proteins scattered among the structural genes [34, 35] . the first orfs (orf1a and 1b) comprise two-thirds (approximately 67%) of the genome length and encode 16 nonstructural polyproteins (nsps [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] and are directly translated from the genomic rna [17] . there is a −1 ribosomal frameshift between orf1a and orf1b, leading to the production of two large replicase polypeptides (pp): pp1a and pp1ab. these polypeptides are further processed by two virally encoded cysteine proteases, the papain-like protease (plpro) and a 3-chymotrypsin-like protease (3clpro) into 16 nsps [3, 33, 36] . there are at least four structural proteins encoded by the coronaviral genome: a spike glycoprotein (s), an envelope protein (e), a membrane protein (m) and nucleocapsid protein (n) with short untranslated regions at both termini, required to produce a structurally complete viral particle [37] . the typical coronavirus virion structure and proteins are shown in figure 3 . the m protein is in higher quantities in comparison to any other proteins in the virus particle; with its three transmembrane domains, it shapes virions, promotes membrane curvature and binds to the nucleocapsid [38, 39] . the n protein contains two domains, both of which can bind to nsp3 protein to help tether the genome to replication-transcription complex (rtc) and package viral rna into the viral particle during viral assembly [39, 40] . the e protein is involved in virus assembly and virion release from host cells, while the s protein plays a vital role in attachment to host receptors, viral entry and determines host tropism [41, 42] . additionally, some coronaviruses, such as hcov-oc43 and hcov-hku1, have a hemagglutinin-esterase (he) gene between orf1b and s [43] [44] [45] [46] . this based on the genetic sequence identity and the phylogenetic reports, sars-cov-2 is sufficiently different from sars-cov; thus, who has classified it as a new betacoronavirus that infects humans [30] . the genome of hcovs is a single-stranded positive-sense rna (+ssrna) (~26-32 kb) with 5 -cap structure and 3 -poly a tail, which is among the largest known rna genomes [31] [32] [33] . the typical hcovs gene order is 5 -replicase-s-e-m-n-3 , with numerous (6 to 11) open reading frames (orfs) encoding accessory proteins scattered among the structural genes [34, 35] . the first orfs (orf1a and 1b) comprise two-thirds (approximately 67%) of the genome length and encode 16 nonstructural polyproteins (nsps 1-16) and are directly translated from the genomic rna [17] . there is a −1 ribosomal frameshift between orf1a and orf1b, leading to the production of two large replicase polypeptides (pp): pp1a and pp1ab. these polypeptides are further processed by two virally encoded cysteine proteases, the papain-like protease (plpro) and a 3-chymotrypsin-like protease (3clpro) into 16 nsps [3, 33, 36] . there are at least four structural proteins encoded by the coronaviral genome: a spike glycoprotein (s), an envelope protein (e), a membrane protein (m) and nucleocapsid protein (n) with short untranslated regions at both termini, required to produce a structurally complete viral particle [37] . the typical coronavirus virion structure and proteins are shown in figure 3 . the m protein is in higher quantities in comparison to any other proteins in the virus particle; with its three transmembrane domains, it shapes virions, promotes membrane curvature and binds to the nucleocapsid [38, 39] . the n protein contains two domains, both of which can bind to nsp3 protein to help tether the genome to replication-transcription complex (rtc) and package viral rna into the viral particle during viral assembly [39, 40] . the e protein is involved in virus assembly and virion release from host cells, while the s protein plays a vital role in attachment to host receptors, viral entry and determines host tropism [41, 42] . additionally, some coronaviruses, such as hcov-oc43 and hcov-hku1, have a hemagglutinin-esterase (he) gene between orf1b and s [43] [44] [45] [46] . this hemagglutinin, like the influenza homolog enzyme, binds to sialic acid on host cell-surface glycoproteins and possesses acetyl-esterase activity [47] . besides coronavirus-conserved genes, the sars-cov, sars-cov-2 and mers-cov genomes contain several specific accessory genes including orf3a/b, 4a/b, orf5, orf6, orf7a/b, orf8a/b and 9b ( figure 4) [4, 48, 49] . all the structural and accessory proteins are translated from subgenomic rnas (sgrnas) generated during genome transcription/replication of covs [4] . hemagglutinin, like the influenza homolog enzyme, binds to sialic acid on host cell-surface glycoproteins and possesses acetyl-esterase activity [47] . besides coronavirus-conserved genes, the sars-cov, sars-cov-2 and mers-cov genomes contain several specific accessory genes including orf3a/b, 4a/b, orf5, orf6, orf7a/b, orf8a/b and 9b ( figure 4) [4, 48, 49] . all the structural and accessory proteins are translated from subgenomic rnas (sgrnas) generated during genome transcription/replication of covs [4] . attachment, cell entry, translation of viral replicase, genome replication, translation of structural proteins and virion assembly and release are the phases of coronavirus replication cycle [4, 50] . sars-cov, mers-cov and sars-cov-2 bind to different host receptors to gain entry into host cells [4, 51, 52] . viral entry is mediated by the transmembrane s glycoprotein that comprises two functional subunits (s1 and s2 subunits) responsible for receptor recognition and viral-host cell membranes fusion, respectively [53, 54] . s1 receptor-binding domain (rbd) mediates binding to the cognate host cell receptor; however, the s2 domain mediates the fusion events, between viral envelope and host cell membrane [52, 55, 56] . as recently found, sars-cov-2 uses the same ace2 receptor [57] , as sars-cov, whereas mers-cov uses dipeptidyl peptidase 4 (dpp4, also known as cd26) receptor (table 1 ) [58] . the fusion of the s protein to the plasma membrane of host cell generates a double membrane vesicle in the host cell, thereby allowing release of the nucleocapsid into the cytoplasm, followed by genome transcription [53, 54] . upon entry into the cell, virus-specific rna and proteins are synthesized, probably entirely in the cytoplasm. translation starts with the expression of two polyproteins, pp1a and pp1ab, which undergo co-translational proteolytic processing into the proteins that form the replicase complex. this complex is used to transcribe a 3′-coterminal set of nested subgenomic mrnas, as well as genomic rna that have a common 5′ "leader" sequence derived from the 5′ end of the genome. new virions are assembled by budding into intracellular membranes of the pre-golgi compartment and released through the cell secretory mechanisms [4, 42, 48, 50] . attachment, cell entry, translation of viral replicase, genome replication, translation of structural proteins and virion assembly and release are the phases of coronavirus replication cycle [4, 50] . sars-cov, mers-cov and sars-cov-2 bind to different host receptors to gain entry into host cells [4, 51, 52] . viral entry is mediated by the transmembrane s glycoprotein that comprises two functional subunits (s1 and s2 subunits) responsible for receptor recognition and viral-host cell membranes fusion, respectively [53, 54] . s1 receptor-binding domain (rbd) mediates binding to the cognate host cell receptor; however, the s2 domain mediates the fusion events, between viral envelope and host cell membrane [52, 55, 56] . as recently found, sars-cov-2 uses the same ace2 receptor [57] , as sars-cov, whereas mers-cov uses dipeptidyl peptidase 4 (dpp4, also known as cd26) receptor (table 1 ) [58] . the fusion of the s protein to the plasma membrane of host cell generates a double membrane vesicle in the host cell, thereby allowing release of the nucleocapsid into the cytoplasm, followed by genome transcription [53, 54] . upon entry into the cell, virus-specific rna and proteins are synthesized, probably entirely in the cytoplasm. translation starts with the expression of two polyproteins, pp1a and pp1ab, which undergo co-translational proteolytic processing into the proteins that form the replicase complex. this complex is used to transcribe a 3 -coterminal set of nested subgenomic mrnas, as well as genomic rna that have a common 5 "leader" sequence derived from the 5 end of the genome. new virions are assembled by budding into intracellular membranes of the pre-golgi compartment and released through the cell secretory mechanisms [4, 42, 48, 50] . , the 3′-terminus of the sars-cov-2 and sars-cov genomes contain eight accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14 and 3a, 3b, p6, 7a, 7b, 8a, 8b and 9b, respectively) while mers-cov genome contains only five (3, 4a, 4b, 5 and 8b). the genes encoding accessory proteins are unique in different coronaviruses in terms of number, genomic organization, sequence and functions (data extracted from [35, 49, 57] the coronavirus genomes encode two replicase polypeptides pp1a and pp1ab translated from orf1a and orf1b; four structural genes encoding for four structural proteins including (s) spike, (m) membrane, (e) envelope and (n) nucleocapsid proteins. the single-stranded rna genomes of sars-cov-2 (~29.8 kb), sars-cov (~29.7 kb) and mers-cov (~30.1 kb) harbor two large genes, the orf1a (red) and 1b (blue) genes encoding accessory genes (nsps 1-16, shades of red and blue). encoded nonstructural proteins: 16 nsps (nsp1-nsp16) in sars-cov-2, sars-cov and mers-cov. along with structural proteins (s, e, m and n), the 3 -terminus of the sars-cov-2 and sars-cov genomes contain eight accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14 and 3a, 3b, p6, 7a, 7b, 8a, 8b and 9b, respectively) while mers-cov genome contains only five (3, 4a, 4b, 5 and 8b). the genes encoding accessory proteins are unique in different coronaviruses in terms of number, genomic organization, sequence and functions (data extracted from [35, 49, 57] virologic as well as genetic studies have demonstrated that bats are reservoir hosts of both sars-cov and mers-cov, but also that they can use other species as intermediate hosts before spreading to humans [59, 60] . the detection of two genomes distinct from known swine in ill piglets were reported by two independent groups [61, 62] . the phylogenetic analyses showed that these novel swine enteric alphacoronaviruses (seacovs) were strongly related to the rhinolophus bat coronavirus hku2 isolated in guangdong province, in southern china [61, 62] . this suggests that coronaviruses of bat origin may have 'jumped' the barrier of the species to infect pigs as intermediate hosts. the cd26 receptor sequence alignment between humans and pigs demonstrates a 94.5% overlap, which is sufficient for the possible cross-species transmission [63] . it has also been documented that pigs are susceptible to human sars-cov [64] and mers-cov infections [65] . the large number of mutations within the rbd enabled viruses to infect new hosts, representing a potential threat for both animal and human health. in southern china, the unique climate, the high density of domestic as well as wild pigs, along with the extensive bat distribution and carriage of tremendous quantities of recombinant novel coronaviruses may result in the appearance of more novel coronaviruses in the future [66] . it is generally acknowledged that numerous viruses have existed and were restricted to their natural reservoirs for lengthy times [17] . the consistent spillover of viruses from natural hosts to humans and other species is essentially related to human activities, including urbanization and modern agricultural practices, leading to the constant human exposure to the ever-changing mutant covs from their reservoirs [15, 17] . the close contact between humans and animals and the practice of eating raw meat are both risk factors for causing a new human cov outbreak [15] . hence, covid-19 should be considered as a zoonotic disease that spread from animals to humans. following the first sars-cov-2 outbreak in seafood and wildlife market in wuhan, secondary cases started to be identified after ten days. although these new patients did not have any contact with the market, they had a history of contact with people who attended the market [60] . therefore, similarly to sars-cov and unlikely to mers-cov, human-to-human transmission for sars-cov-2 has been reported and is currently considered as the main type of transmission worldwide [5, 19] . on january 13, 2020, thailand announced the first non-chinese case of infection that spread from the chinese provinces, to the asian continent [60] . this case was a chinese tourist who has traveled to thailand and did not have any epidemiological link to the market [30] . more recently, forster et al., by using phylogenetic analysis based on nucleotide mutations of 160 complete human sars-cov-2 genomes found that three variants of sars-cov-2 (a as the ancestral type, plus b and c) represent the bat outgroup coronaviruses. in particular, the a and c types were found mostly in european-american patients, whereas the b type was common in east asia suggesting that this kind of analysis could help in following the evolution of sars-cov-2 [67] . it was demonstrated that sars-covs have adapted themselves to bind to human ace2 receptor and infect human cells effectively [68] . this form of adaptation required a series of amino acid changes in the rbd within the s protein of sars viruses that circulated in bats [56, 68] . therefore, the human-to-human transmission that was seen in the course of the sars-cov outbreaks is directly attributable to the ability of sars-covs to adapt their s protein to efficiently bind to human ace2, thus infecting ciliated bronchial epithelial cells and type ii pneumocytes [15, 69] . similar to sars-cov, ace2 is also used by sars-cov-2 as the entry receptor in the ace2-expressing cells, suggesting that sars-cov-2 has a life cycle similar to sars-cov [56, 68] . as outlined before, sars-cov s protein regulates the receptor binding and membrane fusion activities determining host tropism and transmission capacity. several evidences highlighted a higher binding affinity of sars-cov-2 rbd to the ace2 receptor. in particular, molecular and in silico analyses demonstrated that sars-cov-2 rbd conformation and amino acid composition enhance the bonding between the s protein and ace2 receptor [51, 70, 71] . a recent biophysical and structural analysis of the sars-cov-2 s protein showed that it binds to ace2 receptor with about 10-to 20-fold higher affinity than the s protein of sars-cov [52, 72] . this high affinity could account for its extreme infectivity among human populations. another feature of the powerful infectivity of sars-cov-2 is that the shedding pattern of viral particles in sars-cov-2 diagnosed patients is similar to that of influenza patients in which viral loads at the time of symptom onset are higher and gradually decrease within days; interestingly, this pattern seems to be different from that reported for sars-cov patients where the highest shedding is reported 10 days after the onset of symptoms [20, 73, 74] . these results indicate that sars-cov-2 can spread more easily than sars-cov in the community even in the absence of symptoms or when only initial mild symptoms are present [75] . the human-to-human transmission of sars-cov-2 mainly occurs by inhalation of respiratory droplets spread by coughing or sneezing from an infected individual, but also by direct contact of contaminated surfaces and then touching the nose, mouth and eyes [24, [76] [77] [78] . the virus was shown to remain stable in favorable atmospheric conditions on different surfaces for days [79] . additionally, transmission in an unventilated environment or closed spaces due to high aerosol concentrations has been suggested [76, 77] . in agreement, the presence of sars-cov-2 in the surfaces of the houses of confirmed patients was reported, further strengthening this mode of contact transmission. moreover, live viruses were also found in the stool of covid-19 patients, as previously found for both sars-cov and mers-cov [77] . given its capacity for survival in feces and the expression of ace2 within intestine, it was demonstrated that sars-cov-2 can infect these tissues and can be released in feces; therefore, water supply contamination and fecal-oral route transmission is also hypothesized [24, 80] . however, at present, there have not been reported cases of fecal-oral transmission of the virus. studies have also indicated that sars-cov-2 transmission via ocular surfaces should not be overlooked, as contaminated droplets and body fluids could easily infect the human conjunctival epithelium [81] . sars-cov-2 is also responsible for cluster transmission, in particular within family clusters [77] . in some cities, 50% to 80% of all reported cases of covid-19 accounted for cluster transmission [82] . based on the current information, there is no evidence for transplacental transmission from infected pregnant women to their fetus, who underwent caesarean section [24, 78] . therefore, whether transmission during vaginal birth can occur remains to be established, neonatal covid-19 disease as postnatal transmission was documented [83] . although, sars-cov-2 may definitely infect infants, it has been reported that neonates, infants and children develop significantly milder forms of the disease than their adult counterparts [24, 84] . coronaviruses are responsible for 5-10% of acute respiratory illness while it has been estimated that 2% of the population is deemed as an asymptomatic carrier of these viruses. the first discovered hcov was ibv that causes respiratory disease in human whereas, hcov-229e and hcov-oc43, which cause the common cold in humans [15, 26, 85] . they were not considered to be highly pathogenic for humans until the outbreak of sars in guangdong state of china in 2002 and 2003. sars-cov infected more than 8000 people worldwide and caused 916 deaths (table 2) , representing a mortality rate by around 10% [86] . ten years later in 2012, mers-cov emerged in saudi arabia and infected over 2494 people with 858 deaths, accounting for a mortality rate approximately of 35% [9, 24, 87] . starting in china in december 2019, there were reports of patients presenting severe viral pneumonia [15, 88, 89] . this public health concern resulted in many unknown pneumonia cases who were admitted to local hospitals [22, 78] (https://www.who.int). primary etiologic investigations performed in those patients showed that they were epidemiologically linked to a huanan wholesale seafood market that also traded live animals and wildlife [17, 24, 90] . by january 7th, 2020 chinese authorities announced that a new type of coronavirus was isolated [60, 91] . the epicenter of infection was probably linked to a zoonotic pathogen being present in the seafood and exotic animal wholesale market [60, 91] . the rapid increasing numbers and rate of fatalities indicated a second mode of transmission, from human-to-human, that allowed viral spreading primarily in other asian countries such as south korea and iran followed by many countries such as italy, spain, germany, france, brazil and usa [24, 60] . it is very intriguing to note that the sars epidemic in southern china in 2002 and the current outbreak of covid-19 had peaked in mid-february due to exposure to live animals sold in markets. furthermore, similar to the sars outbreak, this epidemic has occurred during the spring festival in china, as the most famous traditional countrywide festival in china, gathering nearly three billion people from different areas. these favorable conditions caused the wide transmission of this fatal pneumonia and severe difficulties for disease control and prevention of the epidemic [92] . based on clinical data of diagnosed patients during the sars-cov-2 outbreak, the basic reproduction number (r0) is estimated to range between 2 and 6.47 in various modeling studies [76, 93] . the sars-cov-2 r0 is in line with the one estimated for sars-covs and mers-covs (from 2 to 5) [94, 95] . currently, increasing countries are experiencing clusters of cases and community transmission following sars-cov-2 pandemic. since its emergence, the covid-19 has drawn well-deserved attention from authorities in order to protect their community and stop or slow down transmission of this disease. at the time of this review, according to the daily report of the world health organization, sars-cov-2 has affected over 17,889,134 people with around 228,611 daily new cases and killed more than 686,145 people all over the world, by august 3rd, 2020 (the up to date fatality rate is reported from https://covid19.who.int). we must take into consideration that these data are relative to laboratories and clinically confirmed cases while the actual number including asymptomatic cases, infected undiagnosed and death patients would be much higher than reported cases. the transmission of seasonal respiratory coronaviruses can be affected by several climate parameters such as temperature and humidity [96] . therefore, understanding the relationship between weather and transmission of covid-19 is the key to forecast the intensity and end time of this pandemic. to this regard, emerging evidence suggests that whether climate conditions may influence the transmission of the sars-cov-2 by boosting the spread (much of the data have not been peer-reviewed yet). to date, covid-19 has had a significant expansion in the northern hemisphere (nh) belt, given that it covers cities and populated areas; conversely, in the belt of the southern hemisphere (sh), which covers very low population and landless areas, covid-19 has not been reported yet. based on climate and era-interim reanalysis dataset in nh belt from november 2019 to march 2020, we compared the average rate of humidity and temperature between five cities in european countries with significant community transmission of covid-19 versus five cities of north africa which are expected to be less exposed to covid-19. the information recorded by the meteorological stations has been used, since these are more accurate than satellite data [97] . as shown in table 3 , the average amount of humidity is very close between european and african selected sites. the main reason is the proximity of these cities to the mediterranean sea coastline. in addition, the north wind, which blows from northern europe to european cities (ecs), increases the humidity of these cities. conversely, there are temperature differences between considered ecs and north african cities (table 3) . thus, temperature and humidity should be considered parameters involved in the transmission of covid-19. up to know, few studies have investigated the association of temperature and humidity with covid-19 incidence and death rates. the first meteorological study was done in 100 different chinese cities each having more than 40 cases of covid-19 in a 3-day period during the end of january [98] . this group showed that high temperature and humidity significantly reduces the transmission of covid-19. their results indicate that the increases of 1 • c in temperature and 1% in relative humidity lower r by 0.0225 and 0.0158, respectively [98] . a preprint study on confirmed covid-19 cases collected from 429 cities showed that every 1 • c increase in the minimum temperature of higher-temperature cities reduced the disease incidence and death rates by 0.86 [99] . another preprint study suggested that the average increase of 1 • c in temperature correlates negatively with the predicted number of cases worldwide [100] . these results are in accordance with wu y et al. who showed that among all confirmed covid-19 new cases and new deaths from 166 countries (excluding china), a 1 • c increase in temperature is associated with a 3.08% reduction in daily new cases and a 1.19% reduction in daily new deaths, whereas a 1% increase in relative humidity was associated with a 0.85% reduction in daily new cases and a 0.51% reduction in daily new deaths [101] . a recent study conducted in italy showed a positive correlation of sars-cov-2 spreading and weather conditions including temperatures ranging 4-12 • c and relative humidity of 60-80% [102] . in a geographic and population modeling study conducted in five largest cities in colombia, the transmission of sars-cov-2 seems to be comodulated by temperature and humidity. their observation revealed a strong reduction of transmission in climates with temperatures above 30 • c and relative humidity below 78% which may comodulate the infectivity of sars-cov-2 within respiratory droplets [103] . table 3 . average humidity and temperature in 10 different cities in europe and north africa between november 2019 to march 2020. the first five cities represent significant communities where transmission of covid-19 was reported, whereas the second 5 cities are expected to be less exposed to covid-19 due to different weather conditions. average humidity (%) temperature ( • c) rome 71 65 66 63 63 15 1 11 13 14 paris 78 76 79 74 66 9 8 8 9 9 madrid 70 67 68 63 61 10 10 9 13 12 milan 77 74 69 58 62 11 8 7 11 11 lisbon 75 74 77 76 71 15 14 12 15 15 rabat 72 71 69 72 74 16 17 14 17 16 algiers 64 62 61 61 66 16 17 15 18 16 tunis 61 66 72 65 70 17 16 14 15 15 tripoli 80 83 73 75 71 15 10 8 9 11 cairo 45 52 55 53 46 25 18 16 18 22 overall, these meteorological analyses support that the combination of temperature and humidity could represent a direct influence on the transmission of the covid-19. it can be assumed that the arrival of summer and rainy season in the nh can effectively reduce the transmission of the covid-19. the distribution of covid-19 across different longitudes and latitudes with a range of temperatures and humidity may help to predict the prevalence of this disease in terms of environmental characteristics. this could lead to a better understanding of how the virus spreads around the world ( figure 5 ). it should be noted that apart from the capability of sars-cov-2 to persist on environmental surfaces under favorable atmospheric conditions, the duration of its persistence may be affected by temperature and humidity. however, caution is needed when considering the implications of these findings, which may be subject to confounding. although warmer climates may slow the spread of sars-cov-2, relying on weather changes alone to slow the transmission of covid-19 are unlikely to be enough. however, using this type of dataset and climate analysis modeling is possible to identify areas that are most likely to be at risk of significant covid-19 cases in the future and serve as an alarm signal to various government departments and agencies to adopt the necessary measures to prevent virus spread [104] . moreover, more data are gathering around the world due to the change of the season and all authors agree that the association between temperature/humidity and sars-cov-2 is an appreciable hypothesis, but not a certainty yet. 19 cases in the future and serve as an alarm signal to various government departments and agencies to adopt the necessary measures to prevent virus spread [104] . moreover, more data are gathering around the world due to the change of the season and all authors agree that the association between temperature/humidity and sars-cov-2 is an appreciable hypothesis, but not a certainty yet. the most common symptoms of patients at onset of covid-19 disease are defined as fever, dry cough, fatigue and less often, symptoms of sputum production, headache, sore throat, myalgia; hemoptysis, dyspnea, diarrhea and lymphopenia were also observed [15, 24, 28] (figure 6 ). the spectrum of clinical features of covid-19 has been found ranging from an asymptomatic state to severe respiratory failure and multiorgan dysfunction [24, 76] . symptomatic people are considered to be more contagious, similar to most viral-related respiratory diseases. however, individuals who remain asymptomatic may also transmit the virus and cases infected by an asymptomatic individual in the prodrome period of covid-19 have also been reported [76] . asymptomatic infections can occur because of weakened immune responses and subclinical manifestations or also because the the most common symptoms of patients at onset of covid-19 disease are defined as fever, dry cough, fatigue and less often, symptoms of sputum production, headache, sore throat, myalgia; hemoptysis, dyspnea, diarrhea and lymphopenia were also observed [15, 24, 28] (figure 6 ). the spectrum of clinical features of covid-19 has been found ranging from an asymptomatic state to severe respiratory failure and multiorgan dysfunction [24, 76] . symptomatic people are considered to be more contagious, similar to most viral-related respiratory diseases. however, individuals who remain asymptomatic may also transmit the virus and cases infected by an asymptomatic individual in the prodrome period of covid-19 have also been reported [76] . asymptomatic infections can occur because of weakened immune responses and subclinical manifestations or also because the virus is waiting for opportunities to invade and reproduce. a recent study has shown that a viral load detected in an asymptomatic patient was just like to the one observed in symptomatic patients, indicating the capability of transmission in asymptomatic patients [74] . according to disease presentation, covid-19 can be classified as mild, moderate, severe and critical ( virus is waiting for opportunities to invade and reproduce. a recent study has shown that a viral load detected in an asymptomatic patient was just like to the one observed in symptomatic patients, indicating the capability of transmission in asymptomatic patients [74] . according to disease presentation, covid-19 can be classified as mild, moderate, severe and critical (table 4 ) [57, 76, 105] . in 81% of all confirmed covid-19 cases. dry cough, mild fever, sore throat, nasal congestion, muscle pain, headache and malaise. absence of serious symptoms like dyspnea, also the absence of radiograph features. it may rapidly deteriorate into severe or critical cases, non-pneumonia or mild pneumonia. dry cough, tachypnea and shortness of breath. acute respiratory distress syndrome (ards), severe pneumonia, severe dyspnea, sepsis or septic shock, tachypnea (respiratory frequency) ≥ 30/min, blood oxygen saturation (spo2) ≤ 93%, partial pressure of arterial oxygen to fraction of inspired oxygen ratio (pao2/fio2) < 300, and/or lung infiltrates > 50% within 24 to 48 h. fever can be absent or moderate. in 5% of all confirmed covid-19 cases. respiratory failure, septic shock, rnaemia, cardiac injury and/or multiple organ dysfunction or failure. case fatality rate is 49% (higher case fatality rate for patients with preexisting co-morbidities and lower-case fatality rate (0.9%) for patients without co-morbidities). cardiovascular disease (10.5%), diabetes (7.3%), respiratory disease (6.5%), hypertension (6%) and oncological complications (5.6%). in 81% of all confirmed covid-19 cases. dry cough, mild fever, sore throat, nasal congestion, muscle pain, headache and malaise. absence of serious symptoms like dyspnea, also the absence of radiograph features. it may rapidly deteriorate into severe or critical cases, non-pneumonia or mild pneumonia. dry cough, tachypnea and shortness of breath. acute respiratory distress syndrome (ards), severe pneumonia, severe dyspnea, sepsis or septic shock, tachypnea (respiratory frequency) ≥ 30/min, blood oxygen saturation (spo 2 ) ≤ 93%, partial pressure of arterial oxygen to fraction of inspired oxygen ratio (pao 2 /fio 2 ) < 300, and/or lung infiltrates > 50% within 24 to 48 h. fever can be absent or moderate. in 5% of all confirmed covid-19 cases. respiratory failure, septic shock, rnaemia, cardiac injury and/or multiple organ dysfunction or failure. case fatality rate is 49% (higher case fatality rate for patients with preexisting co-morbidities and lower-case fatality rate (0.9%) for patients without co-morbidities). cardiovascular disease (10.5%), diabetes (7.3%), respiratory disease (6.5%), hypertension (6%) and oncological complications (5.6%). and 95% of patients are likely to experience symptoms within 12.5 days from contact [24, [106] [107] [108] . however, in an asymptomatic carrier the incubation period was 19 days, complicating the challenge to contain the outbreak [109] . the median time between onset of symptoms and dyspnea is 5 days, 7 days for hospitalization and 8 days for acute respiratory distress syndrome (ards) (figure 7 ) [24] (https://www.epicentro.iss.it/coronavirus/sars-cov-2). patients at this stage in intensive care unit (icu) with quarantine facilities may require mechanical ventilation. moreover, bacterial infections can cause a secondary pneumonia [108] . in addition, the period from the beginning of covid-19 symptoms to death varied between 6 and 41 days with an average of 14 days [110] . this period depends on immune system status and the patient's age, being shorter in 70-year-old subjects compared with those younger [78, 110] . in people with compromised immune systems and in elderly patients with underlying health problems, sars-cov-2 is able to infect the lower respiratory tract leading to severe pneumonia [111] . in 25-30% of patients presenting acute lung injury, shock, ards and acute kidney injury, icu admission was absolutely required [24] . recovery started within the 2nd or 3rd weeks with the median duration of hospitalization of 10 days. the virus appears to be more fatal in individuals with underlying co-morbidities (50-75% of fatal cases) [24, 111] . available dataset was obtained from italian istituto superiore di sanità (iss) on 34,026 patients dying in-hospitals ( figure 7) . the mean number of diseases was 3.3 (median 3, sd 1.9). overall, 4.0% of the reported cases has no co-morbidities, 14.0% with a single comorbidity, 20.6% with 2 and 61.4% with 3 or more (https://www.epicentro.iss.it/coronavirus/sars-cov-2). sars-cov-2 infections revealed some unique clinical characteristics that include targeting the lower airway which is evident by symptoms of upper respiratory tract including rhinorrhoea, sneezing and sore throat [78] . chest computed tomography (ct) scans revealed pneumonia in most sars-cov-2 infected patients and several cases showed an infiltrate in the upper lung lobe, which is related to increasing dyspnea with hypoxemia [28, 78, 112] . table 4 describes the full picture of covid-19 clinical manifestation. atypical symptoms include rnaemia, acute cardiac injury, ards and grand-glass opacities that lead to death [113] . it should be noted that covid-19 manifestations such as fever, dyspnea, dry cough and bilateral ground-glass opacities in chest ct scans are similar to the previous betacoronavirus-related diseases [113, 114] . although gastrointestinal symptoms such as diarrhea were reported in sars-cov-2 infected patients, the similar gastrointestinal distress occurred in only a small percentage of mers-cov or sars-cov patients ( figure 6 ) [78] . it was shown that severe cases were characterized by an increased inflammation due to both systemic and localized immune response activation [78, 115] . higher leukocyte numbers, significantly high blood concentrations of cytokines and chemokines were noted in these cases [28, 78] . hence, it is now accepted that high levels of proinflammatory cytokines could worsen the prognosis [28, 113] . the symptoms of sars-cov-2 infection appear after an incubation period of 1 to 14 days, similar to those of sars-and mers-cov infections (median approximately 5.2 days in different studies) and 95% of patients are likely to experience symptoms within 12.5 days from contact [24, [106] [107] [108] . however, in an asymptomatic carrier the incubation period was 19 days, complicating the challenge to contain the outbreak [109] . the median time between onset of symptoms and dyspnea is 5 days, 7 days for hospitalization and 8 days for acute respiratory distress syndrome (ards) (figure 7 ) [24] (https://www.epicentro.iss.it/coronavirus/sars-cov-2). patients at this stage in intensive care unit (icu) with quarantine facilities may require mechanical ventilation. moreover, bacterial infections can cause a secondary pneumonia [108] . in addition, the period from the beginning of covid-19 symptoms to death varied between 6 and 41 days with an average of 14 days [110] . this period depends on immune system status and the patient's age, being shorter in 70-year-old subjects compared with those younger [78, 110] . in people with compromised immune systems and in elderly patients with underlying health problems, sars-cov-2 is able to infect the lower respiratory tract leading to severe pneumonia [111] . in 25%-30% of patients presenting acute lung injury, shock, ards and acute kidney injury, icu admission was absolutely required [24] . recovery started within the 2nd or 3rd weeks with the median duration of hospitalization of 10 days. the virus appears to be more fatal in individuals with underlying co-morbidities (50%-75% of fatal cases) [24, 111] . available dataset was obtained from italian istituto superiore di sanità (iss) on 34,026 patients dying inhospitals ( figure 7) . the mean number of diseases was 3.3 (median 3, sd 1.9). overall, 4.0% of the reported cases has no co-morbidities, 14.0% with a single comorbidity, 20.6% with 2 and 61.4% with 3 or more (https://www.epicentro.iss.it/coronavirus/sars-cov-2). figure 7 . median times, in days, from the onset of symptoms to death, to hospitalization, from hospitalization to death with and without intensive care unit (icu)-admittance (report based on available data on july 9th, 2020 collected from istituto superiore di sanità, iss). sars-cov-2 infections revealed some unique clinical characteristics that include targeting the lower airway which is evident by symptoms of upper respiratory tract including rhinorrhoea, sneezing and sore throat [78] . chest computed tomography (ct) scans revealed pneumonia in most sars-cov-2 infected patients and several cases showed an infiltrate in the upper lung lobe, which is related to increasing dyspnea with hypoxemia [28, 78, 112] . table 4 describes the full picture of covid-19 clinical manifestation. atypical symptoms include rnaemia, acute cardiac injury, ards and grand-glass opacities that lead to death [113] . it should be noted that covid-19 manifestations figure 7 . median times, in days, from the onset of symptoms to death, to hospitalization, from hospitalization to death with and without intensive care unit (icu)-admittance (report based on available data on july 9th, 2020 collected from istituto superiore di sanità, iss). covid-19 clinical evaluation is focused mainly on epidemiological data, clinical symptoms and clinical and laboratory tests. although the scenario is continually changing, several approaches were selected as standard laboratory methods for covid-19 diagnosis. lab tests, differently from clinical-based analyses, immediately reveal sars-cov-2 infected patients. this was particularly important for diagnosis due to the difficulties in detecting specific clinical signs and symptoms in covid-19 patients. moreover, atypical manifestations revealed by pulmonary imaging [116] and the huge number of different clinical signs and symptoms forced the development of molecular-based laboratory tests [117, 118] . lastly, the analysis of personal history of each patient played a fundamental role in covid-19 diagnosis and up to now is considered one of the key information for detecting infected patients also in the early phases of infection. therefore, the epidemiological history together with clinical and laboratory tests are all required for the diagnosis of covid-19. a detailed description focused on clinical diagnostic methods was reviewed by taisheng li [119] . herein, we present an updated overview of the principal techniques used for covid-19 diagnosis. high-throughput sequencing and real time quantitative polymerase chain reaction (rt-qpcr) are the best nucleic acid detection techniques for sars-cov-2. however, in clinical diagnosis, the application of high-throughput sequencing technology is limited due to high cost and its equipment dependency [114, 120, 121] . moreover, to speed up the development of standardized analytic kits for diagnostic application, the quantification of viral load was not considered. therefore, the rt-pcr method was chosen as the gold standard for the detection of sars-cov-2 infections from the commonly used samples such as naso-and oropharyngeal swabs [106, 107, 121, 122] . this molecular method relies on the amplification of up to three sars-cov-2 specific targets including the rna-dependent rna polymerase (rdrp), e and n genes [121] . the who has released numerous rt-pcr protocols for the detection of sars-cov-2 rna at https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance (accessed march 15, 2020). three of those protocols are mentioned below. the us centers for disease control and prevention (cdc) developed an rt-pcr that includes three sets of oligonucleotide primers and probes recognizing three regions of the virus n gene (named n1, n2 and n3) and an additional primer/probe set to detect the human rnase p gene (rp) representing an internal control for rna extraction and retro-transcription. moreover, the positive control consisting in retro-transcribed viral rna is also available at cdc. to report the positivity for sars-cov-2 two out of three n regions must be positive. the chinese center for disease control and prevention (china cdc) recommends the use of specific primers and probes targeting the orf1ab and n gene regions for sars-cov-2 detection by rt-pcr [123] . the positivity is confirmed when both targets are detected. available online: http://ivdc.chinacdc.cn/kyjz/202001/t20200121_211337.html (accessed on 21 january 2020). overall, the who summarized all the primer pairs and probes that can be used to detect sars-cov-2 in clinical specimens with the description of rt-pcr settings and the specificity. apart from the possibility to perform the rt-pcr in house using the selected primer pairs and probes, several ready to use kits were developed for rt-pcr performing. one of the most used is the allplex 2019-ncov (seegene, seoul, south korea) which includes three different viral targets and a positive control [124] . another example is represented by the bgi's real-time fluorescent rt-pcr kit for detecting sars-cov-2 that includes one sars-cov-2 specific target and an internal control of the reaction (bgi, cambridge, ma, usa). both companies declared a sensibility of 100-150 viral copies per ml and a high specificity that excludes most respiratory tract viral and bacterial pathogens. the recommended samples for both in-house and ready-to-use rt-pcr kits include upper and lower respiratory specimens such as throat, nasal nasopharyngeal (np) and/or oropharyngeal (op) swabs, lower respiratory tract aspirates, sputum, bronchoalveolar lavage (bal) fluid and nasopharyngeal wash/aspirate or nasal aspirate. it was observed that samples of the lower respiratory tract provide the higher viral loads [74] . on the other hand, it was shown that in the early stage of infection, the positive rate of rt-pcr was reported to be about 60% for throat swab samples [125] . indeed, although being the gold standard, the rt-pcr presents some drawbacks. one of the most important is related to the sensibility because it was extensively reported that in the presence of low viral load this technique fails in detecting viral genome leading to false negative results [126] . due to this problem, clinical governance as well as kit troubleshooting indicate to retest all the samples showing only single positive target along with patient resampling. to this respect, it should be underlined that operator skills or sampling sources can profoundly affect rt-pcr testing results [22] . finally, during this pandemic several microbiologic labs worldwide are experiencing scarce availability of rna extraction as well as ready-to-use rt-pcr kits increasing the timing of diagnosis confirmation through molecular approaches. very recently, it was reported that the allplex 2019-ncov and the realstar sars-cov-2 rt-pcr kits can amplify the target genes bypassing the rna extraction step for a faster diagnosis [127] . although rt-pcr is specific for the diagnosis of covid-19, its false-negative rate cannot be overlooked due to the severe consequences of missed diagnosis. clinicians have demonstrated the usefulness of ct and chest radiography for the diagnosis of covid-19 associated pneumonia [128] . moreover, the ability of radiologists to diagnose covid-19 pneumonia from chest ct evaluations has been reported to be very high [129] . then a combination between rt-pcr and ct imaging represents the best approach for the correct covid-19 diagnosis. in particular, for early detection and assessment of disease severity, the high-resolution ct (hrct) of the chest is considered necessary [130, 131] . one study analyzed the consistency and diagnostic value of rt-pcr test compared with chest ct in 1014 patients with suspected sars-cov-2 infection. findings indicated that the chest ct sensitivity in suspected patients was 75% based on negative rt-pcr results and 97% based on positive rt-pcr results [132] . moreover, salehi et al. confirmed the higher sensibility of pulmonary imaging with respect to rt-pcr for covid-19 diagnosis and showed a positive correlation between specific ct findings with the different stages of the disease and its severity [116] . the collection of numerous ct images has opened the possibility to build a database of pulmonary images from covid-19 patients. interestingly, the recent progress in integrating artificial intelligence (ai) with computer-aided design (cad) software for diagnostic imaging revealed that ai could be used to support disease diagnosis [133, 134] . ito et al. reviewed the literature on the use of ai for lung diagnostic imaging of covid-19 patients. among the 15 selected studies, 11 used ai for ct and 4 used ai for chest radiography. the number of datasets ranged from 106 to 5941, with sensitivities ranging from 67-100% and specificities ranging from 81-100% for prediction of covid-19 pneumonia. this study revealed the usefulness of ai approach to support the diagnosis of covid-19, but also for future emerging diseases [134] . all the collected knowledge on lung lesions revealed some characteristic ct findings of covid-19 pneumonia: the pulmonary ground-glass opacities in a peripheral distribution and the consolidation referring to an increase in pulmonary parenchymal density [135] [136] [137] . however, chest ct manifestations can vary in different patients and stages of infection, highlighting certain shortcomings of this approach. apart from atypical manifestation that cannot be recognized by radiologists, several lung images are common in viral pneumonia leading to misdiagnosis [138] . soon after the beginning of sars-cov-2 spreading, infected patients underwent antibody research for both basic research and clinical applications. one of the first studies reported the seroconversion of 100% of infected patients (n = 285) within 19 days after symptom onset. seroconversion for igm and igg occurred simultaneously or sequentially and both immunoglobulins titers plateaued within 6 days after seroconversion. importantly, the application of serology testing in surveillance in a cluster of 164 close contacts of covid-19 patients identified 4.6% of positive patients showing negative rt-pcr results [139] . hence, several studies underlined the recommended usage of serology to promote the detection of sars-cov-2 infections where np swab specimens were improperly collected, molecular assays were unsatisfactorily carried out and for determining asymptomatic infections [122] . based on these data, several companies developed kits for igm/igg testing showing a high detection rate of infected patients. basically, there are two different testing methods: the rapid igg-igm test and the classical enzyme-linked immunosorbent assay (elisa)-based test. the rapid test consists in a lateral flow qualitative immunoassay on a strip to detect the presence of both anti-sars-cov-2-igm and anti-sars-cov-2-igg in human specimens such as whole blood, serum and plasma. this igg-and igm-combined antibody test kit has a sensitivity of 88.66% and specificity of 90.63%. results are obtained in 15 min leading to its useful application as point-of-care testing and in supporting rt-pcr-based diagnostic [140] . on the other hand, several elisa-based kits are now commercially available, and their sensitivity and specificity were compared showing an overall high specificity, but a variable sensibility [141] . differently from the rapid tests, the elisa-based test should be performed on serum or plasma samples collected from venous sampling. interestingly, the authors showed the neutralizing capacity of sars-cov-2 specific antibodies on caco-2 cells directly incubating the sera from patients with the cell monolayers [141] . this assay is extremely important for the plasma-based therapies that are successfully used to treat seriously ill patients (see below). finally, recently published papers described the seroconversion of covid-19 patients including the evaluation of iga that seems high in the early stages of infection (about 4 days' post symptom development) [142, 143] . another interesting application of antibody detection is represented by the fluorescence immuno-chromatographic assay for the detection of sars-cov-2 nucleocapsid protein in human specimens such as np swab [144] . it shows the fastness of rapid tests (results in 10 min), the possibility to use the same type of sample that is commonly used for rt-pcr-based diagnosis and high sensibility (detection of the nucleoprotein in all positive samples tested). although these methods were suggested for covid-19 diagnosis, the extent of antibodies production by infected patients is greatly variable. moreover, the delay of antibodies production with respect to the onset of symptoms affects the use of this approach for diagnosis. vice versa, it is reported that several governments, included italy, are using serologic test for population screening to assess the proportion of people that have developed an immunological response against sars-cov-2 (http://www.salute.gov.it/portale/nuovocoronavirus). this screening will help also to detect asymptomatic and/or paucisymptomatic subjects. the rapid spread of sars-cov-2 raises an urgent requirement for effective therapeutic strategies against covid-19. although many efforts have been intended to develop vaccines against hcovs infections in recent years, there is no official and effective treatment against sars-cov-2. however, different considerable options have been applied for possible vaccine validity, efficacy and safety along with speeding up other ongoing searches to discover valuable modalities for dealing with the emerging covid-19 [12, [145] [146] [147] [148] . most of the drugs that are being used to cope with covid-19 epidemic are directed towards specific viral molecular targets and biologic processes through which the virus spreads damaging the host. in line, all available experimental therapies for covid-19 management are based on previous experiences in treating sars-cov and mers-cov infections, such as inhibitors of sars-cov-2 fusion/entry/replication, anti-viral agents against main viral proteases, regulators of sars-cov-2 induced host inflammatory response and direct administration of human monoclonal antibodies (mabs) (figure 8 ) [149] . apart from all these possible therapeutic approaches, it has been reported that the chinese medicine products, as lianhuaqingwen and shufeng jiedu capsules may be helpful for sars-cov-2 treatment [12, 150] . indeed, this product is mainly used to treat upper respiratory tract infections such as the flu, swelling and pain in the throat, mumps and strep throat [151, 152] . moreover, four covid-19 cases have been described to gain improvement after taking combined chinese and western medicine [153] . notably, encouraging progress in deciphering sars-cov-2 genome will lead to new potential therapeutic targets. likewise, more prospective, rigorous population studies are urgently required to confirm the therapeutic effect as well as the safety of new potential therapeutic strategies in order to further implement robust preventive and control measures against sars-cov-2 spread. as outlined above, multiple strategies are aimed at developing covs vaccines, most of which are headed for the surface-exposed spike (s protein) glycoprotein as the major virus-host cell membrane interactor. to this aim, vaccines under study are based on full-length s protein, s1-rbd, expression of virus-like particles (vlp), dna or viral vectors [42, [154] [155] [156] [157] [158] . as outlined above, the s1 includes the rbd that interacts with its host cell receptor, ace2, whereas the s2 mediates fusion between the virus and host cell membranes promoting the entry and subsequent replication of the viral rna into the cytoplasm [158] . the ace2 receptor, as a specific biologic target for vaccine development, is under study in a controlled pilot clinical trial to investigate the effect of recombinant human ace2 (rhace2; gsk2586881) in patients with severe covid-19 (nct04287686) ( figure 8i ) [159, 160] . vice versa, both recombinant proteins containing rbd and the recombinant vectors encoding rbd can be used to generate the effective sars-cov vaccines given the capability of this domain to induce neutralizing antibody [156] . indeed, the first available sars-cov-specific human monoclonal antibody with neutralizing activity against sars-cov, named cr3022, was found to bind potently to sars-cov-2 rbd, in agreement with the high homology shared by this domain with sars-cov homolog [161] . however, it must be taken into account that more than 85% of the rbd antibody epitopes in sars-cov-2 show implicit noticeable changes, indicating the necessity to develop more specific monoclonal antibodies for sars-cov-2 [162] . angiotensin receptor blockers (arbs), such as losartan, valsartan, telmisartan, usually assumed for treating high blood pressure, heart and kidney failure in people with diabetes, have been recently proposed as a novel therapeutic approach to block sars-cov-2 rbd binding to ace2-expressing cells binding, similarly to ace inhibitors [163] . additional targetable epitopes that should be considered are the heptad repeat 1 (hr1) and heptad repeat 2 (hr2) in sars-cov-2 s protein. in fact, the hr2-derived peptides (hr2p) and ek1 (a modified oc43-hr2p peptide), exhibit effective fusion inhibitory activity towards sars-cov-2, suggesting a promising strategy in treating sars-cov-2 infection, although further studies are required to strengthen these hypotheses ( figure 8i ) [164, 165] . lately, immuno-informatics have been employed to identify significant cytotoxic t lymphocyte (ctl) and b-cell epitopes in sars-cov-2 s protein, such as the nucleocapsid (n) protein as well as the potential b cell epitopes of the e protein of mers-cov as likely immunoprotective targets [166, 167] . reverse genetic strategies have been successfully used in live-attenuated vaccines to inactivate the exonuclease effects of non-structural protein 14 (nsp14) or to wipe out the envelope protein in sars [154] . a recent study also revealed that the invasion process requires the priming of the s protein which is facilitated by the host cell produced serine protease tmprss211. the clinically demonstrated serine protease tmprss2 inhibitor camostat mesylate, which partially blocks sars-cov-2 entry into host cells, was shown to be a good target to significantly reduce pulmonary infection in covid-19 affected individuals ( figure 8i ) [168] moreover, it has been suggested that coronavirus entry also involves ph and receptor-dependent endocytosis [169, 170] ; thus, targeting endocytosis may be another assessable option for fighting sars-cov-2 ( figure 8i ). in this view, throughout ai technology, a group of approved drugs, such as the janus kinase (jak) inhibitor baricitinib [171] targeting the ap-2-associated protein kinase 1 (aak1) regulating clathrin-mediated endocytosis, has been developed ( figure 8i ) [172] . furthermore, other drugs such as arbidol (chictr2000029621), a haemagglutinin inhibitor and chloroquine phosphate, a traditional antimalarial drug, have been added to the national health commission of the people's republic of china (nhc) guidelines for covid-19 treatment ( figure 8i ) (http://www.nhc.gov.cn). in particular, in vitro studies have demonstrated that chloroquine as well as hydroxychloroquine could impair the endosome-mediated viral entry or later stages of viral replication [173] . combination of hydroxychloroquine and azithromycin has also been suggested as a valid approach since it showed more rapid resolution of infection than hydroxychloroquine alone [174] ; however, the combined use of azithromycin and hydroxychloroquine seems to be associated with at increased risk of arrhythmias. available online: https://www.acc.org/latest-in-cardiology/articles/2020/03/27/14/00/ventricular-arrhythmia-riskdue-to-hydroxychloroquine-azithromycin-treatment-for-covid-19 (accessed on 29 march 2020). to date, several attempts have also been made in targeting viral main enzymes; in fact, many inhibitory drugs targeting the coronavirus main proteinase 3c-like protease (3clpro) have been validated in clinical trials (e.g., lopinavir/ritonavir; chictr2000029387, chictr2000029468, chictr2000029539) ( figure 8ii ) [175] . moreover, four additional molecules including prulifloxacin, tegobuvir, bictegravir and nelfinavir, detected by high-throughput screening, showed reasonable binding conformations with the viral main protease [176] . moreover, a recent study by performing a virtual screening using a three-dimensional model of the sars-cov-2 3c-like protease (3cl), identified 16 biologic candidates that deserve further consideration. among these, the antivirals ledipasvir or velpatasvir proved to be particularly attracting as therapeutics to combat the new coronavirus showing optimal anti-viral activity and minimal side effects, such as fatigue and headache; also, epclusa (velpatasvir/sofosbuvir) and harvoni (ledipasvir/sofosbuvir) are promising antivirals, not only for their effective and synergic inhibitory activities against two viral enzymes, but also for their minimized possibilities to develop resistance [177] . a certain number of clinical trials on antiviral drugs aimed to arrest sars-cov-2 replication are currently in progress, such as remdesivir (nct04252664, nct04257656) favipiravir (chictr2000029600, chictr2000029544) and asc09 (chictr2000029603) ( figure 8iii ). among these, remdesivir was recently approved for medical use in america and european union and seems to be the most promising antiviral for fighting sars-cov-2 [178] (http://www.who.int), as in vitro studies demonstrated that this molecule, a mono-phosphoramidate prodrug of an adenosine, effectively inhibited sars-cov-2 rna synthesis [179] . targeting the sars-cov-2 rna genome could, therefore, be another potential strategy. in fact, a crispr/cas13d technology, which is an rna-guided rna-targeting crispr system, has been employed to specifically chew up sars-cov-2 rna genome. in this system, a cas13d protein and guide rnas-containing spacer sequences are used to specifically complement the virus rna genome ( figure 8iv ). furthermore, rna genome can be packaged into one adeno-associated virus (aav) vector, making the crispr/cas13d system more efficient for virus elimination and resistance prevention, taking into account that aav has serotypes highly specific to the lung, the main organ infected by sars-cov-2 [180] . in addition to antiviral therapy, a new treatment strategy having a significant impact on clinical outcomes is utmost required. immunomodulatory therapy to downregulate the cytokine storm may provide great benefit to the treatment of covid-19. recently, researchers focused on targeting specific molecular markers involved in inflammatory cytokines-receptors interactions, their correlation in health and disease and drugs in use that can activate or block their actions. a higher concentration of cytokines has been found in the plasma from covid-19 patients in icu compared with the ones from non-icu covid-19 patients, suggesting that the cytokine storm could be linked to the severity of the disease [113] . corticosteroids are among the most commonly used drugs for immunomodulatory therapy of infectious diseases. however, the use of corticosteroids in the treatment of covid-19 can cause host immune suppression and delay of viral clearance. a recent study on 201 patients with ards showed that treatment with methylprednisolone decreased the risk of death (hazard ratio 0.38, 95% confidence interval 0.20-0.72). these findings indicate that using corticosteroids does not influence viral clearance time, length of hospital stays or duration of symptoms in patients with mild covid-19 [181] . thus, the use of corticosteroids is considered beneficial in severe cases of covid-19 (especially in patients with ards), but not in mild cases. accordingly, a recent retrospective study showed the potential benefits from low-dose corticosteroids treatment in a subset of critically sars-cov-2 patients [182] ; these data are in contrast with nhc guidelines that highlight that systematic use of corticosteroids is not recommended for these cases, due to their immunosuppressive effects. however, administration of corticosteroids has been indicated for specific reasons such as exacerbation of asthma or chronic obstructive pulmonary disease (copd), septic shock or severe acute respiratory distress syndrome (ards). further studies are required to find out how and when it is appropriate the use of corticosteroids for covid-19, as there are no available data on the benefits of corticosteroid treatment in sars-cov or mers infection [183] . apart from corticosteroids, il-6 pathway inhibitors such as sarilumab, siltuximab and tocilizumab have been proposed as experimental approach considering the increased il-6 levels that have been observed in patients with severe covid-19 [184] . tocilizumab is a recombinant, humanized monoclonal antibody commonly used for treating patients with rheumatoid arthritis, lupus and psoriasis that binds to il-6 receptors blocking fcr activation; in covid-19 patients, tocilizumab could reduce sars-cov-2-induced inflammatory responses [185] . accordingly, several case reports have referred positive outcomes regarding tocilizumab [113, [186] [187] [188] [189] [190] , but clinical impact of tocilizumab on covid-19 patients as an approved clinical approach has not been evaluated yet. in line, to further investigate the efficacy and safety of tocilizumab in patients with covid-19, a controlled clinical trial is now under way (chictr2000029765) ( figure 8v ). overall, the combination of an immunomodulatory agent to reduce the cytokine storm with an antiviral agent may give physicians more time to provide supportive treatment for patients with covid-19. at the time of writing this review, due to the lack of a specific available therapy, plasma from convalescent patients containing specific antibodies has been proposed as a principal treatment [190, 191] , for patients in rapid disease progression, severe or critical conditions ( figure 8vi ). in a recent retrospective study, one dose (200 ml) of convalescent plasma (cp) collected from 10 severe adult cases has been reported to be tolerated; thus, increasing or maintaining high level of neutralizing antibodies broke down the viral load in seven days, improve clinical symptoms and paraclinical criteria within three days and lung lesions were found to be differently absorbed on radiological examination within seven days [192] . therefore, being cp a promising rescue option for severe covid-19, several clinical trials (chictr2000030010, chictr2000030179, and chictr2000030381) are in progress to investigate the efficacy and safeness of cp direct infusion in covid-19 patients [191] . in addition, combined therapy with mabs and remdesivir seems to be an ideal therapeutic option for covid-19 [193] . pharmaceuticals companies are now focused on searching for specific and effective mabs against covid-19. taking into account that technologies capable of making fully human antibodies such as human single-chain antibody variable fragments (hu-scfvs) or humanized-nanobodies (single-domain antibodies, sdab) able to overpass virus-infected cell membranes (trans bodies) and to interact or interpose with biologic processes required for virus replication are already available [194] . a large number of clinical trials regarding cell-based therapies have been started in china during covid-19 outbreak. among these, mesenchymal stromal cells (mscs)-based therapy displayed strong safety profile and possible efficacy in patients with ards, according to covid-19-related clinical studies listed on the who's international clinical trials registry platform (who ictrp) and national institutes of health's clinical trials.gov databases [195] . nevertheless, further investigations are required to better understand if these therapies could be effective in treating respiratory virus-induced complications. mscs have been largely employed in basic research and clinical trials [196] [197] [198] , and their safeness and effectiveness have been extensively documented especially in immune-mediated inflammatory disorders, such as graft-versus-host disease (gvhd) [199] and systemic lupus erythematosus (sle) [200] . mscs immunomodulatory and differentiation abilities [201] as well as their competency to produce several cytokine types or to directly interact with immune cells have been already described [202] . indeed, they are activated by pathogen-associated molecules (pamps) such as single or double-stranded rnas [203, 204] , priming the immune response during infections. two clinical investigations of systemic msc administration in patients with either covid-19 or avian influenza a (h7n9) have been recently published [205, 206] . the first one, a single-center msc transplantation pilot study, was aimed at exploring mscs therapeutic potentiality in patients with covid-19 pneumonia and conducted at the you'an hospital in beijing, china, from 23 jan 2020 to 16 feb 2020 (chictr2000029990). seven patients with covid-19 pneumonia, sars-cov-2 rna positive, with different degrees of severity, including one critically ill requiring icu care were enrolled and monitored for 14 days after msc injection. a significant improvement of pulmonary function and symptoms were observed two days after msc transplantation characterized by an increase of peripheral lymphocytes and of the anti-inflammatory il-10 levels and a decrease of the c-reactive protein and tnf-α amounts [205] . moreover, an increment of the cd14 + cd11c + cd11b mid regulatory dendritic cell (dc) population and a decrease of cytokine-secreting immune cells such as cxcr3 + cd4 + t cells, cxcr3 + cd8 + t cells, and cxcr3 + nk were detected within 3-6 days in the treated patients compared to the placebo control group [205] . mscs play a role in attenuating cytokine storm, most importantly, because these cells do not express ace2 and tmprss2 viral receptors are insusceptible of sars-cov-2 infection. these observations are in agreement with the knowledge that mscs induce the maturation of dendritic cells into a novel jagged-2-dependent regulatory dendritic cell population [207] , shifting the th1/th2 balance towards th2. thus, from these preliminary results, it seems evident that mscs intravenous transplantation could represent a secure and effective treatment in patients with covid-19 pneumonia, especially those critical. indeed, it inhibits the over activation of the immune system and promotes endogenous repair by preventing pulmonary fibrosis and improving both pulmonary microenvironment and lung function [205] . more than 15 potential vaccine candidates for covid-19 are under development around the world, including inactivated, recombinant subunits, nucleic-acid-based, adenoviral vector, and recombinant influenza viral vector vaccines [208] . moreover, taking into consideration the strong homologies existing among the various coronavirus strains, it was thought that vaccines acting on other coronaviruses, such the avian live ibv vaccine (strain h) directed towards the chicken cov ibv, could be a valuable alternative therapeutic strategy [209] . the coalition for epidemic preparedness innovations (cepi) recently announced that three programs aimed to develop covid-19 vaccines, by utilizing established vaccine platforms, have started [210] . in addition, cepi already financed the company moderna, inc. to compare mrna therapeutics and vaccines, allowing the release of the first batch of mrna-1273 in february 2020, which is an mrna vaccine against sars-cov-2 ready for phase i study in the united states. available online: https://investors.modernatx.com/news-releases/news-release-details/moderna-ships-mrnavaccine-against-novel-coronavirus-mrna-1273 (accessed on 24 february 2020). more recently, scientists from the university of pittsburgh have announced a potential vaccine against sars-cov-2, delivered throughout a fingertip-sized patch, capable of producing sars-cov-2 specific igg antibodies, sufficient for virus neutralization in mice. this vaccine, called pittcovacc (acronym of pittsburgh coronavirus vaccine), is a trimeric recombinant sars-cov-2-s1 subunit vaccine delivered intracutaneously by microneedle arrays (mnas) [211] . delivering vaccine components to a defined skin microenvironment improves safety by reducing systemic exposure, allowing to reach high vaccine concentrations with a relatively low dose of antigen [212, 213] . furthermore, the skin delivery strategy promotes strong and long-lasting antigen-specific antibody responses due to both the high immunogenicity [214] [215] [216] [217] [218] and the redundant immunoregulatory circuits of the skin [217, 219, 220] . given the urgent need for covid-19 vaccines, mnas strategy seems to be a promising immunization approach against coronavirus infection including sars, mers and other emerging infectious diseases. on april 24, the oxford chadox1 ncov-19 vaccine was the first in europe to start human trial stage, with 1110 healthy volunteers enrolled for the tests. oxford scientists have already employed chadox1 in the past to dispense vaccines against ebola, chikungunya, rift valley fever and, above all, mers. chadox1, a chimpanzee-derived adenovirus vector, has been employed to deliver the full-length mers spike gene and shown to induce large amounts of neutralizing antibodies against mers in a mouse model [221, 222] . therefore, the modified chadox1 vaccine, carrying the sars-cov-2 spike gene is under human trial stage. on april 30, the university of oxford has announced a collaboration with the uk-based global biopharmaceutical company astrazeneca for further development, large-scale production and potential delivery of the covid-19 vaccine candidate. available online: https://www. ovg.ox.ac.uk/news/landmark-partnership-announced-for-development-of-covid-19-vaccine (accessed on 30 april 2020). since chadox technology is already available and formerly tested in humans for other vaccines, phase iii will consist in administering vaccine to volunteers following them into their regular environments to ensure that these subjects actually become immune to the disease up to three years. if trials succeed, oxford researchers have proposed to complete testing throughout ring vaccination, namely delivering vaccine to members of the first circle of contacts of covid-19 positive people and then to evaluate if the virus spreads to the second circle, as was previously done during the 2018 ebola epidemic in the democratic republic of the congo. to be a promising immunization approach against coronavirus infection including sars, mers and other emerging infectious diseases. on april 24, the oxford chadox1 ncov-19 vaccine was the first in europe to start human trial stage, with 1110 healthy volunteers enrolled for the tests. oxford scientists have already employed chadox1 in the past to dispense vaccines against ebola, chikungunya, rift valley fever and, above all, mers. chadox1, a chimpanzee-derived adenovirus vector, has been employed to deliver the full-length mers spike gene and shown to induce large amounts of neutralizing antibodies against mers in a mouse model [221, 222] . therefore, the modified chadox1 vaccine, carrying the sars-cov-2 spike gene is under human trial stage. on april 30, the university of oxford has announced a collaboration with the uk-based global biopharmaceutical company astrazeneca for further development, large-scale production and potential delivery of the covid-19 vaccine candidate. available online: https://www.ovg.ox.ac.uk/news/landmark-partnership-announced-fordevelopment-of-covid-19-vaccine (accessed on 30 april 2020). since chadox technology is already available and formerly tested in humans for other vaccines, phase iii will consist in administering vaccine to volunteers following them into their regular environments to ensure that these subjects actually become immune to the disease up to three years. if trials succeed, oxford researchers have proposed to complete testing throughout ring vaccination, namely delivering vaccine to members of the first circle of contacts of covid-19 positive people and then to evaluate if the virus spreads to the second circle, as was previously done during the 2018 ebola epidemic in the democratic republic of the congo. figure 8 . schematic representation of sars-cov-2 infection and virus-induced human immune system response. proposed drugs directed both towards specific sars-cov-2 molecular targets and biologic processes are highlighted: inhibitors of sars-cov-2 fusion/entry targeting ace2 receptor, spike protein, tmprss2 or hr1 and hr2 epitopes and clathrin-mediated endocytosis (i); molecules against sars-cov-2 main protease (ii); molecules against viral genome replication (iii); crispr figure 8 . schematic representation of sars-cov-2 infection and virus-induced human immune system response. proposed drugs directed both towards specific sars-cov-2 molecular targets and biologic processes are highlighted: inhibitors of sars-cov-2 fusion/entry targeting ace2 receptor, spike protein, tmprss2 or hr1 and hr2 epitopes and clathrin-mediated endocytosis (i); molecules against sars-cov-2 main protease (ii); molecules against viral genome replication (iii); crispr technologies targeting sars-cov-2 rna genome (iv); modulators of sars-cov-2 induced inflammatory response (v) and human neutralizing antibodies (vi). ace2, angiotensin-converting enzyme 2; tmprss2, type 2 transmembrane serine proteases; rdrp, rna-dependent rna polymerase; hr1, heptad repeat 1; hr2, heptad repeat 2; hr2p, heptad repeat 2-derived peptides; ek1, a modified oc43-hr2p peptide. adapted from [223] . overall, a joint effort headed to apply both already consolidate and innovative approaches, such as ai to facilitate drug discovery, will be required to develop a broad-spectrum antiviral drugs and vaccines towards existing and potential future coronavirus infections to prevent another highly pathogenic virus epidemic. moreover, continuous collaboration in basic and clinical studies will improve the discovery of new antiviral drugs with therapeutic potentials, decrease the time for drug release on the market and make them affordable for all countries. furthermore, vaccine delivery strategies and cell-based therapies benefit from the significant progresses made by recombinant dna technologies combined with emerging biotechnology and bioengineering methodologies. thus, these approaches can speed up the development and set up of new vaccines and clinical therapies to fight against novel pathogens to protect public health all over the world. this study represents a holistic picture of the current investigations in response to the outbreak of covid-19. the current pandemic is obviously an international public health problem and it remains a challenging task to fight the sars-cov-2 of unknown origin and mysterious biologic features. lesson from the previous two pandemics, mers and sars outbreaks, provide valuable insights about how to manage the current pandemic and provide a reference for future studies to combat disease progression. despite sars-cov-2 rapid transmission, the scale up country readiness, speedy response teams and the capacity of all laboratories are reducing the spread of the virus as well as its mortality rate. as the pandemic is still ongoing and expanding, further studies on all aspects of the disease are needed to better understand the infection, beneficial treatments and development of vaccines. nevertheless, this pandemic, together with the previous ones, have taught us in the harshest possible way that the entire scientific community must be vigilant and ready to advice appropriate containment and screening measures to avoid the spread of any future emerging pathogen. we would like to acknowledge majidi nezhad for providing the meteorological data and climate analyses and gaia scoarughi and adeleh salehi for drawing figures. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: 16 nonstructural polyproteins (nsps 1-16); 2019 novel coronavirus (2019-ncov); 3-chymotrypsin-like protease (3clpro); 3c-like protease (3cl); acute respiratory distress syndrome (ards); adeno-associated virus (aav); angiotensin receptor blockers (arbs); angiotensin-converting enzyme 2 (ace2); ap-2-associated protein kinase 1 (aak1); artificial intelligence (ai); avian infectious bronchitis virus (ibv); basic reproduction number (r0); blood oxygen saturation (spo2); bronchoalveolar lavage (bal); centers for disease control and prevention (cdc); chinese center for disease control and prevention (china cdc); chronic obstructive pulmonary disease (copd); coalition for epidemic preparedness innovations (cepi); computed tomography (ct); computer-aided design (cad); convalescent plasma (cp); coronavirus (cov); cytotoxic t lymphocyte (ctl); dendritic cell (dc); dipeptidyl peptidase 4 (dpp4, also known as cd26); envelope glycoprotein (e); enzyme-linked immunosorbent assay (elisa); european center for medium-range weather forecasts (ecmwf); european cities (ecs); feline infectious peritonitis (fip); graft versus-host disease (gvhd); hemagglutinin-esterase glycoprotein (he); heptad repeat 1 (hr1) and heptad repeat 2 (hr2); high-resolution ct (hrct); hr2-derived peptides (hr2p); ek1 (a modified oc43-hr2p peptide); human coronaviruses (hcovs); human covs (hcovs); human single-chain antibody variable fragments (hu-scfvs); humanized-nanobodies (single-domain antibodies, sdab); intensive care unit (icu); istituto superiore di sanità (iss); janus kinase (jak); membrane glycoprotein (m); mesenchymal stromal cells (mscs); microneedle arrays (mnas); middle east respiratory syndrome coronavirus (mers-cov); monoclonal antibodies (mabs); naso-pharyngeal (np); national health commission of the people's republic of china (nhc); nonstructural protein 14 (nsp14); northern hemisphere (nh); nucleocapsid phosphoprotein (n); open reading frames (orfs); oro-pharyngeal (op); 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dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov therapeutic drugs targeting 2019-ncov main protease by high-throughput screening prediction of the sars-cov-2 (2019-ncov) 3c-like protease (3cl pro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates compounds with therapeutic potential against novel respiratory remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro virus against virus: a potential treatment for 2019-ncov (sars-cov-2) and other rna viruses corticosteroid treatment of patients with coronavirus disease 2019 (covid-19) potential benefits of precise corticosteroids therapy for severe 2019-ncov pneumonia clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury a systematic review of anakinra, tocilizumab, sarilumab and siltuximab for coronavirus-related infections understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression tocilizumab treatment in covid-19: a single center experience tocilizumab, an anti-il-6 receptor antibody, to treat covid-19-related respiratory failure: a case report first case of covid-19 in a patient with multiple myeloma successfully treated with tocilizumab convalescent plasma: new evidence for an old therapeutic tool? convalescent plasma as a potential therapy for covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients remdesivir as a possible therapeutic option for the covid-19 human transbodies that interfere with the functions of ebola virus vp35 protein in genome replication and transcription and innate immune antagonism current status of cell-based therapies for respiratory virus infections: applicability to covid-19 autologous mesenchymal stem cells for the treatment of secondary progressive multiple sclerosis: an open-label phase 2a proof-of-concept study mesenchymal stem (stromal) cells for treatment of ards: a phase 1 clinical trial mesenchymal stromal cell secretome for severe covid-19 infections: premises for the therapeutic use survival after mesenchymal stromal cell therapy in steroid-refractory acute graft-versus-host disease: systematic review and meta-analysis therapeutic applications of mesenchymal stem cells for systemic lupus erythematosus mesenchymal stromal cells: clinical challenges and therapeutic opportunities mesenchymal stromal cells: sensors and switchers of inflammation tumour viruses and innate immunity mesenchymal stem cells: a double-edged sword in regulating immune responses transplantation of ace2-mesenchymal stem cells improves the outcome of patients clinical study of mesenchymal stem cell treating acute respiratory distress syndrome induced by epidemic influenza a (h7n9) infection, a hint for covid-19 treatment mesenchymal stem cells induce mature dendritic cells into a novel jagged-2-dependent regulatory dendritic cell population potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review potential interventions for novel coronavirus in china: a systematic review cepi. cepi to fund three programmes to develop vaccines against the novel coronavirus microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid dissolving undercut microneedle arrays for multicomponent cutaneous vaccination microneedle delivery of autoantigen for immunotherapy in type 1 diabetes the immunological anatomy of the skin professional antigen-presenting cells of the skin cd4 + t cell responses elicited by different subsets of human skin migratory dendritic cells cutaneous immune responses mediated by dendritic cells and mast cells microneedle patches for vaccination in developing countries organization of the skin immune system and compartmentalized immune responses in infectious diseases neuronal regulation of cutaneous immunity a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice pharmacologic treatments for coronavirus disease 2019 (covid-19): a review key: cord-312670-hi3fjne4 authors: corman, v. m.; lienau, j.; witzenrath, m. title: coronaviren als ursache respiratorischer infektionen date: 2019-08-27 journal: internist (berl) doi: 10.1007/s00108-019-00671-5 sha: doc_id: 312670 cord_uid: hi3fjne4 background: there are six human pathogenic coronaviruses (cov), which mainly cause infections of the respiratory system. in everyday clinical practice, it is helpful to know the relevance and characteristics of these pathogens. objective: to present the epidemiology, clinical picture and differences of human pathogenic cov and to provide information on the diagnostics and treatment of patients suspected of having cov infections. material and methods: selective literature search, presentation of results and discussion of fundamental works and expert recommendations, including publications by the world health organization (who), the european centre for disease prevention and control (ecdc) and the robert koch institute. results: the four endemic human covs (hcov-nl63, hcov-229e, hcov-oc43 and hcov-hku1) mainly cause mild respiratory tract infections. in addition to these four endemic hcov, the two epidemic cov, severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov can cause severe pneumonia. the sars-cov has not been detected in humans in the last 15 years and mers-cov has been circulating mainly on the arabian peninsula since 2012; however, neither a specific treatment nor approved vaccines exist for any of the six human pathogenic covs. conclusion: all six human covs can be diagnosed using rt-pcr on respiratory specimens but this is rarely necessary for the four endemic strains. in current clinical practice sars-cov has no importance as it has not been detected in humans for 15 years; however, a possible mers-cov infection should be taken into account in patients with typical symptoms and travel history to endemic regions. in this case, rapid diagnostic and general hygiene practices are important to prevent further transmission. es gibt sechs humanpathogene coronaviren (cov), die vornehmlich respiratorische infektionen im menschen auslösen. vier davon kommen weltweit vor und sind sehr häufig. im regelfall rufen diese virustypen nur leichtere respiratorische infektionen hervor. schwere verläufe sind selten und treten am ehesten bei immunsupprimierten auf. zwei weitere typen sind selten, aber für schwere virale pneumonien verantwortlich: das mers-und sars-cov (mers "middle east respiratory syndrome"; sars "severe acute respiratory syndrome" [schweres akutes atemwegssyndrom]). während sars-cov seit 2004 nicht mehr im menschen gefunden wurde, werden infektionen mit mers-cov seit 2012 kontinuierlich detektiert. cov sind behüllte ribonukleinsäure(rna)-viren mit einem plusstrang-rna-genom. sie werden in die unterfamilie orthocoronavirinae (ordnung nidovirales, familie coronaviridae) klassifiziert. die unterfamilie orthocoronavirinae umfasst eine große anzahl an verschiedenen genera, subgenera und virusspezies [12] . darunter sind viele varianten, die in der veterinärmedizin sowohl für haustiere (etwa bezüglich der felinen infektiösen peritonitis bei katzen) als auch für nutztiere (schwere diarrhöen bei rindern und schweinen) von bedeutung sind. bisher wurden sechs verschiedene cov in menschen gefunden, folglich ist nur ein kleiner teil der bekannten cov humanpathogen (. tab. 1). bei symptomatischen infektionen lösen cov im regelfall respiratorische erkrankungen aus. vier der bisher bekannten sechs humanpathogenen cov werden weltweit im menschen gefunden und sind je nach patientenkollektiv für 10-15 % der akuten respiratorischen erkrankungen (are) verantwortlich [10, 15, 24] . zusätzlich zu diesen ständig im menschen zirkulierenden varianten wurden in den vergangenen jahren zwei cov im menschen gefunden, nämlich sars-cov und mers-cov, die aus dem tierreservoir auf den menschen übergegangen sind und bei einem deutlich größeren anteil der infizierten schwere virale pneumonien mit tödlichem verlauf auslösen [25, 28] . mit hcov-oc43 und -229e wurden zwei der vier endemischen humanpathogenen cov (hcov) bereits in den 1960er-jahrenbeschrieben. die zweiweiteren endemischen cov (hcov-nl63 und -hku1) wurden erst deutlich später, in den 2000er-jahren entdeckt [5] . alle vier cov sind etablierte humanpathogene erreger, die weltweit vorkommen und klassischerweise are auslösen können. je nach patientenkollektiv werden hinweise auf eine aktive cov-infektion bei bis zu 20 % der patienten mit akuten ambulant erworbenen respiratorischen erkrankungen gefunden, sowohl bei kindern als auch bei erwachsenen [10, 15, 24] . asymptomatische infektionen wurden ebenfalls beschrieben [23] . obwohl die mehrzahl der infektionen mit den vier endemischen cov nur leichte atemwegserkrankungen verursacht, können alle hcov auch schwere hier steht eine anzeige. zusätzlich zu den vier endemischen hcov haben in den letzten zwei jahrzehnten die beiden epidemischen cov sars-cov und mers-cov beim menschen schwere atemwegserkrankungen ausgelöst. mers-cov ist das zweite hochpathogene cov, das im menschen gefunden wird. es wurde bei menschen erstmalig 2012 im verlauf einer fatalen viralen pneumonie in saudi-arabien entdeckt [11, 14, 28, 30] . seitdem wurden etwa 2500 humane mers-cov-infektionen gemeldet, wovon etwa 30 % tödlich verliefen. über 90 % der infektionen traten in ländern der arabischen halbinsel auf, besonders in saudi-arabien. importierte fälle wurden aber auch in europäischen ländern einschließlich deutschland dokumentiert, insgesamt sind bisher mehr als 25 länder betroffen (. abb. 1b; [28] ). in manchen ländern kam es ausgehend von einem importierten fall auch zu weiteren autochthonen infektionen. besonders zu erwähnen ist hierbei der mers-cov-ausbruchinsüdkorea im jahr2015, bei dem ausgehend von einem einzelnen infizierten reiserückkehrer mehr als 160 weitere nosokomiale infektionen resultierten, unter anderem auch von medizinischem personal [11] . neben mensch-zu-mensch-übertragungen im häuslichen umfeld und im krankenhaus stellen dromedare auf der arabischen halbinsel die wichtigste infektionsquelle dar [11, 14] . mers-cov zirkuliert seit mindestens 2012 kontinuierlich in dieser geografischen region, sodass eine infektion mit mers-cov bei passender anamnese weltweit differenzialdiagnostisch relevant ist [26] . interessanterweise stellen dromedare auch außerhalb der arabischen halbinsel ein reservoir für mers-cov dar. so wurde das virus auch in dromedaren in west-und ostafrika sowie in asien nachgewiesen (. abb. 1b und 2; [2, 13, 31] ). primäre zoonotische infektionen wurden aus diesen regionen allerdings noch nicht bekannt. seit 2015 stehen die beiden pandemischen cov (sars-und mers-cov) auf der von der weltgesundheitsorganisation (who) aufgesetzten liste der "priority diseases". diese liste verzeichnet die acht infektionskrankheiten, bei denen zukünftig eine weltweite epidemie für möglich gehalten wird und für die ein dringender forschungs-und entwicklungsbedarf zur verhinderung einer ausbreitung gesehen wird [17] . pneumonie · akute respiratorische erkrankungen · middle-east-respiratorysyndrome-coronavirus · schweres akutes atemwegssyndrom · erkältung background. there are six human pathogenic coronaviruses (cov), which mainly cause infections of the respiratory system. in everyday clinical practice, it is helpful to know the relevance and characteristics of these pathogens. objective. to present the epidemiology, clinical picture and differences of human pathogenic cov and to provide information on the diagnostics and treatment of patients suspected of having cov infections. material and methods. selective literature search, presentation of results and discussion of fundamental works and expert recommendations, including publications by the world health organization (who), the european centre for disease prevention and control (ecdc) and the robert koch institute. results. the four endemic human covs (hcov-nl63, hcov-229e, hcov-oc43 and hcov-hku1) mainly cause mild respiratory tract infections. in addition to these four endemic hcov, the two epidemic cov, severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov can cause severe pneumonia. the sars-cov has not been detected in humans in the last 15 years and mers-cov has been circulating mainly on the arabian peninsula since 2012; however, neither a specific treatment nor approved vaccines exist for any of the six human pathogenic covs. conclusion. all six human covs can be diagnosed using rt-pcr on respiratory specimens but this is rarely necessary for the four endemic strains. in current clinical practice sars-cov has no importance as it has not been detected in humans for 15 years; however, a possible mers-cov infection should be taken into account in patients with typical symptoms and travel history to endemic regions. in this case, rapid diagnostic and general hygiene practices are important to prevent further transmission. die symptome einer sars-cov-infektion sind vergleichbar mit den symptomen einer mers-cov-infektion. da in den letzten 15 jahren keine menschlichen sars-cov-infektionen mehr bekannt wurden, verzichtet diese kurze übersichtsarbeit auf eine detaillierte betrachtung der sars-cov-infektionen. virale koinfektionen der lunge, unter anderem mit influenza a und b, parainfluenza, rhinovirus und adenovirus, sind sowohl bei der infektion mit endemischen als auch bei infektionen mit mers-und sars-cov beschrieben worden. koinfektionen mit bakterien kommen auch gelegentlich vor. bei einigen patienten mit sars/mers-cov-infektion wurde zusätzlich von sekundär nosokomial erworbenen nichtviralen infektionen (pneumokokken, staphylokokken, klebsiellen, acinetobacter, candida) berichtet. inwiefern diese sekundären infektionen auf die intensivmedizinische behandlung und beatmung zurückzuführen sind oder ein spezifisches grundsätzliches risiko einer cov-infektion darstellen, ist noch nicht verstanden. neben dem regelhaften nachweis von cov im respirationstrakt sind alle endemischen cov auch in stuhlproben nachgewiesen worden; sie sind jedoch typischerweise keine kausale ursache einer gastroenteritis [9, 20] . zudem gibt es hinweise, dass infektionen mit hcov-oc43 eine rolle bei neurologischen erkrankungen spielen könnten [18, 29] . die unterscheidung zwischen cov und anderen erregern, die eine are auslösen, ist anhand der klinischen symptome grundsätzlich nicht möglich. jedoch ist eine spezifische labordiagnostik bei verdacht auf eine infektion mit endemi-schen cov bei harmlosem verlauf und patienten ohne besonderes risiko für die entstehung von komplikationen auch nicht indiziert. bei komplizierten verläufen und schweren infektionen des respirationstrakts ist eine testung auf cov mittels echtzeit-reverse-transkriptase-polymerase-kettenreaktion (rt-pcr) möglich und sinnvoll. geeignetes probenmaterial sind abstriche oder sekrete aus dem oberen, je nach klinik auch aus dem unteren respirationstrakt. eine diagnostik über den nachweis von antikörpern ist aufgrund der häufigen (re-)infektionen nur in ausnahmefällen sinnvoll und begründet. eine spezifische untersuchung auf eine erkrankung durch mers-cov muss beim vorliegen bestimmter kriterien durchgeführt werden. mittel der wahl ist auch für mers-cov der direktnachweis der viralen rna mittels rt-pcr [3, 14, 26, 27] . geeignete materialien sind vor allem sekrete aus den unteren atemwegen (bronchoalveoläre lavage, absaugsekrete, sputum), da dort die höchsten viruskonzentrationen vorliegen [4] . ist die gewinnung und testung dieser materialien nicht möglich, können auch abstriche aus dem oberen respirationstrakt (nasen/rachen) untersucht werden. die antikörperdiagnostik basierend auf der testung von zwei konsekutiven serumproben, beispielsweise im abstand von 4 wochen, ist für den ausschluss einer akuten infektion ungeeignet und nur in bestimmten situationen (umgebungsuntersuchung, postexpositionsuntersuchung) indiziert [26] . aufgrund der interindividuellen heterogenität der antikörperantwort bei mers-cov-infektionen ist die quantifizierung spezifischer antikörpertiter zum nachweis oder ausschluss einer mers-cov-in-fektion ungeeignet. in . abb. 3 ist dargestellt, wann und wie in einem mers-cov-verdachtsfall vorgegangen werden sollte. bei der typischerweise unspezifischen klinik von mers-cov-infektionen sollte auch die möglichkeit einer infektion mit anderen pathogenen in betracht gezogen werden [26] . bei patienten, für die laut mers-cov-falldefinition eine diagnostik indiziert war und von denen proben zum ausschluss einer mers-cov-infektion an das konsiliarlabor für cov gesendet wurden, werden regelmäßig andere erreger als mers-cov als ursächlich für die respiratorische erkrankung identifiziert. häufig handelt es sich dabei um (eigene unpublizierte daten) 4 » mers-cov ist beim menschen hauptsächlich in den tiefen atemwegen zu finden mers-cov kann auch von einem menschen auf einen anderen übertragen werden. allerdings wurde eine kontinuierliche übertragung in der bevölkerung bislang nicht beobachtet; auch im normalen häuslichen umgang finden nur wenige übertragungen statt [6] . diese beobachtung steht im gegensatz zu den beobachteten gravierenden krankenhausassoziierten ausbrüchen auf der arabischen halbinsel und in südkorea. der scheinbare widerspruch lässt sich dadurch erklären, dass mers-cov beim menschen im gegensatz zum auftreten bei kamelen hauptsächlich in den tiefen atemwegen zu finden ist [4] . im rahmen der behandlung eines infizierten patienten, beispiels-weise mit bronchoskopie, beatmung und absaugung, kann es zum kontakt mit sekreten aus den tiefen atemwegen kommen, insbesondere wenn infektionspräventionsmaßnahmen unzureichend umgesetzt werden. treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-beta1b (miracle trial): study protocol for a randomized controlled trial mers coronaviruses from camels in africa exhibit regiondependent genetic diversity assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection hosts andsourcesofendemichumancoronaviruses transmission of mers-coronavirus in household contacts european centre for disease prevention and control. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) -tenth update incidence, significance, and persistence of human coronavirus infection in hematopoietic stem cell transplant recipients human coronavirusesareuncommoninpatientswithgastrointestinal illness human coronavirus infections in israel: epidemiology, clinical symptoms and summer seasonality of hcov-hku1 middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission international committee on taxonomy of viruses (2019) ictv virus taxonomy detection of distinct mers-coronavirus strains in dromedary camels from kenya mers coronavirus: diagnostics, epidemiology and transmission cocirculation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in 2004 fatal outcome of human coronavirus nl63 infection despite successful viral elimination by ifn-alpha in a patient with newly diagnosed all the who r&d blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts human coronavirus oc43 associated with fatal encephalitis ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study commonly circulating human coronaviruses do not have a significant role in the etiology of gastrointestinalinfectionsinhospitalizedchildren empfehlungen des rki für das management von kontaktpersonen laborbestätigter symptomatischer mers-fälle rki (2015) schwere respiratorische erkrankungen in verbindung mit middle east respiratory syndrome coronavirus (mers-cov). falldefi-nition zur fallfindung, meldung und übermittlung asymptomatic summertime shedding of respiratory viruses frequentdetectionofhumancoronaviruses inclinicalspecimensfrompatientswithrespiratory tract infection by use of a novel real-time reversetranscriptase polymerase chain reaction severe acute respiratory syndrome (sars) -disease outbreak news who (2019) clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance detection of coronavirus in the central nervous system of achildwithacutedisseminatedencephalomyelitis isolation of a novel coronavirus from a man with pneumonia in saudi arabia countrywide survey for mers-coronavirus antibodies in dromedaries and humans in pakistan weitere infos zu e.med finden sie auf springermedizin.de unter "abos" key: cord-314451-mqnqjn0c authors: roberts, anjeanette; deming, damon; paddock, christopher d; cheng, aaron; yount, boyd; vogel, leatrice; herman, brian d; sheahan, tim; heise, mark; genrich, gillian l; zaki, sherif r; baric, ralph; subbarao, kanta title: a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice date: 2007-01-12 journal: plos pathog doi: 10.1371/journal.ppat.0030005 sha: doc_id: 314451 cord_uid: mqnqjn0c no single animal model for severe acute respiratory syndrome (sars) reproduces all aspects of the human disease. young inbred mice support sars-coronavirus (sars-cov) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. they are relatively inexpensive and easily accessible, but their use in sars research is limited because they do not develop illness following infection. older (12to 14-mo-old) balb/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. we adapted the sars-cov (urbani strain) by serial passage in the respiratory tract of young balb/c mice. fifteen passages resulted in a virus (ma15) that is lethal for mice following intranasal inoculation. lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. these observations suggest that mice infected with ma15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. the ma15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant sars-cov, these mutations result in a highly virulent and lethal virus (rma15), duplicating the phenotype of the biologically derived ma15 virus. intranasal inoculation with ma15 reproduces many aspects of disease seen in severe human cases of sars. the availability of the ma15 virus will enhance the use of the mouse model for sars because infection with ma15 causes morbidity, mortality, and pulmonary pathology. this virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals. the occurrence in late 2002 and early 2003 of cases of severe acute respiratory syndrome (sars) in southeast china quickly drew international attention as the disease sickened more than 8,000 people and spread to more than 30 countries within six months. since the identification of the etiological agent, the sars-coronavirus (sars-cov), in 2003, development and characterization of animal models for evaluation of prophylaxis and treatment strategies have been of great interest. although sars-cov has not been associated with a subsequent widespread outbreak since 2002-2003, the potential for such an outbreak remains. identification of a sars-like coronavirus in chinese horseshoe bats (rhinolophus species) that are indigenous across southeast asia suggests that they may represent a natural reservoir from which viruses may be introduced into the human population [1] . the course of infection in animal models is abbreviated compared with the course of sars in humans; however, many aspects of sars-cov-associated disease are reproducible in animal models, including age-dependent susceptibility, re-covery of sars-cov from respiratory tissues and secretions, infection of type i and type ii pneumocytes and bronchial epithelial cells, detection of viral genome in blood and extrapulmonary tissues, and pulmonary pathology (including pneumonitis, edema, necrotic debris, and hyaline membrane formation) [2, 3] . clinical symptoms have been reported in some species, but the findings are not entirely reproducible in outbred species. variability in clinical symptoms seen in outbred species may result from additional factors, including infection with co-pathogens, stress, existence of sub-species of test animals, and use of different virus strains. this variation can be problematic in studies of pathogenesis and vaccine efficacy unless a large enough number of animals are included in each treatment group [2] . the ideal animal model would demonstrate viral replication in respiratory tissues, histopathologic evidence of respiratory disease, and consistent clinical signs of disease, including mortality. a small animal model in which all of these aspects of virus-associated disease are seen would be desirable because reproducible data can be generated in inbred animals, and larger numbers of animals can be included for statistical analysis of biological outcomes. to generate a model that satisfies these criteria, we have serially passaged sars-cov in the respiratory tract of young balb/c mice, resulting in a lethal virus that causes dosedependent weight loss and mortality associated with higher viral titers in the respiratory tract than are seen with the wildtype virus and with histopathologic findings of severe pulmonary disease. the characteristics of this lethal mouseadapted sars-cov, (ma15), are reported here. adaptation of sars-cov (urbani) was achieved by serial passage through lungs of balb/c mice as previously described for influenza a and influenza b viruses [4, 5] . lightly anesthetized mice were inoculated intranasally with 10 5 50% tissue culture infectious dose (tcid 50 ) per mouse of sars-cov (urbani). two to three days post-infection (d.p.i.), when peak viral titers are observed, lungs were harvested from infected mice and clarified homogenates were used as inocula for continued serial passage via intranasal inoculation in mice. to screen for virulence in mice, groups of young naïve mice (n ¼ 5-8) were inoculated with lung homogenates collected at passage 2, 10, and 15 (p2, p10, and p15, respectively), weighed daily, and observed twice daily for signs of morbidity or mortality. deaths were not observed following inoculation with p2 or p10; however, increased morbidity, as indicated by weight loss, was noted in p10inoculated mice at day 3 post-infection (p.i.) (unpublished data). mortality was observed in p15-inoculated mice; 60% of p15-inoculated mice died or displayed extreme morbidity and were euthanized by 5 d.p.i. mortality was not observed in mice infected with intermediate passages (p11-p14), and accompanying morbidity was not measured (unpublished data). supernatant from lung homogenates at p15 contained a heterogeneous virus pool as evident upon sequence analysis of cdna fragments generated by reverse transcriptase pcr (rt-pcr) amplification from purified viral rna (vrna). dual peaks were observed on sequence chromatograms at several nucleotide residues, indicating mixed populations in the virus pool. to obtain a clonal population, p15 virus was biologically cloned by three rounds of terminal dilution in vero cells. five clones were screened for lethality in 6-to 8wk-old balb/c mice; these clones caused mortality from 50% to 100%. one clone, designated ma15, resulted in 100% mortality within 6 d. balb/c mice aged 6 to 8 wk, 4 mo, and 13 mo were all susceptible to ma15 infection, with severe morbidity or death occurring within 3 to 5 d following intranasal inoculation. in order to identify the mutations in ma15 associated with the lethal phenotype, the sequence of ma15 was compared with that of sars-cov (urbani). the sequence of the initial sars-cov (p0) virus was identical to the published sars-cov (urbani) sequence, and six nucleotide substitutions were identified in the ma15 genome compared with that of sars-cov (urbani). all six substitutions were predicted to cause amino acid substitutions. these six mutations were localized to open reading frame (orf) 1ab (four mutations) and orfs s and m (one each) of sars-cov ( figure 1 ). independent analysis conducted by the institute for genomic research (rockville, maryland, united states), under a national institute of allergy and infectious diseases (niaid) contract, confirmed the same six mutations in the ma15 sequence compared with those in the sars-cov (urbani) sequence. to confirm that the six mutations identified in the ma15 virus were responsible for lethality in mice, the mutations were introduced into cdna clones from which recombinant sars-covs were recovered. sequence analysis confirmed that recombinant viruses contained the appropriate mutation sets that were derived from the ma15 virus. in addition to generating a recombinant virus that included the six mutations (rma15), two additional recombinants were generated containing either the two mutations in the structural protein genes or the four mutations in the orf 1ab (rma15 sm and rma15 orf1ab , respectively). the three recombinant viruses demonstrated similar kinetics and levels of viral replication compared with that severe acute respiratory syndrome (sars) is a severe, sometimes fatal respiratory disease caused by a coronavirus (sars-cov). in order to study the disease and evaluate vaccines and antiviral drugs, animal models that mimic the disease are necessary. however, no single animal model for sars reproduces all aspects of the disease as it affects humans. sars-cov replicates in the lungs of young mice, but they do not show signs of illness. adaptation of sars-cov by serial passage in the lungs of mice resulted in a virus (ma15) that is lethal for young mice following intranasal inoculation. lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by hematological changes and pathological changes in the lungs. mice infected with ma15 virus die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes, and ciliated epithelial cells. the ma15 virus has six coding mutations in its genome, which, when introduced into a recombinant sars-cov, confer lethality. the ma15 virus will enhance the use of the mouse model for sars because infection with this virus in mice reproduces many aspects of severe human disease, including morbidity, mortality, and pulmonary pathology. of sars-cov infectious clone (icsars-cov), a wild-type recombinant sars-cov (urbani) generated from cdnas, the biologically derived ma15 virus, and sars-cov (urbani) in in vitro single-cycle growth analyses in vero e6 cells. at a multiplicity of infection (moi) of 0.1, viruses reached peak replication (10 7.0-7.5 pfu/ml) at ;24 h.p.i., with a slight delay in peak titers for rma15 orf1ab ( figure s1 ). northern blot analysis of rna from infected vero e6 cells indicated that genomic vrna and viral mrna and all eight sub-genomic mrnas were present in similar ratios for the recombinant viruses and ma15 virus as for sars-cov (urbani) ( figure 2a ). the level of expression and mobility of the structural proteins (s and n) and an accessory protein (orf 3a, also called x1) of the recombinant viruses and ma15 virus were comparable to those of sars-cov (urbani), as determined by western blot analysis ( figure 2b ). although s, n, and x1 are somewhat reduced in the lane ( figure 2b ) from icsars-covinfected cultures, this is associated with reduced amounts of total protein added to the well, rather than with any specific reduction in x1 expression. thus, the recombinant viruses (rma15 sm , rma15 orf1ab , rma15, and icsars-cov) and the ma15 virus demonstrate no defects in replication or in rna or protein expression compared with sars-cov (urbani) in vero e6 cells. groups of naïve mice were inoculated with serial 10-fold dilutions of ma15 virus, and the dose-dependent weight loss and lethality observed following infection is summarized in table 1 . mice receiving a dose !10 3.9 tcid 50 of ma15 virus died or lost more than 20% of their initial body weight between days 3 and 5 p.i. mice experiencing weight loss in excess of 20% initial body weight are euthanized in accordance with our animal study protocol. the 50% lethal dose (ld 50 ) was 10 4.6 tcid 50 . at an intermediate, non-lethal dose (10 2.9 /mouse), mice lost 8.4% 6 2.5% of initial body weight by day 4 p.i. at a lower dose, weight loss was not significant (4.3% 6 1.0% by day 4 p.i.) similarly, groups of naïve mice were inoculated with serial 10-fold dilutions of rma15, rma15 orf1ab , or rma15 sm , and followed daily for signs of morbidity (indicated by weight loss) and mortality. mortality was observed only in rma15inoculated mice at doses equal to or in excess of 10 4.4 tcid 50 /mouse ( table 1 ). the ld 50 for rma15 of 10 3.9 tcid 50 is similar to the ld 50 observed for the ma15 virus. consistent with observations following infection with ma15 virus, mice inoculated with low concentrations of rma15 (10 0.4-2.4 tcid 50 /mouse) had little to no significant weight loss, but an intermediate dose of rma15 (10 3.4 tcid 50 /mouse) resulted in significant weight loss but no mortality. thus rma15 recapitulated the phenotype of the ma15 virus in mice. inoculation with the highest doses of rma15 sm and rma15 orf1ab (10 6.2 tcid 50 /mouse) did not result in mortality (table 2) , and only rma15 orf1ab -infected mice demonstrated significant morbidity as indicated by weight loss. the rapid lethality observed following administration of ma15 virus (or its recombinant clone rma15) to balb/c mice could result from changes in tissue tropism with or without viremia, increased viral load and subsequent necrosis in pulmonary or extrapulmonary tissues, failure of innate or early adaptive immune responses, immunopathology in pulmonary or extrapulmonary tissues, or a combination of these or other factors. in order to evaluate whether changes in tissue tropism or levels of viral replication could contribute to the lethal phenotype of the ma15 virus, viral titers in lungs, spleen, liver, and brain of balb/c mice were determined at various time points following intranasal inoculation with sars-cov (urbani) or ma15. mice were inoculated intranasally with sars-cov (urbani) (10 5 tcid 50 /mouse, a dose that results in peak viral titers in lungs of mice 2 d.p.i.) or with lethal (10 5.6 tcid 50 /mouse) or sub-lethal (10 3.6 tcid 50 /mouse) doses of ma15 virus. at various time points p.i., mice were sacrificed and tissues harvested for subsequent processing. data from replicate experiments were combined. high titers of virus were detected in the lungs of mice through day 5 p.i. (figure 3 ). one day after inoculation, the amount of sars-cov (urbani) in lungs was ;10 6 tcid 50 /g tissue. following administration of a lethal dose, ma15 virus was found to replicate to significantly higher titers of ;10 9 tcid 50 /g tissue within 24 h (p ¼ 0.0001; figure 3 ). in this study and in previous studies (k. subbarao, a. roberts, l. vogel, e. lamirande, et al., unpublished data), peak virus titers (;10 7.0 tcid 50 /g tissue) occur at day 2 following inoculation with sars-cov (urbani). in comparison, by day 2 p.i., viral titers in ma15-inoculated mice reached, or persisted at, titers of ;10 9 tcid 50 /g tissue, regardless of inoculating dose ( figure 3 ). furthermore, in mice inoculated with a sublethal dose of ma15, virus was detected at 10 7.7 tcid 50 /g lung on day 5 p.i. by comparison, in sars-cov (urbani)-infected mice, virus was detected at titers of 10 5.1 tcid 50 /g lung on day 5 p.i. by 6 d.p.i., virus was no longer consistently detected in mice inoculated with sars-cov (urbani) or with sub-lethal doses of ma15 virus ( figure 3 ). following inoculation with a lethal dose of ma15 virus, virus was also recovered from spleen, brain, and liver from day 1 through day 4 p.i. at titers ranging from 10 1.8 to 10 2.7 tcid 50 /g (brain) and from 10 1.8 to 10 3.3 tcid 50 /g (spleen) ( table 3) . virus was detected more frequently in the liver and at higher titers (10 2.1 -10 4.3 tcid 50 /g) than in brain or spleen. virus was not recovered from any of these organs following inoculation with sars-cov (urbani), except in one mouse, sacrificed 5 d.p.i., in which virus was detected in the spleen at a titer just above the limit of detection (table 3 ). although the levels of viral replication detected in these tissues in ma15-infected mice are modest, the number of mice in which virus was detected is remarkable, and both the frequency and the titers are significantly higher than those observed in sars-cov (urbani)-infected mice. in a separate experiment, infectious virus was also detected in sera of mice infected with ma15 virus or recombinant sars-covs on days 2 and 4 p.i. data from this experiment suggest that the presence of virus in serum may be facilitated by the mutations in the s and m genes, since virus was detected more frequently and at higher titers in sera from mice infected with ma15, rma15, or rma15 sm than in sera from mice infected with icsars-cov or ma15 orf1ab ( figure s2 ). in addition to recovery of infectious virus from these tissues, total rna was also isolated from whole blood, brain, kidney, liver, intestine, spleen, thymus, heart, and lungs of balb/c mice after infection. although quantitative virology holds biological relevance since it indicates viral burden by measuring infectious virus, rt-pcr of vrna or sub-genomic mrnas may be more sensitive assays for the presence of virus. due to the replication strategy of sars-cov, primers that specifically amplify sub-genomic mrnas allow detection of products of viral transcription. furthermore, amplification of virus-specific mrnas allows distinction from vrna that may represent non-viable virus [6] . rt-pcr amplification of genomic, antigenomic, and sub-genomic mrna-specific sequences was employed to confirm the presence of sars-cov nucleic acid or viral transcription products in different tissues. groups of four mice were inoculated with a lethal dose of ma15 virus or with sars-cov (urbani) (10 5 tcid 50 /mouse). mice inoculated with ma15 virus did not survive past day 4 p.i. sars-cov (urbani)-infected mice were followed through day 14 p.i. vrna, and viral mrna were amplified from lungs of ma15-infected mice on days 1-4 p.i. in contrast, vrna was amplified from sars-cov (urbani)-infected mice through day 14 p.i., but mrna, indicating viral transcription, was consistently amplified only through day 5 p.i. mrna was detected as late as day 7 p.i. by rt-pcr in one of four of the sars-cov (urbani)-infected mice (tables 4 and 5) . we were also able to amplify vrna and mrna from blood, brain, thymus, heart, and spleen of ma15-infected mice (tables 4 and 5 ). viral mrna was consistently detected by rt-pcr in the tissues of ma15-inoculated mice from day 2 p.i. through day 4 p.i. and in the thymus and heart of ma15infected mice as early as day 1 p.i. in contrast, vrna and viral mrna were detected transiently in very few sars-cov (urbani)-infected mice and only in the blood, spleen, and thymus. vrna and viral mrna were not detected by rt-pcr analysis in liver, intestines, or kidneys from ma15 or sars-cov (urbani)-infected mice. these tissues may contain factors that inhibit rt-pcr amplification of sars-cov rnas, since virus was isolated from liver homogenates of ma15-infected mice but was not detected by rt-pcr, and viral mrna was detected in intestines by in situ hybridization (unpublished data). in summary, virus or viral-specific rna was detected in blood, lung, thymus, brain, spleen, liver, and heart from almost all ma15-infected mice. in contrast, virus or viral-specific rna was detected in the lungs, and only sporadically in blood, spleen, and thymus from sars-cov (urbani)-infected mice (tables 3-5) . the lungs of mice infected with sars-cov (urbani) showed a rapid progression from focal, mild, perivascular, and peribronchiolar mononuclear inflammatory cell infiltrates to a diffuse, but transient, interstitial pneumonitis on day 3 p.i. ( figure 4a , 4c, 4e, and 4g). occasional ciliated columnar epithelial cells of the bronchioles and alveolar pneumocytes stained for viral antigen by immunohistochemistry (ihc) ( figure 5a , 5c, 5e, and 5g). no viral antigens were detected at day 14 p.i., and most mice at this time point showed no significant pulmonary histopathology (unpublished data). in comparison, the pulmonary pathology of mice infected with ma15 virus showed a rapid progression of inflammatory changes ( figure 4b , 4d, 4f, and 4h), but with more extensive damage to bronchiolar and alveolar epithelial cells ( figure 6a and 6b). these changes were especially evident in pneumocytes, and many detached, pyknotic, and necrotic cells were identified in alveolar spaces ( figure 6a ). inflammatory infiltrates, although consistently identified, were generally similar in intensity to those observed in the lungs of sars-cov (urbani)-infected mice ( figure 4) . however, the distribution and amount of viral antigens identified by ihc was far greater in the lungs of ma15-infected mice than in the lungs of mice infected with sars-cov (urbani) ( figure 5 ). intracellular sars-cov antigens were extensively and abundantly distributed in bronchiolar epithelium, alveolar pneumocytes, and in necrotic debris within the alveoli and bronchiole lumens of ma15-infected mice ( figure 6c and 6d) . no significant histopathologic changes were identified in extrapulmonary organs, including the liver, spleen, thymus, or brain, of mice infected with sars-cov (urbani) or ma15 virus. furthermore, sars-cov antigens were not detected in any of these tissues by ihc staining. infection of young balb/c mice with 10 6.4 tcid 50 /mouse of ma15 virus resulted in elevated levels of the liver enzyme alkaline phosphatase (alp) in sera collected on days 1-6 p.i. alp levels were significantly higher in sera of ma15inoculated mice than they were in pre-infection sera from the same mice. serum alp levels in ma15-inoculated mice were also significantly higher than those of mice inoculated with 10 6.4 tcid 50 /mouse of sars-cov (p ¼ 0.0045; table s5 ). levels of other liver enzymes, including aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, creatine kinase, urea nitrogen, total bilirubin, and albumin, were not significantly altered following inoculation with ma15 virus (unpublished data). although moderately elevated levels of creatinine were observed in ma15inoculated mice, no significant differences were seen between ma15-and sars-cov-inoculated mice, suggesting that this was not associated with the lethal phenotype of the ma15 virus. furthermore, significant lymphopenia and neutrophilia were observed following infection with sars-cov and ma15 virus. although some samples were lost due to coagulation of blood, the alterations in the lymphocyte and neutrophil counts were more severe following infection with ma15 virus than they were following sars-cov infection (table s5) . in order to determine whether ma15 virus can be used as a more stringent challenge in evaluating vaccines and antiviral therapy designed for sars-cov than could the non-lethal sars-cov (urbani), we inoculated eight mice with 50 ll of sars-cov (urbani) at 10 5 tcid 50 /mouse and mock-immunized an additional eight mice. four weeks after inoculation, the mice were bled for determination of sars-cov-specific serum neutralizing antibodies and challenged with 10 6.9 tcid 50 of ma15 virus. mice were followed daily for signs of (figure 7 ). serial passage of sars-cov (urbani) in the lungs of balb/ c mice resulted in a mouse-adapted sars-cov (ma15 virus) that is lethal for young (6-to 8-wk-old) balb/c mice. the virulence and lethality of the ma15 virus result from six mutations in the sars-cov genome that occurred within 15 passages through balb/c mice. introduction of these six mutations into a recombinant sars-cov infectious clone, rma15, conferred a lethal phenotype on the virus for young balb/c mice. five of these six mutations (not including the t67a mutation occurring in the non-structural protein [nsp] 9 of orf 1ab) occur within nsp 5 (two mutations), nsp 13, s, and m. these genes have been reported as ones where sequence evolution occurred during adaptation of sars-cov in humans [7] . the mutations in nsp 9, nsp 5 (h133y and e269a within the main protease, 3cl pro ), and nsp 13 (a4v within the helicase protein) do not occur within any known functional domains and do not alter known cleavage sites utilized in the processing of the orf 1ab polyprotein. furthermore, the mutations in the ma15 virus do not occur at any specific amino acid positions identified by the chinese sars molecular epidemiology consortium [7] . only the mutation in s (y436h) occurs within a known functional domain, the receptor-binding motif (rbm). other reports have indicated that mutations within the rbm may account for increased affinity of the virus for its cellular receptor angiotensin converting enzyme 2 [8] . however, the y436h mutation in ma15 virus does not occur at previously identified sites of the rbm and angiotensin converting enzyme 2 interaction, and preliminary findings suggest that the y436h mutation does not increase binding of the sars-cov (tor2) rbd to murine angiotensin-converting enzyme 2 [9] . reports of adaptation of an influenza a virus for increased virulence in mice indicate that multiple gene products may interact or contribute independently to virulence. in one adaptation of influenza a/fm/1/47 (fm-ma), findings indicated that four viral gene segments contributed to increased virulence [4] , but additional analyses indicated that mutations occurring in at least two and likely three gene products acted synergistically to account for the increased virulence [10] [11] [12] . recombinant icsars-cov produced mild pneumonia identified by x-ray changes in macaques, similar to the clinical disease noted with wild-type sars-cov (urbani) [13] . consistent with these findings, recombinant sars-cov bearing the six novel mouse-adapted mutations, rma15, recapitulated a fatal respiratory disease phenotype in mice, demonstrating the ability of the sars molecular clones to capture complex disease phenotypes. we generated two other recombinant sars-covs, rma15 sm (encoding the two mutations in the s and m genes) and rma15 orf1ab (encoding the four mutations in orf 1ab). neither of these was lethal ( table 2) in balb/c mice, indicating that sars-cov adaptation for balb/c mice involves mutations in at least two and possibly three genes (s þ orf 1ab, m þ orf 1ab, or s þ m þ orf 1ab). although recombinant viruses bearing two or four of the mutations were not lethal, each had a different phenotype than that of sars-cov (urbani). it is possible that the mutations in s and m contribute to increased viremia ( figure s2 ) and that the mutations in orf 1ab contribute to increased pathogenicity indicated by weight loss (table 2) . both quantitative virology and ihc analysis of the lungs of ma15-infected mice indicate extraordinarily high levels of viral replication in pulmonary tissues as early as 24 h.p.i., and the level of replication remains high through day 4 p.i. unlike sars-cov-infected mice, in which viral antigen is detected by ihc staining at modest levels in bronchial epithelium and in alveolar pneumocytes on days 1 and 2 p.i., the bronchial epithelium of ma15-infected mice is replete with viral antigen at day 1 p.i., as are alveolar pneumocytes on days 1 and 2 p.i. by day 3 p.i., viral antigen is rarely detected in the in this model, day 3 to day 4 p.i. seems to be a critical time for the outcome of sars-cov infection. sars-cov (urbani)-infected mice demonstrate a pronounced but transient interstitial inflammation at day 3 p.i. that is absent in ma15infected mice. this transient inflammation is not associated with significant weight loss but coincides with the beginning of viral clearance from the lungs. in contrast, by day 3 p.i., ma15 virus-infected mice lose weight, and necrotic cellular debris begins to fill the bronchioles and alveoli. by day 4 p.i., ma15-infected mice have lost in excess of 20% of their initial body weight, and several die without other overt clinical signs such as ataxia, paralysis, hunched posture, etc. viral titers in lungs of ma15-infected mice are up to 1,000-fold higher than those in sars-cov-infected mice by 24 h.p.i. and remain higher than peak levels in sars-cov-infected mice through day 4 p.i. in situ hybridization further confirms the intense and persistent signal of ma15 (and rma15) vrna in lung tissues at day 4 p.i. when vrna from sars-cov (urbani) and the non-lethal rma15 sm and rma15 orf1ab viruses are less abundant ( figure s3 ). objective data related to respiratory distress such as plethysmography and measurement of blood gases could not be collected because of practical and logistical constraints on experiments carried out in an animal biosafety level 3 laboratory. however, our findings indicate that ma15 virus-infected mice die as a result of overwhelming pulmonary viral infection and destruction of bronchiolar epithelial cells and alveolar pneumocytes. viral load in sars cases was an important determinant of severe disease and death [14] , but the mechanism of disease leading to fatal outcome in human cases of sars may be different than that observed in ma15-infected balb/c mice. the mechanism of death following sars-cov infection in humans, and particularly the relative contribution of virusinduced damage and immunopathology, are not fully underfour weeks after immunization, mice were challenged intranasally with 50 ll ma15 virus (10 6.9 tcid 50 /mouse), weighed daily, and observed twice daily for morbidity and mortality. surviving mice that lost in excess of 20% initial body weight were euthanized. symbols represent mean values for sars-cov-immunized mice (triangles) and mock-immunized mice (circles). error bars indicate standard error. doi:10.1371/journal.ppat.0030005.g007 stood. the histopathology documented in the mice infected with the ma15 virus includes a rapid progression and extensive damage to bronchiolar and alveolar epithelial cells ( figure 6a and 6b ), but it does not show some features such as alveolar edema and hyaline membranes that were reported in many sars-cov-infected patients, or in aged mice infected with the urbani strain of sars-cov [15] . this can be explained by the relative virulence of the ma15 strain, and the time p.i. when the lungs were evaluated. mice infected with the ma15 virus developed severe morbidity and died within 3 to 5 d following infection and did not survive long enough to show progression of the diffuse alveolar damage seen in aged mice or in human patients, who survive for a relatively prolonged length of time before succumbing to complications of the acute infection. in aged mice following sars-cov infection, histopathologic changes indicative of progressive diffuse alveolar damage, including proteinaceous deposits around alveolar walls and intraalveolar edema, are seen beginning on day 5 [15] . the rapid progression of infection and the extensive and persistent pulmonary replication of the virus are accompanied by viremia and detection of virus in extrapulmonary sites, suggesting that other factors may contribute to the increased pathogenicity of the ma15 virus. a prolonged viremic state or a secondary viremia is seen in mice infected with ma15 and rma15 that is not observed following infection with the recombinant sars-covs (urbani), rma15 sm , or rma15 orf1ab . the ma15 virus model captures viremia and multi-organ involvement noted in human sars patients [16] . however, as in all sars cases, the primary site of infection is the lung. perfusion of tissues harvested from mice in these experiments was not performed, and therefore, we cannot be certain that the detection of vrna in various extrapulmonary sites in ma15-inoculated mice is not due to virus carried to and remaining in the organs and blood. however, on days 2 through 4 p.i., ma15-specific mrnas in various tissues are detected at a higher frequency and signal intensity than in whole blood, and the presence of ma15 vrna in extrapulmonary tissues is detected by in situ hybridization. furthermore, infectious virus was detected on day 1 p.i. in homogenates of extrapulmonary tissues when rt-pcr of rna from blood did not amplify any viral specific products (tables 3-5) . taken together, these observations support a hypothesis that virus is present and replicating at low levels in extrapulmonary tissues. in balb/c mice infected with ma15 virus or sars-cov, significant lymphopenia and neutrophilia were observed compared with pre-infection levels. ma15 virus-infected mice had significantly greater lymphopenia and neutrophilia than sars-cov-infected mice, reflecting hematological evidence of increased disease severity following ma15 virus infection. ma15 virus-infected mice also had significantly higher levels of alp than uninfected or sars-cov-infected mice. elevated levels of alp, an enzyme found in all tissues, but especially concentrated in the liver, may indicate cell destruction in the liver, intestines, or other tissues. reports of elevated alp levels in sars patients are rare [17, 18] , but neutrophilia and lymphopenia have been reported frequently in human cases of sars [19] [20] [21] [22] [23] [24] [25] . although steroid treatment and secondary bacterial infections may be speculated to be the cause of neutrophilia, some patients with sars had neutrophilia prior to initiation of steroid treatment and in the absence of bacterial infections [26] . additionally, elevated absolute neutrophil counts at presentation were independent indicators of severe outcome of sars in human cases [21, 27, 28] . the definitive causes of lymphopenia, neutrophilia, and elevated alp levels have not been identified in human cases of sars or in this mouse model. the mouse-adapted sars-cov ma15 virus will be a valuable tool in evaluating sars-cov vaccines and antiviral therapy. quantitative virology was the only outcome measure available in young balb/c mice challenged with sars-cov (urbani). because the ma15 virus replicates rapidly to high titer and is lethal for young balb/c mice, this virus provides a more stringent challenge than the sars-cov (urbani) virus in evaluating the efficacy of therapeutic interventions or prevention strategies. prophylaxis that is able to rescue ma15-infected mice from a lethal outcome must lower viral burden in lungs very rapidly (e.g., within the first 24 h). prophylaxis that accomplishes such reduction in the burden of ma15 virus may also reduce the severity of immunopathology that follows. a reduction of immunopathology was demonstrated in a sars-cov hamster model when a monoclonal antibody specific to sars-cov spike protein administered the day after infection was able to arrest a further rise in viral titer in the lungs [29] . if similar protection can be demonstrated against challenge with ma15 virus in balb/c mice, the efficacy of intervention will be well proven. infection of young balb/c mice with the ma15 virus provides a model that is small and accessible and that can be evaluated extensively at an immunological level. finally, and most importantly, ma15 virus infection of young balb/c mice provides many elements that replicate observations in acute (and chronic) cases of sars infection in humans, including sequence changes during adaptation in several genes (nsp 5, nsp 13, s, and m); viral replication and histopathological changes in lungs of infected animals; viremia; detection of vrna in extrapulmonary sites, including the intestines; clinical indicators of illness, including mortality; and changes in blood counts, including lymphopenia and neutrophilia. the availability of a molecular clone of the ma15 virus, and the ability to generate additional sars-cov recombinant viruses that recapitulate the in vivo phenotype, will allow for detailed probing of the mechanisms mediating sars-cov pathogenesis and acute lung damage. all animal and viral experiments were conducted in biosafety level 3 laboratories or animal facilities, and all personnel wore personal protective equipment, including tyvek suits and hoods and positive pressure hepa-filtered air respirators. all animal protocols employed in these studies have been approved by niaid's animal care and use committee. serial passage of sars-cov in mice. a dose of 10 5 50% tcid 50 of sars-cov (urbani) was administered intranasally to a lightly anesthetized, 6-wk-old female balb/c mouse in a total volume of 50 ll [30] . two days after inoculation, the mouse was euthanized, and its lungs were removed and homogenized with an ex-gen omni glh homogenizer (omni international, http://www.omni-inc.com) as a 10% w/v suspension in leibovitz's l15 medium (invitrogen, http:// www.invitrogen.com), supplemented with the following antibiotics: 0.4 mg/l piperacillin (sigma, http://www.sigmaaldrich.com), 0.1 mg/l gentamicin (invitrogen), and 5 mg/l amphotericin b (quality biological, http://www.qualitybiological.com). the lung homogenate was clarified by low-speed centrifugation at 2,000 rpm (650g) for 5 min, and the supernatant was administered intranasally to three naïve mice. the process of intranasal inoculation of three female balb/c mice with pooled, clarified supernatants of 10% lung homogenates collected 2 to 3 d.p.i. was repeated 14 times. identification of lethal phenotype and biological cloning of a lethal virus. the supernatant from p15 lung homogenates was subjected to three rounds of terminal dilution on vero cells. then, 5-fold serial dilutions were added to cells in 96-well plates (one dilution/plate). two to three days later, when cytopathic effect was evident in no more than nine wells per plate, supernatants from infected wells were collected, diluted, and transferred to fresh monolayers of vero cells. after the third round of terminal dilution, 50 ll of five independent clones were screened for lethality in four 8-wk-old female balb/c mice. mortality associated with these five clones was between 50% and 100%. one of these five clones (ma15) that caused 100% lethality within 6 d was expanded by two more passages in vero cells and used in further studies. virus replication in the respiratory tract of mice. young (6-to 8wk-old), female balb/c mice were lightly anesthetized and inoculated intranasally with 50 ll of serially diluted virus. for sars-cov (urbani), a dose of 10 5 tcid 50 /mouse was administered unless noted otherwise. for lethal doses of ma15 virus, the dose administered was equal to10 5.6 tcid 50 /mouse; for sub-lethal doses, the dose administered was equal to 10 3.6 tcid 50 /mouse. at various time points p.i., mice were euthanized and tissues collected for analyses. for determination of viral titers, tissues were homogenized to a final 10%w/v suspension in leibovitz's l15 medium supplemented with antibiotics. tissue homogenates were clarified by low-speed centrifugation, and virus titers were determined in vero cells on 24-and 96-well plates as previously described [30] . virus titers are expressed as tcid 50 /g of tissue with a lower limit of detection of 10 1.5 tcid 50 /g. purification of vrna. tissues homogenates were clarified by lowspeed centrifugation; supernatants were transferred to eppendorf tubes (http://www.eppendorf.com) and further clarified by centrifugation at 16,000g for 3 min. vrna was extracted from 600 ll of supernatant using the qiaamp viral rna mini kit (qiagen, http:// www1.qiagen.com) eluted in 50 ll of water, quantified by measurement of optical density at 280 nm on a nanodrop nd-1000 spectrophotometer (nanodrop technologies, http://www.nanodrop.com), and stored at à80 8c. rt-pcr and sequence analysis. first-strand cdnas were generated by reverse transcription from rna purified from clarified cell supernatants of vero cells infected with either sars-cov ma15, sars-cov (urbani) (p0), or from the clarified supernatants of p15 lung homogenates using reagents from the brilliant qrt-pcr plus core reagent kit (stratagene, http://www.stratagene.com). in brief, 11 ll of vrna was heat denatured at 95 8c for 2 min and quenched on ice before addition of 10x core rt buffer, 0.2 lg random primers, 0.08 lmol dntp mixture, and 20u stratascript reverse transcriptase for a 20-ll reaction mixture. reverse transcription was performed with an initial incubation of 25 8c for 10 min, followed by a 30-min incubation at 45 8c and a 3-min denaturation at 95 8c. first-strand cdnas were amplified in overlapping pcr products spanning the entire genome. three microliters of each cdna reaction were amplified by pcr using the advantage-hf pcr kit (bd biosciences, http://www.bdbiosciences.com) or herculase enhanced dna polymerase (stratagene) as per manufacturers' protocols. fifteen pairs of primers were used to generate the overlapping pcr products (tables s1 and s2). amplification products were visualized on agarose gels and purified by use of the qiaquick gel extraction kit (qiagen). pcr products were sequenced in both forward and reverse directions, using 69 forward and 62 reverse primers (table s3) . automated sequencing was performed utilizing the bigdye terminator version 3.1 cycle sequencing kit (applied biosystems) as per manufacturer's instructions on an abi prism 3730 dna analyzer (applied biosystems). sequences were assembled and analyzed with vector nti and auto assembler dna sequence assembly software (abi prism; applied biosystems). when a mutation was identified in comparison with the sars-cov (urbani) published sequence, independent rt-pcr reactions were run and subsequent rt-pcr products were sequenced through the region containing the putative mutation to confirm the mutation. detection of sars-cov by rt-pcr. total rna was isolated from various tissues or whole blood and purified using an rneasy mini kit (qiagen) with an on-column dnase digestion (rnase-free dnase set; qiagen), as per manufacturer's protocol. approximately 0.8 g of tissue, cut into pieces no larger than 0.5 cm on any one side, was collected into 1 ml of rnalater (ambion, http://www.ambion.com) and stored at room temperature for 24 h and subsequently at 4 8c until processed. any unused tissue remaining after one month was moved to à20 8c. approximately one-half or 0.2-0.4 g of tissue was homogenized with a disposable probe (omni international) in 900 ll of rlt buffer (rneasy mini kit; qiagen), supplemented with 1% (v/v) b-mercaptoethanol (sigma). rlt suspensions were transferred to 1.8-ml eppendorf microcentrifuge tubes and centrifuged at 4 8c for 3 min at 16,000g. rna was purified further as per manufacturer's protocol. rna from blood (;0.5 ml per sample) was purified using a qiaamp rna blood mini kit (qiagen) as per manufacturer's protocol with the additional dnase on-column digestion. rnas were quantified and 100 ng were reverse transcribed and amplified as described above. in brief, total rna was heat denatured at 95 8c for 2 min and quenched on ice before addition of 10x first strand buffer, 20u rnase block, 0.3 lg random primers, 0.08 lmol dntp mixture, 25u stratascript reverse transcriptase, and water for a 20-ll reaction. reverse transcription was performed with an initial incubation at 25 8c for 10 min, followed by a 30-min incubation at 45 8c and a 3-min denaturation at 95 8c. three microliters from each rt reaction were subsequently used in a 50-ll pcr with sars-covspecific primers for amplification of a 314-bp sequence of the nucleocapsid gene (sars-cov forward (59-ggtgacggcaaaatgaaagagc-39), sars-cov reverse (59-ggagaatttcccctactg-39)). b 2 microglobulin (b 2 m) primers were used in separate reactions as rna quantity and pcr controls (b 2 m forward (59-atgggaagccgaacatactg-39), b 2 m reverse (59-cagtctcagtgggggtgaat-39)). pcr reactions were performed for a total of 35 cycles with 50 x advantage cdna polymerase (clontech, http://www.clontech.com), 0.04 lmol dntp mixture (stratagene), and 10x cdna pcr reaction buffer (clontech) or opti-prime 10x buffer 4 (stratagene) supplemented with 0.02 lmol mgcl 2 (quality biological) for rna amplification of b 2 m and sars-cov, respectively (table s4) . construction of sars-cov cdna plasmids for reconstructing the mouse-adapted recombinant virus rma15. six codon changes (10384t.a, 10793a.c, 16177c.t, 12814a.g, 22797t.c, and 26428e.k) identified in ma15 compared with the published sars-cov (urbani) sequence, were inserted into cdna clones and used to construct and rescue infectious recombinant clones of sars-cov (urbani) as described previously [31] . in brief, for each mutation two overlapping amplicons were generated by pcr, joined at primerintroduced bsmbi sites, and ligated into a cdna of icsars-cov using two unique restriction sites that flanked the mutation of interest. pcr reactions were performed with expand long taq (roche applied sciences, http://www.roche-applied-science.com) in 30 cycles of 94 8c for 30 s, 55 8c for 30 s, and extensions at 68 8c for 1 min using the following primers on plasmids encoding portions of the icsars-cov genome: for the mutation at nucleotide position 10384, primer pairs 59-10124 (59-catgt cattt gcaca gcag) and 39-10364c (59-attag gtctc atggc acac) and 59-10384t.a (59-agacc taatt atacc attaa ag) and 39-11091c (59-caagc acaag aatgc gtgc) were used. a second pcr amplification using primers 59-10124 and 39-11091c produced a pcr product that was digested with the appropriate restriction enzymes (bglii, mfe1) and ligated into the icsars-cov cdna plasmid. similarly, the 10793 mutation was constructed using primer pairs 59-10124 and 39-10770c (59-ctttc aaagc agcac acata tc) and 59-10793a.c (59-gaaag cgctg ctgca gaatg) and 39-11091c, followed by amplification with primers 59-10124 and 39-11091c. this pcr product was digested with the appropriate restriction enzymes (bglii and mfe1) and ligated into the icsars-cov cdna plasmid. the 12814 mutation was similarly constructed using the primer pairs 59-m13r3 and 39-sars d mu1(à) (59-nnncg tctcg ttcca gttct gcgta aattg tacct gtacc) and 59 sars dmu1(þ) (59-nnncg tctct ggaac cacct tgtag gtttg) and 39 d1500(à) (59-ccctg tagac gacat cagtac). the two resulting amplicons were joined following digestion (with bsmbi) and purification (qiaquick pcr purification kit; qiagen). the product dna was digested with bamhi and acli and inserted into an icsars-cov cdna. the 16177 mutation was constructed using primers 59-16004 (59-catcctaatcaggagtatgc) and 39-16157c (59-caccta-c a g c c t g c a a g a c ) a n d p r i m e r s 5 9-1 6 1 7 7 c .t (59-gctgtaggtgtttgtgtattg) and 39-18044c (ctttatat-caacgctgaggtg). primers 59-16004 and 39-18044c were used to amplify the appropriate fragment that was purified and digested with pflmi and bbvci, and ligated into an icsars-cov cdna. the 22797 mutation was constructed using primer pairs 59 #38 (59-agagg aactg ctgta atgtc tc) and 39 smas mus(à) (59-nnncg tctct atgat tacca gttga agtag catc) and 59 smas smus(þ) (59-nnncg tctca tcataa ttata aatat aggta tctta gacat gg) and 39 sars e 4592(à) (59-ctagc acaaa tgcca gctcc). the two resulting amplicons were joined following digestion (with bsmbi) and purification and ligation. the full-length product was digested and inserted into an icsars-cov cdna utilizing restriction endonuclease sites agei and sali. the 26428 mutation was constructed using primer sets 59 #44 (59-tgatcctctgcaacctgagc) and 39 smas mum(à) (59-nnncgtctcacggtaatagtaccgttgtctgc) and 59 smas mum(þ) (59-nnncgtctctaccgttgagaagcttaaacaactcc) and 39 sars x5(à) (59-nnnnnttaattaattaatttgttcgtt-tatttaaaacaaca). resulting amplicons were ligated following digestion with bsmbi. the mutation-containing product was digested and inserted into an icsars-cov cdna at swai and ndei restriction sites. in all cases, the final plasmids and the mutations in the plaquepurified viruses were verified by rt-pcr and sequencing. assembly of full-length cdnas and recovery of recombinant viruses. the full-length cdnas of wild-type sars-cov (icsars-cov) or mouse-adapted sars-cov recombinants were produced as previously described [31] . rna transcripts from full-length cdnas were added to 800 ll of the vero e6 cell suspension (8.0 3 10 6 ) in an electroporation cuvette and four electrical pulses of 450 v at 50 lf were given with a gene pulser ii electroporator (bio-rad, http://www. bio-rad.com) similar to protocols previously described [32, 33] . the presence of full-length cdnas and transcripts was verified by separation on agarose gels and visualization by uv light. the transfected vero e6 cells were seeded in a 75-cm 2 flask and incubated at 37 8c for 2 d. viruses were plaque purified in vero e6 cells. in vitro growth of recombinant viruses. sars-cov (urbani), icsars-cov (urbani infectious clone), rma15 (icsars-cov with all six mutations found in ma15), rma15 orf1ab (icsars-cov with the four mutations found in the replicase genes of ma15 orf 1ab), and rma15 sm (icsars-cov with the two mutations found in the structural genes of ma15 s and m) viruses were propagated on vero e6 cells in eagle's mem supplemented with 10% fetal calf serum, kanamycin (0.25 lg/ml), and gentamicin (0.05 lg/ml) at 37 8c in a humidified co 2 incubator. cultures of vero e6 cells were infected in duplicate at an moi of 0.1 for 1 h. cell monolayers were washed twice with 2 ml of pbs and overlaid with complete mem. supernatants were collected at various times p.i. and virus was quantified by plaque assay on vero e6 cells in 60 mm 2 dishes. plaques were visualized by neutral red staining and counted at 48 h. northern blot analysis. cultures of vero e6 cells were inoculated with sars-cov viruses at an moi of 1 and incubated for 1 h at 37 8c. at 10.5 h.p.i., intracellular rna was isolated using trizol reagent (invitrogen) as directed by the manufacturer, and 0.1 lg of total mrna was treated with glyoxal and separated on agarose gels using northernmax-gly according to the manufacturer's directions (ambion). the rna was transferred to brightstar-plus membrane (ambion) for 3.5 h and then cross-linked to the membrane by uv light. the blot was prehybridized and probed with an n gene-specific oligodeoxynucleotide probe (59-cttgact gccgcct ctgct b t b ccct b ct b gc b -39), where biotinylated nucleotides are designated with a subscript b. blots were hybridized overnight, and washed with low and high stringency buffers as recommended by the manufacturer. filters were incubated with strepavidin-ap, washed, and then incubated with chemiluminescent substrate cdp-star (ambion). the blots were overlaid with film and developed. western blot analysis. ten hours p.i. with sars-cov (urbani), ma15 (the biologically derived clone), icsars-cov, rma15, rma15 or-f1ab, or rma15sm cells were washed once in pbs and lysed in buffer containing 20 mm tris-hcl (ph 7.6), 150 mm nacl, 0.5% deoxycholine, 1% nonidet-p-40, and 0.1% sodium dodecyl sulphate (sds). supernatants clarified of nuclei were added to an equal volume of 5 mm edta/0.9% sds, resulting in a final sds concentration of 0.5%, and samples were heat inactivated for 30 min at 90 8c prior to transfer to biosafety level 2. after transfer to biosafety level 2, samples were again heat inactivated for 30 min at 90 8c before use. equivalent sample volumes were loaded onto 7.5% ready gels (bio-rad) and transferred to a pvdf membrane (bio-rad). for detecting sars-cov antigens, blots were probed with polyclonal mouse antisera directed against venezuelan equine encephalitis virus replicon particles (vrps) that expressed the sars-cov orf 3a (vrp-orf3a), s (vrp-s), or n (vrp-n) proteins diluted 1:200 for sars-cov orf 3a and 1:500 for s and n antisera. dilutions were done in 5% blotto in pbs/0.5% tween 20 and developed using ecl chemiluminescence reagents (amersham, http://www.amershambiosciences.com). in situ hybridization. paraffin-embedded sections, 5-mm thick, were probed with 35 s utp-labeled riboprobes complementary to the n gene of sars-cov (urbani) or the sindbis virus genome, as a negative control, using previously described methods [34] . in brief, following treatment to prevent nonspecific probe binding, the tissues were incubated overnight with either probe at 5 3 10 4 cpm/ll in hybridization buffer at 42 8c. the slides were then washed, dehydrated, and coated with nbt emulsion (eastman kodak, http:// www.kodak.com), and incubated at à80 8c for 5 d prior to development. positive signal, as determined by silver grain deposition, was evaluated by light microscopy. statistics. log-transformed virus titers were compared in a mann-whitney u test, and statistical significance was assigned to differences with p-values ,0.05. histopathology and immunohistochemistry. lungs, liver, spleen, thymus, and brain were obtained from individual mice euthanized at various time points, fixed in 10% neutral buffered formalin, and processed for histopathologic and ihc examination as described [35, 36] . tissue sections (3 lm) were stained with hematoxylin and eosin and by using an immunoalkaline phosphatase technique with a hyperimmune rabbit anti-sars-cov nucleocapsid protein antibody at a dilution of 1/1000. figure s1 . i. lungs and sera were collected for viral titration. virus titers are expressed as log 10 pfu/g lung (circles) or log 10 pfu/ml serum (squares) from individual mice; bars represent the geometric mean for the group. limits of detection for viral titers from lung and sera are 250 pfu/ml and 50 pfu/ml, respectively. found at doi:10.1371/journal.ppat.0030005.sg002 (35 kb pdf). figure s3 . in situ hybridization of sars-cov rna in lungs of balb/c mice balb/c mice were infected with sar-cov (urbani), rma15 sm , rma15 orf1ab , rma15, or ma15 virus. lungs were harvested on days 2 and 4 p.i., and in situ hybridization was performed as described in materials and methods. no specific signal was observed with a control riboprobe at any time point, while sars-cov-n specific signal was readily apparent within the lungs for all five viruses at day 2 p.i. in contrast, at day 4 p.i., abundant sars-cov-specific in situ signal was observed in lungs of mice infected with the lethal ma15 virus or the lethal recombinant rma15, but not in lungs of mice infected with the non-lethal sub-clones or sars-cov (urbani). found at doi:10.1371/journal.ppat.0030005.sg003 (6.7 mb pdf). the genbank (http://www.ncbi.nlm.nih.gov/genbank) accession numbers for the viruses and sequences discussed in this paper are mouse-adapted sars-cov ma15 sequence (dq497008) and sars-cov (urbani) (ay278741). author contributions. ar performed passage of the sars-cov through balb/c mice, and all other experimental work except generation of recombinant viruses and in situ analysis of lungs. ar generated related figures, materials and methods, figure legends, and interpretation of findings, and wrote and revised the primary manuscript. dd contributed generation and characterization of recombinant clones, generation of related figures, recording of data, and data interpretation. dd made written contributions to figure legends, materials and methods, and review of primary manuscript. cdp performed all histopathological processing and evaluation of sars-cov-and ma15-infected tissues, interpretation of findings, generation of related figures, materials and methods, and figure legends, as well as review of primary manuscript. ac performed partial sequencing of ma15 genome, evaluation of mouse tissues for sars-cov mrnas and vrnas by rt/pcr, generated supporting tables s1-s5, and wrote related materials and methods, and reviewed the primary manuscript. by contributed to the generation and characterization of recombinant clones, generation of figures, recording of data, and data interpretation. by contributed to the writing of materials and methods and the review of primary manuscript. lv performed passage of the sars-cov through balb/c mice and all other experimental work except generation of recombinant viruses and in situ analysis of lungs. lv's written contributions include data recording, generation of materials and methods, and review and editing of primary manuscript. bdh performed partial sequencing of sars-cov, p15, and ma15 genomes, and primary manuscript review and editing. ts contributed preparation of sars-cov-infected and recombinant virus-infected tissues for in situ hybridization analysis. ts contributed to the writing of materials and methods and the review of primary manuscript. mh contributed experimental design and evaluation of sars-covinfected and recombinant virus-infected tissues by in situ hybridization techniques. mh made written contributions that include generation of materials and methods, the figure s3 legend, and a review of primary manuscript. glg performed all preparation of sars-cov-and ma15-infected tissues for histopathological processing and evaluation, generation of related figures, materials and methods, figure legends, and interpretation of findings, as well as review of the primary manuscript. srz contributed review of all histopathological evaluation of sars-cov-and ma15-infected tissues, generation of related figures, materials and methods, figure legends, and interpretation of findings, as well as review of the primary manuscript. rb contributed experimental design, characterization of recombinant clones, generation of related data and figures, and data evaluation and interpretation. rb made written contributions to figure legends, materials and methods, and the drafting and review of the primary manuscript. ks provided data interpretation and review and editing of the manuscript. funding. this research was supported in part by the intramural research program of the us national institutes of health (nih), niaid, and in part by nih/niaid grants ai059136 and ai059443. competing interests. the authors have declared that no competing interests exist. bats are natural reservoirs of sars-like coronaviruses animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting 25-26 is there an ideal animal model for sars? increased virulence of a mouse-adapted variant of influenza a/fm/1/47 virus is controlled by mutations in genome segments 4, 5, 7, and 8 a single amino acid change in the c-terminal domain of the matrix protein m1 of influenza b virus confers mouse adaptation and virulence mechanisms and enzymes involved in sars coronavirus genome expression molecular evolution of the sars coronavirus during the course of the sars epidemic in china receptor and viral determinants of sars-coronavirus adaptation to human ace2 structure of sars coronavirus spike receptor-binding domain complexed with receptor genetic analysis of mouse-adapted influenza a virus identifies roles for the na, pb1, and pb2 genes in virulence the influenza virus variant a/fm/1/47-ma possesses single amino acid replacements in the hemagglutinin, controlling virulence, and in the matrix protein, controlling virulence as well as growth mutations in the hemagglutinin and matrix genes of a virulent influenza virus variant, a/fm/1/47-ma, control different stages in pathogenesis cynomolgus macaque as an animal model for severe acute respiratory syndrome initial viral load and the outcomes of sars aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus clinical significance of hepatic derangement in severe acute respiratory syndrome viral loads in clinical specimens and sars manifestations a prediction rule for clinical diagnosis of severe acute respiratory syndrome pathogenesis of severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong difference and significance of t-lymphocyte subsets in differential diagnosis between severe acute respiratory syndrome and common atypical pneumonia clinical and laboratory features of severe acute respiratory syndrome vis-a-vis onset of fever coronavirus as a possible cause of severe acute respiratory syndrome haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis temporal patterns of hepatic dysfunction and disease severity in patients with sars severe acute respiratory syndrome among children severe acute respiratory distress syndrome (sars): a critical care perspective therapy with a severe acute respiratory syndrome-associated coronavirusneutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice sars cov replication and pathogenesis in human airway epithelial cultures reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus a single amino acid change in nsp1 attenuates neurovirulence of the sindbis-group alphavirus s.a.ar86 replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in a fatal case of severe acute respiratory syndrome in taiwan characterization of a novel coronavirus associated with severe acute respiratory syndrome we would like to thank jadon jackson for his expertise and assistance in all animal studies carried out at niaid. we would like to thank maria giovanni (microbial genomics, niaid) and elodie ghedin (the institute for genomic research) for contributions in confirming ma15 mutations. we would like to thank other members of the subbarao lab for their support. key: cord-313505-2lr4xara authors: resende, paola cristina; delatorre, edson; gräf, tiago; mir, daiana; motta, fernando do couto; appolinario, luciana reis; da paixão, anna carolina dias; ogrzewalska, maria; caetano, braulia; dos santos, mirleide cordeiro; de almeida ferreira, jessylene; santos junior, edivaldo costa; da silva, sandro patroca; fernandes, sandra bianchini; vianna, lucas a; da costa souza, larissa; ferro, jean f g; nardy, vanessa b; croda, júlio; oliveira, wanderson k; abreu, andré; bello, gonzalo; siqueira, marilda m title: genomic surveillance of sars-cov-2 reveals community transmission of a major lineage during the early pandemic phase in brazil date: 2020-06-18 journal: biorxiv doi: 10.1101/2020.06.17.158006 sha: doc_id: 313505 cord_uid: 2lr4xara despite all efforts to control the covid-19 spread, the sars-cov-2 reached south america within three months after its first detection in china, and brazil became one of the hotspots of covid-19 in the world. several sars-cov-2 lineages have been identified and some local clusters have been described in this early pandemic phase in western countries. here we investigated the genetic diversity of sars-cov-2 during the early phase (late february to late april) of the epidemic in brazil. phylogenetic analyses revealed multiple introductions of sars-cov-2 in brazil and the community transmission of a major b.1.1 lineage defined by two amino acid substitutions in the nucleocapsid and orf6. this sars-cov-2 brazilian lineage was probably established during february 2020 and rapidly spread through the country, reaching different brazilian regions by the middle of march 2020. our study also supports occasional exportations of this brazilian b.1.1 lineage to neighboring south american countries and to more distant countries before the implementation of international air travels restrictions in brazil. introduction 59 covid-19, the disease caused by severe acute respiratory syndrome coronavirus-2 60 (sars-cov-2), is leading to high rates of acute respiratory syndrome, hospitalization, and death 61 genomes (> 10% of ambiguous positions), we obtained a final dataset of 7,674 sequences. because 160 most sequences recovered (75%) were from the united kingdom (uk), we generate a "non-161 redundant" global balanced dataset by removing very closely related sequences (genetic similarity 162 > 99.99%) from the uk. to achieve this aim, sequences from the uk were grouped by similarity 163 with the cd-hit program 27 and one sequence per cluster was selected. with this sampling 164 procedure, we obtained a balanced global reference b.1.1 dataset containing 3,764 sequences that 165 were aligned with the new b.1.1 brazilian sequences generated in this study using mafft v7.467 166 28 and then subjected to maximum-likelihood (ml) phylogenetic analyses. the ml phylogenetic 167 tree was inferred using iqtree v1.6.12 29 (mc) and compared to a null hypothesis generated by tip randomization. results were considered 182 significant for p < 0.01. 183 the age of the most recent common ancestor (tmrca) and the spatial diffusion pattern of the 185 brazilian sars-cov-2 sequences here obtained were classified as clade b.1 (95%, n = 90), and 210 particularly within the sub-clade b.1.1 (92%, n = 87) (fig. 1b) . the prevalence of the sub-clade 211 b.1.1 in our sample (92%) was much higher than that observed in other brazilian sequences 212 available in gisaid (36%) (fig. 1c) phylogenetic tree, consistent with the hypothesis of multiple independent introductions (fig. 2) (fig. 2) . we also detected two other well-supported (sh-alrt 228 > 80%) monophyletic clades of small size (n = 2-11) mostly composed by brazilian sequences 229 ( supplementary fig. 1) . 230 in addition to sharing the three nucleotide mutations (g28881a, g28882a, g28883c) 231 characteristic of the clade b. fig. 2 ). despite the low genetic diversity, analyses of 253 geographic structure rejected the null hypothesis of a panmixed population (supplementary 254 january -20 th february) and its dissemination to brazil at 19 th february (95% hpd: 4 th february 260 -28 th february) (fig. 3a) from western europe into brazil before 2 nd february and that synapomorphic mutations t29148c 283 and t27299c were fixed at sequential steps during subsequent virus local spread (fig. 4b) the authors wish to thank all the health care workers and scientists, who have worked hard to deal 499 with this pandemic threat, the gisaid team and all the submitters of the database. gisaid a novel coronavirus from patients with pneumonia in china the 393 species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov 394 and naming it sars-cov-2 an interactive web-based dashboard to track covid-19 397 in real time brazilian ministry of health. brasil confirma primeiro caso da doença -covid-19 brazilian ministry of health nextstrain: real-time tracking of pathogen evolution a dynamic nomenclature proposal for sars-cov-2 to assist genomic 406 epidemiology. biorxiv global initiative on sharing all influenza data -from 408 vision to reality revealing covid-19 transmission by sars-cov-2 genome 410 sequencing and agent based modelling. biorxiv tracking the covid-19 pandemic in australia using genomics a phylodynamic workflow to rapidly gain insights into the dispersal 415 history and dynamics of sars-cov-2 lineages. biorxiv sars-cov-2 transmission chains from genetic data: a danish case 418 study. biorxiv introductions and early spread of sars-cov-2 in france. biorxiv spread of sars-cov-2 in the icelandic population full genome viral sequences inform patterns of sars-cov-2 spread into 424 and within israel. medrxiv rapid sars-cov-2 whole genome sequencing for informed 426 public health decision making in the netherlands. biorxiv phylodynamics of sars-cov-2 transmission in spain. biorxiv the emergence of sars-cov-2 in europe and the us. biorxiv introductions and early spread of sars-cov-2 in the new 433 genomic surveillance reveals multiple introductions of sars-cov-2 into 435 northern california the ongoing covid-19 epidemic in minas gerais, brazil: insights from 437 epidemiological data and sars-cov-2 whole genome sequencing. medrxiv importation and early local transmission of covid-19 in brazil genomic and phylogenetic characterization of an imported case of sars-cov-2 in 444 detection of 2019 novel coronavirus (2019-ncov) by real-time rt-447 pcr sars-cov-2 genomes recovered by long amplicon tiling multiplex 449 approach using nanopore sequencing and applicable to other sequencing platforms seqinr 1.0-2: a contributed package to the r project for statistical 452 computing devoted to biological sequences retrieval and analysis. structural 453 approaches to sequence evolution cd-hit: a fast program for clustering and comparing large sets of 457 protein or nucleotide sequences mafft multiple sequence alignment software version 7: 460 improvements in performance and usability iq-tree: a fast and 463 effective stochastic algorithm for estimating maximum-likelihood phylogenies modelfinder: fast model selection for accurate phylogenetic estimates exploring the temporal 469 structure of heterochronous sequences using tempest (formerly path-o-gen) correlating viral phenotypes with phylogeny: 472 accounting for phylogenetic uncertainty bayesian phylogenetic and phylodynamic data integration using 475 beast 1.10 improved performance, scaling, and usability performance computing library for statistical phylogenetics bayesian coalescent 480 inference of past population dynamics from molecular sequences bayesian phylogeography 483 finds its roots key: cord-288484-qy619tfg authors: bernard‐valnet, r.; pizzarotti, b.; anichini, a.; demars, y.; russo, e.; schmidhauser, m.; cerutti‐sola, j.; rossetti, a. o.; du pasquier, r. title: two patients with acute meningoencephalitis concomitant with sars‐cov‐2 infection date: 2020-05-30 journal: eur j neurol doi: 10.1111/ene.14298 sha: doc_id: 288484 cord_uid: qy619tfg in december 2019, a cluster of patients with pneumonia of unknown cause led to the identification of a new strain of pandemic coronavirus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2). since the first sars-cov outbreak, human coronaviruses are known for their neurological tropism. if respiratory complications are at the forefront of clinical presentation of sars-cov-2, neurological involvement remains poorly described and understood. we report here two patients infected with sars-cov-2 who presented with neurological symptoms and signs. in december 2019, a cluster of patients with pneumonia of unknown cause led to the identification of a new strain of pandemic coronavirus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] . since the first sars-cov outbreak, human coronaviruses have been known for their neurological tropism [2, 3] . respiratory complications are at the forefront of the clinical presentation of sars-cov-2 and neurological involvement remains poorly described and understood. we report here two patients infected with sars-cov-2 who presented with neurological symptoms and signs. the clinical and ancillary test descriptions were personally retrieved by the authors, who examined the patients. this report was conducted in compliance with the swiss federal act on research involving human beings, which waives ethical approval for case reports of less than five patients. both patients gave written informed consent for clinical and biological data to be used for this report. viral/bacterial detection was performed using the filmarray meningitis/ encephalitis panel (biofire diagnostics, salt lake city, ut, usa) and confirmed by traditional polymerase chain reaction. patient 1 was a 64-year-old woman without psychiatric history, known to have had contact with sars-cov-2 (her husband tested positive 15 days before) and presenting for 5 days with flu-like symptoms (mild asthenia, myalgia, cough) without fever, acutely developed psychotic symptoms. she was first admitted to a psychiatric ward, but presented a tonico-clonic seizure motivating her admission to an external hospital. a routine electroencephalogram revealed nonconvulsive, focal status epilepticus (abundant bursts of anterior low-to mediumvoltage irregular spike and waves superimposed on an irregularly slowed theta background) that was managed with intravenous clonazepam and valproate. she was immediately referred to our center. the patient appeared disoriented, with strong attention deficit, verbal and motor perseverations and bilateral grasping, alternating with psychotic symptoms (hyper-religiosity with mystic delusions, visual hallucinations). there was no neck stiffness or focal signs on neurological examination. cerebral magnetic resonance imaging was normal, but her lumbar puncture was compatible with viral meningoencephalitis (table 1) and sars-cov-2 was detected in her nasopharyngeal swab. however, neither sars-cov-2 nor classic viral/bacterial pathogens were detected in the cerebrospinal fluid (csf) ( table 1. ). anti-nmethyl-d-aspartate antibodies were tested negative in csf. treatment by acyclovir was transiently administered until herpes simplex/varicella zoster virus polymerase chain reaction results came back negative. a follow-up electroencephalogram 24 h after admission showed a moderate theta background slowing, without epileptiform features. the patient markedly improved 96 h after admission with resolution of her symptoms. a 67-year-old woman, already diagnosed with sars-cov-2 infection for 17 days with mild respiratory symptoms, presented an intense wake-up headache. a few hours later, she was found drowsy and confused, lying on the floor of her bathroom. she was referred to our hospital. on neurological evaluation, she was disoriented with motor perseverations, bilateral grasping, aggressiveness and left hemianopia and sensory hemineglect; there was no neck stiffness. sars-cov-2 pneumonia was diagnosed by a positive nasopharyngeal swab and an ultrasound showing subpleural condensation. brain magnetic resonance imaging was normal and her lumbar puncture revealed lymphocytic pleocytosis (table 1) . however, csf sars-cov-2 and viral/bacterial pathogen polymerase chain reaction tests were negative (table 1 ). the patient transiently received ceftriaxone, amoxicillin and acyclovir. neurological symptoms resolved within 24 h, except for a mild headache. the patient was discharged 72 h after admission with no symptoms. we report on two patients who developed meningoencephalitis a few days after a diagnosis of sars-cov-2 infection. both had a 'benign' form with only mild respiratory and general symptoms. however, they suddenly developed severe neuropsychological symptoms and one developed a status epilepticus. the csf *these authors contributed equally to the article. profiles being compatible with viral meningoencephalitis, a large screening for the usual pathogens, including sars-cov-2, was performed but was negative. although proof of a direct involvement of sars-cov-2 is missing, we hypothesize that it was responsible for this neurological presentation. firstly, the usual pathogens that cause viral meningoencephalitis were negative. second, the neurological picture occurred in the wake of proven sars-cov-2 infection. third, coronaviruses are known for their neurological tropism and for inducing encephalitis. it is of note that csf detection of coronavirus rna seems infrequent [3] . a possible mechanism accounting for the encephalitic presentation in these patients may be a para-infectious one, somewhat reminiscent of the association of coronaviruses with acute disseminated encephalomyelitis and (for sars-cov-2) guillain-barr e syndrome [4, 5] . such a mechanism would explain the rapid clinical recovery of both patients and the absence of magnetic resonance imaging lesions, suggesting a limited viral process, contrary to a previous report showing severe encephalitis and viral rna in the csf, although, in this case, herpes simplex virus encephalitis was not formally excluded [6] . to conclude, we report the first temporal association between acute sars-cov-2 infection and aseptic encephalitis with focal neurological symptoms and signs. further studies are needed to identify the spectrum of neurological complications of this pandemic outbreak and the underlying pathophysiological mechanisms. a novel coronavirus from patients with pneumonia in china multiple organ infection and the pathogenesis of sars neurologic alterations due to respiratory virus infections detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis guillain-barre syndrome associated with sars-cov-2 infection: causality or coincidence? a first case of meningitis/encephalitis associated with sars-coronavirus-2 we would like to thank prof. pierre-alexandre bart, dr david gachoud, dr jan novy, dr nicola marchi and dr sergiu vijala for taking care of these patients at different steps during their hospitalization. we would also like to acknowledge the work of dr onya opota, dr katia jaton and prof. gilbert greub in molecular biology diagnostic testing. anti-n-methyl-d-aspartate receptor, anti-contactin-associated protein-like 2, anti-leucine-rich glioma-inactivated 1, anti-dipeptidyl-peptidaselike protein 6, anti-gamma aminobutyric acid b receptor, anti-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, anti-immunoglobulin-like cell adhesion molecule 5, anti-metabotropic glutamate receptor 5 and anti-glycine receptor. key: cord-322908-e3gok0ot authors: huang, fangfang; li, ying; leung, elaine lai-han; liu, xiaohua; liu, kaifeng; wang, qu; lan, yongqi; li, xiaoling; yu, haibing; cu, liao; luo, hui; luo, lianxiang title: a review of therapeutic agents and chinese herbal medicines against sars-cov-2 (covid-19) date: 2020-05-20 journal: pharmacol res doi: 10.1016/j.phrs.2020.104929 sha: doc_id: 322908 cord_uid: e3gok0ot the epidemic of pneumonia (covid-19) caused by novel coronavirus (sars-cov-2) infection has been listed as a public health emergency of international concern by the world health organization (who), and its harm degree is defined as a global “pandemic”. at present, the efforts of various countries focus on the rapid diagnosis and isolation of patients, as well as to find a treatment that can combat the most serious impact of the disease. the number of reported covid-19 virus infections is still increasing. unfortunately, no drugs or vaccines have been approved for the treatment of human coronaviruses, but there is an urgent need for in-depth research on emerging human infectious coronaviruses. clarification transmission routes and pathogenic mechanisms, and identification of potential drug treatment targets will promote the development of effective prevention and treatment measures. in the absence of confirmed effective treatments, due to public health emergencies, it is essential to study the possible effects of existing approved antivirals drugs or chinese herbal medicines for sars-cov-2. this review summarizes the epidemiological characteristics, pathogenesis, virus structure and targeting strategies of covid-19. meanwhile, this review also focus on the re-purposing of clinically approved drugs and chinese herbal medicines that may be used to treat covid-19 and provide new ideas for the discovery of small molecular compounds with potential therapeutic effects on novel covid-19. broad-spectrum antiviral drug in the prevention and treatment of malaria [34] . cq/hcq block viral from entering into cells by inhibiting glycosylation of host receptors, proteolytic processing, and endosomal acidification, as well as regulate immunity through attenuation of cytokine production, inhibition of autophagy and lysosomal activity in host cells [35, 36] . cq can inhibit sars-cov-2 infection at a low-micro molar concentration and hcq is more potent than cq [37, 38] . a multicenter clinical trial involving more than a dozen hospitals in china showed that cq can improve radiologic findings, enhance viral clearance and reduce disease progression in the treatment of patients with covid-19, so china has included cq in the recommendations regarding the prevention and treatment of covid-19 [37, 39, 40] . at the same time, another clinical trial showed that hcq can significantly shorten the clinical recovery time and promote the absorption of pneumonia among patients with covid-19 [41] . notably, azithromycin reinforced the effect of cq/hcq in covid-19 patients, but the publishing journal's society subsequently declared that the trial did "not meet the society's expected standard" [42, 43] . conversely, the higher cq dosage should not be recommended for critically ill patients with covid-19 because of its potential safety hazards, especially when taken concurrently with azithromycin and oseltamivir [44, 45] . in summary, although cq/hcq have shown anti-sars-cov-2 efficacy both in vivo and in vitro trials as well as relatively well tolerated, some clinical trial designs and outcome data have not been submitted or published to peer review [46] . it is not recommended in the use of cq/hcq for covid-19 outside of the hospital or a clinical trial due to lack of reliable efficacy data and potential toxic effects [47, 48] . remdesivir (gs-5734), a prodrug of gs-441524 developed by the american pharmaceutical company gilead sciences, showed promise at the peak of the ebola virus outbreak due to its low ec50 and host polymerase selectivity against the ebola virus [49, 50] . subsequently, research about it also showed significant anti-sars-cov and mers-cov activity [51, 52] . as a nucleoside analog with exonuclease resistance, remdesivir is metabolized to active nucleoside triphosphates that effectively prevents the elongation of the rna chain by inhibiting rna polymerase, but will not be digested with a viral exonuclease (nsp14) with proofreading activity [53] . compared with ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine and favipiravir (t-705), remdesivir j o u r n a l p r e -p r o o f has the best efficacy and the lowest toxic side effects on anti-sars-cov-2 in vero e6 cells [32] . the united states first reported the clinical case of remdesivir in the treatment of sars-cov-2 associated pneumonia [54] . currently, a number of clinical trials are ongoing, aiming to verify the safety and antiviral activity of remdesivir in the treatment of covid-19. clinical findings of the team of professor cao bin of the china-japan friendship hospital suggested that the remdesivir is adequately tolerated but do not provide significant clinical or antiviral effects in severe patients with covid-19 [55] . however, the results of the global clinical trial are believed that remdesivir can relieve symptoms and reduce mortality, especially for patients in intensive care who require mechanical ventilation [56] . meanwhile, the clinical trials in chicago have suggested that early covid-19 patients benefit more due to the reduction of lung damage [57] . in conclusion, remdesivir is still in the consideration of one of the most promising drugs for treatment covid-19 currently [58] . lopinavir/ritonavir (lpv/r), also known as kaletra, is an oral combination agent for treating hiv approved by the fda, which has shown anti-coronavirus efficacy in studies of sars and mers [59] [60] [61] [62] . as a new protease inhibitor, lpv/r interrupts viral nucleic acid replication via inhibition of 3clpro [63] . xushun guo's team at sun yat-sen university school of medicine derived a homology modeling to confirm that lpv/r significantly inhibited the function of cep_c30 to prevent the sars-cov-2 reproduction cycle [64] . in addition, two groups in china and korea have reported lpv/r can improve the clinical symptoms of patients with covid-19 [65, 66] . besides, lpv/r can achieve better antiviral effects when used with interferon or ninavir than alone [67] . however, the latest evidence suggests that it may cause liver damage and prolong hospital stay in the covid-19 infected patients [68] . furthermore, no benefit was observed with lpv/r treatment beyond standard care in hospitalized adult patients with severe covid-19 [69] . therefore, whether lpv/r can become an important adjuvant drug in anti-sars-cov-2 therapy and improve the clinical outcome of patients remains to be determined. j o u r n a l p r e -p r o o f previously, we summarized small molecules currently used/planned to treat covid-19, which may be an important short-term strategy for the treatment of covid-19, but their efficacy and safety in covid-19 need to be further confirmed by clinical trials. drug development against covid-19 appears to be crucial in the context of a rapidly evolving epidemic, however, the conventional development of new drugs is time-consuming with safety concern. therefore, it seems unrealistic to synthesize new drugs and perform safety and toxicity tests over a short period of time. antiviral therapy with chinese herbal medicines have been recorded for a long time in chinese history, and previous studies have shown that chinese herbal medicines have great potential for preventing sars transmission [105] . given the low toxicity and availability of chinese herbal medicines, screening active compounds targeting viral or host targets from chinese herbal medicines may be a potential strategy for treating covid-19. in this review, we summarized potential chinese herbal medicines ( table 2 ) that may treat covid-19 by targeting proteins such as spike protein, ace2, 3clpro, plpro and rdrp. we also predicted the binding affinities between these compounds and covid-19 related targets by molecular docking, with a focus on six compounds: quercetin, andrographolide, glycyrrhizic acid, baicalin, patchouli alcohol, and luteolin. and the binding patterns of these six compounds to the key targets of sars-cov-2 are shown in figure 2 . quercetin, a flavonoid compound, is widespread in fruit and vegetables. as a dietary source compound, quercetin exerts diverse biological activities including anti-inflammatory, anti-oxidant, anti-viral, anti-allergic, anti-cancer, mood-improving as well as vasoprotective [106] [107] [108] . studies have found that quercetin exhibits antiviral properties against a variety of viruses, including influenza a virus (iav) [108] , hepatitis c virus (hcv) [109] , enterovirus 71 (ev71) [110] , and sars-cov, etc [111, 112] . it has been confirmed that quercetin showed a good inhibitory effect on sars-cov 3clpro expressed in pichia pastoris, with an inhibition rate of 82% [111] . in addition, enzyme inhibition assays in vitro also showed that quercetin had inhibitory activity against sars-cov 3clpro [112] . since the 3clpro sequence of sars-cov-2 is highly similar to that of sars-cov [10, 25] , we speculated that quercetin may also exhibit antiviral effects on sars-cov-2. however, it has not been documented whether quercetin inhibits sars-cov-2, so we docked quercetin to 3clpro as well as other key targets, and the docking results showed that quercetin bound well to each target, with a binding energy of -5.6 kcal/mol to 3clpro. surprisingly, we found that quercetin bounds better to spike protein, ace2, rdrp and plpro indicating good potential against sars-cov-2. in addition, it has a wide range of sources with relatively low cost, so it is worth testing its efficacy against sars-cov-2 infection. andrographolide, the main active component isolated from the extract of the herb andrographis paniculata, has a wide range of biological activities including immunity regulation, anti-virus, anti-bacteria, anti-parasite, anti-tumor, and anti-hyperglycemia [113, 114] . previous studies have shown that andrographolide has a broad spectrum of antiviral properties, which inhibits various virus infections including influenza a virus (iav) [115] , human immunodeficiency virus (hiv) [116] , chikungunya virus (chikv) [117] , dengue virus (denv) [118, 119] , and enterovirus d68 (ev-d68) [120] . atchara paemanee et al. suggested that andrographolide may exert j o u r n a l p r e -p r o o f broad-spectrum antiviral activity by interfering a variety of cellular pathways (including autophagy, unfolded protein response (upr) pathway and oxidative stress, etc.). they further found the anti-dengue virus activity by acting on grp78, a key regulator of unfolded protein response [119] . in addition, andrographolide exerts antiviral activity against h1n1 by inhibiting the activation of rlrs signaling pathways and thereby improving h1n1 virus-induced cell death [121] . to test the anti-viral activity against sars-cov-2, we docked andrographolide with key targets, and the results also showed that andrographolide bound well to the key targets including spike protein, ace2, 3clpro, rdrp and plpro, which indicated that andrographolide has potential efficacy against sars-cov-2. moreover, enmozhi, s. k. et al. proved andrographolide as a potential inhibitor of sars-cov-2 3clpro through in silico studies [122] . overall, as a plant-derived compound, andrographolide is widely distributed with low cytotoxicity, but its potent antiviral activity against a variety of viruses calls for further investigation. glycyrrhizic acid is a plant product isolated from the traditional chinese medicine licorice rdrp and spike is -6.9 kcal/mol, -7.3 kcal/mol, -7.2 kcal/mol, -6.5 kcal/mol, respectively. it can be seen that glycyrrhizic acid also has strong binding affinity to other targets. given the antiviral effect of glycyrrhizic acid on sars-cov, and its potential interaction with ace2, we speculated that glycyrrhizic acid may have potential to treat sars-cov-2. moreover, glycyrrhizic acid plays an important role in inhibiting immune hyperactivation and cytokine storm factor development [129] , therefore, we believe it is worth testing its efficacy against sars-cov-2 infection. baicalin and specific binding to proteases by itc map, native electrospray ionization mass spectrometry (esi-ms) and its chemical structure [28] . at the same time, we used molecular docking to study the docking of baicalin to other key targets of sars-cov-2, in which the binding energy of baicalin to the target plpro was -8.5 kcal/mol. the results of docking showed that baicalin bounds strongly to other targets of sars-cov-2 (table 2) . therefore, it can be reasonably speculated that baicalin is one of the potential drugs for covid-19 treatment. in view of the low toxic effect of baicalin, its effect against sars-cov-2 warrants further study. patchouli alcohol (pa), a tricyclic sesquiterpene compound extracted from the traditional chinese medicine patchouli, has a wide range of pharmacological and biological effects including antiviral, immunomodulatory, anti-inflammatory, antioxidative, and antitumor [134] . pa has been found to have anti-influenza a (iav) effect in vitro, while h1n1 virus is the most sensitive to pa [135] . in addition, yunjia yu et al. found that intracellular pi3k/akt and erk/mapk signaling pathways may be involved in the anti-iav effect of pa and pa significantly inhibits the in vitro proliferation of different iav, suggesting that pa may block iav infection by directly killing viral particles and interfering with some early stages after viral adsorption [136] . another study showed that pa also has an effect against influenza virus (ifv) in vivo and enhances protection against ifv infection in mice by enhancing host immune responses and attenuating systemic and pulmonary inflammatory responses [137] . to investigate the anti-sars-cov-2 activity of pa, we investigated the possibility of pa binding to sars-cov-2 related targets using molecular docking ( table 2 ). the docking results showed that the binding effect of pa and rdrp was satisfactory, which provided some support for the antiviral effect of pa. the above study results showed that patchouli alcohol had antiviral effect and also modulated the levels of inflammatory cytokines, suggesting that pa may be a novel and effective antiviral and anti-inflammatory drug for covid-19. luteolin, a natural flavonoid extracted from chinese herbal medicine, displays multiple biological activities, including anti-inflammatory, anti-cancer, antioxidant, antiviral, and heart protective [138] . it was reported that luteolin can interfere with the virus in early virus life cycle, to a certain extent, block the absorption and internalization of influenza virus, thereby inhibited the replication of iav [139] . the above experiments suggested that luteolin is a potential antiviral drug that inhibits viral replication by regulating host proteins. in addition, minhua peng et al. confirmed luteolin inhibited the dengue virus ns2b/ns3 protease activity by analyzing the nucleotide sequence of the luteolin-resistant escape mutant [140] . it also has been documented luteolin has an anti-epstein-barr virus (ebv) effect, and in immunoblot analysis, 20 μg/ml of luteolin showed a significant inhibitory effect on ebv lytic cycle [141] . another study showed that luteolin extracted from torreya nucifera is an effective sars-cov 3clpro inhibitor [112] . to interrogate the anti-sars-cov-2 effect of luteolin, we performed molecular docking of luteolin to j o u r n a l p r e -p r o o f key targets of sars-cov-2. the docking results showed that luteolin bound well to the key target of sars-cov-2. among them, the binding energy of luteolin to ace2 was -7.1 kcal/mol ( table 2 ). taken together, luteolin has a good antiviral effect, which suggests that luteolin may be a potential drug for the treatment of covid-19. the results of molecular docking are shown in table 2 . from the target point of view, the binding effect of ace2 and plpro with these natural compounds was more prominent; while from the natural compounds, the lowest binding energy was -9.0 kcal/mol for cryptotanshinone and plpro, while the highest was -4.3 kcal/mol for lignan and 3clpro, that is to say, the range of binding energy was from -9.0 kcal/mol to -4.3 kcal/mol, which indicated that the natural compounds had a good binding effect with the target. our aim of docking was to select natural compounds with high potential efficacy against sars-cov-2, but it should be pointed out that these compounds cannot be considered to treat covid-19 only by such a screen which is aimed to provide priority to focus. furthermore, the 3d structure of the targets we used were based on the reported gene sequences. if the virus mutates during transmission, new screening is recommended. in conclusion, our review summarizes more than a dozen of natural compounds classified as antiviral/pneumonic protectors, which may directly inhibit sars-cov-2. however, their actual effect in the treatment of covid-19 needs to be verified by further studies. covid-19 poses a great threat to global health and safety. it is an urgent task for us to control the spread of the epidemic and reduce the mortality rate as soon as possible. but so far, the specific mechanism of the virus is still unclear, and no specific drug has been developed for the virus. at treatment. this review also has limitations. the large and rapidly published literature on covid-19's treatment means that the findings and recommendations are constantly evolving as new evidence arises. it is not uncommon that drugs that proved effective at an early stage based on small-scale clinical trials later turned out to be ineffective. we look forward to the cooperation of all scientists around the world to develop effective drugs to treat current and future potential 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syndrome coronavirus infection as identified by temporal kinome analysis inhibition effects of patchouli alcohol against influenza a virus through targeting cellular pi3k/akt and erk/mapk signaling pathways oral administration of patchouli alcohol isolated from pogostemonis herba augments protection against influenza viral infection in mice novel extraction techniques and pharmaceutical activities of luteolin and its derivatives luteolin decreases the yield of influenza a virus in vitro by interfering with the coat protein i complex expression luteolin escape mutants of dengue virus map to prm and ns2b and reveal viral plasticity during maturation bioactive constituents of lindernia crustacea and its anti-ebv effect via rta expression inhibition in the viral lytic cycle flavonoid baicalin hiv-1 infection at the level of viral entry anti-sars coronavirus 3c-like protease effects of isatis indigotica root and plant-derived phenolic compounds discovery of potential multi-target-directed ligands by targeting host-specific sars-cov-2 structurally conserved main protease($) emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction emodin inhibits current through sars-associated coronavirus 3a protein effective inhibition of mers-cov infection by resveratrol kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus tanshinones as selective and slow-binding inhibitors for sars-cov cysteine proteases structure-based drug design, virtual screening and high-throughput screening rapidly identify antiviral leads targeting covid-19 clinical efficacy of matrine and sodium chloride injection in treatment of 40 cases of covid-19 decoction in the treatment of novel coronavirus pneumonia lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (sars-cov-2) traditional chinese medicine for treatment of coronavirus disease 2019: a review covid-19: an update on the epidemiological, clinical, preventive and therapeutic evidence and guidelines of integrative chinese-western medicine for the management of 2019 novel coronavirus disease multidrug treatment with nelfinavir and cepharanthine against covid-19 key: cord-315685-ute3dxwu authors: ehaideb, salleh n.; abdullah, mashan l.; abuyassin, bisher; bouchama, abderrezak title: evidence of a wide gap between covid-19 in humans and animal models: a systematic review date: 2020-10-06 journal: crit care doi: 10.1186/s13054-020-03304-8 sha: doc_id: 315685 cord_uid: ute3dxwu background: animal models of covid-19 have been rapidly reported after the start of the pandemic. we aimed to assess whether the newly created models reproduce the full spectrum of human covid-19. methods: we searched the medline, as well as biorxiv and medrxiv preprint servers for original research published in english from january 1 to may 20, 2020. we used the search terms (covid-19) or (sars-cov-2) and (animal models), (hamsters), (nonhuman primates), (macaques), (rodent), (mice), (rats), (ferrets), (rabbits), (cats), and (dogs). inclusion criteria were the establishment of animal models of covid-19 as an endpoint. other inclusion criteria were assessment of prophylaxis, therapies, or vaccines, using animal models of covid-19. result: thirteen peer-reviewed studies and 14 preprints met the inclusion criteria. the animals used were nonhuman primates (n = 13), mice (n = 7), ferrets (n = 4), hamsters (n = 4), and cats (n = 1). all animals supported high viral replication in the upper and lower respiratory tract associated with mild clinical manifestations, lung pathology, and full recovery. older animals displayed relatively more severe illness than the younger ones. no animal models developed hypoxemic respiratory failure, multiple organ dysfunction, culminating in death. all species elicited a specific igg antibodies response to the spike proteins, which were protective against a second exposure. transient systemic inflammation was observed occasionally in nonhuman primates, hamsters, and mice. notably, none of the animals unveiled a cytokine storm or coagulopathy. conclusions: most of the animal models of covid-19 recapitulated mild pattern of human covid-19 with full recovery phenotype. no severe illness associated with mortality was observed, suggesting a wide gap between covid-19 in humans and animal models. in part the easy transmission from person-to-person, and its dissemination within the body in severe and fatal cases [11] [12] [13] [14] [15] [16] [17] [18] . accordingly, sars-cov-2-induced covid-19 has led to a pandemic that overwhelmed the capacity of most national health systems, resulting in a global health crisis [19] . so far, an estimated 11,280 million persons in 188 countries were infected, of which 531,000 died [20] . the clinical spectrum of covid-19 is complex and has been categorized as mild, severe, and critical, representing 81, 14, and 5% [2, 3] . the mild pattern comprises patients with either no signs and symptoms or fever and radiological evidence of pneumonia [3] . the severe pattern manifests as rapidly progressive hypoxemic pneumonia involving more than half of the lung with a full recovery phenotype [2, 3] . the critical pattern consists of ards requiring respiratory assistance and mosd that result in death in approximately half of the patients [2, 3, 7, 21] . mortality was associated with host factors such as old age, comorbidities, and immune response [4] . viral and immunopathological studies revealed distinct patterns between mild and severe or critical forms of covid-19 [4, 5, 9, [21] [22] [23] [24] [25] [26] [27] . both severe and critically ill patients displayed higher viral load in the upper respiratory tract than mild cases, together with delayed clearance overtime [21, 22] . likewise, they presented with lymphopenia due to a decrease in cd4+ and cd8+ t cells, as well as t cell exhaustion accompanied by a marked inflammatory response [5, 9, [24] [25] [26] [27] . pro-and anti-inflammatory cytokines and chemokine concentrations were increased systemically and locally in the lung and correlated with severity [5, 9, 24] . in contrast, in the mild illness, the lymphocyte count was normal, with no or minimal inflammatory response [5, 23] . together, these suggest that the viral load and dynamic together with the host inflammatory response may play a pathogenic role. clinical and post-mortem studies of fatal cases of covid-19 demonstrated major alteration of coagulation and fibrinolysis [17, 18] . this was associated with widespread thrombosis of small and large vessels, particularly of the pulmonary circulation contributing to death in a third of patients [8, [28] [29] [30] [31] [32] [33] . these observations suggest that dysregulated coagulation may be an important mechanism of covid-19 morbidity and mortality [34] . in this context, animal models appear crucial to a better understanding of the complex biology of covid-19. animal models of sars-cov-2-induced covid19 have been rapidly reported since the start of the pandemic [35] . however, whether they express the full phenotype of covid-19, particularly the severe and critical patterns associated with lethality, remains to be determined. in this systematic review, we examined whether the newly created animal models reproduce the phenotype of human covid-19. moreover, we examined the knowledge generated by these models of covid-19 including viral dynamic and transmission, pathogenesis, and testing of therapy and vaccines. we conducted a systematic review according to the preferred reporting items for systematic reviews and meta-analysis (prisma) statement [36] to identify studies describing the creation of an animal model of covid-19 as an endpoint (table 1 and additional file 1). additional file 1 shows the data extraction and appraisal approach as well as the selected outcome. the systematic search identified 101 studies and 326 preprints, of which 400 articles were excluded because they were reviews, non-original articles, unrelated to the covid-19 infection, or experimental animals that do not support sars-cov-2 replication such as pigs, ducks, and chickens ( fig. 1 and additional file 2). additional file 2 displays all the excluded studies and the rationale for their exclusion. thirteen peer-reviewed studies and 14 preprints were included in the analysis. the studies used nonhuman primates (n = 13) [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] , mice (n = 7) [50] [51] [52] [53] [54] [55] [56] , hamsters (n = 4) [56] [57] [58] [59] , ferrets (n = 4) [60] [61] [62] [63] , cats, and dogs (n = 1) [63] (tables 2, 3 , 4, and 5). male and female, as well as young and old, were included but with no associated comorbidities. the aims were to investigate the pathogenesis of covid-19 (n = 15), testing drugs and vaccines (n = 14), the host table 1 search strategy and selection criteria we searched the medline, as well as biorxiv and medrxiv preprint servers for original research describing or using an animal model of sars-cov-2 induced covid published in english from january 1, 2020, to may 20, 2020. we used the search terms (covid-19) or (sars-cov-2) and, (animal models), (hamsters), (nonhuman primates), (macaques), (rodent), (mice), (rats), (ferrets), (rabbits), (cats), and (dogs). the preprint servers were included in the search as the field of covid-19 is developing quickly. inclusion criteria were the establishment of animal models of covid-19 as an endpoint. other inclusion criteria were assessment of prophylaxis, therapies, or vaccines, using animal models of covid-19. exclusion criteria consisted of reviews, non-original articles, and unrelated to the covid-19 infection or experimental animals that do not support sars-cov-2 replication. 101 studies and 326 preprints were screened of which 13 peer-reviewed studies and 14 preprints were included in the final analysis (fig. 1) . the variables extracted were the population type, study aim, the virus strain used, clinical response, pathology, viral replication, and host response as well as the effects of prophylaxis, drugs, or vaccines. the outcomes were organized according to species and categorized into phenotype (signs or symptoms; histopathology, timecourse of the illness and outcome), viral (titer in each tissue organ, detection methods, duration of positivity), and host response (dynamic of seroconversion, inflammatory, and hemostatic markers), therapy, and vaccine (efficacy and safety) immune response (n = 6), and the virus dynamic and transmission (n = 4) (tables 2, 3, 4, and 5). all the experimental animals were inoculated with sars-cov-2 with various strains, doses, and route of administration that differed across studies (tables 2, 3 , 4, and 5). likewise, the time-point for tissue collection and pathological assessment were variables. these together precluded any comparisons between the animal models either intra-species or inter-species. nonhuman primate models viral model rhesus macaques (n = 10) [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] , cynomolgus (n = 3) [46] [47] [48] , and african green model (n = 1) [49] and common marmoset (n = 1) [46] were assessed as models for covid-19 (table 2) . sars-cov-2 strains, dose, and route of inoculation were different across studies. different doses of virus inoculum were compared in a single study and showed that viral load in the upper and lower respiratory tract, fever, weight loss, respiratory distress, and mortality were comparable regardless of the doses except for mild transient neutropenia and lymphopenia in the high dose group [43] . in contrast, the route of administration resulted in different pathological response as the intratracheal route elicited severe interstitial pneumonia, as compared with mild interstitial pneumonia and no pneumonia from the intraconjunctival and intragastric routes, respectively [45] . the animals were euthanized at different time-points post-inoculation ranging from 3 to 33 days. the animals displayed variable clinical manifestations from none to fever, altered respiratory patterns, and igg antibody anti-sars-cov-2 spike s1 subunit evaluation of medical interventions reticulonodular opacities pet scan: fdg uptake lung and regional lymph nodes (2), mediastinal lymph nodes and . § dpi day post-inoculation, ¶ crp c-reactive protein, || na not available. **vaccine encoding spike protein variants: full-length sars-cov-2 s protein, s.dct deletion of the cytoplasmic tail of sars-cov-2 s protein, s.dtm deletion of the transmembrane domain and cytoplasmic tail reflecting the soluble ectodomain, s1 s1 domain with a fold on trimerization tag, rbd receptor-binding domain with a fold on trimerization tag, s.dtm.pp a prefusion stabilized soluble ectodomain with deletion of the furin cleavage site, two proline mutations, and a fold on trimerization tag, im intramuscular other general signs ( table 2 ). the clinical manifestations were not different between old and young macaques [46] [47] [48] . structural and ultrastructural examination of the respiratory tract were also variables including mild to moderate interstitial pneumonitis, edema, foci of diffuse alveolar damage with occasional hyaline membrane formation, and pneumocytes type ii hyperplasia ( table 2) . old rhesus macaques exhibited more diffuse and severe interstitial pneumonia than young ones [47] . the extrapulmonary injury was investigated in five studies [40, 42, 43, 46, 49] . these revealed pathological changes in two studies [46, 49] including distention and flaccidity of the intestine, inflammatory cells infiltrating the jejunum, and colon, steatosis of the liver, and alteration of myocardial fiber architecture with increased mitochondrial density [46, 49] . no mortality was observed in any of the nonhuman primate models. comparisons between species of nonhuman primates were not possible except in one study, which suggested that rhesus macaques were superior to cynomolgus and common marmoset as models of human covid-19 [46] . other comparisons suggested that sars-cov elicited more severe lung pathology than sars-cov-2 and middle east respiratory syndrome (mers-cov) [48] ( table 2) . the virus replicated rapidly and at higher titers in the upper airway and lung in all four species [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . the virus was detected in pneumocytes type i and ii and ciliated epithelial cells of nasal, bronchial, and bronchiolar mucosa [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . this differs from mers-cov where the virus was mainly present in type ii pneumocytes [46] ( table 2 ). replication of the virus was also demonstrated in jejunum, duodenum, colon, and rectum [37, 38, [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . viral genome was detected in the blood of rhesus macaques, cynomolgus, and marmoset in one study [46] . viral replication of nasopharyngeal as well as anal swabs, and the lung in old macaques was higher than in young ones [47, 48] . sars-cov-2 infection-induced igg antibodies response against the sars-cov-2 spike was noted in all species [37, 46, 48, 49] except in marmoset [46] . the antibodies were protective against a second exposure to the virus [43, 44] . there was no difference between males and females [37, 39-43, 46, 47] ; however, young rhesus macaques had lower antibody titers than the old macaques [46] . the innate immune response to sars-cov-2 infection was variable with normal, high, or low leucocytes and lymphocyte counts [37, 46] . occasional reduction of cd4 + and cd8 + t cell concentrations was documented [37] as well as the transitory release of various cytokines and chemokines at different days postinoculation [37, 46, 49] . dna and inactivated virus-based vaccines were evaluated and showed protection in these nonhuman primates. however, the dna vaccine did not reduce the virus presence in the upper airway, while there was a residual small interstitial pneumonitis in the macaques that received the inactivated virus [40, 41] . this suggests that none of the virus tested so far displayed a comprehensive protection against sars-cov-2 infection. several candidate dna vaccines based in various forms of the sars-cov-2 spike (s) protein were also tested in rhesus macaques [39] . the findings revealed that only the vaccine encoding the full-length (s) offered optimal protection against sars-cov-2 [64] . nonhuman primates served also for the evaluation of antiviral therapies and medical interventions such as ct-and petscanners [47] . wild type mice (balb/c, c57bl/6), immunodeficient mice (scid), chimeric mouse expressing human angiotensinconverting enzyme 2 (hace2), and the rna-dependent rna polymerase (sars1/sars2-rdrp) were evaluated as models of covid-19 (table 3) . moreover, knockout (ko) mice were generated to test specific immunological pathways or therapy, including ablation of type i (ifnar1−/−), iii interferon (ifn) receptors, (il28r−/−), signal transducer and activator of transcription 2 (stat2−/−), and serum esterase (ces1c−/−). patient isolates of sars-cov-2 from different sources and variable times of passaging on various cell cultures or balb/c mice were employed (table 3) . mouseadapted sars-cov-2 was developed using two methods. the first by serial passaging (up to 6) through the lungs of balb/c mice until the virus spike receptor-binding domain (rbd) adapted to the murine ace-2 [54] . in the second, using genetic engineering, the sars-cov-2 rbd was remodeled to enhance its binding efficiency to murine ace2 [52] . the clinical signs and symptoms varied from none to mild weight loss, arched back, and slight bristles. whole-body plethysmography was used to measure the respiratory function of the animals and showed a mild to moderate reduction in old more than in young (table 3) . likewise, the pathological changes varied according to the experimental models and included peribronchiolar inflammation, lung edema, moderate multifocal interstitial pneumonia, lymphocyte infiltration, and intraalveolar hemorrhage. survival of hace2 mice was decreased at 5-day post-inoculation and was attributed to high viral replication in the brain, while it was weight and minimal in the lung, suggesting a different pathogenic mechanism of death from human covid-19 [52] . wild type mice showed no pathology as compared to hace2 mice, indicating that the lack of human ace2 receptor cannot be infected or inefficiently with sars-cov-2 [50, 56] . on the other hand, mouse-adapted sars-cov-2 exhibited more severe pathology, particularly in the aged mouse than hace2 transgenic mouse, suggesting that these models may be more relevant for the study of human covid-19 [52, 54] . however, whether the pathogenesis induced by the mouse-adapted sars-cov-2 is translatable to humans warrants further studies [52, 54] . the virus replicated to high titers in the upper and lower respiratory tract in most of the genetically modified mice models but not in wild type. viral replication was detected outside the respiratory tract in the intestine of hace2 mice [50] as well as in the liver, and heart in mouse modified sars-cov-2 rbd [52] . increased viral replication in ko mice ifnar1−/− suggested that interferon limited the viral replication [56] . specific igg antibodies against sars-cov-2 were documented in two studies ( table 3 ). the igg antibodies were found to cross-react in their binding to the spike protein of sars-cov, however, with no crossneutralization, hence suggesting the conservation of the same spike protein epitopes among coronaviruses [53] . proinflammatory cytokines and chemokines were demonstrated in mouse-adapted sars-cov-2 and ko mouse ( table 3 ). the inflammatory response was significantly higher in the old than young mice. antiviral therapies, including remdesivir [55] , ifn lambda [52] , and human monoclonal igg1 antibody against rbd [50] , were tested in these mouse models and produced a protective effect. likewise, vaccines using viral particles expressing sars-2-s protein [52] or an rbd-based vaccine were tested and showed protection [55] . wild type syrian hamsters and knockout hamsters for signal transducer and activator of transcription 2 (stat2−/− lacking type i and iii interferon signaling) and interleukin 28 receptors (il28r−/− lacking ifn type iii signaling) were reported as models for covid-19. patient isolate of sars-cov-2 from different sources and different passages on various cell cultures was used (table 4) . sars-cov-2 was administered intranasally at different titers to anesthetized hamsters. viral transmission between hamsters was demonstrated either through direct contact or indirectly via airborne transmission. the clinical manifestations included weight loss, which was consistently observed. other clinical signs and symptoms such as rapid breathing, lethargy, ruffled furs, and hunched back posture were reported in one study [57] . the histopathological findings were variables according to the experimental models and ranged from lung consolidation to multifocal necrotizing bronchiolitis, leukocyte infiltration, and edema. stat2−/− hamsters exhibited attenuated lung pathology as compared with il28r-a−/− hamsters [56] . the virus replicated to high titer in the upper and lower respiratory tract in most of the hamsters' models. viral replication was detected in the blood and kidney with a low concentration (table 4 ). stat2−/− hamsters had higher titers of infectious virus in the lung, viremia, and high levels of viral rna in the spleen, liver, and upper and lower gastrointestinal tract in comparison with wild type and il28r-a−/− hamsters. specific igg antibodies against sars-cov-2 were documented in the sera of hamsters at different time-points from virus inoculation ranging from 7 to 21 days. increased expression of proinflammatory and chemokine genes was demonstrated in the lungs of the sars-cov-2 infected animals, however with no increase in circulating levels of proteins such as tnf, interferon-γ, and il-6. immunoprophylaxis with early convalescent serum achieved a significant decrease in viral lung load but not in lung pathology [57] . ferrets, cats, and dogs were administered intranasally or intratracheally with various doses and strains of sars-cov-2 (table 5 ). ferrets displayed elevated body temperature for several days associated with signs that differed according to the studies. these include decreased activity and appetite, sporadic cough, and no body weight loss [60] [61] [62] [63] . no clinical signs were reported either in cats or in dogs. ferrets exhibited acute bronchiolitis [61, 63] , with perivasculitis and vasculitis [63] , but with no discernible pneumonia. cats disclosed lesions in epithelial nasal, tracheal, and lung mucosa (table 5 ). the virus replication and shedding were demonstrated in the upper airways and rectal swabs in ferrets and cats, but the extent to other tissues varied in ferrets from none to multiple organs, including the lung, blood, and urine. no viral rna was detected in cats' lungs. dogs showed rna-positive rectal swab but none in the upper or lower airways. viral transmission between ferrets and cats was demonstrated either through direct contact [55] or indirectly via airborne route [62] . ferrets, cats, and dogs exhibited specific antibody response against sars-cov-2 [60, 62, 63] . a study of the ferret immune response to sars-cov-2 revealed a subdued low interferon type i and type iii response that contrasts with increased chemokines and proinflammatory cytokine il6, which is reminiscent of the human response [61] . this systematic review of experimental animal models of sars-cov-2 induced-covid-19 identified 13 peerreviewed studies and 14 preprints that reported data on nonhuman primates [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] , mice [50] [51] [52] [53] [54] [55] [56] , hamsters [56] [57] [58] [59] , ferrets [60] [61] [62] [63] , cats, and dog [63] models of covid-19. the main findings indicate that most of the animal models could mimic many features of mild human covid-19 with a full recovery phenotype [3] . they also revealed that older animals display relatively more severe illness than the younger ones [38, 46, 48, 52, 54] , which evokes human covid-19 [3, 6] . however, none of the animal models replicated the severe or critical patterns associated with mortality as observed in humans with covid-19 [3] . the results of this systematic review are consistent with studies of animal models of sars-cov and mers-cov, which failed to replicate the full spectrum of humans' illness [65, 66] . nonetheless, several features of mild covid-19 in humans could be mirrored. high viral titers in the upper and lower respiratory tract and lung pathology were demonstrated in both large and small animal models. the pathology encompassed mild interstitial pneumonia, consolidation, and diffuse alveolar damage, albeit localized to a small lung area, edema, hyaline membrane formation, and inflammation. sars-cov-2 elicited specific antibody response against various viral proteins in the sera of most of the animal models. this systematic review revealed that none of these newly established animal models replicated the common complications of human covid-19 such as ards and coagulopathy [6, 8, 28-33, 67, 68] . ards can be particularly severe and results in refractory hypoxemia requiring maximum respiratory supportive measures in the intensive care unit [6, 67, 68] . the coagulopathy can lead to severe complications such as massive pulmonary embolism, cerebrovascular stroke, and mesenteric infarction, including in younger people [8, 28, 32, 33] . the pathology underlying these two complications were recently revealed by post-mortem studies disclosing diffuse alveolar damage involving the whole lung, hyaline membrane formation, and infiltration with inflammatory cells, thus leaving no air space open for ventilation [17, 18, 64, 69, 70] . it also detected the presence of diffuse and widespread thrombosis in the micro-and macro-circulation, including the pulmonary circulation compromising the lung perfusion [17, 18] . this double hit affecting the ventilation and perfusion simultaneously underlies the intractable hypoxemia that contributed to the high mortality. none of the animal models replicated the respiratory failure, thromboembolic manifestations, and their the mechanisms of the lung injury and coagulopathy are not well understood, although several known pathways were postulated including cytokine storm leading to upregulation of tissue factor [5, 9, 24] , activation/injury of the endothelium infected by the virus [30, 67, 71] , complement activation [72] , alveolar hypoxia promoting thrombosis [73] , and autoantibodies against phospholipid and lupus anticoagulant [74, 75] modulating the hemostasis and coagulation cascade directly. hence, the development of animal models that replicate the dysregulation of the inflammation and coagulation could be important, as these would allow the deciphering of the intimate mechanisms at play. this, in turn, may aid in identifying therapeutic targets and the testing of immunotherapy, anticoagulation, and thrombolytic interventions and thereby may improve the outcome. both antiviral and vaccine therapies were tested in rhesus macaques and mice infected with sars-cov-2 [40] [41] [42] . the antiviral drug stopped the viral replication and improved the pneumonitis [42, 55] . the vaccines induced an increase in titers of neutralizing antibodies in the sera that correlated with the decrease of viral replication and prevented the lung pathology [39] [40] [41] . these results represent a substantial proof of the concept of antiviral or vaccine efficacy against sars-cov-2 in animal models. however, because of the lack of overt clinical illness, the rapid clearance of the virus, and spontaneous improvement of the pneumonitis without lethality, the models do not permit the full assessment of the duration of the protection of the vaccines, or the effect of antiviral therapy on survival. since the emergence of sars-cov infection in 2003 [76] , followed by the mers-cov in 2012 [77] , and now with covid-19, researchers have not been able to develop a model of coronavirus infection that reproduces the severity and lethality seen in humans [65, 66] . one of the well-known reasons lies in the difference of ace-2 receptor binding domain structure across species [78] . human and primates have conserved a comparable structure that allows binding with high affinity to the sars-cov-2 [78] . the hamsters, ferrets, and cats maintained an intermediate affinity, while mice exhibit very low affinity [78] . the latter explains why wild-type mouse does not support sars-cov-2 replication, and hence, the necessity to create a chimera that expresses human ace-2, to enable the use of this species as a model of covid-19 [50] . more recently, a study applying single-cell rna sequencing to nonhuman primate uncovered another explanation that may underlie the difference between nonhuman primates and humans in expressing the complex phenotype of covid-19 [79] . the study reveals that the cellular expression and distribution of ace2 and tmprss2 which are essential for virus entry in the cells and its spread inside the body differ in the lung, liver, and kidney between the two species. ace2 expression was found lower in pneumocytes type ii and higher in ciliated cells in nonhuman primate lung as compared to humans [40] . this is particularly significant as type ii pneumocytes are critical targets of sars-cov-2 in humans and the pathogenesis of lung injury/damage. finally, the innate immune response including the defense system against viruses diverged during evolution both at the transcriptional levels and cellular levels, which may also explain why the sars-cov-2 hardly progresses in these animals outside the respiratory system [80] . taken together, these fundamental differences represent a real challenge to the successful development of an animal model that reproduces human covid-19. this systematic review has a few limitations. first, it is the high number of preprints included in this study that have not been peer-reviewed. second, the animal models from the same species were difficult to compare across studies, as they used different viral strain, inoculum size, route of administration, and timing of tissue collection. this systematic review revealed that animal models of covid19 mimic mild human covid-19, but not the severe form covid-19 associated with mortality. it also disclosed the knowledge generated by these models of covid-19 including viral 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manuscript. there was no funding source for this study.availability of data and materials all data generated or analyzed during this study are included in this published article [and its supplementary information files]. the authors declare that they have no competing interests.received: 13 july 2020 accepted: 21 september 2020 key: cord-310017-c8rd714a authors: popa, alexandra; genger, jakob-wendelin; nicholson, michael; penz, thomas; schmid, daniela; aberle, stephan w.; agerer, benedikt; lercher, alexander; endler, lukas; colaço, henrique; smyth, mark; schuster, michael; grau, miguel; martinez, francisco; pich, oriol; borena, wegene; pawelka, erich; keszei, zsofia; senekowitsch, martin; laine, jan; aberle, judith h.; redlberger-fritz, monika; karolyi, mario; zoufaly, alexander; maritschnik, sabine; borkovec, martin; hufnagl, peter; nairz, manfred; weiss, günter; wolfinger, michael t.; von laer, dorothee; superti-furga, giulio; lopez-bigas, nuria; puchhammer-stöckl, elisabeth; allerberger, franz; michor, franziska; bock, christoph; bergthaler, andreas title: mutational dynamics and transmission properties of sars-cov-2 superspreading events in austria date: 2020-07-17 journal: biorxiv doi: 10.1101/2020.07.15.204339 sha: doc_id: 310017 cord_uid: c8rd714a superspreading events shape the covid-19 pandemic. here we provide a national-scale analysis of sars-cov-2 outbreaks in austria, a country that played a major role for virus transmission across europe and beyond. capitalizing on a national epidemiological surveillance system, we performed deep whole-genome sequencing of virus isolates from 576 samples to cover major austrian sars-cov-2 clusters. our data chart a map of early viral spreading in europe, including the path from low-frequency mutations to fixation. detailed epidemiological surveys enabled us to calculate the effective sars-cov-2 population bottlenecks during transmission and unveil time-resolved intra-patient viral quasispecies dynamics. this study demonstrates the power of integrating deep viral genome sequencing and epidemiological data to better understand how sars-cov-2 spreads through populations. graphical abstract the sars-cov-2 pandemic has already infected more than 13 million people in 188 countries, causing 570,375 global deaths as of july 13 th 2020 and extraordinary disruptions to daily life and economies (1, 2). the international research community rapidly started to establish diagnostic tools, assess immunological and pathological responses and define risk 5 factors for covid-19 (3) (4) (5) (6) . clustered outbreaks and superspreading events of sars-cov-2 pose a particular challenge to pandemic control (7) (8) (9) (10) . however, we still know comparatively little about the fundamental underlying properties of sars-cov-2 genome evolution and transmission dynamics within the human population. during its sweep across the globe, the 29.9 kb-long sars-cov-2 genome has accumulated 10 mutations at a rate 2-3 fold lower than those for the sars, mers and influenza a viruses (11) . acquired fixed mutations enable phylogenetic analyses and have already led to important insights into the origins and routes of sars-cov-2 spread (12) (13) (14) (15) . conversely, low frequency mutations and their changes over time within individual patients offer deep insights into the dynamics of intra-host evolution. the resulting viral quasispecies represent 15 diverse groups of variants with different frequencies that form structured populations and maintain high genetic diversity, contributing to fundamental properties of infection and pathogenesis (16, 17) . with a population of 8.8 million people, austria is located in the center of europe and 20 operates a highly developed health care system that utilizes a national epidemiological surveillance program. as of june 15 th 2020, contact tracing was performed for all 17, 082 reported cases that tested positive for sars-cov-2, and 6,287 cases could be linked to 502 epidemiological clusters (18) (methods) . due to its prominent role in international winter tourism, austria has been implicated as a superspreading transmission hub across the 25 european continent. tourism-associated spread of sars-cov-2 from austria may be responsible for up to half of all imported cases in denmark, norway and a considerable share of cases in many other countries including iceland and germany (12, 19, 20) . in this study we phylogenetically and epidemiologically reconstructed major sars-cov-2 infection clusters in austria and analyzed their role in transcontinental virus spread. 30 moreover, we combined our deep viral genome sequencing data with epidemiologically identified chains of transmissions and family clusters together with biomathematical analyses to study genetic bottlenecks and the dynamics of genome evolution of sars-cov-2. our results provide fully integrated genetic and epidemiological evidence for continental spread 5 of sars-cov-2 from austria and establish fundamental transmission properties of the sars-cov-2 in the human population. phylogenetic-epidemiological reconstruction of sars-cov-2 infection clusters in austria 5 we sequenced 576 sars-cov-2 rna samples from cases originating from different geographical locations across austria. the main focus was on the austrian provinces of tyrol and vienna (fig. 1a) , given that these two regions led to numerous positive cases (fig. s1a) (18) . the samples presented in this study capture the first phase of the outbreak (mid-february to mid-march 2020) as well as the peak of infections in austria (fig. 1a) , and 10 cover a balanced sampling across multiple epidemiological and clinical parameters including age, sex and viral load ( fig. s1b-c) . samples from both swabs (nasal, oropharyngeal) and secretions (tracheal, bronchial) were included, in order to investigate not only the evolutionary dynamics within the population, but also within individuals (fig. s1d) . we assembled sars-cov-2 genome sequences, constructed phylogenies and identified low 15 frequency mutations based on high-quality sequencing results with >5 million reads per sample and >80% of mapped viral reads (fig. s2a-b) . our pipeline was validated by experimental controls involving sample titration and technical sample replicates ( fig. s2c to investigate the link between local outbreaks in austria and the global pandemic, we 20 performed phylogenetic analysis of 305 sars-cov-2 genomes from the austrian cases (>96% genome coverage, >80% aligned viral reads) and 7,695 global genomes from the gisaid database (fig. 1b, table s1 ). our analysis revealed six distinct phylogenetic clusters defined by fixed mutation profiles that were mainly present in the tyrol region (tyrol-1, tyrol-2, tyrol-3) and in vienna (vienna-1, vienna-2, vienna-3) (fig. 1b) . these 25 clusters are related to the global clades 19a, 20a, 20b and 20c (fig. s3a) . our largest phylogenetic cluster, "tyrol-1" (fig. s3b) , whose cases are closely linked to the ski-resort ischgl, was assigned to clade 20c (fig. 1b) . this clade is predominantly populated by strains from north america (fig. s3a) . integration of the phylogenetic analysis of austrian sars-cov-2 sequences with 30 epidemiological data from contact tracing resulted in strong overlap of these two lines of evidence (fig. 1c, table s2 ). all sequenced samples from the epidemiological cluster a mapped to the relatively homogenous phylogenetic cluster vienna-1 (fig. 1c) with an index patient with reported travel history to italy. of note, both clusters tyrol-1 and vienna-1 6 originate from crowded indoor events (i.e. apré ski bar; spinning class), by now known origins for superspreading events. the epidemiological cluster s, which includes resident and travel-associated cases to the skiresort ischgl or the related valley paznaun, largely mapped to the phylogenetic cluster tyrol-5 1 (fig. 1c) . while different sars-cov-2 strains circulated in the region of tyrol, this data suggests that the epidemiological cluster s originated from a single strain with a characteristic mutation profile leading to a large outbreak in this region (fig. 1d) . to elucidate the possible origin of the sars-cov-2 strain giving rise to this cluster, we searched for strains matching its mutation profile among global sars-cov-2 sequences 10 ( fig. 1d-e, fig. s3c ). we found that all strains in clade 20c matched the mutation profile of the tyrol-1 cluster in our phylogenetic analysis (fig. 1e, fig. s3c ). to reveal possible transmission lines between european countries at that time, we performed phylogenetic analysis using all 7,675 high-quality european sars-cov-2 sequences sampled before march 31 that were available in the database gisaid. using this approach, we identified 15 several samples matching the tyrol-1 cluster mutation profile from a local outbreak in the region hauts-de-france in the last week of february (fig. 1e, fig. s3c ) (21) . introduction events of this sars-cov-2 strain to iceland by tourists with a travel history to austria were reported as early as march 2 (fig. 1e, s3c ) (12), indicating that this strain was already present in ischgl in the last week of february. these findings suggest that the emergence of 20 the cluster tyrol-1 coincided with the local outbreak in france (fig. 1e) and with the early stages of the severe outbreak in northern italy (22) . the viral genomes observed in the tyrol-1 cluster were closely related to those observed among the icelandic cases with a travel history to austria (fig. s3d-e) (12). vice versa, many of the icelandic strains with a "tyrol-1" mutation profile had reported an austrian or icelandic exposure history (fig. s3f) . 25 together, these observations and epidemiological evidence support the notion that the sars-cov-2 outbreak in austria propagated to iceland. moreover, the emergence of these strains coincided with the emergence of the global clade 20c (fig. s3c) . one week after the occurrence of sars-cov-2 strains with this mutation profile in france and ischgl, an increasing number of related strains based on the same mutation profile could be found 30 across continents (fig. 1e) where they established new local outbreaks, for example in new york city (13). as a popular international destination attracting thousands of tourists from european countries and overseas, ischgl may have played a critical role as transmission hub for the spread of clade 20c strains to europe and north america (fig. s3g-h) . 7 dynamics of low frequency and fixed mutations in clusters next, we sought to gain insights into the fundamental processes of sars-cov-2 infection by integrative analysis of viral genomes. mutational analysis of sars-cov-2 genomes from austrian cases revealed that more than half of the observed substitutions were non-5 synonymous, with the most frequent non-synonymous mutations occurring in nsp6, orf3a and orf8 (fig. s4a-b ). an analysis of the mutational signatures in the 7,695 global strains and the austrian subset of sars-cov-2 isolates showed a non-homogeneous mutational pattern dominated by c>u, g>u and g>a substitutions (fig. s4c) . moreover, we observed increased mutation rates in the 3' and 5' utrs ( fig. s4d) . we found that 30% of the 10 positions in the genome (9, 194 total positions) harbored variants among the sequenced strains from austria and we identified mutational hotspots for both high and low-frequency mutations ( fig. 2a, fig. s5a alleles being fixed in more than three samples and exhibiting a frequency <0.50 in at least two other samples ( fig. 2a) . we confirmed the non-homogeneous mutational pattern of fixed mutations in our low-frequency data, suggesting that the same biological and evolutionary forces are at work for both types of mutations ( fig. s5c-e) . based on our phylogenetic analysis, we found a sub-cluster inside the phylogenetic tyrol-1 cluster, defined by a fixed non-synonymous g>u mutation at position 15,380 (fig. 2b) . this mutation is absent from most of the other austrian cases but was detected at low and intermediary frequencies in other cases of the tyrol-1 cluster. interestingly, around the emergence of this mutation, sequences sharing the same mutational profile (i.e. tyrol-1 25 haplotype and g>u at position 15,380) appeared in other european countries including denmark and germany (fig. 2c) . similarly, a synonymous fixed c>u mutation at position 20,457 defines a subcluster inside the phylogenetic vienna-1 cluster (fig. 2d) . potential functional effects of this mutation are unknown although it is predicted to slightly alter the locally stable rna region with a destabilized central fold ( fig. s6c-d) . the cases from this 30 subcluster intersect with members of two families (families 1 and 7) (fig. 2e , see also 8 conversely, four members of family 7, who tested positive for sars-cov-2 between march 16 th and 22 nd , were epidemiologically assigned to cluster al and also show a fixed u nucleotide at position 20,457 ( fig. 2d-e) . this data indicates the emergence of a fixed mutation within a family, and provides phylogenetic evidence to link previously disconnected epidemiological clusters. together, these results from two superspreading events (tyrol-1, 5 vienna-1) demonstrate the power of deep viral genome sequencing in combination with detailed epidemiological data for observing viral mutation on their way from emergence at low frequency to fixation. impact of transmission bottlenecks and intra-host evolution on sars-cov-2 mutational 10 dynamics the emergence and potential fixation of mutations in the viral quasispecies depend on interhost bottlenecks and intra-host evolutionary dynamics (23, 24) . to test the individual contributions of these forces, we first investigated the transmission dynamics between known pairs of donors and recipients by inferring the number of virions initiating the infection, also 15 known as the genetic bottleneck size (23, 25) . our set of sars-cov-2 positive-tested cases comprised twenty-three epidemiologically confirmed donor-recipient pairs (fig. 3a, fig. s7a ). using a beta-binomial method to quantify the bottleneck size (25), we found a median bottleneck size of 514 virions (ranging from 3 to greater than 5000) (fig. 3b) . the observed relatively large bottleneck size of sars-cov-2 is driven by the stability of mutation 20 frequencies across transmissions (fig. s7a ). next, we investigated the dynamics of intra-host evolution by using time-resolved viral sequences from nine longitudinally sampled patients. these patients were subject to different medical treatments and four of them succumbed due to covid-19 related complications 25 (table s3) . we observed diverse mutation patterns across individual patients and time (fig. 3c, fig. s7b ). samples from patients 2, 3, and 10 showed a small number of stable lowfrequency mutations (≥ 0.02 and ≤0.50), while patients 1, 4, 5, 7, 8 and 9 exhibited higher variability including the fixation of individual mutations (fig. 3c, fig. s7b ). the patientspecific dynamics of viral mutation frequencies may indicate influences of both host-intrinsic 30 factors such as immune responses and overall state of their health as well as extrinsic factors such as different treatment protocols. finally, we examined the genetic distance between samples obtained across donor -recipient pairs and serially acquired patient samples. this analysis revealed that genetic divergence was greater between the consecutive samples within 9 individual patients than during inter-host transmission (fig. 3d) , suggesting that viral intrahost evolution has a potentially strong impact on the landscape of fixed mutations. unprecedented global research efforts are underway to match the rapid pace of the covid-5 19 pandemic around the globe and its pervasive impact on health and socioeconomics. these efforts include the genetic characterization of sars-cov-2 to track viral spread and to dissect the viral genome as it undergoes changes in the human population. here, we leveraged deep viral genome sequencing in combination with national-scale epidemiological workup to reconstruct austrian sars-cov-2 clusters that played a substantial role in the 10 international spread of the virus. notably, our time-resolved study shows how emerging lowfrequency mutations of sars-cov-2 become fixed in local clusters with subsequent spread across countries, thus connecting viral mutational dynamics within individuals and across populations. exploiting epidemiologically well-defined clusters and families, we were able to determine the inter-human genetic bottleneck for sars-cov-2, i.e. the number of virions 15 that start the infection and produce progeny in the viral population, at around 10 1 -10 3 . our quantified bottlenecks are based on a substantial number of defined donor-recipient pairs and in agreement with recent studies implying larger bottleneck sizes for sars-cov-2 compared to estimates for influenza a virus (23, (26) (27) (28) (29) . these bottleneck sizes correlate inversely with higher mutation rates of influenza virus as compared to sars-cov-2. of note, estimates of 20 viral bottleneck size are likely influenced by many parameters including virus-specific differences and stochastic evolutionary processes (29) . successful viral transmission also depends on other factors including the rate of decay of viral particles, availability of susceptible cells, the host immune response and co-morbidities (23, 30) . to better understand the mechanisms at work, future investigations will need to probe these factors in the context 25 of viral intra-host diversity across body compartments and time (31) (32) and assess how the underlying viral population structures act together and influence genome evolution of sars-cov-2 (33, 34) . this study underscores the value of tightly integrated epidemiological and molecular 30 sequencing approaches to provide the high spatiotemporal resolution necessary for public health experts to track pathogen spread effectively. this enables the retrospective identification of sars-cov-2 chains of transmissions and international hotspots such as the phylogenetic cluster tyrol-1 (15, (35) (36) (37) . our data also show that all but cluster tyrol-2 carry 10 the prevalent d614g mutation in the s protein (38), supporting the notion of multiple introduction events to tyrol. moreover, our phylogenetic analysis of cluster vienna-1 allowed us to uncover previously unknown links between epidemiological clusters. this observation was subsequently confirmed by extended contact tracing, demonstrating deep viral genome sequencing directly contributes to public health efforts. 5 the time has come to implement the technical capacities and interdisciplinary framework for prospective near real-time tracking of sars-cov-2 infection clusters by integrating approaches which combine viral phylogenetic and epidemiological evidence as well as possible complementary data such as serological testing (39) . such inter-disciplinary 10 platforms will be particularly relevant for the prevention of superspreading events and the assessment of the effectiveness of pandemic containment strategies in order to improve the preparedness for anticipated recurrent outbreaks and resurgences of sars-cov-2 as well as future pandemics (40). sequencing data processing and analysis following demultiplexing, fastq files containing the raw reads were inspected for quality criteria (base quality, n and gc content, sequence duplication, over-represented sequences) using the fastqc (v. 0.11.8) (42). trimming of adapter sequences was performed with bbduk from the bbtools suite (http://jgi.doe.gov/data-and-tools/bbtools). overlapping read 5 sequences in a pair were corrected for with bbmerge function from the bbtools. read pairs were mapped on the combined hg38 and sars-cov-2 genome (genbank: mn908947.3) using the bwa-mem software package (v 0.7.17) (43). only reads mapping uniquely to the sars-cov-2 viral genome were extracted. primer sequences were removed after mapping by masking with ivar (44) . from the viral reads bam file, the consensus 10 fasta file was generated using the samtools (v 1.9) (45) #102024, twist biosciene) were titrated in increasing ratios (0.1%, 1%, 5%, 10%, 90%, 20 100%) and subjected to cdna synthesis and pcr amplification as described. these controls are important for assessing the limit of low frequency detection across samples. rna was extracted and processed independently from sample cemm0001 to serve as a technical control for pcr processing. amplicons from the cemm0008 sample were sequenced twice in order to assess the potential biases introduced by the sequencing step. phylogenetic analysis was conducted using the augur package (version 7.0.2) (51). we compiled a randomly subsampled dataset of 7695 full length viral genomes with high coverage (<1% ns) that were available from gisaid (https://www.gisaid.org/, 2 nd of june) and 305 sequences obtained in this publication. sequences from gisaid were filtered for entries from human hosts with complete sampling dates. metadata information for patient age 20 and sex were excluded from the analysis. multiple sequence alignments (msa) were performed using mafft (52). a masking scheme for homoplasic and highly ambiguous sites was applied to avoid bias in the following phylogenetic analysis as discussed elsewhere [n. de maio, c. walker, r. borges, l. weilguny, g. slodkowicz, and n. goldman, "issues with sars-cov-2 sequencing data," virological.org.]. we reconstructed the phylogeny with the 25 augur pipeline using iq-tree (52) and further processed the resulting trees with treetime to infer ancestral traits of the nodes (53) . phylogenetic trees were rooted with the genome of "wuhan-hu-1/2019". the same workflow was repeated for phylogenetic reconstruction of all high-quality european strains before march 31 st 2020 available in the gisaid database by june 7 th 2020 (7675). clade annotations for global trees were adapted from nextstrain.org 30 (https://github.com/nextstrain/ncov/blob/master/config/clades.tsv), clusters of austrian strains were identified based on shared mutation profiles and patient location from epidemiological data. 14 inter-host mutations were reconstructed using the augur pipeline to infer nucleotide changes at the internal nodes (51) . positions reported as highly homoplasic were masked, including the first 55 and the last 100 nucleotides [n. de maio, c. walker, r. borges, l. weilguny, g. slodkowicz, and n. goldman, "issues with sars-cov-2 sequencing data," virological.org.]. 5 the consequence type of the mutations was annotated using a customized implementation of the ensembl variant effect predictor (vep version 92) using the first sars-cov-2 sequenced genome (ncbi id: nc_045512v2) as a reference. the mutational profile was obtained as the normalized count of the number of mutations in each of the 192 trinucleotide changes. to account for the genomic composition of the sars-cov-2 virus we also divided 10 each triplet probability by the total number of available triples in the sars-cov-2 reference genome. for the intra-host analysis, the process to obtain the mutational spectra panels was the same as intra-host but using the low frequency variant calling output (3090 mutations across 404 austrian samples with alleles frequencies between 0.05 and 0.5). the mutational profile was computed following the same rationale as for the inter-host variants. 15 we aimed to assess whether the variation in the rate of single nucleotide substitution along the sars-cov-2 genome can be solely explained by its tri-nucleotide composition. we devised a statistical test performing local estimations of the deviation from the expectation of the observed number of mutations with respect to the expected based on the tri-nucleotide 20 composition of a particular region of the genome. we first counted the total number of nonprotein affecting mutations (i.e., synonymous variants and upstream/downstream gene variants) that has been observed across sequenced viral genomes of infected individuals. the focus on non-protein affecting mutations aims to lessen the potential positive selection bias derived from their effect into the coding parts of the viral genome. we did not consider 25 mutations in masked sites (see filtering of mutations for further information about masked sites). we next assigned to each reference nucleoside a probability of mutation of the three alternatives based on its tri-nucleotide context (5' and 3' nucleosides) and the relative probability of mutation derived from the 7,695 samples from gsaid. then we performed n (n=10 6 ) randomizations of the same number of observed mutations distributing them along 30 the sars-cov-2 genome according to their mutational probability. protein-affecting mutations were not randomized, and masked sites were not available to the randomization. we then divided the 29,903 bp of the viral genome into 10 windows of 1kb (except the last 15 window with 903 bps). analogously, in the zoom-in analysis, we divided the first and last 1kb window of the viral genome into 10 windows of 100 bp. for each window we estimated the mean and standard deviation number of simulated mutations within the window. finally, for each window we estimated the deviation from the expectation using a log-likelihood test (i.e., g-test goodness of fit), where we compared the observed number of mutations in the 5 window versus the mean simulated number. to address the question whether mutations that have been observed in the austrian sars-cov-2 samples have an influence on the rna structure of the virus we performed 10 computational predictions at the secondary structure level with the viennarna package (54) . we started with characterizing locally stable rna structures in the sars-cov-2 reference genome nc 045512.2 with rnalfold. we required that the underlying sequences were not longer than 150 nt and we targeted thermodynamic stability by selecting only regions whose free energy z score was at least -3 among 1000 dinucleotide shuffled sequences of the same 15 sequence composition. this approach yielded 386 locally stable rna secondary structures spread throughout the sars-cov-2 genome, which were then intersected with 12 unique fixed mutations found in austrian samples, i.e. positions 241, 1059, 3037, 12832, 14408, 15380, 20457, 23403, 24642, 25563, 27046, and 28881-28883 . this approach resulted in seven hits which were subsequently analyzed in detail. we performed single sequence 20 minimum free energy (mfe) structure predictions for both the reference and the mutation variants. in addition, we assessed for each region the level of structural conservation within a set of phylogenetically related viruses. here we were particularly interested in finding evidence for covariation in stacked helices. typical covariation patterns are compensatory mutations, i.e. cases where a mutation in one nucleotide is compensated by a second mutation 25 of its pairing partner, such as a gc base-pair being replaced by an au pair. likewise, consistent mutations comprise cases where only one nucleotide is exchanged, thereby maintaining the base-pair, e.g. gc to gu. we characterized orthologous regions in selected sarbecovirus species with infernal (55), produced structural multiple sequence nucleotide alignments with locarna (56) and computed consensus structures with rnaalifold (57) . in 30 addition, each block was analyzed for structural conservation by rnaz (58) . bottleneck estimation. 16 we first set out to estimate the transmission bottleneck sizes for each donor-recipient pair. our analysis is based on the beta-binomial method presented in leonard et al (25) . for shared mutations with a defined donor to recipient transmission (fig. 3b) , we determined those mutations present in both samples and calculated their absolute difference in frequency. similarly, we made the same computations between time consecutive pairs for serially 17 sampled patients. if multiple samples were obtained on the same day, the sample with lowest ct value was considered. note that the time-consecutive pairs had differing number of days between samples. to these genetic distances obtained from the shared variants we added the sum of the frequencies of the variants detected in only one of the pairs of shared samples; i.e. we calculated the l1-norm of the variant frequencies. statistical difference between the 5 genetic distances from transmission pairs versus consecutive pairs from serially sampled patients, was determined by a wilcoxon (one-sided) rank-sum test. hörmann for providing samples and tobias pahlke for support with computing cluster. we thank the tourism office paznaun -ischgl for statistical data. competing interests: authors declare no competing interests. 27 data and materials availability: virus sequences are deposited in the gisaid database (see table s1 ). all phylogenetic trees used in this study are available for visualization under the following urls: global build: https://nextstrain.org/community/bergthalerlab/sars-cov-2/nextstrainaustria ; european build with european strains before 31 march: 5 https://nextstrain.org/community/bergthalerlab/sars-cov-2/earlyeurope build with austrian strains used for phylogenetic analysis: https://nextstrain.org/community/bergthalerlab/sars-cov-2/onlyaustrian (transmission chains, n=23) and intra-patient consecutive time points (n=39) (fig. 3c, fig. 15 s7b). only variants seen in two samples are considered. s1: acknowledgements for sars-cov-2 genome sequences derived from gisaid table s2 : epidemiological clusters referred to in this study. table s3 : clinical information of covid-19 patients relating to figure 3 and figure s7 . a pneumonia outbreak 5 associated with a new coronavirus of probable bat origin immunology of covid-19: current state of the science deep immune profiling of 25 covid-19 patients reveals patient heterogeneity and distinct immunotypes with implications for therapeutic interventions viral and host factors related to the clinical outcome of covid-19 genomewide association study of severe covid-19 with respiratory failure superspreading and the effect of individual variation on disease emergence epidemiology of covid-19 in a long-term care facility in king county novel coronavirus-infected pneumonia in wuhan, china high sars-cov-2 attack rate following exposure at a choir practice -skagit county emergence of genomic diversity and recurrent mutations in sars-cov-2 spread of sars-cov-2 in the icelandic population introductions and early spread of sars-cov-2 in the new york city area investigation of three clusters of covid-19 in singapore: implications for surveillance and response 10 measures genomic surveillance reveals multiple introductions of sars-cov-2 into northern california quasispecies diversity determines pathogenesis through cooperative interactions in a viral population viral quasispecies. virology. 479-480 emergence of coronavirus disease 2019 (covid-19) in austria sars-cov-2 transmission chains from genetic data: a danish case study superspreading and exportation of covid-19 cases from a ski area in austria estimating the burden of sars estimation of covid-19 outbreak size in italy matters of size: genetic bottlenecks in virus infection and their potential impact on evolution pathogen population bottlenecks and adaptive landscapes: overcoming the barriers to disease emergence transmission bottleneck size estimation from pathogen deep-sequencing data, with 15 an application to human influenza a virus shared sars-cov-2 diversity suggests localised transmission of minority variants quantifying 23 influenza virus diversity and transmission in humans stochastic processes constrain the within and between host evolution of influenza virus temporal dynamics in viral shedding and transmissibility of covid-19 virological assessment of hospitalized patients with covid-2019 sociovirology: conflict, cooperation, and 20 communication among viruses evolutionary dynamics in structured populations coast-to-coast spread of sars-cov-2 during the early epidemic in the united 30 investigation of a covid-19 outbreak in germany resulting from a single travel-associated primary case: a case series a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity connecting clusters of covid-19: an epidemiological and serological investigation projecting the transmission dynamics of sars-cov-2 through the postpandemic period a proposal of alternative primers for the artic network's multiplex pcr to improve coverage of sars-cov-2 genome sequencing fastqc a quality control tool for high throughput sequence data fast and accurate short read alignment with burrows-wheeler transform an 5 amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar the sequence alignment/map format and samtools the sequence alignment/map format and samtools lofreq: a sequence-quality aware, ultra-15 sensitive variant caller for uncovering cell-population heterogeneity from highthroughput sequencing datasets a statistical framework for snp calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data a program for annotating and predicting the effects of single nucleotide polymorphisms, snpeff: snps in the genome of drosophila melanogaster strain w1118 using 25 drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program nextstrain: real-time tracking of pathogen evolution effective stochastic algorithm for estimating maximum-likelihood phylogenies treetime: maximum-likelihood phylodynamic analysis viennarna package 2.0 infernal 1.1: 100-fold faster rna homology searches inferring noncoding rna families and classes by means of genome-scale structure-based clustering rnaalifold: improved consensus structure prediction for rna alignments from the cover: fast and reliable prediction 15 of noncoding rnas the two technical replicates of this titration are depicted with different symbols. (d) comparison of variant detection for two independent full processing (pcr amplification, library preparation, sequencing) of the same patient sample, cemm0001. (e) comparison of variant detection for two independent sequencing runs of the same patient sample cemm0008. geographic distribution of clades bases on information for 8,000 strains in the global phylogenetic analysis in this study. (b) distribution of sars-cov-2 from austrian covid-19 sequences over the six phylogenetic clusters identified in this publication. (c) clade 20c of time-resolved phylogenetic trees reconstructed from 7,695 randomly subsampled global strains and 305 austrian strains (left) or all 7675 european high-quality sequences dated before 31 st of march. (d) statistics of foreign exposure history of icelandic covid-19 cases as reported in gisaid. (e) icelandic strains with austrian exposure history matching austrian cluster profiles. (f) exposure history of all sars-cov-2 sequences from icelandic covid-19 cases available on gisaid that match the mutation profile of the phylogenetic cluster tyrol-1. (g) international tourists visiting ischgl between mutational analysis of fixed mutations in sars-cov-2 sequences. (a) ratio of non-synonymous to synonymous mutations in unique mutations identified in austrian sars-cov-2 sequences. (b) frequencies of synonymous and non-synonymous mutations per gene 7 or genomic region normalized to length of the respective gene, genomic region or gene product (nsp1-16). (c) mutational spectra panel. mutational profile of interhost mutations. relative probability of each trinucleotide change for mutations across sars-cov-2 sequences in 7,695 global sequences obtained from gisaid samples plus 305 austrian samples (top) or 305 sars sars-cov-2 sequences from gisaid compared to the expected distribution (based on 10 6 randomizations) according to their tri-nucleotide context. the grey line indicates the mean number of simulated mutations in the window, the colored background represents the distribution of expected mutations (+/-standard deviation with respect to the mean) and the red dots indicate a significant difference (g-test goodness of fit shows a zoom analysis of low-frequency mutations. (a) number of variants detected across the different sample types. (b) number of variants per variant class. (c) mutational profile (relative probability of each trinucleotide) of 6,685 intra-host mutations across austrian 9 mutational profile (relative probability of each trinucleotide) of 1,318,413 intra-host mutations across austrian samples (alleles frequencies less than 0.01) (lower panel). (d) analysis of the mutation rate (analogous to the interhost mutation rate panel) across the sars-cov-2 genome using 2465 rna secondary structure prediction of the upstream 300nt of the sars-cov-2 reference genome (nc 045512.2), comprising the complete 5'untranslated region (utr) and parts of the nsp1 protein nucleotide sequence. the canonical aug start codon is located in a stacked region of sl5 (highlighted in gray). mutational hotspots observed in the austrian sars-cov-2 samples are highlighted in color. two fixed mutations at positions 187 and 241, respectively, are marked in red. low-frequency variants with an abundance between 2% and 50% in individual samples are shown in orange s7: viral intra-host diversity in individual patients. (a) two examples of low key: cord-325959-uqg2xkie authors: bundschuh, christian; egger, margot; wiesinger, kurt; gabriel, christian; clodi, martin; mueller, thomas; dieplinger, benjamin title: evaluation of the edi enzyme linked immunosorbent assays for the detection of sars-cov-2 igm and igg antibodies in human plasma date: 2020-06-08 journal: clin chim acta doi: 10.1016/j.cca.2020.05.047 sha: doc_id: 325959 cord_uid: uqg2xkie background: besides sars-cov-2 rt-pcr testing, serological testing is emerging as additional option in covid-19 diagnostics. aim of this study was to evaluate novel immunoassays for detection of sars-cov-2 antibodies in human plasma. methods: using edi(tm) novel coronavirus covid-19 enzyme linked immunosorbent assays (elisas), we measured sars-cov-2 igm and igg antibodies in 64 sars-cov-2 rt-pcr confirmed covid-19 patients with serial blood samples (n=104) collected at different time points from symptom onset. blood samples from 200 healthy blood donors and 256 intensive care unit (icu) patients collected before the covid-19 outbreak were also used. results: the positivity rates in the covid-19 patients were 5.9% for igm and 2.9% for igg ≤5 days after symptom onset; between day 5 and day 10 the positivity rates were 37.1% for igm and 37.1% for igg and rose to 76.4% for igm and 82.4% for igg after >10-15 days. after 15-22 days the “true” positivity rates were 94.4% for igm and 100% for igg. the “false” positivity rates were 0.5% for igm and 1.0% for igg in the healthy blood donors, 1.6% for igm and 1.2% for igg in icu patients. conclusions: this study shows high “true” vs. low “false” positivity rates for the edi(tm) sars-cov-2 igm and igg elisas. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a novel coronavirus that causes coronavirus disease 2019 (covid-19), has recently emerged to cause a human pandemic. currently, detection of sars-cov-2 with rt-pcr testing from upper or lower respiratory specimens is gold standard method for the confirmation of suspected covid-19 patients [1] [2] [3] . besides sars-cov-2 rt-pcr testing, serological testing is emerging as additional option in covid-19 diagnostics [4] . preliminary data show a potential use of specific sars-cov-2 antibody tests to aid in the diagnosis of suspected covid-19 patients [5, 6] . furthermore, there is great interest in antibody testing of healthcare workers to evaluate hospital transmission and in addition antibody tests could ultimately help to see how far the transmission is in the general population [4] . recently, epitope diagnostics inc. has developed the edi tm coronavirus covid-19 igm and igg enzyme linked immunosorbent assay (elisa) kits. to our knowledge there is only very limited peer reviewed published data in the literature on the performance of these novel immunoassays [7] . before these novel immunoassays can potentially be implemented in clinical routine, they need thorough evaluation in appropriate sized "positive" and "negative" cohorts. therefore, the aim of this study was to perform an analytical and clinical evaluation of the edi tm elisas for the detection of sars-cov-2 igm and igg antibodies in human plasma. this work was performed at the konventhospital barmherzige brueder linz and ordensklinikum linz barmherzige schwestern in linz, austria. the study protocol was approved by the local ethics committee in accordance with the declaration of helsinki,. using vacuette® polyethylene terephthalate glycol blood collection tubes (greiner bio-one, kremsmuenster, austria) edta and lithium-heparin anticoagulated blood were collected and plasma aliquots were frozen at -80°c until further analysis. data were statistically analyzed with the medcalc 13.1.2.0 (medcalc software) and spss 23.0 (spss inc.). we measured sars-cov-2 igm and igg antibodies fully-automated on an immunomat instrument (serion diagnostics) with the edi tm novel coronavirus covid-19 igm and igg enzyme linked immunosorbent assay (elisa) kits (epitope diagnostics inc.). the edi tm novel coronavirus covid-19 igm and igg elisa kits are based on the nucleocapsid protein of sars-cov-2 (n), are ivd ce-marked, and are approved for the qualitative detection of sars-cov-2 igm and igg antibodies in human plasma. the measurement of the sars-cov-2 igm and igg antibodies was performed following the manufacturer´s instruction. the following interpretation rules of the patient results (single run) for the sars-cov-2 igm and igg assays were used: if the patient sample od was below the negative cutoff the result was reported negative (-); if the patient sample od was above the negative cut off but below the positive cutoff the result was reported borderline (+-); if the patients sample od was above the positive cutoff the patient was reported as positive (+). to evaluate the precision of the sars-cov-2 igm and igg assays in our laboratory, we performed a replication study according to the clinical and laboratory standards institute (clsi) guideline ep5-a [8] . one negative patient lithium heparin plasma pool and one positive lithium patient heparin plasma pool were aliquoted into 10 1.5 ml plastic tubes for each concentration level and frozen at -80°c. we analyzed these samples in duplicates in one run per day for 10 days on an immunomat instrument. within-run and total analytical imprecision (cv) was calculated with the clsi single-run precision evaluation test [8] . the detection limits for the sars-cov-2 igm and igg assays were determined by assaying a diluted lithium heparin plasma sample of a healthy individual in replicates of 20 and was calculated as 3 sd added to the mean response of the diluted sample. the diluted sample was prepared by mixing a fresh plasma sample in a 1:10 ratio with sample dilution buffer. this prediluted plasma sample was then treated equally with all other patient samples with respect to the assay procedure. the 20 replicates were assayed on the same microwell plate. for the clinical evaluation we measured sars-cov-2 igm and igg antibodies in three different cohorts: first in a "positive cohort" of patients with sars-cov-2 rt-pcr confirmed 5 covid-19 with serial blood samples at different time points from symptom onset. in these confirmed covid-19 patients sars-cov-2 igm and igg antibodies were clinically suspected. second a cohort of healthy blood donors and third a cohort of patients admitted to an intensive care unit (icu). the healthy blood donors and the icu patients were recruited prior to the covid-19 outbreak (december 3 rd 2019) and serve as "negative cohorts" were no sars-cov-2 igm and igg antibodies were clinically suspected. the sars-cov-2 igm assay had a within-run cv of 7% and a total cv of 10% at a mean optical density (od) of 0.103 (negative patient pool), and within-run cv of 4% and a total cv of 10% at a mean od of 0.297 (positive patient pool). the sars-cov-2 igg assay had a within-run cv of 4% and a total cv of 9% at a mean od of 0.173 (negative patient pool), and within-run cv of 7% and a total cv of 9% at a mean od of 0.537 (positive patient pool). the detection limit was an od of 0.095 for the sars-cov-2 igm and an od of 0.083 for the sars-cov-2 igg assay. 3.2. rates of "true" vs. "false" positivity the final study population consisted of 64 patients (53 males, 11 females) with sars-cov-2 rt-pcr confirmed covid-19 with serial blood samples (n=104) collected at different time points from symptom onset. the median age was 65 years (range 14-95, iqr 56-87, years). table 1 shows low "true" positivity rates (i.e. seroconversion rates) for igm (5.9%) and igg (2.9%) sars-cov-2 antibodies within the first 5 days after symptom onset in patients with covid-19. between day 5 and day 10 the "true" positivity rates were 37.1% for igm and 37.1% for igg. the "true" positivity rates rose to 76.4% for igm and 82.4% for igg after 10-15 days. after 15-22 days the "true" positivity rate was 94.4% for igm and 100% for igg sars-cov-2 antibodies (table 1) . of the 64 patients with confirmed covid-19 two patients (3%) were immune compromised. one patient had an active hemato-onoclogical disease (a diffuse large b-cell lymphoma). this patient did not develop antibodies against sars-cov-2 until 13 days after symptom onset and eventually died of covid 19. the second confirmed covid-19 patient suffered from immune thrombocytopenia and was treated with immune modulating/suppressing therapies. this patient developed sars-cov-2 antibodies on day 13 and fully recovered from covid-19. in the cohort of 200 healthy blood donors the "false" positivity rate was 0.5% for igm and 1% for igg sars-cov-2 antibodies ( table 2 ). the "false" positivity rate in the cohort of 256 icu patients (was 1.6% for igm and 1.2% for igg sars-cov-2 antibodies (table 3 ). in the supplementary data we report the quantitative values of od and the ratio od/negative control in the cohort of patients with sars-cov-2 rt-pcr confirmed covid-19, in the healthy blood donors as well as in the icu patients ( supplementary table 1-3 ). our evaluation of the edi tm novel coronavirus covid-19 elisas for the qualitative detection of sars-cov-2 igm and igg antibodies in human plasma confirms that these novel immunoassays meet the quality specification for laboratory medicine. the cvs were ≤10%, which is adequate for elisas. the detection limits of the sars-cov-2 igm and igg assays were below the negative control and therefore also appropriate. furthermore, the clinical evaluation study demonstrated high "true" positivity rates in the sars-cov-2 rt-pcr confirmed covid-19 patients with symptom onset after 15 days with 94.4% for igm and 100% for igg sars-cov-2 antibodies. finally, we reported low "false" positivity rates in the healthy blood donors with 0.5% for igm and 1% for igg as well as in the icu patients with 1.6% for igm and 1.2% for igg sars-cov-2 antibodies. prior approaches to serologic detection of infection with emerging coronaviruses including sars and middle east respiratory syndrome (mers) have focused on the spike glycoprotein and the nucleocapsid protein, which are considered the immunodominant antigens for these viruses [11] . the edi tm sars-cov-2 igm and igg assays are based on the nucleocapsid protein of sars-cov-2 (n). in agreement with the proposed sars-cov-2 antibody response after symptom onset we found very low seroconversion rates before day 5. whereas after 15 days we found very high seroconversion rates in our sars-cov-2 confirmed covid-19 patients [5, 6, [12] [13] [14] . in line with our findings, it has recently been reported that serological diagnosis is especially important for patients who may present late, beyond the first 2 weeks of illness onset. at this late stage of disease sars-cov-2 rt-pcr might already be negative [15] . it would be tempting to only use sars-cov-2 igg antibody tests to aid in the diagnosis of suspected covid-19 patients. however, when using igm in addition to igg assays, this might allow differentiating an early from a late stage or even past infection. in addition, the combined "false" positivity rates for the edi tm sars-cov-2 igm and igg assays we reported in the healthy blood donors (<2%) and intensive care patients (<3%) was rather low. of note, with the edi tm sars-cov-2 igm and igg immunoassays we only report antibody binding to the recombinant nuceleocapsid protein of the sars-cov-2 and we did not perform neutralization assays in our sars-cov-2 rt-pcr confirmed patients. since the edi tm sars-cov-2 igm and igg is currently approved as qualitative assay, we did not test the linearity of the assays. however, when interpreting the quantitative data (od values 8 and the ratios od/negative control) in supplementary table 1 , we found a clear antibody response in sars-cov-2 antibody positive covid-19 patients from <5 days until >15 days with increasing median od and the values ratios od/negative control, demonstrating that the edi tm sars-cov-2 igm and igg assays are suitable for serial measurements. this study is the first thorough evaluation of the edi tm sars-cov-2 igm and igg elisas and we reported high "true" vs. low "false" positivity rates, indicating the suitability of these novel immunoassays for clinical routine. competing interests: none declared. laboratory testing for coronavirus disease (covid-19) in suspected human cases intermin guidance 19 th detection of 2019 novel coronavirus (2019-ncov) by realtime rt-pcr causing an outbreak of pneumonia report from the american society for microbiology covid-19 international summit profiling early humoral response to diagnose novel coronavirus disease (covid-19) antibody responses to sars-cov-2 in patients of novel coronavirus disease use of convalescent plasma therapy in two covid-19 patients with acute respiratory distress syndrome in korea evaluation of precision performance of clinical chemistry devices; approved guideline. clsi document ep5-a analytical and clinical evaluation of a novel high-sensitivity assay for measurement of soluble st2 in human plasma -the presagetm st2 assay prognostic value of soluble st2 in an unselected cohort of patients admitted to an intensive care unit -the linz intensive care unit (licu) study evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses virological assessment of hospitalized patients with covid-2019 analytical performances of a chemiluminescence immunoassay for sars-cov-2 igm/igg and antibody kinetics interpreting diagnostic tests for sars-cov-2 key: cord-311214-eqwxkwqa authors: kumar, roshan; verma, helianthous; singhvi, nirjara; sood, utkarsh; gupta, vipin; singh, mona; kumari, rashmi; hira, princy; nagar, shekhar; talwar, chandni; nayyar, namita; anand, shailly; rawat, charu dogra; verma, mansi; negi, ram krishan; singh, yogendra; lal, rup title: comparative genomic analysis of rapidly evolving sars-cov-2 viruses reveal mosaic pattern of phylogeographical distribution date: 2020-04-16 journal: biorxiv doi: 10.1101/2020.03.25.006213 sha: doc_id: 311214 cord_uid: eqwxkwqa the coronavirus disease-2019 (covid-19) that started in wuhan, china in december 2019 has spread worldwide emerging as a global pandemic. the severe respiratory pneumonia caused by the novel sars-cov-2 has so far claimed more than 60,000 lives and has impacted human lives worldwide. however, as the novel sars-cov-2 displays high transmission rates, their underlying genomic severity is required to be fully understood. we studied the complete genomes of 95 sars-cov-2 strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. we show that there is no direct transmission pattern of the virus among neighboring countries suggesting that the outbreak is a result of travel of infected humans to different countries. we revealed unique single nucleotide polymorphisms (snps) in nsp13-16 (orf1b polyprotein) and s-protein within 10 viral isolates from the usa. these viral proteins are involved in rna replication, indicating highly evolved viral strains circulating in the population of usa than other countries. furthermore, we found an amino acid addition in nsp16 (mrna cap-1 methyltransferase) of the usa isolate (mt188341) leading to shift in amino acid frame from position 2540 onwards. through the construction of sars-cov-2-human interactome, we further revealed that multiple host proteins (phb, ppp1ca, tgf-β, socs3, stat3, jak1/2, smad3, bcl2, cav1 & specc1) are manipulated by the viral proteins (nsp2, pl-pro, n-protein, orf7a, m-s-orf3a complex, nsp7-nsp8-nsp9-rdrp complex) for mediating host immune evasion. thus, the replicative machinery of sars-cov-2 is fast evolving to evade host challenges which need to be considered for developing effective treatment strategies. downloaded on march 19, 2020 from ncbi database and based on quality assessment two 126 genomes with multiple ns were removed from the study. further the genomes were annotated 127 using prokka [22] . a manually annotated reference database was generated using the genbank 128 file of severe acute respiratory syndrome coronavirus 2 isolate-sars-cov-129 2/sh01/human/2020/chn (accession number: mt121215) and open reading frames (orfs) 130 were predicted against the formatted database using prokka (-gcode 1) [22] . further the gc 131 content information was generated using quast standalone tool [23] . 132 to determine the evolutionary pressure on viral proteins, dn/ds values were calculated for 9 134 orfs of all strains. the orthologous gene clusters were aligned using muscle v3. team, 2015) . 139 to infer the phylogeny, the core gene alignment was generated using mafft [27] present 141 within the roary package [28] . further, the phylogeny was inferred using the maximum 142 likelihood method based and tamura-nei model [29] in megax [30] and visualized in 143 interactive tree of life (itol) [31] and grapetree [32] . 144 to determine the single nucleotide polymorphism (snp), whole-genome alignments were 145 made using libmuscle aligner. for this, we used annotated genbank of sars-cov-146 2/sh01/human/2020/chn (accession no. mt121215) as the reference in the parsnp tool of 147 harvest suite [33] . as only genomes within a specified mumi distance threshold are recruited, 148 we used option -c to force include all the strains. for output, it produced a core-genome 149 alignment, variant calls and a phylogeny based on single nucleotide polymorphisms. the snps 150 were further visualized in gingr, a dynamic visual platform [33] . further, the tree was 151 visualized in interactive tree of life (itol) [31] . 152 sars-cov-2 protein annotation and host-pathogenic interactions 153 updated in any protein database, we first annotated the genes using blastp tool [34] . the 156 similarity searches were performed against sars-cov isolate tor2 having accession no. 157 ay274119 selected from ncbi at default parameters. the annotated sars-cov-2 proteins 158 were mapped against virusite [35] and interaction databases such as virus.string v10.5 159 [36] and intact [37] for predicting their interaction against host proteins. these proteins were 160 either the direct targets of hcov proteins or were involved in critical pathways of hcov 161 infection identified by multiple experimental sources. to build a comprehensive list of human 162 ppis, we assembled data from a total of 18 bioinformatics and systems biology databases with 163 five types of experimental evidence: (i) binary ppis tested by high-throughput yeast two-hybrid 164 (y2h) systems; (ii) binary, physical ppis from protein 3d structures; (iii) kinase-substrate 165 interactions by literature-derived low-throughput or high-throughput experiments; (iv) 166 signaling network by literature-derived low-throughput experiments; and (v) literature-curated 167 ppis identified by affinity purification followed by mass spectrometry (ap-ms), y2h, or by 168 literature-derived low [36, 38] . 169 filtered proteins (confidence value: 0.7) were mapped to their entrez id [39] based on the 170 ncbi database used for interactome analysis. hpi were stimulated using cytoscape v.3.7.2 171 [40] . 172 next, functional studies were performed using the kyoto encyclopedia of genes and genomes 174 (kegg) [41, 42] and gene ontology (go) enrichment analyses using uniprot database [43] 175 to evaluate the biological relevance and functional pathways of the hcov-associated proteins. 176 all functional analyses were performed using string enrichment and stringify, plugin of 177 cytoscape v.3.7.2 [40] . network analysis was performed by tool networkanalyzer, plugin of 178 cytoscape with the orthogonal layout. 179 general genomic attributes of sars-cov-2 181 in this study, we analyzed a total of 95 sars-cov-2 strains (available on march 19, 2020) 182 isolated between december 2019-march 2020 from 11 different countries namely usa (n=52), 183 china (n=30), japan (n=3), india (n=2), taiwan (n=2) and one each from australia, brazil, 184 oronasopharynges or lungs, while two of them were isolated from faeces suggesting both 186 respiratory and gastrointestinal connection of sars-cov-2 (table 1) . no information of the 187 source of isolation of the remaining isolates is available. the average genome size and gc 188 content were found to be 29879 ± 26.6 bp and 37.99 ± 0.018%, respectively. all these isolates 189 were found to harbor 9 open reading frames coding for orf1a (13218 bp) and orf1b (7788 190 bp) polyproteins, surface glycoprotein or s-protein (3822 bp), orf3a protein (828 bp our analysis revealed that strains of human infecting sars-cov-2 are novel and highly 201 identical (>99.9%). a recent study established the closest neighbor of sars-cov-2 as sarsr-202 cov-ratg13, a bat coronavirus [45] . as covid19 transits from epidemic to pandemic due 203 to extremely contagious nature of the sars-cov-2, it was interesting to draw the relation 204 between strains and their geographical locations. in this study, we employed two methods to 205 delineate phylogenomic relatedness of the isolates: core genome ( figure 1a & c) and single 206 nucleotide polymorphisms (snps) ( figure 1b ). phylogenies obtained were annotated with 207 country of isolation of each strain ( figure 1a & b). the phylogenetic clustering was found 208 majorly concordant by both core-genome ( figure 1a ) and snp based methods ( figure 1b) . 209 the strains formed a monophyletic clade, in which mt093571.1 (south korea) and 210 mt039890.1 (sweden) were most diverged. focusing on the edge-connection between the 211 neighboring countries from where the transmission is more likely to occur, we noted a strain 212 from taiwan (mt066176) closely clustered with another from china (mt121215.1). with the 213 exception of these two strains, we did not find any connection between strains of neighboring 214 countries. thus, most strains belonging to the same country clustered distantly from each other 215 and showed relatedness with strains isolated from distant geographical locations ( figure 1a & 216 b). for instance, a sars-cov-2 strain isolated from nepal (mt072688) clustered with a strain 217 from usa (mt039888). also, strains from wuhan (lr757998 and lr757995), where the 218 virus was originated, showed highest identity with usa as well as china strains; strains from 219 india, mt012098 and mt050493 clustered closely with china and usa strains, respectively 220 ( figure 1a & b) . similarly, australian strain (mt007544) showed close clustering with usa 221 strain ( figure 1a & b) and one strain from taiwan (mt066175) clustered nearly with chinese 222 isolates ( figure 1b ). isolates from italy (mt012098) and brazil (mt126808) clustered with 223 different usa strains ( figure 1a & b) . notably, isolates from same country or geographical 224 location formed a mosaic pattern of phylogenetic placements of countries' isolates. for viral 225 transmission, contact between the individuals is also an important factor, supposedly due to 226 which the spread of identical strains across the border of neighboring countries is more likely. 227 but we obtained a pattern where indian strains showed highest similarity with usa and china 228 strains, australian strains with usa strains, italy and brazilian strains with strains isolated 229 from usa among others. this depicts the viral spread across different communities. however, 230 as genomes of sars-cov-2 were available mostly from usa and china, sampling biases is 231 evident in analyzed dataset as available on ncbi. thus, it is plausible for strains from other 232 countries to show most similarity with strains from these two countries. in the near future as 233 more and more genome sequences will become available from different geographical locations; 234 more accurate patterns of their relatedness across the globe will become available 235 snps in all predicted orfs in each genome were analyzed using sars-cov-237 2/sh01/human/2020/chn as a reference. snps were determined using maximum unique 238 matches between the genomes of coronavirus, we observed that the strains isolated from usa 239 although the primary mode of infection is human to human transmission through close contact, 281 which occurs via spraying of nasal droplets from the infected person, yet the primary site of 282 infection and pathogenesis of sars-cov-2 is still not clear and under investigation. to 283 explore the role of sars-cov-2 proteins in host immune evasion, the sarscov-2 proteins 284 were mapped over host proteome database ( figure 3b & table 3 ). we identified a total of 28 285 proteins from host proteome forming close association with 25 viral proteins present in 9 orfs 286 of sars-cov-2 ( figure 3c ). the network was trimmed in cytoscape v3.7.2 where only 287 interacting proteins were selected. only 12 viral proteins were found to interact with host 288 proteins ( figure 3a ). detailed analysis of interactome highlighted 9 host proteins in direct 289 association with 6 viral proteins. further, the network was analyzed for identification of 290 regulatory hubs based on degree analysis. we identified mitogen activated protein kinase 1 291 similarly, the viral protein papain-like proteinase (pl-pro) which has deubiquitinase and 309 deisgylating activity is responsible for cleaving viral polyprotein into 3 mature proteins which 310 are essential for viral replication [57] . our study showed that pl-pro directly interacts with 311 ppp1ca which is a protein phosphatase that associates with over 200 regulatory host proteins 312 to form highly specific holoenzymes. pl-pro is also found to interact with tgfβ which is a 313 beta transforming growth factor and promotes t-helper 17 cells (th17) agreement with our prediction where we found il6 as an interacting partner. our study also 322 showed jak1/2 as an interacting partner which is known for ifnγ signaling. it is well known 323 that tgfβ along with il6 and stat3 promotes th17 differentiation by inhibiting socs3 324 [62]. th17 is a source of il17, which is commonly found in serum samples of covid-19 325 patients [61, 63] . hence, our interactome is supported from these findings where we found 326 socs3, stat3, jak1/2 as an interacting partner [64] . the results suggested that 327 proinflammatory cytokine storm is one of the reasons for sars-cov-2 mediated 328 immunopathogenesis. 329 in the next cycle of physical events the viral protein nc (nucleoprotein), which is a major 330 structural part of sarv family associates with the genomic rna to form a flexible, helical 331 nucleocapsid. interaction of this protein with smad3 leads to inhibition of apoptosis of sars-332 cov infected lung cells [65] , which is a successful strategy of immune evasion by the virus. membranes. cav1 has been reported to be involved in viral replication, persistence, and the 346 potential role in pathogenesis in hiv infection also [71] . thus, orf3a interactions will 347 upregulate viral replication thus playing a very crucial role in pathogenesis. multiple 348 structural proteins were observed to target the specc1 proteins and linked with cytokinesis 350 and spindle formations during division. thus, major viral assembly also targets the proteins 351 linked with immunity and cell division. taken together, we estimated that sars-cov-2 352 manipulate multiple host proteins for its survival while, their interaction is also a reason for 353 immunopathogenesis. 354 as covid-19 continues to impact virtually all human lives worldwide due to its extremely 356 contagious nature, it has spiked the interest of scientific community all over the world to 357 understand better the pathogenesis of the novel sars-cov-2. in this study, the analysis was 358 performed on the genomes of the novel sars-cov-2 isolates recently reported from different 359 countries to understand viral pathogenesis. with the limited data available so far, we observed 360 no direct transmission pattern of the novel sars-cov-2 in the neighboring countries through 361 our analyses of the phylogenomic relatedness of geographical isolates. the isolates from same 362 locations were phylogenetically distant, for instance, isolates from the usa and china. thus, 363 there appears to be a mosaic pattern of transmission indicative of the result of infected human 364 travel across different countries. as covid-19 transited from epidemic to pandemic within a 365 short time, it does not look surprising from the genome structures of the viral isolates. the 366 genomes of six isolates, specifically from the usa, were found to harbor unique amino acid 367 snps and showed amino acid substitutions in orf1b protein and s-protein, while one of them 368 also harbored an amino-acid addition. this is suggestive of the severity of the mutating viral 369 genomes within the population of the usa. these proteins are directly involved in the 370 formation of viral replication-transcription complexes (rtc). therefore, we argue that the 371 novel sars-cov-2 has fast evolving replicative machinery and that it is urgent to consider 372 these mutants to develop strategies for covid-19 treatment. the orf1ab polyprotein protein 373 and s-protein were also found to have dn/ds values approaching 1 and thus might confer a 374 selective advantage to evade host responsive mechanisms. the construction of sars-cov-2-375 human interactome revealed that its pathogenicity is mediated by a surge in pro-inflammatory 376 cytokine. it is predicted that major immune-pathogenicity mechanism by sars-cov-2 377 includes the host cell environment alteration by disintegration by signal transduction pathways 378 and immunity evasion by several protection mechanisms. the mode of entry of this virus by 379 s-proteins inside the host cell is still unclear but it might be similar to sars cov-1 like 380 viruses. lastly, we believe as more data accumulate for covid-19 the evolutionary pattern 381 will become much clear. 382 isolates. highly similar genomes of coronaviruses were taken as input by parsnp. whole-591 genome alignments were made using libmuscle aligner using the annotated genome of 592 mt121215 strain as reference. parsnp identifies the maximal unique matches (mums) among 593 the query genomes provided in a single directory. as only genomes within a specified mumi 594 distance threshold are recruited, option -c to force include all the strains was used. the output 595 phylogeny based on single nucleotide polymorphisms was obtained following variant calling 596 on core-genome alignment. c) the minimum spanning tree generated using maximum 597 likelihood method and tamura-nei model showing the genetic relationships of sars-cov-2 598 isolates with their geographical distribution. 599 table 1 : general genomic attributes of sars-cov-2 strains. 618 structural and functional basis of 402 sars-cov-2 entry by using human ace2 structure, function, 404 and antigenicity of the sars-cov-2 spike glycoprotein genome composition and 406 divergence of the novel coronavirus (2019-ncov) originating in china sars3a/orf3a sars3b/orf3b sars7a/orf7 sars7b/orf8 key: cord-305587-xtqvtleb authors: ma, cuiqing; su, shan; wang, jiachao; wei, lin; du, lanying; jiang, shibo title: from sars-cov to sars-cov-2: safety and broad-spectrum are important for coronavirus vaccine development date: 2020-05-11 journal: microbes infect doi: 10.1016/j.micinf.2020.05.004 sha: doc_id: 305587 cord_uid: xtqvtleb the global pandemic of covid-19 caused by sars-cov-2 (also known as 2019-ncov and hcov-19) has posed serious threats to public health and economic stability worldwide, thus calling for development of vaccines against sars-cov-2 and other emerging and reemerging coronaviruses. since sars-cov-2 and sars-cov have high similarity of their genomic sequences and share the same cellular receptor (ace2), it is essential to learn the lessons and experiences from the development of sars-cov vaccines for the development of sars-cov-2 vaccines. in this review, we summarized the current knowledge on the advantages and disadvantages of the sars-cov vaccine candidates and prospected the strategies for the development of safe, effective and broad-spectrum coronavirus vaccines for prevention of infection by currently circulating sars-cov-2 and other emerging and reemerging coronaviruses that may cause future epidemics or pandemics. in december 2019, the outbreak of an unexplained pneumonia similar to severe acute 32 respiratory syndrome (sars) in wuhan, china was reported by the health commission of hubei 33 province, china. this severe respiratory illness was identified by multiple diagnostic methods as an 34 infection by a novel coronavirus (1) (2) (3) (4) , which was temporarily denoted as 2019-ncov by world 35 health organization (5) ， and renamed "severe acute respiratory syndrome coronavirus 2" against sars-cov using their own vaccine platforms (33) (34) (35) (36) (37) . however, most vaccines are in preclinical studies, and only a few of them have been reported to under assessment in clinical trials 111 (https://clinical trials.gov/ct2/show/nct03615911; https://clinical trials.gov/ct2/show/nct0339578). 112 currently, no vaccine has been approved for the prevention of sars. since effective antiviral 113 strategies to control sars-cov and sars-cov-2 infections are still lacking, vaccination is still 114 regarded as the major approach for preventing potential re-emergence of sars-cov, and more 129 it is known that most of the current influenza vaccines are inactivated vaccines, which plays a and is crucial for determining host tropism and transmission capacity (61, 62) . generally, the s protein 189 of coronavirus is functionally divided into the s1 subunit, responsible for receptor binding, and the generating s1 and s2 subunits is located at r694/s695 (67) . in addition, it has been reported that s 193 protein has strong immunogenicity and can induce high titer neutralizing antibody (63) . rbd of 194 sars-cov s1 is located in s318-510 and the key rbm is s425-494, of which r453 is critical for for the whole protein, around 73%-76% for the rbd, and 50%-53% for the rbm (7, 30) . in s1 subunit, sars-cov-2 and sars-cov shared around 50 conserved amino acids, and the 204 three-dimensional structure of sars-cov-2 rbd was composed of a core and an external 205 subdomain, which was more similar to that of sars-cov (29) . we aligned the sequence of s protein protective immunity (35, (78) (79) (80) . for example, the expression of full-length s protein and its trimer of recombinant s1 or s2 proteins, respectively, and found that anti-s1 and anti-s2 iggs were able to 304 abolish the binding between s protein and its cellular receptor(s), although anti-s1 igg showed a 305 significantly higher blocking efficiency (106) . as shown in figure 2b , the amino acid sequences of 306 hr1 and hr2 are highly conserved and homologous between sars-cov and sars-cov-2. in fact, 307 these domains are also highly conserved in other coronaviruses (104) , thus the s2 subunit has potential 308 to be used as a target for the development of pan-cov vaccine against divergent virus strains. altogether, these results suggested that the s2 domain of sars-cov s protein, as a vaccine rbds. the red number indicates the core amino acids in rbd when it binds to receptor ace2. the green frame is the amino acid sequence of rbm. b. sequence alignment of sars-cov and sars-cov-2 hr1. the red frame is the location where the variable amino acid residues between sars-cov and sars-cov-2 hr1. importation and human-to-human 437 transmission of a novel coronavirus in vietnam a novel coronavirus (2019-ncov) causing pneumonia-associated respiratory syndrome nowcasting and forecasting the potential 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coronaviruses as the gene source of alphacoronavirus 463 and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus epidemiology, genetic recombination, and pathogenesis of 466 coronaviruses molecular evolution of human coronavirus genomes origin and evolution of pathogenic coronaviruses the 2019-new coronavirus epidemic: 471 evidence for virus evolution structural and functional basis of sars-cov-2 entry by using 482 human ace2 inhibition of sars-cov-2 infections in 484 engineered human tissues using clinical-grade soluble human ace2 sars vaccine development characterization of humoral responses in mice 488 immunized with plasmid dnas encoding sars-cov spike gene fragments the spike protein of sars-cov -a target for vaccine and therapeutic 491 development peptide nanoparticles as novel 493 immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine development of subunit vaccines against severe acute respiratory syndrome the covid-19 vaccine 498 development landscape immunogenicity of sars inactivated vaccine in 500 balb/c mice a subcutaneously injected uv-inactivated 502 sars coronavirus vaccine elicits systemic humoral immunity in mice inactivated sars-cov vaccine prepared from whole virus induces a 504 high level of neutralizing antibodies in balb/c mice identification and characterization of novel neutralizing epitopes in the 506 receptor-binding domain of sars-cov spike protein: revealing the critical antigenic determinants in inactivated 507 sars-cov vaccine inactivated sars-cov vaccine elicits high titers of spike protein-specific antibodies 509 that block receptor binding and virus entry immunization with sars 511 coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus glycan arrays lead to the discovery of autoimmunogenic activity of sars-cov evaluation of antibody-dependent enhancement of sars-cov infection in 515 rhesus macaques immunized with an inactivated sars-cov vaccine rationalizing the development of live attenuated virus vaccines engineering attenuated virus vaccines by controlling replication fidelity severe acute respiratory syndrome 521 coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice adenoviral expression of a truncated s1 intranasal vaccination of recombinant adeno-associated virus 533 encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (sars-cov) spike protein induces 534 strong mucosal immune responses and provides long-term protection against sars-cov infection immunization with modified vaccinia virus 537 ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in 538 ferrets a live, impaired-fidelity coronavirus vaccine 540 protects in an aged, immunocompromised mouse model of lethal disease evaluation of a recombination-resistant coronavirus as a 542 broadly applicable, rapidly implementable vaccine platform safety and immunogenicity from a phase i trial of inactivated severe acute 544 respiratory syndrome coronavirus vaccine a sars dna vaccine induces 546 neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial structure, function, and evolution of coronavirus spike proteins a pan-coronavirus fusion inhibitor targeting the hr1 550 domain of human coronavirus spike bat-to-human: spike features determining 'host jump'of coronaviruses sars-cov, mers-cov, 552 and beyond mers-cov spike protein: targets for vaccines and therapeutics receptor-binding domain of sars-cov spike protein induces highly 556 potent neutralizing antibodies: implication for developing subunit vaccine fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 559 domain in spike protein functional analysis of an epitope in the s2 subunit of the murine coronavirus spike protein: 69 cross-host evolution of severe acute respiratory 571 syndrome coronavirus in palm civet and human identification of two critical amino acid residues of the 573 severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double 574 substitution strategy structural analysis of major species barriers between humans and palm civets for severe acute respiratory 576 syndrome coronavirus infections receptor recognition and cross-species infections of sars coronavirus mechanisms of host receptor adaptation by severe acute respiratory 579 syndrome coronavirus a 193-amino acid fragment of the sars coronavirus s protein 581 efficiently binds angiotensin-converting enzyme 2 a single amino acid substitution (r441a) in the receptor-binding domain of sars coronavirus 583 spike protein disrupts the antigenic structure and binding activity inhibition of sars-cov-2 (previously 2019-ncov) infection by a 585 highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate 586 membrane fusion antibodies against trimeric s glycoprotein 588 protect hamsters against sars-cov challenge despite their capacity to mediate fcγrii-dependent entry into b cells in 589 vitro a 219-mer cho-expressing receptor-binding domain of sars-cov s 591 protein induces potent immune responses and protective immunity recombinant receptor-binding domain of sars-cov spike 593 protein expressed in mammalian, insect and e. coli cells elicits potent neutralizing antibody and protective immunity antigenic and immunogenic characterization of recombinant 596 baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design anti-severe acute respiratory syndrome coronavirus 599 spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcγr pathway antibody-dependent sars coronavirus infection is 602 mediated by antibodies against spike proteins sars cov subunit vaccine: antibody-mediated 604 neutralisation and enhancement anti-spike igg causes severe acute lung injury by skewing 606 macrophage responses during acute sars-cov infection the role of cd4 and cd8 t cells in mhv-jhm-induced demyelination immunodominant sars coronavirus epitopes in humans 610 elicited both enhancing and neutralizing effects on infection in non-human primates recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies 613 primarily targeting the receptor binding region identification of a critical neutralization determinant of severe acute 615 respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines identification of murine cd8 t cell epitopes in 617 codon-optimized sars-associated coronavirus spike protein antigenicity and immunogenicity of sars-cov s protein 619 receptor-binding domain stably expressed in cho cells receptor-binding domain of severe acute respiratory syndrome coronavirus 621 spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies. the 622 mers-cov spike protein: a key target for antivirals advances in mers-cov vaccines and therapeutics based on the 626 receptor-binding domain receptor-binding domain of sars-cov spike protein induces 628 long-term protective immunity in an animal model priming with raav encoding rbd of sars-cov s protein and 630 boosting with rbd-specific peptides for t cell epitopes elevated humoral and cellular immune responses against 631 sars-cov infection searching for an ideal vaccine candidate among different 633 mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design cross-neutralization of human and palm civet severe acute 636 respiratory syndrome coronaviruses by antibodies targeting the receptor-binding domain of spike protein potent binding of 2019 novel coronavirus spike protein by a sars 639 coronavirus-specific human monoclonal antibody sars-cov fusion peptides induce membrane surface 641 ordering and curvature expression and characterization of recombinant s2 subunit of sars-coronavirus s 643 fusion protein evaluation of candidate vaccine approaches for 645 mers-cov a novel neutralizing monoclonal antibody targeting the 647 n-terminal domain of the mers-cov spike protein human monoclonal antibodies against highly conserved 649 hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing elicitation of immunity in mice after immunization with the s2 651 subunit of the severe acute respiratory syndrome coronavirus quantitative comparison of the efficiency of antibodies 653 against s1 and s2 subunit of sars coronavirus spike protein in virus neutralization and blocking of receptor binding: 108 specific asparagine-linked glycosylation sites are critical for dc-sign-and 661 l-sign-mediated severe acute respiratory syndrome coronavirus entry current advancements and potential strategies in the development of mers-cov vaccines identification of an ideal adjuvant for 665 receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus virus detectives seek source of sars in china's wild animals isolation and characterization of viruses related to the sars 669 coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus 673 in chinese horseshoe bats alveolar surfactant homeostasis and the pathogenesis of pulmonary disease cgas produces a 2'-5'-linked cyclic dinucleotide 677 second messenger that activates sting cyclic gmp-amp is an endogenous second messenger in innate 679 immune signaling by cytosolic dna pivotal roles of cgas-cgamp signaling in antiviral defense and 681 immune adjuvant effects pulmonary surfactant-biomimetic nanoparticles potentiate 683 heterosubtypic influenza immunity prospects for a dengue virus vaccine don't rush to deploy covid-19 vaccines and drugs without sufficient safety guarantees key: cord-321855-7b1c2xdh authors: alshami, alanoud; alattas, rabab; anan, hadeel; alhalimi, abdulbary; alfaraj, ahmed; al qahtani, hadi title: silent disease and loss of taste and smell are common manifestations of sars-cov-2 infection in a quarantine facility: saudi arabia date: 2020-10-30 journal: plos one doi: 10.1371/journal.pone.0241258 sha: doc_id: 321855 cord_uid: 7b1c2xdh objectives: in this study, we aimed to study the clinical presentations, and viral clearance of sars-cov-2 positive quarantined individuals. design: cross-sectional study. setting: governmentaldesignated facility in the eastern province, saudi arabia. participants: 128 laboratory-confirmed covid-19 quarantined individuals who had a history of travel abroad in the last 14 days before the quarantine or were in direct contact with laboratory-confirmed cases. the study was from march 18th-till april 16th. primary and secondary measures: the clinical presentation, prevalence of asymptomatic carriers among sars-cov-2 positive quarantined subjects, and the difference between virus clearance among symptomatic and asymptomatic individuals. results: sixty-nine of the 128 residents (54%) were completely asymptomatic until the end of the study. the remaining 59 residents (46%) had only mild symptoms. the most common symptom was a sudden loss of smell and taste, accounting for 47.5%. the median time to virus clearance was significantly different between the two groups. symptomatic residents cleared the virus at a median of 17 days (95% ci, 12.4–21.6) from the first positive pcr vs. 11days (95% ci, 8.7–13.3) in the asymptomatic group (p = 0.011). false-negative test results occurred in 18.8% of the total residents and false-positive results in 3%. conclusion: the prevalence of asymptomatic carriers among quarantined travelers and those identified by contact tracing is high in our study. therefore, testing, tracing, and isolating travelers and contacts of laboratory-confirmed cases, regardless of symptoms, were very effective measures for early disease identification and containment. loss of taste and smell were the most common presentations in our mild symptomatic residents and should be used as a screening tool for covid-19. the persistent positive pcr beyond 14 days observed in the mild symptomatic residents despite being symptoms free, warrant further studies to determine its implications on disease spread and control. introduction a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first discovered on december 31st, 2019, in wuhan city, china, after a cluster of atypical pneumonia was observed. this infection was labeled as corona virus disease 2019 (covid 19) [1] . this has led to an outbreak of infection in china that spread globally over a few months and was declared a pandemic by the who on march 11th, 2020. currently, most reported cases are from outside china. as of july 19th, 2020, there are 14 270 805 confirmed cases worldwide, and 250,920 of these cases are in saudi arabia, with 2486 reported deaths. although we have gradually gained some insight into the clinical presentation spectrum of covid-19 from other countries' experiences and published data, we still do not have enough information about the real spectrum of the disease and the prevalence and clinical characteristics of asymptomatic carriers. goa et al. have conducted the first and largest covid-19 epidemiological study in china. their study included 72, 314 subjects, and they described a mild disease course in about 80% of their cohort, severe disease in 14%, critical in 5%, and asymptomatic in only 1.2% [2] . the possibility of developing asymptomatic infection has further been reported in larger percentages in multiple small reports [3, 4] however large data is still lacking. hu et al. have examined 24 asymptomatic infected individuals with a history of close contact with sars-cov-2 confirmed cases and found that only 20% of them developed symptoms. the importance of detecting asymptomatic carriers relies on their ability to spread the disease. this assumption has been illustrated by different reports [4] [5] [6] . however, it has not been validated yet in large scale studies. a recent study has shown that the viral load detected in asymptomatic patients was similar to the viral load of symptomatic patients suggesting that asymptomatic patients can also transmit the disease [7] . the fact that there is a group of asymptomatic carriers who are roaming around unrecognized and might be able to transmit the disease imposes a serious public health threat if not early identified and contained. identifying the asymptomatic carriers early on will help us better understand the dynamics of the disease spread and guide us to find better measures of disease containment and mitigations. in this study, we aimed to study the clinical characteristics among quarantined individuals with positive sars-cov-2 pcr and viral clearance in quarantined individuals with covid-19. this study was approved by the institutional review board at king fahad specialist hospital-dammam. as this study does not carry more than minimal risk, we only obtained a verbal consent that was taken in the beginning of the phone call conversation. the verbal approval to participate was documented by the research team using a web-based data collection tool (red-cap). for all minors involved in the research, verbal consent and clinical information were taken from their parents. this is a cross-sectional study conducted in a quarantine facility in al-khobar city in the eastern province of saudi arabia. the facility was designated only for sars-cov-2 positive cases confirmed by real-time polymerase chain reaction (rt-pcr) using combined nasopharyngeal and oropharyngeal samples. residents were travelers who tested positive, confirmed cases with mild disease transferred from hospitals, or confirmed cases admitted directly from the community after being traced by the regional public health authority. all residents admitted from march 16th until april 6th, whether adults or children, were included. admission to the quarantine was restricted to low-risk stable patients with mild symptoms only. elderly patients >65 years of age with more than one comorbidity were not allowed in this quarantine. all residents were followed up daily by nurses with symptoms screening checklist. following up, residents on daily bases have allowed us to differentiate between 3 categories of patients: true asymptomatic carriers (no symptoms at all), pre-symptomatic (developed symptoms in the quarantine), and symptomatic patients who had symptoms during or before admission. all data was collected upon presentation to the quarantine. data were collected prospectively by nurses using the standardized case report forms, generated by modified who/ international severe acute respiratory and emerging infection consortium case record form for severe acute respiratory infections [8] . all data collected was verified by the research team through a short phone survey to ascertain the accuracy of the data, especially the symptoms presented within the last 14 days before admission to the quarantine. the collected data included symptoms-based screening checklists, such as a new onset of cough, sore throat, runny nose, fever, and myalgia-atypical symptoms like headache, nausea and vomiting, diarrhea, and loss of taste and smell. history of comorbidities was taken by self-reported medical history, and it included: hypertension, diabetes mellitus, chronic lung disease, renal disease, and cardiovascular disease. history of pregnancy and current smoking status were also recorded. pcr samples were taken for each resident upon admission and were repeated every 72 hours as long they were positive. these samples were sent to the regional laboratory for processing, and the results were uploaded on the hesn database (saudi public health electronic database). patients were deemed infection-free if they had two negative pcrs 24 hours apart (as per saudi cdc guideline). we also calculated the percentage of false-negative and falsepositive tests. a false-negative test was defined as a negative test that was preceded and followed by a positive test within 24-72 hours' time frame. a false-positive result was defined as a positive test that was preceded and followed by a negative test within 24-72 hours' time frame. we mainly used descriptive statistics. quantitative/continuous variables were presented as mean ± standard deviation (sd) or median with inter-quartile range (iqr). frequencies, proportions were used to describe qualitative data. bivariate analysis was performed using an independent sample t-test or mann-whitney u test, which is appropriate. survival analysis was used to determine the time from the first positive pcr until the first truly negative one. rt-pcr test and sampling. detection of sars-co-v-2 in respiratory samples was performed by rt-pcr using the altona kit (real star sars-cov-2 rt-pcr kit) (usa). the kits include detection of e gene sequence specific for all b-beta-corona virus and sars-cov-2 specific sequence of s gene. samples for pcr were collected simultaneously from the nasopharynx and oropharynx following the who instructions for sample taking. each set of the test was reported as screening and confirmatory. rna was extracted and reverse-transcribed to cdna. then real-time pcr was performed as instructed by the manufacture using specific primers and probes targeting the sars-cov-2 genomes. internal control is included in the assay to rule exclude reaction inhibition. no patients or public were involved in this study during the design, conduct, or analysis phases. among the 128 patients included in the study, 69 (53.9%) were female table 1. the mean age was 39.6 years, with a range of (8-76 years). among the 128 residents, four children <16 years were included. three out of the four children were asymptomatic, and one child presented with mild fever and cough. sixty-five residents (50.8%) had a history of travel outside the kingdom within the last 14 days before admission to the quarantine. iran and iraq being the most visited countries accounting for (68.2%) followed by the uk (10.61%) and the rest of countries accounting for the remaining 21.2%. sixty-two residents (49.2%) had a history of direct exposure to a lab-confirmed case, and one resident was screened based on symptoms and tested positive without a clear history of travel or contact with a suspected or lab-confirmed case. sixty-nine patients (54.3%) did not exhibit any symptoms (asymptomatic carriers/ silent disease), while the other 59 patients (45.7%) had only mild symptoms. the most common reported symptom among our residents was a complete and sudden loss of smell and taste, 28 (47.5%). these symptoms appeared at a median of 6 days (iqr, 4-9 days) after the onset of fever or upper respiratory tract symptoms. however, it was the only clinical symptom in 18.65% of patients. residents also reported a history of cough in (40.7%), myalgia in (39%), and headache in (37.3%). the median time for symptoms resolution was five days (iqr 3-11days) . out of the 59 symptomatic patients, 11(18.64%) were pre-symptomatic, who subsequently developed symptoms at a mean of 7.78 days (+/-5.7 sd) after the first positive pcr while in quarantine. the median incubation period in symptomatic residents was seven days (iqr, 3.75-12 days). a total of 664 pcr tests have been done for the 128 residents. the median number of tests per person was 5 (iqr, 4-6 tests). twenty-four residents had at least one false negative test (18.8%) in (table 2) . symptomatic and asymptomatic residents were significantly different in the timing of clearing the virus. the median time to develop a real negative pcr (2 negative pcrs 24 hours apart) in the symptomatic group was 17 days (95% ci, 12.38-21.6) vs. 11 days (95% ci, 8.66-13.34) in the asymptomatic group (p = 0.01)) (fig 1) . the first confirmed case of sars-cov-2 in saudi arabia was on march 2nd, 2020, for a saudi traveler who came back from iran. in response to the covid19 pandemic, saudi arabia was one of the first countries that applied early and stringent mitigation measures while the number of confirmed cases was still low (300 cases) [9] . on march 15th, 2020, the saudi cdc (weqaya) imposed new regulations on all travelers coming from outside the kingdom to prevent importing infection from these countries. all travelers and close contacts of laboratoryconfirmed cases were tested and quarantined for 14 days in a designated facility regardless of the presence or absence of symptoms. this regulation has helped us to better understand the prevalence of sars-cov-2 infection in asymptomatic patients. in our cohort, the majority of residents were either truly asymptomatic (54%) or had mild symptoms (46%). the proportion of asymptomatic carriers in our cohort is considered to be one of the highest reported so far. however, these findings don't reflect the true prevalence of asymptomatic carriers in the general population as particularly vulnerable groups who were expected to have a more severe course were excluded. having said that, people > 65 years of age constitute only 3.41% of the general saudi population, while the majority (71.72%) are between 15-64 years of age. our proportion of asymptomatic carriers was somehow close to the reported proportion of the diamond princess cruise ship passengers in japan (50%) [10] . however, a lower prevalence has been reported in other studies. a report from germany on 116 travelers coming back from wuhan city to frankfort has shown a prevalence of 1.7% [11] . the most common symptoms were sudden and complete loss of taste and smell occurring in almost half of them (47.5%). more interestingly, these symptoms were found to be the only presenting symptoms in 18.7% of our residents, and this in itself should be considered as a red flag and should be reported as one of the common symptoms of mild disease. a similar prevalence of isolated loss of smell was demonstrated by varia et al. in a survey of 2428; he found a 74% prevalence of loss of smell, of which 17.3% were isolated [12] . furthermore, isolated olfactory dysfunction was also reported as the only clinical manifestation of covid-19 by simon b et al. [13] . the majority of our symptomatic patients report that they could not smell even strong odors like perfumes and could not differentiate between sweet and sour food. loss of taste and smell was reported as a rare clinical manifestation of covid-19 in a chinese study, accounting only for 5% of the symptoms [14] . however, subsequent studies have shown that these symptoms were more prevalent than previously reported. our findings are in light with a recent study that reported a 59% prevalence of loss of taste and smell in a cohort of covid-19 patients [15] . these symptoms were also detected in 70% of covid-19 patients in the early disease phase in a study from italy [16] . a multi-center european cohort of 417 covid-19 patients with mild to moderate disease has also reported a very high prevalence of olfactory (85.6%) and gustatory dysfunction (88%) [17] . although these symptoms were reported more commonly in mild disease, these findings might be subjected to selection and reporting bias. our study only included patients with mild disease and excluded those with severe disease who might not be able to report these symptoms. most of the residents who were quarantined because of contact with lab-confirmed cases were identified by contact tracing programs. although this information might be subjected to recall bias, none of our patients with a history of direct contact with lab-confirmed sars-cov-2 infection (including household contacts) reported exposure to symptomatic individuals. therefore, it indicates that they probably contracted the infection from asymptomatic or pre-symptomatic individuals. however, our study cannot confirm these findings. our study is unique in terms of the numbers of pcr performed per patient and also unique in terms of frequently testing the asymptomatic residents. a median of 5 tests per resident was done for both groups. this has allowed us to assess the virus clearance time accurately and to shed light on some of the pcr tests short comes. because we have enrolled asymptomatic residents in our study, we elected to use the first available positive pcr as the starting point for virus clearance time calculation. we are keeping in mind that those asymptomatic residents might have contracted the virus a few days or weeks before the quarantine. although our symptomatic group had mild symptoms, they have shed the virus for a longer period of time when compared with the asymptomatic group (17 vs. 11 days). nevertheless, we had a few outliers. we had a 32 years old asymptomatic lady who cleared the virus after 36 days of admission. the clinical implications for persistently positive pcr in true asymptomatic and mildly symptomatic patients need to be further studied. as a pcr test will not differentiate between a person who is contagious from a person who is not, further studies utilizing virus cultures and neutralization assays to help dictate the best duration of the quarantine. our study result is different than what was reported by lui et al. in which he found that majority of patients with mild symptoms cleared the virus at 10 days, while those with severe symptoms lasted for more than 10 days [18] . a false-negative test in a previously positive patient was not an uncommon finding in our cohort. this is most likely related to sampling techniques and specimen source. so, if the negative test was not repeated, we would have 18.8% of the time mistakenly reported these results as negative. so, we probably need to always do a repeat confirmatory test before deeming that the patient is infection-free. we also had about 3% of false-positive test results, which might be secondary to the detection of dead virus rna particles rather than re-infection, as described recently by the south korea group. this study is one of the few studies that assessed the prevalence of asymptomatic disease among quarantined individuals. it is unique in terms of the numbers of pcr performed per patient and the frequent tests performed in the asymptomatic group. this study is a cross-sectional study, so symptoms ascertainment before admission was subjected to recall bias. however, we find that majority of patients were well aware of their symptoms, and they were able to give us the date of onset of symptoms precisely. this study results cannot be generalized on all covid-19 patients as we have excluded some high-risk groups and people with moderate to severe disease. in conclusion, the prevalence of true asymptomatic carriers among covid-19 quarantined individuals in our study was high. however, this high prevalence might not represent the true prevalence in the general population. these findings re-enforce the importance of using testbased strategies and contact tracing rather than symptoms-based screening checklist when dealing with travelers and subjects with direct contact with a lab-confirmed case. a testingbased screening strategy perhaps should be extended to first-line healthcare workers and atrisk groups. also, countries planning for mitigation measures release should enhance their testing abilities to prevent new outbreaks. sudden onset of loss of smell and taste were prevalent in our study and were key symptoms of mild disease. these symptoms alone or in combination with other symptoms might as well be utilized as a marker for testing patients who may be positive for the virus. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china the epidemiological characteristics of 2019 novel coronavirus diseases (covid-19 covid-19 in south korea-challenges of subclinical manifestations transmission of 2019-ncov infection from an asymptomatic contact in germany clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing asymptomatic cases in a family cluster with sars-cov-2 infection sars-cov-2 viral load in upper respiratory specimens of infected patients laboratory testing for coronavirus disease 2019 (covid-19) in suspected human cases preparedness and response to covid-19 in saudi arabia: lessons learned from mers-cov estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship sars-cov-2 infection among travelers returning from wuhan olfactory and gustatory function impairment in covid-19 patients: italian objective multicenter-study isolated sudden onset anosmia in covid-19 infection. a novel syndrome? neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan loss of smell and taste in combination with other symptoms is a strong predictor of covid-19 infection remote psychophysical evaluation of olfactory and gustatory functions in early-stage coronavirus disease 2019 patients: the bologna experience of 300 cases olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multi-center european study viral dynamics in mild and severe cases of covid-19 key: cord-294912-xl0wzi16 authors: alteri, claudia; cento, valeria; antonello, maria; colagrossi, luna; merli, marco; ughi, nicola; renica, silvia; matarazzo, elisa; di ruscio, federica; tartaglione, livia; colombo, jacopo; grimaldi, chiara; carta, stefania; nava, alice; costabile, valentino; baiguera, chiara; campisi, daniela; fanti, diana; vismara, chiara; fumagalli, roberto; scaglione, francesco; epis, oscar massimiliano; puoti, massimo; perno, carlo federico title: detection and quantification of sars-cov-2 by droplet digital pcr in real-time pcr negative nasopharyngeal swabs from suspected covid-19 patients date: 2020-09-08 journal: plos one doi: 10.1371/journal.pone.0236311 sha: doc_id: 294912 cord_uid: xl0wzi16 since sars-cov-2-based disease (covid-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription pcr (rtpcr) results, raises as a major clinical need. here we evaluated the performance of a ddpcr-based assay to quantify sars-cov-2 titer in 55 suspected covid-19 cases with negative rtpcr results thanks to in-house ddpcr assay (targeting rdrp and host rnasep). samples were collected at asst-gom niguarda between february and may 2020 at hospital admission. clinical and imaging data were obtained for clinical staging and definition of disease severity. patients were mainly female (45.5%) with a median age of 73 (57–84) years. ddpcr-based assay detected sars-cov-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/ml, iqr: 72–345). in 15 of them (78.9%), chest ct showed a classical covid-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe covid-19 manifestations. ddpcr did not identify any trace of sars-cov-2 genome in the respiratory samples of the remaining 36 patients. the serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of sars-cov-2 antibodies in all patients tested positive for sars-cov-2 in ddpcr (100%). contrariwise, negative tests were observed in 95.0% ddpcr negative patients (p<0.001). thanks to a ddpcr-based assay, we achieved a rapid and accurate sars-cov-2 diagnosis in rtpcr-negative respiratory samples of individuals with covid-19 suspect, allowing the rapid taking care and correct management of these patients. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the pandemic of covid-19, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has posed a serious threat to global public health, calling for the development of reliable diagnostic tests, able to identify (and possibly quantify) sars-cov-2. currently recommended diagnostic tests for sars-cov-2 detection are real-time pcr (rtpcr) assays [1, 2] , which are able to detect viral rna through the amplification of 2 or 3 distinct genomic regions. despite the availability of these methods, the diagnostic optimum is far to be reached. the dynamic range of these tests is limited, and their diagnostic sensitivity on nasopharyngeal and oropharyngeal swabs has been shown to be insufficient in a number of sars-cov-2 related pneumonia cases [3] [4] [5] . first evidences suggest false negative results in 20%-30% of cases [3, 6] . the persistently negative rtpcr results in patients with clinical suspects of covid-19, and no alternative diagnosis, could be related to the lower viral titers that characterize the most widely used nasopharyngeal samples [7] in comparison to other respiratory specimens, such as sputum [8] , as well as to the replication kinetics of the virus. initial data are indeed suggesting that viral loads in throat swab and sputum samples peak at around 5-6 days after symptoms onset [9] , with consequent risk of uncontrolled sars-cov-2 transmission in the time period preceding the viral load peak. the prompt diagnosis of covid-19 patients is therefore a clinical need that is only partially met, which advocates for more sensitive and accurate diagnostic technologies. droplet digital pcr (ddpcr) is a highly sensitive assay for the direct detection and quantification of dna and rna targets. it has been increasingly used in infectious disease settings, especially thanks to its ability to consistently and reliably detect down to few copies of viral genomes [10] [11] [12] [13] [14] . whether the detection of low-level and/or residual viral presence is required, quantitative data obtained by ddpcr are far more informative than those provided by standard rtpcr assays [13] . standing the necessity of a limitation (as much as possible) of false negative results in covid-19 diagnosis, the use of ddpcr could provide a critical support [8] . its use for sars-cov-2 detection, however, is still very poorly investigated in clinical settings, and no data are currently available for european patients. in this study, the presence of sars-cov-2 genome was evaluated in 55 sars-cov-2 rtpcr negative nasopharyngeal swabs from covid-19 suspected patients thanks to a quantitative ad hoc designed assay based on ddpcr. negative nasopharyngeal swabs tested for sars-cov-2 by rtpcr (genefinder tm covid-19 plus realamp kit, elitech; allplex tm 2019-ncov assay, seegene) were collected during february-april 2020 from 55 covid-19 suspected patients at asst gom niguarda admission. for each patient, demographic and clinical information such as age, gender, clinical manifestations and symptoms were retrieved and stored in an anonymous database ad hoc built for the study. the severity of the covid-19 was classified into mild, moderate, or severe, if showing i) mild clinical symptoms without sign of pneumonia on imaging, ii) fever and respiratory symptoms with radiological findings of pneumonia, iii) respiratory distress, with oxygen saturation �93% at rest, mechanical ventilation, or presence of multiorgan failure (septic shock) and/or admission to intensive care unit (icu) hospitalization. the authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. medical records were registered and processed by using pseudonymization measures and assuring that it was not (and it is no longer) permitted the identification of data subjects. the study protocol was approved by the asst grande ospedale metropolitano niguarda research ethics committee (prot. 92-15032020). signed informed content was obtained for each participant. this study was conducted in accordance with the principles of the 1964 declaration of helsinki. total rna was extracted from 280 ul of nasopharyngeal swabs using qiaamp viral rna mini kit (qiagen) following manufacturer's instruction. sars-cov-2 genomic rna was quantified by means of the qx200™ droplet digital™ pcr system (ddpcr, biorad) using an home-made protocol targeting the rna dependent rna polymerase (rdrp) of sars-cov-2 (forward: to verify the correct performance of the sars-cov-2 rna quantification and housekeeping gene quantification, two independent experiments using different dilutions of the sars-cov-2 rna from a reference patient (rtpcr cycle n. 20 corresponding to 10 7 copies/ml in ddpcr) were performed. in particular, six serial dilutions were performed in order to deposit 10 5 , 10 4 , 10 3 , 10 2 , 10, 2 copies per reaction. all samples were repeated in duplicate, with exception of the last point repeated in quadruplicate. coefficient of determination (r 2 ) of sars-cov-2 quantification was assessed by linear regression analysis by plotting the standards' measured copies and comparing them with expected values of serial dilutions. negative and positive controls, consisting of 40 rna samples obtained from human nasopharyngeal swabs collected before september 2019, and 60 rna samples obtained from human sars-cov-2 positive nasopharyngeal swabs collected during the pandemics were included in each experiment. negative samples for sars-cov-2 were retrospectively selected on the basis of their availability, the storage at -80˚c in order to preserve eventual presence of rna, and the collection date (september 2019-january 2019). an alternative confirmed diagnosis was available for most of these samples (influenza a, n = 15; bacterial infections, n = 9; influenza b, n = 3, rsv, n = 2; hcov-oc43, n = 1). in order to further confirm the performance of the ddpcr-based assay, igg against sars--cov-2 were tested by using a commercial chemiluminescent microparticle immunoassay igg against sars-cov-2 (https://www.corelaboratory.abbott/us/en/offerings/segments/ infectious-disease/sars-cov-2, sensitivity:99.9% specificity:100%) [16] in a subgroup of 34 patients for those serum samples at the later stage of illness (from 3 days to 100 days after) were available (sars-cov-2 ddpcr positive: 14; sars-cov-2 ddpcr negative: 22). for ddpcr methods characterization, coefficient of determination (r2) was assessed by linear regression analysis, whereas the limit of detection (lod), defined as the lowest concentration at which 95% of positive samples were detected, was determined by probit regression analysis. reproducibility of sars-cov-2 quantification methods was assessed by intra-and inter-run tests using serial standard dilutions. the coefficient of variation (cv) was calculated as the standard deviation (sd) of sars-cov-2 copies/reaction divided by replicates mean. descriptive statistics are expressed as median values and interquartile range (iqr) for continuous data and number (percentage) for categorical data. to assess significant differences, fisher exact test and wilcoxon test were used for categorical and continuous variables, respectively. a p-value <0.05 was considered statistically significant. statistical analyses were performed with spss software package for windows (version 23.0, spss inc., chicago, il). baseline demographic and clinical characteristics of the 55 patients included in the study are reported in table 1 . patients were mainly female (45.5%) with a median age of 73 (iqr: 57-84) years. all patients were symptomatic with exception of one (covid-19 contact) and thus immediately hospitalized and tested for sars-cov-2 rtpcr, whose result was negative. thirty-five patients reported fever (63.6%) and 36 had pulmonary involvement (65.5%). further chest ct showed a bilateral interstitial pneumonia in 16 patients (29.1%). for the remaining 20, other lung diseases were diagnosed ( table 1) . the method showed a good linear correlation between expected and observed sars-cov-2 quantification (r2 = 0.996) (fig 1a and s1a fig) . by intra-run tests, the differences between the expected and observed sars-cov-2 copy number per reaction in the two experiments were 121,200±566 and 124,400±1,697 for 10 5 copies, 10,080±113 and 12,560±2,715 for 10 4 copies, 1,035±26 and 1,580±254 for 10 3 copies, 108 ±11 and 120±3 for 10 2 copies, 16±3 and 26±3 for 10 copies, 2.2±3.3 for 2 copies. for inter-run tests, the mean (+sd) sars-cov-2 copy number per reaction was 122,800 ±2,262 for 10 5 copies, 11,320±1,753 for 10 4 copies, 1,307±385 for 10 3 copies, 114±8 for 10 2 copies, 21±7 for 10 copies, 1.1±1.5 for 2 copies (fig 1b) . mean cv was 0.09 and 0.15 for intrarun and inter-run tests, respectively. probit analysis predicted a limit of detection (lod) of 2.9 (95% ic 2.0-11.5) copies per reaction. no signal was detected in any of the 40 sars-cov-2 negative samples tested, all collected between june and september 2019, before sars-cov-2 pandemic (s1b the rtpcr-negative nasopharyngeal swab samples obtained at first visit from the 55 patients included in the analysis were tested with ddpcr in blind. the quality of all swab samples was confirmed by rnasep quantification (median, iqr, copies/ml: 136,286 [84,548-256,714]). our home-made ddpcr assay detected sars-cov-2 in nasopharyngeal swab samples belonged to 19 patients (table 1 and s1 table) . median viral load was 128 copies/ml (iqr: 72-345); the highest value detected was 1800 copies/ml (s1c and s1d fig) . of note, for 12 patients the subsequent nasopharyngeal swabs gave multiple negative rtpcr results (median number of negative swabs: 3 [3] [4] ). for the remaining 7 patients sars-cov-2 was later on detected also by rtpcr, during the follow-up investigation performed within 3 days since the initial negative test. ddpcr provided negative results in the remaining 36 nasopharyngeal swabs. table 1 reported demographic and clinical characteristics respect to ddpcr results. patients with a ddpcr confirmed sars-cov-2 infection were more recently admitted to the hospital (march 18 vs. april 28, p = 0.008), were more frequently affected by cough and dyspnea sars-cov-2 detection by ddpcr (p = 0.047 and <0.001, respectively), and showed more frequently a classical bilateral interstitial pneumonia (p<0.001) respect to patients with a negative ddpcr result. of note, a severe covid-19 manifestation characterized 14 of the 19 patients with a ddpcr confirmed sars--cov-2 infection (73.7%, s1 table) . median time from symptoms-onset to hospital admission was not significantly different between the two groups (p = 0.425). in order to further confirm the performance of the ddpcr-based assay in sars-cov-2 detection, igg against sars-cov-2 were tested by using a commercial chemiluminescent immunoassay [16] . sars-cov-2 was detected at later time points also by standard qualitative rtpcr for three of these five patients (id 1, 4, 8, s1 table) . contrariwise, negative antibody findings were observed in 19/20 (95.0%) patients tested negative for sars-cov-2 in ddpcr ( table 1 ). the single patient tested negative for sars-cov-2 by ddpcr, but positive at the serological assay was characterized by a time from symptoms-onset to nasopharingeal swab of 28 days, and a time from symptomsonset to serological assay of 48 days, thus suggesting a late-stage disease at hospital admission, when probably the viral clearance already occurred. this proof-of-concept study shows that an in-house ddpcr-based assay can allow an efficient detection of sars-cov-2 at low copy number in symptomatic cases resulted negative by standard rtpcr. the effective prevention, treatment and control of covid-19 cannot be achieved without an early, sensitive, and reliable diagnosis. the laboratories play a critical role in confirming the initial clinical suspicion of this disease, as confirmation of sars-cov-2 presence is essential to ensure the prompt initiation of containment and treatment protocols. this is of utmost importance to avoid further spread of the pandemic, and to assure the best clinical and therapeutic management of the infected patients in the hospital setting. unfortunately, currently used rtpcr assays lack of the necessary sensitivity to identify all cases of sars-cov-2 infection (20% of false negative results [5, 6] ). complementary laboratory assays are therefore strongly needed. here, we applied an in-house ddpcr-based assay for sars-cov-2 detection in rtpcr negative swab samples. thanks to this rdrp-based assay, sars-cov-2 genome could be accurately identified and quantified down to only 2.9 (95% ic 2.0-11.5) copies per reaction corresponding to a viral load of 28 (95% ic 13-118) copies/ml, highlighting the great sensitivity of the method. moreover, being the quantification per ml highly dependent on the quality of sampling and the extraction procedure, the presence in this assay of an internal reference gene (rnasep) as a quality control, excludes any eventual pcr inhibition and confirms successful rna extraction, thus dramatically reducing the risk of false negative results. rnasep showed a quantification close to 10 5 copies/ml in the majority of samples, with few peaks of 10 6 , suggesting a relatively stable expression of this gene in our population. of note, when the sars-cov-2 amount was normalized according to the rnasep quantification (per 100,000 copies of rnasep) viral load results did not differ significantly respect to quantifications per ml in the majority of patients (73.6%, 14/19, s1 table) . a 1 log decrease was observed only for 5 samples, all characterized by an rnasep expression at least 0.5 log higher respect to the mean value (s1 table) . by using this reliable, reproducible and highly sensitive assay, we could improve the efficiency of covid-19 diagnosis on nasopharyngeal swabs even at a very early stage of viral replication, or in anatomical districts where the virus is present at lower levels, such as the oropharyngeal or the nasopharyngeal samples [8, 9] . this bridges a significant diagnostic gap left by commercial rtpcr systems used to date as a bulwark of molecular diagnostics of sars--cov-2. this provides a significant clinical advantage in terms of prompt identification of subjects with sars-cov-2 infection, and for the consequent implementation of all appropriate containment and therapeutic measures. indeed, all the 19 patients tested positive by ddpcr were characterized by generally low viral loads (never above the 2,000 copies/ml) that potentially justified the negative contextual rtpcr results. the sars-cov-2 detection by ddpcr in these patients was also confirmed by the presence of sars-cov-2 igg at the later stage of illness (from 3 days to 90 days after, 14/14 patients with a serum sample available). on the other side, ddpcr did not detect sars-cov-2 genomes in 36 patients hospitalized during the pandemic and for whom (out one, probably hospitalized at a late-stage disease, when the viral clearance was already occurred) subsequent serological assays excluded a previously contact with sars-cov-2. taken together, our results support the superiority of our ddpcr-based assay to currently used rtpcr tests for the molecular detection of sars-cov-2. the ability of ddpcr to generate accurate quantitative data has had a huge impact on the study of viral agents of infectious disease [8, 10-12, 14, 18] . thanks to an extremely high sensitivity, this next generation pcr platform is providing a consistent help in all those clinical scenarios in which is of great importance to identify even the smallest amount of viral particles [10, 12, 14] . in the context of covid-19 diagnosis, two recent studies highlight the best performances of ddpcr in detecting low viral load samples [19, 20] . in particular, by targeting orf1ab and n genes, these papers demonstrated that negative rtpcr samples could be identified as positive by the optimized ddpcr (4/96 samples in [19] and 25/27 samples in [20] ). in the suo et al paper, the sars-cov-2 positivity by ddpcr was confirmed by rtpcr in subsequent follow-up investigations for all the 25 patients [19] . differently, our study showed the ddpcr advantages over currently available rtpcr approaches also in the setting of persistently and repeatedly negative rtpcr results. in particular, without this assay, no molecular laboratory confirmation of sars-cov-2 would have been available at hospitalization for 12 patients, all characterized by a severe covid-19 manifestation and with evidence of bilateral interstitial pneumonia, and for two patients affected by pneumonia with pleuritis (n = 1), and lung cancer (n = 1), for whom the mere clinical evaluation excluded the covid-19. our study has some limitations. a) the sample size was small (the study has been designed as a proof of concept, though); b) the correlation of the sars-cov-2 viral load with the timing of infection was not feasible; c) the infectivity of the virus was not assessed. moreover, the ddpcr turnaround time (longer than for qpcr), and the difficulty to convert this system in a high throughput individual assay makes it necessary to apply this system only in specific clinical scenarios. as this assay could potentially be used on different samples, the evaluation of its performances in different laboratories will be necessary for the inter-laboratory reproducibility, and thus the cross-validation of the methods. overall, ddpcr should be a complement to the current standard rtpcr-based diagnosis, in order to improve as much as possible the rapid identification of sars-cov-2 infection (making possible the diagnosis long before the viral load peak is reached and antibodies appear) and thus the definitive containment of the pandemics. even more relevant, this test could be used for treatment monitoring, or for all supposed covid-19 patients, who are negative for nasopharyngeal swab nucleic acid tests twice by rtpcr, but probably at risk of carrying sars--cov-2. in conclusion, we have provided preliminary results to prove that ddpcr is a promising molecular diagnostic tool to detect low levels of sars-cov-2 rna. it possesses all the critical characteristics that would allow its use to improve and accelerate covid-19 diagnosis in clinical samples. as its use could be foreseen in routine clinical practice, additional data on a larger number of clinical samples, and possibly from multicenter studies, are required to further confirm its sensitivity, specificity, and reliability. diagnosis and clinical management of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: an operational recommendation of peking union medical college hospital (v2.0). emerging microbes & infections 2020 laboratory testing for coronavirus disease (covid-19) in suspected human cases covid-19 disease with positive fecal and negative pharyngeal and sputum viral tests negative nasopharyngeal and oropharyngeal swab does not rule out covid-19 false-negative results of real-time reverse-transcriptase polymerase chain reaction for severe acute respiratory syndrome coronavirus 2: role of deep-learning-based ct diagnosis and insights from two cases antibody responses to sars-cov-2 in patients of novel coronavirus disease sars-cov-2 viral load in upper respiratory specimens of infected patients quantitative detection and viral load analysis of sars-cov-2 in infected patients viral load of sars-cov-2 in clinical samples a sensitive and accurate quantification method for the detection of hepatitis b virus covalently closed circular dna by the application of a droplet digital polymerase chain reaction amplification system clinical utility of droplet digital pcr for human cytomegalovirus highly precise measurement of hiv dna by droplet digital pcr absolute quantification by droplet digital pcr versus analog real-time pcr quantification of hiv-dna and residual viremia in patients starting art by droplet digital pcr: their dynamic decay and correlations with immunological parameters and virological success performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in use of digital droplet pcr to detect mycobacterium tuberculosis dna in whole blood-derived dna samples from patients with pulmonary and extrapulmonary tuberculosis ddpcr: a more sensitive and accurate tool for sars-cov-2 detection in low viral load specimens quantitative detection and viral load analysis of sars-cov-2 in infected patients we thank bio-rad italia for providing technical support, particularly dr. laura sard, dr. marilisa marinelli and dr. marco tronconi and medical affairs bio-rad team, for their valuable assistance in the implementation of the study. the authors also thank dr. silvia nerini and all the staff of the microbiology and virology laboratory of asst grande ospedale metropolitano niguarda for outstanding technical support in processing swab samples, performing laboratory analyses and data management. conceptualization: luna colagrossi, chiara vismara, carlo federico perno. key: cord-312652-zhccmfgw authors: hu, xiumei; zhang, ruyi; an, taixue; li, qiang; situ, bo; ou, zihao; wu, changmeng; yang, biao; tian, peifu; hu, yuhai; ping, baohong; wang, qian; zheng, lei title: impact of heat-inactivation on the detection of sars-cov-2 igm and igg antibody by elisa date: 2020-06-20 journal: clin chim acta doi: 10.1016/j.cca.2020.06.032 sha: doc_id: 312652 cord_uid: zhccmfgw background: to establish a safe and accurate method for detecting sars-cov-2 igm and igg, we assessed the impact of sera after heat-inactivation on the sars-cov-2 igm and igg levels measured by elisa-immunoassay. methods: the serum samples of 62 patients with covid-19 and 18 healthy controls were collected in hankou’s hospital of wuhan from february 27 to march 6, 2020. before and after the samples were inactivated, the levels of igm and igg antibodies were measured. results: the indexes of antibodies after inactivated were significantly higher than those in fresh sera, while the positive rates in all participants or in patients with covid-19 did not change. the positive coincidence rate, negative coincidence rate and total coincidence rate of igm antibodies before and after inactivation were 100.00% (55/55), 96.00% (24/25) and 98.75% (79/80), respectively (κ=0.971, p<0.001), while those for igg antibodies were 98.21% (55/56), 91.67% (22/24) and 98.75% (79/80) respectively (κ=0.910, p<0.001). these results showed a good consistency. conclusions: heating-activation does not decrease the diagnostic efficacy of sars-cov-2 igm or igg antibodies. sera inactivated by heating at 56℃ for 30 minutes should be recommended to minimize the risk of virus contamination of laboratory staff. as the largest known rna viruses, conronaviruses (covs) are further divided into four genera: α-covs, β-covs, γ-covs, and δ-covs, among which α-and β-covs are able to infect mammals [1, 2] , whereas the other two genera can infect birds and could also infect mammals. according to the previous researches, covid-19 was caused by sars-cov-2 [3] , a new member of β-covs that was recently isolated from human airway epithelial cells, characterized by next-generation sequencing in january 2020 [4] . this public health concern intense attention not only within china but internationally. although the covid-19 outbreak has led to implementation extraordinary public health measures to reduce further spread of the virus within china and elsewhere. on march 11, 2020, the coronavirus outbreak was declared a global pandemic by the who. the spread and potential lasting existence of the covid-19 pose severe threats to the world [5] . consequently, how to increase the detection of the virus and find more potential therapeutic strategies become of vital importance for scientists and clinicians globally. the nucleic acid of sars-cov-2 rt-pcr test has become the standard method of diagnosis of covid-19 [6] , although these real-time pcr test kits have many limitations such as high false negative rates and long turnaround times [7] . some companies have developed specific antibodies of sars-cov-2 testing kit to quickly identify infected patients to prevent virus transmission and to assure timely treatment. it is widely accepted that igm provides the first line of defense during viral infections, prior to the generation of adaptive, high affinity igg responses that are important for long term immunity and immunological memory [8] . according to chinese diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7), sars-cov-2 specific igm becomes detectable around 3-5 days after onset and igg reaches a titration of at least 4-fold increase during convalescence compared with the acute [9] . therefore the detection of igm and igg antiviral antibodies in the serum samples from patients can be additional evidence to confirm covid-19. to reduce the risk of accidental exposure of laboratory workers, sera of patients will be heat-inactivated. previous study showed that coronaviruses are vulnerable to heat and heating at 56℃ for 25 minutes could reduce more than 4 log 10 tcid 50 /ml of mers virus (the sixth human coronavirus identified) [10] . furthermore, a recent study showed that exposure to 56℃ for 30 minutes could decreased the viral titer of sars-cov-2 significantly although the number of detectable rna copies of virus was not affected [11] . however, the potential impact of heat-inactivation on the levels of sars-cov-2 igm or igg antibodies is not published. in order to establish a safe and accurate method for detecting sars-cov-2 specific antibodies, we retrospectively assessed the impact of sera heat-inactivation at 56℃ for 30 minutes on the levels of sars-cov-2 igm or igg antibodies. for this retrospective, single-center study, we recruited 62 patients at hankou hospital in wuhan, china from february 26 to march 6, 2020. all patients were diagnosed as having covid-19 according to who interim guidance were enrolled in this study [12] . and 18 healthy controls in hospital were added to help the assessment. the study was approved by hankou hospital ethics committee (no. hkyy-2020-028). throat-swab specimens from the upper respiratory tract that were obtained from all the cases and controls were maintained in viral-transport medium. and serum samples taken from peripheral venous were maintained in 5ml vacuum blood collection tubes with separation gel. laboratory measurements were conducted on collected specimens. sars-cov-2 infection was confirmed by viral nucleic acid detection with real-time rt-pcr using the same protocol described previously [13] . the igm and igg antibodies of sars-cov-2 were measured by elisa-immunoassay using sars-cov-2 antibody detection kit (guangzhou darui biotechnology co, ltd). the assay is based on a double-antigen sandwich principle that detects antibodies biding sars-cov-2 spike protein receptor binding domain (rbd) in sera. serum samples were centrifuged at 3000rpm for 15 minutes to obtained serum samples. then 500μl fresh serum was dispensed in b2 biosafety cabinet. the rest serum was incubated at 56℃ for 30 minutes in a water bath and cooled to room temperature by uv irradiation in the biosafety cabinet for 30 minutes afterwards. serum was diluted in dilution buffer by 1:100. then the diluted serum and 100μl negative or positive controls were added to the wells coated with recombinant sars-cov-2 antigen and incubated for 40 minutes at 37℃. wells were washed by washing solution in the kit for 5 times followed by the addition of hrp-conjugated sars-cov-2 antigen and subsequent incubation for 20 minutes at 37℃. wells were washed five times and urea peroxide and tetramethylbenzidine were added for colorimetric reaction. following 10 minutes of incubation at 37℃, the reaction was stopped by 2 mol/l h 2 so 4 solution and the resultant absorbance was read on a microplate reader at 450nm. the cut-off value of igm antibody was defined as the mean index of negative control duplicate wells plus 0.25, while that of igg antibody was defined as the mean index of negative control duplicate wells plus 20% of the mean index of positive control duplicate wells. and the results determined by the manufacture are: negative<cut-off value; cut-off value≤weak positive<0.5; >0.5 positive. all testing was done in accordance with the manufacturer's guidelines [14] . we present continuous measurements as mean (sd) if they are normally distributed or median (iqr) if they are not, and categorical variables as count (%). for laboratory results, we also assessed whether the measurements were outside the normal range. we used spss (version 24.0) for all analyses. wilcoxon test was used to compare nonparametric variables before and after heating. categorical variables was compared with kruskal-wallis test. kappa test was used to evaluate the consistency of detection between fresh sera and heated sera. statistical significance was defined as p<0.05. the cut-off values of different dates for the assay were shown in table 1 . baseline characteristics of 80 participants are shown in table 2 , stratified by cases/healthy controls. 77.50% of participants were patients. 60% of participants were male. the median age was 65 ranged from 26 to 94 in all participants. the optical density (od) at 450 nm of antibodies measured after heating were significantly higher than those in fresh sera (wilcoxon test: z=-3.729 for igm, p<0.001; z=-7.609 for igg, p<0.001). the specific data were shown in figure 1a and figure 1b . among the 18 healthy controls, the indexes of igg antibody increased when the sera were heat-inactivated (wilcoxon test: z=-3.224, p=0.001), while there were no statistical differences for igm in both conditions(wilcoxon test: z=-1.136, p=0.256). however, the detection results of both of igm and igg antibodies remained negative whether the sera heated or not . when 62 sera of patients analyzed, as seen in table 3 , the indexes of igm or igg antibody increased when the sera were heat-inactivated (wilcoxon test: z=-3.390 for igm, p=0.001; z=-6.797 for igg, p<0.001). additionally, the positive detection rate in patients did not change when sera were heat-inactivated. ( test： =0.086, p=0.769 for igm before and after inactivation; =0.100, p=0.752 for igg before and after inactivation). then we analyzed the biological interpretation of igm and igg before and after heating. there was one sample changed from negative to positive after heating in igm detection. one positive sample became negative while two negative samples became positive after heating in igg detection. there was 1 sample changed from negative to positive both in igm and igg detection. the data were shown in figure 1c and 1d and the specific changed samples were plotted in red color. furthermore, table 4 showed that the positive coincident rates, negative coincident rates and total coincident rates of sars-cov-2 antibodies in patients before and after inactivation. the kappa value of the test for igm was 0.971, and the positive coincidence rate, negative coincidence rate and total coincidence rate of igm antibodies before and after inactivation were 100.00% (55/55), 96.00% (24/25) and 98.75% (79/80), respectively. in addition, the kappa value of the test for igg was 0.910, and the positive coincidence rate, negative coincidence rate and total coincidence rate of igg antibodies before and after inactivation were 98.21% (55/56), 91.67% (22/24) and 98.75% (79/80). the results above showed that the consistency of the antibodies detection before and after heating was high although the specific indexes of antibodies changed slightly. as the outbreak and spread of covid-19, scientists and clinicians globally are working swiftly to combat this disease. the diagnostic assays have been developed rapidly in china and other countries, and have played significant roles in diagnosis, monitoring, surveillance and infection control [15] . the nucleic acid of sars-cov-2 rt-pcr test has become the standard method of diagnosis of covid-19 [6] . however, there are many limitations for the real-time pcr test kits: 1) the pcr tests require certified laboratories, expensive equipment and trained technicians to operate. 2) the high false negative rate of pcr makes it difficult to discover all of the covid-19 patients and the asymptomatic infection [7] . the process of blood sample collection is more controllable making the detection of antibody more reliable. furthermore, elisa has a high sensitivity of igm detection, which is beneficial to early diagnosis of covid-19 patients. thus some companies have developed specific antibodies of sars-cov-2 testing kit to quickly identify infected patients to prevent virus transmission and to assure timely treatment. rapid detection of both igm and igg antibodies will play vital role in diagnosis and treatment of covid-19. evidence showed that sars-cov-2 could be detected in blood, raising the possibilities of blood transmission [16] . according to previous data, sars-cov rna was detected in 50% of plasm and 78% of serum samples during the first week of illness [17] . heating treatment at 56℃ for 30 minutes is widely used to inactivate the virus for further detection or research. it has been demonstrated that during detection, inactivation of virus could reduce the risk of accidental exposure of laboratory workers [18, 19] . however, the potential impact of heat-inactivation of sera for sars-cov-2 igm or igg is not validated. in our study, the indexes of sera igm and igg increased after heating-activation using elisa. the positive detection rate in patients did not change when sera were heatinactivated. the positive coincidence rate, negative coincidence rate and total coincidence rate of igm antibodies before and after inactivation were 100.00% (55/55), 96.00% (24/25) and 98.75% (79/80), respectively. in addition, the positive coincidence rate, negative coincidence rate and total coincidence rate of igg antibodies before and after inactivation were 98.21% (55/56), 91.67% (22/24) and 98.75% (79/80). the kappa test result showed that the consistency of the antibodies detection before and after heating was high using sars-cov-2 antibody detection kit (elisaimmunoassay). previous study indicated that heating inactivation of sera may bring to false negative results in anti-toxoplasma igm testing [20] . the main reason for this may be the production of aggregates composed of iga, igm, igg an albumin resulting from heatinduced aggregation of serum proteins [21] . however, some other studies showed that heating sera may lead to false positive results or eliminate heat-labile factors which could give false-positive results. for instance, using the abbott hiv eia assay with heating sera may cause false-positive hiv results [22] [23] [24] , while heat-inactivation could reduce the heat-labile factor presented in many sera that interfere with binding of the human papillomavirus (hpv) multiplexed competitive luminex immunoassay [25] . the possible reasons for the high consistency of the antibodies detection before and after heating may be as followed: 1) elisa immunoassay has a high sensitivity in detection, which may be not affected by sera with heat-induced aggregations. 2) heating sera may destroy the advanced structure of the antibody, which may interfere with the binding to the assay; however, heating treatment could also inactivate the complement system in sera, which could avoid non-specific reaction and decrease the background in immunological assays [26, 27] . in addition, there was 1 sample changed from negative to positive both in igm and igg detection, which did not interfere the whole study interpretation. therefore, our analysis provide evidence that sera inactivated by heating at 56℃ for 30 minutes could reduce the risk of virus contamination, and would not impair the detection efficacy of sars-cov-2 igm or igg testing using this elisa assay. nevertheless, the indexes of igm and igg in sera with discrepancy changed sharply before and after heating-activation especially the coefficients of variation were higher than 30% in indexes of igg, and additionally, the positive rate were relatively low in sera from these patients, which were probably due to the small samples. as a result, more participants should be recruited and more experiments should be conducted to validate the current data. in summary, sera inactivated by heating at 56℃ for 30 minutes could minimize the risk of virus contamination and did not impair the positive detection rate using sars-cov-2 antibody detection kit (elisa-immunoassay) and eventually represents a valuable contribution to a safer covid-19 serological diagnosis. however, as the small samples in our study, more experiments and samples in other laboratories are needed to validate the results. sars and other coronaviruses as causes of pneumonia emerging coronaviruses: genome structure, replication, and pathogenesis a novel coronavirus outbreak of global health concern a novel coronavirus from patients with pneumonia in china world health organization detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis igm in microbial infections: taken for granted? diagnosis and treatment plan for new coronavirus pneumonia (trial version seventh) heat inactivation of the middle east respiratory syndrome coronavirus. influenza other respir viruses evaluation of heating and chemical protocols for inactivating sars-cov-2 clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance coronaviruses and the human airway: a universal system for virus-host interaction studies world health organization. laboratory testing for coronavirus disease 2019 (covid-19) in suspected human cases the sars-cov-2 outbreak: diagnosis, infection prevention, and public perception consistent detection of 2019 novel coronavirus in saliva quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome heat treatment of whole blood and serum before chemical analysis effect of heat inactivation of hiv on specific serum proteins and tumour markers impact of heatinactivation on anti-toxoplasma igm antibody levels mechanism of aggregates generated by heating human serum effect of using heat-inactivated serum with the abbott human t-cell lymphotropic virus type iii antibody test effects of heat inactivation on hiv antibody screening and confirmatory test systems heat inactivation of serum may interfere with htlv-iii/lav serology optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18 the effect of heat inactivation of serum on aggregation of immunoglobulins interferences in immunoassay data curation, investigation ruyi zhang: visualization, investigation, writing-original draft preparation taixue an and qiang li: investigation, data curation bo situ and zihao ou: software, data curation changmeng wu and biao yang: visualization peifu tian: editing yuhai hu: methodology, investigation conceptualization, methodology, supervision and fuding highlights:  heating-activation at 56℃ for 30 minutes does not impair the antibodies of sars-cov-2 heating-activation would not decrease the diagnostic efficacy of sars-cov-2 igm or igg antibodies (elisa-immunoassay)  sera inactivated by heating should be recommended to minimize the risk of virus contamination of laboratory staff the elisa-immunoassay detection kits were supported by prof. songying wu and prof. jinlin hou. author contributions: all authors have accepted responsibility for the entire content of this manuscript and approved its submission. research funding: this work was supported by funds from the national natural science foundation of china (81601819), outstanding youths development scheme of nanfang hospital, southern medical university (2016j013) and medical science and technology research foundation of guangdong province (a2016280). competing interests: the authors declare that they have no competing interests. ethical approval: the study was approved by hankou hospital ethics committee (no. hkyy-2020-028). key: cord-304306-rxjahqwh authors: vlachakis, dimitrios; papakonstantinou, eleni; mitsis, thanasis; pierouli, katerina; diakou, io; chrousos, george; bacopoulou, flora title: molecular mechanisms of the novel coronavirus sars-cov-2 and potential anti-covid19 pharmacological targets since the outbreak of the pandemic date: 2020-10-08 journal: food chem toxicol doi: 10.1016/j.fct.2020.111805 sha: doc_id: 304306 cord_uid: rxjahqwh the novel coronavirus sars-cov-2 has emerged as a severe threat against public health and global economies. covid-19, the disease caused by this virus, is highly contagious and has led to an ongoing pandemic. sars-cov-2 affects, mainly, the respiratory system, with most severe cases primarily showcasing acute respiratory distress syndrome. currently, no targeted therapy exists, and since the number of infections and death toll keeps rising, it has become a necessity to study possible therapeutic targets. antiviral drugs can target various stages of the viral infection, and in the case of sars-cov-2, both structural and non-structural proteins have been proposed as potential drug targets. this review focuses on the most researched sars-cov-2 proteins, their structure, function, and possible therapeutic approaches. coronaviruses (covs) are enveloped, positive-sense, single-stranded ribonucleic acid (rna) viruses that belong to the family coronaviridae. these viruses harbor the largest genome among rna viruses, specifically 26 to 32 kilobases . based on phylogenetic analyses, covs can be classified into four genera, which include alpha coronaviruses, beta coronaviruses, gamma coronaviruses, and delta coronaviruses (jaiswal and saxena, 2020) . out of those genera, beta-covs contain the majority of viruses that infect humans, with the aforementioned genera being subdivided into four lineages (a, b, c, d) . generally, covs can infect the respiratory, hepatic, gastrointestinal, and central nervous system of mammals, birds, and fish . there are seven covs known to cause human disease. four of those covs (hcov229e, nl63, oc43, and hku1), known as non-severe acute respiratory syndrome (sars)-like covs, induce mild diseases and are globally endemic, while three highly pathogenic covs (sars-cov-1, mers, and sars-cov-2) can lead to lethal disease (raoult et al., 2020) . out of the latter three covs, sars-cov-2, responsible for the coronavirus disease 2019 , has proved a serious threat against public health and global economies in 2020 (zheng, 2020) . sars-cov-2 is a beta-coronavirus of the b lineage with a diameter of approximately 60 to 140 nm (cascella et al., 2020) . its genome appears to encode as many as 14 open reading frames (orfs), with the 5'orf1a/orf1ab encoding polyproteins, which are autoproteolytically processed into 16 non-structural proteins (nsps) (gordon et al., 2020) . those nsps (nsp1-16) form the replicase/transcriptase complex (rtc). this complex is composed of various enzymes, including a papain-like protease (nsp3), the main protease (nsp5), an nsp7-nsp8 primase complex, a primary rna-dependent rna polymerase (rdrp/nsp12), a helicase/triphosphatase (nsp13), a 3'-5' exoribonuclease (nsp14), a uridine-specific endonuclease (nsp15), and n7-and 2'o-methyltransferases (nsp10/nsp16). the remaining orfs encode four structural proteins and nine putative accessory factors (gordon et al., 2020) . these structural proteins include the spike (s) glycoprotein, the small envelope (e) glycoprotein, the membrane (m) glycoprotein, and a nucleocapsid (n) protein (astuti and ysrafil, 2020) . coronaviruses entry into host target cells is dependent on the binding of s glycoprotein to a specific cell receptor and the subsequent priming of the aforementioned protein by host cell proteases. sars-cov-2 uses the angiotensin-converting enzyme 2 (ace2) host receptor for internalization and, mainly, the type ii transmembrane serine protease (tmprss2) for spike glycoprotein priming (kumar et al., 2020) . specifically, the s glycoprotein binding domain, which is present at 331 to 524 residues, attaches strongly to the ace2 receptor (astuti and ysrafil, 2020) . after attachment, tmprss2 and a number of lysosomal proteases cleave and activate the s glycoprotein leading to the entrance of sars-cov-2 into the cell through endocytosis or direct fusion of the viral membrane with the host membrane (romano et al., 2020; shang et al., 2020) . once inside the host cytoplasm, the viral rna undergoes translation, where the orfs that encode nsps are firstly translated and produce the polyproteins pp1a and pp1ab that are further cleaved by virus-encoded proteases into nsps (kumar et al., 2020) . the aforementioned proteases are the papain-like proteases (plpro) and the chymotrypsin-like protease (3clpro) (astuti and ysrafil, 2020) . these nsps have a great role in viral rna replication. specifically, the rna replication machinery of sars-cov-2 involves the rdrp (nsp12), the zinc-binding helicase (nsp13), and enzymes, such as a bifunctional 3′→5′ mismatch exonuclease and a cap ribose 2′-o methyltransferase, which are related to viral rna modification, such as messenger rna (mrna) capping and rna proofreading (viswanathan et al., 2020) . the accessory and structural proteins are translated by a specific set of subgenomic rnas (romano et al., 2020) . some of those, including m, s, e, are insulated in the endoplasmatic reticulum and then transported to the endoplasmatic reticulum-golgi intermediate compartment (ergic) (astuti and ysrafil, 2020) . the replicated genome has the ability to join the n protein to the nucleocapsid form and move into the ergic (astuti and ysrafil, 2020) . the virions are later assembled at ergic and are subsequently released through exocytosis out of the cell (kumar et al., 2020) . patients infected with sars-cov-2 were first reported in december 2019, and, since then, cases have risen to the point that as of 30 august 2020, this virus has led to 24,854,140 cases and 838,924 deaths worldwide (who, 2020; zheng, 2020) . the dominant clinical features of covid-19 include cough, shortness of breath or difficulty breathing, fever or chills, muscle or body aches, vomiting or diarrhea, and new loss of taste or smell (cdc, 2020) . person to person spread of sars-cov-2 via respiratory droplets, when a patient coughs, sneezes, or talks is the main way of covid-19 transmission, while the mentioned disease can also occur through indirect means such as touching objects contaminated with sars-cov-2 (lotfi et al., 2020) . this virus primarily affects the respiratory system, with respiratory symptoms of covid-19 being quite heterogeneous, varying from minimal symptoms to significant hypoxia with acute respiratory distress syndrome (ards) (yuki et al., 2020) . moreover, severe covid-19 patients display an acute inflammatory response. specifically, transcriptomic rna-seq analysis of covid-19 patients showed that the viral infection induced several immune pathways and pro-inflammatory cytokines, implying sustained inflammation and cytokine storm (wu et al., 2020b) . covid19 could be fatal with risk factors, including the co-existence of chronic diseases, age, sex, obesity, and smoking (michelozzi et al., 2020; petrakis et al., 2020) ). obesity, particularly, is considered a chronic inflammatory state with obese individuals showcasing high concentration of pro-inflammatory cytokines like interleukin 6 (il-6). with regard to covid-19, il-6 has been shown as an independent predictor of mortality (yadav et al., 2020) . the currently available antiviral option for hospitalized patients is remdesivir, which may inhibit the replication process by targeting the rdrp. previously proposed treatments for hospitalized patients included hydroxychloroquine, which thought to disrupt virus endocytosis, and lopinavir/ritonavir, which thought to inhibit sars-cov-2 main protease (astuti and ysrafil, 2020; magro, 2020) . the aforementioned facts have made the development of therapeutic molecules a necessity in the scientific community. spike is a transmembrane glycoprotein in covs that forms homotrimers that protrude from the viral surface . spike is the structural protein that gives cov viral particles their characteristic crown-like shape, from which their original name was coined (coutard et al., 2020) . spike is essential in viral entry, a process which is achieved through binding with the ace2 host receptor . the above has led to considering s a promising drug target, therefore the study of this glycoprotein is important in the development of novel therapeutics against covid-19. structure-wise, each monomer of the s glycoprotein contains two subunits termed s1 and s2, which mediate attachment and membrane fusion, respectively (ou et al., 2020) . the s1 is divided into the n-terminal domain (ntd), followed by the receptor-binding domain (rbd) and two structurally conserved subdomains termed sd1 and sd2. the rbd constantly switches between a standing-up position, associated with receptor binding, and a lying down position associated with immune evasion (shang et al., 2020) . the sd1 and sd2 subdomains cap the s2 subunit and, thus, protect the conserved fusion machinery (henderson et al., 2020) . the s2 subunit contains the fusion peptide (fp), the heptad repeat 1 (hr1), the heptad repeat 2 (hr2), the transmembrane domain (tm), and the cytoplasmic domain (cp) (xia et al., 2020) . viral entry requires both binding of s to the cellular receptor and s glycoprotein priming by cellular proteases (hoffmann et al., 2020) . this priming includes a cleavage at the boundary between the s1 and s2, for the majority of coronaviruses, plus a cleavage at the s2' site located immediately upstream of the fp, a process present in all coronaviruses (hoffmann et al., 2020; mccallum et al., 2020; walls et al., 2020) . the latter cleavage has been implicated in activating the glycoprotein for membrane fusion through extensive irreversible conformation changes (mccallum et al., 2020) . specifically, after cleavage, the exposed fusion peptide inserts itself into the plasma membrane while the hr1 and hr2 can interact to form the six helical bundle (6hb) fusion core, which eventually leads to the fusion of the viral membrane with the host plasma membrane (joshi et al., 2020) . the above priming is accomplished through proteases such as tmprss2 and lysosomal cathepsins (jaimes et al., 2020) . sars-cov-2 s differentiates from covs of the same clade since it features a novel furin-like cleavage upstream of the cleavage site 1 which seems to be cleaved during biosynthesis. this characteristic potentially allows for more efficient spreading of sars-cov-2 in the human population compared to other lineage b beta coronaviruses coutard et al., 2020) . while using the spike glycoprotein as a potential drug target, a researcher can focus on viral binding to host receptor or membrane fusion. as prime example, monoclonal antibodies can be used to tightly bind the spike glycoprotein's rbd in order to neutralize sars-cov-2. such a case is the monoclonal antibody cr3022, which binds to s rbd while not competing with the binding of ace2, allowing the monoclonal antibody to interfere with ace2 binding (huo et al., 2020) . another approach, this time focusing on membrane fusion, is ek1c4. ek1c4 is a lipopeptide which disrupts membrane fusion by targeting hr1 (xia et al., 2020) . on the other hand, interrupting s function could also be achieved by targeting host cell proteins with the use of drugs such as ace2 and tmprss2 inhibitors (mckee et al., 2020) . unfortunately, ace inhibitors, which are used for the treatment of hypertension and chronic heart failure, do not inhibit ace2. moreover, scientists are concerned that such drugs could increase the likelihood of severe covid-19 illness, though current data tends to provide no link between the use of ace inhibitors and increased severity of covid-19 (hippisley-cox et al., 2020; who, 2020) . all the above can be considered the first steps towards covid-19 therapeutics and indicate that the spike glycoprotein may indeed be the most promising drug target against sars-cov-2 infection. coronaviruses' nucleocapsid protein, or n protein, is the most abundant viral protein that can be detected in the human host from the early stages of infection (che et al., 2004) . its main function is to bind to viral rna to form a ribonucleoprotein nucleus, which contributes to its entry into the host cell as well as to the interaction with cellular processes after fusion, such as cell cycle regulation, and antigen processing and presentation of the virus (huang et al., 2004; wang et al., 2010) . the sequence of n protein of sars-cov-2 is approximately 90% similar to the same protein of sars-cov (gralinski and menachery, 2020) . the n protein genome comprises a serine-rich binding region (sr) between an n terminal domain (ntd) and a c terminal domain (ctd). n structural protein forms the replication transcription complexes (rtc), which play an important role in the synthesis of the genome virus (xia et al., 2020) . in sars-cov, the ν protein contributes to the activation of cyclooxygenase-2 (cox-2), thus promoting inflammation in the lungs (yan et al., 2006) . in addition, it participates in processes that affect the cell cycle, such as inhibiting the phosphorylation of phosphoprotein b23, a protein that is essential for cell cycle evolution during centrosome replication (zeng et al., 2008) . moreover, studies have shown the inhibitory effect of n protein on the cyclin-cyclin-dependent kinase complex (c-cdk), resulting in a reduction of s-phase progression (surjit et al., 2006) . a different function of the n protein is its interaction with the protease subunit p42, which degrades viral proteins . specifically, this interaction may impair proteolysis of viral proteins and their presentation to cytotoxic t-lymphocytes, thus promoting viral evasion from immune effectors . concerning the immune system, n protein causes limitations in the immune responses generated by the body due to viral infections as it inhibits type i interferon (ifn) (lu et al., 2011) . according to another study, due to the aggregation of the human elongation factor 1 a (ef1a) induced by the n protein, the proliferation of the cells of the cell line under study, specifically 293 t cells, was reduced (zhou et al., 2008) . in contrast, n protein interacts with heterogeneous nuclear ribonucleoprotein (hnrnpa1), causing an increase in viral rna synthesis (luo et al., 2005) . the structural study of the ntd of sars-cov-2 n protein showed that it resembles a wrist consisting of acidic moieties with a palm of basic components, and the core of the beta-sheet extends like fingers (kang et al., 2020) . the ntd of the n protein binds to the rna genome in the n45-181 region that exists as a monomer (chang et al., 2006) , while the presence of the amino acids arginine at position 94 and tyrosine at position 122 appears to be necessary for viral rna binding (mcbride et al., 2014) . according to further studies, the enhanced binding capacity of viral rna requires the combination of the binding region and ntd and ctd of n protein (chang et al., 2009) . n protein function regulation is mediated through the central linker region, which includes serine and arginine residues (sr region), having essential phosphorylation sites. in this region, the major phosphorylation site is formed, which enhances protein interactions in proteins and the localization of binding proteins within the cell (chang et al., 2014) . the ctd is hydrophobic and rich in helical region in which dimerization occurs as it contains residues that self-bond to form homodimers (mcbride et al., 2014) . structurally, each asymmetric ctd group consists of four individual homodimers from the compound of which an octamer is formed. the arrangement of these sections is schematically similar to the letter "x" which forms symmetrical folding structures, perpendicular to the middle point of the structure. the n terminus appears to be basic due to the positive charges in this region, which could be implied to be a site for nucleic acid binding (chen et al., 2007) . n protein, together with s protein, is a very promising area in the development of effective therapeutics to prevent the proliferation of viral offspring as it participates in activities necessary for the function and proliferation of the virus and is, therefore, another key ingredient after spike proteins (satarker and nampoothiri, 2020) . currently, the only potential drug targeting the n protein is pj34 which inhibits the viral protein's function and has shown some promise both in vitro and in animal studies (saxena, 2020) . the coronaviruses' envelope protein, or e protein, is a vital protein, which is tiny and consists of the n-terminal domain, a hydrophobic domain, and the c terminal domain (ruch and machamer, 2011) . the n terminal extends to the first nine amino acids, the hydrophobic region extends between 10-37 amino acids and the c terminal between 38-76 amino acids, where the first 11 amino acids are located in virion while the hydrophobic tail is in the cytoplasm, which through its oligomerization creates an ionic pore across the membrane (shen et al., 2003; verdia-baguena et al., 2013) . through structural studies, sars-cov-2 form is presented, which includes 35 α-helical regions and 40 looped regions (gupta et al., 2020) . a unique characteristic that occurs in sars-cov-2 e protein and not in other homologous sars-cov e protein is the replacement of the 69 th amino acid of the protein sequence, from arginine to alanine, glutamine, and aspartate. furthermore, a deletion specific to sars-cov-2 envelope protein flanks this position. these changes may have an effect on viral conformation, protein-protein interaction and, possibly, affect the oligomerization process necessary to form a transmembrane ion channel. in addition, at positions 55 and 56 of the amino acid sequence, amino acid valine and threonine have been identified (bianchi et al., 2020) . in coronaviruses, the e protein forms viroporins which are small hydrophobic proteins that are necessary for viral assembly and release, while also participating in pathogenic processes that cause cytotoxicity (ye and hogue, 2007) . the co-expression of m and e proteins facilitates the production of spherical infectious particles, while the heterotypic j o u r n a l p r e -p r o o f interactions of nsp2 and nsp3 cause the desired curvature in the endoplasmic reticulum (er) membrane (schoeman and fielding, 2019) . the hydrophobic tail of the protein in the cytoplasm through the proline residues incorporated in it targets the region of the cis-golgi complex, as well as the n-terminal of protein e targets the golgi complex through additional elements (cohen et al., 2011) . the diffusion of the ionic gradient from the e protein into the ergic and golgi may lead to the exit of the virion (liu et al., 2007) . the last four amino acids of the sequence that comprise the c terminal domain include a pattern called postsynaptic density protein / disc large / zonula occludans-1 (pdz) binding pattern (pdm). therefore, e protein through binding of protein associated with caenorhabditis elegans lin-7 protein 1 (pals1) to the pdm, aids in the disruption of the lung epithelium, thus being a potential pharmacological target for improved treatment against sars-cov-2 (teoh et al., 2010; satarker and nampoothiri, 2020) . regarding therapeutics, in silico studies have proposed a number of drugs that target the e protein, which include belachinal, macaflavanone e, and vibsanol (gupta et al., 2020) . membrane protein, or m protein, is present in greater amounts than all other proteins in coronaviruses (ea and jones, 2019). in coronaviruses, this protein has a short length n terminal domain, as its total length is 220-260 amino acids, belongs to the n-linked glycosylated proteins with a conserved region of 12 amino acids and is attached to tripartite transmembrane regions which are further linked to the c terminal domain (arndt et al., 2010) . according to structural studies, it exists in two forms, one compact and one long. initially, these two forms are homodimers of the n-terminal ectodomain and the c-terminal endodomain, where the endodomain can be elongated or compressed, resulting in a long and compact form. tyrosine residues at position 211 are necessary for the stability of the long form of the m protein, as well as the spike protein is observed mainly in the long form of the m protein, suggesting that this form promotes the establishment of the spike proteins. the long form of the m protein bends the membrane, thus creating a spherical structure surrounding the ribonucleoprotein (neuman et al., 2011) . m protein is organized in a two-dimensional lattice and provides a scaffold in viral assembly, while its translation takes place in the polysomes connected to the membranes following its fusion to the endoplasmic reticulum and transport to the golgi complex, where it interacts with the e proteins to create virions (neuman et al., 2006; tseng et al., 2010) . m protein appears to affect the immune response to pathogens, as it inhibits nuclear factor kappa b (nfkb) through interactions with ikkb (i kappa b kinase) leading to a decrease in cox-2 levels and resulting in an increase in the proliferation of the pathogenic virus (fang et al., 2007) . in addition, the c terminus of the m protein inhibits the interaction of 3-phosphoinositide-dependent protein kinase 1 (pdk1) and protein kinase b (pkb), thus leading to the release of the caspases 8 and 9, which eventually cause cell death or apoptosis (tsoi et al., 2014) . based on the above information regarding the m protein of coronaviruses, sars-cov-2 protein can be a potential pharmacological target for limiting and inhibiting the formation of virions and preventing inflammatory reactions in host cells, as the presence of the m protein is crucial in the viral life cycle (satarker and nampoothiri, 2020) . currently, broad spectrum antiviral drugs that target the membrane protein of coronaviruses, such as jl103, have been proposed for possible use against sars-cov-2 (khan et al., 2020). viruses in the coronaviridae family code for two types of cysteine proteases, including the 3c-like protease (3clpro) and up to two papain-like proteases. with these enzymes' use, the polyprotein generated through the translation of the viral genome of the virus is cleaved into sixteen nonstructural proteins. this processing step is crucial for the generation of the replicase complex that is required for rna replication. sars-cov-2, in particular, encodes a single papain-like protease (henceforth referred to as plpro) (freitas et al., 2020) . the plpro domain is part of the multi-domain nsp3 protein. it processes the nsp1/2, nsp2/3, and nsp3/4 cleavage sites. the plpro domain, which is membrane-associated, recognizes a cleavage sequence in the threshold areas between nsp1/12, nsp2/3, and nsp3/4. after the cleavage, the nsp1 nsp2, nsp3 are separated from the viral polyprotein and the next steps of the formation of the replicase complex may be excecuted, allowing the production of new virions and the establishment of the viral infection. studies on the enzymatic function of plpro have shown its capacity for recognition and hydrolysis of the ubl protein isg15 (interferon-induced gene 15) and the protein ubiquitin (ub), both of which are cellular proteins. thus, plpro constitutes a deubiquitinating (dub) and deisgylating enzyme (báez-santos et al., 2015) . this type of activity has important ties to the course of the innate immune response in the setting of sars-cov infection. numerous instances of ubiquitination and isgylation events are observed in various stages of the innate immune response. the interference of plpro in different parts of these pathways can be achieved through the recognition and the interaction with deisgylating and/or deubiquitinating proteins that exist therein. specifically, plpro appears to act as an antagonist, blocking the generation of cytokines of importance in the host innate immune response. these include type i interferon β (ifnβ), as well as ccl5 and cxcl10 (freitas et al., 2020) . given its crucial role in the virus replication cycle and the suppression of the host immune defenses, plpro has had a significant existing history as a drug target against a number of coronaviruses, such as mers cov and sars cov. similarly, amid the covid-19 pandemic, plpro constitutes a promising pharmacological target. within the scientific community, there is a race for the development of inhibitors against this particular viral enzyme, as well as for the possible employment of existing protease inhibitors. numerous studies are dedicated to the exploration of pre-existing, fda-approved drugs with the goal of repurposing them for the inhibition of sars-cov-2. through a number of methods, among which is molecular dynamics and virtual screening, fda-approved drugs, which offer the advantage of already completed preclinical and clinical phases, are combed through in order to select the best candidates for the inhibition of the sars-cov-2 papain-like protease. a prime example of a drug candidate provided through such an approach is phenformin (kandeel et al., 2020) . as mentioned above, the main protease, also known as 3c-like protease (3clpro), is among popular targets against sars-cov-2. 3clpro executes proteolytic cleavage of the overlapping pp1a and pp1ab translated viral polyproteins, allowing the production of functional proteins during the coronavirus replication process (ullrich and nitsche, 2020) . more specifically, 3clpro (nonstructural protein 5), following its autocleavage, processes the aforementioned polyproteins at their respective cleavage sites (du et al., 2004) . the 3clpro monomer contains three domains, with a long loop connecting the second and third domains. the enzyme's active site is located between the first and the second domain, containing a catalytic pair of cysteine and histidine. the histidine's imidazole attracts the side-chain proton of the cysteine, creating a thiolate nucleophile, which then attacks the amide bond on the enzyme's substrate. through proton abstraction from histidine, the n-terminal peptide product is released, while the cterminal product is released through the hydrolysis of the thioester, allowing the subsequent restoration of the catalytic pair (estrada, 2020) . in order for the enzyme to perform its catalytic activity, dimerization is necessary. the two protomers bind to each other through an n-terminal finger, which aids in the formation of the substrate-binding site . the mediation of nonstructural viral proteins' maturation by 3clpro makes it a very attractive target for the development of anti-coronavirus drugs. it is important to note that 3clpro executes cleavage exclusively of the polypeptide sequences that follow a glutamine residue. this ability offers a significant advantage, given that there are no known host-cell protease enzymes that share this particular specificity of substrate. in essence, inhibitors of the main protease would be unlikely to have off-target effects (ullrich and nitsche, 2020) . sars-cov inhibitors have so far steered towards peptide inhibitors and small-molecule inhibitors (wu et al., 2020a) . studies conducted on the design and screening of 3clpro inhibitors usually focus on cleavage sites, substratebinding sites, the catalytic pair at the enzyme's active center, as well as various reported residues of critical importance for enzyme function. significant conservation of the catalytic site has been observed between the 3clpro structures of the sars-cov, mers-cov, and sars-cov-2 viruses. this discovery allows the examination of already established inhibitors of the other coronaviruses' 3clpro enzyme, under the possibility that they may prove effecting against the 3clpro of the novel sars-cov-2 . furthermore, research groups have been establishing screening processes for the possible repurposing of various drugs. among those, anti-bacterial drugs, antihypertensive drugs, as well as natural compounds with antiviral properties have shown a high binding affinity to 3clpro, making them attractive candidates for the inhibition of the viral 3clpro towards the treatment of covid-19 (wu et al., 2020a) . another key enzyme necessary for the viral replication cycle that has been characterized as potent therapeutic target is the viral helicase (vlachakis et al., 2013c) . sars-cov-2 nsp13 has been shown to possess ntpase and rna helicase activity; it catalyzes the unwinding of double stranded rna in an ntp-dependent manner (shu et al., 2020) . due to its sequence conservation across coronaviruses, targeted inhibition of its enzymatic activity is recognized as a promising strategy for antiviral therapy (habtemariam et al., 2020) . sars-cov-2 nsp13 has a high structural and functional similarity with mers and sars nsp13, belonging to the sf1 helicase family. it consists of 5 domains that form a triangular pyramid shape; the two 'reca-like' domains, domain 1a and 2a, along with domain 1b form the triangular base, while the zinc binding domain (zbd) and stalk domain, that connects 1b and zbd domains, are located at the apex of the pyramid (hao et al., 2017; jia et al., 2019) . domains 1a, 1b and 2a are involved in ntp and nucleic acid binding, while it has been shown that the nsp13 enzymatic activity is enhanced by direct interaction of nsp12 at the zbd-1a interaction site (jia et al., 2019; romano et al., 2020) . a number of compounds have been reported as competitive inhibitors against sars coronavirus (scv) nsp13 activity that could pave the ground for clinical research for sars-cov-2 inhibition. myricetin and scutellarein have been identified to strongly interrupt atpase but not helicase activity, possibly by direct binding at the atpase domain (yu et al., 2012) . aryl diketoacids (adk) and dihydroxychromone derivatives demonstrated selective inhibition against the scv nsp13 duplex dna-unwinding activity in multiple binding sites in the target enzyme, while 2-arylmethyloxy-6-(3chlorobenzyloxy)-5-hydroxychromones were shown to inhibit both ntpase and helicase activities (lee et al., 2009a; lee et al., 2009b; kim et al., 2011) . another class of antiviral compounds, bananins, have been shown to inhibit viral transcription and replication by significantly reducing the viral rna levels whereas they did not affect viral entry. the compounds were tested against scv ntpase/helicase activity and a number of bananin derivatives were found to be potent atpase and helicase inhibitors at concentrations significantly below toxicity levels (tanner et al., 2005) . 3-[(2-nitrophenyl)sulfanylmethyl]-4-prop-2-enyl-1h-1,2,4-triazole-5-thione, ssya10-001, was recognized by adedeji and coworkers as a novel inhibitor of scv that interferes with the dsrna/dsdna activity of nsp13 without competitive inhibition of ntp and nucleic acid binding, but possibly by inducing conformational changes of the enzyme that disrupt the unwinding and translocation mechanisms (adedeji et al., 2012) . bismuth salts have been also characterized as strong inhibitors of both atpase and duplex-unwinding activities through binding to the zinc binding domain (zbd) of scv nsp13 at micromolar concentrations (yang et al., 2007a; yang et al., 2007b) . similarly, a recent study on sars-cov-2 identified inhibitory effects of bismuth salts against nsp13, and specifically, bismuth potassium citrate (bpc) and ranitidine bismuth citrate (rbc) exhibited effective inhibition of ntpase and helicase activity with the same binding mode, interrupting the sars-cov-2 replication cycle (shu et al., 2020) . in silico methods have been proven to be a crucial step for the rapid, cost-efficient identification of potent compounds with inhibitory effects in antiviral research loukatou et al., 2015; papageorgiou et al., 2016) . as such, computational approaches employing molecular modelling and virtual screening against known antivirals, active compounds and/or fda approved drugs for drug repurposing, have been applied for the identification of candidate sars-cov-2 helicase inhibitors (loukatou et al., 2015) . iftikhar et al. identified two compounds, meclonazepam and oxiphenisatin, that bind at β19-β20 loop on the 1a domain of sars-cov-2 helicase, that is directly involved in unwinding double stranded nucleic acids (iftikhar et al., 2020) . vapreotide, an aids-related diarrhea drug, was found to exhibit the lowest binding free energy amongst 23 approved antivirals in a computer-aided drug design pipeline (borgio et al., 2020) . similar approaches have identified cmp1, cmp3a, cmp11 and cmp15 to competitively bind at the ntp binding site (mirza and froeyen, 2020) , and elbasvir, approved for hcv treatment, to inhibit not only helicase activity by blocking ssdna binding, but also rna binding to rdrp (balasubramaniam and reis, 2020). rna viruses encode rna-dependent rna polymerase (rdrp), an essential enzyme for viral replication since it catalyzes rna synthesis, governing the initiation and elongation phase (ng et al., 2008) . rdrps share a similar core structure resembling the shape of a right hand with three subdomains, namely the palm, finger and thumb subdomains (vlachakis et al., 2017; venkataraman et al., 2018) . the catalytic site of rdrps is formed by seven conserved motifs (motif a-g), five of which are located within the most conserved palm subdomain (a-e), while motifs f and g are found in the fingers (vlachakis et al., 2013a; vlachakis et al., 2013b; shu and gong, 2016) . sars-cov-2 polymerase, or nsp12, recently resolved structure consists of a right-hand rdrp domain and a nidovirus-specific n-terminal extension domain, the niran domain. rdrp and niran domains are connected by an interface domain and an additional β-hairpin domain located at the groove formed by niran domain and palm subdomain stabilizes the structure (gao et al., 2020) . due to their high significance in viral life cycle and their conserved mode of function across viral families, rdrps have long been the target of antiviral research with a number of approved therapies targeting viral polymerases (tsai et al., 2006; ng et al., 2008; reznik and ashby, 2017; yao et al., 2018; peersen, 2019) . consequently, during the current covid-19 outbreak, nsp12 is considered as a promising target for antiviral therapeutics. identified nucleoside analogues that were originally developed to target viral rdrps and novel compounds are evaluated as antiviral drug candidates against covid-19 (neogi et al., 2020) . remdesivir is the most promising drug candidate to date. it is a prodrug of an adenosine nucleotide analog that is converted to active nucleoside triphosphate upon cell entry and competes with atp in the rdrp binding site, leading to chain termination (warren et al., 2016; yin et al., 2020) . remdesivir was developed for the treatment of ebola virus disease (evd), but it has not been approved after showing low efficacy in a phase iii clinical trial for evd (warren et al., 2016) . however, in vitro and in vivo studies of remdesivir against mers-cov and sars-cov and recent studies against sars-cov-2 showed inhibitory effects and antiviral activity with low cytotoxicity levels (sheahan et al., 2017; wang et al., 2020) . additionally, remdesivir was tested in a compassionate clinical trial for severe covid-19 patients, and after 10-day treatment an 68% clinical improvement was observed (grein et al., 2020) . several clinical trials have been reported to evaluate remdesivir safety and efficacy, while a number of patents have already been granted (eastman et al., 2020) . favipiravir, a guanine analog developed for treatment of influenza viruses, currently approved in china and japan, is also an anti-covid19 drug candidate (furuta et al., 2017) . favipiravir inhibits polymerase activity, yet the exact mode of action has not been elucidated. two possible mechanisms for chain termination have been proposed, either by inhibition of the atp catalytic site, or by misincorporation in a nascent viral rna (furuta et al., 2005) . clinical trials for favipiravir have shown efficient treatment for moderate covid-19 patients and several clinical trials have been registered and are ongoing (cai et al., 2020; du and chen, 2020; li and de clercq, 2020) . besides remdesivir and favipiravir, several nucleotide analogs have been reported as potential antiviral agents. ribavirin, a ribonucleoside analog that has been tested against several rna viruses but exhibits higher levels of cytotocisity and side-effects, is also on ongoing clinical trial for covid-19 treatment combined with interferon-α2b (falzarano et al., 2013) . jockush et al. have shown in the inhibitory effects of sofosbuvir, alovudine, zidovudine, tenofovir alafenamide and emtricitabine and termination of polymerase rna synthesis through in vitro assays (jockusch et al., 2020) . silibilin is predicted to have a dual activity against sars-cov-2 infection; silibilin can potentially reduce viral replication activity by targeting nsp12 as a remdesivir-like inhibitor, and modulate inflammatory responses by direct inhibition of stat3 (boschbarrera et al., 2020) . stat3 showcases both pro-and anti-inflammatory abilities; it mediates interleukin 10 (il-10) activity, a known anti-inflammatory cytokine. on the other hand, in the case of cancer, persistent activation of stat3 mediates tumor-promoting inflammation (hillmer et al.; yu et al., 2009) . as an alternative strategy, an isonicotinic acid derivative, enisamium and its putative metabolite, vr17-04, have been identified by in vitro assays as drug candidates against sars-cov-2. enisamium was shown to inhibit the activity of the sars-cov-2 rna polymerase and its active metabolite exhibits similar efficacy with remdesivir triphosphate, it is approved in 11 countries and does not require intravenous administration, unlike remdesivir (walker et al., 2020) . in silico approaches have also been used to identify novel inhibitors, either by screening clinically used polymerase inhibitors, or fda approved drugs and/or natural compounds (kar et al., 2020; pokhrel et al., 2020; ruan et al., 2020; zhang et al., 2020b) . it is critical to find ways to block the spread of sars-cov-2 and stop covid-19. research on sars-cov-2 structure and mechanisms of action has provided a number of intriguing drug targets. those targets, which include both non-structural and structural proteins, have an essential role in various stages of viral infection, from viral entry to viral replication and formation of viral vesicles. disrupting their action may have a therapeutic effect against covid-19. although research on the aforementioned targets is intense, and some results may seem promising, the current situation requires being precautious before declaring the uncovering of a sars-cov-2 therapy. nevertheless, specific studies, especially on the spike glycoprotein and viral proteases, allow some restrained optimism regarding the future. as in any therapeutic approach concerning human disease, toxicity is a critical component in the development of safe and effective drugs against sars-cov-2. ace2, as reviewed earlier, is a promising target for the development of therapeutic strategies. a recombinant form of the human ace2 protein was synthesized as a therapeutic treatment for covid-19, functioning as a decoy for sars-cov-2 and essentially preventing the virus from binding to the cell surface ace2 (schuster et al., 2010) . the approach of recombinant proteins carries the risk of off-target binding, which is the binding of the recombinant protein to other cell-receptors, leading to unwanted signaling cascades. furthermore, use of recombinant proteins may lead to the activation of the immune system, which may in turn result in the production of anti-drug antibodies and their deposition in the form of immune complexes, potentially hindering the function of important organs (de groot and scott, 2007) . lastly, this sort of treatment may lead to serum sickness, in the case that the immune system may view the recombinant protein as an antigen (schellekens, 2005) . in the case of viral replication inhibitors, instances of toxicity reported for other nucleoside analogs can provide insight into the possible toxicity of analogs such favipiravir or remdesivir, which are being evaluated for use against sars-cov-2. while inhibiting the viral polymerase enzyme, nucleoside analogs additionally inhibit mitochondrial dna polymerase-gamma, resulting in reduction of mitochondrial protein synthesis (moyle, 2000) . aspects of toxicity include myopathy and pancreatitis (johnson et al., 2001) , severe metabolic acidosis paired with elevated lactate, with significant observed mortality (falcó et al,. 2002 ) and bone marrow suppression in patients treated with 3'-azido-3',3'-dideoxythymidine (azt) (moyle, 2000) . as far as protease inhibitors are concerned, drugs such as ritonavir, an inhibitor of cytochrome cyp3a4, can have an effect on the metabolism of drugs-substrates of cyp3a4, which in turn may result in unpredictable interactions between the drug (rock et al., 2014) . as summarized by chary and colleagues, the use of humanized antibodies as a therapeutic method harbors dangers of hypersensitivity reactions, immunostimulation, which results in chills, fever and other reactions, and finally, immunosuppression, which may often become a window for opportunistic infections (chary et al., 2020) . to date, efforts are being made by the scientific community to develop a vaccine against sars-cov-2. vaccines that have been developed and are in the stage of clinical trials are based on one hand on the virus genome and on the other hand, on the structure of the viral s-protein tu et al., 2020) . one of the vaccines that started developing in early 2020 is moderna's mrna-1273. this vaccine is a synthetic clone of mrna that encodes the viral s-protein. the goal of the vaccine is to provoke an immune response specific for s-protein of sars-cov-2. another characteristic of this vaccine is that the virus is not required for its development, as the synthetic mrna is encapsulated in lipid nanoparticles (tu et al., 2020) . the chadox1 ncov-19 vaccine was developed by the university of oxford and is currently in clinical phase iii. this vaccine consists of a non-replicable adenovirus vector and the s protein sequence of the sars-cov-2 protein. characteristic of this vaccine is that due to the adenovirus-based vector, it can cover both the respiratory and gastrointestinal epithelium, which are the two main sites expressing the sars-cov-2 ace-2 receptor (folegatti et al., 2020) . finally, the university of queensland is developing a vaccine that is equally based on the molecular structure of the virus sars-cov-2. as sars-cov-2 is an enveloped virus, the fusion of its viral membrane with the host cell membrane is required for infection. for this purpose, the structure of the glycoproteins of the viral envelope alter from the pre-fusion form, which is more unstable, to the post-fusion form. thus, this vaccine is a stabilized subunit vaccine based on molecular clamp technology, such as the vaccines against influenza virus and ebola virus, and allows the recombinant viral proteins to remain stable in the pre-fusion 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zoonotic origins of human coronaviruses structural basis for inhibition of the rnadependent rna polymerase from sars-cov-2 by remdesivir stats in cancer inflammation and immunity: a leading role for stat3 identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 covid-19 pathophysiology: a review the nucleocapsid protein of sars-associated coronavirus inhibits b23 phosphorylation crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors novel coronavirus polymerase and nucleotidyl-transferase structures: potential to target new outbreaks sars-cov-2: an emerging coronavirus that causes a global threat the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha the authors are responsible for the choice and presentation of views contained in this article and for opinions expressed therein, which are not necessarily those of unesco and do not commit the organization. key: cord-317355-z5tk3v3b authors: dunker, susanne; hornick, thomas; szczepankiewicz, grit; maier, melanie; bastl, maximilian; bumberger, jan; treudler, regina; liebert, uwe g.; simon, jan-christoph title: no sars-cov-2 detected in air samples (pollen and particulate matter) in leipzig during the first spread date: 2020-10-13 journal: sci total environ doi: 10.1016/j.scitotenv.2020.142881 sha: doc_id: 317355 cord_uid: z5tk3v3b the sars-cov-2 pandemic co-occurred with pollen season in europe 2020 and recent studies suggest a potential link between both. air samples collected at our measuring station in leipzig and purified pollen were analyzed for sars-cov-2 typical signals or for virus-induced cytopathic effects, to test if the virus could bind to bioaerosols and if so, whether these complexes are infectious. the results show that neither air samples nor purified pollen were infectious or could act as carrier for virus particles. the analysis of the first pandemic phase of severe acute respiratory syndrome coronavirus 2 in europe 2020 demonstrated that infections co-occurred with the pollen season (brandstetter et al., 2020) . moreover, pollen may interfere with antiviral immunity, e.g. pollen significantly diminish the epithelial response to rhinovirus infection (gilles et al., 2020) . to date it is not known, if pollen allergy has an impact on the prevalence or severity of the sars-cov-2 pandemic. in china, allergic diseases, asthma, and copd were not shown to be risk factors for sars-cov-2 infection (zhang et al., 2020) , but in this region the maximum peak was reached during winter time, outside the pollen season and patients were not interviewed for any history of pollinosis. importantly, pollen can act as a carrier for various bacteria and moulds (heydenreich et al., 2012; obersteiner et al., 2016; oteros et al., 2019) , and sars-cov-2 remained viable in aerosols for several hours (van doremalen et al., 2020) . furthermore, morawska & cao (2020) (morawska and cao, 2020) speculate that wind-induced dispersion may occur. it was speculated, if bioaerosols may serve as carriers for the virus as regions with high air pollution were more severely affected (conticini et al., 2020) and just recently it could be demonstrated that sars-cov-2 rna can be present on pm (setti et al., 2020) . we therefore aimed at investigating whether sars-cov-2 can bind to pollen or other kind of particulate matter within bioaerosols sampled at our station in leipzig and if so, whether these complexes are infectious. j o u r n a l p r e -p r o o f fresh pollen samples from betula pendula, quercus robur and ostrya carpinifolia were collected (tab. a1). inflorescences were dried in paper bags at 30 c and pollen were sieved through a 70 µm filter (celltrics, sysmex, norderstedt, germany) and stored dry upon analysis. highly purified reference material (alnus glutinosa, betula pendula and corylus avellana) were purchased from allergon ab (ängelsholm, sweden). attempts of sars-cov-2 isolation were made using natural environmental samples (described above, table 1 ). following centrifugation at 11,000 rpm (5 min/ room temperature) supernatant was removed and saved for pcr testing (cov pcr day 0). the pellet was resuspended in dmem supplemented with nystatin/ amphothericin (40 µg/ ml/ 50 µg/ ml), seeded onto vero e6 cells in a 2.5 cm 2 petri dish, incubated at 37 °c/ 5 % co 2 and observed for 5 days for the occurrence of virus-induced cytopathic effects (cpe). negative at a final concentration of 0.2 µm, and 1 µl ssiii/platinum tag. input-rna was 5 µl, and 2.6 µl of water was added to reach a final reaction volume of 20 µl. alternatively rdrp_sarsr-f2/r1 and rdrp_sarsr-p2/p1 were used for amplification and detection of the respective gene. rt was done at 55 °c for 10 min, followed by 3 min denaturation at 94 °c and 45 amplification cycles with denaturation for 15 sec at 94 °c, annealing for 30 sec at 58 °c and elongation for 30 sec at 60 °c. amplification was monitored after each elongation step at 530 nm. the pollen season in leipzig, germany started with a high number of hazel (corylus sp.) pollen at the beginning of february 2020, followed by alder (alnus sp.) one week later (fig. 1 ). nine days after the last higher alnus sp. pollen peak (94 pollen/ m³), the first covid-19 cases were documented. overall, low case numbers were registered in leipzig in contrast to other german cities, most likely related to an early lockdown (red bar in fig. 1 ). the birch (betula sp.) pollen season started during the decrease of registered covid-19 cases in april and was followed by pollen emission from oak (quercus sp.) and pine (pinus sp.). particulate matter (pm 2,5 ) concentrations were partially higher than the threshold level of 10 µg/m³ (annual mean) defined by the who (dotted grey line in fig. 1) . however, the concentrations were still lower than in regions of northern italy (bianconi et al., 2020; conticini et al., 2020) or wuhan (ma and kang, 2020) and could be an additional reason for low registered case numbers in leipzig. air samples containing bioaerosols and particulate matter were collected starting with the first wave of infections (grey bars in fig. 1 ). in none of these samples sars-cov-2 typical for a detailed analysis of a possible correlation between concentrations of the most abundant pollen, particulate matter and registered covid-19 cases, a correlation matrix was created with r (package "performanceanalytics") (fig. 2) . the number of registered cases (green box, fig. 2 ) was positively correlated with the pollen-concentration of corylus sp. and negatively correlated with the concentrations of grasses (poaceae). (table 1) . next, we wished to determine whether pollen can bind sars-cov-2 at all. to address this issue, commercially available, highly purified pollen (alnus glutinosa, betula pendula and corylus avellana) were incubated in vitro with sars-cov-2 (3x10 4 tcid 50 in 0.5 ml dmem, i.e. approx. 1x10 7 genome equivalents) for 1 hr. thereafter, pollen was washed, centrifuged as indicated in the supplement before they were screened for presence of viral rna and infectious sars-cov-2. again, no sars-cov-2 typical signal was detected by rt-pcr or cpe at any time point (table 1) . moreover, we could exclude the possibility that components of the air samples interfered with virus detection since geq of sars-cov-2 spiked to original samples were clearly detected by rt-pcr or cpe (fig. a1 , table a2 ). we are somewhat consoled by this negative finding since a positive result would have had severe implications for sars-cov-2 restrictions, as pollen can be transported over long distances (up to several 100 km) (hjelmroos, 1991) . it should be noted however, that due to a low number of registered covid-19 cases in leipzig, it may be difficult that a measurable virus load can be detected in the air due to low number of emitters, especially since infected persons also had to go into quarantine immediately. therefore, a negative result for sars-cov-2 is not surprising for this case study, but could j o u r n a l p r e -p r o o f potentially be different in cities with much higher infection rates. statistically, it is highly probable to find virus on pollen and particulate matter when the concentration of both is really high also meaning that there is a coalescence effect between droplets and pm. this probably confirms that the spread effect in leipzig was essentially due to the direct contact human-to-human and not mediated by a vehicle, while in regions with higher pm load, transmission via pm could be an additional pathway (setti et al., 2020) . we also considered technical limitations to account for our failure to detect sars-2 cov-2 signals in air samples and in purified pollen preparations. air sampling: several authors have shown, that cyclone samplers are suitable to collect virus particles in the air (d'arcy et al., 2014; kim et al., 2018; verreault et al., 2008; west and kimber, 2015) . d`arcy et al. (2014) (d'arcy et al., 2014) successfully detected airborne virus particle in hospital air with a cyclone sampler, which is in accordance with the fact that this technology was originally developed to collect biological warfare agents in the air. the height of our measurement station was chosen to guarantee a representative measurement in contrast to near-ground stations which show higher variability in pollen concentrations (rojo et al., 2019) . nevertheless, it would be interesting to analyze if sars-cov-2 signals in near-ground traps, e.g. on crowded public places can be detected. sars-cov-2 detection: nucleic acids were analyzed by rt-pcr and infectivity was tested by analysis of cpe on vero indicator cells. we could exclude that suspended air samples or purified pollen interfered with sars-cov-2 replication since sars-cov-2 spiked to the original samples was readily detected (fig. a1 , table a2 ). our in vitro incubation experiments of highly purified pollen and sars-cov-2 were performed in fluid suspension. however, the binding pattern between pollen and virus particles could be potentially different in the air, e.g. due to electrostatic effects. such effects might be of interest in future studies. in summary, this is a first study investigating the relation of pollen and sars-cov-2 pandemic. the results show that neither air samples nor purified pollen of different taxa were infectious or could act as a carrier for virus particles. this leaves open the possibility that pollen or particulate matter within bioaerosols may affect sars-cov-2 susceptibility indirectly for example by perturbing nasal or bronchial epithelial barrier function or anti-viral immunity as shown by others for rhinovirus infection (gilles et al., 2020) . the leipzig e.v. as potential conflicts of interest. note: there was no inhibition of sars-cov-2 replication. * sars-cov-2 pcr; e, rdrp and n gene, measured without prior cell cultivation, day 0 ** sars-cov-2 pcr; e, rdrp and n-gen, measured after cultivation for 8 days j o u r n a l p r e -p r o o f particulate matter pollution and the covid-19 outbreak: results from italian regions and provinces symptoms and immunoglobulin development in hospital staff exposed to a sars-cov-2 outbreak can atmospheric pollution be considered a co-factor in extremely high level of sars-cov-2 lethality in northern italy? detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr. euro surveillance bulletin europeen sur les maladies transmissibles environmental viral contamination in a pediatric hospital outpatient waiting area: implications for infection control pollen exposure weakens innate defense against respiratory viruses grampositive bacteria on grass pollen exhibit adjuvant activity inducing inflammatory t cell responses evidence of long-distance transport of betula pollen electrochemical detection of airborne influenza virus using air sampling system air quality variation in wuhan, daegu, and tokyo during the explosive outbreak of covid-19 and its health effects airborne transmission of sars-cov-2: the world should face the reality pollen-associated microbiome correlates with pollution parameters and the allergenicity of pollen artemisia pollen is the main vector for airborne endotoxin nearground effect of height on pollen exposure sars-cov-2rna found on particulate matter of bergamo in northern italy: first evidence aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 methods for sampling of airborne viruses. microbiol innovations in air sampling to detect plant pathogens clinical characteristics of 140 patients infected with sars-cov-2 in wuhan studies were co-financed by the german science foundation via the idiv-flexpool (project 100269858). we especially want to thank the dr. födisch umweltmesstechnik ag for the author regina treudler indicated sanofi genzyme, novartis, alk-abello, abbvie, shire, fraunhofer institute izi and hautnetz leipzig e.v. as potential conflicts of interest.j o u r n a l p r e -p r o o f key: cord-309074-pys4aa60 authors: huang, victoria w.; imam, sarah a.; nguyen, shaun a. title: telehealth in the times of sars-cov-2 infection for the otolaryngologist date: 2020-05-30 journal: world j otorhinolaryngol head neck surg doi: 10.1016/j.wjorl.2020.04.008 sha: doc_id: 309074 cord_uid: pys4aa60 objective: in response to the american academy of otolaryngology – head and neck surgery’s recommendations to limit patient care activities in the times of sars-cov-2, many elective surgeries have been canceled without patient clinics transitioning to virtual visits. with regulations for telemedicine loosened, new possibilities for the practice of otolaryngology have opened. to address the uncertain duration of this pandemic, a review was conducted of current literature on use of telemedicine services in the current sars-cov-2 pandemic and in previous national emergencies to reveal the role telemedicine can play for otolaryngology practices. data sources: pubmed articles with an independent search query were utilized. methods: literature review performed by one author searched for all published english-language literature on telehealth in the sars-cov-2 era. articles were considered for discussion if they provided relevant developments for telemedicine in the context of the sars-cov-2 pandemic. results: telemedicine can be up-scaled in the current sars-cov-2 pandemic where exposure containment is of the utmost priority. with patient interaction possible through virtual communication, telemedicine allows continued patient care while minimizing the risk of viral spread. in the realm of otolaryngology, telemedicine has been used in the past during disasters with other studies demonstrating high diagnostic concordance with inpatient visits. many institutions have recognized the potential for such care as they begin utilize both virtual visits and in-person care during this pandemic. conclusion: to limit the spread of sars-cov-2, we support the aao-hns recommendation for the adoption of novel ways to employ telemedicine in this era. many emergency departments and health care systems have the infrastructure necessary for synchronous video telemedicine visits that can be leveraged to provide quality care with patients. with the continued need to socially distance, telemedicine can protect both physicians and patients from unnecessary exposure to the virus. socially distance, telemedicine can protect both physicians and patients from unnecessary exposure to the virus. the practice of medicine is changing as the coronavirus infection, also known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), continues to spread. currently, without a vaccine, social distancing and isolation have become the most effective measures to decrease transmission 1,2 .otolaryngologists appear to be at higher risk than their colleagues of contracting sars-cov-2 according to the director of the intensive care unit at peking union medical college hospital 3 . this may be due to high viral load in the nasal cavities and nasopharynx 4 . additionally, the frequent use of irrigation and anesthetic sprays in otolaryngology may aerosolize these viral particles. this can dramatically increase exposure to sars-cov-2 as the virus can remain airborne viable for longer than three hours 5, 6 .these compounding risks have resulted in the cancellation of elective surgeries, rescheduling of nonurgent office visits, and even alterations to how standard physical exams and diagnostic tests are performed 7 . however, high-quality patient care must still be provided while minimizing the risk of exposure of patients and providers to the virus. in response to these evolving needs, the american academy of otolaryngology -head and neck surgery (aao-hns) telemedicine committee has put forth new recommendations to prioritize novel applications of telehealth to help limit coronavirus disease pandemic spread while maintaining quality care 8 . telehealth and telemedicine are defined as direct exchanges of medical information from one site to another through secure electronic communication to improve a patient's health 9 . many health systems in the u.s. already have telemedicine programs in place, allowing clinicians to see patients who are at home. these services have traditionally been used for chronic disease management with increasing research in mental health and counseling use especially during times of crisis 10, 11 . these virtual sessions can be either synchronous with real time video exchange or asynchronous (store-and-forward) with clinical data stored and forwarded to a remote clinician for further analysis. in the current sars-cov-2 pandemic, the medicare population continues to be at high risk and a transition to telemedicinecare for these patients may greatly reduce exposure and decrease risk of infection. there are three main types of virtual services practitioners can provide to medicare beneficiaries: medicare telehealth visits, virtual check-in, and e-visits 12 . medicare telehealth visits require audio and video telecommunication systems that allow real-time communication between the patient and the distant site. in the past, these visits required a prior relationship with the provider, but in this declared national emergency, the u.s.department of health and human services (hhs) stated that they will not conduct audits to confirm a relationship with the provider. another major barrier to wide-spread adoption was reimbursement 12 . to address this, the hhs has waived certain telehealth regulations to allow for its expanded use for medicare patients. retroactive to march 6, 2020, medicare will pay for office, hospital, and other visits through telehealth across the country at the same rate as regular, in-person visits. additionally, the hhs office for civil rights has also made flexibilities on hipaa guidelines, allowing clinicians and patients to communicate through any non-public facing remote communication product including apple facetime, facebook messenger video chat, google hangouts video, and skype, though hipaa-compliant video communication products such as skype for business, updox, vsee, zoom for healthcare, doxy.me, and google g suites hangouts meet are still recommended 13 . as infection rates continues to change, the duration of this pandemic remains uncertain and necessitates plans to utilize available structures. in this article, we review the current literature on use of telemedicine services in the current sars-cov-2 pandemic and its use in previous national emergencies to help realize the true value of telemedicine and change the way otolaryngologists can provide care for patients. with the aao-hns telemedicine committee advocating for novel applications of telemedicine in this era of sars-cov-2, a review of its previous applications revealed telemedicine has long played a role in medical response to disasters 14 . telemedicine can deploy large numbers of providers and facilitate triage to prevent the overwhelming of front-line providers. this makes it an ideal model to up-scale in a pandemic where exposure containment is of the utmost priority. pandemic responses in the united kingdom and australia have already called upon the increased use of telemedicine 11 . however, clinician willingness and acceptance of telehealth, reimbursement, and organization of the current health care systems continue to be major barriers to increased implementation outside of the emergency department 11, 15 . in the u.s., many hospital systems already have telemedicine departments embedded within their systems. recognizing that much of medicine is cognitive, many neurologic intensive care units across institutions like cleveland clinic, university of pittsburgh and jefferson health provide virtual icu visits through which neurointensivists monitor patients remotely while bedside critical care nurses examine patients with their guidance 16, 17 . the use of electronic consults(econsults),which allow primary care physicians to consult specialists through the electronic medical record, has been documented in the canadian health system 18, 19 .this was recently studied in the otolaryngology practice at ucsd and resulted in improved access to specialists for timely advice and reduced unnecessary face-to-face specialist referrals 20 . many major university hospital sites are now adopting more technology to improve their response to the sars-cov-2 pandemic. ucla and new york presbyterian hospitals have followed singapore's response by providing rapidly accessible information through a chatbox on their websites to answer any questions regarding sars-cov-2 11, 21, 22 . as testing in the u.s. becomes more available in this era of sars-cov-2, telemedicine continues to take the main role of "forward triage", evaluating patients with respiratory symptoms before they arrive in hospitals 23 with the aao-hns recommending all otolaryngologists to limit patient care activities to time-sensitive, urgent, and emergent medical conditions, elective surgeries have been canceled with many outpatient clinics rescheduling appointments and transitioning to virtual visits 7, 8 . at institutions like johns hopkins and ohio state, providers are asking patients to change appointments to virtual ones 29, 30 .telemedicine appointments can be conducted with both the clinician and patient at home, limiting travel and exposure while maintaining uninterrupted quality care with established patients. as a part of the covid-19 public health emergency response, medicare has stated that it will pay for office, hospital, and other visits through telehealth across the country at the same rate as regular, in-person visits 12 this presents a unique opportunity for clinicians to make use of telemedicine. the otolaryngology field has been slow to widely adopt telemedicine, though it has been gaining ground as the frequent archiving of audio and visual images from exams makes the field uniquely suited to adopt advances in telemedicine. in 2010,the hurricane katrina disaster left louisiana state university health science center with no neurotology service. a store-andforward model of telemedicine was implemented and a previously established telemedicine neurotology clinic in baton rouge forwarded clinical materials to a neurotologist in pittsburgh, which resulted in positive anecdotal patient responses 33 .to identify specific areas in otolaryngology that would be most suitable for telemedicine visits, a study on veterans in the new england area identified that 62% of visits did not require specialized procedures and could be conducted with the help of a health technician that could synchronously communicate with a remote otolaryngologist 34 . taking advantage of telemedicine and remote centers can also greatly reduce travel burden as the study found 42% of patients were driving over 3 hours for otolaryngology services. to address concerns of diagnostic accuracy through telemedicine visits, ohio state piloted studies assessing diagnostic concordance of an on-site and remote practitioner receiving synchronous information through video 35 .for the21 cases, there was a 95% diagnostic concordance for patients presenting with a variety of diagnoses such as vocal cord leukoplakia, acute otitis externa, and septal deviation. of note, only 62% of patients indicated they would travel to see an otolaryngologist, suggesting that about one third of patients may not have pursued otolaryngology care if there was not a nearby telemedicine clinic. physician and patient satisfaction of the video and audio quality were both over 95% and both agreed that care was more accessible with this technology. while all these applications of telemedicine resulted in high patient and provider satisfaction, they still required patients to travel to a telemedicine clinic and interact with a healthcare provider, making them less ideal in the era of sars-cov-2. some hospitals have employed telemedicine in ways that do not require any travel to a clinic to limit patient exposure to providers. following head and neck free tissue transfer, residents at ucsf performed60 flap checks through video. evaluations of skin color, skin turgor, capillary refill and doppler signal were similar between telemedicine and in-person groups 36 . however, if patients experience flu-like symptoms, they are urged to call the clinic or hospital and asked to call another number to assess if they will need further testing. anecdotal evidence from physicians noted better reimbursement when compared to telephone visits and higher physician satisfaction with patient interaction. however, as the aao-hns still allows for inperson care of "time-sensitive, urgent, and emergent medical conditions", hospitalized patients and post-operative visits after major surgery are still seen in-person with proper protection. seattle's slowing infection rates highlights how adherence to early social distancing and quarantine can reduce infection rates of sars-cov-2 40 . even as elective surgeries are cancelled and non-urgent outpatient visits are postponed, there is still a way to provide quality care for patients. with the need for social distancing, telemedicine has become the ideal tool to allow communication between a physician and a patient. to limit the spread of sars-cov-2, we support the aao-hns recommendation for the adoption of novel ways to employ telemedicine in this era. as many emergency departments and health care systems have the infrastructure necessary for synchronous video telemedicine visits, we propose the adoption of novel uses for existing telemedicine portals. for those without an existing structure, it is possible to outsource telemedicine services to programs such as teladoc health or american well 23 . we have reviewed the available methods of employing telemedicine and continue to encourage new applications and integration into otolaryngology practices. current use of telemedicine is targeted toward the screening and management of suspected sars-cov-2 patients. as the sars-cov-2 pandemic continues to unfold, recommendations on social isolation may evolve and require adjustments to traditional patient care workflow. we support the aao-hns recommendation to limit all in-person care to urgent cases, but propose the use of telemedicine to continue quality care with established patients. with the continued need to socially distance, telemedicine can protect both physicians and patients from unnecessary exposure to the virus. none. none. none. scientific and ethical basis for social-distancing interventions against covid-19 world health organization. guidance for health workers europe's doctors repeat errors made in wuhan sars-cov-2 viral load in upper respiratory specimens of infected patients aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 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comparison of video and in-person free flap assessment following head and neck free tissue transfer telemedicine in otolaryngology outpatient setting-single center head and neck surgery experience detection of sars-cov-2 among residents and staff members of an independent and assisted living community for older adults physical distancing is working and still needed to prevent covid-19 resurgence in king not applicable. not applicable. the datasets supporting the conclusion of this article are included within the article.authors' contributions san, vwh, sai drafted the manuscript;all authors read and approved the final manuscript. this study requires no ethics approval due to public data-based analysis.the authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. not applicable all authors declare no conflict of interest in this study. this article does not contain any studies with human participants or animals performed by any of the authors. key: cord-315064-2mgv9j6n authors: escher, felicitas; pietsch, heiko; aleshcheva, ganna; bock, thomas; baumeier, christian; elsaesser, albrecht; wenzel, philip; hamm, christian; westenfeld, ralph; schultheiss, maximilian; gross, ulrich; morawietz, lars; schultheiss, heinz‐peter title: detection of viral sars‐cov‐2 genomes and histopathological changes in endomyocardial biopsies date: 2020-06-12 journal: esc heart fail doi: 10.1002/ehf2.12805 sha: doc_id: 315064 cord_uid: 2mgv9j6n aims: since december 2019, the novel coronavirus sars‐cov‐2 has spread rapidly throughout china and keeps the world in suspense. cardiovascular complications with myocarditis and embolism due to covid‐19 have been reported. sars‐cov‐2 genome detection in the heart muscle has not been demonstrated so far, and the underlying pathophysiological mechanisms remain to be investigated. methods and results: endomyocardial biopsies (embs) of 104 patients (mean age: 57.90 ± 16.37 years; left ventricular ejection fraction: 33.7 ± 14.6%, sex: n = 79 male/25 female) with suspected myocarditis or unexplained heart failure were analysed. emb analysis included histology, immunohistochemistry, and detection of sars‐cov‐2 genomes by real‐time reverse transcription polymerase chain reaction in the ikdt berlin, germany. among 104 embs investigated, five were confirmed with sars‐cov‐2 infected by reverse real‐time transcriptase polymerase chain reaction. we describe patients of different history of symptoms and time duration. additionally, we investigated histopathological changes in myocardial tissue showing that the inflammatory process in embs seemed to permeate vascular wall leading to small arterial obliteration and damage. conclusions: this is the first report that established the evidence of sars‐cov‐2 genomes detection in embs. in these patients, myocardial injury ischaemia may play a role, which could explain the ubiquitous troponin increases. emb‐based identification of the cause of myocardial injury may contribute to explain the different evolution of complicated sars‐cov‐2‐infection and to design future specific and personalized treatment strategies. in december 2019, a novel coronavirus with potential zoonotic origin, named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was identified as the causative agent of a cluster of suspicious pneumonia cases in wuhan, hubei, china. the incredible fast worldwide spread of the coronavirus disease 2019 (covid-19) prompt the world health organization (who) to declare covid-19 as a pandemic on 11 march 2020. 1 more than 1 776 867 confirmed cases of covid-19 and more than 111 828 fatalities in 185 countries have been attributed to sars-cov-2 as of 14 april 2020 (https://who.sprinklr.com/). molecular tests (real-time reverse transcriptase polymerase chain reaction, rt-qpcr) were generally used to confirm the clinical diagnosis of covid-19. a recent report showed that sars-cov-2 could be detected in different types of clinical specimens such as broncho alveolar lavage, sputum, nasal swabs, feces, blood, and urine. 2 the ubiquitous distribution of the main viral entry receptor angiotensin converting enzyme 2 (ace2) for sars-cov-2 entry into the target cells led to the hypothesis of the involvement of other potential target organs for sars-cov-2 besides the respiratory tract, for example, the heart, the liver, the brain, the pancreas, or the kidneys. 3 infection with the sars-cov-2 is associated with systemic illness by hyper-inflammation. 4 cardiovascular complications with embolism due to covid-19 have been reported recently. [5] [6] [7] [8] acute myocardial injury associated with covid-19 manifested as an increase of high-sensitivity cardiac troponin levels. 1 however, direct sars-cov-2 rna in the heart muscle has not been demonstrated so far. the damage caused by sars-cov-2 to the cardiovascular system and the underlying mechanisms remain to be investigated. accordingly, we prospectively analysed endomyocardial biopsies (embs) from a cohort of 104 samples of patients with suspected myocarditis or unexplained heart disease for the presence of sars-cov-2 rna by rt-qpcr and hints for histopathological injury. up to 8 embs each of 104 patients [mean age: 57.90 ± 16.37 years; left ventricular ejection fraction (lvef): 33.7 ± 14.6%, sex: n = 79 male/25 female] with suspected myocarditis or unexplained heart failure were analysed between 3 february and 26 march 2020 in german clinical centres in accordance with sars-cov2 spread in germany. embs were routinely taken from left ventricle. in 60.4% a hypertrophy was seen according possible due to a cardiac oedema. coronary artery disease was excluded angiographically in all patients prior to emb. the suspected diagnosis had been made by clinicians. embs were send for further diagnosis to the laboratory ikdt (institute for cardiac diagnostic and therapy berlin, germany). analysis included histology, immunohistochemistry, and molecular virology. following emb extraction, samples were transferred to formalin for histological analyses and to rnalater ™ solution (thermo fisher scientific, waltham, ma, usa) for immunohistological and molecular analyses. dna was extracted by puregene core kit a (qiagen, hilden, germany) according to manufacturer's instructions. total rna from one emb was isolated using trizol reagent ™ (thermo fisher scientific, waltham, ma, usa), solubilized in depc-h 2 o, and treated with dnase (peqlab, erlangen, germany) to remove any traces of dna followed by reverse transcription with high-capacity cdna reverse transcription kit (thermo fisher scientific, waltham, ma, usa). random hexamer primers (5 μm) were used in addition to specific primers targeting the e-gene of sars-cov2 (0.2 μm each). dna and cdna concentrations were measured by pcr-based quantifiler ™ human dna quantification kit (thermo fisher scientific, waltham, ma, usa) or by expression of housekeeping gene hprt, respectively. detection of other cardiotropic viruses (enteroviruses, adenoviruses, human herpesvirus 6, epstein-barr virus, parvovirus b19) using (rt-) qpcr or nested pcr was applied as described elsewhere. 9,10 commercially available rt-qpcr kits targeting the e-gene and rdrp-gene (tib molbiol, roche diagnostics, germany) and the assay n2 (n-gene) published by the cdc were chosen to initially screen samples for presence of sars-cov2 genomes. as aforementioned assays were proven to be the most robust, rdrp-gene assay was used for confirmation of results. 11 all rt-qpcr assays were performed using taqman universal pcr mastermix (thermo fisher scientific, waltham, ma, usa) in a 20 μl reaction mix consisting of 2× pcr buffer including enzyme mix, primers, and probes concentrations as recommended by manufacturers and 21.5 μl and 5 μl of cdna. thermal cycling was carried out as recommended by manufacturers on either abi quantstudio 12k flex or biorad cfx96 thermal cyclers. in brief, real-time rt-pcr was performed with 45 cycles using 1.5 μl of cdna. however, for validation of our pcr results additional pcr runs were performed with 5 μl of cdna not altering the results obtained by the 1.5 μl approach. synthetic in vitro rna of e-gene and rdrp-gene assays were diluted 1:10 prior to cdna synthesis and plasmid positive control of n-gene assay was diluted 1:104 prior to rt-qpcr to account for an expected low yield in total rna extracted from emb samples. histology was developed from formalin-fixed tissue by haematoxylin & eosin (he); azan, and periodic acid-schiff (pas) staining in light microscopy. for immunohistological evaluation, specimens were rnalater fixed, embedded in tissue tec (slee, mainz, germany) and immediately snap-frozen in methyl butane which had been cooled in liquid nitrogen and then stored at à80°c until processing. embedded specimens were cut into cryosections placed on 10% poly-l-lysine-precoated slides. myocardial inflammation was diagnosed by cd3 + tlymphocytes/mm 2 (dako, glostrup, denmark), cd11a + /lfa-1 + lymphocytes/mm 2 (immuno tools, friesoythe, germany), cd11b + /mac-1 + macrophages/mm 2 (immuno-tools, friesoythe, germany), cd45r0 + t memory cells (dako, glostrup, denmark), perforin + cytotoxic cells/mm 2 (bd bioscience, san jose, california). in addition, we stained intercellular adhesion molecules and mhc class ii cell surface receptor (cd54/icam-1 and hladr, immunotools, friesoythe, germany). staining were quantified by digital image analysis. 12 approval was not required. endomyocardial biopsy results of total patient cohort are summarized in table 1 . out of the 104 emb samples, five patients were positive for sars-cov-2 e-gene specific sequences indicating the first description of sars-cov-2 presents in a case series. besides latent infection with parvovirus b19, no other viral pathogens were detectable in sars-cov-2 positive samples. based on the clinical history, the clinicians expressed a suspicion of a previous covid-19 infection, but they were not tested with throat swab sample during admission to the hospital. the clinical courses of the five patients were different and showed highly acute to mild forms. patient 1 was a 48-year-old male with newly diagnosed heart failure and significantly reduced systolic function (ef 22%). suspected diagnosis was acute myocarditis. he described sudden onset of high-grade fever and dyspnoea within a few days. in addition, he suffered from thrombi and embolia. he reported a prior vacation in tyrol, austria. this patient showed a highly acute status was admitted to the intensive care unit (icu) and due to severe infection. the diagnosis of a small-vessel vasculitis was established, and cyclophosphamide and additional steroids were initiated. the patient recovered adequately. after receiving emb results, immunosuppressive treatment was stopped immediately. patient 2 was a 62-year-old male with mildly reduced ef (40%) and moderate lv-hypertrophy, and without respiratory infect. this patient had a new cardiac impairment of lv function since january 2020. the cause was unknown, so a possible myocarditis was assumed. with the exception of cardiac symptoms, this patient had a mild course and did not need to be monitored by icu. patient 3 was a 60-year-old female with heart failure symptoms but preserved ef (60%) with pronounced lvhypertrophy. initially, she was admitted to the icu with severe acute respiratory syndrome. blood tests revealed elevated levels of markers of myocyte injury (see table 2 ), which remained positive during the first days of her hospitalization. after respiratory improvement the emb was carried out 4 weeks after onset of syndromes. in this interesting case, the cardiac symptoms occurred with a pronounced relapse after the initial event. patient 4 was a 36-year-old male with a significantly reduced systolic function (ef 25%) with a history of mild respiratory infect 3 weeks ago. the clinical course developed without complications and icu surveillance. during hospitalization, the levels of troponin decreased laboratory values on day 15 were in reference range, and he recovered during this time. patient 5 was a 39-year-old male with heart failure symptoms but preserved ef with suspected diagnosis of acute myocarditis. the patient had a history of upper airway infection with headache and fever up to 4 weeks before admission. he suffered from shortness of breath, t-wave inversions in the anterolateral leads in ecg, elevated cardiac troponin i, and cardiac magnetic resonance imaging compatible with myocarditis. the course of this patient was acute and required icu treatment. in patients 2-5, treatment strategies were not modified after receiving the result of sars-cov-2 rt-qpcr in emb. they were treated symptomatically, in part with initiation of guideline-directed medication for heart failure. patient characteristics and emb results are summarized in table 2 . sars-cov-2 loads determined in the embs were low (ct values: 36.66 ± 1.99) corresponding to less than 100 to 500 viral copies/reaction. viral loads were determined from the internal sars-cov-2 positive control with a ct value of 32.73 ± 1.12 corresponding to approximately 10e + 4 copies/reaction while the ct values of sars-cov-2 negative samples were below 40 cycles and thus below detection limit. results from rt-qpcr are shown in table 3 . histological assessment of embs revealed an active myocarditis according to the dallas criteria in patient 1 13,14 ( figure 1a) . histological analysis could also show necrosis of myocytes and interstitial tissue and granulation tissue in the periphery of necrosis of the type observed after an infarction ( figure 1a) . immunohistochemical emb analysis confirmed pronounced intramyocardial inflammation. analysis of immune cell infiltrates of sars-cov-2 genome positive embs showed elevated number of t-cells, macrophages, lymphocytes, and t-memory cells (cd45r0) in four of the five patients ( figure 1a-c, e, f) . moreover, all sars-cov-2 patients exhibited an elevated number of cell adhesion molecules (cd54/icam-1). patient 2 showed inflammatory response on limit values. we could show that the inflammatory process in cardiac tissue seemed to permeate vascular wall. the inflammatory process was leading to arterial obliteration and damage (figure 1b-d) . the final mechanism of tissue damage in consequence of vascular obliteration appears to be similar to systemic forms of vasculitis leading to ischaemia. the neighbouring myocardium displayed vacuoles in myocytes as a sign of restricted metabolism. perivascular fibrosis with variation of fibre densities could be seen in cases 2 to 5 (not shown in figures). this phenomenon indicated relicts of previous damage. in this study, we established for the first time the evidence of sars-cov-2 genome detection in 5 of 104 embs of patients with suspected myocarditis or unexplained heart failure. after the first cases describing pneumonia of unknown origin in wuhan, china, sars-cov-2 rapidly spread worldwide with critical challenges for the public health and medical communities. cardiovascular involvement in covid-19 seems to be a notable complication. first single case reports could show viral particles in interstitial cytopathic macrophages and their surroundings in emb of a severe covid-19 shock patient by electron microscopic analyses. whether direct myocardial injury due to viral involvement or the effect of systemic inflammation appear to be the most common mechanisms responsible for cardiogenic shock situation needs to be further investigated. 19 in the analytic stage, real-time rt-pcr assays remain the molecular test of choice for the aetiologic diagnosis of sars-cov-2 infection. specificity of e-gene and rdrp-gene assays tested with clinical respiratory samples and sars-cov and mers-cov did not result in cross-reactivity and false positive results. high sensitivity of both assays as indicated by low pcr limit of detection for purified rna could also be confirmed for rna spiked into and extracted from swab samples. 15 however, direct sars-cov-2 rna detection in the myocardium has not been demonstrated so far. herein, we demonstrated by rt-qpcr that sars-cov-2 genomes is present in different cases. in this study, we described series of different histories of cardiovascular patients admitted to the hospital. one main clinical finding is that cardiac involvement with positive sars-cov-2 genomes in embs can either occur acutely or with latency after onset of symptoms of infection. based on the results of currently published research, it seems important to discuss the manifestations and characteristics of myocardial damage induced by covid-19. 16 herewith, we validated the direct cardiac involvement associated with intramyocardial inflammation in patients with sars-cov-2 genome positivity in embs. in patient 1, we could show an active myocarditis and in patient 5 a borderline-myocarditis according the dallas criteria. in the remaining patients, an inflammatory cardiomyopathy was determined. recent literature data have shown that cardiac troponin i concentration is increased in all patients with sars-cov-2 infection, and values exceeding the 99th percentile in the upper reference limit can be observed in 8-12% of positive cases. 17 moreover, patients with covid-19 are known to be at higher risk of acute pulmonary embolism, and elevated d-dimer levels on admission are predictive of adverse outcomes for patients with covid-19. 5 the first vascular sign has been referred to as 'vascular thickening' or 'vascular congestion' in the lung. bai et al. 18 reported vascular thickening to be significantly associated with covid-19 compared with non-covid-19 pneumonia (59% vs. 22%, p < 0.001). the physiopathologic mechanisms behind these changes remain unclear, but their role in diagnosis and possible future treatment strategies is substantial. in this regard, a very recent report showed that pericytes demonstrating high ace-2 expression might act as target cells for sars-cov-2, while pericyte injury can result in endothelial cell dysfunction. 22 recent reports showed that besides pericyctes ace-2 is expressed to different levels also in cardiomyocytes, endothelial cells, fibroblasts, and leucocytes. 23 however, ace-2 expression does not argue for permissive infection of a respective target cell by sars-cov-2. on the other hand, recent reports have demonstrated that sars-cov-2 genomes could be detected besides airway epithelium cells also in the intestinal enterocytes, spleen, liver, kidney, and heart. 2, 24 in addition, recent histologically post-mortem analyses in covid-10 positive patients revealed lymphocytic endotheliitis in different organs with evidence of direct viral infection, indicating endothelial dysfunction as a possible principle determination of microvascular dysfunction by shifting the vascular equilibrium towards more vasoconstriction with subsequent organ ischaemia and inflammation. 21 although nearly all organs seemed to be affected by covid-19, we currently do not know in-depth details about the organ-specific infection by sars-cov-2. in this regard, tavazzi and coworkers have shown recently in their case description using electron microscopy on embs of a patient with covid-19 in cardiogenic shock that sars-cov-2 particles could be localized to interstitial macrophages and their surroundings but not in cardiomyocytes. 19 as to whether this observation is due to a transient viraemia or infected macrophage migration from the lung has to be evaluated. our finding of sars-cov-2 genome detection in embs of patients suffering from myocarditis/inflammatory cardiomyopathy cannot rule out or confirm the infection of cardiac cells but revealed incremental insights into organ-specific infection of sars-cov-2 using possibly macrophage migration as a shuttle from the lung to the heart. in this study, we investigated histopathological changes in myocardial tissue in the series of sars-cov-2 positive embs. in line with the recently published study, we could show that the inflammatory process in embs seemed to permeate vascular wall leading to small arterial obliteration. the final mechanism of tissue damage in consequence of vascular obliteration appears to be similar to systemic forms of vasculitis. we therefore hypothesize that in these patients, myocardial injury ischaemia may play a role, which could explain the ubiquitous troponin increases. as a result, this ischaemia could trigger possible cardiac arrhythmias. a limitation of this study is that we did not had enough material for sars-cov-2 genome in depth analysis to certainly exclude cross reaction with other corona virus strains due to the limited material available by the embs. however, the high sensitivity and specificity of the used pcr systems to detect solely sars-cov-2 genomes have been demonstrated recently. 15 another limitation is that we cannot completely exclude that the detection of sars-cov-2 genomes in the heart might result from contamination of circulating blood. unfortunately, we have no blood samples to the corresponding embs on hand to analyse this aspect. however, sars-cov-2 load in blood seemed to be low in comparison with other clinical types of specimens. 2 nevertheless, sars cov-2 can potentially bind to its cellular ace2 receptor in heart tissue cells and can therefore be detected in the heart muscle. in this regard, a recent report showed that pericytes in the heart demonstrating high ace-2 expression might act as target cells for sars-cov-2 while pericyte injury can result in endothelial cell dysfunction. 20, 22 if sars cov-2 can replicate in these target cells of the heart, this has to be investigated in subsequent analysis. the low detection rate and low viral loads of sars-cov-2 genomes may be due to the limited number, size, and quantity of embs. heart tissue cells (e.g. pericytes) are not the main target cells of sars-cov-2 while specimens of the main target the lung of infected patients are easier and in larger quantity to obtain than embs and may contribute to the low detection rate in embs. however, we showed that sars-cov-2 is detectable in the heart muscle but can only speculate about the clinical relevance of sars-cov-2 infection of the heart. as to whether sars-cov-2 infection may induce myocarditis is questionable, however, may trigger an ongoing progress to myocarditis of other reason. in conclusion, in this study, we could show for the first-time evidence of sars-cov-2 genome detection in 5 of 104 patients with suspected myocarditis or unexplained heart failure with different history of symptoms and time duration. in addition to inflammation and consequential damage, one possible histopathological mechanism may be vascular involvement with arterial obliteration which can lead to ischaemia. a possible sars-cov-2 infection should therefore be considered in patients with acute unexplained heart failure or new cardiac arrhythmias. we believe that recognition by the scientific community of myocarditis as a possible complication associated with covid-19 may be helpful for strict monitoring of affected patients. emb-based identification of the cause of myocardial injury may contribute to explain the different evolution of complicated sars-cov-2-infection and to design future specific treatment strategies. an antiviral therapy is not yet available. based on our histopathological results, possible anticoagulant/antiaggregation therapy should be investigated. clinical features of patients infected with 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circulating human herpes simplex viruses dilated cardiomyopathy myocarditis: the dallas criteria european society of cardiology working group on myocardial and pericardial diseases. current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr potential effects of coronaviruses on the cardiovascular system: a review cardiac troponin i in patients with coronavirus disease 2019 (covid-19): evidence from a meta-analysis performance of radiologists in differentiating covid-19 from viral pneumonia on chest ct sepe sars-cov-2 genomes in endomyocardial biopsies 7 myocardial localization of coronavirus in covid-19 cardiogenic shock. myocardial localization of coronavirus in covid-19 cardiogenic shock the ace2 expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov-2 endothelial cell infection and endotheliitis in covid-19 what is a pericyte? cell type-specific expression of the putative sars-cov-2 receptor ace2 in human hearts sars-cov-2 productively infects human gut enterocytes all nurses, clinicians, and infectious diseases specialists working hard in this difficult period are greatly acknowledged for their efforts and daily care for patients suffering from sars-cov-2 infection. this work was done within a profit grant of the investitionsbank berlin (profit no. 10169028, berlin, germany). for their excellent technical assistance, we thank k. winter, c. seifert, s. ochmann, c. liebig, and k. errami (ikdt berlin, germany). none declared. key: cord-308252-qwoo7b1l authors: cardinale, vincenzo; capurso, gabriele; ianiro, gianluca; gasbarrini, antonio; arcidiacono, paolo giorgio; alvaro, domenico title: intestinal permeability changes with bacterial translocation as key events modulating systemic host immune response to sars-cov-2: a working hypothesis date: 2020-09-16 journal: dig liver dis doi: 10.1016/j.dld.2020.09.009 sha: doc_id: 308252 cord_uid: qwoo7b1l the microbiota-gut-liver-lung axis plays a bidirectional role in the pathophysiology of a number of infectious diseases. during the course of severe acute respiratory syndrome coronavirus 1 (sars-cov-1) and 2 (sars-cov-2) infection, this pathway is unbalanced due to intestinal involvement and systemic inflammatory response. moreover, there is convincing preliminary evidence linking microbiota-gut-liver axis perturbations, proinflammatory status, and endothelial damage in noncommunicable preventable diseases with coronavirus disease 2019 (covid-19) severity. intestinal damage due to sars-cov-2 infection, systemic inflammation-induced dysfunction, and il-6-mediated diffuse vascular damage may increase intestinal permeability and precipitate bacterial translocation. the systemic release of damageand pathogen-associated molecular patterns (e.g. lipopolysaccharides) and consequent immune-activation may in turn auto-fuel vicious cycles of systemic inflammation and tissue damage. thus, intestinal bacterial translocation may play an additive/synergistic role in the cytokine release syndrome in covid-19. this review provides evidence on gut-liver axis involvement in covid-19 as well as insights into the hypothesis that intestinal endotheliitis and permeability changes with bacterial translocation are key pathophysiologic events modulating systemic inflammatory response. moreover, it presents an overview of readily applicable measures for the modulation of the gut-liver axis and microbiota in clinical practice. the outbreak of coronavirus disease 2019 (covid19) , which is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has rapidly diffused worldwide and is now considered a pandemic by the world health organization [1] . sars-cov-2 is a positive-sense single-stranded rna virus and a member of the betacoronavirus genus. coronaviruses bind to their target cells through the angiotensin 2-converting enzyme (ace2), a monocarboxypeptidase that cleaves numerous peptides within the renin-angiotensin system and other substrates [1] . the cell entry of coronavirus depends on the binding of viral spike (s) proteins to cellular receptors and on s protein priming by host cell proteases [1] . ace2 is expressed constitutively by the epithelial cells of the lung, intestine, kidneys, and blood vessels and is present in the 3 terminal ileum and colon in concentrations that are among the highest in the body [2] . intestinal involvement and intestinal cell infection were previously demonstrated in the severe acute respiratory syndrome coronavirus 1 (sars-cov-1) epidemic [3] . a considerable number of sars-cov-1 patients experienced gastrointestinal symptoms [4, 5] . while sars-cov-1 can infect the lungs and intestine, tissue response in these two organs differs. small pathology findings can be observed in the intestine with optical microscopy, both in biopsies taken during the initial stages [4] and in autopsy samples [6] . however, electron-microscopy [4] and immunohistochemistry [3, 6] studies have revealed the presence of sars-cov-1 in surface enterocytes and in small vessels in the intestine. therefore, although changes in the intestine are usually milder than those seen in the lungs, an increase in intestinal permeability to lipopolysaccharides (lpss) and the translocation of intestinal bacteria are likely to occur. the liver is also a target of coronavirus. viral nucleic acids of sars-cov-1 have been found in liver tissue, and percutaneous liver biopsies of sars-cov-1 have shown conspicuous atypical features of liver injury, including acidophilic bodies, hepatocyte ballooning, and lobular activities without fibrin deposition or fibrosis [7] . the present review will discuss different pathophysiological mechanisms through which the gastrointestinal tract may contribute to sars-cov-2 infection progression. covid-19 presents with fever and typical respiratory symptoms, but also with gastrointestinal symptoms and liver damage [8] [9] [10] [11] . as previously demonstrated in sars-cov-1, gastrointestinal manifestations are significant extrapulmonary complaints in covid-19 patients [8] [9] [10] [11] . in particular, diarrhoea is frequent [8] [9] [10] , while nausea and vomiting have been associated with lung involvement [11] . the potential role of the gut in sars-cov-2 infection has been demonstrated in adult and paediatric subjects [12] [13] [14] [15] . in particular, the faecal excretion of sars-cov-2 has been observed in more than half of infected subjects [13, 15] , and this excretion is prolonged [14, 15] and persists for nearly 5 weeks, even after patient respiratory samples test negative for sars-cov-2 rna [15] . conversely, although sars-cov-2 is detectable in stools, there is no clear evidence regarding the ability of faecal virus excretions to induce infection and thus facilitate faecaloral transmission. sars-cov-2 uses the sars-cov receptor ace2 for entry and the transmembrane serine 4 protease 2 (tmprss2) for s protein priming. interestingly, the evaluation of ace2 and tmprss2 expression according to anatomic sites and cell types revealed that ace2 expression in the small intestine and colon is respectively ~40x and ~3x higher with respect to the lungs, whereas tmprss2 expression in the small intestine and colon is respectively ~2x and ~20x higher with respect to the lungs [16] (fig. 1 ). since vascular involvement may determine the systemic consequences of the infection on different districts, it is important to keep in mind that the extension of the epithelial/blood barrier is 50-100 m 2 in the lungs and 250-400 m 2 in the intestine [17] (fig. 1) . besides what can be inferred by data on the expression of ace2 and tmprss2 [13] [14] [15] [16] [17] [18] 19] , direct infection of intestinal cells by sars-cov-2 has been demonstrated through the intestinal organoid technique [20] . since inflammation seems to upregulate ace2 expression [17] , it is important to understand whether patients with inflammatory bowel disease (ibd) are more susceptible to covid-19 and the cytokine release syndrome (crs) associated with lung injury and fatal outcome [21] . while the risk of sars-cov-2 infection in ibd patients depends on several universal risk factors, including social distancing [22] , older age and comorbidities have been associated with a negative outcome in ibd, whereas ibd treatments have not, highlighting that acute ibd flare prevention and inflammation reduction may avoid severe covid-19 [23] . sars-cov-1 and sars-cov-2 infections are characterized by intense systemic symptoms triggered by an exuberant host immune response [25, 26] . however, sepsis is not evident in most cases [5] [6] [7] [8] . excessive systemic inflammatory responses with or without the pulmonary counterpart (acute respiratory distress syndrome/ards) that are characterized by high levels of a wide range of proinflammatory cytokines and chemokines have been associated with severe morbidity and mortality in a wide variety of viral diseases [25] . similar features have been reported during the course of acute lung injury caused by respiratory syncytial virus, influenza a virus, and sars-cov-1 [25, 26] . the reason for this unbalanced inflammatory response in some viral infections is not well understood and is probably multifactorial [25] . although the role of bacterial lps has not been demonstrated in human sars-cov-1 and sars-cov-2, the cellular receptor for degradation products of the intestinal bacterial flora (pathogen-associated molecular pattern 5 molecules, in particular lps), i.e. the toll-like receptor 4 (tlr4), has already been confirmed to induce harmful inflammatory responses during acute viral infections [25] . furthermore, the peculiar phenotype of covid-19 inflammatory-cytokine response points toward the activation of t helper 17 lymphocytes, a particular subset of t helper lymphocytes characterized by a high production of il-17 and other proinflammatory cytokines. this is the same systemic inflammatory response observed in intestinal bacterial translocation [26] . cross-talk between the lungs and intestinal barrier has been described, which may be relevant in the pathogenesis of ards and sepsis [27] . in ards and critically ill patients with sepsis, bacterial translocation is widely documented and can be viewed as an advanced event that precipitates lung disease and rapidly worsens systemic inflammation [27] . in other words, intestinal barrier dysfunction can be considered part of multiorgan failure, and contributes to further lung damage and systemic inflammation in ards and sepsis [27] . in sars-cov-2 infection, bacterial translocation may be an early event related to intestinal damage due to tissue infection, systemic inflammation-induced dysfunction, and il-6-mediated diffuse vascular damage. a reconciliatory working hypothesis is presented in fig. 2 . sars-cov-2 infection and replication culminate in the activation of innate [28] and adaptive immunity [29] and complement [30] , with systemic and local effects, e.g. the release of lymphocyte-derived (ifn gamma, il-6, sil-2r alpha, sil-6r, and gm-csf) and monocyte/macrophage (il1-receptor antagonist, il-10, il-6, ip-10, monokine, ifn alpha, mip-1 alpha, mip-1 beta, and sil6r) cytokines and neutrophil and macrophage accumulation in tissues [31] . in addition, il-6-mediated endothelial cell damage (tissue factor exposure), coagulation [32] , and vasculature leakage induce further tissue damage and perpetuate systemic inflammation, e.g. crs [33] . this complex systemic and local pathologic condition may induce intestinal damage and increase the intestinal permeability precipitating intestinal barrier failure and bacterial translocation, with the systemic release of damage-associated molecular patterns (damps) and pathogenassociated molecular patterns (pamps), e.g. lpss and consequential tlr activation, which in turn auto-fuel vicious cycles of systemic inflammation and tissue damage (fig. 2) . intestinal bacterial translocation may play an additive/synergistic role in the crs underlying the unfavourable evolution of covid-19 [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . cross talk between lpss and il-6 that determines heartburn and systemic inflammatory response independent of prostaglandins has been previously described [34] . liver cells express ace2 [35] , particularly cholangiocytes, 6 which have easily been experimentally infected by sars-cov-2 [36] . the liver injury demonstrated in sars-cov-2 and the negative clinical significance of liver test alterations [10] suggest a reduced clearance capacity of the liver filter as compared to bacterial degradation products and other toxins (pamps and damps) during sars-cov-2. covid-19 patients are at a high risk of both venous and arterial thromboembolism [37] . thrombocytopenia and increased d-dimer levels are common and associated with worse clinical outcomes in covid-19 patients [38] . indeed, the majority of covid-19 patients who die present disseminated intravascular coagulation [39] . however, further investigations are needed to determine whether these marked prothrombotic changes are specific for this viral agent or secondary to the cytokine storm. viruses can stimulate systemic inflammatory response and cause an imbalance between procoagulant and anticoagulant mechanisms. platelets themselves are activated upon antigen recognition and interact with white blood cells to form clots to facilitate the removal of pathogens [38] . sars-cov-1 was reported to be associated with specific autoptic changes including proliferation, swelling, and apoptosis of endothelial cells, in addition to edema, infiltrates of inflammatory cells, and necrosis of small vessel walls in the lungs, heart, brain, kidneys, liver, and gastrointestinal tract [40] . strikingly similar histological pictures have recently been reported in covid-19 patients with diffuse severe endotheliitis of the submucosal vessels in the small bowel [41] . in a way, this kind of endothelial injury is similar to that commonly reported in intestinal transplant-associated thrombotic microangiopathy, which is observed after hematopoietic stem cell transplant [42] . indeed, in these cases ischemic enterocolitis presents with abdominal pain and gastrointestinal bleeding. a similar picture can also be seen in type ii/iii cryoglobulinemic vasculitis, a well-known extrahepatic feature of chronic hepatitis c and other viral infections [43] due to immune complex deposition along medium and small vessel walls, or of antiphospholipid syndrome. of note, covid-19 cases with multiorgan ischemic damage due to antiphospholipid syndrome presenting with fever, diarrhoea, and respiratory symptoms have been reported [44] . for this reason, it may be speculated that sars-cov-2-associated diarrhoea and enterocolitis is due to a plethora of mechanisms leading to microischemic damage of the gi tract, rather 7 than to direct viral damage of enterocytes resulting in malabsorption [45] . unfortunately, data from endoscopic or autoptic reports on the pattern of covid-19 damage to the gi tract are limited. one of the few initial reports on endoscopic findings in patients with covid-19 highlighted segmental haemorrhagic colitis in the absence of virocytes [46] . one study found that bowel wall abnormalities were highly frequent findings in covid-19 patients (13/42 or 31% of ct scans) and were associated with intensive care unit (icu) admission (or 15.5) [24] . pathology correlates demonstrated ischemic enteritis with patchy necrosis and fibrin thrombi in the arterioles of operated patients. interestingly, in the same study 54% of covid-19 patients who underwent abdominal ultrasound had a dilated sludge-filled gallbladder suggestive of cholestasis, a finding that may reflect the reduction in bile flow and biliary stasis observed in intestinal function failure [24] whether ischemic damage in the gastrointestinal tract is more relevant than direct viral damage is an intriguing question. recently, effenberger et al. [47] investigated faecal calprotectin (fc) levels in 40 patients with covid-19 stratified according to the presence of active diarrhoea, ceased diarrhoea, or no experience of diarrhoea. they found increased fc levels in covid-19 patients with ceased or ongoing diarrhoea as compared with covid-19 patients without diarrhoea. notably, while fc levels correlated with circulating il-6 levels, there was no correlation between fc levels and faecal sars-cov-2 rna. these findings also suggest that intestinal damage is not directly caused by the virus. the host response to sars-cov-2 is certainly attributable to the biological characteristics of the virus. however, pre-existing conditions that may affect host response can exacerbate the crs and lead to negative outcomes (severe ards, icu admission, organ failure, and death). pre-existing endothelial dysfunction is a common denominator among people who have an increased risk of a severe form of covid19 [48] . indeed, hypertension, diabetes, obesity, cardiovascular disease, cancer, chronic kidney disease, chronic liver disease, alzheimer's disease, advanced age, and male sex increase the risk of a moderate or severe covid-19 outcome [49] . in each of these conditions, endothelial dysfunction may be present. 8 intriguingly, endothelial dysfunction and leaky gut coexist in preventable noncommunicable chronic diseases [48, 50] . indeed, increased intestinal permeability has been described in many gastrointestinal, liver, and systemic inflammatory and autoimmune diseases [50] . increased intestinal permeability has been reported in 10.5-42.9% of ulcerative colitis patients, 36% of crohn's disease patients, 34% of systemic sclerosis patients, 30% of patients with type 1 diabetes, 25% of patients with primary biliary cirrhosis, 65% of patients with chronic liver disease and type 2 diabetes, 35% of patients with liver cirrhosis, 31% of patients with non-alcoholic fatty liver disease, and 36% of autistic patients. these conditions, particularly metabolic and liver diseases, were found to be highly prevalent among hospitalised covid-19 patients at the highest risk of negative outcomes [51] [52] [53] [54] [55] [56] . interestingly, one of the first reports concerning the covid-19 epidemic in the us described a population of 24 hospitalised and severely ill overweight/obese (median bmi 33.2+/-7.2) subjects with type ii diabetes (58%) and obstructive sleep apnoea (21%) and reported a 50% mortality rate [51] . according to a meta-analysis of 18 studies [10] , almost 40% of covid-19 patients had one or more comorbidities, with hypertension, cardiovascular disease, and type ii diabetes mellitus being the most frequent [10] . furthermore, noncommunicable preventable diseases, such as metabolic syndrome, seem to have an unfavourable prognostic role [9] . a negative prognostic role has also been attributed to systemic inflammation markers such as white blood count and c-reactive protein levels [8, 11] , gastrointestinal symptoms such as nausea and vomiting [11] , and liver test abnormalities [9] . patients with severe covid-19 had higher rates of gastrointestinal symptoms and liver injury as compared to those with non-severe disease [52] . although histomorphological studies are lacking, liver test abnormalities, including transaminase and gamma-glutamyl transferase elevation, were recorded in about 30% of covid-19 patients [8] [9] [10] , while the presence of cardiovascular or metabolic comorbidities was present in about 40% of patients, thus suggesting a high prevalence of non-alcoholic fatty liver disease in patients with a severe covid-19 phenotype [8-10, 53, 54] . in a large series of 5700 hospitalised patients from new york-area hospitals [55] , older patients, men, and those with pre-existing conditions were highly prevalent, similar to what has been reported in china [55] . lighter and colleagues found that patients under 60 with a bmi over 35 were at least twice as likely to be admitted to the icu and were 3x more likely to die than patients with lower bmi [56] . the aforementioned intestinal permeability changes associated with metabolic disorders 9 may account for the clinical severity of these middle-aged patients [7] [8] [9] [10] [11] [12] [13] [14] 18] . furthermore, the different prevalence of metabolic syndrome, which is higher in western countries, may contribute to the higher infection fatality ratio [ifr] attributed to the united kingdom [57] as compared to china [58] . a pre-existing increase in intestinal permeability, dysbiosis, and low-grade latent systemic inflammation may help trigger and fuel the inflammatory-cytokine storm in covid-19 through the systemic release of damps and pamps (lps) and tlr activation, which in turn maintains a vicious cycle of systemic inflammation and tissue damage (fig. 2) . based on the aforementioned hypothesis, the gut-liver axis should be investigated as a possible therapeutic target in covid-19 patients. simple indications such as avoiding alcohol and the non-clinically justified intake of drugs with potential negative effects on the intestinal mucosa and/or microbiota, such as nsaids, antibiotics, proton-pump inhibitors, and drugs and supplements potentially associated with drug-induced liver injury, may prove beneficial in terms of preventive measures. furthermore, as recommended in other serious acute infections, enteral nutrition should be preserved. beyond drug stewardship and dietary indications, the gut-liver axis can be targeted through other treatment options including antibiotics, prebiotics, probiotics, symbiotics, postbiotics, and faecal microbiota transplantation [59] [60] . these treatment options could involve different therapeutic pathways based on different phases of sars-cov-2 infection. first, some probiotics have been shown to act against other viruses (e.g. rotavirus) in vitro and to decrease the duration of the virus in human stool samples [61] [62] . therefore, their administration may reduce the permanence of sars-cov-2 in patient faecal samples. this could result in a reduced reservoir for the infection, a reduced risk of transmission through the faecal-oral circuit, and reduced gastrointestinal symptoms. microbiome therapeutics could also act at the initial phase of the disease before it potentially evolves into a critical clinical picture. several microbiome modulators are known to beneficially influence the immune system and the release of anti-inflammatory cytokines [63] [64] , with a potential effect on the development of sars-cov-2-related crs, which is one of the key drivers of worsening covid-19 clinical severity. specifically, elevated tissue and serum levels of il-6 are implicated in the pathogenesis of various inflammatory and autoimmune disorders and crs. inflammatory response in sars-cov-2 infection is largely characterized by an increase in il-1 and il-6 [65] . a combination of bifidobacterium animalis ssp. lactis and lactobacillus rhamnosus gg (lgg) daily was able to decrease il-6 production in 290 children receiving vaccines for streptococcus pneumoniae and bordetella pertussis [66] . moreover, in mice the administration of immunobiotic lactobacillus plantarum to the respiratory tracts in acute pneumonia virus infection promoted survival in association with diminished levels of il-6 [67] . in addition, treatment with lgg led to changes associated with decreased il-6 levels [68] . moreover, some microbiome modulators, mainly prebiotics and probiotics, helped maintain intestinal barrier integrity through the production of short-chain fatty acids, which have a trophic activity on enterocytes and tight junctions [69] . finally, microbiometargeted therapeutics may also play a role in covid-19 patients already admitted to icu [70] . in patients admitted to the icu as a result of traumatic injury, microbiome î²-diversity has been associated with different outcomes, including mortality risk, hospital length of stay, icu length of stay, number of days on the ventilator, hospital-acquired infections, and ards (p<0.05) [71] . probiotics have been shown to reduce the risk of icu-acquired infections in aggregate [72] and several meta-analyses have also found reductions in icu-acquired pneumonia (or, 0.59; 95% ci, 0.42-0.79) and icu length of stay [73] . notably, in a doubleblind randomised controlled trial of 80 children with acute lung injury in a paediatric icu, lactobacillus acidophilus was able to decrease serum tnf-î± and il-6 levels and lead to a higher volume to peak tidal expiratory flow and improved mean arterial and pulmonary arterial pressure, with significant differences as compared with placebo [74] . furthermore, faecal microbiota transplantation was able to increase overall survival and decrease the risk of bloodstream infection in fragile inpatients with c. difficile infection [75] . however, despite these promising results, the use of microbiome modulators should be carefully considered in critically ill patients due to the potential severe adverse events that can occur in this setting [76] . overall, different strategies aimed at modulating the intestinal microbiota while preserving biodiversity and eubiosis should be taken into consideration from the onset of sars-cov-2 infection. indeed, the attempt to reach or maintain homeostasis in the gut-liver axis may be seen as a prophylactic strategy favouring a milder course of sars-cov-2 infection, while rapidly targeting gut-liver involvement may favour recovery. however, additional randomized controlled trials are warranted. the evidence 11 summarised above also supports the theoretical therapeutic role of gut microbiota and permeability modulators in sars-cov-2 infection. there is convincing preliminary evidence linking microbiota-gut-liver axis perturbations, proinflammatory status, and endothelial damage in noncommunicable preventable diseases with covid-19 severity. the reduction of the mortality induced by the use of dexamethasone in patients hospitalized with covid-19, supports furtherly the clinical relevance of the propagation of the systemic inflammation in covid-19 [77] . intestinal damage caused by sars-cov-2 may further contribute to a disequilibrium and crs. these mechanisms may account for the clinical severity of covid-19 observed in patients with certain chronic disorders. while solid clinical data and a proven pathophysiological link between the gut, systemic inflammatory response, and the lungs in the progression of sars-cov-2 are necessary, the gut-liver axis and microbiota should be considered likely key players and possible therapeutic targets. sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme exploring the pathogenesis of severe acute respiratory syndrome (sars): the tissue distribution of the 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probiotics and the immunological response to infant vaccinations; a double-blind randomized controlled trial critical adverse impact of il-6 in acute pneumovirus infection effect of probiotic lactobacillus rhamnosus gg intervention on global serum lipidomic profiles in healthy adults the effect of probiotics on the production of short-chain fatty acids by human intestinal microbiome microbiome: what intensivists should know the gut microbiome distinguishes mortality in trauma patients upon admission to the emergency department probiotics in the critically ill: a systematic review of the randomized trial evidence impact of the administration of probiotics on mortality in critically ill adult patients; a metaanalysis of randomized controlled trials effects of probiotics on ghrelin and lungs in children with acute lung injury: a double-blind randomized, controlled trial incidence of bloodstream infections, length of hospital stay, and survival in patients with recurrent clostridioides difficile infection treated with fecal microbiota transplantation or antibiotics: a prospective cohort study probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial dexamethasone in hospitalized patients with covid-19 -preliminary report the authors thank dr. melissa kerr for the english editing, and dr. antonella cardinale for assistance in drafting the figures. key: cord-305704-grzrkff9 authors: almutairi, abdulelah; alfaleh, mohammed; alasheikh, muath title: dermatological manifestations in patients with sars-cov-2: a systematic review date: 2020-07-28 journal: cureus doi: 10.7759/cureus.9446 sha: doc_id: 305704 cord_uid: grzrkff9 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been initially defined as a disease of the respiratory tract; however, with the increasing number of patients and announcing that the virus became a pandemic, new systemic clinical manifestations are observed, including dermatological manifestations. however, the identification and characteristics of these manifestations are still controversial. this review article aims to evaluate the medical literature and explore the dermatological clinical manifestations in patients with sars-cov-2. the literature was reviewed through medline®, ovid, pubmed®, and embase®. searching terms included were a combination of "dermatological" or "skin" and "symptoms" or "manifestations" and "sars-cov-2". the following step was filtering the results to include only original research studies investigating the different types of skin and dermatological clinical manifestations in patients with sars-cov-2. a total of 879 studies were retrieved. following the exclusion of studies on animals and including only studies on humans, 32 studies emerged. altogether, seven studies were identified as eligible, covering 555 patients with sars-cov-2 who had dermatological symptoms. three studies were retrospective, two studies were prospective, and two studies were case series. different types of dermatological lesions can occur in patients with sars-cov-2, most commonly erythema, urticaria, and varicella-like rash. dermatological manifestations with sars-cov-2 can be misdiagnosed with other conditions. further studies with robust design are needed. coronaviruses are defined as a class of viruses that commonly lead to mild to moderate respiratory tract infections [1] . moreover, in the last few years, there were some mutations that occurred in coronaviruses leading to transmission from animals to humans [2] . furthermore, the virulence of the virus has increased, leading to increased mortality. examples of these viruses are the middle east respiratory syndrome-related coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov), and the recently explored severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [3] . sars-cov-2 virus has been primarily identified in wuhan city in china, in november 2019 [4] . the transmission rate of the virus started to increase rapidly and progressively till being announced as a pandemic by the who in february 2020 [5] . signs and symptoms of the new viral infection might range from an absence of symptoms to severe and sometimes, life-threatening condition [6] . at the beginning of this wave of infection, it was thought that sars-cov-2 affects only the respiratory tract; however, with the increasing number of new cases globally, other systemic symptoms have been reported, which varied in severity [7] . one of these systemic symptoms is dermatological manifestations [8] . some patients with sars-cov-2 were observed to have some cutaneous symptoms such as urticaria spreading over the body, erythematous rash, skin vesicles, similar to chickenpox infection [9] . these dermatological symptoms were commonly reported all over the body, particularly over the trunk [10] . also, patients with sars-cov-2 complained of itching of varying severity. however, there is still a debate on these symptoms and whether there are other symptoms identified in patients with sars-cov-2 [11] . our review aims to examine the current medical literature to explore the different types of dermatological clinical manifestations in patients with sars-cov-2. this systematic review of the literature was performed in compliance with the preferred reporting items for systematic reviews and meta-analyses (prisma) checklist recommendations for systematic review and meta-analysis [12] . this systematic review was carried out through searching electronic databases to include eligible studies in four databases, including medline®, pubmed®, ovid, and embase®. searching terms included "dermatological" or "skin" and "symptoms" or "manifestations" and "sars-cov-2". all the titles, as well as abstracts that emerged from this exploration, were reviewed completely to prevent missing any eligible studies. the results were then filtered to include only original research studies examining the different types of skin and dermatological clinical manifestations in patients with sars-cov-2. additionally, all study designs from different countries were included. only studies written in the english language were listed as related studies, which can be further assessed in the second step. following this stage, the inclusion criteria to choose the studies that will be recognized in the systematic review were determined. abstracts were reviewed manually to determine the appropriate abstracts to be considered. the inclusion criteria included discussing enough data on the dermatological symptoms with sars-cov-2. moreover, only studies done among adult patients were included. furthermore, references of the chosen studies were assessed to distinguish any related studies. lastly, the required data sets were collected from the final record of eligible studies and summarized. studies were eliminated in case of in vitro or animal involvement, overlapped or incomplete data, and unavailability of full-text studies or inappropriate study design. entire details on the search strategy are shown in figure 1 . the first step included a preparatory review, a specially designed excel sheet was used for data extraction. chosen data from eligible studies were then reviewed through the excel sheet. any studies published by one research group examining alike variables were evaluated for any potential duplication. cochrane, a quality assessment tool, was used to evaluate the quality of the included clinical studies [13] . data were then statistically expressed in terms of frequencies (number of cases) and percentages for categorical variables. mean, standard deviations, medians, and interquartile ratios were used to represent the numerical variables. all statistical calculations were performed by ibm spss version 26 (statistical package for the social science; ibm corp, armonk, usa). after searching the abstracts and reviewing the eligibility criteria in identified potential abstracts, a total of seven studies [14] [15] [16] [17] [18] [19] [20] were considered as eligible to be included in the present systematic review, covering a total of 555 patients with sars-cov-2 who had dermatological symptoms in the form of skin lesions. out of the seven studies, two studies were prospective [14, 16] , three studies were retrospective [17, 19, 20] , and two studies were case series [15, 18] . additionally, all the included studies considered the objective of describing the dermatological manifestations of patients with sars-cov-2 with varying severity levels in different countries. due to the scarcity of data and lack of experience with the newly identified virus, all study designs were included [14] [15] [16] [17] [18] [19] [20] . furthermore, all the studies identified the types of dermatological manifestations in patients. erythema and urticaria were common in four studies [14, [17] [18] [19] , varicella-like lesions were defined in three studies [15, 17, 19] , while other types of rash were identified in four studies [16] [17] [18] 20] . the included studies are discussed in detail in table 1 . thailand. one patient out of the 48 patients had a skin rash with petechiae. because dengue is very common in thailand, petechiae rash is a frequent clinical manifestation in dengue. the patient had thrombocytopenia. dengue was misdiagnosed in the beginning. there was no image, and a biopsy is not routinely done for dengue diagnosis in thailand. the patient was initially misdiagnosed as dengue, which has led to a delayed diagnosis there is a chance that a patient with sars-cov-2 can primarily have a skin rash that can be misdiagnosed as another common disease. the novel sars-cov-2 virus was identified in late 2019, with minimal knowledge and experience regarding its diagnosis and treatment until present [4] . although the symptoms of sars-cov-2 are mainly affecting the respiratory tract, other systemic features have been identified [13] . dermatological manifestations, such as urticaria, erythema and rash, were also identified in some patients with sars-cov-2 in different countries. however, the description of these manifestations and their correlation to sars-cov-2 is still controversial [18] . the present review evaluated the description and characters of dermatological manifestations in patients with sars-cov-2. it has been shown that different types of cutaneous lesions occur in patients with sars-cov-2, even in patients with mild respiratory symptoms. hence, the misdiagnosis of dermatological lesions in sars-cov-2 is common. furthermore, the most frequent types of lesions were varicella-like rash and rash resembling recent viral infection, in addition to urticaria and erythema. it is worth noting that these lesions might be itching or non-itching and are usually mildly severe in most patients, resolving at the end of the course of infection. dermatological lesions can also develop at a later stage of the infection. erythema and urticaria in sars-cov-2 patients have been described in some studies. the most robust study design described dermatological manifestations in 375 spanish sars-cov-2 patients. the study described the lesions as commonly occurring in the acral areas, with 19% having erythema in the form of pseudo-chilblain, which developed in later stages in the infection, and another 19% having urticaria lesions. also, the dermatological lesions related to sars-cov-2 were severe in the acral area, with less severe lesions in other parts. additionally, the maculopapular rash was also characteristic in spanish patients with a prevalence of 47% [14] . another study that had a retrospective design reviewed 14 patients and showed that dermatological lesions appeared at a later stage of the infection, with inflammatory lesions occurring in 50% of patients in the form of exanthema, chickenpox-like vesicles. additionally, the study identified that the remaining 50% of patients had vascular lesions in the form of porcelain-like macules, livedo, necrotic purpura, chilblain, and eruptive cherry angioma [17] . another study from italy included 88 patients in a retrospective design and demonstrated that 20.4% of patients had cutaneous manifestations, with eight patients developed the dermatological symptoms at an earlier stage of the onset of the sars-cov-2 infection, while ten patients developed the lesions after hospitalization due to sars-cov-2 due to severe infection. importantly, there was no correlation between the occurrence of dermatological lesions and the severity of sars-cov-2 infection, where some patients had dermatological lesions without developing respiratory symptoms for sars-cov-2. the study also highlighted that the lesions are resembling those occurring in common viral infections [19] . this finding was also supported by a varicella-like rash observed by two studies [15, 17] . it is worth to mention that other frequent conditions can misdiagnose dermatological lesions occurring in patients with sars-cov-2. in thailand, one study described one out of 48 sars-cov-2 patients who had a rash with petechiae that was misdiagnosed with dengue disease, especially with the absence of routine biopsy in this setting [20] . the included studies also had some limitations; most of the included studies were performed in one center, which may affect the external validity of outcomes. also, the sample size in each study was relatively small, which could reduce the reliability of their outcomes. these limitations should be considered in future studies. finally, this is considered the first systematic review to identify the dermatological manifestations occurring in patients with the novel sars-cov-2 infection. this should be a guide for clinicians to prevent the misdiagnosis of these manifestations with other dermatological conditions. dermatological lesions are frequent in patients with sars-cov-2, especially erythema, urticaria, and varicella-like rash. differential diagnosis should be thoroughly considered before deciding that the present rash is related to sars-cov-2 infections. till present, the rash is not correlated to the severity of sars-cov-2 infection, which needs to be confirmed with studies with more robust design and larger sample sizes. these findings should be considered by clinicians working with patients with sars-cov-2 in order not to misdiagnose the occurrence of dermatological lesions, which may delay therapy or increase the risk of complications. covid-19: attacks the 1-beta chain of hemoglobin and captures the porphyrin to inhibit human heme metabolism (preprint) cutaneous manifestations in covid-19: a new contribution urticarial eruption in covid-19 infection covid-19 pandemic and the skin-what should dermatologists know? (in press) comment on: cutaneous manifestations in covid-19: a first perspective. safety concerns of clinical images and skin biopsies a dermatologic manifestation of covid-19: transient livedo reticularis covid-19 in singapore-current experience: critical global issues that require attention and action alert for non-respiratory symptoms of coronavirus disease 2019 (covid-19) patients in epidemic period: a case report of familial cluster with three asymptomatic covid-19 patients (preprint) covid-19 can present with a rash and be mistaken for dengue": petechial rash in a patient with covid-19 infection dermatology staff participate in fight against covid-19 in china covid-19 and cutaneous manifestations the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration the cochrane collaboration's tool for assessing risk of bias in randomised trials classification of the cutaneous manifestations of covid-19: a rapid prospective nationwide consensus study in spain with 375 cases varicella-like exanthem as a specific covid-19-associated skin manifestation: multicenter case series of 22 patients chilblain-like lesions on feet and hands during the covid-19 pandemic vascular skin symptoms in covid-19: a french observational study (preprint) a case of covid-19 presenting in clinical picture resembling chilblains disease. first report from the middle east cutaneous manifestations in covid-19: a first perspective covid-19 can present with a rash and be mistaken for dengue in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. key: cord-313517-5ipj2z86 authors: fung, joshua; lau, susanna k. p.; woo, patrick c. y. title: antigen capture enzyme-linked immunosorbent assay for detecting middle east respiratory syndrome coronavirus in humans date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_7 sha: doc_id: 313517 cord_uid: 5ipj2z86 the middle east respiratory syndrome (mers) is the second novel zoonotic disease infecting humans caused by coronavirus (cov) in this century. to date, more than 2200 laboratory-confirmed human cases have been identified in 27 countries, and more than 800 mers-cov associated deaths have been reported since its outbreak in 2012. rapid laboratory diagnosis of mers-cov is the key to successful containment and prevention of the spread of infection. though the gold standard for diagnosing mers-cov infection in humans is still nucleic acid amplification test (naat) of the up-e region, an antigen capture enzyme-linked immunosorbent assay (elisa) could also be of use for early diagnosis in less developed locations. in the present method, a step-by-step guide to perform a mers-cov nucleocapsid protein (np) capture elisa using two np-specific monoclonal antibodies is provided for readers to develop their in-house workflow or diagnostic kit for clinical use and for mass-screening project of animals (e.g., dromedaries and bats) to better understand the spread and evolution of the virus. the middle east respiratory syndrome (mers) is the second novel zoonotic disease infecting humans caused by coronavirus (cov) in this century. to date, more than 2200 laboratory-confirmed human cases have been identified in 27 countries, and more than 800 mers-cov associated deaths have been reported since its outbreak in 2012 [1] . rapid laboratory diagnosis of mers-cov is the key to successful containment and prevention of the spread. nucleic acid amplification test (naat, e.g., real-time reverse transcription quantitative polymerase chain reaction [real-time rt-qpcr]), virus isolation, transmission electron microscopy, immunohistochemistry, and serological methods (e.g., antigen capture enzyme-linked immunosorbent assay [elisa] and immunofluorescence assay [ifa] ) have been developed and used for mers-cov diagnosis [2] [3] [4] [5] [6] [7] . while the "gold standard" for mers-cov diagnosis is naat of the upper region of the envelope gene (up-e) or the nucleocapsid (n) gene as suggested by the world health organization (who), antigen capture elisa assay for mers-cov can also be informative when naat is not available or when the serological assay is used to confirm the findings and aid treatment decision [2, 3] . further to diagnosing possible human infection of mers-cov, this method is also useful for screening the virus in the wildlife or agricultural applications. government agencies and research groups may find serological tests like antigen capture elisa to be more economical than naats for routine screening of mers-cov in farm-held or city-dwelling animals. the antigen capture elisa described in this method offers four significant advantages over traditional naats. firstly, serological screening requires less space in facilities and can be performed in point-of-care locations to minimize sample transporting and reduce turnover time. to avoid crosscontamination from amplicons in naats, the workflow usually requires four separate physical locations: (1) sample preparation (lysis, extraction of nucleic acids, and reverse transcription), (2) naat master mix preparation, (3) template addition, and (4) amplification and analysis. though technologies like real-time rt-qpcr simplify the workflow, such requirements limit the assay to be performed in regional laboratories designed or designated for this application. antigen capture elisa, on the other hand, can be performed on open bench in a single location after virus inactivation, allowing it to be performed in even the most minimally designed facility. secondly, antigen capture elisa can be performed with simple equipment and can be established with limited initial investment. for performing naats at a modern standard, uv cabinets or workstations for master mix preparation and sample addition, thermal cyclers, agarose gel running, and visualization equipment are the least requirement. for more stringent testing and faster turnaround, it calls for a real-time pcr thermal cycler (e.g., roche's lightcycler systems or bio-rad touch detection systems) which requires a fair amount of initial investment and limits the assay from being performed in remote or less developed locations. in contrast, antigen capture elisa and other serological methods can be performed with much simpler equipment. multichannel pipettes, automatic plate washer, and plate reader are the only specialized tools needed for this application and can be purchased with ease if those are not already available. thirdly, much less training is required for technicians to handle serological testing than naats. though naats and elisa are some of the most basic assays performed in a medical laboratory and minimal training is needed for an experienced worker to perform such task, to allow quicker and broader surveillance of mers-cov in human and animal population, it would be beneficial to set up more surveillance facilities in the less developed parts of the world. the time and resources needed to train a novice laboratory worker to perform elisa are much less, as only dilution and pipetting skills are required. fourthly, common nucleic staining chemicals used in naats for amplicon visualization are a possible mutagen and post potential health risk to workers and the surrounding environment; while chemicals and solutions used in elisa are relatively safer. to visualize the amplicons after agarose gel electrophoresis or during the qpcr thermal cycles, dyes like ethidium bromide (etbr), sybr green, or gel red are used; while etbr is a known mutagen, others are a relatively new addition to the market and extensive safety data is not widely available [8] . in comparison, the chemicals and solutions used in elisa are commonly found in clinical and research laboratories and are generally safe when used properly. finally, and most importantly, antigen capture elisa can offer high sensitivity and specificity for mers-cov diagnosis in even early infection and animal samples. we have previously demonstrated that by using two mers-cov nucleocapsid protein (np) specific monoclonal antibodies (mabs) in performing capture elisa, the test can accurately detect mers-cov virus down to 10 tcid 50 /0.1 ml and has a specificity of 100% [3] . as the nasopharyngeal aspirate viral load from patients during acute infection are around 10 6 copies/ml and nasal samples in dromedaries are usually around 10 4 -10 6 copies/ml, this test offers sufficient sensitivity for mer-cov diagnosis and screening [9] [10] [11] . other forms of mers-cov serological diagnostic test have also been developed based on different principles and are designed to fulfill different purposes, one should also review those options and evaluate their needs. to detect seroconversion from previous infection of mers-cov, the who suggests laboratories to perform ifa or elisa together with neutralization assay, the result alone can be used to determine if it is a confirmed case, regardless of the results from naat assay [2] . for rapid on-site diagnosis of mers-cov, we have previously reported the adaptation of the antigen capture elisa in the format of lateral flow immunoassay (lfia). this assay can yield results in under half an hour, requires minimal equipment, training, and can be stored at room temperature, thus allowing it to be performed in the field [12] . this lfia is also able to detect mers-cov-like viruses (e.g., tylonycteris bat cov hku4 and pipistrellus bat cov hku5) and is useful for the research to understand the evolutional history of mers-cov [13, 14] . in the current manuscript, the method for performing np capture elisa using two mers-cov-np-specific monoclonal antibodies (mabs) will be introduced. the general workflow of the assay is summarized in a figure for quick referencing [3, 15] (fig. 1 ). the antigen capture elisa is also known as sandwich elisa and makes use of a "capture" antibody and a "detection" antibody. the capture antibody is coated onto the wells of a microtiter plate before the assay. then following sample processing, the lysate is incubated in the wells of the microtiter plate. if the sample contains peptides from mers-cov (specifically nucleocapsid protein), they will bind with the coated antibody and be "captured" onto the microtiter plate. even minute amount of viral peptide can be retained in the well if the capture antibody has a high affinity to the peptide and was coated at high concentration. unbonded proteins are then washed away before the addition of the second, "detection" antibody. the secondary mab also recognizes the mers-cov np, presumably binds to a distinct epitope, and is conjugated with horseradish peroxidase for detection. the combination of two mabs in an elisa assay offers increased sensitivity for mers-cov np. on the other hand, this "sandwich" approach also allows improved specificity for the mers-cov nucleocapsid protein by combining the specificities of the two mabs, allowing it to differentiate and identify mers-cov spiked sample from other samples from healthy and patients who contracted various respiratory tract infections, as previously demonstrated [3] . in this assay the nucleocapsid protein was selected as the target for generating antibodies to detect mers-cov. according to previous experience when working with sars-cov, we observed that the np is a highly immunogenic and abundantly expressed structural protein, and a more preferable target than the spike (s) protein [16, 17] . working with the hypothesis that the np protein of mers-cov might also be a desirable target when developing an antigen capture elisa for it, we have shown that the assay offers high specificity and sensitivity, as mentioned above. the steps related to the cloning and purification of (his) 6 -tagged recombinant np (rnp) of mers-cov for the generation of anti-mers-cov-rnp mabs will not be described, as there are commercially available antibodies readily available for purchase. the horseradish peroxidase (hrp) system was used for the colorimetric visualization at the final stage of the assay. commercial elisa kits may utilize other detection methods; optimization may be needed. for readers who would like to generate their own hrp conjugated detection antibody, there are also kits available. when preparing solutions and buffers for the assay, investigators should be aware that "old" buffers may be more likely to be contaminated. the accuracy and reproducibility of the assay can be affected, as the peptides from fungus or other microorganisms may compete with the target antigen. prepare fresh solutions periodically (~1 month); autoclave or filter sterilize the buffers if available. if contaminations are a common occurrence, the addition of 0.05% sodium azide (nan 3 ) as a preservative is an option. microtiter plates with antibody 1. dilute the mers-cov-rnp mab 1f6 in blocking buffer. (see note 2). 2. coat the microtiter plates by adding 100 μl of the solution prepared per well. 3. cover the plate with an adhesive plastic cover and incubate at 37 c overnight (see note 3). 4. discard the adhesive plastic cover and remove the solution. 5. wash the plate with 300 μl of washing buffer per well for five times using an automatic microplate washer. 6. dry the plate by patting the plate on a paper towel. 7. allow the plate to air-dry. proceed to the next step or cover the plate with adhesive plastic cover and store at 4 c until use. all processes with potentially infectious mers-cov materials should be handled according to institutional, local, and international regulations, guidelines, and standard operating procedures (sop) to avoid spreading and contamination of the facility. all work with infectious mers-cov was performed inside a biosafety level-2 cabinet with sop in approved biosafety level-3 facilities during development and evaluation of the assay [3, 18, 19 ]. 1. aliquot 50 μl of viral lysis buffer to new 1.5 ml conical screw cap tubes according to the number of samples and controls (see note 4). 2. pipette 100 μl of specimen from the sample collection tube to the 1.5 ml conical screw cap tubes with viral lysis buffer, mix well. allow sufficient time for inactivation. 3. transfer the inactivated sample out of the biosafety cabinet to the general laboratory area, according to established sop. 4. there are many viral lysis buffers available for purchase from bio-reagents vendors, e.g., buffer al from qiagen. readers could request samples and perform their own testing on the conditions required to efficiently inactivate mers-cov. 5 . tmb solutions are normally purchased from bio-reagents vendors at ready-to-use dilations, follow manufacturer's instructions. world health organization: world health organization, 20 avenue appia laboratory testing for middle east respiratory syndrome coronavirus a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt are other fluorescent tags used instead of ethidium bromide safer clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia a highly specific rapid antigen detection assay for on-site diagnosis of mers rapid detection of mers coronavirus-like viruses in bats: pote1n-tial for tracking mers coronavirus transmission and animal origin genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus differential diagnosis of pandemic (h1n1) 2009 infection by detection of haemagglutinin with an enzyme-linked immunoassay sars coronavirus detection methods detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests 1. serially dilute the inactivated sample in sample dilution buffer, add 100 μl of the mixture into the wells in duplicates.2. gently shake the plate for 2 min to mix well, and then incubate at 37 c for 30 min while being covered with an adhesive plastic cover.3. discard the adhesive plastic cover and remove the solution.4. wash the plate with 300 μl of washing buffer per well for five times using an automatic microtiter plate washer. 1. dilute the secondary detection antibody (mab 7c4 conjugated with hrp) in enzyme dilution buffer immediately before use.2. add 100 μl of the diluted detection antibody to each well using an 8-channel pipette.3. cover the plate with an adhesive plastic cover and incubate at 37 c for 30 min.4. discard the adhesive plastic cover and remove the solution.5. wash the plate with 300 μl of washing buffer per well for five times using an automatic microtiter plate washer. 1. add 100 μl of 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb) substrate solution to each well (see note 5).2. cover the plate with aluminum foil to protect from light, incubate for 10 min at room temperature.3. add 50 μl of stop solution to each well to stop the reaction.4. read the plate using an automatic plate reader at wavelength 450 nm.5. analyze the data by using the predetermined cutoff value. 1. an automated microtiter plate washer-dispenser would be a good addition to the workflow as the washing steps can be performed in a shorter amount of time and with greater consistency. but multichannel or even single-channel pipettes can be used instead.2. the actual dilution of antibodies depends on the batch and quality of the mabs used. evaluations and characterization to determine the specificity and sensitivity have to be performed to establish the optimal dilution for the highest signal-to-noise ratio.3. prevent the microtiter plates to dry up by placing the plates in a box with some moist tissue paper laying under while storing in an incubator or on a rack in a warm water bath. key: cord-320619-r466dc5t authors: chand dakal, tikam title: sars-cov-2 attachment to host cells is possibly mediated via rgd-integrin interaction in a calcium-dependent manner and suggests pulmonary edta chelation therapy as a novel treatment for covid 19 date: 2020-11-05 journal: immunobiology doi: 10.1016/j.imbio.2020.152021 sha: doc_id: 320619 cord_uid: r466dc5t sars-cov-2 is a highly contagious virus that has caused serious health crisis world-wide resulting into a pandemic situation. as per the literature, the sars-cov-2 is known to exploit humanace2 receptors (similar toprevious sars-cov-1) for gaining entry into the host cell for invasion, infection, multiplication and pathogenesis. however, considering the higher infectivity of sars-cov-2 along with the complex etiology and pathophysiological outcomes seen in covid-19 patients, it seems that there may be an alternate receptor for sars-cov-2. i performed comparative protein sequence analysis, database based gene expression profiling, bioinformatics based molecular docking using authentic tools and techniques for unveiling the molecular basis of high infectivity of sars-cov-2 as compared to previous known coronaviruses. my study revealed that sars-cov-2 (previously known as 2019-ncov) harbors a rgd motif in its receptor binding domain (rbd) and the motif is absent in all other previously known sars-covs. the rgd motif is well known for its role in cell-attachment and cell-adhesion. my hypothesis is that the sars-cov-2 may be (via rgd) exploiting integrins, that have high expression in lungs and all other vital organs, for invading host cells. however, an experimental verification is required. the expression of ace2, which is a known receptor for sars-cov-2, was found to be negligible in lungs. i assume that higher infectivity of sars-cov-2 could be due to this rgd-integrin mediated acquired cell-adhesive property. gene expression profiling revealed that expression of integrins is significantly high in lung cells, in particular αvβ6, α5β1, αvβ8 and an ecm protein, icam1. the molecular docking experiment showed the rbd of spike protein binds with integrins precisely at rgd motif in a similar manner as a synthetic rgd peptide binds to integrins as found by other researchers. sars-cov-2 spike protein has a number of phosphorylation sites that can induce camp, pkc, tyr signaling pathways. these pathways either activate calcium ion channels or get activated by calcium. in fact, integrins have calcium & metal binding sites that were predicted around and in vicinity of rgd-integrin docking site in our analysis which suggests that rgd-integrins interaction possibly occurs in calcium-dependent manner. the higher expression of integrins in lungs along with their previously known high binding affinity (∼k(d) = 4.0nm) for virus rgd motif could serve as a possible explanation for high infectivity of sars-cov-2. on the contrary, human ace2 has lower expression in lungs and its high binding affinity (∼k(d)= 15nm) for spike rbd alone could not manifest significant virus-host attachment. this suggests that besides human ace2, an additional or alternate receptor for sars-cov-2 is likely to exist. a highly relevant evidence never reported earlier which corroborate in favor of rgd-integrins mediated virus-host attachment is an unleashed cytokine storm which causes due to activation of tnf-α and il-6 activation; and integrins role in their activation is also well established. altogether, the current study has highlighted possible role of calcium and other divalent ions in rgd-integrins interaction for virus invasion into host cells and suggested that lowering divalent ion in lungs could avert virus-host cells attachment. ever since the recent emergence of novel coronavirus (sars-cov-2, earlier known as 2019-ncov) in the wuhan city of china and its subsequent transmission in other countries has resulted into serious heatth crisis as well as breakdown of socio-economic development. scientific community from all over the world is industriously engaged in and committed to find a potent therapeutic solution for the treatment of covid-19. as of 10 th april, 1,521,252 confirmed cases and 92,798 deaths were recorded as a cumulative data from different parts of the world (who situation report no. 81 available at who.int accessed on 12-04-2020). at the onset of the disease, the infected symptomatic patients experience hyperthermia, pharyngeal congestion, cough, and anosmia (in some cases); however, as disease progress more than fifty percent patients develop severe labored breathing, clinically referred to as dyspnoea or tachypnoea qui et al., 2020; . advance stages are characterized by severe pulmonary inflammation, fibrosis and obstructions of the bronchioles resulting in a pneumonia-like pathophysiological condition qui et al., 2020; . in both symptomatic and asymptomatic covid-19 patients, sars-cov-2 manages to cause significant damages to multiple organs before any patients could realize they are infected with sars-cov-2. this is because neither any neurological indications nor any signs of heart, kidney and liver failure are seen at the onset of disease in these patients (qui et al., 2020). as of today, there is no specific antiviral therapy available in any form, either vaccine or drug or others, to combat covid-19 and its infection. the neutralizing antibodies that were previously tested and found successful against sars-cov-1 have displayed inappreciable cross-reactivity against sars-cov-2.for some antibodies (such as cr3022) that could bind to sars-cov-2, their neutralizing efficacy has not been tested and appreciated yet yuan et al., 2020; wrapp et al., 2020) . it has been asserted that the binding sites for these monoclonal antibodies on sars-cov-2 are vulnerable and it is only an assumption that antibodies that could bind strongly would possibly neutralize the sars-cov-2 also. moreover, cross-reacting neutralizing antibodies are also doubted for their ability to confer prolonged protection against sars-cov-2. so far,we have no other choice than to use previously tested drugs or to solely oblige known tactics as precautionary measures against covid-19. recently, some researchers suggested the use of combination of remdesivir (a broad-range antiviral drug) and chloroquine for effective control of sars-cov-2 infection under in vitro condition (devaux et al., 2020; . similarly, hydroxychloroquine and azithromycin has also been referred to as a potent therapeutic weapon against the sars-cov-2 virus . drug repurposing approach, which involved a screening of a thousand of molecules showed that hiv protease inhibitors, rna-dependent rna polymerase (rdrp) inhibitors and some other inhibitors and agonists such as methisazone (an inhibitor of protein synthesis), cgp42112a (an angiotensin at2 receptor agonist) and abt450 (an inhibitor of the non-structural protein 3-4a) could become promising treatment options for covid-19 (gordon et al., 2020; li et al., 2020; shah et al., 2020) . lu (2020) suggested some treatment options for covid-19 that include use of nucleoside analogues, neuraminidase inhibitors, lopinavir or ritonavir, remdesivir, 3tc/tdf/efv monotherapy or combination therapy (dna polymerase inhibitors), anti-inflammatory or immune-suppressive drugs, just to name a few. besides this, some traditional chinese medicine, for instance,shufengjiedu and lianhuaqingwen capsules could also be useful (lu, 2020) . using virtual screening (kandeel and al-nazawi, 2020), epigenetic dysregulation (sawalha et al., 2020) , proteinprotein interaction mapping (cava et al., 2020) , integrated network pharmacological approach , and similar such approaches, a number of other drugs have also been repurposed for covid-19 treatment. however, most of the drugs are either in developmental stages or under clinical trials (https://clinicaltrials.gov/). in the midst of this pandemic situation, it is obvious that there exists no preventive therapy for highly contagious sars-cov-2 and this is strikingly alarming to all of us. a number of studies demonstrated the key role of human ace2 in virus attachment to host cells (wan et al., 2020; zhao et al., 2020) . the three-dimensional crystal structure of sars-cov-2 spike receptor binding domain complexed with its receptor, human ace2 (angiotensin converting enzyme 2), has been already solved .all other structural, functional and antigenicity related information related to sars-cov-2 are also available (walls et al., 2020) . based on biophysical data, it has been demonstrated that the human ace2 binds to sars-cov-2 with greater affinity than sars-cov-1 (wrapp et al., 2020) . however, owing to their low copy number (protein expression) (chen et al., 2020), their high affinity (for sars-cov-2 than sars-cov-1) alone could not manifest reasonable virus-host cell attachment, at least in lung cells. when this manuscript study was under progress, sigrist and coauthors (2020) showed that sars-cov-2 harbors a rgd motif and integrins (that display high affinity for rgd motifs) may be involved in facilitating virus entry into host cells. however, the study did not explain the full mechanistic state of affairs involved in rgd-integrins interaction and virus entry into the host cells.i also found rgd motif in the spike receptor binding domain of sars-cov-2 and studied mechanistic basis of rgd-integrin (and other ecm protein such as icam1) mediated virus invasion into host cells. this study is the first study to present striking evidence (substantiated by existing facts in literature) favoring the role of calcium and other divalent ions (magnesium, manganese etc.) in rgd-integrins mediated virus attachment with the host cells for and that lowering the concentration of calcium and other divalent ions in lungs could be a possible mechanism to avert sars-cov-2 infection and invasion. herein, i did comparative protein sequence analysis, motif scanning, gene expression data analysis and bioinformatics based molecular docking using most trustworthy tools and techniques. the the coronavirus related nucleotide and protein sequences were retrieved from genbank (https://www.ncbi.nlm.nih.gov/), uniprotkb (https://www.uniprot.org) and sars coronavirus 2 data hub of the ncbi (https://www.nih.gov/coronavirus). the protein sequence of sars-cov-1 and sars-cov-2 coronaviruses were subjected to pair-wise the pfam, prosite and hamap profiles of the s protein from the coronavirus was ascertained using myhits motif scan (sib, switzerland) (https://myhits.isb-sib.ch/cgi-bin/motif_scan). the putative nlinked glycosylation and other post-translation modification sites were predicted as frequent pattern in myhits outputs (pagni et al., 2007) . the expression profile of ace2 receptor protein and integrins in lungs was ascertained using gene expression database at ebi (https://www.ebi.ac.uk/gxa/home) and the human protein atlas in order to understand the role of divalent ions in rgd-integrins mediated virus-host cells attachment. a composite calcium and magnesium binding-site in integrins were predicted using ioncom (https://zhanglab.ccmb.med.umich.edu/ioncom/), which used an integrated approach based onab initio training and template-based transferals (hu et al., 2016) and predicts calcium binding sites in a given protein by searching four or more oxygen atoms on protein surface arranged in a spherical manner. the input pdb file of integrins such as α5β1 (pdb id: 3vi3) and αvβ6 (5ffg) were subjected for ion binding sites' prediction to specifically predict calcium and magnesium binds sites. the pdb file of the spike receptor binding domain (pdb id: 6lzg) and integrins such as α5β1 (pdb id: 3vi3 and 3vi4) and αvβ6 (pdb id: 5ffg) were retrieved from protein data bank protein-ligand binding modes were clustered according to their rank based on average fullfitnessin output data (grosdidier et. al., 2007) . pairwise sequence alignment of sars-cov-1 and sars-cov-2 spike protein revealed that the nterminus (especially upto 250 aa) of sars-cov-2 is highly divergent than that of the sars-cov-1 ( figure 1 ). the s1 glycoprotein (from 268-304 aa in sars-cov-1 and from 282-317 aa in sars-cov-2) domain which plays an important role in recognition of host cell receptor were found divergent at 10 amino acid positions. the spike receptor binding domain is of 253 amino acid residues in sars-cov-1 and ranges from 317 to 569 aa; whereas, it is of 254 amino acid residues in sars-cov-2 and stretch from 330 to 583 aa. there were 49 mismatches and 1 insersion/deletion that render sars-cov-1 and sars-cov-2 approximately 80% sequence similarity. the s2 glycoprotein domain is a large 544 amino acid residues long region at the c-terminus of the spike protein which plays crucial role in virus fusion with host cells. this region has been predicted to be less divergent than s1 glycoprotein and rbd. there i also found two insertion sequences with the stretch hvsgtngtkrfd 69-80 and rfqtllalhrsyltpgdsssgwtag 236-261 at the n-terminus region of spike protein and these were seen as discontinuous in the amino acid sequence as predicted by the pair-wise sequence alignment. however, considering that these inserts are very short and appeared in the hypervariable region of viral spike protein, the most likely assumption for their origin is that they might have arisen naturally. besides this, the receptor binding domain of the spike protein in sars-cov-2 has a stretch of sequence (teiyqagstpcngvegf 470-486 ) showing mismatch with sars-cov-1. the number and position of putative n-linked glycosylation sites, post-translation modification sites that can induce camp, ck2, tyr, pkc signaling pathways and myristyl sites were predicted to be varying in sars-cov-1 and sars-cov-2 (table 1) . although both coronaviruses have equal number of n-linked glycosylation sites but the position of sites differs (table 1) the spike protein sequence of sars-cov-1 and sars-cov-2 were subjected to motif scanning and prediction using myhits motif scan at sib server. a number of motifs were predicted in the spike protein sequence such as rgd (from 403-405 aa in receptor binding domain of sars-cov-2) ( table 2 ). the rgd motif (k403r substitution) was predicted in spike receptor binding domain of sars-cov-2 and the same was not predicted in the sars-cov-1 spike protein and other previous known coronaviruses. this motif was originally found in fibronectin, which is an extracellular matrix protein and this motif plays a major role in cell adhesion and attachment. a number of proteins are known to interact with these rgd motifs. one such group of proteins are integrins that have strong affinity for rgd motif (~kd=4.0nm) and they employ this motif to efficiently mediate cell adhesion. eight out of approximately twenty known integrins recognize and exploit rgd motifs for cell adhesion (ruoslahti, 1996 (table 2) . presence of rgd motif in the spike receptor binding domain of the sars-cov-2 and the fact that integrins display a strong affinity for proteins harboring rgd motifs (liu, 2009) have been found to be affected by sars-cov-2 infection (figure 2a-c) . these three integrins were used for molecular docking experiments. i also found that expression of an ecm protein icam1 is significantly higher (even higher than integrins) in lungs ( figure 2d ) suggesting that sars-cov-2 spike protein might be using these integrins and/or ecm proteins such as icam1 (or others) for invading host cells. on the contrary, the ace2 rna is detected in very low quantity (almost unidentifiable) in lungs and detectable expression peaks were detected in other organs such as duodenum, small intestine, colon, gallbladder, kidney, testis, and heart muscles ( figure 3 ). in congruence with this, xu and coauthors (2020) found high expression of ace2 in oral mucosa and zhang and co-authors (2020) demonstrated digestive route as a potential mechanism of ace2 mediated virus infection based on the single-cell transcriptomic analysis. on the contrary, chen and coauthors (2020) observed relatively low levels of ace2 mrna expression in lungs which further supports my claims. the rgd motif is the minimal indispensable requirement for integrins to bind with any viral protein which virus can use for attachment with host cells (hussein et al., 2015) . nagae figure 4b ). icam1 is also known to bind integrins and this mechanism is exploited by some viruses (such as rhinoviruses) to gain entry into respiratory system for pathogenesis (bella et al., 1998) . the rgd motifs are well known in the field of cell biology, cell therapy and tissue engineering because (table 3 ). in any disease or pathological condition where integrin expression would be high, i can expect patient to proteinurea and renal insufficiency are known to associate with edta chelation therapy and i suggest that a strict monitoring protocol, comprising routine cytokine profiling, blood examination, urine test and ct scanning etc., must be practiced while treating patients with pulmonary edta chelation therapy. in combination with the edta/egtabased pulmonary chelation therapy, modulation of integrins expression using integrins inhibitors or anti-integrin antibodies could also serve as a mechanism to treat . the docking was done using hdock server (hdock.phys.hust.edu.cn/). spike protein is shown in red and synthetic rgd peptide is shown in green. figure 5. pulmonary edta chelation therapy which can be clinically executed through a nebulizer or inhaler to allow sodium-edta to pass into the lungs. the sodium-edta will chelates ca +2 ions and other divalent ions making them unavailable for rgd-integrin mediated virus attachment to the host cells. a novel strategy for safe, technically simple, quick, cost-effective and efficient treatment of covid 19 patients. the integrin alphavbeta6 binds and activates latent tgfbeta3 integrin structure and functional relation with ion channels pulmonary function response to edta, an additive in nebulized bronchodilators metal chelation therapy in rheumathoid arthritis: a case report. successful management of rheumathoid arthritis by metal chelation therapy the structure of the two amino-terminal domains of human icam-1 suggests how it functions as a rhinovirus receptor and as an lfa-1 integrin ligand bicyclic rgd peptides with exquisite selectivity for the integrin αvβ3 receptor using a "random design" approach high-affinity rgd-knottin peptide as a new tool for rapid evaluation of the binding strength of unlabeled rgd-peptides to αvβ3, αvβ5, and α5β1 integrin receptors calcium signallingremodelling and disease integrins in the spotlight of cancer identifying protein phosphorylation sites with kinase substrate specificity on human viruses comparison of chelating agents dmps, dmsa and edta for the diagnosis and treatment of chronic metal exposure role of transforming growth factor-β in human disease covid-19: immunopathology and its implications for therapy in-silico discovery of candidate drugs against covid-19 coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein structure analysis of the receptor binding of 2019-ncov chloroquine and hydroxychloroquine as available weapons to fight covid-2019 new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? an rgd sequence in the p2y2 receptor interacts with αvβ3 integrins and is required for gomediated signal transduction rationale for the successful management of edta chelation therapy in human burden by toxic metals edta chelation therapy for the treatment of neurotoxicity ethylenediaminetetraacetic acid induces antioxidant and anti-inflammatory activities in experimental liver fibrosis remdesivir is a direct-acting antiviral that inhibits 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tools apis in 2019 covid-19: consider cytokine storm syndromes and immunosuppression. the lancet effects of high glucose on integrin activity and fibronectin matrix assembly by mesangial cells crystal structure of α5β1 integrin ectodomain: atomic details of the fibronectin receptor severe acute respiratory syndrome coronavirus e protein transports calcium ions and activates the nlrp3 inflammasome ouabain mediates integrin switch in human lung cancer cells myhits: improvements to an interactive resource for analyzing protein sequences integrin α1β1 differentially regulates cytokine-mediated responses in chondrocytes the fmrfamide-like peptide family in nematodes structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody α-lipoic acid sensitizes lung cancer cells to chemotherapeutic agents and anoikis via integrin β1/β3 downregulation dysregulation of immune response in patients with covid-19 in wuhan clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study integrin overexpression induced by high glucose and by human diabetes: potential pathway to cell dysfunction in diabetic microangiopathy edta chelation therapy, without added vitamin c, decreases oxidative dna damage and lipid peroxidation fibronectin and its receptors rgd and other recognition sequences for integrins epigenetic dysregulation of ace2 and interferon-regulated genes might suggest increased covid-19 susceptibility and severity in lupus patients silico studies on therapeutic agents for covid-19: drug repurposing approach. life science, 117652 the role of integrins in pulmonary fibrosis immunopathological characteristics of coronavirus disease a potential role for integrins in host cell entry by sars-cov-2 integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium integrins as therapeutic targets for respiratory diseases potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody tissue-based map of the human proteome a genome-wide transcriptomic analysis of protein-coding genes in human blood cells structural basis for pure antagonism of integrin αvβ3 by a high-affinity form of fibronectin structure, function, and antigenicity of the sars-cov-2 spike glycoprotein receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro utilizing integrating network pharmacological approaches to investigate the potential mechanism of ma xing shi gan decoction in treating covid-19 cryo-em structure of the 2019-ncov spike in the prefusion conformation disintegration of retroviruses by chelating agents high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa pathological findings of covid-19 associated with acute respiratory distress syndrome a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes. biorxiv single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 i acknowledge mohanlal sukhadia university, udaipur for the infrastructure facility. the work has been conducted in absence of any funding. key: cord-303741-1ou0cy5k authors: stafstrom, carl e.; jantzie, lauren l. title: covid-19: neurological considerations in neonates and children date: 2020-09-10 journal: children (basel) doi: 10.3390/children7090133 sha: doc_id: 303741 cord_uid: 1ou0cy5k the ongoing worldwide pandemic of the novel human coronavirus sars-cov-2 and the ensuing disease, covid-19, has presented enormous and unprecedented challenges for all medical specialists. however, to date, children, especially neonates, have been relatively spared from the devastating consequences of this infection. neurologic involvement is being increasingly recognized among adults with covid-19, who can develop sensory deficits in smell and taste, delirium, encephalopathy, headaches, strokes, and peripheral nervous system disorders. among neonates and children, covid-19-associated neurological manifestations have been relatively rare, yet reports involving neurologic dysfunction in this age range are increasing. as discussed in this review, pediatric neurologists and other pediatric specialists should be alert to potential neurological involvement by this virus, which might have neuroinvasive capability and carry long-term neuropsychiatric and medical consequences. severe and at times fatal symptoms caused by the novel human coronavirus, severe acute respiratory syndrome (sars)-cov-2, and the associated coronavirus disease 2019 (covid19) , are ravaging the world. while symptoms of covid-19 are primarily pulmonary (fever, dry cough, fatigue, pneumonia), it is becoming increasingly recognized that multiple organ systems can be affected, including the brain, with neurological involvement affecting up to~36% of patients [1] [2] [3] [4] [5] . information gained from studies of related coronaviruses in recent epidemics of severe acute respiratory syndrome (sars, 2002) and middle east respiratory syndrome (mers, 2012) suggests that all three coronaviruses might have neurologic consequences [6, 7] , though the relative severity and frequency of neurologic involvement caused by coronaviruses varies and thus the extent to which sars and mers epidemics inform our understanding of covid-19 remains unclear [5] . nevertheless, the possibility has been raised that sars-cov-2 could invade the brain and cause neurological disease [2, 8] . while appealing conceptually, data supporting the idea that the sars-cov-2 virus can infect the peripheral and central nervous systems (pns, cns) are limited, as discussed below. table 1 lists definitions of relevant terms that are often used in the literature. neurotropic viruses vary in their invasiveness, virulence, and propensity to cause inflammation [9] . the purpose of this review is twofold: (1) to discuss the available data about covid-19 infections in neonates and children, and (2) to provide a perspective about potential neurologic involvement in neonates and children with covid-19 infections, in view of neurobiological development. a few points need clarification up front. first, data about the virus and its effects are accumulating rapidly and our understanding of its consequences will evolve over time. second, much of the literature about covid-19 currently exists as case reports or small series; obviously, the impact of such publications is limited, and greater understanding will emerge only as large, rigorous studies are published. third, we must be mindful that a positive test for sars-cov-2 in a patient with a neurological symptom does not necessarily imply that the virus caused the symptom. the covid-19 epidemic has escalated rapidly and spread widely across the globe, with cases continuing to accrue at an alarming rate. the first case was reported from wuhan, china in mid-december 2019 and three months later, in march 2020, the world health organization declared covid-19 a pandemic. as of this writing (late august, 2020), more than 23 million cases of covid19 have been documented worldwide (with many more mildly symptomatic cases likely not reported), with over 800,000 deaths, and more than 175,000 deaths in the united states alone (www.cdc.gov, accessed 24 august 2020). documentation of the numerous clinical presentations, manifestations, and disease course have proliferated in the medical literature. a pubmed search (23 july 2020) using the keyword covid-19 revealed an astounding number of published reports already (34, 310) , over the course of only a few months. of those citations, only 501 reports (1.5%) also included the keyword neonate, attesting to the low published incidence in newborns. when searching pubmed with the key terms covid-19, neonate and neurological or brain, fewer than 10 articles emerged. therefore, at least so far, covid-19 does not seem to be affecting neonates very often from a neurological point of view; pubmed counts probably underestimate the occurrence of neurological involvement in children, being biased toward areas of the world with greater medical resources. these observations do not preclude the potential for neonatal brain involvement in covid-19 nor exclude the possibility of long-term medical, neurodevelopmental, and psychosocial consequences of the disease. indeed, as time goes on, a wider spectrum of neurologic manifestations will likely emerge with as yet undetermined long-term sequelae. as the covid-19 pandemic continues, certain trends are becoming evident. first, while the number of cases and deaths continue to rise, the disease does not affect infants and children nearly as frequently as adults [9, 10] . to date, approximately 2-5% of cases of covid-19 involve children, who appear to be less severely affected than adults, mainly with pulmonary symptoms [11] [12] [13] . second, disease severity in children who develop covid-19 is usually milder than in adults, and children with severe disease often have an underlying co-morbidity such as immunosuppression [10, 14] . indeed, there is accumulating evidence that adults with covid-19 infection manifest with multiple organ system involvement, including the cns and pns, and that older and sicker individuals carry a higher risk for neurologic problems [1, 4, 15] . these age-dependent differences in disease expression and severity have clear implications for healthcare professionals who deal with the pediatric population because children remain at risk for incurring and spreading the virus, yet many remain asymptomatic. several hypotheses have been posited as to why children are less affected by covid-19, including age-related differences in immune responses [16] , a neutralizing antibody response due to prior exposure to coronaviruses [17] , lower prevalence of co-morbidities in children, and age-specific differences in sars-cov-2 receptor function [18] , neurovirulence, intrinsic biological protective mechanisms, and other host factors [19] . none of these hypotheses is supported compellingly by extant data at present. while the number of affected neonates and children remains small, pediatric practitioners cannot become complacent about the potential for neurologic involvement in covid-19. furthermore, the recently described multisystem inflammatory syndrome-children (mis-c), raises the specter that covid-19 or its after-effects also target children (see section 4) [20, 21] . as data from china, europe and other areas affected early by the covid-19 pandemic are reported, some patterns are emerging regarding pregnant women and neonates. first, it is clear that vertical transmission covid-19 from a pregnant mother to her fetus occurs quite rarely. more than a dozen publications attest to this observation, together encompassing over 100 patients (table 2 lists a few relevant publications, selected from the largest available series and omitting small series and single case reports). none of these reports documents unequivocal vertical transmission. in many of the babies, onset of symptoms occurred in the neonatal period but not immediately at birth, so the exact timing of infection remains uncertain. overall, the data does not support robust transplacental transfer of sars-cov-2, but recent case reports are providing proof-of-principle that the virus can be transmitted intrauterine from infected mother to fetus [22, 23] . an especially apropos case demonstrated maternal viremia, placental infection shown by immunohistochemistry, and high placental viral load with subsequent neonatal viremia, implying transplacental transfer of sars-cov-2 from pregnant mother to fetus [24] ; this newborn presented with neurological symptoms as discussed in section 3. of all the infants reported during the first month of life, most had documented exposure to affected family members [13, 32] which emphasizes the importance of controlling horizontal virus transmission from affected family members to the neonate [33] . while this trend may need revision [27, 34] , so far, it is encouraging that most covid-19-positive pregnant women do not transmit the disease to their unborn children. similarly, there is no evidence that covid-19-positive pregnant women incur covid-19 or develop more severe disease than similar-aged women who are not pregnant. however, the impact of chronic inflammation caused by maternal viral infection on fetal development, pregnancy outcomes and long-term neurodevelopment is unknown. similarly, the timing of maternal viral infection with respect to major milestones of in utero neurodevelopment (i.e., second trimester vs. late third trimester) is an unknown and critical consideration for further research and long-term neurodevelopmental followup. there is legitimate concern about the impact of acute and chronic stress during the pandemic (i.e., worry about the medical complications of sars-cov-2 infection, family disruption, job loss, economic pressures, educational uncertainties, food availability, etc.) on the pregnant patient and developing fetus [35] [36] [37] [38] . furthermore, it is reassuring that there is no definitive evidence that the virus is present or can be transmitted in the breast milk of covid-19-positive women [28, 39, 40] . the current american academy of pediatrics recommendation is that covid-19-positive mothers can breast feed directly while wearing a mask or feed expressed breast milk, using appropriate breast and hand hygiene [41] . extensive guidelines are available regarding principles of management of pregnant women with covid-19 and their newborns [40] [41] [42] . again, all of these observations are preliminary and subject to modification over time. although covid-19 primarily affects the pulmonary system, it is a multisystem infection (e.g., gastrointestinal tract, kidneys, liver, heart) and involvement of the pns and cns are increasingly recognized [43] [44] [45] [46] . data on neurological signs and symptoms are limited but increasing, with a wide spectrum of acute and chronic manifestations becoming apparent [47] . in a series of 214 hospitalized adults with covid-19, 88 of whom had "severe" infections, 36.4% of the entire group was reported to manifest some neurologic involvement, including alteration of consciousness, encephalopathy, headache, cerebrovascular disease, and skeletal muscle injury (myalgia, weakness) [1] . ischemic strokes, many affecting young adults with large vessel occlusions, have garnered considerable concern that the etiology may be a prothrombotic state caused by virus-induced inflammation of the vascular epithelium [48, 49] . many of the young adult stroke victims had other vascular risk factors such as diabetes or hypertension, which emphasizes the importance of comorbidities with systemic inflammatory conditions in disease manifestations and severity. stroke has not been reported in children with covid-19 [50] . the occurrence of encephalitis remains controversial, as virus has not been recovered from cerebrospinal fluid (csf) [51] and overall, a surprisingly small number of covid-19 patients develop classic encephalitic symptoms. autopsy studies are beginning to be published. the wide spectrum of postmortem findings include mostly secondary changes to the cns such as hypoxemia and ischemia, rarely localized perivascular and interstitial neuroglial activation with neuronal loss and axonal degeneration [52, 53] , and no other major cns abnormalities [54] . no pediatric autopsy cases have reported neuropathological involvement. clearly, more data are needed before this issue can be clarified. in the chinese series [1] , only 2 out of 214 patients had seizures (1%), which is not greater than the general population, so it is uncertain whether infected patients are at higher risk. the few patients with seizures are reported mainly in case reports [55, 56] . the lack of frequent seizures is rather curious, especially if encephalopathy is indeed a frequent complication of covid-19; this data may be related to sampling bias rather than actual non-occurrence. a few reports of seizures have appeared in adults using electroencephalography (eeg) [57, 58] , but a more concerted effort to evaluate the brain electrophysiology of children with covid-19 would be informative. the examples of seizures in children with covid-19 described in section 3.6 appear to be largely anecdotal. many additional cases are necessary to conclude whether there is increased seizure susceptibility in the pediatric population. other cns symptoms include headache, dizziness and delirium, all of which can occur as a nonspecific consequence of systemic infection or inflammation of the respiratory tract as well as via a cns mechanism. although headaches are reported frequently, the pain often appears to be nonspecific or associated with inflammation or migraine exacerbation rather than meningeal irritation [59] . the most commonly reported symptoms related to the pns are decreased taste (hypogeusia or ageusia) and smell (hyposmia or anosmia) [60, 61] . a neural mechanism is suspected for hyposmia in covid-19, because decreased smell is often the first symptom experienced and occurs in mild disease in the absence of significant local inflammation or mucosal congestion that are typical of the more benign coronavirus or non-coronavirus nasal infections [62] . a few adolescents with covid-19 have been reported with decreased taste or smell [63] ; these symptoms appear to be very uncommon in children but deserve a more concerted ascertainment effort. several cases of guillain-barré syndrome (gbs) have been reported in adults with covid-19, raising the possibility of post-infectious autoimmune responses against the pns [64, 65] . two case reports of children with gbs who developed covid-19 symptoms about 3 weeks later confirms that gbs can occur with covid-19, though this association remains quite rare given the widespread prevalence of covid-19 [66, 67] . finally, the possibility of central demyelination has been raised, e.g., in the form of multiple sclerosis (ms), among patients with covid-19 [68] ; this concern is relevant in that various disease-modifying agents used to treat ms could theoretically exacerbate ms symptoms [69] . fortunately, there is no evidence that covid-19 triggers central demyelinating disease in children [50] . the lack of unequivocal reports of sars-cov-2 being recovered from the csf of individuals affected with presumed neurological involvement nor in brain tissue from the limited number of autopsied cases strengthens the possibility that the virus does not often directly cause the symptoms but rather, that the neurological sequelae are secondary to hypoxia, cytokine involvement, or some other non-direct mechanism (see section 6). it is appropriately concerning that chronic neurologic diseases such as epilepsy, amyotrophic lateral sclerosis, multiple sclerosis, etc., might be exacerbated during concurrent covid-19 infection or that covid-19 may unmask preexisting cns pathology that might have been unrecognized or asymptomatic. as just discussed, neurologic involvement in children, and in particular neonates, with covid-19 appears to be scarce but may be under reported [70] . a few selected examples of case reports of neurologic involvement in neonates and children are presented in table 3 ; such case reports have limited generalizability, and many lack sufficient details to ascribe causality between sars-cov-2 and neurologic symptoms. most children were assumed to have contracted covid-19 from a family member and some children had concurrent infection with other viruses, confounding any argument for causality. importantly, csf was negative for sars-cov-2 in all children on whom spinal fluid was obtained. all children recovered within a few days or weeks, contrasting with the severe and prolonged courses in many adults. available evidence does not allow distinction between a direct effect of sars-cov-2 causing neurologic dysfunction, versus the symptoms instead being secondary to an over activated immune response (see section 5) . in summary, case reports of neurological involvement in babies and children are rare but accumulating, and the recovery of most infants with early neurologic symptoms implicates some virus-or host-related factors that minimize massive neurological devastation. this newly recognized kawasaki syndrome-like hyperinflammatory disorder presents with acute hypotension and cardiogenic shock and is proliferating across the globe. it is likely a post-infectious syndrome or inflammatory reaction following asymptomatic or mildly symptomatic covid-19 [76] . children can develop toxic shock-like symptoms, hypoxia-ischemia, and significant end organ damage to the heart, kidneys, and other organs. while definitive data is not available, there is concern that the inflammation that hallmarks mis-c may have adverse consequences on the developing brain. while no consistent neurologic picture has emerged, several mis-c patients have had cns involvement as part of their course. in a small series of 6 children with mis-c, 4 patients had neurologic symptoms, including headache, altered mental status, and aseptic meningitis [77] . headache was the most common symptom in a series of 58 children with mis-c associated with sars-cov-2, affecting 26% of patients [78] . a series 21 children from france notes that 57% of patients were "irritable" and another 29% had "other neurological features", though these were not specified [79] . in a larger survey of 186 children, 5-11% had neurologic involvement, depending on age, including encephalitis, seizures or mental status alteration, but details are not provided [80] . finally, 4 of 27 children with covid-19 associated mis-c developed new neurologic symptoms including encephalopathy, headache, weakness, ataxia, and dysarthria [81] ; two patients had lumbar punctures and csf was negative for sars-cov-2 in both. three of the four patients had an eeg; each showed diffuse slowing. brain mri scans of all four children showed abnormal signal intensities of the splenium of the corpus callosum (a finding seen in previous cases of encephalopathy and thought to indicate inflammation-induced focal myelin edema [81, 82] . a recent systematic review of eight studies notes neurological symptoms in~25-50% of children with mis-c, depending upon inclusion criteria [83] . a putative impact on the immature cns and developing immune system, including neural-immune maturation, cannot be overlooked, and the long-term neurologic impact of both covid-19 and mis-c on the developing brain need urgent elucidation. sars-cov-2 is a highly contagious and pathogenic rna virus that is transmitted via droplet, contact, or aerosol routes. the virus gains entry into epithelia of the pulmonary system (upper or lower respiratory tracts), digestive tract, or nasopharynx. the virus is composed of single-stranded rna enveloped by surface proteins (s, e, m, n). the spike (s) glycoprotein serves as the attachment site onto angiotensin converting enzyme type 2 (ace-2) receptors on epithelial membranes. the normal function of ace-2 receptors is to provide protection against pulmonary and endothelial injury [84] . sars and sars-cov-2 share~79% genome sequence identity and both viruses infect epithelium by the binding of spike proteins to ace-2 receptors. while most prevalent on airway and pulmonary epithelium [85] , ace-2 receptors are also reportedly present to a lesser degree on neurons and perhaps glia [2] . binding of the s protein to ace-2 receptors leads to proteolytic cleavage of s by the transmembrane protease tmprss2 [5] . viral rna then enters the epithelial cell and replicates rapidly, translating viral proteins and inducing a host immune response. this immune response can be adaptive, attacking and inactivating the virus. by contrast, the immune response can be maladaptive and induce a massive immune reaction, accompanied by a hyper-inflammatory response hallmarked by excessive cytokine secretion and signal transduction (cytokine storm), and robust cellular immune activation and recruitment [86] . the large-scale cytokine storm consists of a massive release of pro-inflammatory humoral agents such as interleukin-6 (il-6), interferon gamma (ifn-y), mcp-1/ccl2 (monocyte chemoattractant protein 1/chemokine ligand 2), il-1, il-12, il-8, tnfα (tumor necrosis factor alpha), and cxcl 10 (c-x-c motif chemokine ligand 10) that exacerbate the underlying pathophysiology [87, 88] . this cytokine release subsequently feeds forward an overactive and dysregulated cellular immune response defined by macrophage, monocyte, neutrophil, and t-cell hyperactivation and recruitment [89] . the impact of this systemic cytokine storm on neurodevelopment is under investigation in preclinical models [90] and should be the focus of future prospective studies. subsequently, the replicated viruses exit the cell, leading to further infection. it is unknown why children, and neonates in particular, seem to be relatively resistant to covid-19 and its severe symptoms, including neurological manifestations. the cytokine response to coronavirus infection appears to be less robust in young children although the recognition of mis-c may suggest host-dependent genetic susceptibility to enhanced cytokine and/or inflammatory responses [91] , but other mechanisms are also plausible [19] . it remains controversial whether ace-2 inhibitors would provide symptomatic relief or prevent the covid-19 disease, and evidence for the effectiveness of these agents in children and neonates is not yet available [92] . the cellular and molecular basis of sars-cov-2 neurotropism, neuroinvasiveness, and neurovirulence are poorly understood [9] : does the virus get into the brain and if so how, and what does it do in the cns once there (e.g., infects neural cells? causes disease?). neurological involvement in covid-19 might be associated with at least four potential mechanisms: 1. a direct neurotropic or neuroinvasive effect of sars-cov-2 (e.g., anosmia, encephalopathy), 2. a secondary effect of the systemic inflammatory responses triggered by the viral infection (e.g., encephalopathy), 3. a secondary effect associated with the vascular and prothrombotic effect of the viral infection on the cns or pns vasculature (e.g., strokes, necrotizing leukoencephalopathy), 4. an immune-mediated para-infectious or post-infectious autoimmune effect in response to the viral infection (e.g., gbs, acute disseminated encephalomyelitis). figure 1 summarizes, in schematic fashion, some hypothetical possibilities about how the virus may infect the brain directly, whether the neurological symptoms and signs may be related to systemic or hyperactivation of immune responses, or both [87] . it is important to consider the mechanisms associated with neurological manifestations of covid-19, with an aim toward developing therapeutic options. the possibilities of direct neurotropism and hyper-responsiveness to immune activation (cytokine storm) are considered separately below, though these mechanisms might work synergistically. the sars-cov-2 virus attaches to olfactory epithelium using the ace-2 receptor. after cell entry, the virus replicates and induces a massive immune response leading to excessive cytokine release, comprising a maladaptive immune response. theoretically, virus particles may reach the cns retrogradely via cranial nerve pathways: v from corneal epithelium or oropharyngeal cutaneous sensory receptors; i via the cribiform plate, infecting olfactory sensory neurons; vii and ix from tongue chemoreceptors; x via pulmonary mechanoreceptors. once reaching cns nuclei including brainstem and cortex, a variety of neurologic signs and symptoms are possible. however, it must be noted that the virus has not been recovered from csf or brain tissue, making all of these pathways hypothetical at this point. abbreviations: np, nasopharynx; gi, gastrointestinal; ace-2, angiotensin converting enzyme type 2 receptor; pns, peripheral nervous system; cns, central nervous system; ich, intracranial hemorrhage; gbs, guillain-barre syndrome; bbb, blood-brain barrier. definitive demonstration of direct viral invasion would require a positive csf reverse transcriptase-polymerase chain reaction (rt-pcr) for sars-cov-2, recovery of infective virus from the csf as demonstrated by viral cultures or "plaque assay" [93] , intrathecal synthesis of antibodies to sars-cov-2, or autopsy-demonstrated sars-cov-2 antigen or rna in brain tissue [5] . current published evidence meeting these strict criteria is minimal. while it is plausible that the virus infects the brain through one of the anatomical pathways discussed below, the lack of viral recovery from the cns gives pause to that notion. neuroinvasion has been demonstrated for the related sars and mers viruses [94] , but sars-cov-2 has not been recovered from the csf or brain tissue. animal models of sars and mers have shown that the virus can enter through epithelium of the nasopharynx and travel retrogradely to the cns [95, 96] . interestingly, wild type mice are not vulnerable to infection and disease by human coronaviruses, but transgenic mice with human ace-2 receptors do develop respiratory and neurological symptoms when infected [95, 97] . in such transgenic mice, intranasal exposure to sars or mers leads to brain infection. one of the proposed portals of entry is via olfactory sensory neurons, crossing the cribiform plate into the olfactory bulb, with subsequent retrograde travel along the olfactory nerve (cranial nerve i) to the brainstem, thalamus, and basal ganglia, all areas that are connected to the olfactory cortex. please note that it has yet to be proven that sars-cov-2 infects olfactory sensory neurons. emerging animal models may clarify whether sars-cov-2 is similarly neuroinvasive as sars and whether this isage dependent the sars-cov-2 virus attaches to olfactory epithelium using the ace-2 receptor. after cell entry, the virus replicates and induces a massive immune response leading to excessive cytokine release, comprising a maladaptive immune response. theoretically, virus particles may reach the cns retrogradely via cranial nerve pathways: v from corneal epithelium or oropharyngeal cutaneous sensory receptors; i via the cribiform plate, infecting olfactory sensory neurons; vii and ix from tongue chemoreceptors; x via pulmonary mechanoreceptors. once reaching cns nuclei including brainstem and cortex, a variety of neurologic signs and symptoms are possible. however, it must be noted that the virus has not been recovered from csf or brain tissue, making all of these pathways hypothetical at this point. abbreviations: np, nasopharynx; gi, gastrointestinal; ace-2, angiotensin converting enzyme type 2 receptor; pns, peripheral nervous system; cns, central nervous system; ich, intracranial hemorrhage; gbs, guillain-barre syndrome; bbb, blood-brain barrier. definitive demonstration of direct viral invasion would require a positive csf reverse transcriptase-polymerase chain reaction (rt-pcr) for sars-cov-2, recovery of infective virus from the csf as demonstrated by viral cultures or "plaque assay" [93] , intrathecal synthesis of antibodies to sars-cov-2, or autopsy-demonstrated sars-cov-2 antigen or rna in brain tissue [5] . current published evidence meeting these strict criteria is minimal. while it is plausible that the virus infects the brain through one of the anatomical pathways discussed below, the lack of viral recovery from the cns gives pause to that notion. neuroinvasion has been demonstrated for the related sars and mers viruses [94] , but sars-cov-2 has not been recovered from the csf or brain tissue. animal models of sars and mers have shown that the virus can enter through epithelium of the nasopharynx and travel retrogradely to the cns [95, 96] . interestingly, wild type mice are not vulnerable to infection and disease by human coronaviruses, but transgenic mice with human ace-2 receptors do develop respiratory and neurological symptoms when infected [95, 97] . in such transgenic mice, intranasal exposure to sars or mers leads to brain infection. one of the proposed portals of entry is via olfactory sensory neurons, crossing the cribiform plate into the olfactory bulb, with subsequent retrograde travel along the olfactory nerve (cranial nerve i) to the brainstem, thalamus, and basal ganglia, all areas that are connected to the olfactory cortex. please note that it has yet to be proven that sars-cov-2 infects olfactory sensory neurons. emerging animal models may clarify whether sars-cov-2 is similarly neuroinvasive as sars and whether this isage dependent [97, 98] . however, since mice are not naturally susceptible to the clinical and immunopathological manifestations of coronaviruses affecting humans, translational studies of pathogenic mechanisms and vaccine development become complicated. extensive efforts to modify mice with transgenic approaches have begun to provide informative models. as mentioned, the olfactory epithelium has been touted as a potential site of viral entry into the brain, and hence explain hyposmia [99] .detailed genetic and immunohistochemical examinations of cell types of the olfactory system reveal that ace-2 and tmprss2 are present on olfactory epithelial cells (especially supporting or "sustenacular" cells) but not on olfactory sensory neurons themselves [54, 85, 100] . moreover, there is some evidence of virus-induced cell death in other coronavirus infections but not yet for sars-cov-2 [84, 101] . likewise, the virus might enter via the sensory system of the tongue that mediates taste, with transmission via cranial nerves vii, ix, and x to the nucleus tractus solitarius, thalamus and eventually, brain. finally, trigeminal nociceptors via cranial nerve v from either the corneal epithelium or buccal epithelium could theoretically reach the cns. these potential pathways could explain the symptoms of hypogeusia and altered vision. however, sars-cov-2 has not been recovered from the brain. transynaptic transport from lower respiratory tract mechano-and chemoreceptors to the brainstem medullary cardiorespiratory centers has been proposed as a hypothetical mechanism that could exacerbate brainstem dysfunction and perhaps even worsen respiratory effort [102] ; however, this hypothesis lacks objective validation and remains controversial. other potential routes for virus to enter the cns are through the bloodstream (hematogenous) or via disruption of the blood brain barrier (bbb). from the systemic circulation, the virus might travel to the cerebral circulation where it can damage capillary epithelium and access the brain. interestingly, there is scant evidence that sars-cov-2 produces a significant or sustained viremia [103] . the bbb is essential for transport of molecules into the brain and exclusion of pathogens and overall maintenance of cerebral homeostasis [104] . the bbb is a dynamic structure, consisting of several cell types and proteins, each with its own maturational profile-astrocyte foot processes, pericytes, tight junction proteins, and extracellular matrix, providing structural and functional support. virus attachment to ace-2 receptors at the bbb might facilitate trafficking of the virus into the cns, facilitating endothelial damage and edema [89] . notably, while the bbb is structurally complete at birth and is sufficiently functional in the neonate to provide protection against many pathogens, its full physiological maturation may take several months [105] . in the context of covid-19 infection, the bbb may be dysfunctional, disrupted either by inflammatory response or the virus itself, allowing transmission of the virus or activated immune cells from the circulation into the cns [8, 84] . the release of inflammatory cytokines by activated glia and neural mast cells exacerbate the inflammation [89] . similarly, flow of the virus through lymphatic channels of the interstitial space of the brain could breach the blood-csf barrier and permit virus entry [106] . to date, there is no evidence for the presence of the virus in pathological specimens of the pns or cns, in part due to the dearth of comprehensive autopsies [52] [53] [54] . obviously, patient care has focused on critical pulmonary and life-support measures so neuropathologically-focused autopsy studies have been uncommon. animal studies of covid-19 will be crucial to complement information gained from prior studies of the other coronaviruses. such animal models will provide more information about mechanisms of virus entry into the nervous system and how the virus affects neural function, neural-immune maturation and neurodevelopment, as well as the critical and yet unanswered question of long-term neurological sequelae of covid-19 [107] . that is, if there is predilection of the virus for certain neural structures or chronic neuroinflammation, long-term consequences may arise in various neural functions such as learning, memory, cognition, seizure predisposition, and other functions. all of this is speculative at present. another essential question, alluded to above, is whether cns disease contributes to the respiratory failure seen in covid-19 patients. ongoing or severe hypoxia can exacerbate ongoing symptoms in other organs. in particular, cns respiratory control centers in the brain stem, nearby the vagus nerve, has been speculated to play a role in respiratory failure [102, 108] . at present, there is some reason for guarded optimism for young patients within the devastating covid-19 pandemic. children, particularly neonates, are less likely to become infected and develop severe symptoms, and their propensity to spread the virus is controversial [109] . there is at best slight evidence for vertical transmission of sars-cov-2 or covid-19 disease from pregnant mother to fetus; rather, neonates are more likely incur the disease by exposure to affected individuals postnatally, and breast milk transmission has not been shown (table 4) . a variety of practical guidelines have been developed for the care of pregnant women who have or are suspected to have covid-19 positivity. analogous guidelines for the care of adult covid-19 patients with neurologic problems are also available and need to be developed for children [110] . table 4 . summary of covid-19 infections in children. severe infection caused by the novel coronavirus, sars-cov-2, has predominant pulmonary involvement but can also affect multiple other organ systems, including the cns and pns. symptoms are less frequent and usually less severe in children and particularly in neonates. vertical transmission of sars-cov-2 from pregnant mother to fetus is rare but anecdotal case reports support this possibility. most cases of covid-19 in early life are due to exposures to infected patients (horizontal transmission). there is no reported transmission of sars-cov-2 via breast milk. regarding neurologic involvement in covid-19, there are plausible mechanisms by which the virus can gain entry into the cns and subsequently incur acute neurologic symptoms, either directly or through immune dysfunction ( table 5 ). the occurrence of long-term medical and neuropsychiatric sequelae is unknown. children can be resilient and yet remain vulnerable to coping with the challenges of covid-19 in the context of other acute and chronic diseases. youngsters may not understand the need for social distancing, prolonged quarantine, and other preventative measures, and it is anticipated that stress-related post-traumatic symptoms will develop in some young people, whether or not they actually acquire symptoms. in children with comorbid chronic conditions and developmental disabilities, the challenges are even more profound. therefore, neuropsychological surveillance and studies of the long-lasting effects of this pandemic on neurodevelopment are critical [111] . finally, the emergence of the hyper-inflammatory multisystem syndrome (mis-c) supplants any conclusion that covid-19 is benign or negligible in the pediatric age range. therefore, it behooves neurologists and other pediatric specialists who deal with neonates and young children to be aware of the potential neurologic involvement of this novel, potentially devastating virus. future animal models should evaluate the impact of sars-cov-2 on maternal infection, inflammatory signal transduction through the maternal-placental-fetal axis, and brain development. the importance of large-scale immunization should a vaccine become available, cannot be over emphasized as should the role of systemic inflammation, neuroinflammation, and neural-immune interactions in novel pathophysiology and symptomology. additionally, mechanism-specific targeted therapies could emerge from basic science studies of sars-cov-2 infection. table 5 . neurological involvement in covid-19. acute neurological involvement in adults with covid-19 can include decrease taste/smell, headache, confusion, peripheral nerve dysfunction, strokes, and 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and their potential influence on neuropsychological outcomes in children this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license research in the laboratory of stafstrom is supported by the mathias koch memorial fund, the sandra and malcolm berman foundation, and the paine foundation. research in the laboratory of jantzie is supported by the national institutes of health (1r01hl139492) and the department of defense (w81xwh-18-1-0167) . we thank carlos pardo-villamizar for his insightful comments and critique of the manuscript. the authors declare no conflict of interest. key: cord-314793-kb319t4c authors: borroni, barbara; gazzina, stefano; dono, fedele; mazzoleni, valentina; liberini, paolo; carrarini, claudia; russo, mirella; pontolillo, michela; vecchiet, jacopo; onofrj, m.; bonanni, laura title: diaphragmatic myoclonus due to sars-cov-2 infection date: 2020-10-22 journal: neurol sci doi: 10.1007/s10072-020-04766-y sha: doc_id: 314793 cord_uid: kb319t4c a wide range of neurological signs and symptoms have been associated with sars-cov-2 infection. in the present report, we described two italian patients diagnosed with diaphragmatic myoclonus after covid-19. in both cases, mild lymphocytosis at cerebrospinal fluid analysis and no structural brain changes were reported. the pathophysiological origin of the myoclonus in the two cases was different. in case 1, electroencephalogram did not reveal any cortical correlates and brain imaging of the spine was unremarkable, while in case 2, cortical origin of myoclonus was demonstrated. with the present two cases, we confirm and extend the neurological manifestations of sars-cov-2 infection. electronic supplementary material: the online version of this article (10.1007/s10072-020-04766-y) contains supplementary material, which is available to authorized users. the 2019 new coronavirus (sars-cov-2) is a respiratory virus which has become increasingly prevalent worldwide, reaching a pandemic stage on march 2020 according to who, with more than 6 million people affected at 1st june, according to who data. the clinical manifestations of sars-cov-2 include fever, dry cough, fatigue, headache, gastrointestinal discomfort, dyspnea, muscle ache, and a severe pneumonia causing respiratory distress [1] . neurological signs in patients with sars-cov-2 have been widely reported, suggesting a neuroinvasive nature of virus [2, 3] . with the present observation, we report two cases of diaphragmatic myoclonus as neurological subacute manifestation of sars-cov-2 infection. case 1 a 54-year-old woman with sars-cov-2 presented on march 16, 2020, to the emergency room in brescia, one of the epicenter of sars-cov-2 infection in italy [4] , with fever, dry cough, and dyspnea. thoracic ultrasound was consistent with the suspicion of viral pneumonia, but the nasopharyngeal swab resulted negative. the initial treatment was supportive, and azithromycin was added as empirical therapy. however, in the following 2 weeks, fever and dry cough persisted and she started complaining episodic chest pain, associated with involuntary rapid contractions of the abdominal wall, especially at night. her past medical history was unremarkable except for hypertension. for these symptoms, on april 1, 2020, she returned to the emergency room. the body temperature was 36.8°c (98°f), and oxygen saturation was 95% at room air. myocardial infarction was excluded. nasopharyngeal swab for sars-cov-2 was repeated and she tested positive. she began to receive treatment with hydroxychloroquine and low molecular weight heparin. on may 7, 2020, the patient was admitted to the neurological unit for the persistency of the jerky contractions of abdominal muscles, causing disabling shortness of breath, despite a complete recovery of fever and cough. on examination, she presented with paroxysmal involuntary movements of her abdomen, lasting a few minutes, and presenting from 10 to 15 times a day (see supplementary video 1). the electronic supplementary material the online version of this article (https://doi.org/10.1007/s10072-020-04766-y) contains supplementary material, which is available to authorized users. movements did not affect her chewing or swallowing, nor impact her bowel function. they were not provoked or worsened by any positions or triggered by any maneuvers. moreover, not disabling, small-amplitude twitches of left limbs on maintaining a posture were observed. electromyographic recording confirmed rhythmic and synchronous contractions of the diaphragm at 3-hz frequency as the cause of her abdominal movements (see fig. 1 ), thus making the diagnosis of diaphragmatic myoclonus or van leeuwenhoek's disease [5] . phrenic nerve conduction was normal bilaterally, as well as magnetic resonance imaging of the head and spine, electroencephalogram (eeg), somatosensory evoked potentials, and cerebrospinal fluid analysis. cerebrospinal fluid (csf) evaluation showed mild lymphocytosis (8 cell/mm 3 ) with normal level of glucose and proteins. clonazepam was titrated with significant benefit (0.5 mg tid). case 2 an 80-year-old man was admitted on may 10, 2020, to the emergency room in chieti due to mild dyspnea, persistent fever (38°c), and dry coughing. his past medical history was unremarkable. arterial blood gas analysis showed normal blood oxygen saturation (so 2 = 95.6%), mild hypoxemia (71.9 mmhg), hypocapnia (22.1 mmhg), and alkalosis (ph = 7.534). thoracic computer tomography scan showed a ground-glass nodule at the left apical pulmonary lobe segment. oropharyngeal and nasopharyngeal swabs supported the diagnosis of sars-cov-2. the patient was then empirically treated with azithromycin and supportive therapy with a relief of the symptoms in 10 days with no need of ventilation throughout the entire period. on june 2, 2020, jerky myoclonic contractions of the diaphragm appeared, causing disabling shortness of breath, despite the complete recovery of previous respiratory symptoms (see supplementary video 2). radiography of the thorax showed a complete resolution of the pulmonary infection. patient appeared conscious and collaborative, although was not well oriented in space and time. computed tomography brain scan did not show any acute brain abnormalities. the eeg recording showed lateralized periodic discharges (lpds) synchronous and asynchronous with the diaphragmatic myoclonic movements (see fig. 2a ). csf evaluation showed lymphocytosis (24 cell/mm 3 ) with normal level of glucose and slight increase of proteins (46.2 mg/dl). polymerase chain reaction (pcr) for sars-cov-2 as well as herpes viruses, cytomegalovirus, epstein-barr fig. 1 surface recording of left and right hemidiaphragms in case 1. a patient at rest, ecg artifact. b deep inspiration. c threehertz rhythmic synchronous discharges during involuntary contraction of abdominal muscles. electrophysiological study of the phrenic nerve was performed by placing one monopolar needle (active electrode) two fingerbreadths above the xiphoid process and two monopolar needles (reference electrodes) over the anterior costal margin 16 cm from the active electrode. the same electrodes were used to derive electromyography activity of the diaphragm. l, left; r, right virus, and varicella zoster virus in the csf was negative. treatment with levetiracetam (1000 mg bid) was started with resolution of the myoclonus and improvement of the eeg features after 3 days (see fig. 2b ). diaphragmatic myoclonus is a rare condition and has been associated with many central and peripheral disorders including, but not limited to, encephalitis, stroke, osmotic demyelination, metabolic abnormalities, trauma, and phrenic nerve irritation, its etiology being still debated [6] . in our cases, the temporal sequence suggests, but does not prove, that sars-cov-2 was a causal factor of diaphragmatic myoclonus and cannot address the pathogenetic mechanism leading to the clinical picture. however, a number of similar cases are described in the literature where isolated myoclonus developed following viral infections of the upper respiratory tract or following an influenza-like illness, with no evidence of structural damage of the central nervous system [7] . myoclonus can be a clinical feature of many infectious encephalitis and was notable in the acute and chronic phases of epidemic encephalitis lethargica, which arose almost no myoclonic jerk at diaphragm has been detected as shown in the surface emg recording. eog, electro-oculogram; emg1, right hemidiaphragm electrode; emg2, left hemidiaphragm electrode contemporaneously with the 1918 spanish influenza outbreak, caused by h1n1 virus [8] . although the etiology of encephalitis lethargica remains uncertain, the spanish flu virus has been implicated in the pathogenesis. segmental myoclonus following herpes zoster infection is also well known and is usually transient [9] . here, we draw attention to two patients who developed focal diaphragmatic myoclonus after sars-cov-2 infection. in our two cases, the diaphragmatic myoclonus was not accompanied by evidence of structural damage of the central nervous system. the pathophysiological origin of the myoclonus in our two cases is likely different. in case 1, eeg did not reveal any cortical correlates suggesting that the myoclonus was probably not of cortical origin. in case 2, eeg showed lpds synchronous and asynchronous with the diaphragmatic myoclonic movements, with good response to levetiracetam. by looking at the time interval between cortical alterations and myoclonic jerks, these appear to be irregular (i.e., the cortical abnormalities were not invariably corresponding in time to myoclonic jerks); therefore, as a spinal myoclonus can be ruled out, a subcortical origin of the myoclonus cannot be excluded, even though the good response to levetiracetam would suggest an at least partial involvement of the cortex. this eeg pattern might be related to a widespread inflammatory effect due to sars-cov-2 cns infection. in line with previous reports in the literature [10] , even though pcr for sars-cov-2 performed on csf was negative, we could not exclude the role of sars-cov-2 in the genesis of the cerebral inflammation. it appears that myoclonus associated with sars-cov-2 infection is a heterogenous entity and that different sites of the central nervous system can be affected by the virus. indeed, three cases of generalized myoclonus due to sars-cov-2 infection have been recently reported [10] . with the present report of two cases, we confirm and extend the neurotropic properties of sars-cov-2 virus and point out to a further neurological clinical manifestation of the infection. conflict of interest the authors declare that they have no conflict of interest. ethics approval the work was approved by local ethic committees of brescia hospital and chieti hospital. consent to participate patients signed the informed consent to participate. clinical and computed tomographic imaging features of novel coronavirus pneumonia caused by sars-cov neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china neurological insights of covid-19 pandemic clinical characteristics and outcomes of inpatients with neurologic disease and covid-19 in brescia antony van leeuwenhoek and the description of diaphragmatic flutter (respiratory myoclonus) diaphragmatic flutter isolated postinfectious myoclonus 1918 influenza, encephalitis lethargica, parkinsonism spinal myoclonus following herpes zoster radiculitis lifting the mask on neurological manifestations of covid-19 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-307701-fujejfwb authors: gaurav, shubham; pandey, shambhavi; puvar, apurvasinh; shah, tejas; joshi, madhvi; joshi, chaitanya; kumar, sachin title: identification of unique mutations in sars-cov-2 strains isolated from india suggests its attenuated pathotype date: 2020-06-07 journal: biorxiv doi: 10.1101/2020.06.06.137604 sha: doc_id: 307701 cord_uid: fujejfwb severe acute respiratory syndrome coronavirus-2 (sars-cov-2), which was first reported in wuhan, china in november 2019 has developed into a pandemic since march 2020, causing substantial human casualties and economic losses. studies on sars-cov-2 are being carried out at an unprecedented rate to tackle this threat. genomics studies, in particular, are indispensable to elucidate the dynamic nature of the rna genome of sars-cov-2. rna viruses are marked by their unique ability to undergo high rates of mutation in their genome, much more frequently than their hosts, which diversifies their strengths qualifying them to elude host immune response and amplify drug resistance. in this study, we sequenced and analyzed the genomic information of the sars-cov-2 isolates from two infected indian patients and explored the possible implications of point mutations in its biology. in addition to multiple point mutations, we found a remarkable similarity between relatively common mutations of 36-nucleotide deletion in orf8 of sars-cov-2. our results corroborate with the earlier reported 29-nucleotide deletion in sars, which was frequent during the early stage of human-to-human transmission. the results will be useful to understand the biology of sars-cov-2 and itsattenuation for vaccine development. severe acute respiratory syndrome coronavirus-2 (sars-cov-2) is the cause of the novel human corona virus disease covid-19, first reported on november 17 th , 2019 in wuhan, china [12] . it has spread rapidly creating a global public health emergency situation in the last few months and the world health organization (who) on march 11 th , 2020 declared it a pandemic. till date, sars-cov-2 has infected close to five million people and caused more than 328,000 deaths globally. although started late, india showed ramping of more than 112,000 reports of sars-cov-2 infection including more than 3,400 deaths, as of may 20 th , 2020. as compared to the other recent coronavirus epidemics sars, 2003 and mers (middle east respiratory syndrome), 2012 having mortality rates of 11% [4] and 35% [8] , respectively, sars-cov-2 has a current mortality rate of about 3.4% [7] . however, the transmissibility of sars-cov-2 recorded far greater than the previous coronavirus epidemics. sars-cov-2 has a positive-sense 29.9 kbp rna genome, encoding 12 open reading frames (orfs) [6] . it codes for four structural proteins, namely spike glycoprotein (s), envelope glycoprotein (e), membrane glycoprotein (m), and nucleocapsid protein (n) [12] . s protein is a large membrane protein which helps in endocytosis of the viral particle into the host cell by binding to the angiotensin-converting enzyme 2 (ace2) receptor [15] . e protein is a small viral transmembrane protein which helps in the assembly of the virions by forming ion channels inside the host cell [5] . m protein is the most abundant protein present on the viral membrane, important for morphogenesis and viral assembly [2] . n protein is responsible for the packaging of the viral genome and plays an important role in viral assembly [13] . the genome also codes for 2 overlapping polyproteins, namely polyprotein 1a (pp1a) and polyprotein 1ab (pp1ab); pp1a being a truncated version of pp1ab. these code for 16 non-structural proteins (nsps) including proteases (3c-like protease and papain-like protease) and rna-processing enzymes like rnadependent rna polymerase, helicase, 3'-5' exonuclease, endoribonuclease, guanine n7methyltransferase, 2'o-ribose methyltransferase and adp ribose phosphatase [12] . in addition to structural and nsps, sars-cov-2 genome also codes for at least two other viroporin candidates (other than the e protein), namely orf3a and orf8 [3] . the function and the possible contribution of other orfs in the viral infection are largely unknown. there is a global race to sequence the sars-cov-2 to understand its variability among the population and identify the unique mutation. gujarat biotechnology research centre (gbrc), gujarat india, actively involved in the complete genome sequencing of sar-cov-2 strains from india. more than 144 complete genome sequences of sars-cov-2 strains from the state have been completed. the samples were collected from the covid-19 testing center following the guidelines of indian council of medical research. the complete genome sequencing of the samples were carried out by the gene specific primer sets using the ion s5™ next-generation sequencing system (thermo fisher scientific, usa) following manufacturer's protocol. in this study, we are reporting the unique mutations in the sars-cov-2 genome isolates from india. the two samples from a male and female aged 66 (husband and wife) has been procured from the covid-19 testing facility in gujarat, india. husband and wife contracted infection from their son who got it from local/community transmission. both were recovered without having severe symptoms of covid-19. the complete genome sequence analysis of sars-cov-2 isolated from both the patients showed a size of 29902 bp each (accession numbers mt435081 and mt435082), in contrast with 29903 bp size of the wuhan strain. however, the single nucleotide deletions in the genome of virus isolates from these two patients were at different positions. furthermore, the sequence revealed a total of ten mutations each across the genome as compared to sars-cov-2 strain from wuhan. the details of the mutations in the indian sars-cov-2 isolates areprovided in table 1 . the genome sequence of sars-cov-2 from the virus isolates of two patients in gujrat, india, were analyzed and compared with the isolate from wuhan, china. the single nucleotide change in the viral genome could change the encoding amino acid, which might result in the conformational change in the viral structure protein [11] . the genome of virus isolates from both patients showed a point mutation of c241t corresponding to its genome length and lies in the 5' untranslated region (5' utr). it is unlikely that this mutation has any substantial effect on viral replication. the nucleotide change of c1059t in the protein orf1ab has directed threonine, a polar uncharged amino acid, to mutate into a hydrophobic amino acid, isoleucine. threonine residues are well known to form hydrogen-bond interactions with surrounding polar residues in the interior of protein structure. the amino acid residues around this threonine at position 265 corresponding to the polyproteinorf1ab correspond to 262 k-f-d-t-f-n-g-e-c-p 271 suggesting that threonine might form hydrogen bonds with surrounding polar residues. perhaps it might also assist in the formation of protein bend caused due to glycine and proline having the position of i, i+3 with respect to each other. isoleucine being hydrophobic might disturb any conformational protein bend due to its inability to form a hydrogen bond. this mutation might also result in increased hydrophobic interactions with surrounding phenylalanine residues. since very little is known about nsp2 or any of its homologous structure, no assertion at the structural level could be made. in the native form, nsp12 is bound with the cofactors nsp7 and nsp8 to form the rna dependent rna polymerase (rdrp) complex. the mutation of c14408t at nsp12 in indian isolates of sars-cov-2 lead to the substitution of proline with threonine. the proline residue at position 323 corresponding to the polyprotein results in an unusual turn in the alpha-helix in which it resides [14] . interestingly, the α -helix is one of the sites for the interaction of nsp12 with the cofactor, nsp8. however, this mutation of proline causes significant changes into the structure due to the disturbance of that turn in that α -helix (fig. 1a) . this would probably change the entire structure of that interacting site, leading to the decreased affinity of rdrp to nsp8 which likely disturbs its functioning. mutation study in the 3-d structure of nsp12 ( mutation of a23403g causes the substitution of aspartic acid with glycine. this mutation in the s protein lies in the small loop turn between two anti-parallel beta-strands. these betastrands participate actively in the trimerization of spike protein which is vital for binding of s protein to its receptor ace2 [14] . replacing the aspartic acid residue with the much more flexible glycine might end up in disturbing the β -strands. prediction of effect of the mutation on the secondary structure (fig. 2 a) suggests that this mutation is likely to disrupt one of the four beta-strands in that region, thus possibly inhibiting the trimerization of spike protein on the surface of the virus. this could significantly decrease the affinity of s protein to ace2 receptors as well. as a result of the mutation of g25563t, glutamine gets substituted into histidine in the (fig. 2 b) suggests that the c-terminal of this protein (70-118 amino acids) in the wild-type orf8 protein was found to have four uniformly-spaced beta-strands. however, this mutation was found to essentially chop off the fourth beta-strand. the highly organized secondary structure of the c-terminal of the protein is likely to have some function in the viral replication or infectivity. this mutation truncating the fourth beta-strand probably disturbs its function. thus, this mutation might be severely affecting the functionality of the orf8 protein. we also found this same mutation in the virus isolate from an unrelated individual suggesting that this mutation might be relatively common. this result has a striking similarity with that of an in-vivo experiment reported earlier on sars cov-1 where a 29-nucleotide deletion in the orf8 gene was the most obvious genetic change in the early stage of human-to-human transmission of sars cov-1 [9] . moreover, the 29-nucleotide deleted sars cov-1 strain had a 23-fold less viral replication as compared to its wild type, suggesting that this mutation effectively attenuated the virus. the conspicuous similarity with our result might shed light into the attenuation of the indian strain of sars-cov-2 in humans. it would be interesting to further explore its possibility for vaccine development. interestingly, the mutation in orf8 was found when a at 28254 th position was found to be deleted. however, another stop codon was formed 12 nucleotides farther due to this can compromise the glycosylation process. presence of glycine and proline residues around this region is expected to give the structure kink and expose these residues for a potential o-linked glycosylation process. hence, this mutation of serine into isoleucine is expected to heavily inhibit and compromise this modification in this region. the authors declare no conflict of interest. zhang h, penninger jm, li y, zhong n, slutsky as (2020) angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target. intensive care med 46:586-590 comparison between the predicted secondary structure of wild-type orf8 protein (upper) and e110stop mutated orf8 protein (lower). the e110stop mutation essentially truncates off the last beta-strand (110 th to 118 th amino acid) in the secondary structure of the protein. the other column includes, protein pertaining to the mutation site (protein annotation), reference severe acute respiratory syndrome coronavirus 2 (sars-cov-2): an overview of viral structure and host response role of severe acute respiratory syndrome coronavirus viroporins e, 3a, and 8a in replication and pathogenesis sars: prognosis, outcome and sequelae in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel the architecture of sars-cov-2 transcriptome severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity attenuation of replication by a 29 nucleotide deletion in sars-coronavirus acquired during the early stages of human-to-human transmission formation and transfer of disulphide bonds in living cells a new integrated symmetrical table for genetic codes a new coronavirus associated with human respiratory disease in china evidence for gastrointestinal infection of sars-cov-2 structural basis for the recognition of sars-cov-2 by full-length human ace2 we would like to thank the department of science and technology, government of gujarat for financial support. nucleotide at the same position in wuhan sars-cov-2 sequence, the mutated nucleotide (allele nucleotide), and the corresponding amino acid change due to the point mutation (amino acid mutation) in the full length genome sequence of the indian virus isolates. protein annotation key: cord-310624-3kojrkz7 authors: flores-alanis, alejandro; sandner-miranda, luisa; delgado, gabriela; cravioto, alejandro; morales-espinosa, rosario title: the receptor binding domain of sars-cov-2 spike protein is the result of an ancestral recombination between the bat-cov ratg13 and the pangolin-cov mp789 date: 2020-08-27 journal: bmc res notes doi: 10.1186/s13104-020-05242-8 sha: doc_id: 310624 cord_uid: 3kojrkz7 objective: in december 2019 a novel coronavirus (sars-cov-2) that is causing the current covid-19 pandemic was identified in wuhan, china. many questions have been raised about its origin and adaptation to humans. in the present work we performed a genetic analysis of the spike glycoprotein (s) of sars-cov-2 and other related coronaviruses (covs) isolated from different hosts in order to trace the evolutionary history of this protein and the adaptation of sars-cov-2 to humans. results: based on the sequence analysis of the s gene, we suggest that the origin of sars-cov-2 is the result of recombination events between bat and pangolin covs. the hybrid sars-cov-2 ancestor jumped to humans and has been maintained by natural selection. although the s protein of ratg13 bat cov has a high nucleotide identity with the s protein of sars-cov-2, the phylogenetic tree and the haplotype network suggest a non-direct parental relationship between these covs. moreover, it is likely that the basic function of the receptor-binding domain (rbd) of s protein was acquired by the sars-cov-2 from the mp789 pangolin cov by recombination and it has been highly conserved. in the last 20 years, six coronaviruses (covs) causing respiratory disease in humans have been detected, namely, hcov-229e, hcov-oc43, sars-cov, hcov-nl63, hcov-hku1, and mers-cov, all of which have a zoonotic origin. genetic analyzes suggest that hcov-229e, hcov-nl63, sars-cov and mers-cov may originate from covs found in bats, while hcov-oc43 and hcov-hku1 may have its origin in covs found in rodents. some of these viruses have intermediate hosts between their natural original hosts and humans. hcov-oc43 could be transmitted by cattle, hcov-229e and mers-cov by camelids, and sars-cov by civets [1, 2] . in late 2019, a seventh cov (sars-cov-2) was identified as the cause of coronavirus disease 2019 (covid-19) characterized by fever, cough and dyspnea with more severe disease leading to pneumonia and respiratory distress syndrome [3] . genomic analyzes suggest that this new virus originated from bats covs, specifically from ratg13 bat cov, since these covs share high level of genomic similarity (96.2%) [4, 5] . however, pangolins have also been suggested as natural reservoirs of sars-cov-2 due to theirs genomic similarities that range between 85% and 92% [6] [7] [8] . viruses, like other pathogenic microorganisms, are subject to different evolutionary forces that allow them to adapt and jump from one host to another. mutation, gene flow and recombination generate genetic variation that is maintained or removed by natural selection and gene drift. one of the key factors in the process of adaptation by covs to different species is their ability to infect cells of their new host. covs are able to infect cells through a membrane glycoprotein called spike (s) [1] . this protein contains an amino-terminal subunit 1 (s1) and a carboxyl-terminal subunit 2 (s2) [9] . the s1 has a receptor-binding domain (rbd) that allows the virus to bind to different receptors on cells of its different hosts. for example, mers-cov binds to dipeptidyl dipeptidase 4 (dpp4) [10] , while sars-cov and sars-cov-2 bind to the receptor for the angiotensin-converting enzyme 2 (ace2) [4, 11] . although these last two viruses bind to the same receptor, their rbds present variations in the amino acids involved in the ace2 recognition site [12] . previous reports show that protein s is one of the most variable regions of the sars-cov-2 genome that is under natural selection and where recombination signals have been recorded [5, 7, 13] , suggesting that this protein changes constantly and plays an important role in adapting to humans. in our study, we performed a genetic analysis of the protein s of sars-cov-2 and related covs isolated from different hosts in order to trace the evolutionary history of protein s and the adaptation of sars-cov-2 to humans. the sequences of the glycoprotein spike gene from 76 covs isolated from different hosts and 148 clinical isolates of sars-cov-2 were retrieved from ncbi's gen-bank [14] and gisaid [15] databases (additional file 1). the most representative sequence, the china wuhan h1 sequence, was used as a reference sequence in the figures of this study. the sequences were aligned and analyzed with mafft v7.3 [16] and edited using bioedit v7.2 [17] and jalview v2.11 [18] . the nucleotide substitution model of the sequence set was determined with the j model-test2 program [19] and a phylogenetic tree was obtained using the mr. bayes program [20] under a general time-reversible substitution plus proportion of invariable sites and rate of variation across sites (gtr + i+ g). we ran 5 markov chains monte carlo for 500,000 generations and discarded 25% of the initial trees. the consensus tree was edited using the figtree v1.4.3 program [21] . a haplotype network was built using the popart v1.7 program [22] . nucleotide similarity analysis was carried out using the simplot v3.5 program with a 100 bp window at 30 bp step and kimura 2-parameter model [23] . the dnasp v5.1 program [24] was used to perform the mcdonald-kreitman (mk) test. phylogenetic analysis defined two large clades (a and b). within clade a, 5 clusters belonging to the betacoronavirus (βcov) genus and 2 clusters belonging to the gammacoronavirus (γcov) genus were detected (fig. 1a) . the βcov cluster a7 corresponds to human sars-cov-2 (148 analyzed sequences), 6 pangolin covs (manis javanica) (mp789, pcov_gx_p5l, p5e, p1e, p4l and p2v) and 2 bat covs (ratg13 and rmyn02; rhinolophus affinis and r. malayanus, respectively). within this cluster, we found that sars-cov-2 is genetically related to cov ratg13, and both share a common ancestor with cov mp789 (fig. 1a ). this result agrees with previous analyzes made with the complete genome and with s protein [7, 25] . since we did not find any phylogenetic incongruence reflected on the tree, suggesting a lack of recombination between clusters, we focused more specifically on the cluster where sars-cov-2 was found (a7) (fig. 1a) . we analyzed the genealogical relationships between the covs that comprise cluster a7. the haplotype network showed the formation of a loop between the group of pangolins and 4 hypothetical ancestral haplotypes (fig. 1b) , suggesting recombination within this cluster. furthermore, the network suggests that 4 of the isolates analyzed here (sars-cov-2, ratg13, mp789 and rmyn02) diverged from these 4 hypothetical ancestors (fig. 1b) . despite the fact that sars-cov-2 and ratg13 cov share a genomic nucleotide identity of 96.2% [4] , and an s gene nucleotide identity of 93.15% [7] , the divergence showed in the phylogenetic tree and in the haplotype network rules out a direct parental relationship between these two isolates ( fig. 1a and b) . in 2019, various pangolin covs were isolated, among which the isolate mp789 cov is the most interesting because it shares a nucleotide similarity of 85%-92% with sars-cov-2, and 90% with ratg13 cov [7] . the similarity analysis of the s nucleotide sequences of cluster a7 shows a mosaic similarity pattern across the s gene between sars-cov-2, ratg13, mp789 and rmyn02, which suggests a probable ancestral genetic exchange between the 4 hypothetical ancestors of these covs (fig. 1c) . the most notable differences between sars-cov-2 and the rest of the covs s gene were found in the rbd, indicating a hybrid zone between ratg13 and mp789 covs in this region (fig. 1c) . this result suggests a probable ancestral cross-species recombination between bat and pangolin covs. s protein is thought to be under natural selection and plays an important role in cross-species transmission [5, [26] [27] [28] . a recent study reported negative selection in the s gene when sars-cov-2 was compared with ratg13 and a group of pangolin covs [26] . we performed an mk test between sars-cov-2, ratg13 and mp789, the results of which showed that between sars-cov-2 and ratg13 cov there were more synonymous (ds) than nonsynonymous (dn) substitutions, indicating negative selection (ni > 1). whereas, between sars-cov-2 and mp789 cov the contrary was found, indicating positive selection (ni < 1) ( table 1 ). the negative selection predicted for sars-cov-2 is due to its high similarity to ratg13 cov, therefore, the fixation of dn substitutions are not favored. on the other hand, the incongruences found in the pangolin cov results compared to a previous study [26] could be due to differences in the strategies and methods used. the s protein rbd plays a key role during the infection process of sars-cov-2 to human cells because it contains the six amino acids (l455, f486, q493, s494, n501 and y505) that are essential for efficient binding of sars-cov-2 to ace2 [12] . sars-cov-2 rbd shows a closer similarity to mp789 cov rbd (96.8% homology) than to ratg13 cov rbd (89.56% homology) [7] . interestingly, we found that 26 of 33 dn substitutions between sars-cov-2 and ratg13 cov were located in the rbd, while 7 of 505 dn substitutions between sars-cov-2 and mp789 cov were also located in the rbd (table 1) . this indicates that in this region, mp789 cov has suffered less dn changes than ratg13 cov when compared with sars-cov-2. since only one polymorphism was detected in rbd in the 148 sars-cov-2 sequences, the mk test did not determine any value, suggesting that this is a highly conserved region. the comparison between sars-cov-2 and mp789 cov rbd shows that they share the 6 amino acids that are essential for binding to ace2 receptor, while in ratg13 cov these amino acids are missing (fig. 2) . these results could indicate that both the pangolin and humans have similar ace2 at the interacting domain with s protein, as reported by others [29, 30] . as a consequence, the ace2 binding sites and the region in general should be conserved (70% homology), being sufficient for the interaction to take place. a genetic feature that makes sars-cov-2 more infectious is the fact that the s protein harbors an insert of 12-nucleotides between the s1 and s2 subunits that encode for a polybasic cleavage site (rrar) that is recognized by furins (fig. 2) . this cleavage site is related with an increased efficiency of entry during infection [31, 32] . nevertheless, this insertion is not present in all betacoronaviruses, like in sars-cov [13, 31, 33] . however, the human hku1 cov and mers-cov have variants of polybasic insertions that are also recognized by furins [34] [35] [36] . the presence of these polybasic insertions have been seen to increase the pathogenicity of viruses, such as in avian influenza [37] [38] [39] , mers-like cov [40] , and in bovine cov [41] . we also found that rmyn02 cov has an insert in the same position as that in sars-cov-2, but it is not a polybasic cleavage insertion (-aar). there have been suggestions that instead, it could be the product of recombination between wild bat covs [13] . on the other hand, several experiments have shown that this polybasic cleavage site is acquired and fixed during the serial passage of covs in cell cultures or in animals [37, 42] . the aforementioned leads to two possible explanations for the polybasic cleavage insertion in sars-cov-2 and the role in its adaptation to humans: (1) the ancestor of sars-cov-2 acquired it in a host, went through a recombination process in an unidentified intermediary host, and then jumped to humans, or (2) it was acquired in humans during a cycle of human to human transmission that helped its adaptation and virulence process. rambaut et al. [43] determined that the most recent common ancestor of sars-cov-2 appeared in november 2019 and proposes that the virus had enough time to acquire the insert during transmission between humans. whether the ancestral cov that gave rise to sars-cov-2 came from a bat or a pangolin is still not yet known, but based on the analysis of the s gene performed in this study, we suggest that it is more likely to have come from a bat. however, the region essential for human ace2 (rbd) binding is a hybrid between ratg13 and mp789 covs, and it is likely that the basic function of the rbd was acquired by the sars-cov-2 from the mp789 cov by recombination with an ancestral cov, which had a ratg13 genomic background. subsequently, the sars-cov-2 ancestor jumped to humans where the s protein has been maintained by positive selection and where the rbd has been highly conserved. this illustrates the complexity of cov cross-species infection dynamics and the relevance of genetic exchange between covs. in the future, molecular epidemiological surveillance studies in wild isolates will be vital for identifying genetic changes in viruses that could result in novel adaptations to humans and thereby, enabling the development of another pandemic, such as the one we are currently experiencing. here we mentioned two characteristics in s protein as key factors in the adaptation and infectivity of sars-cov-2, the presence of six amino acids essentials for binding to ace2 receptor and the polybasic cleavage insert. nevertheless, the knowledge about this virus has increased very fast and new possible features involved in virus adaptation to human beings has been reported. to fully understand the origin and adaptation of this virus, it would be necessary to encourage the extensive sampling of wild animals associated with coronaviruses and perform deeper and wider genomic analyses. supplementary information accompanies this paper at https ://doi. org/10.1186/s1310 4-020-05242 -8. additional file 1: table s1 . information of the sequences of sars-cov-2 and other covs used in this study. accession number, isolate, origin, date of collection, host, host common name, sequence size and data source of the spike gene sequences used in this study. zoonotic origins of human coronaviruses origin and evolution of pathogenic coronaviruses clinical features of patients infected with 2019 novel coronavirus in wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov-2 identifying sars-cov-2 related coronaviruses in malayan pangolins are pangolins the intermediate host of the 2019 novel coronavirus (sars-cov-2)? probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak cryo-em structure of the 2019-ncov spike in the prefusion conformation cross host transmission in the emergence of mers coronavirus structural biology: structure of sars coronavirus spike receptor-binding domain complexed with receptor receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus a novel bat coronavirus closely related to • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year • at bmc sars-cov-2 contains natural insertions at the s1/s2 cleavage site of the spike protein ncbi sars-cov-2 resources-ncbi disease and diplomacy: gisaid's innovative contribution to global health parallelization of the mafft multiple sequence alignment program bioedit: a user-friendly biological sequences alignment editor and analysis program for windows 95/98/nt sequence analysis jalview version 2-a multiple sequence alignment editor and analysis workbench jmodeltest 2: more models, new heuristics and parallel computing software for systematics and evolution mrbayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space tcs: a computer program to estimate gene genealogies full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination dnasp v5: a software for comprehensive analysis of dna polymorphism data on the origin and continuing evolution of sars-cov-2 emergence of sars-cov-2 through recombination and strong purifying selection recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission computational inference of selection underlying the evolution of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 broad and differential animal ace2 receptor usage by sars-cov-2 a multibasic cleavage site in the spike protein of sars-cov-2 is essential for infection of human lung cells a unique protease cleavage site predicted in the spike protein of the novel pneumonia coronavirus (2019-ncov) potentially related to viral transmissibility. virologica sinica broad and differential animal ace2 receptor usage by sars-cov-2 the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade spike protein, s, of human coronavirus hku1: role in viral life cycle and application in antibody detection proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism generation of a highly pathogenic avian influenza a virus from an avirulent field isolate by passaging in chickens emergence of a highly pathogenic avian influenza virus from a low-pathogenic progenitor genetic predisposition to acquire a polybasic cleavage site for highly pathogenic avian influenza virus hemagglutinin trypsin treatment unlocks barrier for zoonotic bat coronavirus infection comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia the role of viral population diversity in adaptation of bovine coronavirus to new host environments novel 2019 coronavirus/ncov-2019 genomic epidemiology-virological publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions rme, afa and lsm contributed to the study design; afa performed the genetic and phylogenetic analyses and the interpretation of results; afa, lsm and gd wrote the manuscript. rme and ac did the critical review of the manuscript. all authors read and approved the final manuscript. this work was supported by dgapa-papiit grant in213816. the datasets used and/or analyzed during the current study are available in the additional file 1. not applicable, because neither animals nor humans were used for this study. not applicable. the authors declare that they have no competing interests.received: 1 july 2020 accepted: 19 august 2020 key: cord-322129-uyswj4ow authors: melin, amanda d.; janiak, mareike c.; marrone, frank; arora, paramjit s.; higham, james p. title: comparative ace2 variation and primate covid-19 risk date: 2020-10-27 journal: commun biol doi: 10.1038/s42003-020-01370-w sha: doc_id: 322129 cord_uid: uyswj4ow the emergence of sars-cov-2 has caused over a million human deaths and massive global disruption. the viral infection may also represent a threat to our closest living relatives, nonhuman primates. the contact surface of the host cell receptor, ace2, displays amino acid residues that are critical for virus recognition, and variations at these critical residues modulate infection susceptibility. infection studies have shown that some primate species develop covid-19-like symptoms; however, the susceptibility of most primates is unknown. here, we show that all apes and african and asian monkeys (catarrhines), exhibit the same set of twelve key amino acid residues as human ace2. monkeys in the americas, and some tarsiers, lemurs and lorisoids, differ at critical contact residues, and protein modeling predicts that these differences should greatly reduce sars-cov-2 binding affinity. other lemurs are predicted to be closer to catarrhines in their susceptibility. our study suggests that apes and african and asian monkeys, and some lemurs, are likely to be highly susceptible to sars-cov-2. urgent actions have been undertaken to limit the exposure of great apes to humans, and similar efforts may be necessary for many other primate species. i n late 2019 a novel coronavirus, sars-cov-2, emerged in china. in humans, this virus can lead to the respiratory disease covid-19, which can be fatal 1, 2 . since then, sars-cov-2 has spread around the world, causing widespread mortality, and with major impacts on societies and economies. while the virus and its resulting disease represent a major humanitarian disaster, they also represent a potentially existential risk to our closest living relatives, the nonhuman primates. transmission incidences of bacteria and viruses-including another coronavirus (h-cov-oc43)-from humans to wild populations of nonhuman primates have previously been linked to outbreaks of ebola, yellow fever, and fatal respiratory diseases, leading in some cases to mass mortality [3] [4] [5] [6] [7] [8] [9] . such past events raise considerable concerns among the global conservation community with respect to the impact of the current pandemic 10 . infection studies of rhesus monkeys, long-tailed macaques, and vervets as biomedical models have made it clear that at least some nonhuman primate species are permissive to sars-cov-2 infection and develop symptoms in response to infection that resemble those of humans following the development of covid-19, including similar age-related effects [11] [12] [13] [14] [15] [16] . recognizing the potential danger of covid-19 to nonhuman primates, the international union for the conservation of nature (iucn), together with the great apes section of the primate specialist group, released a joint statement on precautions that should be taken for researchers and caretakers when interacting with great apes 17 . however, the risk for many primate taxa remains unknown. here we begin to assess the potential likelihood that our closest living relatives are susceptible to sars-cov-2 infection. while the biology underlying susceptibility to sars-cov-2 infection remains to be fully elucidated, the viral target is well established. the sars-cov-2 virus binds to the cellular receptor protein angiotensin-converting enzyme-2 (ace2), which is expressed on the extracellular surface of endothelial cells of diverse bodily tissues, including the lungs, kidneys, small intestine, and renal tubes 18 . ace2 is a carboxypeptidase whose activities include regulation of blood pressure and inflammatory response through its role in cleaving the vasoconstrictor angiotensin ii to produce angiotensin 1-7 and triggering varied downstream responses [19] [20] [21] [22] . ace2 is made up of a signal sequence at the n terminus (residues 1-17), a transmembrane sequence at the c terminus (residues 741-762), and an extracellular region, which contains a zinc metallopeptidase domain (residues 19-611) and a collectrin homolog (residues 612-740) 23, 24 . characterizations of the infection dynamics of sars-cov-2 have demonstrated that the binding affinity for the human ace2 receptor is high, which is a key factor in determining the susceptibility and transmission dynamics. when compared to sars-cov, which caused a serious global outbreak of the disease in 2002-2003 25, 26 , the binding affinity between sars-cov-2 and ace2 is estimated to be between fourfold 27-30 and 10-to 20-fold greater 31 . recent reports describing structural characterization of ace2 in complex with the sars-cov-2 spike protein receptorbinding domain (rbd) [27] [28] [29] [30] allow identification of the key binding residues that enable the host-pathogen protein-protein recognition. following the initial binding of the virus to the ace2 receptor, humans experience a great deal of variation in response to infection, with some individuals experiencing relatively mild symptoms, while others experience major breathing problems and organ failures, which can lead to death. some of this response is known to be linked to variation in how the immune system responds to infection, with some individuals experiencing a hyperinflammatory 'cytokine storm', which in turn aggravates respiratory failures and increases mortality risk 32, 33 . there may also be some variation among humans in initial susceptibility to infection, such that approaches examining variation in ace2 tissue expression and gene sequences can offer insight into variation in human susceptibility to covid-19 [34] [35] [36] [37] . similarly, we can use such an approach to compare sequence variation across species, and hence try to predict the likely interspecific variation in susceptibility to initial infection. previous analysis of comparative variation at these sites enabled estimates of the affinity of the ace2 receptor for sars-cov in nonhuman species (bats) 38 . here, we undertake such an analysis for sars-cov-2 across the primate radiation. our aim is to investigate the likelihood of initial susceptibility to infection for different major radiations and species while recognizing that downstream processes such as immune responses are likely to determine the extent to which species and individuals develop symptoms and pathologies in response to infection. we compiled ace2 gene sequence data from 29 primate species for which genomes are publicly available, covering primate taxonomic breadth. for comparison, we assessed 4 species of other mammals that have been tested directly for sars-cov-2 susceptibility in laboratory infection studies 39 . we also included in our analysis the amino acid sequence variation at these sites for horseshoe bats, thought to be the original vector of the virus, and pangolins, a potential intermediate host, where viral recombination may have led to the novel viral form sars-cov-2 40 . we assessed the variation at amino acid residues identified as critical for ace2 recognition by the sars-cov-2 rbd and undertook an analysis of positive selection and protein modeling to gauge the potential for adaptive differences and the likely effects of protein variation. our aim was to develop predictions about the susceptibility of our closest living relatives to sars-cov-2 as a resource for stakeholders, including researchers, caretakers, practitioners, conservationists, and governmental and non-governmental agencies. variation in ace2 sequences. the ace2 gene (2418 bp) and translated protein (805 amino acids) sequences are strongly conserved across primates. the average pairwise identity across 29 primate species is 93.6% for the ace2 nucleotide sequence and 90.8% for the protein sequence, with a pairwise similarity (blosum62 ≥ 1) of 95.3% (supplementary data 1-3). out of 2418 bp, 1631 bp (67.5%) are identical, while 401 bp (16.58%) are phylogenetically-informative sites for primates, and gene trees we generated ( supplementary fig. s1a , b) closely recapitulate the currently accepted phylogeny of primates ( fig. 1 ). in particular, the twelve sites in the ace2 protein that are critical for binding of the sars-cov-2 virus are invariant across the catarrhini, which includes great apes, gibbons, and monkeys of africa and asia (fig. 1) . furthermore, catarrhines do not vary at any of the 21 sites identified by alanine scanning (supplementary table s1 and supplementary fig. s2 ). the other major radiation of monkeys, those found in the americas (platyrrhini), have ace2 sequences that are less similar to humans across the length of the protein (91.68-92.55% identical to h. sapiens, supplementary data 2) but conserved within their clade (average pairwise identity 97.2%, supplementary data 2). they share nine of twelve critical amino acid residues with catarrhine primates; the three sites that vary from catarrhines, h41, e42, and t82, are conserved within the platyrrhines. strepsirrhine primates and tarsiers, were more variable in the binding sites and less similar to the human protein across the length of the sequence (81.86-86.93% pairwise identity, supplementary data 2). like platyrrhines, the tarsier (carlito syrichta), mouse lemur (microcebus murinus), and galago (otolemur garnettii) have an h41 residue, while the sifaka (propithecus coquereli), aye-aye (daubentonia madagascariensis), and the blue-eyed black lemur (eulemur flavifrons) have the same allele as humans and other catarrhines, y41. in non-primate mammals, a higher number of amino acid substitutions are evident (77.37-85.22% pairwise identity to h. sapiens, supplementary data 2), including at critical binding sites. all species possess a different residue to primates at site 24. bats are exceptionally variable within the binding sites, with the genus rhinolophus alone encompassing all of the variation seen in the rest of the non-primate mammals. where primates have glutamine (q24), bats have glutamate (e24), lysine (k24), leucine (l24), or arginine (r24) (fig. 1 ). all fasta alignments of ace2 gene and protein sequences are available in supplementary data 4-7, a full-length protein alignment is also shown in supplementary fig. s2 , and distance matrices are provided in supplementary data 1-3. analysis of species-specific residues on ace2-rbd interactions. the ace2 receptors of all catarrhines have identical residues to humans at the rbd/ace2 binding interface across all 12 critical sites, and are predicted to have a similar binding affinity for sars-cov-2. platyrrhines diverge from catarrhines at three of the twelve critical amino acid residues. compared to catarrhine ace2, the platyrrhines' ace2 is predicted to bind sars-cov-2 rbd with a roughly 400-fold reduced affinity (δδg bind = 3.5 kcal/mol) ( table 1 ). in particular, the change at site 41 from y to h found in monkeys in the americas has the largest impact of any residue change examined (table 2) , which alone is predicted to lead to a 25-fold decrease in the binding affinity to sars-cov-2 ( fig. 2 ). this single mutation combined with additional substitutions, especially q42e, found in platyrrhines is predicted to substantially reduce the likelihood of successful viral binding ( table 2) . of the other primates modeled, two of the three strepsirrhines, and tarsiers, also have the h41 residue and furthermore have additional protein sequence differences leading to further decreases in predicted binding affinity. the predicted fig. 1 ace2 protein sequence alignment and evolutionary relationships of study species. branch lengths represent the evolutionary distance (time, in millions of years) estimated from timetree 63 . we outline amino acid residues at critical binding sites for the sars-cov-2 spike receptor-binding domain. solid outlines highlight sites predicted to have the most substantial impact on viral binding affinity. notably, protein sequences of catarrhine primates are highly conserved, including uniformity among amino acids at all binding sites. primate species that are able to be successfully infected with covid-19 are indicated in red. predicted susceptibility to covid-19 for other primates is additionally coded by terminal branch colors. we use the nomenclature cebus capucinus to be consistent with the species name used in the genome annotation but note the recent adoption of cebus imitator for this species. silhouettes are from phylopic.org and available under the public domain dedication 1.0 license, with the exception of cebus (sarah werning; creative commons attribution 3.0 unported). binding affinity of tarsier ace2 is the most dissimilar to humans and this primate might be the least susceptible of the species we examine. in contrast, coquerel's sifaka (propithecus coquereli), the aye-aye (daubentonia madagascariensis), and a blue-eyed black lemur (eulemur flavifrons) share the same residue as humans and other catarrhines at site 41 and have projected affinities that are near to humans (table 2 ). other mammals included in our study -ferrets, cats, dogs, pigs, pangolin, and two of the seven bat species (r. pusillus and r. macrotis) -show the same residue as humans (y) at site 41, with accompanying strong affinities for sars-cov-2. the remaining five sister species of bats possess h41 and lower binding affinities (table 2) . adaptive evolution of ace2 sequences. we find evidence that the selective pressures acting on ace2 are not equivalent across the major clades in our analysis. the codeml clade model c provided a better fit than the null model (lrt = 26.726, p < 0.001; table 3, supplementary table s3) (table 3 ). in catarrhines, the three positively selected sites identified by beb calculations are not near the binding sites for sars-cov-2 (residues 249, 653, and 658; table 3 ). our results strongly suggest that catarrhines -all apes, and all monkeys of africa and asia, are likely to be susceptible to infection by sars-cov-2. there is high conservancy in the protein sequence of the target receptor, ace2, including uniformity at all identified and tested major binding sites. indeed, even among the 21 residues identified in our full list of potential binding points, catarrhines are invariant (supplementary table 1 residues between platyrrhines and catarrhines, and two of these, h41y and e42q show strong evidence of being impactful changes. these amino acid changes are modeled to reduce the binding affinity between sars-cov-2 and ace2 by ca. 400-fold. recent clinical analysis of viral shedding, viremia, and histopathology in catarrhine (macaque) versus platyrrhine (marmoset, callithrix jacchus) responses to inoculation with sars-cov-2, show much more severe presentation of disease symptoms in the former, strongly supporting our results 16 . similar reduced susceptibility is predicted for tarsiers, and two of the five lemurs and lorisoids (strepsirrhines). what is concerning is that three of the analyzed lemurs spanning divergent lineages-the coquerel's sifaka, the aye-aye, and the blue-eyed black lemur-are more similar to catarrhines at important binding sites, including possessing the high-risk residue variant at site 41, and as such are also predicted to be susceptible. nonetheless, these are only predicted results based on amino acid residues and protein-protein interaction models. we urge extreme caution in using our analyses as the basis for relaxing policies regarding the protection of platyrrhines, tarsiers or any strepsirrhines. experimental assessment of synthetic protein interactions can now occur in the laboratory, e.g. 41 , and confirmation of our model predictions should be sought before any firm conclusions are reached. emerging evidence in experimental mammalian models appears to support our results; dogs, ferrets, pigs, and cats have all shown some susceptibility to sars-cov-2 but have demonstrated variation in disease severity and presentation, including across studies 39, 42 . substitutions at binding sites might be at least partially protective against covid-19 in these mammals. for example, the limited experimental evidence to date suggests that while cats -which have the same residue as humans at site 34-are not strongly symptomatic, they present lung lesions, while dogs-which have a substitution at this site-do not 39 . the amino acid residue at site 24 differs from primates in all other mammalian species examined. however, our models suggest that the variant residues may confer relatively minor reductions in binding affinity. other sources of variation may affect ace2 protein stability 34 . our results are also consistent with previous reports that ace2 genetic diversity is greater among bats than that observed among mammals susceptible to sars-cov-type viruses. this variation has been suggested to indicate that bat species may act as a reservoir of sars-cov viruses or their progenitors 38 . intriguingly, all but 2 bat species we examined have the putatively protective variant, h41. additionally, results of our codeml branchsite analysis support previous findings of ace2 in bats being under positive selection, including sites within the binding domain of sars-cov and sars-cov-2 43 , which may be evidence of hostvirus coevolution. sites showing evidence of positive selection within catarrhine ace2 sequences were not in or near known cov binding sites (table 3 and fig. 1 ). two (residues 653, 658) fall within the cleavage site (residues 652-659) utilized by the sheddase adam17, known to interact with ace2 44 . however, neither of the residues under selection are the amino acids targeted by adam17 45 leaving the functional significance of evolution at these sites uncertain. further clinical and laboratory study is needed to fully understand infection dynamics. there are a number of important caveats to our study. firstly, all of our predictions are based on interpretations of gene and resultant amino acid sequences, rather than based on direct assessment of individual responses to induced infection. nonetheless, the overall pattern of our results is being borne out by infection studies on a few species that are used as biomedical models. so far, all catarrhine species tested by infection studies, including rhesus macaques, long-tailed macaques, and vervet table 3 results of codeml analyses of adaptive evolution across ace2 gene sequences. monkeys 12, 16, 46 have exhibited covid-19-like symptoms in response to infection, including large lung and other organ lesions 16 and cytokine storms 12 . in contrast, marmosets did not exhibit major symptoms in response to infection 16 . while these results support and validate our findings based on ace2 sequence interpretation, the number of primate species that can and will be tested directly by infection studies will be restricted to just a handful. our study enhances this picture, by allowing inferences to be made across the primate radiation, backed up by the published infection studies on a few target model species. some of our results, such as the uniform conservation of ace2 binding sites among catarrhines, backed up by the demonstrated high susceptibility of humans and other catarrhines to sars-cov-2, should give a good degree of confidence of high levels of risk. given the identical residues of humans to other apes and monkeys in asia and africa at the target sites, it seems unlikely that the ace2 receptor and the sars-cov-2 proteins would not readily bind. our results for other taxa are dependent on modeling, hence should be treated more cautiously. this includes all interpretations of the susceptibility of platyrrhines and strepsirrhines, where the effects of residue differences on binding affinities have been estimated based on protein-protein interaction modeling. another caveat is that we have modeled only interactions at binding sites, and not predictions based on full residue sequence variation. residues that are not in direct contact may still affect binding allosterically. other factors, including proteases necessary for viral entry, and other viral targets, may also impact disease susceptibility and responses 34 . more generally, if adhering to the precautionary principle, then our results highlighting higher risks to some species should be taken with greater gravity than our results that predict potential lower risks to others. another limitation of our study is that we have looked at only 29 primate species, albeit with broad taxonomic scope. analysis of additional species is important, especially among strepsirrhine species, where our coverage is relatively scant. in particular, the residue overlap at important binding sites in the sequences of coquerel's sifaka, the aye-aye, and blue-eyed black lemur with those of catarrhines suggests many lemurs may be highly vulnerable and we underscore the need to assess a wider diversity of lemur species. furthermore, we examine only one individual per species, and intraspecific variation across populations should be considered; however, studies on intraspecific ace2 variation with humans and vervet monkeys suggest ace2 variants are low in frequency [47] [48] [49] . finally, it is also important to remember that our study assesses only the potential for the initial binding of the virus to the target site. downstream consequences of infection may differ drastically based on speciesspecific proteases, genomic variants, metabolism, and immune system responses 50, 51 . in humans, the development of covid-19 can lead to a pro-inflammatory cytokine storm of hyperinflammation, which may lead to some of the more severe impacts of infection 32, 52 . nonetheless, it is evident from the hundreds of thousands of deaths and global lockdown that humans are highly susceptible to sars-cov-2 infection, and our results suggest that all apes and monkeys in africa and asia are similarly susceptible. many endangered primate species are now only found in very small population sizes 53 . for example, there are believed to be only around 1000 mountain gorillas left in their entire range 54 . with such small populations, the introduction of a new highly infectious disease is of serious concern. re-opening access to habituated great ape groups for tourism purposes, which may be critical to local economies 55 , may be fraught with issues. iucn best practices recommend that tourists stay at least 7 meters away from great apes 56 , but in practice, almost all tourists get far closer than this -for example, the average distance that tourists get from mountain gorillas at the bwindi impenetrable national park in uganda is just 2.76 m 57 . a concerted effort may be required by all stakeholders to try to avoid the introduction of sars-cov-2 into wild primate populations 10 . recent measures suggested by the iucn for researchers and caretakers of great ape populations include: ensuring that all individuals wear clean clothing and disinfected footwear; providing hand-washing facilities; requiring that a surgical face mask be worn by anyone coming within 10 m of great apes; ensuring that individuals needing to cough or sneeze ideally leave the area, or at least cough/sneeze into the crux of their elbows; imposing a 14-day quarantine for all people arriving into great ape areas who will come into frequent close proximity with them 17 . the iucn's 'best practice guidelines for health monitoring and disease control in great ape populations' should also be followed 58 . our results suggest that dozens of nonhuman primate species, including all of our closest relatives, are likely to be highly susceptible to sars-cov-2 infection, and vulnerable to its effects. major actions may be needed to limit the exposure of many wild primate populations to humans. this is likely to require coordinated input from all stakeholders, including local communities, international and national governmental agencies, nongovernmental conservation and development organizations, and academics and researchers. while the focus of many at this time is rightly on mitigating the humanitarian devastation of covid-19, we also have a duty to ensure that our closest living relatives do not suffer from devastating infections and further population declines in response to yet another human-induced catastrophe. variation in ace2 sequences. we compiled ace2 gene sequences for 16 catarrhine primates: 4 species from all 3 genera of great ape (gorilla, pan, pongo), 2 genera of gibbons (hylobates, nomascus), and 10 species of african and asian monkeys in 7 genera (cercocebus, chlorocebus, macaca, mandrillus, papio, rhinopithecus, piliocolobus, theropithecus); 6 genera of platyrrhines (monkeys from the americas: alouatta, aotus, callithrix, cebus, saimiri, sapajus); 1 species of tarsier (carlito syrichta); and 5 genera of strepsirrhines (lemurs and lorisoids: eulemur, daubentonia, microcebus, propithecus, otolemur) (supplementary table s2 ). we also included four species of mammals that have been tested clinically for susceptibility to sars-cov-2 infection 39 , including the domestic cat (felis catus), dog (canis lupus familiaris), pig (sus scrofa), and ferret (mustela putorius furo). finally, we included the pangolin (manis javanica) and several bat species, including horseshoe bats (rhinolophus spp., hipposideros pratti, myotis daubentonii). sequences were retrieved from ncbi, either from annotations of published genomes or from genbank entries 38 . we manually checked annotations by performing tblastn searches of the human ace2 protein sequence against each genome. we identified one misannotation for exon 15 in microcebus murinus, which we manually corrected. the ace2 nucleotide sequence for alouatta palliata was obtained from an unpublished draft genome, via tblastn searches using the cebus ace2 protein sequence as a query and default search settings. accession numbers for sequences retrieved from ncbi and genbank are provided in supplementary table s2 and the alouatta palliata sequence is available in supplementary data 4. coding sequences were translated using geneious version 9.1.8 and we aligned both nucleotide and amino acid sequences with mafft 59 . amino acids were aligned with the blosum62 scoring matrix, while the 200 pam scoring matrix was used for nucleotides. a 1.53 gap open penalty and an offset value of 0.123 were used for both. we manually inspected and corrected any misalignments, and verified the absence of indels and premature stop codons. to visualize patterns of gene conservation across taxa and identify the congruence of the ace2 gene tree with currently accepted phylogenetic relationships among species, we reconstructed trees using both bayesian (mrbayes 3.2.6 60 ) and maximum likelihood (raxml 8.2.11 61 ) methods with 200,000 mcmc cycles and 1000 bootstrap replicates, respectively (code available on github 62 ). gene trees were compared to a current species phylogeny assembled using timetree 63 , which is also used to illustrate the evolutionary relationships between study species in fig. 1 . phylogenetically-informative sites along the ace2 sequence were identified with the pis function in the r package ips v. 0.0.11 64, 65 . identification of critical binding residues and species-specific ace2-rbd interactions. critical ace2 protein contact sites for the viral spike protein receptor-binding domain (rbd) have been identified using cryo-em and x-ray crystallography structural analysis methods [27] [28] [29] [30] . the ace2-rbd complex is characteristic of protein-protein interactions (ppis) that feature extended interfaces spanning a multitude of binding residues. experimental and computational analyses of ppis have shown that a handful of contact residues can dominate the binding energy landscape 66 . alanine scanning mutagenesis provides an assessment of the contribution of each residue to complex formation [67] [68] [69] . critical binding residues can be computationally identified by assessing the change in binding free energy of complex formation upon mutation of the particular residue to alanine, which is the smallest residue that may be incorporated without significantly impacting the protein backbone conformation 70 . our computational modeling utilizes the human sars rbd/ace2 high-resolution structures, and we make the implicit assumption that the overall conformation of ace2 is conserved among different species. this assumption, which is rooted in the high sequence similarity between ace2 sequences, allows us to use the structure of the complex to predict the impact of mutations at the protein-protein interface. we defined critical residues as those that upon mutation to alanine decrease the binding energy by a threshold value δδg bind ≥ 1.0 kcal/mol. nine of the 21 residues identified by alanine scanning as involved in the ace2-rbd complex met this criterion (supplementary table s1 ). there was a large congruence in the sites identified with those highlighted by other methods. each of the eight sites implicated by cryo-em 27 , were also detected by alanine modeling; five residues were ≥1.0 kcal/mol threshold and 3 were below this threshold. to be cautious, in addition to the 9 critical ace2 sites we identified through alanine scanning, we also examined residue variation at the 3 sites that fell below the ≥1.0 kcal/mol threshold but that were identified as important by structural analyses 27-30 for a total of 12 critical sites. all computational alanine scanning mutagenesis analyses were performed using rosetta software 70 . the alanine mutagenesis approach has been extensively evaluated and used to analyze ppis and design their inhibitors, including by members of the present authorship 71, 72 . we utilized the ssipe program 73 to predict how ace2 amino acid differences in each species would affect the relative binding energy of the ace2/sars-cov-2 interaction. using human ace2 bound to the sars-cov-2 rbd as a benchmark (pdb 6m0j), the program mutates selected residues and compares the binding energy to that of the original. using this algorithm, we studied interactions of all primates across the full suite of amino acid changes occurring at critical binding sites for each species. to more thoroughly assess the impact of each amino acid substitution, we also examined the predicted effect of individual amino acid changes (in isolation) on protein-binding affinity. adaptive evolution of ace2 sequences. we further investigated ace2 and how selective pressures in different clades might be shaping variation at the binding sites, using codeml clade c and branch-site models in paml 74 . we first tested if selection acting on ace2 is divergent between the major clades in our sample (platyrrhine, catarrhine, and strepsirrhine primates, non-primate mammals) with the codeml clade model c, which was compared to the null model (m2a_rel) with a likelihood ratio test 75 . this test shows whether there is a divergent selection (dn/ds ratio = ω) across all clades, but not which clades are experiencing positive selection. we, therefore, followed the clade model with a series of branch-site models, which allow one clade at a time to be designated as a set of "foreground" branches and test whether this clade has experienced episodes of positive selection compared to the remaining sets of "background" branches (ω foreground > ω background ). branchsite models are compared to a null model that fixes ω at 1 with a likelihood ratio test. in the case of the alternative model having a significantly better fit than the null model, indicating positive selection, potential sites under positive selection are identified with a bayes empirical bayes (beb) approach 76 . we completed branch-site models for each primate clade (platyrrhine, strepsirrhine, and catarrhine), as well as bats because previous research has identified ace2 to be under positive selection in this clade, potentially in response to coronaviruses 43 . we had to exclude hipposideros pratti and myotis daubentonii from paml analyses, because only a partial ace2 sequence was available for these two species. input files and control files for paml codeml analyses are available in the github repository 62 . statistics and reproducibility. models in paml were compared with likelihood ratio tests and evaluated for significance with a right-tailed chi-squared distribution. as this was a comparative study of gene sequences across species, we had one representative individual for each species (n = 41) and no replicates. reporting summary. further information on research design is available in the nature research life sciences reporting summary linked to this article. nucleotide and protein sequences used in this study are available from ncbi and are also available as fasta files (supplementary data 4 and 5) and alignments (supplementary data 6 and 7) in the supplemental material. accession numbers are provided in supplementary table s2 . all code used in this project is available via a github repository (https://github.com/ mareikejaniak/ace2). the version of the repository used for this project has been archived in zenodo (doi: 10.5281/zenodo.4018807) 62 . received: 26 august 2020; accepted: 8 october 2020; a novel coronavirus from patients with pneumonia in china emergence of a novel human coronavirus threatening human health impact of yellow fever outbreaks on two howler monkey species (alouatta guariba clamitans and a. caraya) in misiones, argentina ebola outbreak killed 5000 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and disease control in great ape populations. occasional papers of the iucn species survival commission no mafft multiple sequence alignment software version 7: improvements in performance and usability mrbayes: bayesian inference of phylogenetic trees raxml version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies mareikejaniak/ace2: code for primate ace2 project timetree: a resource for timelines, timetrees, and divergence times r: a language and environment for statistical computing (r foundation for statistical computing interfaces to phylogenetic software in r a hot spot of binding energy in a hormonereceptor interface anatomy of hot spots in protein interfaces computational alanine scanning to probe protein-protein interactions: a novel approach to evaluate binding free energies a simple physical model for binding energy hot spots in protein-protein complexes computational alanine scanning of protein-protein interfaces systematic analysis of helical protein interfaces reveals targets for synthetic inhibitors plucking the high hanging fruit: a systematic approach for targeting protein-protein interactions ssipe: accurately estimating protein-protein binding affinity change upon mutations using evolutionary profiles in combination with an optimized physical energy function paml 4: phylogenetic analysis by maximum likelihood an improved likelihood ratio test for detecting site-specific functional divergence among clades of protein-coding genes bayes empirical bayes inference of amino acid sites under positive selection acknowledgements m.c.j. was funded by a natural sciences and engineering council of canada discovery accelerator supplement to a.d.m. and by a postdoctoral fellowship from the alberta children's hospital research institute. p.s.a. thanks the national institutes of health (r35gm130333) for financial support. we thank four reviewers for constructive comments, which improved the manuscript considerably. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s42003-020-01370-w.correspondence and requests for materials should be addressed to a.d.m. or j.p.h.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-328361-hyrke6j2 authors: ithete, ndapewa laudika; stoffberg, samantha; corman, victor max; cottontail, veronika m.; richards, leigh rosanne; schoeman, m. corrie; drosten, christian; drexler, jan felix; preiser, wolfgang title: close relative of human middle east respiratory syndrome coronavirus in bat, south africa date: 2013-10-17 journal: emerg infect dis doi: 10.3201/eid1910.130946 sha: doc_id: 328361 cord_uid: hyrke6j2 nan respiratory syndrome coronavirus in bat, south africa to the editor: the severe acute respiratory syndrome (sars) outbreak of 2002-03 and the subsequent implication of bats as reservoir hosts of the causative agent, a coronavirus (cov), prompted numerous studies of bats and the viruses they harbor. a novel clade 2c betacoronavirus, termed middle east respiratory syndrome (mers)-cov, was recently identified as the causative agent of a severe respiratory disease that is mainly affecting humans on the arabian peninsula (1) . extending on previous work (2), we described european pipistrellus bat-derived covs that are closely related to mers-cov (3) . we now report the identification of a south africa bat derived cov that has an even closer phylogenetic relationship with mers-cov. during 2011-2012, fecal pellets were collected from 62 bats representing 13 different species in the kwazulu-natal and western cape provinces of south africa and stored in rnalater solution (life technologies, carlsbad, ca, usa). details about the bat sample are available in the online technical appendix table ( wwwnc. cdc.gov/eid/article/19/10/13-0946-techapp1.pdf). rna was extracted by using the qiaamp viral rna mini kit (qiagen, hilden, germany). screening for covs was done by nested reverse transcription pcr using broadly reactive oligonucleotide primers targeting a conserved region in the rna-dependent rna polymerase (rdrp) gene (online technical appendix). pcr results were positive for 5 (8%) of the 62 specimens. pcr amplicons for 4 positive specimens yielded alphacoronavirus sequences related to recently described bat alphacoronaviruses from south africa (4) . the other positive specimen, termed pml/2011, was from an adult female neoromicia cf. zuluensis bat sampled in 2011; the specimen yielded a novel betacoronavirus (genbank accession no. kc869678). online technical appendix figure 1 shows the distribution of this bat species. to obtain better phylogenetic resolution, we extended the 398-nt rdrp fragment generated by the screening pcr to 816 nt, as described (5). pml/2011 differed from mers-cov by only 1 aa exchange (0.3%) in the translated 816-nt rdrp gene fragment. thus, pml/2011 was much more related to mers-cov than any other known virus. the amino acid sequence of the next closest known relatives of mers-cov, from european pipistrellus bats (3), differed from mers-cov by 1.8%. the amino acid sequences of viruses from nycteris bats in ghana (3) and the 2c prototype bat covs, hku4 and hku5, from china (6) differed by 5.5%-7.7% from mers-cov. the smaller 152to 396-nt rdrp fragments of 2c bat covs from a hypsugo savii bat in spain (7), bat guano in thailand (8), and a nyctinomops bat in mexico (9) showed no or only partial overlap with the 816-nt fragment generated in this study; thus, a direct comparison could not be done. however, in their respective rdrp fragments, these covs yielded amino acid sequence distances of 3.5%-8.0% and were thus probably more distant from mers-cov than the virus described here. a bayesian phylogenetic analysis of the 816-nt rdrp sequence confirmed the close relationship between pml/2011 and mers-cov (figure) . their phylogenetic relatedness was as close as that of sars-cov and the most closely related bat coronavirus known, rs672 from a rhinolophus sinicus bat (figure) . like pml/2011 and mers-cov, rs672 and sars-cov showed only 1 aa exchange in the translated 816-nt rdrp fragment. to confirm this relatedness, we amplified and sequenced a short 269-nt sequence encompassing the 3′-terminus of the spike gene for pml/2011 (oligonucleotide primers available upon request from the authors). a partial spike gene-based phylogeny using this sequence yielded the same topology as that using the partial rdrp sequence (online technical appendix figure 2 ). again, pml/2011 was most closely related to mers-cov, showing only a 10.9% aa sequence distance in this gene, which encodes the glycoprotein responsible for cov attachment and cellular entry. this distance was less than the 13.3% aa sequence distance between mers-cov and the european pipistrellus covs (3) and less than the 20.5%-27.3% aa sequence distance between mers-cov and hku5 and between mers-cov and hku4 (6) in the same sequence fragment. our results further support the hypothesis that, like human cov-229e and sars-cov, ancestors of mers-cov might exist in old world insectivorous bats belonging to the family vespertilionidae, to which the genera to the editor: extraintestinal pathogenic escherichai coli (expec) bacteria have the ability to cause diverse and serious diseases, such as urinary tract infections (utis) and bacteremia (1-3); incidence of bacteremia is increasing globally (4) . the emergence of multidrug resistance in e. coli is also becoming a global concern, with particular emphasis on e. coli sequence type (st) 131, which is being increasingly reported in utis. drug resistance is mediated by extended-spectrum β-lactamases (es-bls), mainly of the ctx-m family, particularly ctx-m-15 and 14, and less frequently of the shv and oxa families (5, 6) . few studies are available regarding the characterization of e. coli strains causing bacteremia. we characterized 140 e. coli isolates from bacteremia patients treated at nottingham university hospital (nottingham, uk) over a 5-month period, with the aim of developing an epidemiologic profile of the population of expec that causes bacteremia. for context, we compared the isolates with 125 e. coli isolates from urine samples collected during the same period. cases were selected to include isolates from a diverse patient group: patient ages ranged from 1 month to 90 years; patient sex was evenly divided between male and female; infections were community-and hospitalassociated; and suspected sources of infection varied. antimicrobial drug susceptibility tests, pcr detection of esbl genes , multilocus sequence typing using the achtman scheme (http:// mlst.ucc.ie/mlst/dbs/ecoli), and virulence-associated gene (vag) carriage screening by pcr were performed on isolates as described (7). significantly more bacteremia e. coli isolates than urine e. coli isolates were resistant to ciprofloxacin (25.7% isolation of a novel coronavirus from a man with pneumonia in saudi arabia circulation of group 2 coronaviruses in a bat species common to urban areas in western europe human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe coronaviruses in south african bats genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features detection of alpha and betacoronaviruses in multiple iberian bat species group c betacoronavirus in bat guano fertilizer coronaviruses in bats from mexico complete genome analysis of 33 ecologically and biologically diverse rift valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry we thank tobias bleicker, sebastian brünink, and monika eschbach-bludau for technical assistance; thomas seifert, sonja matthee, and conrad mathee for invaluable help; and anna-marie corman for assistance with geographic information processing. key: cord-325614-e9hnhzfg authors: todorov, german; uversky, vladimir n. title: a possible path towards rapid development of live-attenuated sars-cov-2 vaccines: plunging into the natural pool date: 2020-10-14 journal: biomolecules doi: 10.3390/biom10101438 sha: doc_id: 325614 cord_uid: e9hnhzfg severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the causative agent of the coronavirus disease 2019 (covid-19) pandemic spreading around the world, causing massive distress to the world’s economy and affecting healthcare systems worldwide. although some exposed individuals have no symptoms and most symptomatic infections are not severe, covid-19 cases span a wide spectrum, ranging from mild to critical and sometimes resulting in life-threatening complications, such as pneumonia, severe respiratory distress and cardiac problems. currently, there is no curative drug for covid-19 and vaccines are still under development. we are presenting here a strategy for the fast development of natural live-attenuated sars-cov-2 vaccines. our proposed approach is based on screening for, identifying, analyzing and selecting naturally attenuated yet highly immunogenic sars-cov-2 strains, which may lead to a shorter cycle of vaccine development, as well as higher vaccine effectiveness. the most effective way to prevent the epidemiologic infectious diseases is vaccination, which is successfully used to control measles, mumps, poliomyelitis, and rubella [1] , as well as led to the eradication of polio in the usa by 1979. as a result, there are more than 70 vaccines on the market that are targeting approximately 30 infectious agents [2] and helping to prevent morbidity and mortality in millions of people annually [3] . similar to other rna viruses [4, 5] , sars-cov-2 undergoes rapid inter-species evolution leading not only to the accumulation of mutations that makes new sars-cov-2 variants different from the first isolates of the virus obtained in wuhan (https://www.gisaid.org/epiflu-applications/hcov-19-genomic-epidemiology/), but also to the genetic drift resulting in the attenuation of the virus pathogenicity [5] . taking these facts into consideration, it was pointed out that "it is entirely plausible that, at present, some individuals, by chance, have already become infected with attenuated versions of sars-cov-2" [6] . it was also argued that "knowledge of naturally emerging attenuated sars-cov-2 variants across the globe should be of key interest in our fight against the pandemic" [6] . based on similar premises, we are proposing here a strategy for the rapid development of live-attenuated sars-cov-2 vaccines using the targeted search for the attenuated sars-cov-2 variants that can serve as the natural source for the live-attenuated vaccines for sars-cov-2. the use of live-attenuated pathogens is one of the oldest and most effective methods of vaccination. the prominent historic examples of this approach include edward jenner's small pox vaccine based on cowpox virus [7, 8] and albert sabin's live-attenuated polio vaccine [9, 10] . evidence is accumulating that sars-cov-2 infection with the live virus can provide a broad, robust and durable immunity, which, if confirmed, would favor the live-attenuated vaccine approach. an icelandic study suggested durability of the humoral immune response to sars-cov-2 infection (live virus), even in older individuals [11] . other research indicates the development of robust and durable t-cells immunity after sars-cov-2 infection [12] . in the comprehensive analysis of the molecular basis of coronavirus virulence and vaccine development, it was deemed likely that "the main strategy for producing more efficacious vaccines in the near future will be based on live-attenuated vaccines that lack specific virulence markers" [13] . this conclusion was based on the comparative analysis of various forms of vaccines, such as live-attenuated virus vaccines (i.e., vaccines containing whole pathogens that are designed to replicate within the body inducing mild infection and mild disease symptoms), subunit vaccines that contain only the antigenic parts of the pathogen (e.g., vaccines based on recombinant mers-cov spike (s) protein or its receptor-binding domain (rbd) [14] ), replicating recombinant vector vaccines that uses engineered viral vectors with a proven safety record backbone to deliver and express an antigen [15] (e.g., recombinant adenovirus expressing s protein fragments of different size [16] [17] [18] ), and whole-inactivated virus vaccines (i.e., vaccines based on chemically inactivated sars-cov virions [19] [20] [21] [22] [23] or sars-cov-2 virions [24, 25] ). whereas non-living virus vaccines have some advantages, such as stability and good safety profiles, they are known to cause side effects and can be less efficient than the live-attenuated virus vaccines [13] . on the other hand, live vaccines that use a weakened (or attenuated) form of the pathogen tend to have higher immunogenicity than inactivated vaccines (i.e., those using the killed version of the pathogen) as they imitate natural infection more closely and invoke a wider range of immune responses involving both humoral and cellular immunity. also, live-attenuated vaccines tend to require fewer administrations to produce reliable and lasting protection. the main known drawback of live-attenuated vaccines is a potential for the reversion to virulence [13] . nonetheless, all factors considered, a safe and effective live-attenuated sars-cov-2 vaccine(s) would clearly be of significant benefit and may have advantages other types of vaccines do not have. the ability to develop live-attenuated sars-cov-2 vaccine(s) rapidly would be a valuable tool against the current sars-cov-2. furthermore, intra-species evolution of sars-cov-2 potentially leading to future mutations of this virus might cause recurring waves of sars-cov-2-related infections resistant to the originally developed vaccine(s). in that case, new vaccines against the novel sars-cov-2 variants would again be urgently needed. here we propose a possible path towards rapid development of naturally live-attenuated sars-cov-2 vaccines. the proposed approach is based on screening for, identifying, analyzing and selecting naturally attenuated yet highly immunogenic sars-cov-2 strains, potentially leading to a more rapid cycle of vaccine development, as well as higher vaccine effectiveness. identify populations at the highest risk for severe course of sars-cov-2 (e.g., elderly with severe co-morbidities/multiple major risk factors) and find locations of infection clusters in those populations. at or near the location of the infection cluster or simply in very high risk populations (e.g., nursing homes, retirement communities, etc.), screen and test all the highest risk individuals for active sars-cov-2 (pcr test). among those screened, find a subset of individuals with active sars-cov-2 injection (positive pcr test), who show no or minimal symptoms and get the samples of the virus from each such person. then wait till their infection is fully cleared (negative pcr test) and narrow down the selection to those who have not developed any serious symptoms or diagnostic indicators of any long-term health damage associated with covid-19. 4. in the above group (i.e., high risk individuals who have recovered from sars-cov-2 with little or no symptoms/consequences), performs immunological tests and find individuals who have a relatively weak immune system (which would be common in the high risk population you are working with) but still developed a robust immunity against sars-cov-2 (preferably both high level of antibodies as well as cellular immunity). analyze the sars-cov-2 variants from the above subgroup of individuals identified in the previous step. potentially, some of those sars-cov-2 variants would be capable of invoking a robust immune response but are sufficiently attenuated as not to cause major symptoms even in the most vulnerable/highest risk individuals. the analysis should include viral genome sequencing and, ideally, tissue cultures tests of infectivity (to verify attenuation and possibly ascertain its mechanism), etc. as an example, one could test for rates of virus binding to ace2 receptors, for rates of infecting cultured lung cell, etc. subsequent analyses could also include tests in animal models. 6. the above steps should allow to select sars-cov-2 variants with weak infectivity and/or causing low intensity/mild infection. this alone might produce a sufficiently attenuated variant of the virus. otherwise, one should consider additional steps to further attenuate the virus, such as passaging via human tissue culture while applying selection pressure towards survival of the more attenuated forms of the virus, targeted deletions, etc. the result would be the candidates for the live-attenuated sars-cov-2 vaccines. 7. among the candidate variants of the naturally live-attenuated sars-cov-2, identify the ones found in the greatest number of people fitting the above screening criteria. perform additional screening of all the contacts of the individuals found to have carried naturally sars-cov-2 variants. if possible, also screen additional population around the infection cluster under study. check that the additional individuals found to carry the identified attenuated sars-cov-2 variants do not develop a symptomatic infection (or have only mild symptoms) or suffer from any complications. exclude the live-attenuated sars-cov-2 candidates that were found to cause any major issues during this broader screening. the remaining live-attenuated sars-cov-2 candidates could be (potentially) suitable for clinical testing. 8. for the most promising live-attenuated sars-cov-2 candidate(s) identified in step 7, keep recursively tracing the contacts of all individuals infected with the candidate strain, thereby (potentially) collecting data on a significant number of individuals infected with it. the goal of this step is to find as many individuals as possible who were infected with, carried, and recovered from the candidate live-attenuated sars-cov-2 strain and then to accumulate/analyze enough data to (at least tentatively) determine that the candidate strain: (a) does not cause severe or even moderate infection; (b) is robustly immunogenic; (c) does not revert to a more pathogenic form, etc. 9. evaluate whether the thus identified naturally live-attenuated sars-cov-2 candidate(s) is/are suitable for clinical testing as a live-attenuated vaccine. if not, consider additional attenuation steps (e.g., via genetic engineering, such as directed deletion of some specific gene cluster open reading frame(s) [26] or passaging in tissue culture under selection pressure or etc). 10. if the above steps are successful, one would have found a promising live-attenuated sars-cov-2 vaccine candidate and have accumulated a considerable amount of human clinical data on it. the clinical trials still required to ensure the suitability for vaccination may be less extensive than otherwise, potentially providing a faster path to a safe and effective vaccine. apparently, in many cases, attenuated virus strains tends to be transmitted less easily (as they tend to reproduce more slowly, be shed less, etc) than the corresponding non-attenuated strains. natural live-attenuated sars-cov-2 strains, if any, may or may not turn out to be easily transmissible. if the candidate attenuated sars-cov-2 strain is easily transmissible, we would need to evaluate whether a live-attenuated virus that causes a very mild form of infection but is easy to transmit can be considered sufficiently safe for use as a vaccine, or whether it needs to be further attenuated in the lab, which could slow down the development of the vaccine. alternatively, if the candidate attenuated virus stain is not easily transmissible, it may be hard to find (by screening the potentially exposed population) enough people who contracted that particular strain. as a result, it could be difficult to obtain enough of the preliminary human data for that strain, which too could slow down the vaccine development process. even if one only screens the subpopulations most likely to carry live-attenuated virus (i.e., asymptomatic and/or mildly symptomatic individuals in the highest risk groups), finding suitable live-attenuated strains may prove slow and/or difficult. that would depend in part on the rate and patterns of natural evolution of the weaker strains, which in turn depends on a variety of factors, such as mutation rate, selection pressures, differential transmission and so forth. rna viruses tend to have high mutation rates. however, sars-cov-2 mutation rate appears to be lower than that of most rna viruses, possibly due to a "proofreading" enzyme [27] . also, the evolution of weaker strains tends to be rapid in highly lethal infections with short incubation period. since sars-cov-2 has low mortality rate in the younger and healthier groups as well as a relatively long incubation period, its evolution towards the weaker strains may be relatively slow. on the other hand, the current mutation data on sars-cov-2 strains are skewed towards strains obtained from highly symptomatic individuals and may underestimate the prevalence of weaker strains in asymptomatic or mildly symptomatic individuals who never get tested. furthermore, in fairly isolated high-risk high mortality subpopulations, such as nursing facilities and retirement communities, the evolution patterns of sars-cov-2 strains may be closer to those seen in high-mortality infections and favor the emergence of weaker strains more than currently believed. all in all, at present there are too many unknowns to predict how much screening of suitable individuals in high-risk subpopulations will be required to find naturally-evolved candidate strains for the development of live-attenuated vaccines. accurate testing for active sars-cov-2 infections, especially eliminating false-positive results, is critical for the success of the proposed approach. false-positives caused by non-viable viral fragments and/or different coronavirus types are not uncommon. hence, the version of the pcr test with the lowest false-positive rate should be used. it may also be necessary to take multiple samples per patient (from different body sites), use several different pcr primers, correlate pcr test results with antibody tests and t-cell tests, and so forth. additional confirmation of pre-screened live-attenuated sars-cov-2 vaccine candidates would be obtained via culture, full sequencing, passaging and other measures [28, 29] . if sars-cov-2 continues to mutate producing new strains resistant to the original vaccine(s), it may be useful to conduct continuous screening of high risk populations to keep identifying new live-attenuated sars-cov-2 candidates to facilitate continuous and timely "upgrading" of the vaccine. theoretically, there might arise new strains with low virulence in the old but high in the young. perhaps that needs to be ruled out explicitly in the vaccine candidate strains. reversal of live-attenuated virus to more virulent forms is a known (and typically manageable) risk for live-attenuated vaccines. several examples are given by the design of the genetically stable live-attenuated sars-cov vaccines. in the first case, the viral replication fidelity is targeted [30, 31] . replication fidelity of covs, which is~20 times higher than the replication fidelity of other rna viruses, is mediated by 3 → 5 exonuclease (exon) that functions in the rna proofreading [30] [31] [32] [33] . impairing replication fidelity sars-cov by exon inactivation generated species that do not revert to virulence, suggesting that exon inactivation can be utilized in the stable attenuation of covs [31] . in other cases, partial or complete deletion of envelope (e) protein and introduction of mutations in the nonstructural protein 3a (nsp3a) or nsp1 generated attenuated recombinant sars-cov viruses that were able to maintain their attenuation after the in vitro and in vivo passages, being capable of fully protecting mice against challenge with the lethal parental virus [34] [35] [36] . in some patients, vaccines can cause autoimmune reactions and there are reported cases of autoimmune diseases that have been correlated with vaccination [2, 37] . in fact, despite the fact that vaccination is generally a safe procedure that does not cause serious systemic adverse events, in several studies, single or combined multivaccine procedures were shown to precede development of articular (arthritis, rheumatoid arthritis), autoimmune (systemic lupus erythematosus, diabetes mellitus), and neurological (guillain barre syndrome, multiple sclerosis, autism) maladies [37] . however, it seems that live-attenuated vaccines tend to produce fewer autoimmune reactions that non-live vaccines. designing tomorrow's vaccines vaccination and autoimmune diseases: is prevention of adverse health effects on the horizon? vaccine technologies: from whole organisms to rationally designed protein assemblies we shouldn't worry when a virus mutates during disease outbreaks the evolution and emergence of rna viruses the importance of naturally attenuated sars-cov-2in the fight against covid-19 jenner and the history of smallpox and vaccination early clinical pathologists: edward jenner (1749-1823) pioneering figures in medicine: albert bruce sabin-inventor of the oral polio vaccine albert sabin and the coalition to eliminate polio from the americas humoral immune response to sars-cov-2 in iceland broad and strong memory cd4(+) and cd8(+) t cells induced by sars-cov-2 in uk convalescent 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incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus effects of toll-like receptor stimulation on eosinophilic infiltration in lungs of balb/c mice immunized with uv-inactivated severe acute respiratory syndrome-related coronavirus vaccine inactivated sars-cov vaccine prepared from whole virus induces a high level of neutralizing antibodies in balb/c mice development of an inactivated vaccine candidate for sars-cov-2 development of an inactivated vaccine candidate, bbibp-corv, with potent protection against sars-cov-2 attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis the coronavirus is mutating-does it matter? complete genome sequence of a sars-cov-2 strain isolated in northern germany the genetic sequence, origin, and diagnosis of sars-cov-2 coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing discovery of an rna virus 3 -5 exoribonuclease that is critically involved in coronavirus rna synthesis severe acute respiratory syndrome coronaviruses with mutations in the e protein are attenuated and promising vaccine candidates a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo identification of the mechanisms causing reversion to virulence in an attenuated sars-cov for the design of a genetically stable vaccine vaccination as an additional player in the mosaic of autoimmunity funding: this research received no external funding. the authors declare no conflict of interest. key: cord-308110-cco3aq4n authors: okamoto, mika; toyama, masaaki; baba, masanori title: the chemokine receptor antagonist cenicriviroc inhibits the replication of sars-cov-2 in vitro date: 2020-07-30 journal: antiviral res doi: 10.1016/j.antiviral.2020.104902 sha: doc_id: 308110 cord_uid: cco3aq4n cenicriviroc (cvc) is a small-molecule chemokine receptor antagonist with highly potent and selective anti-human immunodeficiency virus type 1 (hiv-1) activity through antagonizing c-c chemokine receptor type 5 (ccr5) as a coreceptor of hiv-1. cvc also strongly antagonizes c-c chemokine receptor type 2b (ccr2b), thereby it has potent anti-inflammatory and immunomodulatory effects. cvc is currently under clinical trials in the patients for treatment of nonalcoholic steatohepatitis, in which immune cell activation and dysregulation of proinflammatory cytokines play an important role in its pathogenesis. in this study, cvc was examined for its inhibitory effect on the replication of sars-cov-2, the causative agent of covid-19, in cell cultures and found to be a selective inhibitor of the virus. the 50% effective concentrations of cvc were 19.0 and 2.9 μm in the assays based on the inhibition of virus-induced cell destruction and viral rna levels in culture supernatants of the infected cells, respectively. interestingly, the ccr5-specific antagonist maraviroc did not show any anti-sars-cov-2 activity. although the mechanism of sars-cov-2 inhibition by cvc remains to be elucidated, ccr2b does not seem to be its target molecule. considering the fact that the regulation of excessive immune activation is required to treat covid-19 patients at the late stage of the disease, cvc should be further pursued for its potential in the treatment of sars-cov-2 infection. the pandemic of severe pneumonia caused by the transmission of the new coronavirus sars-cov-2 is a serious threat to humanity (di gennaro et al., 2020; harapan et al., 2020; helmy, et al., 2020) . at present, no vaccines exist, and the nucleoside analog remdesivir (rdv) has recently been approved for treatment of sars-cov-2 infection (grein et al., 2020) . rdv has broad-spectrum antiviral activity against several viruses, including ebola virus and coronaviruses. in addition to rdv, there is an attempt to treat covid-19 with existing drugs developed for other purposes. these include hydroxychloroquine and chloroquine (pastick et al., 2020) , the anti-influenza virus agent favipiravir (pilkington et al., 2020) , and the human immunodeficiency virus type 1 (hiv-1) protease inhibitor lopinavir/ritonavir . more recently, the anti-parasitic agent ivermectin has been shown to inhibit sars-cov-2 replication in cell cultures (caly et al., 2020) . however, these drugs are not optimized for sars-cov-2, so that they may have inevitable adverse effects due to the requirement of higher dosages. furthermore, these antiviral agents must be used at an early stage of infection, since severe deterioration of pneumonia in some patients is considered to be closely associated with not viral replication but "cytokine storm" caused by dysregulated and excessive cytokine release from activated immune cells (ye et al., 2020) . therefore, the use of anti-inflammatory and immunomodulatory agents is mandatory in the late stage of this disease (alijotas-reig et al., 2020) . we have examined several compounds for their inhibitory effect on sars-cov-2 replication in cell cultures and found that the chemokine receptor antagonist cenicriviroc (cvc) inhibits the replication of sars-cov-2. cvc is a c-c chemokine receptor type 5 (ccr5) antagonist with potent and selective anti-hiv-1 activity (baba et al., 2005) . in addition, cvc antagonizes not only ccr5 but also c-c chemokine receptor type 2b (ccr2b). since ccr2b is the receptor of monocyte chemoattractant protein 1 (mcp-1), cvc exerts anti-inflammatory and immunomodulatory effects in vivo (dawson et al., 2003; xia and sui, 2009) . in fact, cvc is currently under clinical trials for the treatment of nonalcoholic steatohepatitis (nash), in which immune cell activation and dysregulation of proinflammatory cytokines play an important role in its pathogenesis (pedrosa et al., 2020) . veroe6 cell line expressing transmembrane protease serine 2 (veroe6/tmprss2) highly susceptible to sars-cov-2 infection was obtained from japanese collection of research bioresources (jcrb) cell bank in japan (jcrb no. jcrb1819) and used for virus propagation and antiviral assays after removal of mycoplasma contamination. vero cells were also used for experiments. the cells were cultured in dulbecco's modified eagle medium (nacalai tesque, kyoto, japan) supplemented with 5% heat-inactivated fetal bovine serum (fbs), 100 u/ml penicillin g, 100 µg/ml streptomycin, and 1 mg/ml g418 (nacalai tesque). for antiviral assays, the cells were cultured in the absence of g418. sars-cov-2 (wk-521 strain, gisaid database id epi_isl_408667), a clinical isolate from a covid-19 patient, was provided by national institute of infectious diseases, tokyo, japan . the infectious virus titer was determined in veroe6/tmprss2 cells and expressed as 50% cell culture infectious dose (ccid 50 ) per ml. cvc and maraviroc (mrv) were purchased from medchemexpress (monmouth junction, nj). the nucleotide/nucleoside analogs rdv and favipiravir (fpv) were obtained from chemscence (monmouth junction, nj) and selleck chemicals (houston, tx), respectively. mcp-1 was purchased from petpotech (rocky hill, nj). mrv is the ccr5 antagonist clinically approved for treatment of hiv-1 infection (woollard and kanmogne, 2015) . except for mcp-1, these compounds were dissolved in dimethyl sulfoxide (dmso) at a concentration of 20 mm or higher to exclude the cytotoxicity of dmso and stored at -20°c until use. mcp-1 was dissolved in distilled water. veroe6/tmprss2 cells (2 × 10 4 cells/well) were cultured in a 96-well microtiter plate and incubated at 37°c. after 24 h, the cells were mock-infected or infected with sars-cov-2 at a multiplicity of infection (moi) of 0.01 and cultured in the absence or presence of various concentrations of test compounds. after 3 days, the number of viable cells was determined by a tetrazolium dye method. briefly, 100 μl of culture medium was removed from each well, and 10 μl of water-soluble tetrazolium dye solution (dojindo, kumamoto, japan) was added. after incubating at 37°c for 2 h, 100 μl of isopropanol acidified with hydrochloric acid was added, and the absorbance was read at two wavelengths (450 and 620 nm) with a microplate reader (pauwels et al., 1988) . the 50% effective concentration (ec 50 ) of each compound was calculated from a dose-dependent curve based on the viability of infected and uninfected cells. all experiments using sars-cov-2 were conducted in biosafety level 3 (bsl3) facilities of kagoshima university. for immunofluorescence microscopy, vero cells (2 × 10 4 cells/well) were cultured in a microtiter plate and incubated. after 24 h, the cells were infected with sars-cov-2 at a moi of 0.1 in the absence of cvc and incubated. after 2 h, the cells were washed with phosphate buffered saline (pbs) to remove unadsorbed virus particles and further incubated in the absence or presence of 40 μm cvc. after 3 days, the cells were fixed with 4% paraformaldehyde in pbs for 15 min. then, the solution was removed, and the cells were washed with pbs and permeabilized with methanol. after washing with pbs, the cells were treated with pbs containing 1% bovine serum albumin (sigma-aldrich, st. louis, mo) and 0.1% tween 20 (fujifilm wako, osaka, japan) and incubated overnight at 4°c with an anti-sars-cov-2 nucleocapsid rabbit antibody (genetex, irvine, ca). after incubation, the cells were washed with pbs and stained with the secondary antibody goat anti-rabbit igg h&l (alexa fluoro ® 488; abcam, cambridge, uk). the cells were washed with pbs, stained with 4',6-diamidino-2-phenylindole (dapi; bio-rad, hercules, ca), and observed under a fluorescent microscope (bz-x800; keyence, osaka, japan). the inhibitory effect of compounds on sars-cov-2 replication was also evaluated by the viral rna levels in culture supernatants of the infected cells. veroe6/tmprss2 cells were infected with the virus and incubated for 3 days in the absence or presence of test compounds, as described above. ten μl of each culture supernatant was mixed with 10 μl of sidestep lysis and stabilization buffer (agilent technologies, santa clara, ca) and diluted with 80 μl of distilled water. the amount of viral rna was measured by real-time reverse transcription polymerase chain reaction (rt-pcr) using taqman gene expression cells-to-ct tm kit (thermo fisher scientific, waltham, ma), according to the manufacturer's protocol except for its cell lysis step. the primer pair 5'-aaattttggggaccaggaac-3' and 5'-tggcagctgtgtaggtcaac-3' and the probe 5'-fam-atgtcgcgcattggcatgga-tamra-3' were used for real-time pcr reat-time rt-pcr to determine the amount of viral rna, as described above. when veroe6/tmprss2 cells were infected with sars-cov-2 and incubated in the absence of compounds for 3 days, the cells were completely destroyed by the virus-induced cytopathic effect (fig. 1b) . such cell destruction was not observed for the infected cells in the presence of 20 μm cvc, although some morphological changes were identified (fig. 1d ). in contrast, mrv did not inhibit the virus-induced cell destruction even at 40 μm (fig. 1f) . the ec 50 s of cvc and mrv were 19 ± 0.2 and > 40 μm, respectively, based on the inhibition of virus-induced cell destruction (table 1) . both compounds did not show apparent cytotoxicity at concentrations up to 80 μm. dose-dependent protection of the infected cells from virus-induced cell destruction was observed for cvc but not for mrv (fig. 2) . these results indicate that cvc is a selective inhibitor of sars-cov-2 replication. as previously reported , rdv proved to be a potent and selective inhibitor of sars-cov-2 replication in our assay, whereas fpv did not show any selective inhibition even at a concentration of 80 μm (table 1 and fig. s1 ). the anti-sars-cov-2 activity was also examined by the inhibition of viral antigen expression in vero cells. vero cells is less susceptible to sars-cov-2 replication than veroe6/tmprss2 cells. in fact, vero cells were not destroyed by the virus-induced cytopathic effect on day 3 after infection (fig. 3a) . however, viral antigen expression was observed for many cells in the absence of cvc (fig. 3b ). in contrast, the antigen expression was completely inhibited in the presence of 40 μm cvc (fig. 3d) . when the anti-sars-cov-2 activity of cvc was evaluated by the viral rna levels in culture supernatants of the infected cells, cvc significantly and dose-dependently reduced the amount of viral rna in culture supernatants (fig. 4a ). its ec 50 in this assay was 2.9 μm, and more than 95% inhibition was achieved by cvc at a concentration of 10 μm. in contrast, mrv did not reduce the viral rna levels at concentrations up to 40 μm in 2005, we have reported that the small molecule and orally bioavailable ccr5 antagonist cvc inhibits hiv-1 replication at subnanomolar concentrations in vitro (baba et al., 2005) . the anti-hiv-1 activity of cvc is attributed to the inhibition of viral entry using ccr5 as a coreceptor. however, different from other anti-hiv-1 ccr5 antagonists such as mrv, cvc also suppresses the binding of mcp-1 to ccr2b-expressing cells. this unique feature of cvc encouraged its manufacturer to develop this compound as an agent for treatment of nash. a recent randomized, placebo-controlled trial of cvc for treatment of nash demonstrated that twice as many subjects achieved improvement in fibrosis and no worsening of steatohepatitis compared with placebo (friedman et al., 2018; tacke, 2018) . safety and tolerability of cvc were found to be comparable to placebo, suggesting that it can be administered safely to covid-19 patients for preventing severe deterioration of pneumonia due to the cytokine storm. in fact, alveolar macrophage-borne mcp-1 was reported to be a key agent in the initiation of the systemic inflammation of alveolar hypoxia in rats, and a ccr2b receptor antagonist prevented the mesenteric inflammation of alveolar hypoxia (chao et al. 2011) . furthermore, a recent study on transcriptome sequencing of the rnas isolated from the bronchoalveolar lavage fluid and peripheral blood mononuclear cells specimens of covid-19 patients revealed the association between its pathogenesis and excessive cytokine release including mcp-1 (xiong et al. 2020 ). it will be claimed that the anti-sars-cov-2 activity of cvc in vitro is insufficient for the treatment of covid-19 patients. however, our antiviral assay system using (table 1 and data not shown). the broad-spectrum anti-rna virus agent fpv was not inhibitory to sars-cov-2 replication even at 80 μm (table 1 and fig. s1 ), which is consistent with a previous report (choy et al. 2020) . the anti-sars-cov-2 activity of cvc was more obvious, when determined by the inhibition of viral rna levels in culture supernatants. its ec 50 was 2.9 μm (fig. 4) , which was similar to those of rdv (1.9 μm) and ivermectin (2.2-2.8 μm) (data not shown and caly et al., 2020, respectively) . thus, although the anti-sars-cov-2 activity of cvc in vitro is modest, we cannot exclude the possibility that cvc exhibits some antiviral efficacy in covid-19 patients. the target molecule of cvc remains to be elucidated. our preliminary studies on its mechanism of action revealed that cvc did not interfere with the entry of sars-cov-2 (data not shown). unlike hiv-1, ccr5 is not the target of cvc for inhibition of sars-cov-2 replication, since another potent ccr5 antagonist mrv was totally inactive (fig. 2b and fig. 4b ). furthermore, mcp-1 could not block sars-cov-2 infection (data not shown), suggesting that ccr2b is not the target molecule either. further studies, such as a time-of-addition experiment and biochemical approaches, are required to elucidate the mechanism of action, and they are currently in progress. in conclusion, in addition to the potent anti-inflammatory activity of cvc in vivo, the present study has identified its anti-sars-cov-2 activity in vitro. since not only the inhibition of viral replication but also the control of excessive immune activation is mandatory to save covid-19 patients at the late stage of the disease, cvc should be further pursued for its potential in the treatment of sars-cov-2 infection. the data of cvc and rdv represent mean ± range for two separate experiments and mean ± standard deviation for three separate experiments, respectively. immunomodulatory therapy for the management of severe covid-19. beyond the anti-viral therapy: a comprehensive review tak-652 inhibits ccr5-mediated human immunodeficiency virustype 1 infection in vitro and has favorable pharmacokinetics in humans the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 monocyte chemoattractant protein-1 released from alveolar macrophages mediates the systemic inflammation of acute alveolar hypoxia remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro targeting monocyte chemoattractant protein-1 signaling in disease coronavirus diseases (covid-19) current status and future perspectives: a narrative review a randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis compassionate use of remdesivir for patients with severe covid-19 coronavirus disease 2019 (covid-19): a literature review the covid-19 pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control enhanced isolation of sars-cov-2 by tmprss2-expressing cells rapid and automated tetrazolium-based colorimetric assay for the detection of anti-hiv compounds a randomized, double-blind, multicenter, phase 2b study to evaluate the safety and efficacy of a combination of tropifexor and cenicriviroc in patients with nonalcoholic steatohepatitis and liver fibrosis: study design of the tandem trial a review of the safety of favipiravir -a potential treatment in the covid-19 pandemic? development of genetic diagnostic methods for novel voronavirus cenicriviroc for the treatment of non-alcoholic steatohepatitis and liver fibrosis remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro maraviroc: a review of its use in hiv infection and beyond recent developments in ccr2 antagonists transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients the pathogenesis and treatment of the `cytokine storm' in covid-19 we thank national institutes of biomedical innovation, health and nutrition and national institute of infectious diseases for kindly providing veroe6/tmprss2 cells and sars-cov-2 (wk-521 strain), respectively. kagoshima university is applying for a patent of cvc as a sars-cov-2 inhibitor, and m.o., m.t. and m.b. are its inventors. key: cord-314669-lvibjx97 authors: shang, guifang; biggerstaff, brad j.; yang, baocheng; shao, chaopeng; farrugia, albert title: theoretically estimated risk of severe acute respiratory syndrome transmission through blood transfusion during an epidemic in shenzhen, guangdong, china in 2003 date: 2007-11-26 journal: transfus apher sci doi: 10.1016/j.transci.2007.09.004 sha: doc_id: 314669 cord_uid: lvibjx97 background: severe acute respiratory syndrome (sars) is a newly recognized infectious disease that caused an outbreak in south china in 2003. the cause of sars was identified as a novel coronavirus (cov). the existence of asymptomatic seroconvertors and the detection of the sars-cov rna in plasma during the course of infection all suggest that sars could, as least theoretically, be transmitted by transfusion. an estimate of the risk of sars transmission through blood transfusion will contribute to decisions concerning blood safety monitoring and may be useful in the design of strategies to decrease the risk of transfusion-transmitted infections. study design and methods: case onset dates from the 2003 shenzhen sars epidemic and investigational results from taiwan on viremia in humans are used to estimate the number of cases that were viremic throughout the epidemic. estimates of the asymptomatic-to-clinically confirmed sars-cov infection ratio, the proportion of asymptomatic infections reported in a seroprevalence survey in hongkong, and the population size of shenzhen are used to infer the sars-cov transfusion–transmission risk. statistical resampling methods are used. results: based on data from shenzhen, hongkong and taiwan, the maximum and mean risk (per million) of sars-cov transmission from donors in shenzhen were estimated as 23.57 (95% ci: 6.83–47.69) and 14.11 (95% ci: 11.00–17.22), respectively. the estimated risk peaked on april 02, 2003. conclusions: although there are currently no confirmed reports of the transmission of sars-cov from asymptomatic individuals, recent research data indicate that transfusion-transmitted sars-cov is at least theoretically possible. although the risk is low, with its rapid spread of the disease, appearance of alarmingly high infectivity and high fatality rate, public health authorities need to consider strategies for blood donor recruitment and virus inactivation during an epidemic to further ensure blood safety. theoretically estimated risk of severe acute respiratory syndrome transmission through blood transfusion during an epidemic in shenzhen, guangdong, china in 2003 new pathogens and antimicrobial-resistant forms of older pathogens continue to emerge, some with the potential for rapid, global spread, and high morbidity and mortality. three examples of pathogens that are current causes for human health concern are avian influenza viruses, west nile virus (wnv) and the severe acute respiratory syndrome (sars) coronavirus [1] . sars is a newly described respiratory infection with a potential threat to the health of people throughout the world. the etiological agent is a novel coronavirus (cov), the sars-associated cov (sars-cov). the first sars case was identified in foshan municipality on november 16, 2002 , in guangdong (gd) province, china. the patterns of spread of sars suggest droplet and contact transmission. close proximity of persons and handling of human secretions (respiratory secretions, faeces, and the like) enhance the risk for transmission [2] . because of its relative high transmissibility and mortality, sars raised a great concern over the public health. epidemiological investigations showed that a total of 8422 probable cases, with 916 deaths were reported from 29 countries during the outbreak (data at august 7, 2003) , while world health organization (who) announced that the last chain of human transfusion was broken on july 5, 2003 [3] . almost every newly emerging human pathogen is of concern for the safety of the blood supply during and after an epidemic crisis. the potential for transmission of sars-cov through blood and blood products is unknown. the possibility of viremia before the onset of clinical symptoms and/or after symptom resolution remains an important concern regarding blood safety. mildly symptomatic or asymptomatic infections can occur as documented by seroconversion in healthcare workers (hcws) and animal traders [4] [5] [6] . in april 2003, drosten et al. reported that viral rna was detected at extremely low concentrations in plasma during the acute symptomatic phase of infection [7] . in 2004, singapore researchers reported that sars-cov can be detected in the blood of infected patients [8] , and, also in 2004, woo et al. investigated the relative rates of non-pneumonic sars-cov infection (mildly symptomatic infections, not asymptomatic cases) and sars-cov pneumonia [9] . in 2005, tai-wan researchers first reported that the patterns of viremia differ among different sars patients and that some patients have a more protracted viremia [10] . therefore, although there are no confirmed reports of the transmission of sars-cov from asymptomatic individuals, and we are unaware that sars-cov has been transmitted by the transfusion of blood components, the possibility of such transmission during a donor's viremic phase is at least theoretically possible. detection by nucleic acid amplification (nat) of sars-cov in blood specimens from persons acutely infected with sars-cov has been reported in a number of patients [7] . patients with a mild course of sars recover approximately 10 days after the development of clinical symptoms [12] . antibodies to sars-cov were measured in patients on day 16 of the disease [7, 13] , and nat may therefore be the only way to identify patients with mild symptoms or in the early phase of disease who might nevertheless be viremic [7, 14] . therefore, persons with sars could potentially be viremic before the onset of symptoms and/or after symptom resolution. transmission of sars-cov via human cells, tissues, cellular and tissue-based products (hct/ps) recovered during these time periods may be possible [15] . because sars occurred in 2002 and 2003 suddenly and disappeared quickly, however, data on sars viremia prior to symptom onset is very rare. our risk estimate is theoretical and would overestimate the true risk if viremia does not develop before symptom onset. using seroepidemiologic data from the outbreak of sars in shenzhen, guangdong province, china, in 2003, we estimated the temporal trend in the proportion of viremic, asymptomatic individuals throughout this outbreak using a statistical resampling approach [16] . we then scaled these estimates to estimate the risk of transfusion-associated sars-cov transmission during the outbreak. we further compared this result with the estimated risk based on data from taiwan [10] , singapore [6] and guangdong center for disease control (cdc) [2] . the supplemental data for the former estimation is mainly based on a large epidemiology investigation done by woo et al. [9] and the latter estimation is based on a relatively small scale investigation (described further below). our study population was the population of shenzhen, guangdong province, china during the outbreak in 2003. guangdong is where the first case of sars was identified, followed by 1511 clinically confirmed cases, including 58 deaths; of the 1511 confirmed cases, 46 lived in shenzhen [2] . we first estimated how many of the 46 infected individuals with known symptom onset dates were viremic at each time point throughout the outbreak period. this was done using a statistical resampling approach that incorporated these 46 symptom onset dates, an assumed distribution of the length of the time between the onset of viremia and the onset of symptoms, and an estimated distribution of the length of viremia. the assumed distribution of the duration between onset of viremia and symptom onset was derived from data on the incubation period of sars and the timing of viremia onset relative to symptom onset during this incubation period [10] . the estimated distribution of the duration of viremia was derived using data from an extensive seroprevalence study and mass screening for detection of subclinical and non-pneumonic infection in hongkong [9] . we assumed that the dates of infection of 46 individuals with known symptom onset dates were similar to those of all shenzhen residents who became infected that year. we then used the estimate of the number of viremic cases over time to infer the number of asymptomatic, viremic sars-cov infections in the population over time by using seroepidemiologic survey results from guangdong cdc and hongkong that provided estimates of the proportion of sars-cov-infected individuals who are clinically confirmed and of the proportion of asymptomatic sars-cov infections. finally, we used the population size of shenzhen, guangdong province (4,670,000, http://www.demographia.com) to scale the estimated number of viremic, asymptomatic sars-cov infections in the population to estimate the risk or probability of a sars-cov infected blood donation. onset dates for the 46 clinically confirmed cases in the 2003 outbreak were obtained from shenzhen municipal hospital. symptom onset times (in days) of the 46 cases are shown in the pin plot in fig. 1 , with time t = 0 corresponding to february 9, 2003 , the date of the first reported onset, and time t = 88 corresponding to the last reported onset on may 9. the duration of this epidemic was thus considered to be 88 days. wang et al. reported on the course of sars-cov infection and viremia in humans, using results obtained from plasma [10] . the time from infection to symptom onset, the incubation period, is not precisely known but seems to be relatively short (approximately [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . who reported the incubation period as follows [3] : the maximum likelihood estimate of the mean and variance of the time from infection to onset were 6.37 days and 16.69 days, respectively (95% ci 5.29-7.75); therefore 95% of the patients would experience the onset of symptoms within 14.22 days of infection, based on the assumption that the incubation period followed a gamma distribution. there is approximately a 1-2-day lag between infection with the virus and the detection of virus in the blood [10, 14] so that the duration from onset of viremia to symptom onset is roughly 1-2 days shorter than the incubation period. efforts to detect sars-cov rna in plasma during the course of infection found that the highest detection rate, 72%, was found between day 4 and day 11 of illness [10] . analysis of sequential sars-cov load in plasma from six cases revealed different patterns of viremia, with the peak between day 4 and day 8. we calculated the infection dates from the day these patients were diagnosed with sars using sequential plasma samples tested for sars-cov infection. twenty-nine samples were tested, with a mean of 6.8 days and standard deviation of 3.9 days of viremia. we now describe the statistical method we used; a formal development along the lines of the method used here can be found in [16] , where the methods were developed for west nile virus. assume that the rate of blood donation is constant over the course of the epidemic, and assume that potential blood donors have the same risk of infection with sars-cov as the general population, which we anticipate may overestimate the risk. this latter assumption was based on the findings of a serosurvey of 400 healthy blood donors conducted in hongkong [9] , in which three seropositives were identified. finally, we assumed that transfused blood components of sars-cov infected blood donors transmit the infection to recipients with 100% efficiency. our strategy was to view the symptom onset times of the cases (n = 46) as anchor times, then to use the information about how viremia relates to symptom onset to estimate the number of cases with viremia at any time t during the outbreak. then, using this information and information on the asymptomatic or subclinical sars-cov infection-to-clinically confirmed sars ratio (r), the proportion of infected individuals who remain asymptomatic (a), and the population size, we estimated the risk of sars-cov transmission by transfusion from a unit of blood donated at time t during the epidemic. we used monte carlo simulation to estimate the number of asymptomatic, viremic individuals at a fixed time t as follows. because individual case onset times are recorded to the day as discrete times, but the underlying infection process is instead continuous, we first smoothed the observed case onset times by adding a smoothing component. next, simulated viremia time spans were computed for each case. to do this, the onset of viremia, relative to the case onset time, was chosen by taking a random sample from an assumed distribution for the duration from infection to symptom onset, based on the historic information provided above. the duration of viremia was then chosen by taking a random sample (with replacement) of the duration times from the data obtained in taiwan [10] . we have assumed in this procedure that the relative timing and duration of viremia for a case is independent of the symptom onset time. next, to reflect uncertainty in r, a random deviate r from the gamma distribution with mean r = 17.44 and variance var[r] = 100.9 [9] was generated, and each case's simulated viremia time was replicated [[r]] + 1 times to reflect the number of viremic individuals in the population over time. (here, [[r] ] denotes the largest integer in r.) subsequently, to discount viremia times for symptomatic infections and account for uncertainty in a, a random deviate, a, from a beta distribution with mean a = 7.50 · 10 à3 and variance var[a] = 1.86 · 10 à5 [10] was generated, and a random proportion a of the replicated viremia times were truncated to their onset times. at the end of each such run, the number of viremia times occurring at time t were counted over an equally spaced grid of 100 time points spanning the epidemic. this monte carlo sampling process was repeated 5000 times, and the resulting counts of the number of asymptomatic, viremic individuals at each time t on the 100-point grid were averaged. graphing these counts for all grid point times t yielded a curve representing the expected number of asymptomatic, viremic individuals over the course of the epidemic. finally, this curve was divided by the population size of potential donors to yield the estimated risk or probability of sars-cov transfusion transmission. the population size of potential donors was estimated to be 80% of the total population size of shenzhen, as approximately 80% of the population is at least 18 years of age (http://61.144.227/jingji/ tongji/2003yxqk/200411230056.htm, accessed on december 21, 2006) . we called the resulting curve the expected risk curve (erc). we assumed that the possibility of sars infection for the donors is the same as the whole eligible population. we computed two summary measures of the erc to aid interpretation, the maximum value and the mean value over the duration of the epidemic. the maximum is simply the point at which the curve is highest, and the timing of this maximum estimates when the expected transfusion-associated sars-cov risk was highest. the mean, computed by dividing the area under the erc by the duration of the epidemic, provides a measure of the expected transmission risk over the whole course of the epidemic. confidence bands for the true risk curve (trc) around the erc can be computed in various ways. we used a simultaneous percentile-t approach, the details of which are outlined in [16] . a 95% ci for the true maximum is read from the confidence bands for the trc, while the 95% ci for the true mean is computed using a percentile-t approach similar to that for the trc [16] . because of the relatively little available information on the epidemiology of sars and sars-cov infection, we performed a second analyses using different sources of data for key parameters in our modelling approach. in addition to the analysis reported above, in the second analysis we used data drawn from singapore [6] and guangdong cdc [2] , from which r = 0.22 (var[r] = 0.0046), a = 0.012 (var[a] = 0.00014). in the singapore study, wilder-smith et al. [6] reported that of 80 hospital staff, 45 (56%) were positive by sars-cov serology. of the 45 sars-cov-positive study participants, 37 (82%) were classified as having pneumonic sars, 2 (4%) as having subclinical sars, and 6 (13%) as having asymptomatic sars-cov infection. guangdong cdc found one seropositive adult from 84 healthy adults at the study clinic [2] . all computations were performed with the statistical software s-plus, version 7 (insightful corporation, seattle, wa, usa) using either built-in functions or functions written by one of the authors (b.j.b.). five thousand simulations combined to produce the erc, shown as the dark, solid line in fig. 2 . the dashed lines are 95th percentile-t simultaneous confidence bands. also shown in fig. 2 are 100 randomly selected realizations of the erc from the 5000 generated. the scale on the left axis is the risk (per million) of sars-cov transmission from transfusion of a single unit of blood. for results displayed in fig. 2 , estimation was based on the estimates obtained from woo et al. results from the secondary analysis are reported in table 1 , using the given values of r and a. although donor screening and testing have nearly eliminated the risk of transfusion-acquired infections attributable to hiv and hepatitis viruses, the potential emergence and spread of other pathogens, particularly those associated with asymptomatic illness, could result in unrecognized transmission through blood transfusion [1, 17] . our results indicate a small but non-zero risk of sars-cov transmission from transfusion of blood components during an epidemic, at least theoretically. based on data from different published literature, we calculated that during an epidemic of sars in shenzhen in 2003, the risk peaked at approximately 23.57 per million donations in late march/early april, with a mean risk over the course of the outbreak of 14.11 per million donations. the calculated risk was highly limited in time, with the near-zero or zero risk among donations before february and after august, during times of no reported community sars-cov transmissions in shenzhen, guangdong, china. there were 10,766 donations in shenzhen during the time of the epidemic, so based on our average risk estimate of 14.11 per million donations, the expected number of infected donations is 0. 152 (1 in 71,000) . further, based on a simple binomial model, the probability that there was at least one infected donation in the 10,766 donations made is 1 à (1 à 14.11/1,000,000) 10,766 = 0.14, or roughly 14%. the ratio r is a crucial factor in our risk estimation approach. from table 1 , it is clear that our estimates are sensitive to the assumptions about this quantity. good epidemiologic data strengthen inference, and improvements in the understanding of the epidemiology of sars will provide opportunities to refine the estimates we report. for blood safety, surveillance should emphasize the existence of subclinical or non-pneumonic sars-cov infections, since persons with severe symptoms would be less likely to donate blood. the comparison between the different estimates reported in table 1 showed almost the same peak risk time, the 1st or 2nd of april, which is in line with the government report on the outbreak [19] . as the estimate of the timing of the peak in risk is driven by the case onset times, and not really affected by specification of r or a, this result is expected. this is further consistent with the sars epidemic more broadly, as it was rampant in march, april and may 2003 in the mainland of china [19] . our estimated mean transmission risk of 14.11 per million may be too high for several reasons. we assumed a rate of 100% transmission from viremic donors, and the true rate is probably lower. there are also uncertainties about the duration of viremia. on the other hand, we used 80% of the total population of shenzhen (persons aged between 18 yr and 55 yr) as the denominator of our risk estimates, and the true number of potential donors is probably lower; this is expected to underestimate the true risk. furthermore, there are no population-based seroepidemiological data available to inform specification of asymptomatic rates and the other, relevant parameters used in our modelling approach. while the estimates we used for the various parameters in our modelling are the best available, should there be biases in them, these biases would directly impact our results. as we took care in our analyses to use all available, relevant and published data, it is impossible without further (possibly population-based) estimates to adjust for such unknown biases. our sensitivity analysis showed that the values assumed for a and, particularly, for r are critical to the results we obtain. since data from singapore were based on a relatively small sample of healthcare workers, and data from hongkong were based on an extensive seroprevalence study and mass screening for detection of subclinical and non-pneumonic infections, we consider our primary results to be those based on values for a and r obtained from woo et al. [9] . even with no confirmed cases of sars-cov transmission by blood transfusion, several precautionary measures were implemented by chinese blood centers in spring 2003 to prevent the potential risk of sars-cov transmission through blood [20] . these measures included: (1) new questions were added to the donor questionnaire about predonation contact history with suspected sars patients or people with contact history with sars; (2) normal body temperature was added as a criterion for donor qualification; (3) all donors were required to notify the blood collection facilities if fever, cough, or other suspected sars symptoms occurred within two weeks after donation; and (4) a sars-cov antibody testing research project was soon started using available tests on donor samples [20] . kumar and humar [21] reported that transplant patients are uniquely predisposed to emerging infections, and this applies to sars. lessons learned from west nile virus [18] and sars-cov in transplantation should be applicable to future outbreaks of other emerging infectious diseases [21] . in the meantime, on the basis of current knowledge a return of sars cannot be ruled out. particularly, the origin of the virus is assumed to be the civet cat, which could not be quarantined [22] . furthermore, the existence of asymptomatic sars-cov infections and the detection of sars-cov rna in plasma during the course of infection suggest that the transmission of the sars-cov by transfusion is at least theoretically possible. our analyses suggest, however, that this probability is very low during an outbreak similar in magnitude to the one in shenzhen in 2003, with a maximum estimated risk of 23.57 per million donations. in case sars does recur, preventive measures considered during the epidemic should include strategies for blood donor recruitment, and the estimates we provide herein may be used in a quantitative evaluation of such measures. the quantification of these effects will likely not be available unless and until sars recurs. safety of the blood supply in the united states: opportunities and controversies prevalence of igg antibody to sars-associated coronavirus in animal traders -guangdong province, china seroprevalence of antibody to severe acute respiratory syndrome (sars)-associated coronavirus among health care workers in sars and non-sars medical wards mild illness associated with severe acute respiratory syndrome coronavirus infection: lessons from a prospective seroepidemiologic study of health-care workers in a teaching hospital in singapore asymptomatic sars coronavirus infection among healthcare workers identification of a novel coronavirus in patients with severe acute respiratory syndrome detection of severe acute respiratory syndrome coronavirus in blood of infected patients relative rates of nonpneumonic sars coronavirus infection and sars coronavirus pneumonia detection of severe acute respiratory syndrome coronavirus rna in plasma during the course of infection clinical course and management of sars in health care workers in toronto: a case series infectious diseases: deferring competition, global net closes in on sars world health organization. case definitions for surveillance of sars eligibility determination for donors of human cells, tissues, and cellular and tissue-based products (hct/ps) estimated risk of west nile virus transmission through blood transfusion during an epidemic in queens the risk of transfusion-transmitted viral infections. the retrovirus epidemiology donor study transmission of west nile virus from an organ donor to four transplant recipients current status of severe acute respiratory syndrome in china epidemiological study of sars cov among volunteer blood donors in beijing emerging viral infections in transplant recipients pet theory comes to the fore in fight against sars key: cord-325234-skshcrh1 authors: jin, tingxu; li, jun; yang, jun; li, jiawei; hong, feng; long, hai; deng, qihong; qin, yong; jiang, jiajun; zhou, xuan; song, qian; pan, chunliu; luo, peng title: sars-cov-2 presented in the air of an intensive care unit (icu) date: 2020-08-15 journal: sustain cities soc doi: 10.1016/j.scs.2020.102446 sha: doc_id: 325234 cord_uid: skshcrh1 as coronavirus disease 2019 (covid-19) is spreading worldwide, there have been arguments regarding the aerosol transmission of its causative agent, severe acute respiratory syndrome coronavirus 2 (sars-cov-2). moreover, some re-detectable positive (rp) patients have been reported. however, little attention has been given to the follow-up of recovered patients, and there is no environmental evidence to determine whether these patients continue to shed the virus after they test negative. therefore, with an objective to test the hypothesis of airborne transmission of sars-cov-2, it is necessary to 1) determine whether sars-cov-2 particles are present in the indoor air and 2) determine whether recovered patients are still shedding virus, thus providing much-needed environmental evidence for the management of covid-19 patients during the recovery period. in this study, surface and air samples were collected from an intensive care unit (icu) containing one ready-for-discharge patient. all surface samples tested negative, but the air samples tested positive for sars-cov-2. this implies that sars-cov-2 particles may be shed in aerosol form for days after patients test negative. this finding may be one of the reasons for the observation of rp patients; therefore, there is a need for improved clinical and disease management guidelines for recovered covid-19 patients. acute upper respiratory infection (auri) is one of the most widespread infections among humans. respiratory viruses are a common cause of auri, which is responsible for approximately 200 million cases of pneumonia worldwide annually (he et al.,2017; sande, njunge, ngoi, mutunga, & pollard,2019) . the common cold is the most widespread auri caused by a virus (ludwig et al.,2013) ; other viruses that cause auri include influenza a, measles, rubella, etc. auris are a significant public health problem and a source of increased socioeconomic burden worldwide, as evident by the serious global public health crises caused by multiple auri pandemics during the course of j o u r n a l p r e -p r o o f human history. one hundred two years ago, the first wave of the spanish influenza (spanish flu) pandemic occurred in the spring and summer of 1918. thereafter, a serious epidemic occurred from september through december, spreading widely from france across the globe, thus causing the second pandemic wave. the following year, the spanish flu spread from eurasia to oceania, new zealand, and australia, constituting the third wave (crosby,1989) . during the spanish flu pandemic, approximately 600 million people were infected (the total world population at the time was 2 billion people) (lamb,2001) . the morbidity rate was 20% to 40%; the death toll approximately 20 to 50 million; and the mortality rate was 2.5% to 5%, far exceeding the number of deaths that occurred during the first world war (taubenberger & morens,2006) . another auri pandemic was severe acute respiratory syndrome (sars), which occurred in guangdong, china, in 2002 . it first spread to southeast asia and then across the globe (china,2003) . the sars pandemic was not eliminated until mid-2003. according to statistics released by the world health organization (who) in july 2003, there were 8,098 cases of sars worldwide, involving 32 countries and regions. the global death toll due to sars was 774, with a case fatality rate of nearly 11% (department of communicable disease, who,2004) . sars also caused the collapse of patient care services in health care systems due to staff shortages. since medical and nursing staff were infected with sars, hospital intensive care units (icus) could not be run safely, forcing emergency departments to close down (caulford,2004; ahmad,2003; parry,2003) . this was followed by the middle east respiratory syndrome coronavirus (mers-cov) outbreak that occurred in saudi arabia j o u r n a l p r e -p r o o f in july 2012. according to statistics from the who, there were 2,494 cases of mers worldwide from september 2012 to november 2019, involving 24 countries and regions (who,2019) . the global death toll due to mers was 858, with a case fatality rate of 34.4%, which was even higher than that for sars (who mers-cov research group,2013; assiri et al.,2013) . since auris are a serious threat to the health of the world's population and are a large significant obstacle to achieve the goal of building healthy cities via the who healthy cities project (tsouros,1995) , strengthening preventive and control measures for auri diseases is vital to reducing the global socioeconomic burden and improving human health globally, paving the way towards building healthy and smart cities. the transmission of auris in humans is generally believed to occur in three ways: 1) inhalation of liquid droplets containing the virus; 2) close contact with infected persons; and 3) contact with surfaces contaminated with respiratory viruses. moreover, aerosol transmission has been known to play an important role within enclosed spaces. many traditional auris, such as influenza and tuberculosis, as well as many emerging infectious diseases are spread by the airborne route. a retrospective cohort study conducted in hong kong in 2003 suggested that aerosol transmission may have been an important transmission route during the sars epidemic (yu et al.,2004) . other studies reported that mers-cov infection may have been airborne (, , o, zhang, ma, & zhou,2020; ergenekon, mustafa, & vedat,2014) . since the coronavirus disease 2019 j o u r n a l p r e -p r o o f pandemic has spread rapidly, aerosol transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has come into focus. who health officials stated that there is still insufficient evidence that sars-cov-2 is transmitted through the air, except in certain medical settings such as during intubation. to date, some studies have reported the presence of sars-cov-2 particles in the air in isolation rooms from hospitals treating covid-19 patients (yuanfang j,2020; guo, wang, zhang, li, & chen,2020; joshua l. santarpia, 2020; liu, 2020) , but other similar studies in singapore and hong kong have failed to detect sars-cov-2 particles in the air (cheng, 2020; ong, 2020) . additional retrospective studies conducted as the sars-cov-2 epidemic progressed demonstrated that airborne transmission was one of the most likely mechanisms explaining the spatial pattern of infections. this contention has been highlighted in an open letter signed by 239 scientists from 32 countries that was featured in the journal of clinical infectious diseases on july 6. three days later, the who published a scientific brief on the web and said that the transmission of sars-cov-2 by the aerosol route has not been demonstrated, and much more research is needed. therefore, it is crucial to find more evidence to confirm whether sars-cov-2 is transmitted through the air. although all patients in the previous studies had tested positive for sars-cov-2 and had covid-19, little attention has been paid to the follow-up of recovered patients. there have been some reported cases of patients who recovered from covid-19 but tested positive again a few days later, even though they were quarantined after being discharge, and the re-detectable positive (rp) rate was 14.5% (lan et al.,2020) . the reason for this j o u r n a l p r e -p r o o f observation remains unclear, and it is uncertain whether rp patients could cause new infections after being discharged. however, even though the pandemic has been largely controlled in several countries such as china and south korea, rp patients may increase the risk of infection in cities because there are a growing number of patients who tested negative and were then discharged. in addition, there is no environmental evidence confirming whether recovered patients are still shedding sars-cov-2 particles; therefore, urgent investigations are needed because it could be one of the reasons for the observed rp patients. therefore, our study aims to 1) determine whether sars-cov-2 particles are present in the indoor air, with an objective to test the hypothesis of airborne transmission of sars-cov-2, and 2) determine whether recovered patients are still shedding sars-cov-2 particles, thus providing much-needed environmental evidence for the management of covid-19 patients during the recovery period. guizhou province is located in southwest china. the first covid-19 patient was reported on january 22, 2020, and the last patient was reported on march 16, 2020 ( changing gloves between units that are not removed before exiting the unit, limiting entry and exit into the icu by staff, and outfitting all units with negative pressure equipment. the icu was routinely cleaned three times daily at 7:00, 12:00, and 17:00. routine cleaning included sweeping the floor, wiping the tables with 1,000 mg/l chlorine-containing disinfectant, clearing rubbish, and sterilizing the indoor air for 30 min using an ozone disinfection machine. additionally, the surfaces of all objects were wiped using 75% medicinal alcohol when staff were free. the sampling was performed on march 11, 2020 at 19:00, two hours after the completion of routine cleaning, when only one ready-for-discharge patient was isolated and under observation in the icu at jiangjunshan hospital. two types of samples were taken as shown in fig. 2 : surface samples were taken from locations a, b, c, d, and e, and high-volume air samples were taken from locations f and g. high-volume air samples were collected using a wa 400 portable viral aerosol sampler (dingblue tech, inc., http://www.dingbluetech.cn/) at 400 l/min for 15 mins (23±1°c, -15 pa, rh 45%), while the patient was present and was not wearing a mask. air was pumped across an air collection tube and collected into a sterile tube containing 3 ml of viral transport media. each air collection tube was collected independently to avoid cross-contamination. according to the code for indoor environmental pollution control j o u r n a l p r e -p r o o f of civil building engineering gb50325-2010 (2013 (china, 2013) published by the ministry of housing and urban-rural development of the people's republic of china and the number and density of patients in the icu at jiangjunshan hospital, the two sampling points (f and g) were designed as 1) 0.5 m away from the patient's bedside in the isolation room and 2) in the middle of the staff ppe dressing room. the sampling height for both locations was 1.5 m. sampling points were chosen to avoid medical equipment, walls, doors, aisles, and air conditioning vents to prevent obstruction and interference. to evaluate the efficacy of routine cleaning, we used sterile synthetic fibre swabs with plastic shafts to collect surface environmental samples. swabs were premoistened with viral transport media and wiped over the surface of the object for a few seconds and then placed immediately into sterile tubes containing 3 ml of viral transport media. each swab was collected independently to avoid cross-contamination. all samples were stored at 4°c and shipped to the testing laboratory in ice packs within four hours of sampling to test for sars-cov-2. test results were available on the same day. quantitative analysis of the sars-cov-2 ribonucleic acid (rna) genome was carried out by quantitative real-time polymerase chain reaction (qrt-pcr) according to the otherwise, the sample was considered to be negative if both targets had no apparent logarithmic phase or if the ct value was ≥40 or indeterminate. in this study, an "intense positive" constituted a positive result from both the orf1ab gene and the n gene of sars-cov-2. in addition, tests were repeated when a sample tested positive. the sample was confirmed to be positive when the second test was also positive. assessment of the patient epidemiological history revealed that the patient, a 64-yearold female, returned to guizhou from wuhan, china, on january 12, 2020, and developed symptoms, including low fewer and cough, on february 11, 2020. on february 12, 2020, she was hospitalized after sars-cov-2 infection was confirmed at jiangjunshan hospital. the first post-admission chest computed tomography (ct) imaging showed that the j o u r n a l p r e -p r o o f patient had 1) lobular patchy hyperdense shadows in both lungs, 2) emphysema, 3) cardiomegaly, and 4) aortic sclerosis. a routine blood test revealed the following counts: total white blood cells, 7. 4×109/l; neutrophils, 77.2%; and lymphocytes, 13.4%. on february 19, 2020, the patient was transferred to the icu due to deterioration in her condition. after treatment, her condition improved, and she tested negative twice for sars-cov-2 on march 8 and 9, 2020. she then continued to be isolated and under observation for an additional 14 days (fig. 3) . a total of seven samples were taken, and the positive test rate for sars-cov-2 was 14.29% (1/7). in this study, all samples taken from surface and air environments from the staff ppe dressing room tested negative, but the air sample from the isolation room was intensely positive, as shown in table 1 . in this study, we found that sars-cov-2 particles were present in the air of an isolation room in the icu. the findings agreed with those reported by other studies that found that sars-cov-2 was present in indoor air (yuanfang j,2020;guo, wang, zhang, li, & chen,2020;joshua l. santarpia, 2020; liu, 2020) . this result is an underlying piece of evidence supporting the hypothesis of airborne transmission of sars-cov-2. moreover, our findings constituted the first piece of environmental evidence that patients who tested negative for sars-cov-2 may still be shedding the virus. since reported median estimates of the half-life of sars-cov-2 in aerosols range from approximately 1.1 to 1.2 hours (95% confidence intervals of 0.64 to 2.64) (van doremalen et al.,2020), our j o u r n a l p r e -p r o o f samples were taken 48 hours after the patient had tested negative for sars-cov-2 and two hours after the last disinfection operation at jiangjunshan hospital. it may be inferred that the aerial presence of sars-cov-2 in our study was not a result of retention. it is known that recovered patients who are still discharging the virus may have an increased risk of rp. although the reason for rp remains unclear, potential major factors include virology, immunology, and sampling methodologies. a recent pathological research report found that sars-cov-2 could remain in the lung tissue of a ready-for-discharge patient (yao,2020) , and we believe this study also supported the current virologic understanding of rp occurrence among covid-19 patients, i.e., the false negatives caused by faulty sampling methodologies, viral residues in the patient's body, intermittent viral release in the patient's body (xing y-h,2020), and viral distribution (wölfel r,2020; xia, tong, liu, shen, & guo,2020) . negative nasopharyngeal swab tests may result in disregarding the above factors. another study reported that supplementing negative results with an anal swab test at discharge failed to reduce rp occurrence in covid-19 patients (liao, jianghong an xuejiao, et al., 2020) . this study also analysed the differences in anti-sars-cov-2 igg and igm antibody levels in both rp and non-rp patients at the time of discharge but did not find any significant differences. according to the guidelines for the diagnosis and treatment of pneumonia caused by sars-cov-2 (sixth edition) published by the national health commission of the people's republic of china (china b,2020) , the discharge criteria for recovered patients included a return to normal temperature for more than three days, a significant improvement in respiratory symptoms, a significant absorption of pulmonary lesions on chest ct unlike other studies, sars-cov-2 was not detected on object surfaces in our study (cheng et al.,2020b; guo, wang, zhang, li, & chen,2020 ;joshua l. santarpia,2020; liu,2020; ong et al.) , which may be due to differences in routine cleaning operations that could lead to different levels of sars-cov-2 in the environment; for j o u r n a l p r e -p r o o f example, the frequency of disinfection at jiangjunshan hospital was higher than that reported in other locations (guo, wang, zhang, li, & chen,2020; liu,2020; ong et al.) . additionally, the number and density of patients in the icu at jiangjunshan hospital were much lower than those at other hospitals, indicating a markedly reduced risk of sars-cov-2 contamination in the environment. moreover, many studies have shown that factors such as temperature and humidity can affect the spread of the virus in enclosed indoor environments (feng, bi, zhang, cai, & huang,2020; zhang et al.,2019) . some studies reported that the rate in which sars-cov-2 spread was accelerated in cold and dry conditions (casanova, jeon, rutal, , weber, & sobsey,2010; chin, chu, perera, hui, & poon,2020) . within an infected person, respiratory droplets with high humidity are produced under high humidity conditions by the atomization of human secretions containing the virus as they pass the air passage through out lung during coughing and sneezing. these droplets undergo size reduction due to the drying of water content if the surrounding air humidity and temperature are very low. in contrast, the size of the droplet is not reduced if the surrounding air humidity and temperature are high (chen, liu, lin, & chen,2015; liu, wei, li, & ooi,2017; wu, zhang, & zhang,2016) . at the same time, the virus could become diluted with the surrounding water content in the air and become less active. therefore, a humid environment may be beneficial for maintaining droplet size and reducing viral activity and may therefore play a larger role than the temperature of the indoor environment where a sars-cov-2-infected patient is located. in our study, the relative humidity in the icu at jiangjunshan hospital was 45%, which may imply that the droplets shed by the ready-for-discharge patient may undergo size j o u r n a l p r e -p r o o f reduction due to the surrounding air conditions being dry. as the expiratory droplet size of the sars-cov-2 distribution had a peak size between 0.25 and 1.0 μm (liu,2020) , viruses with aerodynamic diameters smaller than 0.5 μm can remain suspended in the air instead of quickly settling onto the surfaces of objects, and exhaled droplet nuclei can be transmitted between occupants via the airborne route (ai, huang, & melikov,2019) . additionally, although our sampling was performed two hours after the last routine cleaning, the frequency of disinfection of object surfaces was higher than that of the air in the icu at jiangjunshan hospital, which may have resulted in the air samples but not the object surfaces testing positive for sars-cov-2. to solve the problem of the longterm presence of sars-cov-2 viral aerosols in enclosed environments and reduce the risk of sars-cov-2 infection, we believe that it is necessary to increase the frequency of indoor air disinfection and to keep the relative humidity of the indoor environment at a high level to maintain droplet size, thereby reducing viral activity. our study has several limitations. first, viral cultures were not performed to demonstrate its viability. second, the sample size was small due to the low incidence of covid-19 in guizhou province. moreover, due to the lack of consumables (air collection tubes), our research began during the late stage of the epidemic in guizhou, and we were unable to continue performing air sample tests. the concentration of airborne sars-cov-2 was not quantified. the aerodynamic size distribution of sars-cov-2 aerosols was not evaluated. additional studies are needed to confirm our observations. our findings revealed the presence of sars-cov-2 in the indoor air of the icu and indicate that the virus may be shed via aerosol for days, even after a patient has tested negative. this finding may be one of the reasons for the observation of rp patients. this finding provides valuable empirical information and environmental evidence for the effective management of covid-19 patients during the convalescent period. we suggest that it is necessary to extend the detection time for covid-19 patients at the time of discharge and to continually monitor the air surrounding recovered covid-19 patients for the presence of sars-cov-2. additionally, the frequency of indoor air disinfection needs to be increased, and the indoor relative humidity should be kept at a high level. our findings can be used to improve both routine cleaning during the outbreak and clinical and disease management guidelines for discharged covid-19 patients. since icus are required to maintain negative pressure, close off the isolation ward, and isolate patients in the wards for days at a stretch, it is not possible to use traditional architectural design measures to reduce airborne virus concentrations such as adequate and effective ventilation and ultraviolet germicidal lamps. additionally, it is difficult to limit the number and density of patients in icus and avoid overcrowding during emergency situations like the early stage of the epidemic in wuhan. therefore, to enhance the isolation and care facilities for infectious diseases, smart designs for health and quarantine facilities and icus are receiving more attention. attention should be given to the physical elements and environments of buildings that had been designed to reduce the risk of infectious diseases such as yersinia pestis, tuberculosis, typhoid, polio, and flu during historical epidemics prior to the development of medicines (megahed & ghoneim,2020) . in recent years, smarter health and quarantine facilities have been j o u r n a l p r e -p r o o f designed. in late 2014, a total of 56 hospitals in the united states of america were designated as specially designed, high-level isolation units (hlius) equipped with an advanced infrastructure, advanced laboratory capabilities, and well-trained staff by state governments and federal public health authorities to care for patients with highly hazardous communicable diseases (hhcds) (herstein et al.,2018) . to improve the ability to prevent, treat, and manage viral respiratory infections and prevent future crises similar to covid-19, we suggest that the following points be considered when designing smart buildings and hospitals in the future: 1) development of new air disinfection equipment with better disinfection efficiency and observable sustained effects; 2) strengthening of the cooperation and communication between scientists belonging to different fields such as environmental engineering, architecture, clinical medicine, and public health to design smarter and more effective icus that conform to health and isolation standards; 3) integration of information communication technology with hospital operations; and 4) implementation of the concept of the internet of things within smart hospitals in smart cities (silva, khan, & han,2018) . the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. we would like to thank prof. maosheng yao from peiking university and dingblue tech, inc. for their support in providing the sampling devices and technical support in this study airborne transmission of exhaled droplet nuclei between occupants in a room with horizontal air distribution clinical characteristics of the recovered covid-19 patients with re-detectable positive rna test (257): newsrx llc comparing 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effects towards sustainable smart cities: a review of trends, architectures, components, and open challenges in smart cities the who healthy cities project: state of the art and future plans aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 mers monthly summary virological assessment of hospitalized cases of coronavirus disease applied thermal engineering, 99, 938-943 secretions of patients with sars-cov-2 infection pathological evidence for residual sars-cov-2 in pulmonary tissues of a ready-for-discharge patient evidence of airborne transmission of the severe acute respiratory syndrome virus distribution of droplet aerosols generated by mouth coughing and nose breathing in an air-conditioned room this research did not receive any specific grant from funding agencies in the public, j o u r n a l p r e -p r o o f key: cord-325902-33pxylb3 authors: hemida, maged gomaa title: middle east respiratory syndrome coronavirus and the one health concept date: 2019-08-22 journal: peerj doi: 10.7717/peerj.7556 sha: doc_id: 325902 cord_uid: 33pxylb3 middle east respiratory syndrome coronavirus (mers-cov) is one of the major threats to the healthcare systems in some countries, especially in the arabian peninsula. mers-cov is considered an ideal example of the one health concept. this is due to the animals, especially dromedary camels, play important roles in the transmission and sustainability of the virus, and the virus can be transmitted through aerosols of infected patients into the environment. however, there is some debate regarding the origin of mers-cov either from bats or other unknown reservoirs. the dromedary camel is the only identified animal reservoir to date. these animals play important roles in sustaining the virus in certain communities and may act as an amplifier of the virus by secreting it in their body fluids, especially in nasal and rectal discharges. mers-cov has been detected in the nasal and rectal secretions of infected camels, and mers-cov of this origin has full capacity to infect human airway epithelium in both in vitro and in vivo models. other evidence confirms the direct transmission of mers-cov from camels to humans, though the role of camel meat and milk products has yet to be well studied. human-to-human transmission is well documented through contact with an active infected patient or some silently infected persons. furthermore, there are some significant risk factors of individuals in close contact with a positive mers-cov patient, including sleeping in the same patient room, removing patient waste (urine, stool, and sputum), and touching respiratory secretions from the index case. outbreaks within family clusters have been reported, whereby some blood relative patients were infected through their wives in the same house were not infected. some predisposing genetic factors favor mers-cov infection in some patients, which is worth investigating in the near future. the presence of other comorbidities may be another factor. overall, there are many unknown/confirmed aspects of the virus/human/animal network. here, the most recent advances in this context are discussed, and the possible reasons behind the emergence and sustainability of mers-cov in certain regions are presented. identification of the exact mechanism of transmission of mers-cov from camels to humans and searching for new reservoir/s are of high priority. this will reduce the shedding of the virus into the environment, and thus the risk of human infection can be mitigated. the main reason behind developing this article is to summarize the current understanding about mers-cov in the context of the one health concept. in this article, i highlight the known information about the mers-cov infection and its pathogenesis in humans, the patterns of mers-cov in dromedary camels, the potential roles of other animals in the transmission cycle of mers-cov, and the interaction of mers-cov/humans/animals. i elaborate on how some strategies can be used to stop or reduce the frequencies of mers-cov outbreaks based on the one health concept, identified some gaps in the literature, and drew conclusions. the one health concept established to ensure the good health and well beings of the human, animal and the environment. human health is mainly affected by environment health as well as animal health (lerner & berg, 2015) . for this review article, i conducted a literature search of the most up-to-date published articles on mers-cov in the past 7 years. first, i focused the introduction section on the historical background of coronaviruses and the one health concept. then, i highlighted the most up-to-date literature from pubmed central, google scholar and researchgate on the interaction of mers-cov/humans/animals. i identified some important gaps in the research dealing with mers-cov/human/environment in the context of the one health concept. i also summarized the current acceptable theories on the emergence and evolution of mers-cov. finally, i highlighted progress to date in the control of mers-cov. historically, mers-cov was first identified in saudi arabia in a patient suffered from severe pneumonia and shortening of breath. the virus was called the novel coronavirus at that time (zaki et al., 2012) . another retrospective study conducted in jordan early 2012 revealed the detection of this novel coronavirus in 11 patients. eight out of them were from the health care workers (hijawi et al., 2013) . coronaviruses are a large group of viruses causing many health problems (respiratory, enteric, and nervous syndromes) in various species of animals and humans. six human coronaviruses that have been identified to date (hcov-229e, hcov-oc43, hcov-nl-63, hcov-huk-1, sars-cov, and mers-cov). two out of them emerged in the past 15 years (lau & chan, 2015) , namely, the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). sars-cov emerged in 2003 in china and spread to many countries throughout the world (peiris et al., 2003) . approximately 8,000 people were infected, and 10% of them died (aronin & sadigh, 2004) . only 9 years later, mers-cov emerged in saudi arabia (zaki et al., 2012) . this is a relatively short period for the emergence of a new coronavirus. one of the main reasons behind the rapid emergence of new coronaviruses is the poor proofreading capability of their rna polymerases (hofer, 2013) . this is in addition to the possibility of the recombination of different coronaviruses (makino et al., 1986) , and it will not be surprising if new coronaviruses emerge in the near future. mers-cov continues to pose great challenges to the healthcare system of some countries in the middle east and arabian peninsula. since its discovery late in 2012 (zaki et al., 2012) , there are ongoing reports to the world health organization (who) from some countries in the middle east, especially the arabian peninsula, with spread to other countries around the globe. according to the latest who statistics, there have been a total of 2,428 laboratory-confirmed cases of mers-cov infection including at least 838 deaths (reported case fatality rate of 35.0%) (who, 2018) . the continuous ongoing reports on mers-cov suggesting the presence of some factors favor its sustainability in certain regions. there are many uncertain aspects of the virus evolution, pathogenesis, and transmission cycle. unfortunately, recently, there was some decline in the rate of research on the virus from different aspects (hemida et al., 2017b) . this hampered the production of new data about the mers-cov from different aspects. below, i summarize the current understanding of the virus in the context of the one health concept. the one health concept is an interesting concept outlining the close interaction among humans, animals and the environment (destoumieux-garzon et al., 2018) . currently, there are two coronaviruses candidates representing the one health concept, sars-cov and mers-cov. animals play important roles in the transmission cycle of both viruses (alshukairi et al., 2018; wang et al., 2005) . both viruses were proven to be of zoonotic origin (gao et al., 2016) . the palm civet cat played important role in the transmission cycle of sars-cov (wang et al., 2005) . some patients proved to visit one restaurant serving the civet cats as a meal (wang et al., 2005) . culling of the civet cats resulted in marked decline in the reported sars-cov cases and now become extinct. many studies made a direct link between the exposure to camel and its meat and milk products and mers-cov human cases . several studies reported the presence of mers-cov specific antibodies in sera of human came in close contact with camels reusken et al., 2016) . meanwhile, mers-cov was isolated from air pf positive dromedary camel herds in saudi arabia (azhar et al., 2014) . mers-cov may infect a wide group of people ranging from very young ages, even infants less than one year of age, to 109 years of age (cdc, 2016) . however, children are less likely to be infected with mers-cov when compared to adults and, if infected, they tend to have asymptomatic or mild disease (arwady et al., 2016) . the reason for this is still not entirely clear and requires further study. the case fatality rate is always very high in case of the immunocompromised infected patients especially those who are suffering from chronic diseases such as cancer, diabetes, blood pressure, kidney problems, etc. (arwady et al., 2016) . human-to-human transmission is reported in many cases. mers-cov replicates efficiently in various in vitro and ex vivo models (chan et al., 2014) . moreover, many family clusters and hospital outbreaks were reported in the past 5 years (arwady et al., 2016; drosten et al., 2014; memish et al., 2013) . this confirms the potential spread of mers-cov among those who are in close contact in the population (mollers et al., 2015) . the most at-risk groups are healthcare workers including nurses, medical doctors and other hospital staff and the elderly with underlying chronic diseases (arabi et al., 2014) . the prevalence rate of mers-cov in primary cases among males is relatively higher than that of females (darling et al., 2017) , which may be because exposure to infected dromedary camels is much higher in males than in females. mers-cov infection triggers some unique interferons and cytokine gene expression profiles. the virus seems to be a poor interferon inducer (chan et al., 2014) . this suggests the potential immune evasion strategies triggered by the virus to hijack the host immune system and may be responsible for the high fatality rate, at least in part. viral spreading among people seems to not yet be very efficient. those in close contact are among the at-risk groups for infection (drosten et al., 2014) , as observed in many hospital outbreaks as well as family clusters (alfaraj et al., 2018; choi et al., 2017; xiao et al., 2018) . this suggests that transmission of the virus among people requires exposure to a high viral load, which will sometimes produce active infection in people who are in close contact. several mers-cov family clusters have been reported (drosten et al., 2014) . interestingly, mers-cov is reported in the dromedary camels in many african countries (egypt, nigeria, tunisia, and ethiopia), but no primary human cases have been reported in these countries to date (ali et al., 2017; roess et al., 2016; van doremalen et al., 2017) , which may be related to some variation in the circulating asian and african strains of mers-cov. some important deletions in the mers-cov currently circulating in dromedary camels from africa were recently reported (chu et al., 2018) . these deletions may explain at least in part the reason behind the variations in the pathogenesis among the asian and african strains of mers-cov. another potential reason behind the absence of human cases in the african countries is the diverse cultural habits among people in africa and the arabian peninsula (fao, 2016) . people in arabian peninsula get in more close touch with camels during the camel show, sports, trade than in africa. this make the human risk of exposure much higher in the ap than africa. mers-cov infection varies from severe respiratory illness accompanied by a high fever and respiratory distress to mild asymptomatic cases. patients are usually admitted to the intensive care unit (icu) and provided with a source of oxygen. most cases result in pneumonia, which is fatal in almost 40% of the affected patients (hong et al., 2017; rubio et al., 2018) . some patients may develop renal failure [13] . several mers-cov travel-associated infections were in many cases associated with the middle east (bayrakdar et al., 2015; rubio et al., 2018) . among these reported was the korean outbreak in early 2015 (choi et al., 2017; kim, andrew & jung, 2017; xiao et al., 2018) . one korean citizen visited some countries in the middle east and then returned home ill. this person visited several healthcare facilities in korea. this resulted in the largest mers-cov human outbreak outside the arabian peninsula (ap) (xiao et al., 2018) . this outbreak confirmed the human-to-human transmission. during this outbreak, mers-cov was isolated from air samples from the hallways of the healthcare facilities close to the hospitalized patients (xiao et al., 2018) . this at least explains in part the rapid development of mers-cov hospital outbreaks. since the discovery of mers-cov late in 2012 (zaki et al., 2012) , many research groups have searched for its potential animal reservoir/s. dromedary camels are the only currently proven reservoir for mers-cov (hemida et al., 2014; hemida et al., 2017b; reusken et al., 2014; reusken et al., 2016) . interestingly, others were able to trace the virus back 30 years ago in dromedary camel specimens in retrospective studies (corman et al., 2014; hemida et al., 2014; reusken et al., 2014) . all these data suggest that the virus has been circulating for decades without being recognized. although the actual and typical clinical features of the mers-cov natural infection in dromedary camels is not well documented to date, very few studies reported these patterns under experimental infection approaches (adney et al., 2014) . based on these findings, camels do not show any pathognomonic signs despite a subtle fever and mild nasal discharge for up to 6 days post-infection (dpi) (hemida et al., 2014) . meanwhile, shedding of the infectious virus was reported in the experimentally infected camels started at 2 dpi up to the 7th dpi (adney et al., 2014) . interestingly, viral rna was still detected at 35 dpi (adney et al., 2014) , though it is not known whether the viral rnas may act as potential sources of infection similar to some other positive-sense rna viruses. no viral shedding in the oral secretions, rectal swabs, urine, or sera of these animals has been reported (adney et al., 2014) , in contrast to the detection of the virus in the fecal specimens and swabs under natural viral field infection (hemida et al., 2014) . these finding suggesting differential patterns of mers-cov infection between natural and experimental approaches. further studies are required to understand the natural mers-cov infection in dromedary camels, which may be achieved by conducting long-term longitudinal studies as well as careful monitoring of the virus infection in large populations of camels. on necropsy examination of mers-cov, experimentally infected dromedary camels revealed only mildto-moderate inflammatory reactions in the upper respiratory tract (khalafalla et al., 2015) . detection of the viral antigens in the tissue sections of the turbinate bone and the upper respiratory tract was reported (adney et al., 2014) . interestingly, seroconversion of the inoculated animals was reported to begin at 14 dpi (hemida et al., 2014) , indicating that mers-cov induces a robust humoral immune response after infection. more recently, one longitudinal study reported the possibility of mers-cov infection in seropositive animals. this raises concern about the role of the antibodies in the protection of the mers-cov infection (hemida et al., 2017a) . it seems that all the members of the family camelidae (dromedary, alpaca, and llamas) are susceptible to mers-cov infection (corman et al., 2014; vergara-alert et al., 2017) . david et al. (2018) reported the presence of antibodies against mers-cov in some alpacas and llamas in israel but only used commercial elisa kits, and they did not address the possibility of cross-reactivity with other coronaviruses especially bcov. it had been previously showed that there is clear cross-reactivity between mers-cov and bcov in dromedary camels (david et al., 2018) . interestingly, one study showed an absence of any detectable antibodies of mers-cov in the sera of bactrian camels (chan et al., 2015) , though this is the only study to report this finding concerning the seronegativity of bactrian camels to mers-cov. it is not well known whether the absence of detectable mers-cov antibodies in the sera of bactrians camels is due to the geographical location of the tested animals in mongolia, far from the middle east and africa. this may be supported by similar findings in dromedary camels in australia and the canary islands (crameri et al., 2015) . another possibility is that this might be due to some genetic factors, which contribute to the resistance of bactrians to mers-cov infections; this point is worthy of further investigation. experimental mers-cov infection in both alpacas and llamas showed a similar pattern to that of dromedary camels (crameri et al., 2016; vergara-alert et al., 2017) , which suggested that both animals might act as a model animal for the study of mers-cov in vivo. however, the experimental infection of pigs with mers-cov did not reveal as much infection as that reported in alpacas and llamas (vergara-alert et al., 2017) . active mers-cov particles were neither retrieved from the experimentally infected animals nor from the close contact non-infected animals during the duration of this study (vergara-alert et al., 2017) . this result suggested that pigs might not play an active role in the transmission of mers-cov. although bats are considered the main reservoir for many coronaviruses, their roles in the mers-cov still need further clarifications. one study reported the presence of mers-cov in one specimen collected from bats in saudi arabia (memish et al., 2013) . the genome sequence of this particular virus showed almost 100% identity to a mers-cov index case (memish et al., 2013) . more recently, jamaican fruit bats were found to be permissible for mers-cov infection (munster et al., 2016) . however, mers-cov-infected bats did not show any apparent clinical signs; however, viral shedding was reported in the swabs from bats up to 9 dpi. the clinical profiles and viral shedding curve during the course of the mers-cov infection in these bats look similar to that of dromedary camels (munster et al., 2016) , yet the amount of infectious viral shedding in bats is less compared to that in dromedary camels. this species of bat is not the most relevant for mers-cov infection, but this study offers some insights into the molecular pathogenesis of mers-cov in bats. interestingly, another study revealed the expression of mers-cov receptors (dipeptidyl peptidase-4, dpp4) in the respiratory and digestive tracts of some insectivorous bats ). an interesting study tested the potential roles of other species in the transmission of mers-cov such as cattle, sheep, goats, donkeys, buffaloes, mules, and horses from egypt, tunisia and senegal (kandeil et al., 2019) . this study revealed the presence of neutralizing antibodies in the sera of some sheep and goats. meanwhile, viral rna was detected in swabs collected from some sheep, goats and donkeys from these countries (kandeil et al., 2019) . several attempts were made to identify an appropriate experimental animal model for mers-cov. the syrian hamster was non-permissive to mers-cov infection (de wit et al., 2013) . experimental infection of this animal neither develops any clinical signs or pathology nor produces any cytokines after infection (de wit et al., 2013) . this was in contrast to new zealand white rabbits, which showed active infection after inoculation with the mers-cov (monchatreleroy et al., 2017) . furthermore, both rhesus macaques and common marmosets supported mers-cov infection (yu et al., 2017) . additionally, both the transgenic and the transduced mice expressing the dipeptidyl peptidase 4 human receptors worked as a model for mers-cov studies (zhao et al., 2015) . interestingly, a new study reported the seropositivity of some sheep and goat to mers-cov from tunisia, senegal and egypt (kandeil et al., 2019) . same study reported the detection of the viral rnas in samples from cow, sheep, goat and donkeys from egypt (kandeil et al., 2019) . this highlights the importance of continuous surveillance and searching for new reservoir/s for the mers-cov transmission cycle. it is now well accepted that human exposure to mers-cov-infected dromedary camels is a predisposing factor to human infection, particularly in immunocompromised people (zumla et al., 2015) . based on the latest who reports, the prognosis of mers-cov infection is poor for elderly patients who have chronic diseases such as cancer, diabetes, kidney failure, etc. (arabi et al., 2014) . transmission of mers-cov from dromedary camels to humans has been proven indirectly in some previous reports (azhar et al., 2014) . one study showed strong evidence of direct transmission of mers-cov from an infected camel to its owner, which was confirmed by comparing the viral genome sequencing of the virus isolated from the infected dromedary camel to that isolated from its owner. both viruses were almost a 100% match (azhar et al., 2014) . meanwhile, this study reported the detection of mers-cov nucleic acid in air samples from the indicated dromedary camel barn during the active course of the viral infection (azhar et al., 2014) . there is a debate about the role of the dromedary camel's milk and meat products and by-products in the transmission of mers-cov. experimental introduction of mers-cov to raw milk revealed little difference between the virus stock in milk and that kept in dmem (van doremalen et al., 2014) . due to their culture, some people in the middle east would drink raw camel milk in efforts to seek treatment for some diseases such as diabetes. the authors acknowledge that mers-cov was introduced into the dromedary camel milk at a high dose and that the viral rna was detected in a limited concentration in the camel's milk (van doremalen et al., 2017) . thus, drinking the raw camel milk poses a great risk to the people consuming this milk without any heat treatment or pasteurization (van doremalen et al. 2014; (zhou et al., 2017) . one study connected the infection of some people to the drinking of the milk from one infected camel (memish et al., 2015) . however, another study was conducted in qatar to assess the possibility of acquiring the infection from contaminated teats and udders of infected she-camels during the process of milking , though no active mers-cov shedding in milk has yet to be reported. further studies are encouraged to come to a conclusion about the potential role of raw camel milk in the transmission of mers-cov. nonetheless, the role of camel meat in the transmission of mers-cov has not been studied carefully to date. thus, special attention should be paid to the efficient cooking of camel meat and its products as well as thorough boiling of camel milk. it is suggested that people not drink raw camel milk to avoid any risk of infection not only with mers-cov but also with other pathogens such as brucellosis (garcell et al., 2016) . some studies reported that mers-cov is one of the occupational zoonotic viral diseases, as was claimed based on some studies investigating the seroconversion of some at-risk group of people to mers-cov. this study reported the presence of specific mers-cov antibodies in approximately 3% of the workers in some slaughterhouses in qatar (farag et al., 2015) . on the other hand, some studies reported the absence of any detectable antibodies in the sera of some herdsmen, veterinarians, and slaughterhouses in saudi arabia (hemida et al., 2015) . one possible explanation for the variations between the two studies is the difference in the sensitivity of the techniques used. those two studies used two different techniques to report the presence/absence of the mers-cov antibodies in the sera of at-risk people (farag et al., 2015; hemida et al., 2015) . regardless, further investigation on a large-scale basis is required to solidify this conclusion about mers-cov. there is more to be known about the molecular biology of mers-cov. identification of the dpp-4 as viral receptors does not rule out the presence of other co-receptors or transcription/translation factors that favor the virus infection in a certain host. there are many immune evasion strategies triggered by mers-cov to hijack the host immune responses, and the mechanisms of such strategies have not been well studied. moreover, there are many unknown aspects especially in the context of the mers-cov/human/animal interaction. meanwhile, some studies were conducted on a small scale or with low numbers of animals/specimens and reported some important conclusion. these studies need further confirmation, and refinement of some of these observations is urgently needed in the near future. here, we highlight some gaps in the research regarding the evolution and transmission of mers-cov. presumably, there might be an unidentified reservoir in the transmission cycle of mers-cov. although respiratory infection still is the main route of mers-cov infection, the exact mechanism of transmission of mers-cov from dromedary camels to humans is still not well understood. the possibility of another reservoir in the transmission cycle of mers-cov has not been ruled out. thus, there might be a missing link in the chain of the human/camel interaction. meanwhile, the exact modes of transmission of mers-cov from dromedary camels to humans have not been well clarified, and the typical pattern of the natural mers-cov infection in dromedary camels has not been well studied. additionally, the potential role of most camel secretions and excretions has not been fully understood. the seroprevalence of mers-cov was reported in the dromedary camels from different countries in africa and asia (ali et al., 2017; hemida et al., 2014) , though feral camels in australia and the canary islands were found to be seronegative (crameri et al., 2015) . the reason behind this phenomenon may be due to these regions being isolated lands and away from the mena region as described above; it may also be due to the absence of an active camel movement between the middle east and africa and these regions of the world. very few studies reported the cross-reactivity between mers-cov and other coronaviruses such as the bovine coronavirus (bcov) that might infect dromedary camels. it is unknown if this might be the reason behind the high seroprevalence of mers-cov among the dromedary camel population. this may be due to the high frequency of exposure to the mers-cov infection during the camel's life, the cross-reactivity of other coronaviruses, or an unknown mechanism related to the dromedary camel's immune system. these considerations require further studies. there is ongoing demand for the development of novel diagnostic assays for coronaviruses, and special interest should be paid to those techniques that enable the simultaneous detection of the viral nucleic acids and those that can simultaneously distinguish between the antibodies for several coronaviruses. furthermore, it is not well understood why only the bactrian camels among the family camelidae did not seroconvert to mers-cov infection (chan et al., 2015) . the genetic susceptibility of certain human populations, especially blood-related people, is not clear in the context of mers-cov infection. there are several levels of human exposure to dromedary camels, such as camel attendants, workers in camel abattoirs, veterinarians inspecting their carcasses, and camel owners. those groups of people are in close contact with camels for various amounts of time and are considered to be a high-risk group of people due to the long time they spend in close contact with the dromedary camels. meanwhile, there is an urgent need to develop a risk scoring system for human exposure to the dromedary camels. it is believed that there is some unidentified reservoirs in the context of mers-cov transmission presenting the virus to the community. this virus is able to infect dromedary camels, which act as an amplifier host for the virus, favoring the circulation of the virus in some camel herds. the virus has the ability to circulate among the animals in the same herd and the camel herds in close proximity to them (fig. 1) . mers-cov in camels has the full potential to infect the human especially immunocompromised persons. once the virus infects a human, there is always a possibility of infecting other people, especially closelyrelated individuals (fig. 1) , including household relatives and workers plus healthcare workers such as doctors and nurses. infection depends on the level of exposure to the infected person, and mers-cov infection in humans ranges from very severe cases of pneumonia to death. currently available data indicate that severely infected individuals can shed the infectious virus into the environment (kim et al., 2016) , though there are few data regarding the capacity of mildly infected individuals to transmit the virus. asymptomatic individuals, however, are unlikely to transmit the virus (moon & son, 2017) . there are many factors behind the emergence, sustainability and spread of mers-cov. the presence of an unidentified mers-cov reservoir in the transmission cycle is still considered, and this unknown reservoir may contribute substantially to the suitability of the virus in certain regions. dromedary camels remain the amplifier of the virus; the close contact of these animals to the human population in certain regions of africa and asia may pose a great risk for human infection and indirectly contribute to the spread of the virus. additionally, public animal markets, especially for dromedary camels, may act as an amplifier of the virus. this poses a great risk to the surrounding community. interesting study addressed the mapping of mers-cov cases in association with the environmental conditions and camel exposure (reeves, samy & peterson, 2015) . this study revealed that the virus is transmitted to dromedary camels through an unknown mechanism. the dromedary camels act as amplifying hosts for the virus. mers-cov is transmitted from dromedary camels to humans through the respiratory aerosols and some other unknown mechanisms. the virus is then transmitted among the human population through respiratory routes. the human-to-human transmission has been confirmed. the human-to-camel transmission still needs further clarification. question marks indicate the non-confirmed phenomenon. full-size doi: 10.7717/peerj.7556/ fig-1 camel exposure is a key predisposing factor for some of mers-cov human cases (reeves, samy & peterson, 2015) . the lack of active surveillance programs for respiratory viruses, especially coronaviruses, may result in many subclinical or mild cases of mers-cov being missed in a certain population. these patients may shed the virus in their secretions and may act as a source of infection to other persons in close contact with them. although many mers-cov vaccine and drug candidates are being researched, none are available yet. all these factors may favor the sustainability of mers-cov in certain regions. interestingly, the case fatality rate of mers-cov among the affected population dropped from almost 50% in 2012 to 34% early 2019 (who, 2018), and we may relate this progress in the control of mers-cov over the past 7 years to many factors. first, identification of the main reservoir of the virus, namely, the dromedary camel (hemida et al., 2014) . second, continuous molecular and serological surveillance of mers-cov among the dromedary camel population in the arabian peninsula and africa (corman et al., 2014; farag et al., 2015; hemida et al., 2017a; hemida et al., 2017b; khalafalla et al., 2015; reusken et al., 2014) . currently, testing the population of camels in regional camel markets is associated with shutting down of the market in case of positive shedding of mers-cov by the animals. i believe this will substantially minimize the risk of community-acquired infections through these positive populations. third, vaccination of dromedary camels, especially animals under two years of age, will have a great impact on the reduction of the viral shedding from these animals to the surrounding community. this will also have a great positive impact on the reduction of the number of reported human infections. fourth, there has been progress in the current understanding of viral tropism, pathogenesis, and mode of transmission in the past five years (chan et al., 2014; widagdo et al., 2017) . fifth, new strategies have been adopted to reduce the spread of infection in health care units (rajakaruna et al., 2017) . sixth, some therapeutic and control approaches for mers-cov such as cyclosporine, ribavirin and interferon show promising trends for the treatment of mers-cov-infected patients (al-tawfiq et al., 2014; de wilde et al., 2013) . meanwhile, good progress has been made in screening large numbers of drugs/therapies for the treatment of mers-cov (han et al., 2018; he et al., 2019; niu et al., 2018; totura & bavari, 2019) . this may lead to the development of some effective novel drugs against mers-cov infection in the near future. to stop mers-cov outbreaks, there are several strategies to be adopted in the context of the one health concept. some strategies are related to the animal, while others are related to human health. the main objective is to minimize or stop the viral shedding from dromedary camels to the environment (fig. 2) . this may be achieved in many ways including regular monitoring of the population of dromedary camels. active animal shedders need to be identified, and quarantine measures should applied until they stop shedding the virus. vaccination of young dromedary camel calves should occur during their first 6 months of life, which will minimize the chances of these animals becoming infected and actively passing the virus to older animals and then to the environment. reorganization and reshaping of the camel industry includes allocating the camel markets away from the cities. global awareness concerning the necessity of thorough boiling and cooking of the camel milk and meat products, respectively, should take place. animal abattoirs should be established far away from large cities. they should not use mixed-animal platforms, and each platform should deal with one species of animal. thorough decontamination of animals' biological wastes in abattoirs should occur using the appropriate standard protocols. regular surveillance of mers-cov among the population especially during the active peak of virus shedding by the animals should occur during november to april every year. people who are in close contact with the camels should wear proper personal protective equipment at all times. at almost 7 years after its emergence, there are ongoing reports of mers-cov infection from time to time. this may be related to many unknown aspects of the viral evolution and pathogenesis. some of these unknown aspects are the following. this work was funded by a grant from the king abdul-aziz city for science and technology (kacst), through the mers-cov research grant program (number 20-0004), which is part of the targeted research program (trp). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the following grant information was disclosed by the author: king abdul-aziz city for science and technology (kacst). mers-cov research grant program: 20-0004. targeted research program (trp). syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters the one health concept: 10 years old and a long road ahead transmission of merscoronavirus in household contacts high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases from sars to mers: evidence and speculation outbreaks of brucellosis related to the consumption of unpasteurized camel milk neutralizing monoclonal antibodies as promising therapeutics against middle east respiratory syndrome coronavirus infection enhanced ability of oligomeric nanobodies 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technology to prevent hospital-acquired infections: lessons from sars, ebola, and mers in asia and west africa mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels mers-cov infection of alpaca in a region where mers-cov is endemic camels, mers-cov, and other emerging infections in east africa definitive diagnosis in suspected middle east respiratory syndrome coronavirus cases broad-spectrum coronavirus antiviral drug discovery stability of middle east respiratory syndrome coronavirus in milk high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan livestock susceptibility to infection with middle east respiratory syndrome coronavirus sars-cov infection in a restaurant from palm civet tissue distribution of the merscoronavirus receptor in bats middle east respiratory syndrome cornavirus (mers-cov) a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of korea comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus host-directed therapies for improving poor treatment outcomes associated with the middle east respiratory syndrome coronavirus infections the author declares there are no competing interests. â�¢ maged gomaa hemida conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. the following information was supplied regarding data availability: this is a literature review; there is no raw data. key: cord-332080-923jpec0 authors: lai, chih-cheng; wang, cheng-yi; ko, wen-chien; hsueh, po-ren title: in vitro diagnostics of coronavirus disease 2019: technologies and application date: 2020-06-05 journal: j microbiol immunol infect doi: 10.1016/j.jmii.2020.05.016 sha: doc_id: 332080 cord_uid: 923jpec0 abstract laboratory-based diagnostic measures including virological and serological tests are essential for detecting severe acute respiratory syndrome coronavirus 2 (sars-cov-2). real-time reverse transcription-polymerase chain reactions (rrt-pcr) can detect sars-cov-2 by targeting open reading frame-1 antibodies (orf1ab), envelope protein, nucleocapsid protein, rna-dependent rna polymerase genes, and the n1, n2, and n3 (3n) target genes. therefore, rrt-pcr remains the primary method of diagnosing sars-cov-2 despite being limited by false-negative results, long turnaround, complex protocols, and a need for skilled personnel. serological diagnosis of coronavirus disease 2019 (covid-19) is simple and does not require complex techniques and equipment, rendering it suitable for rapid detection and massive screening. however, serological tests cannot confirm sars-cov-2, and results will be false-negative when antibody concentrations fall below detection limits. balancing the increased use of laboratory tests, risk of testing errors, need for tests, burden on healthcare systems, benefits of early diagnosis, and risk of unnecessary exposure is a significant and persistent challenge in diagnosing covid-19. (hangzhou bigfish bio-tech co., ltd., zhejiang, china) was recently registered 127 as a ce-ivd for detecting sars-cov-2 orf-1ab and n genes in 128 nasopharyngeal swabs, sputum, and bal fluids. this kit has a reported lod of 129 < 2 × 10 2 copies/ml. 25 130 argene® sars-cov-2 r-gene® (biomérieux sa., marcy-l'étoile, 131 france) was developed to detect sars-cov-2 rdrp, n, and e genes in 132 nasopharyngeal swabs. the lod was determined from an inactivated viral 133 strain spiked in a nasopharyngeal swab sample at 0.43 tcid 50 /ml. this test 134 kit is presently available only for research purposes (table 2) . 26 135 the novel coronavirus (2019 ncov) rt-pcr assay (dynamiker 136 biotechnology (tianjin) co., ltd., tianjin, china) can detect the target genes, 137 orf-1ab, n, and actin, within 1.5 h from oropharyngeal and nasopharyngeal 138 swabs, sputum, bal fluid, serum, plasma, conjunctival swabs, and feces. the 139 reported detection range is 2 × 10 2 copies/ml (lod) to 2 × 10 8 copies/ml. 27 140 the performance of this assay is presently under evaluation. 141 the product, xpert ® xpress sars-cov-2 (cepheid inc.) detects 142 sars-cov-2 rna (n2 and e genes) in nasopharyngeal swabs, aspirates or 143 wash specimens within 45 minutes, and received eua from the usfda during 144 march, 2020. 28 the claimed lod for the assay is 250 copies/ml. the 145 performance of the xpress sars-cov-2 test was clinically evaluated in 146 patients with respiratory illnesses from whom contrived nasopharyngeal swab 147 samples were collected into viral transport media. the samples were then 148 spiked with accuplex sars-cov-2 (a recombinant sindbis virus particle 149 containing the target sequences of the sars-cov-2 genome) at ~2-, 3-, and 150 5-fold lod concentrations. the results were in 100% agreement with the 151 predicted results of the accuplex sars-cov-2 spiked, and negative samples. 152 the biofire® covid-19 test includes assays for sars-cov-2a, 153 sars-cov-2d, and sars-cov-2e to detect sars-cov-2 orf1ab and or8 154 sequences in nasopharyngeal swabs, and is a qualitative test on filmarray® 155 2.0 or filmarray® torch systems. the lod was 3.3 e+02 gc/ml. this 156 product has received usfda eua to detect sars-cov-2 rna. 29 although co-infection in patients with covid-19 is rare, it has been 208 reported. 8, 9, 40 therefore, tests that can screen for multiple pathogens, including 209 sars-cov-2, provide the additional benefit of detecting possible co-infections, 210 leading to the administration of appropriate antimicrobial agents. serological tests that can detect sars-cov-2 igg-igm antibodies are simpler 248 than rrt-pcr, and do not require complicated equipment and protocols 249 (table 3) . thus, these tests can be used for rapid detection and massive 250 screening, particularly for asymptomatic carriers. during the previous sars 251 epidemic, the igm antibody was the first line of defense during viral infections 252 and was detectable in blood samples from patients after 3 -6 days. the igg 253 antibody is responsible for long-term immunity and immunological memory, 254 and was detectable after 8 days. 11 (table 4) . novel coronavirus: implications for virus origins 527 and receptor binding detection of 529 sars-cov-2 in different types of clinical specimens sars-cov-2 532 viral load in upper respiratory specimens of infected patients molecular and 535 serological investigation of 2019-ncov infected patients: implication of 536 multiple shedding routes saliva is a reliable tool to detect sars-cov-2 presumed asymptomatic 603 carrier transmission of covid-19 chest ct for typical 606 2019-ncov pneumonia: relationship to negative rt-pcr testing covid-19 and pneumonia returning from macau in taiwan: clinical course 610 and anti-sars-cov-2 igg dynamic dynamics of 617 anti-sars-cov-2 igm and igg antibodies among covid-19 patients four 620 point-of-care lateral flow immunoassays for diagnosis of covid-19 and 621 for assessing dynamics of antibody responses to sars-cov-2 evaluation of a covid-19 igm and igg rapid test; an efficient tool 627 for assessment of past exposure to sars-cov-2 evaluation of nine commercial sars-cov-2 638 immunoassays a case of 645 transient existence of sars-cov-2 rna in the respiratory tract with the 646 absence of anti-sars-cov-2 antibody response viral load of sars-cov-2 in 649 clinical samples washington state 2019-ncov case investigation team. first case of 652 2019 novel coronavirus in the united states two-year 655 prospective study of the humoral immune response of patients with severe 656 acute respiratory syndrome disappearance of 658 antibodies to sars-associated coronavirus after recovery duration of 661 antibody responses after severe acute respiratory syndrome. emerg infect 662 prolonged 664 virus shedding even after seroconversion in a patient with covid-19 recent advances 667 in the detection of respiratory virus infection in humans virus 670 isolation from the first patient with sars-cov-2 in korea laboratory diagnosis of emerging human 673 coronavirus infections -the state of the art diagnosis of 676 acute respiratory syndrome coronavirus 2 infection by detection of 677 nucleocapsid protein global 682 epidemiology of coronavirus disease 2019: disease incidence, daily 683 cumulative index, mortality, and their association with country healthcare 684 resources and economic status vashist sk. in vitro diagnostic assays for covid-19: recent advances and 687 emerging trends policy for diagnostics testing in 690 laboratories certified to perform high complexity testing under clia prior to 691 emergency use authorization for coronavirus disease-2019 during the 692 public health emergency y-testing-under-clia-prior 8-13 days: 86.1% ≥14 days: 100% specificity: 99 days: 88.1% ≥14 days: 100%/ specificity: 99.8% 18 min ce-ivd conformité européenne in vitro diagnostic device emergency use authorization; lfia, lateral flow immunoassay; na, not available; ruo, research use only; tat, turnaround time; us fda, food and drug administration of the 52 united states key: cord-310507-5h6egve4 authors: van doorn, amarylle s.; meijer, berrie; frampton, chris m. a.; barclay, murray l.; de boer, nanne k. h. title: systematic review with meta‐analysis: sars‐cov‐2 stool testing and the potential for faecal‐oral transmission date: 2020-08-27 journal: aliment pharmacol ther doi: 10.1111/apt.16036 sha: doc_id: 310507 cord_uid: 5h6egve4 background: since the start of the covid‐19 pandemic, there have been many scientific reports regarding gastrointestinal manifestations. several reports indicate the possibility of viral shedding via faeces and the possibility of faecal‐oral transmission. aims: to critically assess the clinical relevance of testing stool samples and anal swabs and provide an overview of the potential faecal‐oral transmission of sars‐cov‐2. methods: a systematic literature search with mesh terms was performed, scrutinising the embase database, google scholar, medline database through pubmed and the cochrane library, including articles from december 2019 until july 7 2020. data were subsequently analysed with descriptive statistics. results: ninety‐five studies were included in the qualitative analysis. 934/2149 (43%) patients tested positive for sars‐cov‐2 in stool samples or anal swabs, with positive test results up to 70 days after symptom onset. a meta‐analysis executed with studies of at least 10 patients revealed a pooled positive proportion of 51.8% (95% ci 43.8 ‐ 59.7%). positive faecal samples of 282/443 patients (64%) remained positive for sars‐cov‐2 for a mean of 12.5 days, up to 33 days maximum, after respiratory samples became negative for sars‐cov‐2. viable sars‐cov‐2 was found in 6/17 (35%) patients in whom this was specifically investigated. conclusions: viral shedding of sars‐cov‐2 in stool samples occurs in a substantial portion of patients, making faecal‐oral transmission plausible. furthermore, detection in stool sample or anal swab can persist long after negative respiratory testing. therefore, stool sample or anal swab testing should be (re)considered in relation to decisions for isolating or discharging a patient. since december 2019, the world has been dealing with the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) leading to corona virus disease 2019 (covid-19) that emerged in wuhan, china. the outbreak in this city led to a major world crisis, the covid-19 pandemic. 1, 2 sars-cov-2 is a non-segmented positive-sense rna virus causing the third betacoronavirus outbreak of this century, which appears to have a higher transmission rate but is less deadly than the previous two; sars-cov 2003 and middle east respiratory syndrome (mers) 2012. 3, 4 prior studies demonstrated that the genome sequence of sars-cov-2 is 79.5% identical to sars-cov, whereas it shares 96.2% of its identity to the coronavirus ratg13 found in bats, but the intermediate reservoir has yet to be identified. 5 while patients infected with sars-cov-2 typically present with fever and respiratory symptoms, a rapidly increasing number of studies report patients presenting with a variety of gastrointestinal symptoms such as diarrhoea, vomiting and abdominal pain. 6 the established transmission route of sars-cov-2 is through respiratory droplets (aerosols), mainly during close person-to-person contact, 7 whereas numerous reports also mention the transmission by infected surfaces. based on the spread through aerosols, the diagnosis of active covid-19 infection primarily relies on the detection of sars-cov-2 viral rna in specimens from the upper respiratory tract (urt; nasopharyngeal and oropharyngeal cavity) and/or lower respiratory specimens (lrt; sputum and/or bronchoalveolar lavage). 8, 9 knowledge about sars-cov-2's other potential routes of transmission and the significance of different methods of testing is relatively sparse, 10 partly as a result of the novelty of this virus. however, there is a growing body of studies in which sars-cov-2 rna was detected in stool samples (including anal swabs) from covid-19 patients. 11 these findings support the possibility of a faecal-oral route of transmission. interestingly, stool tests seem to remain positive when respiratory tests are, or have become, negative. [12] [13] [14] a few articles have briefly reviewed the rapidly increasing body of knowledge on the potential for faecal-oral transmission. 11, 15, 16 this study aims to (1) critically assess the clinical relevance of testing stool samples and anal swabs and (2) provide a critical overview of the available literature regarding the faecal-oral transmission of sars-cov-2. this systematic literature search was performed following the prisma guidelines and conducted using the embase database, google scholar, the medline database through pubmed and the cochrane library from the outbreak in december 2019 until the 17 june 2020. the search strategy can be found in online supplement 1. all articles were imported to mendeley (version 1.17.6), and duplicates were removed. extensive cross-checking of reference lists of the included articles and other reviews was performed. as a result of the rapidly evolving research field concerning covid −19, we also included journal pre-proof articles. all articles were screened based on title and abstract. studies were included when the following inclusion criteria were met: we excluded articles written before december 2019, when the article or abstract/outcomes were not available in english, dutch or german and when the results or quality of data were ambiguous. papers written in chinese, of which the abstract contained sufficient data to provide answers to our research questions, were included for analysis and data extraction. we excluded articles in which follow-up data were insufficient (ie when results of stool testing were not mentioned). review articles were not included, however, reference lists were scrutinised for additional articles. we collected the following data from the eligible original articles: study design, geographic location, study period, number of patients, age, types of tested specimens, number of tested specimens, methods of the performed tests, duration and prevalence of positive test results in different specimens, disease severity, gastrointestinal symptoms, endoscopic results, specific evidence supporting faecal-oral transmission and remarkable patient/population characteristics. data were subsequently analysed with descriptive statistics. relevant data were tabulated with a subdivision by study population size. all studies with population of at least 10 patients were included in the meta-analysis. a weighted pooled estimate of the proportion testing positive from the stool samples was calculated using the freeman-tukey arcsine square root transformation under a random effects model. this analysis was undertaken using medcalc® v19.4.0. the heterogeneity in the estimates between studies was statistically tested using cochran's q statistic and summarised as i 2 . the search strategy resulted in 300 articles suitable for title and abstract screening. after the exclusion of articles which met the exclusion criteria, we included a total of 95 articles for final analysis. figure 1 shows details of the selection procedure. the majority of the included studies were performed in china (74 (77%)), other studies were conducted in korea (6), singapore (2), the united states of america (5), italy (4), france (1), germany (1), thailand (1) and austria (1). all included studies had a case report/ case series design. in most study populations, the subpopulation on which stool and/or anal testing were conducted was considerably lower. in total, stool samples or anal swabs (from now on collectively named as gi specimens) from 2175 patients were tested for sars-cov-2 rna. four studies were included for qualitative analysis, but due to the lack of necessary (follow-up) information, these studies were excluded before final quantitative analysis. [17] [18] [19] [20] therefore, 2149 patients were included for final analysis. seventeen (18%) studies tested sars-cov-2 presence in anal swabs and 81 (85%) in stool samples. in three studies, both specimens were tested. in all studies but one, real-time reverse transcription polymerase chain reaction (rt-pcr) was used to detect sars-cov-2. one study performed inoculation of stool suspension into vero cells followed by virus detection through electron microscopy. 21 in 91/95 (96%) of the included studies, sars-cov-2 rna was identified in gi specimens from at least one of the included patients (tables 1 & 2 twelve studies discussed the association between positive gi specimens and gi symptoms. 13, 14, 19, [38] [39] [40] [41] [42] [43] [44] [45] [46] in all studies, the majority of patients with gi symptoms tested positive in gi specimens, but the association was not statistically significant in most studies. in the study by han et al, it was observed that patients with gi symptoms were significantly more likely to test positive for sars-cov-2 in a stool test (p = 0.033). 44 furthermore, cheung et al found that the proportion of positive stool tests and the stool viral load was higher in patients with diarrhoea than without (p = 0.019 and 0.06 respectively). 46 in addition to the clinical symptoms and the positive gi specimens testing, two studies detected sars-cov-2 rna in endoscopic specimens of the oesophagus, stomach, duodenum and rectum in 1/1 and 2/6 patients. 41, 45 viability of sars-cov-2 was investigated and detected in five studies, in which six patients (6/17 (35%)) had live active virus in their gi specimens using vero cell testing. 19, 21, [47] [48] [49] in this study, we performed a systematic review of the rapidly expanding body of literature to assess the performance and accuracy the results of this study may have various consequences for the diagnosis, prognosis and spread of covid-19. first and foremost, worldwide the decision to isolate or discharge a patient is primarily based on relevant clinical symptoms, focusing on the respiratory tract, and (sequential) negative test results on respiratory specimens collected more than 24 hours apart. 57 we observed that in 64% of patients who tested positive for sars-cov-2 in gi specimens, their gi specimens remained positive for a mean of 12.5 days after respiratory samples became negative. as a result, a number of patients were discharged up to a month before the absence of sars-cov-2 in gi specimens could be guaranteed. the (additional) use of gi specimen testing may provide a more appropriate rationale for isolation and discharge. a major concern could be continuing person-to-person transmission by the faecal-oral route, which argues for closer attention to hand and sanitation hygiene. this should be considered when determining diagnosis and isolation policies. in general the risk to health care professionals from patient exposure is well known, specifically in high aerosol-generating procedures. currently, medical management protocols include measures to mitigate the aerosol transmission risks from procedures related to respiratory tract. 8 as a result, our analyses were based on relatively small patient groups (median 9; range 1-401 patients) and inconsistent methods, parameters, sample timing, sample frequencies and study endpoints differing widely between the included studies, impeding comparisons and robust conclusions. in the early response to the emerging covid-19 outbreak, only respiratory specimens were required for the detection of sars-cov-2 according to initial clinical guidelines. a lot of studies, therefore, refrained from obtaining gi specimens from the patients during their first few days of hospitalisation or observation and could not determine whether respiratory and gi specimens were positive on rt-pcr analysis simultaneously. furthermore, the phenomenon that viral rna of sars-cov-2 can remain positive in gi specimens after respiratory samples became negative was not identified in all studies. this resulted in inadequate (follow-up) information, potentially causing a (outcome) measurement bias. the sole four studies that reported no positive tests in gi specimens were all based on small sample size (1-4 patients) and the testing was performed at an early stage of the disease course. our review demonstrated that there seems a tendency for sars-cov-2 to be more detectable in the respiratory tract at an early stage of the disease and later on, more likely to be detected in gi specimens, which could explain the early negative testing. our review confirms that sars-cov-2 is commonly present in stool samples or anal swabs in which the virus can persist long after respiratory testing has become negative and that the virus may be viable. this suggests the possibility of faecal-oral transmission and that stool sample or anal swab testing should be (re)considered in relation to decisions for isolating or discharging a patient. 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doc_id: 324919 cord_uid: ciamusjs despite the unprecedented effort of the scientific community, the novel sars-cov-2 virus has infected more than 46 million people worldwide, killing over one million two hundred thousand. understanding the mechanisms by which some individuals are more susceptible to sars-cov-2 infection and why a subgroup of them are prone to experience severe pneumonia, and death should lead to a better approach and more effective treatments for covid-19. here, we focus our attention on ace2, a primary receptor of sars-cov-2. we will discuss its biology, tissue expression, and post-translational regulation that determine its potential to be employed by sars-cov-2 for cell entry. particular attention will be given to how the ace2 soluble form can have a great impact on disease progression and thus be used in a potential therapeutic strategy. furthermore, we will discuss repercussions that sars-cov-2/ace2 binding has on the renin–angiotensin system and beyond. indeed, although mostly neglected, ace2 can also act on [des-arg 937]-bradykinin of the kinin–kallikrein system regulating coagulation and inflammation. thorough comprehension of the role that ace2 plays in different pathways will be the key to assess the impact that sars-cov-2/ace2 binding has on organismal physiology and will help us to find better therapies and diagnostic tools. in the last 20 years, humanity has witnessed an increasing number of pandemics that have hospitalized or killed hundreds of thousands of people, leaving our healthcare systems under unprecedented pressure. severe acute respiratory syndrome (sars) in 2002 and middle east respiratory syndrome in 2012 (mers) [1] , and more recently the novel infection named covid-19, detected in wuhan china in 2019 are caused by closely related coronaviruses, sars-cov, mers-cov and sars-cov-2, respectively [2] . their unexpected and periodic appearance combined with a high level of human-to-human transmission has made coronaviruses a threat to our societies and economies, forcing us to think not if but when another pandemic will arise. from this perspective, more needs to be done to better understand the mechanism of infection, if and how the hyperactivation of the immune reaction can be prevented, and which treatments can be provided, especially for people with comorbidities. in this review, a focus will be given to the metallocarboxyl peptidase angiotensin receptor (ace)2 that is used by sars-cov-2 to gain entry into human cells [3] . there are two forms of ace2 [4] . the full-length mace2 is located on cell membranes and consists of a transmembrane anchor and an extracellular domain. it is the receptor site for the spike (s) proteins of sars-cov-2. the s proteins on the envelope of sars-cov-2 are cleaved into s1 and s2 subunits, the s1 protein/receptor interaction being the pivotal determinant for sars-cov-2 to infect a host species [3] . the second form, sace2, is a soluble form that is shed into the circulation [4] . this form of ace lacks membrane anchors and circulates in low concentrations. we will assess whether the level of expression of ace2 and the ratio between mace2 and sace2 could explain why some people experience more severe symptoms than others. furthermore, we will discuss the key role that ace2 plays in regulating molecular pathways that go beyond the renin-angiotensin-aldosterone system (raas) and have been mostly ignored. we believe that efforts towards the full comprehension of the intricate roles that ace2 plays in maintaining organismal physiology will be the key to better understand the multisystemic covid-19 disease and help us to develop better therapies and diagnostic tools. cell entry receptors are undoubtedly the key factors determining the tropism and influencing the severity of infection of a specific virus. furthermore, the high rate of mutations to which these viruses are subject can allow them to change their specificity or binding affinity for a specific receptor. for instance, cov-nl63, sars-cov, and sars-cov-2 use ace2, but cov-nl63 leads to mild respiratory tract illness, probably because of its low-affinity interaction with the receptor [5] . although belonging to the same genus of its related sars-cov/-2 (table 1) , mers-cov binds to dipeptidyl peptidase-4 (dpp4) [6] that plays an important role in glucose metabolism, apoptosis, and the immune system. coronaviruses are not the only family of viruses able to cause respiratory tract illness in humans. the a(h1n1) pdm09 influenza virus that caused the pandemic in 2009 shows a different tropism binding to α2-6-and α2-3-linked sialyl glycans [7] that are present on different cell types and play key roles in cellular communication and cell signaling among others. finally, human rhinoviruses (hrv), which can cause severe illness if in combination with pre-existing conditions [8] , use mainly the membrane form of the intercellular adhesion molecule-1 (icam-1) that is expressed in nasal and bronchial epithelial cells [9] . this interaction has been demonstrated to regulate differently the two soluble and membrane-bound forms, suggesting a different role during the infection [10] . biology ace2 was initially identified in 2000 as a homolog of the ace receptor, sharing 40% identity and 60% similarity, respectively [11, 12] . located on the x chromosome and precisely mapping on chromosomal location xp22, it is formed by 18 exons and 20 introns generating 6 variants through alternative splicing [13] . ace2, which consists of 805 amino acids, has only a single extracellular n-terminal domain containing the active catalytic site domain, a c-terminal membrane anchor, and a conserved hexxh zinc-binding domain [11] . it acts as a carboxypeptidase removing single amino acids from the c-terminus of its substrates [14] . experimental evidence has demonstrated that in the situation of cell energy stress, sirtuin 1 (sirt1) can mediate ace2 transcriptional activation [15] . similarly, the reduced ace2 level in apelin deficient mice was corrected by apelin treatment [16] suggesting that apelin acts as a positive regulator of ace2 expression and might have a role in affecting other pathways regulated by ace2. apelins are a family of peptides that activate the apelin receptor, which physically interacts with the angiotensin type i receptor (at1r) [17] , forcing it into a low-affinity state and reducing the binding and signaling of angiotensin ii (angii) [18] . conversely, the microrna 421 (mir421) has been shown to target a binding site in the 3′-utr of ace2 transcript, negatively regulating the receptor [19] . due to the wide spectrum of symptoms occurring in people affected by covid-19, efforts have been made to study ace2 tissue expression in both mrna and protein levels, identifying the organs more susceptible to this infection. surprisingly, the analysis of ace2 expression in experimental models and in human transcriptome by using different databases revealed that it is very low in the lung, mainly limited to a small fraction of type ii alveolar epithelial cells [20] [21] [22] . since most people present respiratory difficulties in response to sars-cov-2 infection, these findings were explained by the fact that the release of inflammatory cytokines, such as interferons (ifns) caused by sars-cov-2, can increase the expression ace2 and potentiate the infection [23, 24] . however, onabajo and colleagues recently showed that ifns stimulation upon viral treatment induced the expression of a truncated ace2 isoform designate as δace2, which lacks 356 n-terminal amino acids, is not able to bind sars-cov-2 and, therefore, does not contribute to the potentiation of the infection [25] . furthermore, the analysis of ace2 mrna and protein expression in experimental models, and evaluation of three different databases have demonstrated that small intestine, testis, kidney, heart muscle, colon, and thyroid gland are the overlapping tissues with the highest level of ace2 expression, with no expression detected in blood cells [20, 26] . these data explain why people affected by covid-19 experience gastrointestinal problems and kidney dysfunction [27, 28] . several clinical reports have suggested that male sex combined with increasing age [29] , smoking, and pre-existing comorbidities [30] represent risk factors for a poor outcome from the infection. whether there is upregulation of ace2 expression in smokers that could increase their sensitivity to infection is still a matter of debate. in the literature, there are reports supporting [31] [32] [33] and refuting [34] [35] [36] this hypothesis. since the beginning of the covid-19 pandemic, hypertension and diabetes have been correlated with higher risk of mortality, and initial reports speculated that angiotensin converting enzyme inhibitors (acei) and angiotensin receptor blockers (arbs), which are commonly used therapeutic agents for these conditions, would up-regulate ace2 expression, thus increasing the risk of severe illness [37] . recent evidence has challenged this hypothesis, demonstrating both mechanistically and in large cohort studies that acei and arbs do not up-regulate ace2 and are not associated with an increased mortality [35, 36] . interestingly, asthma has not been reported as a risk factor for covid-19 illness and disease progression. kimura and colleagues showed that interleukin (il)-13, a cytokine up-regulated in type 2 asthma, down-regulates the level of ace2 [38] . furthermore, jackson and colleagues demonstrated a negative correlation between ace2 expression and allergen sensitization and asthma [39] . intriguingly, peters and colleagues found that asthmatic subjects treated with inhaled corticosteroids (ics) had low ace2 expression compared with untreated subjects, a finding suggesting that ics treatment could be a predictor of decreased susceptibility to sars-cov-2 infection [40] . ace2 is normally localized on the plasma membrane (mace2) with the n-terminal containing the catalytic site protruding on the extracellular space using as substrate different active peptides present in the interstitium. ace2 can undergo proteolytic shedding by different proteases such as a disintegrin and metalloproteinase domain-containing protein (adam)10, adam17, and transmembrane protease, serine 2 (tmprss2). binding of s1 subunit of the spike protein of sars-cov-2 to the ace2 receptor triggers the cleavage of ace2 by adam17/tumor necrosis factorconverting enzyme (tace) at the ectodomain sites [41] and a soluble form that retains its catalytic activity (sace2) is produced [42] . interestingly, tace inhibitors can decrease viral entry in vitro and in vivo demonstrating their key role in determining sars-cov infectivity and their potential use as a target for antiviral therapies [43] . the significance of ace2 shedding has not been fully elucidated, but in the context of the covid-19 pandemic, the comprehension of the mechanism leading to ace2 shedding, sace2 function, and its plasma level can lead to the development of better therapies and diagnostic tools to follow disease progression and severity. for example, it has been shown that the binding of sars-cov with ace2 induced adam17-dependent shedding, promoting sars-cov uptake into the cells [44] . tmprss2 is able to promote ace2 proteolytic cleavage using different targets in the protein sequence. it cleaves ace2 at the intracellular c-terminal domain and, differently from adam17 [45] , does not produce a soluble form that retains the catalytic function [41] . however, like adam17, it is essential for sars-cov entry into the cell [45, 46] . ace2 shedding can be stimulated by proinflammatory cytokines such as il-1β and tumor necrosis factor (tnf)-α, and endotoxin [47] that could result in a positive effect reducing sars-cov-2 entry, but at the same time, may cause an increase in angii and further activation of the angii/at1r axis worsening inflammation (discussed below) (fig. 1) . different studies have demonstrated that an indeed, without the counterbalance action of ace2, angii is not converted in ang1-7 and able to overactivate its receptor at1r promoting vasoconstriction, production of proinflammatory cytokines such as tnfα, il6, il1, and ros generation through nadph oxidase. ace2 also plays a key role in the regulation of the kks by inactivating ldeabk and deabk making them incapable to bind the receptor brb1. the overactivation of brb1 receptor has been shown to promote inflammation and coagulation increased level of sace2 correlates with disease severity [48, 49] , possibly due to an increase in angii. furthermore, an increase in ca 2+ caused by ionomycin treatment induces adam10-dependent ace2 shedding [47] , while calmodulin, a calcium modulated protein, by binding to ace2, inhibits its shedding [50] . these findings highlight the interconnection between ca 2+ signal transduction pathways and ace2 regulated pathways. the evidence that ace2 can be cleaved from multiple proteases upon different stimuli indicates that the post-translational regulation of this ectoenzyme is of great importance in managing tissue homeostasis. this explains why its dysregulation caused by sars-cov-2 binding has such an intense effect on organismal physiology. due to the main role of ace2 in mediating sars-cov-2 entry into cells, many studies have speculated whether ace2 expression and polymorphism and serum sace2 levels could explain why some people are more prone to experience a severe phenotype while others remain asymptomatic. previous studies have demonstrated that specific residues in the ace2 protein are essential for binding with sars-cov [51] . therefore, genomic variability in those specific residues could modulate the binding between ace2 and the sars-cov-2 spike protein, accounting for a broad range of symptoms exhibited by people affected by covid-19. studies that aimed to find mutations in the ace2 sequences and allele frequency in different populations have demonstrated the existence of specific variance that could affect sars-cov-2 binding [52] [53] [54] . furthermore, a bioinformatic approach has identified new ace2 exons that could participate in alternative splicing by producing a truncated ace2, lacking the n-terminus that could not bind the spike protein, or the c-terminus generating a soluble ace2 that would again reduce virus/cell binding and infectivity [55] . identification of a genomic variability in tmprss2 that affects its expression is worthy of mention because it suggests that european and american populations could be more susceptible to sars-cov-2 infection [56] . we do not yet know whether a specific variability in the adam17 sequence is associated with a high level of sace2 and whether there is a correlation with the sars-cov-2 infection. indeed, since the beginning of this pandemic, opposite assumptions have been made. some researchers think that elevated levels of sace2 may be protective because they are capable of inhibiting the binding of sars-cov-2 to membrane-bound ace2 [4] and would explain why women and children are less susceptible [57] . conversely, recent studies have shown that high levels of sace2 could be evidence of increased ace2 expression, elevated adam17 activity or both and, therefore, greater susceptibility to sars-cov-2 infection [58, 59] . dissecting the roles that ace2 has in maintaining organismal physiology will help us to better comprehend why its dysregulation caused by sars-cov-2 can have such a devastating effect, especially in the elderly with comorbidities. in the next sections, we will describe the molecular pathways in which ace2 plays an important role. we will focus on the role that ace2 plays in counterbalancing the raas but also on its important function in the kinin-kallikrein system (kks) that has been mostly neglected although it plays an essential part in regulating the inflammatory process. despite having a high degree of homology with ace, ace2 shows a remarkable difference in substrate selection, catalyzing with high efficiency the conversion of the vasoconstrictor angii in ang1-7 that binds and activates its own seven-transmembrane g protein-coupled receptor (gpcr) called mas to exert anti-inflammatory and anti-remodeling effects or, with less efficiency, the formation of ang1-9 from angi, which can be converted to ang1-7 by ace [60] . in doing so, ace2 plays a counterbalance action in the raas, which is a critical regulator of blood volume and systemic vascular resistance and contributes to sodium reabsorption, inflammation, and fibrosis, preventing the possible adverse effect of angii accumulation. the interaction between sars-cov2 and ace2 could negatively regulate the receptor [61] causing, in turn, an accumulation of angii that through the unopposed raas activation via the angii/at1r can cause vasoconstriction, oxidative stress, inflammation, atrophy, and fibrosis [62] . there is evidence that at1r can induce apoptosis in lung alveolar epithelial cells in response to angii in rat and human alveolar epithelial cells [63] . furthermore, angii promotes endothelial dysfunction through cyclooxygenase-2 (cox-2) activation, which generates vasoactive prostaglandins and reactive oxygen species (ros) [64] . excessive production of ros upon overactivation of the angii/at1r/ nicotinamide adenine dinucleotide phosphate (nadph) oxidase axis has been correlated with hypertension [65] , and atherosclerosis [66] , and can induce apoptosis through the release of cytochrome c from damaged mitochondria, activation of caspase 3 or p38 mitogen-activated protein kinase (mapk)/jun n-terminal kinase (jnk) cascade. other inflammatory mechanisms activated by an accumulation of angii can also involve the activation of nuclear factor (nf)-kb and transcription of the proinflammatory cytokines il-6, il-1β, and tnfα30 [67] (fig. 1) . therefore, the activation of the immune response caused by the infection in combination with the high level of angii could result in the hyperinflammatory state that is seen in the late phase of sars-cov-2-infected patients. ang a differs from angii by a single amino acid (alanine) at the n-terminal. it shows affinity to at1r and possibly activates all aforementioned pathways in the context of sars-cov-2 infection. as in the case of angii, ace2 is responsible for providing a further layer in raas regulation by catalyzing the conversion of anga in alamandine. this conversion highlights again the key role of ace2 in the regulation of raas. in fact, the produced peptide alamandine is able to bind to the mas-related gpcr member d (mrgd) and exerts a protective action promoting vasorelaxation and an antiproliferative effect [68] . the kks plays a key role in the regulation of several physiological processes such as coagulation, inflammation, and pain [69] . it exerts its action through the production of active peptides such as bradykinin (bk), lys-bk, [des-arg9]-bk (deabk), and lys-[des-arg9]-bk (ldeabk) [70] . by binding to bradykinin receptor-b2 (brb2), bk and lys-bk induce a local increase in nitric oxide production that has a potent vasodilator effect counterbalancing the vasopressor effect of the raas [71] . moreover, bk positively regulates tissue plasminogen secretion (tpa) and, therefore, plays an important role in thrombus formation [72] . deabk and ldeabk act on bradykinin receptor-b1 (brb1) and have important role in the inflammation process [73, 74] . in fact, differently from brb2, brb1 is expressed at very low level on endothelial cells but is induced upon tissue injury and is up-regulated by proinflammatory cytokine release such as il-1b, tnfα, il-2, and ifnγ [73] . evidence has demonstrated that activation of brb1 is able to aggravate the inflammation by causing the further release of proinflammatory cytokines and promoting neutrophil infiltration [75] . therefore, deabk and ldeabk can act as proinflammatory factors that exert their function through the brb1 receptor. it is interesting to note that ace2, which does not inactivate bk, can cleave the terminal residue in deabk and ldeabk, rendering them unable to interact with brb1 [76, 77] . therefore, ace2 internalization due to sars-cov-2 infection will create an imbalance in the kks system causing an overactivation of the deabk/ldeabk/brb1 axis, contributing to increased inflammation and rendering the lung environment more prone to local vascular leakage, leading to angioedema [76] . consequently, approaches aimed at targeting the raas system, but not the kks, will not be as effective in limiting the state of hyperinflammation typical of severe cases of sars-cov-2 infection (fig. 1) . the role of a dysregulated kks remains theoretical in covid-19 [78] . nonetheless, an approach that could target both pathways to fight against covid-19 has already been suggested [79] . ace2 is attracting much interest as a therapeutic target in the fight against covid-19 [4] . as described earlier, ace2 can be shed from the cell and released into the circulation by adam17 while maintaining its catalytic activity and ability to bind sars-cov-2. it has been suggested to use human recombinant ace-2 (hrace2) protein to saturate the viral s-protein and thus prevent cellular entry of sars-cov-2 [4] . soluble hrace2 (shrace2) retains attractive physiological features due to its ability to inactivate sars-cov-2 present in the extracellular milieu. differently from antiviral or anti-inflammatory therapies, shrace2 can act upstream by decreasing the binding between sars-cov-2/mace2, thus reducing infectivity [80] . furthermore, it can also counteract the increase in angii and deabk/ldeabk preserving lung function. administration of hrace2 is well tolerated in healthy subjects [81] , and it has been successfully used to treat patients with ards [82] . apn01 is a rhace2 that is fully glycosylated (7 glycosylation sites) and occurs as a stable noncovalent homodimer. its enzymatic activity relies on incorporated zn 2+ . the use of twice-daily doses of apn01 infusion resulted in a rapid decrease in plasma angii levels and an increase in ang1-7 and ang1-5 levels, as well as a trend to a decrease in plasma il-6 concentrations [82] . recently, monteil and colleagues demonstrated that shrace2 can decrease sars-cov-2 infection in vitro [83] , and clinical trials are already underway (clinicaltrials. gov #nct04335136). furthermore, a more effective way for the delivery of shrace2 that would decrease protease degradation has already been suggested [84] . although our knowledge of the role that endogenous sace2 plays in human physiology is still limited, there is promising evidence that shrace2 could be used as a valid therapy for sars-cov-2 infection. other potential therapeutic approaches include a sars-cov-2 spike protein-based vaccine, a tmprss inhibitor to block the priming of the spike protein, and blockage of the surface ace2 receptor by using anti-ace2 antibody or peptides [81] . camostat mesylate, approved in japan to treat pancreatic inflammation, has been shown to block tmprss2 activity and prevent cellular infection by sars-cov-2 [45] . another strategy that is being investigated in clinical trials is the administration of an antibody or a singlechain antibody fragment (scfv) that binds ace2 and blocks the interaction of spike protein on the virion to ace2 [85] . furthermore, beyond the raas and kks, ace2 has been shown to act on other substrates such as apelin-13/17, neurotensin (1-11), β-casomorphin-(1-7), dynorphin a (1-13), and ghrelin among others [86] . these substrates are interesting potential therapeutic targets for covid-19 [77] , but we will not discuss them because their involvement in covid-19 is still highly speculative. since the first outbreak in wuhan, china, sars-cov-2 has spread worldwide, killing over one million two hundred thousand people. the rapid rate of adaptive mutations combined with high transmissibility renders coronaviruses an ongoing threat of causing future pandemics, with especially high risk for the elderly [87] . although the scientific world is making an extraordinary effort, we are still far from fully understanding whether the overactivation of the immune system and the state of hyperinflammation due to sars-cov-2 infection can be controlled [88] . this would be particularly important in people with comorbidities such as diabetes and hypertension, which are conditions already associated with an inflammatory state. it is likely, even if not yet fully ascertained, that when these pathological states combine with the viral infection, the dysregulation of both the raas and the kks due to ace2 internalization mediated by sars-cov-2 could create the conditions for severe illness and a fatal outcome. in conclusion, we believe that further efforts should be made to fully understand ace2 transcriptional regulation and post-translational modification during the course of covid-19 and in response to treatments. in fact, as already discussed, contrasting hypotheses have been formulated concerning the effect of therapies taken by millions of people that might increase ace2 expression and susceptibility to sars-cov-2 infection. furthermore, although covid-19 is still primarily described as a respiratory viral illness, it is increasingly evident that it is a multisystemic disease. therefore, it is essential to acquire a greater knowledge of the role that ace2 plays in different organs and physiological pathways because of its widespread tissue expression. this may shed light on factors that modulate cell surface ace2 receptor affecting viral cell entry and consequently susceptibility to sars-cov-2 infection. this, in turn, could have significant implications for identifying better therapies and screening tools to assess disease progression and severity. conflict of interest the authors declare that they have no known competing financial 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disentangling the hypothesis of host dysosmia and sars-cov-2: the bait symptom that hides neglected neurophysiological routes date: 2020-06-05 journal: front physiol doi: 10.3389/fphys.2020.00671 sha: doc_id: 300716 cord_uid: urmogf97 the respiratory condition covid-19 arises in a human host upon the infection with sars-cov-2, a coronavirus that was first acknowledged in wuhan, china, at the end of december 2019 after its outbreak of viral pneumonia. the full-blown covid-19 can lead, in susceptible individuals, to premature death because of the massive viral proliferation, hypoxia, misdirected host immunoresponse, microthrombosis, and drug toxicities. alike other coronaviruses, sars-cov-2 has a neuroinvasive potential, which may be associated with early neurological symptoms. in the past, the nervous tissue of patients infected with other coronaviruses was shown to be heavily infiltrated. patients with sars-cov-2 commonly report dysosmia, which has been related to the viral access in the olfactory bulb. however, this early symptom may reflect the nasal proliferation that should not be confused with the viral access in the central nervous system of the host, which can instead be allowed by means of other routes for spreading in most of the neuroanatomical districts. axonal, trans-synaptic, perineural, blood, lymphatic, or trojan routes can gain the virus multiples accesses from peripheral neuronal networks, thus ultimately invading the brain and brainstem. the death upon respiratory failure may be also associated with the local inflammationand thrombi-derived damages to the respiratory reflexes in both the lung neuronal network and brainstem center. beyond the infection-associated neurological symptoms, long-term neuropsychiatric consequences that could occur months after the host recovery are not to be excluded. while our article does not attempt to fully comprehend all accesses for host neuroinvasion, we aim at stimulating researchers and clinicians to fully consider the neuroinvasive potential of sars-cov-2, which is likely to affect the peripheral nervous system targets first, such as the enteric and pulmonary nervous networks. this acknowledgment may shed some light on the disease understanding further guiding public health preventive efforts and medical therapies to fight the pandemic that directly or indirectly affects healthy isolated individuals, quarantined subjects, sick hospitalized, and healthcare workers. the respiratory condition covid-19 arises in a human host upon the infection with sars-cov-2, a coronavirus that was first acknowledged in wuhan, china, at the end of december 2019 after its outbreak of viral pneumonia. the full-blown covid-19 can lead, in susceptible individuals, to premature death because of the massive viral proliferation, hypoxia, misdirected host immunoresponse, microthrombosis, and drug toxicities. alike other coronaviruses, sars-cov-2 has a neuroinvasive potential, which may be associated with early neurological symptoms. in the past, the nervous tissue of patients infected with other coronaviruses was shown to be heavily infiltrated. patients with sars-cov-2 commonly report dysosmia, which has been related to the viral access in the olfactory bulb. however, this early symptom may reflect the nasal proliferation that should not be confused with the viral access in the central nervous system of the host, which can instead be allowed by means of other routes for spreading in most of the neuroanatomical districts. axonal, trans-synaptic, perineural, blood, lymphatic, or trojan routes can gain the virus multiples accesses from peripheral neuronal networks, thus ultimately invading the brain and brainstem. the death upon respiratory failure may be also associated with the local inflammation-and thrombi-derived damages to the respiratory reflexes in both the lung neuronal network and brainstem center. beyond the infection-associated neurological symptoms, long-term neuropsychiatric consequences that could occur months after the host recovery are not to be excluded. while our article does not attempt to fully comprehend all accesses for host neuroinvasion, we aim at stimulating researchers and clinicians to fully consider the neuroinvasive potential of sars-cov-2, which is likely to affect the peripheral nervous system targets first, such as the enteric and pulmonary nervous networks. this acknowledgment may shed some light on the disease understanding further guiding public health preventive efforts and medical therapies to fight the pandemic that directly or indirectly affects healthy isolated individuals, quarantined subjects, sick hospitalized, and healthcare workers. keywords: smell, olfactory bulb, coronavirus, sars-cov-2, covid-19, infections, virulence, host pathogen interactions the sniffing out of coronaviruses named after their crown-like spikes, coronaviruses are large non-segmented single-stranded positive-sense enveloped rna viruses that may spill out from animals to infect humans and cause respiratory diseases. in 2003, the severe acute respiratory syndrome (sars-cov-1) spilled out from civet cats (ksiazek et al., 2003) and, in 2012, the middle east respiratory syndrome (mers-cov) spilled out from camels (zaki et al., 2012) . at the end of december 2019, a new strain of familial coronavirus (sars-cov-2) caused an outbreak of viral pneumonia in wuhan, hubei province in china, but the animal source is still under investigation (probably bats). similarly to -or even better thanits most closely relative sars-cov-1 (shang et al., 2020) , this strain uses the angiotensin-converting enzyme 2 (ace2) as its host receptor to enter targeted cells and to replicate and infect adjacent cells. the ubiquitous presence of this receptor is associated with the systemic affections of covid-19 (patel and verma, 2020) . infected patients experience mild to severe systemic, respiratory, and enteric manifestations, such as fever, myalgia, lethargy, dry cough, dyspnoea, anorexia, abdominal pain, and diarrhea. transmission is mainly by human respiratory droplets carrying the virus, which enters the airways of the host and infects epithelial cells (zhu et al., 2020) . however, there is the need to further investigate the mode of sars-cov-2 transmission and its early and late symptoms, as a matter of disease understating and human conservation. new information comes out every day that could radically change the understanding of the viral nature of the disease, such as the susceptibility of domestic animals to contagion or the role of pollution for viral spreading (setti et al., 2020) . the environmental stability of sars-cov-2 is similar to that of sars-cov-1 (van doremalen et al., 2020) , indicating that differences in the epidemiologic characteristics of these viruses probably arise from other factors, including the contagion from infected individuals that are unaware because asymptomatic (bai et al., 2020) . on march 21st, the british association of otorhinolaryngology released a statement that dysosmia could be associated with sars-cov-2 contagion (hopkins and kumar, 2020) , highlighting the possibility of the nasal-nervous route as alternative access of the virus (baig et al., 2020) . interestingly, a report of april 1st from king's college london researchers stated that 59% of infected individuals participating in their survey reported dysosmia or dysgeusia (covid-19_symptomtracker, 2020) . still, most of the beliefs are anecdotal and not evidencebased. contrariwise, strong evidence supports the notion that respiratory viruses are neurotropic and can access the central nervous system via peripheral nerves, including the olfactory bulb (mori et al., 2005; van riel et al., 2015) . sars-cov-2 shares similar infection pathways compared to its predecessors and therefore the infection mechanisms previously found for other coronaviruses may also be applicable for this new strain. it is urgent to discuss whether sars-cov-2 can gain access to the central nervous system through a nasal-nervous pathway or other routes and if the fatal respiratory failure may be associated with a neuronal injury in critical brain areas of the host. it is believed that the distribution of the targeted host receptor in tissues is consistent with the tropism of the infective agent (to and lo, 2004) . the receptor ace2 is expressed primarily in human airway epithelia, lung parenchyma, and small intestine cells, but also in the heart, kidney, and nervous tissues. despite the ace2 pathway possibly being not the only one preferred by the virus, the receptor itself is located in the brainstem, being particularly expressed in the cardiorespiratory center (xia and lazartigues, 2008) . other than the receptor distribution, two other factors have to be considered when referring to the viral tropism: the path of contagion and the host's abilities to fight the infective agents, which might function as a double-edged sword. the main anatomical district that contacts the sars-cov-2 is the respiratory mucosa and here it is believed to occur the primary in-host proliferation that may indeed be associated with the smell disorders. once inside the human host, the host's abilities come into play against the virus, thus allowing the categorization of sars-cov-2-associated insults in two forms: (i) damages directly associated with viral proliferation (e.g., cytotoxicity) and (ii) damage indirectly associated with host's infection, being mainly hypoxia, inflammation (often an overactive host's ability), and disseminated intravascular coagulation (lillicrap, 2020) . that is, three main hypothesis can be advanced regarding the dysosmia of infected subjects: • hypothesis 1: the impairment is caused by the nasal blockage after the pooling of blood in the capacitance vessels upon viral upper respiratory infection. smell impairment is known to be associated with nasal congestion (akerlund et al., 1995) and the mucosal swelling easily impedes the physical access of odors to the olfactory region. • hypothesis 2: olfactory receptors in the neuroepithelium are disrupted upon excessive local inflammatory responses (doty and mishra, 2001) . in fact, it was suggested that the olfactory function could be independent of nasal congestion, but subjected to recovery after the viscous phase onset (hummel et al., 1998) . • hypothesis 3: dysosmia follows a direct viral insult by the virus because of the peripheral destruction of olfactory receptor cells (perlman et al., 1990) . if this is the case, it is also probable that the virus can invade the central nervous system through the retrograde axonal transport from the olfactory neuroepithelium and olfactory pathways. since sars-cov-2 has not been associated with upper respiratory symptoms , it seems that the first two hypotheses can be excluded. the third hypothesis is the most fascinating, and lately, it has sparked much interest because of the potential public health implications. whatever the nature of dysosmia, it could be difficult to define its underlying etiology since co-viral infections are very common in the outbreak season, patients with mild symptoms are rarely tested and, if admitted to clinics, the concomitant pharyngodynia and fever may blur a proper anamnesis. if dysosmia had late-onset, patients might recognize the olfactory loss long after the insult, when precise identification of the putative cause could be no longer feasible (seiden, 2004) . of note, the deficit in olfactory function after infection should not be permanent instead depend on the severity of the initial insult (duncan and seiden, 1995) . in fact, these neurons are subjected to continual turnover to offer a large spectrum of computational functions throughout different developmental and environmental stages (benito et al., 2018) . although some authors have observed that the damage to the olfactory epithelium was also associated with affections in central olfactory pathways (mohammed et al., 1990; yamagishi et al., 1994) , the other possible access routes cannot be ruled out. with particular emphasis on the nasal cavity as the assumed early route of the host's neuroinvasion, we discuss the hypothesis that sars-cov-2 neuroinvasion of various tissues can be considered the underlying pathway of the virus spreading and associated fatality of the human host. unfortunately, sars-cov-2 may be not always confined to the respiratory or gastrointestinal tract, but it may also invade the annexed nerve tissues. early studies after the outbreak of sars-cov-1 demonstrated the presence of viral particles in the brain, where they were located almost exclusively in the neurons. in light of the high pathophysiological pathway similarity between sars-cov-1 and sars-cov-2 (glass et al., 2004) , it is quite likely they share a similar neuroinvasive potential. putative routes for sars-cov-2 neuroinvasion can be summarized as follows: • neuronal route. sars-cov-2 could enter the nervous system through peripheral nerve fibers, not only including the olfactory receptors in the nasal epithelium, but also the pulmonary network and the enteric nervous system. • pericellular route. being extremely important for the infection of adjacent cells, the extracellular route of major interest for our discussion is the cleft between the efferents of olfactory sensory neurons and their ensheathing cells. other routes could comprise the diffusion through the mucous layer of the respiratory tract or the cerebrospinal fluid. • hematogenous route. the blood guarantees direct access to nervous tissues. specific barriers normally restrict the spread of viruses into the central nervous system, but direct infection of the neurovascular unit or the triggering of neuroinflammation can compromise the integrity. • lymphatic route. it can be considered the third widespread system after the brain-nervous and the cardio-circulatory networks. viral particles can infiltrate through the lymphatics to the lymphoid tissues subsequently infecting cells in close contact with the blood flow. • trojan route. infected migrating leukocyte can carry intracellular viruses and then migrate into the central nervous system. in fact, the infected neuronal cell populations often recruit non-specific leukocytes to fight the infective agent (ransohoff and engelhardt, 2012) , which can be later released and freed to infect local neuronal cells. in humans, the nasal cavity comprises the anterior vestibule, the more extensive respiratory epithelium, and the posterodorsal olfactory epithelium, which comprises distinct cell types and that houses the olfactory neurons with their non-motile cilia (cranial nerve i: odors). underlying the olfactory epithelium is the lamina propria, which contains blood vessels, lymphatics, branches of the trigeminal nerve (cranial nerve v: burning, cooling, irritation, tickling), and the ensheathing cells surrounding the efferents of olfactory neurons (i.e., fila olfactoria) that cross the deep cribriform plate. of note, the trigeminal sensory system constitutes the largest nervous passage that directly innervates the brainstem medulla and, among its three branches, it is important to consider that both the ophthalmic and the maxillary fiber gets in close contact with potential viral accesses, being the conjunctiva (chen et al., 2020b) and the nasal mucosa. opportunely, this mucosa has an extensive surface area that collides with thousands of different particles every day, but whose access is evaded by tight and adherence junctions. the disruption of these junctions causes irreversible functional damages, still without fully being associated with an altered mucosa permeability (ganger and schindowski, 2018) . given the humidity and temperature of the nasal cavity together with the stability of sars-cov-2, it is plausible that the virus has the characteristic features of having a good replication rate in the olfactory mucosa. in fact, higher viral loads of sars-cov-2 were found in the nose compared to the throat (zou et al., 2020) , and this fact could possibly be associated to the unware transmission of the virus by asymptomatic individuals. however, subjects infected with sars-cov-2 resulted to be recurrently coinfected with other respiratory viruses (conger, 2020) , rendering the identification of smell disorders as an early covid-19 sign hard to recognize. any viral particles proliferating in the nasal cavity provokes an inflammatory response at the level of the olfactory epithelium, easily impairing the odor perception upon mucosal swelling and nasal blockage. nevertheless, these events should be accompanied with upper respiratory tract symptoms, such as rhinorrhea, nasal discharge, or sneezing, but these symptoms were not reported during sars-cov-2 infection nor the attachment viral pattern so far. even if the vascular or lymphatic routes have been investigated only for drug administrations rather than infectious diseases (veronesi et al., 2020) , it is reasonable to assume that the coronavirus would get in close contact with these vessels, thus accessing the systemic circulation. however, the lymphatic network in the nasopharynx (i.e., the mucosa-associated lymphoid tissue) is in charge of the immunotolerance and reaction alike in any other tissue. tonsils, which project to the surface of submucosal lymphoid tissue or emerge into the pharynx, and their reticular epithelium are featured with different cell types including the microfold cells and leucocytes. these sites are nevertheless well-patrolled. however, even if an underlying inflammatory mucosa was not manifested through clinical signs, it should be considered that the sars-cov-2 affecting the olfactory epithelium could get access not only to the abovementioned non-nervous tissues but also to a variety of neuronal routes to access the brain. the respiratory system has two though not completely distinct vascular systems and depends for its function on both local and systemic sensory receptors, involuntary central processing, and voluntary breathing, with widespread lymphatic and nervous networks permeating the airways. in particular, the nerve fibers comprise those of the vagus nerve and the dorsal root ganglia. two important cells are the ciliated that propel the mucus toward the nasopharynx and the neuroendocrine cells, whose putative sensor function at the levels of the local airway ganglion may be involved in the fine-tuning of blood flow in response to hypoxia (adriaensen et al., 2003) . in fact, both sympathetic and parasympathetic perivascular nerve fibers extend to small intrapulmonary vessels and adapt the local vascular impedance to diverse conditions. the proper endothelial responses and vascular remodeling in the lungs are known to be highly dependent on the proper functioning of ace2 (li et al., 2013a) , whose expression appears to be increased in smokers and those with chronic obstructive pulmonary disease (leung et al., 2020) . the respiratory muscles have also a vital role, aiding the movement of the thoracic cavity to offer space for inhalation and air expulsion. giving the way of contagion, the host's receptor, and symptoms upon infection, sars-cov-2 is considered a respiratory virus. dry cough and dyspnea arise upon the destruction of epithelial cells of the airways and host's local, hence still controlled, immunoresponse. the potential to affect pneumocytes appears to be a typical feature of the sars viruses, with a replicative potential of sars-cov-2 being higher than its predecessor (chu et al., 2020) . the neuroinvasive potential may be a common denominator as well. giving the high innervation of the lungs, the possibility that the virus may access the nerve fibers of the airways should be of great concern. other coronaviruses were shown to be able to spread to the central nervous system by accessing from dorsal root ganglia (li et al., 2012) in parallel with the widespread blood and lymphatic networks, possibly preferring the circulating leucocytes as vectors rather than freely migrating in the fluids. this preference was indeed observed for sars-cov-1 (gu et al., 2005) , which was found in pulmonary macrophages . the alveoli are highly sprayed with blood and represent the most accessible spot before the endothelial system, where the pumping flow would ensure the rapid dissemination of viral particles. if the coronavirus was able to exploit the blood flow, the infiltration in endothelial or epithelial cells of brain barriers would be plausible (desforges et al., 2019) . the central nervous system appears to be accessed by viruses through not only the hemotogenous, olfactory, trigeminal, or enteric routes. the nerve fibers in the lungs may be subjected, similarly to the enteric network, to two affections. first, the viral interference on critical sensory receptors for the autonomic regulation of breathing can cause a vagal dysregulation, which is known to be associated with negative changes in the reflex control of the airways (e.g., in chronic obstructive pulmonary disease) (undem and kollarik, 2005) . second, like in asthmatic lungs (el-chemaly et al., 2008) , the immuno-and thrombi-derived lesions during covid-19 can easily contribute to progressive lymphatic damage and alteration in the bronchial circulation that impair the nutrient-waste balance for the walls of the larger airways. the respiratory difficulties may require increased involvement of accessory muscles, with the progressive mental and physical exhaustion, hypoxemia, and pulmonary injury. patients infected with sars-cov-2 showed increased circulating levels of il-6, which could originate from the infected epithelial cells of the lungs (zhang et al., 2004) . importantly, the systemic flows of this cytokine have been associated with glial cell activation, brain injury, and infiltrated leukocytes (zhang et al., 2015; rothaug et al., 2016) , which may indeed offer the ideal trojan horse while the host's immune system is committed to counteracting the cytokine flow. a single, multi-component (vergnolle and cirillo, 2018) , epithelial barrier of cells protects the host against the xenobiotics located in the gut and intercellular junctions tightly seal the space in-between cells. the gut-associated lymphoid tissue with the mesenteric lymph nodes and the microfold cells are in charge of the sampling, phagocytosis, and transcytosis of luminal molecules, viruses, bacteria, parasites, and dietary supplements (briguglio et al., 2020a) , being critical for the first-line immune responses of the host (mabbott et al., 2013) . that is, sars-cov-2 faces a dual obstacle represented by both the physical barrier and the immune barrier before being able to fully affecting the enterocytes. however, the virus may be driven by the extensive presence of its host receptor, the ace2, whose expression in the gut reflects the dual role in mediating both the viral infiltration and the host's immunocompetence against the manifestations of covid-19 . most of the aspects of gut functions are controlled by the enteric nervous system, which comprises a network of neuronal cells deep in the wall of the bowel, with the proper homeostatic synchrony being guaranteed by the communication with the intestinal epithelium, dietary factors, the microbiota, and the mucosal immune system. the central nervous system is directly connected to the enteric neuronal network mainly through the parasympathetic vagus nerve that arises from the hindbrain and the sympathetic nerve fibers that arise from the spine, with the main neurotransmitters being acetylcholine and the catecholamines, respectively. the possibility of a fecal-oral transmission for sars-cov-2 has not to be excluded (yeo et al., 2020) as it can be found in stool samples of infected patients alike sars-co-v-1 and mers-cov (shi et al., 2005; corman et al., 2016; holshue et al., 2020) , with commonly referred gastrointestinal symptoms being abdominal pain or diarrhea. the neuroinvasive potential of coronaviruses could be reflected by the absence of tissue destruction or inflammatory lesions in the intestines of infected patients with sars-cov-1 , thus possibly suggesting the propensity of coronaviruses to possess a targeting pattern capable to avoid eliciting host's immunoresponses that could compromise the viral invasion at the early steps. alike the infiltration in the peripheral nerve endings of the olfactory system, coronaviruses were shown to be capable to invade the central nervous system also by making use of alternative peripheral pathways through the spinal cord (li et al., 2013b) . the presence of the virus in the gut may be due to the self-ingestion of mucus from the airways (li et al., 2020a) . undeniably, the enteric invasion can affect both the lymphatic and blood networks. hypoxic conditions in covid-19 patients can easily impair the proper regulation of the trafficking via lymphatic vessels, further contributing to increasing the inflammatory condition (miller et al., 2010) . the excessive infected lymphatic return to the general circulation for degradation in the liver may have a role in the liver damage observed in covid-19 patients, other than the virus-induced cytotoxicity of t cells and the cytokine flow (bangash et al., 2020) . moreover, the fine-tuning of the nervous reflex arcs that regulate important gastrointestinal functions, such as the fluid exchange, may be also impeded (yoo and mazmanian, 2017) . microthrombosis was indeed found in multiple organs (zhou et al., 2020) . it is important to consider that the eventual gut invasion by sars-cov-2 and the subsequent vagal affection could disturb not only the local abovementioned functions but also compromise the immunotolerance ( the axons of olfactory receptor cells that pass the cribriform plate contact the second-order neurons of the olfactory bulb within the spherical glomeruli. this route currently represents the most discussed around the reported dysosmia of patients infected with the coronavirus. in fact, this hypothesis, which we previously raised as the hypothesis 3, implies that sars-cov-2 is able to infiltrate in the olfactory bulb, possibly traveling through neuronal cells (transneuronally) or around neuronal cells (perineurally) to enter the brain. most of the studies that showed the possibility of a central nervous system infiltration from the nasal-nervous route were performed on mice, which have very important differences in the olfactory system (mcgann, 2017) . for instance, the bulbs of rodents are large and positioned prominently at the very front of the cerebrum whereas the counterparts in humans are proportionally smaller, flattened, and positioned underneath the frontal lobe. humans lack the accessory olfactory system, have 8-fold the number of glomeruli processing inputs from each receptor type compared to rodents, and possess more elaborate orbitofrontal cortical regions for interpreting odor inputs. it is vital to ponder the anatomy of the olfactory bulb and the different viral tropism across species. for instance, some authors reported different infiltration mechanisms for the same drug between humans and rodents, being the bulk flow (perineural route) for the first and the rostral migratory stream (transneuronal route) for the second (lochhead et al., 2015) . it should be also considered that the diverse microcircuitry and structure of the olfactory bulb between males and females (oliveira-pinto et al., 2014) may have a role in the nasal proliferation and nervous-access. pioneering studies supporting the olfactory bulb infiltration started in the first half of the twentieth century (faber and gebhardt, 1933) and the first human reports about a possible olfactory affection by sars-cov-1 came out 3 years after the 2003 outbreak (hwang, 2006) . from there on, clinicians and researchers posed much more attention to the possible neuroinvasive feature of viruses, mostly due to the potential fatal consequences upon infection of critical brain areas, such as the brainstem or the thalamus. undeniably, sars-cov-1 was found in both regions after being intranasally injected in laboratory mice (mccray et al., 2007) . this possibility should arise concern as much as that offered by adjacent nerves. trigeminal branches could allow a similar retrograde (toward the soma) and anterograde (toward the synapse) axonal transport toward the brain. however, the transitive relationship between nasal injection and brain infiltration does not assume that the nasal-nervous route is the putative pathway. the transneuronal pathway implies that the virus is endocytosed and transported in endocytic vesicles along the nerve fibers until reaching of the synaptic cleft in the olfactory bulb. the subarachnoid space may be reached here given its adjacent position to the olfactory bulb. however, in order to travel through the olfactory pathway the envelope of sars-cov-2 should be stable all along with the neuronal transport. the induction of apoptosis of infected olfactory receptors is in fact a well-known defense mechanism to prevent anterograde transport of pathogens (mori et al., 2002) . some authors reported cases of encephalopathy with no respiratory manifestations after the contagion with influenza (de jong et al., 2005; simon et al., 2013) . is it possible that the coronavirus was so virulent that it entered the central nervous system directly without passing through the airways? collateral damages from the cytokine flow are known to elicit a sequela of neuroendocrine immunoreactions in the infected host (silverman et al., 2005) , which if not properly controlled could cause permanent damages . the inflammatory-derived disruption of the olfactory epithelium may not only allow sars-cov-2 to proliferate in the lamina propria, but it could also alter the local milieu for proper nervous homeostasis (steinke et al., 2008) , thus allowing the virus to access the central nervous system perineurally through the diffusion in the cleft between the fila olfactoria and their ensheathing cells. these bulk flow processes imply that junctions between cells are more porous than normal, which may be due to either local inflammation or rapid turnover of olfactory neurons that leave a gap after their death. the time between cell death and the repositioning of a new olfactory sensory neuron may be sufficient to allow sars-cov-2 passage, especially if the viral load was very high in the nasal mucosa. the virus could even use the time it takes for new tight junctions to form (lochhead and thorne, 2012) . the absorption into blood vessels or lymphatics connected to the cervical nodes may be parallel routes as well. the brain serves any possible function in humans and, for the most part, remains separate from the other systems of the body, which keeps under control with the extensive network of nerve fibers. unfortunately, neuronal cells express the entry protein ace2 and therefore represent a predefined goal during the progressive sars-cov-2 host invasion. since the trigeminal nerve is the highway among nervous routes -particles up to 100 nm can enter the brain via this route (oberdorster et al., 2004 )-, it is likely that sars-cov-2 uses it as the nervous route. it is used also by bacteria to target central nervous system areas (st john et al., 2016) and it represents a delivery system of interest for far smaller drugs (crowe et al., 2018) . in the past, different viruses that shared the common nasal cavity as access site were observed to follow distinct noradrenergic, dopaminergic, or serotoninergic pathways (barnett et al., 1993; mclean et al., 1993) . indeed, is also the terminal nerve (cranial nerve 0: hormonal responses) that running medially to the olfactory tract enters the brain independently. there is no current report that ever investigated its role in host contagion, but the observed dysosmia in infected patients may recall the smell disorders typical of the kallmann syndrome, in which the terminal nerve could play a critical role in terms of hormonal disruption (taroc et al., 2017) . importantly, the affection of the terminal nerve may affect the hypothalamuspituitary-adrenal axis (sonne and lopez-ojeda, 2020), thus further compromising the host's immunocompetence. other than severe acute respiratory viruses, also prototype human coronaviruses, such as oc43 and 229e, had been found in the cerebrum (dube et al., 2018) . there are plenty of possible mechanisms that can be used by viruses to colonize the brain parenchyma, naming the trans-synaptic transport, microfusion of two adjacent neuronal cells, actin tails, or many others (van riel et al., 2015) . it is likely that sars-cov-2 is capable to make use of one of these (steardo et al., 2020) , but the fact that it can be transferred between neurons is of major concern (li et al., 2020b) . cases of encephalitis and acute flaccid paralysis had been reported not only from the well-known endemic coronaviruses (turgay et al., 2015; morfopoulou et al., 2016) , but also for sars-cov-2 zhao et al., 2020) . viral agents are known to possibly reach the cerebrospinal fluid by diffusing under the ensheathing cells of the fila olfactoria bodewes et al., 2011) . whether via this perineural route or nervousconnected pathways, after initial infection with sars-cov-2 maybe it is not as important as it gets to the brain, the problem is that it gets there. sars-cov-1 was found in brain tissues (xu et al., 2005) , showing necrotic neuronal damages, widespread gliosis, and leukocyte infiltration in the brain mesenchyme. once in the brain, sars-cov-2 is likely to cause similar damages. conversely, if sars-cov-2 could have not entered the brain, then the brain congestion, edema, and neuronal degeneration, which have been found in patients infected with sars-cov-2 (zhou et al., 2020) , would have been due to other causes. the cerebrospinal fluid that recirculates through the brain forms the glymphatic system together with the interstitial fluid (nedergaard, 2013) , and currently represent the main topic of interest that undermines the dogma regarding brain tolerance and the immune privilege. several points of communication give access to the central nervous system, comprising the circumventricular organs (e.g., the median eminence and the subfornical organ), which are in contact with the bloodstream, and the meninges and cerebrospinal fluid, which are in contact with the lymphatics (louveau et al., 2015) . the openings between the blood circulation and the brain parenchyma usually allow the access of circulating signaling molecules important for monitoring the periphery, but they also permit the passage of cytokines and infective agents, exposing local neural and nonneuronal cells. neurological damages are likely caused by the virus-induced inflammatory responses and hypoxic conditions typical of covid-19. the largest pial vessels and smallest brain capillaries allow the virus to spread, and injuries to the lymphatic system may lead to the accumulation of toxic waste products. microglial cells that reside in the brain are capable of phagocytosis and cytotoxic mechanisms that destroy foreign particles, unfortunately eliciting a cytokine flow that opens the blood-brain barrier (yin et al., 2017) . gliosis and glial cell apoptosis is accompanied by the infiltration of leucocytes from the perivascular region to the brain parenchyma where infected neuronal cells reside. if persistently-infected by sars-cov-2, these immune cells could serve as a reservoir and trojan horse for neuroinvasion that increases the viral load. the brainstem comprises many important structures critical for a variety of functions, such as body temperature and cardio-respiratory automation, and it is in charge of integrating humoral, neuronal, and physical responses, such as nausea and vomiting upon toxin detection. in particular, the medulla oblongata drives the respiration with sensorial outputs from metabolic receptors -mostly vascular-and mechanoreceptorsmostly pulmonary-further adjusting the inspiration and expiration. an important convergence point of many neural pathways at the level of the brainstem is the solitary tract, which is a myelinated fiber bundle that connects the trigeminal nerve rostrally and the vagus nerve caudally, being the initial relay for pulmonary receptors. among the circumventricular organs, the area postrema is an important local site that allows the touching of circulating substances with the brain parenchyma (briguglio et al., 2018) . brainstem infiltration of viral agents is known to be associated with medulla oblongata invasion and worst neurological consequences, such as neurogenic pulmonary edema (davison et al., 2012) . the brainstem was shown to be highly infected by both sars-cov-1 (netland et al., 2008) and mers-cov (li et al., 2016) . hence, its widespread anatomical interconnections render the brainstem a coveted and easily invaded prey even for sars-cov-2, which was indeed found in the cerebrospinal fluid (sun and guan, 2020) and therefore suggested to directly or indirectly affect the cardiorespiratory center in critically ill covid-19 patients. it is not clear which nervous routes are preferred by neurotropic viruses to access the brainstem, but both the cranial nerves and the motor components of spinal nerves were shown to be feasible (tan et al., 2014) . as we mentioned above, two types of damages are associated with sars-cov-2 infection, but much of the neuropathology may be associated with the host's inflammatory response to the viral infection. being connected to putative nervous routes of access, the solitary tract may be the intersection that causes direct brainstem infiltration and the cascade of systemic consequences. if we assume the early invasions of pulmonary nerve fibers, then sensory information that the nucleus of the solitary tract receives would be artificial. the sars-cov-2 is known to trigger a substantial systemic inflammatory storm with a subsequent significant break of the blood-brain barrier. following the sequela of neuronal damages that have been discussed after brain infiltration, we know that the inflammation-derived impairment of cerebral microcircuitry in the brainstem, the necrotizing leukoencephalopathy, the neuronal apoptosis of autonomic nuclei, and electrolyte disturbances are all features that can further impede a proper brainstem functioning. of note, these manifestations, at different levels of severity, are typical of septic conditions (benghanem et al., 2020) and therefore likely to occur in severe covid-19 patients. unfortunately, these subjects are often under anesthesia, which is known to increase the glymphatic fluxes (xie et al., 2013) , possibly favoring the trojan route (stamatovic et al., 2005) . the circulating cytokines that get in close contact with the area postrema could further affect the vagal signals together with the bulbospinal pathway that drives the automation of respiratory muscles. the more severe are the brainstem lesions the more complicated would be the ventilator management of critically ill patients. it is therefore conceivable that a brainstem-related neuro-immune impairment upon sars-cov-2 contagion can contribute to higher viral loads in critical neuroanatomical districts, multiple organ dysfunction, immunoincompentence, and fatal consequences. neuropsychiatric implications, such as hallucinations and depression disorders, were observed in individuals infected with other coronaviruses (severance et al., 2011) and sars-cov-1 was indeed found in brain tissues. neurological manifestations, such as polyneuropathy, were assumed to be mostly due to the cytokine flow (stainsby et al., 2011) , which often accompany the viral damage and long-lasting hypoxia in nervous tissues ultimately trigger neurotoxic pathways (de chiara et al., 2012) . similarly, both uncomplicated and severe patients with sars-cov-2 may show peripheral and central nervous system symptoms (mao et al., 2020) , and the inflammatory condition in the most severe individuals is known to be associated with acute delirium but also with the possible persistence of neuropsychiatric conditions, such as stress or depression, in those who will survive (lam et al., 2009) . higher incidence of swallowing disorders (zielske et al., 2014) and aspiration pneumonia (prescott et al., 2015) has been reported in critically ill survivors, thus rendering extremely important the consideration of long-term consequences also in covid-19 patients. the loss of the neuronal population in critical brain areas might underlie these conditions, which could also interest peripheral neuromuscular districts (tsai et al., 2004) . undeniably, this pandemic has shocked everyone, from the infected patients and healthcare workers to the families of the victims and those isolated at home watching the media reporting almost one victim of covid-19 every 2 min in italy. the implications of the "olfactory vector hypothesis" in a number of neurodegenerative diseases, such as alzheimer's and parkinson's diseases, has been presented years ago (doty, 2008) , but it should still be worthy of consideration. patients infected with sars-cov-2, if it was able to enter the central nervous system, would easily encounter neuronal damages over time that might contribute to the development of neuropsychiatric disorders. if we assume that sars-cov-2 could not infiltrate the brainstem, then the neuroinflammation and hematological disruptions would still promote both acute and chronic neuropsychiatric consequences. are also collateral mental conditions to be considered when setting the mental rehabilitation paradigm. subjects isolated or in quarantine experience loneliness, boredom, and anger whereas infected patients mostly experience fear (carvalho et al., 2020) . that is, individuals may feel low, bored, frustrated, lonely, worried for themselves of their kin, anxious, concerned about their finances or health. on the other side, the mental health of the hospital personnel, who is not usually used to deal with emergencies like covid-19, should not be neglected as well. long-term physical and emotional statuses were both showed to be impaired in this population group after the sars-cov-1 outbreak (lam et al., 2009; ngai et al., 2010) . moreover, it was observed that these healthcare workers with or at risk of neuropsychiatric conditions at the time of the outbreak have been more likely to quit their job at the end of the emergency. the travel of sars-cov-2 into the human host is likely to follow no specific route, but several in parallel. considering the infiltration in distinct neuroanatomical sites, we can infer a model of symptom progression (figure 1 ) that is in line with our personal experience but also with other authors' reports about the early stages of the disease (mason, 2020) . the nasal mucosa of humans seems a perfect environment for sars-cov-2 proliferation making it a potential reservoir for infection, like the salivary glands . a possible implication of the glossopharyngeal and facial nerve fibers has not to be figure 1 | the symptom progression during the invasion of the human host by the sars-cov-2 upon nasal (olfactory bulb) access. the severe acute respiratory syndrome coronavirus that was discovered in hubei province, china at the end of december 2019 (sars-cov-2) is a single-strand positive-sense rna virus with the encoding potential of four structural proteins: the spike, the envelope, the membrane, and the nucleocapsid. the human host presents many potential accesses, but the inhalation of infected respiratory droplets exposes the airways first. (n) assuming a nasal access, the olfactory mucosa may represent the primary site of the host for viral proliferation. it is reasonable to believe that sars-cov-2 needs to replicate several times before it can reach a harmful potential to infiltrate in the olfactory bulb, with the smell alterations being reported as one of the earliest symptoms. (o) the terminal nerve fibers extend in the olfactory epithelium colonized by the coronavirus. here, not only axons of the fila olfactoria can be used as routes for central access, but also the trigeminal branches, the terminal nerve, blood or lymphatic vessels, or the cleft under the ensheathing cells of olfactory axons. (p) upon inhalation, alveoli get in close contact with the virus, and it is reasonable to assume that the local inflammation could affect the different nerve fiber populations comprising the neuroendocrine cells and the annexed vagal contacts. lung infection, which might manifest even few days after the contagion, can therefore provide direct access to the solitary tract and other parts of the central nervous system. (e) once the virus has been accessed the epithelial barrier of gut cells through mucus or other vector ingestion, it can colonize the intestines that are permeated with the lymphatic system that drains the villi, the blood capillaries, and both the extrinsic and intrinsic neural networks. a fluctuating but chronic symptomatic onset is easily found. (b) it is still not clear which is the main route for the central nervous system access that may have a critical role in sars-cov-2 nervous infiltration, but both the brain and brainstem are likely to be infiltrated. high temperature is one of the earliest symptom after infection when more robust immune response is triggered, thus subsequently being associated with a permeabilization of the blood-brain barrier and immune cell infiltration. cardiorespiratory centers in the medulla are critical districts that, if affected by either direct viral damages or indirect neuroinflammation and hypoxia, can lead to irreversible damage and fatal outcome. excluded though. all three hypotheses previously advanced may be partially true regarding the smell disorders of infected patients. a partial nasal blockage for co-viral infections, an inflammation of the olfactory epithelium, and a viral insult at the level of olfactory receptor cells may all be concomitantly present. however, some authors recently suggested that the primarily targeted cells appear to be not the neurons, but those cells that would have been in charge of its early airway clearance through sputum expectoration, being the secretory and ciliated epithelial cells (sungnak et al., 2020) . the third hypothesis may be postponed though, thus making the hypothesis of inflammation and mucosal swelling in the back of the nose of primary importance (brann et al., 2020) . it is therefore still too early to acknowledge the nasal-nervous pathway as a potential route for host access and the local immunocompetence of the olfactory bulb may be sufficient to block the sars-cov-2 entering (durrant et al., 2016) . moreover, there are many other neglected routes for neuroinvasion: the trigeminal nerve, the terminal nerve, the enteric and pulmonary nervous networks, the vagus nerve, the dorsal root ganglia, the hematogenous flow through the circumventricular organs of the brain and brainstem, the peripheral lymphatic vessels, the central glymphatic system, the cerebrospinal fluid, the migrating trojan horses, and the conjunctiva. that is, no viral passage across the olfactory bulb of the host has been confirmed so far and how sars-cov-2 gain access to and spread in the central nervous system remains to be explored. on the contrary, if dysosmia was an early sign of infection it could be too late to avoid neuronal affections as viral transport along axons is possibly moving about 2 µm/s in the retrograde direction (bearer et al., 2000) . because of the spatial distribution at the level of the head, it is much more likely that the preferred retrograde pathway is the one involving the shortest nerves at the level of the head, rather than the more systemic ones, such as the vagus nerve. italy is one of the worst-hit countries in europe with a death rate of 13.9% (who, report of may 8th, 2020). among the currently infected, 72,157 are quarantined at home, 14,636 hospitalized in acute covid-19 wards, and 1,168 hospitalized in intensive care units. deceased patients had a mean age of 80 years old, 60.9% were males, 60.9% had three or more comorbid conditions (most common had been hypertension in 68.2% and ischemic heart disease in 28.4% of cases). at admission, the main symptoms were fever (76%), dyspnea (73%), dry cough (38%), diarrhea (6%), and hemoptysis (1%). a mean of 5 days passed from the onset of symptoms to hospitalization and the other 5 days from hospitalization to death (sars-cov-2 surveillance group of italy, report of may 7th, 2020). our direct experience in managing covid-19 patients is in line with the abovementioned records. in addition, we observed also dysosmia being reported by over 80% of our patients. however, symptom reports at admission should not be taken for granted but contextualized on the feverish subject. in fact, all patients admitted to our hospitals are being classified as level 2 or higher (internal classification of covid-19 patients compriseslevel 0: asymptomatic-, -level 1: mild symptoms, pharyngodynia, dry cough, mild fever, -level 2: moderate symptoms, high fever, persistent cough, asthenia, dyspnea, non-invasive oxygen therapy-, -level 3: severe symptoms, invasive oxygen therapy, intensive care). our infected patients were all malnourished (briguglio et al., 2020b) and disoriented in time and space, thus rendering it very difficult to make an exhaustive history about the dysfunction of the olfactory capacity and its onset in reference to the disease. these symptoms may also derive from excessive exposure to disinfectants due to the pandemic (keyhan et al., 2020) , but also from medicines that are known to interfere with the smelly sensations and the aftertaste. on the other hand, many of our front-line colleagues infected with sars-cov-2 often reported a loss of taste and smell, which preceded the systemic symptoms, such a fever, loss of appetite, and diarrhea. since numerous microthrombi are also being observed on peripheral limbs of our hospitalized patients, being likely to occur in pulmonary and cerebral capillaries as well, the neuronal damages should be of great concern. other than antibiotics, antivirals, or corticosteroids, a therapy with lowmolecular-weight heparin at daily doses over 4,000 iu should be considered for hospitalized covid-19 patients. neuronal injuries in critical brain areas of patients that will survive may carry permanent consequences, as was observed for sars-cov-1 survivors (hui et al., 2005) . moreover, the risk of an impaired sympatho-vagal signal is worrying since its proper functioning is important for the peripheral immune response and central microglial polarization (wang et al., 2018) . some authors even suggested that neuronal cells could serve as reservoirs for the virus (kabbani and olds, 2020) . to the best of our knowledge, sars-cov-2 has not been found in nervous tissues so far. in italy, brain autopsies are currently suspended due to biohazard security concerns (previtali et al., 2020) . however, other authors reported the presence of the virus in the cerebrospinal fluid (wu et al., 2020) and others observed multiple cerebral infarcts in vascular territories . dead-end signs of the coronavirus cycle may reflect respiratory signs in the severe covid-19 host, such as ataxic breathing, altered rate and synchrony, apnea, or hyperventilation. overall, the survival of the virus in the nasal cavity plus its neuroinvasive potential is not a novel pathway of contagion among the infective agents. this double survival in two sites of the same host permits the virus to efficiently exploit its neurotropism while still preserving the host-to-host transmission (macgibeny et al., 2018) . the risk of contracting sars-cov-2, like any other infection, primarily depends on the degree of exposure to the pathogen. whether the infected host protects himself and others from contact might contrariwise depend on the level of awareness and, currently, there is no recognition of smell disorders being an early symptom of sars-cov-2 infection (soler et al., 2020) nor it is established the association between dysosmia and sars-cov-2 olfactory bulb invasion. however, as the sars-cov-2 virus spreads, so changed the feature of its pandemic (brussow, 2020) and the possibility that individuals can carry the virus without knowing is a risk that our society cannot take. some individuals may show symptoms of sars-cov-2 infection 10 days after exposure (lauer et al., 2020) , which is a remarkable amount of time during which asymptomatic individuals can be carriers without knowing. to conclude, we can say that: • some infected individuals show dysosmia, which may imply nasal proliferation of sars-cov-2. • public health actions should consider smell disorders when defining social distancing criteria. • axonal, trans-synaptic, blood, lymphatic, or trojan transport are routes for viral neuroinvasion. • viral access in the peripheral or central nervous system 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leaving no stone unturned: allosteric targeting of sars-cov-2 spike protein at putative druggable sites disrupts human angiotensin-converting enzyme interactions at the receptor binding domain. date: 2020-10-16 journal: inform med unlocked doi: 10.1016/j.imu.2020.100451 sha: doc_id: 332948 cord_uid: h297ukuu the systematic entry of sars-cov-2 into host cells, as mediated by its spike (s) protein, is highly essential for pathogenicity in humans. hence, targeting the viral entry mechanisms remains a major strategy for covid-19 treatment. although recent efforts have focused on the direct inhibition of s-protein receptor-binding domain (rbd) interactions with human angiotensin-converting enzyme 2 (hace2), allosteric targeting remains an unexplored possibility. therefore, in this study, for the first time, we employed an integrative meta-analytical approach to investigate the allosteric inhibitory mechanisms of sars-cov-2 s-protein and its association with hace2. findings revealed two druggable sites (sites 1 and 2) located at the n-terminal domain (ntd) and s2 regions of the protein. two high-affinity binders; zinc3939013 (fosaprepitant – site 1) and zinc27990463 (lomitapide – site 2) were discovered via site-directed high-throughput screening against a library of ∼1500 fda approved drugs. interestingly, we observed that allosteric binding of both compounds perturbed the prefusion s-protein conformations, which in turn, resulted in unprecedented hace2 displacement from the rbd. estimated δg(binds) for both compounds were highly favorable due to high-affinity interactions at the target sites. in addition, site 1 residues; r190, h207, k206 and k187, i101, r102, i119, f192, l226, v126 and w104 were identified for their crucial involvement in the binding and stability of zinc3939013. likewise, energy contributions of q957, n953, q954, l303, y313, q314, l858, v952, n953, and a956 corroborated their importance to zinc27990463 binding at the predicted site 2. we believe these findings would pave way for the structure-based discovery of allosteric sars-cov-2 s-protein inhibitors for covid-19 treatment. the novel coronavirus disease also referred to as covid-19 is caused by the sars-cov-2 (severe acute respiratory syndrome coronavirus 2), with incidences first reported in wuhan china in december 2019. 1 this disease has, however, persisted till mid-2020, spreading across 212 countries with over 3,513,507 cases reported coupled with increasingly high casualties numbering over 245,544 globally. 2 sars-cov-2 belongs to a large group of coronaviruses which are known to cause respiratory infections and related complications. these rna viruses are spherical, pleomorphic, positive-sensed, single-stranded and polyadenylated. 3 of all known viruses, coronaviruses (covs) have the largest rna genome 4 , with diverse pathogenic effects in animals and humans. this virus class is divided into four genera namely: alpha-cov, beta-cov, gamma-cov and delta cov [5] [6] [7] , with the beta-cov class prominent for their disease-causing effects in humans (hcovs). seven hcovs have been characterized to date [6] [7] [8] ; among which four (hcov-hku1, hcov-oc43, hcov-nl63 and hcov-229e) cause very mild respiratory symptoms. 9, 10 on the other hand, mers-cov, sars-cov, and sars-cov-2 cause severe respiratory and gastrointestinal infections which, in most cases, can be fatal. 11 although sars-cov-related infections were zoonotically transmitted into human populations, 12, 13 human to human transmissions has further contributed towards viral super-spread via respiratory aerosols. 14 the entry of sars-cov-2 coupled with its replication process in target human cells is achieved by the functionalities of a cohort of components, majorly non-structural and structural proteins, that make up the virus. generally, about 16 non-structural proteins (nsps) mediate diverse pro-pathogenic functions such as replication, processing and proof-reading of genomic frames, host immune evasion among many others, as previously reported. [15] [16] [17] more so, covs comprises of four major structural proteins that are integral to their pathogenesis. [18] [19] [20] these are the nucleocapsid (n), envelope (e), membrane (m) and spike (s) proteins. the n protein makes up the nucleocapsid and other viral genome-related processes 21 while the m protein is the most abundant of the four, playing major roles in maintaining viral structural integrity as well as coordinating other structural proteins. 22 e protein, on the other hand, is crucial to the maturation of the virus [23] [24] [25] [26] [27] while the trimeric s protein mediates viral entry into the host cell via the endosomal or non-endosomal route. 28 two domains make up the s protein namely the n-terminal s1 domain and the c-terminal s2membrane-anchored domain. the s2 region is extensively conserved in covs while constituent s1 region residues are highly diverge across the cov strains. 29 these domains have been further characterized into subdomains due to specific functionalities with respect to host receptor recognition and binding (s1), coupled with membrane fusion and entry (s2) (figure 1 ). similar to sars-cov architecture, some recent reports have sub-categorized the sars-cov-2 s1 ectodomain into the n-terminal domain (ntd), a conserved receptor-binding domain (rbd) which recognizes the human angiotensin-converting enzyme 2 (hace2), 30 and subdomains 1 and 2 (sd1 and sd2). during infection, proteolytic cleavage or priming of the s protein is crucial for viral fusion and entry into host cells, a process mediated by host cell proteases such as the transmembrane serine protease 2 (tmprss2) and cathepsin l, [31] [32] [33] at the s1/s2 (boundary between s1 and s2 subunits) and s2' (immediately upstream s2 fusion peptide -fp) cleavage sites. [34] [35] [36] the s protein primarily exists in a metastable prefusion complex prior to cleavage, after which notable conformational arrangements occur in order to fuse the viral membrane into j o u r n a l p r e -p r o o f the host cell. [37] [38] [39] in addition, the rbd adopts disparate conformational motions to engage the host cell receptor. 40, 41 conformations. [42] [43] [44] the up conformation corresponds to the hace2 accessible state while the down state cannot engage the host cell receptor. 44 the s2 domain, on the other hand, consists of the functionally important fusion peptide (fp), which is critical for viral fusion and formation of the post-fusion complex; heptad repeats 1 and 2 (hr1 and hr2); transmembrane domain (tm) and cytoplasmic tail (ct). the hrs of the s-protein trimer interact to form a fusion core of sixhelical bundle which helps bring the membranes of the virus and host cell in close proximity for fusion and entry. 42 therefore, the roles of sars-cov-2 s-protein present it as an important therapeutic target, which would enable the prevention of viral entry and fusion in host cells. numerous studies have been reported over the past months with regards to the possibility of blocking direct interactions between sars-cov-2 s-protein and hace2. most of these studies were aimed at targeting the s protein rbd domain with antibodies, peptide-based or small molecule compounds that binds with a much higher affinity to block s-protein-hace2 interactions. [45] [46] [47] [48] [49] [50] also, targeting host proteases such as tmprss2 was explored in a recent study, with consequential impediments on sars-cov-2 entry. 30 identification of other functional (allosteric) sites on the prefusion s protein could present another dynamic and effective approach of preventing sars-cov-2 infectivity relative to its interaction with the host cell ace2 and proteases. this alternative target approach for sars-cov-2 s protein is important because its rbd (similar to other covs) has been associated with a high mutational propensity which may in turn alter the affinity of small molecule inhibitors or peptide designed to bind therein. 51 allosteric targeting was explored in a recent study wherein the cov-conserved s2 hr1 region was identified as an important target site for the development of broad-spectrum inhibitors of human covs. the resulting peptide inhibitor (ek1) was evaluated in vivo and exhibited desirable safety and efficacy 52 . more so, the protein contact network (pcn) paradigm was used to map functional allosteric loci on sars-cov s protein. 53 relatively, this study was implemented to (i) identify potential druggable sites across the s1 and s2 domains of the sars-cov-2 s protein other than the rbd-hace2 interface (ii) perform high-throughput (virtual) screening of ~1500 fda approved drugs against the most druggable site(s) (iii) investigate the binding dynamics and interaction mechanisms of the compounds and their consequential effects on the s-protein rbd-ace2 complex. we believe this systematic study will be able to provide structural and molecular insights into possible allosteric sites on sars-cov-2 s protein suitable for selective targeting and structure computational methodologies the three-dimensional structure of sars-cov-2 s-protein (prefusion) was retrieved from pdb with entry 6vsb. 44 this, as previously reported, represents the s-protein rbd conformation in its up (open) state, which is most suitable for hace2 binding. also, to model binding interactions between the prefusion sars-cov-2 s-protein (s1/s2) and the hace2, a crystalized structure with pdb entry 6m0j 54 was separately retrieved. this complex depicts binding between the rbd domain (truncated) of sars-cov-2 s-protein and the protease domain (pd) of hace2. co-crystallized molecules not relevant to this study were removed while missing residues (gaps) in the structures were filled using the modeller algorithm. 55 this preparation was performed on the ucsf chimera graphic user interface (gui). 56 subsequently, using the structural superposition method, we were able to model a complex between prefusion s-protein (s1/s2) monomer (rbd -up conformation) and the hace2 protein ( figure 2 ). j o u r n a l p r e -p r o o f possible druggable sites other than the sars-cov-2 rbd interface were predicted using approaches previously reported. [57] [58] [59] [60] herein, we employed multiple tools for site identification and validation, which include sitemap 61 , fpocket 62 , discovery studio 2016 client 63 and prankweb. 64 sitemap is an exhaustive tool which ranks protein pockets based on properties such as druggability, surface exposure, hydrophobicity and hydrophilicity among others [65] [66] [67] . these details were then used to characterize the predicted pockets after which other predictive algorithms were used complementarily for cross-validation. two highly ranked sites were then selected for further analyses. furthering on the rationale of the study, we mapped out the two most druggable sites on the target protein and virtually screened against them a large chemical library of fda approved drugs (~1500 compounds) derived from the zinc repository (http://zinc.docking.org/substances/subsets/fda/). this screening was performed using highperformance computing-integrated autodock vina 68 prior to which coordinates of the predicted sites were mapped using gridboxes. corresponding binding scores were retrieved from the resulting .pdbqt files and were used to filter down to the topmost 20 compounds for each predicted sites 1 and 2. subsequently, two compounds with the highest binding scores (most negative) were selected for the two predicted sites yielding complexes that were subjected to further simulation studies. as explained in 2.1, the prefusion s-proteins (ligand-bound and unbound) were superimposed with the rbd-hace2 complex (6m0j) after which the single j o u r n a l p r e -p r o o f truncated rbd was removed. by so doing, we obtained models of allosterically-bound and unbound pre-fusion s-protein-ace2 complex. this, as aimed in this study, would provide structural and dynamical insights into the mechanistic effects of allosteric targeting on sars-cov-2 host entry machinery. although computationally expensive (1673 residues), we proceeded with long-timescale md simulation runs for the systems on amber18 graphical processing unit (gpu) using its embedded modules. 69 protein parameters were defined using the ff14sb forcefield while ligand parameters were generated with the antechamber and parmchk modules. likewise, the leap program was used to define coordinate and topology files for the ligand-bound and unbound protein complexes. this program, also, was used to neutralize (addition of counter-ions; na + and cl -) and solvate the systems in a tip3p water box of size 10å. structural minimization was first carried out partially for 5000steps with a restraint potential of 500kcal mol -1 . å 2 followed by another 100000 steps of full minimization with no restraints. a canonical (nvt) ensemble with a 5kcal mol -1 å 2 harmonic restraints was used to heat the systems gradually from 0 -300k for 50ps, after which the systems were equilibrated for 10000ps at a constant 300k temperature without restraints in an npt ensemble. atmospheric pressure was maintained at 1bar with a berendsen barostat 70 while each protein system was subjected to a production run of 350ns. studied systems include zinc3939013-s-protein-hace2 (allosteric site 1), zinc27990463-sprotein-hace2 (allosteric site 2), and unbound s-protein-hace2. corresponding trajectories were saved at every 1ps time-frame until the end of the simulation followed by data plot analyses using microcal origin software. 71 snapshots were also taken and analyzed to monitor structural events and ligand interaction dynamics across the trajectories on the ucsf chimera user j o u r n a l p r e -p r o o f interface (gui) and discovery studio client. 63 the molecular mechanics/generalized born surface area (mm/gbsa) method was used to evaluate binding affinities of the predicted allosteric s-protein binders at their target sites. binding energy profiles for both compounds, inclusive of their energy components, were estimated using 1000 snapshots from the terminal 30ns of md trajectories where conformational stabilities were visible. this approach was important in order to minimize the effects of conformational disorder or entropy on ligand interactions. the equations below mathematically express binding energy calculations: as shown, internal (∆e int ), electrostatic (∆e ele ) and van der waals (∆e vdw ) energies sum up the gas-phase energy (∆g gas ) while the solvation free energy (∆g sol ) is defined by the polar solvation (∆g ele,sol ) and non-polar contribution to solvation (∆g np,sol ) terms. the mm/gbsa method was used to estimate the generalized born (gb) for ∆g ele,sol while the linear relationship between the surface tension proportionality constant (γ = 0.0072 mol -1 å -2 ), solvent accessible surface area (sasa, å 2 ), and β constant was used to solve ∆g np,sol . furthermore, estimated ∆g bind was decomposed into individual residue energies, most especially those that constitute the predicted allosteric pockets where the ligands were bound. this method was essential to identify specific residues that contribute crucially to the stability and inhibitory activities of potential allosteric inhibitors. j o u r n a l p r e -p r o o f based on the study rationale, we set out to identify possible sites for drugging the target protein table 1 ). the architectures of these pockets are shown in figure 3 . furthermore, defining the druggability of a site on target proteins depends on the size (volume) and hydrophobicity (with minimal hydrophilicity) while, on the other hand, high hydrophilicity, reduced hydrophobicity, small pocket size and shallowness characterize "difficult-to-drug" and undruggable pockets 61, [65] [66] [67] . while large hydrophilicity could have repulsive effects on ligand mobility at the binding site, a small or shallow cavity would impede ligand access, fitness, optimal binding and stability. j o u r n a l p r e -p r o o f from table 2 , sites 1 → 3 ranks above the 0.83 halgren dscore threshold making them suitable for therapeutic targeting. relatively, site 1 appears to be highly surface-exposed with a score of 0.933 while a large pocket size and volume for site 2 could favor the use of large-molecule compounds. taken together, high surface-exposure coupled with relatively large volumes, hydrophobicity and favorable donor/acceptor properties for sites 1 and 2 could account for their suitability as targetable allosteric regions on the s-protein other than the rbd (figure 3 ). these presumptions are also reflected by the estimated dscore and sitescore values. in addition, since these predicted sites are highly functional, particularly the overlapping fp, hr1 and cr, targeting them could high-throughput screening and identification of potential allosteric binders to the predicted sites 1 and 2 high-throughput screening using a library of ~1500 fda approved drug compounds (http://zinc.docking.org/substances/subsets/fda/) were performed against the two predicted allosteric sites. results for the top 20 compounds with the highest binding scores are presented in supplementary table s1 and supplementary table s2 for sites 1 and 2 respectively. from the screening results, overall highest scores were estimated for zinc3939013 (-10 j o u r n a l p r e -p r o o f kcal/mol) at site 1 and zinc27990463 (-9.3 kcal/mol) at site 2. as highlighted in our methods, md simulations were performed for the prefusion s-protein-hace2 complexes bound distinctly at two potential allosteric sites. this approach was essential to investigate the likely effects of allosteric targeting on the entry/fusion mechanisms of sars-cov-2 via host hace2. however, this conformation appeared distorted the allosterically-bound s-proteins and could account for displacement motions of the interacting hace2 from the rbd interface. therefore, the allosteric-mediated disruption of sars-cov-2 s-protein rbd and its interaction with hace2, as reported herein, is a major finding that could indicate the viability of allosteric targeting in sars-cov-2 therapy. furthermore, we measured structural stabilities across the ligand-protein complexes relative to the unbound system using the rmsd metrics. as shown in figure 6 , structural instability was highest in the unbound s-protein while its associated hace2 was relatively stable compared to this could indicate the structural effects of allosteric targeting on the s-protein and its interaction with hace2. estimated mean rmsds, as presented in table 2 , corroborates conformational variations among the unbound and bound protein complexes. to minimize the effects of structural disorderliness (entropy) in our calculations, we selected, from the md trajectories, terminal time-frames (270-300ns) from which the systems appeared to relatively stabilize. these were defined as the finally equilibrated (fe) time-frames and were used for subsequent structural analyses ( table 2 ). from the resulting fe-rmsd plots, unbound s-protein was highly unstable while its associated hace2 exhibited low structural motion in line with the rmsd calculations, which could also imply that the binding of s-protein stabilized hace2. in contrast, the allosterically-bound sproteins (sites 1 and 2) were notably stable while their corresponding hace2 showed high structural instability that could correlate with their systemic motions at the s-protein rbd as earlier mentioned. structural analyses of ligand orientations at the respective allosteric sites of sars-cov-2 sprotein were performed using averaged structures from the md trajectories ( figure 9 ). findings reveal that the allosteric binding of zinc3939013 (fosaprepitant) was stabilized at the ntd. fosaprepitant contains a terminal triphosphate group that orients towards residues such as n99, k187, n188, r190 and h207. likewise, its trifluoromethyl group oriented towards d102 while constituent -o and -nh groups mediate interactions with q173 and n121, among others. these altogether could facilitate high-affinity interactions accountable for its stability and allosteric inhibitory effects against the sars-cov-2 and associated hace2. j o u r n a l p r e -p r o o f binding affinities of the compounds were determined using the mm/pbsa technique, which also allowed us to measure the energy contributions of interactive residues at the predicted allosteric sites. energy calculations, as presented in table 4 were performed using relatively stable time-frames (270-300ns) to minimize entropical effects that may interfere with ligand binding activities. in addition, we observed that electrostatic effects contributed most notably to the allosteric binding of zinc3939013 at the ntd region while van der waals contributions had the highest effect on the binding of zinc27990463 at the predicted site 2 pocket. electrostatic contributions at site 1 could be due to the high number of electropositive residues that constitute the pocket, as shown in figure 10 , which may form high-affinity interactions with electronegative moieties of the compound. calculations further revealed that ∆e vdw and ∆e ele were more favorable in the gas phase for zinc3939013 while polar solvation energies were more favorable for zinc27990463 j o u r n a l p r e -p r o o f at the s2 region of the s-protein. this could imply that while the former was buried in the deep hydrophobic pocket of the ntd, the latter was surface exposed due to its trans-domain binding activity as earlier reported. to understand the mechanistic binding of the compounds at both predicted sites, we decomposed the binding free energies into individual contributions of the interacting residues. these were juxtaposed with structural analysis that showed the type and (π-alkyl) interactions. more so, π-π stacked interaction between y313 and a benzene ring (of the 4-tri-fluoromethyl-1,1'-biphenyl group) could be highly crucial for the stability of the compound. taken together, electrostatic energies favored the binding of zinc3939013 at site 1 while vdw energies favored zinc27990463 binding at site 2, which consequentially, were able to perturb the s-protein rbd and allosterically disrupt hace2 interactions. the systemic entry of sars-cov-2 into the human host cell is a crucial process that underlies its virulence and pathogenicity in humans and other animals it infects. this mechanism is mediated by its interaction with the host ace2 (hace2) via attachment and fusion. potential intervention approaches in sars-cov-2 treatment include therapeutic strategies that could prevent sars-cov-2 s-protein binding to hace2. in this study, we implemented an exhaustive approach to identify drug molecules that could potentially bind to sars-cov-2 s-protein at other sites other than the rbd. pertinent to the allosteric targeting approach implemented herein j o u r n a l p r e -p r o o f was the identification of highly druggable sites inherent in the s-protein (s1/s2), which was carried out using multiple pocket prediction algorithms for identification and validation of possible allosteric sites. predicted pockets were then characterized based on their attributes after which two highly probable pockets were selected. these were then screened distinctly against a library of ~1500 fda approved drugs retrieved from the zinc database. amongst all, thermophoresis (mst) can be employed for further validation. these implementations will provide additional insights into the targetability and suitability of these pockets for novel covid-19 therapeutics. findings from this study paves way for novelty in the structure-based design of high-affinity allosteric inhibitors or disruptors of sars-cov-2 association with host hace2 thereby preventing viral entry. authors thank the college of health sciences, university of kwazulu-natal, south africa for providing infrastructural support and we also acknowledge the center for high performance computing (chpc), capetown, south africa, for providing computational resources. authors declare no conflict of 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the gap: a critical step forward towards the design and discovery of potential drugs possible allosteric binding site on gyrase b, a key target for novel anti-tb drugs: homology modelling and binding site identification using molecular dynamics simulation and binding free energy calculations can we rely on computational predictions to correctly identify ligand binding sites on novel protein drug targets? assessment of binding site prediction methods and a protocol for validation of predicted binding sites identifying and characterizing binding sites and assessing druggability fpocket: an open source platform for ligand pocket detection prankweb: a web server for ligand binding site prediction and visualization new method for fast and accurate binding-site identification and analysis therapeutic target-site variability in α 1-antitrypsin characterized at high j o u r n a l p r e -p r o o f resolution silico assessment of potential druggable pockets on the surface of ??1-antitrypsin conformers autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading amber 18 molecular dynamics with coupling to an external bath originpro 9.1: scientific data analysis and graphing software-software review structural basis for the recognition of sars-cov-2 by full-length human ace2 mapping allosteric communications within individual proteins the following information is required for submission. please note that failure to respond to these questions/statements will mean your submission will be returned. if you have nothing to declare in any of these categories then this should be stated. all sources of funding should be declared as an acknowledgement at the end of the text. authors should declare the role of study sponsors, if any, in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. if the study sponsors had no such involvement, the authors should so state. studies on patients or volunteers require ethics committee approval and fully informed written consent which should be documented in the paper.authors must obtain written and signed consent to publish the case report from the patient (or, where applicable, the patient's guardian or next of kin) prior to submission. we ask authors to confirm as part of the submission process that such consent has been obtained, and the manuscript must include a statement to this effect in a consent section at the end of the manuscript, as follows: "written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal on request".patients have a right to privacy. patients' and volunteers' names, initials, or hospital numbers should not be used. images of patients or volunteers should not be used unless the information is essential for scientific purposes and explicit permission has been given as part of the consent. if such consent is made subject to any conditions, the editor in chief must be made aware of all such conditions. even where consent has been given, identifying details should be omitted if they are not essential. if identifying characteristics are altered to protect anonymity, such as in genetic pedigrees, authors should provide assurance that alterations do not distort scientific meaning and editors should so note. please specify the contribution of each author to the paper, e.g. study design, data collections, data analysis, writing, others, who have contributed in other ways should be listed as contributors.this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.not applicable to this study.fao conceptualized, implemented, analyzed, interpreted and wrote the manuscript, kfo performed molecular dynamics simulation, while mes revised and approved the manuscript for submission. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests:j o u r n a l p r e -p r o o f key: cord-312664-tgpaidhp authors: liang, julia; pitsillou, eleni; karagiannis, chris; darmawan, kevion k; ng, ken; hung, andrew; karagiannis, tom c. title: interaction of the prototypical α-ketoamide inhibitor with the sars-cov-2 main protease active site in silico: molecular dynamic simulations highlight the stability of the ligand-protein complex date: 2020-05-28 journal: comput biol chem doi: 10.1016/j.compbiolchem.2020.107292 sha: doc_id: 312664 cord_uid: tgpaidhp the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) causes an illness known as covid-19, which has been declared a global pandemic with over 2 million confirmed cases and 137,000 deaths in 185 countries and regions at the time of writing (16 april 2020), over a quarter of these cases being in the united states. in the absence of a vaccine, or an approved effective therapeutic, there is an intense interest in repositioning available drugs or designing small molecule antivirals. in this context, in silico modelling has proven to be an invaluable tool. an important target is the sars-cov-2 main protease (m(pro)), involved in processing translated viral proteins. peptidomimetic α-ketoamides represent prototypical inhibitors of m(pro). a recent attempt at designing a compound with enhanced pharmacokinetic properties has resulted in the synthesis and evaluation of the α-ketoamide 13b analogue. here, we performed molecular docking and molecular dynamics simulations to further characterize the interaction of α-ketoamide 13b with the active site of the sars-cov-2 m(pro). we included the widely used antibiotic, amoxicillin, for comparison. our findings indicate that α-ketoamide 13b binds more tightly (predicted glidescore = -8.7 and -9.2 kcal/mol for protomers a and b, respectively), to the protease active site compared to amoxicillin (-5.0 and -4.8 kcal/mol). further, molecular dynamics simulations highlight the stability of the interaction of the α-ketoamide 13b ligand with the sars-cov-2 m(pro) (δg = -25.2 and -22.3 kcal/mol for protomers a and b). in contrast, amoxicillin interacts unfavourably with the protease (δg = +32.8 kcal/mol for protomer a), with unbinding events observed in several independent simulations. overall, our findings are consistent with those previously observed, and highlight the need to further explore the α-ketoamides as potential antivirals for this ongoing covid-19 pandemic. effective therapeutic, there is an intense interest in repositioning available drugs or designing small molecule antivirals. in this context, in silico modelling has proven to be an invaluable tool. an important target is the sars-cov-2 main protease (m pro ), involved in processing translated viral proteins. peptidomimetic α-ketoamides represent prototypical inhibitors of m pro . a recent attempt at designing a compound with enhanced pharmacokinetic properties has resulted in the synthesis and evaluation of the α-ketoamide 13b analogue. here, we performed molecular docking and molecular dynamics simulations to further characterize the interaction of α-ketoamide 13b with the active site of the sars-cov-2 m pro . we included the widely used antibiotic, amoxicillin, for comparison. our findings indicate that α-ketoamide 13b binds more tightly (predicted glidescore = -8.7 and -9.2 kcal/mol for protomers a and b, respectively), to the protease active site compared to amoxicillin (-5.0 and -4.8 kcal/mol). (1) . a novel coronavirus was isolated and designated severe acute respiratory syndrome coronavirus 2 (sars-cov-2), causing coronavirus disease 2019 . as of april 16, 2020 , this ongoing global health emergency has resulted in over 2,000,000 confirmed cases in 185 countries and regions, with more than 25% of confirmed cases in the united states (2) . the global mortality rate has been estimated to be 5.7%, with higher mortality occurring among the elderly (3) . the majority of deaths have occurred among adults aged greater than 60 years and those with serious underlying health conditions, with the highest fatality in those aged greater than 85 years ranging from 10% to 27% in the united states (4, 5) . differences in disease prevalence are affected by sex, with data indicating that there is a higher prevalence of covid-19 among men (6, 7) . the majority of early cases were linked to exposure to the huanan seafood wholesale market, potentially through zoonotic transmission (8) . human-to-human transmission of sars-cov-2 was subsequently found to occur, with an attack rate within families of 83% suggestive of its high transmissibility (9, 10). the current outbreak of sars-cov-2 follows that of recent outbreaks of severe acute respiratory syndrome coronavirus (sars-cov) in 2002 and the middle east respiratory syndrome coronavirus (mers-cov) in 2012 (11) . these coronaviruses are both zoonotic pathogens, with bats serving as the primary reservoir (12) . masked palm civets were the intermediate reservoir for sars-cov, and dromedary camels for mers-cov, where zoonotic transmission to humans subsequently occurred (12) . while sars-cov-2 appears to have lower fatality rates than sars-cov (9.5%) and mers-cov (34.4%), it has a greater ability to spread (11, 13) . like sars-cov, the pathogenesis of sars-cov-2 involves the binding of its spike protein to angiotensin converting enzyme-2 (ace2) in the host (14, 15) . when cleavage occurs between the s1 and s2 subunits, the spike protein becomes activated for j o u r n a l p r e -p r o o f membrane fusion for entry into the host cell (14, 15) . ace2 is expressed on numerous tissues in the nasopharynx and intestinal epithelia, particularly in type ii alveolar cells in the lung (16) (17) (18) . following entry of the virus into the host cells, viral rna attaches to the host ribosome for translation of large polyproteins that are processed via proteolysis into components for new virions (19, 20) . along with the papain-like protease, the coronavirus main protease (m pro ) is responsible for this proteolysis (19) . encoded by open reading frame 1 (orf1) of the genome as non-structural protein 5 (nsp5), m pro cleaves at 11 sites in the polyproteins (19) . to date, there is an absence of a vaccine and a lack of effective antiviral therapeutics against sars-cov-2. therefore, there is an intense interest in identifying compounds that may interact with key viral molecular targets. due to their functional importance and high degree of conservation among coronaviruses, m pro s have become an important target in the design of anti-coronaviral drugs (19, 21) . the structure of the sars-cov-2 m pro was initially solved by jin et al. in late january of this year (22) , accelerating the search for drugs that may act as lead compounds. following the 2002 sars outbreak, work by hilgenfeld at al. aimed at designing compounds with broad-spectrum anti-coronaviral activity, focussing on main proteases (19, 23) . previously, they found that peptidomimetic α-ketoamides were potential candidates for broad-spectrum inhibitors of coronavirus and enterovirus replication (24) . most recently, work aimed at improving the biological properties to produce an inhibitor specific for the sars-cov-2 m pro resulted in the potential antiviral agent α-ketoamide 13b (25) . it was found that compound 13b demonstrates binds to the substrate-binding cleft and exhibits antiviral activity in vitro, inhibiting the sars-cov-2 m pro with ic50 = 0.67 ± 0.18 µm (25) . here, our aim was to further investigate the interaction of the α-ketoamide 13b with the sars-cov-2 m pro in silico. to highlight the importance of molecular dynamics simulations, we j o u r n a l p r e -p r o o f compared the properties of α-ketoamide 13b, with one of the most widely prescribed antibiotics, amoxicillin. amoxicillin was chosen for comparison for two reasons: 1) although it does not possess antiviral properties, it remains a mainstay as a frontline therapy for viral infections, including covid-19, presumably to protect from opportunistic secondary bacterial infections, and 2) our initial screening indicates that it binds to the active site of the sars-cov-2 m pro akin to -ketoamide 13b, albeit with lower affinity. preparation of systems and docking calculations were carried out using the schrodinger suite crystallographic water molecules were removed. the homodimer protein complex was prepared using the protein preparation wizard (28) . this was used to assign bond orders, add hydrogens, create zero-order bonds to metals, and create disulphide bonds. hydrogen bonds were assigned and optimised, followed by restrained energy minimization. ligand structures were pre-processed using ligprep (29) , for the generation of ionization and stereoisomer variants of input molecules to obtain structures with optimised geometry. two receptor grids of 20 x 20 x 20 å in size were generated around the active site of the protease, centroid to residues gly-23, thr-24, gly-143, his-163, thr-190, and ala-191 on each chain. ligands were docked to each chain separately. docking was carried out using the quantum mechanics-polarized ligand docking (qpld) workflow (30) of schrodinger. initial docking was performed using the extra precision (xp) scoring function of glide (31) . partial charges on ligand atoms were then calculated using quantum mechanical methods using j o u r n a l p r e -p r o o f the 'accurate' setting in jaguar (32) . ligands were re-docked using the calculated charges with xp docking mode of glide, and the final pose was selected based on glidescore. classical md simulations were performed using gromacs 2018.2 software(33, 34) with the charmm27 force field (35, 36) . ligand topology was generated using swissparam (37) . protein-ligand complexes were solved using tip3p water (38) in a dodecahedral box with a minimum of 2.0 nm distance between any protein atom to the closest box edge. sodium ions were added to the solvated system to neutralise the charge. energy minimisation was performed using a steepest-descent gradient method for a maximum of 50,000 steps. each complex was then restrained using an isothermal-isochloric (nvt) ensemble and isothermal-isobaric ensemble (npt) for 100 ps. temperature was maintained at 310 k with a modified berendsen thermostat (39) , and pressure at 1.0 bar with the parrinello-rahman barostat (40) . bond lengths were constrained using the lincs algorithm (41) , with long-range electrostatic forces calculated using the particle-mesh ewald scheme (pme) (42) (grid spacing 0.16 nm). cutoff ratios of 1.2 nm for coulomb and van der waals potentials were used for the calculation of short-range nonbonded interactions. simulations were carried out for 100 ns with a time-step of 2 fs in triplicate, with random generation of velocities according to a maxwell distribution. visual molecular dynamics 1.9.3 (43) was utilised for analysis and visualisation of trajectories. molecular mechanics-poisson boltzmann surface area (mm-pbsa) was utilised for the quantification of free energy calculations (44) . this was performed using the g_mmpbsa tool (45) . mm-pbsa calculations were performed on 1 ns segments of the triplicate stabilised trajectories (46) . energy contributions from electrostatic, van der waals, and polar solvation terms were calculated using the adaptive poisson-boltzmann solver (apbs) (47) . grid spacing was set to 0.05 nm, and values of 80 and 2 were used for solvent dielectric constant and solute j o u r n a l p r e -p r o o f dielectric constant, respectively. solvent-accessible surface area (sasa) was used to approximate the non-polar energy contribution, with the probe radius set to 0.14 nm. entropic energy terms were excluded from the calculations. the α-ketoamide 13b ligand binds with relatively high affinity to the active site of the sarsdocking was performed using the qpld workflow of schrodinger to obtain a rigorous estimate of ligand binding affinities to the active site. the receptor grid was centred around the active site residues for a comprehensive search of binding poses using partial atomic charges calculated using quantum mechanical methods. the α-ketoamide 13b ligand bound to the protease with a glidescore of -8.8 kcal/mol to the monomer, compared to -5.2 kcal/mol for amoxicillin. for binding to the dimer structure of the protease (table 2) it is noted that from visual inspection, while α-ketoamide 13b remains strongly bound to the active site of the protease throughout the trajectory (media s1), amoxicillin does not remain table 2 indicates that van der waals interactions are the predominant driving force for binding of αbinding. polar solvation energy was also significantly more unfavourable compared to binding with α-ketoamide 13b. while mm-pbsa is a commonly used method for estimating free energy, it should be noted that entropy terms are excluded from calculations. while mm-pbsa methods have been used to rescore poses obtained through molecular docking, it should be noted that values produced should be treated as relative differences across a set of ligands, rather than absolute (48, 49). nevertheless, the results presented here demonstrate that αketoamide 13b is able to bind strongly to the substrate binding site of the protease. residue energy contributions are decomposed in figure 4 , where the average energy contributions are shown on a per-residue basis for the entire protease. for α-ketoamide 13b, energy contributions are largely confined to the protomer on which they are bound. residue energy contributions are mostly favourable, shown as negative peaks below the x-axis. the residue with the most negative energy contribution corresponding to the most favourable interaction is met-49 on both protomers. this residue lies within the region where the large fluctuations in rmsf were observed in figure 2 . a more favourable energy contribution was observed for this residue on protomer a on the dimer of -2.40 kcal/mol compared to -1.26 kcal/mol for protomer b. this is in line with the slightly stronger δg of for α-ketoamide 13b binding to protomer a compared to protomer b (table 2) . met-49 is located in the s2 subsite of the substrate binding pocket, previously found to form a 'lid' in the closely related sars-cov m pro s2 site facilitating hydrophobic interactions that enable inhibition of the enzyme with α-ketoamides (24) . another residue contributing favourably to binding to both protomers where they found that glu-166 adopted an inactive conformation in protomer b (25) . glu-166 is a key residue essential for catalytic activity, interacting with nh2 terminal residues of each protomer at the dimer interface, shaping the s1 pocket of the substrate binding site (25, 52) . tck, kn, and ah conceptualized the aims and methodology and were involved in supervision. jl performed formal data analysis, was involved in data curation, and produced the first draft of the manuscript. ck performed formal data analysis and was involved in data curation. ep and kkd performed formal data analysis and validation. all authors contributed to editing and reviewing the manuscript. amoxicillin -5.0 -4.8 situation report an interactive web-based dashboard to track covid-19 in real time real estimates of mortality following covid-19 infection. the lancet infectious diseases severe outcomes among patients with coronavirus disease 2019 (covid-19)-united states novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases zhonghua liu xing bing xue za zhi= zhonghua liuxingbingxue zazhi sex difference and smoking predisposition in patients with covid-19. the lancet respiratory medicine a novel coronavirus outbreak of global health concern. the lancet early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster sars-cov-2 and covid-19: the most important research questions a novel coronavirus emerging in china -key questions for impact assessment. the new england journal of medicine sars and mers: recent insights into emerging coronaviruses the many estimates of the covid-19 case fatality rate. the lancet infectious diseases sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov-2 spike glycoprotein regulation of alveolar epithelial cell survival by the ace-2/angiotensin 1-7/mas axis sars-cov replicates in primary human alveolar type ii cell cultures but not in type i-like cells high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design structure of mpro from covid-19 virus and discovery of its inhibitors coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structure-based design, synthesis, and activity assessment crystal structure of sars-cov-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors protein interfaces, surfaces and assemblies service pisa at european bioinformatics institute identifying and characterizing binding sites and assessing druggability protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments importance of accurate charges in molecular docking: quantum mechanical/molecular mechanical (qm/mm) approach extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes berendsen hjc, van der spoel d, van drunen r. gromacs: a message-passing parallel molecular dynamics implementation high performance molecular simulations through multi-level parallelism from laptops to supercomputers implementation of the charmm force field in gromacs: analysis of protein stability effects from correction maps, virtual interaction sites, and water models a force field for drug-like molecules compatible with the charmm all-atom additive biological force fields swissparam: a fast force field generation tool for small organic molecules comparison of simple potential functions for simulating liquid water molecular dynamics with coupling to an external bath crystal structure and pair potentials: a molecular-dynamics study lincs: a linear constraint solver for molecular simulations particle mesh ewald: an n⋅log(n) method for ewald sums in large systems vmd: visual molecular dynamics electrostatics of nanosystems: application to microtubules and the ribosome g_mmpbsa-a gromacs tool for high-throughput mm-pbsa calculations assessing the performance of the mm/pbsa and mm/gbsa methods. 1. the accuracy of binding free energy calculations based on molecular dynamics simulations brown sp, muchmore sw. large-scale application of high-throughput molecular mechanics with poisson−boltzmann surface area for routine physics-based scoring of protein−ligand complexes investigation of mm-pbsa rescoring of docking poses the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra αhelical domain we key: cord-312305-ll29frwc authors: sun, shihui; gu, hongjing; cao, lei; chen, qi; yang, guan; li, rui-ting; fan, hang; ye, qing; deng, yong-qiang; song, xiaopeng; qi, yini; li, min; lan, jun; feng, rui; guo, yan; qin, si; wang, lei; zhang, yi-fei; zhou, chao; zhao, lingna; chen, yuehong; shen, meng; cui, yujun; yang, xiao; wang, xinquan; wang, hui; wang, xiangxi; qin, cheng-feng title: characterization and structural basis of a lethal mouse-adapted sars-cov-2 date: 2020-11-11 journal: biorxiv doi: 10.1101/2020.11.10.377333 sha: doc_id: 312305 cord_uid: ll29frwc the ongoing sars-cov-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. herein, we generated and characterized a novel mouse-adapted sars-cov-2 strain named mascp36 that causes acute respiratory symptoms and mortality in standard laboratory mice. particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe covid-19, and the 50% lethal dose (ld50) of mascp36 was ∼100 pfu in aged, male balb/c mice. deep sequencing identified three amino acid mutations, n501y, q493h, and k417n, subsequently emerged at the receptor binding domain (rbd) of mascp36, which significantly enhanced the binding affinity to its endogenous receptor, mouse ace2 (mace2). cryo-electron microscopy (cryo-em) analysis of mace2 in complex with the rbd of mascp36 at 3.7-angstrom resolution elucidates molecular basis for the receptor-binding switch driven by amino acid substitutions. our study not only provides a robust platform for studying the pathogenesis of severe covid-19 and rapid evaluation of coutermeasures against sars-cov-2, but also unveils the molecular mechanism for the rapid adaption and evolution of sars-cov-2 in mice. one sentence summary a mouse adapted sars-cov-2 strain that harbored three amino acid substitutions in the rbd of s protein showed 100% mortality in aged, male balb/c mice. coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has resulted in a public health crisis (1) . the symptoms of covid-19 are similar to those of sars-cov and mers-cov infections, ranging from fever, fatigue, dry cough and dyspnea, and mild pneumonia to acute lung injury (ali) and the acute respiratory distress syndrome (ards) in severe cases. in fatal cases, multi-organ failures accompanied by a dysregulated immune response have been observed (2) (3) (4) . numerous studies have highlighted age and gender related discrepancies in the distribution of covid-19 cases where the elderly and men tend to have a higher case-fatality ratio when compared to the young and females, suggesting that aged man are more likely to succumb to covid-19 (5, 6) . coronaviridae family, and is an enveloped, single stranded positive-sense rna virus. human angiotensin-converting enzyme 2 (ace2), has been demonstrated as the functional receptor for sars-cov-2 (7, 8) . sars-cov-2 cannot infect wild-type laboratory mice due to inefficient interactions between its s protein and the ace2 receptor of mouse (mace2). (9) . so, several hace2 expressing mouse models such as hace2 transgenic mice (10) , aav-hace2 transduced mice (11) and ad5-hace2 transduced mice (12) have been developed. furthermore, mouse adapted strains of sars-cov-2 have also been developed via either in vivo passaging or reverse genetics (13) (14) (15) . however, all these models cause only mild to moderate lung damage in mice. a small animal model capable of recapitulating the severe respiratory symptoms and high case fatality ratio of covid-19 remains to be established. in this study, we have developed and characterized a new lethal strain of sars-cov-2 named mascp36 by using the in vivo passaging method. previously, we reported the development of mascp6, a mouse-adapted sars-cov-2 strain using a similar strategy (16) . remarkably, intranasal injection of mascp36 caused 100% fatality in aged balb/c mice. prior to death, all infected animals developed severe malfunctions of the respiratory system, including acute respiratory distress syndrome in our previous study, we generated a mouse-adapted strain of sars-cov-2 (mascp6) by 6 serial passages of a sars-cov-2 in the lung of aged balb/c mice, which caused moderate lung damage and no fatality in mice. herein, we further serially passaged for additional 30 times to generate a more virulent sars-cov-2, and the resulting virus at passage 36 ( named as mascp36) was used for stock preparation and titration. to characterize the pathogenicity of mascp36, groups of balb/c were subjected to intranasal injection of varying doses of mascp36. strikingly, survival curve analysis showed that high doses of mascp36 caused almost 100% mortality within ten days in all aged mice, while young mice were resistant to mascp36 challenge and no animals developed disease and died in this group (fig. 1a-d) . aged mice challenged with high dose of mascp36 developed typical respiratory symptoms and exhibited features like ruffled fur, hunched back, and reduced activity. of particular note, tachypnea was common in all moribund animals (supplemental video). for the aged mice, male animals were more susceptible to mascp36 in comparison to female ones, and the ld50 was calculated to 58 pfu and 690 pfu, respectively (fig. 1a, b) . additionally, this unique gender-dependent mortality was also recorded in aged c57bl/6 mice challenged with mascp36 ( fig. s1 ) where 80% of the males challenged with the virus died from respiratory symptoms. thus, serial passaging of mascp6 generated a more virulent mascp36, which was sufficient to cause 100% mortality in aged balb/c mice at low dose (~1200 pfu). we further characterized the in vivo replication dynamics of mascp6 in both young and aged mice, and the results from qrt-pcr showed that high levels of sars-cov-2 subgenomic rnas were persistent in the lung and tracheas till 4 day post infection (dpi) in aged mice (fig. 1e) . marginal viral rnas were also detected in the intestine, heart, liver, spleen, brain, and kidney. the young mice had a similar tissue distribution as the aged ones upon mascp36 challenge, and lung and tracheas represented the major tissues supporting viral replication (fig. 1f ). multiplex immuno staining showed that mascp36 predominantly targeted the airway cc10+ club cells and spc+ at2 cells, while foxj1+ ciliated cells and pdpn+ alveolar type 1 (at1) cells detected few positive signals (fig. 1g ). more sars-cov-2-infected cells were detected in the lung from aged mice than those from young animals at 1 dpi (fig. 1h) , which was in agreement to the high ratio of spc+ at2 cells that co-express ace2 in aged mice ( fig. s2 ). consequently, a striking loss of spc+ at2 cells with apoptosis were observed in the lung from aged mice ( fig. s3 ). collectively, aged male mice that developed severe respiratory symptoms and 100% fatality serve as the most suitable animal host for mascp36 and were chosen for subsequent analysis. to further characterize the pathogical outcome in mascp36 infected aged male mice, the lung as well as other organs were collected at 4 dpi and subjected to histopathological and immunostaining analysis. when observed by the naked eye, lung tissue samples from mascp36 infected animals showed visible lung injury characterized with biolateral cardinal red appearance. furthermore, a lot of sticky secretion was seen at the lung surfaces when compared with the control animals ( fig. 2a ). according to the metrics of acute lung injury (ali) laid out by the american thoracic society (ats), mascp36 infection induced necrotising pneumonia and extensive diffuse alveolar damages (dad) and even ards on day 4 (17) . the microscopic observation showed large quantities of desquamative epithelial cells in bronchiole tubes (yellow arrow) and a large area of necrotic alveoli epithelial cells, fused alveoli walls with inflammatory cells infiltration especially neutrophils in alveolar septae or alveolar airspace, serious edema around vessels (cyan arrow) and scattered hemorrhage (blue arrow) ( fig. 2a ). in addition, foamy cells, polykaryocytes, fibrin cluster deposition, and hyaline membrane formation were common in the mascp36 infected animals (fig. 2b) , indicative of acute respiratory distress syndrome (ards), which is well characterized in severe covid-19 patients (4). interestingly, typical viral inclusion bodies were also observed occasionally in lungs of infected mice (fig. 2b) . comparatively, lung damage in young mice infected with mascp36 were much milder than those of the aged mice; less visual lung damage were seen in gross necropsy, and only thickened alveolar septa and activated inflammatory cell infiltration (19, 20) . these pathological damages caused by mascp36 in mice recapitulated most spectrums of seriously ill covid-19 patients caused by sars-cov-2 infection. additionally, immunostaining of lung sections showed a significant infiltration of cd68 + macrophages and ly-6g + neutrophils in mascp36 infected mice ( fig. s7 ). interestingly, more cd68 + macrophages and ly-6g + neutrophils were detected in young mice than that in aged mice 1 dpi, and reversed on 4 dpi, which indicated rapid and short-lived immune response to limite viral replication in young mice. to test the utility of mascp36 infected mouse model for evaluation of antiviral candidates, h014, a known human monoclonal antibody targeting the rbd of sars-cov-2 (21), was examined for its ability to confer benefit during the infection. to deduce the genetic basis for the lethal phenotype of mascp36, deep sequencing was performed to identify the mutations emerged during the in vivo passaging history. and fig. s8b ). to clarify the potential role of these mutations, the rbd of these different adaptive strains were expressed to assay their binding affinities to mace2 ( fig. s9 ). as expected, the wt rbd presented no detectable binding, but rbds from mouse-adapted strains (rbdmascp6, rbdmascp25 and rbdmascp36) gain gradually enhanced binding abilities to mace2 with affinities ranging from ~500 μm to 2 μm (fig. 4b ). the increased affinity between mace2 and the rbd of mouse-adapted strains probably contribute to the enhanced virulence in mice. to further elucidate the molecular basis for the gradual change in specificity of mascp36, structural investigations of the mace2 in complex with rbdmascp25 or rbdmascp36 were carried out. two non-competing fab fragments that recognize the rbd beyond the mace2 binding sites were used to increase the molecular weight of this complex for pursuing an atomic resolution by cryo-em reconstruction (fig. s9-s12). interestingly, cryo-em characterization of the mace2 in complex with rbdmascp25 revealed that the complex adopts three distinct conformational states, corresponding to tight binding (state 1), loose binding (state 2) and no binding modes (state 3) (fig. s13), indicative of a quick association and quick dissociation interaction manner between the mace2 and rbdmascp25. however, only the tight binding conformation was observed in the mace2-rbdmascp36 complex structure, reflecting a more stable/mature binding mode for the rbdmascp36 to mace2, akin to that of the rbdwt and hace2. we determined asymmetric cryo-em reconstructions of the mace2-rbdmascp36 complex at 3.7 å and three states of the mace2-rbdmascp25 complex at 4.4 to 8.2 å (figs. s11-s12 and table s1). the map quality around the mace2-rbdmascp36 interface was of sufficient quality for a reliable analysis of the interactions ( fig. 4c and fig. s9 ). the overall structure of the mace2-rbdmascp36 complex resembles that of the rbdwt-hace2 complex with a root mean square deviation of 1.0 å (fig. 4c ). the rbdmascp36 recognizes the helices (α1 and α2) located at the apical region of the mace2 via its receptor binding motif (rbm) (fig 4c-4e ). the interaction area on the mace2 could be primarily divided into three patches (pi, pii and piii), involving extensive hydrophilic and hydrophobic interactions with three regions separately clustered by three adaptation-mediated mutated residues (k417n, corresponding to clus1; q493h, corresponding to clus2; and n501y, corresponding clus3) in the rbm ( fig 4c-4e ). coincidentally, a number of amino acid substitutions, such as q493k, q498y and p499t, in the rbm identified in other reported mouse-adapted sars-cov-2 isolates (15, 22, 23) were included either in the clus2 or clus3, underlining the putative determinants for cross-transmission (fig 4f-4g ). an extra clus1 is further accumulated in the mascp36 to gain utmost binding activity and infection efficacy ( fig 4f-4g ). the extensive hydrophobic interactions in clus3 constructed by y501 (or y498 or h498 in other mouse-adapted sars-cov-2 isolates), y505 in the rbdmascp36 and y41, h353 in the mace2, hydrogen bonds in clus2 formed h493 (k493 in other mouse-adapted strain) in the rbdmascp36 and n31, e35 in the mace2 and hydrophilic contacts constituted by n417 in the rbdmascp36 and n30, q34 in the mace2 contribute to the tight binding of the mascp36 to mace2. contrarily, structural superimposition of the rbdwt over the mace2-rbdmascp36 complex reveals the loss of these interactions, leading to the inability of the rbdwt to bind mace2 (fig 4g) . these analysis pinpoints key structure-infectivity correlates, unveiling the molecular basis for adaptation-mediated evolution and cross-transmission of sars-cov-2. clinically, the severe covid-19 disease onset might result in death due to massive alveolar damage and progressive respiratory failure (24) (25) (26) . distinct from all currently reported animal models which mimic the mild to moderate clinical symptoms of covid-19, the mascp36 infected mouse model could manifest many of the severe clinical syndromes associated with covid-19 disease such as pulmonary oedema, fibrin plugs in alveolar, hyaline membrane, and scattered hemorrhage (25, 27) . the as the functional receptor of sars-cov and sars-cov-2, ace2, is highly expressed on vascular endothelial cells and smooth muscle cells in multiple organs, which probably leads to the observed viral tropism contributing to cellular injury. in this model, closely correlated with the higher ratio of ace2-positive cells in type ii pneumocytes in aged mice when compared to those in young mice, massive injury of (at2 cells) type ii pneumocytes was observed in aged mice. therefore, age-related ace2 expression pattern in lungs might contribute to the severe phenotype observed in aged mice. although sars-cov-2 viral antigen has been detected in kidney of postmortem specimens (28) , no viral antigen or viral rna were detected in our model. so in this mascp36 infected mouse model, the kidney injury may arise due to secondary endothelial injury leading to proteinuria. in addition, although sars-cov-2 has also been implicated to have neurotropic potential in covid-19 (29), we did not find typical characteristics of viral encephalitis in this model. importantly, the imbalanced immune response with high-levels of proinflammatory cytokines, increased neutrophils and decreased lymphocytes, which were in line with sars and mers infections (30), playing a major role in the pathogenesis of covid-19 (31) , were also observed in this model. the skewed age distribution of covid-19 disease was reproduced in the mascp36 infected mouse model where more severe symptoms were observed in aged mice when compared to young mice. different from h1n1 pandemic(32), covid-19 appears to have a mild effect on populations under 30 years, and the elderly are more likely to progress to severe disease and are admitted to intensive care unit (icu) worldwide (33) . ace2, the functional receptor of sars-cov-2, expressed increasingly in the lungs with age, which might provide an explanation to the higher disease severity observed in older patients with covid-19 (34) . more importantly, the host immune response may determine the outcome of the disease. our immune system is composed of innate immunity and adaptive immunity. the innate immunity comprises of the first line of defense against pathogens and is acute as well as short lived. however, aging is linked with insufficient, prolonged and chronic activation of innate immunity associated with low-grade and systemic increases in inflammation (inflamm-aging) which can be detrimental for the body (35) . the delicate co-operation and balance are interrupted by the chronic activation of innate immunity and declined adaptive immune responses with increasing age in covid-19 (36) . in the mascp36 infected mouse model, the young mice presented acute inflammatory response with more innate immune cells infiltration on day 1, while lagged and sustained immune response in aged mice. the different immune response in mice model may be vital in limiting virus replication at early times and contribute to different outcome on day 4 in young or aged mice. in addition to the age-related skewed distribution of covid-19, gender-related differences in distribution of covid-19 disease is also recapitulated in this mascp36 infected mouse model with increased susceptibility and enhanced pathogenicity observed in male mice when compared to their female counterparts. biological sex is an important determinant of covid-19 disease severity (37) . in china, the death rate among confirmed cases is 2.8% for women and 4.7% for men (34) . in italy, half of the confirmed covid-19 cases are men which account for 65% of all deaths (38) . this pattern is generally consistent around the world. the skewed distribution of covid-19 suggests that physiological differences between male and female may cause differential response to infection. so the hypothesis that females display reduced susceptibility to viral infections may be due to the stronger immune responses they mount than males (39) . it has been studied that androgens may lower and estrogens may enhance several aspects of host immunity. in addition, androgens facilitate and estrogens suppress lymphocyte apoptosis. furthermore, genes on the x chromosome important for regulating immune functions, and androgens may suppress the expression of disease resistance genes such as the immunoglobulin superfamily (40) . in the mascp36 infected mouse model, we found out that it presented higher mortality of the male than the female infected with the same dose of virus, indicating the successful recapitulation of covid-19 and also its potential application in the study of the pathogenesis of the disease. learning from sars-cov ma15 with increased virulence in mice, multiple gene products may contribute independently to the virulence. unlike the sars-cov ma15, three subsequent emerged mutations (n501y, k417n, and q493h) in the mascp36 were located in rbd, which breaks a barrier for cross-species transmissions of sars-cov-2, enabling gradually adapted recognition of sars-cov-2 to mace2 during the in vivo passages in mice. the serially increased affinities between mace2 and the rbd of mouse-adapted strains confer to enhanced infections in mice. cryo-em structures of the mace2 in complex with rbdmascp25 and rbdmascp36 define preciously the atomic determinants of the receptor-binding switch, providing novel insights into adaptationmediated evolution and cross-transmission of sars-cov-2. in addition, there are also 9 amino acid substitutions outside the s protein of mascp36 (fig. s8) . at present, we cannot rule out the contribution of these mutations, and further validation with reverse genetic tools will help understand the biological function of each single mutation (41) . figs. s1 to s13 center, beijing institute of microbiology and epidemiology (approval number: iacuc-dwzx-2020-002). mouse adapted strain of sars-cov-2 (mascp6) was developed in our previous study (16) . additional serial passage of 30 times was performed as previously described (16) . multiplex immunofluorescent assay. the multiplex immunofluorescence assay was conducted as previously described (16) . briefly, the retrieved sections were incubated with primary antibody for 2 h followed by detection using the hrp-conjugated secondary antibody and tsa-dendronfluorophores (neon 7-color allround discovery kit for ffpe, histova biotechnology, nefp750). afterwards, the primary and secondary antibodies were thoroughly eliminated by heating the slides in retrieval/elution buffer (abcracker®, histova biotechnology, abcfr5l) for 10 sec at 95°c lung homogenates from mascp36-infected mice or mock treated mice were processed as previously described (16) and subjected to rna-seq. total rna from lung tissue were extracted using trizol (invitrogen, usa) and treated with dnase i (neb, usa). sequencing libraries were generated using nebnext® ultratm rna library prep kit for illumina® (neb, usa) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. the clustering of the index-coded samples was performed on a cbot cluster generation system using hiseq pe cluster kit v4-cbot-hs (illumina) according to the manufacturer's instructions. after cluster generation, the libraries were sequenced on illumina novaseq6000 platform and 150 bp paired-end reads were generated. after sequencing, perl script was used to filter the original data (raw data) to clean reads by removing contaminated reads for adapters and low-quality reads. clean reads were aligned to the mouse genome (mus_musculus grcm38.99) using hisat2 v2.1.0. the number of reads mapped to each gene in each sample was counted by htseq v0.6.0 and tpm (transcripts per kilobase of exon model per million mapped reads) was then calculated to estimate the expression level of genes in each sample. deseq2 v1.6.3 was used for differential gene expression analysis. genes with padj≤0.05 and |log2fc| > 1 were identified as differentially expressed genes (degs). degs were used as query to search for enriched biological processes (gene ontology bp) using metascape. heatmaps of gene expression levels were constructed using heatmap package in r (https://cran.rstudio.com/web/packages/pheatmap/index.html). dot plots and volcano plots were constructed using ggplot2 (https://ggplot2.tidyverse.org/) package in r. production of fab fragment. the b8 and d14 fab fragments (43) were generated using a pierce fab preparation kit (thermo scientific). briefly, the antibody was mixed with immobilized-papain and then digested at 37 ˚c for 3-4 h. the fab was separated from the fc fragment and undigested iggs by protein a affinity column and then concentrated for analysis. surface plasmon resonance. mace2 was immobilized onto a cm5 sensor chip surface using the nhs/edc method to a level of ~600 response units (rus) using biacore® 3000 (ge healthcare) and pbs as running buffer (supplemented with 0.05% tween-20). wtrbd, rbdmascp25 and rbdmascp36, which were purified and diluted, were injected in concentration from high to low. the binding responses were measured, and chip surfaces were regenerated with 10 mm glycine, ph 1.5 (ge healthcare). the apparent binding affinity (kd) for individual antibody was calculated using biacore® 3000 evaluation software (ge healthcare). for the competitive binding assays, the first sample flew over the chip at a rate of 20 ul/min for 120 s, after which the second sample was injected at the same rate for another 120s. all antibodies were evaluated at a saturation concentration of 500 nm, while mace2 was applied at 1000 nm concentration. all surfaces of chips were regenerated with 10 mm glycine, ph 1.5 (ge healthcare). the response units were recorded at room temperature and analyzed using the same software as mentioned above. for cryo-em sample preparation, the quaternary complex (rbdmascp25/rbdmascp36-fabb8-fabd14-mace2) was diluted to 0.8 mg/ml. holy-carbon gold grid (quantifoil r0.6/1.0 mesh 300) were freshly glow-discharged with a solarus 950 plasma cleaner (gatan) for 60s. a 3 μl aliquot of the mixture complex was transferred onto the grids, blotted with filter paper at 22℃ and 100% humidity, and plunged into the ethane using a vitrobot mark iv (fei). for rbdmascp25/rbdmascp36-fabb8-fabd14-mace2 complex, micrographs were collected at 300 kv using a titan krios microscope (thermo fisher), equipped with a k2 detector (gatan, pleasanton, ca), using serialem automated data collection software (43) movies (32 frames, each 0.2 s, total dose 60 e − å −2 ) were recorded at final pixel size of 1.04 å with a defocus of between -1.25 and -2.7 μm. image processing. for rbdmascp25-fabb8-fabd14-mace2 complex, a total of 2,109 micrographs were recorded. for rbdmascp36-fabb8-fabd14-mace2 complex, a total of 2,982 micrographs were recorded. both sets of the data were processed in the same way. firstly, the raw data were processed by motioncor2, which were aligned and averaged into motion-corrected summed images. then, the defocus value for each micrograph was determined using gctf. next, particles were picked and extracted for two-dimensional alignment. the partial well-defined particles were selected for initial model reconstruction in relion. the initial model was used as a reference for three-dimensional classification. after the refinement and postprocessing, the overall resolution of rbdmascp36-fabb8-fabd14-mace2 complex was up to 3.69å, on the basis of the gold-standard fourier shell correlation (threshold = 0.143) (44) . for rbdmascp25-fabb8-fabd14-mace2 complex, the class i complex the resolution achieved was 7.89 å, classii complex had a resolution of 8.17 å, while class iii complex was reconstructed to a resolution of 4.4 å. the quality of the local resolution was evaluated by resmap (45) . model building and refinement. the wtrbd-hace2 (pdb id: 6m0j) structures were manually docked into the refined maps of rbdmascp36-fabb8-fabd14-mace2 complex using ucsf chimera (46) and further corrected manually by real-space refinement in coot (47) . the atomic models were further refined by positional and b-factor refinement in real space using phenix (48) . validation of the final model was performed with molprobity (49) . the data sets and refinement statistics are shown in table s1. statistical analyses were carried out using prism software (graphpad). all data are presented as means ± standard error of the means (sem). statistical significance among different groups was calculated using the student's t test, fisher's exact test, two-way anova or mann-whitney test *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively. dying with sars-cov-2 infection-an autopsy study of the first consecutive autopsy in suspected covid-19 cases the pathological autopsy of coronavirus disease 2019 (covid-2019) in china: a review. pathogens and disease considering how biological sex impacts immune responses and covid-19 outcomes clinical features of covid-19 in elderly patients: a comparison with young and middle-aged patients structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor characteristics of sars-cov-2 and covid-19 comparative analysis reveals the species-specific genetic determinants of ace2 required for sars-cov-2 entry the pathogenicity of sars-cov-2 in hace2 transgenic mice mouse model of sars-cov-2 reveals inflammatory role of type i interferon signaling. biorxiv a sars-cov-2 infection model in mice demonstrates protection by neutralizing antibodies a mouse-adapted model of sars-cov-2 to test covid-19 countermeasures adaptation of sars-cov-2 in balb/c mice for testing vaccine efficacy a mouse-adapted sars-cov-2 induces acute lung injury and mortality in standard laboratory mice adaptation of sars-cov-2 in balb/c mice for testing vaccine efficacy an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals covid-19 makes b cells forget, but t cells remember the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) directly decimates human spleens and lymph nodes. medrxiv human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. medrxiv structural basis for neutralization of sars-cov-2 and sars-cov by a potent therapeutic antibody mouse-adapted sars-cov-2 replicates efficiently in the upper and lower respiratory tract of balb/c and c57bl/6j mice a mouse-adapted model of sars-cov-2 to test covid-19 countermeasures histopathology and ultrastructural findings of fatal covid-19 infections in washington state: a case series pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies. modern pathology : an official journal of the united states and canadian academy of pathology clinical features of covid-19 and factors associated with severe clinical course: a systematic review and meta-analysis. ssrn pathological findings of covid-19 associated with acute respiratory distress syndrome. the lancet identification of a potential mechanism of acute kidney injury during the covid-19 outbreak: a study based on singlecell transcriptome analysis neurological associations of covid-19 lung pathology of fatal severe acute respiratory syndrome a new coronavirus associated with human respiratory disease in china pandemic preparedness and response--lessons from the h1n1 influenza of report of the who-china joint mission on coronavirus disease 2019 (covid-19) sars-cov-2: virus dynamics and host response chronic inflammation in the etiology of disease across the life span disparities in age-specific morbidity and mortality from sars-cov-2 in china and the republic of korea characteristics of covid-19 patients dying in italy report based on available data on the lethal sex gap: covid-19 sex differences in immune responses spike mutation d614g alters sars-cov-2 fitness acknowledgements: this work was in memory of prof this work was supported by the national key plan for scientific research and development of china quantifying the local resolution of cryo-em density maps visualization system for exploratory research and analysis tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions towards automated crystallographic structure refinement with phenix.refine molprobity: all-atom structure validation for macromolecular crystallography relion: implementation of a bayesian approach to cryo-em structure determination automated electron microscope tomography using robust prediction of specimen movements chadox1 ncov-19 vaccine prevents sars-cov-2 pneumonia in rhesus macaques data and materials availability: all requests for resources and reagents should be directed to c.-f.q. (qincf@bmi.ac.cn and qinlab313@163.com) and will be fulfilled after completion of a materials transfer agreement. key: cord-309876-l0xginsa authors: vena, antonio; berruti, marco; adessi, andrea; blumetti, pietro; brignole, michele; colognato, renato; gaggioli, germano; giacobbe, daniele roberto; bracci-laudiero, luisa; magnasco, laura; signori, alessio; taramasso, lucia; varelli, marco; vendola, nicoletta; ball, lorenzo; robba, chiara; battaglini, denise; brunetti, iole; pelosi, paolo; bassetti, matteo title: prevalence of antibodies to sars-cov-2 in italian adults and associated risk factors date: 2020-08-27 journal: j clin med doi: 10.3390/jcm9092780 sha: doc_id: 309876 cord_uid: l0xginsa we aimed to assess the prevalence of and factors associated with antisevere acute respiratory syndrome coronavirus-2 (sars-cov-2) positivity in a large population of adult volunteers from five administrative departments of the liguria and lombardia regions. a total of 3609 individuals were included in this analysis. participants were tested for anti-sars-cov-2 antibodies [immunoglobulin g (igg) and m (igm) class antibodies] at three private laboratories (istituto diganostico varelli, medical center, and casa della salute di genova). demographic data, occupational or private exposure to sars-cov-2-infected patients, and prior medical history consistent with sars-cov-2 infection were collected according to a preplanned analysis. the overall seroprevalence of anti-sars-cov-2 antibodies (igg and/or igm) was 11.0% [398/3609; confidence interval (ci) 10.0%–12.1%]. seroprevalence was higher in female inmates than in male inmates (12.5% vs. 9.2%, respectively, p = 0.002), with the highest rate observed among adults aged >55 years (13.2%). a generalized estimating equations model showed that the main risk factors associated with sars-cov-2 seroprevalence were the following: an occupational exposure to the virus [odd ratio (or) = 2.36; 95% ci 1.59–3.50, p = 0.001], being a long-term care facility resident (or = 4.53; 95% ci 3.19–6.45, p = 0.001), and reporting previous symptoms of influenza-like illness (or = 4.86; 95% ci 3.75–6.30, p = 0.001) or loss of sense of smell or taste (or = 41.00; 95% ci 18.94–88.71, p = 0.001). in conclusion, we found a high prevalence (11.0%) of sars-cov-2 infection that is significantly associated with residing in long-term care facilities or occupational exposure to the virus. these findings warrant further investigation into sars-cov-2 antibody prevalence among the italian population. in italy, the first case of pandemic severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection was reported on 20 february, 2020. since then, the number of cases increased rapidly in the north of the country, with the lombardia and liguria regions being heavily affected by the infection [1] . by the end of april 2020, approximately 85,000 laboratory confirmed cases -of sars-cov-2 infection were reported in this geographical area of the country [2] . however, these data included only a fraction of the real number of sars-cov-2 infections, since not all infected patients were symptomatic [3] [4] [5] , required hospitalizations, or provided specimens for laboratory testing. the extent to which surveillance data reflect the true burden of the disease can also be affected by changes in laboratory testing recommendation [1] . serology can represent a key element to overcoming these limits and to better understanding the infection statistics at a population level. the primary outcome of this study was to estimate the prevalence of sars-cov-2 antibodies. the secondary outcome was to evaluate possible factors associated with anti-sars-cov-2 positivity in a large population of individuals from five administrative departments of the liguria and lombardia regions. this was an observational study designed to evaluate the prevalence and factors associated with sars-cov-2 infections among voluntary, unpaid individuals tested for sars-cov-2 antibodies in three private institutions (istituto diagnostico varelli, medical center, and casa della salute di genova) during march and april 2020. these institutions altogether include approximately 5,784,974 inhabitants living in five administrative departments (milano, varese, pavia, genova and savona) of the liguria and lombardia regions. each laboratory process, about 500,000 samples per year, offers a comprehensive range of tests including clinical biochemistry, serology, and genetic analysis. we included non-hospitalized participants (aged > 18 years) who voluntarily tested for sars-cov-2 antibodies in an outpatient setting. after providing informed consent, a sample of venous blood was collected from each participant, all of whom also completed a questionnaire on potential risk factors for developing sars-cov-2 infection. recorded data included age, sex. and occupational or private exposure to sars-cov-2 infected patients. in addition, information regarding stays at a long-term care facilities or prior medical history consistent with sars-cov-2 infection (influenza-like illness defined according to who criteria [6] or loss of smell or taste) within the previous month, were also collected. the primary goal was to assess the prevalence of sars-cov-2 antibodies [either immunoglobulin m (igm) and g (igg)] positivity among the study population. the secondary goal was to investigate the association between positive tests and demographics (age and sex), occupational and private contact with sars-cov-2 infected patients, living in long-term care facilities, and prior symptoms consistent with sars-cov-2 infection. blood samples were analyzed for serological detection at each participating laboratory by trained staff, unaware of the clinical details of the tested patients. the first laboratory (istituto diagnostico varelli) used a chemiluminescent quantitative immunoassay detecting antibodies against nucleocapsid protein and spike protein (the maglumitm 2019) [7] . according to the manufacturer's recommendations, samples were considered positive above a threshold of 1.1 au/ml for igm and igg. this cut-off resulted in clinical sensitivities/specificities of 78.6%/97.5% and 91.2%/97.3% for igm and igg, respectively [7, 8] . the second laboratory (medical center) applied a rapid chromatographic immunoassay for the qualitative detection of igg and igm antibody against spike protein (realy tech ® 2019 ncov/covid-19 igg/igm rapid test device). the manufacturer's reported a clinical sensitivity of 92% for igm; 96% for igg; and a specificity of 100% for igm and igg. the third laboratory (casa della salute di genova) assessed anti-sars-cov-2 antibodies using a commercially available point-of-care lateral flow immunoassay (biosynex ® covid-19 bss, fribourg, switzerland) that can simultaneously detect igm and igg in human blood, with an overall sensitivity of 88.7% and specificity of 90.6% [9] . this qualitative test detected antibodies against nucleocapsid and spike proteins. all laboratories used internal procedures to validate the diagnostic performance of serological tests. in all cases, the results showed values of sensitivity and specificity consistent with those reported by each manufacturer. all statistics were analyzed using spss software. prevalence of anti-sars-cov-2 antibodies (igm or igg) was calculated and the exact binomial distribution was used to calculate 95% confidence intervals (cis). the association between positive sars-cov-2 antibodies and study variables was estimated in two steps. first, a general linear univariate analysis was performed using a chi-squared test. the second step used a generalized estimating equation (gee) model to consider laboratory provenience, with sars-cov-2 seropositivity used as a dependent variable. only differences with a p-values < 0.05 were considered statistically significant. the study protocol was approved by the ethics committee of liguria region (pi prof. matteo bassetti-n. cer liguria 381/2020-id 10770). between 1 march and 30 april 2020, 3609 individuals agreed to participate in the study. the mean number of screened individuals per administrative department was 721 (52-1430), representing 12 people per 100,000 inhabitants. the patients' demographics are outlined in table 1 . overall, 55.6% (2007/3609) were women and 44.4% were men (1602/3609). the median age was 51 years [interquartile range (iqr) 41-63], with the age group >55 years being most represented of the 3609 individuals included in the study population, 398 tested anti-sars-cov-2 positive [11.0% (ci 10.0%-12.1%)]. seroprevalence was higher among women vs. men (12.5% vs. 9.2%, p = 0.002) and varied with age. the rate was highest among adults aged >55 years (13.2%), followed by adults aged 18-35 years (11.9%). as for geographical distribution, the highest prevalence of anti-sars-cov-2 positivity was reported in the administrative departments of savona ( figure 1 ). of the 3609 individuals included in the study population, 398 tested anti-sars-cov-2 positive [11.0% (ci 10.0%-12.1%)]. seroprevalence was higher among women vs. men (12.5% vs. 9.2%, p = 0.002) and varied with age. the rate was highest among adults aged >55 years (13.2%), followed by adults aged 18-35 years (11.9%). as for geographical distribution, the highest prevalence of anti-sars-cov-2 positivity was reported in the administrative departments of savona (figure 1 ). table 2 shows estimated prevalence according to the three different laboratories. several factors showed an association with anti-sars-cov-2 antibodies positivity with univariable analysis ( table 3 ). the variables that showed a p-value < 0.10 were also included in the gee model ( table 4 ). the model showed that the main risk factors associated to sars-cov-2 seroprevalence were the following: occupational exposure to the virus (or = 2.36; 95% ci 1.59-3.50, p = 0.001), living in a long-term care facility (or = 4.53; 95% ci 3.19-6.45, p = 0.001), and reporting previous symptoms of influenza-like illness (or = 4.86; 95% ci 3.75-6.30, p = 0.001) or loss of sense of smell or taste (or = 41.00; 95% ci 18.94-88.71, p = 0.001). in the present observational study performed on a large sample of subject in northern italy, we found the following: (1) the overall seroprevalence of anti-sars-cov-2 antibodies (igg and/or igm) was 11.0%; (2) occupational exposure to the virus, long-term care facility residency, as well as previous symptoms of influenza-like illness or loss of sense of smell or taste were independently associated with anti-sars-cov-2 positivity. to the best of our knowledge, this is one of the first reports that attempts to describe the prevalence of coronavirus disease and to evaluate the potential circulation of sars-cov-2 in north italy. the findings of our study showed that in a definite geographical area of italy, approximately 630,000 people might have developed antibodies (11.0% of 5,784,974 inhabitants). this figure is significantly higher than the number of molecular-confirmed sars-cov-2 infections (~32,600 cases in the five administrative departments) reported by the protezione civile and the italian national institute of health as of 30 april 2020 [2]. the high observed seroprevalence is consistent with recent studies (table 5 ) performed in other heavily affected areas of europe: 9.7% in geneva, switzerland [10] and 10.0% in madrid, spain [11, 12] . table 5 . summary of articles published in the literature reporting data regarding prevalence of sars-cov-2 antibodies in the general population. petersen m.s. [13] faroe islands; nationwide study 1075 0.6% biggs h. [14] u.s.; two metropolitan atlanta counties 696 2.5% menachemi n. [15] u.s; indiana 3658 2.79% fischer b. [16] germany; three federal states 3186 0.91% pollan m. [11] spain; nationwide study 61,075 5.0% havers f. [17] u.s; 10 sites 16,025 from 1.0% (san francisco) to 6.9% (new york city) amorim filho l. [18] brazil; rio de janeiro 2857 4.0% percivalle e. [19] italy; lodi area 390 23.0% soriano v. [12] spain, madrid 674 13.8% stringhini s. [10] switzerland, geneve 2766 9.7% sood n. [20] u.s., los angeles 1702 4.3% living in a long-term care facility was the strongest predictors of sars-cov-2 infection and was reported by 21.6% of anti-sars-cov-2-positive participants (n = 86/398). this connection was not unexpected [21] [22] [23] , since long-term care facilities often have limited or no infection control programs [24, 25] and are usually congregative settings where elderly people have greater exposure to infected patients in the case of respiratory outbreaks [26] [27] [28] . therefore, our results emphasized the importance of implementing strategic bundles for infections prevention in long-term care facilities [29] . in this regard, educational interventions on healthcare providers' knowledge, as well as active surveillance of suspected cases and implementation of barrier precautions, were shown to play a vital role in limiting the spread of other respiratory outbreaks [26] [27] [28] . reporting an occupational exposure to the virus also emerged as an independent factor associated with sars-cov-2 infection and was reported by 8.7% of anti-sars-cov-2-positive participants (n = 35/398). however, approximately two-thirds of anti-sars-cov-2-positive participants did not report any apparent risk depicting the widespread circulation of the virus in the italian community, where it has become endemic. as for clinical symptoms, we found that the prevalence of sars-cov-2 antibodies depends on the type of clinical manifestation reported by the patient, being particularly high in people who reported loss of smell or taste [30, 31] . interestingly, 8.6% of participants (n = 277/3224) who did not report any symptoms presented antibodies positivity. this finding suggests that non-apparent infection is relatively common in a healthy, active population, thus supporting the hypothesis that, as is true for other coronavirus infections [32] , sars-cov-2 infection might also be asymptomatic or pauci-symptomatic and resolves spontaneously without any complications in many cases. in our opinion, the findings of our study could have several implications for pandemic management. because the real number of patients with sars-cov-2 infection is significantly higher than the pcr-confirmed cases, stringent lockdown strategies might possibly be re-implemented only when the intensive care units' capacities to handle emergencies are overwhelmed. since a large proportion of patients with sars-cov-2 infection are asymptomatic, contract tracing methods to limit the spread of the infection could be particularly challenging. thus, screening strategies beyond a symptoms-driven approach will be necessary for italy (e.g., use of mobile applications) to identify enough infected persons to reach sars-cov-2 elimination targets [33] ; our data could also be useful for vaccine design and implementation. there are several limitations that should be discussed. firstly, we do did have any information regarding previous sars-cov-2 molecular testing among those patients who tested positive. accordingly, we cannot provide valuable estimates of antibody prevalence in people positive and negative in pcr testing. secondly, we analyzed serum samples from patients who voluntarily decided to be tested. therefore, the clinical characteristics of the sample might differ from those of the general italian population. thirdly, geographical prevalence of anti-sars-cov-2 antibodies might have been influenced by the type of serological tests used. however, the diagnostic performances of each test are similar to each other; in addition, the highest percentage of infected patients in the liguria region agrees with recent evidence, suggesting the presence of anti-sars-cov-2 antibodies among blood donors from savona and genova since december 2019 (unpublished data reported by the ligurian regional health authority alisa). fourthly, all tests we used are non-fda approved and are yet to be validated. therefore, prevalence estimates could change once new information on the accuracy of tests are available. fifthly, the interpretation of the test is still under discussion, because even patients with confirmed sars-cov-2 infections have low or non-detectable antibodies titles several weeks after acute infection [34] . lastly, based on the specificities of testing kits, we cannot exclude that some participants had false positive results due to past or present infection with other viruses, including non-sars-cov-2 coronavirus strains [35] . in addition, antibody response may be impaired in elderly, immuno-compromised or immunosuppressed participants, and may produce false negative serology test results [36] . in conclusion, the results of the present study demonstrate that infection rates based on surveillance data considerably underestimated the infection rates during the sars-cov-2 virus pandemic in italy. the seroprevalence was much higher among people living in long-term care facilities or those with occupational exposure. in our opinion, these findings warrant further investigation into sars-cov-2 antibody prevalence among the italian population. outside the submitted work, m.b. (matteo bassetti) has participated in advisory boards and/or received speaker honoraria from achaogen, angelini, astellas, bayer, basilea, biomeérieux, cidara, gilead, menarini, msd, nabriva, paratek, pfizer, roche, melinta, shionogi, tetraphase, venatorx, and vifor and has received study grants from angelini, basilea, astellas, shionogi, cidara, melinta, gilead, pfizer, and msd. outside the submitted work, d.r.g. reports honoraria from stepstone pharma gmbh and unconditional grants from msd italia and correvio italia. the authors declare no conflict of interest. case-fatality rate and characteristics of patients dying in relation to covid-19 in italy the novel chinese coronavirus (2019-ncov) infections: challenges for fighting the storm clinical characteristics of asymptomatic and symptomatic patients with mild covid-19 estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship working towards a simple case definition 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outpatients cov-2: olfaction, brain infection, and the urgent need for clinical samples allowing earlier virus detection asymptomatic coronavirus infection: mers-cov and sars-cov-2 (covid-19) sixty seconds on the contact tracing app neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications the laboratory's role in combating covid-19 covid-19 serological tests: how well do they actually perform? diagnostics 2020 key: cord-312160-2820aftb authors: ibrahim, mahmoud a.a.; abdelrahman, alaa h.m.; hussien, taha a.; badr, esraa a.a.; mohamed, tarik a.; el−seedi, hesham r.; pare, paul w.; efferth, thomas; hegazy, mohamed elamir f. title: in silico drug discovery of major metabolites from spices as sars-cov-2 main protease inhibitors date: 2020-10-08 journal: comput biol med doi: 10.1016/j.compbiomed.2020.104046 sha: doc_id: 312160 cord_uid: 2820aftb coronavirus disease 2019 (covid-19) is an infectious illness caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), originally identified in wuhan, china (december 2019) and has since expanded into a pandemic. here, we investigate metabolites present in several common spices as possible inhibitors of covid-19. specifically, 32 compounds isolated from 14 cooking seasonings were examined as inhibitors for sars-cov-2 main protease (m(pro)), which is required for viral multiplication. using a drug discovery approach to identify possible antiviral leads, in silico molecular docking studies were performed. docking calculations revealed a high potency of salvianolic acid a and curcumin as m(pro) inhibitors with binding energies of −9.7 and −9.2 kcal/mol, respectively. binding mode analysis demonstrated the ability of salvianolic acid a and curcumin to form nine and six hydrogen bonds, respectively with amino acids proximal to m(pro)'s active site. stabilities and binding affinities of the two identified natural spices were calculated over 40 ns molecular dynamics simulations and compared to an antiviral protease inhibitor (lopinavir). molecular mechanics-generalized born surface area energy calculations revealed greater salvianolic acid a affinity for the enzyme over curcumin and lopinavir with energies of −44.8, −34.2 and −34.8 kcal/mol, respectively. using a string database, protein-protein interactions were identified for salvianolic acid a included the biochemical signaling genes ace, mapk14 and esr1; and for curcumin, egfr and tnf. this study establishes salvianolic acid a as an in silico natural product inhibitor against the sars-cov-2 main protease and provides a promising inhibitor lead for in vitro enzyme testing. coronaviruses belong to the coronaviridae family and are named for distinctive protein spikes covering the virus' outer membrane surface. several members of the family are known to cause respiratory tract infections in humans ranging from mild common colds to severe sars and mers infections [1, 2] . coronavirus disease 2019 (covid19) was first observed in wuhan province and identified by the chinese centre for disease control and prevention as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [3, 4] . the viral genome harbors 11 genes encoding 29 proteins and peptides; (www.ncbi.nlm.nih.gov/nuccore/nc_045512.2?report=graph). four proteins constitute the viral structure, including the spike or s protein [5] . in sars-cov-2, the s protein binds to an angiotensin-converting enzyme 2 (ace2), a necessary step for viral entry into the host cell. studies thus far indicate that the virus' s protein binds stronger to ace2 than the one of sars-cov, providing a rationale why covid-19 so easily spreads and is highly infectious. another group of sars-cov-2 proteins controls how the virus replicates as well as avoids the host's immune system. these non-structural proteins initially expressed as two large polyproteins are processed into 16 peptide components. the main protease (m pro or 3clpro), cleaves the polyproteins into 11 fragments, whose structures were recently elucidated and an inhibitor that blocks the m pro catalytic activity identified [6] . from this work, m pro appears to be a promising target for designing small molecule inhibitors. covid-19 rapidly spreads due to the global mobility of humans and is currently present in more than 200 countries. patients mainly suffer from fever, dry cough, labored breathing, and bilateral lung infiltrates. the causative agent is diagnosed from throat or nasal swabs j o u r n a l p r e -p r o o f with nucleic acid sequence similarity. rapid disease spreading coupled with high mortality rates makes covid-19 a major global public health threat [7] . recently, emergency use of remdesivir has been issued by the u.s. food and drug administration for treatment of covid-19. with few treatment options available, there is an urgent need to seek out effective strategies for prophylaxis for such viral outbreaks. using experimental methods of drug discovery is time-consuming and costly. therefore, structure-based computational modeling of ligand-receptor interactions can be used to identify potential m pro inhibitors to block viral replication. herbal extracts and spices are natural immune boosters and/or anti-infective agents currently utilized in many parts of the world [8] . in traditional folk medicine, spices, botanical detoxifiers [9] , antioxidants [10] and plant haematinics [11] are used as antiviral mediators to prevent/minimize disease. low toxicity makes such metabolites well suited as drug leads for viral diseases such as covid-19. in this study, selected spices with documented, biologically activity (e.g. cinnamon, clove, ginger, mustard and others) were exemplarily chosen to generate a metabolite library for the screening of m pro -specific drug candidates with presumable effectiveness against covid-19. the resolved crystal structure of the main protease (m pro ) of sars-cov-2 in complex with n3 inhibitor (pdb code: 6lu7 [12] ) was used for molecular docking as well as molecular dynamics calculations. water and spectator ions were deleted. h++ server was j o u r n a l p r e -p r o o f used to study the protonation state of m pro and to add all missing hydrogen atoms [13] . in h++ calculations, the following physical conditions were applied: ph=6.5, internal dielectric=10, external dielectric= 80 and salinity=0.15. the chemical structures of the 32 investigated natural spices were retrieved from the pubchem database and their 3d structures were generated using omega2 software [14, 15] . all generated structures were minimized using merck molecular force field 94 (mmff94s) with the assistance of available software (szybki) [16] . the 2d chemical structures of the investigated compounds are illustrated in table 1 . for molecular docking calculations, autodock4.2.6 software was utilized [17] . the pdbqt file of sars-cov-2 m pro was prepared according to the autodock protocol [18] . in autodock4.2.6, default parameters were employed, except the numbers of genetic algorithm (ga) run and energy evaluations (eval). ga and eval were set to 250 and 25,000,000, respectively. the grid was defined to cover the active site of the sars-cov-2 m pro . the grid size and spacing value were 60 å × 60 å × 60 å and 0.375 å, respectively. the grid center coordinates were −13.069, 9.740, 68.490 (xyz assignments, respectively). the atomic charges of studied natural spices were assigned using the gasteiger method [19] . the predicted binding poses for each compound were processed by the built-in clustering analysis amber16 software was utilized to conduct molecular dynamics (md) simulation for the natural spices in complex with sars-cov-2 m pro [20] . the details of the employed md simulations are described in ref. [21, 22] . in brief, general amber force field (gaff) [23] and amber force field 14sb [24] were applied to describe spices compounds and m pro , respectively. restrained electrostatic potential (resp) approach [25] was utilized to assign the atomic partial charges of the natural spices using gaussian09 software [26] . docked spice-m pro complexes were water solvated with 15 å distances between the box edge and atoms of the spice-m pro complexes. solvated spice-m pro complexes were minimized by 5000 steps and afterward smoothly heated from 0 k to 300 k over a brief interval (50 ps). using periodic boundary conditions and npt ensemble, the spice-m pro systems were simulated for 10 ns of equilibration and 40 ns of production. all molecular dynamics simulations were carried out with pmemd.cuda implemented in amber16. all molecular docking and molecular dynamics calculations were performed on compchem gpu/cpu cluster (hpc.compchem.net). the binding energies of the investigated spices compounds with sars-cov-2 m pro were estimated using molecular mechanical-generalized born surface area (mm-gbsa) approach with modified gb model (igb=2) implemented in amber16 software [27] . for the mm-gbsa calculations, uncorrelated snapshots were collected over the production run, and a single-trajectory approach was employed, in which compound, receptor, and complex j o u r n a l p r e -p r o o f coordinates were retrieved from a single trajectory. the binding energy (δg binding ) was estimated as follows: where the energy term (g) is estimated as: the physicochemical parameters of the most promising natural spices as sars-cov-2 m pro inhibitors were predicted using the online molinspiration cheminformatics software %abs was estimated as follows [28] : j o u r n a l p r e -p r o o f the online web-based tools of swisstargetpredicition (http://www.swisstargetprediction.ch) were applied to predict the biological targets for the most promising natural spices as sars-cov-2 m pro inhibitors. the disgenet online database (https://www.disgenet.org) was utilized to collect the available database for sars diseases. venn diagram was designed using interactivenn online tool [29] . protein-protein interaction (ppi) network was generated using a string functional database for top predicted targets [30] . cytoscape 3.8.0 was employed to investigate target-function relation based on the network topology [31] . lack of treatments against covid-19 pinpoints a critical need to systematically screen and identify compounds that can block viral reproduction. since the main protease of sars-cov-2 (m pro ) plays a critical role in the viral replication process, structure-based computational modeling of ligand-receptor interactions and molecular dynamics has been used to screen metabolites from common spices as potential m pro inhibitors. indeed several herbal plants have already been reported as antiviral entities against hepatitis b, respiratory syncytial virus and influenza [32] . (table 1) . most docked natural products shared the same binding modes, forming hydrogen bonds with key amino acid residues in the active site such as thr190, gly143, cys145, and glu166. 2d binding modes for each of the compounds are displayed in figure s1 . the peptidomimetic molecule lopinavir, which functions as an antiretroviral protease inhibitor against hiv was used as a positive control [33, 34] as it has recently been clinically figure s1 . b no hydrogen bond was observed. since the reliability of ligand-enzyme binding energies using molecular docking scores have been questioned due to complicating environmental factors such as a lack of ligand-receptor flexibility, solvent effects, and dynamics [37, 38] table 2 . for salvianolic acid a, binding energy was dominated by e ele interactions with an average value of −65.5 kcal/mol which was three times higher than that of lopinavir and curcumin, with an average value of −26.1 and −19.8 kcal/mol, respectively. this is attributed to a higher number of hydrogen bonds for salvianolic acid a with the key amino acids inside m pro 's active site, compared to lopinavir or curcumin (table 1) while drug-likeness is a qualitative measure utilized in drug discovery to evaluate pharmacokinetic properties such as oral bioavailability. physicochemical parameters were evaluated using molinspiration cheminformatics, (http://www.molinspiration.com) online software calculation toolkit. the predicted parameters are summarized in salvianolic acid a and curcumin protein targets were predicted and classified using a swisstargetprediction ( figure s2 ). one hundred and seventeen genes were identified using disgenet online tools for severe acute respiratory syndrome diseases (sars, c1175175). utilizing venn diagram comparison analysis, commonly shared genes for salvianolic acid a included ace, casp3, casp1, esr1 and mapk14, and for curcumin tnf, egfr and adam17 ( figure 5 ). angiotensin-converting enzyme 2 (ace2) is a host protein and the receptor for sars-cov-2 entry [39] . mapk14 inhibition is predicted to block the ace2 signaling pathway, and in turn, reduce cell internalization of sars-cov-2. for sars-s adam17-dependent shedding of ace2, a process coupled with tnf-α production, reduced viral reproduction [40] . salvianolic acid a and curcumin predicted j o u r n a l p r e -p r o o f genes targets were also analyzed via a string ppi network and visualized by cytoscape 3.8.0. the top 10 scored genes for salvianolic acid a included ace, mapk14 and esr1 and for curcumin egfr and tnf (table s1 ). the covid-19 pandemic has had a catastrophic impact on human health and global economies. sars-cov-2 main protease (m pro ) may well prove to be the achilles heel of coronavirus genomics and bioinformatics analysis mers, sars and other coronaviruses as causes of pneumonia a novel coronavirus from patients with pneumonia in china world health organization. who director-general's remarks at the media briefing on 2019-ncov on 11 composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 crystal structure of sars-cov-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors live): 2,418,429 cases and 165,739 deaths from covid−19 virus pandemic-worldometer stay safe: helpful herbal remedies in covid-19 infection hidden treasures of ethnobotanical medicine. a faculty lecture delivered at the faculty of science in vitro anti-radical activities of extracts of solanum nigrum (l.) from south africa nutritional composition of ten ethnobotanicals used for the treatment of anaemia in southwest nigeria structure of m(pro) from sars-cov-2 and discovery of its inhibitors h++: a server for estimating pkas and adding missing hydrogens to macromolecules conformer generation with omega: algorithm and validation using high quality structures from the protein databank and cambridge structural database autodock4 and autodocktools4: automated docking with selective receptor flexibility computational protein-ligand docking and virtual drug screening with the autodock suite iterative partial equalization of orbital electronegativity -a rapid access to atomic charges in-silico drug repurposing and molecular dynamics puzzled out potential sars-cov-2 main protease inhibitors natural-like products as potential sars-cov-2 m(pro) inhibitors: in-silico drug discovery development and testing of a general amber force field simmerling, ff14sb: improving the accuracy of protein side chain and backbone parameters from ff99sb a well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the resp model gaussian 09 combined molecular mechanical and continuum solvent approach (mm-pbsa/gbsa) to predict ligand binding rate-limited steps of human oral absorption and qsar studies interactivenn: a web-based tool for the analysis of sets through venn diagrams anti-colorectal cancer targets of resveratrol and biological molecular mechanism: analyses of network pharmacology, human and experimental data cytoscape: a software environment for integrated models of biomolecular interaction networks antiviral natural products and herbal medicines lopinavir/ritonavir in the treatment of hiv-1 infection: a review the metabolic effects of lopinavir/ritonavir in hiv-negative men a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 clinical efficacy of lopinavir/ritonavir in the treatment of coronavirus disease 2019 binding affinity via docking: fact and fiction software for molecular docking: a review sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor adam17 inhibition may exert a protective effect on covid-19 key: cord-321358-plxz5mkg authors: zheng, jun title: sars-cov-2: an emerging coronavirus that causes a global threat date: 2020-03-15 journal: int j biol sci doi: 10.7150/ijbs.45053 sha: doc_id: 321358 cord_uid: plxz5mkg an ongoing outbreak of pneumonia caused by a novel coronavirus, currently designated as the severe acute respiratory syndrome coronavirus-2 (sars-cov-2), was reported recently. however, as sars-cov-2 is an emerging virus, we know little about it. in this review, we summarize the key events occurred during the early stage of sars-cov-2 outbreak, the basic characteristics of the pathogen, the signs and symptoms of the infected patients as well as the possible transmission pathways of the virus. furthermore, we also review the current knowledge on the origin and evolution of the sars-cov-2. we highlight bats as the potential natural reservoir and pangolins as the possible intermediate host of the virus, but their roles are waiting for further investigation. finally, the advances in the development of chemotherapeutic options are also briefly summarized. on 23 feb 2020, the lock-down of wuhan, a central city in china, has alarmed people all over the world of an emerging novel coronavirus that is posing a major public health and governance challenges. the novel virus, previously called the 2019-novel coronavirus (2019-ncov), is currently designated as the severe acute respiratory syndrome coronavirus-2 (sars-cov-2). as of 27 feb, this emerging infection has been reported in 47 countries, causing over 82,294 infections with 2,804 deaths ( fig. 1 ) [1] . this novel virus is also becoming a mounting threat to chinese and global economies. coronaviruses (covs) are members of the family coronaviridae, the enveloped viruses that possess extraordinarily large single-stranded rna genomes ranging from 26 to 32 kilobases in length [2] . covs have been identified in both avian hosts and various mammals, including bat, camels, dogs and masked palm civets, and are previously regarded as pathogens that only cause mild diseases in the immunocompetent people until the emergence of the coronavirus causing severe acute respiratory syndrome (sars-cov) in late of 2002 [3] [4] [5] [6] . currently, at least seven coronavirus species are known to cause diseases in humans. the viruses of 229e, oc43, nl63 and hku1 only cause common cold symptoms, which are mild. severe illness can be caused by the remaining three viruses, namely sars-cov, which resulted in the outbreak of sars in 2002 and 2003 [3, 4] ; the coronaviruses that are responsible the middle east respiratory syndrome (mers-cov), which emerged in 2012 and remains in the circulation in camels [7] ; and sars-cov-2, the viruses emerged in december 2019 in wuhan of china and a great effort is being undertaken to contain its spreading [8] . in this review, we will briefly introduce the outbreak history of sars-cov-2, the signs and symptoms of the infected patients, its transmission dynamics, the advances in the understanding on its evolutional origin and the chemotherapeutic options being developed for the treatment of its infection. the key events of sars-cov-2 outbreak and the pathogen characteristics since december 2019, an increasing number of patients with pneumonia of unknown etiology in wuhan, a city with 11 million people, have alarmed the local hospital. on 29 december 4 cases were linked to huanan seafood wholesale market [9] , where non-aquatic live animals, including several kinds of wild animals, were also on the sales. the local center for disease control (cdc) then found additional patients linked to the same market after investigation, and reported to china cdc on 30 dec 2019 [9] . the second day, world health organization (who) was informed of the cases of pneumonia of unknown etiology by china cdc [10]. on 6 jan 2020, a level 2 emergency response was launched by china cdc [11] . the causal agent was not identified until 7 jan 2020; a new type of coronavirus was isolated by chinese authority [10] . the genome sequence of sars-cov-2 (wh-human_1) was first released and shared by china on 10 jan [12] . the isolation and identification of sars-cov-2 apparently facilitated the development of molecular diagnostic methods and the confirmation of the infected patients. as of 21 jan, there are 270 cases were confirmed from wuhan [13]. on 23 jan, wuhan city was locked down by local government. on 30 jan, who declared a "public health emergency of international concern" (fig. 1) . subsequently, the viruses were successfully isolated from several laboratories [8, 14, 15] . the virion of sars-cov-2 looks like a solar corona by transmission electron microscopy imaging: the virus particle is in a spherical shape with some pleomorphism; the diameter of the virus particles range from 60 to 140 nm with distinctive spikes about 8 to 12 nm in length [8] . the observed morphology of sars-cov-2 is consistent with the typical characteristics of the coronaviridae family. the genome sequence of sars-cov-2 from clinical samples has been obtained by several laboratories with deep sequencing [8, [14] [15] [16] [17] [18] . the viral genome of sars-cov-2 is around 29.8 kilobase, with a g+c content of 38%, in total consisting of six major open reading frames (orfs) common to coronaviruses and a number of other accessory genes [14, 16] . the sequences analysis showed that the genome sequences of viruses from different patients are very conserved [14, 15, 19] , implying that the human virus evolves recently. a typical characteristic of the sars-cov-2 infected patient is pneumonia, now termed as coronavirus disease 2019 , demonstrated by computer tomographic (ct) scan or chest x -ray [3, 8, 18] . in the early stages, the patients showed the acute respiratory infection symptoms, with some that quickly developed acute respiratory failure and other serious complications [20] . the first three patients reported by the china novel coronavirus investigating and research team all developed severe pneumonia and two of these three patients with available clinical profiles showed a common feature of fever and cough [8] . a subsequent investigation of a family of six patients in the university of hong kong-shenzhen hospital demonstrated that all of them had pulmonary infiltrates, with a variety of other symptoms [18] . the chest x-ray and ct imaging in a study showed that 75% of 99 patients demonstrated bilateral pneumonia and the remaining 25% unilateral pneumonia [21] . overall, 14% of the patients showed multiple mottling and ground-glass opacity [21] . the first cases of coronavirus infection in the united states also showed basilar streaky opacities in both lungs by chest radiography. however, the pneumonia for this patient was only detected on the day 10 of his illness [22] . it is also of note that one of patients among the family of six patients did not present any other symptoms and signs, but had ground-glass lung opacities identified by ct scan [18] . at least four comprehensive studies on the epidemiological and clinical characteristics of sars-cov-2 infected patients have been performed [21, [23] [24] [25] . the most common signs and symptoms of patients are fever and cough [21, [23] [24] [25] . fatigue was complained by 96% of patients (n=138) in one study [24] , but was less outstanding (18%, n=44) in another report [23] . a combinational analysis of the common recorded signs or symptoms of the reported cases found that fever was observed in around 90% of the sars-cov-2 infected patients; the number of patients with cough is relatively less (68%) compared to fever (table 1 ). in addition, shortness of breath or dyspnea, muscle ache, headache, chest pain, diarrhea, haemoptysis, sputum production, rhinorrhoea, nausea and vomiting, sore throat, confusion, and anorexia were also observed in a proportion of the patients [21, [23] [24] [25] (table 1) . a common feature of patients of sars, mers or covid-19 is the presence of severe acute respiratory syndrome; however, the estimated fatality rate of covid-19 (2.3%) is much lower than sars (~10%) and mers (~36%) [26, 27] . furthermore, the viruses responsible for above three diseases are evolutionary distinct (see below for details) [19] . it is clear now that sars-cov-2 can be transmitted by human-to-human despite the majority of the early cases had contact history with the huanan seafood market [11, 18, 28] . analysis of 425 patients with confirmed covid-19 showed that the incubation period is 3 to 7 days. the mean was 5.2 days (95% ci: 4.1 to 7.0), and the 95 th percentile of the distribution is 12.5 days (95% ci: 9.2 to 18) [11] . notably, it was reported that the incubation period could be as long as 24 days in a rare case [25] . the basic reproductive number (r 0 ) up to the period of 4 jan 2020 was estimated based on the study of 425 patients to be 2.2 (meaning that one patient has been spreading infection to 2.2 other people) [11] , slightly smaller than the value of 2.68 by a modelling in another [29] . the r 0 of sars-cov-2 from both of these two studies is smaller than that of sras, which are 3 before public health measures were implemented [30] . however, subsequent investigation based on the analysis of high-resolution real-time human travel and infection data estimated that the r 0 is much larger, ranging from 4.7 to 6.6 before the control measures [31] , implying that sars-cov-2 is highly contagious and more infectious than initially estimated. this conclusion is consistent with the wide spread of sars-cov-2 within a short period time and was also echoed by the finding that sars-cov-2 spike (s) protein had 10-to 20-fold higher affinity to human angiotensin-converting enzyme 2 (ace2) receptor than that of sars-cov based on the cryo-em structure analysis of s proteins [32] . similar to sars-cov, the entry of sars-cov-2 into host cells depends on the recognition and binding of s protein to ace2 receptor of the host cells [14, 33] . the high affinity of s protein to ace2 receptor likely contributes to the quick spreading of virus. the finding of ace2 as the receptor of sars-cov-2 also indicates that human organs with high ace2 expression level, such as lung alveolar epithelial cells and enterocytes of the small intestine, are potentially the target of sars-cov-2 [34] . as a new coronavirus, it is not known yet about how sars-cov-2 spreads. current knowledge for sars-cov-2 transmission is largely based on what is known from the similar coronaviruses, particularly sars-cov and mers-cov, in which human-tohuman transmission occurs through droplets, contact and fomites. sars-cov is predominantly transmitted through indirect or direct contact with mucous membranes in the mouth, eyes, or nose [35] . it has been shown that unprotected eyes and exposed mucous membranes are vulnerable to sars-cov transmission [36] . a member of the national expert panel on pneumonia was infected by sars-cov-2 after the inspection in wuhan [37] . as he wore a n95 mask but not any eye protector, and experienced eye redness before the onset of pneumonia, it was thus suspected that unprotected exposure of the eyes to sars-cov-2 might be another transmission pathway [37] . however, sars-cov-2 was not detected from the conjunctival swab sample in a confirmed covid-19 patent with conjunctivitis [38] , suggesting that more evidences are needed before concluding the conjunctival route as the transmission pathway of sars-cov-2. the mode of transmission by mers-cov is not well understood but is believed to spread largely via the respiratory close contact route [39, 40] . based on the transmission mode of sars-cov and mers-cov, a serial of preventive measures have been recommended, including avoiding close contact with people suffering from acute respiratory infections and frequent hand-washing [41] . the viruses of sars-cov-2 were also detected in the stool samples in some patients but not all [18, 22] , suggesting that a possible fecal-oral transmission occurs [42] . a systematic study showed that viruses could be detected in oral swabs, anal swabs and blood samples of the patients, and the anal swabs and blood could test positive when oral swab tested negative [43] . furthermore, a trend of shift from more oral positive in the collected samples during the early period of patient infection to more anal positive during later period of infection was also found [43] . therefore, a multiple shedding routes of sars-cov-2 might exist. one of the challenges for preventive control of sars-cov-2 spreading is that the viruses are likely transmitted by asymptomatic contact. a german businessman was found infected by sars-cov-2 after attending a conference together with a colleague, who had no signs or symptoms of infection but had become ill due to the sars-cov-2 infection later [44] . this observation suggests that infected patients likely start to shed viruses before the onset of any symptom, which undoubtedly will bring great challenge to the current practice of preventive control by measuring body temperature. despite the claim of the transmission by asymptomatic contact has been challenged [45] , other asymptomatic carriers were also observed to transmit the viruses of sars-cov-2 [46, 47] . consistently, a study found that an asymptomatic patient had a similar vial loads in the samples of nasal and throat swabs to that of the symptomatic patients [48] . it is critical to identify the origin, native host(s) and evolution pathway of the virus that causes an outbreak of a pandemic. this information can help understand the molecular mechanism of its cross-species spread and implement a proper control measure to prevent it from further spreading. the association of initially confirmed sars-cov-2 cases with huanan seafood market suggested that the marketplace has played a role in the early spreading [11, 23] , however, whether it is the origin of the outbreak and what is the native host(s) of sars-cov-2 remain uncertain. in fact, the firstly documented patient was not linked to huanan seafood market [23] . the analysis of sars-cov-2 origin was firstly performed based on the genome sequence of virus isolates from six patients [19] . when compared with sars-cov and mers-cov, the nucleotide sequences of sars-cov-2 showed a higher homology with that of sars-cov while was relatively poor with that of mers-cov [19] . despite some of the six major ofrs of sars-cov-2 genes share less than 80% identity in nucleotide acids to sars-cov, the seven conserved replicase domains in orf1ab has 94.6% sequence identity in amino acids between sars-cov-2 and sars-cov [14] , suggesting that these two viruses might belong to the same species. the origin of sars-cov has been extensively investigated. masked palm civets were initially considered to transmit sars-cov to humans as a close variant of sars-cov was detected from palm civets [49] . this conclusion was supported by the fact that three of the four patients had the record of contact with palm civets during the two small-scale of sars outbreaks occurred in late 2003 and early 2004 [50, 51] . however, a deep investigation based on the genome sequence of isolated viruses showed that sars-cov-like virus in civet had not been circulating for long [52] . subsequently, coronaviruses with high similarity to the human sars-cov or civet sars-cov-like virus were isolated from horseshoe bats, concluding the bats as the potential natural reservoir of sars-cov whereas masked palm civets are the intermediate host [53] [54] [55] [56] . it is thus reasonable to suspect that bat is the natural host of sars-cov-2 considering its similarity with sars-cov. the phylogenetic analysis of sars-cov-2 against a collection of coronavirus sequences from various sources found that sars-cov-2 belonged to the betacoronavirus genera and was closer to sars-like coronavirus in bat [19] . by analyzing genome sequence of sars-cov-2, it was found that sars-cov-2 felled within the subgenus sarbecovirus of the genus betacoronavirus and was closely related to two bat-derived sars-like coronaviruses, bat-sl-covzc45 and bat-sl-covzxc21, but were relatively distant from sars-cov [15, 18, [57] [58] [59] . meanwhile, zhou and colleagues showed that sars-cov-2 had 96.2% overall genome sequence identity throughout the genome to batcov ratg13, a bat coronavirus detected in rhinolophus affinis from yunnan province [14] . furthermore, the phylogenetic analysis of full-length genome, the receptor binding protein spike (s) gene, and rna-dependent rna polymerase (rdrp) gene respectively all demonstrated that ratg13 was the closest relative of the sars-cov-2 [14] . however, despite sars-cov-2 showed high similarity to coronavirus from bat, sars-cov-2 changed topological position within the subgenus sarbecovirus when different gene was used for phylogenetic analysis: sars-cov-2 was closer to bat-sl-covzc45 in the s gene phylogeny but felled in a basal position within the subgenus sarbecovirus in the orf1b tree [57] . this finding implies a possible recombination event in this group of viruses. of note, the receptor-binding domain of sars-cov-2 demonstrates a similar structure to that of sars-cov by homology modelling but a few variations in the key residues exist at amino acid level [15, 19] . despite current evidences are pointing to the evolutional origin of sars-cov-2 from bat virus [15, 57] , an intermediate host between bats and human might exist. lu et. al. raised four reasons for such speculation [15] : first, most bat species in wuhan are hibernating in late december; second, no bats in huanan seafood market were sold or found; third, the sequence identity between sars-cov-2 and bat-sl-covzc45 or bat-sl-covzxc21, the closest relatives in their analyses, is lower than 90%; fourth, there is an intermediate host for other humaninfecting coronaviruses that origin from bat. for example, masked palm civet and dromedary camels are the intermediate hosts for sars-cov [49] and mers-cov respectively [60] . a study of the relative synonymous codon usage (rscu) found that sars-cov-2, bat-sl-covzc45, and snakes had similar synonymous codon usage bias, and speculated that snake might be the intermediate host [61] . however, no sars-cov-2 has been isolated from snake yet. pangolin was later found to be a potential intermediate host for sars-cov-2. the analysis of samples from malytan pangolins obtained during anti-smuggling operations from guangdong and guangxi customs of china respectively found novel coronaviruses representing two sub-lineages related to sars-cov-2 [62] . the similarity of sars-cov-2 to these identified coronaviruses from pangolins is approximately 85.5% to 92.4% in genomes, lower than that to the bat coronavirus ratg13 (96.2%) [14, 62] . however, the receptor-binding domain of s protein from one sub-lineage of the pangolin coronaviruses shows 97.4% similarity in amino acid sequences to that of sars-cov-2, even higher than that to ratg13 (89.2%) [62] . interestingly, the pangolin coronavirus and sars-cov-2 share identical amino acids at the five critical residues of rbd of s protein, while ratg13 only possesses one [62] . the discovery of coronavirus close to sars-cov-2 from pangolin suggests that pangolin is a potential intermediate host. however, the roles of bat and pangolin as respective natural reservoir and intermediate host still need further investigation. as an emerging virus, there is no effective drug or vaccine approved for the treatment of sars-cov-2 infection yet. currently, supportive care is provided to the patients, including oxygen therapy, antibiotic treatment, and antifungal treatment, extra-corporeal membrane oxygenation (ecmo) etc. [21, 22] . to search for an antiviral drug effective in treating sars-cov-2 infection, wang and colleagues evaluated seven drugs, namely, ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine, remdesivir (gs-5734) and favipiravir (t-750) against the infection of sars-cov-2 on vero e6 cells in vitro [63] . among these seven drugs, chloroquine and remdesivir demonstrated the most powerful antiviral activities with low cytotoxicity. the effective concentration (ec 50 ) for chloroquine and remdesivir were 0.77âµm and 1.13âµm respectively. chloroquine functions at both viral entry and post-entry stages of the sars-cov-2 infection in vero e6 cells whereas remdesivir does at post-entry stage only. chloroquine is a drug used for an autoimmune disease and malarial infection with potential broad-spectrum antiviral activities [64, 65] . an ec90 (6.90 âµm) against the sars-cov-2 in vero e6 cells is clinically achievable in vivo according to a previous clinical trial [66] . remdesivir is a drug currently under the development for ebola virus infection and is effective to a broad range of viruses including sars-cov and mers-cov [67, 68] . functioning as an adenosine analogue targeting rdrp, remdesivir can result in premature termination during the virus transcription [69, 70] . the ec90 of remdesivir against sars-cov-2 in vero e6 cells is 1.76 âµm, which is achievable in vivo based on a trial in nonhuman primate experiment [63, 69] . encouragingly, in the first case of sars-cov-2 infection in the united states, treatment with remdesivir was provided intravenously to the patient on the day 7 without any adverse events observed. the patient's clinical condition was improved on day 8 and the previous bilateral lower-lobe rales disappeared, implying the remdesivir might be effective to the treatment of sars-cov-2 infection [22] . this result, however, should be interpreted with caution as this is only single case study and a proper trial control was lacking. in addition, baricitinib, a janus kinase inhibitor, was also predicted to reduce the ability of virus to infect lung cell by an analysis of benevolentai [71] . currently, chloroquine and remdesivir are under phase 3 clinical trial and open-label trial for treatment of sars-cov-2 infection respectively (table 2 ) [72] . preliminary results showed that chloroquine phosphate had apparent efficacy in treatment of covid-19 [73] . however, caution must be taken during clinical use of chloroquine as its overdose is highly fatal without known antidote [74] . despite the lack of documented in vitro data supporting the antiviral efficacy on sars-cov-2, several antiviral chemotherapeutic agents have been registered for the clinical trials for the treatment of covid-19 (table 2 ) [72] . sars-cov-2 is an emerging pathogen, without any effective drug available for treatment at the moment. it spreads quickly and can result in death of the infected patients. despite the current mortality rate is 2.3% [26] , the emergence of large number of infected patients within short period of time could result in the collapse of health care system, and thus the mortality rate might be elevated. effective preventive measures must be implemented to control it from global spreading. in addition, great effort should be made on the development of vaccine and antiviral drugs. meanwhile, the 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infected patients: implication of multiple shedding routes transmission of 2019-ncov infection from an asymptomatic contact in germany study claiming new coronavirus can be transmitted by people without symptoms was flawed presumed asymptomatic carrier transmission of covid-19 evidence of sars-cov-2 infection in returning travelers from wuhan, china sars-cov-2 viral load in upper respiratory specimens of infected patients isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars-cov infection in a restaurant from palm civet cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human a review of studies on animal reservoirs of the sars coronavirus bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor discovery of a rich gene pool 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an animal model dose refinements in long-term therapy of rheumatoid arthritis with antimalarials broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio baricitinib as potential treatment for 2019-ncov acute respiratory disease. the lancet therapeutic options for the 2019 novel coronavirus (2019-ncov) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies the effects of acute chloroquine poisoning with special reference to the heart mechanism of action of t-705 against influenza virus mechanisms of action of ribavirin against distinct viruses molecular dynamic simulations analysis of ritonavir and lopinavir as sars-cov 3cl(pro) inhibitors darunavir: a nonpeptidic antiretroviral protease inhibitor tmc310911, a novel human immunodeficiency virus type 1 protease inhibitor, shows in vitro an improved resistance profile and higher genetic barrier to resistance compared with current protease inhibitors an endocytosis blocking agent, inhibits zika virus infection in different cell models arbidol: a broad-spectrum antiviral compound that blocks viral fusion neuraminidase inhibitors: zanamivir and oseltamivir the authors have declared that no competing interest exists. key: cord-332723-rz1iilsv authors: creager, hannah m.; cabrera, barbara; schnaubelt, andy; cox, jesse l.; cushman-vokoun, allison m.; shakir, salika m.; tardif, keith d.; huang, meei-li; jerome, keith r.; greninger, alexander l.; drobysheva, daria; spaulding, usha; rogatcheva, margarita; bourzac, kevin m.; hinrichs, s.h.; broadhurst, m.j.; fey, p.d. title: clinical evaluation of the biofire® respiratory panel 2.1 and detection of sars-cov-2 date: 2020-07-06 journal: j clin virol doi: 10.1016/j.jcv.2020.104538 sha: doc_id: 332723 cord_uid: rz1iilsv we evaluated the performance of the biofire® respiratory panel 2.1 (rp2.1) in the detection of sars cov-2 in comparison against three other sars cov-2 eua assays. in these studies, the rp2.1 panel had 98% positive percent agreement (48/49) and 100% negative percent agreement (49/49). since 30% of nasopharyngeal swab specimens have a sars cov-2 ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other eua approved sars cov-2 assays. these data suggested that the biofire® rp2.1 panel, along with four other sars cov-2 assays (roche cobas, cepheid xpert xpress, biofire® defense covid19, and necov19), consistently detected viral rna at the 10-7 dilution. overall, these studies suggest that the biofire® rp2.1 assay can be used to detect acute cases of sars cov2 in addition to patients with low viral titer later in disease presentation. the gold standard for sars-cov-2 diagnosis is detection of viral rna in nasopharyngeal (np) swab specimens. sample-to-answer nucleic acid amplification assays for the detection of sars-cov-2 rna are available for a limited number of high-throughput diagnostic platforms including the roche cobas 6800/8800 (1, 2) , the hologic panther and panther fusion, (3) (4) (5) (6) and the abbott m2000 (7) . high-throughput platforms are mostly utilized in larger reference laboratories, state public health laboratories, and academic medical centers, but these assays are not well-suited to use in other settings that lack large testing volumes or the capacity to perform high complexity tests. this has led to the centralization of sars-cov-2 testing, meaning that turnaround time may be prolonged by the need to transport specimens over long distances. as sars-cov-2 prevalence increases, decentralized testing capability is needed to facilitate rapid identification of sars-cov-2 cases. to date, the biofire covid-19, cepheid xpert xpress sars-cov-2 (8), diasorin simplexa (9) and the genmark eplex sars-cov-2 tests (6) have emerged as rapid covid19 testing platforms that fill this niche. the biofire ® filmarray ® system (biofire diagnostics, llc, salt lake city, ut, "biofire") is another testing platform that is widely used in multiple laboratory environments. this multiplex, residual natural nasopharyngeal swab in transport media (nps) specimens leftover from sars-cov-2 testing performed as part of patient care were collected during march and april of 2020 at the university of washington (seattle, wa), university of nebraska medical center (omaha, ne), and arup laboratories (salt lake city, ut). original specimen testing for sars-cov-2 was conducted according to manufacturer's instructions at arup laboratories using the hologic panther fusion sars-cov-2 assay (fda eua), at the university of nebraska medical center using the roche cobas sars-cov-2 assay (fda eua), or at the university of washington using a laboratory developed test based on the cdc n1 and n2 sars-cov-2 assays (washington eua) conducted as described in perchetti et al. (10) . specimens were frozen upon study enrollment to allow for storage and shipping. additional nps specimens collected before december 2019 and therefore presumed to be negative for sars-cov-2 were provided by biofire diagnostics. ten-fold serial dilutions of a natural nasopharyngeal swab specimen with known high positivity for sars-cov-2 rna (e gene detected at a cycle threshold (ct) of 16.6 by the cobas sars-cov-2 assay) were prepared with a diluent of pooled nps. diluent was prepared from samples that tested negative for sars-cov-2 using an assay developed at nebraska medicine (necov19; fda eua) and was confirmed to be pcr negative prior to use. these samples were not tested for other respiratory viruses prior to pooling. on two separate subsequent occasions, an aliquot of the 10 -4 or 10 -5 diultions was thawed and added to newly generated pools of nps to create additional intermediate dilutions between 10 -6 and 10 -8 . single-use aliquots of each dilution were stored at -80°c and thawed immediately prior to use. testing by commercial assays (biofire rp2. performed according to manufacturer's instructions with the exception of the abbot id now. the instructions for use for this assay have been revised and now limit testing to swab specimens that can be used to directly inoculate the sample cup. because comparison between platforms required use of a liquid specimen, we followed the instructions for use associated with the original j o u r n a l p r e -p r o o f eua for the abbot id now assay and used transfer pipettes from the kit to add 200 μl of np swab specimens in transport medium to the sample cup. table 1 ). the remaining fifty specimens were expected to test negative for sars-cov-2 because they were collected prior to december 2019. testing of one positive and one negative specimen yielded invalid results due to instrument errors; these could not be retested according to the instructions for use due to insufficient specimen volume. these specimens were excluded from the analyses, resulting in reduction of sample size to 49 valid positive and 49 valid negative specimens. the 49 negative specimens tested negative for sars-cov-2 on the biofire rp2.1 assay (100% negative percent agreement, table 1 ). the biofire rp2.1 assay detected sars-cov-2 in 48/49 positive specimens (98% positive percent agreement, table 1 performance of the sars-cov-2 assay on the biofire rp2.1 was comparable to that of necov19, cobas, genexpert, and biofire defense assays ( table 2, supplemental table 1 ) with detection of sars-cov-2 in all replicates down to the 10 -7 dilution. applying ct values from the ldt to a standard curve generated from extracted sars-cov-2 quantitated rna standard showed that this dilution contains approximately 10 3 copies/ml. virus detection was inconsistent at lower concentrations. sars-cov-2 detection dropped off below the 10 -6 dilution and 10 -5 j o u r n a l p r e -p r o o f dilutions for the hologic aptima assay and abbott id now assay, respectively (table 2, supplemental table 1 ). our studies show that the biofire rp2.1 has similar performance to high throughput shedding at levels unlikely to result in transmission, a false negative result would be less consequential (13, 14) . the addition of a sars-cov-2 test to a commonly used multiplex pcr panel will expand the number of laboratories able to test for sars-cov-2 and will allow detection of coinfection as well as of alternative diagnoses. once the northern hemisphere respiratory season arrives, the ability to test for influenza, rsv, and sars-cov-2 simultaneously on the biofire rp2.1 will greatly benefit hospitals as an important infection control management tool. the corresponding author has acted as a consultant and received grant funding from biofire diagnostics. ct values are shown for each assay used for characterizing clinical specimens, as indicated on the x axis. horizontal bars represent ct median values for each assay. j o u r n a l p r e -p r o o f the detection of sars-cov-2 using the cepheid xpert xpress sars-cov-2 and roche cobas sars-cov-2 assays comparison of sars-cov-2 detection from nasopharyngeal swab samples by the roche cobas(r) 6800 sars-cov-2 test and a laboratory-developed real-time rt-pcr test rapid random access detection of the novel sarscoronavirus-2 (sars-cov-2, previously 2019-ncov) using an open access protocol for the panther fusion comparison of the panther fusion and a laboratory-developed test targeting the envelope gene for detection of sars-cov-2 comparison of commercially available and laboratory developed assays for in vitro detection of sars-cov-2 in clinical laboratories comparison of four molecular in vitro diagnostic assays for the detection of sars-cov-2 in nasopharyngeal specimens comparison of abbott id now and abbott m2000 methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from symptomatic patients multicenter evaluation of the cepheid xpert xpress sars-cov-2 test rapid and sensitive detection of sars-cov-2 rna using the simplexa covid-19 direct assay validation of sars-cov-2 detection across multiple specimen types detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr research use only 2019-novel coronavirus (2019-ncov) real-time rt-pcr primers and probes virological assessment of hospitalized patients with covid-2019 this work was funded by a grant from biofire diagnostics to pdf. we would like to thank key: cord-279255-v861kk0i authors: dhama, kuldeep; khan, sharun; tiwari, ruchi; sircar, shubhankar; bhat, sudipta; malik, yashpal singh; singh, karam pal; chaicumpa, wanpen; bonilla-aldana, d. katterine; rodriguez-morales, alfonso j. title: coronavirus disease 2019–covid-19 date: 2020-06-24 journal: clin microbiol rev doi: 10.1128/cmr.00028-20 sha: doc_id: 279255 cord_uid: v861kk0i in recent decades, several new diseases have emerged in different geographical areas, with pathogens including ebola virus, zika virus, nipah virus, and coronaviruses (covs). recently, a new type of viral infection emerged in wuhan city, china, and initial genomic sequencing data of this virus do not match with previously sequenced covs, suggesting a novel cov strain (2019-ncov), which has now been termed severe acute respiratory syndrome cov-2 (sars-cov-2). although coronavirus disease 2019 (covid-19) is suspected to originate from an animal host (zoonotic origin) followed by human-to-human transmission, the possibility of other routes should not be ruled out. compared to diseases caused by previously known human covs, covid-19 shows less severe pathogenesis but higher transmission competence, as is evident from the continuously increasing number of confirmed cases globally. compared to other emerging viruses, such as ebola virus, avian h7n9, sars-cov, and middle east respiratory syndrome coronavirus (mers-cov), sars-cov-2 has shown relatively low pathogenicity and moderate transmissibility. codon usage studies suggest that this novel virus has been transferred from an animal source, such as bats. early diagnosis by real-time pcr and next-generation sequencing has facilitated the identification of the pathogen at an early stage. since no antiviral drug or vaccine exists to treat or prevent sars-cov-2, potential therapeutic strategies that are currently being evaluated predominantly stem from previous experience with treating sars-cov, mers-cov, and other emerging viral diseases. in this review, we address epidemiological, diagnostic, clinical, and therapeutic aspects, including perspectives of vaccines and preventive measures that have already been globally recommended to counter this pandemic virus. o ver the past 2 decades, coronaviruses (covs) have been associated with significant disease outbreaks in east asia and the middle east. the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) began to emerge in 2002 and 2012, respectively. recently, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), causing coronavirus disease 2019 (covid19) , emerged in late 2019, and it has posed a global health threat, causing an ongoing pandemic in many countries and territories (1) . health workers worldwide are currently making efforts to control further disease outbreaks caused by the novel cov (originally named 2019-ncov), which was first identified in wuhan city, hubei province, china, on 12 december 2019. on 11 february 2020, the world health organization (who) announced the official designation for the current cov-associated disease to be covid-19, caused by sars-cov-2. the primary cluster of patients was found to be connected with the huanan south china seafood market in wuhan (2) . covs belong to the family coronaviridae (subfamily coronavirinae), the members of which infect a broad range of hosts, producing symptoms and diseases ranging from the common cold to severe and ultimately fatal illnesses, such as sars, mers, and, presently, covid-19. sars-cov-2 is considered one of the seven members of the cov family that infect humans (3) , and it belongs to the same lineage of covs that causes sars; however, this novel virus is genetically distinct. until 2020, six covs were known to infect humans, including human cov 229e (hcov-229e), hcov-nl63, hcov-oc43, hcov-hku1, sars-cov, and mers-cov. although sars-cov and mers-cov have resulted in outbreaks with high mortality, others remain associated with mild upper-respiratory-tract illnesses (4) . newly evolved covs pose a high threat to global public health. the current emergence of covid-19 is the third cov outbreak in humans over the past 2 decades (5) . it is no coincidence that fan et al. predicted potential sars-or mers-like cov outbreaks in china following pathogen transmission from bats (6) . covid-19 emerged in china and spread rapidly throughout the country and, subsequently, to other countries. due to the severity of this outbreak and the potential of spreading on an international scale, the who declared a global health emergency on 31 january 2020; subsequently, on 11 march 2020, they declared it a pandemic situation. at present, we are not in a position to effectively treat covid-19, since neither approved vaccines nor specific antiviral drugs for treating human cov infections are available (7) (8) (9) . most nations are currently making efforts to prevent the further spreading of this potentially deadly virus by implementing preventive and control strategies. in domestic animals, infections with covs are associated with a broad spectrum of furthermore, it acts as a critical factor for tissue tropism and the determination of host range (45) . notably, s protein is one of the vital immunodominant proteins of covs capable of inducing host immune responses (45) . the ectodomains in all covs s proteins have similar domain organizations, divided into two subunits, s1 and s2 (43) . the first one, s1, helps in host receptor binding, while the second one, s2, accounts for fusion. the former (s1) is further divided into two subdomains, namely, the n-terminal domain (ntd) and c-terminal domain (ctd). both of these subdomains act as receptorbinding domains, interacting efficiently with various host receptors (45) . the s1 ctd contains the receptor-binding motif (rbm). in each coronavirus spike protein, the trimeric s1 locates itself on top of the trimeric s2 stalk (45) . recently, structural analyses of the s proteins of covid-19 have revealed 27 amino acid substitutions within a 1,273-amino-acid stretch (16) . six substitutions are located in the rbd (amino acids 357 to 528), while four substitutions are in the rbm at the ctd of the s1 domain (16) . of note, no amino acid change is seen in the rbm, which binds directly to the angiotensinconverting enzyme-2 (ace2) receptor in sars-cov (16, 46) . at present, the main emphasis is knowing how many differences would be required to change the host tropism. sequence comparison revealed 17 nonsynonymous changes between the early sequence of sars-cov-2 and the later isolates of sars-cov. the changes were found scattered over the genome of the virus, with nine substitutions in orf1ab, orf8 (4 substitutions), the spike gene (3 substitutions) , and orf7a (single substitution) (4) . notably, the same nonsynonymous changes were found in a familial cluster, indicating that the viral evolution happened during person-to-person transmission (4, 47) . such adaptive evolution events are frequent and constitute a constantly ongoing process once the virus spreads among new hosts (47) . even though no functional changes occur in the virus associated with this adaptive evolution, close monitoring of the viral mutations that occur during subsequent human-to-human transmission is warranted. the m protein is the most abundant viral protein present in the virion particle, giving a definite shape to the viral envelope (48) . it binds to the nucleocapsid and acts as a central organizer of coronavirus assembly (49) . coronavirus m proteins are highly diverse in amino acid contents but maintain overall structural similarity within different genera (50) . the m protein has three transmembrane domains, flanked by a short amino terminus outside the virion and a long carboxy terminus inside the virion (50) . overall, the viral scaffold is maintained by m-m interaction. of note, the m protein of sars-cov-2 does not have an amino acid substitution compared to that of sars-cov (16) . the coronavirus e protein is the most enigmatic and smallest of the major structural proteins (51) . it plays a multifunctional role in the pathogenesis, assembly, and release of the virus (52) . it is a small integral membrane polypeptide that acts as a viroporin (ion channel) (53) . the inactivation or absence of this protein is related to the altered virulence of coronaviruses due to changes in morphology and tropism (54) . the e protein consists of three domains, namely, a short hydrophilic amino terminal, a large hydrophobic transmembrane domain, and an efficient c-terminal domain (51) . the sars-cov-2 e protein reveals a similar amino acid constitution without any substitution (16) . the n protein of coronavirus is multipurpose. among several functions, it plays a role in complex formation with the viral genome, facilitates m protein interaction needed during virion assembly, and enhances the transcription efficiency of the virus (55, 56) . it contains three highly conserved and distinct domains, namely, an ntd, an rna-binding domain or a linker region (lkr), and a ctd (57) . the ntd binds with the 3= end of the viral genome, perhaps via electrostatic interactions, and is highly diverged both in length and sequence (58) . the charged lkr is serine and arginine rich and is also known as the sr (serine and arginine) domain (59) . the lkr is capable of direct interaction with in vitro rna interaction and is responsible for cell signaling (60, 61) . it also modulates the antiviral response of the host by working as an antagonist for interferon (ifn) and rna interference (62) . compared to that of sars-cov, the n protein of sars-cov-2 possess five amino acid mutations, where two are in the intrinsically dispersed region (idr; positions 25 and 26) , one each in the ntd (position 103), lkr (position 217), and ctd (position 334) (16) . besides the important structural proteins, the sars-cov-2 genome contains 15 nsps, nsp1 to nsp10 and nsp12 to nsp16, and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14) (16) . all these proteins play a specific role in viral replication (27) . unlike the accessory proteins of sars-cov, sars-cov-2 does not contain 8a protein and has a longer 8b and shorter 3b protein (16) . the nsp7, nsp13, envelope, matrix, and p6 and 8b accessory proteins have not been detected with any amino acid substitutions compared to the sequences of other coronaviruses (16) . the virus structure of sars-cov-2 is depicted in fig. 2 . sequence percent similarity analysis. we assessed the nucleotide percent similarity using the megalign software program, where the similarity between the novel sars-cov-2 isolates was in the range of 99.4% to 100%. among the other serbecovirus cov sequences, the novel sars-cov-2 sequences revealed the highest similarity to bat-sl-cov, with nucleotide percent identity ranges between 88.12 and 89.65%. meanwhile, earlier reported sars-covs showed 70.6 to 74.9% similarity to sars-cov-2 at the nucleotide level. further, the nucleotide percent similarity was 55.4%, 45.5% to 47.9%, 46 .2% to 46.6%, and 45.0% to 46.3% to the other four subgenera, namely, hibecovirus, nobecovirus, merbecovirus, and embecovirus, respectively. the percent similarity index of current outbreak isolates indicates a close relationship between sars-cov-2 isolates and bat-sl-cov, indicating a common origin. however, particular pieces of evidence based on further complete genomic analysis of current isolates are necessary to draw any conclusions, although it was ascertained that the current novel sars-cov-2 isolates belong to the subgenus sarbecovirus in the diverse range of betacoronaviruses. their possible ancestor was hypothesized to be from bat cov strains, wherein bats might have played a crucial role in harboring this class of viruses. splitstree phylogeny analysis. in the unrooted phylogenetic tree of different betacoronaviruses based on the s protein, virus sequences from different subgenera grouped into separate clusters. sars-cov-2 sequences from wuhan and other countries exhibited a close relationship and appeared in a single cluster (fig. 1 ). the covs from the subgenus sarbecovirus appeared jointly in splitstree and divided into three subclusters, namely, sars-cov-2, bat-sars-like-cov (bat-sl-cov), and sars-cov (fig. 1) . in the case of other subgenera, like merbecovirus, all of the sequences grouped clinical microbiology reviews than italy. a john hopkins university web platform has provided daily updates on the basic epidemiology of the covid-19 outbreak (https://gisanddata.maps.arcgis.com/ apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6) (238) . covid-19 has also been confirmed on a cruise ship, named diamond princess, quarantined in japanese waters (port of yokohama), as well as on other cruise ships around the world (239) (fig. 3) . the significant events of the sars-cov-2/covid-19 virus outbreak occurring since 8 december 2019 are presented as a timeline in fig. 5 . at the beginning, china experienced the majority of the burden associated with covid-19 in the form of disease morbidity and mortality (65), but over time the covid-19 menace moved to europe, particularly italy and spain, and now the united states has the highest number of confirmed cases and deaths. the covid-19 outbreak has also been associated with severe economic impacts globally due to the sudden interruption of global trade and supply chains that forced multinational companies to make decisions that led to significant economic losses (66) . the recent increase in the number of confirmed critically ill patients with covid-19 has already surpassed the intensive care supplies, limiting intensive care services to only a small portion of critically ill patients (67) . this might also have contributed to the increased case fatality rate observed in the covid-19 outbreak. the novel coronavirus was identified within 1 month (28 days) of the outbreak. this is impressively fast compared to the time taken to identify sars-cov reported in foshan, guangdong province, china (125 days) (68) . immediately after the confirmation of viral etiology, the chinese virologists rapidly released the genomic sequence of sars-cov-2, which played a crucial role in controlling the spread of this newly emerged novel coronavirus to other parts of the world (69) . the possible origin of sars-cov-2 and the first mode of disease transmission are not yet identified (70) . analysis of the initial cluster of infections suggests that the infected individuals had a common exposure point, a seafood market in wuhan, hubei province, china (fig. 6 ). the restaurants of this market are well-known for providing different types of wild animals for human consumption (71) . the huanan south china seafood market also sells live animals, such as poultry, bats, snakes, and marmots (72) . this might be the point where zoonotic (animal-to-human) transmission occurred (71) . although sars-cov-2 is alleged to have originated from an animal host (zoonotic origin) with further humanto-human transmission (fig. 6 ), the likelihood of foodborne transmission should be ruled out with further investigations, since it is a latent possibility (1). additionally, other clinical microbiology reviews potential and expected routes would be associated with transmission, as in other respiratory viruses, by direct contact, such as shaking contaminated hands, or by direct contact with contaminated surfaces (fig. 6) . still, whether blood transfusion and organ transplantation (276) , as well as transplacental and perinatal routes, are possible routes for sars-cov-2 transmission needs to be determined (fig. 6 ). from experience with several outbreaks associated with known emerging viruses, higher pathogenicity of a virus is often associated with lower transmissibility. compared to emerging viruses like ebola virus, avian h7n9, sars-cov, and mers-cov, sars-cov-2 has relatively lower pathogenicity and moderate transmissibility (15) . the risk of death among individuals infected with covid-19 was calculated using the infection fatality risk (ifr). the ifr was found to be in the range of 0.3% to 0.6%, which is comparable to that of a previous asian influenza pandemic (1957 to 1958) (73, 277) . notably, the reanalysis of the covid-19 pandemic curve from the initial cluster of cases pointed to considerable human-to-human transmission. it is opined that the exposure history of sars-cov-2 at the wuhan seafood market originated from humanto-human transmission rather than animal-to-human transmission (74) ; however, in light of the zoonotic spillover in covid-19, is too early to fully endorse this idea (1). following the initial infection, human-to-human transmission has been observed with a preliminary reproduction number (r 0 ) estimate of 1.4 to 2.5 (70, 75) , and recently it is estimated to be 2.24 to 3.58 (76) . in another study, the average reproductive number of covid-19 was found to be 3.28, which is significantly higher than the initial who estimate of 1.4 to 2.5 (77) . it is too early to obtain the exact r 0 value, since there is a possibility of bias due to insufficient data. the higher r 0 value is indicative of the more significant potential of sars-cov-2 transmission in a susceptible population. this is not the first time where the culinary practices of china have been blamed for the origin of novel coronavirus infection in humans. previously, the animals present in the liveanimal market were identified to be the intermediate hosts of the sars outbreak in china (78) . several wildlife species were found to harbor potentially evolving coronavirus strains that can overcome the species barrier (79) . one of the main principles of chinese food culture is that live-slaughtered animals are considered more nutritious (5) . after 4 months of struggle that lasted from december 2019 to march 2020, the covid-19 situation now seems under control in china. the wet animal markets have reopened, and people have started buying bats, dogs, cats, birds, scorpions, badgers, rabbits, pangolins (scaly anteaters), minks, soup from palm civet, ostriches, hamsters, snapping turtles, ducks, fish, siamese crocodiles, and other animal meats without any fear of covid-19. the chinese government is encouraging people to feel they can return to normalcy. however, this could be a risk, as it has been mentioned in advisories that people should avoid contact with live-dead animals as much as possible, as sars-cov-2 has shown zoonotic spillover. additionally, we cannot rule out the possibility of new mutations in the same virus being closely related to contact with both animals and humans at the market (284) . in january 2020, china imposed a temporary ban on the sale of live-dead animals in wet markets. however, now hundreds of such wet markets have been reopened without optimizing standard food safety and sanitation practices (286) . with china being the most populated country in the world and due to its domestic and international food exportation policies, the whole world is now facing the menace of covid-19, including china itself. wet markets of live-dead animals do not maintain strict food hygienic practices. fresh blood splashes are present everywhere, on the floor and tabletops, and such food customs could encourage many pathogens to adapt, mutate, and jump the species barrier. as a result, the whole world is suffering from novel sars-cov-2, with more than 4,170,424 cases and 287,399 deaths across the globe. there is an urgent need for a rational international campaign against the unhealthy food practices of china to encourage the sellers to increase hygienic food practices or close the crude live-dead animal wet markets. there is a need to modify food policies at national and international levels to avoid further life threats and clinical microbiology reviews economic consequences from any emerging or reemerging pandemic due to close animal-human interaction (285) . even though individuals of all ages and sexes are susceptible to covid-19, older people with an underlying chronic disease are more likely to become severely infected (80) . recently, individuals with asymptomatic infection were also found to act as a source of infection to susceptible individuals (81) . both the asymptomatic and symptomatic patients secrete similar viral loads, which indicates that the transmission capacity of asymptomatic or minimally symptomatic patients is very high. thus, sars-cov-2 transmission can happen early in the course of infection (82) . atypical clinical manifestations have also been reported in covid-19 in which the only reporting symptom was fatigue. such patients may lack respiratory signs, such as fever, cough, and sputum (83) . hence, the clinicians must be on the look-out for the possible occurrence of atypical clinical manifestations to avoid the possibility of missed diagnosis. the early transmission ability of sars-cov-2 was found to be similar to or slightly higher than that of sars-cov, reflecting that it could be controlled despite moderate to high transmissibility (84) . increasing reports of sars-cov-2 in sewage and wastewater warrants the need for further investigation due to the possibility of fecal-oral transmission. sars-cov-2 present in environmental compartments such as soil and water will finally end up in the wastewater and sewage sludge of treatment plants (328) . therefore, we have to reevaluate the current wastewater and sewage sludge treatment procedures and introduce advanced techniques that are specific and effective against sars-cov-2. since there is active shedding of sars-cov-2 in the stool, the prevalence of infections in a large population can be studied using wastewater-based epidemiology. recently, reverse transcription-quantitative pcr (rt-qpcr) was used to enumerate the copies of sars-cov-2 rna concentrated from wastewater collected from a wastewater treatment plant (327) . the calculated viral rna copy numbers determine the number of infected individuals. the increasing reports of virus shedding via the fecal route warrants the introduction of negative fecal viral nucleic acid test results as one of the additional discharge criteria in laboratory-confirmed cases of covid-19 (326) . the covid-19 pandemic does not have any novel factors, other than the genetically unique pathogen and a further possible reservoir. the cause and the likely future outcome are just repetitions of our previous interactions with fatal coronaviruses. the only difference is the time of occurrence and the genetic distinctness of the pathogen involved. mutations on the rbd of covs facilitated their capability of infecting newer hosts, thereby expanding their reach to all corners of the world (85) . this is a potential threat to the health of both animals and humans. advanced studies using bayesian phylogeographic reconstruction identified the most probable origin of sars-cov-2 as the bat sars-like coronavirus, circulating in the rhinolophus bat family (86) . phylogenetic analysis of 10 whole-genome sequences of sars-cov-2 showed that they are related to two covs of bat origin, namely, bat-sl-covzc45 and bat-sl-covzxc21, which were reported during 2018 in china (17) . it was reported that sars-cov-2 had been confirmed to use ace2 as an entry receptor while exhibiting an rbd similar to that of sars-cov (17, 87, 254, 255) . several countries have provided recommendations to their people traveling to china (88, 89) . compared to the previous coronavirus outbreaks caused by sars-cov and mers-cov, the efficiency of sars-cov-2 human-to-human transmission was thought to be less. this assumption was based on the finding that health workers were affected less than they were in previous outbreaks of fatal coronaviruses (2) . superspreading events are considered the main culprit for the extensive transmission of sars and mers (90, 91) . almost half of the mers-cov cases reported in saudi arabia are of secondary origin that occurred through contact with infected asymptomatic or symptomatic individuals through human-tohuman transmission (92) . the occurrence of superspreading events in the covid-19 outbreak cannot be ruled out until its possibility is evaluated. like sars and mers, covid-19 can also infect the lower respiratory tract, with milder symptoms (27) . the basic reproduction number of covid-19 has been found to be in the range of 2.8 to 3.3 based on real-time reports and 3.2 to 3.9 based on predicted infected cases (84) . coronavirus infection in humans is commonly associated with mild to severe respiratory diseases, with high fever, severe inflammation, cough, and internal organ dysfunction that can even lead to death (92) . most of the identified coronaviruses cause the common cold in humans. however, this changed when sars-cov was identified, paving the way for severe forms of the disease in humans (22) . our previous experience with the outbreaks of other coronaviruses, like sars and mers, suggests that the mode of transmission in covid-19 as mainly human-to-human transmission via direct contact, droplets, and fomites (25) . recent studies have demonstrated that the virus could remain viable for hours in aerosols and up to days on surfaces; thus, aerosol and fomite contamination could play potent roles in the transmission of sars-cov-2 (257) . the immune response against coronavirus is vital to control and get rid of the infection. however, maladjusted immune responses may contribute to the immunopathology of the disease, resulting in impairment of pulmonary gas exchange. understanding the interaction between covs and host innate immune systems could enlighten our understanding of the lung inflammation associated with this infection (24) . sars is a viral respiratory disease caused by a formerly unrecognized animal cov that originated from the wet markets in southern china after adapting to the human host, thereby enabling transmission between humans (90) . the sars outbreak reported in 2002 to 2003 had 8,098 confirmed cases with 774 total deaths (9.6%) (93) . the outbreak severely affected the asia pacific region, especially mainland china (94) . even though the case fatality rate (cfr) of sars-cov-2 (covid-19) is lower than that of sars-cov, there exists a severe concern linked to this outbreak due to its epidemiological similarity to influenza viruses (95, 279) . this can fail the public health system, resulting in a pandemic (96) . mers is another respiratory disease that was first reported in saudi arabia during the year 2012. the disease was found to have a cfr of around 35% (97) . the analysis of available data sets suggests that the incubation period of sars-cov-2, sars-cov, and mers-cov is in almost the same range. the longest predicted incubation time of sars-cov-2 is 14 days. hence, suspected individuals are isolated for 14 days to avoid the risk of further spread (98) . even though a high similarity has been reported between the genome sequence of the new coronavirus (sars-cov-2) and sars-like covs, the comparative analysis recognized a furin-like cleavage site in the sars-cov-2 s protein that is missing from other sars-like covs (99) . the furin-like cleavage site is expected to play a role in the life cycle of the virus and disease pathogenicity and might even act as a therapeutic target for furin inhibitors. the highly contagious nature of sars-cov-2 compared to that of its predecessors might be the result of a stabilizing mutation that occurred in the endosome-associated-protein-like domain of nsp2 protein. similarly, the destabilizing mutation near the phosphatase domain of nsp3 proteins in sars-cov-2 could indicate a potential mechanism that differentiates it from other covs (100) . even though the cfr reported for covid-19 is meager compared to those of the previous sars and mers outbreaks, it has caused more deaths than sars and mers combined (101) . possibly related to the viral pathogenesis is the recent finding of an 832-nucleotide (nt) deletion in orf8, which appears to reduce the replicative fitness of the virus and leads to attenuated phenotypes of sars-cov-2 (256) . coronavirus is the most prominent example of a virus that has crossed the species barrier twice from wild animals to humans during sars and mers outbreaks (79, 102) . the possibility of crossing the species barrier for the third time has also been suspected in the case of sars-cov-2 (covid19) . bats are recognized as a possible natural reservoir host of both sars-cov and mers-cov infection. in contrast, the possible intermediary host is the palm civet for sars-cov and the dromedary camel for mers-cov infection (102) . bats are considered the ancestral hosts for both sars and mers (103) . bats are also considered the reservoir host of human coronaviruses like clinical microbiology reviews hcov-229e and hcov-nl63 (104) . in the case of covid-19, there are two possibilities for primary transmission: it can be transmitted either through intermediate hosts, similar to that of sars and mers, or directly from bats (103) . the emergence paradigm put forward in the sars outbreak suggests that sars-cov originated from bats (reservoir host) and later jumped to civets (intermediate host) and incorporated changes within the receptor-binding domain (rbd) to improve binding to civet ace2. this civetadapted virus, during their subsequent exposure to humans at live markets, promoted further adaptations that resulted in the epidemic strain (104) . transmission can also occur directly from the reservoir host to humans without rbd adaptations. the bat coronavirus that is currently in circulation maintains specific "poised" spike proteins that facilitate human infection without the requirement of any mutations or adaptations (105) . altogether, different species of bats carry a massive number of coronaviruses around the world (106) . the high plasticity in receptor usage, along with the feasibility of adaptive mutation and recombination, may result in frequent interspecies transmission of coronavirus from bats to animals and humans (106) . the pathogenesis of most bat coronaviruses is unknown, as most of these viruses are not isolated and studied (4) . hedgehog coronavirus hku31, a betacoronavirus, has been identified from amur hedgehogs in china. studies show that hedgehogs are the reservoir of betacoronavirus, and there is evidence of recombination (107) . the current scientific evidence available on mers infection suggests that the significant reservoir host, as well as the animal source of mers infection in humans, is the dromedary camels (97) . the infected dromedary camels may not show any visible signs of infection, making it challenging to identify animals actively excreting mers-cov that has the potential to infect humans. however, they may shed mers-cov through milk, urine, feces, and nasal and eye discharge and can also be found in the raw organs (108) . in a study conducted to evaluate the susceptibility of animal species to mers-cov infection, llamas and pigs were found to be susceptible, indicating the possibility of mers-cov circulation in animal species other than dromedary camels (109) . following the outbreak of sars in china, sars-cov-like viruses were isolated from himalayan palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides) found in a live-animal market in guangdong, china. the animal isolates obtained from the live-animal market retained a 29-nucleotide sequence that was not present in most of the human isolates (78) . these findings were critical in identifying the possibility of interspecies transmission in sars-cov. the higher diversity and prevalence of bat coronaviruses in this region compared to those in previous reports indicate a host/ pathogen coevolution. sars-like coronaviruses also have been found circulating in the chinese horseshoe bat (rhinolophus sinicus) populations. the in vitro and in vivo studies carried out on the isolated virus confirmed that there is a potential risk for the reemergence of sars-cov infection from the viruses that are currently circulating in the bat population (105) . the disease caused by sars-cov-2 is also named severe specific contagious pneumonia (sscp), wuhan pneumonia, and, recently, covid-19 (110) . compared to sars-cov, sars-cov-2 has less severe pathogenesis but has superior transmission capability, as evidenced by the rapidly increasing number of covid-19 cases (111) . the incubation period of sars-cov-2 in familial clusters was found to be 3 to 6 days (112) . the mean incubation period of covid-19 was found to be 6.4 days, ranging from 2.1 to 11.1 days (113) . among an early affected group of 425 patients, 59 years was the median age, of which more males were affected (114) . similar to sars and mers, the severity of this ncov is high in age groups above 50 years (2, 115) . symptoms of covid-19 include fever, cough, myalgia or fatigue, and, less commonly, headache, hemoptysis, and diarrhea (116, 282) . compared to the sars-cov-2-infected patients in wuhan during the initial stages of the outbreak, only mild symptoms were noticed in those patients that are infected by human-to-human transmission (14) . the initial trends suggested that the mortality associated with covid-19 was less than that of previous outbreaks of sars (101) . the updates obtained from countries like china, japan, thailand, and south korea indicated that the covid-19 patients had relatively mild manifestations compared to those with sars and mers (4). regardless of the coronavirus type, immune cells, like mast cells, that are present in the submucosa of the respiratory tract and nasal cavity are considered the primary barrier against this virus (92) . advanced in-depth analysis of the genome has identified 380 amino acid substitutions between the amino acid sequences of sars-cov-2 and the sars/sarslike coronaviruses. these differences in the amino acid sequences might have contributed to the difference in the pathogenic divergence of sars-cov-2 (16) . further research is required to evaluate the possible differences in tropism, pathogenesis, and transmission of this novel agent associated with this change in the amino acid sequence. with the current outbreak of covid-19, there is an expectancy of a significant increase in the number of published studies about this emerging coronavirus, as occurred with sars and mers (117) . sars-cov-2 invades the lung parenchyma, resulting in severe interstitial inflammation of the lungs. this is evident on computed tomography (ct) images as ground-glass opacity in the lungs. this lesion initially involves a single lobe but later expands to multiple lung lobes (118) . the histological assessment of lung biopsy samples obtained from covid-19-infected patients revealed diffuse alveolar damage, cellular fibromyxoid exudates, hyaline membrane formation, and desquamation of pneumocytes, indicative of acute respiratory distress syndrome (119) . it was also found that the sars-cov-2infected patients often have lymphocytopenia with or without leukocyte abnormalities. the degree of lymphocytopenia gives an idea about disease prognosis, as it is found to be positively correlated with disease severity (118) . pregnant women are considered to have a higher risk of getting infected by covid-19. the coronaviruses can cause adverse outcomes for the fetus, such as intrauterine growth restriction, spontaneous abortion, preterm delivery, and perinatal death. nevertheless, the possibility of intrauterine maternal-fetal transmission (vertical transmission) of covs is low and was not seen during either the sars-or mers-cov outbreak (120) . however, there has been concern regarding the impact of sars-cov-2/covid-19 on pregnancy. researchers have mentioned the probability of in utero transmission of novel sars-cov-2 from covid-19-infected mothers to their neonates in china based upon the rise in igm and igg antibody levels and cytokine values in the blood obtained from newborn infants immediately postbirth; however, rt-pcr failed to confirm the presence of sars-cov-2 genetic material in the infants (283) . recent studies show that at least in some cases, preterm delivery and its consequences are associated with the virus. nonetheless, some cases have raised doubts for the likelihood of vertical transmission (240) (241) (242) (243) . covid-19 infection was associated with pneumonia, and some developed acute respiratory distress syndrome (ards). the blood biochemistry indexes, such as albumin, lactate dehydrogenase, c-reactive protein, lymphocytes (percent), and neutrophils (percent) give an idea about the disease severity in covid-19 infection (121) . during covid-19, patients may present leukocytosis, leukopenia with lymphopenia (244), hypoalbuminemia, and an increase of lactate dehydrogenase, aspartate transaminase, alanine aminotransferase, bilirubin, and, especially, d-dimer (244) . middle-aged and elderly patients with primary chronic diseases, especially high blood pressure and diabetes, were found to be more susceptible to respiratory failure and, therefore, had poorer prognoses. providing respiratory support at early stages improved the disease prognosis and facilitated recovery (18) . the ards in covid-19 is due to the occurrence of cytokine storms that results in exaggerated immune response, immune regulatory network imbalance, and, finally, multiple-organ failure (122) . in addition to the exaggerated inflammatory response seen in patients with covid-19 pneumonia, the bile duct epithelial cell-derived hepatocytes upregulate ace2 expression in liver tissue by compensatory proliferation that might result in hepatic tissue injury (123) . coronavirus can cause disease in several species of domestic and wild animals, as well as humans (23) . the different animal species that are infected with cov include horses, camels, cattle, swine, dogs, cats, rodents, birds, ferrets, minks, bats, rabbits, snakes, and various other wild animals (20, 30, 79, 93, 124, 125, 287) . coronavirus infection is linked to different kinds of clinical manifestations, varying from enteritis in cows and pigs, upper respiratory disease in chickens, and fatal respiratory infections in humans (30) . among the cov genera, alphacoronavirus and betacoronavirus infect mammals, while gammacoronavirus and deltacoronavirus mainly infect birds, fishes, and, sometimes, mammals (27, 29, 106) . several novel coronaviruses that come under the genus deltacoronavirus have been discovered in the past from birds, like wigeon coronavirus hku20, bulbul coronavirus hku11, munia coronavirus hku13, white-eye coronavirus hku16, night-heron coronavirus hku19, and common moorhen coronavirus hku21, as well as from pigs (porcine coronavirus hku15) (6, 29) . transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and porcine hemagglutinating encephalomyelitis virus (phev) are some of the coronaviruses of swine. among them, tgev and pedv are responsible for causing severe gastroenteritis in young piglets with noteworthy morbidity and mortality. infection with phev also causes enteric infection but can cause encephalitis due to its ability to infect the nervous system (30) . bovine coronaviruses (bocovs) are known to infect several domestic and wild ruminants (126) . bocov inflicts neonatal calf diarrhea in adult cattle, leading to bloody diarrhea (winter dysentery) and respiratory disease complex (shipping fever) in cattle of all age groups (126) . bocov-like viruses have been noted in humans, suggesting its zoonotic potential as well (127) . feline enteric and feline infectious peritonitis (fip) viruses are the two major feline covs (128) , where feline covs can affect the gastrointestinal tract, abdominal cavity (peritonitis), respiratory tract, and central nervous system (128) . canines are also affected by covs that fall under different genera, namely, canine enteric coronavirus in alphacoronavirus and canine respiratory coronavirus in betacoronavirus, affecting the enteric and respiratory tract, respectively (129, 130) . ibv, under gammacoronavirus, causes diseases of respiratory, urinary, and reproductive systems, with substantial economic losses in chickens (131, 132) . in small laboratory animals, mouse hepatitis virus, rat sialodacryoadenitis coronavirus, and guinea pig and rabbit coronaviruses are the major covs associated with disease manifestations like enteritis, hepatitis, and respiratory infections (10, 133) . swine acute diarrhea syndrome coronavirus (sads-cov) was first identified in suckling piglets having severe enteritis and belongs to the genus alphacoronavirus (106) . the outbreak was associated with considerable scale mortality of piglets (24,693 deaths) across four farms in china (134) . the virus isolated from the piglets was almost identical to and had 95% genomic similarity with horseshoe bat (rhinolophus species) coronavirus hku2, suggesting a bat origin of the pig virus (106, 134, 135) . it is also imperative to note that the sads-cov outbreak started in guangdong province, near the location of the sars pandemic origin (134) . before this outbreak, pigs were not known to be infected with bat-origin coronaviruses. this indicates that the bat-origin coronavirus jumped to pig by breaking the species barrier. the next step of this jump might not end well, since pigs are considered the mixing vessel for influenza a viruses due to their ability to be infected by both human and avian influenza a viruses (136) . similarly, they may act as the mixing vessel for coronaviruses, since they are in frequent contact with both humans and multiple wildlife species. additionally, pigs are also found to be susceptible to infection with human sars-cov and mers-cov, making this scenario a nightmare (109, 137) . it is only a matter of time before another zoonotic coronavirus results in an epidemic by jumping the so-called species barrier (287) . the host spectrum of coronavirus increased when a novel coronavirus, namely, sw1, was recognized in the liver tissue of a captive beluga whale (delphinapterus leucas) (138) . in recent decades, several novel coronaviruses were identified from different animal species. bats can harbor these viruses without manifesting any clinical disease but are persistently infected (30) . they are the only mammals with the capacity for self-powered flight, which enables them to migrate long distances, unlike land mammals. bats are distributed worldwide and also account for about a fifth of all mammalian species (6) . this makes them the ideal reservoir host for many viral agents and also the source of novel coronaviruses that have yet to be identified. it has become a necessity to study the diversity of coronavirus in the bat population to prevent future outbreaks that could jeopardize livestock and public health. the repeated outbreaks caused by bat-origin coronaviruses calls for the development of efficient molecular surveillance strategies for studying betacoronavirus among animals (12) , especially in the rhinolophus bat family (86) . chinese bats have high commercial value, since they are used in traditional chinese medicine (tcm). therefore, the handling of bats for trading purposes poses a considerable risk of transmitting zoonotic cov epidemics (139) . due to the possible role played by farm and wild animals in sars-cov-2 infection, the who, in their novel coronavirus (covid-19) situation report, recommended the avoidance of unprotected contact with both farm and wild animals (25) . the live-animal markets, like the one in guangdong, china, provides a setting for animal coronaviruses to amplify and to be transmitted to new hosts, like humans (78) . such markets can be considered a critical place for the origin of novel zoonotic diseases and have enormous public health significance in the event of an outbreak. bats are the reservoirs for several viruses; hence, the role of bats in the present outbreak cannot be ruled out (140) . in a qualitative study conducted for evaluating the zoonotic risk factors among rural communities of southern china, the frequent human-animal interactions along with the low levels of environmental biosecurity were identified as significant risks for the emergence of zoonotic disease in local communities (141, 142) . the comprehensive sequence analysis of the sars-cov-2 rna genome identified that the cov from wuhan is a recombinant virus of the bat coronavirus and another coronavirus of unknown origin. the recombination was found to have happened within the viral spike glycoprotein, which recognizes the cell surface receptor. further analysis of the genome based on codon usage identified the snake as the most probable animal reservoir of sars-cov-2 (143) . contrary to these findings, another genome analysis proposed that the genome of sars-cov-2 is 96% identical to bat coronavirus, reflecting its origin from bats (63) . the involvement of bat-derived materials in causing the current outbreak cannot be ruled out. high risk is involved in the production of bat-derived materials for tcm practices involving the handling of wild bats. the use of bats for tcm practices will remain a severe risk for the occurrence of zoonotic coronavirus epidemics in the future (139) . furthermore, the pangolins are an endangered species of animals that harbor a wide variety of viruses, including coronaviruses (144) . the coronavirus isolated from malayan pangolins (manis javanica) showed a very high amino acid identity with covid-19 at e (100%), m (98.2%), n (96.7%), and s genes (90.4%). the rbd of s protein in cov isolated from pangolin was almost identical (one amino acid difference) to that of sars-cov-2. a comparison of the genomes suggests recombination between pangolin-cov-like viruses with the bat-cov-ratg13-like virus. all this suggests the potential of pangolins to act as the intermediate host of sars-cov-2 (145) . human-wildlife interactions, which are increasing in the context of climate change (142) , are further considered high risk and responsible for the emergence of sars-cov. covid-19 is also suspected of having a similar mode of origin. hence, to prevent the occurrence of another zoonotic spillover (1), exhaustive coordinated efforts are needed to identify the high-risk pathogens harbored by wild animal populations, conducting surveillance among the people who are susceptible to zoonotic spillover events (12) , and to improve the biosecurity measures associated with the wildlife trade (146) . the serological surveillance studies conducted in people living in proximity to bat caves had earlier identified the serological confirmation of sars-related covs in humans. people clinical microbiology reviews living at the wildlife-human interface, mainly in rural china, are regularly exposed to sars-related covs (147) . these findings will not have any significance until a significant outbreak occurs due to a virus-like sars-cov-2. there is a steady increase in the reports of covid-19 in companion and wild animals around the world. further studies are required to evaluate the potential of animals (especially companion animals) to serve as an efficient reservoir host that can further alter the dynamics of human-to-human transmission (330) . to date, two pet dogs (hong kong) and four pet cats (one each from belgium and hong kong, two from the united states) have tested positive for sars-cov-2 (335) . the world organization for animal health (oie) has confirmed the diagnosis of covid-19 in both dogs and cats due to human-to-animal transmission (331) . the similarity observed in the gene sequence of sars-cov-2 from an infected pet owner and his dog further confirms the occurrence of human-to-animal transmission (333) . even though asymptomatic, feline species should be considered a potential transmission route from animals to humans (326) . however, currently, there are no reports of sars-cov-2 transmission from felines to human beings. based on the current evidence, we can conclude that cats are susceptible to sars-cov-2 and can get infected by human beings. however, evidence of cat-to-human transmission is lacking and requires further studies (332) . rather than waiting for firmer evidence on animal-to-human transmission, necessary preventive measures are advised, as well as following social distancing practices among companion animals of different households (331) . one of the leading veterinary diagnostic companies, idexx, has conducted large-scale testing for covid-19 in specimens collected from dogs and cats. however, none of the tests turned out to be positive (334) . in a study conducted to investigate the potential of different animal species to act as the intermediate host of sars-cov-2, it was found that both ferrets and cats can be infected via experimental inoculation of the virus. in addition, infected cats efficiently transmitted the disease to naive cats (329) . sars-cov-2 infection and subsequent transmission in ferrets were found to recapitulate the clinical aspects of covid-19 in humans. the infected ferrets also shed virus via multiple routes, such as saliva, nasal washes, feces, and urine, postinfection, making them an ideal animal model for studying disease transmission (337) . experimental inoculation was also done in other animal species and found that the dogs have low susceptibility, while the chickens, ducks, and pigs are not at all susceptible to sars-cov-2 (329) . similarly, the national veterinary services laboratories of the usda have reported covid-19 in tigers and lions that exhibited respiratory signs like dry cough and wheezing. the zoo animals are suspected to have been infected by an asymptomatic zookeeper (335) . the total number of covid-19-positive cases in human beings is increasing at a high rate, thereby creating ideal conditions for viral spillover to other species, such as pigs. the evidence obtained from sars-cov suggests that pigs can get infected with sars-cov-2 (336). however, experimental inoculation with sars-cov-2 failed to infect pigs (329) . further studies are required to identify the possible animal reservoirs of sars-cov-2 and the seasonal variation in the circulation of these viruses in the animal population. research collaboration between human and animal health sectors is becoming a necessity to evaluate and identify the possible risk factors of transmission between animals and humans. such cooperation will help to devise efficient strategies for the management of emerging zoonotic diseases (12) . rna tests can confirm the diagnosis of sars-cov-2 (covid-19) cases with real-time rt-pcr or next-generation sequencing (148, 149, 245, 246) . at present, nucleic acid detection techniques, like rt-pcr, are considered an effective method for confirming the diagnosis in clinical cases of covid-19 (148) . several companies across the world are currently focusing on developing and marketing sars-cov-2-specific nucleic acid detection kits. multiple laboratories are also developing their own in-house rt-pcr. one of them is the sars-cov-2 nucleic acid detection kit produced by shuoshi biotechnology (double fluorescence pcr method) (150) . up to 30 march 2020, the u.s. food and drug administration (fda) had granted 22 in vitro diagnostics emergency use authorizations (euas), including for the rt-pcr diagnostic panel for the universal detection of sars-like betacoronaviruses and specific detection of sars-cov-2, developed by the u.s. cdc (table 1) (258, 259) . recently, 95 full-length genomic sequences of saras-cov-2 strains available in the national center for biotechnology information and gisaid databases were subjected to multiple-sequence alignment and phylogenetic analyses for studying variations in the viral genome (260) . all the viral strains revealed high homology of 99.99% (99.91% to 100%) at the nucleotide level and 99.99% (99.79% to 100%) at the amino acid level. overall variation was found to be low in orf regions, with 13 variation sites recognized in 1a, 1b, s, 3a, m, 8, and n regions. mutation rates of 30.53% (29/95) and 29.47% (28/95) were observed at nt 28144 (orf8) and nt 8782 (orf1a) positions, respectively. owing to such selective mutations, a few specific regions of sars-cov-2 should not be considered for designing primers and probes. the sars-cov-2 reference sequence could pave the way to study molecular biology and pathobiology, along with developing diagnostics and appropriate prevention and control strategies for countering sars-cov-2 (260) . nucleic acids of sars-cov-2 can be detected from samples (64) such as bronchoalveolar lavage fluid, sputum, nasal swabs, fiber bronchoscope brush biopsy specimen, pharyngeal swabs, feces, blood, and urine, with different levels of diagnostic performance (table 2) (80, 245, 246) . the viral loads of sars-cov-2 were measured using n-gene-specific quantitative rt-pcr in throat swab and sputum samples collected from covid-19-infected individuals. the results indicated that the viral load peaked at around 5 to 6 days following the onset of symptoms, and it ranged from 10 4 to 10 7 copies/ml during this time (151) . in another study, the viral load was found to be higher in the nasal swabs than the throat swabs obtained from covid-19 symptomatic patients (82) . although initially it was thought that viral load would be associated with poor outcomes, some case reports have shown asymptomatic individuals with high viral loads (247) . recently, the viral load in nasal and throat swabs of 17 symptomatic patients was determined, and higher viral loads were recorded soon after the onset of symptoms, particularly in the nose compared to the throat. the pattern of viral nucleic the results of the studies related to sars-cov-2 viral loads reflect active replication of this virus in the upper respiratory tract and prolonged viral shedding after symptoms disappear, including via stool. thus, the current case definition needs to be updated along with a reassessment of the strategies to be adopted for restraining the sars-cov-2 outbreak spread (248) . in some cases, the viral load studies of sars-cov-2 have also been useful to recommend precautionary measures when handling specific samples, e.g., feces. in a recent survey from 17 confirmed cases of sars-cov-2 infection with available data (representing days 0 to 13 after onset), stool samples from nine cases (53%; days 0 to 11 after onset) were positive on rt-pcr analysis. although the viral loads were lower than those of respiratory samples (range, 550 copies per ml to 1.21 ϫ 10 5 copies per ml), this has essential biosafety implications (151) . the samples from 18 sars-cov-2-positive patients in singapore who had traveled from wuhan to singapore showed the presence of viral rna in stool and whole blood but not in urine by real-time rt-pcr (288) . further, novel sars-cov-2 infections have been detected in a variety of clinical specimens, like bronchoalveolar lavage fluid, sputum, nasal swabs, fibrobronchoscope brush biopsy specimens, pharyngeal swabs, feces, and blood (246) . the presence of sars-cov-2 in fecal samples has posed grave public health concerns. in addition to the direct transmission mainly occurring via droplets of sneezing and coughing, other routes, such as fecal excretion and environmental and fomite contamination, are contributing to sars-cov-2 transmission and spread (249) (250) (251) (252) . fecal excretion has also been documented for sars-cov and mers-cov, along with the potential to stay viable in situations aiding fecal-oral transmission. thus, sars-cov-2 has every possibility to be transmitted through this mode. fecal-oral transmission of sars-cov-2, particularly in regions having low standards of hygiene and poor sanitation, may have grave consequences with regard to the high spread of this virus. ethanol and disinfectants containing chlorine or bleach are effective against coronaviruses (249) (250) (251) (252) . appropriate precautions need to be followed strictly while handling the stools of patients infected with sars-cov-2. biowaste materials and sewage from hospitals must be adequately disinfected, treated, and disposed of properly. the significance of frequent and good hand hygiene and sanitation practices needs to be given due emphasis (249) (250) (251) (252) . future explorative research needs to be conducted with regard to the fecal-oral transmission of sars-cov-2, along with focusing on environmental investigations to find out if this virus could stay viable in situations and atmospheres facilitating such potent routes of transmission. the correlation of fecal concentrations of viral rna with disease severity needs to be determined, along with assessing the gastrointestinal symptoms and the possibility of fecal sars-cov-2 rna detection during the covid-19 incubation period or convalescence phases of the disease (249) (250) (251) (252) . the lower respiratory tract sampling techniques, like bronchoalveolar lavage fluid aspirate, are considered the ideal clinical materials, rather than the throat swab, due to their higher positive rate on the nucleic acid test (148) . the diagnosis of covid-19 can be made by using upper-respiratory-tract specimens collected using nasopharyngeal and oropharyngeal swabs. however, these techniques are associated with unnecessary risks to health care workers due to close contact with patients (152) . similarly, a single patient with a high viral load was reported to contaminate an entire endoscopy room by shedding the virus, which may remain viable for at least 3 days and is considered a great risk for uninfected patients and health care workers (289) . recently, it was found that the anal swabs gave more positive results than oral swabs in the later stages of infection (153) . hence, clinicians have to be cautious while discharging any covid-19infected patient based on negative oral swab test results due to the possibility of fecal-oral transmission. even though the viral loads in stool samples were found to be less than those of respiratory samples, strict precautionary measures have to be followed while handling stool samples of covid-19 suspected or infected patients (151) . children infected with sars-cov-2 experience only a mild form of illness and recover immediately after treatment. it was recently found that stool samples of sars-cov-2-infected children that gave negative throat swab results were positive within ten days of negative results. this could result in the fecal-oral transmission of sars-cov-2 infections, especially in children (290) . hence, to prevent the fecal-oral transmission of sars-cov-2, infected covid-19 patients should only be considered negative when they test negative for sars-cov-2 in the stool sample. a suspected case of covid-19 infection is said to be confirmed if the respiratory tract aspirate or blood samples test positive for sars-cov-2 nucleic acid using rt-pcr or by the identification of sars-cov-2 genetic sequence in respiratory tract aspirate or blood samples (80) . the patient will be confirmed as cured when two subsequent oral swab results are negative (153) . recently, the live virus was detected in the selfcollected saliva of patients infected with covid-19. these findings were confirmative of using saliva as a noninvasive specimen for the diagnosis of covid-19 infection in suspected individuals (152) . it has also been observed that the initial screening of covid-19 patients infected with rt-pcr may give negative results even if they have chest ct findings that are suggestive of infection. hence, for the accurate diagnosis of covid-19, a combination of repeated swab tests using rt-pcr and ct scanning is required to prevent the possibility of false-negative results during disease screening (154) . rt-pcr is the most widely used test for diagnosing covid-19. however, it has some significant limitations from the clinical perspective, since it will not give any clarity regarding disease progression. droplet digital pcr (ddpcr) can be used for the quantification of viral load in the samples obtained from lower respiratory tracts. hence, based on the viral load, we can quickly evaluate the progression of infection (291) . in addition to all of the above findings, sequencing and phylogenetics are critical in the correct identification and confirmation of the causative viral agent and useful to establish relationships with previous isolates and sequences, as well as to know, especially during an epidemic, the nucleotide and amino acid mutations and the molecular divergence. the rapid development and implementation of diagnostic tests against emerging novel diseases like covid-19 pose significant challenges due to the lack of resources and logistical limitations associated with an outbreak (155) . sars-cov-2 infection can also be confirmed by isolation and culturing. the human airway epithelial cell culture was found to be useful in isolating sars-cov-2 (3). the efficient control of an outbreak depends on the rapid diagnosis of the disease. recently, in response to the covid-19 outbreak, 1-step quantitative realtime reverse transcription-pcr assays were developed that detect the orf1b and n regions of the sars-cov-2 genome (156) . that assay was found to achieve the rapid detection of sars-cov-2. nucleic acid-based assays offer high accuracy in the diagnosis of sars-cov-2, but the current rate of spread limits its use due to the lack of diagnostic assay kits. this will further result in the extensive transmission of covid-19, since only a portion of suspected cases can be diagnosed. in such situations, conventional serological assays, like enzyme-linked immunosorbent assay (elisa), that are specific to covid-19 igm and igg antibodies can be used as a high-throughput alternative (149) . at present, there is no diagnostic kit available for detecting the sars-cov-2 antibody (150) . the specific antibody profiles of covid-19 patients were analyzed, and it was found that the igm level lasted more than 1 month, indicating a prolonged stage of virus replication in sars-cov-2-infected patients. the igg levels were found to increase only in the later stages of the disease. these findings indicate that the specific antibody profiles of sars-cov-2 and sars-cov were similar (325) . these findings can be utilized for the development of specific diagnostic tests against covid-19 and can be used for rapid screening. even though diagnostic test kits are already available that can detect the genetic sequences of sars-cov-2 (95), their availability is a concern, as the number of covid-19 cases is skyrocketing (155, 157) . a major problem associated with this diagnostic kit is that it works only when the test subject has an active infection, limiting its use to the earlier stages of infection. several laboratories around the world are currently developing antibody-based diagnostic tests against sars-cov-2 (157). chest ct is an ideal diagnostic tool for identifying viral pneumonia. the sensitivity of chest ct is far superior to that of x-ray screening. the chest ct findings associated with covid-19-infected patients include characteristic patchy infiltration that later progresses to ground-glass opacities (158) . early manifestations of covid-19 pneumonia might not be evident in x-ray chest radiography. in such situations, a chest ct examination can be performed, as it is considered highly specific for covid-19 pneumonia (118) . those patients having covid-19 pneumonia will exhibit the typical ground-glass opacity in their chest ct images (154) . the patients infected with covid-19 had elevated plasma angiotensin 2 levels. the level of angiotensin 2 was found to be linearly associated with viral load and lung injury, indicating its potential as a diagnostic biomarker (121) . the chest ct imaging abnormalities associated with covid-19 pneumonia have also been observed even in asymptomatic patients. these abnormalities progress from the initial focal unilateral to diffuse bilateral ground-glass opacities and will further progress to or coexist with lung consolidation changes within 1 to 3 weeks (159). the role played by radiologists in the current scenario is very important. radiologists can help in the early diagnosis of lung abnormalities associated with covid-19 pneumonia. they can also help in the evaluation of disease severity, identifying its progression to acute respiratory distress syndrome and the presence of secondary bacterial infections (160) . even though chest ct is considered an essential diagnostic tool for covid-19, the extensive use of ct for screening purposes in the suspected individuals might be associated with a disproportionate risk-benefit ratio due to increased radiation exposure as well as increased risk of cross-infection. hence, the use of ct for early diagnosis of sars-cov-2 infection in high-risk groups should be done with great caution (292) . more recently, other advanced diagnostics have been designed and developed for the detection of sars-cov-2 (345, 347, (350) (351) (352) . a reverse transcriptional loopmediated isothermal amplification (rt-lamp), namely, ilaco, has been developed for rapid and colorimetric detection of this virus (354) . rt-lamp serves as a simple, rapid, and sensitive diagnostic method that does not require sophisticated equipment or skilled personnel (349) . an interactive web-based dashboard for tracking sars-cov-2 in a real-time mode has been designed (238) . a smartphone-integrated home-based point-of-care testing (poct) tool, a paper-based poct combined with lamp, is a useful point-of-care diagnostic (353) . an abbott id now covid-19 molecular poct-based test, using isothermal nucleic acid amplification technology, has been designed as a pointof-care test for very rapid detection of sars-cov-2 in just 5 min (344) . a crispr-based sherlock (specific high-sensitivity enzymatic reporter unlocking) diagnostic for rapid detection of sars-cov-2 without the requirement of specialized instrumentation has been reported to be very useful in the clinical diagnosis of covid-19 (360) . a crispr-cas12-based lateral flow assay also has been developed for rapid detection of sars-cov-2 (346) . artificial intelligence, by means of a three-dimensional deep-learning model, has been developed for sensitive and specific diagnosis of covid-19 via ct images (332) . tracking and mapping of the rising incidence rates, disease outbreaks, community spread, clustered transmission events, hot spots, and superspreader potential of sars-cov-2/covid warrant full exploitation of real-time disease mapping by employing geographical information systems (gis), such as the gis software kosmo 3.1, web-based real-time tools and dashboards, apps, and advances in information technology (356-359). researchers have also developed a few prediction tools/models, such as the prediction model risk of bias assessment tool (probast) and critical appraisal and data extraction for systematic reviews of prediction modeling studies (charms), which could aid in assessing the possibility of getting infection and estimating the prognosis in patients; however, such models may suffer from bias issues and, hence, cannot be considered completely trustworthy, which necessitates the development of new and reliable predictors (360) . recently emerged viruses, such as zika, ebola, and nipah viruses, and their grave threats to humans have begun a race in exploring the designing and developing of advanced vaccines, prophylactics, therapeutics, and drug regimens to counter emerging viruses (161) (162) (163) 280) . several attempts are being made to design and develop vaccines for cov infection, mostly by targeting the spike glycoprotein. nevertheless, owing to extensive diversity in antigenic variants, cross-protection rendered by the vaccines is significantly limited, even within the strains of a phylogenetic subcluster (104) . due to the lack of effective antiviral therapy and vaccines in the present scenario, we need to depend solely on implementing effective infection control measures to lessen the risk of possible nosocomial transmission (68) . recently, the receptor for sars-cov-2 was established as the human angiotensin-converting enzyme 2 (hace2), and the virus was found to enter the host cell mainly through endocytosis. it was also found that the major components that have a critical role in viral entry include pikfyve, tpc2, and cathepsin l. these findings are critical, since the components described above might act as candidates for vaccines or therapeutic drugs against sars-cov-2 (293) . the majority of the treatment options and strategies that are being evaluated for sars-cov-2 (covid-19) have been taken from our previous experiences in treating sars-cov, mers-cov, and other emerging viral diseases. several therapeutic and preventive strategies, including vaccines, immunotherapeutics, and antiviral drugs, have been exploited against the previous cov outbreaks (sars-cov and mers-cov) (8, 104, (164) (165) (166) (167) . these valuable options have already been evaluated for their potency, efficacy, and safety, along with several other types of current research that will fuel our search for ideal therapeutic agents against covid-19 (7, 9, 19, 21, 36) . the primary cause of the unavailability of approved and commercial vaccines, drugs, and therapeutics to counter the earlier sars-cov and mers-cov seems to owe to the lesser attention of the biomedicine and pharmaceutical companies, as these two covs did not cause much havoc, global threat, and panic like those posed by the sars-cov-2 pandemic (19) . moreover, for such outbreak situations, the requirement for vaccines and therapeutics/drugs exists only for a limited period, until the outbreak is controlled. the proportion of the human population infected with sars-cov and mers-cov was also much lower across the globe, failing to attract drug and vaccine manufacturers and clinical microbiology reviews producers. therefore, by the time an effective drug or vaccine is designed against such disease outbreaks, the virus would have been controlled by adopting appropriate and strict prevention and control measures, and patients for clinical trials will not be available. the newly developed drugs cannot be marketed due to the lack of end users. the s protein plays a significant role in the induction of protective immunity against sars-cov by mediating t-cell responses and neutralizing antibody production (168) . in the past few decades, we have seen several attempts to develop a vaccine against human coronaviruses by using s protein as the target (168, 169) . however, the developed vaccines have minimal application, even among closely related strains of the virus, due to a lack of cross-protection. that is mainly because of the extensive diversity existing among the different antigenic variants of the virus (104) . the contributions of the structural proteins, like spike (s), matrix (m), small envelope (e), and nucleocapsid (n) proteins, of sars-cov to induce protective immunity has been evaluated by expressing them in a recombinant parainfluenza virus type 3 vector (bhpiv3). of note, the result was conclusive that the expression of m, e, or n proteins without the presence of s protein would not confer any noticeable protection, with the absence of detectable serum sars-cov-neutralizing antibodies (170) . antigenic determinant sites present over s and n structural proteins of sars-cov-2 can be explored as suitable vaccine candidates (294) . in the asian population, s, e, m, and n proteins of sars-cov-2 are being targeted for developing subunit vaccines against covid-19 (295) . the identification of the immunodominant region among the subunits and domains of s protein is critical for developing an effective vaccine against the coronavirus. the c-terminal domain of the s1 subunit is considered the immunodominant region of the porcine deltacoronavirus s protein (171) . similarly, further investigations are needed to determine the immunodominant regions of sars-cov-2 for facilitating vaccine development. however, our previous attempts to develop a universal vaccine that is effective for both sars-cov and mers-cov based on t-cell epitope similarity pointed out the possibility of cross-reactivity among coronaviruses (172) . that can be made possible by selected potential vaccine targets that are common to both viruses. sars-cov-2 has been reported to be closely related to sars-cov (173, 174) . hence, knowledge and understanding of s protein-based vaccine development in sars-cov will help to identify potential s protein vaccine candidates in sars-cov-2. therefore, vaccine strategies based on the whole s protein, s protein subunits, or specific potential epitopes of s protein appear to be the most promising vaccine candidates against coronaviruses. the rbd of the s1 subunit of s protein has a superior capacity to induce neutralizing antibodies. this property of the rbd can be utilized for designing potential sars-cov vaccines either by using rbd-containing recombinant proteins or recombinant vectors that encode rbd (175) . hence, the superior genetic similarity existing between sars-cov-2 and sars-cov can be utilized to repurpose vaccines that have proven in vitro efficacy against sars-cov to be utilized for sars-cov-2. the possibility of cross-protection in covid-19 was evaluated by comparing the s protein sequences of sars-cov-2 with that of sars-cov. the comparative analysis confirmed that the variable residues were found concentrated on the s1 subunit of s protein, an important vaccine target of the virus (150) . hence, the possibility of sars-cov-specific neutralizing antibodies providing cross-protection to covid-19 might be lower. further genetic analysis is required between sars-cov-2 and different strains of sars-cov and sarslike (sl) covs to evaluate the possibility of repurposed vaccines against covid-19. this strategy will be helpful in the scenario of an outbreak, since much time can be saved, because preliminary evaluation, including in vitro studies, already would be completed for such vaccine candidates. multiepitope subunit vaccines can be considered a promising preventive strategy against the ongoing covid-19 pandemic. in silico and advanced immunoinformatic tools can be used to develop multiepitope subunit vaccines. the vaccines that are engineered by this technique can be further evaluated using docking studies and, if found effective, then can be further evaluated in animal models (365) . identifying epitopes that have the potential to become a vaccine candidate is critical to developing an effective vaccine against covid-19. the immunoinformatics approach has been used for recognizing essential epitopes of cytotoxic t lymphocytes and b cells from the surface glycoprotein of sars-cov-2. recently, a few epitopes have been recognized from the sars-cov-2 surface glycoprotein. the selected epitopes explored targeting molecular dynamic simulations, evaluating their interaction with corresponding major histocompatibility complex class i molecules. they potentially induce immune responses (176) . the recombinant vaccine can be designed by using rabies virus (rv) as a viral vector. rv can be made to express mers-cov s1 protein on its surface so that an immune response is induced against mers-cov. the rv vector-based vaccines against mers-cov can induce faster antibody response as well as higher degrees of cellular immunity than the gram-positive enhancer matrix (gem) particle vector-based vaccine. however, the latter can induce a very high antibody response at lower doses (167) . hence, the degree of humoral and cellular immune responses produced by such vaccines depends upon the vector used. dual vaccines have been getting more popular recently. among them, the rabies virus-based vectored vaccine platform is used to develop vaccines against emerging infectious diseases. the dual vaccine developed from inactivated rabies virus particles that express the mers-cov s1 domain of s protein was found to induce immune responses for both mers-cov and rabies virus. the vaccinated mice were found to be completely protected from challenge with mers-cov (169) . the intranasal administration of the recombinant adenovirus-based vaccine in balb/c mice was found to induce long-lasting neutralizing immunity against mers spike pseudotyped virus, characterized by the induction of systemic igg, secretory iga, and lung-resident memory t-cell responses (177) . immunoinformatics methods have been employed for the genomewide screening of potential vaccine targets among the different immunogens of mers-cov (178) . the n protein and the potential b-cell epitopes of mers-cov e protein have been suggested as immunoprotective targets inducing both t-cell and neutralizing antibody responses (178, 179) . the collaborative effort of the researchers of rocky mountain laboratories and oxford university is designing a chimpanzee adenovirus-vectored vaccine to counter covid-19 (180) . the coalition for epidemic preparedness innovations (cepi) has initiated three programs to design sars-cov-2 vaccines (181) . cepi has a collaborative project with inovio for designing a mers-cov dna vaccine that could potentiate effective immunity. cepi and the university of queensland are designing a molecular clamp vaccine platform for mers-cov and other pathogens, which could assist in the easier identification of antigens by the immune system (181) . cepi has also funded moderna to develop a vaccine for covid-19 in partnership with the vaccine research center (vrc) of the national institute of allergy and infectious diseases (niaid), part of the national institutes of health (nih) (182) . by employing mrna vaccine platform technology, a vaccine candidate expressing sars-cov-2 spike protein is likely to go through clinical testing in the coming months (180 (298) . the process of vaccine development usually takes approximately ten years, in the case of inactivated or live attenuated vaccines, since it involves the generation of long-term efficacy data. however, this was brought down to 5 years during the ebola emergency for viral vector vaccines. in the urgency associated with the covid-19 outbreaks, we expect a vaccine by the end of this year (343) . the development of an effective vaccine against covid-19 with high speed and precision is the combined result of advancements in computational biology, gene synthesis, protein engineering, and the invention of advanced manufacturing platforms (342) . the recurring nature of the coronavirus outbreaks calls for the development of a pan-coronavirus vaccine that can produce cross-reactive antibodies. however, the success of such a vaccine relies greatly on its ability to provide protection not only against present versions of the virus but also the ones that are likely to emerge in the future. this can be achieved by identifying antibodies that can recognize relatively conserved epitopes that are maintained as such even after the occurrence of considerable variations (362) . even though several vaccine clinical trials are being conducted around the world, pregnant women have been completely excluded from these studies. pregnant women are highly vulnerable to emerging diseases such as covid-19 due to alterations in the immune system and other physiological systems that are associated with pregnancy. therefore, in the event of successful vaccine development, pregnant women will not get access to the vaccines (361) . hence, it is recommended that pregnant women be included in the ongoing vaccine trials, since successful vaccination in pregnancy will protect the mother, fetus, and newborn. the heterologous immune effects induced by bacillus calmette guérin (bcg) vaccination is a promising strategy for controlling the covid-19 pandemic and requires further investigations. bcg is a widely used vaccine against tuberculosis in high-risk regions. it is derived from a live attenuated strain of mycobacterium bovis. at present, three new clinical trials have been registered to evaluate the protective role of bcg vaccination against sars-cov-2 (363) . recently, a cohort study was conducted to evaluate the impact of childhood bcg vaccination in covid-19 pcr positivity rates. however, childhood bcg vaccination was found to be associated with a rate of covid-19-positive test results similar to that of the nonvaccinated group (364) . further studies are required to analyze whether bcg vaccination in childhood can induce protective effects against covid-19 in adulthood. population genetic studies conducted on 103 genomes identified that the sars-cov-2 virus has evolved into two major types, l and s. among the two types, l type is expected to be the most prevalent (ϳ70%), followed by the s type (ϳ30%) (366) . this finding has a significant impact on our race to develop an ideal vaccine, since the vaccine candidate has to target both strains to be considered effective. at present, the genetic differences between the l and s types are very small and may not affect the immune response. however, we can expect further genetic variations in the coming days that could lead to the emergence of new strains (367) . there is no currently licensed specific antiviral treatment for mers-and sars-cov infections, and the main focus in clinical settings remains on lessening clinical signs and providing supportive care (183) (184) (185) (186) . effective drugs to manage covid-19 patients include remdesivir, lopinavir/ritonavir alone or in a blend with interferon beta, convalescent plasma, and monoclonal antibodies (mabs); however, efficacy and safety issues of these drugs require additional clinical trials (187, 281) . a controlled trial of ritonavirboosted lopinavir and interferon alpha 2b treatment was performed on covid-19 hospitalized patients (chictr2000029308) (188) . in addition, the use of hydroxychloroquine and tocilizumab for their potential role in modulating inflammatory responses in the lungs and antiviral effect has been proposed and discussed in many research articles. still, no fool-proof clinical trials have been published (194, 196, 197, (261) (262) (263) (264) (265) (266) (267) (268) (269) (270) (271) (272) . recently, a clinical trial conducted on adult patients suffering from severe covid-19 revealed no benefit of lopinavir-ritonavir treatment over standard care (273) . the efforts to control sars-cov-2 infection utilize defined strategies as followed against mers and sars, along with adopting and strengthening a few precautionary measures owing to the unknown nature of this novel virus (36, 189) . presently, the main course of treatment for severely affected sars-cov-2 patients admitted to hospitals includes mechanical ventilation, intensive care unit (icu) admittance, and symptomatic and supportive therapies. additionally, rna synthesis inhibitors (lamivudine and tenofovir disoproxil fumarate), remdesivir, neuraminidase inhibitors, peptide (ek1), antiinflammatory drugs, abidol, and chinese traditional medicine (lianhuaqingwen and shufengjiedu capsules) could aid in covid-19 treatment. however, further clinical trials are being carried out concerning their safety and efficacy (7) . it might require months to a year(s) to design and develop effective drugs, therapeutics, and vaccines against covid-19, with adequate evaluation and approval from regulatory bodies and moving to the bulk production of many millions of doses at commercial levels to meet the timely demand of mass populations across the globe (9) . continuous efforts are also warranted to identify and assess viable drugs and immunotherapeutic regimens that revealed proven potency in combating other viral agents similar to sars-cov-2. covid-19 patients showing severe signs are treated symptomatically along with oxygen therapy. in such cases where the patients progress toward respiratory failure and become refractory to oxygen therapy, mechanical ventilation is necessitated. the covid-19-induced septic shock can be managed by providing adequate hemodynamic support (299) . several classes of drugs are currently being evaluated for their potential therapeutic action against sars-cov-2. therapeutic agents that have anti-sars-cov-2 activity can be broadly classified into three categories: drugs that block virus entry into the host cell, drugs that block viral replication as well as its survival within the host cell, and drugs that attenuate the exaggerated host immune response (300) . an inflammatory cytokine storm is commonly seen in critically ill covid-19 patients. hence, they may benefit from the use of timely anti-inflammation treatment. anti-inflammatory therapy using drugs like glucocorticoids, cytokine inhibitors, jak inhibitors, and chloroquine/hydroxychloroquine should be done only after analyzing the risk/benefit ratio in covid-19 patients (301). there have not been any studies concerning the application of nonsteroidal anti-inflammatory drugs (nsaid) to covid-19-infected patients. however, reasonable pieces of evidence are available that link nsaid uses with the occurrence of respiratory and cardiovascular adverse effects. hence, as a cautionary approach, it is better to recommend the use of nsaids as the first-line option for managing covid-19 symptoms (302) . the use of corticosteroids in covid-19 patients is still a matter of controversy and requires further systematic clinical studies. the guidelines that were put forward to manage critically ill adults suggest the use of systemic corticosteroids in mechanically ventilated adults with ards (303) . the generalized use of corticosteroids is not indicated in covid-19, since there are some concerns associated with the use of corticosteroids in viral pneumonia. stem cell therapy using mesenchymal stem cells (mscs) is another hopeful strategy that can be used in clinical cases of covid-19 owing to its potential immunomodulatory capacity. it may have a beneficial role in attenuating the cytokine storm that is observed in severe cases of sars-cov-2 infection, thereby reducing mortality. among the different types of mscs, expanded umbilical cord mscs can be considered a potential therapeutic agent that requires further validation for managing critically ill covid-19 patients (304) . repurposed broad-spectrum antiviral drugs having proven uses against other viral pathogens can be employed for sars-cov-2-infected patients. these possess benefits of easy accessibility and recognized pharmacokinetic and pharmacodynamic activities, stability, doses, and side effects (9) . repurposed drugs have been studied for treating cov infections, like lopinavir/ritonavir, and interferon-1␤ revealed in vitro anti-mers-cov action. the in vivo experiment carried out in the nonhuman primate model of clinical microbiology reviews common marmosets treated with lopinavir/ritonavir and interferon beta showed superior protective results in treated animals than in the untreated ones (190) . a combination of these drugs is being evaluated to treat mers in humans (miracle trial) (191) . these two protease inhibitors (lopinavir and ritonavir), in combination with ribavirin, gave encouraging clinical outcomes in sars patients, suggesting their therapeutic values (165) . however, in the current scenario, due to the lack of specific therapeutic agents against sars-cov-2, hospitalized patients confirmed for the disease are given supportive care, like oxygen and fluid therapy, along with antibiotic therapy for managing secondary bacterial infections (192) . patients with novel coronavirus or covid-19 pneumonia who are mechanically ventilated often require sedatives, analgesics, and even muscle relaxation drugs to prevent ventilator-related lung injury associated with human-machine incoordination (122) . the result obtained from a clinical study of four patients infected with covid-19 claimed that combination therapy using lopinavir/ritonavir, arbidol, and shufeng jiedu capsules (traditional chinese medicine) was found to be effective in managing covid-19 pneumonia (193) . it is difficult to evaluate the therapeutic potential of a drug or a combination of drugs for managing a disease based on such a limited sample size. before choosing the ideal therapeutic agent for the management of covid-19, randomized clinical control studies should be performed with a sufficient study population. several classes of routinely used antiviral drugs, like oseltamivir (neuraminidase inhibitor), acyclovir, ganciclovir, and ribavirin, do not have any effect on covid-19 and, hence, are not recommended (187) . oseltamivir, a neuraminidase inhibitor, has been explored in chinese hospitals for treating suspected covid-19 cases, although proven efficacy against sars-cov-2 is still lacking for this drug (7) . the in vitro antiviral potential of fad-approved drugs, viz., ribavirin, penciclovir, nitazoxanide, nafamostat, and chloroquine, tested in comparison to remdesivir and favipiravir (broad-spectrum antiviral drugs) revealed remdesivir and chloroquine to be highly effective against sars-cov-2 infection in vitro (194) . ribavirin, penciclovir, and favipiravir might not possess noteworthy in vivo antiviral actions for sars-cov-2, since higher concentrations of these nucleoside analogs are needed in vitro to lessen the viral infection. both remdesivir and chloroquine are being used in humans to treat other diseases, and such safer drugs can be explored for assessing their effectiveness in covid-19 patients. several therapeutic agents, such as lopinavir/ritonavir, chloroquine, and hydroxychloroquine, have been proposed for the clinical management of covid-19 (299) . a molecular docking study, conducted in the rna-dependent rna polymerase (rdrp) of sars-cov-2 using different commercially available antipolymerase drugs, identified that drugs such as ribavirin, remdesivir, galidesivir, tenofovir, and sofosbuvir bind rdrp tightly, indicating their vast potential to be used against covid-19 (305). a broadspectrum antiviral drug that was developed in the united states, tilorone dihydrochloride (tilorone), was previously found to possess potent antiviral activity against mers, marburg, ebola, and chikungunya viruses (306) . even though it had broad-spectrum activity, it was neglected for an extended period. tilorone is another antiviral drug that might have activity against sars-cov-2. remdesivir, a novel nucleotide analog prodrug, was developed for treating ebola virus disease (evd), and it was also found to inhibit the replication of sars-cov and mers-cov in primary human airway epithelial cell culture systems (195) . recently, in vitro study has proven that remdesivir has better antiviral activity than lopinavir and ritonavir. further, in vivo studies conducted in mice also identified that treatment with remdesivir improved pulmonary function and reduced viral loads and lung pathology both in prophylactic and therapeutic regimens compared to lopinavir/ritonavir-ifn-␥ treatment in mers-cov infection (8) . remdesivir also inhibits a diverse range of coronaviruses, including circulating human cov, zoonotic bat cov, and prepandemic zoonotic cov (195) . remdesivir is also considered the only therapeutic drug that significantly reduces pulmonary pathology (8) . all these findings indicate that remde-sivir has to be further evaluated for its efficacy in the treatment of covid-19 infection in humans. the broad-spectrum activity exhibited by remdesivir will help control the spread of disease in the event of a new coronavirus outbreak. chloroquine is an antimalarial drug known to possess antiviral activity due to its ability to block virus-cell fusion by raising the endosomal ph necessary for fusion. it also interferes with virus-receptor binding by interfering with the terminal glycosylation of sars-cov cellular receptors, such as ace2 (196) . in a recent multicenter clinical trial that was conducted in china, chloroquine phosphate was found to exhibit both efficacy and safety in the therapeutic management of sars-cov-2-associated pneumonia (197) . this drug is already included in the treatment guidelines issued by the national health commission of the people's republic of china. the preliminary clinical trials using hydroxychloroquine, another aminoquinoline drug, gave promising results. the covid-19 patients received 600 mg of hydroxychloroquine daily along with azithromycin as a single-arm protocol. this protocol was found to be associated with a noteworthy reduction in viral load. finally, it resulted in a complete cure (271) ; however, the study comprised a small population and, hence, the possibility of misinterpretation could arise. however, in another case study, the authors raised concerns over the efficacy of hydroxychloroquine-azithromycin in the treatment of covid-19 patients, since no observable effect was seen when they were used. in some cases, the treatment was discontinued due to the prolongation of the qt interval (307) . hence, further randomized clinical trials are required before concluding this matter. recently, another fda-approved drug, ivermectin, was reported to inhibit the in vitro replication of sars-cov-2. the findings from this study indicate that a single treatment of this drug was able to induce an ϳ5,000-fold reduction in the viral rna at 48 h in cell culture. (308) . one of the main disadvantages that limit the clinical utility of ivermectin is its potential to cause cytotoxicity. however, altering the vehicles used in the formulations, the pharmacokinetic properties can be modified, thereby having significant control over the systemic concentration of ivermectin (338) . based on the pharmacokinetic simulation, it was also found that ivermectin may have limited therapeutic utility in managing covid-19, since the inhibitory concentration that has to be achieved for effective anti-sars-cov-2 activity is far higher than the maximum plasma concentration achieved by administering the approved dose (340) . however, ivermectin, being a host-directed agent, exhibits antiviral activity by targeting a critical cellular process of the mammalian cell. therefore, the administration of ivermectin, even at lower doses, will reduce the viral load at a minor level. this slight decrease will provide a great advantage to the immune system for mounting a large-scale antiviral response against sars-cov-2 (341). further, a combination of ivermectin and hydroxychloroquine might have a synergistic effect, since ivermectin reduces viral replication, while hydroxychloroquine inhibits the entry of the virus in the host cell (339) . further, in vivo studies and randomized clinical control trials are required to understand the mechanism as well as the clinical utility of this promising drug. nafamostat is a potent inhibitor of mers-cov that acts by preventing membrane fusion. nevertheless, it does not have any sort of inhibitory action against sars-cov-2 infection (194) . recently, several newly synthesized halogenated triazole compounds were evaluated, using fluorescence resonance energy transfer (fret)-based helicase assays, for their ability to inhibit helicase activity. among the evaluated compounds, 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-iodophenyl) hydrazinyl]-4h-1,2,4-triazole-3-thiol and 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-chlorophenyl) hydrazinyl]-4h-1,2,4-triazole-3-thiol were found to be the most potent. these compounds were used for in silico studies, and molecular docking was accomplished into the active binding site of mers-cov helicase nsp13 (21) . further studies are required for evaluating the therapeutic potential of these newly identified compounds in the management of covid-19 infection. monoclonal antibodies (mabs) may be helpful in the intervention of disease in clinical microbiology reviews cov-exposed individuals. patients recovering from sars showed robust neutralizing antibodies against this cov infection (164) . a set of mabs aimed at the mers-cov s protein-specific domains, comprising six specific epitope groups interacting with receptor-binding, membrane fusion, and sialic acid-binding sites, make up crucial entry tasks of s protein (198, 199) . passive immunization employing weaker and strongly neutralizing antibodies provided considerable protection in mice against a mers-cov lethal challenge. such antibodies may play a crucial role in enhancing protective humoral responses against the emerging covs by aiming appropriate epitopes and functions of the s protein. the cross-neutralization ability of sars-cov rbd-specific neutralizing mabs considerably relies on the resemblance between their rbds; therefore, sars-cov rbd-specific antibodies could cross-neutralized sl covs, i.e., bat-sl-cov strain wiv1 (rbd with eight amino acid differences from sars-cov) but not bat-sl-cov strain shc014 (24 amino acid differences) (200) . appropriate rbd-specific mabs can be recognized by a relative analysis of rbd of sars-cov-2 to that of sars-cov, and cross-neutralizing sars-cov rbd-specific mabs could be explored for their effectiveness against covid-19 and further need to be assessed clinically. the u.s. biotechnology company regeneron is attempting to recognize potent and specific mabs to combat covid-19. an ideal therapeutic option suggested for sars-cov-2 (covid-19) is the combination therapy comprised of mabs and the drug remdesivir (covid-19) (201) . the sars-cov-specific human mab cr3022 is found to bind with sars-cov-2 rbd, indicating its potential as a therapeutic agent in the management of covid-19. it can be used alone or in combination with other effective neutralizing antibodies for the treatment and prevention of covid-19 (202) . furthermore, sars-cov-specific neutralizing antibodies, like m396 and cr3014, failed to bind the s protein of sars-cov-2, indicating that a particular level of similarity is mandatory between the rbds of sars-cov and sars-cov-2 for the cross-reactivity to occur. further assessment is necessary before confirming the effectiveness of such combination therapy. in addition, to prevent further community and nosocomial spread of covid-19, the postprocedure risk management program should not be neglected (309) . development of broad-spectrum inhibitors against the human coronaviral pathogens will help to facilitate clinical trials on the effectiveness of such inhibitors against endemic and emerging coronaviruses (203) . a promising animal study revealed the protective effect of passive immunotherapy with immune serum from mers-immune camels on mice infected with mers-cov (204) . passive immunotherapy using convalescent plasma is another strategy that can be used for treating covid-19-infected, critically ill patients (205) . the exploration of fully human antibodies (human single-chain antibodies; huscfvs) or humanized nanobodies (single-domain antibodies; sdab, vh/vhh) could aid in blocking virus replication, as these agents can traverse the virus-infected cell membranes (transbodies) and can interfere with the biological characteristics of the replicating virus proteins. such examples include transbodies to the influenza virus, hepatitis c virus, ebola virus, and dengue virus (206) . producing similar transbodies against intracellular proteins of coronaviruses, such as papain-like proteases (plpro), cysteinelike protease (3clpro), or other nsps, which are essential for replication and transcription of the virus, might formulate a practical move forward for a safer and potent passive immunization approach for virus-exposed persons and rendering therapy to infected patients. in a case study on five grimly sick patients having symptoms of severe pneumonia due to covid-19, convalescent plasma administration was found to be helpful in patients recovering successfully. the convalescent plasma containing a sars-cov-2specific elisa (serum) antibody titer higher than 1:1,000 and neutralizing antibody titer more significant than 40 was collected from the recovered patients and used for plasma transfusion twice in a volume of 200 to 250 ml on the day of collection (310) . at present, treatment for sepsis and ards mainly involves antimicrobial therapy, source control, and supportive care. hence, the use of therapeutic plasma exchange can be considered an option in managing such severe conditions. further randomized trials can be designed to investigate its efficacy (311) . potent therapeutics to combat sars-cov-2 infection include virus binding molecules, molecules or inhibitors targeting particular enzymes implicated in replication and transcription process of the virus, helicase inhibitors, vital viral proteases and proteins, protease inhibitors of host cells, endocytosis inhibitors, short interfering rna (sirna), neutralizing antibodies, mabs against the host receptor, mabs interfering with the s1 rbd, antiviral peptide aimed at s2, and natural drugs/medicines (7, 166, 186) . the s protein acts as the critical target for developing cov antivirals, like inhibitors of s protein and s cleavage, neutralizing antibodies, rbd-ace2 blockers, sirnas, blockers of the fusion core, and proteases (168) . all of these therapeutic approaches have revealed both in vitro and in vivo anti-cov potential. although in vitro research carried out with these therapeutics showed efficacy, most need appropriate support from randomized animal or human trials. therefore, they might be of limited applicability and require trials against sars-cov-2 to gain practical usefulness. the binding of sars-cov-2 with ace2 leads to the exacerbation of pneumonia as a consequence of the imbalance in the reninangiotensin system (ras). the virus-induced pulmonary inflammatory responses may be reduced by the administration of ace inhibitors (acei) and angiotensin type-1 receptor (at1r) (207) . several investigations have suggested the use of small-molecule inhibitors for the potential control of sars-cov infections. drugs of the fda-approved compound library were screened to identify four small-molecule inhibitors of mers-cov (chlorpromazine, chloroquine, loperamide, and lopinavir) that inhibited viral replication. these compounds also hinder sars-cov and human covs (208) . therapeutic strategies involving the use of specific antibodies or compounds that neutralize cytokines and their receptors will help to restrain the host inflammatory responses. such drugs acting specifically in the respiratory tract will help to reduce virus-triggered immune pathologies in covid-19 (209) . the later stages of coronavirus-induced inflammatory cascades are characterized by the release of proinflammatory interleukin-1 (il-1) family members, such as il-1 and il-33. hence, there exists a possibility that the inflammation associated with coronavirus can be inhibited by utilizing anti-inflammatory cytokines that belong to the il-1 family (92) . it has also been suggested that the actin protein is the host factor that is involved in cell entry and pathogenesis of sars-cov-2. hence, those drugs that modulate the biological activity of this protein, like ibuprofen, might have some therapeutic application in managing the disease (174). the plasma angiotensin 2 level was found to be markedly elevated in covid-19 infection and was correlated with viral load and lung injury. hence, drugs that block angiotensin receptors may have potential for treating covid-19 infection (121) . a scientist from germany, named rolf hilgenfeld, has been working on the identification of drugs for the treatment of coronaviral infection since the time of the first sars outbreak (19) . the sars-cov s2 subunit has a significant function in mediating virus fusion that provides entry into the host cell. heptad repeat 1 (hr1) and heptad repeat 2 (hr2) can interact and form a six-helix bundle that brings the viral and cellular membranes in close proximity, facilitating its fusion. the sequence alignment study conducted between covid-19 and sars-cov identified that the s2 subunits are highly conserved in these covs. the hr1 and hr2 domains showed 92.6% and 100% overall identity, respectively (210) . from these findings, we can confirm the significance of covid-19 hr1 and hr2 and their vital role in host cell entry. hence, fusion inhibitors target the hr1 domain of s protein, thereby preventing viral fusion and entry into the host cell. this is another potential therapeutic strategy that can be used in the management of covid-19. other than the specific therapy directed against covid-19, general treatments play a vital role in the enhancement of host immune responses against the viral agent. inadequate nutrition is linked to the weakening of the host immune response, clinical microbiology reviews making the individual more susceptible. the role played by nutrition in disease susceptibility should be measured by evaluating the nutritional status of patients with covid-19 (205) . for evaluating the potential of vaccines and therapeutics against covs, including sars-cov, mers-covs, and the presently emerging sars-cov-2, suitable animal models that can mimic the clinical disease are needed (211, 212) . various animal models were assessed for sars-and mers-covs, such as mice, guinea pigs, golden syrian hamsters, ferrets, rabbits, nonhuman primates like rhesus macaques and marmosets, and cats (185, (213) (214) (215) (216) (217) (218) . the specificity of the virus to hace2 (receptor of sars-cov) was found to be a significant barrier in developing animal models. consequently, a sars-cov transgenic mouse model has been developed by inserting the hace2 gene into the mouse genome (219) . the inability of mers-cov to replicate in the respiratory tracts of animals (mice, hamsters, and ferrets) is another limiting factor. however, with genetic engineering, a 288-330 ϩ/ϩ mers-cov genetically modified mouse model was developed and now is in use for the assessment of novel drugs and vaccines against mers-cov (220). in the past, small animals (mice or hamsters) have been targeted for being closer to a humanized structure, such as mouse dpp4 altered with human dpp4 (hdpp4), hdpp4-transduced mice, and hdpp4-tg mice (transgenic for expressing hdpp4) for mers-cov infection (221) . the crispr-cas9 gene-editing tool has been used for inserting genomic alterations in mice, making them susceptible to mers-cov infection (222) . efforts are under way to recognize suitable animal models for sars-cov2/covid-19, identify the receptor affinity of this virus, study pathology in experimental animal models, and explore virus-specific immune responses and protection studies, which together would increase the pace of efforts being made for developing potent vaccines and drugs to counter this emerging virus. cell lines, such as monkey epithelial cell lines (llc-mk2 and vero-b4), goat lung cells, alpaca kidney cells, dromedary umbilical cord cells, and advanced ex vivo three-dimensional tracheobronchial tissue, have been explored to study human covs (mers-cov) (223, 224) . vero and huh-7 cells (human liver cancer cells) have been used for isolating sars-cov-2 (194) . recently, an experimental study with rhesus monkeys as animal models revealed the absence of any viral loads in nasopharyngeal and anal swabs, and no viral replication was recorded in the primary tissues at a time interval of 5 days post-reinfection in reexposed monkeys (274) . the subsequent virological, radiological, and pathological observations indicated that the monkeys with reexposure had no recurrence of covid-19, like the sars-cov-2-infected monkeys without rechallenge. these findings suggest that primary infection with sars-cov-2 could protect from later exposures to the virus, which could help in defining disease prognosis and crucial inferences for designing and developing potent vaccines against covid-19 (274). in contrast to their response to the 2002 sars outbreak, china has shown immense political openness in reporting the covid-19 outbreak promptly. they have also performed rapid sequencing of covid-19 at multiple levels and shared the findings globally within days of identifying the novel virus (225) . the move made by china opened a new chapter in global health security and diplomacy. even though complete lockdown was declared following the covid-19 outbreak in wuhan, the large-scale movement of people has resulted in a radiating spread of infections in the surrounding provinces as well as to several other countries. large-scale screening programs might help us to control the spread of this virus. however, this is both challenging as well as time-consuming due to the present extent of infection (226) . the current scenario demands effective implementation of vigorous prevention and control strategies owing to the prospect of covid-19 for nosocomial infections (68) . follow-ups of infected patients by telephone on day 7 and day 14 are advised to avoid any further unintentional spread or nosocomial transmission (312) . the availability of public data sets provided by independent analytical teams will act as robust evidence that would guide us in designing interventions against the covid-19 outbreak. newspaper reports and social media can be used to analyze and reconstruct the progression of an outbreak. they can help us to obtain detailed patient-level data in the early stages of an outbreak (227) . immediate travel restrictions imposed by several countries might have contributed significantly to preventing the spread of sars-cov-2 globally (89, 228) . following the outbreak, a temporary ban was imposed on the wildlife trade, keeping in mind the possible role played by wild animal species in the origin of sars-cov-2/covid-19 (147) . making a permanent and bold decision on the trade of wild animal species is necessary to prevent the possibility of virus spread and initiation of an outbreak due to zoonotic spillover (1) . personal protective equipment (ppe), like face masks, will help to prevent the spread of respiratory infections like covid-19. face masks not only protect from infectious aerosols but also prevent the transmission of disease to other susceptible individuals while traveling through public transport systems (313) . another critical practice that can reduce the transmission of respiratory diseases is the maintenance of hand hygiene. however, the efficacy of this practice in reducing the transmission of respiratory viruses like sars-cov-2 is much dependent upon the size of droplets produced. hand hygiene will reduce disease transmission only if the virus is transmitted through the formation of large droplets (314) . hence, it is better not to overemphasize that hand hygiene will prevent the transmission of sars-cov-2, since it may produce a false sense of safety among the general public that further contributes to the spread of covid-19. even though airborne spread has not been reported in sars-cov-2 infection, transmission can occur through droplets and fomites, especially when there is close, unprotected contact between infected and susceptible individuals. hence, hand hygiene is equally as important as the use of appropriate ppe, like face masks, to break the transmission cycle of the virus; both hand hygiene and face masks help to lessen the risk of covid-19 transmission (315) . medical staff are in the group of individuals most at risk of getting covid-19 infection. this is because they are exposed directly to infected patients. hence, proper training must be given to all hospital staff on methods of prevention and protection so that they become competent enough to protect themselves and others from this deadly disease (316) . as a preventive measure, health care workers caring for infected patients should take extreme precautions against both contact and airborne transmission. they should use ppe such as face masks (n95 or ffp3), eye protection (goggles), gowns, and gloves to nullify the risk of infection (299) . the human-to-human transmission reported in sars-cov-2 infection occurs mainly through droplet or direct contact. due to this finding, frontline health care workers should follow stringent infection control and preventive measures, such as the use of ppe, to prevent infection (110) . the mental health of the medical/health workers who are involved in the covid-19 outbreak is of great importance, because the strain on their mental well-being will affect their attention, concentration, and decision-making capacity. hence, for control of the covid-19 outbreak, rapid steps should be taken to protect the mental health of medical workers (229) . since the living mammals sold in the wet market are suspected to be the intermediate host of sars-cov-2, there is a need for strengthening the regulatory mechanism for wild animal trade (13) . the total number of covid-19 confirmed cases is on a continuous rise and the cure rate is relatively low, making disease control very difficult to achieve. the chinese government is making continuous efforts to contain the disease by taking emergency control and prevention measures. they have already built a hospital for patients affected by this virus and are currently building several more for accommodating the continuously increasing infected population (230) . the effective control of sars-cov-2/covid-19 requires high-level interventions like intensive contact tracing, as well as the quarantine of people with suspected infection and the isolation of infected individuals. the implementation of rigorous control and preventive measures together might control the r 0 number and reduce the transmission risk (228) . clinical microbiology reviews considering the zoonotic links associated with sars-cov-2, the one health approach may play a vital role in the prevention and control measures being followed to restrain this pandemic virus (317) (318) (319) . the substantial importation of covid-19 presymptomatic cases from wuhan has resulted in independent, self-sustaining outbreaks across major cities both within the country and across the globe. the majority of chinese cities are now facing localized outbreaks of covid-19 (231) . hence, deploying efficient public health interventions might help to cut the spread of this virus globally. the occurrence of covid-19 infection on several cruise ships gave us a preliminary idea regarding the transmission pattern of the disease. cruise ships act as a closed environment and provide an ideal setting for the occurrence of respiratory disease outbreaks. such a situation poses a significant threat to travelers, since people from different countries are on board, which favors the introduction of the pathogen (320). although nearly 30 cruise ships from different countries have been found harboring covid-19 infection, the major cruise ships that were involved in the covid-19 outbreaks are the diamond princess, grand princess, celebrity apex, and ruby princess. the number of confirmed covid-19 cases around the world is on the rise. the success of preventive measures put forward by every country is mainly dependent upon their ability to anticipate the approaching waves of patients. this will help to properly prepare the health care workers and increase the intensive care unit (icu) capacity (321) . instead of entirely relying on lockdown protocols, countries should focus mainly on alternative intervention strategies, such as large-scale testing, contract tracing, and localized quarantine of suspected cases for limiting the spread of this pandemic virus. such intervention strategies will be useful either at the beginning of the pandemic or after lockdown relaxation (322) . lockdown should be imposed only to slow down disease progression among the population so that the health care system is not overloaded. the reproduction number (r 0 ) of covid-19 infection was earlier estimated to be in the range of 1.4 to 2.5 (70); recently, it was estimated to be 2.24 to 3.58 (76) . compared to its coronavirus predecessors, covid-19 has an r 0 value that is greater than that of mers (r 0 ͻ 1) (108) but less than that of sars (r 0 value of 2 to 5) (93) . still, to prevent further spread of disease at mass gatherings, functions remain canceled in the affected cities, and persons are asked to work from home (232) . hence, it is a relief that the current outbreak of covid-19 infection can be brought under control with the adoption of strategic preventive and control measures along with the early isolation of subsequent cases in the coming days. studies also report that since air traffic between china and african countries increased many times over in the decade after the sars outbreak, african countries need to be vigilant to prevent the spread of novel coronavirus in africa (225) . due to fear of virus spread, wuhan city was completely shut down (233) . the immediate control of the ongoing covid-19 outbreaks appears a mammoth task, especially for developing countries, due to their inability to allocate quarantine stations that could screen infected individuals' movements (234) . such underdeveloped countries should divert their resources and energy to enforcing the primary level of preventive measures, like controlling the entry of individuals from china or countries where the disease has flared up, isolating the infected individuals, and quarantining individuals with suspected infection. most of the sub-saharan african countries have a fragile health system that can be crippled in the event of an outbreak. effective management of covid-19 would be difficult for low-income countries due to their inability to respond rapidly due to the lack of an efficient health care system (65) . controlling the imported cases is critical in preventing the spread of covid-19 to other countries that have not reported the disease until now. the possibility of an imported case of covid-19 leading to sustained human-to-human transmission was estimated to be 0.41. this can be reduced to a value of 0.012 by decreasing the mean time from the onset of symptoms to hospitalization and can only be made possible by using intense disease surveillance systems (235) . the silent importations of infected individuals (before the manifestation of clinical signs) also contributed significantly to the spread of disease across the major cities of the world. even though the travel ban was implemented in wuhan (89) , infected persons who traveled out of the city just before the imposition of the ban might have remained undetected and resulted in local outbreaks (236) . emerging novel diseases like covid-19 are difficult to contain within the country of origin, since globalization has led to a world without borders. hence, international collaboration plays a vital role in preventing the further spread of this virus across the globe (237) . we also predict the possibility of another outbreak, as predicted by fan et al. (6) . indeed, the present outbreak caused by sars-cov-2 (covid-19) was expected. similar to previous outbreaks, the current outbreak also will be contained shortly. however, the real issue is how we are planning to counter the next zoonotic cov epidemic that is likely to occur within the next 5 to 10 years or even sooner (fig. 7) . several years after the global sars epidemic, the current sars-cov-2/covid-19 pandemic has served as a reminder of how novel pathogens can rapidly emerge and spread through the human population and eventually cause severe public health crises. further research should be conducted to establish animal models for sars-cov-2 to investigate replication, transmission dynamics, and pathogenesis in humans. this may help develop and evaluate potential therapeutic strategies against zoonotic cov epidemics. present trends suggest the occurrence of future outbreaks of covs due to changes in the climate, and ecological conditions may be associated with humananimal contact. live-animal markets, such as the huanan south china seafood market, represent ideal conditions for interspecies contact of wildlife with domestic birds, pigs, and mammals, which substantially increases the probability of interspecies transmission of cov infections and could result in high risks to humans due to adaptive genetic recombination in these viruses (323) (324) (325) . the covid-19-associated symptoms are fever, cough, expectoration, headache, and myalgia or fatigue. individuals with asymptomatic and atypical clinical manifestations were also identified recently, further adding to the complexity of disease transmission dynamics. atypical clinical manifestations may only express symptoms such as fatigue instead of respiratory signs such as fever, cough, and sputum. in such cases, the clinician must be vigilant for the possible occurrence of asymptomatic and atypical clinical manifestations to avoid the possibility of missed diagnoses. the present outbreak caused by sars-cov-2 was, indeed, expected. similar to clinical microbiology reviews previous outbreaks, the current pandemic also will be contained shortly. however, the real question is, how are we planning to counter the next zoonotic cov epidemic that is likely to occur within the next 5 to 10 years or perhaps sooner? our knowledge of most of the bat covs is scarce, as these viruses have not been isolated and studied, and extensive studies on such viruses are typically only conducted when they are associated with specific disease outbreaks. the next step following the control of the covid-19 outbreak in china should be focused on screening, identification, isolation, and characterization of covs present in wildlife species of china, particularly in bats. both in vitro and in vivo studies (using suitable animal models) should be conducted to evaluate the risk of future epidemics. presently, licensed antiviral drugs or vaccines against sars-cov, mers-cov, and sars-cov-2 are lacking. however, 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origin and continuing evolution of sars-cov-2 covid-19: the race for a vaccine all authors substantially contributed to the conception, design, analysis, and interpretation of data and checking and approving the final version of the manuscript, and we agree to be accountable for its contents.this compilation is a review article written, analyzed, and designed by its authors and required no substantial funding to be developed.all authors declare that there are no existing commercial or financial relationships that could, in any way, lead to a potential conflict of interest. with 25 years of research and teaching experience in the areas of microbiology, immunology, virology, public health, medicine, and biomedicine as an eminent researcher, he has developed several diagnostics, vaccines, immunomodulatory modules, and hypotheses to counter infectious diseases of animals, poultry, and public health concerns. he has to his credit 600 publications, 6 books, and 65 book chapters. dr. dhama has been recognized as an extremely productive researcher in the journal nature. he has been honored with 50 best paper awards and other recognitions. he is an naas (national academy of agricultural science, india) associate and has worked as nodal officer, wto, and member, wildlife health specialist group (iucn). he is actively serving as editor-in-chief, co-eic, editor, and member, editorial board, of nearly 20 scientific journals. his google scholar h-index is 47 and scopus h-index is 31. sharun khan, m.v.sc., is currently working as a researcher in the stem cell laboratory, division of surgery, icar-indian veterinary research institute, izatnagar, india. his area of interest is regenerative medicine with a focus on understanding cell biology and molecular pathways involved in the maintenance and differentiation of stem cells originating from different tissues. he has particular interest and knowledge in the fields of veterinary medicine, pharmacology, infectious diseases of animals, wildlife diseases, diagnosis and therapy of animal diseases, nutrition, and biomedicine. with excellent academic records, he has received awards and recognitions (fellowships and scholarships) and participated in national and international workshops, training programs, and courses. he has a keen interest in learning excellent scientific writing skills and has published 30 papers, including in international journals of repute. he is highly enthusiastic about gaining knowledge of advancements in educational and scientific research areas.ruchi tiwari is currently working as assistant professor in the department of veterinary microbiology, college of veterinary sciences, duvasu, mathura, india. she is currently pursuing her ph.d. (hons) degree from duvasu. with an excellent academic record and 10 years of research and teaching experience, she has expertise in the field of diagnosis, prevention, and control of important livestock/poultry diseases/pathogens having public health significance, along with particular reference to veterinary microbiology, immunology, ethnoveterinary medicine, alternative and complementary therapies, and bacteriophage therapy. dr. tiwari has published 150 research/review articles and 5 book chapters. she has been honored with the young scientist award, best paper awards (10) key: cord-315288-fcx4q6mp authors: hussain, mohammed hassan; siddiqui, saad; mahmood, sara; valsamakis, theodoros title: tracheal swab from front of neck airway for sars-cov-2; a bronchial foreign body date: 2020-08-27 journal: bmj case rep doi: 10.1136/bcr-2020-237787 sha: doc_id: 315288 cord_uid: fcx4q6mp we report the case of a bronchial foreign body, following a tracheostomy site swab for sars-cov-2, aiming to raise awareness and vigilance. a qualified nurse was performing a routine sars-cov-2 swab on a 51-year-old woman, fitted with a tracheostomy in the recent past following a craniotomy. this was part of the discharging protocol to a nursing home. during the sampling, part of the swab stylet snapped and was inadvertently dropped through the tracheostomy site. initial ct imaging was reported as showing no signs of a foreign body but some inflammatory changes. bedside flexible endoscopy through the tracheostomy site revealed the swab in a right lobar bronchus. this was subsequently removed by flexible bronchoscopy. this case highlights the need for clear guidance on how samples for sars-cov-2 are taken from patients with front of neck airways (laryngectomy/tracheοstomy) and the potential pitfalls involved. we report the case of a bronchial foreign body, following a tracheostomy site swab for sars-cov-2, aiming to raise awareness and vigilance. a qualified nurse was performing a routine sars-cov-2 swab on a 51-year-old woman, fitted with a tracheostomy in the recent past following a craniotomy. this was part of the discharging protocol to a nursing home. during the sampling, part of the swab stylet snapped and was inadvertently dropped through the tracheostomy site. initial ct imaging was reported as showing no signs of a foreign body but some inflammatory changes. bedside flexible endoscopy through the tracheostomy site revealed the swab in a right lobar bronchus. this was subsequently removed by flexible bronchoscopy. this case highlights the need for clear guidance on how samples for sars-cov-2 are taken from patients with front of neck airways (laryngectomy/tracheοstomy) and the potential pitfalls involved. the novel covid-19, sars-cov-2, is currently a pandemic. while in most cases, only a mild illness ensues, severe disease can be complicated by acute respiratory distress syndrome, septic shock, cardiac injury and death. 1 the risk of spread of this virus has led to stringent measures being implemented both in hospitals and society in general. at our university, all admissions are being swabbed for sars-cov-2. real-time reverse transcriptase pcr (rrt-pcr) of a combined oropharyngeal and nasopharyngeal swab is used to confirm diagnosis. patients with front of neck airways, either in the form of a laryngectomy or tracheostomy stoma site, present a challenge in terms of testing for sars-cov-2. there is no current clear guidance on how these patients should be tested. in addition, questions remain around whether the exclusion of the upper airway in laryngectomy patients affects the sensitivity of rrt-pcr testing in nasopharyngeal and oropharyngeal swabs. the potential pitfalls of taking a swab from a tracheostomy site are highlighted clearly by this case. a 51-year-old woman presented with temporal lobe thrombosis complicated by haemorrhagic transformation. this required neurosurgical intervention in the form of a craniotomy and evacuation of haematoma and she was transferred to our institute with a tracheostomy tube in situ. prior to discharge to a nursing home, a swab was taken to test for sars-cov-2, as per protocol prior to transfer. a mucosal swab was attempted through the trachesotomy tube. during this process, the nurse felt the swab stylet snap with the distal end falling into the trachea, although she was not certain. the patient became momentarily unsettled with her oxygen requirements increasing to 4 l/min from 2 l/min. she quickly returned to her normal, with her oxygen saturation levels maintained at baseline levels of 88%-92% on 2 l/min of o 2 , considering her background of chronic obstructive pulmonary disease. the culture swab used at our institute is a sigma virocult, a small vial with 1.0 ml medium and a standard sigma swab (figure 1). the bud type is cellular foam. the swab's stylet length is 15 cm and this breaks into two parts with the distal part (bud end) inserted in the vial. a plain radiograph was performed (figure 2), which was unremarkable. a ct scan was then performed and initially reported as showing no signs of a foreign body, but rather signs of infective changes in the posterior segment of the right lower lobe. later, an addendum report of the ct scan raised suspicion of a foreign body as subtle signs were identified (figures 3-5). a decision had been made for a flexible endoscopy, a high-risk procedure in sars-cov-2 era, to take place. this was performed through the tracheostomy site using a disposable flexible ambu ascope 4 rhinolaryngo slim device. the swab was identified on the right side, in a lobar bronchus (video 1) and was subsequently removed by flexible bronchoscopy. accurate and prompt detection of sars-cov-2 is essential to controlling outbreaks both in hospitals and in the community. diagnosis is usually confirmed by rrt-pcr of combined nasopharyngeal and oropharyngeal swabs. 2 3 there have been recent studies into whether the sars-cov-2 virus can be detected from other tissue samples. one study which included 1070 tissue samples from 205 patients with covid-19 found that brochoalveolar lavage fluid showed the highest positive rates (93%). this was compared with 63% for nasal swabs and 32% for pharyngeal swabs. 3 there is currently no guidance on how patients with front of neck airways should be tested. the question arises as to how the biodistribution of sars-cov-2 is affected in these patients, especially in laryngectomy patients where there is an exclusion of the upper airway. the us's centers for disease control and prevention recommends a lower respiratory aspirate in special clinical circumstances such as patients on mechanical ventilation. 4 the national tracheostomy safety project's statement on considerations for trachestomy reiterates that tracheal aspirates are preferable to mucosal swabs but does not outline when tracheal aspirates should be taken. 5 the above case highlights the potential dangers of taking a mucosal swab from a trachesotomy site. hightened concerns around sars-cov-2 and wearing full personal protective equipment increase the probability of human error occurring. there is a need for clear guidance on how to test patients with front of neck airways for sars-cov-2. this will be dependent on two main factors. first, how a front of neck airway affects the biodistribution of sars-cov-2 in the mucosa of the oropharynx and nasopharynx and second, understanding of the risk of increased aerosolisation associated with taking any sample from a tracheostomy site. further studies are needed to shed light on the above. contributors mhh: conception of idea and drafting of the manuscript. ss and sm: literature review and drafting the manuscript. tv: review of the final manuscript. funding university hospitals of leicester nhs trust (866108). competing interests none declared. provenance and peer review not commissioned; externally peer reviewed. this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. mohammed hassan hussain http:// orcid. org/ 0000-0002-5988-3405 clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected: interim guidance clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china detection of sars-cov-2 in different types of clinical specimens interim guidelines for collecting, handling, and testing clinical specimens from persons under investigation (puis) for coronavirus disease ntsp. ntsp considerations for tracheostomy in the covid-19 outbreak ► there is lack of guidance on how to test patients with front of neck airway for sars-cov-2. ► mucosal tracheal swab through a tracheostomy tube carries an increased risk and appropriately designed sampling devices, which among else would be radiopaque, should be used. ► despite the sensitivity of a tracheal aspirate being higher than that of an oropharyngeal/nasopharyngeal swab, further research is needed to clarify the increased risk of aerosolisation in this cohort of patients. ► ct imaging cannot always exclude a foreign body bronchus and communicating detailed clinical information to radiology colleagues is, as always, of paramount importance if there is suspicion of a foreign body. ► visualisation of the airway should always be considered as the examination of choice in the absence of any contraindications.copyright 2020 bmj publishing group. all rights reserved. for permission to reuse any of this content visit https://www.bmj.com/company/products-services/rights-and-licensing/permissions/ bmj case report fellows may re-use this article for personal use and teaching without any further permission.become a fellow of bmj case reports today and you can: ► submit as many cases as you like ► enjoy fast sympathetic peer review and rapid publication of accepted articles ► access all the published articles ► re-use any of the published material for personal use and teaching without further permission if you have any further queries about your subscription, please contact our customer services team on +44 (0) 207111 1105 or via email at support@bmj.com.visit casereports.bmj.com for more articles like this and to become a fellow key: cord-327654-9g8zcxaa authors: chi, xiaojing; liu, xiuying; wang, conghui; zhang, xinhui; li, xiang; hou, jianhua; ren, lili; jin, qi; wang, jianwei; yang, wei title: humanized single domain antibodies neutralize sars-cov-2 by targeting the spike receptor binding domain date: 2020-09-10 journal: nat commun doi: 10.1038/s41467-020-18387-8 sha: doc_id: 327654 cord_uid: 9g8zcxaa severe acute respiratory syndrome coronavirus 2 (sars-cov-2) spreads worldwide and leads to an unprecedented medical burden and lives lost. neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. here, using sars-cov-2 spike receptor-binding domain (rbd) as a bait, we generate a panel of humanized single domain antibodies (sdabs) from a synthetic library. these sdabs reveal binding kinetics with the equilibrium dissociation constant (k(d)) of 0.99–35.5 nm. the monomeric sdabs show half maximal neutralization concentration (ec(50)) of 0.0009–0.07 µg/ml and 0.13–0.51 µg/ml against sars-cov-2 pseudotypes, and authentic sars-cov-2, respectively. competitive ligand-binding experiments suggest that the sdabs either completely block or significantly inhibit the association between sars-cov-2 rbd and viral entry receptor ace2. fusion of the human igg1 fc to sdabs improve their neutralization activity by up to ten times. these results support neutralizing sdabs as a potential alternative for antiviral therapies. c oronavirus disease 2019 is caused by infection of emerging severe acute respiratory syndromeassociated coronavirus 2 (sars-cov-2) and had been declared by world health organization as the first coronavirus pandemic in human history 1 . the severity of covid-19 symptoms can range from asymptomatic or mild to severe with an estimated mortality rate from less than 2% to up to 10% of patients depending on various factors 2 . sars-cov-2 is spreading rapidly and sustainably around the world, urging prompt global actions to develop vaccines and antiviral therapeutics. sars-cov-2 polyprotein shares~86.15% identity with sars-cov (genbank id: aas00002.1) and is classified into the genus betacoronavirus in the family coronaviridae 3 . sars-cov-2 is an enveloped, positive-sense, single-stranded rna virus with a large genome of approximately 30,000 nucleotides in length. the virusencoded membrane (m), spike (s), and envelope (e) proteins constitute the majority of the protein that is incorporated into sars-cov-2 envelope lipid bilayer. the s protein can form homotrimers and protrudes from envelope to show the coronal appearance, invading susceptible cells by binding potential sars-cov-2 entry receptor angiotensin converting enzyme 2 (ace2) 3 . recently, researchers have figured out the molecular structure of sars-cov-2 s protein 4 . it is composed of 1273 amino acids and structurally belongs to the type i membrane fusion protein with two areas s1 and s2. the s1 region mainly includes the receptor binding domain (rbd), while the s2 region is necessary for membrane fusion. the rbd structure determines its binding efficiency with ace2 and provides an important target for neutralizing antibody recognition. single domain antibodies (sdabs), namely nanobodies, were initially identified from camelids or cartilaginous fish heavy-chain only antibodies devoid of light chains, where antigen-binding is mediated exclusively by a single variable domain (vhh) 5 . therefore, sdabs are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs, imaging probes and diagnostic reagents 6 . the advantages of short development time, flexible formatting and robust production efficiency make sdab a powerful means to defeat infectious disease pandemics. for therapeutic purpose, relatively sophisticated humanization techniques have been adopted to modify the camelid-specific amino acid sequences in the framework to their human heavy chain variable domain equivalent, without altering sdab's biological and physical properties and reducing species heterogeneity 7 . as sars-cov-2 is an emerging human virus, the whole population is susceptible due to the lack of protective antibodies. the existing neutralizing antibodies in convalescent plasma have been adopted as powerful therapeutic alternatives for covid-19 patients. in this study, using a synthetic humanized sdabs discovery platform, we obtain several high-affinity sars-cov-2 rbd targeting sdabs with desired neutralization activities. the results illustrate the potential of synthetic sdab library as a resource for antiviral molecules that can be rapidly accessed in a pandemic. these sdabs offer a potential hope for future anti-sars-cov-2 antibody cocktails. identification of sars-cov-2 rbd binding sdabs. sars-cov-2 makes use its envelope s glycoprotein to gain entry into host cells through binding ace2. recent cryo-em research revealed that the s protein shows an asymmetrical homotrimer with a single rbd in the "up" confirmation and the other two "down" 4 . antibodies may take advantage of this rbd structure to block virus entry. to enrich for sars-cov-2 rbd binding sdabs, we performed four rounds of biopanning using a lab owned, full synthetic, humanized phage display library with recombinant rbd protein. after phage elisa identification of 480 clones, a number of sdabs exhibited an excellent affinity for sars-cov-2 rbd (supplementary table 1 ). five distinctive sdad sequences (1e2, 2f2, 3f11, 4d8, and 5f8) were cloned into a prokaryotic expression vector and recombinant sdab proteins were purified by nickel-charged sepharose affinity chromatography (fig. 1a) . humanized sdabs obtained in this study are about 125 amino acids with a single vhh domain in average molecular weight less than 15kda (fig. 1a) . the sdabs consist of three complementarity determining regions (cdrs), as well as four framework regions (frs). the amino acids in the frameworks have been maximally humanized, except for residues phe-42 and ala-52 (numbers refer to the international immunogenetics information system amino acid numbering (imgt.cines.fr)) in framework-2 to maintain proper antigen affinity and best stability 7 . framework residues are illustrated in supplementary fig. 1 . surface plasmon resonance (spr) technology is widely accepted as a golden standard for characterizing antibodyantigen interactions. to determine the kinetic rate and affinity constants, detailed analysis of spike rbd-binding to purified sdab proteins was carried out by spr. the sars-cov-2 or sarc-cov rbd protein was immobilized on the surface of biacore chip cm5, respectively. then, various concentrations of purified sdabs were prepared and injected to pass over the surface. the sensorgram data were fitted to a 1:1 steady-state binding model. spr results demonstrated that the equilibrium dissociation constant (k d ) for the sars-cov-2 rbd protein against sdabs 1e2, 2f2, 3f11, 4d8, and 5f8 were 35.52 nm, 5.175 nm, 3.349 nm, 6.028 nm, and 0.996 nm, respectively ( fig. 1b-f, h) . however, the sdabs showed no binding with sars-cov rbd, except for the clone 5f8 demonstrating a relatively low affinity with k d = 239.2 nm (fig. 1g, h) . overall, as monovalent antibody fragment, the sdabs identified in this study reveals a satisfactory binding performance in a sars-cov-2 specific manner. neutralization of sars-cov-2 by rbd-specific sdabs. to further evaluate the neutralization activity of these sdabs, sars-cov-2 spike-pseudotyped particle (sars-cov-2pp) infectivity assay was first established. pseudotyped particles are chimeric virions that consist of a surrogate viral core with a heterologous viral envelope protein at their surface, which can be operated in biosafety level 2 (bsl-2) and frequently used tool for studying virus entry mechanism and neutralizing antibodies 8 . we observed that all five sdabs showed inhibition potency of sars-cov-2pp infection with ec 50 (half maximal neutralization concentration) ranging from 0.0009 to 0.069 µg/ml (fig. 2a) . we next tested the neutralization activity of the sdabs with sars-cov-2 live virus (fig. 2b) . the copy number of viral rna that was present in the cell culture supernatant was used as a proxy for viral replication. similarly, these sdabs showed comparable neutralization efficiency, with ec 50 at approximately 0.13-0.51 µg/ml. totally, these monovalent sdabs demonstrated encouraging neutralization activity against both pseudotyped and authentic virus, although the neutralization potency is not completely matched (fig. 2c) . this phenomenon was normally reported in middle east respiratory syndrome coronavirus (mers-cov) neutralizing antibodies and may be likely explained by the difference in sdab recognized rbd spatial epitope or the steric hindrance formed by antigen-antibody complex 9, 10 . interference of the ace2-rbd interaction by the sdabs. within sars-cov-2 rbd, the receptor binding motif (rbm) directly contacts ace2. recent report demonstrating that sars-article nature communications | https://doi.org/10.1038/s41467-020-18387-8 cov-2 uses ace2 as its receptor with a much stronger affinity (10-fold to 20-fold higher) than sars-cov 4 . to determine whether sdabs targeted different antigenic regions on the sars-cov-2 rbd surface, we performed a competition-binding assay using a real-time biosensor (fig. 3) . we tested all five sdabs in a competition-binding assay in which human ace2 was attached to a cm5 biosensor. compared with a non-related isotype control sdab (fig. 3a) , addition of 1e2 and 4d8 completely prevent binding of sars-cov-2 rbd to ace2 (fig. 3b, e) . whereas, sdabs 2f2, 3f11, and 5f8 could partially compete the rbd/ receptor association (fig. 3c , d, f). these data suggested that these sdabs can be divided into rbm targeting or non-rbm targeting groups though it is not directly associated with either affinity or virus neutralization activity, which has laid a solid foundation for further development of bispecific neutralizing antibodies to overcome potential virus mutation in the future. inhibition of sars-cov-2 entry by fc-fused sdabs. sdabs can be readily fused to human igg fc-domain to overcome the limitations of the monovalent sdabs, such as the short blood residential time and lacking antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity 11 . in addition, bivalent sdabs can be obtained via the disulfide bond formation in fc hinge area, which was reported to increase sdab's activity 12 . to further explore the possibility of sdab-based antiviral therapeutics and enhance neutralization activity, we constructed human heavy chain antibodies by fusing the human igg1 fc region to the c-terminus of sdabs ( fig. 4a, b) . these fc fusion sdabs were produced in mammalian cells with supernatant yields around 25-50 µg per milliliter in shaking flask. fc fusion sdabs in culture supernatants were affinity purified with hitrap protein a hp antibody purification columns ( supplementary fig. 2 ) and analyzed in both reducing and non-reducing conditions in western blot using an anti-human igg to detect fc. as shown in fig. 4c , the size of the constructed intact sdab-fc is around 80 kda in the non-reducing condition, but a 40 kda monomer was observed by prior treatment in reducing condition to break disulfide bonds. this suggests a correct expression and secretion of heavy chain antibodies in consistence with our design. neutralization assay results showed that genetic fusion of human fc could maintain or increase the neutralization activity of these sdabs for up to 10-fold in molar concentration of ec 50 using the sars-cov-2pp entry assay ( fig. 4d and supplementary fig. 3 ). importantly, all fc-fused sdabs demonstrated potency with ec 50 at sub-nanomolar level (fig. 4d) . finally, we showed that some of the sdabs are suitable for immunofluorescence staining (supplementary fig. 4) and western blot to detect ectopically expressed sars-cov-2 s protein ( supplementary fig. 5 ). given the disease severity and rapid global spread of covid-19, there is an urgent need for development of vaccines, monoclonal antibodies, and small-molecule direct-acting antiviral medications. neutralizing antibodies directly target viral envelope protein, precisely block the virus-receptor association, and inhibit virus entry through a variety of molecular mechanisms. in this study, we isolated and characterized several humanized neutralizing sdabs that exhibit one-digit to two-digit nanomolar or even subnanomolar ec 50 against sars-cov-2 using both pseudotyped and infectious viruses. sdabs have been investigated as important therapeutic alternatives against viral infection because of their high yield, low cost and intrinsic stability. for mers-cov, neutralizing sdabs were isolated from immunized dromedary camels or llamas and demonstrated ec 50 value between 0.001 and 0.003 µg/ml with low k d values (0.1-1 nm) 13, 14 . comparable inhibition efficiency on sars-cov-2pp and affinity kinetics were obtained for the sdabs identified in this study using a nonimmune library, which can speed up the discovery of neutralizing antibodies in an emergent outbreak. with further optimization and increase of library size and diversity, the synthetic sdab library technology will promote the discovery speed of powerful therapeutic antibodies 15, 16 . fda approved the first sdab-based medicine for adults with acquired thrombotic thrombocytopenic purpura in 2019 [17] [18] [19] [20] . considering the cost and potential risks of full human antibody in some viral diseases, such as dengue virus infection, sdab fragments are a novel category of therapeutic molecules and can be readily reconstructed in a tandemly linked way to increase their blood residential time, biological activity, and eliminate underlying concerns about antibody-dependent enhancement (ade) of viral infection 21 . in addition to being used as an injectable drug, the stable sdabs can be also developed into aerosolized inhalations and disinfection products for the prevention of covid-19. besides, prior to the success of covid-19 vaccines, the construction of sdab-based adenovirus or adeno-associated virus gene therapy might provide long-term passive immune protection in vulnerable population, health care workers, or in severely affected areas. since the mature covid-19 animal models have not been developed, this study did not involve in vivo studies. as a next step, the crystal structure analysis of antigen-antibody complexes will be put on the agenda. in conclusion, the discovered neutralizing antibodies in this study could lead to new specific antiviral treatments and shed light on the design and optimization of covid-19 vaccines. library design and construction. a synthetic sdab phage display library was used for the screening of sars-cov-2 neutralizing antibodies. to minimize a possible antigenic effect from camelid sequences, sdab frameworks (frs) for library construction were determined according to a universal humanized scaffold architecture 7 , and the sequences of the frs were illustrated in supplementary fig. 1 . briefly, residues in frs 1, 3, and 4 were mutated based on human heavy chain vh in maximum. in fr 2, humanization of residues at positions 49 and 50 was adopted to increase stability of sdabs, whereas residues 42 and 52 are maintained in camelid due to their critical impact on antigen affinity and/or stability (supplementary fig. 1 ). for the design of variable regions, we analyzed a robust cdr repertoire from immune or naïve llama vhh clones. a synthetic diversity was introduced in the three cdrs by the positioned nucleotide assembly with cysteine and stop codon avoided. a constant length of 8 amino acids was selected for cdr1 and cdr2, and 18 amino acids for cdr3 (supplementary fig. 1 ). frameworks and cdrs were assembled using only 8 cycles of overlapping polymerase chain reaction (pcr) to prevent drift during amplification. diversified sdab mixture was cloned in phagemid vector fadl-1 (antibody design labs, san diego, ca, usa) using sfii/bgli sites with the pelb peptide leader sequence fused with the sdabs at nterminus. the ligation product was purified and used to transform electrocompetent e. coli tg1 cells. a total 50 electroporations was performed in the condition of 1800 v, 10 mf, 600 w. each electroporation was resuspended with 2 ×yt and incubated with a shaking agitation for 1 h at 37°c, and then combined and plated onto more than a thousand agar petri dishes (140 mm) to ensure enough size of the library. library size was calculated by plating serial dilution aliquots and at least 1.2 × 10 10 individual recombinant clones were obtained. quality control was carried out by sequencing more than 1000 clones. more than 950 clones are full length and unique sdabs and less than 50 clones show various errors, such as vector self-ligation, reading frame shift and fragment deletion. antibody selection by phage display. screening for sars-cov-2 rbd targeting antibodies was performed by panning in both immunotubes and native condition using a proprietary full-synthetic library of humanized sdabs with high-diversity, according to a standard protocol. briefly, for the 2nd and 4th panning rounds, the purified sars-cov-2 rbd protein fused with mouse fc was coated on nunc maxisorp immuno tubes (thermofisher) at around 5 µg/ml in pbs overnight. for the 1st and 3rd panning rounds, rbd protein was first biotinylated with ez-link™ sulfo-nhs-lc-biotin (thermofisher) and then selected with streptavidin-coated magnetic dynabeads™ m-280 (thermofisher). the tubes or beads were blocked using 2% w/v skimmed milk powder in pbs (mpbs). after rinsing with pbs, about 1 × 10 13 flowed over the chip surface. after each cycle, the sensor surface was regenerated with10 mm glycine-hcl ph 2.5. the data were fitted to a 1:1 interaction steadystate binding model using the biaevaluation 1.0 software. for competition-binding assays, the ace2 protein was diluted in 10 mm sodium acetate buffer, ph4.5, and was immobilized on the chip at about 650 response units. for the analyses, the his-tagged sars-cov-2 rbd protein was diluted in hbs-ep buffer or hbs-ep buffer with 100 nm antibody (1e2, 2f2, 3f11, 4d8, or 5f8). the rbd in different buffer at gradient concentrations (0, 6.25 nm, 25 nm, 100 nm and, 400 nm) was flowed over the chip surface. after each cycle, the sensor surface was regenerated with 10 mm glycine-hcl ph 2.5. the binding kinetics was analyzed with the software of biaevaluation using a 1:1 binding model. sars-cov-2 spike pseudotyped particle (sars-cov-2pp). to produce sars-cov-2pp, hek293t cells were seeded 1 day prior to transfection at 2.5 × 10 6 cells in a 10-cm plate. the next day, cells were transfected using lipofectamine 2000 (thermofisher). the plasmid dna transfection mixture (1 ml) was composed of 15 µg of pnl-4.3-luc-e − r − and 15 µg of pcdna-sars-cov-2-s that was purchased from sino biologicals and reconstructed by deletion of 18 amino acid cytoplasmic tail. a nonenveloped lentivirus particle (bald virus) was also generated as negative control. 16 h after transfection, the media was replaced with fresh media supplemented with 2% fbs. supernatants containing sars-cov-2pp were typically harvested at 36-48 h after transfection and then filtered through a syringe filter (0.22 µm) to remove any cell debris. sars-cov-2pp was freshly used or allocated and frozen at −80°c. to conduct the virus entry assay, 293t cells were transiently transfected with human ace2 expression plasmid and 1 × 10 4 cells and seeded in each well of a 96-well plate at 1 day prior to transduction. the next day, 100 µl of supernatant containing sars-cov-2pp was added into each well in the absence or presence of serially diluted sdabs or human igg1 fc-fused sdabs. forty-eight hours after transduction, the cells were lysed in 100 µl of passive lysis buffer and 50 µl lysate was incubated with 100 µl of luciferase assay substrate according to the manufacturer's instructions (promega, madison, wi, usa). ethics statement and virus isolation. sars-cov-2 was isolated from bronchoalveolar lavage fluid (balf) from a covid-19 patient in the jin yin-tan hospital of wuhan as reported previously 22 . briefly, the patient was a 65-year-old man who reported a high fever and cough, with little sputum production, at the onset of illness. he had a continuous fever and developed severe shortness of breath 16 days later. balf sample was collected from this hospitalized patient by nurses according to a standard procedure in which a bronchoscope is passed through the mouth into the lungs to obtain cells and other components from bronchial and alveolar spaces. a clinical protocol was conducted in accordance with the declaration of helsinki and was approved by the national health commission of china and ethics commission of the jin yin-tan hospital of wuhan (no. ky-2020-01.01). written informed consent was waived by the ethics commission of the designated hospital for emerging infectious diseases. clearing the airway and collection of balf were as standard of care and for clinical etiological diagnosis. therefore, the requirement for written informed consent was waived given the context of emerging infectious diseases. for the isolation and identification of potential pathogens, the balf specimens were filtered and inoculated onto vero cells. all cultures were observed daily for a cytopathic effect (cpe). cpe were observed in 30% of vero cells after two passages. the viral particles in culture supernatants were characterized by negative staining electron microscope. the isolated sars-cov-2 was obtained from the patient by dr. lili ren and the virus full length sequence was deposited in gisaid database with accession id of epi_isl_402123, which is completely as same as genbank accession number mn908947. gisaid is a globally recognized virus database and more than 56,000 viral genomic sequences of hcov-19 have been shared via gisaid since the start of the covid-19 outbreak. sars-cov-2 neutralization assay. the 50% tissue culture infectious dose (tcid 50 ) assay was performed for sars-cov-2 in vero cells. briefly, cells were seeded 24 h before infection in a 24-well plate at a density of 8 × 10 4 cells/well. viruses were serially diluted at 1:10 dilution. after 72 h of incubation, the media were removed, and cells were fixed and stained with crystal violet. the tcid 50 /ml titer was determined. for antibody neutralization assay, vero cells were seeded in 96-well plates at 1 day prior to infection. serially diluted sdabs were mixed with sars-cov-2 at 100 tcid 50 per well and incubated at 37°c for 1 h. the antibodyvirus mixture was incubated on vero cells at 37°c for 1 h. unbound sars-cov-2 virions were removed by washing cells with fresh medium, then incubated for 24 h at 37°c. the culture supernatants were collected for viral nucleic acid quantification. viral rna quantification was carried out by taqman real-time rt-pcr as reported with plotted standard curves using in vitro transcribed rna. briefly, the viral rna was isolated using trizol ls reagent (invitrogen, carlsbad, ca) according to the manufacturer's protocol. rna was extracted from 100 µl culture supernatants and eluted in 50 µl dnase/rnase-free water. the viral nucleocapsid gene-based quantification assay was developed using the taqman production of human igg1 fc fusion sdabs. the sequences of selected sdabs were cloned into a mammalian expression vector under the control of hef1-htlv promotor and fused with n-terminal interleukin-2 signal peptide and c-terminal fc region, comprising the ch2 and ch3 domains of human igg1 heavy chain and the hinge region. maxiprepped plasmids were transiently transfected into 293-f cells (thermofisher) and the cells were further cultured in suspension for 6 days before harvesting antibody-containing supernatant. fc-fused sdabs were prepared with prepacked hitrap® protein a hp column (ge healthcare). the produced fcfusion protein was analyzed by sds-page and the western blot using standard protocols for dimerization, yield and purity measurement. the primary antibody used for western blot was a horseradish peroxidase conjugated goat anti-human igg (sigma-aldrich, st. louis, mo, usa). immunofluorescence microscopy and western blot. cultured 293t cells on coverslips were transfected with either sars-cov-2 s expression plasmid or empty vector for 24 h and then fixed using 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% triton x-100 (sigma-aldrich) in pbs for 10 min. the cells were then incubated with each sdab overnight at 4°c. after three washes with pbs, the cells were incubated with alexa fluor 488-conjugated 6x-his tag monoclonal antibody (his.h8) (thermofisher, ma1-21315-a488, 1:1000) for fig. 4 inhibition of sars-cov-2 entry by fc-fused sdabs. a representation of the human igg1 fc-fused sdabs in this study. sdab-fc fusion construction generates a bivalent molecule with an approximate molecular weight of 80 kda. b homology modeling of the bivalent 5f8-fc molecule with swiss-model server (https://swissmodel.expasy.org) 23 . the template structure for 5f8 modeling was based on a humanized camelid sdab in the pdb database (3eak). the structure is depicted as cartoons and colored with secondary structure. three cdrs, hinge region and fc were indicated. c five fc-fused sdabs were analyzed by western blot with gradient sds-page in reducing (with β-mercaptoethanol) or nonreducing (without β-mercaptoethanol) condition. d summary of ec 50 value of fc-fused sdabs neutralization against sars-cov-2pp. ec 50 fold increases versus the corresponding monovalent sdabs were calculated. 1 h at room temperature. the nuclei were stained with dapi (1:10,000) diluted in pbs for 5 min and mounted with an antifade reagent (thermofisher). images were acquired with a leica tcs sp5 confocal microscope system. for western blot, 293t cells in 6-well plate were transfected with sars-cov-2 s, sars-cov-2 s or empty vector individually. twenty-four h post transfection, cell lysates were prepared, and the samples were boiled with 2× sds loading buffer and loaded onto a 10% polyacrylamide gel. after electrophoresis, the separated proteins were transferred onto a nitrocellulose membrane (bio-rad, hercules, ca, usa). the resulting blots were probed with a sdab as primary antibody and an hrp-linked 6x-his tag antibody (thermofisher, his.h8, ma1-21315-hrp, 1:1000) as the secondary antibody. antibody against β-actin is from sigma-aldrich (a1978, 1:8000). the ecl reagent (amersham biosciences, piscataway, nj, usa) was used as the substrate for detection. statistics and reproducibility. data were analyzed using graphpad prism 6.01 (graphpad software, san diego, ca, usa). the values shown in the graphs are presented as means ± sd. one representative result from at least two independent experiments was shown. antibody neutralization experiments usually use two to four duplicated wells for each treatment. for sars-cov-2pp entry assay and sars-cov-2 infection, the infectivity data were first inversed to neutralization activity. each neutralization data set was normalized by the background control (no virus) to define the real value for 100% neutralization. after transformation to neutralization, the lowest concentration point of antibody treatment was set to 0% neutralization. then, a 4-parameters neutralization nonlinear regression model was fitted to report ec 50 values. all experiments were performed independently at least twice and similar results were obtained. one representative data of one experiment were shown. covid-19: towards controlling of a pandemic clinical features of patients infected with 2019 novel coronavirus in wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin cryo-em structure of the 2019-ncov spike in the prefusion conformation naturally occurring antibodies devoid of light chains nanobodies as therapeutics: big opportunities for small antibodies general strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold production of pseudotyped particles to study highly pathogenic coronaviruses in a biosafety level 2 setting prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies fusion of higg1-fc to 111in-anti-amyloid single domain antibody fragment vhh-pa2h prolongs blood residential time in app/ps1 mice but does not increase brain uptake fusion of the mouse igg1 fc domain to the vhh fragment (arp1) enhances protection in a mouse model of rotavirus chimeric camel/human heavy-chain antibodies protect against mers-cov infection a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov nali-h1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies construction of synthetic antibody phage-display libraries caplacizumab as an emerging treatment option for acquired thrombotic thrombocytopenic purpura caplacizumab treatment for acquired thrombotic thrombocytopenic purpura (hercules trial) clinical pharmacology of caplacizumab for the treatment of patients with acquired thrombotic thrombocytopenic purpura. expert rev caplacizumab treatment for acquired thrombotic thrombocytopenic purpura molecular mechanism for antibody-dependent enhancement of coronavirus entry identification of a novel coronavirus causing severe pneumonia in human: a descriptive study swiss-model: homology modelling of protein structures and complexes reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the sequences of sdabs have been deposited in genbank with the accession numbers mt813428-mt813432. the isolated sars-cov-2 full length sequence was deposited in gisaid database with accession id of epi_isl_402123, which is completely as same as genbank accession number mn908947. all other data are available from the corresponding author upon reasonable requests. source data are provided with this paper.received: 29 march 2020; accepted: 12 august 2020; this work was supported by cams initiative for innovative medicine grant 2020-i2m-2-010 and 2016-i2m-3-020. w.y., x.c., l.r. q.j., and j.w. designed experiments and interpreted the data. w.y., x.c., x.l., c.w., x.l., j.h., and x.z. performed experiments and analyzed the data. w.y. conceived the study, supervised the work, and wrote the paper. all authors read and approved the final manuscript. a patent application has been filed on 17 march 2020 on single domain antibodies targeting sars-cov-2 (china patent application no. 202010185593.9). supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-18387-8.correspondence and requests for materials should be addressed to q.j., j.w. or w.y.peer review information nature communications thanks the anonymous reviewers for their contribution to the peer review of this work. peer reviewer reports are available.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-324557-4u8dja0n authors: leblanc, jean‐françois; germain, marc; delage, gilles; o’brien, sheila; drews, steven j.; lewin, antoine title: risk of transmission of severe acute respiratory syndrome coronavirus‐2 by transfusion: a literature review date: 2020-08-15 journal: transfusion doi: 10.1111/trf.16056 sha: doc_id: 324557 cord_uid: 4u8dja0n severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) is a novel human coronavirus responsible for coronavirus disease 2019 (covid‐19). the emergence of this virus in wuhan (china) at the end of 2019, and its worldwide spread to reach the pandemic stage, has raised concerns about the possible risk that it might be transmissible by transfusion. this theoretical risk is further supported by reports of the detection of viral rna in the blood of some infected individuals. to further address this risk, a thorough pubmed literature search was performed to systematically identify studies reporting data on the detection of sars‐cov‐2 rna in blood or its components. complementary searches were done to identify articles reporting data on the in vitro infectivity of blood components. at least 23 articles presenting data on the detection of sars‐cov‐2 rna in blood, plasma, or serum were identified. of these, three studies reported on blood donors with covid‐19 infection identified post‐donation, and no cases of transfusion transmission were identified. a few studies mentioned results of in vitro infectivity assays of blood components in permissive cell lines, none of which were able to detect infectious virus in blood or its components. complementary searches have identified reports demonstrating that the correlation between the presence of viral rna in a biological sample and infectivity requires a minimal rna load, which is rarely, if at all observed, in blood components. overall, the available evidence suggests that the risk of transmission of sars‐cov‐2 by transfusion remains theoretical. in january 2020, chinese health authorities reported several cases of a new acute respiratory illness arising in december 2019 in the city of wuhan, hubei province. [1] [2] [3] [4] symptoms of this novel illness are typical of respiratory infections of viral origin; fever, fatigue, myalgia and dry cough are commonly observed. [2] [3] [4] [5] although most patients experience mild to moderate symptoms and recover within a few days, some 20% of identified patients exhibit more severe forms of the disease requiring prolonged hospitalization, and in some cases acute care and ventilation. 6 the exact mortality rate is difficult to assess, as varying proportions of asymptomatic or presymptomatic cases have been reported, and broad serosurveys to understand the true burden of disease have been hampered by a variety of logistic and scientific issues. 7 however, the detailed study of wu and mcgoogan, reporting on 72 314 suspected cases from the chinese center for disease control and prevention, including 44 672 confirmed cases, provides an estimated case fatality rate of 2.3%. 6 simultaneously to the primary reports of cases of covid-19, a virus was isolated from bronchoalveolar lavage fluids of affected patients. characterization of the virus and elucidation of the nucleotide sequence of its genome identified an enveloped, non-segmented positive single-stranded rna virus, a novel member of the betacoronavirus family, subfamily orthocoronaviridae. this new virus, referred to by the acronym sars-cov-2, shares 79.6% genomic sequence identity with severe acute respiratory syndrome coronavirus (sars-cov). 2, 8 the latter is a coronavirus that was responsible for an outbreak of a severe acute respiratory syndrome which affected several countries in 2003. that outbreak was successfully managed through strict confinement of infected individuals and quarantine of their contacts. during that outbreak, there was no evidence of transfusion transmission. 9 conversely, sars-cov-2 rapidly spread on a broad scale as a result of air travel and the relative ease by which the virus is transmitted by respiratory droplets from coughing and sneezing. on march 11, 2020, the world health organization (who) officially declared that covid-19, the disease acronym caused by sars-cov-2 infection, had reached the pandemic level. 10 as of june 30, 2020, more than 10.4 million cases of sars-cov-2 infection, and more than 509 000 deaths from covid-19, have been reported worldwide. [11] [12] [13] the emergence of this novel infectious agent has forced blood component suppliers to raise their level of awareness and to quickly assess the potential risk to blood safety. this article aims to evaluate the available evidence on the theoretical risk of sars-cov-2 transmission by transfusion, including attempts at determining infectiousness of blood components. the pubmed public biomedical literature database (https://pubmed.ncbi.nlm.nih.gov/) was searched for references that pertain to the risk of transmission of covid-19/sars-cov-2 by transfusion. more specifically, pubmed was interrogated with a series of queries aimed at identifying references that relate to covid-19/sars-cov-2 and the detection of viral genomic material in blood, plasma, or serum. as this enveloped virus would not be expected to survive the fractionation process, key words associated with purified plasma products were not included in the search. queries were built from a basic search script from this core script, queries focused on the detection of viral genomic material in blood were built. queries and their respective search results are shown in table 1 . titles and abstracts from the nonoverlapping 734 references from searches #2 and #5 (equivalent to search #4) were examined. from this screen, 23 references reporting any data or stating any information on the detection of sars-cov-2 genomic material in human blood, plasma, or serum, were selected ( table 2) . while examining the above 734 references, some references pertaining to in vitro or animal models of sars-cov-2 infectivity were intercepted and saved. additional searches specifically targeting the in vitro infectivity of blood, plasma, or serum samples were performed to complete the list of references on that topic. additional searches were performed to identify references pertaining to covid-19/sars-cov-2 and infection of endothelial cells. a non-exhaustive, restricted list of relevant references were selected and are discussed in the text. an exhaustive search strategy led to the identification of 23 references reporting data on the detection of sars-cov-2 genomic material in blood components (table 2) . 4, 8, as correctly pointed out by huang et al., 4 the presence of sars-cov-2 genomic material in the blood of asymptomatic/presymptomatic individuals or covid-19 patients should be referred to as rnaaemia, as opposed to viremia, which refers to the presence of intact, infectious virions in blood. we shall adhere to this terminology throughout the text. several observations can be made from the data summarized in table 2 . first, rnaaemia, when present, is close to the limit of detection of rrt-qpcr, with cycle threshold (ct) values well above 30 in the vast accepted article majority of cases. second, rnaaemia tends to be associated with more severe disease. 18, 20, 26, 28, 30, 32 third, 18 of the 23 identified studies report on cases of patients diagnosed with covid-19. even when rna testing is done on whole blood, plasma, or serum from a preselected cohort of covid-19 patients, the prevalence of rnaaemia is generally low. in this context, the article of zheng et al. (2020) 28 the viral infectivity of a biological sample, including blood, plasma, or serum, can be determined in vitro using cells that are known to be susceptible to infection. in such a cellular model, infection results in either detectable cytopathic effects, cell lysis, intracellular replication of the virus and production of viral particles in the culture supernatant, or a combination of these manifestations. infectivity can also be demonstrated in a susceptible animal model, in which viral infection will result in signs and symptoms similar to those observed in humans. several cell lines can support sars-cov-2 replication. [36] [37] [38] [39] in fact, any cell line which expresses the cognate angiotensin-converting enzyme 2 (ace2) and capable of sustaining replication of the virus can be used to assess infectivity. 40 among the most commonly used cell lines are vero and its derivatives. originally derived from african green monkey kidney epithelial cells, the vero cell line is broadly used for the study of human respiratory viruses. this in vitro model permitted confirmation of the infectivity of respiratory samples collected from suspected covid-19 cases. 2, 8, 22 other cell lines (huh7, calu3, caco-2) have also been shown to be permissive for sars-cov-2 replication. 37 various animal models of infection have also been identified and characterized. [41] [42] [43] as stated earlier, attempts at detecting infectivity in blood have been so far unsuccessful. in fact, infectivity in biological samples outside of the respiratory tract has not been demonstrated. although infectivity can be detected in respiratory samples, a minimal rna load, in terms of equivalent rna copy number, appears to be necessary for in vitro infection of cell lines to occur. the data of la scola et al. (2020) 44 suggest that individuals whose respiratory samples yield ct values above 34 are no longer contagious. bullard et al.'s results support the idea that the quantitative criterion for infectivity could be even higher: their data suggest that the infectivity of samples with ct values > 24 might be below the limit of detection of an in vitro infectivity assay. 45 the recent article by huang et al. (2020c) is consistent with these observations. 46 given that rnaaemic blood samples generally give ct values in the high 30's, the above reports on the relationship between rnaaemia in respiratory samples and infectivity are consistent with the idea that blood is unlikely to be an infectious source of sars-cov-2. aside from a respiratory infection, sars-cov-2 appears to induce systemic effects which likely contribute to the pathological mechanisms observed in the most severe cases of infection. 3, 4 furthermore, some reports have suggested that the sars-cov-2 virus could infect endothelial cells lining the interior of blood vessels, [47] [48] [49] [50] raising the possibility that infectious virions might be present in the circulation. however, some of these findings have been challenged. 51 furthermore, these findings are based on case accepted article reports of covid-19 patients or post-mortem analysis of deceased covid-19 patients, and the presumed detection of sars-cov-2 virions was performed by electron microscopy and immunohistochemistry, which are prone to artifacts and misinterpretations. 51 in addition, two of these articles report on deceased patients that had comorbidities that were directly involved with the organ origin of the suspected observation of sars-cov-2 virions, namely the kidney of a kidney transplant patient 47 and the brain of a parkinson's disease patient. 50 sars-cov-2 rna has also been detected in five out of 104 endomyocardial biopsy samples from patients exhibiting signs of myocarditis or unexplained cardiac failure, suggesting that the virus might leach into the myocardium. 52 however, escher et al. did not report the detection of virions by electron microscopy or immunohistochemistry, nor were they able to demonstrate that the rna, detected after  33 cycles of rrt-qpcr, was infectious. thus, this observation could be a bystander detection or leaching/contamination of the biopsy sample with lung tissue. there is some evidence that sars-cov-2 can infect human primary cd4+ t cells in culture and drive the expression of viral proteins in these cells. however, the relevance of these infections is not known, as these infections did not appear to be productive in terms of live viral particles. it is also expected that the burden of infected t cells, if it were to occur in vivo, would be substantially reduced through leukoreduction. 53 to this day, there has not been a single reported case of transmission of a respiratory virus by transfusion. accordingly, the long historical track record on the mode of transmission of respiratory viruses predicts that sars-cov-2 would not be transmissible by transfusion. so far, this hypothesis appears to be true, as there has been no documented case of transfusion-transmitted sars-cov-2. this article is protected by copyright. all rights reserved. as stated by katz (2020) , 54 given that some asymptomatic/presymptomatic individuals appear to be infectious (through their respiratory secretions), and that some of these individuals must have donated blood since the beginning of the pandemic, if indeed sars-cov-2 was hematogenous, then it is likely that some cases of transmission by transfusion would have been identified among transfused patients on a worldwide scale. furthermore, rnaaemia is generally associated with a more severe disease course; accordingly, the majority of rnaaemic individuals are not healthy enough to donate blood, which further reduces the theoretical risk of transmission by transfusion. the fact that epidemiological investigations and contact tracing indicate that new covid-19 cases are generally related to close contacts with infected individuals, and that no cases have been linked to transfusion, is reassuring from a blood safety standpoint. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. this article is protected by copyright. all rights reserved. a novel coronavirus genome identified in a cluster of pneumonia cases -wuhan a novel coronavirus from patients with pneumonia in china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan, china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention infectious diseases society of america. idsa covid-19 antibody testing primer a pneumonia outbreak associated with a new coronavirus of probable bat origin emerging infectious disease agents and their potential threat to transfusion safety. appendix 2. sars coronavirus world health organization. who director-general's opening remarks at the media 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model for sars-cov-2 infection and countermeasure development viral rna load as determined by cell culture as a management tool for discharge of sars-cov-2 patients from infectious disease wards predicting infectious sars-cov-2 from diagnostic samples culture-based virus isolation to evaluate potential infectivity of clinical specimens tested for covid-19 endothelial cell infection and endotheliitis in covid-19 clinical and pathological investigation of severe covid-19 patients direct endothelial damage and vasculitis due to sars-cov-2 in small bowel submucosa of covid-19 patient with diarrhea central nervous system involvement by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) electron microscopy of sars-cov-2: a challenging task detection of viral sars-cov-2 genomes and histopathological changes in endomyocardial biopsies isolation, sequence, infectivity, and replication kinetics of severe acute respiratory syndrome coronavirus 2 is sars-cov-2 transfusion transmitted? accepted article references are presented in ascending order of online publication date *for those studies which detected rnaaemia, this column shows the timing of collection of positive samples. for those studies which did not detect rnaaemia, this column shows the entire range of times when blood samples were collected. †abbreviations: ct, cycle threshold; nd wb, whole blood; lod, limit of detection of the article) mentions that a total of 31 samples were tested; fig. 1a (p. 466) suggests that a total of 51 serum samples were tested. §there is ambiguity regarding the total number of sars-cov-2 rna-positive serum samples. the text (p. 114) states that nine serum samples were positive; the data of fig. 1 (p. 116) indicate that eight serum samples were positive. ǁthere is an ambiguity regarding the mean rna concentratios in sars-cov-2-positive serum samples. the abstract (p. 112) mentions a concentration of 1,210 ± 1,861 copies/µl in positive samples, whereas the results and discussion section (p. 114) mentions a concentration of 127 copies/µl in positive samples timing of blood sample collection* this article is protected by copyright. all rights reserved. timing of blood sample collection* this article is protected by copyright. all rights reserved. key: cord-319877-izn315hb authors: de wit, emmie; van doremalen, neeltje; falzarano, darryl; munster, vincent j. title: sars and mers: recent insights into emerging coronaviruses date: 2016-06-27 journal: nat rev microbiol doi: 10.1038/nrmicro.2016.81 sha: doc_id: 319877 cord_uid: izn315hb the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 marked the second introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. the continuing introductions of mers-cov from dromedary camels, the subsequent travel-related viral spread, the unprecedented nosocomial outbreaks and the high case-fatality rates highlight the need for prophylactic and therapeutic measures. scientific advancements since the 2002–2003 severe acute respiratory syndrome coronavirus (sars-cov) pandemic allowed for rapid progress in our understanding of the epidemiology and pathogenesis of mers-cov and the development of therapeutics. in this review, we detail our present understanding of the transmission and pathogenesis of sars-cov and mers-cov, and discuss the current state of development of measures to combat emerging coronaviruses. supplementary information: the online version of this article (doi:10.1038/nrmicro.2016.81) contains supplementary material, which is available to authorized users. mers 12 , and a cluster of three cases of mers in the uk was identified in september 2012 (ref. 13 ). mers-cov continued to emerge and spread to countries outside of the arabian peninsula as a result of travel of infected persons; often, these imported mers cases resulted in nosocomial transmission. in may 2015, a single person returning from the middle east started a nosocomial outbreak of mers in south korea that involved 16 hospitals and 186 patients 14 . as of 26 april 2016, there have been 1,728 confirmed cases of mers, including 624 deaths in 27 countries 15 . this review highlights the pandemic and epidemic potential of emerging coronaviruses and discusses our current knowledge of the biology of sars-cov and mers-cov, including their transmission, their pathogenesis and the development of medical countermeasures. key features of these viruses are the dominance of nosocomial transmission, and pathogenesis that is driven by a combination of viral replication in the lower respiratory tract and an aberrant host immune response. several potential treatments for sars and mers have been identified in animal and in vitro models, including small-molecule protease inhibitors, neutralizing antibodies and inhibitors of the host immune response. however, efficacy data from human clinical trials are lacking but are needed to move these potential countermeasures forward. respectively (fig. 1a) . similarly to all viruses in the order nidovirales, sars-cov and mers-cov have a unique coding strategy: two-thirds of the viral rna is translated into two large polyproteins, and the remainder of the viral genome is transcribed into a nested set of subgenomic mrnas 16, 17 (fig. 1b) . the two polyproteins, pp1a and pp1ab, encode 16 non-structural proteins (nsp1-nsp16) 18 that make up the viral replicase-transcriptase complex. the polyproteins are cleaved by two proteases, papainlike protease (plpro; corresponding to nsp3) and a main protease, 3c-like protease (3clpro; corresponding to nsp5). the nsps rearrange membranes that are derived from the rough endoplasmic reticulum (rer) into double-membrane vesicles, in which viral replication and transcription occur 19 . one unique feature of coronaviruses is the exoribonuclease (exon) function of nsp14 (ref. 20) , which provides the proofreading capability required to maintain a large rna genome without the accumulation of detrimental mutations 21, 22 . sars-cov and mers-cov transcribe 12 and 9 subgenomic rnas, er-golgi intermediate compartment (ergic) . a cellular compartment that facilitates transport between the endoplasmic reticulum (er) and the golgi complex. infected individuals who each infect a disproportionately large number of secondary cases. respectively, and these encode the four structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as several accessory proteins that are not involved in viral replication but interfere with the host innate immune response or are of unknown or poorly understood function. the envelope spike glycoprotein binds to its cellular receptor, angiotensin-converting enzyme 2 (ace2) for sars-cov and dipeptidyl peptidase 4 (dpp4) for mers-cov 23 . after membrane fusion, either directly with the host cell membrane or with the endosome membrane, the viral rna genome is released into the cytoplasm, and the rna is uncoated to allow translation of the two polyproteins, transcription of the subgenomic rnas and replication of the viral genome (fig. 1b) . newly formed envelope glycoproteins are inserted in the rer or golgi membranes; genomic rna and nucleocapsid proteins combine to form nucleocapsids, and the viral particles bud into the er-golgi intermediate compartment (ergic). virion-containing vesicles subsequently fuse with the plasma membrane to release the virus 24 . the first indication of the source of sars-cov was the detection of the virus in masked palm civets and a raccoon dog and the detection of antibodies against the virus in chinese ferret badgers in a live-animal market in shenzhen, china 25 . however, these animals were only incidental hosts, as there was no evidence for the circulation of sars-cov-like viruses in palm civets in the wild or in breeding facilities 26 . rather, bats are the reservoir of a wide variety of coronaviruses, including sars-cov-like and mers-cov-like viruses 27 (fig. 2) . thus, the search for the reservoir of mers-cov initially focused on bats, but a serological survey in dromedary camels from oman and the canary islands showed a high prevalence of mers-cov-neutralizing antibodies in these animals 28 . in addition, mers-cov rna was detected in swabs that were collected from dromedary camels at a farm in qatar that was linked to two human cases of mers, and infectious virus was isolated from dromedary camels in saudi arabia and qatar [29] [30] [31] [32] . serological evidence for the circulation of a mers-cov-like virus in dromedary camels has been obtained in the middle east, eastern africa and northern africa, dating back as far as 1983 (ref. 33 ). dromedary camels in saudi arabia harbour several viral genetic lineages 34 , including those that have caused human outbreaks. taken together, these data strongly point to the role of dromedary camels as a reservoir for mers-cov. the ubiquity of infected dromedary camels close to humans and the resulting continuing zoonotic transmission may explain why mers-cov continues to cause infections in humans, whereas sars-cov, without the continuing presence of an infected intermediate host and with relatively infrequent human-bat interactions, has caused no more infections in humans. human-to-human transmission of sars-cov and mers-cov occurs mainly through nosocomial transmission; 43.5-100% of mers cases in individual outbreaks were linked to hospitals, and very similar observations were made for some of the sars clusters 35, 36 . transmission between family members occurred in only 13-21% of mers cases and 22-39% of sars cases. transmission of mers-cov between patients was the most common route of infection (62-79% of cases), whereas for sars-cov, infection of health care workers by infected patients was very frequent (33-42%) 35 . the predominance of nosocomial transmission is probably due to the fact that substantial virus shedding occurs only after the onset of symptoms 37, 38 , when most patients are already seeking medical care 39 . an analysis of hospital surfaces after the treatment of patients with mers showed the ubiquitous presence of viral rna in the environment for several days after patients no longer tested positive 40 . moreover, many patients with sars or mers were infected through super spreaders 14, 35, 37, [41] [42] [43] . the clinical courses of sars and mers are remarkably similar, although there are subtle differences (box 1) . owing to the current sparsity of data on human mers-cov infections 44 , the pathogenesis of this virus is poorly understood; however, similar mechanisms may underlie the pathogenesis of both mers and sars. the binding of spike protein to ace2 and the subsequent downregulation of this receptor contribute to lung injury during sars 45 . although it seems counterintuitive that receptor downregulation would increase pathology, it has been shown that ace2 can protect against acute lung injury. the downregulation of ace2 results in the excessive production of angiotensin ii by the related enzyme ace, and it has been suggested that the stimulation of type 1a angiotensin ii receptor and middle east respiratory syndrome coronavirus (mers-cov) encode two large polyproteins, pp1a and pp1ab, which are proteolytically cleaved into 16 non-structural proteins (nsps), including papain-like protease (plpro), 3c-like protease (3clpro), rna-dependent rna polymerase (rdrp), helicase (hel) and exonuclease (exon). an additional 9-12 orfs are encoded through the transcription of a nested set of subgenomic rnas. sars-cov and mers-cov form spherical particles that consist of four structural proteins. the envelope glycoprotein spike (s) forms a layer of glycoproteins that protrude from the envelope. two additional transmembrane glycoproteins are incorporated in the virion: envelope (e) and membrane (m). inside the viral envelope resides the helical nucleocapsid, which consists of the viral positive-sense rna ((+)rna) genome encapsidated by protein nucleocapsid (n). b | following entry of the virus into the host cell, the viral rna is uncoated in the cytoplasm. orf1a and orf1ab are translated to produce pp1a and pp1ab, which are cleaved by the proteases that are encoded by orf1a to yield 16 nsps that form the rna replicase-transcriptase complex. this complex localizes to modified intracellular membranes that are derived from the rough endoplasmic reticulum (er) in the perinuclear region, and it drives the production of negative-sense rnas ((-)rnas) through both replication and transcription. during replication, full-length (-)rna copies of the genome are produced and used as templates for full-length (+)rna genomes. during transcription, a subset of 7-9 subgenomic rnas, including those encoding all structural proteins, is produced through discontinuous transcription. in this process, subgenomic (-)rnas are synthesized by combining varying lengths of the 3′end of the genome with the 5′ leader sequence necessary for translation. these subgenomic (-)rnas are then transcribed into subgenomic (+)mrnas. although the different subgenomic mrnas may contain several orfs, only the first orf (that closest to the 5′end) is translated. the resulting structural proteins are assembled into the nucleocapsid and viral envelope at the er-golgi intermediate compartment (ergic), followed by release of the nascent virion from the infected cell. one of a panel of recombinant inbred mouse strains derived from a genetically diverse set of founder strains and designed for the analysis of complex traits. the aggregation of leukocytes around blood vessels. (agtr1a) increases pulmonary vascular permeability, thus potentially explaining the increased lung pathology when the expression of ace2 is decreased 46 . immunopathology. the immune response is essential for the resolution of an infection, but it can also result in immunopathogenesis. one indication that immunopathogenesis may contribute to sars was the observation that viral loads were found to be decreasing while disease severity increased 39, 47 . it is unclear whether a similar trend applies to mers 48, 49 . moreover, progression to acute respiratory distress syndrome (ards) is associated with the upregulation of pro-inflammatory cytokines and chemokines, particularly interleukin-1β (il-1β), il-8, il-6, cxc-chemokine ligand 10 (cxcl10) and cc-chemokine ligand 2 (ccl2) 50, 51 ; increased plasma levels of these molecules have been detected in patients with sars [52] [53] [54] [55] . retrospective longitudinal studies in patients who recovered from sars versus those who succumbed to the disease have shown an early expression of interferon-α (ifnα), ifnγ, cxcl10, ccl2 and proteins that are encoded by ifn-stimulated genes (isgs) in all patients, but only patients who survived then had gene expression profiles that are indicative of the development of an adaptive immune response. by contrast, patients who succumbed maintained high levels of cxcl10, ccl2 and isg-encoded proteins, whereas spike-specific antibodies were present at low levels or were absent 56 , which suggests that severe disease is related to the lack of a switch from an innate immune response to an adaptive immune response. experiments using collaborative cross mouse lines and mouse-adapted sars-cov identified one host gene, trim55, as important for sars pathogenesis. although there was no difference in clinical signs or viral replication in trim55 −/− mice compared with wild-type mice, perivascular cuffing and the number of inflammatory cells in the lungs were reduced in the trim55 −/− mice 57 . a biological process in which small rna molecules induce the degradation of specific mrna molecules, thereby inhibiting gene expression. systems in which a dna molecule is produced that contains the viral leader and trailer sequences, with an assayable reporter replacing the viral orfs. when combined with the expression of viral proteins in trans, this system can be used to model the viral life cycle without the necessity of using infectious virus. the involvement of the host immune response in the pathogenesis of sars, and most likely also that of mers, suggests that drugs which inhibit viral replication will need to be combined with treatments that control detrimental immune responses. immune evasion. sars-cov and mers-cov use several strategies to avoid the innate immune response. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna (dsrna) or uncapped mrna, are detected by pattern recognition receptors (prrs), such as retinoic acid-inducible gene i protein (rig-i; also known as ddx58) or melanoma differentiation-associated protein 5 (mda5; also known as ifih1) 58 . this triggers complex signalling cascades involving myd88 that lead to the production of type i ifns and the activation of the transcription factor nuclear factor-κb (nf-κb). in turn, active nf-κb induces the transcription of pro-inflammatory cytokines (fig. 3a) . type i ifns signal through ifnα/β receptor (ifnar) and downstream molecules such as signal transducer and activator of transcription (stat) proteins to stimulate the production of antiviral proteins that are encoded by isgs, such as ifn-induced protein with tetratricopeptide repeats 1 (ifit1 ; fig. 3b ). collectively, this establishes an antiviral immune response that limits viral replication in infected and in neighbouring cells (fig. 3) . infection of knockout mice revealed the importance of innate immunity. infection of myd88 −/− and stat1 −/− mice, but not mice that were deficient in ifn receptors, with a mouse-adapted strain of sars-cov resulted in more severe disease than infection with a non-adapted sars-cov strain 59, 60 . moreover, mers-cov infection of wild-type mice that were transduced with human dpp4 caused mild disease, but symptoms were more severe in myd88 −/− mice and ifnar1 −/− mice 61 . sars-cov and mers-cov avoid host detection of their dsrna by replicating in virus-induced doublemembrane vesicles that lack prrs 19, 62, 63 . moreover, the recognition of sars-cov mrnas, for example, by mda5 and ifit1 is prevented by capping of the viral mrnas by nsp14 and the nsp10-nsp16 complex 64 . recombinant sars-cov that lacks the methylation activity of nsp16 is attenuated and exhibits increased sensitivity to type i ifns. this effect is dependent on ifit1 or mda5, as the same virus is not attenuated in mice that are deficient in either molecule 65 . although mrna capping has not yet been shown for mers-cov, structural similarity between the mers-cov nsp10-nsp16 complex and the sars-cov nsp10-nsp16 complex suggests that a similar mechanism exists to avoid host recognition of mers-cov mrnas by cytosolic prrs 66 . sars-cov encodes at least eight proteins that interact with the signalling cascades downstream of prrs; in mers-cov, several proteins have been identified with similar functions (fig. 3) . the nucleocapsid protein of sars-cov has been associated with the suppression of rnai in mammalian cells 67 . furthermore, this protein antagonizes ifn induction, probably early in the signalling cascade, as downstream signalling molecules relieve the inhibition 68 . mers-cov orf4a has a similar ifn-antagonistic function, involving the binding of dsrna and subsequent inhibition of mda5 activation 69 , potentially through interaction with ifn-inducible dsrna-dependent protein kinase activator a (prkra; also known as pact), which interacts with mda5 and rig-i 70 . moreover, mers-cov orf4a, orf4b, orf5 and membrane protein inhibit the nuclear trafficking of ifn-regulatory factor 3 (irf3) and activation of the ifnb promoter 71 . these viral proteins, except for orf5, also inhibit the expression of genes that are under the control of an ifn-stimulated response element (isre), and orf4a reduces the expression of genes that are stimulated by nf-κb 71 . finally, mers-cov orf4b interacts with tbk1 and inhibitor of nf-κb kinase-ε (ikkε), thereby suppressing the interaction between ikkε and mitochondrial antiviral-signalling protein (mavs) and inhibiting the phosphorylation of irf3 (ref. 72 ). the membrane protein of sars-cov inhibits the formation of a signalling complex that contains ikkε, thus repressing the activation of irf3 and irf7 and their induction of type i ifn expression. the membrane protein of mers-cov inhibits irf3 function and the expression of genes that are regulated by an isre, including ifnβ 71 , but whether this occurs through a mechanism similar to that of sars-cov is unclear. sars-cov plpro disrupts nf-κb signalling 73 and blocks the phosphorylation of irf3 indirectly 73, 74 . furthermore, sars-cov plpro inhibits the induction of type i ifns, potentially through the deubiquitylation of phosphorylated irf3 (refs 73, 75) . similar functions have been described for mers-cov plpro 76 . experiments involving recombinantly expressed proteins, in vitro translation, protein overexpression and minireplicon systems have shown that nsp1 of sars-cov blocks the ifn response through the inhibition of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have an incubation period of ~5 days, and 95% of patients develop disease within 13 days of exposure 14, 38, [144] [145] [146] . common early symptoms are fever, chills, coughing, malaise, myalgia and headache, and less common symptoms include diarrhoea, vomiting and nausea 2, 6, 39, 89, 90, 92, 95, 144, [146] [147] [148] . upper respiratory tract symptoms and viral shedding are rare, which explains difficulties in obtaining a laboratory diagnosis from nasal or nasopharyngeal swabs 149 . abnormal chest x-rays are more common in patients with mers (90-100%) 144,148 than in those with sars (60-100%) 39, 89 . accordingly, only 20-30% of patients with sars require intensive care and subsequent mechanical ventilation, whereas 50-89% of patients with mers require intensive care 2, 39, 89, 90, 95, 144, 147, 148 . the higher incidence of acute respiratory distress syndrome (ards) in individuals with mers is reflected in the case fatality rate: this is ~36% for mers compared with ~10% for sars 15, 145 . comorbidities have an important role in both sars and mers. several risk factors are associated with poor disease outcome, especially advanced age and male sex 2, 14, 39, 144, 146, 148, 150, 151 . for mers, additional risk factors for a poor outcome include diabetes mellitus, hypertension, cancer, renal and lung disease, and co-infections 14, 144, 146, 148, 150, 151 . health care settings seem to increase the risk of viral transmission owing to aerosol-generating procedures such as intubation and bronchoscopy. appropriate hospital hygiene practices and awareness are crucial to limit future nosocomial outbreaks. both nsp7 and nsp15 from sars-cov were also suggested to be ifn antagonists, but the underlying mechanism is unknown 73 . nsp15 is an inhibitor of mavsinduced apoptosis; however, this occurs through an ifn-independent mechanism 84 . finally, transcriptomic and proteomic analysis of human airway cell cultures showed that mers-cov but not sars-cov induces repressive histone modifications that downregulate the expression of certain isgs 85 . it should be noted that most of the interactions of sars-cov and mers-cov proteins with innate immune pathways were established in in vitro systems, which rely on the overexpression of viral and, . following prr-mediated detection of a pamp, the resulting interaction of prrs with mitochondrial antiviral-signalling protein (mavs) activates nuclear factor-κb (nf-κb) through a signalling cascade involving several kinases. activated nf-κb translocates to the nucleus, where it induces the transcription of pro-inflammatory cytokines. the kinases also phosphorylate (p) ifn-regulatory factor 3 (irf3) and irf7, which form homodimers and heterodimers and enter the nucleus to initiate the transcription of type i interferons (type i ifns). both severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have developed mechanisms to interfere with these signalling pathways, as shown; these subversion strategies involve both structural proteins (membrane (m) and nucleocapsid (n)) and non-structural proteins (nsp1, nsp3b, nsp4a, nsp4b, nsp5, nsp6 and papain-like protease (plpro); indicated in the figure by just their nsp numbers and letters). b | binding of type i ifns to their dimeric receptor, ifnα/β receptor (ifnar), activates the janus kinase (jak)-signal transducer and activator of transcription (stat) signalling pathway, in which jak1 and tyk2 kinases phosphorylate stat1 and stat2, which form complexes with irf9. these complexes move into the nucleus to initiate the transcription of ifn-stimulated genes (isgs) under the control of promoters that contain an ifn-stimulated response element (isre). collectively, the expression of cytokines, ifns and isgs establishes an antiviral innate immune response that limits viral replication in infected and in neighbouring cells. again, viral proteins have been shown to inhibit these host signalling pathways to evade this immune response. iκbα, nf-κb inhibitor-α. a broadly active antiviral nucleoside analogue with several direct and indirect mechanisms of action; mainly used for the treatment of hepatitis c, in combination with interferon. having polyethylene glycol (peg) attached, to a drug for example; this moiety improves the solubility, decreases the immunogenicity and increases the stability, of the drug of interest, thereby allowing a reduced dosing frequency to be used. (table 1) and sars-cov, including the use of antibodies, ifns, inhibitors of viral and host proteases, and host-directed therapies. in the absence of a clinically proven effective antiviral therapy against sars-cov and mers-cov, patients mainly receive supportive care, which is often supplemented by different combinations of drugs. ribavirin 86 and various types of ifn have been given to patients with mers in saudi arabia 87 and china 88 , typically in combination with a broad-spectrum antibiotic and oxygen. the efficacy of treatments for sars-cov and mers-cov infection currently remains unclear. in addition, treatment for mers is typically started only in a late disease stage, when immunopathology predominates and antiviral drugs are likely to provide little benefit. ribavirin was used most frequently during the sars outbreak, often in combination with corticosteroids, which have an anti-inflammatory effect 2,89-92 . ifnα was also given, usually in combination with immunoglobulins or thymosins, which stimulate the development of t cells, and in a small number of cases in combination with ribavirin 93, 94 . none of these treatments was tested in a clinical trial, which makes it difficult to assess their efficacy. in fact, retrospective analysis did not yield a treatment combination that was clearly effective. moreover, the data from patients are contradictory about whether ribavirin, when used alone, provided a benefit or was possibly even detrimental 89, 90, 92, 95 . in vitro coronaviruses have a lower sensitivity to ribavirin than other viruses. deletion of the nsp14-encoding sequence increases the sensitivity of coronaviruses to ribavirin; however, the underlying mechanism is unclear and is not related to the proofreading function of nsp14 (ref. 96 ). therefore, ribavirin should be considered only in combination with other antiviral treatments. although ifns are effective against mers-cov in vitro [97] [98] [99] , their effect in humans has yet to be proved. the effectiveness of ifn is increased in vitro if ribavirin is added 98, 100 , and a combined use of the two drugs reduces disease severity in a rhesus macaque model of mers 101 . the potential side effects of these treatments, such as fatigue, depression and anaemia, have inhibited their use as a first-line treatment for mers, and they are generally administered only after a patient's condition starts to deteriorate. for example, one study of five patients who were infected with mers-cov indicated no survival following ribavirin and ifnα2b therapy; however, therapy was not started until 10 days after admission 87 . a separate study found an improvement in survival 14 days after mers diagnosis and the start of treatment, but not 28 days after 102 . in a third study, a combination of ifnα2a and ribavirin or ifnβ1a and ribavirin did not improve survival; however, some of the patients were more than 50 years old and had preexisting renal failure 103 . in a single case in which ribavirin and ifnα2b were started shortly after admission to hospital, the patient started to improve on day 6 after admission and made a complete recovery 104 . ifnβ1b is a more potent inhibitor of mers-cov replication in vitro than other types of ifn 97, 99 , and an improved outcome of disease was observed in common marmosets after challenge with mers-cov 105 . thus, the type of ifn that is used for treatment in humans should be reconsidered (usually, ifnα is used). furthermore, ribavirin and/or ifns should be tested in clinical trials to determine their efficacy in mers treatment and to establish treatment protocols. additional antiviral treatments. the protease inhibitors lopinavir and ritonavir, which are used in combination to treat infection with hiv, improved the outcome of patients with sars when combined with ribavirin, compared with patients who were treated with ribavirin alone 106, 107 . lopinavir showed no clear antiviral activity against mers-cov in vitro 97 , and it is thus rarely used in patients with mers. however, lopinavir and ritonavir improve the outcome in common marmosets when treatment is initiated 6 hours after infection with mers-cov 105 . thus, the testing of lopinavir and ritonavir in clinical trials in patients with mers should be reconsidered. one patient who received pegylated ifnα, ribavirin, lopinavir and ritonavir in combination had undetectable levels of mers-cov in the blood 2 days after the initiation of therapy; however, this patient did not survive 108 . the combination of ifnα, ribavirin, lopinavir and ritonavir was also used for mers treatment in south korea, but efficacy data are not yet available. however, three case reports indicate recovery in five out of seven patients who were treated with this combination [109] [110] [111] . as 3clpro and plpro are essential for cleavage of the viral polyproteins and are distinct from cellular proteases, they are ideal drug targets, in particular plpro, compounds that mimic biologically active peptides or proteins. a complement-activated molecule that is important for the recruitment to and activation of inflammatory cells in the lungs. which is involved in both viral replication and ifn antagonism. indeed, most antiviral drug-like molecules have been developed against 3clpro and plpro, which was aided by the rapid report of crystal structures of these proteases 112 . plpro was initially identified as a drugable target for sars-cov; recently, it has been noted that some of the compounds that target plpro from sars-cov are also active against plpro from mers-cov. for example, both 6-mercaptopurine and 6-thioguanine inhibit mers-cov and sars-cov in vitro 113 ; however, the efficacy of these molecules has not yet been tested in vivo. mycophenolic acid also inhibits the replication of mers-cov in vitro 97, 99 through the inhibition of plpro 113 , but it had no effect in marmosets 105 . as new coronaviruses are likely to emerge from bats, protease inhibitors were designed against bat coronaviruses with the goal of developing a universal antiviral compound against emerging zoonotic coronaviruses. this approach yielded an inhibitor of tylonycteris bat coronavirus hku4 (hku4-cov), which is closely related to mers-cov 11 . this inhibitor, named sg85, indeed inhibits mers-cov replication in vitro 112 . similarly, peptidomimetics that target and inhibit 3clpro of mers-cov, hku4-cov and pipistrellus bat coronavirus hku5 (hku5-cov) have also been identified, but have not yet progressed beyond the in vitro stage 114 . several other drugs that were approved for use in humans were shown to inhibit the replication of mers-cov in vitro, notably chloroquine, chlorpromazine, loperamide and cyclosporine a 62, 99, 113, 115 , although their mechanisms of action are unknown, and the benefit of cyclosporine a in patients is debatable owing to the immunosuppressive effect of the drug. although cyclophilin inhibitors that do not result in immunosuppression are available, their activity against mers-cov has not yet been tested. antibody and plasma therapy. plasma from convalescent patients and/or antibody therapies have been the leading proposed treatment for mers so far 116 . there are several potential advantages to this approach. for example, as case numbers increase, the pool of survivors becomes larger; provided these individuals have sufficiently high antibody titres and are willing and able to donate plasma, this is a low-tech, reasonably safe treatment option. furthermore, generation of monoclonal antibodies for use in humans is well established, with a fairly straightforward path to safety and efficacy testing. however, to date, there are very few reports on the use of convalescent plasma and none on the use of monoclonal antibodies as treatments for acute, severe respiratory disease in humans. a post hoc meta-analysis of 32 studies of either sars or severe influenza found a significant reduction in the pooled odds of mortality when convalescent plasma was used 117 . however, study design was rated as low or very low quality, as there were generally a lack of control groups and a moderate-to-high risk of bias, which suggests that a properly designed clinical trial of convalescent plasma use in severe respiratory infections is needed 117 . potent monoclonal antibodies that neutralize the mers-cov spike protein in vitro have been developed [118] [119] [120] [121] . however, with a few exceptions, in vivo data relating to the use of convalescent plasma or monoclonal antibodies in the treatment of mers are currently lacking. serum from high-titre dromedary camels decreased mers-cov loads in the lungs of mice that had been transduced with human dpp4 (ref. 122 ). human polyclonal antibodies against the spike protein were generated by vaccinating transchromosomic bovines, and treatment with these antibodies reduced viral titres in the lungs of dpp4-transduced mice when treatment was administered 24 or 48 hours after challenge with mers-cov 123 . dpp4-transduced mice were also treated with humanized neutralizing monoclonal antibody 4c2h, which is directed against the receptor-binding domain of the mers-cov spike protein, 1 day after mers-cov challenge, and this treatment also decreased viral titres in the lungs 124 , as did the neutralizing antibody lca60, which was obtained from a convalescent patient and produced recombinantly 125 . human neutralizing monoclonal antibodies regn3048 and regn3051 also provided a benefit in mice that expressed human dpp4 and were challenged with mers-cov 126 . the human neutralizing monoclonal antibody m332 reduced mers-cov replication in the lungs of rabbits following prophylactic, but not therapeutic, treatment 127 . treatment of rhesus macaques with the human monoclonal antibody 311b-n1 day after challenge resulted in reduced lung pathology 128 . in all of these studies, viral replication was not completely inhibited, and there were some pathological alterations to the lungs, despite the therapy. furthermore, none of the studies addressed the potential emergence of escape mutants in vivo. alternatively, antibodies that target the region of dpp4 that binds to the spike protein could be used to prevent entry of mers-cov; this approach was successful in vitro 129 . however, whether such a treatment would be feasible and would not have substantial adverse effects in humans remains to be determined. host-directed strategies can also limit viral replication. for example, the spike protein of sars-cov is cleaved by cathepsin b and cathepsin l, transmembrane protease serine 2 (tmprss2) and possibly other host proteases 130, 131 . inhibition of host serine proteases by camostat reduced the entry of sars-cov and increased survival in a mouse model 132 . however, the targeting of host proteases is more likely to result in undesirable side effects than the targeting of viral proteases. another underappreciated strategy is attenuation of detrimental host responses. the development of such treatments would require a thorough understanding of the host responses that are involved in acute lung injury and ards, processes that are unfortunately poorly understood at the moment. nonetheless, in vitro studies and limited studies in animal models with other respiratory viruses have shown that anaphylatoxin c5a is important for the development of acute lung injury, and blocking anaphylatoxin c5a can reduce lung pathology 133 . vaccines that contain immunogenic parts of a pathogen rather than the entire pathogen. vaccines based on the direct introduction of a plasmid encoding an antigen; following in situ production of this antigen, an immune response is mounted against it. changes in gene expression during in vitro mers-cov infection were used to predict potential effective drugs. one of the drugs with predicted efficacy, the kinase inhibitor sb203580, modestly inhibited sars-cov and mers-cov replication following the treatment of cells prior to infection, but treatment after infection inhibited replication of only sars-cov and not mers-cov 134 . vaccines. vaccination could be used to prevent infection or to reduce disease severity, viral shedding and thereby transmission, thus helping to control mers outbreaks. several vaccination strategies were developed against sars-cov and tested in animals, such as an inactivated virus, a live-attenuated virus, viral vectors, subunit vaccines, recombinant proteins and dna vaccines 135, 136 . similar approaches have been used for the development of experimental mers-cov vaccines 137 . to date, three mers-cov vaccines have been evaluated in non-human primates. in one study, rhesus macaques were primed with dna encoding the spike protein, followed by boosts with spike dna and with recombinant protein consisting of the spike subunit containing the receptor-binding domain, or primed and boosted once with the subunit protein. both approaches reduced pathological changes in lung function in animals that were infected with mers-cov 19 weeks after the last vaccination 138 . moreover, three vaccinations with a recombinantly expressed protein that contains the receptor-binding domain of the spike protein reduced viral loads and lung pathology in rhesus macaques that were infected 2 weeks after the last vaccination 139 . three dna vaccinations with a construct encoding the full-length spike sequence reduced viral loads and pathology in the lungs after challenge with mers-cov 5 weeks after the last vaccination 140 . one concern of vaccination in humans is vaccinemediated enhancement of disease, a process in which the disease following infection is more severe in vaccinated individuals than in unvaccinated individuals. although this was observed in only a small subset of vaccine studies that were carried out for sars-cov 136 and has not yet been observed in any of the published mers-cov vaccine studies, it is an important concern. moreover, it is unclear who to vaccinate against mers-cov, as healthy individuals seem to be at little risk of severe disease. older patients or patients with underlying disease, who have the highest risk of severe mers, would be important target populations. however, vaccination in such patients can be problematic owing to their poor immune responses, as has been established for influenza virus 141 . in addition, vaccination of people with a high risk of exposure to mers-cov, such as health care workers, slaughterhouse workers and camel herders, is advisable 142 . as our understanding of the pathogenesis of emerging coronaviruses increases, so will the opportunities for the rational design of therapeutics that target viral replication or immunopathology. the rational design of new drugs and the repurposing of existing compounds have already resulted in the development of plpro inhibitors and the identification of kinase inhibitors that inhibit the replication of sars-cov and mers-cov in vitro. however, only a few potential treatments have progressed past the identification of an effect in vitro, and in vivo studies to select the most promising treatment options are required. the development of several mouse models of mers is thus an important step forward (box 2) . owing to the acute nature of mers and the important role of immunopathology, combination therapies aimed at simultaneously inhibiting viral replication, limiting viral dissemination and dampening the host response are likely to yield the best results. furthermore, treatment should be started as early as possible, rather than waiting until the patient has already developed extensive lung damage. the development of therapies against sars and mers needs to focus on testing in humans, in properly controlled clinical trials. the current non-standardized, uncontrolled approach to treatment is not informative and may not be beneficial to the patient. the recent ebola outbreak has demonstrated that rapid clinical trial design and approval are possible and that exceptional situations call for deviations from normal procedures . while treatments are being developed and evaluated, the prevention of viral transmission is key to reducing the burden of mers. the large proportion of nosocomial mers-cov infections indicates that preventive measures in hospitals are currently either not fully implemented or insufficient. prevention of zoonotic transmission from dromedary camels is another possibility to decrease the number of mers cases. the most of our understanding of the pathogenesis of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) comes from animal studies. ideally, these models recapitulate all or specific aspects of human disease. several mouse models have been established, for example by using mouse-adapted sars coronavirus (sars-cov) or expressing human receptors in mice 152 . although it has been recognized that mice might poorly mimic specific human responses to infection, the availability of knockout and transgenic mice enables the targeted study of virus-host interactions. several non-human primate models have been developed for sars-cov and mers coronavirus (mers-cov) 152 . the close relationship of non-human primates to humans often allows faithful recapitulation of a disease and the host response. however, these benefits are countered by the need for specialized husbandry, the sometimes limited availability of the animals and reagents, and high costs. the pathogenesis of sars-cov and mers-cov in their respective reservoir hosts is not nearly as well studied as that in humans. currently, only one experimental-infection study has been carried out in bats with mers-cov 153 , and none has been carried out for other coronaviruses. thus, data are mostly limited to the detection of coronaviruses in naturally infected bats. the detection of coronaviruses mainly in faecal samples from bats and not in oral swabs suggests that replication in bats occurs predominantly in the gastrointestinal tract 9,154,155 . by contrast, a combination of intratracheal and intranasal inoculation of masked palm civets with sars-cov resulted in interstitial pneumonia, with oral and rectal viral shedding 156 . the pathogenesis of mers-cov in dromedary camels has been studied experimentally in a limited number of animals. these animals developed transient mild disease; however, large quantities of mers-cov were shed from the upper respiratory tract, in line with the predominant replication of mers-cov in the nasal turbinates and larynx in these animals, which explains the frequent zoonotic transmission 157 . first vaccines against mers-cov have been tested in dromedary camels 140, 143 ; indeed, when camels were vaccinated with a modified vaccinia virus that expresses the mers-cov spike protein, subsequent challenge of these animals with mers-cov resulted in less viral shedding than in unvaccinated animals 143 , thereby potentially limiting the transmission to naive animals or to humans. certainly, there has been progress in the development of vaccines and therapies against emerging coronaviruses, but more research and rigorous testing is required if we are to successfully combat these novel pathogens. when the severe acute respiratory syndrome (sars) outbreak developed into the first pandemic of the twenty-first century, it became clear that the medical and scientific communities were not adequately prepared for the emergence of highly pathogenic viruses. whereas several months elapsed and several thousand cases of sars were observed before the causative agent was identified as sars coronavirus (sars-cov) 4-6 , subsequent advances in molecular diagnostic tools, such as next generation sequencing, meant that middle east respiratory syndrome coronavirus (mers-cov) was identified before it caused a large outbreak of mers 11 . the availability of the full-length genome of mers-cov enabled the rapid development and distribution of diagnostic assays. the first animal models of disease, several treatment efficacy studies and the identification of the reservoir followed soon after. unfortunately, the sars pandemic did not yield solid clinical data on the efficacy of treatment regimens. these data are urgently needed for the treatment of mers, as well as to prepare for novel coronaviruses that may emerge. several studies have used synthetic biology to study the zoonotic transmission potential of sars-cov-like viruses from bats 9,10,158,159 . the ebola virus outbreak in west africa has highlighted the need for fast-tracking of potential treatments, as several clinical trials were started only towards the end of the outbreak. the combined experiences of the outbreaks of sars, mers and ebola provide a blueprint for the response to emerging pathogens: after the identification of the causative agent, diagnostic assays need to be developed and distributed rapidly, and simultaneously, awareness of the new syndrome and reporting of (suspected) cases 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syndrome coronavirus isolates the first description of mers-cov replication and shedding in the respiratory tract of dromedary camels synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice sars-like wiv1-cov poised for human emergence the work was supported by the intramural research program of the national institute of allergy and infectious diseases (niaid), us national institutes of health. the authors declare no competing interests. key: cord-328175-4i3cz20j authors: van doremalen, neeltje; falzarano, darryl; ying, tianlei; de wit, emmie; bushmaker, trenton; feldmann, friederike; okumura, atsushi; wang, yanping; scott, dana p.; hanley, patrick w.; feldmann, heinz; dimitrov, dimiter s.; munster, vincent j. title: efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets date: 2017-07-31 journal: antiviral research doi: 10.1016/j.antiviral.2017.03.025 sha: doc_id: 328175 cord_uid: 4i3cz20j abstract cases of middle east respiratory syndrome coronavirus (mers-cov) continue to be identified and with a lack of effective clinical treatment and no preventative strategies, treatment using convalescent plasma or monoclonal antibodies (mabs) is a potential quick route to an intervention. passive immunotherapy via either convalescent plasma or mabs has proven to be effective for other infectious agents. following infection with mers-cov, common marmosets were treated with high titer hyperimmune plasma or the mab m336, at 6 and 48 h post inoculation. both treatments reduced signs of clinical disease, but reduction in viral loads in the respiratory tract were only found in the hyperimmune plasma group. a decrease in gross pathology was found only in the mab-treated group, but no histological differences were observed between treated and control animals. while both hyperimmune plasma and the m336 treatments reduced the severity of disease in the common marmoset, neither treatment resulted in full protection against disease. middle east respiratory syndrome coronavirus (mers-cov) was first detected in 2012 in a resident of saudi arabia, and has since resulted in > 1900 cases with a case fatality rate of 36% (who, 2015) . the severity and the epidemic potential of mers-cov highlights the importance of the development of treatment options. as of yet, no specific vaccine or antiviral treatment against mers-cov is available. few studies have been published investigating the effectiveness of existing antiviral treatments, and no treatments have been thoroughly assessed in clinical trials as of yet. convalescent plasma has been identified by the world health organization (who) and the international severe acute respiratory and emerging infection consortium (isaric) as a potential treatment against mers-cov to reduce clinical consequences of mers-cov infection (2013 , who, 2014 and recently a study protocol was developed to investigate the feasibility of convalescent plasma treatment in mers patients (arabi et al., 2015) . in vivo, the administration of convalescent sera obtained from dromedary camels resulted in dose-dependent decreased lung viral titers and disease severity in an adenovirus-hdpp4 mouse model . several monoclonal antibodies (mabs) have been developed against mers-cov, which show neutralizing capacity in vitro (jiang et al., 2014; tang et al., 2014; ying et al., 2014) . efficacy of mabs has been assessed in several mers-cov mouse models generally showing reduction in virus replication (corti et al., 2015; li et al., 2015; pascal et al., 2015; luke et al., 2016) . these studies suggest that mabs have potential as mers-cov treatment. the mab m336, identified from a large phage-displayed antibody library panned against recombinant mers-cov spike protein receptor binding domain, inhibited 90% mers-cov pseudovirus infection at a concentration of 0.039 mg/ml (ying et al., 2014) . m336 was shown to almost completely overlap with the binding site of dpp4 and mimic critical interactions between dpp4 and the mers-cov spike protein (modjarrad et al., 2016) . it has therefore been speculated that the potential for viral escape mutants might be limited by the requirement of the spike protein to bind to dpp4 (ying et al., 2015) . prophylactic treatment with m336 resulted in significantly reduced viral titer in rabbit lung tissue (houser et al., 2016) and both prophylactic and therapeutic treatment with m336 protected mice against lethality by mers-cov infection (agrawal et al., 2016) . here we assess the effect of treatment with marmoset-derived hyperimmune plasma as well as the human mab m336 on disease outcome in the recently developed marmoset mers-cov infection model, which recapitulates severe respiratory disease (falzarano et al., 2014) . approval of animal experiments was obtained from the institutional animal care and use committee at rocky mountain laboratories. all experiments were performed in an association for assessment and accreditation of laboratory animal care-approved facility by certified staff, following the guidelines and basic principles in the united states public health service policy on humane care and use of laboratory animals, the nih guide for the care and use of laboratory animals and the animal welfare act, united states department of agriculture. the institutional biosafety committee (ibc) approved work with infectious mers-cov strains under bsl3 conditions. sample inactivation was performed according to ibc-approved standard operating procedures for removal of specimens from high containment. hyperimmune plasma was obtained from a convalescent common marmoset (callithrix jacchus) from a previous experiment (falzarano et al., 2014) inoculated with 5.2 â 10 6 tcid 50 mers-cov via the intratracheal, intranasal, ocular and oral route, then inoculated with 5.2 â 10 6 tcid 50 mers-cov on 20 dpi via the intratracheal route and finally inoculated with 5.2 â 10 6 tcid 50 mers-cov adjuvated with titermax gold (sigma aldrich) on 41 dpi via the intramuscular route. the final virus neutralizing (vn) titer was 3840. this method was chosen as sera collected after the initial infection did not contain sufficient neutralizing antibodies (vn titer ¼ 40). as a control, plasma was obtained from an uninfected common marmoset (internal collection), vn titer <20. the common marmoset mers-cov infection model was used; mers-cov infection results in the development of more severe respiratory disease than observed in the rhesus macaque model (de wit et al., 2013; munster et al., 2013; falzarano et al., 2014) . common marmosets were procured from an usda-approved source (worldwide primates inc). animals were monitored for the presence of disease by clinical observation and serology for the presence of disease at worldwide primates inc. additionally, when animals arrived at rocky mountain laboratories they were placed in quarantine and clinically evaluated by serum chemistry, complete blood counts and thoracic radiography to confirm absence of previous infection. three different groups were created; the hyperimmune plasma group (h), the monoclonal antibody group (m), and the control group (c). three animals were randomly assigned per group and inoculated as described previously (falzarano et al., 2014) . briefly, inoculation with mers-cov strain emc/2012 was performed intranasally (100 ml per nare), orally (500 ml), intratracheally (500 ml) and ocular (50 ml per eye) with dmem containing 4 â 10 6 tcid 50 mers-cov/ml (total dose 5.2 â 10 6 tcid 50 ). the hyperimmune plasma and monoclonal antibody groups, consisting of one female and two male common marmosets each, received 1 ml hyperimmune plasma or m336 diluted in pbs (5 mg/ml) intravenous (i.v.) at 6 hpi, and 1 ml hyperimmune plasma or m336 subcutaneous (s.c.) at 2 dpi (marmosets h1-3, m1-3). two out of three animals in the control group (all male common marmosets), received 1 ml control plasma i.v. 6 hpi, and 1 ml control plasma s.c. 2 dpi (marmosets c1-2). the third animal received 1 ml of pbs (diluent of the mab) via the same routes (marmoset c3). the animals were observed twice daily for clinical signs of disease, using a scoring system as described previously (falzarano et al., 2014) . breathing was scored as normal (<60/minute), increased (60e100/ minute), or severely increased (>100/minute). based on the scoring sheet, euthanasia was indicated at a clinical score of 35 or more. clinical exams were performed on 0, 2, 5, and 7 dpi on anaesthetized animals using isoflurane and ketamine. x-rays were taken and nasal, oral, fecal, and urogenital swabs were collected in 1 ml dmem with 50 u/ml penicillin and 50 mg/ml streptomycin. blood samples were collected on à5, 2, and 7 dpi and examined using the piccolo xpress chemistry analyzer (abaxis). the blood sample collected on 2 dpi was obtained before treatment was administered. temperature was monitored with iptt-300 temperature probes (bmds) that were injected interscapularly prior to the start of the experiment. all animals were euthanized at 7 dpi (fig. 1a) . terminal blood samples were obtained and samples of the following tissues were collected: conjunctiva, nasal mucosa, tonsil, trachea, four lung lobes, mediastinal lymph node, liver, spleen, kidney, and bladder. gross pathology (surface area of the lung which was either consolidated and/or hyperemic) per lung lobe was documented as percentage area affected by lesions. radiographic images acquired included ventrodorsal, right lateral and left lateral thoracic images. thoracic radiographs were obtained using a mobile digital radiography unit with a flat panel digital detector (sound technologies tru/dr, sound-eklin carlsbad, ca). each set of radiographs was graded according to a published scoring paradigm (brining et al., 2010) as follows: 0, normal examination; 1, mild interstitial pulmonary infiltrates; 2, moderate interstitial infiltrates, perhaps with partial cardiac border effacement and small areas of pulmonary consolidation (alveolar patterns and air bronchograms); 3, pulmonary consolidation as the primary lung pathology, seen as a progression from grade 2 lung pathology. grading per animal was done independently and blinded by two veterinarians. hcov-emc/2012 was provided by the erasmus medical center, rotterdam, the netherlands. virus propagation was performed in veroe6 cells (provided by the bowen laboratory, colorado state university) in dmem supplemented with 2% fetal calf serum, 1 mm l-glutamine, 50 u/ml penicillin and 50 mg/ml streptomycin (2% dmem). veroe6 cells were maintained in dmem supplemented with 10% fetal calf serum, 1 mm l glutamine, 50 u/ml penicillin and 50 mg/ml streptomycin. marmoset tissues were evaluated for pathology and the presence of viral antigen. all tissues were fixed for a minimum of 7 days in 10% neutral-buffered formalin and subsequently embedded in paraffin. lungs were perfused with 10% formalin and processed for histologic review. the lung is divided into right upper, right lower, left upper, and left lower lobe. each of these four sections are then sampled at the hilus, at mid-lobe and at the periphery of the lobe for a minimum of 12 sections per animal. this method is used for all non-human primate studies at rocky mountain laboratories. hereafter, tissue sections were stained with hematoxylin and eosin. for the detection of viral antigen immunohistochemistry was performed using an in-house produced rabbit polyclonal antiserum against hcov-emc/2012 (1:1000). grading was done blinded by a board-certified veterinary pathologist. to obtain morphometrical data of immunohistochemistry staining, stained sections were scanned with an aperio scanscope xt (aperio technologies, inc., vista, ca) and analyzed using the imagescope positive pixel count algorithm (version 9.1). between 30 and 105 mm squared were evaluated at 2â magnification. the default parameters of the positive pixel count (hue of 0.1 and width of 0.5) detected antigen adequately. tissue for rna analysis was collected in triplicate. lung tissue was obtained from the hilar and mid-lobe region of the lung. samples were analyzed independently in duplicate. tissues (30 mg) were homogenized in rlt buffer and rna was extracted using the rneasy method (qiagen) according to the manufacturer's instructions. rna was extracted from swabs using the qiaamp viral rna kit on the qiaxtractor. the upe mers-cov detection assay was used for the detection of mers-cov viral rna (corman et al., 2012) . 5 ml rna was tested with the rotor-genetm probe kit (qiagen) according to instructions of the manufacturer. dilutions of mers-cov with known titer were run in parallel. dilutions of in vitro transcribed upe mers-cov rna were run on the digital droplet pcr (biorad) in quadruplicate to determine genome copies. hereafter, dilutions were run on the rotor-genetm in quadruplicate. the last dilution to give a ct-value in all replicates was defined as the limit of detection (lod) in genome copies. finally, the number of genome copies was determined in the mers-cov dilutions with known titer, and lod was calculated in tcid 50 equivalent. small tissue samples (up to 100 mg) in 1 ml of 2% dmem were homogenized. hereafter, mers-cov was titrated in quadruplicate in veroe6 cells; cells were inoculated with ten-fold serial dilutions of tissue homogenate, incubated 1 h at 37 c, washed twice with pbs, and scored for cytopathic effect 5 days later. tcid 50 was adjusted for tissue weight and calculated by the method of spearman-karber. two-fold serial dilutions of heat-inactivated (30 min, 56 c) marmoset sera were prepared in 2% dmem, after which 100 tcid 50 of mers-cov virus was added. after 1hr incubation at 37 c, virus was added to veroe6 cells. at 5 dpi, cytopathic effect was scored. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, which still inhibited mers-cov virus replication. student's t-test (unpaired, one-tailed) was used to test for significance. p values of <0.05 were considered significant. all values are reported as mean ± sd. the final vn titer of hyperimmune plasma was 3840, control plasma was <20 and m336 was 491530. animals were inoculated with 5.2 â 10 6 tcid 50 mers-cov strain emc/2012 and treated iv 6 hpi and sc 48 hpi. these two different administration routes were chosen to achieve high systemic bioavailability; i.v. administration immediately results in high circulating neutralizing antibody titers whereas s.c. administration results in a slower systemic distribution of antibodies, therefore allowing a longer bioavailability (wohlrab, 2015) . animals treated with hyperimmune plasma reached a neutralizing titer between 40 and 160 at 2 dpi and maintained these levels throughout the experiment. animals treated with m336 reached neutralizing titers between 10,240 and 20,480 at 2 dpi, which decreased to 3840e7680 at 7 dpi. in contrast, serum obtained from animals treated with control plasma or pbs did not contain detectable levels of neutralizing antibodies at any time point (fig. 1b) . upon inoculation with mers-cov strain emc/2012, all animals developed clinical disease. clinical scores of control group animals were found to be higher than clinical scores of treated animals post inoculation ( fig. 2a) . no changes in body temperature were observed. all animals showed loss of appetite and decreased activity, often seen combined with a hunched posture. no changes in respiratory rate were reported for treated animals m1 and m2. increased respiration rate (>60 breaths/minute) was observed in all other animals, and progressed to labored breathing (>100 breaths/ minute) in all three control animals (c1-c3) and one treated animal (h1). this was accompanied by open mouth breathing in all control animals, but not h1. all radiographs were independently and blindly graded by clinical veterinarians (brining et al., 2010) ) and in all views were normal (score ¼ 0) prior to inoculation on day 0. within the control group, all three animals were graded at 2 at 5 dpi with severe interstitial infiltrates seen early that progressed to pulmonary consolidation within the caudal and middle lung lobes on 7 dpi (score ¼ 3). both the hyperimmune plasma treatment and the mab treatment group on average had lower graded radiographs in comparison to the control group. on 7 dpi, one hyperimmune plasma-treated animal was characterized as having grade 1 (h2, mild interstitial pulmonary infiltrates in the left caudal lung lobes), while the remaining two hyperimmune plasma-treated animals had either moderate (h1, grade 2) or severe (h3, grade 3) radiographic signs. one mab-treated animal had normal radiographic findings (m2, grade 0) throughout the duration of the study, whereas the other two animals (m1, m3) were scored as presenting with mild radiographic findings (grade 1) ( fig. 2b and c and fig. s1 ). blood chemistry values were investigated using the piccolo xpress chemistry analyzer on à5, 2, and 7 days post inoculation. overall, no apparent differences in measured clinical chemistries were noted between the control and the treated groups. the values of the investigated parameters (bun, creatinine, alt, ast, alp, ggt, total protein, glucose and calcium) were found to be within the normal ranges for marmosets, and no consistent patterns or trends were noted between groups (fig. s2 ). gross pathology of the lungs was scored by a board-certified veterinary pathologist for each lung lobe, both dorsal and ventral. for the control animals, the median percent lesions was 32.5% (c3), 55% (c1) and 67.5% (c2). no abnormal pathological findings were observed in one of the hyperimmune plasma-treated animals (h2), whereas the median of gross pathology of the lungs in the other two treated animals was 25% (h1) and 77.5% (h3). animals treated with m336 showed relatively little gross pathology (median ¼ 0% (m1), 2.5% (m2) and 22.5% (m3)). this difference between control and treated animals was found to be significantly different using an unpaired one-tail student's t-test (hyperimmune plasma-treated p ¼ 0.0352; mab-treated p ¼ 0.0007) (figs. 2c and 3a) . as a measure of pulmonary edema and inflammation, lung to body weight ratios were calculated for each animals. no significant differences were observed between the control and hyperimmune plasmatreated group, however the mab-treated group had a significantly lower lung to body weight ratio than the control group (p ¼ 0.0039) (fig. 3b) . no histological differences were observed in the severity or nature of the pneumonia or distribution of viral antigen between the control and treated animals. all marmosets, with the exception of one treated marmoset (h2) which developed no lung pathology, developed multifocal to coalescing, moderate to marked subacute bronchointerstitial pneumonia with type ii pneumocyte hyperplasia. the adjacent alveolar interstitium was thickened by congestion, edema and fibrin and moderate numbers of macrophages and neutrophils. alveolar spaces contained moderate to marked numbers of pulmonary macrophages and neutrophils. immunohistochemistry demonstrated small numbers of pneumocytes and macrophages positive for viral antigen. these cells were predominantly associated with areas of pneumonia (fig. 3c ). no differences in the amount of viral antigen between groups could be found using morphometrical analysis. on 0, 2, 5, and 7 dpi nasal, oropharyngeal, urogenital and fecal swabs were collected and the presence of viral rna was examined via quantitative reverse transcription polymerase chain reaction (qrt-pcr). viral rna presence was often below detection limit, except for two and one nasal swabs for hyperimmune plasmatreated animals and control animals, respectively, and one oral and fecal swab for one control animal on 7 dpi. in all instances, urogenital swabs were negative (fig. s3) . upon necropsy of the animals on 7 dpi, 13 tissue samples were collected and the presence of viral rna in these tissues was analyzed using qrt-pcr. as observed in previous infection of marmosets (falzarano et al., 2014) , the highest viral loads were found in the lower respiratory tract of the animals (fig. 4a ). viral loads in lung tissues from hyperimmune plasma-treated compared to control animals were found to be significantly lower using a onetailed unpaired student's t-test (average of 4.0 â 10 4 and 1.2 â 10 6 tcid 50 equivalent/gram, respectively, p-value ¼ 0.008). this difference was found to be significant even when negative values from animal h2 were excluded from analysis (average of 5.9 â 10 4 tcid 50 equivalent/gram, p-value ¼ 0.0263). however, treatment of marmosets with m336 did not significantly reduce viral load in lung tissue (average of 5.4 â 10 5 and 1.2 â 10 6 tcid 50 equivalent/gram, respectively, p-value ¼ 0.0864) (fig. 4b) . viral rna was found in the nasal turbinates, trachea, conjunctiva, tonsils and mediastinal lymph nodes of some but not all animals with no pattern related to treatment. viral rna in the liver, spleen, kidney and bladder was below the limit of detection (fig. 4c) . no infectious virus could be found in any tissue samples. currently no specific antivirals or vaccines are approved for mers-cov treatment, and limited information is available on the efficacy of potential treatment options in vivo. a potential ventral-dorsal and lateral thoracic radiographs as well as gross pathology images of marmosets taken 7 dpi. shown are animals h3, m1 and c1. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) treatment for mers-cov would be the use of neutralizing antibodies, either via convalescent plasma or monoclonal antibodies. severity of disease has been associated with a delayed adaptive immune response (park et al., 2015) , and thus antibody-based therapy might be beneficial. administration of neutralizing antibodies early in disease onset when patients were infected with respiratory pathogens such as sars-cov and influenza a virus was reported to be beneficial (mair-jenkins et al., 2014) . treatment of sars-cov-infected patients with convalescent plasma early in disease progression resulted in a higher likelihood of disease remission and survival (cheng et al., 2005) and administering convalescent plasma in two sars-cov-infected healthcare workers resulted in complete recovery (yeh et al., 2005) . convalescent plasma treatment of patients with severe a(h1n1)pdm09 influenza virus infection resulted in reduced mortality and respiratory tract viral load (hung et al., 2011) . meta-analysis of studies on the treatment of spanish influenza h1n1 1918 with convalescent plasma showed an absolute reduction of 21% in case-fatality rate (luke et al., 2006) . a recent study suggests that the availability of donors with sufficiently high mers-cov antibody titers might be limited; only 12 out of 443 tested sera obtained from patients, healthcare workers and household contacts were positive on elisa (arabi et al., 2016) . furthermore, it has been suggested that severity of disease is linked to serological response; in patients who developed severe disease upon infection with mers-cov higher neutralizing antibody levels were detected, whereas in patients with mild or subclinical disease lower and potentially short-lived neutralizing antibody levels were detected (drosten et al., 2014; park et al., 2015) . it has been well-established that severity of disease is linked to the prevalence of comorbidities (badawi and ryoo, 2016) , the existence of which might contradict the collection of convalescent serum. thus, the pool of healthy subjects with sufficient neutralizing antibody titer in convalescent plasma might be very limited. finally, the hyperimmune plasma used in this study had a high neutralizing antibody titer of 3840, whereas at best human convalescent plasma has neutralizing antibody titers of 320e800 (park et al., 2015; arabi et al., 2016) (these studies use different methods to measure neutralizing antibodies). it is unclear whether convalescent plasma with lower neutralizing titers would have a similar effect on disease progression, as lower neutralizing antibody titers in the circulation will likely be reached. monoclonal antibodies could provide an alternative; mab treatment of healthy volunteers inoculated with influenza a virus h3n2 resulted in a reduction in median viral load in nasal swabs and a 35% reduction in median total symptoms (ramos et al., 2015) . as of yet, very few studies have been done to evaluate the efficacy of convalescent plasma treatment of mers-cov. administration of camel sera to mice sensitized to mers-cov infection resulted in a decrease in viral titer in lungs. in addition, when repeating the study with type i interferon receptor-deficient (ifnar à/-) mice, which lose weight upon inoculation with mers-cov, a decrease in weight loss as well as less severe histological changes in lung tissue was observed . so far, only one documented mers-cov case has received convalescent plasma treatment and this patient was still hospitalized on day 77 of illness (park et al., 2015) . the effect of mabs against mers-cov has been shown both in vitro (jiang et al., 2014; tang et al., 2014; ying et al., 2014) and prophylactic and therapeutic administration of mabs to mers-cov mouse models has been shown to decrease the viral titer and load found in lung tissue (corti et al., 2015; li et al., 2015; pascal et al., 2015; luke et al., 2016) . prophylactic treatment with m336 via the i.v. or i.n. route resulted in significantly reduced viral titer (40e9000 fold) in rabbit lung tissue. in contrast, administration of m336 1 dpi via the same routes did not reduce viral rna titers in the lungs of rabbits (houser et al., 2016) . reduced mortality and morbidity was observed in a mers-cov mouse model upon intraperitoneal prophylactic and therapeutic treatment with m336, and therapeutic treatment resulted in a decrease in viral titer as well as viral rna in lung tissue (agrawal et al., 2016) . it can be argued that neither the rabbit nor the mouse model have a high predictive value for potential mers therapies in humans. rabbits remain asymptomatic upon inoculation with mers-cov and infection appears to be more prominent in the upper respiratory tract, which suggests that disease progression in rabbits differs considerably from that observed in patients who will require antiviral therapy most (haagmans et al., 2015) . the mouse as a model is valuable as an initial validation method of a therapy, but the predictive value of mouse models for therapeutic applications in humans is relatively limited as opposed to non-human primate models (seok et al., 2013) . in this study, hyperimmune plasma treatment of marmosets inoculated with mers-cov resulted in a small (0.5e1 log) but significant reduction in respiratory tract viral loads, as well as reduced disease severity such as observed with radiographs, compared to marmosets treated with non-convalescent plasma or pbs. however, the observed differences were relatively minimal and no differences in histopathology were observed. in contrast, treatment with m336 in the common marmoset did not result in a significant decrease in respiratory tract viral loads compared to control animals, however a significant reduction in disease severity was observed. this was most pronounced when comparing diseaseassociated signs. only one animal treated with m336 showed increased respiratory rates (>60 breaths/minute), and all animals had no evidence of infiltration radiographically, showed decreased levels of gross pathology and the lowest lung to body weight ratio. however, no changes in histology were observed compared to control animals. qrt-pcr and in situ staining for mers-cov were negative in the lungs of one marmoset treated with hyperimmune plasma, although mers-cov viral loads and associated pathology were detected in the conjunctiva of this animal. it is possible that in this case, inoculation with mers-cov did not result in a successful infection of the respiratory tract. the remaining two marmosets treated with hyperimmune plasma were found to have viral loads of up to 1.1 â 10 5 tcid 50 equivalent per gram lung tissue. the marmosets utilized in this study are outbred, and the significance of genetic factors or differences in immunology cannot be excluded. importantly, when statistical tests were performed excluding the marmoset with no viral load in the respiratory tract, differences in tissue of marmosets 7 dpi. mean values ± sd were calculated. statistical significance was calculated using a one-tailed unpaired student's t-test; p-values: * > 0.05. (c) viral load in extra-respiratory tissues from marmosets 7 dpi. mean values ± sd were calculated. red ¼ hyperimmune plasma-treated marmosets; blue ¼ mab-treated marmosets; green ¼ control marmosets. dotted line ¼ limit of detection. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) viral load in the lobes of the lower respiratory tract between control and hyperimmune plasma-treated animals were still significant (p ¼ 0.0263). we show that treatment of mers-cov infected animals with hyperimmune plasma or m336 results in lower clinical scores. treatment with mabs reduced the occurrence of severe respiratory symptoms further than hyperimmune plasma did, combined with less gross pathology. mabs might therefore be a better therapy if the goal is to elevate symptoms associated with severe mers disease. hyperimmune plasma reduced viral titers, but if a reduction in viral lung load does not result in less severe symptoms, the question is raised of what the importance is of such a change. regardless, the observed differences are small and most treated animals showed mild-to-moderate respiratory symptoms, suggesting that the effect of both treatments is limited. passive transfer of a germlinelike neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and metaanalysis thoracic radiography as a refinement methodology for the study of h1n1 influenza in cynomologus macaques (macaca fascicularis) use of convalescent plasma therapy in sars patients in hong kong detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques transmission of mers-coronavirus in household contacts infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits prophylaxis with a mers-cov-specific human monoclonal antibody protects rabbits from mers-cov infection convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis a roadmap for mers-cov research and product development: report from a world health organization consultation pneumonia from human coronavirus in a macaque model kinetics of serologic responses to mers coronavirus infection in humans pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection efficacy and safety of treatment with an anti-m2e monoclonal antibody in experimental human influenza genomic responses in mouse models poorly mimic human inflammatory diseases identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution coronavirus infections from who-isaric joint mers-cov outbreak readiness workshop: clinical management and potential use of convalescent plasma 10e12 pharmacokinetic characteristics of therapeutic antibodies experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection the authors would like to thank drs. bart supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.antiviral.2017.03.025. key: cord-300399-21xozruq authors: jayamohan, harikrishnan; lambert, christopher j.; sant, himanshu j.; jafek, alexander; patel, dhruv; feng, haidong; beeman, michael; mahmood, tawsif; nze, ugochukwu; gale, bruce k. title: sars-cov-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations date: 2020-10-18 journal: anal bioanal chem doi: 10.1007/s00216-020-02958-1 sha: doc_id: 300399 cord_uid: 21xozruq the unprecedented global pandemic known as sars-cov-2 has exercised to its limits nearly all aspects of modern viral diagnostics. in doing so, it has illuminated both the advantages and limitations of current technologies. tremendous effort has been put forth to expand our capacity to diagnose this deadly virus. in this work, we put forth key observations in the functionality of current methods for sars-cov-2 diagnostic testing. these methods include nucleic acid amplification–, crispr-, sequencing-, antigen-, and antibody-based detection methods. additionally, we include analysis of equally critical aspects of covid-19 diagnostics, including sample collection and preparation, testing models, and commercial response. we emphasize the integrated nature of assays, wherein issues in sample collection and preparation could impact the overall performance in a clinical setting. in december 2019, health officials in wuhan, prc, reported a disease outbreak involving a cluster of cases of "pneumonia of unknown cause." since then, the 2019 coronavirus disease (covid-19 or sars-cov-2) outbreak has been characterized as a pandemic, spread to 188 countries worldwide, causing 9,30,000 fatalities and more than 29 million confirmed cases (as of september 15, 2020) . the pandemic has proven to be a significant challenge to our ability to reduce the global spread of the sars-cov-2. given the global scale of infections due to the novel virus and the lack of approved therapeutics and vaccines, the covid-19 pandemic has drawn comparisons to the deadly 1918 spanish flu pandemic. a key difference is current advances in molecular diagnostic technology that has enabled us to rapidly characterize the novel virus, identify infectious (including asymptomatic) patients, and potentially isolate them to control the disease spread. however, initial delays in assay design and supply-chain bottlenecks prevented the deployment of accurate diagnostic tests at scale globally. this was found to be a critical gap in arresting the spread of this devastating disease worldwide. nevertheless, recent developments have led to broader deployment of diagnostic tests, albeit ones that require at least a dedicated sample collection setup. nucleic acid (na) amplification tests (e.g., polymerase chain reaction (pcr)) and immunoassays (e.g., enzymelinked immunosorbent assay (elisa)) are among the most utilized tools in today's covid-19 diagnostic testing landscape. this review paper examines current molecular diagnostic tools (fig. 1) , such as amplification-based (including crispr-cas based), antibody and antigen tests, and sequencing, utilized for the detection of sars-cov-2. we discuss the capabilities offered by each of these molecular diagnostic tools and challenges encountered in utilizing them in the current pandemic, and provide an assessment of future directions in research that could help remedy some of the identified shortcomings. we also report how each of these methods plays a complementary role suited for different stages of the pandemic. several review papers in the literature describe diagnostic approaches for covid-19 detection. this paper underscores the importance of the integrated nature of these diagnostic tools, wherein improvements in sample collection and preparation are needed to complement advances towards sensitive and accurate diagnostic methods. this knowledge is relevant to the current scenario wherein differences in analytical sensitivity and clinical sensitivity have hampered the effectiveness of covid-19 diagnostics. this situation reflects the challenges faced to develop and thoroughly assess the assays, reagents, and sample handling processes required for a reliable test. also, this situation generates the need to mobilize a largescale production pipeline to meet the enormous demand for assays for a new, largely unknown pathogen, sars-cov-2. furthermore, this paper discusses the challenges in ramping up testing. as an example, we discuss the problem of false-negatives that has impacted covid-19 testing and how improvements in sample collection could help remedy the situation. we underscore the lack of research into ensuring repeatability of respiratory sample collection. we also discuss various clinical samples that are utilized for diagnostics and how viral loads (amount of viral proteins or viral nucleic acids in a sample) vary with disease onset in the sample types. we compare and contrast the utility of each sample type from the perspective of sensitivity to utilization in self-sampling formats. in addition, we also discuss sample preparation aspects that are relevant to wider utilization and point-of-care (poc) deployment of covid-19 diagnostic tests (pcr, isothermal amplification, and sequencing-including library preparation). finally, we identify environmental sampling, surveillance opportunities, and advanced detection technologies being developed for commercial applications. nucleic acid (na) amplification and its subsequent detection are the most widely used method for the diagnosis of viral agents. these methods have been extensively reported for the detection of past outbreaks caused by coronaviruses such as mers and sars-cov [1] . in particular, reverse transcription polymerase chain reaction (rt-pcr) is the current standard for the detection of active sars-cov-2 infections [2] . rt-pcr broadly involves four steps-lysis of sars-cov-2 in the sample, purification of the viral rna, reverse transcription to complementary dna (cdna), and amplification of specific regions of the cdna, and finally, optical detection of the amplified cdna. the development of rt-pcr assays for a novel virus such as sars-cov-2 involves sequencing the genome and design of primers and probes. a variety of rt-pcr assays utilizing different primer/probe sets have been developed. these assays utilize different primer/probe sets targeting different regions of the sars-cov-2 genome. the selection of the primer/probe set affects the sensitivity of the assay [3] [4] [5] . for instance, a study from the university of washington reported e gene [6] and n2 gene primer/probe sets (us centers for disease control and prevention) to have better sensitivity for sars-cov-2 detection assay [7] . a recent report by anantharajah et al. evaluated rt-pcr assays utilizing who-recommended primer/probe sets on clinical samples. they found substantial differences in the assay sensitivity depending on the choice of primer and probe [8] . the difference was more pronounced for samples with low viral load (median cycle threshold, ct > 30). a recent study by jung et al. compared the performance of seven primer-probe sets for the n gene and three primer-probe sets for the orf1 gene for the detection of sars-cov-2 rna. the study evaluated the specificity and sensitivity of amplification at three different reaction temperatures (55, 58 , and 60°c). the primer-probe sets from the japan niid (niid_2019-ncov_n) and china cdc (orf1ab) were reported to exhibit the best performance without any non-specific amplification or crossreactivity for other coronaviruses (hcov-229e, hcov-oc43, and mers-cov rna) [9] . as an alternative to rt-pcr, other amplification-based formats such as digital pcr (dpcr) and isothermal amplification-based assays (rt-lamp) are being utilized for covid-19 diagnostics. digital droplet pcr involves partitioning the sample into multiple droplets, with each droplet serving as a reactor for independent pcr. dpcr utilizes endpoint amplification for direct quantification of viral load, is less susceptible to amplification inhibitors in the sample matrix, and, unlike qpcr, does not require calibration curves. dpcr has been shown to have higher sensitivity and lower false-negative results when compared with rt-pcr [10, 11] . thus, dpcr could be helpful in scenarios wherein low viral load or rna degradation has resulted in false-negative results using rt-pcr [12] . in addition, dpcr assays could be used to identify insufficient samples by quantifying a reference gene from human rna as a control [13] . isothermal amplification-based methods such as loopmediated isothermal amplification (lamp) and recombinase polymerase amplification (rpa) are well suited for rapid poc detection. in contrast to rt-pcr, isothermal amplification is carried out at a constant temperature, does not require thermal fig. 1 overview of covid-19 diagnostic workflow-samples are collected and stored in a transport medium, lysed, rna extracted, reverse transcribed to complementary dna (cdna), and then amplified (via pcr or isothermal amplification). the amplified viral sequence is detected/quantified using fluorescent dyes or colorimetric readout. crispr-cas-based detection (sars-cov-2 detectr) works by the activation of cas12 due to the presence of a target rna sequence. the activated cas12 subsequently cleaves reporter labels generating a fluorescent signal. the sequencing workflow converts the cdna into a form compatible with the sequencer (library preparation) and then determines the cdna sequence via digital images (sequencing-by-synthesis) or using electrical signals (nanopore sequencing). antigen-based lateral flow assays detect the sars-cov-2 antigen using an immunoassay format. viral antigen forms a sandwich bound by capture and detection antibodies. the presence of the labeled detection antibody indicates the presence of antigen in the sample (image created with biorender.com) cycling, and can be carried out using minimal instrumentation. in addition, due to the higher amplification efficiency, the test is rapid in comparison to rt-pcr. abbott's id now poc platform for covid-19 is based on isothermal amplification and has the shortest turnaround time among fda-authorized diagnostic products [14] . in comparison to sars-cov, the increased transmissibility of sars-cov-2 has been attributed to asymptomatic carriers and presymptomatic patients [15] . the viral load of asymptomatic or presymptomatic cases has been found similar to that of symptomatic covid-19 patients, suggesting comparable transmissibility [16] . there is increasing research suggesting the need for large-scale population-wide testing to identify and isolate asymptomatic and presymptomatic covid-19 cases [17] [18] [19] [20] . centralized and high-throughput, automated rt-pcr assay platforms [21] are well suited to test a large number of samples in a cost-effective and timely manner. recently, a shortage of commercial reagents for na extraction and amplification has hindered efforts to scale up the number of covid-19 tests. alternative reagents, buffers, and protocols have been explored to remedy such shortages [22] and will need to be utilized to support mass testing strategies. these include open-source reagents and extraction-free rt-pcr protocols. it is important to validate such alternative reagents and protocols in clinical settings on automated assay platforms before use. for instance, due to the proprietary nature of reagents used in commercial rt-pcr platforms, it is critical to evaluate the effect of alternative reagents on the sensitivity and specificity of high-throughput, automated assays. the pooling of samples is an alternative method for scaling covid-19 tests and could be useful for populationwide screening of covid-19 cases. the strategy has been recently utilized to screen millions of people for active infections in wuhan, china. these involve the pooling of multiple samples into a single rt-pcr. various approaches (such as array testing [23] ) have been proposed for the pooling of samples for covid-19 testing [23] [24] [25] [26] . optimum pool size and approach depend on factors such as the prevalence of the covid-19 in the population, and the sensitivity of the test utilized [23, 27] . dpcr, which has shown to have higher sensitivity in comparison to rt-pcr, has been proposed as an alternative for testing pooled samples [28] . despite its wide use, amplification-based covid-19 assays have faced numerous practical challenges [2] . these include vulnerabilities in the preanalytical and analytical aspects of the assay [29] . analytical issues include those attributed to variation in viral load or timing of sample collection in relation to disease progression [29] . a study by kucirka et al. notes that the probability of false-negatives using rt-pcr (from nasopharyngeal (nps) or oropharyngeal (ops) swabs) decreases from the day of infection (100%) to symptom onset (38%). the lowest false-negatives are observed at 3 days after symptom onset before increasing again [30] . preanalytical issues, such as inadequate respiratory sample collection or improper sample handling, could result in falsenegative results [29] . in comparison to the significant advances in analytical aspects such as automated operation and sensitivity of rt-pcr assays, respiratory sample collection using swabs is a manual process and prone to errors (see "sars-cov-2 clinical sample collection and preparation"). issues in the collection of respiratory samples can manifest as an inadequate quantity of human respiratory epithelial cells in a sample. such issues can be avoided by including a primer/ probe set to detect the presence of the human rnase p gene in the collected samples. however, quantification of human rnase p in respiratory samples (rather than a qualitative presence) might be necessary to identify inadequate biological sampling [13] . besides, the faulty design of the primer/probe set for the human rnase p gene in the rt-pcr assay has the potential to cause false-negative results [31, 32] . proper interpretation of rt-pcr assays as a proxy for infectivity is another aspect that needs attention. recently, there have been multiple reports of recurrent positive rt-pcr results in covid-19 patients, even after the resolution of symptoms [33, 34] . a positive rt-pcr test does not necessarily indicate a patient is infectious. for instance, wölfel and team were unable to isolate sars-cov-2 in culture from respiratory patient samples 8 days after symptom onset, despite positive rt-pcr results [34] . amplification-based tests detect the viral rna (including degraded viral rna) and not necessarily the presence of an infectious virus. more work is necessary to understand the implications of positive rt-pcr results and the potential for disease transmission. this is important from the perspective of relying on rt-pcr tests as a criterion for determining return to work conditions for healthcare personnel and other essential workers [35] . assays that utilize droplet dpcr to measure sars-cov-2 rna in its intact form could be valuable to detect potentially infectious covid-19 samples [36] . rt-pcr is the most widely used method for the detection of viral pathogens, including the sars-cov-2 virus. however, given the current challenges with rt-pcr, alternative techniques involving crispr-cas and isothermal amplification are being explored. for instance, there are severe limitations associated with the availability, costs, and the need for trained personnel to run rt-pcr tests. diagnostic capabilities with lower cost, faster turnaround times, and portability are critical, given the global scope and magnitude of the pandemic. crispr, well known as a gene-editing tool, has been leveraged for rapid, poc detection of viral pathogens [38, 39] . figure 2 describes the schematic for a molecular diagnostics assay utilizing the crispr/cas system. the crispr-cas adaptive immune system consists of a guide rna (grna) and a crispr-associated nuclease (cas). the grna consists of a nucleotide sequence, called crispr rna (crrna), which is complementary to the target sequence. the cas13 (a type vi crispr-cas) nuclease is activated by the presence of an rna sequence (target rna) complementary to the crrna. the cas13 then performs a targeted cleavage and also triggers a non-specific rnase activity, leading to degradation of nearby rna (regardless of the presence of a complementary sequence). this "collateral cleavage" activity of cas13 is utilized to cut rna reporter labels, which then release a fluorescent signal. other crispr-cas systems such as cas9 or cas12, which target dna, have also been utilized for the detection of viral targets [40, 41] . crispr-based diagnostics (crispr-dx) have been utilized for the detection of viral targets, including dengue virus, zika virus, and lentiviruses [39] . these assays enable multiplexing and increased sensitivity in comparison to stand-alone isothermal amplification methods. crisp-dx combines conventional na amplification with the crispr-cas13 system to achieve better sensitivity and specificity. the specificity is achieved through the activation of cas13 via the binding of crrna to target rna. in addition, sensitivity is achieved by signal amplification due to the "collateral cleavage" of reporter labels [42] . chiu and team reported a rapid lateral flow-based assay (sars-cov-2 dna endonuclease-targeted crispr trans reporter (detectr)) for detection of sars-cov-2 [43] . the assay can detect the virus from respiratory swab samples with sensitivity comparable to that of the us centers for disease control and prevention (cdc) sars-cov-2 real-time rt-pcr assay in 30-40 min. the assay combines reverse transcription loop-mediated isothermal application (rt-lamp), crispr-cas12-based detection, and a readout on a lateral flow strip. the current protocol can be incorporated into a poc instrument, which would involve microfluidic automation to achieve accurate metering and mixing of reagents along with an integrated isothermal heater [44] . however, the rna extraction from swab samples is performed using a cdc-recommended protocol that involves the use of a conventional spin column (qiagen dsp viral rna mini kit) and a magna pure 24 instrument. fig. 2 schematic of a crispr/ cas-based molecular diagnostic test. adapted from [37] with permission these steps involve bulky equipment and are not suitable for poc use. other recent reports have demonstrated the utility of crispr-cas for covid-19 diagnostics in clinical samples [45] [46] [47] . however, rna extraction is performed manually, proving to a critical limitation to its poc use. the automation of this key rna extraction step using poc sample preparation methods and integration with coupled isothermal rt-lamp+crispr-based lateral assay readouts will enable a fast, field-deployable platform that can be used in current and potential future pandemics. another advantage of crispr-based assays is the use of lyophilized reagents and the capability to run assays directly on raw samples. gootenberg et al. developed a multiplexed crispr-cas13 assay (sherlockv2-specific high-sensitivity enzymatic reporter unlocking-version 2) capable of detecting four targets in a single reaction [41] . this multiplexing is achieved by using distinct cleavage preferences of different cas13 enzymes on homopolymer reporters. in addition, they utilized lyophilized cas13 reagents on a lateral flow strip format. the cost of a single paper-based sherlock test was estimated to be as low as usd 0.61 [48] . the multiplexing capability reported in this work is relevant to the current pandemic due to reports of co-infection among sars-cov-2-infected patients [49, 50] . multiplexed assays could be useful to identify potential false-negative assay results. for instance, the multiplexed assay could be used to detect infection from alternative respiratory pathogens with clinical symptoms similar to covid-19 [51, 52] . building on the capabilities of sherlockv2, myhrvold et al. developed a protocol (hudson) that can detect multiple viral targets directly from clinical samples, including saliva with minimal sample processing, which is relevant to the current pandemic since saliva is being explored as an alternative sample for covid-19 testing [42] . despite a lower observed sars-cov-2 viral load in comparison with nps and ops, saliva samples have several advantages for poc screening and selfadministered tests (see "covid-19 testing models") [53, 54] . hudson uses heat to lyse viral particles and chemical reduction to inactivate rnases subsequently. this approach obviates the need for centrifuges used in columnbased extraction and can be used in poc platforms [55] . a protocol utilizing crispr-cas13 sherlock assay has been reported for the detection of sars-cov-2 [56] . curti et al. reported a crispr-cas12-based method to detect synthetic sars-cov-2 rna fragments from spiked saliva [57] . however, the limit of detection was much lower (10 5 copies/μl) in comparison to sars-cov-2 rnaspiked buffer samples (10 copies/μl). recently, shine (sherlock and hudson integration to navigate epidemics), a diagnostic assay combining sherlock and hudson, was utilized to detect sars-cov-2 from unextracted clinical specimens (np swabs and saliva) [58] . crispr-dx for covid-19 testing has shown to have comparable accuracy to cdc-recommended quantitative rt-pcr tests. in addition, these tests are simpler, are less expensive, and have a faster turnaround time. the integration of these assays with sample preparation on an automated poc format utilizing lyophilized reagents is needed, which will greatly enhance the clinical utility of crisp-dx for the covid-19 pandemic as an alternative to rt-pcr. given their lower cost, faster turnaround time, and higher sensitivity, crispr-dx could also be used in population-wide diagnostic screening or poc settings. next-generation sequencing (ngs) plays a critical role in identifying and monitoring a viral outbreak. unbiased ngs is the key first step in identifying a novel viral strain [59] . the sequencing data has also enabled the determination of possible origins [60] and transmission patterns of viral pathogens, such as sars-cov-2 [61] . additionally, the identification of the sequence is the first step towards the design of primers and probes for na-based rt-pcr tests. sequencing will continue to give us important information about how the virus is evolving, guiding the development of potential vaccines and therapies [62] . recently, sequencing has been approved as a clinical diagnostic test (under fda emergency use authorization guidelines) for covid-19 [63] . sequencing approaches can be broadly divided into shortread and long-read technologies [64] . short-read technology generates sequence data from na fragments shorter than 1000 base pairs. illumina sequencing, a short-read technology, is based on a sequencing-by-synthesis approach. the workflow involves three steps: library preparation (fragmentation of the dna to be sequenced and ligation of adapters), the amplification of dna fragments, and finally, sequencing of the amplified dna strands based on the sequencing-bysynthesis approach. the collective fluorescent signal resulting from synthesizing a large number of amplified identical dna strands allows the inference of nucleotide identity. long-read technologies include single-molecule real-time sequencing (pacbio) and nanopore sequencing (oxford nanopore), which are capable of generating sequencing data from high molecular weight na (> 1000 base pairs). nanopore sequencing works by measuring the electric current across an engineered protein nanopore, as a single strand of na translocates through it. ngs is an unbiased approach to the identification of novel infectious agents since it does not require a priori knowledge of the pathogen. ngs was used to identify the novel pathogen (2019-ncov, now known as sars-cov-2) in bronchoalveolar lavage fluid (blf) from three patients in wuhan, china, exhibiting symptoms of severe pneumonia [59] . sequencing was utilized after conventional pcr using a multiplexed rt-pcr panel failed to identify the presence of known pathogens (including human coronaviruses). ngs was able to identify the genome sequence of the causative pathogen in blf from nine patients in wuhan, exhibiting similar clinical symptoms [65] . the sequence was shown to be 99.98% identical to each other, which confirmed the presence of a novel, single virus. when similar symptoms were detected in patients in australia [66] , india [67] , and cambodia [68] , sequencing was used to confirm that the symptoms were indeed caused by the same pathogen. timely availability of viral genome sequences is a key step in the design of rt-pcr-based assays for novel viruses such as sars-cov-2 [6] . early on, sars-related sequencing information allowed for the release of primers and probes for covid-19 [69] . since then, improved covid-19 primers have been proposed [70] , and a bait capture hybridization probe sequence has been released [71] . ngs is also an important tool that can provide insights into the temporal or geographic traits of pathogens. for instance, sequencing was an important tool in understanding the nature of early undetected community transmission of covid-19 in the usa [72, 73] . in the past, researchers have tracked the evolution and spread of the zika virus in the americas [74, 75] , and of the ebola virus in west africa [76] . in the case of covid-19, sharing of this genomic data has already been very helpful [77] , with existing consortiums employed. early reports showed 99.9% sequence homology [78] , but, more recently, higher sequence diversity has been reported [79, 80] . recent research has shown that mutations in the sars-cov-2 spike protein led to the emergence of a more transmissible form of the virus [81, 82] . understanding of sars-cov-2 mutations is also important to understand the effectiveness of future vaccines and therapeutics [83] . recently, the cdc launched a nationwide genomics consortium to utilize real-time sequencing data to support such efforts [84] . metagenomic ngs has been proposed as a diagnostic tool for detecting a broad spectrum of pathogens [75] , including sars-cov-2 in clinical samples [85] . these include direct rna sequencing of sars-cov-2, avoiding inherent amplification bias associated with rt-pcr [86, 87] . however, clinical samples with low viral titers will require targeted sequencing approaches [75] , such as amplicon-based [88] , hybridization-based capture [71, 89] , and crispr-casbased enrichment [90] . in addition to its ability to detect coinfections, ngs can offer better sensitivity compared with rt-pcr [85] . the ability of ngs to detect small variations in the viral genome that could guide clinical decisions is also notable [85] . in comparison to rt-pcr, ngs has not been used extensively as a diagnostic tool in the current pandemic. this is possibly due to complex sample and library preparation protocols, expensive platforms requiring expertise to operate and analyze results, and relatively long turnaround time. the greatest difficulty is overcoming other signals in the sample: typically, < 1% reads are non-human, and ngs is particularly prone to contamination [91] . ngs has also traditionally been slower (a standard illumina instrument takes > 18 h [92] ) and more expensive than other methods. in large-scale testing, ngs is not currently competitive with pcr-based methods; however, there is ample evidence to imply that it could become a viable testing method in the future. ngs diagnostic tests can be scaled using approaches utilizing reverse transcription (rt) primers to barcode up to 19,200 patient samples in a single sequencing run [93] . viral genomes have been recovered directly from clinical samples [94] , including testing of bacterial, fungal, viral, and parasitic rna from multiple body fluids and tissues for multiplexed pathogen detection [91, 95] . additionally, chemiluminescence enzyme immunoassay (clia)-certified laboratories are increasingly utilizing ngs for diagnosis of encephalitis, meningitis [96, 97] , sepsis, and pneumonia [92] . in one test specific to covid-19, nanopore sequencing was used to identify sars-cov-2 from nps within 8 h [88] . nanopore sequencing has a lower raw-read accuracy in comparison to sequencing-by-synthesis approaches. however, nanopore sequencers such as the minion are inexpensive, have faster turnaround time, and are portable. further advances in automation and integration of sample and library preparation (see "sample preparation") will enable the widespread adoption of its capabilities. table 1 , summarizes the various approaches we have described in this section on the utilization of sequencing for covid-19 pandemic management. given the increasing global need for covid-19 tests, rapid and inexpensive assays are required to supplement current na amplification-based assays. these include antigen-based tests and often in the form of an immunoassay. in sars-cov-2 antigen detection, targets could be the nucleocapsid, spike, envelope, or membrane proteins of the virus [105] [106] [107] . viral antigens bind specifically to a corresponding antibody, and the event can be detected using optical, magnetic, electrochemical, and surface plasmon resonance-based techniques, among other methods (see "nanomaterials for poc covid-19 diagnostics") [108] . one of the challenges in developing effective antigen tests is the lack of antibodies for specific proteins of the sars-cov-2 virus. as an alternative to antibodies, selex (systematic evolution of ligands by exponential enrichment)-based strategies are useful in identifying affinity ligands such as aptamers specific to sars-cov-2. recently, aptamers target the receptor-binding domain (rbd) of the sars-cov-2 spike protein [109] . aptamers are significantly easier and cheaper to produce in comparison to antibodies. such efforts will enable the development of reliable and more accessible antigen-based assays. the viral load is found to be variable in covid-19 patients [110, 111] , depending on factors such as time between disease onset and sample collection, type and quality of the sample, disease severity, and patient age [112] . zou et al. reported ct values in the range of 19-40 in upper respiratory specimens of infected patients [110] . antigen tests are less sensitive than rt-pcr and could be less reliable in the clinical diagnosis of covid-19 patients with low viral load. such challenges have affected the clinical sensitivity of antigen tests for influenza and other respiratory viruses [112] . recent studies on four different commercial antigen tests demonstrated a wide range of sensitivities from 16.7 to 85% (with 100% specificity) in covid-19 clinical samples [113] . other studies have shown sensitivity as high as 93.9% (ci 95%: 86.5-97.4). however, the reported sensitivities were lower and more variable (72.2%, ci 95%: 49.1-87.5) in samples with low viral load (ct value > 25.1) [114] . table 2 lists recent work utilizing the detection of sars-cov-2 antigens along with the sensitivity/ limit of detection (lod). despite these limitations, rapid antigen tests can be used as a simple and inexpensive screening tool for active covid-19 infections. for instance, rapid antigen tests have been proposed as a screening tool for covid-19 at airports and border checkpoints. sample collection remains a significant challenge in antigen testing. more ideal poc sample types, such as saliva, are less invasive, and their adoption is expected to accelerate the use of antigen tests as a much-needed screening tool. a recent evaluation reported a low sensitivity of 11.7% for self-collected covid-19positive saliva samples using antigen tests [115] . the wide variation in the sensitivities of rapid antigen tests needs to be evaluated and understood [120] . based on the current understanding of the sensitivity challenges associated with antigen tests for influenza, we believe advances in respiratory sample collection, antigen preconcentration, and detection modalities will drive the adoption of antigen-based screening [122] . antibody-based tests detect antibodies generated as a result of a sars-cov-2 infection. antibodies are proteins generated by the immune system in response to an infection. the level of antibody response can vary with age, gender, and presence of comorbidities [123, 124] . immunoglobulin g, m, and a (igg, igm, and iga) are being used as potential markers for covid-19. igm is shown to be an indicator of early-stage infection, while higher igg levels are observed during late stages or post-recovery [124] [125] [126] [127] [128] . covid-19 antibody can be detected based on existing methods (table 3 ) such as colloidal gold-based immunochromatographic assay (gica) [129] [130] [131] [132] , magnetic chemiluminescence enzyme amplicon-based target sequencing using nanopore sequencer to detect sars-cov-2 and other respiratory organisms in clinical samples [85, 88] target (amplicon-based and hybridization-based capture) sequencing using bgi sequencer to detect sars-cov-2 and other respiratory organisms in clinical samples [89] direct rna sequencing of sars-cov-2 (from clinical specimens grown in cell culture) using nanopore sequencer [86, 87] proposed scaled testing protocol using rt primers to barcode up to 19,200 patient samples in a single sequencing run [93] outbreak surveillance analysis of sequencing data from clinical samples (using illumina sequencer) unveiled undetected community transmission of covid-19 in the state of washington [72] analysis of sequencing data from clinical samples (using illumina sequencer) unveiled multiple routes of introduction of covid-19 into the state of california [73] amplicon-based targeted sequencing from clinical samples (using nanopore sequencer) unveiled multiple routes of introduction of covid-19 into the netherlands [102] platforms and global consortiums utilizing genomic data to track real-time spread and evolution of pathogens including covid-19 [84, 103, 104] immunoassay (clia) [123] [124] [125] , and enzyme-linked immunosorbent assay (elisa) [126, [133] [134] [135] [136] . pan et al. [137] and li et al. [130] reported the detection of sars-cov-2 antibodies from whole blood. the sensitivity and specificity were found to be consistent with tests performed on serum samples [130] . antibody tests are useful in epidemiological studies to assess the number of asymptomatic cases in the population [138, 139] . however, cross-reactivity of antibody is observed between human coronaviruses, such as sars-cov-2, sars-cov, and mers-cov. antibody-based rapid diagnostic tests are performed on serum, plasma, or whole blood (fingerstick) poc settings. however, false-positives could lead to an inflated estimate of covid-19 prevalence [139] . in addition, the temporal antibody response could vary depending on the individuals tested. also, by design, antibody-based rapid diagnostic tests do not detect the presence of sars-cov-2 neutralizing antibodies. tests that detect antibodies that have a high affinity for sars-cov-2 virus are more likely to indicate the presence of neutralizing antibodies. more research is needed to understand if a positive antibody test is indicative of immunity against future sars-cov-2 infections. despite these limitations, given the low cost and ease of use, with improvements in clinical sensitivity and specificity, antibody tests could be used for mass-screening of covid-19 prevalence. it could also play a role in the evaluation of vaccines in the future [140] . monitoring the prominence of infection throughout a pandemic is of critical importance in all stages of a pandemic. each stage of a pandemic, as defined by the world health organization, has unique testing needs [141] . in early and peak phases, rapid turnaround times and scale-up are of great importance. in the post-peak phases of a pandemic, easily accessible, low-complexity, inexpensive, and high-throughput monitoring is needed. current virus testing technologies and methods have proven effective in various capacities. however, each method has unique strengths and limitations, which must be considered and uniquely paired with the pandemic phase, population, available resources, and virus characteristics if an optimal response is to be achieved. while testing continues inside hospitals and clinics, other unique methods are proving to be highly effective testing models for the sars-cov-2 pandemic. several unique testing models have been incorporated in the current pandemic response and will undoubtedly play a role in future pandemic response planning. here, we describe these methods and their relation to diagnostic testing, and provide insight into their application. the testing of infectious viruses presents several unique challenges. traditionally, a patient visits a hospital for evaluation and diagnostic testing. the limitations of this method include increased risk of exposure due to travel and interaction with healthcare workers and social concentration of infected and non-infected persons. other concerns include overloading healthcare workers and facilities, as well as the financial cost of in-person testing. several unique testing models are being utilized to reduce exposure and resource consumption. testing can be categorized by the location of the sample collection and analysis as well as if a healthcare worker aided in this process. the combination of these possibilities results in several unique testing models. drive-thru models have distinct advantages over traditional testing and have proven effective in rapid community testing [142] . while this model is limited in the number of communities worldwide that could utilize it, many urban and rural areas of the world could incorporate such a method. this method minimizes interaction while enabling more oversight on proper sample collection. the need for reporting of selfillness has been well argued [143, 144] . self-testing models involve the collection and possibly the analysis of a sample at home by the patient. recent work by tu et al. has shown the clinical utility of patient self-collected swabs from the tongue, nasal, and mid-turbinate for covid-19 diagnostic testing [145, 146] . the fda has recently authorized at-home selfcollection of saliva and nasal swabs for covid-19 diagnostics [147] . the advantages of this method include expanded testing and reduced risk of nosocomial transmission in overcrowded healthcare settings. the primary limitation is the adaption of established testing methods to a home collection model. additionally, accuracy in sample collection and processing in self-testing could be of concern. to address this concern, a home testing method where a healthcare worker visits a patient's home to collect samples has found success in treating patients [148] . healthcare workers can aid in sample collection and analysis to reduce the risk for error at the expense of increased risk of exposure. several companies, including labcorp, letsgetchecked, and nurx, have presented home-based poc collection devices for testing. it is encouraging to note that recent studies have reported equivalent sensitivity in self-collected samples [142, 149] . point-of-care diagnostic methods with an emphasis on portability and speed are seeing a large increase in development; these tests are predominantly immunoassay based [107] . aside from the assay itself, sample collection methods are also seeing progress. spectrum solutions llc, a developer of biosample collection devices, developed the sdna-1000 for collecting saliva biosamples, which is currently being used in sars-cov-2 testing and analysis. such technologies enable self-testing models to become highly feasible. yang et al. proposed a home-based testing model wherein the patient collects and processes their sample and then images the result from a paper-based assay using a cell phone for subsequent cloud upload and evaluation [150] . the integration of poc diagnostic assay with self-collection of samples will reduce the risk associated with the shipping of infectious samples and avoid potential issues due to sampling degradation during transport. though all the test methods are fundamentally different, the quality of the sample is crucial for successful detection. this is [151] . increasingly, inaccuracies in rt-pcr tests have been attributed to sample collection [13, 152] and handling. upper respiratory samples are the primary specimen used for covid-19 diagnostic testing [153] . in comparison to advances in amplification-based tests, methods for the collection of respiratory samples need improvement. given the scale of testing required, improper sampling by untrained personnel has contributed to false-negative results [152] . there have been efforts to develop a robot for respiratory swab sampling [21] . however, the device was not tested in vivo or clinical settings. alternative samples like saliva or nasal swabs are simpler, less invasive, and could potentially be used to avoid improper sampling associated with respiratory swab samples. sample preparation (or preprocessing) refers to the sequence of steps required to convert a clinical sample into a form compatible with downstream analysis and detection such as amplification or sequencing. sample preparation has been a major bottleneck in widespread testing during the covid-19 pandemic. the availability of rna extraction kits and lysis reagents and low-throughput rna extraction protocols have resulted in increased turnaround times and delays. a rapid surge in demand for tests is threatening to overwhelm the diagnostic capabilities. a review by esbin et al. describes alternative covid-19 diagnostic protocols involving lysis reagents and direct addition of samples (extraction-free protocols) into the amplification reaction mix [22] . there have been recent advances in extraction-free protocols for covid-19 amplification [154] [155] [156] [157] [158] [159] and sequencing-based tests [160] . however, such protocols involve trade-offs between lower sensitivity and simplicity [22, 161, 162] . automated extraction platforms utilizing liquid handling robots are useful for high-throughput, centralized covid-19 diagnostic testing. in addition, decentralized point-of-care (poc) diagnostic tests also have a role to fulfill. integrated poc platforms with "sample-in answer-out" capability will enable covid-19 diagnostic capability in doctor's office and clinics. such platforms will ensure reduced turnaround time, repeatability, and potentially lower costs by reducing reagent consumption. these will involve automation of sample preparation utilizing microfluidics and their integration with the detection assay. such methods have been extensively reviewed [44, 163, 164] and will need to be validated with covid-19 clinical samples. similar approaches are required to push increased utilization of sequencing to support the covid-19 pandemic. for instance, the complexity and labor-intensive nature of sample and library preparation workflow could limit the widespread adoption of sequencing [165] [166] [167] . efforts towards the automation and integration of sample and library preparation steps will enable increased adoption of sequencing platforms for covid-19 pandemic management. expanding sequencing capabilities will enable us to track mutations of the sars-cov-2 virus in real-time with potential implications to the efficacy of future covid-19 vaccines and therapeutics. it is important to treat a diagnostic assay as an integrated unit, with each step of the workflow from sample collection and preprocessing to the final detection and analysis, impacting the overall sensitivity. we have summarized some of the key factors impacting each step in the diagnostic workflow (from sample collection to detection) with reference to relevant literature (fig. 3 ). nasopharyngeal/oropharyngeal sample nasopharyngeal/oropharyngeal swabs are generally used as the collection mode for upper respiratory samples in early detection of sars-cov-2 by pcr. in particular, flocked swabs are especially useful when the total volume of the sample matrix is low. these special types of nylon swabs help in collecting and retaining more analyte than traditional spunfiber swabs made from cotton, polyester, or rayon [168] . swabs are generally taken from the nasopharynx or oropharynx [169] , with a nasopharyngeal sample being more sensitive than oropharyngeal [16, 170, 171] . usually, a healthcare worker collects these samples, but it has been shown that the collection can be done at home by the patient [172, 173] . selfsampling reduces the risks to healthcare workers (see "covid-19 testing models"). following swab collection, swabs are placed into a viral rna extraction medium and subsequently amplified (e.g., by rt-pcr) [169] . depending on the disease progression and the number of days passed since the onset of symptoms, the viral load varies considerably [169] . in such cases, swabs from different sources can be combined to increase the probability of detection. sputum is mucus produced by the act of coughing up and spitting out the material produced in the respiratory tract (the trachea and bronchi) [174] . it has been shown that, for covid-19, sputum contains more viral rna than nps, resulting in a higher chance of detection [170, 175, 176] . sputum collection is a much simpler process than swab sampling and can easily be done by the patient. this reduces the chances of healthcare providers getting infected while collecting samples. this process involves rinsing the mouth with water, followed by expectorating of deep cough sputum directly into a sterile, leak-proof, collection cup. however, not every patient produces sputum. alternatively, sputum can be clinically induced [177] . han et al. showed that sars-cov-2 could be detected from such samples [178] . saliva is an alternative sample that can be easily be collected by the patient at home. zheng et al. showed that saliva had a higher detection rate (88.09%) for sars-cov-2 in comparison to throat swabs (45.24%) and nps (76.19%) [171] . while most studies have found saliva to be as sensitive as nps for covid-19 diagnostic tests [54, 179, 180] , few have reported slightly lower sensitivity [181] . blf is an excellent sample for covid-19 diagnostic with a high detection rate (93%) compared to sputum (73%) and nps (63%) [170] . it is generally collected from patients with severe illness or undergoing mechanical ventilation. collecting blf involves the instillation of sterile normal saline into a subsegment of the lung, followed by suction and collection [182] . due to the complexity involved and the aerosol-generating nature of the procedure, blf is more useful in determining the recovery of admitted patients. stool samples can be collected and tested concurrently with other samples to both monitor disease progression and limit the instance of false-positives. like many other coronaviruses, large viral loads of sars-cov-2 can be found in stool [170, 183, 184] . however, it should be noted that stool samples tend to be among the most difficult samples to process for viral detection. stool samples are difficult to process due to the presence of many pcr inhibitors, such as bile, polysaccharides, hemoglobin, and bilirubin [185] . processing of stool samples often requires highly trained technicians, and results are highly susceptible to user error. therefore, the viral detection rate could be variable [183, [186] [187] [188] . viral loads can be found in stool samples at early onset through the convalescent stage of illness. viral loads have been found in stool samples from as early as the onset of symptoms to as late as 33 days after onset of symptoms [175, 186] . stool samples may provide a non-invasive alternative to nps and ops. the collection of stool samples is simple and can often be performed at home. sample collection involves the capture of a stool sample in a clean, dry container and transfer of the sample into a sterile specimen cup. serum and whole blood are primarily used in antibody tests for tracking disease progression, epidemiological studies, and patient immunity. these have been discussed in the section "antibody-based tests." table 4 lists the various clinical samples used in covid-19 diagnostics and important factors such as peak viral load, time to peak viral load, and average detection rate. these factors should enable guidance on their utility for different testing models such as at-home or poc testing. covid-19 is primarily spread via human-to-human transmission, though environmental transmission has also been reported [194] . unlike other enveloped viruses, sars-cov-2 are less susceptible to environmental stresses [194] . depending on the environment, temperature, and type of surfaces, it can survive up to 14 days [195] . hence, environmental surveillance for sars-cov-2 is useful in covid-19 containment and preventing transmission. air sampling can be done using an air sampling pump [196] (e.g., skc biolite+, zefon bio-pump® plus). these pumps can sample dust/particulates, vapors/gases, bioaerosols, or environmental air samples. they use cassettes and filters to collect samples for further analysis. air samplers are efficient, adaptable, and easy to use. but for preserving the viability of samples, quick and subsequent handling of sampling is needed. air samplers are used for confined spaces such as hospital rooms and apartments. researchers are now focusing on the sampling of sewage water for covid-19 surveillance [197] [198] [199] . this can be used to detect the presence of sars-cov-2 in certain populations and also to quantify the scale of infection [200] . the persistence of sars-cov-2 on surfaces is important to understand the risk of infection from fomites. for instance, sampling from high-touch surfaces will help uncover modes of transmission and also the effectiveness of disinfection protocols [201] . who has provided guidelines on potential sampling sites for such studies at covid-19 healthcare facilities [202] . premoistened swabs are used to collect samples from such surfaces with rt-pcr used to detect the presence of sars-cov-2 [203] [204] [205] . viral cultures could demonstrate the viability of sars-cov-2 on such surfaces. surface sampling using swabs is labor-intensive, and a large number of samples are required in surveillance studies. in april this year, the national institutes of health (nih) launched the rapid acceleration of diagnostics-radical (radx-rad) initiative to accelerate the development of novel approaches to covid-19 diagnostics [206] . this initiative is also aimed at the repurposing of current diagnostic techniques towards covid-19 and future pandemics. the initiative plans to enable rapid covid-19 detection platforms for homebased and poc use. in this section, we review some of the recent work on advanced techniques utilizing nanostructured materials, developed for the detection of active covid-19 infections at poc. these include platforms utilizing fieldeffect transistors (fet), plasmonics, and breath-based detection of sars-cov-2 virus, associated antigens, or rna. fet biosensors consist of an electrode (gate) functionalized with receptors or probe molecules to specifically bind with the analyte of interest. the binding of the target analyte causes a change in electrostatic surface potential (electrostatic gating effect), resulting in a change in the current flowing between the source and drain [211] . seo et al. developed a graphene-based fet sensor for the detection of sars-cov-2 in clinical samples (fig. 4a) [207] . the sensor consists of graphene sheets conjugated with the sars-cov-2 spike antibody (receptor) that specifically binds with the sars-cov-2 spike protein (analyte). the binding causes a real-time change in the current response, indicating the presence of sars-cov-2 spike protein or sars-cov-2 virus in the sample. the platform was shown to be highly specific (exhibiting no response to mers-cov spike proteins) and was able to detect clinical samples with sars-cov-2 viral load as low as 242 copies/ml. researchers from the university of maryland, [170, 193] baltimore, developed a colorimetric assay utilizing thiolmodified antisense oligonucleotides (aso) capped on the surface of gold nanoparticles (aunps) for the detection of isolated sars-cov-2 rna (fig. 4b ) [208] . the aso-capped aunps agglomerate in the presence of sars-cov-2 viral rna, causing a change in surface plasmon resonance (spr) of the agglomerates in solution. further addition of rnase h causes the aso-capped aunps to precipitate, resulting in further amplification of the spr signal and enabling a visual detection of sars-cov-2 rna in 10 min. qiu and coworkers developed a dual-functional plasmonic biosensor incorporating localized surface plasmon resonance (lspr) and plasmonic photothermal (ppt) effect for the detection of sars-cov-2 ( fig. 4c ) [209] . the biosensor consists of two-dimensional gold nanoislands (aunis) functionalized with complementary dna (cdna) receptors that hybridize with the target sars-cov-2 rna sequence. the hybridization event leads to an lspr phase change (response) that can be detected. in addition, when the aunis are illuminated at the plasmonic resonance frequency, a localized thermoplasmonic heat is generated, promoting the selective hybridization of the target sars-cov-2 rna sequence to cdna receptors. biosensors based on electrochemical sensing [212, 213] , quartz crystal microbalance [214] , and magnetoresistance [215] have been utilized in the past for viral detection [108] . these techniques can, in principle, be applied to covid-19 detection at poc. haick et al. developed an interesting alternative to covid-19 diagnostics involving the detection of volatile organic compounds (vocs) from the exhaled breath of patients (fig. 4d ) [210] . the chemi-resistive sensor array consists of aunps linked to organic ligands that swell or shrink upon exposure to various vocs, causing a change in fig. 4 schematic of novel covid-19 diagnostics platforms utilizing nanostructured materials for potential poc use. a a graphene-based fet sensor for sars-cov-2 detection. the device consists of the sars-cov-2 spike antibody conjugated to graphene sheets. the specific binding of spike antibody to sars-cov-2 spike protein causes a change in current (response) between source and drain. (modified with permission from [207] . copyright © american chemical society) b a colorimetric assay based on spr utilizing thiol-modified antisense oligonucleotides capped on the surface of gold nanoparticles for the detection of isolated sars-cov-2 rna. (modified with permission from [208] . copyright © american chemical society) c a dual-functional lspr biosensor utilizing gold nanoislands for sensitive detection of target sars-cov-2 rna sequence from a multigene mixture. (modified with permission from [209] . copyright © american chemical society) d a chemi-resistive sensor utilizing array consisting of aunps for detection of vocs from the exhaled breath of covid-19 patients (reproduced from [210] with permission from the royal society of chemistry) the electric resistance. although the technique will need to be validated in a clinical study with a larger sample size, it could prove very useful as a rapid screening tool for covid-19. rapid and reliable commercial screening of sars-cov-2 is critical to limit the 2019 covid-19 pandemic. research labs and medical device companies across the world made major shifts in manufacturing desperately needed test kits at a rate that matches the rapidly increasing demand during the 2019-2020 pandemic. to produce effective covid-19 tests, companies must overcome the challenges associated with scalability, accuracy, cost, testing supply-demand, and sample collection. the usa alone has performed over 5 million covid-19 tests in the first 3 months of testing [216] . reducing storage, transportation, and testing time are of utmost importance. covid-19 tests generally fall into two categories, virus rna testing (using mostly rt-pcr) to diagnose current infections or serological tests for anti-sars-cov-2 antibodies to determine if the patient was exposed. china first released the covid-19 genome on jan 11, 2020, and the malaysian institute for medical research successfully produced "primers and probes" for sars-cov-2 the same day [217] . because of the early and rapid deployment of tests, south korea stands out for its response to the covid-19 pandemic. south korean company, seegene, was one of the first commercial companies to provide large-scale testing with the development of the allplex 2019-ncov assay, which was deployed in an impressive 3 weeks within the release of the covid-19 genome. allplex™ 2019-ncov assay is a multiplex real-time rt-pcr assay that targets three specific genes for sars-cov-2, which include rdrp and n genes and e gene for all sarbecovirus, including sars-cov-2. in the usa, the cdc was the first to produce a one-step process that tests samples on a 96-well plate and completes a full test in~45 min using rrt-pcr [218] . a major issue that hindered pcr testing is the supply chain for reagents and swabs. samples for covid-19 pcr are collected from the nasal, nasopharyngeal, or oropharyngeal areas using specialized synthetic fiber swabs with plastic shafts. guidelines set by the us centers for disease control and prevention (cdc) state that the sample be suspended in viral transport media and stored between 2 and 8°c for up to 72 h and after 72 h must be stored at − 70°c, which poses challenges for widespread sample collection [219] . collected samples are combined with primers and polymerase and then passed through a thermocycler for amplification and optical detection. considering the scale of required tests and the highly infectious nature of covid-19, sample processing is well suited for automated devices such as roche's (basel, switzerland) rapid test polymerase chain reaction detection devices. in an 8-h period, these devices can reportedly process 384 and 1058 patient samples for the cobas 6800 and 8800, respectively [220] . point-of-care devices remove the need for storage and transportation, saving substantial testing time. bosch (waiblingen, germany) has developed a portable point-of-care rt-pcr device called vivalytic, which is fully automatic and can complete a full in situ test in 2.5 h. serological testing [221] provides surveillance of sars-cov-2 antibodies identifying who has been in contact with the virus and information on immunity. the development of antibodies typically takes 2 weeks after infection, and for this reason, serological testing is not the preferred method for diagnosing infection. common commercialized serological tests include rapid diagnostic tests (rdts) and enzymelinked immunosorbent assay (elisa). rdts are typically point-of-care qualitative lateral flow assays where samples are collected from a finger prick, saliva sample, or nasal swab [52] . elisa tests whole blood, plasma, or serum and is typically performed in a laboratory. patient samples are incubated with an immobilized pathogen in which antibodies in the sample can form bonds. a secondary immunolabel with a fluorescent signature is used to quantifiably and qualitatively identify the pathogen [222, 223] . the 2019 sars-cov-2 pandemic brought to light the weaknesses of many health organizations worldwide in meeting the challenges of a global pandemic. the global financial and health costs associated with this pandemic are sure to total trillions of dollars. global pandemics will likely continue to be a significant threat in the modern world, and the pandemic response strategy is worthy of a thorough evaluation. viral diagnostic testing has been observed to be one of the areas in the sars-cov-2 pandemic response in need of assessment and improvement. assessment of the global response with a focus on the diagnostic methods and instruments utilized will provide recognition on what should be done to better prepare for future pandemics. we reviewed aspects of viral diagnostic tools relevant in the sars-cov-2 pandemic and provided insight into their current state, including advantages, limitations, and scalability, and then provided direction on future work that would enable diagnostics to be better prepared for a future pandemic response. rt-pcr remains the gold standard in sars-cov-2 infection testing. rt-pcr has encountered limitations in sensitivity. improved primer and probe design, along with better control over preanalytical variables such as sample collection and handling, is expected to improve performance in clinical settings. other na amplification techniques, including dpcr and isothermal amplification, are being used to a lesser degree and have distinct advantages in sensitivity and turnaround time, respectively. crispr-dx shows promise in achieving higher sensitivity and specificity over na amplification alone. additionally, crispr-based technologies appear to be more conducive to multi-target reactions, raw body sample compatibility, and poc applications. in addition to being the first step in the identification of a novel virus strain, sequencing can provide insight into the evolution, transmission, and unique characteristics of a virus. while not commonly used as a diagnostic method due to higher turnaround time and costs over traditional methods, the fda has recently granted eua to a high-throughput sars-cov-2 clinical testing (illumina covidseq) [63] . the advent of nanopore sequencers combined with advances in integrated sample and library preparation will enable the widespread adoption of its sequencing for covid-19 pandemic management. although antibody and antigen detection methods were initially limited by low sensitivity and specificity as well as a longer development cycle in comparison to rt-pcr, they promise a cost-effective approach to population-wide massscreening and are amenable to poc use. commercial testing has relied on rt-pcr and serological testing, aiming to diagnose a current infection and previous exposure, respectively. the commercial response was limited by supply chain issues rather than by technology constraints. rt-pcr-based assays were available within a few weeks of genome discovery, while serological testing methods became available within a few months. beyond the viral diagnostic technologies themselves, the methodologies involving the collection and analysis of samples have played and will continue a critical role in effectively diagnosing viral infections during a pandemic. methods, including self-and drive-thru testing, are expected to limit transmission while reducing overall per-patient testing costs. as diagnostic testing technologies develop, it will be important to incorporate the unique user needs and requirements associated with pandemic response methodologies. finally, biological sample collection and preparation remain essential regardless of the sample detection method. the type of sample, viral load, collection method, illness phase, and preprocessing are all factors that impact the sensitivity of the diagnostic method and must be appropriately matched for ideal results. improvements to preanalytical vulnerabilities (such as ensuring repeatable sample collection and processing) should move in tandem with advances in the analytical performance of an assay to ensure the optimum clinical performance of a covid-19 assay. table 5 provides a summary of various techniques reviewed in this work and their potential role in disease control and pandemic management. in conclusion, given the global spread and scale of covid-19 infections, the diagnostic ecosystem has encountered various bottlenecks. in spite of these challenges, various diagnostic tools will continue to play a critical and complementary role in the management of various stages of the covid-19 pandemic. 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remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest harikrishnan jayamohan is a former employee of roche sequencing solutions inc. and owns shares in roche holding ag. all other authors have no conflicts to declare. key: cord-299093-zp07aqpm authors: harrison, andrew g.; lin, tao; wang, penghua title: mechanisms of sars-cov-2 transmission and pathogenesis date: 2020-10-14 journal: trends immunol doi: 10.1016/j.it.2020.10.004 sha: doc_id: 299093 cord_uid: zp07aqpm the emergence of sars-coronavirus 2 (sars-cov-2) marks the third highly pathogenic coronavirus to spill over into the human population. sars-cov-2 is highly transmissible with a broad tissue tropism that is likely perpetuating the pandemic. however, important questions remain regarding its transmissibility and pathogenesis. in this review, we summarize current sars-cov-2 research, with an emphasis on transmission, tissue tropism, viral pathogenesis, and immune antagonism. we further present advances in animal models that are important for understanding the pathogenesis of sars-cov-2, vaccine development, and therapeutic testing. when necessary, comparisons are made from studies with sars to provide further perspectives on covid-19, as well as draw inferences for future investigations. infection. first, viral entry may heavily depend on the expression of tmprss2, as nearly undetectable amounts of ace2 still support sars-cov entry, so long as tmprss2 is present [27] . second, the mrna expression of cellular genes such as escrt (endosomal sorting complex required for transport) machinery gene members (including chmp3, chmp5, chmp1a, and vps37b) related to pro-sars-cov-2 lifecycle is higher in a small population of human type ii alveolar cells with abundant ace2, relative to ace2-deficient cells [28] . this suggests that sars-cov-2 hijacks a small population of type ii alveolar cells with high expression of ace2 and other pro-viral genes for its productive replication. third, the lungas the main tropism of sars-covs-may be contingent on the regulation of ace2 at the transcriptional and protein levels [24, 29, 30] [25, 31] . for example, in human airway epithelial cells, ace2 gene expression is upregulated by type i and ii interferons [25, 31] during viral infection. lastly, compared to other sars-covs, sars-cov-2 spike contains a unique insertion of rrar at the s1/s2 cleavage site [17] [18] . this site can be pre-cleaved by furin, thus reducing the dependence of sars-cov-2 on target cell proteases (tmprss2/cathepsin l) for entry [17] [18] and potentially extending its cellular tropism, given that proteolytically active furin is abundantly expressed in human bronchial epithelial cells [32, 33] . one of the distinctions between sars-cov and sars-cov-2 is the latter's ability to efficiently infect the upper respiratory tract (urt), such as nasopharyngeal (np) and/or oropharyngeal (op) tissues, possibly due to its higher affinity for ace2, which is expressed in human nasal and oral tissues [34] [35] [23, 25, 36] . the readily detectable titers of sars-cov-2 in the urt mucus of covid-19 patients during prodromal periods might contribute to explaining the more rapid and effective transmissibility of sars-cov-2 relative to sars-cov [37] . human covs often cause enteric infections, with variable degrees of pathogenicity [38] . indeed, ace2 and tmprss2 are abundantly expressed within the human and many other mammalian intestinal tracts, specifically the brush border of intestinal enterocytes [23, 25, 26, 39] . accordingly, gastrointestinal illness has been frequently reported in covid-19 patients [40, 41] , consistent with the recovery of sars-cov from sars patients' stool samples [42] , suggesting a potential fecal-oral route of transmission for these two covs. of note, ~20% of covid-19 patients examined have had detectable sars-cov-2 rna in feces, even after respiratory symptoms subsided, suggesting that sars-cov-2 titers might be prolonged in the intestinal tract [41] . although further testing is warranted, these data suggest the possibility that fecal-oral transmission of sars-cov-2 might occur. evidently, robust epidemiological studies are needed to conclusively demonstrate if covid-19 patients recovering from respiratory illness are able to spread sars-cov-2. human covs are transmitted primarily through respiratory droplets, but aerosol, direct contact with contaminated surfaces, and fecal-oral transmission were also reported during the sars epidemic [43] [44] [45] . early reports of patients with cough, lung ground glass opacities, and symptom progression to severe pneumonia, suggested communicability of sars-cov-2 via the respiratory route (figure 2 ) [1] [2] [3] . direct transmission by respiratory droplets is reinforced by productive sars-cov-2 replication in both the urt and lrt, and the increasing number of reports indicating human-to-human spread among close contacts exhibiting active coughing ( figure 2 ) [35, [46] [47] [48] . so far, the basic reproduction number (r 0 ) is ~2.2, based on early case j o u r n a l p r e -p r o o f journal pre-proof tracking in the beginning of the pandemic, with a doubling time of 5 days [47] [49] . furthermore, there is now evidence for non-symptomatic/pre-symptomatic spread of sars-cov-2, which is in contrast to the transmission dynamics of sars-cov [50] . this finding underscores the ability of sars-cov-2 to colonize and replicate in the throat during early infection [37, 51, 52] . based on these apparent disparities in virus transmission, one study modeled the transmission dynamics of sars-cov-2 in pre-symptomatic individuals, and indicated that the pre-symptomatic r 0 has approached the threshold for sustaining an outbreak on its own (r 0 >1); by contrast, the corresponding estimates for sars-cov were approximately zero [49] . similarly, asymptomatic spread of sars-cov-2 has been documented throughout the course of the pandemic [48] [51, [53] [54] [55] [56] . understanding the relative importance of cryptic transmission to the current covid-19 pandemic is essential for public health authorities to make the most comprehensive and effective disease control measures that include mask-wearing, contact tracing, and physical isolation. for sars-cov-2, various modes of transmission have been proposed, including aerosol, surface contamination, fecal-oral route, representing confounding factors in the current covid-19 pandemic; thus, their relative importance is still being investigated (figure 2 ) [57] . aerosol transmission (spread >1m) was implicated in the amoy gardens outbreak during the sars epidemic, but the inconsistency of these findings in other settings suggested that sars-cov was likely an opportunistic airborne infection [43, 58] . similarly, no infectious sars-cov-2 virions have been isolated, though viral rna was detectable in the air of covid-19 hospital wards [59] . generation of experimental aerosols carrying sars-cov-2 (comparable to those that might be generated by humans) have offered the plausibility of airborne transmission, but the aerodynamic characteristics of sars-cov-2 during a natural course of infection is still an area of intense inquiry [60] . nonetheless, deposition of virus-laden aerosols might contaminate j o u r n a l p r e -p r o o f objects (e.g. fomites) and contribute to human transmission events [59, 61] . finally, fecal-oral transmission has also been considered as a potential route of human spread, but this route remains an enigma despite evidence of rna-laden aerosols being found nearby toilet bowls, along with detectable sars-cov-2 rna in rectal swabs during the precursor epidemic of covid-19 in china [41, 59, 62] . in general, common cold covs tend to cause mild urt symptoms and occasional gastrointestinal involvement (figure 3) . on the contrary, infection with the highly pathogenic covs, including sars-cov-2, causes severe 'flu'-like symptoms that can progress to acute respiratory distress (ards), pneumonia, renal failure, and death [46, 48, 63, 64] . the most common symptoms are fever, cough and dyspnea, accounting for 83%, 82% and 31% of covid-19 patients (n=99), respectively in one epidemiological study [65] . the incubation period in covid-19 is rapid, ~5-6 days, versus 2-11 days in sars-cov infections [38, 47, 48] . as the pandemic is progressing, it has become increasingly clear that covid-19 encompasses not only rapid respiratory/gastrointestinal illnesses, but can have long-term ramifications such as myocardial inflammation [66] . furthermore, severe covid-19 is not restricted to the aged population as initially reported; children and young adults are also at risk [67] . from a diagnostic perspective, covid-19 presents with certain 'hallmark' laboratory and radiological indices, which can be helpful in assessing disease progression ( table 1) it is widely accepted that the aging process predisposes individuals to certain infectious diseases [68] . in the case of covid-19, older age is associated with greater covid-19 morbidity, admittance to the icu, progressing to ards, higher fevers and greater mortality rates [69] [70, 71] . moreover, lymphocytopenia, neutrophilia, elevated inflammation-related indices, and coagulation-related indicators have been consistently reported in older (≥65 years old) relative to young and middle-aged covid-19 patients [ table 1 ;( [72, 73] ) [46, 65, 71, 74, 75] . at the cellular level, a lower capacity of cd4 + and cd8 + t-cells to produce ifn-γ and il-2, as well as an impairment in t-cell activation from dendritic cells (dcs) in acute covid-19 patients (≥55 years old), might potentially compromise an optimal adaptive immune response [76] . based on examples from mice, a productive cd4 + t-cell response relies heavily on lung resident dcs (rdcs) and abates sars-cov infection [77, 78] . however, whether a reduction in the dc population in the lungs of older, more severe patients causes sub-optimal t-cell activation during sars-cov-2 infection remains to be robustly investigated. higher proportions of proinflammatory macrophages and neutrophils have also been observed in the bronchoalveolar lavage fluid (balf) of covid-19 patients with severe symptoms compared with those exhibiting mild symptoms (key figure, figure 4 ) [79] . accordingly, proinflammatory cytokines (e.g. il-6, il-8) are elevated in the balf of severe j o u r n a l p r e -p r o o f journal pre-proof covid-19 patients, along with higher expression of inflammatory chemokines (e.g. ccl2) in macrophages relative to non-severe covid-19 patients [79] [80] [81] [82] . indeed, similar inflammatory milieux have been associated with severe lung pathology in sars patients, along with the notable 'cytokine storm' that can present in critically ill covid-19 patients [83, 84] [71, [85] [86] [87] . these proinflammatory mediators can, in turn, perpetuate lung disease by elevating creactive protein (crp) from the liver ( table 1) products [93] . specifically, covs can avoid immune sensing via i) the formation of dmvs that sequester viral nucleic acid from being recognized by prrs and ii) direct ablation of the functionality of immune signaling molecules by viral proteins [11, 94] . the structural and functional conservation of these proteins across the betacoronavirus genus and in nsps between sars-cov and sars-cov-2, suggests that some of these suppressive mechanisms might be employed by sars-cov-2 (see below) [1] . indeed, patients with severe covid-19 have reported an imbalanced immune response with high concentrations of inflammatory cytokines/chemokines, but little circulating ifn-β or ifn-λ, resulting in persistent viremia [95] . of note, among several respiratory viruses tested, sars-cov-2 has demonstrated to most potently suppress type i and type iii ifn expression in both human bronchial epithelial cells and ferrets [81] . thus, evasion of ifn signaling by sars-cov-2 and impaired ifn production in j o u r n a l p r e -p r o o f human peripheral blood immune cells might contribute to the productive viral replication, transmission, and severe pathogenesis during covid-19, although further testing is warranted to fully dissect these putative evasion pathways [95] . with regard to functional conservation of viral proteins, sars-cov and mers-cov nsps and accessory proteins circumvent viral rna-sensing pathways at multiple stages (e.g. rig-i, mda-5) through proteasomal degradation and/or prevention of protein activation ( figure 5 ) [94] . functional conservation between sars-cov and mers-cov pl pro (encoded by nsp3) proteins has been reported, where these proteins target the initial prr signaling cascade at multiple levels of the pathway includingbut not limited to-rig-i, mavs, tbk1, irf3 and nf-k b ( figure 5 ) [96] [97] [98] . the sars-cov pl pro also targets the dna-sensing pathway at sting ( figure 5) ; antagonizing this pathway might be important as mitochondrial stress during dengue virus infection triggers ifn-β production that is dependent on sting activation [99, 100] . recent evidence suggests the sars-cov-2 pl pro might also inhibits ifn-i expression in human kidney epithelial cells, yet the mechanisms remain to be defined [101] . moreover, nsp1 of highly pathogenic hcovs, including sars-cov and mers-cov displays a pleiotropic effect, targeting several components of ifn-i signaling ( figure 5 ) [102, 103] . this potent suppressive function of nsp1 also appears to be maintained in sars-cov-2, primarily through shutdown of translational machinery and prevention of immune gene expression [101, 104, 105] . furthermore, because there are only five accessory genes in the mers-cov genome compared to eight and seven in the sars-cov and sars-cov-2 genomes, respectively, similar immunosuppressive mechanisms may exist but appear to be mediated via different proteins [106, 107] . for example, sars-covs orf6 can inhibit irf3 activation and stat1 nuclear translocation, whereas this same effect is obtained by orf4a/b and orf5 of mers-cov j o u r n a l p r e -p r o o f ( figure 5 ) [118, 119] . coincidently, the apparent loss of these proteins may provide evidence for why mers-cov is more sensitive to ifn treatment than sars-covs in primary and continuous cells of the human airways [108] . the sars-cov-2 proteins appear to have stronger inhibitory effects than their counterparts of highly pathogenic sars-and mers-cov [105] . in light of these findings, sars-cov-2 has replicated more efficiently than sars-cov in ex vivo human lung explants, possibly through the greater suppression of ifn-i/iii cytokines [109] ; further work given that sars-cov-2 uses the same ace2 entry receptor as sars-cov, rapidly deploying mouse models for pathogenesis studies were well underway within weeks of the pandemic's inception. however, various impediments remain for sars-cov-2 in productively infecting mice in these models, as it is unable to bind mouse ace2 (mace2) [111] . to overcome these prerequisites, several mouse models have been developed that recapitulate certain components of j o u r n a l p r e -p r o o f human covid-19. one of these strategies is to genetically modify mice to express human ace2 (hace2) (humanized mice) under the epithelial cell-specific cytokeratin-18 (krt 18 ) promoter [112] , a universal chicken beta-actin promoter [113] , or the endogenous mace2 promoter [111] . all these mice are susceptible to sars-cov-2 infection, but phenotypic disease varies because of differential hace2 tissue expression [112] [113] [111] . for instance, krt18-hace2 and betaactin-hace2-transgenic mice rapidly succumb to sars-cov-2 infection with lung infiltration of inflammatory immune cells inducing severe pulmonary disease, accompanied by evident thrombosis and anosmia, which partially recapitulate human covid-19 [114] [115] . as the onset of severe histopathological changes occurs days after peak virus infection, these models recapture the delayed morbidity seen in covid-19 patients as a result of inflammatory cell infiltration [115] . therefore, employing humanized mouse models of severe sars-cov-2 infection might be useful for testing the efficacy of antiviral drugs, vaccines, and immune therapeutics that ablate hyperinflammation [114] . however, the broad expression of hace2 in these models significantly expands sars-cov-2 tissue tropisms and might alter its pathogenic mechanisms [114] [115] . for example, both sars-cov and sars-cov-2 infection lead to encephalitis in these mouse models, which is not common in covid-19 patients [113, 115, 116] . considering the fact that the majority of human sars-cov-2 infections are asymptomatic or mild, mice originally bearing mace2 that is replaced by hace2 may be more appropriate for assessing pathogenesis and tissue tropism [111] . this model develops mild lung pathology, with sars-cov-2 infection being restricted to the lung and intestine [111] . in addition to the transgenic modification, mice can also be sensitized to sars-cov-2 infection via transient transduction of adenovirus (ad5)-or adeno-associated virus (aav)-expressing hace2 in respiratory tissues, akin to the approach previously used for mers-cov infection [117] [118] [119] . these mice develop viral pneumonia, weight loss, severe pulmonary pathology, and a high viral load in the lung, consistent with human covid-19 [119] . this approach might be quickly adapted to many genetically modified mouse strains that might provide mechanisms of sars-cov-2 pathogenesis and protective immune responses. this model is limited, however, by the transient ectopic expression of hace2 from the ad5/aav vector that can induce mild bronchial inflammation and expand cell tropism of sars-cov-2 and thus, presumably alter disease pathogenesis [120] . rather than genetic modification in host animals, viruses can also be genetically modified and be used in model animals [121, 122] . for instance, in one study, serial passaging of sars-cov-2 in mice led to enrichment of a n501y viral mutant that elicited interstitial pneumonia and inflammatory responses in both aged and young wild-type balb/c mice [123] . another mouseadapted sars-cov-2 strain (ma10) carrying three mutations in the rbd of spike protein caused severe lung pathology and ards in mice, characteristic of severe covid-19 [124] . despite the three mutations in the rbd of the mouse-adapted spike, vaccination with full length sars-cov-2 spike elicited robust neutralizing antibody titers and complete protection against a secondary challenge with ma10 [124] ; these findings suggest that this strain may be applicable to pathogenesis studies, as well as antiviral drug and vaccine testing in rodents. the role of non-human primates (nhp) in evaluating coronavirus pathogenesis cannot be understated. depending on the nhp model utilized, clinical signs/symptoms may be mild or absent entirely [125] [126] [127] . in rhesus macaques, several studies have noted reduced appetite, j o u r n a l p r e -p r o o f transient fevers (1 day post infection: dpi) and mild weight loss without overt signs of respiratory distress or mortality [125] [126] [127] . by contrast, cynomolgus macaques did not display any observational signs of disease in another study [126] . although certain nhps appear to only mimic mild disease (if any), rhesus macaques have exhibited high viral loads in nasal swabs, throat samples, and balf early post inoculation, and viral rna was still measurable by qpcr in the trachea and lung on day 21 p.i., highlighting the apparent tropism of sars-cov-2 for the urt and lingering viral nucleic acid in respiratory tissues after resolution of disease [51, 125] . sars-cov-2 has also been detected in nasal swabs at 10 dpi in nhps, consistent with the prolonged urt shedding of virus in covid-19 patients at ~9 dpi [51, 125, 126, 128] . the tropism of sars-cov-2 for the lrt in nhps has also been recapitulated by the development of multifocal lesions and interstitial pneumonia, supporting the hypothesis that lung injury is driven by increased infiltration of neutrophils and macrophages into the lung following viral infection such as thickened alveolar septum and diffuse severe interstitial pneumonia when compared to young macaques (3-5 years old) [129] . therefore, these studies highlight the importance of also considering the age factor, as an additional variable, when selecting animal models that might closely, or accurately, recapitulate human disease. evaluating efficacious vaccine candidates in nhps will also be important for understanding correlates of protection against sars-cov-2. accordingly, reports of antibodydependent enhancement, as well as of non-neutralizing humoral responses to the conserved regions of sars-cov-2, raise concerns on our future ability to effectively administer an immunogen without inducing immunopathology [130, 131] . furthermore, upon viral challenge, lymphocytes have expanded in rhesus macaque models around 5 dpi with complementary b-cell responses against sars-cov-2 spike appearing 10-15 dpi in blood samples [125] ; expansion of these adaptive immune compartments was analogous to those observed in covid-19 patients [37, 125, [132] [133] [134] . subsequent re-challenged rhesus macaque have presented a rapid anamnestic immune response characterized by significantly higher neutralizing antibody (nab) titers than the primary infection macaques [127] . thus, protective efficacy seems to depend primarily on nab titers, at least in nhps, and so far, t-cell numbers have not substantially increased following re-challenge in the serum of these animals, and in a secondary study, cd4 + and cd8 + cytokine (e.g. ifn-γ) responses did not correlate with immune protection from dna vaccines with different components of the sars-cov-2 spike protein [135] [127] . although these animals have failed to manifest overt signs of infection and respiratory compromise, nhps still represent the 'gold standard' for evaluating the protective efficacy of human-bound sars-cov-2 vaccines based on parallels to humans in terms of viral tropism, immunopathology, and correlates of protection [127] . further research is urgently needed to j o u r n a l p r e -p r o o f explore the durability of immune responses to sars-cov-2, considering reports of waning immunity to other covs and the detection of pre-existing cross-reactive 'common-cold' cov tcells with sars-cov-2 in naïve humans (see outstanding questions) [136, 137] . the emergence of sars-cov-2 as the most recent example of zoonotic virus spillovers into in the last 24 hours leading up to death, all 13 patients which were included for this metric had a prothrombin time of >12.1s. aerosol: suspension of fine solid or liquid droplets in the air (or a gas medium), such as dusts, mists, or fumes. anamnestic immune response: memory immune response to a previously encountered antigen. angiotensin-converting enzyme 2 (ace2): cell surface enzyme of endothelial, epithelial, and other cells, with a well-defined function in maintaining normal blood pressure. anosmia: partial or complete loss of the sense of smell. antibody-dependent enhancement: phenomenon by which antibodies against a virus are suboptimal to the virus and enhance its entry into host cells. convalescence period: the time of gradual recovery after an illness or injury. correlates of protection: quantifiable parameters such as antibodies, indicating that a host is protected against microbial infection. cytokine storm: severe immune reaction in which the body releases too many cytokines into the blood too quickly. d-dimer: fibrin degradation product in the blood after a clot is degraded by fibrinolysis. disseminated intravascular coagulation (dic): condition in which blood clots form throughout the body and block small blood vessels, leading to multiorgan failure. fecal-oral transmission: route of disease transmission by which an infectious agent in fecal materials is passed to the mouth of another. fomite: inanimate object (clothes, utensil, and furniture etc.) that, when contaminated with an infectious agent, can transfer the infectious agent to a new host. furin: proprotein convertase that cleaves a precursor protein into a biologically active state. incubation period: timeframe elapsed between when a host is first exposed to an infectious agent and when signs or symptoms begin to appear. lung ground glass opacity: nonspecific radiological description of an area of increased opacity in the lung through which vessels and bronchial structures are still visible. neutralizing antibody (nab): an antibody that binds a pathogen with high affinity and prevents the latter from exerting its biological effect. neutrophil extracellular traps (nets): networks of extracellular fibers, primarily composed of dna from neutrophils due to chromatin decondensation, which can 'trap' extracellular pathogens. pattern recognition receptor: germline-encoded host sensor that recognizes a signature pattern in microbial molecules. prodromal period: the time immediately following the incubation period of a microbial infection in which a host begins to experience symptoms or changes in behavior/functioning. prothrombin time: measurement of the extrinsic pathway of coagulation. r 0 (reproductive number): the expected number of new disease cases generated by one case. an r 0 > 1 indicates the outbreak will expand; r 0 <1 the outbreak will die out. respiratory droplet: small aqueous droplet produced by exhalation, consisting of saliva or mucus and other matter derived from respiratory tract surfaces. zoonotic disease: infectious disease caused by a pathogen that has crossed a species barrier from animals to humans. the ongoing covid-19 pandemic has resulted in numerous accounts of different transmission routes between humans. droplet transmission (>5μm) is the most pronounced and heavily implicated mode of transmission reported during the pandemic. direct contact spread from one infected individual to a second, naïve person has also been considered a driver of human-to-human transmission, especially in households with close interactions between family members. the contagiousness of sars-cov-2 after disposition on fomites (e.g. door handle) is still under investigation, but is likely a compounding factor for transmission events, albeit less frequently than droplet or contact-driven transmission. both airborne and fecal-oral human-to-human transmission events were reported in j o u r n a l p r e -p r o o f the precursor sars-cov epidemic but have yet to be observed in the current crises. solid arrows show confirmed viral transfer from one infected person to another with a declining gradient in arrow width denoting the relative contributions of each transmission route. dashed lines show the plausibility of that transmission type but have yet to be 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infection model in mice demonstrates protection by neutralizing antibodies rapid generation of a mouse model for middle east respiratory syndrome generation of a broadly useful model for covid-19 pathogenesis vaccination, and treatment adenovirus vectors for gene therapy, vaccination and cancer gene therapy a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice a mouse-adapted sars-cov-2 model for the evaluation of covid-19 medical countermeasures a sars-cov-2 protein interaction map reveals targets for drug repurposing a mouse-adapted sars-cov-2 induces acute lung injury (ali) and mortality in standard laboratory mice respiratory disease in rhesus macaques inoculated with sars-cov-2 comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model sars-cov-2 infection protects against rechallenge in rhesus macaques cynomolgus macaque as an animal model for severe acute respiratory syndrome age-related rhesus macaque models of covid-19 immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection antibody responses to sars-cov-2 in patients of novel coronavirus disease antibody responses to sars-cov-2 in patients with covid-19 deep immune profiling of covid-19 patients reveals distinct immunotypes with therapeutic implications dna vaccine protection against sars-cov-2 in rhesus macaques seasonal coronavirus protective immunity is short-lasting targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals type-i, type-iii ifns then signal in an autocrine or paracrine manner through the janus kinase 1 (jak1)/signal transducer and activator of transcription 1 and 2 (stat1/2) pathway, culminating in antiviral interferon-stimulated gene (isg) transcription. listed here are sars-cov (abbreviated cov), sars-cov-2 (abbreviated cov-2) and mers-cov (abbreviated m-cov) ifn-i antagonists, which make these viruses resistant to interferon responses. ifn-iii is also implicated in exhibiting potent antiviral effects in lung/intestinal tissues, but the underlying evasion strategies of this pathway for these viruses are currently unknown. sars-cov proteins are highlighted in blue, while functions of sars-cov-2 and mers-cov proteins are highlighted in red and green, respectively. question mark symbol (?) denotes sars-cov-2 protein bound a member of that signaling pathway in [123], but further work is necessary to confirm its immunological mechanism. sars-cov-2 proteins with * denotes functional conservation with sars-cov which animal(s) serves as the natural reservoir of sars-cov-2?  does active replication of sars-cov-2 in the upper respiratory tract contribute to enhanced transmissibility in humans?  is intestinal sars-cov-2 infection a source of virus transmission?  which sars-cov-2 proteins antagonize innate and adaptive immune responses? do the sars-cov-2 proteins with more potent antagonistic immune functions increase virulence in humans compared to other hcovs?  why do some recovered patients fail to develop neutralizing antibodies?  what are the host and/or viral factors driving  what are the underlying mechanisms contributing to an inadequate ifn response to sars-cov-2?  what are the correlates of immune protection for sars-cov2 and will they provide sterilizing immunity?  will candidate vaccines against sars-cov2 also be effective in elderly subpopulations this work was supported by a national institutes of health grant r01ai132526 to p.w.the authors declare no competing financial/non-financial interest.j o u r n a l p r e -p r o o f journal pre-proof j o u r n a l p r e -p r o o f key: cord-335155-x9az3twa authors: qi, zhen; hu, yu; li, wei; chen, yanjun; zhang, zhihua; sun, shiwei; lu, hongchao; zhang, jingfen; bu, dongbo; ling, lunjiang; chen, runsheng title: phylogeny of sars-cov as inferred from complete genome comparison date: 2003 journal: chin sci bull doi: 10.1007/bf03183930 sha: doc_id: 335155 cord_uid: x9az3twa sars-cov, as the pathogeny of severe acute respiratory syndrome (sars), is a mystery that the origin of the virus is still unknown even a few isolates of the virus were completely sequenced. to explore the genesis of sars-cov, the fdod method previously developed by us was applied to comparing complete genomes from 12 sars-cov isolates to those from 12 previously identified coronaviruses and an unrooted phylogenetic tree was constructed. our results show that all sars-cov isolates were clustered into a clique and previously identified coronaviruses formed the other clique. meanwhile, the three groups of coronaviruses depart from each other clearly in our tree that is consistent with the results of prevenient papers. differently, from the topology of the phylogenetic tree we found that sars-cov is more close to group 1 within genus coronavirus. the topology map also shows that the 12 sars-cov isolates may be divided into two groups determined by the association with the sars-cov from the hotel m in hong kong that may give some information about the infectious relationship of the sars. sars-cov, as the pathogeny of severe acute respiratory syndrome (sars), seems to be the first coronavirus that is lethal to humans. coronavirus (family coronaviridae, genus coronavirus) is an enveloped, single-stranded plus sense rna virus whose genome has approximately 30 kb size. whereas coronaviruses may cause severe disease in animals, coronaviruses human strains only cause mild diseases until sars-cov was discovered. to date, sars-cov genomes from 12 isolates have been completely sequenced and released [i-4] . preliminary analysis of sars-cov genome indicated that the virus is not phylogenetic closely related to any previously identified coronaviruses. few obvious clues were given by the genome sequence to answer an important question: what is the origin of sars-cov? based on alignment of amino acid sequences or nucleotide acid sequences, some hypotheses were brought forward to elucidate the origin of sars-cov. however, the distant relationship of sars-cov to any known virus inferred from very low score of alignment makes these assumptions worthless. coronaviruses were classified into three groups a;cording to the serotypes: groups 1 and 2 contain mammalian viruses, while group 3 contains only avian viruses [5, 6] . based on the analysis of phylogeny from predicted proteins of sars-cov, rota et alp] claimed that sars-cov does not closely resemble any of these three groups and suggest the 4th group for sars-cov. some other authors arrived the same conclusion by analyzing proteins of other isolates[i, 3, 4] . to resolve the uncertain infectious relationship among different sars-cov strains, ruan et alp] compared genomes from 14 sars-cov isolates and identified 129 sequence variations among them. combined with the knowledge of contact source history and geography, common variant sequences were used as genetic signatures to reconstruct a probable lineage map of the sars-cov infections. they concluded that the case associated with infections originating in hotel m in hong kong form a group while other isolates form the other one. however, some details are still unclear. meanwhile, due to the limitation of data, ruan et al. have to restrict their research to 26140 loci. in addition, some of these 129 mutations might have occurred during in vitro expansion and might be sequencing errors rather than true ones [7] . to address such issues, a theoretical method (named fdod) that we previously developed based on shannon's defmition of information, entropy and degree of disagreement is used. fdod calculates species specific complete information set (cis) from its primary sequence of whole genome, thus circumambulate alignment and avoid any bias that may be associated with particular genomic regions. primary sequence of a genome is the result of its evolutionary history. the more closely phylogenetic related two species are, the more similar sequences they should have. hence, cis can be regarded as a reasonable measure of species distance [8, 9] . the software we developed and the supplementary material are available upon request. to date, genomes from 12 sars-cov isolates and 12 previously identified coronaviruses have been completely sequenced. we download these genomes from anonymous ftp server (ftp://130.14.22.5/genbank/genomes). table 1 gives the related information such as accession number, host, source, group, etc. the primary sequences of coronavirus genomes are subjected to fdod software to calculate the distance matrix based on their discrepancy of cis[8j. then, the neighbor based on neighbor joining algorithm in phylip 3.6 package was used to construct the unrooted tree from distance matrix. to generate multiple data sets for evaluating robustness of the branches of the tree, we adopted the jackknife algorithm to randomly resample[loj. finally, the consensus tree is produced using consense in phylip 3.6. the unrooted phylogenetic tree was constructed for genomes from 12 sars-cov isolates and that from 12 previously identified coronviruses (fig. 1) . it can be split into two parts at the point indicated by the arrow in fig. 1 . all sars-cov isolates are located at one side while 12 coronviruses are at the other part. consistent with the result of rota et alyj, the three groups of coronaviruses depart from each other clearly in our tree. the bootstrap value at the divergent point of these three groups is 92% (bootstrap values higher than 70% correspond to a probability higher than 95%[1i j ). our result indicated that sars-cov is closer to group 1 of the coronaviruses than to the other two groups (fig. 2 ) by the support with the high bootstrap value (bootstrap value is 97% for clad of group 1 and 81 % at divergent point of cpig_l and sars_tor2). differently, 1176 rota et al. regard sars-cov as a distinct group within the genus coronavirus based on alignment of amino acid sequences[i-4 j . prevenient paper shows that poorly conserved or variable-length region is not reliable for phylogeny construction based on aligmnent [12,13 j . since the similarity between sars-cov and coronaviruses was very 10w[2 j , the new method would be needed to build the phylogenetic tree. the results are based on our new method that circumambulate alignment of sequences and the results are supported moderately by some evidence related to serology. ksiazek et aly4j performed immunohistochemical assays with various antibodies reactive with coronaviruses from three groups and with the immune serum specimen from a sars patient. their result demonstrated strongly cytoplasmic and membranous staining of infected cells with antibodies related to coronaviruses of group 1 while no staining was identified with any antibodies related to coronaviruses of groups 2 and 3. hemagglutinin esterase gene, which presents in all coronaviruses of group 2 and some of group 3, does not exist in sars-covand coronaviruses of group 1[2 j , that also support our result that sars-cov is closer to coronaviruses of group 1. from the result one may assume that the origin of sars-cov may be more related to the coronaviruses of group 1 than to those ofthe other two groups. coronaviruses of group 1 cover a wide range of hosts, .. however, we cannot determine which one may be the normal host carrying sars-cov. possibly, sars-cov might come from an unknown animal that is not the host ofpreviously identified coronaviruses. since the taxon sampling is an important factor nfluencing the branching pattern of a tree [15] , we also construct unmoted trees from different samplings to inspect the robustness of our results and underlying method. very similar results were acquired that corroborate their robustness ofthe method (data not shown). the result shows an infectious relationship map for 12 sars-cov isolates which is very similar to that drawn by ruan et alp] (fig. 3) . 12 sars-cov isolates form two main groups (designated "local spread" and "global spread") determined by the association with the exposure at hotel m in hong kong. the sars_sg_3, as a secondary contact case, looks obviously like an ancestral strain among 5 isolates that came from singapore. however, it is also consistent with the results of ruan et al. and they attributed it to a potentially back mutational event during the transmission of the virus. sars_twi is placed within the cluster "global spread" which includes the isolates that are directly or indirectly associated with the infection at hotel m such as sars_tor2, sars_ hk_2, sars_urban, etc. we cannot decide which one of two main groups sars_hk_1 should be located in, since its contact history is not available. the genome se-1178 quence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection a complete sequence and comparative analysis of a sars-associated virus (isolate bjoi) coronaviridae, in virus taxonomy, classification and nomenclature of viruses coronavmidae: the viruses and their replication effects of passage history and sampling bias on phylogenetic reconstruction of inman influenza a evolution the characterization of a measure of infonnation discrepancy phylogeny based on whole genome as inferred from complete infonnation set analysis jackknife, bootstrap and other resampling plans in regression analysis bull, 1. 1., an emprical test of bootstrapping as a method for assessing confidence in phylogenetic analysis alignment-ambiguous nucleotide sites and the exclusion of systematic data elision: a method for accommodating multiple molecular sequence alignments with alignmentambiguous sites a novel coronavirus associated with severe acute respiratory syndrome taxon sampling and the accuracy of large phylogenies key: cord-322051-89wgv100 authors: tanasa, ingrid andrada; manciuc, carmen; carauleanu, alexandru; navolan, dan bogdan; bohiltea, roxana elena; nemescu, dragos title: anosmia and ageusia associated with coronavirus infection (covid-19) what is known? date: 2020-05-28 journal: exp ther med doi: 10.3892/etm.2020.8808 sha: doc_id: 322051 cord_uid: 89wgv100 in 2020 a new pandemic caused by the sars-cov-2 coronavirus is affecting the lives of millions of patients and healthcare workers worldwide. the clinical picture of this infection is in a dynamic process of discovery, and more symptoms emerge as the clinicians observe and diagnose manifestations that affect multiple organs. anosmia (loss of smell), and ageusia (loss of taste) become more frequently cited as independent symptoms or in association with the most common manifestations of the disease, such as fever, cough and dyspnea. a thorough screening program will prevent most nosocomial and community-acquired infections by promoting efficient triage and specific measures such as isolation of the patients. therefore, it is important to include frequent symptoms in the anamnesis and questionnaires to select those patients who might benefit from testing, isolation, and treatment. this study summarizes the existing data regarding the association of anosmia and ageusia with the sars-cov-2 infection. it also aims to describe manifestations of these, particularly in the clinical picture of all symptomatic patients. the clinical picture of sars-cov-2 infection (17) (18) (19) . an increasing volume of empirical data indicates that anosmia, hyposmia, ageusia, and dysgeusia may be possible symptoms of sars-cov-2 infection, regardless of, or alongside, classic symptoms. this report summarizes the evidence regarding the association of anosmia and ageusia with sars-cov-2 infection. it also aims to describe these manifestations' particularly in the clinical picture of symptomatic patients. a literature search was conducted in medline, embase, and cochrane databases using the following keywords, including synonyms, and all the possible combinations of them: anosmia, ageusia, sars-cov-2, covid-19, coronavirus. studies were included that documented symptoms associated with sars-cov-2 infection (up to may 5, 2020) . viral upper respiratory tract infections frequently determine olfactory dysfunction and taste disfunction. coronaviruses are the main determinants of the common cold, along with other viruses such as rhinoviruses, influenza, and parainfluenza. therefore, it is no surprise that the sars-cov-2 virus would determine smell alteration in some infected patients. in a retrospective observational study, klopfenstein et al (20) reported that 54 patients (47%) with confirmed sars-cov-2 infection developed anosmia, 4.4 (±1.9) days after infection onset, and that was the third symptom to manifest in 38% (22/52) of the cases. the mean duration of anosmia was 8.9 (±6.3) days, and one patient had not recovered at the end of the follow-up (after 28 days). anosmia was associated with dysgeusia in 85% of cases (n=46). mao et al (21) published a retrospective case series at three hospital centers in wuhan, china. it included 214 patients confirmed with sars-cov-2 infection and evaluated the presence of neurological symptoms (central, peripheral) and musculoskeletal symptoms. as for the peripheral symptoms (8.9%), the authors reported that the most common were hypogeusia and hyposmia with 5.6 and 5.1%, respectively. although currently there are no guidelines that recommend testing of the persons with symptoms such as anosmia or ageusia, some authors suggest testing and isolation for those patients who experience these symptoms alongside with hyposmia and dysgeusia in the absence of other explanatory conditions (22, 23) . recent studies suggested that the different variants of the virus could determine the extent of susceptibility and clinical course for the infected patient, so that different cohort of patients may have a polymorphic clinical presentation (24) . li et al (25) demonstrated that some ace2 variants might prevent the association with sars-cov-2 s-protein. however, scarce data are currently available, and more research could lead to a proper understanding of this topic. on the other hand, cao et al (26) demonstrated the genetic polymorphisms of the ace2 receptor and the specific prevalence in the european and asian populations suggesting the possibility of different clinical courses for these patients. few studies described the pathophysiological mechanisms of the olfactory and gustatory dysfunction in the sars-cov-2 infection. available data indicate the virus has a neural spread and a cytopathic effect on the neurons. it affects mainly neurons in the cortex and hypothalamus (27) . some authors reported three mechanisms for anosmia in covid-19 patients: i) local infection of support cells and vascular pericytes in the nose and olfactory bulb that may affect the function of bipolar neurons or mitral cells; ii) damage to support cells in the sensory epithelium that may indirectly influence the signaling pathway from sensory neurons to the brain; and iii) damage to sustentacular cells and bowman's gland cells that could lead to diffuse morphological damage to the olfactory sensory epithelium and altering of smell perception (28, 29) . in scientific literature, two syndromes with different outcomes are described. one is the olfactory cleft syndrome, which involves a conductive loss due to mucosal obstruction of the olfactory cleft. the other is post-viral anosmia syndrome, which implies a neural loss, due to the destruction of the olfactory sensory neurons (30) . the first syndrome has a relatively rapid resolution of anosmia, while the second one can be associated with a persistent olfactory deficit. netland et al (31) detected the coronavirus 60 h after exposure to sars-cov-2 in the olfactory bulb and after four days, its dissemination to the pyriform cortex and dorsal nucleus of the rafe. this aspect suggests a rapid viral propagation to the brain structures. recent studies found that sars-cov-2 could be detected in saliva and that it is possible to measure the viral load in this fluid (32, 33) . hu et al (32) studied the cellular distribution of taste cells and ace2 receptor distribution. they found that the percentage of ace2 positive cells was higher in taste cells, which indicated that sars-cov-2 might invade them and lead to ageusia in these patients. however, data regarding the exact mechanism by which sars-cov-2 determines ageusia is limited. the virus may bind to the sialic acid receptors and occupy and accelerate the degradation of the gustatory particles, leading to a decrease in taste sensation (34) . medical imaging and neuropathology studies evaluate changes in the olfactory bulb, cranial nerves, and brains of the infected patients. thus, magnetic resonance (mr) imaging of the olfactory bulb can be used to assess the patients with anosmia (35) . the main mr findings in anosmia secondary to upper respiratory infection are reduced olfactory bulb and tract volume, which correlate with the olfactory function (35) . anamnesis and personal reports of anosmia and ageusia are also necessary. one can use questionnaires for clinical diagnosis and follow-up in anosmia or ageusia patients. in a prospective study, lechien et al (24) evaluated anosmia and ageusia using questionnaires based on smell and taste component of the national health and nutrition examination survey, and the short version of the questionnaire of olfactory disorders-negative statements (sqod-ns). these authors found a substantially higher prevalence of olfactory and gustatory dysfunction in european covid-19 patients. more, the olfactory disorder may appear before the rest of the complaints. they identified a significant association between olfactory and gustatory dysfunctions. in a cross-sectional study by bagheri et al (36) , 10,069 participants responded to an online checklist that evaluated the sense of smell and taste. the results indicated a significant correlation between anosmia and sars-cov-2 infection, decreased taste sensation, and decreased quality of life. we found numerous validated questionnaires and tests for assessing anosmia and ageusia. however, more data are needed to provide quantitative data on the incidence and severity of these symptoms in association with sars-cov-2 infection. according to a report of the french society of otolaryngology, patients must avoid corticosteroids for the treatment of the sars-cov-2 infection (37) . on the other hand, clinicians frequently use empirical oral steroids for the treatment of anosmia, to decrease inflammation and edema. we suggest that individualized case management and treatment should be applied, considering the increased risks of immunosuppression associated with these drugs. the relative risk seems to be reduced in the case of intranasal steroids use. here, the main concern is the exacerbation of an upper respiratory tract viral infection (38) . lechien et al (24) reported that the most frequently used treatments for olfactory dysfunction in sars-cov-2 infected patients were nasal saline irrigations, followed by nasal and oral corticosteroids. as for gustatory dysfunction, clinicians used l-carnitine or trace elements and vitamins. smell training is a simple, safe, and readily available method in the context of social distancing for smell recovery in different forms of anosmia (39) (40) (41) . there is no therapeutic regime for ageusia. the condition may either improve gradually on its own or may remain the same. in patients with associated xerostomia, a therapeutic option could be artificial saliva. scarce data reported in the literature suggest that most patients will achieve smell recovery up to 14 days after resolving the disease. in the study conducted by lechien et al (24) , 67.8% of the anosmic patients recovered olfactory function within the first eight days following the resolution of the disease, and it seems that, at least, 25.5% of the patients recovered both olfactory and gustatory functions throughout the two weeks after the resolution of general symptoms. further research is needed to demonstrate the association between anosmia and ageusia with sars-cov-2 infection, the clinical manifestations determined by variants of ace2 receptor, and recovery rates of olfactory and gustative dysfunction, and specific treatment protocols of these manifestations. anosmia and ageusia are symptoms that require further investigation during a clinical consultation, due to the increasing evidence of their association with the new coronavirus. careful screening and prevention must be offered to avoid nosocomial and community infection. not applicable. no funding was received. all data generated or analyzed during this study are included in this published article. iat and dn designed the study. iat, dn and ac retrieved the data. cm, ac and dbn contributed to data extraction and quality assessment. cm, dbn and reb were responsible for the analysis and discussion of data. iat and dn drafted the manuscript. cm, ac, dbn and reb revised critically the manuscript and made substantial intellectual contributions to the study. all authors read and approved the final manuscript. not applicable. the coronavirus disease 2019 (covid-19) pandemic a pneumonia outbreak associated with a new coronavirus of probable bat origin cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding a new threat from an old enemy: re emergence of coronavirus (review) towards effective covid 19 vaccines: updates, perspectives and challenges (review) stabilized coronavirus spikes are resistant to 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different populations multiple organ infection and the pathogenesis of sars a single-cell atlas of the airway epithelium reveals the cftr-rich pulmonary ionocyte morphologic changes in the nasal cavity associated with sialodacryoadenitis virus infection in the wistar rat inflammatory obstruction of the olfactory clefts and olfactory loss in humans: a new syndrome? severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study reduced salivary mucin binding and glycosylation in older adults influences taste in an in vitro cell model mri detection of olfactory bulb and tract coincidence of covid-19 epidemic and olfactory dysfunction outbreak covid-19 and treatment with nsaids and corticosteroids: should we be limiting their use in the clinical setting topical therapy in anosmia: relevance of steroid-responsiveness effects of olfactory training in patients with olfactory loss olfactory training is helpful in postinfectious olfactory loss: a randomized, controlled, multicenter study olfactory training changes electrophysiological responses at the level of the olfactory epithelium the authors declare that they have no competing interests. key: cord-336057-tj9qcuf8 authors: lv, yantian; gu, binbin; chen, ying; hu, siming; ruan, ting; xu, guopeng; ding, jian; xu, xiao; shen, xinghua title: no intrauterine vertical transmission in pregnancy with covid-19: a case report date: 2020-08-05 journal: j infect chemother doi: 10.1016/j.jiac.2020.07.015 sha: doc_id: 336057 cord_uid: tj9qcuf8 abstract the coronavirus disease 2019(covid-19)has been a worldwide pandemic diseases, nearly 400,000 people died at now. the data of status of pregnant women and neonates after infection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is limited. we report a case of pregnant woman in her third trimester with critical covid-19, and amniotic fluid, umbilical cord blood, placenta, and neonatal gastric fluid were retained during cesarean section. the sars-cov-2 nucleic acid test results of these specimens were negative. there is no evidence of intrauterine vertical transmission during delivery in the third trimester, but the data are limited and need to be further explored. since the sars-cov-2 virus was discovered in china in december 2019, as of june 05, 2020, who confirmed 6535354 cases of (sars-cov-2) infections worldwide, and nearly 400000 deaths were declared 1 . as a special population, pregnant women have attracted much attention, and the presence of intrauterine vertical transmission is critical to the outcome of newborns. here, we report a pregnant woman with critical covid-19 to provide some help for the treatment of pregnant women. a 28-year-old 31-week pregnant woman was admitted to the fifth people's hospital of suzhou on february 6,2020 for recurrent fever and cough in 2 weeks. epidemiological history showed that the patient went to wuhan to visit relatives on january 4, and returned to suzhou on january 23. she developed fever on january 25, with a maximum body temperature of 38.3℃. she went to the fever clinic of suzhou municipal hospital on february 2. the blood test showed a normal white blood cells, lymphopenia and elevated crp j o u r n a l p r e -p r o o f (table). the result of the sars-cov-2 nucleic acid test was negative, however, given her epidemiological history, the possibility of sars-cov-2 infection remained to be considered. a chest ct scan on february 4 revealed multiple peripheral ground-glass exudates with partial consolidation in bilateral lungs ( figure) . on february 6, after the 4th sars-cov-2 nucleic acid test returned to be positive, she was transferred to the fifth people's hospital of suzhou for treatment. blood test showed 10.60×10 9 /l of wbc, 0.85×10 9 /l of lymphocytes, 124.1mg / l of crp, 0.288 ng/ml of pct. abg analysis showed that po 2 was 120.6mmhg, pco 2 was 38.2mmhg, and po 2 /fio 2 index was 294.according to the sars-cov-2 pneumonia protocol (5th edn) 2 , the pregnant woman was classified as critical type of covid-19. we gave her high flow oxygen, lopinaviri/rionavir for antiviral, cefoperazone tazobactam for anti-infection, dexamethasone for fetal maturation, and symptomatic management. on february 8, the fetal heart rate was 100 beats/min, the patient's abg analysis showed that pf index was 208, and ct scan indicated pulmonary lesions deteriorated. the cesarean section was performed under lumbosacral combined anesthesia immediately, and a 1830g live-born male infant was delivered successfully. amniotic fluid, umbilical cord blood, placenta, and neonatal gastric fluid were collected during the operation and tested for the sars-cov-2 nucleic acid, and the mother and infant were separated after the operation. j o u r n a l p r e -p r o o f on february 9, a blood test showed an increase in pct of 1.63 ng/ml, linezolid was added to enhance anti-infection. lopinaviri/ritonavir was replaced by abidol because of nausea and vomiting. on february 12, ct scan indicated that the lesions absorbed than before and her clinical symptoms improved continually. on february 18, chest ct showed that the lesions almost disappeared. in addition, the sputum sars-cov-2 nucleic acid test was negative for the fourth time, and the anal swab sars-cov-2 nucleic acid was also negative. the patient was discharged on february 20. as for the newborn, his apgar scores were 8, 8 and 10 at 1, 5, and 10 minutes after birth, with weight of 1830g, blood pressure of 77/48mmhg, and pulse oxygen 95%.he was admitted to the negative pressure isolation ward after birth, ncpap breathing support, given sulbenicillin sodium, azithromycin for anti-infection treatment. in addition, not only sars-cov-2 nucleic acid test results were negative in 4 times pharyngeal swabs, but also the anal swab, amniotic fluid, umbilical cord blood, placenta, and neonatal gastric fluid were negative. on march 28, the neonatal weight had grown to 2530g, with no difficulties of breathing and pulse oxygen of 99%, he was discharged. the common syndrome of covid-19 are fever, dry cough, sore throat, muscle soreness, shortness of breath, occasional diarrhea and other symptoms [3] [4] [5] . there was no significant difference in symptoms between j o u r n a l p r e -p r o o f pregnant women and non-pregnant women 6 . it should be noted that this case was diagnosed based on multiple nucleic acid tests. for nucleic acid testing, sputum specimens are recommended because the positive rate of sputum(74.4%~88.9%) is higher than that of the nasopharynx swap (53.6%~73.3%) 7 .in treatment, lopinavir/ritonavir has been shown to be safe in hiv-infected pregnant women, but due to adverse reactions, we used arbidol instead, which was proven to be safe and effective. another important question is whether there is a possibility of intrauterine vertical transmission. chen 6 .reported the delivery of 9 cases of pregnant women with covid-19. sars-cov-2 tests were performed on amniotic fluid, umbilical cord blood, neonatal throat swabs and breast milk samples from six of these patients. the results were negative. zhu 8 described 10 neonates in pregnant women with covid-9, including two cases of vaginal delivery. 9 cases had pharyngeal swab specimens taken 1-9 days after birth for sars-cov-2 nucleic acid tests, all of which were negative. li 9 also reported a 35-week pregnant woman with covid-19, whose amniotic fluid, cord blood and placenta, breast milk samples as well as neonates swab sars-cov-2 nucleic acid were all negative. however, dong 10 reported a case of a neonate by cesarean, whose sars-cov-2 igg and igm were positive 2 hours after birth. before her born, her mother had been infected sars-cov-2 for more than 20 days. it is known that igm antibodies are not transferred to the fetus via the placenta. it seemed that the elevated igm in the neonate indirectly thank you very much for giving us an opportunity to revise our manuscript. we appreciate the editor and reviewers very much for their constructive comments and suggestions on our manuscript entitled" no intrauterine vertical transmission in pregnancy with covid-19：a case report". those comments are very helpful for revising and improving our paper.we have studied the comments carefully and made corrections which we hope meet with approval. the main corrections are in the manuscript and the responds to the reviewers' comments are as follows (the replies are highlighted in blue). kind regards. situation report-1 new coronavirus pneumonia prevention and control program e13a/files/ab6bec7f93e64e7f998d802991203cd6.pdf hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama. published online epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of covid-19 in china clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infection clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia lack of vertical transmission of severe acute respiratory syndrome coronavirus 2 possible vertical transmission of sars-cov-2 from an infected mother to her newborn pregnant women with new coronavirus infection: a clinical characteristics and placental pathological key: cord-331193-33cyvidx authors: mawhinney, jamie a; wilcock, catherine; haboubi, hasan; roshanzamir, shahbaz title: neurotropism of sars-cov-2: covid-19 presenting with an acute manic episode date: 2020-06-14 journal: bmj case rep doi: 10.1136/bcr-2020-236123 sha: doc_id: 331193 cord_uid: 33cyvidx a 41-year-old man with no significant medical history presented with acute behavioural disruption on the background of a 1-day history of severe headache and a 10-day history of dry cough and fever. he was sexually disinhibited with pressured speech and grandiose ideas. his behaviour worsened, necessitating heavy sedation and transfer to intensive care for mechanical ventilation despite no respiratory indication. investigations confirmed that he was positive for severe acute respiratory syndrome coronavirus 2 (sars-cov-2). neuroimaging and a lumbar puncture were normal. initial screening for sars-cov-2 in the cerebrospinal fluid was negative although no validated assay was available. the patient’s mental state remained abnormal following stepdown from intensive care. psychiatric assessment found features consistent with acute mania, and he was detained under the mental health act. this case indicates the need to consider covid-19 in a wider series of clinical presentations and to develop a validated assay for sars-cov-2 in the cerebrospinal fluid. a 41-year-old man with no significant medical history presented with acute behavioural disruption on the background of a 1-day history of severe headache and a 10-day history of dry cough and fever. he was sexually disinhibited with pressured speech and grandiose ideas. his behaviour worsened, necessitating heavy sedation and transfer to intensive care for mechanical ventilation despite no respiratory indication. investigations confirmed that he was positive for severe acute respiratory syndrome coronavirus 2 (sars-cov-2). neuroimaging and a lumbar puncture were normal. initial screening for sars-cov-2 in the cerebrospinal fluid was negative although no validated assay was available. the patient's mental state remained abnormal following stepdown from intensive care. psychiatric assessment found features consistent with acute mania, and he was detained under the mental health act. this case indicates the need to consider covid-19 in a wider series of clinical presentations and to develop a validated assay for sars-cov-2 in the cerebrospinal fluid. covid-19 is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). 1 the virus was identified as the cause of an outbreak of pneumonia in hubei province, china, in december 2019 and has spread globally, so far responsible for over 2 million cases and 150 000 deaths worldwide. 2 sars-cov-2 is the seventh coronavirus known to infect humans and is a member of the orthocoronavirus subfamily. 3 it appears to be principally transmitted via respiratory droplets and contact with surfaces conveying the pathogen. 3 entry into the host cells is reportedly via the ace-2 receptor. 4 the most common initial symptoms of covid-19 are fever, dry cough, fatigue and myalgia. 5 the primary complication is acute lung injury resulting in type 1 respiratory failure, with a significant proportion requiring intensive care unit admission. 6 7 however, in addition to these respiratory features, the disease affects multiple organs including the cardiovascular system and gastrointestinal system. 4 6 8 there have been few case reports of primarily neurological presentations of the disease. [9] [10] [11] [12] this article outlines a case of covid-19 presenting with an acute manic episode necessitating emergency intubation and discusses potential mechanisms for the development of neuropsychiatric disease. a 41-year-old man with no significant medical history other than congenital nystagmus presented to the emergency department in the early hours of the morning. he had woken at night restless, agitated and reported feeling like his 'brain was racing'. he told his wife that he felt like he was 'going to die'. he also confessed to numerous hitherto undisclosed homosexual encounters and other sexual behaviours described as uncharacteristic by his wife. this acute presentation was preceded by a 1-day history of severe occipito-parietal headache, described as the 'worst headache ever', and a 10-day history of a dry cough and fever. his wife also reported similar but milder symptoms. he did not report anosmia. he had no significant smoking or alcohol history and denied any illicit drug use. he did however report a severe transient mood reaction with some possible paranoid features to cannabis in 2004 with no further use since then. his sister had a previous episode of postpartum psychosis and was subsequently diagnosed with bipolar disorder. physical examination revealed fine bibasal inspiratory crepitations and a nystagmus in all directions. neurological examination was otherwise normal. his mental state examination however was abnormal. he was loud and highly aroused with sexual disinhibition and overfamiliar behaviour, inappropriately questioning and touching members of staff. his speech was pressured, and his mood subjectively and objectively elevated. his thoughts were grandiose with persecutory elements, and he had persistent strong religious ideas, manifestations of which included attempts to anoint fellow patients with water. he also obsessively wrote down every personal interaction and bodily sensation. he said he found this experience 'liberating'. he did not report visual or auditory hallucinations. this abnormal behaviour worsened while in the emergency department to the point where it was deemed necessary for him to be transferred to intensive care for sedation and mechanical ventilation despite no respiratory indication. a nose and throat swab taken on admission subsequently tested positive for the presence of sars-cov-2 viral rna and chest x-ray showed features consistent with a covid-19 pneumonitis. bloods results during admission are shown in table 1. his crp and neutrophils were raised initially but new disease settled shortly after admission, as did his mild lymphopaenia. liver function tests were initially normal, but he developed a mild transaminitis soon after admission. thyroid stimulating hormone, b12 and folate were normal. serological screening was negative for hiv antigen and antibody, hepatitis b surface and core antigen, hepatitis c igg antibodies and treponema pallidum antibodies. no common autoantibodies were found in the serum, apart from mildly raised hep2 antinuclear antibodies of uncertain significance. a coeliac screen was also negative, as were n-methyl-d-aspartate receptor (nmda) and voltage-gated k+ channel autoantibodies. ct and mri brain showed no acute intracranial pathology or evidence of encephalitis. a lumbar puncture with normal opening pressure demonstrated gin-clear cerebrospinal fluid (csf) and no erythrocytes, leucocytes or other organisms. glucose level in the csf has 4.8 mmol/l (plasma glucose 8.4 mmol/l) and protein level 0.19 g/l. the sample was also was negative for herpes simplex virus dna, varicella zoster virus dna, enteroviorus rna and parechovirus rna not detected. initial screening for sars-cov-2 in the csf were also negative, although a validated assay was not yet available making interpretation difficult. empirical antimicrobial and antiviral treatment to cover for bacterial meningitis, community-acquired pneumonia and viral encephalitis were commenced but ceased after 48 hours in the absence of any evidence of ongoing infection on clinical and biochemical investigation. the patient was extubated after less than 24 hours of mechanical ventilation and moved to a level one ward environment. the patient's respiratory symptoms settled within 2 days of step down, but his mental state remained abnormal. an addenbrokes cognitive examination scored 90/100 with 18/18 in attention tasks and 25/26 in memory tasks while a frontal assessment battery scored 14/18 losing points for inhibitory control and lexical fluency and motor assessment. by day 8, his behaviour had escalated further culminating in a security call and emergency sedation for the safety of himself, the ward staff and other patients. he was subsequently detained under section 2 of the mental health act 1983, transferred to an acute inpatient psychiatric hospital and commenced on regular olanzapine. during this admission, he continued on regular antipsychotics and benzodiazepines for sedation. he required transfer to the emergency department while still an inpatient where he was investigated for severe left-sided chest pain. a ct pulmonary angiogram confirmed ongoing inflammatory changes consistent with covid-19 pneumonitis but no other pathology. as the ambulance came, i confessed to my wife that i had sex with men (most of which before marriage), although i am heterosexual. i felt that i was incapable of lying or hiding the truth and thought i was dying. i was in hospital for a total of 20 days with psychosis and mania, which i experienced as fascinating. this may seem strange from an outside perspective, but i was, in my mania, trying to help the doctors as much as i could, while at the same time trying to make sense of my condition. i began to think that i was part of a tv show, in which i was sent back from the future to save the nhs, and i was curious to see how this would end. for my family and friends it was frightening. luckily, they had a lot of support from each other, and from the great team of doctors at st. thomas hospital. ► covid-19 manifests in a number of ways affecting multiple systems including the central nervous system (cns). ► the neuroinvasive potential of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (neurotropism) has been reported, but the pathophysiology remains unclear with uncertainty over its long-term consequences. ► there are multiple effects of sars-cov-2 virus on the cns, and currently, there are no specific treatment options available particularly for the neurotropic sequelae. ► further research is needed to develop a validated assay for sars-cov-2 in the cerebrospinal fluid. his mania improved on return to the psychiatric unit, and he was discharged 12 days after instigation of the section 2 order. his medications on discharge included olanzapine 10 mg daily with a plan to reduce this to 7.5 mg within the next week and 1 mg twice daily of clonazepam also to be weaned in the community. at follow-up 23 days from the original presentation, he was well at home, and he and his wife reported that he was now at his baseline level of function. this report outlines a rare case of acute mania associated with sars-cov-2 infection. this was particularly severe, necessitating emergency intubation and subsequent inpatient psychiatric admission. although this may represent a first episode of a primary psychiatric condition such as bipolar disorder, it is also important to consider other organic disease given the simultaneous diagnosis of covid-19. although it is not yet possible to confirm here due to the lack of a validated csf-pcr assay, previous reports have implicated sars-cov-2 in the development of viral encephalitis, and this remains an important differential. 9 12 in one of the initial reports on patient presentations and outcomes from wuhan, confusion accounted for 9% of reported symptoms, although the nature of these episodes was not expanded on. 7 in addition to this, there are further reports of covid-19 causing acute haemorrhagic necrotising encephalopathy and acute inflammatory neuropathy in guillain-barre syndrome. 10 11 an acute delirium was also considered, although the absence of a fluctuating course and inattention as key features made this less likely. neurotropism of sars-cov-2 has been tentatively reviewed in the literature. the entry of sars-cov-2 into human host cells is mediated mainly using ace-2 as a receptor, and although the lungs and the gastrointestinal tract are the principle sites of expression of ace-2 in the body, the protein is also expressed throughout endothelial cells in the brain providing a theoretical route of entry into the central nervous system. 13 14 more specifically, the amygdala, which has key functions in emotional intelligence as well as those related to sexual arousal, has been demonstrated to express ace-2 in animal models thus providing a focus to which the spike proteins of the virus may bind. 15 it has also been hypothesised that the virus may enter via peripheral nerve terminals. 16 neurological invasion of the virus therefore may represent another potential aetiology for this acute illness in absence of any other biological, psychology or social precipitating factors. this is, to the best of our knowledge, the first report of an acute episode of mania or psychosis as a result of sars-cov-2 infection. the pathophysiology of this is yet to be discerned, but given the temporal relation, we are led to assume that viral infection mediated this presentation. the ideal treatment modality for neuropsychiatric manifestations of covid-19 and the long-term prognosis of such cases remain to be seen. this case indicates the need to consider testing for and diagnosis of covid-19 in a wider series of clinical presentations including new onset psychiatric and neurological disorder. more research is required to look at the neurological manifestations of covid-19, as well as a need to develop a validated assay for sars-cov-2 in the csf in order to determine the neuroinvasive potential of the virus. pathological findings of covid-19 associated with acute respiratory distress syndrome world health organisation. who covid-19 dashboard the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak covid-19 and the cardiovascular system clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study covid-19 and the gastrointestinal tract: more than meets the eye neurological complications of coronavirus disease (covid-19): encephalopathy permissions/ bmj case report fellows may re-use this article for personal use and teaching without any further permission. become a fellow of bmj case reports today and you can: have any further queries about your subscription, please contact our customer services team on +44 (0) 207111 1105 or via email at support@bmj.com. visit casereports.bmj guillain-barré syndrome associated with sars-cov-2 infection: causality or coincidence? covid-19-associated acute hemorrhagic necrotizing encephalopathy: ct and mri features a first case of meningitis/encephalitis associated with sars-coronavirus-2 tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating central mas receptors the neuroinvasive potential of sars-cov2 may be at least partially responsible for the respiratory failure of covid-19 patients contributors all authors were the acute medical team managing the patient. jam wrote the manuscript and cw, hh and sr reviewed the manuscript and provided insight into further research in the field.funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.competing interests none declared. patient consent for publication obtained.provenance and peer review not commissioned; externally peer reviewed.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. orcid id jamie a mawhinney http:// orcid. org/ 0000-0002-7851-7953 key: cord-323666-t7cshj05 authors: cegolon, l.; javanbakht, m.; mastrangelo, g. title: nasal disinfection for the prevention and control of covid-19: a scoping review on potential chemo-preventive agents. date: 2020-08-18 journal: int j hyg environ health doi: 10.1016/j.ijheh.2020.113605 sha: doc_id: 323666 cord_uid: t7cshj05 background: neither pre-exposure nor post-exposure chemo-prophylaxis agents are currently available to prevent covid-19. on the other hand, high loads of sars-cov-2 are shed from the nasal cavity before and after symptoms onset. objective: to conduct a scoping review on the available evidence on tolerable nasal disinfectants with encouraging health outcomes against sars-cov-2, i.e., agents effective against at least two different viruses beyond sars-cov-2. methods: online databases were searched to identify papers published during 2010–2020. publications were selected if they were relevant to the scoping review. the review was narrative, describing for each treatment the mechanism(s) of action, tolerability, in vitro and in vivo evidence of the effects against sars-cov-2 and whether the product had been marketed. results: eight treatments were scrutinized: hypothiocyanite, lactoferrin, n-chlorotaurine, interferon-alpha, povidone-iodine, quaternary ammonium compounds, alcohol-based nasal antiseptics and hydroxychloroquine. in vitro viricidal effect against sars-cov-2 was reported for povidone-iodine. inhibition of other coronaviruses was described for lactoferrin, hydroxychloroquine and quaternary ammonium compound. no treatment has been tested against sars-cov-2 in randomized controlled clinical trials thus far. however, interferon-alpha, lactoferrin and hydroxychloroquine were tested in one-arm open label uncontrolled clinical trials. oxidant activity (hypothiocyanite, n-chlorotaurine and povidone-iodine), enhancement of endocytic and lysosomal ph (quaternary ammonium compounds and hydroxychloroquine) and destruction of the viral capsid (quaternary ammonium compounds, alcohol-based nasal antiseptics) were the main mechanisms of action. lactoferrin and interferon-alpha had subtle biological mechanisms. with the exception of n-chlorotaurine, the others are products available on the market. conclusions: effective and safe chemo-prophylactic drugs against sars-cov-2 do not exist yet but most eligible candidates are already in the market. whilst the human nasal cavity is the port of entry for sars-cov-2, the mouth is involved as exit site through emission of respiratory droplets. the well-known hand-to-nose-to-hand cycle of contamination requires appropriate additional strategies for infection control. to narrow down the subsequent laboratory and clinical investigations, a case-control approach could be employed to compare the use of candidate drugs among individuals testing positive and negative to covid-19 swabs. but most eligible candidates are already in the market. whilst the human nasal cavity is the port of entry for sars-cov-2, the mouth is involved as exit site through emission of respiratory droplets. the well-known hand-to-nose-to-hand cycle of contamination requires appropriate additional strategies for infection control. to narrow down the subsequent laboratory and clinical investigations, a case-control approach could be employed to compare the use of candidate drugs among individuals testing positive and negative to covid-19 swabs. j o u r n a l p r e -p r o o f background the current coronavirus disease 2019 , caused by a novel beta coronavirus (sars-cov-2), has been declared a pandemic affecting 213 countries as of 12 th june 2020 [1] . unlike sars-cov, sars-cov-2 seems to replicate efficiently in the upper airways during the incubation period, which is estimated to last up to 14 days [2, 3] . during this prodromal stage, asymptomatic and pre-symptomatic individuals release large amounts of viruses from infected cells [3] . as a result, viral transmission is more effective with sars-cov-2 than with sars-cov, which conversely was contagious only during the active/critical phase of the disease [3] . furthermore, human coronaviruses which may cause common cold are known to cause re-infection regardless of pre-existing humoral immunity [4, 5] . therefore, there are two issues with covid19: high transmissibility of the virus and variable clinical pattern of the disease, which can range from pre-symptomatic/asymptomatic condition to life threatening pneumonia featured by severe acute respiratory syndrome (sars) and hypercoagulable state [3, 5] . the high risk of contagion from sars-cov-2 has been tackled so far by infection prevention and control (ipc) measures such as self-isolation, social distancing, quarantine of contacts, use of personal protective equipment (ppe), travel restrictions and other limitations to the freedom of movement of individuals. however, the effect of each intervention in containing the spread of sars-cov-2 has not been evaluated yet [6] . currently, the world health organization and the european centre for disease prevention and control guidelines recommend testing only symptomatic individuals and close contacts of a confirmed covid-19 case [7, 8] . whilst in the initial stage of the epidemic a symptom-based screening strategy was useful with the aim to treat and isolate infected cases, covid-19 outbreaks in care homes and hospitals suggest that the current ipc measures are inadequate and have failed j o u r n a l p r e -p r o o f [9, 10] . furthermore, the above ipc measures are not sustainable in the long run, as they would severely ruin the economies of countries heavily affected by covid-19. the clear need to relax/avoid the lock down measures enforced in many countries prompts for a revised approach to reduce the transmission of sars-cov-2 from asymptomatic/pre-symptomatic individuals, until a pharmacological intervention (ideally a vaccine or a chemoprophylaxis) will hopefully become available [9] . as explained above, the problem with covid-19 is transmission from asymptomatic/pre-symptomatic individuals, a phenomenon particularly critical in care homes and hospitals, where more than half residents testing positive might not develop symptoms [2, 9] . in los angeles families have been advised by the public health department to remove their relatives from care homes to reduce health risk of community outbreaks of covid-19 [11] . mass testing has been considered in various countries as a possible strategy to reduce the transmission of sars-cov-2 from asymptomatic individuals [9] . nonetheless, mass testing on defined and contained outbreak clusters (as done in south korea) may be sensible, not in the current global scenario though, with an ongoing pandemic [12] . not to mention that mass testing is probably far beyond the capacity of microbiology laboratories even in high-income countries. however, restricting mass testing to high risk congregate settings, such as care homes, hospitals, mental health facilities and prisons may be appropriate [9] . in an outbreak reported from a care home in washington state, hosting a total of 76 residents, 27 of the 48 residents testing positive for covid-19 at real time pcr were asymptomatic during the 14 days preceding the test [10] . high loads of sars-cov-2 are shed from the nasal cavity into the environment also before symptoms onset [2, 9] and transmission of sars-cov-2 from asymptomatic individuals has been described [13] [14] [15] . unnoticed, asymptomatic cases of covid-19 might therefore constitute an important source of contagion, as endorsed by recent evidence from the china's national health commission, reporting that the vast majority of infections (four out of five) were totally j o u r n a l p r e -p r o o f asymptomatic. in particular, 130 out of the total 166 new infections identified in china on 1st april 2020 had no symptoms whatsoever [16] although asymptomatic individuals testing positive for sars-cov-2 are enforced to quarantine for 14 days, mass testing would yet not resolve the endemic presence of the virus in the general population, a condition posing the risk of repeated resurgences of outbreaks, especially with cold weather conditions [17] . there is in fact evidence that the transmissibility and viability of sars-cov-2 reduces importantly with hot and humid climate [5, 18, 19] , as confirmed by the diminished spread of the novel coronavirus with increasing relative humidity from 23.33% to 82.67% (p-value = 0.002) and with increased weather temperature from −13.17°c to 19 °c (p-value = 0.003) observed for chinese cities during january-march 2020 [20] . humoral immunity seems not protective against human coronaviruses, and some evidence even points out a potential antibody dependent enhancement (ade) triggered by re-infections featuring the severe/critical forms of covid-19, as with other viral diseases such as dengue, sars-cov, mers-cov and the west nile virus. [5] . given the potential of reversed and untoward consequences following relaxation of the above ipc measures, especially during cold months, and in absence of a specific registered treatment or vaccine against sars-cov-2, there is a clear and urgent need to find alternative solutions to prevent and control the replication of the virus and the spread of covid-19 among humans [14] . covid-19 is a rapidly evolving pandemic. asymptomatic individuals testing positive for sars-cov-2 and asked to self-isolate at home were in italy about 40% of all positive individuals at the beginning of the epidemic, then becoming >80% following the end of full lockdown [21] . figure 1 reports the corresponding changes as percentage or odds; the latter detects the improvement of the index score better than the former because it is able to overcome the ceiling effects j o u r n a l p r e -p r o o f therefore, in addition to an effective treatment for symptomatic patients, there is an urgent need to abate the carriage of sars-cov-2 in the human nasal cavity of asymptomatic/pre-symptomatic individuals, in order to contain the transmission of the novel coronavirus within the community. to find tolerable topical nasal disinfectants featured by encouraging efficacy against sars-cov-2, e.g. drugs effective against at least two different viruses beyond sars-cov-2, with similar viral/molecular structure. pubmed was investigated using "anti-infective agents, local" and "anti-infective agents, nasal" as key words and applying the filters "age: 19+ years", "humans" and "english" to identify papers published during 2010-2020. the 254 publications returned by the system were scrutinized by inspection of title and abstract. the majority of papers dealt with drugs against methicillin resistant staphylococcus aureus (generally, mupirocin in nose associated with either chlorhexidine or j o u r n a l p r e -p r o o f hexachlorophene body wash). the pharmacological agents relevant for the present scoping review were: povidone-iodine solution (reported by 7 papers), alcohol-based nasal antiseptics (2 papers), quaternary ammonium compounds (1 paper) and n-chlorotaurine (1 paper). furthermore, "nasal disinfection" was used as search term in three online repositories of preliminary not peer-reviewed reports. the treatments found were: povidone-iodine solution and interferon-alpha (2 papers from medrxiv), hypothiocyanite (2 papers, of which 1 from ssrn) and alcohol-based nasal antiseptics (1 paper from arxiv). every cited treatment was used as key term to find additional information from different electronic databases. the abstracts of the original articles were explored for the following terms: mechanism(s) of action, tolerability and any evidence of toxic effects or selection of resistant strains, whether the treatment was tested in vitro (in particular against sars-cov-2), or reached the clinical trials stage, or is currently marketed/promoted/sold. in figure 2 , r-sh is a peptide or protein with a thiol moiety essential for the activity of numerous enzymes and proteins. sulphydryl oxidation of r-sh by oscn − or oi − generates sulfenyl thiocyanate (r-s-scn) or sulfenyl iodide (r-s-i), determining inhibition of bacterial glycolysis, respiration and glucose transport. inhibition of the pentose phosphate pathway was also observed only for oi − , with sulfenic acid (r-s-oh) as well as scn − or i − formed at the end of the cycle. either scn − or i − reacts with native lpo and the cycle restarts. the anti-microbial activity of the entire system (enzyme plus substrates) is known to be more effective than hypothiocyanite or hypoiodite alone and has been explained by the production of short-lived, highly reactive intermediates [22] . furthermore, the activity of the i − peroxidase system is more effective against e. coli than the scn − system, in that lower i − concentrations are necessary. the i − peroxidase system deserves to be tested in vitro against the new coronavirus [22] . additionally, oscn − seems able to alter the surface proteins of different respiratory viruses, probably by oxidizing free thiol radicals and creating disulfide bonds [23] , thus contrasting the j o u r n a l p r e -p r o o f binding of viruses with the human airways mucosae. oscn − may also arguably hamper the synthesis and assemblance of viral proteins and nucleic acids, thus interfering with the release of viruses from infected cells [16, 24, 25] . the products of lpo extracellular oxidative complex is part of the human natural protective system of central airways against pathogen threats [16, [23] [24] [25] . due to the need of maximizing gas exchange, alveolar epithelial cells cannot contain strong protective structures. the latter cells are fragile and vulnerable to infectious agents, as shown by the diffuse alveolar damage discovered in two patients undergoing surgical resections for lung adenocarcinoma, later discovered to be affected also by covid-19 pneumonia [26] . bronchi were not reported to be as vulnerable as alveoli in the same study [26] . the lack of lpo/h2o2/scn − system in nasal and eye secretions of humans may explain the survival and proliferation of bacteria and respiratory viruses in the mucosae of conjunctiva and nose and their subsequent shedding in the environment [27] [28] [29] [30] [31] [32] . at micromolar concentration, the reactive mixture lpo/h 2 o 2 /oscn − proved cidal activity against a range of bacteria (gram positive and negative), fungi (candida albicans and candida krusei) and viruses as hiv, herpes-simplex virus (hsv-1), adenovirus, echovirus, respiratory syncytial virus (rsv) and influenza virus [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . in a recent experiment in-vitro, oscn − produced by the oxidase/lpo/ h 2 o 2 /thiocyanate system rapidly and effectively inactivated a/swine/illinois/02860/09 (swh1n2) influenza a virions, successfully preventing the infection of both primary human and male sprague-dawley rat tracheabronchial epithelial cells [43] . in a more recent in-vitro cell-free experiment, all 12 different strains of influenza a and b viruses (the major circulating serotypes and species causing epidemics) were effectively inactivated by the lpo/h 2 o 2 /(scn − /i − ) system [39] . considering the strain-independent effect, the authors of the j o u r n a l p r e -p r o o f latter study encouraged a pharmaceutical application of the lpo/h 2 o 2 /(scn − /i − ) system in vivo to contribute to the clearance of influenza virus [39] . another laboratory experiment also challenged enzyme free oscn − against a/h1n1 2009 pandemic influenza virus in-vitro, showing an evident dose-dependent viricidal activity, without cell toxicity [24] . considering a demonstrated wide spectrum cidal effect [17, 39] , not targeting specific proteins, it could be reasonably argued that oscn − may be effective also against sars-cov-2 and would therefore deserved to be tested in vitro [24, 25] . since it is already naturally present in human airways secretion and considering its large spectrum of action, a low level of resistance due to viral mutations and limited adverse reactions can be predicted with oscn − due to its similarities to transferrin, lf possesses iron binding capabilities; its iron is not released even at ph 3.5. this property ensures iron sequestration in infected tissues where the ph is typically acidic, preventing the utilization of iron by pathogenic bacteria [45] . lf is a constituent of the innate immune response and during viral infections the expression of genes encoding lf was elevated by approximately 150 fold in sars-cov patients compared with healthy controls [46] . lf possesses strong antiviral activity against a broad spectrum of rna and dna viruses, such as hiv, zika virus, chikungunya, hepatitis c virus, cytomegalovirus, rotavirus, among others [47] [48] [49] [50] [51] [52] [53] [54] . the antiviral activity of lf takes place particularly when the virus attacks the host cell, since lf prevents the virus from anchoring and then entering the host cell [56] . it has been reported that lf binds to heparan sulfate proteoglycans (hspgs), which are cell-surface and extracellular matrix macromolecules that are composed of a core protein decorated with covalently linked glycosaminoglycan chains [45] . hspgs could be the preliminary docking sites on the host cell membrane and play an important role in the process of entry of sars-cov into the cell [56] . as shown in figure 3 , lf blocks the infection of sars-cov by competing with the virus for hspgs [56] . this mechanism could prevent the viral concentration on the cell surface, as well as to the specific entry receptors (ace2). it is not presently known whether lf binds to ace2 [56] . it is likely that lf can inhibit sars-cov-2 invasion at micromolar concentrations and in a dose dependent fashion, just as for sars-cov [56] . however, there is no current confirmed information that sars-cov-2 binds to hspgs. whether sars-cov-2 also enters host cells via hpsgs in the same way clearly warrants further investigation. lf is available as an oral supplement and is widely used as a nutritional additive for infants at doses ranging from 100 mg to 4.5g a day for various indications without apparent toxicities [55] . there is a list of 24 commercial products available by the american corporation amazon. a randomized, open-label, parallel-group clinical trial was conducted to examine the effectiveness of sucking tablets containing lf and lpo (lf+lpo) in alleviating symptoms of the common cold and/or influenza infection. treatment and non-treatment groups (overall 407 subjects) were further classified into subgroups habitually wearing a face mask, washing their hands, or gargling [58] . the incidence and duration of common cold, influenza infection and gastrointestinal symptoms were not statistically different between treatment and non-treatment groups. (lf+lpo) tablets were moderately effective in significantly reducing the duration of fever higher than 38°c in the subgroup that did not wear a protective face mask [58] . lf in its free form is degraded within the stomach by the action of hydrochloric acid and hydrolytic enzymes. newer formulations of lf including encapsulation and liposomalization have been explored. another interesting observation is that zinc saturated lactoferrin can apparently exert a a. in presence of lactoferrin b. in absence of lactoferrin more potent antiviral effect. this is of particular relevance in covid-19 as zinc supplementation has been proposed as a possible intervention for the disease [58] . an oral supplement combining a liposomal bovine lactoferrin (llf) syrup (32 mg of lf/10 ml plus 12 mg of ascorbic acid) and a zinc solution 10 mg/10 ml 2-3 times a day and 4-6 times/day was administered for 10 days to 75 symptomatic covid-19 patients tested positive for igm/igg rapid test and self-isolated at home. a control group was treated only with llf. all patients, who were followed up for one month, resolved their symptoms within 4-5-days since treatment inception and a half dose of llf on 256 household contacts was effective to prevent the infection [48] . last but not least, out of seven authors, six worked for sesderma laboratories, the producer of all treatments used in the trial [48] . as mentioned above, lf, alone or in combination with oscn − , is already being tested in a phase 1 clinical trial (rct02598999) on healthy volunteers and patients affected by cystic fibrosis [23, 25] . n-chlorotaurine (nct) is a natural oxidant part of the human defense system which is produced in the body (hocl + taurine → nct + h 2 o) by the strongly toxic hypochlorous acid and the aminoacid taurine [59] . recently nct was obtained by synthesis and large quantities were available. this compound revealed an optimal compromise between sufficient disinfecting power and good tissue tolerability. nct can be stored long-term at low temperatures, and has killing activity against bacteria, fungi, parasites and viruses (herpes simplex virus type 1; herpes simplex virus type 2; adenovirus; influenza virus a and hiv-1). nct can be applied to sensitive body regions as an endogenous antiseptic. tolerability was very good in the eye, skin, mucous membranes and paranasal sinuses. the ciliary beat frequency of epithelial cells of the nasal mucosa, a very sensitive parameter for toxicity, was decreased only moderately and reversibly by 1% nct [60] . a special field of application of nct is inhalation for treatment of various infections. a phase i double-blind randomized clinical study with test group (inhalation of 1% nct) and a parallel control group (0.9% nacl as placebo) was carried out in two austrian centers. the forced expiratory volume in j o u r n a l p r e -p r o o f the first second (fev1), was not reduced and blood analyses showed no abnormalities compared to the baseline and compared to the control arm [60] . overall, there is no evidence of efficacy of nct to clear the nasopharynx from sars-cov-2. the interferon, discovered by virologists in 1957, is part of the human anti-viral innate defenses and plays a critical role in anti-viral immunity, interfering with the viral replication and spread with different mechanisms, including inhibition of the cell metabolism and downregulating the secretion of cytokines which stimulate the adaptive immunity [61] . an in vitro test confirmed the efficacy of ifn-α against sars-like coronavirus infections [62] . animal tests have confirmed that ifn-α nasal spray can effectively block or reduce sars-cov infection-related damage in monkeys [63, 64] . sars-cov-2 can inhibit the endogenous secretion of ifn by host cells, thus in turn reducing their ability to suppress viral infections. therefore, the use of exogenous ifn is critical with the severe form of covid-19 [65] . a recent open-label prospective study tested nasal drops of recombinant human interferon alpha (rhifn-α) on 2,944 health care workers in china during sars-cov-2 epidemics as follows [66] : • 2,415 subjects (997 doctors and 1,418 nurses with average ages of 37.38 and 33.56 years respectively) included in a "low-risk group" were administered 2-3 drops/nostril/time of rhifnα, 4 times/day for 28 days. • 529 subjects (122 doctors and 407 nurses with average ages of 35.24 and 32.16 years respectively) were included in a "high-risk group" and administered 2-3 drops/nostril/time of rhifn-α, 4 times/day for 28 days, in addition to thymosin-α 11.6 mg hypodermic injection once a week. at day 28 no study subject was positive for covid-19 and nobody developed respiratory symptoms [66] . the study was not a clustered, randomized study. the control group used was j o u r n a l p r e -p r o o f medical staff with covid-19 pneumonia in the epidemic area during the same period reported in the literature, rather than a strictly parallel, placebo-controlled group. in addition, the paper in its current form is a preliminary report which has not been certified by peer review. although ifn is seemingly effective to disinfect the upper airways from covid-19, its use may be more appropriate for front-line individuals such as health care staff, especially considering its likely high cost. since the protocol entails also the injection of thymosin-α 11.6 mg once a week, ifn does not seem of practical use in the general population. polyvinylpyrrolidone polymer with iodine (pvp-i), also known as povidone iodine, was discovered and marketed as disinfectant since 1955. the air-liquid interface of human nasal epithelial cells cultures collected from patients affected by chronic rhinosinusitis were recently exposed in vitro to a 0.5% solution of pvp-i (nasodine ® licensed by firebrick pharma) [67] . no cell toxicity was observed on paracellular permeability or cilia beat frequency. the trans epithelial electrical resistance of cultured cells was significantly reduced only after 30 minutes exposure to nasodine ® [66] . consequently, pvp-i could be considered as a safe therapy when used as a mouthwash or taken nasally or used during ophthalmic surgeries. the viricidal activity in-vitro of topical and oral pvp-i products against sars-cov-2 was recently reported [68] . a phase iii clinical trial (actrn12619000764134) is ongoing to assess the safety and efficacy of povidone-iodine nasal spray (nasodine®) in the treatment of subjects affected by common cold, potentially caused by human coronaviruses [69] . pvp-i awaits clinical trial data confirming an effective activity of drug against sars-cov-2. although it is an effective antiseptic, pvp-i may not be optimal for pregnant women and children though. hypothyroidism has been reported with povidone-iodine antiseptics in neonates. transient hyper-thyrotropinemia can occurs in neonates whose mothers had been exposed to povidone-iodine as a skin disinfectant during and after labour [70] . hydroxychloroquine (hcq), a less toxic derivative of chloroquine (cq), at micromolar concentration proved anti-viral activity against sars-cov-2 in vitro [71] , and its mechanism of action entails the increase of ph of intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion [72] . in addition, cq could inhibit sars-cov entry through changing the glycosylation of ace2 receptor and spike protein [73] . along with the derivative hcq, cq has even entered multiple clinical trials. the evidence on the efficacy of hcq to clear the nasopharynx from sars-cov-2 is relatively low, being founded only on one small open-label non-randomized clinical trial, which did not confirm clinical improvement of covid-19 patients though [74] . furthermore, hcq if featured by risk of side effects in terms of qtc prolongation and haemolysis associated with deficiency of glucose-6-phosphate dehydrogenase [75] . on this note, the mayo clinic recommended baseline electrocardiogram monitoring before commencing treatment of covid-19 patients with hcq [76] . the efficacy of hydroxychloroquine, also in combination with azithromycin has been questioned too, since it was not associated with a significantly reduced j o u r n a l p r e -p r o o f mortality among 1,428 randomly sampled covid-9 patients admitted to 25 hospitals of the new york city metropolitan region [77] . lastly, nasal antimicrobials such as hcq are typically not used on a regular basis to reduce subclinical colonization in individuals because this strategy might lead to increased development of resistant bacteria/viruses. sars-cov-2, sars-cov and mers-cov are lipophilic enveloped viruses relatively easy to inactivate by exposure to alcohols. ethanol 62-71% is among the reagents already proven effective to disinfect fomites from sars-cov-2 within 1 minute [78] . shintake recently proposed a controlled inhalation of ethanol vapor obtained from readily available alcoholic beverages (whisky or japanese sake), to disinfect the human airways from sars-cov-2 [79] . two additional studies tested the effectiveness of alcohol-based antiseptic in reducing nasal bacterial carriage in health care professionals at an urban hospital center [80] or in colonized patients [81] . further clinical research is however necessary to investigate the preventive and protective effects against sars-cov-2 because the above papers were not peer-reviewed. a commercially available, non-prescription product, nozin nasal sanitizer antiseptic was used as test agent in both studies. the safety-tested formulation is composed of 70% ethanol active combined with a mixture of natural oil emollients and the preservative benzalkonium chloride. sterile phosphate-buffered saline with 0.017% peppermint oil as a masking agent was used as placebo treatment control. cov-2. the amounts of qacs used in households (including detergents for personal care, soaps and liquid hand washes), workplace, and industry settings has likely increased a lot, and their usage will continue to be elevated considering the trend of covid-19 pandemic [82] . inactivation of sars-cov-2 by formulated detergents is believed to occur as a result of disruption of the virally modified, host-cell-derived, phospholipid bilayer glycoproteinaceous envelope, and the associated spike glycoproteins that interact with the ace2 receptor for infection of host cells. another mechanism entails raising the endocytic and lysosomal ph [82] . using a text mining approach, baker [83] identified the following three classes of qacs having a possible antiviral activity against coronaviruses. • ammonium chloride. various uses, including metabolic acidosis. viral activity against murine coronavirus and hepatitis c. • cetylpyridinium chloride. used as antiseptic, mouthwash, personal care products, cleaning agents etc. it is also being used as an antimicrobial agent for meat and poultry products. it was tested in a clinical trial as a treatment against respiratory infections [84] . cetylpyridinium chloride has anti-viral activity against influenza, hepatitis b, poliovirus 1. • miramistin. used as antiseptic, has antiviral activity against hiv, influenza, herpes and sars-cov. qacs such as cetylpyridinium chloride and miramistin have not been tested yet against sars-cov-2 in vitro or in clinical trials. the clear and severe threat posed by covid-19 prompts an elevated use of qacs as a reasonable strategy to mitigate and contain the spread of the infection. it is important, however, to assess the potential environmental impact of elevated qac use, which may include disruption of wastewater treatment unit operations, proliferation of antibiotic resistance, formation of nitrosamine j o u r n a l p r e -p r o o f disinfection byproducts, and negative effects on biota of surface waters. exploration of potential technologies to minimize the environmental releases of qacs is also warranted [82] . the viricidal effect in vitro against sars-cov-2 has been described for povidone-iodine only. in vitro inhibition of other coronaviruses was reported for lf, hcq and aqcs. no treatment was tested against sars-cov-2 in randomized controlled clinical trials. the lack of effective and safe drugs against sars-cov-2 implies primarily that the relevant literature on this topic does not exist. most drug candidates are already in the market. a pointed out by baker et al. "as simple as it sounds, it is entirely possible that we should be looking in our bathroom cupboards for potential remedies against covid-19" [83] . whilst the nasal cavity is the port of entry for sars-cov-2, the mouth is also relevant as exit site of the virus through emission of saliva droplets. the well-known hand-to-nose-to-hand cycle of contamination requires appropriate additional methods for infection control. the present scoping review identified knowledge gaps more than available evidence on the topic of chemoprevention of covid-19. since speed is essential in responding to the covid-19 pandemic [85] , undertaking clinical trials on all the candidate agents identified could be too demanding and requires too much time. taking everything into account, these data suggest that the search for convenient non-antibiotic drug(s) could be pursued with a case-control study, comparing the use of candidate drugs among individuals positive and negatives to coronavirus swab tests. the results of such study could help in narrowing down the subsequent laboratory and clinical investigations. none to declare. j o u r n a l p r e -p r o o f covid-19: what is next for public health? coronavirus disease 2019 (cvoid-19) antibodies to coronaviruses are higher in older compared with younger adults and binding antibodies are more sensitive than neutralizing antibodies in identifying coronavirus-associated illnesses hypothesis to explain the severe form of the disease invisible spread of sars-cov-2 guidance for health system contingency planning during widespread transmission of sars-cov-2 with high impact on healthcare services world health organization. laboratory testing for coronavirus disease (covid-19) in suspected human cases-interim guidance asymptomatic transmission, the achilles' heel of current strategies to control covid-19. nejm pre-symptomatic sars-cov-2 infections and transmission in a skilled nursing facility consider pulling residents from nursing homes over 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(covid-19) pneumonia in two patients with lung cancer antimicrobial factors in whole saliva of human infants effect of hormone replacement therapy on lacrimal fluid peroxidase activity in woman exposure to hydrogen peroxide and eye and nose symptoms among workers in a beverage processing plant exposure to hydrogen peroxide at tlv level does not induce lung function changes: a longitudinal study survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids a novel host defense system of airways is defective in cystic fibrosis the lactoperoxidase system links anion transport to host defense in cystic fibrosis bactericidal and cytotoxic effects of hypothiocyanite-hydrogen peroxide mixtures lactoperoxidase, peroxide, thiocyanate antimicrobial system: correlation of sulfhydryl oxidation with antimicrobial action the effects of different (pseudo) halide substrates on peroxidase-mediated killing of actinobacillus actinomycetemcomitans nonspecific bactericidal activity of the lactoperoxidase-thiocyanate hydrogen peroxide system of milk against escherichia coli and some gram-negative pathogens susceptibility of influenza viruses to hypothiocyanite and hypoiodite produced by lactoperoxidase in a cell-free system inhibition of candida albicans by the peroxidase/scn/h2o2 system inhibition of herpes simplex virus type 1, respiratory syncytial virus and echovirus type 11 by peroxidase-generated hypothiocyanite inhibition of hiv infectivity by lactoperoxidase-produced hypothiocyanite hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular h1n2 influenza a virus lactoferrin-a multifunctional protein with antimicrobial properties the biology of lactoferrin, an iron-binding protein that can help defend against viruses and bacteria expression profile of immune response genes in patients with severe acute respiratory syndrome a nutritional supplement containing lactoferrin stimulates the 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coronaviruses on inanimate surfaces and their inactivation with biocidal agents possibility of disinfection of sars-cov-2 (covid-19) in human respiratory tract by controlled ethanol vapor inhalation. arxiv -cornell university reduction of nasal staphylococcus aureus carriage in health care professionals by treatment with a nonantibiotic, alcohol-based nasal antiseptic evaluation of an alcohol-based antiseptic for nasal decolonization of methicillin-resistant staphylococcus aureus in colonized patients increased use of quaternary ammonium compounds during the sars-cov-2 pandemic and beyond: consideration of environmental implications. environmental science & technology letters repurposing quaternary ammonium compounds as potential treatments for covid-19 randomized, double-blind, placebo controlled clinical trial to assess the safety and effectiveness of a nove dual-action oral topical formulation against upper respiratory infections moving quickly in pandemics key: cord-321918-9jwma2y6 authors: xiu, siyu; dick, alexej; ju, han; mirzaie, sako; abdi, fatemeh; cocklin, simon; zhan, peng; liu, xinyong title: inhibitors of sars-cov-2 entry: current and future opportunities date: 2020-06-15 journal: j med chem doi: 10.1021/acs.jmedchem.0c00502 sha: doc_id: 321918 cord_uid: 9jwma2y6 [image: see text] recently, a novel coronavirus initially designated 2019-ncov but now termed sars-cov-2 has emerged and raised global concerns due to its virulence. sars-cov-2 is the etiological agent of “coronavirus disease 2019”, abbreviated to covid-19, which despite only being identified at the very end of 2019, has now been classified as a pandemic by the world health organization (who). at this time, no specific prophylactic or postexposure therapy for covid-19 are currently available. viral entry is the first step in the sars-cov-2 lifecycle and is mediated by the trimeric spike protein. being the first stage in infection, entry of sars-cov-2 into host cells is an extremely attractive therapeutic intervention point. within this review, we highlight therapeutic intervention strategies for anti-sars-cov, mers-cov, and other coronaviruses and speculate upon future directions for sars-cov-2 entry inhibitor designs. coronaviruses (covs) are enveloped positive-stranded rna viruses. they belong to the order of nidovirales and are classified into four genera: α, β, γ, and δ. 1 coronaviruses are animal viruses with circulating reservoirs in mammals and birds. for most coronaviruses, the lifecycle can be dissected into four steps, including viral entry, replication, assembly, and release. 2 until last year, six strains of coronaviruses have been identified that are pathogenic to humans. among them are cov-nl63, cov-oc43, cov-hku1, and cov-229e that could cause mild respiratory tract diseases. 3 however, two of the β-covs, the severe acute respiratory syndrome coronavirus (sars-cov), and the middle east respiratory syndrome coronavirus (mers-cov) have caused severe epidemics in the past. 4, 5 in april 2003, sars-cov was responsible for 8098 infections, with a fatality rate of ∼10% by the end of september 2003. 6 mers-cov emerged from its zoonotic reservoir in 2012 and infected 2494 people with a fatality rate of ∼34% by the end of 2019. 7 both outbreaks having such high fatality rates, highlight the need for surveillance of coronavirus emergence. while efforts for the development of antivirals against sars-cov or mers-cov are still in process, a new coronavirus (sars-cov-2) has emerged from an epicenter located in wuhan, china, in december 2019. 8 sars-cov-2 is highly contagious and has quickly spread in and beyond china. as of may 28, 2020, there have been more than 5 596 550 diagnosed cases around the world, with 353 373 confirmed deaths (figure 1 ). 9 the united states of america and brazil reporting the majority of the confirmed cases in the americas, with 1 658 896 and 391 222 cases, respectively. recently the genome of sars-cov-2 was determined, which revealed 80% identity with that of some sars-cov strains (gz02, bj01, tor2, sz3, pc4-227) and interestingly 96% identity to the bat coronavirus batcov ratg13. 11 the receptor-binding spike (s) protein is highly divergent from other covs and displays nucleotide sequence identities of 75% or less to all other previously described sars-covs. however, again, the new sars-cov-2 s protein shares 93.1% identity to the ratg13 s protein. 11 the glycoprotein or s protein is responsible for receptor recognition and viral entry into host cells. the spike protein can be divided into two domains; s1 is responsible for angiotensin-converting enzyme ii(ace2) recognition, the recently identified host cell receptor, and s2 mediates membrane fusion (figure 2 ). 12 structural alignment of sars-cov-2 s protein with sars-cov s protein shows that both s proteins are similarly with a root-mean-square deviation (rmsd) of 3.8 å over 959 cα atoms, while the s2 domain, responsible for membrane fusion, display the most substantial similarities with an rmsd of 2.0 å ( figure 2c ). engagement of the host cell receptor ace2 is important for viral entry; however, subsequent entry steps can vary and are cell-type specific. sars-cov can enter the host cell via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon ace2 binding. 13, 14 the clathrin-mediated pathway includes the s protein binding to ace2 and subsequent dynamin/clathrinmediated internalization of endosomal vesicles that maturate to late endosomes. within the late endosomes and lysosomes, acidification of the internalized endosomes and h + -dependent activation of the cellular cathepsin l proteinase takes place that cleaves and activates the s protein, therefore initiating viral fusion with the endosomal/lysosomal membrane (figure 3 ). in the case of sars-cov, cell culture studies revealed that the entry process is delayed with a lag phase of around 30 min, suggesting substantial maturation requirements. 15 in accordance with findings that mouse hepatitis coronavirus (mhv) and feline coronavirus (fcv) infections of hela cells are also heavily dependent on endosomal maturation, the clathrindependent entry and endosomal maturation are key to entry across coronaviridae. 16 for sars-cov-2, a recent study also confirms that virus can use host cell receptor cd147 to gain entry into the host cells besides ace2. 17 in addition to the endosome-mediated entry pathway, host proteases also play critical roles in the nonendosomal entry of coronaviruses. 5 host proteases such as the transmembrane protease serine 2 (tmprss2) and tmprss11d can cleave the s protein at the s1/s2 cleavage site (figure 2 ) to prime and activate the s protein for membrane fusion during the nonendosomal pathway. 18 a recent study also confirms that tmprss2 expressing veroe6 cells are highly susceptible to sars-cov-2 infection, highlighting the importance of tmprss2 in the replication cycle. 19 mers-cov can also be activated by furin (serine endoprotease) to initiate the nonclathrin mediated membrane fusion event. 20 interestingly, in the new sars-cov-2 s figure 3 . entry model of sars-cov-2 into the host cell. binding of the s1 domain within the spike (s) protein to the cellular ace2 receptor triggers conformational changes in the s2 domain that results in internalization and subsequent membrane fusion ((a) endosomal/clathrindependent pathway). the endosomal pathway is facilitated by a low ph and the ph-dependent cysteine protease cathepsin l. alternatively, sars-cov-2 can enter the cell via the nonendosomal/clathrin-independent pathway (b). during this route, ace2 recognition by the sars-cov-2 s protein (comparable to route a) is followed by additional activation/cleavage of the s protein into s1 and s2 domains by cell membrane-associated serine proteases such as tmprss2 and tmprss11d. the figure was prepared with https://biorender.com/. . genome organization of sars-cov-2. genome organization of the sars-cov-2 and location the central genes within the genome (numbers in brackets). 29 the figure was prepared with https://biorender.com. protein, additional amino acid insertions at the s1/s2 cleavage site results in an "rrar" furin recognition site absent in sars-cov s protein. 21 this polybasic insertion sequence has possible implications for the sars-cov-2 replication cycle and its increased pathogenicity. indeed, polybasic furin sites have been observed in hemagglutinin (ha) proteins of highly virulent avian and human influenza viruses, and similar furinlike processing events are also observed for other rna viruses such as ebola virus and marburg virus, human immune deficiency virus (hiv), and flaviviruses. 22 to activate the s protein for membrane fusion with the cellular membrane, structural rearrangements within the s2 domain are required. two heptad repeats, hr1 (dark blue in figure 2 ) and hr2 can interact to form a six-helix bundle (6-hb), a common postfusion structure shared by all type i viral glycoproteins, to bring viral and cellular membranes in close proximity. additionally, the s2 domain contains a membrane interacting domain or fusion peptide that is exposed upon specific triggers such as receptor binding or low endosomal ph. to date, three membrane interacting regions with hostmembrane destabilizing effects have been identified in the sars-cov s protein: two conserved sequences across coronaviridae, with residues 798−815 23 and residues 864− 886, 24 both c-terminal positioned at the second cleavage site in the s protein termed s2′ at arg 797 and a less conserved third region with membrane disordering properties residues 770−788. 25 once in the host cell, the viral particle uncoats and is ready for transcription and translation. 26 the first orf codes for approximately 67% of the genome and is separated into open reading frames (orf) 1a and 1b (figure 4 ). orf1a and orf1b are translated into polyproteins pp1a (4382 amino acids) and pp1ab (7073 amino acids) that are processed by 3-c-like protease (3clpro) and papain-like protease (plpro). the processing of these polyproteins produces a variety of nonstructural proteins (nsps), including rna-dependent rna polymerase (rdrp) and helicase, to catalyze viral genome replication and protein synthesis. 27 the remaining orfs in the sars-cov-2 genome code for accessory and structural proteins. following further assembly, the mature virions are transported to the cell surface in vesicles and released by exocytosis. 28 any protein involved in the replication process could be a potential target for the development of antiviral agents. as mentioned previously, zhang et al. determined the fulllength genome sequence of sars-cov-2 and revealed that the virus was very similar (89.1% nucleotide similarity) to a group of sars-like coronaviruses. 30 simultaneously, shi et al. found that sars-cov-2 shares 96% sequence identity at a wholegenome level to a bat coronavirus, and importantly, they confirmed that sars-cov-2 utilizes the same cell entry receptor, ace2, as sars-cov. 11 recently, the cryo-em structure of full-length human ace2 bound to the rbd of the sars-cov-2 was solved, providing an important structural foundation for intervention strategies. 31 conservation analysis also revealed that the rdrp and the 3clpro are highly conserved between sars-cov-2 and sars-cov. 32 therefore, it is widely accepted that sars-cov-2 would behave similarly to sars-cov with regards to viral entry and replication. being the first step in the infection process, the entry of pathogenic viruses into susceptible cells is an extremely attractive intervention point. as with other well-known viruses, such as hiv-1 and ebola, viral entry of coronaviruses is a complex multiple-step process with numerous interactions and processing points that, in theory, could be targeted. 33 in this review, we summarize case studies and highlight efforts in designing entry inhibitors against sars-cov, mers-cov, and other coronaviruses that can provide important information to combat the current sars-cov-2 outbreak. ■ host cell ace2 receptor recognition by the sars-cov-2 spike (s) as a promising antiviral target binding of the sars-cov-2 spike (s) protein to the cellular ace2 receptor represents the first encounter (in both the endosomal and nonendosomal pathway) in the viral replication cycle and provides prophylactic intervention opportunities. 34 sars-cov-2 spike (s) recognizes with its rbd the cellular ace2 receptor with high affinity (k d = 14.7 nm) 12 as judged by surface plasmon resonance (spr) interaction analysis, and intervention at the rbd-ace2 interface can potentially disrupt infection efficiency. recently the cryo-em and crystal structures of sars-cov-2's rbd in complex with ace2 were solved and provide important structural guidance for inhibitor design ( figure 5 ). 31 the interface can be divided into three contact sides, mainly polar in nature, and is similar to the sars-cov-ace2 complex. 35, 36 in this structure, an extended loop of the rbd contacts an arch-like helix α1 of the proteolytic domain (pd) of ace2 via an n-(cluster 1), central (cluster 2), and cterminal (cluster 3) portion ( figure 5 purple box). additionally, helix α2 and loop 3−4 (connecting β3 and β4) of ace2 provide limited contacts. at the n terminus of α1 (cluster 1), gln498, thr500, and asn501 of the rbd interact via hydrogen bonds with tyr41, gln42, lys353, and arg357 from ace2. the middle portion (cluster 2) of the rbd loop contacts via tyr453, the ace2 pd at residue his34. at the c terminus of α1 (cluster 3), gln474 of rbd contacts gln24 of ace2, and phe486 of rbd interacts with met82 of ace2 through van der waals interactions ( figure 5 ). the structures of the rbds from the sars-cov-2-ace2 complex and the sars-cov-ace2 complex are quite similar, with an rmsd of 0.68 å over 139 cα atoms ( figure 6 ). 31 a comparison of both structures, however, also highlights some deviations at all three clusters summarized in table 1 . these deviations need to be considered carefully during the inhibitor design process. in addition, helix α2 and the loop 3−4 connecting β3 and β4 are also contributing to the interface. sars-cov-2 s protein monomer was obtained from pdb 6vsb and rbd-ace2 complex from pdb 6vw1. boxes 1, 2, and 3 highlight polar clusters 1, 2, and 3, respectively. ■ targeting the rbd peptide analogues, monoclonal antibodies, and protein chimeras as rbd inhibitors. both sars-cov and sars-cov-2 use ace2 to gain entry into the host cells. as such, this critical interaction can be blocked to stop viral entry. 19 this strategy was first demonstrated by hsiang et al. using a biotinylated enzyme-linked immunosorbent assay (elisa), hsiang et al. reported the disruption of the sars-cov s protein-ace2 interaction by small peptides. from a total of 14 designed peptides, peptides sp-4, sp-8, and sp-10 ( figure 7 and table 2 ) significantly blocked the interaction of the sars-cov s protein with ace2 with ic 50 values of 4.30, 6.99, and 1.88 nm, respectively. additional immunofluorescence assay (ifa) studies with s-protein-pseudotyped retroviruses, revealed a novel mechanism of infection inhibition of vero e6 cells by sp-10. 37 38 in light of the successful inhibition of sars-cov with this linked peptide, a similar strategy could potentially be effective against the new sars-cov-2. the recently solved cryoem structure of sars-cov-2 in complex with the human ace2 receptor can provide a structural rationale for the peptide design. 31 monoclonal antibodies (mab) have potential applications for diagnosis, prophylaxis, and treatment of established and evolving viral infections. 39−41 prabhakar et al. isolated specific antibodies from b cells in xenomouse immunized with sars-cov. further investigation revealed that several abs directly react with the rdb domain, and a combination of two abs (4d4 and 3c7) displayed near-complete neutralization efficiency as compared to a single ab application. 42 two additional potent monoclonal antibodies, mab201 and mab68, could be isolated from transgenic mice immunized with the soluble ectodomain of sars-cov s protein. 43 this mab could bind sars-cov s protein directly with affinities of 34 nm (mab 201) and 83 nm (mab 68) as judged by spr analysis. mice that received 40 mg/kg of mab 201 or mab 68 before sars-cov infection showed complete protection from reinfection of lung tissues. 43, 44 cross-reactivity of mabs is highly desirable, and dimitrov et al. identified the human mab m396 that binds sars-cov with high affinity (k d = 20 nm). 45 mice that received 200 μg of m396 were nearly completely protected from infection by urbani and gd03 virus strains. 46 m396 did compete with the sars-cov receptor, ace2, for binding to the rbd, suggesting that m396 inhibits sars-cov-ace2 binding as the predominant mechanism of action. 45 however, sars-cov-2 showed some complexities for rbd directed antibodies. for instance, wrapp et al. tested crossreactivity of three antibodies, including s230, m396, and 80r, against sars-cov-2 rbd. despite the partly high degree of structural homology between the sars-cov-2 and sars-cov, no binding to the sars-cov-2 rbd was detected for any of the three antibodies at the concentration of 1 μm. it can be concluded that sars-cov antibodies will not necessarily be cross-reactive for sars-cov-2. 12 in a different approach, hu et al. generated a novel chimeric recombinant protein recently by connecting the extracellular domain of human ace2 to the fc region of human immunoglobulin igg1. these chimeric constructs displayed high-affinity for the sars-cov-2 and sars-cov rbd binding and potently neutralized sars-cov and sars-cov-2 in vitro, with ic 50 values between 0.8 and 0.1 μm, respectively. these journal of medicinal chemistry pubs.acs.org/jmc perspective recombinant chimeras also showed cross-reactivity and could have, therefore, useful applications for diagnosis, prophylaxis, and treatment of sars-cov-2. 47 using the velocimmune platform, pascal et al. generated several human, noncompeting monoclonal antibodies that target mers-cov s protein and block viral entry into host cells. among them, two antibodies, regn3051 and regn3048, can significantly inhibit mers-cov pseudoparticles, with ic 50 values of 460 and 180 pm, respectively. 48 in addition, regn3051 and regn3048 showed a good performance in a novel transgenic mouse model, which was developed by replacing the mouse dpp4 coding sequence with that encoding human dpp4. results suggested that both regn3051 and regn3048 were able to potently reduce mers-cov specific rna levels in the lungs at a 200 μg per mouse dose compared with the isotype control antibody. at the 20 μg dose, regn3051 was more effective at decreasing mers-cov rna levels compared with regn3048 at the same dose. 48 recently, in the common marmoset model of mers-cov infection, de wit et al. tested the prophylactic and therapeutic efficacy of regn3051 and regn3048. data demonstrated that their protection might be more effective in a prophylactic treatment process rather than treatment of mers-cov. 49 in the latest attempt, chen et al. identified sars-cov-2 rbd specific antibodies from samples of 26 recovered covid-19 patients using an rbd-specific elisa binding study. among them, 311mab-31b5 and 311mab-32d4 effectively neutralized pseudovirus entry, with ic 50 values of 0.0338 and 0.0698 μm, respectively. 50 recently, in an elisa based (cross)reactivity assay, assessing antibody-containing supernatants of a collection of 51 sars-s hybridoma's derived from immunized transgenic h2l2 mice that encode chimeric immunoglobulins, wang et al. identified a chimeric mab 47d11 that targets rbd. 47d11 exhibited cross-neutralizing activity of sars-cov-s protein and sars-cov-2-s protein pseudotyped vsv infection with ic 50 values of 0.19 and 0.57 μm, respectively. 51 brouwer et al. used cross-sectional blood samples from three pcr-confirmed sars-cov-2-infected individuals to screen for binders to a soluble prefusion-stabilized s protein of sars-cov-2 using an elisa-based approach. all three blood samples did bind to the prefusion-stabilized s protein and prompted subsequent sorting of sars-cov-2 s proteinspecific b cells for mab isolation. nineteen nabs could be identified that target a diverse range of antigenic sites on the s protein and showed remarkable picomolar inhibiting activities with the two most potent ic 50 values of 0.010 and 0.007 μg/ ml (cova1-18 and cova2-15, respectively) against live sars-cov-2 virus. 52 large antibody libraries are crucial in response to rapidly emerging pathogens. using eight large phage-displayed vh, scfv, and fab libraries and panning against the rbd of the sars-cov-2, li et al. identified an exceptional potent (k d to rbd of 160 pm as judged by biolayer interferometry) mab igg1 ab1 that competes with ace2 in vitro and protected transgenic mice expressing hace2 from high-titer intranasal sars-cov-2 challenge. 53 in two different assays using replication-competent sars-cov-2 in a microneutralizationbased assay, 100% neutralization at <400 nm, and in a luciferase reporter gene assay, an ic 50 of 200 nm was reported. moreover, transgenic mice expressing human ace2 administrated with 0.3 mg of ig1 ab1 prior intranasal infection with sars-cov-2 did not show any detectable replicationcompetent virus, demonstrating the preventive effect of igg1 ab1. 53 small molecules targeting the rbd. besides peptides, mab, and protein chimeras, small molecules are still the preferred modality for a drug. this is due to improved pharmacokinetics, stability, and dosage logistics compared to proteins or peptides. 54, 55 in addition, small molecules have advantages compared to peptides/proteins regarding dissemination logistics in remote areas and the high expenses of peptide/protein production. 54, 55 to identify small molecule entry inhibitors against the sars-cov s protein, sarafianos et al. screened a chemical library composed of 3000 compounds according to lipinski's rule of five 56 and identified an oxazole-carboxamide derivative, ssaa09e2 (1, table 3), that blocks the binding of the rbd of 57 lundin et al. screened a library of 16 671 diverse compounds and found a small molecule inhibitor, k22 (2), which was able to inhibit hcov-229e with an ic 50 value of 0.7 μm and cc 50 value of 110 μm. studies for mechanism showed that k22 targeted a very early step in the hcov-229e life cycle and may interact with viral particles, thus inactivating their binding. 58 ■ targeting the cellular receptor peptide analogues as ace2 inhibitors. human angiotensin-converting enzyme (ace) is a highly glycosylated type i integral membrane protein and has been identified as a fundamental regulator of the renin−angiotensin system (ras) in humans and is an important target in regulation of blood pressure homeostasis. ace2 is a human homologue of ace. 59 it contains a single zinc-binding catalytic domain, which is 42% similar to the human ace active region. 60 ace2 can catalyze the cleavage of angiotensin i into angiotensin 1-9, and angiotensin ii into the vasodilator angiotensin 1-7 and its organ-and cell-specific expression also suggests a role in the regulation of cardiovascular and renal function and fertility. 60 ace2 is a functional receptor to the sars-cov during viral entry, and recent research demonstrated that sars-cov-2 also utilizes ace2 for infection. 61 however, ace2 cannot be inhibited by ace inhibitors, so there is an urgent need to develop specific ace2 inhibitors that would prevent infection by both sars-cov and sars-cov-2. one of the first efforts to target the ace2 receptors was documented by liu et al. using a novel epitope assembling assay, liu et al. identified linear b-cell immuno-cross-reactive epitopes of sars-cov s protein by synthesizing 22 longer peptides. five of these peptides showed serologically highly cross-reactivity in all tested sars patients sera. among them, peptide s 471-503 could significantly block the binding of rbd to ace2. s 471-503 , derived from the s1 fragment ( figure 7 and table 2 ) could target ace2, and showed antiviral activity against sars-cov infection in vitro, with an ec 50 value of 41.6 μm, providing an important basis to explore the antiviral potential of s 471-503 against sars-cov-2. 62 another peptide derived from the rbd, rbd-11b, located in s1 of the sars-cov s protein, is crucial for binding to the host cells ace2 receptor 62 (figure 7 and table 2 ). given the vital role of this motif, meyer et al. confirmed the binding to ace2 of a synthesized peptide mimicking this region ( 438 ykyryl 443 ) with a k d of around 46 μm. moreover, rbd-11b displays no toxicity, as judged by an mtt (3-(4,5)dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumbromide) cell proliferation assay, on veroe6 cells. in addition, rbd-11b showed antiviral activity to hcov-nl63 at a peptide concentration of 7 mm in caco2 cells, which also used ace2 as a functional receptor. 63 constrained peptides are receiving more attention in the drug development field, combining the best attributes of antibodies and small molecules. linear peptides are often highly flexible and unstructured in solution, only forming structures upon target binding. this can sometimes reduce the affinity of such peptides for their target by an entropic penalty mechanism. however, stabilization methods such as cyclization or hydrocarbon stapling can increase the physicochemical characteristics and drug-like properties while negating the entropic penalty of binding and having a positive impact on affinity. 64 using a constrained peptide library displayed on filamentous phages, ladner et al. identified several peptides inhibiting ace2 function with the most potent being dx600 (table 2) . dx600, an n-terminal acetylated and c terminal amidated peptide, was a potent ace2 peptide inhibitor with an ic 50 value of 10 nm and a k i value of 2.8 nm. dx600 did not inhibit ace activity and thus is specific to ace2. in addition, dx600 was chemically stable and not hydrolyzable by ace2. 65 although it is not clear whether dx600 can inhibit coronavirus, as an effective ace2 inhibitor, anticoronavirus tests should be conducted in the future. small molecule as ace2 inhibitors. as discussed previously, peptide and constrained peptide inhibitors have inherent caveats concerning their use as drugs. 64 therefore, screening for small molecule inhibitors, guided by information gleaned from the previous studies is the next logical step. a virtual screen targeting the ace2 catalytic site with around 140 000 compounds combined with a molecular docking approach led to the identification of naae (n-(2-aminoethyl)-1 aziridine-ethanamine) (3, table 3 ). 3 showed a dosedependent inhibition of ace2 catalytic activity with an ic 50 value of 57 μm and a k i of 459 μm. despite its micromolar potency in inhibiting a sars-cov pseudotyped virus, cytotoxicity data is not available to date. 66 chloroquine (4) currently has applications for malaria and amoebiasis treatment. interestingly, nichol et al. showed that chloroquine could also block the interaction of rbd of sars-cov to ace2 under cell culture conditions with an ed 50 value of 4.4 μm. 67 recently, wang et al. found that 4 blocked sars-cov-2 virus infection, with an ic 50 value of 1.13 μm and a cc 50 > 100 μm in vero e6 cells. 68 chloroquine possibly increases endosomal ph required for virus/cell fusion as well as impairs with the terminal glycosylation of the cellular ace2 receptor, thereby reducing the affinity of sars-cov/sars-cov-2 to ace2. besides its antiviral activity, chloroquine may synergistically enhance its antiviral effect with immunemodulating activity in vivo. 68 at present, chloroquine is carried out in clinical research in china for the treatment of sars-cov-2 (chictr2000029609). 69 hydroxychloroquine (5) is an analogue of chloroquine, which shares the same mechanism of action as chloroquine but displays a more tolerable safety profile. 70 71 recent studies suggest that 4 and 5 could cause ventricular arrhythmias, 72 qt prolongation, 72,73 retinopathy, 74 and other cardiac-related toxicity, which may pose a particular risk to critically ill patients. although both show antiviral activity, safety, and effectiveness, they require further clinical research. turner et al. identified that the sars-cov receptor, ace2, undergoes proteolytic shedding, releasing an enzymatically active ectodomain during viral entry. 75 further research identified that a disintegrin and metalloproteinase (adam17) is responsible for shedding regulation of ace2. inhibiting adam activity with the adam-specific inhibitor gw280264x (6) reduced shedding of ace2 at 1 nm against sars-cov. 76 another enzyme involved in ace2 shedding is tace (tnf-α converting enzyme, a member of the adam family). two tace inhibitors, tapi-0 (7) and tapi-2 (8), reduced ace2 shedding against sars-cov, with ic 50 values of 100 and 200 nm, respectively. 75 perhaps the most promising small molecule described to date is the very potent ace2 inhibitor mln-4760 (9). 9 can inhibit the catalytic activity of ace2 with an ic 50 of around 440 pm. 77 the crystal structure of the apo and 9 bound ace2 complex revealed a significant subdomain movement of the nterminal and c-terminal subdomains of ace2 upon 9 binding. this movement is important to position critical residues to stabilize the bound inhibitor. its high potency makes 9 a very attractive candidate for sars-cov-2 interference; however, no antiviral coronavirus data is available at this time. milewska et al. synthesized several polymer-based compounds showing prominent anticoronaviral activity. among them, a cationically modified chitosan derivative, n-(2hydroxypropyl)-3-trimethylammonium chitosan chloride (htcc, 10), and hydrophobically modified htcc (hm-htcc, 11) were found that could inhibit hcov-nl63 replication. for both tested polymers, their ic 50 values were relatively low in llc-mk2 cells, amounting to ∼50 nm for 10 and ∼230 nm for 11. cc 50 values were ∼0.8 and ∼1 μm for 10 and 11, respectively. 78 recent research showed that 10 and 11 blocked the interaction of hcov-nl63 with its ace2 receptor and thus interfered with the process of viral entry. 79 despite the availability of many compounds with inhibitory effects on ace2, the corresponding admet data in a preclinical model is not available. regardless, direct inhibition of ace2 is probably not a viable therapeutic modality, however. this is due to its important normal physiological roles, in addition to its lung injury protective role in acute respiratory distress syndrome from a variety of causes, including sars-cov infection. 80, 81 as such, directly inhibiting ace2 as an antiviral strategy appears to be physiologically unsound, and virally targetted blockers of its interaction with the sars-cov/sars-cov-2 s protein hold greater promise. membrane fusion is a crucial step in the mers/sars infection cycle in both described pathways (see section 1). within the endosomal/clathrin-dependent route, internalized viral particles need to fuse with the endosomal membrane to escape the endosomal/lysosomal environment. this is achieved via a conformational change of the s protein (s2 domain) within the acidic milieu followed by membrane fusion activation by the host protease cathepsin l. membrane fusion is also essential during the nonendosomal/clathrin-independent route to fuse with cellular membranes facilitated by host protease cleavage of the s protein by cell membrane-associated proteases such as tmprss2. 19 in conclusion, the s2 domain of the sars-cov s protein and host proteases such as 84, 85 hr1 and hr2 can interact with each other to form a 6-hb to bring viral and cellular membranes close (for exact location, see figure 2 ). on the basis of this requirement, bosch et al. obtained peptides corresponding to region hr2 within the hr. hr2-8 displayed in an infection inhibition assay with pseudotyped sars-cov s protein in vero cells an ec 50 value of 17 μm (figure 7 and table 2 ). 84 moreover, hr2-8 demonstrated concentration-dependent inhibition of hcov-nl63 infection with an ic 50 value of 0.5 μm and a cc 50 value of 20 μm. 86 on the basis of these initial results, further development of the hr2-8 peptide is necessary to develop a more potent human coronaviruse (hcov) peptide inhibitor. similarly, ngai et al. obtained three hr derived peptides, including hr1-a, gst-removed-hr2, and hr2 peptide, with remarkable inhibitory activity against sars-cov ( figure 7 and table 2 ). virus entry inhibition studies suggested that hr1-a, derived from the hr1 region, had an ec 50 value of 1.61 μm. gst-removed-hr2 peptide and hr2 peptide, derived from the hr2 region, had ec 50 values of 2.15 and 0.34 μm, respectively. 87 hr2p, spanning residues 1251−1286 in hr2 domains, could effectively inhibit mers-cov infection and s protein-mediated membrane fusion (figure 7 and table 2 ). this study indicates that hr2p could specifically inhibit mers-cov in vero cells, with an ic 50 value of ∼0.6 μm and a cc 50 value of >1000 μm. hr2p also demonstrated high selectivity, as indicated by its high selectivity index (si > 1667). importantly, the introduction of arg, lys, or glu residues into the hr2p peptide increased stability, solubility, and anti-mers-cov activity. 88 to improve the stability, solubility, and antiviral activity of hr2p, channappanavar et al. designed and synthesized an hr2p analogue named hr2p-m2. hr2p-m2 strongly blocked s protein-mediated cell−cell fusion in a dose-dependent manner at ic 50 values of 0.55 μm in vitro. in vivo, hr2p-m2 intranasal administration to ad5/ hdpp4 transgenic mice protected them from mers-cov infection and reduced the lung viral titers by more than 1000fold. moreover, combination treatment with ifn-β was demonstrated to enhance the protective effect. 89 the development of a drug with broad-spectrum hcov inhibitory activity is increasingly becoming an attractive approach. xia et al. found that the ek1 peptide showed pan-cov fusion inhibitory activity against multiple hcovs ( figure 7 and table 2 ). 90 further investigation revealed that ek1 directly reacts with the hr1 region and can competitively inhibit viral 6-hb formation. the pseudovirus assay suggested that the antiviral activity of ek1 against hcov-oc43, hcov-nl63, and hcov-229e infection with ic 50 values of 1.81, 6.02, and 3.35 μm, respectively. in vitro cytotoxicity assay determined that ek1 is not cytotoxic at concentrations up to 1 mm. mice that received 5 mg/kg of ek1 were nearly completely protected from infection by hcov-oc43 and 200 μg of ek1 against mers-cov infection. recently, this team found that ek1 could also potentially inhibit sars-cov-2 with an ic 50 value of 2.38 μm in pseudovirus assay and an ic 50 value of 0.19 μm in fusion inhibitory assay. 91 to improve the inhibitory activity of ek1 against sars-cov-2, they conjugate the cholesterol molecule to the ek1 peptide and found that a new peptide, ek1c4, exhibited highly potent inhibitory activity inhibit sars-cov-2 s-mediated membrane fusion and pseudovirus infection with ic 50 values of 1.3 and 15.8 nm, the cc 50 of ek1c4 was 5 μm, and the selectivity index was >136. in the oc43-infected mouse model, mice that received 0.5 mg/kg of ek1c4 were nearly completely protected from infection by hcov-oc43. these data suggested that ek1c4 could be used for inhibition and treatment of infection by currently circulating sars-cov-2. 92 mers-5hb, a polypeptide derived from the hr1 and hr2 region, was synthesized by gong et al., and affinity analysis demonstrated a low k d value of 0.24 nm, and an ic 50 value of 1 μm against mers-cov and cc 50 > 50 μm. hr derived peptides is a highly promising strategy for viral fusion inhibition. successful hr peptides have been used in the past to block entry of other virus families such as the hiv with the gp41 derived peptide fuzeon (t20), the only approved fusion inhibitor for hiv-1 treatment to date. 93 therefore, hr derived peptides highlight a promising strategy for inhibitor development combating the new sars-cov-2. xia et al. reported that two peptide-based membrane fusion inhibitors, 229e-hr1p and 229e-hr2p (figure 7 and table 2 ), targeting the hcov-229e s protein hr1 and hr2 domains, could competitively inhibit the viral autologous 6-hb formation and inhibit hcov-229e s protein-mediated viruscell membrane fusion with ic 50 values of 5.7 and 0.3 μm, respectively. moreover, neither 229e-hr1p nor 229e-hr2p had significant cytotoxicity to huh-7 and a549 cells at concentrations up to 1000 μm. in addition, 229e-hr2p potentially inhibited pseudotyped and live hcov-229e infection with ic 50 values of 0.5 and 1.7 μm, respectively. 94 the s2 domain is the most conserved motif between the sars-cov and the new sars-cov-2 s protein. 92 it represents an ideal immunogen for the generation of a novel or repurposing sars-cov s2 domain targeting mabs with cross-reactive potential. 95 sasazuki et al., for example, could successfully isolate the human mab 5h10 from immunized kunming (km) mice. 5h10 displayed an anti-sars-cov neutralizing activity of around 5 μg/ml. cell fusion assays indicate that 5h10 can inhibit viral fusion and entry rather than viral attachment to the surface of host cells or cleavage of the s protein. consequently, the s protein of sars-cov might be the direct target of 5h10; however, further studies are required to confirm this hypothesis. 96 tan et al. identified mab 1a9 (ic 50 value between 25 and 50 μg/ml), an anti-sars-cov s2 domain mab, that binds to a conserved loop region between the hr1 and hr2 domains of the s2 domain. 97 tsunetsugu-yokota et al. found that antibody skot20 can inhibit sars-cov with an ec 50 value of 5 μg/ml in vero e6 cells sars-cov. mutational studies indicate that skot20 restrict conformational changes within the s2 domain, essential for viral entry. 98 99 on the basis of this approach, they identified two small molecules, tgg (12, table 4 ) and luteolin (13) , that can bind avidly to the sars-cov s2 protein and inhibit viral entry of sars-cov into vero e6 cells with ic 50 values of 4.5 and 10.6 μm, respectively. cytotoxicity assay showed that the cc 50 of 12 and 13 were 1.08 and 0.155 mm, respectively. therefore, the selectivity index of 12 and 13 were 240.0 and 14.62, respectively. further acute toxicity suggested that the 50% lethal doses of 12 and 13 were ∼456 and 232.2 mg/kg, respectively. these indicated that these small molecules could be used at relatively high concentrations in mice. 98 quercetin (14), an analogue of 13, also showed antiviral activity against sars-cov, with an ic 50 arbidol (16), a broad-spectrum drug, has been licensed for decades in russia and china against influenza by binding to the ha protein to block the viruses−cell fusion. 103 recently, wang et al. identified that 16 efficiently inhibited sars-cov-2 virus infection in vitro with an ic 50 value of 4.11 μm, a cc 50 value of 31.79 μm, and an si of 7.73. 104 vankadari compared protein sequence analysis and found that a small region of the s2 domain (aa947−aa1027) of the sars-cov-2 spike glycoprotein resembles that of the influenza virus h3n2 ha. so the mechanism of 16 was to target the sars-cov-2 spike glycoprotein and blocked its trimerization, which may inhibit host cell adhesion and hijacking. 105 in january 2020, in wuhan, china, a clinical pilot trial conducted with 36 patients with sars-cov-2 virus infection received 400 mg 16 three times a day for 9 days; 31 untreated sars-cov-2 patients served as a control group. in this trial, patients with 16 showed a tendency to decrease viral load as determined by rt-pcr and reduced mortality (0% vs 16%), as compared to the control group. 106 the hr regions of sars-cov and sars-cov-2 s protein share a high degree of conservation, and the described small molecules as fusion inhibitors can have potential applications in inhibiting sars-cov-2 fusion. indeed, targeting virus surface protein is a promising antiviral strategy, whether inhibiting rbd or s2 domain. during clathrin-dependent viral entry, the host cellular cathepsin l protease plays a key role in infection efficiency by activation of the s protein into a fusogenic state to escape the late endosomes, and cathepsin l (lysosomal endopeptidase) cleavage is believed to expose a hydrophobic fusion peptide essential to initiate membrane fusion. 107 in light of its vital role in the sars cov infection cycle, cathepsin l is a desirable target to interfere with virus−cell entry. 83 cathepsin l consists of a pro-and a mature-domain. in a low ph milieu, the pro-domain is autocatalytically cleaved to obtain the papain-like folded mature-domain consisting of an n-terminal helical domain and a c-terminal β-sheet domain (figure 8 ). 108 a well conserved cys-his-asn triad in the active site is crucial for substrate binding and catalysis. in light of its importance in the sars-cov-2 replication cycle, cathepsin l is a highly desirable target that will be described in the following section. 109 teicoplanin is a glycopeptide antibiotic, with applications in the treatment of serious infections caused by gram-positive bacteria such as streptococcus and staphylococcus aureus. 110 interestingly, teicoplanin was shown to block the entry of sars, mers, and ebola virus by specifically inhibiting the cathepsin l activity. 111 more recently, zhang et al. showed that teicoplanin could also block the entry of the new sars-cov-2 pseudoviruses with an ic 50 value of 1.66 μm. as a routinely used clinical antibiotic, teicoplanin could be potentially used immediately to combat the current sars-cov-2 outbreak. 112 small molecules as cathepsin l inhibitors. human cathepsin l plays numerous critical roles in diverse cellular settings associated with human diseases. 113 previous studies also highlighted the feasibility of targeting this cysteine endopeptidase with small molecules with implications for possible intervention strategies of sars-cov-2 infection. 113 a high-throughput screen (hts) of a 1000-compound library that resulted in the identification of mdl28170 (17 , table 4 ) by bates et al., and in an antiviral activity assay, 17 specifically inhibited cathepsin l-mediated substrate cleavage and blocked sars-cov viral entry, with an ic 50 value of 2.5 nm and ec 50 value in the range of 100 nm. however, despite its potent inhibitory activity, no cytotoxicity data for 17 is currently available. 83 two small molecules, cid 16725315 (18) and cid 23631927 (19) , were reported by diamond et al. as viral entry inhibitors of the sars-cov. in a cathepsin l inhibition assay, 19 could block cathepsin l with an ic 50 value of 6.9 nm, while 18 showed slightly weaker potency with an ic 50 value of 56 nm. interestingly, besides inhibiting sars-cov, compound 19 (ec 50 value of 273 nm) showed some inhibition activity for ebola virus infection (ec 50 value of 193 nm) of human embryonic kidney 293t cells. importantly, 19 did not show any sign of toxicity to human aortic endothelial cells at 100 μm. this data offers a new promising point for the treatment of sars and ebola virus infections. 114 recently, in a cell-based assay screen of ∼14 000 compounds, ssaa09e1 (20) was identified that could specifically bind to the cathepsin l proteinase and interference sars-s protein during viral entry, with an ic 50 value of 5.33 μm. in a pseudotype-based assay in 293t cells, the ec 50 value of 20 was around 6.4 μm, and no cytotoxicity was detected below 100 μm. 57 using sars-cov entry assays, zhou et al. screened 2100 cysteine protease inhibitors with confirmed activity to inhibit human cathepsins. among them, k11777 (21) demonstrated the most robust activity. results demonstrated that 21 blocked sars-cov pseudovirus entry at an ic 50 value of 0.68 nm while no toxicity was observed, cc 50 value >10 μm. interestingly, for other coronaviruses, 21 showed broadspectrum antiviral activity with ic 50 values of 1.48, 6.78, and 46.12 nm against hcov-229e, hcov-nl63, and mers-cov, respectively. 115 inhibitors of cell membrane-associated tmprss2. either the endosomal cysteine proteases cathepsin l or the cell membrane-associated serine protease tmprss2 can facilitate sars-cov virus entry into host cells by cleavage of the viral s protein. 19 this cleavage exposes fusion-competent motifs known as fusion peptides, and importantly, for sars-cov, the interference of both proteases is required for efficient inhibition of virus replication. 19 matsuyama et al. identified camostat (22 , table 4 ), a commercially available serine protease inhibitor that can efficiently prevent sars-cov infections at 10 μm by inhibiting tmprss2 activity. however, even at high concentrations (100 μm) of 22, the inhibition of viral entry via sars s protein-mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that despite the inhibition of tmprss2, 35% of virus entry takes place via the endosomal cathepsin pathway. therefore, they examined the activity of pseudotyped viruses when treated with a combination of (23,25)trans-epoxysuccinyl-l-leucylamindo-3methylbutane ethyl ester (est, a cathepsin inhibitor) and 22. the results suggested that simultaneous treatment with est and 22 remarkably blocked infection (>95%). 116 similarly, poḧlmann et al. reported that 22 could prevent the viral entry of sars-cov-2. importantly, full inhibition efficiency was attained when treated with both 22 and e-64d (a cathepsin inhibitor). both studies indicate that sars-cov and sars-cov-2 enter cells via a similar mechanism, showing the potential of 22 as a promising candidate for further development as a sars-cov-2 treatment. 19 inhibitors of the furin cleavage site in the coronavirus spike proteins. elevated levels of furin expression were able to facilitate mers-cov pseudovirion infection, and viral entry could be reduced by furin sirna silencing. 20 decanoyl-rvkr-chloromethylketone (23, dec-rvkr-cmk), a furin inhibitor, was shown to block mers-cov s protein-mediated entry as well as virus infection, with journal of medicinal chemistry pubs.acs.org/jmc perspective an ic 50 value of 75 μm in hek-293t cells. furthermore, when cathepsin inhibitor camostat was used in combination with 23, a significant inhibition in infectivity was characterized compared to camostat alone. 20 recently, bestle et al., showed that the potent peptidomimetic inhibitor mi-1851 (24) could prevent proteolytic processing of the s protein from sars-cov-2 by endogenous furin in hek293 cells. however, no antiviral data is available for 24 yet. 117 the peculiar furin-like cleavage site (s1/s2-site in figure 2 ) in sars-cov-2 that is absent in the sars-cov and other sars-like covs indicates that furin inhibitors could play a significant role in blocking the viral entry process. 117, 118 ■ host factor inhibitors sars-cov-2 cell entry also relies on host cell factors. therefore, these host cell factors can play an essential role as targets for sars-cov-2 inhibition. 119 chlorpromazine (25 table 5 ) is an antipsychotic drug developed for the treatment of schizophrenia. it has also been reported to inhibit the infection of hepatic c virus (hcv), 120 mouse hepatitis virus (mhv-2), 27 and alphavirus. 121 (28), an abelson kinase signaling pathway inhibitor that could inhibit abelson tyrosine−protein kinase 2 (abl2) to block mers-cov virion fusion with endosomal membranes with an ic 50 value of 10 μm. 28 showed no cytotoxic effects in vero cells at 100 μm. 123, 124 another abl inhibitor, dasatinib (29) , was active against both mers-cov and sars-cov, with ic 50 values of 5.4 and 2.1 μm, respectively. 125 on the basis of an hts assay using cytopathic-effect ( phenotypic screening methods are usually used to identify firstin-class drugs without knowing the actual target and mechanism of action of the drug, while target-based screening identifies best-in-class drugs. 127−129 although the phenotypic screening approach often is limited in terms of capacity compared to in silico target-based screening, it can have advantages in identifying cell-active compounds providing information on drug solubility or cell uptake. 127−129 many drugs, especially natural products, have an unknown mechanism of action but were shown to inhibit coronavirus entry. 130 hsiang et al. screened a library of 121 chinese herbs using a biotinylated enzyme-linked immunosorbent assay to search for active compounds that could potentially inhibit sars-cov s protein binding to ace2. further studies identified emodin (31 , table 5 ), the active component from polygonum multiflorum and rheum officinale, could block the interaction of sars-cov s protein to ace2, with an ic 50 value of 200 μm in an s protein-pseudotyped retrovirus assay using vero e6 cells. however, the mechanism of action of 31 still needs to be determined. 131 sarafianos et al. found that ssaa09e3 (32), a benzamide derivative, could prevent sars-cov virus−cell membrane fusion in pseudotyped-based and antiviral-based assays, with an ic 50 value of 9.7 μm, but a cc 50 value of 20 μm indicates additional unknown cellular targets. 57 out of an hts, ve607 (33) was identified using a phenotype-based screen from a 50 240 structurally diverse small-molecule compound library. pseudotype virus entry assay suggested ve607 can specifically inhibit sars-cov virus entry into cells with an ec 50 value of 3 μm and inhibited sars-cov plaque formation with an ic 50 of 1.6 μm. 132 a similar hts approach was employed by zhang et al. for screening a compound library consisting of 727 structurally diverse small molecules. eighty-four compounds were identified with significant anticoronavirus potential. further studies revealed that 51 compounds inhibited virus entry, while 19 others interfered with viral replication. 133 natural products should, however, be considered with caution due to their unknown mechanism of action and possible toxic side effects. the recent sars-cov-2 outbreak, with its high fatality rate, has raised global concerns and was declared as a global pandemic by the who. the number of infections continues to rise, and numerous research groups around the globe have prioritized the identification and development of new covid-19 treatments. still, there are no effective treatments to date. viral entry is the first step in the viral life cycle and represents an attractive intervention point by blocking the coreceptor interaction or the virus−cell membrane fusion event. sars-cov-2 and other coronaviruses have similar infection mechanisms. this is especially true for sars-cov and cov-nl63, which share the same human ace2 receptor crucial for viral entry. therefore, already developed inhibitors against known hcovs could potentially be used to combat sars-cov-2. these efforts identified a large number of inhibitors, including peptides, antibodies, small-molecule compounds, and natural products with anticoronavirus activity. although many inhibitors demonstrated efficacy in inhibiting coronavirus virus infection, no specific prophylactic or postexposure therapy is currently available for hcovs. one of the main reasons causing this is that most of the potenial agents were not adequately evaluated for in vitro and in vivo studies. most drugs are in the preclinical stage and stopped in animal models due to poor bioavailability, safety, and pharmacokinetics so that few entered human trials. in light of the urgency of the current outbreak, repositioning of already approved drugs is increasingly becoming a promising approach, especially with toxicity and safety data in hand. the most effective measure to prevent viral diseases is vaccination. coronavirus vaccine development mainly focused on s protein, and some of them reported can inhibit sars, 134−136 and mers. 137 although vaccination strategies were developed in the context of previous epidemics, no vaccine for sars-cov-2 infections is yet available. since the recent sars-cov-2 outbreak, research groups around the world are now stepping up to develop vaccines targeting sars-cov-2, and vaccine research routes include nucleic acid vaccines, viral vector vaccines, inactivated vaccines, and recombinant protein vaccines. typical vaccine development is time, resource, and financially consuming, although this pandemic has created initiatives that hope to speed the development of a sars-cov-2 vaccine. even the most optimiztic views regarding an effective sars-cov-2 vaccine being created are at least one year away. even after creation, other hurdles for the sars-cov-2 include global implementation and distribution, and different strategies for containing this contagion should be explored simultaneously as the vaccine efforts. in addition to small-molecule inhibitors, monoclonal antibodies, and vaccine development, convalescent sera from sars-cov-2 survivors (convalescent-phase sera) is an additional option for covid-19 treatment. passive immunization was well established for viral infection prophylaxis. 138 by metaanalysis of studies about the 1918 influenza, h1n1 influenza epidemic demonstrated that early treatment of convalescent blood products decreased the risk ratio caused by pneumonia from 37% to 16%. 139 nevertheless, the appropriate titer of the convalescent-phase sera antibody remains to be determined, which was required for therapeutic efficacy to inhibit sars-cov-2. research carried out with mers-cov suggested that sera from patients recovering from infections did not contain sufficient antibody titers for therapeutic use. 140 recent initiatives such as the governmental (usa) operation warp speed (ows) to support the development, manufacturing, and 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protectively immunizes mice recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region sars coronavirus spike polypeptide dna vaccine priming with recombinant spike polypeptide from escherichia coli as booster induces high titer of neutralizing antibody against sars coronavirus prospects for a mers-cov spike vaccine passive immunization. prim care meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol key: cord-308583-vtmwv8zl authors: du, qishi; wang, shuqing; wei, dongqing; sirois, suzanne; chou, kuo-chen title: molecular modeling and chemical modification for finding peptide inhibitor against severe acute respiratory syndrome coronavirus main proteinase date: 2005-02-15 journal: analytical biochemistry doi: 10.1016/j.ab.2004.10.003 sha: doc_id: 308583 cord_uid: vtmwv8zl abstract severe acute respiratory syndrome (sars) is a respiratory disease caused by a newly found virus, called sars coronavirus. in this study, the cleavage mechanism of the sars coronavirus main proteinase (mpro or 3clpro) on the octapeptide nh2-avlq↓sgfr-cooh was investigated using molecular mechanics and quantum mechanics simulations based on the experimental structure of the proteinase. it has been observed that the catalytic dyad (his-41/cys-145) site between domains i and ii attracts the π electron density from the peptide bond gln–ser, increasing the positive charge on c(co) of gln and the negative charge on n(nh) of ser, so as to weaken the gln–ser peptide bond. the catalytic functional group is the imidazole group of his-41 and the s in cys-145. nδ1 on the imidazole ring plays the acid–base catalytic role. based on the “distorted key theory” [k.c. chou, anal. biochem. 233 (1996) 1–14], the possibility to convert the octapeptide to a competent inhibitor has been studied. it has been found that the chemical bond between gln and ser will become much stronger and no longer cleavable by the sars enzyme after either changing the carbonyl group co of gln to ch2 or cf2 or changing the nh of ser to ch2 or cf2. the octapeptide thus modified might become an effective inhibitor or a potential drug candidate against sars. nucleotides. the replicase gene alone encompasses more than 21,000 nucleotides, 2/3 of the whole genome sequence. the replicase gene encodes two overlapping polyproteins, pp1a (4377 a.a.) and pp1ab (7072 a.a.). sars cov main proteinase (m pro ) cleaves the polyprotein at no less than 11 conserved sites, a process initiated by the enzymeõs own autolytic cleavage from pp1a and pp1ab [7, 8] . the released polypeptides mediate all of the functions required for sars viral replication and transcription. the functional importance of the m pro 0003-2697/$ -see front matter ó 2004 elsevier inc. all rights reserved. doi:10.1016/j.ab.2004. 10.003 in the viral life cycle makes it an attractive target for the drugs design against sars [7] [8] [9] [10] . anand et al. [7] suggested that the rhinovirus 3c pro inhibitor ag7088 could well serve as a starting point for an anti-sars drug based on the theoretical homology model of sars cov m pro . chou et al. [9] found the fitting problem of ag7088 to the binding pocket of sars cov m pro , and they suggested its derivative kz7088 as a better starting point. sirois et al. [11] did virtual screening for sars-cov protease based on kz7088 pharmacophore points. chou et al. [9] also suggested an octapeptide nh 2 -avlq fl sgfr-cooh that can be converted to an inhibitor of sars cov m pro by some chemical modification. this octapeptide was synthesized, proved to be a cleavable peptide, and showed strong inhibiting activity in a tube test [12] . du et al. [13] analyzed the cleavage mechanism of the octapeptide by sars cov m pro and discussed the possible chemical modification of the octapeptide using molecular dynamical and quantum mechanical simulations based on the homology structure of sars cov m pro , to convert it into a stable and effective drug. this progress on finding inhibitors against the sars enzyme is also summarized in a recent review [10] . the experimental structure of sars cov m pro was first determined by raoõs research group [8] . they provided several crystal structures of sars cov m pro at different ph conditions and the complex structure of sars cov m pro with ligand hexapeptidyl cmk [7, 8] . the experimental structure and the theoretical homology structure of sars cov m pro share many structural similarities, such as the catalytic active region and the cys-his cleavage dyad [7, 8] . however, there are several differences between the theoretical and the experimental structures. for examples, the experimental structure of sars cov m pro is a dimer, the protomer a has catalytic activity, and the protomer b has no activity. however, the n finger of protomer b, which plugs in the active cleft of protomer a, plays an important role in both dimerization and maintenance of the active form of the enzyme [8] . these new findings from the experimental structure of sars cov m pro provide a reliable basis for inhibitor design. the octapeptide nh 2 -avlq fl sgfr-cooh shows strong inhibiting activity in vitro [12] . however, the use of native peptide for clinical applications has been hampered by their intrinsic metabolic instability (indigestion by enzymes) and poor absorption through membrane. a cleavable peptide of sars cov m pro means that it possesses a competent binding with its receptor and a breakable peptide bond in the cleavage site. to convert a cleavable peptide into a stable and effective inhibitor, the peptide has to be modified according to the ''distorted key'' theory [14, 15] . one important step for the chemical modification is a good understanding of the cleavage mechanism, particularly the changes in chemical bonds [16] [17] [18] [19] [20] [21] [22] . the protease-susceptible sites in a given protein or peptide usually extend to an octapeptide region [14, 15] . the corresponding amino acid residues are sequentially symbolized by eight subsites r 4 , r 3 , r 2 , r 1 , r 1 0 , r 2 0 , r 3 0 , r 4 0 and the eight matching positions in the protease by s 4 , s 3 , s 2 , s 1 , s 1 0 , s 2 0 , s 3 0 , s 4 0 , respectively (see fig. 3 of [15] ). occasionally, the susceptible sites in some proteins may contain one subsite less or one more. however, eight amino acid residues are the most common case. although the protein being cleaved contains much more than eight amino acid residues, only an octapeptide segment fits in the active region of the protease. therefore, our research will focus on the cleavability of octapeptides. as shown in fig. 1 , the cleavage point is always on the peptide bond between r 1 and r 1 0 . in fig. 1a we use the combination of a thin line and a dashed line to represent the conjugate p property in the peptide bond. in fig. 1b the breakable peptide bond between r 1 and r 1 is converted to a strong ''hybrid peptide bond'' through a chemical modification and hence cannot be cleaved by the protease. the modified octapeptide can still bind to the active site, but it cannot be cleaved by the enzyme, just like a distorted key that can neither open the lock nor be pulled out. the octapeptide thus modified will automatically become a desired inhibitor candidate. the octapeptide avlqsgfr is the first suggested [9] based on the molecular structure of sars cov m pro and has been proved cleavable by experiments. in this research we study the cleavage mechanism, the properties of the relevant chemical bonds, and the catalytic interactions between the octapeptides and the sars cov m pro using molecular mechanical and quantum chemical simulations to provide useful insights for the chemical modification. the study was carried out in the following four steps: (1) docking the octapeptide to sars cov m pro followed by an energy minimization for the complex structure using molecular mechanics, (2) computing the atomic charge distribution in the binding pocket for the energy-minimized structure using ab initio quantum mechanics, (3) computing the molecular energy, chemical bond properties, and atomic charges of the octapeptide with the background charge distribution [23] of sars cov m pro using ab initio quantum chemistry, and (4) with the same background charge distribution, computing the molecular energy, chemical bond properties, and atomic charges of the modified octapeptide. the computations have been carried out with the molecular dynamics and standard ab initio quantum chemistry at the hf/6-31g* level [23] . the experimental structure (1uj1.pdb) of sars cov m pro [8] is used in the calculation and shown in fig. 2a . each protomer structure of the sars cov m pro dimer has three domains. the two protomers a and b form a right angle and the join part is in the domain iii. the n finger of protomer b plugs into the domain ii of protomer a and plays an important role in the catalytic activity. the pdb data of sars cov m pro contains 300 hydration water molecules, which provide the solvation ion environment with some water molecules joining the catalytic reaction. in fig. 2a the green signs ''<'' represent the solvation water molecules. the docking calculation between sars cov m pro and the octapeptide avlqsgfr was performed using the molecular although still bound to the active site, the peptide has lost its cleavability after its scissile bond was modified from a hybrid peptide bond to a strong bond. adapted from chou [15] with permission. operating environment program package [24] . a total of 25 docking conformations were obtained, and the one with the lowest energy was used for further energy minimizing calculation. fig. 2b shows the complex structure of sars cov m pro with the ligand octapeptide. the positions of catalytic dyad his-41 and cys-145 are indicated in fig. 2b . according to anand et al. [7] , yang et al. [8] , and chou et al. [9] , the catalytic active region is in the pocket between domains i and ii of protomer a. during calculation the catalytic active region on which we were focusing contains the amino acid residues cys-22, gly-23, thr-24, thr-25, leu-27, his-41, val-42, cys-44, thr-45, ala-46, glu-47, asp-48, met-49, leu-50, asn-51, pro-52, tyr-54, phe-140, leu-141, asn-142, gly-143, ser-144, cys-145, glu-163, his-164, met-165, glu-166, his-172, asp-187, arg-188, and gln-189 from promoter a and the residues phe-3, arg-4, lys-5, met-6, and ala-7 from promoter b. but in the practical calculation, the actual region involved contains much more than the above-listed amino acid residues. it includes all the amino acid residues within 5 å surrounding the octapeptide [9, 18] . water molecule plays an important role in the proteolytic reaction. these results are derived from the complex structure obtained by following the aforementioned docking and energy-minimization procedures. the hydrophilic and hydrophobic complementary interaction is important in the ligand-receptor interaction. according to the a sphere model [25, 26] , the active region of proteinase consists of a series of a spheres. each interaction site between the ligand and receptor contains one or more a spheres. there must be at least one hydrophobic interaction site. figs. 4a and b are the hydrophilic and hydrophobic surfaces of octapeptide avlqsgfr and the catalytic active region of sars cov m pro , respectively. in the figures the green parts are hydrophobic surfaces and the blue parts are hydrophilic surfaces. there is good hydrophilic and hydrophobic complementary interaction between ligand and receptor: the hydrophilic parts of the ligand correspond to the hydrophilic parts of the receptor and the hydrophobic parts of the ligand correspond to the hydrophobic parts of the receptor. the octapeptide nh 2 -avlq fl sgfr-cooh has four hydrophobic amino acid residues: r4(ala), r3(val), r3 0 (phe), and r4 0 (arg). among them r4(ala) and r3(val) are exposed in solvent and r3 0 (phe) and r4 0 (arg) are covered by hydrophobic surfaces of the proteinase. the peptide bond to be cleaved between r1(gln) and r1 0 (ser) is just in the join point between the exposed part and the covered part. the calculations were focused on the effect of sars cov m pro on the peptide bond to be cleaved. how to properly treat a biomacromolecule and the solvent effects is a nontrivial problem in quantum chemical calculations. electrostatic interaction plays the dominant role in ligand-receptor combinations and must be taken into account in the quantum mechanical study. in view of this, we treated the sars cov m pro as a background with many point charges. the amino acid residues in the catalytic region between domains i and ii of protomer a were divided into seven segments, and their atomic charges were separately calculated using the ab initio quantum chemical hf/6-31g* method. the 5 amino acid residues of the n finger of protomer b were taken into account also. the 76 amino acid residues in eight segments are listed in table 1 . there are 1148 atomic charges in the background, including all atoms in the catalytic cleft; the overlapped atoms are not counted. another difficult problem for quantum mechanical calculation for a biological system in solution is the solvent effect. in the pdb data [8] of sars cov m pro there are 300 solvation water molecules. these water molecules not only provide the solvation environment but also indicate that some water molecules are involved in the catalytic reaction. in our calculation the 300 solvation water molecules were treated as the background charges. each water molecule has 3 point charges. therefore, there are 1148 + 300 · 3 = 2048 background point charges. in table 2 we list the mulliken atomic charges q mull and electrostatic potential equivalent charges q esp of his-41, which are obtained by fitting atomic charges to the electrostatic potential at the van der waals surface. the atomic charges from semiempirical method am1 are quite different from the results by ab initio hf/6-31g* [23] ; in particular; the q esp charges of am1 do not appear reasonable. because the hf/6-31g* q esp charges reproduce the quantum mechanical electrostatic potential on the molecular surface, in this research we use q esp from the hf/6-31g* calculations to illustrate our points. the imidazole group of his-41 is a nucleophilic agent and its pk = 6.0, near the concentration of proton [h + ] in pure water. the imidazole ring is both a proton donor and a receptor. it has good catalytic activity in the physiological condition [27] . it can be seen from table 2 that the negative charge of n d1 (à0.4094) and the positive charge of h þ d1 (0.4318) are large in magnitude and are close to the peptide bond gln-ser. therefore, the proton h þ d1 attracts the p electron density from the peptide bond gln-ser so as to weaken this chemical bond. the polar nitrogen n d1 attracts one hydrogen h 1 of bridge water and makes the water molecule dissociated, causing the nucleophilic attack of hydroxyl group oh à to the c(co) and the electrophilic attack of h + to n(nh) on the two sides of peptide bond gln-ser. the sulfur atom s in cys-145 forms a hydrogen bond with h(nh) and helps the proteolytic reaction (fig. 3b) . the peptide bond is considered a pseudo p bond. the six atoms in the peptide bond (gln)ca co-nhca (ser) are almost in a plane. fig. 5a shows the electron density contour map of the peptide bond gln-ser in the gaseous phase on the p plane containing the carbonyl group co of gln and the nh group of ser. the electron density contours surrounding atoms n(nh) and c(co) form two triangles reminiscent of the hybrid orbits of the sp 2 type, and the 2p z orbits, which are perpendicular to the plane and form a p 4 3 bond system. in table 3 , we list the calculated atomic charges of the six atoms on both sides of the peptide bond gln-ser obtained from the ab initio hf/6-31g* calculations in the gaseous phase with the background charges of sars-cov m pro . the negative charge of n(nh) of serine in the octapeptide increases to à0.8612 in the background of proteinase charges from the original value of à0.8534 in the gaseous phase. similarly, the corresponding positive charge of the carbonyl carbon c(co) in glutamine of the octapeptide increases to 0.8190 from the original 0.8050. this is favorable to the electrophilic attack of oh à to c(co) and the nucleophilic attack of h + to n(nh) in the proteolytic reaction. fig. 5b is the contour map of differential electronic density of the peptide bond gln-ser in the octapeptide avlqsgfr, obtained by subtracting the electron density in the gaseous phase from the electron density in the background [23] of sars cov m pro and solvent water molecules. in fig. 5b the bold gray line is the 0 value line where the electron densities in the gaseous phase and in the protease background are equal. the solid thin lines show the regions where the computed electron densities are greater in the sars cov m pro background than in the gaseous phase, and the dashed thin lines show the areas where computed electron densities are smaller in the proteinase background than in the gaseous phase. we find that along the peptide bond between (gln)c-n(ser) the electron densities increase on the n(ser) side and decrease on the c(gln) side. this change is favorable for a nucleophilic attack of anion oh à to (gln)c and for an electrophilic attack of cation h + to n(ser). sars cov m pro has very high cleavage specificity and is highly conserved in the coronavirus family [7] . the cleavage specificity of sars cov m pro concentrates on the sites r2, r1, and r1 0 . the development of peptides as clinically useful drugs is limited by their poor stability and bioavailability, which is due in part to their inability to readily cross membrane barriers such as the intestinal and blood-brain barriers. systematic chemical modification strategies that convert peptides into drugs are an attractive research topic in current medicinal chemistry [28] . for the peptide inhibitor of proteinase, the chemical modification to cleavable octapeptide should focus on the scissile peptide bond between r 1 and r 1 0 to be cleaved by sars cov m pro . a successful example shows that after the peptide bond co@nh is replaced by reduced single bond ch 2 anh, the modified octapeptide rennin inhibitors have shown greatly increased affinity for rennin and strong resistance to enzyme hydrolysis [29] . in this study we suggest a similar modification to octapeptide avlqsgfr. in the peptide bond (gln) co@nh(ser) the carboxyl group co of glutamine on subsite r 1 is replaced by ch 2 or cf 2 or the nh of serine on subsite r 1 0 is replaced by ch 2 ; then the p bond system is broken and the modified octapeptide avlq sgfr may become a stable inhibitor for sars-cov m pro and an effective drug candidate against sars. here we show the possibility of the chemical modification through computational modeling. table 4 lists the atomic charges of the six atoms on both sides of the hybrid peptide bond gln-ser after the chemical modification. comparing with table 3 , we find that after co is replaced by ch 2 , the charge of the carbon atom in ch 2 turns negative (à0.6572) from originally a positive value (0.8612) in the co group; hence the nucleophilic attack by oh à becomes impossible. furthermore, the negative charge of n(nh) on the ser side decreases to à0.2892 from à0.8190 in table 3 , and hence the electrophilic attack by h + also becomes more difficult. likewise, the modifications co fi cf 2 and nh fi ch 2 will make the proteolytic reaction more difficult also. searching for cleavable peptides is the first step in finding potential inhibitors against the proteinase. even within a limited range of octapeptides, it is by no means an easy job because the possible different patterns of octapeptides are as many as 20 8 = 2.56 · 10 10 . however, some algorithms, such as the vectorized sequence-coupled algorithm [14] and the discriminant function algorithm [15] , can be successfully used to narrow the search scope. meanwhile, similar methods that can be used to search the cleavable peptides by sars enzyme have been developed [5, 6] . the octapeptide nh 2 -ala-val-leu-gln-ser-gly-phe-arg-cooh is the first designed on the basis of the molecular structure of sars-cov m pro [9] and it has been experimentally demonstrated to be a cleavable peptide with high bioactivity [12] . a detailed study of the cleavage mechanism needs a great deal of experimental work. however, computational simulation may provide useful insights for an in-depth understanding of the mechanism. the cleavable octapeptide could be changed into an effective inhibitor of sars-cov m pro or a drug candidate against sars after a proper chemical modification to replace the cleavable peptide bond with a noncleavable bond. the octapeptide thus modified will lose its susceptibility although it can still tightly bind to the active site and become a competent inhibitor, just like the role of a distorted key in blocking the access of other keys to a lock, as elucidated by chou [15] in a review paper. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome and influenza: virus incursions from southern china n-terminal domain of the murine coronavirus receptor ceacam1 is responsible for fusogenic activation and conformational changes of the spike protein zcurve_cov: a new system to recognize protein coding genes in coronavirus genomes, and its applications in analyzing sars-cov genomes prediction for proteinase cleavage sites in polyproteins of coronaviruses and its applications in analyzing sars-cov genomes coronavirus main proteinase (3cl pro ) structure: basis for design of anti-sars drugs the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars review: structural bioinformatics and its impact to biomedical science virtual screening for sars-cov protease based on kz7088 pharmacophore points synthesis and biological evaluation of a novel sars-cov m pro inhibitor polyprotein cleavage mechanisms of sars covm pro and chemical modification of the octapeptide a vectorized sequence-coupling model for predicting hiv protease cleavage sites in proteins review: prediction of hiv protease cleavage sites in proteins prediction of the tertiary structure and substrate binding site of caspase-8 solution structure of the raidd card and model for card/card interaction in caspase-2 and caspase-9 recruitment a model of the complex between cyclin-dependent kinase 5(cdk5) and the activation domain of neuronal cdk5 activator solution structure of bid, an intracellular amplifier of apoptotic signaling prediction of the tertiary structure of a caspase-9/inhibitor complex prediction of the tertiary structure of the beta-secretase zymogen identification of the n-terminal functional domains of cdk5 by molecular truncation and computer modeling exploring chemistry with electronic structure methods: a guide to using gaussian a computational procedure for determining energetically favorable binding sites on biologically important macromolecules functionality maps of binding sites: a multiple copy simultaneous search method converting a peptide into a drug: strategies to improve stability and bioavailability a potent new renin inhibitor. in vitro and in vivo studies this work was supported by grants from the chinese national science foundation under contract no. 20373048 and the tianjin commission of sciences and technology under contract nos. 033801911 and 023618211. key: cord-324978-9qfhsj3n authors: alagaili, abdulaziz n.; briese, thomas; mishra, nischay; kapoor, vishal; sameroff, stephen c.; de wit, emmie; munster, vincent j.; hensley, lisa e.; zalmout, iyad s.; kapoor, amit; epstein, jonathan h.; karesh, william b.; daszak, peter; mohammed, osama b.; lipkin, w. ian title: middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia date: 2014-02-25 journal: mbio doi: 10.1128/mbio.00884-14 sha: doc_id: 324978 cord_uid: 9qfhsj3n the middle east respiratory syndrome (mers) is proposed to be a zoonotic disease; however, the reservoir and mechanism for transmission of the causative agent, the mers coronavirus, are unknown. dromedary camels have been implicated through reports that some victims have been exposed to camels, camels in areas where the disease has emerged have antibodies to the virus, and viral sequences have been recovered from camels in association with outbreaks of the disease among humans. nonetheless, whether camels mediate transmission to humans is unresolved. here we provide evidence from a geographic and temporal survey of camels in the kingdom of saudi arabia that mers coronaviruses have been circulating in camels since at least 1992, are distributed countrywide, and can be phylogenetically classified into clades that correlate with outbreaks of the disease among humans. we found no evidence of infection in domestic sheep or domestic goats. clusters of human infection indicate that human-to-human mers-cov transmission can occur (4, 5) . however, the origin of the infection in most cases remains unknown. analysis of human mers-cov sequences by cotten et al. has revealed the presence of at least three circulating genotypes within the ksa alone (6) . phylogenetic analyses of 13 complete and 8 partial genome sequences enabled estimates of the timing and geographic origins of individual viral clades. the authors proposed that mers-cov emerged in humans in 2011 and noted that sequence divergence between clades is consistent with several sporadic introductions of the virus into the human population, presumably from an animal reservoir. efforts to identify an animal reservoir have focused on bats and camels. bats harbor a wide range of betacoronaviruses (7) ; furthermore, bat cell lines display the mers-cov receptor, dipeptidyl peptidase 4 (8) , and can be experimentally infected. a short sequence fragment consistent with mers-cov was reported in a bat in bisha, ksa, collected in close proximity to the home and workplace of the 2012 index case patient from whom the initial virus isolate was obtained (9) . that same patient owned four pet dromedary camels (dc). serological analysis of those dc revealed the presence of antibodies reactive with mers-cov; however, no mers-cov sequences were found by pcr analysis of nasal or rectal swabs or serum. additional human cases have been associated with exposure to dc, and in some instances, investigators have described both serologic and genetic evidence of mers-cov infection in dc. memish and coworkers reported pcr detection of mers-cov sequences in a dc with respiratory illness owned by an individual with mers-cov who had no history of contact with other infected humans (10). haagmans et al. investigated an outbreak of the disease among humans on a qatari farm and found mers-cov sequences in nasal swabs from 6 of 14 seropos-itive dc. analysis of open reading frame 1a (orf1a) and fragments representing orf1b, spike, and orf4b revealed similarity but not identity to sequences obtained from the mers-covinfected humans at the same farm. the authors provide evidence that mers-cov can infect dc but cautiously conclude that data are insufficient to determine whether the infection spread from dc to humans, from humans to dc, or via another host to both species (11) . several groups have reported serological reactivity with mers-cov or a closely related virus in dc in the middle east (12) (13) (14) (15) (13) . in two regions of the ksa, hemida and colleagues detected antibodies to mers-cov in 90% of 310 dc but not in sheep, goats, cattle, or chickens. the seroprevalence was lower in dc ͻ1 year of age (72% versus 95%), suggesting widespread infection in early life (15) . to determine the prevalence of mers-cov infection in dc throughout the ksa, we undertook a nationwide survey by using both serological and molecular methods. serum, whole blood, and rectal and nasal swabs were freshly collected from dc, sheep, and goats in november and december of 2013 in the southwestern (gizan), western (taif), northwestern (tabuk), eastern (hofuf), and central (unizah, riyadh) regions of the ksa (table 1) . we also collected archived serum samples obtained from dc in 1992 through 2010 (table 2 ). sera were initially tested for the presence of antibodies reactive with mers-cov by using a cell enzyme-linked immunosorbent assay (elisa) based on vero cells infected with mers-cov. subsets of sera positive by elisa were tested in western blot assays that used extracts of vero cells infected with mers-cov and a luciferase immunoprecipitation system (lips) assay based on recombinant mers-cov nucleoprotein. potential serologic cross-reactivity with bovine coronavirus (bo-cov) was addressed by testing for reactivity with bo-cov nucleocapsid protein by lips assay. the presence of viral nucleic acids in rectal and nasal swabs and a subset of serum and whole blood samples was assayed by reverse transcriptionquantitative pcr (rt-qpcr) with primers targeting the upe and orf1a genome regions of mers-cov (16, 17) . one hundred fifty (74%) of 203 dc sampled countrywide in 2013 were found to have antibodies to mers-cov by elisa. the prevalence of seropositivity was higher in adult dc (ͼ2 years of age; 93/98, 95%) than in juvenile dc (յ2 years of age; 57/104, 55%) (p ͻ 0.0001, 2 test). the lowest prevalence of seropositive juveniles was found in the southwestern region, in proximity to the city of gizan (1/21, 5%) (fig. 1a) . a higher prevalence of antibodies to mers-cov in older animals was also seen in samples obtained in years prior to 2012. in 2010, the seroprevalence was 76% (16/21) in juveniles versus 91% (21/23) in adults in the central region (riyadh, kharj). in 2009, the seroprevalence was 72% (40/56) in juveniles versus 92% (24/26) in adults (riyadh, rumah) ( table 2) . seroreactivity was also found in samples collected from dc as early as the 1990s: 1/1 (100%) in 1992, 2/2 (100%) in 1993, 114/123 (93%) in 1994, 6/6 (100%) in 1996, and 6/6 (100%) in 2003. analysis of selected seropositive dc sera by western immunoblot assay indicated two distinct reaction patterns that showed reactivity either to mers-cov spike glycoprotein alone or to spike glycoprotein and nucleocapsid protein. these results were concordant with results of mers-cov nucleocapsid lips assays ( fig. 2 ; see table s1 in the supplemental material). potential serologic cross-reactivity to bo-cov was addressed by analyzing bo-cov nucleocapsid reactivity, which has been shown to be subgroup specific in covs (18) . overall, 17% (35/ 203) of dc were positive for bo-cov, ranging from 3% in the southwest (gizan) to 25% in the east (hofuf) and 20% in adult animals versus 14% in juvenile animals; 2 animals (juveniles in taif) were seropositive for bo-cov exclusively, while the remaining 16% (33/203) were reactive to bo-cov and mers-cov, and 58% (117/203) were reactive to mers-cov alone (see table s1 in the supplemental material). rectal and nasal swabs collected in parallel with serum samples from the same animals were assayed for mers-cov nucleic acids by rt-qpcr. nucleic acids were most frequently detected in nasal swabs; rectal swabs were found positive in only three cases; in two of them, the nasal sample was also positive. the regional distribution of pcr-positive animals is shown in fig. 1b . in contrast to serology, where mers-cov-reactive antibodies were more prevalent in adults than in juveniles (95% versus 55%), mers-cov nucleic acids were found more frequently in juveniles (36/104, 35%) than in adults (15/98, 15%) (p ϭ 0.003, 2 test). the five samples with ͼ10 6 copies were all from juveniles, four of them from seronegative animals. the prevalence of pcr-positive dc ranged from 66% in taif in the west to 0% in gizan in the southwest. pcr analysis of a random selection of serum and whole blood samples collected from nasal or rectal swab pcr-positive, seropositive, and seronegative dc revealed no evidence of viremia (see table s1 in the supplemental material). these included 13 adults and 29 juveniles phlebotomized in 2009, 15 adults and 14 juveniles phlebotomized in 2010, and 8 adults and 13 juveniles phlebotomized in 2013. these animals included the five juveniles with the highest viral genome sequence load in nasal swabs. serum samples collected from goats (n ϭ 36) and sheep (n ϭ 112) in 2013 in the central region (unizah, riyadh) were not immunoreactive with mers-cov but were immunoreactive with bo-cov (25% of goats, n ϭ 36; 54% of sheep, n ϭ 24). nasal swabs from 36 goats and 78 sheep were negative in rt-qpcr assays for mers-cov upe. to test the validity of rt-qpcr results and determine phylogenetic relationships of viral sequences found in dc in the ksa to previously reported sequences, we amplified and sequenced longer regions of the spike, orf1ab, and nucleocapsid genes from rt-qpcr-positive samples (for the sequences of the primers used, see table s2 in the supplemental material). eleven of 13 swab samples with ͼ10 5 copies in upe rt-qpcr yielded products for sequencing. no suitable products were obtained from samples with lighter viral sequence loads (ͻ10 4 copies) (see table s1 in the supplemental material). phylogenetic analysis of a 1,044nucleotide (nt) region of the spike gene and a 2,004-nt region of the orf1ab gene indicated ͻ1% divergence from previously published mers-cov sequences (fig. 3 ) (genbank accession no. kj396756 to kj396771). the nucleocapsid sequence was identical to the previously reported mers-cov sequences. mers-cov is posited to be a zoonosis. however, the evolutionary history of mers-cov and the reservoirs and vectors for human infection remain obscure. early anecdotal reports that some mers-cov victims had exposure to dc led to serologic investigation of dc in spain and oman (14) , jordan (12), egypt (13) , and the ksa (15) that revealed antibodies to mers-cov. definitive evidence that dc can be infected with mers-cov was obtained when viral sequences were detected in nasal swabs from dc sampled in close proximity to outbreaks of the disease among humans in qatar (11) and jeddah, ksa (10) . nonetheless, as noted by nishiura and colleagues, current data do not fulfill the two criteria required to implicate dc as a significant reservoir species in the epidemiology of mers-cov (19) , i.e., (i) that dc are sufficient to maintain mers-cov and (ii) that the presence of dc is essential to the continuous transmission of infection. results presented here do not establish the latter; however, they do provide evidence for the former. our study is the first comprehensive countrywide survey of dc from the ksa, the country with the most recorded mers-cov cases, to use both serological and molecular diagnostic methods. analysis of specimens from western regions of the country, from tabuk in the northwest, taif in the west, and gizan in the southwest, revealed regional differences. although the seroprevalence was high in adults throughout the country at ͼ80%, in juveniles, it ranged from 90% in the east to 5% in the southwest. the sero-prevalence in dc յ2 years of age was lower than that in older animals, confirming the results of hemida et al. (15) . molecular analysis of nasal and rectal swab specimens indicated the highest prevalence of mers-cov sequences in dc in the west and northwest. nasal swabs with heavy sequence loads (ͼ10 5 copies) also clustered in the taif region. a second sample collection in the west (taif) separated from the first by an interval of 2 months confirmed the presence of heavy viral sequence loads in nasal swabs collected from juvenile animals sampled in this area (data not shown). these findings suggest that continuous, longer-term surveillance is necessary to determine the dynamics of virus circulation in dc populations. lower prevalence rates of both mers-cov and bo-cov were evident in samples from the southwest. this may relate in part to the enforcement of restrictions of livestock movement in and out of gizan province implemented after the rift valley fever outbreak in 2000 but also to the generally lower dc population density in this region than in other regions of the ksa. viral nucleic acids were more commonly detected in nasal swabs than in rectal specimens and were more frequent in juvenile than in adult animals. these findings, together with the absence of viremia and the known respiratory tract tropism of several other coronaviruses, suggest that airborne transmission is the most likely mode of mers-cov transmission. although nucleic acid copy numbers were commonly highest in juvenile animals that were seronegative or had low antibody titers, positive findings were also obtained with specimens from highly seropositive and adult animals. our findings in archived dc specimens, although restricted to serology, strongly suggest that mers-cov or a closely related virus has been circulating in dc in the ksa for at least 2 decades. complete genomic sequences of mers-cov found in contemporary dc in the ksa are identical to sequences of viruses recovered from human mers-cov victims (unpublished data). although we speculate that dc are potential reservoirs for human transmission, we cannot prove this relationship from the current data. rigorous epidemiological investigation of the potential for exposure to dc in sporadic cases of mers-cov (those where there is no opportunity for human-to-human transmission) is required to test this model. if dc can be implicated, other questions will arise. did mers-cov truly emerge as a human pathogen in 2012, or were cases of cryptic infection not appreciated because of a lack of suitable diagnostic tests? we may be able to address this conundrum by using archived human materials. if evidence of human mers-cov infections cannot be detected prior to 2012, we must entertain the possibility that mutation facilitated cross-species transmission. however, we see no path to address this possibility absent access to historical dc respiratory tract specimens. the only archived dc specimens we have been able to locate are dc sera; our efforts to recover mers-cov sequences from camel blood have been unsuccessful. what are the roles of bats, if any, as reservoirs of mers-cov? these limitations notwithstanding, the most urgent public health concern, raised in work we and others have reported that focuses on dc infection, is to determine the role of these animals in sporadic human infection. the evidence is clearly sufficient to support targeted investigation of direct or indirect exposure to dc in the disease among humans. sample collection. samples included dc, sheep, and goat sera; whole blood and nasal and rectal swabs freshly collected in 2013; and archived serum samples from 1992, 1993, 1994, 1996, 2004, 2009, and 2010 . two rectal and two nasal swabs were obtained from each animal. one rectal swab and one nasal swab were placed into rnalater (life technologies, carlsbad, ca), and one rectal swab and one nasal swab were placed into viral transport medium (becton dickinson, franklin lakes, nj). all were stored at ϫ80ºc. after 24 h in fixative, plates were rinsed three times with phosphate-buffered saline (pbs) and placed in pbs for storage at 4°c. plates were loaded with infected and noninfected cells in alternating rows to generate a differential reading for each serum tested. positivity was defined as an infected-cell optical density of ͼ0.6 and ͼ3ϫ the noninfected-cell optical density. test sera were diluted 1:3,000 in pbs-0.05% tween 20 -1% bovine serum albumin; secondary antibodies were rabbit anti-goat igg (hϩl)-horseradish peroxidase conjugate (1:3,000; bio-rad, hercules, ca), rabbit anti-sheep igg (hϩl)-horseradish peroxidase conjugate (1:3,000; bio-rad), and anti-llama igg-horseradish peroxidase conjugate (1:10,000; bethyl laboratories, montgomery, tx). western blot. extracts of noninfected vero cells or vero cells infected with mers-cov strain emc were generated at rocky mountain laboratories, loaded onto discontinuous 3 and 7.5% sds gels (bio-rad), and transferred onto nitrocellulose with iblot transfer stacks (invitrogen iblot; life technologies). lanes were loaded with alternating infected and noninfected extract samples, and a pair were cut for incubation with dc sera (1:800 in blocking solution) after blocking of the membrane in pbs-0.05% tween 20 -5% dry milk blocking solution for 1 h. membranes were washed three times with pbs-0.05% tween 20 after a 2-h incubation with serum and then incubated for another 1.5 h with secondary antibody (1:7,000 in blocking solution; anti-llama igg-horseradish peroxidase conjugate; bethyl laboratories). following three more washes, the membranes were developed with westernsure premium chemiluminescent substrate (li-cor, lincoln, ne) and read on a c-digit blot scanner (li-cor). lips assay. the nucleocapsid proteins of bo-cov and mers-cov were pcr amplified with primers introducing appropriate restriction sites for cloning into vector pren-2 fused to the c terminus of the renilla luciferase reporter (20) . sequence-confirmed construct dna was purified from escherichia coli cultures (qiagen, hilden, germany), and transfected into cos-1 cells (african green monkey kidney, atcc crl-1650; 1 g; lipofectamine; invitrogen, life technologies). cells were harvested at 48 h posttransfection in lysis buffer (50 mm tris [ph 7.5], 100 mm nacl, 5 mm mgcl 2 , 1% triton x-100, 50% glycerol, protease inhibitors), and the protein extract was clarified by two rounds of centrifugation at 12,000 ϫ g. the target concentration was determined in relative light units (rlu), and approximately 10 ϫ 10 6 rlu were incubated with test serum (1:100) in a final volume of 100 l buffer a (50 mm tris [ph 7.7], 100 mm nacl, 5 mm mgcl 2 , 1% triton x-100) per well in 96-well plates (catalog no. 249944; thermo/nunc, waltham, ma). after 1 h of incubation at room temperature, protein a/g beads (catalog no. 53135; pierce, junction city, or) were added and the mixtures were transferred to 96-well filter plates (catalog no. msbvn1b50; millipore, billerica, ma) for another hour of incubation at room temperature with shaking. bead-bound anti-gen was washed eight times with buffer a and three times with pbs (tecan hydroflex, maennedorf, switzerland) and then read with coelenterazine substrate (renilla luciferase assay system; promega, madison, wi) on a centro lb960 luminometer (berthold, bad wildbad, germany). nucleic acid extraction and pcr. total nucleic acids were extracted from nasal swabs, serum, and whole blood on a qiacube with cador reagent kits (qiagen) or rneasy reagent kits for extraction of rna from rectal swabs. real-time qpcr used a onestep real-time qpcr buffer (invitrogen, life technologies) and primer/probes upe and orf1a (16, 17) . products for sequencing were generated by rt-pcr. cdna was reverse transcribed with superscript iii and random hexamer primers. pcr was performed with amplitaq gold (life technologies) and primers designed to amplify a 1,044-nt region of the spike gene (heminested pcr), a 913-nt region of the n gene (nested pcr) or a 2,004-nt region of the orf1ab region (heminested pcr). for the sequences of the primers used, see table s2 in the supplemental material. products were purified by agarose gel electrophoresis and with qiaquick gel extraction kits (qiagen) and subsequently sequenced on both strands by the dideoxynucleotide chain termination method (genewiz, south plainfield, nj). supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi:10.1128/mbio.00884-14/-/dcsupplemental. middle east respiratory syndrome coronavirus (mers-cov)-update. world health organization isolation of a novel coronavirus from a man with pneumonia in saudi arabia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus in bats, saudi arabia mers-cov-eastern mediterranean (85): animal reservoir, camel, suspected, official. promed-mail middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses missing information in animal surveillance of mers-cov antibody-profiling technologies for studying humoral responses to infectious agents we are grateful to his highness, prince bandar bin saud al saud, president, saudi wildlife authority, for his unlimited support and encouragement; to devon welsh and kawthar muhammad for mobile laboratory coordination; to andrew schultz for statistical analysis; to ellie kahn for editorial contributions; to aaloki shah, parisa zolfaghari, mia r. key: cord-319241-div9rzax authors: singh, bhuchitra; gornet, megan; sims, holly; kisanga, edwina; knight, zachary; segars, james title: severe acute respiratory syndrome‐corona virus‐2 (sars‐cov‐2) and its effect on gametogenesis and early pregnancy date: 2020-09-23 journal: am j reprod immunol doi: 10.1111/aji.13351 sha: doc_id: 319241 cord_uid: div9rzax sars‐cov‐2 infection and pregnancy has been the topic of hundreds of publications over the last several months, however, few studies have focused on the implications of infection in early pregnancy and reproductive tissues. here we analyzed available evidence pertaining to sars‐cov‐2 infection, early pregnancy, and reproductive tissues. we searched pubmed and embase databases in accordance with guidelines of preferred reporting items for systematic reviews and meta‐analyses (prisma) for publications from inception to june 4, 2020. four reviewers screened titles and abstracts, and obtained full text articles for analysis. 62 studies were included in the review. biological plausibility for infection with sars‐cov‐2 exists in testis, ovaries, and placenta as they express ace2 receptor activity. in males, sars‐cov‐2 infection could lead to functional abnormalities leading to spermatogenic failure and male infertility. in females, an alteration of the ace2 cascade via sars‐cov‐2 infection could lead to impairment in important follicular and luteal processes. there is also evidence of significant placental pathology in sars‐cov‐2 infection, but it is unclear what effects there may be for early pregnancy, though available data suggest less severe effects compared to other respiratory virus outbreaks. further investigation is needed regarding sars‐cov‐2 in reproductive function and early pregnancy. pregnant women and their fetuses may be impacted disproportionally by emerging infections [1] as exemplified by the 2009 h1n1 influenza pandemic [2] and the severe fetal complications of zika virus [3, 4] . the novel coronavirus infection, severe acute respiratory coronavirus 2 (sars-cov-2), this article is protected by copyright. all rights reserved was first reported in wuhan, china, on dec 31, 2019 and was declared a pandemic by the who on march 11, 2020 [5] . since then, the number of worldwide reported cases of covid-19 has increased to 6.11 million and 370,000 deaths as of may 31, 2020. [6] to combat the rapidly spreading virus, nations have implemented strategies to suppress and mitigate the community acquired infections such as social distancing, availability of rapid testing, contact tracing, and vaccine development [7, 8] . coronaviruses are single-stranded rna, enveloped, non-segmented viruses which cause complications ranging from common cold to pneumonia and death [9] . common symptoms of sars-cov-2 infection include fever, cough, myalgia, headache, and diarrhea [10] . highly infective, the virus is transmitted to 2-3 people for every infected person, with a reproduction number (r0) of 2.2 [11] . while an estimated 80% of covid-19 cases are mild and can be managed with home-care, 15-20% require hospitalization, with 5% of cases severe enough to require mechanical ventilation [6] . the total mortality rate, including those from asymptomatic and mild cases, seems to be approximately 1% [12] . the primary mode of transmission occurs similarly to that of other respiratory infections; droplet particles are inhaled through close physical contact with an infected person when coughing or sneezing [13] . it does not appear that vertical transmission from pregnant women to fetuses occurs [14] , however, this has not been determined conclusively. other coronaviruses of the previous two decades, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), have caused severe respiratory infections in humans. the novel sars-cov-2 shares 82% of the genome of the virus that causes sars-cov [15] . since its first identification, more cases of covid-19 have been counted than that of mers and sars combined. limited studies on sars-cov-1 reported increased morbidity and mortality in pregnant women, while some recent studies have shown women with sars-cov-2 are less likely to have complications comparatively [16, 17] . however, very little data are available regarding the effects of coronaviruses in early pregnancy. much of the available data focuses solely on covid-19 infections in the third trimester and the possibility of vertical transmission. though there is a lack of concrete data, sars-cov-2 does have the potential to cause pregnancy complications in the first trimester including miscarriage and this article is protected by copyright. all rights reserved congenital abnormalities [18] . in this paper, we aimed to summarize the available data related to the effects of sars-cov-2 infection on the ovaries, testicles, gametes, and early pregnancy. in addition to current sars-cov-2 literature, we also investigate the potential effects of covid-19 in early pregnancy using published data and outcomes from the sars-cov and mers-cov pandemics. a systematic search in the literature published in the pubmed and embase databases was conducted in accordance with the guidelines of the preferred reporting items for systematic reviews and meta-analyses (prisma). additional citations were identified from the reviewed literature. an academic data specialist developed the search strategy and searched the databases of pubmed and embase from inception to june 4, 2020. we searched for articles that contained information related to sars-cov-2 and reproductive tissues (ovaries, testes), gametes, placentation, and early pregnancy in humans. our search phrases included: "severe acute respiratory syndrome coronavirus 2", "2019 ncov", "sarscov 2", "sars-cov-2", "pregnancy", "gravidity", "abortion", "germ cells", "oocytes", "gametes", "embryonic structures", "embryo", "fertility", "testes", "miscarriage"(see appendix 1 for completed list of databases search strategy and figure 1 for prisma table). due to limited number of publications available we did not consider a sample size as an eligibility criterion. majority of the literature composed of case reports, guidelines, and editorials. we did not use the cochrane rob 2.0 tool due to absence of randomized clinical trials. similarly, due to lack of cohort studies, we did not use the newcastle-ottawa scale to rate the studies. using a standardized form, 4 reviewers (hs, mg, ek, and bs) screened titles, abstracts, and full-text articles reporting potentially eligible studies. any disagreements were resolved with a third reviewer (j.s.). we acknowledge the possibility of bias in this literature review due to inclusion of the limited research data available. sars-cov-2 related reporting is increasing exponentially and this review is limited to the date of the literature search was conducted. the search strategy described above identified 2,828 articles, of which 131 were considered for full review. 62 articles met the inclusion criteria and were used to support this review. table 1 summarizes case series and studies of sars-cov-2 infection within various reproductive tissues in patients who tested positive for sars-cov-2. spermatogonia, sertoli, and leydig cells are predominantly enriched in ace2 (19, 20) . sars-cov-2 and mers-cov use ace2 receptors to gain entry into host cells [21] . wang et al. [19] performed uniform manifold approximation and projection (umap) and marker gene analyses to identify nine major cell cluster in testicular cells. ace2 expression was determined by analyzing the rna expression profile of ace2 at single-cell resolution. spermatogonia and spermatids showed concentrated expression of transmembrane serine protease 2 (tmprss2) for viral spike (s) protein priming. both zhao and wang [19, 20] also reported almost 3-fold higher percentage expression of ace2 in leydig and sertoli cells compared to spermatogonia (4.25% vs. 1.40%). sars-cov-2 uses tmprss2 and sars-cov-2 ace2 receptors to gain entry into host cells [22] . therefore, there is a high potential for sars-cov-2 infection in human testes. in situ hybridization, morphological, and immunohistochemical analysis were performed on 6 testis obtained at autopsy from confirmed sars-cov-2 infected males and from 4 non-sars autopsy control testis samples [23] . all control testis had normal morphology, whereas the sars-cov-2 infected testis showed peritubular fibrosis and vascular congestion along with extensive germ cell destruction. there were significantly more cd 3 + t lymphocytes and cd68+ macrophages (0.65% and 2.11% in control vs. 4.49% and 11.72%) infected. however, the virus did not affect the testis directly since in situ hybridization, using both sense and antisense rna probes, showed no staining [23] . a cohort study by li enrolled 38 sars-cov-2 patients for semen testing using rt-pcr and found 6 patients had positive results for sars-cov-2 (4 in acute stage of the disease and 2 in recovery. however, no information is known about virus shedding, survival time, and concentration in semen [24] . a cross-sectional observational study by pan et al. identified presence of sars-cov-2 by qrt-pcr in a single ejaculated sample of 34 confirmed cases of sars-cov-2 in china. sars-cov-2 was not detected in the semen 1 month after diagnosis. they also reported sparse expression of ace2 and tmprss 2 using single-cell transcriptome analysis, with almost no overlapping gene expression [25] . this article is protected by copyright. all rights reserved both jung and carlsen reported that male fertility may be diminished due to fever associated with the sars-cov-2 infection for up to 72-90 days following infection as fever can lead to decreased sperm concentration and motility [26, 27] . in addition, sars-cov-2 patients may develop cytokines storm syndrome (hemophagocytic lymphohistiocytosis), which can impact testicular function and maintenance, as cytokines microenvironment is critical to testicular function, and alterations may be tumorigenic [28, 29] . as stated earlier, sars-cov-2 uses tmprss2 and sars-cov-2 ace2 receptor to gain entry into host cells [21] . both reproductive-age women and post-menopausal women have ace2 mrna transcripts expressed abundantly in the ovary [30] . therefore, the female reproductive system is potentially at a high risk of sars-cov-2 infection. there are no studies that evaluate sars-cov-2 infection inhuman ovarian tissue and cells. the ace2 receptor is widely expressed in the placenta [31] . overall, there is limited evidence regarding sars-cov-2 and transplacental transmission, though most reviews and the american college of obstetrics and gynecology have stated there is no conclusive evidence of transplacental transfer of sars-cov-2 from infected mothers [32, 33] . comparable to other more well-known transplacental infections, which are known to have hematogenous infectivity, sars-cov-2 has been shown to produce rnaemia, and therefore plausibly could be transmitted via a transplacental route [34] . at present, the literature regarding vertical transmission is mixed; several recent studies have concluded there is no definitive evidence of vertical transmission [35, 36, 37] , while others have findings that suggest in utero transmission may occur, as evidenced by presence of neonatal igm antibodies (which cannot otherwise be transferred transplacentally) [38, 39, 40] and neonatal nasopharyngeal and anal sars cov-2 rna isolation [41] . while this evidence of transplacental transmission is compelling, dedicated study of placental histopathology and pcr as it relates to sars-cov-2 is needed to better distinguish transplacental versus intrapartum versus postpartum vertical transmission. this article is protected by copyright. all rights reserved our review identified two dedicated studies of placental histopathology in sars-cov-2. in one study investigating placentas of sars-cov-2 positive mothers, 45% showed low grade fetal vascular malperfusion, though notably all infants tested negative [42] . interestingly, shanes et al. [43] noted higher rates of arteriopathy and maternal vascular malperfusion than controls, whereas findings of fetal malperfusion was nonsignificant compared to controls. both studies noted significant increase in intervillous thrombi, which may be a direct correlate to the thromboembolic disorders believed to occur in sars-cov-2. data regarding placental viral isolates in specifically sars-cov-2 is scarce, and with mixed results. to date, there are few published reports of presence of sars-cov-2 rna in placenta or membrane samples [44, 45] . in the study by penfield et al. [44] 3 of 11 placental or membrane swabs were positive within 30 minutes of delivery in women with severe to critical covid-19 at time of delivery. notably, there were no signs of vertical transmission, and all infants were negative, raising the distinct possibility of contamination. a study by patane et al. [45] is the first to specifically describe sars-cov-2 rna on the fetal side of the placenta. in two other published reports, viral pcr was performed on placental tissue and/or all products of conception in mothers diagnosed with symptomatic sars-cov-2 infection following third trimester delivery. all samples were negative [36, 46] . to date, there appears to be a single study directed at investigating the intrauterine environment of early pregnancy and sars-cov-2 with several limitations [47] . specifically, the study retrospectively reviewed records and labs results for two symptomatic women with covid-19 in the first trimester. the first woman, at 8 weeks gestation, was given a clinical diagnosis given husband's diagnosis at home as well as radiologic findings. the second woman was diagnosed at 10 weeks gestation via nasopharyngeal test. on the day of amniocentesis, both patients were negative for sars-cov-2 rna via throat swabs, but positive for sars-cov-2 igg antibodies in serum (the first woman also had igm antibodies). sars-cov-2 rna was not isolated in the amniotic fluid, though notably amniotic samples were collected over a month after symptom resolution and testing positive. however, according to center for disease control, it is anticipated that sars-cov-2 infection will affect fetal development like other respiratory coronaviruses. this article is protected by copyright. all rights reserved the status of sars cov-2 within the vaginal environment is currently unknown. many viruses have been previously isolated within vaginal fluid, such as hepatitis c virus and zika virus, which are both rna viruses. at present, it is not believed that sars-cov-2 is able to be sexually transmitted, which is consistent with other historical coronaviruses. however, inability for sexual transmission does not preclude sars-cov-2 from having a significant clinical impact. to our knowledge, there is one study investigating the presence of sars-cov-2 in the vaginal fluid of women with severe sars-cov-2. other studies that tested vaginal swabs were tested in the peripartum period, and were negative [36, 46] . specifically, 10 women with severe covid-19 were tested or sars-cov-2 in vaginal fluid, with all samples negative for virus [48] . notably, these women were postmenopausal and swabs were taken 17 or more days after onset of disease. animal studies have demonstrated that ace2 is present within murine, leporine, bovine, and equine ovarian tissue (including but not limited to stromal cells, granulosa cells, and oocytes). furthermore, in these studies, ace2 is known to be an essential protein involved in multiple cascades responsible for steroid secretion [49, 50] , oocyte maturation and ovulation, and resumption of meiosis in the accepted article oocyte [51, 52] through its interplay with ang ii and ang-(1-7). most importantly, in regards to early pregnancy, ang-ii and ang-i (and by extension ace2) have been shown to be key proteins involved in formation, maintenance, and regression of corpus luteum [53] . alterations in this cascade of proteins responsible for normal development of the corpus luteum may explain how sars-cov-2 can negatively impact early pregnancy. in the human ovary, there is evidence that folliculogenesis, oocyte maturation, and ovulation involve the renin-angiotensin aldosterone (ras) system, an important component which involves ace2 enzyme expression [49] . therefore, sars-cov-2 could theoretically damage ovarian tissue, and decrease ovarian function and oocyte quality, resulting in female infertility or even miscarriage. the role of ace2 with ang-ii and ang(1-7) , and their demonstrated importance in the formation and maintenance of corpus luteum, could explain how alterations in ace2 may negatively impact early pregnancy [24, 53] . specifically, the impact of sars-cov-2 may be two-fold: the effects of sars-cov-2 viral infection and local immune reaction itself, as well as the downstream consequences of altering the essential ace2 and ace2 receptor mechanism. specifically, as the ace2 and ace2 receptor cascades are involved in multiple basic ovarian processes, it may be reasonable to infer that their normal function and availability could be altered due to sars-cov-2 use for entry. notably, placental pathology in the setting of respiratory illness is difficult to interpret, as protracted this article is protected by copyright. all rights reserved the effect of sars-cov-2 on early pregnancy is still unknown, and any potential effects are extrapolations from cell-level plausibility and experiences from other recent viral respiratory syndromes, such as sars and mers. very limited data may suggest that there are higher miscarriage rates with sars and mers versus covid-19 [35] . fluid were all negative [55] . while the relationship between sars-cov, sar-cov-2, and third trimester placental tissues has been investigated, it is unclear what the implications may be for infection of early placental tissue and early pregnancy, and furthermore the specific consequences related to gestational age at time of infection. one study investigating ace2 expression in human placental tissue, which is a known main protein for sars-cov and sars-cov-2 viral transmission [31] , showed differential expression of ace2 based on gestational age. for example, the expression of ace2 was at very low levels in placental trophoblastic cells in the first trimester, with increased expression at 24 weeks gestational age. this lower level ace2 expression in the first trimester could suggest a decreased vulnerability to transplacental infection in early pregnancy. in comparison to zika virus (zikv), which is known to be transmitted transplacentally and cause significant adverse pregnancy outcomes, axl (an essential zikv entry cofactor) is more concentrated in the early maternal-fetal interface. this may explain how zikv is more readily transmitted in early pregnancy [56] . interestingly, although sars-cov-2 and sars-cov seem to share this same pathogenic receptor, and additionally have up to 85% sequence similarity [57, 58] , by all published reports the effect on early pregnancy appears very different between the two infections. while a study by ng. et al. [54] described normal placental pathology findings in women convalescent from sars-cov who had spontaneous miscarriage in the first trimester, the miscarriage rate overall with sars-cov infection appears more severe. specifically, data regarding miscarriage rate in sars and mers outbreaks may suggest a more severe effect on early pregnancy physiology, e.g. higher spontaneous pregnancy loss, than in sars-cov-2 infection. one study demonstrated a 2% miscarriage/stillbirth rate for sars-cov-2 versus 18% and this article is protected by copyright. all rights reserved 25% for mers-cov and sars-cov, respectively [35] . another study performed during the 2002-2003 sars pandemic showed that 4 of 7 (57%) pregnant women infected with sars-cov had a spontaneous miscarriage in the first trimester of pregnancy [55] , though notably no viral inclusion bodies or particles were detected in the products of conception. additional biochemical studies do not aid in further clinical understanding, as biophysical and structural evidence show that sars-cov-2 actually binds ace2 with higher affinity than sars-cov, and therefore could plausibly transmit to placenta to a greater degree than sars-cov [59] . this does not appear to be consistent with the better outcomes seen with sars-cov-2 infection and pregnancy, and it is clear that further investigation is needed. notably, only transient positive results in amniocentesis have been reported for other rna virus infections (zika), and these patients were outside of the standard gestational age range for traditional amniocentesis. in the event it is discovered that sars-cov-2 may be isolated in vaginal secretions, the implications, though unknown, could represent a mechanism for ascending infection. interestingly, an animal study investigating rat coronavirus (rcv) found that in addition to infecting respiratory epithelium, it also infects the genital tract of females, reportedly causing perturbations of the hormonal cycle and miscarriage [60, 61] . isolation of virus within vaginal fluid could also theoretically represent a disruption of a microbiome or "virome", of which the consequences of disruption may be reproductive failure, predisposition to other infection, and pregnancy complications. one study showed that higher viral diversity in the vagina was significantly associated with preterm birth [62] . notably, viruses isolated in this study included adenoviruses, which are an important cause of respiratory illness globally. as such, it is important to understand the relationship of sars-cov-2 within vaginal fluid, as understanding implications of sars-cov-2 in early pregnancy may alter counseling, work up, and approach to pre-pregnancy counseling and intervention. the covid-19 outbreak continues to increase in the number of cases, deaths, and countries affected. current data regarding reproductive tissues and early pregnancy in sars-cov-2 infection this article is protected by copyright. all rights reserved are limited. this may be a result of restricted capabilities of research institutions in time of pandemic, decreased patient interaction within hospitals and institutions due to social reasons and policy, and absolute gestational age relative to time and testing abilities within the pandemic, as many women are just now completing their first trimester. in light of the currently available evidence, specific recommendations beyond standard personal protective equipment, social distancing accommodations, and hygienic practices can be made. specifically, it can be suggested that clinicians practicing assisted reproductive technology should be particularly cautious, as sars cov-2 infection and systemic effects may impact testicular tissues, ovarian tissue, and granulosa cells and therefore testicular and ovarian function, spermatozoa, oocyte quality, and pregnancy outcomes [24] . as standard reproductive health, pre-pregnancy, and pregnancy patient care resumes, surveillance qiu et al. [56] yes no ten non-pregnant women swabbed liu et al. [53] yes no three vaginal swabs tested (peripartum). fan et al. [41] yes no two vaginal swabs tested (peripartum) this article is protected by copyright. all rights reserved accepted article public health approach to emerging infections among pregnant women pandemic 2009 influenza a (h1n1) virus illness among pregnant women in the united states characterizing the pattern of anomalies in congenital zika syndrome for pediatric clinicians zika vrus and birth defectsreviewing the evidence for causality who declares covid-19 a pandemic world health organization accessed 31 the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus genetic recombination, and pathogenesis of coronaviruses pregnancy : what obstetricians need to know early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia report 4: severity of 2019-novel coronavirus (ncov) who collaborating centre 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and neonates at birth accepted article this article is protected by copyright. all rights reserved coronavirus disease 2019 (covid-19) during pregnancy: a case series no sars-cov-2 detected in amniotic fluid in midpregnancy sars-cov-2 is not detectable in the vaginal fluid of women with severe covid-19 infection angiotensin-(1-7), its receptor mas, and the angiotensin-converting enzyme type 2 are expressed in the human ovary in vitro development of pig preantral follicles cultured in a serum-free medium and the effect of angiotensin ii angiotensin ii signaling promotes follicle growth and dominance in cattle angiotensin-(1-7) induces ovulation and steroidogenesis in perfused rabbit ovaries angiogenesis in the human corpus luteum: changes in expression of angiopoietins in the corpus luteum throughout the menstrual cycle and in early pregnancy the placentas of patients with severe acute respiratory syndrome: a pathophysiological evaluation pregnancy and perinatal outcomes of women with severe acute respiratory syndrome single-cell rna expression profiling of ace2 and axl in the human maternal-fetal interface all rights reserved 57. zhang yz. novel 2019 coronavirus genome genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan cryo-em structure of the 2019-ncov spike in the prefusion conformation encyclopedia of virology reproductive abnormalities associated with a coronavirus infection in rats the vaginal eukaryotic dna virome and preterm birth ncov' or 'sars cov 2' or 'corona virus'/exp or 'corona virus' or (corona and ('virus'/exp or virus)) or 'coronavirus'/exp or coronavirus or 'covid 19' or covid19) and (pregnant or 'pregnancy'/exp or pregnancy or 'live birth'/exp or 'live birth' or (live and ('birth'/exp or birth)) or 'miscarriage'/exp or miscarriage or 'abortion'/exp or abortion or 'birth'/exp or birth or 'ivf'/exp or ivf or iui or 'sex'/exp or sex or 'fertility'/exp or fertility or 'infertility'/exp or infertility or 'gamete'/exp or gamete or 'oocyte'/exp or oocyte key: cord-334313-v2syspu6 authors: long, s. wesley; olsen, randall j.; christensen, paul a.; bernard, david w.; davis, james r.; shukla, maulik; nguyen, marcus; ojeda saavedra, matthew; cantu, concepcion c.; yerramilli, prasanti; pruitt, layne; subedi, sishir; hendrickson, heather; eskandari, ghazaleh; kumaraswami, muthiah; mclellan, jason s.; musser, james m. title: molecular architecture of early dissemination and evolution of the sars-cov-2 virus in metropolitan houston, texas date: 2020-05-03 journal: biorxiv doi: 10.1101/2020.05.01.072652 sha: doc_id: 334313 cord_uid: v2syspu6 we sequenced the genomes of 320 sars-cov-2 strains from covid-19 patients in metropolitan houston, texas, an ethnically diverse region with seven million residents. these genomes were from the viruses causing infections in the earliest recognized phase of the pandemic affecting houston. substantial viral genomic diversity was identified, which we interpret to mean that the virus was introduced into houston many times independently by individuals who had traveled from different parts of the country and the world. the majority of viruses are apparent progeny of strains derived from europe and asia. we found no significant evidence of more virulent viral types, stressing the linkage between severe disease, underlying medical conditions, and perhaps host genetics. we discovered a signal of selection acting on the spike protein, the primary target of massive vaccine efforts worldwide. the data provide a critical resource for assessing virus evolution, the origin of new outbreaks, and the effect of host immune response. significance covid-19, the disease caused by the sars-cov-2 virus, is a global pandemic. to better understand the first phase of virus spread in metropolitan houston, texas, we sequenced the genomes of 320 sars-cov-2 strains recovered from covid-19 patients early in the houston viral arc. we identified no evidence that a particular strain or its progeny causes more severe disease, underscoring the connection between severe disease, underlying health conditions, and host genetics. some amino acid replacements in the spike protein suggest positive immune selection is at work in shaping variation in this protein. our analysis traces the early molecular architecture of sars-cov-2 in houston, and will help us to understand the origin and trajectory of future infection spikes. we sequenced the genomes of 320 sars-cov-2 strains from covid-19 patients in metropolitan houston, texas, an ethnically diverse region with seven million residents. these genomes were from the viruses causing infections in the earliest recognized phase of the pandemic affecting houston. substantial viral genomic diversity was identified, which we interpret to mean that the virus was introduced into houston many times independently by individuals who had traveled from different parts of the country and the world. the majority of viruses are apparent progeny of strains derived from europe and asia. we found no significant evidence of more virulent viral types, stressing the linkage between severe disease, underlying medical conditions, and perhaps host genetics. we discovered a signal of selection acting on the spike protein, the primary target of massive vaccine efforts worldwide. the data provide a critical resource for assessing virus evolution, the origin of new outbreaks, and the effect of host immune response. [keywords] sars-cov-2, covid-19, genome sequencing, remdesivir, molecular evolution significance covid-19, the disease caused by the sars-cov-2 virus, is a global pandemic. to better understand the first phase of virus spread in metropolitan houston, texas, we sequenced the genomes of 320 sars-cov-2 strains recovered from covid-19 patients early in the houston viral arc. we identified no evidence that a particular strain or its progeny causes more severe disease, underscoring the connection between severe disease, underlying health conditions, and host genetics. some amino acid replacements in the spike protein suggest positive immune selection is at work in shaping variation in this protein. our analysis traces the early molecular architecture of sars-cov-2 in houston, and will help us to understand the origin and trajectory of future infection spikes. [introduction] pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus is now responsible for massive human morbidity and mortality worldwide (1-4). the virus was first documented to cause severe respiratory infections in wuhan, china, beginning in late december 2019 (5) (6) (7) . global dissemination occurred extremely rapidly, and has affected major population centers on many continents, especially in asia, europe, and north america (8) (9) (10) . in the united states, seattle and the new york city region have been especially important centers of covid-19 disease caused by sars-cov-2. similarly, in seattle and king county, 4,620 positive patients and 303 deaths have been reported as of april 15, 2020 (12) . the houston metropolitan area is the fourth largest and the most ethnically diverse city in the united states, with a population of approximately 7 million (13) . the 2,000-bed houston methodist health system has eight hospitals and serves a large multinational and socioeconomically diverse patient population throughout greater houston. although the city is well-known as the energy capital of the world, houston also has a very large port, and the george bush international airport is a major transportation hub for domestic and international flights to asia, europe, and central and south america. many of these flights provide direct city-to-city service to diverse countries, including global population centers. the first covid-19 case in metropolitan houston was reported on march 5, 2020 (14) . community spread was suspected of occurring one week later (14). many of the first cases in our region were associated with national or international travel, including areas known to have covid-19 virus outbreaks (14). these facts, coupled with the existence of a central molecular diagnostic laboratory that serves all houston methodist hospitals and our very early adoption of a molecular test for the sars-cov-2 virus, permitted us to rapidly interrogate genomic variation among strains causing infections in the greater houston area. we here report that sars-cov-2 was introduced to the houston metropolitan region many independent times from diverse geographic regions, including europe, asia, and south america. the virus spread rapidly and caused disease throughout the metropolitan region. we identified clear genomic signals of person-to-person transmission. some events were known or inferred based on conventional public health maneuvers, but many were not. in addition, spatialtemporal mapping found evidence of rapid and widespread community dissemination soon after covid-19 cases were reported in houston. analysis of the relationship between distinct genomic viral clades and hospitalization did not reveal significant evidence of more virulent genome types, underscoring the need for a better understanding of the relationship between severe disease, underlying medical conditions, gender, and host genetics. description of metropolitan houston. houston, texas, is located in the southwestern united states, 50 miles inland from the gulf of mexico. it is the most ethnically diverse city in the united states (15 viral genome sequencing and phylogenetic analysis. we sequenced the genomes of sars-cov-2 strains dating to the earliest stages of confirmed covid-19 disease in houston. phylogenetic analysis identified the presence of many diverse viral genomes that in the aggregate represent many of the major clades identified to date (16) (fig. 2) . clades a2a, b, and b1 were the three more abundantly represented phylogenetic groups (fig. 2 ). geospatial and time series. we examined the spatial and temporal mapping of the genome data to investigate community spread (fig. 3) . the figure illustrates evidence of widespread and rapid community dissemination soon after the initial covid-19 cases were reported in houston. there is also evidence to suggest there were multiple independent strains introduced into metropolitan houston, followed by local spread throughout all regions of the community. epidemiologically linked patients. we investigated the relationships among some of the genomes that were obtained from patients known to share common epidemiologic associations, such as living in the same household. in all instances, individuals known to be epidemiologically associated had identical or nearly-identical sars-cov-2 genomes, a finding consistent with person-toperson transmission of the virus. geospatial relationships of genetically similar isolates. we next tested the hypothesis that genetically related viruses were constrained to particular geographic areas of metropolitan houston. although in some instances this was the case, an important observation was that most of the individual related subclades were comprised of strains distributed over broad geographic areas (fig. 4) . these findings are consistent with the known propensity of respiratory virus sars-cov-2 to spread rapidly from person to person. patients with shared viral genomes likely constitute epidemiologic clusters, as a consequence of direct transmission to one another, shared transmission through an unknown third party, or via a reticulate network. patients, and other metadata. it is possible that sars-cov-2 genetic subtypes may have different clinical characteristics, analogous to what was thought to have occurred with the ebola virus (17) (18) (19) and is known for other infectious agents. as an initial examination of this issue in sars-cov-2, we tested the hypothesis that patients with disease severe enough to warrant hospitalization were infected with a non-random subset of virus genotypes. we also tested the hypothesis of non-random association between virus clades and disease severity based on in-patient versus out-patient status, the need for mechanical ventilation, and the number of days on a ventilator. we found no apparent simple relationship between viral clades and disease severity using these indicators of disease severity. similarly, there was no simple relationship between viral clades and other metadata, such as gender, age, or length of stay ( fig. s1-s7 ). machine learning analysis. machine learning models were built to predict mortality, length of stay, in-patient status, or icu admission based on the viral genome sequence. f1 scores (the evaluation metric used in classification algorithms) for most classifiers ranged between 0.5 to 0.6, indicative of classifiers that are performing similarly to random chance. outcome (lived versus died) was only weakly correlated with age in this data set (pcc = 0.318), and similarly regression models built to predict age and length of stay based on the viral genome sequence also had poor performance, with r 2 values near zero. classifiers were also trained to predict host characteristic, gender and ethnic group. the two largest ethnic groups in the patient population were african american and caucasian, as recorded in the electronic medical record. the binary classification model had an f1 score of 0.67 [0.51-0.83, 95% ci], likely reflecting social networks in early person-to-person transmission. a table of classifier accuracy scores and performance information is provided as table s1 . analysis of the nsp12 polymerase gene. the sars-cov-2 genome encodes an rna dependent rna polymerase (rdrp) used to replicate this rna virus. two amino acid substitutions (phe476leu and val553leu) in the nsp12 rdrp have been reported to confer significant resistance in vitro to remdesivir, an adenosine analog (20) . remdesivir is inserted into rna chains by rdrp during replication, resulting in premature termination and inhibition of virus. this compound has shown prophylactic and therapeutic benefit against mers-cov experimental infection in rhesus macaques (21) . a recent publication describing results from a compassionate use protocol reported that remdesivir may have therapeutic benefit in some hospitalized patients (22) . if efficacy is confirmed by a randomized controlled trial, this drug may be be widely used in large numbers of patients worldwide. to acquire baseline data about allelic variation in the gene encoding nsp12 rdrp, we analyzed the 320 sequenced viral genomes. the analysis identified 12 nonsynonymous single nucleotide polymorphisms (snps) in nsp12, resulting in 11 amino acid replacements throughout the protein ( table 1) . the most common amino acid change was pro322leu, identified in 224 of the 320 houston isolates. this amino acid replacement is present in genomes from the a2a clade, and distinguishes the a2a clade from other sars-cov-2 clades. the other amino acid changes in nsp12 were mainly present in single isolates from individual houston strains, and some have been identified in other strains in the global gisaid collection (23, 24) . a prominent exception was the identification of 29 strains with a met600ile polymorphism. all 29 strains were phylogenetically very closely related members of the a2a clade, and they all also had the p322l amino acid replacement characteristic of this clade (fig. s8) . these data indicate that the met600ile change is the derived state among strains with the pro322leu replacement. importantly, none of the observed amino acid polymorphisms in nsp12 were located precisely at the two sites associated with remdesivir in vitro resistance (20) , and 10 of the 11 polymorphic amino acids are located on the surface of this rdrp. however, importantly, the ala448val replacement occurs in an amino acid that is located directly above the nucleotide substrate binding site that is comprised of lys545, arg553, and arg555, as recently shown by structural studies (25, 26) (fig. 5) . the ala448 position is comparable to val553 (fig. 5) , and a val553leu mutation in sars-cov was identified to confer resistance to remdesivir (20) . coronavirus such as sars-cov and mers gain entry into susceptible host cells using a densely glycosylated viral-surface molecule knows as spike (s) protein. the s protein of sars-cov-2 virus and its close coronavirus relatives binds directly to the host angiotensin-converting enzyme 2 (ace2) to enter host cells. thus, s protein is a major translational research target, including small molecule inhibitors and extensive vaccine efforts globally (27, 28) . analysis of the gene encoding the s protein identified 41 snps, including 25 that produce amino acid changes (table 2, fig. 6a ). seven of these replacements (a263v, f456l, s967r, t1027i, m1050v, k1157m, and q1208h) are not represented in the gisaid database as of april 15, 2020. f456 makes contact with the human ace2 receptor, occupying a pocket formed by ace2 residues thr27, asp30 and lys31 ( fig. 6b ) (29) . the f456l substitution is expected to detrimentally affect binding to ace2, as the shorter leu side chain would not fill the pocket well. we note that seven of the amino acid replacements map to the periphery of the s1 subunit n-terminal domain (ntd). four of these replacements (a27s, t29i, g261v, and a263v) can be mapped to the recently determined cryo-em structure of the sars-cov-2 spike (27), whereas three other amino acid changes (l18f, t22i, and h69y) are located in flexible ntd loops that could not be modeled (fig. 6a) . the clustering of amino acid replacements to a distinct region of the protein, together with the occurrence of two different amino acid replacements occurring at the same residue is a strong signal of positive selection. inasmuch as infected patients make antibodies against the ntd region, we favor the idea that host immune selection is a key force contributing to amino acid variation in this region, resulting in an enhanced fitness phenotype of virus variants. also of note, the d614g replacement was observed in 70% (224 of 320 strains) of the strains sequenced in this study. residue d614 is located in subdomain 2 (sd-2) of the s protein, and it forms a hydrogen bond and electrostatic interaction with two residues in the s2 subunit of a neighboring protomer (fig. 6c) . replacement of aspartate with glycine would eliminate both interactions, thereby substantively weakening the contact between the s1 and s2 subunits. we speculate that this weakening produces a more fusogenic spike protein, because s1 must first dissociate from s2 before the s2 subunit can refold and mediate fusion of viral and cell membranes. stated another way, virus strains with the d614g variant may be better able to enter host cells, potentially resulting in enhanced spread. we discovered substantial genetic diversity of the sars-cov-2 viruses causing covid-19 disease in houston, texas. the majority of cases studied are progeny of strains that cause widespread disease in european and asian countries. we interpret these data as demonstrating that the sars-cov-2 virus was introduced into houston many times independently, likely by individuals who had traveled to different parts of the world. in support of this interpretation, the first cases in metropolitan houston were associated with a travel history to a known covid-19 region (14). the data are consistent with the fact that houston is a large international city characterized by a multi-ethnic population. the diversity present in our 320 viral genomes contrasts somewhat with data reported recently by gonzalez-reiche et al. (30) , who studied 84 sars-cov-2 isolates causing disease in patients in the new york city region. those investigators concluded that the vast majority of disease was caused by progeny of strains imported from europe. similarly, bedford et al. (31) reported that much of the covid-19 disease in the seattle, washington area was caused by strains that are progeny of a virus strain recently introduced from china. the viral diversity present in metropolitan houston permitted us to test the hypothesis that distinct viral clades were nonrandomly associated with hospitalized covid-19 patients or disease severity. although our sample size is relatively small, we found little evidence to support this hypothesis. clearly, this important matter warrants further study with a larger sample size, and this analysis is currently underway. we used machine learning classifiers in an attempt to identify snps that contribute to increased infection severity or otherwise affect virus-host outcome. the models could not be trained to accurately predict these outcomes from the viral genome sequence data, which may be due to the small sample size and class imbalance. as such, no particular snps were identified that are predictive of disease severity or infection outcome. classifiers were also trained to search for predictors of host characteristics such as age by decade, gender, and ethnic group. we found that the african american versus caucasian populations had a predictive signal, and hence potentially significant snps, which may be borne out with increased sampling in these two groups. however, examination of the geographic distribution of the viral isolates classified using this model largely coincided with the demographic distribution of ethnic groups in the houston metropolitan region. as such, the underlying snps found by the model may reflect social networks present early in the spread of sars-cov-2 in the houston metropolitan area, rather than distinct viral or human genetic factors. remdesivir is a nucleoside analog that has been reported to have activity against mers-cov, a coronavirus related to sars-cov-2 (21) . recently, grein et al. (22) reported that this drug shows promise in treating covid-19 patients in a relatively small compassionate use protocol. because in vitro resistance of sars-cov to remdesivir has been reported to be caused by either of two amino acid replacements in rdrp (phe476leu and val553leu), we interrogated our data for polymorphisms in the nsp12 gene. although we identified 11 different inferred amino acid replacements in the 320 genomes analyzed, none of these were located at the two positions associated with resistance in vitro. these findings suggest that if remdesivir proves to be efficacious in covid-19 patients, and is deployed widely as a treatment, the majority of sars-cov-2 strains currently circulating should be susceptible to this drug. however, the ala448val polymorphism we identified occurs at an amino acid site that intriguingly is located directly above the nucleotide substrate entry channel and nucleotide binding residues lys545, arg553, and arg555 (20, 25) (fig. 5) . one possibility is that substitution of the smaller alanine residue with the bulkier valine may impose structural constraints for the modified nucleotide analog to bind and thereby disfavor remdesivir binding. this in turn leads to reduced incorporation of remdesivir into the nascent rna, increased fidelity of rna synthesis, and thus drug resistance. a similar mechanism has been proposed for a v553l change (20) . we also identified one strain with a lys477asn replacement in nsp12. this substitution is located very close to a phe476leu replacement reported to produce partial resistance to remdesivir in vitro in sars-cov patients from 2004, although the amino acid positions are numbered differently in sars-cov and sars-cov-2. structural studies have suggested that this amino acid is surfaceexposed, and distant from known key functional elements. our observed lys477asn change is also located in a conserved motif described as a finger domain of rdrp (fig. 5) . one speculative possibility is that lys477 is involved in binding a yet unidentified cofactor such as nsp7 or nsp8, an interaction that could modify nucleotide binding and/or fidelity at a distance. these data warrant additional study in larger patient cohorts, especially individuals treated with remdesivir. analysis of the gene encoding the spike protein identified many new inferred amino acid replacements not present in available databases. these data, coupled with structural information available for s protein, raises the possibility that many of the amino acid variants have functional consequences. for example, clustering of several amino acid changes to the ntd suggests varying residues in this region bestow a fitness phenotype. regardless, we are now beginning to acquire critical information about the location and extent of amino acid replacements occurring in the s protein in natural populations of sars-cov-2. these data permitted generation of many biomedically relevant hypotheses now under study. our work represents analysis of the largest sample of sars-cov-2 genome sequences to date from patients in the southern united states. this investigation was facilitated by the fact that we had rapidly assessed the suitability and performance of the sars-cov-2 molecular diagnostic test in january 2020, more than a month before the first covid-19 patient was diagnosed in houston. our large healthcare system has eight hospitals and many clinics located in geographically diverse areas of the city. these facilities serve patients of very diverse ethnicities and socioeconomic status, thus our data likely represent a broad overview of virus diversity causing infections throughout metropolitan houston. we acknowledge that not every "twig" of the sars-cov-2 evolutionary tree in houston is represented in these data because the samples studied were not comprehensive. the genomes reported here represent the first strains documented to cause covid-19 disease in the houston area. thus, they are an important resource that will underpin further and our ongoing study of sars-cov-2 molecular evolution and dissemination in houston. as of april 15, 2020, there were 5,602 reported cases of covid-19 in metropolitan houston, with the number of cases growing daily. we are now sequencing virus genomes essentially in real time from infected patients, which will permit us to provide important data that can be exploited to facilitate an enhanced public health response to this pandemic. sars-cov-2 genome sequence analysis. consensus viral genome sequences from the houston isolates were generated using the artic ncov-2019 bioinformatics pipeline. publicly available genomes and metadata were acquired through gisaid on april 13, 2020 (16) . gisaid sequences containing greater than 1% n characters, and houston sequences with greater than 5% n characters were removed from consideration. identical gisaid sequences originating from the same geographic location with the same collection date were also removed from consideration to reduce redundancy. nucleotide sequence alignments for the combined houston and gisaid strains were generated using mafft version 7.130b with default parameters (32) . sequences were manually curated in jalview (33) to trim the ends and to remove sequences containing spurious inserts. phylogenetic trees were generated using fasttree with the generalized time-reversible model for nucleotide sequences (34) . trees were parsed, annotated, and visualized using the r packages treeio and ggtree (35) (36) (37) . clc genomics workbench (qiagen) was used to generate the trees in the bi filled map visualization. the home address for patients whose isolates were sequenced was matched to a dictionary of addresses downloaded from openaddresses.io (https://openaddresses.io/) to obtain the latitude and longitude geocoding data. because addresses are transcribed and subject to manual error, the fuzzywuzzyr package was used to match the best address in the dictionary. all address matches were manually reviewed for accuracy. the latitude/longitude coordinates were plotted onto a map using the microsoft power bi desktop map visualization. to examine geographic relatedness among genetically similar isolates, the geospatial maps were filtered to four small independent clades. the home address of the patients was again visualized using the microsoft power bi desktop map visualization. time series. the geospatial data were filtered into three time intervals (3/5/2020-3/16/2020, 3/5/2020-3/23/2020, and 3/5/2020-3/30/2020) to illustrate the spread of confirmed sars-cov-2 positive patients we identified over time. machine learning analysis. machine learning models were trained to predict patient metadata categories including mortality, length of stay, inpatient versus outpatient status, icu admission, overall outcome, gender, age, and ethnicity from viral sequence data. models were trained with the consensus whole genome fasta files by dividing each viral genome into 15-mer oligonucleotide k-mers, which were used as features to train xgboost models (38) as described previously (39, 40) . models were assessed by computing f1 scores for classifiers and r 2 scores for regression models. unless otherwise stated, data for the first 5folds of a 10-fold cross validation are shown. pdf?sfvrsn=fcf0670b_4. world health organization coronavirus disease 2019 (covid-19) situation report substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) a novel coronavirus outbreak of global health concern another decade, another coronavirus clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a new coronavirus associated with human respiratory disease in china european centre for disease control and prevention (ecdc) covid-19) pandemic: increased transmission in the eu/eea and the uk -seventh update severe outcomes among patients with coronavirus disease 2019 (covid-19) -united states houston region grows more racially/ethnically diverse gisaid: global initiative on sharing all influenza data -from vision to reality ebola virus glycoprotein with increased infectivity dominated the 2013-2016 epidemic human adaptation of ebola virus during the west african outbreak functional characterization of adaptive mutations during the west african ebola virus outbreak coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection compassionate use of remdesivir for patients with severe covid-19 the establishment of reference sequence for sars-cov-2 and variation analysis remdesivir and sars-cov-2: structural requirements at both nsp12 rdrp and nsp14 exonuclease active-sites structural basis for the inhibition of the rna-dependent rna polymerase from sars-cov-2 by remdesivir structure of the rna-dependent rna polymerase from covid-19 virus cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural and functional basis of sars-cov-2 entry by using human ace2 introductions and early spread of sars-cov-2 in the new york city area cryptic transmission of sars-cov-2 in washington state mafft multiple sequence alignment software version 7: improvements in performance and usability jalview version 2--a multiple sequence alignment editor and analysis workbench fasttree 2--approximately maximum-likelihood trees for large alignments treeio: an r package for phylogenetic tree input and output with richly annotated and associated data ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data two methods for mapping and visualizing associated data on phylogeny using ggtree xgboost: a scalable tree boosting system developing an in silico minimum inhibitory concentration panel test for klebsiella pneumoniae using machine learning to predict antimicrobial mics and associated genomic features for nontyphoidal salmonella key: cord-319194-ukuia48s authors: liò, pietro; goldman, nick title: phylogenomics and bioinformatics of sars-cov date: 2004-02-04 journal: trends microbiol doi: 10.1016/j.tim.2004.01.005 sha: doc_id: 319194 cord_uid: ukuia48s tracing the history of molecular changes in coronaviruses using phylogenetic methods can provide powerful insights into the patterns of modification to sequences that underlie alteration to selective pressure and molecular function in the sars-cov (severe acute respiratory syndrome coronavirus) genome. the topology and branch lengths of the phylogenetic relationships among the family coronaviridae, including sars-cov, have been estimated using the replicase polyprotein. the spike protein fragments s1 (involved in receptor-binding) and s2 (involved in membrane fusion) have been found to have different mutation rates. fragment s1 can be further divided into two regions (s1a, which comprises approximately the first 400 nucleotides, and s1b, comprising the next 280) that also show different rates of mutation. the phylogeny presented on the basis of s1b shows that sars-cov is closely related to mhv (murine hepatitis virus), which is known to bind the murine receptor ceacam1. the predicted structure, accessibility and mutation rate of the s1b region is also presented. because anti-sars drugs based on s2 heptads have short half-lives and are difficult to manufacture, our findings suggest that the s1b region might be of interest for anti-sars drug discovery. tracing the history of molecular changes in coronaviruses using phylogenetic methods can provide powerful insights into the patterns of modification to sequences that underlie alteration to selective pressure and molecular function in the sars-cov (severe acute respiratory syndrome coronavirus) genome. the topology and branch lengths of the phylogenetic relationships among the family coronaviridae, including sars-cov, have been estimated using the replicase polyprotein. the spike protein fragments s1 (involved in receptor-binding) and s2 (involved in membrane fusion) have been found to have different mutation rates. fragment s1 can be further divided into two regions (s1a, which comprises approximately the first 400 nucleotides, and s1b, comprising the next 280) that also show different rates of mutation. the phylogeny presented on the basis of s1b shows that sars-cov is closely related to mhv (murine hepatitis virus), which is known to bind the murine receptor ceacam1. the predicted structure, accessibility and mutation rate of the s1b region is also presented. because anti-sars drugs based on s2 heptads have short half-lives and are difficult to manufacture, our findings suggest that the s1b region might be of interest for anti-sars drug discovery. can phylogeneticists and bioinformaticians help virologists to tackle sars-cov (severe acute respiratory syndrome coronavirus)? phylogenetic methodology has progressed almost beyond recognition in the past decade and the study of phylogenetic relationships among species is now a valuable source of information in a variety of biological fields. the widespread use of a reliable statistical formalisation in phylogenetic and bioinformatic studies is necessary to extract the maximum information from sequence data [1] . recent work, although reporting impressive insights into the mechanisms of pathogenesis of sars-cov, has assessed the phylogeny of proteins of this virus using overly simple algorithms [2, 3] . the distance methods used to assess sars-cov phylogeny present several disadvantages. first, by converting a sequence alignment to pairwise distances we necessarily lose some of the evolutionary information contained within the analysed sequences [1] . second, distance methods are known to compromise the accuracy of estimates of evolutionary divergence, which are fundamental to understanding the rate and mode of viral evolution. third, there are no known methods to test evolutionary models and estimated trees produced using the pairwise distance methodology [1] . here we use state-of-the-art phylogenetic methods to analyse all the available coronaviridae and sars-cov sequence datasets to gain an insight into the origin and evolution of sars-cov and to narrow down the list of potential regions of its genome that might be interesting targets for drug design. the statistically most robust method that can be used to achieve these aims is to consider the phylogenetic inference problem in a likelihood framework, using a valid model of evolution for viral genomes [1] . the choice of such a model for single-stranded rna virus genomes is difficult. a parametric model, based on chemical or biological properties of rna, might underestimate or completely miss important unknown constraints; for example, packaging of single-stranded rna genomes that requires interaction with coat proteins [4] . an alternative approach is to use empirical models that are generated through comparisons of observed sequences; for example, simply counting apparent replacements between closely related sequences. given that sequence databases are biased towards mammalian and bacterial dna sequences, there are relatively few coding single-stranded rna sequences to be aligned, and non-coding rna sequences, such as rrna or trna, might be subjected to different selective (e.g. structural) constraints. because the proteins encoded by rna genes might be subjected to functional constraints in a similar manner to non-viral proteins, it might be better to use empirical amino acid substitution models that describe the probability of fixation of amino acid changes rather than rna models. furthermore, relative to primary structure, the secondary structure of homologous proteins persists long after any statistically significant sequence similarity has vanished; sequences with 25% amino acid identity probably have the same secondary structure [5] . amino acid models of evolution that incorporate protein structural information perform better than simple amino acid models [6] . moreover, selection pressures act on protein function, which in turn is closely related to structure. therefore, incorporating structure information into evolutionary analysis can assist in incorporating selective constraints. here, the programme passml-tm, which implements protein secondary structure-based models of evolution, has been used for analyses [7, 8] . the first undertaking is the determination and rooting of the coronavirus phylogeny, that is, the putative origin of the sequences of interest. rooting the coronaviridae and sars-cov phylogenies a large fraction (, 70%) of the sars-cov genome encodes a replicase polyprotein, which has significant sequence homology to all of the replicase polyproteins that have been sequenced to date from the order nidovirales (comprising the coronavirus, torovirus, okavirus and arterivirus genera). therefore, this protein is a good choice for investigating the phylogenetic relationships among the family coronaviridae. viral sequences have high mutation rates and, consequently, alignments are usually difficult to prepare. here, clustalw is applied using standard parameters [9] , followed by careful refinement of the alignments both by eye and by using the protein secondary structural information for each nidovirus sequence as predicted by phd (http://cubic.bioc.columbia.edu/predictprotein/) [10] and psi-pred (http://bioinf.cs.ucl.ac.uk/ psipred) [11] . figure 1 shows the maximum likelihood tree produced using a set of homologous replicases from five sars-cov strains, 12 other coronaviruses representing both groups 1 and 2 of the genus [2, 3] , one torovirus (breda virus) and one okavirus [yellow head (yh) virus], which were determined to most closely represent the consensus coronavirus sequence by a psi-blast search [12] . the coronavirus sequences allow the determination of the root of the sars-cov strains, whereas the torovirus and okavirus sequences provide insights into the rooting of the family coronaviridae phylogeny, which is closer to group 1 than group 2 of the coronavirus genus. as found previously by marra et al. [2] and rota et al. [3] , the root of sars-cov is closer to coronavirus group 2. all sars-cov strains are almost completely identical in sequence (,99% dna sequence homology) and therefore it is not possible to get any meaningful phylogeny within the sars-cov group. the avian infectious bronchitis virus (ibv), which causes respiratory disease in chickens, and the turkey virus (tcv), which causes enteric disease, are clustered together; their ancestor divides groups 1 and 2 of the mammal-infecting coronaviruses. the close clustering of the chicken and turkey viruses suggests that the difference between enteric and respiratory tropisms might require only a few amino acid changes. experiments have shown that just two point mutations in the spike (s) glycoprotein can change porcine transmissible gastroenteritis coronavirus (tgev), a mostly enteric virus that can kill piglets, into a non-deadly virus that excels at the respiratory route but replicates poorly in the gut [13 -15] . to infect enteric tract cells with tgev, two different domains of the s protein of tgev, mapping to between amino acids 522 and 744 and close to amino acid 219, are involved [13] . the first domain binds to aminopeptidase n (papn); many viruses use co-receptors, and it is probable that the second domain maps a co-receptor essential for the enteric tropism of tgev [14, 15] . the clustering of murine hepatitis virus (mhv) and rat coronavirus (rtcov) might reflect the relatively close proximity in which the hosts reside and perhaps the similarity of murine and rat target receptors. note that mhv receptors, including ceacam1, have recently been identified [16, 17] . the clustering of the human oc43 (hcov-oc43), bovine (bcov) and porcine haemagglutinating encephalomyelitis (hev) coronaviruses might reflect conditions contributing to cross-infection in farming. the breda torovirus is enteric, whereas the yh okavirus infects gill tissue in prawns. this indicates that the switch between enteric and respiratory tropism is a general characteristic of the order nidovirales. coronaviruses attach to host cells through the s glycoprotein [15, 18] . this protein is translated as a large polypeptide that is subsequently cleaved into a receptorbinding peripheral subunit (s1) that remains non-covalently associated with a fusion-inducing membrane-spanning (s2) fragment [18] . studies have shown that the entry of the porcine coronavirus tgev into cells is mediated by the interactions of s1 with papn, an ectoenzyme abundantly expressed at the apical membrane of enterocytes covering the villi of the small intestine [19] . the fact that the s protein mediates the first interaction of the virus with human cells suggests that it might represent an excellent target for effective anti-sars-cov drugs; unfortunately, structural information is only available for the 3clpro proteinase, which is part of the coronavirus replication complex (pdb accessions 1q2w, 1p9u, 1p9s and 1lvo) [20] . the s protein shows relatively high sequence homology within two of the major coronavirus groups (. 60% within group 1; . 38% within group 2), whereas the homology between groups is lower (15-21% between groups 1 and 2; 15-21% between sars-cov and group 1 or 2). comparative sequence analysis between the sars-cov sequences and sequences from groups 1 and 2 of the coronavirus genus reveals three regions of varying sequence conservation: from amino acid positions 1 to , 400, 401-680 and 681-1255. interestingly, the first two regions correspond to the s1 fragment (we subsequently refer to these regions as s1a and s2a) and the third to the s2 fragment. figure 2a shows a cartoon of the gene for the s protein. the region s1a is poorly conserved between groups 1 and 2 and sars-cov, whereas s1b is flanked by two very well conserved motifs making it easier to align the internal sequences. notably, this region is homologous to a tgev spike region that is reported to contain determinants for tissue tropism [13 -15] . the region s2 is more conserved than s1a and s1b and consequently the alignment is easily determined; passml-tm, which uses the evolutionary relationships of the sequences analysed to improve its predictions of secondary structure and accessibility, predicts a transmembrane helix at location 1196-1218 of the s protein. the maximum likelihood trees of s1b and s2 are shown in figures 2b and 2c , respectively. the length of the trees and the distances between sequences reflect the sequence homologies according to the model of evolution, explaining why the s2 tree is much shorter than s1b tree. the phylogenetic tree produced from the analysis of the spike s2 fragment from several coronaviruses indicates that sars-cov is closer to group 2 of the coronavirus genus than to group 1. phylogenetic analysis of the s1 fragment from several coronaviruses indicates that sars-cov has an even closer relationship to group 2 viruses than the previous analysis suggests. phylogenetic analysis of a 300 amino acid region at the terminus of s1, which we denote s1b, indicates that sars-cov belongs to group 2 and is closely related to mhv and rtcov. the differences between these results might be a result of recombination events involving sars-cov or convergent evolution, or they might also be caused simply by chance; however, it is apparent that sars-cov is most closely related to group 2 of the coronavirus genus. because the s1 fragment of mhv binds to ceacam1, we suggest that the s1b region of the sars-cov spike might bind to a human ceacam1 receptor instead of papn. holmes and collaborators, who have identified receptors for mhv (murine cea-cam1a), hcov-229e (human papn) and feline coronaviruses (feline papn) [16, 17] , are currently investigating this hypothesis (k. holmes, pers. commun.). in support of the proposal that s1b is involved in receptor-binding is the fact that this region is homologous to a domain of the tgev s protein, which is located between amino acids 522 and 744 and is also involved in receptor-binding [13, 16] . notably, tgev mutants that lack sialic acid-binding activity contain single point mutations in the s protein (cys155phe, met195val, arg196ser, asp208asn or leu209-pro) [21, 22] . sialic acid-binding activity might help tgev to resist detergent-like substances encountered during gastrointestinal passage and therefore facilitate infection of the intestinal epithelium [23] . we found that only cys155 is conserved in sars-cov; this is in agreement with clinical findings that show that 20 -50% of sars patients present gastrointestinal symptoms [24] . the low conservation of the s1a region among coronavirus sequences suggests that once more strains of sars-cov or other closely related species are available it will become possible to use innovative comparative sequence analyses to examine positive selection that acts in this region [1] . several important functional determinants have been discovered in fragment s2. it contains a cytoplasmic tail enriched in cysteine residues (1217 -1236; figure 2a ); this is a common feature among coronaviruses and appears to be related to membrane fusion [25] . several authors have discovered that sars-cov s2 contains two conserved regions of heptad repeats (913 -1000 and 1151-1185; figure 2a ) [26, 27] (see also a press release by w.r. gallaher and r.f. garry http://www.virology.net/sars/ s2model.html). these heptads suggest that sars-cov uses mechanisms to gain entry to a cell that are similar to those used by human immunodeficiency virus (hiv), the virus that causes aids (acquired immunodeficiency syndrome), orthomyxoviruses and paramyxoviruses, and also ebola. it is known that peptides derived from these repeat regions in hiv and the paramyxoviruses can specifically inhibit virus entry and subsequently viral replication [26] . currently, sars treatment is modeled after the drug known as t20 (http://www.hivmedicine. com/textbook/drugs/t20.htm). this drug is a complex peptide that is difficult to manufacture, has a short halflife in the human body and must be injected. this suggests that other regions of the genome, such as s1b, which might be of some interest for drug design, should be described. we report in figure 2d the consensus protein structure estimate of s1b obtained using passml-tm, phd [10] and psi-pred [11] . passml-tm also estimates the distribution of mutation rates along the protein and the sitewise mutation rate (figure 2d ). the sars-cov genome is at rich (59%). asymmetries in strand composition can reveal mutation bias (for example, cytosine deamination) or selection [28] . we found that the gc [(g 2 c)/(g þ c) ¼ 0.02] and at skew [(a 2 t)/ (a þ t) ¼ 0.037] in the sars-cov genome are smaller than those of the hiv genome (gc skew is 0.15 and at skew is 0.23), which has a double-stranded rna genome with a similar at content. this suggests the existence of some selection on g and c distribution along the sequence to control the types of rna secondary structure that form [29] . cg is the only dinucleotide statistically underrepresented {f(cg)/(f(c)f(g) ¼ 0.46, where f(cg) is the frequency of cg dinucleotides and significance is assessed according to refs. [30, 31] }. because this depletion is also found in the hiv genome but not in that of tobacco mosaic virus, it might occur as a result of mutational bias in vertebrate cells. comparison of the mutation patterns in the sars-cov genome sequences from 16 sites occur within or near to single base and dinucleotide repeat stretches. despite the absence of a pairing rule, the ratio of rates of transition and transversion mutations is , 2, as is often found in double-stranded dna. the low gc and at skews and the low number of mutations suggest that evolvability of sars-cov might be restricted by selective constraints acting on the rna structure and packaging of the genome, and therefore it might also be restricted by the low fitness of its mutational neighbours. sequence features that form stems and loops that are potentially involved in coronavirus genome packaging have been described [32, 33] . mutational neighbours with ; and sars-covs from strains tor2, bj01, bj02, bj03 and cuhkw1], one torovirus (breda) and one okavirus (yellow head virus, yh). coloured areas indicate coronavirus groups 1 (blue) and 2 (yellow). (d) predicted structure and mutation rate for the sars-cov s1b region. secondary structure has been predicted (row pred) in three classes: helix (h), sheet (e) and coil (c). accessibility has been predicted (row acc) in three classes: buried (b), exposed (e) and intermediate (i). mutation rates (row mut) are partitioned into eight classes (classes 1-8 have relative rates of evolution 0.10, 0.27, 0.44, 0.64, 0.88, 1.19, 1.65 and 2.83, respectively), inferred using the empirical bayes method [37] . the sequence homologous to the region 522-744 of the tgev spike protein is shown in red. maximum likelihood trees and mutation rate analyses were computed using passml-tm [7] . different fitness might explain why, although some rna viruses evolve at high rates, some rna viruses are highly stable [34, 35] . interestingly, the ease of tropism switching as exemplified by the closeness between turkey and chicken coronaviruses (figure 1) is favoured by the large number of viral particles in each host, by their mutation rates, by the large populations of hosts (birds and other species) and by the aerial mode of viral spread (for instance, through sneezing and faeces). these factors suggest that birds might act as powerful engines for virus evolution. in this review we have highlighted that the s1 and s2 fragments of the sars-cov s protein have different mutational patterns. on the basis of phylogenetic evidence ( figure 2b ) and the homology with the tgev 522-744 region, it is suggested that a short region of the s1 fragment, which we denote s1b, located at positions 400-680, might be of particular interest to virologists, structural biologists and biotechnologists. the sitewise secondary structure, solvent accessibility and mutation rate of this region have been estimated; our work in progress includes further structural characterization and fold family determination. at the moment, sars appears to be under control despite doctors having neither drugs nor a vaccine to protect against it. because it could reappear in the future, research should proceed and hopefully our findings might assist in maintaining a feed-forward loop on sars-cov research between bioinformatics analysis and experimental work from microbiologists and virologists. as a final comment, it is notable that, in all the phylogenies, human coronaviruses hcov-229 and hcov-oc43 always cluster with porcine coronaviruses. because ericsson and collaborators [36] reported the identification of two homologous human proteins that act as receptors for porcine endogenous retrovirus, the benefits and risks of porcine -human xenotransplantation should be carefully balanced. those of us who make our living studying microorganisms often take their metabolic versatility for granted. however, we are reminded of this versatility yet again with the recent publication by larimer et al. [1] on the complete genome sequence of rhodopseudomonas palustris, a purple photosynthetic bacterium that is a member of the alpha group of proteobacteria. r. palustris was used in some of the earliest studies that characterized aromatic degradation in microbial species and is found in very diverse environments, an observation that reflects its spectacular metabolic capabilities and ability to grow using any one of four different modes of metabolism: photosynthetic, photoheterotrophic, chemoheterotrophic or chemoautotrophic. the ability of this organism to adjust its metabolism so dramatically in response to changes in energy and nutrient availability sets r. palustris apart from many of its close relatives. the r. palustris genome is composed of a single circular chromosome (5.46 mb) and one 8.4 kb plasmid and encodes 4,836 genes. the relatively large size of the r. palustris genome is reminiscent of other ubiquitous environmental bacteria for which we have complete genome sequences, including streptomyces coelicolor (8.7 mb), streptomyces avermitilis (9.0 mb) and pseudomonas putida (6.6 mb). the streptomycete genomes contain an unprecedented number of gene clusters that encode known or predicted proteins involved in secondary metabolism. these gene clusters might represent dna that was acquired through lateral gene transfer at some point in the evolution of the streptomycetes. by contrast, r. palustris has a minimal number of insertion sequence elements, and few regions of atypical nucleotide composition which, taken together, suggest that lateral gene transfer has not played a major role in shaping this genome. the genes that encode components of major metabolic pathways are also randomly distributed throughout the r. palustris genome. in r. palustris, key genes that code for bacteriochlorophyll and carotenoid biosynthesis, as well as those for membrane-bound reaction centre complexes of photosynthesis were found to be clustered in a 55 kb region of the genome. forms i and ii of rubisco, the key enzyme of the calvin -benson -bassham pathway of carbon dioxide fixation are encoded by the genome, as well as two rubisco-like proteins (so far only identified in the photosynthetic chlorobium tepidum) [2] . based on the biochemical characterization of the rubisco-like protein in c. tepidum [3] , in r. palustris these proteins probably play a role in sulfur metabolism. approximately 31% of the predicted coding sequences in the genome are devoted to energy metabolism. for example, many predicted protein sequences for the oxidation of inorganic compounds including thiosulfate and hydrogen, which provide reducing energy for carbon dioxide and nitrogen fixation are encoded, and also carbon monoxide and formate dehydrogenases. analysis of the genome also highlights the ability of r. palustris to degrade heterocyclic aromatics and chlorinated benzoates, and reveals the presence of four-ring cleavage pathways. as with the soil bacteria for which we have complete genome sequences, just over 9% of the r. palustris genome is devoted to regulation. this is probably a reflection of the need of these species to sense variations in environmental conditions and respond accordingly. this diversity is also reflected in the high percentage of the genome that is devoted to transport capabilities and chemotaxis. as , 30% of the r. palustris genome consists of genes that are either hypothetical or conserved hypothetical proteins, or of unknown function, the biochemical characterization of these predicted protein sequences will undoubtedly reveal additional new physiological capabilities for this species. r. palustris has been studied for its metabolic potential for close to forty years [4] , however, the genome sequence revealed a greater set of predicted coding sequences for the biodegradation and metabolism of aromatic compounds than had been expected, as well as an ability to combine these pathways under anaerobic and aerobic conditions. it is anticipated that, as with the genomes of other metabolically diverse environmental species such as deinococcus radiodurans, the genome of r. palustris can serve as a model to increase our understanding of how organisms can use diverse metabolic capabilities to respond to changes in substrate, light and other environmental cues. corresponding author: claire m. fraser (cmfraser@tigr.org). molecular phylogenetics: state-of-the-art methods for looking into the past the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome self-assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed recognition of analogous and homologous protein folds: analysis of sequence and structure conservation combining protein evolution and secondary structure combining protein secondary structure prediction and evolutionary inference using protein structural information in evolutionary inference: transmembrane proteins clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice predict protein the psipred protein structure prediction server iterated profile searches with psi-blast -a tool for discovery in protein databases two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence identification of a receptor binding domain of the spike glycoprotein of human coronavirus hcov-229e conformational changes in the spike glycoprotein of murine coronavirus are induced at 378c either by soluble murine ceacam1 receptors or by ph 8 the coronavirus surface glycoprotein interspecies aminopeptidase-n chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus severe acute respiratory syndrome and its lesions in digestive system coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor cloaked similarity between hiv-1 and sars-cov suggests an anti-sars strategy asymmetric substitution patterns: a review of possible underlying mutational or selective mechanisms equal g and c contents in histone genes indicate selection pressures on mrna secondary structure comparative dna analysis across diverse genomes compositional differences within and between eukaryotic genomes nucleocapsid-independent specific viral rna packaging via viral envelope protein and viral rna signal identification of probable genomic packaging signal sequence from sars-cov genome by bioinformatics analysis evolvability of an rna virus is determined by its mutational neighbourhood evolution of sex and the molecular clock in rna viruses identification of receptors for pig endogenous retrovirus likelihood models for detecting positively selected amino acid sites and applications to the hiv-1 envelope gene $ -see front matter q fraser the institute for genomic research we thank rodrigo lopez and ivo cozzani for helpful suggestions. p.l. was partially supported by a bbsrc grant. n.g. is supported by a wellcome trust fellowship in basic biomedical research. key: cord-335122-8s3bcyo8 authors: marshall, steve; duryea, michael; huang, greg; kadioglu, onur; mah, james; palomo, juan martin; rossouw, emile; stappert, dina; stewart, kelton; tufekci, eser title: covid-19: what do we know? date: 2020-09-21 journal: am j orthod dentofacial orthop doi: 10.1016/j.ajodo.2020.08.010 sha: doc_id: 335122 cord_uid: 8s3bcyo8 evidence regarding provision of orthodontic care during covid-19 pandemic is examined. droplets (e.g. from sneezing, agps) tend to settle on surfaces, or unprotected mucosa of close contacts, and may be the source of direct or indirect virus transmission (also termed droplet transmission). 43 in experimentally simulated aerosolization of sars-cov-2, the virus maintains viability on surfaces for up to 72 hours indicating indirect (droplet) transmission can occur long after droplets establish contact with surfaces. [44] [45] [46] in contrast, small droplets (e.g. from coughing, talking, exhaling, or agps) produced by similar experimental aerosolization can evaporate into "droplet nuclei" and remain in the air for many hours. 44, 45, [47] [48] [49] the amount of viable sars-cov-2 in droplet nuclei remains unclear, but in subjects infected with other respiratory viruses, such as influenza, experiments comparing coughing and breathing suggest an equivalent production of viral rna and replication-competent virus, detected at close range (< 12 inches). 50, 51 although this has not been adequately studied for sars-cov-2, similar findings might be anticipated. [52] [53] [54] moreover, saliva can be aerosolized during agps and is a known source of sars-cov-2 in infected individuals. 55 a timeline suggesting when infected individuals are most contagious has been informed by studies assessing viral shedding of covid-19 patients by two methods: detection of sars-cov-2 rna and sars-cov-2 replication in cultured cells. 56 viral rna can be detected 1-3 days prior to the onset of covid-19 symptoms, with the highest viral load in the upper respiratory tract occurring near the onset of symptoms followed by a decreasing viral load that is time-dependent based on disease severity. viral rna is shed for 1-2 weeks in asymptomatic cases and 3 or more weeks for mild to moderate cases of covid-19. [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] more severe symptoms require a longer time to reduce viral load. 57, 59, 60, [67] [68] [69] [70] [71] reduction in viral load is accompanied by increases in neutralizing antibodies. 57 the findings from a limited number of studies evaluating virus viability during the course of covid-19 illness suggest it is rare to find infected symptomatic individuals shedding viable virus after 9 days of symptom onset. 57, 61, [72] [73] [74] [75] a study of nursing home residents found viral rna and viable virus in pre-symptomatic and asymptomatic subjects. 73 taken together, these data suggest viral shedding, detected by viral rna, may be an indicator of sars-cov-2 transmissibility prior to the onset of covid-19 symptoms, but not later in the course of covid-19 illness. however, measuring virus viability is more complex and may not be as sensitive as rna detection. 57 includes agps and medical care without agps (non-aerosol generating procedures, non-agps) have not revealed a clear consensus on the risk of airborne transmission of sars-cov-2. 78 airborne transmission of viable sars-cov-2 virus during medical agps on infected patients is suggested from previous studies of sars-cov-1, but has not been confirmed. 78 in situations where healthcare workers wearing personal protective equipment (ppe) attend to patients with covid-19 and do not perform medical agps, direct airborne transmission of replicationcompetent sars-cov-2 has not been confirmed. 79 the results of hospital studies evaluating aerosolization of body fluids and respiratory droplets of sars-cov-1 infected patients generated during certain medical agps (tracheal intubation, non-invasive ventilation, bronchoscopy, etc.), suggest that airborne transmission of sars-cov-2 may be possible during these procedures. 79 however, the "possibility" is not clearly defined. high quality studies using consistent methodology to assess virus transmissibility during medical agps are lacking. 78 a 2012 systematic review of five case-control studies and five retrospective cohort studies on the transmissibility of sars-cov-1 during medical agps found a weak association with tracheal intubation across multiple studies and could draw no conclusions regarding other medical agps. 80 subsequent studies on sars-cov-1 transmissibility during medical agps produced variable results. 43, 81 similar controversial findings were found for influenza a h1n1. 82 to date, although there is evidence suggesting that sars-cov-2 is likely transmitted via bioaerosol, 83 there is no direct evidence of airborne transmission of sars-cov-2 during medical agps when healthcare workers are wearing appropriate ppe, the risk of airborne transmission is not clearly defined. 78 using precaution as the guiding principle in risk management, the cdc and who have adopted guidelines for barrier and environmental protection based on the hypothesis that airborne transmission can occur, even though a detailed understanding remains to be elucidated. from the cdc guidelines: development of a comprehensive list of agps for healthcare settings has not been possible, due to limitations in available data on which procedures may generate potentially infectious aerosols and the challenges in determining if reported transmissions during agps are due to aerosols or other exposures. there is neither expert consensus, nor sufficient supporting data, to create a definitive and comprehensive list of agps for healthcare settings. 84 scientific consensus on the transmission of sars-cov-2 during medical non-agps is not yet available. results from studies designed to sample air for sars-cov-2 rna, in hospital rooms where infected patients were cared for without medical agps, produced variable results. [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] the studies finding the presence of viral rna reported very low amounts. [85] [86] [87] [88] [89] [90] this experimental design assesses the presence of sars-cov-2 rna but does not assess virus viability. currently there are no studies reporting airborne viable (replication-competent) sars-cov-2 virus j o u r n a l p r e -p r o o f in hospital settings where infected patients are cared for, but not subjected to medical agps, by healthcare workers wearing surgical masks. 81 in contrast to medical procedures in hospital and clinical settings, the risk of airborne sars-cov-2 infection during treatment of orthodontic patients has not been studied. additionally, there are no reports of transmission of the virus in an orthodontic setting to elucidate clues regarding risk and transmission. guidelines to mitigate the risk of sars-cov-2 airborne transmission during orthodontic treatment must be inferred from studies of sars-cov-2 infected patients during agps and non-agps in other healthcare settings. however, the extent to which agps and non-agps differ between the practice of medicine and the practice of orthodontics, and the impact of these differences on the risk of sars-cov-2 transmission has not been adequately addressed. as potential sars-cov-2 transmission from pre-symptomatic and asymptomatic individuals remains a possibility, covid-19 screening procedures will not prevent the unintentional treatment of some contagious individuals. this poses an unknown risk of airborne transmission for both agps (mechanically-generated bioaerosol) and non-agps (patient sneezing, coughing, talking, exhaling) in orthodontic practice. in orthodontic practice, agps include the use of rotary instruments (high-speed and slow-speed hand piece), airwater syringes (produce both splatter and aerosol), ultrasonic scalers, or air abrasion/polishing instrumentation on the tissues within the oral cavity. use of these instruments generates aerosolized particles including particulates from dental materials and bioaerosol from aerosolized saliva and respiratory droplets. the particles/droplets generated range from 0.1-50 µm. the bioaerosol contents include live bacteria, fungus and viruses that increase the contamination of the air and surfaces in the area of patient treatment. [97] [98] [99] [100] many reports have characterized the bacterial content of this bioaerosol, but there is a lack of research characterizing the production of viable airborne virus from agps used in orthodontic practice. 99, 101 of particular importance is the size difference between viruses and bacteria. for example, bennett et al. 102 found that bacteria (oral streptococci) in aerosols generated during dental agps dissipate within 30 minutes of their peak concentration. however, streptococci are 10 -fold larger in diameter compared to sars-cov-2, which may limit their maintenance in aerosol compared to that seen for coronaviruses. 44, 45, [47] [48] [49] 103 how much aerosol is generated during orthodontic debonding? although the composition of particulates and bioaerosol generated during debonding of fixed orthodontic appliances has been widely studied (viruses excluded), the amount of bioaerosol generated during orthodontic debonding is not exactly known, and remains unknown for virus and virus particles. [98] [99] [104] [105] [106] [107] [108] [109] (for a j o u r n a l p r e -p r o o f comprehensive review, see zemouri et al. 99 eliades et al. 101 ). evaluating various dental procedures in situ, polednik 98 established that compared to background levels, airborne particulates increase approximately 6-fold for composite grinding, compared to a 2.5-fold increase for ultrasonic scaling. composite grinding produced the most particulate aerosol of any dental agp tested. levels of bacterial aerosolization compared to background were ~1.5 -fold greater across dental agps tested. does aerosol generation differ when debonding is performed with a slow-speed vs. high-speed hand piece, or when debonding is performed with water vs. without water? there are no studies addressing this question in situ, and no studies quantifying the amounts of bioaerosol generated during orthodontic debonding. the production of aerosol containing bonding adhesive and enamel particulates has been measured during removal of orthodontic adhesive from human teeth under laboratory conditions. 104, 105, 107, 109 one study suggests particle size differs between slow-speed and high-speed hand pieces, and the addition of water spray to the procedure results in a reduction of particle size generated during debonding. slow-speed handpieces, with or without water spray, produced particles ~5-15 µm. high speed handpieces without and with water spray produced particles ~3 µm and ~0.5-1.3 µm respectively. 105 a second study by the same research group suggests debonding with a high-speed hand piece and water spray generates approximately 2-fold more adhesive and enamel particulates compared to debonding with a slow-speed hand piece without water spray. 107 results from another research group evaluating smaller diameter particulates suggests the addition of water spray during slow-speed hand piece reduction of bulk composite reduces, by onehalf, the amount of airborne particulates smaller than 0.1 µm in diameter. 110 taken together, these studies suggest that slower speed and water spray may reduce the amount of particulate aerosol produced. additional studies are needed to confirm this. it has been proposed that the use of water spray during orthodontic debonding improves debonding efficiency and thereby reduces the time that bioaerosol is produced. 101 additional studies are needed to confirm this hypothesis. at the present time, we cannot extrapolate from these laboratory studies to understand the amount of viable sars-cov-2 present in bioaerosol produced by various permutations of hand piece use during orthodontic debonding. by and large, the clinical evidence for reduction of aerosols by the use of high volume extra-oral evacuation (hve) comes from studies detecting the bacterial load produced during ultrasonic scaling. 111 results from these studies have not been consistent. significant bacterial load reductions (83-94%) 112, 113 or no reduction 114 laboratory studies generating aerosol by various dental agps have suggested aerosol is reduced by the use of hve. 107, 115, 116 however, the generalizability of findings from laboratory ultrasonic scaling studies to orthodontic debonding in situ is not fully understood. additionally, a recent meta-analysis of randomized and non-randomized trials assessing interventions to reduce bacterial aerosolization during dental agps suggests the use of hve is not more effective than pre-procedural rinses with chlorhexidine or chlorine dioxide. 111 however, it is uncertain how this pertains to aerosolized viable virus. at present, no studies have assessed the effectiveness of hve during orthodontic debonding on the reduction of transmissible sars-cov-2. the use of hve should be considered an prudent adjunctive measure to reduce the risk of virus transmission via bioaerosol during orthodontic debonding, but the efficacy of hve use remains unclear. new evacuation instrumentation (e.g. high-flow extractor 117 ) are being developed and studied for their efficacy in reducing mechanically generated bioaerosol. ongoing research will determine the benefit of their use during dental agps. 114 currently, cdc recommendations for reducing the risk during agps in a dental setting include: 37 • four-handed dentistry • use of hve • dental dams when practical • ppe including n95 mask, face shield, gown, gloves • portable hepa air filtration system properly situated are air filtration/air purification systems effective in reducing aerosols generated during orthodontic debonding? are hepa filters required in air filtration/air purification systems for effective reduction of aerosols generated during orthodontic debonding? to lessen the risk of airborne transmission of sars-cov-2 from the bioaerosol produced during dental agps, portable hepa filtration systems (known as high-efficiency particulate air, high-efficiency particulate absorbing and high-efficiency particulate arrestance systems), are recommended by the cdc as a supplement to the barrier protection of ppe. 37 there is ample evidence that hepa air purification reduces the concentration of airborne particles in the size range associated with airborne sars-cov-2, 118-121 but direct evidence for reduction of viable virus has not yet been reported. cdc guidelines suggest best practices for the positioning and use of portable hepa systems in the operatory during dental agps, which is a subject of ongoing research. 37, 122 the use of ppe during orthodontic procedures j o u r n a l p r e -p r o o f as previously discussed, the infective potential of patient bioaerosol is not fully understood. bioaerosol generated from coughing, sneezing, exhaling, or by mechanical aerosolization of saliva during patient procedures occurs as a range of drop and droplet sizes, all of which are potentially infective, by direct or indirect droplet contact with uncovered mucosal surfaces, or by inhalation of droplets or droplet nuclei. ppe is an important part of a system protecting doctors, staff and other patients by reducing the spread of viral respiratory infection. other parts of that system are equally important: patient pre-screening, patient isolation from other patients, minimizing the number of staff caring for a patient, appropriate donning, doffing, and disposal of ppe, appropriate decontamination of surfaces and equipment, and appropriate biohazard waste management. 123 the who and cdc have recommended the use of ppe to match the potential mode of sars-cov-2 transmission during patient care. 37, 124 high-filtration masks (n95 or equivalent) are recommended as protection during aerosol generating procedures because of their barrier capability. however, in practice, uncertainty remains regarding the effect mask training, mask type and the re-use of masks and gowns on the true nature of protection . 123, 125, 126 reports of headache among healthcare personnel during prolonged use of n95 respirators 127, 128 has prompted investigation of powered air-purifying respirators as a possible improvement in potential side effects of n95 respiratory use. 129 there is a lack of high-quality research comparing the effectiveness of the n95 respirator and the surgical mask in preventing transmission of sars-cov-2 to a healthcare worker under conditions of varying transmission risk. a recent systematic review and meta-analysis of 4 randomized trials compared protection from respiratory illness for surgical masks vs. n95 respirators in healthcare workers potentially exposed to patients with acute viral respiratory illness (influenza). 130 due to the heterogeneity of methods and outcome measures, the findings of no difference between surgical masks and n95 respirators are weakly supported with low or very-low levels of evidence. comparing surgical masks and n95 respirators for protection from other respiratory viruses have produced similar findings. 126, 131 no trials have tested n95 respirator protection against sars-cov-2 transmission directly. this evidence should be interpreted with caution. laboratory studies indicate n95 respirators are far superior in blocking penetration of 10 -80 nanometer virions compared to surgical masks. 132 trials conducted in healthcare settings suffer from variation in mask training, mask fitting, mask use, and mask removal that is absent in wellcontrolled laboratory studies. 125, 133 it is not yet clear that surgical masks offer equivalent protection to n95 respirators in situ. what ppe is most appropriate during aerosol generating procedures vs. non-aerosol generating procedures? the cdc recommends the use of face shields, gowns, and gloves during both agps and non-agps. 37 the cdc recommends the use of an n95 respirator, or a respirator offering equivalent or greater barrier protection to the inhalation of bioaerosol, during dental agps, and a surgical facemask during dental non-agps. 37 n95 respirators are recommended to limit inhalation of potentially infectious aerosol. surgical facemasks offer a more limited "protection for the wearer against exposure to splashes and sprays of infectious material from others." 134 when should ppe be discarded and replaced during patient care? the cdc recommends discarding gloves, gowns, surgical masks between each patient. 135 the cdc recommends n95 respirators be disposed after each use, but have provided guidance for extended use, or re-use after decontamination, during periods of reduced n95 availability. 135 limited re-use is defined as using the same n95 respirator for multiple patients, but removing (doffing) after each patient encounter. the respirator is stored between encounters. extended use is defined as using the same n95 respirator continuously during encounters with multiple patients. there are strict guidelines for extended use and limited re-use of n95 respirators. 135 the cdc has issued strategies for dealing with supply shortages of ppe that include decontaminating nioshapproved n95 filtering facepiece respirators (ffrs) without exhalation valves. although knowledge gaps remain in the efficacy of ffr decontamination, moist heat, ultraviolet germicidal irradiation, and vaporous hydrogen peroxide, appear to be appropriate decontamination methods. however, ffr decontamination is meant to be implemented under strict guidelines. these guidelines should be thoroughly understood prior to implementing this strategy. 135 two recent meta-analyses of randomized controlled trials, studying the effect of pre-procedural mouth rinses (ppmrs) on bacteria produced during dental agps, concluded there is moderate evidence that ppmrs (chlorhexidine, cetylpyridinium chloride, providone-iodine, or essential oils) significantly reduce aerosolized bacteria. 115, 136 there is no direct evidence for a similar effect of these oral antiseptics on aerosolized viruses. according to the cdc, "there is no published evidence regarding the clinical effectiveness of ppmrs to reduce sars-cov-2 viral loads or to prevent transmission. although sars-cov-2 was not studied, ppmrs with an antimicrobial product (chlorhexidine gluconate, essential oils, povidone-iodine or cetylpyridinium chloride) may reduce the level of oral microorganisms in aerosols and spatter generated during dental procedures." 37 a number of narrative reviews suggest selected oral antiseptic rinses, including 1.0% hydrogen peroxide, have antiviral activity in vitro, but this indirect evidence requires well-designed trials to evaluate clinical efficacy in situ. [137] [138] [139] [140] [141] j o u r n a l p r e -p r o o f how much time should be allocated between patients when aerosol generating procedures are performed? currently, there is not enough information to answer this question directly. cdc guidelines for performing agps on patients known to be infected with sars-cov-2 require treatment in an airborne infection isolation room with a minimum of 6 air changes per hour and a minimum waiting time of 69 minutes to reduce potentially infectious aerosol by 99.9% 35, 142 aerosol-generating treatment of asymptomatic or pre-symptomatic orthodontic patients poses a risk that is not quantifiable. the cdc has suggested evaluating hvac systems for airflow patterns, rates of air exchange, and increased filtration, and the addition of portable hepa filtration systems to reduce this risk. 37 there is no direct evidence for the efficacy of physical partitions reducing the risk of sars-cov-2 transmission in and open operatory facility. as part of 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prevention and control recommendations for healthcare personnel during the coronavirus disease 2019 (covid-19) pandemic implementing filtering facepiece respirator (ffr) reuse, including reuse after decontamination, when there are known shortages of n95 respirators efficacy of preprocedural mouthrinses in the reduction of microorganisms in aerosol: a systematic review transmission routes of 2019-ncov and controls in dental practice in vitro bactericidal and virucidal efficacy of povidone-iodine gargle/mouthwash against respiratory and oral tract pathogens potential role of oral rinses targeting the viral lipid envelope in sars-cov-2 infection virucidal efficacy of different oral rinses against sars-cov-2. the journal of infectious diseases is the oral cavity relevant in sars-cov-2 pandemic? airborne contaminant removal air changes/hour (ach) and time required for airborne-contaminant removal by efficiency key: cord-328003-yovp8squ authors: duan, liangwei; zheng, qianqian; zhang, hongxia; niu, yuna; lou, yunwei; wang, hui title: the sars-cov-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens date: 2020-10-07 journal: front immunol doi: 10.3389/fimmu.2020.576622 sha: doc_id: 328003 cord_uid: yovp8squ the ongoing pandemic of coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. like other coronaviruses, the sars-cov-2 genome encodes spike (s) glycoproteins, which protrude from the surface of mature virions. the s glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. surface location of the s glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. in the light of its crucial roles in viral infection and adaptive immunity, the s protein is the focus of most vaccine strategies as well as therapeutic interventions. in this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the sars-cov-2 s glycoprotein, aiming to provide valuable insights into the design and development of the s protein-based vaccines as well as therapeutics. the coronavirus disease 2019 (covid-19) global pandemic represents an unprecedented public health, social and economic challenge (1, 2) . the etiological agent of covid-19 is a new member of the coronaviridae family that is closely related to severe acute respiratory syndrome coronavirus (sars-cov) and was recently referred to as sars-cov-2 by the coronavirus study group of the international committee on taxonomy of viruses (3) . the virus has spread rapidly and sustainably around the global resulting in over twenty-one million cases and more than 750,000 deaths as of august 15, 2020 (4) . coronaviruses (covs) are enveloped positive-sense rna viruses (5) . enveloped covs entering host cells and initiating infection is achieved through the fusion of viral and cellular membranes (6, 7) . membrane fusion is mediated by the large type i transmembrane s glycoprotein on the viral envelope and the cognate receptor on the surface of host cells (8) (9) (10) . the surfaceexposed location of the s glycoprotein not only allows it to carry out membrane fusion but also renders it a direct target for host immune responses, making it the major target of neutralizing antibodies (11) . because of its central roles in viral infection and eliciting protective humoral and cell-mediated immune responses in hosts during infection (10) , the s protein is the primary target for vaccine design as well as antiviral therapeutics (12) . here, we provide a comprehensive overview of the wealth of research related to the sars-cov-2 s glycoprotein biosynthesis, structure, function, and antigenicity, aiming to provide useful insights into the design and development of the s protein-based vaccines as well as therapeutics to prevent or treat the ongoing global spread of sars-cov-2/covid-19. the sars-cov-2 s glycoprotein is synthesized as a 1273-amino acid polyprotein precursor on the rough endoplasmic reticulum (rer) (figure 1 ) (13) . the unprocessed precursor harbors an endoplasmic reticulum (er) signal sequence located at the n terminus, which targets the s glycoprotein to the rer membrane and is removed by cellular signal peptidases in the lumen of the er (14, 15) . a single stop-transfer, membrane-spanning sequence located at the c terminus of the s protein prevents it from being fully released into the lumen of the er and subsequent secretion from the infected cell (16, 17) . co-translationally, n-linked, highmannose oligosaccharide side chains are added during synthesis (18, 19) . shortly after synthesis, the s glycoprotein monomers trimerize, which might be thought to facilitate the transport from the er to the golgi complex. once in the golgi complex, most of the high-mannose oligosaccharide side chains are modified to more complex forms (20, 21) , and o-linked oligosaccharide side chains are also added (22, 23) . in the trans-golgi network, the sars-cov-2 s glycoprotein is proteolytically cleaved by cellular furin or furin-like proteases at the s1/s2 cleavage site, comprising multiple arginine residues that are not found in the closely related sars-cov (24, 25) . cleavage at the s1/s2 site yields a surface subunit s1, which attaches the virus to the host cell surface receptor, and a transmembrane subunit s2, which mediates the fusion of viral and host cell membranes (10) . the s1 and s2 subunits remain associated through noncovalent interactions in a metastable prefusion state (11) . furin-like cleavage is essential for the sprotein mediated cell-cell fusion and viral infectivity, and is required for efficient sars-cov-2 infection of human lung cells (24) and airway epithelial cells (26) . following cleavage, an er retrieval signal (errs) consisting of a conserved kxhxx motif (27) located at the extreme c terminus ensures that the mature sars-cov-2 s protein accumulates near the er-golgi intermediate compartment (ergic) (27, 28) , where driven by interactions with another structural protein, the membrane (m) protein, the s protein participates in virus particle assembly and is incorporated into virus envelope ( figure 1 ) (29, 30) . besides, a fraction of mature sars-cov-2 s proteins travel through the secretory pathway to the plasma membrane, where they can mediate fusion of infected with uninfected cells to form multinucleated giant cells (syncytia) (24, 31) . this may allow direct spreading of the virus between cells and potentially alter the virulence of sars-cov-2 (24) . notably, a deletion of~20 amino acid containing the errs from the cytoplasmic tail of the sars-cov-2 s protein has been shown to increase the infectivity of single-cycle vesicular stomatitis virus (vsv)-s pseudotypes (9) and replicationcompetent recombinant vsvs bearing the s glycoprotein (32, 33) , which likely could be translated to single-cycle human immunodeficiency virus (hiv)-s or other retrovirus-s pseudotypes straightforward (33) . presumably, this deletion may enhance the cell surface expression of the sars-cov-2 s glycoprotein (32) , thereby facilitating the s protein incorporation into pseudovirions and replication-competent virions. as mentioned above, the sars-cov-2 s glycoprotein plays pivotal roles in viral infection and pathogenesis. mature s glycoprotein on the viral surface is a heavily glycosylated trimer, each protomer of which is composed of 1260 amino acids (residues 14-1273) ( figure 2a) . the surface subunit s1 is composed of 672 amino acids (residues 14-685) and organized into four domains: an n-terminal domain (ntd), a c-terminal domain (ctd, also known as the receptor-binding domain, rbd), and two subdomains (sd1 and sd2) ( figure 2a ) (34) . the transmembrane s2 subunit is composed of 588 amino acids (residues 686-1273) and contains an n-terminal hydrophobic fusion peptide (fp), two heptad repeats (hr1 and hr2), a transmembrane domain (tm), and a cytoplasmic tail (ct), arranged as fp-hr1-hr2-tm-ct ( figure 2a ) (34) . as a typical class i viral fusion protein (35) , the sars-cov-2 s glycoprotein shares common structural, topological and mechanistic features with other class i fusion proteins, including hiv envelope (env) glycoprotein and influenza virus haemagglutinin (ha) (36) (37) (38) . like other class i viral fusion proteins, the sars-cov-2 s glycoprotein is also a conformational machine that mediates viral entry by rearranging from a metastable unliganded state, through a prehairpin intermediate state, to a stable postfusion state (38, 39) . since the first genome sequence of sars-cov-2 became publicly available (40) , a number of structures have been determined for the sars-cov-2 s glycoprotein trimer fragments in both the prefusion and postfusion states ( figures 2b-d) (11, 34, 41) . the overall architecture of the prefusion sars-cov-2 s ectodomain stabilized by two consecutive proline mutations in two conformations determined by single particle cryo-electron microscopy (cryo-em) is a~160 å long trimer with a triangular cross-section, with the s1 subunit adopting a "v" shape contributing to the overall triangular appearance and the s2 subunit forming the stalk ( figures 2b, c) (11, 34) . the structural difference between these two conformations only lies in the position of one of the three s1 rbds ( figures 2b, c) (11) . when all three rbds are in the "down" position, the resulting s ectodomain trimer assumes a closed conformation, in which the receptor-binding surface of the s1 rbd is buried at the interface between protomers and cannot be accessible by its receptor ( figure 2b ) (11) . the s ectodomain trimer with one single rbd in the "up" position assumes a partially open conformation and represents the functional state, as the receptorbinding surface of the "up" rbd can be fully exposed ( figure 2c ) (11, 34) . the structural information provides a blueprint for structure-based design of vaccine immunogens and entry inhibitors of sars-cov-2. in the closed sars-cov-2 s ectodomain trimer, interprotomer interactions occur through the s1 ctd packed against the other two s1 ctds and one ntd from an adjacent protomer because of domain swapping and through s2, primarily between helical interactions formed by the upstream and central helices from each subunit around the trimer axis ( figure 2b ) (11) . the s1 subunits rest above the s2 trimer, the life cycle of sars-cov-2 begins with membrane fusion occurring at the plasma membrane or within acidified endosomes after endocytosis, which is mediated by conformational changes in the s glycoprotein triggered by angiotensin-converting enzyme 2 (ace2) binding. following viral entry, sars-cov-2 releases its genomic rna into the host cell cytoplasm. genome rna is first translated into viral replicase polyproteins (pp1a and 1ab), which are further cleaved by viral proteases into a total of 16 nonstructural proteins. a replication-transcription complex (rtc) is formed based on many of these nonstructural proteins. in the process of genome replication and transcription mediated by rtc, the negative-sense (− sense) genomic rna is synthesized and used as a template to produce positive-sense (+ sense) genomic rna and subgenomic rnas. the nucleocapsid (n) structural protein and viral rna are replicated, transcribed, and synthesized in the cytoplasm, whereas other viral structural proteins, including the s protein, membrane (m) protein and envelope (e) protein, are transcribed and then translated in the rough endoplasmic reticulum (rer) and transported to the golgi complex. in the rer and golgi complex, the sars-cov-2 glycoprotein is subjected to co-translational and post-translational processing, including signal peptide removal, trimerization, extensive glycosylation and subunit cleavage. the n protein is subsequently associated with the positive sense genomic rna to become a nucleoprotein complex (nucleocapsid), which together with s, m, and e proteins as well as other viral proteins, is further assembled and followed by budding into the lumen of the er-golgi intermediate compartment (ergic) to form mature virions. finally, the mature virions are released from the host cell, waiting for a new life cycle to start. this figure is adapted from the template in biorender (https://biorender.com/). stabilizing the later in the prefusion conformation ( figure 2b ) (11) . when the s ectodomain trimer adopts a partially open conformation, the rbd in the "up" position will abolish the contacts with the s2 subunit of an adjacent protomer, destabilizing the partially open conformation ( figure 2c ) (11, 34) . this will be beneficial to the dissociation of the s1 subunit and facilitate conformational rearrangements that the s2 trimer undergoes to mediate viral entry. prefusion structures of human coronavirus hku1 (hcov-hku1) and mouse hepatitis virus s protein ectodomains without two consecutive proline mutations reveal only fully closed conformation (37, 42) , similar to that observed for a full-length, wild-type prefusion form of the sars-cov-2 s glycoprotein (41) . notably, it is well established that trimeric prefusion hiv-1 env primarily resides in a closed configuration that is conformationally masked to evade antibody-mediated neutralization (43, 44) and can spontaneously sample a transient, functional configuration (45) . it can thus be speculated that native cov s glycoproteins on mature and infectious virions share a similar conformational masking feature (46) , concealing the receptor-binding surface (for those utilizing ctds as rbds) ( figure 2c ), which is further discussed below. several lines of research have established that angiotensinconverting enzyme 2 (ace2) is an entry receptor for sars-cov-2 (47) (48) (49) . detailed interactions between the sars-cov-2 rbd and its receptor ace have been revealed by several structures of ace2 in complex with rbd (50) (51) (52) (53) . structurally, rbd consists of two subdomains: a core and an external subdomain (51, 52) . an extended loop (residues 438-506), which lies on one edge of the core subdomain, presents a gently concave surface to cradle the n-terminal helix (a1) of ace2. analysis of the interface between the sars-cov-2 rbd and ace2 reveals that a total of 17 residues in rbd are in contact with 20 amino acids in ace2, forming a network of hydrophilic interactions that are suggested to predominate the virus-receptor engagement (51) . outside this extended loop, residue lys417 located in helix a3 of the core subdomain, was shown to form ionic interactions with asp30 of ace2. as the extended loop contains almost all the amino acids of the sars-cov-2 rbd that contact ace2, it is referred to as the receptor-binding motif (rbm) (51) . it has been proposed that inhibiting the interaction between rbd and ace2 might be useful in treating sars-cov-2 infection. recombinant soluble ace2 (54) and ace2-fc (55, 56) have been shown to have potential applications in the prevention and treatment of sars-cov-2 infection in vitro. as the interaction between the rbd and ace2 is extensive, small molecules probably cannot be used as entry inhibitors to effectively block the virus entry by targeting the interaction interface. however, peptides would be able to engage most of the residues belonging to rbm (57) . a pioneering study demonstrated that a 23-amino acid peptide (residues 21-43), derived from the n-terminal helix (a1) of ace2, specifically associates with the sars-cov-2 rbd with low nanomolar affinity and disables receptor interactions (57), representing a promising strategy for preventing the virus from invading human cells. in another study, a 65-amino acid peptide (residues 19-83), derived from the n-terminal back-to-back helices (a1 and 2) and composed of most of the residues of ace2 that mediate interactions with the s protein, shows a similar but probably more potent inhibitory effect (58) . the formation of a trimer-of-hairpins structure (also known as six-helix bundle) comprising hr1 and hr2 in the postfusion conformation is a unifying feature of class i viral fusion proteins (37) . the crystal structure of a protein construct in which sars-cov-2 hr1 and hr2 were connected by a six-residue hydrophilic flexible linker was determined to be a canonical six-helix bundle structure with a rod-like shape ∼115 å in length and ∼25 å in diameter (59) . three hr1 helices form a parallel central coiled-coil with three hr2 helices packing in an oblique, antiparallel manner against deep hydrophobic grooves on the surface of the central coiled-coil (59) . notably, when a full-length s protein construct bearing the native furin-like cleavage site was transiently expressed by expi293f cells, the purified s proteins contained the dissociated s2 trimer in the postfusion conformation (41) . the cryo-em structure of this trimeric postfusion s2 shows that the central helix (ch) extended regular helices from the central coiled-coil, oriented toward target cells ( figure 2d ) (41) , which forms the longest central triple helical coiled-coil (~180å) among all known class i transmembrane subunit structures. the sars-cov-2 s trimer in the pre-hairpin intermediate state is very unstable and is just transiently present in vivo after triggering by ace2 engagement, stymieing structural characterization of the s protein in this state (60) . however, although this fusion-intermediate phase is very short, it is enough for inhibitory peptides to associate with the prehairpin intermediate and block the six-helix bundle formation (39) . furthermore, it has already been shown that the hr1 regions in various human covs are highly conserved (61) , and therefore could serve as an attractive target for the design and development of potent and broad-spectrum inhibitors of pan-covs, including sars-cov-2. a highly potent pan-coronavirus fusion inhibitor, ek1c4, has been reported to have good prophylactic and therapeutic potential against sars-cov-2 infection (59). as mentioned earlier, the sars-cov-2 s proteins are heavily decorated by heterogeneous n-linked glycans projecting from the s trimer surface. the sars-cov-2 s sequence encodes up to 22 n-linked glycan sequons per protomer, which likely plays an important role in protein folding (19) and host immune evasion as a glycan shield (62) . of the 22 potential n-linked glycosylation sites on the s protein, 14 were identified to be predominantly occupied by processed, complex-type glycans (63) . the remaining eight sites were found to be dominated by oligomannose-type glycans, which are divergent from those founded on host glycoproteins (63) . although glycosylation sites (n165, n234, n343) proximal to the receptor-binding sites on the sars-cov-2 s protein can be observed, ace2 bound to the glycosylated and deglycosylated s ectodomains with nearly identical affinity (1.7 nm vs 1.5 nm) determined by a biolayer interferometry binding assay (64) . this observation suggests that the high binding affinity between the sars-cov-2 s protein and ace2 does not depend on the s protein glycosylation. when the site-specific n-linked glycans are mapped onto the prefusion structure of the sars-cov-2 s ectodomain (63), the resulting model exhibited substantially higher levels of glycanfree surface than that revealed by structures of fully glycosylated, trimeric hiv-1 env ectodomains (65, 66) . this suggests that the sars-cov-2 s protein is covered by a less dense and less effective glycan shield compared to viral glycoproteins from hiv-1 (36, 66) and lassa virus (67) , which may be beneficial for the induction of humoral immunity and could be good news for a sars-cov-2 vaccine (68) . notably, it has been shown that multiple major viral surface antigens have neutralizing epitopes that are partly or even exclusively composed of carbohydrate moieties (69, 70) , exemplified by the hiv-1 env spike, which could be recognized by a large number of carbohydrate-binding antibodies, including 2g12, pg9, pg16, ch04, pgt121, pgt128, pgt135, and pgt145 (70, 71) . in the case of sars-cov-2, more recently a potent neutralizing antibody against both sars-cov and sars-cov-2, s309, has been shown to recognize a highly conserved glycan-containing rbd epitope (72) . these observations suggest that carbohydrate moieties could be immunogenic and highlight the need for immunogens to display the glycans important for the recognition of neutralizing antibodies (73) ; in support of this, specific n-linked glycans on hemagglutinin has been shown to be essential for the elicitation of broadly neutralizing antibodies against influenza (74) . accordingly, there has been mounting interest in exploring the potential of immunogenic glycan moieties as vaccine candidates against multiple viruses, including sars-cov-2 (75, 76) . membrane fusion and viral entry of sars-cov-2 is initiated by binding of rbd in the viral s glycoprotein transiently sampling the functional conformation to ace2 on the surface of target cells (figure 1 ) (10). after receptor engagement at the plasma membrane or ensuing virus endocytosis by the host cell (8), a second cleavage (s2′ cleavage site) is generated, which is mediated by a cellular serine protease tmprss2 (48) or endosomal cysteine proteases cathepsins b and l (10) (figure 1) . protease cleavage at s2′ site frees the fusion peptide from the new s2 n-terminal region, further destabilizes the sars-cov-2 s glycoprotein and may initiate s2-mediated membrane fusion cascade. following the second cleavage, the fusion peptide at the n terminus of the s2 trimer is inserted into the host membrane (8) , forming the pre-hairpin intermediate state (39) . since the pre-hairpin intermediate state is extremely unstable, the s2 fusion protein is refolded quickly and irreversibly into the stable postfusion state (39, 77) . these large conformational rearrangements pull the viral and host cell membrane into close proximity, leading ultimately to the membrane fusion (8, 39) . since sars-cov-2 was identified as the causative agent of covid-19, and its first genome sequence was released immediately and freely by a chinese research group (40), sars-cov-2 vaccine candidates based on various vaccine platforms, such as inactivated or live attenuated vaccines, dna and mrna vaccines, viral vector-based vaccines, and recombinant protein-based vaccines, have been developed (12, 78) . most of these vaccine strategies are based on the full-length s glycoprotein, the major viral surface antigen (12) . when a vaccine strategy requires that the sars-cov-2 s protein be recombinantly expressed in the human body, the errs should be omitted to enhance the cell surface expression level of the resulting protein. theoretically, the native hiv-1 env trimer present on the surface of intact virions is thought to be a most ideal immunogen (60) , as most of the neutralizing antibodies thus far described could recognize and bind to the prefusion form of trimeric hiv-1 env, although it is with great difficulty that such neutralizing antibodies against this glycan-covered, sequence-variable native form are induced (36) . for sars-cov-2, different lines of research have shown that convalescent sera from sars-cov and sars-cov-2 patients showed no or limited crossneutralization activity against these two viruses by pseudotyped and authentic viral infection assays, despite significant crossreactivity in binding to the s glycoproteins of both viruses (9, (79) (80) (81) . similar results were also observed in infected or immunized animals (48, 79, 81) . together with the finding that although the sars-cov-2 s protein shares a high degree of amino acid sequence identity with that of sars-cov (~76% overall), the rbm is less conserved (~47% identity) than any other functional region or domain (82) , it can thus been surmised that the rbm has the most immunodominant neutralizing epitope(s) of the whole s protein, capable of readily eliciting strong neutralizing antibody responses. however, the native trimeric sars-cov-2 s protein could conceal each of its immunodominant rbms by adopting the closed conformation (41, 83) . therefore, sars-cov-2 evades immune surveillance also through conformational masking, which is well-documented for hiv-1 (43, 44) ; while at the same time, the s protein could transiently sample the functional state to engage ace2, consistent with the notion that the fusion glycoprotein of highly pathogenic viruses have evolved to perform its functions while evading host neutralizing antibody responses. another concern for vaccine candidates based on the fulllength s glycoprotein of sars-cov-2 is raised by the observation that the s1 subunit could spontaneously dissociate from the s glycoprotein probably as a trimer that still assumes the rbd closed conformation, leaving only the postfusion s2 trimer (41) . the resulting s1 and s2 subunits might expose immunodominant, nonneutralizing epitopes that are utilized by sars-cov-2 to serve as decoys to distract the host immune system, inducing a large proportion of ineffective antibody responses, as documented for hiv-1 (60) and respiratory syncytial virus (rsv) (84) . it should be noted that although vaccine candidates based on the full-length s protein of the closely related sars-cov could elicit neutralizing antibody responses against infection of sars-cov, they may also induce harmful immune responses, including liver damage of the vaccinated animals, infection of human immune cells by sars-cov, and antibody-dependent enhancement of sars-cov infection (85) (86) (87) (88) (89) . therefore, although the s proteins of both sars-cov and sars-cov-2 are thought to be promising vaccine immunogens for generating protective immunity, optimizing antigen design is critical to ensure an optimal immune response through exposing more neutralizing epitopes and displaying fewer potentially weakly or non-neutralizing epitopes (90) . vaccines containing or expressing the full-length s protein or its soluble ectodomain form should thus be engineered to sample a rbd(s) "up" conformation while the rest is still kept in the prefusion state (91, 92) . apart from recombinant, soluble, stabilized ectodomains that are engineered to expose the immunodominant rbd by adapting the rbd(s) "up" conformation, rbd proteins of sars-cov and sars-cov-2 have also been widely used as recombinant protein-based vaccines (85, (93) (94) (95) . the rbd of sars-cov is highly immunogenic (96, 97) and is targeted by most of the neutralizing monoclonal antibodies that have been characterized (98) . based on the observation that a 193-amino acid fragment (residues 318-510) was previously identified to be the minimal rbd region of sars-cov (99), a corresponding 194-amino acid fragment (residues 331-524) can be readily selected as the minimal rbd region of sars-cov-2 and has already been characterized (100) . this minimal form of rbds of both viruses could serve as a vaccine candidate (100) . however, a conserved cysteine residue is located immediately upstream of the minimal rbd fragments of both viruses and always forms a disulfide bond in nearly all published structures containing this residue (101, 102) ; this is also the case for middle east respiratory syndrome coronavirus (mers-cov) (103, 104) and hcov-hku1 (37), consistent with the observation that all rbds of these viruses share a conserved structural core. the disulfide bond contributes to stabilization of the rbd structure and likely modulates the protein immunogenicity. this notion is consistent with the observation that mice immunized with a longer form of the sars-cov rbd (residues 318-536) produced a higher titer of neutralizing antibodies compared with mice immunized with the minimal rbd region (residues 318-510) (105) . therefore, when each of the minimal rbd fragments of sars-cov and sars-cov-2 is used as vaccine candidates, the critical cysteine residue should not be ignored and thus should be included (106) . besides the rbd, which has been shown to a major target for human neutralizing antibody responses (107), the ntd was recently identified to be a new vulnerable site of the sars-cov-2 s protein for antibody neutralizing and therefore could also serve as a recombinant protein-based vaccine (108) (109) (110) . as expected, ntd-specific neutralizing antibodies could target the s protein in both closed and open conformations (108) . in addition, the apparent accessibility of the fusion peptide and hr1 region in published structures of the sars-cov-2 s ectodomain trimer as well as their high sequence conservation among covs suggests that they would be good immunogen candidates for epitope-focused vaccine design aimed at raising broadly cov neutralizing antibodies (46) . the epitope-focused vaccine design has proven to be successful in generating neutralizing antibodies against rsv fusion glycoprotein (111). however, neutralizing antibodies targeted against these two regions still need to be isolated in infected individuals to support this notion. unlike wild-type full-length s protein of sars-cov-2, the above monomeric fragments do not induce any infection-enhancing antibodies or harmful immune or inflammatory responses (106, 112) , all of which could be potentially avoided through structure-based immunogen design to improve immunogenicity (113, 114) . however, wide-type full-length or soluble ectodomain form of the sars-cov-2 s protein could trigger stronger cellular immune responses (115) , which have been demonstrated to play an important role in controlling diseases caused by covs (116, 117) , including sars-cov-2 (118) , and are probably also an important determinant of effective vaccines against sars-cov-2 (115, 119) . additionally, when more than one rbd of the s protein trimer is engineered to be locked in the "up" conformation (120, 121) , the antigenicity and immunogenicity of the resulting rbds would be significantly enhanced compared to monomeric rbd form (97, 122) . moreover, improved protection is likely to be achieved when vaccinated with full-length or soluble ectodomain form of the sars-cov-2 s protein in that both forms can elicit neutralizing antibodies directed against non-rbd sites, as observed for mers-cov (123) . genetic variation has been used by many viruses that have rna genomes (124) , including hiv and influenza, as a mechanism to avoid antibody-mediated immunity, and is partially responsible for the great difficulty in developing effective and durable vaccines against these viruses (36) . as an rna virus, however, sars-cov-2 has a very low mutation rate overall (125) likely because covs have a genetic proofreading mechanism (126) . all reported variations occurred in the sars-cov-2 s glycoprotein have a prevalence of no more than 1% (127) , with an exception of d614g, which has become the most prevalent genotype in the global covid-19 pandemic (127) . fortunately, although the d614g mutation of the sars-cov-2 s protein has been shown to enhance viral infectivity (128) (129) (130) , until now there is no evidence that infection with sars-cov-2 carrying the g614 mutant will be associated with disease severity (127, 131) . furthermore, assays using both monoclonal and polyclonal antibodies generated from individuals naturally infected with d614-or g614-carrying viruses demonstrated that the d614g mutation retains or even increases viral susceptibility to neutralization (127, 130, 132, 133) . this suggests that the d614g mutant maintains or favors an open, functional conformational state (134) . although at an extremely low frequency, natural variations, including l452r a475v, v483a, and f490l that render the s glycoprotein resistant to certain neutralizing antibodies targeting the rbd, emerged under no selection pressure exerted by approved vaccines or neutralizing antibodies or entry inhibitors (127, 132) . however, it has been shown that sars-cov-2 escape mutants could be easily selected and quickly amplified under the selection pressure of single antibody treatment (135) . these observations suggest that a combination of at least two neutralizing antibodies that recognize and bind to distinct and non-overlapping epitopes on the sars-cov-2 s glycoprotein (e.g., rbd and ntd, as well as hr and glycan) is required to restrict the possible occurrence of viral escape mutants and potential subsequent loss of single antibody-mediated neutralization (135) (136) (137) (138) . when these observations are taken into consideration for vaccine design and development, an ideal sars-cov-2 immunogen should contain as many exposed neutralizing epitopes as possible, although the rbd also possesses extra epitope(s) besides the epitope in the rbm region (72, (139) (140) (141) . sars-cov-2 is a highly contagious pathogen that continues to spread quickly around the globe, causing covid-19 to be one of the worst pandemics in recorded history. a safe and efficacious vaccine represents one of the best ways to reduce or eliminate the covid-19 pandemic (142) . unfortunately, no vaccines for any of the known human covs have been licensed (143, 144) , although several potential sars-cov and mers-cov vaccines have advanced into human clinical trials for years (117, 145) , suggesting the development of effective vaccines against human covs has always been challenging. however, it has been shown that both sars-cov and sars-cov-2 could readily induce neutralizing antibodies following natural infection or immunization (146) (147) (148) (149) . moreover, a growing number of neutralizing monoclonal antibodies targeting the sars-cov-2 s glycoprotein with 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coronavirus vaccines against coronaviruses: the state of the art. vaccines (basel) (2020) 8:309 immunogenic profile of sars-cov-2 spike in individuals recovered from covid-19. medrxiv immunogenicity of a dna vaccine candidate for covid-19 dna vaccine protection against sars-cov-2 in rhesus macaques antibody signature induced by sars-cov-2 spike protein immunogens in rabbits progress and prospects on vaccine development against sars-cov-2. vaccines (basel) (2020) 8:153 what are the most powerful immunogen design vaccine strategies? reverse vaccinology 2.0 shows great promise structure-based vaccine antigen design all authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. we would like to thank prof. xinqi liu for critical reading of the manuscript; and drs. yanbin feng, mengyuan xu, jing ma and jianrong feng for helpful comments and discussions on the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 duan, zheng, zhang, niu, lou and wang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-330343-p7a8chn4 authors: kelly-cirino, cassandra; mazzola, laura t; chua, arlene; oxenford, christopher j; van kerkhove, maria d title: an updated roadmap for mers-cov research and product development: focus on diagnostics date: 2019-02-01 journal: bmj glob health doi: 10.1136/bmjgh-2018-001105 sha: doc_id: 330343 cord_uid: p7a8chn4 diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. ► the middle east respiratory syndrome-coronavirus is a high-priority pathogen identified by the who r&d blueprint because of its high fatality rate, large geographical range of the dromedary camel reservoir and lack of medical interventions. ► accurate and accessible diagnostic tests are essential to outbreak containment and case management, as well as surveillance in both humans and animals, but available diagnostic tests are limited by inconsistent quality assessment, specimen acquisition issues and infrastructure requirements. ► diagnostic research and development (r&d) needs to include point-of-care testing options, syndromic panels for differential diagnosis, a greater understanding of viral and antibody kinetics, improved access to clinical specimens, and establishment of international reference standards. diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. the middle east respiratory syndrome-coronavirus (mers-cov) is an emerging virus associated with severe respiratory illness, first detected in 2012 in saudi arabia. 1 5 provides an overview to the current status of mers-cov diagnostics, including feedback from subject matter expert and developer interviews on the common challenges with test development and implementation, and identifies gaps for further research and development (r&d). mers-cov is a zoonotic virus, and dromedary camels (camelus dromedarius) are the reservoir host and the source of zoonotic transmission to humans. [6] [7] [8] dromedaries appear to be only mildly symptomatic following infection and present a significant reservoir risk for spillover events. 2 6 9 mers-cov rna has been detected in dromedary camels in a number of countries, including egypt, oman, qatar and saudi arabia, with evidence suggesting that mers-cov is also widespread in the middle east, africa and south asia. 5 8 10-35 infection in camels is notifiable to the oie. 36 individuals with close and frequent contact with dromedaries are at a higher risk for mers-cov infection than the general population. 37 38 clinical indications and management coronaviruses are a family of viruses that can cause diseases in humans, ranging from the common cold to severe acute respiratory syndrome (sars). the clinical spectrum of mers ranges from no symptoms (or asymptomatic infection), mild symptoms including fever, cough, gastrointestinal illness and shortness of breath, to severe disease including pneumonia, acute respiratory distress syndrome and death. 2 39 severe cases of mers can result in respiratory failure, requiring mechanical ventilation and support in intensive care. risk factors for severe disease include a weakened immune system, older age (>60 years), and comorbidities such as diabetes, cancer, renal disease and chronic lung disease. 40 41 human-tohuman transmission spreads through close and unprotected human contact, and more than half of reported mers cases have occurred through nosocomial transmission. [42] [43] [44] [45] to prevent nosocomial infections, who and others recommend using standard infection and prevention control measures when caring for patients. [46] [47] [48] who also recommends that contact tracing of all symptomatic and asymptomatic close contacts of the primary patient should be conducted routinely. 49 the molecular epidemiology for mers-cov has not changed significantly since the initial human cases were detected in 2012. the current virus remains 99% identical to the sequences seen in the first human cases from 2012 as well as archived camel sera from 1983, with no increase in pathogenicity observed in the animal host. [50] [51] [52] as genetic mutations could impact detection, bmj global health immunotherapy and vaccine development efforts, 53 sequencing of mers-cov strains from camels and humans (after a zoonotic spillover) is important and is regularly being conducted in affected member states (who, personal communication, 2018). there are currently no prophylactic or therapeutic interventions of proven efficacy for any coronavirus infections. without a specific therapy for mers, treatment is supportive. 5 54 55 effective mers therapeutics are still in the early stages of research and evaluation. several broad-spectrum antiviral agents including nitazoxanide, 56 viral methyltransferase inhibition 57 and nucleotide prodrugs 58 have shown in vitro activity against mers-cov. early results for novel mers-specific therapeutics that inhibit viral replication or have specific neutralising activity are promising. 47 59 60 the who r&d blueprint for mers has called for three types of vaccines: (1) dromedary camel vaccine to prevent zoonotic transmission, (2) human vaccine for long-term protection of persons at high exposure risk and (3) human vaccine for reactive use in outbreak settings. 55 61 mers-cov vaccines are in the early stages of development, 55 62 63 with one candidate vaccine in phase i clinical trials (nct02670187). 64 neutralising monoclonal antibodies have been designed to target the mers-cov spike protein, 53 65 with chadox1 and modified vaccinia ankara vectors also strong vaccine candidates, 60 66 but none have yet advanced to clinical trials. to accelerate the process, the coalition for epidemic preparedness innovation has recently launched a call for proposals for the development of a human mers-cov vaccine in order to engage with developers interested in supporting these efforts. 67 the who laboratory guidelines recommend nucleic acid amplification tests (naat) for diagnosis, using serology for diagnosis only when naat is not available. 68 in suspected patients, a single negative test result does not exclude diagnosis. repeat sequential sampling and testing is strongly recommended. the kinetics of mers-cov infection has been shown to vary widely across cases, 40 69-72 prompting a more detailed investigation of viral and antibody dynamics across the broad range of sample types, disease states and host factors. 73 74 the best naat test sensitivity is achieved using specimens from the lower respiratory tract (sputum, tracheal aspirates or bronchoalveolar lavage), where mers-cov replication occurs at higher and more prolonged levels of mers-cov rna, typically between 10 6 and 10 10 copies/ml. 72 75 mers-cov viral load is generally higher for severe cases, with more prolonged viral shedding than mild cases. viral load concentrations, which may be undetectable at early-stage infection, generally peak in the second week after symptom onset, and then drop to undetectable in survivors by the fourth week from onset. upper respiratory tract specimens (nasopharyngeal or oropharyngeal swabs) may also be used, but demonstrate 100×-1000× lower viral load and can test negative for mild cases. 76 77 if possible, both upper and lower respiratory tract sampling are advised. specimens outside the respiratory tract are not recommended for diagnosis, as they can test negative in both severe and mild presentation. viral rna has been detected in stool samples (10 4 copies/ml), serum samples (10 3 copies/ml) and urine (10 2 copies/ml), more likely an indicator of severity as it typically precedes a poor clinical outcome. 71 76 78 serological diagnosis can be made using paired samples, more often used for research rather than diagnostic purposes, preferably with the initial sample collected in the first week of illness and the second collected 3-4 weeks later. if only a single serum sample can be collected, this should occur at least 3-4 weeks after onset of symptoms for determination of a probable case. table 1 presents an overview of the implementation requirements for mers-cov diagnostics (detailed commercial product information is presented in online supplementary tables s1 and s2). molecular diagnostics such as naat (eg, pcr) typically require sophisticated laboratory infrastructure including biosafety cabinets, 79 while most serological tests (elisa, indirect immunofluorescence test (iift)) can be run on the benchtop in a more modest laboratory environment, depending on sample preparation precautions. 80 81 point-of-care (poc) tests are designed to be used outside of a traditional laboratory; near-poc tests are defined for rapid use in a laboratory near the patient, but are more automated and easy to use than the traditional laboratory test. 72 75 poc tests such as low-complexity rapid diagnostic tests (rdts) can be used at the bedside, typically with non-invasive samples after minimal training. inhouse tests are described in sections below; commercial sources are listed in online supplementary tables s1 and s2. naats are currently the standard for mers-cov diagnosis, as these tests (typically reverse transcriptase pcr (rt-pcr)) have the highest sensitivity at the earliest time point during the acute phase of infection. following the who guidelines, two different targets on the mers-cov need to be detected by rt-pcr to confirm a case. mers-cov assays to detect the upstream envelope gene (upe) followed by confirmation of open reading frame 1a (orf1a), 1b (orf1b) genes or nucleocapsid (n) genes for confirmation have been developed. 55 82 most commercial pcr tests perform parallel screening for the upe gene with confirmation by the orf1a, orf1b or n genes (most commonly upe + orf1a). initial naat tests for mers-cov were developed as inhouse tests, following the first detection of mers-cov in the middle east. [83] [84] [85] [86] inhouse tests are not necessarily subject to quality control or regulation, and may not be rigorously validated; in some cases, inhouse tests are eventually developed into commercial products through collaboration and licensing efforts. 50 83 84 87-89 commercial assays may undergo an international and/or incountry regulatory process; once on the market they can be independently evaluated for sensitivity, specificity and limit of detection. 78 90 as of 2018, there are several commercial naat tests available for mers-cov, including duplex and multiplex panels (see online supplementary table s1). serology is not widely performed for diagnosing acute mers-cov infection; however, it has been a useful tool bmj global health to determine the extent of infection around clusters and in seroepidemiological studies in animals and humans. seroconversion typically occurs during the second and third week after symptom onset; data suggest that low antibody titre in the second week or delayed seroconversion is more closely associated with mortality than high viral load. 71 74 mers-cov seroconversion may not be observed for some patients, notably with mild or asymptomatic infection, and can show cross-reactivity with antibodies to other coronaviruses. 42 69 serological methods for the detection of antibodies against mers-cov include elisa, iift and neutralisation tests. mers-cov serological assays can employ commercial reagents or proprietary monoclonal antibodies as capture agents. 87 91 92 many mers-cov elisa tests are labelled for research use only, with little or no clinical validation data available. similar to the elisa, iift is used when it is difficult to evaluate specific antigens individually by enzyme immunoassays or there is a preference for broader analysis of an immobilised specimen. iift microscopy assay can probe the entire antigen spectrum of the specimen, and is often designed for simultaneous detection of antibodies against biochemically distinct antigens. neutralisation is a method for detecting anti-mers-cov antibody activity via inhibition of infection or replication, 69 93 performed as plaque reduction neutralisation, microneutralisation (mn) and pseudoparticle neutralisation (ppnt). mn is labour-intensive and slow, requiring at least 3-5 days for results; neutralisation techniques other than ppnt require biosafety level 3 containment as they involve live virus cultures. 94 rdts can leverage the same antibody/antigen capture agents as elisa but in a lateral flow strip cartridge. 95 this enables a fast 10-30 min time to result, but with a 100-fold lower detection sensitivity than elisa. 91 92 follow-up confirmatory testing is therefore required. rdts are typically paired with minimally invasive specimen collection (blood, oral fluid, nasal swabs) so that they can be used with minimal training outside of laboratory settings. early prototypes for mers-cov rdts have been developed, 87 92 96 with commercial rdts for detection of mers-cov in camels and humans available (online supplementary table s2). the human mers-cov rdt does not appear to be widely used, perhaps due to the more invasive processing required for lower respiratory specimens, as well as sensitivity issues for upper respiratory specimens. the camel mers-cov rdt is used with upper respiratory specimens; however, test sensitivity varies depending on specimen sampling and infection kinetics. 97 multiplex panels at the early stages, the symptoms of mers-cov infection can mimic diseases such as influenza, pneumonia, sars and other respiratory infections. a syndromic approach involves testing for pathogens based on a syndrome such as fever or acute respiratory distress; a shift from individual tests to multiplex panels can quickly identify or eliminate likely pathogens from a single specimen. for analysis of circulating reservoirs, multiplex microbead-based immunoassays have been used to detect igg antibodies for multiple pathogens. 98 99 multiplex, syndromic panels that include mers-cov have been demonstrated using pcr-based panels including mers-cov, showing similar limits of detection to single assays. 89 100 101 commercial respiratory panel tests including mers-cov have also recently been developed (see online supplementary table s1). there is a need for international consensus and adoption of minimum standards for tests used in diagnosis, surveillance and research, following who's recommended algorithm for human cases 82 and oie recommendation for animal health. 36 harmonisation of the testing process can be achieved by building consensus and capacity across international and incountry laboratories. in order to enable and sustain the capacity for a rapid outbreak response, laboratories must have access to high-quality reagents and instrumentation, along with technical support and cold-chain transport when necessary. in addition, international reference panels would achieve a more standardised training for external quality assessment (eqa) and quality control. building on mandatory case reporting, 102 an international mers-cov data sharing platform that includes case exposure history and sequence data would greatly facilitate the knowledge base across the mers-cov community. [103] [104] [105] [106] clinical validation understanding mers-cov viral dynamics across a broad range of specimen types is critical to establishing the limits of detection and timing of diagnostics in order to make the greatest impact for diagnosis, case management and surveillance. ensuring a test has appropriate sensitivity and specificity is a major challenge in the development of diagnostics for novel and rare pathogens, as there is often a very limited supply of well-characterised clinical material. especially during the early stage of an outbreak, clinical evaluation must often be performed in the affected countries by laboratories working closely with the ministries of health. typically only a small number of patient specimens are shared outside of the affected countries due to strict import and export regulations, particularly for 'dual-use' pathogens. 107 108 specifically, the provisions of the nagoya protocol have significant impact on the access to genetic materials for both commercial and non-commercial applications. 109 110 in particular, the development and validation process for new diagnostics could be accelerated if well-characterised specimens and reference standards could be more easily obtained. eqa can be useful for evaluation of test performance, as shown with evaluations of both inhouse and commercial assays for mers-cov, [111] [112] [113] and bmj global health more recently a global proficiency testing programme used to assess laboratory detection of mers-cov. 114 even after validation, a substantial amount of reference material is required for quality control; often manufacturers must develop their own calibration standards to maintain supply and to control lot-to-lot variability. international reference standards and qualified specimen panels can accelerate the development and validation of diagnostic tests. in particular, the who international biological reference preparations (as provided by member states) serve as reference sources for ensuring the reliability of in vitro biological diagnostic procedures used for diagnosis of diseases and treatment monitoring, including mers-cov. several international institutes also provide specimens for validation; these groups typically have a defined pathogen/disease focus with a corresponding archive of biological reference materials; however, the supplies may be limited (see online supplementary material 1). currently, mers-cov diagnosis by pcr requires a laboratory with sophisticated facilities and biosafety cabinets. the turnaround time to receive a test result can take days to weeks, depending on laboratory proximity, sample transport options and laboratory processing capacity, 72 75 and infrastructure requirements place most pcr systems in reference laboratories, which may not be ideal for diseases like mers-cov that recommend immediate isolation for infections detected across a variety of settings. 81 115 116 a more nimble approach is needed for mers-cov case detection and triage, 92 117 and at border crossings for animal surveillance, quarantine and targeted vaccination. 11 21 87 118 the fao-oie-who mers technical working group has given a clear call for the development of an rdt to improve identification and isolation of primary human cases in healthcare facilities. 5 serological rdts are ideal for low infrastructure settings such as a primary health clinic, home or field testing. however, specimen collection remains a key challenge for mers-cov, as the recommended lower respiratory specimens are difficult to obtain outside of a hospital setting. upper respiratory specimens such as nasal swabs are easy to obtain and work well in conjunction with rdts for camels, but these specimens generally have low virus titre in humans, thus limiting current use of rdts to animal testing. 87 92 96 improvement of the current rdt detection chemistry, if feasible, may support the future use of these tests in humans, at least for rapid triage in highly infectious cases. poc and near-poc microfluidic platforms enable a more flexible, but still highly sensitive approach for near-patient naat testing in decentralised settings. near-poc naat platforms are compact and self-contained, with automated sample preparation for processing in minimal laboratory settings, which most healthcare workers can be trained to operate within a day. [119] [120] [121] recent publications describe mers-cov assays designed for poc pcr, 89 loop-mediated isothermal amplification assay 122 and paper-based sensor detection 123 ; however, no mers-cov assays are currently available for the existing near-poc platforms. given that pcr is now the standard for mers-cov diagnosis, it would be highly desirable to have an automated, self-contained naat assay that can be readily deployed in a field or clinic setting. syndromic testing can be valuable during the early stages of an outbreak, in order to distinguish mers-cov from other respiratory infections or identify cases of coinfection. 100 124 a syndromic panel could be effective in expediting pathogen and outbreak identification, especially with technologies that can screen for multiple pathogens simultaneously. 125 using the panel approach, a definitive diagnosis could enable timely decisions about triage, treatment, infection control and contact tracing. 126 while the per-test cost rises with test complexity, including additional reagents and more sophisticated instrumentation, a rapid and efficient diagnosis scheme can impact intervention and infection control and can be cost-saving overall. 127 128 as respiratory diseases are both regional and seasonal, 129-131 region-specific panels may be more cost-effective. 132 multiplex panels offer the alternative for a 'bundled' testing paradigm; however, if not routinely used (if the market is small), then developers may be reluctant to support the test for diagnostic use, which requires additional investment for validation and regulation. surveillance can be an effective method to identify the initial stages of outbreak, but it requires routine and effective sampling. the impact of surveillance testing depends on the test sensitivity and specificity, sampling rates, kinetics of the disease, and whether the target is animal or human populations. most surveillance sampling is performed in the field, either through population-based or 'hot spot' sampling. for mers-cov, it may be difficult and expensive to implement routine surveillance in dromedary camel stock, as they represent a significantly large reservoir but may suffer only mild effects from mers-cov infection, if any. the ideal surveillance tool would be a highly sensitive and field-appropriate screening test. per-test cost is also an important factor along with ease of implementation. this review has identified diagnostics currently available for mers-cov and highlighted ongoing challenges caused by critical gaps in diagnostics to support outbreak management. rdts offer the potential for rapid poc screening for mers-cov; however, there are practical limits to implementing lower respiratory sample acquisition outside of a hospital setting, limiting feasibility. poc or near-poc naat platforms provide an opportunity for implementation of automated, self-contained bmj global health testing in hospitals and clinics with limited training in endemic-prone areas. expansion of test menu options for existing poc or near-poc naat platforms will strengthen incountry response capacity to endemic diseases and simultaneously ensure countries are prepared for future pandemics. syndromic multiplex panels may expedite differential diagnosis of mers-cov from other endemic respiratory diseases, but further analysis is needed to inform implementation and cost-effectiveness in the context of regional and seasonal detection. there is also a need for more sensitive serological assays with lower cost and minimum cross-reactivity that can be used as surveillance tools. a more detailed understanding of mers-cov viral and antibody kinetics is needed across the broad range of sample types in order to optimise the use of existing assays and to address ongoing technical challenges in the detection of mild and asymptomatic infections. surveillance continues to be important for the detection of mers-cov spillover events; however, questions remain 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diseases in the asia pacific region point-counterpoint: large multiplex pcr panels should be first-line tests for detection of respiratory and intestinal pathogens cost analysis of multiplex pcr testing for diagnosing respiratory virus infections impact of a rapid respiratory panel test on patient outcomes pcr for detection of respiratory viruses: seasonal variations of virus infections global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study prevalence and seasonal distribution of respiratory viruses during the 2014 -2015 season in istanbul development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay acknowledgements we gratefully acknowledge input to the roadmap from all those who attended the fao-oie-who global technical meeting on mers-cov in september 2017. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organisations with which they are affiliated. editorial assistance for later drafts was provided by rachel key: cord-331429-mh2hd5fe authors: srikantiah, padmini; charles, myrna d.; reagan, sarah; clark, thomas a.; pletz, mathias w.r.; patel, priti r.; hoekstra, robert m.; lingappa, jairam; jernigan, john a.; fischer, marc title: sars clinical features, united states, 2003 date: 2005-01-17 journal: emerg infect dis doi: 10.3201/eid1101.040585 sha: doc_id: 331429 cord_uid: mh2hd5fe we compared the clinical features of 8 u.s. case-patients with laboratory-confirmed severe acute respiratory syndrome (sars) to 65 controls who tested negative for sars coronavirus (sars-cov) infection. shortness of breath, vomiting, diarrhea, progressive bilateral infiltrates on chest radiograph, and need for supplemental oxygen were significantly associated with confirmed sars-cov infection. t he clinical course and outcomes of cases of severe acute respiratory syndrome (sars) in asia and canada have been well described (1) (2) (3) (4) (5) (6) . most of these studies defined cases based on clinical and epidemiologic criteria with or without laboratory evidence of sars-associated coronavirus (sars-cov) infection. in the event of a subsequent outbreak, distinguishing clinical features associated with sars-cov infection may help inform decisions regarding patient evaluation and infection control practices while laboratory results are pending. we describe the clinical characteristics of patients in the united states with laboratory-confirmed sars and compare them to persons who tested negative for sars-cov but had similar illnesses we defined a case-patient as a u.s. resident who met the clinical and epidemiologic criteria for suspected or probable sars and had laboratory evidence of sars-cov infection (7) . laboratory evidence of sars-cov infection was defined as 1) isolation of sars-cov, 2) detection of sars-cov rna by polymerase chain reaction (pcr), or 3) detection of antibodies against sars-cov by using enzyme-linked immunosorbant assay or indirect fluorescent-antibody assay (8, 9) . after obtaining verbal consent, health officials used a standard questionnaire to interview by telephone patients with suspected or probable sars and their healthcare providers. data collected included clinical symptoms, past medical history, relevant exposures, physical examination, radiographic and laboratory findings, and clinical course and outcome. case-patients with laboratory-confirmed sars were compared to a convenience sample of persons who met the clinical and epidemiologic criteria for suspected or probable sars but subsequently tested negative for sars-cov infection. controls had negative findings on all testing performed for sars-cov, including the absence of antibody against the virus in convalescent-phase serum samples obtained >21 days after onset of symptoms. statistical analysis was performed with sas software version 8.2 (sas institute, cary, nc). univariate odds ratios, 95% confidence intervals, and p values for association were calculated by using exact likelihood methods. we identified 8 case-patients with laboratory-confirmed sars-cov infection in the united states. dates of onset of symptoms were from february 22 to may 24, 2003. the median age of case-patients was 43 years (range 22-53 years); 4 were women. two case-patients were pregnant (8 weeks' and 19 weeks' gestation) at the onset of their illness. no other major underlying medical conditions were noted. seven case-patients reported travel to an area with community transmission of sars in the 10 days before illness onset, including hong kong (n = 4), toronto (n = 2), and singapore (n = 1). one case-patient returned to the united states 13 days before illness onset after traveling to hong kong with her spouse, who was also a laboratory-confirmed sars patient. three (38%) patients visited a healthcare facility during their travel in the 10 days before illness onset, and 4 patients stayed at a hotel associated with a well-defined sars cluster (7) . over the course of their illness, findings suggestive of a lower respiratory tract infection developed in all 8 patients with laboratory-confirmed sars; these findings included dyspnea (n = 8), rales (n = 5), and hypoxia (n = 5) (table 1) . symptoms indicative of an upper respiratory tract infection, including rhinorrhea and sore throat, were reported less often. the most common symptoms at illness onset included fever (n = 8), chills (n = 6), and headache (n = 5). four (50%) patients reported at least 1 respiratory symptom at illness onset. in the remaining 4 patients, respiratory symptoms began 3-7 days after illness onset. the median duration of symptoms before a patient sought medical evaluation was 6 days (range 3-14 days). when patients were first evaluated, the median recorded temperature was 38.6°c (range 37.0°c-40.0°c); the median recorded oxygen saturation on room air was 95% (range 87%-100%). gastrointestinal symptoms were also prominent. six patients reported diarrhea, and 5 reported vomiting during the course of their illness. when present, diarrhea occurred a median of 3 days after onset (range 2-3 days) and was noted before (n = 4), or within 48 hours (n = 2) of receiving antimicrobial therapy. vomiting began a median of 5 days after onset (range 3-9 days). all 8 case-patients had radiographic evidence of pulmonary infiltrates during the course of their illness ( table 2) . bilateral pulmonary infiltrates developed in 7 patients during the course of illness with both interstitial and alveolar involvement. of these, 6 demonstrated worsening chest radiographic findings in week 2 of illness. the first abnormal chest radiograph was obtained a median of 7 days after onset of symptoms (range 1-14 days). six patients had an abnormal chest radiograph when first evaluated, including 3 with bilateral infiltrates. two patients had unremarkable initial chest radiographs on days 6 and 8 after onset, respectively, but were subsequently noted to have infiltrates on chest imaging obtained on days 8 and 11 of their illness. during the course of their illness, all 8 case-patients received antibacterial therapy. three patients also received oseltamivir; none was treated with ribavirin. one patient received corticosteroids. seven patients were hospitalized for a median of 8 days (range 6-15 days). two patients were admitted to the intensive care unit for 7 and 9 days, respectively; no deaths occurred ( table 2) . antibodies against sars-cov developed in all 8 patients; 3 had positive pcr findings in clinical specimens (1 sputum and 2 stool specimens) (7) . variable levels of clinical laboratory testing were performed ( table 2) . the 8 patients with laboratory-confirmed sars were compared to 65 sars-cov-negative controls (>18 years old), of whom 14 (22%) had radiographic evidence of pneumonia. forty-four (68%) controls tested negative for antibodies to sars-cov on serum obtained >28 days after symptom onset; the remaining 21 (32%) controls had a negative serologic finding for sars-cov 22-28 days after illness onset. patients were similar to controls with regard to age and sex. fifty-eight (89%) controls reported travel to an area with community transmission of sars in the 10 days before illness onset. however, patients were significantly more likely than controls to have visited a healthcare facility during their travel (3/8 vs. 4/65; p = 0.03) or to have stayed at the hotel associated with the sars cluster (3/8 versus 1/65; p < 0.01). univariate analysis of clinical features showed that dyspnea, hypoxia, rales, vomiting, and diarrhea were more common among sars-cov-positive patients than sars-cov-negative controls (table 3) . case-patients were also significantly more likely than controls to report fever as an initial symptom (8/8 vs. 29/65; p < 0.01) and to have an abnormal chest radiograph at the time of first evaluation (6/8 versus 12/52; p < 0.01). when the analysis was limited to patients with radiographic evidence of pneumonia, dyspnea and vomiting remained associated with sars-cov infection. in addition, sars-cov-positive cases were significantly more likely to have bilateral multifocal infiltrates (7/8 cases versus 4/14 controls; p = 0.02) and radiographic progression of pulmonary infiltrates into week 2 of illness (6/8 cases versus 0/14 controls; p < 0.01). we compared the 8 u.s. patients with laboratory-confirmed sars to sars-cov-negative controls who met the clinical and epidemiologic criteria for suspected or probable sars. our findings indicate that sars-cov infection is associated with significant lower respiratory tract disease. patients with laboratory-confirmed sars were more likely than controls to have dyspnea, hypoxia, and rales. patients were also more likely than controls to have an abnormal chest radiograph at the time of first evaluation. these clinical findings are similar to those reported in case series from asia and canada, and contrast the clinical manifestations of sars-cov with most viral respiratory pathogens including other human coronaviruses (1) (2) (3) (4) (5) 10) . when compared to controls with radiographic evidence of pneumonia, patients with sars were more likely to manifest dyspnea and progressive bilateral pulmonary infiltrates. this radiographic progression to multifocal infiltrates has been a prominent finding in several previous studies and may prove to be a hallmark feature of the later stages of this disease (1-3,6,11). among u.s. case-patients, diarrhea and vomiting were also significantly associated with sars-cov infection. while gastrointestinal symptoms were a relatively uncommon feature in some previous reports (1, 3) , diarrhea was frequently reported in other case series, including a major community outbreak at a hong kong apartment block (2, 4, 5, 12) . although previous studies have described the clinical features of patients with laboratory-confirmed sars, none compared the characteristics of these patients with sars-cov-negative controls. our findings suggest that the combination of gastrointestinal symptoms, dyspnea, and bilateral pulmonary infiltrates may warrant a higher level of suspicion for sars-cov infection. by contrast, patients with findings of only upper respiratory tract infection may be unlikely to have sars. although moderate lymphopenia was prominent among u.s. case-patients, it was also a fairly common finding among controls who likely had other viral sources of infection. the small number of persons with laboratory-confirmed sars in the united states limited our power to identify independent clinical predictors of sars-cov infection. further data are needed to describe the full clinical spectrum of sars-cov infection and to clarify when specific clinical findings are most likely to occur during the course of illness (13, 14) . early recognition of possible sars-cov infection and rapid initiation of infection control precautions are currently the most important strategies for controlling sars (15) . identifying persons who warrant further investigation for sars-cov infection may be difficult on the basis of clinical symptoms alone, especially early in the course of illness. appropriate preparedness for sars will thus require vigilant clinicians and public health officials to integrate timely epidemiologic information, astute clinical evaluation, and improved laboratory diagnostic tools. clinical features and short-term outcomes of 144 patients with sars in the greater toronto area outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts patient data, early sars epidemic clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study clinical and laboratory features of severe acute respiratory syndrome vis-a-vis onset of fever sars surveillance during emergency public health response real-time reverse transcription-polymerase chain reaction for sars-associated coronavirus a novel coronavirus associated with severe acute respiratory syndrome clinical manifestations, laboratory findings, and treatment outcomes of sars patients severe acute respiratory syndrome: radiographic appearances and pattern of progression in 138 patients enteric involvement of severe acute respiratory syndrome-associated coronavirus infection the spectrum of severe acute respiratory syndrome-associated coronavirus infection a clinical prediction rule for diagnosing severe acute respiratory syndrome in the emergency department combining clinical and epidemiologic features for early recognition of sars interest is in the epidemiology of hiv and tuberculosis coinfection in the developing world. key: cord-328000-i9tzr13z authors: cockrell, adam s.; leist, sarah r.; douglas, madeline g.; baric, ralph s. title: modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice date: 2018-07-24 journal: mamm genome doi: 10.1007/s00335-018-9760-9 sha: doc_id: 328000 cord_uid: i9tzr13z the emergence of highly pathogenic human coronaviruses (hcovs) in the last two decades has illuminated their potential to cause high morbidity and mortality in human populations and disrupt global economies. global pandemic concerns stem from their high mortality rates, capacity for human-to-human spread by respiratory transmission, and complete lack of approved therapeutic countermeasures. limiting disease may require the development of virus-directed and host-directed therapeutic strategies due to the acute etiology of hcov infections. therefore, understanding how hcov–host interactions cause pathogenic outcomes relies upon mammalian models that closely recapitulate the pathogenesis of hcovs in humans. pragmatism has largely been the driving force underpinning mice as highly effective mammalian models for elucidating hcov–host interactions that govern pathogenesis. notably, tractable mouse genetics combined with hcov reverse genetic systems has afforded the concomitant manipulation of virus and host genetics to evaluate virus–host interaction networks in disease. in addition to assessing etiologies of known hcovs, mouse models have clinically predictive value as tools to appraise potential disease phenotypes associated with pre-emergent covs. knowledge of cov pathogenic potential before it crosses the species barrier into the human population provides a highly desirable preclinical platform for addressing global pathogen preparedness, an overarching directive of the world health organization. although we recognize that results obtained in robust mouse models require evaluation in non-human primates, we focus this review on the current state of hcov mouse models, their use as tractable complex genetic organisms for untangling complex hcov–host interactions, and as pathogenesis models for preclinical evaluation of novel therapeutic interventions. the etiologies of coronaviruses (covs) comprise an extensive host range that includes reptiles, birds, pigs, dogs, cats, cattle, rodents, bats, camels, and humans. the extensive nonhuman host range may serve as reservoirs for pre-emergent coronaviruses poised for zoonotic transmission into human and animal populations (forni et al. 2017; zhou et al. 2018) . there are six known human coronaviruses (oc43, 229e, nl-63, hku1, sars-cov, and mers-cov), four of which hku1, were identified in the last 15 years. considering that bats may be the pinnacle reservoir for the origin of most hcovs (sars-cov, mers-cov, 229e, and nl63), evidence for sars-cov and mers-cov indicates that intermediate zoonotic hosts (civet cat and camel, respectively) may be as important for the evolution of a pathogenic cov to emerge into the human population (forni et al. 2017) . the emergence of novel pathogenic covs into the human population can result in highly lethal respiratory hcovs with global pandemic potential through human-to-human spread, which is underscored by the global spread of sars-cov (~ 10% mortality) in 2002-2003 and the ongoing re-introduction of mers-cov (~ 35% mortality) into humans on the arabian peninsula. human-to-human transmission of mers-cov was most apparent in the south korean outbreak in 2015, established by a single individual returning from a visit to the arabian peninsula, and resulting in 186 individuals infected with an ~ 20% mortality rate (lee 2015) . the high mortality rate in south korea also established a society-wide panic that led to a severe economic toll, anticipated to cut 1 3 0.1% from the gdp rate in 2015 (lee 2015) . once an hcov emerges in humans new tools and robust mouse models are needed to decipher pathogenic mechanisms. ultimately, understanding the hcov-host molecular interaction networks that regulate pathogenesis can guide the engineering of therapeutic countermeasures. an ideal strategy for development of models that recapitulate human respiratory covs clearly includes the development of a genetically tractable mammalian model that effectively recapitulates human pathogenic phenotypes, and an efficient reverse genetics system for seamless manipulation of the hcov genome. genetic control of both host and hcov genomes facilitates the ability to draw causeand-effect relationships between virus/host genetics and pathogenic outcomes. reverse systems genetic tools were developed to manipulate the large hcov genomes (~ 30 kb) that encode 16 non-structural proteins; structural proteins including spike (s), envelope (e), membrane (m), and nucleocapsid (n); and a number of accessory proteins that vary in sequence and function ( fig. 1) [reviewed in (cockrell et al. 2017) ]. a number of in vitro experiments (biochemical and tissue culture based) demonstrated that hcov proteins interact with host cell proteins to ensure fidelity during virus replication or facilitate evasion of host cell immune responses. the most prominent hcov-host cell interaction is between the major determinant of viral tropism, the spike protein, and a host cell-specific receptor. the spike protein forms a trimer on the surface of the virus with each monomer harboring a receptor binding domain (rbd) that interacts with its cognate receptor on the host cell (ace2 for sars-cov and dpp4 for mers-cov) (fig. 2) . the spike protein, especially the rbd, is a major target for development of antibody and vaccine therapeutics. other highly conserved structural and non-structural proteins, chiefly enzymatic proteins, may serve as targets for broadly effective anti-hcov therapeutic targets. the accessory proteins are not conserved between hcovs, and numerous studies revealed that these gene sets encode critical determinants of species-specific pathogenesis by modulating host immune responses (fig. 1) . in vitro studies have shown a number of accessory proteins to be important for evasion of host innate immune responses through interactions with host proteins [reviewed in (de wit et al. 2016; totura and baric 2012) ]. although in vitro studies are imperative for understanding molecular and biochemical mechanisms, only in vivo studies can decipher how complex hcov-host interactions relate to pathogenic phenotypes following a respiratory infection. discerning the fine balance between pathogenesis and protection, that is governed by the relationship between host innate and adaptive immune responses, requires the development of mammalian models that effectively recapitulate the pathogenesis of respiratory hcovs. the challenges associated with modeling pathogenesis of emergent and pre-emergent hcovs are borne out of models developed for sars-cov and mers-cov over the last 15 years (table 1 ; fig. 2 ). mice, ferrets, and non-human primates (nhps) are traditionally used for the studies involving respiratory pathogens and are among the species initially investigated for sars-cov and mers-cov replication and pathogenesis in the lungs. taking into account a number of practical considerations when comparing models for both sars-cov and mers-cov, mouse models appear to be the most pragmatic (table 1) . although we emphasize the practicality of hcov mouse models for the purposes of this review and the absolute critical need for robust primate models for downstream drug and vaccine testing, it is important to note that research (e.g., efficacy of therapeutic countermeasures) established in a robust mouse model should effectively translate to large animal models, such as nhps, that more closely reflect human physiology. in general, mouse models for both sars-cov and mers-cov are affordable, readily available, require fig. 1 emergent and pre-emergent coronavirus genome organization. the orf1a and orf1b genes (green) encode 16 non-structural proteins (nsp1-nsp16) that are highly conserved throughout coronaviruses. the structural genes (red) encode the structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n), which are common to all coronaviruses. the accessory genes (dark shade) are unique to different coronaviruses with regard to number, genomic organization, sequence, and function nominal training for handling, amenable to manipulation under bsl3 conditions, amenable to analysis with commercially available molecular reagents, amenable to large numbers for purposes of experimental reproducibility, and are genetically tractable (table 1) . despite these commonalities, establishing a mouse model for mers-cov presented challenges not encountered with sars-cov (table 1) . even now, 6 years after the first case of mers-cov, one of the biggest challenges remains a very limited understanding of the pathology of mers-cov in humans. only two studies have described the pathology of mers-cov in two fatal human cases (alsaad et al. 2017; ng et al. 2016 ). the two fatal human mers-cov cases were similar to those observed in cases of sars-cov, wherein patients exhibited severe acute respiratory distress with diffuse alveolar damage and formation of hyaline membranes, alveolar fibrin deposits, edema, hemorrhaging, cellular debris, alveolar and interstitial inflammation throughout lungs, and extensive pulmonary tissue necrosis (alsaad et al. 2017; ng et al. 2016) . fatal cases of sars-cov and mers-cov were commonly associated with a gradual loss of respiratory function with available intervention restricted to mechanical respiratory support and oxygen supplementation [reviewed in ]. therefore, effective mouse models for sars-cov and mers-cov should minimally be able to recapitulate fatal respiratory disease having pathology similar to that observed in humans. as described below, a number of mouse models exhibiting fatal respiratory disease were developed for sars-cov and mers-cov; however, a single impediment was realized early in model development for mers-cov that was not confronted for sars-cov. the mouse orthologue of the human receptor for mers-cov, dipeptidyl peptidase 4 (dpp4), did not support interaction with the mers-cov spike glycoprotein rbd (cockrell et al. 2014) . therefore, unlike sars-cov, commercially available mice were not susceptible to mers-cov (towler et al. 2004) ]. in order to establish lethal mouse models, the spike protein, and other genomic determinants, have been modified through adaptive evolution in mouse lungs (day et al. 2009; frieman et al. 2012; roberts et al. 2007a ). thereby, any mouse subspecies that exhibits an unaltered mouse ace2 can be infected with the mouse-adapted sars-cov. wild-type mers-cov column (green). mers-cov infects humans via an interaction of the spike protein [rcsb pdb id: 5x5f (yuan et al. 2017) ] with its cognate receptor, dpp4 [rcsb pdb id: 2onc (feng et al. 2007) ]. the mers-cov spike protein is not able to interact with the mouse orthologue of human dpp4 (cockrell et al. 2014; coleman et al. 2014) . therefore, the mouse dpp4 gene had to be genetically modified in order to allow for infection with mers-cov (cockrell et al. 2016; li et al. 2017; pascal et al. 2015) . pre-emergent covs column (grey). pre-emergent covs can use either known human receptors for covs (ace2 or dpp4) (ge et al. 2013; menachery et al. 2015; wang et al. 2014; yang et al. 2016 yang et al. , 2014 , or novel, unknown receptors to infect their host. the genetically highly diverse panel of mouse strains from the collaborative cross has the potential to provide mouse models for studies of newly emerging covs due to the high genetic variability present in this resource. for all virus images: yellow represents the envelope protein; light/dark blue represents the membrane protein; and red represents the nucleocapsid protein infection and replication (coleman et al. 2014 ). susceptibility and mers-cov pathogenesis was achieved using unique approaches to genetically engineer mice, as described in detail below. establishing mouse models of fatal disease for sars-cov and mers-cov has afforded the necessary knowledge to institute a preclinical in vivo platform that can be used to evaluate pre-emergent hcov respiratory pathogens with unknown disease outcomes (fig. 2) . the strategy is widely portable to other important human pathogens that replicate poorly or not at all in the mouse. the need for sars-cov mouse models was recognized shortly after the sars-cov outbreak that spread globally in 2002-2003. three different strategies were employed for development of sars-cov mouse models: (i) different mouse species (or subspecies) were challenged with wildtype human sars-cov isolates in order to find a model that allows for replication and reflects severe respiratory disease symptoms observed in infected human patients; (ii) mice were genetically engineered to modify the host receptor, which facilitated productive sars-cov replication and pathogenesis; and (iii) adaptive evolution of wild-type sars-cov to a chosen mouse species was done to enhance pathogenesis, and associated clinical phenotypes in vivo. initial studies to develop a mouse model for sars-cov evaluated the susceptibility of various mouse subspecies to clinical isolates of sars-cov. in early 2004, the first study using the mouse as a model organism for sars-cov was reported ). 4-6 weeks old female balb/c mice were intra-nasally infected with a clinical isolate of sars-cov (urbani) and efficient viral replication was observed from the upper and lower respiratory tract with peak titers on day 3 and clearance by day 7, whereas viral replication was not detected in other organs. however, mice continued to gain weight and did not show other signs of clinical disease. passive transfer of immune sera to naïve mice was sufficient to prevent viral replication in the respiratory tract, indicating that wild-type sars-cov could elicit an effective humoral immune response. nonetheless, in the absence of pathogenesis with no clinical signs of disease, it is difficult to properly evaluate the efficacy of therapeutic countermeasures. in a second study, glass et al. used c57bl/6 mice which were known to exhibit a th1-biased immune response in contrast to balb/c mice table 1 practical considerations for establishing an effective hcov animal model a yes-required virus adaptation b yes-after genetically engineering a mouse c affordability-more cost effective relative to ferret and nhp models d availability-developed models that can be readily acquired and studied by the broader scientific community e ease of handling-mice require least amount of specialized training compared to ferrets and nhps f amenable to absl3 conditions-mice are most amenable to absl3 conditions due to space limitations, specialized handling requirements, and personnel limitations g maybe-dependent on species and techniques h n/d-not determined that were considered to be th2-biased (glass et al. 2004) , which may account for differences in pathogenic outcomes. like the balb/c study, 5-6 weeks old c57bl/6 mice were infected with the clinical sars-cov urbani isolate. infected c57bl/6 mice exhibited slower weight gain than mock-infected mice and sars-cov was found not only in the upper and lower respiratory tract but also in the brain, which was not associated with clinical disease in humans. developing a sars-cov model in c57bl/6 mice had the added benefit of expanding the applicability of the model to genetically modified knock-out (ko) mice that are predominantly generated on a c57bl/6 background, thereby facilitating the investigation of host genetics on sars-cov pathogenesis (glass et al. 2004) . utilizing cd-1 −/− and rag −/− knock-out mice, glass et al. demonstrated that nk cells, nk-t cells, or t and b lymphocytes are not required for clearance of sars-cov in the mouse (glass et al. 2004 ). these initial studies to evaluate different murine subspecies as models for sars-cov resulted in replication in the respiratory tract with no indication of the clinical pathogenesis often observed in human cases experiencing severe respiratory distress. knock-out mice deficient in the innate immune response were the first to exhibit clinical alterations that included weight loss and progressively worsening pulmonary disease (hogan et al. 2004 ). 129svev and stat1 −/− mice on a 129svev background infected with a sars-cov clinical isolate (toronto-2) showed that stat1 was required for the resolution of sars-cov infection. clinical signs of disease were only observed in ko mice, whereas efficient viral replication in the lower respiratory tract caused no clinical symptoms in 129svev wild-type mice. in contrast, stat1 −/− mice supported viral replication with peak titers on day 3 and persistence throughout day 22 (hogan et al. 2004 ). most features of pathology that were found in stat1 −/− mice were different from those found in humans. however, some pathological features common to humans, such as bronchiolar injury with focal respiratory epithelial cell necrosis, supported the idea that the mouse species could be a good model for sars-cov-associated clinical findings observed in the respiratory tract of infected human patients. increased fatality as consequence of acute respiratory distress syndrome (ards) from sars-cov infection was often associated with advanced age (> 60 years old) in humans (booth et al. 2003; donnelly et al. 2003; tsui et al. 2003) , which was exploited to improve mouse models of sars-cov pathogenesis. to replicate advanced age in humans, 12-14 months old balb/c (roberts et al. 2005) , c57bl/6, and 129s6 (roberts et al. 2008 ) mice were intra-nasally infected with a clinical isolate of sars-cov (urbani). balb/c and c57bl/6 mice showed comparable levels and kinetics of viral replication while aged 129s6 mice cleared sars-cov by day 5 (roberts et al. 2008 ). the predominant clinical feature regarding mouse studies is weight loss, a consequence of appetite loss and dehydration. all three lines of aged mice showed transient signs of clinical disease including weight loss, ruffled fur, hunching, and dehydration that resolved by day 7. moreover, all three strains exhibited histopathological findings such as perivascular and peribronchiolar infiltrates, necrotic debris in bronchioles, and interstitial pneumonitis. importantly, extensive alveolar damage in aged balb/c mice persisted through day 9 post-infection (p.i.), findings that closely resembled pathology observed in humans. these are some of the first controlled studies to demonstrate that differences in host genetics can significantly influence sars-cov pathogenesis, a topic that is revisited below with regard to using a tractable population of genetically distinct mice referred to as the collaborative cross. concurrent with these studies, rockx et al. demonstrated that pathogenesis related to sars-cov clinical isolates was also dependent upon the genetics of the sars-cov strain evaluated in vivo (rockx et al. 2007 ). using reverse genetics rockx et al. modified the spike protein from the sars-cov urbani strain to encode the spike from middle (cuhk-w1) and early (gz02) phase clinical isolates or a civet strain spike gene from hc/ sz/61/03 (rockx et al. 2007 ). an infectious clone harboring the clinical isolate gz02 or civet hc/sz/61/03 spikes exhibited severe weight loss and death, as well as pathology consistent with acute respiratory distress syndrome in aged, but not young mice (rockx et al. 2007 ). despite these early achievements with aged mice for sars-cov mouse model development, it is impractical to maintain large colonies of aged mice; and immune senescence in aged mice may complicate studies involving pathogenesis and effective evaluation of immune responses to therapeutic countermeasures. in addition to these limitations, full-length clinical isolates of sars-cov were not shown to be lethal in aged mice, a clinical outcome that impacted nearly 10% of all human cases. lethality was not achievable using clinical sars-cov isolates in various young wild-type or immune-incompetent mouse strains. with this in mind, attention turned to genetic modification of mice to acquire a lethal model for sars-cov pathogenesis. in order to continue working with unaltered human clinical isolates of sars-cov, mouse strains constitutively expressing the human receptor for sars-cov, human angiotensin converting enzyme 2 (hace2), were generated. the logic followed that human clinical isolates of sars-cov are evolved to use the hace2 receptor more effectively than the innate mouse ace2 (mace2) receptor; therefore, may elicit pathology consistent with a lethal respiratory infection. different constitutive promotors were used to express hace2 in mice: cytokeratin promotor (mccray et al. 2007; netland et al. 2008) , chicken beta-actin promotor with a cytomegalovirus ie enhancer (tseng et al. 2007) , and the mouse ace2 promotor (yang et al. 2007 ). expression levels of human ace2 correlated with disease severity in all transgenic mouse models. human ace2 overexpression mice generated with the cytokeratin promoter and the mace2 promoter were attempts to limit expression to respiratory cells and cells that exhibited innate mace2 receptor expression, respectively (mccray et al. 2007; netland et al. 2008; yang et al. 2007 ). even though these transgenic mice showed infection of airway epithelium, all of them exhibited high levels of hace2 expression in the brains of transgenic mice which supported increased viral load in brain tissue (mccray et al. 2007; netland et al. 2008; tseng et al. 2007; yang et al. 2007 ). the increased viral loads in the brains of infected animals ultimately led to mortality caused by extensive dissemination and encephalitis in the brain (mccray et al. 2007; netland et al. 2008; tseng et al. 2007; yang et al. 2007) . accordingly, despite the successful generation of lethal hace2 overexpression mouse models for sars-cov pathogenesis, neurological-related mortality confounded their value as models that could effectively mimic lethal respiratory disease often observed in infected humans. adaptive experimental evolution has been a staple of virology for > 60 years (kalter 1949) , providing critical insights into pathogenic mechanisms while bestowing robust, lethal mouse models to interrogate the efficacy of vaccines and therapeutics (bolles et al. 2011; cockrell et al. 2016; rasmussen et al. 2014; roberts et al. 2007a ). adaptive experimental evolution is performed by serial in vivo passages in the tissue of interest to adapt the virus to the host innate and adaptive immune responses. this is similar to the ongoing battle between a virus and the host immune responses occurring in nature, but on an expedited time-scale and in a tissue-specific manner. tissue-specific adaptive evolution places the virus under selective pressure for mutations that allow for more efficient replication. to adapt sars-cov to cause severe acute respiratory disease in mouse lungs, 6-week-old female balb/c mice were intra-nasally infected with the clinical urbani isolate (roberts et al. 2007a ). this process was repeated (15 rounds) until signs of clinical disease were observed, predominantly weight loss that reached clinical endpoints for humane euthanasia (considered humane lethality) (roberts et al. 2007a ). the resulting mouse-adapted sars-cov was designated ma15. rapid weight loss was accompanied by high viral titer in the lungs, viremia and dissemination to extrapulmonary sites, lymphopenia, neutrophilia, and histopathological changes in the lung commonly associated with pneumonitis. accordingly, mortality was a consequence of extensive viral replication which led to virus-associated destruction of pneumocytes and epithelial cells. only six coding mutations were sufficient to render the sars-cov urbani human clinical isolate 100% lethal in 4 weeks old, 6-8 weeks old, and aged balb/c mice (roberts et al. 2007a ). the ma15 sars-cov has since been used in a multitude of studies and proven to not only cause lethal disease in balb/c mice but also in other mouse strains (deng et al. 2014; frieman et al. 2010; gralinski et al. 2015 gralinski et al. , 2017 totura et al. 2015; zhao et al. 2012) . additional passaging of ma15 resulted in the generation of other mouse-adapted sars-cov strains including ma20 (20 passages) (frieman et al. 2012) , while an independent research group acquired a lethal sars-cov model by passaging the human urbani isolate 25 rounds through the lungs of balb/c mice, resulting in v2163 (day et al. 2009 ). in addition to acquiring lethal sars-cov models of disease, comparing various passages of mouse-adapted sars-cov provides the unique advantage of being able to identify which sars-cov proteins acquire specific mutations that can elicit severe respiratory phenotypes. identification of adaptive mutations acquired in specific sars-cov proteins provided insight into virus-host interaction networks that may be used for the development of virusdirected therapeutic countermeasures. combining mouseadapted sars-cov strains with mouse models that support interrogation of the host genome for molecules that influence sars-cov pathogenesis can reveal import interaction nodes amenable to host-directed therapeutic intervention. as mentioned above, a number of early sars-cov mouse model studies showed that disease progression and outcome are mouse strain dependent, indicating that host genetics have a considerable influence on sars-cov pathogenesis. this is not surprising since human gene association studies have indicated that differences in an individual's genetics may govern susceptibility and clinical outcomes of respiratory viral pathogenesis (everitt et al. 2012; forton et al. 2009; kenney et al. 2017; mills et al. 2014; pasanen et al. 2017; patarcic et al. 2015; zhou et al. 2012 ), including retrospective studies of sars-cov-infected patients (chan et al. , 2006 ching et al. 2010; wang et al. 2008; zhu et al. 2011 ). however, human genetic associations do not indicate cause-and-effect, which require the capacity to model the impact of host genetics on respiratory viral pathogenesis, in this case sars-cov pathogenesis. a recently developed, innovative resource for genetic mapping, called the collaborative cross (cc), comprises a panel of recombinant inbred mouse strains containing tractable genetic diversity that approaches the genetic diversity in the human population (churchill et al. 2004; threadgill et al. 2002) . using an octo-parental breeding scheme that includes classical laboratory stains (a/j, c57bl/6j, and 129/svimj), mouse models for human diseases (nod/shiltj for diabetes; nzo/hlltj for obesity), and wild-derived mouse strains (cast/eij, pwk/phj, and wsb/eij), the cc captures 90% of the genetic variation present in the three major mouse subspecies (mus musculus musculus, mus musculus domesticus, mus musculus castaneus) (roberts et al. 2007b) . virus infection studies in cc mouse lines, including sars-cov, have led to mapping of high and low host response alleles as they relate to development of clinical signs of disease following viral pathogenesis (bottomly et al. 2012; brinkmeyer-langford et al. 2017; ferris et al. 2013; gralinski et al. 2015 gralinski et al. , 2017 green et al. 2017; rasmussen et al. 2014) . rapid genetic mapping is most successful through comparative analysis of cc strains that show extreme phenotypes such as high versus low weight loss, or high versus low lung titers. extreme phenotypes in select cc strains may reflect clinical outcomes observed in human disease, as observed in testing mouse-adapted ebola virus in different cc strains . applying the cc technology to sars-cov pathogenesis identified two cc strains (cc003/ unc and cc053/unc) with opposing susceptibility profiles . genetic mapping revealed an adaptor protein (ticam2) in the toll-like receptor pathway as a strong candidate driving severe respiratory disease phenotypes . based on these observations, the cc mouse platform can be used to identify novel mouse models that recapitulate human clinical outcomes resulting from pathogenic viruses. the cc mouse strains are an extraordinarily powerful platform to establish novel mouse models for emergent and pre-emergent isolates of respiratory coronaviruses. harnessing the power of cc host genetic mapping will support the identification of novel hcov-host molecular interaction networks that can be subsequently validated in genetically engineered mice. innovations in gene editing technologies such as crispr/cas9, talens, and synthetic zinc fingers have augmented the efficiency of genetic engineering in mice, making genomic modifications (i.e., allele-specific mutations, allele swaps, knock-outs, knock-ins) feasible in multiple mouse species, on a large-scale (doench 2018; kim and kim 2014) . coincidentally, advances in the crispr/ cas9 technology overlapped with the outbreak of mers-cov in 2012, which was fortuitous for the development of lethal mers-cov mouse models. building on expertise from the sars-cov outbreak, coronavirus researchers immediately recognized the overwhelming need for an effective mers-cov mouse model following the emergence of mers-cov in 2012. however, researchers were perplexed to find that mouse lines conventionally susceptible to sars-cov infection/replication were completely resistant to infection with clinical mers-cov isolates (coleman et al. 2014) . initial studies demonstrated that non-human primates were susceptible to clinical isolates of mers-cov (de wit et al. 2013b; falzarano et al. 2014; johnson et al. 2016 johnson et al. , 2015 munster et al. 2013) ; therefore, the inability to infect mice with mers-cov was likely not due to restriction by the host immune responses. this was validated in immune-incompetent mouse lines that also lacked the capacity for infection/replication of mers-cov (coleman et al. 2014) . concurrent with some of the initial mers-cov studies in mice, raj et al. identified a novel receptor for mers-cov, human dipeptidyl peptidase 4 (hdpp4) (raj et al. 2013) . unlike sars-cov which could infect mice through interaction of the rbd in the spike protein with the mace2 receptor, mouse dpp4 (mdpp4) did not support an interaction with the mers-cov spike protein (cockrell et al. 2014) . crystal structures of the mers-cov spike-hdpp4 interface revealed a number of specific amino acids necessary for an interaction between the rbd in spike and two specific domains, referred to as blades iv and v on the β-propeller structure, of the hdpp4 structure (lu et al. 2013; wang et al. 2013) . ectopic expression studies with the mouse dpp4 receptor revealed that altering a minimum of two amino acids on the mdpp4 (positions a288 and t330) conferred susceptibility of the human fibroblast cell line, 293t, to mers-cov infection (cockrell et al. 2014; peck et al. 2015) . importantly, in mice t330 is an n-linked glycosylation site that may sterically hinder mers-cov infection, and is not present in dpp4 orthologues from susceptible species including human, nhps, bats, and camels (peck et al. 2015) . apparently, ferret and hamster dpp4 may also have similar glycosylation's, which may explain the inability of mers-cov to also infect/replicate in these conventional small animal models (de wit et al. 2013a; peck et al. 2017; raj et al. 2014; van doremalen et al. 2014 ). species-specific challenges utilizing the dpp4 receptor not only limited development of mouse models, but stymied development of all small animal models required for evaluating therapeutic countermeasures. accordingly, the initial development of mouse models for mers-cov required employing a number of methods that focused on engineering mice to express/overexpress the human dpp4 receptor (agrawal et al. 2015; li et al. 2016; zhao et al. 2015 zhao et al. , 2014 (fig. 3) . the first mouse model, developed by zhao et al., established transient expression of human dpp4 in the airway of balb/c mice, c57bl/6 mice, and several knock-out mice using an adenoviral vector to transduce an hdpp4 overexpression cassette (ad-hdpp4) to the lungs of mice (zhao et al. 2014) . transient hdpp4 expression rendered mice susceptible to clinical isolates of mers-cov infection [1 × 10 5 plaque forming units (pfu) dose], supporting viral replication in the lungs and producing transient weight loss with mild pneumonia, particularly in older and immunodeficient mice (zhao et al. 2014) . no mortality or signs of advanced clinical respiratory disease were observed (zhao et al. 2014) . as a platform strategy that could be applied to novel emerging viruses, the ad-hdpp4 model provided a means to rapidly evaluate mers-cov-directed therapeutic countermeasures with regard to interfering with replication in the lungs of infected mice. nevertheless, the ad-hdpp4 model was not an effective means of understanding how therapeutic countermeasures could prevent development of severe respiratory disease that resulted in ~ 35% mortality in infected human cases. to develop more clinically relevant mouse models, a number of groups turned to more conventional hdpp4 knock-in approaches, resulting in ubiquitous, constitutive overexpression of hdpp4 throughout the mouse (fig. 3) . in 2015, agrawal et al. reported development of a robust mers-cov mouse model by placing expression of hdpp4 under the cag promoter (agrawal et al. 2015) . infection with clinical isolates of mers-cov resulted in high viral loads in the lungs that produced severe respiratory disease and was fatal by day 6 post-infection (agrawal et al. 2015) . however, disease was confounded by high viral loads in extrapulmonary tissues including the brain, heart, spleen, kidney, and intestines (agrawal et al. 2015) . in an independent study, zhao et al. demonstrated multi-organ damage in an hdpp4 overexpression mouse model employing the same promoter (zhao et al. 2015) . the mers-cov viral loads were nearly two logs higher in the brains of infected mice than in the lungs and corresponded to viral encephalitis observed in the brain by day 9, with mice exhibiting signs of paralysis (zhao et al. 2015) . li et al. went a step further by generating hdpp4 overexpression models with the goal of restricting expression to epithelial cells . two different models were generated using either the cytokeratin 18 (k18) or surfactant c protein (spc) promoter to drive expression of hdpp4. mouse lines derived from the spc-hdpp4 cassette did not exhibit mortality or signs of respiratory disease. in contrast, infection of k18-hdpp4 mice with 1 × 10 5 pfu of a mers-cov clinical isolate (emc_2012) produced weight loss, lung hemorrhaging and mortality by 6-7 days p.i., and lung pathology consistent with severe respiratory disease . unfortunately, the k18 promoter did not limit mers-cov infection to the lungs, instead high viral loads in the brains of infected mice caused neurological disease that was consistent with the observed mortality . in a novel genetic engineering approach, pascal et al. employed regeneron's velocigene technology to precisely replace the entire mdpp4 coding region (~ 79 kb) with the hdpp4 gene, which included ~ 82 kb of sequence encoding both introns and exons (pascal et al. 2015) (fig. 3) . despite removal of nearly the entire mdpp4 gene, the 5′ and 3′ untranslated regions of mdpp4 were maintained; thereby, retaining the endogenous mdpp4 promoter and rna termination regions conferred expression levels and tissue distribution for hdpp4 similar to that obtained for mdpp4. infection with 2 × 10 5 pfu of a mers-cov clinical isolate resulted in viral replication in the lungs with mild clinical disease and pathology indicative of inflammation, with no weight loss or mortality through day 4 p.i. (pascal et al. 2015) . subsequent studies with the velocigene mice comprised a more detailed evaluation of the model, wherein mice infected with 2.5 × 10 4 pfu achieved dramatic weight loss and mortality (using 20% cut-off) by 7 days p.i. . c dpp4 sequence used in velocigene hdpp4 knock-in mouse model (pascal et al. 2015) . mouse genomic sequence, from exon 2 through the stop codon in exon 26, was deleted and replaced with exon 2 through exon 26 and a portion of 3′ untranslated sequence of human genomic sequence. d dpp4 sequence used in hdpp4 knock-in model (li et al. 2017) . mouse genomic sequence from codon i264 in exon 10 to codon v340 in exon 12 was replaced with the human equivalent. e dpp4 sequence used in 288-330 +/+ mouse model (cockrell et al. 2016) . crispr/cas9 technology used to make a288l substitution in exon 10 and t330r substitution in exon 11 of mouse dpp4. f dpp4 construct used in hdpp4 overexpression mouse models. constructs included cdna from human dpp4 flanked by constitutive promoter, polyadenylation signals, and other regulatory elements (agrawal et al. 2015) ; or by 5′ and 3′ genomic regions of human cytokeratin 18 or human surfactant protein c ) (coleman et al. 2017) . lung pathology indicated that mice developed moderate signs of respiratory infection with little indication of severe respiratory disease. these studies were also the first indication that cd8 + t cells and macrophages play a significant role in mers-cov pathogenesis (coleman et al. 2017) . importantly, mers-cov infection/ replication was primarily in the lungs, with little involvement of extrapulmonary tissues (coleman et al. 2017; pascal et al. 2015) . the lack of brain lesions in this model was a significant advancement over transgenic models that relied on global/ubiquitous hdpp4 expression. nonetheless, this model is not readily available to the scientific community by regeneron, and if the model is obtained it comes with a number of commercial restrictions from regeneron that limit its utility when considering development of therapeutic countermeasures (declaration by the authors of this manuscript). another limitation often overlooked with hdpp4 expression models is the innate functions of dpp4 that have evolved in a species-specific manner to maintain various physiological processes inherent to the host. altered dpp4 activity and/or expression is associated with many pathological conditions that include psycho-neuroendocrine disorders, solid tumor cancers, hematological malignancies, infectious disease, and autoimmune/inflammatory diseases (klemann et al. 2016) . the enormous biological breadth of dpp4 is predominantly through its enzymatic activity wherein it cleaves off the amino terminal dipeptides of various biological substrates having l-alanine or l-proline at the penultimate position (klemann et al. 2016) . one of the more important physiological processes of dpp4, also known as cd26, is the modification of numerous cytokine and chemokines involved in the maintenance of immunological homeostasis (klemann et al. 2016; ohnuma et al. 2008) . as a cell surface molecule on cd4 + and cd8 + t cells, dpp4 influences numerous immunological processes including t cell activation and proliferation, cytokine production, differentiation to immunoglobulin producing plasma cells, and transendothelial migration (ohnuma et al. 2008) . therefore, overexpressing full-length hdpp4 or replacing mdpp4 with full-length hdpp4 in the knock-in mouse models may significantly alter the inherent biological and immunological processes that mdpp4 has evolved to execute in a species-specific manner, in the mouse. ultimately, if hdpp4 expression results in a modified immune response in mice, these could artificially influence the pathogenic outcomes of mers-cov infections and immunological responses that are central to evaluating the efficacy of therapeutic countermeasures. the goal of the most recent generation of mouse models was to genetically modify the mdpp4 receptor so that it would be amenable to interaction with the mers-cov spike rbd and subsequent infection, thereby avoiding the need to introduce the hdpp4 receptor. achieving a mers-cov susceptible mdpp4 with minimal modifications would be central to limiting any functional alteration to mdpp4 that could interfere with its broad influence over multiple host physiological processes. in 2016, cockrell et al. utilized the crispr/cas9 technology to make two amino acid substitutions in the mdpp4 gene (a288l and t330r) of c57bl/6j mice (cockrell et al. 2016) (fig. 3) . as discussed above, in vitro overexpression of the modified mdpp4 was previously found to alter the susceptibility of cell lines to mers-cov infection (cockrell et al. 2014; peck et al. 2015) . mice with the modified dpp4 (referred to as 288-330 +/+ mice) encode both amino acid substitutions on both chromosomes. characterization of this model demonstrated that innate mdpp4 expression profiles and physiological processes influenced by dpp4 (glucose metabolism and t cell activation) were not altered by the two amino acid modifications of mdpp4. the 288-330 +/+ mice infected with various isolates of mers-cov (clinical isolates, a camel isolate, and molecular clones) supported high viral replication in the lungs, but failed to exhibit clinical signs of disease (cockrell et al. 2016) . adaptive evolution through 15 rounds of serial passage in 288-330 +/− mice yielded a mouse-adapted mers-cov capable of achieving lethality, decreased respiratory function, and lung pathology indicative of severe acute respiratory distress syndrome at viral doses of 5 × 10 6 pfu (cockrell et al. 2016) . mers-cov infected 288-330 +/+ mice that exhibited severe respiratory disease were absent of any detectable virus in the brain. the development of severe respiratory disease and mortality could be prevented using a mers-cov spike-directed vaccine, and through prophylactic administration of an antibody therapeutic (cockrell et al. 2016) . although the 288-330 +/+ model is highly effective for studying mers-cov pathogenesis and evaluating therapeutic countermeasures, the high infectious dose (5 × 10 6 pfu) required to achieve severe respiratory disease and mortality left room for improvement. an additional 20 rounds of passaging resulted in a mouse-adapted virus capable of achieving similar severe respiratory disease and mortality, but at substantially lower doses (10 3 -10 5 pfu) (douglas et al. 2018) . overall, limiting changes in the mdpp4 receptor to two amino acids, researchers were able to minimize disruption of the natural expression levels, distribution, and biological functions of mdpp4. the 288-330 +/+ mers-cov mouse model continues to have a central role in studies investigating mers-cov pathogenesis and therapeutic countermeasures (menachery et al. 2017b, c) . the most recent mouse model consists of a genetically modified mdpp4 receptor through replacement of exons 10-12 (including introns) of the mdpp4 locus with the hdpp4 equivalent, resulting in a minimal knock-in mouse model referred to as hdpp4-ki (li et al. 2017) (fig. 3) . modification of mdpp4 in the hdpp4-ki model is much smaller than the regeneron full-length hdpp4 knock-in model, but larger than the two amino acid modifications to mdpp4 in the 288-330 +/+ model, placing it somewhere between the two types of models. regarding biological and immunological function, it is not clear if the modified mdpp4 in the hdpp4-ki model has altered expression, biodistribution, or functional profiles compared to wild-type mdpp4 receptor. nevertheless, the hdpp4-ki mice were susceptible to infection with clinical isolates of mers-cov, resulting in high levels of viral replication in the lungs but no signs of respiratory disease. a mouse-adapted mers-cov was achieved through 31 serial passages of mers-cov, inducing severe respiratory disease accompanied by high mortality at infectious doses of 10 4 -10 5 pfu (li et al. 2017) . similar to the 288-330 +/+ model, the hdpp4-ki model exhibited little involvement of extrapulmonary tissues in mers-cov pathogenesis (li et al. 2017 ). the mouse-adapted viruses acquired by li et al. revealed unique mutations in specific viral proteins that may be critical for achieving and evading host immune responses. comparison of these viruses to those obtained by douglas et al. demonstrated that changes at amino acid 222 in the mers-cov spike protein may be critical to achieve lethal infection at low infectious doses (douglas et al. 2018; li et al. 2017 ). importantly, this mutation was not present in earlier passages by cockrell et al. in the 288-330 +/+ model, which required high infectious doses to achieve severe respiratory disease and lethality (cockrell et al. 2016) . mouse-adapted viruses, such as those used by cockrell et al., douglas et al. and li et al. can help provide insight into adaptive evolution and identify specific mutations or regions of the virus that are important for enhancing virus fidelity in the host and/or evading the host immune responses. adaptations in mers-cov most notably occur in the spike region, which is an important determinant of host tropism. adapted mers-cov strains with mutations in this region may not provide an accurate representation of the observed viral pathogenesis in humans. currently, achieving lethal disease in mice with clinical isolates of mers-cov require expression of full-length hdpp4, as demonstrated for the regeneron hdpp4 knock-in mice. however, altering dpp4 significantly through overexpression or replacement of mdpp4 can disrupt the immunological homeostasis of the animal, making it difficult to analyze disease etiology and immune responses to therapeutic countermeasures. a mers-cov mouse model would ideally involve infection with clinical mers-cov isolates in an unmodified mouse. one possibility is utilizing cc mice, as described above for sars-cov, which offer a wider range of genetic variation than the genetically engineered c57bl/6 mice used in currently established mouse models. however, unlike sars-cov, the cc mice are not susceptible to mers-cov. crossing various cc lines with mice that have minimal alterations to mdpp4 (e.g., 288-330 +/+ mice), or direct genetic engineering of cc lines using crispr/cas9 technologies, can produce mouse lines that maintain native mouse dpp4 expression, distribution, and functional profiles, but can be screened for differential susceptibility to clinical isolates of mers-cov. the capacity to genetically engineer receptors to alter mouse susceptibility to hcov infection, combined with adaptive hcov evolution and genetically diverse cc mouse strains, establishes a platform that can be used to evaluate the pathogenesis of pre-emergent hcovs with unknown etiologies. the mouse tools founded from studies of sars-cov and mers-cov pathogenesis will aid in understanding if specific pre-emergent covs have pathogenic potential in mammalian models of severe respiratory disease. most hcovs (sars, mers, 229e, and nl63) are considered to have their origin in bats [reviewed in (forni et al. 2017; menachery et al. 2017a) ]. metagenomics analysis of various bat species has identified a diverse repertoire of sarslike (sl-bcovs) and mers-like (ml-bcovs) bat coronaviruses (ge et al. 2012 (ge et al. , 2013 he et al. 2014; lau et al. 2015 lau et al. , 2010 lau et al. , 2005 wu et al. 2016; yang et al. 2016) . those with pre-emergent potential include sl-covs wiv1 and wiv16, shown to utilize the ace2 receptor (ge et al. 2013; menachery et al. 2015; yang et al. 2016) , and an ml-cov hku4, demonstrated to utilize the dpp4 receptor for infection yang et al. 2014) . notably, wiv1 and wiv16 are the only pre-emergent covs directly isolated from bat feces on vero e6 cells and shown to replicate in various cell lines expressing the human, or nhp, ace2 receptor (ge et al. 2013; yang et al. 2016) . in contrast to wiv1 and wiv16, isolated hku4 was not demonstrated to infect cells, but rather lentiviral particles pseudotyped with hku4 spike protein supported efficient binding and infection of cells expressing hdpp4 yang et al. 2014) . the structure of cov spike proteins in the context of pseudotyped lentiviral particles may differ to that in cov particles, and may account for the difficulty of hku4 infecting and replicating on cells expressing hdpp4. the spike proteins of many pre-emergent bat covs (bcovs) have amino acid differences that preclude efficient interaction with known human host receptors, such as hace2 or mace2. transmission to intermediate hosts (civets or camels) and humans may require additional adaptations in the spike protein [reviewed in (graham and baric 2010; menachery et al. 2017a) ]. therefore, to evaluate pathogenesis of pre-emergent bcovs in mouse models, the spike proteins were replaced by generating infectious molecular clones with known spike proteins from clinical isolates of sars or mouse-adapted sars-cov becker et al. 2008; menachery et al. 2015 menachery et al. , 2016 . the first of these studies derived an infectious molecular clone sl-bcov from sequences of four previously identified sl-bcovs (hku3-1, hku3-2, hku3-3, and rp3) (becker et al. 2008 ). the chimeric virus was able to replicate in balb/c mouse lungs, but did not elicit signs of respiratory disease commonly associated with sars-cov (becker et al. 2008) . similarly, a chimeric wiv1 (wiv1-ma15) replicated in balb/c mouse lungs, but was attenuated with regard to causing respiratory disease (menachery et al. 2016) . attenuation of wiv1-ma15 was partially overcome by infecting hfh4 mice that overexpress the hace2 receptor (menachery et al. 2016) , indicating that mouse adaptation of the wiv1-ma15 to mace2 could enhance disease in wild-type balb/c mice. indeed, agnihothram et al. demonstrated that mouse adaptation of an infectious molecular clone of an hku5-se chimeric resulted in dramatic respiratory disease in aged balb/c mice . hku5 is closely related to mers-cov, but since there was no mouse model available for mers-cov at the time, it was more practical to replace the hku5 spike with the mouse-adapted sars-cov spike. these studies demonstrated that adaptive mutations outside of the spike are necessary to elicit respiratory disease in different mammalian species. however, this was not the case for the shc014 bcov chimeric virus (shc014-ma15), which caused significant weight loss in balb/c mice and replicated to titers comparable to sars-cov ma15 in the lungs . moreover, wild-type shc014 spike protein supported infection/replication in the lungs of balb/c mice, but did not result in disease. applying the classical approach of adapting shc014 to mice by serial passaging may be beneficial, but can be time-consuming. screening shc014, or other pre-emergent bcovs, in cc mouse strains has the potential to rapidly lead to mouse models capturing different, or even all, aspects of clinical disease in human patients. the ultimate goal for mouse model development is capturing disease etiologies that closely reflect what is observed during human cov infection, so the efficacy of various therapeutic countermeasures can be evaluated for effective resolution of pathogenic outcomes. a number of hcov-directed drug, antibody, and vaccine therapeutics were evaluated for efficacy against sars-cov until 2012 when the focus of therapeutic development shifted to mers-cov [reviewed in (dyall et al. 2017; zumla et al. 2016) ]. currently, there are no fda-approved therapeutics for the specific treatment of sars-cov or mers-cov. various combinations/types of broad spectrum antivirals (e.g., ribavirin) combined with interferon therapies (interferon α or β), and/ or immune modulators (e.g., corticosteroids) were ineffective for sars-cov and mers-cov, and in some cases appeared to worsen disease [reviewed in (dyall et al. 2017; zumla et al. 2016)] . a recent clinical trial seeks to interfere with mers-cov replication using a combination therapy that includes fda-approved protease inhibitors (lopinavir/ ritonavir) (https ://clini caltr ials.gov/ct2/show/study /nct02 84584 3). repurposing fda-approved drugs has been the interest of numerous therapeutic studies, but little is known regarding the efficacy of drug therapeutics against sars-cov or mers-cov outside of cell-based in vitro studies [reviewed in (dyall et al. 2017) ]. mouse models confer distinct advantages for therapeutic examination including cost savings through small scale production for testing, and experimental reproducibility. the recent development of a novel nucleotide prodrug, gs-5734, demonstrated efficacy against ebola virus in nhps (warren et al. 2016) , and has since moved into phase 2 clinical trials for ebola [https ://clini caltr ials.gov/ct2/show/ nct02 81858 2?cond=preva il+iv&rank=1; (dornemann et al. 2017)] . a recent study demonstrated broad spectrum efficacy of gs-5734 against multiple hcovs (sars-cov, mers-cov, and nl63) and pre-emergent bcovs (hku3, hku5, sch014, and wiv1) on primary human airway epithelial cells (sheahan et al. 2017) . importantly, gs-5734 exhibited in vivo efficacy against sars-cov-induced immunopathology if the drug was administered prophylactically, or a therapeutic dose 24-h post-infection (sheahan et al. 2017) . delaying treatment until 48-h post-infection did not ameliorate disease symptoms. although the kinetics of sars-cov pathogenesis will differ between mouse and human, these results indicate that the molecular programs leading to respiratory immunopathology are established shortly after infection, thereby limiting effective therapeutic treatment to a short window, post-infection. the study by sheahan et al. also demonstrates the importance of having mouse models that recapitulate severe respiratory disease often seen in humans (sheahan et al. 2017 ). an effective therapeutic should not only prevent viral replication, but more importantly should effectively restrict respiratory pathogenesis. achieving both may require therapeutic intervention with a combination of an hcov antiviral and a hostdirected therapy that curtails immunopathology associated with acute respiratory distress syndrome. only in the last 3 years have mers-cov mouse models, described above, become available to assess therapeutics; therefore, future studies will include evaluating in vivo efficacy of gs-5734 against mers-cov in the 288-330 +/+ model described above. as an ongoing threat to worldwide public health, a plethora of therapeutic research focused on development of mers-cov-specific antibodies and vaccines, both of which have been extensively reviewed [reviewed in dyall et al. 2017; okba et al. 2017)] . importantly, few studies have evaluated the therapeutic efficacy of mers-cov antibodies and vaccines in mammalian challenge models that elicit severe respiratory disease, often resulting in death (cockrell et al. 2016; munster et al. 2017; tai et al. 2016; van doremalen et al. 2017; wang et al. 2018 ). rather, many preclinical studies exhibited efficacy in the mouse or nhp challenge models with protection defined as reduced viral loads in the lungs by plaque assay or rt-pcr. preclinical results have expedited the process of moving some antibody and vaccine therapeutics into early clinical trials [reviewed in dyall et al. 2017; okba et al. 2017) ]. based on results obtained in studies with gs-5734, it will be critical to determine efficacy of antibody and vaccine therapeutics in lethal respiratory models of mers-cov infection. vaccine countermeasures are not only being considered as intervention strategies for staving off disease in humans, but have also exhibited efficacy in dromedary camels, the mers-cov zoonotic host (haagmans et al. 2016; muthumani et al. 2015) . targeting zoonotic hosts, such as dromedary camels or bat populations, may be an effective means of curtailing transmission into the human population. thinking beyond vaccine countermeasures, we now have the ability to harness genome editing technologies that could allow us to intervene with the process of zoonotic transmission. c57bl/6j mice were made susceptible to mers-cov by using crispr/cas9 technologies to modify two amino acids at positions 288 and 330 on mouse dpp4, to the orthologous amino acids encoded by human dpp4 (cockrell et al. 2016) . genetic modification of the human dpp4 orthologues in camel and bat to look like mouse dpp4 at these positions may result in camels and bats that are now resistant to mers-cov infection. although introduction of mers-cov-resistant bats into the environment may take a long time, camels are considered farm animals in many parts of northern africa and the middle east with controlled breeding practices; therefore, it may be feasible to establish resistant herds that are unable to transmit mers-cov to humans. genome editing of farm animals is clearly achievable as recently demonstrated in pigs modified with crispr/cas9 for genome-wide inactivation of porcine endogenous retroviruses (pervs), thereby eliminating the potential of cross-species transmission of pervs during xenotransplantation of pig organs into humans, with the intention of easing the shortage of available human organs (niu et al. 2017; yang et al. 2015) . the combined power of genetic engineering of mice, reverse genetic systems for covs, mouse-adaptation, and genetically diverse mouse populations afford the tools to develop models that reproducibly recapitulate severe respiratory disease that can be caused by hcovs in humans. establishing mouse models for sars-cov and mers-cov have largely depended on one or more of these tools. regardless of how effective models for sars-cov and mers-cov are for studying pathogenesis and therapeutic interventions, there are drawbacks related to mouse-adapted and transgenic mouse models that alter susceptibility and pathogenesis to hcovs. acquired mutations associated with severe respiratory disease in mouse-adapted hcovs may have no bearing on human pathogenesis or have altered pathogenic outcomes in humans. genetically engineering a specific host species to enhance susceptibility restricts applications of the mouse model, requiring cross-breeding to introduce novel host mutations from knock-out mouse lines and cc mouse strains. this process is labor intensive and requires an extensive timeline. alternatively, one could introduce mutations directly into genetically diverse lines; however, this is not cost effective. additionally, it is difficult to replicate and study co-morbidities in mice that are associated with lethal respiratory disease seen in humans. pre-existing conditions such as advanced age, diabetes, chronic lung diseases, heart disease, and kidney disease have been reported in many of the severe/lethal mers-cov human cases [reviewed in ]. one way to address many of these concerns could be to screen genetically diverse populations of mice (e.g., collaborative cross) with clinical isolates or infectious molecular clones of wild-type strains from emergent, or pre-emergent, hcovs. screening in genetically diverse mouse populations may yield mouse models with a range of clinically relevant phenotypes that more closely reflect the plethora of respiratory disease outcomes observed in the human population. experiences from sars-cov and mers-cov have taught researchers that each hcov may require unique approaches for mouse model development. the mouse tools instituted for sars-cov and mers-cov constitute a versatile preclinical platform for addressing global pathogen preparedness, a directive of the world health organization. open access this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original 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chinese horseshoe bats as the source of human severe acute respiratory syndrome coronavirus mice transgenic for human angiotensin-converting enzyme 2 provide a model for sars coronavirus infection receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus genome-wide inactivation of porcine endogenous retroviruses (pervs) isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections rapid generation of a mouse model for middle east respiratory syndrome multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus a functional variation in cd55 increases the severity of 2009 pandemic h1n1 influenza a virus infection fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin genetic variation of the human alpha-2-heremans-schmid glycoprotein (ahsg) gene associated with the risk of sars-cov infection coronaviruses-drug discovery and therapeutic options key: cord-332448-5fz8ef4f authors: mutnal, m. b.; arroliga, a. c.; walker, k.; mohammad, a. a.; brigmon, m. m.; beaver, r. m.; midturi, j. k.; rao, a. title: early trends for sars-cov-2 infection in central and north texas and impact on other circulating respiratory viruses date: 2020-05-02 journal: nan doi: 10.1101/2020.04.30.20086116 sha: doc_id: 332448 cord_uid: 5fz8ef4f introduction: rapid diagnosis and isolation are key to containing the rapid spread of a pandemic agent like sars-cov-2, which has spread globally since its initial outbreak in wuhan province in china. sars-cov-2 is novel to most parts of the world including usa and the effect on normally prevalent viruses is just becoming apparent. we present our initial data on the prevalence of respiratory viruses in the month of march, 2020. methods: this is a retrospective cohort study post launching of sars-cov-2 testing at bswh, temple tx. testing for sars-cov-2 was performed by real-time rt-pcr assay and results were shared with state public health officials for immediate interventions. results: more than 3500 tests were performed during the first two weeks of testing for sars-cov-2 and identified 168 (4.7%) positive patients. sixty-two (3.2%) of the 1,912 ambulatory patients and 106 (6.3%) of the 1,659 ed/inpatients were tested positive. higher rate of infection (6.9%) were noted in the patients belonging to age group 25-34 years and least number of positive cases were noted in <25 years old (2%) group. the tx state county specific patient demographic information was shared with respective public health departments for epidemiological interventions. incidentally, this study showed that there was a sudden decrease in the occurrence of other infections due to seasonal viruses, perhaps due to increased epidemiological awareness, about sars-cov-2, among general public. authors would also like to share a small study on sars-cov-2 serological assay for the detection of igg antibodies. conclusions: this study was intended to provide an initial experience of dealing with a pandemic and the role of laboratories in crisis management. epidemiological interventions depend on timely availability of accurate diagnostic tests and throughput capacity of such systems during large outbreaks like sars-cov-2. in december 2019, wuhan city, the capital of hubei province in china, became the center of an 50 outbreak of pneumonia of unknown cause. by jan 7, 2020, chinese scientists had isolated a novel 51 coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2; previously known as 2019-52 ncov), from these patients with virus-infected pneumonia. 1 cases have now spread to 190 countries. as 53 of march 23, 2020 there were more than 372,000+ confirmed cases and 16,000+ deaths. 2 although the 54 outbreak is likely to have started from a zoonotic transmission event associated with a large seafood 55 market that also traded in live wild animals, it soon became clear that efficient person-to-person 56 transmission was also occurring. 3 57 the clinical spectrum of sars-cov-2 infection appears to be wide, encompassing asymptomatic 58 infection, mild upper respiratory tract illness, and severe viral pneumonia with respiratory failure and 59 even death, with many patients being hospitalized with pneumonia in wuhan and elsewhere. 4 a global 60 pandemic has erupted due to a high proportion of asymptomatic patients coupled with a high degree of 61 viral shedding, long incubation period, and late clinical manifestations. prolific testing, therefore, 62 remains one of the most effective epidemiological interventions to stop early community spread. 63 unfortunately, the novelty of sars-cov-2 meant that no testing was immediately available making it 64 difficult for public health officials to stay ahead of the pandemic curve. in texas state to adopt sars-co-v2 testing to assist state public health officials for tracing and tracking 71 patients and their immediate contacts. as the pandemic continues to spread across the nation, goal of this 72 study was to share the early clinical trends for covid-19 in north and central regions of texas. the aim 73 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. . https://doi.org/10.1101/2020.04.30.20086116 doi: medrxiv preprint of this study is to encourage other laboratories to consider an early start to testing during pandemics, share 74 initial trends in this part of the world and possible impact of sars-cov-2 on other seasonal respiratory 75 viruses. this report describes the early trends of sars-cov-2 infections in the central and north texas, 76 usa and impact of epidemiological interventions that may have led to the decrease in the incidence of was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. baylor organization will be referred to as bsw hospitals (bswh). all adult patients were prescreened 110 according to who and bswh guidelines to be eligible for sars-cov-2 testing. briefly, patients were 111 prescreened on bswh web portal, phone app and/or through e-visit prior to making appointment for 112 specimen collection at one of the several designated locations. patients were asked for travel history and 113 any other associated symptoms such as fever, cough and shortness of breath. when clinically indicated, 114 sars-cov-2 testing was ordered by the attending physician or by other care providers. 115 as bswh laboratory continues testing, this study included data from the day testing began on 116 march 11, 2020 and until march 23, 2020. these two hospital systems within bswh represent central 117 and north texas population and are limited to these regions of texas due to community outreach. study 118 includes data for sars-cov-2 testing from these two regions and seasonal respiratory virus testing data is 119 limited to central texas region. 120 epidemiological, demographic, clinical and laboratory data were extracted from electronic 122 medical records and laboratory information system. the sars-cov-2 primers were designed by manufacturer of the assay to detect rna targets from 135 the sars-cov-2 in respiratory specimens from patients as recommended for testing by public health 136 authority guidelines. the method employs two primers for amplifying orf1 gene and n gene from 137 sars-cov-2 virus and the assay includes extraction and internal controls built in the same cartridge. 138 internal sample processing controls to verify sample lysis, nucleic acid extraction, and proper system and 139 reagent performance are built into each luminex extraction cartridge. human rnaase p was used as an 140 internal control. luminex aries offers true random-access testing, however, increased demand for testing 141 necessitated validation of a similar assay on the luminex nxtag platform for batched testing, this 142 method includes additional envelope (e) gene target for sars-cov-2 detection. the luminex nxtag 143 platform offers high throughput but on a batched processing using similar primers as luminex aries. 144 both assays had received fda emergency use authorization prior to submission of this manuscript. 145 other respiratory virus testing 146 bswh utilizes respiratory virus syndromic panel, also from luminex, for the diagnosis of upper 147 respiratory infections. this luminex nxtag assay was used as previously described 6 was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. the typical turnaround time for specimen collection to verification of test results was less than 15 hours. 183 individuals were delivered a prescreening questionnaire to be eligible for testing through bswh table 1 . more than 75% of the patients presented in emergency department had fever and 192 a total of 3,571 sars-cov-2 rrt-pcr tests were performed at bswh laboratory, 1,912 194 specimens were received from ambulatory and/or drive-through collection sites, and 1,659 from the 195 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. to 34 years age group (7.4%) followed by 6.9% in 55 years to 64 years age group. data presented here 199 indicated a lower incidence (2%) among the <25 years old (figure 4) . was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. . https://doi.org/10.1101/2020.04.30.20086116 doi: medrxiv preprint ( figure 6) . 221 this study also looked at co-infections rates from sars-cov-2 positive patients. we searched for 222 262 patient records that had concurrent testing requests for sars-cov-2 and other respiratory virus 223 infections. contrary to several other reports from other parts of the nation, this study did not notice any 224 co-infection cases with sars-cov-2. 225 226 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the sars-cov-2 literature is evolving at breakneck pace, but there is a paucity of literature 228 detailing in-house testing solutions to combat the national delays in turn-around time or the shortages of 229 testing kits available. as real-time rrt-pcr is already widely deployed in diagnostic virology 230 laboratories, this study recommends any institution with molecular testing capabilities consider 231 proactively reaching out to manufacturers to improve testing capabilities and turn-around time. in the race 232 against this pandemic, real-time data empower epidemiologists and public health officials to identify, 233 track, and contain spread as much as possible. integrating laboratory-based reporting with epidemiologic 234 surveillance registers will only further improve public health outcomes. 235 the intent of this study was not to assess the performance characters of the rrt-pcr assay for 236 the detection of sars-cov-2 infection. authors are of the opinion that accurate determination of test 237 performance characters will require appropriate distribution of cohorts among the general population 238 especially in the context of virus shedding, transmission dynamics, asymptomatic carriage and specimen 239 requirements are still being debated and investigated. sars-cov-2 has exhibited great degree of plasticity 240 in all of the above characters hence it may take additional time and understanding to determine the 241 performance characters of the assay. 242 the literature data available at the time of the emergency were few for most usa healthcare 243 systems and above all stemming from the only experience available on the outbreak from covid 2019. 244 the only country with published data and epidemiological or management studies was represented by the 245 chinese outbreak. 7 however, the health system and the chinese government represent a very different 246 model from the usa reality where healthcare is regional and private for most part, which enjoys 247 significant autonomy such as the possibilities available to try to improve and optimize diagnosis, 248 management and partnership with public health officials. in this context, bswh ramped up efforts in 249 laboratory diagnosis and collegial collaboration with public health officials for effective epidemiological 250 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. to best of authors' knowledge, this is the first report on sars-cov-2 testing from this part of the 272 world. bswh laboratory would like to share this information with our readers and other laboratories that 273 early adoption of testing for pandemic diseases like covd-19 has long-term implications in management 274 and control measures. bswh laboratory provided test results data on both ambulatory and inpatient 275 population, and shared patient demographics with local public health officials. 276 all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. . https://doi.org/10.1101/2020.04.30.20086116 doi: medrxiv preprint emergency department or admitted for further evaluations. major symptoms included were fever and 278 cough, more than 75% of the patients reported to have these symptoms. khuwara et al 12 reported similar 279 findings in wuhan outbreak, reporting greater than 90% and 75% of the patients exhibiting fever and 280 cough, respectively. 281 interestingly, data mining did not yield any co-infections with sars-cov-2 unlike stanford 282 medicine data 13 . this study attributes the initial trend of not finding co-infections with sars-cov-2 to was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. this study was intended to provide an initial experience of dealing with a pandemic and how 302 laboratories are required to be part of the crisis management. this study demonstrated that proactive 303 collaboration with assay manufacturers would enable laboratories to be prepared for emerging diseases 304 like covid-19. epidemiological interventions depend on availability of accurate diagnostic tests and 305 throughput capacity of such system during large outbreaks like sars-cov-2. it is also important to have a 306 well-organized plan to report the test results to public health officials to initiate counter measures to 307 control the infections. it is also imperative to build a diagnostic algorithm to include testing for other 308 seasonal respiratory viruses, especially most common viruses like influenza and rsv, which may require 309 medical attention. 310 was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted may 2, 2020. the novel coronavirus originating in wuhan, china: challenges for 324 global health governance clinical features of 69 cases with coronavirus disease clinical course and risk factors for mortality of adult inpatients with 331 covid-19 in wuhan, china: a retrospective cohort study clinical evaluation of the luminex nxtag respiratory 336 pathogen panel the novel coronavirus: a bird's eye view rapid viral diagnosis and ambulatory management of 340 suspected covid-19 cases presenting at the infectious diseases referral hospital the european virus archive goes global: a growing resource 344 for research detection of 2019 novel coronavirus (2019-ncov) by real-346 time rt-pcr molecular diagnosis of a novel coronavirus causing an outbreak of pneumonia covid-19: active measures to support community-dwelling older adults. travel 350 med infect dis rates of co-infection between sars-cov-2 and 352 other respiratory pathogens closure of schools during an influenza pandemic priorities for the us health community responding to covid-356 19 authors would like to thank jeffry hunt for help with data extraction and courtney key: cord-308945-i2agpvhk authors: phipps, william s; sorelle, jeffrey a; li, quan-zhen; mahimainathan, lenin; araj, ellen; markantonis, john; lacelle, chantale; balani, jyoti; parikh, hiren; solow, e blair; karp, david r; sarode, ravi; muthukumar, alagarraju title: sars-cov-2 antibody responses do not predict covid-19 disease severity date: 2020-07-15 journal: am j clin pathol doi: 10.1093/ajcp/aqaa123 sha: doc_id: 308945 cord_uid: i2agpvhk objectives: initial reports indicate adequate performance of some serology-based severe acute respiratory syndrome coronavirus 2 (sars-cov-2) assays. however, additional studies are required to facilitate interpretation of results, including how antibody levels impact immunity and disease course. methods: a total of 967 subjects were tested for igg antibodies reactive to sars-cov-2, including 172 suspected cases of sars-cov-2, 656 plasma samples from healthy donors, 49 sera from patients with rheumatic disease, and 90 specimens from individuals positive for polymerase chain reaction (pcr)–based respiratory viral panel. a subgroup of sars-cov-2 pcr-positive cases was tested for igm antibodies by proteome array method. results: all specificity and cross-reactivity specimens were negative for sars-cov-2 igg antibodies (0/795, 0%). positive agreement of igg with pcr was 83% of samples confirmed to be more than 14 days from symptom onset, with less than 100% sensitivity attributable to a case with severe immunosuppression. virus-specific igm was positive in a higher proportion of cases less than 3 days from symptom onset. no association was observed between mild and severe disease course with respect to igg and igm levels. conclusions: the studied sars-cov-2 igg assay had 100% specificity and no adverse cross-reactivity. measures of igg and igm antibodies did not predict disease severity in our patient population. as the covid-19 global pandemic continues, 1 a major priority is the application of serologic testing to determine the scale and rate of exposures. the coronavirus disease 2019 (covid-19) pathogen, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is an enveloped, positive-sense, single-stranded rna betacoronavirus with an approximately 30 kilobase genome. 2 the molecular detection of sars-cov-2 is based on targeting the viral genome (eg, orf1a/b, e, s, n genes) by polymerase chain reaction (pcr) [3] [4] [5] [6] [7] and is currently the gold standard to diagnose acute infection. 3 cellular and humoral immunity resolve the infection, which can be detected by the formation of antibodies specific for the virus. various serologic assays have recently acquired the food and drug administration's emergency use authority for sars-cov-2 antibody testing in covid-19 patients, but the interpretation of antibody data and their clinical significance remains challenging. understanding the time course of antibody response and potential reasons for • pcr-confirmed cases of covid-19 demonstrate high rates of seroconversion beyond 14 days of symptoms unless a patient is severely immunosuppressed. • testing for igg against sars-cov-2 nucleocapsid protein can be performed with high specificity, including in the setting of prior respiratory infection or underlying rheumatic disease. • index values of igg and igm antibodies did not appear to predict disease severity. apparent failure of seroconversion are essential. further, before assessing whether specific antibodies ameliorate sars-cov-2 infection or prevent reinfection, confidence in the analytical specificity of the test is required. antibody assays in general are frequently susceptible to nonspecific reactivity, leading to false positives. this can have dramatic effects when the incidence of exposure is low. thus, a high positive predictive value gained from minimal cross-reactivity towards other pathogen or autoimmune-associated antibodies is critical. long et al 8 have described a variable antiviral igm and igg immune response to sars-cov-2 infection in a chinese population in which seroconversion in a group of 285 patients from 3 hospitals showed igg positivity for all cases beyond 17 to 19 days. bryan et al 9 demonstrated timing of seroconversion for an idaho population. additional studies are lacking for the us population. the goals of this study were to ascertain key performance metrics of analytical specificity and cross-reactivity for a sars-cov-2 igg serologic assay, perform a detailed cross-sectional and serial assessment of igg and igm antibody responses in suspected covid-19 patients, and determine their relation to disease severity. this study was approved by the university of texas southwestern institutional review board. a total of 967 individuals (995 total specimens) were included in this study, including 656 healthy controls, 29 patients with systemic lupus erythematosus, 20 with rheumatoid arthritis, 90 with previous positive respiratory viral pcr panel and/or cytomegalovirus (cmv) igg, and 172 suspected cases of covid-19 ❚figure 1❚. suspected cases comprised instances of new onset or acutely worsening fever, respiratory, or gi symptoms (applied to all pcr positive cases, and 55 of 96 pcr-negative cases), reported exposure to a covid-19 patient, and/or requiring rule-out testing preprocedure. sars-cov-2 igg (abbott 06r86) testing was performed on the abbott architect i2000sr in accordance with manufacturer's specifications. the test is a chemiluminescent microparticle immunoassay (cmia) for qualitative detection of igg antibodies against sars-cov-2 nucleocapsid protein (ncp) in human serum and plasma. strength of response in relative light units reflects quantity of igg present, and is compared to a calibrator to determine the calculated index (specimen/calibrator [s/c]) for a sample (with positive at 1.4 or greater). igm antibody reactivity against sars-cov-2 ncp was measured using a laboratory-developed proteome array as described previously. 10 briefly, ncp expressed in baculovirus insect cells (sino biological) and in escherichia coli (creative diagnostics) along with control proteins (human igm and anti-human igm) were printed onto nitrocellulose membrane-coated slides (grace bio) in sextuple using a nanoplotter np2.1 inkjet printer (gesim). patient serum samples were diluted 1:100 and incubated with the antigens on the array and the igm antibody specificities detected with cy5-conjugated anti-human igm (1:1,000, jackson immunoresearch). the array was scanned using genepix 4400a scanner (molecular device) at wavelength 635 nm. the resulting images were analyzed using genepix pro 7.0 software (molecular devices). the median of the signal intensity for each spot was calculated and the local background around the spot subtracted, and data obtained from sextuple spots were averaged. the background subtracted signal intensity was normalized to the average intensity of the total human igm (internal positive control) to generate normalized signal intensity (nsi). samples with nsi of 25 or higher were considered positive for igm. the nsi of ncp igm was used to generate heat maps using cluster and treeview software (http://bonsai.hgc. jp/~mdehoon/software/cluster/index.html). specificity was evaluated using 240 banked plasma samples collected prior to the covid-19 pandemic (blood donors september through november 2019) and samples from an additional 416 healthy donors without recent illness collected from march to april 2020. cross-reactivity specimens related to prior respiratory illness and/or cmv igg positivity (90 total samples) were collected by cross referencing banked serum in the hla lab (january 1, 2015-september 30, 2019) with patients who had previously tested positive for cmv igg, influenza a/b, respiratory syncytial virus (rsv), or an endemic coronavirus (nl63, 229e, oc43, or hku1) by viral molecular tests. as the patients may have been immunosuppressed, we included only those specimens having normal or high levels of total igg (measured alongside sars-cov-2) with no infusion of intravenous immunoglobulin in the preceding 3 months. likewise, we tested 29 samples from lupus patients (collected 2004-2007) who were positive for multiple autoantibodies (100% ana, 62% anti-dsdna, 75% anti-u1rnp, 55% anti-sm, 34% anti-ro52, and 24% anti-la) and an additional 20 samples (collected 2011-2014) from rheumatoid arthritis patients positive for rheumatoid factor (85% were also anti-cyclic citrullinated peptide positive). agreement with pcr-based molecular testing was determined using 172 plasma samples collected (147 lithium heparin, 13 edta, and 12 sodium citrate) from suspected covid-19 cases with prior or same-day pcrbased nasopharyngeal swab testing on the m2000 abbott realtime sars cov-2 assay or the abbott id now covid-19 assay. testing using the m2000 platform was performed either alone, or as reflex testing in the setting of a negative result using the id now platform. patient charts were reviewed to determine time between symptom onset (fever, respiratory symptoms, or gastrointestinal complaints) and severity of condition (whether or not intensive care was required). a subgroup of 37 pcrpositive cases (17 igg positive, 20 igg negative) selected based on sample availability were additionally evaluated for sars-cov-2 igm. for 15 pcr-positive cases, 2 to 6 serial measurements were performed using available residual plasma samples. igg levels and seroconversion based on the calculated index (s/c) were tracked over time. the calculated index (s/c, igg level) was provided by the instrument. when multiple values of igg s/c were compared, a mean and standard deviation were calculated. student t test was used to compare 2 groups of nonparametrically distributed data, and a p value less than .05 was considered significant. the sars-cov-2 igg assay was calibrated followed by an imprecision study performed over a period of 5 consecutive days and was found to be acceptable. analytical specificity of the assay was evaluated with samples from healthy blood donors, and none of these samples (0/656) were positive for virus-specific igg ❚table 1❚; the mean index value was 0.04, well below the cutoff of 1.4 for a positive index value. the cross-reactivity results for respiratory viral infection appear in table 1 of 172 potential covid-19 cases included in the study described, 76 were confirmed positive by pcr methods. overall, 29 of 76 (38%) tested positive for sars-cov-2 igg. the time course of symptom onset revealed increasing igg positivity rates ❚table 3❚ from less than 3 days (1/15, 7%) to 3 to 7 days (8/27, 30%) and 8 to 13 days (5/15, 33%), to being the highest after 14 days (5/6, 83%). igg positivity was high (10/13, 77%) for patients with indeterminate time from symptom onset. igm testing ❚figure 2❚ performed on 37 pcr-positive specimens showed positivity in 9 of 17 (53%) igg-positive cases and, interestingly, in 7 of 20 (35%) igg-negative samples. igm positivity occurred at larger proportion for less than 3 days (3/6, 50%) compared to igg, but at similar rates overall at days 3 to 7 (4/11, 36%), days 8 to 13 (4/11, 36%), and after 2 weeks (4/5, 80%). igm positivity was low (1/4, 25%) for patients with indeterminate time from symptom onset. sars-cov-2 igg antibody results agreed with the pcr-negative samples for 96 of 97 (99%) of cases, including 55 instances of patients with new or acute-on-chronic symptoms suspicious for covid-19 and with known time of onset. we hypothesized that a more severe disease course was related to an increased immune response, which may result in a higher level of sars-cov-2 igg antibody reactivity. cytokine storm has been implicated as a potential life-threatening event in sars-cov-2 infection, and this would activate many aspects of the immune system including the humoral antibody response. we compared igg levels from all sars-cov-2 pcr-positive patients who had a mild/moderate disease course to those who had severe disease (admitted to the intensive care unit [icu]), and there was no difference in igg antibody levels between the 2 groups ❚figure 3a❚. given the impact of time from symptom onset on igg measurement, igg indices were additionally plotted relative to day post symptom onset ❚figure 3b❚, showing a predominant overlap between mild/moderate and severe groups. similarly, igm levels were not much different in mild/moderate and severely affected patients ❚figure 3c❚ and ❚figure 3d❚. it is possible that the course of igg levels was qualitatively different for severe patients, so data from serially collected igg samples were plotted against day of symptom onset ❚figure 4❚ for a select group with serially available samples. severely affected patients were tracked longer, because they were hospitalized longer, but a similar early increase in antibody indices was observed in mild/moderately affected patients when compared to severely affected patients. interestingly, 1 patient was seronegative even on day 28, but this was attributed to immunosuppression to prevent cardiac transplant rejection. thirty-eight samples were available from 13 patients with known date of symptom onset and 4 samples from 2 patients with indeterminate date of symptom onset. within this group, 77% (10/13) became igg positive, including specifically 0% (0/8) for less than 3 days post symptom onset, 33% (3/9) at 3 to 7 days post symptom onset, 86% (6/7) at 8 to 13 days post symptom onset, and 91% (10/11) at more than 14 days figure 4 . for those where seroconversion was not observed, samples were only available for less than 7 days from symptom onset for 2 cases or the patient was subject to significant here we confirmed the high specificity reported by the manufacturer for a sars-cov-2 igg serologic assay, using comparatively larger groups for certain rheumatologic conditions and infections. notably, cmv igg did not cause assay interference despite potential false positivity reported by the manufacturer. rheumatoid factor is an anti-human antibody (igm or igg) that, if complexed with other human immunoglobulins, could falsely increase positivity of an assay. however, we observed no interference by rheumatoid factor in 20 samples. testing 47 samples with prior endemic coronavirus infection yielded no false positives. negative agreement between igg and pcr indicated only 1 case testing igg positive despite negative pcr testing. this initial pcr result was later determined to be a false negative based on evaluation using an alternative molecular platform. positive agreement with pcr was lower in the early stages of infection, increasing with time from symptom onset, yet not as quickly compared to the manufacturer's report. overall, our results largely corroborate and add to the findings by bryan et al 9 who evaluated the same platform. that study showed high specificity in testing 1,020 specimens submitted for herpes simplex virus western blot serology from before the covid-19 pandemic. as such, specificity and cross-reactivity were not specifically addressed in the setting of underlying rheumatologic disease or previous endemic coronavirus. a possible difference between our findings was sensitivity after 14 days of symptoms. in our study, a single negative case attributed to a patient with marked immunosuppression resulted in reduced sensitivity beyond 14 days. an unknown factor in similar studies is the number of cases included with severe underlying immunosuppression. for instance, a recent publication by long et al 8 also indicated 100% igg positivity at 17 to 19 days. this latter study utilized a different assay and focused on a population in china and was thus not as comparable. however, the same question persists regarding the makeup of comorbidities in the test population and highlights that discrepancies may arise in antibody response when comparing serology in unequal groups. nonetheless, within our serial testing group, given the higher number of cases beyond 14 days, we did encounter sensitivity of 91% (10/11), which was closer to previous findings. 8, 9 as with bryan et al, 9 we have noticed alternative cutoff values for igg level could be used with potentially beneficial diagnostic effects. as an example, lowering the cutoff by half (to 0.7) would capture an additional 4 cases with midrange days (5-11 days) from symptom onset without any loss in specificity based on the pcr result. were positive for igm. these samples were positive for igm earlier than igg, ranging from 0 to 11 days from symptom onset. this increased the sensitivity by 35% within the igg-negative samples tested (7/20) and improved diagnostic utility by 9% overall (7/76). these findings emphasize that results will differ between assays focused on igg alone compared to those that separately measure multiple immunoglobulin classes or test for total antibodies (eg, roche). as described, we segregated our igg and igm results based on severity (icu care vs no icu care). long et al 8 indicated that a severe disease course resulted in a high igg level during the second week of disease that becomes indistinguishable from milder cases after 14 days. we did not observe such a difference using a different cmia method. this could be due to fewer patient samples, but the significance of their finding was very strong, which indicates it should have replicated were it a real phenomenon. a limitation in our study, however, is that, although the s/c values are retrievable for the assay, the package insert only describes qualitative reporting based on the cutoff value. furthermore, it should be noted that antibody levels may correlate with other factors such as how long disease lingers before final resolution, longterm complications, or period of communicability. igm levels in our study based separately on the proteome microarray also showed no significant difference when analyzed by disease severity. thus, antibody levels themselves do not appear to reflect disease severity, although serologic reactions not assessed here could potentially do so. a major hurdle to validation was access to patients after a sufficient period of infection because most patients presented before 14 days from symptom onset. limited resources and self-quarantine measures have impaired repeated testing for serial testing at a later date. consequently, less data on mild and moderate patients existed compared to patients admitted to the icu. we do, however, have the advantage of reviewing medical charts to find examples of false negative by pcr and false negative by serology. these examples indicate that molecular and serologic testing have complementary roles in tracking exposure to sars-cov-2. our data do not provide information on how long igg stays positive in the long term or whether it specifically confers immunity. as communities continue to grapple with the covid-19 pandemic, reliable measures of previous exposure and immunity are essential. several platforms are now coming into broader clinical use, providing a window into the sars-cov-2 antibody response. widespread efforts to track sars-cov-2 patients for antibody development will clarify expectations for when testing should return positive, situations in which seroconversion may fail, and what the antibody response can tell us in patients with active infections. a pneumonia outbreak associated with a new coronavirus of probable bat origin coronaviridae study group of the international committee on taxonomy of viruses. 2020. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 2019-ncov) real-time rt-pcr diagnostic panel for emergency use only: instructions for use detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr world health organization. coronavirus disease (covid-19) technical guidance: laboratory testing for 2019-ncov in humans improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19-rdrp/hel real-time reverse transcription-pcr assay validated in vitro and with clinical specimens molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia antibody responses to sars-cov-2 in patients with covid-19 performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in identification of autoantibody clusters that best predict lupus disease activity using glomerular proteome arrays key: cord-320935-3n157yl4 authors: kumar, manish; mohapatra, sanjeeb; mazumder, payal; singh, ashwin; honda, ryo; lin, chuxia; kumari, rina; goswami, ritusmita; jha, pawan kumar; vithanage, meththika; kuroda, keisuke title: making waves perspectives of modelling and monitoring of sars-cov-2 in aquatic environment for covid-19 pandemic date: 2020-09-12 journal: curr pollut rep doi: 10.1007/s40726-020-00161-5 sha: doc_id: 320935 cord_uid: 3n157yl4 prevalence of sars-cov-2 in the aquatic environment pertaining to the covid-19 pandemic has been a global concern. though sars-cov-2 is known as a respiratory virus, its detection in faecal matter and wastewater demonstrates its enteric involvement resulting in vulnerable aquatic environment. here, we provide the latest updates on wastewater-based epidemiology, which is gaining interest in the current situation as a unique tool of surveillance and monitoring of the disease. transport pathways with its migration through wastewater to surface and subsurface waters, probability of infectivity and ways of inactivation of sars-cov-2 are discussed in detail. epidemiological models, especially compartmental projections, have been explained with an emphasis on its limitation and the assumptions on which the future predictions of disease propagation are based. besides, this review covers various predictive models to track and project disease spread in the future and gives an insight into the probability of a future outbreak of the disease. the unprecedented event of covid-19 pandemic brought by the novel coronavirus (sars-cov-2) with severe acute respiratory syndrome (sars) has claimed more than 0.9 million lives across the globe. the novel coronavirus (ncov) belongs to the same subfamily of orthocoronavirinae as mers-cov and sars-cov. still, it is distinctly disparate from the other members, in terms of its contagiousness and evolution rate [1] [2] [3] . while it is believed that infants and older people are more vulnerable to infection, young asymptomatic carriers are of significant concern as they aid in virus transmission without their knowledge [4] . several reports have already documented the presence of the virus and its genetic material in the faecal matters this article is part of the topical collection on emerging contaminants and urine of infected persons. additionally, several concerns have been raised, including possible leaching and infiltrations of effluents from health care facilities, sewage, and drainage water [5] . thus, in this time of accelerated transmission of the novel coronavirus, it is crucial to have a robust surveillance system to pace the monitoring of the disease spread. wastewater-based epidemiology (wbe) approach can be used to understand and evaluate the degree of establishment, penetrance, and infectivity of a specific infection in the community [6] . in this approach, presence and abundance of specific biomarkers (e.g. viral genes) in wastewater are examined, as they may reflect the general status of inhabitants related to the biomarkers within a given wastewater catchment [7] . this approach has proved useful for studying the epidemiology of various infectious diseases and is a crucial tool for disease prevention, control and intervention. thus, this paper reviews the potential of wbe in establishing a vigilant and robust surveillance system to track down coronavirus spread in the community. although sars viruses are of zoonotic origin and several countries have already facing secondary transmission of sars-cov-2, developing an early warning system always presents a challenge. in addition to wbe, several modelling approaches might aid in assessing the disease spread both at a regional and global scale. while compartmental models have traditionally relied on the researcher's understanding of human behaviour but its relevance deteriorates as a function of limited understanding about the response of the government and knowledge about the virus as depicted in fig. 1 . thus, statistical models have found its relevance at the wrong time as evident from the recent studies that advocated using bacillus calmette-guérin (bcg) and polio vaccination to counter the health implication of covid-19 based on a simple correlation of early covid-19 mortality data of countries like iran and india [8] . though bcg or polio vaccines were helpful, predictions solely based on correlation analysis may compromise the entire response system against the virus. a greater challenge lies in making a wider audience to understand the very foundation on which this assumptive theory of virus predictions is based. our aim through this article is to simplify these complex models into understandable theories so that the conclusions of the various prediction models may be understood in its context. this paper aims to collate information on recent developments on wbe in monitoring the trend of community-scale sars-cov-2 prevalence as well as models to predict virus spread and transmission among populations. the receptor-binding domain (rbd) of the heavily glycosylated s protein of sars-cov-2 interacts with the angiotensin-converting enzyme 2 (ace2) receptor and attaches to the surface of the host cells leading to receptor-mediated endocytosis of the virion [9] . the viral envelope fuses with the endosomal membrane with the help of host proteases and releases the viral genome into the cytoplasm of the host cell [10] . the virus generally binds with ace2 receptors in the lungs and intestinal tract, replicates further, resulting in severe consequences. more than 10% of patients without acute infection experienced diarrhoea and nausea within 1-2 days before the advancement of disease and development of any critical symptoms, including fever and respiratory illness [11] [12] [13] . subsequently, it put forward the possibility of the virus load along with faecal matter either in an active or infective state. this was later confirmed by the presence of the infectious virus and its genetic material (rna) in the faecal matter of several patients infected by sars-cov-2 [14] . the rna was continuously detected in the faecal matter of more than 23% of patients ages that ranged from 10 months to 78 years old [15] . during the infamous sars (2002-03) and mers (2012) outbreak, patients were suffered from several gastrointestinal symptoms, including diarrhoea at the beginning of the disease [16, 17] . it has been reported that 16-73% of patients with sars had diarrhoea throughout the malady, for the most part during the first week of the disease outbreak and the viral rna continued to be found in the faecal matter of the patients even after 30 days of illness [17, 18] . as the disease progressed, mers-cov rna was also detected in the faecal matter of most of the positive patients attributed to replication in human primary intestinal epithelial cells [19, 20] . while the binding ability of ace2 receptors decides the infectivity of sars-cov, sars-cov-2 was found to use human ace2 at a better efficiency compared to other strains of sars-cov, including the strain identified in the year 2003 [21] . although several recent publications have highlighted the presence of sars-cov-2 genetic materials [22] [23] [24] [25] [26] [27] until today, there is no evidence of faecal-oral transmission of sars cov-2. however, presence of virus genetic material in toilet bowls, sinks, saliva and respiratory secretions of infected patients [28, 29] and the raw sewage water in different parts of the globe [22] [23] [24] [25] [26] [27] has raised several concerns including enteric involvement of this virus. available evidence on other sars and mers virus suggest that coronaviruses can survive for several weeks and retain its viability for several days at varying temperature range. it has been reported for sars-cov where the virus remained viable for 5 days at a temperature ranging from 22 to 25°c and relative humidity ranging from 40 to 50% [30] . interestingly, the same virus remained infectious for 2 weeks when the experiment was conducted for 4°c, but its infectivity reduced drastically to 2 days when the temperature was increased from 4 to 20°c [31] . similarly, a recent study on the sars-cov-2 highlighted the computational modelling to study the travel time and survival of the virus from source to wastewater treatment plants (wwtps) [32] . considering the proof of faecal discharge for both sars-cov and mers-cov and their capacity to stay viable under conditions that could encourage faecal-oral transmission, it is conceivable that sars-cov-2 could likewise be transmitted by means of this course. thus, the enteric involvement of sars-cov-2 is of greater concern in developing and underdeveloped countries where poor hygiene practices and open defection are prevalent. several techniques have been developed to detect viruses and their genetic material in various environmental matrices. in general, while some techniques test infectivity of viruses, others focused on nucleic acid isolation. most of the approaches to detect and diagnose sars-cov-2 are based on the isolation of genetic material of the virus to confirm infection among patients (fig. 2) . however, over the past, techniques such as cell culture and pcr-based molecular techniques have been used [33, 34] . cell culture assays investigate both virus presence and its infectivity by monitoring response (cytopathic effects, cells bursting, plaque) of suitable host cells. however, the difficulty of culturing and slow growth rates discourages the application of this method specifically to the novel virus, where expensive cell culture assay and requirement of biosafety level 3 are of additional concern. recently, many researchers have reported the presence of sars-cov-2 rna in the wastewater using reverse transcriptase-pcr (rt-pcr) technique. this technique involves rna concentration, extraction, amplification to detect its presence at the lowest available concentration without giving much information on the infectious nature of the virus. there is much discussion around the infectious nature of sars-cov-2 and the role of its genetic material. however, sars-cov-2 needs much more than its genetic material to spread infections. outside the host, the virus is considered to be non-living, i.e. viron, which is mainly composed of rna, surrounded by a lipid envelop and cannot selfreplicate. infection is only possible due to the presence of viable virus but not due to the presence of its genetic material in the host body. the virus is said to be viable if it replicates and increases the viral load in the host body. sars-cov-2 through its spike proteins (s protein) attach to the host cell receptors (ace2), which is commonly found in the respiratory or intestinal tract of human beings and subsequently enters into the host cell to replicate [10] . as pcr targets specific parts of the virus genetic material, the degree to which a virus was affected by several disinfectants present in the wastewater and its viability cannot be addressed by this technique. hence, the detection of sars-cov-2 rna does not imply the infectious nature of the virus. therefore, assay-based techniques are more appropriate to study the infectious nature of the virus. while several studies have identified the presence of sars-cov-2 in the faecal matter of corona-infected patients [35, 36] , there is a growing concern on the transmission of the virus through water treatment plants (wtps) and wwtps. several studies also detected the genetic material of the virus in raw wastewater across the globe [22, 26, 27] . nevertheless, the viability and infectivity of the virus in faecal matter are yet to be documented; the possibility of virus transmission through wastewater and drinking water is of major concern. previous studies have highlighted the persistence of coronavirus and sars virus in the wastewater, which ranged from hours to several days in the absence of disinfection practices [31, 37] . chlorination is the most commonly used disinfection technique, mainly in both wtps and wwtps in developed and developing countries as a tertiary treatment step [38] . although the effect of chlorine on disinfection of sars-cov-2 has not yet been documented, available data on other enveloped and coronaviruses can provide an insight into the viability and infectious nature of viruses. chlorination was found to be effective against several enveloped viruses, such as vesicular stomatitis virus, african swine fever virus, equine viral arteritis virus and porcine reproductive and respiratory syndrome virus within 10 min of exposure [39] . among the enveloped viruses, equine viral arteritis virus was inactivated 100% when exposed to chlorine (0.015%) for 1 min. similarly, sars was reported to be disinfected adequately at a free chlorine dose ranging from 0.2 to 0.5 mg/l [31] . another enveloped virus, ebola, was reported to be disinfected 100% at chlorine doses of 5 and 10 mg/l, and a 3.5 log 10 reduction was reported in the presence of free residual chlorine 0.16 mg/l under 20 s contact time [40] . free chlorine is known to penetrate the membrane of modelled enveloped virus (pseudomonas phage phi6) and reacted rapidly with the proteins and polymerase complex. instead, the peptides of the enveloped virus are 150 times more reactive than the studied non-enveloped coliphage ms2 virus. in such a scenario, inactivation of the order 4 log 10 was reported for phi6 [41] . recently, acidic electrolyzed water (ew) having a high concentration of free available chlorine has shown strong potential in deactivating sars-cov-2. it was found that virucidal activity of the virus significantly increased with the loss of free chlorine at long residence time [42] . hence, the risk of sars-cov-2 transmission through drinking water and wastewater is low where chlorination is carried out as it is expected to inactivate the virus and its genetic materials, which has been seen for other enveloped viruses. however, community exposure through sewage overflow, building having a faulty plumbing system [43] or aerosol-mode transmission at the wwtps [44] cannot be avoided. who has highlighted that since 1970s, over 1500 novel pathogens have been discovered and close to 40 new transmittable diseases were identified [17] . most of these diseases were reported to have a severe impact at the community level, with reports of many outbreaks happening during the last 20 years, most significantly sars (severe acute respiratory syndrome) during 2002-2003 and covid-19 during 2019-2020 [17] . the consequences of such an epidemic/pandemic have led to the emergence of critical monitoring on the spread and the disease trend. however, there exist various limitations in the surveillance systems mainly to cope with the rapid population growth and changing environmental conditions. wastewater-based-epidemiology (wbe) approach could be applied as a monitoring tool for surveillance and early warning systems of transmittable disease hotspots (fig. 3) . according to recent reports [35, 45] , sars-cov-2 was shed in the patients' faecal matter for much longer (e.g. 22 days) than the duration of virus shedding in the upper respiratory swabs (10 days). hence, identification and quantification of the viral genome in wastewater can be a reliable indicator of disease prevalence among the communities, as revealed for other similar viruses [46] [47] [48] . very recently, studies report detections of rna of sars-cov-2 in wastewater [27, 46, [49] [50] [51] [52] [53] , with detected rna amount being higher than that expected from reported infection cases. moreover, sars-cov-2 was detected in wastewater 7-10 days earlier than clinically reported cases [46, 53, 54] . in summary, wastewater analysis is recommended as a non-invasive initial-warning tool that can help in monitoring the trend and status of covid-19 spread and as a device for tweaking public health response [35, 55, 56] . thus, under the present condition, such environmental surveillance tool also has the potential to be implemented in wastewater treatment systems to help authorities to manage/regulate the exit strategy. earlier knowledge regarding 2019-ncov hinted at transmission being limited between animals and humans, with lesser development and emphasis in terms of inter-human transmission, making earlier epidemiological predictions [57, 58] . recent developments in terms of phylogenesis, molecular epidemiology and refinements in evolutionary models have helped build a broader understanding of the spread and transmission of the current pandemic [59] . phylogenetic evolution can be traced and validated using different mathematical models, primarily aimed at the detection of episodic mutating diversification and pervasive selection, including techniques of projection-based fitting of nucleotide substitution [60] . further, homology models, enabling protein structures to be analysed and compared in a three-dimensional space, provide higher leverage for understanding the structural templates [61] . however, a serious shortcoming in various risk-based projection models is with regards to its predictability of spread of infection, which considerably and dynamically change in response to the social behaviour of the people under time accelerated learning model [62] . this void creates a large scope for the induction of pure statistics-based mathematical models with various epidemiological considerations taking a back seat and so is the accuracy and precision of the prediction. therefore, with regard to the stated facts, we have reviewed the historical, mathematical, epidemiological evolution of various models with regards to their capabilities in early detection of (i) disease spread, (ii) co-variability of the virus with environmental factors and (iii) transmission among and between different multi-cellular species. the potential scale of the spread of a virus can be understood in terms of its basic reproduction number (r o ). however, studies show that r o variability with societal intervention is more severe than previously assumed, as has been the case in wuhan, china, where r o declined to 0.32 from 3.86 post-implementation of lockdown [62, 63] . these models often referred to as compartmental models, segregate the population into various broad categories such as (i) susceptible (s), exposed (e), infectious (i) and recovered (r). it has gained widespread popularity in the field of epidemic spread due to its greater mathematical emphasis on simulating and adapting under real conditions. most of these epidemiological models trace their origin in the theory of [64] kermack and mckendrick (1927) and the early works of ross and hudson [65] , which stressed on finding the critical causal factor which impacts the severity and frequency of the epidemic. in this regard, a brief overview of their work becomes essential in understanding the assumptions as well as the accuracy of these models. kermack and mckendrick formulated a simple hypothesis and opined that epidemics propagates in the community via direct contact with an infected person, while other indirect transmission sources being negligible. with the rapid spread of infection, the number of sick people in the population increases, which further stabilizes on account of increasing deaths and recoveries. also, the chances and prospects of recovery or death or future infection to other persons will change with each passing day in the infection cycle of a sick person. a primary assumption of the theory was that epidemics are usually short-lived, and therefore, cannot change the population at large. thus, the community under scrutiny for disease spread was kept constant for modelling. the study established that for each set of recovery, death and infectivity, there exists a threshold population density. if the current population of the region exceeds the threshold density, then any addition of a newly infected case will result in the start of the epidemic cycle again. a serious observation was concerning predicting the end of the epidemic. kermack and mckendrick believed that infection exists in a region as long as its population exceeds the critical population density with the epidemic trying hard to move the community towards the critical threshold value, where it finally wanes away and dies out. the theory was further modified to compare the virulence and severity of different viruses for the population with identical population density, recovery and death rate. to give a mathematical basis to this developed hypothesis, the two researchers divided the time into a large number of small constant units, with the assumption that infection starts at the beginning of the time instant with no occurrence of any outbreak in between the passage of one-time unit to other (eq. 1). here, t means the time and ⍬ means the total number of time intervals. y t,⍬ shows the total sick population and p t,⍬ denotes the infected people at any given time instant. p 0,0 will indicate the total infected population at the beginning of the modelling, including the newly infected as well as the existing infected people, which are the potential host for the epidemic spread in the population. if we assume the rate of removal as ψ (sum of recovery and death rate), then the total removed population from the consideration will be ψ ⍬ y t,⍬ , which is equal to y t,⍬ − y t + 1,⍬ + 1. further, if we assume the rate of infectivity to be ɸ ⍬ after the passage of ⍬ time intervals and the total unaffected population to be x t , then the total number of people becoming infected in the unit area as a function of unaffected people will be given by eq. 2. further simplifications lead to the expression of these terms in a more complex differential form, drawing heavily upon the concepts of infinite integrations, which are beyond the scope of this review. however, a more straightforward but more accurate modified form of the current model was used by [62, 63] , for evaluating the coronavirus spread in wuhan, china. the study divided the population of wuhan into six categories, including susceptible (s), latent (e), reported infectious (i), unreported infectious (a), hospitalized (h) and recovered (r). further, two key parameters, including transmission rate (b) and ascertainment rate (r), were used to develop a dynamic network of parameters, which could be represented by eq. 3. where di and dh are the infection and hospitalization period, respectively. further, the effective reproduction number of the disease was calculated as per eq. 4 where α is the rate of transmission calculated as the ratio of unascertained vs. ascertained cases and dq is the period from the onset of symptoms of illness to the hospitalization time. further, the distribution parameters were estimated using monte-carlo markov chain (mcmc) simulations using the likelihood and poisson distribution functions. the same model was also used by [66] in predicting the r o of 2019-ncov to be around 2.68. more so, the baseline projection of the study estimated that the mean imported infectious cases in the cities of beijing, shanghai and shenzhen could be 113, 98 and 80, respectively. [67] employed similar compartmental models to divide the population of hubei province into 5 categories, including susceptible population, asymptomatic, infectious with symptoms, isolated, and recovered to estimate a r o value of 6.49. the most conservative estimate of r o for china has been from who, which predicted value between 1.4 and 2.5. [68] used an additional two categories of death and cumulative cases to estimate a r o of 4.08, assuming an average latent period of infection to be 9 days. [69] overestimated the r o value for china to be 6.47, despite being based on the compartmental models and including appropriate social interventions. while these epidemiological models are more flexible in terms of incorporating dynamic changes with regards to the spread of the virus, their variability in prediction has led to increased dependency of researchers on statistical trend-based models. in this regard, the study by [70] used a simple statistical growth function with an assumed incubation and serial interval of 5.2 and 7.5 days, respectively. similarly, [71] used outbreak trajectories from stochastic simulations to obtain a r o value of 2.2. [72] used a more precise estimation method involving epidemiological data concerning hospitalization, death and onset of illness. further, through a doubly interval-censored likelihood function and bayesian methods, different parameter values within the intervals were determined. statistical models have also been used in order to estimate the serial interval (time duration between two successive cases in an epidemiological transmission chain) of ncov using a similar bayesian approach [73] . few studies have relied on fitting data in different probability density functions to estimate the incubation period of 2019-ncov to be around 5.1 days [74] . associating environmental conditions with the spread of 2019-ncov started due to some early work on sars-cov, where it was found that out of the four protein structures of the sars-cov, i.e. (i) spike protein (s), (ii) nucleocapsid (n), (iii) envelope protein (e) and (iv) membrane protein (m), the n protein exhibits the highest hydrophilicity and least stability due to absence of disulphide bonds. the high isoelectric point (pi = 10.1) and the absence of cysteine residue, makes n protein the weakest link of the virus [75] . any disruption in the n protein means inhibition in the replication of viral rna. further, the sub-genomic transcription and translation also suffer, leading to instability of the virus. earlier studies by [76] , suggested that the poliovirus and rhinovirus have also suffered a similar fate due to viral capsid inactivation at 42°-45°c following the denaturation of protein structures. a study by jane-valbuena et al. [77] further showed that the few outer capsid proteins are highly thermal sensitive. these studies provided enough excellent reasons for modelling the spread of 2019-ncov with the external environmental conditions, assuming that the cases of infection will decrease through secondary infection routes due to the inactivation of the virus on different surfaces; however, the possibility of transmission via direct contact remains unchanged. while the 2019-ncov transmission rate is directly dependent upon close contact with the infected person but the possibility of aerosol suspension via droplets from contagious people remains a significant source of secondary infection. a study by [78] showed that some known coronavirus (hcv/229e) could survive and attain a half-life between 27 and 67 h in conditions with humidity varying between 30 and 50%. further evidence suggests that a decrease in temperature by 6°c can increase the half-life of the virus by 3 h, even at 80% humidity [79] . an interesting observation in this regard was noted in sars outbreak, where it was suggested that due to the low humidity conditions inside the airplane, the virus reached beyond 2 m from the infected person [80] . it is worth noting that all the previous studies have explicitly pointed to the role of humidity and air temperature as the controlling variables in virus spread. however, the consensus is still not clear about the magnitude to which these variables impact the virus spread. published articles have stated that warmer weathers have a suppressing impact on the contagion spread, but the previous outbreak never happened to the scale that has occurred in 2019-2020, leaving lesser applicability of the inferences of these researches [81] [82] [83] [84] . we have presented the various modelling studies done in this regard in table 1 . the spread of coronavirus is sporadic through human-tohuman transmission mainly by droplets produced during coughing or sneezing. there are two pathways involved in transmission one via direct contact with the symptomatic patient and another is indirect contact from the environment [92] . direct contact with symptomatic patient implies being in close proximity with covid-19-infected patient that is within the periphery of 1 m. on the other hand, indirect contact happens via the presence of microbes within droplet nuclei (i.e., particle < 5 μm diameter) in the air or over surfaces [35, 36] . these droplet nuclei can survive in the air or on surfaces for a longer period and, therefore, can be transmitted by coming in contact with an infected surface. however, the probability of airborne transfer is dependent upon the retention power of droplet nuclei in the air or on the surfaces. the likelihood of indirect transmission is less likely in comparison to direct transmission. further, there has been some evidence suggesting oral-faecal transmission of the virus as covid-19 infects the intestinal tract, thereby indicating the faecal presence of the virus [93] [94] [95] [96] [97] [98] [99] . however, this paper, as mentioned above, rules out any such established possibility as of now. based on the available evidence, the who has recommended the use of masks to avoid direct and indirect transmission of the virus. the impact of the coronavirus pandemic is devastating. it has raised several concerns among the population due to fear and worries of catching an infection. although several enveloped viruses are reports to survive for many days, even at 25°c, countries with hot weather have reported cases of covid-19. several factors, including virus transmission, infectivity and inactivation, are crucial before assessing wbe. this review raised the concern on the faecal-oral route of virus transmission, especially in developing countries lacking disinfection steps in water and wastewater treatment plants. additionally, countries lacking proper sanitation facilities having open defecation practices are more vulnerable to be infected by sars-cov-2. the high variance in the prediction capability of various statistical models proves less beneficial for government authorities in dealing with the containment of the disease spread. however, the compartmental models can offer higher adaptability under these circumstances and can offer higher leverage in terms of policy planning. the study predicted the lifetime of the virus on a surface as that time, which achieves a 6-log reduction in concentration based on the recommendation of the us fda. the study provides thermal sterilization guidelines to the healthcare workers exposed at the forefronts of the virus outbreak. sajadi et al. 2020 [86] temperature, humidity and latitude impact on covid-19 spread and identification of any seasonality trend across the globe. regions with significant cases of community transmission are located between the 30°-50°n belt. the identified belt has similar meteorological conditions, including temperature (5-11°c) and low absolute humidity ranging between 4 and 7 g/m 3 . study is assisted by era-5 reanalysis product. the study suggested strong seasonal influence on 2019-ncov spread, with the rate of transmission slowing during the summers. shi et al. 2020 [87] impact of temperature and relative humidity on virus transmission rate in china. no association between the relative humidity and the covid-19 incidence was established. however, with increasing temperature, the transmission rate declined. models can be used in order to understand and quantify the rate of transmission of covid-19 incidence with a change in temperature. amin et al. 2020 [88] role of climatic differences on covid-19 spread in iraqi kurdistan region. data analysis of the covid-19 infection with temperature and humidity suggested that high climatic spatial differences may facilitate the infection spread. regions with high seasonal and spatial climatic differences carry high susceptibility in terms of disease spread. bherwani et al. 2020 [89] the impact of covid-19 incidence was analysed using seir model, and the impact of temperature and humidity was statistically analysed using response surface methodology in india the impact of government-imposed lockdown was considered in the seir (compartmental) model. further, anova and higher-order polynomial functions of rsm techniques provide much better estimates. the model predicts 20-day increase in crisis management for the inability to implement a strict lockdown. it also showed that hot weather might significantly reduce the spread of the virus. yao et al. 2020 [90] correlation between covid-19 related death rate and multiple linear regression model was used to establish the said correlation by adjusting parameters such as temperature, gdp, relative humidity and hospital beds per capita. the study speculated that the effect of air pollution is only limited to making symptoms severe from moderate. toppi et al. 2020 [91] associating high atmospheric pollution with increased 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acknowledgments we acknowledge the fund received from kiran c patel centre for sustainable development (kpcsd) and uk-india education and research initiative (ukieri). conflict of interest the authors declare that they have no conflict of interest.human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. papers of particular interest, published recently, have been highlighted as: key: cord-326706-75mjs6vm authors: waterfield, thomas; watson, chris; moore, rebecca; ferris, kathryn; tonry, claire; watt, alison; mcginn, claire; foster, steven; evans, jennifer; lyttle, mark david; ahmad, shazaad; ladhani, shamez; corr, michael; mcfetridge, lisa; mitchell, hannah; brown, kevin; amirthalingam, gayatri; maney, julie-ann; christie, sharon title: seroprevalence of sars-cov-2 antibodies in children: a prospective multicentre cohort study date: 2020-11-10 journal: arch dis child doi: 10.1136/archdischild-2020-320558 sha: doc_id: 326706 cord_uid: 75mjs6vm background: studies based on molecular testing of oral/nasal swabs underestimate sars-cov-2 infection due to issues with test sensitivity, test timing and selection bias. the objective of this study was to report the presence of sars-cov-2 antibodies, consistent with previous infection. design: this multicentre observational cohort study, conducted between 16 april to 3 july 2020 at 5 uk sites, recruited children of healthcare workers, aged 2–15 years. participants provided blood samples for sars-cov-2 antibody testing and data were gathered regarding unwell contacts and symptoms. results: 1007 participants were enrolled, and 992 were included in the final analysis. the median age of participants was 10·1 years. there were 68 (6.9%) participants with positive sars-cov-2 antibody tests indicative of previous sars-cov-2 infection. of these, 34/68 (50%) reported no symptoms prior to testing. the presence of antibodies and the mean antibody titre was not influenced by age. following multivariable analysis four independent variables were identified as significantly associated with sars-cov-2 seropositivity: known infected household contact or=10.9 (95% ci 6.1 to 19.6); fatigue or=16.8 (95% ci 5.5 to 51.9); gastrointestinal symptoms or=6.6 (95% ci 3.0 to 13.8); and changes in sense of smell or taste or=10.0 (95% ci 2.4 to 11.4). discussion: children demonstrated similar antibody titres in response to sars-cov-2 irrespective of age. fatigue, gastrointestinal symptoms and changes in sense of smell or taste were the symptoms most strongly associated with sars-cov-1 antibody positivity. trial registration number: https://www.clinicaltrials.gov (trial registration: nct0434740) on the 15 april 2020. during the first wave of the sars-cov-2 pandemic in england, children accounted for just 1% of confirmed infections, 1 had a milder clinical course and had much lower mortality than adults, 1-4 a pattern similar to other international settings. 3 4 the reasons for this are unknown, but various hypotheses exist. public health measures, such as school closures, may have minimised children's exposure to sars-cov-2. it is also possible that children have a different immune response to the virus, for example, reduced expression of the ace2 gene, the host receptor for sar-cov-2 virus in airway cells. [5] [6] [7] despite existing data, it is impossible to state accurately what proportion of children were infected with sars-cov-2 in the uk. studies based on molecular testing of oral/nasal swabs with realtime reverse transcription pcr (rt-qpcr) underestimate infection due to issues with test sensitivity, timing of testing and selection bias due to only symptomatic individuals undergoing testing. 8 a potentially more reliable method is to test for specific antibodies. existing antibody tests typically detect igg or total antibody to either the nucleocapsid or spike proteins of the virus. 9 antibody testing has greater potential than rt-qpcr to detect previous asymptomatic/mildly symptomatic infection, and is not dependent on coinciding with active infection. current best seroprevalence estimates from adults in the uk indicate that approximately 6.2% have antibodies consistent with previous sars-cov -2 what is already known on this topic? ► children are relatively unaffected by the sars-cov-2 infection with very few requiring hospitalisation. ► a large, but unknown proportion of children with sars-cov-2 infection are asymptomatic. ► molecular testing of oral/nasal swabs underestimates sars-cov-2 infection. ► gastrointestinal upset is a relatively common symptom of covid-19 in children. adding gastrointestinal upset to the list of symptoms triggering a test in children would improve case-finding. ► asymptomatic and mildly symptomatic children are capable of developing an antibody response to sars-cov-2. ► this study did not find a difference in rates of seropositivity or antibody responses according to age in the children of healthcare workers. infection. 10 these findings are similar to other domestic and international seroprevalence studies. [11] [12] [13] [14] it is unclear what proportion of children are asymptomatic and which symptoms are most associated with paediatric sars-cov-2 infection. estimates based on rt-qpcr testing of oral/nasal swabs suggest that cough or fever are the most common symptoms. [15] [16] [17] [18] [19] [20] however, these studies focus on symptomatic cohorts, introducing selection bias, [15] [16] [17] [18] [19] [20] which leads to underestimation of the asymptomatic proportion. the objective of this study was to report the presence, and titres, of sars-cov-2 antibodies in healthy children of healthcare workers across the uk and to report the symptomatology of infection including the asymptomatic rate. this multicentre observational prospective cohort study was designed to determine the seroprevalence of sars-cov-2 antibodies in healthy children, and report the symptomatology of infection. this study has been written in conjunction with the strengthening the reporting of observational studies in epidemiology guidelines. 21 the study protocol has undergone external peer review and is available as an open access publication. 22 participants were recruited from five uk centres, in the four regions of the uk, between 16 april 2020 and 3 july 2020. the sites included tertiary national health service (nhs) hospitals (belfast, cardiff, manchester and glasgow) and a public health england site (london). children of healthcare workers, aged between 2 years and 15 years at the time of recruitment, were eligible to participate. a 'healthcare worker' was defined as an nhs employee. healthcare workers were categorised according to role, including whether that role involved patient facing activities. approximately 150 non-patient facing staff were included to provide a comparison group, and to improve the generalisability of the results. participants were identified at each participating nhs organisation using internal intranet advertisements and email circulars. children were excluded if they were receiving antibiotics, had been admitted to hospital within the last 7 days, were receiving oral immunosuppressive treatment or if ever diagnosed with a malignancy. informed consent was obtained, and assent given by children where possible. participants were free to decline/withdraw consent at any time without providing a reason and without being subject to any resulting detriment. all children underwent phlebotomy performed by experienced paediatric medical and nursing professionals. serum and/or plasma were tested for antibodies to sars-cov-2, in united kingdom accreditation service accredited laboratories using the following assays, which have been validated for use in adults: [23] [24] [25] ► nucleocapsid assays (abbott architect sars-cov-2 igg and roche elecsys anti-sars-cov-2 total antibody) ► spike protein assays (diasorin liaison sars cov-2 s1/ s2 igg assay) the abbott, roche and diasorin assays are highly specific for sars-cov-2 antibodies, using the manufacturer's suggested cut-offs, with specificities of 1.00 (95% ci 0.98 to 1.00), 1.00 (95% ci 0.99 to 1.00) and 0.98 (95% ci 0.96 to 0.99), respectively. [23] [24] [25] they do, however, have lower sensitivities at 0.94 (95% ci 0.86 to 0.98), 0.84 (95% ci 0.75 to 0.91) and 0.64 (95% ci 0.54 to 0.73), respectively. [23] [24] [25] a summary of the tests used is provided in table 1. study data were collected on a case report form (crf) using redcap (research electronic data capture) electronic data capture tools. 26 participants and their parents provided information at enrolment relating to age, sex, previous health and potential predictors of sars-cov-2 seropositivity including; known contact with individuals with covid-19, contact with individuals who have been symptomatic and/or self-isolating and results of any diagnostic testing such as rt-qpcr testing/ antibody testing. participants and their parents also reported any symptoms and illness episodes since the onset of the pandemic in march but prior to the first clinic appointment. data were collected relating to symptoms but not relating to time of onset or duration of illness. to minimise recall bias, data relating to exposures and illness episodes were collected blinded to antibody testing results. copies of the crfs used at enrolment can be found in the online supplemental material. ► presence of antibodies (igg/total antibody) to sars-cov-2 in serum or plasma reported as titres. ► sars-cov-2 seropositivity defined as a positive antibody test using the manufacturer's advised positivity cut-off. ► predictors of sars-cov-2 positivity including reported symptoms. the study was powered to detect a change in seroprevalence of sars-cov-2 antibodies at three time points (enrolment, and 2 months and 6 months following enrolment). to achieve this, 675 participants were required (assuming alpha of, 0.05 and beta of 0.2). allowing for a 30% dropout rate, we aimed to recruit 900 participants from five sites. the data presented in this study reflect only the data collected at enrolment and the study is ongoing. variables including sex, age, parent role, symptomatology, household contacts and sars-cov-2 antibody prevalence were analysed using descriptive statistics (number and proportion for discrete variables, median and iqr for continuous variables). seroprevalence rates between sites were compared using fisher's exact test and antibody titres were correlated with age using the kendall's rank correlation test and mean titres were compared between symptomatic and asymptomatic participants using the wilcoxon rank sum test. variables associated with sars-cov-2 positivity were analysed using univariate and multivariable analyses to identify predictors of sars-cov-2 seropositivity. initially all possible variables were assessed using univariate analysis with fisher's exact testing of categorical data, and the mann-whitney u test for continuous data (continuous data were skewed). all variables were then included in a weighted binary multivariable logistic regression model. participants with incomplete crfs were excluded from univariate and multivariable analyses. analysis was conducted in r (r core team, 2014). a patient and public involvement (ppi) group comprising parents and children was convened. the ppi group met virtually and via socially distanced meetings. the group contributed to the design of the study through online surveys and video discussions. they have also contributed to media interviews on national television and the lead young person has coauthored a manuscript outlining their experience of taking part in the study. 27 this study was registered at https://www. clinicaltrials. gov (trial registration: nct0434740) on 15 april 2020 (last updated 27 may 2020). at the time of registration no patients had been recruited to the study which opened on 16 april 2020. the end of the study will be the last study visit. in total, 1042 potential participants were screened for inclusion, of whom 35 were excluded; 18 were outside the specified age range, 1 met specific exclusion criteria and 16 declined consent. the remaining 1007 children were enrolled, of which 15 were excluded from analysis due to unsuccessful phlebotomy; 992 were included in the final analysis ( figure 1) there were 68/992 participants with positive sars-cov-2 antibodies, giving a seroprevalence of 6.9% (95% ci 5.4% to 8.6%, n=992). of those with positive sars-cov-2 antibody tests, 34/68 (50%) reported no symptoms. the most commonly reported symptoms associated with sars-cov-2 seropositivity were fever 21/68 (31%), gastrointestinal symptoms (diarrhoea, vomiting and abdominal cramps) 13/68 (19%) and headache 12/68 (18%). the presence of fever, cough or changes in a sense of smell/taste were recorded in 26/68 (38%) of participants. no children within this cohort had severe disease requiring hospital admission. a summary of reported symptoms and their frequency can be seen in table 3. seroprevalence of sars-cov-2 antibodies varied between sites. belfast had significantly lower seroprevalence than all other sites at 0.9% (95% ci 0.2% to 3.3%, n=215); p<0.0001, and in london seroprevalence was significantly higher than all other sites at 11.6% (95% ci 7.8% to 16.8% n=199); p=0.0069. the remaining three sites reported seroprevalence rates between 5.6% and 8.9%. the differences between these three sites were not statistically significant (table 2) . the mean antibody titres, for those testing positive, were; ► 4.86 calculated index s/c (95% ci 4.28 to 5.45, n=58) for the abbott architect sars-cov-2 igg assay. ► 65.32 cut-off index coi (95% ci 43.24 to 87.40, n=31) for the roche elecsys anti-sars-cov-2 total antibody assay. ► 64.17 au/ml (95% ci 37.99 to 90.36, n=31) for the diasorin liaison sars cov-2 s1/s2 igg assay. there was no correlation between age and antibody titres ( figure 3) . the results from the abbott architect sars-cov-2 igg assay indicated a small but significant difference in mean antibody titres between asymptomatic 4.3 s/c (95% ci 3.4 to 5.2) and symptomatic participants 5.5 s/c (95% ci 4.7 to 6.2); p=0.04. there was no significant difference in mean antibody titres for the roche elecsys or diasorin liaison assays when comparing symptomatic and asymptomatic participants (p=0.23 and 0.58, respectively) (figure 3). a table of concordance between the three assays used is available in the online supplemental material. the univariate analysis of individual variables associated with sars-cov-2 seropositivity is shown in table 3. in addition to clinical features, variables such as age, gender, the role of the parent (patient facing or not) and known household contacts were included. age and gender were not significantly associated with sars-cov-2 seropositivity (table 3) . parental role showed significant association in the univariate analysis, but this was no longer significant once corrected for site and other variables in the multivariable analysis. contact with a household member with confirmed sars-cov-2 infection was significantly associated with sars-cov-2 seropositivity in the participant in both the univariate and multivariable analyses (table 3) this observational study is one of the largest uk studies of paediatric sars-cov-2 antibody seroprevalence, and the only study to recruit from all regions of the uk. following the first pandemic wave in the uk, 68/992 (6.9%) children of healthcare workers had evidence of prior infection with sars-cov-2. while this is likely to be higher than the general population it is surprisingly similar to the seroprevalence reported by the office for national statistics study of adults from england and wales (6.2%), 10 and similar to international estimates. [11] [12] [13] as expected, there was marked geographical variation, with london reporting the highest seropositivity rates (11.6%) and belfast the lowest (0.9%) p<0.0001. these regional variations are consistent with published adult estimates of seroprevalence from the same time period. 10 in this study there was a near equal number of children under 10 years of age 32/68 (47%) and children over 10 years of age 36/68 (53%) developing antibodies consistent with previous sars-cov-2 infection. age, as a categorical or continuous variable, was not a statistically significant factor in predicting the presence of antibodies, or the overall titres in children irrespective of the assay used ( figure 3) . of the 68 participants with positive antibody tests, 34/68 (50%) reported no symptoms. the most commonly reported symptoms associated with sars-cov-2 seropositivity were fever (21/68, 30%) and gastrointestinal symptoms (13/68, 19%) . these symptoms, in addition to fatigue, and changes in sense of smell or taste, were independently associated with previous sars-cov-2 seropositivity based on the weighted binary multivariable regression modelling. these findings reflect a number of international studies. [15] [16] [17] [18] [19] [20] current uk testing strategies directing testing only for those with fever, cough or changes in smell/taste would have identified 26/34 (76%) of symptomatic participants in this study (assuming 100% sensitivity and specificity of rt-qpcr swab testing). adding gastrointestinal symptoms would have identified nearly all symptomatic cases in this cohort (33/34, 97%). it is, however, important to note that the predictive value of individual symptoms is context-dependent and their utility will vary depending on the season and the symptomatology of other circulating infections. these findings may be useful to policy makers when considering the best approach to screening paediatric populations for sars-cov-2. there is evidence from adult serological studies that those with severe illness develop a significantly greater antibody response than those with mild or asymptomatic disease. [28] [29] [30] this has raised concerns that children, who typically have mild disease, may fail to develop a meaningful antibody response to sars-cov-2 infection. more recently, emerging adult data suggest that even asymptomatic adults are capable of mounting a potentially lasting and protective immune response. 31 32 in our study antibody titres measured using the abbott architect sars-cov-2 igg assay were significantly higher in symptomatic children compared with asymptomatic children (p=0.04). these findings were not replicated with either the roche elecsys anti-sars-cov-2 or diasorin liaison sars cov-2 s1/s2 igg assays. it therefore remains unclear to what extent the severity of symptoms in children influences the antibody response. the strengths of this study are that it is a large multicentre study including children from across the four nations of the uk. the findings are based on systematically screening children for sars-cov-2 antibodies and this removes selection bias from assessment of the asymptomatic proportion and determination of symptomatology. the limitations of this study are: ► the sars-cov-2 antibody tests have not been validated for use in children. ► the absolute sample size of seropositive participants is relatively small. ► there was selection bias towards children of hospital staff and children with only mild disease. ► there is a risk of recall bias due to the retrospective nature of data collection relating to symptomatology. this study demonstrates that approximately half of children with positive antibody tests for sars-cov-2 reported no symptoms. this study also demonstrates that younger children were just as likely to have sars-cov-2 antibodies as older children and that they are capable of mounting a similar antibody response. covid-19 in children: analysis of the first pandemic peak in england defining the epidemiology of covid-19 -studies needed cdc covid-19 response team. coronavirus disease 2019 in children -united states amsterdam: national institute for public health and the environment (rivm) nasal gene expression of angiotensin-converting enzyme 2 in children and adults physiological and pathological regulation of ace2, the sars-cov-2 receptor angiotensin-converting enzyme 2 (ace2), sars-cov-2 and the pathophysiology of coronavirus disease 2019 (covid-19) a cautionary tale of false-negative nasopharyngeal covid-19 testing covid-19: phe laboratory assessments of molecular tests latest data and analysis on coronavirus (covid-19) in the uk and its effect on the economy and society prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study seroprevalence of anti-sars-cov-2 igg antibodies in geneva, switzerland (serocov-pop): a population-based study seroprevalence of sars-cov-2 igg significantly varies with age: results from a mass population screening (sars-2-screen-cda) epidemiology of covid-19 among children in china detection of covid-19 in children in early sars-cov-2 infection in children children with covid-19 in pediatric emergency departments in italy clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study screening and severity of coronavirus disease 2019 (covid-19) in children in the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies seroprevalence of sars-cov-2 antibodies in children of healthcare workers-a prospective multicentre cohort study protocol evaluation of the abbott sars-cov-2 igg for the detection of anti-sarscov-2 antibodies evaluation of roche elecsys antisars-cov-2 serology assay for the detection of anti-sars-cov-2 antibodies evaluation of diasorin liaison sarscov-2 s1/s2 igg serology assay for the detection of anti-sars-cov-2 antibodies research electronic data capture (redcap)--a metadata-driven methodology and workflow process for providing translational research informatics support listening to the voices of children and young people involved in medical research antibody responses to sars-cov-2 at 8 weeks postinfection in asymptomatic patients antibody responses to sars-cov-2 in patients with covid-19 profile of immunoglobulin g and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19 detection, prevalence, and duration of humoral responses to sars-cov-2 under conditions of limited population exposure this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. ethics approval ethical approval was obtained from the london -chelsea research ethics committee (rec reference -20/hra/1731) and the belfast health & social care trust research governance (reference 19147tw-sw).provenance and peer review not commissioned; externally peer reviewed.data availability statement data are available in a public, open access repository. all of the individual participant data collected during this study will be available (including data dictionaries) on the queen's university belfast database within 3 months of completion of the study.supplemental material this content has been supplied by the author(s). it has not been vetted by bmj publishing group limited (bmj) and may not have been peer-reviewed. any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by bmj. bmj disclaims all liability and responsibility arising from any reliance placed on the content. where the content includes any translated material, bmj does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. key: cord-338243-njkhwkwk authors: zhang, dayi; ling, haibo; huang, xia; li, jing; li, weiwei; yi, chuan; zhang, ting; jiang, yongzhong; he, yuning; deng, songqiang; zhang, xian; wang, xinzi; liu, yi; li, guanghe; qu, jiuhui title: potential spreading risks and disinfection challenges of medical wastewater by the presence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) viral rna in septic tanks of fangcang hospital date: 2020-06-23 journal: sci total environ doi: 10.1016/j.scitotenv.2020.140445 sha: doc_id: 338243 cord_uid: njkhwkwk abstract the outbreak of coronavirus infectious disease-2019 (covid-19) pneumonia raises the concerns of effective deactivation of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in medical wastewater by disinfectants. in this study, we evaluated the presence of sars-cov-2 viral rna in septic tanks of wuchang cabin hospital and found a striking high level of (0.5–18.7) × 103 copies/l after disinfection with sodium hypochlorite. embedded viruses in stool particles might be released in septic tanks, behaving as a secondary source of sars-cov-2 and potentially contributing to its spread through drainage pipelines. current recommended disinfection strategy (free chlorine ≥0.5 mg/l after at least 30 min suggested by world health organization; free chlorine above 6.5 mg/l after 1.5-h contact by china centers for disease control and prevention) needs to be reevaluated to completely remove sars-cov-2 viral rna in non-centralized disinfection system and effectively deactivate sars-cov-2. the effluents showed negative results for sars-cov-2 viral rna when overdosed with sodium hypochlorite but had high a level of disinfection by-product residuals, possessing significant ecological risks. the outbreak of coronavirus infectious disease-2019 (covid-19) pneumonia since 2019 is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (lai et al., 2020; li et al., 2020; ralph et al., 2020) , and it has rapidly spread throughout 202 countries around the world. till 20 th june 2020, there are over 8.0 million confirmed cases and around 450,000 deaths globally, and the number is still increasing rapidly (who, 2020a) . there is clear evidence of human-to-human transmission of sars-cov-2 (chan et al., 2020; chang et al., 2020; li et al., 2020; poon and peiris, 2020) . besides direct contact and respiratory routes (carlos et al., 2020; lai et al., 2020; wu et al., 2020) , stool transmission might be an alternative route owing to the survival of sars-cov-2 in patient's stools (holshue et al., 2020; ling et al., 2020; tian et al., 2020; xiao et al., 2020; xing et al., 2020; zhang et al., 2020c) . as municipal wastewater pipe network receives huge amounts of wastewater from asymptomatic patients and treated sewage from hospitals, sars-cov-2 from non-or inefficient-disinfected wastewater might persist for a prolonged time in pipe network, becoming a secondary spreading source (zhang et al., 2020a) . it brings urgent requirement and careful consideration of disinfection strategies to prevent sars-cov-2 from entering drainage pipe network. disinfection is of great importance to eliminate or deactivate pathogenic microorganisms. traditional disinfection strategies include ultraviolet germicidal irradiation and biocidal agents, e.g., gaseous ozone, alcohol, formaldehyde, hydrogen peroxide, peroxyacetic acid, povidone iodine and chlorine-based disinfectant (kampf et al., 2020; tseng and li, 2008; tseng and li, 2007; walker and ko, 2007) . some disinfectants are intensively used in hospitals for nurse personal care (dumas et al., 2019; ioannou et al., 2017) . among them, chlorine-based disinfectants are widely j o u r n a l p r e -p r o o f used for their broad sterilization spectrum, high inactivation efficiency, low price, and easy decomposition with little residue (how et al., 2017) . nevertheless, overuse of chlorine-based disinfectants brings concerns of disinfection by-products (dbps) which are harmful to ecosystems and human health (bull et al., 2011; richardson et al., 2007; wang et al., 2014) . more than 600 kinds of dbps have been observed (richardson, 2011) , such as trihalomethanes (thms), haloacetic acids (haas), halogen acetonitriles (hans), halonitromethanes (hnms) and haloacetamides (hacams) (ding et al., 2020; kozari et al., 2020; luo et al., 2020; zhai et al., 2014) . some of them are reported attributable for bladder cancer (evlampidou et al., 2020; li and mitch, 2018) and adverse reproductive outcomes (nieuwenhuijsen et al., 2000) . for effective centralized disinfection, world health organization (who) has suggested free chlorine ≥ 0.5 mg/l after at least 30 minutes of contact time at ph<8.0 (who, 2020b). additionally, china has launched a guideline for emergency treatment of medical sewage containing sars-cov-2 on 1 st february 2020, requiring free chlorine of ≥ 6.5 mg/l and contact time of ≥1.5 hour in disinfection units (china-mee, 2020) . unfortunately, the performance of chlorine-based disinfectants on sars-cov-2 in real medical wastewater treatment system is not clear yet. in this work, we studied the presence of sars-cov-2 viral rna in septic tanks of wuchang cabin hospital (wuhan, china) to evaluate the disinfection performance and optimize disinfection strategies to prevent sars-cov-2 from spreading through drainage pipelines. further analysis of dbps evaluated the potential ecological risks in the effluents. j o u r n a l p r e -p r o o f wuchang cabin hospital was a temporary hospital designated for covid-19 patients, formerly wuchang stadium located in wuhan (longitude, e114°20'05''; latitude, n30°32'46'') . it was open from 5 th february as the first cabin hospital in wuhan and closed on 10 th march, in total receiving 1,124 covid-19 patients ( figure 1 ). it had eight separate toilets and all sewage from toilets and showers were combined, with daily volume of wastewater ranging from 60 to 200 m 3 . the wastewater was firstly pumped from toilets and showers into the first preliminary disinfection tank, and sodium hypochlorite was unregularly added to a final concentration of 800 g/m 3 when it was 90% full. afterwards, the first preliminary disinfection tank was temporarily enclosed and the wastewater was then pumped into another disinfection tank and disinfected in the same way. on each day, septic tanks outside the hospital were emptied at 8 am to discharge the effluents into pipe network and wastewater treatment plants and received wastewater from preliminary tanks at 10 am. sodium hypochlorite was supplemented for 1.5 hr contact and the mixing was conducted by continuous pumping and stirring. wastewater was then pumped into three septic tanks. before march 5 th , the dosage of sodium hypochlorite in septic tanks was 800 g/m 3 and it increased to 6700 g/m 3 after 5 th march to secure complete deactivation of sars-cov-2. influent and effluent samples were collected from septic tanks of wuchang cabin hospital on 26 th february, 1 st march and 10 th march, 2020 (table 1) . on these sampling days, the number of covid-19 patients in wuchang cabin hospital was j o u r n a l p r e -p r o o f stable, ranging from 200 to 400. around 2.0 l of water was directly collected in a plexiglass sampler and transferred into a sterile plastic bag for biological analysis and a brown glass bottle for dbps analysis. as we found obvious settlement of stool particles and suspended solids in septic tanks, a stratified plexiglass sampler was used to obtain samples from different layers of septic tanks to assess the levels of viruses and dbps along depth, designated as top-layer (0-50 cm) and bottom-layer (50-100 cm) water. free chlorine was measured on site using pcii58700-00 (hach, usa). dbps measurements were carried out on a gcms-qp2020 (shimadzu, japan) equipped with atomx purge, trap autosampler (teledyne tekmar, usa) and an sh-rtx-5ms [(5%)-phenyl-(95%)-methylpolysiloxane] fused silica capillary column (30 m × 0.25 mm, 0.25 μm film thickness). the autosampler operating conditions were as follows: purge for 11 min at 30 °c with high-purity nitrogen gas at a flow rate of 40 ml/min, dry purge for 1.0 min at 20 °c with the flow rate of 40 ml/min, pre-desorption at 180 °c and desorption for 2.0 min at 190 °c, and bake for 6.0 min at 200 °c. collected water samples were placed in 4 °c ice-boxes and immediately transferred into laboratory for rna extraction. after centrifugation at 3,000 rpm to remove suspended solids, the supernatant was subsequently supplemented with nacl (0.3 mol/l) and peg-6000 (10%), settled overnight at 4 °c, and centrifuged at 10,000 g for 30 minutes. viral rna in pellets was extracted using the ez1 virus mini kit one-way anova was used to compare the difference between samples and p-value less than 0.05 refers to significant difference. all data were presented in mean ± standard deviation (n=3). j o u r n a l p r e -p r o o f for all influents of septic tanks received from wuchang cabin hospital, there was no positive result for sars-cov-2 viral rna. on 26 th february and 1 st march, sars-cov-2 viral rna was (14.7±2.2)×10 3 and (7.5±2.8)×10 3 copies/l in the effluents of septic tanks, respectively (table 1) the absence of sars-cov-2 viral rna in the influents of septic tanks suggested that preliminary disinfection in wuchang cabin hospital was satisfactory to remove sars-cov-2 in aqueous phase after 1.5-hour contact. in septic tanks, disinfection achieved free chlorine > 6.5 mg/l for 1.5 hours when the dosage of sodium hypochlorite was 800 g/m 3 , meeting well with the guideline for emergency treatment of medical sewage containing sars-cov-2 suggested by china cdc. however, sars-cov-2 viral rna was surprisingly positive in the effluents after 12 hours when free chlorine declined to nondetectable. it might be explained by the release of embedded sars-cov-2 viral rna from stool particles in septic tanks. sars-cov-2 has been found in patients' stools in many previous studies (wu et al., 2020; xing et al., 2020) , which could escape from disinfection and slowly release into aqueous phase. suspended particles as small as 7 mm can protect viruses from uv exposure and dwindle their vulnerability to direct sunlight inactivation, and 0.3-mm sized particles can shield viruses from disinfection for their prolonged survival (templeton et al., 2005) . as stools are rich in organic compounds and form numerous suspended solids containing sars-cov-2, they are of high risk as the source releasing viruses in septic tanks. it is also evidenced by more sars-cov-2 rna copy numbers in upper-layer waters, explained by more stool residuals and suspended solids in the bottom of septic tanks absorbing sars-cov-2 from aquatic water. our results suggested that current recommended disinfection dosage could effectively remove aqueous sars-cov-2 viruses in wastewater containing limited suspended solids, like wastewater treatment plants (la rosa et al., 2020; zhang et al., 2020b) . but it might be not enough for embedded viruses in suspended solids and require higher level of chlorine-based disinfectant supplement. septic tanks can behave as a long-term source j o u r n a l p r e -p r o o f to release sars-cov-2 viral rna into waters when disinfection is not sufficient and challenges public health via potentially spreading viruses in drainage pipelines. the surprising presence of sars-cov-2 viral rna after disinfection with sodium hypochlorite suggested that free chlorine > 0.5 mg/l after 1.5-hour contact time cannot completely removing sars-cov-2 viral rna, and 800 g/m 3 dosage of sodium hypochlorite might be not enough to secure a complete disinfection of medical wastewaters, particularly for those from cabin, temporary or non-centralized hospitals containing huge amount of stool debris and suspended solids. from the negative results of sars-cov-2 viral rna (table 1) , the complete deactivation of sars-cov-2 was achieved when the dosage of sodium hypochlorite was 6700 g/m 3 . as the number of covid-19 patients were similar on the three sampling days, they produced similar wastewater discharge and viral load. the change of sars-cov-2 in effluents was therefore attributing to the increasing sodium hypochlorite supplement for disinfection. nevertheless, it was an over-dosage and resulted in a significant level of dbps in the effluents, which was about 15 times higher than other hospital wastewater (luo et al., 2020) . the four detected chloroform and trihalomethanes accounted to the majority of total dbps in wastewaters (furst et al., 2019) and their composition was similar as previous studies (luo et al., 2020; zhong et al., 2019) . they show high ecological risks and challenge the surrounding environment receiving disinfected medical wastewater, possessing threats to ecological system and human health (ding et al., 2020; li et al., 2019 give initial information about the potential risks of viral spread and dbps residues during disinfection processes for medical wastewater containing sars-cov-2. our study for the first time reported an unexpected presence of sars-cov-2 viral rna in septic tanks after disinfection with 800 g/m 3 of sodium hypochlorite and current disinfection guideline by who and china cdc might not secure a complete removal of sars-cov-2 in medical wastewater. sars-cov-2 might be embedded in patient's stools, protected by organic matters from disinfection, and slowly release when free chlorine declines. septic tanks in non-centralized disinfection system of journal pre-proof j o u r n a l p r e -p r o o f cabin hospitals or isolation points potentially behave as a secondary source spreading 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pediatric patients during the convalescent phase formation of brominated disinfection byproducts during chloramination of drinking water: new polar species and overall kinetics sars-cov-2 spillover into hospital outdoor environments ultra-fast and onsite interrogation of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in environmental specimens via surface enhanced raman scattering (sers) fecal specimen diagnosis 2019 novel coronavirus-infected pneumonia disinfection byproducts and their toxicity in wastewater effluents treated by the mixing oxidant of clo2/cl-2 key: cord-325324-kh2aal5n authors: teng, shaolei; tang, qiyi title: ace2 enhance viral infection or viral infection aggravate the underlying diseases date: 2020-08-06 journal: comput struct biotechnol j doi: 10.1016/j.csbj.2020.08.002 sha: doc_id: 325324 cord_uid: kh2aal5n ace2 plays a critical role in sars-cov-2 infection to cause covid-19 and sars-cov-2 spike protein binds to ace2 and probably functionally inhibits ace2 to aggravate the underlying diseases of covid-19. the important factors that affect the severity and fatality of covid-19 include patients' underlying diseases and ages. therefore, particular care to the patients with underlying diseases is needed during the treatment of covid-19 patients. severe acute respiratory syndrome (sars). this was also called sars-1 and the virus was named sars-cov-1. the second beta-coronavirus pandemic occurred in middle eastern countries in 2012 and was hence named middle east respiratory syndrome coronavirus (mers-cov) (10) . the infection was transmitted to 25 countries and resulted in 1360 cases and 527 deaths (http://www.emro.who.int/pandemic-epidemic-diseases/mers-cov/mers-situation-update-january-2020.html). the current (third) pandemic of beta-coronavirus (sars-cov-2) has affected almost all countries, resulting in the disease named covid-19. here, we attempt to analyze the available data from publications or from official who and usa cdc resources and underscore the associations between covid-19 and its comorbidities. like other coronaviruses, sars-cov-2 is a single strand positive rna virus with 29, 811 nucleotides that encodes 12 putative open reading frames responsible for more than 26 proteins through ribosomal frameshifting and host proteasomal processing (11, 12) . the first step of viral infection is attachment, which depends on the interaction of the viral surface with cellular receptors. the sars-cov-2 spike protein (s) is cleaved by the human furin enzyme to generate two subunits, s1 and s2, that are arranged to extrude outward from the viral particle. both s1 and s2 play crucial roles for viral entry (3) . the s1 subunit binds to the host receptor angiotensin converting enzyme 2 (ace2) ( table 1) . while its binding to the membrane-bound ace2 promotes viral attachment to infected cells, the soluble ace2 might prevent viral infection by binding to s1 (13) . the s2 subunit, after s1's interaction with ace2, promotes viral fusion with the host cell membrane via interaction with transmembrane protease, serine 2 (tmprss2) and enables viral entry (3) . interestingly, tmprss2 has the proteolysis effect on ace2, which augments the entry of sars-cov-1 and probably cov-2 (14) (15) (16) . after entry, viral particle is endocytosed to the endosome and uncoated in a ph-related manner. viral rna is released to the host endoplasmic reticulum (er). not only do viral protein translation and rna transcription happen in the er, but also viral polyprotein processing: viral subgenomic rnas and stem looped rnas are formed in the er. newly generated viral particles are assembled in the golgi body for exit out of cells. the viral membrane protein (m), important for maintaining viral structure and the viral envelop protein (e) together play roles in viral assembly and release. although the basis of viral replication is outlined as above, many aspects are still not understood, especially for the polyprotein processing, ribosomal frameshifting and formation of subgenomic rnas. we will specifically discuss the interaction of sars-cov-2 with ace2 in this minireview. viral origin has been a standing hot topic of sars-cov-2 for a variety of reasons. here we trace the early published information that are more reliable than those from the mass media. the first two confirmed covid-19 cases occurred in wuhan, china on december 8 th and 10 th of 2019. the patients never went to the huanan seafood market that was disputed to be a potential origin of sars-cov-2. in the next 10 days (december 13 to 23), more cases were found. among the total 25 cases found during these 10 days, 15 patients went to the seafood market; therefore, the seafood market was argued to be the origin of sars-cov-2. an epidemiological analysis of the first 425 cases in wuhan found that most subsequently obtained infections were not originated from the huanan seafood market (17) . it is now more or less denied by epidemiologists that the huanan seafood market could have any link with the virus (18, 19) . however, the seafood market origin of sars-cov-2 became mythologized. the outbreak in wuhan caused the viral spreading to other provinces in china by late january while it was later transmitted to other countries: first to thailand, then usa, france, germany and italy. sothern korea and italy are among the first countries with outbreaks in february (17, 20) . it quickly became a pandemic and affects more than 200 countries now with more than 3 million cases and over 200,000 deaths globally. a previously isolated bat coronavirus (batcov ratg13) has been identified that shares more than 96% homology in nucleotide sequences and more than 97% homology in amino acid sequences with sars-cov-2 (21) . this study implied that the sars-cov-2 might have originated from bats. however, bats are not a common resident animal in the city of wuhan. therefore, an intermediate host for sars-cov-2 is believed to exist assuming it is a zoonotic virus. one study found that a coronavirus isolated from the pangolin is 91.02% and 90.55% identical in nucleotide sequences to sars-cov-2 and batcov ratg13, respectively (22) . another study showed that sars-cov-2 is possibly associated with coronaviruses derived from some wild animals, including paguma larvata, paradoxurus hermaphroditus, in the same branch of the phylogenetic tree (23) . however, they all are unlikely the intermediate host because the genome and orf1a homology show that the virus is not even close to sars-cov-2. given sars-cov-2 is originated from bat ratg13, the virus from the intermediate host should be closer to sars-cov-2 than to the ratg13. although it is difficult to find it, the intermediate host is still the interest of virologists (24, 25) . surprisingly, a research group screened the susceptibilities of different companion animals to sars-cov-2 infection and found that cats and ferrets are very susceptible to sars-cov-2. on the contrary, sars-cov-2 poorly infects and replicates in dogs, pigs, chickens, and ducks (26). the first recorded covid-19 case in the usa was in seattle, washington on january 19 th , 2020 in a patient who traveled back from wuhan, china. two days later, cases were reported in chicago on january 21 st , 2020. another two days later, covid-19 cases were confirmed in california and arizona. these early cases all occurred in patients who traveled from china. due to its highly contagious nature, sars-cov-2 has proven catastrophic in both prevalence and fatality in the united states. the most important difference between sars-cov-2, sars-cov-1 (2002) and (2009) is that sars-cov-2 is highly contagious with a much higher reproductive number (r0) of 5.7 (27) or 3.57 (28) is rapidly spreading to other countries and territories to cause covid-19. more importantly, epidemic data suggests that the covid-19 might become a seasonal pandemic at a similar or even a larger scale (29) . although the final fatality rate is the most accurate and can only be obtained when the pandemic is over, the current data shows that it could be very high in some countries. interestingly, the fatality by covid-19 clearly correlates with whether the patients have one or more underlying diseases. the cdc webpage have listed 10 high-risk underlying diseases; the top comorbidities include hypertension and cardiovascular diseases (cvds) (30) (31) (32) (33) and occur at significantly higher rates in the african american and nonwhite hispanic groups in the usa (34) . the abovementioned comorbidities of covid-19, hypertension, dm and cvds, are closely associated with ras signaling. angiotensin i is generated from angiotensinogen by renin after being stimulated by several conditions such as low blood pressure, bleeding and dehydration (35) (36) (37) (38) . angiotensin i is then converted to angiotensin ii by angiotensin converting enzyme (ace) (39, 40) . angiotensin ii constricts vasculature to elevate the blood pressure. therefore, abnormally high levels of angiotensin ii cause hypertension and also aggravate dm sequelae and induce cvds as shown in fig. 1 have been clinically used to treat the relevant diseases. angiotensin ii can be subsequently hydrolyzed by ace2 to generate angiotensin peptides, mainly angiotensin-(1-7), also called angis a vascular dilator and hence reduces blood pressure (see fig. 1 ). ace ii also hydrolyzes the angiotensin i to other peptide angiotensin such as angiotensin-(1-9) that is another vascular dilator (42) (43) (44) . therefore, the ras signaling is a balanced system that must be maintained for the healthy cardiovascular system with normal blood pressure and normal cardiovascular function. a theory for explaining how the aforementioned comorbidities significantly increase the fatality of covid-19 was based on the fact that these patients have a history of taking drugs such as aceis, arbs and ibuprofen (45, 46) . these drugs not only inhibit ace but increase the level of ace2 as well. the increase of ace2 might elevate the chances of infection of sars-cov-2, which directly relates to the viral infection and fatality. this theory is shown in the left side of fig. 1 as "ace2 enhancing viral infection" (45, 46) . however, this theory does not explain why the onset of covid-19 in these patients is so rapid that an emergent rescue is needed and many of them still die. furthermore, it contradicts the recently published reports that using arbs and aceis improved covid-19 (47) (48) (49) . some critical reviews analyzed the available evidence and found that it does not support a deleterious effect of ras blockers in covid-19 (50) (51) (52) . therefore, it is suggested that there is currently no reason to discontinue ras blocking aceis in stable patients who suffer from the covid-19 (50) . ace2 is a cell membrane protein in lungs, arteries, heart, kidney and intestines (53) (54) (55) . ace2 converts angiotensin ii to peptide ang-(1-7) or angiotensin i to peptide ang-(1-9); both peptides are vasodilators (35, 41, (56) (57) (58) . therefore, ace2 physiologically reduces blood pressure and is anti-hypertensive when the angiotensin ii is elevated. sars-cov-2 spike protein (s) is cleaved by the human furin enzyme to generate s1, which binds to the host receptor, ace-2. it is possible that the released free spike or the cleaved s1 protein in the blood might bind to cellular membrane ace2 of heart, artery and alveolar lung cells to block the conversion of angiotensin ii to ang-(1-7) and/or angiotensin i to ang-(1-9), which is consistent with a previous experimental result on sars-cov-1 (59) . the interaction with sars-cov-2 s protein might exhaust ace-2 or damage ace-2 function. therefore, our hypothesis, as shown in the right side of fig. 1 as "viral aggravating existing diseases", is that comorbidities in covid-19 patients are aggravated by the infection of sars-cov-2 to causes higher fatalities because the viral s protein interacts with ace2 to inhibit ace2 function. this hypothesis is also supported by a clinical finding that an imbalance of the angii-ace2-ang-(1-7) axis occurs in human pulmonary artery hypertension (pah), with reduced ace2 levels implicated in the pathogenesis of severe pah (60) . however, an animal study demonstrated that ace2 level in lung is low and that prolyloligopeptidase (pop) is the main enzyme responsible for ang ii conversion to ang-(1-7) in the lungs (61) . therefore, an alternative mechanism might exist when ace2 is exhausted in covid-19 patients. and 50%, respectively (66) . a novel furin cleavage site was identified at the boundary between the s1/s2 subunits of sars-cov-2 but not in sars-cov-1 and other closely sars-related covs (67) . recent studies revealed that sars-cov-2 uses ace2 as a receptor for cellular entry (67, 68) . sars-cov-2 first binds to ace2 via rbd of its spike protein and then fuses viral and host membranes (67) . the study also showed that sars-cov-1 spike polyclonal antibodies block sars-cov-2 mediated entry into host cells and cross-neutralizing antibodies may provide protection against sars-cov-2 (67). (table 2) . for example, ace2 residue s19 can form a strong polar contact with sars-cov-2 residue a475, where some monoclonal antibodies could not effectively neutralize the viral mutation a475v in this site (71) . d355 in ace2 can form hydrogen-bonds with t500 and g502 in sars-cov-2. molecular dynamics study suggested that d355 is involved in a critical hydrogen-bonding network of rbd-ace2 interaction (72) . ace2 residue m82 and sars-cov-2 residue f486 are involved in hydrophobic interactions at the interface. f486 has been identified as a binding site for neutralizing antibodies (73) . these results explain the efficient transmission of sars-cov-2 in humans. the sequence variants in ace2 have been found to be related to human complex disorders. several case-control association studies identified the ace2 genetic single nucleotide polymorphisms associated with hypertension (74, 75) . the coding mutations in key residues of ace2 may alter the binding affinity of sars-cov-2/ace2 complex and change pathogenicity of ace2. highthroughput sequencing technologies have been used to identify the rare single nucleotide variants (snvs) in different populations. we collected 227 missense variants from the genome aggregation database (gnomad: https://gnomad.broadinstitute.org/). of 227 missense snvs, 6 rare mutations can be mapped onto the contact residues at the sars-cov-2 rbd/ace2 interface (table 2 and figure 2 ). s19p (rs73635825) and t27a (rs781255386) have positive binding energy changes, indicating that these two snvs can increase the ace2-rbd binding affinity. s19p has 63 alleles (0.3% allele frequency) in the african population. s19 is interacting partner of sars-cov-2 residue a475 (69). it's observed the sars-cov-2 mutation a475v can reduce the sensitivity to some monoclonal antibodies (71) . the increased binding affinity may result the sars-cov-2 mutant became resistant to neutralizing antibodies. in addition, s19p in african population is predicted as a possibly damaging variant. in contrast, t27a present in latino population are predicted to be a benign variant. the other four mutations (e35k, e37k, m82i and d355n) have negative binding energy changes and can weaken the binding affinity of sars-cov-2/ace2 complex. one possible explanation is that these mutations can disrupt the interaction network of protein complex. for example, d355 forms a hydrogen bond with sars-cov-2 residues t500 (72), d355n (rs961360700) in european population (non-finnish) can break this hydrogen-bonding network and reduce the protein-protein interaction between sars-cov-2 spike rbd and ace2. these findings indicate that the sequence variants in different populations can have different effects on protein function of ace2 and the protein-protein interaction between ace2 and sars-cov-2 spike protein, which may result in the different health disparities of covid-19. different disparities have been reported in populations with covid-19 whose deaths are related to older age, male sex, and concomitant diseases (34) . first, it was noted that the deaths caused by covid-19 clearly associate with age of the patients. this was first reported for the covid-19 populations in wuhan, china (76) where the median age of patients who died was 75 (range 48 to 89). another study estimated the death rate in china was 0.66% and was increasing by age to 6.4% for age 60 and older, and to 13.8% for age of 80 or older (77, 78) . a similar situation was confirmed in uk with 80% of covid-19-related deaths in those aged 65 years and over and in the usa with those in the 65-84 years age group accounting for 25% of cases, 46% of intensive care unit admissions and 46% of deaths (79, 80) . the link between death rate and age has been observed in many countries (81) . many factors could connect age to covid-19-caused fatalities. one of the important facts is that the aforementioned comorbidities of hypertension, dm and cvds are less common in younger people than that in older people. the underlying diseases links to both the case fatality of covid-19 and the economically disadvantaged groups and/or socially isolated communities. for example, as dr. yancy summarized (34), 1) more than 50% of covid-19 cases and nearly 70% of covid-population in chicago; 2) 70.5% of deaths have occurred among african american persons, who represent 32.2% of the state's population in louisiana; 3) 33% of covid-19 cases and 40% of deaths have occurred among african american individuals, who represent 14% of the population in michigan; and 4) new york city is the epicenter of covid-19 in the usa, african americans and hispanics have accounted for 28% and 34% of deaths, respectively (population representation: 22% and 29%, respectively). dr. yancy also noted that it is likely that some, if not most, of these differences in disease rates and outcomes will be explained by concomitant comorbidities. however, it is possible that the majority of the disparity has to do with access to healthcare, education about the virus and its symptoms, or other well-documented socioeconomic disparities in us healthcare. the claims that covid-19 disproportionately affects the individuals of minority groups and aged people are not only supported by reported data but also by our hypothesis that sars-cov-2 infection generates spike protein that interacts with ace2 to either exhaust ace2 or inhibit ace2 function or both so that the comorbidities are aggravated (figure 1 ). the top comorbidities in covid-19 patients are hypertension, and cardiovascular disease, all of which are directly related to ace2. therefore, we suggest that ace2-sars-cov-2 spike interaction is a specific target not only for treatment of the severe diseases but also for prophylactic control of the infection of sars-cov-2. the strategies of using the target might be better against the spike protein of sars-cov-2 than ace2 because targeting ace2 per se might impose a detrimental effect on the patient who has the underlying diseases. here we reviewed recently published information with regard to covid-19, especially the biological role of ace2 in the pathogenesis of covid-19 and certain comorbid diseases. we hypothesized that ace2 plays a key role in the severity and fatality of covid-19 and viral infection exhausts ace2 or functionally inhibits ace2 to aggravate the comorbidities of covid-19 patients. our hypothesis emphasizes the relationship between covid-19 and the comorbid diseases which not only interpret high fatalities of covid-19 in aged people but may also contribute to socioeconomic disparities of covid-19. the medical intervention strategies of using the target might be better against the spike protein of sars-cov-2 than ace2 because targeting ace2 per se might impose a detrimental effect on the patient who has the underlying diseases. the effects of variants on binding affinity were predicted using mcsm-ppi2 (http://biosig.unimelb.edu.au/mcsm_ppi2/). mutation pathogenicity predictions were extracted from dbnsfp (https://sites.google.com/site/jpopgen/dbnsfp). allele counts in different populations were downloaded from gnomad (https://gnomad.broadinstitute.org/) figure 1 . two theories to explain the severity of covid-19: 1. ace2 enhancing viral infection, and 2. viral aggravating existing diseases. ace catalyzes the conversion of angiotensin i to angiotensin ii, a vasoconstrictor. angiotensin ii is converted by ace2 to angiotensin-(1-7), a vasodilator. theory 1 suggests that using acei/arb increases ace2, which further enhances viral infection. theory 2 suggests that ace2 can be either exhausted or functionally inhibited by s1 so that more angiotensin ii and less angiotensin-(1-7) are produced, which would aggravate underlying diseases. (green), sars-cov-2 spike receptor-binding domain (rbd, yellow orange) and receptor-binding motif (rbm: brown) were shown as cartoons. ace2 contacted residues, including s19 (purple), t27 (red), e35 and e27 (magenta), m82 (orange) and d355 (hot pink), were displayed as sticks. the image was generated using pymol (http://www.pymol.org/) based on pdb id: 6lzg. genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences genomic classification using an informationbased similarity index: application to the sars coronavirus coronaviruses: an overview of their replication and pathogenesis the "common cold" in frail older persons: impact of rhinovirus and coronavirus in a senior daycare center a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes coronavirus infections-more than just the common cold novel 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sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structural and functional basis of sars-cov-2 entry by using human ace2 structural basis for the recognition of sars-cov-2 by full-length human ace2 the impact of mutations in sars-cov-2 spike on viral infectivity and antigenicity enhanced receptor binding of sars-cov-2 through networks of hydrogen-bonding and hydrophobic interactions key residues of the receptor binding motif in the spike protein of sars-cov-2 that interact with ace2 and neutralizing antibodies association of ace2 genetic polymorphisms with hypertension-related target organ damages in south xinjiang ace2 gene polymorphism and essential hypertension: an updated meta-analysis involving 11,051 subjects updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan covid-19: death rate is 0.66% and increases with age, study estimates estimates of the severity of coronavirus disease 2019: a model-based analysis protecting older people from covid-19: should the united kingdom start at age 60? estimating the infection and case fatality ratio for coronavirus disease (covid-19) using ageadjusted data from the outbreak on the diamond princess cruise ship biomarkers of biological age as predictors of covid-19 disease severity human aminopeptidase n is a receptor for human coronavirus 229e structural and functional analysis of the surface protein of human coronavirus oc43 receptor and viral determinants of sars-coronavirus adaptation to human ace2 receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc key: cord-332992-8rmqg4rf authors: de vries, a. a. f. title: sars-cov-2/covid-19: a primer for cardiologists date: 2020-07-15 journal: neth heart j doi: 10.1007/s12471-020-01475-1 sha: doc_id: 332992 cord_uid: 8rmqg4rf in the late autumn of 2019, a new potentially lethal human coronavirus designated severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan, china. the pandemic spread of this zoonotic virus has created a global health emergency and an unprecedented socioeconomic crisis. the severity of coronavirus disease 2019 (covid-19), the illness caused by sars-cov‑2, is highly variable. most patients (~85%) develop no or mild symptoms, while others become seriously ill, some succumbing to disease-related complications. in this review, the sars-cov‑2 life cycle, its transmission and the clinical and immunological features of covid-19 are described. in addition, an overview is presented of the virological assays for detecting ongoing sars-cov‑2 infections and the serological tests for sars-cov-2-specific antibody detection. also discussed are the different approaches to developing a covid-19 vaccine and the perspectives of treating covid-19 with antiviral drugs, immunomodulatory agents and anticoagulants/antithrombotics. finally, the cardiovascular manifestations of covid-19 are briefly touched upon. while there is still much to learn about sars-cov‑2, the tremendous recent advances in biomedical technology and knowledge and the huge amount of research into covid-19 raise the hope that a remedy for this disease will soon be found. covid-19 will nonetheless have a lasting impact on human society. electronic supplementary material: the online version of this article (10.1007/s12471-020-01475-1) contains supplementary material, which is available to authorized users. coronaviruses (covs) were first described in the 1930s and first visualised (by electron microscopy) in the 1960s [1, 2] . covs own their name to the pedal-shaped protrusions emerging from the surface of the virus particles (also called virions), which give them an appearance reminiscent of a crown. covs belong to the family of coronaviridae, which is divided into two subfamilies. the main subfamily-orthocoronavirinae-is subdivided into four genera, designated alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. covs generally have a limited host range and, with a few exceptions, cause either respiratory or enteric infections in mammals and birds. until recently, six different human covs were distinguished, all of which primarily infect the respiratory tract (tab. 1). of these viruses, hcov-229e, hcov-hku1, hcov-oc43 and hcov-nl63 are responsible for some 10-15% of the annual common colds [3] . whereas infections with these viruses usually take a mild course this is not the case for infections with severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov, for which case-fatality rates of~10 and 35%, respectively, have been reported [4] . recently, a new human cov has emerged in wuhan, people's republic of china, which appears genetically very closely related to several bat cov isolates and somewhat more distantly to sars-cov [5, 6] . based on phylogenetic considerations, the new virus was named sars-cov-2, which stands for severe acute respiratory syndrome coronavirus 2 [7] , and the accompanying disease was designated coronavirus disease 2019 . like sars-cov and mers-cov, sars-cov-2 is assumed to be a zoonotic virus with bats serving as reservoir hosts. transmission of sars-cov and mers-cov from bats to humans likely occurred via intermediate hosts (presumably civet cats in the case of sars-cov and dromedary camels for mers-cov) to allow these viruses to adapt in a stepwise fashion to their new host species. spillover of sars-cov-2 from bats to humans probably also involved an intermediate host. whether pangolins have acted as such, as initially proposed, is, however, doubtful [8] . the extraordinary ability of rna viruses to adapt to changing selective pressures and to invade new hosts stems from the low fidelity with which they replicate their genome (i.e. genetic material) in combination with their high replication rate (i.e. short generation time). as a consequence, the genomes of rna viruses are highly heterogeneous, consisting of dynamic populations of genetically related variants, also known as mutant swarms or quasispecies [9] . this not only allows rna viruses to infect new host species but also to escape immune surveillance and to become resistant to antiviral drugs. cov particles are roughly spherical, have a diameter of 120-160 nm and consist of a core (also called nucleocapsid) surrounded by a protective coat or envelope ( fig. 1 ; [10] ). the core contains the viral genome complexed with the nucleocapsid (n) protein. cov genomes consist of a single rna molecule of positive polarity with a length of~26.4 to~31.7 kilobases (kb). the envelope is composed of a lipid bilayer (like the one found at the surface of cells) in which sev-eral transmembrane proteins are inserted. for sars-cov-2 the major envelope proteins are the spike (s) protein, the membrane (m) protein and the envelope (e) protein. the s protein is involved in the binding of the virus to target cells and has a crucial role in the penetration of these cells by mediating membrane fusion. after binding of sars-cov-2 particles to its cellular receptor angiotensin i converting enzyme 2 (ace2) and cleavage of the s protein by transmembrane serine protease 2 (tmprss2), the viral envelope fuses with the plasma membrane of the target cell, resulting in the delivery of the viral genome inside the cell ( fig. 2 ; [11, 12] ). alternatively, upon binding of their s protein to ace2, sars-cov-2 particles are taken up by the target cell in small vesicles called endosomes. next, the s protein is cleaved by the endosomal protease cathepsin l, which initiates the fusion of the viral envelope with the lipid bilayer of the endosome, causing the release of the viral genome into the cytoplasm of the cell. a recent paper suggested that sars may utilise cd147 (also known as basigin or emmprin) as an alternative attachment receptor to enter target cells [13] . after the cov genome, which is actually a very long mrna [(+)grna], is set free inside the cell, it is immediately translated to produce two large polyproteins designated pp1a and pp1ab (fig. 1) . synthesis of pp1ab requires the translation machinery to switch reading frame before encountering the stop codon of open reading frame (orf) 1a by a process called ribosomal frameshifting. autoproteolytic cleavage of pp1a and pp1ab produces 16 different nonstructural proteins (nsps), which together form the viral replicase complex [14] . this complex makes a complementary copy of the viral rna genome called the antigenome [(-)grna], which serves as a template for the synthesis of new cov genomes (replication) (fig. 2) . these (+)grna molecules, in turn, form the templates for the generation of a nested set of so-called subgenomic (sg) rnas of negative polarity these negative-strand sgrnas are subsequently transcribed to produce a nested set of positive-strand sgr-nas (figs. 1 and 2). translation of these (+)sgrnas yields the major structural proteins (i.e. the s, m, e and n protein in the case of sars-cov-2) and a number of accessory proteins [15, 16] . the accessory proteins of covs are involved in the modulation of different cellular processes for the benefit of the virus. most accessory proteins perform multiple functions and several of them are incorporated into virus particles as minor structural proteins [17] . the n protein binds the newly synthesised positive-strand rna genomes. the resulting ribonucleoprotein complexes (i.e. nucleocapsids) associate with membranes of the endoplasmic reticulum (er)-golgi intermediate compartment (ergic) in which the s, m and e proteins are inserted. subsequently, virus particles are formed by budding of the capsids through the ergic membranes (fig. 2) . these virus particles are then transported in vesicles (i.e. exosomes) to the surface of the cell, where they fuse with the plasma membrane, leading to the release of the newly assembled virus particles in the extracellular space (fig. 2) . from there they can spread to new target cells to initiate additional rounds of virus production. since the first report on sars-cov-2 on 31 december 2019 by the wuhan municipal health commission, the virus has spread rapidly across the globe, creating a pandemic with a colossal socioeconomic impact affecting all continents except antarctica. as of 7 july 2020, john hopkins university had registered 11,626,759 confirmed cases of covid-19 and 538,190 covid-19-related deaths, which would correspond to a global case-fatality rate of~6%. due to the limited testing capacity in many countries, especially at the beginning of the pandemic, and the existence of many asymptomatic and paucisymptomatic covid-19 patients, the true incidence of sars-cov-2 is presumably much higher. as a consequence, the true fatality rate will be lower too. yet, excess mortality data indicate that the number of covid-19-related deaths is also considerably higher than the reported death count (https://voxeu.org/ article/excess-mortality-england-european-outliercovid-19-pandemic), which may be caused by misschematic drawing of sars-cov-2 life/ infectious cycle. see running text for explanation. ace2 angiotensin i converting enzyme 2, tmprss2 transmembrane serine protease 2 diagnosis and/or underreporting. for the well-characterised covid-19 outbreak in february 2020 on the cruise ship diamond princess, case-fatality and infection-fatality rates for sars-cov-2 of 2.6% and 1.3%, respectively, were calculated [18] . however, these figures may also not be fully representative of the global situation because of the relatively old age of the passengers, on the one hand, and the relatively high-quality care provided to the patients, on the other. indeed, demographic differences as well as differences in health status, health care, covid-19 treatment and cause of death assessment (i.e. did a person die with or die from covid-19?), may explain the differences in reported case-fatality rates between different countries. by comparison, the global casefatality rate associated with seasonal influenza epidemics is~0.1%. the risk of covid-19 hospital death is positively correlated with age, body-mass index and socioeconomic deprivation, e.g. in people ≥80 years of age an adjusted hazard ratio (hr) of 12.64 has been reported [19] . males are~2-fold more likely to die from covid-19 than females and mortality in caucasians is lower than in the other races [19, 20] . recently, blood group has also been identified as a risk factor for acquiring covid-19 with respiratory failure, i.e. blood group o and a are associated with, respectively, a lower and higher risk of acquiring severe covid-19 than the other blood groups [21] . most comorbidities are associated with a higher risk of covid-19 hospital death, including cardiovascular disease, diabetes, (haematological) cancer, hypertension and respiratory disease [19, 20] although after adjustment for multiple variables the association with high blood pressure was lost and with chronic heart disease was rather weak (hr 1.27) [19] . this illustrates the fact that care should be taken in interpreting the results of univariate analyses. in the absence of a sars-cov-2 vaccine and protective immunity resulting from infections with endemic human covs, current efforts to bring the pandemic to a halt are focusing on the reduction of the basic reproduction number (r0), which is defined as the expected number of secondary cases produced by a typical infected individual during the entire infectious period in a completely susceptible (i.e. non-immune) population and without any deliberate actions to reduce disease transmission. based on early transmission dynamics in wuhan, li et al. estimated the r0 for sars-cov-2 to be 2.2 (95% confidence interval (ci) 1.4-3.9) [22] . since covid-19 is primarily a respiratory infection, respiratory droplets produced (in decreasing numbers) by sneezing, coughing, singing, talking and breathing are the main sources of virus transmission. a treacherous aspect of sars-cov-2 infections is the presymptomatic transmission of the virus, which may have played an important role in the (rapid) spreading of the virus around the globe. by studying 77 transmission pairs and based on a mean incubation time of 5.2 (95% ci 4.1-7.0) days, he et al. inferred that infectiousness started from 2.3 (95% ci 0.8-3.0) days before symptom onset and peaked at 0.7 (95% ci -0.2-2.0) days before onset of illness to gradually decline afterwards [23] . this resulted in an estimated presymptomatic transmission rate of 44%, implying that containment measures based on isolation of virus shedders will only be effective if contact tracing includes the 2-3 days before symptom onset in the index case. in several studies, sars-cov-2 rna has been demonstrated by reverse transcription-quantitative polymerase chain reaction (rt-qpcr) analysis (see below) in stool and sewage, and occasionally infectious virus has been recovered from faecal samples [24, 25] . moreover, sars-cov-2 has been shown to replicate in enterocytes of human small intestinal organoids [26] , and the viral n has been detected by immunofluorescence microscopy in the cytoplasm of gastric, duodenal and rectal epithelial cells but not in oesophageal epithelial cells of endoscopic biopsies [27] . this raises the possibility of faecal transmission of sars-cov-2, although formal evidence for this has not yet been obtained. recently, different species of companion and farm animals including cats, dogs, ferrets, hamsters and minks have been shown to be permissive to sars-cov-2 infection (see, for example, [28] ) and evidence has been obtained suggesting transmission of the virus from humans to these domesticated animals and vice versa (https://www.rivm.nl/en/novelcoronavirus-covid-19/pets). for additional information on sars-cov-2 transmission, see appendix 1 of the electronic supplementary material. the outcome of sars-cov-2 infections is highly variable, which may relate to several factors including (1) infectious dose, (2) general health of the infected individual and (3) appropriateness of his/her immune response. a large percentage (up to 81%) of people are unaware of being infected with sars-cov-2 [29] . analysis of 44,415 confirmed covid-19 cases from china as of 11 february 2020 indicated that 81% had mild disease, 14% were severely ill and 5% developed critical illness, resulting in a case-fatality rate of 2.3% [30] . in mildly affected individuals the infection remains largely restricted to the upper respiratory tract, while in seriously ill patients it subsequently spreads to the lower respiratory tract and, from there, possibly to other organs. the most common (flu-like) symptoms of covid-19 are chills/pyrexia, dry cough, dyspnoea and fatigue or myalgia. other, less common, symptoms include expectoration, nasal congestion/ rhinorrhoea, sore throat, headache, conjunctivitis, abdominal pain, diarrhoea, nausea or vomiting, ageusia, anosmia, skin rash and discoloration of fingers or toes. these symptoms are usually mild and develop gradually (reviewed in [4, 31, 32] ). more seriously affected individuals suffer from pneumonia, accompanied by progressive dyspnoea, chest pain, haemoptysis, crackles and/or respiratory insufficiency. in the most severe cases of covid-19, the pneumonia develops into acute respiratory distress syndrome (ards), which may lead directly to respiratory failure and is a major cause of covid-19-related death. the development of ards is thought to largely result from the hypercytokinaemia (cytokine storm) induced by the virus and, together with secondary microbial infections (e.g. due to loss of intestinal epithelial barrier integrity), can culminate in sepsis and end-organ dysfunction [33, 34] . interestingly, manifestations of covid-19 in children are generally less severe than in adults. the precise reason(s) for this is/are unclear. due to the higher incidence of respiratory infections, the innate immune system of children may be 'trained' and therefore better capable of mounting an efficient first line of defence against the virus, preventing the induction of a cytokine storm due to vigorous replication of sars-cov-2 in the lower airways. alternatively, in children replication of sars-cov-2 could be suppressed by the presence of other respiratory viruses. also, airway epithelial cells of children may be less permissive to sars-cov-2, e.g. because of lower ace2 expression levels [35] . the average incubation period of sars-cov-2 is 5 days with a 95% ci ranging from 2 to 14 days [22, 36] . in a study of 41 hospitalised covid-19 patients, 22 developed dyspnoea with a median time from illness onset to dyspnoea of 8.0 days (interquartile range (iqr) 5.0-13.0 days). in the same study, medium times from onset of symptoms to hospital admission, ards, mechanical ventilation and admission to the intensive care unit (icu) were 7.0 ( chest radiography of 64 chinese covid-19 patients with rt-qpcr confirmation, showed abnormal x-ray images in 69% of the patients. in 6 of these patients, radiographic abnormalities were observed before testing positive for sars-cov-2 rna. consolidation and ground-glass opacities were the dominant findings and mostly involved the periphery of the lower halves of both lungs [39] . computed tomography (ct) scans of 81 confirmed covid-19 patients before and ≤1 week, >1-2 weeks and >2-3 weeks after symptom onset revealed abnormalities in 2.8, 11.1, 13.0 and 12.1 of the 20 lung segments, respectively. ct image abnormalities rapidly evolved from predominantly focal unilateral to diffuse bilateral ground-glass opacities that progressed to or co-existed with consolidations within 1-3 weeks [40] . for more information on this topic, see [41] . while the respiratory tract is the primary target of sars-cov-2, the clinical symptoms mentioned above show that other organs/organ systems, including the cardiovascular system, the digestive tract, the liver, the kidney, the brain and the eyes, can also be affected by covid-19 [42] [43] [44] [45] . although sars-cov-2 particles/components have been detected in, for example, endothelial cells, the digestive tract and the liver, not all extrarespiratory manifestations of covid-19 are necessarily caused by direct viral injury but may also be the consequence of the hypoxaemia, (hyper)inflammatory response, neuroendocrine imbalance and other pathophysiological changes induced by the airway infection [43] . for information on the clinical management of (severe) covid-19 see, for example, nicola et al. [46] and xie et al. [47] . primarily intended for low-and middle-income countries, the clinical care for severe acute respiratory infection toolkit of the world health organization is another excellent resource for managing adult and paediatric patients with severe forms of covid-19 (https://www. who.int/publications/i/item/clinical-care-of-severeacute-respiratory-infections-tool-kit). a brief introduction to antiviral immune responses in general is provided in appendix 1 of the electronic supplementary material. in the large majority of persons, infection with sars-cov-2 is rapidly and efficiently controlled by the immune system and remains (largely) confined to the upper respiratory tract. as a consequence, these people develop no or only mild disease. however, in a small percentage of infected individuals the lower respiratory tract also becomes infected, accompanied by hyperinflammation and overactivation of the immune system, leading to excessive production of cytokines and accumulation of immune cells in the lungs. due to the severe injury caused by the virus and by the immune system to the airway epithelial cells and the underlying endothelial cells, the alveolar-capillary barrier is broken, resulting in vascular leakage, alveolar oedema/collapse and ards. this may be followed by further clinical deterioration and ultimately cause death as a result of multi-organ damage/failure due to secondary sars-cov-2 and bacterial infections and immune-mediated mechanisms. although detailed knowledge about the interactions between the innate immune system and sars-cov-2 is still scarce, innate immune responses are thought to play an important role in limiting the viral infection (to the upper respiratory tract). based on previous research on sars-cov and mers-cov and various animal models of virus-induced acute lung injury as well as haematological and biochemical laboratory findings in covid-19 patients, the following scenarios can be envisioned [33, 34, 48, 49] . infection of upper airway epithelial cells, recognition/uptake of sars-cov-2 particles by dendritic cells and resident macrophages and activation of the complement system in the upper airways trigger the local production and release of pro-inflammatory cytokines and chemokines. these proteins induce chemotaxis of neutrophils, monocytes/macrophages, natural killer (nk) cells and lymphocytes to the site(s) of infection and participate in the activation of these immune cells. in the case of an adequate immune response, the virus infection is subsequently cleared by the concerted action of innate immune cells, the complement system, t and b lymphocytes and sars-cov-2-specific antibodies. however, when the immune response overshoots, a viscous cycle is induced of further pulmonary accumulation of immune cells, production of more pro-inflammatory factors and aggravated immune-mediated lung injury. in patients with severe covid-19, but less so in patients with mild disease, lymphopenia is commonly observed, with drastically reduced numbers of b cells, helper (i.e. cd4 + ) t cells (ths), cytotoxic (i.e. cd8 + ) t cells (ctls) and nk cells. these patients also have an increased percentage of neutrophils and a decreased percentage of monocytes, eosinophils and basophils ( [50] and references therein). in general, neutrophil count and neutrophil-to-lymphocyte ratio positively correlate with disease severity and a worse clinical outcome [51] . in addition, nk cells and cd8 + t cells show signs of functional exhaustion, the extent of which is directly proportional to disease severity. during recovery from covid-19, the numbers of b cells, ths, ctls and nk cells as well as exhaustion marker expression in cytotoxic lymphocytes normalise ( [50] and references therein). the specific b-cell responses (i.e. antibody production) to sars-cov-2 have been studied in detail by long et al. [52] in 285 patients. at 2-4 days after onset of symptoms, sars-cov-2-specific immunoglobulin (ig) g and/or igm antibodies were detected in 6% of the serum samples. after 17-19 days all patients had sars-cov-2-specific iggs, while virus-specific igms reached a peak of 94.1% at 20-22 days after symptom onset. during the first 3 weeks after onset of illness, sars-cov-2-specific igg and igm levels gradually increased. severely diseased patients had significantly higher virus-specific igg titres than mildly affected individuals at 2 weeks after symptom onset. the median day of seroconversion for both igg and igm was 13 days after onset of illness. padoan and colleagues studied the kinetics of sars-cov-2-specific antibodies for 6 weeks after the onset of fever in 19 adult patients with rt-qpcr-confirmed covid-19 [53] . average sars-cov-2-specific iga (i.e. mucosal antibody) and igm levels above background started to be detected at 6 and 8 days after the onset of covid-19, respectively. the average level of sars-cov-2specific antibodies was higher for iga than for igm for the whole observation period. the average igg titre peaked at day 20-22 and remained fairly constant for 1 month, while the average igm level peaked at 10-12 days and gradually dropped thereafter. relatively little is known about the cellular immune response to sars-cov-2. using a large pool of peptides representing predicted t-cell epitopes, sars-cov-2-specific cd8 + and cd4 + t lymphocytes were identified in~70 and 100% of covid-19 convalescent patients, respectively [54] . the majority of the ths and ctls were directed against the highly expressed s, m and n proteins of sars-cov-2. ths to the s protein were robust and showed a positive correlation with sars-cov-2 igg and iga titres. interestingly, sars-cov-2-reactive cd4 + t lymphocytes were detected in~50% of unexposed individuals, indicative of possible cross-reactive t-cell recognition between endemic covs and sars-cov-2. in another study of 14 covid-19 convalescent patients, variable numbers of nsp5-, s-and n-specific t cells were detected and a direct correlation between the neutralising antibody titre and the strength of the n-specific t-cell response was found [55] . diagnostic assays for covid-19 fall into two categories: (1) tests to detect viral components, i.e. sars-cov-2 rna or protein; (2) assays to measure adaptive immunity (i.e. antibodies and t lymphocytes) against sars-cov-2 infection [56, 57] . although not specific, radiography to visualise lung disease, biomarker analysis to assess tissue/organ damage and inflammation, electron microscopy to visualise virus particles and virus infectivity assays to quantify functional virus particles can also aid in the diagnosis of covid-19. rt-qpcr-based laboratory assays on upper or lower respiratory tract samples are presently the standard tests to detect ongoing sars-cov-2 infections. rnavirus detection by rt-qpcr is a multistep procedure involving the extraction of rna from the clinical specimen, the preparation of dna copies of the extracted rna by an enzyme called reverse transcriptase, the logarithmic amplification of viral sequences in a socalled thermocycler using a thermostable dna polymerase and virus-specific dna primers and the realtime detection of the amplification products using fluorescence-based methods (see, for example, https:// www.youtube.com/watch?v=thg_02miq-4 for an illustrative movie). the preferred specimens for the detection of sars-cov-2 rna by rt-qpcr are nasopharyngeal samples collected with a flocked swap and preserved in an appropriate transport medium. besides in airway samples, sars-cov-2 rna is also frequently detected in faeces (reflecting infection of the gastrointestinal tract), occasionally in blood samples (indicative of viraemia) and rarely in urine. an alternative method to confirm active infections is by a sandwich enzyme-linked immunosorbent assay (elisa; fig. 3a ). this assay detects viral protein (i.e. antigen) in clinical specimens after lysis of the cells and virus particles in the sample. the resulting solution is applied to a well coated with a so-called capture antibody specific for one of the virion proteins. next, an antibody recognising another epitope of the same virion protein (the so-called detection antibody) is applied to the well, followed by an enzymeconjugated third antibody directed against the detection antibody. a colourless substrate is then added to the well, which can be converted by the enzyme into a coloured product that is detected with a colorimeter. the amount of coloured product that is generated correlates with the amount of virus present in the clinical specimen. since a sandwich elisa is a laborious procedure requiring multiple washing steps after each addition, which are usually performed automatically, this method is unsuitable for point-ofcare testing. this has led to the development of immunochromatography strips for fast instrument-free testing employing capillary movement (fig. 3b) . these strips contain a sample pad for specimen application followed by a conjugate pad containing an antigenspecific antibody linked to, for example, colloidal gold or latex particles (first antibody). if the sample contains antigens, complexes are formed with the first antibody, which migrate to the so-called test line, where a fraction of these complexes are bound by another antigen-specific antibody covalently attached to the strip (second antibody), resulting in the formation of a coloured line. the remaining complexes and noncomplexed first antibodies migrate further until they reach the so-called control line, which contains covalently attached antibodies recognising the first antibody (third antibody). this results in the formation of a second coloured line, irrespective of the presence of antigen in the clinical specimen. in general, antigen detection assays are less sensitive than genome detection methods, resulting in a higher rate of false-negative results. moreover, whereas rt-qpcr tests and sandwich elisas can provide a reasonable estimate of virus load, immunochromatography tests do not allow accurate quantitative analyses. in early infections, the amount of sars-cov-2 in specimens from the upper part of the respiratory tract may be too low to be detected and repeated sampling may be necessary. virological tests are not only instrumental in determining whether somebody is infected with sars-cov-2 but also can be used for monitoring disease progression (by taking samples at different sites), to identify virus shedders and for taking de-isolation decisions. different studies report somewhat different times after the onset of symptoms at which the first antibodies against sars-cov-2 proteins are detected. moreover, the extent and kinetics of the humoral immune response to sars-cov-2 varies between individuals. this may relate to differences in infectious dose but also be the result of inherited and acquired differences in their immune system. in most studies, igm, igg and iga antibodies directed against the sars-cov-2 s and n proteins are first detected at roughly the same time, i.e. a few days after the onset of symptoms. igm and igg levels peak at 2-3 weeks after illness onset and drop faster afterwards for igms (see also above). serological tests alone can thus not be used to establish with certainty that a person is infected with sars-cov-2. the formats of antibody detection tests are similar to those of the antigen detection assays except that viral proteins (most s or n, but sometimes also whole virus lysates) serve as bait. serological tests are particularly useful in surveillance and containment programmes and for epidemic forecasting. they can also be used for assessment of immunity resulting from natural infection or active immunisation. however, it is not yet clear to what extent ig levels correlate with protective immunity. in this respect, virus neutralisation assays (e.g. plaque reduction and tissue culture infectious dose assays) may be more informative. in these cell culture assays, virus is mixed with (different dilutions) of patient's (or control) serum and added to susceptible cells to determine whether the serum contains antibodies that can inhibit virus infection. a comprehensive overview of (1) diagnostic tests, (2) performance data of commercially available diagnostic assays, including information on their sensitivity and specificity, and (3) use case descriptions of diagnostic tests for covid-19 can be found at https:// www.finddx.org/covid-19/. see also appendix 1 of the electronic supplementary material. in theory, sars-cov-2 could be completely eradicated by strict isolation of all infectious sources similar to the extinction of a predator in the absence of prey. however, in practice such an approach seems unfeasible given (1) the extent of the virus's spread and the huge socioeconomic impact of global containment measures and (2) the required intense and persevering surveillance using virological tests that can identify all virus shedders, including individuals with an asymptomatic sars-cov-2 infection. this has created a huge incentive for the development of a safe and effective sars-cov-2 vaccine (see https://docs.google.com/spreadsheets/d/ 16dbphf9od0mhhtcr12of6yucfirzp_-xgkynebnip ds/edit#gid=1804775590 for an overview) to block infection or, at least, mitigate the consequences of infection. although there are currently no approved vaccines for human covs, the development of a vaccine for covid-19 may benefit from (1) past and ongoing vaccine studies of sars-cov and mers-cov as well as (2) the successes and failures of animal cov vaccines. this will, however, not obviate the need for extensive testing of candidate vaccines in appropriate animal models and clinical studies, especially since correlates of protection (e.g. neutralising fig. 4 overview of the different approaches to developing a vaccine for sars-cov-2 antibody titres) may be different for different covs. the repertoire of sars-cov-2 vaccines that is currently being developed comprises both replicating and non-replicating vaccines ( fig. 4 ; [58] [59] [60] ). the non-replicating vaccines include (1) wild-type sars-cov-2 particles inactivated by chemical treatment or high-energy radiation, (2) non-replicating viral vectors directing the expression of one or more sars-cov-2 antigens, (3) virus-like particles (i.e. assemblies of one or more of the viral envelope proteins often with a lipid envelope), (4) mrna or plasmid dna encoding one or more viral proteins (in particular the s protein as the principal target of virus-neutralising antibodies), (5) subunit vaccines consisting of (parts of) the s protein in monomeric or trimeric form and (6) vaccines based on synthetic peptides representing viral antigenic determinants/epitopes. as these 'dead' vaccines are generally less immunogenic than 'live' vaccines, they are often administered together with an adjuvant to boost the immune response and to stimulate long-term immunological memory. the inclusion of an adjuvant may not be needed for the non-replicating viral vector-and nucleic-acid-based vaccines, as these vaccines will mediate de novo synthesis of sars-cov-2 antigens inside target cells and therefore may induce a stronger immune response. one obvious advantage of especially inactivated virusand nucleic-acid-based vaccines is that they can be produced in large amounts in a relatively short time. the replicating vaccines comprise (1) attenuated ver-sions of sars-cov-2 generated by serial passage in cultured cells or by genetic engineering and (2) replicating viral vectors encoding one or more sars-cov-2 antigens. although 'live' vaccines usually confer more durable and robust protection against disease than 'dead vaccines', their development time is generally much longer. moreover, attenuated virus vaccines require extensive safety assessment to exclude the early appearance in vaccinees of variants/mutants that have regained their (full) disease-causing potential. more information about the pros and cons of the different types of vaccines and their effects in pre(clinical) studies can be found in various recent reviews [58] [59] [60] . a covid-19 vaccine does not necessarily have to contain/express sars-cov-2 proteins. since innate immune responses represent the first line of defence against invading pathogens, boosting these responses by applying live vaccines directed against, for example, tuberculosis, poliovirus or measles virus may reduce the susceptibility to sars-cov-2 infections and the severity of covid-19 [48] . despite the urgent need for a sars-cov-2 vaccine, the safety aspects should not be neglected. one potential danger is that a sars-cov-2 vaccine induces (poorly neutralising) antibodies, which facilitate subsequent virus uptake by myeloid cells via antibody or complement receptors present at their surface. this antibody-dependent enhancement of infection has previously been observed for feline infectious peritonitis virus (fipv) in cats vaccinated with a recombinant poxvirus expressing the fipv s protein [61] [62] [63] . another risk is the induction, by vaccination, of a skewed type 2 th (th2) response, resulting in th2-driven immunopathology accompanied by pulmonary eosinophilia, as observed with certain experimental sars-cov and mers-cov vaccines [62, 63] . moreover, since studies on endemic human covs have suggested immunity against these viruses to be short-lived [64] , it will be important to establish the duration of protection from disease following vaccination against covid-19. finally, one has to consider the possibility that a vaccine based on current sars-cov-2 variants will provide no or only partial protection against future strains of the virus or other novel human covs. nonetheless, active immunisation using an effective vaccine in combination with a rigorous surveillance and containment programme can lead to the complete eradication of a viral disease, as demonstrated for smallpox. which type(s) of vaccine will eventually be used to combat covid-19 will be determined by a combination of factors including safety, efficacy and time-to-market introduction. rather than trying to prevent the development of sars-cov-2-related sickness by arming the immune system through vaccination, another strategy consists of thwarting the virus once an infection has revealed itself by the administration of antiviral drugs to the patient. due to (1) the lack of approved drugs directed against human covs and (2) the long times involved in the development and evaluation of new antiviral agents, the main focus is currently on repositioning existing drugs designed for other (viral) diseases to treat covid-19 patients [65] . in addition, unapproved drugs showing antiviral activity in animal models of sars-cov and mers-cov are being tested for the ability to inhibit sars-cov-2 replication. mechanistically, anti-sars-cov-2 drugs exert their beneficial effect by inhibiting crucial steps in the viral life cycle like receptor binding, envelope fusion, polyprotein processing and rna-dependent rna synthesis ( fig. 5 ; [60, 63] ). recombinant soluble human ace2 (sace2) is being evaluated for its ability to block the interaction between sars-cov-2 and target cells. sace2 could also compensate for the sars-cov-2-related reduction of surface ace2 levels and consequential disturbance of angiotensin ii/angiotensin-(1-7) balance [66] and thereby help to preserve pulmonary vascular integrity. s-protein-specific neutralising monoclonal antibodies can either interfere with the binding of the virus to target cells or prevent fusion of the viral envelope with the plasma membrane or the membrane of endosomes. merging of viral and cellular membranes and subsequent release of the viral genome into the host cell cytoplasm may also be inhibited using peptidomimetic fusion inhibitors or the influenza virus entry inhibitor arbidol (umifenovir). several studies have also suggested a possible beneficial effect of the antimalarial drugs chloroquine and hydroxychloroquine on covid-19 progression, which may at least in part be related to their ability to inhibit endosomal acidification and thereby membrane fusion of internalised virus particles [67, 68] . however, (hydroxy)chloroquine is known to cause qtc prolongation, which may result in a torsades de pointes type of ventricular tachycardia. apart from using monoclonal antibodies for passive immunisation purposes (blood-group-matched) convalescent plasma of recovered covid-19 patients could also be employed to protect newly infected individuals from developing severe disease [69] . sars-cov-2 replication can also be inhibited by treatment with nucleoside analogues (usually administered as prodrug) that interfere with the rna-dependent rna synthesis needed to amplify the viral genome and to produce the subgenomic mrnas. nucleoside analogues under current investigation for the treatment of covid-19 include ribavirin, favipiravir (t-705) and remdesivir (gs-5734). other pharmacological targets could be nsp3 and nsp5, the papain-and chymotrypsin-like proteases encoded by orf 1a and involved in the processing of the sars-cov-2 polyproteins that produce the viral replication and transcription complexes (figs. 1 and 2) . accordingly, clinical trials have been initiated with the human immunodeficiency (hiv) protease inhibitor combination lopinavir/ritonavir. the in vivo effectiveness of these drugs against sars-cov-2 remains to be seen as the hiv protease belongs to the family of aspartyl proteases and nsp3 and nsp5 are cysteine proteases. a highly specific way to inhibit sars-cov-2 would be by rna interference. this method is based on the delivery to virus-infected/target cells of small (modified) rna molecules (so-called small interfering rnas or sirnas) that are binding/complementary to the viral genome and promote its degradation. by simultaneous treatment of covid-19 with multiple sirnas targeting different parts of the sars-cov-2 genome, it will be very hard for the virus to escape from this antiviral mechanism by changing its genetic code through mutation. an alternative to the inhibition of viral proteins/ processes is the suppression of host cell factors/ processes required by the virus. obvious targets are the sars-cov-2 attachment receptor ace2 and the host proteases involved in the processing of the s protein to render it fusion competent. however, care should be taken that antagonists of these host proteins do not interfere with vital physiological processes. early activation of cellular defence systems could also be an effective means to suppress sars-cov-2 replication. one possibility would be to treat patients with interferons, as these cytokines play an important role in the (1) development of early innate immune responses to viral infections, (2) activation of subsequent adaptive immune responses and (3) dampening of immunopathogenic mechanisms [70] . support for this approach is provided by the finding that sars-cov and mers-cov counteract interferon signalling by multiple mechanisms and that replication of these viruses can be inhibited by interferons [15, 63, 71] . there may, however, also be a downside to treatment of covid-19 with interferons given the recent finding that they can increase ace2 expression in human airway epithelial cells [72] . in general, in patients at risk of developing severe disease following infection with sars-cov-2, antiviral drug therapy should be started as soon as possible to minimise virus-induced damage of airway epithelium and to avoid the subsequent development of hypercytokinaemia. in addition, covid-19 patients receiving antiviral drugs must be closely monitored for possible adverse effects and potential interactions with other drugs should be reckoned with. similar to what has been found for hiv and hepatitis c virus, combining different antiviral drugs may have additive or synergistic effects. antiviral drug cocktails will also lower the risk of the selection of sars-cov-2 variants with mutations that resist antiviral drug therapy. antiviral drugs may also be employed as prophylactics to prevent infection or to reduce virus shedding and thereby the risk of virus spreading. immunomodulators covid-19 severity and covid-19-associated mortality are positively correlated with the serum levels of (1) positive acute phase proteins (e.g. c-reactive protein (crp), ferritin and the fibrin degradation product d-dimer), (2) inflammatory cytokines (e.g. interferon γ (infγ), interleukin (il) 1β, 6 and 7 and tumour necrosis factor α (tnfα)) and chemokines (e.g. c-c motif chemokine 2 (ccl2), also known as monocyte chemoattractant protein 1 (mcp1); and c-x-c motif chemokine 10 (cxcl10), also known as interferon gamma-induced protein 10 (ip-10)) and (3) with the neutrophil-to-lymphocyte ratio [33, 37] . this lends support to the idea that sars-cov-2-induced hyperinflammation is a major contributor to covid-19 pathogenesis. accordingly, multiple clinical trials have been initiated to investigate the effect of immunomodulation on the course of covid-19 [33, 60, 73] . in view of the essential role of both innate and adaptive immune responses in the clearance of (corona)viral infections, timing and dosing of the immunomodulatory therapy will have a large impact on the outcome of these studies. this is nicely illustrated by the fact that both inhibition and stimulation of granulocyte-macrophage colony-stimulating factor are considered as therapy for covid-19 patients [33] . other targets of immunomodulation include the il1β, il6, infγ and tnfα signalling pathways. inhibition of these signalling pathways is in most cases accomplished by therapeutic antibodies directed against the cytokine itself or its receptor. in addition, small molecule inhibitors of c-c chemokine receptor type 2 and 5, the il1 receptor and janus kinase (jak)-signal transducer and activator of transcription protein (stat) signalling are being evaluated in clinical studies. other immunomodulatory therapies that are currently being investigated include the administration of mesenchymal stem cells, nk cells, high doses of intravenous immunoglobulins (ivig) or recombinant human surfactant protein d and the inhibition, by a small molecule drug and monoclonal antibody, of the processing of complement factors c3 and c5, respectively [74] . the use of glucocorticoids to curtail sars-cov-2-induced hyperinflammation is still highly controversial [75] , while there are some indications for a suppressive effect of statins on the likelihood of developing covid-19 symptoms in the elderly [76] . a comprehensive overview of drugs under development for covid-19 can be found at https://docs. google.com/spreadsheets/d/16dbphf9od0mhhtcr 12of6yucfirzp_-xgkynebnipds/edit#gid=1206197573. when using antivirals or immunomodulators in the treatment of covid-19, one should be vigilant for drug-induced organ toxicity, especially of the liver, brain, heart and kidney, and for (ventricular) arrhythmias. multiple recent studies have identified venous and arterial thrombosis secondary to sars-cov-2 infection as a major complication of covid-19, which likely contributes to its relatively high mortality rate [77, 78] . the pathophysiological mechanisms involved in the covid-19-associated coagulopathy are incompletely understood. factors that may contribute to the thrombophilia observed in severely ill covid-19 patients include the following: (1) a disturbed balance between pro-and anticoagulant activities due to excessive production of proinflammatory cytokines, activation of complement, formation of neutrophil extracellular traps and activation of platelets; (2) inflammation-related endothelial activation; (3) death of sars-cov-2-infected endothelial cells; (4) endothelial dysfunction caused by unbalanced angiotensin iiangiotensin ii type-1 receptor signalling; (5) formation of prothrombotic antiphospholipid antibodies; (6) immobility-associated reduction of blood flow; (7) hypoxia due to respiratory impairment resulting from sars-cov-2-induced lung injury [79] [80] [81] . based on the high incidence of venous thromboembolism, myocardial infarction and stroke among covid-19 patients and the mortality-reducing effect of empirical therapeutic anticoagulation with heparin in covid-19 patients with a sepsis-induced coagulopathy score ≥4 [82] , clinical trials have been initiated to investigate the impact of (different doses) of antithrombotics on disease outcome [79] . in addition, recommendations have been formulated for (tailored) prophylactic or therapeutic anticoagulation therapy in specific patient groups (see, for example, [80, 83] ). in covid-19 patients receiving antiviral therapy possible interactions with anticoagulants and antiplatelet drugs could occur, which preclude the use of certain drug combinations or require dose adjustments. if use of pharmacological antithrombotics is contraindicated, mechanical therapy could be considered. as heparin not only inhibits blood clotting but also possesses anti-inflammatory and anti-arrhythmic effects and may block sars-cov-2 host-cell interactions, it could benefit covid-19 patients in multiple ways [74] . as already mentioned above, albeit patients with preexisting cardiovascular disease (cvd) may not be more susceptible to contracting sars-cov-2 than healthy individuals, they have a higher chance of developing severe covid-19 and are more likely to die from sars-cov-2 infection. the higher covid-19-related mortality of cvd patients may be directly related to or coincident with their cardiovascular comorbidities. although covid-19 primarily disrupts the respiratory system, the disease frequently affects the cardiovascular system as well, especially in more severe cases. the currently available data suggest that some 20-30% of hospitalised covid-19 patients display cardiac complications [84] , and in a recent study of 671 severely ill covid-19 patients acute myocardial injury and acute heart failure were identified as death-related complications in 30.6% and 19.4% of the 62 deceased patients, respectively [85] . of these patients, 98.4% had ards and 90.3% suffered from acute respiratory failure. a small percentage of hospitalised covid-19 patients develop cardiac disease in the absence of marked pulmonary illness [86] . the cardiovascular complications of covid-19 include acute myocardial injury, heart failure with or without cardiogenic shock, pericardial effusion with or without tamponade, arrhythmias and sudden cardiac death and thrombosis of small and large blood vessels [31, 87] . the most common cardiac complication of covid-19 is acute myocardial injury, as evinced by increased plasma levels of cardiac troponin and echo-/ electrocardiographic abnormalities. in a retrospective study of 416 inpatients, 19.7% displayed cardiac injury defined as high-sensitivity cardiac troponin i (hs-ctni) levels above the 99th percentile of a healthy reference population. the mortality rate among patients with elevated hs-ctni levels was 51.2% versus 4.5% in patients without myocardial injury [88] . moreover, disease severity and risk of death were positively correlated with hs-ctni levels, even after controlling for other comorbidities. interestingly, in another study, plasma troponin t levels demonstrated a high and significantly positive linear correlation with plasma highsensitivity crp levels and n-terminal pro-brain natriuretic peptide levels [89] , suggesting a link between cardiac injury, systemic inflammation and myocardial wall stress. multiple mechanisms may be responsible for the myocardial injury observed in covid-19 patients. it could be the result of atherosclerotic plaque erosion/rupture (i.e. obstructive coronary artery disease) induced by covid-19-related hypoxaemia, systemic hyperinflammation or angiotensin-2/angiotensin-(1-7) misbalance [66] leading to type 1 myocardial infarction. the myocardial injury could also be caused by virus-or immune-mediated myocarditis, stress-induced cardiomyopathy or oxygen supply and demand mismatch resulting in type 2 myocardial infarction (i.e. myocardial infarction without acute coronary syndrome), possible linked to respiratory insufficiency and/or sepsis. sepsis and the sepsis-related cytokine storm together with myocardial injury and ischaemia are likely involved in the occurrence of cardiac arrhythmias in covid-19 patients. the cytokine storm is also thought to be largely responsible for the hypercoagulable state and consequential disseminated intravascular coagulation / thrombotic microangiopathy and venous thrombosis including pulmonary embolism often seen in (severely ill/deceased) covid-19 patients. other factors contributing to the coagulopathy could be endothelial damage due to (1) the hyperinflammatory response typical of severe covid-19 cases, (2) disturbance of the angiotensin-2/angiotensin-(1-7) balance and (3) sars-cov-2 infection of endothelial cells [81] . more extensive discussions about the cardiovascular aspects of covid-19 are provided in some recent reviews [87, 90, 91] . for information on how to manage the various cardiovascular complications of the covid-19 pandemic, see [92, 93] and the guidance provided by the european society of cardiology https://www.escardio.org/static_file/ escardio/education-general/topic%20pages/covid-19/esc%20guidance%20document/esc-guidance-covid-19-pandemic.pdf. after sars-cov and mers-cov, sars-cov-2 is the third zootic betacoronavirus of the 21st century that imposes a serious threat to human health. the outcome of a sars-cov-2 infection is highly variable and dependent on multiple factors, including sars-cov-2 genotype, infectious dose, genetic background, health status, immunological readiness and age. while covid-19 is primarily a respiratory disease and mostly has a mild course, in severe cases other organs are also affected, including the heart and vasculature. to what extent the cardiovascular manifestations of covid-19 are due to direct viral injury of cardiovascular cells or are an indirect consequence of the extensive lung pathology and accompanying (hyper)inflammation remains to be determined. the covid-19 pandemic has once again alerted us to the high adaptive potential of rna viruses and the downsides of globalisation. at the same time, international collaboration between medical professionals, scientists and politicians will be instrumental in tackling the current and foreseeable future (corona)virus outbreaks. indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds 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cardiovascular syndrome association of cardiac injury with mortalityinhospitalizedpatientswithcovid-19inwuhan, china cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) cardiovascular considerations for patients, health care workers, and health systems during the covid-19 pandemic covid-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options sars-cov-2 infection and cardiovascular disease: covid-19 heart management of cardiovascular disease during coronavirus disease (covid-19) pandemic acknowledgements i would like to acknowledge all covidkey: cord-335137-5qt286kc authors: chatterjee, swapan k.; saha, snigdha; munoz, maria nilda m. title: molecular pathogenesis, immunopathogenesis and novel therapeutic strategy against covid-19 date: 2020-08-11 journal: front mol biosci doi: 10.3389/fmolb.2020.00196 sha: doc_id: 335137 cord_uid: 5qt286kc the coronavirus disease 2019 (covid-19), is a highly contagious transmittable disease caused by a recently discovered coronavirus, pathogenic sars-cov-2. followed by the emergence of highly pathogenic coronaviruses in 2003 sars-cov, in 2012 mers-cov, now in 2019 pathogenic sars-cov-2, is associated with a global “pandemic” situation. in humans, the effects of these viruses are correlated with viral pneumonia, severe respiratory tract infections. it is believed that interaction between angiotensin converting enzyme 2 (ace2) cell receptor and viral spike protein mediates the coronavirus entry into human respiratory epithelial cells and establishes the host tropism. ace2 receptor is highly expressed in airway epithelial cells. along with viral-receptor interaction, proteolytic cleavability of s protein has been considered as the determinant of disease severity. several studies highlight the occurrence of impaired host immune response and expression of excessive inflammatory response especially cytokines against viral infection. the mechanisms of sars-cov-2 induced acute lung injury are still undefined; however, the term cytokine storm has now been recognized to be closely associated with covid-19. the levels of inflammatory mediators from cytokine storm cause damage to the host cells. in particular, the proinflammatory cytokine il-6 appears to be the key mediator in early phase of virus-receptor interaction; however, secreted il-6 might not be representative of lung inflammation. understanding the cellular, and molecular factors involved in immune dysregulation and the high virulence capacity of covid-19 will help in potential targeted therapy against it. “drug repurposing” and “molecular docking analysis” is considered as an attractive alternative approach in analyzing suitable drug candidates to combat sars-cov-2 infection. globally, extensive research is in progress to discover a new vaccine for novel covid-19. moreover, our review mainly focuses on the most state-of-the-art therapeutic approach mediated by “mannose-binding lectin (mbl).” one of the most significant molecules of innate immunity is mbl. it plays a major role in the activation of the complement system as an ante-antibody prior to the response of any particular antibody. recombinant human mbl can be used as immunomodulators against sars-cov-2. various viral members belong to the coronaviridae family (covs) under the order nidovirales are continually circulating among the human population and responsible for causing mild to acute respiratory disease (corman et al., 2020; hoffmann et al., 2020) . covs member coronaviruses belong to the subfamily coronavirinae and can be classified into alpha-, beta-, gamma-, and delta-covs. covs family members contain a positive-sense, single-strand rna genome, 26 to 32 kilobases in length. the extensive distribution and infectivity capacity of covs establish them as an important pathogen. besides numerous avian hosts, various member of covs have been recognized in a range of mammals, like bats, mice, masked palm civets, dogs, camels, and cats (guan et al., 2003) and responsible for disease related to hepatic, respiratory, gastrointestinal systems, and nervous system in humans . a new virus called the 2019 novel coronavirus (an enveloped beta-coronavirus) is identified in december 2019 and associated with a de novo contagious respiratory disease which primarily transpired in hubei province, china wang c. et al., 2020) . initially, the infection was emerged from the huanan seafood market and is initiated due to animal contact. consequently, the disease spread through human-human contact within china and later rapidly spread causing new public health crises worldwide (chan et al., 2020; zhu et al., 2020) . in early march 2020, this disease was documented as "pandemic" by the world health organization (cucinotta and vanelli, 2020) . virologists are still uncertain about exactly how covid-19 spreads. medical doctors and scientists tend to agree that covid-19 is transmittable through the inhalation of droplets from a person who has viral infection. however, the droplets discharged from cough and sneeze is heavy enough that might not travel more than 1 m (corman et al., 2019) . unlike airborne particles that are much smaller than droplets and can stay in the atmosphere for a longer time. according to who, covid-19 is not airborne; however, several experts appear to disagree. common disinfectants like sodium hypochlorite; hydrogen peroxide etc., can be used to destroy the virus (kampf et al., 2020) . as per current information, subsequent transmission of this infection is possible through stool and/or contamination of the water supply . although in most people the disease severity is mild, but the clinical manifestation of covid-19 mainly spans from an asymptomatic state to pneumonia, acute respiratory distress syndrome, followed by multi organ dysfunction. fever, headache, cough, fatigue, sore throat, myalgia, breathlessness, and conjunctivitis (in some cases) are the common clinical characteristics of this disease singhal, 2020) . several phylogenetic analysis suggest that bat is likely the animal origin of the sars-cov-2 as it is 89% genomically identical with bat sars-like-covzxc21, 50% with mers-cov and about 82% identical with human sars-cov (zhou p. et al., 2020) . genome sequencing data also suggest that pangolins also have approximately 85.5-92.4% conserved sequence to sars-cov-2 (yang et al., 2015) . other studies also suggest that sars-cov-2 could be a recombinant virus of bat coronavirus and snake coronavirus. the truth behind the origin of sars-cov-2 is yet to be discovered (xie and chen, 2020) . the spike (s) glycoprotein, envelope (e) protein, membrane (m) protein, and nucleocapsid (n) protein are four structural proteins of novel sars-cov-2. the most significant surface protein is spike glycoprotein which interferes in establishing the association between the human respiratory epithelial cells to the virus via cell surface membrane receptor angiotensin-converting enzyme 2 (ace2) and finally establishes the host tropism (li et al., 2003) . in this review, we emphasize the pandemic potential as well as molecular and immunological events involved in the pathogenesis of novel sars-cov-2. as the spike protein of sars-cov-2 plays a major role in the disease spreading, we comprehensively summarize the structure of spike protein and their potential role in the mutation of the virus. we also concisely discuss the potential effective treatments as well as new therapeutic approaches to control the transmission of this covid-19 pandemic. sars-cov-2 are single-stranded rna viruses characterized by two groups of proteins, namely -(a) structural proteinssuch as spike (s) proteins marking all coronaviruses and bind to receptors on the host cell; nucleocapsid (n) that protects the genetic information of virus, matrix (m), and envelope (e); (b) replicase complex encoding non-structural proteins, such as proteases (nsp3 and nsp5) and rdrp (nsp12; zhou p. et al., 2020) . this enzyme is an ideal target as it helps the virus to replicate, meaning the virus depends on protease. recent studies have shown that sars-cov-2 share a similar type of genomic organization with other beta coronaviruses. like others, it produces a ∼800 kda polypeptide upon transcription of genome. the proteolytic processing is mediated by papainlike protease (plpro) and 3-chymotrypsin-like protease (3clpro) and produces various non-structural proteins required for replication of viral particles. it is then predicted that these proteolytic enzymes could serve as the probable target site for anti-coronaviruses inhibitors (yang et al., 2015) . recently, sars-cov showed an open reading frame 3a protein; coded by one of the group specific genes, and showed no sequence similarity to other known structural coronavirus proteins. it interacts with the e and s proteins while the m protein is glycosylated in all coronaviruses. thus, the close homology of the m protein glycosylation and its likeness to 3a protein could result to investigate the glycosylation of these two membrane proteins (song et al., 2018) . viral transmembrane spike (s) glycoprotein is a trimeric class i fusion protein promotes the sars-cov-2 entry into the cell and the main target of neutralizing antibodies upon infection. previous studies have shown that the function of spike protein depends upon its cleavage by host proteolysis enzyme into the s1 and s2 subunit. the attachment of the virus was facilitated by the s1 subunit whereas the s2 subunit assists in the fusion of viral and human cell membrane. additionally, both the n-terminal domain (ntd) and the c-terminal domain (ctd) of the s1 subunit are capable in function as a receptor-binding entity (millet et al., 2012) . although the s1 ctd region is utilized by both sars-cov and mers-cov to identify the receptor [receptor binding domain (rbd)], the region responsible for sars-cov-2 s-protein-hace2 interaction remains unknown. complete genomic analysis of each monomer of s-protein has shown that it has 22 predicted n-linked glycosylation sites and 4 predicted o-linked glycosylation sites. studies conducted with cryo-electron microscopy (cryo-em) have shown that 14-16 n-glycans are present on 22 potential sites in s-protein which are responsible for proper protein folding and priming of the protein by host proteases. the glycosylation pattern of the s-protein is one of the major features as it acts as a possible site for mutation and also facilitates the coronavirus to evade both innate as well as adaptive immune responses (song et al., 2018) . a recent study suggests that prediction of sars-cov-2 spike glycoprotein structure, glycan shield pattern and pattern of glycosylation has great inference on understanding the viral camouflage as well as the outline of cell entry, and also facilitate the development of new small-molecule drugs, vaccines, antibodies, and screening of the human host targets (song et al., 2018) . sequence alignment data done by the clustal-w has shown that the s2 domain region (aa570-aa1278) of sars-cov-2 share a 91% identity with sars-cov spike glycoproteins. though in the s1 domain (aa01-aa550), it shows a larger sequence difference (∼55% identity), which is essential for host cell adhesion, target interaction, and tropism of virulence but it has a conserved domain for ternary folding residue. this finding suggests that sars-cov-2 also capable to interact with sars-cov host targets like ace2, cd26, ezrin, and cyclophilins (vankadari and wilce, 2020) . receptor recognition is one of the major steps in viral infection of host cells and viral infectivity and pathogenesis. sars-cov depends upon ace2 (angiotensin-converting enzyme 2) receptor which is highly expressed in human epithelial, endothelial, renal, cardiovascular tissue, and lung parenchyma (tikellis and thomas, 2012) . human ace2 is a type i integral metallocarboxypeptidase act as a key player in the renin-angiotensin system (ras). ace2 exhibits protective function in the cardiovascular system, and other organs and act as a major target site for the treatment of hypertension. ace2 protein is more profusely expressed on the apical surface of polarized epithelial cells as well as well-differentiated cells and certain progenitor cells in the bronchi (li et al., 2003) . expression of ace2 receptor in progenitor cells of respiratory tract cells with hair-like projections called cilia serves for the coronavirus entry site in the human body. welldifferentiated epithelial cells expressing ace2 are readily infected by coronavirus. the viral infection thus correlates with the cell differentiation condition, ace2 receptors expression, and localization of membrane binding. however, to date, there are still unanswered inquiries remain regarding ace2 expression in human epithelial cells and its modulatory role in coronavirus. questions include the type of epithelial cells involved in disease, the polarity of ace2 expression on epithelial cells, and whether the coronavirus infection is ace2 dependent. interestingly, appearances of ace2 receptor density on the progenitor cell surface increased with age and are generally present higher in men than in women (xie et al., 2006) . a study by wu et al. (2020) , using computer modeling has shown the presence of identical 3-d structures in the receptor-binding domain of the spike proteins of both sars-cov-2 as well as sars-cov. biochemical interaction studies and crystal structure analysis by wan et al. have proved that sars-cov-2 receptor-binding domain (rbd) contain residue 394 (glutamine), which has sequence similarity with sars-cov residue 479, and both can be accepted by the human ace2 receptor on the critical lysine 31 (wan et al., 2020) . angiotensin converting enzyme 2 is an entry receptor for sars-cov-2 and shows 76% amino acid sequence homology with the sars-cov-s. structural configuration study shows that, ace2 contains 17 amino acid n-terminal signal sequences and a 22 amino acid hydrophobic transmembrane sequence near the c-terminus. ace2 also contains 43 amino acid cytoplasmic domain, a potential phosphorylation sites, eight cysteine residues, and seven potential af-linked glycosylation sites (jia, 2016) . the viral spike (s) protein of sars-cov-2 binds to cellular receptor ace2 in a similar way to sars-cov-1 but with a 10to 20-fold higher binding affinity. these findings suggest that increased ace2 expression might confer easier transmissibility and also increase the susceptibility of sars-cov-2 into the host cell (bourgonje et al., 2020) . studies using angiotensin-ii receptor blockers (arb) and ace-inhibitors (ace-i) suggest that the upregulation of cellular ace2 expression facilitates the binding of sars-cov-2 and associated with severe disease manifestation. this receptor recognition by viral cell leads to host cell entry of the virus in combination with s-protein priming by the host cell protease tmprss2. downregulation of ace2 expression by sars-cov-2 could decrease the angiotensin-ii clearance and lead to aggravation of tissue damage. identification of interaction site and the downstream signaling cascade of sars-cov-2 and ace2 receptor in the human cells will help to design the antibody-based therapeutic strategy (gue et al., 2020) . a study by zhou p. et al. (2020) on a mouse model of sars-cov infection, demonstrated that overexpression of human ace2 receptor are associated with the severity of the disease. he also suggested that in pulmonary tissue alveolar epithelial type ii (aecii) cells express 83% of ace2 receptor and provide a suitable site for viral invasion. additionally, gene ontology enrichment analysis of aecii has demonstrated that increased expression of ace2 is associated with high levels of various viral process-related genes, including viral life cycle, genome replication, assembly, and regulatory genes for viral processes, etc. (zhou p. et al., 2020) . these findings imply that the ace2expressing aecii is able to assist coronavirus replication in the lung. like pulmonary tissue some extra-pulmonary tissues such as heart, kidney, endothelium, and intestine express the ace2 receptor (li et al., 2020d) . another observation also suggests that a state of insulin resistance and elevated plasma glucose levels are associated with increased expression of ace2 in lung epithelial cells and act as the risk factor for morbidity and mortality in sars-cov-2 infected patients (finucane and davenport, 2020) . according to a study by huang et al. (2020) suggest that in addition to lung epithelial tissue sars-cov-2 might affect other tissues including male tissues like testis and seminal vesicles. he also reported that sars-cov-2 infection might be related to cardiac injury . a recent study by, holshue et al. (2020) unveiled that stool from a sars-cov-2 infected patient was positive for sars-cov-2, which suggests that this virus might infect the gastrointestinal tract. significantly, high expression of ace2 on the luminal surface of intestinal epithelial cells acts as a co-receptor for amino acid absorption from food. studies also suggest that intestine might act as a major entry site for sars-cov-2 infection and infected epithelium of gut might have significant inference on fecal-oral transmission and viral spread confinement . entry of corona-virus into the cell depends upon the priming of s-proteins by host cell proteases which comprise the cleavage at the arginine-rich site (multi-basic) s1/s2 and the s20 sites. the efficient cleavage of s-protein along with cleavage site sequence establishes the zoonotic potential of coronavirus. various studies have proved that sars-cov-2 infection initiation and spread of disease into the host cells mainly depends upon s protein priming by tmprss2 (transmembrane protease serine type 2), the serine protease. in humans, epithelial tissues, especially those lining the upper airways, bronchi, and lower airways show extensive tmprss2 expression (hoffmann et al., 2020) . the protein sequence analysis demonstrates that tmprss2 is conserved, with 78% sequence identity in human whereas, in mouse embryos and adult tissues, in situ hybridization analyses divulge the presence of tmprss2 in the epithelial cell lining the urogenital, gastrointestinal, and bronchi and bronchioles of respiratory tracts. however, the exact physiological function of tmprss2 within lung epithelial cells is not clear but various data suggest that it helps in proteolytic cleavage of the epithelial sodium channel and regulate sodium currents (iwata-yoshikawa et al., 2019) . according to the study, tmprss2 belongs to the type ii transmembrane serine protease family, and play a major role in the cleavage of hemagglutinin (ha) molecule in the influenza virus upon entry into human airway epithelial cells in human (hatesuer et al., 2013) . however, it can cleave glycoproteins (spike protein) and stimulates it to induce the fusion of virushost cell membrane at the cell surface which in turn assists virus entry into the host cell. various studies demonstrate that certain tmprss2 variants expression increases the threat of disease severity in influenza a (h1n1) infection (hatesuer et al., 2013) . a study by hoffmann et al. (2020) had demonstrated that in infected cells, precleavage at the s1/s2 site by furin might encourage consequent tmprss2-dependent entry and spread of infection. a type i transmembrane protein, furin is an activating protease that plays a critical role in fusion of viral membrane and viral entry within the host cell. thus the role of anti-tmprss2 as active-site inhibitors or inhibition of furin dependent entry might help us to consider them as probable therapeutic targets for influenza viruses and also for coronaviruses (hoffmann et al., 2020) . the s protein present in the membrane of sars-cov-2 has been considered as the most potent virus entry determinant into the host cell through the receptor ace2. a study by belouzard et al. (2009) revealed that a significant cleavage event takes place by proteolytic enzymes at position s20 of sars-cov-2 s protein by tmprss2, results in the fusion of membranes as well as viral infectivity mediated by the release of viral rna. other studies also revealed that entry of viral rna into the host cell depends upon not only membrane fusion, but also on the clathrindependent, and/or clathrin-independent endocytosis (wang et al., 2008) . after released into the cytoplasm viral rna genome is translated into two structural proteins and poly-proteins, which help in viral replication. upon infection with sars-cov-2 genome host cell activates well-coordinated and rapid immune response, i.e., innate and adaptive immune response, which represents the first line of defense against the viral infection. in endosome, membrane specific pattern recognition receptors (prr), like toll-like receptor (tlr3, tlr8, tlr7, and tlr9) or the cytosolic rna sensor, rig-i/mda5 or the secretory type prr like mannose-binding lectin (mbl) and c-reactive protein (crp) can recognize viral rna as pathogen-associated molecular patterns (pamps) (perlman and netland, 2009 ). interferon (ifn) type i activate a potent innate immune response against viral infection and also induce effective adaptive immune response. this recognition initiate a complex signaling cascade by recruiting adaptor proteins like mitochondrial antiviral-signaling protein (mavs), ifn-β (trif), and stimulator of interferon genes protein (sting) and activate downstream cascades molecules, like adaptor molecule myd88. this interaction activates transcription factors like nuclear factor-κb (nf-κb) and interferon regulatory factor 3 (irf3) and helps in nuclear translocation. in the nuclei, these transcription factors induce the production of type i interferons (ifn-α/β) and a plethora of proinflammatory cytokines especially il-6 (li et al., 2020a) . thus, interactions between the host cell and virus fabricate an assorted set of first line defense against the virus at the entry site. type i ifn mediated activation of jak-stat pathway; initiate the transcription of ifn-stimulated genes (isgs) under the control of the ifn-stimulated response element (isre). accumulation of type i ifn can suppress viral replication and act as an immune modulator to promote phagocytosis of antigens by macrophage, as well as nk cells mediated restriction of infected cells. thus, blocking the production of ifns or disorder of the jak-stat signaling pathway or altered expression of macrophages has a direct effect on the survival of the virus within the host cell (zhou z. et al., 2020) . generally, th1 mediated immune response plays a predominant role in adaptive immunity against viral infections. t cell responses majorly depend upon the presence of apc (antigen presenting cells) mediated cytokine microenvironment. cd8 + cytotoxic t cells (ctls) which are capable of secreting a cluster of molecules such as, granzymes, perforin, and ifn-γ are essential in the eradication of virus infected cells. cd4 + helper t cells facilitate the overall adaptive response by assisting cytotoxic t cells. on the other hand, b-cell mediated humoral immune response, plays a protective role by producing the neutralizing antibody, and also impedes re-infection. according to some recent findings, in covid-19 patients, an elevated level of chemokines and plasma cytokines like interleukins (il-1, il-2, il-4, il-7, il-10, il-12, il-13, and il-17), ip-10, macrophage colony-stimulating factor (mcsf), mcp-1, gcsf, hepatocyte growth factor (hgf), ifn-γ, mip-1α, and tnf-α, etc., are associated with disease severity (li et al., 2020e) . like in sars and mers the presence of "lymphopenia" and "cytokine storm" may have a significant role in the pathogenesis of covid-19 (moore and june, 2020). moreover, like in cancer and other chronic infections, persistence of cytokine storm might stimulate necrosis or apoptosis of t cells, and leads to their exhaustion (catanzaro et al., 2020) . this "cytokine storm" is responsible for commencing of viral sepsis followed by lung injury induced by inflammation-which is related to other complications like acute respiratory distress syndrome (ards), pneumonitis, respiratory failure, sepsis shock, organ failure, and potentially death. severity of covid-19 in patients is associated with the marked decrease in the number of circulating b cells, cd8 + cells, cd4 + cells, natural killers (nk) cells, as well as a decrease in eosinophils, monocytes, and basophils (zhou z. et al., 2020 ; figure 1 ). novel drug development is a time consuming process that depends upon costly laboratory as well as clinical trials, possibilities of mistakes, and other conditions. so, now a day's various bioinformatics tools like simulation, molecular docking, chemical stability studies, and target point determination playing an imperative and inventive approach in the designing of new drugs (de ruyck et al., 2016) . among them "molecular docking" of any specific place provide a clearer idea about designing new drugs, examining and comparing their efficacy in any particular disease. in the case of "pandemic" caused by sars-cov-2 where there is no specific medicine or vaccine available, "drug repositioning" or "drug repurposing" plays an attractive role (sheahan et al., 2020) . however, such drugs require a clinical trial to show their effectiveness against the disease. in a recent study, neda shaghaghi used the crystal structure of sars-cov-2 proteinase and herbal medicines for docking analysis. according to the study terpenoids from the herbal medicines be able to impede the enzymatic cavity of important amino acids and inhibit the viral protease (shaghaghi, 2020) . another study was done in virtual high throughput screening of clinically approved drugs and the structure of sars-cov-2 mpro revealed that saquinavir and beclabuvir, lopinavir, ritonavir, and nelfinavir act as the potential candidates for covid-19 therapy (sheahan et al., 2020) . molecular docking study is proved to be an economic method where technology-based ligand-protein interaction for a particularly active site reveals their possibilities as therapeutic candidates before synthesizing them. various in silico studies based on molecular docking has proved us-fda approved drugs like chloroquine, hydroxychloroquine, remdesivir, and arbidol as potential inhibitors of various viral polymerases and proteases and also known to be the most suitable targets for the sars-cov-2 treatment (wang m. et al., 2020) . another interesting study on the sars-cov-2 spike glycoprotein and membrane cd26 docked complex model reveals a great interface among the proteins. this interaction between the loops of the s1 domain and the cd26 surface establishes cd26 as a potential binding site for s-protein along with ace2 (vankadari and wilce, 2020). studies were done on secondary metabolites of various indian medicinal plants like garlic, curcumin, cardamom, ashwagandha, neem, aloe vera, harsingar, etc., have shown effective inhibition properties against sars-cov-2 protease enzyme (srivastava et al., 2020) . these compounds have an effective affinity toward the key amino acids active site and able to inhibit them during the catalytic process. although the results obtained from drug repositioning or secondary metabolites of herbal plants by molecular docking provide information regarding suitable drugs candidate for covid-19 therapy but thorough in vitro studies and in vivo studies can prove their effectiveness against sars-cov-2. providing supportive care to the patients is the best strategy in current situations, as so far no established antiviral treatment is to be useful in controlling an outbreak of novel covid-19 (guo et al., 2020) . so far numerous compounds have been checked in animal models or in cell culture to establish them as a potential therapeutic approach against entry or replication of sars-cov or mers-cov or sars-cov-2, but experiments done in animal or in vitro does not inevitably translate into efficacy in humans (li and de clercq, 2020) . based on the structural as well as the pathogenesis of covid-19, continuous extensive investigations are in progress to determine the effective therapeutic agents for sars-cov-2 infections. studies converge on drug discovery against emerging covid-19 outbreaks can be broadly classified into two different modes of treatment: (a) virus-based therapy and (b) host-based treatment options. (a) virus-based therapy (i) viral nucleic acids are made up of nucleosides and nucleotides. drugs that are capable to target nucleotides or nucleosides and/or viral nucleic acids have a wide-range of activity against covs and other viruses. studies have shown that various nucleoside analog, like ribavirin, favipiravir, and galidesivir have antiviral activity against some animal covs, and in the (guo et al., 2020) . (ii) major enzymes and proteins involved in viral replication of covid-19 are potential targets for anti-viral treatment. the plpro enzymes and papain-like protease of sars-cov and mers-cov exhibit proteolytic, de-ubiquitylating as well as deisgylating activities. studies have proven that among various protease inhibitors, lopinavir-ritonavir acts as the most effective one (guo et al., 2020) . (iii) the key immunogenic antigen of sars-cov-2 is membrane-anchored spike glycoprotein, which plays an essential role in the host cell and virus interaction. studies have shown that some monoclonal antibodies (mabs) can target definite epitopes on the rbd of s1 subunit and restrain virus-cell receptor binding; on the other hand, others attach with the s2 subunit and disrupt virus-cell fusion (jiang et al., 2014) . studies also suggest that mabs exhibit neutralizing activities and also able to reduce viral titers in vitro as well as in small animal models. as s protein of sars-cov-2 displayed high homology to that of sars-cov, cr3022 the neutralizing antibody of sars-cov was found to bind potently with the rbd domain of sars-cov-2 (tian et al., 2020) . previous studies also suggest that adoptive transfers of plasma which contain anti-mers-cov-s antibodies can block virus attachment and also accelerate viral clearance. depending upon this idea, scientists are trying to develop convalescent plasma-based therapy against pandemic sars-cov-2 infection (li et al., 2020c) . studies also imply antiviral peptides as a proposed analog for viral spike protein. analogous peptides for the pre-transmembrane domain, n terminus, or the loop region which separates the hr1 and hr2 domains of sars-cov are competent to inhibit the formation of virus plaque (tang et al., 2014) . some studies also prove that some sirnas have antiviral activities in vitro but still they are in preclinical development (wu et al., 2005) . (iv) along with e, m, and n proteins some accessory proteins are essential for virion assembly and viral replication by suppressing the host immune response. anti-viral therapy against such proteins might have effective role in covid-19 infection (vigant et al., 2013) . studies have proved that lj001 and jl103 (lipophilic thiazolidine derivatives) act as membrane-binding photosensitizers. they are proficient in the production of singlet oxygen molecules which stimulate various changes in lipid membranes results in the prevention of fusion between viral and host cell membranes (zumla et al., 2016) . host-directed therapies are related to the improvement of host immune response, host status, and/or handling of hostrelated factors coupled with viral replication. (i) studies have shown that innate interferon response by host cells is important for viral replication within the host cell. though covs are capable to restrain the interferon response to imply immune evasion, in vitro studies have revealed that treatment with various recombinant interferons might incline the infection severity (menachery et al., 2014) . (ii) to suppress dysfunctional systematic inflammation, corticosteroids have excellent pharmacological effects. these are one of the major imunomodulators which were widely used in the treatment of sers-cov and mers-cov and are also considered as a helpful agent in the management of the current epidemic of sars-cov-2 (li et al., 2020b) . another school of study suggests that combined use of both inducers of interferon along with innate immunomodulators might be efficient as antiviral agents against sers-cov-2. (iii) apart from immunomodulators, other substances like metformin, atorvastatin, fibrates, as well as nutritional supplements might play the most important role in treating the current pandemic by boosting immunity (zumla et al., 2016) . (iv) covs generally utilize specific host factors like host receptors or other enzymes and proteins for viral entry as well as viral replication. thus specific monoclonal or polyclonal antibodies, peptides, or functional inhibitors against host receptors or injecting an excessive liquid form of ace2 receptor or recombinant technology must use to stop host membrane-viral fusion (huentelman et al., 2004) . (v) a recent study by hoffmann et al. (2020) demonstrated that sars-cov-2 spike protein priming depends upon transmembrane protease serine 2 (tmprss2) for viral entry. further studies also revealed that the serine protease inhibitor camostat mesylate, can block tmprss2 activity and considered as an attractive candidate for therapy (hoffmann et al., 2020) . (vi) another study suggests that the entry of coronavirus into the host cell involved ph-and receptor-dependent endocytosis. a host kinase, ap-2-associated protein kinase 1 (aak1) regulates clathrinmediated endocytosis. molecular docking studies reveal that the janus kinase inhibitor baricitinib is a potent aak1-inhibiting drug, is expected to be a suitable drug candidate for covid-19 (richardson et al., 2020 ; table 1 ). carbohydrate-binding agents are considered as anti-cov agents that target spike protein and restrain cov entry. they are capable to bind specifically with the oligosaccharides present on the viral surfaces such as s and hiv glycoprotein (garcia-laorden et al., 2008) . in in vitro condition as well as in mouse model they inhibit a wide range of covs, including hcov-229e, sars-cov, hcov-nl63, and hcov-oc43. glycans have a wide variety of shape, mass, charge, or other physical properties which help them to mediate extensive biological roles. natural proteins called lectins target the sugar moieties of a wide variety of glycoproteins which is not involved in cell adhesion. most lectins belong to glycan families with distinct "carbohydrate-recognition domains" (crds) that conserve specific primary amino acid sequences or 3d structure and evolved from shared ancestral genes. in innate immunity, mbl acts as a key pattern-recognition molecule. it mainly functions as an ante-antibody, a humoral factor, crucial for the first-line host defense prior to the production of antibodies. studies have shown that expression of mbl is associated with the initiation of complement activation via the lectin pathway and also responsible for opsonophagocytosis (eisen, 2010) . a study by hartshorn et al. (1993) demonstrates that mbl function as an opsonin and inhibit hemagglutination as well as infectivity against respiratory viruses, such as influenza a virus. binding of mbl with the n-linked high-mannose carbohydrate side chain present at the tip of s hemagglutinin protein is able to neutralize the infectivity of the influenza a virus. studies also revealed that sars-cov s-protein contains 23 potential n-linked glycosylation sites. binding of mbl with the s protein mannose side chains of sars-cov can be used as the most useful remedial agent (ip et al., 2005) . thus, glycosylation of viral peptides might be considered as a novel therapeutic strategy against current covid-19 pandemic (figure 2 ). since, across the world, covid-19 infection causes severe public health concern, analysis of the characteristics features of sars-cov-2, its interaction with the host receptor and immune responses, the phylogenetic and genomic similarity with other viruses will provide a clearer picture of diseases onset in individuals. several groups of scientists have postulated that just like sars-cov, sars-cov-2 also depends upon the ace2 as a receptor for host cell entry. the interaction of the virus transmembrane spike (s) glycoprotein with host cell receptors act as the determinant of the pathogenesis. a higher degree of disease severity is associated with viral load and age and sex of the patients. in elderly patients, the high viral load is associated with low immunity as well as higher expression of the ace2 receptor in some hematopoietic cells, in cardiopulmonary tissues, and macrophages and monocytes. at the same time, male patients are more susceptible to the sars-cov-2 infection than their female counterparts. "lymphopenia" (low blood lymphocyte count) is correlated with the clinical severity of covid-19 related infection. however, lymphopenia can be considered as a biomarker of poor prognosis of covid-19 which was also correlated with casualty in the influenza a (h1n1) pandemic in the year 2009. the study also, suggests that in one or more host species sars-cov-2 attached with integrins as cell receptors, through a conserved sequence rgd (403-405:arg-gly-asp) with the rbd domain of the spike proteins. pharmacotherapy against integrin can control the association between virus and integrin which is necessary to neutralize pathogenesis. the development of sars-cov-2 infection majorly depends upon the interaction between the individual's immune system and the virus. on one hand viral factors like virus type, mutation potential, virus viability along with the immune system factors of an individual like age, gender, nutritional status, genetics, neuroendocrine-immune regulation, etc., contribute to the severity of the disease. an effective immune response majorly depends upon a crucial part, called inflammation. proper elimination of any infections majorly depends upon inflammation. initial recognition of pathogens is the primary step for the onset of inflammatory response followed by the recruitment of immune cells. these immune cells help in the elimination of the pathogens and eventually lead to tissue repair and restoration of homeostasis. though, in some infected individuals, sars-cov-2 induces extreme and prolonged cytokine/chemokine responses, called "cytokine storm." this cytokine storm is related to multiple organ dysfunction or ards and later leads to physiological deterioration and death. an elevated level of serum cytokine il-6 and c-reactive protein considered as the biomarker of severe β-coronavirus infection. upon infection with β-coronavirus monocytes, macrophages, and dendritic cells get activated and start the secretion of prominent pro-inflammatory cytokine, like il-6 along with other inflammatory cytokines. il-6 can activate either classic cis-signaling or trans-signaling. various pleiotropic effects on the acquired immune system (t and b cells) as well as on innate immune system [macrophages, neutrophils, and natural killer (nk) cells], are correlated with activation of cis-signaling and leads to cytokine release syndrome (crs). on the other hand upon activation of trans-signaling, elevated il-6 level creates the "cytokine storm" which is related with the secretion of monocyte chemoattractant protein-1 (mcp-1), vascular endothelial growth factor (vegf), il-8, and also excessive il-6. it is also associated with reduced expression of adhesion molecule e-cadherin on endothelial cells. reduced expression of e-cadherin and secreted vegf mainly contribute to vascular permeability as well as leakage, which in turn coupled with the pathophysiology of hypotension and pulmonary dysfunction in acute respiratory distress syndrome (ards). an antagonist of il-6 is tocilizumab, previously approved to treat juvenile idiopathic arthritis which is a rheumatic condition, is again "repurposed" for the covid-19 pandemic. this finding suggests that we can potentially use il-6 directed therapies not only in covid-19 but also in other pandemics in the future involving ebola and influenza viruses. in this review, we summaries the different aspect of the therapeutic potential of the various anti-viral derivative. finally, most reasonable options must be evaluated further in clinical trials against the covid-19 pandemic. it might consist of either mono-therapy or combinational therapies comprise of interferon beta-1b, lopinavir-ritonavir, and/or mabs and antiviral peptides. thorough analyses of glycans are essential for the expansion of glycoprotein-based vaccine which might approach to correlate the immunogenicity with structural variations. in different expression systems, glycosylation act as a measure to evaluate antigen quality. basic understanding correlated with rbd domain of the spike protein of sars-cov-2 consist of complex sialylated n-glycans and sialylated mucin type o-glycans will be useful to design suitable immunogens for vaccine development. mbl is a serum c-type lectin, which can bind sars-cov per se or infected cell and also capable to inhibit the infectivity of the virus. studies have shown that "mbldeficient" individuals are at more risk to sars infection. we support, mbl as a potent therapeutic and prophylactic strategy in the prevention of sars-cov-2 pandemics. shortly, it will be possible to develop broad-spectrum, novel, antiviral drugs active against a larger array of coronavirus, and also will be the ultimate treatment strategy for circulating and emerging covid infections. sc contributed to design, editing, and approval of final version of the manuscript. ss drafted and prepared the manuscript, and drew the figures. mm contributed to read and editing the draft of the manuscript. all 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10.1007/s40618-020-01438-8 sha: doc_id: 318164 cord_uid: 6rqi17oz purpose: sperm cryopreservation is fundamental in the management of patients undergoing gonadotoxic treatments. concerns have risen in relation to sars-cov-2 and its potential for testicular involvement, since sars-cov-2-positive cryopreserved samples may have unknown effects on fertilization and embryo safety. this study therefore aimed to analyze the safety of sperm cryopreservation for cancer patients after the onset of the pandemic in italy, through assessment of the risk of sars-cov-2 exposure and viral rna testing of semen samples. methods: we recruited 10 cancer patients (mean age 30.5 ± 9.6 years) referred to our sperm bank during the italian lockdown (from march 11th to may 4th 2020) who had not undergone a nasopharyngeal swab for sars-cov-2 testing. patients were administered a questionnaire on their exposure to covid-19, and semen samples were taken. before cryopreservation, sars-cov-2 rna was extracted from a 150 µl aliquot of seminal fluid in toto using qiaamp viral rna kit (qiagen) and amplified by a real time rt pcr system (realstar sars-cov2 rt pcr, altona diagnostics) targeting the e and s genes. results: the questionnaire and medical interview revealed that all patients were asymptomatic and had had no previous contact with covid-19 infected patients. all semen samples were negative for sars-cov-2 rna. conclusion: this preliminary assessment suggests that a thorough evaluation (especially in the setting of a multidisciplinary team) and molecular confirmation of the absence of sars-cov-2 in seminal fluid from asymptomatic cancer patients may assist in ensuring the safety of sperm cryopreservation. sperm cryopreservation has become a mainstay in the management of cancer patients undergoing genotoxic treatments capable of inducing transient or permanent sterility [1, 2] . the harmful cytological and molecular effects of cancer treatments on male gametes have been extensively studied: the high cell renewal rate of the seminiferous epithelium makes it extremely vulnerable to iatrogenic damage [3] [4] [5] . cryopreservation in liquid nitrogen at − 196 °c can keep sperm alive indefinitely, thus enabling male fertility to be preserved. in fact, in these conditions no chemical reaction can take place, as there is insufficient thermal energy. however, literature studies have reported that viruses stored in liquid nitrogen may maintain their pathogenic properties [6] . this means that sperm cryopreservation might also preserve any viral species contaminating the semen sample. the swift spread of sars-cov-2, and the uncertainty caused by the paucity of data on female and male fertility in patients with covid-19, has pushed assisted reproduction centers to seek common guidelines for both assisted reproduction techniques and gamete cryopreservation. sars-cov-2 shows a marked tropism for respiratory tissues, targeting types i and ii pneumocytes and alveolar macrophages [7] . however, extra-respiratory transmission routes cannot be excluded. sars-cov-2 seems capable of interacting with angiotensin 2 converting enzyme (ace2) [8, 9] . this is thought to enable cell invasion, and the diffuse expression of the virus in various human tissues, including the testis [10] , leads to serious concerns regarding the safety of cryopreserved gametes. an exhaustive risk analysis for cryopreserved gametes and the possible consequences for embryo development is currently impossible, given the almost total lack of knowledge in this area [11] . it might therefore be important to identify sars-cov-2-positive patients before cryopreservation procedures for the duration of the pandemic [12] , given that sars-cov-2 may now be present in semen samples and in liquid nitrogen in sperm banks across the world [13] . in italy, the current recommendation is to continue performing sperm cryopreservation in cancer patients, except for those with symptoms consistent with a severe acute respiratory infection. since nasopharyngeal swab testing was not widespread in the first stage of the pandemic and testing was limited mainly to patients with covid-19 symptoms, it is essential to establish the suitable management of asymptomatic subjects who need to cryopreserve their semen. this study thus aimed to evaluate the safety of sperm cryopreservation of cancer patients referred to our sperm bank after the onset of the pandemic in italy through the assessment of the risk of sars-cov-2 exposure and, in selected volunteers, viral rna testing of semen samples. the study was approved by our university hospital's institutional review board (policlinico umberto i-"sapienza" university of rome ethics committee), and all patients gave their written informed consent. we recruited ten patients referred to the laboratory of seminology -sperm bank "loredana gandini" during the italian lockdown (from march 11th to may 4th 2020) who had not previously undergone nasopharyngeal sars-cov-2 testing. sars-cov-2 rna analysis was carried out in suitable semen samples before cryopreservation: patients with a semen sample volume below 2.0 ml, and patients who did not cryopreserve their semen due to azoospermia, were excluded. information was collected on cancer type and medical history (previous diseases, medication and drug use, smoking habits and andrological diseases). patients were also asked if they had been tested for covid-19 and/or had had any symptoms in the previous 2 weeks. semen samples were collected by masturbation, if possible after 3-5 days of abstinence, but when treatment needed to be started urgently, sperm was stored regardless of the recorded abstinence interval. all samples were allowed to liquefy at 37 °c for 30-60 min and were then assessed according to world health organization laboratory manual [14] . the variables considered were: ejaculate volume (ml), sperm concentration (n × 10 6 /ml), total sperm number (n × 10 6 per ejaculate), progressive motility (%), and morphology (% abnormal forms). staff were provided with adequate personal protective equipment and samples were cryopreserved in high security straws, as normal. sars-cov-2 rna analysis was performed on 150 µl of seminal fluid in toto, aliquoted before the samples were processed for cryopreservation. viral rna was extracted using qiaamp viral rna kit (qiagen) according to the manufacturer's instructions. ten µl of extracted rna was reversetranscribed and simultaneously amplified using a real time rt pcr system (realstar sars-cov-2 rt pcr, altona diagnostics) targeting the e and s genes. a triage questionnaire regarding general health and respiratory symptoms was administered twice to the patients by a medical assessor: first by phone, when the patient requested an appointment, and a second time on the day of sperm cryopreservation (generally 24-48 h later). the questionnaire investigated: • presence and nature of any flu-like and/or acute respiratory disease symptoms (chills, temperature > 37.5 °c, dyspnea, etc.) • history of recent travel to or stay in countries/areas, including italian regions and towns, with a confirmed presence of sars-cov-2 • close contact with a probable or confirmed case of sars-cov-2 infection • recent admittance to a healthcare facility with confirmed cases of sars-cov-2. data from the patients' medical history and analyses are described as counts or percentages for categorical variables and means ± standard deviations for continuous variables. all computations were carried out with statistical package for the social sciences (spss) 25.0 (spss inc., chicago, usa). the patients' mean age was 30.5 ± 6.9 years. the most frequently reported malignancies were hematological in nature (4 hodgkin lymphomas, 1 mediastinal gray zone lymphoma), followed by testicular cancers (four patients) and ewing sarcoma (one patient). a complete caseload description is available in table 1 . semen testing for sars-cov-2-we asked our patients to provide aliquots from their semen samples for sars-cov-2 rna testing. the rt-pcr showed no amplifications for the e and s genes in any samples, indicating the absence of sars-cov-2. clinical evaluation and covid-19 questionnaire-according to our medical interview and the answers to the covid-19 questionnaire, no patients had fever > 37.5 °c or dyspnea on admittance to the sperm bank. in the previous 2 weeks all patients had been asymptomatic and had not recently traveled to or stayed in areas with a confirmed presence of sars-cov-2 spread, nor had any close contacts with confirmed covid-19 cases or people with acute respiratory infections. one patient had been hospitalized, but in a ward with no confirmed cases of covid-19 (table 2 ). in december 2019, wuhan's hospitals started to highlight cases of a severe atypical pneumonia of unknown etiology. on january 7th 2020, researchers identified a virus (now known as sars-cov-2) with a high homology with a bat coronavirus [15] . the swift global spread of sars-cov-2, its high infectiousness and its severe clinical signs [16] forced many countries to apply harsh and restrictive containment policies in an attempt to control the pandemic. in italy, the government declared a country-wide lockdown curbing almost all activities, including those of reproductive medicine centers. the paucity of knowledge, owing to the novelty of the virus, and the unforeseen magnitude of the pandemic, forced assisted reproduction specialists to demand clear and common guidelines in relation above all to medically assisted reproduction and gamete cryopreservation. current restrictions in italy have led reproductive centers to drastically limit their activities, accepting only asymptomatic patients about to undergo potentially gonadotoxic treatments. for sperm banks like ours, this means above all cancer patients who urgently need to begin radio-and/or chemotherapy [17] . however, apart from safety issues for the sperm bank personnel, this novel pandemic forced us to consider three major issues: (1) does the sars-cov-2 virus reach the testis and the seminal fluid? if it does, (2) are there any long-lasting consequences of viral testicular infection? (3) would the use of infected cryopreserved semen affect the fertilization process [18, 19] ? while most of these questions are still lacking a definitive answer, some data in relation to other viruses in seminal fluid has been published. a number of viruses have been isolated from the seminal fluid of infected men, including replicating zika, ebola and marburg viruses [20] . the troublesome possibility is that some may even show a long-term persistence in the seminal fluid, with severe repercussions for assisted reproductive technologies (art): for example, the zika virus has been detected in semen from asymptomatic men for up to 1 year after their recovery [21] . however, there is still little data on sars-cov-2. a recent paper from li et al. reported the presence of viral rna in the semen of around 19% of acute (four patients) and recovering (two patients) covid-19 patients [22] . although the limited caseload and undisclosed methods hinder us from generalizing from their results [23] , the implications for reproductive medicine are alarming. conversely, we recently found that a symptomatic sars-cov-2 positive patient did not harbor viral rna in his semen and urine during the remission phase (about a week after the positive nasopharyngeal swab) [24] , and similar results were observed by song et al. in several recovering patients [25] . pan et al. confirmed, in a longer follow-up of 34 patients, that viral rna was undetectable in their seminal fluid about one month after covid-19 diagnosis [26] . the most evident difference between these studies lies in the patients recruited. li et al. reported a caseload of patients presumably suffering from a more severe disease than those in the previous reports. testing for the presence of sars-cov-2 in different stages of the disease might justify these different results. in more severe covid-19 cases, a higher systemic diffusion of the disease might enable testicular involvement. alternatively, in mild to moderate cases the sars-cov-2 clearance rate from seminal fluid might coincide with the clinical recovery. overall, however, the evidence is still too scarce to be conclusive, and to date no evidence on sexual transmission is available. in any case, how might sars-cov-2 reach the testis? we know that viruses often spread to the male reproductive tract from the blood, as the blood-testis barrier does not seem to constitute an insurmountable obstacle to viruses, especially in the presence of systemic or local inflammation [20] . to date, few studies have investigated the presence of sars-cov-2 in blood. wang et al. found a minimal percentage of positive blood samples through rt-pcr amplification of viral rna [27] , whereas zhang et al. detected the virus in 40% of blood samples [28] . it could be possible that the virus only spreads to the blood under certain circumstances, such as the acute phase or severe disease, and then to other organs such as the testis. the presence of ace2 and tmprss2 may make the testis and male genital tract a viable site for the virus, but the cellular type that might function as reservoir has yet to be determined. it is therefore imperative to establish whether, when and how sars-cov-2 reaches the seminal fluid and how long it persists there, to assess the risks and establish strict procedures [17] for the use of gametes in assisted reproduction (transmission of infection, infection of embryos, congenital diseases, spontaneous abortions, etc.). solving this uncertainty is even more important when we consider cryopreserved semen samples. as noted above, in this period sperm banks are mainly collecting and storing germ cells from male cancer patients. these patients have suffered persistent spermatogenetic damage from various cancer treatments and assisted reproduction may be their only chance to achieve fatherhood [3, 5, 29] . while many concerns have been raised in relation to the collection, shipping and use of these samples [13] , sperm banks in italy are currently permitting sperm cryopreservation for asymptomatic subjects, in accordance with local indications. from the little data available, the risk that sars-cov-2 might spread to the testis and be transmitted through the seminal fluid seems fairly low in mild to moderate covid-19 patients and, although no investigation has been conducted in asymptomatic subjects, the chance of sars-cov-2 being present in our asymptomatic cancer patients should be even lower. however, the prevalence of asymptomatic subjects and their contribution to the transmission of covid-19 is not well characterized [30] , and their role in the spread of the virus poses a tremendous epidemiological challenge [31] . these subjects must be identified, and a careful clinical evaluation and contact history may still be the only way to do so [31] . for this reason, the patients at our sperm bank underwent a detailed medical history and epidemiological questionnaire to minimize the risk. this was further confirmed by testing seminal fluid samples from 10 asymptomatic cancer patients for sars-cov-2 rna. as mentioned above, no viral rna was found in any sample, suggesting that asymptomatic cancer patients with no previously known sars-cov-2 infection have a very low chance of harboring this coronavirus in their semen. in our experience, sars-cov-2 testing of patients referred for sperm banking, while recommended [12] , was inadequate at the onset of the pandemic. while a screening strategy with nasopharyngeal swabs or serological testing is advisable in all cancer patients, the large numbers of symptomatic inpatients at the start of the pandemic made this unfeasible. in the absence of sars-cov-2 testing, it is important to distinguish between asymptomatic patients who have, and those who have not, been at risk of exposure, to select patients whose cryopreserved gametes present the smallest residual risk of harboring the virus. the inclusion of this test in cryopreservation protocols during the pandemic could further ensure the safety of sperm cryopreservation performed in this period. from this perspective, the absence of viral rna in our cryopreserved samples has a double value. first, it comprises a preliminary biomonitoring for sars cov-2 in the semen of asymptomatic cancer patients. second, it underlines the importance of taking a thorough medical history, accompanied in our case by the administration of an epidemiological questionnaire. this careful evaluation (especially in the setting of a multidisciplinary team, where the art specialist works in tandem with the oncologist and infectious disease specialist/virologist), together with the molecular confirmation of the virus' absence in the semen sample, should help ensure the safety of sperm cryopreservation. this precautionary approach, together with suitable personal protective equipment and the use of high security straws, should minimize the risks associated with cryopreserving sperm during the pandemic. furthermore, the current evidence seems increasingly to point to the absence of sars cov-2 in semen. the risk of preserving contaminated semen samples is thus minimal, provided that all necessary precautions and safety procedures are observed. nonetheless, it should be stressed that these results are not conclusive, as they refer to a very small caseload, and need further confirmation, especially as we focused on the safety issues associated with possible asymptomatic covid-19 infections, without investigating whether sars-cov-2 infection is capable of spreading to the testis and seminal fluid. otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. fertility preservation in patients with cancer: asco clinical practice guideline update summary cryopreservation of sperm: effects on chromatin and strategies to prevent them spermatogenesis in hodgkin's lymphoma patients: a retrospective study of semen quality before and after different chemotherapy regimens effects of chemotherapy and radiotherapy on spermatogenesis in humans fatherhood and sperm dna damage in testicular cancer patients biobanking in microbiology: from sample collection to epidemiology, diagnosis and research comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 structure analysis of the receptor binding of 2019-ncov addressing male sexual and reproductive health in the wake of covid-19 outbreak the novel angiotensin-converting enzyme (ace) homolog, ace2, is selectively expressed by adult leydig cells of the testis evaluating risk, safety and efficacy of novel reproductive techniques and therapies through the eurogtp ii risk assessment tool sars-cov-2 pandemic and repercussions for male infertility patients: a proposal for the individualized provision of andrological services covid-19: should we continue to cryopreserve sperm during the pandemic? world health organization (2010) who laboratory manual for the examination and processing of human semen a pneumonia outbreak associated with a new coronavirus of probable bat origin the covid-19 epidemic covid-19: the perspective of italian embryologists managing the ivf laboratory in pandemic emergency the covid-19 pandemics: shall we expect andrological consequences? a call for contributions to andrology sars-cov-2 infection, male fertility and sperm cryopreservation: a position statement of the italian society of andrology and sexual medicine (siams) (società italiana di andrologia e medicina della sessualità) the breadth of viruses in human semen persistence and clinical relevance of zika virus in the male genital tract clinical characteristics and results of semen tests among men with coronavirus disease 2019 sars-cov-2 presence in seminal fluid: myth or reality study of sars-cov-2 in semen and urine samples of a volunteer with positive naso-pharyngeal swab absence of 2019 novel coronavirus in semen and testes of covid-19 patients † no evidence of severe acute respiratory syndrome-coronavirus 2 in semen of males recovering from coronavirus disease 2019 detection of sars-cov-2 in different types of clinical specimens molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes effect of chemo-or radiotherapy on sperm parameters of testicular cancer patients asymptomatic coronavirus infection: mers-cov and sars-cov-2 (covid-19) asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths key: cord-303216-1pbuywz6 authors: das, gaurav; mukherjee, nabanita; ghosh, surajit title: neurological insights of covid-19 pandemic date: 2020-04-22 journal: acs chem neurosci doi: 10.1021/acschemneuro.0c00201 sha: doc_id: 303216 cord_uid: 1pbuywz6 the novel coronavirus sars-cov-2, which was identified after a recent outbreak in wuhan, china, in december 2019, has kept the whole world in tenterhooks due to its severe life-threatening nature of the infection. the virus is unlike its previous counterparts, sars-cov and mers-cov, or anything the world has encountered before both in terms of virulence and severity of the infection. if scientific reports relevant to the sars-cov-2 virus are noted, it can be seen that the virus owes much of its killer properties to its unique structure that has a stronger binding affinity with the human angiotensin-converting enzyme 2 (hace2) protein, which the viruses utilize as an entry point to gain accesses to its hosts. recent reports suggest that it is not just the lung that the virus may be targeting; the human brain may soon emerge as the new abode of the virus. already instances of patients with covid-19 have been reported with mild (anosmia and ageusia) to severe (encephalopathy) neurological manifestations, and if that is so, then it gives us more reasons to be frightened of this killer virus. keeping in mind that the situation does not worsen from here, immediate awareness and more thorough research regarding the neuroinvasive nature of the virus is the immediate need of the hour. scientists globally also need to up their game to design more specific therapeutic strategies with the available information to counteract the pandemic. in this viewpoint, we provide a brief outline of the currently known neurological manifestations of covid-19 and discuss some probable ways to design therapeutic strategies to overcome the present global crisis. c ovid-19 is currently a global pandemic impacting around 212 countries and has already claimed the lives of more than 79,385 individuals worldwide with confirmed cases globally now standing at around 1,356,780. 1 experts across the world warn that the disease, which originated in the wuhan district of china in the december of 2019, will only propagate more and the present numbers are likely to inflate further. the sudden outbreak of covid-19 shook the scientific fraternity, and scientists across the world are working tirelessly to understand the virus and its properties in order to design intervention strategies to combat the disease. so far, we have been able to understand that covid-19 is caused by a virus known as sars-cov-2, which is a single-stranded rna virus (ssrna) with a genome size of 29903 bp. sars-cov-2 belongs to the same beta-coronavirus clade of the previously reported sars-cov and mers-cov and bears sequence similarity to sars-cov. 2 in fact, not only do they bear similarity in sequences, but also the entry point of both the viruses in humans is through the same receptor recognition. the virus sars-cov-2 has been speculated to have been transmitted from bats to human considering the fact that evidence of a similar coronavirus, ratg13, has been reported in bats. 3 sars-cov-2 is made up of three structural proteins, namely, the spike (s), envelope (e), and membrane (m), which makes up the viral envelope, and the nucleocapsid containing the rna genome. it is the spike protein that carries out the frontline action for the virus by performing the initial receptor recognition with the human angiotensin-converting enzyme-2 receptor (hace2). 3 it is this interaction of hace2 with the viral spike protein that is central to the viral infection. the crystal structure of sars-cov-2 has revealed the presence of a core and receptor-binding domain (rbd) that is more specifically involved in recognizing the hace2. though both sars-cov and sars-cov-2 exploit the same receptor hace2 in humans to gain entry into the host, the sars-cov-2 binding is more compact with a four residue motif from 482 to 485 in the hace2 ridge, thus enhancing the binding affinity of sars-cov-2 over sars-cov for hace. 3 moreover, the two viral hot spots, namely, hot spot-31 and hot spot-353 on hace2, are stabilized more by the sars-cov-2 rbd as compared to sars-cov. 3 all this clearly indicates why sars-cov-2 has a selective advantage over sars-cov in causing infection and is a more evolved and lethal strain. the major clinical manifestation of sars-cov-2 is severe pneumonia causing immense respiratory distress in the patients, specially the aged or those with already pre-existing conditions. sars-cov-2 leads to chronic inflammation of the lungs, severe dyspnea, fever, dry cough, and cyanosis and in more vulnerable patients a complete lung failure. 4 ace2, which is the entry point for sars-cov-2, has almost a ubiquitous presence in human organs including lung parenchyma, gastrointestinal tract, nasal mucosa, renal and urinary tract, human airway epithelia, lymphoid tissues, reproductive organs, vascular endothelium, and brain. 4 the virus is believed to enter chiefly through the nasal mucosa or the gastrointestinal tract due to their higher expression of protein hace2. the intriguing part though is that recently reported studies have noted altered mental health in some covid-19 patients showing symptoms like anosmia and ageusia thereby indicating a neuroinvasive nature of the virus. 2 the neurological manifestations of sars-cov-2 have been recently recognized from ct scan images and mri scan of the brain of a patient who contracted covid-19 and showed symptoms of necrotizing hemorrhagic encephalopathy. 5 acute necrotizing encephalopathy (ane) is a rare disorder leading to brain dysfunction mostly caused by viruses, which results in seizures, liver problems, and mental disorientation following infection. 5 the disease is characterized by multifocal symmetric lesions in the brain, which affect the brain stem, thalami, cerebellum, and cerebral white matter. ane causes neuroinflammation resulting from a cytokine storm characterized mainly by the production of the interleukin-6 (il-6), secreted by the macrophages, which in turn have been activated by the granulocyte−macrophage colony-stimulating factor (gm-csf) produced by the helper t cells. 6, 7 the resultant cytokine storm may also cause a surge in interleukin (il)-2, il-7, interferon-γ, inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1-α, and tumor necrosis factor-α leading to hyperinflammation. 6 this systemic inflammation causes severe encephalopathy in the patient, and that may lead even to stroke. specifically, in this patient infected with sars-cov-2 showing ane, the mri images displayed clear evidence of hemorrhage through hypointense signal intensity in the susceptibilityweighted images and increase in the rim on the postcontrast images. 5 the likeliness of covid-19 patients to contract neurological infections can be exacerbated by secondary factors like smoking, which according to a pilot study can enhance the chances of contracting covid-19 based neuroinfections due to the functional interactions between hace2 and the nicotinic receptor (nachr). 8 the same study reports that due to the coexpression of hace2 and nicotinic acetylcholine receptor (nachr) in many cells, there exists a functional link between them. so, when smokers smoke, it augments the expression of the hace2 due to the nicotine stimulation of nachr. 8 hence, while performing autopsies on brains of covid-19 patients, it would be wise to conduct smoker versus nonsmoker based analysis as this will help to shed light on smoking being an additional risk factor in covid-19 patients along with age and already existing ailments. 8 now, as encephalopathy has been identified as one of the symptoms of covid, the neuroinvasiveness of sars-cov-2 needs to be assessed to fully understand the neurological implications of covid-19. the brain reportedly, like most other organs, expresses the hace2 considered to be the entry point of the sars-cov-2 viruses in humans and is therefore not immune to viral infection. though sars-cov-2 is yet to be detected in cerebrospinal fluid, sars-cov with similar structural and functional features has been detected in the cerebrospinal fluid of patients, indicating the ability of the virus to breach the extremely rigid blood−brain barrier. 4 if previous studies with other covs are taken into consideration, sars-cov-2 like its other family members will first infect the peripheral nerve terminals and then slowly crawl its way through the synapse-connected route into the cns. 7 previous studies in the relevant field with sars-cov and mers-cov observed that they infiltrate the brains of transgenic mice when administered intranasally. 4 the infiltration of the virus into the brain took place through the olfactory nerves, eventually affecting the thalamus and the brain stem. the brain stem was eventually observed to be the worst infected. 4 so, following these experimental studies along with the hematologic spread of sars-cov-2 in cns, retrograde neuronal transport of the virus through the vagal nerve afferents from the lungs into the cns must be taken into consideration. 7 also, with reports claiming infection of the gastrointestinal tract by sars-cov-2, the virus could even use the enteric nervous system and its sympathetic afferent neurons to reach the cns. 7 now with the reports of involuntary breathing, hyposmia, and ageusia in covid-19 patients, scientists have started to speculate that not only does sars-cov-2 infects lungs but it has severe implications in neurons, specifically those in the medulla oblongata, which regulates breathing, lung, and heart functions, and any damage to it can result in chronic respiratory distress as reported in covid-19 patients. 4 it has been put forward that the latency period of the virus may be enough to actually destroy the neurons in the medullary region of the brain and can lead to coma or death (figure 1 ). 4 as reports of sars-cov-2 reaching the blood−brain barrier through the circulating blood and breaching it by attacking the endothelial layer to gain access to cns emerge, the virus might just be using an alternating route in the form of the olfactory bulb instead of the common hematological route. 2 if this is to be considered, the virus might just be making its way into olfactory mucosa, mostly consisting of olfactory neurons along with blood vessels and epithelial cells. the olfactory mucosa is connected with the olfactory bulb through the cribriform plate, which is found at the very base of the frontal lobes of the brain. 2 this very much explains the hyposmia and other neurological symptoms that are being increasingly observed in covid-19 patients (figure 1) . the point to note here is that the long-term effects of the neuroinvasive nature of the virus may result in an increased risk of neurodegenerative diseases with involvement in the pathogenesis of neurological disorders like parkinson's disease or multiple sclerosis. 7 the conditions are more likely to be worsened in covid-19 patients with pre-existing neurological disorders. from the evidence accumulated in various studies and the very nature of covs possessing neuroinvasive properties, the neurological manifestations of covid-19 cannot be ignored. those patients reporting early neurological symptoms, like loss of taste or smell or even seizures, must be tested for covid-19 and kept under constant observation of neurologists as we know the latency period may be enough for the virus to completely annihilate the medullary neurons and threaten the life of the patient. 4 also, more autopsies of not only the lungs but brains need to be performed in order to gain deeper insights into the neurotrajectory of covid-19 infection. in this hour of great distress, therapeutic interventions from scientists across the globe are needed in order to fight this pandemic ( figure 2) . as discussed at the beginning of this viewpoint, we have made good progress in understanding the key interactions between the hace2 and the rbd of the spike protein in sars-cov-2. 3 hence, we could utilize that to develop some good antiviral intervention strategies. some antibody drugs could be designed following the epitopes on the sars-cov-2 rbd. also, rbd can itself be used to test its efficacy as a subunit vaccine. 3 since the nature of the interactions between rbd and hace2 are protein−protein interactions, designing of peptide-based therapeutics could also be explored (figure 2 ). 9 many more such structure-guided therapeutics like small molecules or peptides that interfere with the receptor recognition of the virus could possibly halt the progression of the disease (figure 2 ). 9 in fact, inhibition of transmembrane serine protease 2 (tmprss2), the surface protease that cleaves the viral spike protein to unleash the full infective potential of the virus, also known as the priming of the virus, was once considered to be an effective therapeutic intervention to stop the viral spread only later to be dropped owing to the unknown physiological impact of tmprss2 blockade. 7 also patients with reported hyperinflammation caused by the cytokine storm should be administered appropriate steroids, selective cytokine blockers like atlizumab, antibody therapy, and inhibitors of the jak pathway. 6 ■ the geographical topology of covid-19 pandemic another important feature, which we must consider before deploying treatment modalities to patients, is the genetic predisposition and susceptibility of the patient under consideration, which may vary at the population level across variable geographical locations. a recent study published before the massive outbreak of sars-cov-2 in europe and the united states of america (usa) tried to screen for ace2 mutants that would resist attachment of the s-protein of the virus in different populations but was not able to find any direct evidence. but thereafter, as covid-19 slowly engulfed the entire world, a new study has indicated variations observed in the spike surface glycoprotein (ala930val) in the indian sars-cov-2 strain as compared to the strains from the usa, italy, wuhan, and nepal. 10 the same study also reported the presence of an antiviral mirna, has-mir-27b, only unique to the indian host population, which surprisingly even binds to the mutated region of the indian sars-cov-2 strain. 10 mir-27b is known to inhibit hiv-1 replication and is widely regarded as an antiviral mirna. the above claims of the study are further corroborated by the failure of anti-hiv regimens to cure patients in china while the same anti-hiv drugs seem to have worked in some indian patients. along with this, it is now being widely speculated by many that widespread bacillus calmette−gueŕin (bcg) vaccination in countries like india may have boosted the immunity of the country's thriving population and may be now serving as a protective shield against the widespread covid-19 attack across the world. countries without such universal policies on bcg vaccination like italy, the u.s., or the netherlands are among the worst hit by covid-19 infection. unarguably such claims require more elaborate scientific proof to be accepted as facts, but ethnicity and regional diversity do play a role in responding to such pandemics that can be affirmed without doubt. this only highlights the need for more genetic screening and populationbased genome-wide studies in divergent geographical regions in order to better understand the host−pathogen interactions in a region-specific manner, which could pave the way for the genesis of more region-specific therapeutics and treatment regimens. hence, taken together the neurological consequences of covid-19 revealed over the last few days of the outbreak, patients reporting altered mental health or an inability to taste or smell can no longer be ignored and must be tested for covid-19 without fail. moreover, these patients or patients with existing neurological conditions should be monitored closely so that the symptoms do not aggravate over the subsequent days of the infection. this strategy could also be considered as a preliminary screening strategy in case of large scale community transmission of covid-19. the prevailing health care units taking care of infected patients must now include neurologists to gain more perspective into the nature of the infections, which have a high chance of turning out to be neurological. meanwhile, more autopsies on brains of covid-19 patients with neurological symptoms need to be performed to establish the neuroinfection track of the disease so that appropriate measures can be taken. along with these more engrossing therapeutic interventions ranging from passive antibody therapies to sars-cov-2, structure-guided molecular design needs to galvanize the entire scientific community. corresponding author email: sghosh@iitj.ac.in authors gaurav das − organic and medicinal chemistry and structural biology and bioinformatics division thanks mhrd and iit jodhpur for their fellowships. s.g. kindly acknowledges serb india (crg/2019/000670) for financial support and iit jodhpur for initiation grant and infrastructure coronavirus disease (covid-19) pandemic. world health organization evidence of the covid-19 virus targeting the cns: tissue distribution, host−virus interaction, and proposed neurotropic mechanisms structural basis of receptor recognition by sars-cov-2 the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients covid-19-associated acute hemorrhagic necrotizing encephalopathy: ct and mri features covid-19: consider cytokine storm syndromes and immunosuppression letter to the editor regarding the viewpoint "evidence of the covid-19 virus targeting the cns: tissue distribution, host−virus interaction, and proposed neurotropic mechanism does covid19 infect the brain? if so, smokers might be at a higher risk perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease-19 (covid-19). asian pac comparative analyses of sar-cov2 genomes from different geographical locations and other coronavirus family genomes reveals unique features potentially consequential to host-virus interaction and pathogenesis. biorxiv key: cord-327690-di7hfghi authors: yang, xiaobo; yu, yuan; xu, jiqian; shu, huaqing; xia, jia'an; liu, hong; wu, yongran; zhang, lu; yu, zhui; fang, minghao; yu, ting; wang, yaxin; pan, shangwen; zou, xiaojing; yuan, shiying; shang, you title: clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study date: 2020-02-24 journal: lancet respir med doi: 10.1016/s2213-2600(20)30079-5 sha: doc_id: 327690 cord_uid: di7hfghi background: an ongoing outbreak of pneumonia associated with the severe acute respiratory coronavirus 2 (sars-cov-2) started in december, 2019, in wuhan, china. information about critically ill patients with sars-cov-2 infection is scarce. we aimed to describe the clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia. methods: in this single-centered, retrospective, observational study, we enrolled 52 critically ill adult patients with sars-cov-2 pneumonia who were admitted to the intensive care unit (icu) of wuhan jin yin-tan hospital (wuhan, china) between late december, 2019, and jan 26, 2020. demographic data, symptoms, laboratory values, comorbidities, treatments, and clinical outcomes were all collected. data were compared between survivors and non-survivors. the primary outcome was 28-day mortality, as of feb 9, 2020. secondary outcomes included incidence of sars-cov-2-related acute respiratory distress syndrome (ards) and the proportion of patients requiring mechanical ventilation. findings: of 710 patients with sars-cov-2 pneumonia, 52 critically ill adult patients were included. the mean age of the 52 patients was 59·7 (sd 13·3) years, 35 (67%) were men, 21 (40%) had chronic illness, 51 (98%) had fever. 32 (61·5%) patients had died at 28 days, and the median duration from admission to the intensive care unit (icu) to death was 7 (iqr 3–11) days for non-survivors. compared with survivors, non-survivors were older (64·6 years [11·2] vs 51·9 years [12·9]), more likely to develop ards (26 [81%] patients vs 9 [45%] patients), and more likely to receive mechanical ventilation (30 [94%] patients vs 7 [35%] patients), either invasively or non-invasively. most patients had organ function damage, including 35 (67%) with ards, 15 (29%) with acute kidney injury, 12 (23%) with cardiac injury, 15 (29%) with liver dysfunction, and one (2%) with pneumothorax. 37 (71%) patients required mechanical ventilation. hospital-acquired infection occurred in seven (13·5%) patients. interpretation: the mortality of critically ill patients with sars-cov-2 pneumonia is considerable. the survival time of the non-survivors is likely to be within 1–2 weeks after icu admission. older patients (>65 years) with comorbidities and ards are at increased risk of death. the severity of sars-cov-2 pneumonia poses great strain on critical care resources in hospitals, especially if they are not adequately staffed or resourced. funding: none. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pneumonia is a newly recognised illness that has spread rapidly throughout wuhan (hubei province) to other provinces in china and around the world. [1] [2] [3] [4] as of feb 19, 2020, the total number of patients has risen sharply to 74 283 in the mainland of china, with 2009 (2·7%) deceased. the clinical spectrum of sars-cov-2 pneumonia ranges from mild to critically ill cases. previous studies have only described the general epidemiological findings, clinical presentation, and clinical outcomes of patients of sars-cov-2 pneumonia. 1, 2, 5 however, specific information characterising critically ill patients remains unknown. the data on the clinical characteristics and outcomes of critically ill patients with sars-cov-2 infection are scarce, but are of paramount importance to reduce mortality. in this study, we investigated critically ill patients with confirmed sars-cov-2 pneumonia who were admitted to wuhan jin yin-tan hospital. the baseline sars-cov-2-associated morbidity and mortality data from this study will be of considerable value for the early identification of individuals who are at risk of becoming critically ill and who are most likely to benefit from intensive care treatment. this single-centre, retrospective, observational study was done at wuhan jin yin-tan hospital (wuhan, china), which is a designated hospital to treat patients with sars-cov-2 pneumonia. all patients, except infected healh-care workers from jin yin-tan hospital, were transferred from other hospitals. we retro spectively analysed patients from dec 24, 2019, to jan 26, 2020, who had been diagnosed with sars-cov-2 pneumonia, according to who interim guidance, and who were critically ill. 6 laboratory confirmation of sars-cov-2 infection was performed by the local health authority as previously described. 1, 2 critically ill patients were defined as those admitted to the intensive care unit (icu) who required mechanical ventilation or had a fraction of inspired oxygen (fio 2 ) of at least 60% or more. [7] [8] [9] identification of critically ill patients was achieved by reviewing and analysing admission logs and histories from all available electronic medical records and patient care resources. for patients who were alive by jan 26, 2020, their living status was confirmed on feb 9, 2020. the ethics commission of jin yin-tan hospital approved this study (ky-2020-06.01). written informed consent was waived due to the rapid emergence of this infectious disease. we reviewed clinical electronic medical records, nursing records, laboratory findings, and radiological examinations for all patients with laboratory confirmed sars-cov-2 infection. the admission data of these patients were collected. data were evaluated and collected, by using a case record form modified from the standardised international severe acute respiratory and emerging infection consortium case report forms. 10 any missing or uncertain records were collected and clarified through direct communication with involved health-care providers and their families. we collected data on age, sex, exposure history, chronic medical histories (chronic cardiac disease, chronic pulmonary disease, cerebrovascular disease, chronic neurological disorder, diabetes, malignancy, dementia, malnutrition, and smoking), symptoms from onset to hospital admission (fever, cough, dyspnoea, myalgia, malaise, rhinorrhoea, arthralgia, chest pain, headache, and vomiting), vital signs at icu admission (heart rate, respiratory rate, blood pressure), laboratory values on admis sion (haemoglobin concentration, lymphocyte count, platelet count, arterial blood gas analysis, fio 2 , partial pressure of oxygen (pao 2 ), and lactate concen tration), coexisted infection, treatment (oxygen therapy, vaso constrictive agents, antiviral agents, antibacterial agents, cortico steroids, and immuno globulin), as well as living status. during the outbreak of sars-cov-2 infection, the number of critically ill patients exceeded the capacity of icus. therefore, two provisional icus were urgently established in jin yin-tan hospital and hence most mechanical ventilator settings and recordings were not recorded, except records of positive end-expiratory pressure in some cases. as a routine, electronic medical data were archived onto a local server, from which we retrieved these data. the primary outcome was 28-day mortality after icu admission. secondary outcomes included incidence of sars-cov-2-related acute respiratory distress syndrome (ards) and the proportion of patients requiring mechanical ventilation. ards and shock were defined according to the guidance of who for novel coronavirus disease 2019 (covid-19). 6 acute kidney injury was identified on the basis of serum creatinine. 11 cardiac injury was diagnosed if the serum concentration of hypersensitive cardiac troponin i (hstni) was above the upper limit of the reference range (>28 pg/ml), measured in the laboratory of jin yin-tan hospital. the aim of this study is to report the clinical courses and clinical outcomes of critically ill patients being cared for evidence before this study the novel coronavirus disease 2019 is a disease that has affected populations around the world. we searched pubmed for articles published up to feb 11, 2020, using the keywords "2019 novel coronavirus", "2019-ncov", "covid-19", or "sars-cov-2". we identified eight articles that describe the epidemiological and clinical characteristics of patients infected with severe acute respiratory syndrome coronavirus 2 (sars-cov-2). however, none of these studies focused on characterising critically ill patients infected with sars-cov-2, who are at increased risk of death. we report the clinical courses and clinical outcomes of 52 critically ill patients from 710 laboratory-confirmed cases of sars-cov-2. 35 (67%) patients had acute respiratory distress syndrome (ards) and 37 (71%) required mechanical ventilation. 32 (61·5%) patients had died at 28 days, and the median duration from intensive care unit (icu) admission to death was 7 (iqr [3] [4] [5] [6] [7] [8] [9] [10] [11] the mortality of critically ill patients with sars-cov-2 pneumonia at 28 days is considerable. the survival time of nonsurvivors is likely to be within 1-2 weeks after icu admission. older patients (>65 years) with comorbidities and ards are at increased risk of death. the severity of sars-cov-2 pneumonia poses great strain on critical care resources in hospitals, especially if they are not adequately staffed or resourced. in the hospital during the study period. there were, therefore, no formal hypotheses being implemented to drive the sample size calculation and we included the maximum number of patients who met the inclusion criteria. we expressed descriptive data as mean (sd) or median (iqr) for continuous variables and number (%) for categorical variables. we assessed differences between survivors and non-survivors using two-sample t test or wilcoxon rank-sum test depending on parametric or nonparametric data for continuous variables and fisher's exact test for categorical variables. we used a kaplan-meier plot for survival data. tests were two-sided with significance set at α less than 0·05. the stata/ic 15.1 software (statacorp, college station, tx, usa) was applied for all analyses. by jan 26, 2020, 710 patients had been admitted to wuhan jin yin-tan hospital with confirmed sars-cov-2 pneumonia, of whom 658 (93%) were considered ineligible, including three patients who had cardiac arrest immediately after admission. 52 (7%) critically ill patients were included in this study (figure 1). all patients were residents of wuhan city and were transferred from other hospitals. the mean age was 59·7 years (sd 13·3), and 27 (52%) were older than 60 years (table 1). 35 (67%) patients were men. 17 (33%) patients had a history of exposure to the huanan seafood market, and 10 (19%) had exposure to patients with confirmed or highly suspected sars-cov-2 infection. 21 (40%) patients had chronic diseases, including cerebrovascular diseases in seven (13·5%) patients, all of whom died at 28 days. all patients had bilateral infiltrates on chest x-ray. the most common symptoms were fever (98%), cough (77%), and dyspnoea (63·5%; table 2). among 52 critically ill patients, six (11%) did not experienced fever until 2-8 days after the onset of symptoms related to sars-cov-2 infection. the median duration from onset of symptoms to radiological confirmation of pneumonia was 5 (iqr 3-7) days. the median duration from onset of symptoms to icu admission was 9·5 (7·0-12·5) days. the median acute physiology and chronic health evaluation ii (apache ii) score of all patients was 17 (iqr 14-19; table 3). most patients had organ function damage, including 35 (67%) with ards, 15 (29%) with acute kidney injury, 12 (23%) with cardiac injury, 15 (29%) with liver dysfunction, and one (2%) with pneumothorax (table 2). the median hstni was 161·0 (iqr 41·8-766·1) pg/ml. hospital-acquired infection was noted in seven (13·5%) patients, including one (2%) patient who had pulmonary and blood stream infection of carbapenem data are n (%) or mean (sd), unless otherwise specified. sars-cov-2=severe acute respiratory syndrome coronavirus 2. *patients who have confirmed sars-cov-2 infection or are highly suspected of being infected. of the 20 patients who survived, eight patients were discharged. three patients were still on invasive ventilation at 28 days, including one patient who was also on ecmo. one patient was on non-invasive ventilation, two were using high-flow nasal cannula, and six were using common nasal cannula. compared pneumonia or from onset of symptoms to icu admission were different between survivors and non-survivors (table 3) . the ratio of partial pressure of oxygen (pao 2 ) to fio 2 was significantly lower in non-survivors. based on apache ii score and sofa score at icu admission, non-survivors were in a more critical condition than survivors. in the cohort, lymphocytopenia occurred in 44 (85%) patients, with no significant difference between the two groups. compared with survivors, non-survivors were more likely to develop ards, and to receive mechanical ventilation, either invasively or noninvasively. we report on 52 critically ill patients with con firmed sars-cov-2 infection, characterised by severe hypoxaemia. 32 (61·5%) of critically ill patients had died at 28 days. of all included patients, 37 (71%) required mechanical ventilation and 35 (67%) had ards. since no specialised medication to treat sars-cov-2 infection has been identified at this time, the mainstay of treatment has been supportive care. patients are being treated in isolation, and their close contacts are being quarantined. for non-critically ill patients, close followup is likely to be sufficient to manage the disease. 1-3 for critically ill patients, however, aggressive treatments and intensive care are needed. to our knowledge, this is the first study to characterise critically ill patients infected by sars-cov-2. in three previously published studies of crtically ill patients, the patient numbers were too small to summarise the characteristics and mortality of these patients with sars-cov-2 pneumonia. 1, 2, 5 like sars-cov and middle eastern respiratory syndrome (mers)-cov, sars-cov-2 is a coronavirus that can be transmitted to humans, and these viruses are all related to high mortality in critically ill patients. 12 however, the mortality rate in patients with sars-cov-2 infection in our cohort is higher than that previously seen in critically ill patients with sars. in a cohort of 38 critically ill patients with sars from 13 hospitals in canada, 29 (76%) patients required mechanical ventilation, 13 (43%) patients had died at 28 days, and six (16%) patients remained on mechanical ventilation. 8 17 (38%) of 45 patients and 14 (26%) of 54 patients who were critically ill with sars infection were also reported to have died at 28 days in a singapore cohort 13 and a hong kong cohort, 14 respectively. the mortality rate in our cohort is likely to be higher than that seen in critically ill patients with mers infection. in a cohort of 12 patients with mers from two hospitals in saudi arabia, seven (58%) patients had died at 90 days. 15 since the follow-up time is shorter in our cohort, we postulate that the mortality rate would be higher after 28 days than that seen in patients with mers-cov. the fundamental pathophysiology of severe viral pneumonia is severe ards. men and people of an older age (>65 years) are more likely to develop ards than women or those of a younger age. 16 therefore, it is reasonable that the mortality at 28 days of severe sars-cov-2 pneumonia is similar to the mortality of severe ards, which is near 50%. 17 with a substantial increase in the number of critically ill patients infected by sars-cov-2, more provisional icus are being established in wuhan, china. qualified specialists are coming to wuhan from other provinces of china and are currently working in these provisional icus, fever clinics, and newly constructed hospitals. 18 as the clinical capacity to treat patients improves, the mortality of critically ill patients with sars-cov-2 pneumonia is expected to decrease. as mentioned in previous studies, nearly 70% of patients infected by sars-cov-2 were men. 1, 2 the patients are older in our study than in previous studies. 1, 2 we observed that non-survivors were older than survivors. based on previous studies, evidence suggests that older, male patients are the most susceptible to sars-cov-2 infection, 2 which is supported by our data. as previously reported, patients with a history of cerebrovascular disease are at increased risk of becoming critically ill or dying if they have sars-cov-2 infection. 2, 5 in our cohort, fever is the most common symptom in patients with sars-cov-2 pneumonia, which is in accordance with previous studies, but not all patients had fever. 1, 2, 5 we also found that fever was not detected at the onset of illness in six (11·5%), and that it was in fact detected 2-8 days later. the delay of fever manifestation hinders early identification of patients infected with sars-cov-2-if patients are asymptomatic identification of suspected cases is more difficult. 19, 20 the median duration from onset of symptoms to radiological confirmation of pneumonia was 5 (3-7) days, meaning that early or repeated radiological examinations are useful in screening patients with sars-cov-2 pneumonia. 4 as for laboratory tests, lymphocytopenia occurred in more than 80% of critically ill patients in our cohort. lymphocytopenia is a prominent feature of critically ill patients with sars-cov infection because targeted invasion by sars-cov viral particles damages the cytoplasmic component of the lymphocyte and causes its destruction. 21 additionally, lymphocytopenia is also common in the critically ill patients with mers infection, which is the result of apoptosis of lymphocytes. 22, 23 therefore, we postulate that necrosis or apoptosis of lymphocytes also induces lymphocytopenia in critically ill patients with sars-cov-2 infection. in a previous study, mainly in non-critical patients infected with sars-cov-2, 35% of patients had only mild lymphocytopenia, 2 suggesting that the severity of lymphocytopenia reflects the severity of sars-cov-2 infection. mechanical ventilation is the main supportive treatment for critically ill patients. the pao 2 /fio 2 ratio was lower in our patients than in patients admitted to zhongnan hospital. 5 the substantial difference in pao 2 /fio 2 ratio between survivors and non-survivors in our study, indicates this ratio is associated with the severity of illness and thus prognosis. barotrauma seems less severe in patients with sars-cov-2 infection who are being mechanically ventilated than that seen in mechanically ventilated patients with sars-cov. in our study, barotrauma occurred in only one (2%) patient, who had been hospitalised for nearly 1 month, and he is currently on a ventilator and receiving ecmo. in patients with sars, barotrauma occurred in about 25% of patients on mechanical ventilation. 14 the lower occurrence of barotrauma in our cohort is probably related to the widely accepted strategy of protective ventilation in mainland china. 24 at the same time, prone position and ecmo have been used to treat patients with sars-cov-2 pneumonia. without solid evidence, nearly half of the patients were given antiviral agents, and more than half were given intravenous glucocorticoids. patients treated with lopinavir were from an ongoing clinical trial registered on chinese clinical trial registry (chictr2000029308). remdesivir was given to the first patients with sars-cov-2 pneumonia in the usa. 4 trials on remdesivir are about to recruit both mild to moderate patients (nct04252664) and severe patients (nct04257656) infected with sars-cov-2. although, intravenous glucocorticoids were commonly used in patients with severe sars or mers pneumonia, their efficacy remains controversial and their use to treat sars-cov-2 infection is also controversial. 5, 25 an ongoing clinical trial (nct04244591) might shed some light on the safety and efficacy of these drugs as treatment. this study has several limitations. first, only 52 critically ill patients were included. however, the population from which they were sampled was much larger than that of the three studies previously published. 1, 2, 5 we included all the critically ill patients being cared for in the icu of jin yin-tan hospital who met the inclusion criteria. due to the exploratory nature of the study, which was not driven by formal hypotheses, the sample size calculation was waived. instead, we hope that the findings presented here will encourage a larger cohort study or potentially some randomly controlled trials. second, some specific information from the icu was missing, such as mechanical ventilation settings. the data on radiographical examination, supportive treatment, living status, and the duration from icu admission to death, however, are indisputable. third, this is a retrospective study. the data in this study permit a preliminary assessment of the clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia. further studies are still needed. in conclusion, the mortality of critically ill patients with sars-cov-2 pneumonia is high. the survival term of the non-survivors is likely to be within 1-2 weeks after icu admission. older patients (>65 years) with comorbidities and ards are at increased risk of death. the severity of sars-cov-2 pneumonia poses great strain to hospital critical care resources, especially if they are not adequately staffed or resourced. xy, yy, jxu, hs, hl, and ys collected the epidemiological and clinical data. jxi, ywu, lz, zy, mf, and ty summarised all data. xy, yy, hl, jxi, ywa, sp, and ys drafted the manuscript. xz and sy revised the final manuscript. we declare no competing interests. after publication, the data will be made available to others on reasonable requests to the corresponding author. a proposal with detailed description of study objectives and statistical analysis plan will be needed for evaluation of the reasonability of requests. additional materials might also be required during the process of evaluation. deidentified participant data will be provided after approval from the corresponding author and wuhan jin yin-tan hospital. clinical features of patients infected with 2019 novel coronavirus in wuhan, china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a novel coronavirus from patients with pneumonia in china first case of 2019 novel coronavirus in the united states clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical 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limitations date: 2020-06-14 journal: new microbes new infect doi: 10.1016/j.nmni.2020.100713 sha: doc_id: 319184 cord_uid: voc0eqb9 abstract the ongoing pandemic of sars-cov-2 is a one of the most devastating outbreaks witnessed in the last 100 years. the outbreak started in china's hinterland and spread rapidly to almost every country culminating in woefully overwhelmed healthcare systems in most countries. the only approved diagnostic test to accompany radiographic evaluation is the reverse-transcriptase pcr. however, the applicability of this test in diagnosis and surveillance is challenged by global shortage in reagents and unavailability of well-equipped laboratories with specialized staff in several lowand middle-income countries. the need for development of accurate and rapid diagnostic assays became apparent. handful of immunodiagnostic tests and other molecular approaches were developed and tested. other recently developed point-of-care molecular tests are expected to be helpful in pandemic management since no particular skills are required from the operator. fortunately, handful of serological tests have granted authorization to be used under emergency situation by fda in diagnosis of sars-cov-2. medically important coronaviruses were identified from nasal secretions of patients with mild rdrp/hel (rna-dependent rna polymerase/helicase). of note, a recent study found 146 nucleocapsid n2 and envelope e genes to be the most sensitive singleplex reactions and no 147 significant change in cycle threshold (ct) was noted when both assays were combined [44] . the this work is a personal effort and did not receive any specific grant from funding agencies in the 269 public, commercial, or not-for-profit sectors. author declares the absence of any potential or actual conflict of interest. the specimen containing the antigen (analyte) is placed on the sample pad. the antigen, with the fluid, 557 moves to the conjugate pad where it is bound by a labelled antibodies specific to the targeted antigen. the 558 conjugate pad also contains labelled antibodies non-specific to the antigen to be detected. antigen-559 antibody complexes migrate through the nitrocellulose membrane and reach the "test line" area. in such 560 area, antigen-specific antibodies are immobilized to catch the antigen-antibody complexes. when antigen-561 antibody complexes accumulate in this area, the line become visible to the unaided eye. the non-specific 562 antibodies also migrate and surpass the "test line" to the "control line" area and the line also become consistent 367 detection of 2019 novel coronavirus in saliva enteric involvement of coronaviruses: is faecal-oral 370 transmission of sars-cov-2 possible? evaluation of saline, phosphate buffered saline and minimum essential medium as 374 potential alternatives to viral transport media for sars-cov-2 testing centers for disease control and prevention. cdc real-time rt-pcr diagnostic panel molecular diagnosis 379 of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia detection of viral 382 pathogens by reverse transcriptase pcr and of microbial indicators by standard 383 methods in the canals of the florida keys pitfalls of quantitative real-time reverse-transcription polymerase 385 chain reaction laboratory testing for covid-19 molecular diagnosis 391 of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia detection of 394 2019 novel coronavirus (2019-ncov) by real-time rt-pcr clinical 397 evaluation of the cobas sars-cov-2 test and a diagnostic platform switch during 48 398 hours in the midst of the covid-19 pandemic laboratory readiness and response for novel coronavirus (2019-ncov) in expert 402 laboratories in 30 eu/eea countries molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-406 19-rdrp/hel real-time reverse transcription-pcr assay validated in vitro real-time rt-pcr for severe acute respiratory syndrome coronavirus 2 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detection of asian-and african-lineage zika viruses using reverse transcription loop-mediated isothermal amplification combined with a avian-origin influenza a (h7n9) virus by reverse transcription loop isothermal amplification combined with a lateral-flow device rapid and visual detection of 2019 458 novel coronavirus (sars-cov-2) by a reverse transcription loop-mediated isothermal 459 amplification assay transcription loop-mediated isothermal amplification assays targeting sars-cov-2 transcription loop-mediated isothermal amplification method for rapid detection of 467 sars-cov-2 development of a reverse 469 transcription-loop-mediated isothermal amplification as a rapid early-detection method for 470 novel sars-cov-2 rt-lamp) diagnostic platform a novel reverse transcription loop-477 mediated isothermal amplification method for rapid detection of sars-cov-2 clinical applications of crispr-based genome 480 editing and diagnostics cutting-edge infectious disease diagnostics with 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from sars and mers epidemic profile of specific antibodies to sars-cov-2: the first report evaluation of commercial and automated sars-cov-2 igg and iga elisas using 522 coronavirus disease (covid-19) patient samples serological immunochromatographic 525 approach in diagnosis with sars-cov-2 infected covid-19 patients antibody testing for covid-19: a report from the national covid scientific advisory 529 development of a 531 prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus 532 infection key: cord-297332-rzf0cw1x authors: wang, qidi; zhang, lianfeng; kuwahara, kazuhiko; li, li; liu, zijie; li, taisheng; zhu, hua; liu, jiangning; xu, yanfeng; xie, jing; morioka, hiroshi; sakaguchi, nobuo; qin, chuan; liu, gang title: immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates date: 2016-04-11 journal: acs infectious diseases doi: 10.1021/acsinfecdis.6b00006 sha: doc_id: 297332 cord_uid: rzf0cw1x [image: see text] severe acute respiratory syndrome (sars) is caused by a coronavirus (sars-cov) and has the potential to threaten global public health and socioeconomic stability. evidence of antibody-dependent enhancement (ade) of sars-cov infection in vitro and in non-human primates clouds the prospects for a safe vaccine. using antibodies from sars patients, we identified and characterized sars-cov b-cell peptide epitopes with disparate functions. in rhesus macaques, the spike glycoprotein peptides s(471–503), s(604–625), and s(1164–1191) elicited antibodies that efficiently prevented infection in non-human primates. in contrast, peptide s(597–603) induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (esd) mechanism. this peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (s(597–603) and s(604–625)) and a long-term human b-cell memory response with antisera from convalescent sars patients. thus, peptide-based vaccines against sars-cov could be engineered to avoid ade via elimination of the s(597–603) epitope. we provide herein an alternative strategy to prepare a safe and effective vaccine for ade of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection. a novel severe acute respiratory syndrome coronavirus (sars-cov) was characterized in march 2003 after a global effort following the first epidemiological case in november 2002. 1 ten years later (2012), a novel middle east respiratory syndrome (mers) coronavirus emerged, which was a βcoronavirus, similar to sars-cov. 2 sars is a deadly infectious disease and has the potential to seriously threaten public health and socioeconomic stability worldwide. 1 serologic and genetic investigations indicate that sars-cov is of zoonotic origin, 3, 4 with bats as the likely animal reservoir. since the identification of sars-cov in 2003, there have been major advances in understanding sars-cov genetics 1, 5, 6 and molecular epidemi-ology 7 and in identification of host receptors 8, 9 and t-cell epitopes. 10, 11 models in mice, ferrets, and monkeys have been established, each with particular strengths and weaknesses. 12 vaccine candidates have included dna, a live attenuated strain, recombinant proteins, inactivated whole virions, and vector vaccines; 13 however, none have been approved through clinical investigation. 14 safety concerns about a sars-cov vaccine have been raised, given the observation in vitro of antibody-dependent enhancement (ade) of sars-cov infection that could, in theory, exacerbate disease. 15 other observations include evidence of ade reported here for the first time induced by an inactivated sars-cov vaccine in rhesus macaques ( figure 1 ) and by antisera from sars patients (table s1) , as well as ade in other coronavirus infections. 16−18 ade in vitro has been observed for many other viruses, including yellow fever virus, west nile virus, human immunodeficiency virus (hiv), ebola virus, respiratory syncytial virus (rsv), and influenza a virus. 19 −21 hiv-induced ade has been described, 22, 23 but the exact mechanism is still uncertain. 24 among other examples of ade, secondary infection with dengue virus of a heterologous serotype has been associated with an immunopathologic vascular leakage and hemorrhagic syndrome, dengue hemorrhagic fever/dengue shock syndrome (dhf/ dss). 25 most descriptions of ade relate to fc-gamma receptor (fcγr) and/or complement components promoting viral uptake and virus replication pathways. 26−28 goncalvez et al. reported that treatment of juvenile rhesus monkeys with the cross-reactive mab 1a5, which recognizes the fusion loop in dii of dengue virus, enhanced dv4 viremia by several logs within 3−6 days of infection. 26 furthermore, a 9-amino acid (aa) deletion at the nterminus of the ch 2 domain in the fc region abrogated enhancement in vitro and in vivo, confirming that ade occurred through an fcγr pathway for this class of antibodies. another study analyzed the stoichiometric relationship between antibodymediated neutralization and enhancement of west nile virus (wnv) infection in cells expressing fcγr. 29 the results showed that there is an antibody occupancy threshold on the virion for neutralization or enhancement of virus infection. strongly neutralizing diii-lateral ridge-specific mabs inhibit at lower occupancy, whereas weakly neutralizing mabs that bind distinct epitopes require a much higher mab occupancy for neutralization. when mab occupancy falls below the threshold for neutralization, ade can occur. in subsequent studies, the same group showed that the level of antibody occupancy that promotes ade in vivo is also modulated by the binding of c1q, which restricts ade in an igg subclass-specific manner. 30 on the basis of these studies, many neutralizing antibodies may enhance infection in vitro in fcγr + cells when their concentrations fall below a key occupancy threshold, and some abs that poorly neutralize may strongly enhance over a wide dose−response range. however, for antibody isotypes that bind c1q avidly, much of the enhancing effect may be minimized in vivo. other studies suggest that fcγr-dependent ade may not be the only mechanism for antibody enhancement of infection. huang et al. 31 demonstrated ade in vitro of an anti-prm mab against denv in cell lines that lacked expression of fcγr (e.g., baby hamster kidney cell line bhk-21 and murine fibroblast cell linenih3t3). importantly, a peptide in the m3 region (cpflkqnepedidcw) of prm blocked this ade in a dosedependent manner. the antibody appeared to have dual specificity and bound to dengue virus virions and/or a crossreactive hsp60 protein on the cell surface. thus, ade via non-fcγr-or complement-dependent mechanisms is plausible for denv, yet poorly characterized in vitro and in vivo. herein, we discovered that a peptide of the viral sequence simultaneously elicits the antibodies of disparate functions in protection and enhancement against sars-cov infection by the studies with host vero e6 cells in vitro and in non-human primates. vaccine in the lungs of macaques. first, we observed the ade of sars-cov infection in lungs after immunization with the inactivated virus vaccine in macaques. the monkeys with or without the immunization of inactivated sars-cov vaccine were infected by nasal cavity inoculation of live sars-cov virions ( figure 1 ). infected macaques showed lungs with broadened interval and visible macrophage infiltration with alveolar epithelial hyperplasia. however, ade macaques after vaccination showed lungs with broadened interval, lung interval fractured with large amounts of macrophage and lymphocyte infiltration, visible fibrin, and protein-rich edema in the alveolar cavity. protected macaques showed lungs with slightly . the animals were then challenged with nasal cavity inoculation of sars-cov (1 × 10 6 tcid 50 in 4.0 ml/monkey, pumc01 sars-cov strain) 14 days after the boost. the animals were sacrificed 6 days after viral challenge, and lung tissues were sampled for general and pathological observations. pathological examination procedures were as described in ref 37. infected macaque: lung interval broadened, visible macrophage infiltration with alveolar epithelial hyperplasia. ade macaque: lung interval broadened, lung interval fractured with large amounts of macrophage and lymphocyte infiltration, visible fibrin and protein-rich edema in alveolar cavity. protected macaques: lung interval slightly broadened, without visible abnormalities. the arrows indicate the lung lesions of infected and ade animals. broadened intervals without visible abnormalities. the results implied that the simple vaccination with inactivated whole sars-cov virions might be ineffective for protection of virus infection. identification of highly immunodominant b-cell peptides of sars-cov in humans. mapping highly immunoreactive human b-cell peptides by employing sars patient antisera was carried out by a number of research groups. consistent with previous reports, jiang's laboratory reported that the m1−131 and m132−162 peptides 32 from the membrane protein and the n153−178 and n362−412 peptides from the n protein 33 were highly reactive with all convalescent-phase test sera from sars patients. the laboratories of wu and che collaboratively identified three conformational (amino acids 1− 69, 68−213, and 337−422) and three linear (amino acids 1−69, 121−213, and 337−422) epitopes on the full-length n protein that were immunodominant. 34 using a "split and mix" combinatorial strategy, we synthesized an overlapping peptide library spanning all four structural proteins of the sars-cov bj01 strain, including the envelope (e), membrane (m), nucleocapsid (n), and spike (s). we screened this peptide library with antisera from convalescent sars patients and identified dominant peptide epitopes recognized by anti-sars igg. 35 further experiments in this paper indicate that three peptides in the s protein (s 471−503 , s 604−625 , and s 1164−1191 ) were strongly bound by sars-cov-specific human igg from 12.8, 32.6, and 13.9%, respectively, of the 470 sars patients ( table 1) . extension of the peptides (table s2 ) to further characterize the epitopes' n-and c-termini identified a new immunodominant peptide (s 597−625 ), which was recognized by antisera from 64% of the patients ( table 1 ). the s 597−625 peptide specifically reduced the titer of human igg in convalescent sera from sars patients who reacted with sars-cov viral lysates ( figure 2a ). we studied sequentially collected sera from 21 convalescent sars patients whose sera (obtained 3 months after onset of infection) strongly reacted with the s 597−625 peptide. the antisera from these patients continued to recognize the s 597−625 at 12 months [12/21 (57.1%)], 18 months [7/21 (33.3%)], and 24 months [6/21 (28.6%)] after the onset of sars ( figure 2b ), implying that the s 597−625 peptide is a major immunodominant peptide in humans and elicits a long-term b-cell memory response after natural infection with sars-cov. epitope-specific antibodies of sars-cov exhibited enhancing or neutralizing functions in vitro. to study the functional significance of immunodominant peptides of the s glycoprotein, we generated 23 monoclonal antibodies (mabs, table s3 ) by immunization of mice. ten of these strongly bound to the sars-cov virion ( figure 3a ). among them, five blocked sars-cov infection of vero e6 cells ( figure 3b ), including mab4e5, mab4a10, and mab6g5 against s 471−503 , mab11b1 against s 604−625 , and mab126−10 against s 1164−1191 . in contrast, two mabs (mab43-3-14 and mab219-10-28) against s 597−625 , despite sharing some of the same isotypes and constant region configurations with neutralizing antipeptide antibodies (igg1/κ, igg2a/κ, and igg2b/κ; table 1 ), markedly enhanced sars-cov infection of vero e6 cells ( figure 3b ). this was particularly striking given that vero e6 cells lack fcγr, and the best-understood mechanism for ade involves fcmediated internalization and replication of virus. mab43-3-14table 1 . peptides recognized by sars-infected patients, relevant mouse mabs, and functional classification a a *, red-, blue-, and green-colored peptides represent the epitopes that were assembled into immunogenic peptides. ζ, neu (neutralizing) or ade indicates the ability of the individual mab to block or enhance, respectively, sars-cov infection of vero e6 cells. #, the reactivity of individual peptides with antisera from 470 convalescent sars patients (male, 192; female, 278) was determined using an elisa. commercially available elisa kits were used to confirm that 81% of the 470 antisera were positive against viral lysates as antigen (cutoff value = 0.1). the titer of each serum was averaged from duplicate wells. and mab219-10-28-enhanced vero e6 cell infection was reduced in a dose-dependent manner by s 597−625 , but not by an unrelated hbv peptide ( figure 4a ,b), whereas ade of human antisera from sars-cov-infected patients was also specifically blocked by the same peptide ( figure 4c ,d), implying that peptide-based ade occurred during the epidemic period of a sars outbreak. an antibody-peptide binding elisa indicated that mab11b1 against s 604−625 reacts with both s 597−625 and s 604−625 , whereas mab43-3-14 against s 597−625 only bound to s 597−625 ( figure 4e ). the data suggest that mab11b1 was induced by s 604−625 and is a consensus peptide of s 597−625 and s 604−625 , whereas mab43-3-14 was from the n-terminal peptide s 597−603 (lyqdvnc) immunizing antigen. synthesized peptides lyqdvn (s 597−602 ), lyqdvnc (s 597−603 ), and lyqdvnct (s 597−604 ) were all immunoreactive with the tested sera of 24 sars-positive patients, further confirming that the s 597−603 peptide is an immunodominant one in humans ( figure s1 ). alanine scanning mutagenesis of the s 597−604 peptide demonstrated that l597, y598, q599, d600, and c603 are critical amino acid residues for interaction with mab43-3-14 ( figure 4e ; table s4 ). the s 597−603 peptide lies close to the c-terminus of the sars-cov major receptor (ace2)-binding domain (rbd). 8 we speculate that mab43-3-14 may expose specific conformations that catalyze sars-cov attachment to and/or membrane fusion with target cells. neutralization or enhancement of sars-cov infection in non-human primates by epitope-specific antibodies. low-molecular-weight peptides behave like haptens, with relatively low immunogenicity. to make them more immunogenic, we chemically ligated four copies of each peptide to a lysine core [brch 2 co-lys 2 -(lys)-β-ala-conh 2 (brk 2 ka), scheme 1] to prepare a multiple antigen peptide system (map) 36 and purified them by preparative hplc ( figure 5a ; table s7 ). molecular weight was confirmed by lc-ms/ms ( figure 5b ). we used these synthetic maps to vaccinate rhesus monkeys (table s4) as follows: vac1, control group (received 0.9% nacl) of four macaques randomly assigned to two groups for day 2 or day 6 postinfection sacrifice; vac2, map-s 597−625 given to six macaques randomly assigned to two groups for day 2 or day 6 postinfection sacrifice; vac3, a mixture of map-s 471−503 , map-s 604−625 , and map-s 1164−1191 given to six macaques randomly assigned to two groups for day 2 or day 6 postinfection sacrifice; and vac4, a mixture of map-s 471−503 , map-s 597−625 , and map-s 1164−1191 given to six macaques randomly assigned to two groups for day 2 or day 6 postinfection sacrifice. the immunization protocol included one priming injection followed by three boosts at 2-week intervals (chart s1). antipeptide polyclonal abs (monkey igg) monitored by elisa showed high-level responses to all immunized peptides after three boosts ( figure 6 , left). antibodies ( figure 6a ) against the four peptides in sera from vac1-immunized animals were at background level on all blood collection days. vac2 gradually induced anti-s 597−625 peptide antibodies after three boosts. not surprisingly, both vac2 and vac4 immunizations elicited high levels of igg against the s 604−625 peptide because the vaccine components consisted of the s 597−625 or s 604−625 epitopes in these two animal groups. anti-s 604−625 and -s 1164−1191 antibodies in the vac3-immunized animal group reached their highest levels before the second boost (28 days after the first injection). the average titer of anti-s 604−625 igg was greater than 1:10 6 in vac3-immunized monkeys before sars-cov challenge and was 3-or 2-fold times that of vac2 or vac4, respectively. interestingly, when vac3-vaccinated animals were challenged by sars-cov, the igg level against s 604−625 gradually decreased ( figure 6b ) on days 2 and 6 postinfection. this is different from the vac2 and vac4 immunizations, after which igg levels inexplicably increased on day 2 postinfection and subsequently decreased on day 6 postinfection. this observation implies that anti-s 604−625 antibodies are strongly involved in the neutralization of sars-cov in monkeys and that the existing s 597−603 epitope (in vac2 and vac4) may reduce the total titer of anti-s 604−625 antibodies. all immunized macaques were subsequently challenged with sars-cov (pumc01 strain) via nasal inhalation on the 14th day after the last boost, as described. 37 at 2 and 6 days postinfection (dpi), the macaques were sacrificed (chart s1; table s7 ). the data for lung damage and viral burden in these macaques are summarized in table 2 . gross lung pathology was evident at both 2 and 6 dpi in all groups. in mock-immunized macaques (vac1), the average pathology grade was grade iv ( figure 7a ,b; table s5 ), with severe diffuse alveolar damage, including alveolar shrinking and rupture of elastic fibers. massive macrophage infiltration was associated with fusion of alveolar septa. pulmonary edema, fibrin, hemorrhage, and cellular debris contributed to the severe interstitial pneumonitis. damage was similar (grade iv) for vac2, somewhat reduced (grade ii−iii) for vac4, and markedly diminished (grade i−ii) for vac3. immunohistochemistry using mabs against the viral peptides revealed a large number of sars-cov-infected macrophages and alveolar epithelial cells in the vac1 group ( figure 7c ), averaging 13.5 ± 2.6 infected cells per 10 high-power fields (hpf) and 10.0 ± 3.0 infected cells per 10 hpf at 2 and 6 dpi, respectively. quantitative viral burden analysis by quantitative rt-pcr showed an average of 141,000 copies/mg lung tissue (2 dpi) and 136,000 copies/mg lung tissue (6 dpi) of sars-cov in the lungs of the vac1 group. compared with the vac1 group, the vac2 group showed similar histology at 2 dpi, but the interstitial pneumonia was far worse at 6 dpi, characterized by massive numbers of inflammatory cells in hemorrhagic septa, with extensive exudation. sars-cov-infected cells in the lungs averaged 13.8 ± 2.1 per 10 hpf at 2 dpi and 9.8 ± 3.0 per 10 hpf at 6 dpi, whereas viral copies were 116,000/mg lung tissue and 143,000/ mg lung tissue, respectively. thus, even though s 597−625 was immunodominant in patients, antigenic in monkeys, and reactive with the neutralizing mab 11b1, it was nonprotective and even harmful when used as a vaccine. in contrast, the immunized monkeys in the vac3 group had a strongly increased ability to control sars-cov infection in association with induction of high levels of anti-s 604−625 antibodies ( figure 7e ). at 2 dpi, hemorrhage in septa and elastic fibers of the alveolar wall were indicated by apparent inflammation in silver staining results. alveolus septa broadening with increasing infiltration of inflammatory cells were recorded by 6 dpi, indicating that only early, acute diffuse alveolar damage occurred. sars-cov-infected lung cells in the vac3 group averaged 7.4 ± 3.2 per 10 hpf (vs vac1 group: p < 0.01) and 7.0 ± 2.9 per 10 hpf (vs vac1 group: p < 0.01) at 2 and 6 dpi, respectively. the sars-cov burden averaged 6,200 copies/mg lung tissue at 2 dpi and 7,300 copies/mg lung tissue at 6 dpi. this represents a ratio of ∼19 and ∼20 between the number of sars-cov copies in the vac3 and vac1 groups at 2 and 6 dpi, respectively. for animals that received vac4, symptoms of acute diffuse alveolar damage were visible, including fusion of thick septa and ruptured elastic fibers of the alveoli. the average number of sars-cov infected cells in the lung tissues was 8.14 ± 3.32 per 10 hpf at 2 dpi (vs vac1 group: p < 0.01) and 7.8 ± 2.91 per 10 hpf at 6 dpi (vs vac1 group: p < 0.01). the sars-cov burden 46 the cutoff value was set to 0.1 (the average for binding of unrelated mab-hiv-p27). (b) neutralization or enhancement of the sars-cov infection in vero e6 cells by mabs (1.0 μg/ml) was specifically reduced by the corresponding immunopeptide (0.1 μg/ml), but not by a hepatitis b virus (hbv) peptide (0.1 μg/ml). hbv peptide (lldyqgmlpv) is an unrelated control peptide from an hbv surface protein. s 597−625 and hbv peptide were pre-incubated with the corresponding mabs or human antisera for 30 min at 4°c before function was tested. these experiments were performed in triplicate, and the data are presented as the mean ± standard deviation. averaged 31,254 copies/mg lung tissue at 6 dpi, that is, 4.5 times the burden of 6,835 copies/mg lung tissue at 2 dpi. the vac4 group also showed a 4.3-fold increase to vac3 group (31,254 copies/mg vs 7,300 copies/mg) of viral burden at 6 dpi, suggesting that the presence of igg against s 597−603 facilitated sars-cov infection of immunized macaques. in a separate experiment focused on the phenomenon of ade in macaques, rhesus monkeys were divided into six groups (n = 3 per group, table s9 ). three groups were separately sacrificed at 2 or 6 dpi; each time point included a control group and two groups that received the enhancing mab43-3-14 at doses of 0.2 mg/kg or 1.8 mg/kg 1 day prior to challenge with the sars-cov pumc01 strain. gross pathologic changes were again recorded in the control group at a grade iv level ( figure 8a,b) . although clear pathologic changes were observed in one of the three monkeys in the 0.2 mg/kg group, we concluded that previous treatment with a dose of 0.2 mg/kg mab43-3-14 did not, on average, significantly reduce or facilitate sars-cov infection. however, macaques treated with 1.8 mg/kg mab43-3-14 showed a marked increase in lung lesions. the lung lesion area in macaque d4-060060 (4.0 × 2.5 cm 2 ) at 6 dpi was the largest in all dic experimental groups. at 6 dpi, all lungs from the 1.8 mg/ kg group showed larger areas of necrosis, severe sheets of septa fusion, necrotic lesions at the hemorrhagic septa, and massive macrophage infiltration in the alveoli, indicating that the interstitial pneumonia was much more severe in the mab43-3-14-treated group than in the control group ( figure 8c ). there were more sars-cov-infected cells in the lung tissue (12.5 ± 2.3 per 10 hpf at 2 dpi and 13.4 ± 2.6 per 10 hpf at 6 dpi, p < 0.01 vs control group). the average sars-cov burden in the lungs was 911,000 copies/mg lung tissue at 2 dpi and 944,000 copies/mg lung tissue at 6 dpi, a 10-fold enhancement compared with the control group at 2 dpi and a 14-fold enhancement at 6 dpi ( figure 8d ). this result further confirmed that enhancement of the sars-cov infection in macaques was directly related to antibodies against s 597−603 . in this study, we reported for the first time that a sars-cov inactivated vaccine could induce ade and lung pathology in experimental rhesus monkeys. four antigenic peptides (s 471−503 , s 604−625 , s 597−625 , and s 1164−1191 ) from the spike protein of sars-cov were identified by high cross-reactivity with a large number of antisera from convalescent sars patients. ten of 23 mabs alanine scanning mutagenesis indicated that l597, y598, q599, d600, and/or c603 are critical amino acid residues and that s 597−603 is responsible for mab43-3-14-induced enhancement, which may catalyze sars-cov attachment and/or membrane fusion with target cells by exposing specific conformations of the spike protein. s 471−503 is located at the virus rbd 8 and can block sars-cov infection of vero e6 cells in vitro, 35 probably by interfering with high-affinity attachment of sars virions to the human ace2 receptor at residues t487 and n497 of the rbd. 38, 39 the coronavirus spike proteins have been characterized as class i fusion proteins containing highly conserved heptad repeat regions (hr1 and hr2). 40 thus, interfering with hr1 binding to hr2 has become a recent target for prevention of viral fusion and entry into target cells. s 1164−1191 overlaps hr2 (1145−1184 aa) in the sars-cov spike glycoprotein. the mechanism by which anti-s 1164−1191 antibodies reduce the efficiency of sars-cov fusion is likely to be associated with an exposed five-turn αhelix conformation via hr2 binding to hr1 and an extended conformation formed by residues 1160−1177 and 1178−1184 of three antiparallel hr2 helices in an oblique orientation surrounding a parallel, trimeric coiled-coil of three hr1 helices. 41 binding of mab581-39 to the sars-cov virion was the strongest among the mabs ( figure 3a ), which implies that s 1164−1191 peptide may act as a major immunoantigen. severe lung injury occurred in one challenged rhesus monkey that had been immunized with an inactivated sars-cov vaccine ( figure 1 ). very strong serologic reactivity of s 597−625 with antisera from 64.4% of convalescent sars patients was observed; the response was to two tandem epitopes (s 597−603 and s 604−625 ), but each epitope generated antibodies with disparate functions regarding neutralization or enhancement of sars-cov infection. thus, in persons infected by sars-cov, enhancing antibodies and neutralizing antibodies may partly counteract each other's functions. sars-cov's ade was also reported by other research laboratories. using an hl-cz human promonocyte cell line, chen and huang found that higher concentrations of antisera collected from sars-cov-infected patients facilitated sars-cov infection and induced higher levels of virus-induced apoptosis. they further demonstrated that this phenomenon occurred via antispike protein antibodies that mediated ade, but not via anti-n protein antibodies. 42 bruzzone and jaume's laboratory showed that antispike immune serum increased infection of human monocyte-derived macrophages by replication-competent sars-cov as well as by spike-pseudotyped lentiviral particles (sars-covpp), although they did not clarify whether this enhancement was through the fcγrii pathway. 43 reaction a a b-cell epitopes with a free −sh sequence (containing a cys residue) were prepared directly. b-cell epitopes without a free −sh group were anchored by an additional cysteine residue at the c-terminal ( figure s2) . a four-branched, brominated, multiple-antigen core peptide (brk 2 ka, figure s3 ) via lysine two α-amino and side-chain amino groups was prepared that finally conjugated with four copies of individual antigenic peptide. lentiviral particles, as well as by replication-competent sars coronavirus. jaume and collaborators proposed that ade of sars-cov utilizes a novel cell entry mechanism into immune cells. 44 this study demonstrates for the first time that an antibody (mab43-3-14) targeting a specific linear epitope (s 597−603 ) of the sars-cov spike protein can mediate enhancement of virus infection both in vitro and in non-human primates via an epitope sequence-dependent mechanism. we also demonstrate that the viral peptides that induce protection from pathogenesis can be identified and distinguished from those inducing enhancement in primates. these findings reveal a new mechanism of virus evasion of host defense that potentially provides an alternative strategy to prepare safe and effective vaccines against ade of virus infections. peptide design and synthesis. new peptides from the spike glycoprotein were designed on the basis of the information gained in the first round of immunopeptide discovery. 32 peptides were synthesized manually by standard fmoc chemistry protocols on rink-amide mbha resin (novabiochem, san diego, ca, usa; 0.44 mmol/g). all of the l-α-fmocprotected amino acids and coupling reagents (dic and hobt) were obtained from gl biochem (shanghai, china). all solvents were of analytical grade and used without further purification. each amino acid assembly was completed by using 3 equiv of l-α-fmoc-protected amino acids and coupling reagents, and the ninhydrin colorimetric test after each coupling was carried out to ensure no detectable amino remaining. in the case that the peptide-bearing beads were colorized (positive) by ninhydrin test, the free amino group was then blocked by reacting with 15% acetic anhydride in dichloromethane (dcm) (v/v) for 30 min. crude peptides without cys, met, arg, and trp residues were cleaved using a one-step tfa cleavage method under the following conditions: tfa (1.9 ml), deionized water (50 μl), and triethyl silane (50 μl). for peptides containing cys, met, arg, or trp residues, the following conditions were used: tfa (1.63 ml), thioanisole (0.1 ml), phenol (0.1 g), deionized water (0.1 ml), edt (50 μl), and triethylsilane (20 μl). tfa solution was treated with ice-cooled diethyl ether to precipitate the peptide. the crude peptide was washed twice by ice-cooled diethyl ether and dried in the n 2 flow condition. in particular, peptide s 471−503 was synthesized on synphase pa ram d-series lanterns (loading 8 μmol/lantern, mimotopes, raleigh, nc, usa). the lanterns were treated with 20% piperidine in dimethylformamide (dmf) for 15 min for deprotection. the general coupling conditions for lanterns utilized a solution of 80% dmf and 20% dcm, with reagents at the following concentrations: aa, 120 mm; hobt, 144 mm; dic, 120 mm. for each lantern a minimum of 500 μl of the activated amino acid solution was required. alternatively, bromophenol blue (5 μl/ml coupling solution, 5m) was used as an indicator to monitor the reaction. simultaneous side-chain deprotection and cleavage were carried out using 2.5 ml per lantern of a solution of 82.5% trifluoroacetic acid (tfa)/5% thioanisole/5% anisole/5% water/2.5% 1,2-ethanedithiol for 2 h. all crude peptides were purified by hplc to >95% purity on a semipreparative rp c18 column (zorbax, 300sb-c18, 9.4 × 250 mm. agilent, colorado springs, co, usa) eluting at 4 ml/min with a 0−20% ch 3 cn (buffer b) gradient in water (buffer a) containing 0.1% tfa over 5 min followed by a 20−65% ch 3 cn over 30 min. pure peptides were identified by an shimadzu lc-10at analytic hplc system with a c8 column (4.6 mm × 250 mm, 5 μm) and agilent lc-msd tof ( figure s4; s 36 the branched lysine core (brch 2 co-lys 2 -(lys)-β-ala-conh 2 ) scaffold was synthesized manually by standard fmoc chemistry protocols on 0.22 mmol (500 mg) of rink-amide mbha resin (novabiochem, 0.44 mmol/g) as described above. n-α-fmoc-lys (fmoc) was used at the branch points. the fmoc-protected amino acids (5.0 equiv) were assembled by using 5.0 equiv of dic coupling reagent and hobt additive. the n-termini of the four branches of the lysine core were bromoacetylated on the resin with 10 equiv of bromoacetic acid and dic in dmf for 2 h. the bromoacetylated lysine core was cleaved from the resin after treatment with 5% h 2 o/95% tfa for 2 h. it was then precipitated with cold diethyl ether, lyophilized, and purified by hplc on a semipreparative rp c18 column (zorbax, 300sb-c18, 9.4 × 250 mm) eluting at 4 ml/ (table s4 ). the average igg levels against peptides in each vaccine group were monitored by elisa on the day before injection or boost. igg level (b) against s 604−625 is an average of grouped macaques. the titer of anti-s 604−625 igg was greater than 1:10 6 in all relevant immunized monkey groups. a average gross lung pathology was determined by a standard in table s8 . b sars-cov-infected macrophages and alveolar epithelial cells in the vaccine groups were counted by using antipeptide antibodies. c quantitative viral burden analysis by quantitative rt-pcr (viral copies/mg lung tissue). maps were prepared for immunizations using a modified chemical ligation protocol. 45 ligation was achieved by mixing the lysine core and peptide in 500 μl of solvent at certain ph value. the reaction was monitored by an analytical rp c8 column (kromasil c8, the nest group, inc., southborough, ma, usa) under uv214 nm wavelength. after it appeared complete, the reaction was quenched by adding 0.05% tfa to adjust the ph to 7.0. the final product was then purified by semipreparative hplc at a flow rate of 4 ml/min. the ligated peptide was lyophilized and characterized by an agilent lc-msd tof. the preparative conditions of maps are listed in table s10 . detailed . more than 500 antisera of sars patients and 27 sera of healthy volunteers were involved in this study, which was conducted according to the guidelines for treatment of human subjects of the national institutes of health, usa. written informed consent was obtained from all study participants. the pepscan was carried out as described by hu et al. 35 without major modification. the optical density was read at 450 and 630 nm in an optical density reader (labsystem, franklin, ma, usa). the cutoff value was determined as twice the average value of the negative controls. binding of mouse monoclonal antibodies to the sars-cov virion. the sars-cov pumc01 strain 46 was diluted with coating buffer to 4 × 10 −5 tcid 50 /ml (bicarbonate/carbonate coating buffer, 0.05 m, ph 9.6). the virus was then coated on 96well plates with 100 μl (4 × 10 −4 of tcid 50 ) of virus per well for 12 h at 4°c. the coating buffer was removed from the wells, and 100 μl of blocking buffer was added per well and incubated for 12 h at 4°c (2% bsa in pbs, ph 7.4; gibco, carlsbad, ca, usa). the plates were washed twice with pbs (ph 7.2). the mouse monoclonal antibodies against sars-cov and control mab against hiv-p27 were diluted to 0.001 μg/μl in the blocking buffer, added to the wells separately, and incubated for 1 h at 37°c. the plates were washed twice with pbs and the secondary antibody (hrp-conjugated goat anti-mouse igg, (genetex, irvine, ca, usa) diluted 1:1000 in blocking buffer) was added to each well and incubated for 1 h at 37°c. the secondary antibody was removed, the plate was washed three times with pbs, and then 100 μl of tmb substrate solution (promega, madison, wi, usa) was added to each well. after incubation for 30 min at room temperature, the absorbance of the test wells was read at 450 nm (a 450 ). analysis of neutralization using a neutral red staining (nrs)-based assay. mabs were serially diluted in dmem culture medium. human polyclonal antisera from convalescent sars patients were diluted to 1:500 (v/v). the sars-cov pumc01 strain was diluted with dmem to 4 × 10 −4 tcid 50 . the nrs neutralization assay was performed in 96-well flat-bottom plates. the setup is similar to that of the cytopathic effect (cpe)-based neutralization assay. after incubation of 50 μl (4 × 10 −4 tcid 50 ) of virus with 50 μl of antibodies or antisera in a total of 100 μl of dmem medium per well for 1 h at 37°c, vero e6 cells (2 × 10 5 cells in 100 μl, from cell culture center of pumc) were added to each well. on day 5 of infection, when >70% of the cells showed cpe in the viral control wells (with dmem instead of antibody or antisera), the culture medium was removed from the test wells and 100 μl of 0.15% neutral red (sigma, st. louis, mo, usa) in dmem was added to each well. after incubation for 1 h at 37°c, the neutral red medium was removed, the plates were washed twice with pbs (ph 7.2), and then 100 μl of acidified alcohol (1% acetic acid in 50% ethanol) was added to each well. after incubation for 30 min at room temperature, the absorbance of neutral red stained plates was read at 450 nm (a 450 ). percent neutralization was determined by the following formula: % neutralization = (a 450 of antibody test wells − a 450 of viral control wells)/(a 450 of cell control wells − a 450 of viral control wells) × 100. generation, purification, and measurement of the affinity of antipeptide monoclonal antibodies. ganptransgenic mice were originally established in a c57bl/6 strain as described previously. 47 the mice were backcrossed with balb/c mice for at least eight generations and used for immunization to establish mabs. all mice were maintained in specific pathogen-free conditions by the center for animal resources and development (card), kumamoto university. as immunogens, four peptides of s 471−503 , s 604−625 , s 597−625 , and s 1164−1191 were synthesized and conjugated with keyhole limpet hemocyanin (klh) by operon biotechnologies (tokyo, japan). one hundred micrograms of peptide−klh emulsified in complete freund's adjuvant (cfa) was injected subcutaneously as a primary immunization and boosted after 2 weeks with incomplete freund's adjuvant (ifa). sera of immunized mice were measured by elisa using plates coated with free peptides. the mice with highest serum ab titers were further immunized, and 4 days later the spleen cells were obtained for polyethylene glycol-mediated cell fusion with mouse myeloma cell line x63 using standard procedures. 48 the fused cells were selected with hat medium in microculture plates at a concentration of 2 × 10 4 cells/well in the presence of il-6 (5 units/ml). the mabs binding to the immunizing peptide ags were detected with hrp-conjugated goat anti-mouse igg ab (zymed, south san francisco, ca, usa) with the substrate opd (wako chemicals, osaka, japan). the positive signals (a 490 > 1.0) were selected, and hybridomas were cloned by a limiting dilution method, adapted to serum-free media, and purified by protein g-sepharose affinity chromatography (ge healthcare, buckinghamshire, uk). the purity of the antibodies was examined by sds-page and protein staining with coomassie brilliant blue. the heavy and light chain isotypes were determined using an isotyping kit (bd biosciences, franklin lakes, nj, usa). the affinity of the mabs for peptides was determined by the biacore assay. 49 the on-and off-rate constants (k on and k off ) for binding of the mabs to s 471−503 , s 604−625 , s 597−625 , and s 1164−1191 peptides (table 1) were determined using the biacore system (biacore international ab, uppsala, sweden). the carboxylmethylated dextran surface of the sensor chip was activated with n-ethyl-n′(3-dietylaminopropyl) carbodiimide and n-hydroxysuccinimide (edc−ehs). 50 each peptide was immobilized through the free thiol group of a cysteine residue that was deliberately placed at the n-terminus, by injection of 35 μl of a 20 μg/ml solution in 10 mm 2-(n-morpholino)ethanesulfonic acid buffer (ph 6) to the edc−nhs-activated surface that had been reacted with 2-(2-pyridinyldithio)ethaneamine. the excess disulfide groups were deactivated by the addition of cysteine. the mabs were diluted in 10 mm hepes (ph 7.4), 150 mm nacl, 3.4 mm edta, and 0.05% (v/v) biacore surfactant p20 and injected over the immobilized antigen at a flow rate of 5 μl/min. the association was monitored by the increase in the refractive index of the sensor chip surface per unit time. the dissociation of the mabs was monitored after the end of the association phase with a flow rate of 50 μl/min. kinetic rate constants were calculated from the collected data using the pharmacia kinetics evaluation software. 51 the k on was determined by measuring the rate of binding to the antigen at different protein concentrations. macaque experiments. challenges with sars-cov. monkey challenges were performed in a bsl-3 facility. 37 briefly, the animals were anesthetized with ketamine (0.5 ml/kg, im). the tcid 50 10 −6 (4.0 ml/monkey) of the stock pumc01 strain virus was sprayed into the nasal cavity of the animals. pathologic examination. the lung tissues were sampled from the euthanized animals. organs were examined grossly, and photographs were taken to record the lung damage. samples of 5−10 g of lung tissues were collected from each lung and frozen at −80°c. the remaining tissues were fixed in 10% formaldehyde for 1 week in the bsl-3 laboratory. the tissues were then processed in the regular pathology laboratory by dehydration, embedding, and sectioning. for hematoxylin and eosin (h&e) staining, the sections were sequentially treated with xylene i, 10 min; xylene ii, 10 min; 100% alcohol, 5 min; 95% alcohol, 5 min; 90% alcohol, 5 min; 80% alcohol, 5 min; 70% alcohol, 5 min; and pbs, 5 min. the sections were then stained in hematoxylin for 5 min; washed with tap water and double-distilled water for 5 s, with pbs for 5 min, with 95% alcohol for 30 s, with eosin for 30 s, with 95% alcohol for 5 s, with 100% alcohol i for 5 s, with 100% alcohol ii for 5 s, with xylene i for 5 min, with xylene ii for 5 min, and with xylene iii for 5 min; and mounted with mounting buffer (zhongshan inc., guangzhou, china). immunohistochemical analysis of the infected lung tissues. a mixture of the mouse monoclonal antibodies mab-4e5 (0.01 μg/ml), mab-11b1 (0.01 μg/ml), and mab-9a6 (0.01 μg/ml) was used as primary antibody for the immunohistochemical analysis of the infected lung tissues. briefly, tissue sections were treated with the primary antibody at 4°c overnight, washed three times with pbs, incubated with hrp−goat-anti-mouse igg (1:1000, zhongshan inc., prc) at 37°c for 30 min, and then washed three times with pbs. the dab substrate (1:50) was added and incubated at 37°c for 30 min and then stained with hematoxylin for 3 min. rt-pcr for analysis of the sars-cov burden. the tissues from cranial and caudal lobes of the left lung and cranial, middle, and caudal lobes of the right lung from each animal were sampled and pooled together, and the total rna was isolated from the lung tissues using trizol (invitrogen, usa). one hundred milligrams of lung tissue was mixed with 1 ml of trizol extraction buffer and homogenized for 60 s (homogenizer, ika, germany). the rna wan then extracted with chloroform and precipitated with isopropanol. rna was dissolved in 50 μl of depc water, and the rna quality was verified by electrophoresis on agarose gel. for the reverse transcription, 5 μg of total rna was reverse transcribed with a high-capacity cdna kit (recertaid first strand cdna synthesis kit (fermentas, glen burnie, md, usa) with random hexamer primers. the reaction was performed at 42°c for 60 min and terminated at 70°c for 10 min. two microliters of reverse transcription mixture was used as template. the primers for sars-cov were 5′-atgaattac-caagtcaatggttac-3′ and 5′-cataaccagtcggta-cagctac-3′. the pcr was normalized against monkey glyceraldehyde phosphate dehydrogenase (gapdh) (nm002046). the gapdh primers were 5′-tcaacagcg-acacccactc-3′ and 5′-cttcctcttgtgctcttg-ctg-3′ (product size = 201 bp). the pcr was performed with a pcr kit (taq dna polymerase, fermentas). the pcr products were detected by 1.6% agarose gel electrophoresis. reaction conditions were as follows: 10× taq buffer (2 μl), 2 mm dntp (2 μl), forward primer (20 μm, 0.4 μl), reverse primer (20 μm, 0.4 μl), taq dna polymerase (0.8 μl), 25 mm mgcl 2 (2 μl), template dna (cdna, 2 μl), h 2 o (9.4 μl); step 1, 94°c for 3 min; step 2, 94°c for 30 s, 55°c for 30 s, 72°c for 30 s, for 26 cycles, then 72°c for 10 min. qrt-pcr for analysis of the sars-cov burden. the sars-cov fragment was prepared by rt-pcr. a 240 bp fragment of sars-cov was amplified and separated by 1.6% agarose electrophoresis. the dna fragment was recovered from the gel with a gel extraction kit (minelute gel extraction kit, qiagen stanford, valencia, ca, usa). the recovered dna was measured and adjusted to concentrations of 1 × 10 6 , 1 × 10 5 , 1 × 10 4 , 1 × 10 3 , 1 × 10 2 , and 1 × 10 1 copies/μl, which were used to generate a standard curve. this known copy number standard curve was used to calculate the copy number of sars-cov mrna in the infected lung tissues. rt-pcr for the infected samples was performed under the same conditions used for standard curves with the roche light cycler following the instructions for the sybr green realtime pcr master mix (toyobo, osaka, japan). the copy number of the virus was detected as follows. briefly, 2 μl of the reverse transcription mixture from step 1 was used as template. the primers for sars-cov were 5′-atgaattac-caagtcaatggttac-3′ and 5′-cataaccagtcggta-cagctac-3′. the reaction was continued for 40 cycles under the conditions: 2 × quantitect sybr green pcr (10 μl), gorward primer (10 μm, 1 μl), teverse primer (10 μm, 1 μl), rnase-free water (3 μl), yemplate dna (cdna, 5 μl), total volume (20 μl); step 1, 94°c for 3 min; step 2, 94°c for 15 s, 55°c for 30 s, 72°c for 20 s, for 40 cycles and then move to the melt curve procedure. immunization of monkeys. the peptide and/or peptide mixtures were dissolved as 480 μg/ml of each peptide in 0.9% nacl and then formulated with an equal volume of freund's complete adjuvant (fca) (sigma, st. louis, mo, usa) according to the manufacturer's instructions. the injected dose of vaccine was 0.5 ml/4 kg/monkey (120 μg/4 kg/monkey) given through intramuscular (im) injections at four sites on the arms and legs. rhesus monkeys were boosted at 2, 4, and 6 weeks after the first injection with the same dose of peptides and volume of vaccine as for the first injection, but formulated in an equal volume of freund's incomplete adjuvant (ifa) (sigma). venous blood samples (2 ml/each) were collected every 7 days from each monkey. the sera were isolated by centrifugation at 2000 rpm and immediately stored at −80°c. antipeptidic igg was detected by elisa. free peptides were diluted in carbonate buffer (ph 9.6) to a final concentration of 10 μg/ml. one hundred microliters of the peptide solution was added to 96-well polystyrene high bind microplates (corning life science, santa clara, ca, usa) for coating overnight at 4°c . the wells of plates were then washed three times with pbs-tween buffer (10 mm phosphate buffer, 150 mm nacl, 0.05% tween 20, ph 7.2) and subsequently blocked with 3% bsa in pbs-tween buffer for an additional 1 h at 37°c. after three thorough washings of the wells with pbs-tween buffer, the antigen-coated microplates were incubated with diluted monkey sera at room temperature for 1 h. after three washings with pbs-tween buffer, bound antibodies were detected with hrpconjugated goat anti-monkey igg (1:2000 v/v, gene tex). after three washings with pbs-tween, a tmb substrate solution (promega, madison, wi, usa) was added and the color developed for 20 min before the reaction was stopped with 100 μl of 0.2 m h 2 so 4 . the absorbance at 450 nm was read using a microtiter plate reader. macaques treated by mab43-3-14. eighteen rhesus monkeys in six groups (table s6 , 3−4 years old) were examined according to the national microbiologic and parasite spf standard and were verified free of anti-sars antibody. mab43-3-14 was adjusted to concentrations of 0.4 and 3.6 mg/ml with 0.9% nacl, respectively. a dose of 0.2 or 1.8 mg/kg of the antibody given by intravenous (iv) injection was used to treat the different groups. the same volume of 0.9% nacl was used for the control group. the procedures were approved by the animal use and care committee of the institute of laboratory animal science, peking union medical college (animal welfare assurance no. a5655-01, iacuc approval date september 24, 2007). statistical analysis. all data were analyzed using prism software (graphpad software). for neutralization assays and elisa, mean and standard deviation were used to determine significance. for qrt-pcr, geometric mean and standard deviation were used to determine virus burden. from sars to mers: 10 years of research on highly pathogenic human coronavirus identification of a novel coronavirus in patients with severe acute respiratory syndrome bats are natural reservoirs of sars-like coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome the chinese sars molecular epidemiology consortium . molecular evolution of the sars coronavirus during the course of the sars epidemic in china angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus identification of an 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crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins antibodydependent infection of human macrophages by severe acute respiratory syndrome coronavirus anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcγr pathwa synthesis of proteins by native chemical ligation comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection generation of high-affinity antibody against t cell-dependent antigen in the ganp gene-transgenic mouse a novel nuclear phosphoprotein, ganp, is up-regulated in centrocytes of the germinal center and associated with mcm3, a protein essential for dna replication real-time biospecific interaction analysis using surface plasmon resonance and sensor chip technology immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system author contributions ∥ w.q.d., z.l.f., and k.k. equally contributed to this work. the authors declare no competing financial interest. we are grateful to professor carl f. nathan and dr. michael s. diamond for their kind discussion and manuscript preparation. we thank dr. baoxing fan for his serological evaluation of the large number of sars-cov convalescent antisera. the project described in this paper was supported by the national institute of allergy and infectious diseases (u01ai061092). the study was also partially supported for preparation of the monoclonal antibodies by contract research for mext for emerging and reemerging infectious diseases and promotion of fundamental studies in health sciences (04-2) of the nibio and national natural science foundation of china (no. 90713045). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord-337089-ksh62ni0 authors: salajegheh tazerji, sina; magalhães duarte, phelipe; rahimi, parastoo; shahabinejad, fatemeh; dhakal, santosh; singh malik, yashpal; shehata, awad a.; lama, juan; klein, jörn; safdar, muhammad; rahman, md. tanvir; filipiak, krzysztof j.; rodríguez-morales, alfonso j.; sobur, md. abdus; kabir, farrokhreza; vazir, bita; mboera, leonard; caporale, marco; islam, md. saiful; amuasi, john h.; gharieb, rasha; roncada, paola; musaad, sahar; tilocca, bruno; koohi, mohammad kazem; taghipour, ali; sait, ahmet; subbaram, kannan; jahandideh, alireza; mortazavi, pejman; abedini, mohammad amin; hokey, david a.; hogan, unarose; shaheen, mohamed n. f.; elaswad, ahmed; elhaig, mahmoud m.; fawzy, mohamed title: transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) to animals: an updated review date: 2020-09-21 journal: j transl med doi: 10.1186/s12967-020-02534-2 sha: doc_id: 337089 cord_uid: ksh62ni0 covid-19 caused by a novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) originated in wuhan (hubei province, china) during late 2019. it has spread across the globe affecting nearly 21 million people with a toll of 0.75 million deaths and restricting the movement of most of the world population during the past 6 months. covid-19 became the leading health, economic, and humanitarian challenge of the twenty-first century. in addition to the considerable covid-19 cases, hospitalizations, and deaths in humans, several cases of sars-cov-2 infections in animal hosts (dog, cat, tiger, lion, and mink) have been reported. thus, the concern of pet owners is increasing. moreover, the dynamics of the disease requires further explanation, mainly concerning the transmission of the virus from humans to animals and vice versa. therefore, this study aimed to gather information about the reported cases of covid-19 transmission in animals through a literary review of works published in scientific journals and perform genomic and phylogenetic analyses of sars-cov-2 isolated from animal hosts. although many instances of transmission of the sars-cov-2 have been reported, caution and further studies are necessary to avoid the occurrence of maltreatment in animals, and to achieve a better understanding of the dynamics of the disease in the environment, humans, and animals. future research in the animal–human interface can help formulate and implement preventive measures to combat the further transmission of covid-19. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the disease was referred to as coronavirus disease-2019 (covid-19) [3] . the sars-cov-2 is the third coronavirus that has emerged in the last two decades. others are the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov), which emerged during 2002 and 2012, respectively [2, 4] . compared to sars-cov and mers-cov, sars-cov-2 is responsible for the most significant economic loss and the highest number of infections and deaths [5] . until 13 august 2020 around 21 million covid-19 cases and over 0.75 million deaths have been reported. sars-cov-2 belongs to the order nidovirales, suborder coronavirineae, and family coronaviridae. the family coronaviridae contains two subfamilies named lentovirinae and orthocoronavirinae. the latter is further classified into four genera, namely alphacoronavirus (αcov), betacoronavirus (βcov), gammacoronavirus (γcov), and deltacoronavirus (δcov), as shown in fig. 1 . the γcovs and δcovs cause diseases in birds while αcovs and βcovs are mainly found in mammals such as bats, rodents, civets, pigs, horses, cattle and humans [6] [7] [8] [9] . sars-cov-2 clusters with lineage βcov together with sars-cov and mers-cov [8] ; both originating from bats [1, 10, 11] . coronaviruses are enveloped viruses with singlestranded positive-sense rna (+ss) and genome size between 26 and 32 kb in length [12, 13] . these viruses possess four essential structural proteins: spike glycoprotein (s), matrix protein (m), envelope protein (e), and nucleocapsid protein (n) [13] [14] [15] 22] . according to the genomic analysis, sars-cov-2 shares a 96.2% identity with the genome of bat cov; ratg13, indicating a possible origin of the virus in bats [3, 16] . several studies suggested that the pangolin could be the potential intermediate host involved in the evolution of the virus because of the unique receptor-binding domain configuration [17] [18] [19] 22] . among the global fear due to the rapid spread of the covid-19 and absence of specific treatment or vaccine, the first case of human-to-animal transmission was recorded in hong kong, where a 17-years-old pomeranian dog was affected [20] . this case raised concerns about the possibility of sars-cov-2 transmission from humans to animals and vice versa, which would represent the difficulties in fighting the virus significantly. however, based on recently published findings, other authors hypothesized that an immunological cross-protection between sars-cov-2 and canine respiratory coronavirus (crcov) exists due to the high homology between the spike protein epitopes of the two taxonomicallyrelated coronaviruses [21] . the objective of the present study was to gather, present, and discuss information on the reported cases of covid-19 in animals focusing on the virus transmission cases in pets and perform genomic and phylogenetic analyses of sars-cov-2 isolated from animal hosts. further studies on the dynamics of the disease are essential to adopt suitable control measures to reduce transmission of the virus. to discuss the origin of sars-cov-2, it is necessary to analyze the source of other coronaviruses such as sars-cov and mers-cov, as shown in fig. 2 [22] . sars-cov emerged in guangdong province, southern china, in november 2002 and was characterized as a contagious respiratory disease of humans [22, 23] . records showed 8422 registered cases and 916 deaths in humans, spreading to 29 countries during the epidemic with a case fatality rate (cfr) of 9.6% [23] . studies revealed the presence of several coronaviruses in species of horseshoe bats (genus rhinolophus), which are evolutionarily related to sars-cov in their genome organization and sequence [24, 25] . in the further investigations of the initial sars outbreak of 2002 in hong kong, it was recognized that cats (felis domesticus) and ferrets (mustela furo) could be infected with sars-cov [26] . in 2012, another coronavirus causing severe acute respiratory syndrome in humans was first reported in saudi arabia and was later called the middle east respiratory syndrome coronavirus (mers-cov) [27] . mers-cov is βcov of the c lineage with a genotype very similar to that of bats of the same line, such as btcov-hku4 and btcovhku5 [28] . the transmission of mers-cov to dromedaries was identified through the detection of specific antibodies against the virus in these animals [29] . according to corman et al. [30] , mers-cov has been circulating in dromedaries for more than 20 years [30] . therefore, it can be assumed that bats are reservoirs for several coronaviruses, including sars-cov, mers-cov, and sars-cov-2 [25, 31, 32] . according to reports sars-cov-2 is closely related to sars-cov [33, 34] and the similarity between the genomes was about 80% [3, 16, 35] . though sars-cov-2 has most likely originated from bats, it is not yet clear which animal served as an intermediate host and contributed towards the evolution of the virus before the spillover to humans occurred [22] . in another study, it was demonstrated that sars-cov-2 was a chimeric virus between a bat coronavirus and a coronavirus of unknown origin [36] . in a similar research, it was reported a novel bat-derived coronavirus, denoted rmyn02, identified from the metagenomic analysis of samples from 227 bats collected from yunnan province in china between may and october 2019 [37] . notably, rmyn02 shares a 93.3% nucleotide identity with sars-cov-2 at the scale of the complete genome and 97.2% identity in the orf1ab gene. it could, therefore, be considered the closest relative of sars-cov-2 reported to date. in contrast, rmyn02 showed low sequence identity (61.3%) with sars-cov-2 in the receptor-binding domain (rbd), suggesting that rmyn02 might not bind to angiotensin-converting enzyme 2 (ace2). critically, and in a similar manner to sars-cov-2, rmyn02 was characterized by the insertion of multiple amino acids at the junction site of the s1 and s2 subunits of the spike (s) protein. this provides strong evidence that such insertion events can occur naturally in animal βcov [37, 39] . it can be seen that tiger sars-cov-2/usa/ ny-040420 epi_isl_420293 shares 99.96% nucleotide identity with human sars-cov-2 reference genome at the complete genome-scale, followed by mink sars-cov-2/nb04 epi_isl_447634 (99.90%), mouse sars-cov-2/hrb-26 m epi_isl_459910 (99.87%), cat sars-cov-2 epi_isl_437349 (99.85%), and dog sars-cov-2/hkg/20-03695/2020 (99.51%) demonstrating a high level of nucleotide identity between sars-cov-2 isolates from domestic animals and humans (table 1) . also, noteworthy is the shared identity between tiger sars-cov-2/usa/ny-040420 epi_isl_420293 and mink sars-cov-2/nb04 epi_isl_447634 (99.89%), cat sars-cov-2 epi_isl_437349 and tiger sars-cov-2 usa ny-040420 epi_isl_420293 (99.84%), cat sars-cov-2 epi_isl_437349 and mink sars-cov-2/ nb04 epi_isl_447634 (99.78), cat sars-cov-2 epi_ isl_43734 and dog sars-cov-2/hkg/20-03695/2020 (99.39%), tiger sars-cov-2/usa/ny/040420 epi_ isl_420293 and dog sars-cov-2/hkg/20-03695/2020 (99, 54%), and dog sars-cov-2/hkg/20-03695/2020 and mink sars-cov-2/nb04 epi_isl_447634 (99.53%) ( table 1) . regarding the percentage of nucleotide identity of the spike protein, dog sars-cov-2/hkg/20-03695/2020, tiger sars-cov-2 usa ny-040420 epi_isl_420293, and cat sars-cov-2 epi_isl_437349 share 99.97% with human sars-cov-2 ( table 2 ).the amino acid identity of the spike protein between dog sars-cov-2/hkg/20-03695/2020, tiger sars-cov-2 usa ny-040420 epi_isl_42029 and cat sars-cov-2 epi_ isl_437349 with sars-cov-2 is 99.92% (table 3) . however, the spike protein amino acid and nucleotide identity among tiger sars-cov-2/usa/ny/040420 epi_isl_42029, dog sars-cov-2/hkg/20-03695/2020, and cat sars-cov-2 epi_isl_437349 was 100% (tables 2 and 3) . 18:358 this result seems to be related to animal species that have been infected by sars-cov-2 through humans (dog, cat, and tiger, at first). transmission to humans does not seem so likely, however, given the identity between nucleotides and amino acids of the spike protein, transmission between animals seem possible. because it has a similar genome to other animal coronaviruses, sars-cov-2 may have undergone nucleotide mutation when transmitted to animals, expressing amino acids that increased its pathogenicity in animals, especially those related to spike protein [38, 39] . the rbd of sars-cov-2 spike protein, which lies in the s1 domain, is a critical element for determining the susceptibility of the new host species. the studies performed on the interaction between the viral rbd with host cellular receptor (ace2) revealed snakes, pangolins, and turtles as the potential intermediate hosts [40] . turtles, along with other animal species, are favored animals in the huanan seafood wholesale market. however, extended studies are needed to prove their associations scientifically [11] . one of the probable intermediate hosts for sars-cov-2 is a pangolin. pangolin-cov has 91.02% and 90.55% identity to sars-cov-2 and batcov ratg13, respectively [33, 35] . furthermore, the sars-cov-2 spike proteins rbd resembles closely malayan pangolin cov (pangolin-cov) [40] . these findings suggested that pangolins can be the intermediate host for sars-cov-2 transmission. further research is necessary to confirm the origin and transmission dynamics of sars-cov-2. the possible protective effect of pet-ownership against coronaviruses warrants consideration where coronavirus prevalence is high across the pet population. canine respiratory coronaviruses often occur among dogs. ownership of an infected pet can lead to the transmission of viruses from animals to humans. although possible protection caused by the possession of a pet has not yet been found, the frequent occurrence of coronavirus in canines could help the human immune system develop a better response against sars-cov-2 [41] . at the beginning of the sars-cov-2 outbreak, it was thought that pets were not susceptible to the sars-cov-2. however, a natural infection of a cat was reported in belgium with traces of the virus identified in the collected samples by pcr. this cat exhibited respiratory difficulty, vomiting, and diarrhea, which may indicate active replication of the virus inside the animal [39, 41, 42] . however, the animal was not examined by a veterinarian, so further evaluation such as serology was necessary. in another study, shi et al. showed that cats could be not only naturally infected with sars-cov-2 but also that adolescent cats artificially inoculated with the virus presented severe histological lesions and died [18] . however, in further studies, dogs exhibited seroconversion, but the virus could not be isolated. the susceptibility of cats and ferrets to sars-cov-2 could be attributed to the ace2 (sars-cov-2 receptors) [3, 43] . these receptors are expressed in type ii pneumocytes serous epithelial cells of tracheobronchial submucosal glands in ferrets [44] . moreover, the sars-cov-2 spike-contacting regions of ace2 are similar in both ferrets and cats, differing by only two amino acids [5] . the previous reports have shown that sars-cov can infect ferrets and cats [26] , which implies that ferrets and cats may also be susceptible to sars-cov-2. this possibility might be associated with cases of sars-cov-2 transmission to animals. in addition to probably sharing the same origin, bats have significant similarities among their genotypes [3, 25, 31, 32, 35, 36] . based on sars-cov epidemiology, the covid-19 pandemic raises the alarm that animals may become infected and become potential transmitters to humans. the fear of possible transmission of animals to humans and the fact that many residents were forced to leave their animals behind due to evacuation and quarantine, thinking that they would return soon, generated a large number of animal abandonment [45] . authorities in hunan and zhejiang provinces of china also announced that they would start killing pets found in public to prevent the transmission of the virus. this concern intensified in late february 2020, when dogs in hong kong tested positive for the new coronavirus, being considered the first known case of transmission of covid-19 from humans to animals later, felines [20, 39, 46, 47] . in a recent study, it was investigated the possible protection conferred by previous exposure of individuals to naturally infected animals with coronaviruses taxonomically related to the circulating sars-cov-2 [21, 41, [48] [49] [50] . the animals were experimentally infected by sars-cov-2 via the intranasal route through the receptor ace2 [51] . several studies report the use of ace2 by the new coronavirus as its receptor for cell entry [3, 43] . dogs were also inoculated, although seroconversion was observed, no virus could be isolated from the inoculated and uninoculated contacts. further, ferrets showed equal susceptibility to cats, while pigs, chicken, and ducks were found referent to vulnerability [5] . until now, studies based on rbd domain analysis ruled out the probability of mice, rats, and rabbit's involvement in the sars-cov-2 cycle [52] . the findings on ferrets, orangutans, and monkeys showed a higher affinity of ace2 with the rbd domain of sars-cov-2 s protein [53] . a codon-usage based analysis pointed to snakes as a probable host, although these findings were contradicted by subsequent studies [54] . some researchers revealed that cats could naturally be infected with other coronaviruses such as feline coronavirus (fcov) just as canines can be infected with canine coronavirus (ccov) [55, 56] . these animals probably get infection once the virus binds to the receptor, ace2 [51] . another research group conducted a retrospective serological survey on cats infected with sars-cov-2 in wuhan, china. the authors collected 102 samples after the emergence of covid-19 and included 39 samples collected before the outbreak. fifteen (14.7%) of 102 serum samples collected after the outbreak were positive for antibodies against the sars-cov-2 receptor-binding domain (rbd) by elisa test. among the positive samples, 11 had neutralizing antibodies to sars-cov-2 with a titer ranging from 1/20 to 1/1080. twenty-nine out of the 39 serum samples collected before the outbreak were negative. however, no cat that tested positive on serological tests showed any clinical signs. no serological cross-reactivity was detected between sars-cov-2 and type i or ii of the feline infectious peritonitis virus (fipv). as the authors suggested, the cat population studied in wuhan was infected with sars-cov-2 after the beginning of the outbreak [57] . in france, a group of 18 veterinary students investigated the spread of the new coronavirus in 21 pets (9 cats and 12 dogs). eleven cases showed symptoms compatible with covid-19, while only two confirmed positive for the new coronavirus. although three of these cats showed clinical signs of respiratory or gastrointestinal disease, no animal was considered positive by rt-pcr or by the presence of specific antibodies to sars-cov-2 [51] . in a separate study, the researchers demonstrated that ferrets, cats, and dogs could be experimentally infected by sars-cov-2 via the intranasal route [40] . several studies reported that the sars-cov-2 uses the same receptor, ace2, to enter the respiratory mucosa [3, 5, 43] . this probably indicates the possibility of transmission of sars-cov-2 from humans to animals. in a recent experimental study, it was observed that cats infected with sars-cov-2 could transmit the virus to naïve cats that come into contact with them [58] . however, whether they can transmit the virus to humans or else, humans can transmit the virus to pets, or other animals are not yet fully understood. also, the first non-domesticated animal case of sars-cov-2 transmission was from a big cat, nadia, and a 4-year-old malayan tiger infected from the covid-19 positive workers at the bronx zoo, new york, united states. this was the first known animal infection in the us and a tiger anywhere in the world, and was confirmed by the us department of agriculture (usda), the us centers for disease control and prevention (cdc), national veterinary services laboratories, and the wildlife conservation society (wcs) [54, 59, 60] . consequently, covid-19 was detected in four tigers and three lions. mild infection of respiratory distress was also noted in two pet cats in the usa [61] . a new case was reported in moskva, moskovskaya oblast, russia, where a 5-year-old cat tested positive for sars-cov-2. samples (throat and nasal swabs) were taken from the suspect cat to detect sars-cov-2. the lab tests were performed using rt-pcr in real-time with electrophoretic detection of amplification products. the obtained amplification reaction product was sequenced using selected specific primers flanking a 232 bp n gene fragment (orf1ab) of the sars-cov-2. the tests showed 100% identity of the analyzed fragment of the n gene orf1ab of the sars-cov-2. the animals were quarantined [62] . it was previously shown that sars-cov does not infect or cause disease in poultry. because the covid-19 virus belongs to the same group as sars-cov and uses the same ace2 host cell receptor, it is highly unlikely that poultry is susceptible to covid-19. still, it remains to be scientifically proven [63] . for the phylogenetic analysis, complete genome sequences of the viruses falling in the betacoronavirus genus were retrieved from ncbi genbank. the sequences involve sars-cov and sars-cov-2 of sarbecovirus subgenus and few of the sequences from different animal species including the dog, mink, rabbit, rat, pangolin, hedgehog, camel, bats, and wild and domestic felines comprising subgenuses merbecovirus, embecovirus, hibecovirus and nobecovirus within the genus betacoronavirus (fig. 3) . the sequence alignment was done with clustalw in mega 6.02 software, and phylogeny was constructed using the gtr + g + i substitution model applying the maximum likelihood method. in the analysis, canine and feline covs of alphacoronavirus were taken as the outgroup for constructing a phylogenetic tree of betacoronavirus. all the covs of genus betacoronavirus clustered in their respective subgenus clades. notably, the mink and tiger virus isolates showed 99.6-99.9% homology with human sars-cov-2 isolates from different parts of the world. contrarily, the canine betacoronavirus (crcov) from subgenus embecovirus showed 45.8-46.2% similarity with sars-cov-2. furthermore, pangolin cov, sars-like bat covs, and bat covs (ratg13 strain) showed homology between 86.6 and 96.3% with sars-cov-2. the camel isolate of mers-cov showed a 51% similarity with sars-cov-2 at the nucleotide level (fig. 3) . our analysis demonstrated a high divergence between animal origin covs and sars-cov-2 except for a few strains which were isolated from mink and tiger as were the cases of covid-19 infected persons. though the exact ways of transmission of sars-cov-2 from infected humans to animals are vaguely understood, the possible and promising transmission may occur through touching their noses or mouth by infected hands defiled with respiratory droplets or saliva [64] . at the time of sneezing, coughing, or even talking, infected humans can disseminate respiratory droplets, which have a pivotal role in the transmission of the virus to animals [65] . however, the transmission of the virus from affected people can be facilitated by some favorable risk factors, e.g., kissing, petting, licking, or hugging pet animals [66] . according to the government of the netherlands, through a letter issued by the ministry of agriculture, nature and food quality, it is possible that an employee who worked on a mink farm infected with sars-cov-2 contracted the virus, having already been recovered from the disease. also, research shows that minks can be asymptomatic and that cats play an important role in the potential transfer of viruses between investigated farms [67]. there are multiple studies on the origin of coronaviruses and their zoonotic potential. some coronaviruses that infect animals can sometimes be spread to humans and then spread between people as happened in the case of mers and sars. this is also what happened with the virus that caused the current outbreak of covid-19. however, the exact origins of this virus are still unclear. the scientific community has put significant effort into identifying the source of sars-cov-2, and to date, genetic evidence suggests that it was likely acquired from bats. the first infections were linked to a live animal market in wuhan, suggesting a zoonotic origin. the virus is now spreading from person to person and caused one of the most significant pandemics of the recent era. infection with sars-cov-2 has been reported in several animals through serological and molecular analyses and experimental inoculations. however, it is not proved that animals can transmit sars-cov-2 to humans and to what extent humans can transmit this virus to animal species. veterinary public health officials and designated public health officials should work to determine whether animals should be tested for sars-cov-2 when the animals are in the same environment as infected owners using a one health approach. available information suggests that the risk of sars-cov-2 spread from animals to humans is low. even though human-to-animal transmission or vice versa is possible, caution and effective communication with the pet owners are necessary to prevent the abandonment and death of animals indiscriminately. although a case of sars-cov-2 transmission from animals to humans has already been reported, this is only a report, so further research is needed before declaring that animals can transmit the new coronavirus to humans. geographic information system (gis) based on computer and information technology would be useful in analyzing the disease pattern. the collaborative global effort, such as "one-human-environmental-animal health, " is required to reduce the global risk of zoonotic diseases. further studies are essential to understand better the origin of the virus and transmission dynamics, which will help educate people and avoid unnecessary discrimination to animals. preventive measures that have proven to be effective should be 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sars-cov-2 infection and pathogenesis covid-19 preclinical models: human angiotensin-converting enzyme 2 transgenic mice receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars cross-species transmission of the newly identified coronavirus 2019-ncov canine coronavirus highly pathogenic for dogs a tale of two viruses: the distinct spike glycoproteins of feline coronaviruses sars-cov-2 neutralizing serum antibodies in cats: a serological investigation transmission of sars-cov-2 in domestic cats a tiger at the bronx zoo has tested positive for coronavirus centers for disease control and prevention cdc. coronavirus disease 2019 (covid-19). if you have animals confirmation of covid-19 in two pet cats in new york world organization for animal health oie what we know about avian coronavirus infectious bronchitis virus (ibv) in poultry and how that knowledge relates to the virus causing covid-19 in humans pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses practical measures to prevent covid-19: a mini-review the risk of sars-cov-2 transmission to pets and other wild and domestic animals strongly mandates a one-health strategy to control the covid-19 pandemic. one health publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank dr. amir ghorbani of the ohio state university and joão inácio magalhães duarte, for his critical comments on this paper. we gratefully acknowledge the authors, originating, and submitting laboratories of the sequences from the gisaid's epicov ™ database, on which part of the phylogenetic tree reconstruction is based. the initial writing was carried out by sst and pmd; the other authors contributed to the complementation, critical review, and final of the manuscript. all authors read and approved the final manuscript. not applicable. all data analyzed or generated during this study are included in this publication and its additional files. not applicable. not applicable. the authors declare that they have no competing interests. key: cord-304479-uxp1kg86 authors: goodarzi, pedram; mahdavi, farzad; mirzaei, rasoul; hasanvand, hamze; sholeh, mohammad; zamani, farhad; sohrabi, masodreza; tabibzadeh, alireza; jeda, ali salimi; niya, mohammad hadi karbalaie; keyvani, hossein; karampoor, sajad title: coronavirus disease 2019 (covid-19): immunological approaches and emerging pharmacologic treatments date: 2020-08-08 journal: int immunopharmacol doi: 10.1016/j.intimp.2020.106885 sha: doc_id: 304479 cord_uid: uxp1kg86 the sars-cov-2 virus is an etiological agent of pandemic covid-19, which spreads rapidly worldwide. no proven effective therapies currently exist for this virus, and efforts to develop antiviral strategies for the treatment of covid-19 are underway. the rapidly increasing understanding of sars-cov-2 virology provides a notable number of possible immunological procedures and drug targets. however, gaps remain in our understanding of the pathogenesis of covid-19. in this review, we describe the latest information in the context of immunological approaches and emerging current antiviral strategies for covid-19 treatment. in december 2019, many severe respiratory diseases accompanied by pneumonia appeared in wuhan, hubei province, china, with unknown etiology [1] [2] [3] . sequencing examination on lower respiratory tract specimens showed a novel coronavirus named 2019 novel coronavirus (2019-ncov) [4] [5] [6] [7] [8] . data from genome sequencing of sars-cov-2 assist the researchers in approving diagnostic examination, epidemiologic tracking, and advancement of preventive and curative strategies [9] . the clinical sign of covid-19 has a wide range from moderate, self-restraint respiratory tract illness to acute progressive pneumonia, multi-organ collapse, and death [9, 10] . up to the present, there were no licensed therapies for the therapy of 2019-ncov infection. after the rise of the sars-cov-1 in 2003, screening of licensed drugs for the therapy of sars led to the identification of some drugs (as reviewed by vijayanand and colleagues) [11] , such as protease inhibitors, nucleoside analogs, intravenous immunoglobulins, convalescent sera, tumor necrosis factor-alpha blockers, interferons, traditional chinese medicines, and glycyrrhizin. this virus causes sars in humans [11] . there are no confirmed efficacious treatments for the however, researchers made much effort to develop antiviral strategies for the treatment of covid19 . this review will describe the current evidence on significant proposed, repurposed, or experimental therapies for covid-19 and present a summary of contemporary clinical experience and treatment guidelines for this new pandemic sars-cov-2. antibodies are an essential part of host immune reactions to viral infection. due to their unique maturation process, antibodies can emerge extremely particular to viral antigens [12] . the first use of immunoglobulins (antibodies) as a therapy opportunity for viral diseases can be hunted back to the beginning 20th century, using sera of infected people, who had improved from the same condition [13, 14] . this natural therapy regimen ( serum therapy), was slowly replaced by immunoglobulins purified from merged sera, intravenous immune globulin (ivig) [15] . notwithstanding the achievement of both serum therapeutics and ivig, no meaningful advancement was made to produce antibodies as therapeutics. the hybridoma technique was continuously launched, facilitating the separation of monoclonal antibodies (mabs) from immunized mice in 1975 [16] . various approaches have been established since the mid-1980s for the effective separation of mabs toward viruses of human and animal sources [12] . to date, there are no confirmed vaccines or therapeutic medications that are special to covid-19. preventing monoclonal antibodies (mabs), owing to their fantastic antigen specificity, is among the most suitable nominees for compensating viral infection [17] . hence, recognizing and cloning of mabs that can accurately target viral superficies proteins to hinder the viral entrance to host cells is an acceptable strategy for blocking and handling covid-19, mainly when effective vaccines and therapeutics medications are unavailable in the outbreak of the covid-19 pandemic [18] . mabs targeting exposed positions on viral outside proteins are frequently identified as hopeful classes of drugs toward infectious disorders and have conferred therapeutic potency for numerous types of viruses [19, 20] . neutralizing antibodies produced against coronavirus mainly target the spike (s) glycoproteins outside the viral envelope that mediates entrance into host cells. the s protein is composed of two functional subunits, which involve cell binding (s1 subunit) and s2 subunit, which includes membrane fusion. s1 subunit the main target of the humoral immune system, that potent neutralizing antibodies often produced against it [21] [22] [23] [24] [25] [26] . a recent report by wang et al. has shown that human mabs can neutralize both sars-cov-2 and sars-cov in cell culture [27] . another report demonstrated that chen and colleagues separated two human mabs employing sars-cov-2 rbd-specific memory b cells separated from healed cases with covid-19. these two mabs could especially attach to sars-cov-2 rbd, block the interplay within sars-cov-2 rbd and the hace2 receptor, and lead to potent neutralization of sars-cov-2 s protein pseudotyped virus infection. before-mentioned human anti-sars-cov-2 rbd-ace2 blocking mabs are initially described and endure transcendent hope to be employed as specific preventive and therapeutic tools toward underway sars-cov-2 pandemic [18] . this knowledge suggests that mabs have a promising approach for tackling the covid-19 pandemic. the results of using mabs are still premature, and future studies will shed light on the feasibility of this type of biological therapy for patients with covid-19 ( figure) (table) . the sera separated to form recovered patients from the infectious disease called convalescent plasma, which has been employed to prevent and treat various infectious illnesses for longer than one century. antibodies exist in immune or convalescent plasma, intercede their curative effects through multiple mechanisms. the antibody can attach to a given infectious agent by compensating its infectivity directly. another mechanism that antibody may also contribute to its therapeutic effect includes complement activation, phagocytosis, and antibody-dependent cellular cytotoxicity. non-neutralizing antibodies attach to the infectious agent but do not intervene with their capacity to replicate in-vitro systems may also participate in prevention and expedite the rehabilitation process [28, 29] . across the previous two decades, convalescent plasma therapy has been successfully employed to manage sars, mers, and the 2009 h1n1 pandemic with competent potency and safety [30] [31] [32] [33] . a meta-analysis of 32 investigations of sars-cov and severe influenza virus infection explained a statistically meaningful decline in the pooled odds of fatality following treatment with convalescent plasma, contrasted with placebo or no treatment [34] . however, convalescent plasma treatment was unable to considerably enhance the durability of the ebola virus infection, presumably due to the lack of data on neutralizing antibody titration for stratified examination [35] . because the virological and clinical features share similarities between sars-cov-1, mers, and sars-cov-2, convalescent plasma treatment might be a confident strategy choice for covid-19 saving [36] . patients who have healed from covid-19 with a huge neutralizing antibody concentration may be a precious donor reservoir of convalescent plasma. it is worth mentioning that there are potential safety concerns on convalescent plasma therapy, including transmitting other infectious agents and a pathological event called antibodydependent enhancement (ade) [37] . ade attributes to a means of how antibodies increasing throughout a previous infection exacerbate clinical severity as an outcome of disease with a distinct viral serotype. this event is famed for some viruses, prominently the dengue virus [38] . although, in convalescent serum trials, attention, and alertness to recognize any enhanced infection, evidence will be demanded. in a recent report published in china, researchers revealed that in 10% of patients, one dose of convalescent plasma (200 ml) was well-tolerated and significantly increased or maintained neutralizing antibodies at high levels, resulting in the disappearance of viremia within a week. it can also modulate multiple parameters compared to pre-transfusion, including decreased c-reactive protein and increased lymphocyte numbers. besides, convalescent plasma therapy in seven cases who had the former viremia led to the disappearance of viremia (undetectable viral load). another finding that study was that convalescent plasma treatment was well-tolerated and conceivably improved the clinical symptoms, thereby neutralizing viremia in patients with severe covid-19. finally, the authors of this study believe that further investigations are wanted to define the optimal dose and clinical benefits of convalescent plasma therapy [39] . future studies should analyze the efficiency of convalescent plasma treatment in many patients, and the potential risk of this therapeutic method must be deeply assessed (figure) (table) . the increasing knowledge is regarding covid-19 pathogenesis has supported the role of excess inflammatory mediators in patients with covid-19. patients' pathological characteristics with covid-19 include capillary leakage of liquid and entrance of inflammatory cells, including t cells, neutrophils, and macrophages [40] , referring a function for chemokines and cytokines targeting vascular endothelium. the concentration of pro-inflammatory cytokines, including il-1, il-6, tnf-α, and ifn-γ, are elevated in the blood of cases infected with covid-19 [3, 41] . recent studies report different cytokine profiles in patients with severe covid-19 [3, [41] [42] [43] [44] [45] [46] . in a study carried out by huang and colleagues have revealed the higher concentration of il-2, il-7, il-10, tnf, g-csf, ip-10; cxcl10, mcp-1 (ccl2) and mip-1a (ccl3), but not il-6, in cases hospitalized in the intensive care unit (icu) contrasted with non-icu patients (4). another study confirmed that during acute covid-19 disease, some pro-inflammatory cytokines such as il-1β, il-1ra, il-2r, il-6, il-8 (cxcl8), il-17, ifn-γ, gm-csf elevated [42, 43, 45, 46] . in recent exciting research, the concentration of some cytokines/ chemokines, including il-6, il-10, ifnγ, tnf, and ip-10 in icu patients with covid-19, have been higher than mild to moderate non-icu patients [3, [42] [43] [44] . various strategies, including global targeting of the inflammation or compensating a single crucial inflammatory marker, are applied to cope with cytokine storm in covid-19. between key cytokines, il-6 has drawn significant attention levels, and antibodies that hinder the il-6 receptor (il-6 antagonist, tocilizumab, and sarilumab) are now following phase 2/3 clinical trials for the possible therapy of covid-19 [47] . another hopeful strategy is targeting ifn-γ, which has been remarked by beginning a clinical trial for jak-stat inhibitor (ruxolitinib) for managing covid-19 severity [48] . tnf works upstream of il-6, and anti-tnf treatments earlier revealed protecting impacts in deadly sars-cov disease [49] . a recent report by cavalli et al. have shown that the efficacy of anakinra (human interleukin-1 receptor antagonist protein) was notably higher in subjects with covid-19 contrasted with those who received standard treatment for three weeks, led to decreased levels of serum crp, improved respiratory function (72% vs. 50%), increased survival rate (90% vs. 56%). however, the study results demonstrated that bacteremia's risk was elevated in cases receiving anakinra than those receiving standard treatment [50] . although there is no particular antiviral medicine for covid-19, knowledge of cytokine storm mechanisms can help to speculate possible therapeutic interventions ( figure) (table) [51] . it is alleged that corticosteroid treatment is not supported for viral pneumonia [52] . investigations have revealed that the use of systemic corticosteroids for patients with sars-cov and mers-cov was correlated with a higher fatality rate than patients under standard treatment [53, 54] . the same finding was described in cases with influenza virus-associated pneumonia [55] . in a study performed by matsuyama et al. [56] , they utilized the nasal administration of corticosteroids for patients infected with coronaviruses. they indicated that in the cell culture models, the inhaled form of corticosteroids, such as ciclesonide, could be useful for the treatment of coronaviruses. ciclesonide exerts antiviral and anti-inflammatory activity in in-vitro models [56] . furthermore, there are some open clinical trials for the therapeutic assessment of methylprednisolone on covid-19 patients [57] . a systematic review study carried out by tahvildari and colleagues [58] , shows that at least six different published studies on the effect of corticosteroids on covid-19 patients. also, wu et al. [59] indicated that the use of corticosteroids for patients with covid-19 who developed ards could lead to a better outcome and reduce the mortality rate. these results indicate the necessity of checking the clinical conditions of covid-19 patients before prescribing corticosteroids. finally, recently, a case report study from japan shows that orally inhaled ciclesonide alleviates the local inflammation in the lung of patients with covid-19 pneumonia and inhibits the propagation of the virus by antiviral activity [60] . further studies are required to unravel the precise mechanism of corticosteroids in the exacerbation of patients with covid-19 ( figure) (table) . mesenchymal stem cells (mscs) are a subset of non-hematopoietic adult stem cells, readily isolated from various tissues. they show immunoregulatory activity and could be employed for the tissue repair process as they secrete paracrine factors [61] . cell-based therapy, remarkably stem cell therapeutics, has become an encouraging remedial field, in which many perceive possibilities to cure deadly and inflammatory disorders [62, 63] . notwithstanding the notable progress of stem cell-based treatment, immunogenicity, poor cell source, and moral problems are deemed the main therapeutic approach restrictions. among these, mscs have drawn much attention due to origin potential, a high reproduction speed, having a low invasive method, and free of moral problems. there is an extreme advantage in applying msc treatment in contrast with other approaches [64] . the results indicate that following covid-19 infection may start suppressing immune overactivity in the human body. in patients infected with sars-cov-2, the immune system generates massive volumes of inflammatory factors, prompting cytokine storm in which the immune cells produce an extreme amount of cytokines and chemokines [65] . herein, it is the opening of the msc therapy strategy in the treatment of covid-19 patients. msc cure can limit the storm release of cytokines by the immune system and raise endogenous restoration by regenerative features of stem cells [66] . recently, some countries, such as china, the usa, iran, and various other countries, have launched msc therapy, and some reports are currently available in the published literature. mscs, working their immunomodulatory features and their differentiation capacity, can inhibit lung tissue loss by hindering the cytokine storm and restoration and regeneration of damaged tissues [67] . a recent study carried by chen and co-workers indicated that the use of mscs notably improves the survival proportion of h7n9-induced ards and provides a philosophical background for treating h7n9-induced ards preclinical research and clinical research. because h7n9 and covid-19 share similar complications and are associated with multi-organ collapse, msc-based treatment could be a feasible option for the treatment of covid-19 [68] . in the same way, a recent case-report study showed that the adoptive transfer therapy of human umbilical cord blood derived-mesenchymal stem cells (hucmscs) to a chinese female patient afflicted with acute covid19 syndromes improved her laboratory tests and ct images [69] . before receiving any treatments, the percentage of her neutrophils was increased to 87.9% while the number of lymphocytes was decreased to 9.8%. she was operated with antiviral medications, including lopinavir/ritonavir, ifn-α, and oseltamivir, also the intravenous dose of moxifloxacin, xuebijing, methylprednisolone, and immunoglobulins. the case was also curbed to non-invasive mechanical ventilation to expedite breathing and decrease muscle weakness due to weak oxygenation. as the vital symptoms exacerbate, the case was treated with hucmscs solely and with α1 thymosin 5 × 107 cells per three times. the study results explained that following the second injection, serum albumin, crp, alt, and ast were steadily diminished, and other important symptoms were enhanced. after that, the patient was discharged from the ventilator and capable of walking, and the number of neutrophils and white blood cells returned to the baseline levels. most importantly, the abundance of cd3+, cd4+, and cd8+ t cells was significantly enhanced. also, the qualitative outcomes of ct images following the second and third doses of hucmscs revealed that pneumonia was attenuated. after two days of the third injection, the patient was rescued from the icu, and most of the vital symptoms and clinical laboratory parameters returned to the standard ranges. the outcomes recommended that hucmscs could be an excellent strategy choice alone or in combination with other immunomodulatory tools for covid-19 patients [69] . a recent study performed in china in cooperation with the united states recruited seven cases with covid19 pneumonia from january 23 to february 16. patients experienced mscs transplantation, and their clinical signs were consecutively checked for 14 days. the study demonstrated that the transplantation of hucmscs led to a marked decrease in the level of pro-inflammatory cytokines and a substantial improvement in clinical symptoms without any significant adverse effects [70] . the pulmonary function, along with the seven patients' clinical symptoms, were significantly improved after two days of transplantation. the number of peripheral lymphocytes also increased, while crp concentration was diminished after the treatment. additionally, the number of hyperactive cytokine-secreting immune cells, namely cxcr3+cd4+, cxcr3+cd8+, and cxcr3+ nk cells was remarkably lowered within 3-6 days after transplantation of hucmscs. moreover, the frequency of cd14+cd11c+cd11b mid regulatory dc cell population was significantly elevated. the level of tnf-α was significantly reduced, while il-10 was raised in the hucmscs-treated group contrasted with the placebo control group. besides, the gene expression characterization explained that ace2 and tmprss2 genes are not expressed in hucmscs, implying that the coronavirus would not infect these cells. hence, the intravenous transplantation of hucmscs is seemingly safe and efficient for the treatment of cases with covid-19 pneumonia, notably those in critically severe conditions [70] . as multiple clinical trials are launched worldwide, we should not have to wait long to determine if mscs are a viable and valid treatment choice for severe covid-19. considering the need for mitigating the prevailing covid-19 pandemic, with superiority to manage fatality as low as possible, the judgment that msc is reliable and can invert severe critical disease with high power is an invention designing a completely novel biological procedure that needs to be developed urgently (figure) (table) . chloroquine and hcq are both known as antimalarial drugs. clinical studies introduced these two drugs as a possible choice for covid-19 treatment due to having in-vitro antiviral and antiinflammatory properties [71] [72] [73] [74] . several studies suggested that chloroquine could improve the radiological and virological features of covid-19 [53] . chloroquine is a reliable and effective drug for covid-19 in some preclinical trials [75] and other studies [72] . in this regard, smith et al. [72] indicated that cardiac arrhythmia is a significant side effect of chloroquine. in the matter of hydroxychloroquine, reports are controversial [72, 73, 76] . in a study conducted by shamshirian et al. [76] , there was no potential clinical efficacy in prescribing hcq. simultaneously, the in-vitro anti-sars-cov-2 activity of this particular drug seems to be more than chloroquine [72, 73] . fortunately, there are currently several clinical trials being conducted on these drugs [57] . also, regardless of the solo practice for these drugs, gautret, and colleagues [77] suggested a combination of hcq and azithromycin as an effective treatment for decreasing the viral load in patients with covid-19. another aspect of these drugs is the possibility of using them in different conditions such as pregnancy. a majority of studies conducted on hcq did not reflect any serious concerns, and this drug seems to be safe for pregnant women [78] . besides, as mentioned by lother et al. [79] , clinical trials for the assessment of medicines as post-exposure prophylaxis could be helpful. a recent report noted that the severity of covid in patients treated with hcq was higher than those not receiving this medication. also, the report showed that there was no meaningful correlation within the use of hcq and intubation or mortality [80] . besides, another report demonstrated that amongst cases hospitalized in metropolitan new york with covid-19, practice with hcq, azithromycin, or both, matched with neither medication, was not meaningfully correlated with variations in an in-hospital fatality. however, the analysis of these conclusions may be restricted by the observational study [81] . the recent study also indicated that hcq did not substantially decrease symptom severity in outpatients with early, mild covid-19 [82] . at the same time, another study performed by mitjà and colleagues indicated that hcq in patients with mild covid-19 has no benefit beyond routine care [83] . moreover, arshad and co-workers [84] showed that in patients with covid-19 treated with hcq alone and combined with azithromycin, it was correlated with a decline in covid-19 associated mortality. comprehensive systematic review and meta-analysis studies and clinical trials in this field are urgently needed ( figure) (table) . ace2 (angiotensin-converting enzyme-2) is a transmembrane enzyme expressed on the exterior of epithelial cells in the many organs such as lungs, arteries, heart, kidney, and intestines [85, 86] . recently, the new coronavirus is responsible for pandemic covid-19, sars-cov-2 is thought to be mainly or exclusively bound to ace2 [87, 88] . the molecular interplay among ace2 and spike has been created [87, 88] , and manufactured compounds or antibodies, interfering with the interplay of ace2, and the viral spike protein could be produced. another therapeutic approach is the use of soluble ace2 as a virus scavenger and neutralizer. soluble ace2 formed by a proteolytic splitting of the membrane anchor is ordinarily located in the plasma; however, its concentration is shallow. an increase in the availability of soluble ace2 at tissue positions would change the rivalry with membrane-bound ace2 toward the soluble form, leading to the repression of viral entry into the cells. it is also expected that this approach would preserve tissue ace2 [89, 90] . a new study has recently shown that the recombinant form of ace2 reduces the infection and viral growth in cell culture and organoids by acting as a decoy for sars-cov-2 [89] . this study showed that by adding a genetically altered variant of ace2, termed human recombinant soluble angiotensin-converting enzyme 2 (hrsace2), the entry of covid-19 into the lung epithelial cells was halted. in this study, the results of cell culture indicated that hrsace2 decreased the load of sars-cov-2 by 1,000-5,000 times [89] . the authors also used the blood vessels and kidney organoids to explain that sars-cov-2 could directly contaminate and propagate in these tissues, suggesting a potential agent of multi-organ collapse and cardiovascular damages as a result of covid-19. the augmentation of hrsace2, too, diminished the infectivity of sars-cov-2 in these organoids [89] . in engineered models of the human blood artery and kidney organoids developed from human stem cells, it was confirmed that it could straight contaminate and replicate itself in these tissues. these findings provide crucial information about the pathogenesis of covid-19 and explain the reason for multi-organ collapse and cardiovascular injuries. in this engineered human tissues, hrsace2 also diminished the viral load of sars-cov-2. the researchers highlighted that their experiment has only tested the drug efficacy through the initial stages of sars-cov-2 infection. further investigation would be demanded to determine the fidelity of this recombinant therapy for later stages of the disease (figure) ( table) . the prescription of ribavirin for the therapy of coronaviruses returns to sars-cov and mers-cov. reports indicated that this antiviral agent's administration did not show promising results for the treatment of sars-cov [91] . meanwhile, ribavirin antiviral activity was addressed in in-vitro studies in a dose-dependent manner [91, 92] . on the other hand, a group of studies demonstrated a beneficial role of ribavirin in the treatment of mers-cov [93] . simultaneously, some investigations showed that the combination of ribavirin and interferon was unsuccessful in treating mers-cov [94] . there is limited knowledge about the efficiency of ribavirin in the amelioration of covid-19 [58] . a study conducted by elfiky et al. [95] , using bioinformatics approaches, favipiravir (also known as t-705) is an antiviral drug that selectively and robustly hinders the rna-dependent rna polymerase (rdrp) of rna viruses, was licensed in 2014 in japan to cure pandemic influenza virus diseases [97] . interestingly, despite its anti-influenza virus activity, this molecule can also halt the replication of an extensive range of rna viruses (e.g., flaviviruses, alphaviruses, filoviruses, noroviruses, arenaviruses, bunyaviruses, and other rna viruses) [97] . regarding the emergence of sars-cov-2, it is urgently essential to recognize active antiviral [98] . they indicated that patients receiving favipiravir showed improved chest imaging, faster decreased viral load, and fewer adverse effects than the control group [98] . another study performed by chen et al. compared the efficacy of favipiravir versus arbidol [99] . they showed that the clinical recovery rate on day seven and the degree of auxiliary oxygen treatment or non-invasive mechanical ventilation did not significantly vary within the favipiravir-and arbidol-treated groups. besides, the current study demonstrated that favipiravir significantly enhanced the latency to relief for fevers. also, adverse effects caused by favipiravir were mild and manageable [99] . these preliminary clinical results provide useful information about therapeutic options for sars-cov-2 infection (figure) ( table) . remdesivir (gs-5734) is a prodrug (nucleotide) with extensive antiviral action toward viruses from distinct genera in-vitro [100] . it also has therapeutic effects on nonhuman primate models of deadly ebola and nipah virus contaminations [101, 102] . investigations conducted on epithelial cells from human airway explained that remdesivir additionally hinders replicating an extensive range of coronaviruses, including mers-cov [103] . moreover, some reports indicated that remdesivir has robust action toward sars-cov-2 in-vitro [104, 105] . a recent investigation performed on rhesus macaques contaminated with sars-cov-2 noted that the treatment with a 6-day regimen of iv remdesivir launched 12 hours following virus inoculation was correlated with some therapeutic outcomes (lower disease severity rates, less pulmonary infiltrates, lower virus titers in bronchoalveolar lavage samples) contrasted with the control animals. of note, remdesivir medication did not diminish the viral load or the titer of the virus in the nasopharynx or rectal swabs compared to the control of vehicle control [106] . various clinical trials are currently being performed in the us, china, and other countries. a recent clinical trial in hospitalized patients with severe covid-19 in china showed that remdesivir treatment was not correlated with a decline in hospitalized patients' recovery period. the results indicated that patients receiving remdesivir had a lower period of hospital stay than those receiving placebo (18 vs. 23 days); however, such a reduction in hospital stay period was not statistically significant. also, the continuation of invasive mechanical ventilation was more concise (but not statistically meaningful) in the remdesivir-treated group, and only a tiny percentage of patients (0.4%) underwent invasive mechanical ventilation at the time of reception. remdesivir did not significantly reduce the viral load of sars-cov-2 in nasopharyngeal, oropharyngeal, and sputum specimens. remdesivir was stopped in 18 patients (12%) because of adverse effects [107] . however, a phase iii randomized, open-label trial performed on hospitalized patients with severe covid-19 showed that the disease severity was lower in patients who received remdesivir within 10 days after the onset of clinical symptoms compared with those treated after 10 days of the manifestation of clinical signs [108] . notably, patients treated with remdesivir had a more short recovery period than those treated with placebo. also, the mortality rate in remdesivir-treated patients (7.1%) was lower than patients receiving a placebo. however, the difference in mortality rate within the two groups was not statistically meaningful [109] . another clinical trial has been recently established in the us, china, and other countries to explore the efficacy of remdesivir in improving patients with covid-19 (table 1) . further clinical trials are wanted to determine the effect of remdesivir on patients with covid-19 (figure) ( table) . ivermectin is an fda-licensed drug that has a broad spectrum of antiparasitic activity [110] . studies have shown that this drug exerts antiviral action toward an extensive range of viruses invitro [111] [112] [113] [114] . it has been designated that ivermectin hinders the interplay within the human immunodeficiency virus-1 (hiv-1) integrase protein (in) and importin (imp) α/β1 heterodimer accountable for the nuclear import of in [115] . ivermectin has, too, been demonstrated to impede nuclear import and hiv-1 replication [114] . also, ivermectin suppresses explicitly the activity of the ns3 helicase enzyme, required for the replication of flaviviruses [116] . in the same way, ivermectin inhibits the replication of dengue virus type 2 (denv-2) in aedes albopictus [117] . another study indicated that ivermectin prevents the interaction between denv 1 and 2 ns5 with its nuclear transporter importin α/β in-vitro and make effort protection toward denv1-4 [113] . ivermectin is thought to be effective against sars-cov-2, the virus which causes pandemic covid-19 [118] . it has been reported that ivermectin can reduce the replication of sars-cov-2 when added to the cell culture 2 h post-infection. besides, ivermectin can diminish the viral load by ~5000 folds, whiting 48 h post-infection [118] . the next critical step is to examine dosing regimens that mimic the currently recommended use of ivermectin in humans [119] . a current phase iii clinical trial in thailand showed that ivermectin was safe but did not perform any clinical advantage when used for dengue viruses. however, studies recommended that dosing regimens might be improved and expanded, depending on pharmacokinetic analyses [120] . although denv differs from sars-cov-2, the design of future clinical trials should be revisited to provide valuable information about the efficacy of this drug on covid-19. current studies hold great promise for the prescription of ivermectin as a possible antiviral therapy against sars-cov-2 ( figure) (table) . β-d-n4-hydroxycytidine is a ribonucleoside analog called eidd-1931, an orally bioavailable prodrug by wide range antiviral action toward multiple independent rna viruses, which includes influenza, ebola, coronaviruses, and veev [121] [122] [123] [124] . currently, there are no specific licensed therapeutics for sars-cov-2. recently, an interesting study indicated the efficacy of eidd-2801against covid-19 in human cells and mice [123] . eidd-2801 is an orally bioavailable drug that its mechanism of action is similar to remdesivir. these drugs can mimic the function of infections caused by other types of coronaviruses [123] . clinical trials seem to be needed to investigate the probable applicability of eidd-2801 in the clinic against pandemic covid-19 ( figure) (table) . lopinavir and ritonavir are the hiv-1 fda approved drugs, which inhibitors of the hiv protease. this anti-protease activity seems to be active on the sars-cov-2 protease, either. lopinavir and ritonavir could induce adverse effects, such as qt prolongation, and must be carefully prescribed for patients with liver-associated diseases [72] . several clinical trials conducted on the combinatory use of lopinavir/ritonavir was more pronounced in patients with covid-19 than other therapeutic regimens [57, 58] . one study also showed that the combination of lopinavir/ritonavir and arbidol (an antiviral agent against rna viruses) did not significantly protect covid-19 in patients [125] . meanwhile, zhu and colleagues [126] demonstrated that arbidol monotherapy was more effective than the use of lopinavir/ritonavir and arbidol. besides, an in-vitro study performed on lopinavir did not exhibit any direct antiviral activity against sars-cov-2 [74] . regardless of these findings, the administration of lopinavir/ritonavir seems beneficial on sars-cov and mers-cov only when used at the early stage of infection [127] . in conclusion, it seems hard to introduce the lopinavir/ritonavir as a treatment option for covid-19, but further clinical trials are warranted to elucidate the effectiveness of these drugs (figure) ( table) . there is progressing proof that cases with severe covid-19 promote a hypercoagulable status, which has been correlated with weak outcomes such as increased respiratory malfunction, severe respiratory distress syndrome, or mortality [128] [129] [130] [131] [132] [133] [134] . the initial treatment with anticoagulation drugs in patients with severe covid-19 infection may diminish the risk of thrombotic complexities and promote clinical consequences [59, 131, 133, 135, 136] this increasing evidence urged researchers to focus on the potential applicability of using anticoagulating agents for covid-19. heparin is an anticoagulant agent possessing potential benefits beyond anticoagulant activity. it has been shown that heparin decreases coronary thrombosis, pulmonary emboli, and microvascular ischemia. besides, it has anti-inflammatory and antiviral properties, enabling this compound to lower the degree of lung inflammation and improving oxygenation [137] . a new retrospective report by tang et al. confirmed that anticoagulant therapy is correlated with a diminished mortality percentage in covid-19 patients with coagulopathy. also, the results recommend that patients with severe covid-19 disease or considerably elevated levels of d-dimer (>6 x uln) may have declined fatality when they receive preventive doses of heparin. researchers also proposed that anticoagulant treatment seems to be correlated with a more favorable prediction in severe covid-19 patients engaging sepsis-induced coagulopathy (sic) standards extended d-dimer [138] . however, prospective studies are demanded to validate these conclusions because the current retrospective study has limited data ( figure) (table) . rates that humanity has experienced in the 21st century after the pandemic influenza outbreak of 1918. although the gaps remain in our understanding of the pathogenesis covid-19, the velocity and mass of antiviral strategies began to examine possible medicines for covid-19 to highlight both the demand and capacity to provide high-quality data even amid a pandemic. probably the best plan for fighting with the sars-cov-2 is an effective vaccine, which prompts the immune system to produce antibodies against viral proteins or t cells that can eliminate infected cells. however, vaccine development is slower than the spread of the epidemic; therefore, the clinically useful candidate drugs would be necessary and urgent for the treatment of patients with covid-19. among the immunological approaches, cp therapy might be a trusting approach choice for covid-19 saving; however, future investigations should examine the efficacy of cp treatment in many patients, and the potential risk of this therapeutic approach must be profoundly evaluated. besides, many clinical trials are underway across the world; however, currently, no therapies have been shown useful to date for covid-19, or some drugs such as remdesivir have a limited benefit in patients with covid19. there are no conflicts to declare. this work was supported by the iran university of medical sciences. in vero cells hydroxychloroquine was found to be more potent than chloroquine to inhibit sars-cov-2 [73, 139] hcq could significantly shorten ttcr and promote the absorption of pneumonia [140] hcq significantly associated with viral load reduction/disappearance in covid-19 patients and its effect is 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treatment of adults with mild covid-19: a randomized-controlled trial treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid-19 a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis cryo-em structure of the 2019-ncov spike in the prefusion conformation receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus prosper, inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy? sars: systematic review of treatment effects pharmacologic treatments for coronavirus disease 2019 (covid-19): a review clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and meta-analysis ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study anti-hcv, nucleotide inhibitors, repurposing against covid-19 novel coronavirus treatment with ribavirin: groundwork for evaluation concerning covid-19 favipiravir as a potential countermeasure against neglected and emerging rna viruses experimental treatment with favipiravir for covid-19: an open-label control study favipiravir versus arbidol for covid-19: a randomized clinical trial gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys remdesivir (gs-5734) protects african green monkeys from nipah virus challenge broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 remdesivir in adults with severe covid-19: a randomised, double-blind study to evaluate the safety and antiviral activity of remdesivir (gs-5734™) in participants with moderate coronavirus disease (covid-19) compared to standard of care treatment remdesivir for the treatment of covid-19 -preliminary report the pharmacokinetics and interactions of ivermectin in humans-a mini-review influenza a viruses escape from mxa restriction at the expense of efficient nuclear vrnp import nuclear import and export inhibitors alter capsid protein distribution in mammalian cells and reduce venezuelan equine encephalitis virus replication nuclear localization of dengue virus (denv) 1-4 non-structural protein 5; protection against all 4 denv serotypes by the inhibitor ivermectin ivermectin is a 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poor prognosis in patients with novel coronavirus pneumonia isth interim guidance on recognition and management of coagulopathy in covid-19: a comment the procoagulant pattern of patients with covid-19 acute respiratory distress syndrome clinical observation and management of covid-19 patients covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid-19 in coronavirus disease 2019 treatment: a review of early and emerging options, open forum infectious diseases compassionate use of remdesivir for patients with severe covid-19 an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice effect of convalescent plasma therapy on viral shedding and survival in covid-19 patients treatment with convalescent plasma for covid-19 patients in wuhan effective treatment of severe covid-19 patients with tocilizumab tocilizumab treatment in covid-19: a single center experience a single center observational study of the clinical characteristics and short-term outcome of 20 kidney transplant patients admitted for sars-cov2 pneumonia individual risk management strategy and potential therapeutic options for the covid-19 pandemic use of siltuximab in patients with covid-19 pneumonia requiring ventilatory support early, lowdose and short-term application of corticosteroid treatment in patients with severe covid-19 pneumonia: single baricitinib therapy in covid-19: a pilot study on safety and clinical impact are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? indomethacin has a potent antiviral activity against sars coronavirus inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs advances in respiratory virus therapeutics-a meeting report from the 6th isirv antiviral group conference nelfinavir inhibits replication of severe acute respiratory syndrome coronavirus 2 in vitro atazanavir inhibits sars-cov-2 replication and pro-inflammatory cytokine production a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy in vero e6 cells remdesivir inhibits sars-cov-2 replication [104, 105] remdesivir was not associated with statistically significant clinical benefits [107] clinical improvement was observed in 68% of severe covid-19 patients treated with remdesivir in a cohort study [143] remdesivir was associated with shortened recovery time and decreased rate of mortality in patients with covid-19 versus the placebo group [ inhibition of viral entry into the cell in vitro hrsace2 had a dose dependent effect of viral growth of sars-cov-2 and was able to reduce it by a factor of 1,000 to 5,000 in cell cultures [89] currently no known published data regarding efficacy or safety in the treatment of covid-19ivermectin inhibiting impα/β1 which mediated nuclear import of viral proteins in some human and animal viruses.in vitro activity against some human and animal viruses in vitro evidence of activity against sars-cov-2 in infected vero-hslam cells reported with high concentrations of the drug [118] currently no known published data regarding efficacy or safety in the treatment of covid-19 key: cord-326017-qw4qynqv authors: laskar, partha; yallapu, murali m.; chauhan, subhash c. title: “tomorrow never dies”: recent advances in diagnosis, treatment, and prevention modalities against coronavirus (covid-19) amid controversies date: 2020-08-06 journal: diseases doi: 10.3390/diseases8030030 sha: doc_id: 326017 cord_uid: qw4qynqv the outbreak of novel coronavirus disease (2019-ncov or covid-19) is responsible for severe health emergency throughout the world. the attack of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is found to be responsible for covid-19. the world health organization has declared the ongoing global public health emergency as a pandemic. the whole world fights against this invincible enemy in various capacities to restore economy, lifestyle, and safe life. enormous amount of scientific research work(s), administrative strategies, and economic measurements are in place to create a successful step against covid-19. furthermore, differences in opinion, facts, and implementation methods laid additional layers of complexities in this battle against survival. thus, a timely overview of the recent, important, and overall inclusive developments against this pandemic is a pressing need for better understanding and dealing with covid-19. in this review, we have systematically summarized the epidemiological studies, clinical features, biological properties, diagnostic methods, treatment modalities, and preventive measurements related to covid-19. the emergence and spread of 2019 novel coronavirus (2019-ncov) or the severe acute respiratory syndrome (sars) coronavirus 2 (sars-cov-2) has threatened global public health. the coronavirus disease 2019 (covid-2019), which is a transmission of sars-cov-2 to humans, was reported first in wuhan, hubei province, china in december 2019. later, covid-19 rapidly spread worldwide creating a pandemic as there have been around 15,656,884 reported cases and 636,576 reported deaths to date (24 july 2020) affecting 213 countries and territories around the world and two international conveyances (https://www.worldometers.info/coronavirus/). north america, south america, and asia are the most affected (maximum number of cases) continents by this pandemic so far (https:// www.worldometers.info/coronavirus/, data accessed on 24 july 2020) ( figure 1a ). in spite of rapid development of knowledge, precautionary measurements, and clinical trials, researchers, regulatory bodies, and government administrations are facing a great challenge globally in various aspect to prevent covid-19 pandemic. the present situation is leading us to a still unknown future and conflicting paradox in the battle against sars-cov-2. further, a plethora of documents have been published on "coronavirus" and "coronavirus disease" (year-wise keyword search for last 10 years in pubmed on 24 july 2020) research, where a maximum and a very large number of results were obtained in 2020 on both the keywords in comparison to previous years ( figure 1b) . nature index has also reported a hugely increasing global research publishing phenomenon on covid-19 pandemic as evidenced by 66,883 articles and 19,420 preprints (https://www.natureindex.com/news-blog/thetop-coronavirus-research-articles-by-metrics). a thorough and cumulative scientific knowledge about the progress against covid-19 amid various conflicts and controversies is the need of the time to set a clear directive in this battle. through this review article, we provide an overview of updated and rapidly evolving progress against covid-19 pandemic including various conflicts to the readers in a comprehensive manner. considering this, we have summarized diverse research areas covering the current known biological properties of sars-cov-2, diagnostic tools for detection, therapeutic measurements for possible treatment, and prevention techniques to stop further spreading of this pandemic. still unknown future and conflicting paradox in the battle against sars-cov-2. further, a plethora of documents have been published on "coronavirus" and "coronavirus disease" (year-wise keyword search for last 10 years in pubmed on 24 july 2020) research, where a maximum and a very large number of results were obtained in 2020 on both the keywords in comparison to previous years ( figure 1b) . nature index has also reported a hugely increasing global research publishing phenomenon on covid-19 pandemic as evidenced by 66,883 articles and 19,420 preprints (https://www.natureindex.com/news-blog/the-top-coronavirus-research-articles-by-metrics). a thorough and cumulative scientific knowledge about the progress against covid-19 amid various conflicts and controversies is the need of the time to set a clear directive in this battle. through this review article, we provide an overview of updated and rapidly evolving progress against covid-19 pandemic including various conflicts to the readers in a comprehensive manner. considering this, we have summarized diverse research areas covering the current known biological properties of sars-cov-2, diagnostic tools for detection, therapeutic measurements for possible treatment, and prevention techniques to stop further spreading of this pandemic. coronaviruses (subfamily: coronavirinae, family: coronaviridae, order: nidovirales) are enveloped single stranded positive sense rna genomes that range in size from 26 to 32 kilobases [1, 2] . coronavirus consists of four structural proteins: the nucleocapsid, envelope, membrane, and spike forming a core-shell morphology (figure 2 ), whose diameter is in the range from 60 nm to 140 nm with spike like projections on its surface [2] [3] [4] . the name, coronavirus (latin: corona = crown) came out due to the presence of a crown or the sun's corona-like spike (s) glycoprotein on viral surface forming club-shaped protrusions, which is also evidenced through the electron microscope [2] [3] [4] . this transmembrane spike (s) glycoprotein on viral surface mediates the entry into host cells, forming homotrimers protruding from the viral surface [5] . the receptor binding domain (rbd) in the spike (s) glycoprotein is the most mutable part of the coronavirus genome leading to generation of new properties and ability of virus to infect new cell types or even new species [6] . based on phylogenetic relationships and genomic structures, the subfamily coronavirinae is divided into four genera (α-cov, β-cov, γ-cov, and δ-cov). α-cov and β-cov only infect mammals, whereas γ-cov and δ-cov infect generally birds and sometimes even infect mammals. β-cov and γ-cov are responsible for respiratory diseases in humans and gastroenteritis in animals [2, 7] . presence of four coronaviruses (subfamily: coronavirinae, family: coronaviridae, order: nidovirales) are enveloped single stranded positive sense rna genomes that range in size from 26 to 32 kilobases [1, 2] . coronavirus consists of four structural proteins: the nucleocapsid, envelope, membrane, and spike forming a core-shell morphology (figure 2 ), whose diameter is in the range from 60 nm to 140 nm with spike like projections on its surface [2] [3] [4] . the name, coronavirus (latin: corona = crown) came out due to the presence of a crown or the sun's corona-like spike (s) glycoprotein on viral surface forming club-shaped protrusions, which is also evidenced through the electron microscope [2] [3] [4] . this transmembrane spike (s) glycoprotein on viral surface mediates the entry into host cells, forming homotrimers protruding from the viral surface [5] . the receptor binding domain (rbd) in the spike (s) glycoprotein is the most mutable part of the coronavirus genome leading to generation of new properties and ability of virus to infect new cell types or even new species [6] . based on phylogenetic relationships and genomic structures, the subfamily coronavirinae is divided into four genera (α-cov, β-cov, γ-cov, and δ-cov). α-cov and β-cov only infect mammals, whereas γ-cov and δ-cov infect generally birds and sometimes even infect mammals. β-cov and γ-cov are responsible for respiratory diseases in humans and gastroenteritis in animals [2, 7] . presence of four corona viruses (hku1, nl63, 229e and oc43) have been found in human circulation which are generally cause mild respiratory disease [8] . the 2019-ncov is phylogenetically closely related to bat sars-like coronaviruses, hence name sars-cov-2 and belongs to β-cov genus lineage b [9] . regarding pathogenicity and transmissibility, sars-cov-2 may differ from other known sars-cov due to a significant change in its spike glycoproteins (orf8, and orf3b) [10] . doremalen et al. [11] showed that the sars-cov-2 virus remained viable in aerosols (<5 µm) for at least up to 3 h and was more stable on plastic and stainless steel than on copper and cardboard. sars-cov-2 was first identified from the samples (cultured human airway epithelial cells along with the virus from isolated bronchoalveolar lavage fluid) of adult covid-19 patients in wuhan, china. its morphology was analyzed by negatively stained sample under transmission electron microscopy (tem) [12] . zhu et al. [12] made a conclusion based on tem ultra-structural image of sars-cov-2 virus particles. this follows as (i) it is generally spherically shaped with a diameter ranges from 60 to 140 nm, (ii) it has an envelope with quite distinctive 9 to 12 nm protein spikes, and (iii) it has genetic material which matched to the genome from lineage b of the genus β-cov-showing more than 85% identity with a bat sars-like cov (bat-sl-covzc45, mg772933.1). these observations are similar to the overall structure of coronaviridae family viruses. tem image of an isolate from the first united states case of covid-19 is also evidenced the spherically shaped viral particles (colorized blue) containing cross-sections through the viral genome (black dots) [13] . the crystal structures of the unliganded sars-cov-2 main protease (m pro ) and its complex with an α-ketoamide inhibitor was used to provide some knowledge about the drug target for covid-19 [14] . this m pro enzyme is essential one along with the papain-like protease(s) for processing the viral rna translated polyproteins. thus, inhibition of this enzyme may block the viral replication. the three-dimensional crystal structure, at 1.75 å resolution, of the sars-cov-2 m pro is highly similar to the sars-cov m pro , due to the 96% sequence identity. dimerization (necessary for catalytic activity) of the m pro is regulated by domain iii (residues 198 to 303) mainly through a salt-bridge interaction between glu 290 of one protomer and arg 4 of the other. like sars-cov-2, world also has seen an outbreak of worldwide pandemic and a large-scale fatal swine disease during the past two decades because of three other zoonotic (transmitted from animals to human) coronaviruses, such as sars in 2003, middle east respiratory syndrome (mers) in 2012, and swine acute diarrhea syndrome (sads) in 2017. surprisingly, these viruses have been originated from bats, a natural source of various other highly lethal zoonotic viruses (such as hendra, nipah, ebola, and marburg viruses). sars and sads viruses were claimed to be originated in china [15, 16] . based on these facts, scientists including chinese research group from wuhan (primary epicenter of also warned further possible coronavirus outbreaks from bats and with a high probability that outbreak will occur in china [17] . both sars-cov and mers-cov bat β-coronaviruses crossed over to humans through an intermediary host, which was palm civet cats in the guangdong province of china (sars-cov, in 2002-2003, mortality rate 11%) and dromedary camels in saudi arabia (mers-cov, in 2012, fatality rate 34%), respectively [18] . in the case of covid-19, the virus was transmitted to human from bats, but the intermediary host animal(s) are not yet known. a study claimed that the intermediary host animal is pangolin due to the following findings on pangolin-cov, sars-cov-2, and batcov ratg13 viruses: (i) at the whole-genome level, both pangolin-cov and sars-cov-2 share 91.02% similarity among them, (ii) pangolin-cov and sars-cov-2 are reported to be the second closest relative to each other than to batcov ratg13, (iii) in the receptor binding domain (rbd) of spike glycoprotein, five key amino acid residues involved in the interaction with human angiotensin converting enzyme 2 (ace2) of pangolin-cov and sars-cov-2 are consistent, and (iv) only sars-cov-2 contains a potential cleavage site for furin proteases unlike both pangolin-cov and ratg13 [19] . it was also concluded that the transmission of human sars-cov-2 virus from bat may include more than one intermediary host including pangolins [20] . similar to sars-cov, the 2019-ncov is reported to have the capability to transmit efficiently among humans due to familial cluster of pneumonia [9] . several cases were reported person-to-person transmission of this virus not only through family settings, but were also in hospital and infected travelers [9, 21] . person-to-person transmission of the sars-cov-2 infection is occurred via airborne droplets to the nasal mucosa in closed environments, close contact between people, unwashed hands, and touching contaminated surfaces with less possibilities. within the incubation period ranges from 2 to 14 days in general, sars-cov-2 may replicate locally in cells of the ciliated epithelium resulting cell damage and inflammation. primarily, respiratory secretions of any infected person are used to diagnose the presence of virus by special molecular tests including normal/low white cell counts with elevated c-reactive protein (crp) [18] . additionally, abnormal computerized tomographic (ct) chest scan is also proved to be helpful to diagnose any infected person even for those with no symptoms or mild disease [18] . the sars-cov-2 showed lower mortality but faster spreading than sars-cov and mers-cov. isolation of sars-cov-2 from oral swabs, bronchoalveolar lavage fluid, and stool proved them to be highly contagious [22, 23] . sars-cov-2 infects human by interacting with a functional receptor, metallopeptidase named angiotensin converting enzyme 2 (ace2), for its successful cellular entry ( figure 2 ) [22, 24] . crystal structure of the c-terminal domain of spike protein in complex with human ace2 (hace2) revealed an overall similar binding mode as that of sars-cov with hace2 [24] . it was determined that 2019-ncov uses ace2 as a cellular entry receptor in human, chinese horseshoe bats, civets, and pigs but not for mice and cells without ace2 protein expression capability [22] . other coronavirus receptors, such as aminopeptidase n and dipeptidyl peptidase 4 do not play any role for cellular entry of 2019-ncov [22] . previous studies revealed that almost all human organs are known to have ace2 mrna, though the protein expression of ace2 mrna was largely unknown. such ace2 receptor is found to be present in arterial and venous endothelial cells, arterial smooth muscle cells in the lungs, stomach, small intestine, liver bile ducts, colon, skin, kidney parietal epithelial cells, lymph nodes, and in the brain [25] . the surface of lung alveolar epithelial cells and enterocytes of the small intestine also express ace2 protein allowing them to be infected by sars-cov-2 [25] . the tissues of the upper respiratory tract are not the primary site of entrance for sars-cov, as oral and nasal mucosa and nasopharynx did not show ace2 expression on the surface of epithelial cells, rather upper respiratory tract might be susceptible to secondary infections from the infected lower respiratory tract. lower lungs may show higher opacity in the ct scans due to its more ace2 expression [26] . higher viral loads have been recorded in the nose than the in throat, with similar viral loads seen in asymptomatic and symptomatic patients [27] . coronaviruses generally are found to cause acute and chronic respiratory, enteric, and central nervous system diseases in humans as well as in other animals. the symptoms of a covid-19 patient are usually fever, cough, sore throat, breathlessness, fatigue, and feeling of discomfort. for most of the people, it is found mild. for elderly and the patient with comorbidities may develop pneumonia, acute respiratory distress syndrome (ards), and multi-organ dysfunction leading to death. many infected people are found to be asymptomatic causing a problem for early detection and controlling the spread of disease. mortality rate estimated by the world health organization (who) (as of 3 march 2020) is 3.4% (https://www.worldometers.info/coronavirus/coronavirus-death-rate/). further, speculation about the association of human coronaviruses with more serious human diseases (such as multiple sclerosis, hepatitis, or enteric disease in infants) are still under question due to no proper evidence [8] . in absence of any approved therapeutics or vaccines for the treatment of covid-19, who has promoted "test, isolate, and trace" method as a preventive measure. thus, early, rapid, and accurate diagnosis of covid-19 patients is becoming very crucial to control the sources of infection and to prevent further community spread. with a gradual understanding of biological properties of sars-cov-2, various diagnostic methods and device strategy with point of care facilities have been developed for covid-19 detection worldwide. a summary of various diagnostic methods (table 1) are presented for the covid-19 detection. various countries approved and implied different testing methods according to the regulation of their own health agencies based on situation and availabilities. below subsections are summarized the recent developments on diagnostic methods based on (i) nucleic acid, (ii) protein, (iii) chest scan and (iv) autopsy. nucleic acid-based detection strategy has been widely used against detection of various diseases, including coronavirus and recent covid-19. in this section, we review some nucleic acid-based detection methods that are commonly being employed for the diagnosis of covid-19. polymerase chain reaction (pcr) is an enzymatic method widely used in molecular biology to make millions to billions of copies of a specific dna sample [28] . this method involves following steps in a series or cycles of temperature changes: (i) denaturation: separating the two strands of the dna containing the gene segment with the application of heat, (ii) annealing-marking gene segment of each strand of dna with a primer, (iii) primer extension: using a dna polymerase to assemble a copy alongside each segment, and (iv) repeat: continuously copy the copies [28] . various pcr-based methods are an indispensable, common, and rapid techniques for scientist to amplify a minute nucleic acid sample to a large enough amount for a number of applications [29] . due to high sensitivity and high sequence specificity, the pcr-based method has been used as a routine and reliable technique for detecting coronaviruses. coronavirus is a rna virus, so in general reverse transcriptase-pcr (rt-pcr) method is implied as follows: coronavirus rna is transcribed into cdna by reverse transcription, then the pcr is performed on cdna, and finally detection of pcr product through specific detection method(s) (gel visualization and sequencing) [30, 31] . real-time: real-time reverse transcriptase-pcr (rt-pcr) detection method is evolved as a common platform for detection of all kinds of coronaviruses due to its low cost per test, less time-consuming process and more sensitive than the conventional rt-pcr assay [32, 33] . the whole genome sequence of sars-cov-2 enabled to develop pcr-based kits to diagnose covid-19 in laboratory and clinical settings [34] [35] [36] [37] [38] [39] . corman et al. [34] developed a robust diagnostic methodology considering the sars-related virus sequences available in genbank. a close genetic relatedness to the 2003 sars-cov and synthetic nucleic acid technology helped this process to design and validate such strategy without using any virus isolates and samples from infected person. such a technique can successfully discriminate 2019-ncov from sars-cov. this approach provided the first version of the diagnostic protocol to the who from exclusivity testing on 75 clinical samples (13 january 2020). a real-time rt-pcr based test is found to be more sensitive than radiological test for pediatric patients [36] . pediatric patients with milder symptoms, showed no clear clinical signs or chest x-ray findings but their real-time rt-pcr exhibited positive results. further, in this report, real-time rt-pcr showed positive results in rectal swab-testing but negative results in nasopharyngeal swab-testing for eight out of ten pediatric patients suggesting shedding of virus in the gastrointestinal tract and a possible fecal-oral transmission. wang's group [37] reported that the rt-pcr based findings using different types of clinical specimens collected from 82 infected individuals. in their study, it was found that viral loads were significantly correlated among 30 pairs of throat swab and sputum samples. overall, real-time rt-pcr based method enables developing a high-throughput testing for rapid, on-demand, low-cost, reliable, quantitative detection technique against covid-19 in clinical settings [39] . probe free: a team of indian institute of technology, delhi, india reported first probe-free real time pcr assay for covid-19 detection (http://www.iitd.ac.in/content/icmr-approves-probe-freecovid-19-detection-assay-developed-iit-delhi-0). they have used comparative sequence analyses to identify unique regions (short stretches of rna sequences) in the sars cov-2 genome, which are not present in other human coronaviruses. in this highly sensitive assay, primers can specifically target unique regions (conserved in over 400 fully sequenced) of covid-19 genomes, which was reported after extensive optimization using synthetic dna constructs followed by in vitro generated rna fragments. indian council of medical research has approved this technique as it does not require any fluorescent probes (thus low-price) but still useful for high throughput testing. isothermal amplification of nucleic acids is a rapid, efficient, and alternative amplification technique than pcr. this process can be applied at a constant temperature without any thermos-cycling apparatus, unlike in the case of pcr [40, 41] . the isothermal amplification technique can be performed in water bath, on the cell surface, or even inside living cells, making it a superior technique over pcr [40, 41] . based on reaction kinetics of isothermal nucleic acid amplification, it is divided to exponential amplification, linear amplification, and cascade amplification. these are further sub-divided into transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of rna technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of dna, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification, based on the developments in molecular biology of dna/rna synthesis [40, 41] . furthermore, the use of microfluidic chips, capillary platforms, and test paper with isothermal amplification technique has been developed for single-cell or single-molecule analysis. among these, loop-mediated isothermal amplification (lamp) has been implied successfully for coronavirus detection [42] [43] [44] [45] . lamp technique can amplify target nucleic acid sequence using two or three sets of primers and a polymerase at a constant temperature (~60-65 • c) [46] [47] [48] . in comparison to pcr-based technique, lamp can produce considerably higher amount of dna with high strand displacement and replication activity due to the use of additional pair of "loop primers". park et al. [46] developed reverse transcription lamp (rt-lamp) assay(s) to detect genomic rna of sars-cov-2. these rt-lamp assays (in combination with leuco crystal violet colorimetric detection method) can detect as low as 100 copies of sars-cov-2 rna within 30 min. these rt-lamp assays were highly specific towards sars-cov-2 compared to other human coronaviruses (hcov-229e, hcovoc43, mers-cov, and sars-cov). yu et al. [49] also developed a rapid and sensitive isothermal lamp based method (ilaco) for the detection of covid-19 virus rna or cdna samples. in this method, ilaco was used to amplify a fragment of the orf1ab gene using 6 primers, which was proved to be specific for sars-cov-2 species (i.e., low chance for false positives) in comparison to the sequences of 11 related viruses by the help of online tool primer-blast (including 7 similar coronaviruses, 2 influenza viruses and 2 normal coronaviruses). ilaco can detect synthesized rna equivalent to 10 copies of 2019-ncov (performance is comparable to taqman based qpcr detection method), where reaction time varied from 15-40 min based on virus load in the collected samples. another lamp-based colorimetric detection method was reported to identify sars-cov-2 virus rna from purified rna or cell lysis (without an rna purification step) [48] . the sensitivity of this portable method is equivalent to a commercial rt-qpcr test with only heating and visual inspection. zhu et al. [47] demonstrated a successful and accurate diagnosis of covid-19 using one-step rt-lamp coupled with nanoparticles-based biosensor (nbs) assay (rt-lamp-nbs) within approximately 1 h (from sample collection to result interpretation). they have employed two designed lamp primer sets (f1ab-rt-lamp and np-rt-lamp), heating block (to maintain a constant temperature at 63 • c), a real-time turbidity (la-320c) and visual detection reagents (vdr) in addition to nbs interpretation to simultaneously amplify and detect genes of sars-cov-2 in a "one-step" and "single-tube" reaction. the sensitivity of sars-cov-2 rt-lamp-nbs was 12 copies (each of detection target) per reaction, whereas no cross-reactivity was observed for all pathogens of non-sars-cov-2 (virus, bacteria, and fungi). the rt-lamp-nbs assay showed 100% the analytical sensitivity of sars-cov-2 for oropharynx swab samples of clinically diagnosed covid-19 patients and 100% specificity for clinical samples collected from non-covid-19 patients. crispr-cas (clustered regularly interspaced short palindromic repeats-crispr associated) is an adaptive immune system, which was discovered first in escherichia coli in 1987 and later also in other bacteria species. these are found predominantly in archaea (87% of genomes) than in bacteria (50% of genomes) [50, 51] . being an immune system of archaea and bacteria, crispr and crispr-associated proteins deliver protection against invasive nucleic acids (such as dna, or rna from phages, plasmids, and other exogenous dna elements) [50, 51] . scientists later exploited this immune responsive system by reengineering to target parts of genetic material for precise genetic alterations of any particular cellular type, which is the basis of crispr therapeutic and diagnostic platforms for human [52, 53] . this adaptive immune system is also widely used as a tool for sars-cov-2 detection. crispr associated enzyme cas13 has already been utilized for rapid and portable sensing for successful rna-targeting [54] . a specific high-sensitivity enzymatic reporter unlocking (sherlock) platform was developed by combining isothermal preamplification with cas13 to detect single molecules of rna or dna for dengue or zika virus [55] . an updated sherlock protocol has been reported for multiplexable, portable, rapid, and quantitative covid-19 detection (https://broad.io/sherlockprotocol), which can target sequences in a range between 20 and 200 am (10-100 copies per microliter of input). another development of accurate crispr-cas12-based lateral flow assay able to detect sars-cov-2 with 95% positive predictive agreement and 100% negative predictive agreement from respiratory swab rna extracts (less than 40 min) [56] . another newly developed method, sars-cov-2 dna endonuclease-targeted crispr trans reporter (detectr), was found to perform simultaneous reverse transcription and isothermal amplification by (i) rt-lamp for rna extracted (for nasopharyngeal or oropharyngeal swabs), (ii) cas12 detection of predefined coronavirus sequences, and (iii) cleavage of a reporter molecule confirms, which detects the virus [56] . a fncas9 editor linked uniform detection assay (feluda) was developed for detecting nucleotide sequences, classifying nucleobase identity, and inferring zygosity [57] . feluda is able to distinguish clear signatures of sars-cov-2 sequence in synthetic dna within one hour using a specific ribonucleoprotein (rnp) from non-specific rnp (such as h1n1 or hbb). feluda can also clearly distinguish between two sars-cov-2 and sars-cov-1 sequences. this approach further can be developed as lateral flow assay on a paper strip to distinguish sars-cov-2 synthetic dna using sars-cov-2 specific rnp. protein-based testing has become as an alternative and additive detection strategy in addition to nucleic-acid based testing methods for coronavirus [58] . in response to any infected viral protein antigens, antibodies (i.e., a blood protein produced in response to and counteracting a specific antigen) are generated in patient's body resulting a very specific antigen-antibody (ag-ab) serological interaction. detection of this specific antibody level(s) due to the sars-cov-2 infection can be useful for surveillance of covid-19 pandemic. this indirect serological test opens up wide range of possibilities, such as, (i) successful detection of asymptomatic patients, (ii) creating large a window of testing time even with a gradual decrease of viral load, (iii) protect community transmission due to false negative results by other methods, and (iv) proper guidance for individual quarantine period [59] [60] [61] [62] [63] [64] [65] [66] . kwok-yung yuen's group [61] successfully detected antibodies generated in response to sars-cov-2 viral proteins at the time when the detection of the viral proteins become difficult due to gradual declining trend of viral load(s). serological test using enzyme-linked immunosorbent assay (elisa) for antibodies (immunoglobulin m, igm and immunoglobulin g, igg) is more confirmatory and unreliable results from oral swabs for 2019-ncov detection [62] . this test can be applied for respiratory, blood, or fecal samples. guo et al. [63] conducted a covid-19 profiling study on early humoral response based on iga, igm, and igg response. this study found that igm and iga antibody were detected 5 days, while igg was detected 14 days after symptom onset, with a positive rate of 85.4%, 92.7%, and 77.9%, respectively [63] . the detection of igm by elisa was found more efficient than that of qpcr after 5.5 days of symptom onset [63] . a successful immunological field-effect transistor (fet)-based biosensing device was developed for detecting sars-cov-2 in clinical samples, where the sensor was developed by conjugating a specific antibody against sars-cov-2 spike protein to graphene sheet coated fet [64] . this rapid diagnostic device for sars-cov-2 antigen requires no sample pre-treatment or labelling. this is a highly-sensitive detection method for the sars-cov-2 spike protein at concentrations of 1 fg/ml in phosphate-buffered saline and 100 fg/ml in clinical transport medium, 1.6 × 10 1 pfu/ml in culture medium and 2.42 × 10 2 copies/ml in clinical samples. the false positive results in serological tests for covid-19 is a concern due to the presence antibodies generated against other coronaviruses (such as for common cold) irrespective of the presence of sars-cov-2 antibodies. recently, researchers have got a high frequency of cross-reactivity in plasma samples from 15 covid-19 patients against the s protein of sars-cov-2 and sars-cov [65] . however, more accurate antibody-based detection method can be made with additional features of infected patient in serological test, such as (i) combined (igm and igg) antibody assay rather than a single antibody test, (ii) more number of testing, (iii) report of elevated levels of c-reactive protein, d-dimer, lymphocytes, leukocytes, or blood platelets [66] . there are growing concerns regarding the covid-19 testing despite of huge efforts in this direction. due to sudden outbreak and a huge increase of covid-19 cases, a sufficient number of covid-19 test kits are unavailable in hospitals and healthcare centers. further, an automatic detection system with a quick diagnostic capability could be an alternative or auxiliary method to prevent community spreading of covid-19. in this situation, most countries have recommended the rt-pcr-based methods as the standard technique for covid-19 diagnosis. serological tests are also considered to be the primary technique for covid-19 detection. however, small hospitals, health centers in sub-urban and village areas, even private hospitals in sub-urban area may not have an approved rt-pcr testing center or pcr testing infrastructure facilities. on the other hand, chest-scan, a routine technique implied for prominent pneumonia pattern, has been evolved as useful non-invasive technique for covid-19 detection [67] . both chest x-ray and computed tomography (ct)-scan were successful to distinguish the manifestations of typical pneumonia in the case of mers-cov and sars-cov infection [68, 69] . these scans have helped to diagnoses suspected person to isolate and treat more quickly, even when the rt-pcr based test did not respond properly. such tests have proven lung histology (lung damage or holes/honeycomb-like appearance) of covid-19 patients [70] . thus, chest-scan is useful for suspected covid-19 patients with negative rt-pcr result. 3.3.1. x-ray x-rays, a form of high-energy electromagnetic radiation, are shorter wavelengths than uv rays and longer wavelengths than gamma rays. x-ray machines are widely available sophisticated diagnostic imaging technique for body, bone and other dense objects that can block the radiation through a limited exposure to radiation. in addition, x-ray scans can be used for lung infections, pneumonia and tumors. an automatic prediction of covid-19 was successfully reported using chest x-ray images and a deep convolution neural network based pre-trained transfer models (resnet50, inceptionv3 and inception-resnetv) [71] . these pre-trained transfer models helped to obtain a higher prediction accuracy for small x-ray dataset. this model has end-to-end structure without manual feature extraction and selection methods, where the resnet50 is an effective one among all pre-trained models in the small dataset (50 covid-19 vs. 50 normal). computed tomography (ct) scan is a computer-assisted medical imaging device which combines cross-sectional (tomographic) scanned images of specific areas or virtual slices of any organ taken from different angles producing a 3d view of that particular organ. ct-scan is one of the methods used to diagnose various abnormalities of the chest (such as pneumonia, lung cancer etc.) [72, 73] . thus, chest ct-scan is also being used as a fast, painless, non-invasive and accurate auxiliary diagnostic method in addition to the rt-pcr test for the suspected covid-19 patient [74] [75] [76] . the national health commission of china included the chest ct findings as evidence of clinical diagnosis of covid-19 for patients in hubei province at the fifth edition of the diagnosis and treatment program of 2019 new coronavirus pneumonia due to the false-negative rate of rt-pcr test for covid-19 patient [74] . several groups found chest ct scan more sensitive and better diagnostic tool in comparison to rt-pcr for covid-19 detection [75, 76] . recent investigations demonstrated that the ct-scan of covid-19 patient(s) clearly showed bilateral pulmonary parenchymal ground-glass and consolidative pulmonary opacities, with a rounded morphology, crazy-paving pattern, linear opacities, and peripheral lung distribution [77, 78] . in contrast, other study did not find any lung cavitation, discrete pulmonary nodules, pleural effusions, and lymphadenopathy [77] . further, high-resolution ct (hrct)-scan for the chest is reported to be important tool to help clinicians to diagnose quickly and accurately the effected lung disease [79] . artificial intelligence (ai) and deep learning-based automated ct image analysis of lung have been also developed to distinguish covid-19 pneumonia and influenza-a viral pneumonia [80] [81] [82] . these automated deep learning based methods can produce graphical pattern of a particular covid-19 patient which is helpful for clinician to diagnose prior to pathogenic testing [83] . in spite of such clinical diagnostic values, ct scan still fails to come at the forefront of covid-19 diagnosis due to the following reasons: (i) it is expensive, (ii) it requires technical expertise, and (iii) it is incapable of distinguishing sars-cov-2 pneumonia from other viral pneumonia and hysteresis. an autopsy report, through examination of a corpse by dissection, is an important source of information for research purposes to evaluate any disease causing death. autopsy has been proved to be hugely beneficial to diagnose emerging and reemerging infectious diseases, like covid-19 [84] [85] [86] . in contrary, several groups raised their concern that the few autopsies have been performed on patients who died with suspected or confirmed covid-19 infection especially in the primary epicenters of pandemic (such as china and italy) [85, 87] . despite the suggestion by who on performing post-mortem examinations for covid-19 deaths with following recommended safety procedures, many governments including italy discouraged the practice of autopsy during the period of increasing number of death and even some scientific report highlighted that the post-mortem examination does not have any primary diagnostic role, whereas autopsy may still have a clinical role in selected cases [88, 89] . though based on autopsies, physicians can determine a profound change of the view of covid-19 disease not as a pneumonia but a systemic, vascular disease, putatively generated by autoimmunity [85] . thus, a strong recommendation was urged to perform full autopsies on patients who died with suspected or confirmed covid-19 infection with recommended exceptional biosafety guidelines to reduce the further spread of potential infection from any corpse. there is currently no clinically proven therapeutic regimen to prevent and eradicate sars-cov-2 infection [90] . covid-19 is being managed by the supportive treatment (oxygenation and ventilation, conservation fluid management). however, the use of broad-spectrum antibiotics [91] . this section summarizes various treatment modalities for covid-19 ( figure 3 ). important tool to help clinicians to diagnose quickly and accurately the effected lung disease [79] . artificial intelligence (ai) and deep learning-based automated ct image analysis of lung have been also developed to distinguish covid-19 pneumonia and influenza-a viral pneumonia [80] [81] [82] . these automated deep learning based methods can produce graphical pattern of a particular covid-19 patient which is helpful for clinician to diagnose prior to pathogenic testing [83] . in spite of such clinical diagnostic values, ct scan still fails to come at the forefront of covid-19 diagnosis due to the following reasons: (i) it is expensive, (ii) it requires technical expertise, and (iii) it is incapable of distinguishing sars-cov-2 pneumonia from other viral pneumonia and hysteresis. an autopsy report, through examination of a corpse by dissection, is an important source of information for research purposes to evaluate any disease causing death. autopsy has been proved to be hugely beneficial to diagnose emerging and reemerging infectious diseases, like covid-19 [84] [85] [86] . in contrary, several groups raised their concern that the few autopsies have been performed on patients who died with suspected or confirmed covid-19 infection especially in the primary epicenters of pandemic (such as china and italy) [85, 87] . despite the suggestion by who on performing post-mortem examinations for covid-19 deaths with following recommended safety procedures, many governments including italy discouraged the practice of autopsy during the period of increasing number of death and even some scientific report highlighted that the postmortem examination does not have any primary diagnostic role, whereas autopsy may still have a clinical role in selected cases [88, 89] . though based on autopsies, physicians can determine a profound change of the view of covid-19 disease not as a pneumonia but a systemic, vascular disease, putatively generated by autoimmunity [85] . thus, a strong recommendation was urged to perform full autopsies on patients who died with suspected or confirmed covid-19 infection with recommended exceptional biosafety guidelines to reduce the further spread of potential infection from any corpse. there is currently no clinically proven therapeutic regimen to prevent and eradicate sars-cov-2 infection [90] . covid-19 is being managed by the supportive treatment (oxygenation and ventilation, conservation fluid management). however, the use of broad-spectrum antibiotics [91] . this section summarizes various treatment modalities for covid-19 ( figure 3) . viral infection is always a major concern for morbidity and mortality in animals and humans worldwide. development of antiviral drugs have been always a pressing need to treat such viral infections. since the approval of first antiviral drug, idoxuridine in 1963, 90 drugs were clinically approved to treat nine human infectious diseases (human immunodeficiency virus, hiv; hepatitis b virus, hbv; hepatitis c virus, hcv; herpesvirus; influenza virus; human cytomegalovirus; varicella-zoster virus; respiratory syncytial virus; and human papillomavirus) [92] . the antiviral drugs mostly inhibit the viral development rather than destroying the target pathogen unlike most antibiotics. a broad-spectrum antiviral is found to be effective against a wide range of viruses based on drug repurposing strategy [93] . drug repurposing or drug repositioning is a cost-effective and time-efficient alternative strategy, which involves the recycle or re-use of clinically approved drugs for new disease instead of searching of new drugs [94, 95] . in contrary to in vitro phenotypic screening of known drugs, in silico/computational drug repurposing strategy is a hypothesis-driven approach to identify the drugs for the treatment of any disease using big data analysis [96, 97] . the drug-repurposing has been implied for several human diseases including antiviral drug development against coronavirus [95] . various groups have proposed number of drug candidates through drug-repurposing (in vitro and in silico) for covid-19 treatment [98] [99] [100] [101] . who focused and initiated the "solidarity trial" (announced on 18 march 2020) of four existing antiviral compounds/formulations ( figure 4 ) to assess their clinical benefit against covid-19 [102, 103] . viral infection is always a major concern for morbidity and mortality in animals and humans worldwide. development of antiviral drugs have been always a pressing need to treat such viral infections. since the approval of first antiviral drug, idoxuridine in 1963, 90 drugs were clinically approved to treat nine human infectious diseases (human immunodeficiency virus, hiv; hepatitis b virus, hbv; hepatitis c virus, hcv; herpesvirus; influenza virus; human cytomegalovirus; varicellazoster virus; respiratory syncytial virus; and human papillomavirus) [92] . the antiviral drugs mostly inhibit the viral development rather than destroying the target pathogen unlike most antibiotics. a broad-spectrum antiviral is found to be effective against a wide range of viruses based on drug repurposing strategy [93] . drug repurposing or drug repositioning is a cost-effective and time-efficient alternative strategy, which involves the recycle or re-use of clinically approved drugs for new disease instead of searching of new drugs [94, 95] . in contrary to in vitro phenotypic screening of known drugs, in silico/computational drug repurposing strategy is a hypothesis-driven approach to identify the drugs for the treatment of any disease using big data analysis [96, 97] . the drug-repurposing has been implied for several human diseases including antiviral drug development against coronavirus [95] . remdesivir, an antiviral compound, which showed activity against multiple variants of ebola virus in cell-based assays and rhesus monkey model. chloroquine (cq) and its derivative hydroxychloroquine (hcq), antiviral compound(s) have been used to treat malaria and amebiasis. a combination of lopinavir and ritonavir, is co-formulated for hiv-1 treatment. another combination of lopinavir and ritonavir plus interferon-beta (lpv/rtv-ifnb) has been approved for the treatment of relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis. about 115 clinical trials were identified by belhadi et.al., which includes open-label studies (46%), double-blind (13%), and single blind studies (10%) [104] . they also classified the number of trials (n) and total numbers of planned inclusions (n) for lopinavir/ritonavir (n = 15, n = 2606), chloroquine (n = 11, n = 1102), hydroxychloroquine (n = 7, n = 1048), and remdesivir (n = 5, n = 2155). in contrary, several controversial reports including toxic side effects on these promising candidates under clinical trial(s) raised some challenging questions for researchers. those have been presented below: remdesivir, an antiviral compound, which showed activity against multiple variants of ebola virus in cell-based assays and rhesus monkey model. chloroquine (cq) and its derivative hydroxychloroquine (hcq), antiviral compound(s) have been used to treat malaria and amebiasis. a combination of lopinavir and ritonavir, is co-formulated for hiv-1 treatment. another combination of lopinavir and ritonavir plus interferon-beta (lpv/rtv-ifnb) has been approved for the treatment of relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis. about 115 clinical trials were identified by belhadi et.al., which includes open-label studies (46%), double-blind (13%), and single blind studies (10%) [104] . they also classified the number of trials (n) and total numbers of planned inclusions (n) for lopinavir/ritonavir (n = 15, n = 2606), chloroquine (n = 11, n = 1102), hydroxychloroquine (n = 7, n = 1048), and remdesivir (n = 5, n = 2155). in contrary, several controversial reports including toxic side effects on these promising candidates under clinical trial(s) raised some challenging questions for researchers. those have been presented below: a report presents that patients (with symptom duration of 10 days or less) receiving remdesivir showed clinical improvement than those receiving placebo, but it did not make any statistically significant clinical benefits [105] . an open-label non-randomized clinical trial study demonstrated significant decrease in viral load and carriage duration in covid-19 patients receiving hydroxychloroquine (600 mg/day during ten days). this treatment showed enhanced effects in combination with azithromycin, but it identified serious methodological flaws [106, 107] . another randomized clinical study did not make any difference in recovery rates upon hydroxychloroquine treatment in 30 covid patients [108] . however, a hype on cq and hcq has created drug shortages and affected other potential treatments (such as for patients with lupus). there was no significant benefit (clinical improvement) observed with lopinavir-ritonavir treatment [108] . mortality and percentages of patients with detectable viral rna at various time points were similar in the lopinavir-ritonavir group and the standard-care group. it was also reported median time to clinical improvement was shorter by one day for lopinavir-ritonavir group than that observed with standard care. vaccination is one of the most effective and preventive medications against various diseases caused by pathogens (such as virus or bacteria). currently there are about 25 approved vaccinations available against various life-threatening diseases, including measles, polio, tetanus, diphtheria, meningitis, influenza, typhoid, and cervical cancer (https://www.who.int/topics/vaccines/en/). a vaccine typically contains an agent (weakened or killed forms of any microbe, its toxins, or one of its surface proteins), which though resembles a disease-causing microorganism, but provides active acquired immunity to that particular infectious disease [109, 110] . an antiviral vaccine helps to boost our natural immune response to an invading virus by priming it to recognize viral antigens. in general, antiviral vaccines can be classified as follows: (i) inactive or live-attenuated viruses, (ii) virus-like particle (vlp), (iii) viral vectors, (iv) protein-based, (v) dna-based, and (vi) mrna-based vaccines [110, 111] . like many other diseases, the vaccine development is not successful and conclusive for coronavirus disease. until now, there is no proper vaccine is developed or approved for the treatment of human coronavirus diseases (such as sars-cov and mers-cov) [111] [112] [113] . most big pharmaceutical companies also in the race to develop effective vaccines for cov infection [112, 113] . the existing knowledge on previous strategies for cov vaccine developments can benefit the ongoing research as sequence analysis of the sars-cov-2 genome showed close relation to sars (80%) and to one bat ratg13 sars-like cov (96%) than to mers cov (54%) [112] . liu et al. [111] reported that 188 patents (mentioned in cas content collection) are directly associated with anti-sars and anti-mers vaccines (15 patents on inactive and live-attenuated virus vaccines, 28 patents on dna vaccines, 21 patents on viral vector vaccines, 13 patents on vlp vaccines, and three patents on mrna vaccines) with a demonstrated immune response, which could be a huge boost for covid-19 vaccine developers. an accelerated evaluation of next-generation vaccine for covid-19 has been triggered as soon as the genetic sequence of sars-cov-2 is published on 11 january 2020 [112] [113] [114] [115] . as of 28 july 2020, 164 vaccine candidates have been identified (https://www.who.int/who-documents-detail/draft-landscape-of-covid-19-candidatevaccines), of which 25 candidate vaccines are in clinical evaluation ( table 2 ) and 139 candidate vaccines are in preclinical stages. associated problems and pitfalls are surely a major concern resulting huge roadblock for vaccine discovery against covid-19. the pitfalls are many folded, which as follows: (i) antibody-dependent enhancement (ade) of viral replication for vaccinated people in future recurrence due to immune backfiring, (ii) rouge immunization resulting a huge damage on someone's own immune cells (such as neutrophil and basophil), (iii) in addition to immune response malfunctioning, three imperatives of vaccine effort: speed, manufacture and deployment at scale, and (iv) global access for a newly developed vaccine [116, 117] . further, new findings and hypotheses stirred up the debate on covid-19 research making it difficult as well as relevant not only in therapy but also in producing a life-saving vaccine, without any risk of generating immunity-based complications. (i) molecular mimicry: two independent groups (lucchese and flöel; macario and cappello) proposed molecular mimicry as the culprit in determining multi-organ damages (including anosmia, leukopenia, and vascular damage) in covid-19 patients [118] [119] [120] . indeed, there are confirmations that a number of human proteins share common epitopes with sars-cov-2 proteins and these epitopes are highly immunogenic. (ii) biphasic infection: due to (a) the ability of human coronaviruses to cause respiratory re-infections, regardless of pre-existing humoral immunity and (b) evidence on circulation of severe acute respiratory syndrome coronavirus type 2 (sars-cov-2) in italy before the detection of first covid-19 case, an hypothesis was given on biphasic infection, the immunological result of a prior viral infections either by sars-cov-2 or different strains of coronaviruses, or potentially even other respiratory viruses resulting increased susceptibility to more severe forms of covid-19, following a secondary infection with sars-cov-2 [121] . this theory is sustained by anecdotal clinical reports of "biphasic infection" and "cytokine storm" through a possible ade immunological mechanism, which was already observed with infections sustained by other coronaviruses (such as sars-cov and mers-cov) or other viruses (such as west nile virus and dengue). convalescent plasma (cp) therapy involves the administration of antibodies to a susceptible person to prevent or treat an infectious disease providing an immediate immunity [122] . in this therapy, donated blood from the infected person who've recovered from that infection have antibodies in their blood that can fight against the infection. the cp therapies have been tested since 1890s as a possible alternative and/or auxiliary treatment including sars-cov, ebola, influenza a (h5n1) etc. viruses [123, 124] . additionally, cp therapy did not show any serious and immediate adverse effects in earlier cov treatment [125] . the plasma with antibodies is prepared after separation of blood cells from the donated blood which can be extended to combat against covid-19. cp therapy has been generally approved as experimental treatment for patients with critical conditions. duan et al. [126] reported potentially improved clinical outcomes for 10 severe covid-19 patients with a dose of 200 ml of cp (derived from recently recovered donors with the neutralizing antibody titers above 1:640) with additional use of antiviral agents and maximal supportive care. this study confirmed a number of positive outcomes, including, (i) neutralizing antibody level increased rapidly up to 1:640 in five cases, while for other four cases maintained at a high level (1:640), (ii) increased oxyhemoglobin saturation within three days, (iii) increase in lymphocyte counts (0.65 × 10 9 /l vs. 0.76 × 10 9 /l), (iv) decreased c-reactive protein (55.98 mg/l vs. 18.13 mg/l), (v) varying degrees of absorption of lung lesions within seven days, (vi) undetectable viral load in seven patients who had previous viremia. additionally, treatment with human immunoglobulin is reported to increase same-day thrombotic event risk significantly (0.04 to 14.9%) [127] . transfer of blood substances may include inadvertent infection with another infectious disease agent and react to serum constituents resulting immunological reactions such as serum sickness [128] . more high-quality studies, adequate selection of donors with high neutralizing antibody titers, central blood banking facilities to process the donated serum, efficient serological and virological assays, production and the use of cp according to precise ethical and controlled conditions are needed for implementing this therapy further. latest studies showed that ace2, the key functional receptor of sars-cov-2, is highly expressed in kidney (nearly 100 times higher than in lung) resulting it to be a main target organ for sars-cov-2 attack [129] . thus, novel coronavirus infections hugely damage the kidney of any severe covid-19 patient suffering from an immunological damage due to a cytokine storm, the imbalance of pro-inflammatory and anti-inflammatory factors. thus, auxiliary continuous blood purification could be an essential technology to take care of inflammatory factors, cytokine storm, electrolyte imbalance, and acid-base balance for any covid-19 patient resulting a reduced renal work-load and a possible recovery of renal function [130, 131] . at present, extracorporeal blood purification technology is supplied for severely ill patient with novel coronavirus pneumonia as an auxiliary treatment to improve condition [132] . there is an increasing awareness, tendency, and agenda to use traditional (t) medicine (indigenous health traditions) and complementary and alternative medicine (cam) (methods outside the biomedical mainstream, particularly in industrialized countries). globally, half the population uses t/cam as a preventive measure [133, 134] . the national center for complementary and integrative health (nccih) of the national institute of health (nih), usa, has included various medical methods under the umbrella term cam, such as homeopathy, naturopathy, ayurveda, medicinal systems, and products originating from traditional medicine [135] . further, herbalism, aromatherapy, acupuncture, massage, and reflexology are also various name and forms among the most popular branches of cam [134] . the potential use of cam (sometimes along with conventional medicine) has been reported to be efficient therapeutic strategy against several virus associated diseases such as influenza, dengue, japanese encephalitis, hepatitis c, zika, and hiv [134] [135] [136] . considering the efficacy of cam against coronaviruses with minimal reported adverse effects on host cells, ministry of ayush (ayurveda, yoga & naturopathy, unani, siddha and homoeopathy), government of india, recommended scientists, researchers and clinicians to pursue research and use of indian herbal drugs on covid-19. through a memorandum, government of india is now trying to utilize the ayurveda, siddha, homeopathy, and unani system of medicine including prophylactic measures, intervention during quarantine, asymptomatic and symptomatic cases, public health research, survey, laboratory-based research etc. (https://www.ayush.gov.in/). indian traditional medicinal systems, one of the oldest treatments in human history (existed nearly 5000 years ago witnessed and scripted in ancient literature), played a significant role in encountering global health due to its antiviral, anti-inflammatory and antioxidant properties [137, 138] . such a traditional branch of science could be a potential option against covid-19 because of its proved efficacy not only in treatment but also in preventive strategy for several diseases, including respiratory viral infections through immunity boosting, rejuvenating lifestyle, and dietary management [137, 138] . ayush recommended selected traditional drugs for covid-19 as follows: http://ayush.mp.gov.in/sites/default/files/corona%20advisory_3.pdf. [139] . traditional chinese medicine (tcm) is also recently being included as one of the treatment modalities in china for covid-19. previous record on efficacious tcm against respiratory tract infectious diseases, such as lianhua qingwen capsules (exerts independent antiviral effects) and shufeng jiedu capsules (synergistic antiviral effects with western medicine) on influenza viruses, has encouragd the practice of tcm against covid-19 [140, 141] . wang et al. [142] reported massive improvement for four covid-19 patients as a result of combination therapy including chinese and western antiviral medicine, where they used lopinavir/ritonavir (kaletra®), arbidol, and shufeng jiedu capsule (sfjdc) with necessary supportive care. however, lack of high-quality research, standard clinical trials, sufficient research articles, clear mechanism of action, rigorous population studies, prospective business model are also urgently required to establish the therapeutic effect and implementation of t/cam among mass population. in spite of some emphatic results through traditional, complementary, and integrative products, practices, and practitioners against covid-19, north american and european governments are keeping their silence on these practices (https: //www.cdc.gov/coronavirus/2019-ncov/prevent-getting-sick/prevention.html) and rather warned of possible harm and overselling (https://www.fda.gov/news-events/press-announcements/coronavirusupdate-fda-and-ftc-warn-seven-companies-selling-fraudulent-products-claim-treat-or). john weeks, editor-in-chief of the journal of alternative and complementary medicine, mentioned this double standard attitude as he pointed out that "no practices have definitive evidence for benefit against covid-19, yet providers with other stripes are using experimental practices and off-label drugs every day in their desperate to ease patient suffering and elicit hope" [143] . prevention is the utmost important strategy to fight against covid-19 in the present situation. several preventive measurements are taken to reduce the spread and transmission of the covid-19 infection. this is classified as follows (i) contact tracing, (ii) sharing or proper dissemination of information, (iii) precautionary measurements ( figure 5 ). covid-19 [140, 141] . wang et al. [142] reported massive improvement for four covid-19 patients as a result of combination therapy including chinese and western antiviral medicine, where they used lopinavir/ritonavir (kaletra®), arbidol, and shufeng jiedu capsule (sfjdc) with necessary supportive care. however, lack of high-quality research, standard clinical trials, sufficient research articles, clear mechanism of action, rigorous population studies, prospective business model are also urgently required to establish the therapeutic effect and implementation of t/cam among mass population. in spite of some emphatic results through traditional, complementary, and integrative products, practices, and practitioners against covid-19, north american and european governments are keeping their silence on these practices (https://www.cdc.gov/coronavirus/2019ncov/prevent-getting-sick/prevention.html) and rather warned of possible harm and overselling (https://www.fda.gov/news-events/press-announcements/coronavirus-update-fda-and-ftc-warnseven-companies-selling-fraudulent-products-claim-treat-or). john weeks, editor-in-chief of the journal of alternative and complementary medicine, mentioned this double standard attitude as he pointed out that "no practices have definitive evidence for benefit against covid-19, yet providers with other stripes are using experimental practices and off-label drugs every day in their desperate to ease patient suffering and elicit hope" [143] . prevention is the utmost important strategy to fight against covid-19 in the present situation. several preventive measurements are taken to reduce the spread and transmission of the covid-19 infection. this is classified as follows (i) contact tracing, (ii) sharing or proper dissemination of information, (iii) precautionary measurements ( figure 5 ). track, trace, and share of the information on covid-19 are major preventive steps to stop spreading of sars-cov-2. a thorough, quick, and updated report should be always available covering various information (such as number of infected cases, casualties, regions affected in each country etc.) on easily accessible platforms. the promotion of "data science" or big data and data driven interdisciplinary research areas has helped a lot to control such global infectious disease or epidemics through extensive surveillance, sharing of epidemiological data, and patient monitoring [144] . benefits of big data with technological advancement has facilitated the communication among regional, national and international healthcare agencies to monitor future host adaption, viral evolution, infectivity, transmissibility, morbidity, mortality, mental health impact and psychological effects due to covid-19 epidemic. here we have mentioned some authentic responsible agencies and tools along with several controversies aroused from the use of data science as follows: who: who is providing a daily "situation report" as an update on covid-19 pandemic (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports), where who receives the data from national authorities. worldometer: worldometer is a free reference website (managed by an international team of developers, researchers, and volunteers) that provides counters and real-time statistics on various topics such as world population, government, economics, society, media, environment, food, water, energy, and health (https://www.worldometers.info/). now the data in website is currently available in 35 languages and will be available in 11 more languages soon (https://www.worldometers.info/ languages/, accessed on 1 may 2020). this reference website belongs to a united states of america (usa)-based digital company dadax (http://dadax.com/). presently, this website is being used quite popularly to provide various statistics (graphs, countries, death, symptoms, incubation, transmission, news) on covid-19 pandemic around the world (https://www.worldometers.info/coronavirus/) and the data is also trusted and used by several agencies including government organization of various countries. the speed and ease of communication is the heart of management to control the spread of any infectious diseases as it helps to build extensive surveillance, share of epidemiological data, and patient monitoring. mobile phone or especially smartphone with the help of various mobile apps (software application) can improve this purpose due to its speedy connectivity, computational power, remote access, electronic reporting, epidemiological data basing, real-time geospatial information, digitized process of contact tracing, more complete and shareable records, enhanced coordination among regional, national and global health agencies [145, 146] . a past experience on using smartphones for geospatial tracking of infectious diseases such as hiv, ebola, and tuberculosis has helped to build these devices further to control covid-19 epidemics by implementing healthcare strategies, and improving general awareness among masses [147] [148] [149] . further, smartphone embedded point-of-care testing and self-reporting facilities has helped to reduce risk of contracting covid-19 as these help actual and suspected patients with mild symptoms under self-quarantine to report remotely without going to any overcrowded hospital or healthcare centers [150, 151] . who launched mobile app (https://github.com/worldhealthorganization/app) to accurately track and trace covid-19 cases. there are also several official contact tracing apps available for the citizen of various countries to inform a person's own health to help governments in strategic control (e.g., "aarogya setu" in india, "covid watch" in usa, "nhs covid-19" in uk, "alipay health code" in china, etc.,). despite huge effort and help from data science, dueling in data caused also deviation, divisive and neglecting attitude regarding covid-19. this further facilitates the spread of pandemic, shifting epicenters, more casualties, economic loss and so many other secondary problems created due to pandemic. here we have summarized some problems associated with data handling. reproductive number: the basic reproduction number (r 0 ), an indication of the transmissibility of a virus in infectious disease epidemiology, has been used to represent the average number of new covid-19 infections generated by an infectious person in a totally naïve population [152] . it is estimated that (i) the number of infections is likely to increase for r 0 > 1, and (ii) transmission is likely to die out for r 0 < 1. thus, a true estimation of r 0 can be beneficial in terms of prevention of any infectious disease. liu et al. estimated higher reproductive number of covid-19 compared to sars coronavirus and also reported that the average reproductive number for covid-19 (r 0~3 .28) is considerably higher than that of who estimate (r 0 = 1.95) [153] . they mentioned that the current estimates of r 0 for covid-19 are possibly biased due to insufficient data and short onset time. "risk of death" measurement: estimation of the case fatality risk or the risk of death among positive cases is a common epidemiological practice to measure the severity of infection for a given disease. kobayashi et al. [154] mentioned several key epidemiological problems for assessment of the severity of covid-19 as follows-(i) "division of the cumulative number of deaths by that of cases tends to underestimate the actual risk because deaths that will occur have not yet observed, and so the delay in time from illness onset to death must be addressed"; (ii) "the observed dataset of reported cases represents only a proportion of all infected individuals and there can be a substantial number of asymptomatic and mildly infected individuals who are never diagnosed"; (iii) "ascertainment bias and risk of death among all those infected would be smaller when estimated using shorter virus detection windows and less sensitive diagnostic laboratory tests". they further suspected that the total number of reported covid-19 infections will be underestimated due to many undetected mild or asymptomatic cases that go. open data source: in this fight against covid19 , an open data source is a big step forward in the age of big data. amaro et al. [155] urged to make an effort through a "community letter" for sharing bimolecular simulation data on covid-19 to help and improve connection and communication among scientists and investigators working on simulation, experimental and clinical data. they committed to share methods, models, and results of their study openly and quickly to test findings, ensure reproducibility, test significance, eliminate dead-ends, and accelerate discovery on covid-19 applications. they committed to use preprint servers such as arxiv, biorxiv, and chemrxiv, open access data repositories such as zenodo, an open data sharing platforms for models and trajectories such as the molecular sciences software institute (molssi) sars-cov-2 biomolecular simulation data and algorithm store, the open science framework, the european open science cloud and several other agencies. they also make an appeal and encourage others for similar best practices on open data efforts in other research areas to prevent covid-19. cyber-attack: in this grave situation due to covid-19, the revolutionized advancement of information and communications technology is a crucial weapon to control the healthcare infrastructure in addition to businesses, deliveries of food and essential items to remote places, online grocery shopping etc. but there is a growing concern due to an increasing report of malicious attacks on information and communications technology during covid-19 as attackers find this an opportunity for financial gains and promoting evil intents. a 220 times increase in spam email and 260% in malicious urls have been reported from february to march 2020, in which the united states is found to be the epicenter for such targets [156] . healthcare systems, government and media outlets, financial services are some of most important organization and industries, which are identified at the risk of cyber-attack [157] . this team has identified the top ten such deadly cybersecurity threats amid covid-19 pandemic, which are as follows-(i) distributed denial of services (ddos) attack, (ii) malicious domains, (iii) malicious websites, (iv) malware, (v) ransomware, (vi) spam emails, (vii) malicious social media messaging, (viii) business email compromise, (ix) mobile threats, and (x) browsing apps. the worldometer website was reported under cyber-attack in march 2020 and was then hacked a few days later, resulting in incorrect information (dramatic rise on covid-19 statistics in vatican city) for approximately 20 min (https://www.euroweeklynews.com/2020/03/16/false-report-of-900k-dead-in-vatican-city-lastnight-i-nearly-fell-off-my-chair-reading-it/). use of contact tracing covid-19 apps also raised some security and privacy concern as it keeps personal database of any individual [158, 159] . thus there is a continuous debate on this issue and demand of more reliable apps among mass population, whereas governments are trying to make it a mandatory use as experts shown their confidence about these apps [160] . social media activism: containment measures are primary guideline to tackle the novel coronavirus (covid-19) pandemic. due to moving out of physical public spaces, people are using online platforms as even more prominent and powerful tools to communicate social discussion and understand the unprecedented global crisis. however, ferrara reported after tracking and studying 43.3m real-time english tweets about covid-19 (the large-scale data was collected since january 21, 2020, the day the first covid-19 case was reported on us soil to the dataset up to 12 march 2020, the day before the united states government announced the state of national emergency due to the covid-19 pandemic) that the information on social media platforms are populated by bots, automated accounts [161] . this study provided the evidence that high bot score accounts are used to amplify certain topics of discussion at the expense of others, such as (i) promotion of political conspiracies and divisive hashtags alongside with covid-19 content, and (ii) enabling participatory activism to shed light on issues that may otherwise be censored in china. thus, this study demands more nuanced and regulated discussion on social media platforms on covid-19. behavioral changes and use of protective gears have been prescribed as the first and foremost precautionary step to stop the spread of sars-cov-2. gradual understanding of the transmission of this virus and previous experience on successful behavioral imposition to control other epidemics such as aids (changes in sexual behavior, condom promotion, and government interventions) has helped a lot in that direction. though more efficient behavioral changes in daily lifestyle requires better understanding and proper implementation of the rules with time on covid-19 transmission. awareness on the risks from exposure level to respiratory droplets, airborne virus, contamination level from surfaces, concentration of transmission, incubation period, infectivity even before onset of symptoms in the incubation period, transmission from asymptomatic people will definitely be helpful to understand (i) behavioral changes and precautionary guidelines and (ii) use of protective gears. further use of telemedicine and robot can also play a crucial role as precautionary steps to combat against such infectious diseases. behavioral changes and precautionary guidelines: knowledge on covid-19 has helped to set precautionary guidelines in our day to day lifestyle. various health agencies such as who, the centers for disease control and prevention (cdc), national public health institute of the united states and many more recommended following general measures to prevent the spread of covid-19-(i) complete washing of hands often using an alcohol-based hand sanitizer to kill the sars-cov-2, (ii) avoid close contact, (iii) cover mouth and nose with a cloth or mask (e.g., n95 respirators) in public places, (iv) cover coughs and sneezes, (ii) avoid touching eyes, nose and mouth when outside, (iii) avoid travelling or gathering in crowded places, (iv) clean and disinfect frequently touched surfaces, (v) women with infants are encouraged to breastfeed their babies to enhance their immunity (https://www.cdc.gov/coronavirus/2019-ncov/prevent-getting-sick/prevention.html, https://apps.who.int/iris/handle/10665/330376). in addition to hand hygiene practices, who also has given proper guidelines for 1. sanitation and plumbing of covid-19 hospitals, quarantine centers; 2. toilets and the handling of feces of covid-19 patients; 3. safe management of health care waste; 4. environmental cleaning and laundry at healthcare facilities; 5. safe disposal of greywater or water from washing personal protective gears, surfaces and floors; 6. safe management of dead bodies; 7. management of waste generated at home; 8. treatment and handling requirements for excreta (https://www.who.int/publications-detail/water-sanitation-hygiene-and-waste-managementfor-the-covid-19-virus-interim-guidance). the guidance from national center for complementary and integrative health (nccih), national institutes of health prescribed a "healthy waiting" in life style, which includes social distancing (or physical distancing), mild exercise, stress reduction, restriction on smoking and alcohol (https://www.nccih.nih.gov/health/in-the-news-coronavirus-and-alternativetreatments). to prevent such health crisis and boost immunity with special reference to respiratory health, ministry of ayush, india recommended the following self-care guidelines in daily lifestyle (https://www.mohfw.gov.in/pdf/immunityboostingayushadvisory.pdf): further, home isolation or quarantine of suspected cases and those with mild illnesses have been entailed as prevention to reduce the burden on covid-19 hospitals for severe cases [162] . as a preventive measurement, countries have imposed not only inter nation lockdown, but also total or partial lockdown inside their territory to minimize transmission from foreign nationals, social gathering and day to day activity [163] . lockdown area are also classified based on infection progression and hotspot are also indicated to inform mass about the containment zone. thermal screening has become a common strategy to track the probable symptomatic cases at various juncture of transportation. it is obvious that such changes in daily life of general population and healthcare unit need more time to cope with. implementation of such changes is challenging in short time against rapid infectious nature of covid-19. according to the psychiatrists and allied professionals, covid-19 pandemic and such forceful precautionary guidelines created subsyndromal mental health at multiple levels in the general population, among healthcare workers, and in vulnerable populations resulting the symptoms of anxiety and depression, self-reported stress, insufficient sleep [164] . also, it is surely very conflicting to maintain balance between economy and public health during such long lockdown [165] . every section of society specially those belonging to lower socio-economic state is being affected economically sooner or later due to such crisis, which create a desperation to neglect such imposed rules on mass population resulting further increase of covid-19 cases. protective gears: various protective gears are playing an important role to stop the spread pandemic. frontline healthcare professionals, in addition to healthy mass population are at a huge risk in covid-19 transmission from patients, suspected cases. as of 12 april 2020, more than 2200 healthcare workers including doctors have been infected (https://health.economictimes.indiatimes.com/news/industry/whosays-over-22000-healthcare-workers-across-52-countries-infected-by-covid-19/75107238). in 2002 during the sars outbreak, nearly 21% of those virus-affected were healthcare workers [166] . this is an even more warning situation as it is important that the healthcare workers should be protected from infection to ensure uninterrupted medical facility and to prevent virus transmission to other patients. thus, use of protective gears such as personal protective equipment (ppe) kits, mask (tested n95 respirators), gloves and goggles are important accessories for healthcare professionals, whereas for mass population mask (more specifically n95 mask) is foremost important along with other protective guidelines. suddenly, very high demand of such protective gears resulted a huge shortage worldwide, which needs a huge workforce to support healthcare facilities in this crisis. additive manufacturing (i.e., 3d printing), an eminent technology for medical device preparation, came out as an innovative solution for covid-19 protective gears production with the help of other embedded technologies such as-(i) antimicrobial polymers and nanoparticles for making ppe, (ii) angiotensin-converting enzyme 2 coated nanoparticles containing respiratory masks, chewing gums and nasal filters (iii) preparation of recyclable decontaminating nanofiber filtered face masks, etc. [167, 168] . these ideas will surely be advantageous to prepare more effective protective gears for managing such pandemic, though further research is needed to implement such efforts to market. telemedicine: with the advancement of telecommunication technology, telemedicine has emerged as medical activity involving a doctor-patient distant interaction through telecommunication, which have been interchangeably known as telehealth (more politically correct term) or online health and e-health (fashionable term) [169] . such telecommunication-based remote medical health facility is surely advantageous against any infectious disease outbreak and thus it is recommended by various health agencies for covid-19 as it (i) reduces person-to-person contact by obeying social distancing, (ii) maintain balance in facilities with limited workforce, (iii) evolves as an alternative cost effective health provider in comparison to traditional home visit [169] [170] [171] . in spite of a promising future and willingness to use telehealth among patients, telemedicine suffers from various disadvantageous due to (i) less scope outside of emergency situations, (ii) clinician unwillingness and low acceptance among patients, (iii) problem with reimbursement, (iv) organization of the healthcare system. robotic gesture: robots already shown a huge potential as medical device for diagnosis to surgery including pediatric airway operation, oropharyngeal cancer operation etc. [172, 173] . in similar fashion, to combat infectious epidemics like covid-19, robots can play pivotal roles due to its abilities as follows-(i) large area cleaning and disinfection of contaminated surfaces (such as using uv light devices), (ii) diagnosis by automated or robot-assisted nasopharyngeal and oropharyngeal swab or blood testing, (iii) patient care by delivering medications and food to infected/suspected persons under quarantine, (iv) measuring vital signs, (v) assisting controls on transportation of mass population (such using thermal sensors and vision algorithms), (vi) help in telemedicine, (vii) facial tracking of any individual already having infection as a process of contact tracing, (viii) navigation in high-risk areas for sample transfer as well as delivery of medicines using autonomous drones or robot-assisted ground vehicles [174, 175] . such robot-assisted strategy to control epidemic may speed up the process, reduce the risk of infection specially for frontline health care professionals, and increase workforce ( figure 6 ). though despite such help in clinical care, logistics and reconnaissance, proper deployment of robots may face some problem due to privacy and security, incompatible diagnosis with the mutation of virus. tracing, (viii) navigation in high-risk areas for sample transfer as well as delivery of medicines using autonomous drones or robot-assisted ground vehicles [174, 175] . such robot-assisted strategy to control epidemic may speed up the process, reduce the risk of infection specially for frontline health care professionals, and increase workforce ( figure 6 ). though despite such help in clinical care, logistics and reconnaissance, proper deployment of robots may face some problem due to privacy and security, incompatible diagnosis with the mutation of virus. in conclusion, this article presented the current progress of four primary and important sphere of research in the battle against covid-19 pandemic, which are knowledge of the biological properties of virus, diagnostic modalities, treatment modalities, and prevention modalities. the huge development and deployment of advanced level of research and technology has helped to improve severely affected the global health condition due to covid-19. a prior knowledge on previous viral outbreaks and pandemic has really helped researchers, health agencies and administrations a lot in response to covid-19. in spite of such a plethora of research, the progress was hampered in many areas due to conflicting views and controversies about covid-19. several such grey areas in transmission, best diagnostic methods with no false positive reports, possible treatment outcome, figure 6 . possible preventive methodologies against infectious disease in future. in conclusion, this article presented the current progress of four primary and important sphere of research in the battle against covid-19 pandemic, which are knowledge of the biological properties of virus, diagnostic modalities, treatment modalities, and prevention modalities. the huge development and deployment of advanced level of research and technology has helped to improve severely affected the global health condition due to covid-19. a prior knowledge on previous viral outbreaks and pandemic has really helped researchers, health agencies and administrations a lot in response to covid-19. in spite of such a plethora of research, the progress was hampered in many areas due to conflicting views and controversies about covid-19. several such grey areas in transmission, best diagnostic methods with no false positive reports, possible treatment outcome, contact tracing, precautionary steps, proper public health management and many other fields has shattered the progress resulting a chaos and crisis in public health and economy. despite the conflict, differences in opinions are an important part of any constant progress. thus, these conflicting views have helped the field to grow and will create unprecedented opportunities further solving several unresolved questions. governments should encourage and endorse innovative research ideas to beat such pandemics, not only in basic research and biomedicine, but also in engineering, information technology and many unknown corners to get a marketable solution in spite 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review and evaluation. jmir mhealth uhealth 2016, 4, e25 real-time tracking of self-reported symptoms to predict potential covid-19 rapid implementation of mobile technology for real-time epidemiology of covid-19 estimate of the basic reproduction number for covid-19: a systematic review and meta-analysis the reproductive number of covid-19 is higher compared to sars coronavirus a community letter regarding sharing biomolecular simulation data for covid-19 ten deadly cyber security threats amid covid-19 pandemic covid-19 contact tracing and privacy: studying opinion and preferences. arxiv contact tracing mobile apps for covid-19: privacy considerations and related trade-offs. arxiv stiller, b. wetrace-a privacy-preserving mobile covid-19 tracing approach and application. arxiv #covid-19 on twitter: bots, conspiracies, and social media activism. arxiv social distancing during the covid-19 pandemic: staying home save lives inter nation social lockdown versus medical care against covid-19, a mild environmental insight with special reference to india covid-19 and mental health: a review of the existing literature beat covid-19 through innovation protecting health-care workers from subclinical coronavirus infection the role of additive manufacturing and antimicrobial polymers in the covid-19 pandemic angiotensin-converting enzyme 2 coated nanoparticles containing respiratory masks, chewing gums and nasal filters may be used for protection against covid-19 infection telehealth for global emergencies: implications for coronavirus disease 2019 (covid-19) lessons for the future telemedicine in the era of covid-19 artificial intelligence: who is responsible for the diagnosis? robotic-assisted surgery for primary or recurrent oropharyngeal carcinoma evaluation of an ultraviolet room disinfection protocol to decrease nursing home microbial burden, infection and hospitalization rates handling, and testing clinical specimens from persons under investigation (puis) for coronavirus disease this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest.diseases 2020, 8, 30 key: cord-339762-lh8czr0a authors: ng, dianna l.; al hosani, farida; keating, m. kelly; gerber, susan i.; jones, tara l.; metcalfe, maureen g.; tong, suxiang; tao, ying; alami, negar n.; haynes, lia m.; mutei, mowafaq ali; abdel-wareth, laila; uyeki, timothy m.; swerdlow, david l.; barakat, maha; zaki, sherif r. title: clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates, april 2014 date: 2016-03-31 journal: the american journal of pathology doi: 10.1016/j.ajpath.2015.10.024 sha: doc_id: 339762 cord_uid: lh8czr0a middle east respiratory syndrome coronavirus (mers-cov) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal mers-cov infection is unknown. we describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of mers-cov in the world, which was related to a hospital outbreak in the united arab emirates in april 2014. the main histopathologic finding in the lungs was diffuse alveolar damage. evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. double staining immunoassays that used anti–mers-cov antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of mers-cov antigen; double immunostaining with dipeptidyl peptidase 4 showed colocalization in scattered pneumocytes and syncytial cells. no evidence of extrapulmonary mers-cov antigens were detected, including the kidney. these results provide critical insights into the pathogenesis of mers-cov in humans. middle east respiratory syndrome coronavirus (mers-cov) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal mers-cov infection is unknown. we describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of mers-cov in the world, which was related to a hospital outbreak in the united arab emirates in april 2014. the main histopathologic finding in the lungs was diffuse alveolar damage. evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. double staining immunoassays that used antiemers-cov antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of mers-cov antigen; double immunostaining with dipeptidyl peptidase 4 showed colocalization in scattered pneumocytes and syncytial cells. middle east respiratory syndrome coronavirus (mers-cov) was initially isolated from a sputum specimen of a patient who died of respiratory and renal failure in saudi arabia in 2012. 1 recent data have indicated that dromedary camels are likely to transmit mers-cov to humans. 2, 3 human-to-human mers-cov transmission is well documented, particularly in the setting of nosocomial outbreaks. 4, 5 as of july 7, 2015, the world health organization (who) was notified of 1368 laboratory-confirmed cases of mers-cov infection with 487 reported deaths (35.6%) from 26 countries, primarily in men with a median age of 50 years. 6 most cases (>75%) were reported from the kingdom of saudi arabia. 6 mers-cov infection can result in a wide clinical spectrum from asymptomatic infection, upper respiratory tract illness, to severe pneumonia and multiorgan failure. 7e9 mers-cov binds to dipeptidyl supported by cdc operational funds. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. disclosures: none reported. current address of m.g.m., national cancer instituteeshady grove, rockville, md. peptidase 4 (dpp4) receptors that are primarily in the lower respiratory tract but also distributed in other tissues. 10 although there have been numerous cases and fatalities, the pathologic changes and viral distribution in humans associated with severe mers-cov illness are unknown, and knowledge of pathogenesis remains limited. this report provides the first autopsy, clinicopathologic, immunohistochemical (ihc), and ultrastructural description of a fatal case of mers-cov. this patient, who had multiple close contacts, was identified as part of an epidemiologic investigation of a large cluster of mers-cov infections at hospital in the united arab emirates (uae) in april 2014. this outbreak occurred in the context of a surge of cases in the arabian peninsula in which 515 mers-cov cases were identified in saudi arabia from april 11 to june 9, 2014 (world health organization, http://www.who.int/csr/don/2014_06_13_ mers/en, last accessed october 9, 2015). tissues obtained at autopsy were fixed in 10% buffered formalin, paraffin-embedded, sectioned at 4 mm, and stained by routine hematoxylin and eosin. an immunostaining protocol was applied with use of several mouse antibodies and one human antibody to mers-cov as described previously. 11 ihc assays that used mouse anti-mers antibodies ( table 1) lung tissue samples were excised from a paraffin block with the use of a 2-mm punch and processed for electron microscopy examination as previously described. 12 full genome sequencing by the sanger method with the use of direct genome walking pcr and next-generation sequencing (illumina miseq; illumina, san diego, ca) were performed as previously described on confirmed positive lung tissue, 13 named as abu dhabi_uae_8_2014 and deposited into genbank (http://www.ncbi.nlm.nih.gov; genbank accession number kp209306). clustalw was used to align the full genome with the respective available complete or near complete mers-cov genomes in the american journal of pathologyajp.amjpathol.org the patient was a 45-year-old, nonsmoking, obese (body mass index, 30.5 kg/m 2 ) filipino man with no relevant past medical history, recent travel, or exposure to sick contacts or farm animals. he worked in a storage room at a paramedic station with no patient care duties. he shared housing with five roommates from the paramedic department. he presented to an emergency department in abu dhabi, uae, on april 2, 2014, with a 4-day history of fever, rhinorrhea, and productive cough. at evaluation, he was afebrile with an oxygen saturation of 99% on room air, and a chest x-ray showed a left-sided opacity ( figure 1a) . the patient was diagnosed as acute bronchitis, prescribed 20 mg prednisolone daily for 5 days and paracetamol, and discharged. he returned to the emergency department on april 6, 2014, with persistent cough and shortness of breath; a chest x-ray showed a left-sided opacity and air bronchograms, and the patient was diagnosed with pneumonia, given a prescription for levofloxacin, and discharged ( figure 1b) . he returned to the emergency department the same day, with worsening shortness of breath and was admitted. he had a temperature of 38.7 c, pulse of 113 beats per minute, a respiratory rate of 24 breaths per minute, blood pressure of 123/73, an oxygen saturation of 93% on room air, and left basal crackles and rales. an arterial blood gas on room air showed ph of 7.49, partial pressure of carbon dioxide of 27.6 mm hg, partial pressure of oxygen of 52.4 mm hg, and bicarbonate of 20.7 meq/l, consistent with respiratory alkalosis. laboratory evaluation showed a normal white blood cell count with lymphopenia (0.5 k/ml) and creatinine of 0.9 mg/dl (supplemental table s1 ). blood, sputum, and urine cultures were obtained, and a nasopharyngeal swab specimen was sent for mers-cov testing. on april 7, day 1 of admission, oseltamivir, ceftriaxone, and azithromycin were started empirically. the day after admission he was transferred to the intensive care unit for tachypnea and respiratory distress and was intubated for mechanical ventilation; a portable chest x-ray showed multiple patchy airspace opacities ( figure 1c ). the patient became hypotensive, requiring inotropic support, and developed acute kidney injury and renal failure, requiring dialysis. on april 8, rt-pcr assay of the nasopharyngeal swab specimen collected on april 7 was reported to be positive for mers-cov for upe and orf1a gene targets. 16 bacterial cultures of blood, sputum, and urine specimens obtained at admission after antibiotic administration were negative. on april 9, he was given 100 mg hydrocortisone intravenously every 8 hours and started on nitric oxide at 16 ppm. the patient's condition continued to deteriorate, and he died april 10. the body was kept refrigerated at 4 c, and an autopsy was performed 10 days after death. notable findings included massive pleural effusion (5 l), substantial pericardial effusion (150 ml), and abdominal effusion; edematous and consolidated lungs; and generalized congestion. the predominant pulmonary histologic pattern was exudative-phase diffuse alveolar damage with denuding of bronchiolar epithelium, prominent hyaline membranes, alveolar fibrin deposits, type 2 pneumocyte hyperplasia, rare multinucleated syncytial cells, and alveolar septa involved by edema and lymphocytes with fewer plasma cells, neutrophils, and macrophages (figure 2, aec) . dispersed foci of necrotic debris were seen both subpleurally and within alveoli. no viral inclusions were seen, and moderate anthracosis was present. all four antibodies (1510/1513, sections of trachea and bronchi showed mild-to-moderate lymphocytic mucosal and submucosal inflammation with a few neutrophils and plasma cells ( figure 2g ). occasional clusters of noninvasive and extracellular candida yeast and hyphae were seen in septa, alveolar spaces, and overlying respiratory epithelium, suggesting postmortem overgrowth. bronchial submucosal glands were focally necrotic ( figure 2h ). immunostaining for mers-cov antigens was identified in both unremarkable and necrotic bronchial submucosal glands ( figure 2i ). multiple double immunostaining immunoassays were performed to assess for colocalization of mers-cov and cells labeling for cytokeratin, cd68, surfactant, and dpp4. double staining with cytokeratin confirmed the presence of viral antigens within pneumocytes and epithelial syncytial cells, whereas double staining with cd68 showed two distinct populations with no colocalization of mers-cov and macrophages (figure 3, a and b) . surfactant double staining revealed type 2 pneumocytes and syncytial cells with intracytoplasmic viral antigens ( figure 3c ). dpp4 antigens were detected in pneumocytes, syncytial cells, mononuclear leukocytes, and vascular endothelium, although colocalization of mers-cov and dpp4 was observed in scattered pneumocytes and syncytial cells ( figure 3d ). electron microscopy showed infected and degenerated pneumocytes encased between hyaline membranes, composed of fibrin and basement membranes ( figure 3e ). clusters or individualized, predominately spherical, 50 to 150 nm in diameter, viral particles were observed in membrane-bound vesicles ( figure 3f) . the kidney showed increased globally sclerotic glomeruli (5% to 10% of glomeruli), thickened bowman capsules, severe atherosclerosis and hyaline arteriolosclerosis, patchy interstitial inflammation, and intratubular proteinaceous and granular casts. diminished lymphoid follicles and a robust interfollicular proliferation of pleomorphic immunoblasts intermixed with a polymorphous population of reactive lymphocytes were seen in multiple lymph nodes; the spleen also contained numerous immunoblasts and reactive lymphocytes. the bone marrow was normocellular with maturing trilineage hematopoiesis and left-shifted the american journal of pathologyajp.amjpathol.org granulopoiesis. the heart revealed diffuse myocyte hypertrophy, moderate coronary atherosclerosis, and patchy fibrosis. moderate steatosis, scattered calcifications, and mild portal tract and lobular lymphocytic inflammation were identified in the liver. the sections of the cerebrum and cerebellum were unremarkable. mers-cov ihc was negative in multiple specimens from different organs, including kidney, liver, spleen, several lymph nodes, bone marrow, small intestine, and colon. overall the histology was well-preserved; although the sections of brain showed minimal-to-mild autolysis and kidney showed moderate autolysis. the genome sequence is similar (>99%) to other known mers-covs and clustered closely with camel-derived mers-cov strains (kj650295 to kj650297) obtained in al-hasa, saudi arabia, in 2013, suggesting recent origin in camels, although the patient had no known camel exposures. as indicated from the phylogenetic tree (supplemental figure s1 ), the sequences from this case and close contacts cluster most closely in the same clade, further supporting their transmission link between these cases. this report provides invaluable insights as the first description in the published literature of the clinicopathologic, ihc, and ultrastructural findings of a fatal case of mers-cov infection. the histopathologic pattern observed in the lungs was diffuse alveolar damage. mers-cov ihc and double staining techniques showed viral antigens were predominantly localized to type 2 pneumocytes and epithelial syncytial cells. although the pathogenesis of severe and fatal mers-cov infection is unknown, these postmortem findings provide critical insights, including evidence that pneumocytes are important targets, suggesting that direct cytopathic effects contribute to mers-cov respiratory symptoms. an ex vivo study that examined mers-coveinfected human lung tissue found evidence of pneumocyte damage by electron microscopy, including detachment of type 2 pneumocytes, and membrane blebbing, suggestive of apoptosis. 17 however, ihc staining for mers-cov was patchy, implying that other causes such as immune dysfunction may be relevant. acute renal failure is commonly observed in critically ill mers-cov patients 7e9,18 and mers-cov rna was detected in urine 18, 19 ; however, no evidence of extrapulmonary mers-cov dissemination was observed, suggesting that acute renal failure in this patient was not caused by direct renal infection but likely by other factors, such as hypoperfusion or cytokine dysregulation. the presence of mers-cov antigens in submucosal glands provides a mechanism by which the virus enters respiratory secretions and becomes transmissible. the pathologic features of mers-cov are shared by other similar respiratory illnesses, such as severe acute respiratory syndrome (sars)-cov. several reports that evaluated fatal cases of sars-cov describe diffuse alveolar damage in various stages as the most characteristic feature, 20 with ihc staining of sars-cov antigen primarily in alveolar epithelial cells. 20 syncytial cells may also be seen with other coronavirus infections, including sars-cov and paramyxovirus infections. 20 mers-cov entry is mediated by dpp4, which is expressed throughout the lower respiratory tract 17 but also numerous other organs, including the kidney. 10 double staining with mers-cov and dpp4 ihc was seen in pneumocytes and syncytial cells, consistent with the identification of these cells as targets of mers-cov infection. in addition, ex vivo models have detected mers-cov antigen in bronchial epithelial cells, pneumocytes, and endothelium and dpp4 in bronchiolar epithelium, endothelium, pneumocytes, and alveolar macrophages, 17, 21 correlating with our findings. in contrast, the functional receptor for sars-cov is angiotensin-converting enzyme 2. 22 similar to ddp4, angiotensin-converting enzyme 2 has a broad distribution, with heavy expression in alveolar epithelial cells, enterocytes, and endothelial cells. 23 although evidence shows that sars-cov also infects type 2 pneumocytes, 24,25 sars-cov was detected in several extrapulmonary sites by in situ hybridization and electron microscopy, including circulating lymphocytes, lymphoid tissues, renal distal tubules, and small intestinal mucosa. 26 because mers-cov rna was detected in blood, 19 and dpp4 is widely distributed in different tissues, 10 extrapulmonary dissemination could be possible, but we did not detect any evidence of mers-cov spread outside the respiratory tract. pulmonary findings of consolidation, diffuse alveolar damage, and pleural effusions with no evidence of bacterial pneumonia are consistent with the clinical features reported in other critically ill adults with mers-cov infection. 7e9 pathologic evidence of several chronic diseases in this patient, including myocardial fibrosis, atherosclerosis, and hepatic steatosis, correlates with reports of individuals with underlying comorbidities, such as end-stage renal disease, diabetes mellitus, hypertension, or cardiac disease, having an increased risk of developing severe mers-cov infection. 4, 7 the impact of low-dose oral prednisolone before admission and intravenous hydrocortisone on the clinical course and fatal outcome of this mers-cov patient is unknown. follicular depletion in lymph nodes, consistent with corticosteroid use, was noted, and no bacterial coinfections were identified. the hydrocortisone dosing of 100 mg every 8 hours in the intensive care unit before death was higher than recommended for refractory septic shock by the surviving sepsis campaign guidance, 27 but the patient only received a few doses. a systematic review of high-dose corticosteroids for treatment of sars-cov reported evidence of harm without survival benefit. 28 in patients with influenza a(h1n1)pdm09 virus infection, corticosteroid treatment was associated with increased mortality, 29 although further studies are needed. therefore, until further evidence is available, high-dose corticosteroid treatment for mers-cov pulmonary disease should be avoided. 30 current clinical management of mers-cov patients is based on supportive care of complications and adherence to recommended infection prevention and control measures (cdc, http://www.cdc.gov/coronavirus/mers/infection-preventioncontrol.html, last accessed october 9, 2015). our findings provide invaluable and unprecedented insights into the histologic changes, pathogenesis, and viral dissemination of mers-cov in humans. further studies, including additional postmortem examinations, evaluation of the host immune response, and identification of sites of viral replication, are necessary to strengthen knowledge on pathogenesis, transmission patterns, and effective treatment strategies for optimal clinical management and infection control practices. isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation ksa mers-cov investigation team: hospital outbreak of middle east respiratory syndrome coronavirus investigation team: hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description world health organization: middle east respiratory syndrome coronavirus (mers-covesummary of current situation. lit update risk assess 7 epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study albarrak am: clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: 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course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov replicates in primary human alveolar type ii cell cultures but not in type i-like cells immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan multiple organ infection and the pathogenesis of sars surviving sepsis campaign guidelines committee including the pediatric subgroup: surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock sars: systematic review of treatment effects do corticosteroids reduce the mortality of influenza a (h1n1) infection? a meta-analysis world health organization: clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance 2 we thank cynthia goldsmith for reviewing the electron microscopy and azaibi tamin for providing the mers-coveinfected vero cell cultures. supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2015.10.024. key: cord-337835-78i6j11i authors: alfaraj, sarah h.; al-tawfiq, jaffar a.; alzahrani, nojoom a.; altwaijri, talal a.; memish, ziad a. title: the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus date: 2017-02-09 journal: j infect doi: 10.1016/j.jinf.2017.02.001 sha: doc_id: 337835 cord_uid: 78i6j11i nan ho and colleagues recently drew attention to the consequences of co-infection with influenza and hiv. 1 we present four cases of combined infection with influenza and middle east respiratory syndrome coronavirus (mers-cov) infection. nasopharyngeal swabs or tracheal aspirates were tested for mers-cov using real-time reverse-transcription polymerase chain reaction (rt-pcr). 2,3 samples were tested for influenza a, b and h1n1 by rapid molecular test (gen-exper for detection of flu a, b and 2009 h1n1, cepheid). in the first case, a 39 year-old male, health care worker, an engineer who became ill seven days before admission. he had fever >38 c, cough and sore throat. he had no nausea, vomiting, diarrhoea or shortness of breath (sob). he denied history of travel or contact with positive case or camels. he was febrile with a temperature of 39.5 c. chest x-ray showed non-homogenous opacity at the lower right lung zone. a nasopharyngeal swab was positive for mers-cov with ct value upe gene 34, and orf1a 34 ( table 1 ). the test was negative for influenza but a repeat swab after 48 h was negative for mers-cov and positive for h1n1. the patient received azithromycin, ceftriaxone and oseltamivir. the patient was discharged home after two negative swabs of mers-cov and being asymptomatic for 48 h. in the second case, a 61 year-old female with diabetes mellitus and dyslipidemia was admitted with a three-day history of shortness of breath and productive cough. she had no nausea, vomiting, or diarrhoea. she denied history of travel or contact with positive case or camels. she was afebrile with a temperature of 37 c. chest x-ray showed patchy opacities involving middle and lower zones of both lung fields. a nasopharyngeal swab was positive for mers-cov with ct value upe gene 34, orf1a 35 and negative for influenza. a repeat swab after 48 h was negative for mers-cov but positive for h1n1. she required bipap and she was subsequently intubated and was started on mechanical ventilation. she was extubated after 13 days. the patient received piperacillinetazobactam, and erythromycin. the patient was discharged home after she had 2 negative swabs of mers-cov and being asymptomatic for 48 h. in the third case, a 29 year-old housekeeper female was admitted with two days history of fever and cough. she had no nausea, vomiting, diarrhoea nor shortness of breathing. she had a history of contact with mers-cov positive case. she was afebrile with a temperature of 36.9 c. chest x-ray was normal. a nasopharyngeal swab collected upon presentation was positive for mers-cov with ct value upe gene 32 orf1a 32. the swab was negative for influenza. a repeated swab after 48 h was positive mers-cov and positive for h1n1. the patient received oseltamivir, azithromycin and ceftriaxone. the patient was discharged home after she had 2 negative swab of mers-cov and being asymptomatic for 48 h. in the fourth case, the patient was a 73 year-old female with a history of hypothyroidism, heart failure, lymphoma, and lung fibrosis. she has no history of travel or contact with positive case or camels. four days prior to her presentation, she had productive cough and shortness of breath. she had no fever, diarrhoea, vomiting or nausea. she was afebrile with a temperature of 36.7 c. chest x-ray showed bilateral diffuse infiltrate (fig. 1) . a nasopharyngeal swab was positive for mers-cov with ct value upe gene 37; orf1a 36 and negative for influenza. a repeat swab after 3 days was negative for mers-cov but positive for influenza a. the patient was treated with piperacillinetazobactam for six days and oseltamivir for 5 days. the patient was discharged home after two negative mers-cov and being asymptomatic for 48 h. these patients highlight the co-infection with mers-cov and influenza. the exact reason to have a negative influenza test at the time of positive mers-cov is not completely understood. it is possible that the presence of mers-cov inhibits the pcr reaction for influenza virus. however, an earlier case of mers-cov tested initially positive for influenza a(h1n1)pdm09. 4 on the other hand, the positivity of nasal swabs for influenza is specimen type and technique dependent. 5 thus, initially negative influenza tests could be a false test result. positive results for viral respiratory pathogens should not preclude testing for mers-cov because coinfection can occur. 6 only a small number of mers cases had co-infection with influenza a, parainfluenza, herpes simplex, and streptococcus pneumoniae. 7 in one case, a co-infection with herpes simplex virus type 1 dna13 and rhinovirus rna14 were detected by rt-pcr. 8 the investigation of the first 47 cases showed no co-infection with mers-cov. 2 there is a controversy regarding the risk of increased or decreased severity of co-infections. for example co-infections with respiratory syncytial virus (rsv) and human meta-pneumovirus (hmpv) causes more severe infection than either virus alone with longer hospitalization and oxygen requirement. 9 other studies did not demonstrate these effects. 10 the association and the impact of co-infection with mers-cov and influenza viruses deserve further evaluation and studies. all authors have no conflict of interest to report. completeness of case ascertainment and availability of environmental data in legionnaires' disease enhanced surveillance in england, 2012e2014 to the editor, bai et al. 1 previously highlighted the importance of effective communicable disease surveillance in china for the detection of outbreaks to inform infectious disease prevention and control. to achieve similar aims with relation to the control of legionnaires' disease in england a national enhanced surveillance scheme operates. surveillance data must be both complete and as timely as possible, as highlighted by freeman et al. in this journal. 2 therefore we sought to assess the completeness of case ascertainment in addition to the availability of environmental data reported to the national enhanced legionella surveillance scheme (nelss) for residents in england. the value of environmental data is particularly important for legionnaires' disease in order to inform the attribution of potential environmental sources of cases and clusters of this infection. in england, the national legionella surveillance team (nlst) leads on surveillance and control of legionnaires' disease, while field epidemiology services (fes) teams collect surveillance data and investigate sporadic cases, clusters and outbreaks in conjunction with local health protection teams (hpts). the nlst worked with the fes teams in the north west (nw) and west midlands (wmids) regions of england to audit the reporting of legionella cases with onset dates during each calendar year between 2012 and 2014, inclusive. eligible cases were those cases of legionella spp. infections resident in either of these regions in england, which were laboratory confirmed by urinary antigen testing, culture or serological testing. these cases were identified in the month of february following each calendar year by the nlst and were compared to those known by the fes teams for nw and wmids regions. completeness of case ascertainment was calculated as the proportion of cases recorded by fes teams for cases with onset of symptoms between 2012 and 2014 inclusive which were reported to the nlst. the availability of environmental data reported to the national enhanced surveillance scheme was calculated as the proportion of cases during the same time period with any environmental investigation data reported to the nlst. the results are summarised in table 1 ; in the nw region, a mean of 34 cases were reported per year between 2012 and 2014. in the wmids region, the mean number of cases per year was 46. both regions reported 100% of cases to the nlst. mean availability of environmental data reported was 80.8% in the nw and 42.8% in wmids. however, there was a the impact of hiv on the burden and severity of influenza illness in malawian adults: the bash-flu study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections an adult returned traveler from dubai hospitalized with an influenza-like illness (ili): middle east respiratory syndrome (mers) or influenza? infection control implications from a near mers case saudi ministry of health. case definition and surveillance guidance for mers-cov testing in saudi world health organization. who guidelines for investigation of cases of human infection with middle east respiratory syndrome coronavirus (mers-cov) clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection prospective study of human metapneumovirus infection in children less than 3 years of age the role of infections and coinfections with newly identified and emerging respiratory viruses in children key: cord-316126-j51dik7f authors: zhang, x. sophie; duchaine, caroline title: sars-cov-2 and health care worker protection in low-risk settings: a review of modes of transmission and a novel airborne model involving inhalable particles date: 2020-10-28 journal: clin microbiol rev doi: 10.1128/cmr.00184-20 sha: doc_id: 316126 cord_uid: j51dik7f since the beginning of the covid-19 pandemic, there has been intense debate over sars-cov-2’s mode of transmission and appropriate personal protective equipment for health care workers in low-risk settings. the objective of this review is to identify and appraise the available evidence (clinical trials and laboratory studies on masks and respirators, epidemiological studies, and air sampling studies), clarify key concepts and necessary conditions for airborne transmission, and shed light on knowledge gaps in the field. we find that, except for aerosol-generating procedures, the overall data in support of airborne transmission—taken in its traditional definition (long-distance and respirable aerosols)—are weak, based predominantly on indirect and experimental rather than clinical or epidemiological evidence. consequently, we propose a revised and broader definition of “airborne,” going beyond the current droplet and aerosol dichotomy and involving short-range inhalable particles, supported by data targeting the nose as the main viral receptor site. this new model better explains clinical observations, especially in the context of close and prolonged contacts between health care workers and patients, and reconciles seemingly contradictory data in the sars-cov-2 literature. the model also carries important implications for personal protective equipment and environmental controls, such as ventilation, in health care settings. however, further studies, especially clinical trials, are needed to complete the picture. t he world is facing a devastating new infectious disease, with only preliminary scientific data to guide policy. disagreement with the world health organization's stance on personal protective equipment (ppe), guideline changes over time (e.g., european cdc, france) , and inconsistent data on the effectiveness of medical masks have left health care workers (hcws) wondering if they are sufficiently protected. the general consensus is that sars-cov-2 predominantly transmits through droplets and contact (although precise mechanisms for both modes of transmission are yet to be fully understood), but the airborne debate is still raging. this review attempts to summarize current cumulative data on sars-cov-2's modes of transmission and identify gaps in research while offering preliminary answers to the question on everyone's mind: is the airborne route significant and should we modify our covid-19 ppe recommendations for frontline workers in low-risk settings? this review starts by investigating the differences between droplets and aerosols and goes over prerequisites for clinically significant airborne transmission. it then appraises the evidence in support of the airborne hypothesis: trials and experiments on masks, epidemiological studies, data on sars-cov-1, air sampling findings, and aerosol studies. the focus is on low-risk health care settings, in the absence of aerosolgenerating procedures (agps), with a special look at long-term-care facilities where major outbreaks occurred. national and international guidelines are compared, and alternative hypotheses for sars-cov-2's contagiousness are explored, such as presymptomatic transmission, as well as fomite and fecal routes. possible mechanisms behind high hcw infection rates are described, and the limits of the precautionary principle are addressed. finally, a revised model of inhalable particles is proposed to support ppe recommendations and guide future research. determining sars-cov-2's main mode of transmission is essential as it informs clinical guidelines for patient management, prevention practices, and hcw protection. while infectious disease precautions in health care settings are transmission-based (either airborne or droplet), in reality, the distinction is not clear-cut; instead, they are two ends of a spectrum. in the literature, respiratory droplets are usually defined as larger particles (diameter ͼ 5 m) sometimes visible to the human eye, produced during spitting, sneezing, and coughing. these droplets are thought to be the main mode of transmission of covid-19 (1), and they typically travel 1 to 2 m before landing on surrounding surfaces. however, they may be propelled further in the presence of ventilation (2) or forceful ejection (e.g., a violent sneeze) (3) and under certain environmental conditions (e.g., cool and humid) (4) . the sars-cov-2 virus is also thought to be transmitted by direct contact person to person (e.g., exchange of saliva or a handshake) or by indirect contact through intermediate objects (e.g., sharing of cups, doorknobs). generally, contact transmissions occur when contaminated hands are brought to the face and touch mucous membranes (eyes, nose, and mouth). the fate of smaller droplets may be desiccation (evaporation of the liquid) and formation of particles called droplet nuclei, or aerosols, which can contain infectious agents but also secretions, cells, surfactant, and any other product contained in the original droplet. traditionally, aerosols are defined as particles of ͻ5 m that can remain airborne for prolonged periods (several minutes or even hours) and travel long distances with air currents (several meters away). with the potential for direct entry into the lungs, they are the primary mode of transmission for tuberculosis, measles, and varicella. in other communicable diseases, such as influenza, aerosols are considered opportunistic and play a role that is of variable importance depending on the context (5) . conversely, in the field of industrial hygiene, occupational exposure of different body regions to harmful airborne agents is classified into three overlapping categories, according to the median size of penetrating particles (6): 100 m for nose and mouth (inhalable), 10 m for trachea and bronchi (thoracic), and 4 m for alveoli and air exchange regions (respirable). this aerosol classification was recently reviewed and elegantly illustrated by milton (7) . in this model, the concept of aerosol inhalability is defined as the fraction of particles capable of penetrating into the head airways or below, upon inhalation: it excludes larger droplets with ballistic behavior (since inhalation requires suspension in the air) but includes particles that are larger than the traditional 5-m definition of aerosols. throughout our review, this more nuanced conceptualization of airborne transmission will be explored, and the larger inhalable aerosols will be contrasted to the smaller respirable aerosols from the classic airborne model. finally, some procedures, such as intubation, are known to generate aerosols, while others, such as nebulizer therapy, are associated with an uncertain risk of aerosolization (8) . n95s (or similar respiratory protection devices) are unequivocally recommended for hcws working in high-risk settings with agps, although controversy still remains around which interventions constitute an agp. the design protocol for the n95, and the origin of the name, is based on its efficiency at capturing 95% of the most penetrating size range (0.3 m) of respirable aerosols (9) . by default, respirators are therefore capable of blocking the entire spectrum of airborne particles. medical masks, on the other hand, are designed to block droplets and do not undergo aerosol-filtering tests; they are therefore not considered to provide respiratory protection against airborne transmission. given that substantial disagreement persists on the importance of natural aerosol generation by covid-19 patients, and consequently, the necessary level of respiratory protection in non-agp contexts, our review will focus on transmission and ppe in low-risk health care settings. natural respiratory activities such as breathing, talking, and coughing can generate a broad range of particle sizes, from submicron aerosols to large droplets (10) (11) (12) (13) (14) . for the viral aerosols to constitute a clinically significant risk of airborne infection, three conditions are required: viral load (the concentration of infectious particles), infectivity (the ability of a virion to infect a host cell), and tropism (the specificity of a virus for a particular host cell type or tissue). since the amount of sars-cov-2 virus required to infect a host is unknown, and likely varies from one individual to another (preprint article [15] ), it is hard to determine whether typical respiratory activity generates sufficient quantities of infectious aerosols for airborne transmission. in a light-scattering study, stadnytskyi et al. estimated that 1 min of loud speaking generated at least 1,000 virion-containing droplet nuclei that remain airborne for more than 8 min (16) . however, the calculations were based on several theoretical assumptions and data from sputum load was incorrectly applied to saliva, likely overestimating aerosol viral loads. in this model, the probability that a hypothetical speech-generated droplet nucleus of 3 m contains a sars-cov-2 virion is only 0.01%, after aerosolization and desiccation. furthermore, in a mathematical modeling study on viral aerosol emissions, an individual with a high viral load was estimated to emit only modest amounts of virus with regular breathing (1,248 copies/ m 3 ) compared to coughing (7.44 million copies/m 3 ) (17). accordingly, the authors conclude that the infectious risk posed by a typical covid-19 patient is low, especially if symptoms are mild, and only a few individuals with high viral load pose a significant risk. these authors suggest that strict respiratory protection may be needed in the case of prolonged exposure to high emitters in poorly ventilated closed environments. notwithstanding, evidence of aerosol generation during natural respiratory activity or the presence of viral rna in the air are not sufficient to prove that the virus remains infectious once airborne. not all viruses are equally stable in the air, and further aerodynamic and environmental factors may inactivate viruses during aerosolization (18) . therefore, upon detecting sars-cov-2 aerosols, infectivity must then be demonstrated. evaluation of infectivity is usually done with viral cultures: researchers were able to culture rhinovirus (19) and influenza (20) from the fine particles emitted naturally by infected participants, and only recent yet unpublished research has started to achieve the same for sars-cov-2. however, it is important to note that culture methods vary between viruses and false-negative results due to the low sensitivity of commonly used sars-cov-2 cultures could have possibly underestimated infectivity from air samples until now. for instance, clinical samples (e.g., nasopharyngeal swabs) that yield positive cultures typically have low pcr cycle threshold (c t ) values of ͻ25 (samira mubareka, university of toronto, unpublished data), while c t values for environmental samples (including air samples) are often ͼ35. finally, since particles penetrate and deposit in different parts of the respiratory tract depending on size, knowledge of target locations for infection (e.g., viral tropism) can hint at typical size range and mode of transmission. sars-cov-2's main entry into host cells is through ace2 receptors, which seem to be largely expressed in the nose (21, 22) . importantly, the highest and most consistent signs of viral infectivity have been observed for nasal cells, with a gradient along the respiratory tract characterized by a marked reduction in infectivity in the distal bronchioles and alveoli. this may suggest that lower airways are not targets for infection and that transmission via respirable aerosols is not predominant. interestingly, the typical patchy bilateral pneumonia found in covid-19 patients is postulated to be caused by oropharyngeal microaspirations rather than direct viral seeding in the lungs, possibly accounting for the increased risk with age and comorbidities (22) . different types of studies suggest airborne transmission, but their levels of evidence are variable. in this review, given the focus on health care settings and hcw protection, studies are appraised according to clinical relevance: hard outcomes (e.g., morbidity) are markers of higher levels of evidence, while surrogate outcomes (e.g., pathophysiological mechanisms, modeling, and laboratory results) are considered lower levels of evidence, independent of method or design quality (table 1) . the term "mask," as used here, comprises medical masks, surgical masks, procedural masks, fluid-resistant masks, and face masks worn by hcws. the term "respirator" is used interchangeably with n95, which is the equivalent of ffp2 (european standard filtering facepiece) and kf94 (korean filter) respirators. in the absence of clinical trials on sars-cov-2, trials on other viruses with similar infection patterns (i.e., documented droplet and suspected airborne transmission) are the best available alternatives. recent systematic and narrative reviews comparing the effectiveness of respirators versus masks against common viral respiratory infections (including coronaviruses and influenza viruses such as h1n1) come to similar conclusions: both devices offer comparable protection in health care settings (23) (24) (25) (26) (27) (28) (29) (30) (31) . a few reviews (32) (33) (34) favor respirators, on the basis of two randomized controlled trials (rcts) conducted by the same lead authors, macintyre et al. (table 2 ) (35, 36) . individually and in combination (meta-analysis) (33), these two rcts report superiority of continuous n95 use over mask use for a single self-reported outcome: clinical respiratory illness (cri), defined as two or more respiratory symptoms or one respiratory symptom and a systemic symptom. no difference is found for other more rigorous outcomes: influenza-like illness (ili; defined as fever and one respiratory symptom), laboratory-confirmed viral respiratory infection (lvi), or laboratory-confirmed influenza (lci). the difference between the self-reported outcome and the laboratory results could be explained by detection bias in the absence of participant blinding and universal testing: higher symptom reporting rates in the medical mask group, rather than true infection, could have skewed cri results in favor of respirators. furthermore, selection bias is suspected to have occurred during allocation, given the surprisingly uneven distribution of major confounding variables such as agps, age, and handwashing, between the n95 and mask groups. the other two rcts (37, 38) included in the reviews had more robust methodologies and lesser risk of bias (e.g., comparable groups, test results for all participants, and longer follow-up periods). the studies did not find any significant differences between respirators and masks for clinical and laboratory outcomes, in both low and high-risk settings. a recent systematic review of observational studies suggests that "n95 respirators might be more strongly associated with protection from viral transmission than surgical masks" (39) . regrettably, of 10 studies, not a single one directly compared respirators to masks, and nine of them looked at sars or mers rather than sars-cov-2. the lone covid-19 study only compared n95s to no masks and did not include medical masks at all (40) . the researchers drew their conclusions by comparing the pooled results for n95 studies with the pooled results for mask studies, obtaining a p value for interaction by mask type that was borderline significant after partial adjustment. however, the difference between the two groups was not statistically significant (overlapping confidence intervals) and the very high heterogeneity (i 2 ϭ 88%) could have undermined the validity of the meta-analysis. also, the presence of agps was unknown in 7 of 10 studies: since all the studies were done in a hospital setting where agps frequently occur, and n95s are known to be superior in high-risk settings, failure to adjust for agps will skew the results in favor of n95s. finally, all 10 studies were observational and many did not control for important confounding factors, leading the authors themselves to rate the overall certainty for mask data as low. since many trials studied airborne viruses (e.g., influenza) and included exposure to agps, it may seem surprising that the vast majority of reviews, past and present, did not find respirators to be superior to masks. a possible explanation is that, while not designed to filter very fine particles, the medical mask might nonetheless be effective in blocking the low levels of aerosols produced in most health care contexts. a few case reports seem to support this hypothesis. for example, in a study of two severely ill covid-19 patients who were not initially isolated, contact tracing identified 421 hcws, of whom only 8 tested positive (41) . all infected hcws had close and prolonged contact without wearing the mask or ocular protection and had been present during agps. on the other hand, all of the hcws who used droplet and contact precautions did not get infected, leading the authors to conclude that there was no evidence of airborne transmission. similarly, two studies reported on 34 and 41 intensive care hcws exposed to an intubated and mechanically ventilated covid-19 patient: 50 and 85% wore surgical masks, respectively, and the others wore n95s, yet none were infected according to clinical and laboratoryconfirmed results (42, 43) . furthermore, a covid-19 patient who stayed 35 h in an open cubicle of a general ward, coughed frequently, and received high-flow oxygen at 8 liters/min, did not infect any of the 71 staff members and 49 patients, of which 7 and 10, respectively, had close contacts wearing either n95s or masks (44) . finally, strict contact and droplet precautions, as well as the use of masks rather than respirators, completely prevented nosocomial transmission from three community-infected hcws to coworkers and patients in an italian hospital (45) . as for the effectiveness of medical masks as source control (blocking particles emitted by infected individuals), clinical trials are scarce (46) (47) (48) , and they suggest a reduction of clinical but not laboratory-confirmed viral illnesses. therefore, we must turn to lower levels of evidence (e.g., laboratory studies) for further guidance. the ability of protection devices to control either source emission (e.g., infected individuals) or exposure prevention (e.g., hcws) has been the subject of several laboratory studies, whose findings are summarized in table 3 . the majority show high filtration capacity for both masks and respirators. the latter, however, are known to provide better protection against fine particles (ͻ5 m) because of a far superior fit factor. interestingly, source control with masks may be superior to exposure prevention by either respirators or masks. although these studies provide relevant information on the theoretical performances of protection devices, the experimental generation process and particle sizes may not resemble natural respiratory activity. also, many studies suffer from major limitations and inconsistencies in design: the use of different respiratory viruses with distinct behaviors, the lack of information on the size distribution of particles tested, the use of nonstandardized test particles (e.g., in contrast to standard respirator testing protocols), selection bias for ballistic behavior (petri dish sampling) rather than aerosols (air sampling), and confounding biases (e.g., fit factor and variable cough intensities). more importantly, many laboratory studies fail to account for crucial clinical and behavioral factors. for example, studies have reported lower adherence to n95 respirators compared to medical masks, due to higher rates of adverse events (35, 36, 49) . in one study on the tolerability of respirators in hcws, the probability of discontinuing respirator use during an 8-h work shift was around 50 to 70%, despite regular 15-or 30-min breaks every 2 h (50) . other studies show that one of the most challenging steps in donning and doffing is n95 use, which can result in a higher risk of contamination (51, 52) . in addition, an important, yet overlooked factor is the fitting of the device on the face (or the degree of leakage of particles around the edges). the fit factor varies between mask models and is typically very high for respirators, which is probably its main advantage. however, a poorly fitted respirator could perform no better than a loosely fitting mask (53) . seals used in some laboratory studies are poor surrogates for actual fitting on a hcw. finally, during exposure to covid-19 patients, hcws are instructed to wear ocular protection in addition to masks, and yet very few studies examine the combined effects of overall ppe. some experiments have shown that masks integrated with visors (54) and face shields individually (55) are protective not only against droplets but also aerosols (but efficiency decreases with exposure time). the vast majority of epidemiological studies that analyze sars-cov-2 outbreak patterns (case identification, contact tracing, epidemiological curves, and basic reproduction number or r0 estimates), undertaken in a variety of contexts, including health care facilities (41) (42) (43) (44) (45) , homes (56) , churches (57), fitness facilities (58), call centers (59), airplanes (60) , and company conferences and tour groups (61) , are in agreement: contact and droplets were the probable modes of transmission. rather than long-range propagation and frequent mass outbreaks typical of airborne patterns, the distribution of infected individuals was strongly correlated with close encounters and secondary attack rates were estimated be very low, around 5% (62) . rather than high r0 estimates typical of airborne viral pathogens such as chickenpox (5 to 11) (63) and measles (6 to 27) (64), community reproduction numbers fell between 2 and 4 (65, 66) and were easily lowered by droplet and contact precautions (67) . moreover, the who's largescale epidemiological analysis of 75,465 covid-19 patients did not confirm any cases of long-range airborne transmission (68) . in health care settings, the use of medical masks appears to be sufficiently protective of hcws exposed to covid-19 patients, as mentioned previously. several epidemiological reports from hospitals around the world even show little or no nosocomial transmission in the absence of recommended ppe (i.e., no n95s or masks during agps or improper mask use during close contact). combining the findings of six studies, out of a cumulative total of 295 hcws exposed to covid-19 patients without proper protection, only 5 hcws were infected. all five workers either did not wear any mask or used a mask intermittently during an agp or prolonged exposure (ͼ60 min) (69) (70) (71) (72) (73) (74) . these low levels of transmission from nonisolated covid-19 patients to nonequipped hcws are not suggestive of significant airborne transmission and support the effectiveness of basic pci measures beyond ppe. nonetheless, some epidemiological evidence is compatible with short-range airborne transmission. the washington choir outbreak is known for linking aerosolization from loud vocalization (i.e., singing) to rapid spread; however, the index case was symptomatic rather than asymptomatic as reported by the media (75), and multiple opportunities for droplet or fomite transmission were revealed in the published investigation (76) . in turn, the well-known outbreak at the guangzhou restaurant has been the subject of controversy: based on epidemiological data, one research team determined that droplets, expelled further than usual by air conditioning, were the probable source of transmission from an index patient to two neighboring tables (2); a second team, based on computer modeling and a tracer gas (a surrogate for exhaled particles), ruled in favor of airborne transmission (preprint article [77] ). moreover, a recently published study analyzed an outbreak involving two groups who rode separate buses to attend a 128-participant worship event (78) . while no transmission occurred on bus 1, 23 passengers on bus 2 were infected, some of whom were sitting up to 5 m away from the index case. seven other participants who did not ride on the buses were infected, all of whom reported close contact with the index case during the outdoor event. since proximity to source was not correlated with infection risk in the bus, but window and door seats seemed to be protective, the researchers hypothesized that bus 2's closed environment and air recirculation enabled airborne transmission to occur. furthermore, the widely studied diamond princess cruise ship outbreak is still up for debate. based on epidemiological data showing exclusive in-room transmission following imposed quarantine, as well as no correlation between infection patterns and central ventilation system, one research team concluded that close contacts and fomites were the main transmission routes (preprint article [79] ). in support of this view, an environmental study failed to detect any virus in air samples despite widespread positive surface sampling; however, passengers had disembarked at the time of sampling (80) . conversely, a modelization study simulating the cruise ship outbreak found that the epidemic models which best predicted the empirical data suggested predominant short-range and long-range airborne transmission (preprint article [81] ). finally, two studies (82, 83) analyzed the impacts of public health policies on the epidemiological curves of highly impacted regions: the first compared wuhan, italy, and new york city (nyc) while the second compared 15 u.s. states. according to the authors, mask-wearing but not social distancing (quarantine, stay-at-home, and lockdown) policies were effective in curtailing covid-19 outbreaks, suggesting that the main route of transmission is airborne rather than contact and droplets. however, the studies have come under criticism for not accounting for major confounding biases, such as differences between the three regions in terms of timing of lockdown (at ͼ9,000 confirmed cases in italy and nyc [84, 85] compared to 495 confirmed cases in wuhan [86] ), public health policy (e.g., contact tracing efficiency, testing criteria, and access), and population demographics (87) . in addition, using the date of governmentmandated mask-wearing as the start point for regression slopes is misleading, since the impacts of any new policy on epidemiological curves are delayed and nonlinear, especially given uneven compliance to mask-wearing, typically around 50% in the united states. (88) , but variable between states, compared to over 95% in asia (89) . if we further scrutinize nyc (as well as other states), it appears that the number of daily new cases, hospital admissions, and deaths started to fall before the mask-wearing order (84) , thus warranting an alternative explanation for the decline, such as an increasing proportion of immune individuals or the adoption of more aggressive testing. moreover, researchers could not explain why certain states managed to control their outbreaks without mask-wearing policies and others did not show a decline in new or cumulated cases after facemask adoption. beyond the airborne versus droplet debate, there is consensus among epidemiologists: prolonged short-range exposure is the main risk factor. interestingly, the revised airborne model presented in the conclusions: proposed model (below), involving inhalable aerosols, can accurately explain epidemiological observations as well as the dynamics of several contentious outbreaks. despite some caveats, sars-cov-1 studies may be useful to understand sars-cov-2, given that they share around 80% of their genomic sequence (66) . a well-studied outbreak at amoy gardens in hong kong, a high-rise housing estate where ͼ300 tenants were confirmed infected despite little contact between them, was studied by different teams (90, 91) . the majority agree on airborne transmission of sars-cov-1, originating from the aerosolization of feces and urine through hydraulic action (i.e., toilet flushing) of an index patient who presented with diarrhea and high viral load in excrements. this particular outbreak involved primarily environmental and engineering factors such as unsealed floor drain traps, bathroom fans causing negative pressure, bathroom fixtures contributing to drain overload or backflow, and the specific configuration of the exhaust system, which contributed to drawing aerosolized sewer droplets from the plumbing system back into the bathrooms and spreading them throughout the building (92) . the involvement of respiratory aerosols was not hypothesized. more relevant to health care settings is a hong kong hospital outbreak study on medical students exposed to an index sars patient: proximity with the patient was the main risk factor, but the duration of contact did not appear to be associated with transmission. the researchers conclude that the mode of transmission was probably through droplets and contact, but airborne transmission could not be excluded, especially given the presence of a potential agp (30-min nebulizer therapy four times a day) (93) . furthermore, in a canadian study, air samples were collected from 15 sars patient rooms in low-risk and high-risk settings, as well as four adjacent nursing support areas: 2 of the 40 wet air samples and none of the 28 dry air samples were pcr positive (94) . the two positive samples were both from the room of a single recovering sars patient where agps did not appear to be performed. subsequent viral culture; however, turned out negative. as for protection devices, a case-control study in five hong kong hospitals showed no difference in infection rates between hcws wearing a mask or a respirator, when exposed to sars patients (95) . other observational studies (96) (97) (98) done in high-risk settings (including agps) suggest possible n95 superiority, but the studies either did not adequately compare the two equipment types or did not obtain statistically significant results. other lower levels of evidence for sars-cov-1 come to similar conclusions regarding ppe. no nosocomial transmission was found in hcws from eight u.s. hospitals, despite several of them not wearing any masks and 5% of them being exposed to agps (99) . furthermore, no nosocomial transmission was found in vietnamese hcws exposed for 3 weeks to hospitalized cases, wearing only medical masks (100) . however, given the differences between sars-cov-1 and sars-cov-2 (e.g., peak viral load, asymptomatic transmission rates, and mortality rates), direct extrapolations from one virus to the other must be made with caution. similarly to the current pandemic, the significance of airborne transmission for the previous sars remains uncertain to this day, as the prerequisites (viral load, infectivity, and tropism) are not clearly met. unfortunately, sars-cov-1 seems to suffer from the same lack of rigorous clinical trials as its contemporary cousin. data from air and no-touch surface sampling studies (tables 4 and 5) conducted in covid-19 patient rooms and health care facilities are often cited to support airborne transmission. unfortunately, interstudy comparisons are complicated by the diversity of methodological approaches. for instance, positive air samples correlate with patient features (e.g., viral load and symptom intensity and duration), ventilation parameters, and cleaning procedures, but these elements are not always mentioned or detailed. moreover, large variations are reported in terms of total volume of air collected (ͻ100 liters to up to 10,000 liters), flow rates (3.5 to 300 liters/min), sampling duration, and technique (gelatin versus polycarbonate filtration, dry cyclonic sampling versus condensation sampling). furthermore, the sampling of no-touch surfaces, defined as areas typically out of reach of human contact or droplets and therefore assumed to be contaminated by aerosols only, is often poorly described and not always comparable to air samples. given that each design is associated with its own set of advantages and limitations (e.g., longer duration of air sampling may increase detection probability but decrease infectivity), there is no easy conclusion to be drawn when comparing studies. the majority of published and unpublished studies detected viral rna in the air and on no-touch surfaces (table 4 ), but some did not (table 5) . unfortunately, few positive studies included viral cultures. the main limitations of these studies were the lack of information on particle sizes and concentrations, unknown or suboptimal air sampler location, unknown time interval between aerosol production and collection (air or surface), and possible false negatives (e.g., negative pressure, open windows, and insufficient sampling volume or duration). for the studies that calculated viral concentrations from the environmental samples, various protocols, target genes (e.g., orf1ab/ rdrp, e, n, and s), and chemistry detection technology, should caution against direct comparisons. most studies were carried out in both low-and high-risk areas, and frequently in intensive care units (icus) where agps commonly occur and ventilation is optimized. many studies, however, did not specify the general risk level and did not indicate if agps were carried out during sampling. therefore, positive air and no-touch surface samples could not be clearly associated with an emission source (i.e., natural aerosolization versus agps) or risk factors (e.g., ventilation rate). this makes the results hard to generalize to most low-risk health care settings, such as long-term-care facilities. negative results from air sampling studies in home and commercial settings (80, 101) , in the definite absence of agps, also add to the uncertainty. it is worth noting that when researchers modelized aerosol emission during normal breathing, the observed concentrations of airborne particles were low, frequently under the detection limit for most air sampling approaches (102) . this could explain the negative results of many studies (table 5) . nonetheless, air and no-touch surface sampling studies support the presence of natural and/or intervention-generated aerosols in covid-19 health care facilities. however, the infectivity of these aerosols and their significance as a transmission route, beyond the mere detection of viral particles, remain uncertain. indeed, a better understanding of viral resistance to airborne stress is key to estimating infectious risk. three published studies (103) (104) (105) included viral cultures from air samples, all of which were negative; however, the santarpia et al. study (103) observed indirect signs of viral replication in two of their samples, including a mild cytopathic effect upon microscopic inspection after 3 to 4 days. on the other hand, in two unpublished studies, santarpia sars-cov-2 and health care worker protection clinical microbiology reviews et al. (106) and lednicky et al. (107) succeeded in obtaining positive cultures. the former used innovative methods such as detection of viral rna in supernatant and western blotting to yield interesting results. however, data scrutiny is impeded by the absence of c t values in the manuscript. in turn, the latter study would benefit from a thorough peer review process given that its methodology is not clearly detailed, and total and culturable viral counts seem implausible, since they are orders of magnitude higher than previously reported in the literature. the use of a condensation-based air sampler could perhaps explain the unusual results. the fact that few research teams have attempted to culture the virus, and many of those who have did not succeed, could imply that sars-cov-2 aerosols are scarce or weakly infectious. however, multiple other factors could be at play. viral cultures must be done in biosafety level 3 facilities and are therefore not easily accessible to some research teams. even when culturing is possible, viral shedding dynamics may be unpredictable or intermittent, leading to failed detection within the time frame of air sampling (108) . furthermore, the sampling process of aerosols, in itself, may induce substantial damage to viruses and alter their integrity and, consequently, their infectivity (109) . finally, current culture techniques may not be optimal for the low viral concentration found in air samples. increased sensitivity could be achieved with a bioassay or alternative methods such as electron microscopy, detection of viral proteins, and rt-qpcr in culture lysis and supernatants (106) . lastly, studies involving the in vitro generation of sars-cov-2 aerosols with jet collison nebulizers have been widely cited in support of airborne transmission. using this method, the well-known van doremalen et al. letter measured infectious titers per liter of air in a simulated aerosolized environment and showed stability of the sars-cov-2 virus in aerosols for up to 3 h, with a half-life of 1.2 h (110). another similar study made headlines because the aerosols produced were stable for up to 16 h (111) . as with all in vitro models for bioaerosols, while they provide precious information on virus properties in aerosol state, including relative stability (which seems to be high) and comparative viral behavior, it is uncertain whether the mechanically produced sars-cov-2 aerosols exhibit the same properties as naturally generated ones. therefore, such experimental studies are generally considered of low applicability to clinical settings. tragic outbreaks in long-term-care facilities (ltcs) have plagued many countries in europe (112) and north america (113) , with astonishing death tolls. some facilities report 100% resident infection rates, high hcw infection rates, as well as faulty ventilation systems (114), triggering intense debate over potential airborne transmission. while aerosols could have contributed in cases involving inadequate ventilation (115) , other explanations are also conceivable. some have justified the devastating statistics by pointing to higher viral loads (116) or longer infection periods (117) in the elderly, two phenomena likely attributable to the weakening of the immune system with age. notwithstanding, ltcs are fundamentally vulnerable to covid-19 because of an array of predisposing risk factors (118, 119) . unlike the general adult population, covid-infected residents in ltcs are not always capable of communicating their symptoms and frequently have atypical clinical presentations, such as diarrhea, delirium, or falls (120) . on the other hand, between 50 and 75% (121, 122) of them are asymptomatic or presymptomatic at the time of their positive test. these geriatric features complicate and delay case detection. the typical patient profile also leads to poor compliance with infection prevention and control (ipc) practices: most residents have neurocognitive disorders and behavioral symptoms, but some also have mental health disorders or intellectual disability, which means isolation, mask-wearing, and hand hygiene are often impossible. rates of resident noncompliance can reach almost 100% in certain special care units (e.g., wandering ward). moreover, a majority of residents with severe loss of functional autonomy requiring several hours of proximity care per day (e.g., personal hygiene and bath, urinary and bowel elimination, feeding, and medication administration), means close and sustained contact between hcws and infected patients (without source control for the most part) and consequently, higher infection risk on both sides (123) . structural and administrative impediments also come into play. some ltcs have high bed occupancy rates and tight physical spaces (e.g., shared bedrooms and bathrooms), where distancing becomes a challenge and cross-contamination an inevitability (124) . with high population density and limited space, it is very difficult to efficiently segregate patients into zones according to infectious status, leading to mixed units and high infection rates. moreover, some facilities have defective ventilation systems (115) , while others have no mechanical ventilation at all, and must rely on opening windows for air exchange. most importantly, many already understaffed ltcs were hard hit by pandemic-related absenteeism and had to resort to mobilizing staff between units and facilities or calling on lesser-trained external staff to fill in; this element exaggerated all the other risk factors because it hindered the detection and isolation of suspected cases, the deployment of covid-19 units with dedicated staff, the optimal application of ipc practices, and the overall quality of care (125) . unfortunately, despite ltcs being at the epicenter of many regions' epidemic, data are still lacking. studies on transmission modes specific to this geriatric subgroup, where various clinical, administrative, and environmental factors intersect, would be very revealing. most authorities agree with the who recommendations for droplet and contact precautions with covid-19 patients. in the united kingdom (126), canada (127), france (128) , switzerland (30), spain (129), portugal (130) , and australia (131), medical masks are indicated in most situations and respirators are required only in high-risk settings involving agps. recently, the who has acknowledged that "short-range aerosol transmission, particularly in specific indoor locations, such as crowded and inadequately ventilated spaces over a prolonged period of time with infected persons cannot be ruled out" but specifies that the significance of covid-19 airborne transmission has not been convincingly demonstrated and requires further research (1) . while the european society of intensive care medicine and society of critical care medicine (132) is also in line with who ppe recommendations, the european centre for disease prevention and control began by recommending respirators at all times, but backtracked in recent updates and now states that both equipment types are appropriate outside of agps (133) , in agreeance with the infectious diseases society of america (idsa) (134) . on the other hand, the united states (135), south korea (29), singapore (136) , and china (137) recommend respirators for routine care. the u.s. cdc states that hcws should wear an n95, but a facemask is a suitable alternative if a respirator is not available. in summary, most western countries have adopted similar guidelines in line with who recommendations, but comparisons with countries in other parts of the world were not possible due to language barriers. surprising attack rates have been reported. possible explanations include the high presymptomatic contagion of certain individuals (138) , as well as the many asymptomatic or paucisymptomatic cases (139) who seem to have similar viral loads to their symptomatic counterparts (140) . furthermore, unlike sars-cov-1 which reached peak viral load (and therefore contagion) at day 7 to 10 from the start of symptoms (141), viral load seems to peak right before the advent of symptoms (108) . given these data, certain researchers estimate that 44% of transmission happens in the presymptomatic phase (108) . finally, nasopharyngeal viral load appears to be much (up to 1,000 times) higher than that of the first sars (142) . we are therefore faced with a very contagious virus that can silently infect a large number of people. moreover, another possible mode of transmission that remains to be elucidated is through fomites. few studies look at sars-cov-2 survival on surfaces. a widely cited experiment showed that the virus could subsist between 4 h (on copper) and 72 h (on plastic) (110) . however, the study took place under experimental conditions (laboratory surface inoculation, at a stable temperature of 21 to 23°c) which do not represent droplet deposition on surfaces in clinical contexts nor the variations of typical indoor environments. nonetheless, the potentially prolonged stability of coronaviruses on surfaces (143) , as well as the extensive environmental contamination reported by many surface sample studies in health care settings (108, (144) (145) (146) (147) , needs to be confirmed by future research, including viral cultures for infectivity. possible fecal transmission is also worth considering. a significant proportion of patients declare gastrointestinal symptoms before respiratory symptoms, and it is even a predominant form of presentation in some individuals (148) . in addition, severe covid-19 cases appear to have more gastrointestinal symptoms than mild or moderate cases (149) . a meta-analysis of over 4,000 patients reported 48% pcr-positive stool samples, of which 70% remained positive even after nasopharyngeal pcr had turned negative (150) . endoscopic studies also found rna in the esophagi, stomachs, duodena, and recta of patients with severe gastrointestinal symptoms (151) . finally, two studies showed the toilet was among the most contaminated areas in indoor settings (152, 153) : interestingly, the patient who's toilet air sample was positive had a negative exhaled breath sample, warranting the consideration that detectable airborne sars-cov-2 could originate from fecal rather than respiratory aerosols. as with air, a limited number of studies have been able to culture infectious viruses from stools (154, 155) , supporting infectivity. in theory, fecal transmission could occur through different routes, including contact (e.g., while changing incontinence briefs), short-range aerosolization (i.e., inhalation), or long-range aerosolization due to toilet flushing (156) . the latter was well established in the sars-cov-1 amoy gardens outbreak and was recently considered the main mode of transmission in a sars-cov-2 outbreak involving a high-rise building in china, where the nine infected cases lived in three vertically aligned flats connected by drainage pipes in the master bathrooms (157) . hcws constitute a high-risk population for infection (158) . however, the contribution of nosocomial transmission was perhaps overestimated at the beginning of the pandemic, since recent genome-sequencing studies have highlighted the importance of community-acquired infection among hcws (159) . for instance, with epidemiological and genomic data on 50 hcws and 10 patients at hospitals in the netherlands, researchers linked these infections with three different clusters, two of which showed local circulation in the community (160) . within each cluster, "identical or near-identical sequences in health care workers at the same hospital, and between patients and health care workers at the same hospital, were found, but no consistent link was noted among health care workers on the same ward or between health care workers and patients on the same ward." the authors therefore concluded that the patterns observed were consistent with multiple introductions into the hospitals through community-acquired infections. similarly, studies are pointing to community transmission dynamics and public policies (e.g., universal mask-wearing) as the main drivers of hcws infection (161) (162) (163) . nonetheless, given that hcws can both infect patients and get infected from patients, workplace practices deserve a closer look. in the presence of a contagious virus and extensive environmental contamination in health care settings, any breach in protection, as small as it may be, can lead to infection. hcws who work regularly with covid-19 patients, especially those in close contact (e.g., patient attendants, nurse aides) can hardly maintain constant and perfect compliance with ipc practices. besides, risk exposure not only occurs with patients during ppe violations but also with other staff members in shared areas without ppe (e.g., cafeterias and changing rooms). unfortunately, few studies looked at ppe compliance during the covid-19 pandemic: one study reported very poor adherence to mask recommendations due to lack of use (almost 30%) or improper use (164) . before the pandemic, cornerstone practices such as hand hygiene were already poorly applied according to several studies in a variety of hospital departments (including icus) across different countries (165) (166) (167) . a drastic change in a short lapse of time appears improbable, especially in long-term-care facilities where the culture and philosophy are one of "home setting" rather than health care setting. moreover, hcws appear to have a false perception of their own compliance with hygiene practices: a mers-cov study showed an absence of correlation between staff's self-assessment and their observed behavior (168) . the researchers mention that most hcws understood the importance of hand hygiene but did not consistently apply it. even so, proper ppe use does not only depend on individual compliance and technique; it is a multidimensional issue with organizational, systemic, and political ramifications (169) . more importantly, ppe is neither the only nor the best way to protect hcws. in fact, when it comes to protection from occupational hazards, ppe is the last and least effective measure in the niosh hierarchy of controls (170) (see fig. 1b ). for the current pandemic and future ones, our priority should therefore be elimination strategies (e.g., decreasing bed occupancy rates, source control), engineering controls (e.g., segregated red zones and proper ventilation), and administrative controls (e.g., dedicated staff, adequate training, and strict enforcement of ipc regulations), ending with ppe (29) . unfortunately, we have seen, around the globe, many health care systems fail to meet the structural, human, and material challenges brought on by covid-19, and some hcws have paid the price for our collective unpreparedness. one final potential source for hcw infection could be the combination of risk factors for aerosol accumulation in certain exceptional circumstances, such as an overcrowded and underventilated long-term-care facility (115) , or makeshift hospitals such as we have seen built around the world (171) . while the vast majority of home and hospital environments are probably safe (172) , some care homes are located in old substandard infrastructure which relies on natural ventilation and does not allow for optimization of air exchange. it is plausible that under these specific conditions, normally minimal levels of infectious respirable aerosols could reach a threshold where classic airborne transmission becomes significant. while we wait for future research to confirm this scenario, we must strive to control what we can, eliminating physical, environmental, and administrative risk factors to protect frontline workers (173) . drawing the line between precaution and excess is a fundamentally subjective process. many experts agree that current droplet and contact precautions are adequate in low-risk settings. however, some prefer to exercise precaution by recommending respiratory protection with critically ill patients (e.g., severe desaturation or tachypnea), arguing that these clinical features predict progression to agps such as intubation (174) . others consider that the minimum precautionary practice is universal n95 use. finally, some argue that only drastic measures such as full head hoods and full-body suits, often seen in china, are sufficiently protective. in the presence of diverging opinions on the definition of so-called precaution, it seems reasonable to use an evidence-based approach to ppe recommendations. the bulk of evidence, until now, indicates that the medical mask is protective in low-risk settings and the respirator is required only for agps, although higher levels of evidence in the future may tip the balance the other way. long-term care facilities, where the risk level may at times be considered high despite the absence of agps, deserve special attention from researchers. lastly, one could argue that our collective but rather limited energy, time, and resources should be invested in the most impactful areas: proven practices that achieve broad consensus and transmission routes that appear to be predominant. for sars-cov-2, long-winded debates on the gray zones and the applicability of the precautionary principle sometimes distract from crucial measures, such as hand hygiene, source control, and optimal ventilation (175) , which are uncontroversial and highly effective, yet still unevenly applied in some settings such as long-term-care facilities. we are in favor of a return to core ipc principles, which should dominate the scientific conversation around covid-19 management. beyond the alarming statistics, several success stories around the world prove that much can be achieved quickly and efficiently with basic yet effective practices (45, (176) (177) (178) , without the need to resort to elaborate theories or equipment. this article is an in-depth literature overview attempting to answer frequently asked questions about droplet and airborne transmission. although not a systematic review, it goes deeper than current narrative reviews and has important implications for ipc practices, hcw protection, and future research. however, there are several limitations. the first is the controversial distinction between droplets and aerosols, still commonly used in much of the scientific literature, although deemed arbitrary and inaccurate by many experts. natural generation of particles belonging to a broad range of size, containing various concentrations of infectious agents, is probably concurrent rather than mutually exclusive, and transmission patterns are likely on a continuum rather than dichotomous. our proposed model addresses this issue. going forward, we are in favor of adapting public health policies and ppe recommendations to include a broader industrial hygiene-inspired definition of aerosols, as presented above, in order to lessen confusion and better represent the nuanced and complex reality of sars-cov-2 transmission. the second major limitation is the lack of clinical studies on sars-cov-2 transmission and ppe effectiveness, meaning that many conclusions are drawn from lower levels of evidence, extrapolations from other viruses, and laboratory and experimental studies. the available literature, however, is mostly consistent: while airborne transmission exists under certain conditions, there is limited direct evidence of it, especially in low-risk health care settings. given the very high viral load typical of sars-cov-2 infections, it is surprising that, after several months of pandemic, many air samples turn out negative or weakly positive, and subsequent positive cultures remain scarce. this may be attributed to the many logistical and technical limitations associated with air sampling and viral cultures, as mentioned previously, which could underestimate airborne infectivity. we must therefore rely primarily on clinical evidence (trials on masks and epidemiological studies) to study transmission; for now, it suggests that the classic airborne route is not significant. a broader airborne model, involving the short-range inhalation route, could better explain current observations. third, only a few national and international guidelines are compared because of the lack of translated documents. a thorough search of guidelines from comparable countries across different continents would allow for an unbiased comparison but is very challenging in practice. while impatiently waiting for future studies, especially clinical trials, to dispel remaining uncertainties and provide definitive answers to the questions raised here, we would like to propose a revised model for sars-cov-2 transmission, involving inhalable aerosols and favorable conditions for airborne transmission (fig. 1) . the premises of this model are based on cumulative data and clinical observations. in light of the positive air and no-touch surface samples found in health care facilities, respiratory sars-cov-2 aerosols probably occur, but many of their attributes are yet unknown; studies thus far seem to suggest these aerosols are short-range and dilute with distance (102, 103, 144) . similarly, epidemiological studies do not support the existence of long-distance aerosol propagation: the four outbreaks most often cited as evidence of airborne transmission (the washington choir, the guangzhou restaurant, the eastern chinese bus riders, and the diamond princess cruise ship) all involved individuals who were in relatively close contact for a prolonged period of time, in an enclosed space, with the presence of enabling factors (e.g., crowdedness, air currents, and poor ventilation). indeed, these conditions seem necessary for respiratory airborne transmission to occur. fecal aerosols, on the other hand, may be more common due to toilet flushing, but further studies are needed to clarify their role and distinguish them from respiratory aerosols. worst-case scenario: no protection on either the sick patient (source) or the health care worker (exposure), emission of particles of various sizes (droplets and aerosols) during natural respiratory activity (breathing, talking, and coughing), entry of infectious inhalable aerosols, and impaction in the nose where viral receptors are abundant and infectivity is greatest. (b) best-case scenario and niosh hierarchy of controls: source control (mask-wearing by the sick patient), engineering control (optimal ventilation), and exposure control (droplet-contact ppe worn by the health care worker) to prevent short-range droplet and inhalable aerosol transmission. clinical microbiology reviews moreover, to solve the mystery of particle size, we must first acknowledge that airborne transmission is not exclusive to small aerosols: some larger particles typically classified as droplets may remain airborne, especially if suboptimal airflows contribute to their preservation in suspension and reduce their dilution (179) . thus, inhalable aerosols are the ideal candidate to explain current findings, because they exhibit shorter travel distance and air suspension time than respirable aerosols while having greater potential for infection because of their higher probability of containing virions (16) . furthermore, because inhalable aerosols are larger, they are more likely to deposit proximally in upper airways compared to respirable aerosols (180) , which is in line with the robust data suggesting that nasal cells are the main portal for initial infection, with a gradient of infectivity from the proximal (nose) to the distal (lungs) respiratory tract (21, 22) . therefore, transmission of short-range airborne and inhalable aerosols could explain the seemingly contradictory finding that there are viruses in the air and transmission between individuals without contact, but lack of convincing clinical evidence of classic airborne transmission (i.e., long-distance ranges and superiority of respirators). this size range could exhibit behaviors typical of both droplets and aerosols: higher viral load, airborne behavior, inhalation, and deposition in the nose. despite relatively shorter suspension time, inhalable aerosols become especially significant in the case of prolonged exposure and close proximity. in addition, they are less likely to follow air streams through leaks in the nonfitted mask, nor make it down to alveolar space, because of larger size, but rather will remain in nasal cells due to natural impaction processes. consequently, tight seals and superior filtration would not be required in most low-risk settings, as masks (with the help of face shields) could readily block these airborne particles. however, different categories of hcws may not be exposed to the same level of risk: an attendant who spends an hour feeding, bathing and positioning a patient will be at much higher risk of inhaling aerosols compared to a doctor who questions and examines a patient for 10 min. finally, ventilation parameters (air exchange rate, flow direction, and airflow patterns) would play a role, since they could contribute to enhancing or reducing airborne suspension and transmission (181) . this model is difficult to assess given the short-range distance and the short airborne stability, as well as the alteration of particle size during most air sampling processes (desiccation and impaction in liquid). however, we believe this novel paradigm, which departs from the outdated aerosol/droplet dichotomy, more accurately portrays the reality of naturally generated viral particles and the nuances in transmission patterns. broadening the "airborne" definition to inhalable aerosol exposure in the context of proximity care, and considering inhalation as a significant route of entry for the sars-cov-2 virus, could open up new paths of exploration. in summary, traditional droplets (larger particles with ballistic behavior that deposit onto surfaces), as well as our newly defined inhalable aerosols (particles that can be suspended, breathed in, and impacted at the nose, at the location of highest infectivity), could be the predominant modes of transmission of sars-cov-2. classic respirable aerosols, even if present, seem unlikely to be significant in routine health care contexts, possibly due to insufficient quantity, 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the air and on surfaces in the covid-19 hospital indoor air quality monitoring for the detection of sars-cov-2 (covid-19) virus environmental contamination of sars-cov-2 on surfaces, air-conditioner and ventilation systems a field indoor air measurement of sars-cov-2 in the patient rooms of the largest hospital in iran aerosol and environmental surface monitoring for sars-cov-2 rna in a designated hospital for severe covid-19 patients we thank magali-wen st-germain for the original design, creation, and development of fig. 1 , as well as patrick lane, sceyence studios, for the final version of fig. 1 . we thank stéphanie langevin, quoc dinh nguyen, luc trudel, and jean barbeau for their contribution in reviewing the original manuscript.both authors substantially contributed to the conception, design, analysis, and interpretation of data, as well as reviewing and approving the final version of the manuscript. we agree to be accountable for the contents.c.d. is holder of tier-1 canada research chair on bioaerosols. for this review article, we received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.we declare that we do not have commercial or financial relationships that could, in any way, lead to a potential conflict of interest with regard to this publication. key: cord-333547-88dkh6xd authors: hasan, shadi w.; ibrahim, yazan; daou, marianne; kannout, hussein; jan, nila; lopes, alvaro; alsafar, habiba; yousef, ahmed f. title: detection and quantification of sars-cov-2 rna in wastewater and treated effluents: surveillance of covid-19 epidemic in the united arab emirates date: 2020-10-19 journal: sci total environ doi: 10.1016/j.scitotenv.2020.142929 sha: doc_id: 333547 cord_uid: 88dkh6xd testing sars-cov-2 viral loads in wastewater has recently emerged as a method of tracking the prevalence of the virus and an early-warning tool for predicting outbreaks in the future. this study reports sars-cov-2 viral load in wastewater influents and treated effluents of 11 wastewater treatment plants (wwtps), as well as untreated wastewater from 38 various locations, in the united arab emirates (uae) in may and june 2020. composite samples collected over twenty-four hours were thermally deactivated for safety, followed by viral concentration using ultrafiltration, rna extraction using commercially available kits, and viral quantification using rt-qpcr. furthermore, estimates of the prevalence of sars-cov-2 infection in different regions were simulated using monte carlo. results showed that the viral load in wastewater influents from these wwtps ranged from 7.50e+02 to over 3.40e+04 viral gene copies/l with some plants having no detectable viral rna by rt-qpcr. the virus was also detected in 85% of untreated wastewater samples taken from different locations across the country, with viral loads in positive samples ranging between 2.86e+02 and over 2.90e+04 gene copies/l. it was also observed that the precautionary measures implemented by the uae government correlated with a drop in the measured viral load in wastewater samples, which were in line with the reduction of covid-19 cases reported in the population. importantly, none of the 11 wwtps’ effluents tested positive during the entire sampling period, indicating that the treatment technologies used in the uae are efficient in degrading sars-cov-2, and confirming the safety of treated re-used water in the country. sars-cov-2 wastewater testing has the potential to aid in monitoring or predicting an outbreak location and can shed light on the extent viral spread at the community level. coronaviruses (covs), which range from 60 to 220 nm in size, are enveloped positive-sense single-stranded ribonucleic acid (rna) viruses with club-like spikes on their surface, and infect a wide range of hosts including birds and mammals. they were first identified in the mid-1960s, and have traditionally been associated with the common cold in humans. more recently, they gained significant medical importance due to the emergence of three deadly zoonotic strains: (i) severe acute respiratory syndrome coronavirus (sars-cov) in 2002 (mortality rate 11%), (ii) middle east respiratory syndrome coronavirus (mers-cov) in 2012 (mortality rate 35%), and (iii) sars-cov-2 in 2019 (mortality rate still being determined, but estimated at 2-3%) [1] [2] [3] [4] [5] . covs have more detrimental impacts on immunocompromised and elderly individuals and have the potential to create worldwide outbreaks [6] . the first cases of sars-cov-2 infections occurred in either late november or early december 2019, and quickly spread until the world health organization (who) declared it a public health emergency of international concern on january 30 th 2020, and a pandemic on march 11 th 2020. despite international efforts to contain the virus using various policies, it is still posing a serious global challenge today [7, 8] . scientists attribute the difficulty in managing the outbreak to a long incubation period (~5 days) and viral shedding and transmission by asymptomatic infected individuals [9, 10] . most of what we know about viral shedding comes from studies that quantify sars-cov-2 in pharyngeal or nasopharyngeal swabs, with very little work done to understand the shedding of this virus in stool. a limited number of studies have shown that the shedding period of sars-cov-2 in stool samples varies considerably, and can still be detected up to 27.9 ± 10.7 days after infection in some cases [9, 11] . the major transmission routes of sars-cov-2 have been shown to be through aerosol droplets, person to person contact, and contaminated surfaces j o u r n a l p r e -p r o o f journal pre-proof [11] [12] [13] [14] [15] . some studies demonstrated that the virus can replicate in cells that line the human digestive tract, and that a significant number of covid-19 patients have detectable levels of viral rna in their stool [16] [17] [18] . in some cases, the number of viral gene copies in one gram of stool ranges from ~10 4 to 10 8 genomes [16] [17] [18] . even though the virus is detectable in the stool of covid-19 patients, there is limited evidence that it is infectious, as only one study to date has demonstrated this to be the case, while another study demonstrated that it was not possible to isolate infectious viruses from the stool of covid-19 patients [16] [17] [18] . these findings indicate that sars-cov-2 could be detected in the municipal wastewater of a region containing infected individuals. furthermore, it also means that monitoring the virus in municipal wastewater represents an effective low-cost method to track infections in the population. wastewater monitoring has been a successful method of tracking emerging contaminants and circulating antimicrobial/antibiotic resistance genes [19] [20] [21] . furthermore, monitoring different types of viruses, such as hepatitis a, noroviruses, and rotavirus, in wastewater has also been used as a surveillance technique to investigate the distribution of these viruses in a community, a practice termed water-based epidemiology (wbe) [22, 23] . it is thus possible that wbe and the environmental surveillance of sars-cov-2 in wastewater could help track the outbreak in a certain region and identify regions with infected asymptomatic individuals. carrying out sars-cov-2 testing to identify and quarantine affected individuals is a good strategy for managing this outbreak in countries with testing capabilities and capacities, and is the method used here in the uae. however, it is a reactionary strategy that can only be deployed after a case is reported clinically, and by then it is likely that the virus has been spreading through a population at a significant level. in contrast, detecting and quantifying sars-cov-2 in wastewater covers a larger portion of the population as it essentially tests j o u r n a l p r e -p r o o f journal pre-proof everyone who contributed to the wastewater sample analyzed [24, 25] . if successful, it would be a way of mass testing and could lead to early detection before infections manifest as clinical cases or hospital admissions. recent peer-reviewed and non-peer-reviewed studies published by several groups in the netherlands, the usa, france, spain, italy, australia, and turkey have demonstrated that the idea of tracking infection rates by measuring the viral load in wastewater is feasible, cost-effective, and efficient [1, [26] [27] [28] [29] . the authors believe that the viral concentration in wastewater is a good predictor of the number of infected individuals in the population of the united arab emirates (uae), including asymptomatic cases that would otherwise remain undetected. most importantly, monitoring wastewater could be used as a way of tracking the prevalence of the virus and become an early-warning tool for new outbreaks in the future. consequently, the main objectives of this study were: (i) to detect the presence of sars-cov-2 virus in municipal (untreated) wastewater and treated effluents of wastewater treatment plants (wwtps) in the uae; (ii) to quantify the viral concentration in viral gene copies per liter; and (iii) to explore whether these measurements mirror infections in the population in order to comment on the utility of this method to track the epidemiology of the disease. thus far, the frequency and degree of testing individuals and wastewater samples achieved in the uae have been replicated only in a few other jurisdictions. to the best of our knowledge, this is the first study to detect and quantify sars-cov-2 in wastewater in the region. various sewer access points (e.g. manholes located in neighborhoods) and pumping stations. these 11 wwtps implement a series of treatment technologies including preliminary, primary, secondary (asp/clarification), and tertiary (sand filtration, disinfection, chlorination) for the purpose of reusing treated water (tse) for irrigating farmland and watering green spaces. composite samples were collected over a 24-hour period using auto-samplers, and 250 ml of the composite samples were transferred to isolab gmbh sterile polypropylene (pp) plastic bottles, labortechnik gmbh, germany) for 90 min [30] [31] [32] . although this inactivation step might lead to some viral loss, it is an essential safety measure that minimizes the risk of sars-cov-2 transmission to laboratory personnel. studies have demonstrated that thermal deactivation at lower temperatures, similar to the one chosen in this study, resulted in a non-infectious virus that could still be detected by rt-pcr based methods, while deactivation at much higher temperatures (92 o c) resulted in complete degradation of the virus so that detection was not possible by rt-pcr [32, 33] . after heat inactivation, the samples cooled down to 4 o c prior to filtration and viral concentration. in the second method, 8,000 average molecular weight polyethylene glycol (peg) was used to concentrate the viruses [30] . briefly, 50 ml of the filtrate was transferred to a 50 ml conical tube, followed by the addition of peg and nacl to a final concentration of 8 and 1.8%, respectively. the sample was mixed on a nutator for 1 h in order to dissolve the peg and nacl and precipitate viruses. the sample was then centrifuged at 12,000 x g using a fixed angle rotor for 2 h at 4 o c. the supernatant was poured off and the pellet was re-suspended in 1.5 ml of trizol (thermofisher cat#15596). isopropanol (1 ml) was added to the aqueous layer and the mixture was agitated by inverting the tube before incubation at room temperature for 10 min. the rna was pelleted by centrifugation at 12,000 x g for 15 min at 4 o c and washed once in 75% ethanol prior to resuspension in elution buffer (40 µl) from the abiopure viral dna/rna extraction kit. the two protocols described for viral concentration and rna extraction (either ultrafiltration columns/rna extraction kit or peg/trizol) were selected and tested because they were the two methods that were reported for this type of work at the time these experiments were initiated in early april 2020 [30, 34] . the authors wanted to compare them to decide which methodology to adopt as wastewater sampling continued. all the data reported in the tables were obtained using the ultrafiltration method ( fig. 1a) the extracted rna (8 µl the prevalence of sars-cov-2 infection in a certain region was estimated using the measured viral loads in wastewater, the wastewater flow rate, the viral load in the stool of infected individuals, and the estimated daily production of stool per capita according to eq. 2 [27] : the stool viral load (r f ) in log 10 was simulated as a log-normal distribution with a mean of 7.18 and stdev of 0.67 [16] . daily stool production per capita (f) (g/capita.day) was simulated as a log-normal distribution with a mean of 149 and stdev of 95 as per the statistics reported for the high-income countries [35] . the percentage of covid-19 patients who shed virus in their stool (ɛ) was modeled as a uniform distribution from 0.29 to 0.55, as reported by the limited number of studies on this topic thus far [11, 15, 36] . the reported data from the simulations were based on 50,000 calculations. j o u r n a l p r e -p r o o f at the time when the authors initiated this study, only two studies were reported in non-peerreviewed pre-prints; one study utilized ultra-concentration columns for viral concentration followed by rna kit extraction, while the other study used peg to concentrate viruses followed by trizol extraction [30, 37] . therefore, in order to assess the differences between these two methods and determine which one to use for our study, viruses from the same wastewater samples (50 ml) were concentrated using either peg followed by rna extraction with trizol or using a 30 kda mwco column followed by extraction using a viral rna kit as described in the materials and methods. quantification of the viral load was then performed identically on the rna extracted from each method using rt-qpcr. different methods for wastewater virus concentration have been adopted by different groups around the world [27, 30, 37] . the methods fall into three major categories and involve concentrating viruses using either (i) precipitation using chemicals such as peg or al(oh) 3, followed by centrifugation [1, 30, 38, 39] , (ii) ultrafiltration columns with very low molecular weight cut-offs [27, 29, 34] , or (iii) binding of viruses to electronegative membranes [27, 40] . using ultrafiltration columns in combination with the commercial rna kits resulted in higher counts compared to using peg and trizol (fig. 2) . indeed, a reading of 31.7 gene copies/ml was determined using the ultrafiltration method compared to a reading of 2.6 gene copies/ml for the same sample using the peg/trizol method. consequently, all subsequent virus measurements in the wastewater samples were performed using the ultrafiltration and commercial rna extraction kit method. the peg protocol tested here was used in the state of massachusetts in late march 2020, with a significantly higher number of reported cases than what was reported in the uae when j o u r n a l p r e -p r o o f journal pre-proof the measurements reported here were performed. in the massachusetts study, high counts ranging between 50 and 300 gene copies/ml were reported in the wastewater samples [30] . precipitation-based methods that rely on chemicals such as peg to concentrate viruses have a higher throughput since they are cheap and user-friendly compared to the expensive ultrafiltration column method that sometimes requires multiple rounds of centrifugation to concentrate the wastewater sample. however, using ultrafiltration columns in combination with commercial rna extraction kits might represent a more sensitive approach, more suited to detecting lower concentrations of the virus in wastewater. the ultrafiltration method might be more appropriate for long term surveillance as well after the virus concentration drops below the detectable limit of the assay. once that occurs, a more sensitive assay would be able to detect both methods used have advantages and caveats [41] . for example, one drawback of concentrating viruses using the ultrafiltration method is related to the fact that it is often difficult to concentrate all viruses from the sample, as some viruses are adsorbed to the solid phase in the wastewater sample and are thus pre-filtered when the sample is passed through the 0.2micrometer filter. as more groups report the experimental protocols they used to make sars-cov-2 wastewater measurements, scientists need to come together to agree on standardizing the method used to perform this analysis. whatever the method is chosen, there will always be a viral loss that results from different steps in the process. it is the author"s opinion that the most important property of wastewater monitoring is sensitivity, reproducibility, precision, and not j o u r n a l p r e -p r o o f necessarily accuracy. at this point, the value of these measurements is to observe the change over time in order to gain insight into infection rates and kinetics in the population. laboratories with access to bsl-3 labs will lead the way in determining which methods are best suited for this type of analysis in the future. the viral loads in the 11 wwtps and the 38 locations in the uae are summarized in fig. 3 and table 2 j o u r n a l p r e -p r o o f preceded by an elevation of sars-cov-2 concentration in wastewaters [43] . indeed, several studies have explored the detection and quantification of sars-cov-2 in wastewater influents [27, 30, 34, 39, [44] [45] [46] . however, only a few studies have attempted to establish a correlation between the increase/decrease in the viral load in the wastewater influents and the number of infected patients [1, 27, [43] [44] [45] . 2020, 346 new covid-19 cases were reported in the uae, which is less than those reported on the same day of the preceding month [42] . in another study carried out in the murcia region (spain), the epidemiological data on covid-19 revealed a tight correlation between the virus prevalence (cases per 1.00e+05 inhabitants) and the viral load in the wwtp"s influents. their study also confirmed that sars-cov-2 could be detected weeks before the initial confirmed case [1] . these studies, together with our findings, provide strong evidence for the significance of environmental epidemiological surveillance in monitoring sars-cov-2 outbreaks, and for this surveillance method to be used as an early warning tool for possible outbreaks or "second waves". this tool can help decision-makers in developing appropriate policies to manage outbreaks in a certain area. it can also help evaluate the effectiveness of these policies in reducing the widespread transmission of the virus. although mass testing of inhabitants for sars-cov-2 is an excellent option for assessing the virus prevalence, wastewater surveillance of sars-cov-2 viral loads can also be a good indicator of the circumstances on the ground and could direct the mass testing efforts to specific geographic locations. in epidemiology, the reproduction number is the number of individuals an infected person interacts with and infects. this number can be significantly reduced through infection mitigating policies that a jurisdiction implements [47, 48] . the first covid-19 case in the uae was reported on january 29 th 2020, and that person later recovered on february 9 th 2020. the j o u r n a l p r e -p r o o f several months after the virus outbreak, many countries still cannot carry out enough sars-cov-2 tests to identify and isolate infected individuals in order to curb the epidemic. what makes the situation even more complex is that a significant amount of viral transmission is attributed to asymptomatic infected individuals or individuals with very mild symptoms [51] [52] [53] . this also means that the number of actual infections is underestimated. thus, augmenting testing strategies using wastewater epidemiology data represents a promising new tool in estimating prevalence in a given location. different conclusions can be drawn from the wastewater analyses depending on how the data is processed, essentially achieving different levels of insight into the outbreak. at the first level, viral concentration in the wastewater (gene copies/l) is not very helpful on its own, though it can give you a good overview of how an outbreak is progressing from sampling the same area over time. this assumes that the flow of water through the area does not change much between sampling dates and viral load can inform on whether the outbreak is getting better or worse. importantly, different areas cannot be compared to each other using this first metric only. for instance, the viral load in viral gene copies/l for b4 wwtp gives an indication that the region served by this wwtp is significantly more affected than the region served by b2 wwtp, attempted to provide an estimate in different regions using the monte carlo approach and the results were reported with 95% ci based on the simulated variables. as more groups around the world report results and communicate laboratory protocols used to make these measurements, a comprehensive comparison needs to be carried out in order for scientists to agree on standardized methods. overall, this study showed the potential of detecting sars-cov-2 in wastewater as an early warning and prediction tool for the spread of the disease. this monitoring can help authorities take appropriate and prompt actions to contain any potential outbreak of covid-19 in their communities. sars-cov-2 rna in wastewater anticipated covid-19 occurrence in a low prevalence area the deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in 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historic district to slow coronavirus coronavirus: uae to clear streets for disinfection drive who says asymptomatic covid-19 transmission is "very rare iceland lab"s testing suggests 50% of coronavirus cases have no symptoms no symptoms in 80% of covid cases raise concerns the authors are thankful to the ministry of interior (moi) in abu dhabi (uae) for their financial support (award no. cpra-2020-027), their immense efforts in coordination and key: cord-339009-wcoch07b authors: file, thomas m.; tsang, kenneth w. t. title: severe acute respiratory syndrome: pertinent clinical characteristics and therapy date: 2012-08-23 journal: treat respir med doi: 10.2165/00151829-200504020-00003 sha: doc_id: 339009 cord_uid: wcoch07b severe acute respiratory syndrome (sars) is a newly emerged infection that is caused by a previously unrecognized virus–a novel coronavirus designated as sars-associated coronavirus (sars-cov). from november 2002 to july 2003 the cumulative number of worldwide cases was >8000, with a mortality rate of close to 10%. the mortality has been higher in older patients and those with co-morbidities. sars has been defined using clinical and epidemiological criteria and cases are considered laboratory-confirmed if sars coronavirus is isolated, if antibody to sars coronavirus is detected, or a polymerase chain reaction test by appropriate criteria is positive. at the time of writing (24 may 2004), no specific therapy has been recommended. a variety of treatments have been attempted, but there are no controlled data. most patients have been treated throughout the illness with broad-spectrum antimicrobials, supplemental oxygen, intravenous fluids, and other supportive measures. transmission of sars is facilitated by close contact with patients with symptomatic infection. the majority of cases have been reported among healthcare providers and family members of sars patients. since sars-cov is contagious, measures for prevention center on avoidance of exposure, and infection control strategies for suspected cases and contacts. this includes standard precautions (hand hygiene), contact precautions (gowns, goggles, gloves) and airborne precautions (negative pressure rooms and high efficiency masks). in light of reports of new cases identified during the winter of 2003–4 in china, it seems possible that sars will be an important cause of pneumonia in the future, and the screening of outpatients at risk for sars may become part of the pneumonia evaluation. severe acute respiratory syndrome (sars) was first recog-1. definition nized in the guangdong province in southeast china in late 2002, for surveillance purposes and before the availability of laboraand it subsequently spread globally rapidly during the early tory tests to detect the causative agent, sars was originally months of 2003. [1, 2] sars captured worldwide attention as a defined using clinical and epidemiologic criteria from suspect or highly infectious disease with high mortality and as an occupationprobable cases. [3, 4] a suspect case included a respiratory illness of al hazard among healthcare providers. the impact of sars was unknown etiology and with the following criteria: extensive from both a sociological and economical consequence -• measured temperature >100.4ºf (>38.0ºc) particularly in asia. because the causative agent of sars is • one or more clinical findings of respiratory illness (e.g. cough, contagious, preventative measures focus on avoidance of exposhortness of breath, difficulty in breathing, or hypoxia) sure, and infection control strategies for suspected patients and • travel within 10 days of onset of symptoms to an area with contacts. the emergence of sars illustrates the need for global suspected or documented community transmission of sars cooperation of healthcare systems to ensure the public health of (excluding areas with secondary cases limited to healthcare local regions, and the need to be prepared to rapidly institute workers or direct household contacts and close contact within policies to respond to newly emerging infectious threats. 10 days of onset of symptoms with either a person with a respiratory illness or a person under investigation or suspected recombination event between mammalian-like and avian-like parof having sars). ent viruses which may have been responsible for the switch of host of the sars-cov from animals to humans. a probable case was defined as a suspect case with either radiographic evidence of pneumonia or respiratory distress syndrome, or autopsy findings consistent with respiratory distress 3. epidemiology syndrome without an identifiable cause. [4] once the virus was identified and laboratory tests became as of july 2003 the cumulative number of worldwide sars available for detection, the surveillance case definition for sars cases was 8437, with a mortality of 9.6%. [12] of the reported cases was updated to include laboratory criteria for evidence of infection 64% were from china, 19% from hong kong, 8% from taiwan, with the sars-associated coronavirus (sars-cov). initially, 3% from canada, and 2% from singapore. the us has been since it was unclear whether sars infection could be present in relatively spared from the clinical impact of sars. at july 2003, people who were asymptomatic, the definition included the possi-27 probable cases had been reported, of which only eight had bility of asymptomatic ('subclinical') infection. however, subselaboratory confirmation of acute coronavirus infection. [13] quent investigations suggest that asymptomatic infection is very the initial cases reported in hong kong were linked to an index uncommon. [1] as a consequence, the latest surveillance case defipatient, a medical doctor from the guangdong province of china nition for sars by the us center for disease control and who traveled to hong kong to attend a wedding in late february prevention (cdc) has been updated to include clinical criteria for 2003. [14] he had previously treated patients with 'atypical' pneuearly illness, mild-to-moderate illness, and severe respiratory illmonia in guangdong. subsequently several guests who had stayed ness, which are then characterized by epidemiological and laboraat the same hotel became ill with sars. these patients subsetory criteria (table i) . [5] quently infected numerous healthcare workers and family members or became index cases in other countries (canada, vietnam, singapore, etc.) [ figure 1 ]. the mortality has been higher in older patients and those with co-morbidities. the syndrome has been observed primarily in soon after the recognition of the clinical syndrome of sars, adults aged 25-70 years, and children have been relatively spared. several different laboratories identified a novel coronavirus, desig-there appears to be no significant underlying predisposing condinated sars-cov, in vero e6 cell cultures inoculated with respirtion for the development of sars, however the elderly and atory secretions and lung tissue of infected patients. [6, 7] other patients with underlying conditions are at greater risk for mortalitechniques, including electron microscopy, reverse transcriptionty. in one study from hong kong the mortality for those >60 years polymerase chain reaction (rt-pcr), and serovonversion, have of age was 43%. [15] in another study, multivariate analysis showed also pointed to this as the causative agent. sero-epidemiological that age >60 years, presence of diabetes mellitus or heart disease, data indicate the sars-cov was not previously found in and the presence of other co-morbid conditions were independenthumans. [8] preliminary reports of detection of the sars ly associated with mortality. [16] early in the evaluation of this coronavirus in himalayan palm civet cats and a number of other syndrome, most of the descriptions were of patients who required species are suggestive of interspecies transmission of this new hospitalization. however, as more cases were identified, particuvirus. investigators from hong kong reported a coronavirus relarly in the western countries, the majority of patients have not sembling sars virus isolated from several himalayan palm civet required hospitalization. cats and a raccoon dog obtained in a market in the guangdong province (such animals are considered as food delicacies in that sars appears to be transmitted by close contact with patients region. [9] ) they also reported that several of the handlers at the who have illness due to sars virus. [1, 2] the greatest risk of market had antibody to the sars virus. studies of the genetic transmission is most probably via direct contact with respiratory sequence of the two viruses show a similar pattern, suggesting a secretions. there is no evidence of spread from patients before species jump from wild animals to humans. furthermore, experi-they develop symptoms. the majority of cases have been reported mental infection of macaques with sars-cov produced a pneu-among healthcare workers and family members of affected permonia that was pathologically similar to sars in humans. [10] sons. however, evidence of community spread of the disease is stavrinides and guttman compared the sars-cov genome with emerging, suggesting that other modes of transmission, such as related coronaviruses and found about half the dna resembled airborne or direct contact, may also have a role. [1] clusters of cases coronavirus sequences from mammals, while the other half looked in community settings such as hotels and apartment buildings like virus found in birds. [11] these data suggest a possible past demonstrate that transmission can be efficient. many household [5] clinical criteria presence of two or more of the following features: fever (might be subjective), chills, rigors, myalgia, headache, diarrhea, sore throat, or rhinorrhea temperature >100.4°f (>38°c), and one or more clinical findings of lower respiratory illness (e.g. cough, shortness of breath, or difficulty breathing) meets clinical criteria of mild-to-moderate respiratory illness, and one of more of the following findings: radiographic evidence of pneumonia, or acute respiratory distress syndrome, or autopsy findings consistent with pneumonia or acute respiratory distress syndrome without an identifiable cause one or more of the following exposures in the 10 days before onset of symptoms: travel to a foreign or domestic location with documented or suspected recent transmission of sars-cov, or close contact with a person with mild-to-moderate or severe respiratory illness and history of travel in the 10 days before onset of symptoms to a foreign or domestic location with documented or suspected recent transmission of sars-cov one or more of the following exposures in the 10 days before onset of symptoms: close contact with a person with confirmed sars-cov disease, or close contact with a person with mild-to-moderate or severe respiratory illness for whom a chain of transmission can be linked to a confirmed case of sars-cov disease in the 10 days before onset of symptoms detection of serum antibody to sars-cov by a test validated by cdc (e.g. enzyme immunoassay), or isolation in cell culture of sars-cov from a clinical specimen, or detection of sars-cov rna by a rt-pcr test validated by cdc and with subsequent confirmation in a reference laboratory (e.g. cdc) case classification b probable case of sars-cov disease: meets the clinical criteria for severe respiratory illness and the epidemiologic criteria for likely exposure to sars-cov confirmed case of sars-cov disease: clinically compatible illness (i.e. early, mild-to-moderate, or severe) that is laboratory confirmed a tests to detect sars-cov are being refined and, therefore, criteria for laboratory diagnosis of sars-cov are changing. b asymptomatic infection or clinical manifestations other than respiratory illness may be identified in future as more is learned about sars-cov. cdc = center for disease control; rt-pcr = reverse transcription polymerase chain reaction; sars-cov = sars-associated coronavirus. contacts have become ill. epidemiologic evidence indicates that quired sars via contact with a fomite. of still unexplained significance, a few patients have been involved in the transmission the transmission of sars is facilitated by face-to-face contact, of an unusually large number of secondary cases -so-called superand this still appears to be the most common mode of spread in the spreading events. form of droplet transmission. [1] however, airborne or fecal transmission may have a role in some settings, and it could account for peiris et al. [17] studied the viral load of sars-cov over time in the extensive spread within buildings and other confined areas that respiratory secretions from 14 sars patients and found the load in has been observed in some places in asia. transmission via casual nasopharyngeal aspirates increased to a peak on the tenth day after contact is uncommon, but has been documented on an airplane or onset of symptoms, then decreased gradually. notwithstanding the in a taxi. [1] some healthcare workers also appeared to have ac-small sample size and the effects of concomitant administration of ribavirin and corticosteroid, this suggests that sars patients of virology in beijing. the virus was apparently transmitted to the student's mother and a nurse caring for the student (all three had might be most contagious in the second week of illness. laboratory-confirmed cases). at the time of writing, a total of nine after the termination of the initial outbreak of sars in july possible cases of sars have been identified and the health author-2003, there were two further cases of sars-cov infection which ities were involved in active surveillance to identify other possible were likely acquired in a laboratory setting, one case in late august cases. [20] and the other in december. [18] this reinforces the necessity for careful laboratory practices when working with this virus. it 4. clinical manifestations appears there was no evidence of secondary transmission associated with either case, despite the active social activities undertaken the initial descriptions of the clinical manifestations of sars by these two scientists after their exposure to sars-cov. an have come from reports of patients who have required hospitalizaadditional four community-acquired cases from the guangdong tion. [8, [21] [22] [23] [24] in such patients the disease is often reported as a bi-province of china were subsequently diagnosed from december phasic or tri-phasic illness with an initial acute febrile phase 2003 through january 2004. of interest, no close contacts of these followed by a lower respiratory illness phase then progression in cases was found to have fever or respiratory symptoms after home approximately 20-30% of patients to a phase characterized by quarantine to date. [19] more recently, in april 2004, the chinese acute respiratory distress syndrome (ards) necessitating ventilator support. ministry of health reported additional cases of sars which seemed to be initially associated with an index case of a 26-year-it is imperative to appreciate that individual patients do not old female graduate student who worked at the national institute necessarily display these 'phases', which could be highly individpeiris et al. [17] (n = 50) lee et al. [23] (n = 138) poutanen et al. [22] (n = 10) tsang et al. [21] (n = 10) ualized, from hyperacute to indolent presentation in time course. festations as the patients described above (figure 2). although the at the time of writing, the mortality of probable sars cases was majority of patients developed symptoms of respiratory tract inapproximately 10%, but was much higher in older individuals and fection during admission, only a minority of patients had sympthose with significant co-morbidities. toms at the time of admission to the hospital. diarrhea was present the mean incubation period of sars is estimated to be 6 days, in only 10% at the time of admission, but 50% developed this with a usual range up to approximately 10 days after exposymptom while in the hospital. these investigators also observed sure. [1, 2, 15, 25] the illness generally begins with a prodrome of fever, the duration of time from the onset of illness until the evolution of often associated with chills, rigors and myalgia. headache and various endpoints of their disease: fever, 0.3 days; admission to the severe malaise may accompany this phase; rash has been absent in most cases. in one outbreak within an apartment complex in hong kong, diarrhea was found in 66% of cases. [1] after a typical period of 3-7 days, a lower respiratory phase may begin with the onset of non-productive cough and progressive pneumonia. in the initial reports of cases, 20-30% of patients required intensive care unit management and mechanical ventilation. the presenting symptoms of patients admitted to the hospital from four published series are listed in table ii. [8, [21] [22] [23] most patients were admitted to the hospital several days after the onset of symptoms. the most common complaints were fever and chills or rigors. upper respiratory tract symptoms such as rhinorrhea and sore throat were less common. at the time of examination, abnormal auscultatory findings were present in about one-third of patients. although fever and progressive respiratory manifestations are a hallmark of most cases of sars, patients with more indolent characteristics (including absence of fever) have been describedespecially in elderly or immunocompromised patients. [1, 26] the first report of a complete outbreak of sars (from beginning of the outbreak until declaration of containment) was recently published by vu et al. [27] they report a cohort of patients, all of whom required hospitalization, who presented with similar manihospital, 4.3 days; onset of radiographic change, 4.4 days; onset of multivariate analysis, the only factors that were predictive of an respiratory symptoms, 4.5 days; onset to maximal radiographic adverse outcome were advanced age, a high peak lactate change, 10 days; onset to intubation, 10.5 days; onset to end of dehydrogenase level, and a higher absolute neutrophil count. fever, 12.7 days; and onset to death, 18.8 days. [27] 5. diagnosis chest x-ray abnormalities are usually absent during the initial phase of illness but become progressively abnormal during the the initial manifestations of sars are not specific and cannot phase of lower respiratory illness. initially this is characterized by easily be distinguished from those of other respiratory infections. early focal interstitial infiltrates, usually seen as ground glass the first clinical definition developed by the who (see section 1) opacity, and progressing to bilateral disease (figure 3). a typical was found to be only 29% sensitive and approximately 70% ards picture has emerged in many very seriously ill patients. in specific for identifying laboratory documented cases. [30] clinicians these patients, high-resolution computed tomography (hrct) is should conduct thorough diagnostic testing to rule out other etiolomore sensitive in early disease when the chest x-ray could be gies in patients suspected of having sars. initial recommended normal or only showing inconclusive consolidation. characteristidiagnostic testing procedures include chest radiograph and pulse cally, hrct shows peripheral and, most commonly, lower lobe oximetry. since sars often progresses rapidly, repeated chest xconsolidation. [28, 29] although non-diagnostic and closely mimickrays within the first or second day, sometimes twice daily, may be ing the appearance of bronchiolitis obliterans with organizing helpful in documenting the course of disease. indiscriminate use of pneumonia, hrct is also helpful for showing no evidence of hrct to detect 'radiographically occult disease' is to be discourpleural effusion or intrathoracic lymphadenopathy, which are very aged in view of infection control issues, and the rapidly progresrarely seen in sars. [2, 21, 28, 29] spontaneous pneumomediastinum sive nature of sars, thus making it likely that radiographic also occurs as a rare complication with sars. [8] abnormalities would be more apparent within a few days after laboratory abnormalities most often associated with sars hospitalization. [2] tests for evaluation of specific organisms assoinclude absolute lymphopenia, mild neutropenia, and thrombociated with pneumonia should be performed, and include: blood cytopenia. mild to moderately elevated plasma levels of creatine cultures, sputum gram stain and culture, and testing for viral phosphokinase, lactate dehydrogenase, and transaminases were respiratory pathogens -especially influenza and respiratory synseen in 30-80% of cases (table ii) . cytial virus. urinary antigen for both pneumococcus spp. and lee et al. [23] found that advanced age, male sex, and high levels legionella spp. should also be considered. acute and convalescent of serum creatine phosphokinase, serum lactate dehydrogenase, a serum (preferably 28 days after onset of symptoms) should be relatively high initial neutrophil count (i.e. mean 4.6 vs 3.7 × collected from each patient who meets the sars clinical case 10 -9 l), and a low levels of serum sodium were significant predic-definition. however, if acute and convalescent phase sera are tive factors for intensive care unit admission, and death. on collected at least 8-10 days apart, a 4-fold or greater rise in different rt-pcr assays performed on different specimen aliquots identify the coronavirus rna. [33] because of the possibility of false-negative cultures and rt-pcr assays, only the absence of antibody in a serum specimen obtained >21 days after symptom onset is considered by the cdc to be a negative laboratory test for sars coronavirus. the likelihood of detecting sars-cov is increased if multiple specimens (e.g. stool, serum, respiratory tract specimens) are collected during the course of illness. clinicians should consult with their local laboratory personnel and health department about obtaining such tests. the priority of specimens for sars-cov testing and optimal timing for collection are presented in table iii. of the 50 patients with clinical sars described by peiris et al., [8] 45 had serological or pcr evidence of sars-associated coronavirus infection; and of the 5 who were unconfirmed, 4 had serological testing prior to 14 days of onset of illness (possibly prior to the time of seroconversion). another series of 72 cases in hong kong showed that despite significantly high dosages of corticosteroid therapy, a seroconversion of 95.8% occurred on day 21 after onset of illness. [35] this differs from the us experience where of the 74 probable cases of sars reported by 15 july 2003, only 8 had been confirmed by laboratory diagnosis (all by serology); 38 were negative and 28 had no result. b antibody testing should also be carried out on a serum sample collected >28 days after symptom onset. antibody titer when tested in parallel should be considered indicative of a confirmed case. a variety of methods for detection of coronavirus infection are now available. these include culture methods, pcr-based methods, and serological tests. [31] culture of the sars coronavirus is considered solid evidence of infection, but there have been problems with the various generations of rt-pcr assays, both with false-positive results and with inconsistent detection of viral genome in the first days of illness as well as later in the convalescent phase. it is therefore recommended that detection of sars-cov rna by rt-pcr be validated by a second reference laboratory. [32] because antibodies to sars coronavirus have not been found in the general population, background sars coronavirus antibodies do not appear to be a substantial concern. however, the current serologic assays (both elisa and ifa [indirect fluorescent antibody] formats) do not reliably detect antibodies until the titers rise substantially after the second week of illness. according to the uscdc, suspect or probable cases are considered laboratory-confirmed if sars coronavirus is isolated, if antibody to sars coronavirus is detected and confirmed by a second reference laboratory, or if two table iv . a summary of infection control precautions for patients hospitalized with suspected/probable severe acute respiratory syndrome (sars) [reproduced from sampathkumar et al., [39] with permission] place patient in a negative pressure, specially vented, room a number of other agents have been suggested for therapy of sars-cov, including interferon-α, glycyrrhizin, and protease inhibitors. [1] in one preliminary, uncontrolled study by loutfy et al. [38] from toronto, use of interferon 9 μg/day for a minimum of 2 days and increased to 15 μg/day for a total of 10 days, plus corticosteroids (oral prednisone 50mg twice a day, or intravenous methylprednisolone 40mg every 12 hours) was associated with reduced disease-associated impaired oxygen saturation and more rapid resolution of radiographic lung abnormalities. the authors acknowledge, however, that these findings need to be interpreted cautiously in view of lack of randomization, the retrospective dosing, and the limited sample size (a total of 21 patients). table v . a summary of protective measures taken by severe acute respiratory syndrome (sars)-infected and non-infected staff in hong kong hospitals (reproduced from seto et al., [40] c comparing proportion of infected (n = 11) over non-infected staff (n = 72), with those without mask. since the causative agent of sars is contagious, in the absence of effective drugs or vaccines the only currently effective strategy 6. treatment for limiting the impact of sars is implementation of preventive at the time of writing (24 may 2004), no specific therapy is recommended. a variety of treatments have been attempted, but there are no controlled data. most patients have been treated throughout the illness with broad-spectrum antimicrobials, supplemental oxygen, intravenous fluids, and other supportive measures. some clinicians have advocated a combination of ribavirin and corticosteroids, but the efficacy of these drugs has not been established. the use of systemic corticosteroids in sars is controversial, and the efficacy based on controlled studies is unavailable. [36] one study found initial use of pulse-dosed methyl prednisolone (≥500 mg/day) to be more efficacious and equally well tolerated as a lower dose of methyl prednisolone, but this was based on retrospective observational evaluation. [35] the use of corticosteroids in patients with viral infections can be hazardous when not accompanied by an effective anti-viral agent. [36] early testing of ribavirin and other antiviral compounds against the novel coronavirus have not produced evidence of in vitro activity. [31] an evaluation of the use of ribavirin was published by knowles et al., [37] who reported adverse events in 110 patients with suspected or probable sars treated with ribavirin. of those 110 patients 61% had evidence of hemolytic anemia; hypocalcemia and hypomagnesemia were detected in 58% and 46% of patients, respectively. [33] the authors felt the benefits of ribavirin may not outweigh the risk of adverse events. there was a potential for ribavirin to have negative clinical and economic consequences because of the adverse events. it is now considered among the table vi . recommendations for the evaluation of patients with communityacquired respiratory illness in the presence or absence of severe acute respiratory syndrome-associated coronavirus (sars-cov) transmission in the world [34] in the absence of sars-cov transmission anywhere in the world, the diagnosis of sars-cov disease should be considered only in patients who require hospitalization for radiographically confirmed pneumonia and who have an epidemiologic history that raises the suspicion of sars-cov disease. in the absence of sars-cov transmission anywhere in the world, suspicion of sars infection is raised if, within 10 days of symptom onset: the patient had a history of recent travel to mainland china, hong kong, or taiwan; was in close contact with ill people with a history of recent travel to such areas; is employed in an occupation at particular risk for sars-cov exposure, including a healthcare worker with direct contact or a worker in a laboratory that contains live sars-cov; or is part of a cluster of cases of atypical pneumonia without an alternative diagnosis once sars-cov transmission has been documented in the world, a diagnosis of sars should still be considered in patients who require hospitalization for pneumonia and who have an epidemiologic history described above. in addition, all patients with fever or respiratory symptoms should be questioned about whether within 10 days of symptom onset they have had: close contact with someone suspected of having sars-cov disease; a history of foreign travel (or close contact with an ill person with a history of travel) to a location with documented or suspected sars-cov infection; exposure to a domestic location with documented suspected sars-cov (including a laboratory that contains live sars-cov); or close contact with an ill person with such an exposure history fig. 4 . algorithm for evaluating and managing patients requiring hospitalization for radiographically confirmed pneumonia, in the absence of person-toperson transmission of severe acute respiratory syndrome-associated coronavirus (sars-cov) anywhere in the world. [34] measures centered on avoidance of exposure and infection. such hospitalized patients with suspected sars should be isolated measures include global and regional surveillance, early detection in negative pressure rooms; healthcare workers should wear masks of new cases, identification of patient contacts, and strict adher-(a high efficiency mask such as the n-95 respirator used for ence to infection control policies. guidelines for infection control tuberculosis) to prevent air-droplet and airborne acquisition. behave been published and should be consulted for updated recom-cause coronaviruses can survive on environmental surfaces, good mendations. [34, 39] healthcare workers encountering a possible case hand-washing with soap and water or use of an alcohol-based hand of sars (suspected or probable) should take meticulous safety rub is highly recommended as well. environmental surfaces that precautions and seek advice from an expert in sars infection are frequently touched by the patient or are soiled with body fluids control. should be cleaned and disinfected with a household disinfectant. the early symptoms of sars-cov disease usually include fever, chills, rigors, myalgia, and headache. respiratory symptoms often do not appear until 2-7 days after the onset of illness. 2 in settings with more extensive transmission, all patients with fever or respiratory symptoms should be evaluated for possible sars-cov disease. 3 depending on symptoms and exposure history, initial diagnostic testing for patients may include complete blood count, chest x-ray (cxr), pulse oximetry, blood cultures, sputum gram stain and culture, testing for viral respiratory pathogens (notably influenza and respiratory syncytial virus), legionella sp. and pneumococcal urinary antigen. 4 an alternative diagnosis should be based on laboratory tests with high-predictive value (e.g. blood culture, urinary antigen). 5 chest computed tomography (ct) may show evidence of an inflitrate before a cxr. therefore, a chest ct should be considered in patients with a strong epidemiologic link to a known case of sars and a negative cxr 6 days after onset of symptoms. 6 sars isolation precautions should be discontinued after consultation with local public health authorities and the evaluating physician. an important characteristic of the recent sars outbreaks has been infection transmission. table iv lists precautions for patients hospitalized with sars. the predilection for transmission to healthcare providers after patient care. for the most part this has occurred after close, patients with sars-cov disease who do not otherwise need to unprotected contact with symptomatic individuals. healthcare be hospitalized can be managed appropriately as outpatients. workers who have had unprotected exposure and who develop these patients should limit interactions outside the home. they fever or respiratory symptoms should not come to work, and should be instructed to wear surgical masks in the presence of should report their symptoms to the infection control/employee household contacts, contain respiratory secretions in facial tissues, health service and their physician immediately. healthcare workand wash hands frequently. they should stay away from work, ers who have had unprotected exposure during procedures with school, or other public places for 10 days after resolution of fever. high risk of aerosolization (e.g. intubations, bronchoscopy) should household members or other close contacts of these patients should wear gloves and practice good hand hygiene. in the abbe quarantined for a 10-day period, since there is a high risk of sence of fever or respiratory symptoms, they need not limit their sars. [20] this policy has been running since the middle of march 2003 at queen mary hospital of the university of hong kong, activities. despite the disappearance of sars in hong kong since june the importance of sars precautions was demonstrated in a 2003. only authorized and minimum number of staff working in case-control study in five hong kong hospitals, with 241 nonthese wards may enter the premises. all staff entering these infected and 13 infected healthcare providers who had documentrestricted areas follow strict and stepwise 'gowning' and 'degowned contacts with sars patients (table v) . [40] all of the healthcare ing' procedures, and use standard personal protection equipment providers were surveyed concerning the use of masks, gloves, (disposable surgical paper cap, n-95 mask, and reusable eye gowns, and hand-washing, as recommended under droplet precaugoggles and cotton surgical gown). patients are treated with potent tions. no staff member who reported use of all four measures was antibiotics, usually in the form of a combination of cephalosporin infected. in contrast, all 13 infected staff members had omitted at and macrolide, or in the event of allergy to these antibiotics with least one of the measures (p = 0.0224). the authors observed that levofloxacin. patients who improve clinically and radiologically both surgical and high efficiency masks (n-95 masks) were proare unlikely to have sars, and are moved to wards that don't tective against infection, whereas paper masks did not significantrequire the level of intensive care and/or isolation as would be ly reduce the transmission (such masks are easily wet with saliva required for those patients who have sars, for observation for and are not recommended for precautions against droplets). 5-7 days before discharge. in the event of confirmed or suspected in order to be prepared for the recurrence of sars and the need sars, a patient will be diverted to the appropriate wards to for early implementation of control measures, the us cdc reminimize exposure of fellow patients, if no single-room accomleased clinical guidelines for the identification and evaluation of modation could be provided. possible sars-cov disease among patients presenting with community-acquired illness. [34] the key principles upon which control 8. conclusion measures are based have taken into consideration the fact that in the year 2003 a vast majority of patients with sars-cov disease because a new virus causes sars, it is very difficult to predict had a clear history of exposure either to a sars patient or to a the eventual significance of this infection. however, the resetting in which srs-cov transmission occurred (i.e. hospital); emergence of sporadic cases in china, has caused great concern as and developed pneumonia. recommendations for the evaluation to its future impact. it has had enormous economic and political of patients with community-acquired respiratory illness were deimpact on the affected areas of the world. although important veloped for two primary circumstances: firstly in the absence of progress has been made concerning the etiology, epidemiology, sars-cov transmission anywhere in the world, and secondly and prevention of this virus, many important questions remain. once transmission had been documented (these were released prior without answers to some of these questions, the eventual province in china (or close contact with an ill person with a outcome of sars remains unclear. history of recent travel to the area) in the 10 days before onset of symptoms. [19] when such patients are identified, appropriate isolaa major outbreak of severe acute respiratory syndrome references in hong kong outcomes of 144 patients with sars in the greater toronto area management of severe acute respiratory syndrome: the center for disease control and prevention. outbreak of severe acute respiratory 417-24 syndrome: worldwide case definitions for surveillance of severe acute respiratory syndrome sars clinical description of a completed outbreak sever acute respiratory syndrome (sars) [online]. available from url: of sars in vietnam severe acute respiratory syndrome: radiographic high-resolution ct findings of severe acute cases: united states and worldwide a novel coronavirus associated with severe acute respiratory syndrome evaluation of who criteria for identifying patients with severe respiratory syndrome out of hospital: prospective observa identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe (sars) and coronavirus testing: united states center for disease control and prevention. interpreting sars-cov test results the sars coronavirus from animals in southern china koch's postulates fulfilled for sars 20] virus detection of sars coronavirus in plasma by mosaic evolution of the severe acute respiratory real-time rt-pcr available level preparedness and response to severe acute respiratory syndrome (sars) from url high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome the use of corticosteroids in sars severe acute respiratory syndrome common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome clinical progression and viral load in a roids in severe acute respiratory syndrome: a preliminary study sars: epidemiology, clinical 18. center for disease control and prevention / presentation, management, and infection control measures center for disease control and prevention. recent sars cases in china accessed 2004 and contact in prevention of nosocomial transmission of severe acute respirato-may 20] ry syndrome (sars) center for disease control and prevention. china reports ninth recent possible sars case correspondence and offprints: professor thomas m. file, jr, infectious a cluster of cases of severe acute respiratory disease service identification of severe acute respiratory syndrome in canada key: cord-331856-j0gedx43 authors: basile, k.; maddocks, s.; kok, j.; dwyer, d.e. title: accuracy amidst ambiguity: false positive sars-cov-2 nucleic acid tests when covid-19 prevalence is low date: 2020-09-30 journal: pathology doi: 10.1016/j.pathol.2020.09.009 sha: doc_id: 331856 cord_uid: j0gedx43 nan in countries with a low prevalence of covid-19 and a low pre-test probability, confirmation of positive nucleic acid test (nat) results for sars-cov-2 is recommended given the potential for false positive results. as of 24 september 2020, there have been 26,983 confirmed cases and 861 deaths from covid-19 in australia. widespread testing, together with australia's geographic advantage, border controls, social distancing and public health messaging have all contributed to limit the number of infections. australia has one of the highest testing rates in the world with 7,441,327 sars-cov-2 nats performed on 6.4% of the population 1 since 22 january 2020, of which 0.4% were positive. the prevalence of laboratory-confirmed covid-19 has varied between the different jurisdictions in australia since the pandemic was declared, with the highest overall rate of 0.8% in victoria in july 2020. during the early phases of the pandemic, sars-cov-2 nats were mainly performed by public health laboratories using 'in-house' developed tests targeting one or more regions of the sars-cov-2 genome. over time, commercial nats became available, and testing was also undertaken by private laboratories. ideally and prior to intended use, all sars-cov-2 diagnostic assays should be validated to ensure they are fit for purpose. the urgent nature of the pandemic led to expedited assessments of many sars-cov-2 tests by regulatory bodies such as the therapeutic goods association in australia and the food and drug administration in the united states of america, with approvals contingent on the supply of ongoing evidence to support the safety and performance of the assays. [2] [3] [4] initially, the public health laboratory network (phln) australia recommended that confirmatory testing be performed on samples where sars-cov-2 rna had been detected to ensure that the result was a true positive. 5 however, this was not always practical or efficient with substantial testing volumes and in the face of shortages of nucleic acid extraction and testing reagents and consumables. despite the second wave of infections in victoria, the prevalence of covid-19 in australia remains low (<1%), meaning that in the absence of epidemiological risk factors, the pre-test probability will be low and false positive results will occur even with highly specific nats. for example, the positive predictive value (ppv) of sars-cov-2 nats with a specificity of 99% is only 50% when the prevalence of infection is 1%. 6 at the height of the pandemic in australia in late march, the prevalence of covid-19 was 2% (range 0.7-3.4% between the different states and territories), 7 indicating that nats with the same analytical performance will have a ppv of approximately 67% (range 41.2-77.3%). when sars-cov-2 was detected by in-house or commercial nats in nsw health pathology (nswhp) laboratories, samples were sent to nswhp-institute of clinical pathology and medical research, westmead hospital, for supplementary testing as part of nswhp's testing algorithm to minimise false positive results. supplementary testing was performed using real time reverse-transcriptase polymerase chain reaction (rt-pcr) assays targeting the e, rdrp, m, n, orf1ab and orf1b genes 8 using either referred nucleic acid extract and/or nucleic acid re-extracted from the original sample using the magna pure 96 instrument (roche diagnostics, germany). the final result was determined as the consensus of the results from testing the six targets above. of 122 samples referred from both internal and external laboratories for sars-cov-2 confirmatory testing from 14 july to 24 september 2020, we identified a false positive rate of 11% (13/122). only two of the 13 cases defined as false positive had sars-cov-2 serology and/or respiratory tract pcr results available, both testing negative for sars-cov-2-specific serology and one patient testing positive for rhinovirus. false positive results may not always be easily identified, and laboratory staff (alone, or in conjunction with clinicians and/or public health physicians) should remain vigilant for their presence. suspicion should arise if there are discrepant clinico-epidemiological findings (particularly problematic when there are many asymptomatic infections); unexpected laboratory results (such as discordant results where only one sars-cov-2 target is detected in assays with multiple targets, and/or rt-pcr results with high cycle threshold values) or contamination (for example, when a batch of samples test positive); incorrect results from external quality assurance programs; warnings from diagnostic companies about potential contaminated assays or reagents; 9 or when supplemental nats on other platforms, the use of different sars-cov-2 targets, sars-cov-2-specific serology, or genomic sequencing do not concur with the initial nat result. in the context of australia's low prevalence of covid-19 and thus low pre-test probability for infection, we recommend that all positive sars-cov-2 nat results be confirmed by supplementary testing on the original nucleic acid extract and/or re-extraction of nucleic acid from the original sample (if available) and tested using another assay(s) with different gene targets and/or lower limits of detection 10 (fig. 1) . repeat respiratory tract sampling (including sputum if available), especially in asymptomatic individuals with no identified epidemiological links, is an approach that has been implemented by laboratories following the release of the phln guidance document on nat result interpretation for sars-cov-2. 6 sera should also be collected for the detection of sars-cov-2-specific antibodies (fig. 1) . serology testing may not always confirm acute sars-cov-2 infection (particularly if collected early in the illness course, as sars-cov-2-specific antibodies appear around 10 days after disease onset) but can be useful if positive. however, confirmation of infection requires convalescent sera to be collected to demonstrate seroconversion or a four-fold or greater rise in antibody titres between the acute and convalescent samples. serology may also be used for retrospective diagnosis of sars-cov-2 where nat testing was not performed or was inconclusive. a combination of these approaches will assist with the recognition of reinfection, with sars-cov-2-specific antibody assays still to be validated. 11 timely identification of true false positive sars-cov-2 nat results is important as unrecognised false positive results can lead to unnecessary quarantining and contact tracing, delays in the recognition and treatment of the true illness, significant patient anxiety and concern, potential exposure to nosocomial infection from other patients with confirmed covid-19, wastage of personal protective equipment, and inaccurate statistics regarding local prevalence of infection. the authors state that there are no conflicts of interest to disclose. australian government department of health. coronavirus (covid-19) current situation and case numbers covid-19 testing in australia -information for health professionals. covid-19 test performance false negative tests for sars-cov-2 infectionchallenges and implications emergency use authorization (eua) information, and list of all current euas australian government department of health. phln guidance on laboratory testing for sars-cov-2 (the virus that causes covid-19) australian government department of health. phln guidance on nucleic acid test result interpretation for sars-cov-2 covid-19 national incident room surveillance team. covid-19 interpret with caution: an evaluation of the commercial ausdiagnostics versus in-house developed assays for the detection of sars-cov-2 virus rt-qpcr testing of sars-cov-2: a primer sars-cov-2 reference panel comparative data european centre for disease prevention and control. threat assessment brief. reinfection with sars-cov: considerations for public health response key: cord-322837-tqgwgvo0 authors: gable, lance; ram, natalie; ram, jeffrey l title: legal and ethical implications of wastewater sars-cov-2 monitoring for covid-19 surveillance date: 2020-06-24 journal: j law biosci doi: 10.1093/jlb/lsaa039 sha: doc_id: 322837 cord_uid: tqgwgvo0 scientists have observed that molecular markers for covid-19 can be detected in wastewater of infected communities both during an outbreak and, in some cases, before the first case is confirmed. the cdc and other government entities are considering whether to add community surveillance through wastewater monitoring to assist in tracking disease prevalence and guiding public health responses to the covid-19 pandemic. this scientific breakthrough may lead to many useful potential applications for tracking disease, intensifying testing, initiating social distancing or quarantines, and even lifting restrictions once a cessation of infection is detected and confirmed. yet, new technologies developed in response to a public health crisis may raise difficult legal and ethical questions about how such technologies may impact both the public health and civil liberties of the population. this article describes recent scientific evidence regarding covid-19 detection in wastewater, identifying public health benefits that may result from this breakthrough, as well as the limitations of existing data. the article then assesses the legal and ethical implications of implementing policy based on positive sewage signals. it concludes that the first step to implementing legal and ethical wastewater monitoring is to develop scientific understanding. even if reliability and efficacy are established, limits on sample and data collection, use, and sharing, must also be considered to prevent undermining privacy and autonomy in order to implement these public health strategies consistent with legal and ethical considerations. limits on sample and data collection, use, and sharing, must also be considered to prevent undermining privacy and autonomy in order to implement these public health strategies consistent with legal and ethical considerations. the covid-19 pandemic has challenged civil society worldwide in unprecedented ways and has revealed the need for real-world solutions to dire health and economic concerns. during the initial phase of the pandemic, many state and local officials implemented extensive efforts to achieve social distancing and "flatten the curve" of the outbreak through expansive stay-at-home orders and related restrictions. 1 these restrictive measures have been important for controlling the spread of disease, but have also imposed a severe social and economic burden on many people. by contrast, the federal government has ordered the continued operations of meat those locations. these methods may also be a boon to communities where cases of covid-19 have not yet spiked, providing an opportunity to get ahead of the first wave of the outbreak even if widespread testing is not feasible. wastewater monitoring may be one such new community detection method. recent research indicates that the coronavirus that causes covid-19-sars-cov-2-not only infects the respiratory system but also infects the gastrointestinal system and ultimately appears in feces. researchers found that a large amount of virus resides in the gastrointestinal tract, where it adheres to and is retained by epithelial cells that line the intestines and from which it is shed into feces. 15 clinical studies found sars-cov-2 was in feces in the majority of patients where this was studied. in fact, a positive signal for sars-cov-2 in feces was present in adults an average of 12 days after nasal-throat swabs turned negative and in pediatric patients, on average, more than 2 weeks after the virus had completely disappeared from throat swabs. 8 the first positive signal in one of the cities (amersfoort) was obtained on march 5, 18 the day before the first case of covid-19 was confirmed there in two high school students. 19 studies at mit 20 and elsewhere 21 similarly confirmed the sensitive detection of sars-cov-2 markers in wastewater of municipalities where the disease is known to be occurring. scientists say their purposes for carrying out these studies included "to monitor the circulation of the virus in the population," 22 to obtain "population-wide data [to] help inform modeling efforts," 25 and "to detect pathogens in populations when investigations in humans is difficult for logistic, ethical or economic reasons." 26 the question of whether the collection of samples solely for such population-level surveillance and model-building purposes may nevertheless raise individualized privacy concerns is discussed in part ii. moreover, scientists and other commentators have additionally suggested that the results of wastewater monitoring of sars-cov-2 may trigger actions that more directly affect the movement and privacy of people. they aver that sewage-based data will "inform decisions surrounding the advancement or scale-back of social distancing and quarantine efforts," 27 and that community monitoring "at the municipal or community level … may allow for more granular detection of sars-cov-2 … to help preemptively enact public health measures prior to the widespread onset of disease." 28 detecting sars-cov-2 through this method may precede confirmed reports of disease by a week or two, such that "tracking viral particles in wastewater could give public-health officials a head start on deciding whether to introduce measures such as lockdowns." 29 if wastewater-based epidemiology can be applied at a community level, then "effective intervention can be taken as early as possible to restrict the movements of that local the proposed use of wastewater screening to detect sars-cov-2 viral rna has the potential to greatly enhance our technical capabilities to identify, track, pinpoint, and quantify 32 second, public health authorities could use this information to justify increased testing among people living in homes or working at sites close to where the virus has been found in wastewater, or to implement neighborhood-wide screening programs in these areas. the use of these data to target resources to provide voluntary screening programs in areas with sars-cov-2 detection likely would not implicate legal concerns. courts have long recognized that public health powers include the authority to conduct testing and screening programs. 46 in addition, voluntary screening programs facilitate individuals' autonomy and right to refuse testing. by contrast, a conditional screening program, which would require a person subject to a quarantine order to be tested prior to being able to leave their home would necessitate balancing the state's use of police powers against the rights of a specific individual to refuse testing. well-established legal precedent suggests that efforts to require testing or treatment for a high-dangerous infectious disease falls within a state's legal powers. 47 if individuals refuse to cooperate, they would not be physically forced to get tested, but they might be subjected to other restrictions such as quarantine or reasonable monetary penalties. refusing individuals are thus presented 14 with a choice of complying with the testing or treatment, or being subjected to more restrictive powers. the implications of these more restrictive powers are discussed below. conditional screening requirements based on wastewater detection of sars-cov-2 may raise fourth amendment concerns. courts are likely to uphold such requirements, so long as the regulatory framework for conditional screening strikes a reasonable balance between privacy interests and legitimate public health needs. where the government undertakes surveillance activities primarily to advance public health purposes, courts have generally upheld such activities under the special needs doctrine. 48 in this context, the government is not constitutionally required to secure a warrant before engaging in a search. rather, courts will sustain a public health programs so long as they reasonably balance between privacy interests nationality. 52 this concern would be particularly acute with respect to the use of wastewater screening to impose substantial restrictions on movement. if such restrictions were targeted only at urban neighborhoods with sufficient density to detect sars-cov-2 in wastewater, the practical effect might be imposing restrictions disproportionately on areas that are populated predominantly by people of color. government officials must avoid discriminatory applications of restrictive powers. beyond their legal obligations, moreover, government officials bear an ethical obligation to consider whether the impact on the community is just when imposing restrictions and to support communities disproportionately harmed by restrictive orders. 53 fourth, public health authorities could extend or reimpose community-wide social distancing measures based on positive wastewater screening for sars-cov-2. in the current crisis, such measures have included closing businesses, prohibiting gatherings in affected areas, and limiting participation in activities deemed non-essential. courts are likely to be deferential to government-imposed restrictions along these lines. 54 wastewater screening for sars-cov-2 could provide an important tool to detect new outbreaks of covid-19 and to target resources to intervene to stop the spread of the disease; however, scientific research must establish the efficacy of such testing in identifying communitybased covid-19 infections before its use can be considered as the basis for public policy. very little research on the efficacy of sars-cov-2 monitoring in sewage has been accomplished given the recent emergence of the disease and discovery of the virus in feces and sewage. we infections can be established, wastewater data might also feasibly be used to indicate when areas are ready to relax restrictive measures or to detect a reversal or continuation of trends towards recovery. until reliability and efficacy are demonstrated, implementing wastewater screening for sars-cov-2 to target public health resources, to require testing, to impose restrictions on movement, or to remove restrictions based on an absence of virus in the wastewater, is premature. even if reliability and efficacy are established, we recommend that for efforts to implement wastewater screening for disease and other biological markers, legal and ethical considerations must be taken into account, including appropriate limits on sample and data collection, use, and sharing, so as to prevent unduly undermining privacy and autonomy, and to reduce the potential for problematic misuse of data or coercive interventions. as our scientific understanding of the connection between the presence or absence of sars-cov-2 in wastewater improves, however, these data could be the basis for scientifically informed decisions to implement public health intervention strategies consistent with legal and ethical considerations. virus hunters find coronavirus clues in sewage, circle of blue: water news sars-cov-2 titers in wastewater are higher than expected from clinically confirmed cases, medrxiv preprint time course quantitative detection of sars-cov-2 in parisian wastewaters correlates with covid-19 confirmed cases ) (emphasis omitted); see also united states v. spain, 515 f. supp. 2d 860, 861 (n.d. ill. 2007) united states v. hajduk, 396 f see riverdale mills, 392 f.3d at 64 (noting that wheat.) 1 (1824) (articulating a broad scope for state police powers, including "[i]nspection laws, quarantine laws, health laws of every description 11 (1905) (upholding broad public health authority under state police powers) see jacobson, supra note 37 (upholding authority to mandate smallpox vaccination under state police powers) quarantining the law of quarantine: why quarantine law does not reflect contemporary constitutional law norms, 9 wake forest us emergency legal responses to novel coronavirus: balancing public health and civil liberties cal. 1900) (invalidating a racially discriminatory mass quarantine order in san francisco for bubonic plague that only applied to chinese-american neighborhoods) health justice strategies to combat covid-19: protecting vulnerable communities during a pandemic the slaughter-house cases, 83 u.s. (16 wall.) 36 (1873) (upholding a state order moving slaughterhouse downstream in response to cholera outbreaks) the authors would like to acknowledge bobby teng, wayne state medical student, for suggesting sars-cov-2 in wastewater as a topic of research interest at wayne state, and dr.philip pellett, chair of biochemistry, microbiology, and immunology at wayne state, for key: cord-340049-6rqmc89u authors: salvatori, giovanni; luberto, laura; maffei, mariano; aurisicchio, luigi; roscilli, giuseppe; palombo, fabio; marra, emanuele title: sars-cov-2 spike protein: an optimal immunological target for vaccines date: 2020-06-03 journal: j transl med doi: 10.1186/s12967-020-02392-y sha: doc_id: 340049 cord_uid: 6rqmc89u covid-19 has rapidly spread all over the world, progressing into a pandemic. this situation has urgently impelled many companies and public research institutes to concentrate their efforts on research for effective therapeutics. here, we outline the strategies and targets currently adopted in developing a vaccine against sars-cov-2. based on previous evidence and experience with sars and mers, the primary focus has been the spike protein, considered as the ideal target for covid-19 immunotherapies. the outbreak of the novel coronavirus disease covid-19, now officially designated as severe acute respiratory syndrome-related coronavirus sars-cov-2, has progressed rapidly into a pandemic. in just a few months, since december 2019, covid-19 has spread worldwide with over 4.218.212 confirmed cases and more than 290.242 confirmed deaths as of may 14th, 2020 (who, situation). this dramatic situation calls for the rapid development of safe and effective prophylactics and therapeutics against infection of its causative agent. to date, no therapeutics or vaccines against any human-infecting coronaviruses have been approved. currently, ongoing strategies to trigger an effective immune response in humans against sars-cov-2 are taking advantage of previous experiences on other coronaviruses such as sars-cov and mers-cov. since the sars-cov-2 virus shares striking structural similarity and sequence conservation with these two lethal coronaviruses, the immunization strategies exploited against sars and mers viruses have been adopted in guiding the design of new sars-cov-2 vaccines. immunization with one or more sars-cov-2 subunit antigens, either administered as purified protein or expressed by viral, rna or dna vaccine vectors, is one approach to designing a vaccine. among the more likely targets for vaccination are the structural proteins that bedeck the surface of sars-cov-2. these include the envelope spike protein s, the small envelope protein e, the matrix protein m and the unexposed nucleocapsid protein n. an early study on recombinant vectors expressing the s protein of sars-cov found this protein to be highly immunogenic and protective against sars-cov challenge in hamster, while in contrast, the n, m, and e proteins did not significantly contribute to a neutralizing antibody response or protective immunity [1] . evidence of the key role played by the s protein in counteracting coronavirus infection came from studies on human-neutralizing antibodies from rare memory b cells of individuals infected with sars-cov [2] or mers-cov [3] . in such studies, antibodies directed against the s protein of sars-cov were found effective in inhibiting virus entry into the host cells. more recently, it has been found that sars-cov s elicited polyclonal journal of translational medicine antibody responses, and vigorously neutralized sars-cov-2 s-mediated entry into cells, thus further encouraging the use of this molecular target for vaccination and immunotherapies [4] . structural studies of antibodies in complex with sars-cov s and mers-cov s have provided information about the mechanism of competitive inhibition to the host receptor. the receptor-binding domain (rbd) in sars-cov-2 s protein was identified and found to bind strongly to ace2 receptors [5] . sars-cov rbd-specific antibodies cross-react with sars-cov-2 rbd protein, and sars-cov rbd-induced antisera neutralized sars-cov-2, providing additional evidence that targeting this domain of the s protein of sars-cov-2 with a vaccine could be effective in preventive covid-19 [5] . given the above and that the coronavirus s glycoprotein is surface-exposed and mediates entry into host cells by interacting with angiotensin-converting enzyme 2 (ace2), it rapidly became the main target of neutralizing antibodies and the focus of therapeutic and vaccine design. several companies and research institutes have started developing a vaccine that has the sars-cov-2 protein s as its target (see table 1 ), although the various vaccination strategies show a differing ability to induce in the host both an antibody-mediated humoral response and a cell response mediated by cd4 or cd8 t lymphocytes in preclinical models. the evolving molecular heterogeneity of sars-cov has raised concerns about the breadth and efficacy of protection provided by specific vaccine strains and the possible development of immune escape. however, it has been observed that a heterotypical response blocking sars-cov-2 s-mediated entry into host cells is elicited, coinciding with the sequence and structural conservation of sars-cov-2 and sars-cov s protein, suggesting that immunity against one virus can potentially provide protection against related viruses. one of the most perplexing questions regarding the current covid-19 coronavirus epidemic is the possible worsening of the disease by immunotherapies, as the consequence of an antibody dependent enhancement (ade) of infection with sars-cov-2. ade of viral entry has been a major concern for epidemiology, vaccine development, and antibody-based drug therapy. ade viral entry into the target cell of sars-cov-2 is mediated by the fc receptor ii and not by its canonical receptor. it has been suggested that ade may explain geographical differences in the severity of covid-19 due to prior exposure to similar antigenic epitopes [6] . one study showed that the antibody against sars-cov spike protein potentiated infection of monocytes. however, ade-infected macrophage did not support the productive replication of sars-cov, and no detectable release of progeny virus was observed [7] . in a mouse model of vaccination for sars-cov with different approaches including inactivated virus, dna or recombinant spike (s) protein, vaccines lead to pulmonary immunopathology. however, despite deterioration in the pulmonary histopathology profile of the vaccinated mice, all the sars-cov vaccines induced antibody and protection against infection with sars-cov [8] . it has been found that higher concentrations of antisera against sars-cov neutralized infection, while highly diluted anti-sera significantly increased sars-cov infection. results from infectivity assays indicate that sars-cov ade is primarily mediated by diluted antibodies against envelope spike protein [9] . however, the relevance of ade in coronavirus infections remains elusive, as no direct evidence of it has been found in various vaccination models [5] . accordingly, it has been shown that vaccination of the rhesus macaque monkey with an attenuated sars-cov revealed no exacerbation of infection even several weeks after vaccination, when the antibody titer was reduced [10] . it is of relevance that several companies are involved in the development of a vaccine against the spike protein of sars-cov-2, exploiting different strategies such as purified protein or expressed by viral, rna or dna vaccine vectors. this target has been guided by previous preclinical history of the proven efficacy of immunotherapies against the homologous protein of sars-cov. although the ade effect of non-neutralizing antibodies directed against the sars-cov-2 s protein remains controversial, safety testing of covid-19 s protein-based b cell vaccines in animal models is strongly encouraging prior to clinical trials. given the urgency of an effective vaccination to prevent the spread of sars-cov-2, this plurality of approaches in vaccine generation with complementary strategies, paves the way to a wider immunotherapeutic spectrum, thus increasing the chances of success in such a short time frame. abbreviations ace2: angiotensin-converting enzyme 2; rbd: receptor-binding domain; ade: antibody dependent enhancement. contributions of the structural proteins of severe respiratory syndrome coronavirus to protective immunity an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus structure, function, and antigenicity of the sars-cov-2 spike glycoprotein characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine is covid-19 receiving ade from other coronaviruses? microbes infect antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus antibodydependent sars coronavirus infection is mediated by antibodies against spike proteins evaluation of antibody-dependent enhancement of sars-cov infection in rhesus macaques immunized with an inactivated sars-cov vaccine publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. em and gs wrote the manuscript with contributions from all the authors. all authors read and approved the final manuscript. not applicable. not applicable. not applicable. not applicable. key: cord-324480-7u5lh4jx authors: sharma, a.; preece, b.; swann, h; fan, x.; mckenney, r.j.; ori-mckenney, k.m.; saffarian, s.; vershinin, m.d. title: structural stability of sars-cov-2 degrades with temperature date: 2020-10-14 journal: biorxiv doi: 10.1101/2020.10.12.336818 sha: doc_id: 324480 cord_uid: 7u5lh4jx sars-cov-2 is a novel coronavirus which has caused the covid-19 pandemic. other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. sars-cov-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. it has been reported that sars-cov-2 can remain infectious for hours on surfaces. as such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. here we have used atomic force microscopy to examine the structural stability of individual sars-cov-2 virus like particles at different temperatures. we demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. this is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of covid-19 in winter. statement of scientific significance the economic and public health impact of the covid-19 pandemic are very significant. however scientific information needed to underpin policy decisions are limited partly due to novelty of the sars-cov-2 pathogen. there is therefore an urgent need for mechanistic studies of both covid-19 disease and the sars-cov-2 virus. we show that individual virus particles suffer structural destabilization at relatively mild but elevated temperatures. our nanoscale results are consistent with recent observations at larger scales. our work strengthens the case for covid-19 resurgence in winter. sars-cov-2 is a virus of zoonotic origin which was first identified in humans in late 2019 [1] . similar to other coronaviridae [2] , the viral particles are enveloped and polymorphic decorated by a variable number of s protein spikes on their membrane [3] . one of the most confusing and yet urgently pressing questions at the time of this writing is whether the covid-19 pandemic caused by sars-cov-2 will show seasonal character. climate and seasonal dependence was expected early in the pandemic [4] due to similarity with other human coronavirus diseases [5] , however the rates of infections have failed to strongly decline in the summer of 2020, leading to widespread doubts about covid-19 seasonality. at the same time, a mounting body of evidence, from theoretical studies [6] to experimental research on viral populations and their infectivity [7, 8] suggest that seasonality is indeed to be expected. however an understanding of how sars-cov-2 survives different environmental conditions is still incomplete and mechanisms of virus particle degradation are poorly mapped out. this then creates uncertainty for public health policy and its forward projection. a key challenge in studying sars-cov-2 is the extreme level of threat associated with the live virus and the resultant need for high safety standards for such work. aside from the envelope and s proteins, sars-cov-2 also packages the positive sense rna genome encapsidated with thousands of copies of nucleocapsid, n proteins. sars-cov-2 also packages thousands of copies of matrix protein (m) which consists of three membrane spanning helixes with small intraluminal and extra luminal domains. in addition, an unknown number of envelope (e) proteins, which contain a single membrane spanning helix, are also packaged in each virion. we have previously shown that similar to sars-cov [9] , the expression of sars-cov-2 m, e, and s proteins in transfected human cells is sufficient for the formation and release of virus like particles (vlps) through the same biological pathway as used by the fully infectious virus [10] . these vlps faithfully mimic the external structure of the sars-cov-2 virus. the vlps however, possess no genome and thus present no infectious threat which enables rapid studies with reduced safety requirements. the ability to produce non-infectious vlps further enabled us to devise and rapidly validate novel strategies for manipulation of these particles, most notably via the addition of protein tags to the s and m proteins (these findings are detailed in a separate manuscript). here, we report studies of vlps subjected to variable temperature conditions before or after being immobilized and dried out on a functionalized glass surface. we show that exposure of vlps to a mildly elevated temperature (34 c) for as little as 30 minutes is sufficient to induce structural degradation. the effect is weaker for particles exposed to elevated temperatures in solution and stronger for exposure in the dry state. overall, these results provide insight into the viral seasonality of sars-cov-2. during initial refinement of vlp purification strategies and associated vlp characterization [10] , we have found that such particles remain stable for at least a week if stored in liquid buffer at near 0 °c conditions (on ice). we therefore examined whether they would remain stable at room temperature under dry conditions on a model surface (fig. 1 ). spytag-s vlps were adhered to microtubules (mts) are harder to image stably due to high amount of background noise which obscures poly-l-lysine inhomogeneity and mt washout sites (likely due to loose debris on the surface -plausibly a byproduct of particle degradations). features consistent with intact vlps are prominent relative to noise levels and hence easy to resolve (fig. 2 ), but they are so exceedingly rare at 34 °c that they are considered outliers (fig. 3 ). they were seen in large area surveys in which each particle is mechanically probed only a few times. such particles do not survive intact during even a single detailed zoomed-in scan (fig. 2) . it takes 20-30 minutes to install the sample into the afm, approach the surface and validate tip condition. therefore mildly elevated temperature has a rapid effect on vlp integrity. vlps incubated at 34 °c in solution and imaged at room temperature ( fig. 1c ) survive better than particles at 34 °c under dry conditions but still often appear disrupted or aggregated in afm imaging. a common transmission route for sars-cov-2 is through bioaerosols created during sharp exhalation events such as sneezing or coughing. the bioaerosol droplets tend to dry out quickly due to high surface area and small volume [11, 12] . therefore virus particles may be exposed to both wet and dry conditions before coming into contact with and infecting the next host. it is widely recognized that virus particles often spread after their deposition on various surfaces [13] (although direct contact of the next host with contaminated bioaerosol may also be a viable route) and it is therefore also appreciated that virus particles can survive on various surfaces for an extended length of time [14] . the ability to make vlps based on the sars-cov-2 genome, combined with abundant available structural information allowing for high precision design strategies for the vlps, opens a unique opportunity for fast progress and allowed us to overcome the safety concerns associated with experiments on the full virus. here we used this technology to study the stability of the viral envelope and associated proteins (m, e, and s) under different environmental conditions. as might be expected a priori, the vlps do indeed degrade when exposed to elevated temperatures. our afm imaging revealed that negligibly few particles retain their shape and even those exceptional particles degraded nearly instantly during scanning and hence are likely already structurally impaired. the mt washout sites are readily identifiable for scans at room temperature, but are difficult to see with an identical colormap presentation due to elevated background noise. however, artificially extending the z range of the data helps reveal the presence of mt washout sites (right panel, washout site highlighted with red oval). faint modulation in the rightmost image is most likely due to electronic noise in the imaging system although a mechanical vibration noise contribution cannot be excluded. a new coronavirus associated with human respiratory disease in china the molecular biology of coronaviruses structures and distributions of sars-cov-2 spike proteins on intact virions climatic-niche evolution of sars cov-2 temperature, humidity, and latitude analysis to estimate potential spread and seasonality of coronavirus disease 2019 (covid-19) extended lifetime of respiratory droplets in a turbulent vapour puff and its implications on airborne disease transmission environmental stability of sars-cov-2 on different types of surfaces under indoor and seasonal climate conditions stability of sars-cov-2 in different environmental conditions purification and electron cryomicroscopy of coronavirus particles, sars-and other coronaviruses minimal system for assembly of sars-cov-2 virus like particles toward understanding the risk of secondary airborne infection: emission of respirable pathogens droplet fate in indoor environments, or can we prevent the spread of infection? stability of sars-cov-2 and other coronaviruses in the environment and on common touch surfaces and the influence of climatic conditions: a review, transboundary and emerging diseases aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 the effects of temperature and relative humidity on the viability of the sars coronavirus key: cord-337430-c2vdnml7 authors: timpka, toomas title: sports health during the sars-cov-2 pandemic date: 2020-05-02 journal: sports med doi: 10.1007/s40279-020-01288-7 sha: doc_id: 337430 cord_uid: c2vdnml7 nan in december 2019, the chinese city of wuhan reported an outbreak of sars-cov-2 (severe acute respiratory syndrome coronavirus-2) infection that causes the covid-19 disease, an atypical pneumonia [1] . modelling analyses per late january 2020 showed that the outbreak was no longer contained within wuhan, and that other major chinese cities sustained localised outbreaks [2] . cases were thereafter exported across china, as well as internationally. in early march 2020, the world health organisation proclaimed that the outbreak had developed to a pandemic. the pandemic caused by sars-cov-2 has had a major impact on sports, from the cancellation of major events and championships [3] to small sports clubs being forced into bankruptcy [4] . the aim of this commentary is to examine the consequences of the sars-cov-2 pandemic for sports and provide recommendations for response measures from the sports community. the sars-cov-2 is not the first coronavirus to generate concern [5] . healthcare systems across the world have had to manage sars in 2003-2004 and mers (middle eastern respiratory syndrome), which has been ongoing since 2012. infection with sars-cov-2 is established in the upper airways which at times can lead to very high virus production. the body responds first via the innate immune system and subsequently via the adaptive immune system, whereby the virus is eliminated from the body. however, sars-cov-2 may enter the lower respiratory tract, reach the furthest alveoli, damage the alveolar epithelial barrier, and allow fluid flow across the interstitial barrier, thus decreasing oxygenation [6] . when the immune system responds, there is a risk of a cytokine storm syndrome that may cause acute respiratory distress, systemic inflammation, multi-organ failure, and sometimes death [7, 8] . the median incubation time for covid-19 is 4-6 days (2-14 days) [9] . the main symptoms among adults are fever, a new persistent dry cough, shortness of breath, and occasionally loss of sense of smell. no specific symptoms can distinguish covid-19 from other common respiratory tract infections, e.g. influenza. asymptomatic cases or cases with only mild symptoms, e.g. slight cough and sense of illness, may also occur [10] . from early chinese cohorts of symptomatic patients, the majority have been reported to suffer mild progression of the disease (> 80%), 14% severe (dyspnea, decreased oxygen saturation), and 5% a life-threatening condition [11] . diagnosis of covid-19 has thus far (early april 2020) been confirmed by polymerase chain reaction (pcr) analysis of secretions from the nasopharynx (or pharynx) [5] . reliable serological tests are expected to soon be ready for widespread use [12] . these tests will be able to detect past sars-cov-2 infection, which will be important for identifying individuals who have been infected, and to follow the evolution of the pandemic. the impact on the lungs can in most cases be demonstrated by computerised tomography. there are currently only experimental pharmacological treatments available for covid-19. remdesivir is a nucleotide analogue inhibitor of the virus's rna-dependent rna polymerases. it has a wide spectrum of antiviral activity against rna viruses, including sars-cov and mers-cov [13, 14] . the malaria and rheumatism drug, chloroquine phosphate, demonstrates in vitro activity against sars-cov-2, and unverified reports from china also indicate in vivo efficacy in humans [15] . it is predominantly people greater than 70 years of age and individuals with chronic conditions who are at risk of developing severe covid-19 disease [11] . children and young people are reported to contract sars-cov-2 infections and transmit the virus, but they seldom suffer a serious course of illness [16] . the progress of a pandemic is essentially hard to forecast due to lack of knowledge about the infectious agent and population response behaviours [17, 18] . this implies that the planning of response measures must be dynamically adapted to surveillance reports on the disease and immunity status in the population that the measures are intended for [19, 20] . sars-cov-2 is transmitted through droplets from coughs and sneezing and contact with infectious secretions [9] . the most important measure to prevent virus transmission is, therefore, thoroughness with personal hygiene, i.e. washing hands frequently and carefully, coughing and sneezing in the arm-fold, use of hand sanitizer, and, in certain contexts, use of face masks [21] . the second main preventive measure is social distancing, which ranges from keeping at least a 2-m person-to-person distance to cancellations of sports events, school closures, and household quarantine [22] . the national public health agencies choose social distancing regulations based on an overall assessment of how critical certain activities are for society as a whole and whether motivation to comply with the rules can be assumed. the concept of 'proportionate universalism' is generally applied [23] , i.e. that population health interventions are seen as universal, not targeted, with a scale and intensity that is proportionate to the level of collateral disadvantage caused in the community. during the sars-cov-2 pandemic, effectively all population-level interventions include the recommendation that social contacts with the elderly, and especially the senior elderly, are to be reduced to an absolute minimum. domestic and international travel are also generally discouraged, especially to and from metropolitan areas with ongoing disease transmission. sports organisations should develop a pandemic response strategy that addresses the needs of its athletes and coaches, while complying with the regulations and recommendations issued by the government and national public health agency. for a society to function during a pandemic, its members need to trust their fellow citizens as well as the government institutions that are issuing regulations [24] . if sportspeople do not believe that most others are going to play by the temporary restrictive rules, they are unlikely to adhere to them. the pandemic strategy of a sports organisation must therefore be unmistakeably communicated to its memberships. swedish athletic's covid-19 website [25] is an example of a platform used to communicate a temporary framework for sports practice and competitions to a sports community. the website presents links to national government and public health agency regulations, contact information to managers at the federation office, and recommendations specific for athletics. it is updated hourly by the chief information officer, and its contents are discussed every second day within a multi-disciplinary pandemic task force at the federation. the temporary frameworks for organised sports practice and competitions must be developed based on the social distancing and quarantine protocols activated during the pandemic. furthermore, novel arrangements of informal physical exercise for children which conform to prevailing social distancing regulations need to be created. the main principles for the temporary frameworks are that activities should be performed outdoors in small groups and that physical contact is avoided as far as possible. the regulations for football (soccer) practice issued by the norwegian football association demonstrate these principles [26] : maximum 5-person groups, an adult to be present in each group, 2-m person-to-person distance, no physical contact, and balls should not be handled or headed and should be washed after each session. at sports facilities, the sharing of locker rooms by large groups should be avoided and easy access to handwashing facilities provided. in individual sports, virtual competitions can be arranged using internet resources [27] , with athletes participating at the same time and different locations, or at the same location and different times. regarding individual athletes, the risk of developing covid-19 can be reduced by regular sleep, eating a wellbalanced diet, and staying well hydrated, to maintain the capacity of the immune system [28] . a proper intake of fruit and vegetables (7-8 portions per day) is beneficial, since these foods contain polyphenols and flavonoids that support immune function [29] . athletes with asthma should use their prescribed medications meticulously to reduce the risk for a more serious course of illness. there are as yet no clinical studies of covid-19 among athletes. in most cases, young people appear to cope well with covid-19, and the symptoms improve over the course of a week [16] . however, if return-to-sports is made too soon, there is a risk of heart and lung complications [11, 30] . taking at least 10 days of complete rest from exercise is required, or rest for a minimum of 7 days from when symptoms stop [31] . the period of rest should be followed by stepwise return-to-play with careful evaluation before proceeding to the next level. the sars-cov-2 pandemic is challenging for the elite athlete, considering both the risk of infection and the fact that season's goals and aims must be abandoned. the rulesof-play may also have changed, e.g. with regard to how final league tables are determined and qualifying for major championships, such as the olympic games, is decided. individual-level health monitoring can, therefore, be considered among elite athletes during the pandemic period. high levels of stress have a negative effect on mood and can also reduce the capacity of the body to resist infection [32] . continuing training can help to relieve some pressures. season targets should be re-evaluated and new realistic goals determined as soon as possible. sponsorship and other financial support contracts should then be re-negotiated accordingly. in swedish athletics, weekly surveillance of athletes listed for the national team (n = 190) was initiated in march 2020. the monitoring uses web-based self-reports of covid-19 symptoms, if any individuals in the athlete's household or training group have symptoms, and a mood assessment. during the first weeks of monitoring, participation has been satisfactory (about 75%). the collected data are used by the national team coach and medical team for planning of supportive interventions in different areas and at individual and group levels. in societies world-wide, the sars-cov-2 pandemic has serious effects on morbidity and mortality. in response, societies have restricted social contacts and redirected health service resources to covid-19 patients. sports communities are not excluded from the negative consequences of the pandemic. in athletes, covid-19 can not only cause disruption of training and competition programmes, but also can cause more significant health issues [6, 7, 30] . athletes not contracting the disease are impacted by the pandemic through cancellation of competitions and loss of incomes. in april 2020, most professional sports have been locked down and thousands of community sports clubs need immediate support to avoid bankruptcy. these collateral consequences of the pandemic will influence sports participation for a long period of time and require effective countermeasures. to withstand the pandemic, sports organisations should cooperate with national public health agencies, epidemiologists, and sports medical researchers and practitioners to build trust and resilience, protect the elderly and other vulnerable groups, and safeguard sports participation at all levels. author contributions tt conceived the article, conducted the data collection and wrote the commentary. funding no sources of funding were used to assist in the preparation of this article. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia nowcasting and forecasting the potential domestic and international spread 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antibody tests for sars-cov-2. lancet corona virus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies systematic review of covid-19 in children shows milder cases and a better prognosis than adults population-based simulations of influenza pandemics: validity and significance for public health policy use of rapid online surveys to assess people's perceptions during infectious disease outbreaks: a cross-sectional survey on covid-19 requirements and design of the prosper protocol for implementation of information infrastructures supporting pandemic response: a nominal group study monitoring the covid-19 epidemic in the context of widespread local transmission coronavirus disease (covid-19) advice for the public. world health organization. corona virus disease guidance on social distancing for everyone in the uk fair society, healthy lives: the marmot review. london: strategic review of health inequalitites in england post-2010 trust is the key to fighting the pandemic swedish athletics. angående coronaviruset fotballens koronaregler [corona rules for football practice asics, and the runkeeper™ app team up to offer a more rewarding virtual experience recommendations to maintain immune health in athletes effect of flavonoids on upper respiratory tract infections and immune function: a systematic review and meta-analysis covid-19 and the cardiovascular system respiratory health in athletes: facing the covid-19 challenge can exercise affect immune function to increase susceptibility to infection? conflict of interest toomas timpka declares that he has no conflicts of interest relevant to the content of this article. key: cord-328996-3sf2i45r authors: barthélémy, romain; blot, pierre-louis; tiepolo, ambre; le gall, arthur; mayeur, claire; gaugain, samuel; morisson, louis; gayat, etienne; mebazaa, alexandre; chousterman, benjamin glenn title: efficacy of almitrine in the treatment of hypoxemia in sars-cov-2 acute respiratory distress syndrome date: 2020-06-06 journal: chest doi: 10.1016/j.chest.2020.05.573 sha: doc_id: 328996 cord_uid: 3sf2i45r nan am received speaker's honoraria from novartis, orion, and servier and fees as a member of the advisory board and/or steering committee from adrenomed, sanofi, roche, abbott, and 4teen4. bc received fees as a member of an advisory board from roche diagnostics. the other authors have not disclosed any potential conflict of interest. research letter 1 dear editor, 2 critically ill covid-19 patients frequently present profound hypoxemia with acute 3 respiratory distress syndrome (ards) requiring mechanical ventilation (mv) 1 . according to 4 recently published covid-19 guidelines 2 , ventilatory support aims at increasing alveolar 5 oxygen partial pressure with non-invasive methods and eventually mv. ventilator settings 6 are optimized in order to recruit collapsed alveoli and reduce ventilator-induced lung injury. 7 however, in sars-cov-2 ards, it has been hypothesized that recruitment strategies may be 8 hazardous because of a preserved compliance 3 and a poor response to peep 4 whereas 9 physiological measurements rather show increased intrapulmonary shunt 5 . abnormal 10 pulmonary vascular dilation and increased perfusion surrounding areas of lung opacity have 11 been identified with dual-energy ct imaging, suggesting that insufficient hypoxic pulmonary 12 vasoconstriction (hpv) may plays a major role in the onset of hypoxemia 6 . 13 almitrine, a drug that used to decrease intrapulmonary shunt by enhancing hpv, improves 14 gas exchange in ards 7 . we hypothesized that almitrine could improve hypoxemia in sars-15 cov-2 ards patients. 16 this monocenter retrospective study aimed to evaluate the association between almitrine 19 introduction and improvement of oxygenation in sars-cov-2 ards. the study was conducted 20 in a 36-bed icu (hôpital lariboisière, paris, france) fully dedicated to the covid-19 outbreak. 21 4 the medical records of all patients admitted between march 14 th 2020 and april 11 th 2020 22 were reviewed. 23 inclusion criteria in the study were: admission for respiratory failure, a diagnosis of ards 24 according to berlin criteria 8 , laboratory confirmed sars-cov-2 infection, almitrine infusion in 25 icu. the primary endpoint was the arterial oxygen partial pressure (pao 2 ) to fraction of 26 inspired oxygen (fio 2 ) ratio between baseline value and peak value during the 1 st -6 th hour 27 timeframe after introduction of almitrine. pao 2 /fio 2 ratio was measured with fio 2 1. the 28 other endpoints were incidence of treatment failure at 24h, 48h, 72h and 96h, and safety. 29 treatment failure was defined as death, or the need for additional rescue therapy. increase 30 in right atrial pressure (rap) and lactate during the first 6 hours and peak values for liver 31 tests during the first 48h were reported as safety data. 32 this study was approved by an institutional ethics committee: institutional review board 33 (irb 00006477) of hupnvs, paris 7 university. 34 patients were managed according to local protocol based on international guidelines 9 . 36 intrapulmonary shunt is confirmed after exclusion of a patent foramen ovale. pleural 37 effusions are considered for drainage. hemodynamic optimization is performed to address 38 low pvo 2 effect. early respiratory management includes limitation of tidal volume and 39 plateau pressure, systematic neuromuscular blockade and prone positioning session of at 40 least 16h. peep is individualized to improves oxygenation without deteriorating compliance 41 and cardiac output. in case of persistent refractory hypoxemia, we usually consider almitrine 42 infusion (initial dose 2 µg.kg −1 .min −1 ) and/or inhaled nitric oxide (ino). almitrine and ino use 43 are decided after collegial discussion on a case-by-case basis. if severe hypoxemia persists 44 despite the latter treatments, extracorporeal membrane oxygenation (ecmo) team is called 45 to evaluate the indication of the device. 46 continuous variables before and after almitrine infusion were compared by a wilcoxon rank 48 sum test for paired data. all statistical analyses were performed using r statistical software 49 version 3.6.1. 50 patients 52 eighty-six patients were admitted to our icu during the studied period. nineteen of the 20 53 patients that met inclusion criteria had complete data and were analyzed ( eighteen patients (95%) had at least one session of prone positioning before almitrine. 59 the median pao 2 /fio 2 ratio increased from 79 [64-100] at baseline to 117 [81-167] after 61 almitrine (p=0.001) (figure 1 ). 62 this is the first study describing therapeutic effects of almitrine in sars-cov-2 ards. in our 73 observational study, almitrine was associated with an increase in pao 2 /fio 2 ratio after 74 treatment. however, this improvement of hypoxemia seems to be heterogenous amongst 75 patients. furthermore, despite an associated improvement in pao 2 /fio 2 ratio, the majority 76 of patients receiving almitrine went on to needing additional rescue interventions or died. 77 this may be explained by the fact that, in our study, almitrine has been used as a rescue 78 therapy in severe patients with worsening hypoxemia and very low pao 2 /fio 2 ratio. 79 even though this is a small sample study without control group, our data shows that 80 enhancing hpv is an encouraging strategy to reduce hypoxemia in sars-cov-2 ards. this 81 could at least be helpful to secure intra-or inter-hospital transfers in the most severe 82 patients, or gain precious time until a more invasive life support is available. 83 the ideal time to start almitrine also remains to be determined. almitrine could be used 84 earlier in the treatment of sars-cov-2 hypoxemia to reduce the need or duration of mv, a 85 scarce resource in the setting of worldwide outbreak. however, improvement in hypoxemia 7 is not necessarily associated with improved outcome. on the other hand, almitrine has been 87 identified as a potential candidate against targeted proteins of sars-cov-2 with expected 88 inhibitory effect 10 . altogether, we believe that almitrine should be evaluated in clinical trials 89 aiming at improving patient centered outcome for covid-19 patients. 90 in this monocenter retrospective study, we found that almitrine at the dose of 2 92 µg.kg −1 .min −1 was associated with an increased pao 2 /fio 2 ratio in the following 6 hours in 93 sars-cov-2 ards patients. 94 12 -cumulative incidence of treatment failure (n) baseline characteristics and outcomes of 96 patients infected with sars-cov-2 admitted to icus of the lombardy region surviving sepsis campaign: guidelines on 99 the management of critically ill adults with coronavirus disease covid-19 pneumonia: different respiratory 102 treatments for different phenotypes? intensive care med lung recruitability in sars-cov-2 associated acute 105 respiratory distress syndrome: a single-center, observational study analysis of therapeutic targets for sars-cov-2 and 123 discovery of potential drugs by computational methods key: cord-334773-yw2qgv13 authors: lisco, giuseppe; de tullio, anna; giagulli, vito angelo; guastamacchia, edoardo; de pergola, giovanni; triggiani, vincenzo title: hypothesized mechanisms explaining poor prognosis in type 2 diabetes patients with covid-19: a review date: 2020-08-10 journal: endocrine doi: 10.1007/s12020-020-02444-9 sha: doc_id: 334773 cord_uid: yw2qgv13 purpose: epidemiological data suggest that comorbid patients, mostly those with type 2 diabetes (t2d), are predisposed to poor prognosis in coronavirus disease 2019 (covid-19), leading to serious healthcare concerns. the aim of the present manuscript is to review the main relevant mechanisms possibly contributing to worsen the clinical course of covid-19 in t2d. results: poor glucose control, high glycaemic variability and diabetes-related comorbidities at baseline, particularly cardiovascular diseases and obesity, contribute in worsening the prognosis in the above-mentioned cluster of patients. moreover, both a lower efficient innate immune system response and cytokine dysregulation predispose patients with t2d to impaired viral clearance and more serious pulmonary and systemic inflammation once the sars-cov-2 infection occurred. inconclusive data are currently available for specifically indicate or contraindicate concurrent medications for managing t2d and its comorbidities in infected patients. conclusions: t2d individuals should be considered as more vulnerable to covid-19 than general population, and thus require adequate advices about hygienic tips to protect themselves during the pandemic. a careful management of glucose levels and diabetes-related comorbidities remains essential for avoiding further complications, and patient monitoring during the pandemic should be performed also at distance by means of telemedicine. further studies are needed to clarify whether medications normally used for managing t2d and its associated comorbidities could have a protective or detrimental effect on covid-19 clinical course. background firstly identified and characterized as 2019-ncov [1] , human severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been reported as a novel infective agent arisen at the end of the 2019 [2] . sars-cov-2 is a positivesense, single strand, enveloped rna virus belonging to the family of coronaviridae, and is the 7th beta-coronavirus recognized to infect humans [3] . first metagenomic rna sequencing of sars-cov-2 showed the single-strand rna consisted of 29,906 nucleotides, and was closely related to a group of bat sars-like coronaviruses (89,1%) [4] . further observations confirmed that sars-cov-2 was closely related (88%) with two bat sars-like coronavirus (bat-sl-covzc45 and bat-sl-covzxc21), but was distant from other two human coronaviruses responsible for severe infective pneumonia: sars-cov (79%) and middle east respiratory syndrome coronavirus (mers-cov) (50%) [5] . a high grade of homology between genomic sequences of sars-cov-2 form different patients (99.98%) has also been reported, thus confirming a human-to-human transmission of the novel infective agent [5] . phylogenetic analysis suggested that sars-cov-2 progenitors circulated in animal host including bats [6] , snakes, malayan pangolins, civets, mouse [7, 8] , and underwent to a naturally occurred selection before the zoonotic spillover finally adapting to persistently infect the new host [9] . official epidemiological report declared that the early cases of infections were detected in december 2019, and involved people who worked or visited the hua nan south china seafood market [10] , in wuhan capital city (hubei province; people's republic of china). however, it remains still debated whether the zoonotic spillover might have been occurred in other places [11] , particularly in southeast asia [12] , and consequently sars-cov-2 infection might have been imported to wuhan. epidemiological analysis showed a marked widespread of the infection within community places due to a large human-to-human transmission [13] and wuhan rapidly became the hub of a new pneumonia outbreak [14] . due to a consistent widespread of detected cases among several countries, the world health organization declared the state of pandemic on march 11, 2020 when confirmed cases raised up to 118,000, and sars-cov-2 spread into 114 countries [15] . clinical manifestations of the novel coronavirus disease 2019 (covid19) include fever (87%), cough (58%), dyspnoea (38%), muscle soreness (35%), chest distress (31%) in a context of bilateral pneumonia (76%) with ground glass opacification (70%) at ct scan due to lung interstitial involvement [16] . autoptic studies described macroscopic features of pleuro-pericarditis, lung consolidation, oedema with overall increased pulmonary weight; while microscopic hallmarks are characterized by pneumocyte hyperplasia, lymphocytic and multinucleated giant cells infiltration, hyaline membranes [17] [18] [19] . other signs and symptoms of covid-19 include acute conjunctivitis [20, 21] ; diarrhoea, abdominal pain/discomfort and vomiting [22] [23] [24] ; convulsion, headache, muscle soreness [25] ; diffuse erythematous rush and widespread urticaria [26] ; acute kidney injury [27, 28] ; pharyngodynia, nasal congestion with rhinorrhoea and smell/taste impairment [29] . sars-cov-2 may directly affect myocardial tissue, and significantly complicate the prognosis of underlying cardiovascular diseases [30, 31] . sars-cov-2 infection usually occurs asymptomatically or mildly symptomatic form of the disease but in predis posed patients with specific clinical conditions, a serious clinical course could be observed thus leading to worse prognosis or death [12] . worldwide reported case-fatality rate for covid-19 differs considerably among geographical areas [32] , and could be attributable to several variables, such as testing strategies for screen suspected cases and identification of infectious patients (statistical); accessibility to intensive care according to restricted national healthcare system capacities (organization); baseline patient age and comorbidities (medical) [33, 34] . from the latter point of view, a poor prognosis is usually observed in elderly patients [35] and worldwide age-specific case-fatality rate occurred very high among patients with one or more underlying chronic diseases including cardiocirculatory, renal, pulmonary, central nervous system and mental illness, diabetes mellitus (dm) and malignancies [36, 37] . according to the data shared by the italian national institute of health, patients who died while tested positive for sars-cov-2 exhibited an elevated mean age (78.5 years old), mostly men (70%) and with one or more pre-existent chronic diseases (2.7 in mean) [38] . of these, blood arterial hypertension (78%) and dm (34%) were the most commonly reported clinical comorbidities, followed by ischaemic heart disease (30%) and atrial fibrillation (22%). the leading cause of deaths was attributable to acute respiratory distress (97%) [38] . the global estimated prevalence of t2d accounts for more than 450 millions affected patients corresponding to 9.3% of the worldwide population [39] . therefore, the number of patients with t2d who will contract sars-cov-2 infection is expected to be considerable, and should increase over time. t2d per se does not increase the risk of contracting sars-cov-2 infection but could exacerbate the clinical course of covid-19 leading to a detrimental prognosis [40] . indeed, the frequency of diabetes in patients with covid-19 has been reported to 9-12% [41] [42] [43] , raising up to 16-20% in hospitalized patients including those who required intensive care for severe disease [44, 45] . more recently, data collected from nine hospitals from seattlearea in the united states demonstrated that 58% of patients who required hospitalization for respiratory symptoms attributable to covid-19 had t2d [46] . in severely ill patients with covid-19 a pre-existent t2d was observed in about 35% of the cases and, according to the results of an univariate analysis, the presence of t2d resulted a significant risk factor for poor prognosis in this clinical setting (or 8.14; p < 0.0001) [47] . dm has also been reported as the main clinical condition observed in non-survived patients with covid-19 (22%) [48] , thus resulting one of the most frequently associated comorbidity in covid-19 deceased patients [33] . this concern has been further confirmed by the results of a cohort study among 85 fatal cases of covid-19 in wuhan, hence defining dm as a potentially harmful comorbidity predisposing to worse clinical course or death once sars-cov-2 infection occurred [49] . different hypothesis should be considered for explaining this clinical phenomenon, including glucose control at baseline and during the infection course, pathophysiology and immune system response in sars-cov-2 infected patients with t2d, diabetes-related comorbidities and concomitant medications. herein, a point-to-point discussion about these putative mechanisms has been carried out. epidemiological data showed that t2d represents a risk factor for infectious diseases, mostly with bacterial aetiology, particularly at the level of skin and soft tissue, genitourinary, gastrointestinal and respiratory systems [50] . moreover, life expectancy in individuals with t2d may be affected due to infectious diseases and in certain clusters of patients, such as in elderly with t2d, the leading cause of mortality is attributable to severe pulmonary infections rather than other highly prevalent comorbidities, including cardiovascular diseases and malignancies [51] . on the other hand, dm increases the cumulative risk of medical consultation, hospital admission, intensive care requirement and poor prognosis because of pandemic influenza [52] . further data reported that airways and pulmonary infections with different aetiologies, including sars and mers, were more frequently diagnosed in t2d patients, also showing a severe clinical course [53] [54] [55] [56] . in dm, hyperglycaemia is considered one of the most important factor in determining this burden [57] . indeed, osteomyelitis, soft tissues infections, endocarditis, tuberculosis and sepsis are most commonly observed in diabetic patients with poor glycaemic control compared to those who achieve better glucose management, and a worse glucose control contributes to increase the rate of hospitalization and mortality, too [57] . both hyperglycaemia and high glucose variability may consistently complicate the clinical course also in case of influenza a [58] . more recently, a retrospective observational study recruiting more than 7000 cases of covid-19 from hubei province (china) and including 952 patients with a pre-existent t2d displayed a higher mortality rate (hr 1.49), more prevalence of multiorgan damage and a greater requirement of medications (antibiotics, systemic corticosteroids, vasoactive substances, oxygen inhalation and either non-invasive or invasive mechanical ventilation) in patients with dm than in nondiabetics [59] . interestingly, authors also found that, among t2d patients, those with better glucose control (glucose levels between 70 and 180 mg/dl) respective to those with worse glucose control (>180 mg/dl) during hospitalization exhibited a significantly lower rate of mortality (hr 0.14; p < 0.008), and a fewer risk of progression to acute respiratory distress syndrome (hr 0.47; p < 0.009), acute kidney (hr 0.12; p < 0.046) and myocardial (hr 0.24; p < 0.01) injury [59] . similar results were found by another observation in which worse glucose control (glucose levels >110 mg/dl) at the admission and during hospitalization was found to be an independent risk factor for progression to critical ill or death among t2d patients with confirmed covid-19 [60] . therefore, hyperglycaemia represents a relevant matter in patients with covid-19 fostering poor prognosis once the infection occurred. in addition, recent evidences suggest that sars-cov-2 may induce beta-cells damage thus leading to insulin secretion impairment. this phenomenon, in addition to a pre-existent hyperglycaemia and considering that systemic inflammation due to the infection exacerbates the insulin-resistance, is thought to play a significative role to further worsen glucose control and complicate the clinical course of covid-19 [61] . in conclusion, epidemiological data suggested that dm, particularly t2d, is a frequently observed comorbidity in patients with sars-cov-2 infection who require hospitalization, more intensive treatment and exhibit poor prognosis or death. poor baseline and ongoing glucose control in hospitalized patients rather than the presence of t2d per se seems to facilitate covid-19 progression [62] . hence, an optimal and timely blood glucose management during pandemic should be considered as an effective strategy to reduce the probability of hospitalization requirement of infected patients, and for improving the clinical course of those hospitalized for receiving either non-intensive or intensive care. innate and adaptive immune responses play a crucial role against viral infections [63] . immune response against coronaviruses has been reviewed elsewhere, highlighting the role of both innate and adaptive systems to promptly contrast virus replication, facilitate virus clearance, stimulate tissue repair and develop persistent defence [64] . immune response in covid-19 is not still completely understood making necessary further investigation to better control the pandemic evolution [65] . however, seriously ill covid-19 patients exhibit an exaggerate response of neutrophils and alveolar macrophages, and a relevant peripheral lymphocytes dysfunction [66] , which lead to an uncontrolled viral shedding, consequent viremia and further systemic immune-mediated damage, thus triggering a harmful vicious circle [66] . glucose levels may significantly influence immune response as observed in patients with dm. natural killer (nk) cells activity is weakened in case of hyperglycaemia, and is inversely related with fasting plasma glucose, 2-h postprandial glycaemia and hba1c levels [67] . macrophage activation and phagocytosis are both decreased in patients with poor glucose control, but should be restored after an adequate optimization of metabolic control [68] . neutrophil activation and phagocytosis are both impaired by hyperglycaemia as demonstrated in animal models and humans, thus suggesting a relevant impairment of innate immune response in patients with chronic hyperglycaemia [69] . in an animal model, obese and hyperglycaemic mice experienced higher rate of respiratory infection due to influenza and bacterial pneumonia and that was related to a lower efficient alveolar macrophage response against infections. in addition, a defective toll-like receptor 4 signalling has been recognized in neutrophils exposed to the gram-negative lipopolysaccharide, leading to a blunted release of chemokines and cytokine, and decreased myeloperoxidase activity [69]. moreover, t-cells function is significantly dysregulated in t2d and cd4 + lymphocytes preferentially differentiate in thelper 1 and t-helper 17 instead of t-helper 2 with a consequent imbalance between pro-inflammatory and antiinflammatory activities [70] . on the other hand, the levels of interferon gamma-which normally stimulates cd4 + t cells maturation in sense of t-helper 1 rather than t-helper 2-were found to be lower in t2d patients sera, and can contribute in a blunted t-cell response in t2d [71] . nevertheless, lower levels of interleukin (il)-10 have also been described in t2d patients. considering that il-10 is capable to suppress the release of pro-inflammatory cytokines, lower levels of il-10 could be related with higher il-6-to-ifn gamma and tnf-alpha-to-ifn gamma ratios hence suggesting an enhanced activation of circulating monocytes [71] . high levels of il-6 have been detected in diabetic patients respective to those with euglycemia, suggesting that hyperglycaemia play a crucial role in determining this immunological effect [72, 73] . in animal models, hyperglycaemia and insulin-resistance increase the level of circulating proinflammatory cytokines and oxidative stress at baseline [74] . since this pro-inflammatory background usually results reversible after an effective treatment of hyperglycaemia, a low dose endotoxemia consistently enhances systemic inflammation in a animal model [75] . therefore, hyperglycaemia may predispose to an exaggerate immune response even in case of a mild-to-moderate viral load. as known, a hyperinflammatory syndrome with cytokine dysregulation has been well recognized in seriously ill patients [76] , thus highlighting its crucial role in serious manifestations of covid-19 [77] . specific interleukins and chemokines (il-2, il-7, il-6, tnf-alpha, interferon gamma induced protein 10, granulocyte-colony stimulating factor) are upregulated in patients who exhibited a worsen prognosis [78] and particularly high levels of il-6 have been detected in case of serious pulmonary involvement or in patients requiring intensive care [79] . these findings are also more evident in elderly patients who display less vigorous immune response against viral shedding, greater susceptibility to more serious pulmonary and systemic involvement, and are finally predisposed to covid-19 progression [80] . both the number and function of t cells (both cd4 + and cd8 + ) [81, 82] , b cells depletion, and hypercoagulability have also been observed in seriously ill cases and the greater the magnitude of these haematological and biochemical alterations then the greater the severity of the prognosis [83, 84] . in conclusion, diabetic patients especially elderly individuals and those with worse baseline glucose control may exhibit immune system dysregulation that predispose them to a less effective response against sars-cov-2 and to a dysfunctional inflammation that requires to be carefully monitored in confirmed cases of covid-19, for preventing or avoiding a harmful progression of the disease. angiotensin-converting enzyme 2 (ace 2 ) is a carboxypeptidase normally involved in the cleavage of angiotensin i and angiotensin ii, and is the main receptor for sars-cov-2 playing a determinant role in viral entry into the host, and clearly explaining both the transmissibility and severity of covid-19 among humans [85] . ace 2 is expressed at the level of several tissues (transmembrane and soluble forms), such as lung [86] , oral mucosa [87] , intestine [88] , brain [89] , pancreatic islets [90] , testis [91] and kidney [92] . differentiated type 2 pneumocytes normally express ace 2 , which is essential to regulate pulmonary homoeostasis and protects against pulmonary injury [93] . indeed, low levels of ace 2 have been described in severe acute and chronic pulmonary diseases thus predisposing to poor prognosis [94] . however, ace 2 is overexpressed in chronic diseases, including t2d, and this phenomenon could be also related with chronical exposure to several medications [94, 95] . this biochemical condition is believed to facilitate the internalization of sars-cov-2 into pneumocytes, thus contributing to worse prognosis in covid-19 [94, 95] . however, the role of ace 2 overexpression in worsening the prognosis is an emergent issue, and remains currently debated [96] . cardiovascular diseases, including coronary and cerebrovascular artery disease and heart failure, are frequently observed in t2d patients and it has been estimated that about a third of them displayed these kind of complications over time [97] . cardiovascular system is the main extrapulmonary compartment extensively involved in covid-19, as suggested by a frequent myocardial involvement in affected patients especially in those having hypertension, t2d and cardiovascular diseases at baseline [98] . vascular inflammation and endothelial dysfunction [99] , myocardial injury and cardiac arrhythmias are not-infrequently observed in covid-19 confirmed cases, significantly influencing the risk of poor prognosis or death in this cluster of patients [100] [101] [102] . overweight-obesity syndrome is a multifactorial disease which significantly predispose to cardiometabolic risk, and is strictly associated with insulin-resistance, glucose metabolism impairment and t2d [103] [104] [105] . despite obesity is usually associated with decreased risk of death in patients with severe acute respiratory distress (obesity paradox), currently available data suggest that an elevated body mass index should be considered as an independent risk factor predisposing to poor prognosis [106] and death in covid-19 [107] [108] [109] [110] . indeed, the prevalence rate of obesity in this cluster of patients has been reported in 42% of the cases [111] and a bmi greater than 35 kg/m 2 has been usually observed in patients requiring hospitalization and invasive mechanical ventilation [112] also in younger patients (<60 years) [113] . pathophysiological mechanisms possibly related with poor prognosis in obese patients are not completely understood but may be attributable to a greater inflammatory background as similarly found in dm due to hyperglycaemia and insulin resistance [70] . obesity is usually associated to other severe comorbidities with high impact on cardio-metabolic health, such as fatty liver disease, vascular inflammation, cardiovascular atherosclerotic diseases and heart failure [114] that might be related with a higher burden of lethal complications, especially in hospitalized patients with covid-19 [115] . in addition to cardiometabolic risk factors, obese patients are more prone to have a decreased pulmonary ventilation or obstructive sleep apnoea, which predispose them to low levels of blood oxygenation at baseline and consequently to worse respiratory outcomes in case of acute infective respiratory diseases [116, 117] . nutritional patterns usually exhibited by obese patients are frequently characterized by an elevated dietary consumption of processed food rich in saturated fat, cholesterol, sugar and a low consumption of fibres and micronutrients, such as vitamin d [118] . these dietary patterns may affect physiological microbiome composition, weaken the immune response against microbial agent and foster immune system dysfunction [115] . in addition, vitamin d deficiency/insufficiency and sedentary lifestyle, which are highly prevalent among obese patients, should be considered as predisposing factors for worse prognosis in response to acute infections, including covid-19 [119] . abdominal obesity usually leads to a low cardiorespiratory reserve and systemic inflammatory dysfunction which predispose to a worse prognosis in covid-19 [120] . conversely, regular physical exercise, which is normally lacking in an obesogenic lifestyle, is associated to higher levels of cardiorespiratory fitness, and is believed to improve the innate immune response and attenuate cytokine dysregulation often experienced by high risk patients with the so called cytokine storm [121] . visceral obesity is also a risk factor for both the development and progression of cardiovascular diseases [114] considering that it fosters higher level of pro-thrombotic circulating factors and predisposes to thrombotic events [122] . furthermore, visceral obesity is more prevalent in men than women and this biological phenomenon has been hypothesized to have a putative role in driven poor prognosis of covid-19 especially in men. from this point of view, male obesity is usually associated with a functional hypogonadism and these clinical conditions are both associated with one other [123] , according to a complex and multifactorial pathogenesis [124] that fosters higher baseline levels of inflammation and endothelial dysfunction [125] . aromatase gene expression is enhanced by prostaglandin e 2 , whose levels were found to be higher in visceral adipose tissue of obese men [126] . an enhanced aromatase activity at the level of adipose tissue increases local levels of estradiol that is thought to be a defensive mechanism against local inflammation and insulin resistance [127] . whether augmented aromatase levels (and activity) at the level of adipose tissue in men may be responsible for a systemic testosterone-tooestrogen imbalance remains questionable. however, obese men are usually affected by this biochemical condition and body mass index is inversely correlated to testosterone-to-oestrogen ratio [128] . a low testosterone-to-estradiol ratio contributes to increase the cardiovascular risk particularly in elderly [129] and in those with previous cardiovascular diseases [130] [131] [132] . in addition, hormonal pattern associated with visceral obesity may play a relevant role in reducing the efficacy of immune response against infectious agents, predisposing to cytokine dysregulation, endothelial dysfunction, thrombosis, finally driving patients, particularly men, to poor prognosis or death [133] . moreover, ace 2 is also expressed on adipose cells [134] and a larger extension of adipose tissue in obese patients may significantly increase the number of available receptors for sars-cov-2, thus leading to a much greater viral shedding once the infection occurs [135] . in conclusion, multiple diabetes-associated chronic comorbidities, particularly obesity, have been found to worsen the prognosis in covid-19 affected patients by acting as independent risk factors. careful management and prompt interventions are thus required for improve clinical outcome predominantly in type 2 diabetic patients. pathogenetic mechanisms basically involved in sars-cov-2 infection may be exacerbated by the use of concomitant medications for the management of t2d. of note, an exaggerate pulmonary and systemic expression of ace 2 facilitates sars-cov-2 replication and may be responsible for worse clinical course and poor prognosis [94, 95] . in animal model, pioglitazone has demonstrated to increase ace 2 expression particularly at the level of hepatic and adipose tissue [136, 137] . on the other hand, the analysis of potential therapeutic targets for sars-cov-2 assessed by a computational model found pioglitazone to have a potential for inhibiting 3clpro, an essential target for viral replication [138] . these findings did not provide conclusive assumption and diabetologists can safely prescribe pioglitazone taking into account general concerns including fluid retention and heart failure [139] . gliflozins prescription is increasing over time, also considering a long-term beneficial cardiovascular and renal protection [140, 141] . many mechanisms of cardiovascular and renal protection have been proposed, including an enhanced induction of ace 2 expression at the level of heart and kidney but it is unclear whether this effect may negatively influence clinical course in covid-19 [142] . moreover, despite gliflozins reduce inflammatory injury and endothelial dysfunction, none conclusive data are currently available to confirm their potential beneficial effect in diabetics with covid-19 [143] . concerns about gliflozins use in covid-19 are attributable to volume contraction, renal insufficiency and increased risk of ketoacidosis that may be supposed to occur particularly in hospitalized patients, including those severely ills. dipeptidyl peptidase iv (dpp-iv or cd26) is a transmembrane and soluble ectopeptidase largely expressed in human tissues, including airways, lung and leucocytes [144] . particularly, dpp-iv is specifically involved in mers pathogenesis given that it mediate mers-cov-2 internalization in host cells [145] . no data actually support the role of dpp-iv in the internalization of sars-cov-2 and further study are needed to verify this aspect, and for demonstrating clear protective effects [146] . however, dpp-iv inhibitors have shown to potentiate immune response by increasing t-cells survival, consequently enhance immune response [147] , and possibly playing a relevant role in reducing the onset or progression of acute pulmonary manifestations in covid-19 [145] . glucagon-like peptide 1 receptor agonists (glp-1ras) belong to an effective class of medications approved for the treatment of t2d and for preventing diabetes-related cardiovascular and renal complications over time [148] [149] [150] [151] . immune response and systemic inflammation play a crucial role in sars-cov-2 infection, particularly in case of severe clinical course of the disease. in this clinical scenario, it is speculated that the use of glp-1ras could provide both a better glucose control and anti-inflammatory effects with consequent improvement of outcomes in covid-19 affected patients [143] . in patients with pulmonary inflammation, mononuclear circulating cells express lower level of glp-1 receptors than controls and this condition is associated with a blunted production of interferon gamma by t cells and nk cells. in addition, the expression of programmed death cell protein 1, which mediates t-cell apoptosis, is enhanced on t-cell surface and consequently the efficiency of cell-mediated immune response is weaken. liraglutide, a once-daily administered long-acting glp-1ra, demonstrated to restore the expression of glp-1 receptors on macrophage surface, and potentiate immunemediated response [152] . in addition, both dpp-iv pharmacological inhibition (linagliptin) and glp-1ra (liraglutide) were found to contrast pulmonary injury, reduce platelet activation, microvascular thrombosis, and oxidative stress in mice model of endotoxemia [153] . moreover, glp-1ras have been found to reduce the expression of pulmonary il-33, thus playing an interesting role in contrasting a il-33 mediated damage in immune-allergic diseases and viral infections [154] . considering that pancreatic islets express ace 2 , glucose impairment in covid-19 diabetic patients could be attributable to a partial insulin deficiency [56] . however, glucose impairment could also be attributable to sars-cov-2 infection-induced stress or as the consequence of some medications or treatment protocols, including high-dose glucocorticoids, particularly used in hospitalized patients [155] . insulin therapy is recommended in hospitalized patients [156] , including those with covid-19, and a basal-bolus respective to a sliding scale regimen should be preferred in infected patients for avoiding glycaemic excursion and greater glucose variability [157, 158] . hypertension is frequently observed in t2d patients leading to a consistent increase in the risk of atherosclerotic cardiovascular and renal diseases [159] . ace inhibitors and angiotensin receptor blockers compared to other antihypertensive medications demonstrated to be more effective to prevent or delay the onset of cardiovascular and renal complications in people with t2d, and are currently used as the first-line treatment in these patients [160, 161] . evidence from animal models suggested that ace inhibitors are able to increase the expression of ace 2 at the level of lung, thus leading to initial clinical concerns about the management of arterial hypertension in diabetic patients [162] . first suggestions advised to replace these medications with calciumchannel blockers in order to avoid undesirable detrimental effects of covid-19 clinical course [95] . nevertheless, no sufficient data supported the hypothesis that the use of renin-angiotensin system blockers may interfere with the internalization of sars-cov-2 in respiratory cells hence increasing its virulence [163] . in addition, ace inhibitors and angiotensin receptor blockers protect lung, heart and kidney against inflammation, thus playing a potential beneficial role also in t2d patients with covid-19 [164] . statins are usually prescribed especially in t2d patients to protect against cardiovascular complications [165] . the role of statins in covid-19 is not clearly established, however, some observation should be done. first, covid-19 is associated with higher cardiovascular mortality specifically in patients with more risk factors, including hypertension and t2d, and statins could be useful to maintain or optimize lipid management and improve endothelial dysfunction in this clinical setting [166] . in animal models, statins inhibit the myeloid differentiation primary response 88 (myd88) [167] . myd88 is a protein adaptor for inflammatory signalling pathways downstream of members of toll-like receptors and il-1 receptor families, and plays a critical function in the activation and amplification of innate immune response [168] . the inhibition of myd88 seems to reduce pulmonary inflammatory damage, and to improve survival in sars-cov and mers-cov infected mice [167] . patients with established cardiovascular disease, and those at higher risk of atherosclerotic cardiovascular disease including t2d (also in primary prevention) should be advised to maintain current statin treatment also in case of confirmed covid-19. in patient with active covid-19, an increased risk of muscular damage has been described and statin therapy could be interrupted for a short period of time in order to favour muscular recovery [169] . t2d should be considered as a risk factor for poor prognosis in covid-19 due to several reasons, including poor baseline glucose control and high glycaemic variability, diabetes-related immune dysfunction and concurrent comorbidities, also proven to act as independent risk factors for poor prognosis in this clinical setting. considering that respiratory system remains essentially the leading way for sars-cov-2 entering in host, and given that immune barriers are weaken in t2d patients, including resident pulmonary macrophages, neutrophils and t cells [70] , diabetologists could improve clinical outcomes in covid-19 simply by the use of such a medication with potential for positive immune-modulation and anti-inflammatory effects. unfortunately, inconclusive deductions are currently available for considering concomitant medications favourable (as well as detrimental) factors in covid-19 patients with t2d. despite some putative mechanisms have been identified and some speculative hypothesis have also been formulated indicating that some anti-diabetes medications may improve clinical course in covid-19 (pioglitazone, gliflozins, dpp-iv inhibitors, glp-1ra), further studies are needed to clarify the issue. a timely glucose control should be obtained in diabetic patients during pandemic for the purpose to avoid potentially harmful outcomes in case of sars-cov-2 infection [170] . anti-diabetic medications should be used with caution, also considering their impact on patient safety in case of complications related with covid-19 infection (renal failure, cardio-respiratory burden, ketoacidosis, etc). oral and non-insulin injectable medications (glp-1ras) should be considered to maintain or intensify household glucose control, while insulin treatment is currently known to be safer in case of hospitalized and seriously ill patients [171] . given this consideration, in seriously ill patients, immune response should be adequately monitored in order to carefully predict or precociously diagnose a severe disease [172] and cytokines, including il-6, may be considered as a goal for novel and more specific target therapies particularly in t2d individuals [173] [174] [175] . beyond the risks strictly related with covid-19 infections in diabetic patients, further concerns should be taken into account. social distancing and lockdown have been proposed as the most effective action to prevent and reduce spread of sars-cov-2 infection [176] . consequently, more time has been spent in household and access to public and private services has been consistently reduced. thus, several barriers exist for diabetic patients to maintain an adequate fitness status and weight management as well as access to ambulatory care for obtaining adequate treatment adjustments [177] . in the last few months, home confinement led to physical inactivity and low exposure to sunlight, with possible detrimental effects particularly in diabetic and obese patients [119] . physical exercise should be encouraged also at home or outdoor, even respecting social distancing rules, since it promotes weight loss, and improves cardiorespiratory fitness [121] ; reduces levels of cortisol thus reinforcing immune response against infections; induces t-cell differentiation and maturation [178] . importantly, patients should be advised about risks and supported regarding pandemic-related concerns. particularly, they should be elucidated about general hygienic tips to protect themselves and others from sars-cov-2 spread; promptly recognize covid-19 signs and symptoms; access orderly to approved testing if exposed; furthermore, it is important to avoid unnecessary routine appointment in person especially for elderly patients [179] . closely monitoring of glucose control should be recommended at home and glucose reports should be periodically sent to the diabetologist for proper therapy adjustments [180] . the hard lesson form lombardy disaster probably teaches to change prospective in the conception of patients care during the present and in case of further pandemic. implementation of community-centred care system (home care and mobile clinics) may result in a prompt detection and better management of infected patients particularly for frail individuals. moreover, it might contain an unnecessary hospital workload, letting hospital activities to focus mainly on acute cares and limiting contagion risks among inpatients and healthcare personnel. finally, it could provide at home special cares for elderly and comorbid patients [181] . in this historical moment, the use of technologies appears certainly appropriated to better manage chronic clinical conditions, including dm [182] [183] [184] . diabetic outpatients should be conveniently managed by means of telemedicine that includes several services such as emergency phone calls, social media messaging, teleconsulting and ambulatories should be fitted with technological tools according to patients ability, healthcare personnel characteristics and available economic resources [62] . conflict of interest the authors 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(american association of clinical endocrinologists at the epicenter of the covid-19 pandemic and humanitarian crises in italy: changing perspectives on preparation and mitigation. nejm catalyst innovations in care delivery managing diabetes in pregnancy before, during, and after covid-19 acceptability and utilization of newer technologies and effects on glycemic control in type 2 diabetes: lessons learnt from lockdown patient empowerment using electronic telemonitoring with telephone support in the transition to insulin therapy in adults with type 2 diabetes: observational, pre-post, mixed methods study key: cord-323905-ayufx3wv authors: kort, n. p.; barrena, e. gómez; bédard, m.; donell, s.; epinette, j.-a.; gomberg, b.; hirschmann, m. t.; indelli, p.; khosravi, ismail; karachalios, t.; liebensteiner, m. c.; stuyts, b.; tandogan, r.; violante, b.; zagra, l.; thaler, m. title: recommendations for resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic: the european hip society and european knee associates survey of members date: 2020-08-18 journal: knee surg sports traumatol arthrosc doi: 10.1007/s00167-020-06212-0 sha: doc_id: 323905 cord_uid: ayufx3wv purpose: the covid-19 pandemic has disrupted the health care system around the entire globe. a consensus is needed about resuming total hip and knee procedures. the european hip society (ehs) and the european knee association (eka) formed a panel of experts that have produced a consensus statement on how the safe re-introduction of elective hip and knee arthroplasty should be undertaken. methods: a prospective online survey was done among members of ehs and eka. the survey consisted of 27 questions. it includes basic information on demographics and details the participant’s agreement with each recommendation. the participant could choose among three options (agree, disagree, abstain). recommendations focussed on pre-operative, peri-operative, and post-operative handling of patients and precautions. results: a total of 681 arthroplasty surgeons participated in the survey, with 479 fully completing the survey. the participants were from 44 countries and 6 continents. apart from adhering to national and local guidelines, the recommendations concerned how to make elective arthroplasty safe for patients and staff. conclusion: the survey has shown good-to-excellent agreement of the participants with regards to the statements made in the recommendations for the safe return to elective arthroplasty following the first wave of the covid-19 pandemic. in recent months, the sars-cov-2 pandemic has evolved rapidly in europe, disrupting the personal, social, economic and professional lives of health professionals on a large scale. the overall goal of most governments in europe has been to flatten the curve of infected sars-cov-2 patients and prevent a collapse of national health systems. the april 2020 sars-cov-2 survey completed by ehs and eka members in europe has confirmed the impact of sars-cov-2: this pandemic has resulted in a tremendous reduction in primary hip and knee arthroplasty procedures as shown in the survey. a broad consensus is needed about the factors that need to be in place before restarting such procedures. delaying hip and knee arthroplasty in patients with severe osteoarthritis (oa) may lead to increased opioid use. it is associated with lower clinical results and increased readmission rates after the index procedure. moreover, when access to hip and knee arthroplasty is limited, as it is now in the wake of the covid-19 sanitary measures, the direct and indirect costs for our health care and social systems are enormous. many patients suffering from oa have to prolong their absence from work, request temporary unemployment benefits, and burden the public welfare system. we are now entering a new phase in most european countries, where we can consider restarting elective hip and knee arthroplasty in a "post-pandemic" period. to date, the scientific basis for the existing guidelines is not robust; there is much room for an exchange of ideas between surgeons. the current concern is to map out the optimal trajectory for starting up elective hip and knee arthroplasties. as a result, the european hip society (ehs) and the european knee association (eka) formed a panel of experts that have produced a consensus on how the safe re-introduction of elective arthroplasty should be undertaken. they have provided recommendations based on the available evidence [1]. this survey aimed to validate the recommendations by involving arthroplasty surgeons from a wide geographical area to promote then the recommendations for a safe return to elective joint arthroplasty across europe and elsewhere. a prospective online survey was done online using sur-veymonkey (portland, usa: https ://www.surve ymonk ey.com) among members of european hip society (ehs) and european knee associates (eka). a link to the survey was sent by email to all members of the ehs and the eka and affiliated arthroplasty surgeons. the online survey was launched on 23rd may 2020 and concluded on 6th june 2020. the survey consisted of 27 questions. it includes basic information on demographics and details the participant's agreement with each recommendation. the participant could choose among three options (agree, disagree, abstain). the recommendations focus on three time periods; pre-operative, per-operative, and post-operative (table 1) . this survey did not require formal ethical approval with a practice dedicated to adult reconstruction. a total of 681 arthroplasty surgeons participated in the survey, with 479 fully completing the survey. the geographical spread of this survey included surgeons from 44 different countries in 6 continents (fig. 1) . the ehs and eka had a 22.1% and 20.9% response rate, respectively. the mean time in practice for all participants was 20 years (min 1 year-max 46 years). the detailed results are shown in table 2 . the survey has shown good-to-excellent agreement by the participants to the statements made in the recommendations for the safe return to elective arthroplasty following the covid-19 pandemic. although the response rate from both the ehs and eka membership was low, at around 20%, it is notable that the mean time in elective arthroplasty of the participants was 20 years. this means that very experienced surgeons gave their opinions. coupled with the global coverage of the survey, the mean time in elective arthroplasty is a proper validation for the recommendations. resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic 1. when should elective hip and knee arthroplasty be resumed? elective hip and knee arthroplasty can be resumed when appropriate prerequisites concerning facilities, workforce, testing, supplies are met, and approval of local health authorities are obtained. facilities in areas with low, or relatively low and stable incidence of sars-cov-2, can be allowed to provide care for patients needing nonemergent, non-sars-cov-2 healthcare 2. are new triage/patient selection criteria needed for hip and knee arthroplasty patients once elective surgery is resumed? increased demand for hip and knee arthroplasty, coupled with limited hospital resources, will force surgeons to select which patients will receive hip and knee arthroplasties sooner than others. this will entail employing objective, transparent criteria in prioritizing patient selection to identify the patients most in need who also have lower risk factors for disease transmission and post-operative complications 3. what are the priority indications for elective adult hip and knee reconstruction? there is universal agreement regarding the indications for urgent hip and knee arthroplasty procedures, such as femoral neck fractures, periprosthetic fractures, dislocations and acute infection, even in the setting of the sars-cov-2 pandemic. however, priority indications for non-emergency procedures in primary and revision hip and knee arthroplasty are lacking and should be clarified 4. what is the role of outpatient hip and knee arthroplasty/enhanced recovery protocols in light of the sars-cov-2 pandemic? it is universally accepted that shorter hospital stays and treatment in a sars-cov-2-free environment decrease the risk of sars-cov-2 infection in patients undergoing elective surgery. performing hip and knee arthroplasty with enhanced recovery protocols that decrease the length of stay in a facility or can be used in ambulatory care centres to allow for day surgery might be beneficial for reducing the risk of viral transmission. transitioning to enhanced recovery systems requires full support, adoption of all the subspecialties involved and the possible approval of health care payers, which may not be possible in the pandemic setting 5. what is the impact of delaying hip and knee arthroplasty for the patients themselves and for society? delaying elective hip and knee arthroplasty has negative consequences on the quality of life of patients and negatively impacts society as a whole. the benefits of hip and knee arthroplasty should be carefully weighed against the risks of viral transmission and infection, complications and mortality in the mostly elderly population requiring joint arthroplasty resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic: pre-operative phase 1. what is the appropriate pre-operative clinical and laboratory screening and timeline for patients? all patients undergoing surgery should have their temperature and oxygen saturation measured. a thorough medical history should be acquired and patients asked about any symptoms they may have, such as cough, fever, loss of smell or taste, headache or gastrointestinal disorders. additionally, they should be questioned about recent travels and their occupation to stratify them into possible high-risk groups. regarding the laboratory testing, it would be ideal for all patients to undergo rt-pcr testing for sars-cov-2 before the operation. however, local guidelines and the efficacy of tests must also be taken into consideration. if there is a paucity of available tests, then only high-risk patients should be tested. there is no indication for additional chest ct scans in the pre-operative screening. time allowing, all surgical patients should apply social distancing principles for two full weeks prior to testing and the surgical procedure and self-quarantine in their home for the period between test acquisition and day of surgery 2. is pre-operative tracking of patients, staff and relatives necessary? all patients and staff will need to be screened for potential symptoms of sars-cov-2 prior to entering a hospital facility. in particular, staff must be routinely screened for potential symptoms. isolation prior to surgery can be guided. all patients, staff and relatives, especially those with patient contact, need to be investigated for previous symptoms, travels abroad and possible contacts with populations at high-risk for sars-cov-2 3. should spinal anaesthesia be routine? spinal anaesthesia can be considered safer for every negatively screened patient (with or without a sars-cov-2 test) than general anaesthesia, since the latter requires airway manipulation and endotracheal intubation, procedures that can more easily transmit sars-cov-2 -should we only select patients as candidates who also consent to regional/spinal anaesthesia? it cannot be made mandatory that patients consent to regional/spinal anaesthesia. nevertheless, the benefits of regional anaesthesia should be thoroughly explained to the patients, and whenever possible, this should be strongly considered as the preferred means of anaesthesia -if spinal anaesthesia does not occur, should we cancel the operation? if general anaesthesia cannot be avoided, every precaution should be taken to avoid contamination. if a sars-cov-2-negative environment has been achieved that is as secure as possible, then general anaesthesia with informed consent can be considered it is reasonable to assume that patients who test positive for sars-cov-2 should not be operated on and should be discharged from the hospital. these patients should remain in quarantine at least 14 days and until a subsequent test turns out negative (two confirmed negative pcr swab tests with a 24 h interval) and they are free of fever, cough or other symptoms 5. what kind of back-up plan is needed to guarantee quality care and patient safety in this pandemic situation? the treatment of sars-cov-2 patients is placing a huge strain on the resources of many hospitals, to the detriment of treatment of other health problems. safety of hip and knee arthroplasty patients remains of paramount importance. complications of cardiac and respiratory origin are the most common problems needing acute care. as elective operations begin, every hospital should have icu capacity available to accept and accommodate patients with cardiopulmonary or other significant complications. it should be assumed that, after the resumption of normal functions at hospitals and in society in general, there will still be a risk of another outbreak of the disease. in such a situation, hospitals should be prepared to return to a "safe mode" with reorganization of the wards and personnel. every hospital should have in place a plan for emergency distribution of the personnel and wards before elective surgery resumes resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic: perioperative phase 1. is there a need for: -modification or reorganization of hospital wards (patient density, bed density, medical and nursing staff density, etc.)? in most orthopaedic departments, the workforces have been adjusted to accommodate fewer cases, due to the reduction in trauma cases and cancellation of elective procedures. as elective operations restart, the standard social distancing guidelines should be sufficient. patient numbers in the clinical areas (wards, waiting areas, outpatient clinics) should be halved, with the patients spaced at least 2 m apart -separation of elective and trauma orthopaedic surgery (with regard to the general orthopaedic departments)? sars-cov-2 screening practices should ensure the health and safety of patients and staff. sars-cov-2-negative elective hip and knee arthroplasty must be separated from the sars-cov-2-positive trauma unit. since all patients undergoing elective procedures are screened before administration and have tested negative for sars-cov-2, they may be reluctant to go into certain settings for fear of viral transmission. there should be a physical separation of sars-cov-2-positive and sars-cov-2-negative patients and specific wards should be exclusively dedicated to treating patients with sars-cov-2 to reduce the risk of contaminating other patients 2. should relatives/visitors or other supportive personnel be allowed onto the orthopaedic wards and operating suite? until normality returns, the number of visitors should be minimized as much as possible to reduce potential transmission of sars-cov-2 among patients, their family members and medical staff. this includes vendor representatives who travel to facilities to undergo, perform and support procedures. limiting interactions between individuals and social distancing are part of the mainstream management against the spread of sars-cov-2. the number of visitors should be limited to those who are essential for a patient's care. although it is recognized that these rules create considerable anxiety for the patient, keeping all patients, relatives and staff safe from sars-cov-2 is the priority 3. is there a need for extra disinfecting environmental procedures in between surgeries? standard protocols for cleaning and sterilizing instruments need to be meticulously applied moreover, once the patient has departed from the operating room, this should be left empty for a specific period of time and all high-touch surfaces, including the anaesthetic machine and the anaesthetic work area, should be cleaned and disinfected with an environmental protection agency (epa)-approved hospital disinfectant. the length of time in between patients depends on the number of air exchanges per hour in the room or space in question 4. what role do ultra-clean operating theatres, orthopaedic exhaust suits and ultraviolet light systems play in both the wards/beds and the theatre? elective orthopaedic theatres and operative procedures, including personal protective equipment, are designed to reduce the risk of surgical site infection. there are no guidelines or protocols published for managing elective hip and knee arthroplasty procedures with respect to laminar flow in the presence of sars-cov-2. it is recommended that surgical helmets not be used as primary protection against aerosol and airborne disease. the surgeon should consider wearing an n95 mask as an added precaution when using a surgical helmet in patients considered to be sars-cov-2-negative in the pre-operative work-up ultraviolet light has no evidence to support its routine use and poses a hazard to operating staff the survey has shown good-to-excellent agreement of the participants with regards to the statements made in the recommendations for the safe return to elective arthroplasty following the first wave of the covid-19 pandemic. during hip and knee arthroplasty, the use of power tools, burrs or electrocautery generates potentially infective aerosol. the major aim should be to avoid transmission of sars-cov-2 by aerosolization of blood or other body fluids. hence, adequate personal protective equipment should be available and used during surgery. first and foremost, all patients undergoing elective surgery should wear a mask. surgeons and the entire surgical team who scrub during procedures should ideally wear an exhaust suit, including a mask (preferably n95, filtering face piece [ffp2, or p3] and a face shield). in the absence of a face shield, protective eyewear may be used, but this is a compromise. in addition, scrubs should be frequently changed during the surgical day. single-use gowns, single-use gloves and hair and shoe covers can also, theoretically, reduce the transmission of the virus resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic: post-operative phase 1. since the impact on mind and body during joint arthroplasty is significant, our patients may have lower immunity and be more vulnerable to sars-cov-2 infection as sars-cov-2-positive is an absolute contra-indication for elective surgery, the standard enhanced recovery protocol for reducing complications is imperative 2.organization of a shelter-in-place post-operative period, home care, community nurses or informal care ideally, patients should be discharged home with the standard sars-cov-2 precautions being taken and only transferred to a nursing home in cases where that is not possible, since higher rates of sars-cov-2 may exist in those facilities 3. avoid face-to-face contact for the orthopaedic follow-up and physical therapy guidance -consider telemedicine web-based tools and telemedicine are preferred during this sars-cov-2 pandemic and may become the standard in the post-pandemic era -consider a new approach in wound closure technique in the setting of the sars-cov-2 pandemic post-operative in-office visits should be minimized, and digital health programmes allowing health care providers to closely monitor patients remotely should be supported. self-administered wound management should become the standard of care for patients in the post-pandemic era 4. what is the advice for patients who develop sars-cov-2 symptoms? in the event a patient develops sars-cov-2 symptoms or comes into contact with a sars-cov-2-positive person, they should seek health care advice. the patient should use the system in place in their country to seek the relevant advice. this may be through their primary care physician or an online or telephone advice service. their orthopaedic surgeon and hospital should also be notified 5. are special adjustments needed for post-op sars-cov-2 prophylaxis? since sars-cov-2-positive is an absolute contraindication or elective surgery, the standard enhanced f recovery protocol and sars-cov-2 preventive measures are imperative. time allowing, all surgical patients should apply social distancing principles for the first two weeks after the operation and self-quarantine in their home open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic: the european hip society and european knee associates recommendations. knee surg sports traumatol arthrosc publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest nc has provided consultancy services to stryker netherlands (amsterdam), zimmer-biomet (warsaw indiana) andbodycad. (naples usa) no other conflicts of interest have been recorded.funding no funding has been provided for this study.ethical approval this survey did not require formal ethical approval with a practice dedicated to adult reconstruction. (13) 59 (10) 585 (86) 5. what is the impact of delaying hip and knee arthroplasty for the patients themselves and for society?530 (92) 27 (5) 19 (3) 576 (85) resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic: preoperative phase 1. what is the appropriate preoperative clinical and laboratory screening and timeline for patients? 435 (80) 80 (15) 31 (6) 546 (80) 2. is preoperative tracking of patients, staff and relatives necessary?379 (71) 121 (23) 36 (7) 536 (79) 3. should spinal anaesthesia be routine? 420 (79) 74 (14) 40 (8) 534 (78) -should we only select patients as candidates who also consent to regional/spinal anaesthesia?383 (72) 111 (21) 36 (7) 530 (78) -if spinal anaesthesia does not occur, should we cancel the operation?384 (73) 108 (20) 37 (7) 539 (78) 4. if the sars-cov-2 tests are positive, how long should the hip or knee arthroplasty surgery be postponed?474 (90) 29 (6) 24 (5) 527 (77) 5. what kind of back-up plan is needed to guarantee quality care and patient safety in this pandemic situation?476 (92) 19 (4) 24 (5) 519 (76) resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic: perioperative phase 1. is there a need for -modification or reorganization of hospital wards (patient density, bed density, medical and nursing stuff density, etc.)?417 (81) 70 (14) resuming elective hip and knee arthroplasty in the setting of the sars-cov-2 pandemic: post-operative phase 1. since the impact on mind and body during joint arthroplasty is significant, our patients may have lower immunity and be more vulnerable to sars-cov-2 infection 427 (88) 32 (7) 24 (5) 483 (71) 2.organization of a shelter-in-place post-operative period, home care, community nurses or informal care 419 (87) 36 (8) 26 (5) 481 (71) 3. avoid face-to-face contact for the orthopaedic follow-up and physical therapy guidance -consider telemedicine 310 (65) 130 (27) 40 (8) 480 (70) -consider a new approach in wound closure technique in the setting of the sars-cov-2 pandemic 299 (62) 141 (29) 39 (8) 479 (70) 4. what is the advice for patients who develop sars-cov-2 symptoms? 455 (95) 11 (2) 13 (3) 479 (70) 5. are special adjustments needed for post-op sars-cov-2 prophylaxis? 362 (76) 86 (18) 31 (7) 479 (70) key: cord-284867-p4jgyusp authors: schöler, lara; le-trilling, vu thuy khanh; eilbrecht, mareike; mennerich, denise; anastasiou, olympia e.; krawczyk, adalbert; herrmann, anke; dittmer, ulf; trilling, mirko title: a novel in-cell elisa assay allows rapid and automated quantification of sars-cov-2 to analyze neutralizing antibodies and antiviral compounds date: 2020-10-09 journal: front immunol doi: 10.3389/fimmu.2020.573526 sha: doc_id: 284867 cord_uid: p4jgyusp the coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is currently the most pressing medical and socioeconomic challenge. constituting important correlates of protection, the determination of virus-neutralizing antibodies (nabs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. in contrast to standard serological elisas, plaque reduction neutralization tests (prnts) are laborious, time-consuming, expensive, and restricted to specialized laboratories. to replace microscopic counting-based sars-cov-2 prnts by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated sars-cov-2 neutralization assay employing an in-cell elisa (icelisa) approach. after optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, sars-cov-2-infected cells became amenable as direct antigen source for quantitative icelisa. antiviral agents such as human sera containing nabs or antiviral interferons dose dependently reduced the sars-cov-2-specific signal. applying increased infectious doses, the icelisa-based neutralization test (icnt) was superior to prnt in discriminating convalescent sera with high from those with intermediate neutralizing capacities. in addition, the icnt was found to be specific, discriminating between sars-cov-2-specific nabs and those raised against other coronaviruses. altogether, the sars-cov-2 icelisa test allows rapid (<48 h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of nabs and antiviral drugs using reagents and equipment present in most routine diagnostics departments. by the time of writing, more than 29 million people experienced a laboratory confirmed infection with the severe acute respiratory syndrome coronavirus (sars-cov)-2 and more than 930,000 people died while having coronavirus infectious disease . first surveillance studies and calculations of excess mortality rates indicate that the precise number of infections and the true number of fatalities exceed above-mentioned numbers by far. coronaviruses (covs) are positive strand rna viruses widespread among various vertebrate hosts including bats and rodents (1) . together with four seasonal human covs (hcovs) and the two other emerging hcovs sars-cov-1 and mers-cov, sars-cov-2 is the seventh hcov causing widespread human diseases (2) . in december 2019, sars-cov-2 was first recognized in the hubei province in china (3) from where it rapidly spread throughout the world. in addition to its genetic similarity, sars-cov-2 not only shares some clinical characteristics with sars-cov-1 (4) but also exhibits some highly relevant particularities such as an increased spreading efficacy and the length of the course of disease (5) . on january 31, the who declared the sars-cov-2 outbreak a public health emergency of international concern. on march 11, 2020 , who started to denote the outbreak a global pandemic. the center of the pandemic shifts between regions, posing danger of repetitive local and temporal reintroduction circles. thus, even countries that managed the first wave must prepare in terms of diagnostics capacities for potential future re-emergences. most sars-cov-2 infections lead to mild or moderate illnesses. however, a considerable fraction of cases proceeds to severe pneumonia or life-threatening acute respiratory distress syndrome. elderly individuals and people with pre-existing comorbidities such as impaired immunity, chronic respiratory diseases, cardiovascular diseases, and cancer are more prone to suffer from severe covid-19. the case fatality rate (cfr) is difficult to calculate in the midst of the pandemic. depending on the age, cfr estimates of up to 18.4% for individuals older than 80 years and 1.38% (range 1.23-1.53) for the general population have been reported (6) . given the extent, pace, and severity of the covid-19 pandemic, diagnostics departments even in countries with highly developed medical systems struggle to provide sufficient and timely test capacities. since nucleic acid-based pathogen detection has a very short window of opportunity, assays detecting long-lasting immune responses such as antibodies are required to monitor virus spread and to estimate potential herd immunity. with some delay, most infected individuals raise a detectable humoral immune response including specific immunoglobulins (ig) m, iga, and igg (7) (8) (9) . neutralizing antibodies (nabs) bind and abrogate the function of viral proteins such as the sars-cov-2 spike (s) protein that are essential for virus entry into host cells, e.g., through recognition of the cognate receptor ace2 (10) . accordingly, monoclonal nabs exhibit strong therapeutic and prophylactic efficacies in sars-cov-2-infected human ace2-transgenic mice (11) . a recent vaccination study conducted in non-human primates identified nabs as correlate of protection (12) , indicating that a human sars-cov-2 vaccine should also be capable to elicit potent nab responses. accordingly, first human vaccine studies focussed on the question if nabs were induced (13) . additionally, monoclonal nabs and nab-containing hyper-immunoglobulin preparations may be applicable as treatment against covid-19 (14) . nabs are also the backbone of convalescent plasma (cp) therapies (15) (16) (17) that are one of seven clinical recommendations of the idsa (18) . based on the havoc covid-19 causes to the global economy, immunity passports and vaccination certificates, documenting protection through nabs, have been discussed [e.g., (19) ]. taken together, sars-cov-2-specific nabs and their quantification appear to be of central importance for the medical and socio-economic management of the pandemic. different types of neutralization tests (nt) have been developed for sars-cov-2. however, to our knowledge, these assays rely on laborious microscopic counting of virus plaques or antibody-stained foci by trained personnel (20) (21) (22) or on genetically modified viruses such as transgenic sars-cov-2 mutants (23) or pseudo-typed viruses [e.g., vesicular stomatitis virus (vsv) or human immunodeficiency virus (hiv)] expressing the s protein of sars-cov-2 (24, 25) . genetically modified viruses are generally prohibited in routine diagnostics laboratories and usually not applicable in less developed regions. due to the central importance of nabs and the limitations of the currently available methodology, we established a cheap, simple, fast, reliable, and automatable in-cell elisa (icelisa)-based icnt applicable in routine diagnostics departments with access to bsl3 laboratories. caco-2 (atcc htb-37) and vero e6 (atcc crl-1586) were cultivated in roswell park memorial institute 1640 [rpmi-1640 (gibco 21875-034)] and high glucose dulbecco`s minimal essential medium [dmem (gibco 41966-029)], respectively, supplemented with 10% (v/v) fcs, penicillin, and streptomycin at 37°c in an atmosphere of 5% co 2 . sars-cov-2 was isolated from a patient sample using vero e6 and confirmed by sars-cov-2 diagnostic qrt-pcr. viral titers were determined by tcid50 titration. human ifna2 and ifnb were purchased from pbl assay science (#11101) and peprotech (#300-02bc), respectively. the collection of serum samples and the virus isolation have been approved by the ethics committee of the medical faculty of the university of duisburg-essen (20-9208-bo, 20-9511-bo, and 20-9512-bo). anti-sars-cov-2 igg antibodies were detected using the elisa detecting antibodies recognizing the sars-cov-2 spike protein (euroimmun medizinische labordiagnostika, germany) according to manufacturer's instructions. for the analysis of neutralizing antibodies, serum samples were inactivated at 56°c for 30 min. a detailed icelisa/icnt protocol is provided in supplementary file 1. briefly, defined doses of sars-cov-2 were incubated with different serum dilutions for 1 h at 37°c prior to vero e6 infection. at 16-24 h p. i., cells (~5 × 10 4 /well of a 96-well plate) were fixed with 4% (w/v) paraformaldehyde/pbs. cells were permeabilized with 1% (v/v) triton-x-100/pbs and blocked with 3% (v/v) fcs/pbs. the primary antibody was added and incubated for 2 h at room temperature or overnight at 4°c. peroxidase-labelled secondary antibody was incubated for 1-2 h. washing steps were performed with 0.05% (v/v) tween-20/pbs. tetramethylbenzidin (tmb) substrate was added to visualize the enzyme reaction. the reaction was stopped with 0.5 m hcl. subsequently, the absorbance was measured using a microplate multireader (mithras2 lb 943; berthold technologies). the a-n mab1 (abin6952435), a-n mab2 (abin6952433), a-e ab (abin1031551), and pod-coupled secondary antibodies (dianova) were used. in the case that an absolute quantification is necessary, the relative quantification of the icelisa can be transformed into an absolute quantification in terms of the virus dose using virus calibration curves (like in figure 1 ) or using commercially available recombinant n preparations. immunoblotting was performed as described previously (26) using antibodies recognizing the sars-cov-2 n protein (abin6952435) or gapdh (sc-25778, santa cruz). lysates were inactivated by sequential heat incubation (10 min at 70°c and 10 min at 95°c) before they were discharged from the bsl3 laboratory. proteins were visualized using peroxidase-coupled secondary antibodies (dianova) and the ecl chemiluminescence system (cell signaling technology). we hypothesized that virus-encoded proteins expressed by infected cells should be amenable as source of viral antigens for the detection and quantification by elisa. we optimized the experimental conditions such as cell type, virus dose, infection period, cell fixation method, blocking reagent, amd type and concentrations of primary and secondary antibodies ( figure 1 and data not shown). as described in the materials and methods section and provided as detailed laboratory protocol in the supplementary information (supplementary file 1), we compared different commercially available antibodies for the icelisa-based quantification of sars-cov-2 proteins. based on existing data on virus entry (10), we infected human caco-2 cells with graded virus doses (ranging from 0.03125 to 2 pfu/cell). at 3 days post infection (d p.i.), we fixed and stained the cells with different antibodies either recognizing the e or the n protein of sars-cov-2. in accordance with high expression level of the n protein (27, 28) , certain n-specific mouse iggs exhibited a signalto-noise ratio favourable for icelisa ( figure 1a) . a comparison of vero e6 and human caco-2 cells revealed that the icelisa is applicable to different cells ( figure 1b) . since the icelisa signal directly correlated with viral replication and viral antigen expression, we tested its ability to determine antiviral effects. human caco-2 cells were treated with graded concentrations of human interferon (ifn) a2 or ifnb and infected 3 h later with sars-cov-2. at 3 d p.i., viral antigen amounts were quantified by icelisa. in accordance with a recent clinical phase 2 trial (29), ifnb exhibited strong and dosedependent antiviral activity against sars-cov-2 in human cells ( figure 1c) , indicating that the icelisa is applicable for future experiments addressing the efficacy of potential antiviral drugs in human cells. despite different start mois, similar icelisa signals were observed at 72 h p.i. in vero e6 ( figure 1b) , indicating multiple rounds of virus amplification and extraordinary fast replication kinetics of sars-cov-2 in vero e6 cells, consistent with previous studies (30) . to test if shorter infection periods might result in virus dose-dependent signals, we analyzed infected vero e6 cells after 6, 15, and 22 h. sars-cov-2 was readily detectable in vero e6 cells by icelisa already at 6 h p.i. (figures 1d-f) . to evaluate if the icelisa faithfully reports on the amounts of sars-cov-2-expressed antigens, we infected cells with graded infectious doses and performed immunoblots and icelisas in parallel. as expected, icelisa and immunoblot signals correlated very well ( figure 2a ). since we observed icelisa signals already at 6 h p.i. (figures 1a, d) and the n protein is an abundant component of sars-cov-2 particles (31) (32) (33) , the n protein derived from input viruses could have contributed to icelisa signals. to evaluate this, we infected cells in the presence and absence of the translation inhibitor cycloheximide ('chx'). consistent with the notion that the icelisa signal is dominated by de novo n protein expression, the signal was significantly diminished by chx ( figure 2b) . taken together, these data indicate that the icelisa allowed rapid identification and relative quantification of sars-cov-2 replication in vero e6 and human cells and its inhibition by antiviral compounds. since the icelisa allowed simple and automated quantification of sars-cov-2-dependent antigen expression, we tested if an icelisa-based neutralization test (icnt) can be established. we infected vero e6 cells for 6, 15, and 24 h with graded sars-cov-2 infection doses (500, 5,000, or 50,000 pfu/well) in the absence or presence of two convalescent sera in three different concentrations (1/20, 1/40, and 1/80 dilution). using the high infectious dose, viral antigens became dose dependently detectable by icelisa as early as 6 h p.i. ( figure 3a ). both infection. even the strong icelisa signal at 24 h p.i. was dose dependently neutralized by both sera ( figure 3f ). please note that the neutralizing capacity of given sera dilutions was less pronounced at higher virus doses as compared to lower virus doses, as indicated by residual icelisa signals. taken together, the icelisa resulted in a time-and virus dose-dependent signal constituting a surrogate for sars-cov-2 infection and replication. the fact that the infection and the resulting icelisa signal were neutralized by nabs present in immune sera indicated that the fast and automated icelisa format is applicable for icnts. although most sars-cov-2 nts have not been formally validated and certified, classic plaque reduction neutralization tests (prnt) are currently considered to represent the gold standard for the detection of sars-cov-2-specific nabs. various commercially available igm, iga, and igg elisas have been compared to prnts [e.g., (30) ]. to validate the novel icnt, 53 sera-24 positive for sars-cov-2-specific igg as determined using the euroimmun elisa (elisa ratio: 1.13-9.92) and 29 elisa-negative sera (elisa ratio < 0.9)-were compared side-by-side by icnt and standard prnt using 200 pfu/well ( figure 4a ). one set was processed by icnt, while for the other set virus foci were stained using an antibodybased aec staining method and manually counted by microscopy. standard sars-cov-2 nts base on microscopic counting of plaques or antibody-stained virus foci. to enable plaque/foci recognition and individual counting by trained personnel, a countable number of pfu must be applied in prnts. depending on the prnt protocol, 100 (20) to 400 pfu (21) are used to infect each well. based on previous experiences with virus neutralization experiments (34, 35) , we suspected that lower infectious doses might be more susceptible to nabs than higher virus doses, e.g., through altered ratios of nabs and antigenic regions determining neutralization, such as the receptor binding domain (rbd) of the s protein of sars-cov-2. accordingly, graded infectious doses (1,250-10,000 pfu per well) showed virus dose-dependent susceptibilities to the same serum sample (supplementary figure s1 ). before we assessed clinical specimens, we compared icnt signals upon a high moi infection with viral progeny as determined by parallel tcid50 titration. as expected, we observed that icnt signals and residual virus numbers correlated very well (supplementary figure s2) . figures 5a-c) , the neutralizing capacity of the same sera considerably differed at the more restrictive high virus dose ( figures 5d-f infections of cell cultures with well-defined virus preparations in the presence or absence of compounds that may impair the infection. thus, the nt specificity results from the choice of virus applied to the cells. however, antiviral compounds can either be specific for one particular virus species (e.g., most monoclonal neutralizing antibodies), broadly active against virus families (e.g., receptor blocking agents) or even bigger genera (e.g., hyperimmunoglobulin preparations). in addition to sars-cov-2, other hcovs such as the alphacoronaviruses hcov-229e and hcov-nl63 and the betacoronaviruses hcov-oc43 and hcov-hku1 circulate autochthonously in the human population, resulting in high sero-prevalence rates (36) . to address if the experimental sars-cov-2 infection in the icnt might be diminished by cross-reactive antibodies, we tested sera of individuals who have had infections with the alphacoronaviruses hcov-229e or hcov-nl63 or betacoronaviruses such as hcov-hku1 ( figure 6a ) or a combination of hcov-229e and hcov-oc43 ( figure 6b) . in contrast to the positive control containing sars-cov-2-specific nabs, all tested serum samples recognizing other coronaviruses did not neutralize sars-cov-2. although the limited sample size prevents definitive conclusions regarding crossneutralizing capacities of antibodies raised by other hcovs, in conjunction with consistent publications (37, 38) it indicates the specificity of the icnt assay. we established a novel icelisa-based test principle for detection and relative quantification of sars-cov-2. given the excellent signal-to-noise ratio between infected and uninfected cells, the test was applicable to quantify the efficacy of antiviral compounds, here shown for ifnb, and sars-cov-2-specific nabs present in immune sera. compared to icelisa and icnt, standard virus titrations and prnts are far more laborious, time consuming, and expensivenot to speak from subjective and expectation biases upon usage for research. the entire icnt can be processed in less than 2 days including the infection period. given that the protocol includes an early fixation step using 4% (w/v) paraformaldehyde which inactivates sars-cov-2, all subsequent steps can be processed outside the biosafety level 3 laboratory. the actual data acquisition is conducted within seconds, using standard elisa plate readers present in most routine diagnostics departments. we believe that this is advantageous compared to other nt assays that rely on more sophisticated and more expensive devises, e.g., for imaging cytometry (39) . the icelisa and icnt provide increased data quality and precision by generating continuous data sets. since the detection antibodies can be applied in icelisa and icnts in relatively high dilutions (1/5,000 to 1/10,000 and 1/2,000 of primary and secondary antibody, respectively), the assay is relatively cheap (in our case, around 0.10 € per well for both antibodies and the tmb peroxidase elisa substrate). the specificity of the icelisa and icnt is provided by the defined sars-cov-2 added to the cell cultures on purpose. the primary and secondary detection antibodies just serve to visualize and quantify viral antigens. thus, sars-cov-2-specific antibodies can be applied for icelisa detection notwithstanding potential cross-recognition of other covs such as hcov-hku1 or hcov-oc43 -simply because these viruses are not present in the culture. obviously, such antibodies recognizing conserved residues cannot be used for classic antigen-recognizing elisas due to their inability to discriminate coronaviruses. more virus (>1pfu/cell) can be applied to icnts. such high pfu icnts scrutinize the virusneutralizing capacity of sera more strictly, enabling a higher resolution compared to prnt assays that all rely on low virus numbers. it is tempting to speculate that sera exhibiting superior neutralization in high pfu icnt might be more beneficial in cp therapies. nts based on pseudo-typed viruses using heterologous expression of the sars-cov-2-encoded s by non-related viruses may have certain advantages. however, genetically modified viruses are inapplicable by law in various routine diagnostics departments and usually unavailable in less developed countries. recently, an elegant surrogate assay has been designed that determines if antibodies are capable to prevent the interaction of immobilized hace2 with a reporter enzyme-coupled receptor binding domain (rbd) of s (40) . this assay showed a good correlation with conventional nts (r 2 = 0.8591; p value < 0.0001) and nts based on pseudo-particles (r 2 = 0.8374). however, without the use of genuine infectious viruses, assays relying on pseudo-typed viruses do not fully interrogate the full spectrum of antiviral effects for example if other viral proteins influence the system, e.g., by complement activation (41) . accordingly, the icnt showed a superior correlation compared to prnt (r 2 = 0.987; p value 5.81e-50). taken together, we propose the icelisa and icnt for the quantification of sars-cov-2 replication and its inhibition by nabs and antiviral compounds. by changing the detection antibody and, if necessary, the cells according to the viral infection system, the test principle is transferable to all other viruses. all datasets presented in this study are included in the article/ supplementary material. the studies involving human participants were reviewed and approved by ethics committee of the medical faculty of the university of duisburg-essen (20-9208-bo, 20-9511-bo, and 20-9512-bo). the patients/participants provided their written informed consent to participate in this study. ls, vtkl-t, me, and dm performed research. oa, ak, and ah provided essential reagents. ls, vtkl-t, and mt analyzed the data. ls, vtkl-t, ud, and mt interpreted the data. vtkl-t and mt supervised the project. ls, vtkl-t, and mt wrote the manuscript. all authors contributed to the article and approved the submitted version. the authors received support by the stiftung universitätsmedizin essen, kulturstiftung essen, the else-kröner promotionskolleg elan, and the deutsche forschungsgemeinschaft (dfg) through grants rtg 1949/2, tr1208/1-1, and tr1208/2-1. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. vtkl-t and ls contributed equally to this work. we thank benjamin katschinski and kerstin wohlgemuth for excellent technical assistance. mt was supported by the stiftung universitätsmedizin essen, kulturstiftung essen, the else-kröner promotionskolleg elan, and the deutsche forschungsgemeinschaft (dfg) through grants rtg 1949/2, tr1208/1-1, and tr1208/ 2-1. this manuscript has been released as a pre-print at biorxiv, (42) . the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.573526/ full#supplementary-material supplementary figure 1 | the infectious virus dose strongly influences the neutralization capacity of serum samples. graded amounts of sars-cov-2 were incubated with indicated dilutions of a serum sample (chosen to exhibit a low neutralizing capacity) for 1 h before vero e6 cells were infected. neutralization was evaluated by icelisa. the results of the high moi icnt correlate with residual virus after neutralization. sars-cov-2 was incubated with indicated dilutions of serum samples for 1 h before vero e6 cells were infected. (a) neutralizing capacity was evaluated by icelisa. 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injury by masp-2-mediated complement over-activation. medrxiv (2020) a novel in-cell elisa assay allows rapid and automated quantification of sars-cov-2 to analyse neutralizing antibodies and antiviral compounds the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 schöler, le-trilling, eilbrecht, mennerich, anastasiou, krawczyk, herrmann, dittmer and trilling. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-324856-hf969tav authors: abir, tanvir; kalimullah, nazmul ahsan; osuagwu, uchechukwu levi; yazdani, dewan muhammad nur -a.; mamun, abdullah al; husain, taha; basak, palash; permarupan, p. yukthamarani; agho, kingsley e. title: factors associated with the perception of risk and knowledge of contracting the sars-cov-2 among adults in bangladesh: analysis of online surveys date: 2020-07-21 journal: int j environ res public health doi: 10.3390/ijerph17145252 sha: doc_id: 324856 cord_uid: hf969tav this study investigated the perception and awareness of risk among adult participants in bangladesh about coronavirus disease 2019 (covid-19). during the lockdown era in bangladesh at two different time points, from 26−31 march 2020 (early lockdown) and 11−16 may 2020 (late lockdown), two self-administered online surveys were conducted on 1005 respondents (322 and 683 participants, respectively) via social media. to examine risk perception and knowledge-related factors towards covid-19, univariate and multiple linear regression models were employed. scores of mean knowledge (8.4 vs. 8.1, p = 0.022) and perception of risk (11.2 vs. 10.6, p < 0.001) differed significantly between early and late lockdown. there was a significant decrease in perceived risk scores for contracting sars-cov-2 [β = −0.85, 95%ci: −1.31, −0.39], while knowledge about sars-cov-2 decreased insignificantly [β = −0.22, 95%ci: −0.46, 0.03] in late lockdown compared with early lockdown period. self-quarantine was a common factor linked to increased perceived risks and knowledge of sars-cov-2 during the lockdown period. any effort to increase public awareness and comprehension of sars-cov-2 in bangladesh will then offer preference to males, who did not practice self-quarantine and are less worried about the propagation of this kind of virus. this may contribute to a big economic tragedy in a country such as bangladesh, which is still attributed to everyday wages, as seen in other heavily affected regions of the world. since the sheer illness of the whole country is sufficient to destroy the health care system, this current study is to examine changes of individual perception of risk for contracting sars-cov-2, and the awareness level in bangladesh during the early and late lockdowns implemented by the government of bangladesh. the findings of this study will provide an understanding of people's knowledge level, perception of risk and awareness which can be used to implement emergency policies to counter the spread of sars-cov-2. from 26 to 31 march 2020, the first cross-sectional survey entitled "early lockdown" was performed, referring to the week of the lockdown period in bangladesh and the second cross-sectional survey entitled "late lockdown" was carried out from 11 to 16 may 2020. even though a national community-based sampling survey throughout that time was not conceivable, the data were collected electronically using a google form. a standardized synchronized questionnaire was uploaded on social networking sites, such as facebook and whatsapp, which are widely used by investigators and local people throughout the country. emails with the survey link were sent in the second step via contact lists of the researchers to broaden the scope of the survey. participants in the survey received no incentives. the first survey (early lockdown) assumed a 50% proportion with 90% confidence. because the main objective of this research was on sars-cov-2 and there are no previous studies from bangladesh that examined factors associated with this, an online sample size calculator [21] was used and we took a sample size of approximately 300, including a 10% non-response rate. the second survey (late lockdown) assumed a proportion of 31% (very worried about sars-cov-2) reported in the first study (early lockdown) with 90% confidence [21] . the calculation of the total sample size for the second survey was 710, including a 10% non-response rate. the participants responding to a "yes" or "no" question obtained voluntary on-line consent to express their willingness to attend the study via google forms. this study was approved by the ethics committee (approval number: brur/dwrti/a.n.003) of the dr wazed research and training institute, begum rokeya university, rangpur. rangpur-5404, bangladesh. table 1 presents the questionnaire used in this study. the questionnaire was divided into three sections, including demographics, knowledge, and perception. the demographic variables included age, gender, marital status, education, employment, and religion. there were 12 items on the questionnaire that assessed the respondent's knowledge of covid-19, most of which required a "yes" or "no" response. each question used a binary scale. the scores for each item ranged from 0 (no) to 1 (yes). the knowledge score ranged from 0-12 points. these items have been validated elsewhere to have an acceptable internal consistency [22] . the survey tool for the covid-19 knowledge questionnaire was developed based on the guidelines from the world health organization [5, 23] for clinical and community management of covid-19. those that have contact with someone who has covid-19 infection should be isolated in the right place immediately. the observation period is usually 14 days k10 children and young adults should not take steps to prevent the covid-19 virus from infection. k11 covid-19 individuals with no symptoms of fever cannot spread the virus to anyone k12 individuals should stop being crowded to prevent covid-19 infection. please rate your chances of personal risk of infection with covid-19 for each of the following? risk of becoming infected. risk of becoming severely infected p3 risk of dying from the infection p4 how much worried are you because of covid-19? are you currently or have you been in (domestic/home) quarantine because of covid-19? how do you feel about the quarantine? i am worried/anxious/alarmed and frightened by the quarantine. i consider the quarantine as necessary and reasonable. i am nervous about the quarantine. i am bored by the quarantine. i am frustrated by the quarantine. i am angry because of quarantine. revised and adopted from world health organization, 2019: available at https://www.who.int/bulletin/online_first/ 20-256651.pdf). we asked the respondents about risk perception towards covid-19 (p1−p4). each question used a likert scale with five levels. the scores for each item ranged from 1 (lowest) to 5 (highest). the risk perception score ranged from 5 to 20 points. the cronbach's alpha coefficients of the perception items were 0.74 and demonstrated that the internal consistency of perception items was satisfactory. respondents were also asked, "how they felt about the quarantine" (p6−p11). each question used a likert scale with five levels. the scores for each item ranged from 1 (lowest) to 5 (highest) and the cronbach's alpha coefficient of the "how they felt about the quarantine items" was 0.70, indicating acceptable internal consistency. the explanatory (independent) variable included basic characteristics and explanatory factors including gender, age in categories, level of education, marital, employment, and religious status. the question of worrying about quarantine score ranged from 6 to 30 points. the worried about quarantine score was divided into three categories. the bottom 33.3% of the score was arbitrarily referred to as "low quarantine practice", the next 33.3% as "moderate quarantine practice", and the top 33.3% as "high quarantine practice". furthermore, "high quarantine practice", which was derived by combining the moderate quarantine practice (33.3%) with the high quarantine practice (33.3%) and low quarantine practice, was "low quarantine practice scores" 33.3%. data analysis was performed using stata version 14.1 (stata corp. college station united states of america). categorical variables were presented as frequency and percentage. this study used a t-test to compare the differences between means for early and late lockdowns for knowledge and risk perception items. in the univariate linear regression analysis, all confounding variables with a p-value < 0.20 were retained and used to build a multivariable linear regression model and to determine factors associated with the knowledge and perception score towards covid-19. additionally, we performed a similar stage modelling to that employed by dibley et al. [24] , and a two-staged modelling technique was employed in the multivariable modelling. in the first stage, the demography factors were entered into the baseline multivariable model. a manual process of backward elimination was performed, and variables with p < 0.05 were retained in the first model (model 1). in the second and final stage of modelling, perceived risk of covid-19 factors was added into significant variables in model 1, and variables with p-values < 0.05 were retained in the final model. for all regression analyses performed, we checked the homogeneity of variance and multicollinearity using variance inflation factors (vif). the descriptive statistics of the explanatory and dependent variables are shown in table 2 . this summary of responses was obtained from those who participated in the survey during the early lockdown (26-31 march 2020) and late lockdown (11) (12) (13) (14) (15) (16) may 2020) periods. total responses were a combination of both. most of the respondents (53.2%, n = 532) were 18-28 years old with equal representation of males and females. most respondents (58.2%, n=585) were married, and almost all (83.1%, n = 835) completed tertiary education or its equivalent. of the respondents, 88.8% (n = 892) were muslims, about two-thirds of them (65.5%, n = 658) voluntarily quarantined themselves during the study period while about a quarter of them (19.2%, n=193) did not quarantine. regarding their concern for the spread of the sars-cov-2 virus, the majority (68.7%, n = 690) stated that they were very worried. figure 1a,b show the mean and 95% confidence intervals of perceives risk and knowledge towards covid-19, respectively. data of early and late lockdown periods are presented here, correspondingly. figure 1a indicates statistical differences between early and late lockdowns (p < 0.001), with early lockdown reporting the highest mean values. additionally, as indicated in figure 1b , knowledge towards covid-19 for early lockdown significantly reported the highest mean value compared with late lockdown (p = 0.022). the horizontal values in figure 1a ,b are the minimums and maximums of perceived risk and knowledge scores. the unadjusted and adjusted coefficients for factors associated with the perceived risk of contracting sars-cov-2 are presented in table 3 . compared with the early lockdown period, the results indicated that perceived risk scores for contracting covid-19 in late lockdown period reduced significantly (adjusted coefficients (β) −0.85, 95% ci:−1.31, −0.39). other factors associated with perceived risk scores for contracting covid-19 are females, practised high quarantine, very worried about covid-19, and quarantined at the request of public health order during the lockdown period. age stratification was significant in the univariate analysis and the final model, we removed religion and replaced it with age stratification, and the result showed that age stratification was not statistically significant (wald χ 2 = 0.46, p = 0.7137) and similarly, when gender was replaced with age stratification, age stratification was not significant (wald χ 2 = 0.49, p = 0.6908). the factors associated with perceived risk scores for contracting covid-19 in early lockdown and late lockdown period are presented in tables a1 and a2. towards covid-19, respectively. data of early and late lockdown periods are presented here, correspondingly. figure 1a indicates statistical differences between early and late lockdowns (p < 0.001), with early lockdown reporting the highest mean values. additionally, as indicated in figure 1b , knowledge towards covid-19 for early lockdown significantly reported the highest mean value compared with late lockdown (p = 0.022). the horizontal values in figure 1a ,b are the minimums and maximums of perceived risk and knowledge scores. table 4 showed the unadjusted and adjusted coefficients with 95% confidence intervals (cis) of the knowledge level of covid-19. after the adjustment of potential confounding factors, knowledge about covid-19 has decreased but it was not statistically significant [β = −0.22, 95% ci: −0.46, 0.03] in late lockdown period compared to early lockdown period. additionally, comparatively less knowledge of covid-19 was pertinent among those who performed low quarantine and those who had less education (completed secondary or primary education only). increased knowledge of covid-19 was pertinent among the participants who practised high quarantine, held a bachelor and above degree, and the non-muslim participants. age stratification and employment status were significant in the univariate analysis and our final model, we removed religion and replaced it with age stratification, and age stratification was not statistically significant (wald χ 2 = 1.44, p = 0.2293) and when education was replaced with age stratification, age stratification was not significant (wald χ 2 = 2.54, p = 0.055), but when education was replaced by employment status, employment status was associated with increased knowledge of covid-19 [β = 0.26, 95% ci: 0.03, 0.49, p = 0.027 for those employed]. factors associated with the knowledge level of covid-19 for each lockdown periods are reported in tables a3 and a4. this current study reported a higher mean of perception of risk and low knowledge of contracting the sars-cov-2 among adults in bangladesh. the study also revealed factors associated with the perception of risk and knowledge of contracting the sars-cov-2 in bangladesh and found that females and those with a bachelor's degree reported decreased perceived risk and knowledge of contracting sars-cov-2 than males, and master's/higher degree holders, respectively, practised high quarantine, were very worried, and quarantined at the request of public health order during covid-19, and reported higher perceptive risk of contracting covid-19, while non-muslims (christian/hindu) practised high quarantine and quarantined at the request of public health order during covid-19, and reported increased knowledge scores of contracting the infection. the higher mean score of risk perceptions stated in this analysis could be because the bangladesh government has taken exceptional measures to track the rapid spread of the current global covid-19 disease outbreak [25] . when the number of individuals infected and the fatalities from this epidemic escalate, residents will stick to preventive measures because they are influenced by their knowledge, perceptions and practices towards this disease outbreak [26] . in this study, we analyzed the opinions of bangladeshi people about vulnerability and awareness towards covid-19 during the drastic rise period of the disease outbreak. researchers identified that many were extremely concerned about the transmission of the infectious disease in this predominantly well-educated young muslim population and more than one-third considered themselves to be at low risk of contracting the infection. such a high perception of low risk, coupled with the fairly average covid-19 knowledge scores, is extremely important because clear knowledge predicts a positive attitude and appropriate attitude against covid-19 [22] . in this study, males who were worried about contracting sars-cov-2 were more likely to perceive themselves as being at high risk of contracting the infection, as well as those who did not quarantine themselves or only did so at the request of the public health officers. these findings were similar to those reported in the studies conducted in india, china, and jordan. adults with a higher level of knowledge about covid-19 and who were in quarantine were more concerned about the infection and became frustrated as they did not know how long the impact of the pandemic would last [27] . moreover, in india, it was found that a higher level of knowledge on covid-19 was associated with the high-risk perception of contracting the infection during the consistent lockdown period [28] . in jordan [14] , it was found that, with adequate knowledge, people can perceive the importance of lockdown and the risk of contracting the infection caused by sars-cov-2. experience from previous similar virus attacks (sars-cov-1) in china highlighted the fact that, during such a crisis, people's knowledge, attitudes, and perceptions about the situation affects their response to the crisis. to effectively manage a health emergency, citizens need to be conscious of the problem, to be alert, and acknowledge their responsibilities to preserve their steadiness, because circumstances culminating in fear in the public can escalate the situation into misery [22] . a similar survey conducted to test the knowledge, attitudes, and perceptions of people in the hubei province, china, about the covid-19 outbreak found that higher knowledge, attitude and perception scores among residents was related to the ages and socioeconomic statuses of the respondents [22] . it was surprising to find an average score of knowledge against covid-19 among bangladesh residents, considering that this epidemiological survey was performed at the very early stage of the pandemic in bangladesh. we believe this to be partially attributed to the survey being skewed by people with a bachelor's degree or higher, the largest percentage of respondents being 86%. the magnitude of this pandemic and the unprecedented media attention of this public health disaster will have an important effect on people's awareness about this epidemic. television channels, bangladesh health ministry official websites, and all corporation websites had details about this infectious disease during this time. adults with higher levels of education are more likely to seek information which enhances a sense of personal control through mastering content and acquiring stronger skills [29] . similar to previous findings [22, [30] [31] [32] which suggested that men and young adults are more inclined to engage in risk-taking behaviours, the present study found a significant association between male gender and perceived high-risk of covid-19 among respondents after adjusting for other cofounders. adults who were employed at the time of this study were 0.6 times more likely to show adequate knowledge scores compared to those who were unemployed, but this association was significant only when it interacted with other demographic variables in the model. the slightly higher chances of sufficient information among citizens who did not quarantine themselves, relative to those who did so willingly, could be due to the less severe situation of the covid-19 outbreak in bangladesh and the prevalence of younger adults in this sample, resulting in respondents feeling that they had a lower probability of contamination with the sars-cov-2 virus. it is worth noting that, in this analysis, higher covid-19 awareness scores are strongly correlated with not becoming a practising muslim. it is understood that the negative mentality shown by certain religious manipulators is one of the toughest obstacles in attempts to tackle the dissemination of covid-19 awareness. while the government has called for the public to keep social distances to stop the gathering of crowds (physical distance), certain so-called religious leaders might also be preparing to host meetings involving hundreds. resistance from religious communities to physical isolating appeals has been observed across several predominantly muslim countries, such as indonesia, and the trend exacerbates local government attempts to negotiate with covid-19 propagation. research in turkey [33] echoed the significance of religious figures throughout this disease outbreak in positively motivating populations. although some practitioners preferred to seek counsel from their municipal officials, others adopted their religious leader's instructions when it came to debatable questions, such as covid-19, suggesting that religious leaders have strong influence on the respondent's attitude towards covid-19 mitigation practices during the pandemic. the finding of this study indicates the value of strengthening public health knowledge for bangladeshi citizens towards covid-19. this, in effect, would change behaviours and activities towards covid-19. research findings of the demographic variables correlated with knowledge towards sars-cov-2 are broadly compatible with previous research on sars-cov-1 in 2003 [16, 22] , further indicating that the intervention in health education towards covid-19 in bangladesh would become more successful if it had been primarily structured for mass people and those with low educational thresholds. since sars-cov-2 is a new type of coronavirus, and no pharmacologic therapies at this time are available, increased public awareness and caution seem to be the best approaches to preventing community spread. the travel bans and lockdowns placed in many countries, including bangladesh, may have worked, but they also raised the level of panic among residents. this was evident in this study, where approximately 31% of the respondents were very worried, and others were somewhat worried about the situation. in this situation, lai and others showed that educating the public is a very helpful and effective resource [34] . for countries with fragile health care systems who have dense populations, such as the sub-saharan african countries, lack of awareness about the virus and corrupt policies can combine to create a disaster that is impossible to contain [35] . in the case of covid-19, issues with the current response, lack of transparency, travel restriction delay, quarantine delay, public misinformation, and emergency announcement delay contributed to the outbreak. the findings of this study show that many of the respondents in bangladesh were very worried about the spread of covid-19 coupled with their significant inadequacies in the knowledge of the disease. this suggests the need for more awareness to increase public knowledge and reduce the worries of the bangladeshi people regarding the sars-cov-2 virus. in addition to adhering to the government recommendations of routine hand washing and home quarantine, older males of the muslim faith could be targeted to further improve the knowledge and avoid further transmission of this novel coronavirus, even as the lockdown continues. the current study provided the first evidence of the knowledge and perception of people using an appropriately sampled population during a critical period-the early stage of the covid-19 outbreak. however, the online nature of data collection meant that respondents who had an internet connection were more likely to participate, which may lead to bias, including selection bias because of the over-representation of well-educated people in bangladesh compared to the background population [36] and, as such, the findings may not represent the opinion of the less educated population. hence, findings from this study cannot be generalizable to the entire bangladeshi population and lack causal inference because it was an online cross-sectional design. despite this limitation, this was the only feasible way of data collection at the time of this study. additionally, since the virus is novel and already widespread, there is little possibility to undertake extensive social studies in bangladesh. another limitation of this study was the cross-sectional study design, making it impossible to determine causation. further studies across randomly selected populations across the country are needed to confirm these findings. such studies should also assess the social aspects of the condition. despite these limitations, the present study provides relevant information to fill research gaps in the fight for covid-19. the datasets analyzed during this study are available from the authors on reasonable request. deadliest enemy: our war against killer germs inhibition of sars-cov-2 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cross-sectional survey world health organization. covid-19 update iron and folic acid supplements in pregnancy improve child survival in indonesia the financial express who releases guidelines to help countries maintain essential health services during the covid-19 pandemic comparison of prevalence and associated factors of anxiety and depression among people affected by versus people unaffected by quarantine during the covid-19 epidemic in southwestern china study of knowledge, attitude, anxiety & perceived mental healthcare need in indian population during covid-19 pandemic examining associations between health information seeking behavior and adult education status in the us: an analysis of the 2012 piaac data sex differences in everyday risk-taking behavior in humans sex differences in risk taking behavior among dutch cyclists age patterns in risk taking across the world politics and the covid-19 pandemic: the turkish response severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and corona virus disease-2019 (covid-19): the epidemic and the challenges key: cord-321901-zpi7uis1 authors: roberts, anjeanette; wood, john; subbarao, kanta; ferguson, morag; wood, david; cherian, thomas title: animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting 25–26 august 2005, london, uk date: 2006-11-30 journal: vaccine doi: 10.1016/j.vaccine.2006.07.009 sha: doc_id: 321901 cord_uid: zpi7uis1 abstract severe acute respiratory syndrome (sars) emerged in the guangdong province of china in late 2002 and spread to 29 countries. by the end of the outbreak in july 2003, the cdc and who reported 8437 cases with a 9.6% case fatality rate. the disease was caused by a previously unrecognized coronavirus, sars-cov. drawing on experience with animal coronavirus vaccines, several vaccine candidates have been developed and evaluated in pre-clinical trials. available data suggest that vaccines should be based on the the 180kda viral spike protein, s, the only significant neutralization antigen capable of inducing protective immune responses in animals. in the absence of clinical cases of sars, candidate vaccines should be evaluated for efficacy in animal models, and although it is uncertain whether the united states food and drug administration's “animal rule” would apply to licensure of a sars vaccine, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. this report summarizes the recommendations from a who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on 25–26 august 2005 in south mimms, uk, provides guidance on the use of animal models, and outlines the steps to develop standard reagents and assays for immunological evaluation of candidate sars vaccines. severe acute respiratory syndrome (sars) is a severe respiratory illness caused by the sars coronavirus (sars-cov) [1] . the disease emerged in the guangdong province of china in late 2002 [2] and spread to 29 countries mostly within asia, although europe and north america were also affected, notably toronto, canada. the epidemic was finally controlled by july 2003 through strict implementation of prevalence for sars-cov antibody, although they had no history of sars-like disease. sars-cov-like viruses that were isolated from civets and raccoon dogs had more than 99% homology with human sars-cov, with major differences found in orf8, whose deletion has been suggested to represent a sign of adaptation to humans [12] . only four amino acid residues in the receptor glycoprotein ace2binding domain of the viral spike protein differ between the human epidemic sars-cov strains and civet strains, but they cause more than a 1000-fold difference in binding affinity to the ace2 molecule [13, 14] . although a high prevalence of sars-like coronaviruses were found in chinese horseshoe bats [15, 16] , their great genetic diversity makes it difficult to identify which one might be the ancestor of sars-cov and to decide with certainty whether bats indeed are the animal reservoir of the virus. sars-cov infection exhibits a wide clinical course characterized mostly by fever, dyspnea, lymphopenia and lower respiratory infection, often with concurrent gastrointestinal symptoms including diarrhea [17, 18] . pathology in sars patients has been associated with diffuse alveolar damage, epithelial cell proliferation and multinucleated giant cell infiltrates of epithelial or macrophage origin, suggestive of syncytium-like formation in the lung. the virus can be recovered from peripheral blood mononuclear cells, respiratory secretions, stools, urine and even sweat (for a review, see [19] ). sars vaccine development efforts were initiated very rapidly after the identification of the etiologic agent, even though the immune correlates of protection were not known. research efforts to identify protective antigens and to develop animal models were undertaken in parallel with efforts to develop candidate vaccines [20] , drawing on experience with animal coronavirus vaccines and using several vaccine strategies, including inactivated virus vaccines, purified subunit vaccines, plasmid dna and viral vector-based vaccines as well as virus-like particles. much effort has been made to identify appropriate animal models for sars-cov replication and pathogenesis. several research groups have shown that mice [21, 22] , ferrets [23] , hamsters [24] and nonhuman primates [25] [26] [27] [28] [29] [30] support replication of sars-cov with varying degrees of associated disease. these animal models were used for the evaluation of candidate vaccines, and the common conclusion that has emerged from the evaluation of several vaccines is that the 180 kda viral spike protein, s, is the only significant neutralization antigen [31] [32] [33] [34] and the only one to elicit protective immunity in animal models [21, [35] [36] [37] [38] [39] [40] . the s protein can be divided into two domains by analogy with other coronavirus spike proteins: an n-terminal s1 domain, which contains the receptor-binding site and neutralization epitopes and a cterminal s2 domain which forms the membrane-anchored stalk region and contains a putative fusion peptide followed by two heptad repeats predicted to form a six-helix coiled-coil bundle [41] . in the absence of clinical cases of sars, candidate vaccines will have to be evaluated for efficacy in animal models. the united states fda "animal rule" states that, when efficacy studies in humans are not feasible, vaccines may be approved based on animal data alone, provided the pathophysiological mechanism of the disease is reasonably wellunderstood as is its prevention or reduction by the vaccine. moreover, the protective effect of the vaccine should be demonstrated in more than one animal species expected to react with a response predictive for humans. the endpoint of animal studies should be clearly related to the desired benefit in humans (i.e. enhancement of survival or reduction in major morbidity), and the data generated should allow selection of an effective dose in humans. at the present time it is uncertain whether the "animal rule" would apply to licensure of a sars vaccine. however, it is important to develop standardized animal models and immunological assays in preparation for this eventuality. scientists at the who technical meeting on animal models and antibody assays for evaluating candidate sars vaccines held on 25-26 august 2005 in south mimms, uk, discussed many aspects of research pertaining to the use of animal models in vaccine development including available animal models, suitability of the various models, correlates of protection, critical components of potential vaccines, and the potential for disease enhancement in vaccinated animals following exposure to sars-cov. in addition, standardization of antibody assays and the establishment of a who international standard for sars-cov antibody were also discussed. this report endeavors to summarize the recommendations from this meeting, based on consensus agreement. recommendations for use of each animal model are given in section 2 below. correlates of protection, an overview of vaccine development, and observations pertaining to potential disease enhancement are summarized in the following sections 3-6. in selecting animal models for vaccine evaluation, it is important to remember the principle underlying the so called "animal rule", where data from more than one animal species is often required: each animal species should contribute something different to our understanding of disease and protection. at this time, no single model offers a direct reproduction of what was seen in humans with sars. pathology (including pneumonitis, alveolar edema, and diffuse alveolar damage (dad)) in humans is probably the most difficult element to reproduce in an animal model. attention also should be given to the reduction of viral shedding because this would likely correlate with decreased risk of further spread of the disease among humans. in using animal models to study aspects of sars-cov infection, it must be emphasized that the kinetics of viral replication and appearance and resolu-tion of pathological findings are much more rapid in animal models than in humans. whichever animal model is employed, special consideration should be given to the presence of co-existing pathogens, the age of the animals and the route(s) of infection. a sufficient number of time points and large enough number of animals should be used to allow statistical evaluation. the strain of sars-cov used also could be of importance. it should be emphasized that different species may prove useful for studying different aspects of sars-cov. whereas vaccines or antivirals may be addressed in many models, pathogenesis is best evaluated in those animal models for which immunological tools and reagents are available for detailed analysis of the immune response to the vaccine. this includes inbred mice and rhesus and cynomolgus macaques. it may actually be worthwhile to enhance the virulence of a sars-cov isolate by serial passages in an animal model to produce a challenge virus stock for vaccine studies that would elicit more reproducible disease in the animals. if a highly virulent host-adapted virus were to become available, such as a mouse-adapted or a monkey-adapted sars-cov strain, demonstration of the capacity of vaccines to protect against challenge with these more virulent strains would provide an almost ideal animal model. different models may also need to be employed to evaluate pathogenesis versus immunogenicity. for pathogenesis studies in animal models, mortality is not required as a readout. it would be ideal to develop animal models with comparable levels of mortality to that seen in humans (∼10% overall), including the increased mortality at increased age (∼50% > age 60). the optimal result would be to demonstrate efficacy of vaccines or antivirals in sars-cov animal models that mimic human morbidity and mortality and that show protection without vaccine-associated immunopathology. inbred mouse strains (balb/c, c57bl/6, 129svev, stat1−/−) support sars-cov replication and can develop pneumonitis (129s), but pneumonia and clinical symptoms are only observed in older balb/c mice [24] . the mouse model is suitable for immunogenicity and efficacy studies of vaccines. prolonged viral replication, dissemination of virus to liver and spleen and accompanying pathology are seen in stat1−/− mice; these mice, therefore, also are suitable for studies of pathogenesis and evaluation of antiviral drugs. specific pathogen-free (spf) animals must be used. their age can be either 4-8 weeks or over 12 months and should be specified. the number of animals included must be sufficient for statistical analysis, and should include mock-infected controls. a variety of sars-cov strains has been tested in mice, including urbani, frankfurt, hku-39849, tor-2, and the mouse-adapted sars-cov strain ma-15. these were inoculated by the intranasal (in) route under light anesthesia [21, 42] , using a dose of 10 5 tcid 50 in 50 l/mouse. critical time points for specimen collections are days 2 (peak titer) and 5 post-infection (p.i.) for quantitative virology, days 3 and 5 p.i. for the study of interstitial pneumonitis and dad in aged mice (inflate lungs with 10% neutral-buffered formalin), and day 9 for resolution. golden syrian hamsters are an excellent animal model because they demonstrate high levels of sars-cov replication and develop pneumonitis. hamsters are suitable for vaccine efficacy, immunoprophylaxis and treatment studies [24] . in contrast with balb/c mice, in which the virus is detected only in the respiratory tract and is cleared by day 5 p.i., hamsters demonstrate a longer duration of viral shedding from the upper respiratory tract, some transient viremia, spread of the virus to liver and spleen and, most significantly, inflammation of respiratory tissues [21, 24] . spf animals older than 5 weeks of age should be used in sufficient number for statistical analysis and study design should include mock-infected animals. the animals can be housed in pairs or by three if the experiment is to last less than 4-6 weeks. reserve space should be available to separate the animals in case of fights. males and littermates tend to fight less. virus strains that have been tested in hamsters are urbani, frankfurt and hku-39849. virus should be administered by the in route under light anesthesia, using a dose of 10 3 tcid 50 in 100 l/hamster. as an outcome of efficacy studies, quantitative virology should be preferred over quantitative pcr. for pathology studies, one can grade pathology as none, mild, moderate or severe as per roberts et al. [24] . critical time points for specimen collection are days 2 or 3 p.i. (peak viral titer) and 7 (clearance) for quantitative virology studies; and days 5 p.i. (consolidation and pneumonitis) and 14 or 21 (resolution) for pathology studies (lung). there is evidence from one study that ferrets support sars-cov replication and develop pulmonary lesions [23] but according to another study, the animals remain asymptomatic, in the presence of sars-cov replication [38] . in view of these conflicting data, the ferret model needs to be further studied to determine its optimal utility for vaccine efficacy and immune prophylaxis studies. additional studies are needed to define the extent of biological variability of the model and the possible role of co-pathogens that may contribute to the variability observed between different laboratories. animals aged 6 months or older should be used. although not well documented, more consistent viral replication, pathology and clinical symptoms seem to be observed in older animals. the animals should be screened for viral co-pathogens: aleutian mink disease, respiratory viruses, hepatitis viruses, and others. the route of inoculation may be in or it, but not iv. the dose of virus (strains tor-2, hku-39849) sufficient to ensure reproducibility of infection in all animals is likely to be 10 5 pfu or more/ferret. again, quantitative virology is preferred over qrt-pcr. for pathology studies, the same recommendations apply as for nonhuman primate studies (see below): slides should be shared between pathologists to develop a scoring system. regarding specimen collection of respiratory tissues, further studies are needed to establish how much variation occurs in samples from different lobes of the lungs. critical time points are day 3 p.i. for quantitative virology and days 4-5 p.i. for pathology studies (pneumonitis). nhps support sars-cov replication and develop pneumonitis with a variable degree of clinical symptoms depending upon the species employed. no single nhp species is preferred at this time. the number of animals in a given study needs to be large enough to account for animal-toanimal variability: a sample of 4 or 5 animals is not sufficient. in view of the cost of the experiments, challenge studies should be limited to those vaccine candidates that are most promising, using larger sample sizes (10-12 animals/group) and avoiding animals with free-range periods in life if possible. immunological responses are best studied in species for which microarrays and reagents for identifying immune components are available, such as rhesus or cynomolgus macaques. however, the limited viral replication observed in cynomolgus macaques might be a disadvantage in selecting this species for studies. other recommended nhp species are the common marmoset and african green monkeys (agms, chlorocebus aethiops sabeus). the country of origin may play an important role and should be specified, e.g. philippines (cynomolgus macaques, macaca fascicularis); china (rhesus, macaca mulatta); brazil (marmosets, callithrix jacchus), etc. prior to the experiment, the animals should be housed indoor to limit exposure to potential co-pathogens. they should be screened for parasites (strongyloides, pneumonyssus simicola (lung mites)) and for possible viral co-pathogens (retroviruses, respiratory viruses, adenoviruses). the sars-cov strains tested in nhp models are hku-39849 (cynos), pumc (rhesus) and urbani (marmosets and agms). these were inoculated by the respiratory route (in, it) at a dose of 10 6 pfu or more/nhp. here again, quantitative virology is preferred over qrt-pcr. for pathology studies, it would be an obvious advantage that laboratories share pathology slides for review by different pathologists in order to develop a scoring system. specimens of respiratory tissues should be collected, but further studies are needed to establish how much variation occurs in samples from different lobes of the lungs, as was done and reported for african green monkeys (agms) [28] . critical time points are days 2-4 p.i. for quantitative virology in cynomolgus macaques and agms, and later than day 4 p.i. for rhesus macaques of chinese origin. for collection of tissues for histopathological analyses, days 2-4 p.i. are optimal for cynomolgus macaques and agms, and later than day 4 p.i. for rhesus macaques. due to limitations of immunological reagents (including microarray assays now available), research may be limited to rhesus and cynomolgus macaques. data on other animal models are insufficient for consideration for use in sars-cov vaccine and antiviral evaluations [43] [44] [45] . any additional model other than the four listed above (section 2.2.1 to section 2.2.4) would require thorough characterization including viral replication data and histopathological analysis of sars-cov-infected and mockinfected animals of the same age and gender. viral replication and histopathological data in any new animal model should be reminiscent of at least some aspect of sars in humans. although all the correlates of protection from sars associated disease have not been identified in human infections, neutralizing antibodies are present in convalescent human serum. antibodies to sars-cov spike (s) protein have been shown to prevent virus entry and neutralize virus infectivity in vitro [32, 46] . prophylactically administered monoclonal antibodies and passively transferred sars-cov hyper-immune sera have been shown to prevent sars-cov infection and associated disease following sars-cov challenge of naive mice and hamsters [21, 34, [47] [48] [49] . monoclonal antibodies administered therapeutically (i.e. post-infection) also have been shown to limit viral replication and reduce associated disease in hamsters [50] . although cell mediated immunity may have a protective role in viral clearance or resolution of disease, work in animal models shows that antibody alone is effective for prevention and treatment of sars. thus, mice immunized with live-recombinant vaccines expressing the sars-cov spike protein, using rabies virus [51] , vesicular stomatitis virus (vsv) [52] , adenovirus (ad5) [27, 53] or attenuated vaccinia virus mva [36, 38] as a vector, as well as mice immunized with dna vaccines expressing the s gene [37, 54] , developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. similar findings were reported after mucosal immunization of hamsters and agms using a bovine parainfluenza virus type 3 (bpiv3) vector expressing the sars cov s gene [33, 39] . several whole inactivated virus and recombinant protein candidate vaccines also have been developed and shown to elicit a neutralizing antibody response that provided protection against infectious challenge [55] [56] [57] [58] [59] [60] . in addition, passively administered sera from vaccinated animals prevented sars-cov infection upon subsequent challenge of naïve mice, demonstrating that antibodies induced by these vaccines did confer protection [37, 52] . the neutralizing antibody titer that is necessary to achieve protection in humans exposed to sars-cov is, however, still not known. it was recommended that when evaluating vaccine efficacy in future animal experiments, the challenge virus should be administered at two different time-points, once when postimmunization neutralizing antibody titers are high, and later when neutralizing antibody titers have waned or are low. it also was suggested that viral titers and pathology should be evaluated at two different time points. specific times points for sample collection are given for each animal model in section 2 above. previous observations of disease enhancement have been reported for human viral pathogens and shown to be due to antibody-mediated enhancement of virus entry (for reviews see [61, 62] ). enhanced disease and mortality have been observed in kittens immunized against or infected with a type-i coronavirus, feline infectious peritonitis virus (fipv), when subsequently exposed to fipv infection [63] [64] [65] . aggravated fip is apparently a result of enhanced viral entry into macrophages mediated by sub-neutralizing antibody levels [66] . children vaccinated with inactivated respiratory syncytial virus (rsv) vaccines developed serious disease on subsequent exposure to rsv [67] [68] [69] . individuals exposed to one of the four serotypes of dengue virus developed severe disease when subsequently infected with a second, different serotype [70, 71] . enhanced disease following rsv vaccine or dengue infection occur by different mechanisms than fipv. in view of such examples of enhanced disease following infection in a vaccinated host, there has been heightened concern that a similar phenomenon could occur with sars-cov vaccines. it was highly recommended, therefore, that known mechanisms of disease enhancement observed with other viruses and especially with other coronaviruses should be examined in sars-cov infections, especially in vaccinated animals. although none of the studies to date have shown enhanced respiratory disease following sars-cov challenge in previously immunized animals, further studies in this area are warranted in view of some of the available in vitro data. antibodies against human sars-cov isolates were shown to enhance the entry of pseudo-typed viruses expressing the civet sars-like cov-spike protein into a human renal adenocarcinoma cell line (786-o) . enhancement was only demonstrated at the level of entry, but not of replication [72] . this phenomenon was seen with pseudo-typed lentiviruses expressing sars-cov spike protein of civet sequence specificity, but not with pseudo-typed viruses expressing spike proteins of human sars-cov isolates. it also was not observed with human isolates of sars-cov. the role of enhanced viral entry, as observed in these in vitro studies, has not been related to any known component of human disease or infection in vivo. however, given that sars-cov may replicate, albeit poorly, in human pbmcs [73, 74] , in vitro experiments looking for antibody-enhancement of sars-cov replication in human cells (e.g. macrophages and b-cells) should be performed. several groups have studied sars-cov infection in animals in the presence of neutralizing and sub-neutralizing levels of sars-cov anti-sera or anti sars-cov s-protein monoclonal antibodies, but no evidence of enhanced respiratory disease has been observed. however, foci of hepatic necrosis were noted following sars-cov challenge in mva-sars-s immunized ferrets [38] . although these findings are worrisome, several questions were raised regarding the significance of the observation. the mva-sars-s vaccine used in these experiments was poorly immunogenic in the ferrets. the question of whether there could have been any co-pathogen in the animals was raised. it also would be important to know if the observed phenomenon depends on the mva vector or on the animal model. it was strongly urged, therefore, that the experiment be repeated in ferrets. additional experiments, in nonhuman primates and hamsters, looking for evidence of enhanced respiratory and hepatic diseases upon vaccination and challenge were also encouraged. several candidate sars vaccines that are at various stages of pre-clinical and clinical development are being developed worldwide. in china alone, three companies have been given regulatory approval for the clinical evaluation of a candidate sars vaccine. it is important, therefore, to be able to compare data from each of the candidate vaccines, which, in turn, requires international standardization of the immunological assays used for the evaluation of these vaccines. the accepted method of international standardization is to employ a who international standard (is), which allows comparison of results from different laboratories [75] . this is essential for establishing international requirements for vaccines, diagnostics or therapeutics. an is is prepared from material bearing a close resemblance to the samples being assayed; the material is distributed in glass ampoules with high precision and reproducibility and then freeze dried. it is important that a sufficient number of ampoules (2000-3000) be prepared so as to provide for about 10 years of use, and that the activity of the contents remain stable over this period. the process of establishing a who is involves an international collaborative study, in which the candidate is is compared with other samples. if the results of the tests are suitable, the candidate is assigned a provisional arbitrary unitage, a report is distributed for approval by study participants and for eventual approval by the who expert committee on biological standardization (ecbs). the preparation, storage and distribution of over 90% of is have been undertaken by the national institute for biological standards and control (nibsc) at south mimms (uk), which is a who laboratory for biological standards. nibsc has developed in the past several who iss for calibration of the antibody response against virus vaccines, including iss for antibodies against dengue, hepatitis a virus (hav), [76] hepatitis b virus (hbv), measles, [77] polio, rabies, [78] rubella, and smallpox. a candidate standard against human papilloma virus-16 (hpv-16) is under evaluation. the corresponding is's were used for a variety of antibody assays including virus neutralisation (vn), haemagglutination-inhibition, single radial diffusion, enzyme-linked immunoassay and radio-immunoassay. antibody iss are most useful in epidemiological studies and in clinical trials. their use allows correlates of immunity and potency requirements of prophylactic and therapeutic products to be expressed in international units (ius). data from several collaborative studies demonstrate that use of an is generally reduces the level of variability between assay results. however, there may be problems in using iss due to the complex array of antibody populations in each serum and the different sensitivity of different assay systems. examples of potential problems can be found in hbv and parvovirus b19 studies [79] , which showed that different assay kits gave different results even when the is was included in the assays. another issue is the degree of antigenic homology between the viral antigen used for the preparation of the is and the virus used in the assays. in a jev collaborative study, a candidate antibody is, which had been prepared from the sera of vaccinees immunized with an inactivated vaccine that was antigenically different from some of the viruses used in vn assays, demonstrated that the response to at least some inactivated vaccine is strain specific and the candidate is was consequently not established by the who ecbs. whether a panel of monoclonal antibodies to sars-cov could be used to prepare an is is an attractive alternative which should be explored. significant progress with standardisation of sars-cov antibody assays had been made in china with the development of a national antibody standard. in order to develop the national standard, sera were collected from 20 convalescent sars patients who were found to have sars-cov vn antibody titers ranging from undetectable to 1:203. one serum sample was selected for further evaluation based on crossreactivity with four sars-cov strains and on western blot analysis. this serum was freeze dried in 0.5 ml aliquots and was then assessed for stability by vn assays. the chinese standard was assigned a vn titer of 52.7 with 95% confidence limits of 47.6-62.2. a further important development in china was the preparation of human immunoglobulin for treatment of sars patients. national guidelines have been prepared for collec-tion of plasma, quality control testing and standardisation of assays. three chinese manufacturers have been licensed for preparation of sars immunoglobulin. the source material was plasma from convalescent patients at more than 28 days after infection. all were in good health and their plasma tested negative for blood-borne agents. plasma samples were processed by a combination of cold ethanol fractionation and ultrafiltration. in september 2003, three lots of igg were produced and assayed by nationally-agreed procedures. the stock of immunoglobulin currently available is sufficient to treat 100 patients. monoclonal antibodies have not yet been considered for treatment purposes in china. the importance of assessing immunogenicity of candidate sars-cov vaccines using vn assays is well acknowledged, but the variety of vn tests in use is a significant problem since there is at this time no consensus on the most sensitive, specific, and reproducible assay system. it is therefore desirable to establish an antibody is to serve as a basis of comparison in all vn assays. the most important activity at this time is to obtain a suitable source of antibody. a number of options can be considered, such as convalescent human sera, post-immunization human sera, monoclonal antibodies or hyperimmune animal sera. as an example, the availability of a suitable source of serum from convalescent patients in hong kong needs to be explored, although antibody levels in these individuals are probably quite low by now. it also would be important that other assays than vn be included in the collaborative study, and that the impact of sars-cov strain variation be examined by using different sars-cov strains and/or sera with different specificities. the centralized facility for aids reagents (cfar), which is based at nibsc, could be a suitable model for a sars-cov repository [80] . the cfar was established in 1989 to support aids vaccine research and it is now eu-funded [81] . there are currently 2000 reagents available including peptides, recombinant proteins, human sera/plasma, monoclonal and polyclonal antibodies, expression systems, cdna clones and viruses. a comparison can be drawn between sars-cov and hiv vn assays. currently, there are several different hiv neutralization assays formats under consideration and a lack of agreement on the most suitable assay. the cfar is supporting a joint who/eu project (neunet) to evaluate and standardize hiv vn assays in an international collaborative study. in the usa, a sars-cov repository has been established on behalf of the american type culture collection (atcc) in order to meet the needs of biodefence and the threat of emerging infections [82] . the type of reagents stored includes viruses, peptide arrays, monoclonal antibodies and proteins. it is hoped that an active collaboration can be established between niaid and nibsc in order to meet the expanding needs of the sars research community. based on the discussion at the meeting, the following recommendations were made with respect to standardization of the immunological assays for sars vaccine evaluation: 1. a who repository for sars-cov reagents ought to be developed. collaboration between niaid and nibsc is recommended to achieve this goal. 2. consensus must be reached for the reagents to be given priority in the repository. needed. 4. the most suitable source of antibody for the is is convalescent human sera, but post-vaccination human sera could also be used. 5. a protocol for an international collaborative study aimed at validating the is should be developed and distributed to prospective participants. 6. collaborative study participants should be asked about their assay capabilities, e.g. number of sera, virus strains handled, etc. . . 7. the proposed is collaborative study should include a core set of antibody preparations to be distributed and assayed in each laboratory (e.g. monoclonal antibodies, animal sera, other human sera). 8. tests should be conducted using the same strain of sars-cov in each laboratory, but the different genetic lineages of sars-cov should also be represented in the study. biosafety issues associated with sars-cov vaccine development stem from the reports of laboratory-acquired infections in china. sanofi pasteur has adopted bsl 4 practices and bsl 3 equipment (e.g. class 2 or 3 microbiological safety cabinets with respiratory protective equipment) for the preparation of sars-cov vaccines. of note is the fact that sars-cov appears to be quite resistant to normal methods of virus decontamination (jf saluzzo, personal communication). who has developed guidance, both general [83] , and specific for handling sars specimens [84] . the rapid success in the development of immunogenic and protective vaccines against sars using a variety of platforms is encouraging, but should be tempered with concerns about the possibility of enhanced disease following exposure in vaccinated individuals [85] . concerns mainly stem from reports of enhanced disease in fipv-immunized or -infected kittens [63, 66] , from observations that antibodies elicited against certain coronaviruses mediate antibody-dependent enhancement of viral entry [65] , and from the observation of inflammatory foci in liver tissue following sars-cov challenge in mva-sars-s vaccinated ferrets [38] . candidate sars vaccines will need to be evaluated in more than one animal model. they also will need to be thoroughly evaluated for the duration of the antibody response they induce, as well as for the breadth of their protective effi-cacy against different strains of sars-cov. the implications of the sequence heterogeneity among sars-cov strains are difficult to test at this time because the most divergent strains (civet sars-like viruses) have not been recovered in culture. validation and international standardization of immunological assays for the evaluation of candidate sars vaccines are essential to compare data across different trials. this requires the establishment of international standards for sars-cov antibody and a repository for sars-cov reagents, with an international collaborative study to validate the iss. the establishment of the repository by who in collaboration with nibsc and niaid was recommended. severe acute respiratory syndrome who investigates china's fall in 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immunoglobulin: an international collaborative study to establish the second international standard the 1st international standard for anti-measles serum calibration of a replacement preparation for the international standard for rabies immunoglobulin report of a collaborative study to calibrate the second international standard for parvovirus b19 antibody the importance of standardisation of laboratory evaluations in hiv vaccine trials biodefense and emerging infections research resources repository (bei resources world health organization. laboratory biosafety manual who biosafety guidelines for handling of sars specimens caution urged on sars vaccines the authors thank dr. marc p. girard and dr. marie-paule kieny for their invaluable assistance in preparing the manuscript. the contributions of anjeanette roberts and kanta subbarao were supported in part by the intramural research program of nih/niaid. key: cord-321166-nvphu1fm authors: thomson, emma c.; rosen, laura e.; shepherd, james g.; spreafico, roberto; da silva filipe, ana; wojcechowskyj, jason a.; davis, chris; piccoli, luca; pascall, david j.; dillen, josh; lytras, spyros; czudnochowski, nadine; shah, rajiv; meury, marcel; jesudason, natasha; de marco, anna; li, kathy; bassi, jessica; o’toole, aine; pinto, dora; colquhoun, rachel m.; culap, katja; jackson, ben; zatta, fabrizia; rambaut, andrew; jaconi, stefano; sreenu, vattipally b.; nix, jay; jarrett, ruth f.; beltramello, martina; nomikou, kyriaki; pizzuto, matteo; tong, lily; cameroni, elisabetta; johnson, natasha; wickenhagen, arthur; ceschi, alessandro; mair, daniel; ferrari, paolo; smollett, katherine; sallusto, federica; carmichael, stephen; garzoni, christian; nichols, jenna; galli, massimo; hughes, joseph; riva, agostino; ho, antonia; semple, malcolm g.; openshaw, peter j.m.; baillie, j. kenneth; rihn, suzannah j.; lycett, samantha j.; virgin, herbert w.; telenti, amalio; corti, davide; robertson, david l.; snell, gyorgy title: the circulating sars-cov-2 spike variant n439k maintains fitness while evading antibody-mediated immunity date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.04.355842 sha: doc_id: 321166 cord_uid: nvphu1fm sars-cov-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. herein we demonstrate that the immunodominant sars-cov-2 spike (s) receptor binding motif (rbm) is the most divergent region of s, and provide epidemiological, clinical, and molecular characterization of a prevalent rbm variant, n439k. we demonstrate that n439k s protein has enhanced binding affinity to the hace2 receptor, and that n439k virus has similar clinical outcomes and in vitro replication fitness as compared to wildtype. we observed that the n439k mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. immune evasion mutations that maintain virulence and fitness such as n439k can emerge within sars-cov-2 s, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics. sars-cov-2, the cause of covid-19, emerged in late 2019 and expanded globally, resulting in over 41 million confirmed cases as of october 2020. molecular epidemiology studies across the world have generated over 135,000 viral genomic sequences and have been shared with unprecedented speed via the gisaid initiative (https://www.gisaid.org/). these data are essential for monitoring virus spread (meredith et al., 2020) and evolution. of particular interest is the evolution of the sars-cov-2 surface protein, spike (s), which is responsible for viral entry via its interaction with the human angiotensin-converting enzyme 2 (hace2) receptor on host cells. the s protein is the target of neutralizing antibodies generated by infection or vaccination (folegatti et al., 2020; jackson et al., 2020; keech et al., 2020) as well as monoclonal antibody (mab) drugs currently in clinical trials (hansen et al., 2020; jones et al., 2020; pinto et al., 2020) . a sars-cov-2 s variant, d614g, is now dominant in most places around the globe (callaway, 2020) . studies in vitro indicate that this variant may have greater infectivity while molecular epidemiology indicates that it spreads efficiently and likely maintains virulence (hu et al., 2020; korber et al., 2020; volz et al., 2020; . amino acid 614 is outside the receptor binding domain (rbd) of s, the domain targeted by 90% of neutralizing antibody activity in serum of sars-cov-2 survivors (piccoli et al., 2020) . initial studies suggest that d614g actually exhibits increased sensitivity to neutralizing antibodies, likely due to its effects on the molecular dynamics of the spike protein (hou et al., 2020; yurkovetskiy et al., 2020) . therefore, this dominant variant is unlikely to escape antibody-mediated immunity. the low numbers of novel mutations reaching high frequency in sequenced sars-cov-2 isolates may relate to the moderate intrinsic error rate of the replication machinery of sars-cov-2 (li et al., 2020c; robson et al., 2020) and to this new human coronavirus requiring no significant adaption to humans (maclean et al., 2020) . nevertheless, the increasing number of infected individuals and the large reservoir of individuals susceptible to infection increases the likelihood that novel variants that impact vaccine and therapeutic development will emerge and spread. moreover, the full impact of immune selection, which can drive variant selection, likely has not yet had a dominant influence on the pandemic, since herd immunity has not yet been attained. as population immunity increases and vaccines are deployed at scale this might change. the potential for circulating viral variants to derail promising vaccine or antibody-based prophylactics or treatments, even in the absence of selective pressure from the drug or vaccine, is demonstrated by the failure of a phase iii clinical trial of a mab targeting the respiratory syncytial virus (simoes et al., 2020) , and the need for new influenza vaccines on a yearly basis. it is therefore critical to understand whether and how sars-cov-2 may evolve to evade antibody-dependent immunity. here, we examined the immunodominant sars-cov-2 receptor binding motif (rbm), the primary target of the neutralizing ab response within the rbd (piccoli et al., 2020) and found it to be less conserved than the rbd or the entire spike protein in circulating viruses. to understand the implications of this structural plasticity for immune evasion, we defined the clinical and epidemiological impact, the molecular features, and the immune response to an rbm variant, n439k. this variant has arisen independently twice, in both cases forming lineages of more than 500 sequences. as of october 2020, it has been observed in 12 countries and is the second most commonly observed rbd variant worldwide. we find that the n439k mutation is associated with a similar clinical spectrum of disease and slightly higher viral loads in vivo compared with isolates with the wild-type n439 residue, and that it results in immune escape from polyclonal sera from a proportion of recovered individuals and a panel of neutralizing mabs. n439k provides a sentinel example of immune escape, indicating that rbm variants must be evaluated when considering vaccines and the therapeutic or prophylactic use of mabs. long term control of the pandemic will require systematic monitoring of immune escape variants and selection of strategies that address the variants circulating in targeted populations. competing pressures influence the evolution of the spike rbm. first, the rbm mediates viral entry (shang et al., 2020; walls et al., 2020; wrapp et al., 2020b) and therefore it must maintain sufficient affinity to engage the entry receptor hace2. second, it is a major target of neutralizing antibodies (robbiani et al., 2020; rogers et al., 2020; wec et al., 2020) and could be a primary location for the emergence of immune escape mutations. we set out to understand these competing pressures by evaluating the landscape of rbm sequence divergence observed in circulating sars-cov-2 variants and in other viruses of the sarbecovirus lineage. we used published x-ray structures of sars-cov and sars-cov-2 rbd:hace2 complexes (lan et al., 2020; li et al., 2005) to define the rbm residues using a 6 å distance cutoff (figures 1a-c) . we evaluated ~130,000 sars-cov-2 genomic sequences deposited in gisaid as of october 7, 2020 and observed a high number of variants occurring in the rbm (figure 1a) . to understand how the divergence of the rbm compares to the divergence of the entire rbd and the whole spike protein, we divided the spike protein into three non-overlapping regions: the rbm, the rbd outside of the rbm, and the full s protein outside of the rbd. we counted individual variants occurring at least ten times, and quantified substitutions of different amino acids at the same position as separate variants. we found that the rbm is the least conserved region of s ( figure 1b) . to understand this result further, we evaluated a published deep mutational scanning (dms) data set of the rbd and compared it to sequences of circulating viruses. the dms data defines the effect of each possible single amino acid change on both expression of the rbd and its capacity to bind hace2. for each position in the rbm, we compared the dms results for all amino acid substitutions at that position versus only substitutions that have been observed in circulating sars-cov-2 isolates ( figure 1c) . a subset of residues shows the largest loss of hace2 binding upon mutation (top ~1/3 of rbm residues in figure 1c ) and, as would be expected, few natural variants of these residues have been observed to be circulating to date. surprisingly, these conserved residues each contribute weakly to the rbd:hace2 total interaction energy (the sum of pair-wise interaction energies for all residues at the binding interface in the x-ray structure; "binding energy" in figure 1c ). for the majority of the rbm (bottom ~2/3 of rbm residues in figure 1c ), variation in circulating virus sequences confirms the tolerance to mutation predicted by the dms data. notably, several rbm residues forming the strongest interactions with the receptor, e.g. k417 and e484, are not highly conserved despite their predicted importance. these results suggest that the rbm has a degree of structural plasticity whereby it is able to accommodate mutations without disrupting hace2 binding. evolutionary analysis of sarbecoviruses provides further support for rbm plasticity li et al., 2020b; rambaut et al., 2020) . the sars-cov rbm is highly divergent from the sars-cov-2 rbm (figure s1a-b) while maintaining hace2 binding affinity. additionally, there are many sequence changes in the rbm across a panel of related coronaviruses from animal isolates (figure s1a-b, table s1 ). to determine the ability of members of the sarbecovirus lineage to bind hace2, we produced nine recombinant rbd proteins corresponding to seven animal isolates, sars-cov-2, and sars-cov and evaluated their binding to recombinant hace2 ( figure s1c ). we found that three of the rbds from animal isolates showed strong affinity for hace2: gd pangolin, which has a highly similar rbm to sars-cov-2, and gx pangolin and bat cov wiv1, which have highly divergent rbms (figure s1a-b) . this further supports the conclusion that the rbm is structurally plastic, while retaining binding with hace2 as a receptor. given this plasticity, we next considered whether an rbm variant can lead to immune evasion while retaining virulence. the two most commonly observed circulating rbd variants as of october 2020 contain mutations in the rbm (s477n and n439k). we first identified the n439k variant in march 2020, circulating in scotland from lineage b.1 on the background of d614g (da silva filipe et al., 2020) . using phylogenetic analysis, we determined this variant represented a single lineage (figure 2a ) that increased in frequency to 553 sequences by june 20, 2020 (~10% of the available scottish viral genome sequences for this time period). numbers of n439k and all other isolates decreased in scotland concurrent with control of the pandemic by initiation of stringent public health measures and this lineage has not been detected in scotland after june. however, the n439k variant has been observed in >659 sequences in a second lineage in europe, first sampled in romania on may 13, 2020, then norway on june 23, 2020 and is now circulating in 12 countries, as well as arising independently in the u.s. (figure 2a-c) . as of oct 6, 2020, all 1201 n439k variants arose from a c-to-a transversion in the third codon position, though these counts are heavily influenced by sampling frequency which varies widely between countries. as scotland has a high sampling frequency for its population size (~5.5m), it is possible to calculate a growth rate (voltz and frost, 2017) based on a comparison of the scottish lineages. we find that the growth rate is similar to what has already been shown for the d614g background with no evidence for a faster rate of growth than n439 lineages ( figure s2a ). in addition to its frequency and spread, the n439k variant stood out from other circulating rbm variants as having a plausible mechanism for maintenance of viral fitness. the equivalent position to n439k in the sars-cov rbm is also a positively-charged amino acid (r426), which forms a salt bridge with hace2 (li et al., 2005) . we therefore hypothesized that the n439k sars-cov-2 variant may form this additional salt bridge at the rbd-hace2 interface (rbd n439k:hace2 e329). structural modeling supported that this salt bridge could form without disrupting the binding interface, including the two original salt bridges (rbd k417:hace2 d30 and rbd e484:hace2 k31) (figure 3a-c) . a salt bridge is the strongest type of non-covalent bond and the n439k mutation could plausibly increase the number of salt bridges at the binding interface from two to three, presenting the hypothesis that the n439k variant may have enhanced binding for hace2. to test this hypothesis, we used surface plasmon resonance (spr) to evaluate binding of recombinant n439k s or rbd protein to recombinant hace2. we also evaluated the n439r and k417v variants, each of which is found in sars-cov at these positions. across multiple assay formats, we found that the n439k and n439r variants exhibited a ~2-fold enhanced binding affinity for hace2 as compared to the original n439 variant (termed herein wt) ( figure 3d ). the magnitude of this enhancement was paralleled by a ~2-fold loss of binding affinity for the k417v variant relative to wt. lastly, we also tested the effect of the n439k/r and k417v mutations in combination. these double variants form the same number of salt bridges at the hace2 binding interface as compared to wt, but one is at rbd position 439 rather than 417; we found they had an hace2 affinity similar to the wt ( figure 3d ). these data indicate that acquisition of the n439k mutation enhances binding affinity, which could have implications in vivo in the context of natural infection. also, the enhanced affinity could plausibly compensate for other mutations that would otherwise be detrimental (e.g. k417v), further highlighting the plasticity of the rbm. the enhanced hace2 affinity of the n439k variant, its geographical emergence as independent lineages as well as its prevalence among circulating viral isolates is consistent with maintained viral fitness. we set out to directly examine fitness by evaluating clinical data and outcomes of virus carrying the n439k mutation versus wt n439, as well as by direct in vitro viral growth and competition. we used qpcr to evaluate viral load (as measured by cycle threshold, ct) in 1,918 scottish patients whose viral isolates had been sequenced (figures 4a-b ). viral isolates were either n439k/d614g (n=406), n439/d614g (n=978) or ancestral (n439/d614) (n=534). our analysis found strong evidence that the n439k/d614g genotype was associated with marginally lower cycle threshold (ct) than the n439/d614g genotype (mean ct value difference between n439k/d614g and n439/d614g: -0.65, 95% ci: -1.22, -0.07) ( figure 4b ). as ct measurements were carried out in multiple sites, a sub-analysis of viral load using rna standards was carried out with available samples and showed a near-complete correlation with ct ( figure 4b ). d614g has previously been associated with higher viral loads/lower ct values than d614 (korber et al., 2020) but we did not detect this difference in this statistical analysis due to the intercept of the model being imprecisely estimated (table s2) . clinical outcomes were also obtained for a subset of these patients (n=1,591), who were scored for severity of disease based on oxygen requirement: 1. no respiratory support, 2: supplemental oxygen, 3: invasive or non-invasive ventilation or high flow nasal cannulae, 4: death (figures 4c and s2b ). genotype counts for this analysis were n439k/d614g (n=399), d614g (with n439) (n=735) or ancestral (n439/d614) (n=457). analysis based on our ordinal scale indicated that the n439k/d614g viral genotype was associated with similar clinical outcomes compared to d614g or ancestral genotypes (posterior mean: 0.06, 95% ci: -1.21, 1.33) ( table s3 ). all other results from the severity analysis were qualitatively similar to a previous analysis of the d614g mutation (volz et al., 2020) . these clinical data indicate that the n439k virus is not attenuated. we next tested growth of two representative sars-cov-2 isolates, gla1 (wt n439) and gla2 (n439k), both with the d614g background (table s4) . culture was carried out for 72 hours in vero e6-ace2 cells either with or without tmprss2 expression. there was no significant difference between the growth of these strains after inoculation at multiplicities of infection (mois) of 0.005 and 0.01. the n439k strain replicated slightly faster early after inoculation ( figure 4d ). these data indicate that the n439k mutation does not exhibit dominant negative effects on viral growth, and most likely supports normal replication. to further assess fitness for replication in cultured cells, we carried out a cross-competition assay using inoculation of cells at a matched moi followed by quantitation of n439 and n439k by metagenomic ngs over time (figure 4e ). the n439k strain demonstrated similar fitness as the wt n439 strain, with a possible fitness advantage for n439k in cells expressing tmprss2. taken together with the clinical outcomes, these results indicate that the n439k mutation results in viral fitness that is similar or possibly slightly improved compared to the wild-type n439. having established that virus carrying the n439k mutation is fit, we sought to understand whether this mutation evades antibody-mediated immunity by evaluating recognition of the n439k variant by monoclonal antibodies and by polyclonal immune serum from 445 recovered individuals, including 6 donors who were infected by the sars-cov-2 n439k variant. 7.4% of the tested sera showed a greater than 2-fold reduction in binding to n439k rbd as compared to wt rbd (figures 5a-b and s3) . in some individuals the rbd response was diminished to low titers of <1:30 by the n439k mutation. thus, the response to the rbd is significantly influenced by the n439k mutant within the immunodominant rbm domain (piccoli et al., 2020) in a significant portion of persons potentially immune to wt sars-cov-2. the majority of sera demonstrating loss of binding were those that had overall lower responses to wt rbd, indicating lower ab titers. the sera from the six individuals known to have recovered from infection with sars-cov-2 n439k virus showed no change in binding levels to wt rbd as compared to n439k rbd (figures 5a-b and s3) . this may reflect a true variant-specific response or that differential binding could not be measured due to the limited number of samples analyzed. to understand our results at the level of individual antibodies, we evaluated a panel of 144 mabs isolated from individuals recovered from sars-cov-2 infection early in the pandemic (likely with n439 wt virus) (piccoli et al., 2020; tortorici et al., 2020) , as well as clinical-stage mabs regn10933, regn10987, ly-cov555, and s309 (the parent of vir-7831) hansen et al., 2020; chen et al., 2020; pinto et al., 2020) . 15.5% of these mabs demonstrated a >2-fold reduction of rbd binding in response to the n439k mutation ( figures 5c-d and s4 ). for comparison, we also evaluated the k417v mutation which eliminates one salt bridge at the rbm:hace2 interface and the n439k/k417v double mutation. a similar percentage (12.8% for k417v vs 15.5% for n439k) of mabs lost >2-fold binding to these variants, including several (13.5%) which were not sensitive to either single mutant but were sensitive to the double mutant ( figures 5c-d) . the reduced binding of mabs to these rbd mutants were also confirmed by bio-layer interferometry analysis (bli) (figures 5e and s5a) . to define the potential biological importance of these mutations for evasion of antibody-mediated neutralization, we tested mabs against pseudoviruses expressing s variants n439k, k417v or n439k/k417v (figures 5f-h and s5b ). neutralization of pseudoviruses containing these mutations was significantly diminished for certain mabs, including some that are in clinical development. as predicted by its non-rbm epitope , s309 was capable of neutralizing each of these variants. sensitivity of some neutralizing mabs to mutations at these positions have also been reported in other studies greaney et al., 2020; li et al., 2020a; weisblum et al., 2020) but combinations of mutations have not typically been evaluated. overall, our results demonstrate that mutations compatible with viral fitness can result in immune evasion from both monoclonal and polyclonal antibody responses. the evolution of the sars-cov-2 rbm, a critical epitope for vaccine response and therapeutic mabs, will depend on the fitness of rbm variants. the findings herein describe an example of a naturally-occurring rbm variant which can evade antibody-mediated immunity while maintaining fitness. fitness of this variant, n439k, was demonstrated by repeated emergence by convergent evolution, spread to multiple countries and significant representation in the sars-cov-2 sequence databases, the fact that the n439k rbd retains a high affinity interaction with the hace2 receptor, efficient viral replication in cultured cells, and no disease attenuation in a large cohort of infected individuals. the fitness of n439k is consistent with our findings that the rbm is the most divergent region of s. this divergence indicates an ability of sars-cov-2 to accommodate mutations at the rbm while retaining the functional requirement of hace2 binding, and is likely to be linked to immune pressure from neutralizing ab responses. there is precedent for the most immunogenic region of a viral surface protein to be the fastest mutating despite harboring the receptor binding site; for example, the immunogenic globular head domain of the influenza virus hemagglutinin surface protein, which contains the sialic acid receptor binding site, evolves faster than the stalk region (doud et al., 2018; kirkpatrick et al., 2018) . the ability to accommodate mutations in the rbm indicates a high likelihood that immune-evading sars-cov-2 variants compatible with fitness will continue to emerge, with implications for reinfection, vaccines, and both monoclonal and polyclonal antibody therapeutics. in our profile of immune escape from the n439k variant, we observed resistance to a mab currently being evaluated in clinical trials as part of a two-mab cocktail. the promise of using cocktails of mabs is that they should significantly lower the likelihood of drug-induced selection of resistant viruses . however, if circulating viral strains already carry resistant mutations to one antibody in the cocktail, this could reduce the cocktail to a monotherapy. additionally, considering the high level of plasticity of the rbm demonstrated in the present study, there could be many combinations of rbm mutations compatible with viral fitness while leading to immune escape. this is supported by our result that n439k can compensate for a mutation (k417v) that otherwise decreases receptor binding affinity ( figure 3d ). this particular combination of mutations is plausibly compatible with fitness as it parallels sars-cov rbm:hace2 interactions (salt bridge at sars-cov rbd position r426 and no salt bridge at v404, figure 3a) . notably, several mabs which were not sensitive to these mutations individually were sensitive to them in combination, including the two-mab cocktail ( figure 5c-h) . we propose two approaches that will be critical for minimizing the impact of mab escape mutations. one is to develop mabs with epitopes that are highly resistant to viral escape. this may include epitopes outside of the rbm and/or epitopes that are crossreactive across sars-cov and sars-cov-2, indicating conserved epitopes with a low tolerance for mutation wec et al., 2020; wrapp et al., 2020a) . a comparison of epitopes of rbm-targeting mabs with the most conserved regions of the rbm ( figure 1c ) may also identify rbm mabs with a higher barrier to escape. the second approach is to screen patients, likely at the population level, for the presence of potential resistance variants prior to drug administration. the availability of multiple different mab therapeutics in the clinic could provide the opportunity to tailor the choice of therapeutic to local circulating variants. in general, given that access to therapeutic monoclonal antibodies via clinical trials and emergency use authorization is expanding, and as more people develop immune responses to the wildtype virus, monitoring the evolution of sars-cov-2 will be increasingly critical. although sars-cov-2 is evolving slowly and at present should be controllable by a single vaccine (dearlove et al., 2020) , variation accumulating in the rbm could put this at risk, especially for individuals with a moderate ab response to vaccination or infection. while we only report on evasion of antibody-mediated immunity here, it would be surprising to us if similar changes are not observed to evade t cell immunity and innate immunity. wec, a.z., wrapp, d., herbert, a.s., maurer, d.p., haslwanter, d., sakharkar, m., jangra, r.k., dieterle, m.e., lilov, a., huang, d., et al. (2020) . broad neutralization of sars-related viruses by human monoclonal antibodies. science 369, 731-736. weisblum, y., schmidt, f., zhang, f., dasilva, j., poston, d., lorenzi, j.c.c., muecksch, f., rutkowska, m., hoffmann, h.-h., michailidis, e., et al. (2020) . escape from neutralizing antibodies by sars-cov-2 spike protein variants. (2020) . a new coronavirus associated with human respiratory disease in china. nature 579, 265-269. yurkovetskiy, l., wang, x., pascal, k.e., tomkins-tinch, c., nyalile, t.p., wang, y., baum, a., diehl, w.e., dauphin, a., carbone, c., et al. (2020) . structural and functional analysis of the d614g sars-cov-2 spike protein variant. cell. zhang, l., jackson, c.b., mou, h., ojha, a., rangarajan, e.s., izard, t., farzan, m., and choe, h. (2020) . the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity. https://wwwbiorxivorg/content/101101/20200612148726v1. samples from 439 sars-cov-2 infected individuals were obtained from the ticino healthcare workers cohort (switzerland), described previously (piccoli et al., 2020) , and under study protocols approved by the local institutional review board (canton ticino ethics committee, switzerland). all donors provided written informed consent for the use of blood and blood components (such as pbmcs, sera or plasma). in the ticino region of switzerland and during the time period of collection (february-march 2020) no n439k sars-cov-2 isolates were reported. samples from six n439k variant infected individuals were obtained from the isaric4c consortium (https://isaric4c.net/). ethical approval was given by the south central-oxford c research ethics committee in england (reference 13/sc/0149), and by the scotland a research ethics committee (reference 20/ss/0028). the study was registered at https://www.isrctn.com/isrctn66726260. residual nucleic acid extracts derived from the nose-throat swabs of 1918 sars-cov-2 positive individuals whose diagnostic samples were submitted to the west of scotland specialist virology centre between 3 rd march and 30 th june 2020 were sequenced as part of the cog-uk consortium under study protocols approved by the relevant national biorepositories (16/ws/0207nhs and 10/s1402/33) (consortiumcontact@cogconsortium.uk, 2020) . rbm residues were determined based on the rbd:ace2 complex crystal structures 2ajf for sars-cov (li et al., 2005) and 6m0j for sars-cov-2 (lan et al., 2020) . the 2ajf structure was obtained from the pdb-redo server (pdb-redo.eu) and was subsequently prepared in the molecular modeling software moe (v2019.0102, https://www.chemcomp.com) using the structure preparation, protonation and energy minimization steps with default settings. rbd residues within 6.0a distance of any ace2 atoms (determined using moe) were determined for each of the two copies of the complex in the asymmetric unit, and then were combined to obtain the rbm. 6m0j was obtained from the coronavirus structural task force server (https://github.com/thornlab/coronavirus_structural_task_force) and was further refined (using refmac5 v5.8.0258), manually fitted (using coot v0.9) and prepared (using moe, as described above) in multiple iterative cycles. the final structure was analyzed for rbd-ace2 contact residues with a 6.0a cutoff to obtain the rbm (using moe). the final list of rbm residues (figure 1c ) was arrived at by combining the sars-cov and sars-cov-2 results. using moe, the pairwise binding energy between each residue in sars-cov-2 rbd and each residue in ace2, and the total binding energy for all interactions, was determined at cutoff distances 3.0a, 3.5a, 4.0a, 4.5a, 5.0a, 5.5a, 6.0a, 6.5a and 7.0a. the percentage of the total binding energy for each interacting rbd residue was calculated for each distance cutoff and was then averaged over all cutoffs. the resulting values are shown in green in figure 1c . differential accumulation of amino acid variants in the rbm, rbd or spike protein was computed taking into account only the presence or absence of a variant at any residue. each variant called present counts one. a variant is called present if there are at least x number of supporting sequences deposited in gisaid, where x varies from 2 to 20. the number of variants is then normalized to the size of the domain (number of residues). dms data was retrieved from . variant-level dms scores were aggregated to residue-level by taking the minimum (most disruptive variant) or the average score across all variants of a residue, except for the reference residue and the stop codon. alternatively, minimum and average scores are computed only across variants that have been observed as naturally occurring. data were represented as a heatmap annotated with: frequency of non-reference amino acids in deposited gisaid sequences (n ≈ 130,000, at least 4 sequences were required to call a variant as present), in log10 scale; number of countries in which a variant was observed; and percentage of total binding energy computed from an x-ray crystal structure (cf. structural analysis methods section). prefusion-stabilized sars-cov-2 spike protein variants (residues 14-1211, containing the 2p and furin cleavage site mutations with a muphosphatase signal sequence and a c-terminal avi-8xhis-epea-tag in a pd2610-v5 vector (atum bio) were expressed in expi293f cells at 37°c and 8% co2 according to manufacturer's instructions (thermo fisher scientific). cell culture supernatant was collected after four days and purified over a 5 ml c-tag affinity matrix (thermo fisher scientific). elution fractions were concentrated and injected on a superose 6 increase 10/300 gl column with 1x pbs ph 7.4 as running buffer. sars-cov-2 rbd variants (residues 328-531 with a c-terminal thrombin-cleavage site-twinstrep-8xhis-tag, and n-terminal signal sequence) were expressed in expi293f cells at 37°c and 8% co2 in a humidified incubator. transfection was performed using expifectamine 293 reagent (thermo fisher scientific). cell culture supernatant was collected three days after transfection and supplemented with 10x pbs to a final concentration of 2.5x pbs (342.5 mm nacl, 6.75 mm kcl and 29.75 mm phosphates), or 3.2x for rbd n439r. sars-cov-2 rbds were purified using 1 or 5 ml histalon superflow cartridges (takara bio) and subsequently buffer exchanged into cytiva 1x hbs-n buffer or pbs. rbds from other sarbecoviruses were expressed in expi293f cells at 37°c and 8% co2. cells were transfected using pei max. cell culture supernatant was collected seven days after transfection. proteins were purified using a 5 ml strep-tactin xt superflow high capacity cartridge followed by buffer exchange to pbs using hiprep 26/10 desalting columns. for s binding measurements, recombinant ace2 (residues 19-615 from uniprot q9byf1 with a c-terminal thrombin cleavage site-twinstrep-10xhis-ggg-tag, and nterminal signal sequence) was expressed in expi293 cells at 37°c and 8% co2 in a humified incubator. transfection was performed using expifectamine 293 reagent (thermo fisher scientific). cell culture supernatant was collected seven days after transfection, supplemented with buffer to a final concentration of 80 mm tris-hcl ph 8.0, 100 mm nacl, and then incubated with biolock solution for one hour. after filtration through a 0.22 µm filter, ace2 was purified using a 1 ml streptrap hp column (cytiva) followed by isolation of the monomeric ace2 by size exclusion chromatography using a superdex 200 increase 10/300 gl column pre-equilibrated in pbs (gibco 10010-023). for binding measurements with surface-captured rbd, recombinant ace2 (residues 19-615 from uniprot q9byf1 with a c-terminal avitag-10xhis-ggg-tag, and nterminal signal sequence) was expressed in hek293.sus using standard methods (atum bio). protein was purified via ni sepharose resin followed by isolation of the monomeric ace2 by size exclusion chromatography using a superdex 200 increase 10/300 gl column pre-equilibrated with pbs. for binding measurements with surface-captured ace2, recombinant ace2 (residues 18-615 with a c-terminal gs-igg2a-mm-fc tag, and n-terminal signal sequence) was stably transfected in cho-k1 gs knock-down cell line (atum bio). protein was purified via protein a and buffer exchanged into pbs. spr binding measurements were performed using a biacore t200 instrument. s protein was surface captured via anti-avitag pab covalently immobilized on a cm5 chip, rbd protein was surface captured via streptactin xt covalently immobilized on a cm5 chip, and ace2-mfc was surface captured via covalent immobilization of the cytiva mouse antibody capture kit on a c1 chip. running buffer was cytiva hbs-ep+ (ph 7.4) and all measurements were performed at 25 °c. all experiments were performed as singlecycle kinetics, with a 3-fold dilution series of monomeric ace2 starting from 300 nm, each concentration injected for 180 sec, or a 3-fold dilution series of rbd starting from 50 nm, each concentration injected for 240 sec. all data were double reference-subtracted and fit to a binding model using biacore evaluation software. for one representative replicate, capture levels were normalized to wt for visualization. binding data with ace2 as analyte were fit to a 1:1 binding model. binding data with rbd as analyte were fit to a heterogeneous ligand binding model, due to an artifactual kinetic phase with very slow dissociation that arises when rbd is an analyte; the lower affinity of the two kds reported by the fit is reported as the kd of the rbd-ace2 interaction (the two reported kds are separated by at least two orders of magnitude for all fits). the measured kd for ace2 binding to s is likely influenced by conformational dynamics of the rbds in the context of the prefusion s trimer. reported kds are an average of 3-4 replicates measured on at least two separate days, with error given as sem. a national sequencing collaboration formed at the start of the epidemic in the uk, cog-uk consortium (consortiumcontact@cogconsortium.uk, 2020), has facilitated the tracking of sars-cov-2 sequences across scotland since the start of the outbreak in february 2020 (6,825 sequences by oct 6, 2020) and real-time monitoring of genetic changes in the spike gene that might be associated with changes in virulence or transmissibility. sequencing was carried out using an amplicon-based protocol in real-time at a rate of up to 300 genomes per week. 50% of samples were selected as surveillance samples, representing scottish health boards proportionately based on population size, while 50% were selected to allow intervention with local issues such as nosocomial infection in hospitals and nursing homes. a gradual increase in the prevalence of the n439k polymorphism was noted to become increasingly prevalent during april 2020. this was noted to be particularly common in the greater glasgow & clyde nhs health board region but spread to adjacent scottish health boards also. sequencing libraries were prepared according to the artic ncov-2019 described in detail at https://artic.network/ncov-2019. briefly, pcr amplicons were generated using the ncov-2019 primalseq sequencing primers using 25-35 cycles of amplification. generated amplicons were used to prepare either oxford nanopore or illumina sequencing libraries. oxford nanopore libraries were prepared as described in the link above and sequenced in a flow cell r9.4.1 (oxford nanopore technologies, part number flo-min106d), using minknow version 19.12.6. raw fast5 files were basecalled using guppy version 3.2.10 in high accuracy mode with a minimum quality score of 7. reads were size filtered, demultiplexed and trimmed with porechop (https://github.com/rrwick/porechop), and mapped against reference strain wuhan-hu-1 (mn908947). variants were called using nanopolish 0.11.3 and accepted if they had a loglikelihood score of greater than 200 and minimum read coverage of 20. for illumina sequencing, amplicons were used to prepare libraries using the kapa hyperprep kit (kapa biosystems, part number kk8504) and further processed as described in the competition assay sequencing method. sequencing was carried out on illumina's miseq system (illumina, part number sy-410-1003) using a miseq reagent v2 500 cycle kit (illumina, part number ms-102-2003) . reads were trimmed with trim_galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and mapped with bwa (li and durbin, 2009) ) to the wuhan-hu-1 (mn908947) reference sequence, followed by primer trimming and consensus calling with ivar (grubaugh et al., 2019 ) and a minimum read coverage of 10. uk sequences were obtained from the cog-uk consortium, https://www.cogconsortium.uk. global sequences were obtained from the gisaid initiative, https://www.gisaid.org on oct 19 2020. the sequences were mapped using minimap2 and padded against the wuhan/wh04/2020 reference. the sequences were downsampled with weights that normalise sequence count per epiweek, maximise the number of countries and lineages represented, and enriching for sequences with the n439k mutation. a maximum-likelihood phylogenetic tree was constructed using iq-tree with the the following parameters: -czb -blmin 0.0000000001 -m hky --runs 5 and all other parameters set to default. the tree was visualised with custom python code using the baltic library, https://github.com/evogytis/baltic. for the phylodynamic analysis, scottish "introduction" lineages were identified (lycett et al., 2020, in prep and see http://sars2.cvr.gla.ac.uk/risefallscotcovid), and the skygrowth package in r was used to estimate the effective population size over time, and the growth rate of the lineage within scotland (volz and frost, 2017) . clinical samples submitted to the west of scotland specialist virology centre for sars-cov-2 diagnostic rt-pcr testing were selected for sequencing as part of the covid-19 uk genomics uk consortium (cog-uk) project, resulting in 1918 whole genome sequences originating from the nhs greater glasgow and clyde health board region. sequences were linked to electronic patient records and basic metadata including sample date, age, sex, admission to hospital and mortality at 28 days post diagnosis extracted. the electronic patient records of a subset of 1591 patients underwent full casenote review and clinical severity was recorded based on a 4-level ordinal scale: 1. no requirement for respiratory support, 2. treatment with supplemental oxygen via facemask or low-flow nasal cannulae, 3. intubation and ventilation, non-invasive ventilation or oxygen delivery by high flow nasal cannulae devices, 4. death within the 28 days following diagnosis. we modified the who ordinal scale to these 4 points as described previously (volz et al., 2020) to avoid using hospitalisation as a criterion of severity because 1) many patients in nursing homes had severe infection but were not admitted to hospital, and 2) early in the outbreak, all cases were hospitalised irrespective of the severity of their infection. these data had previously been analysed to test for an effect of the d614g mutation on the severity of disease (volz et al., 2020) ; we extend that analysis here using the same methodology to test for an effect of the n439k mutation. additionally, we perform a new analysis using a model with the same structure to test for an effect of both the d614g mutation and the d614g/n439k mutation combination on the viral load of infected patients, as measured by cycle threshold value. in both cases we cannot estimate the marginal effect of the n439k mutation, as we only have the mutation on the 614g genetic background, so the individual effect of n439k cannot be separated from any potential epistatic interactions between the mutations. briefly, the structure of the model used previously (volz et al., 2020) and in the present study is a phylogenetic generalised additive model with mutation being the primary predictor of interest. the model controls for biological sex, age and the number of days since the first reported case in the dataset, with the latter two being included as penalised splines with a maximum of 30 knots. if the patient was part of a cluster of cases, this was included as a random effect, with individuals not part of clusters being assigned their own levels. correlations driven by the rest of the genome are controlled for by a phylogenetic random effect using a correlation matrix generated under a brownian motion assumption from a phylogeny estimated in iq-tree 2 v. 2.0.6 (minh et al., 2020) using a hky + γ model, masking the positions recommended by de maio et al. as of 22/7/2020 (https://virological.org/t/issues-with-sars-cov-2-sequencing-data/473/13), rooted on the first sequenced sars-cov-2 genome (wu et al., 2020) . the priors for the severity model were those used in the previous analysis of this data. the priors for the model of the viral load were a student-t (mean = 20, scale = 10, degrees of freedom = 3) prior on the model intercept, a gaussian (mean = 0, standard deviation = 10) prior over the fixed effects, and an exponential (lambda = 0.1) prior over the random effect, penalised spline and residual standard deviations. there are two key structural differences between the model used previously (volz et al., 2020) and the model used here. firstly, mutation is a three level rather than two level factor (d614/n439, d614g/n439 and d614g/n439k) with the ancestral d614/n439 being the reference level. secondly, as we are now interested in two mutations, we estimated the phylogeny used to control for the effect of the rest of the genome excluding both the nucleotide position underlying the d614g mutation and the nucleotide position underlying the n439k mutation (in addition to the sites from de maio et al mentioned above). the severity model used a cumulative error structure while the model on the ct values used a gaussian error structure. in both cases, the models were estimated in brms v. 2.13.5 (bürkner, 2018) . the presented models had no divergent transitions, rhat values less than 1.01, and appropriate bulk and tail effective sample sizes for all parameters. shortest probability intervals were calculated using the r package spin v. 1.1 (liu et al., 2015) . analysis code is available at https://github.com/dpascall/sars-cov-2-mutationanalysis. all samples were tested in duplicate using the 2019-ncov_n1 assay rt-qpcr assay (https://www.fda.gov/media/134922/download). ready-mixed primers and probe were obtained from idt (leuven, belgium). pcr was carried out using neb luna universal probe one-step reaction mix and enzyme mix (new england biolabs, herts, uk), primers and probe at 500 nm and 127.5 nm, respectively, and 5 µl of rna sample in a final volume of 20 µl. no template negative controls were included after every seventh sample. six ten-fold dilutions of sars-cov-2 rna standards were tested in duplicate in each assay; standards were calibrated using a plasmid containing the n sequence that had been quantified using droplet digital pcr. thermal cycling was performed on an applied biosystems™ 7500 fast pcr instrument running sds software v2.3 (thermofisher scientific) under the following conditions: 55 °c for 10 minutes and 95 °c for 1 minute followed by 45 cycles of 95 °c for 10 s and 58 °c for 1 minute. assays were repeated if the reaction efficiency was <90% or the r2 value of the standard curve was ≤0.998. where possible, testing of samples was repeated if the %cv of the duplicates was <10%. veroe6-ace2 cells (veroe6 cells induced to overexpress ace2) either with or without tmprss2 overexpression (rhin et al., 2020 under review) were seeded in a 12well plate and inoculated with an moi of 0.01 with either the gla1 (n439/d614g) or gla2 (n439k/d614g) virus isolates for 1hr before washing the cells three times in pbs and replacing with 2% dmem. 100ul of media was removed at each timepoint, rna was extracted, and the presence of sars-cov-2 determined using 2019-ncov-n1 assays (idt) with an neb luna universal probe one-step rt-qpcr kit. a standard curve was used to determine the copy number present per ml of cell culture media. 100ul of the fresh media was also tested for the presence of virus, which was undetectable in all wells. three t25 flasks were seeded with veroe6-ace2 or veroe6-ace2-tmprss2 and inoculated with either single viruses or both gla1 and gla2 virus strains at an moi of 0.01 for 1 hr. the flasks were washed three times with pbs, with 100 ul of the final wash being retained to determine the presence of free virus, before adding 5 ml of fresh 2% dmem. at 24, 48, and 72 hrs, 500 ul of media was removed, which was replaced with 500 ul fresh media. 300 ul was used for rna extraction and ngs analysis of the frequencies of the specific positions within the spike protein. the single virus inoculations showed no alternations in the frequency of the amino acid positions and the final wash showing no free virus in the supernatant. we used an unbiased metagenomic ngs sequencing pipeline to quantify variation across the whole viral genome on the illumina ngs next seq platform. briefly, extracted nucleic acid was incubated with dnasei (thermo fisher, part number am2222) followed by cdna synthesis using superscript iii (thermo scientific, part number 18080044) and nebnext ultra ii non-directional rna second strand synthesis module (new england biolabs, part number e6111l). samples were further processed using the kapa ltp library preparation kit for illumina platforms (kapa biosystems, part number kk8232) and indexed with the nebnext multiplex oligos for illumina 96 unique dual index primer pairs (new england biolabs, part number e6442s). libraries were sequenced on illumina's nextseq 550 system (illumina, part number sy-415-1002), generating 10 million pairs of reads per sample. human mabs were isolated from plasma cells or memory b cells of sars-cov or sars-cov-2 immune donors, as previously described (corti et al., 2011; pinto et al., 2020; tortorici et al., 2020) . ly-cov555 mab was obtained from eli lilly and company. regn10933 and regn10987 mabs were produced recombinantly based on published sequences (hansen et al., 2020) . a total of 148 human monoclonal antibodies or 445 human sera were tested for binding to rbd wt and mutants. spectraplate-384 plates with high protein binding treatment (custom made from perkin elmer) were coated overnight at 4 °c with 0.5 µg/ml (for mabs) or 5 ug/ml (for sera) sars-cov-2 rbd wt, n439k, k417v or n439k/k417v in phosphate-buffered saline (pbs), ph 7.2. plates were subsequently blocked with blocker casein 1% supplemented with 0.05% tween 20 (sigma-aldrich) for 1 h at room temperature. the coated plates were incubated with serial dilutions of the monoclonal antibodies or of the sera for 1 h at room temperature. the plates were then washed with pbs containing 0.1% tween-20 (pbs-t), and alkaline phosphatase-goat anti-human igg (southern biotech) was added and incubated for 1 h at room temperature. after 3 washing steps with pbs-t, p-nitrophenyl phosphate (pnpp, sigma-aldrich) substrate was added and incubated for 30 min at room temperature. the absorbance of 405 nm was measured by a microplate reader (biotek). fitting was performed using a 4-parameter logistic (4pl) model, yielding dose-response curves from which the area under the curve (auc) between 5 and 500 ng/ml was computed. the auc allows to capture, in a single metric, shifts of interest in two parameters of the 4pl model: ec50 and upper asymptote. bli binding measurement was performed on a selection of human monoclonal antibodies tested by elisa. antibodies were diluted to 2.7 µg/ml in kinetic buffer (pbs supplemented with 0.05% bsa) and immobilized on protein a biosensors of an octet red96 system (fortébio). antibody-coated biosensors were incubated for 5 min with a solution containing 5 µg /ml of sars-cov2 rbd wt, n439k, k417v or n439/k417v in kinetic buffer. a dissociation step was then performed by incubating the biosensors for 5 min in kinetic buffer. change in molecules bound to the biosensors caused a shift in the interference pattern that was recorded in real time and plotted using graphpad prism 8 software. replication defective vsv pseudovirus (takada et al., 1997) expressing sars-cov-2 spike protein were generated as previously described (riblett et al., 2016) with some modifications. plasmids encoding sars-cov-2 spike variants were generated by site-directed mutagenesis of the wild-type plasmid, pcdna3.1(+)-spike-d19 (giroglou et al., 2004) . lenti-x™ 293t cells (takara, 632180) were seeded in 10-cm dishes at a density of 1e5 cells/cm 2 and the following day transfected with 5 µg of spike expression plasmid with transit-lenti (mirus, 6600) according to the manufacturer's instructions. one day post-transfection, cells were infected with vsv-luc (vsv-g) (kerafast, eh1020-pm) for 1 h, rinsed three times with pbs, then incubated for an additional 24 h in complete media at 37°c. the cell supernatant was clarified by centrifugation, filtered (0.45 µm), aliquoted, and frozen at -80°c. vero e6 cells (atcc crl-1586) were seeded into clear bottom white 96 well plates (costar, 3903) at a density of 2e4 cells per well. the next day, mabs were serially diluted in pre-warmed complete media, mixed at a 1:1 ratio with pseudovirus and incubated for 1 h at 37°c in round bottom polypropylene plates. media from cells was aspirated and 50 µl of virus-mab complexes were added to cells and then incubated for 1 h at 37°c. an additional 100 µl of prewarmed complete media was then added on top of complexes and cells incubated for an additional 16-24 h. conditions were tested in duplicate wells on each plate and at least six wells per plate contained uninfected, untreated cells (mock) and infected, untreated cells ('no mab control'). virus-mab-containing media was then aspirated from cells and 100 ml of a 1:4 dilution of bio-glo (promega, g7940) in pbs was added to cells. plates were incubated for 10 mins at room temperature and then were analyzed on the envision plate reader (perkinelmer). relative light units (rlus) for infected wells were subtracted by the average of rlu values for the mock wells (background subtraction) and then normalized to the average of background subtracted "no mab control" rlu values within each plate. percent neutralization was calculated by subtracting from 1 the normalized mab infection condition. data were analyzed and visualized with prism (version 8.4.3). ic50 curves were calculated from the interpolated value from the log(inhibitor) vs. response -variable slope (four parameters) nonlinear regression with an upper constraint of <100. each neutralization infection was conducted on three independent days. . dms score is the binding or expression fold change over wt on a log10 scale. aggregated dms data is shown for each residue by taking the minimum (most disruptive variant) or the average score across all possible variants of a residue, except for the reference residue and the stop codon ('mutagenesis' columns). alternatively, minimum and average scores are computed only across variants that have naturally occurred ('observed variants' columns). when no natural variants have been observed, cells are grey. the heatmap is annotated with frequency of non-reference amino acids in deposited sequences (at least 4 sequences were required to call a variant), in log10 scale; number of countries in which a variant was observed; and percentage of total binding energy between rbd and hace2 computed from an x-ray crystal structure. data were sorted on the leftmost dms column. legend on next page (h) correlation of elisa-binding fold change and neutralization fold change for each variant relative to wt (where a smaller elisa auc and therefore a smaller ratio represents loss of binding, and a larger ic50 and therefore a larger ratio represents loss of neutralization) a rbm rbd table s1 . details of the sarbecovirus sequences used for figure s1 . the top 8 sequences shaded in gray were used for the similarity plot and all 69 sequences were used for the entropy plot. parameter estimates on the link scale from the model estimating the impact of the n439k mutation on the ct value of patients infected with sars-cov-2 in scotland. credible intervals represent 95% the shortest posterior density intervals. the difference between d614g/n349 and d614g/n349k was estimated by direct subtraction of the hamiltonian monte carlo samples of the d614g/n349k estimate from the d614g/n349 estimate. ct value did not appear strongly correlated with biological sex or age after controlling for the other factors. patients infected with related viral genomes had correlated ct values at testing potentially implying that there are other undescribed mutations in the genome that are affecting the viral load. parameter estimates on the link scale from the model estimating the impact of the n439k mutation on the severity of infection of patients infected with sars-cov-2 in scotland. credible intervals represent 95% the shortest posterior density intervals. thresholds correspond to the positions of the boundaries between the different severity classes. amino acid change gene mutation gla1 c3037t nsp12 p323l c14408t s d614g a23403g e v5a a24388t t26258c gla2 c3037t nsp12 p323l c14408t nsp15 v35a t19724c s n439k c22879a s d614g a23403g orf 10 v6f g29573t table s4 nucleotide differences between gla1 and gla2. snps determined by cov-glue on consensus sequences relative to wuhan-hu-1 (nc_045512.2). antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape seen with individual antibodies evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic advanced bayesian multilevel modeling with the r package brms an integrated national scale sars-cov-2 genomic surveillance network sars-cov-2 neutralizing antibody ly-cov555 in outpatients with covid-19 a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins genomic epidemiology of sars-cov-2 spread in scotland highlights the role of european travel in covid-19 emergence a sars-cov-2 vaccine candidate would likely match all currently circulating variants how single mutations affect viral escape from broad and narrow antibodies to h1 influenza hemagglutinin safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein complete mapping of mutations to the sars-cov-2 spike receptor-binding domain that escape antibody recognition an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar sars-cov-2 d614g variant exhibits enhanced replication ex vivo and earlier transmission in vivo d614g mutation of sars-cov-2 spike protein enhances viral infectivity an mrna vaccine against sars-cov-2 -preliminary report neutralizing antibodies against sars-cov-2 and other human coronaviruses ly-cov555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of sars-cov-2 infection phase 1-2 trial of a sars-cov-2 recombinant spike protein nanoparticle vaccine the influenza virus hemagglutinin head evolves faster than the stalk domain tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structure of sars coronavirus spike receptorbinding domain complexed with receptor fast and accurate short read alignment with burrows-wheeler transform the impact of mutations in sars-cov-2 spike on viral infectivity and antigenicity emergence of sars-cov-2 through recombination and strong purifying selection transmission dynamics and evolutionary history of 2019-ncov simulation-efficient shortest probability intervals natural selection in the evolution of sars-cov-2 in bats, not humans rapid implementation of sars-cov-2 sequencing to investigate cases of health-care associated covid-19: a prospective genomic surveillance study iq-tree 2: new models and efficient methods for phylogenetic inference in the genomic era refmac5 for the refinement of macromolecular crystal structures mapping neutralizing and immunodominant sites on the sars-cov-2 spike receptor-binding domain by structure-guided high-resolution serology cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody a dynamic nomenclature proposal for sars-cov-2 lineages to assist genomic epidemiology a haploid genetic screen identifies heparan sulfate proteoglycans supporting rift valley fever virus infection convergent antibody responses to sars-cov-2 in convalescent individuals coronavirus rna proofreading: molecular basis and therapeutic targeting isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model structural basis of receptor recognition by sars-cov-2 suptavumab for the prevention of medically attended respiratory syncytial virus infection in preterm infants deep mutational scanning of sars-cov-2 receptor binding domain reveals constraints on folding and ace2 binding a system for functional analysis of ebola virus glycoprotein ultrapotent human antibodies protect against sars-cov-2 challenge via multiple mechanisms scalable relaxed clock phylogenetic dating evaluating the effects of sars-cov-2 spike mutation d614g on transmissibility structure, function, and antigenicity of the sars-cov-2 spike glycoprotein top -pairwise similarity to sars-cov-2 (sliding window size of 30 amino acids) for seven related sarbecoviruses (see figure key) across the rbd region of the spike protein. bottom -site-specific entropy plot across the rbd protein alignment of sars-cov-2 and 68 related viruses (data s1). entropy for each position l (h(l)) was calculated using shannon's entropy formula with a natural log as sites constituting the rbm are annotated in blue the x-axis refers to absolute positions in the sars-cov-2 spike protein sequence. rightbox plot of site-specific entropy values for the rbm sites (blue) and remaining non-rbm rbd sites (gray) sequence alignment (left) and identity for rbm and rbd (right) to sars-cov-2 of the rbd sequences showing binding to hace2. rbm residues indicated by blue boxes. (c) binding of hace2 to human, pangolin and bat sarbecovirus rbds by bli. bat cov ratg13 we thank all scottish nhs virology laboratories who provided samples for sequencing and scott arkison for hpc maintenance. we thank chiara silacci-fregni from humabs biomed, sandra jovic, blanca fernandez rodriguez, federico mele, from the institute for research in biomedicine in bellinzona and tatiana terrot from ente ospedaliero cantonale in lugano for the help in collecting sera samples. we thank cindy ng for help with protein production. we thank julia di iulio for help with analyzing gisaid sequences. we gratefully acknowledge the authors, originating and submitting laboratories of the sequences from gisaid, https://www.gisaid.org, on which much of this research is based.the isaric who ccp-uk study protocol is available at https://isaric4c.net/protocols; study registry https://www.isrctn.com/isrctn66726260. this work uses data provided by patients and collected by the nhs as part of their care and support #datasaveslives. we are grateful to the 2648 frontline nhs clinical and research staff and volunteer medical students who collected the data in challenging circumstances; and the generosity of the participants and their families for their individual contributions in these difficult times. we also acknowledge the support of jeremy j farrar, nahoko shindo, devika dixit, nipunie rajapakse, lyndsey key: cord-330597-nftwj0d5 authors: hopfer, helmut; herzig, martin c.; gosert, rainer; menter, thomas; hench, jürgen; tzankov, alexandar; hirsch, hans h.; miller, sara e. title: hunting coronavirus by transmission electron microscopy – a guide to sars‐cov‐2‐associated ultrastructural pathology in covid‐19 tissues date: 2020-09-27 journal: histopathology doi: 10.1111/his.14264 sha: doc_id: 330597 cord_uid: nftwj0d5 transmission electron microscopy has become a valuable tool to investigate tissues of covid‐19 patients because it allows visualisation of sars‐cov‐2, but the “virus‐like particles” described in several organs have been highly contested. because most electron microscopists in pathology are not accustomed to analysing viral particles and subcellular structures, our review aims to discuss the ultrastructural changes associated with sars‐cov‐2 infection and covid‐19 with respect to pathology, virology, and electron microscopy. using micrographs from infected cell cultures and autopsy tissues, we show how coronavirus replication affects ultrastructure and put the morphological findings in the context of viral replication, which induces extensive remodelling of the intracellular membrane systems. virions assemble by budding into the endoplasmic reticulum‐golgi intermediate complex and are characterized by electron dense dots of cross‐sections of the nucleocapsid inside the viral particles. physiological mimickers such as multivesicular bodies or coated vesicles serve as perfect decoys. compared to other in‐situ techniques, transmission electron microscopy is the only method to visualize assembled virions in tissues and will be required to prove sars‐cov‐2 replication outside the respiratory tract. in practice, documenting in tissues the characteristic features seen in infected cell cultures, seems to be much more difficult than anticipated. in our view, the hunt for coronavirus by transmission electron microscopy is still on. sars-cov-2 currently dominates all headlines as a highly contagious pandemic with considerable mortality [1] [2] [3] [4] . while hygiene precautions and lockdown measures have changed our personal and professional daily lives during the last months, many investigators are eager to understand the biological basis of sars-cov-2's contagiousness and pathogenesis leading to respiratory and multi-organ failure. autopsy studies have established the morphological changes associated with covid-19 and have tried to visualise the virus in tissues [5] [6] [7] [8] [9] . transmission electron microscopy (tem) seems a logical tool to look for sars-cov-2 infection, but some of the published results are highly contested (kidney [8, [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] , endothelium [8, 9, [23] [24] [25] [26] [27] [28] , intestine [8] , liver [29] [30] [31] [32] , placenta [33] [34] [35] [36] [37] ). because most of us are not virologists or electron microscopists dedicated to viral diseases, this review aims at combining multidisciplinary expertise of sars-cov-2 pathology, virology, and electron microscopy. pathologists are good at detecting some viral infections -at least in identifying unusual inclusions on an h&e-stained slide. however, unless there is a knowledge of virus morphology (what they look like) and morphogenesis (how and where in the cell they are assembled), it is difficult to identify them. depending on the virus, we use immunohistochemistry targeting viral proteins or in-situ hybridization to highlight their dna or rna. molecular pathology techniques allow us to test for viruses in tissues when in situ-techniques are not yielding results. all of these techniques have been applied successfully in the context of sars-cov-2 (figure 1 and [5, 38, 39] ). it is important to note that any of these tests require an a priori notion of what is present; otherwise, it is difficult to choose the right reagent (e.g. if a herpesvirus is suspected, and an anti-herpesvirus antibody is used, but the infection is caused by an adenovirus, then the test is negative, and the diagnosis is no closer to being made). virus detection by tem is rarely deployed in the routine setting (outside of the viral em diagnostic laboratories that examine tissues and fluids) because it is expensive, time-consuming, covers only minute portions of the tissue, and is not available in most laboratories. nephropathology is one of few areas in pathology that routinely performs tem. therefore, it is not surprising that renal pathologists were among the first to search for sars-cov-2 infection in the kidneys by tem [10] [11] [12] 40, 41] . most changes they routinely look for in kidney biopsies are visible at magnifications between 1,000x and 10,000x (which can be considered as "low power" in the context of tem). thus, the magnifications required to identify viral structures are way out of their normal "comfort zone". as a consequence, "hunting coronavirus by electron microscopy" this article is protected by copyright. all rights reserved takes us to subcellular structures that we usually do not study because they are not important in the context of the standard pathology diagnostic work-up. complicating things further, ultrastructural preservation is limited in autopsy samples with delayed fixation obscuring subtle changes of the intracellular membrane systems associated with replication of enveloped viruses. to better understand the ultrastructural morphology of sars-cov-2 infection and covid-19, we will first briefly discuss the pathogenesis of covid-19 and coronavirus replication in general and then examine the tem findings in more detail. transmission of community-acquired respiratory viruses including sars-cov-2 usually results from close-range contacts through respiratory droplets or aerosols making contact with mucus membranes of the upper respiratory tract [42] [43] [44] [45] . sars-cov-2 infection of host cells involves specific binding of the viral spike glycoprotein s (s protein) to its receptor ace2. the s protein is then cleaved by host cell proteases to allow for conformational changes mediating fusion between the viral envelope and the host cell membrane. the viral rna is uncoated and released into the cytoplasm. host cells are ciliated epithelial cells of the upper and lower airways and pneumocytes type ii [46] [47] [48] [49] [50] . the level of innate immune response appears to increase as the infection progresses to the lower respiratory tract and the patients increasingly show symptoms [51] . this gradual progression may explain the range of the clinical manifestations from asymptomatic to severe disease. in the alveoli, sars-cov-2 infects pneumocytes type ii as well as pneumocytes type i via local cell-to-cell transmission according to preclinical primate models [52] . sars-cov-2 infection impairs production of surfactant and fluid resorption leading to increased transmural microcapillary pressure and microvascular leakage, finally resulting in adult respiratory distress syndrome clinically and diffuse alveolar damage histologically [2, 5, 6, 8, [53] [54] [55] [56] [57] [58] . while direct infection of the cells in the upper and lower respiratory tract drives these initial phases, the innate immune response is mostly responsible for the hyperinflammatory phase ("cytokine storm") characteristic of severe covid-19 [51, 59, 60] . some evidence suggests that sars-cov-2 infection may not be restricted to the respiratory tract but can spread to other organs. viral rna has been detected by quantitative nucleic acid amplification technique (qnat) initially in blood of severely ill patients, in faeces, and rarely in urine samples [61] [62] [63] [64] [65] [66] [67] . it can also be amplified from multiple tissues obtained at autopsies this article is protected by copyright. all rights reserved including heart, liver, kidneys, intestine, skin, and brain [5, 38, 39, 68] . in-situ hybridization and immunohistochemistry studies support the idea of viral spread throughout the body [8, 12, 33, 38, [69] [70] [71] . however, except for lower respiratory fluids such as sputum or bronchoalveolar lavage [61, 64, [72] [73] [74] and very rarely faeces [65] , other viral rna-containing samples have not been reported to allow for a productive infection in cell cultures [64, 75, 76] . these data suggest limited sensitivity of current culture assays and/or low infectiousness. the time point of viral dissemination is unclear but -from a virological point-of-view -would require significant local replication and access to blood, blood cells, and release at new and distant sites. a better knowledge of these events may help to predict the clinical symptoms and their relevance to the disease course. currently, our knowledge of morphological changes in covid-19 is mostly based on autopsy tissue obtained from severely affected individuals in the pulmonary or hyperinflammatory phase making it difficult to differentiate between changes driven by local viral replication, changes due to the systemic inflammatory response and repair, or possibly therapy effects. indisputable detection of sars-cov-2 by tem would confirm viral replication outside the upper respiratory tract and the lungs and firmly establish a role of direct viral infection to some of the organs mentioned above. interpretation of tem findings in tissues of covid-19 patients benefits from a good understanding of coronavirus replication in cells [77] [78] [79] . like all members of this family, sars-cov-2 virions are enveloped infectious particles with a diameter of 60-140 nm [80] . this article is protected by copyright. all rights reserved mers-cov [81] [82] [83] [84] [85] [86] [87] . recent data show that sars-cov-2 replication induces very similar ultrastructural changes [88] . sars-cov-2 infection and replication can be arbitrarily divided into three phases (figure 2), which may occur simultaneously. 1. after binding to its receptor ace2, sars-cov-2 is shuttled into the endosomal pathway, likely by clathrin-coated vesicles [89] . depending on the presence of furin, a serine protease, cleavage of the s protein triggers early fusion of the viral and the endosomal membranes and causes the release of viral genomes into the cytoplasm [46, 47] . cleavage may also occur by other proteases after fusion of the late endosome with a lysosome [90] . ribosomes recognize the positive-sense genomic rna strand (+grna) as mrna and translate the viral proteins making up the replication-transcription complex (rtc) at the er. this initiates an extensive remodelling of intracellular membranes forming a three-dimensional structure referred to as the replication membranous web (rmw) [83, 84, 91, 92] . virus replication within the rmw has several advantages: all factors necessary are concentrated in close proximity to each other making the process very efficient and, additionally, the rmw may hide viral rna from innate immune sensors within the cytoplasm. morphologically, the rmw is a fascinating and confusing structure containing multiple interconnected vesicles with single or double membranes (termed double-membrane vesicles and convoluted membranes). one has to assume that the rmw is a dynamic structure with multiple fission and scission events, which, unfortunately, cannot be this article is protected by copyright. all rights reserved coronaviruses including sars-cov-2 and the morphological changes associated with replication can be visualised by tem in infected cell lines (figure 3a-g) [81] [82] [83] [84] [85] 87, 88] or organoids [96, 97] . non-infected cultures serve as controls (figure 3h-k). for comparison with tissues from covid-19 patients, understanding the morphology of the assembled viruses and the replication membranous web is most important. budding of the nucleocapsid into membranes containing the structural proteins, forms the viruses with a circular shape (figure 3c). thus, assembled sars-cov-2 virions reside within vacuoles and cannot be seen free in the cytoplasm (figure 3b, d-e) [82] . cross-sections of viruses measure 60-140 nm [80] , and show the membrane of the envelope with the helical electron dense nucleocapsid inside as several small granules of approximately 12 nm. frequently, the centre of the viral cross section is electron lucent. unless a negative staining procedure for viewing viruses in fluids or tannic acid staining of tissue for thin sections is used, the s proteins forming the "corona" are not readily discernible [98] . convoluted membranes (cm) are less frequent structures that develop early after infection ( figure 3f ) [83, 84] . they associate with the dmv and the er, forming an unorganized reticular structure measuring 200-600 nm with a single limiting membrane. cm may be involved in the formation of the dmv [83] . another hypothesis suggests that these structures may serve as a storage for or are generated by an excess of non-structural proteins not incorporated into the dmv [84] . although rare, cubic membrane structures (cms) are the most eye-catching membrane rearrangement in coronavirus-infected cells because the membranes are highly organized (figure this article is protected by copyright. all rights reserved 3g) [84] . in cell cultures, they emerge late after infection. cms consist of highly curved membranes arranged in a recurrent (three-dimensional) pattern. their function is unknown; one hypothesis proposes that they are formed from membranes with an excess in s protein [82, 84, 99] . cms are not specific for coronavirus infection [100] . for example, cms resulting from chloroquine therapy may be seen in kidney biopsies of patients with lupus nephritis (termed "curvilinear bodies" in this context) [101] . late after infection, cell cultures frequently contain large virion-containing vacuoles (lvcv, figure 3b ) [81] [82] [83] . these are large circular vesicles belonging to the secretory pathway containing multiple cross sections of assembled virus and evidence of additional virus budding. marker studies suggest that lvcv are ergic/golgi-derived cisternae [84] . based on the cell culture findings outlined above, we expect to find the same sars-cov-2 morphology and distribution in vesicles of autopsy and biopsy tissues of covid-19 patients. there are few reports on upper airway and lung tissue demonstrating assembled sars-cov-2 virions that morphologically replicate the cell culture findings [58, 80] . others, including some of us, have reported on "virus-like particles" by tem not completely matching the cell culture findings in a variety of organs, including lung [5, 8, 26, 56, 102, 104] , kidneys [5, 8, [10] [11] [12] 23, 104] , liver [29] , heart [104] , intestine [8, 103] , skin [71] , and placenta [33, 36, 69, 70] . colleagues have rightfully raised their concerns that these particles are not consistent with sars-cov-2 [14] [15] [16] [17] [18] 24, 27, 30, 31, 34] , but depict other subcellular structures, making identification of sars-cov-2 by tem much more challenging than expected. this article is protected by copyright. all rights reserved physiological structures including coated vesicles, multivesicular bodies and cross-sections of the rough er are morphological look-alikes of genuine corona viruses [105] . coated vesicles (cv) are single-membrane-bound vesicles of variable size (typically 50-150 nm) characterized by "spiny adornments on their limiting membrane" (f. ghadially) (figure 4e) [106] . they are involved in endocytosis and membrane trafficking (reviewed in [107] ). in clathrin-coated vesicles, the best-studied example, the cv bud off so-called coated pits on the cell surface during micropinocytosis. clathrin and other quantitatively minor proteins provide a three-dimensional structural lattice, which is readily seen in electron micrographs. morphologically identical structures with coats provided by the main proteins copi or copii are involved in transport processes of the trans-golgi network. internalization of sars-cov-2, after binding to its receptor ace2 involves this mechanism [46, 47] . while cv may transport viral proteins ,as shown for vesicular stomatitis virus [108] , and may be used for replication of poliovirus [109] , and, further, have a similar size to that of coronavirus, they are not the assembled virus itself. however, although the projections appear as a perfect "corona" in cross-sections, cv lack the nucleocapsid present inside of coronavirus cross sections, and they are located within the cytoplasm and not within vacuoles. the process is used to sort and target membrane-associated proteins to lysosomal degradation, which happens after fusion of a mvb with a lysosome. a divergent pathway for mvb is the release of the ilv as exosomes after fusion with the cell membrane. interestingly, the molecular machinery involved in these processes is also used for the budding of enveloped viruses such as hiv (reviewed in [111] ). mvb are the perfect "decoy" for electron microscopists searching for viral particles. some of us have fallen for them in our covid-19 autopsy series [5] because the ilv have a similar size as sars-cov-2 and are located within vesicles. the key difference is that ilvs do not show the electron dense granules of the nucleocapsid. pathologists regularly performing tem are familiar with the er. two types of this largest closed and interconnected membrane structure in eukaryotic cells are recognized: the smooth er, the site this article is protected by copyright. all rights reserved of lipid synthesis and metabolism, and the rough er, where ribosomes, attached to the outside of the membrane, translate proteins for membranes, organelles, and secretion [112] . dilatation of the er occurs in the context of cell stress of various aetiologies. in covid-19 tissues, the er is frequently swollen, and the accumulation of membrane structures further adds to the confusing ultrastructure ( figure 4c) . a cross-section through rough er can easily be mistaken for a "viruslike particle", but these are located within the cytoplasm and not in vesicles and lack the nucleocapsid structures inside. in kidneys of covid-19 autopsies, we encountered a peculiar subcellular structure closely mimicking sars-cov-2 but likely related to the er (figure 4c, g-i) [5, 13] . larger vesicles with a smooth outside membrane contained several round to oval small vesicles with prominent electron dense granules on the inside. these granules were bigger than sars-cov-2's nucleocapsid seen in our infected cell cultures and had the same size as the ribosomes visible in areas containing rough er (ribosomes: 20-21 nm (range: 17-23 nm) vs. nucleocapsid: 12 nm (range: 9-16 nm). these vesicles with "outside-in" ribosomes are possibly derived from the rough er by membrane invaginations as suggested in some of the tem pictures ( figure 4i ). because the particles inside are larger than nucleocapsid cross sections, we believe that they likely do not represent assembled virions. however, it is theoretically possible that nucleocapsids may have deteriorated and swollen to approach the appearance of ribosomes due to autolysis. this article is protected by copyright. all rights reserved (too late: the virus has already been cleared by the immune system) and sensitivity (too insensitive: there are too few infected cells or too few virions such that detection by tem becomes very unlikely; the low viral loads found by quantitative nucleic acid amplification techniques (qnat) in tissues other than lung would support this argument). we think that the extensive intracellular membrane remodelling seen could be a result of direct infection, but this is difficult to prove in the absence of newly formed viral particles. ideally, tem morphology will be backed by other in-situ techniques in the same case. another important concept to keep in mind is that viral components (rna and proteins) are not produced in balanced amounts (as suggested in figure 1 and [113] ). therefore, surplus viral rna and proteins may be encountered at the site of infection, in the circulation, and at distant sites. detection of viral rna and proteins does not necessarily reflect the presence of intact and infectious particles. it is also conceivable that pathology in non-respiratory organs could be the result of viral disease distantly, due to transport of viral components, and not a direct result of infection. therefore, tem investigation is essential to verify assembled virions in sars-cov-2 infection and covid-19. in 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article this article is protected by copyright. all rights reserved endocytotic vesicles and vacuoles forty years of clathrin-coated vesicles transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles cellular copii proteins are involved in production of the vesicles that form the poliovirus replication complex endosome maturation the regulation of endosomal sorting complex required for transport and accessory proteins in multivesicular body sorting and enveloped viral budding -an overview cell organization, subcellular structure, and cell division comparison of rna in situ hybridization and immunohistochemistry techniques for the detection and localization of sars-cov-2 in human tissues key: cord-327997-noqbcxua authors: wu, kevin e.; fazal, furqan m.; parker, kevin r.; zou, james; chang, howard y. title: rna-gps predicts sars-cov-2 rna residency to host mitochondria and nucleolus date: 2020-06-20 journal: cell syst doi: 10.1016/j.cels.2020.06.008 sha: doc_id: 327997 cord_uid: noqbcxua abstract/summary sars-cov-2 genomic and subgenomic rna (sgrna) transcripts hijack the host cell's machinery. subcellular localization of its viral rna could thus play important roles in viral replication and host antiviral immune response. we perform computational modeling of sars-cov-2 viral rna subcellular residency across eight subcellular neighborhoods. we compare hundreds of sars-cov-2 genomes to the human transcriptome and other coronaviruses. we predict the sars-cov-2 rna genome and sgrnas to be enriched towards the host mitochondrial matrix and nucleolus, and that the 5’ and 3’ viral untranslated regions contain the strongest, most distinct localization signals. we interpret the mitochondrial residency signal as an indicator of intracellular rna trafficking with respect to double-membrane vesicles, a critical stage in the coronavirus life cycle. our computational analysis serves as a hypothesis generation tool to suggest models for sars-cov-2 biology and inform experimental efforts to combat the virus. a record of this paper’s transparent peer review process is included in the supplemental information. covid-19 (coronavirus disease 2019) has become a global pandemic, fueled by the rapid spread of the coronavirus sars-cov-2 (severe acute respiratory syndrome coronavirus 2), a positive strand rna virus (wu et al., 2020a , sanche et al., 2020 . the scientific community is actively trying to understand sars-cov-2's biological mechanisms and effects. here, we computationally analyze the subcellular localization patterns of sars-cov-2 rna transcripts. our results suggest potential avenues for experimental validation and follow-up, while providing a template for in silico analyses of viral rna. rna subcellular localization is critical to a myriad of cellular processes (ryder and lerit, 2018 , chin and lécuyer, 2017 , buxbaum et al., 2015 . researchers have also discovered that rna localization plays a significant role in the life cycle of viruses, with functions ranging from regulating sites of virion assembly (becker and sherer, 2017) to disrupting host mitochondrial function (somasundaran et al., 1994) . however, the subcellular localization of the sars-cov-2 rna, and other coronaviruses, is largely unexplored. gaining a better understanding of the behavior and localization of sars-cov-2's rna genome and transcripts can lead to a better understanding of its function and pathogenicity, potentially revealing targetable mechanisms. to computationally study this aspect of sars-cov-2 biology, we built upon our recent work developing rna-gps, a state-of-the-art computational model predicting high-resolution rna localization in human cells (wu et al., 2020b) . rna-gps was trained on transcriptome-wide localization patterns of human rnas across eight subcellular landmarks (fazal et al., 2019) . rna-gps's strong performance, coupled with viruses' dependence on hijacking and repurposing existing cell machinery for reproduction, suggests that rna-gps could provide insights into sars-cov-2's localization behavior and can focus future experimental efforts. we use rna-gps to interrogate the dominant subcellular residency patterns of sars-cov-2's genome, which spans approximately 30 kilobases of single-stranded positive-sense rna (kim et al., 2020 ) ( figure 1a ). rna-gps predicts that sars-cov-2 and the transcripts it forms have enriched residency at the nucleolus and the mitochondria. we note that our analysis may suggest potential localization mechanisms for sars-cov-2, rather than direct physical localization, particularly with regard to our mitochondrial prediction. comparison of sars-cov-2's predicted residency with that of other human coronaviruses, including strains causing the common cold, middle east respiratory syndrome (mers), and the sars outbreak of 2003, shows that sars-cov-2 exhibits a stronger mitochondrial and nuclear residency signal than a large majority of its coronavirus relatives. we additionally find that this residency signal appears to be driven by the 5' and 3' ends of the viral genome. we conclude by connecting our predictions to known rna and viral biology and proposing possible explanatory mechanisms for previously observed phenomena. our findings entreat experimental validation and serve as a framework for applying machine learning for principled hypothesis generation in viral biology. we leverage our recent work developing rna-gps, a computational model predicting high-resolution rna subcellular localization in human cells (wu et al., 2020b) . our model was built using apex-seq data, which fuses the engineered peroxidase apex2 protein to various protein localization sequences ( figure 1b , table s1 ) to guide apex2 to each subcellular region for subsequent proximity biotinylation of nearby rnas (fazal et al., 2019) . the resultant transcripts captured and measured at the nucleolus, for example, are transcripts proximal to apex2 in the nucleolus, as well as transcripts proximal to apex2 throughout its entire lifecycle including its transport to the nucleolus. such "en route" transcripts constitute a small proportion of total transcripts, except in the notable case of the mitochondrial matrix cox4 marker, which picks up a sizable proportion of nuclear-encoded transcripts as it is imported to the mitochondria ( figure s1a ). though this is surprising, these nuclear-encoded mitochondrial-enriched transcripts are reproducibly distinct from noise (figure s1b, s1c) and actually enrich for cytoskeletal and intracellular transport processes ( figure s1d ). for the sake of brevity, we will refer to these measurements using their final destinations, as confirmed by imaging: the cytosol, endoplasmic reticulum, mitochondrial matrix, outer mitochondrial membrane, nucleus, nucleolus, nuclear lamina, and nuclear pore. rna-gps predicts localization to each of these eight neighborhoods ( figure 1b ). although rna-gps is trained on human, not viral, rna transcripts, its ability to generalize across cell types not used in training, combined with the fact that viruses commandeer human cellular machinery suggest that it offers a reasonable hypothesis of viral transcript localization behavior given currently available data. nonetheless, there is inherent uncertainty associated with generalizing our model across species, and we use the term dominant subcellular residency to indicate this predictive uncertainty where appropriate. we consider viral transcript subcellular residency predictions to each compartment averaged across all released and annotated sars-cov-2 genomes available as of april 6, 2020 (n = 213) on genbank (coordinators, 2018) . sars-cov-2 is believed to enter the cell as a positive strand genomic rna, subsequently forming 11 positive strand sub-genomic rna (sgrna) transcripts encoding different open reading frames and sharing the same 5' leader sequence and 3' untranslated region (utr) ( figure 1a ). within each viral genome, we predict the residency of each sgrna produced from the primary sars-cov-2 genome. to better understand how strong these predicted residency probabilities are in a meaningful biological context, we frame them relative to predictions for other relevant baseline transcript sequences. we consider two such baselines: the distribution of model predictions on transcripts exhibiting significant localization within the human hek293t cell line (n = 366 transcripts) as measured by apex-seq (fazal et al., 2019) , and the distribution of model predictions on transcripts derived from human coronaviruses, excluding sars-cov-2 (n = 191 genomes, spanning diseases from the common cold to mers, table s2 ). the human baseline quantifies the strength of rna residency signals in sars-cov-2 relative to naturally occurring human transcripts with well-characterized localization behaviors. the coronavirus baseline focuses on differences in the transcript residency behavior of sars-cov-2 relative to similar viral specimens -differences that may help researchers focus on the peculiarities of this virus. for both baselines, we calculate the proportion of the baseline distribution that the sars-cov-2 localization prediction exceeds, which we refer to as a rank score. for example, a residency rank score of 0.6 for the nucleolus relative to human transcripts suggests that the particular viral rna is more likely to have been picked up by the nucleolus apex-seq marker compared to 60% of human rnas that are empirically measured to do so. we find that compared to transcripts with known localizations in human cells, sars-cov-2 has a notable residency signal towards the nucleolus, as well as the mitochondrial matrix ( figure 1c ). these residency signals are consistent across different sgrnas encoded by the virus (shown in each row, figure 1c ), and represent statistically significant predicted residency (table s3 ). the nucleolus is known to play a prominent role in the viral life cycle, even for viruses that primarily replicate in the cytoplasm as sars-cov-2 presumably does (salvetti and greco, 2014) . while some rna viruses like human immunodeficiency virus (hiv) exhibit transcript localization to mitochondria (somasundaran et al., 1994) , there has not been direct evidence that sars-cov-2 does this. as previously discussed, since much of the apex-seq mitochondrial data used to train rna-gps actually consists of nuclear-encoded transcripts likely picked up as the apex-cox4 fusion protein is transported to the mitochondria, we hypothesize that our predicted mitochondrial residency is alluding to similarity in localization pathways, rather than localization destination. in addition to framing our localization results in the context of endogenous human transcripts, we also compare predicted residency of sars-cov-2 sgrnas to that of other human coronaviruses ( figure 1d ). here, we observe similar overall trends in our residency predictions. consistent with the comparison to human transcripts, we find the sars-cov-2 mitochondrial matrix residency signal is stronger than that of many other coronaviruses. additionally, we see an overall pattern suggesting that sars-cov-2 may have a greater affinity for nuclear neighborhoods (nuclear pore, nucleus, nucleolus, and nuclear lamina) compared to other coronaviruses. we also compared the dominant subcellular residency patterns of the coronavirus family (excluding sars-cov-2) with human transcripts using rna-gps. we found that the most prominent residency signals for general human coronaviruses pointed towards the nucleolus, mitochondrial matrix, and er membrane ( figure s2 ). overall, our computational analysis suggests that sars-cov-2's predicted sgrna transcript residency enriching for the mitochondrial matrix and nucleolus may be amplifications of behaviors that were already present in coronaviruses. while direct experimental data measuring coronavirus sgrna transcript localization is not currently available, we sought to validate our predictions on other human viruses with known subcellular localizations. after conducting a systematic literature search, we found one such example: the human cytomegalovirus β2.7 mrna transcript, which localizes to the inner mitochondrial membrane (williamson et al., 2012) and is approximately 2.5 kilobases long. rna-gps predicts this transcript to reside at the mitochondrial matrix with a rank score of 0.81; no other compartments have a rank score exceeding 0.5 ( figure 2a ). thus, the algorithm's residency prediction is in close agreement with experimental evidence for β2.7 mrna localization. while large-scale comparisons are not currently feasible due to lack of datasets measuring viral transcript localization, this example provides some reassurance that rna-gps' predicted viral residencies are reasonable. to further validate the robustness of these results, we also trained a different predictive algorithm (a recurrent neural network, see star methods for additional details) on the apex-seq data and performed a similar set of experiments, comparing sars-cov-2 dominant subcellular residency predictions to human and coronavirus baselines ( figure s3a /b). this alternative model also predicts strong mitochondrial matrix and nucleolus residency for sars-cov-2. since this algorithm uses a very different modeling strategy from rna-gps and nonetheless converges to similar findings, this suggests that the mitochondrial matrix and nucleolus residency predictions are not artifacts of a particular computational modelling strategy and increases our confidence in our findings. in addition to evaluating robustness of our results to modelling strategies, we also evaluated robustness with respect to the apex-seq data used to train the models. as we previously mentioned, many apex-seq transcripts used to train rna-gps's mitochondrial predictions are actually nuclear-encoded. these transcripts exhibited relatively low (albeit significant) enrichment compared to transcripts natively encoded in the mitochondrial genome. to ensure our results have not been driven by potentially noisy data, we excluded nuclear-encoded, "non-canonical" mitochondrial matrix transcripts with relatively low apex-seq enrichment signal (lowest 20% of log fold change enrichment scores), and retrained rna-gps on this adjusted dataset. this denoised model recapitulates the same sars-cov-2 residency towards the mitochondrial matrix and nucleolus ( figure 2b ), suggesting that our predictions are robust to noise in the training data. in summary, our predicted residencies are robust across different modelling strategies, and across variation in the data used to train these models. sars-cov-2 negative strand rna also shows residency to mitochondria and nucleolus during their replication life cycle, coronaviruses like sars-cov-2 copy their positive strand rna to create a negative strand rna that serves as the template for viral "transcription" and production of sgrnas (wu and brian, 2010) . we applied rna-gps to the negative strand sars-cov-2 sgrna precursors and discovered that they also exhibit residency to the mitochondrial matrix and nucleolus ( figure s4 ). this result suggests that the sequence features driving these residency patterns are independently present in both positive and negative strand rnas, further boosting the localization capability of sars-cov-2 during different stages of its viral cycle. in addition to predicting residency, our computational model can also help understand which regions of the transcript may be more responsible for driving these predictions. at a high level, this can be done by evaluating which features were most important for rna-gps's predictions. we specifically investigated the potential contribution of the three main regions of the sars-cov-2 sgrnas: the shared 5' leader sequence, the shared 3' utr, and the variable "coding" sequence in the middle. we predicted residency for each of these regions by itself ( figure 1e ). the 5' leader sequence shows the strongest residency signal for the mitochondrial matrix, and relatively low signal for the nucleolus. in contrast, the 3' utr has the strongest residency for the nucleolus and also has a strong signal for the mitochondrial matrix. the coding sequence (cds) also shows specific signals for these two compartments. as the 5' and 3' sequences are shared by the different sars-cov-2 sgrnas, this is likely a strong factor behind the consistent residency patterns we predict across the different sgrnas. we also performed further computational ablation studies of rna binding protein (rbp) motifs in sars-cov-2. however, computational deletions of all instances of each individual rbp motif, repeated across all enriched rbps, did not significantly alter the rna-gps residency predictions. this result suggests that the sars-cov-2 residency signal could be abundant in the viral genome and may involve complex interactions not captured by relatively short single rbp binding motifs. in this work, we apply computational models of human rna transcript localization to better understand the subcellular localization behavior of the sars-cov-2 genome and its constituent sgrnas. this approach builds upon the idea that the virus uses existing human cell machinery to reproduce, and consequently that sequence-based localization signals are likely shared between human and coronavirus transcripts. the strengths of this approach include (1) the potential to understand viral rna localization without the risk of live viral cultures; (2) the ability to examine hundreds of viral isolates and related coronaviruses and thousands of rbp motif ablations; (3) the ability to examine viral genes, utrs, and negative strands individually, which may otherwise require the ability to precisely synchronize and arrest the viral life cycle. we find that sars-cov-2 appears to harbor strong transcript residency signals towards the mitochondrial matrix and nuclear compartments, often comparable to human rnas and more so than other coronaviruses. this intriguing hypothesis suggests future experimental exploration and validation. as we mentioned previously, we believe that our predicted mitochondrial residency signal is more indicative of a localization pathway than a destination -in the context of coronavirus biology, this may specifically be related to double membrane vesicles (dmvs). coronaviruses are known to produce dmvs to serve functions like concealing the virus from cellular defenses (hagemeijer et al., 2012 , knoops et al., 2008 . while these dmvs are generally believed to be formed via viruses manipulating the er membrane (blanchard and roingeard, 2015) , the mechanism for importing and packaging proteins and rna into these miniature organelles is not as clearly understood. one possible mechanism for importing viral rna involves the virus exploiting rna localization mechanisms that the cell already possesses for endogenous double-membrane organelles: namely, the mitochondria. indeed, introducing just two amino acid point mutations in the murine coronavirus causes both a significant drop in the number of dmv structures observed, as well as a sharp increase in viral protein localization at the mitochondria (clementz et al., 2008) . this alludes to a high degree of resemblance between dmv and mitochondrial localization mechanisms -leading to our hypothesis that our mitochondrial matrix residency predictions are capturing this similarity between the dmv and mitochondria. furthermore, dmvs have been shown to contain double-stranded rna (hagemeijer et al., 2012) ; our strand-agnostic residency predictions are concordant with this evidence and might even encourage formation of such complexes. under this model, sars-cov-2's strong mitochondrial residency signal relative to other coronaviruses may even contribute to its similarly high infectivity by increasing its efficacy in forming these dmv structures. another possible interpretation of these predicted residencies is that previously studied viral protein localizations are influenced by transcript-level localizations, a mechanism that is highly prevalent for proteins in normal human cells (blower, 2013) . protein-protein interaction studies performed on sars-cov-2 have found that its nsp5 (within orf1a), nsp13 (within orf1b), orf6, and orf10 proteins interact with host proteins that predominantly localize to nuclear compartments (gordon et al., 2020). the same study found that the orf9b protein, produced by the "n" sgrna, interacts with tomm70, a mitochondrial import receptor that plays a critical role in modulating interferon response -a key antiviral cellular defense pathway (liu et al., 2010) . in both cases, localized viral transcripts could help drive viral protein localization, enabling more focused protein-protein interactions. a limitation of our work lies in that it applies models trained on human rna transcript localization data to viral transcripts. it is possible that sars-cov-2 infection could alter the host subcellular structures and rna transport machinery so drastically that our learned localization patterns from human cells no longer hold. if rna-gps's predictions turn out to be wrong for this reason, this might suggest that coronavirus infection devastates host cell rna trafficking and localization -a previously unrecognized feature of covid-19 pathobiology. after all, the vast majority of rna binding proteins in the host cell, which are key drivers of transcript localization, recognize and process rnas irrespective of whether they are endogenous or foreign, and inability to "properly" localize viral rnas should mirror a similar breakdown for host cell transcripts. as we are unable to use existing experimental evidence to thoroughly evaluate and cross-reference the predictions discussed here, future experiments in this vein are clearly necessary. given the historical scarcity of studies focusing on viral transcript localization, such experiments would likely reveal interesting, crucial insights into viral pathobiology, whether they confirm our specific mitochondrial and nucleolus predictions or not. it is worth pointing out, though, that this is but one of many complex, interconnected viral mechanisms at play. in summary, we build upon recent computational models of rna subcellular localization to study, in silico, the localization properties of sars-cov-2 transcripts. our results suggest that predicted transcript residency signals, specifically towards the nucleolus and mitochondrial matrix, may be important, unique characteristics of sars-cov-2 that warrant additional study. we connect these observations to known viral biology regarding dmv structures in viral replication, as well as sars-cov-2 protein localization patterns. in doing so, we propose potential cellular mechanisms that underpin viral biologymechanisms that warrant experiments validating their accuracy, and perhaps even their potential as therapeutic targets. more broadly, we hope that our study helps define a framework for applying machine learning models to enable focused hypothesis generation, enabling similar studies that leverage data science to rapidly respond to emerging epidemiological challenges. in the interest of transparency, the following changes were made in this paper during review. we used "rna subcellular residency" rather than "rna localization" to describe rna-gps prediction results, as this is more reflective of the underlying apex-seq training data and its inherent differences compared to viral transcripts. we clarified the origin, specificity, and interpretation of nuclear-encoded transcripts enriched by the cox4-apex2 mitochondrial matrix landmark, thus enhancing our interpretation of this predicted localization. we added a positive control showing a cmv mrna with known mitochondrial localization is correctly predicted by rna-gps. we thank the reviewers and editor for insightful comments that have improved this work. for context, the complete transparent peer review record is included within the supplemental information. u01mh098953 and grants from the silicon valley foundation and the chan-zuckerberg initiative. f.m.f. is supported by an nih k99/r00 award from nhgri (hg010910). author contributions h.y.c. and j.z. conceived the idea for this project and supervised its execution. k.e.w. gathered, preprocessed, and analyzed data for this project with input from all authors. f.m.f. and k.r.p. contributed analysis of mitochondrial apex-seq and fish data with input from all authors. all authors contributed to interpreting localization results in the context of coronavirus biology. k.e.w. wrote the manuscript with input from all authors. declaration of interests k.r.p. is a consultant for maze therapeutics. h.y.c. is affiliated with accent therapeutics, boundless bio, 10x genomics, arsenal bio, and spring discovery. j.z. is affiliated with intervenn biosciences. figure 1: depictions of the sars-cov-2 genome (a), the eight compartments that rna-gps predicts viral transcript residency to (b), and the predicted residencies for sars-cov-2 sgrnas (c, d) and its 5'/cds/3' sequence segments (e). the sars-cov-2 genome produces a series of sub-genomic rnas (sgrnas), each encoding one or more genes/proteins (a). these sgrnas share a common leader 5' sequence and a common trailing 3' utr sequence (arrow blocks). for each sgrna, rna-gps predicts residency to each compartment in (b). italicized text indicates the apex2 fusion protein used to measure transcripts corresponding to each localization (see table s1 ). (c) heatmap of rank scores, indicating how strongly each sgrna (rows) is predicted to exhibit subcellular residency at each compartment (columns), compared to endogenous human transcripts measured to localize to that compartment. colors indicate rank scores; color scale is shared across all heatmaps. most sgrnas share similar residency patterns, exhibiting statistically significant enrichment towards the mitochondrial matrix and nucleolus (see table s3 ). we also computed these rank scores against a baseline of other coronavirus residency signals (d). sars-cov-2 exhibits a stronger mitochondrial matrix residency signal than most other coronaviruses, along with greater overall nuclear residency, particularly at the nucleolus. for context, coronaviruses are generally predicted to have residency at the nucleolus, mitochondrial matrix, and er membrane (see figure s2 ). these predictions are also consistent across different models (see figure s3 ) and negativestrand sars-cov-2 sgrna precursors (see figure s4 ). (e) shows the predicted residency rank scores for shared 5' and 3' segments, and an averaged residency rank score for the variable coding segments. even on their own, the short ~90-250 base pair 5' and 3' segments carry mitochondrial and nucleolar residency signals. figure 1e ). for additional analysis of the mitochondrial dataset and predictions, see figure s1 . resource availability further information and requests for resources should be directed to and will be fulfilled by the lead contact, howard chang (howchang@stanford.edu). this computational study did not generate or use new reagents. the data supporting the findings of this study are all available within publicly available repositories as listed in the key resources table. all code required to query and download viral sequences, as well as to reproduce results and figures can be found within the github repository listed in the key resources table. all software dependencies for rna-gps and the sars-cov-2 analysis described herein are freely available as well. within the github repository, most code pertaining to sars-cov-2 analysis can be found under the "covid19" folder; other folders contain supporting data and source code. obtaining viral genomes sars-cov-2 viral genomes were programmatically queried from the ncbi genbank online database using the biopython library's entrez module (cock et al., 2009) . the exact query sequence used can be found within the "covid19/covid19.py" file in the github repository. returned results were then filtered to retain only assemblies that included annotated, named sgrna "genes." we consider the sgrnas corresponding to orf1ab, s, orf3a, e, m, orf6, orf7a, orf7b, orf8, n, and orf10, as these have the most consistent annotations. in cases where the shared 5' leader sequence or the 3' tail were not explicitly annotated, their regions were inferred to be the 5' and 3' trailing bases outside of any coding regions, respectively. as there are many sars-cov-2 genome assemblies that fit these criteria, subcellular residency predictions are averaged across all genomes. viral genomes constituting the coronavirus baseline follow an identical process, save for using a different ncbi genbank query sequence that specifically fetches matches to the six coronaviruses known to infect humans (excluding sars-cov-2): 229e, nl63, oc43, hku1, mers-cov (beta coronavirus that causes middle east respiratory syndrome, or mers), and sars-cov (the beta coronavirus that causes severe acute respiratory syndrome, or sars) (su et al., 2016) . the exact query sequence used can be found in the "covid19/baseline.py" source file in the github repository. a detailed breakdown of the exact number of genomes we use from each strain is in table s2 . the human cytomegalovirus was chosen for additional evaluation based on a systematic literature review of viral rna localization studies. this is the only example we found that associates a specific viral transcript with a consistent experimentally validated localization. the viral sequence for validation of our model predictions was obtained from the ncbi genbank reference sequence nc_006273.2. due to lack of standardized 5' and 3' utr region annotations for this transcript (despite these being referenced in the literature), we manually determined these regions after reviewing literature and the overall genome annotation. rna-gps uses k-mer featurization with k = 3, 4, 5, applied independently to the 5' untranslated region (utr), coding sequence (cds), and 3' utr parts of the transcript (wu et al., 2020b) . this creates a feature space of (4 3 + 4 4 + 4 5 ) x 3 = 1344 x 3 = 4032 dimensions. these features are then consumed by a random forest model (implemented using the scikit-learn python library) to generate localization/residency predictions. extending this definition to the coronavirus sgrna sequences, we consider the shared 5' leader sequence the fixed 5' utr input to our model, shared 3' utr sequence the fixed 3' utr input to our model, and the variable sgrna sequence the "cds" input. for sake of consistency with sgrna transcript mechanisms, this "cds" sequence includes the current reading frame, along with any 3' downstream bases until the shared 3' utr region begins. each sgrna is individually assigned predicted residencies. rna-gps's per-segment featurization also enables the per-segment residency analysis. for this, we selectively provide the model with only features that correspond to a single segment (i.e. the 5' utr, cds, or 3' utr), with zero values for other features. for the deep recurrent model, we implemented and trained a recurrent neural network that consumes raw bases as input, maps these to a 32-dimensional embedding layer, passes these through two 64dimensional gated recurrent units (gru), and finally a fully-connected layer with sigmoid activation producing 8 localization/residency predictions. this flavor of gru network is popular in sequence modelling and uses "gating" mechanisms to improve learning of longer-range sequence dependencies (chung et al., 2014) . the model was implemented in pytorch and was trained to minimize a binary cross-entropy loss using the adam optimizer (kingma and ba, 2014 ) with a batch size of 1, with early stopping based on validation set area under the receiver operating characteristic (auroc). both rna-gps and the gru model are trained and tuned on the same apex-seq data, measuring localization within hek293t cells (fazal et al., 2019) . localization within this dataset is expressed as an enrichment score compared to the rest of the cell. we consider transcripts that exhibit significant enrichment (log fold change (logfc) > 0 and adjusted p-value ≤ 0.05) for at least one of the eight measured compartments (n = 3660). many transcripts contain more than one significant localization. furthermore, due to the nature of the apex-seq technology, transcripts measured at a specific compartment may also contain transcripts that were picked up as the apex2 labelling protein itself was being transported to that compartment. this effect is usually minimal, except for mitochondrial transcripts (see figure s1 ). we use data splits of 80% train (n = 2928), 10% validation (n = 366), and 10% train (n = 366). as is conventional, the validation set was used for hyperparameter tuning and model architecture tuning. when removing potentially spurious mitochondrial examples, we start with the above dataset and remove all transcripts that measured to localize to the mitochondrial matrix but have log fold change enrichment in the bottom 20th percentile of localized mitochondrial matrix transcripts. this removes the bottom 20% of mitochondrial sequences with the lowest enrichment relative to the rest of the cell (n = 61) -this denoised dataset contains 240 mitochondrial matrix transcripts instead of 301, and a total of 3599 transcripts compared to 3660 previously. we use a database of 102 rna binding protein binding motifs (ray et al., 2013) . to identify matches, we use the same methodology as was used in the rna-gps manuscript (wu et al., 2020b) . we start with the position weight matrix (pwm) that describes the motif, adjust its probabilities to account for the background nucleotide composition of each transcript sequence, define a cutoff score slightly lower than the maximum achievable log-likelihood for that pwm, and identify any subsequences that exceed that cutoff. when ablating these pwms, we use the same methodology for identifying hits, and subsequently replace all hits with "n" bases, re-featurizing the ablated sequence as necessary before feeding into the model, thus generating the ablated localization predictions. baseline construction and rank score baseline distributions are constructed by running a set of baseline transcript sequences through a model predicting transcript localization/residency. for each individual model, there is a per-localization baseline derived from human apex-seq measurements, and one derived from human coronaviruses excluding sars-cov-2. for each localization neighborhood within the human baseline, we consider only transcripts that exhibit significant localization to that neighborhood, as defined by having a logfc > 0 and adjusted p-value ≤ 0.05 when running differential expression analysis against the remainder of the cell. additionally, we only use transcripts not used for model training/tuning (i.e. the test data split), as this most closely approximates what the model would predict when presented with novel sequences. for the coronavirus baseline, we do not have systematically measured localization data, so we cannot constrain this baseline using known localizations behaviors. instead, each sars-cov-2 sgrna is compared only to homologous sgrnas from other coronaviruses. for example, the spike protein's residency prediction is only compared against residency predictions of other coronavirus spike proteins. this limits our comparison to the set of genes with easily traceable homology across human coronaviruses, namely orf1ab, spike (s), envelope (e), membrane (m), and nucleocapsid (n) (woo et al., 2010) . for both these baselines, we define a rank score as the proportion of baseline values that a sars-cov-2 sgrna residency prediction exceeds. a hypothetical value of 0.5 would correspond to a median, 0.25 would correspond to the first quartile, etc.; rank score is thus bound between 0 and 1 (inclusive). note that this rank is calculated for each individual compartment separately, as the baselines themselves are compartment specific. as previously discussed, subcellular residency predictions are averaged across all valid sars-cov-2 genomes prior to calculating rank scores. furthermore, since the human baseline is constrained by measured localizations, whereas the coronavirus baseline is constrained by sequence homology, rank scores for these two baselines are not directly comparable. in addition to computing the rank scores described above, we also evaluate whether these rank scores correspond to significant enrichment. to do this, we compare the underlying predicted residency probabilities (not the rank scores) against a "null" distribution of localization probabilities for human transcripts exhibiting no significant localization. we do this using a one-sided wilcoxon rank-sum test (scipy python package (virtanen et al., 2020) , with the hypothesis that residency probabilities exceed that of the null distribution. our data satisfies the wilcoxon rank-sum test's assumptions of independence, and our residency/localization prediction probabilities are naturally ordinal. to address the fact that we do multiple comparisons, we use the holm method (statsmodels python package (seabold and perktold, 2010) ) to correct the resultant p-values. the sequential fish experiments were described in (fazal et al., 2019) , and resulted in data for 29 transcripts retained for further analysis. the analysis was described in (fazal et al., 2019) , and briefly consists of compiling imaging data from 20 fields of view, each with > 20 cells, and with the data processed using matlab. for the quantification of each field of view, a mask was generated for each gene of interest using a uniform threshold cutoff of 0.5-0.998, after removing non-cell pixels. the colocalization score with mitochondria was calculated by interesting the mask of a particular gene with the mask of the mitochondrial-resident transcript mt-nd3, and then dividing the summed intensity of the intersected mask by the summed intensity of the gene masks of interests. the quantification results for all 20 fields of view were then averaged to obtain the final number. to perform gene ontology enrichment analysis, we used the panther tool (mi et al., 2018) provided by the gene ontology consortium (ashburner et al., 2000 , the gene ontology, 2019 . genes were compared in an overrepresentation test against a reference list of all genes in the homo sapiens database using fisher's exact test, with false discovery rate correction. the annotation used was "reactome version 65." plots were generated using a combination of seaborn and matplotlib python packages (hunter, 2007) . highlights (4-5 bullets, 85 characters each) • application of machine learning model of rna subcellular localization to sars-cov-2 • viral rnas show residency signal for host mitochondria and nucleolus • mitochondria prediction suggests viruses repurpose endogenous pathways • predictions may be linked to vesicle formation and viral-host protein interactions etoc blurb (limit 50 words) where the sars-cov-2 genome localizes inside human cells remains understudied but may regulate viral replication and host response. we use a machine learning model to predict subcellular residency of the sars-cov-2 genome and its encoded transcripts, as well as for other coronaviruses. our predictions suggest new hypotheses for sars-cov-2 mechanisms. the table highlights the genetically modified organisms and strains, cell lines, reagents, software, and source data essential to reproduce results presented in the manuscript. depending on the nature of the study, this may include standard laboratory materials (i.e., food chow for metabolism studies), but the table is not meant to be comprehensive list of all materials and resources used (e.g., essential chemicals such as sds, sucrose, or standard culture media don't need to be listed in the table) . items in the table must also be reported in the method details section within the context of their use. the number of primers and rna sequences that may be listed in the table is restricted to no more than ten each. if there are more than ten primers or rna sequences to report, please provide this information as a supplementary document and reference this file (e.g., see table s1 for xx) in the key resources table. please report the information as follows: • reagent or resource: provide full descriptive name of the item so that it can be identified and linked with its description in the manuscript (e.g., provide version number for software, host source for antibody, strain name). in the experimental models section, please include all models used in the paper and describe each line/strain as: • source: report the company, manufacturer, or individual that provided the item or where the item can obtained (e.g., stock center or repository). for materials distributed by addgene, please cite the article describing the plasmid and include "addgene" as part of the identifier. if an item is from another lab, please include the name of the principal investigator and a citation if it has been previously published. if the material is being reported for the first time in the current paper, please indicate as "this paper." for software, please provide the company name if it is commercially available or cite the paper in which it has been initially described. • identifier: include catalog numbers (entered in the column as "cat#" followed by the number, e.g., cat#3879s). where available, please include unique entities such as rrids, model organism database numbers, accession numbers, and pdb or cas ids. for antibodies, if applicable and available, please also include the lot number or clone identity. for software or data resources, please include the url where the resource can be downloaded. please ensure accuracy of the identifiers, as they are essential for generation of hyperlinks to external sources when available. please see the elsevier list of data repositories with automated bidirectional linking for details. when listing more than one identifier for the same item, use semicolons to separate them (e.g. cat#3879s; rrid: ab_2255011). if an identifier is not available, please enter "n/a" in the column. o a note about rrids: we highly recommend using rrids as the identifier (in particular for antibodies and organisms, but also for software tools and databases). for more details on how to obtain or generate an rrid for existing or newly generated resources, please visit the rii or search for rrids. please use the empty table that follows to organize the information in the sections defined by the subheading, skipping sections not relevant to your study. please do not add subheadings. to add a row, place the cursor at the end of the row above where you would like to add the row, just outside the right border of the table s1 (related to figure 1 ): apex2 fusions used to measure localization of transcripts. apex2 is responsible for labelling, while the protein (segments) it is fused to drive its localization. additional information regarding the apex-seq protocol and data can be found in the original apex-seq manuscript (fazal et al., 2019) , particularly figure s2 . transcripts picked up by apex2 (both en route and upon arrival at each fusion's final destination) are used to train the rna-gps model. , and is used to localize apex to the mitochondria as shown in this illustration. many transcripts thus picked up by cox4 that nominally localize at the mitochondrial matrix are actually nuclear-encoded. we hypothesize that these are picked up as the apex2-cox4 fusion is transported from cytosol to mitochondria (final arrow). (b) sequential fish data showing fraction of transcripts colocalizing at the mitochondria (using the mitochondrial-resident mtnd5 rna as a mitochondrial marker, as described in (fazal et al., 2019) ). nuclear transcripts like xist and neat1 do not show mitochondrial enrichment, while transcripts known to localize to the outer surface of the mitochondria like scd and iars2 are enriched, providing negative and positive controls, respectively. within this range, "non-canonical" nuclearshows a plot of apex-seq log fold-change enrichment scores at each compartment for the 251 mitochondrial-enriched, nuclear-encoded "non-canonical" transcripts used to build rna-gps. we see that these transcripts have enrichment centered around 0 for all but the mitochondrial matrix, indicating that while these transcripts are nuclear-encoded, the apex-seq labelling technology consistently and uniquely associates them with the mitochondrial matrix, and are thus not noise. these transcripts are also biologically meaningful, as shown by a reactome ontology analysis of the 100 most enriched (by p-value) nuclear-encoded mitochondrial matrix transcripts (d). there is a clear emphasis for cytoskeletal and intracellular transport terms (e.g. kinesins, post-chaperonin tubulin folding pathway, recruitment of numa to mitotic centrosomes; adjusted p < 0.05). this supports the interpretation that many of these non-canonical transcripts are picked up as the apex-seq protein is itself trafficked to the mitochondria. figure s2 (related to figure 1 ): summary of residency patterns aggregated across all transcripts comprising the human coronavirus baseline. we see that coronaviruses in general primarily exhibit residency towards the nucleolus, mitochondrial matrix, and er membrane -a pattern similar to that seen in sars-cov-2's sgrnas (albeit less dramatic). figure 1c shows that the positive strand sgrna transcripts tend to exhibit residency towards the mitochondrial matrix and nucleolus. here, we look at the negative-strand precursors to those sgrnas and observe that these transcripts share similar mitochondrial matrix and nucleolus residency patterns. this suggests another layer of conservation of this predicted residency signal. gene ontology: tool for the unification of biology. the gene ontology consortium subcellular localization of hiv-1 <em>gag-pol</em> mrnas regulates sites of virion assembly virus-induced double-membrane vesicles chapter one -molecular insights into intracellular rna localization in the right place at the right time: visualizing and understanding mrna localization rna localization: making its way to the center stage empirical evaluation of gated recurrent neural networks on sequence modeling mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles biopython: freely available python tools for computational molecular biology and bioinformatics database resources of the national center for biotechnology information atlas of subcellular rna localization revealed by apex-seq visualizing coronavirus rna synthesis in time by using click chemistry matplotlib: a 2d graphics environment the architecture of sars-cov-2 transcriptome adam: a method for stochastic optimization sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum tom70 mediates activation of interferon regulatory factor 3 on mitochondria panther version 14: more genomes, a new panther go-slim and improvements in enrichment analysis tools a compendium of rna-binding motifs for decoding gene regulation mitochondrial protein synthesis adapts to influx of nuclear-encoded protein rna localization regulates diverse and dynamic cellular processes viruses and the nucleolus: the fatal attraction. biochimica et biophysica acta (bba) -molecular basis of disease 1842 the novel coronavirus, 2019-ncov, is highly contagious and more infectious than initially estimated. medrxiv statsmodels: econometric and statistical modeling with python localization of hiv rna in mitochondria of infected cells: potential role in cytopathogenicity epidemiology, genetic recombination, and pathogenesis of coronaviruses the gene ontology resource: 20 years and still going strong scipy 1.0: fundamental algorithms for scientific computing in python viral product trafficking to mitochondria, mechanisms and roles in pathogenesis coronavirus genomics and bioinformatics analysis a new coronavirus associated with human respiratory disease in china subgenomic messenger rna amplification in coronaviruses rna-gps predicts high-resolution rna subcellular localization and highlights the role of splicing we thank the members of the chang and zou laboratories for helpful discussions. we thank shuo han, alistair boettiger and alice ting for generating and analyzing fish images presented herein. h.y.c. is supported by rm1-hg007735 and r01-hg004361. h.y.c. is an investigator of the howard hughes medical institute. j.z. is supported by nsf ccf 1763191, nih r21 md012867-01, nih p30ag059307, nih key: cord-324102-75v4ebag authors: garcia rodriguez, alejandro; marcos contreras, sergio; fernandez manovel, santiago manuel; marcos vidal, jose miguel; diez buron, fernando; fernandez fernandez, camino; riveira gonzalez, maria del carmen title: sars-cov-2 infection during pregnancy, a risk factor for eclampsia or neurological manifestations of covid-19? case report date: 2020-10-06 journal: bmc pregnancy childbirth doi: 10.1186/s12884-020-03275-2 sha: doc_id: 324102 cord_uid: 75v4ebag background: there are no published cases of tonic-clonic seizures and posterior bilateral blindness during pregnancy and severe acute respiratory syndrome (sars) coronavirus (cov) 2 (sars-cov-2) infection. we do not just face new and unknown manifestations, but also how different patient groups are affected by sars-cov-2 infection, such as pregnant women. coronavirus disease 2019 (covid-19), preeclampsia, eclampsia and posterior reversible leukoencephalopathy share endothelium damage and similar pathophysiology. case presentation: a 35-year-old pregnant woman was admitted for tonic-clonic seizures and sars-cov-2 infection. she had a normal pregnancy control and no other symptoms before tonic-clonic seizures development. after a caesarean section (c-section) she developed high blood pressure, and we initiated antihypertensive treatment with labetalol, amlodipine and captopril. few hours later she developed symptoms of cortical blindness that resolved in 72 h with normal brain computed tomography (ct) angiography. conclusion: the authors conclude that sars cov-2 infection could promote brain endothelial damage and facilitate neurological complications during pregnancy. the vast majority of patients affected by sars-cov-2 infection present flu-like symptoms [1, 2] . however, as the pandemic evolves, a broader, diverse and unknown spectrum of symptoms can be observed. an increasing number of patients with neurological symptoms have been reported [3] [4] [5] [6] . these manifestations can go from mild and common symptoms such as anosmia or ageusia, to severe ones and less common such as a cerebrovascular accident [3] [4] [5] [6] . symptoms of extrapulmonary disease could be explained by the damage of the endothelial tissue caused by the virus [7] . with this new virus, we do not just face new and unknown manifestations, but also how different patient groups are affected by sars-cov-2 infection, such as pregnant women. that is the reason why we present a case report of a pregnant woman infected with sars-cov-2 who showed seizures and sudden blindness. we describe 35-year-old pregnant women with just hypothyroidism treated with thyroxin 88 μg every 24 h as personal background and optimal blood pressure control during pregnancy, in her 40 weeks and 6 days of gestation. pregnancy has been controlled by hospital protocols with no evidence of obstetric risk factors. with no clinical manifestations the previous days, the patient was brought to our hospital due to generalized tonic-clonic seizures at home. the first suspicion was of eclampsia, and that was why an emergency c-section was performed. the same day the diagnosis of sars-cov-2 infection was confirmed by immunoglobulin rapid test (i-rt) and nasopharyngeal polymerase chain reaction (pcr) of the virus. the only abnormal results in the blood test at arrival to the hospital were lactic acid dehydrogenase (ldh) of 336 ui/i and d-dimer of 5781 ng/ml. during her hospitalization, the blood creatinine values were within the normal range. the procedure was performed with general anaesthesia. induction was made with propofol 200 mg and rocuronium 100 mg. after birth, we started fentanyl 300 μg and sevoflurane for maintenance of the anaesthesia without complications, completing postoperative care in the intensive care unit. after c-section, we initiated treatment with intravenous magnesium sulphate aiming levels between 4 to 6 mg/dl, she developed high blood pressure with values of systolic pressure higher than 160 mmhg and diastolic pressure higher than 100 mmhg and we started intravenous labetalol 60 mg every hour for blood pressure control. after that, we introduced oral treatment with labetalol 100 mg every 12 h, captopril 25 mg every 12 h and amlodipine 5 mg every 12 h. it allowed lowering the dose of intravenous labetalol progressively until suspension. twenty-four hours after c-section, the risk index soluble fms-like tyrosine kinase-1/ placental growth factor (sflt-1/plgf) was 51.4988. few hours after c-section, the patient showed sudden blindness without other neurologic symptoms. regarding to visual acuity: she could appreciate lights and shadows, being impossible with her degree of vision loss to achieve a low vision testing such as counting fingers or noticing moving objects with both eyes. also, she had absence of the bilateral menace reflex (blink-to-threat reflex). the rest of the cranial nerves and neurological exam showed no pathological findings. due to these findings, a brain ct-scan and ctangiography were performed, existing no anomalies in these tests. we notified the case to the neurology department who suspected posterior reversible leukoencephalopathy as a primary diagnosis, although there was no nuclear magnetic resonance confirmation. because of this suspicion, we began treatment with subcutaneous enoxaparin 40 mg every 24 h. the vision of the patient was recovered progressively during the first 48 h and the next day she was moved to a covid-19 hospitalization area with antithrombotic and antihypertensive treatment. currently, she has not any neurological symptoms and normal blood pressure without treatment. the main manifestation of covid-19 is influenza-like symptomatology but there are others such as anosmia or ageusia [1] [2] [3] [4] [5] [6] . not much has been said about different affectations in pregnant women or if this virus has any impact on the foetus. but it is important to have in mind that other coronaviruses such as sars or middle east respiratory syndrome (mers) seems to increase complications such as preeclampsia [8] . in this case, we have a pregnant woman with positive covid-19 i-rt and pcr that shows severe neurological symptoms. at first, the suspicion was eclampsia missing a diagnosis of preeclampsia during pregnancy, even when she had all the usual controls of pregnancy done and correct. the patient did not present any blood pressure alterations during those controls or in the ultrasound of the placenta. also, she did not present any other suggestive clinic of preeclampsia. twenty-four hours after c-section a risk index sflt-1/plgf was requested resulting high (51.4988) for diagnosis of preeclampsia in the next 4 weeks, but low for an imminent complication. the halflife of sflt-1 is estimated in 1,4 days, while the half-life of plgf is 3,7 [9] , therefore the risk would not change. we also have the suspicion of neurological manifestations of covid-19, where seizures are present in 0,7% of the cases [10] , but she did not have any other clinical symptoms of covid-19 such as headaches, ageusia or anosmia. seizures are associated with previous history of cognitive impairment, older age, higher creatine kinase levels and higher c-reactive protein [10] . sars-cov-2 has been identified in brain endothelial cells [3] . eclampsia, posterior reversible leukoencephalopathy and covid-19 share a common pathophysiology affecting endothelial tissue [11, 12] . therefore, we consider that sars-cov-2 infection during pregnancy could increase the risk of suffering posterior reversible leukoencephalopathy or preeclampsia/eclampsia syndrome. to conclude, we believe sars-cov-2 infection could promote brain endothelial damage, triggering the cited neurological complications in our patient. to our knowledge, this is the first report of a patient with covid-19 presenting preeclampsia associated with eclampsia versus posterior reversible leukoencephalopathy without alarm signs or symptoms. we consider further studies are needed to confirm that sars-cov-2 infection is a risk factor to develop neurological complications of pregnant woman during pregnancy. the patient is included in other multicentre studies as patient with covid-19, same as the rest of patients with covid-19 in our hospital. but there is no report or description of this particular case submitted. authors' contributions agr contributed to data collection, follow up and writing of the manuscript. smc contributed to data collection, follow up and writing of the manuscript. cff was the obstetrician of the patient, contributed to data collection and interpretation of the risk index sflt-1/plgf. smfm was the anaesthesiologist of the patient during the c-section, contributed to the initial diagnosis of eclampsia, interpretation of the first attention of the patient, as well as answering questions of the reviewers. jmmv and fdb were the anaesthesiologists at critical care department, contributed to data collection, writing of the manuscript and review of the hypothesis of endothelial damage. mcrg was the neurologist who contributed to the differential diagnosis, interpretation of the brain ct-scan and its concordance with the neurological exam. all authors have read and approved the final manuscript. none. availability of data and materials data are available on request due to privacy or other restrictions. the data that support the findings of this study are available on request from the corresponding author smc. the data are not publicly available due to them containing information that could compromise research participant privacy/ consent. written consent was obtained from the patient to participate. according with the care guidelines, case reports do not need ethics committee approval [13] . this manuscript fulfils the declaration of helsinki [14] . written consent was obtained from the patient for publication. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china sars-cov-2: olfaction, brain infection, and the urgent need for clinical samples allowing earlier virus detection selfreported olfactory and taste disorders in patients with severe acute respiratory coronavirus 2 infection: a cross-sectional study sudden and complete olfactory loss function as a possible symptom of covid-19 endothelial cell infection and endotheliitis in covid-19 coronavirus in pregnancy and delivery: rapid review soluble fms-like tyrosine kinase-1 and placental growth factor kinetics during and after pregnancy in women with suspected or confirmed pre-eclampsia neurologic manifestations in hospitalized patients with covid-19: the albacovid registry incidence of posterior reversible encephalopathy syndrome in eclamptic and patients with preeclampsia with neurologic symptoms posterior reversible encephalopathy syndrome in 46 of 47 patients with eclampsia the care guidelines: consensus-based clinical case reporting guideline development world medical association declaration of helsinki: ethical principles for medical research involving human subjects publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors do not have any conflict of interest and this publication does not have any funding. key: cord-341234-2zgfcrwc authors: hallak, jorge; esteves, sandro c. title: concise practice recommendations for the provision of andrological services and assisted reproductive technology for male infertility patients during the sars-cov-2 in brazil date: 2020-09-02 journal: int braz j urol doi: 10.1590/s1677-5538.ibju.2020.06.03 sha: doc_id: 341234 cord_uid: 2zgfcrwc nan the pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars--cov-2), responsible for the disease so-called co-vid-19, represents the most exceptional health, social, economic, and humanitarian crisis known to humankind since the h1n1 flu of 1918. so far, it affected 213 countries, infecting over 100,000 people daily worldwide, with hundreds of thousands of deaths (2, 3) . restrictions to personal freedom and partial or complete lockdowns have been implemented to safeguard public health, with a noticeable impact on urological practice. currently, diagnostic semen analysis, sperm banking in non-oncological patients, elective surgical sperm retrieval (sr), and related fertility procedures are rated as of low priority in most countries due to the covid-19 pandemic (4) . based on expert best judgment, regulatory authorities, urological, and reproductive medicine societies have considered that postponing care in the above scenarios for six months or longer will have an unlikely risk of clinical harm. however, it has been argued that the pandemic may last many months, even years, and experts believe of second and third waves in the months to come. it is therefore evident that in the absence of a vaccine or broad herd immunity, not only urgent short-term responses but also long-term measures are essential in this most uncertain time. recently, a group of 27 experts from 15 countries and five continents has argued that postponing andrological services and male infertility care during the covid-19 pandemic could permanently compromise the prospects of biological parenthood for 'time-sensitive' patients, thus resulting in a devastating psychological impact on men undergoing fertility-related treatment (1) . the group utilized the term 'timesensitive' to categorize patients in whom the fertility 'window' may be transitory, as listed below ( figure-1 ). 1. severe male infertility (e.g., azoospermic/cryptozoospermic men under medical or post-surgical treatment to improve sperm quantity/quality); 2. inflammatory and systemic autoimmune diseases; 3. cancer, chemotherapy, radiation or immunosuppressive therapy; 4. advanced paternal age (e.g., >50 years). 5. practice recommendations for safe care delivery. an evidence-based analysis was conducted concerning the reasons why these patients should be prioritized for care delivery during the pandemic. moreover, the group of experts developed a series of practical recommendations on how to most optimally provide care in a safe environment, considering patients, staff, and the community as a whole (table-1 ). the intended goals were to, first of all, alleviate the adverse impact of the sars-cov-2 pandemic in the months to come, thus offering reproductive urologists and patients alike greater autonomy, and secondly, help authorities and healthcare providers identify which male infertility patients should be prioritized during the sars-cov-2 pandemic for the continuation of care in a safe environment. brazil has become the new epicenter of the sars-cov-2 pandemic, with over 2.5 million confirmed cases making it the second country in absolute number of deaths after the united states. the death toll is growing steadily, with a significant number of daily deaths in the lower four figures. experts believe the situation can deteriorate further due to the typical economic constraints in our country, in particular, if containment measures, including the widespread use of facial masks, and social distancing do not work properly. a recent probabilistic pilot study conducted in seven districts of the city of são paulo to estimate the prevalence of herd immunity showed that about 5.2% individuals had sars-cov-2 igg antibodies, corresponding to an overall infection rate additional remarks 1. before any service is provided, active sars-cov-2 infections and suspected cases should be excluded. patient testing should include rt-pcr and/or blood antibody, preferably using elisa. ideally, only samples from patients with negative pcr results or who have acquired igg antibodies through past infection or herd immunity should be treated or have sperm cryopreserved. 2. andrological services (e.g., diagnostic semen analysis, sperm functional tests and sperm cryopreservation) should not only be available for oncological patients, but also for the group of 'time-sensitive' patients. i. severe male infertility under medical or surgical treatment aiming at improving sperm quantity or quality (e.g., patients with non-obstructive azoospermia (noa) or cryptozoospermic/severe oligozoospermia, including post-varicocele repair, and those with evidence of loss of patency after successful surgical reconstruction of the reproductive tract). ii. men at reproductive age affected by inflammatory diseases or systemic inflammatory diseases (sads), i.e., before initiation of gonadotoxic therapy or if under the 'fertility window' achieved after temporary (at least three months) discontinuation of therapy. iii. infertile men older than 50 years, in particular those with comorbidities who are candidates for intrauterine insemination (iui) or assisted reproductive technology (art), and who are concerned about the risk of acquiring sars-cov-2 and/or the possibility of using anti-viral therapy with possible gonadotoxic effects. 3. surgical sperm retrieval and cryopreservation of testicular sperm or testicular tissue should be considered in specific situations involving men with noa undergoing medical therapy to improve spermatogenesis. in this setting, procedures should be performed only for pcr negative or igg positive patients. the use of electrocautery should be used with caution or used with an air-aspiration negative pressure device; the surgical smoke might carry the virus in case a patient is infected but asymptomatic, despite a negative testing. only essential staff should stay in the operating theater, and personal protection measures should be strictly followed as determined by the local healthcare authorities. in closed-controlled air systems, the airflow might produce an increase in the viral spread from potential asymptomatic patients. thus, special attention should be given to air quality control, including the use of air filtration systems, particularly in surgical and laboratory areas. for providing instructions about testing and sperm banking. none. this advice includes the use of appropriate personal protective equipment (ppe) by healthcare staff, adherence to social distancing measures for healthcare staff and patients, and space out appointments so that no patients are waiting together in the clinic waiting area. the importance of training staff (receptionists, nurses, technicians, doctors) on ppe needs and usage is highlighted (https://www.cdc.gov/coronavirus/2019-ncov/hcp/clinicpreparedness.html). of 5.192 per 100,000 individuals. importantly, the number of infected individuals in the population has been estimated to be 11.9 times higher than the number of confirmed cases by epidemiological vigilance authorities, meaning that for each individual that tests positive for sars-cov-2, another 12 individuals have been infected without knowing it, with evident implications for viral spreading dynamics (5) . along these lines, the first national study estimating the percentage of infected people in brazil found that the number of individuals presenting with igg antibodies for covid-19, irrespective of developing covid-19 clinical manifestations, was seven times higher than reported. if these projections were applied to the entire coun-6. the precautionary principle and good laboratory practices should be strictly applied when handling the seminal fluid by in the andrology and embryology laboratories. this advice includes (i) use of class ii safety cabinets, which gives protection to the specimen handled as well as the operator performing the work, (ii) use of high-security vials for sperm cryopreservation, as routinely recommended by certified andrology labs and sperm banks, and (iii) additional measures to protect the specimens from laboratory staff (e.g., use of googles, n95 mask, gown/coverall, and gloves) -who might be infected but asymptomatic for sars-cov-2. 7. technicians/biologists should, ideally, be tested by rt-pcr and/or elisa blood antibody testing before resuming activities, and only staff with negative results or who became igg positive or have acquired herd immunity should perform laboratory duties. although the risk is minimal, if the staff that manipulated specimens get infected during the pandemic and is actively working in the lab, an aliquot of cryopreserved semen samples should be tested (e.g., by rt-pcr) because semen samples, cryopreservation media, cryovials, and pipette tips could be contaminated by asymptomatic pcr-positive biologists and technicians. 8. a thorough discussion between patients and healthcare providers should be made for responsible shared decisions. this advice includes the development and use of dedicated informed consent, detailing the risks of attending the facility and being treated or banking of sperm during the sars-cov-2 pandemic. furthermore, explanatory material should be made available for patients. psychological support and financial aid might be offered to those in need. the latter might be particularly relevant to patients under economic pressure due to the pandemic who need to afford the costs of semen analysis, sperm functional tests and sperm banking. 9. advanced planning should guide the continuation of andrological services. working groups and quality managers should determine which patients to prioritize and how working lists should be filled, including staff scheduling. a multi-professional group including reproductive urologists, andrologists, technicians and other relevant health care professionals should work together to provide the optimal care in a safe environment. try, the number of infections would skyrocket to something between 10 and 12 million cases. unfortunately, at present, only about 2% of the entire population have been tested. moreover, only 1.4% of the 25,025 tested individuals in 90 cities across the country has developed igg antibodies, thus indicating that we have much to cover before the estimated 82% population needed to be infected for herd immunity in a disease that has a reproduction number (r0) around 5.7, that is, each infected individual has the potential to spread the disease to another 5 to 6 individuals (6, 7). on the other hand, these figures suggest that the quarantine measures might be working, as shown by a recent study that tested 700 individuals in a medium-sized city with high-income under a 70-day quarantine (8) . in this study, the infected ratio was coincidentally 1.2%. the global mortality rate is around 4.7%, similar to that reported in brazil (3). one aspect of covid-19 in our continental country is its unequal impact in different urban settlements. the number of deaths is higher in peripheral city-areas and in regions where the economic crisis had already reached before the pandemic, regions with not enough or inexistent intensive care units, neither enough artificial breathing apparatus nor highly qualified medical staff. on the one hand, for each death notified by health authorities, another 1.67 to 2.72 occur without testing and notification; therefore, the number of deaths estimated by scientific methods of "nowcasting" should be around 1.5 times higher than officially reported (7, 9) . on the other hand, brazil faces a shortage of tests due to a worldwide shortage of laboratory supplies. consequently, mainly health professionals in the frontline and symptomatic patients have been widely tested. therefore, the 4.1% death rate seems to be unrealistic, because if one divides the number of deaths by a more significant number of tested individuals, the actual death rates go proportionally down. the pilot study conducted in the city of são paulo calculated an overall death rate of around 0.95% (5) . the overwhelming majority of brazilian fertility centers, andrology laboratories, and sperm banks are located in major cities, most of which have been profoundly affected by the sars--cov-2 pandemic. also, reproductive urologists are clustered in large cities. thus, infertility patients living in these cities or traveling from other areas to attend these facilities are at risk of getting infected. although the reported morbidity rate in patients at reproductive age is low, there is a real risk of patients and health care providers to spread the infection in the context of asymptomatic viral shedding. moreover, infected men present an age-independent higher risk for adverse outcomes and death (10) . these observations are probably related to the fact that men have more chronic diseases than women of the same age, including obesity, hypertension, heart conditions, diabetes, and metabolic syndrome, all linked to higher se-verity of manifestations (10) . it is well known that male infertility is associated with a diverse range of general medical conditions, including hypertension (11), obesity (12) , ischemic heart disease, diabetes (13) , and a broad range of cancers (14) . thus, this population deserves special attention. while it is critical for reproductive urologists and fertility centers to continue to offer care to this vulnerable population, it is also essential that all efforts are undertaken to deliver services in a safe environment (15) . equally important is to discuss the possible adverse impact of sars-cov-2 on male reproductive health. the "cytokine storm" seems to be a central element in the pathophysiology of severe co-vid-19. it relates to an excessive and uncontrolled release of pro-inflammatory agents, commonly seen in graft-versus-host situations, multiple sclerosis, pancreatitis, and multiple organ failure. recent studies suggest that cellular and molecular mechanisms contribute to the cytokine storm in viral diseases (e.g., influenza and other respiratory viruses, including sars-cov-1). however, its severity is overwhelmingly higher in sars-cov-2 because of the mechanisms involved in viral and cell membrane fusion. cytokines are a group of proteins (e.g., interferons, interleukins, chemokines, tumor-necrosis [tnf], colony-stimulating factors) used for intercellular signaling and communication, with autocrine, paracrine, and endocrine activity, having the capacity of launching a wide array of intracellular responses through receptor-binding in specific target cells (16) . the semen of healthy men contains a variety of cytokines with immunologic roles, including the regulation of t-cell response and macrophage activity. they are involved in immune defense against genital infections; however, elevated levels of interleukin-1, 2 and 6 and tnf--alpha have been associated with defective steroidogenesis, poor semen quality, and male infertility (17) (18) (19) . thus, these molecules work as protectors of normal testicular function when released in limited and controlled physiological conditions. however, that balance is quickly transformed into a deleterious effect if higher amounts of many classes of cytokines, including the transforming growth factor (tgf) superfamily, are released (17) . a critical aspect of the sars-cov-2 infection for male reproductive health relates to the virus cell-attaching mechanism. angiotensin-converting enzyme 2 (ace2) belongs to the angiotensin-converting enzyme family of dipeptidyl carboxypeptidases, which is homologous to human angiotensin 1 converting enzyme. ace2 is a functional receptor for sars-cov-2 (19) . a remarkable finding is that the testes are ranked second in the number of ace2 receptors after the lungs. sars-cov-2 enters the cell through ace2 receptors via the spike-protein (s), which facilitates viral attachments to the surface of target cells after priming by cellular proteases, in particular, transmembrane protease serine 2 (tmprss2). the 's' protein locates in the virus's external surface and interfaces with the cell by using a receptor--binding domain. this way, the virus can survive by completing intracellular replication and then releasing more viruses that will infect other cells and tissues. in parallel, this process produces cytotoxicity, meaning that organs with higher viral load and ace2 receptors with tmprss2 expression are more prone to be heavily affected (e.g., kidneys and testes). ace2 receptors are also found in other reproductive structures and the bladder. although only a few reports found that orchitis might be a consequence of both sars-cov 1 and 2, initial autopsies of few patients who died from sars--cov-1 in china revealed a clear pattern of widespread germ cell destruction, few or no spermatozoon in the seminiferous tubules, thickened basement membrane, and leukocyte infiltration (20) . moreover, the numbers of cd3þ t lymphocytes and cd68þ macrophages were significantly increased in the interstitial tissue compared with controls. the downside of this first publication it that no sars viral genomic sequence was detected in none of the testes analyzed by in situ hybridization. however, immunohistochemistry demonstrated abundant igg precipitation in the seminiferous epithelium of sars testes, suggesting possible immune response as the cause for the damage, but without real proof-of-concept (20) . nevertheless, duarte-neto and co-workers, in a recent paper using histology and rt-pcr for sars-cov-2, identified systemic-organ involvement, including the lungs and testes (21) . in this report, two patients developed orchitis, confirmed by ultrasound-guided minimally invasive autopsy (mia-us). in another study, pan et al. also reported signs suggestive of orchitis in 16% of sars--cov-2 infected patients (22) . in their cohort of 34 patients, however, rt-pcr carried out 30 days after disease recovery did not detect the virus in the semen. by contrast, in a study involving 38 subjects with either acute infections or after recovery, li et al. found the virus in the semen in 15.8% of individuals (23) . while many questions remain concerning the real risk of virus presence in the seminal fluid, it seems prudent to exercise the utmost precautions for sperm handling during diagnostic testing (e.g., semen analysis) and treatments involving sperm handling, including intrauterine insemination (iui), assisted reproductive technology (art), and cryobanking. most enveloped rna viruses remain viable in ultra-low temperature (e.g., influenza), and although the risk of cross-contamination is minimal, practitioners should apply the precautionary principle (24, 25) . it is important to stress that liquid nitrogen cannot kill the coronavirus; therefore, all efforts should be made to avoid contamination. the responsible use of art, which should drive the medical practice in this field, is even more critical at this challenging time to safeguard the health of the couple seeking fertility, the offspring, and future generations (26). given the observations above and the fact that the ace2 receptor is essential for spermatogenesis, sperm maturation, and spermiation in physiological conditions (27) , it seems sound to assume that the sars-cov-2 poses a threat for the male reproductive health. thus, further research is warranted to understand its implications to the male reproductive health as every organ/ tissue/cell that expresses ace2 receptors, and the serine protease tmprss2, is a potential target for viral invasion, including the testis (28), the leydig cell (29) , the spermatozoa (27) and eventually cells dispersed in the seminal fluid (23) . we reiterate that andrological services and male infertility care cannot be considered low priority during the current sars-cov-2 pandemic, particularly for the most vulnerable patients, like those with cancer, patients using immunosuppressive therapy, and the azoospermic/cryptozoospermic men under medical or post-surgical treatment to improve spermatogenesis. postponing care to 'time-sensitive' male infertility patients could permanently compromise the prospects of biological parenthood. however, care should be provided in a safe environment for patients, staff, and community, and we, therefore, not only reaffirm the value of the recently issued practice recommendations but also firmly believe they should be strictly applied in brazilian healthcare facilities that have restarted-or are planning to restart-delivery of relevant services. dedicated certified andrology laboratories are best prepared to deliver both diagnostic and therapeutic services in a safe environment -during an emergency like the current one and also at normal times. we should continue to look critically and objectively at the rapidly growing sars-cov-2 evidence and investigate the implications of the virus to male reproductive and sexual health. sars-cov-2 pandemic and repercussions for male infertility patients: a proposal for the individualized provision of andrological services coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2. version 2 european association of urology guidelines office rapid reaction group: an organisation-wide collaborative effort to adapt the european association of urology guidelines recommendations to the coronavirus disease pesquisa de mapeamento covid-19 em sp primeiro estudo nacional do coronavírus estima número de casos sete vezes maior no brasil high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2 observatório covid-19 br nowcasting by bayesian smoothing: a flexible, generalizable model for real-time epidemic tracking gender differences in patients with covid-19: focus on severity and mortality high blood pressure is a highly prevalent but unrecognised condition in primary infertile men: results of a cross-sectional study men's body mass index and infertility increased risk of incident chronic medical conditions in infertile men: analysis of united states claims data increased risk of cancer in infertile men: analysis of u.s. claims data covid-19 and assisted reproductive technology services: repercussions for patients and proposal for individualized clinical management. version 2 into the eye of the cytokine storm concentrations and significance of cytokines and other immunologic factors in semen of healthy fertile men inflammatory cytokine concentrations are elevated in seminal plasma of men with spinal cord injuries study on the relationship between different cytokines in the semen of infertility patients orchitis: a complication of severe acute respiratory syndrome (sars) pulmonary and systemic involvement of covid-19 assessed by ultrasound-guided minimally invasive autopsy no evidence of severe acute respiratory syndrome-coronavirus 2 in semen of males recovering from coronavirus disease 2019 clinical characteristics and results of semen tests among men with coronavirus disease methods for long-term virus preservation available at. <https:// www.beiresources.org/portals/2/pdfs/long-term%20and%20 short-term%20stability%20of%20viruses.pdf> 26. hallak j. a call for more responsible use of assisted reproductive technologies (arts) in male infertility: the hidden consequences of abuse, lack of andrological investigation and inaction analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics the novel angiotensin-converting enzyme (ace) homolog, ace2, is selectively expressed by adult leydig cells of the testis release of angiotensinconverting enzyme (ace) from human spermatozoa during capacitation and acrosome reaction both authors contributed to the conception, designed the manuscript, and wrote the draft. jh declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. sce declares the receipt of unrestricted research grants and lecture fees from merck outside the submitted work. none declared. key: cord-327063-ea7a1xfl authors: dhama, kuldeep; patel, shailesh kumar; sharun, khan; pathak, mamta; tiwari, ruchi; yatoo, mohd iqbal; malik, yashpal singh; sah, ranjit; rabaan, ali a.; panwar, parmod kumar; singh, karam pal; michalak, izabela; chaicumpa, wanpen; martinez-pulgarin, dayron f.; bonilla-aldana, d. katterine; rodriguez-morales, alfonso j. title: sars-cov-2 jumping the species barrier: zoonotic lessons from sars, mers and recent advances to combat this pandemic virus date: 2020-08-02 journal: travel med infect dis doi: 10.1016/j.tmaid.2020.101830 sha: doc_id: 327063 cord_uid: ea7a1xfl coronavirus disease 2019 (covid-19), caused by sars-cov-2 (severe acute respiratory syndrome coronavirus-2) of the family coronaviridae, appeared in china in december 2019. this disease was declared as posing public health international emergency by world health organization on january 30, 2020, attained the status of a very high-risk category on february 29, and now having a pandemic status (march 11). covid-19 has presently spread to more than 215 countries/territories while killing nearly 0.62 million humans out of cumulative confirmed infected asymptomatic or symptomatic cases accounting to almost 15 million as of july 22, 2020, within a short period of just a few months. researchers worldwide are pacing with high efforts to counter the spread of this virus and to design effective vaccines and therapeutics/drugs. few of the studies have shown the potential of the animal-human interface and zoonotic links in the origin of sars-cov-2. exploring the possible zoonosis and revealing the factors responsible for its initial transmission from animals to humans will pave ways to design and implement effective preventive and control strategies to counter the covid-19. the present review presents a comprehensive overview of covid-19 and sars-cov-2, with emphasis on the role of animals and their jumping the cross-species barriers, experiences learned from sarsand mers-covs, zoonotic links, and spillover events, transmission to humans and rapid spread, and highlights the new advances in diagnosis, vaccine and therapies, preventive and control measures, one health concept along with recent research developments to counter this pandemic disease. in the 21 st century, we have faced a few deadly disease outbreaks caused by pathogenic viruses such as bird flu caused by avian influenza virus h5n1, swine flu caused by reassorted influenza virus h1n1 pandemic 2009 (h1n1pdm2009), severe acute respiratory syndrome (sars) caused by sars-cov (coronavirus), the middle east respiratory syndrome (mers) caused by mers-cov [1] [2] [3] , ebola [4] , zika [5, 6] , nipah virus infections, and the most recent threat [7] , coronavirus disease 2019 (covid19 ) that has been posed by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) of the family coronaviridae, genus betacoronavirus [8] [9] [10] [11] [12] [13] [14] . the sars-cov-2 virus emerged from the city of wuhan, hubei province, china, during december 2019, was declared as public health international emergency by the world health organization (who) on january 31, 2020. consequently, it was categorized in a high-risk category on february 29, 2020, and gained the pandemic status on march 11, 2020 . the disease emerged from wuhan, china, as its epicentre, which moved later to italy, then the usa, and brazil. subsequently, within a short time interval of six months, it has affected nearly 215 countries/territories and claimed near to 0.62 million human deaths out of cumulative confirmed infected asymptomatic or symptomatic cases accounting to almost 15 million. sars-cov-2 has very adversely affected the usa, brazil, india, russia, south africa, peru, mexico, chile, spain, the united kingdom (uk), iran, pakistan, saudi arabia, italy and other countries. the disease incidences are lower in children than adults but exhibit all symptoms of a disease like adults [15] . the lessons learned from earlier threats of sars, mers and the present covid-19 pandemic situations warrants designing and implementing some modified plans and strategies to combat emerging and zoonotic pathogens that could pose pandemic threats/risks while taking away many human lives [11, [16] [17] [18] [19] [20] [21] [22] . researchers and health agencies across the world are putting high efforts to contain/restrain the spread of this deadly disease. they are pacing to develop potential vaccines and therapeutics/drugs [23, 24] . evidence from the initial outbreak indicates earlier cases had links to huanan wholesale seafood market in china [25] and further isolation of sars-cov-2 from different samples of the area (people, animals, birds, discharges, soil, structures) suggests the involvement of intermediate hosts [26] . recently, a literature of review has pointed out the possible potential role of the animal-human interface, zoonotic links and spillover events towards the origin of sars-cov-2/ covid-19 [11, 20, [27] [28] [29] [30] [31] . in the past couple of decade's animal origin viral diseases, especially bats-linked, have increased many folds in humans with noted cross-species transmissions. although many of the illnesses are linked with bats still information on their ecological behaviour, molecular aspects are limited, which could lead to more viral outbreaks shortly [32] . the ongoing covid-19 pandemic has emphasized the importance of understanding the evolution of natural hosts in response to viral pathogens. in a recent study on ace2 receptors, the gene was found under intense selection pressure in bats and positive selection in other selected mammalian hosts [33] . the sars-cov-2 is also thought to have originated from bats, just like sars-cov and mers-cov. civets and dromedary camels are considered as the intermediate host of sars-and mers-cov, respectively, from where they were transmitted to humans [34] . the understanding of genomic signatures of sars-cov-2 with other covs is must for strategic planning through identifying natural or intermediate hosts. using genomic and protein data in a natural vector method (alignment-free approach), phylogenetic analysis revealed the possible transmission path originates from bats to pangolins to humans [35] . however, the likely source of virus origin and the intermediate host of sars-cov-2 are yet to be identified. initially, when the novel virus emerged in china, a hypothesis was put forward, claiming the recent recombination event as the cause of the sars-cov-2 emergence . nevertheless, the phylogenetic and recombination analysis performed within the subgenus of sarbecovirus demonstrated that the novel virus shows discordant clustering with bat-sars-like coronavirus (ratg13) sequences thus rejecting the possibility of a recent recombination event [36] . previously, it was found that the continuous passaging of mers-cov in non-susceptible cells that express viral receptors led to the accumulation of mutations in the spike protein gene. this paid attention to the potential of coronaviruses like mers-cov to undergo mutations that enhance viral entry into novel animal species, thus resulting in cross-species transmission [37] . the covid-19 outbreak is still associated with several unanswered questions like the possibility of shedding of the virus before the onset of clinical signs, whether the transmission is limited to only through respiratory droplets, the possibility of an intermediate host that is responsible for zoonotic spillover, and the possible transmission characteristics [20, 38] . hitherto studies report that the spillover risk remains high from zoonotic viruses and on the same lines a study from north america proposed a hypothesized conceptual model demonstrating sars-cov-2 spillover from humans to naive wildlife host species through the gastrointestinal route where stool from covid-19 infected patient contaminates water bodies and reaches to wildlife hosts [39] . besides, the pandemic imposed a massive blow on the chinese economy, which is not going to heal soon [40] . instead of the current situation, singapore's prime minister lee hsien loong rightly said that the virus might have started in china. however, it does not respect nationality or race. it does not check your passport before it goes into your body, and anybody can be infected. hence, all suspected people need to be tested and quarantined [41] . further research exploring the sars-cov-2 associated zoonosis and mechanisms accounting for its initial transmission from animals to humans, will lead to sort out the spread of this virus as well as design and develop appropriate prevention and control strategies to counter covid-19. the present comprehensive manuscript presents an overview on covid-19, an emerging sars-cov-2 infectious disease while focusing mainly on the events and circumstantial evidences with regards to this virus jumping the species barriers, sharing a few lessons learned from sars-and mers-covs, zoonotic spillover events (zoonosis), acquiring transmission ability to infect humans, and adopting appropriate preventive and control measures [42] . it also highlights the recent advances in sars-cov-2 diagnosis (see supplement material s1), vaccine, drugs and therapies (see supplement material s2), which could aid to counter and restrain this emerging virus at the face of pandemic situations, as well as prevention and control (see supplement material s3). sars-cov-2 is an enveloped virus measuring approximately 50-200 nm in diameter with a single strand positive-sense rna genome ranging from 26 to 32 kilobases in length [43, 44] . it has club-shaped glycoprotein spikes in the envelope, giving it a crown-like or coronal appearance [25] . the genome sars-cov-2 is comprised of 5′ untranslated region (5′ utr) that includes 5′ leader sequence, open reading frame (orf) 1a/b (replicase genes), spike (s) protein, envelop (e) protein, membrane/matrix (m) protein, and accessory proteins (orf 3,6,7a, 7b, 8 and 9b), nucleoprotein (n), and 3′ untranslated region (3′ utr) in their sequence [45] . it has a 50% genetic identity to mers-cov and 80% to sars-cov [43, 46] . the receptor-binding domain (rbd) of virus spikes helps in binding to cellular receptor angiotensin-converting enzyme 2 (ace-2) [47, 48] . orf and rbd of sars-cov-2 may have a role in elucidating cellular interactions and cross-species transmission mechanisms [49] . receptor binding motifs (rbm) have a role in interaction with human receptors, human to human transmission, and cross-species transmission as gln493 provides favourable interaction, and asn501 shows compatibility with human ace-2 [47] . besides, sars-cov-2 has superior transmission competence in comparison to the sars-cov, leading to a continuously increasing number of confirmed cases [50] . sars-cov-2 has the potential to survive in the environment for several days [51] . though believed to be sensitive to environmental factors and alcohol-based sanitizers, bleach, and chloroform, the sars-cov-2 can survive in wet surroundings for days and in closed air conditions up to 12 hours [26, 52] . survival of sars-cov-2 varies with the nature of the surface (glass, fabric, metal, plastic, or paper), environment, and virus load. it can survive on surfaces for hours to several days. it can survive in aerosols for up to 3 hours and on plastic for up to 72 hours [26, 53] . the initial clinical picture of the covid-19 was pneumonia of unknown origin as the first clinical cases were presented with signs of pneumonia [38] . later it was diagnosed as sars-cov-2 infection that was associated with severe pneumonia. hence, initially named as novel coronavirus pneumonia (ncp) [54] . as the outbreak proceeded, a series of cases were produced, developing a wide range of clinical signs with few remaining asymptomatic being in the early incubation stage of the disease. thus covid-19 is characterized by three major patterns of the clinical course of infection, including mild illness producing upper respiratory signs, non-life-threatening pneumonia, and severe pneumonia with acute respiratory distress syndrome (ards) [55, 56] . initially, mild signs appear for 7-8 days, followed by rapid deterioration and ards. it can be mild to moderate in 80% of affected cases, including pneumonia and non-pneumonia cases. in comparison, 13.8% are severe cases, including dyspnea, respiratory distress, hemoptysis, gastrointestinal infection, liver, central nervous system, and lung damage cases [57, 58] . critical cases account for 6.1% and include respiratory failure, septic shock, and multiple organ failure/dysfunction cases. few cases remain asymptomatic and include cases that can become any of the above during infection [26] . thus, the symptoms can be nonspecific and can range from no symptoms (asymptomatic) to severe pneumonia [26] . in this context, a study concluded that the covid-19 is probably overestimated, as around 2.6 million people succumb to respiratory diseases every year in comparison to approximately 400,000 deaths due to the sars-cov-2 infection [59] . the typical clinical signs of covid-19 are fever, chills, cough, fatigue, and chest distress [60, 61] . fever and cough are considered as the most common symptoms in covid-19 patients [62] , followed by headache, dyspnea, sore throat, hemoptysis, myalgia, diarrhoea, nausea, and vomiting are also observed [61, 62] . some patients have shown rhinorrhea, chest pain [25] , nasal congestion [26] , anorexia, pharyngalgia, and abdominal pain [63] . furthermore, neurological symptoms like anosmia and ageusia are also reported as significant clinical symptoms of covid-19 [64] . the characteristic of covid-19 is attacking the lower respiratory tract and producing signs of upper respiratory distress, including rhinorrhea, sneezing, and sore throat [49] . the clinical presentation of individuals infected with sars-cov-2 revealed upper respiratory tract infection, viremia, viral shedding from the nasopharynx, and stool along with the development of nausea, vomiting or diarrhoea after antiviral treatment [65] . on diagnostic imaging using computed tomography (ct scan) and radiography (x-ray) bilateral pneumonia, ground-glass opacity, multiple mottling, pneumothorax, infiltration, consolidation or bronchoinflation sign has been noted in many cases of covid-19 [25, 49, 66] . previously, sars-cov was found to infect the brainstem heavily [67] . even though fever is considered as the most common symptom associated with covid-19 infection, a large proportion of the patients do not express fever during the initial hospital admission [68] . the different transmission routes of sars-cov-2 infections have not yet been entirely ascertained, and are still under investigation. both direct and indirect pathways of transmission are being explored [69] . similar to sars and mers, sars-cov-2 is predominantly spread via the respiratory route [70] . person to person transmission is the main reason for community and global spread. the initial estimated reproduction number (rovalue) of covid-19 was assessed to be from 0.8 to 2.4 in december 2019, which later has been increased to a mean value of 2.6 (range 2.1-5.1) [71] . human-to-human transmission is by face-to-face contact with a sneeze or cough, or from contact with secretions of infected people [56, 70] . nevertheless, the infectivity of other secretions and excretions are not fully understood and may require further study [45] . aerosol and plastic surfaces can sustain virus for hours to days [53] . travelling of infected people is considered as the main reason for the global spread of covid-19 [20, 56] . although the asymptomatic and mild cases are the major hurdle in the evaluation of the real number of infected people, the genuine data on travellers returning from affected countries or areas may prove crucial in estimating the disease incidence [72] . the possible occurrence of super-spreading events is very high at large gatherings, and suspension of gathering during a pandemic may prove crucial in reducing the overall transmission [73] . close contact with any person within 6 feet of the covid-19 patient or anyone having direct contact with secretions of covid-19 patients [74] may set up the infection. unlike sars-cov, most transmissions in covid-19 are during the prodromal period when the infected individuals produce large quantities of virus in the upper respiratory tract, move/travel, and usually work thus spreading virus before illness develops [56] . the rapid spread of covid-19 among the susceptible population can be due to the wide variation in illness degrees that results in a missed diagnosis. heavy viral load in asymptomatic cases and nosocomial transmission is spreading covid-19 unknowingly [75] . recently, high viral load was detected in the sputum of convalescent patients, pointing out the possibility of prolonged shedding of sars-cov-2 even after recovery. this finding, along with the fact that asymptomatic persons can also act as the potential source of infection, may warrant a reassessment in the transmission dynamics of the covid-19 outbreak [76] . the presence of viral nucleic acids in faeces is an important finding, thereby increasing the possibility of faecal-oral transmission. however, symptoms may or may not be manifested [77] . this was found to be the unique feature of covid-19 and was lacking in the previous sars and mers outbreaks. in a study conducted by the chinese cdc, it was found that the majority of patients (80.9%) infected with covid-19 infection were either asymptomatic or had mild pneumonia [56, 66] . furthermore, all forms of sexual contacts have been reported to pose a significantly high risk of disease transmission through respiratory aerosols and fomites. however, to date, no evidence of sexual transmission is available, and further investigation is required in this direction [78] . disease severity is higher in older individuals, especially males with immunocompromised conditions and comorbidities like diabetes, asthma, or cardiovascular diseases [26, 66] . these are considered to be vulnerable to the sars-cov-2 infection. predisposition increases under risk environments where transmission of the virus from affected persons or contaminated fomites to unaffected ones becomes feasible. it was earlier noted that covs are not common to affect immunocompromised patients like other some viral infections (influenza, rhinovirus, adenoviruses, to name a few). the current pandemic has shown sars-cov-2 to affect more lethally than young patients, mainly destroying the lung tissues [79] . till now, evidence regarding the higher susceptibility of pregnant women in comparison to non-pregnant women lacks in covid-19. also, there is no evidence of vertical transmission (mother to fetus/baby transmission) of covid-19 infection [80] . a case study reporting the birth of a healthy infant by a sars-cov-2 infected woman suggests that mother-to-child transmission is unlikely in the case of covid-19. the study also pointed out that on the delivery day, all the samples tested negative except for sputum, which proved positive [81] . however, as per one most recent report, neonates have been found positive for sars-cov-2, indicating the possibility of vertical transmission from infected mothers to their progeny, thus rendering newborns into a high-risk group owing to their immature immune system [82] . individuals harbouring sars-cov-2 may remain asymptomatic for the incubation period [83] . different from sars-cov and mers-cov infection, the median incubation period of covid-19 was found to be four days [68] . the median period from the development of signs to death was 14 days [84] . the case fatality rate (cfr) of covid-19 was found to be lower than mers and sars [62] . however, current disease dynamics with the involvement of many more countries or areas may change the future mortality rate. the recent analysis suggests that the total fatality rate of covid-19 is calculated at 3.46% [75] . however, italy experienced the worst cfr of more than 9% with older people and males suffering from multiple comorbidities as primary victims [85] . sars-cov-2 has shown characteristics of efficient replication in the upper respiratory tract, causing the less abrupt onset of clinical signs just like the common cold and unlike sars-cov [70] . it can also replicate in the lower respiratory tract as has been noted in cases without pneumonia but having lesions in the lungs on radiological examination [70] . the pathogenesis mechanisms of covid-19 are yet to be fully elucidated. however, both cellular and humoral immune responses against sars-cov-2 or its antigenic structures like spike protein (s) are believed to be of importance [86, 87] with disturbed levels of inflammatory mediators playing a mediating role [66] . following receptor binding with angiotensin-converting enzyme 2 (ace2) through receptor binding motif (rbm) of the receptor-binding domain (rbd) of s1 subunit of the sars-cov-2 spike glycoprotein (s), virus gains entry in host cells [47, 88, 89] . s2 subunit helps in the fusion of viral and hosts cell membranes [47, 90] . sars-cov-2 produces cytopathic effects in respiratory and gastrointestinal surface epithelial cells [91] . these include multinucleated syncytial cells, abnormally enlarged pulmonary cells, infiltration with mononuclear cells, lymphocytes infiltration in pulmonary organs, fibrinous exudation, and hyaline deposition [66] . cytokine storm is believed to be involved in this inflammatory pathophysiology of the covid-19 patients producing lung lesions and systemic symptoms [66] . elevated levels of tnf-α, il1b, ifnγ, ip10, gcsf, mip1a, and mcp1, may have stimulated t-helper-1 (th1) cells leading to this inflammatory cascade [66] . however, levels of anti-inflammatory mediators (il4, il10) were also increased, indicating t-helper-2 (th2) stimulation, which suppresses inflammation, unlike what happens in sars [66] . a study documented that the nucleic acid of sars-cov-2 detected in the faecal samples was as accurate as of that of pharyngeal samples obtained from infected patients. moreover, the patients tested positive for sars-cov-2 in stool showed no gastrointestinal symptoms and had no relation to the severity of lung infections [92] . one of the significant clinical signs of covid-19 patients during the initial presentations was gastrointestinal symptoms. hence, the involvement of git in pathogenesis needs to be explored. the significant laboratory findings include lymphopenia, increased values of erythrocyte sedimentation rate, c-reactive protein, lactate dehydrogenase, and decreased oxygenation index [61] . an increase in proinflammatory cytokine and a decrease in antiinflammatory cytokines have also been noted [66] . viral isolation has been achieved from bronchoalveolar lavage of affected persons; however, in the case of pregnant women, serum, faeces, urine, breast milk, umbilical cord blood, placenta, and amniotic fluid were found to be negative for sars-cov-2 [81] . at the same time, the sputum was tested positive [82] . the presence of abnormal coagulation parameters in patients with severe novel coronavirus pneumonia was associated with poor prognosis. the non-survivor patients had higher levels of d-dimer, and fibrin degradation product (fdp) along with longer activated partial thromboplastin time and prothrombin time compared to survivors at the time of admission [93] . though clinical manifestations, pathological changes, and diagnostic laboratory findings can unravel the disease nature helping in devising therapeutic modalities, however, for epidemiological aspects and future prevention and control, simultaneous tracing of the origin and explaining the spillover events can prove beneficial. the sars-cov-2 has first been reported from the pneumonia patients of the wuhan city in hubei province of china. these patients were involved in trading at a wet animal market in the huanan area. it is believed that sars-cov-2 is introduced from the animal kingdom to human populations during november or december 2019, as revealed from the phylogeny of the genomic sequences from the initially reported cases [94] . the spillover of sars-cov-2 from animals to humans took place at the beginning of december 2019 [56] , and the clinical cases appeared around ending december [46, 95] . genetic analysis showed that this novel virus is closely related to bat covs and is similar but distinct from the sars virus [43] . several evidences based on genome sequences, the homology of the ace2 receptor, and the presence of single intact orf on gene 8 indicate bats as a natural reservoir of these viruses. however, an unknown animal is yet to be unravelled as an intermediate host [43, 46, 47, 49, 56] . initial investigations on animal source origin of sars-cov-2 have inconclusively revealed snakes [27] , pangolins, and turtles [96] . the rapid spread of covid-19 followed the initial animal to human spillover through human-to-human transmission. genetic epidemiology had revealed that the spread from the beginning of december when the first cases were retrospectively traced in wuhan was mainly by a human-to-human transmission and not due to continued spillover [56] . these species cross jumping, spillover, and rapid transmission events are linked to viral characteristics, host diversity, and environmental feasibility. coronaviruses being rna viruses have high mutation rates that, besides creating new strains, enable them to adapt to a wide range of hosts. hence, based on genome sequences, all known human covs have emerged from animal sources [97] . this seventh member of the human cov has also been isolated initially from the pneumonia patients who were having direct or indirect links to the huanan seafood market in wuhan china, wherein other animals were also being sold [98] . these include a 49-year-old lady retailer in this wet animal market, a 61-year-old frequent visitor to this market, and a 32-year-old man [74, 98] . further, isolation of the sars-cov-2 from the environmental samples around this market, including people, animals, soil, discharges, or structures, strengthens the claims of involvement of hosts either as a reservoir or intermediate [26, 52, 99] . recently, a pomeranian dog as a probable intermediate host was identified; however, such reports are yet to be validated, and research is underway to explore the emergence of this infectious disease at the animal-human interface [29, 99] . multiple substitutions were observed in ace2 receptors of a dog [96] . in this context, the pomeranian dog of the infected owner found positive for covid-19 suggest the permissiveness of the species for sars-cov-2 as a result of species jumping [99, 100] . among the fifteen dogs tested from different households with confirmed human covid-19 cases in hong kong sar, two dogs were found to be infected with sars-cov-2. the diagnosis was made using quantitative rt-pcr, serology, and viral genome sequencing. virus isolation was also done from the samples obtained from one dog. the genetic sequences of viruses obtained from the two dogs were identical to the ones that were detected from their human cases indicating human-to-dog transmission [101] . moreover, a study reported that the sars-cov-2 might infect the cats and further transmitted by the infected cat to other cats [102] . one cat was tested positive for sars-cov-2 in france that showed mild respiratory and digestive signs. the cat was tested positive by rt-qpcr on the rectal swab, and serological analysis identified the presence of antibodies against sars-cov-2. genome analysis further confirmed that the sars-cov-2 isolated from the cat belongs to the phylogenetic clade a2a seen in french human indicating humanto-animal transmission [103] . this is not the first time that a domestic cat has been found susceptible to zoonotic coronavirus. during the 2003 sars-cov outbreak, domestic cats were tested positive for sars-cov that were living near sars infected humans [21, 104] . even though experimental evidence indicates the possibility of sars-cov-2 transmission from infected to a susceptible cat close, sars-cov-2 transmission between cats or cat-tohumans are not reported under natural conditions [21] . furthermore, along with dogs and cats, the zoo animals like tigers and lions were also reported to get the sars-cov-2 infection and exhibit clinical signs such as vomiting, diarrhoea, dry cough, breathing difficulty and wheezing [105, 106] . spillover of sars-cov-2 was also reported in mink farms of netherlands, further increasing the concern of transmission to humans. outbreaks of sars-cov-2 were reported in two mink farms holding 12,000 and 7500 animals. the virus is suspected to be introduced by a farmworker having covid-19 [107, 108] . host-pathogen interactions and pathogenesis determine the severity and expression of disease [30, [109] [110] [111] . adaptation over time reduces the severity of infection as happened with hcovs; however, the emergence of novel viruses or strains due to genetic alterations or recombinations can enhance hardness producing novel diseases like covid-19 [111, 112] . evolutionarily, the balance of viral-human interaction and immune response against virus enables adaptation, thereby persistence in a host without severe or symptomatic disease when the aggravated pathogenesis results in mortality. hence, loss of sustainable hosts and transmission to novel hosts becomes inevitable for future sustainability [111, 112] . a pathogen cannot kill all its hosts, and for future sustainability, it adapts to some suitable host or spills over to a new host. sars-cov-2 has been implicated to be originated from animals, and associated with animal linkages, spillover events, cross-species barrier jumping and zoonosis [9, 20, 27, [29] [30] [31] 113] . since the beginning of 2002 till the end of 2019, three coronaviruses viz. sars-cov, mers-cov, and sars-cov-2 have caused havoc in the human population globally and will continue to do so. earlier identified betacoronaviruses (sars-cov and mers-cov) were reported in guangdong province of china in november 2002 and saudi arabia in 2012, respectively [114] . sars-cov-2 is the third zoonotic betacoronaviruses recognized in this century. however, the cfr of the sars-cov-2 is lower to date when compared with sars and mers. it should not be overlooked as many asymptomatic cases may remain undiagnosed due to the unavailability of diagnostic kits in china. with nearly 0.62 million deaths till the preparation of the manuscript, sars-cov-2 is proven to be deadliest as far as the number of deaths is concerned in comparison with sars-cov and mers-cov with 774 and 858 associated deaths, respectively [115, 116] . earlier, covid-19 was linked with the exposure to the huanan seafood market. however, individuals with no history of exposure above were also diagnosed with the illness, further supporting the human to human spread through droplets produced by cough and sneeze [66] . the spread of covid-19 that occurred with a high pace and lack of transparency in reporting the disease by the chinese health ministry and failure in the timely implementation of preventive measures has been considered as the primary contributor as stated earlier in sars [19, 117] . both sars-cov and sars-cov-2 showed prominent similarities in their pathogenesis and epidemics. in both cases, bats were considered as the natural host, and the cold temperature and low humidity in cold, dry winter provided conducive environmental conditions that promoted the survival of the virus in the environment [62] . further, moriyama et al. [118] assessed the significance of the environmental factor on host immune system targeting innate and adaptive both responses in the respiratory tract. zoonotic spillover is the transmission of pathogens to humans from vertebrate animals [119] . at present, these spillovers are of significant concern as in the past, many spillovers in the form of nipah, hendra, ebola, sars, mers, and ongoing covid-19 involving many animal species like pigs, horses, monkeys, camels, civets, among others, were documented. bovine covs have been reported to infect children and thus possess zoonotic potential [9, 13, 120, 121] . spillover is governed by the interaction of viral-specific proteins like s protein and host ace2 receptor [30, 31, 111, 112] . these s proteins have rbd in covs, which contain receptor binding motifs (rbm) that help in specific binding to host ace2 receptors [47, 122] . mutations in amino acid sequences of rbds results in a change in specificity of a receptor, interaction and binding, hence alteration in transmissibility, pathogenicity and cross-species jumping with a predisposition to novel and more severe diseases [110, 123] . in the case of sars-cov-2, rbd of s protein has 10-15 times affinity ace2r [88, 110] . it has furin recognition sequence "rrar" at the s1-s2 cleaving site that represents a functional site for the cellular serine protease tmprss2 thus increasing the efficiency of transmission and contagiousness [88, 90, 110] . in addition to enhanced binding affinity, electrostatic complementarity and hydrophobic interactions are critical to enhancing receptor binding and escaping antibody recognition by the rbd of sars-cov-2, thereby further increasing transmission capability and contagiousness [110] . a detailed investigation regarding the emergence of new coronavirus, host range, and transmissibility is crucial to understand such pandemics shortly. the literature revealed that before the appearance of sars-cov and mers-cov, human coronavirus (hcov) strains like hcov-nl63, hcov-229e, hcov-oc43, and hcov-hku1 were the covs strains producing mild infections in humans. however, their natural ancestral hosts were of animal origin, like bats for hcov-nl63, and hcov-229e and rodents were natural hosts for hcov-oc43 and hku1. these four hcovs were initially of low pathogenicity. to enhance the pathogenicity, they used intermediate hosts such as cattle for hcov-oc43 (natural host was rodent), and alpacas for hcov-229e (bats were natural host) and this way acquired the ability to infect human beings with serious health hazards [124] . added to the involvement of bats and pangolins, the recent reports revealing sars-cov-2 infection in cats, dogs, tigers, lions and minks have raised concerns over this virus affecting multiple animal species, and also points out towards the incidences of reverse zoonosis [31, 102, 106, 108, 125, 126] . the ferrets, cats, and primates are suggested to be good candidates for susceptibility to sars-cov-2 [123, 127] . covid-19 research and surveillance in companion and pet animals, livestock animals, zoo animal species, wildlife animal species as well as their handlers, veterinarians, and owners need to be enhanced during the pandemic, which would help to follow better integrated one health strategies [128] and appropriate preventive and mitigation to counter sars-cov-2 effectively [29, 126, [129] [130] [131] [132] [133] . significance of covid-19 monitoring and implementation of suitable public health measures among workers involved in meat and poultry processing facilities/industries has been emphasized, which would protect them as well as aid in preserving the critical meat and poultry production infrastructure and the meat products [134] . the involvement of intermediate hosts in maintaining and transmitting the virus to susceptible host predisposes humans to novel covs leading to the emergence of new diseases in humans. the currently ongoing sars-cov-2 / covid-19 pandemic has put on hold the entire world [111, 135] . the covs have frequently been associated with animal and human diseases and have a zoonotic interface [97, 111] . usually, one or more types of animal hosts are involved in the transmission cycle of covs to humans [30, 97] . that can be natural host, reservoir host, intermediate host or definitive host [31] . bats have been the natural hosts for human covs of alphacoronavirus (hcov-nl63, hcov-229e) and betacoronavirus (sars-cov, mers-cov, sars-cov-2) genera whereas for betacoronavirus members hcov-oc43 and hcov-hku1, rodents are the natural hosts. genome sequence analysis has revealed bats as a natural host for sars-cov-2 [30, 97] . in natural or reservoir hosts, covs adapts well, however, being unstable rna viruses, they keep multiplying continuously without producing disease thereby enabling persistence or survivability and accumulation of mutations over the time resulting in the emergence of newer and novel strains of viruses [97, 111, 136] . these unique strains or viruses occasionally spill over to other species including animals or humans, adapting to their body systems and hence broaden the biological host range for evolutionary sustainability; however, results in epidemiological widening of disease sphere as well [25, 31] . this transmission and adaptation scenario initiates a host-pathogen response resulting in the novel usually severe diseases that can at times be fatal in initial stages or over extended periods until virus pathogen adapts to host or the host develops sufficient immune defence [137, 138] . it has been reported that almost all hcovs have originated from animals like bats (sars-cov, mers-cov, hcov-nl63, and hcov-229e) and rodents (hcov-oc43 and hku1) [139, 140] . additionally, covs have been reported to infect several species of domestic and wild animals either clinically or subclinically [27, 28, 30, 141] . cattle, horses, camels, swine, dogs, cats, birds, rabbits, rodents, ferrets, mink, bats, snakes, frogs, marmots, hedgehogs, malayan pangolin along with other wild animals may serve as a reservoir host of coronavirus [9, 27, 44, 124, [142] [143] [144] [145] [146] [147] . in the context of sars-cov-2, snakes, pangolins and bats have been suspected as intermediate hosts since the first cases of covid-19 had links to huanan sea food market where different animals, birds, and wild animals were being sold along with seafood items [27, 28, 43, 109, 147, 148] . coronaviruses have been reported to cause salivary, enteric and respiratory infections in laboratory animals (mice, rat, guinea pig, and rabbit) and urinary tract infection, respiratory illness and reproductive disorder in poultry [9, 142] . in bovine, canine, feline and swine covs infections have resulted in diarrhoea, enteritis, respiratory illness, gastro-intestinal affections and nervous symptoms [120, 121, [149] [150] [151] . coronavirus, namely-sw1, has been reported in captive beluga whale using a panviral microarray method [152] . among all the assumptions on animal hosts as the intermediate host, genomic and evolutionary information from pangolins reveals the highest closeness to the sars-cov-2 than any other host covs isolates [153] . the spike protein, the main target of many studies searching for a cure of covid-19, has been found highly similar to sars-cov-2 and, thus, could serve as a surrogate system for further evaluations [153] . bats are the natural reservoir host of many covs. as reported earlier, 7 out of 11 alphacoronaviruses and 4 out of 9 betacoronaviruses as per the international committee on taxonomy of viruses (ictv) classification were solely originated from bats [97, 154] . according to the literature, bats have been regarded as a potential wildlife reservoir whereas civets and dromedary camels as intermediate hosts of sars-cov and mers-cov, respectively [155, 156] . the bat coronavirus, batcov ratg13, has shown higher relatedness to sars-cov-2 at the whole genome level and spike gene in particular [153] . coexistence and frequent recombination between highly diversified and prevalent bat sars-related coronaviruses (sarsr-cov) and coronaviruses may suggest the probable emergence of novel viruses shortly [157, 158] . benvenuto et al. [159] analyzed the whole genome sequences of different covs using fast unconstrained bayesian approximation (fubar) to understand the evolutionary and molecular epidemiology of sars-cov-2. the authors concluded that sars-cov-2 clustered with sequences of bat sars-like covs with a few mutations in nucleocapsid and spike glycoprotein, suggesting its probable transmission from the bats [159] . bats, especially horseshoe bats (rhinolophus spp.), are considered to be the known reservoirs of sars-related covs. since the bat origin, covs have always caused outbreaks in humans, studying the diversity and distribution of coronavirus populations in the bats will help to mitigate future outbreaks in humans and animals [160] . interestingly, bats play a crucial role in all the spillovers mentioned above, indicating their importance in the emergence of new viruses. the reason behind the emergence and broad host range of covs in the past and present might be due to unstable rna-dependent rna polymerase (rdrp), lack of proof-reading ability, high frequency of mutations in the receptor-binding domain of spike gene and genetic recombination [140, 161, 162] . bat covs have high diversity and great potential of spillover in different animal species, as reported earlier in civet cat and dromedary camel, leading to well-known pandemics sars and mers, respectively along with the recent spillover in pigs resulted in swine acute diarrhoea syndrome (sads). however, spillover resulted in the emergence of sads-cov, which showed a 95% genomic identity with bat coronavirus, which led to severe mortality with 24,693 deaths in neonatal piglets [160] . fortunately, it did not excel in the form of the third pandemic, and no human cases were reported till date. the spillover responsible for ongoing covid-19 is still under investigation and a matter of great concern for the researchers all around the globe. based on resampling similarity codon usage (rscu), snakes (bungarus multicinctus and naja atra) were suggested as wildlife reservoirs of sars-cov-2 and reported to be associated with the cross-species transmission [27] and later it was disapproved by other researchers [163, 164] . unfortunately, to date, the intermediate host of the sars-cov-2 is abstruse what results in its escalation in the human population around the globe. in this context, analyzing the interaction between the asn501 site in rbd of spike glycoprotein of sars-cov-2 and the residue at 41 sites of ace2 receptor of different hosts (pangolins, turtle, mouse, dog, cat, hamster and bat) revealed that tyrosine has higher receptor binding affinity than histidine suggesting pangolins and turtle be closer than bats to humans and maybe the probable intermediate hosts of sars-cov-2 [96] . however, this hypothesis was also contradicted by li et al. [28] based on an insertion of the unique peptide (prra) in the sars-cov-2 virus, which was lacking in covs from pangolins. moreover, sars-cov-2 showed higher similarity to the betacov/bat/yunnan/ratg13/2013 compared to the ones that were isolated from the pangolins, thereby denied the direct link of the virus from pangolins. however, further studies are required to confirm the role of pangolins in sars-cov-2 spread to humans. the receptor-binding domain of the spike protein of sars-cov interacts with the host receptor ace2 facilitating its potential of cross-species, as well as human-to-human transmission [47] . similarly, the spike protein of sars-cov-2 was reported to recognize ace2 receptors expressed in fish, amphibians, reptiles, birds, and mammals and has a more robust binding capacity (affinity) in comparison to sars-cov [89] . this suggests their involvement as probable natural and intermediate hosts [161] , which may further help in the selection of animal models for epidemic investigation and preventing its spread [47] . bat origin covs have been found to cross the species barrier that favoured their transmission via recombination/mutations in the rbd. the evidence of a virus outbreak that occurred in chinese pig farms suggests its possible cross-species [160, 165] . also, murine cells were found permissive for sars-cov after substitution of his353 with lys353 in the ace2 receptor of a mouse, which suggests the role of residue changes in the cross-species and human-to-human transmission [166] . mutation in residues at position 479 and 487 of receptor binding motif (rbm) of sars-cov was reported to play a role in civet-to-human and human-to-human transmission, respectively [167, 168] . the covs are more prone to recombination and mutations leading to variable host range, and resemblance of receptors in various hosts results in cross-species jumping [31, 112, 169] . genetic divergence due to these genetic alterations results in the evolution of newer viral strains having altered virulence, tissue tropism, and host range [170, 171] . moreover, the presence of threonine at position 487 was reported to enhance the binding affinity of rbm for the ace2 receptor of civet and humans [172] . however, many sarsrelated coronaviruses (sarsr-cov) have been reported in bats and used ace2 receptors for entry into a host cell, which showed its potential to infect humans directly without any intermediate host [173] . in addition to this, no direct transmission of sarsr-cov is reported from bats to humans to date. however, seropositivity on a serological investigation of individuals without prior exposure to sars-cov residing near bat caves in china revealed likely infection of humans by bat sarsr-cov and related viruses [174] . besides, the interspecies transmission potential of sarsr-covs is due to the orf8 gene [175] . as per reports, the sars-cov emerged via recombination of bat sarsr-covs, was transmitted to farmed civets along with other mammals, and these infected civets spread the virus to market civets. the virus was reported to undergo mutations in infected market civets before its spillover to humans. similarly, the mers-cov circulated for 30 years in camels before the pandemic [97, 176] supporting the hypothesis that after species jumping the exogenous viruses opted for adaptation to the environment and host before spillover to humans [177] . moreover, the possible spillover of other circulating bat sarsr-covs to humans from mammalian hosts soon is highly anticipated. the cross-species jumping and adaptation are determined by the presence of specific receptors on host tissues (like ace 2 receptor for hcov-nl63, sars-cov and sars-cov-2, dipeptidyl peptidase-4 for mers-cov, human aminopeptidase n for hcov-229e, 9-oacetylsialic acids for hcov-oc43, hcov-hku1) which help in binding and entry of the virus into host cells [30, 31] . these receptors are present in various body systems in animals and humans, including respiratory and gastrointestinal systems [30] . reservoir host animals including bats and rodents possess these receptors which are similar to those present in camels, masked palm civets (paguma larvata), or bovines, that act as an intermediate host for different covs [30, 110, 111] . presence of some of these receptors in humans like ace2 or dpp4 makes them vulnerable to cov infection like sars-cov and mers-cov causing sars and mers infections, respectively [31, 168] . the mers-cov spike was found to possess the capacity for adapting to species variation in the host receptor dpp4 [37] . the mechanism expressed by mers-cov in adapting to infect cells of new species might be present in the other coronaviruses. ace2 has also been found as a binding receptor for sars-cov-2 [47] . the species-specific variations in the host receptors limit the interaction with cov spike protein, and this is responsible for the development of the species barrier that prevents spillover infection. snakes, civets, and pangolins are considered as the potential intermediate hosts of covid-19. however, further confirmation is required by tracking the origin of the virus. this is critical for preventing additional exposure to this fatal virus [178] . the probability of the sars-cov-2 spread during incubation and convalescent period has been suggested [76] . as per reports, presence of covs has been observed in respiratory droplets, body fluids and inanimate objects with the ability to remain infectious for nine days on contaminated surfaces resulting in its risk of self-inoculation via mucous membranes of the eyes, mouth or nose [179] [180] [181] . nosocomial, as well as human-to-human transmission, have been reported to occur via virus-laden aerosols, contaminated hands or surfaces, and close community contact with an infected person [66, 182, 183] . the ocular route has been reported in the human-to-human transmission of sars-cov-2, as observed in sars-cov, suggesting the involvement of different ways other than the respiratory tract [184, 185] . later on, the probability of the faecal-oral route for potential transmission of the virus was also suggested [186] . the metatranscriptome sequencing of sars-cov-2 in the bronchoalveolar lavage fluid (balf) of infected individuals resulted in polymorphism in few intra-hosts variants, suggesting the in vivo evolution of the virus thereby affecting its virulence, transmissibility, and infectivity [187] . an overview of coronaviruses jumping the cross-species barriers, zoonotic covs transmitted from bats to animals before spillover to humans, and possible prospects for further transmission to mammalian hosts is depicted in figure 1 . the first two decades (2000-2020) of 21 st century have proven a nightmare for the countries around the globe considering the coronavirus zoonosis, including the ongoing crisis of covid-19 which has involved entire fields of global [188, 189] . the countries affected severely by previous covs were even not evolved entirely from the effects of sars and mers when the covid-19 struck almost the entire world. novel coronavirus sars-cov-2 has shaken all the sectors of the countries irrespective of being developed or underdeveloped including healthcare system, economics, trade, infrastructure, service and production sectors [190, 191] . being a zoonotic disease with still unknown intermediate host, undisclosed features of a novel viral pathogen, unclear modes of transmission and ecological aspects, less explored pathogenesis and substantial morbidity and considerable mortality, the safety of all is a matter of great concern, and thus the involvement of various authorities was sought since the inception of disease [26, 31, 192, 193] . the first time the need for one health concept has risen to a level that authorities in various countries implemented coordinated approaches between medical, veterinary, public health, wildlife, food safety, environmental departments and so on [193] [194] [195] . that involved acquiring suggestions, diagnosis and prevention and treatment measures and their implementation in collaboration. non-medical staff in association with the medical staff was employed for initial screening, quarantine, contact tracing when the expertise of molecular biologists or technicians from various disciplines was used in the laboratory diagnosis. medical staff provided the cure and management of the patients when the public health departments, including public health engineering, municipality, food and supplies ensured sanitation, hygiene, food supply and safety. imposing of lockdown was provided by security personnel's and the transport department facilitated the movement of stranded people. thus, this crises management strategy involved various agencies directly or indirectly. however, as the animal, human and environmental health is linked to one another, the prime and future efforts should primarily focus on all these aspects. in addition to regular hand hygiene, respiratory etiquette, social/physical distancing, use of personnel protective equipment (ppe) and food safety recommendations, one health approach encompasses the role of veterinary, medical and environmental specialists for the prevention and control of current covid-19 crises and investigating the animal origin of covid-19, regulating and limiting the sale and farming of wildlife species for food and taking a one health approach to food systems feeding the world for the prevention of future pandemics [193, 196, 197] . considering the contagiousness of the virus, discouraging the working of affected individuals, public health hygiene strategies, and social distancing has been recommended as preventive measures [26, 198] . food hygiene and safety, as recommended by oie [193] and usda [199] , should be followed. as the viral survivability has been demonstrated on various surfaces [53] hence disinfection by using recommended disinfectants is necessary [200] . environmental hygiene and cleanliness are also essential [200] . interaction with animals and improper utilization of animal products during an outbreak should be avoided [193] . though the one health involves mainly public health, animal health and environmental experts, however, for the successful management of current crises and future prevention and control requires the participation of all concerned sectors having a role in public health measures, identifying clinical cases, diagnosis, contact tracing, proper infection control in various settings, isolation, quarantine, cure and management, public awareness, facilitation of infrastructure and other facilities through local administrations [26, 195] . as the human covid-19 cases are on the rise due to efficient human-to-human transmission, there is a subsequent rise in the natural infections of covid-19 among the companion and wild animal species owing to the spillover. this is mainly because of the specific biological and virological characteristics of coronaviruses that gives them the ability to easily cross-species barriers [130] . even though animal-to-human transmission is not reported in covid-19, 'one health' approach is necessary to control this pandemic virus a schematic illustration of covid-19 clinical signs, modes of transmission, important diagnostic methods, and advances in vaccine development along with salient prevention and control strategies are presented in figure 2 . with the rising number and worldwide spread of covid-19, the need for global efforts rely heavily on the investigations carried out at infection sites to trace different aspects of this novel coronavirus outbreak. one of the critical facets and the earliest research must involve determining the root cause, origin, and source of this emerging infectious disease. shreds of evidence have revealed various cross-species jumping or spillover from animals to humans of these zoonotic coronaviruses. detailed serological investigation of all domestic and wild animals residing in the proximity to humans is of utmost necessity to know and prevent likely spillover of many other bat-related covs in the future. rapid detection of spillovers above will only be possible by the implementation of an effective and robust surveillance system for circulating viruses with high zoonotic potential in animals. besides, detection of a pathogen while crossing the species barrier to start circulation among humans and prevention of human-to-human transmission in early-stage may prove crucial in termination of a probable epidemic or pandemic. application of 'one health' concept involving medical, veterinary, wildlife, public health, and other related professionals may help in infection tracing, exploring risk factors and predisposition, minimizing risk to susceptible ones, and finally devising better prevention and control strategies. in the initial stages of the covid-19 outbreak, the steps taken for implementing stringent control and 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especially those with pulmonary failure, are not a consequence of viral burden and/or failure of the ‘adaptive’ immune response to subdue the pathogen by utilizing an adequate ‘adaptive’ immune defense. rather it is a consequence of immunopathology, resulting from imbalanced innate immune response, which may not be linked to pathogen burden at all. in fact, it might be described as an autoinflammatory disease. the kawasaki-like disease seen in children with sars-cov-2 exposure might be another example of similar mechanism. replicate both in the upper and lower respiratory tract might explain why people infected have such different experiences. the virus can start in the throat or nose, producing a cough, disrupting taste and smell, and then end there. or it might work its way down to the lungs and debilitate that organ and the entire organism. it is quite likely that once sars-cov-2 gets down in the lungs, it's probably just as deadly as sars-cov-1. in sum, the assessment expressed in the general media, but sometimes also in professional literature [26] [27] [28] [29] that the virus is entirely -new‖ and that the immune system is -naive‖ namely that it is totally inexperienced when it comes to this virus is exaggerated. a more accurate description of this virus would be a reemergence of a known foe that has a somewhat more optimal organization of its resources making it more suitable to become a pandemic pathogen. the genetic sequence diversity of sars-cov-2 is low. as a consequence of a massive effort to sequence viral samples obtained from humans infected all around the world, there are now thousands of viral sequences available which permits an unprecedented and detailed analysis of the virus's genetic evolution. during the early phase of the outbreak in wuhan two major lineages of sars-cov-2, with different exposure histories, were categorized as l (∼70% of sequences) and s (∼30%) based on two tightly linked single nucleotide polymorphism (snp)s-a synonymous mutation in the orf1ab of the genome, and a non-synonymous s84l amino acid change in orf8. [30] . the s variant was evolutionarily more related to animal cov. the functional consequences of the s84l mutation are not known. nevertheless, the two variants exhibited similar virulence and clinical outcome [31] . a mutation in the spike protein-that mediates sars-cov-2 entry into host cells and potentially of functional importance-was described by several teams of researchers [32] [33] [34] [35] . the d614g mutation at residue 614 of spike (s) protein causes an amino acid change from aspartate to glycine. the mutation that causes the d614g amino change is transmitted as part of a conserved haplotype defined by four snps that almost always track together, although they probably arise independently. beside the d614g mutation the haplotype includes another nonsynonymous mutation (p322l) in the nsp12 viral protein. while the functional consequences of the p322l mutation remain unclear at present, there is strong evidence that the g614 variant is associated with greater infectivity and higher viral loads in the upper airways during infection [29, [32] [33] [34] [35] . the s protein must be cleaved by host proteases to enable membrane fusion, which is critical for viral entry. the g614 mutation creates a novel serine protease cleavage site that can facilitate its cleavage by host serine protease elastase-2 [32] . also g614 mutation increases both spike stability and membrane incorporation [33] . in cell culture, s-g614 pseudovirus infected ace2-expressing cells significantly more efficiently than the s-d614 pseudovirus [29, 34] . even though the g614 variant appears to be more infectious, it did not appear to be more virulent since hospital outcomes were similar with either variant [29, 33] . dynamic tracking of variant frequencies sampled from covid-19 patients from asia, europe, north america, and australia show that the d614 variant was initially dominant, so most subjects infected during december 2019 through the end of february 2020, had this variant [29] . the earliest viral sequence that carried the g614 mutation with the other three snps that characterize the haplotype was sampled in northern italy on february 20 th . during march 2020 both variants could be identified circulating in the population [29, 34] . by april, the g614 variant was circulating almost exclusively in european and in the greater nyc area. this variant continued increasing in frequency over several months so that by june 2020 it has become the dominant variant all over the globe. it is tempting to hypothesize that the successful mitigation of the outbreak in china and several other east asian countries was due, at least in part, to the fact that they faced the less infective d614 variant. the european countries and nyc had to deal with the more infectious g614 variant. similarly, the early outbreak in washington state was caused by the d614 variant [34] . the original march-april outbreak on the western coast of the united states might have been less severe than in the northeastern united states, because it originated from the washington state d614 variant. by june 2020, the g614 became the global dominant variant, and is responsible for the much more infective july phase of the outbreak in the southern and western usa. the viral genetic data presented clearly demonstrate that variants may arise quickly, even in cov, and have profound effects on the spread and consequences of the covid-19 pandemic. prior research has uncovered gene variants that can alter a person's chances of contracting a viral infectious disease. the most famous example is a mutation in the ccr5 gene, which offers protection against hiv. while there are 32 coding variants in human ace2, with some of them in residues considered important for the binding of s-protein in cov [36] , there is no evidence for the existence of cov s-protein-resistant ace2 mutation in any population [37] . neither is there any evidence for ace2 variants that bind more, or less, efficiently to sars-cov-2 s-protein. based on publicly available data from east asia, europe and north america, a group of british epidemiologists conclude that children are half as likely to be infected by sars-cov-2 as adults [38] . the reliability of these estimates are limited by lack of direct assessments of the transmission of the virus between adult to child, and child to child, compared with adult to adult. furthermore, because children tend to have less comorbidities than adults, they experience less disease symptoms, and as such are tested less than adults. hence, the number of children infected may be grossly undercounted. a publication that in this short timeframe has already been cited frequently, seeks to confirm that the incidence of sars-cov-2 infection is lower in children than adults due to a lower density of ace2 receptors in the nasal epithelium of children as compared to adults [39] . the researchers report finding less ace2 rna in cells scraped from the noses of children than in those from adults. the significant difference in this study is reported to be between those under 17, and those aged 18-60. in addition, there was no difference in the amount of ace2 rna detected according to gender, or those with or without asthma. several caveats to consider: the average relative amount of rna ranged from 2.4 for those less than 10 years old, to 3.09 for those 25 and older. the differences are relatively small and the error bars large. while those over 60 are those most affected by covid-19, the study did not include individuals older than 60 years. also, the density of ace2 receptors may not be uniform throughout the nasal mucosa, and no evidence is presented that the entire nasal mucosa was j o u r n a l p r e -p r o o f equally sampled. lastly, it is unknown how many receptors are needed for the sars-cov-2 virus to successfully infect us. in any case, there is no doubt that children of any age can be infected. i am more convinced by the conclusions of dong et al. [40] that children of all ages are susceptible to infection by sars-cov-2 without significant sex differences. although clinical manifestations of children's covid-19 cases seem generally less severe than those of adult patients, young children, particularly infants, are vulnerable to infection. as the aforementioned publication [39] assumes, if ace2 is the sole receptor for viral entry, then the expectation is that high ace2 tissue expression equals higher infectivity and worse outcomes. however, a careful consideration of the role of ace2 in the renin-angiotensin system physiopathology is indicative of it playing a protective role-meaning higher ace2 expression is more likely to protect us from a worse outcome of viral infection. as such, ace2 has an important role in counterbalancing the effects of ace1. angiotensin ii, a product of ace1 cleaving angiotensin i, can cause vasoconstriction, inflammation, and fibrosis. ace2 cleaves angiotensin ii to angiotensin 1-7, which antagonizes the activities of angiotensin ii-hence, it can suppress inflammation, fibrosis, and generate vasodilation. further, a high ace2/ace1 ratio protects the integrity of the endothelium and promotes antithrombotic activity. previous studies have found ace2 playing a protective role in severe lung injury in ace2 knockout (ko) mice [41] . ace2 ko mice challenged with avian influenza h5n1 [42] or h7n9 [43] resulted in severe lung injury, despite that ace2 is not a receptor for avian influenza. in fact infection with avian influenza strains resulted in downregulation of ace2 expression in the lung and increased serum angiotensin ii, both in mice and human subjects infected by the virus [42] . the question is whether ace2 expression levels are pertinent to sars-cov-2 infection only in the tissues relevant to viral entry and the lungs as its major target, [44, 45] or, given that covid-19 in its severe form is a systemic disease with multi-organ disfunction [46, 47] , ace2 expression levels may be important in multiple organs and tissues other than those of the respiratory system. relevant to this question, lungs do not have high expression levels of ace2, and relatively few cell types express ace2 in the lung compared to other tissues [37, 48] . assuming the importance of ace2 expression throughout the human organism, in silico analyses have been undertaken through integrating public genomics, epigenomics and transcriptomics data in multiple tissues, different populations, disease conditions, and age as well as sex considerations. intriguing in silico findings suggest that east asian populations have higher allele frequencies in expression quantitative trait loci (eqtl) variants associated with higher ace2 tissue expression compared to european and african populations [37, 48] . furthermore, ace2 expression increases by estrogens and to a much lesser degree by androgens, which possibly explains the higher ace2 expression in females [48] . the study suggests an inverse age-dependent ace2 expression in both males and females, a reduced expression in type 2 diabetes, and inhibition of ace2 expression by inflammatory cytokines [48] . these interesting suggestions (supporting a protective role of high ace2 expression against sars-cov2 fatality) need to be validated by clinical observations, in vivo, and in vitro experimentation. until such time, the functional consequences of ace2 expression levels to the susceptibility or response to sars-cov-2 remain unclear [49] . in addition to ace2, viral entry requires s-protein cleavage at the s1/s2 and s2' sites allowing fusion of viral and cellular membranes by host proteases. the transmembrane serine protease s2 (tmprss2) is frequently employed for this purpose by sars-cov-1 and sars-cov-2 [20, 50] , but also by mers [51] , and human cov-229e [52] . the functional importance of tmprss2 in cov infections was tested in tmprss2 ko mice infected with sars-cov-1 and mers-cov. results show that lack of tmptss2 in the airways reduced severity of lung pathology after infection by sars-cov-1 and mers-cov, despite that all other host proteases were intact [53] . apart from cov, tmprss2 is an important host protease for influenza viruses, by cleaving the influenza virus hemagglutinin (ha) molecule [54] . furthermore, genetic variants with higher tmprss2 expression increase susceptibility to severe human h1n1(2009) and avian h7n9 influenza [55] . these findings, raise the intriguing question whether genetic variants with higher tmprss2 expression confer higher risk and/or severity of sars-cov-2 infections. a preliminary study from italy suggests that two distinct tmprss2 haplotypes show significant frequency differences between italian and east asian populations [56] . specifically, the rare alleles of these haplotypes predicted to induce higher levels of tmprss2, are more frequent among italians. a snp belonging to one of the haplotypes is the same one found to be associated with increased susceptibility to severe influenza [55] . as will be discussed further, cytokines play an essential role in the pathogenesis of covid-19. while environment, microbiome, genetics, and host factors, each can influence cytokine responses to pathogen stimulation, genetic variations appears to be a main component shaping cytokine responses in humans [57] . for perspective, the microbiome does seem to have a smaller impact on cytokine production capability. it is estimated that microbiome explain only 10% of cytokine production [58] . large-scale studies from the human functional genomics project [57] have shown that different cytokines have different levels of genetic influence. this is an important concept for host defense and disease, as cytokines are fundamental in orchestrating overall immune responses, and can drive pathology when dysregulated, as is the case in covid-19. pertinent to the present discussion, the il-1/il-6 pathway especially, seems to be regulated mainly by genetic factors [57] . my own early work, has provided evidence for the heritability of tnf production capability in mouse and man [59, 60] . notwithstanding the current paucity of such studies in cov, i strongly believe this area of research promises to generate valuable information as for the pathogenesis and potential treatment of covid-19. african americans infected with sars-cov-2 seem to be at a greater risk for severe outcome. while comorbidities and socioeconomic circumstances certainly play a critical role, cytokines may have an important role too. a preliminary study compared expression levels of cytokines and other immune modulators between caucasian americans and african americans using rnaseq data [61] . results show il-1 and il-18 receptor 1 (il18r1), il12r1, tlr7 and tlr9 were significantly higher in african americans suggesting perhaps the tendency to develop higher inflammatory cytokine responses. much more work is needed to validate these observations. genome wide association studies (gwas) allowing the unbiased clustering of genetic variation defining human diseases, require assembly of dna samples from very large number of subjects which usually take a long time to collect. given that we are just six months into the pandemic, it is quite remarkable that a medium size gwas was already completed. it is the first to document a statistically significant association between genetic variants and severe covid-19 [62] . variations at two loci in the human genome were associated with an increased risk of respiratory failure in patients with covid-19; one, j o u r n a l p r e -p r o o f within the abo blood type cluster on chromosome 9, and the other at position 3p21.31. the frequency of the chromosome 3 risk allele was significantly higher in those patients that needed mechanical ventilation, compared to those with less severe disease progress. importantly, this locus is home to six genes, and it is not yet possible to identify which of them is responsible for aggravating the disease course. this locus contains a cluster of chemokine receptors, xcr1, ccr9 and cxcr6 (and several other cytokine receptor genes close by), which have important regulatory functions in the innate immune system. another candidate within the locus, slca20 is an amino acid (proline) transporter expressed at luminal membrane of small intestine and proximal tubule kidney cells and functions in absorption of proline. its expression in rodent intestine depends on the presence of ace2 [63] . however, amino acid transporters have been shown to induce cytokine responses: genetic variation at the slc36a4 amino acid transporter show strong association with pathogen induced il-22 production [57] . also amino-acids or amino acid catabolites have been reported to modulate cytokine production [64, 65] . so it is conceivable that slca20 might influence the course of covid-19 severity by affecting pathogen induced cytokine production, rather than viral entry through ace2. in this respect, the same risk snp in the chromosome 9 abo blood type cluster that affects covid-19 severity has been associated with elevated il-6 levels in childhood obesity in a previous gwas, thus, possibly linking this genetic allele with elevated il-6 levels (with or without full-blown ‗cytokine storm') described in severe covid-19 patients. the study [62] is equally striking for the genes that failed to turn up. pathogen microorganisms, including several viral infections, are controlled by genetic variations at the hla complex at chromosome 6p21 [66, 67] . the class i and class ii gene products of the hla are involved in antigen presentation, a mandatory process to initiate an adaptive immune response geared to restrain a pathogen. but genetic variants at the hla region did not appear to make a difference in the risk of severe covid-19. thus, the so called ‗adaptive' arm of the immune system seems to be less relevant to covid-19 than the ‗innate' immune response. i have offered here a hypothesis for what these genetic associations might actually mean. if correct it has major mechanistic implication as for the pathogenesis. at minimum it suggests avenues for further studies. recognition of a pathogen by the innate immune system triggers the secretion of the crucially important type i/iii interferons (ifn). the result of ifn signaling is the activation of an entire cascade of events that include the release of proinflammatory cytokines which further signal to endothelial cells, which then enable chemokines to spread throughout the blood to recruit innate immune cells to the site of infection. the recruited nk cells, monocytes, and neutrophils interact with activated endothelium to leave the blood stream and migrate toward the site of infection. at the site of infection they can perform effector functions to control infections, such as release of reactive oxygen species (ros) and, where they can perform effector functions to control infections, such as release of reactive oxygen species (ros) and directly killing infected cells, as well activating a pathogen specific adaptive immune response. given the shared sequence homology, the virus-host interactions of sars-cov-2 is likely to be analogous to those involving other cov. however, these interactions might be similar to j o u r n a l p r e -p r o o f other non-cov viruses as well because of limited repertoire and conserved mechanisms of innate immune signaling. sars-cov-2 engages host pattern recognition receptors (prr), and toll-like receptors (tlr) which initiate downstream signaling pathways triggering secretion of cytokines. if present early and properly localized, ifn are considered the most effective in limiting cov infections [68] . ifn induced proteins can interfere with viral entry and s-protein-mediated membrane fusion [69, 70] . however, in a later phase of the infection, ifn can become pathologic (e.g. upregulation of ace2 in airway epithelium [45] and orchestration of inflammatory response contributing to immuno-pathogenesis [127] ). sars-cov-2, similar to other cov, have developed multifaceted mechanisms to inhibit ifn induction and signaling [71] . this is evident by an early impaired ifn signature in severe covid-19 patients [72, 73] , while actively promoting other inflammatory pathways contributing to pathology (e.g. secretion of pro-inflammatory cytokines interleukin (il) 1β (il-1β) and il-18 [74] ). sars-cov-2 is particularly effective in inducing il-6 and il-8, by inhibiting an endogenous nf-kb repressor, nkrf [75] but probably by other mechanisms as well. while the human innate immune system resources remained unchanged, cov employs multiple innate immunity evasion mechanisms as reviewed recently [27, 76] . the earlier cov infections can provide an important road map to understand covid-19 pathogenesis. thus, a clear indication that immunopathogenesis contributes to sars was the observation that sars-cov-1 viral loads were found to be decreasing while disease severity increased [77, 78] . longitudinal in vivo experiments in which ferrets were infected with sars-cov-2 intranasally showed a robust production of cytokines that continued beyond clearance of the virus. by day seven, despite waning viral burden, the cytokine response continued to expend. remarkably, by day fourteen, while the virus was fully cleared, some cytokines and specifically il-6 remained elevated [73] . in fact, il-6 emerges as the dominant cytokine driving the immunopathogenesis. given that old age appears to be an independent risk factor for developing severe covid-19 [79] , it is noteworthy that in a groundbreaking study ter horst et al., have shown a clear and consistent increase in il-6 and il-1ra production in old age [80] . evidence is accumulating in covid-19 patients pointing to dysregulated monocyte driven dendritic cells and macrophage responses, which then drive the characteristic acute respiratory distress syndrome (ards) and cytokine release syndrome (crs) [81] . cd56 dim nk cells, generally thought to contribute to antiviral host defense through cellmediated cytotoxicity, were depleted primarily in severe cases. whereas cd56 bright nk cells, which are considered producers of ifn-γ and tnf-α, were significantly depleted in all covid-19 samples tested [82] . evidence supports recruitment of nk cells from the periphery to the lung. activation of these nk cells in the target tissue in an environment enriched for il-6 (and other cytokines) probably contributes to pathogenesis [76, 83] , as opposed to resolving the infection. severe cases of covid-19 have significant increase in neutrophil levels in circulation [84, 85] and in bronchoalveolar lavage fluid (balf) [86] . together with the upregulation of chemokines, particularly those that act as chemoattractants for neutrophils and monocytes j o u r n a l p r e -p r o o f [73, [86] [87] [88] , these observations support the influx of these cell types into the bronchi. these neutrophils and monocytes probably disrupt the air-blood barrier by causing collateral damage to airway epithelial cells and vascular endothelial cells while increasing cytokine production. the damage to vascular endothelial cells certainly contributes to microthrombosis. though seemingly contradictory to mechanisms of immune evasion, enhanced innate immune activation is central to the morbidity and mortality of covid-19 patients. immune evasion seems to characterize the first phase of infection with sars-cov-2 and this is associated with reduced innate antiviral immunity. however, approximately twenty percent of infected subjects develop an excessive innate immune activation approximately 7-10 days after infection, which i argue is associated only marginally, if at all, with viral load. rather, the massive inflammatory response is a consequence of host dysregulation of the immune system. in line with observations in sars-cov-1 [89] , sars-cov-2 induces a robust cytokine response with low levels of ifn in the early phase, culminating in improper recruitment of inflammatory monocyte-macrophage and neutrophil populations into target organs, resulting in further cytokine production [73] . a recurring theme in covid-19 pathogenesis is that components of the immune system that are generally thought to contribute to antiviral host defense end up promoting disease severity. typically, the complement system can efficiently recognize and eliminate viral pathogens by opsonizing viruses and virus infected cells, inducing an antiviral inflammatory state, increasing virus-specific immune responses, and neutralizing cell-free viruses [90] . however, the activation of multiple complement pathways, dysregulated neutrophil responses, endothelial injury, and hypercoagulability appear to be interlinked with sars-cov-2 infection and instead serve to drive the severity of the disease [91] . the functional importance of complement activation in cov was tested in c3 ko mice infected with sars-cov-1 [92] . the studies showed that complement activation regulates a systemic proinflammatory response and removal of c3 signaling reduced lung injury and respiratory dysfunction, despite equivalent viral loads present in the lungs. this was associated with reduced lung infiltration of neutrophils and monocytes and lower cytokine and chemokine levels in both the lungs and sera [92] . lung biopsy samples from patients with severe covid-19 show widespread complement activation characterized by c3a generation and c3-fragment deposition [93] . the host complement activator masp2, the key serine protease in the lectin pathway of complement activation, was identified as a target of the n (nucleoprotein) protein of sars-cov-1, mers-cov, and sars-cov-2, resulting in aberrant complement activation and aggravated inflammatory lung injury. in mice, lung injury induced by sars-cov-1 or mers-cov n protein was attenuated when its masp2-binding motif was altered, when masp2 was genetically knocked out, or when the masp2-n protein interaction was pharmacologically blocked [93] . earlier i have discussed the gwas that established significant association between severe covid-19 and the abo blood type cluster [62] . having blood type a was linked to an approximately forty-five percent increase in the likelihood that a patient would develop j o u r n a l p r e -p r o o f respiratory failure, while subjects with blood group o were at a thirty-five percent decreased risk for respiratory failure. a possible mechanistic explanation could be that type o patients harbor both anti-a and anti-b natural igm abs. these may help reduce the viral load of their hosts due to early activation of the classical complement pathway followed by viral clearance. such mechanism has been shown to work in vitro, using measles virus produced in cells engineered to express only a-type, b-type or o-type carbohydrate epitopes. measles virus was neutralized by human serum (that did not contain anti-measles ab), utilizing natural abs against the a and b antigens in a strictly complement-dependent manner [94] . these observations support a role for the complement system in enabling natural abo group abs as first line innate immune defense to viral infections. although most early studies concentrated on the lungs as the target organ in sars-cov-2 infection, it is now clear that covid-19 has a wider spectrum of organ involvement. this may be a result of the broad organ tropism of sars-cov-2, but is more likely due to an outof-control host immune response to the virus. indeed, evidence is accumulating in support of vascular cell dysfunction in multiple organs during sars-cov-2 infection [95] . first, sars-cov-2 is able to directly infect engineered human blood vessel organoids [96] . more importantly, histopathological evidence of vasculitis, sometime associated with viral particles, and accumulation of neutrophils and monocytes, and even lymphocytes, in the wall of blood vessels in multiple organs were described [97] . in addition, endothelial apoptosis and pyroptosis might contribute to endothelial cell injury. similarly, bryce et al., found diffuse vascular endothelial inflammation with micro and macro vascular thrombosis in the venous and arterial circulation [98] . the vascular endothelium is indispensable for the regulation of vascular tone and the maintenance of vascular homoeostasis. endothelial dysfunction is a principal determinant of microvascular dysfunction by shifting the vascular equilibrium towards more vasoconstriction with subsequent organ ischemia, inflammation with associated tissue edema, and a pro-coagulant state [99] . vascular endothelial damage could explain why people with pre-existing conditions like hypertension, diabetes, obesity, and cardiovascular disease are at a higher risk for severe complications. all of those conditions, identified as independent covid-19 risk factors, cause endothelial cell dysfunction. the additional damage and inflammation in the blood vessels caused by the viral infection could push them over the edge and cause catastrophic complications [79] . given that vascular endothelial cells express ace2, one likely hypothesis suggests that sars-cov-2 infects endothelial cells directly which induces injury, activates complement, and sets up a perpetual inflammatory state [91] . however, there are alternative mechanisms for activation of endothelial cells and vasculitis induction that do not necessarily require the presence of the virus itself, e.g. neutrophil extracellular traps (nets) [100] and hypoxia [101] . the ability of neutrophils to form nets is considered beneficial in host defense against pathogens, but as observed regarding other innate immune mechanisms, sustained net formation can trigger a cascade of damaging inflammatory reaction. indeed elevated levels of net-specific markers, myeloperoxidase dna, and citrullinated histone h3, were observed in the sera of covid-19 patients [102] . as such, several research groups are supporting a more central role for nets in covid-19 pathogenesis [102, 103] . coagulation disorders in patients with covid-19 were initially thought to be due to systemic disseminated intravascular coagulopathy (dic). however, considerable cross-talk and mutual engagement between the complement and the coagulation cascades is being increasingly documented in covid-19 and seems to be responsible for a prothrombotic environment distinct from dic, leading to serious adverse outcomes [91, 104] . in fact, elevated d-dimer (a fibrin degradation product indicative of hyperactive coagulation) has emerged as a reliable marker of severe covid-19 [105, 106] . interestingly, the interconnected complement and coagulation cascades, which are being increasingly documented in covid-19, has been long recognized in antiphospholipid syndrome (aps) [107] . given that several publications report antiphospholipid antibodies in patients with covid-19 [108] [109] [110] , despite them not fulfilling the sydney criteria for aps [111] , it is difficult to ignore how much the clinical and immunopathological picture of aps resembles that of the autopsy findings of an exaggerated inflammatory state and thrombosis in many covid-19 patients [112, 113] . a study by nicolai et al., provides mechanistic evidence that multisystem disease in severe covid-19 involves coagulopathy driven by dysregulated innate immune response [114] . they show inflammatory microvascular thrombi containing platelets, fibrin, and a large number of neutrophils in the lung, kidney and heart. neutrophils were highly activated in severe cases compared to less severe cases and platelets showed enhanced neutrophil adhesion and net formation in multiple organs. thus, dysregulated immunothrombosis is linked to both ards and systemic hypercoagulability [114] . in sum, an overwhelmed innate immune system is seemingly unable to assemble a balanced response of appropriate cells, cytokines and other molecules to control the infection in a timely fashion. instead, a disoriented, misguided storm of inflammatory cytokines ends up destroying that which it intended to protect. it disintegrated in moderate and severe covid-19-lymphopenia with drastically reduced cd4+ and cd8+ t-cells is the most consistent finding in moderate and severe covid-19 patients, which also correlates with disease severity and mortality [115] [116] [117] [118] . b-cells are decreased as well [119, 120] unlikely to be the case [88] . despite the fact that lymphopenia seems to be a prominent feature of severe covid-19, patients under immunosuppressive therapy are not at a higher risk for infection, and if infected seem not to be destined to have more severe progression. in fact, reviewing the mortality and morbidity reports published on sars-cov-1, mers-cov, and covid-19, no mention is made on immunosuppression as a risk factor for morbidity and mortality. further, no severe complications or fatality is reported to be linked to transplantation, chemotherapy, autoimmune hepatitis, ibd, or other conditions requiring immuno-suppressive treatment for patients at any age [121, 122] . in fact, reviewing the mortality and morbidity reports published j o u r n a l p r e -p r o o f on sars-cov-1, mers-cov, and covid-19, no mention is made on immunosuppression as a risk factor for morbidity and mortality. further, no severe complications or fatality is reported to be linked to transplantation, chemotherapy, autoimmune hepatitis, ibd, or other conditions requiring immuno-suppressive treatment for patients at any age [121, 122] . these observations reinforce the notion that the reduced t and b lymphocytes in covid-19 patients is not the direct cytopathic effect of the virus itself. the possibility that the peripheral lymphopenia observed in patients with covid-19 reflects recruitment of lymphocytes to the respiratory tract or adhesion to inflamed respiratory vascular endothelium has been suggested [31] . although autopsy studies of patients' lungs and single-cell rna sequencing of balf do identify the presence of lymphocytes, the lymphocytic infiltration is modest at best, arguing against sequestration as a main cause of lymphopenia [123] . the most likely scenario is that inflammatory cytokines are key factors behind the observed lymphopenia. indeed, serum levels of il-6 especially have been closely correlated with lymphopenia, while recovered patients show return of lymphocytes numbers towards the normal range with significant reduction in il-6 levels. a likely mechanism is via the downregulation of multiple hla class ii molecules on cd14 monocytes and b-cells by il-6, as demonstrated by multiple studies [82, 119, 120] . hla class i molecule downregulation was less severe and inconsistently observed. a negative correlation between serum levels of il-6 and the number of class ii hla on cd14+ monocytes supports the notion of downregulation of class ii by il-6 [82, 119] . this decrease in hla molecules suggests that severe covid-19 patients might be unable to mount a normal t cell response due to reduced antigen presentation capability to t cell receptors (tcr). such t cells are then eliminated by apoptosis. autopsy studies on lymphoid organs collected from patients who succumbed to the disease revealed massive lymphocyte death, which was linked to high levels of il-6 as well as fas-induced apoptosis [124] . treatment with tocilizumab, an il-6 receptor antagonist, restores, at least partially, the hla class ii molecules on antigen-presenting cells. in addition, it increased the number of circulatory lymphocytes, further suggesting il-6 is a key player in the lymphopenia development [119] . further, nk t-cells are reduced in number and impaired in function in severe covid-19 in an il-6 dependent manner [120] . since cd8+ t cells require antigen presentation by hla class i for activation via tcr, and these molecules are less reduced than class ii molecule, it is unlikely that the same mechanism of apoptosis of inactivated cd4+ t cells would be operating to a similar extent in cd8+ t cells. rather, it is possible that most cd8+ are eliminated due to activation/exhaustion of t cells [116] . intense immune profiling of covid-19 patients show a very heterogeneous immune response. a rigorous comparison between the studies is complicated by major differences in cohort sizes, dissimilar clinical phenotypes used, utilization of different experimental strategies, and emphasis on different parameters by various investigators. the study by giamarellos-bourboulis et al. [119] suggest that severe covid-19 patients developing severe respiratory failure show one of two immune phenotypes: (1) an immune dysregulation phenotype (~75% of patients) characterized by major reduction of hla-class ii molecules on cd14+ monocytes in the absence of elevated ferritin. this is triggered by monocyte j o u r n a l p r e -p r o o f hyperactivation, excessive il-6 production, and profound lymphopenia, but without il-1β elevation; and (2) a mas phenotype (~25%) associated with elevated ferritin, with relatively less reduction of hla class ii molecules on monocytes and triggered by il-1β. further studies will be needed to verify whether these immunophenotypes are generalizable. mathew et al. [125] identified a subgroup of approximately twenty percent of severe covid-19 patients that lack detectable lymphocyte response to the infection, suggesting a total failure of immune activation. further, these investigators emphasize that the typical tcell/b-cell communication and cooperation, the adaptive immune system depends on, is practically nonexistent in some covid-19 patients. lucas et al., [126] confirmed an overall increase in innate cell lineages, reduction in hla class ii molecules on monocytes, and early surge in cytokines with parallel reduction in t cells, observed by many other studies. in addition, according to these investigators the immune responses to pathogens can be roughly grouped into three categories characterized by different sets of immune cells and cytokine signals used: type 1-broadly t h 1 responses directed against viruses and intracellular bacteria; type 2-directed against parasites that do not invade cells; and type 3-directed against fungi and bacteria that can survive outside the cells. type 1 immune response expected in sars-cov-2 infection was seen in mild to moderate cases, while in severe cases the immune system seemed to invest too many resources in non-appropriate type 2 and type 3 immune signaling. this immune disintegration, the investigators have dubbed as -misfiring,‖ seems to extend to the realm of t and b lymphocytes [126] . other investigators describe the catastrophic cov disease as a lack of switch from an innate immune response to an adaptive immune response [13] . these depictions of the immune response in severe covid-19 are symbolically captured in fig.1 . the antigen specificity of sars-cov-2 t cells have just started to be characterized in covid-19 patients [129, 130] and their potential protective role awaits additional research. however, it is already clear that patients who recovered show specificity for multiple sars-cov-2 proteins, not only spike protein. interestingly, cross-reactive memory cd4+ and cd8+ t cells are also found in ~ 50% of subjects who have never been exposed to sars-cov-1/2 [129, 130] . while sars-cov-1/2 unexposed donors can recognize both structural and nonstructural viral proteins, nonstructural orf-1-nsp7/13-specific t cells are often dominant. in contrast, sars-cov-1/2 recovered individual preferentially recognize structural proteins [130] . at present no satisfactory explanation for this phenomenon has been offered. also unclear is how these preexisting memory t cells, which are presumably generated in response to human common cold cov, affect immunity or pathology upon sars-cov-2 infection. heterologous viral t cell immunity and immunopathogenesis-an important consideration that has not yet received sufficient consideration in the current viral pandemic, is highly relevant to the observations of cross-reactive t-cell responses in non-exposed subjects. in fact, only laboratory animals kept in pathogen free conditions are naïve. no human more than a few weeks old is immunologically naïve [131] . the history of previous exposure, not only to related, but also to unrelated microorganisms, can greatly alter the host's immune response to a viral infection and can change the course of disease [131] . in fact, it is very likely that this phenomenon is a common feature of human viral infections. t cells have high levels of j o u r n a l p r e -p r o o f cross-reactivity because the tcr first scans the peptide-hla complex by binding to the hla, and then it molds itself around the peptide. actually, the tcr contacts only 2-4 amino acids in the peptide, so the total energy of the tcr-peptide-hla interaction is heavily influenced by the hla rather than the peptide. the consequence is a highly promiscuous t cell which allows a large variation in peptide sequences without inhibiting the hla-peptide-tcr interaction [132] . hosts that have never experienced a particular virus, might, nevertheless, have memory t cell pools that show specificity for it by virtue of crossreactivity that can shape the repertoire of the primary response. the observation that the same virus can cause widely different pathological manifestations in humans might be due (at least in part) to an established adaptive immunity toward related or unrelated viruses, which results in enhanced protective immunity in some, reduced protective immunity in others, or altered immunopathology, including enhanced disease severity in some hosts [133] . the role of humoral responses in the pathogenesis of covid-19 remains unclear. as in sars-cov-1 infection, most subjects infected by sars-cov-2 seroconvert within 7-14 days after infection and this process is associated with increase in plasma cells, whereas naïve b cells decrease significantly [134] . while recovered patients generate sars-cov-2specific neutralizing abs and spike-binding abs concurrently, their titers are highly variable in different patients [135] . about one third of recovered patients generate very low titers of sars-cov-2-specific neutralizing abs [135, 136] , and some patients (possibly up to 20%) who recover, do not have detectable neutralizing antibodies at all [129, 135, 137] . these observations bring up the question of how the virus was cleared-as it eventually was in all those patients studied-without strong ab responses. we can speculate that t-cell mediated immune responses, or non-specific responses by innate immune cells were responsible for viral clearance. however, since a pathogen that kills off the host that it needs to survive is also threatening its very own existence, the possibility of cov self-clearing should be considered, especially when alternative hosts are plentiful. in a remarkable study [137] the levels of total igg and igg neutralizing antibodies (as measured using a spike protein pseudotyped virus) were quantified in symptomatic and asymptomatic patients eight weeks after release from the hospital (roughly three months after start of infection). the levels of igg and neutralizing antibodies were significantly decreased in the majority of patients in both groups. the decrease in neutralizing abs was more pronounced in asymptomatic (~80%) as compared to symptomatic (~60%) patients. taken together, the finding that ~20% of people infected with sars-cov-2 do not make anti-viral abs, added to the observation that neutralizing abs begin to drop noticeably during convalescence-suggests that infection with sars-cov-2 does not establish long-lasting serological immunity, at least not for those who are asymptomatic or mildly ill (more than 80% of all those infected by sars-cov-2). even more problematic-several studies show significantly higher igg and iga ab responses. this does not correlate significantly with protection, but rather with severity of disease [138] [139] [140] [141] similar to what was seen in sars [142] . at minimum, these studies suggest that a robust ab response is insufficient to protect from severe disease [76] . while it is frequently assumed that anti-sars-cov-2 abs might be either beneficial or irrelevant, there is also the possibility that such abs might actually be detrimental. first, abs can cause immunopathology by binding viral fragments followed by activation of the complement cascade by the ab complex. the consequences of complement activation in the pathogenesis of covid-19 was discussed above (section 4), and extensively reviewed by others [91] . second, ab responses to cov may contribute to pathology via ab-dependent enhancement (ade). ade is mediated by non-neutralizing virus-specific igg engagement of fc-receptors (fcr) expressed on immune cells, particularly monocytes and macrophages, leading to inflammatory activation of these cells. anti-s-igg passive immunization of sars-cov-1-infected rhesus monkeys significantly enhanced the viral induced acute lung injury with massive accumulation of monocytes and macrophages in the lung in an fcγr dependent fashion [143] . further, serum containing anti-s-igg from patients with sars-cov-1 enhances the infection of sars-cov-1 in human monocyte-derived macrophages in vitro [144] . a monoclonal ab isolated from a patient with mers, targeting the s-protein of mers-cov showed fcr dependent adh [145] . high dose iv immunoglobin (ivig) treatment which has shown some efficacy in cov including covid-19 [146] , may diminish adh by blocking fcr mediated activities of monocytes and macrophages. direct evidence for ade was not documented in covid-19 patients so far. however, as argued [147] , ade should be given full consideration in the safety evaluation of emerging candidate vaccines for sars-cov-2. finally, it should be emphasized that at present, neither anti-spike neutralizing abs nor anti-spike t-cell responses have been established as corollaries of protection. patients afflicted with a chronic autoimmune disease have an increased risk of infections including viral infections [148, 149] . likewise, acute viral infections may also exacerbate pre-existing autoimmune disease, and immunosuppressive therapies may render patients with autoimmune disease more vulnerable to viral infections. despite these compounded considerations, patients with systemic or organ specific autoimmune disease are not at increased risk for infection with sars-cov-2. in fact, of the hundreds of reports published, none mention autoimmune conditions as either independent risk factors for disease or as indicative of a more severe outcome if infected. the data actually suggests that the rate of infection with the virus and their clinical course is not any different from that of the general population [150] [151] [152] . those autoimmune afflicted patients that have develop severe covid-19, are likely to have other comorbidities that are independent risk factors for severe disease. while virus infections can cause flares in otherwise stable autoimmune disease, the data suggests that sars-cov-2 infection is not associated with increased incidents of autoimmune flares. i have previously (section 5) discussed and presented evidence that patients under immunosuppressive therapies, including those afflicted with autoimmune conditions are not at increased risk for infection or for more severe outcome [121, 122] . regarding sle, the prototypic systemic autoimmune disease, a group of investigators suggested that inherent epigenetic dysregulation causing hypomethylation and overexpression of ace2, the functional receptor for sars-cov-2, might facilitate viral j o u r n a l p r e -p r o o f entry, viremia, and increased likelihood of cytokine storm in such patients [153] . the aforementioned role of ace2 expression, as discussed above, suggests that higher expression of ace2 might actually benefit the host more than it benefits the virus. in any case, the clinical experience and published data do not support these predictions. moreover, the accumulating scientific information does not support the notion that severe covid-19 is a direct cytopathic viral disease, but rather a disease in which multi-organ insult occurs by the host's own immune system [154] . conditions in which the immune system attacks its own tissues are usually associated with development of autoabs. indeed, gagiannis et al., studied prospectively a group of 22 patients for the possible role of autoimmunity in covid-19 patients [155] . autoab titers ≥1:100 were detected in 10/11 covid-19 patients who required intensive care treatment, and in 4/11 patients with milder clinical course. based on serological, radiological, and histopathological similarities between covid-19-associated ards and acute exacerbation of connective tissue disease induced interstitial lung disease, these authors suggest that sars-cov-2 infection might trigger or simulate a form of organ specific autoimmunity [155] . similarly, zhou et al., report on autoabs in 21 severe covid-19 patients. the prevalence of anti-52 kda ssa/ro ab, anti-60 kda ssa/ro ab, and antinuclear ab (ana) was 20%, 25%, and 50%, respectively [108 ] . ana was reported in over 35% of 45 consecutive covid-19 patients [110] . several studies document different apl abs in covid-19 patients mostly associated with thrombotic phenomena [108-110, 156, 157] . whether apl in covid-19 is transient, as has been documented in many other viral infections, or develops into persistent and pathogenic, is very difficult to judge from the results published so far. pregnancy related morbidity with fetal losses have not been reported in connection with sars-cov-2 infections. however, since an infection often precedes the clinical onset of aps, it is certainly justified to follow up a positive apl test in a covid-19 patient with a repeated test approximately twelve weeks later to further evaluate the possibility of post-infectious aps. a kawasaki-like disease seen in children is the closest link between sars-cov-2 and the appearance of an autoimmune and/or autoinflammatory condition. investigators from italy's pandemic epicenter in bergamo were the first to focus attention on this disorder [158] . d'antiga and his colleagues, quantified the time course and incidence of kawasaki-like disease in children before and after the covid-19 pandemic, documenting a thirty-fold increase in incidence of kawasaki-like disease after the beginning of the pandemic [158] . kawasaki disease is an acute and usually self-limiting vasculitis of medium and small sized arteries with specific predilection for the coronary arteries that affects previously healthy young children typically under the age of five years. in the acute phase of the disease, patients with kawasaki disease might have hemodynamic instability, a condition known as kawasaki disease shock syndrome. same patients with kawasaki disease fulfil the criteria of macrophage activation syndrome (mas). an association between kawasaki disease and various viral infections have been suspected, including viruses of the coronavirus family, however a specific infectious trigger has yet to be identified. sars-cov-2 should be now added to the list of implicated viruses. the most accepted pathogenetic hypothesis supports j o u r n a l p r e -p r o o f an aberrant response of the immune system to one or more unidentified pathogens in genetically predisposed subjects. following the report from bergamo further reports of similar cases from many countries have been published [159] [160] [161] [162] [163] . with approximately 1,000 kawasaki-like cases reported, these studies provide a consistent clinical picture: the disease appears 2-4 weeks after an infection with sars-cov-2 and most patients have serological evidence of infection; patients are on average older than those with classical kawasaki disease; patients experience respiratory and gastrointestinal involvement; signs of hemodynamic instability; greater incidence of myocardial injury; and more intense inflammatory response due to dysregulated immune response. the incidence of coronary aneurism is lower than in kawasaki disease, but this may be a consequence of relatively short follow up. most patients so far have responded well to the same therapies used for classical kawasaki disease. all these results and considerations support the notion that the immune response to sars-cov-2 is responsible for this kawasaki-like disease [158, 163] in susceptible patients. u.k. pediatricians and their national health service defined and named the ‗new', disease -pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 (sars-cov-2),‖ or pims-ts [159] . the cdc in the u.s. and the who subsequently published their own differing definitions of the disorder, which they termed multisystem inflammatory syndrome in children (mis-c) [160] [161] [162] [163] . physicians and scientists in the field of biology are prone to giving names that are often more confusing than helpful. i have worked for some years on a cytokine named tumor necrosis factor (tnf) that had practically nothing to do with necrosis of tumors, and from any sensible perspective is as much an interleukin (il) as any of the approximately 50 ils. i am not aware of any clinical, therapeutic, or long term follow up consideration that would benefit from a more nuanced definition and naming such as pims-ts or mis-c, rather than simply kawasaki-like disease. in fact some authors agree that -until more is known about long-term cardiac sequalae of mis-c, providers should consider following kawasaki disease guidelines for follow up‖ [161] . until a much better understanding of the pathogenesis of kawasaki disease and kawasaki-like disease emerges, i do not see a reason to further confuse the literature. as i am writing this essay, the pandemic seems far from being over. it surely looks like we are not even in the end of the beginning. the long-term impact of covid-19 is too early to evaluate. patients who have recovered from the disease report lingering chronic fatigue, muscle weakness, loss of sense of smell, and difficulties in concentration. but this might be just the tip of the iceberg. we already know that impaired liver function continues for some time after patients have apparently recovered and the virus has cleared. it is also probable that some patients will have lasting pulmonary damage due to fibrosis as has been documented in about 25% of patients recovered form sars [164] . similarly, myocardial j o u r n a l p r e -p r o o f scarring will cause cardiac impairment in certain covid-19 recovered patients. moreover, the long-term consequences of the massive inflammatory response affecting many tissues, and its effect on the competence of the immune system itself, are unknown. it is, however, possible that the intense inflammation in many tissues might cause cellular damage and exposure of self-antigens eliciting auto-reactive t and b cells and generating an autoimmune disease. another question that could take years to answer is whether the sars-cov-2 virus may lie dormant in the human body for years and then launch itself later in a different form. for example, after a chicken pox infection, the herpes virus that caused the illness reemerges after decades in form of shingles. similarly the hepatitis b virus causes the appearance of liver cancer years later. a general comment is pertinent at this point: the culmination of an interaction between an infectious agent and the human host, even when -full recovery-ensues, does not mean the organism is restored to its previous state (before the encounter), but rather the organism acquires a new equilibrium. as the french physician and philosopher george canguilhem wrote some 75 years ago: -contrary to orthodox medical teaching, health is not some absolute state of perfect physical and mental wellbeing. it is the margin of tolerance for the inconsistencies of the environment… disease is not simply disequilibrium or discordance; it is an effort on the part of nature to effect a new equilibrium in man‖ [165] . we have learned a tremendous amount about sars-cov-2 and covid-19 in a short amount of time. the efficient transmission of data exhibited during this time, has been surpassed only by the efficient transmission of the virus itself. although originally conceptualized as a primarily respiratory viral disease, covid-19 is now clearly recognized as a far more complex, multi-organ, and heterogeneous illness. as with sars-cov-1 and mers-cov, the important considerations for the delicate balance of the viral-host interaction that are responsible for covid-19 are now increasingly appreciated: (1) fast and robust initial viral replication; (2) early viral inhibition of ifn induction and signaling causing a delayed ifn expression which drives immunopathology; (3) out of balance antiviral innate immune response becomes immunopathogenic; and lastly (4) disintegration of the adaptive immune response. paul ehrlich's prediction of horror autotoxicus-at the turn of the 20 th century-has been realized by the innate immune response to cov in the 21 st century. the language of immunology is rife with war metaphors. for over a hundred years we have been educated to believe in the metaphor that the immune system acts as an army defending our bodies. as richard lewontin has written: -while we cannot dispense with metaphors in thinking about nature, there is a great risk of confusing the metaphor with the thing of real interest. we cease to see the world as if it were like a machine and take it to be a machine. the result is that the properties we ascribe to our object of interest and the questions we ask about it j o u r n a l p r e -p r o o f reinforce the original metaphorical image and we miss the aspects of the system that do not fit the metaphorical approximation‖ [166] . what has sars-cov-2 revealed? for me, the answer is our immune response to covid-19 serves as proof of everything i have long thought was wrong with viewing the (adaptive) immune system as a defense organization: it reacts too slowly. it fights today's threats with the solutions of past problems. it is susceptible to exploitation. it destroys that which it intended to protect. it is large, complicated, elaborate and wasteful [167] . if you stand back and evaluate how ineffective is the immune system as a defense organization, it is only logical to conclude that it was never intended as one. sars-cov-2 will eventually be contained, but not by our immune systems. rather, by the international brotherhood of the scientific community. epidemics have always played a natural part in the fabric of human history, but there has never been a time in history where so many different and powerful tools were available to accomplish this task. of all human conditions that disseminate virulent diseases, hubris emerges across centuries as a key driving force. we should embrace cesar augustus motto -festina lente‖, make haste, slowly-even or especially when you are feeling the crunch, take your time. this is not the time for us to skip corners. table 1 . comparison of transmissibility expressed as reproduction number, r 0 and case fatality rates of selected human viral diseases. adopted with changes from wang et al., [26] . the disintegration of the persistence of memory (1954) (rt) by salvador dali is an oil on canvas reaction to his original work the persistence of memory (1931) . in this version, the landscape from the first painting has been engulfed by water. disintegration of objects is occurring above and below the water. the block and plane from his original (lt) have now been separated into brick-like objects that float. some of the bricks on the left side of the painting begin to disintegrate. a watch beneath the water is coming to pieces and another one that sunk beneath the layer of bricks, leaves bits of debris behind. while persistence of memory (lt) symbolizes the importance of immunological memory as 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factors associated with covid-19-related death using opensafely epigenetic dysregulation of ace2 and interferon-regulated genes might suggest increased covid-19 susceptibility and severity in lupus patients on the alert for cytokine storm: immunopathology in covid-19 covid-19-induced acute respiratory failure-an exacerbation of organ-specific autoimmunity? medrxiv high risk of thrombosis in patients with severe sars-cov-2 infection: a multicenter prospective cohort study an outbreak of severe kawasaki-like disease at the italian epicenter of the sars-cov-2 epidemic: an observational cohort study clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 multisystem inflammatory syndrome in children in new york state multisystem inflammatory syndrome in u.s. children and adolescents acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov-2 pandemic kawasaki-like multisystem inflammatory syndrome in children during the covid-19 pandemic long term sequalae of sars: physical, neuropsychiatric, and quality-of-life assessment on the normal and the pathological the triple helix some savage cuts in defense i thank tamar jacob-eliasi for invaluable help with editing and proofreading the manuscript. coj is funded by nih grant r01ar072212. key: cord-338468-c0jv3i1t authors: kanduc, darja title: from anti-sars-cov-2 immune responses to covid-19 via molecular mimicry date: 2020-07-16 journal: antibodies (basel) doi: 10.3390/antib9030033 sha: doc_id: 338468 cord_uid: c0jv3i1t aim: to define the autoimmune potential of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. methods: experimentally validated epitopes cataloged at the immune epitope database (iedb) and present in sars-cov-2 were analyzed for peptide sharing with the human proteome. results: immunoreactive epitopes present in sars-cov-2 were mostly composed of peptide sequences present in human proteins that—when altered, mutated, deficient or, however, improperly functioning—may associate with a wide range of disorders, from respiratory distress to multiple organ failure. conclusions: this study represents a starting point or hint for future scientific–clinical investigations and suggests a range of possible protein targets of autoimmunity in sars-cov-2 infection. from an experimental perspective, the results warrant the testing of patients’ sera for autoantibodies against these protein targets. clinically, the results warrant a stringent surveillance on the future pathologic sequelae of the current sars-cov-2 pandemic. it is well known that coronavirus (re)infections have a pathologic immune potential. indeed: • progressive immune-associated injury is a hallmark of sars infection [1] and, since 2003 [2] , the association with hyper-immune inflammation and systemic immunopathology in the sars-cov-infected host has been well illustrated [2] [3] [4] [5] ; • in 2008, it was reported [6] that prior immunization with sars-cov nucleocapsid protein n causes severe pneumonia in mice infected with sars-cov; • in 2012, immunization with sars coronavirus vaccines was found to lead to lung immunopathology on challenge with the sars virus [7] ; • in 2016, agrawal et al. [8] demonstrated that immunization with inactivated middle east respiratory syndrome (mers) coronavirus vaccine leads to lung immunopathology on challenge with live virus. moreover, the authors documented that immunopathology with sars-cov vaccines occurred for whole-virus vaccines, subunit vaccines, different inactivation methods, different preparation substrates, and with recombinant surface spike protein. finally, the authors also underscored that, even if studies with vector vaccines point to the nucleoprotein n as responsible for the immunopathological effects and indicate that the s protein might be free of risk, nonetheless, also recombinant spike protein induced the immunopathology [6] [7] [8] [9] . however, the molecular and cellular basis for how sars-covs might impact on the host immune system resulting in dysfunctional immune responses, severe morbidity, and mortality remain obscure [1, 10] . following the current sars-cov-2 pandemic, cross-reactivity was proposed as a mechanism that might explain the immunopathology associated with the coronavirus infection [11] . the rationale is that the sharing of peptide motifs between sars-cov-2 and human proteins might evoke immune responses capable of hitting not only the virus but also the human proteins with consequent autoimmune pathologic sequelae in the human host. as a matter of fact, the study [11] called attention to a vast and specific peptide sharing between sars-cov-2 spike glycoprotein and alveolar surfactant-related proteins, in this way addressing the issue of why sars-cov-2 so heavily attacks the respiratory system. in the wake of such results, in order to validate (or, as well, invalidate) the cross-reactivity hypothesis, investigation was expanded here by analyzing the peptide sharing between the human host and immunoreactive epitopes that are also present in sars-cov-2. actually, the mere sharing of peptide sequences between pathogens and human proteins might be of little significance whether it remained sterile of cross-reactive autoimmune reactions. on the contrary, a peptide sharing between immunoreactive pathogen-derived epitopes and human proteins would assume the value of a proof of concept of cross-reactivity as a mechanism contributing to the pathogen-induced diseases. in this scientific framework, using the hexapeptide as an antigenic and immunogenic unit [12] [13] [14] [15] , immunoreactive epitopes that are present in sars-cov-2 were analyzed for matches with the human proteome. the results confirm a vast peptide commonality that involves human proteins implied in pulmonary insufficiency, neurological disorders, cardiac and vascular alterations, pregnancy dysfunctions, multiple cancers and anosmia, among others. analyses were conducted on an immunome composed of 233 linear epitopes that were experimentally validated, present in the proteins of sars-cov, and cataloged in the iedb [16] . the experimentally validated 233 epitopes have been described in detail by ahmed et al. [17] , map identically in sars-cov-2 proteins, and are listed in supplementary materials table s1 . the hexapeptide was used as a measurement unit to define minimal epitopic sequences. indeed, 5-6 amino acids (aa) can form a sufficient minimal determinant for epitope-paratope interaction [12, 13] . likewise, t-cell epitopes contain a core of 5-6 aa that are involved in antigenicity and immunogenicity [13] [14] [15] and are flanked by nh 2 -and cooh-terminus aa that act as anchor motifs. the sars-cov-2 epitope sequences were dissected into hexapeptides which overlapped each other by five aa residues. the resulting 733 hexapeptides were analyzed for occurrence(s) within human proteins using the pir peptide match program [18] . human proteins involved in the hexapeptide sharing were analyzed for function/diseases using uniprotkb, pubmed, and omim resources. the expected value for hexapeptide sharing between two proteins can be calculated by considering the number of all possible hexapeptides, n. since in a hexapeptide each residue can be any of the 20 aa, the number of all possible hexapeptides n is given by n = 20 6 = 64 × 10 6 . then, the number of the expected occurrences is directly proportional to the number of hexapeptides in the two proteins and inversely proportional to n. assuming that the number of hexapeptides in the two proteins is n and neglecting the relative abundance of aa, we obtain a formula derived by approximation, where the expected number of hexapeptides is 1/n or 20 −6 . table 1 reports that 230 out of the 733 hexapeptides composing the analyzed 233 immuno-reactive epitopes occurred among 460 human proteins. many of these shared sequences recurred more times. for instance, the zinc finger protein znf265-a splice factor that is important in renin mrna processing and stability [19] -shares the hexapeptide srsssr with the viral epitope sqassrsssr (iedb id: 60380). the hexapeptide srsssr recurs three times in the zinc finger protein, exactly at aa positions: 211-216, 241-246, and 256-261. then, including multiple occurrences, on the whole the hexapeptides shared with the human proteins amount to 505. for reasons of synthesis, table 2 reports the distribution of the shared hexapeptides relatively to a sample (n = 58) of sars-cov-2 epitopes. in the viral epitope sequences, the hexapeptides shared with human proteins are marked in capital letter format. the distribution of the shared hexapeptides throughout the entire set of 233 sars-cov-2 epitopes is described in supplementary materials table s2 . table 2 documents that numerous immunoreactive sars-cov-2 epitopes are composed mostly or, in many instances, uniquely of peptide sequences shared with human proteins. as an example, among the many, the epitope iedb id 4936, which is present in the viral nucleocapsid protein and corresponds to the aa sequence ategalntpk, results from the consecutive succession/overlapping of 6 mers present in human proteins too. taken together, table 1 , table 2 , and table s2 highlight the unexpectedness of the peptide overlap between the human proteome and the immunoreactive viral epitopes derived from iedb, described by ahmed et al. [17] and analyzed here. from a mathematical point of view, if one considers that the probability of a hexapeptide to occur in 2 proteins is~20 −6 (or 1 out of 64,000,000 or 0.000000015625), then the number of hexapeptides (namely, 505, including multiple occurrences) shared between the sars-cov-2 immunome and human proteins is surprisingly high and can be explained only on the basis of evolutionary processes [20] . in the context of autoimmunity, table 1 , table 2 , and table s2 concretize the effective possibility of cross-reactions between sars-cov-2 and the human proteome, given the fact that the immunological information unit in terms of both immunogenicity and antigenicity is contained in a space formed by 5-6 aa residues [12] [13] [14] [15] . as quantified in table 1 , hexapeptides from immunoreactive viral epitopes occur across 460 human proteins. the 460 human proteins are listed and synthetically described in supplementary materials table s3 . the 460 human proteins are involved in metabolic, developmental, and regulatory cellular functions and-when mutated, modified, deficient or, however, improperly functioning-may lead to altered functions and more or less severe pathologies in the human organism. obvious reasons of space prevent a protein-by-protein analysis of all of them and, here, only a few proteins (given by the uniprot name in italic and shared peptides in parentheses) and the related pathologies that could arise in case of cross reactivity are dealt with as follows. molecular mimicry between sars-cov-2 spike glycoprotein and alveolar surfactants-related proteins have already been described [11] . additional proteins that-if hit-can alter lung functions are: mothers against decapentaplegic homolog 9 protein (qassrs), alterations of which may lead to pulmonary hypertension with proliferating endothelial cells in pulmonary arterioles, right ventricular failure, and death [21] ; • phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (ikdlpk) that is deficient in pulmonary vascular endothelial cells of patients with acute respiratory distress syndrome [22, 23] ; • adenylate cyclase type 9 (kqlssn) that is expressed in multiple cells of the lung with expression highest in airway smooth muscle [24] ; • acetylcholinesterase (avlqsg) where imbalances in the neurotransmitter acetylcholine relate to neurological conditions, such as alzheimer's disease, parkinson's disease, and myastenia gravis; irreversible inhibition of acetylcholinesterase may lead to muscular paralysis, convulsions, bronchial constriction, and death by asphyxiation [25] . • endoribonuclease dicer (assrss) which is linked to pleuropulmonary blastoma [26] ; • protein naked cuticle homolog 1 (llpsla), downregulation of which is linked to poor prognosis in non-small-cell lung cancer and breast invasive ductal carcinoma [27, 28] ; • downregulated in multiple cancers 1 (aaeira) is not expressed in multiple human cancers [29] ; • ubiquitin carboxyl-terminal hydrolase bap1 (llsvll) relates to tumor predisposition syndrome; linked to mesothelioma [30] [31] [32] ; • in addition, the tumor suppressor proteins pinin (ssrsss) [33] , tripartite motif-containing protein 35 (sfkeel) [34] , unconventional myosin-ixb (llpsla) [35] , and cyclin-d-binding myb-like transcription factor 1 (aalqip) [36] . • low-density lipoprotein receptor-related protein 8 (lallll), alterations of which can lead to myocardial infarction [37] ; • dol-p-glc:glc(2)man(9)glcnac(2)-pp-dol alpha-1,2-glucosyltransferase (tltlav) is implicated in susceptibility to the long qt syndrome [38] ; • presenilin-2 (tlacfv) relates to dilated cardiomyopathy and heart failure [39] ; • nesprin-1 (llsagi) is involved in dilated or hypertrophic cardiomyopathy [40, 41] : • nuclear receptor coactivator 6 (pslatv) can cause dilated cardiomyopathy [42] ; • latent-transforming growth factor beta-binding protein 3 (lallll) can associate with skin thickening, cardiac valvular thickening, tracheal stenosis, and respiratory insufficiency [43] . • ephrin-b3 (lallll) is involved in blood pressure control and vascular smooth muscle cell contractility [44] ; • endoglin (lvlsvn) is required for normal structure and integrity of adult vasculature [45] ; • elastin (fgagaa) is a major structural protein of tissues, such as aorta and nuchal ligament, and relates to cutis laxa disease, may also, although rarely, lead to pulmonary artery stenosis, aortic aneurysm, bronchiectasis, and emphysema [46, 47] ; • filamin-a (pyrvvv), alterations of filamin-a can cause disorders related to the vascular system and to a large phenotypic spectrum of disorders such as deafness, urogenital defects, malformations, intestinal obstruction, constipation, recurrent vomiting, and diarrhea [48] ; • glomulin (rimasl) is crucial in vascular morphogenesis-especially in cutaneous veins [49] . • tumor necrosis factor receptor superfamily member 1a (gttvll) may associate with fever, abdominal pain, localized tender skin lesions, myalgia, and reactive amyloidosis as the main complication [50, 51] . • thrombomodulin (agaalq), a well-known natural anti-coagulant that acts as a linker of coagulation and fibrinolysis, is involved in disseminated intravascular coagulation, is a predictor of risk of arterial thrombosis and is essential for the maintenance of pregnancy [52] [53] [54] . • in addition, cross-reactive peptide sharing involving additional proteins can further affect the level of thrombomodulin. indeed, cross-reactions with retinoic acid receptor rxr-alpha (lgfstg) and prostaglandin g/h synthase 2 (tvllke) might completely eliminate thrombomodulin from blood circulation because (1) retinoic acid receptor rxr-alpha promotes the thrombomodulin gene transcription [55] and (2) prostaglandin g/h synthase 2 stimulates the expression of functionally active thrombomodulin in human smooth muscle cells [56] . adding up to such pro-thrombotic scenario, it is also worth of mention the potential cross-reactivity with tyrosine-protein kinase jak2 (llddfv) that is involved in myelofibrosis, myeloid leukocytosis, and thrombocytosis with excessive platelet production resulting in increased numbers of circulating platelets, hemorrhages, and thrombotic episodes [57] [58] [59] . • neuronal pas domain-containing protein 2 (assrss), which is highly polymorphic in autism spectrum disorder patients [60, 61] ; • circadian locomotor output cycles protein kaput (assrss) relates to bipolar disorder [62] ; • adenosine receptor a1 (vlppll), where sleep is significantly attenuated by the loss of adenosine a1 receptor expression [63] ; • bdnf/nt-3 growth factors receptor (sanlaa), alterations may cause temporal lobe epilepsy, memory impairment, anorexia nervosa, bulimia, alzheimer's disease [64] [65] [66] ; • calcium/calmodulin-dependent protein kinase kinase 2 (pslatv) is linked to disturbances of higher cognitive functions, such as working memory and executive function. as well as schizophrenia [67] ; • endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase (lafllf) can cause mental retardation [68] ; • glutaminase kidney isoform, mitochondrial (lqelgk) can associate with epileptic encephalopathy, infantile cataract, skin abnormalities leukocytoclasia at the surface of the dermis, focal vacuolar alterations, hyperkeratosis, parakeratosis, glutamate excess, and impaired intellectual development, global developmental delay, progressive ataxia, and elevated glutamine [69] [70] [71] ; • mitochondrial glutamate carrier 1 (rlqslq) relates to neonatal myoclonic epilepsy [72] . moreover, and remarkably, the voltage-gated calcium channel gamma subunits 2, 3, 4, 6, and 8 contain epitopic hexapeptides (table 3) . voltage-gated calcium channels control cellular calcium entry in response to membrane potential changes [73, 74] , and gamma subunits 2, 3, 4, and 8 regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (or ampa receptor or ampar) and are collectively known as transmembrane ampar regulatory proteins (tarps) [75] . ampars are involved in the fast synaptic transmission in the central nervous system and are altered in many psychological and neurological disorders such as schizophrenia, depression and parkinson's disease [76] . table 3 . hexapeptide sharing between sars-cov-2 epitopes and tarps. lsagif, srsssr gamma-2 subunit lsagif gamma-3 subunit srsssr gamma-4 subunit lgagcf gamma-6 subunit srsssr gamma-8 subunit as a final note, this results section closes with one disorder that is one of the first symptoms to appear in covid-19, namely, anosmia. the role of anosmia as a first symptom is clearly justified by the fact that seven olfactory receptors (i.e., proteins related to the smell) [77] share hexapeptides with the viral epitopes (table 4 ). this study shows that hexapeptides from immunoreactive epitopes present in sars-cov-2 are widespread among a high number of human proteins. such a peptide sharing implies the possibility of cross-reactions and, consequently, as discussed in the results (section 3), of a vast phenotypic constellation of diseases, from pneumonia and neurological disorders to cardio-vascular alterations and coagulopathies. hence, this study appears to offer scientific hints to explain the clinical fact that sars-cov-2 infection is capable of triggering so many and so different pathologies in so many and so different organs of the human host [78] . on the whole, the data indicate that-besides possible virus-induced multi-organ direct cytopathic effects and other possible pathogenic mechanisms-self-reactive antibodies may be at the root of the pathologic scenario that accompanies sars-cov-2 infection. in fact, previous research focusing on sars-cov, which similarly to sars-cov-2 causes a respiratory failure syndrome, showed that anti-spike protein iggs can cause lung damage by directly affecting inflammatory mechanisms, promoting the release of pro-inflammatory cytokines, such as il-8, as well as macrophagic tissue infiltration [79] . a similar effect of sars-cov-2 on inflammatory response has already been proposed [80] . then it appears reasonable to hypothesize that autoantibody-mediated macrophagic and complement activation and autoantibody-dependent cell-mediated cytotoxicity mechanisms might be some of the mechanisms behind the well-documented multi-organ damage in covid-19, thus representing one of many possible links between adaptive and innate immunity in the pathogenesis of the disease. moreover, as reviewed by vabret et al. [81] , it has been shown that high antibody response can associate with more severe clinical cases. this had also been seen in the previous sars-cov-1 epidemic, where neutralizing antibody titers were found to be significantly higher in deceased patients compared to patients who had recovered [82] and might lead to suspect antibody-dependent enhancement phenomena. of note, the present data also indicate that most possibly the damage caused by sars-cov-2 might not end with the end of the pandemic. indeed, the peptide sharing between sars-cov-2 epitopes and specific tumor suppressor proteins, and, in general, many tumor-related proteins [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] , theoretically predicts, in the absence of current clinical data, that a morbidity/mortality increase in various cancers might follow the current sars-cov-2 pandemic. as a conclusive note, it is mandatory also to observe that this study largely undervalues the potential risk of cross-reactivity between sars-cov-2 immunome and human proteins. indeed, analyses were conducted on linear epitopes and used the hexapeptide as an immune unit probe. then, if one considered conformational epitopes and used the pentapeptide as a minimal immune determinant [13, 83] , the number of viral versus human commonalities would increase exponentially. given this premise, the present results call researchers and clinicians for a common research effort to study the autoimmune pathogenicity connected to the anti-sars-cov-2 immune response and suggests, once more, that using entire antigens in anti-sars-cov-2 vaccine formulations might lead to autoimmune manifestations and adverse events [84] . actually, the present data further support the fundamental concept that only "non-self" peptides can lead to safe and efficacious immunotherapies [85] [86] [87] . supplementary materials: the following are available online at http://www.mdpi.com/2073-4468/9/3/33/s1, table s1 : sars-cov-2 immunome consisting of 233 immunopositive linear epitopes, assembled from iedb [16] , described by ahmed et al. [17] , and listed according to iedb id number. table s2 : hexapeptide sharing between 233 epitopes present in sars-cov-2 and human proteins. table s3 : list and short description of 460 human proteins that share hexapeptides with the 233 sars-cov-2 epitopes. funding: this research received no funding. the author declares no conflict of interest. human immunopathogenesis of severe acute respiratory syndrome (sars) lung pathology of fatal severe acute respiratory syndrome an interferon-gamma-related cytokine storm in sars patients pathogenesis of severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with inactivated middle east 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comparative biochemistry of viruses and humans: an evolutionary path towards autoimmunity molecular genetic characterization of smad signaling molecules in pulmonary arterial hypertension endothelial p110γpi3k mediates endothelial regeneration and vascular repair after inflammatory vascular injury role of the pi3-kinase signaling pathway in trafficking of the surfactant protein a receptor p63 (ckap4) on type ii pneumocytes adenylyl cyclase type 9 gene polymorphisms are associated with asthma and allergy in brazilian children actions of drugs on the autonomic nervous system dicer1 mutations in familial pleuropulmonary blastoma down-regulation of nkd1 increases the invasive potential of non-small-cell lung cancer and correlates with a poor prognosis nkd1 down-regulation is associated with poor prognosis in breast invasive ductal carcinoma identification of dmc1, a novel gene in the toc region on 17q25.1 that shows loss of expression in multiple human cancers the nuclear deubiquitinase bap1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma germline bap1 mutations predispose to malignant mesothelioma germline mutations in bap1 predispose to melanocytic tumors characterization of the gene encoding pinin/drs/mema and evidence for its potential tumor suppressor function hls5, a novel rbcc (ring finger, b box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor myo9b is a key player in slit/robo-mediated lung tumor suppression the hdmp1 tumor suppressor is a new wt1 target in myeloid leukemias an lrp8 variant is associated with familial and premature coronary artery disease and myocardial infarction a kcr1 variant implicated in susceptibility to the long qt syndrome mutations of presenilin genes in dilated cardiomyopathy and heart failure analysis of selected genes associated with cardiomyopathy by next-generation sequencing novel nesprin-1 mutations associated with dilated cardiomyopathy cause nuclear envelope disruption and defects in myogenesis perturbation of ncoa6 leads to dilated cardiomyopathy mutations in ltbp3 cause acromicric dysplasia and geleophysic dysplasia the role of grip1 and ephrin b3 in blood pressure control and vascular smooth muscle cell contractility endoglin, a tgf-beta binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1 cutis laxa arising from frameshift mutations in exon 30 of the elastin gene (eln) isolated supravalvular aortic stenosis: functional haploinsufficiency of the elastin gene as a result of nonsense-mediated decay vascular and connective tissue anomalies associated with x-linked periventricular heterotopia due to mutations in filamin a mutations in a novel factor, glomulin, are responsible for glomuvenous malformations germline mutations in the extracellular domains of the 55 kda tnf receptor, tnfr1, define a family of dominantly inherited autoinflammatory syndromes heterogeneity among patients with tumor necrosis factor receptor-associated periodic syndrome phenotypes the discovery of thrombomodulin thrombomodulin in disseminated intravascular coagulation and other critical conditions-a multi-faceted anticoagulant protein with therapeutic potential the thrombomodulin-protein c system is essential for the maintenance of pregnancy acceleration of thrombomodulin gene transcription by retinoic acid: retinoic acid receptors and sp1 regulate the promoter activity through interactions with two different sequences in the 5'-flanking region of human gene regulation of thrombomodulin expression in human vascular smooth muscle cells by cox-2-derived prostaglandins germline jak2 mutation in a family with hereditary thrombocytosis acquired mutation of the tyrosine kinase jak2 in human myeloproliferative disorders a gain-of-function mutation of jak2 in myeloproliferative disorders association of per1 and npas2 with autistic disorder: support for the clock genes/social timing hypothesis circadian-relevant genes are highly polymorphic in autism spectrum disorder patients role for the clock gene in bipolar disorder control and function of the homeostatic sleep response by adenosine a1 receptors contribution of ntrk2 to the genetic susceptibility to anorexia nervosa, harm avoidance and minimum body mass index genetic association of neurotrophic tyrosine kinase receptor type 2 (ntrk2) with alzheimer's disease trkb gene)variants and temporal lobe epilepsy: a genetic association study mutations in the alpha 1,2-mannosidase gene, man1b1, cause autosomal-recessive intellectual disability identification of a loss-of-function mutation in the context of glutaminase deficiency and neonatal epileptic encephalopathy gls hyperactivity causes glutamate excess, infantile cataract and profound developmental delay glutaminase deficiency caused by short tandem repeat expansion in gls impaired mitochondrial glutamate transport in autosomal recessive neonatal myoclonic epilepsy structure and pharmacology of voltage-gated sodium and calcium channels dolphin, a.c. the physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential calcium channel gamma subunits: a functionally diverse protein family the role of transmembrane ampa receptor regulatory proteins (tarps) in neurotransmission and receptor trafficking the human olfactory receptor gene family coronavirus diseases (covid-19) current status and future perspectives: a narrative review anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection understanding sars-cov-2-mediated inflammatory re-sponses: from mechanisms to potential therapeutic tools antibody responses against sars coronavirus are correlated with disease outcome of infected individuals homology, similarity, and identity in peptide epitope immunodefinition peptide cross-reactivity: the original sin of vaccines self-nonself" peptides in the design of vaccines immunogenicity, immunopathogenicity, and immunotolerance in one graph from hbv to hpv: designing vaccines for extensive and intensive vaccination campaigns worldwide key: cord-334550-xb0alubj authors: samaddar, arghadip; gadepalli, ravisekhar; nag, vijaya lakshmi; misra, sanjeev title: the enigma of low covid-19 fatality rate in india date: 2020-07-28 journal: front genet doi: 10.3389/fgene.2020.00854 sha: doc_id: 334550 cord_uid: xb0alubj coronavirus disease 2019 (covid-19), an acute onset pneumonia caused by a novel betacoronavirus severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has rapidly evolved into a pandemic. though its origin has been linked to the wuhan city of china’s hubei province in december 2019, recent reports claim that the original animal-to-human transmission of the virus probably happened sometime between september and october 2019 in guangdong province, rather than hubei. as of july 3, 2020, india has reported a case positivity rate of 6.5% and a fatality rate of 2.8%, which are among the lowest in the world. also, the severity of the disease is much less among indians as evidenced by the low rate of icu admission (15.3%) and the need for mechanical ventilation (4.16%). as per the world health organization (who) situation report 165 on july 3, 2020, india has one of the lowest deaths per 100,000 population (1.32 deaths against a global average of 6.04). several factors related to the pathogen, host and environment might have some role in reducing the susceptibility of indians to covid-19. these include some ongoing mutations that can alter the virulence of the circulating sars-cov-2 strains, host factors like innate immunity, genetic diversity in immune responses, epigenetic factors, genetic polymorphisms of ace2 receptors, micro rnas and universal bcg vaccination, and environmental factors like high temperature and humidity which may alter the viability and transmissibility of the strain. this perspective -highlights the potential factors that might be responsible for the observed low covid-19 fatality rate in indian population. it puts forward several hypotheses which can be a ground for future studies determining individual and population susceptibility to covid-19 and thus, may offer a new dimension to our current understanding of the disease. in december 2019, an outbreak of fatal pneumonia, the coronavirus disease 2019 , caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was reported from the wuhan city of china's hubei province. the outbreak was declared as a public health emergency of international concern by the world health organization (who) on january 30, 2020 and a global pandemic on march 11, 2020. 1,2 though its origin has been linked to hubei province, recent phylogenetic analysis claims that the original animal-tohuman transmission of the virus probably happened some 3 to 4 months before december 2019 in guangdong province, rather than hubei (forster et al., 2020) . as of july 3, 2020, the disease has affected more than 10.7 million people across 216 countries and territories with 517,877 deaths. 3 a difference in the case fatality rates (cfr) was observed across countries, possibly due to demographic variations, differences in the virus strains in circulation and the nature of containment measures implemented. the fatality rates in the united states, united kingdom, italy, france, and spain have surpassed that of china by manifolds. india, the second most populous country in the world, with a population of 1.3 billion people, had documented the first case of covid-19 on january 30, 2020, the same day as italy (kaushik et al., 2020) . as of july 3, 2020, india has reported a total of 625,544 cases and 18,213 deaths which reflects a milder trajectory with lower case positivity (6.5%) and fatality (2.8%) rates compared to the global figures. 3 the traditional model for any infectious disease consists of a triad of the causative agent, the host, and an environment in which the agent and host are brought together, causing the disease to occur in the host. little is known regarding the origin of sars-cov-2. there have been speculations that the virus got transmitted to humans from bats or pangolins, but conclusive evidence regarding the same is lacking. genomic sequence data indicate that sars-cov-2 has 96.2% sequence homology with a bat coronavirus ratg13 (zheng, 2020) , and 91% homology with a pangolin coronavirus (zhang et al., 2020) . besides, it also shares 79.5% identity with sars-cov (zheng, 2020) . a recent study suggests that pangolins are the natural reservoirs of sars-cov-2-like coronavirus, with the pangolin-cov having a more closely related s1 protein to that of sars-cov-2 (zhang et al., 2020) . it is unclear how the animal-to-human transmission occurred and whether the virus acquired greater pathogenic potential and transmissibility after having entered the human host. more studies need to be focussed in this aspect for better understanding of the pathogenicity. the pandemic has made a considerable impact on health-care infrastructure worldwide, with medical facilities struggling to cope-up with the increased demands for life-saving medicines, ventilators and personal protective equipments. 1 world health organization (who) emergency committee. statement on the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus (2019-ncov). geneva: who; 30 january 2020. available from: https://www.who.int/news-room/detail/ 30-01-2020-statement-on-the-second-meeting-of-the-international-healthregulations-(2005)-emergency-committee-regarding-the-outbreak-of-novelcoronavirus-(2019-ncov) (accessed may 25, 2020). 2 world health organization (who) emergency committee. who director-general's opening remarks at the media briefing on covid-19 -11 march 2020. geneva: who; 11 march 2020. available from: https: //www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-atthe-media-briefing-on-covid-19---11-march-2020 (accessed may 25, 2020). 3 world health organization (who). coronavirus disease (covid-19): situation report, 165. geneva: who; 03 july 2020. available from: https: //www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/ (accessed july 3, 2020). despite the catastrophic consequences of this pandemic on several affluent nations, its impact on indian population seems to be much lower both in terms of severity as well as cfr. of the total 227,439 active cases of covid-19 till july 03, 2020 in india, 15.3% required icu admission, 4.16% required ventilator support and 15.9% required supplemental oxygen, all of which point toward a less severe disease among indians. 4 so what is that "x" factor apparently safeguarding the indian population from the wrath of this pandemic? this perspective provides an insight into the potential factors that might be responsible for the observed low covid-19 severity and fatality rate in indian population and illuminates the areas of future research which may help in understanding the disease process more vividly. how do the indian sars-cov-2 strains differ from the strains elsewhere? earlier, six genera of coronaviruses were known to cause human disease, of which four (α-cov-229e and nl63, and β-cov-hku1 and oc43) are responsible for mild respiratory infections, mostly in pediatric age group while two (sars-cov and middle east respiratory syndrome coronavirus [mers cov]) are associated with severe outbreaks . sars-cov-2 is a positive sense single-stranded rna virus harboring two major genes, open reading frame 1a (orf1a) and orf1b, which together encode 16 non-structural proteins (nsp1-nsp16). these nsps are organized to form a replication-transcription complex (rtc) that is involved in transcription and replication. nsp3 and nsp5 encode for papain-like protease (plp) and chymotrypsin-like protease (3cl), respectively, which help in peptide cleaving and host innate immune antagonism. nsp12 and nsp15 encode for rna-dependent rna polymerase (rdrp) and rna helicase, respectively. the structural genes encode four structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n), and several accessory proteins (astuti and ysrafil, 2020) . molecular characterization of sars-cov-2 strains based on whole-genome sequencing revealed clustering of the prototype wuhan strain belonging to the o clade (mn908947.3/sars-cov-2/human/chn/wuhan-hu-1/2019), indicating that the virus might have originated in china and eventually spread worldwide fan et al., 2020) . the indian sars-cov-2 strains, unlike the strains elsewhere, are more closely related to bat-cov ratg13 (93% homology) than pangolin cov (83.5% homology). however, the receptor binding domain (rbd) of s protein of indian sars-cov-2 strains are more closely related to pangolin-cov (zhang et al., 2020) . a recent study from national institute of cholera and enteric diseases, west bengal, india analyzed the genome type clusters of 46 indian sars-cov-2 isolates and observed a monophyletic clade of the virus co-existing with the prototype wuhan strain (clade o) and clustering along with the indian isolates, suggesting introduction of the virus in india from several countries. 1 | phylogenetics clades of sars-cov-2 with their order of evolution and the defining mutation(s) (biswas and majumder, 2020) . defining mutation(s) phylogenetic analysis of sars-cov-2 genomes demonstrated two major lineages, l (leucine) and s (serine), based on single nucleotide polymorphisms (snps) at positions 8,782 (orf1ab: t8517c) and 28,144 (orf8: c251t, s84l). the l lineage carries a significantly higher number of derived mutations than s lineage, indicating that these two lineages might have different rates of transmission and replication. also, the l lineage has been described to be more aggressive and contagious than s lineage. in india, the l lineage is more prevalent (95.7%) than s lineage (4.3%) (banerjee et al., 2020) , with reports claiming higher fatality rates at places where the l type is dominant as in the indian state of gujarat (cfr = 5.4%, the highest in india). 5,6 these observations indicate that strain difference has some impact on virulence and pathogenesis. scientists from the national institute of biomedical genomics, india, performed phylodynamic analyses and examined the temporal and spatial evolution of the virus in 3636 sar-cov-2 sequences deposited in global initiative on sharing all influenza data (gisaid) from 55 countries and observed that at least 10 distinct clades (a1a, a2, a2a, a3, a6, a7, b, b1, b2, and b4) of the virus have originated from the ancestral clade o ( table 1) . though initially, the ancestral type was most frequent in all countries due to return of travelers from china, it has gradually been replaced by other types with clade a2a (having non-synonymous d614g mutation in the s1/s2 furin cleavage site of s protein) being the most dominant type in india and also, globally (biswas and majumder, 2020) . phylogenetic analysis has revealed significant regional variation in frequencies of different clades across countries. for example, in the united states, clade b1 is predominant in washington d.c. and west coast, while in new york and east coast, clade a2a is the modal type (biswas and majumder, 2020; brufsky, 2020 ). an analysis of 35 viral sequences submitted to gisaid from india revealed the existence of four clades: ancestral clade o (14.3%) and derived clades a2a (45.7%), a3 (37.1%), and b (2.9%). all individuals 5 available from: https://www.indiatoday.in/india/story/wuhan-s-l-strainmay-be-behind-gujarat-s-high-death-rate-experts-1671346-2020-04-26 (accessed may 25, 2020). 6 available from: https://www.mohfw.gov.in/ (accessed july 03, 2020). infected with a3 had travel history to iran, while those with a2a had no history of international travel. thus, a temporal decline in the diversity of sars-cov-2 clades has been observed globally, which may affect transmission and pathogenicity. a mutational analysis showed co-circulation of two groups (major and minor) of the mutated virus in india. the "major group" (52.2%) represents a2a clade that harbors four co-existing snps: 241c>t (5 utr), 3037c>t (f106f, nsp3), 14403c>t (p323l, rdrp/nsp12) and 23403a>g (d614g, s glycoprotein). the "minor group" (30.4%) consists of strains showing five distinct mutations: 13730c>t (a97v, rdrp/nsp12), 23929c>t (y789y, s), 28311c>t (p13l, n), 6312c>a (t1198k, nsp3) and 11083g>t (l37f, nsp6). all these mutations, except 11083g>t (l37f, nsp6) are unique to indian sars-cov-2 strains (guan et al., 2020; mercatelli and giorgi, 2020; wang et al., 2020) . mutations 22374a>g (q271r), 245 24933g>t (g1124v) and 22444c>t (d294d) in s gene and missense mutations q271r and g1124v in s protein have been observed exclusively in indian strains, which might alter the protein function and thus, affect virulence and pathogenicity (banerjee et al., 2020) . researchers from translational bioinformatics group at international center for genetic engineering and biotechnology (icgeb) in collaboration with the department of biochemistry, jamia hamdard, new delhi, india, performed an integrated mutational analysis of sars-cov-2 genomes from different geographical locations, including india, italy, united states, nepal and wuhan, and observed a novel mutation in s protein (a930v, 24351c>t) of the indian strain, which was absent in other strains (sardar et al., 2020) . a triple site mutation 28881-28883 ggg>aac has been observed at 203/204 region of the n gene that might alter the phosphorylation of serine residue of n protein and interfere with its function, thus reducing the pathogenicity of the strain (ayub, 2020) . besides, some indian strains have displayed unique mutations in the nsp3 gene at positions 6310c>a (s1197r), 7392c>t (p1558l) and 6466a>g (k1249k) (banerjee et al., 2020) . the rna-dependent rna polymerase (rdrp)/nsp12 protein is an integral component of the viral replication machinery and any mutation in this protein might interfere with viral replication and cause accumulation of novel mutations. in indian sars-cov-2 strains, two mutations: 14408c>t (p323l) and 13730c>t (a97v) in rdrp gene have been documented which might have altered the secondary structure of rdrp and resulted in simultaneous emergence of major and minor groups of the virus with characteristic co-evolving mutations (banerjee et al., 2020) . a study from university of bologna, italy, analyzed 10,014 sars-cov-2 genomic sequences in comparison with the reference wuhan genome and revealed a low mutation rate of the virus, with an average of 6.7 mutations per sample with respect to the reference strain. according to the study, snps are the most common mutational events observed worldwide as well as in asia. the most common mutation observed in asia is g11083t, causing a snp at position 37 of thensp6. several other mutations like orf8:l84s (china) and orf3a:g251v (hong kong) have also been documented, with a regional variation in the type and nature of such events (mercatelli and giorgi, 2020) . a comparative genomic analysis of 3067 sars-cov-2 genomes from 59 countries revealed the presence of singleton mutations in 26 countries with united states accounting for the highest number of mutations (44% of total mutations). the mutations g251v (in orf3a), l84s (in orf8) and s5932f (in orf1ab) exist in all continents except africa. also, the genome variability is most prominent in america, australia and new zealand (laamarti et al., 2020) . figure 1 depicts the geographic and genomic distribution of sars-cov-2 mutations. such genomic diversity may give rise to new clades with variations in transcription and replication rates, which in turn may affect virulence and transmissibility. studies combining genomic details with demographic and epidemiological data are essential to identify any ongoing mutation and its impact on virulence and severity of the disease across populations. a comparative analysis of the immune phenotypes in indian and american newborns revealed that indian infants had a higher proportion of dendritic cells, monocytes, natural killer (nk) cells, memory cd4 + t cells, and naïve b cells, compared to american infants (rathore et al., 2018) . such variations in immune systems can be attributed to heritable and nonheritable influences. heritable factors have germline inheritance and have a minor influence on inter-individual and population variation in immune responses (brodin et al., 2015; brodin and davis, 2017) . a population-based cohort study (immvar project) involving subjects of african-american, east asian and european ancestry analyzed the variability in functional responses of t-cells and dendritic cells by gene profiling and concluded that heritable factors accounted for only 22% of the overall variation in gene expression (de jager et al., 2015) . non-heritable factors include environmental influences, such as infections and vaccines, stochastic epigenetic changes arising from imperfect replication machinery, and symbiotic and pathogenic microbes. non-heritable influences are the major factors determining immune variation (brodin et al., 2015; brodin and davis, 2017) . brodin et al. (2015) analyzed various immunological parameters, such as cell population frequencies, cytokine responses, and serum proteins in 210 healthy twins and found that majority of these determinants are dominated by non-heritable influences, indicating that the human immune system is very much shaped by the environment and the variety of microbes that an individual encounters in their lifetime, besides these factors, india has an overwhelming burden of tuberculosis, malaria and hiv, much higher than the developed countries. due to such endemicity, indians are constantly subjected to pathogen-assault, giving rise to a more proactive cell-mediated immune system, than the western population. also, chloroquine and hydroxychloroquine, the two most revered drugs in the context of covid-19 treatment, have been extensively used at community level in india for the prevention and treatment of malaria. 8 given the immunomodulatory and antiviral properties of these drugs, their widespread usage might have contributed to defense against sars-cov-2. second, the epigenetic factors like environment and food habits might have some role in boosting immunity. several kinds of herbs and spices like turmeric, cloves, ginger, mustard, saffron, cardamom, and garlic are essential ingredients of the indian cuisine. these spices are rich in bioactive compounds and phytochemicals which possess medicinal properties (sengupta et al., 2004) . turmeric (indian saffron), a product of curcuma longa, is a rhizomatous herbaceous perennial plant belonging to the ginger family that is routinely used in indian households as a culinary spice and as a component in religious ceremonies. it has also been used as a traditional medicine over centuries. curcumin, the bioactive compound in turmeric has been recognized for its potent antioxidant, anti-inflammatory, antimutagenic, antimicrobial, and anticancer properties. it has demonstrated antiviral activity against a wide range of viruses including human immunodeficiency virus 1 and 2, influenza virus (h1n1, pr8, and h8n1), herpes simplex virus (hsv) 1 and 2, coxsackievirus, hepatitis b and c viruses, human papillomavirus (hpv) types 16 and 18, japanese encephalitis virus, and human t cell lymphotropic virus type 1 (moghadamtousi et al., 2014) . similarly, ginger, another rhizomatous spice used routinely in indian foods contains several bioactive phenolic and terpene compounds that have demonstrated antiviral activity against human respiratory syncytial virus (blocks viral attachment and internalization) (mao et al., 2019) . garlic (allium sativum), a popular condiment used in indian foods, has been shown to possess antiviral activity against hsv-1 and -2, parainfluenza virus type 3, and human rhinovirus type 2 (lee et al., 2016) . capsaicin, an active component of chili peppers, acts as an agonist at vanilloid receptor 1 of dendritic cells and enhances the antiviral activity of cd8 + cytotoxic t-cells by increasing mhc class i-restricted viral antigen presentation in dendritic cells (weber et al., 1992) . it has been shown to inhibit influenza a virus replication in cell cultures in a dose dependent manner (marois et al., 2014) . even, studies from china have highlighted the possible therapeutic role of traditional chinese medicines in the treatment of covid-19 . thus, considering the multi-pharmacological activity of the active components in indian spices, it is possible that they might have some role in defense against sars-cov-2. third, the indian population possesses a high genetic diversity in the immune response genes, collectively referred to as human leukocyte antigen (hla) complex, much more extensive than the caucasian population and this is attributed to the high microbial load to which the indians are exposed during life (mehra, 2010) . the hla complex serves to induce and regulate immune responses. it helps in distinguishing self antigens from non-self foreign proteins. the foreign antigens can be recognized by the t-cells only if they are presented in association with hla proteins. more the number of hla allotypes expressed on the cell surface, broader the range of foreign antigens they can recognize and present to the t-cells. thus, individuals with heterozygous hla alleles possess the ability to present a broader repertoire of antigenic peptides to the immune cells as compared to homozygotes (mosaad, 2015) . according to a study at all india institute of medical sciences, new delhi, india, several novel hla alleles (hla-a * 3303) and unique haplotypes (hla-a2-b50-dr3) have been observed in the indian population, which are not known to occur in other ethnic groups (mehra, 2010) . whether such differences have any influence on the severity of sars-cov-2 infection need to be investigated. recently, nguyen et al. (2020) evaluated the role of cross-protective immunity in covid-19 and reported that individuals with hla-b * 46:01 allele are particularly vulnerable to the disease due to expression of low levels of sars-cov-2 binding peptides, while those with hla-b * 15:03 possess an enhanced capacity to present highly conserved sars-cov-2 peptides that are shared among common human coronaviruses, indicating its potential to elicit crossprotective t-cell based immunity. therefore, genetic diversity of hla alleles can be an important factor in determining the susceptibility to sars-cov-2 infection. fourth, allelic variation in angiotensin converting enzyme 2 (ace2) receptor can be an important factor determining the susceptibility to sars-cov-2 infection. cell culture assays and molecular superimposition studies indicate that sars-cov-2 spike (s) protein has got high affinity for human ace2 receptors and utilizes the same for gaining entry into the target cells. ace2 receptors are widely distributed in body tissues, the highest expression being observed in type ii alveolar epithelial cells (hussain et al., 2020) . recent studies suggest a correlation between the level of ace2 expression on cell membrane and viral infectivity, which in turn governs disease severity and clinical outcomes (jia et al., 2005) . hussain et al. (2020) reported that certain allelic variants of ace2 (s19p and e329g) demonstrates low binding affinity for the viral spike protein and may thus confer resistance to sars-cov-2 infection. a study by calcagnile et al. (2020) found that two snps-s19p (common in africans) and k26r (common in europeans) can potentially affect the interaction between ace2 and sars-cov-2 spike glycoprotein. while the former decreases ace2 affinity for the spike protein and lowers the susceptibility, the latter increases the receptor affinity and predisposes to more severe disease. hence, genetic polymorphisms of ace2 receptors might be an important factor in determining the susceptibility to sars-cov-2. fifth, the micrornas (mirnas) might play a crucial role in defense against sars-cov-2. mirnas are small non-coding rnas that are thought to regulate gene expression and cell differentiation, and have the ability to inhibit viral replication in a sequence-specific manner. considering their unique mechanism of action, mirnas have become attractive candidates as diagnostic biomarkers and therapeutic targets (correia et al., 2017) . several studies have highlighted the potential role of mirnas in the regulation of antiviral defense against h1n1 influenza, hiv-1, hsv-1, hpv, enterovirus-71, hepatitis b and c, and dengue viruses (barbu et al., 2020) . a recent study by translational bioinformatics group at icgeb and department of biochemistry, jamia hamdard, new delhi, india, revealed that nine host mirnas can potentially target sars-cov-2. these are hsa-let-7a, hsa-mir101, hsa-mir125a-5p, hsa-mir126, hsa-mir222, hsa-mir23b, hsa-mir378, hsa-mir380-5 and hsa-mir98. interestingly, a single unique mirna, hsa-mir-27b, has been found to be specifically targeting the indian sars-cov-2 genome, which has not been detected elsewhere. it has been hypothesized that hsa-mir-27b has a specific role in defense against sars-cov-2 in indian population that harbors this unique sequence (sardar et al., 2020) . these findings indicate a possible link between host mirnas and the disease severity as well as treatment outcomes. sixth, the indians have a higher percentage of nature killer (nk) cells, compared to other ethnic groups (rathore et al., 2018) . it has been postulated that the development, tolerance and activation of nk cells are regulated by the killer cell immunoglobulin-like receptors (kir) present on their surface. these receptors interact with hla class i expressed by target cells and regulate cytolytic activity (campbell and purdy, 2011) . while most kirs are inhibitory and tend to suppress nk cell function, activating receptors (kir2ds1-5, kir3ds1) are upregulated in viral infections and host cell aberrations. all activating kirs (except kir2ds4) are encoded by b haplotype (hiby et al., 2010) . it has been observed that b haplotypes are more prevalent in non-caucasian populations such as australia aborigines and asian indians than in caucasian populations, and such populations are likely to be under high selection pressure from infectious diseases, much more than other ethnic groups. kir genes display extraordinary level of polymorphism and diversity among different populations because of which there may be significant differences in ligand affinity or signal transduction. populationbased analysis of kir genes reveal that kir2ds2, an activating kir, has high frequencies (>70%) in australia aborigines and indian population, but low frequencies in other ethnic groups (middleton and gonzelez, 2010) . according to rajalingam et al. (2008) , the indians might have acquired activating kir genes through a process of natural selection that enabled them to survive epidemics during their pre-historic migrations from africa. thus, it is possible that the diversity in kir gene complex occurred as a result of natural selection by pathogens so as to offer a survival benefit. such diversification might be a reason for the indian population to respond differently to sars-cov-2. seventh, live attenuated vaccines like bacillus calmette-guérin (bcg) and mumps, measles, rubella (mmr) commonly administered during childhood, have been found to confer nonspecific protection against unrelated lethal infections by inducing "trained" non-specific innate immune cells for more efficient host response against wide range of pathogens (fidel and noverr, 2020) . the bcg vaccine, given universally to indian babies at birth for protection against tuberculosis, might confer some protection against sars-cov-2, possibly through activation of t-cell mediated immune response. bcg has no direct antiviral action, but it may act as an immunopotentiator (tsiang et al., 1981) . studies have demonstrated the ability of bcg vaccine in reducing viraemia associated with yellow fever vaccine (arts et al., 2018) , and decreasing the severity of encephalomyocarditis virus infection in mice (floc'h and werner, 1976) . researchers from new york institute of technology correlated global covid-19 transmission with bcg vaccine coverage data and observed that countries with a policy for universal bcg vaccination have lower number of covid-19 cases than those without such policy, such as the united states and italy (miller et al., 2020) . japan, where universal bcg vaccination policy is in place since 1947, has maintained a low fatality rate, despite an early spike in covid-19 cases. iran, one of the countries badly hit by this pandemic, started universal bcg vaccination only in 1984, potentially leaving anyone above 36 years of age unprotected. however, according to who, such comparisons are prone to significant bias from many confounders, including variations in demographics and disease burden, covid-19 testing rates and the stage of the pandemic in each country. 9 two randomized controlled trials, bcg-corona (nct04328441) and brace (nct04327206) trials are underway, evaluating the efficacy of bcg vaccine in reducing the viral load and/or sequelae associated with covid-19. one limitation of bcg vaccination is seroconversion, which is the basis for diagnosis of tb in several countries. therefore, mmr, another live attenuated vaccine, might be a potential option for inducing beneficial non-specific effects in human populations and thus provide protection against the catastrophic sequelae of covid-19. a strong correlation has been observed between individuals in geographical locations who received mmr vaccine and reduced covid-19 death rates (gold, 2020) . interestingly, despite children being highly susceptibility to flu, very few children have been affected during the ongoing covid-19 pandemic. also, there has been a striking difference in the covid-19 mortality rates between children and adults. one explanation is that children are protected owing to their recent and more frequent exposures to live attenuated vaccines (bcg, mmr, chickenpox and rotavirus) which can induce the trained myeloid-derived suppressor cells and thus limit disease severity (fidel and noverr, 2020) . though conclusive evidence on this ground is lacking at the moment, considering the positive correlation between the use of live attenuated vaccines and lower covid-19 severity and death rates, hopes remain high for a possible protective action of these vaccines against sars-cov-2. india is a vast country with varied weather conditions. the western part of the country is known for hot dry summers with temperatures exceeding 50 • c, while the himalayan belt in the northern part records temperatures as low as −30 • c. previous studies have shown the impact of weather variables on the transmission dynamics of diseases like influenza and sars. transmission of influenza is facilitated in the presence of cold and/or dry air. a sharp rise in influenza virus activity in northern europe during 2010-2018 was correlated with low temperature and low ultraviolet (uv) radiation indices. based on these facts, it was assumed that covid-19 transmission might decrease or even disappear with the rise in ambient temperature and uv radiation during the summer months. however, a study by yao et al. (2020) incorporating meteorological data and basic reproduction number (r 0 ) of the virus from 224 chinese cities revealed that high temperature, humidity and uv radiation has no significant impact on cumulative incidence rate and transmission of sars-cov-2. infact, countries like iran and australia continued to have rapid disease transmission and exponential rise in the number of cases despite high temperature and humidity, indicating that the inverse relationship between ambient temperature and covid-19 transmission lacks rationality. studies have shown that an interplay between macrodomain (mac) and papain-like protease 2 (plp2) domain of coronavirus nsp3 impacts viral replication, host innate immune antagonism and virulence. strains harboring mutations in these two domains are more rapidly degraded at non-permissive temperatures (37 • c and 40 • c) than the wild-type strains (deng et al., 2019) , indicating that higher temperatures may lower the virulence and pathogenicity of temperature-sensitive sars-cov-2 mutants. the covid-19 fatality rate in india is among the lowest in the world (2.8% against a global average of 4.7%). also, the disease has been observed to be less severe among indians with a significantly higher recovery rate (60.9% versus a global average of 56.6%) and doubling time (20.3 days) as compared to the populations elsewhere. 3,4,10 strict lockdown measures implemented nationwide for more than two months might have kept the transmission and deaths in check. this must have contributed significantly to the much desired flattening of the epidemic curve. there are speculations that india's predominantly young population as compared to the western world (with a higher proportion of elderly people), and the presence of a less virulent strain of the virus in circulation are helping keep fatalities low in india. however, there are no evidences in support of these claims. in fact, phylogenetic analysis has revealed that four clades of the virus: o (ancestral clade from china), b (from china), a3 (from iran) and a2a (from iran, europe, and other countries) are in circulation in india with a2a being the dominant type (biswas and majumder, 2020) . there are reports that the official death counts are much lower as compared to the actual death toll. on april 18, 2020, china revised the death toll to 4632 with a 50% spike in fatality figures. 11 such discrepancies may arise because the official count considers only the hospital deaths, while majority of the deaths still happen at home. also, death due to covid-19 may not be reported in presence of other comorbidities. tracking deaths is far more reliable than cases, which are significantly affected by testing bias. roughly, 86% of the covid-19 patients are asymptomatic who hardly get tested and serve as potential source for transmission of infection. in a densely populated country like india, mass screening of population for covid-19 is not feasible. so there are chances that india might be missing some deaths and not diagnosing every case correctly. however, considering the magnitude of the pandemic, india has ramped-up the testing capacity with nearly 250,000 samples being screened and over 20,000 active cases being detected daily. 6 still, the fatality rate in indian cases is low and none has got a definite answer for that. while this apparent protection among indians is largely attributed to non-heritable influences as discussed earlier, a safe and effective vaccine against sars-cov-2 can reduce disease severity, control transmission, and prevent future infections across all populations. vaccines function by inducing both arms of the adaptive immune response: humoral immunity (producing neutralizing antibodies that prevent virus attachment) and cell-mediated immunity (activating antigen presenting cells, t-cells, and nk cells that recognize and kill virus-infected cells) (mukherjee, 2020) . there are currently ten vaccines undergoing clinical trials against covid-19 based on several technologies, such as, live attenuated, inactivated, mrna, dna, viral vector, and peptide subunit based platforms (mullard, 2020) . a strong global vaccination program with equitable distribution of promising vaccine candidates to all affected regions is needed to tackle the pandemic. this perspective puts forward several hypotheses which may explain individual and population susceptibility to covid-19 which in turn, may help prioritize vaccination in high risk individuals and groups, and thus reduce morbidity and mortality across populations. whether the above factors generate sufficient evidence to provide satisfactory explanation for their apparent protective effects on indian population during covid-19 pandemic, will be clear as the situation unfolds further. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. as and rg: conceptualization, data 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challenge for human beings traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective no association of covid-19 transmission with temperature or uv radiation in chinese cities probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak sars-cov-2: an emerging coronavirus that causes a global threat the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 samaddar, gadepalli, nag and misra. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-344266-ug2uew71 authors: crema, e. title: the sars-cov-2 outbreak around the amazon rainforest: the relevance of the airborne transmission date: 2020-08-07 journal: nan doi: 10.1101/2020.08.06.20169433 sha: doc_id: 344266 cord_uid: ug2uew71 background this paper presents a global analysis of the sars-cov-2 outbreak in brazil. amazonian states have a much higher contamination rate than the southern and southeastern states. so far, no explanation has been provided for this striking difference that can shed light on the airborne transmission of the virus. minimizing airborne transmission, health authorities recommend two meters as a safe distance. however, recent experiments reveal that this can be the main form of contagion. there is a lack of theoretical explanation on how airborne transmission works. methods to investigate the spread of sars-cov-2 in different macro environments, we analyzed the daily official data on the evolution of covid-19 in brazil. we compared our epidemiologic results obtained in states with very different climatic characteristics, and that had adopted, almost simultaneously, similar social isolation measures. to understand the virus spread, it was necessary to calculate theoretically the movement and behavior in the air of saliva droplets. findings the transmission of sars-cov-2 is much faster in the amazon rainforest region. our theoretical calculations explain and support the empirical results observed in recent experiments that demonstrate the relevance of aerial transmission of the coronavirus. interpretation an onset of collective immunity may have been achieved with a contamination rate of about 15% of the amazonian population. if confirmed, this result will have an essential impact on the management of the pandemic across the planet. the airborne transmission played a decisive role in the striking difference in the evolution of the pandemic among brazilian regions. air humidity is the most important climatic factor in viral spreading, while usual ambient temperatures do not have strong influence. there is no safe indoor distance for the coronavirus transmission. so, mask and eye protection are essential. the ray of light that runs through a dark room reveals the existence of numerous small grains of dust that can float in the air for a long time. since antiquity, this phenomenon was already known. a famous observation of this effect is documented in lucretius's poem de rerum natura, written around 50 bc. in addition to the empirical description of the phenomenon, and following the tradition of democritus and epicurus, lucretius also proposed an atomistic explanation for the support of particles in the air, according to which their weight would be compensated by the collisions of air atoms. 1 however, the behavior of tiny bodies immersed in fluids was only understood from the 19th century on owing to the works of robert brown, 2 george gabriel stokes, and finally with einstein's famous work of 1905, on the movement of small particles in suspension within liquids at rest. currently, this phenomenon has gained tragic relevance due to the uncontrolled dispersion of the covid-19 throughout the planet, since airborne transmission is one of the forms of viral contamination, as well as the direct reception of drops exhaled by a contaminated person and the contact with infected surfaces. there is still no consensus among researchers as to which of these forms of contagion is the most important in the case of the coronavirus. despite being the third outbreak of this virus in less than two decades, existing research had not yet fully understood its transmission mechanisms. a similar situation occurred with the influenza virus. while some important books and works drew attention to the relevance of the transmission by aerosols (droplets), [3] [4] [5] other authors argued that short-distance transmission by drops would be the main means of infection, 6, 7 and this latter position prevailed for a long time among health authorities who practically ignored airborne transmission. 8, 9 at the end of march 2020, the world health organization (who) released a bulletin stating that there was insufficient scientific evidence that sars-cov-2 was significantly airborne transmitted. a few months ago, at the beginning of the current pandemic, several governments and the most important health authorities on the planet recommended that only hand washing and a distance of two meters between people would be safe protection procedures and that the use of masks was unnecessary throughout the population. however, with the rapid spread of the coronavirus in countries and in the world, the deadly reality has imposed itself and forced the planetary health authorities to reverse this directive, saving thousands of lives by requiring the use of masks in several countries. from a scientific point of view, this late change in positioning was the authorities' recognition that air transmission of sars-cov-2 is an unquestionable fact. nevertheless, it remains to be understood how this 3 process takes place. in this article we will clarify the physical processes involved in this means of contamination and explain theoretically the results of recent epidemiologic experiments. besides, we will discuss some recent relevant epidemiologic papers and analyze the sars-cov-2 outbreak around the amazon rainforest that may help to understand the relevance of the long-range viral airborne transmission. finally, we will show that there are still some important recommendations that health authorities should indicate to reduce viral transmissibility. the airborne transmission of the coronavirus is now experimentally well demonstrated by important works published during the last months. a relevant study issued in the journal nature revealed the existence of the rna of the sars-cov-2 in aerosols collected from the air of several closed environments and open places of two hospitals in wuhan dedicated only to patients infected with covid-19 (12) . 10 another paper analyzed the air at the nebraska hospital center and also found the sars-cov-2 in most environments occupied by patients with mild and moderate infections. 11 in these two studies it was not possible to confirm if these viruses were active. however, this doubt was finally resolved by a study published in the new england journal of medicine, where the presence of active sars-cov-2 in droplets was observed more than three hours after they were artificially produced in the laboratory (65% of relative humidity and temperatures between 21-23ºc). 12 now, it is certain that under normal day-to-day conditions sars-cov-2 remains active for hours in the droplets suspended in the air. extensive study published in the lancet journal, analyzing empirical data from 16 countries on 6 continents, concluded that the probability of infection by sars-cov-2 decreases by 10·6% when using a protection for the eyes. 13 that is, the risk of contagion through the eyes is very high and continues to be minimized by health authorities, including the who, as had happened in the case of masks. this may be a new mistake in combating the pandemic. on the other hand, further experiments visualized the production of saliva droplets during a person's normal speech, breathing, sneeze and cough. [14] [15] [16] thousands of drops were exhaled and their dispersion in the air was video recorded. they used a laser beam technique of high resolution that was able to identify even submicron droplets. in the video from kyoto university, 17 one can watch the movement of these drops, revealing all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.06.20169433 doi: medrxiv preprint that while the larger ones fall rapidly and settle on the ground and furniture, there are hundreds of micro droplets that remain suspended in the air several hours after being exhaled. and, most seriously, these small drops disperse rapidly and, a few minutes after their production, occupy the entire environment, covering distances greater than eight meters. besides these experiments, there were several empirical situations that put in another new and very relevant epidemiological event has occurred in the current covid-19 pandemic in brazil: the amazonian states that house the forest have presented contamination rates higher than 15%, while in southern states this rate has been less than 1%. to understand this striking difference, we analyzed the official primary data on the pandemic released daily by all states of the country. we will use the number of deaths as an analysis parameter because there is a huge underestimation of the number of cases of the disease due to the extremely low number of tests performed by the brazilian government. it should be kept in mind that the number of deaths is also undervalued. one all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.06.20169433 doi: medrxiv preprint 13% and 15%, respectively. in other cities near the rainforest this percentage attained 20% to 25%. 22 therefore, the contamination rate that could have produced the interruption of the spread of the epidemic observed in fig. 1 was only of the order of 15%. if confirmed by further studies, this result will have an extraordinary impact on the management of the pandemic on the planet. the striking difference observed between the north and south regions could not be explained by the issue of social isolation, since both adopted, almost simultaneously, similar isolation measures. nor is it due to the greater poverty in the northern region, as the southern states also have a large number of inhabitants living in precarious dwellings, including thousands of slums, which would facilitate the rapid spread of the virus. a possible difference in health care system between regions could also not be claimed as an explanation of this phenomenon, as it would not justify the sudden drop in the number of deaths. so, the remarkable disparity in the evolution of the contamination in these regions needs to be deeply investigated. a factor that can play an important role in the spread of sars-cov-2 is the climate. during the months investigated in this work, in the regions surrounding the forest, the average temperatures were always above 30ºc and the average relative humidity, above 80%. in southern states, average temperatures and average relative humidity were not higher than 22ºc and 50%, respectively. therefore, there is no direct relationship between high ambient temperature and decreased transmissibility. on the other hand, some studies showed that, in general, in environments with relative humidity above 60%, approximately, the drops absorb more than evaporate water into the air. 23, 24 so, we believe that the airborne transmission of the sars-cov-2, facilitated by the high humidity of the air, could be a primary factor in the development of the epidemic in brazil. our hypothesis is confirmed by other studies conducted in some cities in brazil at the beginning of the pandemic, although these studies use the number of cases as an analysis variable. 25, 26 to better understand this relationship, it is interesting to know the amount of water vapor that actually exists in the atmosphere, that is, its absolute humidity. one kilo of air with relative humidity 70%, at 30ºc, contains approximately 19g of water in the form of vapor, while at a relative humidity of 40%, at 22ºc, the amount of water is only 6g. that is, the process of water evaporation/absorption is very complex in the amazonian weather conditions, and, on average, the drops expelled by an infected person all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.06.20169433 doi: medrxiv preprint will absorb water from the atmosphere, allowing the viruses to survive much longer in suspension or deposited on surfaces. a similar situation has occurred in abattoirs in france, germany, and the usa that have become huge poles of contamination. the dominant explanation for this phenomenon has been the airborne transmission facilitated by the low temperature of these environments. we agree that the aerial contagion was responsible for the spread of the virus in the abattoirs. however, the pandemic evolution in brazil, middle east, europe, china, and usa has demonstrated that habitual temperatures seem to have little influence on the survival of the virus in the external environment. therefore, we believe that also in the abattoirs the most important factor was the very high humidity of the air during a coughing, sneezing, or speech, thousands of drops of saliva and secretions from the pulmonary tract are expelled with high speeds and penetrate more than two meters into ambient air. these drops have diameters between fractions of micrometers up to fractions of centimeters. [28] [29] [30] [31] depending on their composition and air humidity, they can evaporate or absorb water from the environment, and this process is fundamental to the survival of the virus. after the evaporation or growth of the drop, its equilibrium size will define its dynamics and the progression of the epidemic. the smaller droplets (diameter<10 µm) are responsible for the airborne transmission that has been recently recognized by numerous researchers 32 as decisive in the spread of the sars-cov-2 and the who asks that it be better studied. in an attempt to contribute to this all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.06.20169433 doi: medrxiv preprint scientific effort, this work will also investigate the movement of these droplets to demonstrate how airborne transmission is physically possible. two situations will be considered: they fall into the air at rest; and under action of a vertical airflow upwards which can be produced by an air conditioning system and/or air renewal. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.06.20169433 doi: medrxiv preprint epidemiological implications of this expression, we will calculate the fall of drops of human saliva in the air at a pressure of 700 mmhg and a temperature of 25º c. table 1 shows the values of the time constant τ calculated for drops of radius ranging from 0·1µm to 50 µm. on the other hand, fig. 2 shows the dynamics of two drops, of radii 0·5 µm and 1·0 µm, where are shown the variations of their velocities with time. we observed that the drops start to fall from rest, increase their velocities, and in a few microseconds acquire extremely small constant velocities, vlim. for example, in this ideal situation, with stationary air, a drop of radius 0·1 µm falls with speed 2·2 µm/s, and a drop of radius 0·5 µm falls with speed 35 µm/s, approximately. evidently, these values are negligible when compared to the speeds of the random air currents that exist in real environments, which are in the order of cm/s, according the measurements by matthews et al. 33 these internal currents are produced by local all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. . https://doi.org/10.1101/2020.08.06.20169433 doi: medrxiv preprint differences in temperature and pressure, by the movement of objects or people around the environment, and statistical variation of the pressure. it means that the movement of small suspended particles will be governed by these air currents and they can be dragged until 6 meters or more in 5 minutes. so, these currents are responsible for the movement of dust grains observed by lucretius, and also for the fluctuation and movement of micro drops filmed in the experiments described above. it is important to remember that the coronavirus has a more or less spherical shape with a diameter of the order of nanometers, that is, the drops we dealt with in this work can carry from hundreds to millions of viruses. the last column of table 1 shows the time, ttot, that the drops take to reach the ground starting from a height of 1·80 meters. even considering the air at rest, it shows that drops of diameters 0·2 µm, 1·0 µm, and 2·0 µm would remain suspended for several hours in the air. however, the air currents in indoor environments are even more important when they have an air renewal and/or conditioning system that creates a continuous flow of air in a more or less fixed direction. 33 we will evaluate this phenomenon in the case of an aspiration system placed on the ceiling of the environment, producing an airflow in the vertically upward direction, with speed v. the diagram on the side shows the forces acting on it. in this case, the viscous friction force of the air passing through the droplet can compensate for and/or overcome the weight force, causing it to remain stopped and/or be aspirated towards the ceiling. let's consider the equilibrium limit case, when friction exactly compensates for the weight of the droplet and it stays at rest at a certain height of the ground. disregarding langevin's force, the speed of air that holds a drop at rest can be easily calculated for its different sizes. figure 3 shows the air velocity necessary to balance drops of different radii, where we observe that they are very small velocities, of a few mm/s, even for the largest drops considered. that is, if the air conditioning system is not well dimensioned, it eliminates the smallest drops, but it can keep larger drops in suspension and/or drastically decrease its fall times, precisely the drops that have a greater potential for infection because they can carry a greater amount of viruses. for example, a continuous vertical flow of air with a speed of only 3 mm/s, approximately, keeps drops of diameter 10 µm suspended at rest for an unlimited time. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. . finally, we must discuss the effect of heating systems placed on the ground, as they also create an upward airflow and can keep contaminated droplets floating in the air for a long time, increasing the risk of contamination. perhaps, this may be one of the factors that increase the spread of viral epidemics during the winter. it is now experimentally and theoretically demonstrated that airborne contagion by sars-cov-2 can occur long after an infected person has spoken, coughed, or sneezed in an environment. these scientific results call into question one of the main recommendations of health authorities to contain the outbreak: the distance of 1m to 2m between people would be a safe method of prevention. this indication is based only on old studies about the direct transmission by larger drops, dangerously ignoring the contamination by the virus airborne in droplets that remain suspended in the air for several hours, and even days after the environment has been visited by an infected person. this recommendation created in the population the false idea that, by staying two meters from each other, it is not necessary to use a mask or other protections. even the highest leaders of the who conduct daily interviews without a mask. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 7, 2020. . therefore, an important warning must be made: the distance of two meters is not safe for those who do not wear a mask and, if we consider the infection by the eyes, the distance of two meters is not safe even for those wearing a mask. the very high sars-cov-2 transmission rates in amazonian states in brazil and many abattoirs around the world provide empirical corroboration of the relevance of the airborne way of contagion. these two environments have high air humidity that allows viruses to survive much longer in droplets in suspension or deposited on surfaces. therefore, air humidity seems to be the major climatic factor in the development of the covid-19 epidemic. on the other hand, apparently, there is no direct relationship between high ambient temperature and decreased transmissibility. besides, the collective immunity in amazonian states may have been achieved with a contamination rate of around 15% of the population, much lower than predict conventional statistical studies, and which would have an extraordinary impact on pandemic management across the planet. the argument that the wind disperses the drops has made people feel more protected in open places. however, the same wind that can disperse the drops can also carry them and project them on passersby, whether on the beach, on the street, in the elevator, at home, or on public transport. so, in addition to the mask the use of eye protection should also be recommended. it should not be forgotten that drops in suspension can also be deposited on our face, hair, and clothes. finally, another important alert about air conditioning and heating systems comes from our calculations: if they are poorly positioned and/or sized, they can work as a dangerous spreader of viruses. nature des choses (éditions gallimard, essais folio influenza virus" in douglas and bennett's principles and practice of infectious diseases airborne transmission of sars-cov-2: the world should face the reality who writing group: nonpharmaceutical interventions for pandemic influenza, international measures transmission of influenza a in human beings public health agency of canada canadian pandemic influenza plan (appendix f) department of health and human services pandemic influenza plan aerodynamic analysis of sars-cov-2 in two wuhan hospitals aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19 the airborne lifetime of small speech droplets and their potential importance in sars-cov-2 transmission toward understanding the risk of secondary airborne infection: emission of respirable pathogens environmental transmission of sars at amoy gardens extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards covid-19 outbreak associated with air conditioning in restaurant identifying airborne transmission as the dominant route for the spread of covid-19 mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence absolute humidity modulates influenza survival, transmission, and seasonality evidence that high temperatures and intermediate relative humidity might favor the spread of covid-19 in tropical climate: a case study for the most affected brazilian cities association between climate variables and global transmission of sars-cov-2 survival characteristics of airborne human coronavirus 229e dilution of respiratory solutes in exhaled condensates toward understanding the risk of secondary airborne infection: emission of respirable pathogens atmospheric science, an introductory survey it is time to address airborne transmission of covid-19 air velocities inside domestic environments: an important parameter in the study of indoor air quality and climate key: cord-338544-eph89g47 authors: spuntarelli, valerio; luciani, m.; bentivegna, e.; marini, v.; falangone, f.; conforti, g.; rachele, e. s.; martelletti, p. title: covid-19: is it just a lung disease? a case-based review date: 2020-07-28 journal: sn compr clin med doi: 10.1007/s42399-020-00418-6 sha: doc_id: 338544 cord_uid: eph89g47 due to its extreme virulence, covid-19 virus has rapidly spread, developing a severe pandemic. sars-cov-2 mostly affected the respiratory tract, causing a severe acute lung failure. although the infection of airways, covid-19 can be associated with chronic and systemic damages still not so much known. the purpose of this research is to collect recent evidence in literature about systemic diseases caused by covid-19. the format of the present article has features of a systematic case-based review (level of evidence), and it is structured as a case series report (patients of our covid-19 medicine ward have been selected as cases). data for this review have been selected systematically, taking evidence only from indexed journals and databases: pubmed, scopus, medline, and cochrane systems. papers chosen included systematic reviews, case series, clinical cases, meta-analysis studies, and rcts. we start collecting studies since 2003. the main keywords used were “covid-19” “or” “sars” “or” “sars – cov 2” “and” “systemic disease” / “nephropathy” / “cardiac pathology” / “central nervous system.” clinical cases belong to our covid-19 medicine ward. one of the most severe covid-19 clinical presentations includes cardiovascular problems, like myocarditis, pericarditis, and acute hearth failure. cytokine release syndrome caused by covid-19 develops severe acute kidney failure. it is still unknown the way coronavirus damages the liver, brain, and reproductive system. considering the majority of the new studies about this pathology, it issues that covid-19 is considered to be a multi-organ disease. covid-19 pandemic reached 3.78 million confirmed reported cases worldwide, and it is generally associated to the acronym that precedes its name: severe acute respiratory syndrome (sars). however, the bottom of the iceberg is being progressively unveiled since it is far more than simply a severe interstitial pneumonia. there is a gap in knowledge of pathophysiological process that allows covid-19 to be considered a multi-organ disease in all respects. we looked for main papers about sars and covid-19 as systemic diseases, focusing on cardiovascular, renal, liver, nervous, and reproductive systems. the sars-cov-2 virus not only causes viral pneumonia; however, it also has major implications for the cardiovascular system, but the extent, severity, and duration are still to be defined. in a study of 75 patients, two patients had succumbed to acute myocardial infarction [1] . a study from huang et al. demonstrated that myocardial injury, defined by an increase in hs-ctni levels (> 28 pg/ml), occurred on 5 out of 41 covid-19 patients. noteworthy is the fact that 80% of these patients required icu management demonstrating that myocardial damage in sars-cov-2 infection is severe [2] . a prospective study investigating left ventricular performance in 46 patients with severe acute respiratory syndrome showed subclinical diastolic impairment without systolic involvement [3] . another study in 121 patients with sars identified cardiovascular complications including tachycardia (72%), hypotension (50%), bradycardia (15%), transient cardiomegaly (11%), and transient paroxysmal atrial fibrillation in only 1 patient, although usually self-limiting [4] . a study from singapore reported postmortem examinations: the presence of pulmonary embolism (pe) and deep vein thrombosis and acute myocardial infarction is of great clinical interest, but the generalizability of this limited study is not established [5] . anyway, it is well recognized that sars-cov-2 infection alters coagulation pathway. zhou et al. demonstrated that nonsurvivor covid-19 patients show significant higher levels of plasma d-dimer, activated partial thromboplastin times, and prothrombin times compared with survivors [6] . pathological findings of covid-19 associated with acute respiratory distress syndrome showed few interstitial mononuclear inflammatory infiltrates, but no other substantial damage in the heart tissue [7] . shi s. et al [8] reported that between 57 out of 416 patients, 10.6% had coronary heart disease, 4.1% had heart failure, and 5.3% had cerebrovascular disease underlying the importance of cardiac injury in covid-19. a case report highlights myocarditis as a complication associated with covid-19, even without symptoms and signs of interstitial pneumonia in an otherwise healthy 53-year-old white woman [8] . another case report [9] of an infected 69year-old man, from lombardy, italy, concluded that covid-19 infection was the most likely cause of myocarditis since tests for common causes of myocarditis (parvovirus b19, human herpes virus, epstein-barr virus, enterovirus, cytomegalovirus, adenovirus, hiv, and hepatitis c virus) were negative. hydrocortisone improved its clinical picture. also, very recently, an association between covid-19 and medium size vasculitis-kawasaki disease-has emerged. according to older studies, a suspected association between hcovs and kawasaki disease could not be confirmed. however, shirato k. et al [10] postulated that hcov-229e is a possible causative agent for kawasaki disease. future case control studies are needed to settle the issue. clinical case a 56-year-old male arrived to emergency for chest pain, tachycardia, and dyspnea. blood gas analysis showed mild respiratory failure. sars-cov-2 buffer was positive. blood analysis pointed out an increase of serum myoglobin, high sensitive troponine, and mb creatine kinase. electrocardiogram was typical for myocarditis ( fig.1 ). three potential pathogenic mechanisms of kidney injury secondary to covid-19 have been identified. first, cytokine release syndrome (crs)-aka cytokine storm-has been implicated in the development of intrarenal inflammation and increased vascular permeability. il-6, markedly elevated in ards patients, plays a major role in the pathogenesis of the disease. in crs patients who have undergone car-t cell therapy, the cytokine is the target of the therapy with tocilizumab and, thus, is now also being used empirically in patients with covid-19. second, alveolar-tubular and cardiorenal crosstalk has been implicated. acute kidney injury (aki) is the most frequent extra-pulmonary organ failure in acute respiratory distress syndrome (ards), according to a recently confirmed bidirectional organ crosstalk, possibly ilfig. 1 moderate st depression in most precordial leads 6 mediated. il-6 is not a risk factor for ards development, but high levels are associated to increased mortality rate in patients with ards. independent risk factors are older age, disease severity, diabetes mellitus, and acidosis. finally reninangiotensin-aldosterone system increases both glomerular capillary and systemic hypertension, inducing hemodynamic injury to the vascular endothelium and glomerulus. angiotensin ii and aldosterone may also promote kidney damage with direct profibrotic and proinflammatory actions. as shown in the clinical case below, the further renal impairment caused by covid, in already nephropathic patients, can be considered a mortality predictor. clinical case a 62-year-old woman with chronic kidney failure was hospitalized in emergency ward for metabolic acidosis. sars-cov-2 buffer was positive. blood exams showed an important increase of serum creatinine. during observation, patient developed an important ards with lung edema (fig. 2) . she died after 2 days of noninvasive ventilation. it is less clear whether covid-19 damages the liver. case reports have described increased levels of alanine aminotransferase (alt), aspartate aminotransferase (ast), and bilirubin, indicating liver involvement, in which frequency is related to the severity of covid-19 illness. the same was observed during previous sars and mers epidemics. on the other hand, mansoor n.b. et al [11] reassure about the relationship, describing that the previous studies on the issue actually suggest that clinically significant liver injury is uncommon, even when data for the most severely ill patients are selected. liver injury can also alternatively due to hypoxemia, dysregulated immune response, or drug toxicity. one-third of patients affected with covid-19 have nervous system involvement. virus can be detected in brain specimens and in cerebrospinal fluid. acute onset smell and/or taste disorders are significantly more frequent in cases of covid-19 which is a common influenza and should be considered a focus for clinical suspicion and consideration to selfisolation procedures. anosmia and ageusia are frequently early manifestation of covid-19. although pathogenetic mechanisms are still unclear, vaira l. a. et al [12] proposed a potential explanation. ageusia could be related to the highly expression of ace2, identified as the cellular receptor for sars-cov-2, on the oral mucosa and tongue. anosmia seems not to be related to a mere local inflammatory condition fig. 2 emergency chest x-ray with severe interstitial edema in the nasal mucosa: in the absence of rhinitis symptoms, one hypothesis could be that the alterations are due to direct damage caused by the virus to the olfactory pathway. although many other neurological symptoms, such as headache, dizziness, conjunctivitis, and trigeminal neuralgia can be recorded in these patients, recent studies identified association with many neurological diseases. guillain-barre-strohl syndrome (gbs) is an acute demyelinating polyradiculoneuropathy affecting cranial nerves, dorsal and ventral radices, spinal ganglia, and peripheral nerves. a type 4 hypersensitivity disorder is usually preceded by a respiratory (m. pneumoniae, h. influenzae, cmv, ebv) or gastrointestinal (c. jejuni). sedaghat z. et al [13] , on 15 april 2020, described the first case of gbs apparently associated with covid-19 infection. on the 24 april, another italian case report [14] speculates the association between the acute polyradiculoneuropathy and covid-19. the patient tested negative the most common abovementioned infections classically related to gbs. although the study could not exclude the possibility of an autoimmune or paraneoplastic polyradiculoneuropathy mimicking gbs, the postinfectious etiology, the acute clinical course, and the typical neurophysiologic findings on electroneurography make alternative diagnosis less likely. poyiadji n. et al [15] found out that a woman with covid-19 presenting with altered mental status had features on brain images consistent with acute necrotizing hemorrhagic encephalopathy (ane). the authors concluded that the presence of the characteristic features of symmetric, multifocal lesions with thalamic involvement suggests that this is a case of acute necrotizing hemorrhagic encephalopathy associated with covid-19. ane is a rare complication of influenza and other viruses resulting from intracranial cytokine storm and resulting in blood-brain barrier damage, plausible with covid-19 pathogenetic signature. although metabolic and electrolyte derangements, especially in thalamocortical pathways, secondary to covid-19 are plausible causes of clinical or subclinical acute symptomatic seizures and status epilepticus [16] , lu l. et al [17] concluded that neither the virus nor potential risk factors for seizures increased the likelihood of acute symptomatic seizures in covid-19. clinical case a 72-year-old male arrived to emergency ward for altered brain status. sars-cov-2 buffer was positive. blood exams were quite normal. chest ct scan showed fig. 3 normal brain ct scan at the entrance interstitial pneumonia. brain ct was normal at the entrance (fig. 3) . patient was treated with antiviral therapy for 10 days. at discharge, another brain ct showed new periventricular ischemic lesions (fig. 4) . subject presented a mild cognitive impairment (mini mental state examination was 23). little is still known about an association with reproductive system. ace expression in testes poses patients at risk of orchitis. pathologic analyses of testes of six patients who died of sars revealed widespread germ cell destruction few or no spermatozoon in the seminiferous tubule, thickened basement membrane, and leukocyte infiltration. it further suggests that the reproductive functions should be followed and evaluated in recovered male sars patients [18] . conclusion covid-19 virus still represents a health and socioeconomic problem all over the world. clinicians must consider that it is no longer just a respiratory virus, as it has got a multi-organ trophism. it is important to consider extrapulmonary symptoms immediately to allow for early diagnosis and medical treatment. conflict of interest the authors declare that they have no conflict of interest. ethical approval na. consent written informed consent was obtained by patient for publication of this case report and accompanying images. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study clinical features of patients infected with 2019 novel coronavirus in wuhan left ventricular performance in patients with severe acute respiratory syndrome cardiovascular complications of severe acute respiratory syndrome analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan china: a retrospective cohort study pathological findings of covid-19 associated with acute respiratory distress syndrome association of cardiac injury with mortality in hospitalized patients with covid-19 in wuhan cardiac involvement in a patient with coronavirus disease 2019 (covid-19) possible involvement of infection with human coronavirus 229e, but not nl63, in kawasaki disease covid-19 and the liver: little cause for concern potential pathogenesis of ageusia and anosmia in covid-19 patients. int forum allergy rhinol guillain barre syndrome associated with covid-19 infection: a case report guillain-barré syndrome following covid-19: new infection, old complication? covid-19-associated acute hemorrhagic necrotizing encephalopathy: ct and mri features central nervous system manifestations of covid-19: a systematic review new onset acute symptomatic seizure and risk factors in coronavirus disease 2019: a retrospective multicenter study orchitis: a complication of severe acute respiratory syndrome (sars)1 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-321851-ku4z34lu authors: alosaimi, bandar; hamed, maaweya e.; naeem, asif; alsharef, ali a.; alqahtani, saeed y.; aldosari, kamel m.; alamri, aref a.; al-eisa, kholoud; khojah, taghreed; assiri, abdullah m.; enani, mushira a. title: mers-cov infection is associated with downregulation of genes encoding th1 and th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract date: 2020-02-29 journal: cytokine doi: 10.1016/j.cyto.2019.154895 sha: doc_id: 321851 cord_uid: ku4z34lu abstract mers-cov, a highly pathogenic virus in humans, is associated with high morbidity and case fatality. inflammatory responses have a significant impact on mers-cov pathogenesis and disease outcome. however, cd4+ t-cell induced immune responses during acute mers-cov infection are barely detectable, with potent inhibition of effector t cells and downregulation of antigen presentation. the local pulmonary immune response, particularly the th1 and th2-related immune response during acute severe mers-cov infection is not fully understood. in this study, we offer the first insights into the pulmonary gene expression profile of th1 and th2-related cytokines/chemokines (th1 & th2 responses) during acute mers-cov infection using rt2 profiler pcr arrays. we also quantified the expression level of primary inflammatory cytokines/chemokines. our results showed a downregulation of th2, inadequate (partial) th1 immune response and high expression levels of inflammatory cytokines il-1α and il-1β and the neutrophil chemoattractant chemokine il-8 (cxcl8) in the lower respiratory tract of mers-cov infected patients. moreover, we identified a high viral load in all included patients. we also observed a correlation between inflammatory cytokines, th1, and th2 downregulation and the case fatality rate. th1 and th2 response downregulation, high expression of inflammatory cytokines, and high viral load may contribute to lung inflammation, severe infection, the evolution of pneumonia and ards, and a higher case fatality rate. further study of the molecular mechanisms underlying the th1 and th2 regulatory pathways will be vital for active vaccine development and the identification of novel therapeutic strategies. middle east respiratory syndrome (mers) is a novel viral respiratory illness caused by the mers-cov that was first described in june 2012 in a patient who was hospitalized with signs of severe respiratory tract infection. the patient later died from respiratory and renal failure [1] [2] [3] . according to the statistics provided by the world health organization (who) on june 10, 2019, a total of 2428 laboratory-confirmed cases of human mers-cov infection have been reported, with an estimated 838 deaths in 27 countries. the accumulated evidence suggested that mers-cov infections are characterized by high levels of systemic inflammatory cytokines/chemokines as well as immunopathology [4] [5] [6] [7] [8] [9] [10] [11] . high inflammatory cytokines and chemokines levels have been strongly correlated with poor disease outcomes, immunopathology and massive infiltration of the immune inflammatory cells into the lungs [10, 12] . moreover, the stimulation of various cytokines, chemokines and innate immune cells has been associated with the appearance of cytokine storms, which occur during infections with pathogenic coronavirus (cov) and influenza virus [6, 13, 14] . in addition, infection, viral bronchiolitis pathogenesis, severe immunopathology, and disease enhancement during respiratory syncytial virus (hrsv) infection [10, 12, [15] [16] [17] . likewise, increased levels of inflammatory il-1β and il-1α cytokines have been associated with tissue damage and acute inflammatory responses leading to mortality and severe pathogenesis, as well as induction of the inflammatory loop [14, 18, 19] . mers-cov evades and antagonizes the antiviral immune response, as well as the nuclear factor-κb (nf-κb) signaling pathway and the ifn signaling cascade [20] [21] [22] . the disease pathogenesis of mers-cov infection is complex, with various factors involved in the onset of severe pulmonary damage and dissemination of the virus to other organs. th1 cells cytokines ifn-γ, tnf-α, tnf-β and il-2 play a key role in antiviral immunity by enhancing the toxic effects of cd8 t cells, stimulate nk cells and contribute to the regulation of cellular immunity [9, [23] [24] [25] [26] [27] [28] [29] . th2 cells secrete il-4, il-5, il-6, il-9, il-10, and il-13 which mediate humoral immune response and stimulate antibody production [9, [23] [24] [25] [26] [27] [28] [29] . imbalanced th1/th2 cytokines/chemokines can contribute to the pathological change, inefficient clearing infections, and host susceptibility to immune-mediated diseases. in vivo data highlights the important roles of t cells in controlling and reducing the pathogenesis and decreasing mers-cov titers [30] . there have been no reports describing the complete cellular immune response of the lower respiratory tract to mers-cov. therefore, it is still unclear whether the cd4 + t-helper (th1/th2)-induced cytokines/chemokines are beneficial or harmful for mers-cov-infected patients. moreover, the host immune responses which are activated in the lower respiratory tract during a mers-cov infection also remain unclear. in this study, we offer the first insight into the pulmonary molecular gene expression profile of th1 and th2 cytokines/chemokines (th1 & th2 responses) during acute mers-cov infection using rt 2 profiler pcr. we also describe the expression level of primary inflammatory cytokines and chemokines in the lower respiratory tract. the institutional review board at king fahad medical city reviewed and approved the study protocol (irb register number 17-182). the handling of respiratory samples, as well as aliquoting and viral and cellular rna extraction were executed using appropriate personal protective equipment in a biosafety level 3 laboratory (riyadh regional laboratory, ministry of health, riyadh, saudi arabia) the lower respiratory samples were collected from 39 mers-cov positive patients and 30 healthy non-infected individuals. to exclude effects of antiviral therapy on the expression of cytokines/chemokines understudy, the samples were collected before the initiation of any antiviral treatment for mers-cov. for the sputum sample collections, the patients were asked to rinse their mouth and gargle with water twice immediately before obtaining the sample and instructed not to expectorate saliva or postnasal discharge into the container during collection to reduce the possibility of contamination with upper respiratory tract fluids. subsequently, the patient was requested to breathe and expectorate deep cough sputum directly into a sterile sputum cup (screw cap). tracheal aspirate (ta) was collected via the endotracheal tube as reported elsewhere [31, 32] . bronchoalveolar lavage (bal) was collected by following a standardized protocol (technique, sampling, and procedure) as previously described [33] [34] [35] . the first bal fluid aliquots recovered were discarded to reduce cross-contamination with upper respiratory tract fluids. in order to exclude the effect of viral co-infection with influenza a h1n1 on cytokines/chemokines expression levels, all mers-cov positive patients were screened for h1n1 by genexpert flu (cepheid, sunnyvale, ca) according to the manufacturer′s instructions. samples were centrifuged at 1000 rpm at 4°c for 5-10 min. the cell-free supernatants were frozen at −80°c until they were used for subsequent analysis of the inflammatory cytokine and chemokine by elisarray, as well as for the mers-cov viral load test. the pellets were used for total cellular rna extraction using the rneasy mini kit (qiagen, valencia, ca) to detect mers-cov rna concentrations in the lower respiratory tract, viral rna was isolated from 140 µl of lower respiratory specimens using the qiaamp viral rna extraction mini kit (qiagen) according to the manufacturer′s instructions. the rna was dissolved in 60 µl of elution buffer and stored in separate aliquots at −80°c. an internal control (ic) was included as a control for the procedure used for sample preparation (viral rna purification). the extracted viral rna was tested by real-time rt-pcr duplex assays targeting the upe and orf1a regions of the mers-cov genome, using the realstar® mers-cov rt-pcr kit 1.0 (rt-pcr duplex assay). this system involves two independent assays, one targeting and quantifying a region upstream of the e gene (upe) and the other targeting the open reading frame regions 1a (orf1a) of the mers-cov genome. the analysis was performed using an abi prism® 7500 (applied biosystems). the main 12 human proinflammatory cytokines and chemokines (il-1α, il-1β, il-2, il-4, il-6, il-8, il-10, il-12, il-17a, ifn-γ, tnf-α, and gm-csf) were measured in lower respiratory samples from both mers-cov infected patients and the healthy non-infected control group, using multi-analyte elisarray (qiagen, germantown, md, usa) according to the manufacturer′s protocol. the absorbance of the obtained products after 30 min was measured at 450 nm, and cytokine/ chemokine levels were determined by comparing them to the negative (assay buffer) and positive control (cocktail containing all standard 12 cytokines or chemokines, respectively). the presence of a determined cytokine/chemokine was based on an absorbance value above the negative control. the mean absorbance of the samples and all standards was calculated as replicates. the concentrations were calculated using standard curves. cytokine/chemokines levels were expressed as pg/ml. pulmonary expression genes involved in the th1 and th2 response was assessed. cellular rna was isolated from lower respiratory tract samples using the rneasy mini kit (qiagen, valencia, ca). the concentration (ng/μl) and purity (a260/a280) of the rna was assessed using a nanodrop2000 (thermo-scientific). cdna synthesis and genomic dna elimination were performed using an rt 2 first-strand synthesis kit (qiagen, germantown, md, usa), using 1.5 µl (0.5 μg) of rna. mrna expression levels of th1/th2 immune genes were measured using an rt 2 -pcr profiler array kit (qiagen, germantown, md, usa) on an abi prism 7500 system (applied biosystems) with the rt 2 sybr green rox qpcr master mix (qiagen, germantown, md, usa). briefly, the cdna template was combined with the rt 2 real-time sybr green master mix and rnase-free water. a final reaction volume of 25 μl was added to each well of the rt 2 -pcr profiler array. cyclethreshold (ct) values were obtained using a constant baseline for all real-time rt-pcr runs as previously described [36] . five endogenous control genes provided by the array (actb, b2m, gapdh, gusb, and hsp90ab1) were used to calculate the arithmetic mean, which was then set as the ct value for normalization. the online rt 2 -pcr profiler data pcr array analysis software was used to identify the most appropriate reference genes to use for the normalization of the rt-pcr array gene expression data. real-time rt-pcr array results were analyzed using the online rt 2 -pcr profiler data pcr array analysis software (https://dataanalysis.qiagen.com/pcr/arrayanalysis.php). each sample was assessed for reverse transcription efficiency, pcr array reproducibility, and genomic dna contamination using the quality control function of the software. experimental gene expression was quantified as δδct and calculations were normalized to the housekeeping genes. statistical analysis was performed using spss version 16 (spss, inc., chicago, il). variables were compared between mers cov infected patients and healthy non-infected controls. the t-test and one-way anova analysis were used as appropriate. the data were presented as mean ( ± standard deviation) and counts (percentages). a p-value of < 0.05 was considered statistically significant. the demographic data, outcomes, preexisting conditions, underlying chronic diseases, clinical characteristics of mers cov infected patients and disease outcomes are presented in table 1 . the real-time rt-pcr results showed high viral loads in all mers-cov patients regardless of the type of specimen. the mean mers-cov rna ct values and standard deviations were 25.30 ± 5.1 and 26.3 ± 5.02 for upe and orf1a, respectively ( table 3) . the viral load of mers-cov and the expression levels of inflammatory cytokines/ chemokines were found to be significantly correlated with the case fatality of mers-cov infected patients (p < 0.002). furthermore, we found a significant correlation (p < 0.0001) between mers-cov viral load and expression levels of inflammatory cytokines il-1α, il-1β and neutrophils chemoattractant chemokines il-8 (cxcl8). no influenza a h1n1 co-infections were detected among mers-cov positive patients. the lower respiratory tract samples from mers-cov infected patients and healthy non-infected controls were used to quantify expression levels of the main 12 human pro-inflammatory cytokines and chemokines (il-1α, il-1β, il-2, il-4, il-6, il-8, il-10, il-12, il-17a, ifn-γ, tnf-α, and gm-csf). among the panel of 12 inflammatory cytokines/chemokines, the mean expression levels of il-1α (1148 pg/ml) (p < 0.001) and il1-β (1230 pg/ml) (p < 0.001) were significantly higher in the lower respiratory tract of mers-cov infected patients when compared to healthy non-infected controls ( table 2) . furthermore, our results showed a significant correlation between il and 1α (p < 0.035) and il-1β (p < 0.013) levels and the case fatality of mers-cov infected patients. ifn-γ expression levels were detected in only four patients and tnf-α expression levels were increased only in five patients. the expression of il-8 (cxcl8) was found to be significantly increased (1956 pg/ml) (p < 0.001) in all mers-cov patients when compared to healthy controls (table 3) . our results showed a significant correlation between the expression level of il-8 and the case fatality of mers-cov infected patients (p < 0.003). in order to study the pulmonary th1 and th2 responses in mers-cov infected patients, molecular cytokine/chemokine profiles were assessed using a rt 2 -pcr array. only genes with at least a two-fold change in expression when compared to the housekeeping gene were selected for further analysis. a side-by-side analysis of the data derived from the rt 2 -pcr profiling of pulmonary th1/th2 responses showed that genes encoding th1 and th2-related cytokines and chemokines were largely downregulated in the lower respiratory tract of mers-cov infected patients. a total of 26 genes involved in regulating the th1 and th2 immune response were downregulated in mers-cov infected patients ( table 4 ). the mrna expression levels of 10 th1-related cytokines/chemokines were downregulated. in contrast, the expression of only 6 th1 cytokine/chemokine mrnas, namely il-18, il-18r1, socs5, ccr2, cd4, and cxcr3, were upregulated (table 4) . with regards to the th2 immune response, we identified 15 cytokines/ chemokines mrnas expression levels were downregulated in mers-cov infected patients (table 4) . we did not observe any modulation or differences in th1/th2 responses and inflammatory cytokines/chemokines expression levels between mers-cov infected patients with underlying chronic medical conditions and mers-cov infected patients without underlying chronic medical conditions. the disease outcome, as well as the immunopathology and pathogenesis of mers-cov, sars, and other respiratory viral infections in humans and other animals are strongly linked to the expression levels of inflammatory cytokines and chemokines [16, 37, 38] . various cytokines, chemokines, and innate immune cells have been associated with the immunopathology during coronavirus (cov) and influenza virus infection [5, 6, 13, 14] . this study offers the first insight into the lung molecular gene expression profile of th1 and th2-related cytokines/ chemokines (th1 & th2 responses) during acute mers-cov infection. previous studies have shown that dysregulated and excessive immune responses may cause immunopathology and fatal disease [6, [39] [40] [41] . as such, direct cytopathic effects, high virus titers, and viral evasion of host immune responses are believed to play a major role in the severity of the diseases resulting from sars-cov and mers-cov infections [6, 39, 40] . our results showed high viral load (ct) values in all mers-cov infected patients regardless of the specimen type (table 3) . several pro-inflammatory cytokines, such as il-8 and il-1β, contribute to ards pathogenesis [6, 15, [42] [43] [44] . ards was shown to be the primary cause of death among patients with sars-cov or mers-cov infections [6, 45] . in this study, the majority of mers-cov infected patients progressed to ards, subsequently developing pneumonia and requiring admission to an intensive care unit (icu). in vitro studies have revealed that mers-cov induces significant but delayed modifications in the expression of both ifn and pro-inflammatory cytokines, including il-1β, il-6, il-8, and other chemokines [7, 10, 46] . our results showed high expression levels of il-1α, il-1β and il-8 (cxcl8) in the lower respiratory tract of mers-cov infected patients. evidence from studies investigating sars and respiratory syncytial virus (hrsv) has revealed that il-8 was associated with acute sars infection, bronchiolitis, immunopathology and disease enhancement during hrsv infections [10, 12, [15] [16] [17] . il-8 (cxcl8) is a critical chemokine involved in neutrophil recruitment, activation, and local neutrophil accumulation, and was shown to induce the formation of neutrophil extracellular traps (nets) [44, 47] . nets are highly immunogenic and extremely toxic to the host tissue, being able to directly cause inflammation, pathological changes, and epithelial and endothelial cell death [44, 48] . as such, lung nets can increase inflammation by stimulating il-8 expression, which leads to the recruitment of more neutrophils [44, 47] . we hypothesize that high il-8 expression levels may cause netosis, which results in severe mers-cov infection and increases immunopathology. il-1β has been associated with tissue damage, neutrophil infiltration, acute inflammatory responses, higher case fatality and severe respiratory viral infection [10, 14, 19, 49, 50] . in this study, high expression levels of il-1β were measured in the lower respiratory tracts of mers-cov infected patients. previous studies have shown that the expression of il-1β during influenza a h1n1 infection increased lung inflammation, and subsequent treatment with an il-1β antagonist significantly reduced both inflammation and lung tissue damage, suggesting that il-1β is a critical cytokine that contributes to lung inflammation [51, 52] . likewise, another study has shown that il-1β mrna levels were upregulated in calu-3 cells infected with mers-cov and/or sars-cov [10] . il-1α is an inflammatory cytokine that can be rapidly induced and quickly reaches high levels in response to a range of stimulants, such as il-1β and pathogenic agents [18, 53] . in mouse models, il-1α was shown to be a key player in triggering neutrophilic inflammation [18, 54] . likewise, our results revealed high il-1α expression levels in mers-cov infected patients. therefore, we think that high levels of il-8, il-1α, and il-1β may play a critical role in the immunopathology, severity, and case fatality of mers-cov infected patients. il-1α and il-1β establish an inflammatory loop which activates downstream signaling and enables the il-1α-il-1r1 interaction, leading to further il-1α and il-1β production. thus, a loop of continued and self-perpetuating inflammation occurs, resulting in extensive tissue damage and pathological changes [18] . as such, elevated il-1β and il-1α levels during acute mers-cov infection may cause an inflammatory loop that contributes to the extensive tissue damage and pathological changes associated with this disease, it is important to note that the il-1α and il-1β expression levels were not timely monitored at different intervals in this study. thus, the proposed inflammatory loop may not be generalizable to cases with severe disease or patients treated by antiinflammatory/antiviral drugs. it has been shown that effective control of host inflammatory response and antiviral treatment were associated with a reduction in inflammatory cytokine levels, steady improvement in disease condition, and pulmonary function [55] [56] [57] [58] [59] . consequently, studying il-1α and il-1β expression levels at different intervals could assist in a better understanding of mers-cov immunopathology. we speculate that, monitoring of il-1α and il-1β expression levels at different time points during mers-cov infection may alter the proposed inflammatory loop. tnf-α is an important innate and antiviral cytokine. high levels of tnf-α were detected in an in vitro study of sars-cov and mers-cov at both 24 and 30 h [10] . in our study, the induction of tnf-α was largely absent. similarly, in a recent study, tnf-α expression was not detected in most patients with mers-cov infections in the acute or convalescent stages [11] . this may indicate the limited early in vivo tnf-α response during mers-cov infections. one explanation for the differences between in vitro and in vivo studies could be the different kinetics of the mers-cov responses in the in vitro model, where there is a gradual increase in gene expression over time. however, a more likely explanation is that the complex interplay of target cell infection, mers-cov replication, viral load and time of sample collection. we measured cytokines/chemokines levels and viral load at a single time point in the early phase of infection; assessing cytokines/chemokines concentrations and viral load at different time points during mers-cov infection to create a kinetic profile of cytokines/chemokines might yield additional information. overall, the distinct tnf-α responses in vitro and in vivo might impact on the in vivo pathogenesis and viral amplification. cd4 + t-cell immune responses during respiratory viral infections characterized by the production of signature cytokines, ifn-γ, tnf-α, and il-2 for th1 cells and il-4, il-5, il-6, il-9, il-10, and il-13 for th2 cells [9, 28, 29] . the balance between the th1 and th2 responses is critical for the outcome of viral infections [60, 61] . to our surprise, we found that the expression of genes encoding th1 and th2 cytokines/chemokines were largely downregulated in the lower respiratory tract of mers-cov infected patients ( table 4 ). the pulmonary th1 and th2 responses showed the downregulation of 26 genes encoding th1 and th2 cytokines/chemokines in mers-cov infected patients. ifnγ plays an important role in early immunity, inducing the apoptosis of infected cells and stimulating both cd8 + and natural killer (nk) cells [62, 63] . in this study, both ifnγ and main th1associated cytokine mrna expression levels were downregulated in the lower respiratory tract of mers-cov infected patients. on the other hand, only 6 th1-related cytokines and chemokines (il-18, il-18r1, socs5, ccr2, cd4, and cxcr3) were overexpressed in the lungs of mers-cov infected patients (table 4 ). in regards to th2-induced immune responses, 15 th2 cytokines were downregulated. th2 cytokines play an important role in the humoral immune response and promote antibody production. mers-cov infection completely downregulated the th2 response in this study. we could not detect the mrnas for the specific antiviral immune response in mers-cov infected patients. reghunathan et al. also found a strong inflammatory response in acute sars despite complete downregulation of the mrna expression of genes involved in specific antiviral immune response [15] . downregulation of the major th1/th2 cytokines/chemokines can contribute to the pathological change, inefficient clearing infections, and host susceptibility to immune-mediated diseases. the upregulated th1 cytokines/chemokines (il-18, socs5, ccr2, and cxcr3) have been shown to play an important role in virus-induced immunopathology and usually associated with acute lung inflammation. il-18 was associated with acute lung inflammation [64] . in addition, il-18 was shown to enhance the severity of hrsv disease [65] . socs proteins affect antiviral signaling pathways, allowing viruses to evade the host immune response, and facilitate viral replication. moreover, socs5 has been suggested to shape the presentation of viral disease [66, 67] . previous data showed that, socs5 mrna was significantly upregulated in response to h1n1, h3n2, h5n1, and h11n9. suppressing socs signals improved influenza infection and inhibited hrsv replication [68, 69] . ccr2 mrna upregulation was correlated with hrsv-disease severity [70] . ccr2 antagonism reduced pathology, morbidity, and mortality during influenza a (h1n1) infection [71] . during hrsv infection increased cxcr3 was associated with the underlying pathology [72] . in addition, it has been shown that blocking cxcr3 reduced immunopathology during respiratory virus infections [73] . therefore, we think il-18, socs5, ccr2, and cxcr3 overexpression may play a critical role in immunopathology, severity, and case fatality of mers-cov infected patients. factors contribute to cd4 molecules upregulation during mers-cov infection in this study need further study. previous data showed that, cd4 and cd8 expression on t cells were not altered by mers-cov infection [74] . mers-cov could persistently induce the expression of pro-inflammatory cytokines/chemokines such as il-8, which would up-regulate cd4 molecules to enhance helper t cell infection. expression of high levels of il-8 and the net formation could have important effects on the cd4 expression. we propose a mechanism, which can explain the role of lower respiratory tract cytokines/ chemokines during acute mers-cov infection. il-1β and il-8 (cxcl8) recruit and activate neutrophils. subsequently, activated neutrophils undergo degranulation and netosis, which contributes to pathological changes and leads to the recruitment of more inflammatory. elevation of il-1β and il-1α during acute mers-cov infection may cause an inflammatory loop that contributes to extensive tissue damage and pathological changes. il-8 and il-1β are essential mediators in the development and pathogenesis of ards [6, 15, [42] [43] [44] . moreover, overexpression of il-1α, il-1β, il-8 (cxcl8), il-18, cxcr3, socs5, and ccr2 may play a vital role in the severity, immunopathology, and case fatality of mers-cov infected patients. it is important to note that the samples in this study were screened only for h1n1. we were not able to screen other respiratory viruses. viral co-infections could result in specific virus-virus interactions [75] . however, the inflammatory response induced by one virus may not be significantly changed by the infection of a second or third virus [76] . we did not examine the influences of co-infections with other respiratory viruses on cytokines/chemokines expression levels. any changes in immune profiles in mers-cov infected patients due to other respiratory pathogens are unknown. however, there is a possibility that other respiratory viral infections, which were not screened in our study, might skew immune profiles in mers-cov infected patients. we think that simultaneous study of other variables (ie, the interplay between mers-cov and other respiratory viruses), which could potentially skew the data is likely required to assess any alterations of the immune profiles in mers-cov infected patients. four limitations of this study should be noted. first, we measured cytokines/chemokines expression levels and viral load at a single time point in the early phase of infection; assessing cytokines/chemokines expression levels and viral replication at different time points during mers-cov infection to create a kinetic profile might yield additional information. second, in our study design, we did not include controls from patients infected with other respiratory viral pathogens. third, we have screened mers-cov positive samples only for h1n1, we did not screen our samples for other respiratory viruses. fourth, we were not able to recruit health non-infected individuals with similar mers-cov infected patients underlying diseases/pre-existing conditions. future study designs to close these gaps need to be considered. taken together, the high expression of inflammatory cytokines/ chemokines il-1 α and il-1β, il-8 (cxcl8), il-18, cxcr3, socs5, and ccr2 in the lower respiratory tracts of mers-cov infected patients confirmed the lung immunopathology [46, [77] [78] [79] [80] . therefore, the high expression of inflammatory cytokines and downregulation of the th1 and th2 immune responses in the 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cancers (basel) doi: 10.3390/cancers12082237 sha: doc_id: 332778 cord_uid: rf47ptj6 the coronavirus disease 2019 (covid-19) is currently representing a global health threat especially for fragile individuals, such as cancer patients. it was demonstrated that cancer patients have an increased risk of developing a worse symptomatology upon severe acute respiratory syndrome associated coronavirus-2 (sars-cov-2) infection, often leading to hospitalization and intensive care. the consequences of this pandemic for oncology are really heavy, as the entire healthcare system got reorganized. both oncologists and cancer patients are experiencing rescheduling of treatments and disruptions of appointments with a concurrent surge of fear and stress. in this review all the up-to-date findings, concerning the association between covid-19 and cancer, are reported. a remaining very debated question regards the use of an innovative class of anti-cancer molecules, the immune checkpoint inhibitors (icis), given their modulating effects on the immune system. for that reason, administration of icis to cancer patients represents a question mark during this pandemic, as its correlation with covid-19-associated risks is still under investigation. based on the mechanisms of action of icis and the current evidence, we suggest that icis not only can be safely administered to cancer patients, but they might even be beneficial in covid-19-positive cancer patients, by exerting an immune-stimulating action. since december 2019, a severe pneumonia outbreak diffused from a fish market in wuhan (hubei province, china) to all over the world, being declared a pandemic on the 11 march 2020 by the world health organization (who, geneva, switzerland) [1] . the pandemic is caused by the severe acute respiratory syndrome associated coronavirus-2 (sars-cov-2), identified as the causal agent of the so-called coronavirus disease 2019 (covid-19) [2] . a vaccine is under investigation and 13 clinical trials are currently opened at clinicaltrials.gov, with the common aim to test the efficacy of several different vaccinal strategies [22] . analogously, an effective anti-sars-cov-2 therapy does not exist and a very high number of clinical trials are presently evaluating the efficacy of combining standard of care procedures with molecules that might show either a direct anti-viral effect or the ability to reactivate the immune system of the host against the virus. this immune system remodulation might either reduce the cs (e.g., anti-inflammatory molecules, antibodies blockers of pro-inflammatory cytokines) or reactivate the cellular-mediated defective immune response (e.g., immune checkpoint inhibitors (icis)) [23] [24] [25] [26] . the consequences of covid-19 may be really heavy for people with an immunocompromised immune response, including cancer patients. in this review, the issues of treating cancer during this difficult time of pandemic will be reported. the overall impact of covid-19 on cancer management will be also analyzed, with reference to all the up-to-date published findings. one really yet debated question is the use of icis in cancer care during the pandemic, as they represent molecules which directly target and modulate the immune response. the present knowledge regarding this topic will be deeply dissected. moreover, our personal perspective, sustained by the available clinical findings, will be also disclosed. the outcome of covid-19 has been reported to be worse in patients with coexisting pathologies which are correlated with an impaired immune response to pathogens [7] . for example, elderly subjects or individuals with coexisting comorbidities including hypertension, obesity, diabetes or cancer, have a defective immune system that cannot efficiently contrast the sars-cov-2 infection. in those cases, covid-19 might quickly degenerate towards a severe or critical status [7] . in particular, cancer patients are highly sensitive to infections in general, and they may be therefore vulnerable to sars-cov-2 too. cancer itself may be linked with an extensive immunosuppressive status [27] . additionally, the immunosuppressive condition might be exacerbated as a consequence of strong myelosuppressive therapies, such as chemotherapy or radiotherapy [28] . given their immune-compromised status, cancer patients infected by sars-cov-2 might be at a higher risk of developing severe and critical consequences upon covid-19, including ards, septic shock and acute myocardial infarction [29] [30] [31] . cancer care at the time of covid-19 has been defined as a war on two fronts, mainly due to the reorganization of resources against the covid-19 emergency. indeed, the data regarding cancer patients are still very few and incomplete. consequently, oncologists are faced with a dilemma about how to balance the risk of covid-19 exposure with delivering an effective therapy and assistance to patients [32] . on the other end, cancer patients are directly affected by covid-19 healthcare system disruptions, as they may suffer from an increased perception of their fragility. this is mainly caused by the distance from care centers, with telemedicine substituting the in-presence visits. additionally, problems of cancellation and rescheduling might arise, plus difficulties coming with the imposed self-isolation during the lockdown [32] . mauri et al., on behalf of the international oncology panel and european cancer patient coalition collaborators, on the 25 march 2020, published a summary of all the main international recommendations for patients with cancer during the covid-19 pandemic, translated in 23 diverse languages [33] . the international scientific panel revised 63 different oncology guidelines from all over the world, thereby producing a univocal document, accessible from many different countries, as well as not english-speaking people. the panel of medical experts listed six main areas of recommendation, including: the relationship with the physician, the indications of hygiene and protective measures anti-covid-19, the features of sars-cov-2 infection and the restrictions operated by cancer centers to prevent the spread of the virus [33] . alongside, the oncologists need to reorganize their activity to respond to the threat and to protect their sensitive patients. patients with cancer, including hematological cancers and those who received hematopoietic stem cell transplantation, are generally more vulnerable to pathogens because their immune system is impaired. in the case of covid-19, as a consequence of the immunosuppression, they have a higher risk to develop severe and critical complications following sars-cov-2 infection [34] . often the oncologists work in stressful conditions, given the reorganization of infrastructures and instrumentation for the management covid-19 patients who need intensive care. the advice for oncologists is to adapt to these difficult challenges, hoping that the long-term consequences will be not too devastating for fragile individuals [34] . to help the oncologists, the main medical oncology societies, including american society of clinical oncology (asco), european society for medical oncology (esmo) and italian association of medical oncology) aiom, published several guidelines addressed to both oncologists and cancer patients, in order to safely guarantee the continuation of treatments and to ensure high standards of care. overall, all the societies agreed on the design of specific therapeutic protocols to direct the route of accession to the hospitals for cancer patients. when possible, they are advised to suspend all the unnecessary treatments and to use the telemedicine for the follow-up visits or other evaluations [35, 36] . in the same direction, the "cancer core europe" (cce), which includes seven european cancer institutes, such as the italian istituto nazionale dei tumori di milano, recently published a perspective in nature. cce conceived a general policy to ensure the best standard of care for cancer patients during the pandemic, even with shortages in personnel, medications, beds and infrastructures [37] . two main goals are evidenced in the guidelines: (1) to minimize hospital visits and hospitalization and (2) to prevent anti-cancer treatment complications. in details, to reduce the time spent by oncological patients in the hospital, it has been suggested: (1) to convert, where possible, to intravenous treatments to subcutaneous or oral administration, that can be pursued at home and (2) to postpone cancer non-emergency surgery or replace it with radiotherapy. moreover, in order to prevent insidious therapy-linked side effects, such as neutropenia and lymphopenia, it was advised to reduce cytotoxic chemotherapy or to use palliative care instead of proper treatment where possible [37] . as it will be largely discussed later, it is unlikely that anti-cancer treatments, per se, increase covid-19 infection risk. nevertheless, cancer patients, when infected by sars-cov-2 might develop more severe outcomes, if anti-cancer treatments induce a weakening of the host immune health [38] . to help clinicians, consortia and registries play a pivotal role in collecting and ease the analyses of data coming from cancer patients infected with sars-cov-2. the us covid-19 and cancer consortium (ccc19) is a multicenter registry which is currently helping to fill the gap in cancer care during this pandemic. in particular, ccc19 is assessing the number of cancer patients affected by covid-19. at the moment ccc19 comprehends 90 institutions across 28 different us states plus canada and spain [39] . furthermore, the uk coronavirus cancer monitoring project (ukccmp), is a monitoring project aimed to centralize the data deriving from 55 different institutions within the whole uk. ukccmp goal is the collection and analysis of clinical information from cancer patients affected by covid-19 [40] . in cancer patients, multiple factors may contemporarily contribute to the observed increased prevalence and severity of covid-19. given the often-chronic nature of the disease, such individuals may be older or smokers. furthermore, they might have comorbidities (preexisting, related to cancer and/or medications). altogether, these risk factors may collectively contribute to the individual fragility of people who develop a tumor. in addition, myelosuppression and lymph-suppression in these patients might be triggered by the usage of cytotoxic drugs [41] . given this very intricated clinical situation, the oncologist must evaluate case-by-case what to do to preserve cancer patients from covid-19, as well as to guarantee a successful anti-cancer treatment. sometimes delaying the treatment can be helpful, while other times the postponing may be even more deleterious [41] . since the beginning of the pandemic, already 70 clinical studies carried on cancer patients and related to covid-19 have been registered at clinicaltrials.gov. these trials include both observational and interventional studies, aimed to shed more light on the association occurring between cancer and covid-19. the search criteria employed were: (1) condition or disease: "cancer" and (2) other terms: "covid-19" (search date: 30 june 2020). all the registered studies are reported in table 1 . despite the very short time passed since the beginning of this noxious pandemic, some clinical observations have been already published, although more robust data on bigger cohorts are further needed to confirm the preliminary assessments. in an earlier study published by liang et al., in lancet oncology, on the 14 february 2020, an analysis of the feature of sars-cov-2 infection in 18 cancer patients was conducted [42] . the study was performed in a very small group of patients in china, during the first appearance of the covid-19. the limit of this study is, of course, the reduced number of cases analyzed. overall, the authors observed an increase of the death outcome in infected cancer patients compared with the general sars-cov-2 infected population. moreover, infected cancer patients were at higher risk of severe complications from covid-19, if compared to patients without cancer. the authors concluded that, where possible, the chemotherapy or surgery may be postponed. alternatively, where the delay is not possible, a stronger personal protection and other protective measures have been suggested [42] . although the study conducted by liang et al. was the first one to examine the correlation existing between cancer and covid-19, it was afterward criticized because the cases analyzed were very few (only 18) and highly heterogeneous, therefore the results have been considered overall not conclusive [43] . subsequently, in a larger study published by yu and colleagues, in jama oncology, on the 25 march 2020, the transmission of sars-cov-2 in cancer patients was examined. wherein, 1524 chinese patients with cancer were analyzed and the incidence and outcome of covid-19 infection were evaluated. only 12 out of the 1525 patients contracted sars-cov-2 and developed covid-19 (0.79%). although a smaller number (of only 12 cases) have been analyzed, the overall incidence of covid-19 in patients with cancer was found to be higher than in patients without cancer (0.32%, at the time of the study) [44] . moreover, in patients with non-small cell lung cancer (nsclc) over 60 years of age, the incidence of contracting sars-cov-2 infection was higher than in younger ones. no significant variations were observed among cancer patients in relation to their individual anti-cancer regime. the authors suggested, during the pandemic, to reduce the frequency of hospital visits in order to avoid a place where the likelihood to be infected is higher. moreover, they encouraged to self-isolation and lockdown, where possible [44] . a retrospective study on chinese cancer patients was performed, to characterize the clinical features of covid-19-severely infected cancer patients. the study was published in jama oncology by zhang and colleagues, on the 26 march 2020 [45] . it is a very small analysis, conducted on 28 cancer patients with covid-19. tumor-affected patients presented poorer outcomes and a higher mortality rate compared to non-cancer patients. moreover, the anti-cancer treatments, if received within 14 days prior the infection, increased the risk of developing severe symptoms, independently of the type of treatment. given the limited number of subjects implicated in the analyses, the authors suggest the validation of these observations to larger cohorts of cases [45] . in contrast with the reported chinese studies, a new york-based us study, published by miyashita et al., in annals of oncology, on the past 21st of april, reported that there was no significant difference between the incidence and overall mortality rate of covid-19 between cancer and non-cancer patients [46] . the authors analyzed a total of 5688 patients with covid-19, among which 334 had cancer. the authors postulated that the impairment of the immune system in such patients might contrast the possible development of a cs during covid-19 progression, thus explaining the surprising better outcome [46] . contrariwise, in another study, cancer patients were assessed overall more vulnerable to sars-cov-2 infection, in agreement with the previously reported chinese studies. the study, published in cancer discovery the 28 april 2020, by dai et al., analyzed a cohort of 105 cancer patients and 536 normal patients, all affected by covid-19. the 105 sars-cov-2 positive cancer patients (from the hubei province) were all with active cancer at the period of study, between january 1st and february 24th. the analysis demonstrated that patients with cancer had higher risks of severe covid-19 outcomes. in particular, patients with hematological cancer, lung cancer or with metastatic cancer (stage iv) had the highest severe events frequency and death rate [47] . the patients enrolled, received heterogeneous anti-cancer treatments and, in some cases, more than one type in combination. interestingly, patients who underwent surgery or received icis, had a higher chance to develop severe symptoms therefore needing icu hospitalization and controlled ventilation. the limitation of this latter observation is the very low number of analyzed cases (only six ici-treated and four surgery-treated patients). therefore, larger cohorts are needed to have a robust and meaningful indication about risks associated with specific anti-cancer interventions [47] . a clinical study performed by the institute curie (paris, france), analyzed 141 cancer patients with covid-19, amongst the 9842 cancer patients referred to the institute between the beginning of january and early may 2020. this study claims to be undertaken not only on hospitalized subjects, like the others previously published. in line with the new york study conducted by miyashita et al., the conclusions of this analysis are that covid-19 incidence in cancer patients, when adjusted for age, sex and preexisting comorbidities, is not dissimilar to the one observed in the general population. the authors suggested that the outcome of covid-19 is primarily driven by the initial severity of the infection, rather than by the presence of cancer condition [48] . thanks to the previously described us and uk cancer registries, ccc19 and the ukccmp, recent independent studies were published in the lancet journal, on the 28th of may 2020, reporting observations made on bigger groups of cancer patients with covid-19 (around 800 subjects) [49, 50] . the first study, conducted by lee and colleagues, analyzed data coming from the ukccmp, a clinical registry including data coming from all the main cancer centers in the uk. all patients with active cancer were eligible for the study. the primary endpoint was death or dismission from the hospital. the study considered 800 patients, with active cancer and diagnosed positive to sars-cov-2, between march 18th and april 26th 2020. the 52% of them developed mild symptoms, the 28% died. importantly, the risk of severe and critical covid-19 manifestations was mainly associated with the older age [49] . in fact, as described, in cancer patients affected by covid-19, the mortality is driven by a mix of factors such as age, male gender and presence of comorbidities. after adjusting for those variables, the authors found that chemotherapy received in the 4 weeks before covid-19 diagnosis had no incidence on the overall rate of death. furthermore, no significant effect on mortality was found for ici-immunotherapy, hormone therapy, targeted therapy and radiotherapy received within the 4 weeks prior covid-19 diagnosis [49] . although the mortality of cancer patients was higher than the non-cancer population, such discrepancy was not due to the presence of active cancer nor to the received anti-cancer treatment. this difference was correlated to the other risk factors, including: age, sex, smoking and coexisting comorbidities [49] . kuderer and colleagues, analyzed data from usa, canada and spain cancer patients, deposited in the registry of ccc19. they collected data from baseline, since the detection of sars-cov-2 positivity and at 30-days following sars-cov-2 infection diagnosis. the assessment of mortality after the 30-day was the primary endpoint [50] . it is a study still ongoing (nct04354701, see table 1 ), and the results published are preliminary findings. as such, 928 cancer patients with covid-19 were analyzed. the 30-day all-cause mortality was higher than in non-cancer covid-19 affected population. the explanation given by the authors was that cancer patients may be immunocompromised by cancer and/or anti-cancer treatments. older patients with comorbidities have a higher risk to develop severe covid-19. by may 7 , the 13% of the covid-19 positive cancer patients enrolled died within the 30-days diagnosis sars-cov-2 positivity [50] . additionally, the stronger association with 30-days all-cause death was present in patients receiving a combination of azithromycin plus hydroxychloroquine, drugs used to contrast the viral infection during the severe ards manifestations [50] . from these two studies some questions remain unsolved: (1) is any type of anti-cancer therapy a positive or a negative risk factor for sars-cov-2 infection? (2) are icis, in particular, a risk factor for sars-cov-2 infection rate and/or development of severe symptoms? in the uk study, no interaction with any of the anti-tumor therapies was found, and the higher death rate was instead associated with age, sex, smoking and comorbidities. whereas, the us study is still open, and it did not analyze yet the correlation between the risk of infection/mortality and the anti-cancer regime used. in contrast with the earlier observations derived from the chinese smaller studies, the two novel studies associated the increased risk of covid-19 complications with age, sex and coexisting pathologies instead of cancer pathology itself. the main conclusion coming out from these two bigger studies published in the lancet journal, is that oncological care should be offered to cancer patients during covid-19 emergency, including chemotherapy [51] . the latest published clinical study is a us one, conducted on new york city cancer patients, hospitalized at the memorial sloan kettering cancer center. a total of 423 cancer patients with covid-19 over a total of 2035 were enrolled. in which, 40% of the positively tested were hospitalized, and 20% of them developed severe covid-19, while 12% died. factor predictors for icu, oxygen need and severe outcomes were: age and previous or ongoing treatment with icis. in contrast, chemotherapy and major surgery did not show any significant difference. importantly, the endpoint considered was the hospitalization and admission to icu, and not death rate. this association was found independently from age, sex and cancer type. this observation was confirmed in a heterogeneous subset of cancers, including lung cancer [52] . since the beginning of this pandemic, nine independent clinical studies have been published about the risks possibly related to sars-cov-2 infection in patients with cancer. as reported, the risks analyzed often differed between the diverse studies, and included: (1) the risk of contracting the virus; (2) the probability of developing a severe symptomatology; and (3) the percentage of death [42, [44] [45] [46] [47] [48] [49] [50] [51] [52] . these studies showed a number of limitations. first of all, the considered control group differs between studies. while some studies compared cancer patients with or without covid-19, other ones considered covid-19 patients with and without cancer. additionally, the earlier results derived from very small cohorts, therefore being not very conclusive. although more recent reports included a bigger number of cases, the conclusions reported were often not comparable between each other, as the endpoints examined were often not the same. for example, while kuderer et al. contemplated the 30-days death toll as primary endpoint, the sloan kettering study investigated the incidence of icu hospitalization [50, 52] . although the association between anti-cancer therapy and covid-19 is still unclear, the observation reported consistently by the majority of the studies is that comorbidities, often co-existing in cancer patients, may predispose not necessarily to an increased incidence of the viral infection, but to develop more severe symptoms following the infection (such as ards). in general, since cancer patients have an overall heterogeneous clinical history, it is very difficult to discriminate between the selected effects that cancer and other pre-existing comorbidities might have on the individual immune response against sars-cov-2. in order to gain more robust data and fill this gap, further bigger (and more controlled) studies are ongoing, with the goal to shed light on the link existing between covid-19 and cancer (table 1) . the latest bigger studies deriving from ccc19 and ukccmp data-registries evidenced that none of the anti-cancer therapeutic regimens may affect neither the rate of severe covid-19 nor the mortality rate in cancer patients [51] . while a latest study conducted at the memorial sloan kettering cancer center, highlighted, specifically for icis, an association with increased icu admission rate, but not death rate [52] . so, the question remains still debated: is icis administration harmful or beneficial for cancer patients during the covid-19 pandemic? the discovery of icis literally revolutionized the cancer management. the icis are monoclonal antibodies directed against the so-called immune checkpoints which are activated by cancer cells in the attempt to suppress the immune system and its anti-cancer activity [53, 54] . icis include anti-pd-1, anti-pd-l1 and anti-ctla-4 antibodies and they represent a revolutionary type of immunotherapy for the treatment of solid tumors, including melanoma, lung cancer, renal carcinoma, urothelial cancers and head and neck cancers, as well as several hematological cancers, alone or in combination between each other or with other cytotoxic chemotherapies [55] [56] [57] . for their nature, icis restore the cellular-mediated immunocompetence. by blocking the checkpoints pd-1/pd-l1 and ctla4/b7, the cellular mediated anti-tumor activity of the immune system is then successfully restored and the cancer cells can be efficiently eliminated by the immune system [58] . a case study was conducted on two cancer patients with metastatic melanoma, both treated with anti-pd-1 and anti-ctla4 in combination. they developed immune-related pneumonitis as side effect of the therapy during the pandemic. importantly, they tested negative to sars-cov-2. both patients, were successfully treated and dismissed from the hospital within a week, and still testing negative to the virus. the authors warranted caution in administering the icis to cancer patients (especially if administered as a combination of more than one type), although they did not develop covid-19 [59] . another case study conducted on two cancer patients treated with icis, who were infected with sars-cov-2, developed mild covid-19 symptoms and they positively recovered from the infection. the finding suggests that treatment with icis must not be stopped during the covid-19 pandemic and the eventuality of a sars-cov-2 infection does not obstacle to grant cancer patients with the best standard of care treatment against the infection, which is not associated with a severe symptomatology [60] . do icis compromise cancer patients' immunity and increase the vulnerability to covid-19 infection? or, on the contrary, do icis potentiate their immune system? a recent review on this topic has been published on april 15th in immunotherapy journal, by kattan et al., highlighting the benefits of administering icis in responder cancer patients. indeed, the patients showed a significant improvement in the cancer prognosis of otherwise untreatable malignancies. thus, in such cases, icis must be regularly administered [61] . as described above, currently, very few data are available on cancer patients affected by covid-19 and treated with icis, so the oncologists are left alone to balance risks and benefits of interruption or continuation of such therapy, on a case-by-case basis. two main issues with icis are that: (1) a percentage of treated patients do not respond to them and (2) the individual tolerability of cancer patients may differ, as some subjects, in sporadic cases, may develop immune-related adverse events (iraes) which may vary from mild to life-threatening [62] . the adverse events are rare and extremely heterogeneous, including: gastrointestinal toxicity, mucosal inflammation, endocrine dysregulation and dermatological abnormalities. additionally, a 1-5% of iraes are associated with the development of interstitial pneumonitis, which for some extents, resembles the pneumonitis caused by sars-cov-2 [63] . the iaaes-related pneumonitis risk is very little, but not negligible, for that reason many clinicians prefer to suspend ici-based treatment in cancer patients during this acute phase of pandemic. additionally, sporadic data indicate that viral infection or reactivation may be a complication of icis administration [64] . a recently opened clinical study (which will be open until the end of the pandemic) is currently collecting data in an open register called teravolt, specific for patients affected by thoracic cancer which are infected with sars-cov-2. importantly, preliminary analyses of the available data suggest that ici-based therapy does not increase the overall mortality of thoracic cancer patients affected by covid-19, nor their overall hospitalization rate [30] . as said, cytotoxic chemotherapies and radiotherapies may induce myelosuppression, therefore they lower the overall humoral, as well as the cellular mediated immune response [65] . on the contrary, cancer patients treated with icis have been demonstrated able to restore their immunocompetence during hiv, hepatitis b or hepatitis c viral infection, thereby suggesting that those individuals might be highly immunocompetent if compared to the other cancer patients subjected to cytotoxic regimens [66] . a summary recapitulating the main advantages and disadvantages of administering icis to cancer patients during covid-19 pandemic, is reported in figure 1 . shown rare immune-related adverse events, in particular interstitial pneumonitis (red box); on the other side cancer patients that do not postpone the immunotherapy, continue to adhere to the program therefore having more chance of positive outcomes. moreover, they reactivate the immunocompetence, in particular the t-cell mediated immunity, not only towards cancer cells, but also against viral infection (green box). the measures adopted for icis are currently based on a case-by-case approach. data regarding the association of icis administration and covid-19 outcomes are currently very few and contrasting. since the pandemic started, scientific opinions have been diffused, but not enough solid observational data have been generated yet [37] . importantly, there is no direct evidence of iciinduced toxicity and increased viral infection risk in cancer patients, prior or during the pandemic. in fact, most cancer care centers agree on continuing icis, especially for responder patients with lung cancer [49] . the cytokine release syndrome, which triggers the cs, is a severe complication in patients with covid-19, as it may induce ards [19] . as described above, following a first hyper-inflammatory phase triggered by the cs, sars-cov-2 prolonged infection might induce t-cell hyperactivation and finally exhaustion, associated with concurrent patients' lymphopenia, as well as sepsis [67] . in case of severe sars-cov-2 infection, although both cd4+ and cd8+ t-cells in covid-19 patients start to differentiate, such cells are reduced in abundance and less activated. consequently, the virus clearance is delayed, because the excessive exhaustion of cd8+ t-cells in severe covid-19 patients may reduce their cellular-mediated immune response against the virus [68, 69] . in detail, impaired peripheral t-cells upregulate both the mucin-3 and pd-1 immunosuppressive markers [67] . as a consequence, severe and critical covid-19 patients might end up with viral sepsis [70] . finding a therapy to restore the t-cell functionality in covid-19 patients might avoid the development of viral sepsis and ards complications. independent data in mice and in patients, demonstrated that pd-1/pd-l1 blocking antibodies administered to subjects chronically infected with viruses (i.e., lymphocytic choriomeningitis virus in mice or hiv in humans), enhance the viral control and virus-specific cd8+ t-cell responses. this observation demonstrates that the pd-1 inhibitory pathway is particularly important in exhausted tcells formation. in fact, pd1-blockage restored both cd4+ and cd8+ t-cells abundance and functionality [71, 72] . based on these observations, is it acceptable to continue icis to cancer patients during covid-19 acute pandemic phase? from the above-reported clinical studies, it is not demonstrated that there figure 1 . balance between harmful and beneficial effects of immune-checkpoint immunotherapy in cancer patients during covid-19 pandemic. if on one side immune checkpoint inhibitors (icis) have shown rare immune-related adverse events, in particular interstitial pneumonitis (red box); on the other side cancer patients that do not postpone the immunotherapy, continue to adhere to the program therefore having more chance of positive outcomes. moreover, they reactivate the immunocompetence, in particular the t-cell mediated immunity, not only towards cancer cells, but also against viral infection (green box). the measures adopted for icis are currently based on a case-by-case approach. data regarding the association of icis administration and covid-19 outcomes are currently very few and contrasting. since the pandemic started, scientific opinions have been diffused, but not enough solid observational data have been generated yet [37] . importantly, there is no direct evidence of ici-induced toxicity and increased viral infection risk in cancer patients, prior or during the pandemic. in fact, most cancer care centers agree on continuing icis, especially for responder patients with lung cancer [49] . the cytokine release syndrome, which triggers the cs, is a severe complication in patients with covid-19, as it may induce ards [19] . as described above, following a first hyper-inflammatory phase triggered by the cs, sars-cov-2 prolonged infection might induce t-cell hyperactivation and finally exhaustion, associated with concurrent patients' lymphopenia, as well as sepsis [67] . in case of severe sars-cov-2 infection, although both cd4+ and cd8+ t-cells in covid-19 patients start to differentiate, such cells are reduced in abundance and less activated. consequently, the virus clearance is delayed, because the excessive exhaustion of cd8+ t-cells in severe covid-19 patients may reduce their cellular-mediated immune response against the virus [68, 69] . in detail, impaired peripheral t-cells upregulate both the mucin-3 and pd-1 immunosuppressive markers [67] . as a consequence, severe and critical covid-19 patients might end up with viral sepsis [70] . finding a therapy to restore the t-cell functionality in covid-19 patients might avoid the development of viral sepsis and ards complications. independent data in mice and in patients, demonstrated that pd-1/pd-l1 blocking antibodies administered to subjects chronically infected with viruses (i.e., lymphocytic choriomeningitis virus in mice or hiv in humans), enhance the viral control and virus-specific cd8+ t-cell responses. this observation demonstrates that the pd-1 inhibitory pathway is particularly important in exhausted t-cells formation. in fact, pd1-blockage restored both cd4+ and cd8+ t-cells abundance and functionality [71, 72] . based on these observations, is it acceptable to continue icis to cancer patients during covid-19 acute pandemic phase? from the above-reported clinical studies, it is not demonstrated that there is any occurring synergy between icis-induced pneumonitis and the inflammatory ards determined by sars-cov-2 that can be lethal. therefore, we suggest to not suspend the ici-programmed therapy in cancer patients during the pandemic. additionally, we advise to consider on a case-by-case basis the eventual association of icis with immunosuppressive corticosteroids or chemotherapies. finally, when possible, we propose to concentrate the therapy sessions, so the cancer patient may limit the entrances in the hospital, which can represent a place at higher risk to contract the sars-cov-2 infection. in general, not only the icis-based therapy is pivotal for cancer eradication, especially in patients that are responding to this therapeutic approach. moreover, we would like to highlight that icis might be a possible game-changer for cancer patients during this covid-19 pandemic. in fact, given the scientific considerations reported above, cancer patients subjected to anti-pd-1 or anti-pd-l1 antibody therapy might restore their t-cell anti-cancer (and possibly anti-viral) immune response. in turn, this can help to fight the sars-cov-2 infection. in support to our opinion, a registered clinical study is currently ongoing, conducted on metastatic and advanced cancer patients, affected by covid-19 and which are not eligible to be transferred into an icu. the study already enrolled 384 patients and, once finished, it will uncover the differential efficacy to eradicate sars-cov-2 infection in covid-19 patients treated either with anti-pd-1 antibody nivolumab in association with standard care protocol, or with standard care alone (nct04333914, see table 1 ). a summary of the potential immune-related benefits of ici-immunotherapy is schematized in figure 2 . cancers 2020, 12, x 17 of 22 patient may limit the entrances in the hospital, which can represent a place at higher risk to contract the sars-cov-2 infection. in general, not only the icis-based therapy is pivotal for cancer eradication, especially in patients that are responding to this therapeutic approach. moreover, we would like to highlight that icis might be a possible game-changer for cancer patients during this covid-19 pandemic. in fact, given the scientific considerations reported above, cancer patients subjected to anti-pd-1 or anti-pd-l1 antibody therapy might restore their t-cell anti-cancer (and possibly anti-viral) immune response. in turn, this can help to fight the sars-cov-2 infection. in support to our opinion, a registered clinical study is currently ongoing, conducted on metastatic and advanced cancer patients, affected by covid-19 and which are not eligible to be transferred into an icu. the study already enrolled 384 patients and, once finished, it will uncover the differential efficacy to eradicate sars-cov-2 infection in covid-19 patients treated either with anti-pd-1 antibody nivolumab in association with standard care protocol, or with standard care alone (nct04333914, see table 1 ). a summary of the potential immune-related benefits of iciimmunotherapy is schematized in figure 2 . icis might restore individual cellular-mediated immunocompetence and this lesson from cancer may be even transferred to non-cancer covid-19-affected subjects. in fact, icis have already been used beyond cancer to treat, for example, sepsis-induced immunosuppression [73, 74] . moreover, icis have been confirmed to be safe when administered to cancer patients vaccinated for influenza virus [75, 76] . in line with this concept, three additional independent clinical studies are currently enrolling icis might restore individual cellular-mediated immunocompetence and this lesson from cancer may be even transferred to non-cancer covid-19-affected subjects. in fact, icis have already been used beyond cancer to treat, for example, sepsis-induced immunosuppression [73, 74] . moreover, icis have been confirmed to be safe when administered to cancer patients vaccinated for influenza virus [75, 76] . in line with this concept, three additional independent clinical studies are currently enrolling non-cancer covid-19 patients to test the efficacy of administering icis to reshape the impaired immune system of sars-cov-2 infected individuals (i.e., nct04268537; nct04356508 and nct04413838). importantly, in order to detect sars-cov-2 levels during the progression of covid-19 and, hence, to evaluate the efficacy of the icis-based immunotherapy, the research is currently trying to identify effective and fast viral detection strategies. the rt-pcr based methodologies are the currently recommended standard strategies suggested by the who for both sars-cov-2 diagnosis and prognosis [77] . in addition, novel high-sensitive molecular methods based on the use of the droplet digital pcr have been proposed for the effective virus detection in covid-19 patients, even with low viral load [78, 79] . the pandemic is currently rising and the daily reports from the who do not leave space to hope for any shortcoming improvement, as 10 million cases and nearly 500,000 deaths of covid-19 have now been reported globally (situation report 161, 29 june 2020) [80] . cancer patients are considered subjects with a higher risk of covid-19 poorer outcome. the recent clinical data evidenced that the risk to develop severe or critical symptoms of covid-19 is correlated to factors co-occurring in a cancer patient (e.g., elderly age, sex and presence of many comorbidities), and not to the cancer condition per se. moreover, the most recent and bigger reports (in terms of cohort size) evidenced that none of the anti-cancer therapy significantly affected the overall risk of infection or worsen of the covid-19 symptomatology. currently, it seems that any anti-cancer regime might be followed without any concern during the pandemic, in contrast with the initial suggestions to interrupt or 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hiv-specific t cells is associated with t-cell exhaustion and disease progression immune checkpoint inhibition in sepsis: a phase 1b randomized study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of nivolumab immune checkpoint inhibition in sepsis safety of inactivated influenza vaccine in cancer patients receiving immune checkpoint inhibitors immunogenicity and safety of influenza vaccination in cancer patients receiving checkpoint inhibitors targeting pd-1 or pd-l1 diagnostic strategies for sars-cov-2 infection and interpretation of microbiological results sensitivity assessment of droplet digital pcr for sars-cov-2 detection quantitative detection and viral load analysis of sars-cov-2 in infected patients world health organization (who) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to acknowledge the italian league against cancer (lilt) for its support. the authors declare no conflict of interest. key: cord-328289-3h3kmjlz authors: iadecola, costantino; anrather, josef; kamel, hooman title: effects of covid-19 on the nervous system date: 2020-08-19 journal: cell doi: 10.1016/j.cell.2020.08.028 sha: doc_id: 328289 cord_uid: 3h3kmjlz summary neurological complications have emerged as a significant cause of morbidity and mortality in the ongoing covid-19 pandemic. beside respiratory insufficiency, many hospitalized patients exhibit neurological manifestations, ranging from headache and loss of smell, to confusion and disabling strokes. covid-19 is also anticipated to take a toll on the nervous system in the long term. here we will provide a critical appraisal of the potential for neurotropism and mechanisms of neuropathogenesis of sars-cov-2, as they relate to the acute and chronic neurological consequences of the infection. finally, we will examine potential avenues for future research and therapeutic development. neurological complications have emerged as a significant cause of morbidity and mortality in the ongoing covid-19 pandemic. beside respiratory insufficiency, many hospitalized patients exhibit neurological manifestations, ranging from headache and loss of smell, to confusion and disabling strokes. covid-19 is also anticipated to take a toll on the nervous system in the long term. here we will provide a critical appraisal of the potential for neurotropism and mechanisms of neuropathogenesis of sars-cov-2, as they relate to the acute and chronic neurological consequences of the infection. finally, we will examine potential avenues for future research and therapeutic development. there is increasing evidence that the nervous system is frequently involved in patients hospitalized with coronavirus disease 2019 . this is not surprising since neurological manifestations have long been described also in infections from other respiratory viruses, including coronaviruses (bergmann et al., 2006) . however, the neurological manifestations of covid-19 are common and disabling enough to have attracted widespread attention in the scientific and lay press for their short-and long-term impact on population health (pleasure et al., 2020; wenner moyer, 2020) . a large body of clinical data from tertiary referral centers is rapidly accumulating on this topic worldwide, often with conflicting observations, partly reflecting the preliminary and incomplete nature of the available data. here, we provide a succinct summary of the nervous system involvement in covid-19. in particular, we will focus on the mechanisms of pathogenicity, on the acute and delayed neurological manifestations reported to date, and on how the nervous system involvement compares to that of other respiratory viruses. finally, we will attempt to flesh out caveats and unanswered questions that may help gain a better appreciation of this critical aspect of covid-19 and chart a path forward to minimize its harmful nervous system involvement. an ace2-dependent manner (song et al., 2020) . in brain cells derived from human pluripotent stem cells, dopaminergic neurons, but not cortical neurons or microglia, were particularly susceptible to sars-cov-2 infection . clinical-pathological studies that have tested for the presence of the virus in the brain or the cerebrospinal fluid (csf) have had mixed results. some studies have shown sars-cov-2 rna in brain post-mortem or in the csf in patients with encephalopathy or encephalitis, but at very low levels (moriguchi et al., 2020; solomon et al., 2020) . other studies could not detect viral invasion, even though there was evidence of csf inflammation (bernard-valnet et al., 2020; ye et al., 2020) . considering the inconsistent data and the low levels of viral rna, when detected, the possibility of artifact or contamination has been raised (solomon et al., 2020) . potential routes of brain entry: examination of how the virus could enter the nervous system may help assess the likelihood for direct invasion and pathogenicity. based on other coronaviruses, several potential routes of entry for sars-cov-2 have been proposed (bergmann et al., 2006) . olfactory route: infection of olfactory system is consistent with the observation that loss of smell is a frequent neurological manifestation in covid-19 (see neurological manifestations of and with evidence of increased mri signal in the olfactory cortex suggestive of infection (politi et al., 2020) . the virus could be internalized in nerve terminals by endocytosis, transported retrogradely, and spread trans-synaptically to other brain regions, as described for other coronaviruses (dubé et al., 2018) . ace2 and tmprss2 have been detected in the nasal mucosa at the rna and protein levels, but they seem to be localized to epithelial cells (sustentacular cells), not olfactory neurons (brann et al., 2020) , although another report suggests neuronal involvement (nampoothiri et al., 2020) . therefore, it is unclear if the virus is restricted to the olfactory epithelium or reaches olfactory neurons. blood-brain barrier: the bbb is a common route of entry of blood-borne viruses into the brain (bergmann et al., 2006) . in covid-19, dissemination of the virus into the blood has been described, albeit with widely ranging frequencies (1% to 41%) (wang et al., 2020c; zheng et al., 2020) , and the virus could access the brain by crossing the bbb. crossing the intact bbb would require internalization and transport of the virus across the cerebral endothelium, in which the expression of sars-cov-2 docking proteins remains unclear ( figure 1 ). ace2 immunoreactivity was observed in brain vessels of a patient who died with multiple ischemic infarcts but the cellular localization was not determined (bryce et al., 2020) . the possibility of entry through other putative sars-cov-2 receptors expressed more widely in the cerebral vasculature, such as nrp1 and bsg, cannot be ruled out (cantuti-castelvetri et al., 2020) . on the other hand, sars-cov-2-associated cytokines, including il-6, il-1β, tnf, and il-17 disrupt the bbb (erickson and banks, 2018) and could facilitate the entry of the virus ( figure 2 ). sars-cov-2 has been postulated to induce endothelial infection and inflammation in peripheral vessels (teuwen et al., 2020) , but direct evidence in cerebral endothelial cells has not been thus far provided. rather, a lack of florid cerebrovascular inflammation has been noted in several autopsy studies (bryce et al., 2020; kantonen et al., 2020; reichard et al., 2020; solomon et al., 2020) . comorbidities often seen in covid-19, including cardiovascular risk factor or pre-existing neurological diseases, could, alone or in combination with cytokines, increase bbb permeability (erickson and banks, 2018) . for example, in a covid-19 patient with parkinson's disease, electron microscopy revealed viral particles in frontal lobe microvessels and neurons, suggesting trans-endothelial entry (paniz-mondolfi et al., 2020) . another parkinson's disease patient with obesity, hypertension and diabetes, exhibited at autopsy, in addition to hypoxic-ischemic neuronal damage, microhemorrhages, white matter lesions and enlarged perivascular spaces, but no evidence of sars-cov-2 in the brain (kantonen et al., 2020) . sars-cov-2 could also enter the brain through the median eminence of the hypothalamus and other circumventricular organs, brain regions with a leaky bbb due to openings (fenestrae) in the capillary wall (kaur and ling, 2017) . although the size of the viral particle (80-120nm) is larger than endothelial fenestrae (sarin, 2010) , preliminary data suggest j o u r n a l p r e -p r o o f that median eminence capillaries and tanycytes express ace2 and tmprss, which could allow virus entry into the hypothalamus (nampoothiri et al., 2020) . owing to its widespread connection, the hypothalamus could serve as a gateway to the entire brain. infiltration of infected immune cells: viruses can enter the brain carried by infected immune cells, which can also serve as reservoir (bergmann et al., 2006) . monocytes, neutrophils and tcells traffic into the brain through the vasculature, the meninges and the choroid plexus (engelhardt et al., 2017) , and these sites could be entry points for infected immune cells. conclusive evidence of infection of immune cells by sars-cov-2 has not been provided thus far (merad and martin, 2020) . sars-cov-2 envelope np protein immunoreactivity was observed in cd68+ cells in lymphoid organs (chen et al., 2020a) , while single-cell rna seq data showed viral rna in macrophages in bronchoalveolar lavage of covid-19 patients (bost et al., 2020) . but it remains unclear if this is due to actual virus propagation in macrophages or to phagocytic uptake of virus infected cells or extracellular virions (bost et al., 2020; merad and martin, 2020) . furthermore, several autopsy series have revealed a notable lack of immune cell infiltration (kantonen et al., 2020; reichard et al., 2020; solomon et al., 2020) . in summary, sars-cov-2 can infect neurons in vitro and cause neuronal death, but data from csf and autopsy studies do not provide consistent evidence of direct cns invasion. however, effects on the median eminence and other circumventricular organs, cannot be ruled out and may play a role in the systemic manifestations of the disease. lung damage and respiratory failure: the lung is the organ most affected in covid-19, with massive alveolar damage, edema, inflammatory cell infiltration, microvascular thrombosis, microvascular damage and hemorrhage (carsana et al., 2020) . sars-cov-2 has been detected mainly in pneumocytes and epithelial progenitors (bost et al., 2020; carsana et al., 2020) . the respiratory failure resulting from lung damage leads to severe hypoxia (adult respiratory distress syndrome, ards), requiring assisted ventilation (grasselli et al., 2020) . consistent with hypoxic brain injury, autopsy studies in covid-19 have shown neuronal damage in brain regions most vulnerable to hypoxia, including neocortex, hippocampus and cerebellum (kantonen et al., 2020; reichard et al., 2020; solomon et al., 2020) . a key feature of covid-19 is a maladaptive immune response characterized by hyperactivity of innate immunity followed by immunosuppression (diao et al., 2020; qin et al., 2020; vabret et al., 2020; zhou et al., 2020) . improvement of t-cell function coincides with remission of symptoms and declining viral loads (thevarajan et al., 2020) , attesting to the link between immuno-suppression and disease severity. in patients with severe disease, the cytokine release syndrome can develop (qin et al., 2020; xu et al., 2020) . most covid-19 patients exhibit increased circulating levels of il-6 il-1β, and tnf, as well as il-2, il-8, il-17, g-csf, gm-csf, ip10, mcp1, and mip1α2, and serum levels of il-6 and tnf reflect disease severity (diao et al., 2020) . even in the absence of sars-cov-2 brain invasion, viral proteins shed in the circulation and molecular complexes from damaged cells, such as the nuclear protein high mobility group box 1 (hmgb1) (chen et al., 2020b), could enter the brain through a compromised bbb ( figure 2 ). after brain entry, these molecules could act as pathogen associated molecular patterns (pamps) and damageassociated molecular patters (damps), and induce an innate immune response in pericytes, brain-resident macrophages and microglia, which express toll-like receptors (tlr) (figure 2 ). tlr2 mediates the pro-inflammatory effects of sars-cov spike protein on human macrophages through nf-κb (dosch et al., 2009) . such innate immune response increases cytokine production and impair brain function (dantzer, 2018) . in mice, viral infections increase circulating levels of ifnα/β leading to activation ifnr1 on cerebral endothelial cells and cxcl10-cxcr3-mediated cognitive impairment (cytokine sickness behavior) (dantzer, 2018 ). an ifn type i response does occur in covid-19 and is thought to be protective (merad and martin, 2020) , but could contribute to the alterations in consciousness (see neurological manifestation of the hypothalamus: target and culprit of immune dysregulation: the brain, the hypothalamus in particular, could also contribute to the immune dysregulation ( figure 3 ). several cytokines upregulated in covid-19 (il-6, il-1β, tnf) are powerful activators of the hypothalamic-pituitary-adrenocortical (hpa) axis (dantzer, 2018) . the hpa axis is central to the regulation of systemic immune activity and is activated by bbb dysfunction and neurovascular inflammation (dantzer, 2018). as mentioned above, covid-19 is associated with immunosuppression and lymphopenia. in stroke and brain trauma adrenergic stress involving βadrenergic receptors results in massive systemic immunosuppression . the mechanisms of these effects involve activation of the hpa, leading to the release of norepinephrine and glucocorticoids. these mediators act synergistically to induce splenic atrophy, t cell apoptosis, and nk cell deficiency. in the bone marrow, tyrosine hydroxylase and norepinephrine trigger a response in mesenchymal stromal cells, most likely through β3adrenergic receptors, resulting in a reduction of cell retention . downregulation of these factors, in concert with calprotectin release from damaged lungs, may increases hematopoietic stem cell proliferation skewed towards the myeloid lineage (emergency myelopoiesis) (schulte-schrepping et al., 2020; silvin et al., 2020) , which results in lymphopenia and neutrophilia, two key hematological features of covid-19 (chen et al., 2020a; moriguchi et al., 2020; qin et al., 2020) (figure 3 ). importantly, in sars, hpa activation and glucocorticoid levels are correlated with neutrophilia and lymphopenia (panesar et al., 2004) . hypercoagulable state: another key feature of covid-19 is a profound coagulopathy responsible for some of the most frequent and harmful complications of the disease. in a multicenter study, 88% of patients exhibited evidence of a hypercoagulable state (helms et al., 2020b) . covid-19 coagulopathy is characterized by a distinctive pro-coagulant state with increased cloth strength, increased d-dimers (fibrin breakdown products indicative of intravascular thrombosis), and increased fibrinogen, without significant changes in the number of platelets or prolongation of clotting time parameters (helms et al., 2020b) . coagulopathy and thrombosis may start in the lungs and other infected organs with endothelial damage, complement activation, the procoagulant action of il-6, and neutrophil recruitment (goshua et al., 2020; ramlall et al., 2020) . in turn, neutrophils release extracellular traps (nets) in covid-19 (middleton et al., 2020) , a lattice of chromatin and histones that activates clotting, which contributes to intravascular thrombosis by trapping cells and platelets in many organs including the brain. systemic organ failure: covid-19 also damages other organs. metabolic and pathological evidence of damage to the kidney, heart, liver, gastrointestinal tract, and endocrine organs has been provided inciardi et al., 2020; pal and banerjee, 2020; pan et al., 2020; su et al., 2020) . the resulting systemic metabolic changes, including water and electrolyte imbalance, hormonal dysfunction, and accumulation of toxic metabolites, could also contribute to some of the more non-specific nervous system manifestations of the disease, like confusion, agitation, headache etc. cardiac involvement could impact the brain by reducing cerebral perfusion or, as discussed in the next section, could be an embolic source leading to ischemic strokes. numerous neurological abnormalities have been described in patients with covid-19. these involve the central and peripheral nervous system, range from mild to fatal, and can occur in patients with severe or otherwise asymptomatic sars-cov-2 infection. neurological abnormalities have been described in approximately 30% of patients who required hospitalization for covid-19, 45% of those with severe respiratory illness, and 85% of those with ards (helms et al., 2020a; mao et al., 2020) . in patients with mild covid-19 neurological symptoms are mostly confined to nonspecific abnormalities such as malaise, dizziness, headache, and loss of smell and taste (mao et al., 2020) , routinely observed in respiratory virus infections such as the influenza (chow et al., 2019) . while serious neurological complications have been reported in patients with otherwise mild covid-19 (oxley et al., 2020) , the most severe complications occur in critically ill patients and are associated with significantly higher mortality yaghi et al., 2020) . encephalopathy and encephalitis: alterations in mental status (confusion, disorientation, agitation, somnolence), collectively defined as encephalopathy, have been consistently reported in various cohorts with covid-19. altered mental status occurs rarely (<5%) even in covid-19 patients requiring hospitalization for respiratory illness (mao et al., 2020) , but affects the majority of critically ill covid-19 patients with ards (helms et al., 2020a) . a key question is whether this alteration in mental status represents an encephalopathy caused by systemic illness or an encephalitis directly caused by the sars-cov-2 virus itself. several cases have been reported of covid-19 patients (efe et al., 2020; farhadian et al., 2020; huang et al., 2020b; moriguchi et al., 2020; pilotto et al., 2020) who appear to meet established diagnostic criteria for infectious encephalitis, which include altered mental status, fever, seizures, white blood cells in the csf, and focal brain abnormalities on neuroimaging (venkatesan et al., 2013) . in at least two reported cases, sars-cov-2 was detected in the csf (huang et al., 2020b; moriguchi et al., 2020) although, as discussed in the previous section (nervous system invasion), only modest amounts of viral rna were detected. in at least one covid-19 case, the diagnosis of temporal lobe encephalitis was confirmed by biopsy which showed perivascular lymphocytic infiltrates and hypoxic neuronal damage (efe et al., 2020) , but the presence of sars-cov2 or other viruses in brain or csf was not documented. indeed, most samples of csf in patients with neurological abnormalities in the setting of covid-19 have not revealed evidence of sars-cov-2 (kandemirli et al., 2020) and most samples of brain tissue from autopsies of covid-19 patients have not revealed evidence of encephalitis (see nervous system invasion). besides encephalitis, most covid-19 patients have other reasons for their altered mental status. delirium, confusional states, and coma appear most common in covid-19-related critical illness (helms et al., 2020a; mao et al., 2020; rogers et al., 2020) , which is often marked by hypoxia, hypotension, renal failure, the need for heavy doses of sedatives, and prolonged immobility and isolation (cummings et al., 2020)-all factors well known to cause encephalopathy (maas, 2020) . the rarity of cases clinically consistent with encephalitis, the paucity of histopathological evidence of encephalitis, and the many alternative explanations for the altered mental status suggest that sars-cov-2 brain invasion is a possible but rare cause of encephalopathy. ischemic stroke: stroke is not uncommon among patients hospitalized with covid-19, with reported rates ranging from 1-3% in hospitalized patients and up to 6% of critically ill patients (mao et al., 2020; merkler et al., 2020; yaghi et al., 2020) , 7-fold higher than in patients hospitalized with influenza even after adjustment for illness severity . early case reports described unusual embolic strokes in otherwise young healthy individuals with covid-19 (oxley et al., 2020) , but in subsequent case series patients were generally older and had numerous vascular comorbidities (lodigiani et al., 2020) . therefore, it remains unclear whether these strokes were caused by sars-cov-2 or represented the background incidence of stroke in this high-risk populations that also happened to be infected at the time. it is plausible that sars-cov-2 infection does play some role in causing stroke, given that infections in general increases stroke risk . the covid-19-related hypercoagulability would be expected to increase susceptibility to cerebrovascular events, as reported in an autopsy series in which widespread microthrombi and patches of infarction were observed some brains (bryce et al., 2020) . patients with covid-19 may be at risk of cardioembolic stroke. acute cardiac injury and clinically significant arrhythmias have been reported in approximately 10% of hospitalized covid-19 patients and 20-40% of those requiring intensive care huang et al., 2020a; wang et al., 2020a) . sars-cov-2 infection may rarely cause myocarditis and heart failure even in the absence of significant pulmonary involvement (inciardi et al., 2020) . myocardial injury and arrhythmias, such as atrial fibrillation, in the setting of severe infection may result in cardiac embolism and brain infarction (inciardi et al., 2020) . a substantial proportion of critically ill patients with covid-19 may also develop secondary bacteremia in addition to the primary viral illness. in one case series, approximately 10% of patients requiring mechanical ventilation had bacteremia , which increases the risk of stroke by over 20 folds (dalager-pedersen et al., 2014) . septic emboli to the brain often result in bleeding and in a postmortem magnetic resonance imaging study10% of brains had evidence of hemorrhage (coolen et al., 2020) .taken together, these clinical findings suggest that sars-cov-2 may adversely affect the brain via multiple pathophysiological pathways that culminate in vascular brain injury. post-infectious neurological complications: sars-cov-2 unleashes a dysregulated systemic immune response (see systemic inflammation and immune dysregulation), which can have delayed effects on the nervous system. these immune-mediated manifestations involve both the central and peripheral nervous system and occur typically after the acute phase of the infection subsides. in the cns, reported cases in covid-19 resemble classic post-infectious inflammatory conditions such as acute disseminated encephalomyelitis (parsons et al., 2020) and acute necrotizing hemorrhagic encephalopathy (poyiadji et al., 2020) . peripherally, several cases of guillain-barre syndrome, a neuropathy caused by an immune attack on peripheral nerves, have been reported in patients with recent covid-19 (toscano et al., 2020) . most reported cases describe classic features of this syndrome, such as generalized weakness, evidence of demyelination on nerve conduction studies and elevated proteins without white blood cells in csf (toscano et al., 2020) . the miller-fisher variant of guillain-barre syndrome, characterized by cranial nerve involvement, has also been reported, including at least one case with detectable anti-ganglioside antibodies suggesting an immune attack on the peripheral nerves (gutierrez-ortiz et al., 2020) . sars-cov-2 was not detected in any of the csf samples (toscano et al., 2020) , supporting an immune mechanism rather than direct infection. intensive care related neurological manifestations: the relatively high frequency of altered mental status in hospitalized covid-19 patients is congruent with the severity of their illness. most critically ill covid-19 patients require mechanical ventilation (cummings et al., 2020) and an agitated confusional state (delirium) occurs in more than 80% of mechanically ventilated patients in intensive care units (ely et al., 2001) . patients with ards, which frequently complicates severe covid-19, are at particularly high risk of delirium, likely because of hypoxemia heavy doses of sedatives, administration of paralytic agents or other causes (hopkins et al., 2005; ouimet et al., 2007) . comparison with other viral respiratory infections: many neurological abnormalities seen in covid-19 mirror those of other viral respiratory illnesses. all of the reported covid-19 related post-infectious inflammatory conditions of the nervous system, such as guillain-barre syndrome, acute necrotizing hemorrhagic encephalopathy and acute disseminated encephalomyelitis, are classically seen after infections, including other coronaviruses (gerges harb et al., 2020) . influenza is occasionally associated with an encephalopathy or full blown encephalitis, with evidence of influenza virus in the cerebrospinal fluid (surtees and desousa, 2006) . comparing the large numbers of patients infected by sars-cov-2 worldwide and the relative paucity of reported encephalitis cases, sars-cov-2 seems more similar to other common respiratory viral pathogens like influenza than to neurotropic pathogens that target specifically the brain, such as the herpes simplex virus. in general, however, covid-19 is more debilitating than other common viral respiratory illnesses. physicians have been struck by the frequency of thrombotic complications observed in critically ill covid-19 patients, to the point that some hospitals instituted protocols for empiric, high-dose anticoagulation in patients with elevated d-dimer levels (paranjpe et al., 2020) . emerging data seem to confirm this observation: in one multicenter study, patients with covid-19 and acute respiratory distress syndrome had twice the incidence of thrombotic complications compared to a matched cohort with ards from other causes (helms et al., 2020b) . this also applies to thrombotic complications affecting the brain, since the proportion of covid-19 related hospitalizations complicated by stroke seems much higher than that seen in influenza . based on neuroinflammation-associated abnormalities in the clotting cascade in brain (han et al., 2008) , activated protein c or thrombin inhibitors could also be of therapeutic value. the findings reviewed above indicate that neurological manifestations are common in covid-19 and constitute a defining aspect of the symptomatology of the disease. a caveat is that most clinical data is derived from case series on patients ill enough to require hospitalization at tertiary care centers, providing a biased representation of the frequency and type of the neurological manifestations. similarly, basic science investigations exploring the mechanism of disease have largely emphasized concepts and findings that emerged from other coronaviruses, and there is limited new data on the interaction of sars-cov-2 with the brain and its vasculature. therefore, conclusions based of existing literature have to be considered preliminary and subject to further scrutiny, verification and validation. here are some of the outstanding questions: do the neurological manifestations of covid-19 reflect brain invasion? the encephalopathy is most likely a consequence of systemic factors, such as cytokine sickness, hypoxia and metabolic dysfunction due to peripheral organ failure, while the strokes seem to be related more to hypercoagulability and endothelial injury than to sars-cov-2 vasculitis affecting brain vessels. the loss of taste and smell has been attributed to invasion of the olfactory neural system, but consistent evidence is lacking. in some cases, the possibility of a sars-cov-2 encephalitis could not be ruled out based on the potential for the virus to infect neurons (song et al., 2020) , but definitive clinical and pathological evidence of neurotropism is lacking. a major problem is that the molecular mechanisms of cellular entry for sars-cov-2 are not entirely clear. while ace2 is thought to be the main receptors in some cell types, its expression levels do not seem to correlate with the infectivity potential. for example, the virus gains access to human pluripotent stem cell-derived dopaminergic neurons despite low levels of ace2 . systematic investigation of non-canonical docking and accessory proteins for sars-cov-2 (figures 1, s1 ), their cellular localization and function in human neurons, glia and vascular cells would help address this question. does the brain contribute to the immune dysregulation? sars-cov-2 and inflammatory mediators may gain access to the median eminence and activate hypothalamic neurohumoral pathways that mediate immune dysregulation through the adrenergic system, as described in other brain diseases (figure 3 ). considering the importance of the immune dysregulation in covid-19 severity and outcome, a better understanding of the contribution of the hypothalamus may suggest pharmacological approaches to dampen the immune dysregulation . does the brain contribute to respiratory failure and hypertension? similarly, entry of the virus and/or proinflammatory molecules through the subfornical organ and the area postrema could also affect brainstem autonomic pathways controlling blood pressure and breathing (kaur and ling, 2017) . alterations in blood pressure, both hypertension and severe hypotension in critically patients , are highly prevalent in covid-19. furthermore, it has been suggested that involvement of brainstem respiratory nuclei may contribute to the respiratory failure , but no alterations in respiratory centers or chemoreceptors (carotid bodies) was observed at autopsy in a patient with respiratory dysregulation (kantonen et al., 2020) . to date, evidence of central autonomic involvement is lacking. what are the long-term neurological and neuropsychiatric consequences of covid-19? respiratory virus infections are associated with neurological and psychiatric sequelae, including parkinsonism, dementia, depression, post-traumatic stress disorder and anxiety (limphaibool et al., 2019; rogers et al., 2020) . brain infection is not required for these long-term effects. inflammation and cytokine elevation in sepsis survivors are linked to subsequent hippocampal atrophy and cognitive impairment (iwashyna et al., 2010) . experimental studies suggest a link between activation of the nlrp3 inflammasome, which may occur in covid-19, and alzheimer j o u r n a l p r e -p r o o f pathology (ising et al., 2019) . ards survivors also exhibit increased incidence of long-term depression, anxiety and cognitive impairment (hopkins et al., 2005) . whether these late manifestations are related to non-resolving inflammation or a low-grade immune process driven by molecular mimicry or dysregulated adaptive immunity remains to be established. chronic damage to systemic organs can also harm the brain through chronic hypoxia, metabolic dysfunction and hormonal dysregulation. based on these considerations, significant long-term neurological and psychiatric sequelae have to be anticipated in covid-19, especially in survivors of severe disease. experimental models: models would help address these outstanding questions and facilitate therapeutic development. unfortunately, mice, the most popular laboratory animals, are not susceptible to sars-cov-2 due to differences between mouse and human ace2 (lakdawala and menachery, 2020) . mice expressing human ace2 have been developed and show evidence of brain infection, but only minimal symptoms of disease (song et al., 2020) . hamsters, ferrets, cats and non-human primates could be more viable models (lakdawala and menachery, 2020) . reproducing the systemic effects of the disease would be critical for studying the neurological aspects of covid-19. in vitro approaches involving human pluripotent stem cells organoids and co-cultures are useful to examine infectious mechanisms in brain cells (song et al., 2020; yang et al., 2020) , but do not provide insight into the harmful systemic effects. therefore, there is a pressing need to develop animal models that are amenable to investigate not only the effects of sars-cov-2 on brain cells, but also the systemic effects of the infection and the long-term neuropsychiatric consequences. therapeutic considerations: until safe and effective vaccines are developed, therapeutic efforts have to focus on antiviral agents and on how to best manage respiratory insufficiency, organ failure, hypercoagulable state and immune dysregulation. there is no specific treatment for the neurological manifestations, which are managed according to standard protocols. however, since the neurological complications emerge mainly in severe systemic disease, minimizing hypoxia and protecting the brain from cytokines, damps, pamps and thromboembolic complications are important therapeutic goals. immunosuppression with steroids improves mortality in patients with severe disease, but not in those with milder forms (hayden, 2020) . furthermore, more nuanced approaches to counteract the immune dysregulation, such as targeting specific cytokines or inflammatory pathways are also being tested (vabret et al., 2020) . whether these interventions reduce the short-and long-term neurological and psychiatric complications remain to be established. in conclusion, the neurological manifestations of covid-19 constitute a major public health challenge not only for the acute effects on the brain, but also for the long-term harm to brain health that may ensue. these delayed manifestations are anticipated to be significant since they are likely to also affect patients who did not show neurological symptoms in the acute phase. therefore, clinical and laboratory efforts aiming to elucidate the mechanisms of the acute effects on the brain of sars-cov-2 need to be coupled with investigations on the deleterious delayed neuropsychiatric sequelae of the infection. these efforts should be driven by a close cooperation between clinical and basic scientists and take advantage of the wealth of clinicalepidemiological data and biological specimens that are accumulating worldwide. considering that covid-19 is still raging in many countries, including the us, and that there might be a seasonal resurgence of infection, it is imperative that a such a concerted effort is implemented swiftly and on a large scale. dr. iadecola serves on the scientific advisory board of broadview ventures. dr. kamel serves as co-pi for the nih-funded arcadia trial (ninds u01ns095869) which receives in-kind study drug from the bms-pfizer alliance for eliquis® and ancillary study support from roche diagnostics, serves as deputy editor for jama neurology, serves as a steering committee member of medtronic's stroke af trial (uncompensated), serves on an endpoint adjudication committee for a trial of empagliflozin for boehringer-ingelheim, and has served on an advisory board for roivant sciences related to factor xi inhibition. dr. anrather has no conflict of interests to declare. j o u r n a l p r e -p r o o f in a single nuclear rna-seq profile of human cortical brain tissue (https://celltypes.brain-map.org) (hodge et al., 2019) there was no evidence of ace2 expression in any brain cell type. basigin (bsg) was prominently expressed in pericytes and endothelial cells, while neuropilin-1 (nrp1) was detected in endothelial cells and in several classes of excitatory neurons. low expression of tmprss11a and furin was found in neurons, while cstb was moderately expressed in most cell types with the exception of astrocytes and oligodendrocytes and their precursors. endothelial cells and pericytes also express lymphocyte antigen 6 family member e (ly6e), and the interferon (ifn)-induced transmembrane proteins-1 and 3 (ifitm1, ifitm3), that have been shown to restrict sars-cov-2 cell entry (hachim et al., 2020; pfaender et al., 2020; zhao et al., 2020) . ifn type i receptors (ifnra1 and ifnra2) showed higher expression in endothelial cells than other cell types. cell cluster annotations are from (hodge et al., 2019) . cpm, transcript counts per million within the cell cluster; fraccellexpr, fraction of cells in which the transcript is detected. circulating virus, cytokines, damps and pamps could act on endothelial cells leading to inflammation and opening of the bbb. once in the perivascular space and these factors could induce inflammation in vascular mural cells and brain resident myeloid cells (microglia and macrophages). the resulting cytokine production could affect neuron neuronal function leading to the cytokine sickness, a potential cause of encephalopathy in covid-19. cytokine and sars-cov-2 entry into the median eminence of the hypothalamus could lead to activation of the autonomic nervous system and release of adrenal catecholamines and steroids. in analogy with stroke, brain trauma and myocardial infarction , these neurohumoral effectors could act on the bone marrow to release of immunosuppressor neutrophils and myeloid cells (emergency myelopoiesis), as described in covid-19 (schulte-schrepping et al., 2020) , leading to immunosuppression and lymphopenia. in addition, release of 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priorities in encephalitis: consensus statement of the international encephalitis consortium clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in sars-cov-2 invades host cells via a novel route: cd147-spike protein detection of sars-cov-2 in different types of clinical specimens can covid damage the brain? pathological findings of covid-19 associated with acute respiratory distress syndrome sars-cov-2 and stroke in a new york healthcare system a human pluripotent stem cell-based platform to study sars-cov-2 tropism and model virus infection in human cells and organoids encephalitis as a clinical manifestation of covid-19 ly6e restricts the entry of human coronaviruses, including the currently pandemic sars-cov-2 viral load dynamics and disease severity in patients infected with sars-cov-2 in zhejiang province heightened innate immune responses in the respiratory tract of covid-19 patients the authors are supported by nih grants r01-ns34179, r01-ns100447, r37-ns089323, r01-ns095441, r01-ns/hl37853 (ci), r01ns097443 (hk), ns094507, ns081179 (ja), key: cord-329190-kv9n2qj3 authors: rabaan, ali a.; alahmed, shamsah h.; bazzi, ali m.; alhani, hatem m. title: a review of candidate therapies for middle east respiratory syndrome from a molecular perspective date: 2017-09-01 journal: journal of medical microbiology doi: 10.1099/jmm.0.000565 sha: doc_id: 329190 cord_uid: kv9n2qj3 there have been 2040 laboratory-confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) in 27 countries, with a mortality rate of 34.9 %. there is no specific therapy. the current therapies have mainly been adapted from severe acute respiratory syndrome (sars-cov) treatments, including broad-spectrum antibiotics, corticosteroids, interferons, ribavirin, lopinavir–ritonavir or mycophenolate mofetil, and have not been subject to well-organized clinical trials. the development of specific therapies and vaccines is therefore urgently required. we examine existing and potential therapies and vaccines from a molecular perspective. these include viral s protein targeting; inhibitors of host proteases, including tmprss2, cathepsin l and furin protease, and of viral m(pro) and the pl(pro) proteases; convalescent plasma; and vaccine candidates. the medline database was searched using combinations and variations of terms, including ‘middle east respiratory syndrome coronavirus’, ‘mers-cov’, ‘sars’, ‘therapy’, ‘molecular’, ‘vaccine’, ‘prophylactic’, ‘s protein’, ‘dpp4’, ‘heptad repeat’, ‘protease’, ‘inhibitor’, ‘anti-viral’, ‘broad-spectrum’, ‘interferon’, ‘convalescent plasma’, ‘lopinavir ritonavir’, ‘antibodies’, ‘antiviral peptides’ and ‘live attenuated viruses’. there are many options for the development of mers-cov-specific therapies. currently, mers-cov is not considered to have pandemic potential. however, the high mortality rate and potential for mutations that could increase transmissibility give urgency to the search for direct, effective therapies. well-designed and controlled clinical trials are needed, both for existing therapies and for prospective direct therapies. middle east respiratory syndrome coronavirus overview middle east respiratory syndrome coronavirus (mers-cov) was first isolated in jeddah in the kingdom of saudi arabia (ksa) from a 60-year-old male hospital patient, who died 24 june 2012, 11 days after presenting with acute pneumonia and subsequent renal failure [1] . since then, the who have been notified of 2040 laboratory-confirmed cases, including 712 deaths [2] . while most cases have arisen in the middle east, cases have also emerged in 27 countries worldwide in travellers from the middle east and/ or in their contacts [2] . most human mers-cov infections are considered to be the result of multiple zoonotic transfers. bats are the most likely mers-cov natural reservoir, as with other mammalian coronaviruses (covs), while camels are likely to be the major zoonotic source for human infections [3] [4] [5] . secondary human-to-human transmission is considered to be limited, occurring mainly within family and healthcare settings. the first cluster of cases in humans was retrospectively identified to have occurred in a public hospital in jordan in april 2012 [6] . multiple healthcare facility-associated outbreaks have since occurred in the middle east, most notably in ksa, often linked to deficiencies in infection control procedures [7] [8] [9] [10] [11] [12] [13] . although cases outside the middle east have mainly been isolated, a large outbreak occurred in korea in june 2015, in which human-human transmission resulted in 186 cases and 36 deaths [14] . increased vulnerability to either cross-species or trans-human transmission could result from viral adaptations [15] . mers-cov infection is often accompanied by acute viral pneumonia, and sometimes gastrointestinal symptoms. clinical severity varies from asymptomatic to death, and the extent of asymptomatic spread is unclear. the high mortality rate is mainly accounted for by acute respiratory distress syndrome (ards) [7, 15, 16] . higher mortality is observed among vulnerable patients, such as older individuals and those suffering from comorbid illness, and is also associated with high viral load [7, 11, 15, 17] . in one study in a ksa hospital, intensive care unit (icu) admission among mers-cov patients was associated with a mortality rate of 74.2 % [11] . while mers-cov is not currently considered to have pandemic potential, it is clear that human-human transmission does occur. the exact mechanisms by which mers-cov is transmitted from animals to humans have not been fully elucidated. in the south korean outbreak, the virus emerged in second-and third-generation contacts, resulting in the first human case to be imported into china [18] . this raised concern that viral mutations were contributing to humanhuman transmission. given its high mortality and poor outcomes for vulnerable patients, and the potential for viral mutations, there is no room for complacency in the search for therapeutic options for mers-cov. there is currently no specific therapy. many of the therapeutic options used have been adapted from approaches used to treat severe acute respiratory syndrome (sars-cov) during the outbreak of 2003, and/or the h1n1 influenza virus during the outbreak of 2009 [19] . however, while mers-cov and sars-cov are phylogenetically related betacoronaviruses, they differ in many important respects. mers-cov utilizes human dipeptidyl peptidase 4 (dpp4; cd26) receptors, with binding mediated by the viral spike (s) protein, not the angiotensin-converting enzyme 2 (ace-2) receptors used by sars [20] [21] [22] [23] [24] . mers-cov also has a wider cellular tropism [24] [25] [26] . therapies currently used include broad-spectrum antibiotics, corticosteroids and anti-viral treatments, such as interferons (ifn), ribavirin, lopinavir-ritonavir, or mycophenolate mofetil [19, 22, [27] [28] [29] [30] [31] . however, the efficacy and/or safety of many of these therapies is unclear, and none are specific to mers-cov. ribavirin monotherapy, for example, is associated with multiple side-effects in the treatment of other viral illnesses, including sars-cov, has uncertain efficacy, and has not been tested in animal studies or randomized control trials for mers-cov [22, 31, 32] . corticosteroids have not been successful in the treatment of respiratory distress or lung fibrosis in mers-cov [31, 32] . meanwhile, studies in sars-cov and h1n1 patients suggest that corticosteroid use may in fact increase viral replication in airways, and sars patient and animal studies indicate that it contributes to immunosuppression [33] [34] [35] . mycophenolate mofetil has been associated with fatal disease and high viral loads in a marmoset model of mers-cov infection [36] . ifn therapy, alone or in combination with ribavirin or lopinavir-ritonavir, has shown greater promise in in vitro, animal and human studies [37] [38] [39] [40] . however, clinical studies on ifns vary with respect to factors such as time of administration and type of patient [19, 22, 40] . overall, there is a lack of randomized control trials (rcts) designed to test the safety and efficacy of any potential therapies specific to mers-cov, and much of the information available for existing therapies is based on in vitro and/or animal studies [22, 40, 41] . a position paper on the evidence base for specific mers-cov therapies, published by public health england (phe) and the world health organization-international severe acute respiratory and emerging infection consortium (isaric-who), suggested that benefit was likely to exceed risk for convalescent plasma, lopinavir-ritonavir, ifns and monoclonal/polyclonal antibodies, while, by contrast, for ribavirin monotherapy and corticosteroids it was considered that the risks would outweigh the benefits [42] . for interferon/ribavirin combination therapy, nitazoxanide and chloroquine, the available data were considered to be inadequate for assessment [42] . in this review, we consider potentially effective mers-cov therapies, including ifns, lopinavir-ritonavir and inhibitors of proteases, including tmprss2 and cathepsin l, as well as mers-cov-specific potential therapies, including convalescent plasma, monoclonal antibodies (mabs), antiviral peptides and candidate vaccines. these therapies will be considered from a molecular perspective, in the context of the infection and replication mechanisms of mers-cov. the therapies are summarized in table 1 . mers-cov lineage and structure mers-cov is a betacoronavirus belonging to clade c (lineage 3) of the betacoronaviruses [43] . its closest known coronavirus relatives are the prototypic clade c betacoronaviruses, tylonycteris bat virus hku4, pipistrellus bat hku5 virus and neoromicia zuluensis bat pml/2011 (neocov) virus [1, [43] [44] [45] [46] [47] . in common with other coronaviruses, the genome of mers-cov is a single, positive-stranded rna of over 30 000 nucleotides. it encodes 10 predicted open reading frames (orfs) and genes for 4 structural proteins, namely the spike (s), nucleocapsid (n), membrane (m) and envelope (e) proteins (figs 1 and 2) [48] [49] [50] . orf 1a and 1b encode virus replication-related proteins (pp1a, pp1ab), which are cleaved to give 16 non-structural proteins (nsps) involved in synthesis of viral rna and recombination ( fig. 2) [48] [49] [50] . these include nsp-14, which contains a 39-to-59 exoribonuclease (exon) domain that is important in viral proofreading and in determining the sensitivity of rna viruses to mutagens. thus small-molecule inhibitors references s1/dpp4 binding antibody (mouse): s1 rbd in vitro [76] antibody (human): s1 rbd m336, m337, m338 in vitro/in vivo (mouse, rabbit -m336) [77] [78] [79] antibody (human): s1 rbd in vitro [80, 81] antibody (mouse-humanized): s1 rbd in vitro/in vivo (mouse) prophylactic and therapeutic [82] antibody (mouse-humanized): s1 rbd hms-1 in vitro/in vivo (mouse) [83] antibody (human): s1 rbd in vitro/in vivo (mouse) potential for more mers-specific agents [61] of exon activity could be candidates for mers-cov and other coronavirus therapies [51] . as with other coronaviruses, the mers-cov s protein is critical to host cell receptor binding and cell entry, and is considered to have been under strong positive selection pressure when the virus was transmitted to humans [52, 53] . hence the s protein is a major target for potential anti-mers-cov therapies [53] . the s protein of mers-cov is composed of s1 and s2 subunits ( fig. 2 ) [53] . in common with other coronaviruses, entry into host cells depends on the s1 subunit, which contains a receptor-binding domain (rbd) comprising a core subdomain and a receptor-binding motif (rbm). the mers-cov rbm differs from that of sars-cov and dictates that mers-cov uses the dpp4 receptor, as opposed to the ace-2 receptor [20, 21] . the infection process is shown in fig. 2 . dpp4, which is widely expressed in tissues, including the lung and kidneys, is critical in the species tropism of mers-cov infection; bat, human, camel, non-human primate and swine cells, for example, are permissive for mers-cov infection, whereas mouse, hamster and ferret are not [54, 55] . host species restriction has been attributed to differences in five amino acids involved in dpp4-rbd binding, with glycosylation of the mouse dpp4 also identified as being important in the inhibition of mers-cov infection [54] [55] [56] . the human dpp4 receptor is therefore a potential target for mers-covspecific therapeutics, in particular anti-dpp4 mabs (fig. 2 , table 1 ) [53] [54] [55] [56] . adenosine deaminase (ada), which is a dpp4-binding protein, competes with mers-cov for dpp4 binding and hence is a natural mers-cov antagonist; this gives potential insights for the development of therapeutic antagonists [55] . mers-cov binds to the permissive host cell dpp4 via the rbd of the s1 domain, one of the major targets for potential mers-cov therapies [53] . similarly to other coronaviruses, mers-cov then uses the s2 subunit for virus-host membrane fusion (fig. 2) . fusion results in cleavage of the s protein at the s1/s2 boundary by host proteases [57] . the s2 subunit contains the fusion peptide, two heptad repeat domains termed hr1 and hr2, and a transmembrane (tm) domain ( fig. 2 ) [57] . membrane fusion requires conformational rearrangement of s2, the formation of a sixhelix bundle (6hb) fusion core, of which hr1 and hr2 are essential elements, and exposure of the fusion peptide, which inserts itself into the host cell membrane [52, 57, 58] . hr2-derived peptides have been identified as potentially effective anti-viral agents for treatment of mers-cov [52] ( fig. 2 ; table 1 ). the availability of host cell proteases is essential for mers-cov entry into cells [23, 53] . the host proteases responsible for s protein cleavage at the s1/s2 boundary include the serine protease tmprss2, endosomal cathepsins such as cathepsin l, and furin protease [23, 53, [59] [60] [61] . in vitro studies suggest that uncleaved mers pseudovirus can enter host cells by cathepsin l-dependent endocytosis, but that cleavage of virus during maturation by host proteases such as tmprss2 results in viral entry at neutral ph and the formation of massive syncytia [58] . host cell proteases are therefore potential molecular therapeutic targets for mers-cov prophylaxis and/or treatment (fig. 2 , table 1 ). the tmprss2 inhibitor camostat, for example, has been identified as a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [59] . following host cell entry, mers-cov pp1a and pp1ab are synthesized and then cleaved by two viral proteases, the main protease (mpro/3clpro) and the papain-like protease (plpro) (fig. 2) [57, 63] . thus viral proteases represent further potential molecular targets for therapy ( table 1) . the recently described mers-cov mpro crystal structure resembles other coronavirus mpro proteases [60] . the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg), can inhibit mers cov protease activity in vitro, as can the immunosuppressant drug mycophenolic acid [61] . however, caution is required, as the results of studies on marmosets have associated use of mycophenolate mofetil with fatal disease and high viral loads [36] . other mers-cov proteins involved in helping the virus to circumvent the immune system also present potential molecular targets (fig. 2) . for example, the accessory protein products of orf 4a, 4b and 5 are interferon (ifn) antagonists [62, 63] . the orf 4a protein both inhibits type i ifn production via cytoplasmic and nuclear mechanisms, and interferes with the ifn-mediated interferon-stimulated response element (isre) promoter element signalling pathways [62, 63] . this gives a molecular level rationale for the use of ifn as a therapeutic option in mers-cov treatment. mers-cov can also infect dendritic cells and macrophages [26, 64, 65] . endosomal uptake of mers-cov by dendritic cells following binding via dpp4 prompts these cells to produce abundant amounts of type i and iii ifns [65] . this gives context to the ifn antagonism exhibited by mers-cov accessory proteins. recently, mers-cov has also been shown to infect t cells, which are rich in dpp4, both in vitro and in marmoset spleen [66] . this results in t cell apoptosis and could contribute significantly to viral pathogenesis and further emphasizes the potential therapeutic utility of molecular targeting of dpp4 and/or the mers-cov s protein. both convalescent plasma containing virus-specific antibodies and the use of specific mabs provide options for targeting mers-cov infection at a molecular level. the use of convalescent plasma [or hyperimmune iv immunoglobulin (hvig) from the plasma of convalescent donors] for infectious disease treatments has a long history, including in the treatment of respiratory diseases [67] [68] [69] [70] . for influenza and sars-cov infection, early convalescent plasma treatment within 4-5 days of symptoms is associated with decreased viral load and reduction in mortality [67] [68] [69] [70] . however, for sars-cov the quality of studies has been inconsistent and the results have been inconclusive, with a lack of adequate clinical trials [69, 70] . according to the phe and isaric-who position paper, convalescent plasma (or high neutralizing antibody titre products) is indicated for the treatment of serious mers-cov infection [42] . one rct on 35 critically ill patients with h1n1 infection identified a significant reduction in viral load and mortality in patients who received hvig within 5 days of the onset of symptoms [68] . to date, no rcts have been completed on the use of convalescent plasma/hvig in mers-cov patients. in the light of results from sars and influenza patients, an ongoing clinical trial on the safety and efficacy of convalescent plasma treatment for critically ill mers-cov patients was initiated in may 2014 and is due to report in june 2017 [75; clinicaltrials.gov identifier: nct02190799]. this trial is being carried out in ksa. however, as is common for convalescent plasma therapies, the trial has been affected by logistical and technical issues, including the availability of sufficient donors [71, 72] . issues can also arise in the collection of convalescent plasma that has sufficient levels of mers-cov antibodies, particularly outside the middle east [22, 72] . while there are two case reports in which intravenous immunoglobulin (ivig) was used in treatment of mers-cov, it is uncertain as to whether this contributed to patient recovery [73, 74] . thus, while convalescent plasma is a promising potential therapy for mers-cov, the available clinical evidence is very limited and the results of the ongoing clinical trial will be vital in guiding any future use [71] . focused development of neutralizing monoclonal antibodies targeted against specific mers-cov proteins has meanwhile yielded promising in vitro and/or in vivo results. monoclonal antibodies: s1-dpp4 binding a number of mouse and human neutralizing mabs against the s1 region of mers-cov have been developed and tested in vitro and/or in animal models [52] . targeting of s protein for therapeutic purposes was recently comprehensively reviewed by du et al. [53] [52] . in particular, the s1 rbd is a popular target, as mabs directed against this region have the most potent neutralizing capacity. however, in terms of vaccine development, neutralizing antibodies raised by immunization with full-length s or s1 protein expression vectors may produce a more effective immunogenicity through the targeting of multiple epitopes and the reduction of the possibility of escape mutations [75] . nevertheless, many mouse and human mabs targeting the s1 rbd have given promising results in vitro and in mouse models [19] . (table 1) . a mouse monoclonal antibody, mersmab1, blocks mers pseudovirus cell entry in vitro by binding to the rbd and preventing s1 binding to dpp4 [76] . meanwhile, the human monoclonal antibodies m336, m337 and m338, which target overlapping epitopes in the rbd, all potently neutralize pseudovirus and live mers-cov in vitro [77] . significantly, intraperitoneal injection of m336 has also been shown to have both prophylactic and therapeutic protective effects against mers-cov infection in a wellestablished human dpp4 (hdpp4)-expressing transgenic mouse model, and in rabbits [78, 79] . other anti-rbd human antibodies, mers-4 and mers-27, which recognize distinct rbd regions and block binding to dpp4, likewise have potent in vitro neutralizing activity against pseudovirus and live virus infection, and they also act synergistically [80, 81] . mers-4 has anti-syncytia formation activity [80] . the crystal structure of mers-27 bound to the dpp4 receptor revealed two critical rbd residues [81] . the crystal structure of another anti-rbd antibody 4c2, which was raised in mice, has also been elucidated. this has allowed the identification of an epitope that partially overlaps the rbd receptor binding unit [82] . 4c2 was consequently humanized to give an antibody with prophylactic and therapeutic properties, shown by a reduction of mers-cov lung viral titres in an ad5-hcd26 (hdpp4) transgenic mouse model [82] . another humanized anti-rbd antibody, hms-1, similarly has potent in vivo protective properties against fatal mers-cov infection in a transgenic hdpp4 mouse model [83] . the human antibody lca60 targets both the n-terminal domain (ntd) and the rbd of the s1 region, and was isolated from b cells of a mers-cov-infected human donor before being used to rapidly establish a stable cho cell line that can be used to reliably produce clinical grade antibody [84] . this is a promising candidate for clinical development, given the antibody's potent prophylactic and therapeutic activities against mers-cov infection in ad5/hdpp4 transgenic mice and type i interferon receptor (ifnar)-ko mice [84] . the human anti-rbd antibody 3b11-n is another promising candidate that prophylactically reduces lung pathology in rhesus monkeys infected with mers-cov [85] . targeting the s1-dpp4 interaction from the host side through the development of anti-dpp4 (cd26) antibodies is another possible therapeutic option. the anti-cd26 antibodies 2f9, 1f7 and ys110 target the mers-cov entry into cells in vitro [86] . the 2f9 epitope maps close to the binding site of ada, a natural dpp4 binding protein and mers-cov antagonist, while the 1f7 and ys110 epitopes lie outside this region [55, 86] . thus, targeting of the s1-dpp4 interaction by use of mabs is a promising strategy for the clinical development of molecular therapeutics against mers-cov. another molecular approach involves targeting of the s2-mediated mers-cov-host cell fusion element of the mers-cov infection cycle by use of antiviral peptides. the role of hr1 and hr2 in viral fusion makes them potentially effective molecular therapeutic targets. this has been borne out by in vitro and in vivo results obtained using hr2 peptides (table 1 ). hr1 peptides are ineffective antivirals as they aggregate in physiological solutions [87] [88] [89] . a peptide named hr2p, which spans residues 1251-1286 of hr2, effectively inhibits viral replication and s proteinmediated cell fusion in vitro [87] . a hr2p analogue named hr2p-m2 is an even more potent fusion blocker in vitro, and inhibits mers cov-expressing pseudovirus infection [90] . hr2p-m2 interacts with an hr1 peptide to effectively block 6hb bundle formation. in vivo hr2p-m2 intranasal administration to ad5/hdpp4 transgenic mice protected them from mers-cov infection, as evidenced by the reduction of the lung viral titres by more than 1000-fold [90] . the addition of ifn-b along with hr2p-m2 enhanced the protective effect [90] . thus, s2 hr2 peptides have potential as mers-cov intranasal antiviral treatments. the s protein is also the focus of a number of candidate vaccines (table 1 ) [75, [91] [92] [93] . a fusion product combining truncated rbd and the fc portion of human igg can bind human dpp4 and inhibit mers-cov infection in an in vitro cell culture model [91] . importantly, this rbd-igg fusion product can induce a humoral response in mice vaccinated by subcutaneous injection, hence blocking rbd-dpp4 binding and inhibiting mers-cov infection [91] . further in vivo studies have indicated that intranasal administration to mice induces similar long-term igg humoral responses to those achieved with subcutaneous injection, but superior cellular immune responses and local mucosal responses in lungs [92, 93] . this suggests that this type of construct is both potentially effective and readily deliverable by intranasal means. use of an adjuvant, particularly mf59, significantly improves the humoral and t cell immunogenicity of the rbd s377-588-fc igg fusion construct in subcutaneously immunized mice [94] . the possibility of using the s1 rbd as a vaccine molecular target for a range of divergent mers-cov strains and escape mutants has also been explored recently [95] . the use of five recombinant rbds with mutations observed in different mers-cov outbreaks or in camel strains induced potent neutralizing antibody responses against several mers-cov pseudoviruses [95] . however, while the rbd of the s1 subunit is a logical and promising target for mers-cov vaccine development, the epitope scope is relatively limited and full-length s protein may be a preferable option [75] . technical difficulties in stably expressing abundant quantities of full-length s protein have presented a barrier. however, studies on delivery options, including the use of adjuvants and nanoparticles, may help in overcoming such issues. one study undertaken by novavax (gaithersburg, maryland, usa) showed that the inoculation of mice by intramuscular injection with fulllength s protein proprietary nanoparticles produced a relatively low neutralizing antibody response after 21 days [96] . however, the addition of the adjuvants alum or matrix m1 resulted in a robust and sustained anti-mers-cov neutralizing antibody response [96] . [98] . these viruses are expected to enter clinical trials as a proposed prophylactic mers-cov vaccine. likewise, intramuscular injection of ad5 or ad41 expressing full-length s protein induces both antigen-specific t cell and neutralizing antibody responses in mice [99] . finally, intraperitoneal injection of measles virus expressing either membraneanchored, full-length s protein or soluble s protein lacking the tm domain induces robust mers-cov antigen-specific neutralizing antibody and cytotoxic t lymphocyte in interferon-a/b receptor (ifnar)-deficient mice [100] . recently, an mva-based vaccine expressing s protein has been shown to induce mucosal immunity in mers-cov-infected dromedary camels, with a reduction in excreted virus and viral transcripts [101] . this has potential for veterinary use and the reduction of cross-species infection of humans by camels [101] . dna plasmids gls-5300 is a dna-plasmid vaccine that encodes mers-cov s protein (table 1 ) [102, 103] . it was co-developed by inovio, geneone life science, inc. and the walter reed army institute of research, and has become the first potential mers-cov vaccine to enter human testing [102, 103] . a phase i clinical trial in healthy volunteers commenced in 2016 for the evaluation of its safety and ability to generate antibody and cellular immune responses over a 1-year period, using one of three dosages in a three-injection regimen [102] . the vaccine has already undergone pre-clinical trials in mice, camels and macaques [103] . it induced robust and antigen-specific cytotoxic t lymphocyte and neutralizing antibody responses, which effectively protected animals against viral infection [103] . gls-5300 and other potential vaccine candidates provide an opportunity to develop a prophylactic mers-cov vaccine. however, the barriers to development of a prophylactic vaccine include the current relatively low mers-cov incidence in humans, as well as sourcing suitable small animal models [75, 97, 104] . these factors complicate the definition of a target population for mass prophylactic vaccination and pre-clinical demonstration of vaccine efficacy [104] . in this context, the monoclonal antibodies described above may be invaluable resources in an outbreak situation [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] . in vitro and animal studies while mers-cov-specific therapies are offering promising pre-clinical results, and gls-5300 has entered clinical trials, there is as yet no specific evidence-based therapy or vaccine clinically available for mers-cov. as described in the mers-cov infection and replication section, mers-cov accessory protein products are ifn antagonists [62, 63] . attenuation of the ifn response is an important mers-cov immune response circumvention mechanism [105] . the orf4a in particular inhibits ifn-b production via the inhibition of interferon regulatory transcription factor (irf)-3 and nuclear factor (nf)-kb actions, and thus irf-3-activating small molecules, for example, may be potential therapeutic agents for restoring ifn responses [62, 63] . toll-like receptor-3 (tlr-3) is also involved in the immune response of mice to sars-cov and mers-cov, recognizing viral molecular patterns and initiating the innate response that leads to ifn production (fig. 2 ) [106] . thus, tlr-3 agonists are another possible candidate for mers-cov-specific anti-viral agents [106] . therapeutically, ifn itself is particularly useful prophylactically or during the early days of viral exposure, including for coronaviruses [105, 107] . in vitro and animal studies have confirmed the potential efficacy of ifns in mers-cov therapy, in particular in combination with other therapeutic agents such as ribavirin and/or lopinavir. in vitro, mers-cov was substantially more susceptible to ifn-a than sars-cov [107] . while mers-cov in vero or llc-mk2 cells was sensitive to both ifn-a2b and ribavirin separately, relatively high concentrations were required to reduce viral replication [108] . however, combination therapy allowed the concentrations of each to be substantially reduced [108] . combination therapy of ifn-a2b and ribavirin in macaques administered 8 hours after mers-cov infection reduced systemic and local viral effects, and reduced viral genome copy number and gene expression levels [109] . bioinformatics data from microarray analysis recently showed that ifn-a2b and ribavirin treatment impacts on mers-cov gene expression in 10 different pathways, including genes involved in recognition of pathogens, immune responses and release of cytokines [110] . both ifn-b1b and lopinavir treatment, alone or in combination, also protected marmosets from the adverse clinical, radiological and pathological effects of mers-cov infection [111] . clinically, the use of ifn monotherapy, or ifn therapy in combination with ribavirin and/or lopinavir/ritonavir, has shown some promise (table 1 ) [37] [38] [39] [40] . however, the interpretation of clinical studies has been complicated by variability in factors such as the stage of infection at which therapy was administered. the available data are limited to case studies and retrospective cohort studies [22, 40] . in one case study on a patient who died in a greek hospital, pegylated ifn along with ribavirin and lopinavir was administered as part of the treatment regime, but not until the thirteenth day of the illness [39] . by contrast, in another preliminary study on two patients, the first patient was treated with ifn-a2b and ribavirin within a day of admission prior to mers-cov diagnosis, but he was also being treated with antibiotics, steroids and non-invasive ventilation [37] . patient 2, the wife of patient 1, was treated prophylactically after developing a low-grade fever and poorly defined lung infiltrates, but a diagnosis of mers-cov was not formally made [37] . thus, while patient 1 survived and patient 2 had only a mild course of illness, it is difficult to draw any firm conclusions regarding the efficacy of the treatment. in another case study on a patient in korea, administration of pegylated ifn-a2a along with ribavirin and lopinavir 4 days after hospital admission was deemed to have been effective in viral clearance and patient survival [38] . these case studies do not overall provide firm evidence for the efficacy or otherwise of ifn combination therapy for mers-cov. in one case involving a series of five patients who were critically ill with mers-cov infection and on mechanical ventilation and corticosteroids, ifn-a2b and ribavirin was administered on average 19 days after admission [27] . all five patients died, but they may not have benefited, as they were treated late in their illness and were already critically ill [27] . the benefit of earlier treatment in less vulnerable patients was suggested in another series of six patients in which three who received ifn-a2b and ribavirin early in the illness survived, while three other patients who were older and had comorbid conditions received the combination treatment later and all died [112] . however, in another study in which 20 mechanically ventilated patients with severe mers-cov infection who received pegylated ifn-a 2a and ribavirin early in treatment were compared to 24 patients who did not receive the combination therapy, the 14-day survival rate was significantly higher in the treatment group, but the 28-day survival rate was equivalently low in the two groups [113] . in another retrospective analysis of results from a series of 32 patients who received either ifn-a2a or ifn-b1a in combination with ribavirin, no significant difference in outcome between the two types of ifn was shown, and there was no survival benefit due to use of either ifn [29] . however, most of the patients in this study were aged more than 50 years and some had comorbid conditions, including end-stage renal disease [29] . thus the retrospective studies that have been carried out are heterogeneous in terms of type of patient, stage of disease and type of ifn used, including whether or not it was pegylated or short-acting. there is an urgent need for well-controlled clinical trials for ifn combination therapy in mers-cov, preferably early in the illness, as ifns are routinely available agents whose safety and efficacy is established for other viral illnesses and whose use has a sound molecular basis for mers-cov treatment. another type of therapy with a logical molecular basis for mers-cov treatment is the targeting of proteases, both host and viral (table 1 ; fig. 2 ) [19, 23, 53, 59, 60, [114] [115] [116] [117] . camostat, an inhibitor of tmprss2, is a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [59] . in a pathogenic mouse model of sars-cov infection, viral spread and pathogenesis was effectively blocked by camostat, and it is likely that it would have a similar impact on mers-cov [59] . as camostat is already in clinical use for the treatment of chronic pancreatitis, it represents a potentially safe and effective therapeutic option. recently, another tmprss2 inhibitor, nafamostat, was identified in a split protein-based cell-cell fusion assay as a potent inhibitor of mers-cov s protein-mediated hostviral membrane fusion in vitro [118] . nafamostat is already clinically approved for use by the us food and drug administration (fda) and is used as an anticoagulant [118] . the cathepsin l inhibitor teicoplanin, a glycopeptide antibiotic, was recently shown, via high throughput screening of fda-approved drugs, to block entry of mers-cov, sars-cov and ebola pseudoviruses into the cytoplasm [119] . teicoplanin is currently used clinically as an antibiotic in both prophylaxis and the treatment of serious grampositive bacterial infections. it also has derivatives, including dalbavancin, oritavancin and telavancin, all of which also block viral entry. the viral proteases, mpro (3clpro) and pl(pro), also represent potential molecular therapeutic targets [57, 60] . as well as its role in viral maturation, the mers-cov pl(pro) causes deubiquitination of ifn regulatory factor 3 (irf-3), and hence suppression of ifn b production, which contributes to viral suppression of the innate immune response (fig. 2) [120, 121] . the x-ray 3d crystal structure of mers-cov pl(pro) is similar to that of sars-cov, and includes ubiquitin-like and catalytic core domains [120] . thus the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg), can inhibit mers cov protease activity in vitro [61] . however, the mers-cov pl pro crystal structure also has unique aspects, including the oxyanion hole, and s3 and s5 subsites, which may be viable molecular targets for antivirals specifically designed against mers-cov [120] . a commercial compound termed compound 4 (commercial code f2124-0890,life chemicals) has been identified as an inhibitor of mers-cov and sars-cov plpro activity [122, 123] . the critical binding interactions and mode of inhibition differ between the two viral proteases, with the compound acting as a competitive inhibitor against mers-cov pl(pro), but an allosteric inhibitor of sars-cov pl[pro) [122] . however, f2124-0890 may lose potency in physiological reducing environments [123] . lopinavir is a protease inhibitor with activity against the sars-cov main protease m pro [124] . in a screen of a library of 348 fda-approved drugs to identify anti-mers-cov activity in cell culture, lopinavir emerged as one of four compounds that inhibited viral activity in a low micromolar range [125] . however, the clinical efficacy of lopinavir in mers-cov treatment has not yet been fully established. as mentioned above, it has usually been used clinically in combination with ifn and data are only available from case studies and series. however, notably, lopinavir-ritonavir treatment resulted in better clinical, radiological and pathological outcomes and reduced mortality in marmosets infected with mers-cov [36] . lopinavir has also been identified in a position paper from phe and isaric-who as a potential mers-cov therapy whose benefits are likely to exceed its risks [47] . thus far, mers-cov has not been considered to have pandemic potential. most cases have occurred in the middle east, particularly in ksa. outbreaks have been primarily linked to healthcare institutions, and shortcomings in infection control and prevention procedures [6] [7] [8] [9] [10] [11] [12] [13] [14] . however, potential viral mutations could facilitate expanded viral host range and enhance cross-species and human-human transmission [20, 58, 114] . the outbreak in korea resulted in mers-cov emergence in second-and third-generation contacts, highlighting the potential for mutational changes that could increase the likelihood of human-human transmission [14, 18] . mers-cov also exacts a high mortality rate, mainly due to the development of ards [15] [16] [17] . these factors emphasize the importance of developing targeted therapies and/or vaccines. the most promising advances in the development of specific molecular mers-cov therapies relate to targeting of the viral s protein by means of anti-s1monoclonal antibodies, hr-targeted antiviral peptides and viruses or plasmids bearing s protein as potential vaccine candidates [52, 55, 58, [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] . the use of ifns, usually in combination with other therapies such as ribavirin or lopinavir, also has a logical molecular basis given that ifn antagonism is an important mechanism by which the virus circumvents the innate immune system [62, 63, 105, 107] . targeting of host and viral proteases is also a sound molecular approach, as host proteases are important in viral-host membrane fusion, while viral proteases are key to viral maturation and are also involved in targeting ifns [23, 53, [59] [60] [61] [114] [115] [116] [117] . the therapies currently used for mers-cov have mainly been extrapolated from those used for sars-cov treatment, regardless of the important differences in receptor usage and cellular tropism between the viruses [20] [21] [22] [23] [24] [25] [26] . none of these therapies have been subject to well-controlled trials, and in some cases the risks are likely to outweigh any poorly defined benefits [19, 22, [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . in general, the clinical research response to mers-cov may have been too slow [126] . thus, while there are many promising lines of research in terms of specific molecular targeting of mers-cov, no potential therapies have yet been subject to welldesigned clinical trials, and none have been approved for clinical use, apart from the gls-5300 dna-plasmid vaccine [102, 105] . continuing outbreaks of mers-cov, with possible increases in human-human transmission, are likely to galvanize the research community to push ahead with the design and performance of clinical trials for some of the available monoclonal antibodies and/or antiviral peptides for use in outbreak situations. there are various challenges inherent in the development of specific mers-cov therapies. these include the difficulty of identifying a target population for potential prophylactic vaccines, limited small animal model availability and dependence on transgenic mouse models, and the current relatively low incidence of infection, which complicates the performance of adequate clinical trials [75, 96, 97, 104] . for example, one currently ongoing trial on convalescent plasma therapy has been affected by logistical and technical issues, including insufficient available donors and difficulty in collecting convalescent plasma containing sufficient mers-cov antibody levels [71, 72] . thus, while numerous monoclonal antibodies have been raised with anti-mers activity, in particular against the s protein [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] , and promising antiviral hr2 peptides have been synthesized [87, 90] , the available data are thus far limited to in vitro and animal studies. despite these issues, there is cause for optimism, given the many candidate antibody and peptide therapies. there is also some promising in vitro and animal model evidence suggesting that use of ifns, which are well-established therapies in other viral illnesses, may be of benefit if used sufficiently early in mers-cov treatment, or as a prophylactic, especially in combination with other therapies, including ribavirin or lopinavir-ritonavir [108] [109] [110] [111] . likewise, other drugs that are currently in clinical use for other conditions have been shown to be potentially useful for mers-cov treatment, including camostat and nafamostat; teicoplanin and its derivatives dalbavancin, oritavancin and telavancin; and the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg) [59, 61, [118] [119] 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cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein the heptad repeat region is a major selection target in mers-cov and related coronaviruses identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease inhibitor recognition specificity of mers-cov papain-like protease may differ from that of sars-cov x-ray structure and enzymatic activity profile of a core papain-like protease of mers coronavirus with utility for structure-based drug design small molecules targeting severe acute respiratory syndrome human coronavirus screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture towards improving clinical management of middle east respiratory syndrome coronavirus infection repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection mechanisms of coronavirus cell entry mediated by the viral spike protein the authors received no specific grant from any funding agency. the authors declare that there are no conflicts of interest. this is a review article and no experimental work with humans was performed. five reasons to publish your next article with a microbiology society journal 1. the microbiology society is a not-for-profit organization. 2. we offer fast and rigorous peer reviewaverage time to first decision is 4-6 weeks. 3. our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as 'excellent' or 'very good'. 5. your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord-343827-jo61t3m0 authors: qian, qun; fan, lifang; liu, weicheng; li, jin; yue, junqiu; wang, mingwei; ke, xianliang; yin, yan; chen, quanjiao; jiang, congqing title: direct evidence of active sars-cov-2 replication in the intestine date: 2020-07-08 journal: clin infect dis doi: 10.1093/cid/ciaa925 sha: doc_id: 343827 cord_uid: jo61t3m0 background: currently, there is no direct evidence to prove the active sars-cov-2 replication in the intestinal tract and relevant pathological changes in the colon and rectum. we investigated the presence of virions and pathological changes in surgical rectal tissues of a clinically confirmed covid-19 patient with rectal adenocarcinoma. methods: here, the clinical data were collected during hospitalization and follow-up of this patient. quantitative rt-pcr was performed on the rectal tissue specimens obtained from surgical resection, succus entericus and intestinal mucosa of ileostomy, and rectal mucosa during follow-up after recovery. ultrathin sections of surgical samples were observed for sars-cov-2 virions using electron microscopy. histopathological examination was performed using hematoxylin-eosin stain. immunohistochemical analysis and immunofluorescence were carried out on rectal tissues to evaluate the distribution of sars-cov-2 antigen, and immune cell infiltrations. results: the patient had fever and cough on day 3 postoperatively, was diagnosed with covid-19 on day 7, and was discharged from the hospital on day 41. rna of sars-cov-2 was detected in surgically resected rectal specimens, but not in samples collected on 37 day after discharge. notably, coincidence with rectal tissues of surgical specimens tested nucleic acid positive for sars-cov-2, typical coronavirus virions in rectal tissue were observed under electron microscopy. moreover, abundant lymphocytes and macrophages (some are sars-cov-2 positive) infiltrating the lamina propria were found with no significant mucosal damage. conclusions: we firstly reported that direct evidence of the active sars-cov-2 replication in the patient's rectum during the incubation period, which might explain sars-cov-2 fecal-oral transmission. the current coronavirus disease identified in 2019 , caused by a novel coronavirus, has become a global public health problem [1, 2] . as of may 27, 2020, a total of 5,451,532 cases of covid-19 have been confirmed globally, including 345,752 deaths [3] . there are many reports suggesting that sars-cov-2 rna can be detected and identified in anal/rectal swabs [4, 5] and stool specimens [6, 7] . in fact, one recent small sample study found that rna was consistently detected in rectal swabs, even after viral clearance from the upper respiratory tract, indicating extended duration of viral shedding in fecal samples and raising the possibility of fecal-oral transmission of sars-cov-2 [5] . similar results were reported in another study with more cases involved, raising the possibility of prolonged presence of sars-cov-2 in stools. notably, fecal samples remained positive for sars-cov-2 rna nearly 5 weeks after the viral clearance from the upper respiratory tract in covid-19 patients [8] . considering a high degree of sequence homology between the sars-cov-2 and sars-cov, angiotensin-converting enzyme ii (ace2) has been identified as the entry receptor of sars-cov-2. since this receptor is highly expressed on the epithelial cells from the ileum and colon [9] , the intestinal tract may be a potential route for sarapatients with cancer are considered to be more susceptible to sars-cov-2 [10, 11] . one patient with rectal cancer was admitted to zhongnan hospital of wuhan university for radical operation. on postoperative day 3, the patient began to develop cough and a c c e p t e d m a n u s c r i p t fever; chest ct revealed radiologic characteristics of viral pneumonia. on postoperative day 7, the patient was confirmed to be infected with sars-cov-2. although live sars-cov-2 had been successfully isolated from the fecal sample of a laboratory confirmed sars-cov-2 patient [12] , until now, there has been no direct evidence to prove active sars-cov-2 virus replication in the intestinal tract. it remains unknown whether there are pathological changes related to sars-cov-2 infection existing in colorectal mucosa in covid-19 patients. to clarify the above questions, we performed a retrospective study to detect the presence of sars-cov-2 virions and determine the pathological changes in rectal tissues of this patient. the patient's clinical information is described in supplementary samples of rectal tissues, succus entericus and intestinal mucosa of ileostomy, and rectal mucosa were tested for sars-cov-2 nucleic acid using qrt-pcr. the qrt-pcr analyses were performed following a previously described method [13] . the qrt-pcr test kits (biogerm) were recommended by the chinese center for disease control a c c e p t e d m a n u s c r i p t and prevention. the rectal tissue obtained by resection was soaked in rnalater solution overnight, the solution was discarded and the tissue was frozen at -80°c. the rectal tissue was cut into 1 mm thick sections and fixed in 2.5% glutaraldehyde and 1% osmium tetroxide in a biosafety cabinet with level 2 protection, and subsequently dehydrated using different ascending concentrations of alcohol (30% to 100%), and immersed and embedded in epoxy resin. ultrathin sections (80-100 nm) were prepared on formvar-coated copper grids (200 mesh). the virions were observed with a tecnai g2 20 twin electron microscope (fei company) under 200 kv. immunohistochemical staining was performed on formalin-fixed and paraffinembedded tissue sections (4-μm). sections of rectal tissues were immunostained to evaluate the expression and distribution of the sars-cov-2 antigen. briefly, sections were deparaffinized with xylene and alcohol and subsequently heated in citrate buffer (ph 6.0) for antigen retrieval. after blockage with 3% bsa in pbs for 30 minutes, a rabbit antibody against rp3 np protein (kindly provided by dr. zhengli shi, wuhan institute of virology, chinese academy of science [14] ) was incubated with the sections overnight at 4°c. for immunochemistry study, the slides were subsequently figure 1 . the patient's clinical information is shown in supplementary table 1 clinical characteristics of coronavirus disease 2019 in china the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status world health organization. coronavirus disease (covid-2019) situation reports diarrhea may be underestimated: a missing link in 2019 novel coronavirus characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding the presence of sars-cov-2 rna in the feces of covid-19 patients epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore prolonged presence of sars-cov-2 viral rna in faecal samples the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes clinical features of patients infected with 2019 novel coronavirus in wuhan sars-cov-2 transmission in patients with cancer at a tertiary care hospital in wuhan, china isolation of 2019-ncov from a stool specimen of a laboratory confirmed case of the coronavirus disease 2019 (covid-19) positive rt-pcr test results in patients recovered from covid-19 a pneumonia outbreak associated with a new coronavirus of probable bat origin molecular biology, and pathogenesis of avian influenza a (h5n1) infection in humans influenza viremia and the potential for blood-borne transmission influenza viral rna detection in blood as a marker to predict disease severity in hematopoietic cell transplant recipients the fate of influenza a virus after infection of human macrophages and dendritic cells viral infection triggers rapid differentiation of monocytes into dendritic cells influenza virus a infection of human monocyte and macrophage subpopulations reveals increased susceptibility associated with cell differentiation from influenza-induced acute lung injury to annual update in intensive care and emergency medicine evidence for gastrointestinal infection of sars-cov-2 we are grateful to center for instrumental analysis and metrology, the corefacility and technical support, wuhan institute of virology, cas. we declare that we have no conflicts of interest.a c c e p t e d m a n u s c r i p t key: cord-325966-0g7a9s5z authors: shih, hsin-i.; wu, chi-jung; tu, yi-fang; chi, chia-yu title: fighting covid-19: a quick review of diagnoses, therapies, and vaccines date: 2020-05-30 journal: biomed j doi: 10.1016/j.bj.2020.05.021 sha: doc_id: 325966 cord_uid: 0g7a9s5z the covid-19 pandemic caused by a novel coronavirus, sars-cov-2, has infected more than 4.9 million individuals and resulted in over 300,000 deaths globally. the rapid spread of the virus and the precipitously increasing numbers of cases necessitate the urgent development of accurate diagnostic methods, effective treatments, and vaccines. here, we review the progress of developing diagnostic methods, therapies, and vaccines for sars-cov-2 with a focus on current clinical trials and their challenges. for diagnosis, nucleic acid amplification tests remain the mainstay diagnostics for laboratory confirmation of sars-cov-2 infection, while serological antibody tests are used to aid contact tracing, epidemiological, and vaccine evaluation studies. viral isolation is not recommended for routine diagnostic procedures due to safety concerns. currently, no single effective drug or specific vaccine is available against sars-cov-2. some candidate drugs targeting different levels and stages of human responses against covid-19 such as cell membrane fusion, rna-dependent rna polymerase, viral protease inhibitor, interleukin 6 blocker, and convalescent plasma may improve the clinical outcomes of critical covid-19 patients. other supportive care measures for critical patients are still necessary. advances in genetic sequencing and other technological developments have sped up the establishment of a variety of vaccine platforms. accordingly, numerous vaccines are under development. vaccine candidates against sars-cov-2 are mainly based upon the viral spike protein due to its vital role in viral infectivity, and most of these candidates have recently moved into clinical trials. before the efficacy of such vaccines in humans is demonstrated, strong international coordination and collaboration among studies, pharmaceutical companies, regulators, and governments are needed to limit further damage due the emerging sars-cov-2 virus. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which emerged in wuhan, china in late 2019, has rapidly spread throughout china and globally. although belonging to the same family, sars-cov-2 has different clinical and epidemiological characteristics from sars-cov and middle east respiratory syndrome coronavirus (mers-cov). its high transmissibility resembles that observed for pandemic influenza viruses; however, tools for diagnosis, treatment, and vaccines still need tremendous work to achieve the levels needed to respond to a pandemic flu. although other measures designed to respond to and control a pandemic such as surveillance, quarantine, and social distancing work efficiently to flatten the curve at a major cost to the economy, the development and deployment of effective tests, drugs, and vaccines to protect lives and limit disease spread are still urgent. emergency use authorizations (eua) expedite the availability of drugs to prevent serious or life-threatening diseases or conditions when there are no adequate, approved, and available alternatives. for many drugs that are already marketed for other conditions, off-label use can increase access for patients who need them. currently, thousands of clinical trials are ongoing to test clinical outcomes. adequate clinical trials will soon confirm or refute the usefulness of several candidate drugs and vaccines in treating and preventing covid-19. here, we review the state of diagnostic tests, initial clinical experience on accessible drugs and convalescent plasma administered to patients with covid-19, and updated information on vaccine development against covid-19. for covid-19 patients, fever and cough are the two most common symptoms, and some patients might also suffer from sputum production, sore throat, headache, myalgia/arthralgia, rhinorrhea, and diarrhea [1] . shortness of breath and dyspnea occur in cases that have progressed to pneumonia. of note, a substantial proportion of patients reported olfactory and gustatory disorders, and thus sudden anosmia or ageusia may represent a clinical screening tool to identify covid-patients [2] . most patients had normal or decreased leukocyte count, lymphopenia, and elevated c-reactive protein, and some also had thrombocytopenia and elevated d-dimer, lactate dehydrogenase, and alanine aminotransferase [1] . in patients with pneumonia, ground-glass opacity is the typical radiological finding on chest computed tomography (ct) scan, and it may be obscure on chest x-ray; in patients with severe pneumonia, local or bilateral patchy consolidation has also been seen on ct images [1] . however, these clinical, laboratory, and imaging findings are nonspecific and cannot differentiate covid-19 from other viral respiratory infections; viral diagnostic methods specific for sars-cov-2 should be applied for disease confirmation. the current standard diagnostics for covid-19 are based on detection of the sars-cov-2 rna by nucleic acid amplification tests (naats), usually through real-time reverse transcription polymerase chain reaction (rt-pcr) with conformation by sequence analysis when necessary [3] . reverse transcription loop-mediated isothermal amplification (lamp) assays also appear to be a simple and sensitive diagnostic tool without a requirement high-level facilities and instruments [4] . samples recommended for testing are those from the lower respiratory tract, including sputum, bronchoalveolar lavage (bal), and endotracheal aspirates when possible [5] . sputum, nasopharyngeal swab (np), and oropharyngeal swabs (op) are the most common sample types taken from patients with mild to moderate illness. if both np and op are collected, they can be placed in the same tube and tested simultaneously to save reagents [5, 6] . in general, bal showed the highest positive rates, followed by sputum, np, and op in order of decreasing sensitivity [7] [8] [9] . throat gargling samples are an alternative specimen, although they are less sensitive than sputum [9] . the laboratory confirmation of cases in regions without covid-19 virus circulation requires detection of two different genetic targets of the covid-19 viral genome, while in regions with established covid-19 virus circulation, confirmation through detection of a single genetic target is considered sufficient [3, 5] . many national laboratories have established and published their diagnostic protocols, which are summarized on the world health organization (who) website [10] . for example, the charité protocol from germany recommended detecting the e (envelope) gene for screening, followed by confirmation of e gene-positive samples through detection of the rna-dependent rna polymerase (rdrp) gene, where the e assay is specific for all sars-cov related viruses (i.e., sars-cov, sars-cov-2, and bat-derived sars-related cov) and the rdrp assay using the p2 probe only detects sars-cov-2 [5] . pharyngeal virus shedding is very high during the first week of symptoms, and viral rna shedding from sputum persists even after resolution of symptoms and seroconversion [9, 11] . in a study with most samples (>90%) taken from the lower respiratory tract, the median duration of rna detection was 17 days (interquartile range, 13-22 days) after illness onset, and independent risk factors for prolonged sars-cov-2 rna shedding (>15 days) included male sex, delayed hospital admission, and invasive mechanical ventilation [12] . however, viral rna does not equate to a live virus, and more data are needed to realize whether viral rna load correlates with infectivity [6] . it has also been noted that a negative naat result does not mean that covid-19 is absent, since several factors can lead to false-negative results. these factors include inappropriate sample collection or transportation, sample collection at time when the patient was not shedding sufficient virus, and technical reasons [3, 5] . periodically sequencing the evolving viruses is also suggested to monitor any mutations in the regions targeted by the assays that might affect test performance [3, 6] . moreover, the presence of a non-sars-cov-2 pathogen does not preclude the possibility of covid-19; approximately one-fifth of specimens positive for sars-cov-2 were positive for one or more additional common respiratory viruses [13] . virus isolation is essential to obtain isolates for characterization and to support the development of antivirals and vaccines. however, although sars-cov-2 can be cultured in selected cell lines, such as vero cells and llc-mk2 cells, in a biosafety level-3 laboratory (bsl-3), viral isolation is not recommended as a routine diagnostic procedure due to biosafety concerns and time constraints [3, 9, 11] . studies have indicated that infectious virus can be readily isolated during the first week of symptoms, with sputum having a higher culture yield than np or op; however, infectious virus could not be isolated from samples taken 8 days after onset despite ongoing high viral load detected by rt-pcr [11] . it appears that virus isolation success depends on viral load, as culture failed to yield virus when samples contained <10 6 copies per ml or per sample [11] . further studies elucidating the duration of culture-positivity would provide a rationale for proposing strategies of isolation of infected patients. serological antibody detection is the other broad category of tests to diagnose covid-19, and this method detects igm, igg, or total antibodies (typically in the blood) against sars-cov-2. techniques used for antibody detection include virus neutralization assay, enzyme-linked immunosorbent assay (elisa), immunochromatographic assay, chemiluminescent immunoassay, etc. [11, [14] [15] [16] [17] . most tests are designed to capture antibodies, which recognize the nucleocapsid (n) protein and the s1 subunit and receptor biding domain (rbd) of spike (s) proteins, as n and s proteins are the two major coronavirus immunogens [15, 16] . rbd-specific monoclonal antibodies derived from two b cell clones of one covid-19 patient have demonstrated impressive binding and neutralizing activity against live sars-cov-2 [18] . of importance, these serologic tests should not cross-react with other seasonal coronaviruses. nevertheless, the use of antibody tests is limited to settings of acute illness because it takes time for hosts to mount an adequate immune response [3] . studies indicate that the majority of patients have seroconversion 2 weeks after symptom onset [11, 16, 19] . less than 40% of patients had detectable antibodies within 1 week of onset, but this percentage rapidly increased to 94.3% (igm) and 79.8% (igg) 15 days after onset, with the median seroconversion time of 12 and 14 days, respectively [16] . igm began to decline 4 weeks after symptom onset, while igg remained at high levels after 7 weeks [17, 19] . based on the time course of seroconversion, serological tests could be used as complementary tools to identify patients presenting late in their illness [16] . as mentioned earlier, seroconversion has not usually been followed by a rapid decline in viral rna load [11] . serological tests may also aid in (i) contact tracing, (ii) assessment of prior infection and immunity to sars-cov-2 (if there is protective immunity), (iii) determining the extent of the pandemic with seroprevalence data, and (iv) vaccine evaluation studies [3, 6] . to date, it is not known whether antibodies elicited by sars-cov-2 provide protective immunity against reinfection and how long the protective immunity lasts. a rhesus macaque study does suggest protective immunity after recovery from primary infection, since reinfection did not occur in convalescent monkeys rechallenged with the same dose of sars-cov-2 strains [20] . further studies are necessary to elucidate the situation in humans. rapid antigen-detection methods using immunoassays targeted at n or s proteins are under development, although with the same challenge of low sensitivity observed in influenza virus antigen tests. to date, a number of laboratory-developed assays and commercially available kits (mostly naats and serological antibody tests) have been granted an eua by the us food and drug administration (fda), which greatly strengthens the diagnostic capability of frontline clinical laboratories [21] . currently, there are no drugs or other therapies approved by the us fda to treat covid-19. the major clinical treatment and management approaches emphasize the importance of life supportive care and relief of complications. oxygen-based therapy has been applied when patients experience dyspnea, and advanced sepsis management has been warranted for patients who progressed to severe sepsis and acute respiratory distress syndrome. no existing antiviral drugs have sufficient evidence that they efficaciously treat covid-19 pneumonia. some drugs have been selected to treat covid-19 pneumonia patients ( table 1) ; most of these drugs were designed for other purpose such as ebola, influenza, parasites, human immunodeficiency virus (hiv) infections, and immune therapy for some autoimmune and inflammatory diseases. clinical trials have been conducted in which potential antiviral therapy targets were tested, such as blocking viral entry to human cells, inhibiting viral enzymes that were responsible for genome replication. others focus on the human immune system to boost the innate response and inhibit the inflammatory process to relieve rapid progressed acute lung injuries. global, large-scale, randomized clinical trials are still ongoing to test the safety and clinical outcomes of these drugs. here, we summarize the mechanisms of potential therapeutic options that may combat the emerging sars-cov-2 ( figure) . chloroquine/hydroxychloroquine chloroquine and hydroxychloroquine have a long-standing history in the prevention and treatment of malaria and the treatment of chronic inflammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis. chloroquine and hydroxychloroquine block viral entry into cells by inhibiting the glycosylation of host receptors, proteolytic processing, and endosomal acidification. immunomodulatory effects through attenuation of cytokine production and inhibition of autophagy and lysosomal activity in host cells have also been reported [22, 23] . the experiences of the us, china, and europe have indicated clinical effects of chloroquine and hydroxychloroquine in the early phase but limited effects in the late phase [24] . an earlier study demonstrated that hydroxychloroquine was significantly associated with viral load reduction/disappearance in covid-19 patients and its effect was strengthened by azithromycin [25] . in addition, a recent meta-analysis indicated hydroxychloroquine alone, or in combination with azithromycin had benefits in positive-to-negative conversion of sars-cov-2 and reduction of progression rate though being associated with higher mortality [26] . nevertheless, an us study which evaluated hospitalized patients with covid-19 in metropolitan new york showed no significant difference in in-hospital mortality among patients treated with hydroxychloroquine, azithromycin, both, and neither of them [27] . safety data and data from larger, randomized, placebo-controlled trials with longer follow-up are urgently needed. hydroxychloroquine can prolong the pr, qrs and qtc intervals, especially in patients with underlying risk factors or use in combination with other qt-prolonging drugs; cautious monitoring of ecg changes during treatment is important. chloroquine and hydroxychloroquine are substrates of cyp2c8 and cyp3a4; therefore, co-administration with moderate and strong cyp2c8 and cyp3a4 inhibitors may result in increased plasma concentrations of hydroxychloroquine. experiences and interim guidelines for clinical management of sars-cov-2 infection in taiwan advised that early administration of hydroxychloroquine may be considered to be given for 7 days after thoroughly evaluating potential risks/benefits and ethical issues [28] . lopinavir, an hiv type 1 aspartate protease inhibitor, has in vitro inhibitory activity against sars-cov [29] . ritonavir is combined with lopinavir to increase its plasma half-life through the inhibition of cytochrome p450. an open-label study published in 2004 suggested, by comparison with a control group that received only ribavirin, that the addition of lopinavir-ritonavir (400 mg and 100 mg, respectively) to ribavirin reduced the risk of adverse clinical outcomes (acute respiratory distress syndrome or death) and viral load among patients with sars [29] . lopinavir also has activity, both in vitro [30] and in an animal model [31] , against mers-cov, and case reports have suggested that the combination of lopinavir-ritonavir with ribavirin and interferon alfa resulted in virologic clearance and survival [32] . a randomized controlled trial enrolled covid-19 patient with dyspnea and desaturation in china and suggested that treatment with lopinavirritonavir was similar to standard care in the time to clinical improvement. gastrointestinal adverse events were more common in the lopinavir-ritonavir group, but serious adverse events were more common in the standard-care group. lopinavir-ritonavir treatment was stopped early because of adverse events such as nausea, diarrhea and hepatotoxicity [33] . additionally, as a cyp3a inhibitor, significant drug-drug interactions have been reported before. ivermectin is an fda-approved broad spectrum anti-parasitic agent. it was identified as an inhibitor in vitro and has been demonstrated to limit infection by a broad spectrum of rna viruses such as dengue virus (denv), west nile virus, venezuelan equine encephalitis virus, and influenza by binding to inhibitors of importin α/β-mediated transports of proteins and rna during infection. in the phase iii clinical trial in thailand in 2014-2017 against denv infection with a single daily oral dose, ivermectin was observed to be safe and resulted in a significant reduction in serum levels of viral ns1 protein, but no change in viremia or clinical benefit was observed [34] . ivermectin has been shown to reduce viral rna up to 5,000-fold after 48 h of infection with sars-cov-2 [35] . this drug is extensively metabolized in human liver microsomes by cyp3a4. for patients with liver function abnormality, drug effects and interactions should be closely monitored during treatment. remdesivir remdesivir, a nucleotide analogue prodrug that inhibits rdrp, results in premature termination of the viral rna chain and consequently halts replication of the viral genome. it has shown broad spectrum activity against members of several virus families, including filoviruses (e.g., ebola) and coronaviruses (e.g., sars-cov and mers-cov), and has shown prophylactic and therapeutic efficacy in nonclinical models of these coronaviruses [36] [37] [38] . remdesivir in vitro testing has also shown that remdesivir has activity against sars-cov-2 [36] . a compassionate-use study reported 68% oxygen improvement and 13% overall mortality. a total of 32 patients (60%) reported adverse events during follow-up. the most common adverse events were increased hepatic enzymes, diarrhea, rash, renal impairment, and hypotension, and some of the patients discontinued remdesivir treatment prematurely. higher improvement rate in chest imaging in the favipiravir arm (91.43% versus 62%) [39] . another multicentered randomized clinical study also suggested that the 7 day clinical recovery rate increased in the favipiravir treatment group, along with shorter defervescence time and cough in patients with hypertension and/or diabetes [40] . although few adverse effects were reported during treatment, potential drug and drug interactions should be kept in mind because favipiravir undergoes metabolism in the liver by aldehyde oxidase and xanthine oxidase to produce an inactive oxidative metabolite and is excreted by the kidney [41] . no hepatic or kidney adjustments are recommended at this time, but initiation is not recommended in patients with an estimated glomerular filtration rate less than 30 ml/min. the humoral immune response mediated by antibodies is crucial for preventing viral infections. therefore, the development of specific surface epitope-targeting neutralizing antibodies is a more long-term, albeit more specific, approach to target covid-19 [42] . sars-cov-2-specific neutralizing antibodies (nab) have been detected in patients from day 10-15 after the onset of the disease and remained thereafter. the titers of nab among these patients correlated with the spike-binding antibodies targeting the s1, rbd, and s2 regions. studies in china indicated that one or two doses of 200 ml of convalescent plasma derived from recently recovered donors with neutralizing antibody titers above 1:640 were well tolerated and could significantly increase or maintain the neutralizing antibodies at a high level; this outcome leads to disappearance of viremia, improvement of clinical symptoms, and absorption of lung lesions in radiological examination when administered, in addition to supportive care and antiviral agents such as lopinavir/ritonavir and favipiravir. the donors must be symptom-free for at least 14 days, have a negative sars-cov-2 pcr test after recovery to provide abo-compatible and test negative plasma for major blood-borne diseases at the time of blood donation. known general reactions such as transfusion-associated circulatory overload and transfusion-associated acute lung injury in patients with already severe lung damage still exist and need to be closely monitored [43] [44] [45] [46] . evidence suggests that cytokine release syndrome (crs) might play a major role in severe covid-19 [23, 47] . inflammatory cytokines and chemokines, including il-6, il-1β, induced protein 10 and monocyte chemoattractant protein-1, are significantly elevated in covid-19 patients, especially in severe patients with life-threatening multiple organ dysfunction. in covid-19 patients with elevated inflammatory cytokines, postmortem pathology has revealed tissue necrosis and interstitial macrophage and monocyte infiltrations in the lung, heart, and gastrointestinal mucosa [48] . moreover, severe lymphopenia with hyperactivated proinflammatory t cells [48] and decreased regulatory t cells [49] is commonly seen in critically ill patients, suggesting dysregulated immune responses. tocilizumab is a recombinant humanized monoclonal anti-il-6 receptor antibody. it binds both soluble and membrane-bound il-6r to inhibit il-6-mediated cis-and trans-signaling [50] . given the efficacy of tocilizumab in crs and the pivotal role of il-6 in covid-19, clinical use should consider evaluation of patients with the following criteria: (i) h score, a diagnostic score for hlh, to discriminate patients with crs; (ii) chinese guidelines for covid-19 grade patients into mild, moderate, severe, and critical by vital signs, radiographic findings, and complications; (iii) il-6 measurement, as il-6 levels are significantly elevated in covid-19 patients, especially in icu patients [51] . crp, an acute-phase inflammatory protein synthesized by il-6-dependent hepatic biosynthesis, is a reliable marker of il-6 bioactivity and is used to predict crs severity and monitor il-6 blockade efficacy. most studies suggested that elevated crp levels are associated with severe covid-19 [33, 52, 53] . clinicians should add antivirals and cautiously evaluate the possibility of secondary infection thereafter [54] . adverse hepatic effects have been reported previously, and clinicians should consider discontinuation of drug in cases of marked elevation of liver enzymes and hyperbilirubinemia. other drug-drug interactions should be monitored because tocilizumab interferes with the serum concentration of cyp3a4 substrates. arbidol hydrochloride (umifenovir) umifenovir targets the s protein/ace2 interaction and inhibits membrane fusion of the viral envelope [55] . the agent is currently approved in russia and china for the treatment and prophylaxis of influenza and is being tested in some clinical trials treating covid-19 based on in vitro data suggesting activity against sars [56] . the current dose of 200 mg orally every 8 hours for influenza is being studied for covid-19 treatment [57] . limited clinical experience with umifenovir for covid-19 in china in a nonrandomized study of 67 patients with covid-19 showed that treatment with umifenovir for a median duration of 9 days was associated with lower mortality rates (0% [0/36] vs 16% [5/31] ) and higher discharge rates compared with patients who did not receive the agent [58] . another randomized chinese study compared umifenovir (arbidol) (200 mg*3/day) and favipiravir (1600 mg*2/first day followed by 600 mg*2/day) and indicated that favipiravir, compared to umifenovir, did not significantly improve the clinically recovery rate at day 7 [57] . camostat mesylate is an inhibitor of the cellular serine protease tmprss2 [59] , which is used by sars-cov-2 for s protein priming [60] . it was developed to treat chronic pancreatitis (600 mg daily in three divided doses) and reflux esophagitis (300 mg daily in three divided doses after each meal). sars-cov-2 is surrounded by an envelope composed of a lipid bilayer and envelope proteins. sars-cov-2 initiates human cell entry after the s protein present on the envelope binds to a cell membrane receptor called ace2. the s protein is cleaved into two subunits, s1 and s2, by a human cell-derived protease, which is thought to be furin. s1 then binds to its receptor, ace2. the other fragment, s2, is cleaved by tmprss2, a human cell surface serine protease, resulting in membrane fusion. both ace2 and tmprss2 are therefore thought to be essential in airway cells for sars-cov-2 infection. an in vitro study found that nafamostat and camostat suppressed sars-cov-2 s protein-initiated fusion in 293ft cells (derived from the human fetal kidney) ectopically expressing ace2 and tmprss2. nafamostat, a new developed iv form drug, was found to inhibit sars-cov-2 s protein-initiated fusion at a concentration less than one-tenth of that needed by camostat. adverse effects included mild gastrointestinal upset, dizziness, skin rash, thrombocytopenia, and elevated liver enzymes [61] . vaccination probably offers the best option for blocking infectious disease circulation. table 2) ; they are further discussed in the following section. platform mrna-based vaccines comprise mrna that encodes a protein antigen. conventional mrna-based vaccines encode the antigen of interest and contain 5′ and 3′ untranslated regions, whereas the virally derived, self-amplifying rnas encode not only the antigen but also the viral replication machinery that enables intracellular rna amplification and abundant protein expression [62] . recent mrna vaccine designs have improved the stability and protein translation efficiency for enhanced innate and adaptive immunogenicity [62] . delivery of the mrna vaccine has been optimized by use of lipid nanoparticles for intramuscular or intradermal administration [63] . additionally, unlike conventional vaccines, which are made from either inactivated pathogens or the small subunit of live pathogens, no infectious virus needs to be handled for mrna vaccines. therefore, testing is relatively safe, efficient, cost effective, and rapid. an mrna vaccine for covid-19, mrna-1273, was the first to advance to a phase i clinical trial in the us, which is currently recruiting healthy volunteers aged between 18 to 55 years to assess the safety, reactogenicity, and immunogenicity. dna vaccines, another type of nucleic acid-based vaccines, consist of plasmid-dna encoding one or several antigens that will be expressed in host cells. dna vaccines can be produced rapidly and at low cost. however, the need for specific delivery systems to achieve good immunogenicity and possible genomic integration and persistence in host cells is a remaining concern [63] . dna generation of an inactivated whole-virus (iwv) vaccine is the quickest approach for vaccine production following a new outbreak. such vaccines have successfully been developed for influenza virus and enterovirus 71 [66, 67] . iwv vaccines are usually made by exposure of a virulent virus to chemical or physical agents, e.g., formaldehyde or gamma irradiation, to destroy infectivity while retaining immunogenicity. the need to use large amounts of antigen to elicit an adequate antibody response and the possibility of causing th2-bias hypersensitivity are major concerns for iwv vaccines [68] . one inactivated vaccine candidate that displayed good cross-neutralization to different covid-19 strains has received approval for testing in human trials [69] . viral vector vaccines are also potential tools for vaccine development. these vaccines can specifically deliver genes to target cells, enhance immunogenicity without an adjuvant, and induce a robust cytotoxic t cell response to eliminate virus-infected cells. although the results of viral vector-based vaccines have been encouraging in animal models, some obstacles need to be overcome before use in humans. these obstacles include genetic stability, ability to evade pre-existing immunity, and genotoxicity. adenovirus serotype 5 (ad5) is the most widely used vector because this vector can be easily produced and has high levels of transgene expression and a broad range of viral tropism [70] . the ability to enhance mucosal immunity through targeting epithelial cells of the upper respiratory tract and gut, two main sites that express high levels of the ace2 receptor for sars-cov-2, makes ad5 an advantageous viral vector against covid-19. recombinant ad5 vector-based vaccines have been examined in clinical trials against infectious diseases [71, 72] . the work for covid-19 has been accelerated based on the experiences of previous trials. a candidate vaccine known as ad5-ncov, which encodes a full-length s protein of sars-cov-2, is the first demonstrated to be safe for humans and to proceed to a phase ii clinical trial in china. another viral vector vaccine, chadox1 ncov-19, is composed of a nonreplicating chimpanzee adenovirus vector and genetic sequence of s protein. the vector represents an attractive alternative to the human adenoviral vector due to its good safety profile and lack of pre-existing immunity in human population [73] . the vaccine candidate has entered a phase i/ii clinical trial. lentivirus vector (lv) systems represent an attractive technology for vaccine development. in addition to their ability to effectively deliver genes or antigens of interest into cells and to generate humoral and cellular mediated immune response against the encoded transgenes [74] , lvs can transduce antigen presenting cells (apcs), the main cell types mediating the immune response, at high efficiencies with little to no cytotoxicity [75] . through up or downregulation of immune modulatory genes in apcs by lvs, the genetically modified apcs may potentially activate a strong protective immunity against infections [75] . two vaccine candidates, covid-19/aapc vaccine and lv-smenp-dc vaccine, which were made by modifying artificial apcs and dendritic cells with lvs expressing multiple viral genes and immune modulatory genes, act as 'trojan horses' against sars-cov-2 virus. clinical trials are currently underway to evaluate their safety and immune reactivity. bifidobacterium is one of the domestic, nonpathogenic anaerobic bacteria found in the intestine of humans. these organisms are believed to have health-promoting properties for their host, including increasing the immune response and protecting the host against viral infection [76] . as vaccine vectors, they offer several advantages including low cost, low resistance to antibiotics, noninvasive administration, and high safety levels. the most attractive feature is that bifidobacterium tends to elicit high levels of mucosal antibodies against the expressed foreign antigen following uptake via the mucosal immune system [77] . some strains of bifidobacterium have been used as a delivery vector for the development of vaccines against hepatitis c virus and enterovirus 71 [78, 79] . the bactrl-spike vaccine candidate contains live bifidobacterium longum, which contains synthetic plasmid dna encoding the s protein of sars-cov-2. the ongoing trial is designed to evaluate the safety and tolerability of orally delivered bactrl-spike vaccine in healthy adults. antibody-dependent enhancement (ade) is a condition in which subneutralizing or nonneutralizing antibodies are produced following primary infection or vaccination, and they enhance the infectivity of subsequent infections [80] . ade modulates the immune response and elicits sustained inflammation, lymphopenia, and/or cytokine storm [81] . ade has been observed for a variety of viruses, most notably flaviviruses (e.g., dnev) [82] . ade also occurs in sars-cov infection [83, 84] . diluted anti-sera against sars-cov promotes sars-cov infection, and this enhancement is significantly mediated by anti-s protein antibodies [83, 84] . similarly, vaccination with recombinant s protein of sars-cov elicits both neutralizing and ade-inducing igg antibodies [85] . as described above, many potential vaccine candidates against sars-cov-2 have focused on the full-length s protein. attributed to the taxonomic and structural similarities between sars-cov and sars-cov-2, ade is a critical issue that should be considered seriously during the practical application of sars-cov-2 vaccines. three approaches have been suggested to mitigate the adverse effects of ade. the first one is shielding the nonneutralizing epitopes of the s proteins by glycosylation. the second approach is immunofocusing, which aims to direct the adaptive immune responses to target only the critical neutralizing epitope to elicit a more robust protective immunity. supporting evidence for the latter is that a vaccine candidate based on the shorter rbd induced higher neutralizing activity than based the full-length s protein [10, 86] . the third approach is eliminating epitope sequences that mediate enhancement of infection. protein sequences responsible for ade have been identified at s 597−603 of the sars-cov s protein [85] , a region that is also conserved in sars-cov-2 [40] . thus, vaccines against covid-19 could be engineered to minimize ade via elimination of the epitope. bacillus calmette-guérin (bcg), the most commonly administered vaccine worldwide, contains a live attenuated strain of mycobacterium bovis to protect against tuberculosis (tb). universal vaccination at birth with a single dose of bcg is recommended in many countries where tb is highly endemic or where there is high risk of exposure to tb, such as japan, china, and taiwan. other countries, such as spain, france, and switzerland, have discontinued their universal vaccine policies because of the declining incidence of tb infection and the proven variable effectiveness in preventing adult tb. countries such as the united states, italy, and the netherlands have yet to adopt universal vaccine policies [87] . although developed to prevent severe forms of tuberculosis in children, bcg vaccination has been shown to induce heterologous or nonspecific immune effects against nonmycobacterial pathogens, a phenomenon termed 'trained immunity'. trained immunity refers to the ability of innate immune memory to mount an enhanced subsequent response to diverse microbes [88] . favorable effects of bcg have been observed in mouse and human studies for distinct viral pathogens [89, 90] . epidemiological studies have also linked bcg vaccination to the reduction in all-cause mortality in neonates and respiratory infections in elderly [91, 92] . nod2-and mtor-mediated changes in the epigenetic landscape of immune cells is proposed to underly such protection to increase the secretion of pro-inflammatory cytokines, particularly il-1β, and enhance anti-viral immunity [88, 93] . recent preprint studies suggested significant associations of bcg vaccination with prevalence, progression of disease, and mortality due to covid-19 [94, 95] . the authors indicated that countries without universal policies for bcg vaccination have been more severely affected compared to countries with routine use of the vaccine in neonates. the national immunization program in taiwan has included neonatal bcg vaccination since 1965, and the coverage rate has remained at 97% since 2001 [96] . as of may 20, 2020, a cumulative total of 440 covid-19 cases were confirmed in taiwan with a case fatality rate was of 1.6%. the low morbidity and mortality rate are attributed to the government's quick response, border control, case identification, containment, and resource allocation to protect public health [97] . it is not known whether bcg vaccination plays a protective role against covid-19 infection in taiwan. in addition to bcg, live attenuated influenza vaccine has been shown to promote nk cellmediated heterologous immunity [98] . previous studies also suggest that the heterologous beneficial effects of bcg vaccination may vary by bcg formulation, age, and route of administration [91, 99] . although these vaccines may bridge the gap until a vaccine specifically for sars-cov-2 is available, their protective effects and clinical relevance need to be further characterized. clinical trials have been initiated to study the effects of bcg vaccination given to healthcare workers who are at the frontline of the covid-19 pandemic ( table 2) . before the evidence is available, the who is not likely to recommend bcg vaccination for the prevention of covid-19 [100] . in the face of a pandemic, the rapid development, production, and deployment of diagnostic tools, drugs and vaccines are critical. scientific advancements since the sars and mers pandemics have accelerated our understanding of the epidemiology, pathogenesis, and diagnosis of sars-cov-2, as well as the development of therapies to treat viral infection. rigorous and adequate clinical trials for drug safety and effectiveness in randomized, controlled trials remain fundamental measures to protect the public from drugs that are ineffective, unsafe, or both. some available candidate drugs targeting different levels of human responses to covid-19, such as cell membrane fusion, rna-dependent rna polymerase, viral protease inhibitor, il-6 blocker and convalescent plasma, may improve the clinical outcomes of critical covid-19 patients. as no effective treatment against sars-cov-2 is currently available, the best action is to develop vaccines to prevent the infection. some potential vaccine candidates have progressed to phase i and ii clinical trials, but a year and a half are likely to pass before an effective vaccine is vetted through trials and is ready for marketing for humans. therefore, considerable efforts should be given to limit or hinder the spread of the virus. in addition, pandemics will generate simultaneous demand for drugs and vaccines around the world. the elderly and those with underlying diseases or chronic comorbidities are at greater risk of severe disease or mortality. clinical and serologic studies will be needed to confirm which populations remain at the highest risk once effective treatments or vaccines are available. strong international coordination and collaboration among studies, pharmaceutical companies, regulators, and governments are needed to 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antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor mrna vaccines -a new era in vaccinology new vaccine technologies to a dna vaccine induces sars coronavirus neutralization and protective immunity in mice safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase 1, open-label, single-arm, dose-escalation trial inactivated whole virus influenza a (h5n1) vaccine formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus 71 encephalomyelitis against disease immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus rapid development of an 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generates cytokine-induced memory-like nk cells: impact of human cytomegalovirus infection bacille calmette-guerin vaccine strain modulates the ontogeny of both mycobacterial-specific and heterologous t cell immunity to vaccination in infants antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro key: cord-348192-ibohbjfb authors: odih, erkison e.; afolayan, ayorinde o.; akintayo, ifeoluwa; okeke, iruka n. title: could water and sanitation shortfalls exacerbate sars-cov-2 transmission risks? date: 2020-06-09 journal: am j trop med hyg doi: 10.4269/ajtmh.20-0462 sha: doc_id: 348192 cord_uid: ibohbjfb sars-cov-2, the etiologic agent of covid-19, is shed in stool. sars coronaviruses have been detected in wastewater during outbreaks in china, europe, and the united states. in this perspective, we outline the risk fecal shedding poses at locations without safely managed sanitation, as in most of nigeria where we work. we believe that feco-oral transmission could occur if community transmission becomes high and sustained in densely populated cities without proper sanitation in nigeria and many other african and asian settings. in the absence of basic sanitation, or where existing sanitation is not safely managed, groundwater, which is often drawn up from wells and boreholes for drinking and household use, can become contaminated with enteric bacteria and viruses from fecal matter. endemic and epidemic transmission of multiple feco-oral pathogens via this route continues to be documented in areas without safely managed sanitation, and, therefore, the risk of sars-cov-2 transmission needs to be evaluated, tracked, and forestalled in such settings. we suggest that fecal matter from treatment facilities and recovered patients should be carefully and properly disposed. furthermore, environmental surveillance of sars-cov-2 in wastewater and accumulated human waste, as well as efforts to mitigate the virus’ entry into unprotected household water sources, should be a priority part of the covid-19 response in settings without safely managed sanitation for the duration of the pandemic. every effort must be deployed to limit continued spread of the etiologic agent of the covid-19 pandemic. mounting evidence shows that sars-cov-2 is amplified in the gastrointestinal tracts of infected people, excreted in stool, and detectable in wastewater at high levels [1] [2] [3] (rimoldi et al., 2020 ; medrxiv preprint doi: https://doi.org/10.1101/2020. 05.01.20086009). we are concerned that, in areas without safely managed sanitation, drinking and household water supplies could become contaminated with the virus. this potential risk of feco-oral transmission is highest in densely populated urban centers. sars-cov-2 has largely been transmitted via respiratory droplets and fomites from infected persons to the respiratory systems of susceptible individuals. 4 however, the virus replicates in gut enterocytes 1 and is detected in stool from patients with severe or mild covid-19, as well as from presymptomatic and asymptomatic individuals. 2, 3 recovered covid-19 patients may continue to shed virus for as long as 42 days after symptoms have ceased, even after they test negative by conventional respiratory tests. 3, 5, 6 considerable concern has been expressed in the literature that the feco-oral transmission potential for sars-cov-2 places endoscopists, caregivers of diapered children who shed the virus, 7 and fecal transplant recipients 8 at high risk of contracting the infection. for intestinal sars-cov-2 to transmit via fecal matter, it would have to be viable when shed and persist in the environment until a count greater than an oral infective dose is ingested by a susceptible individual. the cycle could potentially be shortcut by direct dissemination of fecal matter inadvertently from person-to-person or by pests like flies and cockroaches, or it could be broken if wastewater or domestic water treatment inactivates sars-cov-2. because wastewater treatment eradicates sars-cov-2 and most of the worst affected countries have robust water purification systems, community feco-oral transmission has been less extensively discussed. feco-orally transmitted pathogens are endemic in nigeria, which is among the top five countries worldwide contributing to diarrhea-derived under-five mortality. 9 a principal reason why the burden from feco-oral pathogens is so high is that for most nigerians, sewage systems are nonfunctional, incompletely functional, or nonexistent. of six major northern nigeria cities conducting polio virus environmental surveillance, only abuja operates a sewage plant. 10 in lagos, there are multiple sewage treatment plants, but their performance is suboptimal, and they therefore pose a risk of enteric pathogen transmission to surrounding areas. 11, 12 sewage handling capacity has not grown in tandem with the explosive and continued growth of this megacity so that coverage does not extend to all residents. this situation prevails in many african urban centers and in some south asian settings: between 60% and 95% of african city dwellers are not connected to sewerage and instead use a range of autonomous solutions or resort to open defecation. 13 as a result, fecal matter can be deposited into the open environment, pour from toilets unconnected to sewerage into surface water, or be buried underground in soakaways and pits from where, if these receptacles are not adequately protected, it can seep into shallow wells used for irrigation, drinking, and household purposes. 10, [13] [14] [15] according to the who/unicef, 663 million people in africa and asia do not have access to safe water, and diarrheal disease is a major cause of illness and death in those populations. 16 in recent years, surveillance of household water at a number of african and asian locations has revealed frequently found indicators of recent fecal contamination, such as escherichia coli, or outright pathogens, including enteric viruses. in each case, links have been made to human or animal open defecation, proximal latrines, or improperly processed wastewater. [17] [18] [19] in those settings, even pathogens known not to persist or thrive in the environment are among those recovered from contaminated household water or causing outbreaks. 20, 21 although there are as yet no reports of transmission of sars-cov-2 via sewage or fecal matter in settings without safely managed sanitation, or recovery from household water, these examples demonstrate that feco-oral transmission by endemic pathogenic organisms is commonplace in these settings. there is now convincing evidence, from countries with adequate sanitation, that sars-cov-2 is present in feces, around toilets, and in wastewater. 2, 22, 23 both sars-cov and sars-cov-2 nucleic acid have been detected in wastewater during outbreaks. 22, 24, 25 replicable virus is less commonly reported, although it is less commonly sought, and has been found. in laboratory studies, wang et al. 25 found that sars-cov, the agent of the 2003 sars epidemic, remains viable in water for 14 days at 4°c but for only 2 days at 20°c, suggesting that survival in tropical regions may be inadequate to sustain viability in stored water. however, coronaviruses are protected by organic matter, 26 and this will greatly affect their survival under real-world conditions. sars-cov-2 rna has been detected in substantial concentrations in wastewater and downstream water bodies. 27 rimoldi et al. reported that sars-cov-2 is susceptible to wastewater treatment and that viral infectiveness in wastewater is negligible; viral rna was amplified in untreated but not treated wastewater. 28 zhang and coworkers 29 in another preprint, however, found the china cdc-recommended sodium hypochlorite treatment of wastewater to be ineffective for the removal of sars-cov-2 rna. these studies await peer review, and further investigation is needed to clarify risks. as noted by lodder and de roda husman, 22 early finding of a covid-19 case in the united states with no known exposure to an infected case suggests that a form other than human-tohuman respiratory transmission of covid-19 may be possible. additional evidence comes from a recently published systematic review from wuhan, which spotlighted a small number of patients with diarrhea but no respiratory symptoms. 30 however, most patients in this pandemic who could have had the opportunity to be infected feco-orally to date have also been exposed to respiratory droplets or fomites; thus, the magnitude of the risk is challenging to gauge. fecooral transmission nonetheless remains a valid, if untested, hypothesis. 1, 2, 22, 26, [30] [31] [32] [33] our review of the evidence suggests that the risk of this mode of transmission in communities without basic sanitation may be high. unfortunately, countries without effective sanitation and water purification are also those least likely to have the wherewithal to detect live virus in environmental samples (detection of viral nucleic acid does not infer infectivity) and therefore measure this risk. 10, 34 as at the time of writing, most african and asian cities without basic or safely managed sanitation had reported relatively few covid-19 cases. however, case numbers are increasing, and, as they rise, the viral load in untreated fecal waste pools could escalate. this is particularly true of urban settings experiencing rapid rises in case numbers such as lagos and kano, two nigerian megacities. as at may 23, 2020, the nigerian centre for disease control had confirmed 3,357 and 883 cases in these states, respectively, together representing 56.3% of the number of cases nationally. because of occupancy pressures on isolation facilities, most nigerian cities have erected makeshift isolation and treatment facilities for patients who have tested positive, to supplement the few facilities that were available at the start of the pandemic. these new facilities often lie outside hospitals that manage their own wastewater. the same pressures on treatment facilities mean that sars-cov-2-infected persons in nigeria are discharged as soon as two consecutive respiratory swabs test negative: symptom free but likely shedding the virus when they return to their communities. other factors could additionally combine to alter the risk of feco-oral sars-cov-2 transmission. in kathmandu, nepal, where salmonella enterica typhi and paratyphi have been shown to leach into the municipal water system, breaches occur more heavily during the rains. 35 we note that african countries on the upswing of their covid-19 epidemics are just beginning the rainy season. rainy season sewage overflows can overwhelm even properly managed wastewater plants, leading to heightened enteric virus transmission. 36 on the other hand, it is possible that only very high counts of sars-cov-2 would yield orally infectious doses. thus, feco-oral transmission may only occur when the epidemic reaches an as yet unknown threshold. either way, those at risk within those settings are poor urban communities and informal settlements, which have the worst sanitation options and access to health care. individuals could become infected even if they were able to implement physical distancing recommendations, which themselves are a challenge, and need to be decisively protected from fecal sars-cov-2. 37 possible options for halting feco-oral sars-cov-2 transmission are disinfection of known open defecation sites, intensifying handwashing messages, encouraging boiling or chemical treatment of household water, and explicitly treating waste from isolation and treatment facilities. safer sewage management should be instituted or reinstituted, as priority where possible. these include ensuring that standard operating procedures are followed, and there are no interruptions in sewage decontamination, as well as quality assurance to ensure that decontamination goals are met. 11 stalled or slowed sanitation projects should be expedited, and new ones could be explored. individuals who have to work in or close to wastewater handling facilities, particularly those operating suboptimally, should be informed of their risk and provided with protection where feasible. in those situations, as research from the 2003 sars outbreak demonstrated, aerosolized virus poses a risk for respiratory transmission in addition to any feco-oral risk that may exist. 38, 39 on the positive side, fecal shedding of sars-cov-2 can be exploited for community surveillance of wastewater or human waste using similar methods that would be required for risk evaluation. 22, 40 enteric pathogen, polio, and antimicrobial resistance environmental surveillance could be leveraged, where these have been initiated, 10, 41, 42 but sites with no access to sewerage, typically not used for surveillance, must also be included. in high-risk settings, waste and wastewaterbased epidemiology could help balance sampling biases inherent in case-and contact-tracing-based human testing for covid-19 and consequently predict prevalence. 42, 43 it would also preemptively identify epidemic foci and ascertain the exact risk of community transmission via the fecal-oral route. indeed, it could represent a dedicated strategy to protect the poor and marginalized in whom outbreaks in this pandemic have typically been detected with significant lags. 44 although sanitation shortfalls risk sars-cov-2 feco-oral transmission much is focused on the current emergency, the potential risk from feco-oral sars-cov transmission should motivate and even initiate concrete steps toward lasting wastewater and sewage systems wherever possible. this would leave a post-covid-19 development legacy that could impact disease transmission, extend the value of other disease control strategies, 45 and improve the quality of life in the long term. sars-cov-2 productively infects human gut enterocytes the presence of sars-cov-2 rna in the feces of covid-19 patients characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding who, 2020. modes of transmission of virus causing covid-19: implications for ipc precaution recommendations: scientific brief prolonged presence of sars-cov-2 viral rna in faecal samples asymptomatic sars-cov-2 infected case with viral detection positive in stool but negative in nasopharyngeal samples lasts for 42 days faecal-oral transmission of sars-cov-2: practical implications screening faecal microbiota transplant donors for sars-cov-2 by molecular testing of stool is the safest way forward estimates of the global, regional, and national morbidity, mortality, and aetiologies of diarrhoea in 195 countries: a systematic analysis for the global burden of disease study contribution of environmental surveillance toward interruption of poliovirus transmission in nigeria assessment of wastewater discharge impact from a sewage treatment plant on lagoon water isolation and identification of enteroviruses from sewage and sewage-contaminated water in assessment of domestic wastewater management practices in the communal district i of maradi city, niger republic waste of a nation: garbage and growth in india we need to talk about crapping progress on sanitation and drinking water: 2015 update and mdg assessment safe drinking water and waterborne outbreaks bacterial contamination of drinking water sources in rural villages of mohale basin, lesotho: exposures through neighbourhood sanitation and hygiene practices the burden and characteristics of enteric fever at a healthcare facility in a densely populated area of kathmandu the dengue virus in nepal: gaps in diagnosis and surveillance a large and persistent outbreak of typhoid fever caused by consuming contaminated water and street-vended beverages sars-cov-2 in wastewater: potential health risk, but also data source aerodynamic analysis of sars-cov-2 in two wuhan hospitals coronavirus in water environments: occurrence, persistence and concentration methods -a scoping review concentration and detection of sars coronavirus in sewage from xiao tang shan hospital and the 309th hospital survival of coronaviruses in water and wastewater how sewage could reveal true scale of coronavirus outbreak presence and vitality of sars-cov-2 virus in wastewaters and rivers potential spreading risks and disinfection challenges of medical wastewater by the presence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) viral rna in septic tanks of fangcang hospital review article: gastrointestinal features in covid-19 and the possibility of faecal transmission covid-19 faecal-oral transmission: are we asking the right questions? enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? alert for sars-cov-2 infection caused by fecal aerosols in rural areas in china divining without seeds: the case for strengthening laboratory medicine in africa the ecological dynamics of fecal contamination and salmonella typhi and salmonella paratyphi a in municipal kathmandu drinking water the impact of combined sewage overflows on the viral contamination of receiving waters slum health: arresting covid-19 and improving well-being in urban informal settlements evidence of airborne transmission of the severe acute respiratory syndrome virus environmental transmission of sars at amoy gardens leveraging africa's preparedness towards the next phase of the covid-19 pandemic global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage pathogen surveillance in the informal settlement, kibera, kenya, using a metagenomics approach letter to the editor: wastewater-based epidemiology can overcome representativeness and stigma issues related to covid-19 the moment to see the poor impact of rotavirus vaccination varies by level of access to piped water and sewerage: an analysis of childhood clinic visits for diarrhea in peru sanitation shortfalls risk sars-cov-2 feco-oral transmission key: cord-323061-0i5w7vm9 authors: kharel sitaula, ranju; khatri, anadi; janani, m k; mandage, rajendra; sadhu, soumen; madhavan, h n; upadhyay, madan prasad; biswas, jyotirmay title: unfolding covid-19: lessons-in-learning in ophthalmology date: 2020-09-28 journal: clin ophthalmol doi: 10.2147/opth.s259857 sha: doc_id: 323061 cord_uid: 0i5w7vm9 importance: an observant chinese doctor li wenliang became the first physician to alert the world about covid-19. being an ophthalmologist himself, he has put the additional onus on us. the fact that the ocular manifestation could be the first presenting feature of novel coronavirus pneumonia should not be ignored and the possibility of spread of sars-cov-2 through the ocular secretions cannot be ruled out. however, with breakthroughs still evolving about this disease, the calls are now louder for closer examination on the pathogenesis of conjunctivitis associated with it. hence, we conducted a scoping review of all available literature till date to fill in the “potential” gaps in currently available knowledge on ocular manifestations of sars-cov-2 infection in an attempt to establish continuity in the “chain of information” from december 2019 till april 2020. we also summarize a possible hypothesis on much less understood and highly debated topics on regard to the etiopathogenesis of ocular involvement in sars-cov-2 based on either presence or absence of ace2 receptor in the ocular surface. methods: we conducted a scoping review search of published and unpublished sars-cov-2-related english language articles from december 2019 till mid of april 2020 from the online databases. the findings were summarized using text, tables, diagrams, and flowcharts. results: the commonest ocular manifestation in sars-cov-2 infection is follicular conjunctivitis and has been the first manifestation of sars-cov-2 infection in 3 reported cases till date. the ocular surface inoculated with the sars-cov-2 leads to the facilitation of the virus to the respiratory system via the lacrimal passage. rt-pcr analysis of the ocular secretions has shown the presence of the sars-cov-2 nucleotides indicating the possibility of infection of ocular secretions. ace2 receptors and its expression on the ocular mucosal surface are linked behind the etiopathogenesis of conjunctivitis. conclusion: conjunctivitis can be the presenting manifestation but may go unnoticed due to its mild nature. the ocular surface could serve as the entry gateway for the virus and ocular secretions could play a role in virus shed. the eye care personnel, as well as the general people, need to be more vigilant and adopt protective eye measures. the world health organization (who) initially used the term 2019 novel coronavirus to refer to the coronavirus disease that affects the lower respiratory tract of patients causing severe pneumonia in wuhan, china in december 2019 and revised it officially as covid-19. [1] [2] [3] the virus has received the final reference name and is now also commonly known as a severe acute respiratory syndrome coronavirus 2 (sars-cov -2), which is due to its similarity with severe acute respiratory syndrome coronavirus (sars-cov). [4] [5] [6] covid-19 outbreak turned to be pandemic hitting the 210 countries and its territories around the world and 2 international conveyances over the past 14 weeks till 17th april 2020. 7 sars-cov-2 is capable of active replication in the upper respiratory tissues, with continuous pharyngeal shedding of the virus. though the respiratory system is the primary site of insult, various reports have suggested that the eyes can be affected in the early or late stage of the disease process. [8] [9] [10] currently, our understanding of the possible ocular involvement in sars-cov-2 infection is very limited and gradually expanding. nevertheless, the fact that the ocular manifestation could be the first presenting feature of the novel coronavirus pneumonia should not be neglected by the general population, health professionals as well as policymakers and teams. 8, 9, 11, 12 there are recent reports even on the nosocomial sars-cov-2 infection with conjunctivitis in a nurse, suggesting eye as the potential route of nosocomial transmission of the sars-cov-2. 10 multiple respiratory droplets may be released when an infected person coughs, sneezes or speaks. these respiratory droplets contact host ocular mucus membranes. upon contact with an epithelial cell, the virus particle gains entry inside using the sars-cov-2 spike (s) glycoprotein which binds to the cell membrane protein angiotensin-converting enzyme 2 (ace2). the s glycoprotein is composed of 2 subunits s1 and s2. the s2 subunit is shown to be highly conserved with a fusion peptide, a transmembrane domain and a cytoplasmic domain. 13 whether the eyes are one of the preliminary sites of virus transmission from the environment to the lungs via the lacrimal passage or the virus reaches the eyes in retrograde fashion through the airways passage via the lacrimal system to eyes is still an issue to look upon. the presence or absence of angiotensin-converting enzyme (ace) 2 receptors on the ocular surface is still a topic of debate. the conjunctiva is directly exposed to the extraocular pathogens, and the mucosa of the ocular surface and upper respiratory tract is connected by nasolacrimal duct and may share some common entry receptors for these various respiratory viruses. 14 given the rapid worldwide spread of the novel coronavirus and its high impacts on morbidity and mortality on the human health, the ophthalmic professionals have to focus upon the research to understand the pathogenesis of conjunctivitis in covid-19 infection and to identify its impact on the eyes and shedding of the virus via ocular secretions. the knowledge on the sensitivity and specificity of rt-pcr in ocular secretion, the prevalence of sars-cov-2 nucleotides in the eye, and the viral load still needs full exploration. to fulfill this gap of knowledge, we aimed to conduct a scoping review to summarize and critically analyze all the published scientific articles regarding the sars-cov-2 virus and its infection in the eye till the mid of april 2020. this review aims to provide the possible hypothesis on the etiopathogenesis of the ocular involvement, routes of entry of the virus in the eye, presence/ absence of ace2 receptors in ocular surface, rt-pcr analysis in ocular secretions, review of various articles related with sars-cov-2 infections and brief preventive and protective measures of sars-cov-2 infection in ophthalmic practices to avoid the occupational hazard. we planned a scoping review following the methodological framework suggested by arksey and o'malley. 15 the following five stages were followed to conduct this scoping review: i) identifying clear research objectives and search strategies, ii) identifying relevant research articles, iii) study selection of research articles, iv) charting and extraction of data, and v) discussing, collating, summarizing and reporting the results. to achieve a comprehensive literature search (published and unpublished articles) and reviews, we adopted a strategy that involved searching the following online databases: pubmed, google scholar medrxiv, biorxiv, medline, cnki and wanfang data (the two primary databases for biomedical research in mainland china). also, some white papers published online by the national health commission of the people's republic of china, centre for disease control (cdc) and the world health organization were included in the analysis. we searched scientific publications from 1st january till the mid of april 2020. the search terms were 'sars-cov', 'sars-cov-2ncov', '2019 novel coronavirus', "2019-ncov", 'novel coronavirus', "pneumonia", 'covid-19ʹ, "ocular surface involvement", "conjunctivitis", "rt-pcr" and "ace2 receptor". we included all the relevant scientific publications written in english in the review (prospective/retrospective studies, case-series, case report, letter to the editor). around 1/3rd of these papers were at the press (preprint version) and rest were published in high impact peer-reviewed journals, including the new england journal of medicine, jama, and the lancet. we checked the bibliographies of these studies found through the database searches to ensure they can be included in the scoping exercise. the articles were scrutinized and selected by the common consent of the authors after the evaluation of its information regarding ocular involvement in sar-cov-2 infection. the data were summarized using text, tables, diagrams, and flow charts. this scoping review was approved by the local ethical committee of birat eye hospital and adhere to the declaration of helsinki. the ocular manifestations reported till now during sars-cov-2 infection are conjunctivitis, including conjunctival hyperemia, chemosis, foreign body sensation, increased secretions, and epiphora. 8 normal visual acuity, intact corneal epithelium, quiescent anterior chamber, and no tenderness or enlargement of the preauricular lymph node has been mentioned along with the conjunctival findings. 10 epiphora and conjunctival redness had been the first manifestation of sars-cov-2 infection in 3 reported cases till date which includes a member of national expert on pneumonia during his visit to endemic areas of wuhan and an anesthesiologist contracting the virus from a known patient of novel coronavirus pneumonia during intubation in italy; similarly, it was reported in a nurse working in the emergency department of ophthalmology who presented with viral conjunctivitis and watering as a first sign. 12, 16 all reported subjects were fully gowned with personal protective equipment but with no or dislocated protective eyewear. these incidences of anesthesiologist and nurse are only some of the proof of the nosocomial spread of sars-cov -2 in the eyes. 3, 8, 9, 12, 14, 16 dr. li wenliang, the chinese ophthalmologist and the first whistleblower about this disease himself is assumed to get infected by sars-cov-2 from an asymptomatic glaucoma patient and died on february 7, 2020. 17 similarly, dr. jay m galst, a renowned ophthalmologist lost his life on 12th april due to sars-cov -2 infection. both have put the additional onus on us ophthalmologists. the clinical presentation of conjunctivitis is usually characterized by signs and symptoms of viral conjunctivitis. it was recently reported that in a hospitalized patient of covid-19, who complained of ocular redness and watering post 13 days of illness showed bilateral moderate conjunctival injection along with watery discharge and inferior palpebral conjunctival follicles which resembles signs of acute viral conjunctivitis. his conjunctival swabs were also tested positive for sars-cov-2 rna. he was treated with ribavirin eyedrops four times a day and showed symptomatic and clinical resolution of conjunctivitis within 5 days. 18 present understanding shows that sars-cov-2 conjunctivitis has no specific manifestation, can be follicular, and can present in one eye or both. in the early stage, it might appear as common conjunctival hyperemia with fewer watery secretions and akin to thin mucus. occasionally conjunctival hemorrhage in few is also mentioned. 9 the ocular symptoms of the patients can be mild and self-healing such that many covid-19 patients might not bother to report it. so the symptoms may be noted in the early or late phase of the disease. however, correlations between conjunctivitis and covid-19 could also be confounded by co-correlation with unobserved factors. 9 the presence of binding receptors in the ocular surface, the binding affinity between the binding receptors and sars-cov-2 virus, the viral tropism and viral load all are responsible for the manifestations in the form of conjunctivitis. it has also been proven that rhesus monkeys can be infected with sars-cov-2 via the conjunctival route viral load was comparatively high in the nasolacrimal system and lesions in the lung were relatively mild and local when the sars-co-2 entered via the conjunctival route. 19 however, at present, there has been no proven literature to support that the ocular surface abnormalities in covid-19 patients are related to primary eye infection or secondary eye infection. hence, our knowledge and understanding about the sars-cov-2 virus, modes of entry to the eye, hypothesis on the interaction with the renin-angiotensin system (ras) system and ace2 receptor and ocular pathogenesis and rt pcr analysis from the ocular secretions have been summarized below using text, tables, diagrams, and flowcharts. we also tabulated 12 articles' papers related to the ocular manifestation of sars-cov-2 infection and compared the ocular and laboratory findings with the past sars outbreak. coronaviruses (cov) belong to the subfamily coronavirinae, in the family coronaviridae of the order nidovirales. there are four genera: alphacoronavirus, (figure 1 ). coronaviruses are enveloped single-stranded positivesense rna possessing the largest genome of rna viruses (26-32 kb bp in size) with varying g + c contents (32% to 43%). 19 the genomes are polyadenylated at the 3ʹ end and those are the largest genome among rna virus. through genomic study, it is established that sars-cov -2 and sars-cov share the same cell receptor in the human while mers-cov uses dipeptidyl peptidase 4 (dpp4) as a gateway to enter into human cells. 22 over two decades, the virus form beta coronovirinae has been causing many epidemics and pandemic outbreak. these viruses are believed to be zoonotically transmitted and cause secondary transmission from human-to-human. previously known coronaviruses like human coronavirus (hcov) −229e, hcov-nl63, hcov-oc43, and hcov-hku1 were known to cause mild self-limiting symptoms until the identification of three more opportunistic strands that can cause severe pneumonia named acute respiratory syndrome (sars)-cov-1 in 2002, middle eastern respiratory syndrome (mers)-cov in 2012 and more recently sars-cov-2 in december 2019 as shown in table 1 . the morphology and the properties of the viral protein of sars-cov-2 virus and its replication after binding with the target ace2 receptor of the target cell are depicted in figures 2 and 3 . through the ocular route the exposed ocular surface can serve as a gateway to acquiring respiratory viruses. after a detailed review of the various manuscripts, we hypothesize the following probable entry routes of the virus to the eye which is shown in table 2 . besides the nasal mucosa, the sars-cov-2 virus may have alternate assess to the lungs through the eyes where they can manifest the ocular disease or further travel down to the lungs over a week as shown in figure 4 . in the primary stage of the infection, the virus coming into the contact with mucosal region of the eye could be absorbed by the attachment of viral spike protein to the host cell receptor present in the contact region forming antigen receptor complex. this complex is proteolytically processed by type 2 transmembrane protease (tmprss2) leading to subsequent cleavage of ace-2 receptor and activation of viral spike protein thus facilitating the viral entry. [22] [23] [24] the virus may be partly absorbed through conjunctiva or cornea via the nasolacrimal duct or directly enter into the host cells of the upper respiratory tract. this causes primary viremia. the virus replicated in the upper the efficacy of virus entry into host cells depends on three points: the invasiveness of the virus, viral receptors on the host cell membrane, and the immune status of the host. the virus invades the host cell by binding to the angiotensin-converting enzyme 2 (ace 2) receptor via viral spike proteins. the type ii transmembrane serine protease (tmprss2) binds and cleaves the ace-2 receptor. 22 the renin-angiotensin system (ras) plays an important role in the regulation of blood pressure mainly via fluid and electrolyte homeostasis. 25 besides this, ras is also responsible for the tissuespecific regulatory system and is accountable for long term "adaptive" regional changes. these are also found in various glands of the body including intricate regulation of homeostasis in the special sensory -the eye. 26 although ras is associated with various enzymes and peptides, angiotensin-converting enzyme 2 has gained special interest recently. this has been studied well and was found to be the "portal" of entry for the sars-co-v virus during the outbreak of 2003-04. 27 ace 2 is mainly found in the smooth muscles of the upper and lower respiratory tracts including the alveolar wall. while many of the previous literature agree on the presence and its distribution in certain tissues of the eye, the growing debate of its presence in the conjunctiva has become controversial. 9 and with this, the pathogenesis of conjunctivitis in sars-cov-2 infection seems to be much less understood. 25 if the virus were to inflict the cascade of inflammatory reaction on the conjunctiva as per our understanding of its pathogenesis in the respiratory system, one would, without doubt, expect the presence of ace2 receptors on the conjunctiva. some reports also agree to the fact that ace2 receptors are present in the conjunctiva and hence make the above theory more convincing. 9, 25 but after the reports from the centre for evidence-based medicine (cebm) suggested that conjunctiva is free of the ace2 membrane protein, the theory seems to dismantles itself. 28 with reports now suggesting that the virus merely get shed from tears, the enigma only gets more difficult. 11, [29] [30] [31] 33 so far, we know that the chromosome of sars-cov-2 is 82% similar to that of sarscov. 34 if the ace2 receptor exists in conjunctival mucosa, the interaction between the sars-cov-2 virus and the conjunctiva can produce conjunctivitis with features of hyperemia, chemosis, and watery secretion. but the nature of inflammation of the conjunctiva could be milder than the respiratory tract. some reports have predicted the presence of the sars-cov-2 virus in conjunctival samples as early as 2-3 days of systemic disease onset. 13, 31 though present, the positive expression of ace2 in the human ocular surface might be much less than in human lung and kidney tissues. 13 thus, in short, if the ace2 receptor exists in conjunctival mucosa, the interaction between the sars-cov-2 virus and the conjunctiva can produce conjunctivitis with features of hyperemia, chemosis, and watery secretion. hypothesis 2: it is known that even the swab from the oropharynx, despite having ace2 receptors is not a very good site for collecting specimens for detecting the virusrather it's the nasopharynx. 9, 35 contemplating, same could hold true and the conjunctiva may not be a proper specimen to analyze for detection of the virus. the transmigration of the virus could make the virus an "intrinsic" pathogen and hence may not be present on the conjunctival surface or even in the tears. the squamous cells which are shed from the conjunctiva or a conjunctival tissue may harbor the virus and could be studied in much detail. 25, 32 if ace2 receptors are absent in the conjunctiva hypothesis 3: even if ace2 receptor may be absent in the conjunctiva, the ocular surface may harbor the sars-cov thus in short, if we assume that conjunctival mucosa lacks ace2 receptors, these ace2 receptors are also found on the various cell lineages of the macrophages and other immune cells including the eye. if such cells are activated, the degranulation process which follows with the release of inflammatory markers could produce conjunctivitis-like symptoms which share the common pathogenesis with allergic rhinitis and conjunctivitis. based upon the literature published till mid of april 2020, the hypotheses of entry of the virus into the eyes of its interaction with the target tissue are summarized in figure 4 . hypothesis 4: even if we assume that conjunctival mucosa lack ace2 receptors, these ace2 receptors are found on the various cell lineages of the macrophages and other immune cells-including the eye. 36 if such cells are activated, the degranulation process which follows with the release of inflammatory markers could produce conjunctivitis like symptoms which share the common pathogenesis with allergic rhinitis and conjuncitivitis. these hypotheses of entry of the virus into the eyes, its interaction with the target tissue are summarized in figure 5 . we have analyzed and tabulated the 12 articles published about the ocular involvement and outcome of ocular fluid sampling in sars-cov-2 outbreak from dec 2019 till mid of april 2020. we have compared their ocular and laboratory findings with a sars article published in 2003 (table 3) . univariate analysis by few authors has shown that the patients with ocular symptoms were more likely to have higher white blood cell, neutrophil counts, procalcitonin, c-reactive protein, and lactate dehydrogenase than the patients without ocular symptoms. 8 they also suggested that ocular manifestations are more common amongst covid-19 patients with more severe pnuemonia. 8 besides conjunctivitis, other ocular surface abnormalities like dry eyes, pterygium, corneal abrasions, and ulcerations due to sars-cov-2 tropism are still an area to explore in the future. the evaluation of retinal involvement along with the impact of retinal and choroidal circulation in covid-19 cases with ocular involvement is an arena to work upon. the conventional rt-pcr techniques using primer probes targeting the envelope (e) gene for screening and amplification targeting the rna-dependent rna polymerase (rdrp) genes for the confirmation are considered as the gold standard method for diagnosis of sars-cov-2 in respiratory secretions and this may hold true for the ocular samples from conjunctival secretion and tears. 2, 36 there are reports mentioning the concentration of sars-cov-2 nucleotide in the nasopharynx and throat to be of 10 4 -10 10 rnas/swab but the literature to document the exact concentrations in the ocular secretions is limited. 35 hence, the knowledge on the sensitivity and specificity of rt-pcr in ocular secretion, the prevalence of sars-cov-2 nucleotides in the eye and the viral load still needs to be explored in the coming days. the present understanding of the outcome of the rt-pcr analysis in the ocular samples is mainly based on the following-conjunctival specimen pcr analysis -one report has mentioned low prevalence (5.2%; 95% ci, 0.617.8) of sars-cov-2 nucleotides in conjunctival specimens of patients with covid-19 but many have reported negative presence. 8, [28] [29] [30] tear film pcr analysis -few reports have documented the presence of sars-cov-2 in tear fluid while others have not detected sars-cov-2 nucleotides in the tear specimen. [28] [29] [30] [31] 37 the reports of nil prevalence to an extremely low positive rate of sars-cov-2 nucleotides in the tears and conjunctival secretions from the covid-19 patients need to be analyzed cautiously. the inappropriate timing of ocular sample collection (too early or too late of systemic disease), poor specimen site, faulty technique, unknown sensitivity of current rt-pcr technique in ocular fluid, local ocular immune system activation with a significant increase in lactoferrin and secretory iga levels in tears could be some of the factors contributing to the low positive rate of rt-pcr analysis of the ocular samples. since reports have suggested that conjunctivitis in sars-cov-2 infection cases could be both early or late presentation, we suggest that it would be better to perform the rt-pcr analysis of the ocular secretions in both early and late stage of infection to minimize the chance of missing the false negative report of rt-pcr results in tears and conjunctival secretions. the lower of the viral concentration and diverse genome fraction in the eye compared to the tracheal aspirates could be one of the contributing factor. 10 however, the study conducted among the rhesus monkey eyes showed that after direct inoculation of the sars-cov-2 via ocular conjunctival inoculation; the viral load was detectable in several nasolacrimal system associatedtissues, especially in the conjunctiva, lacrimal gland, nasal cavity and throat, which drew the outline of the anatomical bridge between ocular and respiratory tissues. 19 thus, lacrimal duct serving as a conduit to collect and drain the sar-cov-2 from ocular to respiratory tract tissues via inferior meatus. 19 until proven via large-scale analysis, it is always better to urge on the worst side and consider the ocular secretions as an infectious and potential source for the spread of contamination. the infectivity of the tears and conjunctival secretion from affected patients may have impacts not only in the daily ophthalmic practice but also on the universal infection control measures adopted by the general public and health-care professionals as it may have the potential to catalyze the spread. 39 hence, a safe ophthalmic practice is needed for the ophthalmologists and their patients to protect themselves. 15 thus, the best option, for now, is the adoption and improvisation of the precaution measures to break the chain of the spread of thesars-cov-2 virus and infection in the community and ophthalmic practices. 46 some of the effective measures are shown in figure 6 . the ocular surface mucosa could represent a target organ and a "portal" of entry or transporter of sars-cov-2 to infect the respiratory tract. the presence or absence of the ace2 receptors in the conjunctival tissue and nasolacrimal passage is still debatable but may not even be required for producing ocular manifestations. the possibility of lesser affinity to the receptors to the eye compared to other organs such as nose and airway passage could also mean an "unpreferred" gateway for sars-cov-2. the ocular involvement either as the first manifestation or late feature during sarsfigure 6 preventive and protective measures to avoid the entry of contaminated droplets and aerosols in the eyes. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia coronavirus disease (covid-19) technical guidance: laboratory testing for 2019-ncov in humans. who; 2020 novel coronavirus outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle novel coronavirus (2019-ncov) situation report-5 christening of new coronavirus and its disease name create confusion updated characteristics of ocular findings of patients with coronavirus disease 2019 (covid-19) in hubei province ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva the evidence of sars-cov-2 infection on ocular surface 2019-ncov transmission through the ocular surface must not be ignored peking university hospital wang guangfa disclosed treatment status on weibo and suspected infection without wearing goggles epidemiology, genetic recombination, and pathogenesis of coronaviruses role of the eye in transmitting human coronavirus: what we know and what we do not know. front public health scoping studies: towards a methodological framework the covid-19 pandemic from an ophthalmologist's perspective ocular manifestations of a hospitalised patient with confirmed 2019 novel coronavirus disease rhesus macaques can be effectively infected with sars-cov-2 via ocular conjunctival route emerging coronaviruses: genome structure, replication, and pathogenesis an analysis based on decade-long structural studies of sars 3, jvi accepted manuscript posted online 29 evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury many faces of renin-angiotensin system-focus on eye the ocular renin-angiotensin system: a therapeutic target for the treatment of ocular disease ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia medicine (cebm) cfe-b. spreading sars-cov-2 through ocular fluids assessing viral shedding and infectivity of tears in coronavirus disease 2019 (covid-19) patients can the coronavirus disease 2019 (covid-19) affect the eyes? a review of coronaviruses and ocular implications in humans and animals evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov-2 infection the severe acute respiratory syndrome genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan comparative accuracy of oropharyngeal and nasopharyngeal swabs for diagnosis of covid-19. oxford covid-19 evidence service team centre for evidence based medicine high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa assessing viral shedding and infectivity of tears in coronavirus disease 2019 (covid-19) patients the severe acute respiratory syndrome coronavirus in tears covid-19: limiting the risks for eye care professionals clinical characteristics of coronavirus disease 2019 in china there may be virus in conjunctival secretion of patients with covid-19 comparisons of viral shedding time of sars-cov-2 of different samples in icu and non-icu patients ocular manifestation, eye protection, and covid-19. graefe's archive clin exp ophthalmol novel coronavirus disease 2019 (covid-19): the importance of recognising possible early ocular manifestation and using protective eyewear screening for novel coronavirus related conjunctivitis among the patients with corona virus disease-19 covid-19 and ophthalmology: an underappreciated occupational hazard we would like to thank dr. gunjan prasai for proofreading and the linguistic support. there is no funding to report. the authors report no conflicts of interest in this work. cov-2 infection should also not be under looked. therefore, it may be advisable to perform rt-pcr analysis of ocular secretion in both the early and late phases.at the same time, the infectivity of the ocular secretions of the covid-19 infection should not be ignored. due to close doctor-patient distance in ophthalmic practice, social distancing is virtually impossible with greater apt for transmitting sars-cov-2 virus by droplets, aerosols, ocular secretions, and contaminated ocular instruments. thus, hand hygiene and personal protection are recommended for health care workers to avoid hospitalrelated viral transmission during ophthalmic practice.since there are newer reports of sars-cov-2infections coming with recurrence, it would be better to instruct the patients are instructed to report early if they have any ocular problem, however trivial for being a harbinger of more severe disease. key: cord-338775-gh3a0wuf authors: gulersen, moti; prasannan, lakha; tam, hima tam; metz, christine n.; rochelson, burton; meirowitz, natalie; shan, weiwei; edelman, morris; millington, karmaine a. title: histopathological evaluation of placentas after diagnosis of maternal sars-cov-2 infection date: 2020-08-15 journal: am j obstet gynecol mfm doi: 10.1016/j.ajogmf.2020.100211 sha: doc_id: 338775 cord_uid: gh3a0wuf abstract background the impact of maternal sars-cov-2 infection on placental histopathology is not well known. objectives to determine if significant placental histopathological changes occur after diagnosis of sars-cov-2 infection in pregnancy and whether these changes are correlated with the presence or absence of symptoms associated with infection. study design retrospective cohort study of women diagnosed with sars-cov-2 infection who delivered at a single center from april 9th to april 27th, 2020, and had placental specimens reviewed by pathology. women with singleton gestations and laboratory-confirmed sars-cov-2 infection were eligible for inclusion. historical controls selected from a cohort of women who delivered 6 months prior to the study period were matched in a 1:1 fashion by week of gestation at delivery. histopathological characteristics were evaluated in each placenta and the incidence of these findings were compared between placentas after diagnosis of maternal sars-cov-2 infection and historical controls, as well as between placentas from patients with or without typical symptoms related to infection. statistical analysis included use of wilcoxon rank sum test and fisher’s exact test for comparison of categorical and continuous variables. statistical significance was defined as p value < 0.05. results a total of 50 placentas after diagnosis of maternal sars-cov-2 infection and 50 historical controls were analyzed. among placentas from patients diagnosed with sars-cov-2 infection, 3 (6%) were preterm (33 3/7, 34 6/7 and 36 6/7 weeks of gestation), 16 (32%) were from patients with typical symptoms related to infection and 34 (68%) were from patients without typical symptoms related to the infection. all patients had diagnosis of sars-cov-2 infection in the third trimester. decidual vasculopathy was not visualized in any of the placentas from patients diagnosed with sars-cov-2 infection. there was no statistically significant difference in placental histopathological characteristics between the groups. sars-cov-2 testing for all neonates at 24 hours of life was negative. conclusions based on our data, there are no significant placental histopathological changes that occur after diagnosis of sars-cov-2 infection in the third trimester of pregnancy compared to a gestational age-matched historical control group. similar incidences of histopathological findings were also discovered when comparing placentas from patients with sars-cov-2 infection with or without the presence of symptoms typically related to infection. with sars-cov-2 infection have been reported thus far, including miscarriage 2,3 , intrauterine 98 fetal demise 4 , preeclampsia 5,6 , preterm delivery 7-9 , maternal critical illness 7-10 and death 11 , as well 99 as neonatal death 12, 13 . evidence regarding the occurrence of antepartum or peripartum vertical 100 transmission has been conflicting to date. 14-18 101 the placenta represents a highly specialized organ that is crucial for maintaining an optimal 102 environment for fetal development. 19,20 placental evaluation after delivery provides useful 103 information such as the identification of disease processes in the mother or infant that require 104 attention or diagnoses that provide a specific explanation for an adverse outcome. 21 105 characteristic histopathological findings in placentas from mothers with viral infections have 106 been reported. 22-25 however, reports on placental evaluation in women with sars-cov-2 107 infection have been limited to few case series [26] [27] [28] [29] and the association between infection and 108 abnormal placental findings is not well known. therefore, the objective of this study was to 109 determine if any significant placental histopathological changes occur after diagnosis of sars-110 cov-2 infection in pregnancy and whether these changes are correlated with the presence or 111 absence of symptoms typically related to infection. 112 this was a retrospective cohort study with historical controls. cases included placentas from all 116 women diagnosed with sars-cov-2 infection and delivered at a single center (long island 117 jewish medical center -northwell health, queens, ny) from april 9 th to april 27 th , 2020 118 (during the peak of the pandemic in new york). the northwell health institutional review 119 board approved this study as minimal-risk research using data collected for routine clinical 120 practice and waived the requirement for informed consent. women with singleton gestations 121 who had laboratory-confirmed sars-cov-2 infection during their pregnancy were eligible for 122 inclusion. diagnosis of sars-cov-2 infection was confirmed using qualitative real-time 123 polymerase chain reaction (pcr) on maternal nasopharyngeal swab specimens. prior to the study 124 period, universal testing for sars-cov-2 infection had been implemented for all obstetrical 125 patients admitted to labor and delivery. after delivery, placentas were submitted to the 126 pathology department for evaluation. placentas from women with a high clinical suspicion for 127 placenta accreta diagnosed during the antepartum period, on either ultrasound or magnetic 128 resonance imaging, were excluded. placental specimens from cases with accreta had morbidly 129 adherent uterus. as a result, several gross examination characteristics, such as placental weight, 130 could not be obtained, and standardized sampling of each placenta for histologic evaluation 131 would not be consistent with that of other placentas in the study. 132 historical controls were selected from a cohort of women who had placentas submitted to the 133 pathology department (including specimens obtained from delivery at north shore university 134 hospital -northwell health) during november 2019, at least 3 months prior to the first reported 135 case of sars-cov-2 infection in new york. the decision for pathological examination at that 136 time was at the discretion of the delivery physician. however, suggested criteria provided by the 137 pathology department were generally consistent with the college of american pathologists 138 guidelines. 30 historical controls were used, as placentas from patients who tested negative for 139 sars-cov-2 infection during the study period may not have represented an appropriate control 140 group due to reported high false negative rates. 31 controls were matched in a 1:1 fashion by 141 week of gestation at delivery. gestational age at delivery was selected as the matching variable 142 health. 33 mild cases were defined as those who have symptoms typically related to sars-cov-172 2 infection, but without dyspnea or abnormal imaging. moderate cases were defined as those 173 who had evidence of lower respiratory disease by clinical assessment or imaging and a blood 174 oxygen saturation > 93% on room air. severe cases were defined as those with respiratory rate ≥ 175 30 breaths per minute, blood oxygen saturation ≤ 93% on room air, partial pressure of arterial 176 oxygen to fraction of inspired oxygen < 300 and/or lung infiltrates > 50%. critical cases were 177 defined as those that exhibited respiratory failure, septic shock and/or multiple organ dysfunction 178 or failure. 179 two comparative analyses evaluating histopathological characteristics of placentas were 180 performed. the first comparison was between placentas from women after diagnosis of sars-symptoms related to the infection and those who did not have such typical symptoms. statistical 184 analysis included use of wilcoxon rank sum test and fisher's exact test for comparison of 185 categorical and continuous variables. statistical significance was defined as p value < 0.05. 186 during the study period, a total of 52 placentas from singleton gestations diagnosed with sars-189 cov-2 infection were submitted to the pathology department. two cases with placenta accreta 190 were excluded from the analysis. therefore, the study cohort included 50 placentas after cov-2 testing via pcr for all neonates at 24 hours of life was negative. 201 maternal age, race/ethnicity, parity, gestational age at delivery, mode of delivery, neonatal 202 birthweight, and the number of antepartum or intrapartum complications were similar between 203 patients with sars-cov-2 infection and historical controls (table 1) . histopathological findings 204 such as accelerated villous maturation or decidual vasculopathy were not visualized in any of the 205 placentas from patients with sars-cov-2 infection. there was no statistically significant 206 difference in maternal vascular malperfusion histopathological characteristics such as distal 207 villous hypoplasia (4% vs. 2%), excessive infarction (8% vs. 8%) and old hemorrhage in 208 membranes (2% vs. 4%) between the two groups (table 2 ). there was also no statistically 209 significant difference in fetal vascular malperfusion (8% vs. 12%), increased perivillous fibrin 210 (12% vs. 14%), intervillous thombi (26% vs. 16%), chorangiosis (6% vs. 2%) or presence of 211 meconium staining (18% vs. 10%) between the two groups ( table 2) . 212 on subgroup analysis, maternal age, race/ethnicity, parity, gestational age at delivery, mode of 213 delivery, neonatal birthweight, and the number of antepartum or intrapartum complications were 214 similar between patients with sars-cov-2 infection who had typical symptoms related to the 215 infection and those who did not have such typical symptoms (table 3) . time interval from 216 diagnosis of sars-cov-2 infection to delivery was significantly higher among patients who had 217 typical symptoms related to the sars-cov-2 infection compared to those who did not have such 218 typical symptoms (12.5 days vs. 0.5 days; p value < 0.001). there was no statistically significant 219 difference in maternal vascular malperfusion histological characteristics such as distal villous 220 hypoplasia (0% vs. 5.9%), excessive infarction (18.8% vs. 2.9%) and old hemorrhage in 221 membranes (6.2% vs. 0%) between the two groups (table 4 ). there were also no statistically 222 significant differences in fetal vascular malperfusion (6.2% vs. 8.8%), increased perivillous 223 fibrin (12.5% vs. 11.8%), intervillous thombi (37.5% vs. 20.6%), chorangiosis (6.2% vs. 5.9%), 224 or presence of meconium staining (25% vs. 14.7%) between the two groups (table 4) . 225 the results of our study did not demonstrate significant placental histopathological changes 229 occurring after diagnosis of sars-cov-2 infection in the third trimester of pregnancy compared 230 to a gestational-age-matched historical control group with a similar incidence of antepartum or pathology for examination or due to history of melanoma. 26 compared to all historical controls, 240 placentas from women infected with sars-cov-2 in their cohort showed a higher incidence of 241 decidual arteriopathy (47% vs. 16%; p value = 0.04), delayed villous maturation (27% vs. 4%; p 242 value < 0.001), chorangiosis (27% vs. 5%; p value = 0.001), and intervillous thrombi (40% vs. 243 9%; p = < 0.001). 26 rates of delayed villous maturation, chorangiosis, and intervillous thrombi 244 were lower in our cohort of placentas from pregnancies with sars-cov-2 infection, and reassuring. however, in the absence of placental sars-cov-2 testing and standardized serology 294 testing for neonates, we cannot assume or refute vertical transmission. furthermore, whether 295 these pathology findings would be similar after first or second-trimester sars-cov-2 infection 296 and whether earlier infection increases likelihood of vertical transmission is unknown and 297 requires further study. 298 there are several strengths to this study. to our knowledge, and based on a review of the 300 literature, this is the largest reported cohort of examined placentas from pregnancies with sars-301 cov-2 infection and first report comparing histopathological findings in women with and 302 without symptoms related to infection. our control group was matched by gestational age and 303 had the same median maternal age, thus limited possible confounders. our population is 304 diversified in terms of demographics and derived from new york, where the total number of 305 novel coronavirus infections is among the highest worldwide. lastly, all placentas were reviewed 306 by one pathologist subsequent to the initial histopathologic examination, which reduced the 307 impact of interobserver variability. 308 there are also several limitations to this study. the pathologist was not blinded to the clinical 309 history or status of sars-cov-2 infection for each placenta examined, which may have 310 introduced bias regarding interpretation. this is unlikely since no pattern of significant 311 histopathological changes were found. despite a larger sample size of cases relative to other 312 reports in the literature, several outcomes had relatively low frequencies. this may have limited 313 power to detect significant differences, particularly when histopathological characteristics in 314 placentas from infected patients with or without symptoms related to sars-cov-2 infection 315 were compared. we also utilized a historical control group with several clinical diagnoses that 316 may have contributed to the abnormal pathological findings, as placentas are not routinely sent 317 for examination at our institution. however, rates of these diagnoses were similar between cases 318 from infected pregnancies and controls. we did not test each placenta for the presence of sars world health organization. who coronavirus disease 2019 (covid-19) situation report -348 139 coronavirus disease 2019 (covid-19) in pregnant women: a 351 report based on 116 cases radiological findings and clinical 353 characteristics of pregnant women with covid-19 pneumonia maternal death due to covid-19 preeclampsia treatment in severe acute respiratory 358 syndrome coronavirus 2 clinical characteristics of 46 pregnant women 367 with a sars-cov-2 infection in washington state clinical course of severe and critical covid-370 19 in hospitalized pregnancies: a us cohort study intensive care unit admissions for pregnant and 373 nonpregnant women with coronavirus disease maternal death due to covid-19 covid-19) on maternal, perinatal and neonatal outcomes: a systematic review clinical manifestations and outcome of sars-cov-2 mechanisms and evidence of vertical 383 transmission of infections in pregnancy including sars-cov-2 factors preventing materno-fetal transmission of sars vertical transmission of covid-19: sars-cov-2 rna on the fetal side of the placenta in pregnancies with covid-19 positive mothers and 391 neonates at birth evidence for and 393 against vertical transmission for sars-cov-2 (covid-19) pathology of the human placenta the human placenta project: placental structure development, and function in real time classification of placental lesions placental morphology in cytomegalovirus 401 infection notes from the field: evidence of zika virus 403 infection in brain and pla-cental tissues from two congenitally infected newborns and two fetal 404 losses-brazil pathology of congenital zika 406 syndrome in brazil: a case series placental pathology in 410 covid-19 placental pathology in covid-19 positive mothers: preliminary 412 a mechanistic analysis placental 414 intravascular thrombus formation in covid-19 patients pregnancy with new coronavirus infection: clinical 417 characteristics and placental pathological analysis of three cases practice guideline for examination of the 420 placenta false negative tests for sars-cov-2 infection challenges and implications national institutes of health. management of covid-19 early transmission dynamics in wuhan, china coronavirus-infected pneumonia the incubation period of coronavirus disease covid-19) from pubicly reported confirmed cases: estimation and application. ann intern 435 med attention should be paid to venous thromboembolism 437 prophylaxis in the management of covid-19 coronavirus disease 2019 in pregnancy: consider thromboembolic 439 disorders and thromboprophylaxis genomic characterisation and epidemiology of 2019 novel 441 coronavirus: implications for virus origins and receptor binding the sars-cov-2 receptor ace2 expression of 443 maternal-fetal interface and fetal organs by single-cell transcriptome study we would like to acknowledge the contributions of the northwell health covid-19 research 344consortium. 345 key: cord-340305-jtvn9tlm authors: cimolai, nevio title: a minimalist strategy towards temporarily defining protection for covid-19 date: 2020-09-19 journal: sn compr clin med doi: 10.1007/s42399-020-00533-4 sha: doc_id: 340305 cord_uid: jtvn9tlm until either efficacious therapy or vaccination for covid-19 is achieved, there will be a need to regain world economic stability while yet controlling the pandemic with current approaches. for those infected thus far, there is a prevailing perspective that devising recognition for protective immunity will progressively allow segments of society to return to some functionality more than is existing. at this time, the best correlates with protection from natural coronavirus infections are systemic neutralizing antibody and mucosal iga. serum neutralizing antibody more easily fulfills the latter requisite, but current live virus methods for neutralization prevent large-scale application. it is conceivable that the exposure of previously infected individuals can allow for the definition of protective thresholds of neutralizing antibody. thereafter, many other antibody assays will be able to screen for surrogate protection after correlations with protective neutralizing antibody are made. specificity of common antibody tests would benefit from confirmatory blocking systems or confirmatory immunoblotting fingerprints with well-defined antigen(s). the opportunity for the scientific community to make these assessments is evident in the current context of the covid-19 epidemic given the large numbers of infected individuals worldwide. such information will also be vital to guide vaccine development and/or immunotherapy. the clinical burden of covid-19 in the current pandemic is undoubtedly considerable, but the socioeconomic burden must equally weigh in determining how the world will move forward until either an effective chemotherapy and/or vaccine is devised [1] . pending that such success in treatment and/or prevention are achieved, the continuous or relapsing lockdown of societies and hence economies has the potential to cause more damage than may be initially apparent [2] . indeed, it could be prognosticated that the relative economic standstill may cause more damage to humanity than the disease itself. many countries have attempted to partially restore pre-pandemic functions only to experience yet second waves of increasing infections during july and august, 2020. to some, the answer may be to tolerate the expectations of herd immunity with lesser in the way of resistance to infection spread [3, 4] . to others at the other end of the spectrum, a more cautious approach has been to create the best environment for disease prevention while patching holes for or propping up failures in the economy as they appear [3] . somewhere in the midst of this maelstrom, there will need to be practical strategies for achieving success with both the pandemic and the economies simultaneously. contexts of disease and economy will no doubt vary along a spectrum and may require somewhat different approaches in their detail. in this review, a minimalist strategy is proposed to in part provide some solutions towards regaining economic focus while preventing disease. these steps are a modest beginning from the perspective of devising recognition for protective immunity that will progressively allow segments of society to proceed with their lives as they once were or nearly so. such a strategy will thereafter be enhanced as treatment or vaccine developments arise. studies with passive immunity are highly suggestive that antibody has a significant role in protection of coronavirus infection [5] [6] [7] [8] [9] [10] [11] [12] . the latter includes passive administration of anti-sars-cov-2 human monoclonal antibody in animal systems [13] . these findings are critical to our theme since direct antibody in some way may be used as the correlate with neutralization if not directly then by association. therefore, the prospects of finding a serological assay based on antibody detection that defines in some way neutralization and then after disease prevention are considerable. infection with coronaviruses generally protects against reinfection [12, [14] [15] [16] [17] [18] [19] [20] [21] [22] . analogous to the role of passive immunity, pre-existing immunity can potentially be defined with the right measures. both parenteral neutralizing antibody and secretory iga (siga) are associated with protection in model systems [14, 15, [23] [24] [25] [26] [27] . siga has logistical problems with collection and analysis, although technological advances are quite likely capable of overcoming the latter. therefore and in the interim, serum neutralizing antibody measurements will generally be applied as the standard, and other correlates of the latter could be put into common use. for mers, mild infection was associated with decreased antibody levels [28] [29] [30] . severe infections are associated with long-lived neutralizing antibody. increased neutralizing antibody is associated with decreased viral shedding, but there have not been enough natural infections to allow for study and analysis of natural protection as would be desirable. likewise for sars, field studies for the practical protective effects of neutralizing antibody could not be studied due to the short-lived spread of the virus. in studies for sars-cov-2, of over 700 control sera from uninfected patients, no neutralizing antibodies were found [31] . severe disease was associated with earlier generation of anti-sars-cov-2 antibody, and peak neutralizing antibody was measured at approximately 2 ½ weeks. stereotypic antibody responses to sars-cov-2 led to hypotheses that there should be cross-reactivity with sars-cov in the constant rbd [32] . in both humans and animals, such cross-reactivity was suspected to be due to non-neutralizing anti-s antibody again likely relating to a conserved region [33] . crossneutralization was however uncommon, but conceivably there could be other reasons for any such non-specificity [34] [35] [36] . among humans, s-specific antibody dominates and may correlate better with neutralization [34, 35, 37] . furthermore, the correlation between antibodies for different antigens (e.g., s and n proteins) may not be as initially conceived [35, 38] . human monoclonal antibodies that neutralize virus or pseudovirus especially recognized the rbd [39] . collaborative groups have found a strong association between neutralizing antibodies and igg antibodies to the rbd [36, 40] . others have found strong correlations between neutralizing antibodies and eia-detected antibodies to various sars-cov-2 antigens [41, 42] .some have found diversity in immune responses contingent on the nature of presenting disease [38, 43] . the latter would be consistent with the variable antibody responses now determined for the spectrum of pediatric disease [44] . for eia-based antibody studies otherwise, we have learned so far that the type and severity of disease and age may have bearing on the quality and quantity of antibody responses [34, [45] [46] [47] [48] . gender may also possibly affect antibody responses or other relevant immune responses [34, 49] . does neutralization antibody ensure protection? at this time, we can reasonably conclude that the existence of neutralizing antibody and its quantitation associate with protection against coronavirus infections. what we cannot do, however, is guarantee that the presence of all such neutralizing antibody, as measured by conventional live virus neutralization methods in vitro, will ensure protection. that is, laboratory methods measure neutralization as so defined but not protection directly. therefore, it can be held that not all neutralization methods duly measure the same effect that leads to that neutralization outcome [50] . in essence, what creates an in vitro neutralization may or may not truly block viral entry, prevent intracellular events, or ameliorate disease. one potential and theoretical approach to vetting sera that have a higher likelihood of indication for protection is to choose those with higher neutralizing titres. there are several limitations with the application of neutralization as a surrogate for protection [25, 51] . infections with coronaviruses do not always lead to a major increase in such antibody. previously acquired antibody is not always protective for subsequent infection, but low levels of antibody better correlate with susceptibility. resistance is best to the homologous coronavirus, but susceptibility can be had to heterologous strains. there is also the prospect that subsequent changes in viral genome may be accompanied by changes in epitopes that afford neutralization with an accompanying change in neutralization capacity of post-infection or post-vaccination sera [52] . the latter could be the prelude to neutralization escape mutants should they occur [53] . the latter may also be the prelude to emerging reports of possible sars-cov-2 repeat infections [54] . the traditional approach to determining antibody responses is likely to dominate the search for protective immunity, but due consideration and open-mindedness should be maintained for measuring t cell post-infection immunity and its correlates [55] [56] [57] [58] . with the availability of viral antigen, most scientists in the know-how would be able to fashion a test for antibody determination in short order and most would likely choose an enzyme immunoassay (eia) (or nearly equivalent non-enzymebased assay) for its potential of automation and widespread use. the latter is especially likely in the current context where a majority of serodiagnostic tests are of that methodology. the expeditious derivation of an eia or equivalent is a common goal when serodiagnosis is deemed to be of potential value. whereas the creation of an eia is seemingly a relatively easy task, the application of any such is where the test of the matter lies. historically, whether for antibody or antigen detection, eia approaches had a number of pitfalls that were repetitively realized for nearly every such system that was subsequently developed [59, 60] . as would naturally be desirable, an assay with very high sensitivity at the appropriate timing is one evident target. furthermore, an assay with a highly specific result is another evident target. the reality of serodiagnostic systems, however, is that there is generally a gray or indeterminate zone where sensitivity and specificity collide [60] . there is an intersection of true positive results and false positive results more or less in almost every system with few exceptions. for whatever maneuver is conceived to diminish that overlap, eia or an equivalent for antibody detection can be modulated to enhance predictive values in one direction or another, but commonly realizing that there will be a trade-off. the application of a serodiagnostic assay for antibody postinfection or vaccine is furthermore complicated in its evaluation. typically, a standard set of known or presumed "negative" sera are tested as is a standard set of known or presumed "positive" sera. many such analyses, however, do not thereafter assess the assay in the context of the working world prospectively. the jeopardy here is that the prevalence of true disease in the population of patients from whom sera are acquired can vary considerably. there is an inherent bias in such assessments to ensure that the tested serum pool has a considerable number of true positive samples. biases in this regard can be greater than those engendered by convenience sampling [61, 62] . given the functionality of these assays, the predictive values of positive or negative tests vary considerably depending on the prevalence of the infection in the population so studied [63] . for example, even for a given high sensitivity and specificity, a reduction in prevalence from 30 to 3% can lead to a situation where the false positive assays nearly equal the number of true positive assays. the latter scenario can occur when assessments are performed on preselected sera rather than on sera from patients prospectively collected in a larger population. therefore, unless there is considerably widespread infection leading to a proportionately high seroprevalence, serodiagnostic tests will likely suffer with their predictive values. how would this dilemma then be potentially overcome? one such approach to improving specificity would be to conduct blocking assays (direct or competitive) for positive sera in order to reduce the number of false positive tests [64] . this is a time-honored approach that is often forgotten when new serodiagnostic assays are initially created. the approach was also germane to some antigen detection eia versions. effectively, the specificity is enhanced when it can be shown that the antigen of choice significantly reduces the assay quantitation after absorption. the implementation of a blocking step can be entered into an automated procedure in tandem from the start or may be performed as a confirmatory test. the latter is especially suited to a scenario where the proportion of positive tests is low. the simplicity of a blocking test mandates only a brief turnaround time if it is performed in an algorithmic second step. there may be various approaches to the definition of antigen used in the blocking test, but it would be best in the inaugural stages to choose antigen(s) that are deemed inherent to the neutralization effects seen in postinfectious sera. although it could prove that one antigen, e.g., rbd or spike protein sequence otherwise, may alone suffice, pilot studies would be better to work backwards after establishing the antigen-neutralization correlates as described above. purity of the blocking antigen is essential to avoid non-specific competition of non-viral elements if virus is acquired from tissue cultures. another approach to a blocking assay would be the competition between antibody in the human serum specimen and a known monoclonal antibody with affinity to sars-cov-2. choice of a specific monoclonal antibody, or several for this purpose, requires specific characterization. there are many commercial assays for sars-cov-2 antibody that have emerged, but for a survey sample of several so assessed thus far, none has blocking assays as potentially part of the procedure [65] [66] [67] [68] [69] [70] [71] . developmental assays also commonly do not include blocking assays [72] [73] [74] . igm assays are particularly an issue with non-specificity since apparent igm rises may relate in part to solid phase (e.g., polystyrene) absorption or non-specific igm antibody (e.g., rheumatoid factor). of note, early reports have now emerged of blocking assay variations for systems relating to either mers-cov or sars-cov-2 [75] [76] [77] . as suggested above, antigen detection can also suffer from non-specificity, and this dilemma has now been found in some systems [78] [79] [80] . again, a blocking assay in any such configuration for antigen detection has potential to resolve this critical issue especially when raising the threshold for the enhancement of sensitivity is being considered. a second approach, alone or alongside an eia-blocking assay, could be immunoblotting. again, it could prove that one antigen substrate may suffice, but it would be prudent to work backwards after establishing the antigen-neutralization correlates. as experienced pointedly with lyme disease or hiv infections, the immunoblot response can be considerably variable, but a confirmatory fingerprint or several fingerprints can be defined as minimal diagnostic thresholds. a higher threshold stringency can enhance serodiagnostic specificity. guan and others provided some key insights in this regard with sars-cov immunoblotting, and it is only highly probable that similar findings will be had with sars-cov-2 [81] . not all antigens identified in immunoblotting may have functions in neutralization, and likewise, there may be more antigens that are capable of inducing neutralization than are identified by resolving antigens in denaturing electrophoresis procedures [50] . given advances as detailed by olvera et al. and several others, the fingerprint patterns could also conceivably be assessed with peptide sets rather than larger antigens which may bear non-specific epitopes [50, 81, 82] . the protein micro-array concept bears relevance to the ingenuity of how a complex recognition pattern could occur and be thereafter automated [82, 83] . furthermore, we should not restrict ourselves to common antigens that are currently thought relevant since novel key antigens may yet arise [55] . if serological methods of choice later prove to be a surrogate for infectious virus neutralization, e.g., viral pseudoparticles, blocking test components can also be built into those processes [84] [85] [86] [87] [88] . the latter approaches too are capable of automation design or large batch processing. if conventional neutralization assays yield titres in lower dilution than pseudotype virus neutralization assays (e.g., s protein-bearing pseudoviruses with murine leukemia virus), the latter may set higher thresholds as the correlation dictates [89] . if neutralizing antibody correlates with protection, is there a threshold at which the prediction can be confident? whereas there are no guarantees, if the presence of neutralizing antibody correlates with protection, it can be reasonably hypothesized that more protection correlates with higher titres. such a hypothesis is easily testable in the current pandemic given preliminary consensus for the type of neutralization assay to be used. it is proposed that the 1:32-1:40 standard of conventional neutralizing antibody be used as the minimal threshold in inaugural studies. the threshold could be raised or lowered depending on the outcome of field assessments. prospective follow-up of study subjects with the established minimum neutralizing antibody and its potential change in titre and/or protection would be determinable over time. there is then a role for other measures of antibody thereafter. as the initial neutralizing antibody threshold for protection is established, correlates with other tests as surrogates can then begin. in the latter, it would also be best to choose higher thresholds of positivity rather than any positive test per se. such thresholds could thereafter vary pending correlations that become apparent. such an approach provides an abundance of precaution rather than the converse. the test of a minimalist strategy and thereafter its refinement is currently feasible in the milieu of the current pandemic. given the magnitude of infected individuals throughout the world, the numbers required to test for protection could be easily acquired from a multi-centered approach with highly endemic foci or with even a regional study again in a highly endemic area. there will be concern in allowing previously infected, neutralizing antibody-positive individuals to be naturally exposed once again in the community, but such an approach has little difference to the broad testing of candidate vaccines. indeed, the answers may come sooner than the answers of whether vaccine candidates will succeed. infection exposures in the community for those previously infected with or without such antibody markers will also slowly answer other questions relating to those with low level neutralizing antibody (e.g., below threshold), those that do not have any measurable antibody, anamnestic responses, and susceptibility to changing virus. furthermore, the minimalist strategy will potentially set the goal posts for vaccine evaluations. reports of repeat sars-cov-2 infections have already begun to emerge as the pandemic continues [54, [90] [91] [92] . undoubtedly, much more similar information will be collated anecdotally or in larger series and observations. an analysis of these is relevant in considering any minimalist strategy. gousseff et al. present a case series of healthcare workers and community patients who appeared to have a relapsing pattern of covid-19 [90] . the establishment of re-infection within such a short period of time is tenuous at best. early and later positive diagnostic samples could be assessed with viral sequencing. serological profiling with both igg and igm and correlation with viral neutralization are additional tools of interest. the persistence of viral genome after acute infection, and as determined with genetic amplification technology, is well-known and can be measured in weeks. accordingly, comparison of diagnostic cycle thresholds can be of value as was done. co-determination of other pathogens, especially viral for co-infection, is essential. that some patients may clinically relapse with a complicating respiratory illness after an initial non-complicated illness is in keeping with many other respiratory infections. viral culture to confirm live virus provides an additional step in confirmation but evidently is difficult to achieve without support from reference level 3 laboratories. as the authors suggest, are these examples simply ones of persistence or re-infection? bentivegna et al. describe a 69-year female with possible re-infection in which repeat rt-pcr positive samples of the respiratory tract were obtained in the context of four negative samples in between over a 6-7-week period [91] . immunoglobulin g serology was reactive on each occasion. igm serology was reactive only late in the second putative infection. blocking assays for the second serology could have potential use if devised. neutralization tests would have been of value to correlate with putative protection. an established fingerprint of immunoblotting if devised could be sought for in both early and later blood samples to further characterize the quality of immune reactivity. this case report emphasizes the role for a better understanding of applicable diagnostic and confirmatory serology. tomassini et al. highlight a case series of six patients who were possibly re-infected [92] . igm serology, blocking for igg serology, neutralization correlates, and comparison of diagnostic ct values all have their potential merit. to and colleagues describe a repeat infection in a 33-year male in which two distinct isolates were believed to have been identified by whole-genome sequencing [54] . the episodes occurred some 5 months apart. measurable igg was not detected within 10 days of the first episode. igm diagnostics, blocking for igg serology, and neutralization correlates all have their potential merit. the authors initiate relevant dialog about the relevance of genetic drift and possible re-infection. van elslande et al. provide some evidence for a repeat infection after a 3-month interval [93] . a comparison of diagnostic samples suggested repeat infection with phylogenetically distinct strain detections. diagnostic serology with blocking studies, neutralization correlates, and comparison of diagnostic ct value all have their potential merit. these authors repeat the theme on genetic drift and its implications. in any of the aforementioned, sample collection and recollection validation shortly after the initial diagnostic specimen test positive can also contribute to overall accuracy of determining re-infections. while such an approach to defining protective immunity in the interim reaches our goals if that is possible, the otherwise sensible approaches to disease prevention should continue to be enforced. given the current evidence on person-person direct transmission, person-person aerosol transmission, and environment-person indirect transmission, adherence to the basic principles of abrogating infection spread should be maintained as is practical. facets of physical distancing, appropriate decontamination and disinfection, and contextappropriate use of masking all have their roles in minimizing risk. it will be the study populations where some of these preventions may be less stringent in order to test the hypotheses of natural protection after infection or vaccine prevention. nevertheless, if we do define a highly probable and preventative serodiagnostic correlation, the modes of prevention may eventually be seen in alternative fashion or stringency. the overall approach to disease control will be fluid and will adapt as the potential arises. the global economic outlook during the covid-19 pandemic: a changed world after less than 2 months, the simulations that drove the world to strict lockdown 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genes dis a simple protein-based surrogate neutralization assay for sars-cov-2 neutralizing antibody and soluble ace2 inhibition of a replicationcompetent vsv-sars-cov-2 and a clinical isolate of sars-cov-2 longitudinal dynamics of the neutralizing antibody response to sars-cov-2 infection development of a safe neutralization assay for sars-cov and characterization of s-glycoprotein clinical recurrences of covid-19 symptoms after recovery: viral relapse, reinfection or inflammatory rebound? new igm seroconversion and positive rt-pcr test after exposure to the virus in recovered covid-19 patient setting the criteria for sars-cov-2 reinfection-six possible cases symptomatic sars-cov-2 reinfection by a phylogenetically distinct strain publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments this review is dedicated to those healthcare workers and researchers who have had the commitment to protect the populace and energetically pursue the science. key: cord-337812-arivkkj0 authors: chu, ling-hon matthew; choy, wai-yan; tsai, sau-na; rao, zihe; ngai, sai-ming title: rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (sars) coronavirus 3c-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry date: 2006-03-07 journal: protein science doi: 10.1110/ps.052007306 sha: doc_id: 337812 cord_uid: arivkkj0 severe acute respiratory syndrome coronavirus (sars-cov) 3c-like protease (3cl(pro)) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-sars drug development. here we present a rapid and high-throughput screening method to study the substrate specificity of sars-cov 3cl(pro). six target amino acid positions flanking the sars-cov 3cl(pro) cleavage site were investigated. each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the “cartridge replacement” approach and was subjected to enzymatic cleavage by recombinant sars-cov 3cl(pro). susceptibility of each peptide substrate to sars-cov 3cl(pro) cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms). the hydrophobic pocket in the p2 position at the protease cleavage site is crucial to sars-cov 3cl(pro)-specific binding, which is limited to substitution by hydrophobic residue. the binding interface of sars-cov 3cl(pro) that is facing the p1′ position is suggested to be occupied by acidic amino acids, thus the p1′ position is intolerant to acidic residue substitution, owing to electrostatic repulsion. steric hindrance caused by some bulky or β-branching amino acids in p3 and p2′ positions may also hinder the binding of sars-cov 3cl(pro). this study generates a comprehensive overview of sars-cov 3cl(pro) substrate specificity, which serves as the design basis of synthetic peptide-based sars-cov 3cl(pro) inhibitors. our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis. 2003; peiris et al. 2003) . coronaviruses are large, enveloped, single-stranded positive-sense rna viruses that replicate in the cytoplasm of the infected host cell (siddell et al. 1983) . phylogenetic studies reveal sars-cov does not belong to any of the three known groups of the coronaviridae family; thus, it is suggested that sars-cov defines a distinct fourth group of coronaviruses (marra et al. 2003; rota et al. 2003) . the rna genome of sars-cov is ,30 kb in length, with five major open reading frames (orfs) encoding the replicase polyprotein, the spike (s) protein, the envelope (e) protein, the membrane (m) protein, and the nucleocapsid (n) protein (marra et al. 2003; rota et al. 2003) . investigations of various sars-cov proteins can facilitate the development of vaccines and drugs against the sars disease. the pivotal role of sars-cov surface s protein in mediating viral entry sheds light on the generation of s protein-specific neutralizing antibodies (gallagher and buchmeier 2001; choy et al. 2004) , and some of the experimental vaccines have been tested in the murine viral replication model recently (bisht et al. 2004; yang et al. 2004) . meanwhile, the 33.8-kda sars-cov main protease, also known as the sars-cov 3c-like protease (sars-cov 3cl pro ) is being considered extensively as an attractive and promising target for anti-sars drug design, owing to its important biological role in the viral life cycle (anand et al. 2003) . sars-cov 3cl pro mediates extensive proteolytic processing of two overlapping replicase polyproteins, pp1a (486 kda) and pp1ab (790 kda), to yield the corresponding functional polypeptides that are essential for sars-cov replication and transcription processes (herold et al. 1993; ziebuhr et al. 1995; rota et al. 2003) . there are at least 11 conserved sars-cov 3cl pro cleavage sites on the sars-cov polyprotein pp1ab, in which the substrate specificity involves preferentially the leu-glnfl sequence in the p2 and p1 positions at the cleavage site, respectively (ziebuhr et al. 2000; heygi and ziebuhr 2002; anand et al. 2003) . sars-cov 3cl pro comprises three domains, including the catalytic domains i and ii that form the nterminal chymotrypsin fold, and the unique extra helical domain iii in c-terminal that is important for regulating the activity and specificity of sars-cov 3cl pro (shi et al. 2004 ). the reported x-ray crystal structures of the sars-cov 3cl pro allows the investigation of possible interactions of sars-cov 3cl pro with its substrates, which contributes to the development of specific protease inhibitors as potential anti-sars drugs (anand et al. 2003; yang et al. 2003) . a better understanding of the specific enzyme substrate-binding mechanism during the sars-cov 3cl pro proteolytic cleavage process is required for the rational design of an effective protease inhibitor with strong binding affinity to sars-cov 3cl pro . to screen the substrate specificity of sars-cov 3cl pro in a rapid and highthroughput manner in contrast to the traditional tedious procedures, we applied the matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (maldi-tof ms) analysis in combination with the novel "cartridge replacement" solid-phase peptide synthesis approach to investigate the biological significance of amino acid residues in the p2, p3, p4, p1¢, p2¢, and p3¢ positions that are flanking the conserved gln residue in the p1 position at the sars-cov 3cl pro cleavage site (schechter and berger 1967; fan et al. 2004 fan et al. , 2005 . owing to the sensitivity of maldi-tof ms, the protease cleavage progress of a mixture of different peptide substrates can be monitored simultaneously to generate comprehensive data on the substrate specificity of sars-cov 3cl pro . therefore, all of the 20 possibilities of amino acid substitutions in a particular position of the peptide substrate can be easily investigated in the same reaction. our study can provide insights into the molecular mechanism of the sars-cov 3cl pro cleavage process and reveal the feasibility of developing synthetic peptide-based protease inhibitors as potential drugs against sars-cov and other coronavirus infections. furthermore, the rapid approach we described in this study could be widely applicable for monitoring the cleavage activities of other proteases, such as allowing the rapid screening of potential protease inhibitors of the human immunodeficiency virus (hiv) as the basis of drug development. in addition, automations are highly compatible and feasible with our experimental procedures to cope with the increasing demand of high-throughput analysis in proteomic research. substrate specificity preferences of sars-cov 3cl pro positive control of the sars-cov 3cl pro cleavage assay the ps01 peptide was amidated and acetylated at the c and n termini, respectively. the corresponding amino acid sequence of ps01 was based on one of the sars-cov 3cl pro cleavage sites on the sars-cov bj01 polyprotein pp1ab (residues 3232-3247) and was used as the positive control in the enzymatic cleavage assay to assess the activity of the recombinant sars-cov 3cl pro . the original ps01 peptide peak with m/z value of 1739.03 before the enzymatic assay was absent from the mass spectrum after the cleavage reaction ( fig. 1) as it was being cleaved into two peptide fragments with their corresponding m/z values (data not shown), which validated the enzymatic activity of the recombinant sars-cov 3cl pro that we prepared. in this study, we used maldi-tof ms analysis in combination with the solid-phase peptide synthesis approach to examine the biological significance of amino acid residues in a total of six target positions at the sars-cov 3cl pro cleavage sites, including the p2, p3, and p4 positions at the amino side of the p1 position; and the p1¢, p2¢, and p3¢ positions at the carboxyl side of the p1 position (table 1) . based on the flexibility in changing the contents of each amino acid cartridge, we designed a novel peptide synthesis strategy that we named as the "cartridge replacement" solid phase peptide synthesis. our "cartridge replacement" strategy consists of two screening procedures: (1) primary screening and (2) secondary screening. 1. primary screening using the "cartridge replacement" strategy by using the "cartridge replacement" strategy, the corresponding amino acid cartridge at the target position under investigation was deliberately replaced by another peptide peak (*) was resolved before cleavage assay (a-1) and was absent from the mass spectrum after the reaction (a-2). (b) p2 position. for ps02 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (b-1), and only the peptide peaks corresponding to leu (*) and phe (**) substitutions in the p2 position were absent, whereas all other peaks remained after the cleavage reaction (b-2). (c) p3 position. for ps03 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (c-1), and only the peptide peak with pro (*) substitution in the p3 position remained after the reaction (c-2). (d) p4 position. for ps04 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (d-1), which were all absent from the mass spectrum after the reaction (d-2). (e) p1¢ position. for ps05 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (e-1), and the peptide peaks with pro (*), asp (**), and glu (***) in the p1¢ position remained after the reaction (e-2). (f) p2¢ position. for ps06 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (f-1), and the peptide peaks with pro (*) and ile/leu (**) in the p2¢ position remained after the reaction (f-2). (g) p3¢ position. for ps07 peptide substrates, all 20 peptide peaks were resolved on the mass spectrum before cleavage assay (g-1), which were all absent from the mass spectrum after the reaction (g-2). cartridge containing a mixture of 20 standard amino acids in equal molar ratios during the solid-phase peptide synthesizing process so as to generate a mixed pool of 20 different peptide substrates with single amino acid residue alteration at the target position. each batch of the mixed peptide substrates was subjected to in vitro enzymatic cleavage assay by recombinant sars-cov 3cl pro in a single reaction and then analyzed by maldi-tof ms to detect the susceptibility of each of the 20 peptides to cleavage by sars-cov 3cl pro . the results from the primary screening of the in vitro enzymatic cleavage assay of the six batches of synthetic peptide substrates (namely ps02, ps03, ps04, ps05, ps06, and ps07) with amino acid substitutions in their corresponding target positions are shown in figure 1 and summarized in table 2 . before the enzymatic assay, the corresponding peaks of the 20 peptides in each substrate pool were resolved on the mass spectrum with their exact m/z values, which demonstrated the validity of solid-phase peptide synthesis using the "cartridge replacement" approach to generate a pool of 20 peptides with single amino acid difference at the target position. the results also demonstrated the capability of maldi-tof ms to discriminate most of the peptide species with single amino acid difference. p2, p3, and p4 positions at the amino side of the sars-cov 3cl pro cleavage site for ps02 peptide substrates with amino acid substitutions in the p2 position, only the original peptide (m/z value of 1739.03) and the peptide with phe substitution for leu in the p2 position (m/z value of 1773.01) were cleaved, with their corresponding peptide peaks absent from the mass spectrum after the enzymatic assay, the amino acids flanking the sars-cov 3cl pro cleavage sites are labeled from the amino terminus to the carboxyl terminus as follows: -p3-p2-p1 fl p1¢-p2¢-p3¢, as described previously (schechter and berger 1967) . b the control peptide substrate is based on the amino acid sequence of one of the sars-cov 3cl pro cleavage sites on the sars-cov bj01 polyprotein pp1ab (residues 3232-3247). c the corresponding amino acid at this position is substituted by a mixture of 20 standard amino acids in equal molar ratios. the positions flanking the sars-cov 3cl pro cleavage sites are labeled from the n terminus to the c terminus as follows: -p3-p2-p1 fl p1¢-p2¢-p3¢, as described previously (schechter and berger 1967) . b the original amino acid residue in the position under investigation before the substitution with 20 standard amino acids. c the identity of the amino acid in the particular peptide that substituted the residue at the position under investigation. d all peptides other than those in the opposite column were not cleaved by sars-cov 3cl pro . e all peptides other than those in the opposite column were cleaved by sars-cov 3cl pro . f all 20 peptides were cleaved by sars-cov 3cl pro . whereas all other peptides were not cleaved, with their corresponding peaks remaining. for ps03 peptide substrates with amino acid substitutions in the p3 position, only the peptide with pro substitution for val in the p3 position (m/z value of 1737.01) was not cleaved, with its corresponding peptide peak remaining in the mass spectrum after the enzymatic assay, whereas all other peptides were cleaved successfully. for ps04 peptide substrates with amino acid substitutions in the p4 position, all of the 20 peptides were cleaved completely, with their corresponding peaks absent from the mass spectrum after the enzymatic assay. p1¢, p2¢, and p3¢ positions at the carboxyl side of the sars-cov 3cl pro cleavage site for ps05 peptide substrates, only three peptides with pro substitution (m/z value of 1749.05), asp substitution (m/z value of 1767.02), and glu substitution (m/z value of 1781.04) for ser in the p1¢ position, respectively, were not cleaved. for ps06 peptide substrates, only two peptides with pro substitution (m/z value of 1779.06) and ile/leu substitution (m/z value of 1795.09) for gly in the p2¢ position, respectively, were not cleaved. for ps07 peptide substrates with amino acid substitutions in the p3¢ position, all of the 20 peptides were cleaved completely, with their corresponding peaks absent from the mass spectrum after the enzymatic assay. 2. secondary screening using the "cartridge replacement" strategy after the primary screening procedure, we generated the comprehensive cleavage profiles of the corresponding 20 peptide substrates of each target amino acid position simultaneously, based on the "cartridge replacement" strategy. however, there were peptides (carrying ambiguous residues like ile/leu and gln/lys that carry the identical or close masses) in which true cleavage might not be identified with absolute confidence. a secondary screening procedure was necessary, and we proceeded to the synthesis of target peptide substrates into defined batches, particularly for those peptides with identical or close molecular masses, which resulted in ambiguous peaks for detection on the mass spectra in the primary screening procedure. the results of the in vitro enzymatic cleavage assay of five peptide batches (namely, ps02-1, ps02-2, ps02-3, ps06-i, and ps06-l) are shown in figure 2 and summarized in table 3 . for ps02-1 peptide batches, only the original peptide with leu in the p2 position (m/z value of 1739.03) was cleaved, with its corresponding peptide peaks absent from the mass spectrum after the enzymatic assay, whereas the other three peptides with pro, cys, and glu substitutions for leu in the p2 position were not cleaved, with their corresponding peaks remaining. for ps02-2 and ps02-3 peptide batches, not all of the peptides were cleaved, with their corresponding peaks remaining. for ps06-i and ps06-l peptide batches, both the peptides with ile substitution (m/z value of 1795.09) and leu substitution (m/z value of 1795.09) for gly in the p2¢ position, respectively, were not cleaved, with their corresponding peaks remaining. based on the mass spectrometry results from the in vitro enzymatic cleavage assay (table 2) , four target positions including p2, p3, p1¢, and p2¢ positions located at the sars-cov 3cl pro cleavage sites were further analyzed by the insight ii molecular modeling platform, and the molecular docking results are shown in figures 3 and 4 . the control peptide, ps01, was docked to the predefined active site of sars-cov 3cl pro , with the conserved gln residue located at close proximity to cys145-his41 residues at the catalytic dyad of sars-cov 3cl pro (fig. 4) , in which this total energy data (182.74 kcal/mol) generated from energy minimization was used as the reference for comparing the docking results using other peptide substrates. this slightly positive total energy value is due to the relatively high repulsive van der waals energy attributed by the narrow binding pocket on sars-cov 3cl pro . for the ps02 peptide with phe substitution in the p2 position, the total energy of 186.98 kcal/mol was similar to that of the ps01 control peptide (182.74 kcal/mol), which further confirmed the tolerance of phe substitution at the p2 position at the cleavage site (fig. 4) . on the other hand, the total docking energy for ps03 peptide with pro substitution in the p3 position (1028.33 kcal/mol); ps05 peptides with pro substitution (1385.02 kcal/mol), asp substitution (1384.69 kcal/mol) and glu substitution (1384.91 kcal/mol) in the p1¢ position; and ps06 peptides with pro substitution (1383.25 kcal/mol), ile substitution (1381.35 kcal/mol), and leu substitution (1387.60 kcal/mol) in the p2¢ position were at least fivefold higher than the total energy of the ps01 control peptide (182.74 kcal/mol). the relatively high total docking energy revealed the unfavorable cleavage reactions of these peptides by sars-cov 3cl pro (fig. 4 ). we have introduced a rapid and high-throughput screening method to generate a comprehensive overview of the substrate specificity preferences of sars-cov 3cl pro via a combinatory approach that uses maldi-tof ms and the novel "cartridge replacement" solidphase peptide synthesis strategy. research on the substrate specificity preferences of sars-cov 3cl pro is gaining increasing attention, owing to the biological significance of sars-cov 3cl pro in the viral life cycle and the feasibility to design specific sars-cov 3cl pro inhibitors as potential anti-sars drugs. it has been explicitly demonstrated that sars-cov 3cl pro cleaves the coronavirus replicase polyproteins at no less than 11 conserved sites, preferentially involving the leu-glnfl sequence in the p2 and p1 positions at the cleavage site, respectively (ziebuhr et al. 2000; heygi and ziebuhr 2002; anand et al. 2003) . to further extend our knowledge of the substrate specificity of sars-cov 3cl pro , we aimed to comprehensively study the biological importance of amino acid residues in six selected positions (namely p2, p3, p4, p1¢, p2¢, and p3¢) flanking the conserved p1 position at the sars-cov 3cl pro cleavage site by a rapid and high-throughput approach (table 1) . from the primary screening procedure, the results of the ps03, ps04, ps05, and ps07 peptide substrates can be interpreted precisely. from the results of ps04 and , and gln (q; m/z=1753.91) substitutions were resolved on the mass spectrum before cleavage assay (c-1), which all were remaining after the reaction (c-2). the 1740.88 monoisotopic peak with high intensity (*) belongs to the mother peak of the peptide with asp (d) substitution. (d) ps06-i peptide batch. the peak of peptide with ile (i; m/z=1795.09) substitution was resolved on the mass spectrum before cleavage assay (d-1), which remained after the reaction (d-2). (e) ps06-l peptide batch. the peak of peptide with leu (l; m/z=1795.09) substitution was resolved on the mass spectrum before cleavage assay (e-1); which remained after the reaction (e-2). ps07 peptide substrates (fig. 1) , all 20 peptide peaks were absent from the mass spectra after the sars-cov 3cl pro cleavage reaction: the sars-cov 3cl pro can cleave all of the 20 peptides regardless of the types of amino acid substitutions at the p3¢or p4 positions. for ps03 and ps05 peptide substrates, the corresponding peptide peaks (m/z value of 1737.01 for ps03 and m/z values of 1749.05, 1781.04, and 1767.05 for ps05) retained on the mass spectra could be clearly resolved after the cleavage reaction (fig. 1) ; the sars-cov 3cl pro cannot cleave those peptides for which the nonoverlapping m/z values were shown, and hence, those uncleaved peptides can be easily identified. for ps02 peptide substrates, there were only two peptide peaks that vanished after the cleavage reaction. although these two peaks can be identified as corresponding to their exact m/z values, several remaining peptide peaks with close masses introduced ambiguity in the mass spectra. in addition, for ps06 peptide substrates, there were only two peptide peaks found on the mass spectra after the cleavage reaction. one of the peptide peaks (m/z value of 1795.09) may contain two other peptide entities with exact m/z values, i.e., the ile/ leu containing peptide. in fact, this result was also observed in one of the cleaved peaks of ps02 peptide substrates (m/z value of 1739.03). therefore, a secondary screening procedure using the defined batch synthesis approach (table 3 ) was used to solve the above problem. in addition to the well-known conserved gln residue in the p1 position at the sars-cov 3cl pro cleavage site, the p2 position, which is exclusively occupied by leu residue, also serves as another important determinant of substrate specificity. our peptide cleavage results indicated that only the peptide substrate with phe replacement in the p2 position was also favorable for sars-cov 3cl pro cleavage ( fig. 1; table 2 ), with similar total energy level (186.98 kcal/mol) in molecular docking when compared with the total energy of the positive control peptide (182.74 kcal/mol) with leu in the p2 position. also, the results of the three peptide batches (ps02-1, ps02-2, and ps02-3) in the secondary screening procedure further confirmed the results obtained from the primary screening procedure. the ps02-3 peptide batch showed that ile was intolerant in the p2 position for sars-cov 3cl pro . our results demonstrated consistency with other findings (fan et al. 2004 (fan et al. , 2005 , suggesting that the p2 position serves as an important hydrophobic pocket for sars-cov 3cl pro -specific binding; thus, the peptide substrate with replacement of the original leu with the hydrophobic phe in the p2 position was still susceptible to sars-cov 3cl pro cleavage (fig. 4) . from the results of a previous time course cleavage experiment (data not shown), we observed that the figure 3 . interaction between sars-cov 3cl pro and ps01 control peptide as predicted by molecular docking. overview (left) of the interaction between the sars-cov 3cl pro (purple) and peptide substrate ps01 (red) and the zoom-in view (right) of the conserved gln residue (gray) in the p1 position at the cleavage site of the ps01 control peptide being docked into the catalytic dyad of sars-cov 3cl pro , which is composed of the cys145 (orange) and his41 (yellow) residues. sars-cov 3cl pro proteolytic cleavage rate of the positive control peptide was higher than that of the peptide with phe substitution in the p2 position, which accounted for the dominant occurrence of leu in the p2 position. other studies also demonstrated that sars-cov 3cl pro favorably tolerates phe and val substitutions, and to a lesser extent, for met and ile substitutions in the p2 position (fan et al. 2004 (fan et al. , 2005 , hence revealing the significance of the p2 hydrophobic pocket in the determination of sars-cov 3cl pro substrate specificity. furthermore, the p1¢ position, which is frequently occupied by ser residue, also contributes to the substrate specificity of sars-cov 3cl pro considerably. our peptide cleavage results revealed that the p1¢ position is highly unfavorable to the substitution by pro, asp, and glu residues ( fig. 1; table 2 ), which is consistent with molecular docking results showing their total energy levels being higher than that of the positive control peptide by more than fivefold. our findings on the intolerance of asp or glu substitutions in the p1¢ position suggest that the acidic residues at the binding interface of the sars-cov 3cl pro active site are probably in close proximity and sterically complementary to the p1¢ position of the substrate, leading to electrostatic repulsions between the negatively charged acidic residues that hinder the substrate binding to sars-cov 3cl pro . our molecular docking results also demonstrated that the p1¢ position is proximal to the glu47 and asp48 residues at the sars-cov 3cl pro active site (fig. 4) , which further confirmed our interpretations. also, the unfavorable substitution by pro in the p1¢ position could be accounted for by the steric hindrance arising from its cyclic side chain (fig. 4) , which obstructs the substrate binding to sars-cov 3cl pro . previous studies also demonstrated that small aliphatic residues such as ser, ala, and gly are favored in the p1¢ position (fan et al. 2005) . moreover, our results demonstrated that the substrate specificity of sars-cov 3cl pro are less dependent on the p2¢ and p3 positions at the cleavage site. the substitutions of pro, ile, or leu in the p2¢ position and the substitution of pro in the p3 position in the peptide substrates are unfavorable for sars-cov 3cl pro cleavage ( fig. 1; table 2 ), in which these bulky pro residue and bbranching ile and leu residues may cause steric hindrance to binding of sars-cov 3cl pro with its substrates (fig. 4) . the results of ps06-i and ps06-l peptide batches further confirmed the results of the primary screening: neither ile nor leu is tolerant in the p2¢ position for sars-cov 3cl pro . on the other hand, the peptide cleavage results showed that the p3¢ and p4 positions have no effect on determining the substrate specificity preferences of sars-cov 3cl pro ( fig. 1; table 2 ). in contrast to providing quantitative measurements on the kinetic data of the interaction between sars-cov 3cl pro and the substrates, the scope of our studies mainly focuses on the description of a useful tool for rapid and comprehensive screening of substrate specificity for sars-cov 3cl pro , which is also applicable to the studies of other proteases. by combining the maldi-tof ms and synthetic peptide-based approaches, peptide-cleavage studies on the defined equal molar mixture of a batch of 20 peptide substrates before and after the protease cleavage reaction could be performed simultaneously on a single maldi-tof ms analysis. the maldi-tof mass spectrometer is figure 4 . comparison between the molecular models of different selected peptide substrates docked to the active site of sars-cov 3cl pro . the cys145 (orange) and his41 (yellow) residues in the catalytic dyad of sars-cov 3cl pro (purple) and the conserved gln residue (gray) in the p1 position at the cleavage site of the peptide substrate (red) were shown. (a) ps02 peptide with phe (green) in the p2 position was in close proximity to the active site of sars-cov 3cl pro . peptide substrates that were unfavorable for sars-cov 3cl pro cleavage were deviated away from the active site of sars-cov 3cl pro , which include ps03 peptide with pro (blue) in the p3 position (b); ps05 peptide with pro (blue) in the p1¢ position (c); ps05 peptide with asp (blue) in the p1¢ position that was in close proximity to the acidic residues (brown) at the active site (d); ps05 peptide with glu (blue) in the p1¢ position that was in close proximity to the acidic residues (brown) at the active site (e); ps06 peptide with pro (blue) in the p2¢ position (f); ps06 peptide with ile (blue) in the p2¢ position (g); ps06 peptide with leu (blue) in the p2¢ position (h). a delicate and sensitive instrument that enables clear and precise discrimination of different peptides, even with single amino acid difference. therefore, each of the 20 peptide substrates with equal molar ratios in the same mixture can be monitored simultaneously on a single maldi-tof ms analysis before and after the protease cleavage reaction. our approach is advantageous over the traditional methods that are restricted to testing single peptides with defined condition one at a time and the setup of a total of 20 identical experimental conditions for every analysis. after generating the comprehensive cleavage profile on the 20 peptides simultaneously based on the primary screening procedure of the "cartridge replacement" strategy, we can easily identify some of the biologically important residues taking part in the recognition process. nevertheless, we employed the secondary screening procedure using the defined batch-synthesis approach to synthesize target peptides in different batches for exact identification purpose (ambiguous residues like ile/leu and gln/lys that carry identical or close masses). our approach provides an option in which the resolution of the maldi-tof mass spectrometer instrument is not sensitive enough to distinguish between species with 1-da mass difference. also, maldi-tof ms analysis is tolerant to impurities; therefore, the reaction mixture can be analyzed directly after a simple desalting procedure, which reduces the workload of subsequent downstream purifications of cleavage products prior to analysis. for the synthesis of peptide substrates, we deliberately applied the novel "cartridge replacement" strategy, which was based on the flexibility on modifying the solid-phase peptide synthesis process, in which each amino acid cartridge can be filled with different contents of amino acids in defined ratios, to generate the desired combinations of mixed peptide substrates according to experimental needs. by the incorporation of maldi-tof ms analysis and "cartridge replacement" peptide synthesis approach, we presented a useful tool that is widely applicable to studies that involve the monitoring of protease cleavage activity, which includes the rapid screening of potential protease inhibitors of other coronaviruses and the human immunodeficiency virus (hiv). on the basis of our studies, future experiments will be carried out to address the effectiveness of various synthetic peptidebased inhibitors in blocking the protease activity, with the ultimate aim of anti-sars drug development. in this study, we have combined the sensitivity, the high resolution of maldi-tof ms, and the flexibility of the "cartridge replacement" solid-phase peptide synthesis approach to generate sars-cov 3cl pro proteolytic cleavage data rapidly in a comprehensive manner. our studies definitely provide insights into the full automation of such experimental procedures to meet the increasing demand of high-throughput and automated proteomic studies. the coding sequence of sars-cov 3cl pro used for cloning was based on the sars-cov bj01 strain complete genome sequence downloaded from genbank (accession no. ay 278488; http://www.ncbi.nlm.nih.gov/entrez). the construction of the pgex-3cl pro plasmid has been described previously by dr. rao's team at tsinghua university (yang et al. 2003) . the pgex-3cl pro plasmid was transformed into escherichia coli strain bl21(de3)-competent cells and plated on luria-bertani (lb) agar containing ampicillin (100 mg/ml)/ chloramphenicol (25 mg/ml) and grown overnight at 37°c. a single colony was inoculated into 50 ml of lb-broth containing ampicillin (100 mg/ml) and grown overnight at 37°c with shaking. this overnight culture was then transferred into 5 l of lb-broth containing ampicillin (100 mg/ml) for large-scale protein expression at 37°c with shaking. when the culture was grown to an od 600nm of 0.6, it was induced with 0.1 mm isopropyl-1-thio-b-d-galactopyranoside (iptg) and grown for an additional 6 h at 25°c. cells were harvested by 20-min centrifugation at 3200g at 4°c, and the bacterial cell pellet was resuspended in lysis buffer containing 10 mm tris-hcl (ph 7.5), 500 mm nacl, 1 mm dithiothreitol (dtt), 0.5 mm edta, and 0.2 mm phenylmethylsulfonyl fluoride (pmsf), and homogenized by sonication. the lysate was centrifuged at 48,000g for 45 min at 4°c, and the supernatant was loaded onto a glutathione sepharose 4b affinity column (amersham biosciences) equilibrated with the lysis buffer. the column was then washed with 10· column volume of lysis buffer. to cleave the gst-tag from the gst-3cl pro fusion protein, prescission protease (amersham biosciences) was added into the column, and the mixture was incubated overnight at 4°c. the sars-cov 3cl pro recombinant protein was eluted with the lysis buffer, concentrated to 10 mg/ml by microcon centrifugal filter devices (millipore), and then stored at -80°c. "cartridge replacement" solid-phase peptide synthesis the sequence of peptide substrate used in this study was based on one of the sars-cov 3cl pro cleavage sites on the sars-cov bj01 polyprotein pp1ab (residues 3232-3247) ( table 1) . the biological significance of amino acid residues in the p2, p3, p4, p1¢, p2¢, and p3¢ positions that are flanking the conserved gln residue in the p1 position at the cleavage site was investigated one at a time using the novel "cartridge replacement" solid-phase peptide synthesis strategy. in a standard procedure of solid-phase peptide synthesis, each amino acid cartridge that contains a particular type of amino acid is placed side-by-side in the peptide synthesizer according to the target peptide sequence, and the peptide synthesis begins with the cartridge containing the first amino acid at the carboxyl terminus of the sequence. in this study, we utilized flexibility in changing the contents of each amino acid cartridge and designed a novel peptide synthesis strategy that we named as the "cartridge replacement" solid-phase peptide synthesis. prior to peptide synthesis, the corresponding amino acid cartridge at the target position under investigation was replaced by another cartridge containing a mixture of 20 standard amino acids in equal molar ratio, such that a mixed pool of 20 synthetic peptides differing by a single amino acid residue was generated as the end product after complete synthesis of the substrate sequence. six sets of such peptide substrate pools together with the original peptide substrate were synthesized with this approach (table 1) using a standard solid-phase peptide synthesis protocol as described previously (choy et al. 2004 ). the ps02 and ps06 peptide substrates require further analysis by a secondary screening. for the ps02 peptide substrates, the peptides with close masses were separated into three batches: ps02-1, ps02-2, and ps02-3. for the peptide synthesis of the ps02-1 batch, the corresponding leu cartridge at the p2 position was replaced by a cartridge containing a mixture of cys, glu, leu, and pro in an equal molar ratio. for the peptide synthesis of the ps02-2 batch, the corresponding leu cartridge at the p2 position was replaced by a cartridge containing a mixture of asn, lys, and val in an equal molar ratio. for the peptide synthesis of the ps02-3 batch, the corresponding leu cartridge at the p2 position was replaced by a cartridge containing a mixture of asp, gln, ile, and thr in an equal molar ratio. for the ps06 peptide substrates, only the ile and leu substitutions were further investigated and separated into two batches: ps06-i and ps06-l. for the peptide synthesis of ps06-i and ps06-l batches, the corresponding gly cartridge at the p2¢ position was replaced by a cartridge containing ile or leu, respectively. all of the peptide substrates were synthesized by the solidphase technique with an in-house applied biosystems 433a peptide synthesizer on preloaded resins using standard 9-fluorenylmethoxycarbonyl (fmoc) synthesis chemistry with piperidine deprotection and 2-(1h-benzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (hbtu)/n-hydroxybenzotriazole (hobt) activation. the amino (nh 2 ) termini of peptides were acetylated on the resin with acetic anhydride capping solution containing 0.5 m acetic anhydride, 0.125 m n,n-diispopropylethylamine (diea), 0.015 m n-hydroxybenzotriazole (hobt) in n-methylpyrrolidone (nmp). the acetylated peptides were cleaved from the resins, and side-chain protecting groups were removed by cleavage solution containing 25 g/l ethanedithiol (edt) and 50 g/l thioanisole in trifluoroacetic acid (tfa). the cleaved peptides were then precipitated with cold diethyl ether, washed, and lyophilized. the identities of the synthetic peptides were confirmed by mass spectrometry on an ettan maldi-tof pro mass spectrometer (amersham biosciences). the in vitro peptide cleavage assay was performed in a total reaction volume of 25 ml (ph 7.6), containing 1.5 mm recombinant sars-cov 3cl pro , 0.5 mm sum total concentration of the peptide substrate batch, 20 mm na 2 hpo 4 , 200 mm nacl, 1 mm edta, and 1 mm dtt, with overnight incubation at 25°c. cleavage products were desalted by ziptip u-c18 (millipore), and the corresponding peptide peaks before and after the cleavage reactions were resolved by mass spectrometry on an ettan maldi-tof pro mass spectrometer (amersham biosciences) or an abi 4700 maldi tof/tof analyzer (applied biosystems). for acquisition of mass spectra, 0.5-ml aliquots of samples were dispensed onto the maldiplate, followed by a 0.5-ml matrix solution (5 g/l a-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid). all spectra were acquired in a reflectron-positive ion mode and accumulated from 1000 laser shots with acceleration of 20 kv and pulsed extraction. angiotensin iii (m/z value of 897.523) and adrenocorticotropic hormone (m/z value of 2465.199) were used as standards for internal calibration of maldi-tof ms spectra. all three-dimensional molecular modeling studies were performed on an insight ii molecular modeling platform (accelrys software inc.) run on silicon graphics octane2 workstations (silicon graphics inc.). the consistent valence force field (cvff) was selected in all simulations. the x-ray crystal structure of the sars-cov 3cl pro (1uk4) was downloaded from the protein data bank (pdb; http://www.rcsb.org/pdb) and used as a starting model. the catalytic active site of chain a in the sars-cov 3cl pro structure without the bound cmk peptide was used in the study. hydrogen atoms were added for the structure of sars-cov 3cl pro chain a using the builder module. then, the force field potentials and partial charges were assigned. the resulting structure was subsequently subjected to energy minimization using the dis-cover module and was used as the initial structures for molecular docking. since the proteolytic activity sars-cov 3cl pro mainly relies on the active nucleophile cys145 and the acid-base catalyst his41, the region containing all residues (10.0 å radius) around this cys-his dyad was selected as the target site and was used to set up as a grid with the docking module. the nonbond energies of this region were precalculated on the grid. different peptide substrates were then docked into the target site by manual docking using the docking module. the best docking position was based on the docking energy of the peptide/sars-cov 3cl pro complex. thousands of orientations of peptides were searched by maneuvering in the cys-his dyad region of sars-cov 3cl pro manually until reaching the energy minimum. the resultant complex was subjected to energy minimization and molecular dynamics simulation using the discover module. a dielectric constant of 1.0 was used throughout the energy minimization and molecular dynamics simulation. the system was equilibrated at 300 k for 100 psec, during which time the potential energy of the system reached a stable value. a time step of 1 fsec was used to integrate the equation of motion. the final conformation of the structure was obtained through 5000 iterations of steepest descent energy minimization. ultimately, the total docking energy between the peptide substrate and sars-cov 3cl pro in the energy-minimized complex was calculated using the docking module. the docking energy included the van der waals and electrostatic energies, which were the components of the intermolecular energy. coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice synthetic peptide studies on the severe acute respiratory syndrome (sars) coronavirus spike glycoprotein: perspective for sars vaccine development identification of a novel coronavirus in patients with severe acute respiratory syndrome biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase the substrate specificity of sars coronavirus 3c-like proteinase coronavirus spike proteins in viral entry and pathogenesis conservation of substrate specificities among coronavirus main proteases nucleotide sequence of the human coronavirus 229e rna polymerase locus a novel coronavirus associated with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong the genome sequence of the sars-associated coronavirus coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome on the size of the active site in proteases dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors the biology of coronaviruses the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor a dna vaccine induces sars coronavirus neutralization and protective immunity in mice characterization of a human coronavirus (strain 229e) 3c-like proteinase activity virus-encoded proteinases and proteolytic processing in the nidovirales we thank dr. zihe rao's research team at tsinghua university, in particular haitao yang, xiaodong zhou, and xiaoyu xue, for their generosity in providing us with the pgex-3cl pro construct. this project is supported by the sars special rgc grant and special sars funding from greaterchina technology group ltd. key: cord-333682-ktbnrkwh authors: dong, yunzhu; chi, xiangyang; hai, huang; sun, liangliang; zhang, mengyao; xie, wei-fen; chen, wei title: antibodies in the breast milk of a maternal woman with covid-19 date: 2020-07-03 journal: emerging microbes & infections doi: 10.1080/22221751.2020.1780952 sha: doc_id: 333682 cord_uid: ktbnrkwh a maternal woman was positive for sars-cov-2 tested in throat swabs but negative tested in other body fluids, and she had igg and iga detected in breast milk. her infant negative for sars-cov-2 at birth had elevated igg in serum but quickly decayed. these findings suggest that breastfeeding might have the potential benefit to the neonates. the ongoing novel coronavirus (covid-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has posed a global public health concern [1] . the number of pregnant women and neonate with covid-19 is also on the rise [2, 3] . a recent study reported a newborn with elevated igm antibodies to sars-cov-2 born to a mother with covid-19 [4] . vertical transmission of sars-cov-2 and transplacental transmission of antibodies have been proposed with controversial opinions [5] [6] [7] . although clinical and laboratory characteristics, and outcomes of pregnant women with covid-19 have been reported [4], there are no continuously monitored data about the viral loads in several body fluids of the maternal women that would bring potential risks of sars-cov-2 infection to neonates [8] . here, we follow up the viral loads and antibody titers of sars-cov-2 in a maternal woman and the neonate since be hospitalized to discharged. on february 26, 2020, a 33-year-old primiparous woman (38 weeks 2 days of gestation with irregular lower abdominal pain with vaginal fluid for 6 h) suffering from cough and chest tightness 2 weeks ago was admitted to hospital for childbirth. chest radiography showed patchy ground-glass opacities in the periphery of left lung (figure 1, panel a) , and a throat swab was positive for sars-cov-2 through reverse-transcription real-time polymerase chain reaction (rt-pcr) at admission (figure 1, panel b) . laboratory test abnormalities included the decrease of lymphocyte percentage (15.6%), increase of neutrophil percentage (80%), and elevated serum levels of hepatic enzymes (alanine aminotransferase 90 u/l, aspartate aminotransferase 81.8 u/l; supplementary table). the maternal woman was considered as an ordinary case base on mild symptoms and radiologic imaging. due to the pregnancy, there was neither antiviral nor antibiotic treatment for the patient. the woman gave birth in a negative-pressure operating room. all persons in the room wore protective suits and the maternal woman wear an n95 mask immediately after labor. the infant girl was delivered by left occiput anterior (loa) in isolation delivery room and quarantined in the neonatal intensive care unit (icu). the infant's birth weight was 2950 g, and apgar scores were 9 at 1 min and 10 at 5 min. an oropharyngeal swab specimen, obtained immediately after she was taken from the uterus, showed a negative result for the detection of sars-cov-2 rna. the infant was then sent to the negative-pressure ward. after delivery, the woman was transferred to the icu isolation ward to continue treatment with anti-infective medication (azithromycin and ornidazole). on march 7, the woman was transferred to guanggu branch of hubei maternal and child health hospital, a designated hospital for covid-19 treatment in wuhan city. with the use of rt-pcr assays, the mother's throat swabs were continuously positive, and the ct value remains low for sars-cov-2 by rt-pcr at march 8, 12 and 15, and turned negative since march 18, while all samples of breast milk, urine, vaginal secretion, feces, tear, sweat and blood serially collected during the same period were negative (figure 1, panel b) . through enzyme-linked immunosorbent assays (elisas) using sars-cov-2 spike protein as antigen, the titers of igg antibody to sars-cov-2 in serum were 4509.5, 4025.4, 3683.6, 5838.6, 3690.1, respectively ( figure 1, panel c) . the titers of igg antibody in breast milk were 2.34, 3.02, 2.84, 2.79, and 3.35, respectively, when three sars-cov-2 negative maternal woman's breast milk were tested as control (mean titer 0.98) (figure 1, panel d) . breastfeeding protects infants against infections mainly via secretory iga (siga) antibodies. in the early stages of lactation, iga, anti-inflammatory factors and, more likely, immunologically active cells provide additional support for the immature immune system of the neonate. here we determined the antibody to sars-cov-2 in breast milk by elisa. the titers of iga antibody in breast milk were 2.18, 2.23, 2.37 and 2.69, respectively, when three sars-cov-2 negative maternal woman's breast milk was tested as control (mean titer 0.9) (supplementary figure) . on march 22, the mother was discharged, with her abnormal chest radiography and laboratory test findings recovered to normal, and the neonate had a negative result for detection of sars-cov-2 rna in the throat swab and igg titer of 1675.5 in serum, which was lower than her mother (figure 1 ). on april 9, the mother's igg antibody remained a titer of 4066.6 in serum and elevated to 13.9 in breast milk, but the neonate's igg turned negative (figure, panel d and e). and the titer of iga antibody was slightly elevated to 4.01 in breast milk (supplementary figure) . although the sars-cov-2 has been detected in breast milk in recent publication [9] , in our study there is no detection of sars-cov-2 in the mother's body fluids serially collected after delivery when her throat swabs showing positive results for sars-cov-2, especially in breast milk. the neonate had negative result for sars-cov-2 rna at the birth and her igg antibody to sars-cov-2 was observed only within one and half month after birth, indicating the placenta transmission of covid antibody. the mother's igg antibody usually remains in neonate for more than 6 months after birth. infants incapable of producing immunoglobulins are protected by maternal antibodies for up to 12 months after birth [10] , however, our follow-up identified that igg to sars-cov-2 in the neonate maintained less than one and half month, suggesting the potential risk for subsequent covidtiters of igg antibody to sars-cov-2 in the maternal woman's serums determined using elisa. data were shown as mean ± sd of three duplicates. (d) titers of igg antibody to sars-cov-2 in maternal woman's breast milk determined using elisa. data were shown as mean ± sd of three duplicates. (e) titers of igg antibody to sars-cov-2 in the neonate's serums determined using elisa. data were shown as mean ± sd of three duplicates. 19 in neonates. more importantly, the igg and iga antibodies were detected in breast milk indicate the potential immune protection for the neonates. nonetheless, data from large-scale cohort are warranted. a novel coronavirus from patients with pneumonia in china clinical characteristics of pregnant women with covid-19 in wuhan, china clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records possible vertical transmission of sars-cov-2 from an infected mother to her newborn clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china. ma pediatr antibodies in infants born to mothers with covid-19 pneumonia lack of vertical transmission of severe acute respiratory syndrome coronavirus 2, china. merg infect dis detection of sars-cov-2 in human breastmilk maternal antibodies, childhood infections, and autoimmune diseases we thank tao jiang, phd, beijing institute of microbiology and epidemiology, beijing, people's republic of china and hao li, phd, beijing institute of microbiology and epidemiology, beijing, people's republic of china, for the assistance. we thank the patient for granting permission to publish this information. no potential conflict of interest was reported by the author (s). this work is supported by grants of the national key r&d program of china (2020yfc0841400) and national nature science foundation of china (81803429). key: cord-348696-86nbwon2 authors: güemes-villahoz, noemi; burgos-blasco, barbara; vidal-villegas, beatriz; garcia-feijoo, julián; arriola-villalobos, pedro; martínez-de-la-casa, jose maría; diaz-valle, david; konstas, anastasios g. title: novel insights into the transmission of sars-cov-2 through the ocular surface and its detection in tears and conjunctival secretions: a review date: 2020-08-18 journal: adv ther doi: 10.1007/s12325-020-01442-7 sha: doc_id: 348696 cord_uid: 86nbwon2 sars-cov-2 is a highly transmissible virus that spreads mainly via person-to-person contact through respiratory droplets, or through contact with contaminated objects or surfaces from an infected person. at present we are passing through a phase of slow and painful understanding of the origin, epidemiological profile, clinical spectrum, and risk profile of the virus. to the best of our knowledge there is only limited and contradictory evidence concerning sars-cov-2 transmission through other routes. importantly, the eye may constitute not only a potential site of virus replication but also an alternative transmission route of the virus from the ocular surface to the respiratory and gastrointestinal tract. it is therefore imperative to gain a better insight into the potential ophthalmological transmission route of the virus and establish directions on best practice and future models of care for ophthalmological patients. this review article critically evaluates available evidence on the ophthalmological mode of viral transmission and the value of earlier identification of the virus on the eye. more evidence is urgently needed to better evaluate the need for protective measures and reliable ocular diagnostic tests to diminish further pandemic spread. a novel coronavirus epidemic caused by the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has spread to nearly every country in the world since the virus first emerged in china in december 2019. this virus causes the coronavirus disease 2019 (covid19) , which has at the time of writing been responsible for more than 10 million confirmed cases and 500,000 global fatalities, and has had an extraordinary socioeconomic impact worldwide. at present we are going through a phase of slow and painful understanding of the origin, epidemiological profile, clinical spectrum, and risk profile of the virus. sars-cov-2 is a highly transmissible virus that spreads mainly through person-to-person contact via respiratory droplets, or through contact with contaminated objects or surfaces from an infected person. evidence regarding sars-cov-2 transmission through other routes, such as the fecal-oral route and through the ocular surface is currently under investigation. at present, the scientific evidence concerning ophthalmological transmission of the virus is limited and, in many cases, contradictory. it is therefore imperative to gain a better insight into the ophthalmological transmission route of the virus and to establish directions on best practice to plan future models of care for ophthalmological patients. we decided to critically review available evidence on the ophthalmological route of transmission and the value of earlier identification of the impact of covid-19 upon the eye. the current work highlights the urgent need to delineate optimal protective measures and to evaluate the role of any potentially reliable future ocular diagnostic tests which could prevent further pandemic spread. we reviewed the pertinent literature through pubmed. a literature search was conducted under the topics covid-19; sars-cov-2; transmission; eye; rt-pcr; tears; and conjunctiva. relevant references for papers related to ''sars-cov-2/covid-19, ocular transmission and ophthalmology'' published till 8 may 2020 were selected. the websites of the world health organization (who), the centers for diseases for control and prevention (cdc), the american society of ophthalmology, spanish society of ophthalmology, and american and european societies of cataract and refractive surgery have been consulted. the literature search was conducted by all the authors, who selected, summarized, and agreed to the recommendations. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. the new coronavirus sars-cov-2 belongs to the same betacoronavirus family as the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndromerelated coronavirus (mers-cov), which were responsible for the outbreaks of severe acute respiratory syndrome (sars) in 2002 and 2012, respectively [1, 2] . the genome of this enveloped positive-sense unsegmented single-strand rna virus is 29,891 nucleotides in size, encoding 9860 amino acids. overall, the genome of sars-cov-2 has 89% nucleotide identity with bat sars-like-covzxc21 and 82% with that of human sars-cov [3] . viral genomic features are currently under intense scientific scrutiny in order to determine optimal diagnostic and therapeutic strategies for this emerging health threat. although the aforementioned viruses were also highly transmissible, the sars-cov-2 has spread more rapidly than its predecessors owing to a combination of extensive globalization of the economy and worldwide travel and the characteristics of the organism. this had led to a major global healthcare problem with severe global economic repercussions and a marked international recession. unquestionably one of the most powerful potential tools to slow the spread of the virus is to deepen current knowledge of its transmission routes. sars-cov-2 spreads primarily through respiratory droplets and through contact with contaminated objects or surfaces from an infected person. there is more limited evidence regarding sars-cov-2 transmission through other routes, such as the fecal-oral route, which relies upon the detection of the virus in different types of tissue and fluids, including sputum, blood, and feces [4] . moreover, sars-cov-2 has been detected in the tears and in conjunctival specimens collected from infected patients, suggesting that the ocular surface and the tears might represent a potential route for sars-cov-2 infection [5, 6] . epidemiological evidence supports this theory. a multicenter study which documented potential risk factors for sars-cov-2 transmission in patients requiring intubation [7] reported that unprotected eye contact with secretions from infected patients was the most predictive variable for transmission to healthcare workers. this observation highlighted the importance of using protective eyewear as an integral part of personal protective equipment to avoid infection. a similar case was reported earlier this year, when a doctor working in wuhan, who wore an n95 mask, but did not wear eye protection, was subsequently infected with sars-cov-2 and presented with marked conjunctival hyperemia, suggesting that unprotected exposure of the eyes to sars-cov-2 might be a route for disease transmission [8] . cumulative evidence suggests that sars-cov-2 infection can cause conjunctivitis, either as an early sign of the infection or during hospitalization for covid-19. the frequency of conjunctivitis in patients with covid-19 has not been accurately quantified to date with reports indicating markedly variable prevalence. a study analyzing a sample of 1099 patients hospitalized for covid-19 disease in china reported conjunctivitis symptoms in only 0.8% of afflicted patients (see fig. 2 in [9] ). in contrast, wu and co-workers [10] reported a prevalence of conjunctival involvement as high as 31.6%. the ocular symptoms related to covid-19 are in most cases non-specific and resemble those of other forms of viral conjunctivitis. recently, we evaluated the clinical characteristics of conjunctivitis in 14 spanish patients with covid-19 [11] . nearly half of them presented with signs of unilateral conjunctivitis. overall, 11 patients (79%) demonstrated mild ocular hyperemia and in 8 patients (57%) moderate secretions were observed. none of our patients showed conjunctival petechiae, corneal infiltrates, membranes, or pseudomembranes, which may be found in other forms of conjunctivitis (e.g., adenoviral conjunctivitis). none of the spanish patients experienced decreased vision and the mean duration of the conjunctivitis was 3 days (range 1-7 days). the presence of ocular pathology other than conjunctivitis in patients with sars-cov-2 infection has yet to be described. yuen et al. [12] evaluated 45 patients during the epidemic outbreak of sars in hong kong and did not observe any ocular manifestations. however, retinal disorders such as retinal vasculitis, retinal degeneration, and blood-retinal barrier breakdown have all been reported in experimental animal models of coronavirus infection [13, 14] . moreover, it is important to highlight that sars-cov-2 infection appears to predispose patients to thrombotic disorders [15] . therefore, the appearance of ocular vascular thrombotic events as a possible complication of the disease cannot be ruled out at this moment. the viral tropism to the ocular tissue could be partially explained by the presence of receptors for sars-cov-2 in the eye. the mechanism of sars-cov-2 cell entry depends on the viral spike s proteins binding to cellular receptors and on s protein priming by host cell proteases. with regard to virus receptors, sars-cov-2 appears to employ angiotensin-converting enzyme 2 receptor (ace2) for entry and transmembrane serine protease 2 (tmprss2) for s protein priming [16] . these receptors for sars-cov-2 have been identified in a wide range of human tissues, including lungs, small intestine, brain, kidney, and blood vessels among others [17] . importantly, ace2 and tmprss2 receptors have been identified in the eye not only on the ocular surface (cornea and conjunctiva) but also in deeper intraocular tissues, such as retina and choroid [18, 19] . a recent study has established that the expression level of these receptors in the conjunctiva is significantly higher than that in the cornea [18] . however, ace2 expression levels on the ocular surface tissues are lower than those documented in the lung or the brain. moreover, the binding capability of ace2 protein on conjunctival and corneal epithelial cells to the spike proteins of sars-cov has been shown to be inferior to that of the lung [20] . the eye possesses mechanical, immunological, and anatomical defense mechanisms [21] . mechanical barriers comprise the eyelids and eyelashes, as well as the tight intercellular junctions of the corneal epithelium and conjunctival mucosa. moreover, tears contain antimicrobial proteins such as lactoferrin and immunoglobulins. lactoferrin can inhibit the binding of sars-cov to ace2, and tear iga has proven to provide an effective immune defense against different kinds of viruses [22, 23] . the lacrimal drainage system acts as a physical self-cleaning system that clears pathogens from the ocular surface. however, this mechanism may prove counterproductive for sars-cov-2 infection, since passage of infected tears through the nasolacrimal drainage system may serve as an entry route of the virus from the infected ocular surface to the respiratory and digestive tract [24] . the opposite passage of the virus from the nasal and oral mucosa to the eye seems less likely, but cannot be ruled out entirely. a recent study evaluated the ocular tropism of sars-cov-2 in patients with confirmed covid-19. of the 56 subjects investigated there was only one patient who gave a history of prior pterygium surgery, with conjunctivitis and a positive pcr result from the conjunctival swab highlighting the importance of an intact ocular surface in resisting virus invasion [25] . these data could explain the virus' ocular tropism and account for the limited ocular involvement in a healthy eye. there is good evidence for the presence of sars-cov-2 rna in the tears of patients with covid-19 and conjunctivitis [5, 9] . interestingly, reports describing the presence of the virus in tears and conjunctival secretions from patients with conjunctivitis did not detect the virus in patients without conjunctivitis. however, another study from wuhan comprising 63 subjects with confirmed covid-19 found that the only patient with conjunctivitis exhibited a negative conjunctival swab for sars-cov-2 rna, whereas one patient without conjunctivitis had a positive conjunctival swab [26] . undoubtedly, the most appropriate laboratory test for an accurate sars-cov-2 diagnosis is real-time polymerase chain reaction (rt-pcr) of a nasopharyngeal specimen [27] . overall, this test demonstrates high specificity, and its sensitivity depends on the specimen and the time of testing during the course of the disease. importantly, rt-pcr has also been used to detect viral nucleic acid in conjunctival samples. our research team from hospital clinico san carlos, madrid, spain collected conjunctival swab samples from 36 patients with laboratory-confirmed covid-19. of those, 18 patients exhibited conjunctivitis and 18 did not. sars-cov-2 rna was detected in the conjunctival swab of two patients (5.5%). among the 18 patients with conjunctivitis, only one (5.5%) showed a positive result. likewise, sars-cov-2 rna was detected in only one patient without conjunctivitis (5.5%) [28] . therefore, our study revealed the same rate of positive results amongst the group with and without conjunctivitis, suggesting that detecting sars-cov-2 in ocular fluids is not dependent upon the clinical presence of conjunctivitis. however, this extremely low positive rate of sars-cov-2 rna test by rt-pcr in tears and conjunctival secretions from patients with laboratory-confirmed sars-cov-2 infection raises several doubts for its implementation. firstly, the viral load in the conjunctival sample may prove to be insufficient for successful rt-pcr detection of the virus. the viral load obtained with a conjunctival specimen has been consistently shown to be less than that from a nasopharyngeal sample [9] . therefore, it is possible that the viral load does not reach the threshold of detection of the virus, which could explain rt-pcr's low sensitivity in detecting sars-cov-2 rna in tears and conjunctival secretions. indeed, a recent paper by zhou et al. [29] found that the proportion of positive results from conjunctival sars-cov-2 swabs was only 2.5% (only 3 patients from a cohort of 121 patients with confirmed covid-19 tested positive for sars-cov-2 rna with conjunctival swabs). this disconcerting finding is consistent with other studies that have reported a relatively low likelihood of detecting the virus from tear specimens of patients with covid-19 [30] . table 1 summarizes the relevant published articles which report rt-pcr results from tears and conjunctival secretions. since rt-pcr testing is highly specific, but appears to lack sufficient sensitivity, negative test results could in fact be false negative and thus may not exclude the presence of the virus. in this context it is noteworthy that sensitivity can be improved if multiple specimens are collected. a study by xia et al. [5] tested tear and conjunctival secretions twice at an interval of 2-3 days and the results showed better detection and good consistency. however, it should be noted that limited resources and inadequate clinical access to patients with covid-19 by and large limit the possibility of collecting multiple samples. on the other hand, it is possible that the virus is only present in the tears and conjunctival secretions for a brief period of the disease duration, and thus the negative results may be due to incorrect timing of sample collection. a recent experimental study on rhesus macaques confirmed the short-lived presence of the virus in tears after conjunctival inoculation, indicating that although the virus-containing fluid can be found in ocular tissues, the majority of this fluid is drained into the nasopharyngeal space, or swallowed [31] . another factor that may influence the negative results of conjunctival swabs is the stage of the disease at which the conjunctival sample is collected. in a study by chan et al. [32] , tears swabs and conjunctival scrapings tested positive for sars-cov only when collected during the early stage of the infection, whereas the results were negative when specimens were collected at a later stage of the disease. in contrast, a case report of a 30-year-old man, who developed conjunctivitis on day 13 of the infection, demonstrated positive conjunctival swabs for sars-cov-2 over a period of 5 days until day 18 of his illness [9] . similarly, in a case from italy, sars-cov-2 rna was detected in conjunctival secretions up to day 21 following hospital admission, and then 5 days after it became undetectable, but the virus was detected again in the ocular swab sample collected at day 27 [33] . despite ocular complications not being a common clinically detectable manifestation of sars-cov-2 infection, recent evidence suggests that ocular exposure may represent a major transmission route for the virus. thus, the eye not only constitutes a potential site of virus replication but more alarmingly also an insidious route for the transmission of the virus from the ocular surface to the respiratory and gastrointestinal tract. these findings highlight the relevance of appropriate use of personal protective gear for all healthcare personnel, including eye wear protection as a integral part of safety procedures against virus contamination. as a direct result of the pandemic, the healthcare burden of patients without covid-19 has been reduced, and only urgent ophthalmological procedures are performed in many countries. as a result of the proximity needed for ophthalmological examination and the n number of patients with positive rt-pcr form nasopharyngeal swabs, rt-pcr real-time polymerase chain reaction unavoidable exposure to tears and ocular discharge during the exam, ophthalmologists are high-risk personnel for sars-cov-2 infection. many ophthalmic instruments such as gonioscopy and laser lenses as well as ophthalmic ultrasound probes require direct or close contact with patients, which presumably increases the risk of viral transmission. a non-contact tonometer may create an aerosol when measuring intraocular pressure by punching air onto the cornea of patients; hence, it may also facilitate virus spread by aerosol transmission [34] . moreover, since sars-cov-2 has been detected in tears and ocular fluids [11, 28] , nasolacrimal duct syringing, probing, and intubation, which can cause splashing of tears and nasolacrimal secretions, may be considered aerosol-generating procedures [35] . in fact, many intraocular surgical procedures have been deemed aerosol-generating procedures. as such, endoscopic dacryocystorhinostomy should be classified as a high-risk procedure [36, 37] . aerosol generation has been evaluated during phacoemulsification and suggestions have been made to minimize visible aerosol production by using a 2.2-mm phacoemulsification tip and reapplying hydroxypropyl methylcellulose (hpmc) coating of the cornea every minute during phacoemulsification [38] . another study revealed that phacoemulsification did not generate aerosols of 10 lm or smaller with continuous power using 80% longitudinal, 100% torsional, or 80% longitudinal setting with hpmc on the surface [39] . visible aerosol generation during vitrectomy surgery has been also evaluated using a physical model, although this study did not find evidence of visible aerosol generation during simulated vitrectomy surgery [40] . likewise, lasers, electrical cutting, and coagulation systems during surgery may generate an aerosol [41, 42] . therefore, it is essential to adjust the equipment, surgical techniques, and work protocols to diminish the risk of exposure as much as possible. a modification in the design of the surgical fields as well as the use of three-dimensional display systems for ophthalmic surgery may increase the physical distance between surgeon and patient. several ophthalmological societies, such as the american society of ophthalmology, the american and european societies of cataract and refractive surgery, and the spanish society of ophthalmology have issued a series of recommended standard operating protocols on the protective measures to be employed that have been updated with emerging evidence. all agree that it is safest to assume that any ophthalmic patient is potentially at risk of having been infected with sars-cov-2, especially in regions currently facing significant outbreaks of covid-19. overall, the recommended protocols include access control systems with interim guidance for triage, physical distancing measures, frequent and meticulous disinfection of common areas and equipment, the use of slit lamp barriers, or breath shields, a surgical mask for the patient, and personal protective equipment, including eye protection for healthcare personnel [43] . the role of telemedicine during the epidemic is also valuable in diminishing risk. ideally, necessary surgery should be performed only after a negative diagnostic test for sars-cov-2 with pcr prior to surgery for patients and operating room staff alike, as well as requesting a signed informed consent from the patient explaining the risks arising from sars-cov-2 infection. sars-cov-2 is a highly contagious virus that causes significant morbidity and mortality worldwide. understanding its transmission routes is essential to reducing further spread of the pandemic. the eye appears to play a role in virus replication and downstream transmission. accurate and consistent detection of ophthalmic involvement with conjunctival swabs remains problematic and variable since the virus load can remain below the threshold of detection and the timing of taking the swab sample is critical. better understanding and detection of viral transmission from the ocular surface to the respiratory and gastrointestinal tract is required. further large controlled studies are warranted to evaluate the precise rate and role of ocular involvement in sars-cov-2 infection. the clinical value of tear and conjunctival testing and the risk of infection for ophthalmic personnel requires further elucidation. funding. no funding or sponsorship was received for this study or publication of this article. authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. disclosures. noemi güemes-villahoz, barbara burgos-blasco, beatriz vidal-villegas, julián garcía-feijoó , pedro arriola-villalobos, jose maría martínez de la casa and david diaz-valle declare that they have no conflict of interest. anastasios g. konstas is a member of the journal's editorial board. compliance with ethics guidelines. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. data availability. data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. commons attribution-non-commercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http:// creativecommons.org/licenses/by-nc/4.0/. the severe acute 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generation during endonasal procedures in the era of covid-19: risks and recommendations. otolaryngol head neck surg ophthalmology in the time of covid-19: experience from hong kong eye hospital reducing visible aerosol generation during phacoemulsification in the era of covid-19. eye (lond) aerosol generation through phacoemulsification assessing visible aerosol generation during vitrectomy in the era of covid-19. eye (lond) clinical evidence based review and recommendations of aerosol generating medical procedures in otolaryngology-head and neck surgery during the covid-19 pandemic the characterization of surgical smoke from various tissues and its implications for occupational safety covid-19 disease and ophthalmology: an update. ophthalmol ther key: cord-326013-5i35zdmv authors: carpinteiro, alexander; edwards, michael j.; hoffmann, markus; kochs, georg; gripp, barbara; weigang, sebastian; adams, constantin; carpinteiro, elisa; gulbins, anne; keitsch, simone; sehl, carolin; soddemann, matthias; wilker, barbara; kamler, markus; bertsch, thomas; lang, karl s.; patel, sameer; wilson, gregory c.; walter, silke; hengel, hartmut; pöhlmann, stefan; lang, philipp; kornhuber, johannes; becker, katrin anne; ahmad, syed a.; fassbender, klaus; gulbins, erich title: pharmacological inhibition of acid sphingomyelinase prevents uptake of sars-cov-2 by epithelial cells date: 2020-10-29 journal: cell rep med doi: 10.1016/j.xcrm.2020.100142 sha: doc_id: 326013 cord_uid: 5i35zdmv the acid sphingomyelinase/ceramide system plays an important role in bacterial and viral infections. here we report that either pharmacological inhibition of acid sphingomyelinase with amitriptyline, imipramine, fluoxetine, sertraline, escitalopram, or maprotiline, or genetic downregulation of the enzyme prevents infection of cultured cells or freshy isolated human nasal epithelial cells with sars-cov-2 or pseudoviral pp-vsv-sars-cov-2 particles expressing spike, a bona fide system mimicking sars-cov-2 infection. infection activates acid sphingomyelinase and triggers a release of ceramide on the cell surface. neutralization or consumption of surface ceramide reduces infection with pp-vsv-sars-cov-2 spike. treating volunteers with a low dose of amitriptyline prevents infection of freshly isolated nasal epithelial cells with pp-vsv-sars-cov-2 spike. the data justify clinical studies investigating whether amitriptyline, a safe drug used clinically for almost 60 years, or other antidepressants that functionally block acid sphingomyelinase prevent sars-cov-2 infection. infections with a novel member of the coronaviridae family were reported in late 2019 in wuhan, china 1 . the virus was named severe acute respiratory syndrome coronavirus-2 (sars-cov-2). subsequently, the virus spread globally and is responsible for the coronavirus disease 2019 (covid19) pandemic. infection with sars-cov-2 often results in mild respiratory tract disease, but a substantial number of patients also experience severe symptoms and pneumonia, and ∼70% of these critically ill patients require intensive care and ventilator treatment, with a mortality rate of 62% 2 . even when the large number of only mildly affected patients are included, the mortality rates are higher than those associated with seasonal influenza 3,4 . cellular infection with sars-cov-2 is initiated by the binding of the surface unit s1 of the viral spike glycoprotein to its cellular receptor angiotensin-converting enzyme 2 (ace2), resulting in cleavage of the viral spike protein by the activity of transmembrane serine protease 2 (tmprss2) or cathepsin l, and in viral entry [5] [6] [7] [8] . although the binding of the virus to its receptor has been elucidated in detail [6] [7] [8] , the changes that occur in the host cell membrane during viral processing and entry require definition. membrane changes that mediate viral entry may be a very promising target for preventing the infection. previous studies have used replication-deficient vesicular stomatitis virus (vsv) pseudoviral particles (pp-vsv) presenting coronavirus spike protein (pp-vsv-sars-cov-2 spike) on their surface. studies have shown that these particles accurately reflect key aspects of the entry of coronavirus into host cells 5 . these particles were previously shown to bind to ace2 for infectious entry, and entry was inhibited by anti-ace2 antibodies 5 . thus, these particles are a bona fide model for studying the events of sars-cov-2 entry. we have previously shown that acid sphingomyelinase and ceramide play an important role in receptor signaling and infection biology 9, 10 . acid sphingomyelinase (ec 3.1.4.12, sphingomyelin phosphodiesterase; optimal ph 5.0) is a glycoprotein that functions as a lysosomal hydrolase, catalyzing the degradation of sphingomyelin to phosphorylcholine and ceramide. acid sphingomyelinase is present in lysosomes, j o u r n a l p r e -p r o o f 5 but because these compartments are constantly recycling to the plasma membrane it can also be found on the cell surface 9, 10 . the activity of acid sphingomyelinase on the cell surface results in the formation of ceramide in the outer leaflet of the cell membrane. the generation of ceramide molecules within the outer leaflet alters the biophysical properties of the plasma membrane, because the very hydrophobic ceramide molecules spontaneously associate with each other to form small ceramide-enriched membrane domains that fuse and form large, highly hydrophobic, tightly packed, gel-like ceramide-enriched membrane domains [10] [11] [12] [13] . in addition, ceramide has been shown to directly bind to a variety of proteins, including cathepsin d 14 , phospholipase a 2 15 , ceramide-activated protein serine/threonine phosphatases (capp) 16 , protein kinase c isoforms 17, 18 , and lc3b-ii 19 . many antidepressants functionally inhibit acid sphingomyelinase activity [20] [21] [22] [23] [24] [25] . these cationic amphiphilic drugs indirectly inhibit acid sphingomyelinase activity by displacing the enzyme from lysosomal membranes, in particular intralysosomal vesicles, thereby releasing the enzyme into the lysosomal lumen and causing its partial degradation [20] [21] [22] [23] [24] [25] . we have previously shown that rhinovirus infections activate acid sphingomyelinase and lead to the formation of ceramide and ceramide-enriched membrane domains. amitriptyline, sertraline, and other functional inhibitors of acid sphingomyelinase activity (fiasmas) inhibit cellular infection with rhinovirus 26 . similar observations have been made regarding infections with ebola virus 27 demonstrating that amitriptyline and other fiasmas inhibit infection with ebola virus in vitro 27 . because some antidepressants are widely used in clinical practice and have a very favorable safety profile, we investigated whether these drugs could be repurposed to treat or prevent infections with sars-cov-2. our results show that acid sphingomyelinase is activated within 20 to 30 minutes to determine whether acid sphingomyelinase can be targeted to prevent the infection of cells with sars-cov-2, we treated vero cells with the fiasma amitriptyline [22] [23] [24] or left the cells untreated. we then exposed the cells to pp-vsv-sars-cov-2 spike next, we determined whether amitriptyline also affects the infection of human caco-2 cells with authentic sars-cov-2. amitriptyline-pretreated caco-2 cells were infected with sars-cov-2, and the replication of sars-cov-2 was monitored by reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr) using two separate primer sets and a plaque assay. the results showed that amitriptyline also reduced the infection of human cells with sars-cov-2 by approximately 90% (fig. 2 ). j o u r n a l p r e -p r o o f 8 the results of these studies suggested that acid sphingomyelinase serves an important function in sars-cov-2 infection. to confirm the role of acid sphingomyelinase in infection with sars-cov-2 independent of pharmacological inhibition, we used shrna-mediated suppression of the expression of acid sphingomyelinase in caco-2 cells. we confirmed the downregulation of acid sphingomyelinase by measuring the activity of the enzyme in lysates of uninfected cells (fig. 3a) . the results showed that genetic downregulation of acid sphingomyelinase prevented infection with pp-vsv-sars-cov-2 spike (fig. 3a ). control shrna constructs had no effect on cellular infection with pp-vsv-sars-cov-2 spike. we further tested whether neutral sphingomyelinase may also be involved in cellular infection with sars-cov-2. to this end, we transfected caco-2 cells with shrna targeting neutral sphingomyelinase 2 and control shrna. transfection reduced neutral sphingomyelinase activity by approximately 60% but had no effect on cellular infection with pp-vsv-sars-cov-2 spike (fig. 3a) . we next investigated whether infection with pp-vsv-sars-cov-2 spike activates the acid sphingomyelinase/ceramide system. the results showed that treating vero cells with pp-vsv-sars-cov-2 spike resulted in rapid activation of acid sphingomyelinase and a concomitant release of ceramide (fig. 3b, fig. 4a ). in contrast, treating vero cells with pp-vsv lacking sars-cov-2 spike or with pp-vsv-g particles did not result in the activation of acid sphingomyelinase or in a release of ceramide (fig. 3b, fig. 4a ), a finding indicating that the activation of acid sphingomyelinase and the release of ceramide are specifically induced by the spike protein. pretreatment of the cells with 5, 10, 20, or 25 µm amitriptyline prevented the activation of acid sphingomyelinase and the release of ceramide upon infection with pp-vsv-sars-cov-2 spike for 30 min (fig. 3b, fig. 4a ). controls showed that amitriptyline reduced both the constitutive and the viral-induced activity of acid sphingomyelinase (fig. 3b ). treating vero cells with neutralizing antibodies to spike or with recombinant ace2 protein prevented the activation of acid sphingomyelinase and the release of ceramide upon infection with pp-vsv-sars-cov-2 spike (fig. 3b, fig. 4a amitriptyline and other drugs with similar structure and properties have been clinically used for many years (since 1962) to treat patients with depressive disorder. in the volunteer studies we used a low dose of amitriptyline, i.e., 0.5 mg/kg, which is lower than the dose usually used to treat patients with major depression (75-150 mg/day, i.e., approximately 1-2 mg/kg). at this dose, the drug has only mild effects, such as dry mucosa and tiredness. however, at higher dosages (higher than 100 mg/day, twice the dose we used or higher), amitriptyline and many other antidepressants may induce a cardiac qt elongation as a possible (but rare) adverse effect. qt elongation can result in cardiac arrhythmia and serious cardiac adverse effects 29 . thus, it may be advisable to perform an electrocardiogram (ecg) before the first administration of amitriptyline or any of the other fiasmas used here to exclude a pre-existing qt elongation. amitriptyline and other antidepressants acting as fiasmas exert no known adverse effects on the immune system. our studies demonstrate that even low dosages of amitriptyline provide long-lasting and very efficient protection against infection. our findings also indicate that other drugs, such as imipramine, desipramine, fluoxetine, sertraline, escitalopram, and maprotiline, with concentrations similar to that of amitriptyline 22 our studies are strongly supported by a recently pre-printed study 30 that showed a marked beneficial effect of antidepressants on the clinical course of covid-19. hospitalized patients receiving antidepressants had a much better outcome, determined as decreased medical necessity of intubation or death, than patients receiving no antidepressants. best results were obtained with venlafaxin, fluoxetine, escitalopram and mirtazipine, drugs that were also shown in the present study to inhibit acid sphingomyelinase and ceramide release upon pp-vsv-sars-cov-2 spike infection. it is important to note that the dose of antidepressants used in this j o u r n a l p r e -p r o o f 13 retrospective study was not optimized. amitriptyline was used at a medium dose of 26 mg/day, which is a rather low dose. amitriptyline is often used at these low dosages for sedation and not as an antidepressant, while other drugs such as fluoxetine and escitalopram are always used as antidepressants and applied at higher dosages. further, fluoxetine also inhibits sars-cov-2 replication 31 , an effect that was not investigated in the present study, since the pseudoviral particles do not replicate. amitriptyline and many other antidepressants are weak bases that are protonated in lysosomes and thereby trapped in lysosomes, but they also localize in acidic subcompartments of the cell membrane. because of their physicochemical properties (for instance for amitriptyline: high lipophilicity and weak basicity; logp = 4.92, pka = 9.4, www.drugbank.ca), these drugs accumulate in acidic intracellular compartments 32 . this accumulation leads to a high tissue concentration and accordingly to a high volume of distribution (for instance for amitriptyline: vd = 16 l/kg, www.drugbank.ca). thus, high tissue concentrations of these drugs can be detected in all organs. in fact, the highest uptake of amitriptyline among all tissues with a lung/blood concentration gradient of approximately 50 is found in the lungs of mice and rats 33, 34 . in humans, even higher concentration gradients have been found between lung and blood 35 . because of the particularly high accumulation in the lung, the antiviral in vitro concentrations (up to 25 µm) that we found to be effective are likely to be achieved with conventional oral therapy of amitriptyline. we also expect a considerably longer elimination half-life of amitriptyline in lung tissue than in blood because of the high concentration of these drugs in deep compartments, such as lysosomes. the organic ring system of amitriptyline and other fiasmas may bind to the lipid membrane, whereas the protonated tertiary amine displaces acid sphingomyelinase from lysosomal membranes or from the plasma membrane within acidic subdomains. thus, these weak bases do not directly inhibit acid sphingomyelinase activity but rather functionally inhibit it. this mode of action also explains why antidepressants do not completely inhibit acid sphingomyelinase activity and do not induce severe adverse effects in clinical use. in addition, our studies indicate that the administration of anti-ceramide antibodies or neutral ceramidase also protects against sars-cov-2 infections. anti-ceramide j o u r n a l p r e -p r o o f 14 antibodies and neutral ceramidase are not approved for clinical use, but could be easily tested for systemic or local adverse effects before a potential treatment of sars-cov-2 infections. furthermore, both anti-ceramide antibodies and neutral ceramidase could potentially be administered via inhalation or as a nasal spray to prevent sars-cov-2 infections. the molecular mechanisms how ceramide mediates infection of epithelial cells with sars-cov-2 remain to be determined. ceramide has been shown to directly activate several enzymes and to form large, highly hydrophobic ceramide-enriched membrane domains that serve to re-organize receptor and signalling molecules [10] [11] [12] [13] . it remains to be determined whether sars-cov-2 also induces these platforms and whether ceramide and/or these platforms are required for internalization or activation of tmprss2 and cathepsin l that mediate processing of spike require for fusion with the cell membrane 36 . high ceramide levels have been linked with hypertension and obesity 37, 38 the authors declare no competing financial interests. further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, erich gulbins (erich.gulbins@uni-due.de). this study did not generate new unique reagents. the published article includes all datasets generated or analyzed during the study. human nasal epithelial cells were obtained from 6 healthy volunteers, 4 women and 2 men. their ages were 20, 21, 43, 48, 54, and 56. we did not observe any differences in infection between cells from women or men. sample size was planned for the continuous variable difference in viral uptake and was based on two-sided we obtained nasal epithelial cells from these volunteers immediately before oral administration of 0.5 mg/kg amitriptyline (neuraxpharm, germany) and again 1. caco-2 colon epithelial cells (atcc htb-37) were cultured in dmem supplemented as described above. pseudotyped viral particles based on a replication-deficient vsv that codes for egfp and firefly luciferase instead of parental vsv-g (vsv*∆g-fluc) 39 a further aliquot of the nasal epithelial cells was immediately shock frozen in liquid nitrogen after removal for determination of acid sphingomyelinase activity (see below). nasal epithelial cells were also obtained from untreated volunteers as described above and were treated with 10 µm amitriptyline (sigma; # a 8404) dissolved in 0.9% nacl, anti-ceramide or control antibodies, neutral ceramidase (see below) or left untreated for 60 min ex vivo and then infected with pp-vsv-sars-cov-2 spike particles for 60 min. cells were then washed and further incubated for 24 h to allow expression of egfp. infection was analyzed as above. vero cells were grown on glass coverslips, treated with 10 or 25 µm amitriptyline as ceramide amounts were determined by comparison with a standard curve using c16 to c24 ceramides as substrates. to investigators were blinded to the identity of the samples in all microscopy experiments. all data are available upon request from the authors. authors confirm that all data are included in the manuscript. a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor cryo-em structure of the 2019-ncov spike in the prefusion conformation structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structural and functional basis of sars-cov-2 entry by using human ace2 cd95 signaling via ceramide-rich membrane rafts host defense against pseudomonas aeruginosa requires ceramide-rich membrane rafts raft ceramide in molecular medicine compartmentalization of ceramide signaling: physical foundations and biological effects observation of topical catalysis by sphingomyelinase coupled to microspheres cathepsin d targeted by acid sphingomyelinase-derived ceramide ceramide binds to the calb domain of cytosolic phospholipase a2 and facilitates its membrane docking and arachidonic acid release ceramide-activated protein phosphatase: partial purification and relationship to protein phosphatase 2a pkc zeta is a molecular switch in signal transduction of tnf-alpha, bifunctionally regulated by ceramide and arachidonic acid selective ceramide binding to protein kinase c-alpha and -delta isoenzymes in renal mesangial cells targeting flt3-itd signaling mediates ceramide-dependent mitophagy and attenuates drug resistance in aml the tricyclic antidepressant desipramine causes proteolytic degradation of lysosomal sphingomyelinase in human fibroblasts interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine identification of new functional inhibitors of acid sphingomyelinase using a structure-property-activity relation model acid sphingomyelinase/ceramide system mediates effects of antidepressant drugs antidepressants act by inducing autophagy controlled by sphingomyelin-ceramide emerging mechanisms of drug-induced phospholipidosis rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection sars-cov-2 receptor ace2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues increase of heart rate and qtc by amitripytline, but not by venlafaxine, is correleated to serum concentration association between ssri antidepressant use and reduced risk of intubation or death in hospitalized patients with coronavirus disease 2019: a multicenter retrospective observational study the serotonin reuptake inhibitor fluoxetine inhibits sars-cov-2 quantitative modeling of selective lysosomal targeting for drug design distribution and fate of c14-amitriptyline in mice and rats postmortem release of amitriptyline from the lungs; a mechanism of postmortem drug redistribution postmortem distribution of tramadol, amitriptyline, and their metabolites in a suicidal overdose efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 ceramide is upregulated and associated with mortality in patients with chronic heart failure the unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multispecies type i interferon mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization key: cord-341287-i1hyk962 authors: smith, trevor r. f.; patel, ami; ramos, stephanie; elwood, dustin; zhu, xizhou; yan, jian; gary, ebony n.; walker, susanne n.; schultheis, katherine; purwar, mansi; xu, ziyang; walters, jewell; bhojnagarwala, pratik; yang, maria; chokkalingam, neethu; pezzoli, patrick; parzych, elizabeth; reuschel, emma l.; doan, arthur; tursi, nicholas; vasquez, miguel; choi, jihae; tello-ruiz, edgar; maricic, igor; bah, mamadou a.; wu, yuanhan; amante, dinah; park, daniel h.; dia, yaya; ali, ali raza; zaidi, faraz i.; generotti, alison; kim, kevin y.; herring, timothy a.; reeder, sophia; andrade, viviane m.; buttigieg, karen; zhao, gan; wu, jiun-ming; li, dan; bao, linlin; liu, jiangning; deng, wei; qin, chuan; brown, ami shah; khoshnejad, makan; wang, nianshuang; chu, jacqueline; wrapp, daniel; mclellan, jason s.; muthumani, kar; wang, bin; carroll, miles w.; kim, j. joseph; boyer, jean; kulp, daniel w.; humeau, laurent m. p. f.; weiner, david b.; broderick, kate e. title: immunogenicity of a dna vaccine candidate for covid-19 date: 2020-05-20 journal: nat commun doi: 10.1038/s41467-020-16505-0 sha: doc_id: 341287 cord_uid: i1hyk962 the coronavirus family member, sars-cov-2 has been identified as the causal agent for the pandemic viral pneumonia disease, covid-19. at this time, no vaccine is available to control further dissemination of the disease. we have previously engineered a synthetic dna vaccine targeting the mers coronavirus spike (s) protein, the major surface antigen of coronaviruses, which is currently in clinical study. here we build on this prior experience to generate a synthetic dna-based vaccine candidate targeting sars-cov-2 s protein. the engineered construct, ino-4800, results in robust expression of the s protein in vitro. following immunization of mice and guinea pigs with ino-4800 we measure antigen-specific t cell responses, functional antibodies which neutralize the sars-cov-2 infection and block spike protein binding to the ace2 receptor, and biodistribution of sars-cov-2 targeting antibodies to the lungs. this preliminary dataset identifies ino-4800 as a potential covid-19 vaccine candidate, supporting further translational study. c ovid-19, known previously as 2019-ncov pneumonia or disease, has emerged as a global public health crisis, joining severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) in a growing number of coronavirus-associated illnesses which have jumped from animals to people. there are at least seven identified coronaviruses that infect humans. in december 2019 the city of wuhan in china became the epicenter for an outbreak of the novel coronavirus, sars-cov-2. sars-cov-2 was isolated and sequenced from human airway epithelial cells from infected patients 1, 2 . disease symptoms range from mild flu-like to severe cases with life-threatening pneumonia 3 . the global situation is dynamically evolving, and on 30 january 2020 the world health organization declared covid-19 as a public health emergency of international concern (pheic), and on 11 march 2020 it was declared a global pandemic. as of 1 may 2020 there are 3,321,402 people confirmed infected and 237,180 deaths 4 . infections have spread to multiple continents. human-to-human transmission has been observed in multiple countries, and a shortage of disposable personal protective equipment 5 , and prolonged survival times of coronaviruses on inanimate surfaces 6 , have compounded this already delicate situation and heightened the risk of nosocomial infections. advanced research activities must be pursued in parallel to push forward protective modalities in an effort to protect billions of vulnerable individuals worldwide. currently, no licensed preventative vaccine is available for covid-19. to address the urgent need for a medical countermeasure to prevent the further dissemination of sars-cov-2 we have employed a synthetic dna-based vaccine approach. synthetic dna vaccines are amenable to accelerated developmental timelines due to the ability to quickly design multiple candidates for preclinical testing, scalable manufacturing of large quantities of the drug product, and the possibility to leverage established regulatory pathways to the clinic. synthetic dna is temperaturestable and cold-chain free, important features for delivery to resource-limited settings 7 . specifically for the development of a covid-19 vaccine candidate, we leveraged prior experiences in developing vaccine approaches to sars-cov 8 , and our own experience in developing a mers-cov vaccine (ino-4700) 9 ,10 , as well as taking advantage of our vaccine design and manufacturing pathway previously utilized for the zika vaccine candidate, gls-5700 11 , which was advanced to the clinic in under 7 months. ino-4700 and gls-5700 vaccines are currently in clinical testing. prior work has demonstrated that a dna approach for sars and mers can drive neutralizing antibody (nab) responses and provide protection in challenge models 8, 10 . our previous studies indicated immunization of small and large animal models with dna vaccines encoding mers-cov spike (s) protein provided protection against disease challenge with the matched virus. in subjects immunized with ino-4700 (mers-cov s protein dna vaccine) durable neutralizing antibodies (nabs) and t cell immune responses were measured, and a seroconversion rate of 96% was observed and immunity was followed for 60 weeks in most study volunteers 9 . ino-4700 phase 1/2a testing is continuing in south korea, and a larger phase 2 study is being planned to begin in the middle east, both areas which have been most affected by mers infections. the sars-cov-2 spike is most similar in sequence and structure to sars-cov spike protein 12 , and shares a global protein fold architecture with the mers-cov spike protein ( fig. 1 ) allowing us to build on our prior vaccine construct design 10 . unlike glycoproteins of hiv and influenza, the prefusion form of the coronavirus trimeric spike is conformationally dynamic, fully exposing the receptor-binding site infrequently 13 . the receptor-binding site is a vulnerable target for nabs. in fact, mers nabs targeted at the receptor-binding domain (rbd) tend to have greater neutralizing potency than other epitopes 14 . a recent report demonstrated that an anti-sars antibody could cross-react to the rbd of sars-cov-2 15 . these data suggest that the sars-cov-2 rbd is an important target for vaccine development. recent data has revealed sars-cov-2 s protein binds the same host receptor, angiotensin-converting enzyme 2 (ace2), as sars-cov s protein 12 . here, we describe the design and initial preclinical testing of covid-19 synthetic dna vaccine candidates. we show the expression of the sars-cov-2 s antigen rna and protein after in vitro transfection of cos-7 and 293t cells, respectively, with the vaccine candidates. we followed the induction of immunity by the selected immunogen in mice and guinea pigs, measuring sars-cov-2 s protein-specific antibody levels in serum and in the lung fluid, and antibody functionality through competitive inhibition of ace2 binding, pseudovirus and live virus neutralization. the ino-4800 vaccine induces cellular and humoral host immune responses that can be observed within days following a single immunization, including cross-reactive responses against sars-cov. the data demonstrate the immunogenicity of this covid-19 synthetic dna vaccine candidate targeting the sars-cov-2 s protein, supporting further translational studies to advance the development of this candidate in response to the current global health crisis. design and synthesis covid-19 dna vaccine constructs. four spike protein sequences were retrieved from the first four available sars-cov-2 full genome sequences published on gisaid (global initiative on sharing all influenza data). three spike sequences were 100% matched and one was considered an outlier (98.6% sequence identity with the other sequences). after performing a sequence alignment, the sars-cov-2 spike glycoprotein sequence was generated and an n-terminal ige leader sequence was added. the highly optimized dna sequence encoding sars-cov-2 ige-spike was created using inovio's proprietary in silico gene optimization algorithm to enhance expression and immunogenicity. the optimized dna sequence was synthesized, digested with bamhi and xhoi, and cloned into the expression vector pgx0001 under the control of the human cytomegalovirus immediate-early promoter and a bovine growth hormone polyadenylation signal. the resulting plasmids were designated as pgx9501 and pgx9503, designed to encode the sars-cov-2 s protein from the three-matched sequences and the outlier sequence, respectively (fig. 2a) . in vitro characterization of covid-19 dna vaccine constructs. we measured the expression of the encoded sars-cov-2 spike transgene at the rna level in cos-7 cells transfected with pgx9501 and pgx9503. using the total rna extracted from the transfected cos-7 cells we confirmed expression of the spike transgene by rt-pcr (fig. 2b) . in vitro spike protein expression in hek-293t cells was measured by western blot analysis using a cross-reactive antibody against sars-cov s protein on cell lysates. western blots of the lysates of hek-293t cells transfected with pgx9501 or pgx9503 constructs revealed bands approximate to the predicted s protein molecular weight, 140-142 kda, with slight shifts likely due to the 22 potential n-linked glycans in the s protein (fig. 2c) . in immunofluorescent studies the s protein was detected in 293t cells transfected with pgx9501 or pgx9503 (fig. 2d) . in summary, in vitro studies revealed the expression of the spike protein at both the rna and protein level after transfection of cell lines with the candidate vaccine constructs. humoral immune responses in mice. since candidate design, it has been observed that newly published sars-cov-2 spike protein sequences match pgx9501 with >99.9% amino acid sequence identity (supplementary data 1). pgx9501 was therefore selected as the vaccine construct to advance to immunogenicity studies, due to the broader coverage it would likely provide compared with the outlier, pgx9503. pgx9501 was subsequently termed ino-4800. the immunogenicity of ino-4800 was evaluated in balb/c mice, postadministration to the tibialis anterior muscle using the cellec-tra® delivery device 16 . the reactivity of the sera from a group of mice immunized with ino-4800 was measured against a panel of sars-cov-2 and sars-cov antigens (fig. 3a) . analysis revealed igg binding against sars-cov-2 s protein antigens, with limited cross-reactivity to sars-cov s protein antigens, in the sera of ino-4800 immunized mice. we measured the serum igg binding endpoint titers (epts) in mice immunized with pdna against recombinant sars-cov-2 spike protein s1 + s2 regions (fig. 3b, c) and recombinant sars-cov-2 spike protein receptor binding domain (rbd) (fig. 3d, e) . epts were observed in the sera of mice at day 14 after immunization with a single dose of ino-4800 (fig. 3c, e) . we developed a neutralization assay with a pnl4-3.luc.r-ebased pseudovirus displaying the sars-cov-2 spike protein. neutralization titers were detected by a reduction in relative luciferase units (rlu) compared with controls which had no decrease in rlu signal. balb/c mice were immunized twice with ino-4800, on days 0 and 14, and sera was collected on day 7 post-second immunization. the pseudovirus was incubated with serial dilutions of mouse sera and the sera-virus mixture was added to 293t cells stably expressing the human ace2 receptor (ace2-293t) for 72 h. neutralization id50 average titers of 92.2 were observed in ino-4800 immunized mice (fig. 4a, b) . no reduction in rlu was observed for the control animals. neutralizing titers were additionally measured against two wildtype sars-cov-2 virus strains by prnt assay. sera from ino-4800 immunized balb/c mice neutralized both sars-cov-2/wh-09/human/2020 and sars-cov-2/australia/vic01/2020 virus strains with average nd50 titers of 97.5 and 128.1, respectively (table 1) . live virus neutralizing titers were also evaluated in c57bl/6 mice following the same ino-4800 immunization regimen. sera from ino-4800 immunized c57bl/6 mice neutralized wildtype sars-cov-2 virus with average nd50 titer of 340 (table 1) . humoral immune responses in guinea pigs. we assessed the immunogenicity of ino-4800 in the hartley guinea pig model, an established model for intradermal vaccine delivery 17, 18 . one hundred micrograms of pdna was administered by mantoux injection to the skin and followed by cellectra® delivery device on day 0 as described in the methods section. on day 14 anti-spike protein binding of serum antibodies was measured by elisa. immunization with ino-4800 revealed an immune response in respect to sars-cov-2 s1 + 2 protein binding igg levels in the sera (fig. 5a, b) . the endpoint sars-cov-2 s protein binding titer at day 14 was 10,530 and 21 in guinea pigs treated with 100 µg ino-4800 or pvax (control), respectively (fig. 5b) . we next evaluated antibody neutralizing activity following intradermal ino-4800 immunization in the guinea pig model. guinea pigs were treated on days 0, 14, and 28 with pvax or ino-4800, and sera samples were collected on days 35 or 42 to measure sera neutralizing activity against pseudovirus or wildtype virus, respectively. sars-cov-2 pseudovirus neutralizing activity with average nd50 titers of 573.5 was observed for the ino-4800 immunized guinea pigs (table 1) . wildtype sars-cov-2 virus activity was also observed for the ino-4800 immunized guinea pigs with nd50 titers >320 by prnt assay observed in all animals ( table 1) . inhibition of sars-cov-2 s protein binding to ace2 receptor. the induction of antibodies capable of inhibiting spike protein engagement of host receptor is considered relevant for sars-cov-2 vaccine development. we therefore examined the receptor inhibiting functionality of ino-4800-induced antibody responses. we recently developed an elisa-based ace2 inhibition assay as a surrogate for neutralization. the assay is similar in principle to other surrogate neutralization assays which have been validated for coronaviruses 19 . as a control in our assay, we show ace2 can bind to sars-cov-2 spike protein with an ec 50 of 0.025 µg/ml (fig. 6a) . balb/c mice were immunized on days 0 and day 14 with 10 µg of ino-4800, and serum igg was purified on day 21 post-immunization to ensure inhibition is antibodymediated. we compared inhibition of the spike-ace2 interaction using serum igg from a naïve mouse and from an ino-4800 and pgx9503 (outlier (ol)) containing the ige leader sequence and sars-cov-2 spike protein insert. b rt-pcr assay of rna extracts from cos-7 cells transfected in duplicate with pgx9501 and pgx9503. extracted rna was analyzed by rt-pcr using pcr assays designed for each target and for cos-7 β-actin mrna, used as an internal expression normalization gene. delta c t (δ c t ) was calculated as the c t of the target minus the c t of β-actin for each transfection concentration and is plotted against the log of the mass of pdna transfected (plotted as mean ± sd). c analysis of in vitro expression of spike protein after transfection of 293t cells with pgx9501, pgx9503 or mock plasmid by western blot. 293t cell lysates were resolved on a gel and probed with a polyclonal anti-sars spike protein. blots were stripped then probed with an anti-β-actin loading control. d in vitro immunofluorescent staining of 293t cells transfected with 3 µg/well of pgx9501, pgx9503 or pvax (empty control vector). expression of spike protein was measured with polyclonal anti-sars spike protein igg and anti-igg secondary (green). cell nuclei were counterstained with dapi (blue). images were captured using imagexpress pico automated cell imaging system. scale bars are 80.15 µm (left), 66.8 µm (middle) and 77.31 µm (right). vaccinated mouse (fig. 6b) . we repeated the receptor inhibition assay with a group of five immunized mice, and demonstrating that ino-4800-induced antibodies competed with ace2 binding to the sars-cov-2 spike protein ( fig. 6c and supplementary fig. 1 ). ace2 binding inhibition was further evaluated in the guinea pig model. sera collected from ino-4800 immunized guinea pigs inhibited binding of sars-cov-2 spike protein over range of concentrations of ace2 (0.25 µg/ml through 4 µg/ml) (fig. 6d) . furthermore, serum dilution curves revealed sera collected from ino-4800 immunized guinea pigs blocked binding of ace2 to sars-cov-2 in a dilution-dependent manner (fig. 6e ). sera collected from pvax-treated animals displayed negligible activity in the inhibition of ace2 binding to the virus protein, the decrease in od signal at the highest concentration of serum is considered a matrix effect in the assay. ace2 is considered to be the primary receptor for sars-cov-2 cellular entry and blocking this interaction suggests ino-4800-induced antibodies believed important to prevent host infection. in summary, humoral immunogenicity testing in both mice and guinea pigs revealed the covid-19 vaccine candidate, ino-4800, was capable of eliciting functional blocking antibody responses to sars-cov-2 spike protein. biodistribution of sars-cov-2 reactive igg to the lung. lower respiratory disease (lrd) is associated with severe cases of covid-19. the presence of antibodies at the lung mucosa targeting sars-cov-2 could potentially mediate protection against lrd. therefore, we evaluated the presence of sars-cov-2 specific antibody in the lungs of immunized mice and guinea pigs. balb/c mice and hartley guinea pigs were immunized, on days 0 and 14 or 0, 14 and 28, respectively, with ino-4800 or pvax control pdna. bronchoalveolar lavage (bal) fluid was collected following sacrifice, and sars-cov-2 s protein elisas were performed. in both balb/c and hartley guinea pigs which received ino-4800 we measured a statistically significant increase in sars-cov-2 s protein binding igg in bal fluid compared with animals receiving pvax control (fig. 7a-d) . taken together, these data demonstrate the presence of anti-sars-cov-2 specific antibody in the lungs following immunization with ino-4800. fig. 3) . balb/c mouse sars-cov-2 epitope mapping. we performed epitope mapping on the splenocytes from balb/c mice receiving the 10 µg ino-4800 dose. thirty matrix mapping pools were used to stimulate splenocytes for 20 h and immunodominant responses were detected in multiple peptide pools (fig. 9a) . the responses were deconvoluted to identify several epitopes (h2-k d ) clustering in the receptor binding domain and in the s2 domain (fig. 9b) . interestingly, one sars-cov-2 h2-k d epitope, phgvvflhv, was observed to be overlapping and adjacent to the sars-cov human hla-a2 restricted epitope vvflhvtyv 20 . in summary, t cell responses against sars-cov-2 s protein epitopes were detected in mice immunized with ino-4800. the novel coronavirus, sars-cov-2, and associated covid-19 disease has become a global pandemic with a significant morbidity and mortality toll. currently, there are no covid-19 vaccines available, and global dissemination of sars-cov-2 may continue until there is a high level of herd immunity within the human population. here we have described the preclinical development of a synthetic dna-based covid-19 vaccine, ino-4800 to combat this emerging infectious disease. synthetic dna vaccine design and synthesis was immediately initiated upon public release of the sars-cov-2 genome sequences on 11 january 2020. our data support the expression and immunogenicity of the ino-4800 synthetic dna vaccine candidate in multiple animal models. humoral and t cell responses were observed in mice. in guinea pigs we employed clinical delivery parameters and observed sars-cov-2 s protein binding antibody titers and blocking of ace2/sars-cov-2 s protein interaction in serum samples from ino-4800-treated animals. nabs were also measured in both species. halting a rapidly emerging infectious disease requires an orchestrated response from the global health community and requires improved strategies to accelerate vaccine development. in response to the 2019/2020 coronavirus outbreak we employed a synthetic dna medicine platform. the design and manufacture of this synthetic dna vaccine represents a plug and play process in which we insert the target antigen sequence into a highly characterized and clinically tested plasmid vector backbone (pgx0001). the construct design and engineering parameters have been optimized for in vivo gene expression, and previously applied to mers, ebov, zika, and lassa dna vaccine constructs which are all undergoing clinical testing 7, 9, 11, 21, 22 . based upon our previous experience developing a vaccine against mers coronavirus, and previous published studies of sars vaccines, sars-cov-2 s protein was chosen as the antigen target. the sars-cov-2 s protein is a class i membrane fusion protein, which the major envelope protein on the surface of coronaviruses. initial studies have already been performed which indicate sars-cov-2 interaction with its host receptor (ace2) can be blocked by antibodies 23 . in vivo immunogenicity studies in both mouse and guinea pig models revealed levels of s proteinreactive igg in the serum of ino-4800 immunized animals. in addition to full-length s1 + s2 and s1, ino-4800 immunization induced rbd binding antibodies (fig. 3) , a domain known to be a target for nabs from sars-cov convalescent patients 24, 25 . we further demonstrate the functionality of these antibodies through neutralization of sars-cov-2 wild-type virus and pseudovirus (table 1) , and competitive inhibition of sars-cov-2 spike protein binding to the ace2 receptor in the presence of sera from ino-4800 immunized animals (fig. 6) . importantly, anti-sars-cov-2 binding antibodies were detected in lung washes of ino-4800-immunized mice and guinea pigs (fig. 7) . the presence of these antibodies in the lungs has the potential to protect against infection of these tissues and prevent lrd, which is associated with the severe cases of covid-19. in addition to humoral responses, cellular immune responses have been shown to be associated with more favorable recovery in mers-cov infection 26 , and are likely to be important against sars-cov infection 27 . here, we showed the induction of t cell responses against sars-cov-2 as early as day 7 post-vaccine delivery. rapid cellular responses have the potential to lower viral load and could potentially reduce the spread of sars-cov-2 and the associated covid-19 illness. we believe synthetic dna medicine platform has several synergistic characteristics which position it well to respond to disease outbreaks, such as covid-19. as mentioned previously, the ability to design and immediately synthesize candidate vaccine constructs means that in vitro and in vivo testing can potentially begin within days of receiving the viral sequence. the dna plasmid manufacture process allows for scalable manufacture of drug product, which has the potential to circumvent the complexities of conventional vaccine production in eggs or cell culture. additionally, we have published on the stability profile afforded to these products through the use of our optimized dna formulation 7 . the stability characteristics mean that our dna drug product is non-frozen and can be stored for 4.5+ years at 2-8°c, room temperature (rt) for 1 year and 1 month at 37°c, while maintaining potency at temperatures upwards of 60°c. in the context of a pandemic outbreak, the stability profile of a vaccine plays directly to its ability to be deployed and stockpiled in an efficient and executable manner. in this study, we observed seroconversion after a single intradermal administration of the ino-4800 in guinea pigs (fig. 6) . whether a single immunization will be sufficient in humans will be investigated in clinical trials. although vaccine-induced immunopathology has been raised as a potential concern for sars and mers vaccine candidates, and possibly for sars-cov-2 vaccines, these concerns are likely vaccine-platform dependent and, to-date, no evidence of immune pathogenesis has been reported for mers dna vaccines in mice or non-human primate models 10 or sars dna vaccines in mice 8 . lung immunopathology characterized by th2-related eosinophilia has been reported for whole inactivated virus (iv), recombinant protein, peptide, and/or recombinant viral vector vaccines following sars-cov challenge [28] [29] [30] [31] [32] , and more recently in a mers-cov challenge model 33 . however, in the majority of studies protective efficacy without lung immunopathology has been reported for sars-cov and mers-cov vaccines 8, 10, [34] [35] [36] [37] [38] [39] [40] . it is important to note the majority of studies demonstrating cov vaccine-induced immunopathology utilized the balb/c mouse, a model known to preferentially develop th2-type responses. the dna vaccine platform induces th1-type immune responses and has demonstrated efficacy without immunopathology in models of respiratory infection, including sars-cov 8 , mers-cov 10 , and rsv 41 . sars-cov-2 animal challenge studies will assess ino-4800-mediated protection against disease, and vaccineenhanced disease. here, we report functional neutralization of ino-4800 immune sera using a sars-cov-2 pseudovirus assay (fig. 4 , table 1 ), and prnt assay against two wild-type sars-cov-2 strains ( table 1) . as well, we show that ino-4800 induced antibodies block sars-cov-2 spike binding to the host receptor ace2, using a surrogate neutralization assay (fig. 6 ). this study highlights the immunogenicity of ino-4800, and further animal studies will test protection against infection. in summary, these initial results describing the immunogenicity of covid-19 vaccine candidate, ino-4800 are promising, and it is particularly encouraging to measure functional antibodies and t cell responses in multiple animal models. this study supports the further evaluation of ino-4800 as a vaccine candidate for covid-19. cell lines. hek-293t (atcc® crl-3216™) and african green monkey kidney cos-7 (atcc® crl-1651™) cell lines were obtained from atcc (old town manassas, va). all cell lines were maintained in dmem supplemented with 10% fetal bovine serum (fbs) and penicillin-streptomycin. in vitro rna expression (qrt-pcr) in vitro mrna expression of the plasmid was demonstrated by transfection of cos-7 with serially diluted plasmids followed by analysis of the total rna extracted from the cells using reverse transcription and pcr. transfections of four concentrations of the plasmid were performed using fugene® 6 transfection reagent (promega) which resulted in final masses ranging between 80 and 10 ng per well. the transfections were performed in duplicate. following 18 to 26 h of incubation the cells were lysed with rlt buffer (qiagen). total rna was isolated from each well using the qiagen rneasy kit following the kit instructions. the resulting rna concentration was determined by od 260/280 and samples of the rna were diluted to 10 ng per µl. one hundred nanograms of rna was then converted to cdna using the high capacity cdna reverse transcription (revt) kit (applied biosystems) following the kit instructions. revt reactions containing rna but no reverse transcriptase (minus rt) were included as controls for plasmid dna or cellular genomic dna sample contamination. eight microliters of sample cdna were then subjected to pcr using primers and probes that are specific to the target sequence (pgx9501 forward -caggac aagaacacacaggaa; pgx9501 reverse -caggcaggatttgggagaaa; pgx9501 probe -acccatcaaggactttggagg; and pgx9503 forward -aggacaagaacacacaggaag; pgx9503 reverse -caggatctggga gaagttgaag; pgx9503 probe -acaccacccatcaaggactttgga). in a separate reaction, the same quantity of sample cdna was subjected to pcr using primers and a probe designed (β-actin forward -gtgacgtggacatccgta aa; β-actin reverse -cagggcagtaatctccttctg; β-actin probe -tac cctggcattgctgacaggatg) for cos-7 cell line β-actin sequences. the primers and probes were synthesized by integrated dna technologies, inc. and the probes were labeled with 56-fam and black hole quencher 1. the reaction used abi fast advance 2×(cat. no. 4444557), with final forward and reverse primer concentrations of 1 µm and probe concentrations of 0.3 µm. using a quantstudio 7 flex real time pcr studio system (applied biosystems), samples were first subjected to a hold of 1 min at 95°c and then 40 cycles of pcr with each cycle consisting of 1 s at 95°c and 20 s at 60°c. following pcr, the amplifications results were analyzed as follows. the negative transfection controls, the minus revt controls, and the ntc were scrutinized for each of their respective indications. the threshold cycle (c t ) of each transfection concentration for the ino-4800 covid-19 target mrna and for the β-actin mrna was generated from the quantstudio software using an automatic threshold setting. the plasmid was considered to be active for mrna expression if the expression in any of the plasmid transfected wells compared with the negative transfection controls were greater than 5 c t . in vitro protein expression (western blot). human embryonic kidney cells, 293t were cultured and transfected as described previously 42 . 293t cells were transfected with pdna using turbofectin8.0 (origene) transfection reagent following the manufacturer's protocol. forty-eight hours later cell lysates were harvested using modified ripa cell lysis buffer. proteins were separated on a 4-12% bis-tris gel (thermofisher scientific), then following transfer, blots were incubated with an anti-sars-cov spike protein polyclonal antibody (novus biologicals) then visualized with horseradish peroxidase (hrp)-conjugated anti-mouse igg (ge amersham). immunofluorescence of transfected 293t cells. for in vitro staining of spike protein expression 293t cells were cultured on 4-well glass slides (lab-tek) and transfected with 3 µg per well of pdna using turbofectin8.0 (origene) transfection reagent following the manufacturer's protocol. cells were fixed 48 h after transfection with 10% neutral-buffered formalin (bbc biochemical, washington state) for 10 min at rt and then washed with pbs. before staining, chamber slides were blocked with 0.3% (v/v) triton-x (sigma), 2% (v/v) donkey serum in pbs for 1 h at rt. cells were stained with a rabbit anti-sars-cov spike protein polyclonal antibody (novus biologicals) diluted in 1% (w/v) bsa (sigma), 2% (v/v) donkey serum, 0.3% (v/v) triton-x (sigma) and 0.025% (v/v) 1 g ml −1 sodium azide (sigma) in pbs for 2 h at rt. slides were washed three times for 5 min in pbs and then stained with donkey anti-rabbit igg af488 (lifetechnologies, a21206) for 1 h at rt. slides were washed again and mounted and covered with dapi-fluoromount (southernbiotech). antigen binding elisa. elisas were performed to determine sera antibody binding titers. nunc elisa plates were coated with 1 µg ml −1 recombinant protein antigens in dulbecco's phosphate-buffered saline (dpbs) overnight at 4°c. plates were washed three times then blocked with 3% bovine serum albumin (bsa) in dpbs with 0.05% tween 20 for 2 h at 37°c. plates were then washed and incubated with serial dilutions of mouse or guinea pig sera and incubated for 2 h at 37°c. plates were again washed and then incubated with 1:10,000 dilution of horse radish peroxidase (hrp) conjugated anti-guinea pig igg secondary antibody (sigma-aldrich, cat. a7289) or (hrp) conjugated anti-mouse igg secondary antibody (sigma-aldrich) and incubated for 1 h at rt. after final wash plates were developed using sureblue tm tmb 1-component peroxidase substrate (kpl, cat. 52-00-03) and the reaction stopped with tmb stop solution (kpl, cat. 50-85-06). plates were read at 450 nm wavelength within 30 min using a synergy htx (biotek instruments, highland park, vt). binding antibody epts were calculated as previously described 43 . binding antigens tested included, sars-cov-2 antigens: s1 spike protein (sino biological 40591-v08h), s1 + s2 ecd spike protein (sino biological 40589-v08b1), rbd (university of texas, at austin (mclellan lab.)); sars-cov antigens: spike s1 protein (sino biological 40150-v08b1), s (1-1190) (immune tech it-002-001p) and spike c-terminal (meridian life science r18572). ace2 competition elisa. for mouse studies, elisas were performed to determine sera igg antibody competition against human ace2 with a human fc tag. nunc elisa plates were coated with 1 µg ml −1 rabbit anti-his6x in 1× pbs for 4-6 h at rt and washed four times with washing buffer (1× pbs and 0.05% tween 20) . plates were blocked overnight at 4°c with blocking buffer (1× pbs, 0.05% tween 20, 5% evaporated milk and 1% fbs). plates were washed four times with washing buffer then incubated with full length (s1 + s2) spike protein containing a c-terminal his tag (sino biologics, cat. 40589-v08b1) at 10 µg ml −1 for 1 h at rt. plates were washed and then serial dilutions of purified mouse igg mixed with 0.1 µg ml −1 recombinant human ace2 with a human fc tag (ace2-ighu) were incubated for 1-2 h at rt. plates were again washed and then incubated with 1:10,000 dilution of hrp conjugated anti-human igg secondary antibody (bethyl, cat. a80-304p) and incubated for 1 h at rt. after final wash plates were developed using 1-step ultra tmb-elisa substrate (thermo, cat. 34029) and the reaction stopped with 1 m sulfuric acid. plates were read at 450 nm wavelength within 30 min using a spectramax plus 384 microplate reader (molecular devices, sunnyvale, ca). competition curves were plotted and the area under the curve (auc) was calculated using prism 8 analysis software with multiple t-tests to determine statistical significance. for guinea pig studies, 96-well half area assay plates (costar) were coated with 25 µl per well of 5 µg ml −1 of sars-cov-2 spike s1 + s2 protein (sino biological) diluted in 1× dpbs (thermofisher) overnight at 4°c. plates were washed with 1× pbs buffer with 0.05% tween (sigma). hundred microliters per well of 3% (w/v) bsa (sigma) in 1× pbs with 0.05% tween were added and incubated for 1 h at 37°c. serum samples were diluted 1:20 in 1% (w/v) bsa in 1× pbs with 0.05% tween. after washing the assay plate, 25 µl/well of diluted serum was added and incubated 1 h at 37°c. human recombinant ace2-fc-tag (sinobiological) was added directly to the diluted serum, followed by 1 h of incubation at 37°c. plates were washed and 25 µl per well of 1:10,000 diluted goat anti-hu fc fragment antibody hrp (bethyl, a80-304p) was added to the assay plate. plates were incubated 1 h at rt. for development the sureblue/tmb stop solution (kpl, md) was used and o.d. was recorded at 450 nm. sars-cov-2 pseudovirus neutralization assay. sars-cov-2 pseudotyped viruses were produced using hek293t cells transfected with genejammer (agilent) using ige-sars-cov-2 s plasmid (genscript) and pnl4-3.luc.r-e-plasmid (nih aids reagent) at a 1:1 ratio. forty-eight hours post transfection, transfection supernatant was collected, enriched with fbs to 12% final volume, steri-filtered (millipore sigma), and aliquoted for storage at −80°c. sars-cov-2 pseudotyped viruses were titered and yielding >50 times the relative luminescence units (rlu) to cells alone after 72 h of infection. mouse sera from ino-4800 vaccinated and naive groups were heat inactivated for 15 min at 56°c and serially diluted threefold starting at a 1:10 dilution for assay. sera were incubated with a fixed amount of sars-cov-2 pseudotyped virus for 90 min. hek293t cells stably expressing ace2 were added after 90 min and allowed to incubate in standard incubator (37% humidity, 5% co 2 ) for 72 h. post infection, cells were lysed using britelite plus luminescence reporter gene assay system (perkin elmer catalog no. 6066769) and rlu were measured using the biotek plate reader. neutralization titers (id 50 ) were calculated as the serum dilution at which rlu were reduced by 50% compared with rlu in virus control wells after subtraction of background rlu in cell control wells. sars-cov-2 wildtype virus neutralization assays. sars-cov-2/australia/ vic01/2020 isolate neutralization assays were performed at public health england (porton down, uk). neutralizing virus titers were measured in serum samples that had been heat-inactivated at 56°c for 30 min. sars-cov-2 (australia/vic01/2020 isolate 44 ) was diluted to a concentration of 933 pfu ml −1 and mixed 50:50 in 1% fcs/mem containing 25 mm hepes buffer with doubling serum dilutions from 1:10 to 1:320 in a 96-well v-bottomed plate. the plate was incubated at 37°c in a humidified box for 1 h before the virus was transferred into the wells of a twice dpbs-washed 24-well plate that had been seeded the previous day at 1.5 × 10 5 vero e6 cells per well in 10% fcs/mem. virus was allowed to adsorb at 37°c for a further hour, and overlaid with plaque assay overlay media (1× mem/1.5% cmc/ 4% fcs final). after 5 days incubation at 37°c in a humidified box, the plates were fixed, stained and plaques counted. median neutralizing titers (nd50) were determined using the spearman-karber formula relative to virus only control wells. sars-cov-2/wh-09/human/2020 isolate neutralization assays were performed at the institute of laboratory animal science, chinese academy of medical sciences (cams) approved by the national health commission of the people's republic of china. seed sars-cov-2 (sars-cov-2/wh-09/human/ 2020) stocks and virus isolation studies were performed in vero e6 cells, which are maintained in dulbecco's modified eagle's medium (dmem, invitrogen, carlsbad, usa) supplemented with 10% fetal bovine serum (fbs), 100 iu ml −1 penicillin, and 100 µg ml −1 streptomycin, and incubated at 36.5°c, 5% co 2 . virus titer were determined using a standard 50% tissue culture infection dose (tcid50) assay. serum samples collected from immunized animals were inactivated at 56°c for 30 min and serially diluted with cell culture medium in two-fold steps. the diluted samples were mixed with a virus suspension of 100 tcid 50 in 96-well plates at a ratio of 1:1, followed by 2 h incubation at 36.5°c in a 5% co 2 incubator. 1-2 × 10 4 vero cells were then added to the serum-virus mixture, and the plates were incubated for 3-5 days at 36.5°c in a 5% co 2 incubator. cytopathic effect (cpe) of each well was recorded under microscopes, and the neutralizing titer was calculated by the dilution number of 50% protective condition. bal collection. bal fluid was collected by washing the lungs of euthanized and exsanguinated mice with 700-1000 μl of ice-cold pbs containing 100 μm edta, 0.05% sodium azide, 0.05% tween-20, and 1× protease inhibitor (pierce) (mucosal prep solutions (mps) with a blunt-ended needle. guinea pig lungs were washed with 20 ml of mps via 16 g catheter inserted into the trachea. collected bal fluid was stored at −20c until the time of assay. ifn-γ elispot. spleens from mice were collected individually in rpmi1640 media supplemented with 10% fbs (r10) and penicillin/streptomycin and processed into single cell suspensions. cell pellets were re-suspended in 5 ml of ack lysis buffer (life technologies, carlsbad, ca) for 5 min rt, and pbs was then added to stop the reaction. the samples were again centrifuged at 1500 × g for 10 min, cell pellets re-suspended in r10, and then passed through a 45 µm nylon filter before use in elispot assay. elispot assays were performed using the mouse ifn-γ elispot plus plates (mabtech). 96-well elispot plates precoated with capture antibody were blocked with r10 medium overnight at 4°c. 200,000 mouse splenocytes were plated into each well and stimulated for 20 h with pools of 15-mer peptides overlapping by nine amino acid from the sars-cov-2, sars-cov, or mers-cov spike proteins (five peptide pools per protein). additionally, matrix mapping was performed using peptide pools in a matrix designed to identify immunodominant responses. cells were stimulated with a final concentration of 5 μl of each peptide per well in rpmi + 10% fbs (r10). the spots were developed based on manufacturer's instructions. r10 and cell stimulation cocktails (invitrogen) were used for negative and positive controls, respectively. spots were scanned and quantified by immunospot ctl reader. spot-forming unit (sfu) per million cells was calculated by subtracting the negative control wells. flow cytometry. intracellular cytokine staining was performed on splenocytes harvested from balb/c and c57bl/6 mice stimulated with the overlapping peptides spanning the sars-cov-2 s protein for 6 h at 37°c, 5% co 2 . cells were stained with the following antibodies from bd biosciences, unless stated, with the dilutions stated in parentheses: fitc anti-mouse cd107a (1:100), percp-cy5.5 anti-mouse cd4 (1:100), apc anti-mouse cd8a (1:100), vivid dye (1-40) (live/dead® fixable violet dead cell stain kit; invitrogen, l34955), apc-cy7 anti-mouse cd3e (1:100), and bv605 anti-mouse ifn-γ (1:75) (ebiosciences). phorbol myristate acetate (pma) were used as a positive control, and complete medium only as the negative control. cells were washed, fixed and, cell events were acquired using an facs canto (bd biosciences), followed by flowjo software (flowjo llc, ashland, or) analysis. structural modeling. the structural models for sars-cov and mers-cov were constructed from pdb ids 6acc and 5×59 in order to assemble a prefusion model with all three rbds in the down conformation. the sars-cov-2 structural model was built by using sars-cov structure (pdb id:6acc) as a template. rosetta remodel simulations were employed to make the appropriate amino acid mutations and to build de novo models for sars-cov-2 loops not structurally defined in the sars-cov structure 45 . amino acid positions neighboring the loops were allowed to change backbone conformation to accommodate the new loops. the structural figures were made using pymol. statistics. all statistical analyses were performed using graphpad prism 7 or 8 software (la jolla, ca). these data were considered significant if p < 0.05. the lines in all graphs represent the mean value and error bars represent the standard deviation. no samples or animals were excluded from the analysis. randomization was not performed for the animal studies. samples and animals were not blinded before performing each experiment. reporting summary. further information on research design is available in the nature research reporting summary linked to this article. a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china clinical features of patients infected with 2019 novel coronavirus in wuhan covid-19 global cases by johns hopkins csse chinese healthcare workers are facing a surgical mask shortage amid coronavirus panic inactivation of surrogate coronaviruses on hard surfaces by health care germicides intradermal syncon(r) ebola gp dna vaccine is 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elicited both enhancing and neutralizing effects on infection in non-human primates immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus evaluation of antibody-dependent enhancement of sars-cov infection in rhesus macaques immunized with an inactivated sars-cov vaccine immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine immunogenicity and protective efficacy in mice and hamsters of a beta-propiolactone inactivated whole virus sars-cov vaccine enhanced protection in mice induced by immunization with inactivated whole viruses compare to spike protein of middle east respiratory syndrome coronavirus identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo severe acute respiratory syndrome coronavirus infection in vaccinated ferrets development of an intradermal dna vaccine delivery strategy to achieve single-dose immunity against respiratory syncytial virus enhanced cellular immune responses elicited by an engineered hiv-1 subtype b consensus-based envelope dna vaccine immunotherapy against hpv16/18 generates potent th1 and cytotoxic cellular immune responses isolation and rapid sharing of the 2019 novel coronavirus (sars-cov-2) from the first patient diagnosed with covid-19 in australia rosettaremodel: a generalized framework for flexible backbone protein design the authors declare that all data supporting the findings of the study are available in this article and its supplementary information files, or from the corresponding author the source data underlying figs. 2b-d, 3a-e, 4a, b, 5a, b, 6a-e, 7a-d, 8a-c, 9a and supplementary figs. 1, 3b are provided as a source data file.received: 25 february 2020; accepted: 8 may 2020; the studies described in this manuscript were funded by a grant from the coalition for epidemic preparedness innovations (cepi). the authors would like to additional thank stacy guzman at the wistar institute, and olivia bedoya, gloria mendez, francisco vega vega and jon schantz at inovio pharmaceuticals for their assistance. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-16505-0.correspondence and requests for materials should be addressed to k.e.b.peer review information nature communications thanks the anonymous reviewers for their contribution to the peer review of this work. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-329011-spiuqngp authors: huang, yuan; yang, chan; xu, xin-feng; xu, wei; liu, shu-wen title: structural and functional properties of sars-cov-2 spike protein: potential antivirus drug development for covid-19 date: 2020-08-03 journal: acta pharmacol sin doi: 10.1038/s41401-020-0485-4 sha: doc_id: 329011 cord_uid: spiuqngp coronavirus disease 2019 is a newly emerging infectious disease currently spreading across the world. it is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the spike (s) protein of sars-cov-2, which plays a key role in the receptor recognition and cell membrane fusion process, is composed of two subunits, s1 and s2. the s1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the s2 subunit mediates viral cell membrane fusion by forming a six-helical bundle via the two-heptad repeat domain. in this review, we highlight recent research advance in the structure, function and development of antivirus drugs targeting the s protein. the epidemic of novel coronavirus disease 2019 (covid-19) was caused by a new coronavirus occurred in december 2019, and now has spread worldwide and turned into a global pandemic [1] . the covid-19 was quickly discovered to be caused by a coronavirus later named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] , which belongs to the β coronavirus family. it is the seventh known coronavirus to infect humans; four of these coronaviruses (229e, nl63, oc43, and hku1) only cause slight symptoms of the common cold. conversely, the other three, sars-cov, mers-cov, and sars-cov-2, are able to cause severe symptoms and even death, with fatality rates of 10%, 37%, and 5%, respectively. although a large number of studies and clinical trials are being launched on covid-19 around the world [2, 3] , no evidence from randomized clinical trials has shown that any potential therapy improves outcomes in patients [4] . as the epidemic spreads, it is critical to find a specific therapeutic for covid-19, and vaccines targeting various sars-cov-2 proteins are under development. sars-cov-2 is a single-stranded rna-enveloped virus [5] . an rna-based metagenomic next-generation sequencing approach has been applied to characterize its entire genome, which is 29,881 bp in length (genbank no. mn908947), encoding 9860 amino acids [6] . gene fragments express structural and nonstructural proteins. the s, e, m, and n genes encode structural proteins, whereas nonstructural proteins, such as 3-chymotrypsinlike protease, papain-like protease, and rna-dependent rna polymerase, are encoded by the orf region [7] . a large number of glycosylated s proteins cover the surface of sars-cov-2 and bind to the host cell receptor angiotensinconverting enzyme 2 (ace2), mediating viral cell entry [8] . when the s protein binds to the receptor, tm protease serine 2 (tmprss2), a type 2 tm serine protease located on the host cell membrane, promotes virus entry into the cell by activating the s protein. once the virus enters the cell, the viral rna is released, polyproteins are translated from the rna genome, and replication and transcription of the viral rna genome occur via protein cleavage and assembly of the replicase-transcriptase complex. viral rna is replicated, and structural proteins are synthesized, assembled, and packaged in the host cell, after which viral particles are released (fig. 1d) [9] . these proteins are critical to the viral life cycle and provide potential targets for drug therapies. for example, ace2-based peptide, 3clpro inhibitor (3clpro-1), and a novel vinylsulfone protease inhibitor have been experimentally demonstrated to be effective against sars-cov-2 [10] . the sars-cov-2 s protein is highly conserved among all human coronaviruses (hcovs) and is involved in receptor recognition, viral attachment, and entry into host cells. due to its indispensable functions, it represents one of the most important targets for covid-19 vaccine and therapeutic research. in this review, we summarize advances in research of the sars-cov-2 s protein and its therapeutic targeting. with a size of 180-200 kda, the s protein consists of an extracellular n-terminus, a transmembrane (tm) domain anchored in the viral membrane, and a short intracellular c-terminal segment [11] . s normally exists in a metastable, prefusion conformation; once the virus interacts with the host cell, extensive structural rearrangement of the s protein occurs, allowing the virus to fuse with the host cell membrane. the spikes are coated with polysaccharide molecules to camouflage them, evading surveillance of the host immune system during entry [12] . the total length of sars-cov-2 s is 1273 aa and consists of a signal peptide (amino acids 1-13) located at the n-terminus, the s1 subunit (14-685 residues), and the s2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. in the s1 subunit, there is an n-terminal domain (14-305 residues) and a receptor-binding domain (rbd, 319-541 residues); the fusion peptide (fp) (788-806 residues), heptapeptide repeat sequence 1 (hr1) (912-984 residues), hr2 (1163-1213 residues), tm domain (1213-1237 residues), and cytoplasm domain (1237-1273 residues) comprise the s2 subunit ( fig. 2a) [13] . s protein trimers visually form a characteristic bulbous, crown-like halo surrounding the viral particle (fig. 1a) . based on the structure of coronavirus s protein monomers, the s1 and s2 subunits form the bulbous head and stalk region [14] . the structure of the sars-cov-2 trimeric s protein has been determined by cryo-electron microscopy at the atomic level, revealing different conformations of the s rbd domain in opened and closed states and its corresponding functions (fig. 2b , c) [15, 16] . in the native state, the cov s protein exists as an inactive precursor. during viral infection, target cell proteases activate the s protein by cleaving it into s1 and s2 subunits [17] , which is necessary for activating the membrane fusion domain after viral entry into target cells [18] . similar to other coronaviruses, the s protein of sars-cov-2 is cleaved into s1 and s2 subunits by cellular proteases, and the serine protease tmprss2 is used as a protein primer. although the cleavage site of sars-cov is known, that of sars-cov-2 s has not yet been reported [18, 19] . structure of the s1 subunit the binding of virus particles to cell receptors on the surface of the host cell is the initiation of virus infection; therefore, receptor recognition is an important determinant of viral entry and a drug design target. rbd situated in the s1 subunit binds to the cell receptor ace2 in the region of aminopeptidase n. the s1 region contains the ntd and ctd, and atomic details at the binding interface demonstrate key residue substitutions in sars-cov-2-ctd. in addition, the sars-cov-2 s ctd binding interface has more residues that directly interact with the receptor ace2 than does sars-rbd (21 versus 17), and a larger surface area is buried with sars-cov-2 s ctd in complex with ace2 than with sars s rbd. mutations of key residues play an important role in enhancing the interaction with ace2. f486 in sars-cov-2, instead of i472 in sars rbd, forms strong aromatic-aromatic interactions with ace2 y83, and e484 in sars-cov-2-ctd, instead of p470 in sars rbd, forms ionic interactions with k31, which leads to higher affinity for receptor binding than rbd of sars-cov ( fig. 2d) [15, 16, 20, 21] . the rbd region is a critical target for neutralizing antibodies (nabs), and sars-cov-2 and sars-cov rbd are~73%-76% similar in sequence. nine ace2-contacting residues in cov rbd are fully conserved, and four are partially conserved. analysis of the rbm (receptor-binding motif, a portion of rbd making direct contacts with ace2) of sars-cov and sars-cov-2 revealed that most residues essential for ace2 binding in the sars-cov s protein are conserved in the sars-cov-2 s protein. however, some studies showed that murine monoclonal antibodies (mabs) and polyclonal antibodies against sars-rbd are unable to interact with the sars-cov-2 s protein, revealing differences in antigenicity between sars-cov and sars-cov-2 [20] . similarly, a sars-cov rbd-specific antibody failed to block infection mediated by the s protein of sl-cov-shc014 [22] , which suggests that the s1 rbd may not be an ideal drug target due to the highly mutable characteristic of broad-spectrum anti-cov drugs. structure of the s2 subunit the s2 subunit, composed successively of a fp, hr1, hr2, tm domain, and cytoplasmic domain fusion (ct), is responsible for viral fusion and entry. fp is a short segment of 15-20 conserved amino acids of the viral family, composed mainly of hydrophobic residues, such as glycine (g) or alanine (a), which anchor to the target membrane when the s protein adopts the prehairpin conformation. previous research has shown that fp plays an essential role in mediating b-c the s protein rbd closed and opened status. d the s protein binds to ace2 with opened rbd in the s1 subunit. e the six-helix structure formed by hr1 and hr2 of the s2 subunit. membrane fusion by disrupting and connecting lipid bilayers of the host cell membrane [23] . hr1 and hr2 are composed of a repetitive heptapeptide: hpphcpc, where h is a hydrophobic or traditionally bulky residue, p is a polar or hydrophilic residue, and c is another charged residue [24] . hr1 and hr2 form the six-helical bundle (6-hb) (fig. 2e) , which is essential for the viral fusion and entry function of the s2 subunit [13] . hr1 is located at the c-terminus of a hydrophobic fp, and hr2 is located at the n-terminus of the tm domain [25] . the downstream tm domain anchors the s protein to the viral membrane, and the s2 subunit ends in a ct tail [14] . rbd binds to ace2, and s2 changes conformation by inserting fp into the target cell membrane, exposing the prehairpin coiledcoil of the hr1 domain and triggering interaction between the hr2 domain and hr1 trimer to form 6-hb, thus bringing the viral envelope and cell membrane into proximity for viral fusion and entry [26] . hr1 forms a homotrimeric assembly in which three highly conserved hydrophobic grooves on the surface that bind to hr2 are exposed. the hr2 domain forms both a rigid helix and a flexible loop to interact with the hr1 domain. in the postfusion hairpin conformation of covs, there are many strong interactions between the hr1 and hr2 domains inside the helical region, which is designated the "fusion core region" (hr1core and hr2core regions, respectively). targeting the heptad repeat (hr) has attracted the greatest interest in therapeutic drug discovery. the s protein is an important target protein for the development of specific drugs, while the s1 rbd domain is part of a highly mutable region and is not an ideal target site for broad-spectrum antiviral inhibitor development [27] . in contrast, the hr region of the s2 subunit plays an essential role in hcov infections and is conserved among hcovs, as is the mode of interaction between hr1 and hr2 [28] . a synthetic peptide derived from the stem region of the zikv envelope protein was demonstrated in 2017 to potently inhibit infection by zikv and other flaviviruses in vitro [29] , implying antiviral efficiency of peptides derived from conserved regions of viral proteins. peptides derived from the hr2 region of class i viral fusion proteins of enveloped viruses competitively bind to viral hr1 and effectively inhibit viral infection [22] . therefore, hr1 is a promising target for the development of fusion inhibitors against sars-cov-2 infection. the s protein on the surface of the virus is a key factor involved in infection. it is a trimeric class i tm glycoprotein responsible for viral entry, and it is present in all kinds of hcovs, as well as in other viruses such as hiv (hiv glycoprotein 160, env), influenza virus (influenza hemagglutinin, ha), paramyxovirus (paramyxovirus f), and ebola (ebola virus glycoprotein) [30] . similar to other coronaviruses, the s protein of sars-cov-2 mediates receptor recognition, cell attachment, and fusion during viral infection [16, 20, 21, [31] [32] [33] . the trimer of the s protein located on the surface of the viral envelope is the basic unit by which the s protein binds to the receptor [16, 33] . the s1 domain contains the rbd, which is mainly responsible for binding of the virus to the receptor, while the s2 domain mainly contains the hr domain, including hr1 and hr2, which is closely related to virus fusion [34] . receptor binding as mentioned above, the sars-cov-2 s protein binds to the host cell by recognizing the receptor ace2 [33] . ace2 is a homolog of ace, which converts angiotensin i to angiotensin 1-9 [35] . ace2 is distributed mainly in the lung, intestine, heart, and kidney, and alveolar epithelial type ii cells are the major expressing cells [36] . ace2 is also a known receptor for sars-cov. the s1 subunit of the sars-cov s protein binds with ace2 to promote the formation of endosomes, which triggers viral fusion activity under low ph (fig. 1a, b) [37] . interaction between the s protein and ace2 can be used to identify intermediate hosts of sars-cov-2, as ace2 from different species, such as amphibians, birds, and mammals, has a conserved primary structure [38] . luan et al. compared the binding affinities between ace2 and sars-cov-2 s from mammals, birds, snakes, and turtles and found that the ace2 of bovidae and cricetidae interacted well with sars-cov-2 s rbd but that ace2 from snakes and turtles could not. the s protein binds to ace2 through the rbd region of the s1 subunit, mediating viral attachment to host cells in the form of a trimer [15] . sars-cov-2 s binds to human ace2 with a dissociation constant (k d ) of 14.7 nm, though that of sars-cov s is 325.8 nm [15] , indicating that sars-cov-2 s is more sensitive to ace2 than is sars-cov s. through the identification of sars-cov-2 proteins, researchers found~24% difference in s between sars-cov-2 and sars-cov, whereas that of rbd is~23% [39] . viral fusion viral fusion refers to fusion of the viral membrane and host cell membrane, resulting in the release of the viral genome into the host cell. cleavage of the sars-cov-2 s1 and s2 subunits is the basis of fusion. the s protein is cleaved into two parts, the s1 subunit and s2 subunit, by host proteases, and the subunits exist in a noncovalent form until viral fusion occurs [40] . researchers have found that the specific furin cleavage site is located in the cleavage site of sars-cov-2 but not in other sarslike covs [41, 42] . mutation of the cleavage site in sars-cov-2 or sars-like covs has revealed that the s protein of sars-cov-2 exists in an uncleaved state but that the others are mainly in a cleaved state. sars-cov-2 s has multiple furin cleavage sites, which increases the probability of being cleaved by furin-like proteases and thereby enhances its infectivity [43, 44] . the furin-like cleavage domain is also present in highly pathogenic influenza virus and is related to its pathogenicity, as observed in the avian influenza outbreak in hong kong in 1997 [45, 46] . in addition, host cell proteases such as tmprss2 are essential for s protein priming, and they have been shown to be activated in the entry of sars-cov and influenza a virus [18, 47, 48] . another host cell protease that has been proven to cleave viral s protein is trypsin [49] . in summary, the s protein of sars-cov-2 is similar to that of sars-cov, and host cell proteases are essential for promoting s protein cleavage of both sars-cov-2 and sars-cov. the presence of a specific furin cleavage site on sars-cov-2 s might be one reason that sars-cov-2 is more contagious than sars-cov. the formation of 6-hb is essential for viral fusion. the fp in the n-terminus of sars-cov-2 and the two hr domains on s2 is essential for viral fusion [50] . after cleavage of the s protein, the fp of sars-cov-2 is exposed and triggers viral fusion. under the action of some special ligands, the fusion protein undergoes a conformational change and then inserts into the host cell membrane (fig. 1c) [51] . for example, the ligand for influenza a virus is h + , while the ligand for hiv is a coreceptor such as ccr5 or cxcr4 [14] . the distance between the viral membrane and host cell membrane is shortened, and the hr1 domain of the s protein is in close proximity to the host cell membrane, whereas the hr2 domain is closer to the viral membrane side. then, hr2 folds back to hr1, the two hr domains form a six-helix structure in an antiparallel format of the fusion core, the viral membrane is pulled toward the host cell membrane and tightly binds to it, and the two membranes fuse [52] . the fundamental role of the s protein in viral infection indicates that it is a potential target for vaccine development, drug development targeting sars-cov-2 s y huang et al. antibody-blocking therapy, and small molecule inhibitors. considering the similarity with sars-cov and mers-cov, potential nabs and inhibitors targeting sars-cov-2 s are summarized below (fig. 3) . antibodies based on the sars-cov-2 s protein the s protein is the main antigen component in all structural proteins of sars-cov-2. unlike other functional proteins of sas-cov-2, it is responsible for inducing the host immune response, and nabs targeting the s protein can induce protective immunity against viral infection. similar to sars-cov and mers-cov, research on nabs of sars-cov-2 mainly includes mabs, antigenbinding fragments, single-chain variable region fragments, and single-domain antibodies (nbs), which target s1 rbd, s1-ntd, or s2 regions to prevent s2-mediated fusion [53, 54] . on the other hand, multiple sars-cov-2 vaccine types are under development, including rna/dna-based formulations, recombinant viral epitopes, adenovirus-based vectors, and purified inactivated virus [55] . the sequence and striking structural similarity between the sars-cov-2 and sars-cov s proteins emphasize the close relationship between these two viruses, which provides the possibility to treat covid-19 with antibodies targeting the sars-cov s protein [56] . compared with sars-cov-2 rbd, sars-cov-2 interacts with hace2 via the c-terminal domain (sars-cov-2-ctd), showing higher affinity for receptor binding. rbd can induce highly potent nab responses and has the potential to be developed as an effective and safe subunit vaccine against sars-cov-2. sars-cov s polyclonal antibodies obtained from immunized mice completely inhibited the invasion of sars-cov s-mlv (murine leukemia virus), whereas the invasion rate of sars-cov-2 s-mlv was reduced to~10% [20] . the polyclonal anti-sars s1 antibody t62 inhibits the entry of sars-cov s but not that of sars-cov-2 s pseudovirus particles [49] . consistently, recent studies have reported similar results, showing that three sars rbd-directed mabs, s230, m396, and 80r, were unable to bind to sars-cov-2 rbd [16, 20, 21] . on the other hand, several mabs have shown promising results in neutralizing sars-cov-2. cr3022, a sars-cov-specific human mab, binds potently with sars-cov-2 (k d of 6.3 nm, measured by bli in octetred96), suggesting that cr3022 has the potential to be developed as candidate therapeutic, alone or in combination with other nabs, for the prevention and treatment of sars-cov-2 infection [57] . a mab targeting s1 prepared from immunized transgenic mice expressing human ig variable heavy and light chains has recently been shown to neutralize both sars-cov-2 and sars-cov infections via an unknown mechanism that is independent of the blockade of rbd-hace2 interaction [58] . recently, many human blocking mabs (311mab-31b5, 311mab-32d4, 47d11, n3130, n3088, s309, p2c-1f11, p2b-2f6, b38, h4) have been successfully cloned from single memory b cells from recovered covid-19 patients [58] [59] [60] [61] [62] [63] . these mabs specifically bind to sars-cov-2 s to effectively neutralize infection. in addition, sera from sars patients during rehabilitation or animals specifically immunized with sars-cov s1 may cross-neutralize sars-cov-2 and reduce s protein-mediated sars-cov-2 entry (fig. 3) [18] . the stability of the sars-cov-2 s protein is lower than that of sars-cov s [42] . the mapping of multiple s sequences of the subgenus sarbecovirus underscores that the s2 fusion region is more conserved than the s1 subunit and that the s1 subunit is more exposed at the viral surface [16] . the sars-cov s2 subunit plays a key role in mediating virus-cell fusion and its integration into host cells, where hr1 and hr2 interact to form 6-hb, thus enabling the virus to bind to and fuse with the cell membrane [28] . sequence alignment shows that sars-cov-2 hr2 has the same sequence as sars-cov hr2. therefore, sars-cov-2 hr2p (1168-1203 residues) was designed to inhibit sars-cov-2 fusion and entry into a target cell. surprisingly, hr2p showed inhibitory activity against sars-cov-2 s-mediated fusion and sars-cov-2 pseudovirus, with ic 50 values of 0.18 and 0.98 μm, respectively [13] . notably, ek1 is a pancoronavirus fusion inhibitor targeting the hr1 domain of hcov s [22] . the x-ray crystal structure of the 6-hb core of the sars-cov-2 s2 subunit hr1 and hr2 domains has been solved, indicating that several mutant residues in the hr1 region may be related to enhanced interaction in the hr2 region [64] . subsequently, ek1c4, a lipopeptide derived from ek1, was generated and verified to inhibit sars-cov-2 s-mediated cell-cell fusion. as expected, the entry of sars-cov-2 s pseudovirus was also inhibited by ek1c4, with an ic 50 of 15.8 nm,~149-fold more potent than the original ek1 peptide. another sequence-based lipopeptide fusion inhibitor, ipb02, potently inhibits sars-cov-2 s protein-mediated cell-cell fusion and pseudovirus infection [65] . in addition to peptide fusion inhibitors, nelfinavir mesylate (viracept), a currently prescribed anti-hiv protease inhibitor, suppresses both sars-cov-2 s and sars-cov s-mediated cell-cell fusion. viracept is the first reported small molecule fusion inhibitor in addition to peptide fusion inhibitors. moreover, nelfinavir may inhibit the function of tmprss2 involved in activation of the s protein [66] . this discovery makes possible clinical applications of anti-sars-cov-2 therapeutics, especially in the early stage of infection. protease inhibitors targeting sars-cov-2 s cleavage sites sars-cov-2 entry requires cleavage of the s protein at the s1/s2 and s2 sites. proteolysis by tmprss2 and cathepsin b and l plays an important role in priming sars-cov-2 s for entry. camostat mesilate is a potent serine protease inhibitor of tmprss2. utilizing research on the sars-cov and sars-cov-2 cell entry mechanism, it has been demonstrated that sars-cov-2 cellular entry can be blocked by camostat mesilate [18, 67] . there are currently five clinical trials registered to evaluate the efficacy of camostat mesilate (clinicaltrials.gov identifier: nct04321096, nct04353284, nct04338906, nct04355052, nct04374019). in addition, cathepsins in lysosomes are crucial for sars-cov entry via endocytosis. e-64d, an inhibitor of cathepsin l, blocks infection with sars-cov and sars-cov-2 psv [68] [69] [70] . future trials with covid-19 patients may help to confirm the efficacy of e-64d therapy. phosphatidylinositol 3-phosphate 5-kinase (pikfyve) is the main enzyme synthesizing pi (3, 5) p2 in early endosomes [71] . apilimod, a potent inhibitor of pikfyve35, can significantly reduce the entry of sars-cov s pseudovirus into 293/hace2 cells via early endosomes in a dose-dependent manner [49] . treating 293/ hace2 cells with another pikfyve inhibitor, ym201636 [72] , also had a similar effect. moreover, a major downstream effector of pi (3,5)p2, two-pore channel subtype 2 (tpc2) [73] , is important for sars-cov-2 entry, and tetrandrine (an inhibitor of tpc2) inhibits the activity of sars-cov-2 s pseudovirus. furin (proprotein convertase (pc) subtilisin kexin 3, pcsk3), as a member of the pc family, catalyzes the hydrolysis of peptide and protein substrates at paired basic residues [74] . strikingly, sars-cov-2 s harbors a furin cleavage site (682-685 residues) at the s1/ s2 boundary, which may increase the efficiency of sars-cov-2 transmission [75] . the furin-like cleavage site in the s protein of sars-cov-2 may have implications for the viral life cycle and pathogenicity. therefore, furin inhibitors can be used as a drug therapy for sars-cov-2 [41] . patent literature since 1994 describes the use of furin or its inhibitors in the treatment of diseases, and some furin inhibitors that have been reported, including α-1-pdx (α1-antitrypsin portland) [76] , hexa-d-arginine(d6r) [77] , serpin proteinase inhibitor 8 (pi8) [78] , and a peptidomimetic furin inhibitor [79] . the sars-cov-2 s protein binds to the host cell receptor and induces virus-cell membrane fusion, which plays a vital role in the process of virus invasion. moreover, the high affinity between the s protein and ace2 increases the infectivity of sars-cov-2. mammals including pangolins, pets (dogs and cats), and members of cricetidae may be important for determining key residues for association with s from sars-cov and sars-cov-2 [80] . further drug development targeting sars-cov-2 s y huang et al. understanding of the structure and function of sars-cov-2 s will allow for additional information regarding invasion and pathogenesis of the virus, which will support the discovery of antiviral therapeutics and precision vaccine design. structural information will also assist in evaluating mutations of the sars-cov-2 s protein and will help in determining whether these residues have surface exposure and map to known antibody epitopes of s proteins from other coronaviruses. in addition, structural knowledge ensures that the proteins produced by constructs are homogeneous and participate in the prefusion conformation, which should maintain the most neutralizationsensitive epitopes when used as a candidate vaccine or b-cell probe for isolating neutralizing human mabs. furthermore, atomic-level details will enable the design and screening of small molecules that inhibit fusion. since sars-cov-2 and sars-cov rbd domains share 75% amino acid sequence identity, future work will be necessary to evaluate whether any of these abs neutralize newly emerged coronavirus. overall, interaction between the s protein of sars-cov-2 and ace2 should be further studied to contribute elucidation of the mechanism of sars-cov-2 infection. similarly, focusing on high expression of the s protein or its receptor binding region is also of great significance for the development of vaccines. the s2 subunit of sars-cov-2 shows 88% sequence homology with the sars-cov s2 domain and is structurally conserved. therefore, the development of antibodies targeting this functional motif may cross-bind and neutralize these two viruses and related covs. antiviral peptides prevent sars-cov-2 membrane fusion and can potentially be used for the prevention and treatment of infection. it is worth mentioning that ek1c4, which targets the highly conserved hr1 domain of the s2 subunit, is expected to have therapeutic potential against sars-cov-2. more importantly, ek1c4 can be used as a nasal drop, which increases its medicinal properties, it possesses a high genetic barrier to resistance, and does not easily induce drug-resistant mutations. on the other hand, peptide fusion inhibitors may not be widely used clinically and have low bioavailability. therefore, the development of oral small molecule fusion inhibitors is a major direction. in the course of virus epidemics, the ability to adapt to external pressure is an important factor affecting the spread of the virus. regarding the envelope s protein, recombination or mutation in the gene of its rbd can occur to promote transmission between different hosts and lead to a higher fatality rate [81] . mutation of the aspartate (d) at position 614 to glycine (g614) results in a more pathogenic strain of sars-cov-2 [82] , which makes it more difficult to develop antibodies or vaccines that 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potent neutralizing human antibody reveals the n-terminal domain of the spike protein of sars-cov-2 as a site of vulnerability inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion design of potent membrane fusion inhibitors against sars-cov-2, an emerging coronavirus with high fusogenic activity the anti-hiv drug nelfinavir mesylate (viracept) is a potent inhibitor of cell fusion caused by the sars-cov-2 spike (s) glycoprotein warranting further evaluation as an antiviral against covid-19 infections camostat mesilate therapy for covid-19 alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model but not hcov-nl63, utilizes cathepsins to infect cells-viral entry. nidoviruses: toward control of sars and other nidovirus glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) the phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection inhibition of pikfyve using ym201636 suppresses the growth of liver cancer via the induction of autophagy two-pore channels control ebola virus host cell entry and are drug targets for disease treatment proprotein convertases in health and disease drug development targeting sars-cov-2 clinical features of patients infected with 2019 novel coronavirus in wuhan furin inhibition reduces vascular remodeling and atherosclerotic lesion progression in mice furin inhibitor d6r suppresses epithelial-mesenchymal transition in sw1990 and patu8988 cells via the hippo-yap signaling pathway the serpin proteinase inhibitor 8: an endogenous furin inhibitor released from human platelets peptidomimetic furin inhibitor mi-701 in combination with oseltamivir and ribavirin efficiently blocks propagation of highly pathogenic avian influenza viruses and delays high level oseltamivir resistance in mdck cells alteration of brain network topology in hiv-associated neurocognitive disorder: a novel functional connectivity perspective the establishment of reference sequence for sars-cov-2 and variation analysis sars-cov-2 viral spike g614 mutation exhibits higher case fatality rate perspectives on therapeutic neutralizing antibodies against the novel coronavirus sars-cov-2 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro this project was supported by grants from guangzhou science and technology program (#201803040006 to wx), the fund of natural science foundation of guangdong province (#2018a030313056 to wx), and grants from major scientific and technological projects of guangdong province (#2019b020202002 to swl). competing interests: the authors declare no competing interests. key: cord-350557-7i7122zi authors: rawlings, stephen a; ignacio, caroline; porrachia, magali; du, pinyi; smith, davey m; chaillon, antoine title: no evidence of sars-cov-2 seminal shedding despite sars-cov-2 persistence in the upper respiratory tract date: 2020-08-07 journal: open forum infect dis doi: 10.1093/ofid/ofaa325 sha: doc_id: 350557 cord_uid: 7i7122zi rna viruses (eg, zika, ebola, hiv) are often shed in male genital secretions. we evaluated the presence and level of sars-cov-2 rna in semen, nasal secretion, and saliva collected after confirmed infection. sars-cov-2 rna was not detected in semen 6–17 days after the onset of symptoms despite concomitant shedding in oral secretions. while it is known that severe acute respiratory coronavirus 2 (sars-cov-2) infection often starts with 1-2 days of asymptomatic shedding before a person gets sick, there are limited data about the biological source and duration of viral shedding after symptom resolution in recently infected individuals. besides respiratory droplets, sars-cov-2 rna has been isolated in other biological samples including urine, blood, feces, and saliva [1, 2] . the expression of sars-cov-2 receptor angiotensin 2-converting enzyme (ace2) across multiple tissues also suggests that sars-cov-2 could be found in other tissues and body fluids [3] , including in the testis and male genital tract [4, 5] . very few studies exist on the presence of sars-cov-2 rna in semen, and these studies have been limited by considerable time between semen sampling and diagnosis of active infection. in particular, when semen has been examined ~4-6 weeks after sars-cov-2 infection, the virus was not detected, and a case report of a single male acutely infected revealed no sars-cov-2 in seminal fluid [6] [7] [8] . whether sars-cov-2 can be detected in semen during the acute phase of sars-cov-2 infection when the virus is concomitantly present in nasal and/or oral secretions remains a concern. here, we evaluated the presence and level of sars-cov-2 in paired semen, nasal secretion, and saliva samples collected in the short and medium term after confirmed sars-cov-2 symptomatic infections. all participants provided informed, written consent. this study was conducted under a protocol for collecting samples from persons with known sars-cov-2 infection approved by the institutional review board of university of california san diego. men who had been diagnosed with sars-cov-2 based on a combination of medical history, symptoms, and the presence of sars-cov-2 rna in the upper respiratory tract were invited to enroll in this study. all participants were outpatients at the time of enrollment and sample collection. they were instructed to self-collect saliva (passive drool), nasal swab, and semen by masturbation without lubricant after 24 hours of abstinence. samples were collected within 1-3 weeks after the onset of symptoms. semen was processed as in butler et al. [9] . briefly, viral transport medium (2 ml of rpmi 1640 with 2 mmol/l of glutamine and 10% fetal bovine serum [fbs] , with the addition of 100 u/ml of penicillin, 100 μl/ml of streptomycin, and 200 u/ml of nystatin) was added to seminal samples at collection. seminal plasma was separated from seminal cells by centrifugation at 700 × g for 12 minutes within 4 hours of collection and stored at −80°c and −150°c, as previously described [9] . rna was extracted from seminal plasma, saliva, and viral transport media that contained the nasopharyngeal (np) swab; 140 µl of each type of sample was extracted for rna using qiagen's qiaamp viral rna mini kit (cat# 52904) according to the manufacturer's recommendation. cdna from sars-cov-2 rna was generated using the bio-rad one-step rt-ddpcr advanced kit for probes (cat# 186-4021). qualitative tests for sars-cov-2 were performed on the first collected nasal/np specimen for diagnostic confirmation using the fluxergy platform (irvine, ca, usa), which is currently available as a research use only (ruo) or investigational use only (iuo) device for the development of new diagnostic products. quantitative measure of sars-cov-2 in saliva and semen was performed using digital droplet polymerase chain reaction (ddpcr; bio-rad qx200 droplet reader). copy numbers were calculated as the mean of 3 replicates. briefly, we have adapted our validated protocol [10] to quantify sars-cov-2 in various samples following the centers for disease control and prevention recommendations and the 2019-ncov real-time rt-pcr diagnostic panel targeting the virus nucleocapsid (n) and the orf gene [11] . based on testing and dilutions with known positive and negative clinical samples (data not shown), the current level of detection is 0.05 copies/µl. a total of 6 participants aged 28-45 years were enrolled in this study (supplementary table 1 ). all initially presented with clinical symptoms compatible with sars-cov-2 and/or recent history of close contact with a confirmed case. symptoms included cough, shortness of breath, fever, myalgia, fatigue, anosmia, headache, anorexia, and diarrhea. before enrollment, 5/6 participants tested positive for sars-cov-2 rna on np swab samples collected within 1-3 days following the onset of symptoms. id16 was not initially tested by his clinical team, but the diagnosis was supported by both clinical symptoms and recent close contact with a confirmed case. upon enrollment in the study, the diagnosis of active sars-cov-2 infection was confirmed by positive pcr on np swab on day 6 post-symptom onset. the clinical conditions of all participants improved, with complete resolution of initial symptoms, within 1-3 weeks from the onset. paired saliva and semen samples were collected a mean of 12 days (6-17 days) after the onset of symptoms, and ddpcr was performed to quantify the sars-cov-2 level in all samples. half of the participants also had research nasal swabs performed. all 6 semen samples were negative for sars cov-2 (≤0.03 copies/µl), while sars-cov-2 was still detected in all saliva samples (6 participants) and all research nasal swabs (3 participants). saliva levels of virus were quantified and varied from 0.05 to 679 copies/µl of input saliva (figure 1, table 1 ). the temporal dynamic of viral shedding and transmissibility of sars-cov-2 remains a major concern to control the spread of the virus and directly impacts control measures such as isolation, contact tracing, and enhanced hygiene or use of face masks for symptomatic persons. recent studies have shown persistant viral detection in the upper respiratory tract up to 37 days after the onset of symptoms [12] . ace2 expression across tissues and body fluid-including seminal fluid-suggests possible extrarespiratory transmission routes [13] . here, we investigated the presence of sars-cov-2 in the semen of men in whom virus was demonstrably present in the respiratory tract. we found no evidence of sars-cov-2 in semen collected 6-17 days after the onset of symptoms despite all men having concomitant shedding of virus in oral secretions up to 792 copies/µl. though the study is small in number, it adds to available literature that the male genital tract does not appear to be a site where sars-cov-2 is shed in either the acute or late phase of infection. identifying whether sars-cov-2 can table 1 for quantitative measures via digital droplet polymerase chain reaction. abbreviation: np, nasopharyngeal. be shed in semen has implications for public health, as all predominant modes of transmission (ie, droplet, sexual contact, airborne, fomite, etc.) need to be identified in order to help curb the spread of the virus. with the small number of participants in this study, it is difficult to quantify the absolute probably of spread in a large population, but the complete absence of virus using ultrasensitive methods suggests that shedding of the virus in semen is certainly not common. a larger study would be needed to demonstrate and quantify rare events of shedding. whether sars-cov-2 can be detected in the semen at the very early phase of infection or during the incubation period when it is present in the upper respiratory tract [12] would require further investigation, but collective evidence [6] [7] [8] and our study suggest that sars-cov-2 is not present in semen. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. (7) ≤0.03 (7) abbreviation: pcr, polymerase chain reaction. persistence and clearance of viral rna in 2019 novel coronavirus disease rehabilitation patients comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa scrna-seq profiling of human testes reveals the presence of the ace2 receptor, a target for sars-cov-2 infection in spermatogonia, leydig and sertoli cells ace2 expression in kidney and testis may cause kidney and testis damage after 2019-ncov infection absence of 2019 novel coronavirus in semen and testes of covid-19 patients † study of sars-cov-2 in semen and urine samples of a volunteer with positive naso-pharyngeal swab no evidence of severe acute respiratory syndromecoronavirus 2 in semen of males recovering from coronavirus disease 2019 the origins of sexually transmitted hiv among men who have sex with men highly precise measurement of hiv dna by droplet digital pcr 2019-ncov) real-time rt-pcr diagnostic panel temporal dynamics in viral shedding and transmissibility of covid-19 structure analysis of the receptor binding of 2019-ncov fluxergy, inc. the authors thank the participants and their families. author contributions. s.a.r. performed study procedures, analyzed data, and reviewed the manuscript. b.s., l.l., m.p., and c.i. performed study procedures and reviewed the manuscript. a.c. and d.s. analyzed data and wrote the manuscript.financial support. this work was supported by the john and mary tu foundation and the translational virology core of the san diego center for aids research (cfar) grant number ai036214, a national institutes of health-funded program. s.a.r. was supported by the national institutes of health (grant number 5t32ai007384). a.c. was supported by the national institutes of health (grant number ai131971) and the university of california office of the president (ucop r00rg2725).potential conflicts of interest. a.c., s.a.r., b.s., l.l., m.p., and c.i. have no conflicts. d.s. is a consultant for fluxergy, bayer, aids healthcare foundation and arena pharmaceuticals. all other authors report no conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-333703-1ku3jc9s authors: kraus, aurora; casadei, elisa; huertas, mar; ye, chunyan; bradfute, steven; boudinot, pierre; levraud, jean-pierre; salinas, irene title: a zebrafish model for covid-19 recapitulates olfactory and cardiovascular pathophysiologies caused by sars-cov-2 date: 2020-11-08 journal: biorxiv doi: 10.1101/2020.11.06.368191 sha: doc_id: 333703 cord_uid: 1ku3jc9s the covid-19 pandemic has prompted the search for animal models that recapitulate the pathophysiology observed in humans infected with sars-cov-2 and allow rapid and high throughput testing of drugs and vaccines. exposure of larvae to sars-cov-2 spike (s) receptor binding domain (rbd) recombinant protein was sufficient to elevate larval heart rate and treatment with captopril, an ace inhibitor, reverted this effect. intranasal administration of sars-cov-2 s rbd in adult zebrafish recombinant protein caused severe olfactory and mild renal histopathology. zebrafish intranasally treated with sars-cov-2 s rbd became hyposmic within minutes and completely anosmic by 1 day to a broad-spectrum of odorants including bile acids and food. single cell rna-seq of the adult zebrafish olfactory organ indicated widespread loss of expression of olfactory receptors as well as inflammatory responses in sustentacular, endothelial, and myeloid cell clusters. exposure of wildtype zebrafish larvae to sars-cov-2 in water did not support active viral replication but caused a sustained inhibition of ace2 expression, triggered type 1 cytokine responses and inhibited type 2 cytokine responses. combined, our results establish adult and larval zebrafish as useful models to investigate pathophysiological effects of sars-cov-2 and perform pre-clinical drug testing and validation in an inexpensive, high throughput vertebrate model. 2 species that have been reported as naturally susceptible to sars-cov-2 include rhesus and 47 cynomolgus macaques (munster et al., 2020; rockx et al., 2020) , ferret (kim et al., 2020) , cat 48 (shi et al., 2020) , and syrian hamster (chan et al., 2020) . mice, by contrast, are not 49 spontaneously permissive to the virus, but mice expressing the human ace2 receptor provide a 50 useful animal model (bao et al., 2020; jia et al., 2020; lutz et al., 2020) . all these mammalian 51 models have unique advantages and disadvantages for the study of immune responses to sars-52 cov-2 and other host-pathogen interactions but do not allow rapid, whole organismal, high 53 throughput, and low-cost preclinical testing of drugs and immunotherapies. 54 55 as model vertebrates, zebrafish are permissive to human viral pathogens including influenza a 56 (gabor et al., 2014) , herpes simplex virus type 1 (burgos et al., 2008) , chikungunya virus 57 (palha et al., 2013) and human noroviruses gi and gii (van dycke et al., 2019). zebrafish offer 58 many advantages over other animal models due to their high reproductive ability, rapid 59 development, low maintenance costs, and small transparent bodies. importantly, zebrafish 60 olfactory, immune, and cardiovascular physiology share a significant degree of conservation 61 with humans (postlethwait et al., 2020; saraiva et al.) . genetically, more than 80% of disease 62 related genes have a zebrafish orthologue (howe et al., 2013) . the zebrafish innate immune 63 system is already developed in the transparent larval stages and members of all major groups of 64 mammalian cytokines have been identified in the zebrafish genome (gomes and mostowy, 2020; 65 zou and secombes, 2016) . academic laboratories and the pharmaceutical industry use zebrafish 66 larvae in preclinical studies for assessing efficacy and toxicity of candidate drugs for several 67 diseases (taylor et al., 2010) . zebrafish is a proposed model for covid-19 and has recently 68 been used in one vaccination study (galindo-villegas, 2020; ventura-fernandes et al., 2020). 69 this study aims to elucidate the physiopathology of wildtype zebrafish in response to sars-70 cov-2. 71 72 sars-cov-2 infection causes a wide litany of symptoms, ranging from asymptomatic to mild or 73 severe disease (menni et al., 2020) . apart from respiratory symptoms, multi-organ pathologies 74 are often reported with heterogeneous symptoms such as olfactory and taste loss, cardiac 75 dysfunction, renal pathologies, neurological damage, muscle and joint pain, gastrointestinal 76 symptoms, clotting disorders and others (tabata et mudd et al., 2020), and elevated type 2 cytokine levels (lucas et al., 2020) . importantly, this 84 cytokine pattern is in sharp contrast to that found in patients experiencing mild or moderate 85 symptoms, who are able to control exacerbated type 1 and type 3 cytokine responses (lucas et 86 al., 2020). 87 88 sars-cov-2 enters the human host cells when sars-cov-2 spike (s) protein receptor binding 89 domain (rbd) binds to angiotensin-converting enzyme 2 (ace2) on a permissive host cell, then 90 a serine protease, such as tmprss2, cleaves the spike protein s1/s2 site to facilitate fusion of 91 the virion with the host cell membrane (hoffmann et al., 2020a (hoffmann et al., , 2020b . ace2 is expressed in 92 many different cell types across many organs in the human body including lung, olfactory 93 sustentacular cells, enterocytes, and endothelial cells (albini et ace2, the use of ace inhibitors is being considered as a therapeutic intervention in covid-19 112 patients (lopes et al., 2020) . importantly, drugs currently used to treat the covid-19 can be 113 pro-arrhythmic and therefore there is a need to incorporate cardiovascular damage into the list of 114 targets of therapeutic interventions in covid-19 and for models that replicate human cardia 115 physiology (kochi et al., 2020) . 116 117 a hallmark of sars-cov-2 infection is acute loss of smell (cooper et al., 2020) . viral-induced 118 anosmia is not unique to sars-cov-2 infections since viruses such as rhinoviruses, influenza, 119 parainfluenza and coronaviruses are known to be the main cause of olfactory deficits in humans 120 (suzuki et al., 2007; imam et al., 2020) . in mice and humans, ace2 expression is detected in 121 sustentacular cells, olfactory stem cells known as horizontal and globose basal cells in the 122 olfactory epithelium, and vascular cells (pericytes) in the olfactory bulb (brann et al., 2020 the present study reports for the first time that zebrafish larvae exposed to sars-cov-2 appear 134 to mount innate immune responses that resemble cytokine responses of mild covid-19 patients. 135 recombinant sars-cov-2 s rbd is sufficient to cause olfactory, renal and cardiovascular 136 pathologies in larvae and adult zebrafish. we also identify potential mechanisms of sars-cov-137 2 induced anosmia by scrna-seq. our findings support the use of zebrafish as a novel 138 4 vertebrate model to elucidate sarscov-2 pathophysiology and to screen drugs and other 139 therapies targeting covid-19. 140 141 142 143 results 144 145 phylogenetic analyses of ace2 molecules in vertebrates 146 comparative analysis of ace2 molecules in vertebrates indicated that ace2 molecules are well 147 conserved in vertebrates with a 72%-73% similarity and 57.5%-58% identity between zebrafish 148 ace2 and human ace2, respectively (table s1 ). examination of ace2 amino acid motifs in 149 the region involved in binding sars-cov-2 s protein revealed zebrafish ace2 has 50%/64% 150 sequence similarity with the corresponding human ace2 region compared to 71%/78% in 151 macaques ace2 or 57%/71% in ferret ace2 ( systemic injection of recombinant sars-cov-2 protein into adult zebrafish has been shown to 159 induce some toxicity (ventura-fernandes et al., 2020). in order to determine whether 160 recombinant sars-cov-2 s rbd protein causes inflammatory responses in zebrafish larvae, we 161 exposed 5 dpf larvae to sars-cov-2 s rbd recombinant protein for 3 hours (h) and measured 162 cytokine responses by qpcr. as shown in figure 1a , 3 h immersion with sars-cov-2 s rbd 163 protein induced a significant downregulation in ifnphi1 expression and significant increase in 164 expression of ccl20a.3, a pro-inflammatory chemokine. no changes in ace2, tnfα, il1b and 165 il17a/f3 expression were observed ( figure 1a ). these results indicate that rapid immune 166 responses occur in zebrafish larvae exposed to sars-cov-2 s rbd. we next evaluated the 167 effects of sars-cov-2 rbd s on zebrafish larva heart function to validate zebrafish larvae as a 168 model for covid-19 cardiac manifestations. we immersed 7-and 5-days post fertilization (dpf) 169 zebrafish larvae with sars-cov-2 s rbd, or with vehicle, and measured heart rate after 3 h. 170 as shown in figure 1b , 7 dpf and 5 dpf zebrafish treated with sars-cov-2 s rbd had 171 significantly higher heart rates compared to vehicle treated controls. as a positive control for the 172 recombinant protein, we used animals treated with the same dose of recombinant infectious 173 hematopoietic necrosis virus (ihnv) glycoprotein (r-ihnvg), a rhabdovirus known to cause 174 severe endothelial damage in zebrafish (ludwig et al., 2011) . r-ihnvg caused a severe decrease 175 in larval zebrafish heart rate compared to control treated animals ( figure 1b -c). to determine if 176 increased heart rate induced by sars-cov-2 s rbd was dependent on ace2 binding, we co-177 incubated 5 dpf larvae with captopril and reverted sars-cov-2 s rbd induced heart 178 dysfunction. captopril had no effect on r-ihnvg induced bradycardia ( figure 1b ). ventricular 179 trace analyses showed marked differences in rhythm patterns in each treatment group ( figure 180 1d). importantly, the captopril and sars-cov-2 s rbd treated animals, despite having similar 181 heart rates to those of the vehicle treated controls, displayed a unique ventricular trace pattern, 182 warranting future studies regarding the potential cardioprotective role of captopril in combined, these results indicate that zebrafish exposed to sars-cov-2 s rbd protein 184 5 experience tachycardia and suggest that zebrafish larvae constitute a valuable pre-clinical model 185 to test the effects of drugs for covid-19 on cardiac activity in vivo. 186 187 zebrafish 189 anosmia is one of the earliest manifestations of sars-cov-2 infection in humans (cooper et 190 al., 2020) . we have previously shown that ihnv glycoprotein protein is sufficient to induce 191 rapid nasal immune responses as well as neuronal activation in teleost fish (sepahi et ( figure 2d ). loss of the epithelial mosaic structure characteristic of the teleost olfactory 202 epithelium was observed on days 1, 3 and 5 post-treatment ( figure 2e ). by day 5, loss of entire 203 apical lamellar areas due to severe necrosis was observed in the olfactory lamellae of all treated 204 animals compared to controls ( figure 2f ). significant loss of olfactory cilia was recorded in all 205 animals treated with sars-cov-2 s rbd at all time points ( figure 2h ). these results indicate 206 the sars-cov-2 s rbd is sufficient to cause inflammation, edema, hemorrhages, ciliary loss, 207 and necrosis in the olfactory organ of zebrafish. hence, olfactory damage can be caused by 208 indirect mechanisms and in the absence of active sars-cov-2 replication in this tissue. 209 210 toxicity effects of intranasal sars-cov-2 s rbd delivery were also evaluated in distant tissues 211 such as the kidney, a target organ of sars-cov-2. acute kidney injury (aki) incidence varies 212 from 0.9% to 29% in covid-19 patients (su et al., 2020) . renal damage, especially aki, is also 213 common in patients with ras dysfunction such as diabetic patients who suffer from 214 hypertension (ribeiro-oliveira et al., 2008; advani, 2020). a recent study in zebrafish injected 215 with the n-terminal part of sars-cov-2 s protein reported inflammation and damage in several 216 tissues of adult zebrafish including kidney 7 and 14 d post-injection (ventura-fernandes et al., 217 2020). in the present study, histological examination of the head-kidney of zebrafish who 218 received sars-cov-2 s rbd intranasally revealed renal tubule pathology characteristic of aki 219 3 h post-treatment ( figure s1 ). pathology was not as severe at later time points, but vacuolation 220 of the renal tubule epithelium was still visible 5 days post-treatment ( figure s1 ). we did not 221 observe signs of glomerulopathology in treated animals compared to controls. together, these 222 results indicate that intranasal delivery of sars-cov-2 s rbd is sufficient to cause 223 nephropathy in adult zebrafish but that pathology is not as severe as when the protein is delivered 224 by injection. 225 226 intranasal delivery of sars-cov-2 s rbd causes anosmia in adult zebrafish 227 228 adult zebrafish exposed to sars-cov-2 s rbd had a significant reduction of olfactory 229 responses to food extracts of ~50% of preexposure olfaction within minutes as measured by 230 6 electro-olfactogram (eog), indicating an instant effect of the protein on olfactory function (fig. 231 3a). reduction of olfaction was sustained for least one hour of recording, but the olfactory organ 232 remained still semi functional. zebrafish treated with pbs never lost olfaction at any time point. 233 to further quantify the degree of olfactory reduction due to sars-cov-2 s rbd, we took 234 advantage of the two easily accessible and isolated olfactory chambers present in zebrafish. we 235 exposed one naris to the sars-cov-2 s rbd protein and the other naris, from the same animal, 236 to pbs and waited 3 h or 1d before measuring olfaction by eog. at 3 h we observed a 37-70% 237 reduction in food and bile olfactory responses between the treated and untreated naris and a 238 complete loss of olfactory function to both odorants 1 d post-treatment (fig. 3b) . the reduction 239 of olfactory sensitivity for food extract was smaller than that found for bile, probably due to the 240 lower number of osns involved in bile acid detection compared to amino acids found in food 241 (hansen et al., 2003) . our results indicate that sars-cov-2 s rbd-induced-anosmia is not 242 specific for a subset of osns, since both food extracts and bile olfactory signals were suppressed 243 in sars-cov-2 s rbd treated zebrafish. this fact, together with the 1d time to develop 244 complete anosmia and disrupt olfactory epithelial structure, support the hypothesis that sars-245 cov-2 s rbd damage may occur first on sustentacular cells, with subsequent impacts on osn 246 viability and function. 247 single-cell analysis of the zebrafish olfactory organ 249 250 to understand the impact of sars-cov-2 s rbd on zebrafish oo, we performed single cell clusters, 3 endothelial cell (ec) clusters and 7 leucocyte (lymphoid and myeloid) clusters ( figure 258 4a-b). 259 of the 8 ncs, neuron1 and neuron2 corresponded to mature osns. neuron1 expressed markers 260 of ciliated osns (ompa and ompb) in addition to several olfactory receptor (or) genes 261 (buiakova et al., 1996) . neuron2, on the other hand, expressed markers of microvillus osns 262 (trpc2b, s100z, and gnao) and many vomeronasal receptors (vr) such as v2rh32 as well as or 263 gene ( figure s2 ) (kraemer et al., 2008 neuron7 and neuron8 expressed cell cycle and early neuronal progenitor markers as well as 278 tmprss13b and tmprss4a, however the majority of cluster identifying genes in these two clusters 279 are undescribed ( figure s2 ). 280 scs are supporting cells that exist in the neuroepithelium around osns and in humans, they 281 express ace2 (bryche et al., 2020) . scs in the olfactory epithelium can directly arise from 282 horizontal basal cells (hbcs) (yu and wu, 2017). we found 5 clusters of scs in our datasets. subpopulation closely related to the sustentacular4 cluster ( figure s2 ). 292 we identified three clusters of ecs that all express tmprss13 and tmprss4. while we did not 293 detect ace2 expression in any cell clusters, we detected ace2 mrna in adult zebrafish olfactory 294 organ, and at low levels in the olfactory bulb ( figure s3 ). ace2 expression levels have previously 295 shown to be low in neuronal tissues and therefore may be hard to detect by scrna-seq (song et 296 al., 2020). endothelial1 and 2 clusters expressed the endothelial markers sox7 and tmp4a (yao et 297 al., 2019). all three clusters broadly expressed genes associated with tight junctions (tjp3, jupa, 298 ppl, cldne, and cgnl1) as well as many keratin genes ( figure s2 ). interestingly, endothelial3 299 cluster also expressed the calcium channel trpv6 and a slew of non-annotated genes. 300 there are copious amounts of immune cells in the teleost olfactory organ ( intranasal delivery of sars-cov-2 s rbd induces inflammatory responses and 318 widespread loss of olfactory receptor expression in adult zebrafish olfactory organ 319 320 the cellular landscape of the zebrafish olfactory epithelium was affected by sars-cov-2 s 321 rbd treatment and time ( figure 4a -d). this was especially evident in the proportions of 322 8 neuronal cell types 3 d post-treatment when the proportion of mature, omp + ciliated osn was 323 much lower compared to controls and the 3 h treated group. in contrast, neuronal progenitors 324 expressing cell cycle markers (aubk, ecrg4, and mki67) and neuronal differentiation and 325 plasticity markers (neurod1, neurod4, gap43, sox11, and sox4) were expanded 3 d post-treatment 326 ( figure 4b -c). further, we detected a noticeable decrease in the proportion of cells belonging to 327 the lymphocyte2 cluster, a cluster that expressed markers of treg cells (foxp3b) 3 h post 328 intranasal delivery of recombinant sars-cov-2 s rbd but this change was not noticeable at 329 day 3 ( figure 4c ). at 3 d, we observed a third lymphocyte cluster, not detected at 3 h, highly 330 expressing the tcr subunit zap70 as well as plac8 onzin related protein (ponzr1), a molecule 331 that has immunoregulatory roles during th1 type immune responses in mammals ( figure s2 in olfactory neuronal clusters were enriched in processes such as neuron differentiation, sensory 367 system development, and sensory organ morphogenesis, whereas downregulated genes belonged 368 9 to sensory perception of smell, detection of chemical stimulus and gpcr signaling pathway 369 ( figure 5f ). functional enrichment analyses in metascape showed that the top non-redundant 370 enriched clusters in both upregulated and downregulated genes in zebrafish osns 3 h post-371 treatment was sensory perception of smell ( figure 5g ). the same was true 3 days post-treatment, 372 but processes such as regeneration, neuron development, neuron fate commitment and the p53 373 signaling pathway were also enriched within the upregulated genes ( figure 5h ). combined, 374 these results suggest that presence of sars-cov-2 s rbd in the olfactory organ instigates 375 harmful effects on osns within hours and that the magnitude of the osn damage increases by 3 376 days post-treatment. further, these analyses indicate that neuronal regeneration and 377 differentiation processes were initiated by day 3 in order to begin repair of olfactory damage. our study allowed us to dissect how each cell type in the zebrafish olfactory organ responds to 387 sars-cov-2 s rbd. our results indicated unique responses by sc clusters and ec clusters to 388 treatment (figures 4 and 6) . at 3 h, we detected increased expression of apoeb, of transcription 389 factors foxq1a and id2b, the transcriptional regulator nfil3-5, two tumor necrosis factor receptor 390 superfamily members (tnfrsf11b and tnfrsf9a) as well as tcima (transcriptional and immune 391 response regulator), whose mammalian ortholog pcim regulates immune responses as well as 392 endothelial cell activation and expression of inflammatory genes ( figure 6a ) (kim et al., 2009 ). 393 further, at 3 h we observed downregulation of the pro-inflammatory cytokine il17af/3 as well as 394 glutathione peroxidase gpx1b, the transcription factor notch1b, basal cell adhesion molecule 395 bcam, guanine nucleotide-binding protein subunit gamma gng8, and the calcium binding s100z 396 in sc and ec from sars-cov-2 s rbd treated olfactory organs relative to vehicle treated. at 3 397 d post-treatment, we observed significant increased expression of the gene that encodes brain 398 natriuretic peptide (nppc), a vasodilating hormone, the pro-inflammatory chemokine ccl19a.2, 399 the m2 macrophage marker arg2, the transcription factors foxq1a and sox11a, tubulin beta 5 400 tubb5, and the epithelial mitogen epgn, among others ( figure 6b ). downregulated genes at 3 d 401 post-treatment included hsp70.3, apoeb, the osteoblast specific factor b postb, the desmosomal 402 component periplakin (ppl), the vasoconstricting endothelin 2 (edn2), the heparin binding 403 molecule latexin (ltx) involved in pain and inflammation, and cd74b, a part of the mhc-ii 404 complex ( figure 6b ). combined, these data indicated immune regulatory responses in sc and 405 ec clusters early after sars-cov-2 s rbd treatment, followed by transcriptional changes with 406 potential vasoactive effects by day 3. 407 408 go and enrichment set analyses indicated that sc and ec clusters initially undergo 409 transcriptional changes enriched in metabolic responses, response to stress, and cell 410 differentiation ( figure 6c ). later on, at day 3, sc and ec responses were enriched in genes 411 involved not only in the stress response but also in immune responses and responses to wounding 412 ( figure 6d ). similar results were identified using metascape, which showed that the 413 inflammatory response to wounding was moderately enriched in the downregulated genes at 3 h, 414 whereas by day 3, response to wounding became the top enriched set among the upregulated 415 genes ( figure 6e -f). 416 417 exposure of wildtype zebrafish larvae with sars-cov-2 does not support viral replication 418 419 zebrafish larvae have been used as models to investigate several human viruses. infecting 420 zebrafish larvae in a bsl-3 laboratory by immersion in contaminated water is comparable to 421 infecting a cell line. we first checked the stability of sars-cov-2 in zebrafish water overtime, 422 in the absence of any animals. we found that sars-cov-2 viral loads in the water remained 423 stable throughout the experiment ( figure 7a -b). we exposed wildtype ab zebrafish larvae to 424 live sars-cov2 and examined viral mrna abundance over time to determine if zebrafish 425 larvae can support viral replication. we detected no increases in the viral n copy numbers over 426 time and a steady decline in e gene copy numbers in both water from wells containing larvae and 427 virus as well as in the larval tissue ( figure 7c -f). these results indicate that wild-type zebrafish 428 larvae cannot support efficient sars-cov-2 replication as suggested by the in silico 429 comparative sequence analyses of the zebrafish ace2 molecule. 430 431 exposure of zebrafish larvae to sars-cov-2 decreases ace2 expression and triggers pro-432 inflammatory cytokine responses 433 434 in order to determine whether exposure of zebrafish larvae with live sars-cov-2 causes 435 changes in ai, we measured ace2 mrna levels in control and sars-cov-2 exposed larvae over 436 time. ace2 expression was significantly downregulated as early as 6 h post-infection. ace2 437 expression inhibition was sustained over the time course of the experiment with the greatest 438 decrease occurring 2 days post-infection (dpi) ( figure 8a ). we next evaluated changes in 439 expression of cytokine and chemokine genes to establish whether zebrafish mount inflammatory 440 responses that resemble the patterns of mild or severe sars-cov-2 infection. il1β expression 441 was significantly upregulated at 6h, 1 dpi (2-3 fold) and 2 dpi (10 fold) and significantly 442 downregulated at 4 dpi ( figure 8b ). we detected a significant increase in tnfa expression in 443 sars-cov-2 exposed larvae 2 dpi ( figure 8c ). ifnphi1 and ifnphi3 are the two main type i ifn 444 genes involved in larval zebrafish antiviral responses (levraud et al., 2019) . we detected a 445 significant up-regulation of ifnphi1 at 1 and 2 dpi, whereas expression was inhibited at 4 dpi. 446 interestingly, ifnphi3 expression followed a very different pattern compared to ifnphi1, which 447 was significantly downregulated 2 dpi but significantly upregulated at 4 dpi ( figure 8d -e). mxa 448 expression was significantly downregulated at all time points ( figure 8f ). il17af/3 expression 449 was significantly elevated 1, 2 and 4 dpi ( figure 8g ). expression levels of il22, a member of the 450 il10 family, were downregulated 6 hpi and 1 dpi ( figure 8h ) whereas the type ii cytokine 451 il4/il13b was downregulated at 6 hpi, 1 dpi and 2 dpi ( figure 8i ). further, a significant increase 452 in the expression of the chemokine ccl20a.3 was detected in infected larvae at 1 and 2 dpi 453 compared to controls ( figure 8j) . a moderate increase in ccl19a.1 expression was observed at 2 454 dpi followed by a strong down-regulation (15 fold) at 4 dpi ( figure 8k ). taken together, these 455 data indicate that exposure to sars-cov-2 induces a significant antiviral and pro-inflammatory 456 immune response in wildtype zebrafish larvae. this response involved type i ifn, tnfa, il1b, il17 457 and ccl20, reminiscent of covid-19 patients with mild disease. 458 459 the current covid-19 pandemic has propelled the investigation of sars-cov-2 and the 461 development of animal models that help identify therapeutic interventions and vaccines for 462 covid-19. thus far, all animal models reported are mammals, and therefore breeding, genetic 463 manipulation, and animal housing in bsl-3 laboratories make these models costly and not 464 readily available in large numbers. zebrafish can overcome many of the limitations of 465 mammalian models thanks to their transparent bodies, short life-span, low maintenance costs and 466 production of large numbers of embryos. we therefore performed the simplest infection 467 procedure, where sars-cov-2 was added to the water of zebrafish larvae. in this manner, bsl-468 3 trained personnel with no experience in zebrafish microinjection can readily expose larvae to 469 sars-cov-2 without the need of animal protocols in a similar fashion to in vitro cell culture 470 infections. exposure of wildtype zebrafish larvae to sars-cov-2 in the water did not however 471 result in any detectable viral replication. downregulated in response to sars-cov-2 exposure. combined, these data suggest the sars-505 cov-2 induces some type i ifn responses in zebrafish larvae while inhibits others. future 506 12 studies are clearly needed to ascertain the role of teleost type i ifn in the anti sars-cov-2 507 immune response. 508 509 s protein is a structural protein of sars-cov-2 and therefore the target of several vaccine trials. 510 therefore, we exposed zebrafish larvae to sars-cov-2 s rbd protein and investigated 511 transcriptional and physiological responses. rapid changes in gene expression were detected in 512 treated larvae, including up-regulation of the chemokine ccl20a.3 and the down-regulation of 513 ifnphi3. the ccl19/ccl20 axis appears to be critical in teleost antiviral innate responses, as 514 previous studies have shown very rapid responses in larvae exposed to the rhabdovirus svcv 515 (sepahi et al., 2019) . this change was also detected in the larvae that were exposed to the live 516 sars-cov-2 virus in the present study. we further detected a significant down-regulation of 517 type i ifn ifnphi1 gene in larvae exposed to sars-cov-2 s rbd protein. 518 519 examination of ace2 transcriptional changes in zebrafish larvae exposed to sars-cov-2 520 revealed a consistent down-regulation in expression throughout the course of infection. 521 interestingly, we did not observe any changes in ace2 expression after 3h immersion with sars-522 cov-2 s rbd protein. recently, enterocytes were found to be the main cell type expressing 523 ace2 in 5 dpf-old zebrafish larvae (postlethwait et al., 2020); and therefore it is possible that the 524 down-regulation in ace2 expression observed in our experiments was the result of enterocyte 525 responses to sars-cov-2. however, an olfactory epithelial cell cluster was not identified in this 526 dataset, probably because these cells constitute too small a fraction of the cells of an entire larva. 527 importantly, we exposed larvae to the virus at 3 dpf, when the olfactory pit is already sampling 528 the surrounding water, while the gut fully opens only at 4 dpf. thus, changes in ace2 expression 529 levels in the olfactory pit of the zebrafish larvae cannot be ruled out at this point. 530 531 previous work has shown that ace2 knockdown in mice protects from sars-cov infection 532 (kuba et al., 2005) . thus, down-regulation of zebrafish ace2 expression may have protected 533 larvae from sars-cov-2 infection in our experiments. our data agree with studies in mouse 534 lungs, where suppression of ace2 gene expression was consistently observed following sars-535 cov-2 infection (chen et al., 2020). interestingly, changes in ace2 levels can occur in response 536 viruses that do not require ace2 for host entry (chen et al., 2020). thus, although further 537 studies are warranted, our data suggest that ace2 is involved in antiviral sars-cov-2 responses 538 in zebrafish. 539 540 we took advantage of the zebrafish fish model which allows for easy live imaging of heart beats 541 in transparent larvae. we detected in vivo cardiac/heart responses in larval zebrafish exposed to 542 sars-cov-2 s rbd protein characterized by tachycardia. cardiac arrhythmia is a common 543 symptom among covid-19 patients and current research efforts aim to understand how sars-544 cov-2 infection impacts cardiovascular function (libby, 2020) . our findings underscore that 545 sars-cov-2 s rbd is able to cause tachycardia in the zebrafish larval model and that this 546 model can be used for rapid evaluation of drug treatments for covid-19. as a proof of concept, 547 we used captopril, an ace inhibitor currently being evaluated in human clinical trials 548 (nct04355429). captopril treatment ameliorated tachycardia in zebrafish larvae exposed to 549 sars-cov-2 s rbd recombinant protein. our studies therefore suggest the beneficial use of 550 captopril in covid-19 patients undergoing cardiac arrhythmia, but clearly further studies are 551 13 required to fully translate these findings to the clinic and to determine the duration and timing of 552 captopril treatment in covid-19 patients. 553 554 a recent report in zebrafish adults indicated that injection of recombinant sars-cov-2 s n 555 terminal protein caused histopathology of several tissues including the liver, kidney, brain and 556 ovary (ventura-fernandes et al., 2020). additionally, some animals succumbed to injection with 557 the recombinant protein. we did not detect any mortalities neither in larvae nor in adults in any 558 of our experiments, perhaps suggesting that mortalities were due to the injection procedure rather 559 than the protein treatment itself. of note, the dose used in the present study was considerably 560 lower than the dose delivered in the ventura-fernadez study, perhaps explaining the differences 561 in toxicity between both studies. we observed histological damage following a single intranasal 562 delivery of sars-cov-2 protein, specifically at the site of delivery, the olfactory organ, whereas 563 more transient and moderate damage was detected in the renal tubules. renal damage may have 564 occurred by direct uptake of sars-cov-2 s rbd by the kidney once the protein reached the 565 bloodstream following intranasal administration or, alternatively, by activation of ras or 566 inflammatory cascades at the olfactory organ. thus, toxicity of sars-cov-2 s protein in adult 567 zebrafish may be less severe when delivered intranasally than by injection, and future studies 568 should evaluate whether current vaccine candidates also exert similar effects and whether 569 different administration routes cause the same side-effects or not. this is particularly important 570 as the intranasal route appears promising for some vaccine candidates study did not determine when zebrafish recover olfactory function following sars-cov-2 s 597 14 rbd intranasal treatment, but based on our histopathological observations, the olfactory organ 598 was still severely damaged 5 days after treatment, suggesting that recovery of olfactory function 599 may take several weeks in our model. our findings therefore indicate that similar to humans, 600 zebrafish suffer from olfactory pathology and loss of smell in response to sars-cov-2 s rbd 601 protein. thus, olfactory pathophysiology appears to occur even in the absence of viral replication 602 raising the possibility that nasal vaccines for covid-19 may also cause transient anosmia in 603 humans. 604 605 in conclusion, the present study reports that both adult and larval wild-type zebrafish can be 606 useful models to advance our understanding of covid zebrafish larvae in responses to sars-cov-2 s rbd. animals were exposed to sars-cov-2 s 639 rbd protein (r-spike, 2 ng/ml) for 3 h at 28.5°c or vehicle. changes in gene expression were 640 measured by rt-qpcr using rps11 as the house-keeping gene. each data point represents a pool 641 of 4 larvae/well. data are expressed fold-change compared to vehicle controls using the pffafl 642 method. 643 (b) average heart beat per minute of 7 dpf (n=6) zebrafish larvae after 3 h of incubation with 644 vehicle, 2 ng/ml r-spike, or 2 ng/ml r-ihnvg. 645 (c) average zebrafish heart beats per minute in 5 dpf zebrafish larvae (n=8) after 3 h of 646 incubation with vehicle, 2 ng/ml r-spike, or 2 ng/ml r-ihnvg with and without treatment with 647 12mm of captopril. heart beats were recorded for 3 min at 20 under a nikon ti microscope 648 and (c-d) mean viral loads quantified as log of sars-cov-2 n gene and sars-cov-2 e gene copy 726 numbers in control supernatants from well with larvae not exposed to virus, and supernatants 727 from wells with larvae that were exposed to 10 4 pfu of sars-cov-2 for 6 h, 1 d, 2 d and 4 d. 728 each sample represents the supernatant of one well containing 12 larvae. 729 (e-f) mean viral loads quantified as log of sars-cov-2 n gene and sars-cov-2 e gene copy 730 numbers in control larvae and larvae exposed to 10 4 pfu of sars-cov-2 for 6 h, 1 d, 2 d and 4 731 d. each sample point represents one well containing 12 larvae. 732 larval infections began at 2 dpf after mechanical dechorionation at 1 dpf. * p-value<0.05; ** p-733 value<0.01 *** p-value<0.001. results are representative of two independent experiments. 734 735 for all experiments, wild type ab zebrafish were obtained from zirc (oregon, usa). for the 764 intranasal delivery of sars-cov-2 s rbd protein into adult zebrafish, female and male adult 765 zebrafish were obtained from dr. wong's laboratory at the university of nebraska due to 766 lockdown of zirc during the pandemic. all fish were maintained in a filtered aquarium system 767 at 28℃ with a 14 h light and 10 h dark cycle at the university of new mexico aquatics animal 768 facility. all experiments with adults utilized a mix of male and female animals, and the larvae 769 sex is indeterminable. animals were fed ad libitum gemma complete nutrition (skretting). ab 770 larvae were obtained by batch-crossing ab adults allowing for natural fertilization. the morning 771 of fertilization, larvae were collected at n=50 per petri dish and kept in e3 medium containing 772 0.002% methylene blue. in the afternoon, larvae were placed in fresh e3 medium without 773 methylene blue and non-surviving embryos were removed. larvae were maintained at 28.5℃ in 774 e3 medium until 4dpf when they are slowly changed to system water. 775 776 sars-cov-2 777 the sars-cov-2 isolate, a cdc isolate from a us patient (usa-wa1/2020), was obtained 778 from bei resources. the strain was grown at a low moi to minimize generation of 779 noninfectious particles and low passaged virus was used in all experiments described. the virus 780 was propagated on vero e6 cells and viral loads quantitated by rt-qpcr and by plaque forming 781 assays as we have described previously (bradfute et al, 2020). 782 783 intranasal delivery of sars-cov-2 s rbd recombinant protein to adult zebrafish 785 adult zebrafish were anesthetized for 1 min in 0.04 mg/ml tricaine-s (syndel) solution and then 786 moved to an absorbent boat where their gills were still covered with anesthetic solution for 787 administration of solutions to nares. using a microloader tip (eppendorf, 930001007), 5 μl of 20 788 ng/μl sars-cov-2 s rbd (kindly provided by dr. f. krammer) was directly pipetted into each 789 naris, while 5 μl of sterile pbs was applied in control fish. after inoculation, animals were 790 recovered in a separate tank supplemented with o2 before returning to their rearing tank until the 791 end of the experiment. euthanasia was performed on ice to ensure rapid death without perturbing 792 the combined supernatants were centrifuged for 10 min at 400g in supplemented neurobasal medium 820 and cells were counted with a hemocytometer. viability was estimated by trypan blue staining. 821 cells were then strained twice through flowmi 10 μm strainers and loaded onto the chromium 822 controller with a viability of > 85%. cell libraries were generated according to 10x genomics 823 protocols at the university of new mexico cancer center genomics core facility and sequenced 824 on an illumina novaseq 6000 at the university of colorado genomics and microarray core 825 facility. sequencing depth and statistics of the scrna-seq run are shown in figure s2 . sras for 826 this project can be found on ncbi under bioproject #prjna668529. 827 fastqs were run through the cell ranger v3.0 pipeline with default settings using the grcz11 828 zebrafish genome. output matrices were loaded to r (v1.2.5001) as a seurat object (package 829 seurat v3.1.1). first, cells with less than 200 or greater than 2500 features, and greater than 5% 830 mitochondrial features were removed. after counts were normalized using the "lognormalize" 831 method and a scale factor of 10000, 2000 variable genes were selected using the 'vst" method. 832 data was scaled, and pca dimensional reduction was run. jackstraw analysis determined the 833 vehicle control to have 38 significant principal components (pcs) and the treated samples to 834 have 40 significant pcs which were used for clustering analysis. sars-cov-2 s rbd treated 835 samples were integrated with the vehicle treated sample and clustered together using 30 836 significant pcs and a resolution of 0.5. cluster markers were identified with "findallmarkers" 837 in seurat and exported for cluster identification. differential expression analysis was done with 838 seurat "findmarkers" in default settings for each cluster and exported for gene ontology 839 analysis. 840 gene ontology (go) analysis was done with web-based guis metascape and shinygo v0.61 841 which draw multiple currently maintained databases (ensembl, entrz, kegg among others). 842 biological process webs were created using biological process output from shinygo v0.61 in 843 prism graphpad. biological processes bar graphs were produced by metascape. 844 845 electro-olfactogram recordings 846 adult ab zebrafish were anesthetized and received 2 µl of recombinant sars-cov-2 s rbd 847 protein (50 ng/µl) in pbs or pbs alone. after 3 h or 1 day, zebrafish were anaesthetized (0.1 g 848 ms222/l), placed in a v-shape stand and supplied with aerated water containing ms222 849 anesthetic (0.05 g/l). the nasal flap was removed with sterile fine forceps to expose the 850 olfactory rosettes to a continuous tank water source. olfactory responses to zebrafish food 851 extract or goldfish bile were measured by electrical recordings as detailed in sepahi et al., 2019. 852 the food extract was prepared as a filtered solution of 1l tap water and 0.1 g of dry food pellets. 853 water food extracts were separated in 200 ml aliquots and kept frozen until the recording day. a 854 0.5 ml mix of bile fluid from 50 adult goldfish was aliquoted in 10 µl and kept frozen until the 855 day of the recording. before each recording, bile aliquots were diluted 1:1000 in water from the 856 eog system. there were no significant differences in olfactory responses between males and 857 females, hence responses of both sexes were averaged together. the percentage reduction in 858 olfactory activity was calculated by dividing the amplitude of the olfactory signal at time x by 859 amplitude of the olfactory signal at time 0 *100. percentage of olfactory signal reduction 860 between control and treated naris was calculated as follows (amplitude response to odorant in 861 control naris (mv) -amplitude response to odorant in treated naris (mv))/amplitude response to 862 odorant in control naris (mv)) * 100. 863 (sigma) at 42℃, stabilized for 5 min at rt and then imaged to record heart-beat activity. 872 873 zebrafish larvae heart-beat recordings and analysis 874 as the agarose solidified, animals were adjusted to the microscope stage (approx. 5 min) then 875 hearts were recorded using brightfield avi for 3 min at 16.667 frames/s at rt. avi images were 876 then opened in imagej with the time series analyzer v3 plugin. circular rois were drawn in 877 either the atrium or ventricle and average intensity was extracted. the maximum average 878 intensity peaks were identified and counted per 60s as bpm. data were analyzed by one-way 879 anova with tukey's post hoc. 880 881 infection of zebrafish fish larvae with sars-cov-2 882 ten animals were placed in each well in 12-well plates containing 2 ml of tank water and 883 transferred to bsl3 facility the day before infection. gene expression analyses by rt-qpcr 903 whole larvae rna was extracted using trizol. for tissue homogenization bead beater tubes are 904 preloaded with 1.5 g 1.0 mm dia zirconia beads, 1.5 g 2.0 mm zirconia beads and 500 µl trizol. 905 samples were loaded into the tubes and bead beat at 4350 rpm for 45 s. tubes were then 906 centrifuged at 7,000 rpm for 7 min. the homogenate/lysates were transferred to clean 1.5 ml 907 microfuge tubes and spun at 7,000 rpm for 7 min to pellet debris. supernatants were then 908 processed to extract the total rna using a standard chloroform/phenol extraction protocol. rna 909 was quantified by nanodrop and samples were normalized and 1 µg of rna was used to 910 synthesize cdna using the superscript iii first strand system (thermofisher, 18080051). qpcr 911 was performed using ssoadvanced supermix (biorad, 1725270) and primers listed in table s3 912 (supplemental methods). gene expression changes were quantified using the pfaffl method 913 (pfaffl, 2001 glycerophospholipid biosynthesis apoptosis microtubule polymerization or depolymerization gpcr signaling, coupled to cyclic nucleotide 2nd messenger negative regulation of cell differentiation iron uptake and transport circadian regulation of gene expression sensory perception of smell developmental process anatomical structure development response to wounding response to stress immune system response cell chemotaxis multicellular organism development acute kidney injury: a bona fide complication of diabetes the sars-cov-2 990 receptor, ace-2, is expressed on many different cell types: implications for ace-inhibitor-and 991 angiotensin ii receptor blocker-based cardiovascular therapies myocardial injury and covid-19: possible mechanisms savalan the pathogenicity of sars-cov-2 in hace2 transgenic mice the establishment 1000 of neuronal properties is controlled by sox4 and sox11 imbalanced host response to sars-cov-2 1007 drives development of covid-19 macrophage-mediated neuroprotection and neurogenesis in the olfactory 1010 epithelium cov-2 entry genes in the olfactory system suggests mechanisms underlying covid-19-1015 associated anosmia massive transient damage of the olfactory 1019 epithelium associated with infection of sustentacular cells by sars-cov-2 in golden syrian 1020 hamsters olfactory marker protein (omp) gene 1024 deletion causes altered physiological activity of olfactory sensory neurons zebrafish as a new 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1309 sars-cov-2 infection on the diamond princess cruise ship: a retrospective analysis small molecule screening in 1313 zebrafish: an in vivo approach to identifying new chemical tools and drug leads proinflammatory cytokines in the olfactory mucosa result in covid-19 induced anosmia a robust human norovirus 1323 replication model in zebrafish larvae zebrafish 1327 studies on the vaccine candidate to covid-19, the spike protein: production of antibody and 1328 adverse reaction a rampage through the body neuronal wiskott-aldrich syndrome protein regulates 1335 tgf-β1-mediated lung vascular permeability receptor recognition by the 1338 novel coronavirus from wuhan: an analysis based on decade-long structural studies of 1339 sars coronavirus clinical characteristics of 138 hospitalized patients with coronavirus-infected pneumonia in wuhan, china remdesivir and chloroquine effectively inhibit the recently emerged novel 1348 coronavirus (2019-ncov) in vitro the zebrafish activating immune receptor nitr9 signals via 1352 dap12 sars and mers: 1355 recent insights into emerging coronaviruses sox transcription factors in endothelial 1358 differentiation and endothelial-mesenchymal transitions spatiotemporal photolabeling of neutrophil trafficking 1361 during inflammation in live zebrafish regeneration and rewiring of rodent olfactory sensory neurons severe acute respiratory syndrome coronavirus 2 infects and 1368 damages the mature and immature olfactory sensory neurons of hamsters angiomotin-like protein 1 controls endothelial 1374 polarity and junction stability during sprouting angiogenesis metascape provides a biologist-oriented resource for the analysis of 1378 systems-level datasets clinical 1381 characteristics of 3062 covid-19 patients: a meta-analysis sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell 1386 subsets across tissues the function of fish cytokines single-cell rna-seq data 1391 analysis on the receptor ace2 expression reveals the potential risk of different human organs 1392 vulnerable to 2019-ncov infection key: cord-331472-kd4uxcve authors: shahid, zainab; kalayanamitra, ricci; mcclafferty, brendan; kepko, douglas; ramgobin, devyani; patel, ravi; aggarwal, chander shekher; vunnam, ramarao; sahu, nitasa; bhatt, dhirisha; jones, kirk; golamari, reshma; jain, rohit title: covid‐19 and older adults: what we know date: 2020-04-20 journal: j am geriatr soc doi: 10.1111/jgs.16472 sha: doc_id: 331472 cord_uid: kd4uxcve severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), a novel virus that causes covid‐19 infection, has recently emerged and caused a deadly pandemic. studies have shown that this virus causes worse outcomes and a higher mortality rate in older adults and those with comorbidities such as hypertension, cardiovascular disease, diabetes, chronic respiratory disease, and chronic kidney disease (ckd). a significant percentage of older american adults have these diseases, putting them at a higher risk of infection. additionally, many adults with hypertension, diabetes, and ckd are placed on angiotensin‐converting enzyme (ace) inhibitors and angiotensin ii receptor blockers. studies have shown that these medications upregulate the ace‐2 receptor, the very receptor that the sars‐cov‐2 virus uses to enter host cells. although it has been hypothesized that this may cause a further increased risk of infection, more studies on the role of these medications in covid‐19 infections are necessary. in this review, we discuss the transmission, symptomatology, and mortality of covid‐19 as they relate to older adults, and possible treatments that are currently under investigation. j am geriatr soc 68:926–929, 2020 c lusters of pneumonia cases occurring in the city of wuhan in december 2019 led to the eventual identification of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). 1, 2 through an epidemiological investigation, the chinese government narrowed down the origin of the virus to the huanan seafood market in wuhan. the viral sequence had a 96% similarity to a bat coronavirus, and, with no evidence of bat-to-human transmission, it was hypothesized that the virus spread to humans through an intermediate host. 1, 3 genomic sequence studies from malaysia later suggested that the intermediate hosts were pangolins that were smuggled into china from malaysia and sold at the huanan seafood market. 4 the subsequent human-to-human spread set off what later turned into a pandemic. the world health organization (who) declared sars-cov-2 as a pandemic on march 11, 2020. as of march 23, 2020, at 13:25 est, there were 362,019 confirmed cases of sars-cov-2 reported from 168 different countries, with 15,488 deaths and an overall projected case fatality rate (cfr) of 4.3%. 5 the centers for disease control and prevention (cdc) reported that although individuals older than age 65 comprise 17% of the total population in the united states, they make up 31% of covid-19 infections, 45% of hospitalizations, 53% of intensive care unit admissions, and 80% of deaths caused by this infection. 6 this suggests that older individuals are more likely to get covid-19 and have worse outcomes compared with the general population. on 10 pediatric patients with sars-cov-2 infections, 8 continuously tested positive for the virus on rectal swabbing, despite testing negative on nasopharyngeal swabs. 10 given these findings, patients who test negative on a nasopharyngeal swab could potentially still have an active infection. the current proposed mechanism for cell entry is via the angiotensin-converting enzyme-2 (ace-2) receptor found in the lungs, endothelium, heart, kidneys, and gastrointestinal system. 1 spike proteins on the exterior of sars-cov-2 anchor the virus to ace-2 receptors on cells in the lower respiratory tract. this specific mechanism of action may propose a higher risk of infection for older adults. according to the cdc, 63.1% of adults older than age 60 have hypertension, 38% of people older than 65 years have chronic kidney disease (ckd), and 26.8% of adults older than age 65 have diabetes. [11] [12] [13] many of these patients use ace inhibitors and angiotensin-receptor blockers (arbs) that upregulate the ace-2 receptor. 14 thus it is hypothesized that older individuals with such comorbidities may have an elevated risk of and experience a more severe course of infection with sars-cov-2. the most common presenting symptoms in the general population are fever (98%), cough (76%), dyspnea (55%), and myalgias or fatigue (up to 44%). 15, 16 these symptoms are also common in older adults; one study on 21 critically ill patients with sars-cov-2 infection, with a mean age of 70 years, found that the most common presenting symptoms were shortness of breath (76%), fever (52%), and cough (48%). up to 86% of older adults presented with comorbidities, and the most significant ones were ckd (48%), congestive heart failure (43%), chronic obstructive pulmonary disease (copd) (33%), and diabetes (33%). 17 most older adults have some form of organ damage occurring due to sars-cov-2 including acute respiratory disease syndrome (71%), acute kidney injury (20%), cardiac injury (33%), and liver dysfunction (15%), and 67% required vasopressor support for treatment. 17 in all age groups, chest computed tomography imaging of patients with sars-cov-2 revealed ground glass opacities (ggos) (87%), mixed ggos and consolidation (65%), vascular enlargement (72%), and traction bronchiectasis (53%). among these, lesions had peripheral distribution (87.1%), bilateral lung involvement (82.2%), lower lung predominance (54.5%), and multifocality (54.5%). 18 comparatively, chest radiograph findings in older adults showed bilateral reticular-nodular opacities (58%), ggos (48%), pleural effusions (about 33%), peribronchial thickening (about 25%), and focal consolidations (20%). 17 the mortality of the sars-cov-2 pandemic in older adults has been striking. according to the joint who-china factfinding mission, the overall cfr of 17.3% in january decreased to .7% in february, whereas the cfr in adults older than age 80 had increased to 21.9%. 19 another analysis of 72,314 cases indicated an overall cfr of 2.3%, but a cfr of 8% in patients aged 70 to 79 years and 14.5% in patients older than age 80. 20 a report on 355 patients with sars-cov-2 found that patients who died had an average age of 79.5 years. 21 another report on 4,226 cases in the united states indicated a cfr less than 1% in patients younger than age 54 but a cfr of 3% to 11% in patients aged 65 to 84 and 10% to 27% in patients older than age 85. more than 80% of deaths among adult patients occurred in those older than age 65. 6 most of the fatal cases to date have involved older adults and patients with comorbidities. 20, 22 many older adults in the united states have cardiovascular disease (17%), diabetes (26.8%), hypertension (63.1%), copd (23.7%), and ckd (38%). 13,23-26 an analysis by the joint who-china fact-finding mission found that patients older than age 60 and those with comorbidities had the highest risk for severe disease and death. the cfr in patients without comorbidities was 1.4%, whereas the cfr was 13.2% for patients with cardiovascular disease, 9.2% for patients with diabetes, 8.4% for patients with hypertension, 8% for patients with chronic respiratory disease, and 7.6% for patients with cancer. 19 one study on 46 fatal cases of sars-cov-2, in which 84% of patients were older than age 60, found that diabetes is likely associated with increased mortality. 15 another study on critically ill older patients with sars-cov-2 found that 86% of patients had comorbid conditions such as ckd, congestive heart failure, copd, and diabetes. 17 this likelihood of having multiple comorbidities places older adults at an even greater risk of increased mortality from sars-cov-2. treatment sars-cov-2 can be described as a superspreading event that has a rapidly early growth that is then sustained. 27 the best precautions are maintaining regular hand hygiene (because viral stool shedding and viability on surfaces can last from 2 hours to 9 days), decreasing social contact, and, for healthcare workers, wearing personal protective equipment. [27] [28] [29] the reproductive number (r 0 ) for the virus dropped from 3.86 to .32 in a 5-week period once these precautions were taken in china. 27 for patients with covid-19 infection, treatment is focused on supportive care. although there is currently no fda-approved treatment, many medications are being studied for effectiveness against sars-cov-2. chloroquine, a drug approved by the food and drug administration (fda) for malarial and autoimmune diseases, has shown efficacy against sars-cov-2 in vitro. it works by increasing the endosomal ph required for viralcell fusion and by interfering with the terminal glycosylation of ace-2. 30 more than 20 clinical trials are currently ongoing in china to assess chloroquine as a possible treatment for covid-19, and the state council of china has stated that chloroquine has demonstrated marked efficacy in treating covid-19-associated pneumonia in multicenter clinical trials conducted in china. 31, 32 a nonrandomized clinical trial on 20 patients with confirmed covid-19 infection showed that after a daily dose of 600 mg hydroxychloroquine, a less toxic derivative of chloroquine, 57.1% of patients were virus free in 6 days. 33 another drug with promising results is remdesivir, an intravenous drug that inhibits sars-cov-2 replication through premature termination of viral rna. remdesivir is a non-fda-approved investigational drug that has been effective against covid-19 in vitro and has been used on an expanded access, or compassionate use, basis in the united states. 34, 35 in one case, the patient received remdesivir on day 7 of hospitalization due to his worsening condition, and he subsequently had an improvement in symptoms, no longer required oxygen supplementation, and had no adverse effects due to treatment. 36 currently six clinical trials for remdesivir are ongoing. [37] [38] [39] [40] [41] [42] there is also currently an ongoing phase i clinical trial sponsored by the national institute of allergy and infectious diseases testing the safety and immunogenicity of a vaccine for sars-cov-2. 43 there is no benefit of the influenza vaccine for prevention of sars-cov-2 infection, and the cdc recommends all individuals older than age 6 months receive the influenza vaccine to prevent influenza and unnecessary evaluation for sars-cov-2. 44 in conclusion, the sars-cov-2 pandemic has a much higher mortality rate in older adults, and older adults who have certain comorbidities and take ace inhibitors or arbs may have a greater risk of infection and worse outcomes. although many medications and a vaccine are currently under investigation, no fda-approved treatments or vaccines are available for this virus. conflicts of interest: the authors have declared no conflicts of interest for this article. author contributions: zs, rk, bm, dk, dr, rp, and csa assisted in article concept and design, acquisition of data, drafting of the manuscript, and final approval. zs, rv, ns, db, kj, rg, and rj assisted in article concept and design, analysis and interpretation of data, revision of the manuscript for important intellectual content, and final approval. zs, rk, rg, and rj further assisted in revisions of the final manuscript. sponsor's role: we have no sponsors to disclose. a pneumonia outbreak associated with a new coronavirus of probable bat origin the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status the proximal origin of sars-cov-2 probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak coronavirus covid-19 global cases by the center for systems science and engineering (csse) at johns hopkins university severe outcomes among patients with coronavirus disease 2019 (covid-19)-united states epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid-19) during the early outbreak period: a scoping review the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 gong s characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding centers for disease control and prevention (cdc). national diabetes statistics report 2020 hypertension prevalence and control among adults: united states us department of health and human services covid-19 and the cardiovascular system clinical features of patients infected with 2019 novel coronavirus in wuhan initial clinical features of suspected coronavirus disease 2019 in two emergency departments outside of hubei characteristics and outcomes of 21 critically ill patients with covid-19 in washington state relation between chest ct findings and clinical conditions of coronavirus disease (covid-19) pneumonia: a multicenter study who-china joint mission. report of the who-china joint mission on coronavirus disease characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention report sulle caratteristiche dei pazienti deceduti positivi a covid-19 in italia il presente report è basato sui dati aggiornati al 17 marzo 2020 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study hypertension prevalence and control among adults: united states heart disease, myocardial infarction, and stroke-a public health issue chronic obstructive pulmonary disease among adults-united states identifying and interrupting superspreading eventsimplications for control of severe acute respiratory syndrome coronavirus 2 air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents chloroquine is a potent inhibitor of sars coronavirus infection and spread ? title=chloroquine&officialname=&subjectid=&secondaryid=&applier=& studyleader=&ethicalcommitteesanction=&sponsor=&studyailment=& studyailmentcode=&studytype=0&studystage=0&studydesign=0& minstudyexecutetime=&maxstudyexecutetime=&recruitmentstatus=0& gender=0&agreetosign=&secsponsor=&regno=&regstatus=0&country=& province=&city=&institution=&institutionlevel=&measure=&intercode=& sourceofspends=&createyear=0&isuploadrf=&whetherpublic=&btngo=btn& verifycode=&page=1 breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel coronavirus in the united states covid-19) compared to standard of care treatment adaptive covid-19 treatment trial (actt) mild/moderate 2019-ncov remdesivir rct severe 2019-ncov remdesivir rct safety and immunogenicity study of 2019-ncov vaccine (mrna-1273) to prevent sars-cov-2 infection update: public health response to the coronavirus disease 2019 outbreak-united states key: cord-338973-73a7uvyz authors: xu, jiabao; zhao, shizhe; teng, tieshan; abdalla, abualgasim elgaili; zhu, wan; xie, longxiang; wang, yunlong; guo, xiangqian title: systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov date: 2020-02-22 journal: viruses doi: 10.3390/v12020244 sha: doc_id: 338973 cord_uid: 73a7uvyz after the outbreak of the severe acute respiratory syndrome (sars) in the world in 2003, human coronaviruses (hcovs) have been reported as pathogens that cause severe symptoms in respiratory tract infections. recently, a new emerged hcov isolated from the respiratory epithelium of unexplained pneumonia patients in the wuhan seafood market caused a major disease outbreak and has been named the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this virus causes acute lung symptoms, leading to a condition that has been named as “coronavirus disease 2019” (covid-19). the emergence of sars-cov-2 and of sars-cov caused widespread fear and concern and has threatened global health security. there are some similarities and differences in the epidemiology and clinical features between these two viruses and diseases that are caused by these viruses. the goal of this work is to systematically review and compare between sars-cov and sars-cov-2 in the context of their virus incubation, originations, diagnosis and treatment methods, genomic and proteomic sequences, and pathogenic mechanisms. coronaviruses (covs) are a group of viruses that co-infect humans and other vertebrate animals. cov infections affect the respiratory, gastrointestinal, liver, and central nervous systems of humans, livestock, birds, bats, mice, and many other wild animals [1] [2] [3] . for example, severe acute respiratory syndrome (sars) in 2002 and the middle east respiratory syndrome (mers) in 2012 were both coronaviruses that transmitted from animals to humans [4, 5] . the source of unexplained pneumonia was first discovered in wuhan in dec, 2019, and sars-cov-2, a new coronavirus, was isolated from the respiratory epithelium of patients. it belongs to a new evolutionary branch within the cov. on feb. 11th, 2020, the new coronavirus was officially renamed "sars-cov-2" from "2019-ncov" [6] . the disease caused by sars-cov-2 was called "coronavirus disease 2019" (covid-19) [7] . according to on nov. 27th, 2002, a respiratory illness erupted in guangdong province, china [19] . in feb, 2003, the chinese ministry of health announced that this acute respiratory syndrome had thus far resulted in 305 cases and five deaths [20] . the following month, there were clusters of atypical pneumonia reported in other parts of mainland china, hong kong [21] , canada [22] , and singapore [23] . in jul, 2003, sars-cov spread across 26 countries in six continents, and caused a cumulative 8,096 cases and 774 deaths (9.6%) [24] . in particular, a higher mortality (21%) was found in hospital personnel [25, 26] . on dec. 29th, 2019, the health departments of hubei province received a report that four employees of the south china seafood wholesale market were diagnosed with unknown-caused pneumonia in a local hospital, which was the first report of sars-cov-2 [27] . on dec. 31st, 2019, the national health commission of people republic of china and chinese center for disease control and prevention (china cdc) participated in the investigation and case-searching work [27] . on the same day, the government of wuhan released information about the disease outbreaks to society [28] . nowadays, the number of patients infected with sars-cov-2 continues to climb worldwide. by the date of this paper's submission, a cumulative 67,081 cases and 1,526 deaths (2.1%) were reported worldwide. in wuhan, china, the number is 37,914. the main timeline of sars and covid-19 epidemic development were shown in figure 1a ,b, respectively. glucocorticoid and interferon lopinavir/ritonavir (in testing) on nov. 27th, 2002, a respiratory illness erupted in guangdong province, china [19] . in feb, 2003, the chinese ministry of health announced that this acute respiratory syndrome had thus far resulted in 305 cases and five deaths [20] . the following month, there were clusters of atypical pneumonia reported in other parts of mainland china, hong kong [21] , canada [22] , and singapore [23] . in jul, 2003, sars-cov spread across 26 countries in six continents, and caused a cumulative 8,096 cases and 774 deaths (9.6%) [24] . in particular, a higher mortality (21%) was found in hospital personnel [25, 26] . on dec. 29th, 2019, the health departments of hubei province received a report that four employees of the south china seafood wholesale market were diagnosed with unknown-caused pneumonia in a local hospital, which was the first report of sars-cov-2 [27] . on dec. 31st, 2019, the national health commission of people republic of china and chinese center for disease control and prevention (china cdc) participated in the investigation and case-searching work [27] . on the same day, the government of wuhan released information about the disease outbreaks to society [28] . nowadays, the number of patients infected with sars-cov-2 continues to climb worldwide. by the date of this paper's submission, a cumulative 67,081 cases and 1,526 deaths (2.1%) were the initial symptoms of sars patients were fever (100%), cough (61.8%), myalgia (48.7%), dyspnea (40.8%), and diarrhea (31.6%) [29] , and the prognosis of patients was associated with host characteristics (including age, gender, etc.) [30] . during hospitalization, respiratory distress occurred in 90.8% of sars patients [29] . the duration from disease onset to severe respiratory distress was an average of 9.8 ± 3.0 days [29] . during the disease course, some patients developed leukopenia, lymphopenia, and thrombocytopenia with an upregulation of aspartate transaminase (ast), alanine aminotransferase (alt), lactic dehydrogenase (ldh), and c-reactive protein (crp) [29] . in comparison, covid-19 showed similar trends with sars patients [28] . fever, fatigue, and dry cough are the main manifestations of the patients, while nasal congestion, runny nose, and other symptoms of the upper respiratory tract are rare. beijing centers for diseases control and prevention indicated that the typical case of covid-19 has a progressive aggravation process. covid-19 can be classified into light, normal, severe, and critical types based on the severity of the disease [31] : (1) mild cases-the clinical symptoms were mild, and no pneumonia was found on the chest computed tomography (ct); (2) normal cases-fever, respiratory symptoms, and patients found to have imaging manifestations of pneumonia; (3) severe cases-one of the following three conditions: respiratory distress, respiratory rate ≥ 30 times/min (in resting state, refers to oxygen saturation ≤ 93%), partial arterial oxygen pressure (pao2)/oxygen absorption concentration (fio2) ≤ 300 mmhg (1 mmhg = 0.133 kpa); (4) critical cases-one of the following three conditions: respiratory failure and the need for mechanical ventilation, shock, or the associated failure of other organs requiring the intensive care unit [32] . the current clinical data shows that the majority of the deaths occurred in the older patients. however, severe cases have been documented in young adults who have unique factors, particularly those with chronic diseases, such as diabetes or hepatitis b. those with a long-term use of hormones or immunosuppressants, and decreased immune function, are likely to get severely infected. the incubation period of sars is 1-4 days [33] . however, in a small number of patients, the incubation period may be longer than 10 days [34] . it has been demonstrated that the latency of covid-19 varies from 3-7 days on average, for up to 14 days [35] . during this incubation period, patients are contagious, and it has been reported that each case infected on average 3.77 other people (uncertainty range 2.23-4.82) [36] . by comparison, we found that the average latency of covid-19 is slightly longer than that of sars. according to the demographic information of sars patients, infection occurred in all age groups (the average age was dyspnea (40.8%), and diarrhea (31.6%) [29] , and the prognosis of patients was associated with host characteristics (including age, gender, etc.) [30] . during hospitalization, respiratory distress occurred in 90.8% of sars patients [29] . the duration from disease onset to severe respiratory distress was an average of 9.8 ± 3.0 days [29] . during the disease course, some patients developed leukopenia, lymphopenia, and thrombocytopenia with an upregulation of aspartate transaminase (ast), alanine aminotransferase (alt), lactic dehydrogenase (ldh), and c-reactive protein (crp) [29] . in comparison, covid-19 showed similar trends with sars patients [28] . fever, fatigue, and dry cough are the main manifestations of the patients, while nasal congestion, runny nose, and other symptoms of the upper respiratory tract are rare. beijing centers for diseases control and prevention indicated that the typical case of covid-19 has a progressive aggravation process. covid-19 can be classified into light, normal, severe, and critical types based on the severity of the disease [31] : (1) mild cases-the clinical symptoms were mild, and no pneumonia was found on the chest computed tomography (ct); (2) normal cases-fever, respiratory symptoms, and patients found to have imaging manifestations of pneumonia; (3) severe cases-one of the following three conditions: respiratory distress, respiratory rate ≥ 30 times / min (in resting state, refers to oxygen saturation ≤ 93%), partial arterial oxygen pressure (pao2)/oxygen absorption concentration (fio2) ≤ 300 mmhg (1 mmhg = 0.133 kpa); (4) critical cases-one of the following three conditions: respiratory failure and the need for mechanical ventilation, shock, or the associated failure of other organs requiring the intensive care unit [32] . the current clinical data shows that the majority of the deaths occurred in the older patients. however, severe cases have been documented in young adults who have unique factors, particularly those with chronic diseases, such as diabetes or hepatitis b. those with a long-term use of hormones or immunosuppressants, and decreased immune function, are likely to get severely infected. the incubation period of sars is 1-4 days [33] . however, in a small number of patients, the incubation period may be longer than 10 days [34] . it has been demonstrated that the latency of covid-19 varies from 3-7 days on average, for up to 14 days [35] . during this incubation period, patients are contagious, and it has been reported that each case infected on average 3.77 other people (uncertainty range 2.23-4.82) [36] . by comparison, we found that the average latency of covid-19 is slightly longer than that of sars. according to the demographic information of sars patients, infection occurred in all age groups (the average age was ≦45) [37] . there was a proportional difference between male and female (female predominance) [37] , with a male-to-female ratio of 1:1.25 [38] . in addition, hospital staff had a higher risk due to the proximal interactions with large numbers from the infected population. for example, hospital staff accounted for 22% of all cases in hong kong and 22.8% in guangdong [37] . the mortality caused by sars increased with age (> 64 years) [37] , and the overall mortality rate during the outbreak of sars was estimated at 9.6% [39, 40] . li et al. reported that people who have not been exposed to sars-cov-2 are all susceptible to covid-19 [41] . among the 8,866 patients who have been confirmed with covid-19, nearly half of the patients have been aged 50 years or older (47.7%) [36] . the male-to-female ratio is about 2.7:1 [38] and the average incubation period is 5.2 days [42] . however, severe covid-19 cases and deaths have mostly been in the middle-aged adults and the elderly with long smoking histories or other 45) [37] . there was a proportional difference between male and female (female predominance) [37] , with a male-to-female ratio of 1:1.25 [38] . in addition, hospital staff had a higher risk due to the proximal interactions with large numbers from the infected population. for example, hospital staff accounted for 22% of all cases in hong kong and 22.8% in guangdong [37] . the mortality caused by sars increased with age (> 64 years) [37] , and the overall mortality rate during the outbreak of sars was estimated at 9.6% [39, 40] . li et al. reported that people who have not been exposed to sars-cov-2 are all susceptible to covid-19 [41] . among the 8,866 patients who have been confirmed with covid-19, nearly half of the patients have been aged 50 years or older (47.7%) [36] . the male-to-female ratio is about 2.7:1 [38] and the average incubation period is 5.2 days [42] . however, severe covid-19 cases and deaths have mostly been in the middle-aged adults and the elderly with long smoking histories or other basic diseases, such as heart disease and hypertension [43, 44] . at the time that this paper was been submitted, covid-19 patients mortality rate was 2.1% [45] . in 2010, shi et al. isolated a sars-like coronavirus which was highly homologous to the sars-cov, confirming that rhinolophus sinicus (chinese rufous horseshoe bat) was a natural host of the sars-cov [46] . bats are known to be hosts for 30 coronaviruses based on complete genomic sequences analysis [47] . epidemiological investigations have shown that civet cats in the wildlife market were the direct source of sars-cov [48] . among the four patients with sars discovered during the winter of 2003-2004, two were waitresses at a restaurant in guangzhou, china, and one was a customer who viruses 2020, 12, 244 5 of 17 ate in the restaurant a short distance from a civet cage. all six civets in this cage were tested positive for sars-cov [48] . the new emerging sars-cov-2 shares about 80% of the gene sequence of sars-cov, released by the military medical research institute of nanjing military region in 2003 [28] . recently, shi et al. reported that the sequence similarity of coronavirus between sars-cov-2 and the coronavirus isolated from rhinolophus affinis is 96.2%, and suggested that bats may be the source of the virus [49] . so far, the intermediate hosts of sars-cov-2 are elusive and have been reported to be snakes, minks, or variable others [50, 51] . recently, a research group of south china agricultural university reported that pangolins may be one of the intermediate hosts for sars-cov-2, by analyzing more than 1,000 metagenomic samples, because they found that 70% of pangolins are positive for the coronavirus. moreover, the virus isolate from pangolin shared 99% sequence similarity with the current infected human strain sars-cov-2 [52] . taking this recent research into consideration, we agreed that pangolin is more likely to be one of intermediate hosts of sars-cov-2. according to the who data on jul. 31th, 2003 [24] , a total of 8,096 clinically diagnosed cases of sars were reported worldwide, with 774 deaths and 26 countries and regions affected (figure 2a ). most cases were in asia, europe, and america. the main countries in asia were china (including mainland, macao, hong kong, and taiwan), singapore, and so on. the total number of cases in mainland china was 5,327, with 349 deaths [53] . the cases were mainly concentrated in beijing, guangdong, and shanxi ( figure 2b ) [54] . in total, 2,102 patients were from hong kong, macao, and taiwan, with 336 deaths [24] . viruses 2020, 12, x for peer review 5 of 18 basic diseases, such as heart disease and hypertension [43, 44] . at the time that this paper was been submitted, covid-19 patients mortality rate was 2.1% [45] . in 2010, shi et al. isolated a sars-like coronavirus which was highly homologous to the sars-cov, confirming that rhinolophus sinicus (chinese rufous horseshoe bat) was a natural host of the sars-cov [46] . bats are known to be hosts for 30 coronaviruses based on complete genomic sequences analysis [47] . epidemiological investigations have shown that civet cats in the wildlife market were the direct source of sars-cov [48] . among the four patients with sars discovered during the winter of 2003-2004, two were waitresses at a restaurant in guangzhou, china, and one was a customer who ate in the restaurant a short distance from a civet cage. all six civets in this cage were tested positive for sars-cov [48] . the new emerging sars-cov-2 shares about 80% of the gene sequence of sars-cov, released by the military medical research institute of nanjing military region in 2003 [28] . recently, shi et al. reported that the sequence similarity of coronavirus between sars-cov-2 and the coronavirus isolated from rhinolophus affinis is 96.2%, and suggested that bats may be the source of the virus [49] . so far, the intermediate hosts of sars-cov-2 are elusive and have been reported to be snakes, minks, or variable others [50, 51] . recently, a research group of south china agricultural university reported that pangolins may be one of the intermediate hosts for sars-cov-2, by analyzing more than 1,000 metagenomic samples, because they found that 70% of pangolins are positive for the coronavirus. moreover, the virus isolate from pangolin shared 99% sequence similarity with the current infected human strain sars-cov-2 [52] . taking this recent research into consideration, we agreed that pangolin is more likely to be one of intermediate hosts of sars-cov-2. according to the latest data on feb. 14th, 2020 [55, 56] , there have been a total of 67,081 clinically diagnosed cases of covid-19 in worldwide, with 1,526 deaths. a total of 25 countries and regions have infected people. due to the spring festival transportation peak, the disease has been spread more rapidly across china (figure 2c ). as the origin area of covid-19, hubei province has been the most severely infected area, with 54,406 cumulative diagnosis cases. wuhan city has 37,914 cases. guangdong, henan, and zhejiang province have 1,294 cases, 1,212 cases, and 1,162 cases, respectively (figure 2d ). at present, the covid-19 outbreak has been spread to all parts of china and around the world, including the united states, thailand, and japan. it has been noticed that most of these patients have ever been to wuhan or contacted with people who had been in wuhan. the distribution of covid-2019 patients in china (including hong kong, macao and taiwan) and hubei province is shown in figure 3 . sars were reported worldwide, with 774 deaths and 26 countries and regions affected (figure 2a) . most cases were in asia, europe, and america. the main countries in asia were china (including mainland, macao, hong kong, and taiwan), singapore, and so on. the total number of cases in mainland china was 5,327, with 349 deaths [53] . the cases were mainly concentrated in beijing, guangdong, and shanxi (figure 2b ) [54] . in total, 2,102 patients were from hong kong, macao, and taiwan, with 336 deaths [24] . according to the latest data on feb. 14th, 2020 [55, 56] , there have been a total of 67,081 clinically diagnosed cases of covid-19 in worldwide, with 1,526 deaths. a total of 25 countries and regions have infected people. due to the spring festival transportation peak, the disease has been spread more rapidly across china (figure 2c ). as the origin area of covid-19, hubei province has been the most severely infected area, with 54,406 cumulative diagnosis cases. wuhan city has 37,914 cases. guangdong, henan, and zhejiang province have 1,294 cases, 1,212 cases, and 1,162 cases, respectively (figure 2d ). at present, the covid-19 outbreak has been spread to all parts of china and around the world, including the united states, thailand, and japan. it has been noticed that most of these patients have ever been to wuhan or contacted with people who had been in wuhan. the distribution of covid-2019 patients in china (including hong kong, macao and taiwan) and hubei province is shown in figure 3 . as the number of covid-19 patients in china has been growing rapidly, preventing the spread of sars-cov-2 is the most important and urgent task [57] . it was shown that human-to-human transmission of sars-cov-2 has spread via droplets or close contacts [58, 59] , but aerosol and fecal-oral transmission still need further study [60, 61] . to reduce virus transmission, early detection and isolation are essential. in addition, close monitoring in crowded places is also important [62] . the possible pathogens of sars and covid-19 are both derived from wild animals [63] . therefore, hunting, selling, and eating wild animals not only seriously damage the ecosystem, but also lead to the spread of epidemic diseases [64] . thus, banning all wildlife trade is an effective measure to prevent viral prevalence. wearing level-d protective clothing can protect medical staff from infection of respiratory viruses [65] . a vaccine against sars-cov has not been described in any published articles [66] . however, on jan. 26th, 2020, the china cdc started to develop a new vaccine for sars-cov-2. the virus has been successfully isolated and seed strains have been screened [67] . the early symptoms of sars and covid-19 are very similar to winter influenza, and the most important way to distinguish flu and pneumonia is to take throat swabs for viral testing [68] . current diagnostic tests for coronavirus include rt-pcr, real-time reverse transcription pcr (rrt-pcr), reverse transcription loop-mediated isothermal amplification, as well as real-time rt-lamp [69] [70] [71] [72] . national medical products administration has approved seven new nucleic acid test reagents for coronavirus, which were developed based on fluorescence pcr by feb. 1st, 2020 [73] . suspected infections can be detected accurately and quickly for timely isolation and treatment to avoid infecting others by using these test reagents. both sars-cov and sars-cov-2 are covs; hence, the treatment strategies of sars could be relevant for covid-19 [74] . in 2003, sars was mainly treated by isolation of the patients, hormones treatment, antiviral and symptomatic treatments, and many drugs such as glucocorticoid [29] and interferon [75] . now, isolation, antiviral, and symptomatic treatments are still mainly adopted for covid-19 treatment. as effective drugs for sars, hormones and interferons can also be used to treat covid-19 [74] . lopinavir is one kind of protease inhibitor used to treat hiv infection, with ritonavir as a booster. lopinavir and/or ritonavir has anti coronavirus activity in vitro. hong kong scholars found that, compared with ribavirin alone, patients treated with lopinavir/ritonavir and ribavirin had lower risk of acute respiratory distress syndrome (ards) or death caused by sars-cov [76, 77] . lopinavir/ritonavir has also been clinically tested in treatment of covid-19, and showed wonderfully effective treatment for some patients, but the general clinical effect has not been determined [78] . more effective treatments are still under continuing exploration: on jan. 25th, 2020, a joint research team from the shanghai institute of materia medica, chinese academy of sciences, and shanghai tech university screened and identified 30 potential drugs that are reported to be effective against sars-cov-2 [79] . a high-resolution crystal structure of sars-cov-2 coronavirus 3cl hydrolase (mpro) was announced after the outbreak of covid-19 in the world [80] , and human coronaviruses (hcovs) have been treated as severe pathogens in respiratory tract infections. nelfinavir was predicted to be a potential inhibitor of sars-cov-2 main protease [81] . the first patient in the us had been trial-treated with intravenous remdesivir (a novel nucleotide analogue prodrug in development) due to a severe infection [82, 83] . no adverse reactions were observed during the administration, and the patient's condition was effectively improved [84] . clinical trials of remdesivir for treatment of covid-19 just started on feb. 5th and 12th, 2020 in wuhan and beijing, respectively, and the experimental results remain unclear [85, 86] . covs are rna viruses and contain the largest genomes of all rna viruses [87] . covs belong to the subfamily coronavirinae in the family of coronaviridae of the order nidovirales, and this subfamily includes four genera: α-coronavirus, β-coronavirus, γ-coronavirus, and δ-coronavirus [88] . both sars-cov-2 and sars-cov are in the coronavirus family, β-coronavirus genera [89] . the genome of sars-cov-2 is more than 85% similar to the genome of the sars-like virus zc45 (bat-sl-covzc45, mg772933.1), and together these types of viruses form a unique orthocoronavirinae subfamily with another sars-like virus zxc21 in the sarbecovirus subgenus [35] . all the three viruses show typical β-coronavirus gene structure. human sars-cov and a genetically similar bat coronavirus (bat-sl-covzxc21, mg772934) from southwest of china have formed another clade within the sarbecovirus [35] . we also performed comparative genomic analyses of sars-cov-2 and sars-cov by zpicture. the results showed that the genomic sequences of sars-cov-2 and sars-cov have extremely high homology at the nucleotide level (figure 4a,b) . there are six regions of difference (rd) in the genome sequence between sars-cov and sars-cov-2, and the rds are named according to the order of discovery. rd1, rd2, and rd3 (448nt, 55nt, and 278nt, respectively) are partial coding sequences of the orf lab gene; rd4 and rd5 (315nt and 80nt, respectively) are partial coding sequences of the s gene; rd6 is 214nt in size and is part of the coding sequence of the orf7b and orf8 genes. these rds may sequences of the orf lab gene; rd4 and rd5 (315nt and 80nt, respectively) are partial coding sequences of the s gene; rd6 is 214nt in size and is part of the coding sequence of the orf7b and orf8 genes. these rds may provide new molecular markers for the identification of sars-cov-2 and sars-cov, and also help to develop new drugs against sars-cov-2. to analyze the homogeneity of sars-cov, mers-cov, and sars-cov-2, an evolutionary tree was constructed based on the genomes of 35 sars-cov-2 strains from different data submission units, one sars-cov strain, and two mers-cov strains ( figure s1 ). phylogenetic analysis showed that the distance of sars-cov (ay274119) is closer to sars-cov-2 strains than mers-cov (kc164505, jx869059). to analyze the homogeneity of sars-cov, mers-cov, and sars-cov-2, an evolutionary tree was constructed based on the genomes of 35 sars-cov-2 strains from different data submission units, one sars-cov strain, and two mers-cov strains ( figure s1 ). phylogenetic analysis showed that the distance of sars-cov (ay274119) is closer to sars-cov-2 strains than mers-cov (kc164505, jx869059). to further explore whether all encoded proteins of sars-cov-2 are homologous to that of sars-cov, we performed a protein sequence alignment analysis using blastp. the results showed that most of sars-cov-2 proteins are highly homologous (95%-100%) to the proteins of sars-cov virus, indicating the evolutionary similarity between sars-cov and sars-cov-2 (table 2) . however, two proteins (orf8 and orf10) in sars-cov-2 have no homologous proteins in sars-cov. the amino acid sequence of orf8 in sars-cov-2 is different from sequences of conserved orf8 or orf8b derived from human sars-cov [11] . orf8 protein of sars-cov-2 does not contain known functional domain or motif. an aggregation motif vlvvl (amino acid 75-79) has been found in sars-cov orf8b which was shown to trigger intracellular stress pathways and activate nod-like receptor family pyrin domain-containing-3 (nlrp3) inflammasomes [90] . therefore, it will be clinically meaningful to analyze the biological function of these two specific proteins (orf8 and orf10) in sars-cov-2. as the most abundant protein in covs, nucleocapsid (n) protein is highly conserved across covs [91] . the n protein in sars-cov-2 shares~90% amino acid identity with that in sars-cov [28] , which indicates that antibodies against the n protein of sars-cov would likely recognize and bind the n protein of sars-cov-2 as well. n antibodies do not provide immunity to sars-cov-2 infection, but the antibodies have a cross reactivity with sars-cov n protein viruses, which would allow a serum-based assay to identify the asymptomatic sars-cov-2 infected-cases [28] . although previous studies have found serum reactivity to group sars-cov n proteins in chinese populations [92] , exposure to sars-cov-2 should increase the dilution factor if the infection had occurred. this information has important implications for preventing the spread of asymptomatic infections. in addition, spike stalk s2 in sars-cov-2 is highly conserved and shares 99% identity with those of the two bat sars-like covs (bat-sl-covzxc21 and bat-sl-covzc45) and human sars-cov [11] . thus, the broad spectrum antiviral peptides against s2 has the potential to be effective treatment [93] . many studies have been performed to study the pathogenesis of sars-cov [94] [95] [96] . the spike (s) protein and n protein confer stability to the viral particle [97] . the n protein is a structural protein involved in virion assembly, and plays a pivotal role in virus transcription and assembly efficiency [97] . s protein can bind to the cellular receptors of sensitive cells and mediate infection of their target cells, after which it begins to replicate in the cytoplasm [98] . sars-cov mainly targets the lungs, immune organs, and small systemic blood vessels and causes systemic vasculitis and decrease of immune function [99] . more seriously, the infection leads to extensive pulmonary consolidation, diffuse alveolar damage, and the formation of a transparent membrane, finally deteriorating to respiratory distress [100] . cov can enter host cells through the interaction between cov s protein and its host receptor angiotensin-converting enzyme 2 (ace2), which is isolated from sars-cov-permissive vero-e6 cells. the receptor-binding motif (rbm) of s protein can directly contact ace2 [47, 101, 102] . aec2 have been identified to be key binding residues and functional receptor for sars-cov, and it can also protect alveolar cells [103] . the binding of spike protein to ace2 and the subsequent downregulation of this receptor contribute to severe alveolar injury during sars [49] . the downregulation of ace2 results in the excessive production of angiotensin ii by the related enzyme ace, and the stimulation of type 1a angiotensin ii receptor (agtr1a) can lead to the increase of pulmonary vascular permeability, which potentially explains the increased lung pathology when the expression of ace2 is decreased [104] . ace2-transfected t cells form multinucleated syncytia with cells expressing s protein. the virus was shown to replicate effectively in ace2-transfected, but not in mock-transfected t cells. antibodies targeting ace2 can block viral replication in vero-e6 cells [105] . recently, ji et al. demonstrated that the receptor binding domain of sars-cov-2 was capable of binding ace2 in the context of the sars-cov spike protein [51] . among sras-cov spike protein's fourteen residues predicted to interact directly with human ace2 as the receptor for sars-cov, eight amino acids are well conserved in homology sars-cov-2 spike protein [26, 102] . at the same time, wan et al. showed that sars-cov-2 uses ace2 receptors to infect humans, bats, civets, monkeys, and swine, but not mice [106] . compared to previously reported sars-cov strains, sars-cov-2 uses ace2 receptors more efficiently than human sars-cov (year 2003), but less efficiently than human sars-cov (year 2002) [106] . the mutation of proteins determines two important characteristics of the sars-cov-2: a higher ability to infect and enhanced pathogenicity than the bat-like sars-cov, but a lower pathogenicity than sars-cov [43] . as a large number of people have left wuhan, the control of the epidemic situation is extremely urgent, and the treatments of covid-19 are imminent. on feb. 14th, 2020, there were more than 54,000 confirmed patients in hubei province, china [55] . due to the lack of effective antiviral drugs, the prognosis of patients solely depends on their age and physical condition [74] . although it was reported that the clinically recovered patients exceed the number of dead, the majority of the patients are still not cured in hospital. in addition, the potential adaptive mutation of sars-cov-2 makes it difficult for vaccine development. therefore, it is urgent for us to develop more sensitive inspection methods and effective drugs. seven type of covs have been identified to cause human disease [107] . the two highly pathogenic viruses, sars-cov and mers-cov, cause severe respiratory syndrome in humans. the other four human covs (hcov-nl63, hcov-229e, hcov-oc43 and hku1) induce only mild upper respiratory diseases, although some of them can cause severe infections in infants, young children, and elderly individuals [4, 108] . the latest one is sars-cov-2. it has been reported that sars-cov-2 shared almost 80% of the genome with sars-cov [11] . our results also showed that almost all encoded proteins of sars-cov-2 are homologous to sars-cov proteins ( table 2) . hence, clinical drugs and therapies for treating sars may be used as a reference for covid-19 treatment [74] . in addition to the well-known sars-cov, mers-cov, as one merbecovirus subgenus of β-covs, is also extremely invasive. mers-cov is the pathogen of the middle east respiratory syndrome, which can infect both humans and animals, and can be transmitted through camels [109] . it mainly occurs in saudi arabia and has a high mortality rate [66] . studies had demonstrated that the clinical course of sars and mers was highly similar, and sars and mers may have similar pathogenesis [66] . the genome sequence of sars-cov-2 also shows some similarities to that of mers-cov. it will be very interesting to study the relationship among sars-cov, mers-cov, and sars-cov-2 that may be exploited for future developing broad-spectrum antiviral therapies. although more and more studies for sars-cov-2 have sprung up since the outbreak of this epidemic covid-19, based on our comparison, we propose some key questions to be clarified in future studies (table 3 ). in-depth understanding the underlying pathogenic mechanisms of sars-cov-2 will reveal more targets for better therapy of covid-19. table 3 . proposed questions to study sars-cov-2 for future studies. what is the effect of the surface epitope and receptor binding domain of s protein of sars-cov-2 on the virus' infectivity? is there any effect of sars-cov vaccine designed according to s protein on sars-cov-2? does sars-cov-2 orf8 and orf10 proteins, which have no homology proteins in sars-cov, play roles in the infectivity and pathogenicity of sars-cov-2? can the susceptibility of asymptomatic carriers be judged by detecting the serum reactivity level of n protein? apart from droplet transmission and contact transmission, are there other methods to transmit sars-cov-2? what is the percentage of covid-19 patients have been infected with sars and produced antibodies? is there an effective specific anti-sars-cov-2 solution? does traditional chinese medicine have any effect on the treatment of covid-19 caused by sars-cov-2? do ethnic differences affect the transmissibility and pathogenicity of sars-cov-2? do any environmental factors, such as regional conditions or climate, affect sars-cov-2 transmission? supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/2/244/s1, figure s1 : phylogeny of sars-cov-2, sars-cov and mers-cov. unrooted phylogenetic relationships are represented by constructing neighbor-joining tree. table s1 : the genomic information of latest sars-cov-2 strains author contributions: conceptualization, l.x., y.w. and x.g.; methodology, j.x. and w.z.; software, s.z. and w.z.; validation, j.x., s.z. and t.t.; formal analysis, j.x. and s.z.; writing-original draft preparation, j.x. and s.z.; writing-review and editing, l.x., y.w., w.z. and x.g.; 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fighting the storm epidemiology, genetic recombination, and pathogenesis of coronaviruses acknowledgments: all the genomic data of sars-cov-2 is from gisaid, ncbi/genbank, nmdc, cngb/cngbdb and 2019ncovr (table s1 ). here, we would like to express our gratitude to all units and individuals who are responsible for the sample collection and data submission. the authors declare no conflict of interest. key: cord-323093-u3ozc9ry authors: rathnayake, athri d.; zheng, jian; kim, yunjeong; perera, krishani dinali; mackin, samantha; meyerholz, david k.; kashipathy, maithri m.; battaile, kevin p.; lovell, scott; perlman, stanley; groutas, william c.; chang, kyeong-ok title: 3c-like protease inhibitors block coronavirus replication in vitro and improve survival in mers-cov–infected mice date: 2020-08-19 journal: sci transl med doi: 10.1126/scitranslmed.abc5332 sha: doc_id: 323093 cord_uid: u3ozc9ry pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and the newly emerged sars-cov-2, the causative agent of coronavirus disease 2019 (covid-19). we describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3c-like protease (3clpro), an enzyme essential for viral replication. the optimized compounds were effective against several human coronaviruses including mers-cov, sars-cov, and sars-cov-2 in an enzyme assay and in cell-based assays using huh-7 and vero e6 cell lines. two selected compounds showed antiviral effects against sars-cov-2 in cultured primary human airway epithelial cells. in a mouse model of mers-cov infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. these results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses. coronaviruses are a large group of viruses that can cause a wide variety of diseases in humans and animals (1) . human coronaviruses generally cause the common cold, a mild upper respiratory illness. however, global outbreaks of new human coronavirus infections with severe respiratory disease have periodically emerged from animal reservoirs, including severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and, most recently, sars-cov-2, the causative agent of coronavirus disease 2019 . sars-cov-2 emerged in china in december 2019 and subsequently rapidly spread throughout the world. genetic analysis of sars-cov-2 revealed that it is closely related to sars-like betacoronaviruses of bat origin, bat-sl-covzc45 and bat-sl-covzxc21 (2) . despite the periodic emergence of new coronaviruses capable of infecting humans, there are currently no licensed vaccines or antiviral drugs against any coronaviruses, underscoring the urgent need for the development of preventive and therapeutic measures against pathogenic coronaviruses. the coronavirus genome contains two overlapping open reading frames (orf1a and orf1b) at the 5′ end terminal, which encode polyproteins pp1a and pp1ab. the polyproteins are processed by a 3c-like protease [3clpro or main protease (mpro)] (11 cleavage sites) and a papain-like protease (plpro) (3 cleavage sites), resulting in 16 mature nonstructural proteins, including an rna-dependent rna polymerase (rdrp). both 3clpro and plpro are essential for viral replication, making them attractive targets for drug development (3) (4) (5) (6) (7) . coronavirus 3clpro is a cysteine protease that has two n-terminal domains containing two -barrel chymotrypsin-like folds (8) (9) (10) . the active site of 3clpro is located in the cleft between the two domains and is characterized by a catalytic cys-his dyad. we have developed broad-spectrum inhibitors of an array of viruses, including coronaviruses and noroviruses (11) (12) (13) (14) (15) (16) (17) (18) that use 3clpro for viral replication and picornaviruses that use 3c protease (19) . we have shown efficacy of the coronavirus 3clpro inhibitor gc376 (currently in clinical development) in animal models of coronavirus infection (20, 21) . specifically, administration of gc376 to cats with feline infectious peritonitis (fip), a coronavirus-induced systemic disease that is 100% fatal, reversed the progression of fip and resulted in clinical remission (20, 21) . we have recently reported the results of exploratory in vitro studies using a dipeptidyl series of mers-cov 3clpro inhibitors that embody a piperidine moiety as a new design element, as well as pertinent structural and biochemical studies (17) . here, we report the development of 3clpro inhibitors against multiple coronaviruses, including sars-cov-2, and demonstrate in vivo efficacy against mers-cov in a mouse model. the synthesis scheme for compound series 6a to 6k and 7a to 7k is shown in fig. 1 and described in supplementary materials and methods. the activity of compounds 6a to 6k and 7a to 7k against the 3clpro enzymes of mers-cov, sars-cov, and sars-cov-2 was evaluated in a fluorescence resonance energy transfer (fret) enzyme assay (table 1 and table s1 ). several compounds in this series (6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j, and 7j) were also tested in cell-based assays ( table 2) . table 1 and table s1 show 50% inhibitory concentration (ic 50 ) values in a fret enzyme assay for select compounds (6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j, and 7j) and gc376. fifty percent inhibitory concentration (ec 50 ) values and 50% cytotoxic concentration (cc 50 ) values for select compounds and gc376 were measured in cell culture assays (table 2) . cell culture assays included huh-7 cells infected with mers-cov, vero e6 cells infected with sars-cov-2, the crandell-rees feline kidney (crfk) cells infected with fip virus (fipv), and l929 cells infected with mouse hepatitis virus (mhv) ( table 2 ). inhibitors with a p 2 leucine (leu) residue were more potent than those with a cyclohexylalanine against mers-cov 3clpro (compounds 6h and 7h versus 6i and 7i), with submicromolar ic 50 values (table 1 and table s1 ). the compounds tested against mers-cov in cell culture (7a, 6c, 7e, 7h, and 6j) also displayed submicromolar ec 50 values. among these compounds, 6j showed the most potent antiviral activity against mers-cov with an ec 50 value of 0.04 m. gc376 with a p 2 leu residue and a nonfluorinated benzyl cap exhibited 20-fold lower potency against mers-cov in cell culture compared to compound 6j ( table 2) . the compounds were also effective against sars-cov-2 with ec 50 values ranging from 0.15 to 0.9 m in vero e6 cells (table 2) . these compounds were also found to be potent against fipv and mhv, with ec 50 values ranging from 0.07 to 0.22 m. in the fret enzyme assay, these compounds were active against the 3clpro of sars-cov and sars-cov-2 ( table 1 ). the ic 50 values of these compounds against sars-cov-2 3clpro ranged from 0.17 to 0.82 m. among these compounds, 6e showed the most potent antiviral ac-tivity against sars-cov-2 in the enzyme assay (ic 50 , 0.17 m) and cell-based assay (ic 50 , 0.15 m) (tables 1 and 2) . gc376 also exhibited activity against the 3clpro of sars-cov-2 with an ic 50 value of 0.62 m in the enzyme assay ( table 1) . the antiviral effects of compounds 6j and 6e against sars-cov-2 were confirmed in cultured primary human airway epithelial cells from three donors. in the absence of a 3clpro inhibitor, viral titers in the infected cultured primary human airway epithelial cells reached 10 7.3 (donor 1), 10 7.1 (donor 2), and 10 8.4 (donor 3) plaque-forming units (pfu) per milliliter of culture medium. in the presence of compound concentrations that were about two-to threefold higher than the ec 50 values obtained in vero e6 cells (2 m, 6j or 0.5 m, 6e), viral titers were reduced to 10 6.4 (donor 1), 10 6.1 (donor 2), and 10 6.3 (donor 3) pfu/ml for compound 6j or 10 6.1 (donor 1), 10 6.5 (donor 2), and 10 8.1 (donor 3) pfu/ml for compound 6e (table 3) . although there was some variation in viral replication among infected cells from the three donors (especially donor 3), the antiviral effects of both 6j and 6e were evident at the tested concentrations. for infected cells from donors 1 and 2, both compounds inhibited viral replication about 10-fold at the tested concentrations. for infected cells from donor 3, 6e inhibited virus replication about 50%, whereas 6j inhibited virus replication 100-fold at the tested concentrations. we determined multiple high-resolution cocrystal structures of compounds 6b, 6d, 6g, 6h, 7i, or 7j with the 3clpro of mers-cov (fig. 2 , a to f, and figs. s1 to s3). these inhibitors bound to the active site of mers-cov 3clpro, demonstrating that the vicinity of the s 4 pocket is encompassed by an array of primarily hydrophobic residues, including phe 188 , val 193 , ala 171 , and leu 170 (fig. 2 , c and f, and fig. s3 ). hydrophobic and hydrogen-bonding functionalities were incorporated into the 3clpro inhibitors to capture additional interactions, and the position of the cyclohexyl moiety was also examined using appropriate congeners. the bisulfite adducts reverted to the corresponding aldehydes, which subsequently reacted with cys 148 to form nearly identical covalent complexes with a tetrahedral arrangement at the newly formed stereocenter (fig. 2 , a to f, and figs. s1 to s3). the backbone of compound 6h (table 1 and fig. 2 , a to c) engaged in h-bond interactions with amino acid residues gln 192 , gln 167 , and glu 169 . three additional side-chain h-bonds between the -lactam ring and his 166 , phe 143 , and glu 169 also were clearly evident (fig. 2b) fig. 1 . synthesis scheme for compound series 6a to 6k and 7a to 7k. stepwise compound synthesis with intermediate compounds is shown for 3c-like protease (3clpro) inhibitors of the 6a to 6k and 7a to 7k series. the alcohol inputs were reacted with (l) leucine isocyanate methyl ester or (l) cyclohexylalanine isocyanate methyl ester to yield products, which were then hydrolyzed to the corresponding acids with lithium hydroxide in aqueous tetrahydrofuran. subsequent coupling of the acids to glutamine surrogate methyl ester "8" furnished compounds "4". lithium borohydride reduction yielded alcohols "5", which were then oxidized to the corresponding aldehydes "6" with dess-martin periodinane reagent. the bisulfite adducts "7" were generated by treatment with sodium bisulfite in aqueous ethanol and ethyl acetate. a amino acid methyl ester isocyanate/tea/ch 3 was ensconced in the hydrophobic s 2 pocket (fig. 2c) . the extra methylene group in compound 7j, which was converted to an aldehyde and thus became identical to 6j, resulted in the reorientation of the difluorocyclohexyl group and the formation of three h-bonds between gln 195 and ala 171 and the fluorine atoms, with concomitant loss of one of the gln 192 hydrogen bonds and the displacement of phe 143 (fig. 2e ). the substitution of the p 2 leu with p 2 cyclohexylalanine (compound 7i; table s1) resulted in the loss of an h-bond with gln 192 but otherwise adopted the same interactions as observed for compound 6h (fig. s2a ). the electron density, hydrogen bond interactions, and electrostatic surface representations for mers-cov 3clpro in complex with compounds 6b, 6g, and 6d are shown in figs. s1 to s3. next, compound 7j was cocrystallized with sars-cov or sars-cov-2 3clpro, and the structures were compared to that for mers-cov 3clpro and 7j. in the sars-cov 3clpro-7j complex (fig. 2 , g to i), the backbone of compound 7j formed direct h-bonds with cys 145 , his 163 , his 164 , glu 166 , and gln 189 . compound 7j also formed an additional h-bond with his 41 and a water-mediated contact with gly 143 (fig. 2h ). however, there was a loss of the three h-bonds between gln 195 and ala 171 and the fluorine atoms, compared to the cocrystal structure of 7j with mers-cov 3clpro. the electron density map was consistent with both possible enantiomers at the new stereocenter formed by covalent attachment of the s atom of cys 145 in the cocrystal structure of sars-cov 3clpro with 7j. the electron density map for compound 7j in complex with sars-cov-2 3clpro was most consistent with a single enantiomer, although it adopted a similar binding mode and hydrogen bond interactions as observed in the sars-cov 3clpro-7j cocrystal structure (fig. 2 , j to l). superposition of compound 7j with mers-cov 3clpro, sars-cov 3clpro, and sars-cov-2 3clpro ( fig. s4 ) revealed a very similar binding mode for 7j among all three viral proteases. the most potent compound of the series, 6j, was identified in a cellbased assay and had an ec 50 value of 0.04 m against mers-cov (fig. 3a) . we determined the efficacy of compounds 6j and 6h in transgenic hdpp4-ki mice expressing human dipeptidylpeptidase 4, a model of mers-cov infection. first, hdpp4-ki mice were infected with the mouse-adapted mers-cov (mers ma -cov) virus strain and then were treated with compounds 6h and 6j (50 mg/kg per day, once a day) or vehicle as a control starting 1 day post virus infection (1 dpi) and continuing until 10 dpi. all mice treated with vehicle control died by 8 dpi (fig. 3b ). in contrast, 40% of mice treated with compound 6h survived, and all mice treated with compound 6j were alive at the end of the study (15 dpi) (fig. 3b ). the survival of mice treated with compound 6j or 6h was increased compared to the vehicle control (p < 0.05), and the 6j-treated mice had an improved survival rate compared to 6h-treated mice (p < 0.05). all mice treated with compound 6j rapidly recovered from body weight loss starting at 3 dpi (fig. 3c ). the mice that survived after 6h treatment continued to lose body weight until 6 dpi but then started to gain weight from 9 dpi (fig. 3c ). after we observed that treatment with compound 6j resulted in the survival of mers ma -cov-infected hdpp4-ki mice, we conducted another study by delaying treatment initiation until 3 dpi. similar to the first study, no untreated mice or mice given vehicle (control) survived, and there was no statistical difference between these two groups (0% survival) (fig. 3d ). when 6j treatment was started on 1 dpi, four of the five mice survived (80% survival), and there was a statistically significant increased survival in mice treated starting at 1 dpi compared to untreated or vehicle control-treated mice (p < 0.05; fig. 3d ). when 6j treatment was delayed by one additional day (2 dpi), the survival of mice treated with 6j decreased to 40%, but this was still higher than the 0% survival for untreated or vehicle controltreated mice. however, there was no statistical difference between the 6j treatment group starting at 2 dpi and the untreated or vehicle control-treated groups (fig. 3d ). treatment with 6j starting at 3 dpi also failed to improve the survival of mice compared to the untreated or vehicle control-treated groups (fig. 3d ). all mice lost body weight after virus infection, but surviving mice treated with 6j regained the lost weight by 15 dpi (fig. 3e ). recovery of body weight was faster in mice treated with 6j starting at 1 dpi than table 3 . antiviral effects of compounds 6j and 6e against sars-cov-2 in cultured primary human airway epithelial cells. the study was a single experiment, and viral titers were measured in duplicate with a plaque-forming assay. vehicle control at 2 dpi (fig. 3e) . these results show that survival of mice markedly increased when 6j was given at 1 dpi. the lung pathology caused by mers ma -cov infection of hdpp4-ki mice resembles that seen in severe cases of human mers-cov infection, with diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and infiltration of lymphocytes into the alveolar septa (22) . a group of hdpp4-ki mice were infected with mers ma -cov and treated with compound 6j or vehicle as a control starting at 1 dpi. mouse lungs were collected for determination of virus load at 3 and 5 dpi and for histopathology at 6 dpi. lung virus titers decreased in the 6j-treated mice compared to control mice at both 3 and 5 dpi (p < 0.01) (fig. 4a) . edema in the lungs of the treated mice was reduced compared to control mice (p < 0.01) (fig. 4b) . scores for hyaline membrane formation were reduced in 6j-treated mice but were not statistically different from control mice. mers ma -cov-infected hdpp4-ki mice treated with vehicle showed patches in lung tissue variably composed of cellular inflammation, vascular congestion, and atelectasis (fig. 4 , c and e). the airways of these animals were generally intact, with only scattered, uncommon sloughed cells (fig. 4 , c and e). in some lungs from these mice, lymphatic vessels were filled with degenerative cells and cellular debris (fig. 4 , c and e). alveolar edema was detected in some lung tissue sections (fig. 4 , c and e). in contrast, there were few observed lesions in the lungs of mers ma -cov-infected hdpp4-ki mice treated with compound 6j starting at 1 dpi (fig. 4 , d and f). there are currently no approved vaccines or small-molecule therapeutics for the treatment of mers-cov, sars-cov, or sars-cov-2 infection. however, numerous preventive and therapeutic options are under development (3) (4) (5) (6) (7) . the most clinically advanced antiviral compound with a broad spectrum of activity is remdesivir (gs-5734). this nucleoside analog was originally developed as an antiviral drug against ebola virus and has been shown to be effective against both mers-cov and sars-cov in cell culture assays and in animal models of coronavirus infection (23) (24) (25) (26) . prophylactic treatment or early therapeutic treatment of infected mice with remdesivir reduced mers-cov-or sars-cov-mediated weight loss and decreased lung virus titers and lung injury scores compared to those of vehicle-treated animals (23, 26) . remdesvir also showed potent activity against sars-cov-2 in cell culture assays and animal models (27) and was recently issued an emergency use authorization by the u.s. food and drug administration as an investigational antiviral drug for covid-19. another nucleoside analog, eidd-2801, which is a broad-spectrum inhibitor against multiple viruses including influenza viruses, was also shown to be effective against mers-cov and sars-cov in mouse models (28) . our group has been engaged in the discovery of broad-spectrum inhibitors targeting the 3clpro of multiple human and animal coronaviruses. we initially generated dipeptidyl and tripeptidyl series of compounds (29) and observed that the dipeptidyl compound series had superior pharmacokinetic (pk) profiles compared to the tripeptidyl compound series (20) . a representative compound of the dipeptidyl series is gc376, which is currently in clinical development for fip in cats and covid-19. the pk characteristics of multiple dipeptidyl compounds similar to compound 6j, including gc376, were examined through an intraperitoneal or subcutaneous administration in animals, including mice. it was determined that their maximum plasma concentration (c max ) values were >100-fold of the ec 50 for the target virus and that the elimination half-life (t 1/2 ) was 3 to 5 hours. the in vivo efficacy of gc376 against mice infected with mhv or murine norovirus has been demonstrated (11, 15) . inspection of previously obtained crystal structures of the dipeptidyl compounds in a complex with coronavirus 3clpro (17, 19) revealed the potential to achieve enhanced binding interactions with the s 4 subsite by introducing diverse functionalities at the cap position in the inhibitors. in the current study, a new dipeptidyl series focusing on the design of structural variants in the cap substructure were synthesized and evaluated for their activity against coronavirus 3clpro. the ec 50 of gc376 against mers-cov was determined to be ~1 m. one of our goals was to generate compounds with near or below 0.1 m potency against mers-cov and other target coronaviruses. all synthesized compounds displayed varying degrees of inhibitory activity against multiple coronaviruses in the fret enzyme assay and cell-based assays. among these compounds, 6e showed the most potent antiviral activity against sars-cov-2 3clpro in a fret enzyme assay (ic 50 , 0.17 m) and cell-based assays (ec 50 , 0.15 m), and 6j showed the most potent antiviral activity against mers-cov with an ec 50 value of 0.04 m (table 2) . it was previously demonstrated that optimal potency is attained when the p 1 and p 2 residues are a glutamine surrogate and leu, respectively, and that replacement of the p 2 leu with a cyclohexylalanine is inimical to potency (17, 18) . this is clearly evident when comparing the relative potencies of 6h and 7h versus 6i and 7i (table 1 and table s1 ). furthermore, compounds with leu at the p 2 position showed higher cc 50 values compared to those with cyclohexylalanine at p 2 (table s1). x-ray crystallography confirmed the mechanism of action of the inhibitors, which involves formation of a covalent bond between the active site cysteine and the carbonyl carbon of the aldehyde. x-ray crystallography also identified the structural determinants associated with binding, accounting for the observed differences in potency. the high-resolution cocrystal structures of 3clpro inhibitors 7j and 7i with mers-cov 3clpro also confirmed that the difference in activity arose from the loss of a h-bond with gln 192 and the loss of two additional h-bonds from the displacement of gln 167 and phe 143 with 7i ( fig. s2a ). the nature of the interaction of 7j with the s 4 subsite is unique among the compounds examined and provides strong support for our approach, vis-à-vis our focus on the cap position for enhancing binding affinity and potency. compound 7j was cocrystallized with mers-cov, sars-cov, and sars-cov-2 3clpro. superposition of compound 7j with these 3clpro enzymes revealed a similar binding mode among all three proteases. however, a key difference lies with the different conformation adopted by the difluorocyclohexyl ring in the mers-cov 3clpro s 4 subsite, enabling it to engage in additional h-bond binding interactions (fig. 2e and fig. s4 ). compound 7j had a moderately lower potency against sars-cov 3clpro and sars-cov-2 3clpro compared to mers-cov 3clpro in the fret enzyme assay, suggesting that moieties forming h-bonds that were accommodated at the s 4 subsite had an important impact on potency. the barrier to the development of drug resistance increases when an inhibitor engages in h-bond interactions with the backbone of the 3clpro. we used a robust mouse model of mers-cov infection (30-32) to evaluate the efficacy of compounds 6j and 6h. hdpp4-ki mice expressing human dipeptidylpeptidase 4 were infected with a mouseadapted mers-cov virus (mers ma -cov). the infected mice develop fatal lung disease with severe inflammation and weight loss (32) . furthermore, the lung pathology caused by mers ma -cov infection of the hdpp4-ki mice closely resembles that of severe human mers-cov infection and is characterized by diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and infiltration of lymphocytes into the alveolar septa (22) . in the current study, we demonstrated that survival rates in this mouse model were higher with 6j treatment compared to 6h treatment (fig. 3b) . compounds 6j and 6h share a near-identical structure except for the extra methylene group present in compound 6j. compound 6h showed potent anti-3clpro activity, whereas the antiviral activity of compound 6h in cell culture was lower than that of compound 6j (tables 1 and 2) , which may have been the reason for its diminished therapeutic efficacy in the mouse model (fig. 3b) . our findings indicate that therapeutic treatment of infected mice with compound 6j was associated with a reduction in lung viral load and lung pathology (fig. 4) . moreover, treatment of mice with 6j at 1 dpi resulted in the survival of infected mice, whereas delaying treatment initiation until 3 dpi resulted in decreased survival. overall, mouse survival was markedly increased only when 6j was given to mice at 1 dpi (p < 0.05; fig. 3 , d and e). treatment with compound 6j starting at 2 dpi resulted in moderately increased survival of infected mice, but this was not statistically significant (p > 0.05). these results emphasize the importance of early therapeutic intervention in attaining a positive clinical outcome. earlier studies from our group showed that gc376 can cure fatal feline coronavirus disease in cats (20, 21) , demonstrating that a specific coronavirus protease inhibitor can be effective therapeutically against coronavirus disease in a natural host. the mers-cov mouse model used here provides proof of principle regarding the therapeutic potential of our protease inhibitors for treating severe human respiratory coronavirus disease. limitations of the current study include differences in host receptor usage, mortality, and transmissibility between mers-cov and sars-cov-2. thus, further evaluation of our protease inhibitors in mice, hamsters, or nonhuman primates experimentally infected with sars-cov-2 will be crucial to assess these inhibitors as potential therapeutic options for covid-19. our study showed that these compounds were broadly active against the 3clpro of several coronaviruses, with compound 6j displaying the highest activity against mers-cov and compound 6e displaying the highest activity against sars-cov-2. clinical efficacy is influenced by many factors, including drug bioavailability, pk, metabolism, and the chemical stability of a compound. this poses a major challenge with respect to reliably predicting whether the difference in potency against different coronaviruses in assays in vitro can be translated to differences in clinical efficacy. therefore, further research is needed to establish whether one protease inhibitor can be an effective therapeutic for both mers-cov and sars-cov-2 infections in humans. the results from our previous and current studies suggest that the dipeptidyl compound series can serve as a platform suitable for the structure-guided design of one or more inhibitors against highly virulent human coronaviruses. we have generated potent inhibitors of the 3clpro of several coronaviruses, including sars-cov-2, and tested their efficacy in cultured cells and primary human airway epithelial cells. furthermore, we have demonstrated proof-of-concept therapeutic efficacy for one 3clpro inhibitor 6j in hdpp4-ki mice infected with mers ma -cov. our study lays the foundation for advancing this compound series further along the drug development pipeline. the goal of this study was to evaluate the efficacy of 3clpro inhibitors against human coronaviruses, including sars-cov-2, in a fret enzyme assay and cell culture assays, as well as in a mouse model of mers-cov infection. initial antiviral screening was performed with recombinant 3clpro from sars-cov, mers-cov, and sars-cov-2 in the fret enzyme assay. antiviral activity was then assessed in cultured huh-7 cells infected with mers-cov and vero e6 cells infected with sars-cov-2. selected 3clpro inhibitors were further examined using x-ray cocrystallization with mers-cov, sars-cov, and sars-cov-2 3clpro to elucidate the mechanism of action and identify the structural determinants of potency. last, two selected compounds were evaluated for in vivo efficacy in a mouse model of mers-cov infection (hdpp4-ki mice expressing human dipeptidylpeptidase 4 infected with a mouse-adapted mers-cov). age-and sex-matched mice were randomly assigned into various groups for virus infection and treatment studies. microscopic analysis of lung lesions was conducted in a blinded manner; other experiments were not blinded. no mice were excluded from analysis. in vivo studies were performed in animal biosafety level 3 facilities at the university of iowa. all experiments were conducted under protocols approved by the institutional animal care and use committee at the university of iowa according to guidelines set by the association for the assessment and accreditation of laboratory animal care and the u.s. department of agriculture. the studies with mers-cov and sars-cov-2 were performed in biosafety level 3 facilities at the university of iowa. all experiments were conducted under protocols approved by the institutional biosafety committee at the university of iowa according to guidelines set by the biosafety in microbiological and biomedical laboratories, the u.s. department of health and human services, the u.s. public health service, the u.s. centers for disease control and prevention, and the national institutes of health. compounds 6a to 6k and 7a to 7k were readily synthesized as illustrated in fig. 1 and are listed in table 1 and table s1 . briefly, the alcohol inputs were reacted with l-leucine isocyanate methyl ester or l-cyclohexylalanine isocyanate methyl ester to yield dipeptides "2", which were then hydrolyzed to the corresponding acids with lithium hydroxide in aqueous tetrahydrofuran. the subsequent coupling of the acids to glutamine surrogate methyl ester "8" (33, 34) furnished compounds "4". lithium borohydride reduction yielded alcohols "5", which were then oxidized to the corresponding aldehydes "6" with dess-martin periodinane reagent. the bisulfite adducts "7" were generated by treatment with sodium bisulfite in aqueous ethanol and ethyl acetate (35) . the expression and purification of the 3clpro of mers-cov and sars-cov were performed by a standard method described previously by our lab (11, 19, 20) . we also cloned and expressed the 3clpro of sars-cov-2. the codon-optimized complementary dna (cdna) of the full length of 3clpro of sars-cov-2 (genbank accession number mn908947.3) fused with sequences encoding six histidine at the n terminus was synthesized by integrated dna (coralville, ia). the synthesized gene was subcloned into the pet-28a(+) vector. the expression and purification of sars-cov-2 3clpro were conducted after a standard procedure described by our lab (19) . briefly, stock solutions of compounds 6a to 6k and 7a to 7k were prepared in dimethyl sulfoxide (dmso) and diluted in assay buffer, which was composed of 20 mm hepes buffer (ph 8) containing nacl (200 mm), edta (0.4 mm), glycerol (60%), and 6 mm dithiothreitol (dtt). the protease (3clpro of mers-cov, sars-cov, or sars-cov-2) was mixed with serial dilutions of each compound or with dmso in 25 l of assay buffer and incubated at 37°c for 30 min (mers-cov) or at room temperature for 1 hour (sars-cov and sars-cov-2), followed by the addition of 25 l of assay buffer containing substrate (fam-savlq/sg-qxl 520, anaspec, fremont, ca). the substrate was derived from the cleavage sites on the viral polyproteins of sars-cov. fluorescence readings were obtained using an excitation wavelength of 480 nm and an emission wavelength of 520 nm on a fluorescence microplate reader (flx800, biotek, winooski, vt) 1 hour after the addition of substrate. relative fluorescence units (rfu) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously (19) . the dosedependent fret inhibition curves were fitted with a variable slope by using graphpad prism software (graphpad, la jolla, ca) to determine the ic 50 values of the compounds. some compounds in the 6a to 6k and 7a to 7k series were also investigated for their antiviral activity against the replication of mers-cov, fipv, or mhv-1 in huh-7, crfk, or l929 cells, respectively (19) . briefly, medium containing dmso (<0.1%) or each compound (up to 100 m) was added to confluent cells, which were immediately infected with viruses at a multiplicity of infection (moi) of 0.01. after incubation of the cells at 37°c for 24 hours, viral titers were determined with the median tissure culture infectious dose (tcid 50 ) method (fipv or mhv) with the crfk or l929 cells or plaque assay with vero81 cells (mers-cov). for sars-cov-2, confluent vero e6 cells were inoculated with ~50 to 100 pfu per well, and medium containing various concentrations of each compound and agar was applied to the cells. after 48 to 72 hours, plaques in each well were counted. ec 50 values were determined by graph-pad prism software using a variable slope (graphpad, la jolla, ca) (19) . to confirm that these inhibitors also inhibit sars-cov-2 in primary human cells, differentiated human airway epithelial cells from three donors were used as previously described (36, 37) . two compounds (6j and 6e) were tested for their antiviral effects against sars-cov-2. briefly, airway epithelial cells were washed with phosphate-buffered saline (pbs), and sars-cov-2 was inoculated at a moi of 0.1 for 1 hour. after the inoculum was removed, media containing 6j (2 m) or 6e (0.5 m) was added. after 48 hours, cells were subjected to a freeze/thaw cycle, and virus titers were determined by plaque assay on vero e6 cells. the 50% cytotoxic concentration (cc 50 ) for compounds 6a to 6k and 7a to 7k was determined in huh-7, crfk, or l929 cells. confluent cells grown in 96-well plates were incubated with various concentrations (1 to 100 m) of each compound for 72 hours. cell cytotoxicity was measured by a cytotox 96 nonradioactive cytotoxicity assay kit (promega, madison, wi), and the cc 50 values were calculated using a variable slope by graphpad prism software. the in vitro therapeutic index was calculated by dividing the cc 50 by the ec 50. protein purification, crystallization, and data collection in x-ray crystallographic studies mers-cov 3clpro and sars-cov 3clpro were purified as described previously (17, 19) . an escherichia coli codon-optimized construct encoding residues ser 3264 to phe 3568 of the orf1ab polyprotein (sars-cov-2 3clpro, genebank accession number qhd43415.1) was cloned into a pet his6 sumo tev lic cloning vector (2s-t, addgene). expression and initial ni-column purification were performed as described for mers-cov 3clpro and sars-cov 3clpro. the small ubiquitin-like modifier protein (sumo) fusion elution fractions of sars-cov-2 were dialyzed against 20 mm tris (ph 8.0) and 100 mm nacl and treated with the tobacco etch virus (tev) protease (1:10, w/w) overnight. this mixture was loaded onto a 5-ml histrap hp column (ge healthcare) equilibrated with 20 mm tris (ph 8.0) and 100 mm nacl and eluted with 20 mm tris (ph 8.0), 100 mm nacl, and 500 mm imidazole using an äkta pure fast protein liquid chromatography (fplc). the flow-through fractions containing sars-cov-2 3clpro without the sumo fusion were loaded onto a superdex 75 10/300 gl size exclusion column equilibrated with 20 mm tris (ph 8.0) and 100 mm nacl. the fractions were pooled and concentrated to 9.6 mg/ml for crystallization screening. note that four residues from cloning (ser-asn-ile-gly) remain at the n terminus after treatment with tev protease. purified mers-cov 3clpro, sars-cov 3clpro, and sars-cov-2 3clpro in 100 mm nacl and 20 mm tris (ph 8.0) were concentrated to 10.6 mg/ml (0.3 mm), 22 mg/ml (0.64 mm), and 9.6 mg/ml (0.28 mm), respectively, for crystallization screening. all crystallization experiments were set up using an nt8 drop-setting robot (formulatrix inc.) and uvxpo mrc (molecular dimensions) sitting-drop vapor diffusion plates at 18°c. one hundred nanoliters of protein and 100 nl of crystallization solution were dispensed and equilibrated against 50 l of the latter. stock solutions (100 mm) of compounds 6b, 6d, 6g, 6h, 7i, and 7j were prepared in dmso, and complexes were generated by mixing 1 l of the ligand (2 mm) with 49 l (0.29 mm) of the protease and incubating on ice for 1 hour. crystals of the mers-cov 3clpro inhibitor complexes were obtained from the following conditions: compounds 6b, 6d, 6g intensities were integrated using xds (38, 39) using autoproc (40) , and the laue class analysis and data scaling were performed with aimless (41) . structure solution was conducted by molecular replacement with phaser (42) (46), respectively. disordered side chains were truncated to the point for which electron density could be observed. structure validation was conducted with molprobity (47) , and figures were prepared using the ccp4mg package (48) . crystallographic data are provided in table s2. the two best compounds (6j and 6h) in the series were examined for their in vivo efficacy using 10-week-old male hdpp4-ki mice infected with mers ma -cov (30) . in the first study, animals were divided into three groups (n = 5 to 6) and were lightly anesthetized with ketamine/xylazine and infected with 50 l of 750 pfu mers ma -cov via intranasal inoculation. compound 6j or 6h were formulated in 10% ethanol and 90% peg400 and given to mice from 1 to 10 dpi at 50 mg/kg per day (once per day) via intraperitoneal administration. the control mice received vehicle. animals were weighed daily and monitored for 15 days. animals were euthanized when an animal lost 30% of initial weight or at 15 dpi. in the next study, treatment with compound 6j was delayed up to 3 dpi to determine the impact of delayed treatment on mouse survival. animals were divided into five groups (n = 5), and compound 6j (50 mg/kg per day, once per day) was administered to mice starting at 1, 2, or 3 days after virus challenge (1, 2, or 3 dpi, respectively) until 10 dpi. mice were monitored for weight loss and survival as described above for 15 days after virus challenge. as controls, vehicle (10% ethanol and 90% peg400) was administered equivalently to the experimental compound, or animals received no treatment (untreated). the third study was conducted to assess the effects of therapeutic treatment of compound 6j in the lungs. for lung harvest and virus titration, animals were divided into three groups (n = 4) of mice, and compound 6j (50 mg/kg per day, once per day) or vehicle was administered to mice starting at 1 dpi until euthanasia. animals were euthanized at 3 or 5 dpi, and lungs were removed aseptically, disassociated with a manual homogenizer in 1× pbs, briefly centrifuged, and supernatants removed. samples were titered on vero81 cells as reported elsewhere (49) . for lung histopathology analyses, animals were divided into two groups (n = 5), and compound 6j (50 mg/kg per day, once per day) or vehicle was administered to mice starting at 1 dpi for 5 days. mice were euthanized at 6 dpi, lungs were fixed with 10% formalin, and hematoxylin and eosinstained tissues were examined by a veterinary pathologist using the postexamination method of masking (50) . briefly, tissues were scored in an ordinal manner for edema and hyaline membrane formation using the following scale: 0, none; 1, rare (<5 alveoli); 2, <33% of lung fields; 3, 34 to 66% lung fields; and 4, >66% lung fields (30) . the analysis of survival curves in groups was performed using a log-rank (mantel-cox) test and a gehan-breslow-wilcoxon test using graphpad prism software (san diego, ca). log-transformed viral titers in the lungs and lung edema and hyaline membrane formation in groups of mice were analyzed with multiple t tests using graphpad prism software. stm.sciencemag.org/cgi/content/full/12/557/eabc5332/dc1 materials and methods table s1 . compound ic50 in the fret enzyme assay and cc50 in a cell culture assay for mers-cov 3clpro. table s2 . cocrystal structure data for compounds with the 3clpro of mers cov and sars cov. data file s1. individual-level data for all figures and tables. references (51) (52) (53) (54) (55) view/request a protocol for this paper from bio-protocol. genomic characterisation and epidemiology of 2019 novel 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infection depend on differentiation of human airway epithelia automatic indexing of rotation diffraction patterns data processing and analysis with the autoproc toolbox an introduction to data reduction: space-group determination, scaling and intensity statistics phaser crystallographic software n y times web china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china towards automated crystallographic structure refinement with phenix.refine features and development of coot molprobity: all-atom structure validation for macromolecular crystallography developments in the ccp4 molecular-graphics project rapid generation of a mouse model for middle east respiratory syndrome principles and approaches for reproducible scoring of tissue stains in research scaling and assessment of data quality improved r-factors for diffraction data analysis in macromolecular crystallography global indicators of x-ray data quality resolving some old problems in protein crystallography linking crystallographic model and data quality we thank rudragouda channappanavar for performing the initial assays demonstrating drug efficacy against mers-cov. we thank d. george for technical assistance. key: cord-342220-lrqt2gcw authors: dearlove, bethany; lewitus, eric; bai, hongjun; li, yifan; reeves, daniel b.; joyce, m. gordon; scott, paul t.; amare, mihret f.; vasan, sandhya; michael, nelson l.; modjarrad, kayvon; rolland, morgane title: a sars-cov-2 vaccine candidate would likely match all currently circulating variants date: 2020-09-22 journal: proc natl acad sci u s a doi: 10.1073/pnas.2008281117 sha: doc_id: 342220 cord_uid: lrqt2gcw the magnitude of the covid-19 pandemic underscores the urgency for a safe and effective vaccine. many vaccine candidates focus on the spike protein, as it is targeted by neutralizing antibodies and plays a key role in viral entry. here we investigate the diversity seen in severe acute respiratory syndrome coronavirus 2 (sars-cov-2) sequences and compare it to the sequence on which most vaccine candidates are based. using 18,514 sequences, we perform phylogenetic, population genetics, and structural bioinformatics analyses. we find limited diversity across sars-cov-2 genomes: only 11 sites show polymorphisms in >5% of sequences; yet two mutations, including the d614g mutation in spike, have already become consensus. because sars-cov-2 is being transmitted more rapidly than it evolves, the viral population is becoming more homogeneous, with a median of seven nucleotide substitutions between genomes. there is evidence of purifying selection but little evidence of diversifying selection, with substitution rates comparable across structural versus nonstructural genes. finally, the wuhan-hu-1 reference sequence for the spike protein, which is the basis for different vaccine candidates, matches optimized vaccine inserts, being identical to an ancestral sequence and one mutation away from the consensus. while the rapid spread of the d614g mutation warrants further study, our results indicate that drift and bottleneck events can explain the minimal diversity found among sars-cov-2 sequences. these findings suggest that a single vaccine candidate should be efficacious against currently circulating lineages. the magnitude of the covid-19 pandemic underscores the urgency for a safe and effective vaccine. many vaccine candidates focus on the spike protein, as it is targeted by neutralizing antibodies and plays a key role in viral entry. here we investigate the diversity seen in severe acute respiratory syndrome coronavirus 2 (sars-cov-2) sequences and compare it to the sequence on which most vaccine candidates are based. using 18,514 sequences, we perform phylogenetic, population genetics, and structural bioinformatics analyses. we find limited diversity across sars-cov-2 genomes: only 11 sites show polymorphisms in >5% of sequences; yet two mutations, including the d614g mutation in spike, have already become consensus. because sars-cov-2 is being transmitted more rapidly than it evolves, the viral population is becoming more homogeneous, with a median of seven nucleotide substitutions between genomes. there is evidence of purifying selection but little evidence of diversifying selection, with substitution rates comparable across structural versus nonstructural genes. finally, the wuhan-hu-1 reference sequence for the spike protein, which is the basis for different vaccine candidates, matches optimized vaccine inserts, being identical to an ancestral sequence and one mutation away from the consensus. while the rapid spread of the d614g mutation warrants further study, our results indicate that drift and bottleneck events can explain the minimal diversity found among sars-cov-2 sequences. these findings suggest that a single vaccine candidate should be efficacious against currently circulating lineages. sars-cov-2 | evolution | vaccine s evere acute respiratory syndrome coronavirus 2 (sars-cov-2), the virus that causes covid-19, is a member of the coronaviridae family, a diverse group of virus species, seven of which are known to infect humans. four are considered endemic and typically cause mild upper respiratory illnesses; two of these, nl63 and 229e, are within the alphacoronavirus genus, and two, hku1 and oc43, are betacoronaviruses. the latter genus comprises the three highly pathogenic human coronaviruses, including sars-cov-2, as well as middle eastern respiratory syndrome (mers) cov and severe acute respiratory syndrome (sars) cov. sars-cov is the most closely related human virus to sars-cov-2, which is a single-stranded positive-sense rna virus, with an ∼30,000-base pair genome. the genome is split into 10 open reading frames (orfs) that include 16 nonstructural proteins and four structural proteins. the latter category includes spike (s), membrane (m), envelope (e), and nucleocapsid (n). s is the basis for most candidate vaccines, as it mediates virus attachment and entry to host cells and is the target of neutralizing antibody responses (1) (2) (3) (4) . s is cleaved into two subunits, s1 and s2: the former contains the receptor binding domain (rbd), which enables the virus to attach to the angiotensin-converting enzyme 2 (ace2) receptor on host cells. in the span of 7 months, the covid-19 pandemic has caused a devastating global health crisis with significant mortality and socioeconomic implications. as of july 23, 2020, more than 15 million cases and 622,000 attributable deaths have been reported worldwide (5-8) (https://coronavirus.jhu.edu/map.html). phylogenetic analyses suggest that sars-cov-2 is likely derived from a clade of viruses found in horseshoe bats (9) . in s, the bat genome ratg13 has more than 97% amino acid identity with sars-cov-2 (6) . interestingly, the rmyn02 sequence, which is the closest to sars-cov-2 in the long orf1ab but more distant than ratg13 in s, showed the insertion of multiple amino acids at the cleavage site between the s1 and s2 subunits of the s protein (this s1/s2 insertion is a characteristic feature of sars-cov-2) (10). highly similar sequences, especially in the rbd, were also identified in malayan pangolins (11, 12) , emphasizing the plasticity of coronavirus genomes and their propensity to switch hosts. although the closest currently available bat sequences are fairly divergent from sars-cov-2, their characteristics (insertion at s1/s2 cleavage site, high diversity, and similarity between specific gene fragments and particular strains) together with their known adaptive properties (high recombination and host-switching rates and evidence of positive selection) support that these bat viruses constitute the rapid spread of the virus causing covid-19, sars-cov-2, raises questions about the possibility of a universally effective vaccine. the virus can mutate in a given individual, and these variants can be propagated across populations and time. to understand this process, we analyze 18,514 sars-cov-2 sequences sampled since december 2019. we find that neutral evolution, rather than adaptive selection, can explain the rare mutations seen across sars-cov-2 genomes. in the immunogenic spike protein, the d614g mutation has become consensus, yet there is no evidence of mutations affecting binding to the ace2 receptor. our results suggest that, to date, the limited diversity seen in sars-cov-2 should not preclude a single vaccine from providing global protection. a generalist lineage where a specific virus is likely the natural origin of sars-cov-2. we did not study the transmission of the virus from its animal reservoir and focused our analysis on the evolution of sars-cov-2 since its introduction in humans. while the scale of the pandemic attests to the high transmissibility of sars-cov-2 between humans, with a basic reproduction number r 0 estimated to be 2.2 (95% ci, 1.4 to 3.9) in wuhan, china (13), we wanted to investigate evidence of further adaptation of sars-cov-2 to its host, as adaptive processes could interfere with vaccine efficacy. developing a vaccine against sars-cov-2 is a high priority for preventing and mitigating future waves of the pandemic (14) . vaccine candidates typically include an insert that corresponds to one or more virus antigens, either derived computationally or from one or multiple sequence(s) sampled from infected individuals. the first viral sequence derived during the covid-19 outbreak, wuhan-hu-1 (available from the global initiative on sharing all influenza data, gisaid, accession epi_isl_402125), was published on january 9, 2020. as many vaccine programs were initiated at that time, it is likely that this sars-cov-2 sequence, sampled in december 2019 in wuhan, china, is the foundation for many vaccine candidates currently in development. compared to other rna viruses, coronaviruses have a more complex molecular machinery resulting in higher replication fidelity. early evolutionary rate estimates for sars-cov-2 were ∼1 × 10 −3 substitutions per nucleotide per year (15), a rate comparable to that observed during the sars-cov-1 outbreak (16) and in the range for other rna viruses (1 × 10 −3 to 1 × 10 −5 substitutions per nucleotide per year) (17) . while the evolutionary rate is likely to decrease over time (18) , it is important to monitor the introduction of any mutation that may compromise the potential efficacy of vaccine candidates derived from the first available sars-cov-2 sequences. new mutations will be observed as the virus spreads in humans. the viral evolutionary dynamics can be characterized by analyzing viral sequences sampled from individuals who became infected. the accumulation of mutations can be a marker of viral fitness: an increase in viral fitness as the virus adapts to its host will be associated with pervasive mutations at specific sites, whereas a neutral evolution context will be associated with a minimal number of fixed mutations distributed stochastically across the genome. indicators of viral evolution have been shown to be robust predictors of transmission dynamics for several pathogens, such as influenza (19) , lassa (20) , and ebola (21) viruses. typically, the evolution of a virus is driven by genotypic and phenotypic changes in its surface protein. in the case of sars-cov-2, mutations in s are most likely to confer fitness to the virus as it adapts to humans. however, adaptive changes can occur in structural and nonstructural proteins, and these changes, as well as different patterns across structural and nonstructural proteins, may provide insights into the near-and long-term evolutionary dynamics of sars-cov-2, as it spreads in humans. here we analyzed sars-cov-2 sequences sampled since the beginning of the pandemic and found that mutations were rare, indicating that potential vaccine candidates should cover all circulating variants. limited diversity across 18,514 sars-cov-2 genomes. to characterize sars-cov-2 diversification since the beginning of the epidemic, we aligned 27,977 sars-cov-2 genome sequences isolated from infected individuals in 84 countries. the alignment was curated to retain independent sequences that covered over 95% of the orfs. in addition, because sequences from the united kingdom constituted 47% of the dataset (n = 12,157), we sampled a representative set of 5,000 uk sequences, yielding a final dataset of 18,514 sars-cov-2 genomes (si appendix, fig. s1 and fig. 1a ). there were 7,559 polymorphic sites (that is, sites where at least one sequence has a change relative to the reference sequence) across the genome (total length: 29,409 nucleotides). most substitutions were found in a single sequence; only 8.41% (n = 2,474) of the polymorphic sites showed substitutions in two or more sequences (fig. 1b) . only 11 mutations were found in >5% of sequences, and only 7 were found in >10% of sequences (3 of which were adjacent). the mean pairwise diversity across genomes was 0.025%, ranging between 0.01% for e to 0.11% for n. a phylogenetic tree reconstructed based on all genome sequences reflected the global spread of the virus: samples from the first 6 wk of the outbreak were collected predominantly from china (fig. 1c) . as the epidemic has progressed, samples have been increasingly obtained across europe and from the united states ( fig. 1 a and c) . the tree shows numerous introductions of different variants across the globe, with introductions from distant locations seeding local epidemics, where infections sometimes went unrecognized for several weeks and allowed wider spread (23) . prior to the severe travel restrictions that were seen in march 2020, intense travel patterns between china, europe, and the united states allowed transmission of a myriad of variants, which is currently reflected by different lineages in the tree. yet, the tree topology shows minimal structure, even at the genome level, indicating that sars-cov-2 viruses have not diverged significantly since the beginning of the pandemic. to compare how genomes differed from one another, we calculated hamming distances (which correspond to the number of differences between two genomes) across all pairs of sequences. these hamming distances showed a narrow distribution, with a median of seven substitutions between two independent genomes, while linked sequences sampled in cruise ships had a median of two substitutions (si appendix, fig. s2) . surprisingly, hamming distances across genomes sampled in the united states did not show a similar quasinormal distribution but instead a bimodal distribution, observed despite the large number of sequences compared (n = 5,398). we identified that this bimodal distribution was driven by sequences from washington state, possibly reflecting separate introductions in that state. nonetheless, such a bimodal distribution could also indicate a bias in the sampling of sequences (si appendix, fig. s2 ). one s mutation (d614g) has become dominant. since the beginning of the pandemic, two mutations across the genome have become consensus: p4715l in orf1ab (nucleotide 14,143, c to t) and d614g in s (nucleotide 23,403, a to g) ( fig. 1b ) (a third consensus mutation, at nucleotide 3,037, is not reported as the site was masked during our sequence-filtering procedure). these mutations were found in 69.3% and 69.4% of sequences, respectively, and are in linkage (fig. 2b ). given the importance of s for virus entry and as a target of the host neutralizing response, the biologic implications of the d614g mutation are under intense scrutiny (24) (25) (26) (27) (28) . this mutation was first observed in a sequence from china dated january 24, with seven more sequences sampled until february 8. then, the d614g mutation was not observed in china until march 13. in contrast, the d614g mutation was introduced in europe at the end of january (first sequence identified in germany, dated january 28), and it rapidly became dominant on that continent and at every location where the virus subsequently spread ( fig. 2a) . the phylogenetic tree (fig. 2b ) and the distribution of sequences (fig. 2c ) are suggestive of a founder effect. the rapid spread of sequences carrying the d614g mutation implies that the growing viral population should become more homogeneous, that is, the frequency of sequences with shared polymorphisms will increase. we found a median of seven substitutions (based on a comparison of 18,514 sequences) between two independent sars-cov-2 genomes (si appendix, fig. s2 ). yet, genomes with the d614g mutation showed a median of five substitutions, whereas those with d at position 614 differed by eight substitutions (fig. 2d) . to test whether this site was under selection, we used likelihood-based, phylogenetically informed models that assume branch-specific substitution rates (29) and implemented a sampling strategy to circumvent computational limitations imposed by the large number of sequences. subsampled alignments (100 times at a 10% sampling fraction) had diversity estimates statistically similar to the complete alignment for each gene (mann−whitney u test, p > 0.09; si appendix, fig. s3 ). in s, only site 614 was estimated to be under diversifying selection in a majority of subsampled alignments (58%); evidence of diversifying selection indicates that genetic diversity increases in the viral population (i.e., there was a higher proportion of mutations causing an amino acid change than not at site 614, or, the nonsynonymous/synonymous substitution rates ratio, dn/ds, was over 1, p < 0.1) (si appendix, fig. s4 ). because diversifying selection is often associated with the host adaptive response, we considered whether the d614g mutation coincided with targets of antibody and t cell responses. site 614 is at the interface between the s1 and s2 subunits and is thus not highly accessible to antibodies (si appendix, fig. s5 ). therefore, we predict that antibodies to the native s protein would cross-react with s containing the d614g mutation, in agreement with recent reports (24, 25, 27, 28) . many known neutralizing antibodies target the rbd, yet we found little evidence that mutations could affect binding to the ace2 receptor, as only five shared mutations were identified at contact sites with the ace2 receptor, and all were found in 10 or fewer sequences. of these, one mutation, at position 489, was synonymous and found in three sequences (0.02%). the others were nonsynonymous: g476s (n = 10 sequences, 0.05%), y453f (n = 5, 0.02%), g446v (n = 3, 0.02%), and a475v (n = 2, 0.01%). to predict the potential immune pressure linked to t cell responses, we developed a t cell immunogenicity index which takes into account the cd8 and cd4 epitope repertoires in the structural proteins of sars-cov-2 (s, n, m, e) and the frequency of human leukocyte antigen (hla) alleles or haplotypes in a given population. we found that sites with mutations, including 614 in s, were not colocalized with t cell epitopes frequently identified in different populations (si appendix, figs. s6 and s7), and there was no significant relationship between the number of mutations and the immunogenicity index (si appendix, fig. s8 ). most sites in the sars-cov-2 genome were under purifying selection. using phylogenetically informed models (as described above), we identified two sites, residue 614 in s and 13 in n, that were under diversifying selection in a majority of subsampled alignments. for each protein, subsampled alignments tended to have more sites under purifying selection (median = 7.34 ± 4.06% [±sd]) than under diversifying selection (3.10 ± 1.92%) (mann−whitney u test, p = 0.057; si appendix, fig. s4 ) (purifying selection is indicative of a decrease in genetic diversity in the population). likewise, for each codon separately, the proportion of each phylogeny (i.e., the percentage of total branch length) with dn/ds > 1 was small, indicating diversifying selection was episodic and limited (fig. 3a) . global measures of dn/ ds varied across genes, ranging from 0.35 ± 0.02 (m) to 1.43 ± 0.24 (orf10), and were significantly lower for structural genes compared to nonstructural genes (mann−whitney u test, p = 0.042) (fig. 3b) . per-lineage nonsynonymous substitution rates were comparable (student's t test, p = 0.218) in structural (0.0011 ± 0.021) and nonstructural (0.0012 ± 0.028) genes, although some subsampled alignments showed rates that could be a hundred times higher than the median over all alignments (fig. 3c) . across structural proteins, mutations were disproportionately neutral: >70.3% of branch length evolved under neutral (or negative) selection for all sites, and over half of all branch length evolved under neutral (or negative) selection for >82.8% of sites (fig. 3d ) (29) . no evidence of differentiation of the viral population. while there was only limited evidence of diversification at selected sites, we also assessed whether subpopulations among the globally circulating viral population had become genetically differentiated over time. to do so, we used two measures of population differentiation, the g st and d statistics, which characterize changes in allele frequency across populations and can show fitness differences between subpopulations (30) (31) (32) . genetic distances between two subpopulations can range between 0 and 1, indicating no and complete differentiation, respectively. we initially compared 30 genomes sampled from the initial outbreak in wuhan, china, with subsampled alignments of the 18,484 genomes sampled subsequently across the globe. although distances varied across genes, the median genetic distance between these subpopulations was small for both g st (0.0049 ± 0.0047) and d (0.0053 ± 0.0272), indicating little differentiation between the initial outbreak and its global derivatives in the pandemic (fig. 4a) . we then compared subpopulations sampled before and after each consecutive week. similarly, genetic distances between subpopulations were small for both g st (0.0058 ± 0.0096) and d (0.0098 ± 0.0650) and tended to narrow over time rather than diverge (fig. 4 b and c) . signatures of host adaptation can also be seen in the branching patterns of viral phylogenies. bursts in transmissibility are emblematic of increases in relative viral fitness and are reflected in imbalances in the phylogeny, which can be estimated at each internal node (si appendix, figs. s9 and s10) (33) (34) (35) . we estimated phylogenetic η (36, 37) at each internal node of the sars-cov-2 phylogeny reconstructed from subsampled (10%) alignments and compared the distribution of estimates through time to phylogenies simulated under models of neutral and positive time-dependent rates (b(t) = be α(t) ). simulation analyses demonstrated that this metric was robust against sampling fraction (si appendix, fig. s10 ). the distribution of η in the sars-cov-2 phylogenies adhered to expectations of the neutral model and deviated significantly (student's t test, p < 0.001) from those of positive timedependent rates for selection coefficients α ≥ 0.2 ( fig. 4 d and e) . together, the sars-cov-2 population and phylogenetic dynamics showed little evidence that the global spread of sars-cov-2 was related to viral fitness effects. sequence identity with potential vaccine candidates. typical vaccine design strategies rely on either 1) selecting sequences sampled from infected individuals or 2) computationally deriving sequences that cover the diversity seen across circulating sequences and are, in theory, optimal compared to an individual isolate (38) . computationally derived sequences include consensus and ancestral sequences, such as the most recent common ancestor (mrca) of a set of sequences. we inferred the mrca corresponding to 1) sars-cov-2 s sequences sampled from wuhan within the first month of the epidemic, 2) all currently circulating sars-cov-2 sequences, and 3) all sars-cov-2 sequences together with closely related sequences sampled from pangolins (n = 6) and a bat. there were 17 mutations between the human mrca and the human−bat mrca and 44 mutations between the human mrca and human−pangolin mrca. overall, three segments in s reflected significant variability across species (aa 439 to 445, 482 to 501, and 676 to 690) ( fig. 5 a and c and si appendix, fig. s11 ) (39) . in contrast, when considering only human sequences, sars-cov-2 diversity was limited: both mrcas (derived from early sequences from wuhan or from all circulating sequences) were identical to the initial reference sequence wuhan-hu-1. comparing these sequences to the consensus sequence derived from all of the sequences sampled to date, there was only one mutation: d614g (fig. 5b) . fig. 5d illustrates that mutations found across circulating s sequences were rare: besides d614g (found in 69.4% of sequences), the next most frequent substitution is found in 1.96% of sequences (synonymous), with sequences sampled from infected individuals, on average, 0.55 mutations away from the consensus sequence (consisting of 0.12 synonymous and 0.43 nonsynonymous mutations). across the genome, there were, on average, 4.05 nucleotide mutations per individual genome when compared to the consensus, with only p4715l and d614g found in >50% of sequences. there remains an urgent need for a sars-cov-2 vaccine as a primary countermeasure to mitigate and eventually contain the spread of covid-19. the virus's s glycoprotein makes an attractive vaccine target because it plays a key role in mediating virus entry and is known to be immunogenic (40) . neutralizing antibody responses against s have been identified in sars-cov-2−infected individuals (2), and several clinical trials for a sars-cov-2 vaccine will test s as an immunogen. while we focused on s, our comparative analyses of other proteins yielded similar conclusions: a randomly selected sars-cov-2 sequence could be used as a vaccine candidate, given the similarity of any sequence to the computationally derived optimum vaccine candidate (as defined by the mrcas or consensus sequence based on all circulating sequences). vaccines developed using any of these sequences should, theoretically, be effective against all circulating viruses. vaccine developers could consider designing a vaccine insert with the d614g mutation in s, as this mutation has become dominant worldwide. while mutations that become fixed are often linked to the host immune pressure, this seems unlikely for the sars-cov-2 mutation d614g. because this residue lies at the interface between two subunits, it would not be expected to be part of a critical epitope for vaccine-mediated protection (fig. 4 ). as such, pseudoviruses with d614g were as susceptible to neutralization as those with the initial residue d614 (25) . a mutation, s612l, that emerged in mers-cov after passaging the virus in the presence of two antibodies (in 5/15 clones after 20 passages) (41) warrants the evaluation of the analogous d614g mutation in sars-cov-2 for its ability to interfere with the recognition of a distal epitope. a more direct path to viral escape from antibody recognition would be mutations in the rbd, as described for influenza (42, 43) . importantly, we found no mutation in the rbd that was present in more than 1% of sars-cov-2 sequences (highest frequency was 0.2% n439k); such rare variants are unlikely to interfere with vaccine efficacy. in the context of rare sars-cov-2 mutations, the rapid spread of the d614g mutation is singular and has led authors to hypothesize that viruses with d614g may have enhanced fitness (24) . the strongest evidence of a biological effect for this mutation comes from recent reports of an increase in in vitro infectivity or cell entry for pseudoviruses with d614g (25-28). additional work is needed to evaluate whether the increase in infectivity in vitro translates to increased transmissibility (spread) of sars-cov-2 across humans, as there is not necessarily a linear relationship between the two. for example, sars-cov mediates cell entry more efficiently than sars-cov-2 (with or without the d614g s mutation) (26) . hence, it would be important to understand whether, controlling for epidemiological factors, there are higher reproduction numbers associated with viruses carrying the d614g mutation. while a preliminary comparison of the lineages with either d or g in washington state did not indicate an obvious advantage for d614g mutants, as they found similar maximal values for the effective reproduction number (https:// github.com/blab/ncov-wa-phylodynamics), additional comparisons in different geographic locations should be informative. correlating in vitro findings with clinical phenotypes can be complicated. during the ebola outbreak of 2013-2016, some fixed mutations were suspected to confer an advantage to the virus. specifically, an a82v mutation in the glycoprotein, which, like s for sars-cov-2, is critical for the virus entry into host cells, was associated with an increase in infectivity (44) (45) (46) . yet, effects varied across cell types (47) , and no phenotypic differences were associated with the mutations when viruses were evaluated in vivo in mouse and nonhuman primate models (48) , highlighting the difficulty in linking biological mechanisms to outcomes at the population level. so far, no causal association has been identified between the presence of d614g and disease severity (24) . these findings, together with our results, illustrate that mutations can spread through the population without necessarily having a selective advantage, especially at the beginning of an epidemic when most individuals are susceptible. mutations occur more frequently after a host switch, and even slightly deleterious mutations may have an opportunity to spread. hence, the main signal in our study was one of purifying selection that can ultimately eliminate mildly deleterious mutations. our analyses showed limited evidence of diversifying selection, with comparable substitution rates in structural proteins versus nonstructural proteins (under a selection paradigm, structural proteins which are essential for viral entry and the target of the host immune response would have higher rates than the nonessential proteins), low estimates of genetic differentiation following the sites with mutations are shown as spheres. (b) near-identity of potential vaccine candidates. the mrca and wuhan-hu-1 reference sequences were identical, while the consensus derived from all circulating sequences showed a mutation (d614g). site 614 is located at the interface between two subunits. (c) sequence segments that differed between human and pangolin or bat hosts. amino acid segments 439 to 455 and 482 to 501 impact receptor binding, while the 574 to 690 segment corresponds to the s2 cleavage site. (d) sites with shared mutations across sars-cov-2 circulating sequences. the colors of the spheres correspond to the proportion of sars-cov-2 sequences that differed from the wuhan-hu-1 sequence (gisaid: epi_isl_402125, genbank: nc_045512). mutations that were found only in one or two sequences were not represented. initial outbreak, and phylogenetic patterns adhering to a neutral process of evolution. these data indicate that epidemiologic factors could be sufficient to explain the global spread of mutations such as d614g. a founder effect means that these mutations were likely exported to sars-cov-2 naive areas early in the outbreak and therefore given the opportunity to spread widely. as such, on january 28, 2020, a virus carrying the d614g mutation, which was rare among sequences from china, was identified in germany. host and environmental factors permitted the establishment of a sustained cluster of infections that propagated this mutation until it became dominant among european sequences and then globally (fig. 2) . we found no evidence that the frequent identification of this mutation was caused by convergent selection events that would have occurred in multiple individuals. further analyses are needed to characterize the biologic mechanisms behind the spread of the d614g mutation. in summary, our results indicate that, so far, sars-cov-2 has evolved through a nondeterministic, noisy process and that random genetic drift has played a dominant role in disseminating unique mutations throughout the world. yet, it is important to note that founder effects do not exclude that the d614g can confer distinguishing properties in terms of protein stability, infectivity, or transmissibility. sars-cov-2 was only recently identified in the human population-a short time frame relative to adaptive processes that can take years to occur. although we cannot predict whether adaptive selection will be seen in sars-cov-2 in the future, the key finding is that sars-cov-2 viruses that are currently circulating constitute a homogeneous viral population. viral diversity has challenged vaccine development efforts for other viruses such as hiv-1, influenza, or dengue, but these viruses each constitute a more diverse population than sars-cov-2 viruses (si appendix, fig. s12 ). we can therefore be cautiously optimistic that viral diversity should not be an obstacle for the development of a broadly protective sars-cov-2 vaccine, and that vaccines in current development should elicit responses that are reactive against currently circulating variants of sars-cov-2. sequence data. sequences were downloaded from gisaid (https://www. gisaid.org/). a full list, along with the originating and submitting laboratories (gisaid_acknowledgment_table_20200518.xls), is available at https:// www.hivresearch.org/publication-supplements. sequence processing and filtering. all sars-cov-2 sequences available on gisaid as of may 18, 2020 (n = 27,989) were downloaded and deduplicated where possible, and those missing accurate dates (that is, only recording the month and/or year) were removed. sequences were processed using the biostrings package (version 2.48.0) in r (49) . sequences known to be linked through direct transmission were removed, and only the sample with the earliest date (chosen at random when multiple samples were taken on the same day) was retained. sequences were then aligned with mafft v7.467 using the -addfragments option to align to the reference sequence (wuhan-hu1, gisaid accession epi_isl_402125) (50) . insertions relative to wuhan-hu-1 were removed, and the 5′ and 3′ ends of sequences (where coverage was low) were excised, resulting in an alignment consisting of the 10 orfs. any sequences with less than 95% coverage of the orfs (i.e., >5% gaps) were removed, and 30 homoplasic sites likely due to sequencing artifacts identified by de maio et al. were masked (https://github.com/w-l/ problematicsites_sars-cov2/blob/master/archived_vcf/problematic_sites_ sarscov2.2020-05-27.vcf). to identify individual sequences that were much more divergent than expected, given their sampling date, which likely reflected sequencing artifacts rather than evolution, we obtained a tree using fasttree v2.10.1 compiled with double precision under the general time reversible (gtr) model with gamma heterogeneity (51) . this tree was rooted at the reference sequence, and root-to-tip regression was performed following tempest using the ape package in r (52, 53) . outliers were defined as sequences that had studentized residuals greater than 3, and were removed. sequences from the united kingdom corresponded to nearly half of the sequences (n = 12,157/25,671, 47%) of this filtered dataset. to avoid overrepresentation of the uk sequences and bias in subsequent analyses, we investigated the effect of downsampling sequences on the mean hamming distance and identified the minimum number of sequences required to recover the mean corresponding to the full distribution (si appendix, fig. s1 ). a subsample of 5,000 sequences satisfied these criteria, and also ensured that there were fewer sequences from the united kingdom than from the united states (n = 5,398), reflecting the epidemiology. these 5,000 sequences were sampled randomly, with weight proportional to the number of uk sequences collected on that day. after these filtering steps, the alignment used for subsequent analyses included 18,514 sequences. global phylogeny and evolution. the global phylogeny was reconstructed in fasttree v2.10.1 compiled with double precision under the gtr model with gamma heterogeneity (51) , and rooted at the reference sequence. the tree was visualized using ggtree in r (54) . lineages were defined using pan-golin (phylogenetic assignment of named global outbreak lineages), with lineages with >200 taxa as of the may 19 summary being highlighted in the tree (22) (https://github.com/cov-lineages/lineages). the number of polymorphic sites was calculated as the number of sites which had at least one mutation relative to the reference sequence, wuhan-hu-1, ignoring gaps and ambiguities. pairwise distance comparisons. for each pair of sequences, we calculated the hamming distance as the number of sites that are different after removing sites with ambiguities and/or gaps. for computational efficiency, given the size of the alignment, this was implemented in parallel in c++, using bazel (https://bazel.build/) to build on a linux system. this implementation is available to download at https://www.hivresearch.org/publicationsupplements. subsampling gene alignments. alignments for each gene were subsampled for sequence and phylogenetic analyses. each gene alignment was randomly subsampled 100 times per collection date at 5%, 10%, 20%, 30%, and 40%. when fewer than 10 sequences were available for a collection date, all sequences were taken. median hamming distances were computed for each set of subsampled alignments. these were bootstrapped 100,000 times, and 95% cis were estimated and compared to the median hamming distance for the fully sampled alignment. global and site-specific nonsynonymous and synonymous substitution rates. alignments subsampled at 10% 100 times were used to estimate substitution rates. for the set of subsampled alignments for each gene, a mixed-effect likelihood method was used to estimate nonsynonymous (dn) and synonymous (ds) substitution rates globally and at each codon (29) . maximumlikelihood phylogenies were constructed for each alignment using the software iq-tree (55) under a best-fit model determined with modelfinder (56) to prime the dn and ds estimates before branch length optimization. this step serves to expedite the optimization process. branch length optimization was done with a mg94 model [which is the only model available for this analysis (29) ]. the proportion of each phylogeny evolving under neutral (or negative) selection was determined from the mixture density across lineages for each site, assuming different dn and ds along each branch (57) . on the same set of subsampled alignments and phylogenies, a fixed-effects likelihood method was used on internal branches to identify sites under pervasive diversifying selection and to estimate global dn/ds (58) . known biases associated with calculating dn/ds on exponentially growing populations (59) were counterbalanced by subsampling phylogenies, as the typical approach to address this bias, which is to ignore terminal branches, would considerably diminish the power of the analysis to detect any significant result. as p values from the fixed-effect likelihood method are uncorrected, results were not averaged over p values; rather, given that p value calculations are conservative for this analysis (58), sites were considered to be under pervasive diversifying selection if their p value was <0.1 in ≥50% of alignments, which would account for a typical 5% false discovery rate (58) . global and gene-specific population differentiation. alignments subsampled at 10% 100 times were used to estimate population differentiation. the genetic differentiation of subpopulations within sampled sequences was calculated on each gene separately using nei's (30) g st . because comparisons between subpopulations of different sizes can bias genetic differentiation estimates (60), genetic differentiation was also calculated using jost's (31) d, which accounts for differences in genetic heterogeneity between subpopulations and is intended to correct for biases in the size of the subpopulations. both statistics were computed with the mmod package (32) in r (v3.6.1). for each gene, statistics were calculated over 100 bootstrapped samples for each subsampled alignment. subpopulations were defined in two ways. first, sequences originating from the initial outbreak in the hubei province (30 sequences) were compared to all other sequences within a subsampled alignment. second, a 1-wk sliding window was designed to compare all sequences sampled prior to a collection date (subpopulation 1) to all sequences sampled after the same collection date (subpopulation 2). the first collection date for subpopulation 1 was february 14, 2020, the week after the last sequence from the hubei province was sampled (february 8, 2020), the window was designed to terminate when <30 sequences were available in subpopulation 2. time-dependent estimates of phylogenetic diversification. time-dependent estimates of phylogenetic diversification were measured by extracting the branches descending from each internal node (above the root) of each phylogeny and calculating the peak height (η) of the spectral density profile of the graph laplacian of each subtree, which is a measure of the density of branching events (36, 37) . the code to perform the analysis is available for download at https://www.hivresearch.org/publication-supplements. simulation analyses. phylogenies were simulated using a time-forward branching process under constant birth rates (b(t) = b) and timedependent birth rates (b(t) = be αt ) for b = 0.01, 0.03, 0.05, 0.07, and 0.09 and α = ±0.01, ±0.11, ±0.21, ±0.31, and ±0.41, for 20, 220, 420, 620, and 820 tips, and for 1, 11, 21, 31, and 41 time units. simulated phylogenies were downsampled at 0%, 10%, 30%, 50%, and 70%. for each scenario, 100 phylogenies were simulated. time-dependent diversification (i.e., η across subtrees) was calculated for each phylogeny simulated under each scenario. simulations were conducted using the r packages rpanda (r phylogenetic analyses of diversification) (61) and ape (53) . comparisons to sars-cov-2 phylogeny. phylogenies downsampled at 10% from the full (18,514 tips) sars-cov-2 genome phylogeny (following the subsampling strategy described above) were used to calculate the phylogenetic η for each subtree (above the root) for each downsampled phylogeny. neutral phylogenies were simulated under stochastic branching by randomly sampling from the distribution of branch lengths from one downsampled sars-cov-2 phylogeny. this was iterated across all downsampled sars-cov-2 phylogenies. positive time-dependent phylogenies were simulated using a time-dependent process (b(t) = 0.01e αt ) for α = 0.001, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 1, with branch lengths restricted to the distribution of branch lengths from one downsampled sars-cov-2 phylogeny. this was iterated across all downsampled sars-cov-2 phylogenies for each α. neutral and positive time-dependent phylogenies were simulated with a 10% sampling fraction. polytomies were randomly resolved. simulations were conducted using the r packages rpanda (61) and ape (53) . ancestral s protein sequence reconstruction. ancestral s protein sequences were reconstructed from an amino acid alignment of 30 sars-cov-2 sequences sampled from the hubei province, a coronavirus sampled from bat (yunnan ratg13), and six sars-cov-2-like coronaviruses sampled from pangolins using maximum posterior probability and returning a unique residue at each site assuming a jones-taylor-thornton (jtt) model with gamma heterogeneity (62) . the jtt model was the most appropriate model available in the software (62) . the bat sequence was retrieved from gen-bank, and the pangolin sequences were retrieved from gisaid (63) . a sliding window of 10 amino acids (and a step of 1 amino acid) was used to compare the cumulative number of mutations in the human−bat and human− bat−pangolin ancestors with respect to the human ancestral sequence. median values for each window were compared to a null window (computed as a normal distribution of 10 values with a mean equal to the mean value across the entire s protein, 0.046 mutations) using a one-tailed t test. an alignment including the reconstructed sequences is available at https:// www.hivresearch.org/publication-supplements. prediction of cd4+ and cd8+ t cell epitopes. cd4+ and cd8+ t cell epitopes were predicted for four sars-cov-2 structural proteins: s (accession yp_009724390), n (accession yp_009724397), m (accession yp_009724393), and e (accession yp_009724392). cd4+ t cell epitopes were predicted using a server that predicts binding of peptides to any mhc molecule of known sequence using artificial neural networks, netmhciipan 4.0 (64) with a peptide length of 15. mhc class ii hla alleles of hla-dqb1, plus the haplotypes of hla-dpa1-dpb1 and hla-dqa1-dpb, were selected for predictions if they had frequencies of >1/1,000 in known allele/haplotype distributions (http://17ihiw.org/17th-ihiw-ngs-hla-data/). if multiple peptides had the same core, the peptide with the strongest binding score was selected for analysis. cd8+ t cell epitopes were predicted using netmhcpan 4.1 (64) with a peptide length of 9. mhc class i hla alleles of hla-a, hla-b, and hla-c were selected if they were classified as common (frequency ≥ 1/ 10,000) in any of the populations in the database ciwd 3.0 (common, intermediate and well-documented hla alleles in world populations) (65) . epitopes predicted as strong binders (with predicted binding affinities below 50 nm) were selected for analyses. t cell immunogenicity index. for each site in a predicted epitope, the immunogenicity index was defined as the sum of the frequency of the hla alleles or haplotypes restricting the corresponding epitope (multiple epitopes can be predicted at a given site in a protein). total frequencies from ciwd 3.0 were used as the frequencies of the corresponding mhc class i hla alleles (hla-a, hla-b, and hla-c), and the global frequencies from http://17ihiw. org/17th-ihiw-ngs-hla-data/ were used as the frequencies of the corresponding mhc class ii hla alleles or haplotypes (hla-dqb1, hla-dpa1-dpb1, and hla-dqa1-dpb). this procedure was repeated using the frequencies of mhc alleles or haplotypes in different subpopulations listed in the above hla frequency dataset. statistical analyses. for comparisons of mean values in normally distributed data, student's t test was used. when data were not normal, the mann−whitney u test was used. shapiro−wilk tests were used to determine normality. differences in data distributions were estimated using the kolmogorov−smirnov test. data availability. data and code are available at https://www.hivresearch.org/ publication-supplements. all study data are included in the article and si appendix. characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients cross-reactive antibody response between sars-cov-2 and sars-cov infection a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention an interactive web-based dashboard to track covid-19 in real time origin and cross-species transmission of bat coronaviruses in china a novel bat coronavirus closely related to sars-cov-2 contains natural insertions at the s1/s2 cleavage site of the spike protein identifying sars-cov-2-related coronaviruses in malayan pangolins isolation of sars-cov-2-related coronavirus from malayan pangolins early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia projecting the transmission dynamics of sars-cov-2 through the postpandemic period temporal signal and the phylodynamic threshold of sars-cov-2 moderate mutation rate in the sars coronavirus genome and its implications rates of evolutionary change in viruses: patterns and determinants analyses of evolutionary dynamics in viruses are hindered by a time-dependent bias in rate estimates a predictive fitness model for influenza viral hemorrhagic fever consortium, clinical sequencing uncovers origins and evolution of lassa virus ebola virus epidemiology, transmission, and evolution during seven months in sierra leone a dynamic nomenclature proposal for sars-cov-2 to assist genomic epidemiology the emergence of sars-cov-2 in europe and the us tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity naturally mutated spike proteins of sars-cov-2 variants show differential levels of cell entry structural and functional analysis of the d614g sars-cov-2 spike protein variant d614g mutation of sars-cov-2 spike protein enhances viral infectivity detecting individual sites subject to episodic diversifying selection analysis of gene diversity in subdivided populations gst and its relatives do not measure differentiation mmod: an r library for the calculation of population differentiation statistics predicting evolution from the shape of genealogical trees measuring asymmetry in time-stamped phylogenies a metric on phylogenetic tree shapes characterizing and comparing phylogenies from their laplacian spectrum a non-parametric analytic framework for within-host viral phylogenies and a test for hiv-1 founder multiplicity diversity considerations in hiv-1 vaccine selection structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars vaccines: where are we? importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on the middle east respiratory syndrome coronavirus spike glycoprotein to avoid neutralization escape antibody landscapes after influenza virus infection or vaccination mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin biochemical basis for increased activity of ebola glycoprotein in the 2013-16 epidemic functional mutations in spike glycoprotein of zaire ebolavirus associated with an increase in infection efficiency functional characterization of adaptive mutations during the west african ebola virus outbreak naturally occurring single mutations in ebola virus observably impact infectivity recently identified mutations in the ebola virus-makona genome do not alter pathogenicity in animal models biostrings: efficient manipulation of biological strings mafft multiple sequence alignment software version 7: improvements in performance and usability fasttree 2-approximately maximum-likelihood trees for large alignments exploring the temporal structure of heterochronous sequences using tempest (formerly path-o-gen) schliep, ape 5.0: an environment for modern phylogenetics and evolutionary analyses in r two methods for mapping and visualizing associated data on phylogeny using ggtree iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies mod-elfinder: fast model selection for accurate phylogenetic estimates a random effects branch-site model for detecting episodic diversifying selection not so different after all: a comparison of methods for detecting amino acid sites under selection the population genetics of dn/ds rpanda: an r package for macroevolutionary analyses on phylogenetic trees a fast algorithm for joint reconstruction of ancestral amino acid sequences data, disease and diplomacy: gisaid's innovative contribution to global health netmhcpan-4.1 and netmhciipan-4.0: improved predictions of mhc antigen presentation by concurrent motif deconvolution and integration of ms mhc eluted ligand data common, intermediate and well-documented hla alleles in world populations: ciwd version 3.0.0 acknowledgments. we gratefully acknowledge the authors and originating and submitting laboratories of the sequences from gisaid's epicov database on which this research is based. we thank lionel condé, robert gramzinski, joshua herbeck, mélanie merbah, thembi mdluli, lydie trautmann, douglas whalin, and suzanne wollen-roberts. we also thank two reviewers for critical improvements of the original manuscript. key: cord-333326-n9ifhw5s authors: wardell, hanna; campbell, jeffrey i; vanderpluym, christina; dixit, avika title: severe acute respiratory syndrome coronavirus 2 infection in febrile neonates date: 2020-07-09 journal: j pediatric infect dis soc doi: 10.1093/jpids/piaa084 sha: doc_id: 333326 cord_uid: n9ifhw5s most severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infections in pediatric patients are mild or asymptomatic. however, infants have emerged at higher risk of hospitalization and severe outcomes in pediatric coronavirus disease 2019 (covid-19). we report a case series of 4 full-term neonates hospitalized with fever and found to have sars-cov-2 infection with a spectrum of illness severities. two neonates required admission to the intensive care unit for respiratory insufficiency and end organ involvement. half of the patients were found to have a coinfection. one neonate received antiviral therapy with remdesivir and is, to our knowledge, the youngest patient to receive this drug for covid-19. all neonates had favorable outcomes. the majority of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infections in children are mild or asymptomatic. however, infants have been observed to have a higher risk of hospitalization and severe outcomes in pediatric coronavirus disease 2019 . herein we present a case series of 4 full-term neonates who were hospitalized with fever and found to be infected with sars-cov-2. a 19-day-old full-term previously healthy male infant presented with 1 day of fever and fussiness. he was born via normal spontaneous vaginal delivery at 41 weeks' gestation following an uncomplicated pregnancy. his mother had a history of genital infection with herpes simplex virus (hsv) for which she received prophylactic valacyclovir throughout pregnancy. he resides with his mother, father, 4 siblings, and maternal grandmother and her partner. the latter 2 had recently been diagnosed with covid-19 1 week prior to his presentation, and the grandmother required hospitalization. the mother also reported upper respiratory symptoms concurrently but had not been tested. upon presentation, his vital signs were notable for lack of fever (37.4°c, rectal), with normal heart rate (134 beats per minute [bpm]), respiratory rate (30 breaths per minute), blood pressure (95/39 mm hg), and oxygen saturation (97% in room air). his physical examination was normal, notable for an open, soft, and flat anterior fontanelle, good tone, and nonlabored breathing with lungs clear to auscultation bilaterally. he underwent a full neonatal sepsis evaluation, with laboratory evaluation significant for elevated procalcitonin but otherwise unremarkable (table 1) . sars-cov-2 by reversetranscription polymerase chain reaction (rt-pcr) from a nasopharyngeal (np) swab was positive. a chest radiograph was normal. he was initiated on empiric therapy with ampicillin, gentamicin, and acyclovir. empiric antimicrobials were discontinued when blood, urine, and cerebrospinal fluid (csf) cultures and hsv pcr from csf resulted negative at 48 hours. however, just prior to discharge the patient developed respiratory distress with tachypnea, suprasternal and subcostal retractions, and tracheal tugging. he was not hypoxemic but was placed on 0.25 l supplemental oxygen via nasal cannula for comfort. a chest radiograph showed new increased bilateral opacities without focal consolidation and a prominent cardiac silhouette. an electrocardiogram (egg) was obtained and demonstrated normal sinus rhythm with a right axis deviation. laboratory evaluation was significant for an elevated highsensitivity troponin t (58 ng/l [ref: 0-14 ng/l]) and n-terminal portion of pro-brain natriuretic peptide (bnp) (1260 pg/ml [ref: 0-450 pg/ml]). due to concern for evolving myocardial injury, he was transferred to our hospital for further management on inpatient day 3. on arrival, he was admitted to the step-down intensive care unit (icu) where he was noted to be afebrile with normal vital signs including oxygen saturation of 98% on 0.25 l via nasal cannula. physical examination was significant for intermittent mottling while crying, but overall well appearance with good distal perfusion, strong pulses, and a normal cardiac examination. an echocardiogram exhibited normal anatomy with mildly depressed left ventricular function, estimating an ejection fraction of 49% (z score -2.8). laboratory evaluation was delayed secondary to persistent clot formation of all peripheral blood draws, requiring placement of a peripherally inserted central catheter. laboratory studies ultimately demonstrated down-trending troponin t (0.04 ng/ml) and bnp (105 pg/ml), elevated d-dimer (0.8 μg/ml), with normal prothrombin time, partial thromboplastin time, and fibrinogen. rt-pcr from an np swab was negative for influenza a/b, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, and parainfluenza viruses 1-4. multidisciplinary evaluation commenced and he was felt not to meet criteria for multisystem inflammatory syndrome in children potentially associated with covid-19 [1] . due to the concern for end-organ involvement with possibly evolving acute myocardial injury as well as a supplemental oxygen requirement, the patient was initiated on therapy with remdesivir on inpatient day 4 via an expanded-access program from the manufacturer after approval from the us food and drug administration and local institutional review board, with informed consent. he received an initial loading dose of 5 mg/ kg via intravenous administration, followed by 2.5 mg/kg daily. inflammatory and coagulation studies were initially trended daily, and on hospital day 5 d-dimer continued to rise (0.99 μg/ml) prompting initiation of enoxaparin (0.8 mg/kg daily) for thromboprophylaxis. further evaluation by thromboelastography with platelet mapping was consistent with hypercoagulability, with an elevated clot strength (74.5 mm [ref: 50-70 mm]) and g-parameter (14.6 kg/sec [ref: <11 kg/sec]), for which he was started on low-dose aspirin (20.25 mg daily) on hospital day 6. he subsequently tolerated discontinuation of supplemental oxygen. two follow-up echocardiograms remained stable, with unchanged mildly depressed left ventricular systolic function. cardiac markers normalized, and d-dimer declined although remained elevated. the patient received a total of 7 doses of remdesivir, which he tolerated well, with stable creatinine and liver function tests throughout therapy. enoxaparin was discontinued on the final day of hospitalization, and he was discharged home on inpatient day 9 to continue daily low-dose aspirin. at 3 weeks postdischarge, an echocardiogram demonstrated normal systolic function and ejection fraction (z score -1.4). laboratory evaluation was normal aside from persistently elevated d-dimer (0.6 μg/ml) and clot strength (80 mm). he remains on aspirin with close monitoring. notably all 3 of the patient's siblings have tested positive for sars-cov-2 infection since his hospitalization. a 24-day-old full-term male infant presented with fever, lethargy, and decreased oral intake. he was born via scheduled maximum respiratory support repeat cesarean delivery and briefly required phototherapy for neonatal hyperbilirubinemia following birth, but was otherwise healthy. he lives with both parents. several days before his presentation, his father developed fever, muscle aches, and malaise, but had not undergone sars-cov-2 testing. upon developing symptoms, the infant presented to the emergency department (ed) where he was found to be lethargic, febrile (temperature 38.3°c), and tachycardic (heart rate 180-200 bpm) with normal respiratory rate and oxygen saturation. a full sepsis evaluation was initiated, with laboratory evaluation significant for mild leukopenia and lymphopenia, elevated procalcitonin, and unconjugated hyperbilirubinemia (total bilirubin 8.6 mg/dl), but otherwise unremarkable (table 1) . np respiratory viral pcr testing demonstrated coinfection with human metapneumovirus and sars-cov-2. due to tachycardia and signs of dehydration, he received intravenous fluid resuscitation and was admitted to a general pediatrics floor on empiric therapy with ampicillin and ceftazidime. shortly after admission, the patient demonstrated persistent tachycardia despite fluid resuscitation, as well as desaturations requiring initiation of oxygen therapy delivered by nasal cannula. a chest radiograph showed perihilar opacification but no focal consolidations. an electrocardiogram showed sinus tachycardia with diminished left ventricular forces. laboratory testing demonstrated an elevated bnp and d-dimer, with normal troponin t (table 1 ). due to clinical concern for evolving myocarditis, he was transferred to the icu for close monitoring. an echocardiogram was normal, with no evidence of ventricular dysfunction. he remained on a maximum supplemental oxygen support of 1l via nasal cannula during his icu stay. notably, he remained febrile through the first 24 hours of his hospitalization. immunophenotyping, obtained per our hospital's protocol for patients admitted to an icu with sars-cov-2 infection, demonstrated slightly elevated interleukin 10 (20 pg/ml; upper limit of normal [uln], 18 pg/ml) but an otherwise normal interleukin profile and normal t-cell subsets, with mildly decreased thymic immigrants (50.7%). immunoglobulin analysis showed borderline low immunoglobulin g (599 mg/dl; uln, 700 mg/dl). after 24 hours in the icu, tachycardia had improved and bnp decreased, and he was ultimately discharged home on hospital day 3. approximately 4 hours after discharge, he became febrile and drowsy at home. he was brought back to the ed where he was again febrile (temperature 38.9°c), but the remainder of his vital signs were normal. during observation in the ed, he experienced sustained oxygen desaturations to as low as 88% in room air. repeat laboratory testing was obtained and showed normal white blood cell count (9.07 k cells/μl) with elevated lymphocyte count (7.26 k cells/μl) but marked neutropenia (0.18 k cells/μl), elevated procalcitonin (0.18 ng/ml), and ferritin (487.7 ng/ml). a repeated chest radiograph was remarkable for persistent mild bilateral perihilar peribronchial thickening and increased asymmetric bibasilar opacities. he was restarted on 1 l of supplemental oxygen by nasal cannula and readmitted to the general pediatrics floor. cefepime was initiated due to concern for potential bacterial superinfection of a viral pneumonia. his drowsiness and oxygenation improved over 24 hours of hospitalization, and he remained afebrile following admission. antibiotics were transitioned to amoxicillin-clavulanate, with a plan to complete a 7-day course of antibiotics for pneumonia. he was discharged home on hospital day 2. blood, urine, and csf cultures remained negative from both his first and second hospitalizations. notably, his mother began to experience fever and respiratory symptoms while with him in the hospital; she was advised to obtain sars-cov-2 testing in coordination with her primary care provider. a 21-day-old full-term previously healthy male infant presented with fussiness and decreased oral intake for 4 days. he resides in a multigenerational home that includes a maternal aunt who was diagnosed with sars-cov-2 infection 1 week prior to his presentation. in the ed, he was febrile (temperature 38.3°c) and tachycardic (heart rate 172 bpm) with otherwise normal vital signs and physical examination. he underwent a full neonatal sepsis evaluation, significant for a urinalysis with 6 to 10 white blood cells and positive leukocyte esterase as well as positive sars-cov-2 rt-pcr (table 1 ). in the absence of respiratory symptoms, chest imaging was not obtained. he was initiated on empiric therapy with ceftriaxone and admitted to our hospital. the patient defervesced, remained hemodynamically stable, and did not require supplemental oxygen. he received intravenous fluids for 1 day due to decreased oral intake. blood and csf cultures remained negative. urine culture ultimately grew 50 000-100 000 colony-forming units of escherichia coli, and on hospital day 3 he was discharged home to complete a 10-day course of cephalexin. over a period of 4 weeks postdischarge, he returned to the ed on 5 occasions for recurrent fever and was readmitted once for observation following a brief resolved unexplained event. notably, sars-cov-2 pcr from an np swab remained positive when retested 20 days after his initial test. a 21-day-old full-term male infant presented with fever, cough, and congestion for 2 days. he was born via normal spontaneous vaginal delivery to a group b streptococcus-positive mother who received inadequate peripartum antibiotic prophylaxis. he lives with his parents and sibling who reportedly had recent upper respiratory symptoms, but did not seek medical care nor sars-cov-2 testing. upon presentation he was found to be febrile (rectal temperature 38°c) and mildly tachycardic (heart rate 162 bpm), with otherwise normal vital signs and physical examination. he underwent a complete neonatal sepsis evaluation, with laboratory results significant for elevated procalcitonin but otherwise unremarkable (table 1) . sars-cov-2 pcr from an np swab showed positive results. he was initiated on empiric therapy with ceftriaxone and admitted to our hospital. throughout his 2-day hospitalization he remained afebrile without respiratory distress and did not require supplemental oxygen. ceftriaxone was discontinued when blood, urine, and csf cultures were negative at 48 hours, and he was discharged home. these cases illustrate a common pediatric condition-neonatal fever-reevaluated within the paradigm of the covid-19 pandemic. sars-cov-2 has emerged as a significant cause of morbidity and mortality in adults, while most infections in pediatric patients are mild or asymptomatic [2] [3] [4] [5] [6] [7] . infants are noted to be a higher-risk group within pediatric covid-19 [2, 5, 7] . yet, to date few neonatal cases have been described [8] [9] [10] [11] [12] . similarly, at present there is very little known about the outcomes of neonates born to mothers with covid-19 or those infected during this vulnerable neonatal period. in this report, we present 4 febrile neonates hospitalized with sars-cov-2 infection with favorable outcomes. our patients were admitted between april 17 and may 6, 2020, a period that aligned with peak incidence of sars-cov-2 infection in our state. it is likely that our patients were infected postnatally given their ages and the presence of symptomatic individuals in their households. indeed, the incidence of transmission through familial exposure in pediatric covid-19 infections has been estimated between 45% and 91% [3, 5, 7] . however, we cannot definitively exclude perinatal transmission from asymptomatic mothers since they were not tested at the time of delivery. since all febrile neonates require hospitalization for evaluation of serious bacterial infections, even mild symptoms carry significant repercussions. half of the neonates in our series were found to have a coinfection, possibly providing an alternative explanation for fever: patient 2 had another respiratory viral infection (human metapneumovirus) and patient 3 had a urinary tract infection. notably, both of these patients experienced relatively protracted illness courses, including readmission for recurrent fever following initial hospitalization. one patient demonstrated prolonged viral shedding. it remains unclear whether sars-cov-2 was responsible for recurrent symptoms. historical studies have shown that the occurrence of serious bacterial infections is lower in infants with an identified viral infection than in those without a viral infection, but not low enough to obviate the need for sepsis evaluation [13, 14] . equally, as our series suggests, the identification of sars-cov-2 infection in an infant should not preclude evaluation for invasive bacterial infection. myocardial involvement in adults with covid-19 has been shown to be associated with worse outcomes [15] . myocardial involvement during acute infection appears to be rare in children [16] . patient 1 developed elevated cardiac biomarkers, elevated d-dimer, elevated clot strength, and mild systolic dysfunction, initially concerning for early viral myocarditis. antiviral therapy with remdesivir was promptly initiated and he fortunately did not progress to acute myocarditis. it is unknown whether lack of progression was secondary to the antiviral activity of remdesivir or the natural course of his systemic viral infection. direct sars-cov-2 myocardial injury was felt to be unlikely. a previously suggested pulmonary immunovascular coagulopathy model [17] provides another possible explanation of his abnormal cardiac laboratory and imaging findings. in this model, circulating d-dimer reflects pulmonary microvascular thrombosis, and elevated cardiac enzymes reflect ventricular stress induced by pulmonary hypertension. epidemiologic studies of covid-19 in adults have highlighted the disproportionate effect on populations already at risk of health inequity [18, 19] . we note the presence of at least 2 significant socioeconomic risk factors in each of our patients, also noted by mithal et al [12] . these include latinx ethnicity, children of recent immigrants, housing and food insecurity, residence in multigenerational homes, and children of adolescent parents. in developing strategies to protect this vulnerable neonatal population, guidance and management must take into account the neonate as part of the larger family unit to address those risk factors that may make them more susceptible to infection and adverse outcomes. author contributions. all authors contributed significantly to the manuscript in acquisition and interpretation of data, drafting of the initial manuscript, and review and revision. all authors have approved the final manuscript as submitted. financial support. health alert network: han archive-00432 epidemiology of covid-19 among children in china chinese pediatric novel coronavirus study team. sars-cov-2 infection in children • jpids 2020:xx (xx xxxx) • wardell et al screening and severity of coronavirus disease 2019 (covid-19) in children in madrid cdc covid-19 response team. coronavirus disease 2019 in children-united states characteristics and outcomes of children with coronavirus disease 2019 (covid-19) infection admitted to us and canadian pediatric intensive care units children with covid-19 in pediatric emergency departments in italy late-onset neonatal sepsis in a patient with covid-19 sars-cov-2 infection (covid-19) in febrile infants without respiratory distress novel coronavirus in a 15-day-old neonate with clinical signs of sepsis, a case report novel coronavirus infection in febrile infants aged 60 days and younger sars-cov-2 infection in infants less than 90 days old risk of serious bacterial infection in young febrile infants with respiratory syncytial virus infections serious bacterial infections in febrile infants 1 to 90 days old with and without viral infections the impact of 2019 novel coronavirus on heart injury: a systemic review and meta-analysis complete heart block, severe ventricular dysfunction and myocardial inflammation in a child with covid-19 infection immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia variation in covid-19 hospitalizations and deaths across new york city boroughs covid-19 and african americans key: cord-343528-5283jsnu authors: zhang, zhao; shen, libing; gu, xun title: evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission date: 2016-05-04 journal: sci rep doi: 10.1038/srep25049 sha: doc_id: 343528 cord_uid: 5283jsnu middle east respiratory syndrome coronavirus (mers-cov) belongs to beta group of coronavirus and was first discovered in 2012. mers-cov can infect multiple host species and cause severe diseases in human. we conducted a series of phylogenetic and bioinformatic analyses to study the evolution dynamics of mers-cov among different host species with genomic data. our analyses show: 1) 28 potential recombinant sequences were detected and they can be classified into seven potential recombinant types; 2) the spike (s) protein of mers-cov was under strong positive selection when mers-cov transmitted from their natural host to human; 3) six out of nine positive selection sites detected in spike (s) protein are located in its receptor-binding domain which is in direct contact with host cells; 4) mers-cov frequently transmitted back and forth between human and camel after it had acquired the human-camel infection capability. together, these results suggest that potential recombination events might have happened frequently during mers-cov’s evolutionary history and the positive selection sites in mers-cov’s s protein might enable it to infect human. acid substitutions in its s protein receptor binding domain along with its switching host from civet to human 20 . moreover, two amino acid substitutions in the s protein's c-terminal of hku4, a bat beta-coronavirus, enable its entry to human cells and the same amino acid substitutions are also found in mars-cov 21 . furthermore, heptad repeat regions in c-terminal of mers-cov and related coronaviruses also play important roles in viral adaptive evolution 22 . in summary, those studies introduced above suggested that s protein plays a vital role in mers-cov's cross-species transmissibility. however, the evolutionary mechanism of how mers-cov's s and other proteins facilitated the cross-species transmission of mers-cov remains to be investigated. here, we performed a series of phylogenetic and bioinformatic analyses for mers-covs. we systematically investigated the recombination events in mers-covs, the potential transmission route of mers-covs in five different host species and the evolutionary pressure of each mers-cov's protein during cross-species transmission. our study might offer some insight in explaining the possible mechanism in mers-cov's adaptive evolution. epidemic description and phylogenetic analysis of mers-cov. by far, the largest mers-cov outbreak is in saudi arabia and almost all human cases have a direct or indirect link to arabian peninsula. in this study, we collected 74 human mers-cov whole genome sequences from 9 countries (supplementary table 1 ). the geographic distribution of these samples is shown in supplementary fig. 1a . the majority of them are from the countries in arabian peninsula (78.4%, 58/74) and more than half of them are from saudi arabia (64.9%, 48/74) (supplementary fig. 1a) . the peak season for mers is between april (26.5%) and may (25.0%) (supplementary fig. 1b) . based on the whole-genome alignment of our collected sequences (supplementary table 1 ), we performed the phylogenetic analysis for these sequences with two sras-covs serving as the outgroup. our phylogenetic tree shows that all camel and human mers-covs are clustered together. the bat and hedgehog mers-covs formed a basal paraphyletic group to all camel and human mers-cov clade (fig. 1a) . a single camel mers-cov isolated in egypt (gi: 589588051) forms a single basal clade to human and the other camel mers-covs (fig. 1b) . the human-camel mers-cov cluster can be further divided into two clades-clade a and clade b, as previously reported 16 . clade a contains four human strains isolated in jordan and saudi arabia while 70 human and 17 camel mers-covs are mixed in clade b. there are five groups in clade b and we named them as group i to group v as the previous study 15 . there are 25, 17, 14, 2 and 29 mers-cov sequences in group i to group v, respectively (fig. 1b) . we performed the recombination analysis on the collected full-length mers-cov sequences. we find that there are 28 of them experienced potential recombination events (30.4%, 28/92), including three camel mers-covs and 25 human mers-covs (supplementary table 1 ). we divided 28 potential recombinant sequences into seven different types and named them as type 1 to type 7 ( fig. 1btable 1 ). type 1 means the recombination happened between group ii and group v, which includes 3 sequences and is about 11% of total recombinant sequences. type 2 means the recombination happened between group iii and group v, which includes 6 sequences (22%). interestingly, the mers-covs newly found in 2015 in south korea and china are type 2 recombinants 15, 23 . type 3 means the recombination happened between group i and group iii, which includes 2 sequences (7%). type 4, 5 and 6 are the recombination happened between different genomic regions of group iv and group v, which include 7, 4 and 4 sequences (25%, 14% and 14%), respectively. type 7 is the recombination happened among three groups (group i, iv and v), which includes 2 sequences (7%). our phylogenetic analysis showed type 1 belongs to phylogenetic group ii while type 2 and 3 belong to phylogenetic group iii, and type 4 to 7 belong to phylogenetic group v. there is no recombination found in phylogenetic group i and group iv (fig. 1b) . we also reconstructed the phylogenetic tree using non-recombinant sequences only and found that its topology is consistent with the tree based on all sequences (supplementary fig. 2) . we also performed the snp (single-nucleotide polymorphisms) analyses for each recombinant types and found the large recombination segments in type 2, 4, 6, 7 are conspicuous but in type 1, 3, 5 are obscure (supplementary fig. 3 ). in order to explore the selection pressure on the mers-cov proteins when it transmitted from animal host to human, we performed the adaptive evolution analyses for all mers-cov protein in absence of recombinant strains. firstly, we set camel and human mers-covs as the foreground branch and bat and hedgehog mers-covs as the background branch to preform branch-site test in codeml of paml program (see fig. 1a ). the strong positive selection is detected in spike (s) glycoprotein between these two branches (p < 0.001), while there is no significant positive selection in the other mers-cov genes ( table 1) . we find nine positive selection sites in mers-cov spike (s) glycoprotein and eight of them are statistically significant (table 1) . six significant positive selection sites are located in the receptor binding domain of s protein (fig. 2a) . we utilized a published crystal structure (pdb id 4l72 in rcsb protein data bank), the receptor binding domain (rbd, aa 367-606, fig. 2b ) of mers-cov spike glycoprotein complexed with the human receptor dipeptidyl peptidase 4 (ddp4), to demonstrate their locations in a 3d environment (fig. 2b) . the receptor binding domain of mers-cov s protein can be further divided into a receptor-binding sub-domain and a core sub-domain. two significant positive selection sites, k511r and g521n, are in the receptor-binding sub-domain and k511r is in direct contact with human receptor ddp4. q419s, g436n, d472s and r479l are in the core sub-domain. moreover, we also detected a positive selection site in s protein's c-terminal, l775s. secondly, we screened the positive selection sites among human-camel mers-covs (table 2 ). five significant (table 3 ). the genome-wide average nucleotide substitution rate of camel and human mers-covs was 4.81 × 10 −4 substitutions per site per year. open reading frame 3 (orf3) has the fastest substitution rate while orf5 has the slowest substitution rate. the orf4b, nucleocapsid (n) glycoprotein, and spike (s) glycoprotein, have a similar substitution rate which is faster than the whole-genome substitution rate. in order to study the temporal and spatial pattern of mers-cov transmission, a maximum clade credibility (mcc) tree was constructed using mers-cov whole genome sequences without recombinant strains (fig. 3c) . the ancestral host state with time reference was estimated for each tree node and marked with different colors. we named six important nodes in mers-cov divergence on mcc tree for node a to f (fig. 3c ). the possible transmission time for each node and its 95% highest posterior density (hpd) are shown in fig. 3b . we found that the origin time of human-camel mers-cov is relatively late (node d). furthermore, the tmrca for clade b is in ~2012 (fig. 3b , node f) and clade a and clade b are divergent in ~2011 (fig. 3b , node e). interestingly, the mcc tree shows that there are six cross-species transmission events with high posterior probabilities in clade b. five of them are human-to-camel transmission events and one of them are camel-to-human transmission events (fig. 3c ). additionally, with the mers-cov of human/camel and bat/hedgehog mers-cov together, we inferred the ancient mers-cov exists for decades of years (fig. 3b,c) . the tmrca of the mers-covs for vespertilio superans, neoromicia capensis or erinaceus europaeus can be traced back to 2006 (node c), 2003 (node b) and 1996 (node a), respectively. before the emergence of human-camel mers-cov, the estimated tmrca for all mers-covs appeared in ~1996 (fig. 2c , node a). we also preformed root-to-tip analysis using the consistent dataset (fig. 3a) , and the result shows that the origin time of tmrca is in ~1995 with high statistical supports (r 2 = 0.874, p value < 0.001). together these results suggest that the ancient mers-cov should have existed for decades in animal host and got the ability to infect human or camel recently. mers-cov belongs to coronavirus, beta-coronavirus, lineage c. since it was discovered in 2012, mers-cov has attracted extensive attention due to its human-to-human infection capability and high mortality rate. recombination events have been confirmed in human mers-cov 23 . the fact that mers-cov can be found in multiple species proposes its cross-species transmissibility [4] [5] [6] [7] 11 . by far, the evolutionary details of how mers-cov transmitted to human are still unknown. based on the most comprehensive collection of mers-cov genome sequences so far, we tried to elucidate the evolution and transmission of mers-cov among different species. mers-cov has been reported in five species including european hedgehog, two species of bats, dromedary camel, and human. we used the ml method to reconstruct the whole-genome phylogenetic tree of mers-covs isolated from these species. the ml tree shows that the hedgehog mers-covs are basal to all the other mers-covs and two bat mers-covs are basal to camel and human mers-covs. this result suggests that the ancestor of camel and human mers-covs may be from other animal host, such as the hedgehog or bat. we also reconstructed the phylogenetic tree of mers-cov using nj method or based on each mers-cov protein. these trees show a consistent topology, which proposes that the phylogenetic relationship estimated in our study is credible (supplementary fig. 5) . we divided clade b into five groups as pervious study to detect the recombination of mers-cov 15 . because the evolutionary distances among mers-covs are close (table 3) , no large segment recombination could be detected among them. thus, according to discontinuous recombination segments, we defined potential recombination events in mers-covs. this method has been used in the previous study to label potential recombination 24 . in our study, we found 28 strains form seven recombinant types, which took more than 30% of all isolated mers-covs in human and camel. among them, we found 26 strains in six recombinant types (type 1 to type 6) between two phylogenetic groups and two strains in one type (type 7) among three phylogenetic groups. for now, the recombination of mers-cov was confirmed in previous study, but no report about the recombination among more than two groups of mers-cov. interestingly, most recombinant types (type 1, 2, 4, 5, 6 and 7) are related to group v and they make up 92.9% of total recombinant strains (26/28). the result suggests that recombination events might happen frequently and the recombinant types involving group v might happen broadly. additionally, multiple recombination events indicate that double infection and super infection likely existed during the transmission history of mers-cov. we failed to detect possible large recombination segments in type table 3 . evolutionary distance, nucleotide substitution rate and coefficient of variation of substitution rate for human and camel mers-cov proteins. 1, 3 and 5. by comparing the snps (single-nucleotide polymorphisms) of reference sequences with recombinant sequences, we reckoned that specific nucleotide mutation might influence the results of recombination analysis. this problem can be solved by discovering more mers-cov sequences or developing more detailed genotype classification for mers-covs in the future. we also performed phylogenetic analyses for potential recombinant region and got similar results (supplementary fig. 3) . interestingly, the east asian mers-cov strains (china and south korea) belong to type 2 recombinant and the previous study show that their tmrca might be a result of potential recombination event 23 , which indicates the recombinant strains have transmitted broadly. moreover, one recombinant mers-cov lineage has led to the large-scale outbreak in both camel and human 26 . it proposes that recombinant mers-covs have experienced cross-species infection. additionally, our study reveals that the number of recombinant strains is large and the potential recombinant types are abundant. together these findings highlight that we should take more attention to recombinant mers-cov transmission. although how the mers-cov transmitted from its natural host to human is still unknown, it is confirmed the mers-cov have been found in many animal hosts, such as bats and hedgehog. to study the evolutionary pressure on each mers-cov's protein during its potential cross-species transmission, we conducted a comprehensive scan for positive selection sites in mers-cov's proteins. recombinant strains were excluded in this analysis. we set camel-human mers-covs as the foreground branch and hedgehog-bat mers-cov as the background branch and estimated the relative evolutionary pressure on the foreground branch compared to the background branch. we only found that mers-cov's s protein underwent strong positive selection and there are nine significant positive selection sites in s protein. it suggests that s protein was under strong evolutionary pressure during the transmission from its natural host to human. among significant positive selection sites, six of them are located in s protein's receptor binding domain (rbd). rbd is crucial for virus to enter host cells and it comprises of one binding region and one core region. based on the rbd's 3d model, we find that two sites are located in the binding region of rbd, which suggests that these amino acid substitutions might change mers-cov's binding capability to host cells and thus facilitate its cross-species transmission. the other four sites are located in the core region of rbd. these amino acid substitutions might change the structure of core region and indirectly influence mers-cov's cross-species capability. in order to eliminate the sample bias between human/camel group and bat/hedgehog group, we also did random sampling for the 68 non-recombinant human and camel sequences. we tried 10, 20 and 50 random sampling, respectively. using random sampled sequences together with bat and hedgehog mers-cov sequences, we performed branch-site analyses and got the same results as our aforementioned (data not show). we also estimated the nucleotide substitution rates of camel-human mers-covs to investigate its evolutionary dynamics after it infected camel and human. our estimated nucleotide substitution rate for mers-cov's whole genome is 4.81 × 10 −4 , which is slower than the previous estimation 9 . one explanation for the phenomenon is that we used a larger dataset than the previous study, which includes all available mers-cov whole genome sequences. our estimated confidence interval of the substitution rate of mers-cov genome is largely overlapped with the result from another study 16 . the estimated nucleotide substitution rates show that four proteins experienced the accelerated evolution. through evolutionary pressure analysis, we found camel-human mers-cov's four proteins underwent positive selection and detected five significant positive selection sites. one of them is located in m proteins. there is evidence that m proteins are powerful interferon antagonist 26 , which proposes that the evolutionary pressure on m proteins are from host's immune system. two out of five significant sites are found in n protein which is fundamental for mers-cov self-assembly. coronavirus n protein is able to bind to different host cell proteins and demonstrated to have various functions, one of which is also counteracting host interferon as shown in sars-cov [27] [28] [29] [30] . it is reasonable to speculate that mers-cov n protein under intensive selection because its functions were similar to those of sars-cov n protein. the results above suggest that the arm race between mers-cov proteins and host's immune system might be the main evolutionary driving force behind mers-cov's adaptive evolution after it began to infect camel and human. mers-cov spike (s) glycoprotein evolves slightly faster than the genome-wide average rate, which indicates that the nucleotide substitution rate of mers-cov s protein still maintains a fast speed even after it crossed the species boundary. the positive selection site we found in mers-cov s protein with site-specific test is identical to the previous study's result 3 . this site is located in heptad repeats 1 which is a key component in membrane fusion architecture and required for mers-cov entering host cells 31 . in absence of recombinant strains, we performed the mcc analysis using mers-cov whole-genome sequences in order to infer the time and source species when mers-cov crossed the species boundary. the topology of the mcc tree is highly congruent with that of the whole-genome phylogenetic tree. we defined six nodes (a-f) to explain transmission. the posterior probability for the ancestral sate of node a, b or c is not very high in our mcc analysis and the 95% highest posterior density (hpd) of the divergence time for these three nodes is quite long. so these results are weak for demonstrating the exact origin time or ancestor state of mers-cov. however, these estimations still provided the evidence that the ancestor mers-cov should have been infected a number of animal hosts, such as bat or hedgehog, for decades of years (supplementary fig. 4) . the x-intercept (tmrca) in root-to-tip is ~1995 with high statistical supports, which is close to the estimated time of tmrca in mcc analysis. this hypothesis is in agreement with the result of serological studies 2,3 . the appearance of the common ancestor of human-camel mers-cov is in 2010 and the appearance of the tmrca of clade a and b is in 2011, which are exactly the same as the previous report 16 . in clade b, we detected five possible human-to-camel transmission events and one camel-to-human transmission event. it suggests that mers-cov frequently transmitted back and forth between human and camel after it acquired the capability of infecting both hosts. actually, there is at least one confirmed case of camel-to-human mers-cov transmission 32 . scientific reports | 6:25049 | doi: 10.1038/srep25049 in conclusion, we found that potential recombination events are common in mers-cov's evolutionary history and potential recombinant mers-covs can be divided into seven types. the amino acid sites under positive selection in mers-cov s protein, especially those in its receptor binding domain, might have facilitated its cross-species transmission from animal host to human. we detected the strong positive selection in four proteins of camel-human mers-covs, which indicates that they probably experienced strong adaptive evolutionary pressure from host's immune system. additionally, we also found six possible cross-species transmission cases between human and camel. our study investigated the evolutionary dynamics of mers-cov, which shall provide a basis for mers-cov control and treatment. sequence data. the complete genomic nucleotide sequences of 91 mers-covs and two sars-covs were downloaded from ncbi nucleotide database. among 91 mers-cov genomic sequences, 68 of them are from human, 18 of them are from dromedary came, two of them from two bat species neoromicia capensis and vespertilio superans, and 3 of them are from european hedgehog erinaceus europaeus. two sars-cov genomic sequences are from human and bat rhinolophus ferrumequinum, respectively. we used sequences 453061240 as reference to extract open reading frames from each mers-cov genome in this study. genomic sequence alignment and phylogenetic analysis. total 93 collected genomic sequences were aligned using the muscle software with default parameters 33 . clustalw and mafft used to validate the muscle result 34, 35 . alignments were refined manually in bioedit (http://www.mbio.ncsu.edu/bioedit/ bioedit.html). only unambiguously aligned positions were used for subsequent phylogenetic analyses (supplementary files). we used the jmodeltest 3.1 to estimate the best nucleotide substitution model for our alignment 36 , which is gtr+ i+ g. we used the phyml 3.1 to perform the phylogenetic analysis for 93 collected genomic sequences based on their genomic sequence alignment 37 . the branch support values were calculated with shimodaira-hasegawa test integrated in phyml. in clade b, we estimated the consensus sequences for every phylogenetic group using cons tool in emboss explorer (http://bioinfo.nhri.org.tw/cgi-bin/emboss/cons). five consensus sequences were set as reference and every sequence in clade b was used as the query to detect the possible recombination using simplot software 25 . the window is set to 200 bp and the step is set to 20 bp. positive selection analysis. we extracted the coding region of each mers-cov protein using mers-cov 453061240 strain as a reference template. the codeml program implemented in paml 4.7 package was used to detect the positive selection in the codon alignment of each mers-cov protein set 38 . in the branch-site model, the group of human-camel mers-covs was set to be foreground, the group of bat-hedgehog mers-covs was set to be background, and model a with estimated ω value was compared with the null model (model a') with fixed ω value. to reduce the bias from sample size, we performed random sampling on 68 human and camel mers-covs (clade a and b) which are all non-recombinant sequences. we used 10, 20 and 50 as the random sample size with and without replacement. the random sampled sequences together with bat and hedgehog mers-covs were used to generate the datasets for branch-site model analysis as aforementioned method (script see supplementary files). moreover, for each random sample size, we repeatedly drew five times in order to make results robust. we also used the site-specific model to detect positive selection in the human-camel clade. the site-specific model was performed by comparing the models m2a (positive selection) and m8 (beta & ω ) vs. the null models the crystal structure of the receptor binding domain (rbd) of mers-cov spike (s) glycoprotein in complex with the human receptor dipeptidyl peptidase 4 (ddp4) was displayed using jmol (jmol: an open-source java viewer for chemical structures in 3d we estimated the transmission of mers-cov among different hosts or geographic areas. the sampling time and host/geographic location of each sequence were also used in analysis. the nucleotide substitution rates and the origin time of most recent common ancestor (mrca) on various nodes of mcc tree were also estimated using the beast package. a relaxed molecular clock with an uncorrelated log-normal distribution, and a constant population size model were used in bayesian coalescence analysis. according to the outcome of jmodeltest3.1, the gtr+ gamma+ i model of nucleotide substitution was employed in mcc analysis. statistical uncertainty in parameter estimations was reflected by the 95% highest posterior density (hpd) values. mcmc analysis was run for 500/100 million generations for hosts/geographic transmission with sampling every 50,000/10,000 generations to achieve parameter convergence and adequate effective sample sizes (ess > 200). we summarized the trees using treeannotator implemented in the beast v1.8.2 package. the initial 25% samples were discarded as burn-in, leaving 75% trees per run to produce the consensus tree middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group mers coronavirus neutralizing antibodies in camels antibodies against mers coronavirus in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat mers-related betacoronavirus in vespertilio superans bats characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs isolation of a novel coronavirus from a man with pneumonia in saudi arabia spread, circulation, and evolution of the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description evidence for camel-to-human transmission of mers coronavirus molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku14, from domestic rabbits origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus the heptad repeat region is a major selection target in mers-cov and related coronaviruses the recent ancestry of middle east respiratory syndrome coronavirus in korea has been shaped by recombination full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a1 sars-cov nucleocapsid protein binds to hubc9, a ubiquitin conjugating enzyme of the sumoylation system sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus evidence for camel-to-human transmission of mers coronavirus muscle: multiple sequence alignment with high accuracy and high throughput clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform jmodeltest: phylogenetic model averaging new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 paml 4: phylogenetic analysis by maximum likelihood mega6: molecular evolutionary genetics analysis version 6.0 bayesian phylogenetics with beauti and the beast 1.7 this work was supported by the grant from the national science foundation of china (31571355). we thank the anonymous reviewers. z.z., l.s. and x.g. designed the experiments; z.z. and l.s. collected viral sequences, performed phylogenetic analyses, adaptive evolutionary analyses and transmission analyses. z.z., l.s. and x.g. wrote the manuscript. all authors reviewed the manuscript. supplementary information accompanies this paper at http://www.nature.com/srep key: cord-332595-874tpi09 authors: salehi, najmeh; amiri-yekta, amir; totonchi, mehdi title: profiling of initial available sars-cov-2 sequences from iranian related covid-19 patients date: 2020-09-08 journal: cell j doi: 10.22074/cellj.2020.7524 sha: doc_id: 332595 cord_uid: 874tpi09 the etiologic agent sars-cov-2 has caused the outbreak of covid-19 which is spread widely around the world. it is vital to uncover and investigate the full genome sequence of sars-cov-2 throughout the world to track changes in this virus. to this purpose, sars-cov-2 full genome sequence profiling of 20 patients in iran and different countries that already had a travel history to iran or contacts with iranian cases were provided from the gisaid database. the bioinformatics analysis showed 44 different nucleotide mutations that caused 26 nonsynonymous mutations in protein sequences with regard to the reference full genome of the sars-cov-2 sequence (nc_045512.2). r207c, v378i, m2796i, l3606f, and a6407v in orf1ab were common mutations in these sequences. also, some of the detected mutations only were found in iranian data in comparison with all the available sequences of sars-cov-2. the position of s protein mutations showed they were far from the binding site of this protein with angiotensin-converting enzyme-2 (ace2) as the host cell receptor. these results can be helpful to design specific diagnostic tests, trace the sars-cov-2 sequence changes in iran, and explore therapeutic drugs and vaccines. coronaviruses (covs) are related to the family of coronaviridae. they contain a single-stranded rna of 26 to 32 kilobases. pathogenic human covs usually cause mild respiratory diseases (1) . in contrast, two highly pathogenic human covs were identified that transmitted from animals to humans. severe acute respiratory syndrome (sars) coronavirus (sars-cov) as the first one was reported in guangdong, china, in november 2002 that caused more than 8,096 human infections and 774 deaths in 37 countries (2, 3) . the second one was the middle east respiratory syndrome (mers) coronavirus (mers-cov), which was first reported in saudi arabia in june 2012 that infected 1,728 cases and expired 624 patients in 27 countries (2). in december 2019, a new human coronavirus, sars-cov-2, was identified in patients in wuhan, hubei province, china (4, 5) . this new infectious respiratory disease is called coronavirus disease 19 , which is quickly spread around the world. the covid-19 outbreak has a total of 2,072,113 infections and 138,475deaths in 210 countries and territories around the world until 15 th april 2020. as it can be seen in figure 1a , the full genome sequence of sars-cov-2 has ten open reading frames (orfs) that contain four structural proteins; the spike-surface glycoprotein (s), the small envelope protein (e), the membrane glycoprotein (m), and the nucleocapsid protein (n), as well as several nonstructural proteins. in all covs, the s protein plays a crucial role in binding to the host cell receptors (6, 7) . a pairwise sequence alignment between the sars-cov-2 with sars-cov and mers-cov showed about 79% and 50% identity, respectively (8) . the complete genome profile of sars-cov-2 revealed a high overall genome sequence identity to bat-cov-ratg13, pangolin-cov, bat-sarsr-cov-zc45, and bat-sarsr-cov-zxc21 by 96.2%, 91.02%, 87·99%, and 87·23%, respectively (8-10) therefore, sars-cov-2 genome is highly similar to ratg13 genome (9) . however, genes such as orf1b, the s protein, orf7a, and orf10 in pangolin-cov depict higher identity with sars-cov-2 than bat-cov-ratg13 (10) (11) (12) . the genome sequence of sars-cov-2 is being generated by a lot of laboratories around the world, and these are freely available at the global initiative on sharing all influenza data (gisaid) database (13) . these data can be helpful to design more specific diagnostic tests, trace the ongoing outbreak, and explore therapeutic processes. by 15 th april 2020, twenty-three sequences of sars-cov-2 with a length of 87 to 595 bases and one full sequence with a length of 29,828 were available at the gisaid database of iran's location. fifteen and eight of these twenty-three sequences were associated with a part of the n gene and the orf1ab, respectively. three, four, and one (of eight) sequences of orf1ab coded a portion of leader protein, rna polymerase, 3´-to-5´ exonuclease, respectively. all of these twenty-three sequences encoded the related proteins as same as the reference one (nc_045512.2) if we masked the first and last few bases. on the other hand, nineteen sequences of the full genome sequence of sars-cov-2 on the gisaid database from patients in different countries that had a travel history to iran or contacts with iranian cases were retrieved from the database. the one full sequence with iran's location and these iranian related sequences were translated to the protein sequence in six frames. the multiple nucleotide and protein sequence alignments of these initial available data were performed by muscle and clustal omega programs with default parameters, respectively. the mutation results of nucleotide and protein sequences are available in table s1 and s2 (see supplementary online information at www.celljournal.org), respectively. as it can be seen in the table s1 , these sequences totally revealed 44 different nucleotide mutations that have made 26 nonsynonymous mutations in protein sequences (table. s2) regarding the full genome of the sars-cov-2 sequence isolate wuhan-hu-1 (nc_045512.2). these nucleotide mutations should be noticed in designing diagnostic tests to reduce the false-negative results of no binding of primers and probes in qpcr-based tests. figure 1b presents nucleotide mutations that lead to nonsynonymous mutations in protein sequences. a six-nucleotide and two-amino-acid insertions were detected in the full genome sequence of sars-cov-2 with iran's location. the number of mutation events depicts that some of these mutations occurred more than three times among these 20 sequences such as r207c, v378i, m2796i, l3606f and a6407v in orf1ab which are highlighted in the light orange columns in figure 1b . also, the entropy values of these mutations among the 3927 full genome sequences of the sars-cov-2 have retrieved from nextstrain (14) (https://nextstrain.org/) analyses. the entropy values quantify the uncertainty or variability of amino acid mutations for each position in protein sequences. a position on the protein sequence without any mutation in the whole genome of the sars-cov-2 sequence has an entropy of zero (15) . the entropy values show that some mutations have occurred just once in iranian sequences were also rare in the 3927 full genome sequences of sars-cov-2 with 0.002 entropy value (fig.1b) . furthermore, the corresponded protein name for each mutation is identified in figure 1b . accordingly, nsp2, nsp4, nsp6, 3´-to-5´ exonuclease, endornase, and s protein contain more mutation positions with higher events in data from these 20 iranian related patients. among these proteins, the s protein facilitates viral entry into host cells (6, 7) . similar to sars-cov, angiotensin-converting enzyme-2 (ace2) is used as a cellular entry receptor for sars-cov-2 (16, 17) . the viral replication rates and disease severity depend on the binding affinity between the s protein and the ace2 receptor (17) . the 3d structure of the sars-cov-2 receptor-binding domain (rbd) in complex with human ace2 protein receptor (pdb id: 6m17) was superimposed on the s protein of sars-cov-2 with a single rbd up (pdb id: 6vsb) by vmd1.9.3 (18) in figure 1c . as can be seen, the s protein amino-acids variants in our cases are far from the binding site of the s-ace complex. so, none of these mutations cause any disruption on the binding of s protein with ace2. on the other hand, for the sars-cov-2 vaccine and drug designing the s protein is a perfect target on the surface of this virus (19, 20) which its mutations should be observed. in this study, the full genome sequences of sars-cov-2 from the 20 iranian related covid-19 patients were profiled in detail. the results showed some significant mutations such as r207c, v378i, m2796i, l3606f, and a6407v in orf1ab which occur more than three times among these 20 iranian related sequences. also, some rare mutations were found that only happened in these sequences in comparison with all 3927 full genome sequences of sars-cov-2. the structural analysis of s protein showed the s protein mutations in iranian related sequences were away from the binding site of s protein with ace2. these data can be of great help for performing researches to trace the sars-cov-2 sequence changes, designing more specific diagnostic tests to reduce the false-negative results of no binding of primers and probes in qpcrbased tests, as well as exploring specific therapeutic drugs and vaccines in iran. it is certainly needed to generate more full genome sequences of sars-cov-2 from iranian patients to find more certain changes of this virus in iran. . the numbers of the encoded amino acid residues are specified in parenthesis. b. the nucleotide and protein mutations, the number of mutation events in data from the 20 iranian related patients, the entropy values of these mutations in all 3927 sars-cov-2 sequences, and the corresponded proteins are depicted. light orange columns and cyan cells show the common mutations and their corresponded proteins in iranian related sequences, respectively. c. the sars-cov-2 and ace2 complex structure. ace2, chain a, b, c of s protein, and mutated residues are depicted in magenta, green, gray, cyan, and yellow, respectively. epidemiology, genetic recombination, and pathogenesis of coronaviruses sars and mers: recent insights into emerging coronaviruses world health organization. summary of probable sars cases with onset of illness from 1 a novel coronavirus outbreak of global health concern a novel coronavirus from patients with pneumonia in china the spike protein of sars-cov--a target for vaccine and therapeutic development structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins. biorxiv insights into the cross-species evolution of 2019 novel coronavirus global initiative on sharing all influenza data -from vision to reality nextstrain: real-time tracking of pathogen evolution entropy and information approaches to genetic diversity and its expression: genomic geography angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-cov-2 cell entry depends on ace2 and tm-prss2 and is blocked by a clinically proven protease inhibitor vmd: visual molecular dynamics sars-cov-2 vaccines: status report preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies the authors would like to thank the genetic department of royan institute for financially support. there is no conflict of interest. key: cord-328395-2cakgmsj authors: oxford, alexandra e.; halla, fabio; robertson, evan b.; morrison, brad e. title: endothelial cell contributions to covid-19 date: 2020-09-25 journal: pathogens doi: 10.3390/pathogens9100785 sha: doc_id: 328395 cord_uid: 2cakgmsj understanding of the clinical, histological and molecular features of the novel coronavirus 2019 (severe acute respiratory syndrome coronavirus 2 (sars-cov-2)) has remained elusive. coronavirus disease 2019 (covid-19) caused by this virus has unusual clinical presentation with regard to other related coronaviruses. recent reports suggest that sars-cov-2, unlike other related viruses, infects and replicates within endothelial cells, which may explain a significant portion of the observed clinical pathology. likewise, mounting evidence associates vascular and endothelial cell dysfunction with increased mortality. this review focuses on understanding how endothelial cell pathology is caused by sars-cov-2 at the molecular and cellular levels and how these events relate to covid-19. a detailed examination of current knowledge regarding canonical inflammatory reaction pathways as well as alteration of endothelial cell-derived exosomes and transdifferentiation by sars-cov-2 is included in this assessment. additionally, given an understanding of endothelial contributions to covid-19, potential therapeutic aims are discussed, particularly as would affect endothelial function and pathology. the new strain of coronavirus (severe acute respiratory syndrome coronavirus 2 (sars-cov-2)) has created a global health crisis that is unlike any pandemic witnessed since the spanish flu in 1912. it is now reported that 40% of coronavirus disease-19 (covid-19)-related deaths come from cardiovascular complications, with most of the remaining 60% attributed to respiratory failure separate from myocardial injury and unknown causes [1, 2] . many cases of sars-cov-2 infection are asymptomatic, causing subclinical or mild symptoms [3] . however, immunologic overdrive has been strikingly common in those who progressed to a later stage of coronavirus disease , presenting some similar clinical findings as those associated with macrophage activation syndrome (mas) [4] . the associated increase in serum cytokines (termed a "cytokine storm") progresses to cause acute respiratory distress syndrome (ards). in addition, endothelial cell infection promotes the formation of thrombus associated with an elevated neutrophil count [5] . it is important to note that though endothelial infection has been validated, the virus remains highly replicative in the throat and upper respiratory tract (as is symptomatically consistent with respiratory viruses of this type), and has been found to be inactive (un-replicative) within the blood [6] . while the cardiovascular complications in patients with covid-19 are becoming more apparent, the underlying mechanisms leading to these issues are not fully understood. this review will focus on the concept of endothelial cell infection and dysfunction as an active driver of covid-19, which begins as a respiratory illness, with vascular pathology contributing significantly to the most negative patient outcomes. to better explain the varying symptoms and severities that arise from sars-cov-2 infection, the stepwise progression of covid-19 is utilized. disease progression consists of three phases: the early infection phase, the pulmonary phase, and the hyperinflammation phase [7] . initial infection by sars-cov-2 begins the incubation period (during which infectivity may peak), and continues until the onset of symptom presentation [8] . beginning at the point of initial infection, and continuing through the incubation period and infectious course, sars-cov-2 binds to the angiotensin-converting enzyme 2 (ace2) receptor on human lung epithelium. as described in the following, ace2 is found on a variety of other cell types, including the vascular endothelium and intestinal epithelium (see table 1 ). the widespread expression patterns of this receptor may contribute to the varied, systemic symptoms observed. additionally, infection by sars-cov-2 may result in the disease state of covid-19. the pulmonary phase (phase ii) of covid-19 is associated with an established pulmonary disease, caused by infection of local epithelium and viral multiplication. this stage is commonly associated with viral pneumonia, cough, and fever. stage ii may be further subdivided into stages iia (pulmonary disease without hypoxia) and iib (pulmonary disease with hypoxia) [9] . the hyperinflammation phase is when additional systemic, inflammatory symptoms coincide with sars-cov-2 infection and antiviral type i and iii interferon production [10] . this phase of the disease aligns with the idea that covid-19 is associated with severe endothelial cell infection and inflammation leading to an increase in cardiovascular complications. it is important to note that disease severity may be highly dependent on patient characteristics, including incidences of comorbidity (particularly of inflammatory conditions such as cancer and diabetes) [11] . a "cytokine storm" is defined as an excessive immune response towards an external stimulus. cytokines play an essential role in the innate immune system against viral infections. however, an excessive first-line response to viruses can sometimes become more harmful than beneficial [12] . cytokine storms progress rapidly in a very complex manner, resulting in a high mortality rate. huang et al. [13] showed a history of increased proinflammatory cytokines in sars-cov-2-related viruses (including sars and middle east respiratory syndrome-cov (mers-cov)), with these patients displaying increases in il-1β, il-6, il-12, ifnγ, ip10, and mcp1 similar to those found in mas. similarly, the study reported that covid-19 patients also had an increase in proinflammatory cytokines, leading to the activation of t helper 1 cell responses. however, these cytokines led to a further increase in additional cytokines (gcsf, ip10, mcp1, mip1a, and tnfα), which were associated with increased icu admission and disease severity. essentially, the release of copious amounts of pro-inflammatory cytokines induces and sustains the systemic inflammatory response in severe covid-19. following excessive cytokine release, sars-cov-2 infections lead to the activation of additional immune cells that harm healthy cells and disturb normal physiological functions [6, 12] . for example, the release of interferons i and iii, though antiviral in function, may cause significant damage to the lung epithelium following extended exposure, as may be observed in the cytokine storm. this tissue damage may worsen clinical outcomes [10] . an increased neutrophil to lymphocyte ratio, serum levels of inflammatory cytokines and chemokines, and inflammatory markers in blood have been associated with increased disease severity and death. however, the observed neutrophil insurgence is likely not an effective defense against viral pathogens and could be suppressing t cell-mediated antiviral activities [14] . in addition, increased inflammatory markers in blood of covid-19 patients (including c-reactive protein, ferritin, and d-dimers) have been reported, suggesting that there is hyperactivity of the coagulation system [5, 15, 16] . an elevated neutrophil count in covid-19 patients suggests the pathogenic role of neutrophil extracellular traps (nets), which have been found to contribute to thrombosis in other similar pandemic viruses h1n1, sars-cov, and mers-cov [5] . nets function to disarm pathogens through the use of molecular complexes targeting dna and promoting its destruction. common proteins such as neutrophil elastase, cathepsin g, and specific histones are associated with nets [17] . nets are commonly understood as the immune system's first line of defense towards infection, either by the secretion of antimicrobial substances or the engulfment of the pathogen. however, it has shown that an overactive netosis state can lead to more harm than help to the host. moreover, nets are responsible for initiating thrombotic events in arteries, veins, and microvasculature by activating the contact pathway of coagulation via electrostatic interactions [18] and the intrinsic pathway by presenting tissue factor [19, 20] . serum samples in patients with covid-19 display large amounts of net remnants including mpo-dna complex, citrullinated histone h3, and cell-free dna, promoting venous thrombosis and inflammation of the vessel wall [5] . the serum of covid-19 patients seems to have contents which stimulate netosis; the exact contents are yet to be understood and encourages more research concerning nets in covid-19 infected individuals. when added to control neutrophils, covid-19 sera are potent stimulators of netosis [5] . the presence of net remnants, and the activation of netosis via covid-19 sera, may suggest that patients suffer from a pro-netosis stage. this stage may contribute to virally damaged epithelial cells, activated platelets, activated endothelial cells, and excessive release of inflammatory cytokines. consequently, inhibitors of nets are of interest as a potential treatment to reduce the severity of sars-cov-2 infection [21] . table 1 . cell type and tissue distribution of ace2 and transmembrane serine protease 2 (tmprss2) protein expression. detection of ace2 or tmprss2 is indicated by a plus (+) and unsuccessful detection is also shown (-). infection of blood vessel endothelium leading to systemic circulation of the virus is virtually unheard of in previous viral outbreaks. neither taxonomic sister virus sars, nor additional pandemic virus h1n1 (influenza a of the family orthomyxoviridae, the cause of both the 1918 influenza and the 2009 swine flu pandemics), showed such endothelial disruption [5, 22] . nevertheless, sars-cov-2 exhibits an ace-mediated mechanism of endothelial infection. the coronavirus spike protein attaches to ace2 to enter the host cell [23] . this receptor is commonly found on epithelial type ii cells, renal, intestinal, cardiac, and endothelial cells [15, 16] . as analyzed by varga et al., the distribution of this receptor is largely consistent with the clinical findings for target organ failure (e.g., cardiac and renal) but does not fully correlate with the broad symptomatology [23] . this study found the presence of viral bodies and host inflammatory cells in several organs, suggesting that sars-cov-2 leads to the induction of endotheliitis [23] . endothelial cell infection that proceeds via ace2 shows how sars-cov-2 can replicate into a wide range of cells, which may explain some of the clinical symptoms found in covid-19 patients. however, how tissues with restricted serum, and thereby viral particle, access become infected is unknown. following the entry of sars-cov-2 into the host cell, host ribosomes bind to and facilitate the translation of the positive-sense viral rna genome. translation of the viral genome results in the production of polyproteins capable of forming the replication-transcription complex subsequent to processing by viral protease activity. the replication-transcription complex then acts to produce positive-sense genomic rna for future packaging, as well as mrna transcripts for viral structural and accessory proteins (nucleocapsid, envelope, membrane, and spike proteins). these viral mrna transcripts are then trafficked to, and translated by, the endoplasmic reticulum of the host cell. the newly produced viral proteins, as well as the genomic rna, are then packaged by the golgi apparatus and exocytosed as a fully functional viral particle [24, 25] . sars-cov-2 has been demonstrated to use the ace2 receptor on human endothelial cells for cell entry. in fact, it has been shown that only coronaviruses which express the sars-cov spike protein can utilize the ace2 receptor [26] . this was originally reported in a 2003 study conducted using the sars-cov permissive african green monkey kidney cell line vero e6. vero e6 have been subsequently used extensively to characterize this family of viruses and the molecular features of sars-cov spike protein-ace2 receptor interaction [29] [30] [31] . sars-cov and sars-cov-2 both utilize a conserved spike protein (also called the s protein). in sars-cov, the s protein has two distinct subunits-s1 and s2. s1 was determined to have a high-affinity association with respective receptors in particular the ace2 receptor [27, 29] . using a carboxy terminally tagged form of the s1 domain as bait, vero e6 cell lysate was incubated and s1 immunoprecipitated. after gel purification, interacting proteins were identified by mass spectrometry. three proteins were identified as putative s1 interactors with ace2 (8 fragments which comprised 17% of human ace2) being the strongest cell surface receptor candidate. this finding piqued the interest of researchers studying sars as the tissue distribution and localization of ace2 make it an appropriate receptor for sars-cov [30] . this work was then validated in a vero e6 cell line cloned from human-lung endothelial cells where ace2 -s1 binding was confirmed using blocking antibodies [31] . in a separate study, transmembrane serine protease 2 (tmprss2) was shown to activate the viral s protein which affects viral entry but not replication [32] . tmprss2 accomplishes this by cleaving the glycoprotein hemaglutenin which is necessary for the fusion of viral and host cell membranes. this forces a conformational change after endocytosis and allows for viral invasion into the host cell cytoplasm [33] . betacoronaviruses (such as sars-cov, sars-cov-2, and mers) have only two distinct methods of entering endothelial cells. the first is through the endosomal pathway. it has been shown that mers, which binds to and enters cells in a similar method (though with the utilization of the dipeptidyl peptidase-4 receptor) as sars, can bind to cathepsin l in the endosome in the absence of tmprss2 and facilitate entry into the host cell cytoplasm [34] . however, this method results in a greater than 100-fold reduction in infectivity compared with tmprss2-mediated entry [32] . notably, tmprss2 and ace2 are found in their highest concentrations in endothelial cells located in the kidneys, as well as the lung and heart, where sars and mers coronaviruses produce the most life-threatening damage [1, 23] . ace2 contains two virus-binding locations which provide a tight bond between this protein and sars-cov-2 [35, 36] . sars-cov-2 forms a larger binding surface with ace2 than with sars-cov, increasing the binding affinity. binding hotspots within ace2 are designated as hotspot 31 and hotspot 353, which likely form weak salt bridges (electrostatic interaction between amino acid side chains at secondary and tertiary levels of organization) between amino acids in the native protein that are then broken upon binding of the virus spike protein [37] . hotspot 31 breaks and forms two hydrogen bonds with the virus, whereas hotspot 353 breaks the weak salt bridge and forms a single hydrogen bond with the main chain of sars-cov-2 while maintaining one weak salt bridge. both hotspots exhibit stronger and more stable binding through the sars-cov-2-ace2 interface than the native ace2 intramolecular interactions [35, 36] . while ace2 and tmprss2 are the most well-characterized viral entry factors, it is important to note that these proteins are not the only ones that have been reported to facilitate this process [38] . sars-cov-2 portrays an evolutionary strategy that distinguishes it from other zoonotic origin strains, illustrating the possibility of human epithelial sodium channel alpha subunit (enac-alpha) mimicry in covid-19. enac is associated with the renin-angiotensin-aldosterone system (raas) and regulates sodium ion concentration and water homeostasis in distal lung airway tissue [39] . contrary to other areas of the human body, enac sodium transport is not involved in direct salt balance, but rather in maintaining appropriate hydration of the inner surface epithelial layer [39] . therefore, the key role of enac in alveolar tissue is to control the volume of airway-surface liquids. in a study performed by stoner et al., enac was evaluated in flat epithelia from toad urinary bladder and frog skin. the study portrayed the great specificity of enac for sodium; however, little was known about the exact mechanism of activation [40] . importantly, enac needs to be proteolytically activated to function in controlling fluid reabsorption in the airways [40] . it is now reported that furin cleaves enac between arginine and serine residues in the 4th and 5th position (rsar/sass) to become activated [41] . when compared to sars-cov strains, a unique sequence insertion at the s1/s2 site of the spike protein is found in sars-cov-2 [42] . sars-cov-2 is coated with spike glycoprotein (s) that requires proteolytic cleavage for activation [41] . after interaction of s with the ace2 receptor, sars-cov-2 entry into host cells occurs in two steps. once the s1 subunit of the s protein engages the ace2 receptor of the host cell, viral entry into the host cell is facilitated via s1/s2 cleavage [42] . the s2 site is then cleaved to allow the viral-host cell membranes to fuse [42] . the ability of the virus to facilitate the use of host mechanisms to cleave at s1/s2 furthers belief that sars-cov-2 has developed an evolutionarily beneficial insertion to achieve increased host infection. walls et al. show that the furin-like cleavage motifs at the s1/s2 site are present in other viruses, but the exact mimicry of enac-alpha is unique to sars-cov-2 [42] . as explained earlier, the ace2 receptors are present in a variety of different locations ranging from alveolar to intestinal cells [23] . remarkably, enac-alpha are present in apical membranes of similar cells as those of ace2 [23, 41, 43] leading to the hypothesis that sars-cov-2 leverages the protease network that is responsible for the cleavage of enac, which could subsequently lead to excessive sodium and liquid buildup in the lungs explaining clinical findings of ards. anand et al. found that the viral receptor ace2, enac-alpha, and furin were coexpressed in the colon (immature enterocytes) and pancreas (ductal cells) of human tissue and tongue (keratinocytes) of mouse cells [41] . moreover, pcsk5 and pcsk7 are expressed in the intestinal, pancreas, nasal cavity, tracheal epithelial cells, and podocytes of the kidney, similarly to that of the ace2 receptor [41] . thus far, we have discussed the viral mechanisms of sars-cov-2 and resultant covid-19 sequelae as they relate to endotheliitis and endothelial cell infection mediated by viral spike protein-ace2 interaction. we now turn our focus towards potential therapies that can be derived from an understanding of infection and disease progression as fundamentally concerned with endothelial cells and their subsequent pathophysiology. when considering potential treatments for viral infections of this nature, targets may vary based upon treatment intention. the following discussion will concern both the prevention of cellular infection and symptomatic amelioration following infection. prevention of initial cellular entry and infection should be considered separately from ameliorative therapies, as it may negate the need for further symptomatic treatment. as discussed previously, sars-cov-2 finds its receptor in the highly expressed ace2 [44, 45] . this transmembrane receptor functions primarily in the raas, as part of the body's innate homeostatic balance, acting to maintain fluid balance and blood pressure [44, 46] . an understanding of this initial viral binding event allows for the proposition of recombinant ace2 as a competitor against viral binding to the endogenous receptor. if successful, recombinant soluble receptor domains serve to "distract" the virus from endogenous ace2, thereby preventing or reducing cellular infection [44] . this approach has seen initial success in an april 2020 study of endothelial organoids. using an anatomically and physiologically analogous endothelial tissue model, monteil et al. was able to show a significant decrease (4-5-fold decrease) in viral rna upon treatment with human recombinant soluble ace2 (hrsace2) at doses of 50-100 µg/ml [44] . the validity of this approach is bolstered by the recent successful completion of phase i and ii safety trials of clinical grade hrsace2 (gsk2586881) in the treatment of ards. it is important to note that the trial of gsk2586881 concerned itself with the ability of recombinant ace2 to attenuate lung injury through its innate physiological actions, and not as a competitor for viral binding [44, 47] . however, the safety of the drug as demonstrated in phase ii trials, considered in conjunction with the proven ability to reduce viral load in an endothelial model, points to potential applications in this additional therapeutic role. the viral spike protein itself may also present a therapeutic target. published in 2020, in response to the current pandemic, a study conducted by wang et al. has confirmed the first known human monoclonal antibody against sars-cov-2. human monoclonal antibody 47d11 is a neutralizing antibody against the s1 subunit of the spike protein, and has been shown effective in the inhibition of cellular infection of veroe6 cells in vitro. additionally, as 47d11 shows cross-reactivity and neutralization of the s1 in both sars-cov and sars-cov-2, wang et al. propose that this antibody may be effective in the treatment of future coronavirus strains. it is important to note that the specific mechanism of action of 47d11 remains unknown, and is thought to be unrelated to receptor-binding interference, which serves to further differentiate 47d11 from other similar potential therapeutics [48] . in addition to prevention of initial ace2 binding, our previous discussion of sars-cov-2 activation allows for investigation into additional targets. of these targets, the most prescient appears to be the tmprss2 [45, 49] . as of june 2020, tmprss2 inhibitor camostat mesylate is undergoing phase ii trials [50] . this is a placebo-controlled, double-blinded study, with an intervention group of 200 mg oral dosing three times daily for seven days in patients over 18 years of age [50] . the primary outcome measure of this study is viral load as assessed by qpcr from days 0 to 4. secondary measures include changes in viral load at different time points after treatment, changes in symptom severity as assessed by the covid-19 daily self-score tool, changes in infection status, body temperature, and symptom frequency [50] . investigation into camostat mesylate for covid-19 is based upon previous studies showing the efficacy of the drug candidate in sars-cov-1 [49] these studies were primarily interested in the epithelial cells of the lungs, and not the vasculature [49] . however, as direct endothelial infection relies upon the action of this serine protease similarly to epithelial cell infection, it remains possible that camostat mesylate may be efficacious in the prevention of endothelial cell infection. however, additional research into the potential effects on the vasculature is needed. another successful strategy to inhibit sars-cov-2 has been through the use of remdesivir [51] . this drug has an adenosine nucleoside triphosphate analog that interferes with viral rna polymerase and is classified as a delayed chain terminator [52] . remdesivir has exhibited promise in the clinic in early studies resulting in a median recovery time of 11 days versus 15 days for placebo [51] . in addition, mortality was reduced from 11.9% to 7.1% by remdesivir [51] . this drug has shown promising results. however, the morbidity and mortality rate is still cause for concern. therapeutic approaches concerned with the abruption of viral replication processes (including remdesivir) may present relevant targets for future research not reliant on ace2 and tmprss2. these targets may include those relating to transcription, metabolism, and biomolecule synthesis [53] . endothelial pathology as a result of covid-19 progression can be roughly broken into three subcategories: coagulopathy, inflammation (including cytokine storm), and edema. as such, potential endothelial concerned treatments for covid-19 may be subdivided similarly. though both coagulative and edemic pathologies have their roots in inflammation, there are additional considerations that warrant the division as follows. a primary vascular concern of covid-19 appears to be that of rampantly dysregulated thrombotic events [54, 55] . these clotting abnormalities have resulted in stroke and disseminated intravascular coagulation (dic) in severe covid-19 patients, and may represent a significant cause of mortality [54, 56] . though blood thinners, such as heparin, have been shown to reduce mortality in severe covid-19 cases, this acts only after the development of the clotting pathology, and not to prevent the dysregulation of the clotting cascade [54, 55] . to prevent aberrant thrombosis, it is helpful to investigate the endothelial pathology that allows for upregulation of the clotting cascade. in covid-19, sars-cov-2 infection of endothelial cells and subsequent cell death, allows for pathological exposure of blood to the thrombogenic basement membrane of the blood vessel which contains collagen [57] [58] [59] . collagen exposure activates the clotting cascade. as such, actors within the clotting cascade present potential therapeutic targets. of these targets, p-selectin may be appropriate, as antibodies to this cell adhesion molecule have been used to prevent thrombosis [58] . additionally, pro-inflammatory cytokines interleukin 1 beta (il-1β) and tumor necrosis factor (tnf), are integral to the clotting cascade as they function cyclically to trigger platelet production and the upregulation of additional adhesion molecules [56, 60] . as such, antibodies against these cytokines may help to temper the hypercoagulative state and prevent fatal dic. as this clotting pathology is often inflammatory in nature, many of the proposed therapies in the treatment of the cytokine storm may be efficacious in the treatment of the clotting abnormalities and will be discussed in more detail in the following section. a cytokine storm refers to a hyperinflammatory condition related to the uncontrolled release of cytokines [12] . this condition is seen as controlled inflammation (pain, heat, redness, and swelling) that occurs as a healing response to injury and infection, becomes overactive and systemic as opposed to strictly regulated and locally active [61] . in covid-19, the cytokine storm is seen to worsen the inflammatory symptoms of the disease, including the aforementioned clotting pathologies and subsequent multi-system organ damage [59, 62] . therapeutic candidates to treat this inflammatory condition include corticosteroids and monoclonal antibodies, among others [59, 63] . an ongoing trial, currently in phase iii, tests the effects of cytokine-targeting medications in individual and combination therapy [63] . this study tests il-6 antibodies tocilizumab (anti il-6r) and siltuximab (anti il-6), as well as il-1β receptor antagonist anakinra. intervention groups include individual treatment with each of the three pharmaceuticals, as well as combination therapy with anakinra and either tocilizumab or siltuximab. the primary outcome measure of this trial is time to clinical improvement as assessed by hospital discharge or improvement in clinical indicators. secondary outcome measures include, among others, incidences of adverse events, mean change in oxygenation, and mean change in clinical scores [63] . the successful use of anti-interleukin drugs to treat the inflammatory symptoms seen in severe covid-19 would have marked effects on endothelial pathology as these cells are highly responsive to cytokine signaling [59] . this ameliorative effect would be seen in the downregulation of the prothrombotic cascades as initiated by cytokine signaling, as well as decreased inflammatory leukocytic cascades. as mentioned, corticosteroids may be effective in the treatment of inflammatory pathology. a randomized controlled trial conducted by the recovery collaborative group tests the efficacy of dexamethasone in the treatment of hospitalized covid-19 patients. the primary outcome measure of this study is mortality at 28 days. patients received oral or intravenous dosing at 6 mg/day or continued care without corticosteroid intervention. this study found that dexamethasone was effective in lowering mortality at 28 days when compared to control patients receiving mechanical ventilation or supplemental oxygen. however, the mortality rate among patients included in the intervention group was not found to be lower when compared to hospitalized patients not receiving ventilation or external oxygen [64] . edema, or swelling, is a fundamental inflammatory response that occurs primarily due to vascular leakage. similar to other hallmark inflammatory responses of pain, redness, and heat; edema occurs primarily as a result of endothelial cell function or dysfunction [61] . as reviewed by teuwin et al., in covid-19, the overactive edemic response may occur due to a weakening of the endothelium as a result of cell death secondary to direct endothelial infection [59] . additionally, the actions of cytokines il-1β and tnf weaken intercellular tight junctions and result in the overproduction of hyaluronic acid (ha) in the extracellular matrix of endothelial cells [59] . increases in vascular leakage due to cell death following direct endothelial infection may be lessened with the use of antiviral medications. these pharmaceuticals may prevent intracellular viral replication and resultant cell death [59, 65] . a potential agent in this approach is ribavirin, which may inhibit viral rna polymerase and subsequent viral replication. ribavirin was tested in both sars and mers and found significant safety concerns limiting its efficacy, though as of now, suffers from a dearth of sars-cov-2 research [65] [66] [67] . additionally, previous clinical trials showing toxicity related to necessary dosage were concerned mainly with the complete cessation of viral activity, and not with symptomatic amelioration. lower dosages that were shown to be ineffective when used as the sole antiviral treatment in sars, may be effective in combination therapy or in purely symptomatic aims [65] [66] [67] . the weakening of the endothelium may also occur as a result of increased contractility and the loosening of intercellular tight junctions due to the inflammatory properties of cytokines [59, 68] . again, edema may be treated similarly to cytokine storm pathology as the causative agents of inflammation are cytokines such as il-6 [11] . as mentioned, edema may be inflammatory in origin. in covid-19, the actions of cytokines il-1β and tnf can be seen to upregulate ha-synthase-2, resulting in increased ha concentrations in the extracellular matrix of endothelial cells. this leads to marked water retention and vascular leakage and may therefore contribute to severe respiratory pathology and ards [59, 69] . potential therapeutics in this aim include anti-inflammatory drugs against il-1β as discussed previously. through this review, we have discussed the mechanisms, pathologies, and proposed treatments of sars-cov-2 infection with a focus on the effects of direct endothelial infection and dysfunction. though the vasculature itself appears to play a large role in the pathogenesis of the disease, there is additional merit in a discussion of non-vascular infection and pathology that occurs as a function of initial endothelial infection. the following will discuss potential endothelial-derived mechanisms of extravascular infection and pathology. the healthy endothelium has roles beyond those of a vascular nature. in uninfected cells, these functions allow for and maintain the health of endothelial tissue, as well as the overall health of the organism [70, 71] . however, upon endothelial infection, innate processes may become factors driving further pathogenesis. the transdifferentiative capacity of endothelial cells, as well as the innate exocytotic machinery of these cells, may be such infectious considerations [71] [72] [73] . transdifferentiation describes the process whereby a mature cell undergoes transformation into another cell type [70, 74] . endothelial cells retain this ability into maturity and have been found to assume multiple extravascular phenotypes, as shown by fluorescent fate mapping [72, 74] . evidence suggests that descendent cell types may include cerebellar granule neurons, pancreatic acinar and β cells, duodenal and ileal crypt cells, ependymal cells, and skeletal myocytes among others [74] . additionally, genetic fate mapping in rhabdomyosarcoma has given further credence to the transdifferentiative role of endothelial progenitors in disease pathogenesis [72] . this propensity for transdifferentiation does not, on its own, imply a further infectious route for sars-cov-2 from endothelium to descendent cell. however, when considered in conjunction with the extravascular labeling that results from intravenous adeno-associated virus injection (a common labeling method in model systems), relevant conclusions may be drawn. notably, if the ability of intravenous adenovirus to label extravascular cells is due to endothelial transdifferentiation, then this labeling (adenovirus infection in descendant cells upon transdifferentiation) may allow for the inference of a similar route for sars-cov-2 infection [73] [74] [75] . in other words, the transdifferentiation of endothelial cells infected with sars-cov-2 may result in the creation of extravascular descendent cells that are similarly infected (much in the same way that adenovirus injection results in extravascular labeling), thereby furthering systemic sars-cov-2 infection. this analysis should not be interpreted to imply significant similarity between sars-cov-2 and adenovirus beyond that as a model of endothelial infection and viral propagation subsequent to transdifferentiation. the final consideration of endothelial transdifferentiation is that of process and function. due to the limited conditions under which transdifferentiation successfully occurs, endothelial sars-cov-2 infection may alter or disrupt this process [70] . alternatively, successful transdifferentiation of an infected cell may result in a descendent cell with pathologies independent from those occurring due to an active infection, though more research is needed. in addition to transdifferentiation, exocytosis is a possible route for endothelial-derived extravascular sars-cov-2 infection. similar to transdifferentiation, exocytosis can be considered as an innate function of healthy endothelial cells, which may become a driver of further infection and pathology following viral infection with sars-cov-2 [71, 73, 76] . as reviewed by hromada et al., non-pathological endothelial extracellular vesicles have functions relating to immunity, cell-to-cell communication, and tissue regeneration [71] . as discussed, sars-cov-2 is a membrane-bound virus that binds to ace2 [44, 45] . viral binding and enzymatic activities support viral membrane fusion with the endothelial cell membrane, thereby allowing viral rnas, transcription factors, and additional viral proteins to enter the endothelial cell and begin the viral replicative processes that signify an active infection [45] . exocytotic mechanisms may persist during a viral infection, and thus contribute to pathogenesis. in this case, a portion of the exocytosed cell membrane in any vesicle may be of viral origin, and thus may contain the spike proteins needed for viral binding [73, 77] . additionally, during exocytosis of the extracellular vesicle, intracellular endothelial cell contents may be packaged within the vesicle. the inclusion of these components within an exocytosed vesicle is a non-pathogenic mechanism that allows for cell-to-cell communication and transport [76] . however, in the case of an active sars-cov-2 infection, viral components within the endothelial cell may also be packaged within the vesicle [76] . as a result, there is potential for the creation of an endothelial extracellular vesicle possessing membrane-bound spike proteins externally, as well as viral rna and transcription factors internally [73, 76] . this may allow for an endothelial extracellular vesicle to effectively become a virus-like particle capable of transmitting sars-cov-2 to other vascular and extravascular cells. additionally, uninfected endothelial cells may play a role in increasing susceptibility to sars-cov-2 infection. this increased susceptibility may occur due to the exocytosis and subsequent fusion of endothelial vesicles possessing ace2 within their membrane [73, 77] . conferred sars-cov-2 susceptibility may subject the recipient cells to the viral pathology upon infection. combating the covid-19 pandemic will require a comprehensive understanding of the sars-cov-2 pathology caused at the molecular and cellular levels. given the high vaccine failure rate for many common seasonal viruses, researchers should continue to explore both vaccine approaches, and additional antiviral strategies. in this review, we have focused our attention on endothelial cells as an emerging covid-19 contributor and potential therapeutic target. this review has covered the clinical covid-19 manifestations that could be linked to endothelial cell dysfunction, the molecular biology of sars-cov-2 entry into ace2-expressing endothelial cells, immune reactions that are both dictated by and perpetuated upon endothelial cells, current covid-19 therapies reported to address aspects of endothelial cell biology, and perturbation of canonical and non-canonical endothelial cell biological processes (exosome production and transdifferentiation) by this unique virus. therefore, harnessing endothelial cell biology to prevent infection, reduce inflammatory effects and avoid sars-cov-2-mediated disturbances of natural endothelial cell physiology could provide important avenues for improving patient 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editing restores dystrophin expression in a canine model of duchenne muscular dystrophy cellular stress conditions are reflected in the protein and rna content of endothelial cell-derived exosomes exosome-mediated transfer of ace2 (angiotensin-converting enzyme 2) from endothelial progenitor cells promotes survival and function of endothelial cell this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-342538-5bwsm290 authors: izquierdo lara, r. w.; elsinga, g.; heijnen, l.; oude munnink, b. b.; schapendonk, c. m. e.; nieuwenhuijse, d.; kon, m.; lu, l.; aarestrup, f. m.; lycett, s.; medema, g.; koopmans, m. p. g.; de graaf, m. title: monitoring sars-cov-2 circulation and diversity through community wastewater sequencing date: 2020-09-22 journal: nan doi: 10.1101/2020.09.21.20198838 sha: doc_id: 342538 cord_uid: 5bwsm290 the current sars-cov-2 pandemic has rapidly become a major global health problem for which public health surveillance is crucial to monitor virus spread. given the presence of viral rna in feces in around 40% of infected persons, wastewater-based epidemiology has been proposed as an addition to disease-based surveillance to assess the spread of the virus at the community level. here we have explored the possibility of using next-generation sequencing (ngs) of sewage samples to evaluate the diversity of sars-cov-2 at the community level from routine wastewater testing, and compared these results with the virus diversity in patients from the netherlands and belgium. phylogenetic analysis revealed the presence of viruses belonging to the most prevalent clades (19a, 20a and 20b) in both countries. clades 19b and 20c were not identified, while they were present in clinical samples during the same period. low frequency variant (lfv) analysis showed that some known lfvs can be associated with particular clusters within a clade, different to those of their consensus sequences, suggesting the presence of at least 2 clades within a single sewage sample. additionally, combining genome consensus and lfv analyses we found a total of 57 unique mutations in the sars-cov-2 genome which have not been described before. in conclusion, this work illustrates how ngs analysis of wastewater can be used to approximate the diversity of sars-cov-2 viruses circulating in a community. introduction particular city or county, where the titers in sewage seem to correlate with the number of reported cases in the population, suggesting a potential role for sewage surveillance as an early warning tool 18, [20] [21] [22] . therefore, sewage testing is currently considered globally as an adjunct to patient-based surveillance, and has promise as an early warning indicator of increasing virus circulation. enhanced surveillance is a key pillar of the current containment strategy aiming to control the spread of sars-cov-2 and includes frequent testing of people with mild symptoms, investigation of clusters of infection to identify possible common exposures, and monitoring of hospital and icu admissions. whole genome sequencing of sars-cov-2 directly from clinical samples has been developed as an additional tool, to provide information on diversity of circulating strains as a basis for cluster identification. particularly in areas with minimal circulation, sequencing of viruses from patients can help to identify a possible source, provided that sufficient background sequencing has been done. so far, little work is done trying to correlate the sars-cov-2 diversity in sewage and patients 23, 24 . here we aimed to evaluate the potential of next generation sequencing (ngs) of sars-cov-2, from rt-pcr positive wastewater samples, to assess if they reflect the diversity of sars-cov-2 circulating within the population of the netherlands and belgium. previously, sewage samples were collected from different locations in the netherlands and belgium to investigate the levels of sars-cov-2 in sewage using rt-qpcr 18 . to further investigate the genetic diversity of sars-cov-2 a total of 55 wastewater samples obtained from 13 different locations in the netherlands (48 samples) and 7 different locations in belgium (7 samples) with ct values of <36 were selected for whole genome sequencing using nanopore sequencing. the samples covered a time span of 70 days (from march 25 th to june 3 rd 2020). two samples (franeker-92719 and amsterdamwest-92852) were sequenced by nanopore twice, while 24 samples were also sequenced by illumina (table 1) . four primers/probe sets targeting the n (n1-n3) 25 and the e genes 26 were used to evaluate the presence and concentration of sars-cov-2 in sewage samples as described previously 18 . all samples and their ct values are shown in table 1 . the percentage of the genome covered by the assembly of nanopore reads (>10x coverage per site) ranged from 0 to 99.2%. we observed an inverse sigmoidal correlation between the percentage of the genome assembled from nanopore sequencing reads and the ct values of both the n2 and the e primers/probe sets (fig. 1) . the inflection point (ct value at which half of the genome can be obtained) for n1, n2, n3 and e primers/probe sets were ct values of 34.6, 33.8, 33.2 . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint and 32.5, respectively. no correlation was observed between ct values and the percentage of the genome assembled from illumina sequencing data ( supplementary fig. s1 ). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint in order to associate specific mutations to particular a clade or cluster, all consensus sequences, including partial sequences, were compared to the wuhan-hu-1 reference sequence. a total of 145 single nucleotide polymorphisms (snps) compared to the wuhan-hu-1 reference sequence were detected in our dataset (supplementary table s1 ). from these, 24 snps were present in more than one sequence. the maximum number of mutations in individual samples compared to the wuhan-hu-1 reference genome were 11 for hcov-19/env/netherlands/amersfoort-92503-n/2020, hcov-19/env/netherlands/delft-92965-n/2020 and hcov-19/env/netherlands/schiphol-94335-n/2020 (supplementary table fig is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint s1). the presence of clade-defining mutations in the consensus sequence suggests the dominance of a certain clade within a sample, but assessing their presence can also be used to check for virus mixtures in a sample. nextstrain has defined each clade by the presence of at least two linked mutations (https://nextstrain.org/). 19a is the root clade and contains the wuhan-hu-1 reference sequence. both 19b and 20a emerged from 19a, where two and three linked mutations define these major clades, respectively: t28144c and c8782t for 19b; and c3037t, c14408t and a23403g for 20a. clades 20b and 20c emerged from 20a, where the trinucleotide substitution ggg > aac at positions 28881-28883 defines 20b, and the linked mutations c1059t and g25563t define 20c. nucleotide substitution a23403g, a signature mutation of clades 20a, 20b and 20c, and that generates the d614g amino acid substitution in the s glycoprotein, was present in 83.6% (51/61) of the samples that were sequenced at this region (supplementary table s1 ). the table s1 ). one of the two mutations defining clades 20c and 19b (c1059t and t28144c) were found in 2 and 3 consensus sequences, respectively. the regions containing the other clade-defining mutations (c25563t for 20c; and c8782t for 19b) were not sequenced at high enough coverage to determine a consensus sequence in these samples. the hcov-19/env/netherlands/amersfoort-92503-n/2020 sequence contained a mix of the clade-defining mutations c1059t, t28144c and ggg28881-28883aac, that define clades 20c, 19b and 20b, respectively. this indicates that the obtained consensus sequence is a combination of several different viruses and does not represent a single strain. in addition to the clade-defining mutations, we detected 49 and 63 snps in sewage samples that were not present in either the dutch (1544 sequences) or belgian (888 sequences) gisaid datasets, respectively, but were present in the global dataset (55074 sequences, as 8 th of july 2020), although with < 1% prevalence (supplementary table s2 ). moreover, we detected a total of 51 novel mutations present in sewage consensus sequences that were not previously reported (supplementary table s2 ), of which 48 were supported by coverage above the set thresholds to be considered as high quality (coverage >30x for nanopore; and coverage >5x and phred score >30 for illumina). additionally, it is noteworthy to mention that some samples presented discrepancies between the consensus sequences obtained by nanopore and/or illumina sequencing. for example, sample amsterdamwest-92852 was sequenced 3 times (twice with nanopore and once with illumina), in which 4 positions with discrepancies were found between the consensus sequences (supplementary table s1 ). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint these differences were not due to an assembly error, since the alignment of the reads were manually checked and corrected for each discrepancy in every sequencing run. these differences were explained by the presence of two different nucleotides in the reads covering a particular position with varying percentages between sequencing runs, resulting in consensus sequences that could differ between each sequencing run. these results were likely caused by both or either the presence of multiple strains and low viral rna titers in the samples, leading to an amplification bias during library preparation. given that sewage samples are likely to contain a mixture of sars-cov-2 strains, we decided to perform a variant analysis with illumina data to determine whether lfvs were confidently present in a sample. using a coverage > 50x, phred score > 30 and a frequency threshold of > 10% as settings, we found a total of 21 positions with at least one sample containing major and minor variants (table 2) table 2 ).the other variants were present at similar frequencies in the dutch, belgian and global datasets ( table 2) . in addition to consensus sequence, lfv analysis is of importance to be able to identify potential local outbreaks from sewage. to try to associate the presence of a minor variant to . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint sequences belonging to unique clusters, we mapped the 4 most highly prevalent lfvs onto both dutch-belgian and global subsample phylogenetic trees (fig 3) . this analysis indicated that for 3 variants (1440a, 11109t and 24862g) there were clear associations between the presence of the mutation and their clustering on the phylogenies (fig 3) . however, when one of these 3 variants was present as an lfv in a sewage sample the consensus sequence (blue arrows in fig. 3 ) of this sample did not group with the cluster of clinical samples that contains the variant (magenta lines in fig. 3 ). for example, 24862g variant in sample tilburg-94339 was present in two unique clusters within clade 20a, while its consensus sequence (hcov-19/env/netherlands/tilburg-94339-i/2020) was clustered within clade 20b (fig 3 and supplementary figs. s2 and s3) , suggesting the presence of both clades in this sample. although mutation 11083t was most prevalent in clade 19a, it was also present scattered along the trees, indicating poor association with a particular clade. given the high chemical and biological complexity of wastewater samples, virus concentration and rna extraction methods are crucial parts of the process to reach enough is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint viral rna yield for sequencing 29 . in this study, we showed that our method was capable of obtaining complete or near complete genomes from wastewater samples with ct values of at least 5 or 6 cts below the limit of detection (lod) (ct < 39) and partial genomes for samples with higher ct values. therefore, only samples with enough viral rna can be used to effectively analyze sars-cov-2 diversity in sewage samples. in order to increase the percentage of genome covered of the samples, we used a threshold of 10x coverage per position to generate the consensus sequences from nanopore reads. based on previous analysis of viral sequencing data, the error rate with this threshold is less than 0.03% 30 table s2 have a coverage of at least 30x, which produces an error rate of 1 in 585,000 nucleotides sequenced 30 . the use of sewage as a tool to understand the epidemiology and diversity of sars-cov-2 at a community level offers many advantages over human sampling. sewage samples are relatively easy to collect, because no invasive sampling is required, there is no sampling bias towards sequences from moderate and severe cases, there are limited ethical issues, and potentially few samples are required to give a picture of the temporal changes of viral infections in the community 28, 29 . nevertheless, comprehensive comparisons with clinical surveillance and other epidemiological approaches are required to determine the extent and limits of using sewage as a surveillance/early warning tool. furthermore, before the broad use of sewage samples to characterize viral diversity within a population, some obstacles need to be overcome, such as: low viral titers that complicate the retrieval of complete genomes and the distinction of multiple strains within a sample. here we have used two of the most common ngs technologies (nanopore and illumina) to study the diversity of sars-cov-2 found in sewage samples, from the netherlands and belgium, and compared these results with the virus diversity found in sequenced clinical samples. in order to evaluate this diversity in a comprehensive fashion, we have used nextstrain clade classification system because it is based on signature mutations to assign a sequence to a clade (https://nextstrain.org/) 5 , facilitating the association of snps or lfv to a particular clade, especially for genome sequences with <75% coverage. sewage samples can contain a mixture of sars-cov-2 viruses reflecting the multiple viruses circulating within a community. they may also partially reflect the presence of sars-cov-2 from animal origin, as sars-cov-2 has now been detected in domestic and livestock animals such rabbits, minks, cats and ferrets [31] [32] [33] [34] [35] . the analysis of a consensus sequence genome from a wastewater sample may identify the predominant virus strain present in a . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint population, which might be suitable for locations where only 1 or few introductions of closely related viruses have occurred, as it seems to be the case for 2 previous studies in italy and usa 23, 24 . nonetheless, the consensus genome approach cannot reflect the diversity of the viruses circulating in a population with a high degree of viral diversity. moreover, in some cases samples containing several diverging strains at significant levels might lead to retrieve artificial consensus genomes that do not represent an existing virus, which seems to be the case for the hcov-19/env/netherlands/amersfoort-92503-n/2020 sequence, where signature mutations of 3 different clades are present at the consensus level. in this study, we could not detect a genome belonging to the least prevalent clades (20c and 19b) , despite the circulation of these viruses in the human population in both countries during the same period of time (figs 2a and b) . although, it is necessary to mention that mutations associated with clades 19b and 20c were found in 2 and 3 samples, respectively (supplementary table s1 ). however, these consensus sequences were either too short or had a mixture of signature mutations that did not allow to confirm whether they belong to clades 20c and 19b by the nextclade tool. another reason to explain why we did not find consensus sequences belonging to these clades is the limited number of locations represented on the phylogeny by our sewage sample dataset compared with that of the clinical samples, especially for belgium (only 2 sequences from sewage). in depth ngs analysis could help to unravel the diversity of viruses within a complex sample such as wastewater, particularly unbiased sequencing of the sewage virome can give a good picture of the general viral diversity contained in a sample 36 . nevertheless, the detection of variants of a particular virus in a single sample can be difficult due to the relative low number of reads obtained for each virus type. targeted amplification of a genome region of the virus taxa of interest is potentially more sensitive and cheaper to perform. recently, examples of this have been described for enteroviruses, human mastadenoviruses and norovirus 16, 37, 38 . in general, for each virus a specific small fragment (< 400 bp) of the genome is amplified and deep-sequenced, then sequencing reads can be aligned and assigned to a particular genotype or serotype, identifying and determining the prevalence of several virus variants within a single wastewater sample 16, 37, 38 . as the diversity of sars-cov-2 is still limited 39 , this approach would not be as useful for this virus because no single small piece of the genome can reliably differentiate between clades or lineages. however, we tried to overcome this issue by using a variant analysis of sewage samples. we showed that some lfvs can be linked to particular clusters or clades within the trees (fig 3) , without the need of a complete genome. although, in order to confidently determine the presence of a particular clade/cluster within a sample, at least 2 or 3 lfvs associated with such clade/cluster should is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint be present at significant levels. furthermore, variant analysis can also be used to monitor the prevalence of biological interesting mutations in a population. one of the most interesting is the d614g (or a23403g) mutation in the s glycoprotein, that has been shown to increase infectivity in vitro by stabilizing the s1/s2 interaction 40 , and has been associated with higher transmission and mortality rates, although the latter is under debate 41, 42 . unfortunately, the region containing the d614g mutation was not sequenced at high enough coverage to perform a variant analysis in most of the tested samples, and we were not able to find any sample with a mixture of both variants (614d and 614g) . the combination of whole-genome sequencing of clinical samples with epidemiological data has shown to be important for public health decision-making 43 wastewater can also be used to monitor novel mutations. our consensus and lfv analyses revealed a total of 57 mutations that were not present in the global database. from these, 51 were found at the consensus level, 8 as lfvs and 2 were common to both analyses. it is possible that these novel mutations were not previously detected due to several reasons: 1) genetic drift eliminated these variants before they could be further spread and detected; 2) these viruses cause only asymptomatic or mild disease that made them to be less likely to be detected through clinical sampling; 3) they are associated with reduced transmission/replication and did not became fixed in the population; 4) they originate from an unknown animal hosts; 5) they are associated with enhanced enteric shedding/replication; 6) they are associated with intra-host defective genomes. the latter has previously been suggested for the detection of lfvs that generate stop codons in orf1ab and s genes 44 is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint polymerase mistake during the initial pcr cycles of the library preparation can be amplified and identified as a variant. phenotypical studies could help to determine the likelihood and biological relevance of some of these novel mutations. in conclusion, this work illustrates how ngs analysis of wastewater can be used as a tool to approximate the diversity of sars-cov-2 viruses circulating in a community. sequencing of wastewater samples could be a powerful tool to complement clinical surveillance or be used as a standalone procedure in settings where wide clinical sequencing is not feasible. additionally, in-depth ngs analysis of wastewater samples can help in assessing changes in virus diversity to determine emergence of epidemiologically or clinically relevant mutations, aiding public health decision making. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint a total of 55 wastewater (ww) specimens were included in this study. rna from 7 of these ww samples (all from march 25 th ) were collected, processed and extracted previously by kwr 18 . the other 48 ww specimens were collected as 24 h flow-dependent composite samples and processed as previously described 18 illumina sars-cov-2 specific multiplex pcr for nanopore sequencing was performed as described by oude munnink, et al. (2020) 43 . in short, primers for 89 overlapping amplicons spanning the entire genome were used in 2 pcr pools. the amplicon length was set to 500bp with 75bp overlap between the different amplicons. the used concentrations and primer sequences have been described previously 43 . libraries were generated using the oxford nanopore's native barcode kits (catalog numbers: exp-nbd104, exp-nbd114 and sqk-lsk109) and sequenced on a r9.4 flow cell multiplexing up to 24 samples per sequence run. illumina sequencing was performed as described by richard, et al. (2020) 45 . amplicons were generated by the sars-cov-2 specific multiplex pcr described above for the whole . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the resulting raw sequence data were processed as previously described by oude munnink et al. 2020 43 . briefly, an automated snakemake script 46 was used to demultiplex fastq raw reads using porechop (https://github.com/rrwick/porechop), trim primers using cutadapt 47 and perform a reference-based alignment using minimap2 to gisaid sequence epi_isl_412973. the run was monitored using rampart (https://articnetwork.github.io/rampart/). the consensus genome was extracted and positions with a coverage < 10x or <30x were replaced with an "n". mutations in the genome were confirmed by manually checking the alignment in ugene 48 and homopolymeric regions were manually resolved consulting reference genomes. based on previous validation studies 30 , mutations with a cut-off of 30x coverage were considered as high quality, whereas mutations with less than 30x coverage were marked as low quality (supplementary table s2 ). all the processing, reference-based alignment and variant analysis of the illumina generated data was performed using a customized workflow on the galaxy eu server (https://usegalaxy.eu/) 49 . first, raw sequencing reads were filtered using fastp 50 to remove adaptor contamination, ambiguous bases (n), low quality reads (phred score <30) and fragments below the length of 50 nt. for mapping purposes, reads were aligned against the gisaid sequence epi_isl_412973 using the default penalty settings of the bwa-mem 51 . reads were re-aligned using the leftalign utility from freebayes package 52 . all reads with mapping scores of less than 30 were discarded. both consensus sequences and variants were generated using ivar 53 . final consensus sequences (frequency >50%) were . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint constructed using all mapped sequence reads that covered each site at least 5 times and had a minimum quality phred score of 30. for detection of low-frequency variants (lfv), parameters were set as follows: a minimum coverage of 50x, phred score >30 and a minimum frequency threshold of 10%. manual inspection of the aligned reads was also performed to confirm or dismiss the variant calling in ugene 48 . variant positions are given with respect to the wuhan-hu-1 strain (genbank accession number: mn908947) 1 . two reference datasets were used to perform the phylogenetic analysis. the first dataset included all dutch and belgian full-length sars-cov-2 genomes (1544 and 888 sequences, respectively) from gisaid database (https://www.gisaid.org/) publicly available up to the 8 th of july 2020. the second dataset is a subsample of all sars-cov-2 sequences available in gisaid (https://www.gisaid.org) covering the global diversity of sars-cov-2 genomes up to the 1 st of june 2020. this global 'backbone' dataset contains 2552 subsampled high-quality sequences (full length, with ns <5%) to include one unique genome per country/state per week. for the maximum-likelihood (ml) trees, only sequences in this study with >75% genome coverage were included in the analysis. our sequences were aligned with both datasets using mafft (https://mafft.cbrc.jp/alignment/server/). the alignment was manually checked for discrepancies and the ends were trimmed, after which iq-tree 54 was used to perform a ml phylogenetic analysis under the gtr + f + r3 model for the global subsample and the gtr + f + r2 model for the dutch-belgian dataset as the best predicted models using the ultrafast bootstrap option with 1,000 replicates. the phylogenetic trees were visualized using figtree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). clades were assigned by using the nextclade tool within nextstrain (https://clades.nextstrain.org/). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 22, 2020. . https://doi.org/10.1101/2020.09.21.20198838 doi: medrxiv preprint a new coronavirus associated with human respiratory disease in china global initiative on sharing all influenza data -from vision to reality a dynamic nomenclature proposal for sars-cov-2 lineages to assist genomic epidemiology nextstrain: real-time tracking of pathogen evolution sars-cov-2 productively infects human gut enterocytes evidence for gastrointestinal infection of sars-cov-2 infectious sars-cov-2 in feces of patient with severe covid-19 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structure, function, and antigenicity of the sars-cov-2 spike glycoprotein angiotensin-converting enzyme 2 (ace2) is a key modulator of the renin angiotensin system in health and disease monitoring human enteric viruses in wastewater and relevance to infections encountered in the clinical setting: a one-year experiment in central france detection of sars-cov-2 in different types of clinical specimens presence of sars-coronavirus-2 rna in sewage and correlation with reported covid-19 prevalence in the early stage of the epidemic in the netherlands surveillance of influenza a and the pandemic influenza a (h1n1) 2009 in sewage and surface water in the netherlands sars-cov-2 titers in wastewater foreshadow dynamics and clinical presentation of new covid-19 cases sars-cov-2 rna in wastewater anticipated covid-19 occurrence in a low prevalence area time course quantitative detection of sars-cov-2 in parisian wastewaters correlates with covid-19 confirmed cases presence and infectivity of sars-cov-2 virus in wastewaters and rivers temporal detection and phylogenetic assessment of sars-cov-2 in detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr architecture and self-assembly of the sars-cov-2 nucleocapsid protein wastewater and public health: the potential of wastewater surveillance for monitoring covid-19 sewage analysis as a tool for the covid-19 pandemic response and management: the urgent need for optimised protocols for sars-cov-2 detection and quantification validating whole genome nanopore sequencing, using usutu virus as an example susceptibility of rabbits to sars-cov-2 sars-cov-2 infection in farmed minks, the netherlands transmission of sars-cov-2 in domestic cats sars-cov-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 setting a baseline for global urban virome surveillance in genetic diversity and quantification of human mastadenoviruses in wastewater from sydney and melbourne detection of norovirus epidemic genotypes in raw sewage using next generation sequencing geographic and genomic distribution of sars-cov-2 mutations the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity sars-cov-2 amino acid substitutions widely spread in the human population are mainly located in highly conserved segments of the structural proteins sars-cov-2 genomic variations associated with mortality rate of covid-19 rapid sars-cov-2 whole-genome sequencing and analysis for informed public health decision-making in the netherlands sars-cov-2 exhibits intra-host genomic plasticity and lowfrequency polymorphic quasispecies sars-cov-2 is transmitted via contact and via the air between ferrets the galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update fastp: an ultra-fast all-in-one fastq preprocessor aligning sequence reads, clone sequences and assembly contigs with bwa-mem haplotype-based variant detection from short-read sequencing an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies key: cord-336150-l8w7xk0b authors: rathore, jitendra singh; ghosh, chaitali title: severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a newly emerged pathogen: an overview date: 2020-08-25 journal: pathog dis doi: 10.1093/femspd/ftaa042 sha: doc_id: 336150 cord_uid: l8w7xk0b coronavirus disease 2019 (covid-19) is a viral pneumonia, responsible for the recent pandemic, and originated from wuhan, china, in december 2019. the causative agent of the outbreak was identified as coronavirus and designated as severe acute respiratory syndrome coronavirus 2 (sarscov-2). few years back, the severe acute respiratory syndrome coronavirus (sarscov) and the middle east respiratory syndrome coronavirus (mers-cov) were reported to be highly pathogenic and caused severe infections in humans. in the current situation sars-cov-2 has become the third highly pathogenic coronavirus that is responsible for the present outbreak in human population. at the time of this review, there were more than 14 007 791 confirmed covid-19 patients which associated with over 597 105 deaths in more then 216 countries across the globe (as reported by world health organization). in this review we have discussed about sars-cov, mers-cov and sarc-cov-2, their reservoirs, role of spike proteins and immunogenicity. we have also covered the diagnosis, therapeutics and vaccine status of sars-cov-2. on december 31, 2019, several cases of severe pneumonia were reported from wuhan, china. the causative agent of the outbreak was identified as betacoronavirus. genome sequencing revealed that it is closely related to the sars-cov (severe acute respiratory syndrome coronavirus) which had emerged in 2003, and is designated as sars-cov-2 (gorbalenya et al. 2020; zhou et al. 2020) . in a very short duration, more than 80 000 infectious cases including more than 3000 deaths were reported in china as on march 15, 2020. at the time of this review (18 may, 2020), the disease, termed as covid-19 (corona virus disease 2019), had already become pandemic and spread to more than 216 countries and territories, including community transmissions in countries like the united states, germany, france, spain, japan, singapore, south korea, iran, italy and india. as on july 19, more than 14 007 791 cases and 597 105 deaths had been reported globally, with the rapid growth of numbers in many countries. for the up-to-date information about covid-19, visit the world health organization (who) website (https://www.who.int/emer gencies/diseases/novel-coronavirus-2019). the bats are likely to be the origin of sars-cov-2, but the role of an intermediate host cannot be ruled out at this stage. initial studies showed that sars-cov-2, can use angiotensinconverting enzyme 2 (ace2) from bats, cats, civet cats, swine, ferrets, non-human primates (nhps) and humans as a receptor (letko, marzi and munster 2020; wan et al. 2020a; zhou et al. 2020) . a pet dog in hong kong and a tiger in bronx zoo in the united state of america tested positive with sars-cov-2 infection, indicating that canine ace2 can also be recognized by sars-cov-2. pangolins, which are endangered animals and are illegally imported into southern china (guangdong and guangxi provinces), have been considered as a potential intermediate host (lam et al. 2020; zhang et al. 2020b) . the initial reports showed that in most of the covid-19 cases there was mild to moderate infection. however, approximately 20% of the cases were reported severe (chen et al. 2020; wang et al. 2020a) . in this review, we will discuss about sars-cov, mers-cov and sarc-cov-2. we have also discussed about various reservoirs, associated with them. in the end, we have covered the role of spike proteins and their immunogenicity along with the diagnosis, therapeutics and vaccine status of sars-cov-2. zoonotic coronaviruses are becoming a global concern as there was emergence of earlier two coronaviruses, sars-cov and mers-cov (middle east respiratory syndrome coronavirus) which created a havoc and recently emergence of the third highly pathogenic sarc-cov-2. it has been observed that members of the family coronaviridae are known to infect a wide range of vertebrates and humans. before the outbreak of sars (severe acute respiratory syndrome), only two coronaviruses including hcov-229e and hcov-oc43 were known to infect humans. however, post-sars outbreak, the sars coronavirus (sars-cov), human coronavirus hcov-nl63, human coronavirus hcov-hku1 and mers-cov have been isolated from humans. similar to sars-cov and mers-cov, the newly isolated sarc-cov-2 is highly pathogenic in humans and causes severe acute respiratory distress (shi, guo and rottier 2016) . the genomes of coronaviruses consist of a positive and single-stranded rna genome of about 30 kb. the 5 terminus encodes the enzyme viral replicase/transcriptase, which is involved in virus replication, whereas the 3 terminus encodes viral structural proteins and virus group specific accessory proteins. functional studies of these viral proteins in detail are essential for antiviral drug screening and vaccine development. the earliest available genome sequencing data of sars-cov-2 made it possible to compare it with the genomes of sars-cov and other coronaviruses. results showed that sars-cov-2 belongs to the genus betacoronavirus and subgenus sarbecovirus, which also includes sars-cov. however, the mers-cov belongs to another subgenus, merbecovirus (lu et al. 2020; zhou et al. 2020) . the comparison study also showed that there is 79% nucleotide similarity between sars-cov-2 and sars-cov. the essential surface glycoprotein of sars-cov-2 known as spike (s) protein, essential for host cell receptor binding, showed only 72% similarity with sars-cov at the nucleotide level. the genomic organization of sars-cov-2 resembles those of other betacoronaviruses, including 5'-orf1ab-s (surface glycoprotein)-orf3a-e (envelope)-m (membrane glycoprotein)-n-3' as shown in fig. 1 . comparative genome analysis of ratg13, a virus from a rhinolophusaffinis (i.e. horseshoe) bat sampled from yunnan province in china in 2013, with sars-cov-2, showed that sars-cov-2 has 96% similarity at the nucleotide sequence level . although the sars-cov-2 and ratg13 have high similarity, yet they differ in some genomic features, such as sars-cov-2 contains a polybasic (furin) cleavage site insertion (residues prra) between the s1 and s2 subunits of the surface glycoprotein s protein (coutard et al. 2020) . polybasic insertion may increase the infectivity of the virus, as it is absent in other related betacoronaviruses. however, similar polybasic insertions are observed in different human coronaviruses, such as hcov-hku1 and highly virulent strains of avian influenza viruses. therefore, whether polybasic insertion between s1 and s2 subunits of s protein occurs due to the natural evolution in sars-cov-2 or by other means is going to be the topic of debate in the future. however, an independent insertion(s) of the amino acids paa at the s1/s2 cleavage site was also observed in armyn02 virus (having 72% similarity in spike protein and 97% similarity in replicase nucleotide) isolated from rhinolophus bat in yunnan province in mid-2019, indicating that these insertion events may be a natural part of ongoing coronavirus evolution . the receptor-binding domain (rbd) of s protein is essential to interact with the ace2 receptor present on the surface of the target cells of the host. therefore, comparative sequence analysis was performed and results showed that there is 85% similarity in rbd between sars-cov-2 and ratg13, but they share only one amino acid among the six key amino acid residues. further, due to the proteinaceous nature of spike, structural comparisons were also performed, suggesting that the rbd domain of the sars-cov-2 is well suited to interact with the human ace2 receptor. interestingly the same receptor was also utilized by sars-cov to cause infection (wrapp et al. 2020) . the sars-cov-2 is closely related to sars-cov and mers-cov having bat as reservoirs, but there are huge biological differences in the former as compared to the other two. the sars-cov-2 is markedly more infectious and has very different epidemiological dynamics. moreover, the mers-cov has never been able to fully adapt to human transmission (sabir et al. 2016) , whereas there is the remarkable local and global spread of sars-cov-2. as in the case of sars and mers, the intermediate host including civets and camels, respectively, played an important role and may be considered as a true reservoir host (sabir et al. 2016) . therefore, due to the ecological separation between a bat (reservoir) and humans, 'intermediate' or 'amplifying' mammalian host is a must to acquire mutations in sars-cov-2, and is essential for the efficient human transmission. to determine the intermediate host, it is essential to perform a wider sampling of animals that live close to human populations or available in wet markets for human consumption. surprisingly, there was discovery of viruses, closely related to sars-cov-2 from the malayan pangolins (manisjavanica) that are illegally imported into southern china (guangdong and guangxi provinces). it has been observed that rbd domain of guangdong pangolin viruses are particularly closely related to sars-cov-2. there is a 97% amino acid sequence similarity and contain all six critical key mutations that are essential for binding to the ace2 receptor in these viruses. however, the rest of the genome is highly divergent from sars-cov-2. hence, the evolution of coronaviruses in animal reservoirs as well as in intermediate hosts is required to explain the emergence of sars-cov-2 in humans. it might be possible that due to its asymptomatic infection, the virus could have acquired some of its essential mutations during a period the ''cryptic'' spread in humans before it was first detected in december 2019. recombination is another possibility, which cannot be ruled out as sarbeviruses, and coronaviruses experience widespread recombination. the genome of sarbeviruses experience recombination at multiple locations, including spike protein. there are studies, which showed that recombination does occur among sars-cov-2, ratg13 and the guangdong pangolin covs (lam et al. 2020) . the genome of rmyn02 too has been impacted by recombination . because of the small recombinant region, which may likely change as we increase the sample size of viruses related to sars-cov-2, it would be difficult to determine the pattern and genomic ancestry of recombination. a total of 8422 cases with 916 deaths in 29 countries including china were reported due to the human respiratory disease during 2002-2003, caused by the sars-cov. the studies showed that bats acted as a natural reservoir of sars-cov that caused the outbreaks (chan-yeung and xu 2003) . later, the sars-cov like similar antibody and genomic sequences were also discovered in rhinolophus bat, such as in r. ferrumequinum, r. pearsoni, r. sinicus, r. pusillus and r. macrotis (lau et al. 2005) . the comparative study of the genomes revealed that bat sars-like covs (sl-cov) have 78-92% nucleotide sequence identities with sars-cov and also among themselves, and hence display great genetic diversity. further, the phylogenetic analysis pointed out that rhinolophus bat might be the direct progenitor of human sars-cov (hon et al. 2008) . various sars-cov groups were isolated in different epidemic periods and hosts. several methods have been adopted to investigate the selective pressure. results have shown that the most functional proteins of sars-cov adopted the stepwise adaptive evolutionary pathway. for example, the spike protein showed strong positive selection in the early as well as middle phases, and not in the late phase. however, the replicase enzyme experienced positive selection only in humans, and assembly proteins experienced the same in the middle and late phases. interestingly, no such positive selection was observed in any proteins of bat sars-like-cov. however, specific amino acid sites that may be the targets of positive selection in each group were identified (tang et al. 2009 ). later in 2010, a study suggested the presence of two distinct genotypes of bt-slcov in r. sinicus (i.e. rp3/rs672 and hku3/rs806). the results also showed the evidence for the recombinant origin of rp3 and rs672. the phylogenetic study showed that their major parent has a relatively closer relationship with hu-scovs. therefore, there may be a possibility for the presence of a bt-scov lineage in r. sinicus, that may have hu-scovs as their direct ancestor, as reported earlier (hon et al. 2008) . however, these speculations are based on studies done on limited strains only, therefore an extensive analysis for the prevalence of such genotype is required for its credibility (yuan et al. 2010) . in 2012, globally, 64 human cases were confirmed resulting in 38 deaths by june 17, 2013, by a disease having symptoms similar to sars, that emerged in saudi arabia (who 2013). later, it was found that the disease was caused by a virus designated as a novel human coronavirus, mers-cov, phylogenetic data showed that it belonged to lineage c of the betacoronavirusgenus and was highly similar to bat coronaviruses hku4 (tylonycterispachypus) and hku5 (pipistrelluspipistrellus; lau et al. 2013) . comparative genomic results showed that mers-cov has a 50% nucleotide identity in the entire genome with hku4 and hku5. moreover, the rna dependent rna polymerase (rdrp) gene has 82% nucleotide identity. later, while studying the mode of entry into the cell it was confirmed that mers-cov uses dipeptidyl peptidase 4 (dppiv), also known as cd26. as dppiv is evolutionarily conserved among mammals, therefore mers-cov can infect a broad range of mammalian cells (humans, pigs, monkeys and bats) and may be efficient in cross-host transmission (raj et al. 2013) . similar to the case for sars-cov and mers-cov , the bat is still a probable species for origin of sars-cov-2, as it shares 96% whole-genome identity with a bat cov, batcov ratg13, from rhinolophusaffinis from yunnan province ). however, sars-cov and mers-cov before entering humans pass through intermediate hosts, such as civets or camels (cui, li and shi 2019) . this fact indicates that sars-cov-2 was probably transmitted to humans by other animals. by comparing the overall genome identity, it was concluded that pangolin-cov genome sequence is 90.55% identical to ratg13 and 91.02% identical to sars-cov-2. however, there was 96.2% identity between sars-cov-2 and ratg13 . other sar like covs (sl-covs) are also showed similarity with pangolin-cov, as its was 85.65% similar to zxc21 and 85.01% with zc45. in a comparative genome analysis between pangolin-cov and sars-cov-2 (genbank: mn908947), result showed 45.8-100% coverage range (average coverage 76.9%). moreover, pangolin-cov genes shared high average nucleotide (93.2%) and amino acid identity (94.1%) with sars-cov-2 (genbank mn908947). similar results were obtained when pangolin-cov genes were compared with ratg13 where 92.8% nucleotide and 93.5% amino acid identity was observed (zhang, wu and zhang 2020) . interestingly, some of the pangolin-cov genes showed higher amino acid sequence identity to sars-cov-2 genes than to ratg13genes. for example orf1b of pangolin-cov 73.4%, the spike (s) protein 97.5%, orf7a 96.9% and orf10 is 97.3% identical to sars-cov-2. similarly, orf1b 72.8%, the spike (s) protein 95.4%, orf7a 93.6% and orf10 is 94.6% identical to ratg13. the high s protein amino acid identity governs the functional similarity between sars-cov-2 and pangolin-cov. a comprehensive phylogenetic analysis was performed based on the nucleotide sequences of whole-genome sequence, rna-dependent rna polymerase gene (rdrp), non-structural protein genes orf1a and orf1b, and main structural proteins encoded by the s and m genes. results showed that in all phylogenetic trees, pangolin-cov, ratg13 and sars-cov-2 were clustered into a well-supported group designated as ''sars-cov-2 group'' which represents a novel betacoronavirus group. however, within this group, ratg13 and sars-cov-2 were grouped together, and pangolin-cov was their closest common ancestor (zhang, wu and zhang 2020) . recently, an extensive study including localized genomic analysis and the pattern of evolutionary recombination was done. the results showed that the strong purifying selection among coronaviruses from distinct host species as well as cross-species infections is responsible for the origin of sars cov-2 ). therefore, we may summarize the origin and intermediate hosts of sars-cov, mers-cov and sars-cov-2 as shown in fig. 2 . in the s1 domain of s protein, followed by fusion with cell membrane. sars-cov is responsible to cause severe acute respiratory syndrome. sars-cov utilizes angiotensin-converting enzyme 2 (ace2) receptor present on the surface of host cells, as shown in fig. 3 . sarc like-covs and sars-covs have identical genetic organizations with high sequence identities. the schematic representation of spike protein (s) from sars-cov-2 is shown in fig. 4 . however, there is some important exception at the n' terminus of spike protein (s), essential for receptor binding in covs. there is a study to investigate the receptor usage by full-length s of sl-cov, sars-cov and a series of s chimeras. different ace2 receptors from human, civet, or horseshoe bat were expressed in cell lines by using human immunodeficiency virus-based pseudovirus system. several important observations were made in the study. first, the sl-cov s was unable to use any of the three receptors. second, the sars-cov s was unable to enter the cells expressing bat ace2. third, the chimeric s enters the cells with different efficiencies for different constructs via human ace2. fourth, a minimal insert region (amino acids 310-518) was sufficient to convert the sl-cov s from non-ace2 binding to human ace2 binding, indicating that the sl-cov s is largely compatible with sars-cov s protein, both in structure and function (ren et al. 2008) . detailed structural study of human sars-cov rbd complexed with human ace2 receptors was performed. results revealed that it is the truncations in the receptor-binding motif (rbm) region of sl-cov spike protein, which abolished its human ace2-binding ability (li 2008) ). therefore, we may hypothesize that the sl-cov found in horseshoe bats is not the direct ancestor of human sars-cov. moreover, it has been observed that the human sars-cov, as well as its closely related civet sars-cov spike proteins, were not able to use a horseshoe bat (r. pearsoni) ace2 as a receptor for cell entry (ren et al. 2008) . these findings highlight a critical missing link (an intermediate host) in the bat-to-civet/human transmission chain of sars-cov (hou et al. 2010 ). an earlier study showed that ace2 from horseshoe bat could not function as a receptor for sars-cov. however, changing 3 amino acids (40, 41 and 42 amino acids) from she to fyq was found adequate to convert the nonfunctional bat ace2 into a fully active receptor for sars-cov. further, an ace2 molecule from a fruit bat, which naturally has the fyq motif, supports sars-cov entry into the cells thus causing infection. this result indicates that there must be a wide host range for sars-cov-related viruses among different bat populations (yuan et al. 2010) . in the case of sars-cov-2, the structural bioinformatics approaches accurately predicted that sars-cov-2 spikes bind human ace2 (wan et al. 2020b ). when cell lines over-expressed the transmembrane protein 'angiotensin-converting enzyme 2' (ace2) from humans, bats, pig or civet cats and were infected with sars-cov-2, results showed that they became hypersensitized to infection, thus indicating that ace2 is a sars-cov-2 receptor . the binding studies also revealed that receptor-binding domains on the sars-cov-2 s proteins have a high affinity to human ace2 (wrapp et al. 2020 ) which makes it more virulent. however, apart from ace2 interaction, the n-terminal domain (ntd) of the sars-cov-2 s proteins may show binding to alternative host-cell receptors . sars-cov-2 s proteins have also acquired a furin protease cleavage site, by acquiring several basic residues (rrar/s). the sars-cov-2 furin substrate site facilitates the prime cleavage step, which further sensitizes s proteins for subsequent activation of cleavages occurring on susceptible target cells, and finally facilitates virus to enter the cells and cause infection (qing and gallagher 2020) . human sars-cov and sars-like coronavirus (sl-cov) in bats have a similar genomic organization; therefore their corresponding gene products are highly conserved. as far as s protein is concerned, it has only a 63-64% sequence identity at the n-terminal region. it is the n-terminal region of coronavirus s protein that is responsible for receptor interaction. when the immunogenicity of the sl-cov s protein was analyzed and compared with that of sars-cov, results revealed that they shared only a limited number of immunogenic epitopes in their s proteins. moreover, major neutralization epitopes were also different ). in another study, a pseudovirus expressing full-length sl-cov s protein was used to raise mouse sera and monoclonal antibody. series of constructs expressing truncated s protein were prepared and analyzed with elisa, as well as western blot. results showed that amino acids 280-455 and amino acids 561-666 are two immunogenic determinants in mice. further, it was also shown that 280-455 amino acids are more immunogenic, as it was recognized by polyclonal as well as monoclonal antibodies. earlier studies also showed that amino acids 528-635 from sars-cov are immunodominant determinants (he et al. 2004 ). due to the high sequence similarity with sl-cov s protein in the same region, the amino acids 561-666 of s protein also demonstrated immune response in mouse (zhou et al. 2013 ). in a cross-reactivity test with antibodies against rbd domain sars-cov, some of the sl-cov strains (wivi) have shown positive results whereas some strains (shc014) failed too. this difference in reactivity is due to the low sequence identity in the rbd domain of shc014 and high sequence identity in the rbd domain of wivi with rbd domain of sars-cov (zeng et al. 2017 ). detection of novel coronavirus is done by different molecular biology techniques including real-time reverse transcription pcr (rrt-pcr), reverse transcription pcr (rt-pcr), reverse transcription loop-mediated isothermal amplification (rt-lamp), multiplex nucleic acid amplification, real-time rt-lamp and microarray-based assays . who also recommended a pan-coronavirus assay for characterization and confirmation.). viral culture and rt-pcr are among the most efficient and reliable methods for the diagnosis of sars-cov-2 infection. these methods are time consuming and generally takes hours to detect the nucleic acid and many days to isolate the virus from the samples. apart from that, specialized equipments and expertise are also required. to overcome these limitations, rapid diagnosis of sars-cov-2 infection can be done with rapid antigen detection (rad) tests. in rad tests, the immobilized sars-cov-2 antibody on the device can detect viral antigen in the sample. the results of rad tests are prompt and interpreted without specialized instrument. hence, rad tests could be beneficial reduce the workload in diagnostic laboratories and hospitals (mak et al. 2020) . however, as per the who, rad tests for sars â�� cov-2 antigen detection, further needs evaluation and is not recommended for clinical diagnosis (laboratory testing strategy recommendations for covid-19: interim guidance 2020). the immune response to sars-cov2 in the early weeks of the infection can be detected using enzyme-linked immunosorbant assay (elisa), automated chemiluminescence immunoassay (clia), and lateral flow immunoassay (lfia), plaque reduction neutralization tests (prnt), or a combination of these methods (espejo et al. 2020) . the most commonly antigens used in these assays were the spike glycoprotein s1 including the receptor binding domain (jin et al. 2020 ), the nucleocapsid protein or both (pang et al. 2020) . application of inhibitor to halt virus interactions with the host may be one of the prophylactic methods. in this direction, an engineered pan-cov fusion inhibitor has been designed and designated as ek1 peptide. it has shown promising results in mice by inhibiting the infection in five human coronaviruses, including sars-cov, mers-cov and three bat-sl-covs (xia et al. 2019) . it has also been reported that intranasal application of engineered ek1 peptide before or post viral infection showed protection in human dpp4-transgenic mice against mers-cov infection, indicating its potential prophylactic and therapeutic effect. another approach is designing of neutralizing antibody, which may block the interaction with the host cell. the s proteins of sars-cov and mers-cov are immunogenic. the rbd domains of sars-cov and mers-cov are known to have nonsequential epitopes that induce a more potent neutralizing antibody and give protection against sars-cov and mers-cov (du et al. 2009; zhou et al. 2019) . the modification on the structural basis for mers-cov s-rbd amino acid has improved the efficacy against mers-cov infection (zhou et al. 2019) . therefore, we may suggest that sars-cov-2 s-rbd or modified s-rbd of another related coronavirus could be used as target to develop a vaccine against sars-cov-2. recently, neutralizing monoclonal antibodies and nanobodies against the rbd domain of s protein showed protection against sars-cov and mers-cov (du et al. 2009; zhou et al. 2019) . although the ntd and s2 unit of s protein from sars-cov and/or mers-cov was also studied to develop neutralizing antibodies, but the efficacy was found to be very low (du et al. 2009 ). therefore, rbd of s protein sars-cov-2 would be a key target for developing neutralizing antibodies as shown in fig. 5 . cross protection by the antibodies developed against sars-cov, has been observed against bat-sl-cov-w1v1 and bat-sl-cov-shc014 (zeng et al. 2017) . therefore, the development of cross-neutralizing antibodies can be another possible way for urgent prevention and treatment of sars-cov-2 infection. currently, plasma therapy in which polyclonal antibodies from recovered sars-cov-2-infected patients have been used to treat sars-cov-2 infection is also being considered. researchers are working hard to develop monoclonal abs (mabs) and once such antibodies are produced, the next step will involve in vitro testing for neutralizing and/or crossneutralizing activity as well as in vivo evaluation for protective efficacy in available covid-19 animal models. preclinical and clinical trials testing the safety and and efficacy before they are approved for clinical applications are also necessary. recently, 1000 memory b cells specific to sars-cov-2 s1 or rbd (receptor binding domain) have been purified. among these, 178 antibodies showed positive results in antigen binding assays with the top 17 binders having ec 50 below 1 nm specific for rbd. further, among 11 neutralizing antibodies, eight of them have shown an ic 50 value within 10 nm, whereas 414-1 best among all have ic 50 of 1.75 nm. in epitope mapping, three main epitopes recognized by monoclonal antibodies have been identified in rbd domain. interestingly, 515-5 monoclonal antibody from same study, also showed cross-neutralizing property in the sars-cov pseudovirus assay (wan et al. 2020a) . in another study, 61 sars-cov-2-neutralizing monoclonal antibodies were isolated from five infected patients. 19 among them have shown positive result in in vitro neutralization assay and nine among them shown 50% virus-inhibition at the concentrations of 1-9 ng/ml. epitope mapping showed that receptor-binding domain (rbd) and the n-terminal domain (ntd), both are immunogenic in nature. further, structural studies of these monoloclonal antibodies have proven that one is targeting rbd, second one is targeting ntd and a third bridging rbd and ntd. therefore, several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against sars-cov-2 (l et al. 2020). due to the high sequence identity of s protein between sars-cov-2 and its closely related sars-cov , sars-cov nabs have been tested for its cross-reactivity and/or cross-neutralizing activity against sars-cov-2 infection. interestingly, a sars-cov rbd-specific human neutralizing mab, cr3022, have shown the binding of sars-cov-2 rbd with high affinity and may recognize an epitope on the rbd that does not overlap with the ace2-binding site (tian et al. 2020) . further, sars-cov-2 entry and infection may be blocked by crossreacting the sera isolated by convalescent sars patients or from animals specific for sars-covs1 (hoffmann et al. 2020) . moreover, it has been observed that polyclonal antibodies against the rbd domain of sars-cov have been cross-reacted with the rbd protein of sars-cov-2. they cross-neutralized sars-cov-2 infection in hek293t cells expressing the human ace2 receptor. such findings may open new avenues for the potential development of sars-cov rbd-based vaccines that might eventually prevent sars-cov-2 and sars-cov infection (tai et al. 2020) . it may be possible that sars-cov rbd-targeting neutralizing antibodies could be applied for treatment/prophylaxis of sars-cov-2 infection in the current absence of a specific vaccine against sars-cov-2. remdesivir has been recently recognized as a promising antiviral drug in cultured cells, mice and nonhuman primate (nhp) models, against rna viruses including sars-cov and mers-cov (sheahan et al. 2017) . it is currently under clinical trials for the treatment of ebola virus infection (mulangu et al. 2019) . recently studies have shown that ec 90 value of remdesivir against 2019-ncov in vero e6 cells was 1.76 î¼m. these data suggest that its working concentration is likely to be achieved in nhps remdesivir have shown the efficient in vitro antiviral activity against sars-cov-2. however, the controversial evidence of clinical improvement in severe covid-19 patients has been reported recently in france. the five covid-19 patients admitted in icu and treated with remdesivir. treatment showed significant reduction of sars-cov-2 viral load from upper respiratory in most of the cases, however but two patients died with active sars-cov-2 replication in their lower respiratory tract. remdesivir treatment was interrupted for its side effects among four patients due the complexity in such critically ill patients (dubert et al. 2020 ). the first covid-19 case in the united states was intravenously treated with remdesivir (iv) (holshue et al. 2020) . within 24 h of remdesivir treatment, the patient showed recovery sign. as the viral loads was decreasing before remdesivir treatment, therefore it cannot be determined if further viral load reduction and clinical improvement were as the direct result of remdesivir treatment. in another study, the compassionate use of remdesivir (n = 53) reported 68% improved oxygenation, 47% discharge and 13% death. this study was not most significantly as it lacks of a paired control group (grein et al. 2020) . recent study at the national institutes of health (nih), showed preliminary results of the adaptive covid-19 treatment trial (actt, n = 1063). in this randomized controlled trial (rct), remdesivir treatment showed 31% faster time to recovery as comparative to the placebo group (p < 0.001). the mortality rate was also showed reduction in remdesivir group, however it was not statistically significant (8% vs. 11.6%, p = 0.059). so far, remdesivir has not shown any significant benefit in the reduction of mortality rate. currently, remdesivir is recommended by the nih for hospitalized severe covid-19 cases as defined by oxygenation needs (clinical management of covid-19). another drug, like chloroquine (c), has recently been reported as a potential broad-spectrum antiviral drug (savarino et al. 2006) . it inhibits the virus infection by increasing endosomal ph, which is essential for virus/cell fusion, as well as by interfering with the glycosylation of cellular receptors of sars-cov (vincent et al. 2005) . recent studies demonstrated that chloroquine is effective at both entry, as well as at postentry stages of the sars-cov-2 infection in vero e6 cells . apart from antiviral activity, chloroquine also has immune-modulating activity. therefore, it may synergistically enhance antiviral effect in vivo. chloroquine gets widely distributed in the whole body after oral administration, including lungs. the ec 90 value of chloroquinein vero e6 cells against the sars-cov-2 was 6.90î¼m, therefore it could be clinically achievable . the effect of hydroxychloroquine (hcq) and chloroquine (cq) in vitro was also tested by yao et al. in a systematic way, they had divided the experiment into two phases: treatment study and prophylaxis. in the treatment study, they determined the ec 50 values for chloroquine. results showed that it was 23.90 î¼m and 5.47 î¼m at 24 and 48 h, respectively. however, in the case ofhydroxychloroquine,the ec 50 values were 6.14 î¼m and 0.72 î¼m at 24 and 48 h, respectively. on the other hand in the prophylaxis study for chloroquine, the ec 50 values were more than100 î¼m and 18.01 î¼m at 24 and 48 h, respectively. similarly, for hydroxychloroquine, the ec 50 values were 6.25 î¼m and 5.85 î¼m at 24 and 48 h, respectively (yao et al. 2020) . hence, they found that hydroxychloroquine is more effective in vitro than chloroquine for both prophylaxis and treatment. a study in united states, where covid-19 patients hospitalized within 24 h of diagnosis was treated with hydroxychloroquine alone (hcq) or with hydroxychloroquine and azithromycin (hcq + azm) or no hcq as treatments. among patients, there was no significant reduction in mortality rate or in the need of ventilation with hydroxychloroquine alone or with hydroxychloroquine and azithromycin (magagnoli et al. 2020) . a new york hospital stated the qtc prolongation associated with hcq + azm (n = 84; chorin et al. 2020) . it was amplified from a baseline of 435 â± 24 ms to a maximal value of 463 â± 32 ms (p < 0.001) on day 3.6 â± 1.6 of the treatment. till date, researchers present conflicting data's related to the treatment with cq and hcq. therefore, significant randomized control tests (rcts) with improved study designs are required to examine the efficacy and the clinical benefits of hcq/cq treatment over its risks. currently, the nih recommendation are against cq/hcq and hcq + azm treatment for covid-19, except for clinical trials. due to the potential toxicity, nih recommendations are also against the high-dose of cq (600 mg) twice daily for 10 days in all settings (coronavirus disease 2019 (covid-19) treatment guidelines). nitazoxanide has demonstrated potent in vitro activity against sars cov-2, with an ec 50 at 48 h of 2.12 î¼m in vero e6 cells . this potent activity is consistent with ec 50 values for nitazoxanide and its active metabolite, tizoxanide, against mers-cov in llc-mk2 cells where ec 50 values of 0.92 î¼m and 0.83 î¼m respectively, have been demonstrated (rossignol 2016) . dexamethasone, a synthetic glucocorticoid, has antiinflammatory and immunosuppressive properties. there is a hyper inflammatory response involved in the clinical course of patients with pneumonia due to sars-cov-2. the elevated level of c-reactive protein (crp) in sars-cov-2 patients has significantly decreased from 129.52 to 40.73 mg/l at time of discharge. 71% of the patients were discharged home with a mean length of stay of 7.8 days. none of the patients had escalation of care, leading to mechanical ventilation (selvaraj et al. 2020) recently, a randomized, controlled clinical trial in the united kingdom save the lives of people seriously ill with covid-19 when treated with dexamethasone. results showed the reduction of number of death by one-third (ledford 2020) . dexamethasone may be useful for the short-term in severe sars-cov-2 patients as it inhibit the protective function of t cells and block b cells from making antibodies (theoharides and conti 2020). the development of successful vaccine for humans can take several years. as no coronavirus vaccines are available in the market as of now, therefore, the development of a vaccine for the first time can be difficult and time-consuming. however, a mrna-based vaccine has been co-developed by moderna and the vaccine research center at the national institutes of health. in this vaccine, the target antigen's mrna, encapsulated in lipid nanoparticles are injected into vaccinee and antigen expresses in vivo. the phase i clinical trial has been recently started (clini-caltrials.gov: nct04283461). curevac is also using the same platform but they are still in the pre-clinical phase. apart from this, there are various other approaches including dna vaccine, recombinant protein-based vaccine, recombinant vector vaccine, inactivated vaccine and attenuated vaccine. different research companies, universities and institutes are targeting s protein of sars-cov-2 to develop recombinant proteinbased vaccines including novavaxexpress2ion, ibio, sichuan clover biopharmaceutical, baylor college of medicine and the university of queensland. similarly, cansino biologics, geovax vaxart and the university of oxford are using viral-vector-based vaccine platform especially focused on the s protein. applied dna sciences and inovio are using dna vaccines platform again focused on the s protein (amanat fatima 2020). apart from the above-mentioned platforms, the whole microorganism based vaccine platform like inactivated and attenuated virus vaccine is also in consideration. codagenix with the serum institute of india is using live attenuated vaccine platforms. the recombinant vector-based platform (adenovirus vector) is adopted by johnson and johnson, and on the other hand, sanofi is also using the same platform (recombinant influenza vector) to develop a vaccine against sars-cov-2. at this stage, it is difficult to predict the best platform for a vaccine against sars-cov-2 as all the above mentioned platforms have some advantages as well as disadvantages (amanat fatima 2020). coronaviruses have shown the capability to jump species boundaries and adapt to new hosts. therefore, we may face more such kind of outbreaks in the future. role of the intermediate host is also of major importance, as they provide direct pathway for virus transmission in humans. the enormous diversity of viruses in animals and their ongoing evolution makes it important to limit our exposure to animal pathogens as much as possible. based on the metagenomic data it is predicted that the pangolin-cov is most closely related to sars-cov-2. pangolin-cov genome showed 91.02% nucleotide identity with the sars-cov-2 genome. due to a very limited knowledge of this novel virus, it is difficult to explain the significant number of amino acid substitutions that occurred between the sars-cov-2 and sars or sl-covs. for example, in sars-s-cov, six mutations occurred in the regions other than that of the rbd domain, but interestingly no amino acid substitutions were present in the receptor-binding motifs that directly interact with human receptor ace2 protein. therefore, such differences that could affect sars-cov-2 transmission property as compared to sars-cov are of importance for future investigation. sars-cov-2 continues to infect people globally; therefore it is imperative to develop new, safe, accurate, fast and simple new technologies for detecting sars-cov-2. apart from diagnosis, effective prophylactic and therapeutic agents are also required to control and prevent infection. various therapeutic agents including dexamethasone have shown promising results in the in vitro studies to control infection. however, there is an urgency to develop vaccine against coronaviruses. in this direction, studies on neutralizing antibodies from sars-cov and mers-cov against s protein and its many fragments including s1-ntd, rbd and s2 may provide important guidelines for development of vaccine against sars-cov-2. apart from neutralizing antibody against s protein, other approaches including dna vaccine, recombinant vector vaccine, inactivated vaccine and attenuated vaccine are also in pipeline to develop vaccines against sars-cov-2. so far, the traditional public health measures including detection of active cases, isolation of such cases, tracing of all contacts and their quarantine, maintaining social distancing, as well as community quarantine were found to be successful. only after this pandemic ends, we will be in a position to assess the social, health and economic impact of such a massive outbreak. therefore, we must learn lessons for our future from such outbreaks, as new viruses will keep coming. none declared. sars-cov-2 vaccines: status report epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the qt interval in patients with covid-19 treated with hydroxychloroquine and azithromycin clinical management of covid-19 covid-19) treatment guidelines the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade origin and evolution of pathogenic coronaviruses case reports study of the first five patients covid-19 treated with remdesivir in france the spike protein of sars-cov -a target for vaccine and therapeutic development review of current advances in serologic testing for covid-19 the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 compassionate use of remdesivir for patients with severe covid-19 identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor first case of 2019 novel coronavirus in the united states evidence of the recombinant origin of a bat severe acute respiratory syndrome (sars)-like coronavirus and its implications on the direct ancestor of sars coronavirus angiotensin-converting enzyme 2 (ace2) proteins of different bat species confer variable susceptibility to sars-cov entry diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 laboratory testing strategy recommendations for covid-19: interim guidance identification of 2019-ncov related coronaviruses in malayan pangolins in southern china genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coronavirus breakthrough: dexamethasone is first drug shown to save lives functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections potent neutralizing monoclonal antibodies directed to multiple epitopes on the sars-cov-2 spike bats are natural reservoirs of sars-like coronaviruses emergence of sars-cov-2 through recombination and strong purifying selection genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid-19 evaluation of rapid antigen test for detection of sars-cov-2 virus a randomized, controlled trial of ebola virus disease therapeutics potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like cronavirus of bat origin nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia new insights into the antiviral effects of chloroquine short-term corticosteroids in sars-cov2 patients: hospitalists' perspective broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronavirus: epidemiology, genome replication and the interactions with their hosts characterization of the receptorbinding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine differential stepwise evolution of sars coronavirus functional proteins in different host species dexamethasone for covid-19? not so fast potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro human igg neutralizing monoclonal antibodies block sars-cov-2 infection receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus who| novel coronavirus summary and literature update -as of 17 cryo-em structure of the 2019-ncov spike in the prefusion conformation a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans cross-neutralization of sars coronavirus-specific antibodies against bat sars-like coronaviruses recent advances in the detection of respiratory virus infection in humans probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak identification of immunogenic determinants of the spike protein of sars-like coronavirus immunogenicity difference between the sars coronavirus and the bat sars-like coronavirus spike (s) proteins a pneumonia outbreak associated with a new coronavirus of probable bat origin advances in mers-cov vaccines and therapeutics based on the receptor-binding domain key: cord-350737-nrtrhq1f authors: chen, xinchun; zhou, boping; li, meizhong; liang, xiaorong; wang, huosheng; yang, guilin; wang, hui; le, xiaohua title: serology of severe acute respiratory syndrome: implications for surveillance and outcome date: 2004-04-01 journal: j infect dis doi: 10.1086/380397 sha: doc_id: 350737 cord_uid: nrtrhq1f background. severe acute respiratory syndrome (sars) is a novel infectious disease. no information is currently available on host-specific immunity against the sars coronavirus (cov), and detailed characteristics of the epidemiology of sars cov infection have not been identified. methods. elisa was used to detect antibody to sars cov. reverse-transcriptase polymerase chain reaction was used to detect sars cov rna. t cells in peripheral blood of patients were quantified by flow cytometry. results. of 36 patients with probable sars cov infection, 30 (83.3%) were positive for igg antibody to sars cov; in contrast, only 3 of 48 patients with suspected sars cov infection, 0 of 112 patients with fever but without sars, and 0 of 96 healthy control individuals were positive for it. igg antibody to sars cov was first detected between day 5 and day 47 after onset of illness (mean ± sd, 18.7±10.4). conclusion. detection of antibody to sars cov is useful in the diagnosis of sars; however, at the incubation and initial phases of the illness, serological assay is of little value, because of late seroconversion in most patients. severe acute respiratory syndrome (sars) is a novel infectious disease with global impact. since november 2002, an outbreak of sars has affected 33 countries on 5 continents, with 8435 reported cases and 789 deaths at the time when a world health organization (who) report was published in 2003 [1] . a virus from the family coronaviridae, termed "sars coronavirus" (sars cov), has been identified as the cause [2] [3] [4] [5] [6] [7] , and criteria for laboratory confirmation of sars cov infection have been provided by who, on the basis of the following methods: (1) detection of sars cov rna by reversetranscription polymerase chain reaction (rt-pcr); (2) serological detection of sars cov-related antibody; and (3) isolation of sars cov by cell culture [4] . thus, an investigation of the profile and implications of the presence of specific anti-sars cov antibody would be likely to provide information that would be beneficial in diagnostics (confirmation and exclusion of sars cases) and that could function as a valuable indicator to be used in the analysis of host-specific immunity against sars cov and of the character of sars cov infection. the immunology and characteristics of sars cov infection have not yet been fully understood, because there has been such a short span of time since the outbreak of sars and because of the unavailability of such tools as a detection reagent. using an indirect immunofluorescence assay and parallel acute and convalescent serum samples obtained from patients with sars, tested for igg antibody to sars cov, peiris et al. recently documented seroconversion of igg antibody in 93% of patients, at a mean of 20 days [5] . the results of peiris et al.'s study prompted us to study the serology and humoral immunity of sars cov infection. to gain a comprehensive understanding of antibody to sars cov, additional clarification, such as that which would be gained by moredetailed profiles of igg and igm antibodies to sars cov (by such convenient assays as elisa), was required. we also sought to determine the implications of these antibody profiles. included in the present study were 36 patients with probable sars cov infection and 48 patients with suspected sars cov infection. sars was diagnosed on the basis of the case definition provided by who [5] . also included in the study were 112 patients with fever but without sars who were admitted to our hospital during the study's time frame; in addition, 96 healthy individuals, all health-care workers, were included as controls. of the 112 patients with fever but without sars, 35 had an upper-respiratory-tract infection, 46 had pneumonia, 22 had influenza a, and 9 had pulmonary tuberculosis. the 96 individuals in the control group consisted of 36 physicians and 60 nurses, all of whom came into close contact with patients with sars and routinely underwent isolation procedures prior to contact. all of the patients and control individuals who participated in the present study were negative for hiv. the characteristics of the study participants are shown in table 1. all patients and control individuals were informed of the purposes, procedures, and content of our clinical study, and all clinical samples were obtained from them after they had given written informed consent. we obtained clinical specimens of serum, nasopharyngeal aspirate, feces, and whole blood from all patients with probable sars cov infection and all of the patients with suspected sars cov. serum samples were obtained every 3-4 days during the first month after the patients' admission to the hospital and, thereafter, every week until 60 days after the onset of fever. samples of nasopharyngeal aspirate and of feces were obtained on day 0 (i.e., the day of admission) and on day 7 after the patients' admission. whole blood for the measurement of cd4 + and cd8 + t cells was obtained on day 0. for patients with fever but without sars, serum samples were obtained on days 0 and 21, samples of nasopharyngeal aspirate and of feces on days 0 and 7, and samples of whole blood on day 0. for the control group, serum samples were obtained on days 7, 21, and 90 after their first contact with patients with sars; and samples from nasal swabs and samples of feces were obtained on days 7 and 90. complete sars cov particles purified from the supernatant of sars cov-infected vero e6 cell cultures were used as antigen in the detection of igg and igm antibodies to sars cov, by use of an elisa kit (jibiai biotech). all serum samples were stored at ϫ30њc, and the elisas for all samples were performed in parallel. the procedures and the interpretation of the results strictly followed the elisa supplier's instructions. total rna was extracted from the clinical samples, as described elsewhere [6] , by use of a commercial rna-extraction kit. in brief, a qiaamp viral rna mini kit (qiagen) was used for samples from nasal swabs and for samples of nasopharyngeal aspirate, and a qiaamp stool kit (qiagen) was used for samples of feces. for nested rt-pcr, 5 ml of total rna obtained from each clinical sample was reverse transcribed by use of the oligonucleotide 5 -aatgtttacgcaggtaagcg-3 (nt 15627-15608); then the cdna was amplified by use of the outer primers 5 -cagag-ccatgcctaacatg-3 (nt 15239-15257) and 5 -aatgttt-acgcaggtaagcg-3 (nt 15608-15627) and, subsequently, by use of the inner primers 5 -tgttaaaccaggtggaac-3 (nt 15376-15393) and 5 -cctgtgttgtagattgcg-3 (nt 15515-15532) [7] . real-time quantitative rt-pcr assays were performed as described elsewhere [6] , by use of a commercial kit (realart hpa-coronavirus lc rt-pcr reagents; roche biomedical laboratories). rt-pcr was performed on a lightcycler (roche biomedical laboratories), in accordance with the manufacturer's instructions. all samples positive for sars cov rna were confirmed when direct dna sequencing, by use of a dna sequence analyzer (abi 3100; applied biosystems), indicated 99.4% nucleotide-sequence alignment with the sars cov sequence published by genbank (accession numbers gi29826276, gi30027610, and gi30027610). in brief, samples of whole blood were collected in edta and were immunolabeled with monoclonal antibodies cd4-fitc, cd8-pe, and cd3-cy5 (immunotech) at room temperature; mouse igg1-fitc, mouse igg1-pe, and mouse igg1-cy5 (immunotech) were incubated as isotype controls, to allow for subtraction of nonspecific staining. red blood cells were lysed, for 10 min at room temperature, by use of 0.5 ml optilyse c (immunotech), and the cells were then washed 2 times with 0.5 ml of pbs and were resuspended for flow-cytometry analysis by use of a coulter epics xl (beckman-coulter). statistical analysis. continuous variables were compared by student's t test, and correlation was assessed by pearson correlation analysis, both by use of the software program spss version 10.0; was considered to be statistically significant. p ! .05 the production of igg and igm antibodies to sars cov were seen as early as day 3 and day 5, respectively, after the onset of fever. of the 36 patients with probable sars cov infection, 9 (25.0%) had not produced anti-sars cov antibody by day 21, and 6 (16.7%) had not produced it by day 60 (figure 1). of 48 patients with suspected sars cov infection, 3 (6.3%) were positive for igg antibody to sars cov and 2 (4.2%) were positive for igm antibody to sars cov. sars cov sequence published by genbank (accession numbers gi29826276, gi30027610, and gi30027610). moreover, all samples positive for sars cov rna were further confirmed when real-time pcr indicated a viral load of 110 5 copies/ml. no patients with suspected sars cov infection, no patients with fever but without sars, and no control individuals were positive for sars cov rna. in addition, there was no difference between the amount of anti-sars cov antibody produced by patients positive for sars cov rna and that produced by patients negative for sars cov rna. the relationship between the production of igg antibody to sars cov and t cell immunity. we quantified cd3 + cd4 + t cells and cd3 + cd8 + t cells in the peripheral blood of the patients with probable sars cov infection and of those with suspected sars cov infection. our results indicated that all of these patients experienced a dramatic decrease in cd3 + cd4 + t cell percentage (mean ‫ע‬ sd, 19.72% ‫ע‬ 7.78%) and cd3 + cd8 + t cell percentage (mean ‫ע‬ sd, 23.7% ‫ע‬ 6.35%), compared with patients with fever but without sars (p ! .05), whose mean ‫ע‬ sd cd3 + cd4 + t cell percentage and cd3 + cd8 + t cell percentage were and 28.7% ‫ע‬ 7.1%, respectively. in pa-40.6% ‫ע‬ 7.8% tients with probable sars cov infection, there was a significant difference between the cd3 + cd4 + t cell percentage in those positive for igg antibody to sars cov (mean ‫ע‬ sd, ) and that in those negative for it (mean ‫ע‬ 24.07% ‫ע‬ 7.95% sd, ) ( ); however, no significant dif-13.60% ‫ע‬ 10.19% p ! .05 ference was found between the cd3 + cd8 + t cell percentages in these 2 groups (table 2) . also in patients with probable sars cov infection, there was no significant difference, in either the cd3 + cd4 + or the cd3 + cd8 + t cell percentage, between those positive for igm antibody to sars cov and those negative for it ( ). a correlation was found between the day of igg p 1 .05 antibody seroconversion and the cd3 + cd4 + t cell percentages measured on the day of the patients' admission to our hospital ( , ), if we assume day 60 to be the day of r p ϫ0.543 p ! .05 seroconversion for those patients who had not actually seroconverted by then. much progress has been made in sars research, including the identification of the etiologic agent [2] [3] [4] [5] [6] [7] , the complete sequencing of the sars cov genome [8] , the establishment of such diagnostic laboratory methods as rt-pcr and indirect immunofluorescence assay [5, 6] , and the establishment of measures for the prevention of sars and for the management of probable sars cases. however, it is still uncertain whether sars is recurrent and what the impact of recurrence might be, because there is no information on the status of host-specific immunity against sars cov and because the detailed characteristics of the epidemiology of sars cov infection are not known [9, 10] . given this uncertain background, we investigated, for its potentially significant clinical implications, the profile of anti-sars cov antibody in different populations, including patients with probable sars cov infection, patients with suspected sars cov infection, patients with fever but without sars, and healthy control individuals (health-care workers) who came into close contact with patients with sars. the results of our investigations of the presence of anti-sars cov antibody in patients with either probable or suspected sars cov infection show that the use of elisa to detect igg antibody and/or igm antibody to sars cov is a specific and useful method for the diagnosis of sars, especially given that elisa's sensitivity in the detection of anti-sars cov antibody (83.3%) is much better than rt-pcr's sensitivity in the detection of sars cov rna (∼25% in our study). serological assay, however, is of little value during the incubation and initial phases of the illness, because of late seroconversion in most patients ( figure 1a ). in addition, our results indicated that 9 (25.0%) of 36 patients with probable sars cov infection had not produced detectable anti-sars cov antibody by day 21 after the onset of fever; this implies that 25.0% of patients with sars might be misdiagnosed by the laboratory confirmation guidelines that who currently recommends [5] . however, this was not the case in our study, because (1) 3 of these 9 patients were later confirmed, by detection of seroconversion before day 47, to be infected by sars cov and (2) the 6 other patients, who had remained negative for anti-sars cov antibody until day 60, were later confirmed, by fulfillment of who criteria and by exclusion of other pathogenic infections, to be infected by sars cov. specifically, all of the patients with probable sars cov infection came into contact with someone with sars, had documented persistent fever (138њc), showed a consistent clinical course of the illness, and showed evidence of pneumonia, by plain radiography and/or computed tomography; in addition, there was no evidence of infection by other pathogens (including influenza viruses a and b, human parainfluenza viruses 1-3, respiratory syncytial virus, adenovirus, chlamdia pneumoniae, c. psittaci, mycoplasma pneumoniae, and mycobacterium tuberculosis), and there was no conventional pathogenic bacterial infection and no response to antibiotic treatment (0.3 g of levofloxacin/day for 48-72 h). the discrepancy between our diagnosis of sars in these patients and diagnosis on the basis of who guidelines likely reflects the evolution of the identification of definitive sars cases, as more data on the disease accumulate; the current who guidelines are probably not 100% accurate. it is notable that, on 16 july 2003, the centers for disease control and prevention revised its laboratory criteria in its definition of sars, to require that convalescent serum be collected 128 days, instead of 121 days, after the onset of symptoms [11] . the choice of day 28 presumably reflects data indicating that 195% of patients with sars mount a detectable convalescent antibody response [11] . a more technical explanation for our identification of late-seroconverting patients may involve differences between the sensitivity of the elisa used in the present study and the sensitivity of other serological assays, on which the who guidelines are based; this issue should be further clarified as more data are collected. in 6 of our patients with a late antibody response, there may have been other mitigating factors that could have led to a decrease in basic immunity. of these 6 patients, 2 (who were 165 old) had emphysema, 1 had diabetes, 1 was hypertensive and had coronary artery disease, 1 had tetanus, and 1 was pregnant; these conditions may be associated with late seroconversion in these patients. it is notable that a few (2 of 36 [5.6%]) patients with probable sars cov infection were positive for igg antibody to sars cov but, until day 60, were negative for igm antibody to sars cov; the reason for this is uncertain, but one possible explanation is that, in these 2 cases, igm antibody to sars cov persisted for a short time and had disappeared before the patients were admitted to the hospital. we found production of igg antibody to sars cov to be associated with host t cell immunity, because patients who did not produce igg antibody to sars cov had significantly lower cd3 + cd4 + t cell percentages, compared with those in patients who seroconverted. in addition, the day of igg antibody seroconversion correlated with cd3 + cd4 + t cell percentages taken on the day of the patient's admission ( ; ). r p ϫ0.543 p ! .05 these results suggest that the production of igg antibody to sars cov is dependent on cd4 + t cells and that the appearance of igg antibody to sars cov might be an indicator of the production of protective immunity against sars cov. recently, we treated 1 pregnant patient with severe sars cov infection by using convalescent plasma in which the titer of igg antibody to sars cov was 11:500; the igg antibody could be detected until 60 days after the infusion, 2 days before her own igg antibody to sars cov appeared. with combined treatment with the antiviral drug methylprednisolone and convalescent serum, she recovered fully. however, whether the igg antibody to sars cov is itself a neutralizing antibody needs further study. recently, krokhin et al. reported that acute and early convalescent serum, obtained from several patients recovering from sars, can react only with the 46-kda nucleoprotein (which appears to be the major antigen) of sars cov [12] . their results suggest that immune response to this nucleoprotein could serve as an early diagnostic indicator for infection; however, it is unlikely that immune response to this protein offers protection, because it is an internal protein and because neutralizing antibodies are more likely to target cellsurface proteins [13] . nevertheless, it has been shown, for other covs, that some antigenic peptides of the nucleoprotein can be recognized on the surface of infected cells by host t cells [13] ; thus, the appearance of igg antibody in patients with sars will more likely be an indicator of the production of protective immunity against sars cov. our results also indicate that all individuals positive for anti-sars cov antibody should present symptoms, whether severe or not. from 9 feb 2003 to the time when this article was written, our hospital admitted 52 patients with probable sars cov infection and 48 patients with suspected sars cov infection; however, at our hospital there is not a single health-care worker infected with sars cov-a situation that is in stark contrast to that at other hospitals in china, where nearly one-third of patients with sars are health-care workers [14] . that our hospital has no health-care workers infected by sars cov was confirmed by both serological assay and lack of detection of sars cov rna. the 96 health-care workers who participated in our study all came into close contact with patients with sars, for a period of 3 months, but igg and igm antibodies to sars cov were not detected in the serum of the health-care workers. these results may be somewhat difficult to interpret-and it was certainly not the point of our study to investigate this matter-but their implications may be very important; they suggest that the possibility that people can be asymptomatically infected by sars cov-and that such individuals (if they do exist) can transmit the virus-might be very small. the preventive measures taken at our hospital were almost exactly the same as those taken at other hospitals, with the only known difference being that, for prophylaxis, the health-care workers at our hospital took 400 mg of ribavirin/ day. we must caution, however, that these are only anecdotal observations; further study is necessary to determine whether ribavirin has a prophylactic effect against sars cov infection. world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) koch's postulates fulfilled for sars virus coronavirus confirmed as cause of sars updated interim surveillance case definition for severe acute respiratory syndrome (sars)-united states clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study identification of a novel coronavirus in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection sars coronavirus: a new challenge for prevention and therapy modeling the sars epidemic updated interim us case definition for severe acute respiratory syndrome (sars) mass spectrometric characterization of proteins from the sars virus: a preliminary report coronaviridae: the viruses and their replication world health organization. china daily report of sars cases we thank the staff of shenzhen municipal hospital of infectious disease and the staff of shenzhen institute of hepatology, for their technical assistance and management of patients; all of the patients and, especially, the health-care workers who participated in the present study, for their great cooperation; and michael graner (dept. of pediatrics, university of arizona, tuscon), for his helpful comments. key: cord-337712-ylqgraos authors: heinz, franz x.; stiasny, karin title: profile of sars-cov-2 date: 2020-10-30 journal: wien klin wochenschr doi: 10.1007/s00508-020-01763-1 sha: doc_id: 337712 cord_uid: ylqgraos the recent emergence of a new coronavirus (severe acute respiratory syndrome coronavirus‑2, sars-cov-2) that is transmitted efficiently among humans and can result in serious disease and/or death has become a global threat to public health and economy. in this article, we describe some of the most important characteristics of this new virus (including gaps in our understanding) and provide a perspective of ongoing activities for developing virus-specific countermeasures, such as vaccines and antiviral drugs. severe acute respiratory syndrome coronavirus-2 (sars-cov-2) is the seventh coronavirus that has emerged as a human-to-human transmissible agent after zoonotic transfer from wild animals [1] . coronaviruses are found globally in many animal species and are classified into four distinct genera (alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus; fig. 1 ), all sharing the same basic structural and genetic organization as well as replication strategy ( [2, 3] ; figs. 2 and 3). infections of humans and other mammals are caused by the alphacoronaviruses and betacoronaviruses, whereas gammacoronaviruses and deltacoronaviruses have birds as their primary hosts. in animals, coronaviruses cause a range of diseases (primarily respiratory and gastrointestinal infections) in a variety of species, resulting in serious consequences for the livestock industry [4] . of the seven human coronaviruses (hcov), four cause relatively benign respiratory infections and are distributed worldwide [3] . two of them (designated 229e and nl63) are alphacoronaviruses and have their likely origins in bats. the other two (designated oc43 and hku1) are betacoronaviruses with a possible origin in rodents and/or cattle ( [2, 3] ; fig. 1 ). in 2002, the emergence of sars-cov-1 in china and its global spread caused a first coronavirus-related human health crisis of international concern [5] . it became rapidly clear that this virus had jumped to humans from natural reservoirs in bats, likely through intermediate hosts such as civet cats [3] . fortunately, human-to-human transmission could be interrupted completely by hygienic measures of epidemic control within a short period of time, and natural transmission of the virus came to an end by 2003 [6] . a second potentially deadly human coronavirus (middle east respiratory syndrome coronavirus, mers-cov) emerged in 2012 in saudi arabia [7] . in this case, the source of transmission to humans was identified as infected dromedary camels. although human-to-human transmission chains occurred and still do occur, they can be contained and do not result in widespread outbreaks. the risk of zoonotic infection and ensuing limited chains of infection, however, remains because of the enormous viral reservoir in dromedary camels, especially across the arabic peninsula and parts of eastern and northern africa [8, 9] . both sars-1 and mers viruses are betacoronaviruses but belong to two different genetic lineages, b and c, respectively, distinct from lineage a which comprises hcov-oc43 and hcov-hku1 ( fig. 1; [2] ). after the emergence of sars-cov-1, efforts to identify natural reservoirs provided ample evidence for extensive circulation of sars-like coronaviruses in bats fig. 1 dendrogram of coronaviruses assigned to the four genera of alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses (based on percentage amino acid sequence identity in the spike protein s). in betacoronaviruses, genetic lineages are indicated. hku11 and ibv (infectious bronchitis virus) designate avian coronaviruses. with the potential of crossing species barriers and the emergence in humans [10, 11] . corresponding publications were full of warnings that sars-1-like emergences could happen again, unless contact to the natural reservoir of coronaviruses in bats and transmission to humans via intermediate hosts cannot be properly controlled [5] . unfortunately, these warnings did not materialize in practice, and a very similar virus (now called sars-cov-2) emerged at the end of 2019 in wuhan, china. from there, the virus rapidly spread globally and to date (25 october 2020) the pandemic has caused 43,341,451 documented cases of infection and 1,157,509 fatalities/deaths [12] . the genome of sars-cov-2 is~29,800 nucleotides in length [13] and has the characteristic features of coronaviruses in general (fig. 2 ). they all have exceptionally large nonsegmented positive-stranded rna zoom of structural gene region, with accessory genes in red, as described by xie et al. [94] for sars-cov-2 and forni et al. [2] for sars-cov-1 as well as hcov-nl63 [2] . orf open reading frame. designations of structural genes: s spike; e envelope; m membrane; n nucleocapsid genomes (ranging between~26,000 and 32,000 bases [2] ), which contain 3 sets of open reading frames (orf) encoding three groups of proteins with different functions: 1. 16 non-structural proteins (nsp1 to nsp16) that include the viral rna polymerase, proteases and other virus-specific enzymes involved in virus replication. 2. the structural proteins s (spike), e (envelope), m (membrane), and n (nucleocapsid) that are essential building blocks of virus particles (fig. 3a ) (some coronaviruses, e.g. hcov-oc43 have a hemagglutinin-esterase protein as an additional structural component [2] ). 3. a variable number of orfs interspersed with the genes for structural proteins, encoding proteins that are produced in infected cells but are not essential for basic virus replication. these "accessory genes" can differ in number and sequence even among closely related coronaviruses and apparently contribute to the tuning of virushost cell interactions to create an optimal environment for virus replication and transmission, e.g. by counteracting innate antiviral host responses [14] . the genetic mutation rate of sars-cov-2 and coronaviruses in general is lower than that of other rna viruses due to a proofreading activity in the viral rna replication machinery [15, 16] . nevertheless, coronavirus genomes have a high degree of plasticity and mechanisms that allow rapid virus evolution. these include a propensity for recombination (leading to the exchange of whole gene segments among different coronaviruses during double infections of the same cell in the same host) as well as the capacity for acquisition and losses of accessory protein coding genes, facilitating adaptive changes in addition to mutation [2] . together with the structural flexibility of the spike protein, allowing adaptation to different cellular receptors with relative ease, these properties are the foundation for the propensity of coronaviruses for crossing species barriers and jumping hosts. the sars-cov-2 virus particle has the typical coronavirus morphology, with a diameter of~80-120 nm [17] . infectious virions are composed of a helical nucleocapsid (comprising the genomic rna and mul-profile of sars-cov-2 k main topic fig. 3 structural organization of coronavirus particle and the spike protein s. a schematic of virus particle. b ribbon diagrams of the soluble s protein trimer. the three monomers are colored in red, gold, and grey. in the closed state, the receptor-binding domain (rbd) is in the 'down' conformation (protein data bank: pdb 6zgi, [95] ), and in the open state in the 'up' conformation (protein data bank: pdb 6zgg, [95] ), which can interact with the viral receptor ace2 (angiotensinconverting enzyme 2). the interaction site is indicated by a blue ellipse in the right panel tiple copies of the n protein) and a lipid envelope with three membrane-associated proteins, m (membrane), e (envelope), and s, forming the characteristic club-shaped projections at the viral surface (fig. 3a) . the spike protein has attracted special attention, because it provides the essential viral functions for cell entry (receptor binding and membrane fusion) and is the primary target of neutralizing and potentially protective antibodies [18] . it is therefore considered the most important and essential antigen component of vaccines (see section "prospects for vaccines"). each of the spikes is composed of three s protein subunits, and proteolytic cleavage of the s monomers into s1 (membrane-distal part) and s2 (membrane-associated part) by host cell proteases at two closely adjacent sites is required for infectivity [19] . the molecular architecture of s (fig. 3b ) reflects its dual function comprising a receptor-binding domain (rbd) in s1 and a spring-loaded fusion machinery in the membrane-associated s2 [19] . in its prefusion form the s-trimer of sars-cov-2 was found to oscillate between an open and a closed conformation with an up and down orientation of rbd (fig. 3b) , which only in the up conformation is capable of binding its specific receptor angiotensinconverting enzyme 2 (ace2) [20] [21] [22] . fusion of the viral membrane with a cellular membrane is required for the release of the genome into the cytoplasm. this process is driven by a triggered structural change of s2 from its metastable prefusion conformation into a stable postfusion form that is made possible by a combination of receptor binding and proteolytic cleavage between s1 and s2 and provides the energy for membrane fusion [19] . the genome organizations of sars-cov-1 and sars-cov-2 are remarkably similar, with a 79% identity at the nucleotide level [23] . both viruses bind with high affinity to the same receptor, ace2, and are so far the only human coronaviruses in lineage b of betacoronaviruses ( fig. 1 ). despite these similarities, distinguishing features were identified that are likely to contribute to the biological differences observed between the two viruses, including the significantly higher rate of subclinical and mild infections caused by sars-cov-2, which makes control of virus spread currently so difficult. one of the major differences is in the spike protein, which-in contrast to sars-cov-1-has a polybasic cleavage site at the junction between s1 and s2 that is likely to facilitate cleavage activation by the cellular protease furin and/or other proteases [1, 20, 21, 24] . such sequence elements are characteristic of highly pathogenic avian influenza viruses [25] and are also present in the human coronaviruses mers-cov, hcov-oc43, hcov-hku1 [26] . their impact on coronavirus transmissibility and pathogenesis has yet to be established. it is also noteworthy that the adaptive evolutionary changes in s that led to the recognition of ace2 in sars-cov-1 and sars-cov-2, apparently followed different trajectories of mutations, with just one of the six critical amino acids in s that interact with ace2 being identical between the two viruses [27] [28] [29] . the structural evidence thus suggests that these viruses acquired the ability to recognize the same receptor independently in a process of convergent evolution or, alternatively, diverged from a common ancestor a long time ago. potentially important differences are also present in some of the accessory proteins (fig. 2) . these include variations in orf3b, which encodes an interferon antagonist in sars-cov-2 that is more potent than its orthologue in sars-cov-1 [30] . it is also remarkable that the sequence homology of orf8 is exceptionally low between sars-cov-1 and sars-cov-2 (26% at the nucleotide level and 20% at the amino acid level), and also the gene products (8a and 8b) display significant differences [13] . it is currently unknown how precisely these differences shape the biological properties of the two viruses. in the aftermath of the sars-cov-1 epidemic, a large number of related viruses were identified in bats, and conclusive evidence was obtained that the new human virus had originated from this animal reservoir, presumably via adaptation in other mammals, such as civet cats as intermediate hosts [10, 11] . one of these bat virus isolates (designated ratg13) is so far the closest relative of the new sars-cov-2, with approximately 96% identity in almost all genomic regions [31, 32] ; however, ratg13 is unable to recognize human ace2 as a receptor and also does not have the polybasic cleavage site in s. molecular clock analyses indicate that the two viruses are separated by more than 20 years of evolution [27] , and it is therefore unlikely that this bat virus is a direct ancestor of sars-cov-2, which has still to be identified. interestingly, a virus isolated from pangolins was found to be able to interact with human ace2 and to have an rbd which is much more closely related to that of sars-cov-2, although the rest of the genome is significantly more different than that of ratg13 [33, 34] . specifically, the rbd of the pangolin virus contains all six key amino acid mutations required for binding to the ace2 receptor, allowing infection of human cells. it has therefore been speculated that pangolins may have played a role as intermediate hosts in the evolution of sars-cov-2 [34, 35] , but conclusive evidence is still lacking. a second plausible scenario would be that a yet unknown bat virus with the capacity to use human ace2 as a receptor was transmitted directly to humans and remained unidentified as a new human virus for a relatively long time. further adaptive changes during this phase of cryptic spread could have improved transmission among humans until the virus became apparent at the start of the pandemic in wuhan in december 2019. one of the key questions in the current epidemic relates to possible further adaptive changes in sars-cov-2 that could affect its transmission efficiency, disease severity, escape from immunity or other biological properties. two mutations in circulating virus strains have raised special attention. one of them, a replacement of aspartic acid for glycine at position 614 in the spike protein (d614g) (fig. 3b, left panel) , has become dominant and is now found in almost all sars-cov-2 samples worldwide [36] . although in vitro models have provided evidence for a higher infectivity of the mutant virus [37] , there is currently no definitive proof that it arose through a process of natural selection by increased transmissibility and/or immune escape rather than having become dominant as a result of a founder effect [38] . a variant with a dele-tion in orf8 (δ382) was identified in singapore and associated with a milder infection and less systemic release of pro-inflammatory cytokines [39] . as a contrasting example, a virus with a mutation in orf3 that caused more efficient suppression of ifn-i was found in two severely ill patients in ecuador [30] . it will certainly be important to closely follow the emergence of possible adaptive mutations in circulating strains and to study how they might affect transmission and/or disease. the current situation, however, is not characterized by extensive variation but by a rather surprising stability of the virus. this may be due to the relatively low genetic mutation rate of this virus, which is 2-6 times slower than that of influenza virus [16] . on the other hand, the lack of evidence for further adaptive changes might be an indication that the virus had not jumped to humans at the end of 2019 but significantly earlier and that adaptation to its new host after zoonotic transfer had already occurred in this early and cryptic phase of transmission. sars-cov-2 is a cytopathic virus that is transmitted primarily by droplet infections and injures virus-infected cells in airway epithelial cells expressing the viral receptor ace2 [40] . the disease caused by this new virus is called coronavirus disease 2019 (covid-19) [41] . early control of infection by innate immune responses is counteracted by several viral gene products [42, 43] , and competition between virus and host defenses defines whether the infection is readily cleared (asymptomatic and mild respiratory infections) or proceeds to more severe forms including pneumonia, acute respiratory distress syndrome (ards), multiple organ failure and death. the lifethreatening forms of covid-19 are associated with the overproduction of immune cytokines and an uncontrolled systemic inflammatory response, frequently referred to as "cytokine storm" [44, 45] . in addition to early innate immune responses, infection also induces specific t and b cells that can potentially provide immunity against re-infection, at least for a certain period of time [46] . it is still incompletely understood to which extent the different effector arms of adaptive responses (such as cytotoxic t cells, helper t cells and antibodies) contribute to virus clearance and recovery in the acute phase of infection. it is also not yet clear whether t cell responses are only beneficial or contribute to the immunopathology [47] . the case fatality rate strongly increases with age, similar to infections with sars-cov-1 and mers-cov, and may be linked (at least in part) to the phenomenon of immunosenescence that impairs both t cell and b cell responses in aged individuals [48, 49] . the fatality rate is strikingly higher in males than in females [50] , which may be related to the enhanced expression of alleles on the x chromosome (including some toll-like receptors involved in innate immunity profile of sars-cov-2 k main topic but paradoxically also the receptor ace2), stronger b and t cell responses in women, and/or different immunoregulatory functions of sex hormones [50, 51] . it is currently unclear to which extent the different components of adaptive immune responses induced by natural infection will prevent re-infection or at least severe disease and how long such immunity will last. reinfections were shown to be quite common with the four seasonal human coronaviruses at 12 months after primary infection [52] , and reinfection has also been demonstrated with sars-cov-2 [53] . these data indicate that protective immunity after natural infection may not be long-lived and insufficient in certain cases. there is consensus, however, that a sufficiently high concentration of neutralizing antibodies, directed to the s protein and preventing virus entry into cells expressing the virus receptor ace2, play an important role and are essential for conferring immunity [54] . it is unclear, however, which titer of neutralizing antibodies would be protective. immunosenescence is certainly one factor that impairs antibody formation, but current evidence also indicates that antibody responses are lower in individuals with asymptomatic or mild forms of infection and decline more rapidly than in those with more severe disease [55] . recent studies with covid-19 patients indicate a virus-induced impairment of germinal center formation that is likely to hamper the generation of long-lived antibody responses [52, 56] . immune responses may also be influenced quantitatively and qualitatively by pre-existing cross-reactive immunity. sars-cov-2-specific cd4 t cells have been found in 30-50% and cd8 t cells in 20% of healthy people with no evidence of sars-cov-2 infection [55, 57, 58] . these cells were likely induced by past infections with one of the seasonal human coronaviruses and it remains to be investigated, how their presence can influence immune responses, the outcome of ongoing infections with sars-cov-2, and postinfection immunity. development of vaccines against sars-cov-2 has proceeded with an unprecedented pace and breadth [54, 59, 60] , although it is still hampered to a certain extent by the lack of an accepted in vitro correlate of protection. currently, the world health organization (who) lists 198 experimental vaccines in preclinical or clinical development (https://www.who.int/ publications/m/item/draft-landscape-of-covid-19candidate-vaccines, as of 19 october 2020, accessed on 28 october 2020). all technologies available to date are being exploited, including inactivated whole virus vaccines, protein subunit and virus-like particle vaccines, nucleic acid vaccines (dna and rna), different kinds of viral vector vaccines, and live attenuated vaccines. since there is consensus that the induction of neutralizing antibodies (directed to the s protein) is the key to the success of vaccine-induced immunity, virtually all current experimental vaccines build on this protein (or part of it) as a key antigen [55] . at the date of submission of this article, ten vaccine candidates are the most advanced and currently evaluated in phase 3 clinical trials (https://www.who. int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines, accessed 2 october 2020). these front runners include three betapropiolactoneinactivated whole virus vaccines grown in vero cells ( [20, 21] or specific nucleotide modifications for balancing innate immune responses [61, 62] . adenovector vaccines contain defective adenovirus particles in which part of the genome is replaced by the coding sequence of the sars-cov-2 s protein. in some vaccines, these constructs contain mutations like those engineered into the mrna vaccines for stabilizing the s protein. the recombinant adenovirus particles can enter cells and express the s protein in a single round of infection. three of the four experimental vaccines use human adenovirus vectors (based on adeno 5 and/or adeno 26), which have the drawback that pre-existing adenovirus immunity can impair their immunogenicity [55] . in contrast, university of oxford/astra zeneca have made use of a chimpanzee adenovirus, for which almost no immunity exists in humans [63] . nevertheless, vaccination-induced immunity against the vector can still potentially impair immune responses to booster vaccinations, a problem that may be solved by using vaccination schedules with alternative adenovirus serotypes as vectors [54] . in all instances, results from phase 1 and 2 clinical trials have shown the induction of b and t cell responses together with the formation of neutralizing antibodies [54] . the extent of side reactions, although usually mild or moderate and not serious, was quite common with some of the vaccines [54] , and their tolerance in mass vaccination campaigns may become an important criterion for acceptance by the population. since validated in vitro correlates of protection do not yet exist, a true snapshot of vaccine-k profile of sars-cov-2 main topic induced prevention of disease will only become available from the evaluation of phase 3 clinical trials. some of the next important questions will then be how long such protection lasts, how immunogenic the vaccines are in the older and most vulnerable population, whether the vaccines can generate herd immunity, and whether booster vaccinations are necessary to maintain long-term protection. considering the plans for vaccinating millions or even billions of people, special attention will also have to be paid to indications of potentially serious consequences of dysregulated immune responses that would occur late (e.g. upon natural infection), such as antibody-dependent enhancement (ade) of infection [64] [65] [66] . the performance of the vaccines in currently ongoing phase 3 trials cannot be predicted but differences among the candidates are to be expected-both with respect to protection and side reactions-and suboptimal rates of both parameters are possible. in this way, the performance of the front runners will determine the fate of the second line of candidates pushing forward in the race to a covid-19 vaccine. if expectations were too optimistic and results obtained with some of the front runners are disappointing, windows of opportunity will open for an arsenal of alternative developments in progress [54, 59] (https:// www.who.int/publications/m/item/draft-landscapeof-covid-19-candidate-vaccines, accessed 2 october 2020) these include subunit vaccines with s proteins stabilized in their prefusion conformation in combination with potent adjuvants, use of the rbd only as an immunogen instead of the whole s protein [67, 68] , other rationally designed immunogens [69] , other (non-adeno) vector vaccines including replication-competent vectors [55, 70] , self-amplifying rna vaccines [71] , live-attenuated vaccines [55] , dna vaccines [72] , and intranasally applied vaccines with the potential to induce local immunity at the site of virus entry [73] . at the current stage of investigation, it is uncertain, whether and to what extent a covid-19 vaccine can induce durable protection from infection, or more importantly, from severe disease. we also do not yet have information as to the effect of pre-existing cross-reactive coronavirus immunity on vaccine performance, an important aspect to be considered in evaluations of phase 3 clinical trials. one of the most crucial questions relates not only to individual protection, but also to which extent vaccination will be able to confer herd immunity, the death toll of which would be very high when allowed to be induced by natural infection. a positive aspect is certainly the fact that sars-cov-2 appears to be genetically quite stable, and there is currently no evidence for antigenic drift [38, 74] , which would make vaccine development even more challenging. the dramatically increasing load of public health systems with patients suffering from severe covid-19 was paralleled by an intensive search for antivirals that would specifically inhibit virus replication and thus extend treatment options. as a first and most rapid approach, attempts were made to repurpose existing drugs that had already been licensed for the treatment of other infectious diseases [75, 76] . two of the most prominent substances studied in this context were hydroxychloroquin (used for the prophylaxis and therapy of malaria) and remdesivir, which had been developed as a specific treatment of ebola disease and used for the treatment of sars-cov-2 infections [75, 77] . unfortunately, results of clinical application and trials did not meet the high expectations. hydroxychloroquin was shown to have no beneficial effect for covid-19 patients and therefore cannot be recommended for the treatment of sars-cov-2 infections [78, 79] . results from clinical trials with remdesivir are somewhat conflicting and revealed a modest effect in some treatment schedules [80] . additional studies will be needed to resolve existing uncertainties of the incremental benefit achieved by adding remdesivir to standard treatments of covid-19 patients [80] . more specific avenues of drug development may be opened by the determination of high-resolution structures of the sars-cov-2 rna-dependent rna polymerase (rdrp) [81] and the main viral protease (mpro) [82] , responsible for the specific cleavage of protein precursors resulting from primary translation of the viral genome (fig. 2) . both of these enzymes play a pivotal role in the viral life cycle, and knowledge of their atomic structures can inform the specific design of highly potent inhibitors of virus replication. another option for interfering with virus replication is to block interaction of s with its cell entry receptor ace2 (fig. 3) . inhibition of receptor binding is one of the classical modes of antibody-mediated virus neutralization, and convalescent plasma therapy has shown some promise in the treatment of covid-19 patients [83, 84] . in addition, a number of potently neutralizing human monoclonal antibodies have been developed and are currently investigated in the context of phase 3 clinical trials as a more advanced approach compared to plasma therapy (https://www.nih.gov/news-events/news-releases/ clinical-trials-monoclonal-antibodies-prevent-covid-19-now-enrolling). the interaction of s and ace2 may alternatively also be blocked by applying an excess of a soluble form of the receptor itself, produced as a recombinant protein [85] . this molecule was shown to inhibit virus replication in human cells and organoids [86] , although its virus-neutralizing activity was not as high as that of the most potent monoclonal antibodies described so far [87] [88] [89] . it has to be considered, however, that the application of ace2 may profile of sars-cov-2 k main topic have additional beneficial effects by rescuing cellular ace2 activity and thus preventing injury to the lungs [90] . results from ongoing phase 2 clinical trials in europe (eudract number: 2020-001172-15) with recombinant soluble human ace2 for the treatment of covid-19 patients are pending. the global threat by the emergence of sars-cov-2 as a new human pandemic virus has not only led to drastic public health measures for mitigating virus spread, but was also accompanied by an immediate and impressive response of basic and applied research, aiming to understand and combat this new virus. substantial advances have already been achieved in all relevant areas, including genetics and structure of the virus, virus diagnostics, viral pathogenesis, public health measures of infection control, clinical treatment, and vaccine development. despite the successes in all of these areas, science has still to answer a number of crucial and difficult questions. some of the most eagerly expected answers are certainly to the questions of whether protection and herd immunity can be achieved by vaccination, how long vaccine-induced immunity will last, which immediate side reactions have to be tolerated, and whether longterm adverse events will be encountered. the highly divergent spectrum of sars-cov-2-induced disease-ranging from a high proportion of asymptomatic or mild courses to life-threatening and fatal forms of infection-has put some general black boxes of infectious diseases into the spotlight, which are of special medical interest. the large number of subclinical or mild infections is not unique to sars-cov-2 but is frequently encountered with some of the most prominent viruses and represents more the rule than the exception. as examples, poliovirus infections result in poliomyelitis in only 1 of 100 to more than 1 of 1000 individuals infected with the virus [91] , and west nile virus infections lead to central nervous system disease in only about 1 of 150 human infections [92] . many factors are believed to contribute to these differences in individual disease permissiveness and progression, including dose of infecting virus, genetic factors, effectiveness and balance of immune responses, physiological factors and their combinations. the newly made experiences with covid-19 underpin our lack of a detailed understanding of pathogenesis of virus infections in general and will certainly provide a new impetus for research into this fascinating area of virus-host interactions. finally, we certainly do not want this to happen again. the risk of a new outbreak, however, remains real, considering the vast number of coronaviruses circulating in bat populations and their potential of zoonotic transmission and adaptation to humans [11, 93] . investigations of the natural reservoir will therefore be of paramount importance, as well as the further development of strategies to avoid dangerous contacts at the animal-human interface facilitated by illegal and legal trade of wildlife. funding open access funding provided by medical university of vienna. conflict of interest f.x. heinz and k. stiasny declare that they have no competing interests. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. the proximal origin of sars-cov-2 molecular evolution of human coronavirus genomes origin and evolution of pathogenic coronaviruses coronavirusesinfarmanimals: epidemiology and public health implications. veterinary medicine and science a decade after sars: strategies for controlling emerging coronaviruses can we contain the covid-19 outbreak 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efficacy of remdesivir in covid-19 both boceprevir and gc376 efficaciously inhibit sars-cov-2 by targeting its main protease structure of mpro from sars-cov-2 and discovery of its inhibitors structural basis for the inhibition of sars-cov-2 main protease by antineoplastic drug carmofur convalescent plasma treatment of severe covid-19: a propensity score-matched control study sars-cov-2 receptor ace2-dependent implications on the cardiovascular system: from basic science to clinical implications inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 convergent antibody responses to sars-cov-2 in convalescent individuals potently neutralizing and protective human antibodies against sars-cov-2 studies in humanized mice and convalescent humans yield a sars-cov-2 antibody cocktail angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target from emergence to eradication: the epidemiology of poliomyelitis deconstructed longterm, west nile virus-induced neurological changes: a comparison of patients and rodent models sars-cov-2: combating coronavirus emergence an infectious cdna clone of sars-cov-2 sars-cov-2 and bat ratg13 spike glycoproteinstructuresinformonvirusevolutionandfurin-cleavage effects key: cord-317952-4oa9hfb4 authors: bourgonje, arno r.; abdulle, amaal eman; timens, wim; hillebrands, jan‐luuk; navis, gerjan j.; gordijn, sanne j.; bolling, marieke c.; dijkstra, gerard; voors, adriaan a.; osterhaus, albert d. m. e.; van der voort, peter h. j.; mulder, douwe j.; van goor, harry title: angiotensin‐converting enzyme‐2 (ace2), sars‐cov‐2 and pathophysiology of coronavirus disease 2019 (covid‐19) date: 2020-05-17 journal: j pathol doi: 10.1002/path.5471 sha: doc_id: 317952 cord_uid: 4oa9hfb4 angiotensin‐converting enzyme‐2 (ace2) has been established as the functional host receptor for severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), the virus responsible for the current devastating worldwide pandemic of coronavirus disease 2019 (covid‐19). ace2 is abundantly expressed in a variety of cells residing in many different human organs. in human physiology, ace2 is a pivotal counter‐regulatory enzyme to ace by the breakdown of angiotensin ii, the central player in the renin‐angiotensin‐aldosterone system (raas) and the main substrate of ace2. many factors have been associated with both altered ace2 expression and covid‐19 severity and progression, including age, sex, ethnicity, medication and several co‐morbidities, such as cardiovascular disease and metabolic syndrome. although ace2 is widely distributed in various human tissues and many of its determinants have been well recognised, ace2‐expressing organs do not equally participate in covid‐19 pathophysiology, implying that other mechanisms are involved in orchestrating cellular infection resulting in tissue damage. reports of pathologic findings in tissue specimens of covid‐19 patients are rapidly emerging and confirm the established role of ace2 expression and activity in disease pathogenesis. identifying pathologic changes caused by sars‐cov‐2 infection is crucially important as it has major implications for understanding covid‐19 pathophysiology and the development of evidence‐based treatment strategies. currently, many interventional strategies are being explored in ongoing clinical trials, encompassing many drug classes and strategies, including antiviral drugs, biological response modifiers and raas inhibitors. ultimately, prevention is key to combat covid‐19 and appropriate measures are being taken accordingly, including development of effective vaccines. in this review, we describe the role of ace2 in covid‐19 pathophysiology, including factors influencing ace2 expression and activity in relation to covid‐19 severity. in addition, we discuss the relevant pathological changes resulting from sars‐cov‐2 infection. finally, we highlight a selection of potential treatment modalities for covid‐19. this article is protected by copyright. all rights reserved. introduction microbial ecology. in fact, transplantation of gut microbiota from ace2-knockout mice resulted in an increased propensity to develop severe colitis [21] . previously, we investigated the immunolocalization of ace2 in healthy human organs [7] . ace2 was highly expressed on lung alveolar epithelial cells and small intestinal epithelial cells, consistent with potential routes of viral transmission of sars-cov-2, as both respiratory and gastrointestinal systems share interfaces with the external environment. additionally, ace2 was present on vascular endothelial cells and smooth muscle cells in all organs studied. in the kidney, ace2 was strongly expressed in the brush border of proximal tubular cells and moderately or weakly in parietal epithelial cells and podocytes, whereas ace2 staining was weak or negative in glomerular endothelial cells and mesangial cells. ace2 was also present in the basal epidermal layer of the skin and in the oral and nasal mucosa. in contrast, ace2 was absent in lymphoid tissues and hepatobiliary structures [7] . the intense staining on various epithelial cells (small intestine, kidney, skin) strongly suggests raas-independent functions of ace2. these findings trigger alternative hypotheses regarding ace2 involvement in viral transmission pathways. furthermore, we previously noted that endothelial ace2 was upregulated in the glomerular and interstitial capillaries in kidney diseases independent of the initial trigger, indicating that ace2 may also be viewed as a damage marker [22] . summarising, ace2 is widely expressed in human tissues, both in principal target organs of sars-cov-2 and in organs that play a seemingly less important or even unknown role in covid-19 pathophysiology. this article is protected by copyright. all rights reserved. recently, ace2 was unequivocally established as the functional host receptor for sars-cov-2 (figure 3) [8]. binding kinetics revealed a 10-20-fold higher binding affinity as compared to the sars-cov-1 virus [8, 23] . these findings may partially explain the apparently easier transmissibility of sars-cov-2 and that increased ace2 expression may confer increased susceptibility to host cell entry of sars-cov-2. it was previously shown that a specific region within the sars-cov-1 spike protein interacts with ace2, leading to fusion with the host cell membrane [15, 24] . an experimental animal study in ace2-knockout mice further underlined the importance of this receptor in the pathogenesis of sars caused by sars-cov-1 [25] . the authors hypothesised that infection with sars-cov-1 results in ace2 downregulation through its internalisation, induced by binding of sars-cov-1 to ace2, as a mechanism contributing to the severity of lung pathologies [25] . consequently, this would lead to impairment of the protective effect of ace2 on the severity of acute respiratory distress syndrome (ards). this, as well as a harmful effect of ang ii, was previously demonstrated in several animal models of ards [26] [27] [28] [29] . the interaction between ace2 and sars-cov-1 and with sars-cov-2, and further downstream effects, exhibit a high level of similarity between each other [8] . during hypoxia, ang ii-induced pulmonary vasoconstriction occurs, aimed to restore the ventilation-perfusion mismatch, but simultaneously inducing adverse pro-fibrotic effects, which both are ameliorated by concomitant upregulation of ace2 [30] . under similar circumstances, sars-cov-2-induced downregulation of ace2 could impair clearance of ang ii and hence lead to aggravation of this article is protected by copyright. all rights reserved. tissue damage. on the other hand, one may speculate that ace2 downregulation by sars-cov-2 results in decreased opportunity for further viral cell entry, thereby limiting viral spread. however, as one may hypothesise that sars-cov-2 infects ace2-expressing cells with greater efficiency compared to sars-cov-1, presumably through exploiting cellular factors promoting viral attachment and entry, it is likely that sars-cov-2 viruses would need less ace2 to enable viral spread. taken together, the role of ace2 in sars-cov-2 cellular infection is complex and not yet defined, which makes it interesting to study if and how sars-cov-2 interferes with ace2 expression and/or its regulation, and how this influences viral replication. genetic factors ace2 is encoded by a gene located on chromosome xp22 and consists of at least 18 exons and 20 introns, amounting to approximately 40 kb of genomic dna [31] . the genetic architecture closely resembles the structure of the ace gene and may lead to a variety of alternative rna transcripts. the ace2 gene is characterised by a number of polymorphisms, which have been associated with diversity of raas-system pathologies, such as essential hypertension [32] . however, the genetic background of ace2 expression and functionality across different populations in relation to sars-cov-2 is largely unknown. comparative systematic analysis of coding-region variants and expression quantitative trait loci (eqtl) variants of ace2 across different populations, showed higher allele frequencies of eqtl variants associated with higher ace2 tissue expression levels in east asian compared to this article is protected by copyright. all rights reserved. european populations. this may imply a differential susceptibility to sars-cov-2 infection across different populations. however, no evidence supporting potential s-protein bindingresistant ace2 mutants was obtained [33] . structural modelling and superimposition analyses of the native ace2-and ace2-s-protein complex were used to study changes in ace2 variants and the intermolecular interactions with the s-protein. most ace2 coding variants showed high structural similarity and highly similar binding affinity with the s-protein of sars-cov-2. however, two allelic variants were identified that demonstrated considerable variation in intermolecular interaction with the s-protein, showing varying spatial orientation of key interacting residues of ace2 [34] . these ace2 genetic variations may provide a basis for relative or complete potential resistance against sars-cov-2 infection. ace2 expression in the lungs and sars-cov-2 viral load have been suggested to increase with age, which might provide an explanation to the higher disease severity observed in older patients with covid-19 [35] . advancing age is increasingly recognised as one of the strongest predictors for severe covid-19 [6] . older adults (aged above 60 years) are at increasing risk of contracting severe covid-19 with higher complication and case fatality rates [36] . similar to influenza and other respiratory viral infections, gradually decreasing innate and adaptive immune responses may be expected to play an important role in this agerelated increased susceptibility. accumulating data also show the existence of a genderassociated predisposition to covid-19, with men being more prone to develop severe disease than women [37] . ace2 expression may be a contributing factor to this association as single-this article is protected by copyright. all rights reserved. cell transcriptomics demonstrated that ace2 expression was higher among asian men than asian women [38] . observational data indicated higher frequencies of males among critically ill patients [39, 40] . in line, males appeared to be more frequent among deceased patients compared to recovered patients [41] . possible explanations of male predominance among covid-19 patients may be differences in exposure, smoking behaviour, other lifestyle factors, differences in chromosomal ace2 expression, ace2 expression in testicular tissue, sex hormone-driven immune system regulation, or gender differences in raas regulation [37, [42] [43] [44] . interestingly, in two independent cohorts of patients with heart failure, plasma concentrations of ace2 were higher in men than in women [45] . obese patients with covid-19 may have an increased risk of icu-admission and mortality. although obese patients frequently present with mechanical hypoventilation (leading to hypercapnic respiratory failure), those with covid-19 present with hypoxic respiratory failure. this led to discussions about a potential role of fat tissue in covid-19 pathogenesis in relation to ace2 expression. granting that obesity predisposes to developing chronic disease, obesity could also be an independent risk factor for covid-19 [46] . bmi is significantly higher among covid-19 patients with critical disease requiring icu-admission compared to less severe cases [47] . likewise, the proportion of patients with bmi > 25 kg/m 2 was significantly elevated in deceased patients compared to survivors. a chinese multi-centre study reported significantly higher bmi values among patients with severe disease compared to patients having only mild disease [48] . in other emerging large case series, obesity remains this article is protected by copyright. all rights reserved. common and may be a risk factor for respiratory distress, eventually requiring mechanical ventilation [49, 50] . these observations are analogous to other respiratory viral infections, for instance the h1n1 influenza virus infection. during that 2009 pandemic, obesity also emerged as an independent risk factor for hospitalisation and death [51, 52] . this could be attributed to obesity-induced impairment of the immune response, as has been welldocumented for h1n1 influenza [53] . mechanistically, adipose tissue-derived inflammation in obesity leads to substantial metabolic disturbances that could eventually lead to complications such as dyslipidaemia, hypertension, diabetes, cardiovascular disease (metabolic syndrome or syndrome x) and chronic respiratory failure [54] . visceral fat tissue can induce pro-inflammatory effects, which are regulated by adipokines and ang ii. interestingly, ace2 is abundantly present on visceral adipocytes [7, 55] . ace2 on adipocytes exerts systemic effects on the cardiovascular system and experimental studies demonstrated interactions between gender, adipocyte ace2 and complications of obesity, e.g. hypertension [56] . of note, leptin is one of the most important adipokines driving these pro-inflammatory effects and higher leptin availability has been associated with increased ang ii levels as well as decreased ace2 expression and activity [57] . in addition, high leptin levels have been associated with accumulation of alveolar fluid and increased inflammation upon hypoxia and ards [58] . therefore, it may be hypothesised that excess visceral adipose tissue in patients with covid-19 may drive disease progression -whether or not affected by genderespecially through aggravating the cascade of hyperinflammatory reactions in the disease [59] . ultimately, this 'cytokine storm' may lead to multiple organ failure in patients with this article is protected by copyright. all rights reserved. a recent meta-analysis of 46,248 patients diagnosed with covid-19 reported that severe disease was associated with hypertension, chronic respiratory disease and cardiovascular disease [60] . in another report including over 44,000 patients with confirmed covid-19, hypertension, chronic respiratory disease, diabetes mellitus, cardiovascular disease and cancer emerged as the most common comorbidities [1] . many of these comorbidities are characterised by either increased or decreased ace2 expression and/or activity, as well as a shift in ace/ace2 balance in both directions. this could be related to underlying conditions and/or to treatment with raas inhibitors (discussed in: 'pathogenesis and treatment options for covid-19'). however, the relative contribution of each of these underlying conditions to disease severity and mortality remains undetermined. many of the currently available reports were unadjusted for potential confounding factors, including age, sex, and lifestyle factors such as smoking and diet. similarly, many studies were uncontrolled, had relatively short follow-up periods, or were likely affected by inaccurate scoring or under-diagnosis [61] . in general, it is advised that patients using immunosuppressive drugs should not preemptively stop their medication, because there is still much unknown about potential risks or benefits. for instance, transplanted patients frequently use ciclosporin, which has been shown to have antiviral activity against sars-cov-1 [62] . patients with chronic immune-mediated inflammatory diseases (imids, e.g. rheumatoid arthritis [ra] this article is protected by copyright. all rights reserved. [ibd]) who are treated with cytokine inhibitors (e.g. tnf-antagonists, anti-il6r therapy) do not seem to be at an automatically increased risk of developing severe covid-19 [63] . although at first sight these treatments may seem to lead to immune suppression and may therefore be considered potentially harmful in the context of covid-19, they specifically target individual inflammatory cytokines or mediators instead of a broad panel of immune system components. in fact, cytokine inhibitors potentially attenuate the hyperinflammatory state associated with covid-19 and may therefore exert beneficial effects. this concept is supported by observations that the pro-inflammatory cytokines induced in covid-19 seem to be more crucial for the host inflammatory response compared to those involved mainly in viral clearance [63] . patients with solid malignancies treated with immune checkpoint inhibitors (icis), including anti-programmed death(-ligand)-1 (pd-1/pd-l1), anti-cytotoxic t-lymphocyte-associated protein-4 (ctla-4) and chimeric antigen receptor (car) t-cell therapy for certain b-cell related haematologic malignancies, frequently experience t-cellengaging immunomodulatory effects [64] . well-known immune-related adverse events include cytokine release syndrome (crs) and pneumonitis [65, 66] , which in theory might render patients more vulnerable to infections [67] . interestingly, these complications resemble the clinical presentation of advanced covid-19 and respond well to anti-il6r therapy [68] , providing a strong rationale for anti-il6r therapy in covid-19 [69] . as described, ace2 expression and activity is ubiquitously present within the human body, but many of its determinants on tissue level dynamics are unknown. however, in covid-19 this article is protected by copyright. all rights reserved. pathophysiology, there is seemingly huge spatiotemporal heterogeneity in organ involvement, presumably because multiple pathophysiological mechanisms may be causally involved in the observed tissue damage. both sars-cov-2 infection, directly mediated by ace2 expression and activity, and superimposed disease triggers may be responsible for the observed pathological findings. detailed pathological study of tissue specimens is therefore urgently needed to improve our understanding by disentangling the potential origins of tissue damage. the initial clinical presentation of covid-19 consists of respiratory symptoms such as fever, dry cough, shortness of breath, rhinitis, and, additionally, chest pain, myalgia and/or fatigue [70] [71] [72] [73] . in more severe cases needing hospitalisation, viral pneumonia develops with progressive ground-glass opacities on chest computed tomography (ct). in clinically critical cases, this is accompanied by further complications including ards, cardiac pathology and secondary infections. given the similarities between sars-cov-1 and sars-cov-2, lung pathology shows considerable equivalence [74, 75] . hitherto, there are limited reports of mainly autopsy cases describing lung pathological findings [76] [77] [78] [79] . similar to sars, covid-19-associated pathological changes in the lungs generally constitute extensive diffuse alveolar damage with bilateral oedema, proteinaceous or fibrin alveolar exudates and diffuse reactive hyperplasia of type ii pneumocytes (figure 4a-b) . in more advanced pathology, hyaline membrane formation was observed with thickened alveolar septa caused by interstitial fibroblast proliferation consistent with fibrosis ( figure 4b ). in addition, variable presence of patchy mainly interstitial infiltration of mononuclear cells has been reported ( figure 4a) , and in some cases, multinucleated giant cells in alveoli with associated viral changes. in contrast to sars, there is seemingly more thrombo-embolic pathology observed in specimens from patients with covid-19 (discussed in 'thrombo-embolic risk'). also, intra-alveolar deposition of neutrophilic granulocytes was reported in a few instances, most likely due to superimposed bacterial infection. another case report showed immunostaining of the rp3 np protein from sars-cov-2, which was prominently expressed on alveolar epithelial cells, as well as in cell debris within the alveolar space [80] . along the respiratory tract, ace2 has been observed in nasal and bronchial epithelial cells. in addition, ace2 is abundantly expressed on the surface of alveolar type ii pneumocytes, which also co-express several other genes that are involved in the regulation of viral reproduction and transmission, including tmprss2 [38, 81] . type ii pneumocytes usually produce surfactant, maintain their self-renewal and exert immunoregulatory functions. importantly, these cells share the same basement membrane with closely apposed capillary endothelial cells, also expressing high ace2 levels. these data indicate that type ii pneumocytes together with the related capillary endothelium may be a primary site of sars-cov-2 entrance, resulting in damage to those cells and the alveolo-capillary membrane and ongoing reactive hyperplasia of type ii pneumocytes. as type ii pneumocytes remain targets of viral entry and replication, this may lead to a vicious circle of continuing alveolar wall destruction and repair, eventually culminating in progressive severe diffuse alveolar damage. ace2 upregulation has also been described in airways in patients with chronic respiratory disease who are smokers, which, together with disturbed ciliary movement and abnormal this article is protected by copyright. all rights reserved. mucus viscosity, may increase disease vulnerability [82] . however, clinical evidence may indicate that smoking does not necessarily lead to increased vulnerability [83] . recently, it was suggested that the virus could also exploit goblet cells and ciliated cells in the nasal epithelia as entry portals, a plausible primary infection site in many patients [10] . although covid-19 is primarily a severe respiratory illness, acute myocardial injury is frequently observed, manifested by increased levels of high sensitivity cardiac troponin i (ctni) or cardiac troponin t (ctnt) in up to 28% of laboratory-confirmed covid-19 patients [84, 85] . the presence of myocardial injury was associated with worsened outcome, with 7-to 11-fold increased mortality rates. the highest mortality rates were observed in patients with both elevated tnt levels and pre-existing cardiovascular disease. reciprocally, pre-existing cardiovascular disease predisposes for sars-cov-2-induced myocardial injury and covid-19-associated mortality. whereas the relation between myocardial injury (associated with myocardial infarction, heart failure and ventricular arrhythmias) and mortality is evident, the aetiology of acute myocardial injury in response to sars-cov-2 infection is still unresolved. however, several potential mechanisms have been proposed, including sars-cov-2-induced myocarditis, cytokine-mediated injury (i.e. a systemic cardiotoxic cytokine-storm), microvascular injury, or stress-related cardiomyopathy or myocardial infarction [86, 87] . virus-induced myocarditis due to infection of cell populations residing in the heart has also been proposed, though this is still unproven [88] . scattered individual cardiomyocyte necrosis was observed in cardiac tissue from deceased covid-19 patients, however without clear this article is protected by copyright. all rights reserved. signs of myocarditis [88] . given the critical role of ace2 for sars-cov-2 cell entry, resident ace2-expressing cell populations in the heart can be potentially infected. single-cell rna sequencing of discarded donor hearts revealed that pericytes, but not cardiomyocytes, express highest ace2 levels [89] . this suggests that cardiac pericytes form a potential sars-cov-2 target cell, which may cause capillary endothelial cell dysfunction upon infection culminating in myocardial injury. so far, only one case report has been published on the presence of sars-cov-2 in the heart and demonstrated viral particles in interstitial cytopathic cells, most likely macrophages, but not cardiomyocytes or endothelial cells [90] . direct cardiotoxic effects and presence of sars-cov-2 in the heart needs to be confirmed in larger series. as ace2 is abundantly expressed by endothelial cells throughout the body, it loses its ability to prevent thrombosis upon cell entry of sars-cov-2 [12] . in human umbilical vein endothelial cell cultures in vitro, ace2 has been shown to have a role in protection of endothelial function and inhibition of the inflammatory response [91] . in experiments with spontaneously hypertensive rats, ace2 activation reduced thrombus formation and platelet attachment to vessels, while these effects were reversed by inhibition of ace2 [92] . putatively, direct infection of endothelial cells by sars-cov-2 could result in systemic impaired microcirculatory function in different vascular beds. in fact, sars-cov-2 has recently been shown to directly infect engineered human blood vessel organoids in vitro [93] . the permissiveness of endothelial cells in vivo for sars-cov-2 was demonstrated in renal glomerular endothelial cells by electron microscopy [94] . however, since no immunohistochemistry or immune electron microscopy was performed, it remained difficult this article is protected by copyright. all rights reserved. to distinguish between intracellular viral inclusions and normal subcellular organelles, as the latter may masquerade as viruses [95] . furthermore, covid-19 was associated with endotheliitis in various organs such as lung, liver, heart, kidney and small bowel [94, 96] . this suggests that direct infection of endothelium and/or perivascular inflammation may result in endothelial dysfunction, tissue oedema and a pro-coagulant state culminating in microvascular pathology, in particular in patients with pre-existing endothelial dysfunction. thrombo-embolic risk covid-19 patients are at particular risk for developing coagulopathy reminiscent of disseminated intravascular coagulation (dic) which was associated with mortality, possibly due to both venous and arterial thrombosis [97] . arterial thrombosis includes ischaemia of extremities, cerebral infarctions and myocardial infarctions [98] . after initial reports of an increased rate of venous thromboembolism (vte), including deep venous thrombosis (dvt) and pulmonary embolism (pe), a recent dutch study demonstrated a vte incidence of 27% and ~4% arterial thrombosis in covid-19 patients admitted to the icu [99] . in this study, the vast majority (80%) of patients with vte suffered from pe. pe could be an important factor in abrupt worsening of respiratory failure in patients with advanced covid-19 [100] . furthermore, several autopsy studies showed thrombi in the pulmonary vessels, which can be proximal large emboli but are most frequently identified as microthrombi. this microvascular thrombosis is predominantly observed in an environment of marked inflammatory changes including mononuclear cell infiltrates, virally infected cells and diffuse alveolar damage [88] . this article is protected by copyright. all rights reserved. in clinical studies, strongly elevated levels of circulating biomarkers of endothelial activation have been reported [101] . also, a clear picture of hypercoagulability is observed, with elevated d-dimers being most strikingly elevated in patients with severe disease [40, [102] [103] [104] [105] . d-dimer is a fibrin-degradation product that develops after a blood clot is degraded by fibrinolysis. moreover, d-dimer levels at hospital admission predict a worse clinical outcome [6, 97] . although d-dimers are a biomarker for thrombosis, they are also known as strong acute-phase reactants. however, high d-dimer levels seem to persist in advanced covid-19 patients in whom inflammatory markers such il-6 have already decreased, stressing that their elevation is not solely secondary to systemic inflammation [6] . furthermore, as covid-19 patients generally present with normal to slightly elevated platelet levels, strongly increased fibrinogen and normal to only slightly prolonged prothrombin and activated partial thromboplastin time [106] , thromboembolic events in these patients do not seem to be a result of an hypofibrinolytic consumptive diffuse intravasal coagulation as generally observed in sepsis [99] . interestingly, strongly increased levels of antiphospholipid (anticardiolipin and anti-β2glycoprotein i) antibodies have been reported in covid-19 patients with venous and arterial thromboembolisms, which is a feature of the antiphospholipid syndrome (aps) [102, 107] . patients with systemic lupus frequently present with aps and limb ischaemia caused by vasculopathy. in a clinical study in systemic lupus, anti-ace2 antibodies were found to be elevated in almost every patient and correlated with the relative activity of serum ace2 [108] . furthermore, systemic lupus patients overexpress ace2 as a result of hypomethylation, and their vascular complications respond very well to hydroxychloroquine this article is protected by copyright. all rights reserved. treatment, being circumstantial evidence of a speculative link between ace2 and vascular complications in covid-19 [109] . in summary, these observations underline that the hypercoagulable state in covid-19 may be of a systemic nature, and not limited to pe [110] . gastrointestinal (gi) symptoms are commonly observed in patients with covid-19. in a meta-analysis of 4,243 patients, pooled prevalence of gastrointestinal symptoms was 17.6% [111] . moreover, viral rna has been repeatedly detected in stool samples [112, 113] : in the aforementioned study, the pooled prevalence of positive samples was 48.1%. commonly observed gi symptoms include anorexia, diarrhoea, vomiting, and abdominal pain [114] . in this study, diarrhoea as initial disease symptom was reported in 17% of patients, but seemingly no bloody diarrhoea. in addition, patients with digestive symptoms seemed to have a longer time from disease onset to hospital admission and presented with evidence of prolonged coagulation and elevated liver enzyme levels [114] . theoretically, sars-cov-2 could directly invade the gastrointestinal epithelium via ace2. in a single-cell transcriptome study, ace2 was found to be highly expressed in oesophageal upper and stratified epithelium, as well as in absorptive enterocytes derived from both ileum and colon [115] . in addition, ace2 was co-expressed with the tmprss2 prime protein in absorptive enterocytes and upper oesophageal epithelial cells. in our previous study from 2004, we found ace2 to be expressed in enterocytes of all parts of the small intestine, including duodenum, jejunum, and ileum, but not in colonic enterocytes [7] . more specifically, ace2 was densely stained at the villous brush border, but also deeper into the intestinal wall, particularly in smooth muscle this article is protected by copyright. all rights reserved. previously, proteomics analyses demonstrated that ace2 protein is increased in ibd [116] . furthermore, ace2 activity and elevated angiotensin(1-7) concentrations were described in patients with ibd [117] . in that study, it was shown that ang ii and angiotensin (1-7) influence colonic myofibroblast proliferation and collagen secretion, and the use of aceinhibitors (aceis) and angiotensin-receptor blockers (arbs) associated with improved disease outcome in ibd patients [118] . until now, there is no evidence for increased susceptibility for covid-19 in patients with ibd. the implications of covid-19 for immunomodulation in ibd have recently been reviewed [119] . previously, viral rna in faeces could be detected after viral rna in the respiratory tract became negative and evidence for gastrointestinal infection of sars-cov-2 was documented recently, i.e. infectious virus could be isolated from the stool [120, 121] . however, another recent study did not find evidence for the presence of infectious virus in rna-positive stool samples [122] . altogether, these observations suggest that sars-cov-2 actively infects and replicates within the gi tract, implying a possible role for a faecal-oral viral transmission route. liver manifestations have also been reported in covid-19 patients. biochemical signs of mild-to-moderate liver injury are frequently observed, including elevated liver function tests (ast, alt, γ-gt and alp), hypoalbuminaemia, prolonged prothrombin time, and increased crp, ldh and hyperferritinaemia, which may be reflective signs of acute-phase inflammation [123] . liver damage may be primarily attributed to direct viral infection this article is protected by copyright. all rights reserved. causing hepatitis, but may also be interpreted as drug toxicity by administration of high-dose antiviral medications, antibiotics or steroids [124] . ace2 is expressed in the liver, mainly on cholangiocytes instead of hepatocytes, and it has been suggested that ace2 might be upregulated by compensatory hepatocyte proliferation upon cholangiocyte injury [125] . to date, however, little is known about direct viral infection of the liver by sars-cov-2. one study on liver biopsy specimens showed moderate microvascular steatosis and mild lobular and portal activity, though it was unclear whether this was caused by sars-cov-2 infection or by drug toxicity [126] . another study observed mild lobular infiltration by small lymphocytes, patchy necrosis and centrilobular sinusoidal dilation [127] . interestingly, a recent single-cell transcriptomics study found high ace2 expression on cholangiocytes, suggesting that sars-cov-2 may also lead to damage of intrahepatic bile ducts [128] . taken together, one may hypothesise that hepatobiliary involvement in covid-19 primarily results from biliary infection, with secondary injury to hepatocytes. recent evidence points towards significant involvement of the kidney in covid-19. whereas initial studies reported a relatively modest risk for acute kidney injury (aki), subsequent studies reported an incidence rate up to 15% [129] . occurrence of aki in covid-19 patients is associated with higher disease severity in icu-admitted patients, and is an adverse prognostic sign for survival [130] . small studies of covid-19 patients have reported signs of proteinuria and haematuria in about 40% of hospital-admitted patients [131] . ace2 expression has been confirmed on the brush border of proximal tubular cells and on this article is protected by copyright. all rights reserved. podocytes, whereas glomerular endothelial and mesangial cells were weakly positive or negative for ace2 [7] . in the previous sars outbreak, renal involvement was a rare phenomenon, although, if present, aki was often a fatal disease complication [132] . further research provided evidence that this renal involvement, in the form of aki, may be more attributed to processes behind multi-organ failure rather than active viral replication of sars viruses [132, 133] . for instance, crs or cytokine storms have been reported as prior events leading to severe renal damage [134] . more recently, sars-cov-2 viral antigens have been detected in post-mortem specimens, specifically in kidney tubules [135, 136] . in another recent study, histopathological analysis of post-mortem findings revealed diffuse acute tubular injury (ati) with loss of brush border, non-isometric vacuolar degeneration and even necrosis, as well as prominent erythrocyte aggregates occluding the capillary lumina with resulting endothelial damage (figure 4c-d) [137] . in line with the tissue distribution of ace2 in the kidney, coronavirus-like particles were identified in tubular epithelium and in podocytes. based on these recent findings, it is suggested that sars-cov-2 directly targets the kidney parenchyma, especially the renal tubular epithelium and podocytes, with secondary endothelial injury that may induce aki and lead to proteinuria and elevated serum creatinine levels in these patients. moreover, sars-cov-2 infections seem to be more frequently associated with aki compared to sars-cov-1 [131] . the increased binding affinity of sars-cov-2 to ace2 may explain this phenomenon, as it would allow for greater renal infectivity. this article is protected by copyright. all rights reserved. in skin, ace2 has been demonstrated in the basal epidermal layer and eccrine sweat glands [7] . however, reports on skin involvement started to emerge only recently. the extent and origin (reactive, direct viral damage, thrombosis, vasculitis) of skin involvement in covid19 , and the relation to severity of covid-19, remains to be established. following two large covid-19 cohort descriptions only mentioning 'skin rash' without further details in a minority of patients [40, 138] , several case reports have emerged reporting abnormalities ranging from erythematous rash, urticarial plaques, purpura, to chickenpox-like vesicles, without information on histopathology [139] [140] [141] [142] [143] [144] [145] [146] . in addition, recalcati et al. reported skin alterations similar to the aforementioned case reports in 18 of 88 (20.4%) medication-naive covid-19 patients [147] . again, no histopathology was available. very recently, one case report linked covid-19 to the occurrence of immune thrombocytopenic purpura [145] . additionally, another study reported purpura and livedo racemosa in several severely affected covid-19 patients with small vessel thrombosis with co-localization of complement and sars-cov-2 spike proteins on histopathology [148] .this indicates direct viral infection of the small skin vessels. however, the diversity of skin features reported in covid-19 patients suggests other pathogenic mechanisms as well. in healthy skin, the layers above the ace2expressing stratum basale, including the stratum corneum, likely provide a barrier to the virus. however, the clear expression of ace2 in skin suggests that if sars-cov-2 gets the chance to reach its receptor there, for example through damaged skin, it may be able to render itself a porte d'entrée in keratinocytes. given that sars-cov-1 was previously found in sweat [149] , this raises the question whether sars-cov-2 could be excreted in sweat, thereby adding to its transmission potential. in addition, it raises the question whether sars-cov-2 is this article is protected by copyright. all rights reserved. able to infect through binding to ace2 in the eccrine sweat glands of palmar skin, where they are abundantly expressed. pregnancy is a unique physiological state in which a semi-allogeneic fetus (and placenta) is accepted by the maternal immune system, whilst at the same time this system has to maintain the protective capacity for defence against pathogens. due to the necessary adaptations in the immune system and a variety of physiological adaptations (e.g. increased oxygen consumption, mucosal oedema of the respiratory tract), pregnant women are generally characterised by increased susceptibility to respiratory pathogens, and consequently, severe pneumonia. although there is no evidence that pregnant women are more susceptible to sars-cov-2 infection, they may be at increased risk of developing severe illness when contracting sars-cov-2 infection. currently, there is limited evidence regarding the possibility of mother-fetal intra-uterine vertical transmission in covid-19. most descriptions of sars-cov-2 infected pregnant women reported infections during the third trimester of pregnancy [150] [151] [152] [153] . however, uncertainty prevails about whether vertical transmission of covid-19 may occur in any phase of pregnancy [105] [106] . placentas, amniotic fluid samples or newborns (directly after delivery) with positive rt-pcr results have not been described, which means that there is no virological evidence of intra-uterine infection at the maternal-fetal interface [150, [154] [155] . neonatal covid-19 has been reported, but infection could have occurred through other routes as there was no direct evidence for intrauterine vertical transmission [150, 152, 154, 157, 158] . in a small case series, fetal growth restriction this article is protected by copyright. all rights reserved. (fgr) has been described in sars-cov-1-positive women, but no details of the placental histopathological lesions were described [159] . there is one report that described seven placentas that were evaluated histopathologically after maternal infection with sars-cov-1. placentas from infection in the first trimester were normal (n=2). increases in intervillous and subchorionic fibrin deposition were observed once delivered in the acute stage of infection (n=3) which is possibly not sars-cov-specific, but rather related to disturbances in maternal placental blood flow due to hypoxic respiratory disease. third trimester convalescent infection resulted in extensive fetal thrombotic vasculopathy (ftv) with sharply demarcated zones of avascular fibrotic villi resulting in fgr (n=2). the aetiology of the ftv might be related to thrombotic tendency due to sars-cov infection or placental hypoxia [160] . ace2 ould also play a role in this process. however, although placental ace2 expression is found on both the fetal site (umbilical cord, placental villi in the syncytiotrophoblast, cytotrophoblast, vascular endothelium and smooth muscle cells) and on the maternal site (e.g. in the invading and intravascular trophoblast and decidual cells), regulation of placental ace2 expression has not yet been described in relation to sars-cov-2 infection [161, 162] . sars-cov-2 has been implicated to have neurotropic potential in covid-19 [163] . indeed, some patients presented with symptoms that could be attributed to neurological involvement, such as headache, confusion, anosmia, dysgeusia, nausea and vomiting [164, 165] . previous research showed that sars-cov-1 and mers-cov infect the central nervous system with significant involvement of the brainstem [163] . it has been suggested that neuroinvasion of this article is protected by copyright. all rights reserved. the brainstem may be at least partially responsible for respiratory symptoms in covid-19 patients, by compromising neurons within the respiratory centres and chemosensitive neural cells involved in respiratory and cardiovascular regulation [166] . ace2 may play a role in sars-cov-2 neuroinvasion, as it is expressed in the brain on neurons and glial cells, particularly in the brainstem and cardiovascular regulatory areas, including the nucleus tractus solitarius, paraventricular nucleus and the rostral ventrolateral medulla [167, 168] . in addition, ace2 is expressed in the cerebral vascular endothelium, which could lead to endothelial damage subsequently leading to viral access to the brain [169, 170] . in an experimental animal study, it was demonstrated that the sars-cov-1 enters the brain primarily via the olfactory bulb, followed by transneuronal spread of the virus [171] . this could explain the underlying pathophysiology of covid-19-associated anosmia. however, detailed neurological investigation of covid-19 autopsies could further clarify the occurrence and underlying neurological pathology characteristic for sars-cov-2 infection. initially, sars-cov-2 may pass through either the mucous membranes in the upper respiratory tract, primarily the nasal and pharyngeal epithelia, or directly entering the lower respiratory tract and infect bronchial and alveolar epithelial cells [10] . the main symptoms of respiratory infection are fever and cough. in this initial phase, the virus can enter the peripheral bloodstream via the lungs and may result in viraemia [172] . the virus may then proceed to affect other organs expressing ace2, such as the heart and blood vessels, the kidneys and the gi tract. however, the gi tract may also have direct infection by the oral this article is protected by copyright. all rights reserved. route. patients with an increased risk of developing severe disease may experience severe pulmonary involvement resulting in systemic inflammation [6] . the massive inflammatory process at that time results in a severe cytokine storm also affecting other organs in the body. this seemingly occurs in line with other blood-derived viruses entering organs via ace2 on activated endothelium causing, for example, renal or gi problems. in the vasculature, this coincides with red blood cell aggregation and thrombosis. the clinical phase progresses from the initial viraemia to an acute phase (pneumonia), followed by either recovery or severe disease (including ards, aki and eventually multi-organ failure) requiring icu admission [173] . the distinction would depend on patient comorbidity, obesity-induced pre-existent inflammation, immune function and ace/ace2 balance in already affected organs. each phase demands its own treatment regimen ranging from virus entry and replication inhibition in the initial phase to anti-inflammatory and anti-thrombotic medication at later stages. in the following paragraphs, we aim to highlight some of the most commonly advocated treatment strategies being explored to combat covid-19. hydroxychloroquine (hcq) and chloroquine (cq) are two widely used antimalarial, antiviral and anti-rheumatic drugs. recently, in vitro results and small clinical studies emerged that demonstrated antiviral activity of these drugs against sars-cov-2 infection [174] [175] [176] [177] [178] [179] . beneficial effects were presumed to arise from blockage of viral host cell entry by increasing endosomal ph and interference with glycosylation of ace2 [180] . two studies from france this article is protected by copyright. all rights reserved. reported that hcq could lead to viral load reduction within six days, especially when combined with azithromycin. however, these studies were impaired by several methodological constraints [181] . similarly, two chinese trials were performed: one study reported no significant difference in nasopharyngeal viral carriage between hydroxychloroquine treatment and standard supportive care, whereas the other study demonstrated shorter clinical recovery time for patients receiving hcq compared to placebo [178, 179] . for the latter study, however, it was not possible to extrapolate to critically ill patients, which is crucially important because this subgroup of patients may be of particularly increased risk of serious adverse effects upon treatment with hcq/cq, such as ventricular arrhythmias, hepatic failure and cardiac toxicity [181, 182] . indeed, recent studies reported concerns about potential safety hazards as higher dosages were associated with higher mortality and excessive qt interval prolongation, especially when taken concurrently with azithromycin and oseltamivir [183, 184] . another large observational study indicated that hcq/cq may not help in critically ill patients as its administration was not associated with either a significantly lowered or an increased risk of a composite endpoint of intubation or death [185] . thus, currently available data on hcq/cq treatment for covid-19 are inconclusive, but appear far from promising. therefore, upcoming prospective randomised clinical trials will have to determine if treatment with hcq/cq would be a reasonable therapeutic strategy for covid-19 patients and what would be the most suitable timing within the disease course to initiate treatment. this article is protected by copyright. all rights reserved. remdesivir, a rna polymerase inhibitor, was demonstrated to be effective against sars-cov-1 and mers-cov. for instance, remdesivir improved disease outcome and reduced viral load in sars-cov-1 infected mice [186] . in 53 hospitalised patients with covid-19, improvement of clinical status was observed in 36 after receiving at least one dose of remdesivir [187] . furthermore, a recently conducted randomised controlled trial evaluated the role of lopinavir and ritonavir in 199 covid-19 patients, 99 treated with lopinavir/ritonavir while 100 received standard treatment [188] . the authors concluded that patients treated with lopinavir/rotinavir did not demonstrate any significant improvement in hazard ratio for earlier clinical improvement or reduction in mortality at 28 days. in contrast to the primary outcome, patients treated with lopinavir/ritonavir demonstrated clinical improvement 1 day earlier than the control group and were discharged 5 days earlier from the icu. although large clinical trials investigating the therapeutic effect of these antiviral therapies in covid-19 are lacking, it can be hypothesised that the available studies possibly included patients with severe disease alone, and, therefore, future studies may consider evaluating the role of these antiviral drugs earlier in the course of covid-19 [188] . the worldwide growth of sars-cov-2 infections raised serious concerns about the widespread use of antihypertensive drugs, i.e. aceis and arbs, which are also used in treatment of cardiovascular diseases, chronic kidney disease and diabetes mellitus [189] . discussions emerged about whether these drugs may exert beneficial or deleterious effects in covid-19. many opinion papers have been published recently that predominantly state that this article is protected by copyright. all rights reserved. there is no scientific evidence to change the prescription of aceis or arbs for the management of hypertension in the context of preventing or treating sars-cov-2 infection. the use of aceis and arbs as risk factors for developing or aggravating covid-19 has been suggested because of their capacity to upregulate ace2 [190] [191] [192] . however, others have advocated beneficial and protective effects of these drugs in the development of covid-19 [14, 189] . in some animal studies, aceis or arbs increase ace2 levels, whilst other studies failed to demonstrate such shifts in ace2 [12, 19, [193] [194] [195] [196] [197] [198] [199] [200] , although shifts in ace/ace2 balance were noted [19] . therefore, it remains relevant to question whether raas blockers actually increase susceptibility to sars-cov-2 infection by increasing ace2. ace2 is protective against severe lung injury in animal models and ace2 blockade or genetic ace2knockouts result in extensive lung damage and decreased survival after respiratory syncytial virus infection [201] . similarly, at1r blockade by losartan attenuates lung injury in mice administered with the spike glycoprotein of sars-cov-1 [25] . although few human studies have been performed investigating potential effects of raas therapy on ace2 expression and/or activity, it was recently reported that aceis and arbs did not increase plasma ace2 concentrations [45] . similarly, others reported no clear direct effects of aceis on ace2 activity (as evaluated by angiotensin(1-7) levels) [202, 203] . several hypotheses exist about how increased tissue ace2 expression may be protective rather than harmful during sars-cov-2 infection [204] . for example, increased ace2 expression may lead to enhanced sequestration of sars-cov-2, but does not imply automatic activation of further downstream processes essential for viral entry, such as this article is protected by copyright. all rights reserved. involvement of tmprss2, which is required for spike glycoprotein priming, or adam metallopeptidase 17 (adam17), which is required for cleavage of the ace2 ectodomain resulting in increased ace2 shedding. furthermore, arbs lead to competition with ang ii for at1r, resulting in increased ang ii to be processed by ace2. this increases ang(1-7) levels, which results in vasodilating and anti-fibrotic effects, providing crucial protection during coronavirus infections [25] . furthermore, increased binding of ace2 to circulating ang ii could induce a conformational change resulting in less favourable binding of sars-cov-2 to its receptor and decreased internalization of the virus when bound to ace2 [204, 205] . we previously observed a positive shift in plasma ang(1-7)/ang ii balance in favour of the beneficial ang(1-7) peptide, particularly in circumstances of low sodium intake [19] . importantly, however, plasma ace2 levels may be less indicative of the risk of sars-cov-2 infection or membrane-bound ace2 activity, as ace2 shedding by adam17 appears to be regulated separately [206] . interestingly, however, plasma ace2 concentrations appear to be higher in older men with heart failure independent of raas inhibition [45] . clinical trials investigating the potential (side-)effects and safety of aceis and arbs on ace2 expression and activity in covid-19 are ongoing. from a clinical perspective, it may be preferable to await these results instead of discontinuing raas inhibitors, which may lead to clinical derangement especially in patients at high-risk for covid-19 [207] . since currently available evidence indicates that aceis and arbs significantly reduce mortality in cardiovascular disease, reduce progression of ckd, and are crucial in treatment of heart this article is protected by copyright. all rights reserved. failure and hypertension, most clinicians tend to maintain these regimens for their patients, regardless of sars-cov-2 [189] . immunomodulating drugs or biological response modifiers alter the host immune system by interacting with a specific target crucial for disease pathogenesis [208] . many of these compounds enrich the therapeutic armamentarium of several malignancies, autoimmune disorders, transplantation rejection, as well as infectious diseases. especially since vaccine development is time-consuming and antiviral drugs may have a limited therapeutic window, targeted immunomodulators are attractive alternatives. furthermore, these therapies may be crucial to control the hyperactivation of host inflammatory responses and 'cytokine storm' as has been described for covid-19 [209] . however, caution should be taken towards this therapeutic strategy as it will remain challenging to target immune system components without compromising the host defence mechanisms necessary to fight sars-cov-2 infection. in this respect, targeting specific or limited effector mechanisms (e.g. production of pro-inflammatory cytokines or reactive oxygen species), should be preferred over blockage of more proximal immune targets (e.g. pattern recognition receptors) that play a more significant role in regulating host immune defence [209] . the current hypothesis is that a cytokine storm can induce or further aggravate sars-cov-2 infection, and thereby suggests that blocking cytokine pathways could attenuate the disease this article is protected by copyright. all rights reserved. course. among these, interleukin-6 (il-6) is thought to play a prominent role. il-6 is a cytokine with both anti-and pro-inflammatory effects. it can be produced by almost all stromal and immune system cells (monocytes, lymphocytes, macrophages, endothelial cells, mast cells, dendritic cells) and is believed to play a central role in the development of cytokine storm [210, 211] . in line with this reasoning, anti-il6r therapy is a potential therapeutic option in covid-19. currently available humanised monoclonal antibodies against the interleukin-6-receptor (tocilizumab and sarilumab) are being tested in covid-19. a small study demonstrated that tocilizumab ameliorated the increased crp in all 15 patients, which is a direct effect of its pharmacological action. moreover, in critically ill patients with elevated il-6 levels, repeated doses of tocilizumab could be beneficial. however, objective clinical endpoints were not reported [212] . although others have shown comparable results, data on the use of tocilizumab are still preliminary and larger randomised controlled trials are needed [213] [214] [215] . whether anti-il6r therapy should be started early in the course of the disease or restricted to patients with signs of a cytokine storm is still of debate [210] . in addition, other cytokines such as il-1, ifn-γ and tnf-α are abundant in the cytokine storm, and blocking these pathways with appropriate biological being investigated [216] . inhibition of the jak-stat signalling pathway has also been suggested as a potential targeted therapy for covid-19 and several clinical trials are ongoing [217, 218] . inhibitors blocking jak2, such as fedratinib, have been suggested to block viral entry and combating the th17-component of the host inflammatory cytokine storm, without altering interferon this article is protected by copyright. all rights reserved. signalling [219] . sars-cov-2 enters host cells via ace2-mediated endocytosis, which is controlled by upstream regulators including ap2-associated protein kinase 1 (aak1) and cyclin g-associated kinase (gak). one of several high-affinity inhibitors of these regulators is the jak inhibitor baricitinib, which may limit viral host cell entry and intracellular assembly of viral particles through disrupting aak1 and gak. baracitinib may be of particular value during the hyperinflammatory phase, in which high levels of cytokines occur that signal through the jak-stat pathway. however, the optimal time to administer cytokine inhibitors still needs to be determined and results from the aforementioned clinical trials should be awaited. the association between obesity and the progression to hypoxic respiratory failure in patients with covid-19, requiring mechanical ventilation, has led to the assumption that leptin and adipokines may play a key role in this subpopulation of sars-cov-2 infected patients. resveratrol, an antioxidant and food supplement, has been suggested to be of potential therapeutic value because of a triple action. first, in some studies, resveratrol reduces leptin levels [220] . second, resveratrol could suppress ang ii, which might reduce inflammation [221] . third, antioxidant effects in the lung may reduce oxidative stress-induced lung damage [222] . this food supplement is safe in its use (up to 2-3 grams per day) and should be studied in covid-19 patients as an additive to other treatments. this article is protected by copyright. all rights reserved. as a result of the increased risk of thrombotic events in covid-19, guidelines currently advocate liberal use of prophylactic systemic anticoagulation [223] . the international society on thrombosis and haemostasis recently recommended that all hospitalised covid-19 patients, even those not admitted to icu, should receive prophylactic-dose low-molecularweight heparin (lmwh) unless they have contraindications (active bleeding and platelet count <25x10 9 /l) [224] . however, a recent study showed that despite adequate treatment with prophylactic low-dose lmwh, covid-19 patients admitted to icu were still at a substantial risk for pe [99] . this has made the dutch federation of internists decide to recommend a double dose of lmwh in icu patients with covid-19, when bleeding risk allows this strategy [225] . other guidelines advocated prophylactic systemic anticoagulation with unfractionated heparin rather than lmwh [226] , which may be needed in high dosages because of heparin resistance [227] . however, it is unlikely that anticoagulant treatment has a direct disease-modifying effect and it should be stressed that the initial viral load, as well as the systemic inflammatory response, needs to be attenuated since these are the driving forces for vte in covid-19 [228] . future studies are warranted to determine the most suitable approach for thrombosis prophylaxis in covid-19. scientific findings about ace2 and its role in covid-19 pathophysiology, it is crucial to maintain integration of available pathological and molecular evidence to establish the definite role of these potential modulating factors. unraveling the pathologic basis of covid-19 is essential for our understanding of the pathophysiology of the disease. unsurprisingly, severe pathological findings are mainly observed in specific target organs of sars-cov-2, such as the lungs and kidneys. in severe cases, this may lead to ards and multi-organ failure not directly related to ace2 expression and activity. this review focused on the role of widespread ace2 tissue expression, which may become a reasonable therapeutic target together with its effector pathways, for example through implementation of recombinant human ace2 (rhace2) therapy or by targeting bradykinin metabolism in the lungs. however, it will also be important to focus on additional mechanisms that may be involved in cellular infection and may regulate the interaction between sars-cov-2 and ace2. future studies featuring higher numbers of patients are warranted to reliably assess potential differences in ace2 expression, activity and regulation under a variety of physiological circumstances, such as present or lacking interaction with co-receptor or coactivating molecules, as well as in the context of commonly observed underlying conditions, including cardiovascular disease, hypertension, diabetes, obesity, smoking and respiratory disease. in particular, pathological studies of larger series of autopsy findings, probably in human and non-human primate models alike, are required to more accurately determine the relative contribution of each pre-existent co-morbidity and to discriminate between specific sars-cov-2-associated pathology and superimposed pathological changes. furthermore, the this article is protected by copyright. all rights reserved. development of appropriate animal and in vitro models could help to learn more about the sars-cov-2 infection process itself and, most importantly, the disease progression pattern observed in humans. in any case, it is indisputable that devoting scientific efforts to analyse aspects of ace2 in relation to covid-19 pathophysiology is paramount to fuel the development and augmentation of future therapeutic strategies. the current covid-19 pandemic is a major challenge for public health and clinical medicine. for public health, reduction or prevention of virus transmission as well as reduction of predisposing lifestyle factors need to be implemented. for clinical management in the foreseeable future, we should strive to adopt a personalised medicine approach aimed to provide individually tailored treatment in patients affected by covid-19. as highlighted in this review, this should take into account individual patient differences in mutual ace2-sars-cov-2 interactions with their consequences for covid-19 pathophysiology. to achieve this, it is of cardinal importance to carefully register the individual patient phenotype and to integrate this with diagnostic (e.g. laboratory and imaging results) and therapeutic information (e.g. drug toxicity and side-effect profiles). mainly because of low patient numbers in individual studies, currently ongoing trials are challenged to take into account between-subject differences or cohort heterogeneity, which may be considered likely to explain the majority of variation in disease outcome. however, detailed phenotypical stratification of individual patients during their disease course will 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changes in patients with viral pneumonia characteristics and outcomes of 21 critically ill patients with covid-19 in washington state hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: an observational study a pilot study of hydroxychloroquine in treatment of patients with moderate coronavirus disease-19 (covid-19) efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro hydroxychloroquine in the management of critically ill patients with covid-19: the need for an evidence base use of hydroxychloroquine and chloroquine during the covid-19 pandemic: what every clinician should know effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial safety of hydroxychloroquine, alone and in combination with azithromycin, in light of rapid wide-spread use for covid-19: a multinational, network cohort and self-controlled case series study observational study of hydroxychloroquine in hospitalized patients with covid-19 broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses compassionate use of remdesivir for patients with severe covid-19 covid-19 -the search for effective therapy renin-angiotensin-aldosterone system inhibitors in patients with covid-19 are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? preventing a covid-19 pandemic can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? enalapril attenuates downregulation of angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat localization of ace2 in the renal vasculature: amplification by angiotensin ii type 1 receptor blockade using telmisartan perinatally administered losartan augments renal ace2 expression but not cardiac or renal mas receptor in spontaneously hypertensive rats effect of angiotensin ii blockade on a new congenic model of hypertension derived from transgenic ren-2 rats human intestine luminal ace2 and amino acid transporter expression increased by ace-inhibitors upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors myocardial infarction increases ace2 expression in rat and humans combination renin-angiotensin system blockade and angiotensin-converting enzyme 2 in experimental myocardial infarction: implications for future therapeutic directions angiotensin-converting enzyme 2 inhibits lung injury induced by respiratory syncytial virus evidence against a major role for angiotensin converting enzyme-related carboxypeptidase (ace2) in angiotensin peptide metabolism in the human coronary circulation effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension should covid-19 concern nephrologists? why and to what extent? the emerging impasse of angiotensin blockade ace2 x-ray structures reveal a large hingebending motion important for inhibitor binding and catalysis tumor necrosis factor-α convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) ace inhibition and cardiometabolic risk factors, lung ace2 and tmprss2 gene expression, and plasma ace2 levels impact of host genetics and biological response modifiers on respiratory tract infections coronavirus infections and immune responses the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease a combined set of four serum inflammatory biomarkers reliably predicts endoscopic disease activity in inflammatory bowel disease tocilizumab treatment in covid-19: a single center experience favorable changes of ct findings in a patient with covid-19 pneumonia after treatment with tocilizumab rapid and severe covid-19 pneumonia with severe acute chest syndrome in a sickle cell patient successfully treated with tocilizumab off-label use of tocilizumab in patients with sars-cov-2 infection preventing covid-19-induced pneumonia with anticytokine therapy baricitinib for covid-19: a suitable teatment? -authors' reply baricitinib as potential treatment for 2019-ncov acute respiratory disease th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib the inhibitory effect of resveratrol on leptin secretion from rat adipocytes effects of resveratrol on the renin-angiotensin system in the aging kidney resveratrol attenuates intermittent hypoxia-induced lung injury by activating the nrf2/are pathway pulmonary embolism in patients with covid-19: time to change the paradigm of computed tomography isth interim guidance on recognition and management of coagulopathy in covid-19 this article is protected by copyright. all rights reserved leidraad covid-19 coagulopathie isth interim guidance on recognition and management of coagulopathy in covid-19: a comment thromboembolic events and apparent heparin resistance in patients infected with sars-cov-2 thromboinflammation and the hypercoagulability of covid the authors would like to express their gratitude towards else koning for her valuable help in the graphical design of the figures and martin bourgonje for critically proofreading the manuscript. in addition, the authors would like to thank dr. jan von der thüsen (department of pathology, erasmus medical center, rotterdam, the netherlands) and dr. hua su (department of nephrology, union hospital, wuhan, china) for kindly providing us with histological images. key: cord-338923-hc7gagnq authors: jääskeläinen, aj; kuivanen, s; kekäläinen, e; ahava, mj; loginov, r; kallio-kokko, h; vapalahti, o; jarva, h; kurkela, s; lappalainen, m title: performance of six sars-cov-2 immunoassays in comparison with microneutralisation date: 2020-06-15 journal: j clin virol doi: 10.1016/j.jcv.2020.104512 sha: doc_id: 338923 cord_uid: hc7gagnq there is an urgent need for reliable high-throughput serological assays for the management of the ongoing covid-19 pandemic. preferably, the performance of serological tests for a novel virus should be determined with clinical specimens against a gold standard, i.e. virus neutralisation. we compared the performance of six commercial immunoassays for the detection of sars-cov-2 igg, iga and igm antibodies, including four automated assays [abbott sars-cov-2 igg (ce marked), diasorin liaison® sars-cov-2 s1/s2 igg (research use only, ruo), and euroimmun sars-cov-2 igg and iga (ce marked)], and two rapid lateral flow (immunocromatographic) tests [acro biotech 2019-ncov igg/igm (ce marked) and xiamen biotime biotechnology sars-cov-2 igg/igm (ce marked)] with a microneutralisation test (mnt). two specimen panels from serum samples sent to helsinki university hospital laboratory (huslab) were compiled: the patient panel (n=70) included sera from pcr confirmed covid-19 patients, and the negative panel (n=81) included sera sent for screening of autoimmune diseases and respiratory virus antibodies in 2018 and 2019. the mnt was carried out for all covid-19 samples (70 serum samples, 62 individuals) and for 53 samples from the negative panel. forty-one out of 62 covid-19 patients showed neutralising antibodies.the specificity and sensitivity values of the commercial tests against mnt, respectively, were as follows: 95.1%/80.5% (abbott architect sars-cov-2 igg), 94.9%/43.8% (diasorin liaison sars-cov-2 igg; ruo), 68.3%/87.8% (euroimmun sars-cov-2 iga), 86.6%/70.7% (euroimmun sars-cov-2 igg), 74.4%/56.1% (acro 2019-ncov igg), 69.5%/46.3% (acro 2019-ncov igm), 97.5%/71.9% (xiamen biotime sars-cov-2 igg), and 88.8%/81.3% (xiamen biotime sars-cov-2 igm). this study shows variable performance values. laboratories should carefully consider their testing process, such as a two-tier approach, in order to optimize the overall performance of sarscov-2 serodiagnostics. serosurveys are considered essential for creating timely snapshots for global and regional public health management of the ongoing covid-19 pandemic [1] . thus, there is an urgent need for the j o u r n a l p r e -p r o o f development of high-throughput serological assays, which enable population screening, as well as other epidemiological investigations. setting up a serological assay for a completely novel pathogen is challenging in many respects. at present, there is inadequate knowledge as to when and what kind of immune response follows sars-cov-2 infection [2] . we are also yet to learn about factors that may disturb reliable serology, such as potential cross reaction from seasonal coronaviruses. the patient samples consisted of serum specimens sent to the department of virology and immunology, helsinki university hospital laboratory, finland for diagnostic purposes. a subset of these specimens has been included in a previous publication evaluating the euroimmun sars-cov-2 igg and iga assays, and are included here for comparison [3] . the negative panel consisted of 81 serum samples (from 81 individuals) (median age 64 years, range 2-89 years; 33 males, 48 females) ( table 1) the analysis of sars-cov-2 igg or iga antibodies were carried out using the architect plus i2000sr analyzer (abbott, illinois, usa) and sars-cov-2 igg cmia kit (nucleoprotein based antigen; abbott; ce marked), eurolabworkstation (euroimmun, lübeck, germany) and sars-cov-2 igg and iga elisa kits (s1-based antigen; euroimmun) and diasorin liaison ® xl (diasorin, saluggia, italy) and sars-cov-2 s1/s2 igg clia kit (s1/s2 based antigen; diasorin; ruo) according to the manufacturers´ instructions. all samples from the negative panel (n=81) and the patient panel (n=70) were tested with abbott sars-cov-2 igg, and euroimmun sars-cov-2 iga and igg. all samples from the negative panel (n=81) and (due to limited kit supply) 61/70 samples (53/62 individuals) from the covid-19 patient panel were tested with diasorin sars-cov-2 s1/s2 igg. mnt was performed in a bsl-3 laboratory as described previously [5] the growth medium was removed and the virus-serum mixture was added to the cells and incubated for 4 days at 37 °c with 5% co2, after which the cells were stained with crystal violet to detect cytopathic effect (cpe). the neutralisation endpoint titer was determined as the endpoint serum dilution that inhibited the sars-cov-2 induced cpe in at least 2 out 3 parallel wells. the mnt titer ≥40 was considered as positive. for 55 covid-19 patients out of 62, the date of disease onset was available, and disease severity could be rated (mild, moderate or severe; based on siddiqi et al. [2020; 6] ) (table 2, figure 1 ). in the covid-19 patients included in this study, the earliest time point for the mnt to become j o u r n a l p r e -p r o o f positive was 3 days from onset of illness (patient id 50), while the furthest time point for a negative mnt was 16 days from onset (patient id 5) ( figure 1e ; table 2 ). disease severity did not appear to be reflected in the mnt titers of the patients, however, the number of patients in each category was too low to assess significance ( table 2) figure 2 . by using the cut-offs provided by the manufacturers, a trend was observed in which abbott igg yielded positive signals in specimens still negative in euroimmun igg and liaison (ruo) igg ( figure 2 ). rheumatoid factor was detected in five of negative panel specimens (table 1 ). more detailed results are provided in tables 1-3. all of the six immunoassays gave reactive results to a varying degree for the negative panel specimens (table 1) . particularly the acro biotech rapid test and euroimmun iga assay reacted in samples retrieved from patients with autoantibodies. as the sensitivity of the acro biotech rapid test was lower than the other immunoassays tested, we randomly chose an mnt positive specimen (id 61), conducted a dilution series of 1:2 for it, and tested the specimen again with the acro biotech test. an evident prozone effect was detected, and the originally negative test turned igg positive at serum dilution 1:4 up until dilution of 1:16. as serological assays for sars-cov-2 are now becoming available in the market in abundance [7] , assessment of their analytical performance by using clinical specimens is of critical importance. in this study, we assessed the specificity and sensitivity of six commercial immunoassays for the detection of sars-cov-2 antibodies, including two rapid lateral flow tests, in comparison with a neutralisation test. while neutralisation assays are considered to be the gold standard in terms of specificity, they also provide evidence as to development of immunity. eighty-one of the specimens were retrieved in 2018 and 2019 in finland, rendering these specimens as ascertained negative for sars-cov-2 antibodies, and subsequently verifying the very high specificity of the neutralisation test we used (100 % were negative in mnt). we chose serum dilution 1/40 as the limit of detection for the mnt. rf, which is an autoantibody against the fc portion of igg, and a common cause of cross-reactivity in immunoassays [8] , was analysed in the specimens collected in 2018 and 2019. five out of 39 of these specimens were positive for rf; 4/5 were negative in all sars-cov-2 immunoassays, and 1/5 gave a positive reaction in the acro igg and igm test. we conclude that the majority of positive test reactions in the six different immunoassays by using the negative serum panel from 2018-2019 were not due to rf. of note, we observed a prozone phenomenon [9] by diluting specimen id 61 for the acro lateral flow assay. while we did not investigate prozone phenomenon extensively in this study, we do consider it may be an important cause for false negative test results. the prozone phenomenon has been reported for other lateral flow assays previously [10] . of the automated assays included in this study, and by using the cut-off values set by the manufacturers, the best specificity values were observed with abbott igg (95.1%). a previous report from the united states reported a 99.9% specificity [11] . in our study, liaison igg (ruo) assay (94.9%) also showed a good specificity. euroimmun sars-cov-2 iga assay had the best positive agreement (sensitivity) (87.8%), while the positive agreement of the liaison igg (ruo) assay was the lowest (43.8%). the ce marked diasorin liaison sars-cov-2 igg assay was not available for this evaluation. the automated assays from the three manufacturers were all based on different antigen components (s1, s2, nucleocapsid). this is noteworthy, as antibody responses against each of these antigens may develop with varying kinetics, which remains a subject for further investigation. in addition, the immunoassays may detect nonneutralizing antibodies, not detected by neutralization assays. however, the topic of interest in our study was specifically on comparability of the assays with neutralising antibodies. when interpreting sensitivity values, the time from onset of illness in covid-19 patients needs to be accounted for. by using the abbott igg assay, sars-cov-2 igg seroconversion was previously reported in all patients by the day 17 post onset of illness [11] . previous reports suggest a median seroconversion time for sars-cov-2 from 11 days [12] to 13 days [13] . the present study also suggests a relatively long period required for serological response to take place (table 2, figure 1e ). even though extensive conclusions cannot be made from our data, liaison igg (ruo) appears to turn positive at a later point in time from onset of illness in comparison with the other immunoassays evaluated in our study ( table 2 ). perkmann et al (14; 2020) have also reported this phenomenon, and it should be investigated more thoroughly whether antibodies against sars-cov-2 s1/s2 antigen, in general, are detected in later time point. of the two rapid lateral flow assays, the xiamen igg/igm showed a good specificity (97.5% / 88.8%) with a modest positive agreement (sensitivity) (71.9% / 81.3%). in line with a previous report [15] , the performance of the acro biotech igg/igm rapid test appears not to be adequate for the important role of serology for covid-19 control severe acute respiratory syndrome coronavirus 2-specific antibody evaluation of commercial and automated sars-cov-2 igg and iga elisas using coronavirus disease (covid-19) patient samples detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr serological and molecular findings during sars-cov-2 infection: the first case study in finland covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal developing antibody tests for sars-cov-2 rheumatoid factor in acute viral infections: interference with determination of igm, igg, and iga antibodies in an enzyme immunoassay antigen excess in modern immunoassays: to anticipate on the unexpected false-negative serum cryptococcal lateral flow assay result due to the prozone phenomenon performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 antibody responses to sars-cov-2 in patients with covid-19 side by side comparison of three fully automated sars-cov-2 antibody assays with a focus on specificity germany) c liaison® sars-cov-2 igg (diasorin, saluggia, italy) d 2019-ncov igg/igm rapid test cassette microneutralisation assay were carried out according protocol described by table 1. negative serum sample panel consisting of samples collected retrospectively during years 2018-2019, prior the sars-cov-2 epidemic all results were determined according to manufacturers´ instructions diasorin) was research use only (ruo) kit. abbott sars-cov-2 igg chemiluminescent microparticle immunoassay (cmia) all results were determined according to manufacturers´ instructions *two separate samples were available from the patients. first number is the identification code for patient we would like to thank pamela österlund (finnish institute for health and welfare, helsinki, finland) for providing the virus strain. we would also like to thank (in alphabetical order) anu 10-1 * moderate <40 neg neg neg neg 26 severe <40 neg pos neg neg 24 severe <40 neg neg neg neg 38 severe <40 neg pos neg neg 43 severe <40 neg pos neg neg 32 severe *due to limited kit supply, not all specimens tested with mnt could be analysed with these commercial tests. table 3 . specificity and sensitivity of the six commercial immunoassays compared with mnt. mnt titer ≥40 was considered as positive. equivocal results of the commercial assays were regarded as reactive in this analysis. the total number of specimens tested with mnt, and each of the commercial immunoassays, with their respective results are presented in tables 1-2. key: cord-346777-zmmnn9b2 authors: lester, sandra; harcourt, jennifer; whitt, michael; al-abdely, hail m.; midgley, claire m.; alkhamis, abdulrahim m.; aziz jokhdar, hani a.; assiri, abdullah m.; tamin, azaibi; thornburg, natalie title: middle east respiratory coronavirus (mers-cov) spike (s) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies date: 2019-09-11 journal: access microbiol doi: 10.1099/acmi.0.000057 sha: doc_id: 346777 cord_uid: zmmnn9b2 middle east respiratory syndrome coronavirus (mers-cov) is a novel zoonotic coronavirus that was identified in 2012. mers-cov infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately 35 %. the mers-cov spike (s) protein is a major target for neutralizing antibodies in infected patients. the mers-cov microneutralization test (mnt) is the gold standard method for demonstrating prior infection. however, this method requires the use of live mers-cov in biosafety level 3 (bsl-3) containment. the present work describes the generation and validation of s protein-bearing vesicular stomatitis virus (vsv) pseudotype particles (vsv-mers-cov-s) in which the vsv glycoprotein g gene has been replaced by the luciferase reporter gene, followed by the establishment of a pseudoparticle-based neutralization test to detect mers-cov neutralizing antibodies under bsl-2 conditions. using a panel of human sera from confirmed mers-cov patients, the vsv-mers-cov particle neutralization assay produced results that were highly comparable to those of the microneutralization test using live mers-cov. the results suggest that the vsv-mers-cov-s pseudotype neutralization assay offers a highly specific, sensitive and safer alternative method to detect mers-cov neutralizing antibodies in human sera. middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that is associated with respiratory virus infections ranging in severity from asymptomatic to severe respiratory illnesses and death. mers-cov was first isolated from a 60-year-old patient who died with viral pneumonia and acute renal failure in 2012 in saudi arabia [1, 2] . mers-cov cases have been identified in 27 countries, although all cases outside of the arabian peninsula have been associated with exportations from the arabian peninsula [3] [4] [5] [6] [7] . as of 28 february 2019, the world health organization (who) has reported 2374 confirmed cases of mers-cov infection globally, resulting in 823 deaths (https://www. who. int/ emergencies/ mers-cov/ en/). genetically, mers-cov most closely resembles severe acute respiratory syndrome coronavirus (sars-cov), a betacoronavirus known as the first coronavirus to cause severe respiratory infections in humans. sars-cov emerged from an animal host in 2002 and caused a worldwide outbreak comprising an estimated 8000 cases with 800 deaths [7] [8] [9] [10] . similar to sars-cov, mers-cov infections represent zoonotic transmission events [11] [12] [13] . several sero-epidemiology studies have demonstrated that dromedary camels from the arabian peninsula and north africa have high titres of neutralizing antibodies against mers-cov, which can be detected in camel sera collected over 30 years [14] [15] [16] [17] . in contrast to the sars outbreak, which was brief, mers-cov cases continue to occur 7 years after its identification. the continuous spread of mers-cov serves as a constant reminder of the propensity of novel human coronaviruses to emerge from zoonotic reservoirs and cause severe disease in humans, and so continuous serological surveillance efforts remain essential for these viruses. the risk factors for camel-human transmission, humanhuman transmission and hospital outbreaks, and the roles of asymptomatic cases in human-human transmission are not fully understood. the clinical symptoms for mers patients overlap with those for other lower respiratory tract infections, further demonstrating the need to develop low biosafety-level laboratory-based diagnostic assays with high specificity and sensitivity. furthermore, countermeasures for the treatment or prevention of mers-cov infection, including vaccines, are unavailable. therefore, methods for measuring the potency and breadth of neutralization antibodies against mers-cov are essential to address these gaps in our knowledge, as well as to strengthen disease surveillance and disease preparedness platforms. the spike (s) protein of mers-cov is a surface glycoprotein that facilitates receptor binding, membrane fusion and viral entry into host cells via attachment to the cellular receptor dipeptidyl peptidase iv (dpp4), thus playing a pivotal role in mers-cov infection of the host cell [18] . the s protein is a major immunogenic component of covs and is the major target for neutralizing antibodies [19] [20] [21] [22] . laboratory tests, such as immunofluorescence assays (ifas) and conventional enzyme-linked immunosorbent assays (elisas), are commonly used screening tools for detecting anti-mers-cov serum antibodies. however, these assays often yield false-positive reactions due to cross-reactivity with other common human coronaviruses, and so confirmatory tests such as neutralization tests are utilized [23] . the most widely used confirmatory serological assays to detect and measure neutralizing antibody responses to mers-cov utilize neutralization of live virus, through either the plaque reduction neutralization test (prnt) or the microneutralization test (mnt) methods. however, due to the highly pathogenic nature of mers-cov and the absence of treatments and vaccines for mers-cov infection, prnt and mnt tests must be conducted under strict bio-containment procedures in a biosafety level 3 (bsl-3) laboratory. this limits epidemiological and pathogenesis studies globally. the development of an alternative method that can be reliably and conveniently used to determine the prevalence of mers-cov antibodies at a population level is crucial to prevent the spread of mers-cov. a safe and convenient way to detect neutralizing antibodies against mers-cov in serum is the use of viral pseudotypes. viral pseudotype particles have proven to be very valuable tools for studying virus entry pathways, evaluating the efficacy of vaccines, serological surveillance and gene therapy studies [24] [25] [26] [27] [28] [29] [30] [31] . several reports have utilized mers-lentivirus pseudotyped viruses in sero-epidemiological studies and basic research investigations [21, [32] [33] [34] [35] [36] . additionally, vesicular stomatitis virus (vsv) pseudotypes with mers-cov spike glycoproteins have been utilized to investigate the role of the mers s protein in virus-receptor-mediated entry, virus/host tropism and drug screening [37] [38] [39] . however, systematic comparative equivalency analysis between vsv pseudotypes bearing mers-cov spike glycoproteins and the conventional mnt using human sera from confirmed mers-cov patients have not been performed. the purpose of this study was to develop a vsv pseudotypebased neutralization assay to detect mers-cov s protein neutralizing antibodies and calculate its equivalence to traditional neutralization assays, therefore circumventing the use of live virus and the need for widely unavailable high-level containment biosafety facilities. here, we validate a mers-cov neutralization assay using a membraned vsv pseudotype system, where its glycoprotein is replaced with a luciferase reporter gene and its membrane is decorated with mers-cov s proteins. additionally, we perform equivalence testing in comparison with the conventional mnt. we demonstrate that these pseudoparticles are neutralized by sera from mers-cov-infected patients. furthermore, most of the patient sera samples have neutralization activities, and these results were consistent with the neutralization activity that was measured by the conventional microneutralization assay using live mers-cov. these results demonstrate that the mers-cov-s protein pseudotyped vsv particle-based neutralization assay would serve as a safe, reliable and highly specific alternative method to detect mers-cov neutralizing antibodies to be used for future sero-epidemiological studies. baby hamster kidney-21 (bhk-21) cells were cultured and maintained in dulbecco's modified essential medium (dmem) containing 5 % fetal bovine serum (fbs) (life technologies/gibco), and 1× penicillin/streptomycin (p/s) (sigma) at 37 °c under a 5 % co 2 atmosphere. vero (african green monkey kidney epithelial) cells were cultured and maintained in dmem containing 10 % fbs and 1× p/s 37 °c under a 5 % co 2 atmosphere. different vsv-based pseudotypes bearing either mers-cov-s or vsv-g glycoproteins were produced in bhk-21 cells. the pseudoparticle titrations and neutralization assays were carried out in vero cells. pooled normal human serum (pnhs) was purchased from lee biosolutions and used as a negative control serum. human serum from a single patient with laboratory-confirmed mers-cov infection was used as the positive control serum. the positive control serum was collected from the first imported case of mers-cov in the usa during the case investigation, with a neutralizing titre of 320. a laboratory-confirmed sars-cov patient serum sample and a panel of human sera with confirmed high neutralizing antibody titres to human coronaviruses 229e, hku1, oc43 and nl63 were used in this study to evaluate the vsv-mers-cov-s particle-based neutralization assay for potential cross-neutralization. a total of 52 human sera samples from mers-cov-infected patients in saudi arabia were used to examine equivalences. anti-vsv-g monoclonal antibody was purchased from kerafast, inc. a codon-optimized s gene from the mers-cov florida isolate (genbank accession number: kj829365.1) was synthesized by genescript and sub-cloned into pcaggs 2.0 eukaryotic expression vector. vsv-mers-cov-s pseudoparticles were generated as previously described [24] . the pseudoparticle titres were determined in vero cells. vero cells were seeded in 96-well black and white tissue culture-treated plates (perkin-elmer) and were inoculated with 50 µl of pseudoparticles five fold serially diluted in serum-free dmem (1× p/s in triplicate in . after 1 h adsorption, the inocula were removed, replaced with fresh dmem containing 10 % fbs and 1× p/s, and incubated at 37 °c. at 24 h post-inoculation, luciferase activity was determined using the luciferase assay kit (promega, inc.) according to the manufacturer's instructions. the titre of the vsv-mers-cov-s pseudoparticles was defined as the highest dilution yielding 10 5 relative luciferase units (rlu). for the titration of vsv-mers-cov-s pseudoparticles, the pcaggs plasmids encoding vsv recombinant g and the empty vector (ev) pcaggs-ev were used to generate vsv-g pseudoparticles and pseudoparticles without heterologous protein (ev) as positive and negative controls, respectively. ten fold serial dilutions of vsv pseudotyped with mers-s (vsv-mers-s) or negative control pcaggs empty vector containing no glycoprotein (vsv-ev) were used to infect vero cells in 96-well plates. rlu were measured 24 h post-infection. the results are expressed as average rlu±standard deviation (sd) of triplicate wells from an experiment repeated three times with similar results. the error bars indicate the sd. briefly, vero cells were seeded in 96-well black and white tissue culture-treated plates 1 day prior to infection. human sera were heat-inactivated at 56 °c for 30 min, and diluted as two fold serial dilutions with an initial dilution of 1 : 40 in serum-free dmem containing 1× p/s. two hundred microlitres of each serum dilution were mixed thoroughly with 10 5 rlu-equivalent vsv-mers-cov-s pseudoparticles diluted to 200 µl and incubated at 37 °c under a 5 % co 2 atmosphere for 1 h. positive and negative control sera were included with each assay run. in triplicates, 100 μl of the pseudoparticleserum mixtures were transferred onto vero cell monolayers and incubated at 37 °c under a 5 % co 2 atmosphere for 1 h. after adsorption, 100 µl of dmem containing 10 % fbs p/s was added and incubated at 37 °c and 5 % co 2 for 23 h. at 24 h post-inoculation, luciferase activity was determined using the luciferase assay kit according to the manufacturer's instructions. the results are expressed as the percentage neutralization ±sd of three parallel wells from three independent experiments. to calculate neutralization, the luciferase activity from each serial diluted human sera sample was normalized to that from the negative control, pnhs. neutralization was calculated as: (average rlu value of pnhs−average rlu value with test serum/average rlu value of pnhs)×100. reciprocal endpoint titres were defined as the highest dilution of the test serum sample that decreased rlu values by 80 % or more. serum samples that failed to show at least an 80 % reduction at the 1 : 40 dilution were considered to be below the limit of detection. the slides were first coated with poly-l-lys and seeded with hek-293t cells 24 h prior to transfection. the cells were transfected with 1 µg of pcaggs-mers-cov-s plasmid dna or mock transfection. at 24 h post-transfection, the cells' expression of mers-cov-s was detected by convalescent sera from a mers-cov patient with a known neutralizing titre against live virus as a positive control (mers immune sera; 1 : 400), followed by fitc-labelled anti-human iga/igg/igm (1 : 500) (abcam). dapi stain was used and cells were imaged using a zeiss axioimager microscope at 20× magnification. all graphs were generated with the graphpad prism 7 software. all results were calculated and presented as the means±sd obtained from duplicate or triplicate independent experiments. pearson's correlation analysis and the bland-altman method were used to assess the comparative analyses between the vsv-mers-cov-s pseudoparticle neutralization assay and the mnt assay. statistical analyses were performed using graphpad prism 7 software. the specimens used in this study were determined to be nonhuman subject research by the centers for disease control and prevention (cdc) institutional review board (irb). to establish vsv-mers-cov-s pseudotype neutralization assays, we generated vsv-mers-cov pseudoparticles with surface s protein and a firefly luciferase reporter gene as previously described [24] . the expression of mers-cov s proteins was confirmed by immunofluorescence at 24 h post-transfection in hek293t cells (fig. 1a) . it is known that bhk-21 cells do not have the s-binding mers-cov receptor, dpp4, and are therefore not permissible to mers-cov infection, but vero cells do [18, 39] . efficient packaging of mers-cov s on the surface of the vsv pseudoparticles and the ability of the pseudoparticles to enter target cells through receptor binding were confirmed by comparing the titrated inoculation of vero and bhk-21 cells of negative control vsv pseudoparticles containing no viral glycoproteins on their surface (vsv-ev) with that for vsv pseudotyped with mers-cov-s proteins, designated vsv-mers-s (fig. 1b and c) . as expected, in the vero cells the luciferase activity was proportional to the dilution of the mers-s pseudoparticle inoculum (fig. 1) . in comparison to the control pseudoparticles, the mers-s pseudoparticles demonstrated an approximately six fold increase in luciferase activity (fig. 1) . neither the mers-s pseudoparticles nor the control pseudoparticles induced high levels of luciferase activity in bhk-21 cells (fig. 1) , further supporting the notion that bhk-21 cells are resistant to mers-cov infection [39] . based on the rlu values observed at each serial dilution, it was decided to use a 1 : 200 dilution as the input pseudoparticle dose for further experiments for the mers-cov s protein pseudotyped vsv particle-based neutralization assay. to establish optimal parameters for the mers-cov s protein pseudotyped vsv particle-based neutralization assay, the cell density was optimized for maximum luciferase activity. no significant differences in luciferase activities were observed at 10 3 -10 5 cellsper well, although maximum luciferase activity was reached at 10 4 cells per well, and so 10 4 cells per well was selected as the standard assay cell density (fig. s1a , available in the online version of this article). next, we investigated the incubation time for optimal luciferase activity. the maximum rlu values peaked at 24 h post-infection and the rlu values decreased with prolonged incubation times (fig. s1b ). therefore, 24 h post-infection was determined to be the most suitable infection time for mers-cov-s pseudoparticles. to investigate whether anti-mers-cov human sera neutralized vsv-mers-cov-s pseudoparticles, vsv-mers-cov-s pseudoparticles were preincubated with two fold serial dilutions of pnhs serum (negative control), convalescent sera from a mers-cov patient with a known neutralizing titre against live virus as a positive control (mers immune sera) or anti-vsv-g monoclonal antibody. luciferase activity was reduced when vsv-mers-cov-s was preincubated with positive control serum in a dose-dependent manner (fig. 2a) . in contrast, preincubation with the negative control pnhs or anti-vsv-g monoclonal antibody did not significantly reduce luciferase activity at any dilution factor, thus demonstrating that vsv-mers-cov-s pseudoparticles were specifically neutralized by serum from an individual with a confirmed prior mers-cov infection (fig. 2a) . as expected, at any dilution factor, preincubation with anti-vsv-g monoclonal antibody efficiently neutralized vsv-δg pseudoparticles packaged with vsv-g, although neither the mers-cov positive control serum or the negative control pnhs neutralized vsv-δg pseudoparticles packaged with vsv-g (fig. 2b) . neutralization may be assessed as the percentage reduction in infectivity at a single dilution. since our goal is to measure the endpoint neutralization titres of human sera that possibly contain mers-cov neutralizing antibodies, we evaluated the percentage neutralization reduction curve of vsv-mers-cov-s pseudoparticles when preincubated with a mers-cov immune serum with a known live-virus endpoint neutralization titre. the percentage reduction in rlu (% neutralization) was determined by calculating the difference between the average rlu number in triplicate wells with the anti-mers-cov serum and the average rlu number in triplicate wells with the negative control pnhs sample, dividing it by the average rlu number in triplicate wells with the negative control pnhs sample and then multiplying the result by 100. neutralization of vsv-mers-cov-s pseudoparticles was dependent on the serum dilution, reducing rlu between 99 and 53 % at the dilutions tested (fig. 3) . using a target value of 80 % reduction in rlu in comparison to the pnhs control was within the linear range of reduction. our standard positive control mers-cov immune serum crossed the 80 % reduction line between 320 and 640 dilutions. this same serum has a known endpoint titre of approximately 320 in neutralization assays using live virus (unpublished data), demonstrating relative equivalence in the two assays for this serum. we previously measured the endpoint neutralizing antibody titres of 52 human sera collected from mers-cov patients using a live virus mnt. to determine if the vsv-mers-cov-s pseudoparticle neutralization test results in equivalent endpoint titres in comparison to the live virus mnt, we compared the endpoint titres from the live virus mnt to the endpoint titre necessary to achieve an 80 % reduction in rlu with vsv-mers-cov-s pseudoparticles. neutralizing antibody titres ranging from 40 to 1280 from 52 human serum samples against mers-cov were successfully detected by both neutralization assays (fig. 4) . sixty-seven per cent (n=35) of the serum samples had the same endpoint neutralization titres as those measured by either technique. we observed that approximately 29 % (n=15) of the serum samples gave higher neutralizing titres in the vsv-mers-cov-s pseudoparticle neutralization assay than the conventional mnt. furthermore, five sera with neutralization titres below the limit of detection by mnt had neutralizing titres that were detectable by the vsv-mers-cov-s pseudoparticle neutralization assay, thus demonstrating that the vsv-mers-cov-s pseudoparticle neutralization assay may be more sensitive (fig. 4) . the titres from both assays were then analysed statistically. pearson's correlation analysis of all data showed a strong correlation between the two neutralization assays, with r=0.9348, p<0.0001, 95 % confidence interval (ci)=0.8886 to 0.9622 (fig. 4a) . bland-altman method comparison analysis further demonstrated that these two methods are highly comparable. the average of the logarithmic difference of the two methods was 0.3269 (fig. 4b) . collectively, these data indicate that the vsv-mers-cov-s pseudoparticle neutralization assay can be a reliable alternative to the live virus-based mnt. there are six known human coronaviruses that cause respiratory disease in humans. in order to test the specificity of our vsv-mers-cov-s pseudoparticles and their potential cross-reactivity with other common human coronaviruses, we tested 40 serum samples from humans with no known anti-mers-cov exposure. when using the 80 % reduction in rlu as the cut-off value, none of the serum samples neutralized vsv-mers-cov-s pseudoparticles at any dilution factor (fig. s2) . we also tested potential cross-neutralization of vsv-mers-cov pseudoparticles with sera from other humans with confirmed infections with the human coronaviruses 229e, hku1, oc43, nl63 and sars-cov. using the neutralization parameters of 80 % or greater reduction in rlu, we found no cross-neutralization with 229e, hku1, oc43, nl63 or sars-cov anti-sera (fig. 5) . these data confirm the specificity of the vsv-mers-cov-s pseudoparticles. mers-cov continues to circulate on the arabian peninsula and can cause severe infections, including fatalities. at present, there are no antiviral therapeutics or approved vaccines to combat mers-cov infections. the presence of serum neutralizing antibodies ≥20 is a reliable indicator of prior mers-cov exposure. neutralization assays using live viruses are recognized as the gold standard serological assays to confirm the antibody responses to mers-cov infection. however, one major obstacle to performing the mnt assay is the requirement for access to high-level biocontainment laboratories, due to the use of live mers-cov. thus, only a limited number of facilities and researchers are able to perform such neutralization assays. additionally, determining the neutralization of infectious virus in this assay requires the assessment of cytopathic effect in 96-well plates, which is labour-intensive and time-consuming. furthermore, because of the large viral genome, reverse genetics for mers-cov is challenging, and still needs to be used under bsl-3 containment, thereby limiting molecular manipulation and studies. these limitations highlight the need to develop alternative methods for detecting mers-cov neutralizing antibodies. an alternative approach to overcome these shortcomings is the use of viral pseudotypes. generally, lentiviral and vsvbased pseudotypes are used for the study of enveloped viruses. several studies have utilized lentiviral and vsv pseudotype systems harbouring mers-cov spike glycoproteins to study viral entry, viral tropism and other basic science questions [27, 29, [37] [38] [39] [40] . in addition to being utilized to probe basic science questions, pseudoparticle-based neutralization assays have been used for serological surveillance of reservoir species for several other zoonotic viruses in addition to mers-cov [17, 33, [41] [42] [43] [44] [45] [46] . studies have employed lentiviral and env-deficient hiv-1 backbone vector pseudotype systems for mers-cov seroepidemiological surveillance in humans [32] [33] [34] [35] [36] . using dromedary camel sera, hemida et al. demonstrated that lentiviral-based mers-cov s pseudoparticles detected antibodies specific to mers-cov. in our hands, lentiviral particles grew to lower titres than vsv particles, making it challenging to prepare high volumes of stock for use in multiple studies. standardizing many particles for use in multiple studies is important for making comparisons between studies in this context. barlan et al. utilized vsv pseudotyped with mers-cov-s and demonstrated that susceptibility to mers-cov infection is enhanced by cellular proteases that cleave the s protein [37] . fukuma et al. demonstrated that infection with vsv-based pseudoparticles bearing truncated c-terminal mers-cov-s protein and non-truncated mers-cov-s protein was inhibited by rabbit anti-mers-cov s serum and that infection was dependent on the expression of dpp4 [40] . this demonstrated that vsv-based mers-s pseudoparticles can be utilized for neutralization assays. nonetheless, studies featuring seroepidemiological surveillance in humans utilizing a vsv pseudotype system bearing mers-cov spike glycoproteins had not previously been performed. this report describes the first comparison between the neutralization assay using vsv-mers-cov-s pseudoparticles and the mnt using a panel of human sera. this study is limited by only utilizing 52 specimens, which limits its statistical power. although analysing more specimens might yield a more powerful comparative analysis, the complexities of limited availability, diplomatic issues and biosafety concerns make acquiring mers-cov human specimens less feasible. despite this limitation, statistical analyses comparing mnt and vsv-mer-cov pseudoparticle neutralization indicate there was a positive correlation between the two methods. although there was a strong correlation between the two methods, the vsv-mers-cov-s pseudoparticle neutralization assay is slightly more sensitive than the conventional mnt. one potential explanation for this is the surface density of s protein. the generation of pseudoparticles by transient transfection in continuous cell lines, rather than producer cells that are more physiologically relevant, may lead to the production of large amounts of virus with less s protein on its surface. thus, the pseudoparticles may be more susceptible to neutralization because fewer neutralizing antibodies are required to block the entry of mers-cov. however, in corroboration with our data, fukushi et al. previously demonstrated that a neutralization test based on a vsv-based sars-cov-s protein bearing pseudoparticles was also more sensitive than the conventional virus neutralization test using live sars-cov [47] . in comparison with the mnt method, the pseudoparticle neutralization assay required half as much time to test for mers-cov neutralizing antibodies. more importantly, this assay can be performed in a low-biosafety containment laboratory. in addition to being faster and more sensitive than conventional neutralization assays while maintaining specificity, our vsv-mers-cov-s pseudoparticle system offers a convenient approach for examining the antigenic and neutralizing epitope differences among different s proteins from divergent strains of mers-cov. the data we have presented here use one s plasmid, but the pseudoparticles can be packaged using the spike sequence from other virus strains. because s protein is provided in trans in a pcaggs vector, it can be easily mutated or swapped for any spike protein an investigator is interested in studying. there are existing reverse genetics systems for human coronaviruses, including mers-cov, but those systems are more difficult to generate and require bsl-3 facilities [48] [49] [50] . using those full-virus reverse genetics systems are essential for viral replication and pathogenesis studies, but for neutralization studies, the presence of spike alone is sufficient. mers convalescent sera did not neutralize vsv particles packaged with vsv-g, demonstrating that neutralization was due to serum antibodies binding specifically to mers s. one of the main concerns in designing a mers-cov neutralization test is the presence of five other known human coronaviruses, four of which cause common infections. it had previously been shown that anti-coronavirus sera crossneutralize amongst similar coronaviruses [51] . in this study we determined that vsv-mers-cov-s pseudoparticles were not neutralized by antisera from alpha-or betacoronaviruses, further demonstrating the assay's high specificity. in conclusion, we established a neutralization test using a vsv-based pseudotyped virus possessing mers-cov s protein and expressing a luciferase reporter gene. we used the vsv-mers-cov-s pseudoparticle neutralization assay at bsl-2 containment, improving the safety of mers-cov neutralization assays. additionally, we found the system to be more rapid and sensitive and easier to use than a conventional microneutralization assay. these characteristics make the vsv-mers-cov-s pseudoparticle neutralization assay a more attractive, convenient and suitable assay for mers-cov screening and confirmatory serology testing. it offers a safe, rapid alternative method to monitor the potency and breadth of neutralization antibodies against mers-cov exposure in future seroepidemiological studies. isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a 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against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests the authors wish to acknowledge financial support from cdc lassi 2016. the authors declare that there are no conflicts of interest. the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. names of specific vendors, manufacturers, or products are included for public health and informational purposes; inclusion does not imply endorsement of the vendors, manufacturers, or products by the centers for disease control and prevention or the us department of health and human services. the specimens used in this study were determined to be non-human subject research by the centers for disease control and prevention (cdc) institutional review board (irb). key: cord-325014-n7mnhk2v authors: gujski, mariusz; jankowski, mateusz; pinkas, jarosław; wierzba, waldemar; samel-kowalik, piotr; zaczyński, artur; jędrusik, piotr; pańkowski, igor; juszczyk, grzegorz; rakocy, kamil; raciborski, filip title: prevalence of current and past sars-cov-2 infections among police employees in poland, june–july 2020 date: 2020-10-11 journal: j clin med doi: 10.3390/jcm9103245 sha: doc_id: 325014 cord_uid: n7mnhk2v background: coronavirus disease 2019 (covid-19) is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). we aimed to determine the prevalence of current and past sars-cov-2 infections among police employees. methods: this cross-sectional survey was undertaken among 5082 police employees from mazowieckie province, poland. rt-pcr testing for current sars-cov-2 infection and serological tests (elisa) for the presence of anti-sars-cov-2 igm+iga and igg antibodies were performed. results: all rt-pcr tests were negative. the anti-sars-cov-2 igm+iga index was positive (>8) in 8.9% of participants, including 11.2% women and 7.7% men (p < 0.001). equivocal igm+iga index (6–8) was found in 9.8% of participants, including 11.9% women and 8.7% men (p < 0.001). the igg index was positive (>6) in 4.3% and equivocal (4–6) in 13.2% of participants. a higher odds of positive igm+iga index was found in women vs. men (or: 1.742) and police officers vs. civilian employees (or: 1.411). participants aged ≥60 years had a higher odds of positive igg index vs. those aged 20–29 years (or: 3.309). daily vaping also increased the odds of positive igg index (or: 2.058). conclusions: the majority of polish police employees are seronegative for sars-cov-2 infection. vaping and older age (≥60 years) were associated with a higher risk of sars-cov-2 infection. coronavirus disease 2019 (covid-19) is caused by a novel strain of coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which appeared in china in 2019 [1] and evolved into the current pandemic. although the definite laboratory diagnosis of sars-cov-2 infection is currently based on real-time reverse transcriptase-polymerase chain reaction (rt-pcr) testing, and rt-pcr is recommended for clinical testing in cases of suspected covid-19 disease [2], asymptomatic infected individuals (infection carriers) who do not come to medical attention may play an important role in transmitting infection within the population [3] . as the time window for a positive rt-pcr result is short, serological testing, which provides information about whether a person has been exposed to sars-cov-2, may be useful for epidemiological purposes to detect the overall burden of previous infection in a given community. currently, two types of serological assays are available for sars-cov-2 testing [2]. laboratory-based immunoassays, including enzyme-linked immunosorbent assays (elisas), chemiluminescentmicroparticle immunoassays, and immunometric assays, detect various classes of immunoglobulins (ig) against sars-cov-2, including igm, iga, and igg, can be qualitative or quantitative, and are generally performed using serum samples. rapid diagnostic tests are typically lateral-flow assays that can be used for point-of-care testing to detect anti-sars-cov-2 igg, igm, or viral antigens and are usually performed in fingerstick blood samples, although some may use saliva or other specimen types. the rationale for screening using tests detecting sars-cov-2-specific antibodies is that these antibodies develop regardless of symptoms and are present for several months after infection [4] . in addition, population data from spain and iceland indicate that a substantial proportion of infected persons, both asymptomatic and symptomatic, are never tested with rt-pcr in the acute phase [3, 4] . the data obtained during the current sars-cov-2 epidemic in poland indicate that more than 90% of infections within the workforce outbreaks, e.g., among miners, which involve younger and generally more healthy persons, were asymptomatic or oligosymptomatic [5] . poland is a country where the coronavirus epidemic arrived relatively late. the first laboratoryconfirmed covid-19 case was reported on 4 march 2020 [6] . as of 21 september, 78,330 laboratoryconfirmed covid-19 cases and 2282 related deaths were reported in poland [7] . the settings of sars-cov-2 transmission during the first 2 months of the epidemic were mostly hospitals and long-term care facilities, followed by outbreaks in workplaces, including coal mines, furniture factories, and meat-processing plants [8] . polish police officers are at an increased risk of acquiring sars-cov-2 infection due to their duties, including protection of public gatherings and daily verification (by direct visual contact) of compliance with mandatory quarantine rules. nationwide, the number of quarantined persons was >160,000 at the peak in early april 2020 [9] and currently (early september 2020) is about 70,000 [10] . the aim of this study was to determine the prevalence of current and past sars-cov-2 infections among police employees, a high-risk population due to their professional duties, during the covid-19 epidemic. this cross-sectional sars-cov-2 screening survey was carried out from 22 june to 8 july 2020 among police employees (police officers and civilian employees) from mazowieckie province in poland. random-cluster sampling was performed. out of 327 police units (including headquarters/police stations and departments), 170 were randomly selected. the smallest units (<5 people) were excluded from the sampling procedure. the cluster and stratified selection method was applied for sampling procedures to improve accuracy. all police employees from the randomly selected units were invited to participate in the study (8789 individuals from 170 units). due to refusal to participate and exclusion of the smallest units, the study was finally carried out in 122 police units. the questionnaire was completed by 5363 police employees, and biological samples (swab and blood) were effectively collected from 5082 of them. the exclusion criteria include refusal to participate, lack of a signed informed consent, hospitalization, quarantine, leave, and secondment to work in another police unit that was not selected for the survey. participants were invited via email or the police's internal communication system. after completing the questionnaire, each respondent received an individual id code for personal data protection. test samples were collected on the premises of the police units (in a dedicated room) on a day designated by the research team. samples were collected by a nurse or a paramedic equipped with personal protective equipment according to the applicable safety procedures. nasopharyngeal swab samples were collected by a nurse or a paramedic using transport sets specially designed for collecting clinical material for the diagnosis of sars-cov-2 infection. the diaplexq™ novel coronavirus (2019-ncov) detection kit (solgent co., ltd.; daejeon, korea) was used for the detection of sars-cov-2 rna by rt-pcr. virus identification (positive result) was based on the orf1ab and n target gene regions of sars-cov-2. rt-pcr testing was carried out in the national reference laboratory (national institute of public health-national institute of hygiene, warsaw, poland) by qualified clinical laboratory personnel specifically instructed and trained in rt-pcr techniques and in vitro diagnostic procedures. the testing procedure met the requirements of the who recommendations for covid-19 laboratory testing [11] . serum samples (up to 5 ml) were collected for the detection of anti-sars-cov-2 igm+iga and igg antibodies using indirect immunoenzyme assay (elisa). commercially available covid-19 elisa igg and igm+iga kits (vircell s.l., granada, spain) were used, targeting sars-cov-2-specific antigens, spike glycoprotein (s), and nucleocapsid protein (n). serological testing was carried out in the diagnostic laboratory of the central clinical hospital of the ministry of the interior and administration in warsaw by qualified clinical laboratory personnel. the testing procedure was performed according to the test manufacturer's instructions and met the spanish society for infectious diseases and clinical microbiology (seimc) recommendations. an in-house validation was carried out according to the validation protocol for users provided by the test manufacturer. according to the test manufacturer's guidelines, the results were presented in a semiquantitativemanner andthe antibody index was calculated using the following formula: antibody index = (sample optical densities/cutoff serum mean optical densities) × 10. samples with the anti-sars-cov-2 igm+iga index below 6 were considered negative, those with the index between 6 and 8 were considered indeterminate/equivocal, and those with the index above 8 were considered positive. samples with the anti-sars-cov-2 igg index below 4 were considered negative, those with the index between 4 and 6 were considered indeterminate/equivocal, and those with the index above 6 were considered positive. the study used an original 30-item questionnaire adapted to this particular group of police employees. in preparation of the questionnaire, we analyzed the previously published covid-19-oriented research, with special emphasis on the studies and reports published by the who [12] . the questionnaire was made available to the respondents via an internet platform. the computer-assisted web interview (cawi) method was applied. field control was enabled to avoid accidental missing data. the total number of police employees in the mazowieckie province was 17,400. the effective sample size of 5000 was assumed. due to estimated nonresponse rate of 35-45% (the beginning of the holiday season and the presidential elections being held in poland, which influenced the level of police unit involvement), a total of 8789 police employees from 170 police units were invited to take the survey. finally, 5082 individuals took part in both questionnaire and laboratory parts of the study. the questionnaire included several questions related to the personal characteristics, including age, size of the place of residence, living alone or with someone, and the presence of children in the respondent's home. the participants were also asked about self-declared health status (very good, good, fair, or poor), presence of chronic diseases (yes/no), ever and current (past 6 months) tobacco or e-cigarette use, international travel in the past 3 months, and the nature of their work. the type of employment was categorized as a police officer (uniformed and armed force) or a civilian employee (nonuniformed, work closely with uniformed officers, e.g., administrative support). based on the settings of official duties' performance, the following categories were designated: office work, fieldwork, and both office work and fieldwork. participants were also asked about the number of people with whom they had contact during the day, participation in the control of compliance with the quarantine rules, and participation in securing gatherings of more than 50 people. respondents were asked about the presence of 8 symptoms accompanying sars-cov-2 infections in the last 4 months (march-june 2020) (fever, cough, dyspnea/breathlessness, diarrhea, anorexia/lack of appetite, nausea or vomiting, loss of smell, and loss of taste). for the present analysis, only responses regarding the absence of symptoms within the given period were used. for logistic regression analysis, all analyzed variables were recoded into a series of dummy variables (0-1). the questionnaire data were supplemented with available epidemiological data on the number of registered cases and deaths per 10,000 residents in individual poviats (administrative regions) of the mazowieckie province as of 8 july 2020, obtained from the state sanitary inspection [13] . these data were incorporated into the model as continuous variables. the data were analyzed using spss version 26 (ibm, armonk, ny, usa, 2020), r version 4.0.2. (r foundation for statistical computing, vienna, austria, 2020), and h2o version 3.30.0.7 (apache license 2.0). the χ 2 test was used to assess the significance of differences in cross-tables. the associations between continuous variables (igm+iga and igg indexes) were measured by the pearson's linear correlation and the spearman's rank correlation. a logistic regression model was used to determine the strength of the effect of the analyzed factors on the risk of sars-cov-2 infection. machine learning techniques were used to improve the fit of the model-glm with the binominal function with ridge and lasso regularization (with cross-validation). samples were collected by qualified healthcare professionals (nurse or paramedic) in accordance with the standards set out in the ordinances of the polish minister of health. participation in the study was voluntary and free of charge. all participants gave written informed consent before participation in the study. the study protocol was reviewed and approved by the ethics review board at the medical university of warsaw, warsaw, poland (approval number: kb/87/2020). completed questionnaires were obtained from 5363 police employees (61%), and complete samples (nasopharyngeal swab and serum sample) were collected from 5082 police employees (33.5% females; response rate 57.8%). the mean age (sd) was 39.6 years (9.0) overall, 49.7 years (9.6) among women and 39.0 years (8.5) among men. among participants, 79.2% were police officers and 20.8% were civil employees. almost one-third (30.1%) of participants declared office-based work, 17.3% declared fieldwork, and 52.6% declared both office work and fieldwork. a quarter of the participants (25.9%) lived in rural areas, 44.8% lived in cities up to 500,000 inhabitants, and 29.3% lived in the city above 500,000 inhabitants (warsaw). the mean anti-sars-cov-2 igm+iga index was 4.4 ± 0.5 (range: 0.0-43.5). the mean anti-sars-cov-2 igg index was 3.1 ± 0.4 (range: 0.0-44.8) ( table 1) . the anti-sars-cov-2 igm+iga and igg indexes were linearly correlated at r = 0.209 (p < 0.001). using rank correlation, the coefficient rho = 0.355 was obtained (p < 0.001). of those with negative anti-sars-cov-2 igg index (<4), 7.6% had positive anti-sars-cov-2 igm+iga index (>8) and equivocal results were observed in 8.8%. of those with positive anti-sars-cov-2 igg index (>6), 18.0% had positive anti-sars-cov-2 igm+iga index (>8) and equivocal results were observed in 13.8% (table 2 ). the differences were statistically significant (p < 0.001). less than 1% of participants had both positive anti-sars-cov-2 igm+iga and igg indexes. there were no current sars-cov-2 infections among 5082 police employees in this study (all rt-pcr tests were negative). the anti-sars-cov-2 igm+iga index was positive (>8) in 8.9% of participants (95%ci: 8.1-9.7%) overall, in 11.2% (95%ci: 9.7-12.7%) of women, and in 7.7% (95%ci: 6.8-8.6%) of men (p < 0.001). an equivocal (6-8) anti-sars-cov-2 igm+iga index was found in 9.8% (95%ci: 9.0-10.6%) of participants, with a significant difference (p < 0.001) between women (11.9%; 95%ci: 10.4-13.5%) and men (8.7%; 95%ci: 7.8-9.7%) ( figure 1 ). the size of the place of residence also differentiated results in a statistically significant way (p <0.01). no other variable listed in figure 1 was significantly associated with the igm+iga results. overall, 4.3% participants (95%ci: 3.7-4.9%) were igg-seropositive (antibody index > 6). an equivocal (4-6) anti-sars-cov-2 igg index was found in 13.2% (95%ci: 12.3-14.1%) of participants. neither sex (p =0.155) nor other variables listed in figure 2 were significantly associated with the igg results ( figure 2 a logistic regression model predicting a positive anti-sars-cov-2 igm+iga index was developed (cox and snell r square at 0.015 andnagelkerke r square at 0.033). after including all variables listed in figures 1 and 2 along with the number of registered cases and deaths due to covid-19 (per 10,000 inhabitants), only 4 variables showed a correlation with a positive anti-sars-cov-2 igm+iga index. a higher odds of a positive anti-sars-cov-2 igm+iga index was observed among women compared to men (or: 1.742; 95%ci: 1.377-2.203), inhabitants of towns up to 20,000 residents and cities from 20,000 to 500,000 residents (or: 1.526; 95%ci: 1.099-2.119 and or: 1.657; 95%ci: 1.257-2.183, respectively) vs. those living in rural areas, and police officers compared to civilian employees(or: 1.411; 95%ci: 1.004-1.981) ( table 3) . (20-29 years) ; place of residence (rural); living alone; lack of children at home; civil employee; type of employment (fieldwork); average number of contacts per person perday (>100 person);verification of compliance with mandatory quarantine rules (no); protection of public gatherings > 50 persons (no); presence of chronic condition (no); tobacco smoking (no); e-cigarette use/vaping (no); heated tobacco use (no); foreign trip in the first half of 2020 (no). in a logistic regression model predicting a positive anti-sars-cov-2 igg index (cox & snell r square at 0.009, nagelkerke r square at 0.029), only 2 variables showed a correlation with a positive anti-sars-cov-2 igg index. compared to the age group 20-29 years, participants aged ≥60 years had higher odds of a positive anti-sars-cov-2 igg index (or: 3.309; 95%ci: 1.442-7.595). daily vaping (e-cigarette using) also increased the odds of a positive anti-sars-cov-2 igg index (or: 2.058; 95%ci: 1.097-3.861) ( table 3) . of the 217 igg-positive subjects, 56.7% (95%ci: 50.0-63.2%) did not notice any of the 8 most common covid-19 symptomsbetween march and end of june 2020, 18.0% (95%ci: 13.3-23.5%) reported 1 symptom, and 14.7% (95%ci: 10.5-19.9%) reported 2 symptoms. similar responses were obtained in those with negative (n = 4196) and equivocal (n = 669) anti-sars-cov-2 igg index (p = 0.954). the most common symptom was cough (27.4% of all respondents; 95% ci: 26.2-28.6%), but its rates did not differ significantly in relation to the igg result (p = 0.731). of the 8 symptoms, a significant correlation (p < 0.01) was found only for fever, which was reported by 17.1% (95%ci: 12.5-22.5%) of subjects with positive igg index, 12.4% (95%ci: 11.4-13.4%) of those with a negative igg index, and 9.0% (95%ci: 7.0-11.3%) of those with an equivocal igg index. no significant correlations were observed between the iga+igm result and the 8 analyzed covid-19 symptoms between march and end of june 2020, with the difference close to statistical significance only for cough (p = 0.052). our study is the first large cross-sectional sars-cov-2 screening survey performed among the personnel of the uniformed services in europe. in our study population, the anti-sars-cov-2 igm+iga index was positive in nearly 9% of participants, and igg index was positive in over 4% of participants, indicating a previous infection/exposure to sars-cov-2. both indexes were positive in <1% of participants. notably, all rt-pcr tests were negative, indicating no current sars-cov-2 infection, in all 5082 police employees in this study. the relatively low individual overlap between positive results of the igm+iga and igg indexes may be explained by the dynamics of various ig class formation. during the course of sars-cov-2, igm and/or iga are detected first, followed by a longer-lasting igg response. in most patients, seroconversion occurs between 7 and 14 days after the covid-19 diagnosis [14] . however, the speed and strength of individual immune response may be variable, e.g., depending on the viral burden upon exposure, and in some people, detectable antibodies may be not generated after infection due to such factors as an underlying immune disorder, immunosuppression, or other reasons. it is more difficult to explain why we did not have any positive rt-pcr test results despite a 9% rate of positive igm+iga results, presumably indicating early infection. due to the nature of police officers' activities, one may speculate whether our results might reflect a contact, possibly recurrent, with low viral burden that would be insufficient to generate true infection/virus replication (as detectable by rt-pcr) but enough to trigger antibody (iga/igm) production. studies show that while antibody responses may be undetectable or short-lived, memory t cell responses can persist for much longer and, indeed, sars-cov-2-specific t cells were detectable in antibody-seronegative-exposed family members and convalescent individuals with a history of asymptomatic or mild covid-19 [15] . obviously, it also possible that rt-pcr testing might have missed some active cases, and the antibody test used might have yielded some false positives. many of our igg-positive subjects reported some symptoms consistent with covid-19, although only fever was significantly more common in those with positive igg results compared to those without. there is a growing body of scientific data on nonrespiratorysymptoms of sars-cov-2 infection [16] [17] [18] . an analysis of covid-19 symptom profiles showed that gastrointestinal symptoms (diarrhea/lack of appetite), neurological symptoms (loss of smell and/or taste), as well as chills, myalgia, headache, and fatigue, were commonly reported by patients with covid-19 [16] . we can hypothesize that some igg-positive subjects may have observed nonrespiratorycovid-19 symptoms and therefore did not report them to a physician or sanitary inspection. although the sensitivity of rt-pcr is very high, its overall diagnostic accuracy depends on the quality of sampling (nasopharyngeal swab) and subsequent sample handling. antibody tests used might show cross-reactivity with other viruses, such as endemic coronavirus strains [19] . in a dutch summary of various elisa tests, the sensitivity of the vircell igg test at >14 days after illness onset was 97% in severe and 89% in mild/asymptomatic cases and the specificity was 94%. the sensitivity of the vircell igm+iga test at >14 days after illness onset was 97% in severe and 70% in mild/asymptomatic cases and specificity was 82% [20] . in a recent study, the vircell igg test had a sensitivity of 98% and specificity of 83% [21] . in another unpublished study, the sensitivity was much lower (65% for igg and 77% for igm+iga), whereasthe specificity was 96% for the vircell igg test and 83% for the vircell igm+iga test [22] . significant predictors of a positive igm+iga result included female gender, place of residence (town <20,000 inhabitants and city 20,000-500,000 inhabitants), and being a police officer (compared to civilian police employees). both positive and equivocal igm+iga results were significantly more common in women compared to men. although the clinical course of covid-19 is more severe in men [23] and the overall prevalence may be slightly higher in men, italian data indicate that among women aged 20-49 years, the prevalence was higher in women, and only after age 50, women outnumbered by men [24] . this has been explained by the fact that younger women are more represented in jobs (health, education, hotels, and restaurants) exposing them to a higher risk of contagion due to personal contacts. such an explanation seems less valid for police employees, but the higher rate in women may still be explained by gender differences in the nature of social contacts in general. regarding other predictors of positive igm+iga results, association with the local community size might be related to personal contacts with a larger number of people at risk of covid-19, either related or unrelated to police employees' professional activities, and being a police officer might involve a higher risk of exposure to covid-19 compared to civilian employees. however, these might also be chance findings, as other variables potentially reflecting increased exposure, such as the estimated number of persons contacted daily and involvement in the surveillance of quarantined individuals and protection of public gatherings, did not emerge as significant predictors of positive igm+iga and igg results. the two significant predictors of positive igg results were age ≥60 years and daily vaping. use of e-cigarettes, both alone and in combination with conventional cigarette smoking, has been associated with increased virulence and inflammatory potential of respiratory pathogens in general [25] and with covid-19 in particular [26] . however, the opposite results were also found for smoking. in a french cohort study, a lower proportion of participants with confirmed sars-cov-2 infection based on antibody detection was found in smokers compared to non-smokers [27] . regarding the association with age, a number of studies provided somewhat divergent results compared to our findings. in a dutch plasma donor study, antibodies against sars-cov-2 were detected significantly more often in younger people (18-30 years) , which was thought to be related to different social behaviors and higher exposure to the virus before social distancing was implemented [28] . similarly, the seroprevalence in those aged 20-49 years was significantly higher compared to those aged 50 years and older in geneva [29] . in healthy blood donors in milan, seroconversion to igg was noted more commonly among younger subjects, while seroconversion to igm was more common in older subjects [30] . in addition to positive igg and igm+iga results, we also obtained equivocal results of these tests in a significant proportion of participants. in our study population, an equivocal igm+iga index was found in nearly 10% of participants, and igg index was equivocal in 13% of participants. in general, repeated testing is recommended in case of an equivocal serological test result. in our study, testing was performed at a single time-point only, which constitutes a study limitation. an equivocal result indicates that antibodies were detected at a level close to the diagnostic threshold. equivocal results may represent an early infection, detection of decreasing antibody level after a past infection, cross-reactivity with other viruses, an underlying immune disorder, immunosuppression, or other reasons. our findings showed that 17.5% of participants had a positive or equivocal anti-sars-cov-2 igg index. based on this observation, we can hypothesize that due to the asymptomatic course of sars-cov-2 as well as due to the presence of nonrespiratory symptoms, the number of covid-19 cases in the polish population may be underestimated. testing strategies for sars-cov-2 should be regularly revised to include new scientific data on nonrespiratory symptoms of covid-19. regarding comparison of our findings to anti-sars-cov-2 seroprevalence estimates in the general population, no such data are available for poland but they are emerging for other european countries. the estimated seroprevalence was about 5% in spain (by point-of-care lateral-flow assay for anti-sars-cov-2 igm/igg and chemiluminescentmicroparticle immunoassay for igg) [3] and up to 11% in geneva (by another commercially available elisa igg test) [24] , both countries with a several-fold higher per capita covid-19 prevalence in the general population compared to poland. no large population studies have been published with the vircellcovid-19 elisa igg or igm+iga kits. this study has several limitations. the overall response rate was slightly below 58% (based on laboratory test sampling), which might have introduced some bias in terms of potential exposure to sars-cov-2, e.g., employees with more duties and responsibilities might have been less likely to participate. to determine seroprevalence, we relied on a single type of a serological test and a single kit manufacturer, although previous comparisons of the vircellcovid-19 elisa igg or igm+iga kits used do not indicate their inferior performance compared to other tests. the sensitivity and specificity of all currently available serological tests creates some potential for both false negatives and false positives, including cross-reactivity with other (corona) viruses, and false negatives are also possible with rt-pcr due to suboptimal nasopharyngeal swab technique or inadequate sample handling. the study protocol did not allow for retesting in individuals with an equivocal serological test result, which was dictated by feasibility issues and the intent to perform a single-time-point evaluation during a short period of up to 14-21 days. the majority of polish police employees are seronegative for sars-cov-2 infection. most sars-cov-2 infections were asymptomatic or oligosymptomatic, and fever was the only symptom more often reported by igg-positive subjects. e-cigarette use and older age (≥60 years) were associated with a higher risk of sars-cov-2 infection, which emphasizes the importance of quitting smoking to reduce the risk of infection. relatively high proportions of study subjects were igm+iga-positive with negative rt-pcr, or had equivocal igm+iga or igg indexes, an observation requiring further analyses. clinical features of patients infected with 2019 novel coronavirus in wuhan prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study humoral immune response to sars-cov-2 in iceland 95% of infected are asymptomatic). available online epidemiological analysis of the first 1389 cases of covid-19 in poland: a preliminary report covid-19) as of 21 dynamics of the coronavirus disease 2019 outbreak 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sars-cov-2 infected patients evaluation of diagnostic accuracy of 10 serological assays for detection of sars-cov-2 antibodies gender differences in patients with covid-19: focus on severity and mortality. front epidemiological characteristics of covid-19 cases in italy and estimates of the reproductive numbers one month into the epidemic electronic cigarette vapour increases virulence and inflammatory potential of respiratory pathogens association between youth smoking, electronic cigarette use, and coronavirus disease 2019 cluster of covid-19 in northern france: a retrospective closed cohort study herd immunity is not a realistic exit strategy during a covid-19 outbreak seroprevalence of anti-sars-cov-2 igg antibodies in geneva, switzerland (serocov-pop): a population-based study sars-cov-2 seroprevalence trends in healthy blood donors during the covid-19 milan outbreak this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-329010-n0mz098o authors: mckee, dwight l.; sternberg, ariane; stange, ulrike; laufer, stefan; naujokat, cord title: candidate drugs against sars-cov-2 and covid-19 date: 2020-04-29 journal: pharmacol res doi: 10.1016/j.phrs.2020.104859 sha: doc_id: 329010 cord_uid: n0mz098o outbreak and pandemic of coronavirus sars-cov-2 in 2019/2020 will challenge global health for the future. because a vaccine against the virus will not be available in the near future, we herein try to offer a pharmacological strategy to combat the virus. there exists a number of candidate drugs that may inhibit infection with and replication of sars-cov-2. such drugs comprise inhibitors of tmprss2 serine protease and inhibitors of angiotensin-converting enzyme 2 (ace2). blockade of ace2, the host cell receptor for the s protein of sars-cov-2 and inhibition of tmprss2, which is required for s protein priming may prevent cell entry of sars-cov-2. further, chloroquine and hydroxychloroquine, and off-label antiviral drugs, such as the nucleotide analogue remdesivir, hiv protease inhibitors lopinavir and ritonavir, broad-spectrum antiviral drugs arbidol and favipiravir as well as antiviral phytochemicals available to date may prevent spread of sars-cov-2 and morbidity and mortality of covid-19 pandemic. an exopeptidase expressed on epithelial cells of the respiratory tract, may constitute a pharmacological target to limit cell entry of sars-cov-2. the established antimalarial drugs chloroquine and hydroxychloroquine have been shown to inhibit terminal phosphorylation of ace2 and to elevate the ph in endosomes, respectively. chloroquine and hydroxychloroquine constitute candidate drugs against sars-cov infection and covid-19 disease, and are now investigated for their therapeutic efficacy in international clinical trials with covid-19 patients (i. e. solidarity trial). the glycosylated s protein of sars-cov is highly immunogenic to the host immune system, and murine polyclonal antibodies against sars-co-v s protein potently inhibit sars-cov-2 s-mediated cell entry, indicating that cross-neutralizing antibodies targeting conserved s epitopes can be elicited upon vaccination [9] . similar to the earlier sars and mers beta coronaviruses, sars-cov-2 primarily infects alveolar epithelial cell of the lung, leading to a severe bilateral peripheral pneumonia with ground glass opacity in ct images (covid-19 disease), with a mortality rate of 2 % to 5 % [10] . sars-cov-2 also can contribute to multiple organ failure, affecting heart, liver, kidney, central nervous system and gastrointestinal tract [11] . epidemiology thus far suggests that sars-cov-2 is more contagious than sars-cov or mers-cov [12] . multiple mechanisms now identified in the infective and replication processes of sars-cov-2 offer targets for pharmacological interventions. infection of pneumocytes, macrophages and pulmonary mast cells requires viral s protein. this invasion process which involves attachment of s protein to the ace2 receptor is facilitated by host cell derived serine protease tmprss211 [8] . agents that inhibit tmprss211, such as camostat mesilate, may be useful in blocking viral host cell entry. after host cell entry, the viral single-stranded positive rna, is released for replication of virus rna and translation of virus polyproteins that are finally cleaved into mature effector proteins by virus proteases [13] . the s protein interaction with ace2 on host cell cytoplasmic membrane initiates viral infection. strategies capable of disrupting s protein interaction with ace2 could be of significant therapeutic value, because the binding affinity of sars-cov-2 s protein to ace2 is 10-20-fold higher than for the s protein of sars-cov which may contribute to the higher contagiousness of sars-cov-2 as compared to sars-cov [12] . although sars-cov and sars-cov-2 have only 79% genomic sequence similarity, they share a highly conserved receptor binding domain for their s proteins [1] there is also potential for targeting other highly conserved proteins associated with sars-cov and sars-co-v-2, including rdrp and 3clpro (also termed mpro), which share over 95% similarity between the two viruses, despite only 79% genomic sequence sharing. rdrp is an rna-dependent rna polymerase required for replicating the viral genome within the host cell. 3clpro and plpro are both viral proteases which break down viral polyprotein into functional units within host cells that are finally assembled into new viruses. the 3clpro sequences between the two viruses are 96% similar, the plpro sequence identity is 83%, and their active sites show a high degree of conservation [14] . drugs that have recently been shown to target mers-cov in mice [15] , and to inhibit ebola virus rdrp and sars-cov-2 proteases in humans, such as remdesivir and ritonavir/lopinavir, also constitute candidate drugs against sars-cov-2 and are now investigated for their therapeutic efficacy in covid-19 patients in 2 international clinical trials (solidarity trial and discovery trial). finally, certain phytochemicals and natural products with high antiviral activity should be considered for treatment of sars-cov-2 infection and covid-19 disease. results from previous studies reveal that diverse viruses, including ebola virus, sars-coronavirus (sars-cov), mers-coronavirus (mers-cov) and influenza virus employ host cell proteases for activation of their envelope glycoproteins [16] [17] [18] . cleavage and activation of the spike protein (s protein) of sars-cov that is required for membrane fusion and host cell entry is mediated by transmembrane protease/serine subfamily member 2 (tmprss2), an airway and alveolar cell serine protease [19] [20] [21] . pöhlmann and coworkers recently demonstrated that sars-cov-2 also employs tmprss2 for sars-cov-2 s protein priming and s protein-driven cell entry [8] . using camostat mesilate, a clinically proven and commercial serine protease inhibitor that partially blocks infection by [26, 27] , and chronic pancreatitis [28] [29] [30] [31] . camostat mesilate (ni-03) is manufactured as an oral drug by nichi-iko pharmaceutical co., ltd., and ono pharmaceutical, japan, with a three times daily dose recommendation of 100 mg to 300 mg [30, 31] . in a clinical trial investigating camostat mesilate against dyspepsia associated with non-alcoholic mild pancreatic disease, 95 patients received 200 mg camostat mesilate three times daily for 2 weeks and showed only mild, but no severe adverse effects [28] , indicating that camostat mesilate is a well-tolerated drug. nafamostat mesilate (buipel tm ), (6-amidino-2-naphthyl-4-guanidino benzoate-dimethanesulfonate) (fut-175), (cas number: 81525-10-2), is a clinical proven and synthetic serine protease inhibitor approved in japan for the treatment of acute pancreatitis, disseminated intravascular coagulation and for anticoagulation in extracorporeal circulation [32] [33] [34] . in a screening approach of about 1,100 drugs approved by the fda, nafamostat mesilate has been identified to inhibit mers-cov s protein-j o u r n a l p r e -p r o o f mediated viral membrane fusion with tmprss2-expressing lung calu-3 host cells by inhibiting tmprss2 protease activity [35] . since the s proteins of mers-cov and sars-cov-2 share considerable amino acid sequence homology [1, 9] , nafamostat mesilate may also inhibit cell entry of sars-cov-2. in cell culture experiments with simian vero e6 cells infected with sars-cov-2, nafamostat mesilate was shown to be inhibitive against sars-cov-2 infection at ec50 of 22.50 µm [36] , suggesting that nafamostat mesilate is able to prevent sars-cov-2 infection. in a multicenter, randomized, open-label, phase 2 trial in 19 patients with severe acute pancreatitis, nafamostat mesilate was administered intravenously at a daily dose of 240 mg for 5 days without severe adverse effects [34] . sars-cov and related coronaviruses directly interact via their s proteins with angiotensin-converting enzyme 2 (ace2), a host cell exopeptidase and metallocarboxypeptidase that catalyses the conversion of angiotensin i to the nonapeptide angiotensin and the conversion of angiotensin ii to angiotensin 1-7, to initiate s protein-mediated cell entry [37] [38] [39] . it was demonstrated recently that also sars-cov-2 uses ace2 as a receptor for s protein-driven host cell entry [8, 9] . therefore, ace2 constitute a molecular target to inhibit cell entry of sars-cov-2. unfortunately, ace inhibitors as standard drugs for the treatment of hypertension and chronic heart failure fail to inhibit ace2 [40] , but a number of other drugs and compounds have been shown to inhibit ace2. chloroquine phosphate (resochin tm ) and its derivative hydroxychloroquine (quensyl tm , plaquenil tm , hydroquin tm , dolquine tm , quinoric tm ) have been used for decades for the prophylaxis and treatment of malaria and for the treatment of chronic q fever and various autoimmune diseases [41] , and have recently been demonstrated as potential broad-spectrum antiviral drugs [42, 43] . chloroquine phosphate inhibits terminal phosphorylation of ace2, and hydroxychloroquine elevates the ph in endosomes which are involved in virus cell entry [44, 45] , both mechanisms constitute relevant antiviral mechanisms of chloroquine and hydroxychloroquine. in vivo, hydroxychloroquine is metabolized into chloroquine. chloroquine phosphate has previously been shown to inhibit sars-cov infection and spread in vitro [44, 46] , and results from very recent studies reveal that chloroquine phosphate and, more effectively, hydroxychloroquine also inhibit replication of sars-cov-2 in simian vero cells [46, 47] . by using a physiologically-based pharmacokinetic model for chloroquine phosphate and hydroxychloroquine in human lung fluid, it was demonstrated that the concentrations of hydroxychloroquine recommended for treatment of sars-cov-2 infection comprise an oral loading dose of 400 mg twice daily at day 1, followed by an oral maintenance dose of 200 mg twice daily for 4 j o u r n a l p r e -p r o o f days [47] . these results were deduced from in vitro data obtained from sars-cov-2-infected vero cells treated with hydroxychloroquine [47] . a recent pilot trial conducted in more than 10 hospitals in wuhan, jingzhou, guangzhou, bejing, shanghai, chongqing and ningbo, china, with more than 100 patients with covid-19 disease demonstrated that treatment with chloroquine phosphate is superior to control treatment in inhibiting the exacerbation of pneumonia, improving lung imaging findings, promoting laboratory virus-negative conversion, and shortening the course of covid-19 disease [48] . chloroquine phosphate should be administered as an oral daily dose of 250 mg until clinical convalescence [49] . thus, in view of these results and the urgent clinical demand regarding sars-cov-2/covid-19 pandemia, chloroquine phosphate should be recommended to treat covid-19 associated pneumonia in larger populations [48] . a recent open-label non-randomized clinical trial conducted in march 2020 in france with 20 covid-19 patients treated with daily 600 mg hydroxychloroquine for 6 days demonstrated at day 6 a negative viral load (negative nasopharyngeal pcr) in 57% of the hydroxychloroquine-treated patients, as compared to negative viral load in 12.5% of untreated covid-19 patients (control group, n=16) [50] . in a randomized clinical trial conducted in february 2020 in wuhan, china, sixty two covid-19 patients were randomized to receive either daily 400 mg hydroxychloroquine for 5 days (n=31) or no pharmacological treatment (n=31) [51] . improvement and absorption of pneumonia as analyzed in chest ct at day 6 was observed in 80.6% of the hydroxychloroquine-treated patients vs. 54.8% in the untreated patients [51] . the results from these small studies therefore strongly suggest that hydroxychloroquine has therapeutic efficacy in thus, a considerable number of clinical trials investigating therapeutic efficacy of chloroquine phosphate and hydroxychloroquine in patients with sars-cov-2 infection and covid-19 disease have been initiated in china, great britain, spain and thailand [52] [53] [54] [55] [56] . the triple combination of cepharanthine (an anti-inflammatory alkaloid from stephania cepharantha gx_p2v/2017/guangxi (gx_p2v), whose s protein shares 92.2% amino acid identity with that of sars-cov-2 [60] . further, it was demonstrated that gx_p2v also uses ace2 as the receptor for viral cell entry [60] . two libraries of 2,406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on vero e6 cells by gx_p2v, and only the combination of cepharanthine, selamectin and mefloquine hydrochloride was identified as candidate drug combination against sarsj o u r n a l p r e -p r o o f shortly after the identification of the angiotensin-converting enzyme 2 (ace2), a metallocarboxypeptidase that mediates various cardiovascular and renal functions, peptide inhibitors of the enzyme were developed by selection of constrained peptide libraries displayed on phage [61] . the most potent inhibitor, termed dx600, with the amino acid sequence of acvero-e 6 cells, and inhibits infection of engineered human capillary organoids and kidney organoids by sars-cov-2 isolated from a nasopharyngeal sample of a patient with confirmed covid-19 disease [74] , suggesting that hrsace2 can block host cell entry of sars-cov-2 and early stages of sars-cov-2 infections. remdesivir (gs-5734), (cas number: 1809249-37-3), is a novel small-molecule adenine nucleotide analogue antiviral drug that has shown efficacy against ebola virus in rhesus monkeys. once-daily intravenous administration of 10 mg kg(-1) remdesivir for 12 days resulted in profound suppression of ebola virus replication and protected 100% of ebola virus-infected animals against lethal disease [75] . remdesivir displays antiviral activity against other single stranded rna viruses, including filoviruses, pneumoviruses, paramyxoviruses, and the coronaviruses mers-cov and sars-cov [76] [77] [78] . remdesivir is a prodrug that is metabolized into its active form gs-441524, an adenine nucleotide analogue that interferes with the activity of viral rna polymerase and that promotes evasion of j o u r n a l p r e -p r o o f proofreading by viral exoribonuclease, leading to inhibition of viral rna synthesis [78] . remdesivir acts early in infection, and decreases viral rna levels in a dose-dependent manner that parallels impairment of viral load in vitro [78] . these and related mechanisms of action of remdesivir have been demonstrated in vitro for sars-cov [78] , ebola virus [79] and mers-cov [80] . a recent study demonstrates in cell culture experiments with simian vero e6 cells infected with sars-cov-2 that remdesivir is inhibitive against sars-cov-2 infection at ec90 of 1.76 µm, a concentration achieved in vivo in nonhuman primate models [36] . it was further shown that remdesivir efficiently inhibited sars-cov-2 infection of human liver cancer huh-7 cells, which are sensitive to sars-cov-2 infection [36] . [87, 88] , in the usa [89] , and in france [90, 91] . lopinavir (abt-378) is a highly potent inhibitor of the human immunodeficiency virus (hiv) protease essential for intracellular hiv assembly that was developed in 1998 to circumvent hiv resistance towards the protease inhibitor ritonavir (abt-538), caused by mutation of valine at position 82 (val 82) in the active site of hiv protease in response to ritonavir therapy [92] . because the metabolism of lopinavir is strongly inhibited by ritonavir, concomitant oral administration of lopinavir and ritonavir exceeded the in vitro antiviral ec50 of lopinavir by >50-fold after 8 h in rat, dog, and monkey plasma j o u r n a l p r e -p r o o f [92] . coadministration of 400 mg lopinavir with 50 mg ritonavir enhanced in healthy human volunteers the area under the concentration curve of lopinavir in plasma by 77-fold over that observed after dosing with lopinavir alone, and mean concentrations of lopinavir exceeded the ec50 for >24 h [92] . therefore, the combination of lopinavir and ritonavir (kaletra tm ) has been established as an effective oral drug for the treatment of hiv-infected individuals when used in combination with other antiretroviral agents [93, 94] .an initial study in 2003 demonstrated that lopinavir at 4 µg/ml inhibited the cytopathic effect in a plaque reduction assay with fetal rhesus kidney-4-cells infected with sars-cov (hku-39849 isolate) [95] . in this study, newly diagnosed sars patients infected with sars-cov were treated with the combination of lopinavir (400 mg)/ritonavir (100 mg) orally every 12 hours for 14 days. at day 21, sars patients treated with lopinavir/ritonavir had a milder disease course in terms of diarrhea, recurrence of fever, worsening of chest radiographs and reduction of viral load, compared to a historical control group [95] . in a nonhuman primate model of common marmosets infected with mers-cov, lopinavir/ritonavir-treated animals displayed an improved clinical outcome compared to untreated animals, with improved weight loss, lung imaging and pathological findings, and lower mean viral loads in necropsied lung and extrapulmonary tissues [96] . in response to these findings, an ongoing randomized control trial (miracle trial) was initiated to determine the therapeutic efficacy of lopinavir/ritonavir combined with interferon β-1b in patients infected with mers-cov [97] . in a recent [98] . treatment with lopinavir/ritonavir was not associated with a difference from standard care in the time to clinical improvement, and mortality at 28 days was similar in the lopinavir/ritonavir group and the standard-care group [98] . moreover, treatment with lopinavir/ritonavir treatment did not reduce viral rna loads or duration of viral rna detectability as compared with standard supportive care. sars-cov-2 rna was still detected in 40.7% of the patients in the lopinavir/ritonavir group at the end of the trial at day 28 [98] . however, the numbers of lopinavir/ritonavir recipients who had serious complications (acute kidney injury and secondary infections) or requiring noninvasive or invasive mechanical ventilation for respiratory failure were fewer than in those not receiving lopinavir/ritonavir treatment [98] . these results and observations require additional studies to determine whether treatment with lopinavir/ritonavir given at a certain disease stage can reduce some complications in covid-19 patients [98] . china [99] [100] [101] [102] , hong kong [103] , republic of korea [104] , and in europe (discovery trial), investigating remdesivir, lopinavir/ritonavir, and lopinavir/ritonavir plus interferon β-1a [91] . umifenovir [112] . in view of these promising clinical results, clinical trials with umifenovir alone or in combination with lopinavir/ritonavir, chloroquine phosphate or carrimycin have been recently initiated in china [113] [114] [115] [116] . the human interleukin-6 receptor, tocilizumab [123] or favipiravir in combination with chloroquine phosphate and the viral neuramidase inhibitor oseltamivir [124] have been initiated recently in china. 3clpro (also termed mpro) constitutes the main protease of beta coronaviruses that is essential for processing of polyproteins translated from the viral rna [125] . recently, the x-ray structures of the unligated sars-cov-2 3clpro and its complex with α-ketoamides designed as specific inhibitors of 3clpro were reported [126] . two pyridine-containing α-ketoamides, designated 13a and 13b, displayed favorable pharmacokinetic properties in mice and were detected at sufficient concentrations in lung tissue and broncheo-alveolar lavage fluid within 4 hours to 24 hours after subcutaneous administration, demonstrating lung tropism of the compounds [126] . besides subcutaneous administration, inhalation of nebulized 13b by mice resulted in high and long-lasting (24 hours) concentrations in lung tissue, without any adverse effects [126] , pointing out a role of pyridinecontaining α-ketoamides in covid-19 therapy. in a recent study that employed combined structureassisted drug design, virtual drug screening and high-throughput screening, a mechanism-based inhibitor of mpro, termed n3, was identified by computer-aided drug design [127] . n3, a michael acceptor inhibitor that can inhibit the mpros of sars-cov and mers-cov, was shown to form a covalent bond with and to be an irreversible inhibitor of sars-cov-2 mpro [127] . further, in a highthroughput screening approach for identifying inhibitors of sars-cov-2 mpro, ebselen, an organoselenium compound with anti-inflammatory, anti-oxidant and cytoprotective properties, was identified [127] . in a plaque-reduction assay with simian vero cells infected with sars-cov-2, n3 and ebselen displayed antiviral and cell protection efficacy at ec50 values of 16.77 µm and 4.67 µm, respectively [127] , ultimately demonstrating their antiviral potential against sars-cov-2. natural products can inhibit various steps in viral infection and replication, and many of them have broad-spectrum antiviral effects, the mechanisms of which have not been fully characterized. they also can function as immunomodulators, suppressing inflammatory reaction responsible for the major morbidity and mortality of sars-cov-2 infection. phytochemicals, especially flavonoids, which are widely distributed in food plants and botanicals, have been shown to interfere with nlrp3 inflammasome signaling [128] . the respiratory distress syndrome associated with sars coronaviruses develops in part due to viral activation of the nlrp3 inflammasome within activated macrophages and t helper-1 lymphocytes, which causes increased production of inflammatory cytokines [129] . several flavonoids that interfere with activation of the nlrp3 inflammasome may modulate inflammatory response to sars beta coronaviruses: luteolin [130] , myricetin [131] , apigenin j o u r n a l p r e -p r o o f [132] , quercetin [133] kaempferol [134] , baicalin [135] , and wogonoside [136] . these flavonoids have been shown to be active against a wide variety of viruses, via multiple mechanisms [137, 138] , and are available as nutraceutical supplements at a daily dose ranging from 100 mg to 500 mg. emodin (6methyl-1,3,8-trihydroxyanthraquinone) (cas number: 518-82-1) is an anthraquinone compound found in various chinese herbs and is also produced by many species of fungi, including members of the genera aspergillus, pyrenochaeta, and pestalotiopsis. emodin has been shown to inhibit the interaction of sars-cov s protein with its receptor ace2 in a dose-dependent manner [139] . [140] , suggesting that resveratrol may also be effective against sars-cov-2 infection. the emergence of the novel beta coronavirus sars-cov-2 from wuhan, hubei province, china in december 2019 rapidly led to a pandemic involving more than 2,500,000 infected persons and more proven drugs such as camostat mesilate which prevents virus host cell entry by inhibiting tmprss2 [8] , and chloroquine phosphate which inhibits terminal phosphorylation of ace2, or hydroxychloroquine which is metabolized in vivo to chloroquine [44] . for the treatment of ordinary and severe covid-19 pneumonia, and to lower the mortality rate of covid-19 disease, the antiviral drugs remdesivir, favipiravir, umifenovir, and lopinavir/ritonavir plus interferon β-1a should be administered, in particular after the consideration of (preliminary) results from the recent ongoing clinical trials solidarity and discovery [141, 91] cn and dlm conceived the original idea and wrote large parts of the manuscript. as and us 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raw264.7 cells myricetin inhibits nlrp3 inflammasome activation via reduction of ros-dependent ubiquitination of asc and promotion of ros-independent nlrp3 ubiquitination dietary apigenin reduces induction of lox-1 and nlrp3 expression, leukocyte adhesion, and acetylated low-density lipoprotein uptake in human endothelial cells exposed to trimethylamine-n-oxide quercetin and ascorbic acid suppress fructose-induced nlrp3 inflammasome activation by blocking intracellular shuttling of txnip in human macrophage cell lines flavonoids interfere with nlrp3 inflammasome activation baicalin suppresses nlrp3 inflammasome and nuclear factor-kappa b (nf-κb) signaling during haemophilus parasuis infection wogonoside protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting nf-κb and nlrp3 inflammasome activation antiviral efficacy of lavonoids against enterovirus 71 infection in vitro and in newborn mice baicalin, a metabolite of baicalein with antiviral activity against dengue virus emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction effective inhibition of mers-cov infection by resveratrol who launches global megatrial of the four most promising coronavirus treatments use of antiviral drugs to reduce covid-19 transmission virological assessment of hospitalized patients with covid-19 an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice we are grateful to all of the colleagues who have given critical comments on this work.j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f key: cord-321624-z2mntwef authors: kowitdamrong, ekasit; puthanakit, thanyawee; jantarabenjakul, watsamon; prompetchara, eakachai; suchartlikitwong, pintip; putcharoen, opass; hirankarn, nattiya title: antibody responses to sars-cov-2 in patients with differing severities of coronavirus disease 2019 date: 2020-10-09 journal: plos one doi: 10.1371/journal.pone.0240502 sha: doc_id: 321624 cord_uid: z2mntwef background: a greater understanding of the antibody response to sars-cov-2 in an infected population is important for the development of a vaccination. aim: to investigate sars-cov-2 iga and igg antibodies in thai patients with differing severities of covid-19. methods: plasma from the following patient groups was examined: 118 adult patients with confirmed sars-cov-2 infections, 49 patients under investigation (without confirmed infections), 20 patients with other respiratory infections, and 102 healthy control patients. anti-sars-cov-2 enzyme-linked immunosorbent assay (elisa) from euroimmun was performed to assess for iga and igg antibodies. the optical density (od) ratio cutoff for a positive result was 1.1 for iga and 0.8 for igg. additionally, the association of the antibody response with both the severity of disease and the date after onset of symptoms was analyzed. results: a total of 289 participants were enrolled and 384 samples analyzed from march 10 to may 31, 2020. patients were categorized, based on their clinical manifestations, as mild (n = 59), moderate (n = 27), or severe (n = 32). the overall sensitivity of iga and igg from the samples collected after day 7 of the symptoms was 87.9% (95% ci: 79.8–93.6) and 84.8% (95% ci: 76.2–91.3), respectively. compared to the mild group, the severe group had significantly higher levels of spike 1 (s1) antigen-specific iga and igg. all patients in the moderate and severe groups had s1-specific igg, while 20% of the patients in the mild group did not have any igg detected after two weeks after the onset of symptoms. interestingly, in the severe group, the sars-cov-2 igg level was significantly higher in males than females (p = 0.003). conclusion: the serological test for sars-cov-2 has a high sensitivity more than two weeks after the onset of illness. additionally, the serological response differs among patients based on sex as well as the severity of infection. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in late december 2019, an outbreak of initially undiagnosed pneumonia was reported in wuhan, hubei province, china [1] . the causative pathogen was later identified as a novel beta coronavirus closely related to the severe acute respiratory syndrome (sars) coronavirus (cov) family and was recently termed sars-cov-2 [2] . as of july 30, 2020, more than 17 million people were infected with sars-cov-2, and there were up to 670,000 sars-cov-2-associated deaths [3] . the first case in thailand was reported on january 12, 2020 and was a traveler from wuhan [4] . on july 30, 2020, there were 3,304 confirmed sars-cov-2 cases in thailand, with an epicenter in the bangkok metropolitan area. real-time reverse transcription polymerase chain reaction (rt-pcr) diagnostic assays are a goal standard for case ascertainment and diagnosis [5] . however, validated serological tests provide evidence to compliment virological diagnoses, particularly in or after the second week of infection [6] . a greater understanding of the antibody response in an infected population is beneficial for the development of a vaccine. enzyme-linked immunosorbent assay (elisa) is commonly used to access viral-specific antibodies in a quantitative manner, and for decades has been widely accepted as a diagnostic test for antibodies. the sensitive, quantitative measurements of elisa make it suitable to assess dynamic changes in viral-specific antibodies. in principle, antigen-specific igm and iga should be detected in approximately the second week of infection, followed by antigen-specific igg after the second week of infection. there are several serology platforms currently available, which use various antigens. one large nucleocapsid-based elisa study assessing 208 samples reported that igm and iga were detected 3-6 days after the onset of symptoms with a sensitivity of 85.4% and 92.7%, respectively, while igg was detected later, 10-18 days after the onset of symptoms, with a sensitivity of 77.9% [7] . interestingly, another study showed that the seroconversion if igg against the sars-cov-2 nucleocapsid and a peptide from the spike region was detected as early as that of igm and reached its peak within six days after seroconversion [8] . compared to patients with severe cases, a weaker and more rapidly declining antibody response was observed in asymptomatic patients and in those with milder symptoms [9] . the euroimmun anti-sars-cov-2 elisa was one of the first ce-marked (european conformity) diagnostic assays developed and available worldwide. it assesses the response of iga and igg to the spike 1 (s1) protein and has been reported to correlate well with the plaque reduction neutralization test (prnt) [10, 11] . the euroimmun igg assay received emergency use authorization (eua) from the united states (us) food and drug administration (fda). thus far, most of the results have been reported from europe and the us. the objective of this study was to investigate the response of iga and igg antibodies to sars--cov-2 in serial blood samples collected from a population of thai patients with confirmed covid-19, and the association of these responses with the severity of the illness. the present study was conducted at the thai red cross emerging infectious diseases clinical center (trc-eidcc) and the faculty of medicine at chulalongkorn university. the study present was reviewed and approved by the institutional review board of the faculty of medicine (irb number 242/63) and the national blood center, thai red cross society (coa no. nbc 5/2020). confirmed covid-19 cases were defined as those that tested positive for sars-cov-2 rna using real-time reverse transcription-polymerase chain reaction (rt-pcr) testing of combined nasopharyngeal and throat swab (nt) samples. rt-pcr testing was performed in the department of microbiology of the faculty of medicine at chulalongkorn university. sars--cov-2 rna was detected using the cobas1 sars-cov-2 kit (roche diagnostics, basel, switzerland) on a fully automated cobas1 6800 system (roche diagnostics, basel, switzerland) according to the manufacturer's recommendations. nucleic acid was automatically extracted from 400 μl of the nt specimens in viral transport medium (vtm) along with added internal control rna (rna ic). subsequent real-time rt-pcr was performed automatically by the system, targeting orf1a/b and e genes specific to sars-cov-2 and pan-sarbecovirus, respectively. classification of the confirmed case was as follows, according to the covid-19 management guideline of the thai ministry of public health: 1) mild-asymptomatic or upper respiratory tract infection (uri), 2) moderate-pneumonia without hypoxia, and 3) severepneumonia with hypoxia, of which the antiviral treatment was given. the date of the onset of symptoms, disease severity, hospitalization time, and personal demographic information were obtained from hospital medical records. the control group included three subgroups. the first subgroup included 20 plasma samples collected from healthy volunteers in the laboratory and 82 plasma samples leftover from healthy blood donors prior to february 2020. the second subgroup included 49 plasma samples collected from may 1 to may 31, 2020, from patients under investigation (pui) for covid-19 with rt-pcr results that were negative for sars-cov-2. the third control subgroup included 20 serum specimens collected from may 1 to may 31, 2020 from patients with other infections (dengue, hbv, hcv, hiv, mumps, measles, rubella, ebv, cmv, vzv, hsv, and treponema). plasma and serum were aliquoted and stored at -20˚c prior to serological testing. plasma samples of 10 μl were diluted to 1:101 in sample buffer in order to perform sars--cov-2 s1-specific iga and igg assays using anti-sars-cov-2 elisa igg/iga kits (euroimmun, lubeck, germany) according to the manufacturer's instructions. semi-quantitative results were evaluated by calculating the ratio of extinction at 450 nm of each sample over the calibrator. a cutoff ratio of 1.1 was used for sars-cov-2 iga, as suggested by the package insert. the borderline cutoff ratio of 0.8 for sars-cov-2 igg was assigned as positive. demographic characteristics were described for each patient. continuous variables were expressed as the median with an interquartile range (iqr). differences in continuous and categorical variables between the two groups were assessed using the wilcoxon rank-sum test and chi-square test or the fisher exact test, respectively. sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) were also calculated. there were 118 confirmed sars-cov-2 infections from march 10 to may 31, 2020: 59 with mild (upper respiratory symptoms), 27 with moderate (pneumonia without hypoxia), and 32 with severe (pneumonia with hypoxia), with a median age of 38 years (iqr: 27-48). a total of 213 samples collected from 118 patients were tested for antibodies against sars-cov-2, with 36 patients having 1 sample, 69 patients having 2 samples, and 13 patients having 3 samples. a total of 99 samples were collected seven days after the onset of symptoms. there were 49 pui who were negative for sars-cov-2, with a median age of 47 years (iqr: 28-65 years), 25 males and 24 females. the baseline clinical characteristics are summarized in table 1 . there were significant differences in age and sex between the groups, with the patients in the severe group being mostly male (66%) and 40-59 years old. among the 118 confirmed sars-cov-2 patients, 99 had blood samples collected at least once more than 7 days after the onset of symptoms. the overall seroconversion of antibodies after the 7 th day of symptoms is summarized in the specificity, however, varied between the control subgroups. the raw data for all controls are shown in s1 table. there were two false-positive iga and one false-positive igg results out of 102 healthy controls. we were able to obtain and re-analyze seven samples, with an od ratio � 0.8 after two months. the od ratio was quite similar to the initial results, confirming the healthy control group's limited background. of the 49 pui, there were five positive iga and two positive igg results for covid-19 with negative rt-pcr results for sars-cov-2. two of these patient's tests were repeated after 2-4 weeks, and the od ratios returned to normal. of 20 serum specimens collected from patients with other infections, there were two samples with both iga and igg cross-reactivity to cmv-and ebv-positive samples. the seroconversion of the antibodies, stratified by the day of illness, is shown in table 3 . the sensitivity for serological testing within seven days of the onset of symptoms was only 29.7-30.6% for iga and 10.2-16.2% for igg. the iga positivity rate increased to 60% during the 2 nd week and 100% during the 3 rd -4 th weeks, and then declined to 81.9% in the 2 nd month. the igg positivity rate increased to 90% during the 3 rd -4 th weeks of diseases. to investigate the association of antibody levels to the severity of the disease, the antibody levels at the first time point were expressed using the specified cutoff value, stratified by disease severity. the severe group had a significantly higher level of s1-specific iga and igg antibodies compared to the mild group (fig 1) . it should be noted that the two patients in the severe group who did had no detectable s1-specific iga were tested only once, at 31 and 40 days after the onset of symptoms, and therefore it was likely that the iga levels had already declined in these patients. to see the dynamics of each group, we plotted the average antibody level from mild, moderate, and severe groups at five intervals (fig 2) . there were 103, 52, and 58 samples from the mild, moderate, and severe groups, respectively (table 3) . a clear pattern emerged, showing that the severe and moderate groups had significantly higher iga and igg levels 15 days post-symptoms compared to the mild group. of the group with mild symptoms, 20% (7/35) of the samples had no detectable igg antibodies more than 2 weeks after the onset of symptoms. only 1 out of 15 patients from the moderate group had no detectable igg antibodies, while all 15 patients with severe symptoms had high igg levels after the second week (table 3) . since age and sex were associated with the disease outcome, we analyzed the correlation between antibody level and age in the severe group, as shown in fig 3a and 3b ; however, no significant correlation was found. we also compared the antibody levels between males and females within the severe group. interestingly, the levels of both s1-specific iga and igg to were higher in males than in females, with igg being statistically significant (fig 4a and 4b) . the median age of males (51, iqr: 43-59) was also higher than that of females (41, iqr: 24-46) in the severe group. the results of the present study have demonstrated that during the first week of covid-19 infection, the sensitivity of the antibody response to acute viral infection is low. the antibody response seen in the present study started with iga, followed by igg. because it is difficult to compare results using different serological analyses, we have only used data from studies that tested with euroimmun for comparison. the results from 15 studies using euroimmun assays are summarized in s2 table. previous studies have mostly reported that the sensitivity of iga within the first week was less than 60% [12, 13] . in the present study, 30% of covid-19 patients developed positive iga antibodies very early, within 3 days after the onset of symptoms. therefore, the presence of positive iga antibodies might help identify some covid-19 patients in the early stage, however, negative results cannot be used to exclude infection. the seroconversion of iga was 100% in 21 patients at 15-28 days after the onset of symptoms. in a study from france, a 100% sensitivity of iga seroconversion was reported in 82 cases after the second week of symptoms [13] , and in 91 patients after the third week [14] . interestingly, in the present study we noticed a decline in iga after one month, with the sensitivity decreasing to 80%. in regard to igg, it should be noted that for the present study, we used the borderline cutoff to define a positive result to increase the sensitivity of igg. this cutoff did not change the specificity of the test. igg antibodies specific to the sars-cov-2 s1 antigen developed later than iga. the sensitivity of igg was 90% after the second week of symptoms, which is comparable to other studies [13, [15] [16] [17] . in the present study, 20% of the patients with mild symptoms did not develop any igg antibodies specific to covid-19, even after 2 weeks after the onset of symptoms. other studies have found up to 20-30% of cases to be negative for igg [18, 19] . when we analyzed the correlation of antibody levels with clinical severity, it was clear that patients with more severe clinical manifestations had higher antibody levels, for both iga and igg, than patients in the mild group. this observation has been consistently reported in other study populations as well [10, 19, 20] . the explanation behind these findings is not yet clear, however, one current hypothesis is that the elevated inflammatory response in the severe patients might produce a more robust immune response, including antibody production from b-lymphocytes. it also raises concerns about the role of antibody-mediated severity, although there is no evidence to support it. moreover, several studies have reported that there were higher rates of severity and mortality in male patients [21] . in the present study, more females were infected with covid-19 than males (60% female vs. 40% male); however, there were significantly more males (66%) in the severe group. interestingly, we found a significantly higher level of igg in males than in females in the severe group, similar to recent results found by klein et al. [19] . the median age of males was higher than that of females in the severe group. it is possible that higher levels of antibodies might be associated with greater illness severity in male patients. however, there is a speculation that biological sex might affect immunity through various mechanisms [21] . although women seem to have greater antibody responses and are more susceptible to autoimmune diseases than men, other factors, including innate immunity, regulatory t cells, expression of angiotensin-converting enzyme 2 (ace2), or other mechanisms related to sex hormones might explain the greater severity and higher antibody levels observed in male patients. further studies are needed to elucidate the impact of sex on disease severity, which might lead to a better understanding of this challenging disease. although the assessment of specificity was not the main objective of this study, our results confirmed those from previous studies, that the specificity of euroimmun anti-sar-cov-2 iga is lower than that of igg. as summarized in s2 table, the specificity of euroimmun anti-sar-cov-2 iga ranged from 68.3-94.6%, while anti-sar-cov-2 igg ranged from 85-100%. a higher background was observed in the control group with respiratory symptoms. as previously stated, of the 49 pui, there were 5 positive iga and 2 positive igg results that had corresponding negative rt-pcr results for sars-cov-2. two of these patients were repeated after 2-4 weeks, and the od ratio returned to normal. it should be noted that the positive results in these patients could be the result of either a false positive or a true positive case with negative rt-pcr results. however, there was no evidence to support covid-19 infection in these patients. we also observed two samples with both iga and igg cross-reactivity with cmv-and ebv-positive samples. since we did not have serological results for other coronaviruses in these two samples, we did not know definitively if they were directly cross-reactive with cmv and ebv, or the result of cross-reactivity with another coronavirus. however, based on results from other studies summarized in s2 table, there were reports of false positives with various infections, including ebv and cmv seropositives [12, 17] . in summary, the present study extensively reported the serological responses of covid-19 patients in thailand up to 60 days after the onset of symptoms. although most of the samples were tested at two time points, blood samples were collected from patients at different stages and at various intervals. therefore, we did not determine a median time for positive results, which might have been subject to bias. supporting information s1 clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus outbreak of global health concern early transmission patterns of coronavirus disease 2019 (covid-19) in travellers from wuhan to thailand detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr sars-cov-2 antibody testing-questions to be asked profiling early humoral response to diagnose novel coronavirus disease (covid-19) antibody responses to sars-cov-2 in patients with covid-19 clinical and immunological assessment of asymptomatic sars-cov-2 infections severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients clinical performance of different sars-cov-2 igg antibody tests evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-sars-cov-2 antibodies assessment of sars-cov-2 serological tests for the diagnosis of covid-19 through the evaluation of three immunoassays: two automated immunoassays (euroimmun and abbott) and one rapid lateral flow immunoassay (ng biotech) performance characteristics of four high-throughput immunoassays for detection of igg antibodies against sars-cov-2 clinical performance of two sars-cov-2 serologic assays diagnostic performance of seven rapid igg/igm antibody tests and the euroimmun iga/igg elisa in covid-19 patients neutralizing antibodies responses to sars-cov-2 in covid-19 inpatients and convalescent patients sex, age, and hospitalization drive antibody responses in a covid-19 convalescent plasma donor population. medrxiv different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid-19 patients considering how biological sex impacts immune responses and covid-19 outcomes we would like to thank the health care team for at king chulalongkorn memorial hospital, thai red cross, particularly dr. kampol suwanpimolkul, dr. leilanee paitoonpong, and dr. suvaporn anulgulreungkitt. special thanks for the advice from dr. parvapan bhattarakosol and statistical analysis by miss jiratchaya sophonphan. key: cord-344714-0cam9ipf authors: russo, maria; moccia, stefania; spagnuolo, carmela; tedesco, idolo; russo, gian luigi title: roles of flavonoids against coronavirus infection date: 2020-07-28 journal: chem biol interact doi: 10.1016/j.cbi.2020.109211 sha: doc_id: 344714 cord_uid: 0cam9ipf in terms of public health, the 21st century has been characterized by coronavirus pandemics: in 2002-03 the virus sars-cov caused sars; in 2012 mers-cov emerged and in 2019 a new human betacoronavirus strain, called sars-cov-2, caused the unprecedented covid-19 outbreak. during the course of the current epidemic, medical challenges to save lives and scientific research aimed to reveal the genetic evolution and the biochemistry of the vital cycle of the new pathogen could lead to new preventive and therapeutic strategies against sars-cov-2. up to now, there is no cure for covid-19 and waiting for an efficacious vaccine, the development of “savage” protocols, based on “old” anti-inflammatory and anti-viral drugs represents a valid and alternative therapeutic approach. as an alternative or additional therapeutic/preventive option, different in silico and in vitro studies demonstrated that small natural molecules, belonging to polyphenols family, can interfere with various stages of coronavirus entry and replication cycle. here, we reviewed the capacity of well-known (e.g. quercetin, baicalin, luteolin, hesperetin, gallocatechin gallate, epigallocatechin gallate) and uncommon (e.g. scutellarein, amentoflavone, papyriflavonol a) flavonoids, secondary metabolites widely present in plant tissues with antioxidant and anti-microbial functions, to inhibit key proteins involved in coronavirus infective cycle, such as pl(pro), 3cl(pro), ntpase/helicase. due to their pleiotropic activities and lack of systemic toxicity, flavonoids and their derivative may represent target compounds to be tested in future clinical trials to enrich the drug arsenal against coronavirus infections. the 2 nd decade of the 21 st century began with an unprecedented epidemic in human history: the emergence of a new human betacoronavirus strain, first isolated and sequenced in china in early 2020 [1] and called sars-cov-2 (severe acute respiratory syndrome coronavirus-2) as the etiological agent of coronavirus disease 19 (covid-19 ) [2] . globally, at the time of writing (11 july 2020), 12,322,395 confirmed cases of covid-19 have been registered, including 556,335 deaths worldwide except for the antarctic continent, as reported by who (https://covid19.who.int/covid -19) . although awaited by some microbiologists, this pandemic found the health systems of many western countries (italy, spain, france, uk and usa) largely unprepared. however, at the same time, it mobilized the scientific community with the production of over 6000 peer-reviewed scientific articles on pubmed in the last 5 months. waiting for a vaccine, there is currently no specific cure against covid-19. a deeper knowledge of the genetics and biochemistry sustaining sars-cov-2 vital cycle and infectivity will certainly lead to the development of new therapeutic protocols. 7 (camellia sinensis (l.) kuntze) interferes with the replication cycle of dna viruses, such as hepatitis b virus, herpes simplex, and adenovirus [17] . to prepare this review article, especially sections 3 and 4, the pubmed database (www.ncbi.nlm.nih.gov/pubmed/) was consulted up the end of may 2020, to retrieve articles that included the following combination of terms: "coronavirus" and "flavonoid". we selected those papers that convincingly focused on the antiviral activity of defined flavonoids against human coronaviruses, excluding some very recent preprint articles on sars-cov-2 not certified by peer review that, in our opinion, were not of adequate scientific quality. we apologize in advance for possible citations omitted due to space limitations. coronavirus is a family of one strand (+) rna enveloped virus in the order nidovirales. they were originally identified in the sixties in the united kingdom and the united states where scientists isolated two viruses causing common colds in humans [18] . coronaviruses are spherical or pleomorphic, with a diameter of 80-120 nm. in 1968 electron microscopy images revealed the virus crown-like structures resembling the "solar corona" that give rise to the name of this family derived from latin word: "coronavirus" [19] . since then and until last year, two highly pathogenic human strains emerged: sars-cov, in 2003 and mers-cov (middle east respiratory syndrome coronavirus) in 2012 that caused, according to who, severe epidemic outbreaks [20, 21] . they are transmitted to humans from market civets and dromedary, respectively and both originated from bats, a natural reserve of hundreds of still unknown coronavirus [22] . the coronavirus rna genome is bigger than other rna viruses with size ranges from 26,000 to 32,000 bases including from 6 to 11 open reading frames (orf). the first orf (67% of the genome) encodes not structural proteins (nsp), while the remaining orfs give rise to accessory and structural proteins [22] . in particular, the first orf (orf1a/b) translates two polyproteins: pp1a 8 and pp1ab for the presence of a frameshift between orf1a and orf1b. these polyproteins are processed by the main protease (m pro ) also known as 3c-like-protease (3cl pro ) and one or two papain-like proteases (pl pro ) into 16 nsps, which produce viral rna that encodes the four main structural proteins [23] (fig. 2) . the importance of 3cl pro in the viral cycle and the absence of its human homologue makes this enzyme an attractive target for the development of new drugs directed against coronavirus infection. 3cl pro is a three domains cysteine protease with an active site highly conserved among all coronavirus. domain i and ii are six stranded antiparallel β barrels very similar to the architecture of chymotrypsin and picornavirus 3c proteinases. the substrate-binding site is located in a cleft between these two domains. a long loop (residues 184 to 199) connects domain ii to the c-terminal domain (domain iii, residues 200 to 300). this latter domain, a globular cluster of five helices, has been implicated in the proteolytic activity of 3cl pro . anand et al. [24] and dai et al. [23] analyzed substrate-binding pocket of sars-cov and sars-cov-2, respectively to design novel inhibitors for this protease and found that the thiol group of a cysteine residue in the s1' site is important to anchor molecules by a covalent linkage, obtaining efficacious antiviral activity. the s1' site represents one of the four sites (s1', s1, s2 and s4) highly conserved in the catalytic domain of 3cl pro of human coronavirus. the four major structural proteins of coronavirus are: 1. the trimeric spike glycoprotein (s) that localizes on the surface of virus envelope and essential for virus entry into the host cells; 2. the membrane or matrix protein (m); 3. the small envelope protein (e), both essential for the assembly and release of virions; 4. the nucleocapside protein (n), that binds to rna genome forming the helically symmetric nucleocapside [19] . in addition, some coronavirus genome encodes for a glycoprotein of approximately 60-65 kda called hemagglutinin esterase (he). the role of this protein is still unclear, it possesses an acetyl-esterase enzymatic activity able to disrupt sialic acid receptors on the host cells surface, helping the invasion and attachment of virion [22] . genetic and comparative analysis of different known coronaviruses represents a powerful strategy to identify potential drug targets against the current outbreak and the 3cl pro protease is a good example. the first three sars-cov-2 genomes isolated from bronchoalveolar-lavage fluid and sequenced in wuhan (china) showed the typical beta coronavirus organization (subgenus sarbecovirus): a 5' untranslated region (utr), replicase complex (orf1a/b), s, e, m and n genes, 3'utr and several unidentified non-structural orf [1] . comparing rna sequence of 9 chinese patients affected by covid-19 with those of other coronaviruses, lu et al. (2020) performed a phylogenetic analysis to determine the evolutionary history of the virus going back to its likely origin [25] . the authors of this study found that sars-cov-2 rna sequence shared 96% genetic material with a bat virus in a cave of yunnan (china), but was distant from sars-cov (79% identity) and mers-cov (50% identity). homology modelling revealed, however, that the new virus had a similar receptor-binding domain structure (rbd) of spike protein to that of sars-cov, despite amino acid variation at few key residues. for this reason, it was hypothesized that sars-cov and sars-cov-2 shared the same cellular receptor to enter in human cells, the protein angiotensin receptor enzyme-2 (ace2) widely expressed in lung, heart, kidney, testis and gastrointestinal tract [25] . several steps are necessary to start and complete the coronavirus infective cycle: 1. recognition and binding to the cellular receptor(s) (one or two); 2. changes in the conformation and proteolysis of s protein; 3. fusion to cellular membrane; 4. entry of the virus into the host cells by endocytosis [26] . in host cells, the virus uses the endogenous cellular machinery to translate its replicase to transcribe and replicate viral rna. the following steps consist of the translation of the structural proteins by sub-genomic rnas (sgrna) generated during genome transcription/replication and, finally, the virion assembly and release [19] . it is well known that the spike glycoprotein s, located on the surface of the viral phospholipidic membrane, is crucial for coronavirus infection and pathogenesis. it includes two functional domains or subunits, s1, the globular head containing the rbd at the n-terminal, and the s2 subunit at the c-terminal responsible for virus-cell membrane fusion, followed by two heptad regions (hr1 and hr2) and the transmembrane domain (tm). the s protein undergoes several post-translational modifications: its ectodomain is heavily glycosylated and, probably, the oligosaccharides could influence priming by host proteases and determine antibody recognition [26] . in particular, membrane fusion depends on s protein cleavage by host cell proteases at s1/s2 and s2' site responsible for s protein activation (fig. 2) . hoffman et al. (2020) recently demonstrated that the life cycle of sars-cov-2 begins with the rbd of the s protein that makes contact with the ace2 receptor on the host cells [27, 28] . two host serine proteases participate to this event: the transmembrane tmprss2 and the endo-protease furin. the s1/s2 site in sars-cov-2 harbors multiple arginine residues not present on sars-cov, but common to other human coronaviruses, like mers-cov, that are recognized by furin protease (fig. 2) . the authors speculate that the presence of this multibasic cleavage between s1/s2 sites may expand sars-cov-2 tropism and/or enhance its transmissibility compared to sars-cov, due to the ubiquitous presence of furin-like proteases in human tissues, especially lung [27] . inhibition of tmprss2 and furin protease activities can be considered an interesting therapeutic option against coronavirus infection, especially covid-19, allowing the block and/or prevention of sars-cov-2 infection, as recently reported [28] . an alternative therapeutic/preventive strategy may refer to molecules or antibodies capable of disrupting s protein interaction with ace2. this interaction, due to natural selection (mutation and probably a recombination event), is 10-20-fold stronger for sars-cov-2 respect to sars-cov and may explain the higher infectivity of former [28] . ace2 is a type i transmembrane protein, its physiological role consists in the maturation of the peptide hormone angiotensin, essential for the control of vasoconstriction and blood pressure. from a biochemical point of view, ace2 is a dipeptidyl carboxypeptidase with the n-terminal peptidase domain (pd) and the c terminal collectrin (cld) domain ending with a single transmembrane helix and a ~40-residues intracellular segment [29] . the peptidase activity of ace2 is not essential for coronavirus infectivity because virus infecting respiratory tissues use this protein essentially as a receptor, being expressed on the cells of the respiratory tract. in the lung, ace2 is present in alveolar epithelial type ii cells and bronchial epithelial and it was first recognized as a receptor for sars-cov, mers-cov and now for sars-cov-2. mers-cov also recognizes dipeptidyl peptidase 4 (dpp4) or cd26 as entry receptors [30] . the rbd of s protein has a receptor binding motif (rbm) that makes the primary contact with the carboxyl-peptidase domain of ace2 receptor. the amino-acidic sequence of rbm is 50% conserved between sars-cov and sars-cov-2 and structural studies performed by yan et al. demonstrated that the extracellular pd of ace-2 is recognized by rbd through polar residues [31] . in particular, these researchers found that the most prominent alteration is the substitution of val404 in the sars-cov-rbd with lys417 in the sars-cov-2-rbd. structural data on cocrystalized proteins demonstrated that the surface of ace2 contains two "virus-binding hot-spot", two lys residues, essential for sars-cov binding ace2 creating positive charges that need to be neutralized by the coronavirus [32] . two key residues in the rbm of sars-cov-2, gln493 and leu455, bind to these hotspots leading to considerable stabilization of binding and a higher affinity for ace2 than sars-cov. in addition, sars-cov-2 rbd shows a significant higher ace-2 binding affinity due to specific substitutes (residues 482-485: gly-val-glu-gly) that stabilize the interaction between them. finally, phe486, present in the rbm of sars-cov-2, is inserted into a hydrophobic pocket of n terminal α1 helix of ace2, while a leucine residue present in rbm of sars-cov forms probably a weaker contact owing to its smaller chain [32] . virus replication takes place at the level of the cytoplasmic membrane and is mediated by a multisubunit replication/transcription complex (rtc) formed by different viral nsps. after entry, genomic rna (grna) is translated by host ribosomes in polyprotein pp1a and pp1b, which are auto-cleaved to form nsp. these nsps induce a rearrangement of cellular membrane to form double-membrane vesicles where the viral replication complexes are anchored [33] . the core component of rtc complex is the catalytic subunit, nsp12 of an rna-dependent-rnapolymerase (rdrp). for optimal function, this enzyme requires accessory factors: nsp7 and nsp8 that increase rdrp template binding and processivity. nsp3 and nsp5 encode papain like-protease, pl pro , and 3cl pro , essential, as described above, for the cleavage of polyprotein pp1a and pp1b [19] . using the grna as a template, the coronavirus replicase synthesizes full-length negative sense (-) rna, which, in turn, serves as a template for the synthesis of new genomic (+) grna and a set of different sgrna, synthetized by discontinuous transcription. these sgrna encode viral structural and accessory proteins. amino acidic residues involved in rna binding and catalytic active site of rdrp are highly conserved in different rna virus justifying the use of broad-spectrum antiviral inhibitors, such as remdesivir. this drug, that very recently showed its efficacy in a placebocontrolled trial in covid-19 patients [34] , is a prodrug of an adenosine analogue that has been proposed as inhibitor of viral rdrp through non-obligate rna chain termination, a mechanism requiring the conversion of parent compound from mono-phosphate in a tri-phosphate form. in particular, yin et al. (2020) studied the structure of the nucleotide template in complex with rdrp as a model to understand how these drug categories can inhibit sars-cov-2 replicase activity. a new compound, eidd-2801, showed more potent effects than remdesivir in blocking virus replication for the capacity to form two extra hydrogen bonds with the key residue k545 within the catalytic domain of replicase and n4 hydroxyl group off the cytidine ring, and a guanine base in the template strand [35] . although genome replication/transcription is mainly mediated by the viral replicase, other host factors have been involved, as an example, coronavirus n protein, known to act as an rna chaperone to facilitate template switching, and the enzyme glycogen-synthase-kinase-3 (gsk3) [19] . finally, rna helicases (nsp13) represent the second most conserved subunit of the rna synthesis machinery in (+) rna coronaviruses and are involved in diverse steps of their life cycle. they utilize the energy derived from the hydrolysis of nucleoside triphosphates to unwind doublestranded rna [33] . the assembly of viral particles takes place in the er-golgi intermediate complex under the control of m protein through homotypic interactions. in this phase, m protein acts as a scaffold for virus assembly because the interactions between s and m and m and n proteins allow the recruitment of structural proteins to the assembly site. e protein contributes in this phase interacting with m and inducing membrane curvature [19] . finally, mature virions are released in smooth-walled vesicles via the secretory pathway and released by exocytosis. in summary, coronavirus replication takes place in a membrane-protected and nuclease resistant microenvironment that contains (and sequesters) the protein functions required for viral rna synthesis. this strategy is believed to improve duplication/transcription fidelity and, in parallel, repress host defenses triggered by the presence of double-stranded rna [33] . the first appearance in pubmed of flavonoids as potential antiviral agents is dated back in 1951 [36] and in 1966 quercetin was indicated among these compounds [37] . in 1977, the viricidal effect of quercetin, together with other flavonoids (apigenin, pelargonidin, procyanidin), was demonstrated on parainfluenza virus type 3 and herpes simplex virus, but not on poliovirus type 1 and adenovirus type 3. the authors concluded that flavonoids may possess a potent viricidal activity, but only a moderate inhibitory effect on virus multiplication [38] . however, later studies indicated that quercetin was both effective in reducing the infectivity and intracellular replication of herpes simplex virus type 1 (hsv-i), polio-virus type 1, parainfluenza virus type 3 (pf-3), and respiratory syncytial virus (rsv) [39] . hesperetin had no effect on infectivity, but reduced intracellular replication, while catechin and naringin had no or limited effects on either virus infectivity or replication [39] . the second half of the eighties saw a proliferation of studies on the antiviral activities of flavonoids, due to the boost of antiviral researches following the hiv emergency. a review article published in 1991 and focused on the capacity of 3-methoxyflavones to inhibit polio-and rhinoviruses infection, concluded that natural products can interfere with several antiviral mechanisms, from "adsorption of the virus to the host cell to release from it" [40] . one of the first paper exploring the antiviral effect of flavonoids on coronaviruses appeared in 1990 [41] . here, the authors showed that quercetin reduced infectivity of human and bovine coronaviruses, oc43 and ncdcv, respectively, by 50% at a concentration of 60 µg/ml. other flavonoids, such as kaempferol, were ineffective, although the latter, at 10 µg/ml, reduced virus replication by 65% in ncdcv and 50% in oc43 [41] . bovine coronavirus, bcv, was also sensitive to a mixture of theaflavins from black tea (theaflavin, theaflavin-3-monogallate, theaflavin-3'-monogallate, and theaflavin-3,3' digallate) with a mean ec 50 of 34.7 µg/ml in infectivity assays on hrt-18 cell line [42] . on a different coronaviridae of veterinarian interest, the porcine epidemic diarrhea virus (pedv), quercetin 7-rhamnoside inhibited pedv replication in vero cells with an ic 50 of 0.014 µg/ml and a cc50 (cytotoxicity concentration 50%) of 100 µg/ml [43] . other flavonoids, including quercetin, apigenin, luteolin, catechin also showed significant anti-pedv activity, but with ic 50 values from 10-(apigenin) to 800-fold (catechin) higher than quercetin 7-rhamnoside indicating the importance of the o-dihydroxy functional groups at c-3' and c-4' and the rhamnoside at position 7 [43] ( table 1) . in a formulation of traditional chinese medicine (tmc) for the prevention and treatment of sars-cov, one of the component was the flavone glycoside baicalin from scutellaria baicalensis georgi. this compound was tested on frhk4 cell line by a neutralization assay using 10 isolated of sars-cov coronavirus from 10 different patients. baicalin showed an ec 50 of 12.5-25 µg/ml at 48 h without significant cytotoxicity. this value was 200-400-fold higher respect to the c max of 60 ng/ml (half-life about 3 h), considering that, in humans, a standard oral dose corresponds to about 1500 mg. this makes hardly practicable baicalin for antiviral prophylaxis or treatment, although after an intravenous administration of 360 mg in humans, the molecule reached a peak serum concentration of 74 µg/ml [44] . two other flavonoids, lutein and quercetin, showed the capacity to block the entry of sars-cov into host cells [45] . table 1) . the medical herb cinnamomi cortex, obtained from the dried bark of cinnamomum cassia (l.) j. presl, has been used to prepare several organic fractions enriched in bioactive polyphenols. among these, the n-butanol fraction was the most active in inhibiting hiv/sars-cov pseudovirus infection dose-dependently with an ic 50 of 37.3 µg/ml. this result was confirmed also against the wild-type sars-cov infection and the measured ic 50 for the same fraction was of 7.8 µg/ml [46] . the authors attributed the observed antiviral activity to the presence in the extract of procyanidin a2, procyanidin b1 and cinnamtannin b1, which showed an ic 50 ranging between 30-40 µm in the plaque reduction assay on sars-cov. however, none of the procyanidins inhibited the internalization of tfr (transferrin receptor, a marker of clathrin-mediated endocytosis) and they did not affect ace2 expression which, as reported above, is a sars-cov receptor [46] (table 1) . as reported above, sars-cov rna genome encodes for proteinases that are required for replication and transcription. nsp3 and nsp5 are two non-structural regions encoding for pl pro and 3cl pro (also called m pro , see section 2), respectively [47] . the former cleaves the nsp1-nsp3 replicase polyproteins [48] , while the latter is responsible for the processing of nsp4-nsp16 replicase products into individual polypeptides [24] . therefore, both the sars-cov 3cl pro and sars-cov pl pro have been soon considered a potential target for the design and development of antiviral drugs [47] . using two independent assays measuring sars-cov 3cl pro cleavage activity, based on a cell-free and a cell-based method, and aqueous extract from isatis indigotica fortune ex lindl. (synonym of isatis tinctoria l.) root showed a dose-dependent capacity to inhibit the sars-cov 3cl pro proteolytic cleavage activity with an ic 50 of 53.8 µg/ml and 191.6 µg/ml on the cellfree and cell-based assay, respectively [49] . in the same experimental models, several herb-derived flavonoids with accredited antiviral effects were tested (hesperetin, quercetin, and naringenin). among these, only hesperetin dose-dependently inhibited cleavage activity of the 3cl pro in cell-free and cell-based assays (ic 50 60 µm and 8.3 µm, respectively) [49] . it is of interest that quercetin did not show any inhibitory effect on sars-cov 3cl pro , although it was reported to block the entry of sars-cov into host cells [45] . to support the latter conclusion, using a molecular docking approach, it was established that quercetin-3-β-galactoside bound to sars-cov 3cl pro with residue gln189 playing a key role in stabilizing the binding [50] . in an in vitro assay, using his-tag recombinant sars-cov 3cl pro , quercetin-3-β-galactoside inhibited the protease activity competitively with ic 50 of 42.79 µm. mutation of gln189 to alanine did not reduce the enzymatic activity of sars-cov 3cl pro , but lowered of about 2-fold the inhibitory potency of quercetin-3-βgalactoside, due to the reduction in binding affinity [50] (see section 4). quercetin was also used as a reference compound in a different study where several metabolites isolated from the leaves of proteases, although the inhibition was dose-dependent [55] . surface plasmon resonance (spr) indicated that the interaction between papyriflavonol a and sars-cov pl pro was due to a specific binding event with a k d of 212 μm, stimulating the design of more effective coronavirus inhibitors [55] . a more specific study on the inhibition of mers-cov 3cl pro by flavonoids was published later [56] . here, the authors demonstrated that among 40 respectively. also in this case, docking studies demonstrated that the better affinity of rhoifolin could be due to the coordinated binding through the polar s1 site, the hydrophobic s2 and the s3' site with no strong tendency [57] ( table 2) . chalcones also represented an interesting flavonoid group with significant inhibitory activity against the two main coronavirus proteases. in a study based on nine alkylated chalcones isolated from the japanese plant angelica keiskei (miq.) koidz, it was demonstrated a significant capacity to inhibit both sars-cov 3cl pro and sars-cov pl pro protease activity in cell-based and cell-free assays. for the former protease, the cell-free assay indicated a range of ic 50 table 2) . as discussed in the previous paragraphs, the genome of sars-cov encodes structural proteins, including the n protein, an alkaline protein with a lysine-rich region that suggests a nuclear localization signal. the n factor plays a key role in virion assembly through its interactions with the viral genome and m protein. it also participates in viral rna synthesis [59] . an interesting approach was established for the screening of potential inhibitors of sars-cov n protein using a mimicking on glass chip the direct binding of viral rna to n protein. the screening showed that among the 23 polyphenolic compounds tested, only (-)-catechin gallate and (-)-gallocatechin gallate were able to displace the binding of n protein to the rna oligonucleotide. starting from 0.005 μg/ml, both compounds in a concentration-dependent manner attenuated the binding affinity on the designed biochip and at the concentration of 0.05 μg/ml, they showed up to 40% inhibition activity [60] ( table 2) . sars-cov ntpase/helicase (also called nsp13) represents an attractive target for anti-sars therapy since it plays crucial role in the viral life cycle [61] . quercetin and its derivatives have been proposed as a possible inhibitor of the helicase. firstly, using the recombinant protein and a fretbased assay, it was reported that the ic 50 of quercetin towards the duplex dna-unwinding activity of the sars-cov ntpase/helicase was of 8.1 µm, highlighting the importance of the presence of a diketoacid core and a free catechol unit [62] . the same group later published the synthesis of several 7-o-arylmethylquercetin derivatives. three of them, with 3''-cl, 3''-cn, and 4''-cl substituent on arylmethyl group, tested in this study showed inhibitory activity against sars-cov ntpase/helicase ranging between 2.7 and 5.2 µm, the same order of magnitude of the parental compound [63] . the nsp13 helicase possesses a dsdna unwinding activity as well as an atpase activity allowing the helicase to translocate along with the nucleic acids by hydrolyzing atp. a screening of about 64 natural compounds, including several flavonoids (strangely quercetin was not included in this study), all tested at the concentration of 10 µm, did not evidence any compound able to significantly inhibit the dsdna unwinding activity of the nsp13 helicase in a fret-based assay [64] . however, when the same compounds were tested for their capacity to inhibit the atpase activity of the helicase, two of them, myricetin and scutellarein, emerged as a potent inhibitor of nsp13 atpase with ic 50 values of 2.71 and 0.86 µm, respectively. it is of interest that these compounds did not inhibit to the same extent the atp hydrolysis activity of the hepatitis c virus helicase, indicating specificity for the sars-cov enzyme [64] ( table 2 ). following the covid-19 pandemic, the interest of the potential therapeutic use of flavonoids against coronavirus infection focused on sars-cov-2 virus. only few papers have been published up to now, but it is easy to predict that this number will exponentially increase in the next months or weeks. considering the impact of the covid-19 outbreak in china, it was expected that tcm could have a primary role in the therapy of the sars-cov-2 infection or, at least, in the alleviation of its symptoms. in fact, on february 2020, the rate of tcm treatment for covid-19 in china was of about 90% with only 5% of patients that manifested worse clinical signs [65] . the formula qingfei paidu decoction (qfpd), consisting of 21 components (herbs and minerals drugs), showed an effectiveness of 92% in patients at all stages including subjects cured and discharged, cases where clinical symptoms were disappeared, remained stable without aggravation or significantly improved [66] . the beneficial effects of qfpd were evident after 6 days of treatment with chest ct results that ameliorated, tracheobronchial shadow was normal, and inflammation was also absorbed accordingly with the theory of tmc that identifies in the lung the primary target of qfpd against covid-19 [65] . in the attempt to identify the major constituents of qfpd and to investigate its pharmacological mechanism against covid-19 infection, yang et al. [66] applied an integrated multidisciplinary approach (in silico technology that included pharmacological network and molecular networking of lc-ms data). they identified 129 compounds clustered in 14 groups, where flavonoids represented 45% of the total. finally, a recent paper, using a computation approach, demonstrated that narcissoside, an isorhamnetin-3-o-rutinoside flavonoid (glycosyloxyflavone) present in several wild plants, is a potent inhibitor of 6w63, the term that indicates the experimental structure of sars-cov-2 3cl pro protease (https://covid-19.uniprot.org/uniprotkb/p0dtd1). the narcissoside showed a higher affinity than the standard inhibitor x77. the two inhibitors shared the same complex, but narcissoside interacted with three amino acids (leu167, pro52 and pro168) different than those interacting with x77 (his172, leu27 and thr26). finally, the better fit of the glycosyloxyflavone into the active site of the 6w63 was also reinforced by thirteen hydrogen bonds compared to the four established by x77 [67] . from the previous sections, we learned that the bioinformatics approach represents an essential tool to identify antagonist compounds that can specifically target the binding sites of sars-cov viral proteins through complex molecular interactions responsible for viral attachment and replication. this goal can be achieved by targeting structurally important binding sites, highly conserved regions in non-structural proteins including nsp12 rdrp, nsp13 helicase, and the proteases 3cl pro and pl pro [68] . 3cl pro is highly conserved in all coronaviruses, it is essential for the polyprotein cleavage and, as reported above, represents an important target for the development of new inhibitory agents [24] . the analysis of sars-cov-2 3cl pro crystal structure indicated that the enzyme is a homodimer (chains a and b), composed of 306 amino acids and each monomer consists of three different domains [69, 70] . two catalytic domains, i (residues 8-101) and ii (residues 102-184), have an antiparallel β-barrel structure, arranged perpendicular to each other. the active site is located in the cleft between domain i and domain ii with a catalytic dyad (his41 and cys145), connecting to the helical domain iii by a long loop region [71] . domain iii (residues 201-303), a globular cluster of five α-helices, is involved in the dimerization of the 3cl pro , essential for the catalytic activity. nfinger of one subunit is involved in the arrangement of the substrate binding-pocket through a saltbridge interaction between glu290 and arg4 of each protomer. it has also been shown that s1, s2, and s4 subsites are involved in substrate recognition. s1 subsite is the polar site of 3cl pro containing a small aliphatic residue (ser, gly, ala) in the p1 position of the proteolytic site. the hydrophobic s2 subsite contains a large hydrophobic residue in p2, whereas the side chain of val and ala are at p3 and p4 side, respectively, forming a small hydrophobic pocket [69] . due to the covid-19 pandemic, compelling efforts concentrated in identifying and designing new inhibitory compounds targeting the sars-cov-2 proteases and, among these, flavonoids attracted scientists' interest being them promising agents against sars-cov infection (see the section above and these reviews [57, 72] ). accumulating evidence are going in this direction. the inhibitory activity of flavonoids against sars-cov cysteine proteases in the low micromolar range was higher if compared to the effects of peptide-derived inhibitors [55] . the structural features of flavonoids were responsible for their selectivity, suggesting that the two phenyl groups were associated with inhibitory potency, as resulting by ic 50 values of the studies cited above for kaempferol, quercetin, and quercetin-β-galactoside [55] . docking stimulation screening was performed to predict the binding affinity of flavonoids, showing that apigenin, luteolin, quercetin, daidzein, epigallocatechin, and kaempferol were able to inhibit the proteolytic activity of sars-cov 3cl pro [57] (fig. 3) . in particular, the molecular dynamics simulations allow to predict that his41, gly143, and glu166 formed interactions with common functional groups of flavonoids that showed inhibitory activities [73] . for example, fig. 4a shows the predicted interaction between the catalytic site of sars-cov 3cl pro and the phenyl group of kaempferol, which locates in the hydrophilic task of sars-cov through a hydrogen bond with glu166. other hydrogen bonds are formed between the hydroxyl groups and ile188, asp142, while the chromen-4-one scaffold was in the hydrophobic s2 site [57] . a similar association between inhibitory effect and molecular interaction established by docking analysis was described for quercetin-3-βgalactoside against sars-cov 3cl pro [50] . the analysis of structure-activity relationship highlighted crucial pharmacophore features, showing that the specific interactions between quercetin-3-β-galactoside and the active site of the protease occurred by six hydrogen bonds with residues of the catalytic binding pocket. indeed, his41, gly143, ser144, cys145, glu166, and gln189 residues were located near the pharmacophore spheres. in detail, the inhibitory effect of quercetin-3-β-galactoside was strongly associated to the bind with gln189 suggesting the formation of four hydrogen bonds with o and n atoms of the gln189 side chain (fig. 4b) . in addition, the n atom of the main chain of glu166 was involved in the formation of the other two hydrogen bonds with the flavonoid. moreover, quercetin-3-β-galactoside formed hydrophobic interactions with residues leu141, asn142, met165, and glu166 of sars-cov 3cl pro (fig. 4b) . according to this structure, it is possible to speculate that the four hydroxyl groups of quercetin are strongly responsible for its inhibitory role and the spatial conformation based on sugar moiety and 7-hydroxy site allowed structural changes through hydrogen bonding interactions [50] . two other examples can be taken by the works of nguyen et al. [52] and ryu et al. [51] commented in section 3 above. in the former, the gcg interaction with the substrate-binding pocket of 3cl pro involved 7 hydrogen bonds with residues in the catalytic binding pocket and hydrophobic interactions of carbon atoms with his41, cys145, met165, glu166, asp187, arg188, and gln189 of 3cl pro [52] . interestingly, the methylation of hydroxyl groups at c-7 reduced the inhibitory activity, revealing that a biflavone namely amentoflavone showed the highest inhibitory activity by ic 50 value of 8.3 µm ( table 2) . to support the effective inhibitor role of this molecule, computerdocking analysis indicated the interaction with the s1 site of 3cl pro , forming two hydrogen bonds between the c5 hydroxyl group and the nitrogen atom of the imidazole group of his163 and the -oh of leu141. an additional hydrogen bond occurred between the hydroxyl group in the b ring with gln189, involving the s2 site of 3cl pro [51] . the structure-activity relationship analysis of flavones namely apigenin, luteolin and quercetin evidenced that the substitution of c-30 hydroxyl group, as in luteolin, and the hydroxyl group at the c-30 position of quercetin, may play a pivotal role in sars-cov 3cl pro inhibition [51] . this observation was supported by a recent study, which explains by molecular docking study that luteolin forms 5 hydrogen bonds with gln189, leu4, asn142, thr26 and hydrophobic interaction with met49 and val3, in agreement with its lower binding energy [68] . based on these observations, it is not surprising that molecular docking approach, summarized in fig. 3 , supports the role of flavonoids in the inhibition of sars-cov 3cl pro by binding his41 and cys145 of the catalytic site and other active site residues (e.g., met49, gly143, his163, his164, glu166, pro168, and gln89), stimulating their validation by in vitro and in vivo studies. a recent paper confirmed that luteolin can also bind and inhibit the sars-cov 3cl pro [68] . another interesting field of investigation is represented by inhibition of rna viral replication, proposing rdrp as a candidate for targeted drug development. to this purpose, a molecular screening evidenced that theaflavin can interfere with the catalytic pocket of sars-cov-2 rdrp, showing binding energy of -8.8 kcal/mol. by means of molecular docking, it has been demonstrated that hydrophobic interactions are involved in binding of theaflavin to rdrp. in addition, hydrogen bonds were established between functional moieties of theaflavin and residues asp452, arg553 and arg624 of rdrp [74] . from the data reported in the previous sections, the pleiotropic nature of polyphenols and, among them, the flavonoids class, is emerging as a promising and powerful cornucopia of natural compounds with potential antiviral capacity. we reported and commented the results of several studies largely based on docking simulations suggesting the possibility that specific flavonoids can interact with and inhibit key factors responsible for the virus life cycle. in different biological fields, the therapeutic applications of flavonoids are usually accompanied by strengthens and weaknesses, generally shared by other bioactive phytochemicals. as an example, being flavonoids present in our diet, it is easy to associate their beneficial effects with the consumption of foods/dietary patterns enriched in this class of compounds. focusing our analysis on their antiviral properties, recent reviews support this view. in fact, messina et al. (2020) suggested that dietary intervention may ameliorate covid-19 outcomes and polyphenols/flavonoids can contribute to these effects reducing inflammation and blocking nuclear nf-κb translocation [75] . others suggested that "nutraceuticals and functional foods have broad potential for preventing the mechanisms of viral infection and modulating immune responses" [76] , and hypothesize a link between the senolytic activity of some flavonoids (e.g. quercetin) and the higher susceptibility of older people to viral infections, including sars-cov-2. similarly, it has been hypothesized that treating patients affected by covid-19 with senolytics and other anti-aging drugs may represent a prominent approach to prevent viral transmission and quercetin has been included among these potential agents to be tested in clinical trials [77] . we express caution on these interpretations and on too optimist views suggesting that coronavirus viral infections can be ameliorated or prevented by diet. however, although difficult to assess from an experimental and clinical point of view, the concept that strengthening the immune system, reducing inflammation and oxidative stress during pandemic can be achieved by increasing the consumption of food groups and key nutrients remains an attractive hypothesis [78, 79] . obstacles to this assumption are represented by the well-known and largely accepted limits of natural compounds, including flavonoids, in foods and/or in nutraceutical formulations. the extremely low bioavailability, their high bio-transformation due to the intestinal adsorption and complexity of gut microbiota make unlikely that flavonoids and their metabolites can reach blood concentrations in the micromolar range. these limits not only make nutritional supplementation ineffective in humans, but are in clear contradiction with the effective concentrations of flavonoids tested against coronaviruses and viral proteins cited in the previous sections, all in the micromolar range [80] ( table 1-2) . we and others [72] are more prone to believe that the real applicability of flavonoids as antiviral drugs resides in the therapy, more than the prevention. in fact, the large part of the works commented above refers to the direct binding of specific flavonoids to viral targets, although with ic 50 of tens of micromolar. this suggests that, based on in vitro assays and docking models, it will be possible to design and chemically synthesize more efficient compounds based on the flavonoid structures, where key active residues are conserved, while others undergo modifications accordingly to the sar analysis. of course, both natural and synthetic compounds will need functional validations that cannot be limited to in vitro assays based on recombinant proteins ( table 2) . to this aim, the recent covid-19 pandemic is speeding up enormously the commercialization of new reagents and kits to identify sars-cov-2 inhibitors that will probably accelerate the development of innovative methods to assess how coronaviruses infect cells and how to block the infection. the therapeutic application of flavonoids, or their derivatives, as antiviral agents, as pure compounds or in combination with canonical antiviral drugs, presents also the advantage to mitigate the critical issue of their scarce bioavailability. in fact, pharmacological (not nutritional) doses, given for a limited amount of time can be plausibly administered by routes different from oral (e.g., inhalational, intravenous) that preserve the molecules from intestinal metabolism and adsorption, ameliorating their pharmacokinetics and pharmacodynamics parameters. baicalin, cited above in section 3, represents an example of a compound that administered intravenously can reach a serum concentration of 74 µg/ml [44] . of course, clinical studies on infected subjects are the "the great absentee" from the scene, independently if we consider nutritional or pharmacological applications of flavonoids as antiviral agents. to our knowledge, only one trial is present in the database clinicaltrial.gov [81] . it is in the recruitment status and regards the effect of quercetin on the prophylaxis and treatment of covid19 . the trial is based on the assumption that quercetin, being a strong scavenger and antiinflammatory agent, can be effective in covid-19 cases. the quercetin dosage scheduled is 50027 1000 mg for prophylaxis and treatment, respectively [81] . two other studies based on tannins [82] and on an extract obtained from caesalpinia spinosa (molina) kuntze [83] were no yet recruiting. in conclusion, the interest of scientists for the antiviral capacity of flavonoids against human coronavirus infections can benefit of the enormous amount of resources that governments, health agencies, and private companies are pouring in the field, searching for a cure against sars-cov-2. this situation barely resembles what happened in the eighties-nineties following the aids pandemic, when the basic knowledge in the immunological mechanisms controlling the response to hiv infection underwent amazing and unpredictable progresses. waiting for a valuable vaccine against covid-19, the pharmacological approach remains a priority and flavonoids may contribute to it. in this scenario, the "pleiotropic" properties of flavonoids that we mentioned at the beginning of this review, risks to become the passepartout to counteract coronaviruses since they can be effective on both sides, viral and host cells, to inhibit infection. in fact, recent works hypothesized that flavonoids can inhibit both tmprss2 and furin, which cleave the sars-cov-2 spike protein facilitating sars-cov-2 infectivity. molecular docking-based screening and in vitro assays using recombinant proteins indicated that (-)-epicatechin 3-o-(3'-o-methyl) gallate for tmprss2 [84] and baicalein and oroxylin a glycoside for furin [85] can bind and inhibit their respective proteases blocking virus propagation. we hope that the hypotheses and discussions presented here can stimulate scientists to design appropriate experiments to prove that naturally occurring flavonoids or their derivatives can ameliorate anti-coronavirus prophylaxis and therapy. orf1a/b translates two polyproteins: pp1a and pp1b for the presence of a frameshift between orf1a and orf1b. these polyproteins are processed by a main protease known as 3c-likeprotease (3cl pro ) and one or two papain-like proteases (pl pro ) into 16 nsps. nsps produce replicase complex essential for viral replication: nsp12 encodes rna dependent rna polimerase (rdpd) and nsp13 encodes helicase. orfs 2-10 encode viral structural proteins: spike (s), envelope (e), membrane (m), nucleocapside (n) and other auxiliary proteins. in particular, spike protein comprises two regions: s1 with the receptor binding domain (rbd) essential for the recognition of host receptor and s2, essential for membrane fusion and entry. between s1 and s2 subunits there is the polybasic 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activity of natural flavonoids against human and non-human coronaviruses methylbavachalcone neobavaisoflavone psoralidin ic 50 = 4.2-38.4 μm fluorogenic peptide z-rlrgg-amc pl pro papyriflavonol a ic 50 = 3.7 μm fluorogenic peptide z-rlrgg-amc /ml (40% inhibition) quantum dots (qds)-conjugated rna oligonucleotide on biochip we acknowledge the nurses, the medical doctors and the social and health workers around the world who, with their daily work, saved lives and gave their lives during the covid-19 pandemic. ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord-349659-6drnriun authors: grant, benjamin d.; anderson, caitlin e.; williford, john r.; alonzo, luis f.; glukhova, veronika a.; boyle, david s.; weigl, bernhard h.; nichols, kevin p. title: sars-cov-2 coronavirus nucleocapsid antigen-detecting half-strip lateral flow assay toward the development of point of care tests using commercially available reagents date: 2020-07-01 journal: anal chem doi: 10.1021/acs.analchem.0c01975 sha: doc_id: 349659 cord_uid: 6drnriun [image: see text] the sars-cov-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of covid-19. the primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (rt-pcr), which can have good sensitivity and excellent specificity. unfortunately, implementation costs and logistical problems with reagents during the global sars-cov-2 pandemic have hindered its universal on demand adoption. lateral flow assays (lfas) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current rt-pcr testing regime, especially in lowand middle-income countries (lmics). to date, many serology lfas have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. however, on the basis of previously reported results from sars-cov-1, antigen-based sars-cov-2 assays may have significantly better clinical sensitivity than serology assays. to date, only a very small number of antigen-detecting lfas have been developed. development of a half-strip lfa is a useful first step in the development of any lfa format. in this work, we present a half-strip lfa using commercially available antibodies for the detection of sars-cov-2. we have tested this lfa in buffer and measured an lod of 0.65 ng/ml (95% ci of 0.53 to 0.77 ng/ml) ng/ml with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip lfa may serve as a useful starting point for others developing similar tests. i n late 2019, a novel coronavirus, sars-cov-2 was identified in china with significant mortality, morbidity, and infectiousness. 1 by january 2020, sars-cov-2 had spread outside china, including to the united states. 2 rapid testing for sars-cov-2, the virus which causes covid-19, is urgently needed early in the onset of the disease to effectively control the spread of sars-cov-2 within a population. 3, 4 both reverse transcription polymerase chain reaction (rt-pcr) 5−7 and direct viral antigen testing 8, 9 have the potential for diagnosis early in the course of covid-19, unlike serology assays. 10 however, rt-pcr is relatively expensive, and the supply chain required to effectively conduct a population-scale case finding activity using rt-pcr has been severely strained by the sars-cov-2 pandemic. 5 inadequate availability of rt-pcr testing capacity has hindered response efforts even in well-funded health care systems. 11 in low and middle income countries, the situation is even more dire, with testing rates currently orders of magnitude below those of high income countries. lateral flow assay (lfa) antigen tests may be an inexpensive, scalable solution to population scale diagnosis of sars-cov-2 in both high-income countries and lmics. lfas have been shown to be a scalable, easily mass producible test platform, with on the order of a half billion lfas for malaria alone being sold each year, many for approximately usd $0. 25. 12 compared to serology testswhich detect the presence of a target antibodyantigen-based lfas are less sensitive than rt-pcr, but may approach the clinical sensitivity of rt-pcr with further research and development. for example, standard diagnostics recently commercialized a visually read antigen detecting lfa (catalog number 09cov30d) with a selfreported overall sensitivity of 84%, and specificity of 100% compared to rt-pcr. 13 while this is an excellent start, further research is needed to improve upon this sensitivity. it has been postulated that higher viral load may be associated with more severe outcomes and therefore an lfa that can rapidly detect high viremia may have a role in identifying those most at risk of poor outcomes. 14 many lfas for sars-cov-2 are in development, although the vast majority of these are serological tests for previous exposure to sars-cov-2. as of may 1st, 2020, over 300 immunoassays had been reported to the foundation for innovative and new diagnostics (find) as being in development to detect sars-cov-2. however, <20 of these 300 appear to be intended to detect antigens, with the naming of the remaining immunoassays implying they are serology assays. 15 thus, it appears the state of development of antigenbased assays significantly lags behind that of serology tests for sars-cov-2. sars-cov-2 serology assays of all formats, including lfas, may be useful for both epidemiological purposes and to act as an "immunity passport" in populations with a sufficiently high prevalence to allow for acceptable positive predictive values. 16−19 for example, in a pandemic situation where the true local prevalence is 5%, testing with an lfa with a 95% sensitivity and a 95% specificity will yield a positive predictive value of only 56%. however, the inability to detect the early onset of covid-19 means serology lfas are not considered useful for case detection or diagnosis of sars-cov-2 infection, for the purposes of treatment or isolation. for example, in a recent study of the vivadiag covid-19 igm/igg rapid test, a serologic lfa, the clinical sensitivity was only 18.4% vs rt pcr, with a specificity of 91.7%. 10 antigen detecting elisas were previously developed in 2004 for sars-cov-1, with limits of detection of approximately 50 pg/ml and clinical sensitivity as a function of days since onset that was significantly better than the useful time window for the current generation of sars-cov-2 serology assays. these results further imply that there is potential for antigen-based assays for the detection of sars-cov-2. 8, 9 in this work, we describe a half-strip lfa, which is a test format frequently utilized as the first step in assay development for a "full" lfa. 20 a half-strip (also sometimes referred to as a dipstick) lfa has only a nitrocellulose analytical membrane and a wick pad, without sample or conjugate pads. sample and conjugate are premixed in a container, such as a 96-well plate, prior to the insertion of the half-strip. the assay described in this work potentially has utility on its own. however, the assay is primarily intended as a launching point to expedite development for others interested in developing an antigen detecting lfa for sars-cov-2. latex bead conjugation. for the test line conjugate, 400 nm carboxylic red latex beads (car400nm, magsphere, pasadena ca, u.s.a.) were conjugated to rockland 200− 401−a50 polyclonal antibodies (rockland immunochemicals, inc., gilbertsville pa) at a weight/weight ratio of 20:1 (beads/ antibody) using edc/nhs coupling for the test line conjugate. for the control line conjugate, 400 nm carboxylic blue latex beads (cab400nm, magsphere) were conjugated to chicken igy (chrompure 003−000−003, jackson immu-noresearch, west grove pa) at a weight/weight ratio of 10:1 (beads/antibody) using edc/nhs coupling. stock latex particles were washed, activated using fresh stocks of edc and nhs diluted in mes buffer, ph6. latex particles were then conjugated to their respective antibodies, as described above, for 3 h on an orbital shaker at room temperature. the conjugates were then quenched overnight using ethanolamine, again on the orbital shaker. conjugates were then washed and stored in 50 mm borate with 1% 7-day aged casein, as described under nitrocellulose blocking. concentrations for the completed conjugates were determined by measuring the absorbance at 560 nm for red and 660 nm for blue. final stocks were stored at 4°c until use. antibody biotinylation. the antibody was first buffer exchanged into pbs to remove sodium azide using amicon filters (50 kdaa mwco, ufc5050 sigma). specifically, the antibody was concentrated 20-fold and brought back to the original volume with pbs. this was done three times to remove sodium azide. the antibody was biotinylated at 1 mg/ ml with 50 molar excess nhs-dpeg 12 -biotin (10198, quanta biodesign, plain city, oh, u.s.a.). excess biotin was removed using amicon filters again, this time with five total concentration cycles. the biotinylation ratio was determined to be 7.1 mol of biotin per mole of antibody using the quanttag biotin quantification kit (bdk-2000, vector laboratories, u.k.). nitrocellulose striping. twenty mm cn95 was striped with a test line at 8 mm from the edge of nitrocellulose (upstream from the flow direction) and 13 mm from upstream edge of nitrocellulose. the test line was striped at 1 mg/ml polystreptavidin (cat #10 120 050, biotez, berlin, de). the control line was striped at 0.5 mg/ml goat anti-chicken igy (cat #703−005−155, jackson immunoresearch) nitrocellulose blocking. for the preparation of the blocking solution, 6 g casein was dissolved in 80 ml of 50 mm naoh overnight until fully dissolved. the following day, 0.24 g boric acid (b0252, sigma-aldrich, st. louis mo, u.s.a.) and 0.45 g sodium tetraborate (b9876, sigma-aldrich,) were added and the ph was adjusted to ph 8.5. finally, ultrapure distilled water (10977−015, invitrogen, carlsbad, ca) was added to a total volume of 100 ml, yielding 6% casein in 50 mm borate. the solution was heated to 37°c for 7 days and stored at −20°c. nitrocellulose strips were blocked in a solution containing 2% β-lactose (sigma, l3750), 0.5% bovine serum albumin (bsa) (1900−0002, seracare, milford, ma u.s.a.), 0.2 μm filtered 0.05% n-lauryl sarcosine sodium salt (ls777, sigma), 0.3% 7-day aged casein (prepared as described in the methods above), in 20 mm amp at ph 9.0 (a65182, sigma). striped nitrocellulose was dipped into blocking buffer, allowing the blocking buffer to wick the nitrocellulose strip until fully wet. nitrocellulose was then submerged into the blocking buffer for 30 min while rocking, after which it was removed and dried flat for 30 min at 25°c. lfa assembly. blocked nitrocellulose was placed on backing card (miba-020, dcn, u.s.a.). twenty mm ahlstrom 320 (ahlstrom-munksjo oyj, finland) was placed on top of the nitrocellulose with a 3 mm overlap between the two materials ( figure 1 ). excess backing card was cut off. strips were cut to a 4 mm width using a kinematic matrix guillotine cutter (kinematic automaticion, inc., twain harte, ca, u.s.a.). lfa running protocol. to make 1% casein in 50 mm borate, 6% casein (aged 7 days, as prepared above), was diluted in 50 mm borate. nucleocapsid protein samples (genscript cat #z03488 and genemedi gmp-v-2019ncov-n002) were diluted in pbs (gibco 10010−023) with 0.05% tween-20 (seracare 5460−0019). control protein and nucleocapsid antibody conjugated latex stocks were sonicated (qsonica model cl-188) with 30 pulses at 25% power, one second on, one second off, immediately prior to dilution. latex master mix was prepared at a concentration of 0.0645% test-line red latex and 0.0011% control-line blue latex in 50 mm borate and 0.5% casein. biotinylated antibody was diluted to 75 μg/ml in pbs with 0.05% tween-20. immediately prior to running the lfa, 1 μl of biotinylated antibody and 5 μl of latex master mix were added to 69 μl of sample and mixed by pipetting. the combined 75 μl was added to a 96-well plate and an lfa was inserted. after 20 min, the lfa was removed and read using an optical lfa reader to construct a semiquantitative calibration curve (ax-2x-s, axxin, australia). the axxin reader does not typically improve the limit of detection (lod) of the assay compared with the lod by visual read and was used solely for accuracy in lod determination. limit of detection calculation. the data were fitted using a four-parameter logistic fit using the drc (analysis of dose−response curves) package in r. 21 the fit was weighted inversely to the square root of the signal. the test-line lfa reader score corresponding to the limit-of-detection was defined as follows: where μ o is the mean lfa reader score for the negative samples, σ o is the corresponding standard deviations, and σ low positive is the pooled standard deviation for the four lowest nonzero concentrations (0.25, 0.5, 1, 2 ng/ml). 22 the corresponding concentration and associated 95% confidence intervals were then calculated using the fitted curve. a dose response curve was generated for the half-strip lfa using two commercially available sars-cov-2 nucleocapsid (n) proteins, from genemedi and genscript. the limit of detection for the genemedi n protein was 0.65 ng/ml (95% ci of 0.53 to 0.77 ng/ml) and for the genscript n protein was 3.03 ng/ml (95% ci of 0.00 to 7.44 ng/ml). the dose response curve, with 95% ci calculated in r using the drc package, is shown in figure 2 (see the supporting information, si). it has not yet been determined what analytical sensitivity, in either blood or nasal samples, will be required to meet an acceptable level of clinical sensitivity for sars-cov-2. interestingly, a detection limit of 50 pg/ml in an elisa system was shown in 2004 for sars-cov-1 to permit positive rates of n protein detection in sera collected at 1−5, 6−10, 11−15, and 16−20 days after the onset of symptoms for 414 samples from 298 serologically confirmed patients of 92.9, 69.8, 36.4, and 21.1%, respectively. 8, 9 the most significant work that remains for this assay to be usable as a point of care test, if the lod is near clinically useful ranges, will be the addition of a sample and conjugate pad directly on the strip. importantly, the best sample to utilize in a point of care device has not yet been determined. early drafts of target product profiles from who and others for an antigen based lfa have thus far specifically called for nasal swab as the sample matrix. other swab modalities are currently being considered for rt-pcr. 23 and, previous work on sars-cov-1 from 2004 and 2005 in serum showed promising results in an elisa format. 8, 9 it is our intent that duplication of this work, figure 1 . a half-strip was constructed using 20 mm of a nitrocellulose analytical membrane, and 20 mm of wicking pad. notably, as a half strip, no sample preparation, or sample pad, is included in this version of this assay. figure 2 . dose response curve for a half-strip lfa using nucleocapsid protein from two commercially available sources, as measured using a commercially available optical lfa reader. the lod for the genemedi n protein was 0.65 ng/ml (95% ci of 0.53 to 0.77 ng/ml) and for the genscript n protein was 3.03 ng/ml (95% ci of 0.00 to 7.44 ng/ml). raw data for this graph are presented in the si. analytical chemistry pubs.acs.org/ac article and determination of realistic limits of detection for a full strip lfa in multiple sample matrices will help point the way toward the best approach for an antigen detecting lfa for sars-cov-2. potential sample matrices include extracts from anterior nasal swabs, nasopharyngeal swabs, blood, and saliva, as well as more speculative sample types for point of care tests such as stool. further, the best antigen target for an antigen detecting sars-cov-2 lfa has not been determined, though initial work appears to be focusing primarily on nucleocapsid (n) protein. however, future work should be conducted on feasible clinical sensitivities of spike (s) and membrane (m) proteins of sars-cov-2, based on previously reported abundances of these proteins in sars-cov-1. 24 for the direct immuno-detection of intact sars-cov-2 virions, there may also be utility in targeting one antigen for efficient capture and a different antigen for the most sensitive detection. while the antibodies utilized in this half-strip assay were originally developed for sars-cov-1, specificity failures due to cross-reactivity with sars-cov-1 are not considered a significant concern for the current sars-cov-2 pandemic as sars-cov-1 is no longer known to be circulating. while these antibodies have not been used in a published lfa, and significant validation work is still required for any diagnostic application, they have been utilized in several studies specifically investigating sars-cov-2. 25−27 importantly, the antibodies utilized in this assay are polyclonal antibodies, so particular care must be taken in further studies to rule out cross-contamination with other widely circulating coronaviruses and viruses with similar nucleocapsid proteins. while the work within does not assume that this particular antibody pair will be of utility in a commercially available product, even research use of this antibody pair should adequately check for specificity failures due to likely cross-reaction. more specific antibody pairs are under development and will likely be the actual antibodies utilized in commercial assays. additionally, application in commercial assays, and any other related polyclonal antibody, must be carefully considered. other antibodies specific to sars-cov-2, some monoclonal, have also been developed, and further screening of additional antibodies will likely improve assay performance. in this paper, we present a half-strip lfa for the detection of nucleocapsid protein of sars-cov-2. this effort is a first step in the development of a functional lfa with the potential for manufacturing at scale. we utilize only commercially available reagents, and conventional protocols, to allow the straightforward duplication and modification of this approach. further work is required for the development of a practical point of care test for cars-cov-2. further work includes determination of the target antigen(s) that offers greatest sensitivity, screening novel antigen specific antibodies in capture and detector pairs, determination of the optimal sample matrix, and incorporation of the necessary sample preparation into a practical workflow. the supporting information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.0c01975. table of raw data for dose response curve shown in figure 2 for the two different sources of recombinant n proteins evaluated (pdf) case-finding: fast, available, and efficient font-line diagnostics for sars-cov-2 response to covid-19 in taiwan: big data analytics, new technology, and proactive testing quantitative detection and viral load analysis of sars-cov-2 in infected patients diagnostic testing for the novel coronavirus sars-cov-2 viral load in upper respiratory specimens of infected patients singapore claims first use of antibody test to track coronavirus infections development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis antibody responses to sars-cov-2 in covid-19 patients: the perspective application of serological tests in clinical practice patient-collected tongue, nasal, and mid-turbinate swabs for sars-cov-2 yield equivalent sensitivity to health care worker collected nasopharyngeal swabs detection of nucleocapsid antibody to sars-cov-2 is more sensitive than antibody to spike protein in covid-19 patients recapitulation of sars-cov-2 infection and cholangiocyte damage with human liver organoids disparate temperature-dependent virus−host dynamics for sars-cov-2 and sars-cov in the human respiratory epithelium funding provided by the global good fund i, llc (www. globalgood.com). we thank puneet dewan for helpful discussions on the manuscript and clinical needs. pubs.acs.org/ac article key: cord-350451-lf27iuwk authors: benedetti, francesca; pachetti, maria; marini, bruna; ippodrino, rudy; ciccozzi, massimo; zella, davide title: sars‐cov‐2: march toward adaptation date: 2020-07-11 journal: j med virol doi: 10.1002/jmv.26233 sha: doc_id: 350451 cord_uid: lf27iuwk after an initial period of rising numbers of infected subjected by sars-cov-2 and deaths by covid-19, currently some areas of the world are experiencing a reduction of cases. different degrees of lockdown and social distancing measures greatly helped in limiting the spread of the disease. the contribution of other external factors like weather conditions and population density, though not as significant, seemed nonetheless to have played an additional role in shaping the course of the epidemic. a third factor still subject of debate is how and how much the mutations observed in sars-cov-2 provide an indication of viral fitness and adaptation, and their role first into the initial phases of transmission and now during the reduction of viral spreading. aim of this commentary is to provide an overview of the status of the pandemic situation linking together the different factors implicated in current situation, and to summarize some strategies that could help us to better manage either a second wave of this virus or a potential new threat of similar nature. this article is protected by copyright. all rights reserved. the worldwide spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the novel human pathogen, first detected in china quickly became a global health emergency, culminating with the world health organization publicly proclaiming the sars-cov-2 outbreak as a pandemic (11 march 2020) . so far, more than 7 million people have been infected and more than 400 thousand have died, causing profound economic disruption and unsettling consolidated human traditions. sars-cov-2 is an enveloped +single-stranded rna virus, belonging to the betacoronavirus genus, which includes two other rna viruses that have caused recent important epidemics: sars caused by sars-cov and the middle east respiratory syndrome (mers) caused by mers-cov. sars-cov and mers-cov have caused more than 10 000 cumulative cases in the past 2 decades, with mortality rates of 9.6% for sars-cov and 37% for mers-cov, respectively. [1] [2] [3] [4] viral fitness of rna viruses is influenced by their very high frequency of mutation, which, in turn, can affect infection speed and evolution rate. 5 however, the resulting genetic variability needs to be balanced between the maintenance of genetic information integrity and survival within the host. [6] [7] [8] the viral genome mutagenic process is depended on several factors, including viral enzymes responsible for nucleic acids replication, which may have few or no proofreading and/or postreplicative nucleic acid repair capability. in addition, host enzymes, spontaneous nucleic acid damages due to physical and chemical mutagens, and recombination events can contribute to the genomic variability, together with particular genetic elements responsible for the production of new variants. mutation rates are modulated also by additional factors such as determinants of the template sequence and structure involved in viral replication. 5 in most viruses, rna polymerase lacks proofreading capability, with some exceptions such as nidovirales order (to which the betacororonavirus belongs). in nidoviruses, complex machinery originated as cleavage products of the viral orf1a and orf1b, composed of a polymerase (rna-depended rna polymerase) and nonstructural proteins, is responsible for virus replication and transcription. 9,10 biological characterization of viral mutations provide precious insights for assessing viral drug resistance, immune escape, and pathogenesis-related mechanisms. in addition, viral mutation studies are crucial for designing new vaccines, antiviral drugs, and diagnostic assays. several reports result show that sars-cov-2 is rapidly moving across countries, and new mutation hotspots are emerging in different parts of the genome. [11] [12] [13] [14] [15] [16] [17] [18] such variability indeed has implications for control of the pandemic and potential emergence of viral strains with different characteristics, including increased or reduced infectivity and lethality. although sars-cov-2 is less lethal than mers-cov, up to 20% of the infected people develop rapidly a severe disease characterized by interstitial pneumonia and acute respiratory distress syndrome that can ultimately lead to death. this is particularly evident in elderly and in people with underlying medical conditions. 19 however, most of the patients remain asymptomatic or develop mild symptoms, like fever and dry from a public health perspective, these data are important in the case sars-cov-2 reemerges in late fall or early winter of 2020, or another respiratory virus appears in the distant future. timely viral detection, more precise, and reliable tracking methods leading to swift isolation of exposed and infected subjects together with the prompt implementation of measures of social distancing may be complemented with weather pattern analysis and viral sequence analysis, to help to shape more focused interventions for the quick recognition and early containment of new emerging clusters of infection. the authors declare that there are no conflict of interests. http://orcid.org/0000-0003-3866-9239 davide zella http://orcid.org/0000-0001-5576-5770 a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group why are rna virus mutation rates so damn high? viruses at the edge of adaptation quasispecies theory in virology rna virus mutations and fitness for survival -polymerases of coronaviruses: structure, function, and inhibitors. in: gupta sp, ed. viral polymerases structure of rna-dependent rna polymerase from 2019-ncov, a major antiviral drug target the genomic variation landscape of globally-circulating clades of sars-cov-2 defines a genetic barcoding scheme geographic and temporal distributions of sars-cov-2 mutations large scale genomic analysis of 3067 sars-cov-2 genomes reveals a clonal geo-distribution and a rich genetic variations of hotspots mutations emergence of genomic diversity and recurrent mutations in sars-cov-2 emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant the 2019-new coronavirus epidemic: evidence for virus evolution the establishment of reference sequence for sars-cov-2 and variation analysis spike mutation pipeline reveals the emergence of a more transmissible form of sars-cov-2. biorxiv hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-covid-net, 14 states clinical features of patients infected with 2019 novel coronavirus in wuhan real estimates of mortality following covid-19 infection impact of lockdown on covid-19 case fatality rate and viral mutations spread inverse correlation between average monthly high temperatures and covid-19-related death rates in different geographical areas temperature and latitude analysis to predict potential spread and seasonality for covid-19 climate change and infectious diseases: from evidence to a predictive framework absolute humidity modulates influenza survival, transmission, and seasonality roles of humidity and temperature in shaping influenza seasonality on the origin and continuing evolution of sars-cov-2 an 81 nucleotide deletion in sars-cov-2 orf7a identified from sentinel surveillance in arizona discovery of a 382-nt deletion during the early evolution of sars-cov-2. biorxiv attenuation of replication by a 29 nucleotide deletion in sars-coronavirus acquired during the early stages of human-to-human transmission attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2′-omethyltransferase activity the importance of naturally attenuated sars-cov-2in the fight against covid-19. environ microbiol sars-cov-2: march toward adaptation key: cord-339344-qd73h1ie authors: simon, david; tascilar, koray; krönke, gerhard; kleyer, arnd; zaiss, mario m.; heppt, franz; meder, christine; atreya, raja; klenske, entcho; dietrich, peter; abdullah, abdullah; kliem, thorsten; corte, giulia; morf, harriet; leppkes, moritz; kremer, andreas e.; ramming, andreas; pachowsky, milena; schuch, florian; ronneberger, monika; kleinert, stefan; maier, clara; hueber, axel j.; manger, karin; manger, bernhard; berking, carola; tenbusch, matthias; überla, klaus; sticherling, michael; neurath, markus f.; schett, georg title: patients with immune-mediated inflammatory diseases receiving cytokine inhibitors have low prevalence of sars-cov-2 seroconversion date: 2020-07-24 journal: nat commun doi: 10.1038/s41467-020-17703-6 sha: doc_id: 339344 cord_uid: qd73h1ie immune-mediated inflammatory diseases (imids) of the joints, gut and skin are treated with inhibitors of inflammatory cytokines. these cytokines are involved in the pathogenesis of coronavirus disease 2019 (covid-19). investigating anti-sars-cov-2 antibody responses in imids we observe a reduced incidence of sars-cov-2 seroconversion in imid patients treated with cytokine inhibitors compared to patients receiving no such inhibitors and two healthy control populations, despite similar social exposure. hence, cytokine inhibitors seem to at least partially protect from sars-cov-2 infection. i n december 2019, first cases of human infection by a new coronavirus, named severe acute respiratory syndrome corona virus 2 (sars-cov-2), were reported in wuhan leading to a cluster of respiratory infections 1 . this coronavirus disease 2019 (covid-19) rapidly spreads worldwide and the virus increasingly challenges patient groups with enhanced infection risk. the severity of covid-19 is highly heterogeneous and is modulated by yet to be identified host factors. while most individuals experience no or rather mild respiratory symptoms, covid-19 can also lead to severe pneumonia with acute respiratory distress syndrome and death 2 . age, smoking (which increases expression of ace2, the epithelial receptor for sars-cov-2) and certain comorbidities, such as diabetes mellitus, arterial hypertension, and obstructive lung diseases, are associated with severe courses of covid-19 2 . in the context of covid-19, patients with imids are of particular interest, as these patients are characterized by intrinsic immune dysfunction as well as immune modulatory treatments that may enhance infection risk. imids often affect the inner and outer barriers of the body such as the joints (rheumatoid arthritis, spondyloarthritis), gut (inflammatory bowel disease), and skin (psoriasis) 3 . therapeutic interventions for imids target inflammatory cytokines, such as tnf-α, il-6, and il-17 that are both involved in the physiological and pathological host response elicited by sars-cov-2 4 . we therefore questioned whether imid patients receiving cytokine inhibitors might be at higher risk for sars-cov-2 infection. in this study, we analyze the prevalence of anti-sars-cov-2 immunoglobulin g (igg) antibodies in patients with imids receiving cytokine-blocking treatments and show that these patients have a lower seroprevalence of sars-cov-2 than healthy individuals. cohorts and anti-sars-cov-2 igg testing. we documented respiratory and other infectious symptoms as well as social behavior during the outbreak of covid-19 in europe in february up to april 2020 for a period of 12 weeks. this analysis was performed in patients with imids receiving continuous cytokine blockade for their underlying disease (n = 534), in patients with imids receiving no cytokine inhibition (n = 259), in health care professionals involved in the treatment of these patients (healthcare [hc] control, n = 285) and in a cohort of healthy participants unrelated to health care 5 ; (non-health care [nhc] control, n = 971) from the same region. details of imid patients and the control cohorts are depicted in table 1 . to test for exposure to sars-cov-2 we made use of recent insights into the anti-sars-cov-2 immune response: spike proteins and the nucleocapsid are known as the main antigens of corona viruses 6, 7 . the s1 domain of the sars-cov-2 spike protein has been shown to provide good specificity with limited cross-reactivity to antibodies raised during infections with the four endemic coronaviruses 8 . we therefore used an s1 domain-based antibody elisa for the initial testing and confirmed all positive samples by determining the antibody response to the sars-cov-2 nucleocapsid (n) protein. prevalence of anti-sars-cov-2 igg in imid patients. anti-sars-cov-2 igg defined as an od 450 nm of ≥0.8 in the igg antibody test against the spike protein domain s1 was found in 2.27% (95%ci 1.42-3.43%) of the nhc control cohort (fig. 1a table 2 . igg antibody test recognizing the s1 domain of the spike protein in the non-health care (nhc) control cohort, health care (hc) control cohort and immune-mediated inflammatory diseases (imids) with and without cytokine inhibitors (ci); right: risk ratios and 95% confidence intervals of anti-sars-cov-2 igg antibody positivity in the hc control cohort and imids with and without cytokine inhibitors (ci) with the nhc control cohort as reference; b comparison of anti-sars-cov-2 igg positive (anti-s1+; n = 10) and negative (anti-s1; n = 10) samples (in euroimmune elisa) for reactivity in the chemi-luminescent anti-sars-cov-2 spike s1/nucleocapsid igg test (yhlo biotech) and anti-nucleocapsid igg antibody elisa (immundiagnostik inc). c validation with in-house elisa testing reactivities against 1 the s1 domain of the spike protein 2 , the receptor binding domain (rbd) of the s1 domain of the spike protein 3 validation of anti-sars-cov-2 igg testing. positive igg responses against the sars-cov-2 s1 domain were validated by two independent tests, one chemo-luminescence assay for igg against the spike and nucleocapsid protein and an enzyme-linked immunosorbent assay for igg against the nucleocapsid protein only (fig. 1b) . furthermore, the pattern of immune responses against the spike protein s1 domain, the receptor binding domain of the s1 domain, the extracellular domain of the s2 domain and the nucleocapsid of sars-cov-2 were identical in the positively tested samples and patients with rna proven covid-19 but different from patients with endemic hcov infection (fig. 1b) . these data indicate that anti-sars-cov-2 igg responses are derived from covid-19 but not endemic hcov infections. notably, only 6 (13%) of the total 46 sars-cov-2 igg positive participants received a diagnosis of covid-19 during the observation period. this observation is in accordance with recently published data 9 and also reflects the about tenfold difference between confirmed clinical covid-19 cases in bavaria (0.35%) 10 and the seroprevalence of sars-cov-2 in this population study (2.2%). the difference in prevalence of confirmed clinical covid-19 cases and seroprevalence of sars-cov-2 is based on several factors, which include (i) the availability of rna testing, (ii) the sensitivity of rna testing and (iii) the bias toward more symptomatic individuals being hospitalized and tested. the higher prevalence and broader range of symptoms in the anti-sars-cov-2 igg positive participants with diagnosed covid-19 than in those without diagnosed covid-19 supports that notion ( supplementary fig. s1 ). to test whether differences in social exposure between the groups account for the low prevalence of sars-cov-2 igg responses in imid patients treated with cytokine inhibitors, we assessed exposure risk variables (contact with persons with a respiratory infection, presence at workplace outside home, travel to risk areas) of imid patient groups and control groups. the deviation from expected frequencies of social contacts and behavior of imid patients with and without cytokine inhibitors were very similar (fig. 2) , while, not unexpectedly, participants in the hc control cohort showed a pattern of higher exposure risk and higher frequency of symptoms (table 3 ). our data are consistent with the idea that imid patients treated with cytokine inhibitors show reduced susceptibility to sars-cov-2 infection and covid-19. this finding is at first sight counterintuitive as imids are associated with an inherently enhanced infection risk, also pertaining to viral infections 11 . furthermore, inhibition of some of the pro-inflammatory cytokines has shown to increase the risk for infections, in particular bacterial and fungal infections 12 . nonetheless, no increased risk for viral infections, i.e., respiratory infections such as influenza has been reported with the inhibition of tnf-α, il-6, il-17, or il-23, which are the most frequent cytokine inhibitors currently used for the treatment of imids 13 . the low seroprevalence of sars-cov-2 in anti-cytokine treated imids could have two principle explanations: while (i) the four groups were recruited in the same region, (ii) the hc control group having the highest prevalence for sars-cov-2 igg was in direct contact with the imid patients and (iii) all participants were exposed to similar detailed information regarding social behavior during the outbreak of the covid-19 pandemic in germany, imid patients may have followed an even more stringent exposure prophylaxis than healthy individuals. we rigorously tested this hypothesis and showed that deviation from expected frequencies of social contacts and behavior of imid patients with and without cytokine inhibitors was very similar, while, not unexpectedly, hc participants showed a pattern of higher exposure risk and higher frequency of symptoms. since, seroprevalence of sars-cov-2 in imid patients without cytokine inhibitors was similar to healthy participants, other factors need to be considered that account for this very low seroprevalence of sars-cov-2 in cytokine inhibitor-treated imid patients. two recent studies reported that development of a more pronounced anti-sars-cov-2 igg response is linked to more severe disease, suggesting inflammatory responses as important triggers for priming adaptive immunity against sars-cov-2 14, 15 . similar observations have been made for sars-cov 16 . hence, tissue injury and the related release of proinflammatory mediators, which drive the symptoms of covid-19, may also initiate robust antibody responses. infection of epithelial cells by sars viruses triggers the release of cytokines such as tnf-α and il-6 17 , which are key cytokines in imids, where they are known to aggravate inflammation and tissue damage. therefore, treatment with cytokine blockers may dampen the inflammatory tissue damage in response to sars-cov-2, limit clinical onset of covid-19 and also inhibit the formation of anti-sars-cov-2 antibodies. not surprisingly, cytokine blocking strategies, such as il-6 inhibition, are currently tested for the treatment of the hyperinflammatory syndrome associated with covid-19 18 . this study also shows that overall seroprevalence of sars-cov-2 in the general population in germany is low (around 2%), even in bavaria that had the highest number of covid-19 cases in germany. however, this low prevalence may not be generalizable to other places. data from geneva in switzerland, which have been hit more severely by the coronavirus pandemic, show a seroprevalence of around 10% in the general population 10 . even more so, preliminary data from hotspots of coronavirus infection like london and new york city indicate an overall seroprevalence of sars-cov-2 around 20% for the general population and, consistent with our data, even higher rates in healthcare workers 19, 20 . although we have extensively validated anti-sars-cov-2 antibody responses it cannot be fully excluded that some of the responses are due to cross-reacting antibodies raised by endemic coronavirus infections. if this was the case our data would indicate that imid patients treated with cytokine inhibitors also have a reduced susceptibility to infections with endemic coronaviruses. furthermore, virtual absence and/or mild clinical course of clinical covid-19 infection in imid patients [21] [22] [23] [24] argues for reduced susceptibility of these patients to sars-cov-2 and supports the concept of a protective or at least mitigating effect of treatment with cytokine-blocking drugs. in summary, these data suggest that patients with imids receiving cytokine inhibitors may not at enhanced but at lower risk for sars-cov-2 infection compared with the general community and imid patients not receiving such drugs. these data however, only preventive clinical trials and/or larger prospective, observational studies will be the ultimate approach to answer the question of treatment discontinuation. patients. patients with immune-mediated inflammatory diseases (imid; n = 534) were recruited at the fau erlangen-nuremberg, the deutsches zentrum fuer immuntherapie (dzi), the rheumatology clinical practice erlangen, the sozialstiftung bamberg and at the rheumatology practice bamberg. these centers are specialized in the treatment of imid patients in the field of rheumatology, gastroenterology and dermatology. patients were offered to participate if they were on stable (>3 months) treatment with cytokine inhibitors, comprising either therapeutic antibodies and receptors (such as those neutralizing tumor necrosis factor alpha or interleukins-6, −17, or −23) or chemical agents (such as janus kinase inhibitors). in addition, a cohort of imid patients (n = 259) receiving no cytokine inhibitors within the last three months was recruited in the same centers. center distribution of imid patients with and without cytokine inhibitors was identical. healthy control cohorts. in addition, two control groups were analyzed. the first (n = 285) included health care (hc) professionals (doctors, nurses and technicians), who work in the aforementioned institutions and are involved in the treatment, diagnostics and research on imid patients. participants were contacted personally as well as by e-mail and invited to participate in the study. none of hc professionals denied participating in this study as their interest in receiving coronavirus diagnostics was very high. in addition and since the exposure of the hc control to infectious agents may be higher than in the general population, an additional control group with healthy participants not involved in health care was analyzed ("non-health care control", nhc; n = 971). this group was composed of two populations: a cohort of healthy participants (n = 336) from the district of erlangen-höchstadt and the city of erlangen, that has been established via field campaigns to assesses healthy ageing. these participants were not allowed to have a diagnosis of any imid. part of this cohort has been described previously 5 and numbers have been expanded since then. the second part was a cohort of firefighters (n = 635) from the same region (district of erlangen-höchstadt and the city of erlangen), which was recruited by an organized field campaign in march and april 2020. recruitment was centrally organized by the volunteer fire brigade officers, who invited all firefighters of their sub-units to participate (via the corresponding fire brigade mailing lists). all firefighters agreed to participate in this study. as the prevalence of anti-sars-cov-2 antibodies was very similar in the non-firefighter cohort (2.12%) and the firefighter cohort (2.36%), the two nhc control cohorts were pooled. ethical approval. ethical approval (#157_20 b) to conduct this analysis was granted by the institutional review board of the university clinic of erlangen as the responsible ethics committee for all participating institutions. written informed consent was obtained from the study participants. demographic characteristics. age, sex and body mass index were documented in all patients and healthy participants. in addition, history of smoking, arterial hypertension, diabetes mellitus, and chronic lung diseases, which put patients at risk for severe covid-19 infection were documented (table 1) . assessment of symptoms and social contacts. the study groups were exposed to questions on clinical symptoms (cough, rhinitis, throat pain, fever, headache, fatigue, musculoskeletal pain, anosmia, shortness of breath and diarrhea) and social contacts (contact with infected individuals, travel to at risk areas, working outside home) provided by the german federal authority for infectious diseases (robert koch institute) and disseminated through routes of public communication. the following questions were asked to the participants (fig. 2) : (i) contact with infected individuals: have you had contact with people who had a feverish respiratory infection during the last 8 weeks? (ii) travel to risk areas: have you been in a risk area (e.g. northern italy, tyrol) in the last 8 weeks or have you had direct contact with a confirmed or suspected case of covid-19? (iii) working outside home: have you been working in the home office during the last 8 weeks? data about symptoms and social contacts were self-documented (present/absent, complied/ not-complied) using a standardized form during the study period (1st february to 15th april, 2020). these data were self-documented and retrieved when serum analysis was done after 8 weeks. anti-sars-cov-2 antibody testing. the principle of testing igg antibodies against the spike protein of sars-cov-2 has been described previously 25 . serum samples were taken between march 18th and april 30th for anti-sars-cov-2 igg tests. igg antibodies against the s1 domain of the spike protein of sars-cov-2 where tested by the recent ce version (april 2020) of the commercial enzyme-linked immunosorbent assay from euroimmun (lübeck, germany) using the euroimmun analyzer i platform and according to the manufacturers protocol. optical density was determined at 450 nm with reference wavelength at 630 nm. a cutoff of ≥0.8 (od450 nm) was considered as positive. this assay has very high (>99%) specificity (see also https:// www.ukbonn.de/ c12582d3002fd21d/vwlookupdownloads/streeck_-et_al_infection_fatality_rate_of_sars_cov_2_infection2.pdf/%24file/streeck_-et_al_infection_fatality_rate_of_sars_cov_2_infection2.pdf) and has been approved for diagnostic use in the united states (see https://www.centerforhealthsecurity.org/ resources/covid-19/serology/serology-based-tests-for-covid-19.html#sec2). assays were performed in line with the guidelines of the german medical association (rilibak) with stipulated internal and external quality controls. tests used for validation of positive results. two reference tests for igg antibodies against the sars-cov-2 were used, one of them a chemo-luminescent assay for detecting antibodies against the spike and nucleocapsid proteins (shenzhen yhlo biotech, iflash-sars-cov-2, cat #c86095g, shenzhen, china). this assay is based on magnetic beads coated with sars-cov-2 spike and nucleocapsid antigen for the detection of specific igg using a fully automated iflash immunoassay analyzer (shenzhen yhlo biotech). the assays were performed according to the manufacturer's protocols. the igg titer was automatically calculated as arbitrary units (au/ml) and the cutoff value for a positive test was 10 au/ml. in addition, an enzyme-linked immunosorbent assay detecting antibodies against the nucleocapsid protein (immundiagnostik, bensheim, germany). optical density was determined at 450 nm with reference wavelength at 630 nm. a cutoff of ≥0.5 (od 450 nm) was considered as positive. in-house sars-cov-2 elisa. an in-house elisa was prepared according to published coating protocols 24 . in detail, 100 µl of the coating solution, consisting of 1.5 µg/ml of one of the above indicated sars-cov-2 antigens in 1xpbs, were applied to wells of a 96 well plates (costar), incubated overnight at 4°c. the following antigens were used: recombinant sars-cov-2 spike protein, s1 subunit statistics. we summarized participant characteristics using means, standard deviations and percentages as appropriate. for anti-sars-cov2 igg positivity (≥0.8 od450 nm) exact 95% confidence intervals were constructed based on the poisson approximation to the binomial distribution. we estimated relative risks of seropositivity in study groups using the nhc group as the reference and adjusting for age, sex, and sampling-date using a poisson regression model with robust sandwich standard errors 25, 26 . age, sex, and sampling-date were included in the regression as they influence the risk for sars-cov-2 infection 9 . adjustment for sampling date was achieved using the cumulative confirmed covid-19 casecounts reported by the robert koch institute for erlangen and erlangen-höchstadt on the date of serum sampling (https://experience.arcgis.com/experience/ 478220a4c454480e823b17327b2bf1d4; accessed on 07.05.2020 at 16.24). we reasoned that these case counts would approximate the overall risk of exposure to sars-cov-2 from the onset of the pandemic to the date of sampling. we constructed 4 × 2 contingency tables for binary categories of (i) patient-reported history of contact with persons having a febrile respiratory tract infection, (ii) absence from workplace, such as working from home, being unemployed or retired, and (iii) travel to high-risk regions. using these contingency tables we calculated and plotted standardized residuals showing the deviation of the observed frequencies in each study group from expected frequencies of the relevant category. results for chemo-luminescent detection of anti-s1 spike protein antibodies (yhlo biotech) and anti-nucleocapsid antibodies (immunodiagnostik) were compared between s1positive and s1-negative participants using mann whitney u test. a p value of less than 0.05 was considered as significant. all analyses were done using r v.3.5.3 software (r foundation for statistical computing, vienna, austria) and graphpad prism v8.1 (graphpad software, san diego, usa). reporting summary. further information on research design is available in the nature research reporting summary linked to this article. anonymized raw patient data have been deposited in github under the https://doi.org/ 10.5281/zenodo.3929467 (https://github.com/ekoraytascilar/ naturecommunicationscovid. all other data are present in the article and supplementary information files or available from the corresponding author upon reasonable request. source data are provided with this paper. r/sweawe code that reproduces the analyzes and tables reported in this manuscript are desposited in github under the https://doi.org/10.5281/zenodo.3929467 (https://github. com/ekoraytascilar/naturecommunicationscovid). source data are provided with this paper. received: 15 may 2020; accepted: 9 july 2020; a new coronavirus associated with human respiratory disease in china clinical features of patients infected with 2019 novel coronavirus in wuhan how cytokine 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influenza and influenza-related complications: a retrospective cohort study immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid-19 antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocytederived macrophages and dendritic cells tocilizumab treatment in covid-19: a single center experience cumulative incidence and diagnosis of sars-cov-2 infection in new york prevalence of sars-cov-2 antibodies among healthcare workers at a tertiary academic hospital in new york city prevention of covid-19 in patients with inflammatory bowel disease in wuhan, china uneventful course in patients with inflammatory bowel disease during the severe acute respiratory syndrome coronavirus 2 outbreak in northern italy clinical course of covid-19 in a series of patients with chronic arthritis treated with immunosuppressive targeted therapies covid-19 in immune-mediated inflammatory diseasescase series from new york a serological assay to detect sars-cov-2 seroconversion in humans alternatives for logistic regression in crosssectional studies: an empirical comparison of models that directly estimate the prevalence ratio this study was supported by the deutsche forschungsgemeinschaft (dfg-for2886 pandora and the crc1181), the bundesministerium für bildung und forschung (bmbf; project mascara), the h2020 ga 810316 -4d-nanoscope erc synergy project, the imi funded project rtcure, the emerging fields initiative miracle of the friedrich-alexander-universität erlangen-nürnberg as well as the schreiber stiftung gemeinnützige gesellschaft mbh. ds acknowledge funding from the else kröner-fresenius-stiftung (else kröner-memorial-stipendium, no. 2019_ekms.27). the present work was performed in (partial) fulfillment of the requirements for obtaining the degree "dr. rer. biol. hum." for ds at the friedrich-alexander-university erlangen-nürnberg (fau). the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-17703-6.correspondence and requests for materials should be addressed to g.s.peer review information nature communications thanks natalie e. dean, jose scher, and the other, anonymous reviewer(s) for their contribution to the peer review of this work.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-342902-y1v8wzxq authors: yuan, shuofeng; yin, xin; meng, xiangzhi; chan, jasper; ye, zi-wei; riva, laura; pache, lars; chan, chris chun-yiu; lai, pok-man; chan, chris; poon, vincent; matsunaga, naoko; pu, yuan; yuen, chun-kit; cao, jianli; liang, ronghui; tang, kaiming; sheng, li; du, yushen; xu, wan; sze, kong-hung; zhang, jinxia; chu, hin; kok, kin-hang; to, kelvin; jin, dong-yan; sun, ren; chanda, sumit; yuen, kwok-yung title: clofazimine is a broad-spectrum coronavirus inhibitor that antagonizes sars-cov-2 replication in primary human cell culture and hamsters date: 2020-10-07 journal: res sq doi: 10.21203/rs.3.rs-86169/v1 sha: doc_id: 342902 cord_uid: y1v8wzxq covid-19 pandemic is the third zoonotic coronavirus (cov) outbreak of the century after severe acute respiratory syndrome (sars) in 2003 and middle east respiratory syndrome (mers) since 2012. treatment options for covs are largely lacking. here, we show that clofazimine, an anti-leprosy drug with a favorable safety and pharmacokinetics profile, possesses pan-coronaviral inhibitory activity, and can antagonize sars-cov-2 replication in multiple in vitro systems, including the human embryonic stem cell-derived cardiomyocytes and ex vivo lung cultures. the fda-approved molecule was found to inhibit multiple steps of viral replication, suggesting multiple underlying antiviral mechanisms. in a hamster model of sars-cov-2 pathogenesis, prophylactic or therapeutic administration of clofazimine significantly reduced viral load in the lung and fecal viral shedding, and also prevented cytokine storm associated with viral infection. additionally, clofazimine exhibited synergy when administered with remdesivir. since clofazimine is orally bioavailable and has a comparatively low manufacturing cost, it is an attractive clinical candidate for outpatient treatment and remdesivir-based combinatorial therapy for hospitalized covid-19 patients, particularly in developing countries. taken together, our data provide evidence that clofazimine may have a role in the control of the current pandemic sars-cov-2, endemic mers-cov in the middle east, and, possibly most importantly, emerging covs of the future. the current pandemic of novel coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) represents a global public health crisis. sars-cov-2 infection in human has a broad clinical spectrum ranging from mild to severe cases, with a mortality rate of ~ 6.4% worldwide 1 . as of september 29, 2020, over 33 million cases had been reported in 235 countries, areas or territories with more than 1 million deaths, whereas a sizable portion of infected but non-symptomatic people with potential of transmissibility was also reported 2 . the genetically diverse coronavirus (cov) family, currently composed of four genera (α, β, γ, and δ), infects birds, bats and a variety of mammals 3 . within a decade, the world's human population has undergone three major cov outbreaks. sars-cov-1 emerged in guangdong, china in 2002 and, with the aid of commercial air travel, spread rapidly and globally, causing more than 8,000 cases with 10% currently, there are no widely available speci c antiviral therapies for cov in humans. remdesivir exhibited pan-coronavirus inhibitory potential 6 , and was recently granted emergency use authorization by the fda for the treatment of covid-19 based on the signi cant reduced time to recovery 7 . however, the therapy is far from optimal, particularly for severe covid-19 patients, and can only be administered intravenously to hospitalized patients 8, 9 . thus development of additional therapeutic options is urgent, as well as the establishment of combinatorial regimens, such as the triple antiviral combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin, which has been shown to be bene cial in a clinical trial 10 . in efforts to accelerate the development of novel therapies for covid-19, we previously pro led a library of known drugs encompassing approximately 12,000 clinical-stage or fda-approved small molecules 11 . in this study, we focused on the antiviral mechanisms of action and in vivo e cacy of clofazimine, an fda-approved molecule discovered as an anti-tuberculosis drug in 1957 and later used for treatment of leprosy 12 . treating tuberculosis, clofazimine exhibits a minimum inhibitory concentration of 0.016 µg/ml (equivalent to 33.80 nm). the effective concentration of clofazimine against sars-cov-2 (half maximal effective concentration 310 nm) is clinically achievable with standard dosage in patients (peak serum concentration 861 nm) 13 . here, we report the capability of clofazimine to confer protection against sars-cov-2 infection in primary human cell and animal models. most importantly, clofazimine is affordable by covid-19 patients in developing countries which may substantially relieve the critical care pressure caused by continuing pandemic 14 . clofazimine has been found to be well tolerated in humans, showing a desirable safety pro le at doses of 200mg/day in human 13 , a c max of > 861nm, and a selectivity index (cc 50 /ec 50 ) around 30~50 against sars-cov-2 infection 15 ( figure 1a ). these data suggest that therapeutic dosing of clofazimine may be achievable in patients at concentrations likely to have in vivo antiviral activity. using sars-cov-2 infection as a model, we further characterized the antiviral activity of clofazimine in human embryonic stem cell-derived cardiomyocytes that robustly support sars-cov-2 replication 16 . strikingly, and in a dose-dependent manner, clofazimine treatment reduced viral titers in the cell lysate by >3-log10 at a concentration of 10µm when compared with the dmso control ( figure 1b ). next, we assessed the antiviral activity of clofazimine in an ex vivo lung culture system. donor lung tissue was infected with sars-cov-2 for 24 h with drug treatment starting at 2 hours post-inoculation (hpi). our results revealed that clofazimine potently antagonized viral replication in tissues that re ect the primary site of sars-cov-2 replication (figure 1c ). to explore whether clofazimine confers protection against another epidemic cov, we performed a plaque reduction assay for mers-cov. clofazimine reduced mers-cov replication in veroe6 cells with an ec 50 of 1.48±0.17 µm (figure 1d ). immuno uorescence staining for mers-cov np illustrated dramatic suppression of virus infection upon clofazimine treatment (upper panel, figure 1e ), which is supported by the ow cytometry analysis that the percentage of mers-covinfected cells after clofazimine treatment decreased from 44.6% (dmso) to 23.0% (clofazimine) at 24 hpi in huh7 cells (lower panel, figure 1e ). overall, clofazimine exhibited potent broad spectrum anti-cov, and antagonized sars-cov-2 replication in human primary cell and ex vivo lung models. to understand the impact of clofazimine on the virus life cycle, antiviral activity was rst evaluated by a time-of-drug addition assay in a single infectious cycle. treatment with clofazimine during inoculation strongly inhibited sars-cov-2 infection, indicating that clofazimine exerts inhibitory effect on viral entry. intriguingly, clofazimine also blocked sars-cov-2 infection at a post-entry step as evidenced by the observed reduction of viral infection when clofazimine was added at 5 hpi (figure 2a ). to further evaluate the impact of clofazimine on viral entry, we employed vesicular stomatitis virus (vsv)-based sars-cov-2 spike (s) pseudotyped virions. clofazimine treatment dramatically reduced the infectivity of both sars-cov-1 s and sars-cov-2 s pseudotyped virions in veroe6 cells. interestingly, clofazimine did not impact mers-cov s pseudotyped virus particles (figure 2b) , and this lack of entry inhibition may contribute to a lower potency observed for mers-cov. to con rm whether clofazimine also inhibits post-entry steps of viral replication, we evaluated the impact of clofazimine on viral rna production by electroporating in vitro transcribed viral rna into veroe6 cells, which bypasses clofazimine-mediated inhibition on the entry process, and directly measures rna synthesis ( figure 2c ). as expected, remdesivir could effectively reduce the synthesis of negative-stranded rna in a dose-dependent manner ( figure 2d ). intriguingly, viral rna levels were also reduced by 1~1.5 logs in the cells treated with clofazimine at concentrations above 5 µm (figure 2e ). however, no signi cant effect was observed on electroporated gfp mrna translation ( figure 2f) . collectively, these results demonstrated that clofazimine inhibit multiple steps in sars-cov-2 replication by interfering with spike-mediated entry as well as viral rna replication. to explore what is the impact of clofazimine on the transcriptional response of host cells, we employed rna-seq to pro le the transcriptomic-wide changes during clofazimine treatment. we found that in human colorectal caco-2 cells, clofazimine exhibited comparable anti-sars-cov-2 potency as that of remdesivir (figure 3a) , which was chosen for the downstream analysis. transcriptional analysis was performed in caco-2 cells which were either infected with sars-cov-2, treated with clofazimine (10 µm) or both. principal component analysis (pca) on rna-seq results suggested that at 3 hpi, clofazimine treatment (3hpi. cfz) caused overall transcriptome shift towards mock-infection group when compared with the vehicle control group (3 hpi) (figure 3b ), which is consistent with our data indicating that the drug inhibits viral infection at early time point post infection (figure 2 ). at 6 hpi, there were 607 and 448 genes up-and down-regulated by sars-cov-2 infection, respectively (fdr<0.05, fold change>2 or <0.5 compared with mock). the rna level of more than 90% of these genes was reverted by clofazimine treatment, indicating that clofazimine treatment abrogated transcriptomic changes caused by sars-cov-2 infection. this is consistent with the pca plot that treatment with clofazimine at 6 hpi (6 hpi. cfz) caused a dramatic shift towards mock (figure 3b , extended data figure 1a) . interestingly, clofazimine treatment in the absence of infection (6h. cfz) up-regulated genes that were enriched into innate immunity-related pathways, including mapk, interleukin and tnf responses (figure 3c and 3d, extended data figure 1b) . particularly, transcription factors critical for immediate-early cellular response, including ap-1, smad, maff families, were upregulated by clofazimine (figure 3c ). when clofazimine was applied onto infected cells, most of these innate immune pathways were further enriched in upregulated genes (6hpi. cfz, figure 3d , extended data figure 1b and 1c) . these results suggest that clofazimine rewires the transcriptional landscape to prime the innate immunity-related pathways. while de cient early stage innate immune responses have been attributed to poor disease outcome, additional studies are required to determine if this enhanced antiviral response contributes to the in vitro and in vivo e cacy of the drug 17-19 . clofazimine is useful for the treatment of disease due to multidrug resistant mycobacterium tuberculosis, as well as leprosy and certain chronic skin diseases 13 .previous pharmacokinetics studies revealed that clofazimine absorption varies from 45 to 62% following oral administration in leprosy patients. coadministration of a 200mg dose of clofazimine with food resulted in a c max of 0.41 mg/l (equivalent to 861 nm) with a t max of 8h. administered in a fasting state, however, the corresponding c max of clofazimine was 30% lower while the time to c max was 12h 20 . intriguingly, clofazimine exerts antiin ammatory properties due to the suppression of macrophage activity, which may further mitigate the cytokine storm of sars-cov-2 infection in addition to its direct antiviral effects 21 . to determine the in vivo antiviral e cacy of clofazimine, we employed a golden syrian hamster model that serves as a suitable tool to study antiviral effects and disease pathogenesis 22 . since administration of clofazimine with a high fat meal provides better bioavailability 13 , we delivered the drug through oral route utilizing corn oil as vehicle. 25 mg/kg/day of clofazimine given on 3 consecutive days exhibited no signi cant observable toxicity to the animals. remdesivir was included as a positive control drug and dosed at 15 mg/kg/day based on its effective dosage in sars-cov-infected mice 6 . clofazimine has a relatively long duration of action with the mean elimination half-life approximately 25 days, thus we performed prophylactic treatment of hamsters with clofazimine before intranasally challenged with 10 5 pfu of sars-cov-2 ( figure 4a ). expectedly, the dmso-treated control hamsters developed the clinical signs of lethargy, hunched back posture, and rapid breathing starting from 2 dpi, whereas the hamsters treated with clofazimine did not develop any clinical signs. at 2 dpi when the viral loads and histopathological changes were expected to be worse, clofazimine decreased virus plaque forming units in lung tissues by ~1 to 1.5 logs (figure 4b) . consistently, suppression of sars-cov-2 viral load in hamster lungs was con rmed in the clofazimine-treated hamsters (figure 4c ). to explore if the presence of clofazimine in the gastrointestinal tract, after intragastric administration, would prevent sars-cov-2 shedding, animal feces were collected at 2 dpi for viral rna detection. signi cantly less (p=0.0353) viral copies were detectable in clofazimine-treated group when compared with the dmso group, indicating its potential to diminish fecal shedding of sars-cov-2 ( figure 4d ). increased proin ammatory cytokines and chemokines is associated with disease severity of covid-19 patients. to ascertain if the therapeutic effect of clofazimine alleviates virus-induced cytokine dysregulation, we determined the expression levels of interleukin 6 (il-6), tumor necrosis factor alpha (tnf-α), and c-c chemokine receptor type 4 (ccr4), which are prognostic markers for severe covid-19 23 . as shown in figure 4e , mrna expression of il-6 (p=0.0001), tnf-α (p=0.0006), and ccr4 (p=0.0029) were remarkably reduced in the hamsters treated with clofazimine. previous reports have shown that clofazimine can inhibit lymphocyte function 24 . to explore if this is the case in our animal model, hamster sera were collected at 14dpi for measurement of anti-np antibody using an elisa-based enzyme immunoassay. apparently, similarly high levels of antibody responses were triggered in dmso and clofazimine groups, indicating insigni cant suppression of humoral immune response of b lymphocyte by clofazimine ( figure 4i ). taken together, prophylactic administration of clofazimine conferred protection against sars-cov-2 challenge by reducing the virus replication and the associated in ammatory dysregulation. to recapitulate the scenario that most covid-19 patients will receive treatment after diagnosis or disease onset, it was of interest to determine whether therapeutic treatment of clofazimine, with the rst dosing given 24 h after virus exposure, would also ameliorate sars-cov-2 disease. sars-cov-2 infected hamsters were given 3 doses in total before being scari ed at 4 dpi for lung viral yield detection. generally, both therapeutic clofazimine and remdesivir suppressed virus lung titers when compared with the dmso control (figure 4f and 4g). the diminished clinical signs were also associated with substantially decreased il-6 protein amount in the clofazimine (p=0.0119) and remdesivir-treated (p=0.0074) hamster sera (figure 4h ), as increased serum il-6 level has been correlated with respiratory failure and adverse clinical outcome 25 . as for the severity of lung damage, histological examination of hematoxylin and eosin (h&e) stained lung tissues was performed. signi cant amelioration of lung damage was observed after clofazimine treatment (figure 4j ). for prophylactic administration, lung tissues from the dmso group showed severe bronchiolar cell death with massive cell debris lling the lumen, alveolar wall thickened with alveolar exudation; whereas prophylaxis with clofazimine showed no apparent pathological changes. with therapeutic administration, dmso-treated lung sections showed large areas of lung consolidation with alveolar in ltration and exudation, while clofazimine treated lungs exhibited a mild degree of alveolar wall thickening and capillary congestion. generally, prophylactic administration conferred more dramatic improvements of lung pathology when compared with therapeutic administration, which might be attributed to the relatively long t max of clofazimine. nevertheless, both prophylactic and therapeutic treatment with clofazimine reduced sars-cov-2 disease in vivo. since the emergency use authorization by the us fda, remdesivir is considered the standard of care for the treatment of covid-19. to understand the impact of combinatorial treatments of remdesivir and clofazimine on sars-cov-2 replication, we conducted a matrixed dose response analysis. we found that co-application of clofazimine and remdesivir impacts sars-cov-2 replication in a manner that extends beyond the additive combinatorial activity predicted by the bliss independence model (maximal bliss synergy score of 44.28; figure 5a , extended data figure 2) , and indicates these two drugs harbor a synergistic antiviral relationship. clofazimine can be safely dosed at 200 mg/day for the treatment of leprosy, which results in average serum concentrations of 1.79 µm, although the bioavailable fraction of the molecule will be a function of plasma protein binding. the addition of 1.25 µm clofazimine in an in vitro cellular assay with a 10% concentration of fbs resulted in a nearly 20-fold decrease in concentrations of remdesivir required to inhibit viral replication by 90% (figure 5b) . importantly, the combination of drugs did not elicit additional cellular cytotoxicity (figure 5c ). clofazimine was rst used to treat leprosy in 1969, and gained fda approval in 1996 26 . it is an orally bioavailable drug that is included in the who model list of essential medicines. it is generally welltolerated, with adverse events that include skin discoloration, ichthyosis, and gastrointestinal intolerance 27 . besides treating leprosy, clofazimine is an intriguing medication that has implications for multi-drugresistant tuberculosis (mdr-tb) and extensively drug-resistant tuberculosis (xdr-tb). showing good safety evidence, clofazimine is a part of who group c in terms of the treatment guidelines for mdr-tb 28 . this is supported by clinical trials in china, bangladesh, and brazil where patients were receiving clofazimine for 18~21 months at a dose of 100 mg/day 29, 30 . we observed that clofazimine shows pleotropic antiviral activities against sars-cov-2, including inhibition of spike-dependent entry. while it has been reported that clofazimine is internalized through endocytosis, further investigation is required to elucidate if the drug directly impinges on endosomal function to inhibit viral entry 31 , and why the leprosy drug selectively blocks sars-cov, but not mers-cov, entry into cells. importantly, this drug is a lipophilic rhimophenazine dye which inhibits mycobacteria through intercalation into bacterial dna, likely inhibiting dna replication and proliferation 32 . while we observe that clofazimine inhibits the rna replication of sars-cov-2, additional studies are also required to determine if the drug similarly inhibits cov rna unwinding or template function. in sars-cov-2 infection, a delayed innate immune response may result in uncontrollable cytokine storm 19, 33 . clofazimine's effect on rewiring the transcriptional landscape of the cell towards an antiviral status may be important in a disease setting, and understanding the contribution of this activity toward in vivo disease amelioration can provide insight towards its potential to improve viral control through enhancement of innate immune activities. paradoxically, clofazimine has been reported to possess antiin ammatory activity through the inhibition of macrophage function and t lymphocyte activation and proliferation 34 . further elucidation of how clofazimine treatment may balance regulation of innate and adaptive immune responses to improve disease outcome will be important to understand its potential clinical e cacy. in this study, a prophylaxis regimen with three daily doses substantially protected animal from sars-cov-2 infection (figure 4) . in contrast to orally bioavailable clofazimine, remdesivir is currently given through intravenous administration, which makes it di cult to provide on an outpatient or prophylactic basis. moreover, remdesivir requires a complex synthesis process to manufacture, resulting in a high treatment cost (us$520 per vial, or us$3,120 per treatment course) and availability for only several million patients over the next two years 35 . in view of the potentially-long epidemic dynamics and pressures on critical care capacity over the next 5 years, as well as the potential resurgence of sars-cov-2 in the future, clofazimine, which only costs us$1.43/100mg tablets, can be considered as one of the potential countermeasures for global control of the covid-19 pandemic 14 , especially in developing countries. additionally, co-administration with clofazimine could signi cantly reduce costs for remdesivir-based treatment of covid-19, and extend worldwide supplies of remdesivir, and a combinatorial approach can also help mitigate the emergence of drug-resistant viral strains. clofazimine, in conjunction with interferon, is currently being evaluated in clinical trials for the treatment of covid-19 (trial number: nct04465695). the ex vivo and in vivo e cacy of clofazimine suggests that clinical evaluation of the drug as monotherapy in outpatient setting for treatment of early stage disease, or in combination with remdesivir in hospitalized patients, is critical for establishing its potential as a rapidly scalable treatment option for covid-19. human hepatoma huh7 (jcrb, 0403) cells and monkey veroe6 cells (atcc, crl-592 1586) were maintained in dmem culture medium supplemented with 10% heat-inactivated fbs, 50 u/ml penicillin and 50 µg/ml streptomycin. ventricular cardiomyocyte were differentiated from the human embryonic stem cell hes2 (esi) maintained in mtesr1 medium (stemcell technologies) 36 . brie y, hes2 cells were dissociated with accutase (invitrogen) into single cells suspensions on day 0. cells were seeded on low-attachment culture vessels (corning) and cultured in mtesr1 medium supplemented with 40 µg/ml matrigel, 1 ng/ml bmp4 (invitrogen) and 10µm rho kinase inhibitor (rock) (r&d) under hypoxic environment with 5% o 2 . from day 1 to 3, cells were cultured in stempro34 sfm (invitrogen) with 50 µg/ml ascorbic acid (aa) (sigma), 2 mm gluta-max (invitrogen), 10 ng/ml bmp4, and 10 ng/ml human recombinant activin-a (invitrogen). from day 4 to day 7, 5 µm wnt inhibitor iwr-1(tocris) was added. antiviral evaluation in human ex vivo lung tissues human lung tissues for ex vivo studies were obtained from patients undergoing surgical operations at queen mary hospital, hong kong as previously described 38 . the donors gave written consent as approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw13-364). the freshly obtained lung tissues were processed into small rectangular pieces and were rinsed with advanced dmem/f12 medium (gibco) supplemented with 2 mm of hepes (gibco), 1×glutamax (gibco), 100 u/ml penicillin, and 100 μg/ml streptomycin. the specimens were infected with sars-cov-2 hku-001a with an inoculum of 1×10 6 pfu/ml at 500 μl per well. after two hours, the inoculum was removed, and the specimens were washed 3 times with pbs. the infected human lung tissues were then cultured in 1 ml of advanced dmem/f12 medium with 2 mm hepes (gibco), 1×glutamax (gibco), 100 u/ml penicillin, 100 μg/ml streptomycin, 20 μg/ml vancomycin, 20 μg/ml cipro oxacin, 50 μg/ml amikacin, and 50 μg/ml nystatin. supernatants were collected at 24 hours post inoculation (hpi) for plaque assays. male and female syrian hamster, aged 6-10 weeks old, were kept in biosafety level 2 housing and given access to standard pellet feed and water ad libitum as we previously described 22 . all experimental protocols were approved by the animal ethics committee in the university of hong kong (culatr) and were performed according to the standard operating procedures of the biosafety level 3 animal facilities (reference code: culatr 5370-20). experimentally, each hamster was intranasally inoculated with 10 5 pfu of sars-cov-2 in 100 μl pbs under intraperitoneal ketamine (200 mg/kg) and xylazine (10 mg/kg) anesthesia. prophylactic treatment used oral administration of clofazimine given on -3, -2 and -1dpi (25 mg/kg/day), followed by virus challenge at 0dpi, while therapeutic post-exposure and oral administration of clofazimine were performed on 1, 2, and 3 dpi (25 mg/kg/day) with the rst dosage given at 24 hpi. clofazimine was delivered using corn oil (sigma-aldrich, c8267) as vehicle. remdesivir (15 mg/kg/day, medchemexpress) was used as a positive control through intraperitoneal injection. one percent dmso in corn oil was used as a placebo control through oral route. animals were sacri ced at 2 dpi and 4 dpi for virological and histopathological analyses. viral yield in the lung tissue homogenates and/or feces were detected by plaque assay and/or qrt-pcr methods. cytokine and chemokine pro le of the hamster lungs were detected by 2 -δδct method using probe-based one step qrt-pcr (qiagen). elisa kit was utilized to determine the interleukin 6 (il-6) amount in the hamster sera on 4 dpi according to the manufacture's recommendations (elisagenie, hmfi0001). tissue pathology of infected animals was examined by h&e staining in accordance to the established protocol 39 . on 14 dpi, enzyme immunoassay (eia) was utilized to determine the antibody titer of hamster sera against sars-cov-2 np antigen 40 . brie y, 96-well immune-plates (nunc) were coated with 100 μl/well (0.1 μg/well) of sars-cov-2 np in 0.05 m nahco 3 (ph 9.6) overnight at 4°c. after blocking, 100 μl of heat-inactivated serum samples were serial-diluted before adding to the wells and incubated at 37°c for 1 h. the attached antibodies were detected using horseradish-peroxidase-conjugated rabbit anti-hamster igg antibody (invitrogen, a18895). the reaction was developed by adding diluted 3,3',5,5'-tetramethylbenzidine single solution (invitrogen) and stopped with 0.3 n h 2 so 4 . the optical density (od) was read at 450/620 nm using a microplate reader. fastq les from rna-seq were quality examined by fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). reads were processed by cutadapt to remove reads with low quality and to trim adapters. trimmed reads were aligned to hg38 reference genome using tophat2 41 . reads assigned to each gene were counted by featurecounts 42 with refseq gene sets as references. genes without at least 1 read mapped on average in each sample were considered undetectable and were ltered out. read counts were normalized by trimmed mean of mvalues (tmm) method and differential expression was calculated using r package edger and genewise negative binomial generalized linear models with quasi-likelihood tests (glmqlfit) method was used for statistical tests. cut-offs imposed for differential expression analysis was set as false discovery rate (fdr) of 0.05 and fold change >2 or <0. 5 pseudotyping of vsv and pseudotype-based inhibition assay vesicular stomatitis virus (vsv) pseudotyped with spike proteins of mers-cov, sars-cov-1, and sars-cov-2 were generated as previously reported with some modi cations 45 . brie y, bhk-21/wi-2 cells (kerafast, ma) overexpressing the spike proteins were inoculated with vsv-g pseudotyped δg-luciferase vsv (kerafast, ma). after 2 h inoculation at 37°c, the inoculum was removed and cells were refed with dmem supplemented with 5% fbs and vsv-g antibody (i1, mouse hybridoma supernatant from crl-2700; atcc). pseudotyped particles were collected at 24 h post-inoculation, then centrifuged at 1,320 × g to remove cell debris and stored at −80°c until use. to determine the effect of the compounds on viral entry, veroe6 cells were treated with clofazimine at a concentration of 2.5 µm for 1 h prior to inoculation with respective pseudotyped vsv. after 2 h inoculation in the presence of the compounds, the inoculum was removed and cells were refed with fresh medium for further culture. the activity of re y luciferase was measured using bright-glo™ luciferase assay (promega) for quantitative determination at 16 h post-transduction. the full-length sars-cov-2 viral rna transcripts were in vitro synthesized from an infectious clone of sars-cov-2 (kindly provided by pei-yong shi, utmb ) according to a recently published protocol 46 . 10 µg of total rna transcripts and 5 μg sars-cov-2 np gene transcript were mixed with veroe6 cells stably expressing sars-cov-2 np protein and then added into a 0.2 cm cuvette for nucleofection with the 4d-nucleofectortm core unit (lonza) using pulse code v-001. immediately after electroporation, 1000 µl of pre-warmed media was added to the cuvette and cells were subsequently aliquoted into 384-well plates. two hours post-seeding, compounds at different concentrations were added into each well. at 12 hours post-electroporation, intracellular and viral rna was puri ed from the treated cells with turbocapture 384 mrna kit (qiagen) in accordance with the manufacturer's instructions. the puri ed rna was subjected to rst-strand cdna synthesis using the high-capacity cdna reverse transcription kit (applied biosystems, inc) with the following primer (tagrdrp-f: 5'-cggtcatggtggcgaataaccctgtgggttttacacttaa-3'). real-time pcr analysis was performed using taqpath 1-step rt-qpcr master mix (applied biosystems, inc). the following primers and probe were used for negative-stranded rna detection: tag-f: 5'-cggtcatggtggcgaataaccctgt-3', orf1ab-r: 5'-acgattgtgc atcagctga-3', orf1ab-p: time-of-addition assay time-of-drug-addition assay was performed to investigate which stage of sars-cov-2 life cycle clofazimine interfered with as previously described 47 . brie y, veroe6 cells were seeded in 96-well plates (4×10 4 cells/well). the cells were infected by sars-cov-2 usa-wa1/2020 at an moi of 1.5 and then incubated for additional 1 h. the viral inoculum was then removed and the cells were washed twice with pbs. at 1 hpi (i.e., post entry), clofazimine at a concentration of 5 µm was added to the infected cells at time-points indicated, followed by the incubation at 37 °c in 5% co2 until 10 hpi (i.e. one virus life cycle). cells were xed at 10 hpi for quanti cation of the percentage of infected cells using an immuno uorescence assay targeting sars-cov-2 np. complete sequences of sars-cov-2 hku-001a and sars-cov-2 usa-wa1/2020 are available through genbank (accession numbers mt230904 (hku-001a), mt246667 and mn908947 (usa-wa1/2020)). the raw rna-seq data reported in figure 3 have been deposited in geo. other supporting raw data are available from the corresponding author upon reasonable request. source data are provided with this paper. foundation limited, tse kam ming laurence, and norman & cecilia yip foundation. this work was also supported by the grants to the sanford burnham prebys medical discovery institute: dod: w81xwh-20-1-0270; dhipc: u19 ai118610; fluomics/nosi: u19 ai135972, as well as generous philanthropic donations from dinah ruch and susan & james blair. this research was also partly funded by crip (center for research for in uenza pathogenesis), a niaid supported center of excellence for in uenza research and surveillance (ceirs, contract # hhsn272201400008c), by darpa grant hr0011-19-2-0020, by an administrative supplement to niaid grant u19ai142733, and by the generous support of the jpb foundation, the open philanthropy project (research grant 2020-215611 (5384)). the funding sources had no role in the study design, data collection, analysis, interpretation, or writing of the report. 63/010630, entitled methods and compositions for antiviral treatment relates to aspects of this work and was led on 15 april 2020. the corresponding authors had full access to all the data in the study and had nal responsibility for the decision to submit for publication. the other authors declare no competing interests. sars-cov-2 and covid-19: the most important research questions clinical course and molecular viral shedding among asymptomatic and symptomatic patients with sars-cov-2 infection in a community treatment center in the republic of korea middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection sars and mers: recent insights into emerging coronaviruses 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large-scale compound repurposing synergyfinder 2.0: visual analytics of multi-drug combination synergies the authors acknowledge the assistance of the university of hong kong li ka shing faculty of medicine centre for panoromic sciences. this study was partly supported by funding to university of key: cord-347706-r0rs3ls1 authors: roberts, anjeanette; subbarao, kanta title: animal models for sars date: 2006 journal: the nidoviruses doi: 10.1007/978-0-387-33012-9_83 sha: doc_id: 347706 cord_uid: r0rs3ls1 nan in 2002-2003, severe acute respiratory syndrome (sars) was a newly identified illness that emerged in southern china, spread to involve more than 30 countries, and affected more than 8000 people and caused nearly 800 deaths worldwide. although the etiologic agent was rapidly identified to be a previously unknown coronavirus (named sars coronavirus or sars-cov) and the outbreak was controlled by public health measures, no specific options were available for prevention and control of human disease. over the past two years, a number of strategies for vaccines and immunoprophylaxis have been investigated. animal models are essential for preclinical evaluation of the efficacy of candidate vaccines and antivirals, and they are also needed in order to understand the pathogenesis of sars. a number of investigators around the world have evaluated several different animal species as models for sars; this effort is important for two natural reservoir, and second, if the efficacy of vaccines cannot be evaluated in humans, efficacy in two or more animal models may be required for licensure. the ideal animal models would be those in which viral replication is accompanied by clinical illness and pathology that resembles that seen in human cases of sars. however, the consequences of sars-cov infection in different animal models may vary from this picture to one in which viral replication is associated with pathology in the absence of clinical illness or models in which viral replication is present in the absence of clinical illness or histopathologic changes. models that demonstrate clinical illness and pathology can be used to study the disease process as well as to evaluate intervention strategies while models in which virus replication occurs without clinical illness can be used in vaccine or antiviral studies. in these cases, the efficacy of an intervention can be assessed by quantitative virology with or without accompanying pathology. a review of the different animal models that have been reported follows with a summary of the pros and cons and potential applications of the different models. cov intranasally (i.n.), the virus replicates efficiently in the respiratory tract (lungs and nasal turbinates) with a peak of viral replication on day 2 postinfection (mean titer in lungs is 10 6 to 10 7 50% tissue culture infectious doses (tcid 50 ) per gram and mean titer in nasal turbinates is 10 5 to 10 6 tcid 50 per gram following administration of 10 6 tcid 50 of sars-cov i.n.). 1 virus is cleared from the respiratory tract by about day 5. virus replication occurs without signs of illness such as weight loss or ruffled fur and is associated with minimal to mild inflammation in the lungs. viral antigen and nucleic acid are present in the epithelial cells of the large airways on day 2 but are being cleared by day 4. 1 when sars-cov is administered to balb/c mice i.n. and orally, viral nucleic acid can be amplified from lung tissue and small intestines. 2 intranasally administered sars-cov also replicates in the lungs of c57bl/6 (b6) mice, with a peak of viral replication on day 3 and clearance of virus by day 9. 3 sars-cov infected balb/c and b6 mice tend to gain less weight than mock-infected mice 2,3 129svev mice also support replication of sars-cov with a self-limited bronchiolitis that begins with mixed peribronchiolar inflammatory infiltrates, progresses to bronchiolitis with migration of inflammatory cells into surface epithelium and interstitial inflammation in adjacent alveolar septae, that resolves completely over the next 2 weeks. 4 sars-cov infected mice develop a sars-cov specific neutralizing antibody response and are protected from reinfection with sars-cov. antibody alone is sufficient to transfer this protection to naïve mice. 1 mice with targeted defects in the immune system were evaluated in order to determine which arm of the immune system was responsible for clearance of sars-cov in mice. beige, cd1 -/and rag1 -/mice replicated and cleared virus with the same kinetics as b6 mice, without overt signs of clinical disease indicating that nk cells, nk-t cells, and t and b lymphocytes are not required for clearance of sars-cov from the lungs of mice. 3 in young mice, sars-cov induces dramatic upregulation of a subset of inflammatory chemokines (ccl2, ccl3, ccl5, cxcl9, cxcl10) and the chemokine receptor cxcr3 without detectable expression of classic proinflammatory and immunoregulatory cytokines (ifn-γ, il-12, p70, il-4, il-10, and tnf-α) and without evoking marked leukocyte infiltration of the lung. taken together with the observation that beige, cd1 -/and rag1 -/mice clear sars-cov normally, proinflammatory chemokines may coordinate a rapid and highly effective innate antiviral response in the lung. 3 in contrast to young (4 to 6-week-old) balb/c mice that support replication of sars-cov in the absence of clinical illness and pneumonitis, old (13 to 14-month-old) balb/c mice demonstrate illness (weight loss, hunching, dehydration, and ruffled fur from days 3 to 6 postinfection) and interstitial pneumonitis. 5 perivascular lymphocytic infiltrates noted at day 3 were more prominent by day 5 and evidence of alveolar damage was seen, with multifocal interstitial lymphohistiocytic infiltrates, proteinaceous deposits around alveolar walls and intraalveolar edema. at day 9, the perivascular infiltrates persisted and the changes associated with alveolar damage were accompanied by proliferation of fibroblasts in inflammatory foci. a few of these foci persisted through 29 days post-infection and may represent the histologic correlate of fibrosis and scarring seen by high-resolution computed tomography in patients who recovered from sars. 5 mice with a targeted disruption of the stat 1 signaling pathway develop severe sars-when 6 to 8-week-old lightly anesthetized balb/c mice are administered sarsprogresses to diffuse interstitial pneumonia with focal airspace consolidation (unpublished 4 infiltrates. at day 27 postinfection, nodules of dense mononuclear inflammation containing sars-cov infected cells were present in the liver. 4 in stat 1 knockout mice and old balb/c mice, sars-cov replicates to higher titer than in young balb/c mice; old balb/c mice recover from the infection. 5 balb/c and b6 mice in the absence of clinical illness and histopathologic evidence of mild inflammation while 129svev mice show some pneumonitis. mice that recover from infection develop a neutralizing antibody response and are protected from subsequent challenge; antibody alone is sufficient to protect mice from replication of sars-cov in the lower respiratory tract and nk, nk-t, t, and b cells are not required for viral clearance. 3 the efficacy of several vaccines and monoclonal antibodies has been evaluated in balb/c mice. [6] [7] [8] [9] [10] [11] [12] morbidity and mortality and pneumonitis are seen in stat-1 knockout mice 4 and old balb/c mice infected with sars-cov. 5 the pathogenesis of disease in these models is under investigation. infection of balb/c mice and hamsters used the urbani strain of sars-cov while the sars-cov isolate from patient 5688 (hku-39849) was used to infect ferrets (mustela furo). when anesthetized ferrets were infected with 10 6 tcid 50 by the intratracheal (i.t.) route, three of six ferrets were lethargic from days 2 to 4 postinfection and one died on day 4. 14 virus was isolated from pharyngeal swabs on days 2 to 8 postinfection and from trachea, lungs and tracheobronchial lymph nodes at necropsy on day 4. when administered i.t. at a dose of 10 4 tcid 50 of sars-cov, the virus replicates efficiently in the lungs to a titer of 10 6 tcid 50 /ml that peaks at 4 days postinfection. 15 465 observations). v iral antigen was present within cells of the inflammatory pulmonary intranasally administered sars-cov replicates efficiently in the respiratory tract of golden syrian hamsters with a peak of viral replication in the lungs on days 2 or 3 (mean titer approximately 10 7 tcid 50 per gram of lung tissue following administration of 10 3 tcid 50 i.n.) and clearance from the lungs by day 10. this is accompanied by histopathologic evidence of pneumonitis. mild mononuclear inflammatory cell infiltrates are noted in the submucosa of the nasal epithelium and bronchioles at day 3 postinfection. as inflammation in the nasal tissues resolves, inflammatory reaction in the lungs progresses, with confluent areas of consolidation that involve 30-40% of the surface of the lung by day 7 postinfection with resolution by day 14. the lung pathology is not associated with overt clinical illness. in contrast to mice in which replication of intranasally administered sars-cov is restricted to the respiratory tract, transient from infection develop a robust neutralizing antibody response and are protected from subsequent infection with sars-cov. there is no clinical, virologic or histopathologic evidence of enhanced disease upon reinfection even in the presence of sub-neutralizing levels of monoclonal antibodies to the sars-cov spike glycoprotein. 13, 13b cov disease, with weight loss and pneumonitis that begins with acute bronchiolitis and in summary, sars-cov replicates efficiently in the respiratory tract of young viremia occurs 1 to 2 days following infection and virus is detected in the liver and spleen in hamsters. however, inflammation is not observed in these organs. hamsters that recover multifocal pulmonary lesions affecting 5-10% of lung surface area include mild alveolar damage and peribronchial and perivascular lymphocyte infiltration. 15, 16 ferrets are outbred animals that are highly susceptible to viruses that they can acquire from their caretakers. ferrets used as models for sars should be screened to ensure that other intercurrent infections do not modify the disease associated with sars-cov infection. in 2003, sars-cov was isolated from captive civet cats in wild animal markets in guangdong province, china, and several civet cats had detectable antibodies to sars-cov. in a recent report, wu et al. 17 infected farmed civet cats with two strains of sars-cov: five animals were infected with the gz01 virus, the prototype of the virus isolated from civet cats, and 5 animals were infected with bj01, a sars-cov strain that is typical of viruses isolated in hong kong during the sars outbreak. bj01 has a 29nucleotide deletion in its genome compared with gz01. the civet cats were infected with a dose of 3 x 10 6 tcid 50 administered i.t. and i.n. in contrast with the observation that sars-cov infected wild civets appeared healthy, lethargy and a decrease in aggressiveness was noted in the experimentally infected farmed civet cats from day 3 onwards, fever from day 3 to 7, and diarrhea and conjunctivitis in 20-40% of animals. the animals had leukopenia at day 3, but white blood cell counts were normal by day 13. interstitial pneumonitis with alveolar septal enlargement and macrophages and lymphocyte infiltration was noted on days 13 to 35; the histopathological findings are reportedly similar to lesions described in sars-cov infected macaques and experimentally infected ferrets. 17 virus was isolated from throat and anal swabs in 60% of the animals on days 3 and 8 and from organs at necropsy on day 3 (n = 1). viral nucleic acid was detected by reverse transcriptase pcr at necropsy in multiple organs at day 3 and in lymph nodes and spleen at days 13, 23, 34, and 35. the findings in experimentally infected farmed civets support epidemiologic observations made in wild-animal markets 18 that point to civets as a potential source for the transmission of sars-cov from animals to humans. however, further research is required to identify the reservoir(s) of sars-cov in nature. among old world monkeys, rhesus, cynomolgus, and african green monkeys have been experimentally infected with sars-cov and have been used for vaccine immunogenicity and/or efficacy studies. the presence and extent of clinical illness reported in these studies have not been consistent and attempts to isolate sars-cov from tissues have also been variably successful (table 1) . clinical illness and histopathologic findings in sars-cov infected cynomolgus monkeys range from reports of no illness and disease 19, 20 to lethargy, temporary skin rash, and respiratory distress progressing to ards, associated with diffuse alveolar damage, extensive loss of epithelium from alveolar and bronchiolar walls, thickening of alveolar walls, hyaline membranes in some alveoli, and occasional multinucleated giant cells and type 2 pneumocyte hyperplasia at days 4 to 6 postinfection. [21] [22] [23] clinical findings in sars-cov infected rhesus monkeys are absent or mild. histopathologic findings are variable, ranging from no abnormalities to patchy areas of mild interstitial edema and alveolar inflammation interspersed with normal lung parenchyma and occasional areas of intraalveolar edema inflammation at day 14, in 1 of 4 animals 19 to acute interstitial pneumonia through 60 day postinfection with infiltration of lymphocytes and macrophages in nodular areas of lungs. 24 the age of animals may be an important determinant of outcome that can be difficult to ascertain in wild-caught monkeys. in african green monkeys, virus infection in the lungs is patchy and is cleared by day 4 post-infection; the titer of virus in respiratory secretions does not accurately reflect the titer of virus recovered from trachea and lung tissue. 20 histopathological examination of lungs shows diffuse alveolar damage and focal interstitial pneumonitis that parallels virus titers in resolution by day 4 postinfection. 20 three species of new world monkeys have also been evaluated as models for sars (table 1) . although squirrel monkeys and mustached tamarins could not be experimentally infected, common marmosets developed fever and watery diarrhea and histologic evidence of multifocal pneumonitis and hepatitis following sars-cov infection. 25 further evaluation of this model is warranted. the detection of viral rna and neutralizing antibody responses clearly demonstrates that several species of nonhuman primates can be experimentally infected with sars-cov. not surprisingly, the extent of disease in outbred animals is more variable than in inbred animals such as mice. as seen with the other animal models, the course of infection in experimentally infected nonhuman primates is short, with a rapid peak in viral replication and clearance of virus from the lungs by days 4 to 7 in different species. histopathologic changes also resolved rapidly in all but one study. 24 the absence of consistently observed clinical illness and the rapid resolution of viral infection and 468 hepatitis at day 7 and associated pulmonary pathology may limit the role of nonhuman primates to studies of the immunogenicity of vaccines and antivirals rather than pathogenesis or efficacy studies. species can support replication of sars-cov with or without accompanying clinical illness or pulmonary pathology. each model has advantages and disadvantages (table 2) but the common themes are that a number of animal species can be infected when sars-cov is delivered into the respiratory tract, the infection elicits a neutralizing antibody response and the animals are protected from subsequent infection. in comparing reports, readers should bear in mind that the virus used, inoculum and route of virus administration and age of the animal may represent important differences. in studies in mice, the age of the animals and use of anesthesia clearly affect the course of infection. the characteristics of each model should be taken into consideration in determining their utility and application. table 3 lists potential uses of the models discussed above. we thank our colleagues leatrice vogel, elaine lamirande, josephine mcauliffe, brian murphy, jun chen, jadon jackson, and aaron cheng at the nih, sherif zaki, christopher paddock, jeannette guarner, and wun-ju shieh in the infectious disease pathology activity at the cdc, and marisa st. claire at bioqual inc. who have participated in our efforts to evaluate several animal models for sars. this research was supported in part by the intramural research program of the niaid, nih. prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice mice susceptible to sars coronavirus mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice neutralizing antibody and protective immunity to sars coronavirus infection of mice induced by a soluble recombinant polypeptide containing an nterminal segment of the spike glycoprotein rapid and efficient isolation of human monoclonal antibodies neutralizing sars coronavirus from human memory b cells a dna vaccine induces sars coronavirus neutralization and protective immunity in mice evaluation of human monoclonal antibody 80r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based severe acute respiratory syndrome coronavirus infection of golden syrian hamsters sars virus infection of cats and ferrets human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets virology: sars virus infection of cats and ferrets syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and virus burden in golden syrian hamsters civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates isolation and characterization of viruses related to the sars coronavirus from animals in southern china macaque model for severe acute respiratory syndrome replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys aetiology: koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques an animal model of sars produced by infection of macaca mulatta with sars coronavirus pneumonitis and multi-organ system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus key: cord-347460-9vechh4x authors: chang, feng-yee; chen, hsiang-cheng; chen, pei-jer; ho, mei-shang; hsieh, shie-liang; lin, jung-chung; liu, fu-tong; sytwu, huey-kang title: immunologic aspects of characteristics, diagnosis, and treatment of coronavirus disease 2019 (covid-19) date: 2020-06-04 journal: j biomed sci doi: 10.1186/s12929-020-00663-w sha: doc_id: 347460 cord_uid: 9vechh4x on march 11, 2020, the world health organization declared the worldwide spread of the infectious disease covid-19, caused by a new strain of coronavirus, sars-cov-2, as a pandemic. like in all other infectious diseases, the host immune system plays a key role in our defense against sars-cov-2 infection. however, viruses are able to evade the immune attack and proliferate and, in susceptible individuals, cause severe inflammatory response known as cytokine storm, particularly in the lungs. the advancement in our understanding of the mechanisms underlying the host immune responses promises to facilitate the development of approaches for prevention or treatment of diseases. components of immune system, such as antibodies, can also be used to develop sensitive and specific diagnostic methods as well as novel therapeutic agents. in this review, we summarize our knowledge about how the host mounts immune responses to infection by sars-cov-2. we also describe the diagnostic methods being used for covid-19 identification and summarize the current status of various therapeutic strategies, including vaccination, being considered for treatment of the disease. on december 31, 2019, a cluster of cases of pneumonia was announced in wuhan, hubei province, china. subsequently, on january 7, 2020, the chinese health authorities confirmed that this cluster was associated with a novel coronavirus, ncov, which was later named as sars-cov-2, and the ensuing disease was named covid-19. the covid-19 outbreak by the new coronavirus strain was recognized as a pandemic by the world health organization (who) on march 11, 2020. throughout history, there have been a number of pandemic diseases; the more notable and recent ones caused by viruses include the influenza pandemic (spanish flu) in 1918 and another by the influenza virus h1n1 in 2009. the immune system clearly plays a key role in the host defense against the infectious agents during these pandemics. the host is able to mount immune responses upon infection by viruses, as well as other microbes, and control the spread of these pathogens within the body. however, some viral strains are capable of evading the immune attack and proliferate in the body, as well as elicit inflammatory responses, in particular in the lungs, resulting in pneumonia. more importantly, in susceptible individuals, viruses can cause massive inflammatory responses, known as "cytokine storm", resulting in a severe pathological consequence. the advancement in our understanding of the mechanisms of the host immune response are crucial to development of approaches for prevention and treatment of these fast spreading and devastating infectious diseases. the components derived from our immune systems, such as antibodies, can be used to develop sensitive and specific methods for the diagnosis of infectious diseases, as well as novel therapeutic modalities. in this review, we briefly summarize our knowledge about the host immune response upon infection by sars-cov-2. we also discuss the epidemiological aspects of the outbreak, and the potential mechanism of the severe host response, such as cytokine storm. we also describe the antibody-based approaches for diagnosis of covid-19 infection and summarize the current status of various preventive and therapeutic modalities for treatment of the infection. coronaviruses are single-stranded enveloped rna viruses that cause diseases in mammals and birds. in humans, the low pathogenicity strains, including hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku, infect the upper respiratory tract and cause mild to moderate common cold-like symptoms in healthy individuals. they are responsible for 15-30% of all common cold cases. the highly pathogenic strains, including those causing severe acute respiratory syndrome [sars-cov] , middle east respiratory syndrome [mers-cov] , and covid-19 [new sars-cov-2], infect the lower respiratory tract and can cause severe pneumonia [1] . in addition to their rna genetic material, coronaviruses are composed of nucleocapsid (n) and spike (s) proteins, which participate in viral genome assembly, transcription and replication, or mediate viral entry and cause cytopathic effect [2, 3] . the s protein mediates the fusion of viral and host membrane [4] and contains a receptor-binding domain (rbd) that attaches to cells during viral entry. angiotensin-converting enzyme 2 (ace-2) is the receptor for both sars-cov and sars-cov-2 [5] . notably, the four human coronaviruses that cause common cold like symptoms show limited sequence homology in their n (30-67%) and s proteins (9-57%) compared with those of sars-cov-2 [6] . mers-cov also exhibits more distal relationship to sars-cov and sars-cov-2. however, the latter two are more closely related, with their n and s proteins sharing high homology (70-90%) . in 2003, the sars-cov infection, which started in southern china, led to an epidemic; in total, over 8400 cases were reported, which included close to 900 deaths with a case fatality rate of 11% [7] . in 2012, the first case of mers took place in saudi arabia. from that moment on, close to 2500 cases have been reported globally, which included close to 860 deaths, with an estimated case fatality rate of approximately 34% [8] . evidence is mounting that covid-19 spreads via human-to-human transmission of the virus [9] . after exposure to sars-cov-2, the majority of patients recover with little or mild symptoms that include cough and fever [10] . however, it is estimated that approximately 20% of infected individuals develop severe disease, including acute respiratory distress syndrome (ards). according to who, as of april 22, 2020, a total of 2,503, 072 confirmed cases of covid-19 have been detected and 171,791 deaths resulting from the infection have been confirmed worldwide. the case fatality rates in wuhan and worldwide were approximately 4.5 and 6.9%, respectively. the numbers are lower in some countries, for example approximately 5.4% in the united states, 2.2% in south korea, 3.3% in germany, and 1.4% in taiwan. these numbers are likely affected by the extent of screening, thereby implying that covid-19 cases might be underdiagnosed in many other countries. the availability and infrastructure of medical facilities in afflicted countries, especially those seriously affected ones, also likely affect the overall death rates. using public and published information, wu et al. estimated that the overall "symptomatic case fatality risk" (the probability of dying after developing symptoms) associated with covid-19 was 1.4% [11] . the rate of development of severe symptoms and death are clearly associated with the age. it is to be noted this is based on all tested and confirmed cases of covid19, and the true fatality risk is likely lower than 1.4%, since many mildly symptomatic/asymptomatic people might have never got tested. nevertheless, the risk is still higher than that associated with seasonal influenza virus, which is approximately 0.1%. with regard to the spectrum of the severity of the disease, chinese center for disease control and prevention reported of the 44,672 confirmed cases (with the age distribution of > 80 years: 3%; 30-79 years: 87%; 20-29 years: 8%; 10-19 years: 1%; < 10 years: 1%), the spectrum of disease were: mild: 81%; severe: 14%, critical: 5%; and case fatality rate: 2.3% [12] . finally, the basic reproduction number (ro) of the virus has been estimated to be between 1.4 and 3.9, meaning each infection from the virus can result in 1.4 to 3.9 new infections, when no members of the community are immune and no preventative measures, such as vaccination, are taken. by comparison, the median ro value for sars-cov was in the range of 2 to 4 and for 1918 influenza was 1.80. the outcome of clinical infection likely largely depends on the capacity in mounting effective antiviral immune responses in time, to control viral spreading, to limit organ injuries and to speed up recovery. here we summarize the immune response induced by cov. three components are crucial for sars-cov induced diseases: 1) the role of cd8+ t cells in defense against the virus, which causes apoptosis in the infected cells, 2) interactions of the virus with macrophages and dendritic cells, which initiate the early innate and subsequent adaptive immune responses, and 3) type i interferon (ifn) system, an innate response against viral infections, which can inhibit virus replication in the early phase. firstly, the central part of the body's anti-viral immunity is based on the interaction between antigen and antigen presentation cells (apc) when the virus enters the cells. the infected cells are recognized by virus-specific cytotoxic t lymphocytes (ctls) via viral peptides as the antigen presented by major histocompatibility complex (mhc). the antigen presentation of virus mostly depends on mhc i molecules, but mhc ii also has its contribution in some cases. the mhc i molecules display pieces of virus proteins on the surface of infected cells, which creates a signal to activate nearby cd8+ t cells to induce apoptosis in the infected cells. there are many reports on the relationship between various mhc polymorphisms and the susceptibility to sars-cov [13] [14] [15] , but little is known about this association in covid-19. such information could provide beneficial aspects of personalized medicine for treatment or prevention of covid-19. secondly, dendritic cells and macrophages are other first patrolling components of innate immune network, which play important roles in driving both innate and adaptive immune responses to the viral pathogens [16] . the invasion of viruses can be recognized by innate immune cells via pathogen-associated molecular patterns (pamps). in the case of cov, pamps are viral genomic rna, which are recognized by endosomal rna receptors such as tlr3, tr7, tr8, and tlr7 [17] . this can cause rapid responses of the innate immune cells to viruses, resulting in production of a large amount of type i ifn with antiviral functions. lastly, efficient innate immune responses against viruses also depend on type i ifn responses and downstream cascade. type i ifn, by directly interfering with the viruses' replication ability, can prevent reproduction of viruses in infected cells. by mounting type i ifn responses successfully, viral replication and dissemination in an early stage are suppressed. sars-cov and mers-cov use several strategies to avoid the innate immune response, these are probably also employed by sars-cov-2. these include the inhibition of type i ifn recognition and signaling, as well as downregulation of mhc class i and class ii molecules in infected macrophages or dendritic cells, resulting in impaired antigen presentation and diminished t cell activation. moreover, some proteins encoded by sars-cov can interact with the signaling cascades downstream of the pattern recognition receptors. after exposure to sars-cov-2, patients respond to the virus by generating specific igm antibodies within a few days, followed by specific igg production within a week [3, 6, 18] . in the case of sars-cov infection, although the serum anti-viral igm antibody levels decline in a few months, the antiviral igg antibody titers can persist for years. among the many structural and non-structural proteins encoded by sars-cov-2, the n and s proteins are the most immunogenic antigens. antibodies against the n protein are the first to appear and thus can serve as an early and reliable serum marker for virus exposure, whereas antibodies against the s protein develop later and can bind to the viral envelope. recent studies indicated that the convalescent serum contains antibodies that can neutralize sars-cov-2 in cell cultures [2, 3] . therefore, igg against the s protein is both a marker for viral exposure and an indicator of recovery. the potential risk of disease exacerbation by ade, a phenomenon in which pre-existing poorly neutralizing antibodies lead to enhanced infection, has been a serious concern for vaccine development and antibody-based therapeutic strategy. compared to the ade in dengue viral infections, which was supported by a great deal of epidemiological and clinical evidence in the past four decades [19] , this phenomenon in coronaviruses has mainly been observed in cell-based experimental models [20, 21] . to illustrate this further by also using dengue virus as an example: while more severe symptoms, such as dengue hemorrhagic fever (dhf)/dengue shock syndrome (dss), can be observed during primary infection, they are much more frequently developed following a secondary infection with a different serotype (out of the four existing serotypes) [19] . furthermore, it has been well documented that the high level of virus replication seen during secondary infection with a heterotypic virus is a direct consequence of ade of viral replication. this is mediated primarily by the pre-existing, non-neutralizing, or sub-neutralizing antibodies to the virion surface antigens, resulting in enhanced access to target cells, through binding of the virion-antibody complexes to igg fc receptors (fcγr) on these cells [19] . this common underlying theme of ade-based magnification of virus replication also indicates that severe disease is not merely attributable to inherent virulence of virus [22] . interestingly, as described in a later section, several available evidence from the use of convalescent sera in patients with sars, mers [23] and 245 cases with covid-19 [24] suggest the feasibility and safety of convalescent serum trials. here, caution and vigilance to identify any evidence of enhanced coronavirus infection by ade will be required. despite the fact epidemiological and clinical observations supportive of existence of ade in coronavirus infection is not available, a molecular mechanism behind ade of coronavirus has currently been provided [21] . the authors demonstrated that a neutralizing antibody binds to the s protein of coronaviruses like a viral receptor, triggering a conformational change of the spike and mediating a viral entry into fcγr -expressing cells through canonical viralreceptor-dependent pathways. however, an enhanced entry of these pseudovirus-based approaches does not support directly the magnification of viral replication in these cells. compared to dengue viruses, these fcγrbearing cells such as dendritic cells, monocytes and macrophages, even if infected through ade process, would presumably not be so "permissive" for coronaviruses, in terms of their replication and assembly. however, these viewpoints need further investigation. apparently, many host factors could also exacerbate disease during secondary infection. these host factors need to be identified by combined epidemiological and genetic analyses of appropriate patients, and the contribution of underlying host factors to the control of coronavirus replication needs to be determined. for example, ade of replication can possibly occur with the vaccine strains of viruses in the endemic populations, such as attenuated or recombinant coronavirus vaccines. however, the level of replication will likely remain low, and thus this small enhancement of replication will probably not augment the disease. in addition, this could result in heightened vaccine immunogenicity due to a small increase in the virus load. however, further investigation on correlations between immunological responses and disease outcome and the validation of these findings in vaccine trials will be invaluable for developing safe and effective sars-cov2 vaccines (see below). while sars-cov can evade innate immune system, they can also induce intensive inflammatory reactions through innate immune cells. in fact, sars-cov and sars-cov-2 infections are known to activate a massive over-production of cytokines by the host immune systema phenomenon known as "cytokine storm", which usually occurs a few days after the onset of the illness. this also results in increased local and systemic vascular permeability in major organs. cytokine storm is reported in many viral infections, and contributes significantly to the pathogenesis and severity of acute viral infections. the tissue tropism of each virus determines the cytokine profiles of virus-induced cytokine storm (reviewed in [25] ). for example, macrophages produce a higher amount of proinflammatory cytokines than endothelial cells, while virus-infected endothelial cells are the major source of chemokines. however, this may not be generalizable, and it is crucial to elucidate the tropism of sars-cov-2 in order to interpret the data. most proinflammatory cytokines are released from macrophages and severe acute infections are usually associated with the activation of macrophages by enveloped viruses. in addition, activation of neutrophils may also be involved. as mentioned above, viral nucleic acids can induce the production of interferons and proinflammatory cytokines, through engaging endosomal tlrs. these intracellular nucleic acid receptors/sensors have been defined as "protective host factors", as they are critical for host defense against viral infections. however, the identity and contribution of "pathogenic host factors" to virus-induced severe inflammatory reactions and lethality, and how different viruses cause distinct clinical symptoms, remain unclear. some available information related to the cytokine storm induced by sars-cov and mers-cov is summarized below. it is to be noted that while both sars-cov and sars-cov-2 utilize ace-2 as their receptors, mers-cov binds to the receptor dipeptidyl peptidase 4 (dpp4/cd26). the differences in receptor usage may account for the differences in disease patterns, including the organs involved and the extent of the cytokine storm induced. [26] [27] [28] . the clinical course of this infection has three phases: 1) robust virus replication accompanied by fever in the first few days; 2) high fever and pneumonia with progressive decline of virus titers; 3) ards resulting from active host immune responses in the absence of detectable viruses [1] . in addition to infecting and proliferating in the airways and alveolar epithelial cells, sars-cov can also infect dendritic cells, monocytes, and macrophages, without undergoing proliferation (i.e., abortive infection) [29] . sars-cov-infected epithelial cells produce high levels of chemokines such as ccl2, ccl3, ccl5, and cxcl10. in addition, sars-cov-infected dendritic cells [30, 31] and macrophages [29] secrete high levels of proinflammatory cytokines tnf and il-6, and significant amounts of chemokines. it is interesting to note that higher levels of il-1, il-12, ifn-gamma, il-8, and cxcl9, in addition to the cytokines and chemokines mentioned above, were also observed in sars patients with severe diseases. this suggests that other cell types also contribute to sars-cov-induced cytokine storm. the typical pathological changes in the lungs include focal hemorrhage and mucopurulent materials in bronchial trees with diffuse alveolar damage. histological examination shows extensive macrophage and neutrophil infiltration with lower levels of t lymphocytes. existing information suggests that the sars-cov-infected airways and alveolar epithelial cells secrete abundant chemokines to attract immune cell infiltrations to the lungs, including macrophages and neutrophils, thereby causing damage due to high levels of proinflammatory cytokines and other mediators secreted by these cell types. in addition to the airway epithelial cells, mers-cov can also replicate in human monocytes, macrophages, dendritic cells, and activated t cells. the typical lung pathological changes caused by mers-cov is diffuse alveolar damage. in addition, pleural and pericardial effusions associated with generalized congestion and consolidation of lungs have been noted [32] , and the severity of lung lesions were noted to be correlated with extensive infiltration of neutrophils and macrophages [32] . similar to sars-cov, mers-cov can induce high levels of proinflammatory cytokines and chemokines in human monocyte-derived macrophages and dendritic cells. mers-cov infection was also reported to induce increased concentrations of proinflammatory cytokines (ifn-γ, tnf-α, il15, and il17), [33] . the high serum cytokine and chemokine levels in mers patients were correlated with increased infiltration of neutrophil and monocytes along with severe tissue damage in the lungs [32, 34, 35] . thus, the pathological change in the lungs is similar between sars-cov and mers-cov. whereas, the higher mortality rate in mers-cov-infected patients may be due to the higher incidence of pericarditis in infected patients. the first autopsy of covid-19 victims along with immuno-histological staining revealed the presence of sars-cov-2 in the airway epithelia and macrophages, suggesting that the virus can infect both epithelial cells and macrophages [36] . the majority of infiltrating cells are macrophages and monocytes with moderate amounts of multinucleated giant cells and neutrophils. increased levels of cytokines and chemokines, including il-2, il-7, g-csf, m-csf, ifn-γ, ip-10, mcp-1, mip-1α, and tnf-α, were detected in the plasma of covid-19 patients [37] . the most significant predictors of mortality in these patients are serum ferritin level and il-6, suggesting that mortality is due to virus-induced hyperinflammation and cytokine storm during viral infection [38, 39] . compared to the low pathogenic coronaviruses, the common features of high pathogenic coronaviruses include extensive infiltration of leukocytes, which secrete abundant proinflammatory cytokines and other chemical mediators to cause diffuse alveolar damage. also, high pathogenic viruses are associated with abortive infection; for example, in contrast to the less pathogenic strain of influenza virus h1n1, the highly pathogenic influenza virus h5n1 does not replicate in macrophages; the latter is also a more potent inducer of the chemokine cxcl10 [40] the key innate immunity receptors/sensors responsible for high pathogenic coronavirus-induced proinflammatory cytokines are still unclear. finally, notably, more deaths from covid-19 have been caused by multiple organ dysfunction syndrome rather than respiratory failure, which is different from infections caused by sars-cov and mers-cov; the basis for this remains unknown. detection of viral rna in the secretions from the respiratory tract of infected patients by reverse transcription-polymerase chain reaction (rt-pcr) test is currently the standard method for diagnosis of covid-19. they have some limitations, including long turnaround time (typically 2-4 h) and the requirement of specialized facilities. scientists around the world have been devoting effort to developing improved nucleic acid-based, simpler and faster methods. the us fda issued an emergency-use authorization to cepheid's xpert xpress sars-cov-2 test, which became the first pointof-care covid-19 diagnostic test to receive this designation in the us. the test is designed to use the company's automated genexpert systems and has a turnaround time of approximately 45 min. another prominent example is a method for detection of sars-cov-2 by using crispr technologies [41] . these newly developed platforms will clearly require clinical testing before approval for routine use. tests based on antibodies are obviously another option for diagnosis and screening. immediately after infections, viral genome and proteins start to increase, becoming the earliest markers for diagnosis within days. as the host immune responses gear up to confront and reduce viral replications, the viral rna or antigen level declines, but the antiviral igm and igg titers rise up. as patients usually present themselves with cough, fever or shortness of breath, and are already beyond the early stage of infection [10] ; here, nucleic acid tests are expected to pick up only a proportion of patients. a complementary serological test for specific igm or igg will help identify the rest. one report from shenzhen studied 173 patients within 7 days of illness, who were later diagnosed with covid-19 [42] ; in this study rt-pcr could detect two-thirds of the patients, but only 45% tested positive even after 15 days. in contrast, although the serological positive rate within 1 week was less than 40%, the rate increased to 100% after day 15 of disease onset. a combination of nucleic acid and serological test significantly increased the diagnosis rate from 66 to 78% even within 1 week of illness [42] . if these results can be validated, such a combination may become a standard clinical practice in the future. in this regard, li et al., developed an immunoassay that can detect igm and igg antibodies against sars-cov-2 in human blood within 15 min. they tested samples from close to 400 confirmed patients and over 100 negatively-tested patients at 8 different clinical sites and reported a sensitivity of over 88% and specificity of over 90% [43] . in the early stage (containment) of covid-19 pandemic, there was a strong interest for rapid diagnosis and thus prototypes of rapid viral antigen or antibody tests were being developed for point-of-care use. these platforms have the advantage of convenience and a fast turnaround time, but suffer from inadequate sensitivity and specificity, as compared with standard rt-pcr. hence, their results need to be interpreted with caution. preparation of high-quality antibodies and antigens requires years in perfecting such point-of-care tests, judging from the experience in developing such tests for influenza viruses. eventually, the more stringent criteria for a definitive diagnosis will need paired serum samples to demonstrate a true seroconversion [6] . finally, the issue of antibody cross-reactivity with other human coronaviruses warrants discussion. as mentioned above, serological assays usually adopt viral n or s protein as antigens and the protein components from all four human coronaviruses that cause common cold show very limited sequence homology with those of sars-cov-2. thus, despite the fact the majority of the populations have been exposed to the four low pathogenic human coronaviruses, their sera do not react positively in sars-cov-2 elisa [6, 18] . while mers-cov is also more distally related and presents no concerns, the situation for sars-cov-exposed patients is different. as the n and s proteins of the sars-cov strain share high homology (70-90%) with those of sars-cov-2, serum from sars patients can actually cross-react with sars-cov-2 n or s protein in immunoblot or viral neutralization assays [2, 3] . however, as sars-cov epidemic was only transient with a very small proportion of the populations being exposed, this cross-reactivity should not be an important issue. as with many vaccine-preventable viral diseases like measles and chicken pox, the newly emerged sars-cov-2 infection assumes an epidemiological characteristic capable of evading containment measures and facilitating pandemic potential. thus, a high proportion of undetected infections with mild or no symptoms can efficiently sustain viral transmission [44] . while containment and lockdown can serve as temporary control measures, effective vaccines or therapeutic agents are much needed for the ultimate control of the disease. the vaccine research and development thus far have progressed at an unprecedented speed; the first dose of rna-based sars-cov-2 vaccine was administered to test its safety in humans on march 16, 2020, only 2 months after the new virus was first identified. such rapid progress was facilitated by a combination of multiple factors, including advances in vaccine research on sars and mers, as recently reviewed [45] , progress in a number of vaccine technology platforms to the early stage of human trial [46] [47] [48] , and readily available support from the well-orchestrated international collective effort of the coalition for epidemic preparedness innovations (cepi) [49] . the aforementioned innovative aspects that could potentially drive sars-cov-2 vaccine development to market launch within a year or two are summarized below. the coronavirus s protein is a critical target for antiviral neutralizing antibodies and functions to mediate entry into mammalian cell expressing the viral receptor ace2 [3, 50] . moreover, the target neutralizing epitope of sars-cov was further narrowed to a smaller fragment of the s protein, later termed receptor binding domain (rbd) [51] . building on these paradigmatic scientific advances that the s protein is the putative target antigen, sars-cov-2 vaccine candidates are designed to include full or various lengths of the s protein focusing on the rbd. vaccine based on the whole virus may be less preferred due to its association with eosinophilic pulmonary pathology [52, 53] . a number of novel vaccine platforms, including vector-, dna-, and rna-based vaccines, are being developed or improved with innovative technology specifically to combat pandemic-prone outbreaks and have been recently reviewed [54] . nucleic acid vaccines, including both dna and rna, offer the potential to accurately express any protein antigen in host cells and to present the antigen closely resembling antigen expression and presentation during viral infection. dna vaccines against mers and rna vaccine against h7n9 have completed phase i trials that showed these platforms to be safe [46] [47] [48] . the nucleic acid vaccine can be completely synthetic and formulated within a few weeks at sufficient quantities to support clinical trialsa valuable feature when facing potential pandemic. vector-based vaccine consists of a target antigen inserted into a viral genome to render faithful antigen generation, targeting and processing in vivo after vaccination. the first ebola vaccine approved by us fda is a vector-based vaccine using vesicular stomatitis virus expressing a surface glycoprotein of ebola [55] , thus supporting the applicability of this platform in combating emerging infectious diseases. these platforms offer versatile adaptation for antigen of new diseases, and the process development for production is relatively simple and quick, indicating the value of these platforms for the urgent response to new diseases. the rapid infusion of funding from and coordination by cepi in january 2020 was the major driver for the speed of sars-cov-2 vaccine r&d progress (fig. 1) . with its mission being "to stimulate, finance, and coordinate the development of vaccines for epidemic diseases" especially aiming to drive vaccine innovation for highpriority public health threats, cepi has supported a number of "technology platforms". these included a vaccine printer, molecular clamp technology for protein production, and a self-amplifying rna vaccine platform since 2017 (cepi web page https://cepi.net/research_ dev/technology/). an innovated vaccine platform technology pertains to a system that uses the same basic core technological components as a backbone and can be adapted by inserting new genetic or protein sequences to target newly emerging pathogens. it is with the application of this concept that an rna-based sars-cov-2 vaccine, built on the avian flu vaccine platform [46] , is expected to be developed and quickly proceed to human trial within only a few months since covid-19 became an epidemic, and a dna-based vaccine candidate modeling that of mers is soon to follow [46] . the cepi coordinated effort encompassing a wide range of available technologies from industry and research institutes globally has shown preliminary success toward an urgent response to control a pandemic. while coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines also present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology [53, 56] ; this seems to be either exacerbated or eliminated by the formulation of adjuvant selection depending on the th1/th2 bias and induction of durable ifn-γ responses, respectively [57] . in addition, ade, described above, was seen in the lungs of macaques after administration of inactivated sars-cov vaccine or vaccine composed of certain s antigen fragments [58] . these studies highlight the importance of designing the target antigen and selection of adjuvants to ensure both efficacy and safety. considering the novel nature of sars-cov-2 and that an animal model has yet to be established for testing of the vaccine to especially focus on the immunopathological perspectives, the safety concern is anticipated to present most of the hurdles in the development process. herd immunity refers to a state when sufficient proportion of a population becomes immune to sars-cov-2, via natural infection or vaccination, so as to eventually halt further spread of disease, and thus individuals not immune to the virus are protected. in the case of covid-19, the herd immunity was estimated to be 60% of the population. before vaccines are available for mass immunization, the strategy of achieving herd immunity via natural infection has been considered and deemed not advisable when a number of factors were considered. thus, self-isolation and social distancing remain crucial in combating this pandemic so that the initial pressure on our healthcare systems is reduced, and more time is given to us to develop vaccines or effective therapies. the disease spectrum of covid-19 can be divided into mild infection, pneumonia, ards, and even multiple organ failure [37] . after a decade of research on coronavirus, unfortunately, still there are no licensed vaccines, effective specific antivirals, nor drug combinations supported by high-level evidence to treat the infection, especially for newly emerging strains such as sars-cov-2 [59] . several strategies are being considered for the treatment of covid-19, including the use of antimicrobial agents, immunotherapy with virus-specific antibodies in convalescent plasma, monoclonal and polyclonal antibodies produced in vitro or genetically modified antibodies, and interferons. here we focus on immunebased therapies, but for the sake of completeness, we also include therapies using antimicrobial agents as 7supplementary information. the potential interventions for sars-cov infection are summarized in table 1 . biologic drugs composed of monoclonal antibodies (mabs) have been developed for treatment of a variety of diseases. it is thus not surprising that this approach is being considered for the treatment of sars-cov infection and shows promise. a human igg1 mab, cr3022, that binds to sars-cov s protein has been developed [70] . sui et al. found one recombinant human mab (single-chain variable region fragment, scfv, 80r) against the s1 domain of s protein of sars-cov from two nonimmune human antibody libraries. the mab could efficiently neutralize sars-cov and inhibit syncytia formation between cells expressing s protein and those expressing the sars-cov receptor ace2 [71] . a human igg1 mab, cr3014, has been generated and found to be able to neutralize sars-cov and shown to be able to prevent sars-cov infection in ferrets [72] . more recently, ju et al. reported the isolation and characterization of 206 rbd-specific mabs derived from single b cells of eight sars-cov-2 infected individuals [79] . for clones from one patient they demonstrated their ability to neutralize live sars-cov-2. none of these antibodies cross-reacted with rbds from either sars-cov or mers-cov, although the patient plasma exhibited such cross-reactivity. these neutralizing antibodies have the potential to be used for prophylaxis for or treatment of sars-cov-2 infection. agents that directly block the binding of s protein to the functional receptor ace2 also have the potential to be used for prevention of covid-19. guillon et al. demonstrated that binding of sars-cov s protein to ace2 could be inhibited by anti-histo-blood group antibodies, presumably because the virus carries histo-blood group antigen structures of the host [4] . while whether this approach can be developed into effective treatment strategies is uncertain, the findings have a bearing on the effect of the naturally occurring anti-histo-blood group antibodies on the individual variations in susceptibility to sars-cov infection. convalescent plasma can be employed for passive immunotherapy and is usually chosen when there are no specific vaccines or drugs available for emerging infection-related diseases. yeh et al. reported a favorable outcome in the use of convalescent plasma for treatment of sars-cov-infected healthcare workers [73] . arabi et al. tested the feasibility of convalescent plasma therapy as well as its safety and clinical efficacy in critically ill patients suffering from mers-cov infection [74, 80] . if available, convalescent plasma could certainly be considered for the treatment of sars-cov-2-infected critically ill patients. interferons (ifns), including ifn-α and ifn-β, are produced during the innate immune response to virus infection and they are able to inhibit the replication of virus in vitro [75, 81] . as mentioned above, ifn transcription was blocked in tissue cells infected with sars-cov. recombinant ifn-α given on 3 days before the infection could reduce viral replication and lung damage, as compared with the control in monkeys and in a pilot clinical trial [82] . ifn-α inhalation can also be considered. combination of interferon-α-2a with ribavirin was used in treatment of patients with severe mers-cov infection and the survival of these patients was improved [76, 83] . these findings suggest that these fda-approved ifn's could be used for the treatment of covid-19. as mentioned above, cytokine storm is the major underlying pathology in severe cases of covid-19. thus, neutralization of some of the major cytokines are considered as a novel approach for treatment of severely ill cases and reducing morbidity and mortality. huang c et al. reported that increased il-1ß, ifn-γ, ip-10, and mcp-1 in sars-cov-2 infection and higher concentrations of g-csf, ip-10, mcp-1, mip-1a, and tnf-α were found in patients requiring treatment at icu than those not treated at icu [37] . they also noted that cytokine storm was associated with disease severity. conti p et al. reported that pro-inflammatory cytokines of interleukin (il)-1β and il-6 in mild and acute respiratory syndrome are associated with development of lung fibrosis in covid-19 [77] . thus, suppression of proinflammatory il-1 family members and il-6 might have a potential therapeutic effect. il-37, an immunosuppressive cytokine, acts on mtor and increases the activity of adenosine monophosphate kinase, which inhibits protease inhibitors nelfinavir cell a selective post-translational inhibitor [65] nucleotide analog prodrug remdesivir cell and clinical use (first case of covid-19 in the united states) possible inhibitor of rna replication [66, 67] indole-derivative molecule arbidol cell inhibits fusion between viral envelope and cellular membranes [68] immunosuppressive agent cyclosporine a cell block replication via inhibition of nucleocapsid protein [69] monoclonal antibody cr3022 cell and clinical use potently binds the receptor binding domain of s protein [70] monoclonal antibody single-chain variable region fragments, scfv, 80r cell acts against the s1 domain of s protein [71] monoclonal antibody cr3014 cell neutralization of viral infectivity [72] immunotherapeutic potential convalescent plasma cell and clinical use neutralization of viral infectivity [73, 74] interferons ifn-α and ifn-β cell induction of interferon-stimulated genes to suppress viral replication [75, 76] cytokine blocker cytokine il-37 cell inhibits inflammation, by acting on mtor and increasing the activity of adenosine monophosphate kinase [77] cytokine blocker lianhuaqingwen cell anti-inflammation; inhibits il-6 receptor [78] cytokine blocker antibody against il-6 receptor clinical use anti-inflammation; inhibits il-6 receptor inflammation by suppressing production of multiple cytokines downstream of myd88, including il-1β, il-6, tnf and ccl2. il-37 might be a potential therapeutic cytokine for inhibition of inflammation in covid-19 [77] . runfeng l et al. demonstrated that lianhuaqingwen, a traditional chinese medicine, significantly inhibited sars-cov-2 replication by suppressing mrna of il-6 and other pro-inflammatory cytokines in vero e6 cells [78] . cytokine blocker that target interleukin 6 receptor in covid-19 could be potentially developed as therapeutic agents in future. in fact, fda approved mab against il-6 receptor (il-6r) is available for treatment of rheumatoid arthritis. the society for immunotherapy of cancer has issued a statement on access to il-6-targeting therapies for covid-19. it is encouraging that pharmaceutical companies have in fact initiated clinical trials of anti-il-6r for treatment of patients with severe covid-19. there are still a large number of unanswered questions. how fast sars-cov-2 would mutate and would the mutated virus become more infectious or invasive. according to andersen et al. [84] , viruses constantly mutate, but those mutations do not typically make the virus more virulent or cause more serious disease. in fact, most mutations are detrimental to the virus or have no effect. there was a study of the sars-cov in primate cells suggesting that a mutation in this viral strain acquired during the 2003 sars outbreak probably reduced virulence of the virus [85] . another issue is whether sars-cov-2, unlike sars-cov and mers-cov will continue to cause epidemic or even behave like seasonal flu. in fact, we have already witnessed the second wave of outbreak occurring in north america and europe, after the first wave that occurred in asia, and covid-19 may bounce back-and-forth between north and south hemispheres, as influenza virus does each year. finally, factors that determine the individual susceptibility to covid-19 remain to be elucidated. as mentioned above, there are many reports on the relationship between various mhc polymorphisms and the susceptibility to sars-cov. also, what governs the development of severe illness, including cytokine storm, besides the pre-existence of certain diseases and age factor, awaits clarification. despite these uncertainties, scientists in academia and industry around the world have moved at an unprecedented speed to develop improved methods for detection of the virus and treatment of the disease. advancement in immunology over the years has certainly facilitated many of these developments. we shall witness some of the recent advancements in development of vaccines and biologics for treatment of various other serious illnesses being used for fighting against covid-19. we are hopeful these efforts will be sustained even after the pandemic is over, allowing us to be even more ready in the unfortunate event that another epidemic or pandemic, like covid-19, takes place in the future. supplementary information accompanies this paper at https://doi.org/10. 1186/s12929-020-00663-w. additional file 1. supplemental 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a study protocol prevention of experimental coronavirus colds with intranasal alpha-2b interferon interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review the proximal origin of sars-cov-2 attenuation of replication by a 29 nucleotide deletion in sars-coronavirus acquired during the early stages of human-to-human transmission publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank dr. ming-hsiang hong for his assistance in preparation of the manuscript. authors' information none. not applicable.availability of data and materials not applicable. consent for publication not applicable. the authors declare that they have no competing interests. key: cord-350505-uh8r2vyz authors: kalantar-zadeh, kourosh; ward, stephanie a.; kalantar-zadeh, kamyar; el-omar, emad m. title: considering the effects of microbiome and diet on sars-cov-2 infection: nanotechnology roles date: 2020-05-01 journal: acs nano doi: 10.1021/acsnano.0c03402 sha: doc_id: 350505 cord_uid: uh8r2vyz [image: see text] the impact of dietary patterns and the commensal microbiome on susceptibility to and severity of infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus has been largely ignored to date. in this perspective, we present a rationale for an urgent need to investigate this possible impact and therapeutic options for covid-19 based on dietary and microbiome modifications. the mitigating role of nanotechnology with relation to the impact of sars-cov-2 virus is highlighted. s evere acute respiratory syndrome coronavirus 2 (sars-cov-2) is a novel coronavirus that causes coronavirus disease 2019 . since its first detection in december 2019, it has affected millions of people worldwide, carrying a mortality rate much higher than any common flu. while there is an urgent need for its effective treatment based on antivirals and vaccines, it is imperative to explore any other effective intervention strategies that may reduce the mortality and morbidity rates of this disease. it may be possible to look in the gut for a solution to or mitigation of sars-cov-2 infection. the ecosystem of the gut and commensal microbiota can both regulate and be regulated by invading viruses, facilitating either stimulatory or suppressive effects. 1 therefore, it is plausible to consider whether the gut and sars-cov-2 interaction may play significant roles in the intensity of the infection and its clinical outcomes. the integrity of the gut microbiome (the collective genomes of the diverse microbiota that reside in the gastrointestinal tracts of humans) could conceivably be disturbed by sars-cov-2, causing gut dysbiosis in the host (figure 1 ), as with other infectious diseases. there are signs that may connect gut functionality and microbiome responses to sars-cov-2. for instance, the incubation period for sars-cov-2 is typically 5− 6 days, whereas the average incubation period for influenza is 2 days, 2 and diarrhea can be a presenting feature in sars-cov-2 patients. 3 new research indicates that sars-cov-2 may be spread by fecal−oral transmission. 4 the highest sars-cov-2 mortality and morbidity is in the elderly and in those with underlying health problems that are associated with inflammation and other disorders, such as diabetes. 5 interestingly, these cohorts tend to have less diverse gut microbiomes. 6 links between the gut microbiome and age-related health decline have been consistently shown. 7 aging is associated with significant shifts in microbiome diversity and proinflammatory states. the elderly microbiome generally shows a shift away from firmicutes, which dominates in younger adults, toward genera such as alistipes and parabacteroides. 8 a strong interindividual variability has been characterized in the elderly gut microbiome, with fluctuations featuring faecalibacterium and ruminococcus as well as certain clostridium clusters, especially iv and xiva. these may explain, in part, the different impacts of viral infections in elderly individuals. there are also specific trends in microbiome shifts that are seen in asthmatic and diabetic patients. interestingly, asthma appears to be underrepresented among comorbidities for critically ill patients infected with sars-cov-2. 9 in severe asthma, asthma control and sputum neutrophilia are associated with proteobacteria phylum relevant to pathogens such as escherichia, salmonella, vibrio, and helicobacter. 10 additionally, in chronic obstructive airways disease, the phylum bacteroidetes (e.g., prevotella) is decreased. 10 in contrast, the numbers of the h 2 -producing prevotellaceae (e.g., prevotella) were highly enriched in obese individuals prone to type ii diabetes. 11 additionally, an abundance of bif idobacteria (which can produce butyrate) in type ii diabetes patients has been shown to improve glucose tolerance. 12 in relation to this issue, attention should be given to interesting, but limited, reports regarding the abundance of prevotella in sequencing data sets of covid-19 patients. 13−15 an essential step for understanding the effect of the gut on sars-cov-2 is identifying the main gut microbiome species interacting with this virus. in this regard, the possibility that sars-cov-2 can interact with one or many of the 1500 species of microbiota in the gut makes the matter complicated. as such, without any human trials, it is impossible to refer to any specific species that influences sars-cov-2 pathogenesis. however, it is possible to consider the hypothesis of sars-cov-2 gut interaction based on past evidence. many different direct or indirect microbiome pathways could contribute to sars-cov-2-gut interactions. considering the pulmonary inflammation seen in sars-cov-2 patients in the second week of infection, both direct or indirect pathways can be taken into consideration. direct suppression or promotion of viral infection by the microbiome can occur via various mechanisms, such as genetic recombination, alteration of virion stability, driving the proliferation of cells, simulating attachment to permissive cells, and contributing to viral replication suppression; promotion of viral infection may occur by inducing systems' immunoregulatory and perturbing local immune responses. 1 although reports on direct and indirect viral bacterial promotion for influenza viruses are rare, examples of observed suppression are manifold. lactobacillus species, as a result of carbohydrate fermentation, can produce lactic acid, and the consequent ph changes inactivate different viruses. 1 the integrity of epithelial cells in the gut is important, as they produce antiviral compounds that are hostile to viruses. the colonic epithelial cells' functionality relies largely on the luminal presence of butyrate as an energy source, and the main butyrate-producing bacteria in the gut belongs to the phylum firmicutes. one hypothesis regarding microbiome interactions with sars-cov-2 is relevant to the microbiomes' impacts on cytokines. cytokines are small proteins that coordinate the body's response against infection and inflammation. for example, type ii interferon (interferon-γ) classically play important roles in antiviral responses. 16 more importantly, microbial metabolic processes in the gut strongly impact the production of cytokines. microbiota can increase chronic phase proteins and interferon signaling in lung cells to protect against influenza infection. however, as in the case of sars-cov-2, the body's response to infection can go into overdrive. in some patients, the immune response against sars-cov-2 results in excessive levels of cytokines release, leading to hyperinflammation and, clinically, to severe acute respiratory distress syndrome (sards) and multi-organ failure. so far, a cytokine profile associated with sars-cov-2 disease severity has been characterized by increased interferon-γ inducible protein 10 as well as many other cytokines. 2 therefore, the elucidation of host cytokine molecular pathways and microbiota components 17 as well as bacterial reactions in association with cytokine responses may provide novel microbiome-based therapeutic approaches to sars-cov-2 infection. as of yet, no study has been reported to identify the microbiota species that interact with sars-cov-2. considering the presented discussion, nutritional and dietary strategies directed at restoring the well-known beneficial microbiota, which can possibly suppress viral infection in the elderly and those with underlying health problems, may be an effective strategy to mitigate the harmful effects of this virus. one approach, as a whole and to be undertaken prior to any viral infection, could include strengthening the intestinal barrier against pathogens, increasing intestinal motility, and reducing an underlying pro-inflammatory state by adopting a many different direct or indirect microbiome pathways could contribute to sars-cov-2-gut interactions. more varied diet with a moderate increase in high-fiber and plant-based foods. of particular relevance is the enhancement of intestinal butyrate production through the promotion of microbial interactions by dietary changes. this change enhances gut epithelial cell health. in this regard, the shifts around the core microbiome including bif idobacteria, lactobacilli, and prevotella are critical. universally, bif idobacteria and lactobacilli are considered beneficial species regarding butyrate production, while the description of the functionality of prevotella remains controversial ( figure 1) . importantly, prevotella has been abundantly seen in the clinical samples of sars-cov-2 infected patients, 11−13 and the interpretation of its role is challenging and unclear. it is still not known whether prevoltella becomes abundant as a consequence of viral modulation or, conversely, is modulating sars-cov-2. it is unclear if this abundance of prevotella is due to long-standing dietary patterns or originates from modulation of the microbiome after the invasion of the virus. depending on whether prevotella's presence should be amplified or suppressed, the appropriate therapeutic action could be chosen. past studies suggest that high-fat diets increase the abundance of prevotella, whereas plant-based diets and fermented foods result in the opposite. 18 as general advice, frequent snacking between meals may cause dysbiosis and so should be kept to a minimum and only constitute fruit and vegetables, if required. the impact of probiotics should also be investigated. probiotics may help by interacting with the intestinal microbiota and modulating the immune system directly or through modification of the gut microbiota. the most commonly regarded beneficial probiotics in foods are bif idobacteria and lactobacilli species. in this regard, while still not having any full knowledge about beneficial or harmful strains, diets adhering to modest qualities of naturally fermented food are likely to be effective as preventative measures against sars-cov-2 and are of no risk for damaging the integrity of the gut and dysbiosis (figure 1) . without having knowledge about the best acting microbiota strains in response to sars-cov-2, following a healthy, moderate calorie, moderately higher fiber, and more diverse diet is a logical approach to mitigate the severity of this viral infection as a plausible preventive action. an essential investigation into the microbiome of covid-19 patients will be able to reveal the association of this disease to clinical outcomes of such preventative strategies. associations between dietary and microbiome effects and susceptibility to infection and severity of illness should be investigated with different methodologies. the overarching strategy should involve large, adequately powered international studies that recruit covid-19 patients and controls to collect clinical data, detailed dietary assessments, host genetics, immune phenotyping, and multi-site multiomic microbiome markers. the international approach would enable the inclusion of populations from different regions with different backgrounds, various dietary patterns, and environmental exposures. this comprehensive and collaborative approach is essential for unravelling the determinants of clinical outcomes of this infection and for designing targeted therapeutic and preventative measures. the moderating effects of high fiber (especially the choice of the high-fiber food type), freshly fermented, and diverse foods should also be examined as preventative and mitigating measures. in the light of the presented discussion, nanotechnology may play a critical role for rapid diagnosis, monitoring, and the design of effective therapeutic actions for covid-19 with relevance to the gut modulation by sar-cov-2. non-invasive breath tests, with arrays of nanomaterials, can identify the presence of volatile organic compounds with the signatures of modulated microbiota (abundance of prevotella, for example) and, hence, recognize the presence of sar-cov-2 for quick diagnosis and monitoring. 19, 20 ingestible sensors can be designed for the detection of inflammatory proteins associated with covid-19. 21 if the therapeutic strategy relies on the elimination of a specific bacterial strain in the gut, broad spectrum antibiotics would not work, as they also eliminate beneficial bacteria and consequently weaken the gut barrier. nanotechnology can efficiently be implemented in designing intelligent drugs or functional foods, with the possibility of localized delivery in the gut, 22 and also in designing intelligent functional foods. 23 these drugs and foods should target problematic bacterial strains in the gastrointestinal tract and enhance its health by improving gut barriers against pathogens and inflammatory reagents and by providing the base for creating disruptive remedies based on microbiome engineering. 19 nanoscale-enabled tools will likely enable us to observe, to navigate, and to act through the complicated ecosystem of the gut to help in finding either a cure or mitigating procedures for covid-19 and keeping sar-cov-2 under control. nutritional and dietary strategies directed at restoring the well-known beneficial microbiota, which can possibly suppress viral infection in the elderly and those with underlying health problems, may be an effective strategy to mitigate the harmful effects of this virus. without having knowledge about the best acting microbiota strains in response to sars-cov-2, following a healthy, moderate calorie, moderately higher fiber, and more diverse diet is a logical approach to mitigate the severity of this viral infection for now. the commensal microbiota and viral infection: a comprehensive review clinical features of patients infected with 2019 novel coronavirus in wuhan. lancet sars-cov-2 induced diarrhoea as onset symptom in patient with covid-19 evidence for gastrointestinal infection of sars-cov-2 the epidemiological characteristics of 2019 novel coronavirus diseases (covid-19) in jingmen the influence of commensal bacteria on infection with enteric viruses nutrition and the gut microbiome in the elderly composition, variability, and temporal stability of the intestinal microbiota of the elderly do chronic respiratory diseases or their treatment affect the risk of sars-cov-2 infection? aging and the microbiome: implications for asthma in the elderly? krajmalnik-brown, r. human gut microbiota in obesity and after gastric bypass insights into the role of the microbiome in obesity and type 2 diabetes are significant false negatives in the usually sensitive rt-pcr detection of sars-cov2 happening as the usually rna-stranded bacteria is now dna within a bacteria (prevotella?) genome -and won't be detected in the lysogenic state a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster rna based mngs approach identifies a novel human coronavirus from two individual pneumonia cases emerging microbes infect linking the human gut microbiome to inflammatory cytokine production capacity mechanisms by which the gut microbiota influences cytokine production and modulates host inflammatory responses association of dietary patterns with the fecal microbiota in korean adolescents diagnosis and classification of 17 diseases from 1404 subjects via pattern analysis of exhaled molecules ingestible sensors advances in oral nano-delivery systems for colon targeted drug delivery in inflammatory bowel disease: selective targeting to diseased versus healthy tissue application of nanotechnology in food science: perception and overview key: cord-340042-intxyu46 authors: chaudhry, sundas nasir; hazafa, abu; mumtaz, muhummad; kalsoom, ume; abbas, saima; kainaat, amna; bilal, shahid; zafar, nauman; siddique, aleena; zafar, ayesha title: new insight on possible vaccine development against sars-cov-2 date: 2020-09-11 journal: life sci doi: 10.1016/j.lfs.2020.118421 sha: doc_id: 340042 cord_uid: intxyu46 in december 2019, a novel virus, namely covid-19, caused by sars-cov-2, developed from wuhan, hubei territory of china, which used its viral spike glycoprotein receptor-binding domain (rbd) for the entrance into a host cell by binding with ace-2 receptor and cause acute respiratory distress syndrome (ards). data revealed that the newly emerged sars-cov-2 affected more than 24,854,140 people with 838,924 deaths worldwide. until now, no licensed immunization or drugs are present for the medication of sars-cov-2. the present review aims to investigate the latest developments and discuss the candidate antibodies in different vaccine categories to develop a reliable and efficient vaccine against sars-cov-2 in a short time duration. besides, the review focus on the present challenges and future directions, structure, and mechanism of sars-cov-2 for better understanding. based on data, we revealed that most of the vaccines are focus on targeting the spike protein (s) of covid-19 to neutralized viral infection and develop long-lasting immunity. up to phase-1 clinical trials, some vaccines showed the specific antigen-receptor t cell response, elicit the humoral and immune response, displayed tight binding with human-leukocytes-antigen (hla), and recognized specific antibodies to provoke long-lasting immunity against sars-cov-2. immune medical institute), and ino-4800 (inovio) presenting the significant results in initial trials and soon will be tested on human [27] . spike protein (s) of coronavirus are targeted by these vaccines and help in the stimulation of neutralizing antibodies [28] . different subunits of spike proteins like the s1 and s2 subunits, and the receptor-binding domain (rbd) are the critical elements for the formation of a vaccine against the newly emerged virus that helped in producing t cell responses and protective immunity against sars-cov-2 [29] . furthermore, two recombinant proteins that carry a receptor-binding domain (see glossary), and recombinant vectors that code for the receptor-binding domain can also play a useful role in developing a corona-vaccine. besides, the monoclonal antibodies of recovered corona-patients showing better results in neutralizing the antibodies by targeting the specific domains of sars-cov-2 [30, 31] . however, with increasing the cases and deaths of covid-19 day by day, the development of vaccines against sars-cov-2 is also required on an urgent basis. the present review investigated the new insight parameters and components to develop a vaccine against newly emerged covid-19 by targeting the spike protein and epitopes. the present study also aims to share the candidate antibodies and latest development in the formulation of a vaccine against sars-cov-2. further, current challenges and future perspectives, the structure, mechanism, immune response, and different components of covid-19 are studied in the present review for a better understanding. cathepsins b and l in sars-cov-2 [40] . the evidence revealed that the cellular protease, like tmprss2 (see glossary) plays an essential role during the entry and circulation of sars-cov-2. however, the antiviral or vaccine could be a potential therapeutics against covid-19 by blocking the regulation of tmprss2 enzyme [41] . however, the entry of the virus is cell type and protease specific dependent. six hbs are framed by the communication of heptad repeats 1 and 2 (hr1 and hr2), and the areas present in a spike protein formed by the embedment of peptides in the layer of endosomes [42] . as a result, an envelope of virus and plasma film is synthesized by the golgi intermediate compartment. finally, the newly formed virus rna genome is discharged from the cell through exocytosis. the detailed pathway is presented in fig. 2 . to reduce the binding of virus with the host receptor known as ace2, the subunit immunizations for both sars coronaviruses depend on inspiring a resistant reaction contrary to the spike protein [43] . to prevent the viral infection, there is a need for the natural killer cells to perceive the entrance of the virus into a host cell. according to accumulated data, only limited researches are presented yet on the innate immune response to sars-cov-2 infection. recently, an ongoing project (consist of 35 cases) in buali hospital, iran revealed a significant reduction of 45% (1100 u/l) in lymphocytes, improvement in total neutrophil (48%), c-reactive protein (99%), and serum il-6 (58%). the results also reported that the amount of lymphocytopenia and neutrophilia are correlated with infection mortality and disease severity [13, 44] . similarly, another report based on 99 cases in wuhan, china, claimed the 35% reduction in total lymphocytes, 38, 84, and 52% enhancement in neutrophils, c-reactive proteins, and serum il-6, respectively [45] . the immune responses associated with the diseases of the lung may have resulted in a large amount of j o u r n a l p r e -p r o o f cytokines (see glossary) production, which provides the 1 st line defense against viral infection [46] . in addition, the humoral cells of the body are responsible for antibodies production against viral, which could play a significant role in preventing viral infection by killing them [47] . the innate immune system could effectively identify the situation during the incursion of the virus, usually called pathogen-associated molecular patterns (pamps). however, the endosomal rna receptors, rig, cytosolic rna sensor, tlr7, and tlr8 quickly recognized these pamps (present in the form of ssrna on the viral genome) during the replication of the virus and cause stimulation of numerous signaling pathways and transcription factors including nf-κb, irf-3, ap-1 and irf-7 [48, 49] . ultimately, these nuclear factors, especially ap-1 and nf-κb, activated the expression of genes that encoded the chemokines (cxcl8 and ccl2), and cytokines (il-1 and tnf) that lead to the inflammatory responses. similarly, other nuclear factors, namely irf3 and irf7, increased the production of inf-type 1 (ifnα and infβ), which significantly helped in the reduction of viral dissemination and replication (see fig. 3 ) [50] . based on the increasing incidences of newly emerged covid-19, there is necessary to make a successful and safe vaccine against sars-cov-2 to control its present pandemic and future recurrence. the present lethal coronavirus (covid-19) shared the structure homology with previously reported beta-coronaviruses, including mers-cov (50%) and especially sars-cov (79.6%) [27] . based on their structure homology, the genome accession number (genbank), [51] . accumulated data reported that the formation of a vaccine falls into one of the following types; viral vectors (25 projects and one is under clinical trial), dna and mrna-j o u r n a l p r e -p r o o f based (20 projects and one from each type is under clinical trial), protein-based (28 projects and mostly on s protein), virus-like particle (vlp) (5 projects), inactivated or live-attenuated virus (7 projects and two are under clinical trial), and epitope vaccine [52, 53] . antibodies are the best and conservative means to anticipate and control desirable infections. till more than 40 pharmaceutical industries and scholarly foundations all over the world have propelled their plans on immunization improvement against sars-cov-2 [54] . herein, we discussed the new insights and latest developments in each class of vaccines to formulate a strong and effective vaccine against sars-cov-2. the vaccine development gained special attention during the last few decades, including the development of rna, dna, and protein-based vaccines against several viruses like the influenza virus, and ervebo virus. the proteins are the important constituents in the structural activities of coronavirus, which are involved in the transmission, entry, and replication of viruses. the data suggested that proteins could be ideal targets for the development of vaccines [13, 55] . the emerging evidence revealed that the viral spike (s) protein vaccine showed the higher neutralizing titers (see glossary) against sars-cov than other vaccine candidates. based on accumulated data, the s protein is the preferred site of vaccine formation against previously emerged coronaviruses (mers and sars-covs) because s protein could easily be encountered and allow the body to make an immune response more efficiently than other proteins [55, 56] . based on these points, the potential parts of s protein, which are used as antigens in immunization improvement, integrate the entire extent of this protein and vaccine development [57] . pallesen et al. [58] reported that the s ectodomain of coronavirus with g4 showed a glycosylated loop variation in a structure of the virus, and observed the four major j o u r n a l p r e -p r o o f conformational sites in a trimer for the binding of each rbd either relatedly or tightly to a receptor accessible confirmation. however, the structure-based design of coronavirus could provide a prospective way for the formulation of a vaccine. many pharmaceutical industries and universities start working for the development of vaccines against covid-19. recently, the astrazeneca and the university of oxford began to combined effort to develop a spike protein (s) vaccine, namely azd1222 against newly emerged sars-cov-2 in chimpanzee to generate the dna for the spike antigen and to grow the vigorous t cell and b cell responses for better prophylaxis with less dose [59] . besides, another company who made a vaccine against ebola virus, namely cansino biologics (china) started a project to create a safe and efficient s protein antigen-based vaccine (ad5-ncov) against sars-cov-2 that is undergoing the phase-1 clinical trial on individuals aged between 18-60 years under clinicaltrial.gov: nct04313127 [60] . similarly, the two top vaccine producer companies, including glaxosmithkline and sanofi start working on the formation of spike protein-based vaccine against covid-19 to trigger the immune response, and they reported that they would begin phase-1 clinical trials later this year [59] . besides, the subunits of s protein, including receptor-binding domain (rbd), n-terminal domain (ntd), and c-terminal domain (ctd) also consider as a vital target for the progress of a vaccine. the study reported that antibodies that are attached to the n-terminal domain of the s1 subunit of coronavirus demonstrated an active killing movement, which indicates that ntd is applicable in balance for vaccine development. jiaming and his research group [61] experimented on balb/c immunized mice at two different doses (5 and 10 µg) to investigate the effect of recombinant ntd (rntd) for vaccine development and immunogenicity. they revealed that high administration of 10 µg significantly reduced the lung infection caused by a j o u r n a l p r e -p r o o f coronavirus in a recombinant vaccinated mouse model. they also observed a robust t-cell immune retort in the serum of vaccinated mice (rntd) with cpg and aluminum adjuvant. geo et al. [62] experimented on balb/c mice (n=9) to check the rbd, s, and n-protein antibody response after 1-6 weeks of first immunization at 6 µg dose (accessed by eliza). they observed that sars-cov-2 linked rbd and s-protein immunoglobulin g (igg) (see glossary) instantly start to develop inside the immunized mice at 6 th week with the peak titer of 409,600 (100 µg/ml) and 819,200 (200 µg/ml) respectively. interestingly, the peak titer of n-specific igg in antibody response was about 30 times fewer than rbd in vaccinated mice. but recently, another study stated that the n-specific igg is reported as one of the most effective proteins in covid-19 recovered patients as a diagnostic marker and vaccine development [63] . the study revealed that the rbd of specific igg showed about the half of the value of antibody response than s protein, and suggested that the rbd is a meticulously associated immunogen to the recovered patients of covid-19 [62] . however, the s protein and rbd of sars-cov-2 might be an innovative possible target for the formulation of the vaccine. the inactivated vaccine (known as whole killed virus: wkv) is an important vaccine that possesses the ability to cease the replication cycle of the virus, and it is generally prepared by neutralizing the virus through radiation or heat, and chemicals like formaldehyde. according to emerging evidence, the inactivated virus vaccine is one of the safe and effective vaccines, which can easily be prepared with much fewer cost as compared to nucleic acid vaccines [64] . the wkv vaccine could significantly target the subunits of viruses, including s and e proteins, orf, matrix (m), and boost the immune response against viruses [65] . the wkv vaccine has attained j o u r n a l p r e -p r o o f special attention over the decades due to its several advantages against viruses, particularly sars-cov, that could easily neutralize the virus antibodies [64] . most recently, a study experimented on non-human primates (rhesus macaques), rat, and mice (balb/c) at two different doses (3 and 6 µg) for the investigation of protective potential and immunogenicity of purified inactivated virus vaccine candidate (picovacc). they revealed that picovacc significantly neutralized the effect of ten sars-cov-2 strains and demonstrated the partial or complete protective potential against sars-cov-2 in macaques deprived of any development of the antibody-dependent disease. the data suggested that the inactivated virus vaccine candidate (picovacc) could be proved as a novel option for a vaccine development against covid-19 [62] . another study demonstrated that inactivated virus vaccine also showed a protective behavior against previously emerged coronavirus, namely sars-cov-1, but phase-1 clinical trials were dried due to several reasons including a low number of cases and lack of funds, but few neutralizing monoclonal antibodies (see glossary) development by inactivated virus vaccine like cr3022 that could be a potent cross-protection against covid-19 [55, 66] . based on the accumulated data, the live attenuated and inactivated virus vaccines are reported as one of the safest and effective vaccines against previously emerged viruses, namely influenza virus [67] . the live attenuated virus vaccine could be developed by diagnosing the newly emerged transmitted sars-cov-2 through a possible fewer risk of pathogenesis like enhanced anti-inflammatory cytokines, minimum lung infection, and fewer neutrophil influx as compared to wild type virus [51] . the inactivated and live attenuated virus vaccines consider the whole virus as a vaccine. a study evaluated the performance of live attenuated vaccine, by initiating alteration (y6398h) into orf1a/b polyprotein (nsp14) of mice, which showed the significant replication reduction behaviors of coronavirus in mice after day five intracerebral inoculation j o u r n a l p r e -p r o o f [52] . similarly, a recent study suggested that the development of oral live attenuated virus vaccine could be a potential target to diminish the lung infections triggered by sars-cov-2, due to its initial infection in guts which resultingly boosted up the mucosa that associated with immune system during the early immune response against covid-19 [68] . another study also reported that the live attenuated virus vaccine could easily be administrated and serve as a community spread to rapidly develop herd immunity against the pathogens of covid-19 [69] . against sars-cov-2 that is under phase-1 clinical trial [70] . however, few limitations could also be manifested during the expansion of inactivated or live attenuated vaccines against sars-cov-2. first, the live attenuated vaccines could regain its virulent effect in an in vivo or cell culture. second, the coronavirus could prevent seepage from the immunity developed by these vaccines through quick progression. thus, the manufacturer should be aware and careful during the development of these (inactivated and live attenuated) vaccines against sars-cov-2 [71]. antibodies that are made up of dna contained circular dna molecules that encoding at least one foreign particle, and these antibodies are considered as better, as compared to other antibodies [77, 78] . the dna-based vaccine could effectively target several variants of coronavirus including the s1 domain, and prefusion stabilized ectodomain with furring cleavage, spike protein, receptor-binding domain (rbd), cytoplasmic tail, and transmembrane domain [79, 80] . the dna-based vaccine composition is an expensive and sophisticated method, which consists of double-stranded plasmids that are usually designed with the help of a computer/smart device to generate an immune retort against the virus. a specific device, named cellectra is used to generate the electric pulse under the skin to usher the dna-based vaccine [81, 82] . the results revealed no serious adverse effect and immune response was dose-dependent, at 6 mg the candidates showed the maximum immune response against mers-cov. however, future study is required to check the efficiency of dna-based vaccine (dls-5300) against newly emerged sars-cov-2, that could be a novel target. the previously reported studies revealed that the computation strategy is one of the effective techniques for the development of vaccines against many lethal infections, including malaria, cancer, and dengue. this technique worked by recognition of novel t cell epitopes, and mhc 1 and 2 molecules (see glossary) for the development of specific vaccines associated with the transporter of antigen presentation (tap) agents [86, 87] . this vaccine is made up of antibodies related to the segments of immaculate foreign particles and is generally arranged by j o u r n a l p r e -p r o o f substance amalgamation procedures. these antibodies are simpler in readiness and also have efficient control [88] . based on its previous tremendous application in other diseases like dengue, the epitope-based virus could be an essential option in the formulation of vaccines against sars-cov-2. currently, many projects are undergoing vaccine formation. khan subunit immunizations include at least one antigen with solid immunogenicity to prepared to efficiently vaccine that activates the host immune system. this kind of antibody is more secure and simpler to create because it did not contain any live virus for the development of a vaccine. however, it frequently requires the development of adjuvants (see glossary) to induce a solid defensive safe reaction. but like other vaccines like inactivated or live attenuated vaccine, the subunit vaccine is less immunogenic that could be improved by adding the suitable adjuvants [92, 93] . up until few foundations have started working on the formulation of a subunit-based vaccine to target the virus. the university of queensland has begun work to build up this type of subunit body, which depends upon the molecular lamp methodology [94] . similarly, clover biopharmaceuticals inc. started the initial validation in building up an antibody competitor against sars-cov-2 by utilizing the "trimer-tag" innovation [95] . liu and his research group designed a novel s1-subunit based nano-vaccine against sars-cov-2 and stated that nano-vaccine showed the strong immune and humoral response and effectively elicited the t-cell response by triggering cd4 + and cd8 + cells, which to minimize the viral load in affected persons. the results also revealed that the nano-vaccine provoke the iga antibody, which provides the mucosal protection against viral load [96] . kalita the recombinant protein is known as one of the emerging fields for the development of a vaccine against viruses due to several properties including tight binding to specific ace-2 receptor, provoke immune protection against viral infections, increase antibody-dependent viral entry, and promote antigenicity against virus like sars-cov [52] . the accumulated data stated that the recombinant ace-2 (race-2) receptor exerted the potential targets against the protection of many diseases, including acute ang 2-induced hypertension, and severe lung injury [98, 99] . besides, race-2 showed the rapid cure rate with a half-life of about 60 min in mice as well as humans [100] . lei et al. [101] suggested that this vaccine could be a new hope against newly emerged sars-cov-2 due to structure homology with sars-cov-1 [102] . table 1 illustrates some essential vaccine candidates that are presently under clinical trials for the formulation of an active vaccine against the pandemic of sars-cov-2. in contrast, fig. 4 explained the general mechanism of action of different vaccines to provoke the humoral and immune retort against sars-cov-2. j o u r n a l p r e -p r o o f based on the present pandemic situation caused by newly emerged sars-cov-2, there is a need to make a vaccine on urgent bases. after examination, it is revealed that the calculated ro value of sars-cov-2 is 2.2. this indicates that one contaminated person could contaminate more 2.2 persons. however, by using different precautionary measures, isolation, and quarantine activities, the ro value could be decreased [103] . no doubt, the vaccine formulation process is not only expensive and time taking but also very complicated that sometimes gives a very low success rate. currently, more than 40 pharmaceutical industries and various international institutes are working for the development of several types of vaccines. still, all are under phase-1 clinical trials, and no vaccine has been licensed yet [104] . vaccines elicit the immune response against viruses, but currently, one of the major concerns is that no one knows what kind of immune response is required against sars-cov. one of the basic alarms is the disadvantages of several vaccines, including the unstable under physiological condition, low immunogenicity, organ failure, required high dose, and more adjuvants. however, these disadvantages need to be addressed and improved on urgent bases in the next version of vaccine development [105, 106] . however, the delivery of vaccine both in immunization and modality modes are also significant issues which also needs to be revised to minimize the off-target effects. based on data, it is suggested that the aerosol route or oral administration could be a better way for vaccine delivery that might significantly induce a mucosal response. similarly, dna plasmid or vector could also be used for the delivery of the vaccine to its target site [51, 107, 108] . previously due to negligence and improper attention of government towards this task, no proper improvement in vaccine development is seen especially against mers and sars-covs. the government and health departments do not have a proper budget to manage it. in 20 years the sars-cov is considered to be 3 rd epidemic war [7] . based on accumulated data, we suggested that the development of the ideal vaccine should possess some major properties, including 1) the perfect vaccine should be well-coordinated and strongly elicit the t-lymphocyte immunity that could lead to the stimulation of cytotoxic t based on the increasing incidences of covid-19, there is urgently needed to formulate a vaccine against sars-cov-2. based on emerging evidence, we recommended several vaccine categories including subunits, epitopes, mrna, protein, especially s protein, recombinant adenovirus, and fusion proteins, inactivated and live attenuated virus for the formulation of a timely required vaccine against sars-cov-2. more than 40 pharmaceutical industries and institutes are started works on more than 100 projects for the development of different vaccines against covid-19. these vaccines significantly neutralized the sars-cov-2 and developed a strong immune and humoral response against the virus. we suggested that the receptor-binding j o u r n a l p r e -p r o o f domain (rbd), a component of s protein, might show a noteworthy role in neutralizing the antibodies because it inhibits the entrance of the virus into the host by blocking the attachment of the virus to the host membrane and also block the virus membrane fusion which could help in vaccine development. but the site-specific glycan defense creates difficulties in targeting 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tmprss2 and is blocked by a clinically proven protease inhibitor infection and rapid transmission of sars-cov-2 in ferrets key: cord-348821-2u6ki9dv authors: xu, ping; sun, guo-dong; li, zhi-zhong title: clinical characteristics of two human to human transmitted coronaviruses: corona virus disease 2019 versus middle east respiratory syndrome coronavirus. date: 2020-03-10 journal: nan doi: 10.1101/2020.03.08.20032821 sha: doc_id: 348821 cord_uid: 2u6ki9dv after the outbreak of the middle east respiratory syndrome (mers) worldwide in 2012. currently, a novel human coronavirus has caused a major disease outbreak, and named corona virus disease 2019 (covid-19). the emergency of mres-cov and covid-19 has caused global panic and threatened health security. unfortunately, the similarities and differences between the two coronavirus diseases remain to be unknown. the aim of this study, therefore, is to perform a systematic review to compare epidemiological, clinical and laboratory features of covid-19 and mers-cov population. we searched pubmed, embase and cochrane register of controlled trials database to identify potential studies reported covid-19 or mers-cov. epidemiological, clinical and laboratory outcomes, the admission rate of intensive cure unit (icu), discharge rate and fatality rate were evaluated using graphpad prism software. thirty-two studies involving 3770 patients (covid-19 = 1062, mers-cov = 2708) were included in this study. the present study revealed that compared with covid-19 population, mers-cov population had a higher rate of icu admission, discharge and fatality and longer incubation time. it pointed out that fever, cough and generalised weakness and myalgia were main clinical manifestations of both covid-19 and mers-cov, whereas ards was main complication. the most effective drug for mers-cov is ribavirin and interferon. coronaviruses are rna viruses with envelope and non-segmented positive-sense, causing respiratory and intestinal tract infections in humans and other mammals [1] . despite . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 4 although many previous studies have reported clinical characteristics of covid-19 or mers-cov diseases [8] [9] [10] [11] , systematic comparison of clinical features between covid-19 and mers-cov diseases has yet been published. thus, the purpose of this study is to perform a systematic review of epidemiological, clinical and laboratory characteristics of patients infected by covid-19 or mers-cov disease, and to compare covid-19 and mers-cov in the context of their incubation, laboratory features, admission rate of intensive cure unit (icu) and rate of discharge and fatality, which will provide a comprehensive reference for clinical physicians in treatment of coronavirus diseases. a comprehensive and systematic search was performed using pubmed, embase and or (2019 novel coronavirus)). if necessary, we also contacted corresponding author to obtain accurate data. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 5 the study that met following criteria were included: (1) reporting clinical characteristics of covid-19 or mers-cov disease, (2) minimum sample size of five, (3) confirmed covid-19 or mers-cov disease, (4) english literature. studies were excluded as following criteria: duplicate publications, case report, meta-analysis, letter, review, technology report, commentary, animal trial, correspondence, predictive study, guidence, radiograph study and meeting report. at the beginning, 4743 potential publications were identified. we removed 678 duplicates and reviewed the titles and abstracts of remaining 4065 publications. 4015 publications were excluded for following reasons: not involved research point (n = 1378), review (n = 569), no english (n = 33), case report (n = 316), meta-analysis (n = 1), letter (n = 127), technology report (n = 196), commentaries (n = 61), animal study (n = 1107), correspondence (n = 29), predictive study (n = 110), guidence (n = 28), meeting report ( n = 16) and radiograph (n = 44). then, a comprehensive review of full-text was conducted for remaining 50 publications. two reviewers independently screened eligible literature, and any argument was solved by discussion with a third reviewer. finally, thirty-two studies were included in this study . the process was shown in figure 1 . two reviewers extracted independently common, clinical and laboratory characteristics of included studies, with disagreements were solved by discussion with a third reviewer. the extracted data included incubation time, white blood cell (wbc) count, lymphocyte count, creactive protein (crp), alamine aminotransferase (alt), aspartate aminotransferase (ast), . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 6 creatinine, creatine kinase (ck), the admission rate of intensive cure unit (icu), rate of discharge and fatality, symptom, comorbidity, complications and cure rate of drug. for normality distribution data, outcomes were extracted directly. for skewness distribution data, the outcomes was extracted after being converted as specific formula [40] . the quality assessment of included studies was performed through the newcastle-ottawa quality assessment scale (nos), as recommended by the cochrane non-randomized studies [41] . the nos includes three parts for risk of bias, with nine points in total: (1) selection of research groups (four points); (2) inter-group comparability (two points); and (3) ascertainment of exposure and outcomes (three points) for case-control and cohort studies, respectively. study that scored 6 or more was qualified for systematic review [42] . the assessment process was completed by two reviewers independently. all debates were solved by discussion with a third reviewer. all statistical analyses and graphs were generated and plotted using graphpad prism version 7.00 software (graphpad software inc). the p value < 0.05 suggests significant difference. all included studies were retrospective study . among ten studies reported covid-19, one trial was performed in netherlands [12] and remaining nine trials were conducted in china [8-9, 13-16, 37-39] . the sample size ranged from 6 to 425, and had a total . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint [24-26, 31, 33] , one trial was performed in iran [19] and one trial was conducted in japan [10] . the sample size ranged from 5 to 883, and had a total of 2708 patients. the year of publications ranged from 2013 to 2020. clinical and laboratory characteristics of included study were shown in table 1 and table 2 respectively. among thirty-two included studies, four studies obtained 6 points of nos [26, 28, 32, 35] , and remaining twenty-eight studies obtained 7 points of nos or more [8-25, 27, 29-31, 33-34, 36-39] . the result of quality assessment was presented in table 3 . for covid-19 population, the number of patients with fever was 480 (45.2%), cough was 373 (35.1%), generalised weakness and myalgia was 318 (29.9%), stuffy or rhinorrhoea was 6 (0.6%), pharyngalgia was 32 (3%), chest pain was 10 (0.9%), diarrhoea or anorexia was 123 (11.6%), dyspnoea was 140 (13.2%) and dizziness or headache was 60 (5.6%). for mers-cov population, the amount of patients with fever was 404 (14.9%), cough was 424 (15.7%), generalised weakness and myalgia was 337 (12.4%), stuffy or rhinorrhoea was 17 (0.6%), pharyngalgia was 47 (1.7%), chest pain was 27 (1%), diarrhoea or anorexia was 128 (4.7%), dyspnoea was 271 (10%) and dizziness or headache was 131 (4.8%). the above results were shown in table 4 . for covid-19 population, the main complications included shock, arrhythmia, acute respiratory distress syndrome (ards), acute cardiac injury, acute kidney injury and acute . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint liver injury, and the amount of which was 17 (1.6%), 28 (2.6%), 51 (4.8%), 11 (1%), 10 (0.9%) and 2 (0.2%) respectively. for mers-cov population, the number of individuals presented shock was 22 (0.8%), arrhythmia was 11 (0.4%), ards was 83 (3.1%), acute cardiac injury 10 (0.4%), acute kidney injury was 30 (1.1%), acute liver injury was 22 (0.8%) and neurological symptoms was 4 (0.1%). the results were shown in table 5 . table 6 . systematic review was performed for clinical and laboratory outcomes of coronavirus disease. there was no significant difference in age (50.9 ± 2 vs. 53.6 ± 1.5, p = 0.3), wbc table 7 . . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint the higher admission rate of icu was found in mers-cov population than in covid-19 population ( 43.6% vs. 22.4%, figure 2 ). there was a higher discharge rate in mers-cov populations compared with covid-19 population ( 59.9% vs. 33.5%, figure 3 ). the lower fatality rate was found in covid-19 population compared with mers-cov population (6.8% vs. 34.1%, figure 4 ). coronavirus is an important pathogen causing respiratory and intestinal infection. of seven identified coronaviruses, the two very pathogenic viruses, sars-cov and mers-cov, cause severe ards and even acute respiratory failure. with a mortality rate of more than 10% and more than 35% respectively [43] [44] . the four other human coronaviruses in addition, we found that the number of males is more than that of females in either covid-19 or mers-cov population. the possible reason of reduced susceptibility of females to viral infection is that females have a lot of x chromosome and estrogen that are vital components in development of innate and adaptive immunity [47] . meanwhile, numbers of patients with covid-19 infection had chronic comorbidities, mainly hypertension, diabetes and cardiovascular disease, which is similar to mers-cov population. those results indicate that older adult males with chronic underlying disease might be more susceptibility to covid-19 or mers-cov. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 1 1 in terms of laboratory testing, reduced lymphocytes and increased crp were found in both covid-19 and mers-cov patients. this result indicates that covid-19 might be associated with cellular immune response, mainly act on lymphocytes like mers-cov does [48] . the cells infected by viruses induce the release of numbers of pro-inflammatory cytokines and inflammation storm in the body. moreover, increased cytokines might make damage to related organs such as liver [49] . our results demonstrated that abnormal value of ast was found in mers-cov population, but not in covid-19 population. the possible reason is that the follow-up time of covid-19 population was too short, and the liver might remain to be in compensatory stage. for this result, a long follow-up time study is urgently needed. on the other hand, our results suggested that mers-cov population had a higher rate of icu admission and fatality than covid-19 population, indicating that compared with mers-cov, covid-19 has less toxic and more easily cured. however, lower discharge rate was found in covid population than in mers-cov population. the possible explanation is that most of covid-19 patients remained to be hospitalized at the time of manuscript submission, and data on those patients could not be obtained in time. thus, careful interpretation is urged for this result. up to now, no effective strategy has been found for treatment of covid-19 infection [50] . currently, the measure to covid-19 is to control the source of the infection; taking personal protective works to reduce the risk of transmission; and early diagnosing, isolating and supportive treating for confirmed patients. in the present study, our systematic results of cure rate of drug for mers-cov infection indicated that ribavirin and interferon, oseltamivir, antivirals and intravenous immunoglobulin all had been effective for mers-cov infection, with the cure rate of those drugs was 74.2%, 69.2%, 67.1% and 62.5% respectively. thus, we . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 1 2 assume that those drugs might also be effective at covid-19 infection. however, further studies are needed to confirm this idea. this study has several limitations. first, many patients infected by covid-19 remained to be hospitalized at the time of manuscript submission, leading to the unavailable of some data. second, the follow-up time of covid-19 population is too short to get related data from long-term observations of this disease. third, finding of statistical tests and p values between covid-19 and mers-cov populations should be interpreted with caution. fourth, the number of mers-cov patients treated by drugs is small, careful understanding is needed for the cure rate of drug for this disease. finally, as covid-19 is still developing around the world and remains to have many unknowns, the results of this study are staged and need to be carefully understood. more large-sample, multicentre, high-quality research should be performed to update this study. our systematic review reveals that main clinical manifestations of both covid-19 and mers-cov populations are fever, cough and generalised weakness and myalgia. ards is main complication of both two populations. covid-19 population has a shorter incubation time and lower rate of icu admission, discharge and fatality compared with mres-cov population. this study was supported by grants from the national natural science foundation of china (nos.31970862) and guangzhou municipal science and technology project (nos. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint . 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comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis liver manipulation during liver surgery in humans is associated with hepatocellular damage and hepatic inflammation sars and mers: recent insights into emerging coronaviruses author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint key: cord-322811-6lebh7ca authors: baig, mirza s.; alagumuthu, manikandan; rajpoot, sajjan; saqib, uzma title: identification of a potential peptide inhibitor of sars-cov-2 targeting its entry into the host cells date: 2020-06-26 journal: drugs r d doi: 10.1007/s40268-020-00312-5 sha: doc_id: 322811 cord_uid: 6lebh7ca background and objective: coronavirus disease (covid-19) is an ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). due to the incessant spread of the disease with substantial morbidity and mortality rates, there is an urgent demand for effective therapeutics and vaccines to control and diminish this pandemic. a critical step in the crosstalk between the virus and the host cell is the binding of sars-cov-2 spike protein to the angiotensin-converting enzyme 2 (ace2) receptor present on the surface of the host cells. hence, inhibition of this interaction could be a promising strategy to combat the sars-cov-2 infection. methods: docking and molecular dynamics (md) simulation studies revealed that designed peptide maintains their secondary structure and provide a highly specific and stable binding (blocking) to sars-cov-2. results: we have designed a novel peptide that could inhibit sars-cov-2 spike protein interaction with ace2, thereby blocking the cellular entry of the virus. conclusion: our findings suggest that computationally developed inhibitory peptide may be developed as an anti-sars-cov-2 agent for the treatment of sars-cov-2 infection. we further plan to pursue the peptide in cell-based assays and eventually for clinical trials. electronic supplementary material: the online version of this article (10.1007/s40268-020-00312-5) contains supplementary material, which is available to authorized users. the recent severe acute respiratory syndrome coronavirus 2 (sars-cov-2) outbreak has posed a great challenge to human health. during the past 2 decades, we have encountered the outbreak of many deadly viruses, such as ebola [1] , zika [2] and nipah [3, 4] , as well as the evolution of various strains of coronaviruses (cov), mainly sars-cov [5] and mers-cov [6] , which resulted in high morbidity and mortality. after almost 100 years of the deadly influenza virus (h1n1, or spanish flu) pandemic, with millions of deaths worldwide (approximately 40 million) [7, 8] , the recent outbreak of a novel cov, or sars-cov-2, has left the entire world in helplessness and misery. the clinical spectrum of covid-19 ranges from mild fever, cough and shortness of breath, to severe clinical conditions characterized by respiratory failure [9, 10] . old age, together with pre-existing conditions such as lung or heart disease, diabetes, or a compromised immune system, expedite the infection time and severity [11, 12] . multiple recent reviews [13, 14] could brief-up the statistical dynamics of covid-19 cases worldwide. structurally, the cov has the largest known rna genome (26) (27) (28) (29) (30) (31) (32) kb) among other known viruses, characterized by non-segmented, positive-sense, single-stranded rna. this genome encodes four major structural proteins of the virus, including the nucleocapsid (n), envelope (e), membrane (m), and spike (s) proteins [15, 16] . the membrane and envelope proteins are associated with virus assembly, while the spike protein plays the main role in facilitating virus entry via mediating its interaction with the transmembrane surface receptor on the host cells [15, 17] . the spike protein directly interacts with the peptidase domain (pd) of the angiotensin-converting enzyme 2 (ace2) receptor [18, 19] , which technically marks the virus entry inside the cells [20] . the sars-cov-2 shares around 80% sequence identity with the sars-cov genome, suggesting similarity in their host interacting functions [21, 22] . like sars-cov, the spike protein of sars-cov-2 contains s1 and s2 subunits, which are jointly responsible for fusion and entry of the virus [23] [24] [25] inside the host cells. the receptor-binding domain (rbd) in the s1 subunit initiates direct binding with the ace2 pd, whereas the s2 subunit contains basic elements needed for the membrane fusion [19, 20, 23, 24] . ace2, a single-pass type i transmembrane metallocarboxypeptidase enzyme is primarily involved in the maturation of peptide hormone angiotensin (ang), which in turn regulates the vasoconstriction and blood pressure [19, 26, 27] . ace2 is primarily expressed in alveolar epithelial type ii cells and serves as a viral receptor [28, 29] . besides alveolar epithelial type ii cells, it is also expressed in several extrapulmonary tissues, including the heart, kidney and intestine [26, 30] . the full-length structure of ace2 consists of two main domains-the n-terminal pd and the collectrinlike domain (cld) at the c-terminal end [19, [30] [31] [32] . in fact, the spike glycoprotein of sars-cov-2 binds to the homodimer of ace2, which facilitates virus entry into the host cells [19, 33] . in addition to this, studies have been conducted in association with ace2 and the amino acid transporter b0at1 (or slc6a19), and how sars-cov-2 may bind to the ace2-b0at1 complex [19, 34] . ace2 interaction with b0at1 could aid in producing antivirals or a vaccine that can block cov infection by targeting ace2 [35] [36] [37] [38] . with the current epidemiology of sars-cov-2, a vaccine might be considered a highly anticipated therapy. however, given that vaccine development and production is a highly challenging and time-consuming task, the need of the hour is to develop potent therapeutic agents that could effectively curb the infection in the early stages. several approaches, such as decoy-soluble ace2 proteins, antibodies from the serum of infected patients, epitope-based vaccines, repurposing of drugs, and designing blocking peptides are underway [39] [40] [41] [42] [43] [44] [45] [46] . peptides possess several attractive features when compared with small molecules and protein therapeutics, including high structural compatibility with target proteins, the ability to disrupt protein-protein interfaces, etc. computational tools ease the way to reach a therapeutic solution for covid-19. currently, the computational analysis of structural differences in human ace2 impact its binding to the sars-cov-2 spike protein, which thereby lays a foundation for the design and development of ace2-based peptide inhibitors of sars-cov-2 [47] [48] [49] . in the current study, we used computational biology tools to develop a therapeutic strategy utilizing a novel peptide by exploiting the sars-cov-2/ace2 interaction. the protein sequence of sars-cov-2 was retrieved from the rcsb protein data bank (pdb id: 6m17) for peptide design and development. initially, a stretch of 23 amino acid residues (glu23 to leu45) was taken from ace pd (pdb 6m17) and saved as pdb using the 'build structure' module in discovery studio visualizer (biovia, ds, 2019) [50] . this sequence was further optimized for geometry using the 'minimize' functionality in the ucsf chimera software [51] . we performed alanine scanning to better understand the individual roles of the residues within the 23-amino acid peptide in binding with sars-cov-2. alanine substitution of each residue was performed using biovia discovery studio 2020 software [50] . each peptide was then modeled and energy minimized using ucsf chimera software [51] . we also truncated five residues from the n-terminal end of the 23-amino acid (23aa) peptide to get a shorter 18-amino acids peptide (phe28 to leu45). this peptide was further energy minimized using the method described above. the crystal structure of the sars-cov-2 spike protein-ace2 (pdb id: 6m17) complex was retrieved from the protein data bank (https ://www.rcsb.org/) and used as the starting point for the docking studies. molecular docking was performed using pydock (https ://life.bsc.es/pid/pydoc kweb) [52] , haddock 2.4 [53] , and zdock [54] servers, to validate the results for each program, which in turn should be mutually correlated. the docking results were analyzed using the chimera [51] and ds visualizer programs [50] . the results obtained were analyzed for binding energies and peptide conformations in the sars-cov-2 spike protein binding interface. molecular dynamics (md) simulations were performed at 50 ns for the modeled protein. the coordinates of the structure were stored and examined utilizing the analytical tool in the gromacs 4.5.5 package [55] . the toxicity and other relevant molecular mechanistic values of the peptides, such as electrostatics, desolation, and van der waals (vdw) force, were predicted using the 'toxinpred' server (https ://crdd.osdd.net/ragha va/toxin pred/) [56] . toxin-pred is an in-silico method that has been developed to predict and design toxic/non-toxic peptides. the main dataset used in this method consists of 1805 toxic peptides (≤ 35 residues). the physiochemical characteristics of the peptide sequences were determined using the protparam tool (https ://web.expas y.org/protp aram/) of the expasy database server [57] (fig. 1 ). the crystal structure of the sars-cov-2 spike protein with the ace2 pd domain (pdb id: 6m17) was retrieved from the protein data bank (https ://www.rcsb.org/). the interface residues between the sars-cov-2 spike protein and the ace2 pd domain were visualized and interpreted using ucsf chimera software [51] . after a detailed analysis of interface residues, a small stretch of the ace2 pd n-terminal region (23-amino acids: glu23 to leu45) was found to be interacting majorly with the sars-cov-2 spike protein ( fig. 2 and table 1 ). furthermore, we evaluated whether the 23-amino acid chain alone and without the remainder of the ace2 pd domain could bind to the sars-cov-2 spike protein. hence, the 23aa sequence was taken from the ace2 pd domain and docked to the sars-cov-2 spike protein (6m17) using the pydock program [52] . to perform a non-biased analysis, we performed a blind docking run whereby we did not specify the binding site during the docking simulations. the obtained results were clustered and analyzed by comparing the binding energies and docked conformations of the peptide within the sars-cov-2 spike protein (table 2) . interestingly, we found that the peptide binds to the sars-cov-2 spike protein at exactly the same place where the original ace2 pd domain interacts, which shows that the 23-amino acid sequence independently has the potential to inhibit the interaction of the sars-cov-2 spike protein and ace2 complex. after the docking analysis, the sars-cov-2/ace2 peptide interface was determined and the critical interacting amino acids were identified in the ace2 pd domain-derived peptide involved in binding to the sars-cov-2 spike protein ( table 2) . computational alanine (a) scanning was performed to identify the critically important amino acids of the 23aa peptide inhibitor involved in binding to the sars-cov-2 spike protein. the approach of alanine scanning contributes to estimating the binding energy of each residue to the total binding energy of the original peptide [58] . the process involves the substitution of the amino acid with alanine and records the resulting binding energy changes by molecular docking of the substituted three-dimensional structure of the peptide. if substitution by alanine results in a significant drop in overall free binding energy, those residue(s) are considered the critically important residues for binding [59] . hence, we performed alanine scanning of the 23aa peptide to determine the significance of each residue having the inhibitory potential of independently interacting with the ace2 binding region of the sras-cov-2 spike protein. all amino acids in the peptide were independently mutated to alanine using biovia discovery studio 2020 software [50] . alanine-substituted peptides were then modeled and minimized using the ucsf chimera program [51] . (fig. 3) . however, the mutated residues f28a, f32a, h34a, f40a, y41a, and l45a were found to be critically important for binding and stabilizing the peptide-sars-cov-2 spike protein complex (fig. 3) . we observed the change from − 55.873 kcal/mol to − 42.996 drop in the total binding score (fig. 3) . furthermore, we deleted the non-significant (identified through alanine scanning) residues and found the deletion of any residue from f28 to l45 would make an unstable structure. hence, we removed five residues (e23, q24, a25, k26, t27) from the n-terminal region of the peptide and retained residues from f28 to l45, resulting in the final 18aa peptide. in addition to the molecular mechanistic values such as electrostatics and table 1 ). vdw is usually a weak force, a type of intermolecular force enhancing the attraction and binding of a ligand to its receptor. vdw forces are involved in the maintenance and stabilization of a drug-receptor complex. replacing the respective amino acids (f28, f32, h34, f40, y41, l45) with alanine significantly reduces the binding score, either by directly losing the interaction or by changing the peptide conformation, which becomes less compatible with the sars-cov-2 spike protein. hence, based on the results of alanine scanning, the peptide was shortened from the extreme n-terminus due to its dispensability in sars-cov-2 binding. the final peptide derivative (f28, l29, d30, k31, f32, n33, h34, e35, a36, e37, d38, l39, f40, y41, q42, s43, s44, l45) was further docked and analyzed to validate its binding efficiency. we utilized three state-of-the-art programs (pydock [37] , zdock [39] and haddock 2.4 [38] ) to validate our docking studies. the top poses retrieved from each software package resulted in conformations very close to the original 23-amino acid peptide, thereby validating its efficacy as a potent sars-cov-2 inhibitor (fig. 4 ). the final 18aa peptide, with a molecular weight of 2203.39, has an amino acid composition of ala (a) 5.6%, asn (n) 5.6%, asp (d) 11.1%, gln (q) 5.6%, glu (e) 11.1%, his (h) 5.6%, leu (l) 16.7%, lys (k) 5.6%, phe (f) 16.7%, ser (s) 11.1% and tyr (y) 5.6%. the total number of negatively charged residues was four (asp and glu) and the table 2 interacting residues (1) at the interface of sars-cov-2 and the docked 18 aa peptide; and (2) at the interface of sars-cov-2 and the docked 23 aa peptide 1) at the interface of sars-cov-2 (blue) and the docked 18 aa peptide (red). the underlined and bold residues are making strong interactions between the peptides and sars-cov-2 spike protein sars-cov-2 severe acute respiratory syndrome coronavirus 2, ace2 angiotensin-converting enzyme 2, pd peptidase domain total number of positively charged residues was two (lys and his). the extinction coefficient calculated in units of m −1 cm −1 , at 280 nm measured in water, was found to be 1490 ± 10. it is useful to have an estimation of this coefficient for spectrophotometrically following a protein when purifying it. the half-life is a prediction of the time it takes for half of the amount of protein in a cell to disappear after its synthesis in the cell. the presently used expasy tool, protparam, relies on the 'n-end rule', which relates the halflife of a protein to the identity of its n-terminal residue; the prediction is given for three model organisms (human, yeast and escherichia coli). considering the n-terminal of the sequence considered is f (phe), the estimated half-life is 1 .1 h (mammalian reticulocytes, in vitro) . the instability index (ii) is computed to be 68.65, which classifies the protein as unstable. the physical stability of peptides is influenced by both intrinsic and external factors. prediction of peptide stability suggests precautionary steps be taken to make the therapeutically potent unstable peptides stable through certain biochemical analysis. the predicted aliphatic index of the peptide was found to be 70.56, with a grand average of hydropathicity (gravy) of − 0.522. toxinpred was used to predict the toxicity of the 18aa peptide with desired toxicity by mutating the minimum number of amino acids [40] . the 'toxinpred' tool allows us to submit query peptide in the fasta format and to optimize the peptide sequence to obtain the maximum/minimum/desired toxicity based on the quantitative matrix-based position-specific scores by comparing them with the toxic/non-toxic data set. the in-silico predicted 'toxinpred' toxicity results of the 18aa peptide indicate that it is non-toxic compared with the mutated peptides. a25 f28 l29 f32 e33 a36 l39 f40 y41 q42 s43 s44 l45 sars-cov-2: r403 d405 e406 r408 q409 g416 k417 i418 y453 r454 l455 f456 v483 e484 g485 f486 n487 c488 y489 q493 y495 g504 the respective docked complexes of the 23aa and 18aa peptides with target protein were subjected to md simulation studies for analysing their stability in terms of root mean square deviation (rsmd) and potential interactions at 50 ns. the stability of these two protein-peptide complexes was analyzed using the rmsd of backbone atoms. the calculated average rmsd of these complexes was in the range of 0.94-1.48 nm and 0.24-0.8 nm for the 23aa and 18aa peptide complexes, respectively (fig. 5) . by the end of the 50 ns time-scale simulation, both of the protein-peptide complexes attained their stable conformations (fig. 5) . the lower rmsd values obtained after comparing the initial and final peptide complex conformations (fig. 5b) revealed that the docked complexes were stable and hence there was not much difference in their conformations during the course of dynamics simulation. several advantages of peptides, including ease of synthesis and modifications, low toxicity, and high target specificity and selectivity, led us to design a potential therapeutic candidate against covid-19. the present in-silico study is the first step towards designing inhibitory peptides to block sars-cov-2 entry into the host cell. the peptide could inhibit binding of the sars-cov-2 spike protein with the ace2 pd domain, which is the earliest stage of sars-cov-2 infection. the findings of this study suggest that a peptide of 18-amino acids could be considered as a selective therapeutic candidate for covid-19. the study conducted by han and kral shows similar aspects towards designing a peptide inhibitor [49] . the identification of 15 interacting residues of an ace2 pd with an rbd of the sars-cov-2 spike protein at a 3 å region in the crystal structure (pdb 6m17) is in agreement with the findings of han and kral [49] . however, in comparison with our structurally stable 18aa peptide inhibitor comprising the ace2 pd residues ranging from positions 28-45, their inhibitor 1 peptide comprising residues 21-55 is structurally unstable and non-specific; hence, they made multiple modifications to it, which thereby changes its properties as well as its similarity to our peptide. out of a total of 15 interacting residues of ace2 pd, 10 residues are in the stretch of q24 to l45. utilizing this fact in our study, each residue in the 23-45 stretch (23aa peptide) of the ace2 pd was estimated for functional significance and structural stability based on their binding energy and conformational stability. in contrast to inhibitor 1, we obtained a stable 18aa peptide inhibitor where non-significant residues were truncated and the non-interacting residues within the 18aa peptide at the interfacial site were found important, both for binding and stability of the peptide. hence, inhibitor 1 from han and kral differs in structural stability, composition and specificity due to the presence of non-significant residues leading to deformity in its helical structure. provision of experimental investigations of the desired synthesized peptide in combination with our computational studies will be needed to confirm and complete this study. it is recommended to compare our computational results with the designed peptides in vitro and in vivo, and eventually 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open source molecular simulation toolkit in silico approach for predicting toxicity of peptides and proteins protein identification and analysis tools on the expasy server computational prediction of protein hot spot residues unraveling hot spots in binding interfaces: progress and challenges the authors also gratefully acknowledge the indian institute of technology indore (iiti) for providing facilities and other support. the authors are thankful to biovia-dassault systemes for providing sars-cov-2/2020 discovery studio academic research suite license. author contributions msb conceived, designed, and executed the research. ma, us, and sr compiled and analyzed the data. msb has written the manuscript. us reviewed and edited the manuscript. funding this study is supported by the indian institute of technology indore (iiti) india. open access this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by-nc/4.0/. key: cord-339859-anatn295 authors: paret, michal; lighter, jennifer; pellett madan, rebecca; raabe, vanessa n; shust, gail f; ratner, adam j title: sars-cov-2 infection (covid-19) in febrile infants without respiratory distress date: 2020-04-17 journal: clin infect dis doi: 10.1093/cid/ciaa452 sha: doc_id: 339859 cord_uid: anatn295 we report two cases of sars-cov-2 infection (covid-19) in infants presenting with fever in the absence of respiratory distress who required hospitalization for evaluation of possible invasive bacterial infections. the diagnoses resulted from routine isolation and real-time rt-pcr-based testing for sars-cov-2 for febrile infants in an outbreak setting. m a n u s c r i p t 3 sars-cov-2 infection (covid-19) has rapidly emerged as a worldwide cause of severe respiratory disease in adults. [1] early reports indicate that the course of disease is generally milder in children, but fatal cases have been described. [2] [3] [4] even in the setting of asymptomatic or mildly symptomatic infection, children may represent a source of sars-cov-2 spread in community or hospital settings, so understanding the spectrum of covid-19 illness in infants, particularly regarding conditions that result in hospitalization, is crucial to establishment of effective infection control interventions. [5] in the first months of life, infants presenting with fever frequently undergo diagnostic evaluations for invasive bacterial disease, even in the absence of other clinical signs. such evaluations generally include cultures of blood, urine, and, in some cases, cerebrospinal fluid (csf), followed by observation and empiric antibiotic therapy in a hospital setting. [6] a significant percentage of febrile infants have infections with respiratory viruses, including respiratory syncytial virus, enteroviruses, and influenza. [7] these viral infections may occur in the presence or absence of invasive bacterial infections. over a oneweek period in late march 2020 corresponding to a time of widespread community transmission of sars-cov-2 in new york city, we encountered two febrile infants presenting with minimal or no respiratory symptoms who were found to have sars-cov-2 infection without other etiologies despite thorough evaluations. case 1: a 25 day-old full-term male infant was brought to the pediatric emergency department because of fever (38.5°c (101.3°f) on arrival) and irritability. he had had no cough, tachypnea, rhinorrhea, or respiratory distress. there was no change in feeding, and no vomiting or diarrhea was reported. the child had not travelled. both parents were symptomatic with sore throat and subjective fever in the prior two days but had not sought medical attention for themselves. there were no other ill contacts and no known a c c e p t e d m a n u s c r i p t 4 contacts with sars-cov-2 infection. on examination, the patient was alert and active and was noted to have an erythematous, papular facial rash. the remainder of the physical examination was within normal limits. samples of blood, urine, and csf were obtained for analysis. vital signs and pertinent laboratory findings appear in the a real-time rt-pcr assay performed at the new york state department of health detected sars-cov-2 rna in the patient's np sample. empiric therapy with parenteral ampicillin and cefepime was started on admission and continued until the blood, urine, and csf cultures were negative for >48 hours. no antiviral medications were given. the patient was discharged to home in stable condition with infection precautions consistent with centers for disease control and prevention guidelines. [8] case 2: a 56 day-old male infant born at 35 weeks gestation who had had an uneventful perinatal course presented to the hospital with a temperature of 38.2°c (100.8°f) taken rectally at home. the child had no respiratory or gastrointestinal symptoms and had normal oral intake and activity level. his mother, father, and siblings were all well, and there were no other known ill contacts and no history of travel. in the emergency department, the child was febrile but well appearing, with a normal physical examination. vital signs and laboratory findings appear in the table. blood and urine cultures were done, but sampling of the csf was not performed, nor was radiographic imaging. the biofire respiratory pcr panel performed on an np sample was negative, and the np sars-cov-2 real-time rt-pcr (cobas, roche) a c c e p t e d m a n u s c r i p t 5 was positive. the child was treated empirically with parenteral ceftriaxone until the results of blood and urine cultures were negative for >36 hours. no antiviral medication was used, and the patient was discharged in stable condition with infection control guidance. these two cases present a common problem in pediatric medicinethe febrile infantwith an important twist, diagnosis of sars-cov-2 infection during an explosive community-based outbreak in new york city. the epidemic coronaviruses, including mers-cov, sars-cov, and sars-cov-2, have the potential for spread within healthcare settings, making case identification and prompt isolation crucial to protecting patients, physicians, and staff. [9] [10] [11] in the context of an ongoing outbreak, we encouraged routine testing of febrile infants for sars-cov-2, even in the absence of respiratory symptoms. for both cases presented above, because sars-cov-2 testing was sent, and contact/droplet/eye shield precautions were instituted in the emergency department, with n95 masks used during np swab collection because of the potential for aerosol generation. in addition, family members were required to wear surgical masks and upon discharge received instructions for home isolation. we did not find non-sars-cov-2 etiologies for fever in these two infants, and fever without localizing signs has been reported in older children with sars-cov-2. [5] lymphopenia has been described in adult patients with sars-cov-2 infection, and we noted a low absolute lymphocyte count in patient 2. [11] testing for sars-cov-2 in non-np samples was not performed in either child, and both infants had a benign clinical course. however, the need to routinely hospitalize febrile infants for workup of potential invasive bacterial disease may serve as an characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention epidemiology of covid-19 among children in china detection of covid-19 in children in early cdc covid-19 response team. coronavirus disease 2019 in children -united states characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding a prediction model to identify febrile infants </=60 days at low risk of invasive bacterial infection the risk of serious bacterial infection in febrile infants 0-90 days of life with a respiratory viral infection discontinuation of transmission-based precautions and disposition of patients with covid-19 in healthcare settings (interim guidance) hospital outbreak of middle east respiratory syndrome coronavirus sars in healthcare facilities clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china the authors are grateful to the members of the department of pediatrics at nyu grossman school of medicine, nyu langone health/hassenfeld children's hospital, and bellevue hospital center for their dedication to outstanding pediatric care under the extreme conditions of the sars-cov-2 outbreak. the authors received support from the department of pediatrics at nyu grossman school of medicine.there was no external funding for this work. a.j.r. has served as a consultant to pfizer outside the scope of this work. all other authors report no conflicts. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-318316-9unfl966 authors: ortega, joseph t.; zambrano, jose l.; jastrzebska, beata; liprandi, ferdinando; rangel, hector r.; pujol, flor h. title: understanding severe acute respiratory syndrome coronavirus 2 replication to design efficient drug combination therapies date: 2020-10-23 journal: intervirology doi: 10.1159/000512141 sha: doc_id: 318316 cord_uid: 9unfl966 background: the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and its disease co­vid-19 has strongly encouraged the search for antiviral compounds. most of the evaluated drugs against sars-cov-2 derive from drug repurposing of food and drug administration-approved molecules. these drugs have as target three major processes: (1) early stages of virus-cell interaction, (2) viral proteases, and (3) the viral rna-dependent rna polymerase. summary: this review focused on the basic principles of virology and pharmacology to understand the importance of early stages of virus-cell interaction as therapeutic targets and other main processes vital for sars-cov-2 replication. furthermore, we focused on describing the main targets associated with sars-cov-2 antiviral therapy and the rationale of drug combinations for efficiently suppressing viral replication. key messages: we hypothesized that blocking of both entry mechanisms could allow a more effective antiviral effect compared to the partial results obtained with chloroquine or its derivatives alone. this approach, already used to achieve an antiviral effect higher than that offered by every single drug administered separately, has been successfully applied in several viral infections such as hiv and hcv. this review will contribute to expanding the perception of the possible therapeutic targets in sars-cov-2 infection and highlight the benefits of using combination therapies. in the last trimester of 2019 a new coronavirus, named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and responsible for coronavirus disease 2019 , emerged in wuhan, china. there is no yet an approved therapy to treat the infection by sars-cov-2 and its associated disease, covid-19, and the therapeutic approaches used have been changing from the beginning of the outbreak [1] [2] [3] . the standard care of patients with covid-19 is mainly focused on symptomatic and respiratory support according to the diagnosis and treatment of pneumonia and coagulopathies caused ortega by covid-19 [4] . several research groups have been working to determine the possible targets to control the viral infection [5] [6] [7] . three major targets have been identified and can be summarized in the following: early stages of virus-cell interaction, viral proteases, and the viral rna-dependent rna polymerase (rdrp) [7] [8] [9] . the replication cycle of sars-cov-2 and the main targets for the antiviral therapy described in this review are shown in figure 1 . after interaction with its receptor ace2, sars-cov-2 enters the cell through the fusion of its viral membrane with the cellular one ( fig. 1 ) [10] . the viral fusion protein is exposed by priming with a proteolytic enzyme. three different types of proteases have been proposed for this cleavage: (1) priming by the cellular transmembrane serine protease 2 (tmprss2) and also tmprss4 in enterocytes. this priming allows viral entry by the early endosomal pathway, although the exact mechanism of entry is unknown [8, 11, 12] . (2) priming by cathepsin b in the late endosomes [13] . (3) activation during virus egress from the cell by furin [14] . furin-dependent pre-cleavage of the spike has been shown to be necessary for activation through the tmprss2 pathway [15] . in the early stages of infection, different therapeutic agents have been used, including hydroxychloroquine (hcq) [16, 17] , benzoic acid derivatives such as nafamostat and camostat [18] , and neutralizing antibodies [19] . the antiviral effect of hcq is related to an elevation of the ph in endosomes/lysosomes, which is essential for the fusion between the viral and vesicle membranes. in addition, hcq could inhibit sars-cov cellular entry by changing the glycosylation pattern of ace2, the key receptor for sars-cov-2 entry [16, 17] . furthermore, hcq exerts immunomodulatory effects through attenuation of cytokine production [20] . the effects of hcq on cellular targets are considered nonspecific antiviral effects. another early target evaluated against sars-cov-2 is a cellular protease related to the priming of the spike protein (s), which exposes the fusion motive and allows the release of viral rna into the cytosol. the proteolytic processing of the viral spike protein is a key step during the infection process of sars-cov-2. this reaction is driven by tmprss2 and could be inhibited by camostat, nafamostat, and bromhexine [18, 21] . interestingly, some reports showed that sars-cov-2 could gain entry into the host cell without using the late endocytic pathway. the processing of tmprss2 in the extracellular lumen could likely be sufficient to activate the fusion between the viral and host membrane [22, 23] . this alternative entry pathway would explain the low success rates in the use of hcq/chloroquine alone in covid-19 therapy. in addition, chloroquine does not seem to prevent sars-cov-2 entry into human lung cells, suggesting that the tmprss2-primed pathway predominates in the lung [24] . arbidol (umifenovir) is an antiviral agent that targets s/ace2 interaction, inhibiting membrane fusion of the viral envelope [2] . this compound could be considered as an early-stage inhibitor of sars-cov-2. another therapy for covid-19 is the use of convalescent plasma or hyperimmune immunoglobulins (nabs) [25] . antibodies from recovered patients or neutralizing monoclonal antibodies may neutralize the free virus and induce the immune clearance of infected cells. on march 24, 2020 the food and drug administration (fda) released an emergency approval for the use of covid-19 convalescent plasma. the development of neutralizing monoclonal antibodies against sars-cov-2 infection is ongoing [19, 26] . other promising therapeutic targets against sars-cov-2 infection are viral proteases ( fig. 1 ) due to their low homology with host protease and the structural knowledge available for sars-cov [7, 27] . sars-cov-2 has two viral proteases related to the proteolytic processing of the viral polyprotein into mature and functional proteins. these enzymes are the main protease (3cl pro ) and the papain-like protease (pl pro ), both belonging to the family of cysteine proteases. they exhibit a high structural and functional homology with sars-cov proteases [27] [28] [29] . the main protease is responsible for the proteolysis of pre-proteins associated with the viral replication machinery such as the rdrp, the helicase, and the exoribonuclease, among others [29] . several groups have shown by using in silico and in vitro approaches that this protease may be blocked by protease inhibitors effective in hiv therapy [7, 27, 29, 30] . furthermore, lopinavir/ ritonavir, an fda-approved oral drug combination for treating hiv, exhibited in vitro activity against other coronaviruses. however, in the case of sars-cov-2 infection, the timing of the administration of these drugs during the early peak of the viral replication phase (at initial 7-10 days) appears to be critical. delayed initiation of fig. 1 . sars-cov-2 replication. the viral cycle begins with the interaction between the viral spike and the cellular receptor. several membrane proteins have been proposed as possible receptors for sars-cov-2; however, ace2 is likely to be the most important one [10] . after the viral spike has interacted with the receptor, the virus gains entry into the host cytosol by two mechanisms: (1) by late endocytosis, releasing the viral rna after the fusion with the lysosome (this entry is blocked by hcq), or (2) by early endocytosis by fusion of the viral and host membrane without the participation of the lysosome. in these early stages, the priming processing by cellular proteases is the key for the exposure of the viral fusion motive. tmprss2 could act in both early and late endosome entry processes and could be inhibited by benzoic acid derivatives such as nafamostat, camostat, and bromhexine [18, 21] . furthermore, other proteases such as cathepsin b could mediate the entry in the late lysosomal pathway, while only tmprss2 has been related to the early entry endosome [11, 13, 15, 18] . after fusion has occurred, the viral rna is released into the cytoplasm and open reading frame 1 (orf1) is translated to produce the rdrp. subgenomic mrnas are produced by discontinuous transcrip-tion, a process characteristic of this rdrp, which favors recombination. compounds such as remdesivir, favipiravir, and sofosbuvir block this enzyme [39] [40] [41] 45] . the subgenomic mrnas are then translated into protein. the genome has eight orfs. the gene segments that encode nonstructural polyproteins are processed first and translated into orf1a and orf1b producing pp1a and pp1ab proteins, respectively. protein pp1a and pp1ab are cleaved by the viral proteases (3cl pro and pl pro ). the main protease is also the target of protease inhibitors such as lopinavir/ritonavir [7] . the structural proteins -spike, envelope, and membrane proteins -enter into the endoplasmic reticulum/golgi complexes. then, the nucleoprotein combines with the (+) strand genomic rna (nucleoprotein complex) and merges with the other structural proteins in the endoplasmic reticulum-golgi apparatus compartment [64] . finally, the virion is excreted to the extracellular region through the exosomal pathway [64] . hcq, hydroxychloroquine; rdrp, rna-dependent rna polymerase; sars-cov-2, severe acute respiratory syndrome coronavirus 2; tmprss2, transmembrane serine protease 2; orf, open reading frame. ortega/zambrano/jastrzebska/liprandi/ rangel/pujol intervirology 4 doi: 10.1159/000512141 therapy with lopinavir/ritonavir did not result in positive clinical outcomes mainly due to the high viral load [31] . another important concern with lopinavir/ritonavir therapy against sars-cov-2 is related to the administration route. this drug combination is administered to patients with covid-19 by the oral pathway, applicable for drugs with good gastrointestinal absorption and low volume of distribution (the volume of distribution for lopinavir/ritonavir is around 17 l) [32, 33] . however, these compounds bind tightly to plasma proteins (in the plasma, > 98% of the drug is in bound form), which decreases the ability to reach tissues such as the lung [32] . this important pharmacological issue related to the pharmacokinetic parameters of lopinavir/ritonavir needs to be addressed in order to improve the therapeutic outcome of treatment with this drug combination. the second protease related to the sars-cov-2 replication cycle, pl pro , is responsible for the cleavages of the n-terminus of the polyprotein to release nsp1, nsp2, and nsp3, nonstructural proteins associated with viral replication [34] [35] [36] . in addition, pl pro also possesses de-ubiquitination and de-isgylation activities that could antagonize the host's innate immunity [37] . the sars-cov-2 genome replication is carried out by an rdrp and could be one of the best targets in the sars-cov-2 replication (fig. 1) [38] . this enzymatic activity has no parallel process in the eukaryotic cells. thus, attacking this target might reduce side effects that might occur by affecting any similar protein in the host. the sequences of rdrp in sars-cov and sars-cov-2 have a high homology and encode structurally similar proteins. remdesivir is a nucleotide analog inhibitor of rdrp and has shown a broad spectrum of antiviral activity against several rna viruses [38, 39] . remdesivir similarly to sofosbuvir, a direct-acting antiviral used in hcv therapy, is a chain-terminating nucleotide analog. these drugs produce their therapeutic effect by directly interacting with the rdrp and incorporating the active form of the inhibitor into the growing rna strand, preventing the replication to continue [40, 41] . several clinical trials have been conducted evaluating the activity of remdesivir and other direct-acting antivirals used in hcv against sars-cov-2 [42] . only remdesivir reached the fda emergency approval status for its use in patients with severe covid-19. however, the clinical improvements by using remdesivir alone in sars-cov-2 patients are modest. favipiravir, another antiviral agent with broad activity against other rna viruses by inhibiting the rdrp, halting viral replication, was evaluated against sars-cov-2, showing effects in vitro and in vivo [43] [44] [45] . drug combinations that have been widely used in antiviral therapy have become the leading choice for treating hiv and hcv, among other viral pathologies [46] [47] [48] [49] [50] . the use of multiple drugs with different mechanisms of action increases the efficacy of the therapeutic effect. it also allows decreasing the dose of a single drug, thus preventing host toxicity [51] [52] [53] [54] . moreover, one important advantage of combination therapies besides enhancing the selective effect over the target is that this approach minimizes or slows down the development of drug resistance, which is the main issue in antiviral therapy of rna viruses. experimental conditions for drug combinations in vitro can be easily defined. information for most individual drugs used against sars-cov-2 is available, thus conducting in vitro assays for the effect of their combinations could take a very short time (approximately < 2 weeks) by using methods such as the combination indexisobologram method of chou [55] . however, there are some issues associated with the evaluation of drug combinations in clinical trials related to dose, timing, route of administration, efficacy, and toxicity, among others, that make the extrapolation of in vitro data difficult. these factors are associated with, but not limited to, variables in the patient population such as sex, age, race, and disease stage. furthermore, drug combination trials need to fulfill all the ethical concerns to treat patients with placebo or suboptimal therapeutic doses as required for a drug combination study design according to the guidelines of regulatory agencies, such as the fda. as explained above, hcq could produce a non-directacting specific antiviral effect against sars-cov-2 by several mechanisms. however, the results obtained in the clinical trial by using hcq alone were less satisfactory than expected [56] . although hcq affects the early stages of the viral infection, i.e., the endosome-mediated entry, some reports showed that sars-cov-2 could enter the host cells bypassing the pathways blocked by hcq [15] . thus, we hypothesize that this could be avoided by combining hcq and camostat or nafamostat [18] or bromhexine [21, 57] . these tmprss2 inhibitors could block the other mechanism associated with sars-cov-2 entry. in addition to interfering with the different proteases involved in the activation of the fusion peptide of s, a combined scheme might include an inhibitor of this domain [8] . furthermore, we support the fact that other tmprss2 inhibitors such as bromhexine, a drug used as mucolytic, could be an important pharmacological tool to design prophylactic therapies. bromhexine has a well-combination therapies against sars-cov-2 5 intervirology doi: 10.1159/000512141 known drug safety profile and it is marketed as an overthe-counter medication. thus, the design of clinical trials and extrapolation into effective therapy should be less complicated in comparison with drugs as camostat or nafamostat. furthermore, some reports showed that bromhexine could overcome the main pharmacokinetic problem in sars-cov-2 therapy, reaching the pulmonary and bronchial epithelial cells in concentrations higher than those found in the plasma [58] . the above combination targets viral entry into the cell. similarly, neutralizing antibodies and fusion inhibitors could be combined in a triple combination format. most of the targets associated with the early stages of the viral infection are host targets that significantly decrease the probability of developing resistance in comparison with viral targets [51] . the early study published by cao et al. [59] showed that lopinavir/ritonavir could produce a modest positive effect in comparison to the standard care in patients with sars-cov-2. however, several researchers have criticized the structural design of the study of cao et al. [59] , suggesting that the antiviral effect of lopinavir/ritonavir against sars-cov-2 could be higher; thus, more studies are needed [60] [61] [62] . nevertheless, the side effects associated with these protease inhibitors are important. thus, antiviral therapy based on combining these protease inhibitors with another antiviral compound such as remdesivir could decrease the effective dose required to produce the antiviral effect and then decreasing the possibility of side effects. the possibility of the occurrence of viral resistance with this strategy should be kept in mind. of the 1,265 clinical trials based on drugs to treat sars-cov-2 registered in the national institutes of health library, only 113 (< 10%) are based on drug comthe studies shown were retrieved from the website clinicaltrials.gov (see also fig. 2 ). sars-cov-2/covid-19 as condition or disease and the drug names and their combinations were used as keywords for the search. drugs related to blocking early stages of viral replication or viral enzymes are shown in bold. covid-19, coronavirus disease 2019; nih, national institutes of health; sars-cov-2, severe acute respiratory syndrome coronavirus 2. clinical trials using a combination of drugs targeting early replication steps or viral enzymes. an exhaustive revision of the ongoing clinical trials related to sars-cov-2 was performed using the database of the national institutes of health (nih) of the united states. the data were accessed from the nih webserver clinicaltrials.gov. the search terms were "covid" or "sars-cov-2," resulting in 3,009 trials. then, the search parameter drug was applied as a further term, retrieving 1,616 clinical trials. in each search, the trials included those not yet recruiting, recruiting, active, or completed. a visual inspection of each of the 1,616 trials was conducted and only 1,265 included drugs as the main intervention. moreover, the drug combination trials were also reviewed, and only 113 included a combination of pharmacological therapy for the treatment of covid-19 patients. the database search was conducted on august 14, 2020. covid-19, coronavirus disease 2019; hcq, hydroxychloroquine; sars-cov-2, severe acute respiratory syndrome coronavirus 2. ortega/zambrano/jastrzebska/liprandi/ rangel/pujol intervirology 6 doi: 10.1159/000512141 binations. one-third of them include hcq under combination therapy (fig. 2) . table 1 summarizes the ongoing clinical trials combining drugs targeting early viral replication events or viral enzymes. altogether, the virology and pharmacological principles discussed in this review emphasize the importance of early stages of the infection as therapeutic targets and the advantage of simultaneously blocking alternative processes critical for viral replication. the development of effective therapies against this new viral pathogen has proven to be a challenge. useful in vitro and in vivo models are needed to address the effectiveness of drugs or their combination to be used as preclinical data, followed by randomized clinical trials. prior studies with sars-cov might help to develop and validate models for this new infection. more studies will help to fully understand the possible advantages of drug combinations against sars-cov-2 infection. based on the experience obtained with the treatment of other viral infections, the combination of two or more antiviral drugs could produce a better effect than the one reached with a single drug. these antiviral effects could be additive or synergistic, depending on the degree of inhibition of viral load. furthermore, besides the improvement in the antiviral effect achieved, an eventual reduction of the dosage used could represent fewer side effects associated with the therapy. thus, enhancing the antiviral effect and reducing side effects could represent a main change in sars-cov-2 therapy. there are at present many concerns because of the toxicity of chloroquine or its derivative [24] . drug 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coronavirus disease 2019 (covid-19): a state of the art review. otolaryngol head neck surg morphology, genome organization, replication, and pathogenesis of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) the authors have no conflicts of interest to declare. none. key: cord-350352-wgppovfx authors: temmam, sarah; barbarino, alix; maso, djérène; behillil, sylvie; enouf, vincent; huon, christèle; jaraud, ambre; chevallier, lucie; backovic, marija; pérot, philippe; verwaerde, patrick; tiret, laurent; van der werf, sylvie; eloit, marc title: absence of sars-cov-2 infection in cats and dogs in close contact with a cluster of covid-19 patients in a veterinary campus date: 2020-08-29 journal: one health doi: 10.1016/j.onehlt.2020.100164 sha: doc_id: 350352 cord_uid: wgppovfx severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which originated in wuhan, china, in 2019, is responsible for the covid-19 pandemic. it is now accepted that the wild fauna, probably bats, constitute the initial reservoir of the virus, but little is known about the role pets can play in the spread of the disease in human communities, knowing the ability of sars-cov-2 to infect some domestic animals. in this cross-sectional study, we tested the antibody response in a cluster of 21 domestic pets (9 cats and 12 dogs) living in close contact with their owners (belonging to a veterinary community of 20 students) in which two students tested positive for covid-19 and several others (n = 11/18) consecutively showed clinical signs (fever, cough, anosmia, etc.) compatible with covid-19 infection. although a few pets presented many clinical signs indicative for a coronavirus infection, no antibodies against sars-cov-2 were detectable in their blood one month after the index case was reported, using an immunoprecipitation assay. these original data can serve a better evaluation of the host range of sars-cov-2 in natural environment exposure conditions. sars-cov-2 infection has presented unprecedented challenges related to viral disease control and prevention worldwide. while the emergence of the virus is now welldocumented, important questions regarding the transmissibility of the disease among human populations remain to be answered. sars-cov-2 might infect animals [1] , including dogs, cats, ferrets [2] [3] [4] or minks [5] . therefore, the hypothesis that small domestic animals could serve as intermediate or amplification hosts of the virus needs j o u r n a l p r e -p r o o f to be further addressed in a one health approach [6, 7] . to increase the knowledge regarding transmission rates in circumstances of contamination that ensure efficient human-to-human transmission, we have investigated the infection status of dogs and cats living in close proximity with a cluster of veterinary students who developed covid-19 symptoms in winter 2019-2020, i.e. during the initial phase of the first wave of the pandemic in france. we investigated the presence of sars-cov-2 infection of domestic cats (n = 9) and dogs (n = 12) living in close contact with a cluster of french veterinary students, their owners (n = 18), whose median age was 23 years (21-28 years). 14/18 students lived in university residences with common traffic areas and daily outdoor activities to allow animals, especially dogs, to relieve themselves in spaces that were shared by all animals. the other four students lived outside the residence, with reduced contact during lockdown, except for one student who lived with a roommate. all 18 students had contact with at least one sick person within three weeks prior sampling and 11 of them developed symptoms compatible with covid-19 between february 25 th and march 18 th , 2020. the symptoms were mainly cough, with or without fever. headaches, dyspnea and dizziness were reported for one case. among symptomatic students, two were tested positive for sars-cov-2 by rt-pcr. the other nine symptomatic patients were not tested in accordance with french regulations that, at the time of sampling, did not request to test all patients in such clusters. other owners (7/18) were asymptomatic at the time of sampling. all of the owners were in the close vicinity of their pets, e.g. living in the same room -of 12 to 17 square meters -and sharing the same bed (8/8 of cats' owners and 4/12 of dog's owners); accepting j o u r n a l p r e -p r o o f face/hand licking (6/8 and 11/12 of cats' and dogs' owners, respectively). all cats were domestic european shorthair cats. dogs included in the sampled panel were either cross-bred (n = 6) or purebred individuals from the labrador retriever, shetland sheepdog, belgian malinois and white swiss shepherd breeds (n = 6). the average age of these sampled adult animals was 3.3 years for cats (min: 6 months; max: 6.5 years) and 2.7 years for dogs (min: 4 months; max: 8 years). small pets were free of clinical signs, except some respiratory or digestive signs reported for three cats, independently. sera from this per-epidemic cohort of pets (hereafter named "pets epidemic", n = 21) were freshly prepared from blood collected on march 25 th , one month after the index case. a panel of biobanked sera was also obtained from a second cohort (named "pets pre-epidemic" n = 62), composed of animals sampled before the covid-19 pandemicbetween october 2015 and october 2018, containing 55 dogs from 32 popular breeds and seven cats including five european shorthair, one turkish angora and one devon rex. the search for antibodies against sars-cov-2 was carried out on the 79 sera of the two cohorts using a luciferase immuno-precipitation system (lips) assay validated and used previously [8] [9] [10] [11] , using two antigens: (i) the s1 domain of the sars-cov-2 s spike protein and (ii) the c-terminal part (residues 233-419) of the sars-cov-2 n nucleoprotein [12] . viral antigens were produced in hek-293f cells transfected with plasmids expressing the gene for nanoluc fused to the c-terminus of the viral protein. recombinant proteins were harvested from the supernatant (s1, which is a surface glycoprotein secreted in culture supernatant) or cell lysate (n, which is j o u r n a l p r e -p r o o f located inside the virus) without any purification step and incubated with 10 µl of animal serum. the immune complexes were precipitated with protein a/g-coated beads, washed, and the luciferase activity was monitored on a centro xs 3 lb 960 luminometer (berthold technologies, france). the positivity threshold was defined as the mean of 10 negative controls (without serum) + five standard deviations. human patients' sera collected before and during the course of the epidemic were used as negative and positive controls respectively. the lips assay was initially designed to maximize the specificity of the test (>95%) to ensure that human seasonal hcov were not detected when searching for sars-cov-2 ab, assuming a lower sensitivity. the sensitivity of the lips assay has been evaluated in mildly symptomatic humans as 91.4% when compared to the very sensitive cytometric assay [8] . sars-cov-2 specific antibodies were not detected in the "pets epidemic" cohort, and no statistical difference was observed compared to the "pets pre-epidemic" cohort ( figure 1 ). in addition, late nasal and rectal swabs were taken during one week, starting from the day of blood sampling (march 25 th ) and all animals tested negative for the presence of sars-cov-2 by rt-pcr (who.int/docs/defaultsource/coronaviruse/whoinhouseassays.pdf). we concluded that none of the animals included in this study had been or was infected by sars-cov-2, despite repeated close intra-species daily contacts on the campus, and more importantly despite frequent and lasting contacts with covid-19 patients confined to small rooms. several studies have investigated the susceptibility of domestic animals and their putative role in the current covid-19 pandemic (review in [1] ). ferrets, in particular, are used for modeling sars-cov-2 infection in humans [2] , understanding the route of transmission [13] and developing therapeutic strategies [14] . among seven domestic animal species tested, shi et al. [3] have demonstrated that ferrets, cats and dogs can be experimentally infected by the intranasal route, probably through the viral receptor angiotensin-converting enzyme 2 (ace2). accordingly, it has been recently demonstrated that ace2 from these species, as well as from others known to support sars-cov-2 infection, tolerates many amino acid changes with no decrement in its receptor function, a feature that predicts a low species barrier [15] . interestingly, key ace2 residues that distinguish susceptible from resistant species have been identified by alexander et al. [16] , in a scoring model that may also explain the highly contagious nature of the virus amongst humans. dogs, that express low levels of ace2 in the respiratory tract [15] , had a susceptibility score of 23 in the work of alexander et al. and were actually shown by shi et al. to be less susceptible to experimental infection than cats, which had a sars-cov-2 susceptibility score of 27 [3, 16] . cats, during their juvenile post-weaning life (70-100 days), appear to be more vulnerable than older ones [3] . shi et al. also reported that one out of three naïve cats exposed to infected cats became infected, showing that intra-species respiratory droplet transmission can occur in cats, at least from cats experimentally infected with 10 5 pfu by the intranasal route [3] . nasal shedding and subsequent transmission of the virus by direct contact between virus-inoculated cats has also been reported in asymptomatic cats by halfman et al. [17] . consistent with the increased susceptibility of this domesticated species, a study of j o u r n a l p r e -p r o o f the pandemic period, has reported a prevalence of 14.7% seroconverted cats, with cats owned by covid-19 patients (presumably adults cats) having the highest neutralization titers [18] . sars-cov-2 neutralizing antibodies have also been found in two cats and one dog among 10 cats and nine dogs recruited from covid-19 patients admitted to wuhan hospital in march 2020 [19] . on the other hand, a serological survey conducted in china in companion and street cats (n = 87) and dogs (n = 487) sampled from november 2019 to march 2020 has shown no detectable sars-cov-2 specific antibodies [20] . in the latter work, one dog was owned by a sars-cov-2 patient and two dogs were in close contact with it during the quarantine. in a collaborative work, we have described in belgium the first case in cat, identified in march, presenting with diarrhea followed by an acute respiratory syndrome [21] . in the united states, the first two domestic cats displaying respiratory illnesses alongside with sars-cov-2 infection were reported in april 2020 [22] . both cats were owned by persons with suspected or confirmed covid-19. in france, one cat with mild respiratory and digestive signs tested positive by rt-qpcr on rectal swab (but not on nasopharyngeal swab) with antibodies detected in the serum [23] . this case was reported from a series of 22 cats and 11 dogs whose owners were suspected or confirmed positive for covid-19. altogether, these results and the present finding highlight that natural infection of cats is possible but remains a rare event. consequently, the success of detecting sars-cov-2 seropositive pets seems to be linked to well-defined narrowed human/pets interfaces. although based on a small cluster of 21 domestic pets, our cross-sectional study based on the antibody response one month after exposure to the index case points to j o u r n a l p r e -p r o o f journal pre-proof undetectable interspecific transmission of the sars-cov-2 virus between covid-19 patients and domestic dogs or cats under natural exposure conditions. to support the absence of asymptomatic long portage in seronegative animals, late pcrs (sampling starting from march 25 th ) had been performed and were negative. given the susceptibility of cats inoculated intranasally with 10 5 pfu or by direct aerosol transmission from infected cats [3, 17] , it is conceivable that the infected and seroconverted cats identified in wuhan, china [18, 19] , had either been in contact with patients whose viral load was higher than in our study (which is supported by the fact that patients were hospitalized, conversely to our study in which positive students presented mild symptoms), or have had more contacts with infected cats. these questions among others will need to be further addressed. evidence for sars-cov-2 infection of animal hosts infection and rapid 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sars-cov-2 in cats and dogs from households in italy legend to figure 1: lips assay targeting antibodies against sars-cov-2 in domestic cats and dogs in close contact with a cluster of covid-19 patients = 21); from pets sampled before the epidemic (pets pre-epidemic, n = 62) were tested for the presence of antibodies directed to s1 (left panel) and the c-term (residues 233-419) part of the sars-cov-2 nucleoprotein (right panel), using a luciferase immunoprecipitation system assay (lips). the same assay was performed for humans unrelated to this study, either covid-19 human patients (symptomatic human patients epidemic we thank the pets' owners who gave us the authorization of sampling, gwenaelle leseigneur (veterinary nurse at the enva hospital for small animals) for her excellent technical help. we also thank funding from institut pasteur, labex ibeid (anr-10 the authors declare there is no conflict of interest. key: cord-337105-jlmh79qv authors: jacob, fadi; pather, sarshan r.; huang, wei-kai; zhang, feng; hao wong, samuel zheng; zhou, haowen; cubitt, beatrice; fan, wenqiang; chen, catherine z.; xu, miao; pradhan, manisha; zhang, daniel y.; zheng, wei; bang, anne g.; song, hongjun; carlos de la torre, juan; ming, guo-li title: human pluripotent stem cell-derived neural cells and brain organoids reveal sars-cov-2 neurotropism predominates in choroid plexus epithelium date: 2020-09-21 journal: cell stem cell doi: 10.1016/j.stem.2020.09.016 sha: doc_id: 337105 cord_uid: jlmh79qv neurological complications are common in patients with covid-19. while sars-cov-2, the causal pathogen of covid-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood. here we investigated the susceptibility of human induced pluripotent stem cell (hipsc)-derived monolayer brain cells and region-specific brain organoids to sars-cov-2 infection. we found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. we optimized a protocol to generate choroid plexus organoids from hipscs and showed that productive sars-cov-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. together, our findings provide evidence for selective sars-cov-2 neurotropism and support the use of hipsc-derived brain organoids as a platform to investigate sars-cov-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the pathogen responsible for the covid-19 pandemic, has infected over 29 million people and has contributed to over 936,000 deaths world-wide this year so far (who, 2020) . while the disease primarily affects the respiratory system, damages and dysfunction have also been found in other organs, including the kidney, heart, liver, and brain (yang et al., 2020b) . neurological complications, such as cerebrovascular injury, altered mental status, encephalitis, encephalopathy, dizziness, headache, hypogeusia, and hyposmia, as well as neuropsychiatric ailments, including new onset psychosis, neurocognitive syndrome, and affective disorders, have been reported in a significant number of patients (mao et al., 2020; varatharaj et al., 2020) . viral rna has been detected in the brain and cerebrospinal fluid (csf) of some patients with covid-19 and concomitant neurological symptoms (helms et al., 2020; moriguchi et al., 2020; puelles et al., 2020; solomon et al., 2020) . despite numerous reports of neurological findings in patients with covid-19, it remains unclear whether these symptoms are a consequence of direct neural infection, para-infectious or post-infectious immune-mediated disease, or sequalae of systemic disease (ellul et al., 2020; iadecola et al., 2020) . variability in patient presentation and variable timing of testing for viral rna in the csf or brain complicate the interpretation of results in human patients. limited availability of autopsy brain tissue from patients with covid-19 and neurological symptoms and the inability to study ongoing disease pathogenesis in the human brain further underscore the need for accessible and tractable experimental models to investigate sars-cov-2 neurotropism, its functional impact, and to test therapeutics. classic animal models, such as rodents, are limited in their ability to recapitulate human covid-19 symptoms and usually require viral or transgenic mediated overexpression of human sars-cov-2 receptor angiotensin-converting enzyme 2 (ace2) to exhibit symptoms sun et al., 2020) . although cell lines, including many human cancer cell lines, have been used to study sars-cov-2 infection and test drug efficacy (hoffmann et al., 2020b; ou et al., 2020; shang et al., 2020; wang et al., 2020) , they do not accurately recapitulate normal human cell j o u r n a l p r e -p r o o f 4 behavior and often harbor tumor-associated mutations, such as tp53, which may affect sars-cov-2 replication or the cellular response to sars-cov-2 infection (ma-lauer et al., 2016) . furthermore, human tissue and organ systems contain multiple cell types with variable levels of ace2 expression and viral susceptibility, which are not adequately represented in these human cell lines. these limitations support the development of human cellular models for sars-cov-2 infection that better recapitulates the cellular heterogeneity and function of individual tissues. human pluripotent stem cell (hipsc)-based models provide an opportunity to investigate the susceptibility of various brain cell types to viral infection and their consequences. hipscs have been used to generate a variety of monolayer and three dimensional (3d) organoid cultures to study human diseases and potential treatments. for example, hipsc-derived neural progenitors in monolayer and brain organoids were instrumental in studying the impact of zika virus infection on human brain development and solidifying the link between zika virus infection of neural progenitor cells and microcephaly seen in newborns (ming et al., 2016; qian et al., 2016; tang et al., 2016) . additionally, these cultures were useful in screening for drugs to treat zikv infection (xu et al., 2016) . recently, hipsc-derived organoids have been used to model sars-cov-2 infection in many organs, including the intestine, lung, kidney, liver, pancreas, vasculature and brain (lamers et al., 2020; monteil et al., 2020; ramani et al., 2020; yang et al., 2020a; zhou et al., 2020) . these studies have shown that sars-cov-2 can infect and replicate within cells of multiple organs, leading to transcriptional changes indicative of inflammatory responses and altered cellular functions. here, we used hipsc-derived neurons, astrocytes, and microglia in monolayer cultures and region-specific brain organoids of the cerebral cortex, hippocampus, hypothalamus, and midbrain to investigate the susceptibility of brain cells to sars-cov-2 infection. we observed sparse infection of neurons and astrocytes, with the exception of regions of organoids with choroid plexus epithelial cells that exhibited high levels of infectivity. using an optimized protocol to generate choroid plexus organoids (cpos) from hipscs, we showed evidence of productive infection by sars-cov-2 and functional consequences at cellular and molecular levels. to investigate the susceptibility of human brain cells to sars-cov-2 infection, we tested various hipsc-derived neural cells in monolayer cultures and region-specific brain organoids generated using several established (qian et al., 2018; qian et al., 2016) and modified protocols (sakaguchi et al., 2015) ( figure 1a ). monolayer hipsc-derived cortical neurons, astrocytes, and microglia were exposed to either sars-cov-2 virus isolate or vehicle control for 12 hours and analyzed at 24, 48, and 120 hours post-infection (hpi) for immunolabelling using convalescent serum from a patient with covid-19 or sars-cov-2 nucleoprotein antibodies (figures s1a-c). we confirmed the identity of neurons, microglia, and astrocytes by immunostaining for markers map2, pu.1, and gfap, respectively (figures s1a-c). hipsc-derived cortical neurons were co-cultured on hipscderived astrocytes to improve survival, and analysis at 120 hpi showed rare infection of map2 + cells ( figure s1a ). at 48 and 120 hpi, hipsc-derived microglia showed no infection of pu.1 + cells ( figure s1b ), whereas hipsc-derived astrocytes showed sparse infection of gfap + cells ( figure s1c ). as a validation, we tested infection of primary human astrocytes and observed sparse infection at 24, 48, and 120 hpi ( figure s1d ). quantification of hipsc-derived and primary human astrocytes showed infection of 0.02% and 0.18% of all cells at 120 hpi, respectively ( figure s1e ). these results revealed the ability of sars-cov-2 to infect monolayer human cortical neurons and astrocytes, but not microglia, although infection of neurons and astrocytes was rare. to examine the neurotropism of sars-cov-2 in a model system that more closely resembles the human brain, we exposed cortical, hippocampal, hypothalamic, and midbrain organoids to sars-cov-2 or vehicle control for 8 hours and analyzed samples at 24 and 72 hpi. we confirmed the regional identity of cortical, hippocampal, hypothalamic, and midbrain organoids by immunostaining with the markers ctip2, prox1, otp, and th, respectively ( figure s1f ). sars-cov-2 nucleoprotein was detected sparsely in these organoids derived from two different hipsc lines in a range that averaged between 0.6% and 1.2% of all cells at 24 and 72 hpi (figures j o u r n a l p r e -p r o o f 6 1b, 1c, and s1g). co-immunolabeling with dcx and sars-cov-2 nucleoprotein identified most of infected cells as neurons in all organoids ( figure 1d ) and infection of some gfap + astrocytes was found in hypothalamic organoids ( figure s1h ). because these brain organoids contained mostly neurons, the relative susceptibility of neurons compared to astrocytes could not be assessed with certainty. the number of infected cells did not significantly increase from 24 to 72 hpi ( figures 1c), indicating that infection may not spread among neurons in brain organoids within this time window. during development, the choroid plexus develops adjacent to the hippocampus (lun et al., 2015) and some of our hippocampal organoids contained regions with choroid plexus epithelial cells, which were identified by transthyretin (ttr) expression ( figure 1e ). notably, we observed a greater density of infected cells in these regions ( figure 1e ). additionally, we observed colocalization of patient convalescent serum and double-stranded rna (dsrna) immunolabeling in regions with choroid plexus cells, further supporting sars-cov-2 infection ( figure 1f ). together, these results demonstrate that sars-cov-2 exhibits limited tropism for neurons and astrocytes of multiple brain regions, but higher infectivity of choroid plexus epithelial cells. to validate the higher susceptibility and further investigate consequences of sars-cov-2 infection of choroid plexus cells, we sought to generate more pure choroid plexus organoids from hipscs. the most dorsal structures of the telencephalon, including the choroid plexus, are patterned by high wnt and bmp signaling from the roof plate (lun et al., 2015) . an early in vitro study demonstrated the sufficiency of bmp4 exposure to induce choroid plexus fate from neuroepithelial cells (watanabe et al., 2012) . furthermore, exposure of human embryonic stem cell-derived embryoid bodies to the gsk3 antagonist chir-99021 and bmp4 was shown to generate 3d choroid plexus tissue (sakaguchi et al., 2015) . building upon these previous studies, we optimized a simple protocol to generate choroid plexus organoids (cpos) from hipscs ( figure 2a ). undifferentiated hipscs grown in a feeder-free condition were aggregated into embryoid bodies consisting of j o u r n a l p r e -p r o o f 7 approximately 5,000 cells each using an aggrewell plate ( figure 2a ). embryoid bodies were patterned to the anterior neuroectodermal fate using dual-smad inhibition combined with wnt inhibition ( to further characterize these cpos, we performed transcriptome analysis of cpos at 50 div by bulk rna sequencing (rna-seq). gene expression analysis confirmed the expression of choroid plexus markers, including ttr, aqp1, chloride intracellular channel 6 (clic6), keratin 18 (krt18), msx1, and lmx1a, in cpos at high levels comparable to published transcriptomes of adult human choroid plexus tissue (rodriguez-lorenzo et al., 2020) and at much higher levels than those in hippocampal organoids at 45 div ( figure 2d ). in addition, cpos expressed genes related to adherens junction, signaling, ion channels and solute transporters at high levels comparable to those in adult human choroid plexus tissue ( figure 2d ), indicating choroid plexus identity and function. at the whole transcriptome level, cpos, but not hippocampal organoids, showed high correlation to adult human choroid plexus tissue ( figure 2e ). we validated high levels of expression of a group of choroid plexus signature genes in cpos at 50 div and 100 div in comparison to hippocampal organoids at 20 div by qpcr ( figure s2d and table s1 ). given our initial finding of a high rate of infection of choroid plexus cells by sars-cov-2, we examined the expression of known sars-cov-2 receptors. rna-seq analysis showed expression j o u r n a l p r e -p r o o f 8 of ace2 and tmprss2 in cpos at levels similar to the adult human choroid plexus tissue, which were much higher than in hippocampal organoids ( figure 2f ). expression levels of nrp1, a newly identified receptor for sars-cov-2 (cantuti-castelvetri et al., 2020) , were similar in all three samples. immunohistology confirmed expression of ace2 at a much higher level in cpos than in hippocampal organoids ( figure 2g ). qpcr analysis also showed higher levels of ace2 and tmprss2 expression in cpos at 50 div and 100 div than in hippocampal organoids ( figure s2d ) together, these results show that our cpos exhibit a similar transcriptome as adult human choroid plexus tissue and express markers for choroid plexus epithelial cells and sars-cov-2 receptors, representing a suitable experimental model to study sars-cov-2 infection. next, we exposed cpos at 47 div and 67 div to either sars-cov-2 or vehicle control for 8 hours and analyzed samples at 24 and 72 hpi. upon sars-cov-2, but not vehicle treatment, many ttr + choroid plexus cells showed infection as identified by co-localization of patient convalescent serum and sars-cov-2 nucleoprotein immunolabeling in cpos derived from two independent hipsc lines ( figures 3a and s3a ). additionally, we observed co-localization of patient convalescent serum and abundant dsrna immunolabeling in ttr + choroid plexus cells ( figure s3b ). infected cells identified by sars-cov-2 nucleoprotein immunostaining also showed high levels of ace2 expression, consistent with ace2 being a critical cell entry receptor for sars-cov-2 ( figure s3c ). quantification revealed an average of 9.0% and 12.6 % of ttr + cells infected at 24 hpi and 11.9% and 18.6% of ttr + cells infected at 72 hpi for cpos infected at 47 div and 67 div, respectively, across two hipsc lines ( figures 3b and s3d ). an increase in the number of infected cells from 24 to 72 hpi suggested that infection could be productive and spread among cells ( figures 3b and s3d ). to confirm sars-cov-2 productive infection, we examined viral titers using culture supernatants and cpo lysates at 0, 24, and 72 hpi. there was a significant increase in titers of j o u r n a l p r e -p r o o f 9 infectious virus over time after the initial infection ( figure 3c ). the increase of infectious virus present in cpo lysates superseded the increase in culture supernatants, suggesting a slow release of infectious viruses into the culture medium. the increase in viral titers appeared to be bigger than the increase in the number of infected cells, suggesting that the relationship is not linear. consistent with this notion, exposure of cpos at 67 div to different amounts of sars-cov-2 showed a nonlinear increase in infection with higher viral titers ( figures s3e and 3f ). importantly, we observed infection using 10 3 ffu (focus forming units) with an estimated moi (multiplicity of infection) of 0.001, indicating very high susceptibility of cpos to sars-cov-2 infection ( figures s3e and 3f ). next, we examined the cellular consequence of sars-cov-2 infection of cpos. we observed the presence of syncytia in sars-cov-2 infected cells, which significantly increased from an average of 4.6% at 24 hpi to 10.5% by 72 hpi (figures 3d and 3e ). development of syncytia by cell-cell fusion mediated by interaction between sars-cov-2 spike protein and ace2 expressed on adjacent cells has been reported with sars-cov-2 infection of several cell types and is a major mechanism for viral spread (hoffmann et al., 2020a; matsuyama et al., 2020; ou et al., 2020) . in particular, the sars-cov-2 spike protein appears to facilitate membrane fusion at a much higher rate than sars-cov-1 (xia et al., 2020) . by examining individual confocal z-planes we could identify as many as 12 nuclei within a single infected cell at 72 hpi ( figure 3d ). interestingly, in addition to syncytia, we observed a significant increase in cell death in both infected and uninfected ttr + choroid plexus cells in cpos exposed to sars-cov-2 as measured by tunel immunolabeling ( figures 3f, 3g and s3g). cell death increased from an average of 1.4% to 5.9% in infected and from 1.9% to 5.4% in uninfected ttr + cells from 24 to 72 hpi in cpos infected at 67 div across two hipsc lines ( figure 3g ). tunel + uninfected cells tended to appear adjacent to infected cells, suggesting that infected cells may induce adjacent cells to die through an extrinsic mechanism ( figure 3f ). similar results were found for cpos infected at 47 div ( figure s3g ). to confirm the susceptibility of choroid plexus epithelial cells to sars-cov-2 infection, we examined primary human choroid plexus epithelial cells (phcpecs). phcpecs exposed to sars-j o u r n a l p r e -p r o o f cov-2, but not vehicle control, for 12 hours showed many infected cells by immunolabeling with sars-cov-2 nucleoprotein at 72 and 120 hpi ( figure s3h ). the number of infected cells increased with higher moi, with an average of 1.7% and 0.9% of cells infected at 72 and 120 hpi, respectively, using a moi of 5 ( figure s3i ). together, using cpos as a 3d human cellular model, our findings reveal sars-cov-2 tropism for choroid plexus epithelial cells that results in productive infection, increased cell syncytia that may promote viral spread through cell-cell fusion, and increased cell death among both infected and uninfected adjacent cells. to gain additional insight into functional consequences of sars-cov-2 infection in cpos at the molecular level, we performed rna-seq of sars-cov-2 and vehicle-treated 47 div cpos at 24 and 72 hpi. principal component analysis showed clustering of biological replicates within different treatment groups ( figure s4a ). after aligning reads to the sars-cov-2 genome, we detected high numbers of viral transcripts at 24 and 72 hpi, confirming that the virus was replicating within cpos ( figure 4a ). we also confirmed the expression of sars-cov-2 receptors ace2, tmprss2, and nrp1 in cpos at all time points ( figure 4b ). comparison of sars-cov-2 and vehicle-treated cpos revealed 1,721 upregulated genes and 1,487 downregulated genes at 72 hpi, indicating that sars-cov-2 infection leads to large-scale transcriptional dysregulation ( figure s4b and table s2 ). more detailed gene ontology (go) analysis of upregulated genes at 72 hpi showed enrichment for genes related to viral responses, rna processing, response to cytokine, cytoskeletal rearrangement, and cell death ( figure 4c and table s3 ). closer examination of upregulated genes revealed an increase in inflammatory cytokines ccl7, il32, ccl2 (mcp1), il18, and il8 ( figure 4d ). il32 plays important roles in both innate and adaptive immune responses by inducing tnf-alpha, which activates the signaling pathway of nf-kappa-b and p38 mapk. this signaling pathway has been shown to be activated following sars-cov-2 infection in j o u r n a l p r e -p r o o f 11 other cells (bouhaddou et al., 2020) . we validated the upregulation of p38 signaling by immunostaining of the phosphorylated form of p38 in infected cells ( figures s4c and s4d ). interestingly, quantification showed elevated levels of phospho-p38 in nearby uninfected cells, although to a lesser degree than in infected cells, suggesting a substantial non-cell autonomous effects ( figure s4d ). upregulation of many vascular remodeling genes ( figure 4d ), such as nppb and vcan, suggests that infected choroid plexus cells could signal the vasculature to promote leukocyte invasion (shechter et al., 2013) . go analysis of downregulated genes at 72 hpi, on the other hand, showed enrichment for genes related to ion transport, transmembrane transport, cilium, and cell junction ( figure 4c and table s3 ). closer examination of downregulated genes revealed a decrease in the expression of many transporters and ion channels, such as aqp1, aqp4 and slc22a8, which are important for normal csf secretory function ( figure 4d ) (brown et al., 2004; hladky and barrand, 2016) , suggesting functional deficits of choroid plexus cells. downregulation of many cell junction genes suggests a remodeling or break-down of normal csf-blood barrier function, which also occurs during brain inflammation (engelhardt and tietz, 2015) . additionally, the dramatic decrease in ttr production may result in decreased thyroxine transport to the csf, which has been shown to contribute to neuropsychiatric symptoms and "brain fog" or difficulty in concentrating in patients (samuels, 2014) . we validated downregulation of ttr levels in infected cells by immunostaining ( figures s4c and s4d ). we also observed downregulation of ttr levels in uninfected cells at 72 hpi, although to a lesser degree than in infected cells, again suggesting a non-cell autonomous effect ( figure s4d ). transcriptional dysregulation induced by sars-cov-2 in cpos at 24 hpi showed similar gene expression changes as at 72 hpi, although less severe in most cases ( figures 4d and s4e ). comparison of the list of dysregulated genes at 24 and 72 hpi revealed a substantial overlap of 42% and 32% of upregulated and downregulated genes, respectively ( figure s4f ). we validated the dysregulation of a subset of these genes by qpcr ( figure s4g ). j o u r n a l p r e -p r o o f 12 to investigate whether organoids from different tissues exhibit similar transcriptional responses upon sars-cov-2 infection, we compared sars-cov-2-induced transcriptional changes among cpos and published datasets from hepatocyte organoids (yang et al., 2020a) and intestinal organoids . surprisingly, we found little overlap among upregulated or downregulated genes in the three different organoid types, suggesting a predominantly cell typespecific response to sars-cov-2 infection ( figure 4e ). this finding supports the use of a diverse array of human model systems to investigate effects of sars-cov-2 infection. additionally, we found many more dysregulated genes in cpos compared to the other two organoid types, suggesting that sars-cov-2 may exhibit a greater impact on choroid plexus cells ( figure 4e ). together, these transcriptome analyses suggest that sars-cov-2 infection of choroid plexus cells leads to viral rna production and inflammatory cellular responses with a concomitant compromise of normal barrier and secretory functions as well as increased cell death. furthermore, responses to sars-cov-2 infection are largely tissue organoid specific, at least at the transcriptional level. using a platform of hipsc-derived monolayer neurons, microglia, astrocytes, and region-specific brain organoids, we showed limited tropism of sars-cov-2 for neurons and astrocytes with a particularly high rate of infection of choroid plexus epithelial cells. using an optimized protocol for generating cpos from hipscs that resemble the morphology, marker expression, and transcriptome of adult human choroid plexus, we revealed productive infection of sars-cov-2 in choroid plexus epithelial cells, which we confirmed using primary human choroid plexus epithelial cells. analysis of the consequence of sars-cov-2 infection of cpos showed an increase in both cell autonomous and non-cell autonomous cell death and transcriptional dysregulation related to increased inflammation and altered barrier and secretory function. our study provides an organoid platform to investigate the cellular susceptibility, pathogenesis, and treatment strategies of sars-j o u r n a l p r e -p r o o f 13 cov-2 infection of brain cells and further implicates the choroid plexus as a potentially important site for pathophysiology. notably, the choroid plexus is known to be a site of infection for several viruses and agents that affect the brain (schwerk et al., 2015) . although covid-19 primarily manifests as respiratory distress resulting from inflammatory damage to the lung epithelium, there is mounting clinical evidence implicating other organs, such as the kidney, intestine, and brain, in disease pathogenesis (renu et al., 2020) . the currently widely used cellular model systems, such as vero e6 cells or human cancer cell lines, do not adequately recapitulate the cellular diversity and breadth of the disease. although mice engineered to express human ace2 have provided an in vivo model to study sars-cov-2, the system relies on human ace2 overexpression and does not fully recapitulate symptoms of covid-19 in humans. recently, organoids have emerged as a model to study human cells and organogenesis in vitro (clevers, 2016) . organoids for various tissues have been used to better understand sars-cov-2 viral tropism and pathology monteil et al., 2020; ramani et al., 2020; yang et al., 2020a; zhou et al., 2020) . these studies support cell-type specific susceptibility to sars-cov-2 infection. our finding that dysregulated gene expression varies widely among hepatocyte, intestinal, and choroid plexus organoids infected with sars-cov-2 suggests unique responses in different cell types and highlights the need for diverse human cellular model systems when studying the disease. brain organoid models for sars-cov-2 infection are particularly useful since it is not feasible to take brain biopsies from patients with covid-19. the load of sars-cov-2 that may enter the brain is likely to be variable throughout the course of illness and may be difficult to accurately assess in brain samples. for example, viral particles or rna has been detected in the csf or brain of some but not the majority of patients with neurological symptoms (helms et al., 2020; moriguchi et al., 2020; puelles et al., 2020; solomon et al., 2020; virani et al., 2020) , although the amount of viral particles in the csf may be insufficient for detection . j o u r n a l p r e -p r o o f 14 brain organoids allow analyses of the acute and long-term impact of sars-cov-2 infection in real time, with the ability to introduce perturbations, such as therapeutic agents, to assess cellular responses. use of region-specific brain organoids, including cpos, expands the organoid toolset for investigating sars-cov-2 infection, and provides a platform for testing new therapeutics. building upon previous findings (sakaguchi et al., 2015; watanabe et al., 2012) , we optimized a cpo model that is simple, robust and reproducible with the initial goal to study sars-cov-2 infection. these cpos express high levels of sars-cov-2 receptors and exhibit productive infection of sars-cov-2 and pathogenesis at cellular and molecular levels. cpos may provide a better model system than primary human choroid plexus epithelial cells, which are not widely available and often lose some characteristics upon multiple rounds of expansion in culture and showed a lower infection rate than our cpos. unlike a very recently developed protocol that utilizes cultures with matrigel extracellular matrix (pellegrini et al., 2020) , our cpos do not obviously polarize or develop fluid-filled, cystic structures, but do offer the benefit of not relying on matrigel, which has batch-to-batch variation and can lead to increased inter-organoid variability. our protocol uses feeder-free hipscs to further improve organoid reproducibility, and cpos are cultured on an orbital shaker to enhance oxygen and nutrient diffusion throughout the organoids. with these improvements, cpos reproducibly generate projections of cuboidal cells and exhibit uniform expression of choroid plexus markers otx2, aqp1, and ttr in most of the cells within the organoids. they also exhibit a transcriptome similar to adult human choroid plexus tissue. importantly, these cpos express many transporters that are essential for csf secretion. these cpos provide a platform for future investigations of cell type-specific pathogenesis, mechanisms and treatment of sars-cov-2 infection. our cpo platform can also be used to model human choroid plexus development and associated brain disorders in the future (lun et al., 2015) . spread in the cns j o u r n a l p r e -p r o o f 15 ace2 has been identified as a key cell entry receptor for sars-cov-2 (hoffmann et al., 2020b). although moderate ace2 expression has been detected in various brain regions, particularly high levels have been reported in the choroid plexus in humans and mice . the high levels of ace2 expression in cpos may explain their higher susceptibility to sars-cov-2 infection compared to other brain organoids. moreover, the productive infection and high incidence of syncytia in infected choroid plexus cells may contribute to the viral spread. on the other hand, despite relatively low ace2 expression, we did detect sparse infection of neurons by sars-cov-2. recent studies point to the potential for other proteins that are expressed more highly in neural cells, such as nrp1, to serve as receptors for sars-cov-2. we did not observe an obvious increase in the number of infected neurons over time, suggesting that the virus may not spread efficiently from neuron-to-neuron. central nervous system disorders, including seizures and encephalopathy, have been reported with sars-cov-1 with detection of infectious virus and rna in the csf (hung et al., 2003; lau et al., 2004; xu et al., 2005) . a substantial portion of patients with covid-19 exhibit various neurological symptoms, which are highly variable, probably due to many factors (helms et al., 2020; moriguchi et al., 2020; puelles et al., 2020; solomon et al., 2020; virani et al., 2020) . how sars-cov-2 may enter the brain is currently unknown. the main hypotheses are either neuron-toneuron spread via bipolar cells located in the olfactory epithelium that extend axons and dendrites to the olfactory bulb, or a hematogenous route across the blood-csf-barrier (desforges et al., 2014) . neuron-to-neuron propagation has been described for other coronaviruses (dube et al., 2018; netland et al., 2008) , but it was not obvious in our study. our finding that the choroid plexus is particularly susceptible to productive infection with sars-cov-2, raises the possibility that choroid plexus epithelial cells could be infected from virus circulating in closely associated capillaries. sars-cov-2 may replicate within choroid plexus cells and shed viral copies into the csf. indeed, we detected increased viral load in culture supernatants overtime. alternatively, virus may enter the csf via the cribiform plate in nasal passages and then replicate in choroid plexus j o u r n a l p r e -p r o o f 16 cells, increasing viral availability in the central nervous system. viral propagation through the csf may also explain cases of spinal cord pathology in some patients with covid-19 (giorgianni et al., 2020; valiuddin et al., 2020) . more detailed analyses of autopsy brain samples from patients with covid-19 and better animal models of sars-cov-2 infection are necessary to understand potential routes of viral entry into the brain. the transcriptional dysregulation in cpos after sars-cov-2 infection indicates an inflammatory response and perturbed choroid plexus cellular function. our study further suggested both cell autonomous and non-cell autonomous impact of sars-cov-2 infection. many pro-inflammatory cytokines were upregulated, including ccl7, il32, ccl2, il18, and il8, which are important for recruiting immune cells to sites of infection. future studies are needed to validate the release of these cytokines by elisa analyses. notably, one case report examining inflammatory cytokines present in the serum and csf of a patient with covid-19 presenting with severe seizures identified ccl2 as the only cytokine enriched in the patient's csf compared to serum (farhadian et al., 2020) . ccl2 was one of the most highly upregulated genes after sars-cov-2 infection, suggesting that the choroid plexus may be the source of this cytokine in the csf. upregulation of vascular and extracellular matrix remodeling genes also suggests that during infection, choroid plexus cells can signal the adjacent vasculature to recruit inflammatory cells. downregulation of many genes important for csf secretion, including aqp1, and many ion transporters, such as atp1a2, may lead to altered csf production and composition. mutations in many of these dysregulated transporter genes have been implicated in neurological complications in humans, such as migraine and encephalopathy (murphy et al., 2018) . increased expression of cell death genes supports our finding of increased numbers of tunel + cells after sars-cov-2 infection. downregulation of many genes related to the cilium suggests an impaired cytoskeleton and dysfunctional sensing of the extracellular environment. there was also a significant downregulation of ttr, which is necessary for carrying thyroid hormone from the blood into the csf, and lowered ttr production by the choroid plexus has been associated with neuropsychiatric disorders, such as schizophrenia and depression (huang et al., 2006; sullivan et al., 1999) . furthermore, low csf thyroid hormone has been associated with many neurological symptoms, including slowing of thought and speech, decreased attentiveness, and reduced cognition, which have all been reported by patients with covid-19 (ellul et al., 2020) . our study implicates the choroid plexus epithelium as a potential target of sars-cov-2 infection and a contributor to covid-19 disease pathogenesis that warrants further investigation. our study demonstrates clear susceptibility of hipsc-derived neurons, astrocytes, and choroid plexus cells, as well as primary astrocytes and choroid plexus epithelial cells, to sars-cov-2 infection in vitro, however, a thorough analysis of brain samples from patients with covid-19 is necessary to confirm neural susceptibility in vivo. our methodology has a number of constraints that limit result interpretation. first, since brain organoids more closely resemble the developing fetal brain than the mature adult brain, there may exist important differences in sars-cov-2 susceptibility between immature and mature cells. there has been recent evidence of vertical transmission of sars-cov-2 from mother to fetus (dong et al., 2020; vivanti et al., 2020; zeng et al., 2020) , but the impact on the fetal brain remains unclear. brain organoids may serve as a useful model system to explore the potential effects of fetal sars-cov-2 exposure on brain development. second, direct exposure of brain organoid cultures to sars-cov-2 in the culture medium may not accurately recapitulate the physiological amount and duration of viral exposure in humans. different viral titers may reveal different viral tropism in vitro. to avoid the use of high titers that may cause j o u r n a l p r e -p r o o f 18 non-physiological viral infection, we exposed different brain region-specific organoids of the same estimated moi of 0.1 and found a differential infection rate. importantly, sars-cov-2 at an estimated moi as low as 0.001 can lead to detectable infection of cpos, indicating very high susceptibility of choroid plexus cells, at least in vitro. third, brain organoid models lack an intact blood-brain-barrier or blood-csf-barrier which may modulate sars-cov-2 access to the brain. our brain organoids also lack additional cell types, such as oligodendrocytes, microglia, stromal cells, immune cells, and endothelial cells, which may modulate susceptibility to infection and contribute to disease pathogenesis in vivo. immune cells, such as t cells and monocytes, have been shown to mediate host responses to sars-cov-2 infection, including tissue-specific inflammation (tay et al., 2020) . endothelial cells are major targets of sars-cov-2 and may be the primary cause of sars-cov-2-related effects in the brain with neurological symptoms being secondary to vascular changes and hypoxia (ellul et al., 2020) . future studies of sars-cov-2 susceptibility can extend to a more diverse selection of brain cells, as well as peripheral neurons. also see figure s2 and table s1. table s2 for the complete list of differentially expressed genes and table s3 for the complete list of go terms for differentially expressed genes. tmem.: transmembrane. (e) venn diagrams comparing the overlap of upregulated and downregulated genes following s-cov-2 infection in cpos, human hepatocyte organoids (yang et al., 2020a) , and human intestinal organoids . differentially expressed genes at both 24 and 72 hpi were combined for cpos and differentially expressed genes for both expansion and differentiation intestinal organoid types were combined for intestinal organoids. also see figure s4 and tables s1, s2 and s3. further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, dr. guo-li ming (gming@pennmedicine.upenn.edu) . this study did not generate new unique reagents. the access number for the rna-seq data reported in this study is deposited in geo (gse157852). human ipsc lines used in the current study were derived from healthy donors and were either obtained from commercial sources or previously generated and fully characterized (chiang et al., 2011; wen et al., 2014; yoon et al., 2014) . c1-2 hipscs were generated from fibroblasts obtained from atcc (crl-2097). c3-1 hipscs were generated from fibroblasts obtained from an individual in a previously characterized american family (sachs et al., 2005) . wtc11 hipsc were obtained from coriell (gm25256). ht268a hipscs were generated from dermal fibroblasts obtained from coriell (gm05659) (vu et al., 2018) , and pcs201012 hipscs were generated from dermal fibroblasts obtained from atcc (pcs-201-012) (beers et al., 2015) . generation of hipsc lines (bardy et al., 2015) . primary human astrocytes (sciencell research laboratories) were cultured and expanded for two passages, according to the supplier's instructions. after the second expansion, astrocytes were cryopreserved at 250,000 cells per vial. banked astrocytes were thawed directly onto 96 well imaging plates (greiner bio-one) coated with poly-l-ornithine (sigma-aldrich) at 2 âµg/cm 2 and laminin from engelbreth-holm-swarm murine sarcoma basement membrane (sigma-aldrich) at 5 âµg/ml at a density of 16,000 cells per well for shorter infection times (48 hours or less) and 8000 cell per well for 120 hour infections. primary human choroid plexus epithelial cells (sciencell research laboratories) were cultured according to the supplier's instructions. cells were either passaged once or plated directly out of thaw onto 96-well imaging plates coated with poly-l-ornithine at 2 âµg/cm 2 at a density of 4000 cells per well. all studies involving hipscs were performed under approved protocols of the university of pennsylvania and nih. refer to the key resources table for the source of each hipscs. all hipsc lines were confirmed to have a normal karyotype and were confirmed free of mycoplasma, acholeplasma, and spiroplasma with a detection limit of 10 cfu/ml by targeted pcr (biological industries). for cortical, hippocampal, hypothalamic, and midbrain organoid generation, hipscs were cultured on mouse embryonic fibroblast feeder (mef) cells in stem cell medium consisting of dmem:f12 supplemented with 20% knockout serum replacement, 1x mem-neaas, 1x glutamax, 1x penicillin-streptomycin, 1x 2-mercaptoethanol, and 10 ng/ml bfgf in a 5% co 2 , 37â°c, 90% relative humidity incubator as previously described (qian et al., 2018) . culture medium was replaced every day. hipscs were passaged every week onto a new tissue-treated culture plate coated with 0.1% gelatin for 2 hours and pre-seeded with î³-irradiated cf1 mef cells one day in advance. colonies were detached by washing with dpbs and treating with 1 mg/ml collagenase type iv for 30-60 minutes. detached colonies were washed 3 times with 5 ml dmem:f12 and dissociated into small clusters by trituration with a p1000 pipette. for cpo generation, hipscs j o u r n a l p r e -p r o o f 33 were maintained in feeder-free conditions on plates pre-coated with hesc-qualified matrigel (corning) using mtesr plus medium (stemcell technologies). culture medium was replaced every other day and hipscs were passaged every week by washing with dpbs and incubating with relesr reagent (stemcell technologies) for 5 minutes. detached hipscs were broken into smaller clusters by gentle trituration using a 5 ml pipette and seeded onto fresh coated plates. generation of cortical organoids from c3-1 hipscs was performed as previously described (qian et al., 2018; qian et al., 2016) . briefly, hipsc colonies were detached with collagenase type iv, for generation of hypothalamic organoids from c1-2 and c3-1 hipsc lines, hipsc colonies were processed similarly to those for cortical organoids followed by exposure to shh signaling and wntinhibition in induction medium with 50 ng/ml recombinant sonic hedgehog, 1 âµm purmorphamine, 10 âµm iwr1-endo and 1 âµm sag, and then transferred to differentiation medium for further culture. organoids were maintained with media changes every other day. at 70% confluence, undifferentiated hipscs (c1-2 and wtc11 lines) grown in feeder-free conditions were detached using relesr reagent and gently triturated into small clusters using a 5 ml pipette and pipette aid. hipsc clusters were centrifuged at 200 g for 3 minutes and the supernatant was aspirated and replaced with mtesr plus medium supplemented with 10 âµm y-27632. approximately 5,000 hipscs were aggregated into each eb by following instructions using the aggrewell 800 plate and cultured overnight in mtesr plus medium supplemented with 10 âµm y-27632. the next day (day 1), embryoid bodies were gently removed from the aggrewell plate using a p1000 pipettor with a wide-bore tip, washed three times with dmem:f12, and transferred to neural induction medium containing dmem:f12 supplemented with 20% knockout serum on day 30, medium was replaced with differentiation medium containing a 1:1 mixture of dmem/f12 and neurobasal supplemented with 1x n2 supplement, 1x b27 supplement w/o vitamin a, 1x penicillin/streptomycin, 1x non-essential amino acids, 1x glutamax, 10 ng/ml bdnf, and 10 ng/ml gdnf. differentiation medium was replaced every three days and organoids could be maintained for over 100 days. sars-cov-2 usa-wa1/2020 (gen bank: mn985325.1) (harcourt et al., 2020) viral isolate was obtained from bei resources. sars-cov-2 virus was expanded and titered using vero e6 cells. culture cells were infected in a 96-well plate with 100 âµl of medium per well using multiplicity (moi) of 0.2, 1, or 5, and vehicle-treated cells as controls. after virus exposure for 12 hours, cells were washed 3 times with fresh medium and returned to culture. brain organoids were infected in a 24well ultra-low attachment plate (corning) with 0.5 ml of medium per well containing vehicle, 10 3 , 10 4 , or 10 5 focus forming units (ffu) of sars-cov-2 for 8 hours on an orbital shaker rotating at approximately 100 rpm. after virus exposure, organoids were washed 3 times with fresh medium and returned to culture. for infection of organoids, we controlled the virus titer rather than the moi since it is not feasible to obtain an accurate cell count on each batch of organoids before infection. according to our estimate, each organoid contains approximately 250-500,000 cells, corresponding j o u r n a l p r e -p r o o f 36 to an estimated moi of 0.1-0.05 when using 10 5 ffu sars-cov-2 virus and infecting 4 organoids together. at experimental endpoints, brain organoids were placed directly in 4% methanol-free formaldehyde (polysciences) diluted in dpbs (thermo fisher scientific) overnight at 4 o c on a rocking shaker. after fixation, brain organoids were washed in dpbs and cryoprotected by overnight incubation in 30% sucrose (sigma-aldrich) in dpbs at 4â°c. brain organoids were placed in a plastic cryomold (electron microscopy sciences) and snap frozen in tissue freezing medium (general data) on dry ice. frozen tissue was stored at â��80â°c until processing. monolayer cells grown on glass coverslips were fixed in 4% methanol-free formaldehyde (polysciences), diluted in dpbs (thermofisher scientific) for 5 hours at room temperature, washed with dpbs, and stored at 4â°c until ready for immunohistology. serial tissue sections (25 âµm for brain organoids) were sliced using a cryostat (leica, cm3050s), and melted onto charged slides (thermo fisher scientific). slides were dried at room temperature and stored at â��20â°c until ready for immunohistology. for immunofluorescence staining, the tissue sections were outlined with a hydrophobic pen (vector laboratories) and washed with tbs containing 0.1% . tissue sections were permeabilized and nonspecific binding was blocked using a solution containing 10% donkey serum (v/v), 0.5% triton x-100 (v/v), 1% bsa (w/v), 0.1% gelatin (w/v), and 22.52 mg/ml glycine in tbst for 1 hour at room temperature. the tissue sections were incubated with primary antibodies (see the key resources table) diluted in tbst with 5% donkey serum (v/v) and 0.1% triton x-100 (v/v) overnight at 4â°c. after washing in tbst, tissue sections were incubated with secondary antibodies (see the key resources table) diluted in tbst with 5% donkey serum (v/v) and 0.1% triton x-100 (v/v) for 1.5 hours at room temperature. after washing with tbst, sections were washed with tbs to remove detergent and incubated with trueblack reagent (biotium) diluted 1:20 in 70% ethanol for 1 minute j o u r n a l p r e -p r o o f 37 to block autofluorescence. after washing with tbs, slides were mounted in mounting solution (vector laboratories), cover-slipped, and sealed with nail polish. monolayer cultures were imaged with a perkin-elmer opera phenix high-content automatic imaging system with a 20x air objective in confocal mode. at least 9 fields were acquired per well. images were analyzed using columbus image data storage and analysis system. brain organoid sections were imaged as z stacks using a zeiss lsm 810 confocal microscope or a zeiss lsm 710 confocal microscope (zeiss) using a 10x, 20x, 40x, or 63x objective with zen 2 software (zeiss). images were analyzed using either imaris 7.6 or imagej software. images were cropped and edited using adobe photoshop (adobe) and adobe illustrator (adobe). tissue culture supernatants and lysates from sars-cov-2 treated cpos were collected at 0, 24, and 72 hpi for 2 biological replicates per timepoint containing 4 organoids each. tissue culture supernatants were stored at -80â°c until titration. for lysates, organoids were washed with cold pbs, mechanically dispersed and disrupted by freeze-thaw cycles, and the clarified homogenate was stored at -80â°c until titration. viral titers were determined in vero e6 cells using an immune focus forming unit assay. briefly, vero e6 cells (3 x 10 4 cells/96-well) were inoculated with 100 âµl of serially diluted organoid supernatant or lysate in a 96-well plate at 37â°c and 5% co 2 for 20 hours. cells were fixed with 4% methanol-free paraformaldehyde overnight at 4â°c. infected foci were labeled using immunofluorescence staining for sars-cov-2 nucleoprotein. sars-cov-2 nucleoprotein positive cells were counted in each well. readouts were performed on wells with the highest dilution displaying observable infection. j o u r n a l p r e -p r o o f 38 to minimize variability due to sampling and processing, each biological replicate consisted of 3 organoids and replicates for all experimental conditions were processed in parallel for rnaextraction, library preparation and sequencing. at the desired experimental endpoints, organoids were homogenized in trizol (thermo fisher scientific) using a disposable pestle and handheld mortar and stored at -80 c until processing. rna clean-up was performed using the rna clean & concentrator kit (zymo research) after trizol phase separation according to the manufacturer's protocol. rna concentration and quality were assessed using a nanodrop 2000 (thermo fisher scientific). library preparation was performed as previously described with some minor modifications (weng et al., 2017) . about 300 ng of rna in 3.2 âµl was combined with 0.25 âµl rnase inhibitor (neb) and 1 âµl cds primer (5'-aagcagtggtatcaacgcagagtact30vn-3') in an 8-well pcr tube strip, heated to 70 âºc for 2 minutes, and immediately placed on ice. 5.55 âµl rt mix, containing 2 âµl of 5x smartscribe rt buffer (takara), 0.5 âµl of 100 mm dtt (millipore sigma), 0.3 âµl of 200 mm mgcl2 (thermo fisher scientific), 1 âµl of 10 mm dntps (takara), 1 âµl of 10 âµm tso primer (5'-aagcagtggtatcaacgcagagtacatrgrgrg-3'), 0.25 âµl of rnase inhibitor (neb), and 0.5 âµl smartscribe reverse transcriptase (takara) was added to the reaction. rt was performed under the following conditions: 42 âºc for 90 minutes, 10 cycles of 50 âºc for 2 minutes and 42 âºc for 2 minutes, 70 âºc for 15 minutes, and 4 âºc indefinitely. for cdna amplification, 2 âµl of the rt reaction was combined with 2.5 âµl of 10x advantage 2 buffer (takara), 2.5 âµl of 2.5 mm dntps (takara), 0.25 âµl of 10 âµm is pcr primer (5'-aagcagtggtatcaacgcagagt-3'), 17.25 âµl nuclease free water (thermofisher), and 0.5 âµl advantage 2 dna polymerase (takara). thermocycling conditions were as follows: 94 âºc for 3 minutes, 8 cycles of 94 âºc for 15 s, 65 âºc for 30 s, and 68 âºc for 6 minutes, 72 âºc for 10 minutes, and 4 âºc indefinitely. amplified cdna was purified using 0.8x ampure xp beads (beckman coulter), eluted in 15 âµl nuclease-free water, and quantified using qubit dsdna hs assay kit (thermo fisher scientific). cdna was fragmented by combining 100 pg cdna in 1 âµl nuclease free water, 2x td buffer (20 mm tris, ph 8.0; thermo j o u r n a l p r e -p r o o f 39 fisher scientific), 10 mm mgcl 2 , and 16% peg 8000 (milliporesigma), and 0.5 âµl tn5 (lucigen). the mixture was heated to 55 âºc for 12 minutes, and the reaction was terminated upon the addition of 1.25 âµl of 0.2% sds (fisher) and incubated at room temperature for 10 minutes. fragments were amplified by adding 16.75 âµl nuclease free water (thermo fisher scientific), 1 âµl of 10 mm nextera i7 primer, 1 âµl of 10 mm nextera i5 primer, and 25 âµl kapa hifi hotstart readymix (emsco/fisher). thermocycling conditions were as follows: 72 âºc for 5 minutes, 95 âºc for 1 minute, 14 cycles of 95 âºc for 30 s, 55 âºc for 30 s, and 72 âºc for 30 s, 72 âºc for 1 minute, and 4 âºc indefinitely. dna was purified twice with 0.8x ampure xp beads (beckman coulter) and eluted in 10 âµl of 10 mm tris, ph 8 (thermo fisher scientific). samples were quantified by qpcr (kapa) and pooled at equal molar amounts. final sequencing library fragment sizes were quantified by bioanalyzer (agilent) with an average size of ~420 bp, and concentrations were determined by qpcr (kapa). samples were loaded at concentrations of 2.7 pm and sequenced on a nextseq 550 (illumina) using 1x72 bp reads to an average depth of 40 million reads per sample. choroid plexus organoid raw sequencing data were demultiplexed with bcl2fastq2 v2.17.1.14 (illumina) with adapter trimming using trimmomatic v0.32 software (bolger et al., 2014) . alignment was performed using star v2.5.2a (dobin et al., 2013) . a combined reference genome consisting of gencode human genome release 28 (grch38.p12) and ensembl sars-cov-2 wuhan-hu-1 isolate genome (genome assembly: asm985889v3, gca_009858895.3; sequence: mn908947.3) was used for alignment. multimapping and chimeric alignments were discarded, and uniquely mapped reads were quantified at the exon level and summarized to gene counts using star -quantmode genecounts. all further analyses were performed in r v3.6.0. transcript lengths were retrieved from gtf annotation files using genomicfeatures v1.36.4 (lawrence et al., 2013) and raw counts were converted to units of transcripts per million (tpm) using the calculatetpm function in scater v1.12.2 (mccarthy et al., 2017) . a combined tpm normalization using both human and primer sequences are listed in figure s1 . about 0.5-1.5 âµg rna was used to generate the cdna for qpcr with superscriptâ�¢ iii first-strand synthesis system (thermo fisher scientific) according to manufacturer's protocol. for qpcr reaction, 2 âµl of cdna was mixed with 6 âµl of nuclease free water, 1 âµl of 10 mm forward primer, 1 âµl of 10 mm reverse primer and 10 âµl of sybr green qpcr master mix (thermo fisher scientific), and the qpcr reactions were performed on the steponeplus real-time pcr system (applied biosystems). thermocycling conditions were as follows: 95 âºc for 20 s, 44 cycles of 95 âºc for 3 s and 60 âºc for 30 s. the difference between the ct values (â��ct) of the genes of interest and actin gene was calculated for each experimental sample, and 2^(-â��â��ct) was used to calculate the fold-change in expression of the genes of interest between samples using 20 div hos as the reference for figure s2d and 72 hpi vehicle-treated cpos as the reference for figure s4g . shown as mean â± sem or mean + sd as stated in the figure legends . in all cases, the p values are represented as follows: * * * p < 0.001, * * p < 0.01, * p < 0.05, and ns, not statistically significant when p > 0.05. in all cases, the stated ''n'' value is either separate wells of monolayer cultures or individual organoids with multiple independent images used to obtain data points for each. no statistical methods were used to pre-determine sample sizes. for all quantifications of immunohistology, the samples being compared were processed in parallel and imaged using the same settings and laser power for confocal microscopy. for monolayer cultures, cells were segmented by first identifying nuclei, and then cytoplasmic area. mean cytoplasmic intensities for sars-cov-2 nucleoprotein immunostaining were then measured in the segmented cytoplasmic regions, and a mean cytoplasmic intensity threshold was determined to identify sars-cov-2 infected cells. cell numbers were then automatically counted for sars-cov-2 infected dapi + cells, and total dapi + cells. for brain organoids, immuno-positive cells were quantified manually using the cell counter function in imagej (nih) or imaris (bitplane). ming and colleagues tested sars-cov-2 neurotropism using monolayer neural cells and brain organoids generated from human pluripotent stem cells and show minimal neuron and astrocyte infection, but efficient choroid plexus infection, leading to cell death and functional deficits. ccl7 il32 ccl2 il18 il8 casp8 igfbp3 anxa3 anxa2 pawr plau tnc vcan thbs1 nppb ctgf tagln actg2 cdcp1 acta2 cd274 tnfaip6 itgb6 ywhae dynlt1 aqp1 aqp4 atp2b3 slc7a8 slc39a12 rgs9bp arr3 cdhr1 ush2a cngb1 slc22a8 slc5a5 atp1a2 atp8a1 atp2b2 anxa9 ptprc kcna1 slc17a8 camk4 apoe hpgd ttr gene ontology: tool for the unification of biology. the gene ontology consortium the pathogenicity of sars-cov-2 in hace2 transgenic mice neuronal medium that supports basic synaptic functions and activity of human neurons in vitro a cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells 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sars-cov-2 tropism and model virus infection in human cells and organoids clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study encephalitis as a clinical manifestation of covid-19 modeling a genetic risk for schizophrenia in ipscs and mice reveals neural stem cell deficits associated with adherens junctions and polarity antibodies in infants born to mothers with covid-19 pneumonia infection of bat and human intestinal organoids by sars-cov-2 2020) was downloaded from the ncbi gene expression omnibus (geo: accession number gse137619). the data was aligned to grch38.p12 and raw counts tpm-normalized to parallel cpo and ho datasets. tpm expression values were log 10 (tpm + 1) transformed for plotting individual gene expression values 2014) and correlation analysis was performed by calculating spearman correlation coefficients in the space of shared genes across the whole transcriptome of all datasets differential gene expression analysis for cpos between 72 hpi vehicle, 24 hpi sars-cov-2 infection, and 72 hpi sars-cov-2 infection was performed using deseq2 sars-cov-2 viral transcripts were removed for differential gene expression analysis upregulated and downregulated genes for 24 hpi sars-cov-2 compared to 72 hpi vehicle, and 72 hpi sars-cov-2 compared to 72 hpi vehicle were filtered by adjusted p-value < 0.05. volcano plots for 24 hpi sars-cov-2 and 72 hpi sars-cov-2 were plotted using enhancedvolcano v1.7.8. shared and unique signatures for upregulated and downregulated genes between 24 hpi sars-cov-2 and 72 hpi sars-cov-2 were visualized using venndiagram v1 tpm + 1) normalized and converted to row z-scores per gene for visualization. for sars-cov-2 cross-organoid comparison analysis, a list of upregulated and downregulated differentially expressed genes (degs) after sars-cov-2 infection were downloaded for adult hepatocyte organoids at 24 hpi (yang et al., 2020a), intestinal organoids at 60 hpi in expansion (exp) medium (lamers et al., 2020), and intestinal organoids at 60 hpi in differentiation (dif) medium we thank members of ming f.j. contributed to histological analysis of various brain organoids and development of the cpo model. s.r.p. and f.z. contributed to rna-seq data analysis. f.z. contributed to qpcr validation. the authors declare no competing interests.j o u r n a l p r e -p r o o f key: cord-353209-qkhfp66l authors: steiner, daniel j.; cognetti, john s.; luta, ethan p.; klose, alanna m.; bucukovski, joseph; bryan, michael r.; schmuke, jon j.; nguyen-contant, phuong; sangster, mark y.; topham, david j.; miller, benjamin l. title: array-based analysis of sars-cov-2, other coronaviruses, and influenza antibodies in convalescent covid-19 patients date: 2020-06-16 journal: biorxiv doi: 10.1101/2020.06.15.153064 sha: doc_id: 353209 cord_uid: qkhfp66l detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. the urgent need for antibody detection tools has proven particularly acute in the covid-19 era. we report a multiplex label-free antigen microarray on the arrayed imaging reflectometry (air) platform for detection of antibodies to sars-cov-2, sars-cov-1, mers, three circulating coronavirus strains (hku1, 229e, oc43) and three strains of influenza. we find that the array is readily able to distinguish uninfected from convalescent covid-19 subjects, and provides quantitative information about total ig, as well as iggand igm-specific responses. what they do not provide, however, is a broader understanding of the human immune response to sars-cov-2 infection, or illuminate potential relationships between covid-19 infection and previous infections (and immunity to) other respiratory viruses including circulating coronaviruses that cause the common cold. to address these goals, multiplex analytical techniques are required. a bead-based multiplex immunoassay for six coronaviruses infecting humans (pre-sars-cov-2) has been reported, 12 and more recently a 4-plex assay on the quanterix platform focused on sars-cov-2 antigens has been described. 13 despite these advances, there remains a significant need for analytical methods able to rapidly quantify antibodies not only to sars-cov-2, but also to other coronaviruses, and other pathogenic viruses. most importantly, these must be able to discriminate among responses to different closely related viruses and different antigens from the same virus. to address this need, we have developed a prototype 15-plex array on the arrayed imaging reflectometry (air) platform. air is a label-free multiplex sensor method in which the surface chemistry and deposition of capture molecules to form a microarray on a silicon chip are carefully controlled such that s-polarized hene laser light at a 70.6º incident angle to the chip undergoes total destructive interference within the surface film. 14 binding to any probe spot on the array degrades the antireflective condition in proportion to the amount of material bound, yielding an increase in the reflected light as observed by a ccd camera. by comparing the intensity of the reflected light to an experimentally validated model, the thickness change for each spot, and therefore the quantity of each analyte in the sample, may be precisely and sensitively determined. 15 we have previously reported the utility of influenza antigen arrays fabricated on the air platform for assessment of anti-influenza antibodies in human, animal, and avian serum, 16, 17 both as a tool for viral surveillance and for assessment of the efficacy of a candidate vaccine. we have also demonstrated that air is scalable at least to 115-plex assays, used for discriminating different influenza virus serotypes. 18 , 19 we therefore anticipated that the platform would be useful as a way to quantify anti-sars-cov-2 antibodies, antibodies to other coronaviruses including circulating ("common cold") strains, and other respiratory pathogens including influenza. here, we discuss the development and testing of a mixed coronavirus / influenza antigen panel on air, and its application to analyzing the coronavirus antibody profile of a cohort of convalescent covid-19 patients and subjects of unknown disease status. material sources: for air assays, sars-cov-2, sars-cov, mers, and influenza type a and b antigens were obtained from sino biological, inc., and are described in more detail below. most antigens were supplied as lyophilized material and reconstituted at the recommended concentrations using 18-mω water, while the remaining antigens were supplied frozen on dry ice. pbs-et was prepared as phosphate buffer (10 mm monobasic sodium phosphate, 10 mm dibasic sodium phosphate, 150 mm nacl) with 0.02% w/v tween-20 and 5 mm edta. aminereactive substrates for fabrication of air arrays were provided by adarza biosystems, inc. for elisa assays, sars-cov-2 full-length spike and rbd were produced in-house using a mammalian expression system, 20,21 as was influenza a/h1n1/california 2009 hemagglutinin. hcov-229e and hcov-oc43 spike proteins (baculovirus-expressed) were obtained from sino biological. tetanus toxoid (ttd) was obtained from calbiochem. antigen probe formulation: prior to microarray fabrication, antigens were buffer-exchanged and concentrated using amicon centrifugation filters (emd millipore) into phosphate buffer at ph 5.8 and ph 7.2 prior to use. during development, several printing concentrations and/or solution ph values of each antigen were tested, along with sugar additives (glycerol, trehalose) in order to optimize spot uniformity and morphology as well as initial probe thickness. 22 preparation of arrays: arrays were printed on amine-reactive silicon oxide substrates (adarza biosystems, inc.) using a scienion sx piezoelectric microarrayer (scienion, a.g.) with spot volumes of approximately 300 pl. six spots were printed for each antigen, the final layout of which is shown in figure 1 . the number of spots arrayed was not critical to robust analytical performance or statistical analysis. each spot consists of approximately 300 pixels when imaged by the ccd in an air chip reader (adarza biosystems, inc.), with each pixel representing a discrete interrogation of a unique probe surface region. therefore, averaging these pixel values together produces an inherently reliable measure of analyte-to-probe response. dilutions of polyclonal anti-fluorescein (anti-fitc, rockland inc.), were printed as negative intra-array controls. after printing, chips were mounted onto adhesive strips at appropriate spacing for 96-well plates, and then placed into 50 mm sodium acetate buffer (ph 5) for 5 minutes. next, a 1.5% bsa solution was added to each well resulting in a final bsa concentration of 0.5% to passivate the remaining amine-reactive surface functionality. after blocking for 20 minutes, the chips were transferred to new wells containing 20% fetal bovine serum (gibco) in pbs-et as a secondary block, and incubated for 40 min. this step was required to reduce nonspecific binding from human serum at the assay endpoint. the chips were then rinsed briefly (5 min) in new wells containing pbs-et, then transferred to wells containing microarray stabilizer solution (surmodics ivd). after a 30-minute incubation, the chips were dried at 40 °c in an oven for 60 min. this last step renders the sensors shelf-stable, until use in assays performed later. igm, or 20% fbs as a negative control. each of these conditions was produced in duplicate. secondary antibodies were diluted to 1 μg/ml for both goat α-higg (jackson immunoresearch) and rabbit α-higm (rockland, inc.) in adarza diluent. after one hour of incubation with secondary antibodies at room temperature, chips were washed twice for 5 minutes in pbs-et, then rinsed with water and dried with nitrogen as before. data analysis: air images were analyzed using the adarza ziva data analysis tool. probe spots with major defects or debris were manually flagged and eliminated, and minor defects in spot quality were automatically identified and excluded from the median intensity measurement. the median intensity values were converted to median thickness values using a best-fit line to an experimentally derived reflectance model. 14 then, the median thickness values were further processed in microsoft excel as described below, and are referred to simply as "thickness" hereafter. while anti-fitc spots were designed to serve as an intra-chip normalizer, these were not used as such due to the unexpected presence of anti-goat igg antibodies in some single donor human serum samples. therefore, the blank area served an intra-chip normalizer to mitigate any variation in the reactivity of the surface chemistry between air chips. the thickness of the blank area was subtracted from the thickness of each probe spot to produce "normalized thickness" values for each probe spot. all of the normalized thickness values across replicate chips (n=2) were averaged together (maximum of n=24 probe spots) for each antigen, and the standard deviation was calculated. the average thickness for each antigen in the fetal bovine serum (fbs) control was subtracted from the average thickness obtained for each antigen in each subject sample to produce the "normalized thickness change (δ thickness)." in the case of the polyclonal antibody titration, the control chip was incubated in a matrix of fbs and pnhs. elisa assay: serum igg titers specific for sars-cov-2 proteins and selected non-coronavirus proteins were determined by elisa as described previously. 23 human serum standards were used to assign weight-based concentrations of antigen-specific igg as previously described, with the limit of assay sensitivity set at 0.5 μg/ml for all antigens. 23, 24 results: . this is as expected given the prevalence of these viruses in the general population. addition of an anti-sars-cov-2 polyclonal antibody raised against the sars-cov-2 spike protein receptor binding domain (rbd) at 1 μg/ml produced a strong signal on all three rbdcontaining antigens (s1 + s2 ecd, s1, and rbd). overall response to the polyclonal antibody was well-behaved, and titrated to zero as expected ( figure 2c and d) . quantitative data are presented in ångstroms of build. at the highest concentrations, significant cross-reactive binding to the hcov-229e spike protein was observed, as well as some binding to the hcov-oc43 spike protein and mers s1. calculated limits of detection 25 for these data were 43.3 ng/ml (sars-cov-2 s1 + s2 ecd), 40.7 ng/ml (sars-cov-2 s1), and 25.1 ng/ml (sars-cov-2 rbd). however, these should be viewed as provisional, and subject to optimization. response of a commercial anti-sars-cov-2 rabbit polyclonal antibody (pab) on the array. (a) array exposed to array exposed to 20% fbs + 10% pnhs; (b) array exposed to 1 μg/ml anti-sars-cov-2 pab in 20% fbs + 10% pnhs. strong responses to sars-cov-2 s1+s2 ecd, s1, and rbd are observed, as well as smaller cross-reactive responses to hcov-229e, hcov-oc43, and mers spike proteins; (c) quantitative data for the titration. convalescent serum array responses were compared to an elisa assay ( figure 5 ). as elisa values were all igg-specific, and air data discussed thus far (obtained in a "label-free" mode) was a combination of igg and igm-specific responses, these results would not be expected to match precisely. differences in the expression system used for antigen production (baculovirus for commercial antigens used in air; hek 293t cells used for antigens used in the elisa assays) could also lead to differences. however, overall trends for sars-cov-2 antigens correlate well, as shown in figure 6 . to provide further detail with regard to the response, air assays were run using secondary anti-igg and anti-igm antibodies to determine class-specific responses for a subset of samples ( figure 7 ). was the case with assays run using the laboratory air assay, analysis using the ziva system readily discriminated between negative and convalescent samples (figure 8 ). three putative convalescent covid-19 samples gave responses on all sars-cov-2 antigens that were below the threshold for a positive response (two standard deviations above the average of the 16 negative samples). this is analogous to the air and elisa results obtained for sample hd2146, as described above. the remaining 12 convalescent samples gave strong responses on at least one sars-cov-2 antigen, with many responding strongly to both rbd and s2 ( figure 8 ). with at least one sars-cov-2 antigen response above threshold. health and disease result from many factors, including the overall landscape of a person's immune system. as such, methods for profiling antigen-specific antibody titers to a range of diseases in addition to the disease of primary current interest are of utility when studying the disease. to that end, we have presented preliminary data on a 15-plex array on the air platform, developed in response to the need to study sars-cov-2 but incorporating antigens for other coronaviruses and influenza. responses to sars-cov-2 antigens on the array effectively discriminated between serum samples from uninfected and covid-19 convalescent subjects, with generally good correlation to elisa data. follow-up assays demonstrated that exposure of the arrays to anti-igg and anti-igm antibodies enabled discrimination of antibody isotype. an important aspect of this work is the ability to evaluate anti-sars-cov-2 immunity in the context of the individual's overall immune landscape. because available chip real estate allows for substantial expansion of the multiplex capability of the array, in ongoing efforts we will add additional antigens for other strains of influenza (by analogy to our previous work 17 ), as well as other upper respiratory infections such as respiratory syncytial virus and metapneumovirus. other coronavirus antigens including nucleocapsid (n) are also likely candidates for addition to the array, as they are known to produce an immune response (as seen in the elisa results, for example). thus, the flexibility of the air platform will prove useful not only in the current pandemic, but as other viruses inevitably emerge. report from the american society for microbiology covid-19 international summit targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals anti-sars-cov-2 virus antibody levels in convalescent plasma of six donors who have recovered from covid-19 serology assays to manage covid-19 developing antibody tests for sars-cov-2" the lancet sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup diagnostic value and dynamic variance of serum antibody in coronavirus disease development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis profiling early humoral response to diagnose novel coronavirus disease (covid-19) test performance evaluation of sars-cov-2 serological assays development and evaluation of a multiplexed immunoassay for simultaneous detection of serum igg antibodies to six human coronaviruses ultra-sensitive high-resolution profiling of anti-sars-cov-2 antibodies for detecting early seroconversion in covid-19 patients a theoretical and experimental analysis of arrayed imaging reflectometry as a sensitive proteomics technique validation of arrayed imaging reflectometry biosensor response for protein-antibody interactions: cross-correlation of label-free, arrayed sensing of immune response to influenza antigens a multiplex label-free approach to avian influenza surveillance and serology crowd on a chip: label-free human monoclonal antibody arrays for serotyping influenza characterizing emerging canine h3 influenza viruses sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup a serological assay to detect sars_cov-2 seroconversion in humans investigation of non-nucleophilic additives for reduction of morphological anomalies in protein arrays memory b cell expansion by seasonal influenza virus infection reflects early-life imprinting and adaptation to the infecting virus assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot 89-s statistical method for determining and comparing limits of detection of bioassays we thank alicia papalia for assistance with human samples, and florian krammer for the generous donation of plasmids for sars-cov-2 antigen production (s1 + s2 ecd and rbd). key: cord-329493-ueqlhgn0 authors: stadler, konrad; masignani, vega; eickmann, markus; becker, stephan; abrignani, sergio; klenk, hans-dieter; rappuoli, rino title: sars — beginning to understand a new virus date: 2003 journal: nat rev microbiol doi: 10.1038/nrmicro775 sha: doc_id: 329493 cord_uid: ueqlhgn0 the 114-day epidemic of the severe acute respiratory syndrome (sars) swept 29 countries, affected a reported 8,098 people, left 774 patients dead and almost paralysed the asian economy. aggressive quarantine measures, possibly aided by rising summer temperatures, successfully terminated the first eruption of sars and provided at least a temporal break, which allows us to consolidate what we have learned so far and plan for the future. here, we review the genomics of the sars coronavirus (sars-cov), its phylogeny, antigenic structure, immune response and potential therapeutic interventions should the sars epidemic flare up again. a new infectious disease, known as severe acute respiratory syndrome (sars), appeared in the guangdong province of southern china in 2002. it is mainly characterized by flu-like symptoms, including high fevers exceeding 38°c or 100.4°f, myalgia, dry nonproductive dyspnea, lymphopaenia and infiltrate on chest radiography. in 38% of all cases, the resulting pneumonia led to acute breathing problems requiring artificial respirators 1 . the overall mortality rate was about 10%, but varied profoundly with age -although sars affected relatively few children and generally appeared to be milder in the paediatric age group, the mortality rate in the elderly was as high as 50% [2] [3] [4] . although accurate information about the precise origin of the disease is not available, the chinese ministry of health reported an outbreak of unexplained pneumonia to the world health organization (who) on 11 february 2003. during the period from 16 november 2002 to 9 february 2003, 305 cases and five deaths due to atypical pneumonia, which were originally thought to be caused by chlamydia pneumoniae, occurred in the southern chinese province of guangdong 5 . a chinese doctor who had been treating patients in guangdong spread the infection outside of mainland china when he travelled to hong kong on 21 february 2003. there, while resident on the ninth floor of the hotel metropole, the patient developed symptoms and was transferred to the hospital on 22 february where he died the following day. subsequently, ten secondaryinfected guests of the hotel (eight from the ninth floor and two from the eleventh and fourteenth floors) boarded planes and carried the infection to singapore, vietnam, canada and the united states, making sars the first epidemic to be transmitted by air travel. when the epidemic finally waned after more than 100 days, the who counted a cumulative number of 8,098 probable sars cases and 774 deaths worldwide, in a geographical area spanning 29 countries 6 . carlo urbani, a who infectious disease expert who had been called to hanoi, was the first to realize that the chinese doctor in hong kong had suffered from a previously unknown disease and alerted the authorities. following unprecedented collaboration between laboratories and scientists worldwide, a previously unidentified coronavirus was isolated from frhk-4 and vero e6 cells that were inoculated with clinical (nasopharyngeal, oropharyngeal and sputum) specimens from patients 1, 7, 8 . the association of the virus with the disease was confirmed when macaques that were inoculated with the virus developed symptoms similar to those observed in human cases of sars 9, 10 . it is likely that scientists from the chinese academy of military medical science had observed what seemed to be coronavirus particles in an electron micrograph by 26 february 2003. however, with r e v i e w s indicating that this outbreak is the first introduction of sars-cov into the human population. at present, accidental infections of researchers handling the pathogen seem to pose the greatest risk for a renewed spread of the virus as a recent case in singapore has shown 15 . for now, the transmission of this emerging infectious disease has been stopped, but there is no information on when, or if, sars-cov will re-emerge in the human population. coronaviruses were named after their corona solis-like appearance in the electron microscope, which is caused by the club-shaped peplomers that radiate outwards from the viral envelope (fig. 1a) . the spherical capsid contains a positive-strand rna genome of about 30 kb -the largest of its kind. coronaviruses have been subdivided into three groups on the basis of serological and genetic properties 16 . their broad host range extends from man to turkey; where they are typically associated with respiratory, enteric, hepatic and central nervous system diseases. in man, they cause mainly upper-respiratory-tract infections, and are responsible for a large proportion of all common colds. sequence analysis revealed the organization of the 29,740 base (fra isolate; genbank acc.no. ay310120; see sars coronavirus fra in the online links) genome of the sars-cov (fig. 2) to show the characteristic features of coronaviruses [17] [18] [19] [20] (fig. 3) . nucleotides 1-72 contain a predicted rna leader sequence preceding an untranslated region (utr) spanning 192 nucleotides. two overlapping open reading frames (orf1a and orf1b), which encompass approximately two-thirds of the genome (nucleotides 265-21485), are downstream of the utr. a translational read-through by a −1 ribosomal frameshift mechanism allows the translation of the overlapping reading frames into a single polyprotein 20 . virus-encoded proteinases, namely the only a few samples available for analysis, they did not feel confident enough to challenge the authorities and did not publish their findings. close contact with very sick patients facilitates personto-person transmission of the virus -apparently, sars-cov spreads in droplets but its efficiency of infection seems to be low, with an infectivity index of about 3 (refs 11, 12) . in some cases, however, a single person can infect a high number of people but, as yet, there is no satisfactory explanation for this so-called 'superspreader' phenomenon 2 . a virus with close homology to sars-cov was isolated from palm civets and racoon dogs, which are considered delicacies in southern china, indicating that the virus could have jumped recently from these mammals to man 13 . however, a second search by another group failed to reveal any trace of sars-cov in more than 60 animal species, including 76 palm civets 14 . although unlikely, the possibility that humans infected these sars-positive animals cannot be formally excluded. in the meantime, over 30 different sars-cov isolates have been sequenced, which will hopefully allow us to trace the chain of transmission back to its origin. the sars epidemic, which caused global panic and huge economical damage to the asian economy, was contained primarily by aggressive quarantine measures. additional beneficial factors could include the attenuation of the virus following prolonged passage through the human population, or the onset of summer, as higher temperatures generally decrease the incidence of at least some respiratory infections. a natural reservoir for the virus could serve as the launch pad for another sars outbreak during the next winter-spring season. if we are lucky, however, the ability of this virus to cross the species barrier could be the result of a rare series of events that is unlikely to be repeated. a retrospective serological study did not detect anti-sars-cov antibodies in a normal population, assumptions about the function of the remaining proteins can be made by analogy with other coronaviruses, although our knowledge about the biological functions of these proteins is also incomplete (see table 1 for information on the encoded proteins). together with host factors, some of these proteins might form the viral replication-transcription machinery that is associated with the membranous structures in the infected cells 23, 24 . (the peculiar features of the replication cycle of coronaviruses are described in box 1.) the remaining 3′ part of the genome encodes four structural proteins that are arranged in the same order in all papain-like cysteine protease (plpro) and the 3c-like cysteine protease (3clpro), cleave the polyprotein into individual polypeptides, which provide all the proteins that are required for replication and transcription 20, 21 . wheareas group 2 coronaviruses contain two paralogous plpros (pl1pro and pl2pro), only one is found in the orf1a of the sars-cov genome. orf1b encodes a helicase with atpase and dna (and possibly also rna) duplex-unwinding activities, at least when it is expressed in escherichia coli 20 . computational analysis predicted that the carboxy-terminus of orf1b encodes a mrna cap-1 methyltransferase 22 . some finally, at the 3′ end of the genome, a second 340nucleotide utr, which is followed by a poly(a) tract, was found. this 3′ utr contains a 32-nucleotide stem-loop ii-like motif (s2m) motif, which has also been reported in astroviruses, in one equine rhinovirus and avian infectious bronchitis virus (ibv) 28 . a typical feature of coronaviruses is the presence of a transcription-regulatory sequence (trs) that is important in rna transcription and regulation (figs 2 and 3). this short motif is usually found at the 3′ end of the leader rna and, with a few exceptions, precedes each translated orf 29 . when thiel and colleagues 20 isolated one genomic and eight subgenomic rnas from the fra strain and sequenced their 5′ ends, they identified a conserved sequence (5′acgaac3′) that was located in coronaviruses: s, spike protein; e, envelope protein; m, membrane glycoprotein; and n, nucleocapsid protein. furthermore, the structural protein region of the sars-cov genome contains several genes that encode additional non-structural proteins that are known as 'accessory genes' (fig. 3) . some of these molecules seem to be dispensable for virus viability both in vitro and in vivo; their deletion creates viruses that are attenuated [25] [26] [27] . these accessory genes differ significantly among the three coronavirus groups -they are also referred to as group-specific genes. the sars-cov has eight predicted orfs of unknown function in this region of the genome. it lacks the haemagglutinin esterase (he) gene, which is encoded by almost all of the group 2 viruses. the analyses are based on the sequence of the sars-cov fra isolate (genbank accession number ay310120). transmembrane domains (tmds) were predicted using the program psort (threshold is less than −2); the glycosylation sites were predicted using the netnglyc server (see netnglyc in the online links). information on the functional predictions has been taken from refs 20,33. nsp, non-structural protein. coronaviruses contain the largest rna genomes described so far -the positive-sense, single-stranded rna molecule has a 5′ cap and a 3′ poly(a) tail. the life cycle of a coronavirus starts when the spike (s) protein, which forms the distinctive, eponymous crown that is observed with coronaviruses, interacts with a receptor through its s1 domain. the entry, which is probably mediated by the s2 domain, occurs by membrane fusion. the rna genome is then released into the cytoplasm where replication takes place. the host translation machinery translates the overlapping open reading frames orf1a and orf1b by a ribosomal frame-shifting mechanism to produce a single polyprotein. cleavage by virally encoded proteinases yields the components that are necessary to assemble the viral replication complex, which synthesizes full-length negative-strand rna. in addition, a discontinuous transcription strategy during negative-strand synthesis produces a nested set of sub-genomic negative-sense rnas. in this process, the transcription regulatory sequence (trs), which is present at the 3′ end of the leader sequence and, with a few exceptions, upstream of each translated gene, is important. it is postulated to fuse the 3′ ends of the nascent subgenomic minus strands to the antisense leader sequence. these discontinuously synthesized minus strands then act as templates for the synthesis of positive-sense mrnas. an alternative hypothesis proposes that these mrna molecules are generated by discontinuous transcription during positive-strand synthesis (the figure shows the positive-sense mrna products). in almost all cases, only the most 5′ orf is translated. nucleocapsid (n) protein and genomic rna assemble in the cytoplasm to form the helical nucleocapsid. this core structure acquires its envelope by budding through intracellular membranes between the endoplasmic reticulum (er) and the golgi apparatus. the membrane (m), envelope (e) and s proteins, all of which will be accommodated by the lipid bilayer, are transported through the er to the budding compartment, where the nucleocapsid probably interacts with the m protein to trigger assembly. during the transport of the virus through the golgi apparatus, sugar moieties are modified and in some, but not all, coronaviruses the s protein is cleaved into s1 and s2 domains. any s protein that is not incorporated into the virions is transported to the cell surface. finally, the virus is released from the host cell by fusion of virion-containing vesicles with the plasma membrane. the common leader sequence on the 5′ end of each mrna is shown in red. 13 . this observation raises the intriguing hypothesis that the 29-nucleotide deletion that has been observed in most human isolates could have increased the fitness of the virus in human hosts and allowed the spread to the human population. lacking a proof-reading mechanism, rna viruses are generally characterized by a high mutability. the high mutation rate results in continuously evolving viral species, which allow the virus to escape host defences. additionally, coronaviruses have a high frequency of rna recombination that has the theoretical potential of accelerating the emergence of new viral species 29 . owing to the limited number of sequenced isolates, however, it is too early to draw any reliable conclusions concerning the mutation rate of sars-cov. the most interesting amino-acid changes that have been reported so far are two recurrent nonconservative amino-acid substitutions (gly to asp and ile to thr) in the antigenic domain s1 of the spike protein 30 . gly and ile can be found in the hong kong front of nine predicted orfs, and which fitted the description of a trs (figs 2 and 3). by contrast, marra et al. 17 and rota et al. 18 proposed different trss (5′cuaaac3′ and 5′aaacgaac3′, respectively), but these sequences do not precede all predicted genes and no experimental evidence for their function has been provided. although the overall organization of the sars-cov genome is similar to other coronaviruses (fig. 3) , the amino-acid conservation of the encoded proteins is usually low (fig. 2; table 2 ). helped by the groundwork laid by previous generations of coronavirus researchers, the sars epidemic has been the first infectious disease outbreak to fully benefit from the revolutionary technologies of the post-genomic era. less than 1 month after the initial identification of the virus as the infectious agent of sars, two independent genome sequences of the virus had been obtained 17, 18 . within 3 months, the genome sequences of 20 independent clinical isolates were made available in the genbank database (see box 2 for details). comparative analysis of these isolates revealed more than than 99% sequence conservation. the few differences, however, have allowed a straightforward organization of all viral isolates into two families: those originating from mainland china and hong kong and those originating from the index case in the hong kong hotel 30 (fig. 4) . perhaps the most interesting observation was made in the the most important question following identification of the sars-cov was whether it represents a completely new group, a variant of one of the three known groups or a combination of these groups. phylogenetic analysis based on the first available 300 and 405 nucleotides of the highly conserved polymerase gene 7, 31 indicated that sars-cov was distinct from the three known groups. marra and rota also reached the same conclusion 17, 18 , and proposed that sars-cov be placed in its own group (fig. 5a) (fig. 5b) , which indicated that it is more closely related to members of group 2 and might share a common ancestor with them. moreover, this conclusion is corroborated by the striking observation that 19 out of 20 cysteine residues in the s1 domain of the sars-cov spike protein are spatially conserved when compared with the group 2 consensus sequence, whereas only 5 cysteines are conserved when compared with group 1 or 3 consensus sequences (fig. 6) . this analysis supports the conclusion that the sars-cov virus split early from other group 2 viruses and has evolved independently for a long period of time. although very little is known about the sars-cov proteins themselves, homologies with known coronavirus proteins can be used to predict the features of several sars-cov proteins that could be interesting targets for antiviral drugs or vaccines. viral enzymes that are essential for virus replication are the most attractive candidates for the development of small, antiviral molecules, whereas some of the structural proteins represent obvious targets for vaccine development. on the basis of the x-ray crystal structures of 3clpro from transmissible gastroenteritis coronavirus (tgv) 33 and human coronavirus 229e, a three-dimensional model of the corresponding sars virus protein has been proposed 34 . the model can be used to reduce the number of compounds to be tested to find an effective protease inhibitor. indeed, an active form of this protease has already been expressed in e.coli, which can be immediately used for drug screening. the helicase, which is already available in recombinant form, represents another attractive target for highthroughput screens 20 . plpro is another potential candidate for antiviral drugs, although no homologous index case group, whereas asp and thr are found in the mainland isolates. another, non-conservative substitution (gly to arg) in the s1 domain is only found in strains gz01 and bj02. it is tempting to speculate that these mutations represent adaptations to the new host or its immune response. additional information on ongoing variability studies can be obtained directly from the sars coronavirus resource (see online links). genbank database and were available to the scientific community. the 31 sequenced isolates come from china (10 strains), singapore (5 strains), taiwan (12 strains), vietnam (1 strain), germany (1 strain), italy (1 strain) and canada (1 strain), and are derived from patients representing both primary and secondary contacts. place of isolation accession number cov spike protein 39 . all group 1 coronaviruses can use apn as a receptor 40 , whereas the group 2 mouse hepatitis virus (mhv) uses glycoproteins belonging to the carcinoembryonic antigen family as receptors on their target cells 41 . whether the hapn binding domain is the receptor in vivo remains to be shown. the protruding s1 domain is adjacent to the s2 domain, which consists of a stem, a transmembrane region and a short cytoplasmic tail. analogous to other coronavirus s proteins, the sars-cov spike protein contains two heptad repeats that are located in the s2 domain. it is proposed that these repeats might trigger the fusion of the viral envelope with the cell membrane, as has been shown recently for mhv 42 . in some group 2 and group 3 viruses, the spike is cleaved during maturation into two subunits, s1 and s2, which stay non-covalently attached. the exact role of this cleavage process is unclear as it does not seem to influence infectivity; however, it may enhance fusion activity [43] [44] [45] [46] [47] . the mature sars-cov seems to contain uncleaved spike protein, but cleavage after binding to the target cell cannot be excluded. to conclude, the spike protein is a target for the development of agents that block the virus from binding to its cellular receptor and is the docking site for peptides that might inhibit fusion. several additional targets for further study have been identified. first, the 76-amino-acid e protein -computer analysis has predicted a long transmembrane domain close to the n-terminus and two n-terminal glycosylation sites with a very low level of amino-acid similarity to other coronaviruses. second, the m glycoprotein, which is a 221-residue polypeptide that consists of a short n-terminal ectodomain with a n-glycosylation site, three transmembrane segments and a c-terminus located on the interior side of the viral envelope, and which closely resembles m glycoproteins from other group 2 viruses. third, the n protein, which is a 397-residuelong phosphoprotein that interacts with viral genomic rna to form the helical nucleocapsid, and which has a low level of conservation with other coronaviruses. with the notable exception of β-interferon, which has been reported to interfere with the replication of the sars virus in vitro 48 , no licensed drug or vaccine is available. large-scale screening of existing antivirals or big chemical libraries for potential replication inhibitors has not been very successful. at present, the only promising substance is glycyrrhizin, a component of liquorice roots, which also has activity against hiv. this compound was identified during a small-scale experiment involving only a small number of known antiviral compounds 49 . the development of assays that are based on viral enzyme activity and which are amenable to high-throughput screening of existing and new chemical libraries, will likely identify effective compounds in the near future. nevertheless, unless these putative compounds are already licensed or are existing products in the advanced stages of development, they will not be available to treat patients in the structures are available. given its fundamental role in virus biology, the rna-dependent rna polymerase (rdrp) is high on the list of promising targets. of the different possible vaccine targets, the s glycoprotein is the most attractive candidate for exploitation. this protein forms the large surface projections that are characteristic of coronaviruses (fig. 1) , and which are most likely to be composed of homotrimers. the heavily glycosylated 1,255 amino-acid protein 35 contains an amino-terminal, bulbous head (s1), which comprises the receptor-binding domain and is also believed to be responsible for the host and tissue tropism of the virus 36 non-replicating viruses, or viral vectors that are based on adenovirus, canarypox, modified vaccinia virus ankara (mva) or alphavirus. in particular, the devlopment of non-replicating coronavirus-like particles that mimic the structure of native virions could prove promising in the search for a successful vaccine as they display a large repertoire of antigenic sites and discontinuous epitopes. however, each of these approaches, including passive immunotherapy, need to be carefully evaluated as some vaccines that have been developed against feline coronavirus actually exacerbated the disease when vaccinated animals were challenged with the wild-type virus 50 . killed-virus vaccines that are ready to be tested in phase i clinical trials are likely to be available soon. under normal circumstances, a vaccine takes 6-8 years of clinical development after entering phase i clinical trials. these timelines could be shortened considerably should the disease burden and a state of medical emergency induce the regulatory agencies to 'fast track' the approval process. in conclusion, the development of therapeutic strategies against this new coronavirus seems to be possible with the available technologies and is not an unreachable goal to the same level as hiv or hepatitis c virus. given enough time and economic pressure, antiviral drugs, human monoclonal antibodies, vaccines and sirnas that are active against sars-cov are all likely to become available. the remaining question is whether we will have the time to develop effective therapies before another epidemic emerges in the human population. should sars return this winter, we will still need to rely primarily on quarantine measures to contain the disease. equally important is the development of technologies that allow the rapid and simple diagnosis of sars. just as worrying, however, is a scenario in which the virus does not re-emerge for a couple of years, causing the economic incentive for companies to invest in sars to disappear, so none of the above measures will have been developed and implemented. near future. cellular proteins that are essential for virus replication should also not be overlooked as possible targets -new technologies such as double-stranded small interfering rnas (sirnas) have considerable future potential, but as yet, are still riddled with practical difficulties. at present, researchers are working on the development of efficacious delivery systems for sirnas. and following the successful conclusion of this research, they will still need to prove their potential in animal models of infection. antibodies that are able to neutralize viral infection are also likely to be an effective way to prevent and cure this disease. passive immunotherapy using sera from convalescent patients was initially proposed as treatment for disease. given the excellent track record of human monoclonal antibodies in the treatment of cancer and infectious diseases, this should prove a fruitful area for therapeutic development. indeed, monoclonal antibodies obtained from immortalized b lymphocytes of sars convalescent patients have been shown to neutralize virus infection in vitro and to prevent virus replication in a mouse model of sars-cov infection (a. lanzavecchia and r.r., unpublished observations). of course, a safe and effective vaccine would be the ideal solution, because not only would it prevent the disease in vaccinated people but a vaccine would also curtail the spread of the virus. although only a short period of time passed since sars-cov was identified as the infectious agent that was responsible for the epidemic, candidate vaccines based on killed virus are already available. their efficacy still needs to be shown, but our laboratory (and possibly others) are in the process of testing vaccines on the basis of inactivated sars virus in pre-clinical models. in addition to the traditional approach, a number of newer technologies are being used. these include subunit vaccines containing recombinant spike protein expressed in mammalian cells or yeast, either alone or in combination with other sars-cov antigens. alternatively, these antigens could be delivered by dna immunization by figure 6 | the s1 domain of sars-cov spike is structurally related to group 2 coronaviruses. schematic representation of cysteine positions in the s1 domains of group 1, 2 and 3 coronaviruses, compared with the sars-cov spike protein. horizontal bars represent the s1 amino-acid sequences (in the case of sars-cov and ibv) or the consensus profiles (generated from group 1, (g1 cons) and from group 2 (g2 cons)). the bars are drawn to scale. relative cysteine positions are indicated by rectangular bars. only cysteines that are conserved within each consensus are reported. coloured lines connect cysteines that are conserved between the sars-cov s1 domain and the consensus sequence generated from the group 1 (green), group 2 (red) and ibv s1 sequences (blue). coronavirus as a possible cause of severe acute respiratory syndrome epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical presentations and outcome of severe acute respiratory syndrome in children clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study acute respiratory syndrome in china summary of probable sars cases with onset of illness from 1 identification of a novel coronavirus in patients with severe acute respiratory syndrome a nnvel coronavirus associated with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions transmission dynamics and control of severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars in china: tracking the roots of a killer severe acute respiratory syndrome (sars) in singapore virus taxonomy the genome sequence of the sarsassociated coronavirus references 17 and 18 are the first reports of the complete genome sequences of two sars-cov isolates (tor2 and urbani strains, respectively) the complete genome sequence of severe acute respiratory syndrome coronavirus strain hku-39849 (hk-39) mechanisms and enzymes involved in sars coronavirus genome expression the complete genome sequence of a sars-cov isolate (fra) and experimental data on its key rna elements and protein functions are described. the enzymatic activity of the sars-cov helicase and two proteinases is shown viral replicase gene products suffice for coronavirus discontinuous transcription mrna cap-1 methyltransferase in the sars genome the molecular biology of coronaviruses the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system a common rna motif in the 3′ end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early splitoff from the coronavirus group 2 lineage the authors describe the genome organization and expression strategy of the sars-cov. a phylogenetic analysis of the orf1b indicates that the sars-cov represents an early split-off from group 2 coronaviruses structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra α-helical domain coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs on the basis of these structures, the authors provide a three-dimensional model of the main proteinase of sars-cov, and using molecular modelling mass spectrometric characterization of proteins from the sars virus: a preliminary report the coronaviridae retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier switching species tropism: an effective way to manipulate the feline coronavirus genome putative hapn receptor binding sites in sars-cov spike protein feline aminopeptidase n is a receptor for all group i coronaviruses receptor specificity and receptor-induced conformational changes in mouse hepatitis virus spike glycoprotein the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the function of the spike protein of mouse hepatitis virus strain a59 can be studied on virus-like particles: cleavage is not required for infectivity fusiondefective mutants of mouse hepatitis virus a59 contain a mutation in the spike protein cleavage signal fusion formation by the uncleaved spike protein of murine coronavirus jhmv variant cl-2 the virulence of mouse hepatitis virus strain a59 is not dependent on efficient spike protein cleavage and cell-to-cell fusion proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity treatment of sars with human interferons this paper reports the first results obtained for the search of possible sars inhibitors. the authors show that a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination the neighbor-joining method: a new method for reconstructing phylogenetic trees the authors are grateful to the fonds der chemischen industrie and the chiron vaccines cellular microbiology and bioinformatics unit for their continuous support. the authors have declared competing financial interests; see web version for details. key: cord-353392-rqeultbq authors: kumar, govindarajan venkat; jeyanthi, venkadapathi; ramakrishnan, saminathan title: a short review on antibody therapy for covid-19 date: 2020-04-20 journal: new microbes new infect doi: 10.1016/j.nmni.2020.100682 sha: doc_id: 353392 cord_uid: rqeultbq abstract the beginning of the novel sars-cov-2 human coronavirus in wuhan, china, has triggered a worldwide respiratory disease outbreak (covid-19). by april 07, 2020, sars-cov-2 has affected more than 1.36 million people worldwide and caused more than 75,900 deaths. to date, the anti-malaria drug hydroxychloroquine found to be a treatment option for sars-cov-2. in addition to supportive treatment, such as oxygen supply in moderate cases and extracorporeal membrane oxygenation in critically ill patients, unique medications for this condition are also under investigation. here we reviewed the antibody therapy might be an immediate strategy for emergency prophylaxis and sars-cov-2 therapy. the coronavirus is a family of viruses that can cause a range of illnesses in humans with the common cold and more severe forms like severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) which are dangerous. the first identified severe infection caused by a coronavirus arose with the 2003 sars epidemic in china [1, 2] . a second outbreak of severe infection began in 2012 in saudi arabia with the mers [3, 4] . the third outbreak of severe illness caused by the novel sars-cov-2 coronavirus (covid-19) that emerged in the wuhan city, china, is pandemic and spread to more than 200 countries [5, 6, 7] . more than half a million people worldwide have been infected by the novel sars-cov-2 coronavirus. as of 07 april 2020, there have been at least 75,900 confirmed deaths and more than 1.36 million affected people worldwide [7] . every hour the numbers have been increasing, with the united states recording the maximum positive cases in the world. italy, spain, germany, france in europe continue to be the most affected, with more than 16,000, 13000, 1000 and 8,000 deaths respectively till april 7, 2020 [7] . currently, the anti-malaria drug hydroxychloroquine found to be a treatment option for covid-19. a non-randomized study in a small sample size from france shows that the hydroxychloroquine plus azithromycin treatment reduced the viral load in covid-19 patients [8] . following this study, another group from france reported that the hydroxychloroquine plus azithromycin have no strong antiviral activity in severely affected covid-19 patients [9] . clinical studies from china show that the hydroxychloroquine reduced the risk of progression to severe illness in covid-19 patients [10, 11] . chloroquine and hydroxychloroquine are highly toxic in overdose, leading to the rapid onset of central nervous system toxicity (seizures and coma) and cardiovascular failure [12] . hydroxychloroquine received an emergency use authorization from the fda as of 3 april 2020, but there are still a lot of questions about optimal doses and treatments for covid-19. coronavirus virions are spherical with a diameter of approximately 125 nm as revealed by cryo-electron tomography and cryo-electron microscopy [13] . the corona viral genome encodes four main structural proteins namely the surface spike (s) glycoprotein, the membrane (m) protein, the small envelope (e) glycoprotein, and the nucleocapsid (n) protein. all these proteins are required to provide the structure of complete viral particles called virion [14, 15] . the spike protein is ~180kd glycoprotein which is present on the surface of the virus. it is crucial for the entry of coronavirus into the host cell. it contains two subunits namely s1and s2. the s1 subunit binds to the receptor on the surface of the host cell whereas s2 subunit mediates the cell membrane fusion [15, 16] . major research has focused on identifying antibody molecules targeting spike proteins as wuxi biologics, to develop serum therapy that could be useful as first aid for high-risk patients. in this mini-review, we highlight the therapeutic intervention that may have the potential for prophylaxis and sars-cov-2 therapy. convalescent plasma therapy can be considered as one of the way to control the sars-cov-2 pandemic. researchers suggest that this technique is decades old approach which was used early 1930s and the theory is simple. the person who has recovered from viral infection blood is collected and serum is separated. the serum which contains antigen raised antibodies was injected into a newly infected person to combat the virus antigen. antibodies are proteins that are produced by b cells of the immune system. they are able to bind to "antigen" a specific molecule present on the pathogen that invades the human system and directly neutralizes or activates an immune response [17, 18] . based on the previous studies and reports in treating other coronaviruses such as sars and mers, the early administration of convalescent plasma from patients that contains raised antibodies can possibly reduce the spreading of infection and mortality [19, 20, 21, 22] . shen et al. reported that the convalescent plasma transfusion may be beneficial in the treatment of critically ill patients with sars-cov-2 infections. after getting approval from the ethical committee, shenzhen, third people's hospital, they administrated convalescent plasma containing neutralizing antibodies to 5 critically ill patients with sars-cov-2. among those 3 patients discharged from the hospital and 2 patients under an incubation period of 37 days [23] . casadevall and pirofski highlighted the risks of passive administration of convalescent sera, which falls into two categories, serum disease and antibody-dependent enhancement of infection. serum disease is those associated with the transmission of other blood infections, whereas the antibody-dependent enhancement is the theoretical concern that antibodies to one form of coronavirus could enhance infection to another viral strain [24] . hence, it is important to identify the human monoclonal antibody that neutralizes sars-cov-2. those cross-neutralizing antibodies can target a common epitope on these viruses and offers potential for the prevention and treatment of covid-19. targeting the trimeric spike (s) glycoproteins on the sars-cov-2 surface that mediate research reports stating that the sars-cov-2 rbd exhibited a significantly higher binding affinity to the ace2 receptor than sars-cov rbd. this could block the binding of sars-cov-2 rbd to ace2-expressing cells, thus constraining their infection to host cells [26] . yushun wan and his colleagues also reported that the sequence of sars-cov-2 which is similar to sars-cov, including its receptor-binding motif (rbm) that directly contacts ace2, strongly signifying that sars-cov-2 uses ace2 as its receptor [27] . andersen (human) monoclonal antibody that neutralizes sars-cov-2. research reports declaring that the 47d11 binds a conserved epitope on the spike receptor-binding domain and cross-neutralize sars-cov-2. the cross-reactive nature of 47d11 shows that the antibody is more possible to target the conserved core structure of the s1b receptor binding domain. hence these neutralizing antibodies can reduce the course of virus action in the host or defend an uninfected host that is exposed to the virus [33] . therefore, targeting the rbd amino acid in sars-cov-2 treatment will help scientists to develop effective therapeutic agents to treat and prevent this infection. amino acid mutations and recombination in rbd of different host coronaviruses are considered to be associated with host adaptation and infection across species. recent research indicates that recombination and insertion of a cleavage site in the rbd may increase the infectivity and replication capacity of the virus [34] . ou et al. analyzed rbd mutation worldwide and reported 10 mutants identified under high positive selection pressure during spread. they investigated the sars-cov-2 isolates collected from different parts of the world and compared the rbd mutations with the prototype wuhan-hu-1 strain. they identified that the two groups of amino acid mutations in the sars-cov-2 rbd domain: the "similar affinity" group (f342l, r408i) and the "higher affinity" group (n354d d364y, v367f, w436r). the "higher affinity" group rbd mutations under the positive selective pressure enhanced the infection efficiency of the sars-cov-2 [35] . hence, epidemiology data and mutation surveillance are very important to reveal more exact spreading routes of the pandemic sars-cov-2. further, the rbd mutated strains in other countries need great consideration to find the therapeutic. in conclusion, it is very essential to isolate the raised antibodies by sars-cov-2 disease recovered patient's regional wise. raised antibodies should be produced on a large scale for the treatment of sars-cov-2 patients. these antibodies could provide an immediate strategy for emergency prophylaxis and sars-cov-2 therapy, while alternative and more time-consuming development of vaccines and new drugs are underway. as a result, sars-cov-2 neutralizing antibodies may be used to prevent infection in people exposed to sars-cov-2, such as hospital staff caring for suspected sars-cov-2 patients, and may also be used for early treatment of infected individuals to prevent the onset of serious sars-cov-2 disease and to reduce the chance of spreading the virus to exposed individuals. the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status coronavirus infections-more than just the common cold transmission characteristics of mers and sars in the healthcare setting: a comparative study sars and other coronaviruses as causes of pneumonia the proximal origin of sars-cov-2 covid-19: a novel zoonotic disease caused by a coronavirus from china: what we know and what we don't coronavirus covid-19 global cases by the hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection a pilot study of hydroxychloroquine sulfate in patients with common 2019 coronavirus disease-19 (covid-19) efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial toxicokinetics of hydroxychloroquine following a massive overdose cryo-em structure of the 2019-ncov spike in the prefusion conformation coronavirus envelope protein: current knowledge a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china convalescent plasma as a potential therapy for covid-19 convalescent plasma therapy for persistent hepatitis e virus infection therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) -possible lessons from a systematic review of sars-cov therapy feasibility of a randomized controlled trial to assess treatment of middle east respiratory syndrome coronavirus (mers-cov) infection in saudi arabia: a survey of physicians use of convalescent plasma therapy in sars patients in hong kong retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients treatment of 5 critically ill patients with covid-19 with convalescent plasma the convalescent sera option for containing covid19 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody a human monoclonal antibody blocking sars-cov-2 infection. biorxiv genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding rbd mutations from circulating sars-cov-2 strains enhance the structure stability and infectivity of the spike protein key: cord-352123-0bflqj1c authors: csiszar, anna; jakab, ferenc; valencak, teresa g.; lanszki, zsófia; tóth, gábor endre; kemenesi, gábor; tarantini, stefano; fazekas-pongor, vince; ungvari, zoltan title: companion animals likely do not spread covid-19 but may get infected themselves date: 2020-08-07 journal: geroscience doi: 10.1007/s11357-020-00248-3 sha: doc_id: 352123 cord_uid: 0bflqj1c coronavirus disease 2019 (covid-19) is a highly contagious infectious disease caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2). from the epidemiological data, the picture emerges that the more severe etiopathologies among covid-19 patients are found in elderly people. the risk of death due to covid-19 increases exponentially with age. eight out of 10 covid-19 related deaths occur in people older than 65 years of age. older patients with comorbid conditions such as hypertension, heart failure, diabetes mellitus, asthma, chronic obstructive pulmonary disease, and cancer have a much higher case fatality rate. governments and public health authorities all over the world have realized that protections of vulnerable older adults should be a priority during the covid-19 pandemic. covid-19 is a zoonotic disease. the sars-cov-2 virus was originally transmitted likely from a bat or a pangolin to humans. recent evidence suggests that sars-cov-2, similar to other coronaviruses, can infect several species of animals, including companion animals such as dogs, cats, and ferrets although their viral loads remain low. while the main source of infection transmission therefore is human to human, there are a few rare cases of pets contracting the infection from a sars-cov-2-infected human. although there is no evidence that pets actively transmit sars-cov-2 via animal-to-human transmission, senior pet ownership potentially may pose a small risk to older adults by (1) potentially enabling animal-to-human transmission of sars-cov-2 in the most vulnerable population and (2) by increasing the exposition risk for the elderly due to the necessity to care for the pet and, in the case of dogs, to take them outside the house several times per day. in this overview, the available evidence on sars-cov-2 infection in pets is considered and the potential for spread of covid-19 from companion animals to older individuals and the importance of prevention are discussed. coronavirus disease 2019 (covid-19) is a highly contagious infectious disease caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1, 2] . as of june 4, during the 2019/2020 global pandemic, approximately 6.6 million people have contracted the virus globally resulting in over 390,000 deaths [3] . from the beginning of the outbreak of the pandemic, the centers for disease control and prevention (cdc) have stated that sars-cov-2 is a respiratory virus, and, as such, its main route of transmission is via respiratory droplets when an infected individual coughs, sneezes, or just talks in close proximity of others [1] . furthermore, the virus may be also spread via contaminated surfaces, when a person touches a contaminated object (e.g., doorknob), then touches their nose, mouth, or eyes [4] [5] [6] . the mean incubation period of the disease is 5.1 days with 97.5% of patients producing symptoms by day 11.5 [7] . the most common symptoms include fever, cough, fatigue, and shortness of breath. most common cause of death is respiratory failure [8, 9] . there is also increasing evidence suggesting that covid-19 may cause fatal myocardial injury, arrythmia/ cardiac arrest, neurological damage (including stroke), and kidney failure (acute kidney injury occurs in one third of patients) [8] . currently reported case fatality rates vary from 1% to more than 10%, due to country-to-country differences in screening in the whole population. from the epidemiological data, the picture emerges that severe covid-19 is primarily a disease of older people [10] . the risk of death due to covid-19 increases exponentially with age in every country studied [10] . a report of 72,314 cases from the chinese center for disease control and prevention [11] shows that case fatality rates by age are as follows: 0.18% (10-19 years of age); 0.32% (20 to 49 years of age); 1.3% (50 to 59 years of age); 3.6% (60 to 69 years of age); 8% (70-79 years of age); 14.8% (≥80 years of age). a report of 73,780 cases produced by the istituto superiore di sanità (iss) showed that the case fatality rates for males by age in italy are as follows: 0% (10-19 years of age); 0% (20 to 29 years of age); 0.6% (30 to 39 years of age); 1.1% (40 to 49 years of age); 2.4% (50 to 59 years of age); 6.9% (60 to 69 years of age); 19.8% (70-79 years of age); 29.2% (80 to 89 years of age); 30.8% (≥ 90 years of age) [12] . older patients with comorbid conditions such as hypertension, heart failure, diabetes mellitus, asthma, chronic obstructive pulmonary disease, cancer, and chemotherapeutic treatment have a much higher case fatality rate. critical cases have a case fatality rate of 49%, whereas mortality is very low in patients with mild symptoms. in the usa, deaths involving covid-19 by age group are as follows: 1% (≤ 34 year of age), 2% (35-44 years of age), 5% (45-54 years of age), 13% (55-64 years of age), 22% (65-74 years of age), 28% (75-84 years of age), 30% (≥85 years of age) [13] . in total, 79% of all covid-19 related deaths occurred in people older than 65 years of age. the causes of these age-related differences in mortality rates are not yet fully understood. possible causes likely include the less efficient functioning and coordination of both cellular and molecular elements of the immune system, the higher number of comorbidities, and the overall frailty and impaired organismal and cellular resilience of elderly patients [2, 11, 14] . governments and public health authorities all over the world have realized that protections of vulnerable older adults should be a priority during the covid-19 pandemic. the most effective step to prevent further sars-cov-2 infections in older adults is to limit exposure by reducing social activity, avoiding gatherings with many people and public transportation. accordingly, governments and public health authorities ordered nursing homes and assisted living facilities to limit visits, even by family members. formal and informal group social and recreational activities and church services were canceled. although these steps are effective, additional measures are needed to limit spread of covid-19. staying at home all day however is impossible if seniors actually possess a dog as the animal will have to be taken out regularly. yet, pet owners usually take their dogs out alone and social distancing may be well doable. potential role for zoonotic spread of covid-19 in older adults? as coronaviruses generally are zoonotic (i.e., they are transmitted between animals and people), covid-19 as well is a zoonotic disease just like sars. whether the novel coronavirus causing covid-19 came from a bat or a pangolin is still uncertain. the initial event where the virus may have jumped to humans seem to have happened in a so-called "wet" market [15, 16] , where fresh meat as well as living wild animals are commonly sold. because of the observed accumulation of infected people by january 2020 who were in some relation to the "hua nan" market in wuhan, this town of 11million inhabitants located at the yangtze river in central china is commonly considered the "ground zero" for the global pandemic [16, 17] . it should be noted that our understanding of the origin of the pandemic may evolve. a recent phyloepidemiologic analysis of sequenced genomic data of close to 100 sars-cov-2 samples by chinese researchers (published in a non-peer reviewed pre-print form [18] ) suggested that while the crowded market may have boosted spread of the novel virus to the whole city, it may have not originated there. the possibility that the virus may have been introduced from elsewhere clearly warrants more research in the upcoming months. genetic sequence data reveals that the sars-cov-2 virus is closely related to coronaviruses found in rhinolophus bat (horseshoe bat) populations [19] . although bats may be the primary reservoir, the original route of transmission to humans is currently unknown and may have involved an intermediate host, probably a pangolin [20] . sars-cov-2, similar to other coronaviruses, can infect several species of animals. initial data suggested that the sars-cov-2 virus can bind to receptors and infect cells of horseshoe bats and civets, whereas mice are not susceptible. in vivo studies suggest that several species, including cats, can be infected with sars-cov-2 virus, whereas chickens, pigs, and ducks are not susceptible [21] . it is estimated that there are currently 135 to 184 million pet dogs and cats in the usa (according to the us pet ownership & demographics sourcebook by the american veterinary medical association (avma) and the biennial appa national pet owners survey by the american pet products association, respectively). accordingly, 38-48% of us households own at least one dog or cat. studies, including the national poll on healthy aging (https://deepblue.lib.umich. edu/bitstream/handle/2027.42/148428/npha_pets-report_final-040319.pdf?sequence=3&isallowed= y), demonstrate the multifaceted health benefits of senior pet ownership (including increased physical activity such as walking, higher emotional well-being, and significant stress reduction). despite these advantages, pet ownership potentially may pose a minor risk to older adults by enabling animal-tohuman transmission of sars-cov-2 in the most vulnerable population. here, we summarize the available evidence about sars-cov-2 infection in pets. in late march 2020, the federal agency for the safety of the food chain (fasfc) in belgium reported that a pet cat was diagnosed to be infected with sars-cov-2 [21, 22] , showing that felines living in the household of people with covid-19 are at risk of contracting the disease and may potentially spread the virus. the cat became ill 1 week after its owner's return home from italy [22] . susceptibility of cats to sars-cov-2 infection is supported by a recent experimental observation [15] . specifically, it was demonstrated that cats exposed to sars-cov-2 under laboratory conditions can be infected and are able to transmit the disease to other felines. on april 23, it was reported that two pet cats in new york state have tested positive for the sars-cov-2, which are the first confirmed covid-19 cases in companion animals in the usa [22] . one of these two cats became sick approximately a week after a person in its household developed respiratory symptoms. the other cat's owner tested positive for sars-cov-2 before the cat fell ill. both cats developed symptoms of upper respiratory disease, including coughing and nasal discharge. in june 2020, a french study reported that screening of 22 cats and 11 dogs from owners previously infected or suspected of being infected by sars-cov-2 identified a per cat infected by sars-cov-2 [23] . for each animal, rectal, nasopharyngeal swabs, and serum were taken. the infected cat, which exhibited mild respiratory and digestive symptoms, tested positive by rt-qpcr on the rectal swab, whereas the nasopharyngeal swab was tested negative. serological analysis confirmed the presence of antibodies against sars-cov-2. additionally, on april 5, 2020, it was reported that a 4-year-old malayan tiger at the bronx zoo in new york city was tested positive for the sars-cov-2 virus [24, 25] . in addition, six other big cats (another malayan tiger, two amur tigers, and three african lions) were reported to exhibit symptoms, including dry coughs, which are indicative of sars-cov-2 infection. only one tiger was tested for the virus, as collection of the samples in big cats requires anesthesia [25] . it was reported that the felines likely have contracted the virus from a caretaker, who was asymptomatic at the time of contact with the animals [25] . these data support the view that different species of felines are susceptible for sars-cov-2 infection. there are also emerging data that dogs may be also infected by the covid-19 virus [26, 27] . in february 2020 in hong kong, a companion dog was discovered to be positive for sars-cov-2 by pcr testing [24] . this animal is thought to have contracted the sars-cov-2 from its owner who was diagnosed with covid-19 [24] . serological testing on blood samples derived from the dog was performed by a who reference laboratory and yielded a positive result [24] . as of march 25, hong kong's agriculture, fisheries, and conservation department had tested 17 dogs and 8 cats, which lived in households with confirmed covid-19 human cases and, so far, 2 dogs (including the one described above) had tested positive for sars-cov-2. neither of the dogs showed any sign of respiratory disease. one of them, a 17-year-old pomeranian dog died shortly after the diagnosis was made [24] . it should be noted, that in human covid-19 disease, the cause of death often is cardiac arrest. the emerging view is that cardiac damage is present in every fifth covid-19 patients, leading to heart failure and death even among those who do not exhibit respiratory distress syndrome. in addition to hypoxic damage, the heart muscle likely can be infected by the sars-cov-2 virus. sars-cov-2 utilizes angiotensin converting enzyme-2 (ace-2) as a cellular entry receptor and cardiac myocytes as well as coronary arterial endothelial cells express ace-2 abundantly. of note, heart failure is a leading cause of death among older pomeranian dogs [28] . genetically impaired cardiac resilience in this breed may represent increased risk for death associated with covid-19. we propose that veterinarians should be aware of sars-cov-2-induced cardiac pathologies in dogs and look more closely for cardiac symptoms of sars-cov-2 infection in patients, even in the absence of respiratory symptoms. to definitely identify sars-cov-2 as cause of heart failure in dogs from households affected by covid-19, elaborate pathological testing should be performed postmortem in the dogs, most likely without the owners having an interest to cover the costs for these tests. besides cats and dogs, ferrets are also common pet animals in several countries. notably, laboratory animals of the mustelidae family are frequently used models for respiratory diseases [29] , including sars coronavirus. in addition, recent experimental studies verified the possibility of airborne transmission of sars-cov-2 between ferrets (published in a non-peer review, pre-print form [30] ). another study reported strong evidence for the possibility of virus transmission from and between these animals. viral shedding in detectable amount was observed in nasal washes, saliva, urine, and feces of the animals and all of them presented acute bronchitis [31] . minks are close relative of ferrets and are kept for their fur on large mink farms. it has been recently reported that in the netherlands, thousands of minks have been gassed on mink farms to prevent infected mink from becoming a viral reservoir that could cause new outbreaks in humans [32] . it is suspected that rapid spread of the infection occurred via infectious droplets, on feed or bedding, or in dust containing fecal matter [32] . infection in minks looks like covid-19 in humans, from asymptomatic infection to severe pneumonia [32] . mortality was reported to be negligible at one farm and close to 10% at another [32] . alarmingly, feral cats roaming the dutch mink farms and stealing the mink's food were also found to be infected as well [32] . minipigs have also become popular pets in recent years [33] . although recent studies revealed relatively low expression levels of angiotensin converting enzyme2 (ace2) protein (the docking protein for the viral spike protein) in the swine respiratory tract [34] , the susceptibility of pigs to sars-cov-2 transmitted from other pets or humans is still controversial [15, 35] . concluding, there is a small but existing possibility for an animal-human transmission although the main source of infection transmission remains human-human [15] . in theory, a zoonotic disease such as covid-19 could be directly transmitted from companion animals to humans through media such as air or through saliva and bites. contact with pet dogs and cats, including petting, snuggling, being kissed or licked, sleeping in the same location, and sharing food with companion animals is clearly increased during times when the owner is staying at home sick with covid-19, quarantined, or due to governmentally decreed lockdown measures. this common behavior, which, from a medical point of view, may be unfortunate for both the owner's and the pet's health, as it is creating ample opportunities for transmission of the virus in either direction. additionally, as the virus seems to be present in the saliva of infected animals, which lick their paws and fur, every surface in contact with the animal in an affected household should be considered potentially contaminated with sars-cov-2 [36] . it is likely, in the case of the very few positively tested companion animals, that the pets contracted the virus from their owners in an infected household (and not vice versa). actually, there is no evidence for a single case of pet to human transmission to date. cats very often stay within the house of their owners so their risk for contracting the infectious virus from outside is much lower than in dogs, which are taken out multiple times per day while coming into contact with other dogs and humans, sniffing on and touching the ground in parks, open space, or even public transport where previously another human or animal might have infected the area with sars-cov-2. nothing is known yet about the zoonotic potential of sars-cov-2 in seniors or elderly patients who own companion animals. in none of the positively tested cats and dogs was a reference made to the age of the owners or the age of the human commonly caring for the pet. prior to the news of the bronx zoo big cat cases, there have not been any reports of pets or other animals in the usa contracting covid-19, according to the websites of the us department of agriculture and the centers for disease control and prevention (cdc) [25, 36] . since the discovery of the cats infected with sars-cov-2 in new york, the website of the american veterinary association carefully documents each new case [22] . the guidelines of the cdc currently indicate that there is no evidence available that companion animals, including pets, can spread covid-19 [36] . yet, it has to be emphasized that animals have not been routinely tested in the usa and according to the website of the cdc and the american veterinary medical association (avma) [37] as of april 23, routine testing of animals is not recommended by the avma, cdc, us department of agriculture (usda), american association of veterinary laboratory diagnosticians (aavld), national association of state public health veterinarians (nasphv), or the national assembly of state animal health officials [37] . the cdc currently advises that public and animal health officials may decide to test animals that are showing signs of covid-19 and that are known to have been exposed to the virus [38] . veterinarians considering a possible covid-19 infection in the presented patients and performing a test may thus contribute very usefully to the understanding of the zoonotic potential of sars-cov-2. it is now clear that many people infected with sars-cov-2 remain asymptomatic while spreading the disease. if the same holds true also for animals, it cannot be ruled out that asymptomatic household pets and domesticated animals also can shed the virus and infect people without exhibiting actual symptoms of the disease. however, according to shi and coworkers, their viral loads are much lower compared with humans [15] . the reported hong kong case of a sars-cov-2 infected dog also did not have any clinical signs of the disease. furthermore, the sars coronavirus (sars-cov; which is also an animal virus identified in 2003 that causes a zoonotic disease in humans) infects mice, macaques, and marmosets, but does not cause a respiratory disease in them similar to the one it causes in people. middle east respiratory syndrome (mers) similarly is a respiratory illness caused by the coronavirus mers-cov first reported in saudi arabia in 2012. mers-cov can infect primates and hoofed animals, including camels and alpacas. importantly, infected alpacas appear to be asymptomatic, yet they spread the virus. of note is that several coronavirus-mediated diseases exist both in companion animals (including canine respiratory coronavirus, enteric coronaviruses, feline infectious peritonitis) and in livestock (infectious bronchitis virus, alpha coronaviruses causing mild gastrointestinal or respiratory disease, swine enteric coronaviruses, bovine coronaviruses), although most are species-specific and do not affect humans. in the current sars-cov-2 pandemic, the situation is rapidly evolving and in the light of the recent evidence, we should be aware of the possibility that humans can be potentially infected with covid-19 by animals, including by pet cats, dogs, or other domesticated species. the cdc recently issued an interim guidance for public health professionals managing the home care and isolation of people with covid-19 who have pets or other animals (including service or working animals) in the same home [36] . according to these cdc guidelines, state public health veterinarians should be contacted by public health professionals, animal health professionals, or veterinarians that have discovered a household animal with a new, concerning illness and that reside with a person with covid-19 [36] . due to concerns of potential human-to-animal transmission of sars-cov-2, the cdc recommends that people with covid-19 and in-home isolation should limit their interaction with household animals, but rather increase hygiene measures and hand washing after close contact with the pet [36] . it is recommended that while a person infected with covid-19 is symptomatic, they should maintain separation from household animals as they would with other household members, practicing social distancing [36] . dogs are frequently used in veterinary and cardiovascular research as large animal models of heart failure and other chronic diseases [39] . many researchers who are working with dogs on a daily basis in universities and the pharmaceutical industry belong to vulnerable age groups. in the laboratory setting, there is an increased probability for contact with bodily fluids of potential virus carrier animals, especially during procedures that involve invasive surgery [40] . research teams should get adequate safety training to prevent transmission of sars-cov-2 and closely follow laboratory guidelines for handling biological specimens, waste, and hazardous materials. pet health companies are working on developing laboratory tests for companion animals that may be affected by sars-cov-2. as the pandemic is progressing and testing of companion animals by veterinarians will be more often performed, our understanding of sars-cov-2 and its animal-toanimal and animal-to-human transmission will rapidly evolve. as of april 17, two commercial laboratories in the usa reported they had tested using rt-pcr thousands of specimens from dogs and cats for sars-cov-2 and had obtained no positive results [22] . these specimens were submitted from the usa, south korea, canada, and europe for pcr analysis of common pathogens causing respiratory illness in dogs and cats [22] . there is no or only limited information available as to whether these animals had close contact with human covid-19 patients before the testing. the world organization for animal health (oie) considers sars-cov-2 an emerging disease, and therefore the us department of agriculture regularly reports confirmed us animal infections to the oie. for up-to-date information, we advise the reader to consult the online reference sources provided at the end of this paper, including the websites of the avma [22] , cdc [36] , usda [41] , and oie [42] . further and broader veterinary studies are needed to determine which other companion animal species and pets can be infected by sars-cov-2 and elucidate, whether they demonstrate the clinical signs (e.g., upper respiratory infection, lung injury), and whether they develop an immune response. it is crucial to determine where the virus is shed in each species, whether it's in urine, tears, saliva, blood, or feces. it will be also essential to identify the species potentially serving as a reservoir for the virus. future research opportunities include widescale serosurveys of pet animals in contact with confirmed covid-19 patients. it may reveal the extension of this transmission route between various pet animals and humans. taken together, animal-to-human transmission is likely only a minor route of transmission for sars-cov-2. available research suggests that companion animals may theoretically play a role by either establishing a reservoir for sars-cov-2, or even potentially being able to spread covid-19 to other people in the household or people being in close contact with the animals. people should thus be advised to always follow standard handwashing practices before and after interacting with their pets. in view of the large number of fatalities in the older generation, seniors should be advised to adopt an even improved hygiene practice with their pets similar to the "social distancing" recommendations between humans. as per cdc recommendations, elderly people in particular are instructed not to let their pets interact with other people or other pets from outside the household. domestic cats should be kept indoors to prevent them from interacting with other animals or people. dogs should be walked on a leash, maintaining at least 2 m from other people and animals. dog parks or public places as well as public transport, where a large number of people and dogs usually gather, should be avoided. the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak sars-cov-2 and covid-19 in older adults: what we may expect regarding pathogenesis, immune responses, and outcomes aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 2019-ncov transmission through the ocular surface must not be ignored persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application understanding pathways to death in patients with covid-19 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health professionals managing people with covid-19 in home care and isolation who have pets or other animals testing animals for sars-cov-2 confirmation of covid-19 in two pet cats in new york animal models of dilated cardiomyopathy for translational research intracoronary gene delivery of the cytoprotective factor vascular endothelial growth factor-b167 in canine patients with dilated cardiomyopathy: a short-term feasibility study united states department of agriculture world organization for animal health publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments this work was supported by grants from the american heart association, the oklahoma center for the advancement of science and technology, and the presbyterian health foundation. the authors acknowledge the support from the nia-funded geroscience training program in oklahoma (t32ag052363) and the cellular and molecular geroscience cobre (1p20gm125528). the funding sources had no role in the writing of the report and in the decision to submit the article for publication. key: cord-331039-qgom2e3n authors: kavitha, kuppuswamy; sivakumar, subramaniam; ramesh, balasubramanian title: 1,2,4 triazolo[1,5-a] pyrimidin-7-ones as novel sars-cov-2 main protease inhibitors: in silico screening and molecular dynamics simulation of potential covid-19 drug candidates date: 2020-09-22 journal: biophys chem doi: 10.1016/j.bpc.2020.106478 sha: doc_id: 331039 cord_uid: qgom2e3n discovery of a potent sars-cov-2 main protease (m(pro)) inhibitor is the need of the hour to combat covid-19. a total of 1000 protease-inhibitor-like compounds available in the zinc database were screened by molecular docking with sars-cov-2 m(pro) and the top 2 lead compounds based on binding affinity were found to be 1,2,4 triazolo[1,5-a] pyrimidin-7-one compounds. we report these two compounds (zinc000621278586 and zinc000621285995) as potent sars-cov-2 m(pro) inhibitors with high affinity (<−9 kcal/mol) and less toxicity than lopinavir and nelfinavir positive controls. both the lead compounds effectively interacted with the crucial active site amino acid residues his41, cys145 and glu166. the lead compounds satisfied all of the druglikeness rules and devoid of toxicity or mutagenicity. molecular dynamics simulations showed that both lead 1 and lead 2 formed stable complexes with sars-cov-2 m(pro) as evidenced by the highly stable root mean square deviation (<0.23 nm), root mean square fluctuations (0.12 nm) and radius of gyration (2.2 nm) values. molecular mechanics poisson-boltzmann surface area calculation revealed thermodynamically stable binding energies of −129.266 ± 2.428 kj/mol and − 116.478 ± 3.502 kj/mol for lead1 and lead2 with sars-cov-2 m(pro), respectively. affairs, the world economy could contract by 0.9 percent in 2020 as opposed to a previous forecast of 2.5 percent growth [1] . the sars-cov2 is comprised of a positive-strand rna genome of size 29.7 kb and encodes a viral replicase that is associated with the novel genome synthesis and generation of a nested set of sub-genomic messenger rnas, encoding both structural proteins present in all covs: spike (s), envelope (e), membrane (m) and nucleoprotein (n), and a group of proteins specific for sars-cov: 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b [2] . so far, there is neither a drug nor a vaccine for covid-19. the rapid development and identification of efficient interventions against sars-cov-2 remains a major challenge. elfiky showed that sofosbuvir, ribavirin, galidesivir, remdesivir, favipiravir, cefuroxime, tenofovir, and hydroxychloroquine could bind to the rdrp active site tightly and supposed to be good candidates for clinical trials [3] . recently, stilbenoid analogues have been reported to be potential disruptors of the sars-cov-2 spike protein and human ace2 receptor complex [4] . one study suggested hydroxychloroquine and azithromycin as a treatment for covid-19 [5] and immediately refuted by others [6] . remdesivir and chloroquine were shown to inhibit sars-cov-2 in vitro [7] . lopinavir exhibited an anti-cov effect in vitro and is tried for clinical treatment of covid-19 [8, 9] . nelfinavir was shown to inhibit replication of the sars coronavirus (sars-cov), which could reduce the replication of virions from vero cells [10] and was predicted to be a potential inhibitor of sars-cov-2 main protease [11] . attention has been given to the development of furin inhibitors as a potential therapeutic platform against sars-cov-2 infection. however, furinlike enzymes contribute to several pathways and systemic inhibition may lead to some adverse effects [12] . although repurposing of drugs is a good idea, when their effectiveness is not certain, novel drugs are to be designed and developed specifically for novel viruses like sars-cov-2 m pro . structure-based virtual screening and molecular dynamics approaches are particularly suitable to identify novel sars-cov-2 inhibitors [13] . the coronavirus main protease (m pro) is essential for the viral gene expression and replication by the proteolytic cleavage of replicase polyproteins, without which the virus replication is severely hampered and is an important target for anti-cov drug design [14] . m pro has emerged as the most potent antiviral target because of its main role in self-maturation and subsequent maturation of polyproteins [15] . x-ray structures of the unliganded sars-cov-2 m pro and its complexes with various ligands have been reported. since there are no human counterparts with similar cleavage specificity, inhibitors of sars-cov-2 m pro are unlikely to be toxic [16] . sars-cov-2 m pro is a cysteine protease containing cys-145 and his-41 catalytic dyad in its active center. the proteolytic process is believed to be dependant on active site cysteine (cys-145) side chain thiolate nucleophile attack on amide bond of the substrate [17] . the -sh group of cys145 is ion-paired with his41 forming cys145-his41 catalytic dyad, which differs from most serine proteases that have a catalytic ser-his-asp triad in their active sites. in m pro , a stable water molecule occupies the asp position of the typical serine protease triad [18] . both covalent and non-covalent inhibitors of m pro are of immense value as a potent drug against sars-cov-2. covalent inhibitors establish a covalent bond (c-s) with the reactive thiol group of cys145 and form favorable interactions with residues lining the substratebinding site [19] . non-covalent inhibitors mainly act by binding to the active site stronger than the natural substrate by non-covalent bonds like hydrogen bonds, van der walls interactions, and electrostatic interactions. covalent inhibitors are highly selective inhibitors. however, irreversible drug toxicity can be a real challenge related to this class of therapeutics. on the other hand, non-covalent inhibitors could never cause irreversible toxicity but might be less effective. it is evident that both classes have their merits [20] . the objectives of this study were i) to identify evolutionarily important active site amino acids by structure-based sequence alignment of sars-cov-2 and sars-cov m pro enzymes ii) to identify potential non-covalent m pro inhibitors by screening protease-inhibitor-like compounds available in the zinc database by molecular docking studies iii) prediction of absorption, distribution metabolism, excretion and toxicity properties of the top-scoring inhibitors using in silico methods iv) to validate the stable binding of the lead compounds with sars-cov-2 m pro by molecular dynamics (md) simulations and v) to calculate thermodynamic binding energies for each lead compound -sars-cov-2 m pro complex using molecular mechanics poisson-boltzmann surface area (mm/pbsa) calculations. the three-dimensional structures of sars-cov-2 m pro (pdb ids: 6lu7, 6y84, 6yb7, 5re4 and 6w63) were obtained from rcsb-pdb [21] . pdb structures of sars-cov m pro (5nh0, 1p9s and 2zu2) with 95% structural similarities with sars-cov-2 m pro were selected using the jfatcat-rigid algorithm [22] and retrieved. the sequences of all these structures were used for further structure-based sequence alignment of sars-cov-2 and sars-cov m pro enzymes. the crystal structure of sars-cov-2 m pro in complex with an inhibitor n3 determined by x-ray diffraction with 2.16 å resolution pdb 6lu7 [23] was used as the drug target for molecular docking and molecular dynamics (md) studies. ton et al. [24] screened 1.3 billion compounds from the zinc15 [25] library and identified 1,000 probable ligands for sars-cov-2m pro protein. the compounds were made publicly available for further research by the scientific community. all these 1000 ligands for the sars-cov-2 mpro protein were downloaded in sdf format and used as the small molecular library for screening. structure-based sequence alignment was carried out to discern the amino acids that are conserved evolutionarily, particularly in the active site. the sequences of all m pro structures were exported into fasta format and aligned using clustalo [26] and their evolutionary relationship was inferred by the neighbour-joining method [27] using mega x [28] . the bootstrap consensus tree resulting from 500 replicates represented the evolutionary history [29] . the poisson correction method was used to calculate evolutionary distances [30] . this analysis involved 8 m pro sequences. ambiguous positions were removed by the pairwise deletion option and finally 307 positions were included in the dataset. structure-based alignment was performed and important features of the sequences and structures were deciphered using espript [31] . the selected drug target pdb 6lu7 [23] was prepared at ph (7.0), water molecules and the inhibitor were removed from the structure and incomplete residues were fixed using ucsf chimera version 1.14 [32] and swiss-pdb viewer v4.1.0 [33] . druggable binding pockets were predicted by the castp 3.0 server [34] . all the 1000 ligands were protonated, cleaned 3 dimensionally and exported to pdb format using chemaxon marvinview 20.9.0 [35] . the target enzyme 6lu7 and the ligand molecule library were converted into pdbqt format using open babel [36] . autodock vina 1.1.2 [37] in mgltools pyrx virtual screening software [38] was used to screen the ligand library against the target enzyme. ucsf chimera 1.14 [39] was used for analysis and rendering of the docking results. since lopinavir [40] and nelfinavir [41] were shown to be effective in covid-19 patients and also protease inhibitors, they were included as positive controls. absorption, distribution, metabolism, excretion and toxicity predictions were carried out for all the top 10 lead compounds identified from docking results along with positive controls using the swissadme server [42] . ames toxicity, carcinogenicity and acute oral toxicity of lead compounds were predicted by the admetsar 2.0 [43] . the gromacs 5.1.2 software [44] was used to carry out md simulations using the gromos 96 54a7 force field. the topology file was generated from the pdb file through the pdb2gmx program of gromacs. the prodrg2.5 server [45] was used to build the topology parameters of lead1, lead2, lead3, lopinavir and nelfinavir. md simulation of 20 ns with a time step of 2 fs at a 300 k temperature was carried out. a total of 6 systems; one sars-cov-2 m pro apoenzyme (6lu7) and 5 sars-cov-2 m pro complexes viz., 6lu7-lead1, 6lu7-lead2, 6lu7-lead3, 6lu7-lopinavir and 6lu7-nelfinavir were prepared. the apo-protein and protein-ligand complexes were submerged in a solvent box, surrounded by 4 na ions to maintain electro-neutrality. energy minimization was done using the steepest descent algorithm in order to alleviate the bad van der waals interactions strain. after the convergence of the system, equilibration was carried out with nvt and npt ensembles to attain the system temperature and pressure of 300 k and 1 bar, respectively. the electrostatic interaction in the systems was measured with the particle mesh ewald. the gromacs molecular dynamics simulation engine -mdrun‖ program was used to carry out equilibration md simulations. the temperature and pressure of the system were kept constant using the velocity-rescale algorithm and the parrinello-rahman algorithm. the linear constraint solver algorithm was utilized to restrain all the bonding lengths [46] . the simulation trajectories were examined with the visualization molecular dynamics (vmd) software package [47] . root-mean-square deviation (rmsd), root-mean-square fluctuation (rmsf), radius of gyration (rg) and the number of hydrogen bonds were calculated by the gmx rms, gmx rmsf, gmx gyrate and gmx hbond tools of gromacs, respectively. the protein-ligand interaction profiler (plip) web server [48] was used to analyze the docked complexes for types and distances of non-covalent bonds. binding energy of each protein-ligand complex was calculated by the mm-pbsa method [49] using the g_mmpbsa tool [50] . the binding energy of each complex was computed from van der waal energy, electrostatic energy, polar solvation energy and non-polar solvation energy based on the solvent accessible surface area (sasa) model using the script mmpbsastat.py. the final contribution energy of each residue from individual energetic terms obtained from the g_mmpbsa was calculated using mmpbsadecomp.py script. sequence alinement showed some residues that are unique for sar-cov-2 in comparison to sars-cov, which further showed a profound effect on docking studies resulting in new lead compounds, which were not reported for sars-cov m pro . the evolutionary replacement of amino acids from sars-cov to sar-cov-2 were found to be leu3phe, gln8phe, phe12lys, lys15gly, val17met, arg19gln, cys21thr, tyr22cys, asn24thr, val26thr, gly33asp, ile/thr35val, ala44cys, ser/pro45thr, thr47glu, thr48asp, ser/val49met, ile52pro, asp53asn, asp55glu, ile141leu, ala144ser, gln164his, ile165met, gly168pro, ser169thr, gln188arg, and arg189gln. the x-ray crystallographic structure of sars-cov-2 m pro ( fig.2a) the top 1000 viral protease inhibitor-like molecules identified by ton et al. [24] were obtained in sdf format, cleaned three-dimensionally, hydrogenized and used as smallmolecule library for screening. sars-cov-2 m pro structure pdb 6lu7 was prepared by removing water and ligand molecules. two lead compounds lead1-zinc000621278586 and lead2-zinc000621285995 showed maximum binding affinities of -9.3 kcal/mol and -9.1 kcal/mol towards the sars-cov-2 m pro active site, respectively, which are far better than the positive controls lopinavir (-6.8 kcal/mol) and nelfinavir (-7.9 kcal/mol) ( table 1) . binding of three drugs viz. lopinavir, oseltamivir, and ritonavir simultaneously with the protein resulted in a binding affinity of −8.32 kcal/mol [15] . three molecules of natural origin from moroccan medicinal plants crocin, digitoxigenin and β-eudesmol were docked with sars-cov-2 m pro and showed an interaction energy equal to -8.2 kcal/mol, -7.2 kcal/mol and -7.1 kcal/mol, respectively. both lead1 and lead2 were found to surpass these previously reported binding energies. molecular docking of sars-cov-2 m pro with lead1 and lead2 are depicted in fig.3 . lead1 binds to the active site formed by domain-i and domain-ii chymotrypsin-like β barrels, where the active site dyad his41and cys145 is located. khan et al. proposed 5 inhibitors, all of which exhibited significant interactions with the same active site dyads [52] . the binding of lead1 was found to be stabilized by various hydrogen bonds and alkyl bonds. his41, met49 and met165 showed a stronger tendency to form alkyl bonds; on the other hand, phe140, leu141, asn142, gly143, ser144, cys145, his164, glu166 and gln189 formed hydrogen bonds, giving rise to a stronger binding affinity of -9.3 kcal/mol. lead2 bound to the same active site with the his41 and cys145 dyad. his41, met49 and met165 formed alkyl bonds with the lead2, while phe140, ser144 and cys145 were involved in conventional hydrogen bonds with a total binding affinity of -9.1 kcal/mol. it is interesting to note that some amino acids that were unique to sars-cov-2 m pro as identified by the sequence alignment studies were involved in critical bond formation. some of these amino acids were met49, lew141, ser144, his164, and met165 (table-1 ). islam et al. reported that analysis of the non-covalent interactions of a best five phytochemicals with the main protease revealed that the selected compounds interacted with either both (cys145 and his41) or at least one catalytic residue [53] . zhang et al. demonstrated that dimerization of sars-cov-2 m pro is crucial for catalytic activity because the n-finger of each of the two protomers interacts with glu166 of the other protomer. this interaction helps shape the s1 pocket of the substratebinding site [16] . it is interesting to note that almost all 10 lead compounds showed binding preference to the same active site amino acids his41, cys145 and glu166. lead10 showed a binding affinity of -8.7 kcal/mol. lead1, lead2 and lead4 formed a hydrogen bond with glu166. even though the maximum number of interactions of lead4 is the same as lead1, it did not bind to the active site dyad his41and cys145. this makes lead1 and lead2 as the favourable choices. both lead1 and lead2 are pyrimidin-7-one compounds. pyrimidinones are implicated in a wide range of biological activities, including viral infections. the pyrimidone scaffold is the backbone of many of the approved anti-retrovirals e.g. zidovudine, didanosine and zalcitabine [54] . j o u r n a l p r e -p r o o f absorption, distribution, metabolism, excretion and toxicity predictions were carried out for all 10 lead compounds and positive controls. the physiochemical properties like molecular weight, number of hydrogen bond acceptors/donors, topological polar surface area, lipophilicity and solubility were calculated. the pharmacokinetics predictions (table 2) showed that lead1 and lead 2 were nonpermeators of the blood brain barrier and skin with high gastrointestinal absorption. lead1 was predicted to inhibit none of the cytochrome p450 viz., cyp1a2, cyp2c19, cyp2c9, cyp2d6 and cyp3a4. lead2 was inhibitory to cyp1a2 alone. on the other hand, both the positive controls lopinavir and nelfinavir were predicted to inhibit cyp2c19 and cyp3a4. druglikeness screening showed that all lead compounds satisfied all the druglikeness rules viz., lipinski [55] , ghose [56] , veber [57] , egan [58] and muegge [59] , except lead10, which showed violations in the veber and egan rules owing to its high tpsa values. on the other hand, the positive controls showed at least one violation in all the rules except the egan rule. medicinal chemistry analysis showed that all the leads were passed by filters for removal of pan assay interference compounds (pains) [60] and a list of 105 fragments identified by brenk et al. [61] , except lead4, which showed hydantoin alert. the synthetic accessibility scores of lopinavir and nelfinavir were 5.67 and 5.58, respectively, while those of lead1 and lead2 were 3.58 and 3.46, which shows that these leads could be easily synthesized compared to positive controls. drug-induced liver injury probability values of lead1 and lead2 were found to be lower than that of lopinavir; and the acute oral toxicity ld50 values of lead1 and lead2 were predicted to be 2.034 mol/kg and 2.371 mol/kg, respectively. both the leads exhibited negative ames mutagenesis probability scores and were found to be non-carcinogenic. since, both lead1 and lead2 show exceptional druglike properties with good medicinal chemistry properties, they can further be assessed for their in vitro sars-cov-2 m pro inhibitory activities. however, lead1 (zinc000621278586) could be better than lead2 (zinc000621285995) because of its non-inhibitory nature of cytochrome p450, while lead2 inhibits cyp1a2. apart from this minor negative characteristic, lead2 was also found to be a good sars-cov-2 m pro inhibitor. non-carcinogenic #cytochrome p450 inhibitors include inhibitors of cyp1a2, cyp2c19, cyp2c9, cyp2d6 and cyp3a4; all the molecules showed a bioavailability score of 0.55; b pan assay interference compounds alert; c 105 fragments identified by brenk database; d synthetic accessibility score on a scale of 1-10 (1 easy to 10 difficult to synthesize). based on the docking scores and absorption, distribution, metabolism, excretion and toxicity predictions lead1-zinc00062127858, lead2-zinc000621285995 and lead3-zinc000566550443 were selected and their complexes with sars-cov-2 m pro were subjected to md simulations along with complexes of sars-cov-2 m pro -lopinavir and nelfinavir positive controls. the best docking conformation of each of the complexes was chosen and used as the starting point for a 20 ns simulation. the trajectories were analyzed for stability and the complete details of conformations of proteins were observed to reconfirm the results of docking. furthermore, the time-dependent rmsd values of atoms in the unliganded sars-cov-2-m pro , sars-cov-2-m pro -lead1 complex, sars-cov-2-m pro -lead2 complex, sars-cov-2-m pro -lead3 complex, sars-cov-2 m pro -lopinavir and sars-cov-2 m pro -nelfinavir complexes were plotted (fig-4a) . the complexes of all lead compounds and lopinavir were well correlated with unliganded protein with only a few atomic fluctuations in the magnitude. overall mean rmsd values for sars-cov-2-m pro apo protein and sars-cov-2-m pro complexes with lead1, lead2, lead3, lopinavir and j o u r n a l p r e -p r o o f nelfinavir were found to be 0.28±0.034 nm 0.20±0.025 nm, 0.23±0.039 nm, 0.20±0.024 nm 0.21±0.025 nm and 0.26±0.060 nm, respectively. the rmsd of lead1, lead2 and lead3 complexes were less than 0.23 nm, while overall rmsd of all the complexes showed consistency within 0.3 nm over the entire trajectory, which is well within the range of previous reports [54] . similarly, the backbone radiation of gyration (rg) values (fig-4b) for sars-cov-2-m pro apo protein was found to be 2.195±0.016 nm and that of sars-cov-2-m pro complexes with lead1, lead2, lead3, lopinavir and nelfinavir were found to be 2.218±0.014 nm, 2.203±0.016 nm, 2.222±0.017 nm 2.212±0.018 nm and 2.186±0.025 nm, respectively. rg value of 2.2 nm for all lead compounds showed that the binding of these ligands does not cause considerable stress on the backbone of sars-cov-2-m pro . the data revealed that all the systems were compact throughout the simulation, which indicates that the systems are well converged. the binding energies for all protein ligand complexes were calculated for the last 10 ns of md trajectories. all 5 complexes showed negative binding energies (table 3) indicating that j o u r n a l p r e -p r o o f all the complexes were energetically stable. lead1 showed the lowest binding energy (-129.266 ± 2.428 kj/mol) of the lead molecules. the binding energy of lead 1 is lower than lopinavir (-29.410 ± 9.493) and higher than nelfinavir (-140.785 ± 3.989) and was considered as the most stable lead molecule. lead2 and lead3 showed binding energies of -116.478 ± 3.502 and -96.864 ± 3.820, respectively, which is better than that of lopinavir. the lopinavir complex showed a less favorable energy value of -29.410 ± 9.493 kj/mol. the rigorous bootstrapping used in this study resulted in a reduced standard deviation. a previous study calculated δg binding energy for remdesivir, saquinavir, darunavir, nat-1 and syn-16 with target protein chymotrypsin-like protease (3cl pro ) as -45.5240, -36.3026, -48.1041, -41.2565 and -31.5581 kj/mol, respectively, and proposed darunavir as the best protease inhibitor [52] . another study reported -4.62 kcal/mol or -19.33 kj/mol for the mpro-zinc000015988935 complex [62] . the lead compounds of this study, particularly lead1 and lead2, exhibited far better binding energies and hence can be expected to outperform these previously reported drugs and lead compounds. energy decomposition plot was calculated as the energy contribution of each residue (fig.7) . all the lead compounds showed stabilization of the complex around residue number 40 and between residues 140 to 170, indicating that they bind to the active site of sars-cov-2-m pro . lopinavir showed binding in the same region with lower affinity. nelfinavir has been shown to bind in a different region between residues 250 and 300, which makes its usability as an inhibitor questionable. the thermodynamic calculations showed that the binding of both lead1 and lead2 to the active site of sars-cov-2-m pro is energetically favored followed by lead3. hence, they can act as good inhibitors of sars-cov-2-m pro . j o u r n a l p r e -p r o o f structure-based sequence alignment of sars-cov-2 and sars-cov main proteases showed extensive similarities in their secondary and tertiary structures. hence, it can be construed that in silico molecular approaches used for screening sars-cov m pro inhibitors can also be used for finding potent sars-cov-2 m pro inhibitors. this study screened 1000 proteaseinhibitor-like molecules against sars-cov-2-m pro and proposes lead compounds viz., lead1 -2-amino-5-{[(5r)-5-methyl-2,3,4,5-tetrahydro-1h-1-benzazepin-1-yl]methyl}-1h,7h[1, 2, 4] triazolo[1,5-a]pyrimidin-7-one (zinc000621278586) and lead2 -2-amino-5-({1',2'-dihydrospiro[cyclobutane-1,3'-indol]-1'-yl}methyl)-1h,7h-[1,2,4]triazolo[ 1,5-a]pyrimidin-7-one (zinc000621285995) as potent sars-cov-2 m pro inhibitors with better binding properties and less toxicity than existing protease inhibitors like lopinavir and nelfinavir. both these molecules are [1, 2, 4] triazolo[1,5-a]pyrimidin-7-one compounds and their antiviral properties have not been reported previously. md simulation studies showed j o u r n a l p r e -p r o o f that both lead compounds had high binding affinity towards sars-cov-2-m pro . binding free energy calculations by mm/pbsa showed energetically stable negative values of -129.266 ± 2.428 kj/mol and -116.478 ± 3.502 kj/mol for lead1 and lead2, respectively. taken all together, according to docking studies, physicochemical characterizations, adme/tox predictions and molecular dynamics studies, it is safer to conclude that these pyrimidin-7-one lead compounds could be considered as possible sars-cov-2 m pro inhibitors. however, the inhibitory activity of these lead compounds should be further tested in vitro and animal studies. future perspective of this study could be designing a covalent inhibitor based on these pyrimidin-7-one compounds that could form favourable covalent bond with the reactive thiol group of active site cys145.  two lead compounds viz. lead1 (zinc000621278586) and lead2 (zinc000621285995) were found to be potent sars-cov-2 m pro inhibitors based on molecular docking of 1000 protease-inhibitor like molecules derived from zinc database.  both these molecules are [1, 2, 4] j o u r n a l p r e -p r o o f global economy could shrink by almost 1% in 2020 due to covid-19 pandemic: united nations in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel sars-cov-2 rna dependent rna polymerase (rdrp) targeting: an in silico perspective stilbene-based natural compounds as promising drug candidates against covid-19 hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro a. the m. trial group, treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial virtual screening of approved clinic drugs with main protease (3cl pro ) reveals potential inhibitory effects on sars-cov-2 hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus nelfinavir is active against sars-cov-2 in vero e6 cells a review on the cleavage priming of the spike protein on coronavirus by angiotensinconverting enzyme-2 and furin novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors the sars-cov-2 main protease as drug target quaternary structure of the sars coronavirus main protease pharmacoinformatics and molecular dynamics simulation studies reveal potential covalent and fda-approved inhibitors of sars-cov-2 main protease 3clpro covalent versus non-covalent enzyme inhibition: which route should we take? a justification of the good and bad from molecular modelling perspective the protein data bank precalculated protein structure alignments at the rcsb pdb website the crystal structure of covid-19 main protease in complex with an inhibitor n3, pdb release rapid identification of potential inhibitors of sars-cov-2 main protease by deep docking of 1.3 billion compounds zinc 15 -ligand discovery for everyone the embl-ebi search and sequence analysis tools apis in 2019 the neighbor-joining method-a new method for reconstructing phylogenetic trees molecular evolutionary genetics analysis across computing platforms confidence limits on phylogenies: an approach using the bootstrap evolutionary divergence and convergence in proteins deciphering key features in protein structures with the new endscript server ucsf chimera--a visualization system for exploratory research and analysis swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling castp 3.0: computed atlas of surface topography of proteins open babel: an open chemical toolbox autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading small-molecule library screening by docking with pyrx modeller, and imp: an integrated modeling system clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment structural basis of sars-cov-2 3cl pro and anti-covid-19 drug discovery from medicinal plants swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules admetsar: a comprehensive source and free tool for assessment of chemical admet properties phenazine-1-carboxylic acid mediated anti-oomycete activity of the endophytic eil-2 against phytophthora meadii prodrg: a tool for high-throughput crystallography of protein-ligand complexes a parallel linear constraint solver for molecular simulation vmd: visual molecular dynamics plip: fully automated protein-ligand interaction profiler electrostatics of nanosystems: application to microtubules and the ribosome g_mmpbsa-a gromacs tool for high-throughput mm-pbsa calculations structural basis of sars-cov-2 3cl pro and anti-covid-19 drug discovery from medicinal plants ul-haq, identification of chymotrypsin-like protease inhibitors of sars-cov-2 via integrated computational approach a molecular modeling approach to identify effective antiviral phytochemicals against the main protease of sars-cov-2 in-silico homology assisted identification of inhibitor of rna binding against 2019-ncov n-protein (n terminal domain) experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings a knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. a qualitative and quantitative characterization of known drug databases molecular properties that influence the oral bioavailability of drug candidates prediction of drug absorption using multivariate j o u r n a l p r e -p r o o f statistics simple selection criteria for drug-like chemical matter new substructure filters for removal of pan assay interference compounds (pains) from screening libraries and for their exclusion in bioassays lessons learnt from assembling screening libraries for drug discovery for neglected diseases drug repurposing for coronavirus (covid-19): in silico screening of known drugs against coronavirus 3cl hydrolase and protease enzymes the authors express immense gratitude to the google cloud platform for supporting this work with covid hcls research credit 46225154. we are grateful to dr.k.r. venkatesan, principal and the management of sri sankara arts and science college, for providing the facilities and knowledge resources to conduct this research. we are thankful to mr.b.rajganesh, enstoa india private limited, bangalore, india, for providing computational resources for docking studies. the authors declare that they have no conflicts of interest. key: cord-331094-22366b81 authors: ianevski, aleksandr; yao, rouan; fenstad, mona høysæter; biza, svetlana; zusinaite, eva; reisberg, tuuli; lysvand, hilde; løseth, kirsti; landsem, veslemøy malm; malmring, janne fossum; oksenych, valentyn; erlandsen, sten even; aas, per arne; hagen, lars; pettersen, caroline h.; tenson, tanel; afset, jan egil; nordbø, svein arne; bjørås, magnar; kainov, denis e. title: potential antiviral options against sars-cov-2 infection date: 2020-06-13 journal: viruses doi: 10.3390/v12060642 sha: doc_id: 331094 cord_uid: 22366b81 as of june 2020, the number of people infected with severe acute respiratory coronavirus 2 (sars-cov-2) continues to skyrocket, with more than 6.7 million cases worldwide. both the world health organization (who) and united nations (un) has highlighted the need for better control of sars-cov-2 infections. however, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against sars-cov-2 can be time-consuming and costly. convalescent sera and safe-in-man broad-spectrum antivirals (bsaas) are readily available treatment options. here, we developed a neutralization assay using sars-cov-2 strain and vero-e6 cells. we identified the most potent sera from recovered patients for the treatment of sars-cov-2-infected patients. we also screened 136 safe-in-man broad-spectrum antivirals against the sars-cov-2 infection in vero-e6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. we found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. finally, we developed a website to disseminate the knowledge on available and emerging treatments of covid-19. every year, emerging and re-emerging viruses such as sars-cov-2, sars-cov, middle east respiratory syndrome coronavirus (mers-cov), zika virus (zikv), ebola virus (ebov), influenza a virus (fluav) and rift valley fever virus (rvfv) surface from natural reservoirs to infect people [1, 2] . as of june 2020, the number of people infected with sars-cov-2 continues to rise, with more than 6.7 million cases worldwide. we selected subjects among healthy individuals, in-and outpatients, as well as patients recovered from sars-cov-2 or endemic coronavirus infections. hospitalization was determined by whether a patient was able to manage symptoms effectively at home, according to local guidelines. icu admittance was evaluated consistently with the who interim guidance on "clinical management of severe acute respiratory infection when covid-19 is suspected" (who/2019-ncov/clinical/2020.4). the patients gave their informed consent through the koronastudien.no website. for icu patients, consent for sample collection was received after treatment or from relatives. for children, consent for sample collection was received from their parents. donors were recruited through information at the blood collection centers websites and through national media. nasopharyngeal swabs (nps) and blood samples were collected. all patients were treated in accordance with good clinical practice, following study protocols. the study was approved by the national ethical committee (clinical trial: nct04320732; rek: 124170). human telomerase reverse transcriptase-immortalized retinal pigment epithelial (rpe), lung adenocarcinoma a427, non-small-cell lung cancer calu-3 and epithelial colorectal adenocarcinoma caco-2 cells were grown in dmem-f12 supplemented with 100 µg/ml streptomycin and 100 u/ml penicillin (pen-strep), 2 mm l-glutamine, 10% fbs and 0.25% sodium bicarbonate (sigma-aldrich, st. louis, mo, usa). human neural progenitor cells derived from ips cells were generated and maintained as described previously [20] . human large-cell lung carcinoma nci-h460, colon cancer sw620, colorectal carcinoma sw480 and ht29 cells were grown in rpmi medium supplied with 10% fbs and pen-strep. human adenocarcinoma alveolar basal epithelial a549, human embryonic kidney hek-293 cells and kidney epithelial cells extracted from an african green monkey (vero-e6) were grown in dmem supplemented with 10% fbs and pen-strep. the cell lines were maintained at 37 • c with 5% co 2 . the sars-cov-2 strains were isolated and propagated in a biological safety level 3 (bsl-3) facility. two hundred microliters of nasopharyngeal swabs (nps) in universal transport medium were diluted 5 times in culture medium (dmem) supplemented with 0.2% bovine serum albumin (bsa), 0.6 µg/ml penicillin, 60 µg/ml streptomycin and 2 mm l-glutamine and inoculated into vero-e6 cells. after 4 days of incubation, the media were collected, and the viruses were passaged once again in vero-e6 cells. after 3 days, a clear cytopathic effect (cpe) was detected, and the virus culture was harvested. virus concentration was determined by rt-qpcr and plaque assays. viral rna was extracted using the ntnu_mag_v2 protocol, a modified version of the bomb-protocol [21] . the eluate (2.5 or 5 µl) was analyzed by rt-qpcr using a cfx96 real-time thermal cycler (bio-rad, hercules, ca, usa) as described elsewhere [22] , with some modifications. one 20-µl reaction contained 10 µl of qscript xlt one-step rt-qpcr toughmix (2×) (quanta biosciences, beverly, ma, usa), 1 µl each of the primer and probe with final respective concentrations of 0.6 and 0.25 µm and 2 µl of molecular-grade water. thermal cycling was performed at 50 • c for 10 min for reverse transcription, followed by 95 • c for 1 min and then 40 cycles of 95 • c for 3 s and 60 • c for 30 s. for testing the production of infectious virions, we titered the viruses as described in our previous studies [23] [24] [25] . in summary, media from the viral culture were serially diluted from 10 −2 to 10 −7 in serum-free dmem containing 0.2% bovine serum albumin. the dilutions were applied to a monolayer of vero-e6 cells in 6 or 24-well plates. after one hour, cells were overlaid with virus growth medium containing 1% carboxymethyl cellulose and incubated for 96 h. the cells were fixed and stained with crystal violet dye, and the plaques were calculated in each well and expressed as plaque-forming units per ml (pfu/ml). viral rna was extracted using the ntnu_mag_v2 protocol, a modified version of the bomb-protocol. libraries were prepared using a nugen trio rna-seq kit. sequencing was performed on the nextseq500 instrument (ns500528; setup: pe 2 × 75 bp + single index (8 bp)) using a nextseq mid150 sequencing kit and nextseq mid flow cell (ncs version: 2.2.0.4). reads were aligned using the bowtie 2 software package version 2.3.4.1 to the reference viral genome wuhan-hu-1/2019. sequence alignments were converted to binary alignments and sorted using samtools version 1.5. the consensus fastq sequences were obtained with bcftools and vcfutils.pl (from samtools) and converted to fasta using seqtk (https://github.com/lh3/seqtk). viral genomes in fasta formats were submitted to www.gisaid.org. the accession numbers of these genomes are: epi_isl_450352 (hcov-19/norway/trondheim-e10/2020), epi_isl_450351 (hcov-19/norway/trondheim-e9/2020), epi_isl_450350 (hcov-19/norway/trondheim-s15/2020), epi_isl_450349 (hcov-19/norway/trondheim-s12/2020), epi_isl_450348 (hcov-19/norway/ trondheim-s10/2020), epi_isl_450347 (hcov-19/norway/trondheim-s5/2020) and epi_isl_450346 (hcov-19/norway/trondheim-s4/2020). transmission of the strains was visualized using https: //nextstrain.org/ncov/global?f_author=aleksandr%20ianevski%20et%20al. the virus (multiplicity of infection, moi 0.1) was aliquoted in eppendorf tubes and incubated at −80, −20, 4, 20, 37 and 50 • c for 48 h or at 96 • c for 10 min. alternatively, the virus was aliquoted in wells of a 96-well plate without a lid. the virus was exposed to uvc light (λ = 254 nm, ≥125 µw/cm 2 ) for 10, 20, 40, 80, 160, 320 and 640 s using a germicidal lamp in a biosafety cabinet. the thermo and uvc stability of the viral rna was analyzed by rt-qpcr. subsequently, vero-e6 cells were infected with the virus for 72 h, and cell viability was measured using a celltiter-glo assay (promega, madison, wi, usa). luminescence was read using a plate reader. approximately 4 × 10 4 vero-e6 cells were seeded per well in 96-well plates. the cells were grown for 24 h in dmem supplemented with 10% fbs and pen-strep. serum samples were diluted 100-fold, and protein concentrations were quantified using nanodrop. serum samples were prepared in 3-fold dilutions at 7 different concentrations, starting from 1 mg/ml in the virus growth medium (vgm) containing 0.2% bsa and pen-strep in dmem. virus hcov-19/norway/trondheim-e9/2020 was added to the samples to achieve a moi of 0.1 and incubated for 1h at 37 • c. 0.1% dmso was added to the control wells. the vero-e6 cells were incubated for 72 h with vgm. after the incubation period, the medium was removed, and a celltiter-glo assay was performed to measure viability. we have previously published a library of safe-in-man bsaas [26] . supplementary materials table s1 lists these and other potential bsaas, their suppliers and catalogue numbers. the control wells. the cells were mock-or hcov-19/norway/trondheim-e9/2020-infected at a moi of 0.1. after 72 h of infection, the medium was removed from the cells, and a celltiter-glo assay was performed to measure viability. the half-maximal cytotoxic concentration (cc 50 ) for each compound was calculated based on viability/death curves obtained on mock-infected cells after nonlinear regression analysis with a variable slope using graphpad prism software version 7.0a (graphpad software, san diego, ca, usa). the half-maximal effective concentrations (ec 50 ) were calculated based on the analysis of the viability of infected cells by fitting drug dose-response curves using the four-parameter (4pl) logistic function f (x): where f (x) is a response value at dose x, a min and a max are the upper and lower asymptotes (minimal and maximal drug effects), m is the dose that produces the half-maximal effect (ec 50 or cc 50 ) and λ is the steepness (slope) of the curve. a relative effectiveness of the drug was defined as the selectivity index (si = cc 50 /ec 50 ). the threshold of the si used to differentiate between active and inactive compounds was set to 3. area under the dose-response curve auc was quantified as: using the numerical integration implemented in the mess r package (bell laboratories, murray hill, nj, usa), where x max and x min are the maximal and minimal measured doses. serum sensitivity score (sss) was quantified as a normalized version of the standard auc (with the baseline noise subtracted and normalization of the maximal response at the highest concentration often corresponding to off-target toxicity) as where activity threshold t equals 10%. vero-e6 cells were treated with different concentrations of a combination of two bsaas. after 72 h, cell viability was measured using a celltiter-glo assay. to test whether the drug combinations acted synergistically, the observed responses were compared with expected combination responses. the expected responses were calculated based on the zip reference model using synergyfinder web application, version 2 [27] . we measured the igg and igm in human serum using epitope diagnostics enzyme-linked immunosorbent assays (elisa) according to manufacturer specifications (epitope diagnostics, san diego, ca, usa). background-corrected optical density values were divided by the cutoff to generate signal-to-cutoff (s/co) ratios. samples with s/co values greater than 1.0 were considered positive. the pearson correlation coefficients were calculated by means of the stats r package, with the significance determined using a student's t-test. vero-e6 cells were treated with nelfinavir, amodiaquine or both drugs at indicated concentrations. cells were infected with the hcov-19/norway/trondheim-e9/2020 strain at moi 0.1 or mock. after 24 h, total rna was isolated using rneasy plus mini kit (qiagen, hilden, germany). libraries were prepared and sequenced on a nextseq500 (ns500528) instrument (set up: pe 2 × 75 bp + single index 8 bp) using a nextseq mid150 sequencing kit, nextseq mid flow cell, ncs version: 2.2.0.4. reads were aligned using the bowtie 2 software package version 2.3.4.1 to the ncbi reference sequence for sars-cov-2 (nc_045512.2) and to the human grch38 genome. number of mapped and unmapped reads that aligned to each gene were obtained with the featurecounts function from rsubread r-package version 2.10. the gff table for the reference sequence was downloaded from https://ftp.ncbi.nlm.nih.gov/genomes/all/gcf/009/858/895/gcf_ 009858895.2_asm985889v3/gcf_009858895.2_asm985889v3_genomic.gff.gz and flattened to gtf format and given as an additional argument to the rsubread function. the heatmaps were generated using the pheatmap package (https://cran.r-project.org/web/packages/pheatmap/index.html) based on log2-transformed profiling data. we reviewed the current landscape of the available diagnostic tools, as well as the emerging treatment and prophylactic options for the sars-cov-2 pandemic and have summarized the information in a database that can be freely accessed at https://sars-coronavirus-2.info. the information in the database was obtained from pubmed, clinicaltrials.gov, drugbank, drugcentral, the chinese clinical trials register (chictr) and eu clinical trials register databases [28] [29] [30] , as well as other public sources. the website was developed with php v7 technology using d3.js v5 (https://d3js.org/) for visualization. the covid-19 statistics are automatically exported from the covid-19 data repository by the center for systems science and engineering (csse) at johns hopkins university (https://github.com/cssegisanddata/covid-19). we isolated seven sars-cov-2 strains from 22 nps samples of covid-19 patients using vero-e6 cells. the rt-qpcr cycle threshold (ct) values were 18-20 before and 13-15 after propagation of the viruses in vero-e6 cells (figure 1a ,b). we sequenced seven strains and found that the sequences differed from the reference hcov-19/wuhan/wiv04/2019 strain by a few missense mutations ( figure 1c) . phylogenetic analysis showed a close relationship between the strains (figure 1d ). in cross-referencing our sequence data with the pathogen-tracking resource nextstrain.org, we determined that the sars-cov-2 strains isolated in trondheim originated from china, denmark, the usa and canada (figure 1e) . in addition, we tested several cell lines and found that vero-e6 was the most susceptible for virus-mediated death and virus amplification (figure s1a-c). to establish the rationale for safe work, we incubated the virus at different temperatures for 48 h or exposed to uvc radiation for different time periods. the resulting virus preparations were analyzed by rt-qpcr and used to infect vero-e6 cells. virus incubation at 37 • c for 48 h or uvc exposure for 10 sec destabilized the virus and rescued infected cells from virus-mediated death ( figure s2 ). we evaluated the neutralization capacity of seven serum samples obtained from patients recovered from covid-19. we also used sera from patients recovered from endemic coronavirus infections and from healthy blood donors as controls. five samples from patients recovered from covid-19 had serum sensitivity scores (sss) > 5 and rescued >50% cells from virus-mediated death at 1 mg/ml (figure 2a,b) . notably, that serum from a recovered patient with sss = 7.2 neutralized all seven sars-cov-2 strains ( figure s3 ). our neutralization test of 32 samples (table s2) showed a moderate positive correlation with igg (r = 0.59, p < 0.001) and igm (r = 0.43, p = 0.01) s/co values obtained using commercial elisa kits that recognize the sars-cov-2 n protein (figure 2c,d) . however, the correlation between the igg and igm elisa results was only r = 0.28, p = 0.11. furthermore, we found a moderate negative correlation between ssss and time intervals between the sars-cov-2 diagnosis and serum collection for 66 samples (-0.5, p < 0.025; figure 2d ). altogether, these results suggest that patients diagnosed with covid-19 produce different immune responses to the sars-cov-2 infection and that the neutralization capacity of convalescent sera declines with time. we evaluated the neutralization capacity of seven serum samples obtained from patients recovered from covid-19. we also used sera from patients recovered from endemic coronavirus infections and from healthy blood donors as controls. five samples from patients recovered from covid-19 had serum sensitivity scores (sss) > 5 and rescued >50% cells from virus-mediated death at 1 mg/ml (figure 2a,b) . notably, that serum from a recovered patient with sss = 7.2 neutralized all seven sars-cov-2 strains ( figure s3 ). our neutralization test of 32 samples (table s2) showed a moderate positive correlation with igg (r = 0.59, p < 0.001) and igm (r = 0.43, p = 0.01) s/co values obtained using commercial elisa kits that recognize the sars-cov-2 n protein (figure 2c,d) . however, the correlation between the igg and igm elisa results was only r = 0.28, p = 0.11. furthermore, we found a moderate negative correlation between ssss and time intervals between the sars-cov-2 diagnosis and serum collection for 66 samples (−0.5, p < 0.025; figure 2d ). altogether, these results suggest that patients diagnosed with covid-19 produce different immune responses to the sars-cov-2 infection and that the neutralization capacity of convalescent sera declines with time. through the literature review, we made a database to summarize safe-in-man bsaas (https://drugvirus.info/). recently, we have expanded on the spectrum of activities for some of these agents [17] [18] [19] 26, 31] . some of these agents could be repositioned for the treatment of a sars-cov-2 infection. we tested 136 agents against sars-cov-2 in vero-e6 cells. remdesivir was included as a positive control [32] and nicotine as a negative control. seven different concentrations of the compounds were added to virus-infected cells. cell viability was measured after 72 h to determine compound efficiency. after the initial screening, we identified apilimod, emetine, amodiaquine, obatoclax, homoharringtonine, salinomycin, arbidol, posaconazole and nelfinavir as compounds that rescued virus-infected cells from death (auc from 285 to 585; table s1 ). the compounds we identified possessed a structure-activity relationship (figure 3a) . auc for remdesivir was 290. interestingly, 10 µm of nicotine rescued cells from virus-mediated death but altered the cell morphology (auc = 239; figure s4 ). we repeated the experiment with hit compounds, monitoring their toxicity and efficacy. we confirmed the antiviral activity of emetine, amodiaquine, obatoclax, homoharringtonine, salinomycin and nelfinavir (figure 3b ,c). importantly, amodiaquine had a superior si over its analogs chloroquine, hydroxychloroquine, quinacrine and mefloquine ( figure s5 ). thus, we identified and validated anti-sars-cov-2 activities for six bsaas in vero-e6 cells. to test for potential synergism among the validated hits, we treated cells with varying concentrations of a two-drug combination and monitored the cell viability (figure 4a) . the observed drug combination responses were compared with the expected combination responses calculated by means of the zero-interaction potency (zip) model [27, 33] . we quantified synergy scores, which represent the average excess response due to drug interactions (i.e., 10% of cell survival beyond the expected additivity between single drugs has a synergy score of 10). we found that combinations of nelfinavir with salinomycin, amodiaquine, homoharringtonine and obatoclax, as well as the combination of amodiaquine and salinomycin, were synergistic (most synergistic area scores >10; figure 4b ). moreover, the nelfinavir-amodiaquine treatment was effective against all seven sars-cov-2 strains (figure 4c ). thus, we identified synergistic drug combinations against sars-cov-2 infections. we next profiled transcriptional responses to nelfinavir, amodiaquine or both drugs in virus-or mock-infected vero-e6 cells at 24 h. we showed that the addition of nontoxic but effective concentrations of drugs slightly affected the transcription of immune-related genes in virus-infected cells ( figure s6a ). these genes (cxcl1, cxcl2, cxcl3, cxcl8, cxcl10, cxcl11, oasl, ifnl1, mx1 and herc5) are needed for alarming neighboring cells about the ongoing infection and for the protection of the organism from repeated infections. amodiaquine and its combination with nelfinavir lowered the transcription of viral genomic and sub-genomic rnas ( figure s6b) . (a) structure-antiviral activity relation of 136 broad-spectrum antivirals (bsaas). the compounds were clustered based on their structural similarity calculated by ecpf4 fingerprints and visualized using the d3 javascript library. the anti-sars-cov-2 activity of the compounds was quantified using the auc and shown as bubbles. bubble size corresponds to compounds aucs. (b) vero-e6 cells were treated with increasing concentrations of a compound and infected with the hcov-19/norway/trondheim-e9/2020 strain (moi, 0.1) or mock. after 72 h, the viability of the cells was determined using the celltiter-glo assay. mean ± sd; n = 3. (c) table showing half-maximal cytotoxic concentration (cc 50 ), the half-maximal effective concentration (ec 50 ) and selectivity indexes (si = cc 50 /ec 50 ) for selected anti-sars-cov-2 compounds calculated from ctg and plaque assays. mean ± sd; n = 3. figure 3 . anti-sars-cov-2 activity of safe-in man broad-spectrum antivirals in vero-e6 cells. (a) structure-antiviral activity relation of 136 broad-spectrum antivirals (bsaas). the compounds were clustered based on their structural similarity calculated by ecpf4 fingerprints and visualized using the d3 javascript library. the anti-sars-cov-2 activity of the compounds was quantified using the auc and shown as bubbles. bubble size corresponds to compounds aucs. (b) vero-e6 cells were treated with increasing concentrations of a compound and infected with the hcov-19/norway/trondheim-e9/2020 strain (moi, 0.1) or mock. after 72 h, the viability of the cells was determined using the celltiter-glo assay. mean ± sd; n = 3. (c) table showing half-maximal cytotoxic concentration (cc50), the half-maximal effective concentration (ec50) and selectivity indexes (si = cc50/ec50) for selected anti-sars-cov-2 compounds calculated from ctg and plaque assays. mean ± sd; n = 3. to rapidly respond to the covid-19 outbreak, we developed a freely accessible website summarizing novel anti-sars-cov-2 developments and currently approved diagnostic options around the globe. it also tracks the development of therapeutic/antiviral drugs and vaccines. the "treatment" section of the website summarizes 542 in-progress and completed clinical trials that test the efficacy of therapeutic agents to treat covid-19 or complications that arise from covid-19. these trials include over 192 unique therapeutic agents in varying combinations and applications. importantly, we list 71 clinical trials that are already completed or are projected to be completed by the end of june 2020. of note, among these are trials of remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, to rapidly respond to the covid-19 outbreak, we developed a freely accessible website summarizing novel anti-sars-cov-2 developments and currently approved diagnostic options around the globe. it also tracks the development of therapeutic/antiviral drugs and vaccines. the "treatment" section of the website summarizes 542 in-progress and completed clinical trials that test the efficacy of therapeutic agents to treat covid-19 or complications that arise from covid-19. these trials include over 192 unique therapeutic agents in varying combinations and applications. importantly, we list 71 clinical trials that are already completed or are projected to be completed by the end of june 2020. of note, among these are trials of remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, dipyridamole and interferons alpha and beta, which are all phase 3 or 4 clinical trials scheduled to be currently completed. the "prevention" section summarizes 23 current vaccine trials taking place around the globe. although vaccine development lags considerably behind drug development, several repurposed vaccine options have also emerged. this includes trials of the cross-reactivity of the mmr (measles, mumps and rubella) vaccine, as well as several trials of the bacillus calmette-guérin (bcg) vaccine among high-risk populations, such as healthcare workers. finally, the "testing" section of the website provides a summary of 377 currently available laboratory-based and point-of-care diagnostic tests that are approved for clinical diagnosis in at least one country. the website also includes predictions of experimental and approved drugs effective against sars-cov-2, as well as provides a summary of information about the coronavirus pandemic. the website allows interactive exploration of the data with built-in feedback and is available in several languages. the website is updated as soon as novel anti-sars-cov-2 options emerge, or the statuses of existing ones are updated. here, we reported the isolation of seven sars-cov-2 strains from samples of patients suffering from covid-19. full-genome sequencing revealed that the strains were highly similar (98.8%) to one another and to the strains circulating in china, denmark, the usa and canada. all seven strains contain d614g in the s protein. strains with this mutation began spreading in europe in early february and became dominant in other regions (https://doi.org/10.1101/2020.04.29.069054). we screened 12 cell lines for their susceptibility for the sars-cov-2 infection and virus replication. vero-e6 cells appeared to be the most susceptible cell line for virus-mediated cell death and virus propagation. this cell line has been widely used in toxicology, virology and pharmacology research, as well as in the production of vaccines and diagnostic reagents. the cell line is interferon-deficient; i.e., unlike normal mammalian cells, it does not secrete interferon alpha or beta when infected by viruses [34] . moreover, this cell line was used routinely in anti-sars-cov-2 research [35] [36] [37] [38] . we also showed that >10 sec of uvc radiation or 48 h incubation at 37 • c neutralized sars-cov-2, establishing a rationale and methodology for safe work in the laboratory. these results are consistent with previous studies showing that physical factors destabilize sars-cov-2 and other viruses [39] [40] [41] [42] . neutralization tests are crucial tools for the assessment of previous sars-cov-2 exposure and potential immunity [12, 13] . we have developed a test to assess the neutralization capacity of serum samples from patients recovered from sars-cov-2 infections, patients with endemic coronavirus infections and healthy blood donors. our results suggest that covid-19 patients respond differently to the sars-cov-2 infection. moreover, the neutralization capacity of convalescence sera declined with time. thus, the neutralization test allowed us to identify the most potent sera from patients recovered from covid-19 for the treatment of sars-cov-2-infected patients. moreover, results from our neutralization test positively correlated with those from commercial elisa assays. however, the correlation between the igg and igm elisa results was only moderate. the difference could be associated with the time of the sample collection, production of the immunoglobulins or sensitivity that can be attributed to the technique and the antigen in use (i.e., igm is the first immunoglobulin to be produced in response to an antigen and can be detected during early onset of disease, whereas igg is maintained in the body after initial exposure for the long-term response and can be detected after the infection has passed). there were certain concerns regarding the antibody-dependent cell-mediated toxicity of convalescent sera [43] . however, the recent safety study on 5000 hospitalized patients transfused under the u.s. food and drug administration's national expand access program for covid-19 revealed no toxicity (https://doi.org/10.1101/2020.05. 12.20099879) . drug repurposing, also called drug repositioning, is a strategy for generating an additional value from an existing drug by targeting diseases other than that for which it was originally intended [44, 45] . this has significant advantages over new drug discoveries, since chemical synthesis steps, manufacturing processes, reliable safety and pharmacokinetic properties have already been studied in preclinical (animal model) and early clinical developmental phases (phase 0, i and iia). therefore, drug repositioning for covid-19 provides unique translational opportunities, including a substantially higher probability of success to the market as compared with developing new virus-specific drugs and vaccines, as well as significantly reduced costs and timelines to clinical availability [26, 46, 47] . we tested 136 safe-in-man bsaas against sars-cov-2 in cell cultures. we identified salinomycin, obatoclax, amodiaquine, nelfinavir, emetine and homoharringtonine as having anti-sars-cov-2 activity, which we put forward as potential anti-sars-cov-2 drug candidates. nelfinavir (viracept) is an orally bioavailable inhibitor of human immunodeficiency virus hiv-1 (750 mg per os (po) q8hr). it targets hiv protease for the treatment of hiv infections [48] . molecular docking studies predict that nelfinavir binds to the sars-cov-2 protease [49] . nelfinavir could also inhibit cell fusion caused by the sars-cov-2 s glycoprotein (https://doi.org/10.1101/2020.04. 06.026476) [50] . it also inhibits chikungunya virus (chikv), dengue virus (denv), hepatitis c virus (hcv), herpes simplex virus 1 (hsv-1) and sars-cov infections (https://doi.org/10.1039/ c5ra14469h) [51] [52] [53] . amodiaquine is a medication used to treat malaria. the recommended dose for a course of amodiaquine is 30 mg amodiaquine base/kg body weight over three days, i.e., 10 mg/kg/day [54] . [55] [56] [57] [58] [59] [60] . importantly, amodiaquine showed more potent antiviral activity than its analogs chloroquine and hydroxychloroquine. obatoclax was originally developed as an anticancer agent. several phase ii clinical trials have investigated the use of obatoclax in the treatment of leukemia, lymphoma, myelofibrosis and mastocytosis. a continuous 24 h infusion of obatoclax 25-60 mg/day for three days in two-week cycles or 3 h infusions in a 3-day cycle have previously been evaluated in cancer patients [61] . in addition, obatoclax showed antiviral activity against fluav, zikv, wnv, yfv, sinv, junín virus (junv), lasv, herpes simplex virus 2 (hsv-2), echovirus 1 (ev1), human metapneumovirus (hmpv), rvfv and lymphocytic choriomeningitis virus (lcmv) in vitro [18, 24, 25, 62, 63] . it was shown that obatoclax inhibited the viral endocytic uptake by targeting the cellular mcl-1 protein [24] . emetine is an antiprotozoal drug. it is administered by intramuscular or deep subcutaneous injection in a dose of 1 mg/kg/day (maximum 60 mg/day) for 10 days [64] . emetine is also used to induce vomiting. in addition, it possesses antiviral effects against zikv, ebov, rabv, cytomegalovirus (cmv), hcov-oc43, hsv-2, ev1, hmpv, rvfv, fluav, hiv-1 and sars-cov-2 [17, 18, 36, [65] [66] [67] [68] [69] (https://doi.org/10.1101/2020.03.25.008482). emetine was proposed to inhibit viral polymerases, though it could have some other targets [70] . homoharringtonine is an anticancer drug that is indicated for the treatment of chronic myeloid leukemia (2 mg/m 2 iv daily × 7). it also possesses antiviral activities against hepatitis b virus (hbv), mers-cov, hsv-1, ev1, vzv and sars-cov-2 in vitro [18, 36, [71] [72] [73] [74] . homoharringtonine binds to the 80s ribosome and inhibits viral protein synthesis by interfering with the chain elongation [72] . salinomycin is an orally bioavailable antibiotic that is used against gram-positive bacteria in animal husbandry (0.2 mg/kg body weight (bw) po). it also inhibits fluav, respiratory syncytial virus (rsv) and cmv infections [75, 76] . salinomycin was proposed to disrupt the endosomal acidification and to block entry of the viruses into cells [77, 78] . our results have uncovered several existing bsaas that could be repositioned to sars-cov-2 infections. since pharmacokinetic/pharmacodynamic and toxicology studies have already been performed on these compounds, in vivo efficacy studies could be initiated immediately, saving time and resources. combination therapies became a standard for the treatment of hiv and hcv infections. the reasons for using combinations rather than single antiviral are better efficacy, decreased toxicity and the prevention of resistance emergence. here, we found that combinations of nelfinavir with salinomycin, amodiaquine, obatoclax, emetine or homoharringtonine were synergistic against sars-cov-2 in vero-e6 cells. interestingly, synergistic interactions occurred between compounds belonging to different sar clusters (i.e., nelfinavir belongs to a separate cluster than amodiaquine or obatoclax-emetine-homoharringtonine). in particular, the synergy was achieved when a virus-directed drug was combined with host-directed ones. this observation agrees with other studies on such combinations and virus-host interactions [18, 25, 79, 80] (https://doi.org/10.1101/2020.04.14.039925). according to the available pharmacological data for these drugs, the most potent combination could be a combination of orally available nelfinavir and amodiaquine. this was also the combination that exhibited the highest synergy of all the drug combinations we tested, with the synergy score at the most synergistic area being 16.48 (i.e., 16.48% of cell survival beyond the expected additivity between the single drugs). there are no guidelines of what is considered a good synergy, but it is very common to consider synergy >10 as true (significant) synergy. thus, the amodiaquine and nelfinavir combination could result in better efficacy and decreased toxicity for the treatment of sars-cov-2 and perhaps other viral infections. our future goal is to complete preclinical studies and translate our findings into trials in patients. the most effective and tolerable bsaas or their combinations will have a global impact, improving the protection of the general population from emerging and re-emerging viral infections or coinfections and allowing the rapid management of drug-resistant strains. our bigger ambition is to assemble a toolbox of bsaas for the treatment of emerging and re-emerging viral infections. this toolbox can be offered to the who as a means for the fast identification of safe and effective antiviral options. we have summarized the information about the status of currently available and emerging anti-sars-cov-2 options on the freely accessible website (https://sars-coronavirus-2.info). the website is updated regularly and incorporates novel anti-sars-cov-2 options as they emerge or changes the statuses of existing ones as updates occur. in conclusion, sera from recovered patients, bsaas and combinations of bsaas, as well as other available and emerging treatments, could have pivotal roles in the battle against covid-19 and other emerging and re-emerging viral diseases. further development of these options could save time and resources that are required for the development of alternative virus-specific drugs and vaccines. this could have a global impact by decreasing morbidity and mortality, maximizing the number of healthy life years, improving the quality of life of infected patients and decreasing the costs of patient care curtailing to the impact of the current sars-cov-2 pandemic, as well as future viral outbreaks. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/6/642/s1: table s1 : compounds, their suppliers, catalogue numbers and aucs. table s2 . results of neutralization and elisa assays. figure s1 . propagation of hcov-19/norway/trondheim-e9/2020 in cell cultures. figure s2 . effects of temperature and uv radiation on the infectivity of the hcov-19/norway/trondheim-e9/2020 strain. figure s3 . the effects of the serum from patients recovered from the sars-cov-2 infection on the viability of vero-e6 cells infected with 7 sars-cov-2 strains. figure s4 . the effects of nicotine on the viability and morphology of mock-and sars-cov-2-infected vero-e6 cells. figure s5 : the comparison of anti-sars-cov-2 activities of amodiaquine and its analogs. figure s6 . transcriptomic analysis of mock-and sars-cov-2-infected vero-e6 cells treated with nelfinavir, amodiaquine or both drugs. author contributions: all authors contributed to the methodology, software, validation, formal analysis, investigation, resources, data curation, writing and review and editing of the manuscript. d.k. conceptualized, supervised and administrated the study and acquired funding. all authors have read and 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transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry efficacy and mechanism of action of low dose emetine against human cytomegalovirus natural plant alkaloid (emetine) inhibits hiv-1 replication by interfering with reverse transcriptase activity emetine inhibits replication of rna and dna viruses without generating drug-resistant virus variants anti-varicella-zoster virus activity of cephalotaxine esters in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs effect of cantharidin, cephalotaxine and homoharringtonine on "in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication targeting intracellular ion homeostasis for the control of respiratory syncytial virus wnt modulating agents inhibit human cytomegalovirus replication salinomycin inhibits influenza virus infection by disrupting endosomal acidification and viral matrix protein 2 function identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs jnj872 inhibits influenza a virus replication without altering cellular antiviral responses in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov-2 shows synergistic effect acknowledgments: this study is devoted to wenliang li, a chinese doctor who tried to warn about coronavirus, as well as to many other doctors and covid-19 patients. we thank koit aasumets, sergio miguel castañeda zegarra, qindong zhang, simona komarova, nikki upfold, miriam khider, hege hval and kasia kolasa for translation of the sars-coronavirus-2.info website to different languages. we also thank maxim bespalov, sergei shiryaev, pavel uvarov, evgeny kulesskiy and many other people for sharing their ideas on drug candidates. in addition, we thank wei wang, marit bugge and nadra j. nilsen for the cell lines. the authors declare no conflicts of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-343196-vlwzzrgc authors: kiambi, stella; corman, victor m.; sitawa, rina; githinji, jane; ngoci, james; ozomata, abdullahi s.; gardner, emma; von dobschuetz, sophie; morzaria, subhash; kimutai, joshua; schroeder, simon; njagi, obadiah; simpkin, piers; rugalema, gabriel; tadesse, zelalem; lubroth, juan; makonnen, yilma; drosten, christian; müller, marcel a.; fasina, folorunso o. title: detection of distinct mers-coronavirus strains in dromedary camels from kenya, 2017 date: 2018-11-28 journal: emerg microbes infect doi: 10.1038/s41426-018-0193-z sha: doc_id: 343196 cord_uid: vlwzzrgc nan between july 2016 and october 2017, nasal swabs were randomly taken from n = 1421 dromedaries in five counties, namely, turkana (n = 417), marsabit (n = 370), isiolo (n = 403), laikipia (n = 181), and nakuru (n = 50). in addition, monthly repeated sampling was performed on 430 dromedaries from four herds in two different countries (isiolo and nakuru) for a period of 7 months (from april to october 2017). in total, n = 2175 nasal swab samples were collected. all samples were stored frozen in trizol buffer at −80°c. rna extraction (direct-zol™ rna kit, zymo research) and mers-cov nucleic acid detection were performed following the manufacturer's instructions and according to previously established protocols 8 . in seven of 2175 (0.23%) tested nasal swabs, mers-cov rnas were detected by the upe mers-cov rt-pcr screening assay (supplementary table) . for 2/7 samples, which had very low mers-cov rna concentrations (<2 x 10 4 copies/ml), confirmatory rt-pcr testing and sequencing were unsuccessful. the mean viral load for 5/7 samples was 1.1 x 10 7 (range 1.2 x 10 5 -5.0 x 10 7 ) rna copies per ml buffer. four of the five mers-cov rna-positive animals were female and <1 year old, consistent with previous observations that juvenile dromedaries and possibly females may be the main sources for mers-cov excretion 9 . the mers-cov rna-positive animals belonged to two different dromedary camel herds in dabel and lombolio, which are both located within isiolo country. however, the herds neighbor each other and share common pastures and water sources. introduced into the two herds. the dromedaries were sampled on the same day, suggesting simultaneous cocirculation of two different mers-cov strains and perhaps an unexplored infection dynamic in isiolo, which is a camel congregation location. to experimentally confirm the presence of two independently circulating mers-cov strains and to rule out sample cross-contamination, we generated complete mers-cov genome sequences using a previously established protocol 10 . full genome sequences were generated for one specimen of each of the two positive herds using the samples with the highest mers-cov rna concentrations (5.0 x 10 7 and 3.7 x 10 6 copies/ml). other confirmed mers-covpositive samples were assigned to two different mers-cov isolates ("dabel" or "lombolio") by amplifying and sequencing single-nucleotide polymorphisms in the spike gene and the open reading frame 3 (supplementary table) . all three viruses from dabel and both viruses from lombolio shared the same polymorphism patterns. for phylogenetic analysis, we included two representatives of mers-cov lineages representing mers-cov clades a and b, as defined earlier 11 , along with all published clade c (non-a, non-b) mers-cov complete genomes (genbank accessed 2 april 2018). a phylogenetic tree was constructed using the maximum-likelihood method based on the general time reversible model and 500 bootstrap replicates using the phyml plugin in geneious r11 (www.geneious.com, biomatters ltd, new zealand). as shown in fig. 1 , both kenyan dromedary mers-cov isolates clustered with the proposed clade c viruses from ethiopia and egypt in a sister relationship to all arabian mers-covs (clades a and b). the two kenyan mers-cov isolates diverged by 0.02% at the nucleotide level, confirming the circulation of at least two different mers-cov strains in kenya. the next closest mers-cov relative was obtained from a dromedary sampled in egypt in 2014 (nrce-nc163/2014; acc no. ku74020, clade c) and showed 0.23-0.24% nucleotide distance. recombination analysis by rdp v4.95 indicated that none of the two kenyan mers-cov strains had recombined with any of the known clade a, b, or c strains. the previously described clade c african mers-cov strains 4,5 had several mutations in the spike protein, which is responsible for cellular receptor interaction, virus entry, and antibody-directed virus neutralization 12 . an alignment of the amino-acid sequences of all known mers-cov spikes showed that the kenyan mers-cov strains had one unique amino-acid change (s528p) within the core part of the receptor-binding domain (supplementary figure) . as the mutation was not among the 14 amino-acid residues that directly interact with the dipeptidyl peptidase-4 receptor 12 , phenotypic traits of these new clade c mers-cov strains may be comparable to epidemic mers-cov strains as described previously 4 . however, without further extensive experimental assessment, we cannot rule out the possibility that the observed mutation in the spike protein causes differences in the receptor interaction or receptor binding affinity, which may influence virus transmission or host tropism. recently described mers-cov strains from western africa had genomic deletions within open reading frame (orf) 4 a/b that were not seen in eastern african mers-cov strains 4 . both of the encoded proteins, proteins 4a and 4b, have anti-immune functions 13, 14 and may represent important virulence factors in vivo. we provide independent evidence that mers-cov from eastern african dromedaries encode a complete orf4a/b. the observation that mers-cov strains in different parts of eastern africa have a complete orf4a/b suggests the predominance of these strains on the african continent and emphasizes that the orf4a/b deletion is most likely geographically restricted to western africa. taken together, differential spike-receptor interactions and anti-immune activity may influence virus replication and transmission. the limited number of human mers cases in africa would certainly favor the idea that mers-cov strains differ in virulence and transmissibility. further experimental confirmation, preferably by animal transmission experiments in combination with coronavirus reverse genetics 15 , are warranted. the phylogenetic relationship of mers-cov strains from the african continent (clade c) with the strains circulating on the arabian peninsula (clades a and b) hints at the divergence of these clades some time ago. the putative absence of clades a and b mers-covs on the african continent may be explained by a lack of surveillance and testing and/or by the genetic drift of mers-cov on the arabian peninsula. the unidirectional export routes from africa to the arabian peninsula may prevent the reintroduction opportunities of clades a and b mers-covs into african dromedary herds. interestingly, to date, no clade c mers-cov strains from africa have been detected on the arabian peninsula, which is rather surprising, given the continuous and extensive export of african dromedaries to the arabian peninsula. an explanation for this observation may again be a lack of testing of imported animals and/or the fact that previous clade a/b mers-cov infections may have established herd immunity in the arabian dromedary populations. as cov infections do not elicit long-lasting (mucosal) immunity, the introduction of clade cmers-cov strains on the arabian peninsula may be possible in the future and should therefore be monitored. to shed light on possible reasons for the restricted geographic circulation of different mers-cov strains, enhanced virological surveillance of mers-cov is urgently needed in dromedary populations of the affected regions. putative underlying evolutionary and molecular mechanisms that influence the geographic distribution of differentially virulent mers-cov strains should be assessed through phenotypic characterizations of different mers-cov strains. the early detection and characterization of emerging mers-cov strains with new phenotypic features will be highly relevant for future vaccination strategies and the prediction of epidemics in humans. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers and the dromedary camel trade between africa and the middle east mers coronaviruses from camels in africa exhibit regiondependent genetic diversity cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt antibodies against mers coronavirus in dromedary camels mers-cov antibodies in humans detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction time course of mers-cov infection and immunity in dromedary camels rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 mers-cov 4b protein interferes with the nf-kappabdependent innate immune response during infection middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist transgene expression in the genome of middle east respiratory syndrome coronavirus based on a novel reverse genetics system utilizing redmediated recombination cloning key: cord-334220-sqvfr31q authors: messina, francesco; giombini, emanuela; montaldo, chiara; sharma, ashish arunkumar; piacentini, mauro; zoccoli, antonio; sekaly, rafick-pierre; locatelli, franco; zumla, alimuddin; maeurer, markus; capobianchi, maria r.; lauria, francesco nicola; ippolito, giuseppe title: looking for pathways related to covid-19 phenotypes: confirmation of pathogenic mechanisms by sars-cov-2 host interactome date: 2020-11-03 journal: biorxiv doi: 10.1101/2020.11.03.366666 sha: doc_id: 334220 cord_uid: sqvfr31q in the last months, many studies have clearly described several mechanisms of sars-cov-2 infection at cell and tissue level. host conditions and comorbidities were identified as risk factors for severe and fatal disease courses, but the mechanisms of interaction between host and sars-cov-2 determining the grade of covid19 severity, are still unknown. we provide a network analysis on protein–protein interactions (ppi) between viral and host proteins to better identify host biological responses, induced by both whole proteome of sars-cov-2 and specific viral proteins. a host-virus interactome was inferred on published ppi, using an explorative algorithm (random walk with restart) triggered by all the 28 proteins of sars-cov-2, or each single viral protein one-by-one. the functional analysis for all proteins, linked to many aspects of covid-19 pathogenesis, allows to identify the subcellular districts, where sars-cov-2 proteins seem to be distributed, while in each interactome built around one single viral protein, a different response was described, underlining as orf8 and orf3a modulated cardiovascular diseases and pro-inflammatory pathways, respectively. finally, an explorative network-based approach was applied to bradykinin storm, highlighting a possible direct action of orf3a and ns7b to enhancing this condition. this network-based model for sars-cov-2 infection could be a framework for pathogenic evaluation of specific clinical outcomes. we identified possible host responses induced by specific proteins of sars-cov-2, underlining the important role of specific viral accessory proteins in pathogenic phenotypes of severe covid-19 patients. whilst covid-19 predominantly affects the respiratory system, it is a multisystem disease, with a wide spectrum of clinical presentations from asymptomatic, mild and moderate, to severe, fulminant disease (1) . host conditions and comorbidities such as high age, obesity, diabetes, hypertension, organ damages, inflammation and coagulation dysfunctionality, were identified as risk factors for severe and fatal disease courses (2) , but the mechanisms of interaction between host and sars-cov-2 that activate pathological pathways and determine severity, are still unknown. in last months, many studies clearly described several mechanisms of sars-cov-2 infection at cell and tissue level. it was observed that the replication of sars-cov-2, as well as all +rna viruses, occurs in the cytoplasm of the host cell, inducing a membrane rearrangement of rough endoplasmic reticulum (er) membranes into double-membrane vesicles (3, 4) . moreover, nsp8 along with nsp7 and nsp12, which yield the rna polymerase activity of nsp8, are assembled into the replicase-transcriptase complex, that begins to generate anti-sense (-) genomic rna molecules, templates for positive-sense genome (+) and mrna transcripts (5, 6) . during virus entry, a well-known process is the cleavage of s protein by furin on the cell membrane, which lead to split s protein into two subunits, s1 and s2, which the last can interact with ace2 (7, 8) . however, the sars-cov-2-host interaction is not restricted to local infection, but it triggers a systemic reaction, including the activation of the bradykinin storm, as described in many severe covid-19 patients (9) . indeed, sars-cov-2 infection causes from one side a decrease of ace level in the lung cells and, on the other side, an increase of ace2 level, leading to increase bradykinin level (9) . furthermore, microvascular injuries, due to systemic inflammatory response and endothelial dysfunction, were frequently found in severe covid-19 patients (10) . increased circulating d-dimer concentrations, reflecting pulmonary vascular bed thrombosis with fibrinolysis, and elevated cardiac enzyme concentrations, linked to emergent ventricular stress induced by pulmonary hypertension, were associated to early features of severe pulmonary intravascular coagulopathy related to covid-19 (11) . moreover, myocardial injuries were found to be linked to the risk of fatal outcome in covid-19 patients (12) (13) (14) . in 43% severe patients (21% of all covid-19 patients) the myocardial injury was observed and a severe patient had a 4.74-fold increase in the risk of myocardial injury than non-severe patients (15) . although microvascular injuries and microthrombi formation frequently occurred in severe covid-19 patients, the role of sars-cov-2 in this phenotype is not completely explained yet. recently, results of a meta-analysis study on interleukin-6 concentrations in patients with severe or critical covid-19, pointed out that the systemic inflammatory profile of covid-19 is distinct from that of non-covid-19 ards, sepsis, and car t cell-induced cytokine release syndrome, and it might be involved in organ dysfunction in covid-19, such as endovasculitis, direct viral injury and viral-induced immunosuppression (16) . although many aspects of covid-19 pathogenesis and mechanisms of sars-cov-2 have been investigated, only few papers describe the interactions among sars-cov-2 proteins by wet experiments. physical associations in human cells, between sars-cov-2 proteins and human proteins, were found by affinitypurification mass spectrometry (ap-ms), identifying 332 high-confidence sars-cov-2-human proteinprotein interactions (ppis), highlighting an activation of innate immune pathways (17) . then, the influence of sars-cov-2 on transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line, was described through a multi-omic approach. such a multilevel representation highlighted autophagy mechanisms regulation by orf3a and nsp6, the modulation of innate immunity by m, orf3a and ns7b, and the integrin-tgf-β-egfr-rtk signalling perturbation by orf8 of sars-cov-2 (18). virus-host interactome by computational approach has been applied to covid-19 for drug repurposing (17) , allowing the identification of new drug targets (19) and contributing to explain clinical manifestations (20, 21) . the structural information on sars-cov-2 proteins and their interactions with human proteins and other viral proteins, allowed to better understand the mechanisms of sars-cov-2 infection, also comparing it with sars-cov (22) . the interactome based on ppi and gene expression data, have been applied also to uncover the molecular origins of phenotypes of other complex diseases (24, 25) , in order to better define the mechanisms of covid-19 pathogenesis. the understanding of all mechanisms of sars-cov-2 infection also passes by overall visualization of biological reactions and pathways involved in covid-19 and h-cov infections. it can carry out on a disease map, such as covid-19 disease maps, containing many diagrams about host molecular response during the infection (26) (27) (28) . in this context, defining the host response induced by specific viral proteins would be of great importance and can guide the identification of functional viral targets, helping to better define the pathologic phenotypes of the infection. here, we carry out a network analysis on ppi to better identify host biological response induced by sars-cov-2. furthermore, the interactome analysis was applied to design network of sars-cov-2 -host proteins that could lead to a bradykinin storm. we used three different applications to identify interacting proteins: 200 proteins that interact closely with 28 sars-cov-2 proteins, 50 protein interactomes around each sars-cov-2 protein and 200 protein associated with kng1, the pre-cursor of bradykinin (bk). in all three models, pathways associated with proteins were identified, including dna damage/repair, tgf-β signalling, complement cascades ( figure 1 ). to define the massive effect of sars-cov-2 infection on the host cell, an interactome between the entire set of sars-cov-2 proteins and host was carried out. observing the colours' distribution, it is possible to distinguish three different areas, corresponding to as many subcellular districts, where sars-cov-2 proteins seem to be distributed (figure 2 a). in fact, n, nsp1, nsp4 and nsp15 are posed around nuclear proteins, while s, along with nsp5, nsp10, nsp12, nsp13, nsp14, nsp16, orf1a and orf9b, were among cytosolic and membrane proteins, at the bounder of the interactome. protein m and many accessory and not-structural proteins (ns7b, nsp6, nsp7, orf3a, orf7a, orf8, orf10 and orf14) are around mitochondrial and endoplasmic proteins. we described the high values of betweenness centrality and degree for 9 host proteins: two proteins multiorganelle amyloid-beta precursor protein (app) and dual-specificity protein phosphatase (pten); the membrane protein sodium/potassium-transporting atpase subunit alpha-1 (atp1a1); two cytoplasmic proteins, 14-3-3 protein theta (ywhaq) and ubiquitin (ubc); two proteins of endoplasmic reticulum and golgi apparatus, sarcoplasmic/endoplasmic reticulum calcium atpase 2 (atp2a2) and unconventional myosin-vi (myo6); the mitochondrial atp synthase subunit alpha (atp5f1a); one nuclear receptor for export of rnas, exportin-1 (xpo1), suggesting their main role in this infection (sfigure 1 , stable 1 ). in order to further dissect the interactions with the entire proteome of sars-cov-2, enrichment analysis was carried out with wikipathways and kyoto encyclopaedia of genes and genomes (kegg) databases. wikipathways gene enrichment analysis revealed biological pathways of dna replication, ubiquitination and proteasome, with high significance (fdr <0.01%). in addition, kegg pathway enrichment analysis revealed dna and rna replication pathways as the most significant pathways (fdr <0.01%), as well as signalling pathways and viral infection pathways (figure 2 b) . to define the effect of specific viral proteins on host response, interactomes were built, choosing one specific viral protein as seed, imposing to find the 50 closest proteins. these analyses allowed to produce 28 restricted interactomes, which defined the strictest biological interactions associated to seed proteins both among human proteins and other viral proteins. in sfigure for kegg database the gene enrichment analysis on interactomes of ns7b, orf1a, orf3a and orf8 showed pathway clusters highly significant and consistent with possible pathogenic mechanisms, such as the activation of the complement and of the coagulative cascade, (29) and the tgf-β-dominated immune response (30) . in particular, the ns7b interactome revealed snare interactions in vesicular transport pathway, describing the mechanisms of intracellular vesicle trafficking and secretion, as the most significant pathway among all interactome (fdr< 0. 00001%). the orf1a interactome revealed a cluster of pathways, composed by as the virus-host interactome has been efficient to describe the response to specific viral proteins, this method was applied to reconstruct the possible involvement of sars-cov-2 proteins in triggering the bradykinin storm during the infection. in this case, kininogen-1 (kng1), precursor of bradykinin made by proteolysis cleavage, was considered as seed for rwr, imposing 200 closest proteins as limit to stop the algorithm. the resulting interactome showed ns7b, orf3a, orf8 and s as proximal to the seed protein, suggesting their role in the modulation of biological processes around kininogen-1. indeed, it would provide a direct influence of in this study, we built a functional interactome between sars-cov-2 and human proteome through rwr algorithm, to identify biological mechanisms and cell responses during sars-cov-2 infection and to propose a simulated model of infection contributing to a better understanding of covid-19 pathogenesis. districts. the high likelihood of our model to in vivo real mechanisms strengths the representative power of the interactome and the explorative algorithm to simulate biological processes. moreover, the proximity among specific viral proteins into the network, such as nsp7, nsp8, nsp9, nsp10, nsp12, nsp13, nsp14 involved in the viral rna replication, can be a good way to investigate their possible function in viral infection. the closeness among s, nsp5, nsp16, orf9b in the network is difficult to interpret, but s, nsp5 and orf9b, along with n, had significant positive responses of igg antibody in sera of covid-19 patients (31) . in sars-cov infected cell culture, the location of nsp2 in the cytoplasm and to some extent in the nucleus, as well as orf3a, orf7b, orf6 and m in er, seems to be consistent with their location among nuclear and cytoplasmatic, and er proteins respectively (32). the involvement of proteasome and ubiquitination pathways, along with rna replication, represent the principal pathways activated for assembly and replication of sars-cov-2 (18) . in fact, strong increases in rna-modifying proteins were revealed in cell culture after infection with sars-cov-2 (33) . the ubiquitin proteasome system deletes viral proteins to control the infection, but the virus can use them for its propagation (34) . the interactomes, built around a single protein of sars-cov-2, allowed to draw effects on cell, involving specific pathways. vesicular transport mechanism by snare interactions, identified for ns7b, is used by pathogens to penetrate host cells through their membranes and in particular in sars-cov (35, 36) . to study the pathogenesis of covid-19 in systemic context, bradykinin storm was investigated by the interactome approach. we showed ns7b and orf3a interacting with ece1, which can inactivate bk (40) . patients and reinforce the role of orf3a to enhance the bradykinin dysregulation. these host-virus interactions could enhance a better viral fitness during the infection, as suggested by the interaction already observed with sars-cov between ns7b in ece1(18). bradykinin storm in covid-19: the locking of ece1 activity due to ns7b and especially orf3a interaction, could reduce the activation of the vasoconstrictor endotelin-1, and amplify the vasodilatation effect of bk. the ece1 gene is much more expressed in all the tissues than ace2 and ace, especially in the lungs, where ece1 is 128 fold and 3.8 fold expressed compared to ace2 and ace, respectively (41) . similar to the angiotensin peptides, bk is produced from an inactive pre-protein kininogen-1 through the activation by the serine protease kallikrein (42) . furthermore, the excess of bk can lead to vasodilatation, hypotension and hypokalaemia (9, 43) , which is associated with arrhythmia (44, 45) . all these clinical conditions have been widely reported in covid-19 patients (14, 46, 47) . it is also notable that many of the other symptoms reported for covid-19 (myalgia, fatigue, nausea, vomiting, diarrhoea, anorexia, headaches, decreased cognitive function) are remarkably similar to other hyper-bk-conditions that lead to vascular hyperpermeabilization (48) . moreover, the activation of bk is strongly linked to coagulation: active f12 activates plasma kallikrein (pk), that liberates bradykinin by cleavage (49) . the direct interactions between there are many experimental platforms for deriving such physical interactions, such as affinity purification mass-spectrometry (ap-ms) and yeast-two-hybrid (y2h), which enable the accurate identification of interactions with a relatively long time. the scenario reported in this study refers to few experimental data available on public databases and could be different respect to real phenotypes of covid-19 patients. the pathways' analysis did not consider tissue and cell type diversity. finally, the low threshold established for the number of nodes found by rwr (200) limited the reconstruction of the entire pathways. however, this was a software-imposed threshold. although such network-based approach showed great potential in identifying mechanisms not yet observed, experimental tests will be necessary to confirm what we have described. we developed a network-based model for sars-cov-2 infection, which could be a framework for pathogenic evaluation of specific clinical outcomes. here, the ppi interactomes were used to identify mechanistic processes of the viral molecular machinery during sars-cov-2 infection, for viral survival and replication within the host. with this knowledge, proteins interactions crucial for pathogenesis could be discerned. we identified different host response induced by specific proteins of sars-cov-2, underlining the important role of orf3a and orf8 in phenotypes of severe covid-19 patients. the interactome approach applied to identification of biological reactions around kininogen-1, allowed to view ns7b and orf3a interactions with ece1, which might have a role to enhance bradykinin storm. this network-based model of sars-cov-2 -host interaction could guide to develop novel treatments against specific viral proteins, such as monoclonal antibodies. the virus-host interactome was made by merging sars-cov-2 -host protein-protein interaction (ppi) data from intact (51), with data from human ppi databases, such as biogrid, innatedb-all, imex, intact, matrixdb, mbinfo, mint, reactome, reactome-fis, uniprot, virhostnet, biodata, obtained by r packages psicquic and biomart (52, 53) . for sars-cov-2 -host interaction experimentally obtained, 1407 protein interactors and 2305 interactions were download from intact at 12 september 2020. 28 sars-cov-2 proteins are used for all the analyses: e, m, n, s, nsp1, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp10, nsp12, nsp13, nsp14, nsp15, nsp16, orf1a, orf3a, orf6, orf7a, ns7b, orf8, orf9b, orf10, orf14. to obtain functional information about ppi ex vitro, to simulate biological reactions in infected cells and to explore cell response against viral infection, we employed a random walk with restart (rwr) algorithm, a state-of-the-art guilt-by-association approach (54) . this algorithm allows to establish a proximity network from a given protein (seed), to study its functions, based on the premise that nodes related to similar functions tend to lie close to each other in the network. for this study, two types of interactome were performed: the whole sars-cov-2 -host interactome and an interactome per single sars-cov-2 protein. in the first imputation, every protein of sars-cov-2 proteome was used as seeds, with the limit of 200 closest host's proteins to every sar-cov-2 protein, every protein of sars-cov-2 proteome was chosen as seeds for random walk with restart, imposing the limit of 200 closest host proteins to every sars-cov-2 proteins. this analysis allowed to design sars-cov-2-host interactome, where the different colours correspond to the localization of the proteins within the cell. for the second kind of interactome, one sars-cov-2 per time was used as seed, lowering to closest 50 proteins in order to define the induced biological response as better as possible. for each node, a score was computed as a measure of proximity to the seed protein (23) . in total, a large ppi interaction database was assembled, including 13334 nodes and 73584 interactions. graphical representations of networks were performed by gephi 0. 9. 2 (55) . to identify hub protein in the sars-cov-2 -host interactome, the values of betweenness centrality and degree were plotted. betweenness centrality score measure how a specific node is in-between other nodes and then can be considered a hub, while the degree of node corresponds to number of connections. pathways of proteins involved in host response were tested by gene enrichment analysis on kyoto encyclopaedia of genes and genomes (kegg) human pathways and wikipathways databases (56) . to allow gathering of results for every running, the r package enrichr was used, an r interface to web-based tool 'enrichr' for analysing gene sets (57) . the enrichr analysis was performed using these statistical parameters: p-value (fisher exact test), q-value (adjusted p-value for false discovery rate, fdr). results for kegg and wikipathways were considered significant with a revised p-value < 0. 05. to infer pathways involved in single viral interactome, gene enrichment analyses for each viral interactome were collected along with p-values, as reported in enrichr package output. p-values of every single enrichment analyses were transformed by the function x = − log10 (p-value) and the 5% of x values were plotted on heatmap by r package pheatmap (58) . european centre for disease prevention and control (2020) clinical characteristics of covid-19 risk factors for covid-19 severity and fatality: a structured literature review double-membrane vesicles as platforms for viral replication a pneumonia outbreak associated with a new coronavirus of probable bat origin structure of replicating sars-cov-2 polymerase the sars-coronavirus nsp7+nsp8 complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension a multibasic cleavage site in the spike protein of sars-cov-2 is essential for infection of human lung cells sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a mechanistic model and therapeutic interventions for covid-19 involving a ras-mediated bradykinin storm myocardial and microvascular injury due to coronavirus disease immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia association of cardiac injury with mortality in hospitalized patients with covid-19 in wuhan, china characteristics and clinical significance of myocardial injury in patients with severe coronavirus disease 2019 cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) incidence of myocardial injury in coronavirus disease 2019 (covid-19): a pooled analysis of 7,679 patients from 53 studies cytokine elevation in severe and critical covid-19: a rapid systematic review, meta-analysis, and comparison with other inflammatory syndromes a sars-cov-2 protein interaction map reveals targets for drug repurposing multi-level proteomics reveals host-perturbation strategies of sars-cov-2 and sars-cov matrix metallopeptidase 9 as a host protein target of chloroquine and melatonin for immunoregulation in covid-19: a network-based metaanalysis a network medicine approach to investigation and population-based validation of disease manifestations and drug repurposing for covid-19. chemrxiv : the preprint server for chemistry 10 identifying human interactors of sars-cov-2 proteins and drug targets for covid-19 using network-based label propagation structural genomics of sars-cov-2 indicates evolutionary conserved functional regions of viral proteins covid-19: viral-host interactome analyzed by network based-approach model to study pathogenesis of sars-cov-2 infection network medicine: a network-based approach to human disease uncovering disease-disease relationships through the incomplete interactome covid-19 disease map, building a computational repository of sars-cov-2 virus-host interaction mechanisms systems medicine disease maps: community-driven comprehensive representation of disease mechanisms covid-19 disease map, a computational knowledge repository of sars-cov-2 virus-host interaction mechanisms covid-19 related coagulopathy: what is known up to now in severe covid-19, sars-cov-2 induces a chronic, tgf-β-dominated adaptive immune response sars-cov-2 proteome microarray for global profiling of covid-19 specific igg and igm responses analysis of intraviral protein-protein interactions of the sars coronavirus orfeome proteomics of sars-cov-2-infected host cells reveals therapy targets pleiotropic roles of the ubiquitin-proteasome system during viral propagation snare motif: a common motif used by pathogens to manipulate membrane fusion crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core discovery and genomic characterization of a 382-nucleotide deletion in orf7b and orf8 during the early evolution of sars-cov-2 effects of a major deletion in the sars-cov-2 genome on the severity of infection and the inflammatory response: an observational cohort study complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and beta-arrestins the gtex consortium (2020) the gtex consortium atlas of genetic regulatory effects across human tissues the contact activation and kallikrein/kinin systems: pathophysiologic and physiologic activities bradykinin stimulates renal na(+) and k(+) excretion by inhibiting the k(+) channel (kir4.1) in the distal convoluted tubule hypokalemia and sudden cardiac death hypokalemia-induced arrhythmias and heart failure: new insights and implications for therapy. frontiers in physiology clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china kallikrein-kinin blockade in patients with covid-19 to prevent acute respiratory distress syndrome bradykinin: inflammatory product of the coagulation system kinin b1 receptors as a therapeutic target for inflammation intact: an open source molecular interaction database psicquic and psiscore: accessing and scoring molecular interactions biomart--biological queries made easy random walk with restart on multiplex and heterogeneous biological networks gephi: an open source software for exploring and manipulating networks wikipathways: a multifaceted pathway database bridging metabolomics to other omics research enrichr: a comprehensive gene set enrichment analysis web server 2016 update figures and tables figure 1. workflow to describe sars-cov-2 host interactome analysis based on ppi data figure 2. a) ppi interactome, based on human ppi and sars-cov-2-host interactions, with top 200 closest proteins identified by rwr, using together 28 proteins of sars-cov-2 as seeds for only one rwr run. different colours of node and edges represent different locations in the cell wikipathways gene enrichment analyses for 200 proteins identified by rwr algorithm using together 28 proteins of sars-cov-2 heatmap reporting top 5% smallest p values of gene enrichment analysis on list of proteins, obtained by every interactome of single viral seed. the values were reported as -log10 (p value). a) p values of gene enrichment based on kegg 2019. b) p values of gene enrichment based on wikipathways the nodes in red are human proteins, while the nodes in green are virus proteins. b) kegg human pathway and wikipathways gene enrichment analyses for 200 proteins closest to kng-1. c) ppi interactome, where proteins belonged to human complement system wp2806 (modes in purple) and complement and coagulation cascades (nodes in blue) pathways were highlighted. the proteins belonged to both pathways were marked in light blue 1: table with human and viral proteins identified by rwr in all sars-cov-2 proteins interactome are reported, along with statistical parameters and biological information table of gene enrichment analysis obtained by kegg 2019 database all interactions selected by rwr in interactome for single seed viral protein. detailed information about interactors, interaction and database were reported plot betweenness centrality vs. degree of 199 human proteins, retained in all proteins of sars-cov-2 -host interactome e) as seed. the nodes in red are human proteins interactomes based on human ppi and sars-cov-2-host interactions, with top 50 closest proteins identified by rwr, using accessory proteins (orf1a, orf3a, orf6, orf7a, ns7b, orf8, orf9b, orf10, orf14) as seed. the nodes in red are human proteins s-figure 4. interactomes based on human ppi and sars-cov-2-host interactions, with top 50 closest proteins identified by rwr, using accessory proteins key: cord-354398-f3cg8gi1 authors: al-saud, haya; al-romaih, khaldoun; bakheet, razan; mahmoud, lina; al-harbi, najla; alshareef, ibtihaj; judia, sara bin; aharbi, layla; alzayed, abdulaziz; jabaan, amjad; alhadrami, hani; albarrag, ahmed; azhar, essam i.; al-mozaini, maha ahmad title: automated sars-cov-2 rna extraction from patient nasopharyngeal samples using a modified dna extraction kit for high throughput testing date: 2020-09-20 journal: ann saudi med doi: 10.5144/0256-4947.2020.373 sha: doc_id: 354398 cord_uid: f3cg8gi1 background: the pandemic of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has prompted a need for mass testing to identify patients with viral infection. the high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral rna and perform reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr) to detect sars-cov-2. objectives: report on the use of a dna extraction kit, after modifications, to extract viral rna that could then be detected using an fda-approved sars-cov-2 rt-qpcr assay. materials and methods: initially, automated rna extraction was performed using a modified dna kit on samples from control subjects, a bacteriophage, and an rna virus. we then verified the automated extraction using the modified kit to detect in-lab propagated sarscov-2 titrations using an fda approved commercial kit (s, n, and orf1b genes) and an in-house primer-probe based assay (e, rdrp2 and rdrp4 genes). results: automated rna extraction on serial dilutions sars-cov-2 achieved successful one-step rt-qpcr detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. moreover, rt-qpcr detection was successful after automated rna extraction using this modified protocol on 12 patient samples of sars-cov-2 collected by nasopharyngeal swabs and stored in viral transport media. conclusions: we demonstrated the capacity of a modified dna extraction kit for automated viral rna extraction and detection using a platform that is suitable for mass testing. limitations: small patient sample size. conflict of interest: none. background: the pandemic of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has prompted a need for mass testing to identify patients with viral infection. the high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral rna and perform reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr) to detect sars-cov-2. objectives: report on the use of a dna extraction kit, after modifications, to extract viral rna that could then be detected using an fda-approved sars-cov-2 rt-qpcr assay. materials and methods: initially, automated rna extraction was performed using a modified dna kit on samples from control subjects, a bacteriophage, and an rna virus. we then verified the automated extraction using the modified kit to detect in-lab propagated sars-cov-2 titrations using an fda approved commercial kit (s, n, and orf1b genes) and an in-house primer-probe based assay (e, rdrp2 and rdrp4 genes). results: automated rna extraction on serial dilutions sars-cov-2 achieved successful one-step rt-qpcr detection down to 60 copies using the commercial kit assay and less than 30 copies using the inhouse primer-probe assay. moreover, rt-qpcr detection was successful after automated rna extraction using this modified protocol on 12 patient samples of sars-cov-2 collected by nasopharyngeal swabs and stored in viral transport media. conclusions: we demonstrated the capacity of a modified dna extraction kit for automated viral rna extraction and detection using a platform that is suitable for mass testing. t he human community continually faces the threat of emerging pathogens, some of which spread and infect larger populations and can cause pandemics. in the past two decades, we have faced the spread of viral infections caused by new viruses belonging to the coronaviridae family of viruses, commonly known as coronaviruses. coronaviruses are rna viruses that infect birds, mammals, and humans and affect different bodily systems, including the respiratory system. at least six coronaviruses are known to infect humans and cause diseases of variable severities. included among these six coronaviruses are the severe acute respiratory syndrome coronavirus (sars-cov; 2002-2003 outbreak) and middle east respiratory syndrome coronavirus (mers-cov; 2012 outbreak), 1 and the most recently discovered and cause of the ongoing pandemic, severe acute respiratory syndrome coronavirus 2 (sars-cov-2; 2019 outbreak). 2 the spread of sars-cov-2 has prompted the need for widespread rapid diagnostic testing. the method used to test patients for sars-cov-2 is based on reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr), which is performed after rna extraction. 3, 4 the centers for disease control and prevention (cdc) and the world health organization (who) have developed sars-cov-2 assays. the assays are based on pcr primer-probe sets for different genes. the cdc and who assays differ in their targeted genes and both assays are primer-probe based and have high sensitivity and specificity for sars-cov-2. both assays also have minimal cross reactivity with other circulating strains of coronaviruses, and indicate sars-cov-2 positivity with a quantification cycle (c q ) of less than 40 for sars-cov-2 positivity in the pcr test. 5 there are a number of kits that employ these approaches (such as the cobas sars-cov-2 test by roche molecular systems, the realstar sars-cov-2 rt-pcr kit ruo by altona-diagnostic, and the high-throughput automated sample preparation system nucleic acid extraction kit genetic sequencer (dnbseq-g50, dnbseq-g400 fast) by wuhan mgi tech co, ltd, wuhan, china), but the high demand in the last few months has resulted in a shortage and prompted the use of alternative resources. to counter this shortage, we opted for the use of a bead-based dna extraction kit after modifications to the buffers used in the extraction of viral rna followed by a pcr assay based on primers and probes recommended by the who for sars-cov-2 testing. the dna extraction kit we used is manufactured by invitrogen and intended for isolation of genomic dna from forensic specimens, namely the invitrogen forensic dna extraction kit. the modifica-tions of this kit were successful in extracting rna when performed automatically using a robotic system suitable for mass extraction, which provides an alternative to commercial viral rna extraction kits and provides a protocol to increase preparedness for future crises. the detection of the extracted rna was cross validated by kit-based assays and our in-house primer-probe sets, which are based on cdc/who recommendations. our approach provides an alternative to counter the supply chain shortage in sars-cov-2 diagnostic testing for emergency use. the intended use of the chargeswitch forensic dna purification kit (invitrogen life technologies, catalog number cs11200, https://www.thermofisher.com/) is to isolate genomic dna from different types of forensic samples, followed by short tandem repeat analysis. initially we used the kit according to the manufacturer's instructions without modifications to extract rna, but with no success. subsequently, the kit was used according to the manufacturer's instructions with the following modifications to successfully extract human rna as well as virus rna (including sars-cov-2 rna) by the following steps: deactivation step (intended to deactivate sars-cov-2 viruses in the sample) for 30 minutes at room temperature for 500 µl of each sample (nasopharyngeal swabs in transport medium) with 800 µl l13 buffer (cell lysis buffer) adjusted to ph 7, 100% ethanol (300 µl, virus deactivation agent), dithiothreitol (dtt) at a final concentration of 2.5 mm (to preserve rna integrity), β-mercaptoethanol (10 µl/ml, also to preserve rna integrity) and proteinase k (for complete lysis of cells and virus envelopes) at a final concentration of 20 µg/ml. the total volume was 1.3 ml/each sample preparation in deep well plates (prepfiler 96-well processing plates, catalog number 4392904). after the viral inactivation step the plate was loaded on the hamilton automation system for the automated rna extraction. the lysis was performed for 30 minutes at 55°c with agitation at 400 rpm speed followed by cooling for 2 minutes and addition of 200 µl purification buffer and 20 µl of chargeswitch beads per sample and binding with agitation at 400 rpm at room temperature for 10 minutes. after the binding step, centrifugation at 3000 rpm is performed for 2 minutes to ensure complete settling of the bound beads to the bottom of the deep wells followed by 5 minutes on a magnetic plate, and then the supernatant was discarded followed by two washes with w12 buffer (wash buffer) for 5 minutes each and elution with 50 µl of the kit elution buffer and transfer of elution buffer containing rna to a sample plate for subsequent rt-qpcr analysis. vero ccl-81 cells infected with heat-inactivated sars-cov-2 isolates were propagated and then diluted in a biosafety level 3 facility (unpublished data). 10 µl of extracted rna (from the final eluted material at the end of the automated viral rna extraction) was used as input material for the multiplex single-step pcr for three genes using the thermofisher scientific taqpath one-step qrt-qpcr kit (taqpath covid-19 rt-pcr kit, applied biosystems: a48102) as per manufacturer protocol (annealing at 58°c, taqpath one-step rt-qpcr system on the applied biosystems 7500 real time cycler for 25 µl per reaction). the in-house primerprobe assay was run using the conditions and primers indicated in tables 1, 2 and 3. 6 to validate the capacity and sensitivity of the modified invitrogen chargeswitch forensic dna kit (invitrogen, catalogue number cs11200) in extracting total rna from nasopharyngeal swabs, we conducted a series of automated extractions of total rna experiments from healthy donor nasopharyngeal swabs submerged in transport medium and spiked with ms2 (bacteriophage provided in the applied biosystems assay (applied biosystems, taqpath one-step qrt-qpcr, catalogue number a48102) and an rna virus (encephalomyocarditis virus, emcv). a standard two-step rt-qpcr was used to detect human reference genes (rrna) and emcv rna, and single-step rt-qpcr was used to detect bacteriophage ms2 rna after the automated rna extraction (spiking experiments). a stepwise dilution of ms2 (from 10 µl to 0.625 µl) and emcv (10 500 to 325 pfu/µl) were added to the lysis mix and processed using the modified kit on the fully automated extraction. purified rna samples were then screened by rt-qpcr using primers specific for human rrna (forward: atggccgttcttagttggtg, reverse: cgctgagccagtcagtgtag), ms2 (applied biosystems, taqpath one-step qrt-qpcr, catalogue number a48102) and emcv (fwd: aga ggc cgg gta taa ggt tt, rev: tgc aat tcc tca agc tgt tc) . as shown in figure 1 and tables 4a-4c the automated extraction using the modified kit isolated sufficient rna for detection by rt-qpcr to the lowest dilution of ms2 and 325 pfu/µl for emcv. the quantification cycles at which each rna product was detected were from c q of 17.06 to c t of 20.6 for human rrna, c q of 29.7 to 32.3 for ms2 rna, and c q of 24.09 to 27.67 for emcv rna. thus, these results indicate that the modified forensic dna kit is effective for isolation of detectable single-stranded rna from viruses. we validated the modified invitrogen forensic dna purification kit in extracting in-laboratory propagated sars-cov-2 rna by conducting manual and automated extractions on titrations from 15 000 copies to 60 copies of sars-cov-2 followed by rt-qpcr methods: the commercially available taqpath one-step qrt-qp-cr kit (using the n, s, and orf1b genes) and primers and probes from metabion, germany to establish an inhouse rt-qpcr assay based on e, rdrp2 and rdrp4 gene detection as per recommended sars-cov-2 testing from cdc and who. the results for the two assays are shown in figure 2 and indicate detection of sars-cov-2 rna to as few as 60 copies using the commercial kit and 46 copies using the in-house assay. the range of c q values for sars-cov-2 detection were n gene: c q 23.1 (15 000 copies) to c q 29.6 (60 copies), s gene: c q 29.6 (15 000 copies) to c q 36 (60 copies), and orf1b gene: c q 22.3 (15 000 copies) to c q 29.4 (60 copies), for taqpath kit and e gene: c q 31.0 (3000 copies) to c q 37.3 (11 copies), rdrp2 gene: c q 29.3 (3000 copies) to c q 35.3 (11 copies), and rdrp4 gene: c q 30.0 (3000 copies) to c q 38.2 (11 copies), for the in house rt-qpcr assay ( table 5) . we then validated the capacity of the modified invitrogen forensic dna purification kit in extracting sars-cov-2 rna by conducting manual and automated extractions on sars-cov-2 nasopharyngeal swabs submerged in transport medium from patient samples (left over from diagnostic materials and shared by our collaborators in the cdc). a standard single-step rt-qpcr was used to detect sars-cov-2 rna after the rna extraction utilizing single-step rt-qpcr assays (applied biosystems brand, thermofisher scientific). a number of nine samples were processed using the fully automated extraction. purified rna samples were then screened by rt-qpcr using the assay primers-probe sets for 3 genes (orf 1b, s, and n genes). figure 3 and table 6 show the amplification curve and quantification cycle values using the applied biosystems assay. the automated extraction using the modified kit isolated sufficient rna for detection by rt-qpcr. the quantification cycle at which each rna product was detected ranged from c q of 15.9 to c q of 34.8 for the applied biosystems assay. these results indicated that the automated extraction of sars-cov-2 rna using the modified forensic dna kit is effective for isolation of detectable sars-cov-2 rna from patient samples. on the 11th of march, 2020, the world health organization declared the sars-cov-2 virus as a pandemic threat with global reports of more than 2 878 196 confirmed (85 530 deaths) (https:// www.who.int/docs/default-source/coronaviruse/ situation-reports/20200427-sitrep-98-covid-19. pdf?sfvrsn=90323472_4). there are multiple reasons for this high rate of virus transmission, for instance; most of the cases were asymptomatic and considered carriers 7 and responses to the epidemic were delayed. 8 accordingly, it is crucial to screen everyone to confirm and clear positive suspected cases, and this approach will be the key factor to counter global outbreak situations. hence, there has been an unprecedented global effort to increase the mass testing capacity for sars-cov-2 for the sake of both clinical practice and pubfigure 1 . amplification curves produced by rt-qpcr for healthy donor samples prepared from nasopharyngeal swabs submerged in transport medium and spiked with ms2 and ecmv with step-down titrations. the y axis is δrn (the normalized reporter value, also called "rn value", of an experimental reaction minus the "rn value" of the baseline signal generated by the instrument) and the x axis are reference cycles. rrna is the human housekeeping gene, ms2 is a positive-sense single-stranded rna bacteriophage and emcv is the encephalomyocarditis virus, a small nonenveloped single-stranded rna virus. lic health. this worked successfully in two countries: in germany and south korea the case-fatality rates dropped to less than 0.5% probably because of the mass testing that they implemented. 9 in addition, the mass testing in china played an important role in identifying sars-cov-2. 10 yet, very few countries have used the mass testing approach, the most important reason probably being the limited resources for testing. although sars-cov-2 kits are available commercially, the huge world-wide demand over the last few months created a shortage and delay in obtaining them, particularly in the large amounts needed for mass testing. thus, we aimed to use our current available resources, reagents and facility to overcome this shortage. we have adapted the reverse transcription real-time polymerase chain reaction rt-qpcr-based assays, which are the gold standard for sars-cov-2 diagnostics for upper and lower respiratory specimens. this assay utilizes oligonucleotide primers and duallabeled hydrolysis probes (taqman) to increase sensitivity levels. the adapted set of primers and probes are among the lists that have been evaluated and recommended by cdc/who for any in-house assay development for sars-cov-2 testing. the assay also requires an additional step, the use of an rna extraction kit for viral sars-cov-2 extraction prior to the rt-qpcr assay. despite this nucleic acid test several studies have reported false-negative results, 2,11,12 with a reported sensitivity of almost 80%. in order to start our mass testing platform, we first addressed the shortage of rna extraction kits, which is a bottleneck in the testing capacity, at a global level. we opted for the use of a bead-based dna extraction kit after modifications to the buffers used in the extraction, to extract viral rna. the dna extraction kit we used is produced by invitrogen and intended for isolation of genomic dna from forensic specimens. this strategy requires investigating whether these kits after modifications are indeed sufficient in extracting rna in general and viral rna in particular. the invitrogen chargeswitch forensic dna kit was modified to perform automated extraction of rna from nasopharyngeal swab samples, an approach that is suitable for mass extraction. the first step in extracting dna or rna is the cell lysis step in order to release dna and rna. we modified the lysis step by adding agents that protect rna integrity (from rnases) and to lyse virus envelopes more efficiently. we also added ethanol for the purpose of deactivating the highly infectious sars-cov-2. the ultimate method to validate a successful extraction of viral rna is detecting it by rt-qpcr as this is the final output. in our study we successfully valifigure 2 . amplification curves produced by rt-qpcr for healthy donor samples prepared from nasopharyngeal swabs submerged in transport medium and spiked with heat inactivated lab-propagated sars2-cov-2 viral particles. the s, n, and orf1b gene amplification curves were produced after automated rna extraction of decreasing titrations of sars-cov-2 (from 15000 copies/test to ~60 copies/test followed by one-step rt-qpcr using the taqpath assay by applied biosystems. the e, rdrp2, and rdrp4 gene amplification curves were produced after automated rna extraction of decreasing titrations of sars-cov-2 (from 3000 copies/test to 8 copies/test followed by onestep rt-qpcr using the in-house assay. the y axis is δrn and x axis are quantification cycles. figure 3 . amplification curves produced by rt-qpcr for sars-cov-2 samples obtained after diagnostic tests were performed (using leftover specimens), and prepared from nasopharyngeal swabs submerged in transport medium. the s, n, and orf1b gene amplification curves were produced after automated rna extraction sars-cov-2 by one-step rt-qpcr using the taqpath assay by applied biosystems. the samples were spiked by ms2, the internal control for rna extraction. the y axis is δrn and x axis are quantification cycles. dated the detectability of extracted viral rna using an fda-approved assay as well as an in-house developed assay. our initial experiments in which we validated the rna extraction using human reference gene showed efficient detection of rna at low quantification cycle values (high nucleic acid/rna extraction efficiency). moreover, automated extraction using the modified kit on ms2 (bacteriophage provided in the applied biosystems kit for rna extraction control) and ecmv (rna virus) also showed efficient detection of rna at low quantification cycle values (high nucleic acid/rna extraction efficiency). the sensitivity of the detection of ms2 was achieved at as low a titration as 0.625 µl and 325 pfu/µl for ecmv. we compared the sensitivity and specificity of the modified kit using the commercially available taqpath one-step qrt-qpcr kit (uses n, s, and orf1b genes) and an in-house primer-probe based assay (e, rdrp2 and rdrp4 genes) in which the table 6 . rt-qpcr for 9 sars-cov-2 patients after automated rna extraction followed by thermofisher pcr assay. taqpath method detected down to 60 copies and the in-house assay down to 46 copies of sars-cov-2 rna. this prompted us to test the automated extraction on nine sars-cov-2 samples that we obtained after clinical testing was conducted at the centers for disease control and prevention in riyadh, saudi arabia. the rna extracted from these samples was validated by running the pcr using the fda-approved assay from thermofisher scientific taqpath one-step qrt-qpcr (applied biosystems, a48102), in which sars-cov-2 rna was detected in 8 out of the 9 samples. these results indicated the capacity of detecting sars-cov-2 rna after automated extraction with the modified dna extraction kit. the analytical sensitivity and specificity of sars-cov-2 rt-qpcr primer-probe sets varies across different assays. at viral load of 500 copies and higher, the primer-probe sets have comparable sensitivities with quantification cycle values ranging between 30 and 40 cycles, however at a lower copy number some probes have lower sensitivity than others. 6 other factors may also affect detection of sars-cov-2 in samples including sample collection and transport; hence most kit based assays include two or three genes for sars-cov-2 testing. who, for instance, recommends a cocktail of e and rdrp2 and rdrp4 genes for sars-cov-2. two recent studies have evaluated the different sets of primers and probes recommended by cdc/who. one reported that the rdrp and e are among the best to be tested with a high specificity for sars-cov-2, no cross-reactivity with other respiratory viruses with a limit of detection of about 790 viral copies, 6 while another report indicates that the lower sensitivities failed to reach 500 copies. 13 in this study we tested the limit of detection of sars-cov-2 after automated rna extraction by two assays, in which the commercially available assay from applied biosystems had a sars-cov-2 limit of detection of ~60 copies and the in-house assay had a sars-cov-2 limit of detection of 46 copies. however, these different reports on viral load measurement may indicate that no standardized process exists yet. in addition, there is no established threshold for interpretation of viral loads, which may vary in different labs, by assay used and by different targeted genes. in addition, the detection rates in each sample type may vary from patient to patient and may change over the course of individual patients' illnesses. 13 in conclusion, due to the urgent need for high volume sars-cov-2 screening, we report the use of the modified dna extraction kit on an automated platform for sars-cov-2 rna extraction with a cost of 3usd per sample. our findings suggest that use of this protocol will be satisfactory for emergency use after further validation on patient samples. epidemiology, genetic recombination, and pathogenesis of coronaviruses a novel coronavirus from patients with pneumonia in china molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr diagnostic testing for severe acute respiratory syndrome-related coronavirus-2: a narrative review analytical sensitivity and efficiency comparisons of sars-cov-2 qrt-qpcr primer-probe sets presumed asymptomatic carrier transmission of covid-19 impact of international travel and border control measures on the global spread of the novel 2019 coronavirus outbreak an interactive web-based dashboard to track covid-19 in real time positive rate of rt-pcr detection of sars-cov-2 infection in 4880 cases from one hospital in comparative performance of sars-cov-2 detection assays using seven different primer/probe sets and one assay kit report from the american society for microbiology cov-id-19 international summit the study is approved by the institutional research committee and the de-identified samples were left over after completion of diagnostic tests; hence this study requires no consenting as per institutional ethics committee regulations. all authors have given final approval of this manuscript to be published. in addition they have agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. key: cord-343517-vf32wxkx authors: lokman, syed mohammad; rasheduzzaman, md.; salauddin, asma; barua, rocktim; tanzina, afsana yeasmin; rumi, meheadi hasan; hossain, md. imran; siddiki, amam zonaed; mannan, adnan; hasan, md. mahbub title: exploring the genomic and proteomic variations of sars-cov-2 spike glycoprotein: a computational biology approach date: 2020-04-11 journal: biorxiv doi: 10.1101/2020.04.07.030924 sha: doc_id: 343517 cord_uid: vf32wxkx the newly identified sars-cov-2 has now been reported from around 183 countries with more than a million confirmed human cases including more than 68000 deaths. the genomes of sars-cov-2 strains isolated from different parts of the world are now available and the unique features of constituent genes and proteins have gotten substantial attention recently. spike glycoprotein is widely considered as a possible target to be explored because of its role during the entry of coronaviruses into host cells. we analyzed 320 whole-genome sequences and 320 spike protein sequences of sars-cov-2 using multiple sequence alignment tools. in this study, 483 unique variations have been identified among the genomes including 25 non-synonymous mutations and one deletion in the spike protein of sars-cov-2. among the 26 variations detected, 12 variations were located at the n-terminal domain and 6 variations at the receptor-binding domain (rbd) which might alter the interaction with receptor molecules. in addition, 22 amino acid insertions were identified in the spike protein of sars-cov-2 in comparison with that of sars-cov. phylogenetic analyses of spike protein revealed that bat coronavirus have a close evolutionary relationship with circulating sars-cov-2. the genetic variation analysis data presented in this study can help a better understanding of sars-cov-2 pathogenesis. based on our findings, potential inhibitors can be designed and tested targeting these proposed sites of variation. wuhan, hubei province of china in december 2019. the death toll rose to more than 68,000 among 1,250,000 confirmed cases around the globe (until april 4, 2020) [1] . the virus causing covid-19 is named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). based on the phylogenetic studies, the sars-cov-2 is categorized as a member of the genus betacoronavirus, the same lineage that includes sars coronavirus (sars-cov) [2] that caused sars (severe acute respiratory syndrome) in china during 2002 [3] . recent studies showed that sars-cov-2 has a close relationship with bat sars-like covs [4, 5] [7] ]. interestingly, s glycoprotein is characterized as the critical determinant for viral entry into host cells which consists of two functional subunits namely s1 and s2. the s1 subunit recognizes and binds to the host receptor through the receptor-binding domain (rbd) whereas s2 is responsible for fusion with the host cell membrane [ [8] , [9] , [10] ]. mers-cov uses dipeptidyl peptidase-4 (dpp4) as entry receptor [11] whereas sars-cov and sars-cov-2 utilize ace-2 (angiotensin converting enzyme-2) [12] , abundantly available in lung alveolar epithelial cells and enterocytes, suggesting s glycoprotein as a potential drug target to halt the entry of sars-cov-2 [13] . according to recent reports, neutralizing antibodies are generated in response to the entry and fusion of surface-exposed s protein (mainly rbd domain) which is predicted to be an 4 important target for vaccine candidates [ [10] , [14] , [15] ]. however, sars-cov-2 has emerged with remarkable properties like glutamine-rich 42 aa long exclusive molecular signature (dsqqtvgqqdgsednqtttiqtivevqpqlemeltpvvqtie) in position 983-1024 of polyprotein 1ab (pp1ab) [16] , diversified receptor-binding domain (rbd), unique furin cleavage site (prrar↓sv) at s1/s2 boundary in s glycoprotein which could play roles in viral pathogenesis, diagnosis and treatment [17] . to date, few genomic variations of sars-cov-2 are reported [ [18] , [19] ]. there is growing evidence that spike protein, a 1273 amino acid long glycoprotein having multiple domains, possibly plays a major role in sars-cov-2 pathogenesis. viral entry to the host cell is initiated by the receptor-binding domain (rbd) of s1 head. upon receptor-binding, proteolytic cleavage occurs at s1/s2 cleavage site and two heptad repeats (hr) of s2 stalk form a six-helix bundle structure triggering the release of the fusion peptide. as it comes into close proximity to the transmembrane anchor (tm), the tm domain facilitates membrane destabilization required for fusion between virus-host membranes [ [20] , [21] ]. insights into the sequence variations of s glycoprotein among available genomes are key to understanding the biology of sars-cov-2 infection, developing antiviral treatments and vaccines. in this study, we have analyzed 320 genomic sequences of sars-cov-2 to identify mutations between the available genomes followed by the amino acid variations in the glycoprotein s to foresee their impact on the viral entry to host cell from structural biology viewpoint. all available sequences (320 whole genome and surface glycoprotein sequences of sars-cov-2) related to the covid-19 pandemic were retrieved from ncbi virus variation resource repository (https://www.ncbi.nlm.nih.gov/labs/virus/) [22] . in addition, all 40 s glycoprotein sequences from different coronavirus families were retrieved for phylogenetic 5 analysis. the ncbi reference sequence of sars-cov-2 s glycoprotein, accession number yp_009724390 was used as the canonical sequence for the analyses of spike protein variants. variant analyses of sars-cov-2 genomes were performed in the genome detective coronavirus typing tool version 1.13 which is specially designed for this virus (https://www.genomedetective.com/app/typingtool/cov/) [23] . for multiple sequence alignment (msa), genome detective coronavirus typing tool uses a reference dataset of 431 whole genome sequences (wgs) where 386 wgs were from known nine coronavirus species. the dataset was then aligned with muscle [24] . entropy (h(x)) plot of nucleotide variations in sars-cov-2 genome was constructed using bioedit [25] . mega x (version 10.1.7) was used to construct the msas and the phylogenetic tree using pairwise alignment and neighborjoining methods in clustalw [26, 27] . tree structure was validated by running the analysis on 1000 bootstraps [28] replications dataset and the evolutionary distances were calculated using the poisson correction method [29] . variant sequences of sars-cov-2 were modeled in swiss-model [30] using the cryo-em spike protein structure of sars-cov-2 (pdb id 6vsb) as a template. the overall quality of models was assessed in rampage server [31] by generating ramachandran plots (supplementary table 1 ). pymol and biovia discovery studio were used for structure visualization and superpose [32, 33] . 6 multiple sequence alignment of the available 320 genomes of sars-cov-2 were performed and 483 variations were found throughout the 29,903 bp long sars-cov-2 genome with in total 115 variations in utr region, 130 synonymous variations that cause no amino acid alteration, 228 non-synonymous variations causing change in amino acid residue, 16 indels, and 2 variations in non-coding region (supplementary table 2 ). among the 483 variations, 40 variations (14 synonymous, 25 non-synonymous mutations and one deletion) were observed in the region of orf s that encodes s glycoprotein which is responsible for viral fusion and entry into the host cell [34] . notable that, most of the sars-cov-2 genome sequences were deposited from the usa (250) and china (50) (supplementary fig. 1 ). positional variability of the sars-cov-2 genome was calculated from the msa of 320 sars-cov-2 whole genomes as a measure of entropy value (h(x)) [35] . excluding 5′ and 3′ utr, ten hotspot of hypervariable position were identified, of which seven were located at orf1ab (1059c>t, 3037c>t, 8782c>t, 14408c>t, 17747c>t, 17858a>g, 18060c>t) and one at orf s (23403a>g), orf3a (25563g>t), and orf8 (28144t>c) respectively. the variability at position 8782 and 28144 were found to be the highest among the other hotspots ( fig. 1 ). the phylogenetic analysis of a total of 66 sequences (26 unique sars-cov-2 and 40 different coronavirus s glycoprotein sequences) was performed. the evolutionary distances showed that all the sars-cov-2 spike proteins cluster in the same node of the phylogenetic tree confirming the sequences are similar to refseq yp_009724390 (fig. 2) . bat coronaviruses has a close evolutionary relationship as different strains were found in the nearest outgroups and clades (bat coronavirus bm48-31, bat hp-beta coronavirus, bat coronavirus hku9) conferring that 7 coronavirus has vast geographical spread and bat is the most prevalent host (fig. 2) . in other clades, the clusters were speculated through different hosts which may describe the evolutionary changes of surface glycoprotein due to cross species transmission. viral hosts reported from different spots at different times is indicative of possible recombination. the s glycoprotein sequences of sars-cov-2 were retrieved from the ncbi virus fig. 2 ). 8 alterations of amino acid residual charge from positive to neutral (h49y, r408i, h519q), negative to neutral (d111n, d614g, d936y), negative to positive (d1168h, d1259h), and neutral to positive (n74k, s247r) were seen in variants qhw06059, qhs34546, qis61422, qis61338, qik50427, qis30615, qis60978, qis60582, qio04367, and qhr84449 respectively due to substitution of amino acid that differ in charge. the remaining 15 variants were mutated with the amino acids that are similar in charge (fig. 4 a) . the sars-cov-2 spike protein variants were superposed with the cryo-electron microscopic structure of sars-cov-2 spike protein [8] . l5f, n74k, e96d, f157l, g181v, s247r, g476s, v483a, d1168h, and d1259h variants were excluded from superposition due to absence of respective residues in the 3d structure of template (pdb: 6vsb). the superposition showed that most of the residual change were causing incorporation of bulky amino acid residues (t29i, h49y, l54f, s221w, a348t, h519q, a520s, a930v, d936y, and a1078v) in place of smaller size residue except y28n, d111n, r408i, d614g, and f797c (fig. 4 b-p) . fig. 3) . the s2 subunit of spike protein, especially the heptad repeat region 2, fusion peptide domain, transmembrane domain, and cytoplasmic tail were found to be highly conserved in the sars-cov and the sars-cov-2 variants while the s1 subunit was more diverse, specifically the n-terminal domain (ntd) and receptor-binding domain (rbd). covid 19 is one of the most contagious pandemics the world has ever had with 1,250,000 confirmed cases to date (april 4, 2020) and the cases have increased as high as 5 times in less than a month [1] . phylogenetic analysis showed that the sars-cov-2 is a unique coronavirus presumably related to bat coronavirus (bm48-31, hp-betacoronavirus). during this study, we [15] , [38] , [39] , [40] ]. likewise, a number of studies targeting sars-cov-2 spike protein have been undertaken for the therapeutic measures [41] , but the unique structural and functional details of sars-cov-2 spike protein are still under scrutiny. we also found a variant (r408i) at receptor binding domain (rbd) that mutated from positively charged arginine residue to neutral and smaller sized isoleucine residue (fig. 4 i) . this change might alter the interaction of viral rbd with the host receptor because the r408 residue of sars-cov-2 is known to interact with the ace2 receptor for viral entry [42] . similarly, alterations of rbd (g476s, v483a, h519q, and a520s) also could affect the interaction of sars-cov-2 spike protein with other molecules which require further investigations. qia98583 and qis30615 variants were found to have an alteration of alanine to valine (a930v), and aspartic acid to tyrosine (d936y) respectively in the alpha helix of the hr1 domain. previous reports have indicated that hr1 domain plays a significant role in viral fusion and entry by forming helical bundles with hr2, and mutations including alanine substitution by valine (a1168v) in hr1 region are predominantly responsible for conferring resistance to mouse hepatitis coronaviruses against hr2 derived peptide entry inhibitors [43] . this study hypothesizes the mutation (a930v) found in that of sars-cov-2 might also have a role in the emergence of drug-resistance virus strains. also, the mutation (d1168h) found in the heptad repeat 2 (hr) sars-cov-2 could play a vital role in viral pathogenesis. the sars-cov-2 s protein contains additional furin protease cleavage site, prrars, in s1/s2 domain which is conserved among all 320 sequences as revealed during this study ( supplementary fig. 3 ). this unique signature is thought to make the sars-cov-2 more virulent than sars-cov and regarded as novel features of the viral pathogenesis (ref 11). according to previous reports the more the host cell protease can process the coronavirus s can accelerate viral tropism accordingly in influenza virus [[9] , [44] , [45] , [46] ]. apart from that, this could also promote viruses to escape antiviral therapies targeting transmembrane protease tmprss2 (clinicaltrials.gov, nct04321096) which is well reported protease to cleave at s1/s2 of s glycoprotein [47] . comparative analyses between sars-cov and sars-cov-2 spike glycoprotein showed 77% similarity between them where the most diverse region was . coronavirus disease (covid-2019) situation reports severe acute respiratory syndrome-related coronavirus--the species and its viruses, a statement of the coronavirus study group lim, others, a novel coronavirus associated with severe acute respiratory syndrome bats are natural reservoirs of sars-like coronaviruses, science (80-. ) huang, others, a pneumonia outbreak associated with a new coronavirus of probable bat origin pei, others, a new coronavirus associated with human respiratory disease in china genome composition and divergence of the novel coronavirus (2019-ncov) originating in china cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structure analysis of the receptor binding of 2019-ncov fouchier, others, dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc greenough, others, angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses a. nitsche, others, sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor wu, others, potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody an exclusive 42 amino acid signature in pp1ab protein provides 13 insights into the evolutive history of the 2019 novel human-pathogenic coronavirus (sars-cov2) the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade genomic variance of the 2019-ncov coronavirus genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors virus variation resource--improved response to emergent viral outbreaks genome detective coronavirus typing tool for rapid identification and characterization of novel coronavirus genomes muscle: multiple sequence alignment with improved accuracy and 14 proceedings. 2004 ieee comput. syst. bioinforma. conf. 2004. csb bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt mega x: molecular evolutionary genetics analysis across computing platforms the neighbor-joining method: a new method for reconstructing phylogenetic trees bootstrap confidence levels for phylogenetic trees evolutionary divergence and convergence in proteins swiss-model: homology modelling of protein structures and complexes structure validation by calpha geometry: phi, psi and cbeta deviation pymol: an open-source molecular graphics tool receptor recognition mechanisms of coronaviruses: a decade of structural studies a parvovirus b19 synthetic genome: sequence features and functional competence cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein role of changes in sars-cov-2 spike protein in the interaction with the human ace2 receptor: an in silico analysis coronavirus escape from heptad repeat 2 (hr2)-derived peptide entry inhibition as a result of mutations in the hr1 domain of the spike fusion protein host cell proteases controlling virus pathogenicity role of hemagglutinin cleavage for the pathogenicity of influenza virus host cell proteases: critical determinants of coronavirus tropism and pathogenesis coronaviruses: an overview of their replication and pathogenesis receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins laude, others, aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev key: cord-338205-sy91rnse authors: li, chenxi; zhao, chengxue; bao, jingfeng; tang, bo; wang, yunfeng; gu, bing title: laboratory diagnosis of coronavirus disease-2019 (covid-19) date: 2020-07-02 journal: clin chim acta doi: 10.1016/j.cca.2020.06.045 sha: doc_id: 338205 cord_uid: sy91rnse abstract the outbreak of coronavirus disease-2019 (covid-19) caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has threatened health worldwide. as of the end of 2020, there were nearly 10 million confirmed cases and nearly 5 million deaths associated with covid-19. rapid and early laboratory diagnosis of covid-19 is the main focus of treatment and control. molecular tests are the basis for confirmation of covid-19, but serological tests for sars-cov-2 are widely available and play an increasingly important role in understanding the epidemiology of the virus and in identifying populations at higher risk for infection. point-of-care tests have the advantage of rapid, accurate, portable, low cost and non-specific device requirements, which provide great help for disease diagnosis and detection. this review will discuss the performance of different laboratory diagnostic tests and platforms, as well as suitable clinical samples for testing, and related biosafety protection. this review shall guide for the diagnosis of covid-19 caused by sars-cov-2. a novel virus caused an outbreak of pneumonia began from wuhan, hubei province, china. the virus involved in the event was identified as the seventh coronavirus, becoming the third zoonotic human coronaviruses (hcov) of the century, and posing serious threats to international health. the international committee on taxonomy of viruses (ictv) has announced that the novel coronavirus is officially classified as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the world health organization (who) announced that the official name of the disease caused by the virus is coronavirus disease 2019 (covid19) . coronaviruses (covs) are enveloped viruses with a single-stranded, positive-sense rna genome, which is the largest discovered genome of rna virus [1] . before the appearance of sars-cov-2, 6 hcovs have been discovered by researchers, including hcov-229e, hcov-hku1, hcov-nl63, hcov-oc43, severe acute respiratory syndrome (sars) cov and middle east respiratory syndrome (mers) cov [2] . there are currently 7 covs (including sars-cov-2) that can cause human respiratory diseases, but to date, only sars-cov, mers-cov, and sars-cov-2 have caused a large outbreak with high mortality. the ongoing continuation and spread of the covid-19 pose challenges for public health control. on 30 covzc45 and bat-sl-covzxc21) and 79% identity to sars-cov, but only 50% identity to mers-cov [13] . the genome of sars-cov-2 was closely related to that of bat cov ratg13, showing 96.2% overall genomic sequence identity [8] , indicating that human sars-cov-2 and bat cov may share the same ancestor. it was reported covs identified in pangolins with 90% sequence identity to sars-cov-2 by protein sequence alignments and phylogenetic analysis [14, 15] , suggesting pangolins are the most likely intermediate hosts for sars-cov-2. however, the outcome of phylogeny analyses does not necessarily support the view that pangolin is the exact intermediate host of sars-cov-2, and other animals may also serve as intermediate hosts [16] . a recent study reported that similar ace2 receptor residues were found in some species other than pangolin, such as turtles and snakes, which supplies more possibilities for alternative intermediate hosts [17] . in sum, host ranges and animal reservoirs of sars-cov-2 still need to be explored. sars-cov-2 is mainly transmitted through contact, respiratory droplets and the potential route of fecaloral. the estimated reproductive number (r0) of sars-cov-2 ranges from 2.2 to 5.7 [18] [19] [20] , while the reported r0 of sars-cov is around 3 [21] . it is speculated that the primary virus replication occurs in the mucosal epithelium of the upper respiratory tract (pharynx and nasal cavity), and further multiplies in the mucosa of the lower respiratory tract and gastrointestinal tract, causes mild viremia [22] . a study basedhospital survey found that the maximum propagation distance of aerosols containing sars-cov-2 virions might be 4 meters from the patients with covid-19 [23] . neeltje et al. revealed that sars-cov-2 aerosols remained infectious in the tissue culture experiments, and the infectivity only decreased slightly during a 3hour observation period [24] . several recent studies have reported that sars-cov-2 was detected in stool samples [25] [26] [27] . although these evidence indicate that sars-cov-2 may also be an enterovirus that can be transmitted through the fecal-oral route, these discoveries are based on the situation of very few patients and more researches are warranted. similar to other infectious diseases, appropriate specimen collection is the key step in the laboratory diagnosis of covid-19. acceptable specimens include upper respiratory tract specimens, lower respiratory tract specimens, stool specimens, whole blood specimens, and serum specimens, and the respiratory secretions is the most frequently sample for diagnosis [28] . currently, sars-cov-2 has been detected in nasopharyngeal swabs [29, 30] , oropharyngeal swabs [25, 30] , throat swabs [29, 31] , sputum [29, 31, 32] , bronchoalveolar lavage fluid (balf) [10, 11, 33] , whole blood [25] , serum [25] , stool [25] [26] [27] , urine [34, 35] , saliva [36] [37] [38] , rectal swabs [34, 39] and conjunctival swabs [40, 41] . with limited understanding of covid-19, it is difficult to exclude sars-cov-2 infection based on a single negative pcr result, especially when testing was used for upper respiratory tract specimens. collection and detection of lower respiratory tract specimens are strongly recommended even if the upper respiratory tract specimens are negative, especially in patients with severe or progressive conditions [42] . ace2 is mainly distributed in alveolar type ii epithelial cells [17] , suggesting lower respiratory tract specimens (including sputum, tracheal aspirates, balf) may contain high viral rna loads. yu et al. compared the average viral load in sputum, throat swabs and nasal swabs from 127 confirmed or suspected covid-19 patients. the study found that the average viral load in sputum (17429 ± 6920 copies/test) was obviously higher than in nasal swabs (651 ± 501 copies/test) and throat swabs (2552 ± 1965 copies/test) [31] . besides, a high viral load of 10 8 copies per milliliter was detected in the sputum of an asymptomatic patient five days after symptoms onset in germany [32] . these findings indicate a higher viral load in lower respiratory tract samples. zhang et al. analyzed the virus dynamics in oral and anal swabs of 16 covid-19 patients who had been treated for about 10 days. most of the cases were detected positive to sars-cov-2 from oral swabs (8/10, 80%) on the day of the first sampling. however, 5 days after the first sampling, the positive rate of the anal swab (6/8, 75%) was higher than that of the oral swab (4/8, 50%) by nucleic acid test, indicating a change from more oral swabs positive during the early period to more anal swabs positive during the later period [25] . in addition to the most common respiratory specimens, it should be noted that sars-cov-2 has been frequently detected in non-respiratory specimens. it was reported that sars-cov-2 was detected in the urine sample from one confirmed covid-19 patient, although no urinary irritation was found [43] . recently, sars-cov-2 was frequently detected in saliva samples of covid-19 patients during infection [36] [37] [38] . sars-cov-2 was identified in the saliva specimens from 11 out of 12 covid-19 patients by rt-pcr. the viral load monitoring of serial saliva generally showed a downward trend after hospitalization [36] . in another study, sars-cov-2 rna was detected in the posterior oropharyngeal saliva of 20 (87%) patients. the viral load in saliva was highest in the first week after symptom onset and decreased over time [37] . jonathan et al. simultaneously assessed posterior oropharyngeal saliva and nasopharyngeal swab specimens for the detection of sars-cov-2 using an automated xpert® xpress sars-cov-2 assay. in this study, 84.5% (49/58) patients tested positive in both saliva and nasopharyngeal swab, 5.2% (3/58) patients tested positive in saliva only and 10.3% (6/58) patients tested positive in nasopharyngeal swab only [38] . these studies indicated that saliva can be used as a promising non-invasive sample for diagnosis, monitoring and control of covid-19. furthermore, tear and conjunctival swab samples from 2 out of 60 covid-19 patients were detected positive to sars-cov-2 by rt-pcr [41] . however, the virus was not successfully isolated and cultured in the conjunctival secretion, and more studies are needed to be evaluated. currently, the virus has not been detected in cerebrospinal fluid, pericardial effusion, peritoneal effusion, posterior fornix, joint fluid, peritoneal exudate, semen, female reproductive tract secretions and other samples. although no virus has been detected in these specimens until now, it is also recommended that these be treated as infectious specimens. further investigations are needed to identify whether these specimens may spread the virus. according to sars-cov-2 genomes released on public databases, specific primers and probes designed for the target genes can realize the laboratory diagnosis of covid-19. so far, the gene targets were used to detect sars-cov-2 include nucleocapsid (n), envelope (e), spike (s), rna-dependent rna polymerase (rdrp) and open reading frame1ab (orf1ab) genes [44] . as the gold standard test for sars-cov-2 identification, real time quantification rt-pcr (qrt-pcr) is the routine confirmation test recommended by who. however, due to the time-consuming process, the requirements of expensive equipment and biosafety conditions, this test is not suitable for point-of-care diagnosis. point-of-care tests are not only suitable for clinical laboratories, but also can be performed by trained non-laboratory personnel in patient care facilities, such as physicians' offices or emergency departments, making the diagnostic test of sars-cov-2 closer to the patient. the number of commercially available point-of-care tests for sars-cov-2 is increasing. a comparison of different nucleic acid amplification of sars-cov-2, including lab-based tests and point-of-care tests, is shown in table 1 . the who website provides several qrt-pcr protocols for detecting sars-cov-2 in different countries [44] . the gene targets for detection sars-cov-2 are different in china (orf1ab and n genes), germany (rdrp, e and n genes), united states (three targets in n gene), france (two targets in rdrp), thailand (n gene), and japan (pancorona and multiple targets, spike protein). rt-pcr tests/primers for sars-cov-2 in different institutions are shown in table 2 . relative positions of amplified targets on sars-cov-2 genome are shown in fig 1. the centers for disease control and prevention (cdc) established a rt-pcr panel for specific detection sars-cov-2 and universal detection sars-like beta-covs. three sets of different primers were designed for the n gene, one set of primers/probes was used universally detect all beta-covs, while the other two were specific to identify sars-cov-2. the confirmation of covid-19 must be positive for all three targets [45] . two nucleic acid tests for detection rdrp and e genes of sars-cov-2, sars-cov and bat-like beta-covs were developed by charite, germany. both tests are positive could enter the next step of the test, namely the sars-cov-2 specific rt-pcr test for rdrp gene [46] . although various institutions have developed different protocols for sars-cov-2 testing, it remains unclear whether the results from nucleic acid tests based on different targets are comparable. chantal et al. [47] used rna transcripts isolated from a covid-19 patient to compare the analytical sensitivities of four qrt-pcr assays established in the united states, germany, hong kong and china, respectively. the study found that sars-cov-2 could be detected in all primer-probe sets applied in the qrt-pcr tests, but significant discrepancy was observed in the detection limit and the ability to identify negatives and positives with a lower viral load. the primer-probe sets with the highest sensitivity were found in e-sarbeco (germany), 2019-ncov_n1 (united states), and hku-orf1 (hong kong), but the lowest sensitivity was observed in rdrp-sarsr (germany), which may be related to the mismatch in the reverse primer [47] . besides, konrad et al. compared qrt-pcr tests in different pcr systems and a commercial reagent using nasopharyngeal swab or sputum samples from covid-19 patients in germany [48] . when the same primers and probes were used, the distinctions in analytical sensitivities between different pcr systems were also observed. they found that the e gene target was more sensitive than the rdrp target when using a one-step qrt-pcr system. however, the high background of e gene target hindered the clear evaluation of the test, and further optimization of e gene assay may improve the sensitivity. real-time nested rt-pcr assay that connects the time-saving real-time instruments with high sensitivity of nested pcr has proven to be suitable for detecting low-copy-number sars coronaviruses that present in the early stage of disease [49, 50] . in the early days of the outbreak, the detection of sars-cov-2 by nested rt-pcr method has been verified in japan. as of 8 february 2020, the method developed has successfully identified 25 positive patients in japan [51] . recently, ji et al. designed a one-step nested realtime rt-pcr (osn-qrt-pcr) assay for targeting sars-cov-2 orf1ab and n genes. the sensitivity of the assay was 1 copy/test and 10-fold higher than that of a commercial qrt-pcr assay (10 copies/test). among 181 clinical samples, 14 samples with qrt-pcr-negative were confirmed by osn-qrt-pcr. in addition, 7 samples with qrt-pcr-positive in the gray zone were confirmed to positive by osn-qrt-pcr [52] . compared with the qrt-pcr kit, nested rt-pcr analysis showed higher sensitivity and specificity, indicating that it is more suitable for clinical application to detect sars-cov-2 in cases with low viral load. however, nested rt-pcr can cause laboratory cross-contamination, which may lead to falsepositive results [53] . droplet digital pcr (ddpcr) has been shown to improve the lower lod, sensitivity and accuracy in detecting sars-cov-2 [54, 55] . suo et al. analyzed the feasibility of ddpcr for sars-cov-2 rna detection compared with qrt-pcr using the same primer/probe sets issued by china cdc targeting orf1ab or n gene. 26 covid-19 patients with negative rt-pcr results were confirmed positive by ddpcr. the sensitivity and accuracy were improved from 40% and 47% for rt-pcr to 94% and 95% for ddpcr, respectively. within 5-12 days after discharge, 6/14 patients (42.9%) were demonstrated to positive by ddpcr [55] . the study demonstrated that ddpcr could largely reduce the false negatives results caused by qrt-pcr. the team further stringently analyzed the performance of ddpcr and qrt-pcr using 8 primer/probe sets with the same samples and conditions. the results showed that all the 8 primers/probes used in qrt-pcr were unable to significantly distinguish positive and negative at low viral load (10 -4 dilution). false-positive results of qrt-pcr tests of us cdc-n1, n2 and china cdc-n primers/probes sets were discovered. in contrast, the overall performance of ddpcr was significantly better than qrt-pcr, especially for samples with low viral load [54] . however, ddpcr also has some shortcomings. to ensure commutability between molecular diagnostic laboratories, gold standards or calibrant materials still need to be precisely defined. besides, ddpcr is currently more costly than qrt-pcr for each test performed using dedicated instruments and consumables. loop-mediated isothermal amplification (lamp) has the advantages of rapid amplification at a single temperature, which is efficient in the rapid and reliable diagnosis of covs. zhang et al. designed full lamp primers targeting the 5′ region of the orf1a and n genes of sars-cov-2 and detected by a visual, colorimetric rt-lamp alongside a commercial rt-pcr assay. six of seven samples exhibited visible color change suggesting positive amplification, while the single sample remained pink color and was confirmed negative. the colorimetric rt-lamp analysis was 100% consistent with rt-pcr results across a range of cq values, and matched with rt-pcr in the field and point-of-care settings without sophisticated instrumentation [56] . yu et al. [57] developed an isothermal lamp-based detection method for orf1ab gene -(isothermal lamp-based method for covid-19, ilaco). ilaco showed species-specificity by sequence comparison of 11 respiratory viruses (including 7 similar covs, 2 influenza viruses and 2 normal covs). in addition, ilaco's sensitivity was comparable to that of taqman-based rt-pcr, which was able to detect as low as 10 copies of sars-cov-2. besides. mohamed et al. described a highly sensitive, pointof-care test based on lamp and nested-like amplification assay, rapid isothermal amplification assay (penn-ramp) [58] . the sensitivity of ramp was 10 times better than that of lamp and rt-pcr for testing purified targets, and 100 times better than that of lamp and rt-pcr for samples with minimally processed. this method is suitable for use at home, point-of-care and in the clinic with the least trained using and the least instrumentation. it has the potential to reduce false negatives from routine nucleic acid tests. nanoparticles have been introduced to the nucleic acid amplification system to enhance the sensitivity and specificity of sars-cov-2 detection [57, 59, 60] . parikshit et al. designed a naked-eye colorimetric test based on gold nanoparticles (aunps) with thiol-modified antisense oligonucleotides (asos) targeting the sars-cov-2 n-gene. the limit of detection (lod) was found to be 0.18 ng/μl of sars-cov-2 viral load [60] . in addition, a lod of 12 copies/test was observed in a one-step nanoparticles-based biosensor (nbs) coupled with rt-lamp [59] . among laboratory-confirmed covid-19 patients, the analytical sensitivity of sars-cov-2 was 100% (33/33) in the oropharynx swab specimens, and the specificity of the assay was also 100% (96/96) when analyzed the rna templates from non-covid-19 patients. the unique properties of nanoparticles give them an advantage over traditional methods that are usually expensive and laborious. nanoparticles-based amplification is a promising method for diagnosing sars-cov-2 infection in first-line clinical laboratories, especially in areas with challenged medical resources. however, nanoparticles-based amplification is more expensive than qrt-pcr and the pretreatment steps are complex. beside, as with any system using traditional organic carriers, the risk of photobleaching may lead to reduced sensitivity and false-negative results [61] . the automated molecular diagnostic platform demonstrates a highly sensitive, powerful and accurate method for the rapid identification of sars-cov-2. even in a laboratory without pcr training or point-ofcare testing, the assay also can achieve rapid decisions and technological innovation. the inconsistent performance of the different portable benchtop-sized analyzers for sars-cov-2 detection was reported. benoit et al. evaluated the qiastat-dx respiratory sars-cov-2 panel for sars-cov-2 detection. this platform showed a comparable sensitivity to rt-pcr with a lod at 1000 copies/ml. in 69 clinical samples analysis, the overall consistent percentage of qiastat-dx sars and rt-pcr recommended by who was 97%, with a specificity of 93% (27/29) and a sensitivity of 100% (40/40) . no cross-reactions of other respiratory viruses or bacteria were observed in this assay [62] . the results indicated that qiastat-dx respiratory sars-cov-2 panel has a comparable sensitivity to rt-pcr assay. rhoads et al. found that there is a 94% positive percent agreement (ppa) between the id now tests and an improved test developed by the cdc laboratory [63] , while other assessments indicated that id now has a lower ppa, ranging from 75% to 87%, compared with the laboratory-developed reference methods [64] [65] [66] . compared to xpert xpress assay (~46 minutes) and eplex assay (~1.5 hours), id now generates the fastest detection time for each sample (~17 minutes), but it comes at the expense of analysis and clinical performance, with the highest lod and the lowest ppa [67] . in addition, stephanie et al. showed the specificity of this assay was 100%, but 13 of the 46 sars-cov-2 positive samples were false-negative resulting in a lower sensitivity of 71.7%. all of the false-negatives equal to those weak positive samples [7] . this indicates that id now has acceptable performance for samples contain high or moderate viral rna, but may lack sensitivity when performing weakly positive samples. xpress test for targeting sars-cov-2 e-gene and n2-gene with a lod of 100 copies/ml [67] . this difference may be caused by the different methods used to determine the input concentration and requires further verification. in wei's study, xpert xpress had the lowest lod (100 copies/ml) compared to id now (20,000 copies/ml) and eplex (1,000 copies/ml), and highest ppa (98.3%) compared to id now (87.7%) and eplex (91.4%). the information about inconsistent performance in these assays and workflow is vital to make timely and informed decisions on laboratory testing platform. sars-cov-2 consists of multiple virus-encoded proteins, including s, n, e, and m proteins. among those proteins, s and n were the two main antigenic targets of sars-cov-2 antibodies [69] . under the action of a host cell furin-like protease, the s protein is sliced into two separate polypeptides in most covs, s1 and s2 [70] . although s protein is critical for virus entry and exists on the surface of the virus, n protein is the most richly expressed immune dominant protein that interacts with rna and is more conserved than s protein [70] . within s protein, the s1 submit is less conserved and more highly specific to sars-cov-2, suggesting s1 submit is more specific than full-length s or s2 submit as an antigen for covid-19 serologic detection [70] . in addition, the rbd domain of s1 protein, is more conserved than s1 or full-length s, and has much less cross-reactivity with other covs [71] . multiple forms of n protein or s protein (full-length s, s1 domain, s2 domain or receptor-binding domain [rbd]) -are used as targets [70] [71] [72] [73] [74] . immunochromatographic assay is the most commonly used method for the detection of sars-cov-2 antigens [74, 75] . thomas et al. compared four lateral flow antigen-detection kits ( rapigen, liming bio, savant, and bioeasy ) for the detection of sars-cov-2 and showed test performances with significant differences. among these tests, the bioeasy test showed the highest accuracy of 89.2% and kappa coefficient of 0.8, while liming bio test was discontinued during testing because of poor performance. sensitivities of other kits ranged from 16.7% for the savant assay to 85% for the bioeasy test [75] . compared with the lower sensitivity of immunochromatography, highly sensitive biosensors-based tests have been demonstrated in the detection of sars-cov-2. sophie et al. developed a portable, rapid cellbased biosensor with a human chimeric spike s1 antibody for detection of the sars-cov-2 s1 protein [76] . the biosensor allows tests completed within 3 minutes with a detection limit of 1 fg/ml and a semi-linear response range of 10 fg to 1 µg/ml. similarly, subhasis et al. constructed a biosensor device (ecovsens) to target sars-cov-2 s1 protein and compared it with a commercial potentiostat sensor. the lod were found 90 fm for ecovsens and 120 fm for commercial potentiostat sensor in saliva samples [73] . these platforms can be used to monitor sars-cov-2 antigen on a large scale, providing a promising scheme for timely monitoring and eventual control of the global pandemic. with specific reagents, individual antibody types, like igg, igm, and iga, can be determined. after sars-cov-2 infects the human body, igm antibody can be produced within 5-7 days and is most useful for determining recent infection, while igg antibody can be produced within 10-15 days and may remain detectable for months or years [8] . iga is important for mucosal immunity and can be detected in mucous secretions within 6-8 days [77] , though its significance in this disease is still to be determined. in cases where rt-pcr assays are negative and there is a strong epidemiological link to sars-cov-2 infection, paired serum samples (in the acute and convalescent-phase) could support diagnosis once validated serology tests are available with the initial samples collected in the first week of covid-19 and the second collected after 2-4 weeks [28] . tests that detect binding antibodies fall into two broad categories, laboratory tests and point-of-care tests. a comparison of different serology tests for the detection of sars-cov-2 is shown in table 1 . the performance of currently available detection tests is compared in table 3 . niko et al. compared the performance of vircell covid-19 igg (recombinant n protein) and euroimmun sars-cov-2 igg (recombinant s protein) assays at different stages of sars-cov-2 infection. the sensitivity was 70.6% for vircell elisa for and 58.8% for euroimmun elisa on days 5-9 after pcrconfirmed covid-19. during 10-18 days after confirmation, the sensitivity was 100% for vircell elisa and 93.8% for euroimmun elisa [78] . similarly, liu et al. evaluated two elisa kits based on sars-cov-2 s protein and n protein for detecting igg and igm antibodies. the team detected sars-cov-2 igg antibodies in less than 60% of samples using s protein-based elisa test on 6-10 days after the onset of the disease, and the sensitivity of the sample increases to > 90% on 16 to 20 days after onset [79] . in addition, the sensitivity of 74.3% and 77.1% for s protein-based igg and igm elisa, of 70.1% and 68.2% for n protein-based igg and igm elisa, respectively. the overall sensitivity of the s protein-based and n proteinbased elisa tests were 82.2% and 80.4%. a higher sensitivity was observed in s protein-based elisa compared to n protein-based elisa [79] . sars-cov-2 igm and igg antibodies in serum from the first covid-19 case in finland were identified by immunofluorescence assay (ifa). the patient's serum was continuously diluted and incubated for 30 min for igg and 2 h for igm in vero e6 cells. although the antibodies were not detected on day 4 since the first symptoms, igm and igg titer both increased to 1:80 on day 9, 1:32 and 1:128 on day 20, respectively. the serum samples from the control group were not observed specific binding at dilutions greater than 1:20 [80] . niko et al. compared the performance of ifa and neutralization assays at different stages of sars-cov-2 infection. the sensitivity of ifa and neutralization assays increased from 76.5% on days 5-9 after confirmation by pcr to 100% on days 10-18 [78] . although ifa is promising in early diagnosis, non-specific fluorescence may lead to a wrong judgment and the sensitivity of this approach needs to be further evaluated. compared with the euroimmun igg/iga elisa assay, the maglumi™ igg/igm clia assay shows a lower overall sensitivity (84.4% vs. 64.3%). both assays exhibited very similar specificities of igg, which were 99% and 100% for elisa and clia assays, respectively [84] . the qualitative or semi-quantitative detection for sars-cov-2 igm and igg antibodies in serum, plasma and venous blood samples in vitro were proved by lateral flow assay (lfa) [85] [86] [87] [88] igg antibody alone and does not improve diagnostic performance [88] . the study indicated that measuring only lfa igg can avoid false-positive results of igm. the sars-cov-2 antigens coated on this array include n protein, s protein, rbd, s1 and s2 domains. the samples of confirmed covid-19 cases were highly reactive to sars-cov-2 antigens, and the reactivity in igg is more obvious than iga. similar to igg, iga showed higher reactivity to sars-cov-2 n protein, s2 domain, and full-length s, cross-reactivity with sars-cov n protein, but not with mers-cov antigens [89] . the team further developed a modular microarray imaging system for the detection of sars-cov-2 antibody. the imaging platform can produce similar results to the commercial imager (arraycam 400-s, grace biolabs), which are 100 times more expensive. linear regression analysis of microarray fluorescence intensities revealed r-squared values >0.85 between different imaging platforms. this platform has the advantages of low-cost, high-throughput, and can handle more than 100,000 samples, potentially valuable for serosurveillance in covid-19 patients [90] . in addition, tan et al. designed a microfluidic elisa test for quantitative detection of sars-cov-2 s1 protein and anti-sars-cov-2 s1 igg using humanized sars-cov-2 igg and recombinant s1 protein, respectively. the lower lod of 2 ng/ml for anti-sars-cov-2 s1 igg and of 0.4 ng/ml for s1 antigen was achieved in serum [91] . these studies demonstrated high performance of analyzing sars-cov-2 specific antibodies and antigens in covid-19 patients by microarray and microfluidic chip. however, some proteins on the microarray were observed not expressed in mammalian cell systems and need to be further optimized [92] . as the gold standard for virological diagnosis, a culture-based virus detection is an important tool for virus discovery, pathogenesis research and strategy evaluation. neutralization tests evaluated the capacity of serum from covid-19 patients to reduce cytopathic effects (cpe) caused by sars-cov-2 susceptible cells in vitro. it involves incubating the serum or plasma with live virus followed by infection and incubation of cells. it mainly includes virus neutralization tests (vnt) and pseudovirus neutralization tests (pvnt) [93] . the first covid-19 case in finland was confirmed by cytopathic effect (cpe)-based vnt to analyze sars-cov-2 antibody levels. at day 9-10 after the first symptom onset, an increase of at least 4-fold in the neutralizing antibodies was observed, and the titer of antibodies was still increasing on day 20 [80] . wang et al. found that the titers of antibodies bind to n and s proteins was observably higher in severe patients. meanwhile, the average titers of neutralizing antibody against sars-cov-2 pseudovirus and live virus was higher in the sicker cases, by~5-fold and ~7-fold, respectively [94] . renata et al. detected the ability of serum from covid-19 patients to reduce the cpe caused by sars-cov-2 infection of susceptible cells by a micro-neutralization assay. neutralizing antibodies were detected in 16 out of 20 patients, with titers ranging from 10 to 1920. there is a strong positive correlation between neutralizing antibody titers and total sars-cov-2 igg antibody levels in covid-19 patients were observed. in addition, anti-s1-iga, anti-s1-igg and anti-n-igm levels were also related to the neutralizing antibody titers of sars-cov-2. these studies indicating that sars-cov-2 antibody titers in covid-19 patients may reflect the capacity of serum to neutralize sars-cov-2 [95] . focusing on igg antibodies, the performance of in-house plaque reduction neutralization test (prnt), in-house ifa, euroimmun elisa, vircell elisa and fastep lfa were analyzed. in the early stages of infection, the sensitivity to prnt and ifa tests was 76.5%, higher than two elisa tests and lfa test. at the later stage of 10-18 days, ifa, prnt and vircell elisa showed a sensitivity of 100%, while the euroimmun elisa and assure lfa of 93.8% [78] . the study demonstrated the superior sensitivity of the neutralization test in the diagnosis of covid-19. neutralization assay is specific for sars-cov-2 antibodies and could be used to monitor patient immunity to the virus. however, compared with serological tests, neutralization tests are long-period, laborious, expensive, time-consuming, and limited to perform in biosafety level 3 (bsl-3) or bsl-2 laboratories. genomic sequencing of viruses is a powerful tool for analyzing virus evolution, and genetic association to diseases, tracking outbreaks and developing new therapies and vaccines. as of march 2020, hundreds of sars-cov-2 genomes were released on public databases including global initiative on sharing all influenza data (gisaid), ncbi genbank and china national genebank database (cngbdb). at present, sequencing methods of sars-cov-2 include metatranscriptomics sequencing [27] , hybrid capture-based sequencing [96] , amplicon sequencing [96] , and nanopore targeted sequencing [97] . the advantages and disadvantages of sars-cov-2 genomic sequencing are shown in table 1 . the first research team got the sars-cov-2 genomic sequences by metatranscriptomic sequencing, supplemented by pcr and sanger sequencing of a connection between balf and virus culture [8, 13] or from balf directly [11, 33] . lu et al. obtained ten genome sequences of sars-cov-2 from nine patients' balf and culture samples by metatranscriptomic sequencing. the ten genome sequences were nearly identical, displaying more than 99.98% sequence identity [13] . this finding indicates that sars-cov-2 may originate from the same source within a short period. in addition, it was reported that whole-genome sequences of sars-cov-2 from nasopharyngeal and oropharyngeal samples from a covid-19 case by sanger and metatranscriptomic sequencing with both minion and illumina in the united states [27] . the entire genome sequences of the nasopharyngeal and oropharyngeal samples were identical to each other and were highly similar to other available sars-cov-2 sequences. although the amplicon sequencing and hybrid capture sequencing with high sensitivity but low accuracy, and neither of them can be applied to sequence viruses with highly diverse or recombinant due to primers and probes are designed to the known viral genomes [96] . compared with metatranscriptomic sequencing, the results of amplicons and capture sequencing showed that significant increases in the ratio of sars-cov-2 reads out of the total reads, which indicates the enrichment was efficient 5710-fold in amplicon sequencing and 5596-fold in hybrid capture sequencing for each specimen on average. the alleles identified by hybrid capture sequencing showed lower frequencies than amplicon sequencing and metatranscriptomic sequencing, especially for viruses with lower load. capture sequencing neglected a single nucleotide variation (snv) when the cutoff of snv calling was set as 80% allele frequency [96] . combining the strengths of target amplification and long-read, real-time nanopore sequencing, nanopore targeted sequencing was established to simultaneously detect sars-cov-2 and 10 types of other respiratory viruses at lod of 10 copies/ml. nts identified positive to 22 of 61 suspected covid-19 specimens that were either negative or inconclusive by rt-pcr. screening mutations showed that 4 of 19 throat swab samples from covid-19 patients were found single-nucleotide mutations at seven sites [97] . these findings suggested that nts is suitable for the identification and mutation monitoring of sars-cov-2 from clinical samples. laboratory diagnosis of clinical specimens from suspected or confirmed covid-19 patients should be conducted adopting practices and procedures described by who [98] . specimen collection, storage, packaging, and transportation under standard operating procedures. sars-cov-2 was detected in unusual types of specimens such as saliva and conjunctival swabs [36] [37] [38] 40] , so it is recommended that all specimens collected for laboratory investigation should be considered potentially infectious. rna extraction, nucleic acid amplification assays and sequencing should be conducted in bsl-2 laboratory, while neutralization assays and virus culture should be carried out in bsl-3 or bsl-2 laboratory [28] . according to the current evidence, covid-19 virus is primarily transmitted between people via respiratory droplets and contact routes. the droplets can also be deposited directly on individual next to the infected case. hence, washing hands frequently and keeping a distance of at least one meter are considered the main measure to prevent infection [99] . personal protective equipment (ppe) can protect people against infection, especially in susceptible people. ppe recommended by who include medical masks, gloves, gowns, goggles or a face shield, respirators and aprons [100] . among the general public, persons with respiratory symptoms or those caring for covid-19 patients at home should receive medical masks, but n95 respirators is not recommended [101] . using n95 masks compared with the use of medical masks was not related to any statistically significant lower risk of laboratory-confirmed viral infections [102, 103] . with the development of globalization, emerging infectious diseases continually occur and may cause an outbreak globally. understanding biosecurity and personal protection can prepare for the prevention and control of emerging infectious diseases. conflict of interest the authors declare that they have no conflict of interest. genetic recombination, and pathogenesis of coronaviruses a rapid and specific assay for the detection of mers-cov 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review and meta-analysis detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr specific primers and probes for detection 2019 novel coronavirus, china national institute for viral disease control and prevention: beijing real-time rt-pcr assays for the detection of sars-cov-2 detection of second case of 2019-ncov infection in japan; department of virology iii diagnostic detection of novel coronavirus 2019 by real time rt-pcr novel coronavirus (2019-ncov) in suspected human cases by rt-pcr, school of public health high-accuracy multiplexed sars-cov-2 antibody assay with avidity and saliva capability on a nano-plasmonic platform igm and igg 100.0% 13.5% 38.5% 57.9% key: cord-339665-nwwutduy authors: patel, ami; walters, jewell; reuschel, emma l.; schultheis, katherine; parzych, elizabeth; gary, ebony n.; maricic, igor; purwar, mansi; eblimit, zeena; walker, susanne n.; guimet, diana; bhojnagarwala, pratik; doan, arthur; xu, ziyang; elwood, dustin; reeder, sophia m.; pessaint, laurent; kim, kevin y.; cook, anthony; chokkalingam, neethu; finneyfrock, brad; tello-ruiz, edgar; dodson, alan; choi, jihae; generotti, alison; harrison, john; tursi, nicholas j.; andrade, viviane m.; dia, yaya; zaidi, faraz i.; andersen, hanne; lewis, mark g.; muthumani, kar; kim, j joseph; kulp, daniel w.; humeau, laurent m.; ramos, stephanie; smith, trevor r.f.; weiner, david b.; broderick, kate e. title: intradermal-delivered dna vaccine provides anamnestic protection in a rhesus macaque sars-cov-2 challenge model date: 2020-07-29 journal: biorxiv doi: 10.1101/2020.07.28.225649 sha: doc_id: 339665 cord_uid: nwwutduy coronavirus disease 2019 (covid-19), caused by the sars-cov-2 virus, has had a dramatic global impact on public health, social, and economic infrastructures. here, we assess immunogenicity and anamnestic protective efficacy in rhesus macaques of the intradermal (id)-delivered sars-cov-2 spike dna vaccine, ino-4800. ino-4800 is an id-delivered dna vaccine currently being evaluated in clinical trials. vaccination with ino-4800 induced t cell responses and neutralizing antibody responses against both the d614 and g614 sars-cov-2 spike proteins. several months after vaccination, animals were challenged with sars-cov-2 resulting in rapid recall of anti-sars-cov-2 spike protein t and b cell responses. these responses were associated with lower viral loads in the lung and with faster nasal clearance of virus. these studies support the immune impact of ino-4800 for inducing both humoral and cellular arms of the adaptive immune system which are likely important for providing durable protection against covid-19 disease. covid-19 was declared a global pandemic on march 11, 2020 by the world health organization. there are >15.5 million confirmed cases worldwide with the total number of deaths estimated to be >600,000 (july 24, 2020, gisaid.org) . covid-19 presents as a respiratory illness, with mildto-moderate symptoms in many cases (~80%) (chen et al., 2020; huang et al., 2020) . these symptoms include headache, cough, fever, fatigue, difficulty breathing, and possible loss of taste and smell. the factors involved with progression to severe covid-19 disease in 20% of cases are unclear; yet, severe disease is characterized by development of a hyperinflammatory response, followed by development of acute respiratory distress syndrome (ards), potentially leading to mechanical ventilation, kidney failure, and death xu et al., 2020) . the rapid development of vaccine countermeasures remains a high priority for this infection, with multiple candidates having entered the clinic in record time (thanh le et al., 2020) . sars-cov-2 is a positive-sense single stranded rna virus belonging to the family coronaviridae, genus betacoronavirus, the same family as severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). sars-cov-2 is homologous to sars-cov, sharing around 70-80% of its genome (lu et al., 2020) . structural components of sars-cov-2 include the envelope (e) protein, membrane (m) protein, nucleocapsid (n) protein, and surface spike (s) protein, which is a major immunogenic target for humoral and cellular immune responses. virus entry into host cells is mediated through s protein receptor binding domain (rbd) interaction with the host cell receptor angiotensin converting enzyme 2 (ace2) and priming by the serine protease tmprss2 (hoffmann et al., 2020) . recent preprints and published studies also implicate s1 binding to neuropilin 1 (nlp-1) and have identified neutralizing epitopes outside rbd (chi et al., 2020) . sars-cov-2 is the third coronavirus outbreak this century. prior work with the related coronaviruses, sars-cov and mers-cov, delineated that the spike protein of these viruses was an important target for development of neutralizing antibodies, and in animal viral challenges vaccine targeted immunity (reviewed in (du et al., 2009; roper and rehm, 2009; thanh le et al., 2020) (liu et al., 2018; muthumani et al., 2015; van doremalen et al., 2020a) . recently studies have described initial data for vaccines developed from inactivated viruses (gao et al., 2020) , recombinant adenoviral vectors that express the spike antigen (van doremalen et al., 2020b) , or intramuscular (im) delivered dna vaccine candidates in nonhuman primates (nhps). these studies focused on disease protection in acute models with challenge weeks after the final vaccination. two in particular showed the ability of vaccination to lower viral load in the lungs (gao et al., 2020; van doremalen et al., 2020b) , and one reported impact in the lungs and a trend to impact viral loads in the nose . while there are significant questions arising regarding the length of protective immunity from sars-cov2 infection, as well as the long-term protection of vaccination, there have been no studies yet reported of the ability of vaccine memory to impact challenge outcome. here we report on the impact of an id-delivered sars-cov-2 dna vaccine in a nonhuman primate model where the animals were challenged 3 months post-vaccination. recent studies support an important role for both cellular and humoral immunity in protection against covid-19 disease. t cell-mediated immunity has been shown to be important for other beta-coronaviruses, providing both direct protection and help in the generation of effective humoral responses (channappanavar et al., 2014b; gretebeck and subbarao, 2015) . several sars-cov-2 studies have now reported that up to 40% of persons can exhibit mild disease which may be associated with t cell immunity (payne et al., 2020) or humoral responses (robbiani et al., 2020) . this echoes older studies which found that neutralizing antibody levels and memory b cell responses have not been sustained in sars survivors, while responsive t cell populations have proved more durable (le bert et al., 2020; tang et al., 2011; yang et al., 2006) . studies have indicated the levels of sars-cov-2 antibodies wane rapidly in convalescent subjects (long et al., 2020) . we recently described the immunogenicity of a sars-cov-2 dna vaccine (ino-4800), encoding a synthetic spike immunogen in small animal models . this vaccine candidate induced neutralizing and ace2-blocking antibodies, as well as t cell responses in mice and guinea pigs. here, we assess vaccine-induced memory t and b cell responses during the acute expansion and memory phases in rhesus macaques, as well the ability of vaccine-induced memory to impact infection in an nhp viral installation challenge following vaccination by the minimally invasive (id-ep) route. additionally, we addressed the growing concerns regarding the emergence of a dominant sars-cov2 variant g614, which now comprises more than 80% of circulating global viral strains, and is reportedly associated with increased infectivity and spread (korber b et al., 2020) . there have not yet been descriptions of vaccine induced responses driving immunity against g614 variants. we report vaccine-induced humoral and cellular immunity, including neutralizing antibody responses against both the sars-cov-2 d614 virus as well as the new g614 strain. upon sars-cov-2 challenge more than 3 months following final-dose, vaccinated macaques exhibited a rapid recall response against multiple regions of the s protein. this anamnestic response was characterized by expansion of neutralizing antibody responses, including those against the now dominantly circulating g614 variant, as well as the rapid expansion of t cell responses. the immune responses induced by ino-4800 were associated with disease protection. nhps are a valuable model in the development of covid-19 vaccines and therapeutics as they can be infected with wild-type sars-cov-2, and present with early infection that mimics aspects of human disease gao et al., 2020; yu et al., 2020) . rhesus macaques (n=5) received two immunizations of ino-4800 (1 mg), at week 0 and week 4 ( figure 1a ). naïve control animals (n=5) did not receive vaccine. humoral and cellular immune responses were monitored for 15 weeks (~4 months) following prime immunization for memory responses. all animals seroconverted following a single ino-4800 immunization, with serum igg titers detected against the full-length s1+s2 extracellular domain (ecd), s1, s2, and rbd regions of the sars-cov-2 s protein ( figure 1b and c) . cross-reactive antibodies were also detected against sars-cov s1 protein and rbd, but not mers-cov (supplementary figure 1) . sars-cov-2-reactive igg against the ecd and rbd were detected in bronchoalveolar lavage (bal) washes at week 8, 4-weeks following the 2nd immunization dose (supplementary figure 2) . sars-cov-2 pseudovirus-neutralizing antibodies were detected in the serum of vaccinated animals at week 12 following immunization ( figure 1d ). these memory titers were comparable to those observed in other reported protection studies in macaques performed at the acute phase of the vaccine-induced immune response (gao et al., 2020; van doremalen et al., 2020b; yu et al., 2020) and those reported in the sera of convalescent patients (ni et al., 2020; robbiani et al., 2020) . during the course of the covid-19 pandemic, a d614g sars-cov-2 spike variant has emerged that has potentially greater infectivity, and now accounts for >80% of new isolates (korber b et al., 2020) . there is a concern that vaccines developed prior to this variant's appearance may not neutralize the d614g virus. there is also concern that persons infected with earlier strains, may not have immunity against the g614 variant. therefore, we evaluated neutralization against this new variant using a modified pseudovirus developed to express the g614 spike protein ( figure 1e ). similar neutralization id50 titers were observed against both d614 and g614 spikes, supporting induction of functional antibody responses by the vaccine against both sars-cov-2 virus variants. to investigate specific receptor-blocking neutralizing antibody activity, we assessed the capacity to occlude ace2 by vaccine-induced sera. we recently developed a receptor-blocking assay and demonstrated that it correlates with pseudovirus neutralization titers (walker et al. accepted) . in this assay, anti-spike antibodies are bound to sars-cov-2 spike protein in an elisa plate followed by addition of a soluble version of the ace2 receptor. sera containing antibodies which can obstruct the receptor binding site on the spike protein will have lower area under the curve (auc) values in our assay. we observe that sera from 80% of immunized nhps had reduced auc indicating the presence of antibodies that can block the sars-cov-2 spike protein from interacting with ace2 ( figure 1f ). an independent flow cytometry-based assay was also performed to further study the spike-ace2 interaction. ace2-expressing 293t cells were co-incubated with spike with or without the presence of sera. spike binding to ace2 was detected by flow cytometry. in this assay, 100% of macaques inhibited the spike-ace2 interaction, with 53-96% inhibition of the spike-ace2 interaction at a 1:10 dilution and 24-53% inhibition at a 1:30 dilution ( figure 1g ). all 5 peptide pools with t cells responses peaking at week 6, two weeks following the second immunization (0-518 sfu/million cells, with 4/5 nhps showing positive responses) ( figure 1h ). distinct immunogenic epitope responses were detected against the rbd and s2 regions ( figure 1b and c). interestingly, cross-reactive t cells responses were also detected against the sars-cov spike protein (supplementary figure 3a) . however, we did not observe cross-reactivity to mers-cov spike peptides, which is consistent with the lower sequence homology between figure 3b) . ino-4800 immunized macaques and unvaccinated controls were challenged with sars-cov-2 13 weeks (~3 months) post-final immunization (study week 17, figure 2a ). nhps received a challenge dose of 1.1x10 4 pfu of sars-cov-2 isolate usa-wa1/2020 by intranasal and intratracheal inoculation as previously described yu et al., 2020) . one day after viral challenge, 3/5 of ino-4800 vaccinated animals displayed an increase in antibody titers against the sars-cov-2 full-length ecd. by day 7, all vaccinated animals had an increase in antibody titers against the full length ecd, s1, rbd, and s2 proteins ( figure 2b ). seven days post-challenge, robust geometric mean endpoint titers for the ecd ranging from 409,600 -1,638,400 were observed in immunized animals, compared with the naïve group which displayed seroconversion of only 1/5 animals (gmt 100) ( figure 2b ). antibody responses continued to increase against sars-cov-2 proteins 14 days post-challenge. a significant increase in pseudoneutralization titers (>20-fold increase) was observed in all ino-4800 immunized animals against both d614 and g614 spike variants by day 7 post-challenge, compared to unimmunized animals which only developed low neutralizing titers ( figure 2c) . at earlier time points post challenge, viral mrna detection does not discriminate between input challenge inoculum and active infection, while subgenomic (sgmrna) levels are more likely representative of active cellular sars-cov-2 virion replication yu et al., 2020) . as such, sars-cov-2 sgmrna was measured in unvaccinated control and ino-4800 overall, the reduced viral loads following exposure to sars-cov-2 infection at 17 weeks after immunization show an important durable impact mediated by the vaccine. the covid-19 pandemic continues to devastate global health creating a destabilizing environment. although therapies are progressing through multiple stages of development, the need for preventative vaccine approaches for sars-cov-2 remains significant. importantly, multiple vaccines candidates are being advanced to the clinic as potential options for protection against infection and or disease (reviewed in (funk et al., 2020; thanh le et al., 2020) ). recently acute challenge studies in nhps receiving different experimental vaccines have been reported (gao et al., 2020; van doremalen et al., 2020b; yu et al., 2020) . these studies describe reduction in viral loads in the lower airways, and a trend to lower viral loads in the nose by an im dna delivered full length spike ag. however, protective efficacy at time points following contraction of the acute phase immune response and establishment of immune memory have not been previously studied. this is also true looking at prior nhp challenge in the mers or sars models (muthumani et al., 2015; roberts et al., 2008; van doremalen et al., 2020a) . accordingly, understanding the impact of vaccine-induced memory on challenge is of importance. in the current study, we evaluated the durability of an id-delivered full-length spike nhps followed by challenge at 3 weeks following the second immunization. they observed decreases in bal viral loads and a trend to lower nasal swab subgenomic viral loads in some groups, including a group containing a full-length spike ag construct that shares similarity to the construct studied here using a 2 x 1 mg dose by id administration. in a recent clinical study, we reported that id delivery of an hiv vaccine prototype was dose-sparing and demonstrated increased tolerability and improved immune potency compared to im dna delivery (de rosa et al., 2020) . while limited conclusions can be drawn from such comparisons, more study in this context is warranted. in this study, we observed an impact on induction of durable immunity and protective efficacy at a memory time point, 13 weeks post-final immunization. importantly, reduced viral subgenomic rna loads in the lower lung were observed. a trend of lower viral mrna loads was also observed in the nose with more rapid clearance induced in the ino-4800 vaccinated animals. these data support that immunization and memory immunity from such a vaccine in humans might protect from severe disease in the lung as well as limit the duration of viral shedding in the nasal cavity, thus possibly lowering viral transmission. the initial viral loads detected in control unvaccinated animals in this study were approximately 2 logs higher (10 9 pfu/swab in 4/5 nhps on day 1 post-challenge) than in similar published studies performed under identical conditions (~10 7 pfu/swab) . two of the prior reported nhp studies included intranasal delivery as an inoculation route for challenge (van doremalen et al., 2020b; yu et al., 2020) . high-dose challenge inoculums are frequently employed to ensure take of infection, however such high dose challenge may artificially reduce the impact of potentially protective vaccines and interventions (durudas et al., 2011; innis et al., 2019) . despite these limitations, this study demonstrated significant reduction in peak bal sgmrna and overall viral mrna. wolfel et al reported nasal titers in patients of an average 6.5 x 10 5 copies/swab days 1-5 following onset of symptoms . these titers are significantly lower than our nhp challenge dose and support the potential for the vaccine to exhibit great impact in the field against natural sars-cov-2 challenge. t cell immunity has been shown to be an important mediator of protection against betacoronaviruses, and recent studies have specifically identified a role in protection against covid-19 disease (channappanavar et al., 2014a; channappanavar et al., 2014b; sekine et al., 2020; sun et al., 2020) . sekine et al reported sars-cov-2-reactive t cells in asymptomatic and mild covid-19 convalescent patients who were antibody seronegative (sekine et al., 2020) . these results from convalescent patient studies collectively suggest that vaccine candidates which could generate balanced humoral and cellular immune responses could be important in providing protection against covid-19 disease. our study and other published reports show that dna vaccination with candidates targeting the full-length sars-cov-2 spike protein likely increase the availability of t cell immunodominant epitopes leading to a broader and more potent immune response, compared to partial domains and truncated immunogens. in this study, we also observe t cell cross-reactivity to sars-cov-1. further evaluation of these shared responses is likely of interest and may inform development of vaccines targeting related betacoronaviruses. long-lived sars-cov specific t cells have been reported (le bert et al., 2020; yang et al., 2006) . additional studies of sars-cov-2 infected patients will provide important insight towards immunity and long-term protection against covid-19 disease. in addition to t cells, we demonstrated that ino-4800 induced durable antibody responses that rapidly increased following sars-cov-2 challenge. the vaccine induced robust neutralizing antibody responses against both d614 and g614 sars-cov-2 variants. while the d/g 614 site is outside the rbd, it has been suggested that this shift has the potential to impact vaccine-elicited antibodies (korber b et al., 2020) and possibly virus infectivity. our data shows induction of comparable neutralization titers between d614 and g614 variants and that these responses are similarly recalled following sars-cov-2 challenge. we additionally observe a positive relationship between vaccine-induced antibody binding to the rbd and neutralizing antibody titers (supplementary figure 5, r 2 =0.69) . overall, the current challenge study provides a snapshot of anamnestic protective efficacy several months post-vaccination with a small cohort of animals. the data support further study of the sars-cov-2 dna vaccine candidate ino-4800 which is currently in clinical trials. the persistence of sars-cov-2 immunity following natural infection is unknown and recent studies suggest natural immunity may be short lived (long et al., 2020) . given that many people exhibit asymptomatic or mild disease and recover without developing significant antibody responses, additional study of vaccines that induce immunological memory for t and b cell responses is the highly optimized dna sequence encoding sars-cov-2 ige-spike was created using inovio's proprietary in silico gene optimization algorithm to enhance expression and immunogenicity . the optimized dna sequence was synthesized, digested with bamhi and xhoi, and cloned into the expression vector pgx0001 under the control of the human cytomegalovirus immediate-early promoter and a bovine growth hormone polyadenylation signal. use committee at bioqual (rockville, maryland), an association for assessment and accreditation of laboratory animal care (aaalac) international accredited facility. blood was collected for blood chemistry, pbmc isolation, and serological analysis. bronchoalveolar lavage (bal) was collected on week 8 to assay lung antibody levels and on days 1, 2, 4, 7 post challenge to assay lung viral loads. ten chinese rhesus macaques (ranging from 4.55kg-5.55kg) were randomly assigned to be immunized (3 males and 2 females) or naïve (2 males and 3 females). immunized macaques received two 1 mg injections of sars-cov-2 dna vaccine, ino-4800, at week 0 and 4 by the minimally invasive id-ep administration using the cellectra 2000® adaptive constant current electroporation device with a 3p array (inovio pharmaceuticals). animals were challenged with sars-related coronavirus 2, isolate usa-wa1/2020 (bei resources nr-52281. the following reagent was deposited by the centers for disease control and prevention and obtained through bei resources, niaid, nih: sars-related coronavirus 2, isolate usa-wa1/2020, nr-52281. blood was collected at indicated time points to analyse blood chemistry and to isolate peripheral blood mononuclear cells (pbmc), and serum. bronchoalveolar lavage was collected at week 8 and during challenge period to assay viral load. bal from naïve animals was run as control. at week 17, all animals were challenged with 1.2x10 8 vp (1.1x10 4 pfu) sars-cov-2. virus was administered as 1 ml by the intranasal route (0.5 ml in each nostril) and 1 ml by the intratracheal route. nasal swabs were collected during the challenge period using copan flocked swabs and placed into 1 ml pbs. blood was collected from each macaque into sodium citrate cell preparation tubes (cpt, bd biosciences). the tubes were centrifuged to separate plasma and lymphocytes, according to the manufacturer's protocol. samples were transported by same-day shipment on cold-packs from bioqual to the wistar institute for pbmc isolation. pbmcs were washed and residual red blood cells were removed using ammonium-chloride-potassium (ack) lysis buffer. cells were counted using a vicell counter (beckman coulter) and resuspended in rpmi 1640 (corning), supplemented with 10% fetal bovine serum (atlas), and 1% penicillin/streptomycin (gibco). was performed to detect cellular responses. monkey ifn-γ elispotpro (alkaline phosphatase) plates (mabtech, sweeden, cat#3421m-2apw-10) were blocked for a minimum of 2 hours with rpmi 1640 (corning), supplemented with 10% fbs and 1% pen/strep (r10). following pbmc isolation, 200 000 cells were added to each well in the presence of 1) peptide pools (15-mers with 9-mer overlaps) corresponding to the sars-cov-1, sars-cov-2, or mers-cov spike proteins (5 µg/ml/well final concentration), 2) r10 with dmso (negative control), or 3) anti-cd3 positive control (mabtech, 1:1000 dilution). all samples were plated in triplicate. plates were incubated overnight at 37°c, 5% co2. after 18-20 hours, the plates were washed in pbs and spots were developed according to the manufacturer's protocol. spots were imaged using a ctl immunospot plate reader and antigen-specific responses were determined by subtracting the number of spots in the r10+dmso negative control well from the wells stimulated with peptide pools. antigen binding elisa. serum and bal was collected at each time point was evaluated for binding titers. ninety-six well immunosorbent plates (nunc) were coated with 1ug/ml recombinant sars-cov-2 s1+s2 ecd protein (sino biological 40589-v08b1), s1 protein (sino plates were washed four times with pbs + 0.05% tween20 (pbs-t) and blocked with 5% skim milk in pbs-t (5% sm) for 90 minutes at 37°c. sera or bal from ino-4800 vaccinated and control macaques were serially diluted in 5% sm, added to the washed elisa plates, and then incubated for 1 hour at 37°c. following incubation, plates were washed 4 times with pbs-t and an anti-monkey igg conjugated to horseradish peroxidase (southern biotech 4700-5) 1 hour at 37°c. plates were washed 4 times with pbs-t and one-step tmb solution (sigma) was added to the plates. the reaction was stopped with an equal volume of 2n sulfuric acid. plates were read at 450nm and 570nm within 30 minutes of development using a biotek synergy2 plate reader. 96-well half area plates (corning) were coated at room temperature for 3 hours with 1 µg/ml polyrab anti-his antibody (thermofisher, pa1-983b), followed by overnight blocking with blocking buffer containing 1x pbs, 5% sm, 1% fbs, and 0.2% tween-20. the plates were then incubated with 10 µg/ml of his6x-tagged sars-cov-2, s1+s2 ecd (sinobiological, 40589-v08b1) at room temperature for 1-2 hours. nhp sera (day 0 or week 6) was serially diluted 3fold with 1xpbs containing 1% fbs and 0.2% tween and pre-mixed with huace2-igmu at constant concentration of 0.4 µg/ml. the pre-mixture was then added to the plate and incubated at room temperature for 1-2 hours. the plates were further incubated at room temperature for 1 hour with goat anti-mouse igg h+l hrp (a90-116p, bethyl laboratories) at 1:20,000 dilution followed by addition of one-step tmb ultra substrate (thermofisher) and then quenched with 1m h2so4. absorbance at 450nm and 570nm were recorded with biotek plate reader. expressing ace2-gfp were generated using retroviral transduction. following transduction, the cells were flow sorted based on gfp expression to isolate gfp positive cells. single cell cloning was done on these cells to generate cell lines with equivalent expression of ace2-gfp. to detect inhibition of spike binding to ace2, 2.5 µg/ml s1+s2 ecd-his tagged (sino biological, catalog #40589-v08b1) was incubated with serum collected from vaccinated animals at indicated time points and dilutions on ice for 60 min. this mixture was then transferred to 150,000 293t-ace2-gfp cells and incubated on ice for 90 min. following this, the cells were washed 2x with pbs followed by staining for surelight® apc conjugated anti-his antibody (abcam, ab72579) for 30 min on ice. as a positive control, spike protein was pre-incubated with recombinant human ace2 before transferring to 293t-ace2-gfp cells. data was acquired using a bd lsrii and analyzed by flowjo (version 10). sars-cov-2 pseudovirus was produced using hek293t cells transfected with genejammer (agilent) using ige-sars-cov-2 spike plasmid (genscript) and pnl4-3.luc.r-e-plasmid (nih aids reagent) at a 1:1 ratio. forty-eight hours post transfection, supernatant was collected, enriched with fbs to 12% final volume, steri-filtered (millipore sigma), and aliquoted for storage at -80°c. sars-cov-2 pseudovirus neutralization assay was set up using d10 media (dmem supplemented with 10%fbs and 1x penicillin-streptomycin) in a 96 well format. cho cells stably expressing ace2 were used as target cells (creative biolabs, catalog no. vcel-wyb019). sars-cov-2 pseudovirus were titered to yield greater than 20 times the cells only control relative luminescence units (rlu) after 72h of infection. 10,000 cho-ace2 cells/well were plated in 96-well plates in 100ul d10 media and rested overnight at 37˚c and 5% co2 for 24 hours. the following day, sera from ino-4800 vaccinated and control groups were heat inactivated and serially diluted. sera were incubated with a fixed amount of sars-cov-2 pseudovirus for 90 minutes at rt. the sera+virus mix was then added to the plated cho-ace2 cells and allowed to incubate in a standard incubator (37% humidity, 5% co2) for 72h. cells were then lysed using britelite plus luminescence reporter gene assay system (perkin elmer catalog no. 6066769) and rlu were measured using the biotek plate reader. neutralization titers (id50) were calculated using graphpad prism 8 and defined as the reciprocal serum dilution at which rlu were reduced by 50% compared to rlu in virus control wells after subtraction of background rlu in cell control wells. viral rna assay: rt-pcr assays were utilized to monitor viral loads, essentially as previously described (abnink p et al 2019 science). briefly, rna was extracted using a qiacube ht (qiagen,germany) and the cador pathogen ht kit from bronchoalveolar lavage (bal) supernatant and nasal swabs. rna was reverse transcribed using superscript vilo (invitrogen) and ran in duplicate using the quantstudio 6 and 7 flex real-time pcr system (applied biosystems) according to manufacturer's specifications. viral loads were calculated of viral rna copies per ml or per swab and the assay sensitivity was 50 copies. the target for amplification was the sars-cov2 n (nucleocapsid) gene. the primers and probes for the targets were: to generate a standard curve, the sars-cov-2 e gene sgmrna was cloned into a pcdna3.1 expression plasmid; this insert was transcribed using an amplicap-max t7 high yield message maker kit (cellscript) to obtain rna for standards. prior to rt-pcr, samples collected from challenged animals or standards were reverse-transcribed using superscript iii vilo (invitrogen) according to the manufacturer's instructions. a taqman custom gene expression assay sars-cov-2 infection protects against rechallenge in rhesus macaques virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection t cell-mediated immune response to respiratory coronaviruses epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study robust antibody and cellular responses induced by dna-only vaccination for hiv the spike protein of sars-cov--a target for vaccine and therapeutic development differential innate immune responses to low or high dose oral siv challenge in rhesus macaques a snapshot of the global race for vaccines targeting sars-cov-2 and the covid-19 pandemic development of an inactivated vaccine candidate for sars-cov-2 animal models for sars and mers coronaviruses sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor clinical features of patients infected with 2019 novel coronavirus in wuhan convening on the influenza human viral challenge model for universal influenza vaccines tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus sars-cov-2-specific t cell immunity in cases of covid-19 and sars, and uninfected controls a recombinant vsv-vectored mers-cov vaccine induces neutralizing antibody and t cell responses in rhesus monkeys after single dose immunization clinical and immunological assessment of asymptomatic sars-cov-2 infections genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals sars-cov-2 infections and serologic responses from a sample of convergent antibody responses to sars-cov-2 in convalescent individuals animal models and vaccines for sars-cov infection sars vaccines: where are we? robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19. biorxiv immunogenicity of a dna vaccine candidate for covid-19 generation of a broadly useful model for covid-19 pathogenesis, vaccination, and treatment lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study a single dose of chadox1 mers provides protective immunity in rhesus macaques virological assessment of hospitalized patients with covid-2019 pathological findings of covid-19 associated with acute respiratory distress syndrome long-lived effector/central memory t-cell responses to severe acute respiratory syndrome coronavirus (sars-cov) s antigen in recovered sars patients dna vaccine protection against sars-cov-2 in rhesus macaques recall of humoral immune responses after viral challenge. (a) study outline. (b) igg binding elisa. sars-cov-2 s1+s2 and sars-cov-2 rbd protein antigen binding of igg in diluted nhp sera collected prior to challenge and post challenge in ino-4800 vaccinated (right panels) and naïve animals (left panels). (c) pseudo-neutralization assay showing the presence of sars-cov-2 specific neutralizing antibodies against the d614 and g614 variants of sars-cov-2 before and after viral challenge in unvaccinated at week 17 naïve and ino-4800 immunized (5 per group) rhesus macaques were challenged by intranasal and intratracheal administration of 1.1 x 10 4 pfu sars-cov-2 (us-wa1 isolate). (a) log sgmrna copies/ml in bal in naïve (left panel) and ino-4800 vaccinated animals (right panel). (b) peak sgmrna and (c) viral rna in bal 7 days post-challenge. (d) log sgmrna copies/ml in nasal swabs in naïve (left panel) and ino-4800 vaccinated animals (right panel). (e) peak sgmrna and (f) viral rna in nasal swabs 7 days post-challenge key: cord-335652-v98gv5uf authors: salazar, cecilia; díaz-viraqué, florencia; pereira-gómez, marianoel; ferrés, ignacio; moreno, pilar; moratorio, gonzalo; iraola, gregorio title: multiple introductions, regional spread and local differentiation during the first week of covid-19 epidemic in montevideo, uruguay date: 2020-05-10 journal: biorxiv doi: 10.1101/2020.05.09.086223 sha: doc_id: 335652 cord_uid: v98gv5uf background after its emergence in china in december 2019, the new coronavirus disease (covid-19) caused by sars-cov-2, has rapidly spread infecting more than 3 million people worldwide. south america is among the last regions hit by covid-19 pandemic. in uruguay, first cases were detected on march 13 th 2020 presumably imported by travelers returning from europe. methods we performed whole-genome sequencing of 10 sars-cov-2 from patients diagnosed during the first week (march 16th to 19th) of covid-19 outbreak in uruguay. then, we applied genomic epidemiology using a global dataset to reconstruct the local spatio-temporal dynamics of sars-cov-2. results our phylogeographic analysis showed three independent introductions of sars-cov-2 from different continents. also, we evidenced regional circulation of viral strains originally detected in spain. introduction of sars-cov-2 in uruguay could date back as early as feb 20th. identification of specific mutations showed rapid local genetic differentiation. conclusions we evidenced early independent introductions of sars-cov-2 that likely occurred before first cases were detected. our analysis set the bases for future genomic epidemiology studies to understand the dynamics of sars-cov-2 in uruguay and the latin america and the caribbean region. in december 2019, a new coronavirus disease (covid-19) was detected in wuhan, china. its causative agent, known as sars-cov-2, has spread rapidly causing a global pandemic of pneumonia affecting more than 3m people with more than 200,000 deaths to date [1] . global spread of sars-cov-2 was primarily driven by international movement of people, since travel-associated cases of covid-19 were reported outside of china as early as mid-january 2020 [2] . this global health emergency has deployed international efforts to apply genomic epidemiology to track the spread of sars-cov-2 in real time. the recent development of targeted sequencing protocols by the artic network [3] , open sharing of genomic data through the gisaid (www.gisaid.org) database and straightforward bioinformatic tools for viral phylogenomics [4] , provides the opportunity to reconstruct global spatio-temporal dynamics of the covid-19 pandemic with unprecedented comprehensiveness and resolution. south america was one of the last regions in the world to be hit by covid-19. indeed, first cases were confirmed in brazil around 2 months after its emergence in china. then, sars-cov-2 rapidly emerged in neighboring countries like uruguay, that reported first cases in montevideo, its capital city, on march 13 th . population of uruguay is among the smallest in south america (~3.5m), and is mainly concentrated in montevideo and its metropolitan area (~56% of the total population). also, the biggest international airport of the country is placed in this metropolitan area and concentrates more than 90% of international arrivals. these characteristics makes montevideo a suitable place to apply genomic surveillance to uncover epidemiological patterns during the early phase of the covid-19 local outbreak in uruguay. we therefore aimed to characterize the spatio-temporal dynamics of sars-cov-2 by sequencing around 10% of cases occurred during the first week of outbreak in montevideo, allowing us to identify transmission patterns, geographic origins and genetic variation among local strains. ethics statement. residual de-identified nasopharyngeal samples were remitted to the institut pasteur montevideo, that has been validated by the ministry of health of uruguay as an approved center providing diagnostic testing for covid-19. all samples were de-identified before receipt by the study investigators. sample collection, processing and sequencing. nasopharyngeal swabs were obtained from patients residing in montevideo. no other location data was available to researchers to avoid patient identification. swabs were placed in virus transport media (bd universal viral transport medium) immediately after collection. total rna extraction was performed directly from samples (500 μl) using trizol reagent (invitrogen life technologies, carlsbad, ca, usa). an in-house onestep rt-qpcr assay based on taqman probes targeting the n gene and the open reading frame 1b region were used to test for the presence of sars-cov-2 rna. we selected a total of 10 positive samples with cycle-threshold (ct) less than 30 for downstream wholegenome sequencing. sequencing of sars-cov-2 positive samples was performed according to the primalseq [5] approach on the minion sequencing platform (oxford nanopore technologies, united kingdom) using the v2 primer pools. sequencing libraries were prepared based on the pcr tiling of covid-19 virus protocol (artic network) using the ligation sequencing kit (sqk-lsk109) and native barcoding expansion (exp-nbd114) (oxford nanopore technologies, united kingdom), with the following modifications: i) rna was reverse-transcribed using superscript ii reverse transcriptase (invitrogen life technologies, carlsbad, ca, usa) and random hexamer primers following the manufacturer's instructions, ii) phusion hot start ii high-fidelity pcr master mix (thermo scientific) was used for pcr amplification and, iii) blunt/ta ligase (new england biolabs, usa) was used to ligate barcodes to each sample. to detect crosscontamination between samples, a no template sample was added from a patient tested negative for sars-cov-2 from the rna purification step. a total of 15 ng of pooled samples was loaded onto a minion r9.4.1 flow cell and sequenced for 5 hours generating ~2 million reads of average quality of 12. the rampart software from the artic network (https://github.com/artic-network/rampart) was used to monitor the sequencing run in real time, estimate coverage across samples and check barcoding. subsequently, basecalling was performed guppy software v3.2.1 using the high accuracy module. consensus genomes were generated and variants were called using the artic network bioinformatic pipeline (https://artic.network/ncov-2019/ncov2019bioinformatic.sop.html). amplicons that were not sequenced or whose depth was less than 20x were not included in the consensus sequences and these positions were represented by "n" stretches. see supplementary table s1 . phylogenetic and spatio-temporal analysis. to investigate the origin, dynamics and genetic variation of sars-cov-2 in uruguay, we added our 10 genomes to a dataset of 2,443 complete (>29,000 bp.), highquality genomes available from gisaid (https://www.gisaid.org) on april 2 nd 2020. a list of sequences and acknowledgements to the originating and submitting labs is presented in supplementary table s2 . we analyzed this dataset using the augur toolkit version 6.4.2 [3] . briefly, genomes were aligned using mafft [6] and a phylogeny was reconstructed with iq-tree [7] . estimation of ancestral divergence times and geographic locations for internal nodes of the tree, and identification of branch-specific or clade-defining mutations were obtained using augur and treetime [8] . code for performing these analyses can be found at http://github.com/giraola/covid-19-uruguay and results can be visualized at https://nextsrain.org/community/ giraola/covid19-uruguay. genomes (hereinafter referred as cluster c3-uy) were related each other and were placed within clade b (fig. 2) . these clusters were supported by specific, non-homoplasic mutations (table 1) . together, these results indicate three independent introductions of sars-cov-2 into uruguay. early sars-cov-2 circulation and regional spread. to uncover the spatio-temporal dynamics of sars-cov-2 in uruguay, we performed a phylogeographic reconstruction. the most probable ancestral location for c1-uy was australia, however, very similar viruses have been also sequenced from the united kingdom (fig. 3) . the last common ancestor of c1-uy in australia dated back to mar 5 th (confidence interval (ci): feb 28 th -mar 13 rd ). c2-uy was embedded in a cluster of viruses sequenced in canada, the united states, australia and iceland whose emergence dated back to february 28 th (ci: feb 25 th -mar 4 th ) (fig. 4) . c3-uy likely originated from european viruses circulating in spain since jan 21 th (ci: jan 7 th -feb 5 th ). the last common ancestor of c3-uy in uruguay was tracked to mar 4 th (ci: feb 23 rd -mar 11 th ) (fig. 5) . two viruses from chile were intermingled between viruses from spain and uruguay, indicating regional circulation of these variants. together, these results indicate that sars-cov-2 could be circulating in the country previous to its first detection in mar 13 th . local clusters and genetic differentiation. to further characterize uruguayan sars-cov-2, we identified genetic differences among genomes. c1-uy ancestor likely located in australia presented one nonsynonymous mutation c3096t which affected the orf1ab protein (s955l) and genome uy-10 harbored one additional synonymous mutation a29731g. c2-uy ancestor likely located in canada was characterized by one synonymous mutation a24694t and the last common ancestor to uy-7 and uy-8 presented another synonymous change a11782g. additionally, genome uy-8 presented one synonymous mutation c17470t. c3-uy and chilean genomes displayed one synonymous mutations c17470t which distinguished them from the original spanish lineage. additionally, c3-uy differentiated from chilean genomes by one synonymous mutation c25521t. specifically, among c3-uy genome uy-5 presented a single synonymous mutation c6310t and genome uy-6 presented one non-synonymous mutation c7635a in the orf1ab protein (t2457k). overall, we observed mutations within uruguayan clusters supporting rapid local differentiation (table 2) . using portable, low-cost sequencing approaches based on the minion platform (oxford nanopore technologies) we were able to generate whole-genome sequences of sars-cov-2 just 24 hours after receiving the samples in the lab. this allowed us to determine the most plausible spatio-temporal scenario during the first week since covid-19 was detected in uruguay. consequently, we show that real-time molecular epidemiology responses can be implemented locally as previously done by other countries [9] . our results uncovered sars-cov-2 genomes derived from three independent international origins as early as 3 days after the disease was reported in uruguay. international air transport has likely played a central role in the rapid spread of sars-cov-2, contributing to the current covid-19 pandemic. according to air transport statistics from the world bank (https://data.worldbank.org) and the national government we estimated these introductions could have occurred as early as mid-february, around 1 month before first cases were reported in uruguay in march 13 th . this is in line with observations from other geographic regions, for example, in new york first cases were confirmed in march but a recent study suggested that initial virus introductions could be traced back to february 20 th [10] . also, the virus was likely introduced in europe by a traveler that arrived from wuhan to france, but the recent identification of a group of ill chinese tourists in france previous to this case supports earlier introductions [11] . indeed, sars-cov-2 has been proposed to circulate in humans even before its first detection in december 2019 [12] . together, our results highlight the importance of active genomic surveillance during the ongoing covid-19 pandemic to guide informed decisions, based on assessing the epidemiological behavior of sars-cov-2 in real time. the application of rapid genomic epidemiology during local outbreaks allows to identify main routes of virus introduction and estimate spatio-temporal dynamics which can be used to improve contingency measures. remarkably, tracking the emergence of local genetic variants like those we observed in montevideo, is important to evaluate the sensibility of molecular diagnosis and potential impacts on future anti-viral strategies. subsequent generation of a more comprehensive genomic dataset from the ongoing outbreak in uruguay will allow us to identify domestic transmission patterns and spatio-temporal characterization of local clusters. additionally, setting up coordinated efforts to generate genomic data in south america will allow us to perform integrative analyses to uncover sars-cov-2 dynamics at the continental level. an interactive web-based dashboard to track covid-19 in real time novel coronavirus (2019-ncov) situation report -1 multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples | nature protocols nextstrain: real-time tracking of pathogen evolution an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar mafft multiple sequence alignment software version 7: improvements in performance and usability iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies treetime: maximum-likelihood phylodynamic analysis real-time, portable genome sequencing for ebola surveillance introductions and early spread of sars-cov-2 in the new york city area early release -early introduction of severe acute respiratory syndrome coronavirus 2 into europe a genomic perspective on the origin and emergence of sars-cov-2 acknowledgements. we thank josh quick and the artic network for providing sars-cov-2 sequencing primers and support with sequencing protocols. we also thank to everyone who openly shared their genomic key: cord-348384-8cvt1fo6 authors: butsashvili, m.; gulbiani, l.; kanchelashvili, g.; kochlamazashvili, m.; nioradze, g.; kamkamidze, g. title: knowledge of novel coronavirus (sars-cov-2) among a georgian population date: 2020-05-19 journal: nan doi: 10.1101/2020.05.14.20101642 sha: doc_id: 348384 cord_uid: 8cvt1fo6 introduction georgia confirmed its first case of sars-cov-2 infection on february 26, 2020 and by march 31, a total of 110 cases have been reported. limited understanding about epidemics can lead to panic and disrupt public health efforts to contain transmission. it is important to understand perceptions of the population regarding the disease. this study reports results of a survey designed to understand attitudes and knowledge regarding sars-cov-2 virus among georgian population, including health care workers (hcws). materials and methods the online survey was conducted using a facebook advertisement. the target was the whole country and the language used was georgian. we collected information on demographic data, knowledge of the disease, including perceived differences between coronavirus and influenza. we also included questions to capture the populations perceptions about government preparedness to combat the new coronavirus. results the survey was open for three days (march 2-4, 2020). 5228 participants completed the survey. of these, 40.3% were 25-45 years old, 58.2% were female, 20.7% had university degree and 10.3% were hcws. for 25.8% of respondents, covid-19 and influenza are the same diseases; 10.9% did not know if they are different. the majority correctly identified the transmission route and symptoms (96.9% and 98.0%, respectively). 19.1% think that georgia is ready for covid 19 epidemic, while according to 55% the county is not ready, but hcws are trying hard to respond properly. for 18% response is inadequate. there was no difference in knowledge between hcws, non-hcws and unemployed. 20% of hcws as well as other study subjects believe that sars-cov-2 vaccine and medications do exist but are simply not available in georgia. conclusion given that information regarding coronavirus is changing very rapidly, the need to reach people with time-sensitive educational messages as well as prevention strategies is vital. three months have elapsed since discovery of the novel coronavirus causing severe acute respiratory syndrome and classified as sars-cov-2 [1] [2] [3] . georgia, an eastern european country, confirmed its first case of sars-cov-2 infection on february 26, 2020. despite the government's proactive measures during the early stages of the epidemic, such as restriction of air travel, the number of new infections of sars-cov-2 is increasing and by march 31, a total of 110 cases have been reported [4] . limited understanding about epidemics can lead to panic and disrupt public health efforts to contain transmission as people crowd into grocery stores and use public transportation to prepare for an undefined period of lockdown [5] . thus, it is very important to understand the perceptions of the population regarding the disease and pandemic, and the perceived level of government preparedness to fight against the spread of infection. this study reports results of a survey designed to understand attitudes and knowledge regarding sars-cov-2 virus and perceptions of preventive measures among the georgian population, including health care workers (hcws). the online survey was conducted using a facebook advertisement, which included the title, body text, the banner and the link to the questionnaire. the target was the whole country and the language used was georgian. the study was approved by the institutional review board of health research union. we collected information on demographic data (age, gender, marital status, education, employment status), knowledge of symptoms and transmission modes of coronavirus, perceived differences between coronavirus and influenza, availability of antiviral medication and vaccination. we also included questions to capture the georgian population's perceptions about government preparedness to combat the new coronavirus. the survey was open for three days (march 2-4, 2020), during which time 5228 participants completed the survey. of these, 40.3% (n=2106) were 25-45 years old, 58.2% (n=3042) were female, and 46.7% (n=2440) were married. one in five (20.7%, n=1080) of respondents had a university degree (masters or doctoral) and 10.3% (n=536) were working in the health care field (hcws). for 25.8% (n=1348) of respondents, a perception exists that covid-19 and influenza are the same diseases; an additional 10.9% (n=568) did not know if they are different. in response to the question "are you afraid of getting infected with sars-cov-2?" almost half of study participants (46.3%) said "no." the majority of survey respondents correctly identified the transmission route and symptoms of the new coronavirus (96.9% and 98.0%, respectively). respondents had little knowledge regarding antiviral medications and vaccines against covid-19. specifically, 41.4% (n=2164) did not correctly respond about the existence of antivirals and only 65.6% (n=3429) were aware that a vaccine does not exist. nearly half (45.3%) of the respondents reported that covid-19 mortality rates vary from 2 to 5% (table 1) . . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not certified by peer review) the copyright holder for this preprint this version posted may 19, 2020. the majority of participants (95.2%) reported that they would visit a medical facility if symptoms presented. regarding precautions, 58.3% (n=3044) reported using masks in public places. a large proportion of surveyed individuals (75.4%) declared that wearing a mask is partially protective against sars-cov-2. regarding preparations, we asked "have you stocked up on food for current situation related to coronavirus epidemic?" over half of respondents (53.6%, n=2800) reported that they did not think it was necessary. regarding physical distancing strategies to reduce transmission, 13.2% (n=688) indicated they would attend public events if needed even if they had covid-19 symptoms. 19.1% (n=996) of study participants think that georgia is ready for covid 19 epidemic, while according to 55% (n=2898) the county is not ready, but health care institutions are trying hard to respond to this challenge properly. for 18% (n=954) of study subjects, response to the current epidemic in the country is inadequate. very few (1.1%, n=56) of study subjects visited countries with a high prevalence of sars-cov-2 during the last month (february 2020) ( table 2) . . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not certified by peer review) the copyright holder for this preprint this version posted may 19, 2020. . . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not certified by peer review) the copyright holder for this preprint this version posted may 19, 2020. . younger respondents differentiated covid-19 from influenza better than their older counterparts. among those aged 16-24 years, 67.1% (n=1438) indicated that covid-19 and influenza are not the same, followed by 62.4% (n=1314) of participants aged 25 to 45 and 57.1% (n=560) of those older than 45 years (p<0.0001) ( table 3) . . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not certified by peer review) the copyright holder for this preprint this version posted may 19, 2020. we also found that educational attainment was positively associated with awareness regarding a vaccine and antiviral medications. the majority (82.2%, n=888) of respondents who had achieved a masters or doctoral degree correctly reported that a vaccine against sars-cov-2 does not exist compared to 72.1% (n=1282) of those with a bachelor, 56.7% (n=632) of university students, and 50.0% (n=628) of high school graduates. similarly, 70.6% (n=762) of postgraduates correctly responded that antiviral medications against sars-cov-2 do not exist, followed by 62.1% (n=1104) of university graduates, 53.0% (n=590) of university students and 48.4% (n=608) of respondents with a high school education (p<0.0001). health care workers were less apprehensive about infection. in response to the question "are you afraid of getting infected with covid-19?" more than half (55.2%) of hcws responded "no" compared to their unemployed (46.4%) or non-hcw counterparts (44.3%, p<0.0001). there was no detectable difference in knowledge about the virus or therapeutics between hcws, non-hcws and the unemployed. in fact, approximately 20% of hcws as well as other study subjects believe that a sars-cov-2 vaccine and medications do exist but are simply not available in georgia (table 4 ). . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not certified by peer review) the copyright holder for this preprint this version posted may 19, 2020. we chose a convenience sample of facebook participants for our survey because this method is quick, low-cost and reaches many participants [6] [7] [8] . during an epidemic, a face-to-face interview is not appropriate. due to self-quarantine, telephone usage is increased, thus an online survey is most convenient to ensure all potential respondents have access to survey completion. the major limitation of this method is that internet access is differentially apportioned throughout the population. the georgian statistics department estimated internet coverage in 2019 to be 79.3%, with 86% coverage in urban areas and 69.9% in rural areas [9] . we anticipate that facebook accounts are more prevalent among the younger, educated population, as is reflected in our respondent demographics. the level of knowledge was higher among older individuals, which is consistent with previous studies. an online survey about covid-19 conducted in the united kingdom during march 17-18 similarly demonstrated that older adults consider covid-19 to be life-threatening [10]. however, overall awareness and appreciation of the risks appears to be higher among the uk respondents, 77% of whom worried about a coronavirus outbreak, compared to 44% of georgian respondents. according to our study, the media plays important role in disseminating information regarding the coronavirus pandemic, including among hcws. this appears to be a similar trend found during previous outbreaks. for instance, in a study conducted during the sars epidemic, 92% of participants in a kap survey conducted in china reported that their primary source of information about the disease was television [11] . in conclusion, educational attainment and age are correlated with correct information about covid-19. however, misinformation persists. one in five georgians believe that there is a vaccine and medication to treat coronavirus, but that it is not available in the country. training of hcws is essential to improve their awareness level. more than 10% of georgians would still attend a large public event, even with symptoms of covid-19. the media is the primary source of information about covid-19, widely relied upon by the general public as well as hcws. given that information regarding . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not certified by peer review) the copyright holder for this preprint this version posted may 19, 2020. . https://doi.org/10.1101/2020.05.14.20101642 doi: medrxiv preprint evaluation and treatment coronavirus (covid-19) the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status the covid-19 epidemic tbilisi: prevention of coronavirus spread in georgia the pandemic of social media panic travels faster than the covid-19 outbreak using facebook for health-related research study recruitment and program delivery guide to the design and application of online questionnaire surveys considerations for conducting web-based survey research with people living with human immunodeficiency virus using a community-based participatory approach tbilisi: information and communication technologies usage in enterprises a knowledge, attitude and practice survey on sars in a rural area coronavirus is changing very rapidly, the need to reach people with time-sensitive educational messages as well as prevention strategies is vital. key: cord-350903-nwagvvc5 authors: softic, laurent; brillet, rozenn; berry, françois; ahnou, nazim; nevers, quentin; morin-dewaele, margot; hamadat, sabah; bruscella, patrice; fourati, slim; pawlotsky, jean-michel; ahmed-belkacem, abdelhakim title: inhibition of sars-cov-2 infection by the cyclophilin inhibitor alisporivir (debio 025) date: 2020-06-23 journal: antimicrob agents chemother doi: 10.1128/aac.00876-20 sha: doc_id: 350903 cord_uid: nwagvvc5 cyclophilins play a key role in the life cycle of coronaviruses. alisporivir (debio 025) is a nonimmunosuppressive analogue of cyclosporine with potent cyclophilin inhibition properties. alisporivir reduced sars-cov-2 rna production in a dose-dependent manner in vero e6 cells, with a 50% effective concentration (ec(50)) of 0.46 ± 0.04 μm. alisporivir inhibited a postentry step of the sars-cov-2 life cycle. these results justify rapidly conducting a proof-of-concept phase 2 trial with alisporivir in patients with sars-cov-2 infection. i n december 2019, an outbreak of pneumonia emerged in the chinese city of wuhan. a novel coronavirus was identified as the pathogen causing the disease, named covid-19 (for coronavirus disease 2019). this new virus was called severe acute respiratory syndrome coronavirus 2 (sars-cov-2) because of its genetic proximity to sars-cov. at the time of writing, over 3.5 million people have been diagnosed with covid-19 worldwide, while over 250,000 of them have died from complications of the disease. currently, there are no vaccines or effective antiviral drugs targeting sars-cov-2. a pragmatic approach is to assess whether drugs that are approved for other indications or have reached late clinical developmental stages are effective against sars-cov-2 and could be rapidly repurposed for this indication. for instance, chloroquine has been shown to bear potent antiviral properties against sars-cov-2 in vitro, and several clinical trials are under way to assess its efficacy in patients with covid-19. the nucleotide analogues remdesivir and favipiravir, as well as the antiretroviral drug lopinavir in combination with ritonavir, are also under clinical investigation. cyclophilins are cellular peptidyl-prolyl cis-trans isomerases that catalyze the interconversion of the two energetically preferred conformers of the planar peptide bond preceding an internal proline residue. cyclophilins play a key role in the life cycle of many coronaviruses, including human coronaviruses 229e (hcov-229e) and nl-63 (hcov-nl63), feline infectious peritonitis coronavirus (fpiv), sars-cov, and middle east respiratory syndrome coronavirus (mers-cov) (1-7). cyclosporine a (csa), a potent cyclophilin inhibitor, blocks the replication of various coronaviruses in vitro, including hcov-229e, hcov-nl63, fpiv, mouse hepatitis virus (mhv), avian infectious bronchitis virus, and sars-cov (5, (8) (9) (10) . however, csa cannot be used in patients with covid-19 because of its strong immunosuppressive properties. alisporivir (debio 025) is a nonimmunosuppressive analogue of csa that potently inhibits cyclophilins. alisporivir has been administered to more than 1,800 patients with chronic hepatitis c virus infection in phase 2 and 3 clinical trials, alone or in combination with pegylated interferon alpha and/or ribavirin. in vitro, alisporivir inhibits the replication of hcov-229e, hcov-nl63, mhv, sars-cov, and mers-cov at lowmicromolar concentrations without cytotoxic effect (1, 10, 11) . the goal of this study was to assess the antiviral properties of alisporivir against sars-cov-2, with the objective of generating the preclinical proof of concept of antiviral effectiveness required to start a clinical trial in patients with covid-19. the antiviral effectiveness of increasing concentrations of alisporivir was measured in vero e6 cells infected with a clinical isolate of sars-cov-2 at a multiplicity of infection (moi) of 0.02 (fig. 1a) . dimethyl sulfoxide (dmso) was used as a negative control, while chloroquine was used as a positive control of antiviral inhibition. the compounds were added at the beginning of infection, and viral rna was extracted from supernatants at 48 h postinfection and quantified by reverse transcriptase quantitative pcr (rt-qpcr). alisporivir reduced sars-cov-2 rna production in a dose-dependent manner: the 50% effective concentration (ec 50 ) was 0.46 ϯ 0.04 m, and the ec 90 was 3.10 ϯ 1.40 m. the maximum viral rna reduction was 2 log 10 at 5 m. for comparison, the ec 50 of chloroquine was 0.35 ϯ 0.02 m (fig. 1a) . neither alisporivir nor chloroquine was cytotoxic at the effective concentration, with 50% cytotoxic concentrations (cc 50 s) of ͼ20 m and therapeutic indexes of ͼ43 and ͼ57, respectively. we confirmed the anti-sars-cov-2 effectiveness of alisporivir by immunofluorescence. vero e6 cells were infected at an moi of 0.4 for 2 h in the presence of increasing concentrations of alisporivir. after virus removal, infected cells were incubated for 24 h in the presence of alisporivir and immunostained with an anti-double-stranded-rna (dsrna) antibody. alisporivir reduced the number of sars-cov-2-infected cells in a dose-dependent manner, and complete inhibition was attained at 10 m (fig. 1b) . chloroquine also inhibited sars-cov-2 in this assay (data not shown). the next experiment was aimed at identifying the step of the sars-cov-2 life cycle targeted by alisporivir. chloroquine, which inhibits endosome-mediated viral entry, was used as a control. vero e6 cells were infected at an moi of 0.4 for 2 h in the presence of 5 m alisporivir or chloroquine. after virus removal, cells were incubated for 7 h in the absence of the compounds, fixed, and immunostained with the anti-dsrna antibody. no infected cells were detected in the presence of 5 m chloroquine, confirming that chloroquine prevents sars-cov-2 entry into vero e6 cells. in contrast, alisporivir did not inhibit sars-cov-2 entry into vero e6 cells (fig. 1c) . this result was confirmed by a time-of-addition experiment showing that, in contrast to that of chloroquine, the effect of alisporivir was preserved when the compound was added 3 h postinfection. the antiviral effect of alisporivir was abolished when the compound was added 6 h postinfection (fig. 1d) . these results suggest that alisporivir inhibits a postentry step of the sars-cov-2 life cycle. taken together, our results demonstrate that the nonimmunosuppressive macrocyclic cyclophilin inhibitor alisporivir (debio 025) exhibits strong, dose-dependent antiviral properties against sars-cov-2 in vitro. alisporivir inhibits a postentry step of the sars-cov-2 life cycle through mechanisms that remain to be unraveled. these results justify rapidly conducting a proof-of-concept phase 2 trial to assess the antiviral properties and the effect of alisporivir on covid-19 clinical outcomes in infected patients. alisporivir has been shown to be well tolerated when administered as a monotherapy (12) . preclinical pharmacology data indicate that, after oral administration, alisporivir is widely distributed in the whole body, including the lungs, and that its ec 90 against sars-cov-2 in vero e6 cells is clinically achievable in patients. in addition, because alisporivir inhibits all cellular cyclophilins, it also blocks mitochondrial cyclophilin d, a key regulator of mitochondrial permeability transition pore (mptp) opening, a mechanism involved in triggering cell death. therefore, besides its antiviral properties, alisporivir may also be effective in preventing lung tissue damage. a phase 2, proof-of-concept trial with alisporivir in patients with covid-19 is planned to start very soon. human coronavirus nl63 replication is cyclophilin a-dependent and inhibited by non-immunosuppressive cyclosporine a-derivatives including alisporivir function of hab18g/cd147 in invasion of host cells by severe acute respiratory syndrome coronavirus cyclophilins and cyclophilin inhibitors in nidovirus replication nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors feline coronavirus replication is affected by both cyclophilin a and cyclophilin b genetic deficiency and polymorphisms of cyclophilin a reveal its essential role for human coronavirus 229e replication cyclosporin a inhibits the replication of diverse coronaviruses suppression of feline coronavirus replication in vitro by cyclosporin a influences of cyclosporin a and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229e replication alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model alisporivir plus ribavirin, interferon free or in combination with pegylated interferon, for hepatitis c virus genotype 2 or 3 infection this work was funded by the fondation pour la recherche médicale and the agence nationale de la recherche, call anr flash covid-19.alisporivir was kindly provided by debiopharm, lausanne, switzerland. s.f. has served as an advisor and/or speaker for abbvie; j.-m.p. has served as an advisor and/or speaker for abbvie, gilead, merck, and siemens healthcare; a.a.-b. has served as a speaker for abbvie. key: cord-352909-s11tpfoq authors: sun, zhiping; cai, xia; gu, chenjian; zhang, rong; han, wendong; qian, yun; wang, yuyan; xu, wei; wu, yang; cheng, xunjia; yuan, zhenghong; xie, youhua; qu, di title: survival of sars-cov-2 under liquid medium, dry filter paper and acidic conditions date: 2020-08-14 journal: cell discov doi: 10.1038/s41421-020-00191-9 sha: doc_id: 352909 cord_uid: s11tpfoq nan survival of sars-cov-2 under liquid medium, dry filter paper and acidic conditions zhiping sun 1 , xia cai 1 , chenjian gu 2 , rong zhang 2 , wendong han 1 , yun qian 1 , yuyan wang 2 , wei xu 2 , yang wu 2 , xunjia cheng 2 , zhenghong yuan 2 , youhua xie 2 and di qu 1, 2 dear editor, the pneumonia caused by a novel coronavirus was first reported in december 2019 in wuhan of china, and since then has become a pandemic 1, 2 . international committee on taxonomy of viruses (ictv) named the virus as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 3 . sars-cov-2 is transmitted mainly through respiratory droplets and close contact 4 . the fast spread of the coronavirus disease (covid-19) 3 suggests that sars-cov-2 is highly contagious. the virus remained viable in the medium for 7 days at 22°c and 1 day at 37°c 5 . on dry surfaces at room temperature (rt), the virus was reported viable for 1 day on the surface of cloth, for 4 days on stainless steel, and for 7 days on the outer layer of a surgical mask, whereas no infectious virus was recovered from the surfaces of printing and tissue papers after a 3-h incubation 5 . here, we first investigated the infectivity of sars-cov-2 using a plaque-purified strain ncov-sh01 isolated from a patient in shanghai (genbank mt121215) 6 , studied subsequently its stability in liquid medium, on dry filter paper, and under acidic condition (ph2.2) at rt. it would provide guidance on application appropriate measures to control the spread of sars-cov-2 and improve laboratory biosafety management. first the virus stock of ncov-sh01 was quantified on vero-e6 cells by plaque forming assay (plaque forming unit) and tcid 50 assay (tissue culture infection dose), as 6 × 10 5 pfu/ml and 2.8 × 10 6 sars-cov-2 can be shed into wet or dry surrounding by droplets or aerosol 4 . how stable is the virus in different environment? we first determined viral stability in liquid medium. 1.2 × 10 3 pfu (3.75 log 10 tcid 50 ) virus in dmem was added into each well kept in a wet box at rt. after set for 1, 2, 3, 4, 5, 6, or 7 days, respectively, 100 μl of the virus solution was transferred from each sample onto vero-e6 monolayer. cpe were checked daily till day 5. we found that when the virus had been kept in the medium at rt for 1 day, cpe appeared at 24 h.p.i., which was like the untreated virus control. when the virus had been kept for 2 or 3 days, cpe emerged at 48 h.p.i. (table 1b) . by day 3, the virus titer decreased 2 logs (from 3.75 to 1.35 log 10 t-cid 50 ). when the virus had been left in the medium for more than 4 days, no cpe was observed. the loss of infectivity was confirmed by tcid 50 assay, immune florescence staining with the antiserum against viral n protein ( supplementary fig. s2a ) and qrt-pcr (ct value over the cutoff >38, supplementary table s1). we then investigated viral stability on dry filter paper at rt. 1.2 × 10 3 pfu (3.75 log 10 tcid 50 ) virus in 5 μl dmem was added onto sterilized filter paper in plates. after completely dried, the plates were put into a dry box at rt. after set for 1, 2, 3, 4, 5, 6, or 7 days, the virus on the filter paper was eluted with dmem, respectively. the eluted virus titer was 3.42 log 10 tcid 50 after the virus remained on the paper air dried for 1 h (recovery efficiency was 10 3.42 /10 3.75 = 10 −0.33 = 46.77%) and cpe appeared on day 2 post inoculation. when the virus had been kept on dried filter paper for 1 or 2 days at (table 1c) , and the virus titer dropped to 2.17 log 10 tcid 50 with the 2-day incubation. for the 3-day incubation at rt, cpe appeared at day 5 post inoculation but the virus titer could not be determined by tcid 50 assay, and for the 4-day incubation, no cpe was observed. the loss of infectivity was also confirmed by immune florescence staining with the anti-n serum (supplementary fig. s2b ) and qrt-pcr. our results show that covid-19 virus can survive for 3 days in liquid medium or on dry filter paper. for the 3-day incubation in liquid medium at rt, viable virus left only 1.35 log 10 tcid 50 (initial titer was 3.75). for the 3-day incubation on dry filter paper at rt, although cpe was observed, the survived virus could not be quantified by tcid 50 assay. the loss of virus viability in prolonged incubation (>4 days) was confirmed by n protein immunofluorescence staining, qrt-pcr, and further verified by blind passage of the supernatant for three generations. in alex's study, the virus remained viable in the medium for 7 days at 22°c 5 . the reason for the longer survival time in their report might be the higher viral titer they used (~6.7 log 10 t-cid 50 versus 3.75 log 10 tcid 50 in this study). regardless, our results show that sars-cov-2 is highly infectious and relatively stable in the environment, which underscores the importance of environmental disinfection and hand hygiene. since some coronaviruses can cause infectious diseases of digestive tract via gastrointestinal transmission, such as mouse hepatitis virus (mhv), porcine epidemic diarrhea virus (pedv), and feline enteric coronavirus (fecv) [7] [8] [9] . bioinformatics analysis of single-cell transcriptomes revealed that ace2 was expressed in esophagus squamous epithelium cells and enterocytes of ileum and colon 10 , hence sars-cov-2 might be potentially transmitted via the fecal-oral route 11, 12 . we speculate that sars-cov-2 would have to survive the gastric acidic environment if the virus is indeed transmitted via the fecal-oral route. consequently, we determined the survival of sars-cov-2 under acidic condition in vitro. various amount of the virus (1.2 × 10 3 , 1.0 × 10 3 , 5 × 10 2 , 1 × 10 2 , 0.2 × 10 2 , 0.05 × 10 2 , 0.01 × 10 2 pfu) was treated with acidic physiological saline (ph 2.2) at rt for 30 s, 30 min or 60 min, respectively. after treatment, each viral sample was adjusted to ph 7.28 and added onto vero-e6 monolayer. as shown in table 1d , after a 30-s incubation in ph 2.2 saline, significant cpe appeared at 48 h.p.i. with 1.2 × 10 3 , 1.0 × 10 3 , 5 × 10 2 , or 1 × 10 2 pfu of the virus. cpe appeared at 72 or 96 h.p.i. with lower virus titers (1 × 10 2 , 0.2 × 10 2 , or 0.05 × 10 2 pfu). no cpe was seen with 0.01 × 10 2 pfu virus while cpe from the same amount as the virus control (0.01 × 10 2 pfu) was readily observed at 72 h.p.i. after the 30-min or 60-min "+++", 50-75%; "++", 25-50%; "+", 0-25%; "±", not clear-cut; (table 1d and supplementary fig. s3 ) and confirmed by immune florescence staining (supplementary fig. s4 ). transmission of sars-cov-2 through the fecal-oral route is currently uncertain. although sars-cov-2 rna has been detected in patients' stool 11, 13, 14 , infectious virus was not readily isolated from stool. in the present study, when 1.2 × 10 3 pfu virus was treated with the acidic saline of ph 2.2 for 30 or 60 min, virus survival could be observed as manifested by cpe but failed to be quantified, whereas no apparent virus survival was detected with lower virus titers (<1.0 × 10 3 pfu) treated under the same acidic condition. the results suggest that sars-cov-2 at a certain high titer might survive the acidic environment of the stomach for a certain period. although it is unclear whether the virus can replicate in the intestine, the survived virus in the gastrointestinal tract may be excreted in faeces, which would indicate the importance of stool disinfection. whether the virus can be transmitted through the fecal-oral route needs further study. in conclusion, our findings show that sars-cov-2 can survive for 3 days in liquid medium or on dry filter paper, and the virus at a high titer can survive under acidic condition that mimics the gastric environment. our study would provide guidance on application of appropriate measures to control the spread of sars-cov-2 and improve laboratory biosafety. a novel coronavirus outbreak of global health concern the 2019-ncov outbreak joint field epidemiology investigation team, q. l. notes from the field: an outbreak of ncip (2019-ncov) infection in china-wuhan naming the coronavirus disease (covid-19) and the virus that causes it the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status stability of sars-cov-2 in different environmental conditions isolation of a 2019 novel coronavirus strain from a coronavirus disease 19 patient in shanghai pathogenesis of enterotropic mouse hepatitis virus in immunocompetent and immunodeficient mice feline coronaviruses: pathogenesis of feline infectious peritonitis cellular entry of the porcine epidemic diarrhea virus high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa detectable sars-cov-2 viral rna in feces of three children during recovery period of covid-19 pneumonia covid-19: gastrointestinal manifestations and potential fecal-oral transmission viral load of sars-cov-2 in clinical samples virological assessment of hospitalized patients with covid-2019 author contributions z.s., x.c., c.g., and r.z. performed the viral experiment in bsl-3 lab, analyzed the data and participated in writing the paper. w.h., y.q., yy.w., w.x., y.w., and xj.c. participated in experiments in bsl-3 lab. d.q., y.x., and z.y. designed the experiments, planned the approach, wrote and edited the paper. the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.supplementary information accompanies the paper at (https://doi.org/ 10.1038/s41421-020-00191-9). key: cord-334564-bqh9jkds authors: raony, ícaro; de figueiredo, camila saggioro; pandolfo, pablo; giestal-de-araujo, elizabeth; oliveira-silva bomfim, priscilla; savino, wilson title: psycho-neuroendocrine-immune interactions in covid-19: potential impacts on mental health date: 2020-05-27 journal: front immunol doi: 10.3389/fimmu.2020.01170 sha: doc_id: 334564 cord_uid: bqh9jkds coronavirus disease 2019 (covid-19) is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the impacts of the disease may be beyond the respiratory system, also affecting mental health. several factors may be involved in the association between covid-19 and psychiatric outcomes, such as fear inherent in the pandemic, adverse effects of treatments, as well as financial stress, and social isolation. herein we discuss the growing evidence suggesting that the relationship between sars-cov-2 and host may also trigger changes in brain and behavior. based on the similarity of sars-cov-2 with other coronaviruses, it is conceivable that changes in endocrine and immune response in the periphery or in the central nervous system may be involved in the association between sars-cov-2 infection and impaired mental health. this is likely to be further enhanced, since millions of people worldwide are isolated in quarantine to minimize the transmission of sars-cov-2 and social isolation can also lead to neuroendocrine-immune changes. accordingly, we highlight here the hypothesis that neuroendocrine-immune interactions may be involved in negative impacts of sars-cov-2 infection and social isolation on psychiatric issues. in december 2019, a new outbreak of severe acute respiratory syndrome (sars) emerged in wuhan, china. caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the coronavirus disease 2019 (covid-19) caused a national outbreak of severe pneumonia in china and quickly spread worldwide. according to the world health organization (who) official website, on may 6th, 2020, 3,595,662 people have been tested positive for sars-cov-2 infection and 247,652 deaths have resulted from sars-cov-2 worldwide (1). the disease, initially restricted to china, is now a pandemic, comprising all continents so far except for antarctica, thus having become a major planetary health issue (1) . the most common symptoms of covid-19 are fever, cough, dyspnea, sputum production, myalgia, headache, diarrhea, rhinorrhea, anosmia, and ageusia (2, 3) . nevertheless, symptoms of post-traumatic stress disorder (ptsd), anxiety and depression have also been prevalent in patients infected with covid-19 (4, 5) . besides, sars-cov-2 rna was detected in the cerebrospinal fluid of a patient (6) and increasing evidence points out that coronaviruses (covs) may invade the central nervous system (cns) (7) . thus, we describe here the likely routes by which sars-cov-2 can invade the brain. since covid-19 is associated with increased levels of pro-inflammatory cytokines (8) , an immune signature shared with several psychiatric disorders, we propose how the relationship between sars-cov-2/host can possibly impair interactions between the immune, nervous and endocrine systems, leading to psychiatric symptoms. furthermore, once millions of people worldwide are isolated in quarantine to minimize the transmission of sars-cov-2 (9), we also discuss herein evidence on the negative impacts of social isolation measures upon mental health, gathering evidence that explains how social isolation can also lead to neuroendocrine-immune changes, impairing mental health. accordingly, it is likely that both sars-cov-2 infection and social isolation epidemiological measures to contain the pandemic can lead to changes in psychoneuroendocrine-immune circuits with impact on the appearance and/or evolution of mental health impairments in infected subjects, as well as in those individuals that, even though not being infected, are subjected to social isolation due to one or more risk factors. finally, we provide some suggestions for how future research could confirm the hypotheses outlined here, as well as intervention strategies that mitigate the impact of covid-19 pandemic on mental health. coronaviruses (covs) comprise a large enveloped nonsegmented positive-sense rna virus, which belong to the family coronaviridae, within the order nidovirales (10). they are classified in four genera, namely alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, based on their phylogenetic relationships and genomic structures (10) . the α-cov and β-cov are able to infect mammals, whereas the γ-cov and δ-cov tend to infect birds (11) . previously, six covs have been identified as capable of infecting humans (human coronaviruses-hcovs): α-cov hcov-nl63 and hcov-299e, and β-cov hcov-oc43, hcovhku1, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov). the last two hcovs are considered the most lethal among them. however, the novel sars-cov-2 has shown a mortality rate that is presently also expressive (11) . sars-cov-2 infection leads to a clinical picture characterized by highly lethal pneumonia with symptoms similar to those reported for sars-cov and mers-cov (12) . genomic analysis show that sars-cov-2 shares highly homological sequence with sars-cov (13) . although the existence of more than one receptor for this virus cannot be excluded by now, evidence so far reveals that sars-cov-2 enters human host cells using the same receptor of sars-cov, the human angiotensin-converting enzyme 2 (hace2) (14) . consequently, most of the infection mechanisms detailed for sars-cov could be applied to this novel virus. hcovs may enter the cns through distinct routes: hematogenous and/or neuronal retrograde dissemination (7) . the neuronal route can occur through at least two different pathways: (a) via olfactory nerves and/or (b) via enteric nervous system (7, 15 ). an experimental study using k18-hace2 transgenic mice for the expression of hace2 (i.e., human sars-cov receptor) showed that sars-cov, when given nasally, could invade the brain, likely via the olfactory nerves (16) . however, the non-expression of ace2 in neurons in the olfactory system (17, 18) leads to question whether this is really a possible route for sars-cov-2 entry into cns, although it is not yet possible to rule out the possibility that other ace2-independent mechanisms are involved in the entry of sars-cov-2 into host cells. by contrast, ace2 expression is abundant in small intestine endothelial cells (18) , which connect with neurons in the enteric nervous system. in addition, gastrointestinal symptoms are commonly seen in a part of patients with covid-19 (12, 19, 20) and sars-cov-2 was isolated from oral and anal swabs of these patients (21) . in this way, the enteric nervous system, via the vagus nerve, can also be a possible pathway for sars-cov-2 to enter the cns. similarly, the hematogenous route can occur by at least two mechanisms: (a) through infected leukocytes that cross the blood-brain barrier carrying the virus to the brain and/or (b) through direct infection of brain microvascular endothelial cells, which express ace2 (22) . nonetheless, the hematogenous route does not seem to be involved in the cns invasion by sars-cov, since virtually no viral particles were detected in non-neuronal cells of the infected brain areas in the early stage of infection (23) (24) (25) . yet, the precise route(s) by which sars-cov enters the cns remain(s) to be determined. the recent sars-cov-2 rna detection in the cerebrospinal fluid of a patient with covid-19 (6), as well as its similarities with the sars-cov, emphasizes the need to conduct studies aiming at evaluating the neuroinvasive potential of sars-cov-2 in animal models and humans. sars-cov genomic sequences in human brain tissues were found mainly in neurons of the cerebral cortex and hypothalamus, but not in the cerebellum (23, 24) . however, pre-clinical studies with k18-hace2 mice infected by sars-cov revealed viral particles during acute phase in other brain regions besides the cortex and hypothalamus, such as cerebellum, midbrain (e.g., dorsal raphe and substantia nigra), thalamus, amygdala, hippocampus, basal ganglia (e.g., caudate-putamen and nucleus accumbens), cortex (e.g., frontal, infralimbic, and cingulate), and olfactory bulb (16, 26) . in these animals, a rapid spread throughout the brain was accompanied by significant neuronal loss in the cingulate and infralimbic cortices and the anterior olfactory nucleus (26) . interestingly, high levels of cytokines and chemokines, most notably interleukin-6 (il-6) and interferon gamma (inf-γ) were found in brain of k18-hace2 transgenic mice infected by sars-cov (16, 26) . rather surprisingly, minimal signals of local inflammation were observed, and apoptotic or necrotic cells were not detected (16) . considering the high-expression of inflammatory mediators along with a lack of other inflammatory signals, how sars-cov can be leading to neuronal death remains unknown. cell death non-inflammatory processes, such as autophagy, may be an explanation (16) . since autophagy is related to several neurodegenerative and psychiatric diseases (27) , evaluating whether infection by sars-cov-2 can lead to neuronal death by autophagy may also be important for future relationships between sars-cov-2 infection and mental health outcomes. several studies have demonstrated psychiatric manifestations in patients with mers or sars during the acute phase, such as increased stress levels, impaired memory, symptoms of depression, anxiety, ptsd, psychoses, and suicidal behavior (28) (29) (30) (31) (32) (33) . long-term damage has also been seen in these patients. survivors of sars, months or years after the acute phase of the infection, may also exhibit impaired memory, sleep disturbances, increased levels of stress, depression, anxiety, and ptsd symptoms (32, (34) (35) (36) (37) (38) . to date, few studies have evaluated the possible mental health outcomes of sars-cov-2 infection. however, corroborating the data observed in patients with sars, a study recently demonstrated a prevalence of 96.2% of ptsd symptoms in 714 patients with covid-19 during acute phase (4) . another study reported a prevalence of 34.72 and 28.47% of anxiety and depression symptoms, respectively, in 144 patients with covid-19 (5). taken together, these data indicate that infection with these hcov, especially sars-cov-2, can yield a negative impact on mental health, both in the short-and longterm time windows. supplementary table 1 summarizes studies that reported mental health outcomes in patients with mers, sars, or covid-19. many factors can influence the results of studies that have reported symptoms or development of psychiatric disorders in patients with mers, sars, or covid-19. among them (a) the work directly with health care, (b) the presence of family history of psychiatric illnesses, (c) less social support, (d) older age, (e) the isolation, and (f) the use of high doses of steroids during the acute phase (see supplementary table 1) . however, some patients who survived sars displayed psychiatric manifestations that appear to be disproportionate to the extent of lung infection or expected side effects of corticosteroid therapy (25, 28, 39) . furthermore, it has been reported that one patient developed progressive neurological symptoms starting at day 28 after the onset of the disease. this patient eventually died due to the sars-cov infection, and an autopsy revealed the presence of the virus in the brain, together with neuronal necrosis, glial hyperplasia, and edema (25) . although the studies cited above have been conducted with small samples of patients, they suggest that the psychiatric manifestations seen in at least some patients might be a direct effect of the infection of sars-cov. also, studies with humans are important to evaluate and highlight the possible psychiatric outcomes in patients with sars-cov-2 infection. a "cytokine storm" has been proposed as a key mechanism in the sars-cov-2 pathophysiology and related to lung damage and lethality observed in patients bearing covid-19 (8) . accordingly, increased circulating levels of several cytokines have been found in patients with mers, sars, or covid-19 (see table 1 ). interestingly, high levels of pro-inflammatory cytokines (e.g., il-6 and inf-γ) were also found in the cns of k18-hace2 transgenic mice infected by sars-cov (16, 26) . this evidence supports the existence of an immune signature characterized by increased levels of pro-inflammatory cytokines involved in the pathophysiology of different pathogenic sars-cov in humans. furthermore, higher serum levels of pro-inflammatory cytokines (e.g., il-6 and ifn-γ) and chemokines were found in sars patients with severe disease, as compared to individuals with uncomplicated sars (44) (45) (46) . recently, dysregulation of the immune response similar to sars-cov infection has been observed in patients with sars-cov-2 in wuhan (china). particularly, a significant increase in the serum levels of several pro-inflammatory cytokines, or corresponding cytokine receptors, in severe patients (n = 286) than the non-severe ones (n = 166), including il-6, tumor necrosis factor alpha (tnfα) and interleukin-2 receptor (il-2r) (52) . similarly, intensive care unit (icu) patients (n = 13) with severe sars-cov-2 infection displayed higher plasma levels of cytokines, such as il-2 and tnf-α, when compared with non-icu patients (n = 28) (12) . a previous study identified psychiatric manifestations (e.g., psychosis, cognitive impairments, depression, and anxiety symptoms) in patients during the acute phase of sars-cov infection (28) . the authors also found an association between the severity of symptoms and some psychiatric outcomes. if the increase in cytokine levels and the manifestation of psychiatric symptoms are related to the severity of the symptoms of sars-cov infection, the "cytokine storm" might also be related to the "mental health thunderstorms" seen in patients with covid-19? accordingly, a possible mechanism concerning the relationship between sars-cov-2 infection and mental health outcomes is the involvement of neuroimmune networks. table 2 shows that increased levels of various cytokines can be seen in several psychiatric disorders, an immune signature shared with the sars-cov-2 infection. soluble cytokines that reach the brain, or corresponding local altered levels can influence synthesis, release and reuptake of several neurotransmitters, including monoamines, such as dopamine, norepinephrine, and serotonin (78) . changes in the metabolism of neurotransmitters are involved in the pathophysiology of various psychiatric disorders, such as depression, anxiety, ptsd, and obsessive-compulsive disorder (79, 80) . since changes in cytokine levels can lead to a disruption in the metabolism of neurotransmitters, triggering behavioral deficits, we hypothesize than the immune system can be placed as a link between sars-cov or sars-cov-2 infection and mental health impairments. evidence shows that cytokines also play a key role in learning and memory processes. in healthy conditions, an increase in gene il-6 n = 13 icu vs. n = 4 healthy n = 286 severe vs. n = 166 moderate n = 5 critical > n = 9 severe > n = 5 mild n = 2/8 icu n = 15 severe vs. n = 28 mild n = 69 severe vs. n = 11 non-severe n = 11 severe vs. n = 10 moderate n = 7 spo 2 <90% vs. n = 36 spo 2 ≥90% il-1β n = 41 infected vs. n = 4 healthy n = 11 severe vs. n = 10 moderate tnf-α n = 41 infected vs. n = 4 healthy n = 13 icu vs. n = 28 non-icu n = 286 severe vs. n = 166 moderate n = 5 critical vs. n = 9 severe vs. n = 5 mild n = 69 severe vs. n = 11 non-severe n = 11 severe vs. n = 10 moderate il-10 n = 41 infected vs. n = 4 healthy n = 13 icu vs. n = 28 non-icu n = 286 severe vs. n = 166 moderate n = 5 critical vs. n = 9 severe vs. n = 5 mild n = 5/8 icu n = 69 severe vs. n = 11 non-severe n = 11 severe vs. n = 10 moderate n = 7 spo 2 <90% vs. n = 36 spo 2 ≥90% il-2 n = 13 icu vs. n = 4 healthy n = 13 icu vs. n = 28 non-icu n = 69 severe vs. n = 11 non-severe il-2r n = 286 severe vs. n = 166 moderate n = 5 critical > n = 9 severe > n = 5 mild n = 11 severe vs. n = 10 moderate expression of il-1β, il-1 receptor antagonist, il-6, and il-18 occurs in hippocampus during long term potentiation (ltp), a process considered to underlie certain forms of learning and memory (81) (82) (83) . while il-1β is related to ltp maintenance, acquisition of learning and memory consolidation, il-6 has opposite effects. however, during peripheral and central diseases in which the brain levels of il-1β and il-6 are increased, both cytokines tend to inhibit the synaptic plasticity, learning, and memory (84) . importantly, high levels of il-6 were found in blood of sars-cov and sars-cov-2 infected patients (see table 1 ), as well as in cns of k18-hace2 transgenic mice infected by sars-cov (16, 26) . impaired memory has also been observed in both acute and convalescent phases of sars infection in humans (see supplementary table 1 ). therefore, it is possible that the increased levels of il-6 are related to the cognitive impairments observed in sars patients. such issue should be evaluated in future studies. interleukin-6 is a well-known pleiotropic cytokine expressed in low levels in healthy individuals, in the presence of homeostasis alterations it becomes higher and rapidly detected, and even after stress agent removal, its levels can be maintained elevated and cause diseases (81, 85) . accordingly, a dysregulation of this cytokine expression counts for the development of psychiatric disorders (86) , as seen in table 2 . recently, gao et al. (55) showed increased levels of cytokines in patients with sars-cov-2, especially il-6, which seems to be directly related to the severity of the disease. evaluating the blood parameters of 43 adult patients positive to sars-cov-2 and subdivided in groups (mild and severe) they found a significant increase in the combined detection of il-6 and d-dimer specially in the severe cases, pointing out the il-6 and d-dimer combination as a potential biomarker to identify early stages or the prognosis of the covid-19 disease (55). in another study, 29 patients were subdivided in three groups (mild, severe, and critical) and had hematological parameters followed up during disease evolution. it was shown that the more severe the case was, the higher was the il-6 level (53). liu et al. demonstrated that not only increased levels of il-6 related to the severity of covid-19, but also that decreased levels of il-6 were positively correlated with the treatment effectiveness and remission of the disease (56) . in this sense, the humanized anti-interleukin-6-receptor (il-6r) monoclonal antibody (tocilizumab), a drug used against rheumatoid arthritis (85) that inhibits il-6 signaling, has been administered experimentally in treatment of covid-19 (87) . the retrospective evaluation of 21 patients demonstrated that tocilizumab was able to improve the respiratory function and restored the levels of lymphocytes in the blood, which can be promising (87) . in a second vein, a meta-analysis study pointed out that treatment with anti-cytokine drugs, including tocilizumab, may have an antidepressant effect (88) . accordingly, we can conceive that this type of treatment may represent a promising therapeutic alternative to be attempted in humans, not only has beneficial effects for respiratory symptoms associated with covid-19, but also for possible depressive symptoms related to the disease. thus, it would be interesting for future clinical studies to evaluate the effects of tocilizumab and other pharmacological treatments not only on symptoms and tests related to respiratory and immune functions, but also on the psychiatric symptoms. it is important to notice that some individual biological characteristics associated with impaired immunity may influence not only the natural history of covid-19, but also the associated psychiatric outcomes. in this context, obesity, which is linked with systemic inflammation and impaired immunity, can increase vulnerability for covid-19 (89, 90) , contributes to neuroinflammation and constitutes an important risk factor for the development or worsening of psychiatric disorders [for review, see (91) ]. another important factor is aging, which is related to an imbalance in the levels of proinflammatory (high levels) and anti-inflammatory (low levels) cytokines and decrease in t-cell-mediated function (92) . these immunosenescence-dependent changes in the elderly may be associated with higher susceptibility to viral diseases, including covid-19 (93) , as well as neuropsychiatric disturbances, such as cognitive impairments (94) . it has been demonstrated the relationship between aging and symptoms of anxiety and depression in patients infected with sars-cov-2 during the acute phase (5). therefore, both obesity and older age may increase the risk of psychiatric symptoms in patients with covid-19; and one hypothesis is that neuroimmune circuits may be involved in this association. in addition, since poor nutrition and sedentary lifestyle are frequent in the elderly population and in overfat individuals, actions that promote the practice of physical activity and adequate nutrition are crucial, as they can potentially be associated with a lower risk for covid-19 and mental health impairments. pregnancy is another important potential factor that can affect the neuropsychiatric outcomes of covid-19. maternal immune activation (e.g., in response to infection) is a risk factor for neurodevelopmental disorders such as autism spectrum disorder (asd) (95) . autism has a complex etiology, involving environmental, and genetic factors. one of the proposed etiologies for asd is viral infection in early stages of development (96). although the mechanisms by which viral infection can lead to autism are not yet known, it is believed that they may occur through (a) direct infection of the infant cns, or (b) due to the inflammatory response of the mother and/or the fetus, which can lead to neuroinflammation, triggering changes in brain development (96). in fact, clinical evidence supports the participation of the neuro-immune mechanisms in the pathophysiology of asd [for review, see (97) ]. while increasing evidence supports the neuroinvasive potential of sars-cov-2, there is still no consistent demonstration of vertical transmission of this virus. in this sense, a recent study reviewing the effects of sars, mers, and covid-19 on gestational outcomes, including vertical transmission, and demonstrated that fortunately this transmission mechanism does not appear to occur in these betacoronaviruses (98) . however, the controversial data on this aspect and the high expression of ace2 detected in the human placenta (99) revealed that the possibility of vertical transmission needs to be further explored in clinical settings. accordingly, it is important to point out that there is still insufficient evidence to support the association between sars-cov-2 infection during pregnancy and the development of asd. nonetheless, we cannot rule out that changes in the maternal immune response triggered by the sars-cov-2 infection may affect neurodevelopment, another aspect that also deserves the attention of the medical and scientific communities. in any case, since increased levels of cytokines have been observed in covid-19 and in psychiatric disorders, it is likely that changes in neuroimmune axes may be involved in the mental health outcomes occurring in covid-19 patients. although this hypothesis is based mainly on studies with other betacoronaviruses, it will be interesting if future clinical studies, for example, include the search for correlations between the levels of inflammatory markers and psychiatric symptoms in covid-19 patients and survivors. studies in animal models infected with sars-cov-2 may also assist in the investigation of possible pathological mechanisms involved in neurobehavioral disorders related to the viral infection. the activation of the hypothalamic-pituitary-adrenocortical (hpa) axis has been observed during pathologies involving an immune/inflammatory process, including viral infections (100) . the activation of this neuroendocrine axis by pro-inflammatory cytokines causes increased glucocorticoid production, a physiological response that contributes to avoid the deleterious effects of excessive production of inflammatory mediators and a non-specific recruitment of cells with no or low affinity for triggering antigens (101) . in this respect, it seems reasonable to imagine a state hyperactivity of the hpa axis in infected patients, due to the "cytokine storm" observed in these individuals ( figure 1a) . a second aspect deserving discussion is the fact that ace2 overexpression in corticotropin-releasing-hormone (crh)producing neurons in the hypothalamic paraventricular nucleus alters the processing of psychogenic stress in mice, decreasing the crh content in the hypothalamus and corticosterone plasma levels (i.e., less hpa axis activation), as well as anxiety-like behaviors (102) . sars-cov infection decreases the expression of ace2 in the lungs and myocardium of infected mice (103, 104) . also, patients who died from sars and had sars-cov detected in the hearts exhibited reduced ace2 levels, when compared to patients who died from a non-sars related sepsis (104) . although sars-cov genomic sequences have been found in the hypothalamus of humans (24) , it remains to be determined whether the virus also decreases ace2 contents in this brain region. in any case, a downregulation of hypothalamic ace2 levels may be considered as another potential mechanism by which sars-cov/sars-cov-2 induces hyperactivity of the hpa axis with consequent psychiatric disturbances that are observed in these patients, such as the anxiety for example ( figure 1b) . however, the role of ace2 in the sars-cov-2 pathogenesis is still unknown and more studies are needed to test this mechanism. by contrast, in a study that prospectively assessed the presence of hormonal changes in 61 sars survivors (without pre-existing endocrine disorders) 3 months following recovery, 24 patients (39.3%) displayed late hpa axis hypoactivity, with hypocortisolism (105). this alteration appeared to be a pathological effect of sars-cov, since nearly two-third of the patients did not use steroids and the majority were young (mean age: 36.5 years) and previously healthy (105) . retrospective data from sars survivors do not support changes in hpa axis activity during the acute phase, suggesting that sars-associated hypocortisolism is a late onset phenomenon (105) . since the "cytokine storm" is seen in the acute phase of sars (see table 1 ), increased cytokine levels are unlikely to be secondary to hpa axis hypofunction. although proinflammatory cytokines classically increase the activity of the hpa axis (i.e., a downregulation mechanism of the inflammatory figure 1 | hypothetical mechanisms by which sars-cov-2 may lead to changes in the activity of the hypothalamus-pituitary-adrenal (hpa). (a) during a viral infection (e.g., sars-cov-2), pro-inflammatory cytokines are released by immune cells present in the periphery (e.g., macrophages, t and nk cells) and/or in the brain (microglia). these cytokines can act at three levels of the hpa axis: increasing (i) the secretion of the corticotrophin-releasing hormone (crh) in the hypothalamus, (ii) the secretion of adrenocorticotropic hormone (acth) in the pituitary, and (iii) release of glucocorticoids (e.g., cortisol) through the adrenal cortex. by any of these actions, the result is an increased release of glucocorticoids, which bind to their receptors present in immune cells, suppressing the synthesis and release of pro-inflammatory cytokines. therefore, it is possible that increased pro-inflammatory cytokine levels in covid-19 may lead to hyperactivity of the hpa axis. however, due to a dysfunction in the negative feedback between the hpa axis and the immune system, this neuroendocrine axis is not able to reduce the production of inflammatory mediators, a possible explanation for why sars-cov-2 infection leads to cytokine storm. (b) hypothalamic ace2 overexpression decreases the activity of the hpa axis in mice, reducing the crh content in the hypothalamus and corticosterone plasma levels. since sars-cov infection is able to reduce the expression of ace2 in other tissues, one hypothesis (based on molecular similarities between sars-cov-2 and sars-cov) is that sars-cov-2 can induce a decrease in hypothalamic ace2 levels, thus contributing to hpa hyperactivity. (c) although pro-inflammatory cytokines classically increase the activity of the hpa axis, some cytokines (e.g., tgf-β) can decrease the activity of this neuroendocrine axis under specific conditions that remain unclear. this is another mechanism by which the sars-cov-2 infection, inducing an exacerbated inflammatory response, may lead to changes in the hpa axis, in this case, hypoactivity. continuous arrows: stimulation; dashed arrows: inhibition. response), under some conditions, tnf-α and transforming growth factor beta (tgf-β) may induce hpa axis hypoactivity (106). therefore, it is possible that some cytokines that are increased in sars patients play a causative role in sars-associated hypocortisolism. as both hyperactivity and hypoactivity of the hpa axis are associated with depression (107, 108) , hypocortisolism can also be associated with depressive symptoms that can be in sars survivors. in addition, due to the similarities between sars-cov-2 and sars-cov, it is possible that this mechanism involved in hpa axis hypoactivity can also be observed in covid-19 ( figure 1c) . thus, studies that simultaneously evaluate the axis hpa activity, cytokine levels, and psychiatric disturbances in patients and survivors of covid-19 will certainly improve the current knowledge. in the above context, it is noticeable that long-term survivors of the acute respiratory distress syndrome often report traumatic memories from the icu. interestingly, these patients displayed lower baseline cortisol levels and higher incidence of ptsd (109) . such an information leads to questions related to the hypocortisolism observed in sars-cov infected patients, which may reflect an exhaustion of the adrenal cortex function, as a result of the viral infection or distress associated with hospitalization. clearly, future studies are needed to assess whether sars-cov-2 can affect the functioning of the hpa axis and whether this is involved in the association between sars-cov-2 infection and mental health outcomes. in clinical settings, it will be important to observe and measure the stress associated with hospitalization, as well as the presence of traumatic memories, as these factors may also be associated with changes in the hpa axis. a dysfunctional glucocorticoid-immune circuitry has been observed in schizophrenia. after a stress paradigm, while healthy patients experienced an increase in cortisol levels, negatively correlated to the subsequent changes in il-6 levels, patients with schizophrenia had elevated cortisol positively correlated to subsequent changes in il-6 levels, suggesting an inability to down-regulate inflammatory responses to psychological stress in this psychiatric condition (110) . it is well-known that stressful life events may precipitate subsequent exacerbations of the illness (111) . interestingly, elevated levels of circulating il-6 have been found in early episode psychosis patients (112) . increased levels of stress or il-6 have also been described in sars or covid-19 patients (see supplementary table 1 and table 1 ). in addition, several studies reported symptoms of psychosis during the acute or long-term phase in sars patients (see supplementary table 1 ). therefore, it is possible that sars-cov-2 infection and stressors related to hospitalization may increase the risk of psychosis by increasing levels of cytokines and/or by disrupting the glucocorticoid-immune circuits. since infections are associated with increased risk of developing schizophrenia (113) , it seems important that future studies further assess the potential association between sars or covid-19 and the development of schizophrenia, as well as highlighting the importance of measures that prevent or reduce the impact of covid-19 on mental health. therefore, it is possible that increased pro-inflammatory cytokine levels in covid-19 lead to hypoactivity or hyperactivity of the hpa axis and, due to a dysfunction in the negative feedback between the hpa axis and the immune system, this neuroendocrine axis is not able to reduce the production of inflammatory mediators. in this sense, we hypothesize that such a dysfunction in the negative feedback between the hpa axis and production of pro-inflammatory cytokines may also be associated with mental health outcomes of the sars-cov-2 infection, thus conceptually corresponding to a psychoneuroendocrine-immune dysfunction. pre-clinical studies will hopefully provide more consistent clues to define a putative causal association between sars-cov/sars-cov-2 infection and behavioral deficits. in addition, animal models should allow a better control of variables that could also affect this association, such as the isolation of infected patients, since social isolation per se can also lead to both immunological and behavioral dysfunctions. in the current scenario, where social isolation measures are being strongly implemented worldwide, it is also important put into focus the potential damage to the mental health of isolated individuals, infected or not, applied the psycho-neuroendocrine-immune approach discussed herein. the exponential increase in the number of people infected with sars-cov-2 is leading to saturation of health services worldwide. to prevent human-to-human transmission and, in this way, slow down the growth of the pandemic, who has recommended that people avoid getting outside as much as possible (9) . although such a measure is necessary to contain the advance of the pandemic, social isolation can cause negative impacts on mental health of individuals. studies on mental health outcomes of the quarantine during other epidemics, including sars and mers, revealed negative psychological effects, such as symptoms of ptsd, depression, stress, anxiety, and fear. some of the predictors of psychological impact included having a history of psychiatric illness, healthcare work, longer quarantine duration, infection fears, boredom, inadequate supplies, inadequate information, and financial resources (114) . results of an online survey that assessed the levels of psychological impact and stress during the initial stage of covid-19 outbreak were recently reported (115) . the responses of 1,210 subjects showed that 8.1, 28.8, and 16.5% had moderate to severe stress levels, anxiety and depression symptoms, respectively. moreover, the general public with no formal education had a significant greater likelihood of depression during epidemic and higher satisfaction with the health information received was associated with a lower mental health impact of outbreak. people that presented sars-cov-2-related symptoms like coryza, cough, dizziness, and myalgia or reported a history of chronic illnesses showed significant high levels of anxiety, depression, and stress. these results suggest an importance of accurate health information to reduce the impact of rumors and show the need for the media to provide, not only true information, but also information in simple language so that to support those people with less educational background during the epidemic (115) . in addition, these data lead to the urgent need of psychological and psychiatric interventions, together with measures to prevent the spread of sars-cov-2, so that to provide, as much as possible, well-being to both infected and non-infected socially isolated people. several studies show that living alone (vs. living with a family member) is associated with elevated levels of depressive symptoms (116) (117) (118) , higher risk of depression (119), and higher mortality (120). yet, it has been emphasized the need for caution in arguing for a negative association between living alone and mental health (121) . one reason is that other factors may influence the association between living arrangements and mental health, such as social networks (121, 122) , social support (116, 123) and neighborhood environment (117, 118, 124) . in a study using data from more than 20,500 individuals in the united kingdom or england, it was shown that prevalence of common mental disorders was higher in people living alone vs. people not living alone. this association occurred regardless of age and gender but was largely mediated by loneliness. therefore, we believe that people living alone may be more vulnerable to the effects of quarantine on mental health than people living with a family member. accordingly, it would be interesting for future studies to assess the influence of different living arrangements on outcomes of quarantine on mental health. in this framework, loneliness has been associated with several psychiatric disorders, such as depression, anxiety, and suicide behavior (125) . importantly, it has been showed that lonely people present several immune dysregulations, such as upregulated expression of pro-inflammatory cytokine genes (126) . on the other hand, several studies have revealed that changes in the immune system play a key role in mental disorders (127) . therefore, it is possible that changes in the immune system are involved in the negative impacts of loneliness on mental health. accordingly, it is conceivable that inflammatory mediators are also involved in the impact of quarantine on mental health, during covid-19. studies with animal models have provided important clues on the neurobiological and the behavioral consequences of social isolation. in rodents, the stress of social isolation is able to lead to changes in several neurotransmitter systems (e.g., dopaminergic, adrenergic, serotonergic, gabaergic, glutamatergic, nitrergic, and opioid systems). indeed, the synthesis, release and even the corresponding receptor expression can be altered in several brain regions (e.g., hippocampus, cortex) of animals submitted to social isolation stress [for review, see (128) ]. disturbances in neuroplasticity-related signaling pathways are also observed in these models (128) . for instance, rats submitted to chronic social isolation stress displayed brain morphological changes such as decreased number of dendritic spines in the hippocampus and prefrontal cortex, as well as decreased brain-derived neurotrophic factor (bdnf) and phosphorylatedprotein kinase b (p-akt) in the dorsal hippocampus (129) . the bdnf/trkb/pi3k/akt pathway had already been described to be an important pathway in the maintenance of synaptic plasticity through translation and transport of synaptic proteins (130, 131) . in this context, a metanalysis study reported a positive correlation between lower bdnf serum levels and depressive symptoms (132) , and patients who present depressive symptoms may have reduced hippocampal volume (133) , which supports the association between neuroplasticity and depressive disorders. the social isolation stress can also lead to hyperactivity of the hpa axis through an increase in corticosterone production and release in rodents (134) . the abnormal levels of glucocorticoid have been related to depressive-like behavior and can affect the hippocampal neurogenesis (135) . additionally, social isolation stress can lead to neuroinflammation, with higher levels of tolllike receptors, il-6 and tnf-α in the hippocampus (136) , as well as increased plasma levels of tnf-α, il-4, il-10, and acth in isolated rats (137) . a recent systematic review reported that social isolation and loneliness may be linked to systemic inflammation (i.e., high levels of c-reactive protein and il-6) in the general population (138) . accordingly, it is conceivable that nervous, immune and endocrine systems can be interacting with each other, mediating neurobehavior impairments induced by social isolation stress. thus, these interactions may be part of the mechanisms by which social isolation during quarantine, via changes in neuroendocrine-immune circuits, can trigger damage to mental health. yet, future studies are needed to understand the mechanisms associated with the psychological damage caused by quarantine. although the whole population can be affected by the psychological impacts of covid-19, some vulnerable groups may experience the same pandemic scenario differently. a recent study based on a multidisciplinary approach called attention for measures that can support the population susceptibilities such as (1) older adults with multicomorbidities, (2) children and women that stay at home and suffer domestic violence, (3) people with preexisting mental health issues, (4) people with learning difficulties, which might be affected by disruption to support and by loneliness, (5) front-line health care workers that can be affected by the fear of infection, and (6) groups that have hard socio-economic difficulties (22) . as previously mentioned, financial problems may enhance the impact of social isolation on mental health during quarantine (114) . interestingly, studies demonstrated that a worse socioeconomic status is directly related to higher systemic levels of inflammatory markers such as il-6 and c-reactive protein [for review, see (139) ]. thus, it is possible that neuroimmune interactions may also be involved in the impacts of financial stress during covid-19 on mental health. this represents a novel possibility, that for sure requires future investigation. in addition, higher levels of inflammatory markers associated with worse socioeconomic conditions may also explain why lower social support is also associated with symptoms of anxiety and depression in patients infected with sars-cov-2 (5). even though the biological mechanisms involved in the impact of socioeconomic status on mental health are still unclear, actions aiming at reducing socioeconomic inequalities should be a priority, in order to mitigate the impacts of covid-19 on mental health. finally, it is important to note that the evidence highlighted here does not contradict the need for the isolation measures that are necessary to control the pandemic. however, they call attention to the usefulness of strategies aiming at reducing the harmful effects of social isolation on mental health of the general public, including the improvement of psychological intervention and the reduction of socioeconomic inequalities. in summary, previous studies have reported psychiatric manifestations in patients infected with sars-cov-2, such as anxiety, depression and ptsd symptoms (4, 5). since increased levels of cytokines have been observed in covid-19 and in psychiatric disorders, we can place immune/inflammatory pathways as one of the mechanisms involved in mental health outcomes of covid-19. changes in the hpa axis have also been observed in sars patients, indicating that alterations in neuroendocrine-immune circuits may be related to the psychiatric symptoms observed in these individuals. therefore, the hypothesis of the present article is that sars-cov-2 infection can lead to neuroinflammatory and endocrine changes, which in turn may reflect poor mental health. however, it is important to note that related biological factors (e.g., older age, female gender, and overfat), together with other factors inherent to covid-19 (e.g., social isolation, financial stress, and adverse effects of treatments) can influence psychiatric outcomes. accordingly, it is likely that the psychiatric symptoms observed in covid-19 patients are due to processes involved in the virus-host relationship, as well as to psychosocial and therapeutic issues associated with the pandemic. a further important aspect to be pointed out is the impact that the covid-19 pandemic can have on people who are isolated to prevent the transmission of the virus and to prevent health system overload. similar to possible mechanisms involved in the impacts of sars-cov-2 infection on mental health, social isolation may also be associated with dysfunctional psycho-neuroendocrine-immune interactions, which in turn can contribute to the development or the worsening of psychiatric disturbances (figure 2) . it urges to put all ours efforts in understanding the pathophysiology of covid-19, including cns infection and the risk of mental health compromise, but also the effects of this pandemic in the healthy isolated individuals, including children and adolescents, so that to prevent a "new generation" of groups in which the risk of developing mental disturbances, as anxiety or depression, could be increased. if nothing is done, we will probably be doomed to face a new mental health "pandemic" in the future. in terms of social aspects, a number of short term simple attitudes or initiatives, can comprise the encouragement to: (a) strengthen bonds using social media and start thinking positively (140); (b) sleep properly and exercise regularly (141); (c) balance the diet, regular daily routine, relaxation exercise and other healthy lifestyle measures (142) . on the other hand, people should be avoid: substance use, eating too much fast food, excessive online activity, excessive watching television, and believing fake news (142) . it is also important to look for strategies that mitigate the impacts of covid-19 on frontline healthcare providers. for instance, as recommended by ho et al. (143) , healthcare organizations should introduce shorter working periods, regular breaks, and rotating shifts. individuals who experience moderate to severe and/or persistence distress should seek help from mental health professionals or in hospitals in cases of emergency situations (142) . in addition, online consultation can be a potential alternative of delivering therapy (144) . based on the similarity of sars-cov-2 and sars-cov, hematogenic or neuronal retrograde dissemination routes (via olfactory nerve) may be involved in the entry of the sars-cov-2 into the central nervous system (cns). in the cns (left) the virus can lead to increase in cytokines levels (e.g., il-2, il-6, tnf-α, il-1β, inf-γ, and il-10) due to its local or peripheral (right) actions. increased cytokine levels are associated to neuronal death, synaptic plasticity impairments, dysfunction in the neurotransmitter metabolism and in the hypothalamic-pituitary-adrenocortical (hpa) axis. likewise, social isolation can also lead to these neuroendocrine-immune disturbances, for instance: increase in cytokine levels, changes in neurotransmitter systems, hpa axis hyperactivity and disturbances in neuroplasticity-related signaling pathways. through these common mechanisms, both sars-cov-2 infection and social isolation can lead to mental health impairments [e.g., impaired memory, depression, psychoses, anxiety and posttraumatic stress disorder symptoms (ptsd)]. il, interleukin; tnf-α, tumor necrosis factor alpha; inf-γ, interferon gamma. we also believe that art (especially music) can be an ally in the quest to improving mental health, whether for inpatients, health care workers, or isolated people. a meta-analysis study reported that music can modulate cytokine levels (including reducing il-6 levels), as well as neuroendocrine-immune responses triggered by stress, including physical stress caused by viral infection (145) . in addition, it has been reinforced that music interferes positively in the immune system when subjected to acute stress (co 2 stress test), also regulating the function of il-6 and the hpa axis (146) . therefore, music therapy can be a further relevant and simple strategy that might be adopted on a largescale basis, for individuals in social isolation (also including medical staff). overall, it is important that political and health authorities pay attention to the mental health of infected and uninfected individuals during the pandemic, looking for prevention and treatment strategies, since poorer mental health can be associated with shorter life expectancy (147) (148) (149) and high economic burden (150, 151) . beyond the immediate and fundamental task of saving lives during sars-cov-2 pandemic, the due care of his mental health should be timely addressed. protocols aiming at minimizing mental problems during the infection as well as during recovering after hospitalization must be designed. in addition, studies that evaluate the impact of isolation during sars-cov-2 pandemic on mental health are important as they can guide new strategies to preserve population mental health in other critical situations that we can live in the future. finally, it is noteworthy that the approach applied herein, related to psychoneuroimmunology in covid-19, should be convergent with a social sciences approach so that to better understanding and to better tackling this disease. hopefully, future studies may test the hypothesis outlined herein to better understand and consequently mitigate the impacts of covid-19 on mental health. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. ír, cf, and po-s designed the study, managed literature searches, and wrote the manuscript. ws contributed to literature searches and writing of the manuscript. pp, eg, and ws critically reviewed the manuscript. all authors contributed to and have approved the final 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covid-19 the psychoneuroimmunological effects of music: a systematic review and a new model the impact of acute stress on hormones and cytokines, and how their recovery is affected by music evoked positive mood understanding excess mortality in persons with mental illness: 17-year follow up of a nationally representative us survey mental disorders and cause-specific mortality excess mortality, causes of death and life expectancy in 270,770 patients with recent onset of mental disorders in denmark, finland and sweden a review of the economic impact of mental illness the economic burden of mental illness we are grateful to arnaldo p. andrade for his valuable suggestions in writing this article. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.01170/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 raony, de figueiredo, pandolfo, giestal-de-araujo, oliveira-silva bomfim and savino. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-345371-pjbviagq authors: lisi, lucia; lacal, pedro miguel; barbaccia, maria luisa; graziani, grazia title: approaching coronavirus disease 2019: mechanisms of action of repurposed drugs with potential activity against sars-cov-2 date: 2020-07-23 journal: biochem pharmacol doi: 10.1016/j.bcp.2020.114169 sha: doc_id: 345371 cord_uid: pjbviagq on march 11, 2020, the world health organization (who) declared the severe acute respiratory syndrome caused by coronavirus 2 (sars-cov-2) a global pandemic. as of july 2020, sars-cov-2 has infected more than 14 million people and provoked more than 590,000 deaths, worldwide. from the beginning, a variety of pharmacological treatments has been empirically used to cope with the life-threatening complications associated with corona virus disease 2019 (covid-19). thus far, only a couple of them and not consistently across reports have been shown to further decrease mortality, respect to what can be achieved with supportive care. in most cases, and due to the urgency imposed by the number and severity of the patients’ clinical conditions, the choice of treatment has been limited to repurposed drugs, approved for other indications, or investigational agents used for other viral infections often rendered available on a compassionate-use basis. the rationale for drug selection was mainly, though not exclusively, based either i) on the activity against other coronaviruses or rna viruses in order to potentially hamper viral entry and replication in the epithelial cells of the airways, and/or ii) on the ability to modulate the excessive inflammatory reaction deriving from dysregulated host immune responses against the sars-cov-2. in several months, an exceptionally large number of clinical trials have been designed to evaluate the safety and efficacy of anti-covid-19 therapies in different clinical settings (treatment or preand post-exposure prophylaxis) and levels of disease severity, but only few of them have been completed so far. this review focuses on the molecular mechanisms of action that have provided the scientific rationale for the empirical use and evaluation in clinical trials of structurally different and often functionally unrelated drugs during the sars-cov-2 pandemic. on january 30, 2020, the world health organization (who) declared the outbreak of severe acute respiratory syndrome coronavirus 2 (sars-cov-2; initially named 2019 novel coronavirus or 2019-ncov) a public health emergency of international concern, highlighting the need for a coordinated international intervention to limit virus spreading. few weeks later, on march 11, 2020, because of the rapid diffusion of the infection, the who announced that sars-cov-2 infection was a global pandemic. the first cases of respiratory disease caused by 2019-cov-2, thereafter officially named covid-19 (corona virus disease 2019), likely occurred from a zoonotic transmission in china in december 2019 and since then infection has spread across 213 countries and territories. as of july, 2020, sars-cov-2 has infected more than 14,000,000 people and caused more than 590,000 deaths (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/ accessed july 19, 2020). coronaviridae define a family of hundreds of enveloped, positive-sense, single-stranded rna viruses that are known to cause diseases in animals. sometimes these viruses become able to overcome the species barriers (spillover event) and, so far, 7 coronaviruses are known to cause human diseases. among these, four human coronaviruses (i.e., hcov-229e, hcov-nl63, hcov-oc43 and hku1) typically affect the upper respiratory tract and cause relatively minor symptoms. however, the other three coronaviruses [severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and sars-cov-2] are able to replicate in the lower respiratory tract and are responsible for severe forms of pneumonia that can be fatal (1). phylogenetic analysis indicates that sars-cov-2 has high similarity (88-89%) with two coronaviruses circulating in rhinolophus (horseshoe bats) (2) , but it is less closely related to the sars-cov (~79% similarity) and mers-cov (~50% similarity). based on the sequence analysis of the 29.8 kb viral genome and on the presence of bats and live animals in the seafood wholesale market in wuhan (hubei province, china), where sars-cov-2 was detected for the first time, this virus might have arisen from bats or materials contaminated by bat droppings in the chinese seafood market areas and transmitted to humans either directly or through an intermediate host (3) . similar to the other respiratory coronaviruses, sars-cov-2 is transmitted primarily via the respiratory route in the form of droplets, with a possible, though yet unproven, fecal-oral transmission route (4, 5) . the virus is stable for several hours to days in aerosols and on various types of surfaces, suggesting that transmission may occur by person-to-person droplets as well as by contact with fomites in the proximity of infected patients (6) . although many individuals remain asymptomatic, 97.5% of diseased patients display clinical symptoms within 11.5 days (7) . patients with covid-19 may exhibit mild to moderate symptoms, most commonly fever, fatigue, dry cough, anosmia/dysgeusia, or severe pneumonia with dyspnea, tachypnea, and hypoxemia. actually, dyspnea is predictive of severe covid-19 and intensive care unit (icu) admission (8) . other symptoms less frequently reported include muscle and joint pain, headache, diarrhea, nausea or vomiting, hemoptysis (9) . severe covid-19 is associated to acute lung injury (ali) and/or acute respiratory distress syndrome (ards) that generally occur 8-9 days after symptom onset. as with sars-cov infection, an aggressive inflammatory reaction is responsible for the damage to the lung, indicating that the disease severity also depends on dysregulation of the host immune responses. respiratory failure is the most common cause of death (>70%) of fatal covid-19 cases. furthermore, the massive release of cytokines by the immune system can result in cytokine storm and septic shock and/or multiple organs dysfunction syndromes in 28% of fatal cases (10, 11) . other causes of death are cardiac failure, coagulopathy and renal failure (11) . sars-cov-2 appears also to target the central nervous system with anosmia and dysgeusia as early symptoms and convulsions that may develop later on (12) . currently, the standard of care in patients showing ards includes oxygen therapy together with the administration of parenteral fluids. furthermore, many patients with severe respiratory distress, hypoxemia and ards require invasive mechanical ventilation, and, if the situation deteriorates, extracorporeal membrane oxygenation support (13) . therapeutic interventions including administration of drugs may vary from country to country and it is extremely difficult to harmonize the different protocols due also to the different disease stages of the patients (asymptomatic, pre-symptomatic, mild, severe, under mechanical ventilation). so far, there is not a standardized effective pharmacological treatment for covid-19, a part from anecdotal evidence of efficacy. the scarce knowledge of the sars-cov-2 biology and of the host-pathogen interactions leading to covid-19 has markedly hampered the prompt identification of suitable targets for the development of new therapies. a large number of exploratory clinical trials and pivotal studies are being carried out worldwide. among them, the international "solidarity trial" launched by the who on march 2020 with the aim to find an effective treatment for covid-19 patients by comparing four different treatments (i.e., lopinavir/ritonavir, lopinavir/ritonavir plus interferon-β, chloroquine/hydroxychloroquine or remdesivir) against standard of care (see also sections 2 and 3). presently, regulatory authorities all over the world underline the need of common and rigorous approaches to clinical trials in order to generate more robust evidence on the safety/efficacy of the different anti-sars-cov-2 treatments or vaccines that are being tested. here, we review the recently published literature on the pharmacological treatments used so far and/or undergoing evaluation in clinical trials, with focus on the biochemical mechanisms of action of repurposed or investigational drugs, classified as agents directly targeting the virus ( figure 1 and table 1 ) and those used to treat the respiratory distress and inflammation associated with the cytokine release syndrome ( figure 2 and table 2 ). in addition, we summarize the main clinical trials completed or still ongoing in sars-cov-2 infected patients. the first step in any viral infection entails binding of the virus to a host cell through its target receptor. both sars-cov and sars-cov-2 entry into cells requires the interaction of the viral spike (s) glycoprotein (the envelope-associated protein conferring coronaviruses the characteristic crown-like morphology) with the angiotensin-converting enzyme 2 (ace2) (14) (15) (16) . ace2 is a dimeric ectoenzyme with dipeptidyl carboxypeptidase activity. although the ace2 mrna has been detected in a variety of tissues (17) , the protein has not always been analyzed or detected. the ace2 protein is expressed at high levels on the surface of the lung alveolar epithelial cells and enterocytes of the small intestine providing an easily accessible route for sars-cov-2 infection (18) . the ace2 protein is also present in smooth muscle, pericytes and endothelial cells of the vasculature, heart, kidney and this might account for the multi-organ dysfunction observed in severe covid-19 patients (18) (19) (20) (21) (22) (23) . other tissue sites where the ace2 protein was detected include, among others, the basal epithelium of the nasal, nasopharynx oral mucosa, the basal cell layer of epidermis, and testis (18, 24) . the viral s glycoprotein is a trimer and each monomer contains two subunits, s1 and s2, of which s1 is responsible for the virus attachment to the host cell surface though the receptorbinding domain (rbd), whereas s2 is required for the fusion of the viral and cellular membranes. after the attachment step, the entry process requires the s protein priming by cellular proteases, consisting in the proteolytic cleavage at the s1-s2 boundary and at a downstream position in s2; this process leads to the exposure of a peptide that is involved in membrane fusion (25, 26) . the s proteins of coronaviruses can be cleaved by various cellular proteases; in the case of sars-cov-2, the transmembrane protease serine 2 protease (tmprss2) plays a critical role in s protein priming, whilst the endosomal cysteine protease cathepsin l may replace tmprss2 in this function in cells other than those of the lung (27,28). moreover, it has been recently demonstrated that the host cell protease furin can cleave the sars-cov-2 s protein at the s1/s2 site cleavage, an essential step for viral entry into lung cells (29). since ace2 is located within lipid rafts, cell infection by sars-cov-2 also requires interaction of the viral s protein with different raft components, including sialicacid-containing gangliosides; this interaction also facilitates the contact of the s protein with the ace2 receptor (30). after cleavage of the s protein, sars-cov-2 can be induced to fuse at both the plasma membrane and the endosomal membrane (27,31). various endocytic pathways have been described as being used for cell infection by different coronaviruses, including clathrin-coated vesicles, caveolae as well as clathrin-and caveolae-independent mechanisms (32,33). different antiviral agents or other drugs used for indications unrelated to virus infections have been used to block sars-cov-2 entry into the host cells either by i) inhibiting virus attachment and proteolytic cleavage of the s protein, ii) targeting key cellular enzymatic activities or proteins involved in the endocytic processes or iii) using a combination of both mechanisms ( figure 1 ). umifenovir (arbidol) is a small indole-derivative molecule with a broad spectrum of activity against dna/rna and enveloped/non-enveloped viruses that prevents viral entry into the host cell (attachment and internalization), behaving as host-targeting and direct-acting antiviral agent (34,35). in particular, due to its hydrophobicity, umifenovir displays high affinity for the lipids of the host cell membranes altering their fluidity and rendering them less prone to fusion with the virus. this agent is also able to interact with aromatic residues of the viral glycoproteins involved in the attachment and in the membrane destabilization necessary for the fusion process. furthermore, umifenovir markedly affects clathrin-mediated endocytosis by hampering the release of clathrin-coated pits from the plasma membrane with consequent slowing of vesicle intracellular trafficking and accumulation of clathrin-coated structures where the viral particles remain trapped (35). finally, based on structural similarities between the umifenovir binding sites in the hemagglutinin of the h3n2 influenza virus and the s glycoprotein of sars-cov-2, it has been suggested that this drug might block the trimerization of the s glycoprotein, which is essential for the virus cell adherence and entry (36). umifenovir is licensed (only in russia and china) for the prophylaxis and treatment of influenza a and b infections but it has shown in vitro activity against infections by hepatitis c and b (hcv and hbv), ebola and other viruses (37). in a clinical pilot trial conducted in sixty-nine covid-19 patients, oral treatment with umifenovir (n=36) showed a tendency to reduce viral load and mortality rate as compared to the control group receiving interferon or other non-specified antiviral agents (0% vs 16%) (38) . the results of a retrospective cohort study in patients with covid-19, without invasive ventilation, who received umifenovir plus lopinavir/ritonavir (n=16) or lopinavir/ritonavir only (n=17), showed a potential benefit of the triple combination therapy to reduce viral load and delay disease progression. in fact, after 14 days of treatment, in the umifenovir-treated group nasopharyngeal specimens were negative for sars-cov-2 in 94% of patients (vs 53% of the control group) and the chest computed tomography (ct) scans were improved in 69% of cases (vs 29% of the control group) (39) . subsequently, umifenovir was tested as monotherapy (n=34) and its activity compared to that of lopinavir/ritonavir (n=16). on day 14 after treatment, no viral load was detected in the umifenovir group, whereas the virus was still found in 44.1% of patients treated with lopinavir/ritonavir (40) . conversely, in another study with non-icu patients (n=45) umifenovir failed to improve the prognosis and virus clearance compared to the control group receiving symptomatic treatment, including the most appropriate supportive care (n=36) (41) . a similar conclusion was drawn by an observational cohort study on the real-world efficacy and safety of umifenovir used as single agent or in combination with lopinavir/ritonavir. there was no evidence that adding umifenovir to lopinavir/ritonavir could shorten the time to negative conversion of sars-cov-2 nucleic acid in pharyngeal swabs or improve the symptoms (42). however, a retrospective analysis of adverse drug reactions in 217 chinese patients with covid-19, by a hospital pharmacovigilance system, reported a lower incidence of adverse effects (that were mostly at the gastrointestinal and hepatic level) for umifenovir compared to lopinavir/ritonavir (18.1% vs 63.8%) (43) . a small retrospective cohort study has recently suggested the use of umifenovir for post-exposure prophylaxis, based on the significant reduction of infection risk observed in family members (n=66 in 27 families) and health care workers (n=124) who were exposed to patients with confirmed sars-cov-2 infection (44) . further clinical studies are ongoing to evaluate the role of umifenovir in covid-19 management, used as monotherapy [nct04260594] or in combination with other antiviral agents [nct04350684, nct04273763]. in the randomized, double-blind, placebo-controlled clinical nct04350684 trial, umifenovir is added to a therapeutic regimen including interferon-β1a, lopinavir/ritonavir and a single dose of hydroxychloroquine plus standard of care. baricitinib is a potent and selective inhibitor of the janus kinases 1/2 (jak1/jak2), currently used in the therapy of rheumatoid arthritis. based on the results of a benevolentai's knowledge graph, the small-molecule kinase inhibitor baricitinib was predicted to alter virus entry by inhibiting ap2-associated kinase 1 (aak1) and cyclin g-associated kinase (gak), which are likely involved in sars-cov-2 endocytosis (45) . the benevolentai's knowledge graphical method uses machine learning to integrate the scientific information on the biological processes involved in viral infection with that on the mechanisms of action of available drugs in order to identify potential new pharmacological targets and therapeutic indications. besides exerting potential direct antiviral effects, baricitinib might prevent the dysregulated production of pro-inflammatory cytokines typically observed in covid-19 patients via the inactivation of interleukin-6 (il6)-jak-signal transducer and activator of transcription (stat) pathway (this activity will be more deeply discussed in section 3, especially regarding the jak inhibitor ruxolitinib). some clinical trials, also including placebo-controlled studies, are evaluating the safety and efficacy of baricitinb, mostly as 2week add-on therapy in patients with mild to moderate covid-19. results from a small study in 12 patients with moderate covid-19 pneumonia, treated with baricitinib in combination with lopinavir/ritonavir [nct04358614], indicated that a 2-week oral treatment with the jak1/2 inhibitor was well tolerated. moreover, although proper control groups were missing, the authors reported improved clinical and laboratory parameters (46) . a favorable clinical course was also reported in an 87-year-old woman, with a mild-to-moderate covid-19, chronically treated with baricitinib for rheumatoid arthritis, who also received other pharmacological treatments to control viral infection (i.e., lopinavir/ritonavir, hydroxychloroquine) (47) . this patient was part of a familiar cluster of covid-19, and the three other family members (husband, son and daughter) received the same antiviral therapy with the exception of baricitinib. interestingly, the patient's husband (90 chloroquine and hydroxychloroquine are among the most frequently used drugs for the treatment of covid-19 patients in view of their potential inhibitory activity on virus entry. however, other mechanisms appear to contribute to their antiviral activity, including impaired receptor recognition by coronaviruses due to altered terminal glycosylation of ace2 (49) and inhibition of viral attachment to the lipid raft as a consequence of a reduced interaction of the sars-cov-2 s protein n-terminal domain with membrane gangliosides (30). indeed, in vitro studies have demonstrated that chloroquine is able to block sars-cov-2 infection at lowmicromolar concentrations (50) (51) (52) . furthermore, chloroquine and hydroxychloroquine exhibit immunomodulatory activity since they reduce the toll-like receptor (tlr) signaling (that plays a crucial role in the innate immune system) and production of inflammatory cytokines, as well as the expression of co-stimulatory molecules in t cells (for a comprehensive review see 53) . so far, however, there is not conclusive or robust clinical evidence on the usefulness of quinolines in covid-19. starting from mid-february, 2020, chloroquine was included in the sixth version of the covid-19 treatment guidelines by the national health commission of the people's republic of china. according to these guidelines the initial recommended chloroquine dose was 500 mg twice daily for no more than 10 days; however, due to safety concerns the maximum therapy course was reduced to 7 days and a lower dose was recommended for patients weighing less than 50 kg. based on clinical trials conducted in china in more than 10 hospitals, treatment of >100 patients with chloroquine is superior to control treatment in preventing pneumonia exacerbation, improving lung imaging results, accelerating virus-negative conversion, and shortening the disease course (54) . however, detailed information on the study design, patient characteristics or control treatment were not provided. the results of a blinded, randomized, controlled chinese trial for covid-19 pneumonia, reported a significant improvement in terms of symptoms and ct findings in patients treated with hydroxychloroquine (n=31; 400 mg/day for 5 days) compared to the control group (n=31) (55) . conversely, in a previous pilot study in 30 treatment-naïve patients with confirmed covid-19, hydroxychloroquine did not show any clinical benefit (56) . a french study on a cohort of 80 patients with severe covid-19 treated with hydroxychloroquine (600 mg/day for 10 days plus the macrolide antibiotic azithromycin for 5 days) did not reveal antiviral activity or clinical benefit (57). a published interim analysis of a double-blind, randomized, phase iib clinical trial [nct0432352] performed in brazil, after enrollment of the first 81 patients with severe ards treated with high and low chloroquine doses (i.e., 600 mg/twice/day for 10 days, n=41; 450 mg twice daily on day 1 and once daily for 4 days, n=40) indicated that the high-dosage group showed a higher incidence of cardiotoxic effects (qtc interval prolongation) and a higher mortality rate compared to the low-dosage group (39% vs 13%) (58). all these patients also received the macrolide antibiotic azithromycin that may induce cardiotoxic effects. these preliminary data indicate that high chloroquine dosage should not be recommended for treating critically ill covid-19 patients. although hydroxychloroquine is better tolerated than chloroquine, both agents may cause in the long-term life-threatening arrhythmias (an effect increased by the concomitant use of azithromycin), leucopenia, neuropsychiatric effects and retinopathy. in addition, quinoline overdose can lead to cardiovascular collapse, seizures and coma (59). therefore, the use of chloroquine/hydroxychloroquine for covid-19 management requires a careful patient selection and monitoring. based on the initial publication in the lancet of the results of a multinational registry analysis conducted by surgisphere corporation, showing that treatment with hydroxychloroquine/chloroquine (with or without a macrolide) in hospitalized covid-19 patients (n=14,888) failed to induce clinical benefit and was associated with higher risk of death and cardiovascular complications compared to control treatment (n=81,144) (60), on may 23, 2020, the who temporarily halted the solidarity trial arm with chloroquine/hydroxychloroquine. thereafter, the article was retracted by three of the four coauthors of the original article since surgisphere (owned by one of the authors) did not make available to a third-party audit the complete dataset used for the study (61) . thus, on june 3, 2020, the who announced that there was no reason to modify the solidarity trial protocol and the arm with quinolines was resumed. nevertheless, on the basis of a low benefit/risk ratio, the fda retracted the emergency use authorization (eua) previously issued to hydroxychloroquine for use in covid-19 hospitalized patients outside of clinical trials. to have a clear view on the overall risk-benefit ratio of using chloroquine/hydroxychloroquine especially in severely ill covid-19 patients we will have to wait for the conclusion of welldesigned, multi-center, randomized, controlled studies. actually, clinicaltrials.gov lists a number of phase 3 studies testing chloroquine and more frequently hydroxychloroquine, alone six of them also received azithromycin (500 mg on the first day followed by 250 mg daily) to prevent bacterial infection. all patients treated with both drugs showed negative nasopharyngeal sars-cov-2 pcr conversion compared to 57.1% of those treated with hydroxychloroquine as single agent and 12.5% of the untreated ones (62) . the results of a french retrospective non-randomized study in a total of 1061 patients treated for at least 3 days with hydroxychloroquine plus azithromycin showed that early treatment with this drug combination was well-tolerated and associated with a very low fatality rate (0.9%) (63) . the mechanism underlying the potential azithromycin activity against sars-cov-2 still needs to be clarified; recently, it has been hypothesized that this antibiotic might inhibit cd147, a glycosylated transmembrane protein that would serve as additional receptor for sars-cov-2 cell invasion (64) . furthermore, azithromycin might stimulate immune responses against the virus by inducing the synthesis of type i and iii interferons, as demonstrated in epithelial cells collected from patients with chronic obstructive pulmonary disease (65) . as mentioned above, a number of clinical trials are currently evaluating azithromycin mostly in combination with hydroxychloroquine for the treatment of covid-19 or as prophylaxis. another approach to inhibit sars-cov-2 infection consists in inhibiting the protease that cleaves the s protein, thus facilitating viral entry and activation. tmprss2 is an androgen(72, 73) . in addition, nafamostat is able to inhibit the coagulation and fibrinolytic systems, the kallikreinkinin system, the complement cascade, and activation of protease-activated receptors (74) . therefore, their anti-inflammatory, anti-coagulant and fibrinolytic properties might contribute to attenuate the symptoms and complications occurring in covid-19 patients. both agents are approved in japan for the treatment of pancreatitis, and nafamostat is also used for disseminated intravascular coagulation and as anticoagulant in extracorporeal circulation. three case reports of elderly covid-19 patients with pneumonia, all taking antivirals like lopinavir/ritonavir and hydroxychloroquine, showed that the introduction of nafamostat induced clinical and radiological improvement without significant adverse effects (75) inhibition of the viral spike protein cleavage by cathepsin l in the late endosome might also result in decreased sars-cov-2 entry into the cells (80) . once sars-cov-2 reaches the endosomes, the cysteinyl proteinase cathepsin l is the main protease that cleaves the s1 once inside the cell, sars-cov-2, like other coronaviruses, uses two third of its positivesense single-stranded rna genome as template to directly translate two open reading frames (orf1a and orf1ab), connected by a ribosomal frameshift site, into the two overlapping polyproteins, pp1a and pp1ab, which are afterward cleaved by viral proteases into 16 nonstructural proteins (nsps) (85) . some nsps (including rna-dependent rna polymerase, helicase and other enzymatic activities required for the mrna capping and proofreading) eventually contribute to form the replication-transcription complex, which is anchored to double-membrane vesicles integrated into a reticulovesicular network of modified endoplasmic reticulum membranes, also including convoluted membranes (86, 87) . the viral genomic rna is encapsulated by the nucleocapsid n protein that thereafter buds into the ergic and acquires a membrane containing the s, e and m structural proteins. finally, the virus is released by exocytosis ( figure 1 ). the 3cl pro /m pro is highly conserved among various coronaviruses, and mutations in 3cl pro /m pro are often lethal to the virus (88, 89) . therefore, 3cl pro /m pro is indispensable for viral replication and thus represents an attractive therapeutic target for inhibiting the coronavirus infection process (89) . this enzyme is a homodimeric cysteine protease whose recognition sequence at most sites of viral polyproteins is leu-gln↓(ser,ala,gly). several previous reports have indicated that the hiv aspartate protease inhibitors lopinavir and ritonavir have the potential to act also as sars-cov protease inhibitors through their binding to 3cl pro /m pro (90) (91) (92) (93) . for hiv treatment, the two drugs are used in combination, but ritonavir is administered at a dose that does not affect hiv protease activity but rather inhibits the cytochrome p450 3a4-mediated metabolism of lopinavir, thus increasing its plasma levels. both drugs bind to amino acid residues present at the active site of sars-cov-2 furthermore, based on a virtual docking prediction study, some hcv ns3/4a protease inhibitors (e.g., simeprevir, paritaprevir, grazoprevir, boceprevir, telaprevir) might also inhibit the 3cl pro /m pro (101, 102) . however, none of these agents is currently clinically evaluated for covid-19 treatment. concerning the other coronavirus protease pl pro , although considered another potential therapeutic target since it is crucial for viral replication, the development of sars-cov-2 pl pro inhibitors is still at an early stage (103), despite several investigational compounds have been found to efficiently inhibit the corresponding sars-cov and mers-cov enzyme (104). another the adenosine analogue remdesivir is one the most frequently tested anti-sars-cov-2 agents and has been firstly approved in japan for severe covid-19. remdesivir was originally developed for rna virus infections and tested for ebola during the 2018 outbreak in democratic republic of the congo but failed to show clinical benefit. it has a broadspectrum antiviral activity, including mers-cov, sars-cov and sars-cov-2, both in vitro and in vivo in animal models (52, (106) (107) (108) (109) . remdesivir is a prodrug that, after diffusion into the cells, is metabolized to the alanine metabolite gs-704277 and further converted into a nucleoside monophosphate, which is highly polar and remains trapped within the cell (106) . host cell kinases eventually convert the monophosphate derivative into a triphosphate nucleotide that is misincorporated into the nascent rna chain by the rna dependent rna polymerase with consequent inhibition of the rna synthesis (110) . remdesivir has been found to interact with the sars-cov-2 polymerase, competing with the physiological atp nucleotide, and to behave as delayed-chain-terminator, since rna synthesis is terminated after the addition of three nucleotides (105, 111, 112) . it should be noted that the efficacy of remdesivir or of other nucleoside/nucleotide-based agents, whose activity relies on their misincorporation into the viral genome, might be counteracted by a coronavirus proofreading exoribonuclease (nsp14) that would enable the virus to evade the pharmacological inhibition (113) . intravenous remdesivir was used to treat the first covid-19 patient diagnosed in the us with rapid improvement of the clinical conditions (114) and is regarded as one of the most promising agents for sars-cov-2. presently, the drug is included in the national institutes (117) . in an uncontrolled study where 61 patients (64% receiving mechanical ventilation) were treated with remdesivir on a compassionate-use basis, clinical improvement at 28 days was observed in 68% of patients (118) . however, this trial raised several criticisms on the study design and result interpretation, due to lack of control, small sample size, inappropriate data censoring, high variability of disease severity (119) (120) (121) (122) . in another study, remdesivir was administered as compassionate treatment to 32 hospitalized patients (18 of whom in icu) and beneficial effects were observed on sars-cov-2 pneumonia, mainly in non-critically ill patients (123) . remdesvir is usually well-tolerated for short courses. however, concerns were raised about its potential toxicity in patients with kidney dysfunction related not only to the drug-mediated injury of renal tubular epithelial cells, but also to the nephrotoxicity associated with the drug vehicle (i.e., sulfobutylether-β-cyclodextrin) required for the intravenous formulation (124) . another nucleoside analogue used for covid-19 is favipiravir, which acts as a competitive inhibitor of the rna-dependent rna polymerase. this agent was previously approved in japan for the treatment of influenza a and b and, in particular, for novel or re-emerging influenza viruses (125) . presently, the drug has been approved in russia for covid-19 and is investigated worldwide. favipiravir, after undergoing intracellular tri-phosphorylation, exerts antiviral effects as a guanosine analogue, through several mechanisms including chain termination, slowed rna synthesis and lethal mutagenesis (due to c-to-u and g-to-a transitions favored by the low cytosine content of sars-cov-2 genome) (126 high levels of il6 and il8 also contribute to hypercoagulation due to activation of the complement and coagulation cascades, causing disseminated intravascular coagulation (150, 151) . waiting for an effective antiviral therapy or vaccine against the virus, any treatment that can decrease the severe symptoms of covid-19 may help to attenuate the mortality rates and to improve the quality of life of severely ill patients. in this regard, several pharmacological therapies, with different mechanisms of action, have been used in order to improve the symptoms related to covid-19. in the next section, we will consider the agents directed against the: a) cytokine storm and b) cardiovascular damage. the first category includes: i) anti-cytokine drugs, such as tocilizumab, sarilumab, siltuximab, olokizumab, ruxolitinib, baricitinib, anakinra, emapalumab, mavrilimumab; and ii) immunomodulating agents, such as interferon-β, interferon-α or interferon-λ, fingolimod, ozanimod, opaganib, cd24fc, allogenic mesenchymal stem cells and the lately reappraised dexamethasone. the second group comprises: the anti-c5 complement monoclonal antibodies (mabs) eculizumab and ravulizumab; anti-thrombotic and fibrinolytic agents; the phosphodiesterase type 5 inhibitor sildenafil; the vasoactive intestinal polypeptide analog aviptadil; and the anti vegf-a mab bevacizumab ( figure 2) . however, the effects of the two drug sets are closely interconnected, as many drugs that have an impact on the circulatory system may also reduce circulating inflammatory cytokines. the first therapeutic agent used to counteract the inflammatory reaction in patients with (162) . furthermore, a larger study on 100 patients with covid-19 pneumonia accompanied by hyperinflammatory syndrome and acute respiratory failure described improvement or stabilization of the respiratory conditions in 77% of patients (163) . case reports also supported the potential benefit from tocilizumab treatment (164) . a chinese small retrospective study in 21 patients showed that tocilizumab is associated with rapid improvement of the clinical symptoms and hypoxemia, and prevention of clinical worsening in severe covid-19 patients, without serious adverse events (165) . however, tocilizumab did not reduce icu admission and mortality rates in 21 critically ill patients with severe covid-19 pneumonia (166) . tocilizumab treatment was found to be associated with an initial rise in il6 levels, as an expected consequence of the mab-mediated inhibition of il6 interaction with its receptors, followed by a significant decrease of the c-reactive protein inflammatory marker (167). however, the d-dimer, measured as an indicator of intravascular fibrin formation, remained unaffected suggesting that tocilizumab might have limited effect on the activation of the coagulation cascade (163) . furthermore, the clinical response to tocilizumab seems to be negatively affected by hyperglycemia, shown to be associated with increased il6 levels (168) . in regard to tocilizumab safety, concerns have been raised about the risk of candidemia, septic shock and possible occurrence of intestinal perforation (an adverse effects reported in rheumatoid arthritis patients), which may be favored by the altered hemodynamics observed in critically ill covid-19 patients (163, (169) (170) (171) . based on these initial data, the efficacy and safety of tocilizumab in severe covid-19 need to be corroborated by the results of the ongoing randomized controlled clinical trials. clinical studies are also testing the other anti-il6 receptor mab sarilumab (fda-and emaapproved for rheumatoid arthritis) and anti-il6 mabs such as siltuximab (fda-and ema-approved for multicentric castleman's disease) and the investigational mab olokizumab. like tocilizumab, sarilumab is able to bind both il6r and sil6r, but being a fully human mab, has a lower risk of inducing neutralizing antibodies and allergic reactions compared to chimeric/humanized mabs (172 strictly dependent on the jak/stat pathway (173, 174) , jak inhibitors have also been used in covid-19 patients with the aim of reducing the excessive inflammatory reaction. among these, the orally administered ruxolitinib is a jak1/jak2 small-molecule inhibitor approved for the treatment of myelofibrosis, polycythemia vera, and graft-versus-host disease (175) . consistently with its mechanism of action, in patients with myelofibrosis, ruxolitinib was able to reduce il6 and tnfα levels and was well-tolerated (176 (177) . furthermore, in covid-19 patients two case reports of diffuse skin reactions with purpuras and a rapid decrease of hematocrit values were described (178) . baricitinib is another jak inhibitor that besides interrupting the jak1/2-dependent signaling involved in cytokine-mediated inflammatory response to the sars-cov-2 infection, might also exert direct antiviral effects by blocking virus entry (see section 2). anakinra is a recombinant, non-glycosylated form of the natural occurring human interleukininterferon-γ, which inhibits its binding to cell surface receptors and the subsequent activation of intracellular pro-inflammatory signaling pathways. emapalumab is fda-approved to treat the severe inflammatory condition of primary hlh in which serum interferon-γ levels are elevated (186). blockade also immune responses against viruses, through enhancing antigen presentation, costimulation, and cytokine production by the effector cells of innate immune system, leading to enhanced adaptive immune responses (190) . the response mediated by type i interferons is more potent, rapid, transient, diffuse and inflammatory, whereas the type iii interferon response is less potent, slower, sustained, anatomically restricted and less inflammatory (188) . interestingly, the cytokine storm associated with covid-19 is due to an uncontrolled response of the immune system to sars-cov2 viral infection that leads not to only to an excessive production of cytokines but also a diminished/delayed interferon response (191) (192) (193) . based on preclinical studies and observations in sars-cov or mers-cov infected patients, the outcome of the interferon-mediated response to the viral infection seems to depend on the viral load and integrity of the host immune system. in particular, if the initial viral burden is low, type i interferons are promptly released and efficiently clear the infection; conversely, if the viral load is high or in elderly patients the early interferon production is hampered and a delayed interferon-mediated response may not only fail to control the infection but also result in inflammation and lung damage (194) . thus, exogenously administered type i interferons would have protective effects as prophylaxis or in the early stage of sars-cov-2 infection, whereas they may deteriorate tissue injury and pneumonia when administration is delayed. concerning the potential therapeutic role of interferon-λ, the lack of pro-inflammatory systemic effects would allow its safe administration also in an advanced phase of the infection (194) (195) (196) . although better tolerated than type i interferons that may cause severe systemic side effects due to the ubiquitous expression of ifnar, a possible disadvantage of interferon-λ is its lack of antiviral effects on infected alveolar macrophages or endothelial cells that do not express ifnlr1 but may serve as virus reservoir (197) . . in an additional trial, interferon-α1b as nasal drops is assessed for low-or high-risk medical staff exposed to sars-cov2 infected patients, as single agent or combined with thymosin α1, respectively [nct04320238]. in regard to interferon-λ, the safety and efficacy of subcutaneous pegylated interferon-λ is tested as immediate/early therapy in non-critically ill hospitalized or ambulatory patients another immunomodulating agent, fingolimod (fty720), an orally administered drug approved for multiple sclerosis, was evaluated in covid-19 patients (199) . once absorbed, fingolimod undergoes phosphorylation to form an analog of the naturally occurring s1p, a lipid signaling molecule whose activity is mediated by the interaction with four subtypes of g protein-coupled receptors (s1p1 and s1p3-5) (200, 201) . after binding to s1p1, fingolimod initially activates the receptor and thereafter down-regulates its expression, causing retention of naïve t cells and central memory t cells in the lymph nodes and induction of lymphocytopenia (202) . nevertheless, fingolimod does not substantially affect memory effector t cells, which play an important role in the defense against infectious agents (203) . moreover, it does not affect humoral immune responses and does not prevent the generation of virus-specific cytotoxic t cells in the lymph nodes (203) . thus, it has been hypothesized that patients on s1p modulators might have a reduced risk of complications from sars-cov-2 infection. furthermore, due to the s1p role in lung endothelial cell integrity (204) , in covid-19 patients, fingolimod might reduce vascular permeability and consequent lung injury (205) . in clinical trials with fingolimod for multiple sclerosis, conflicting results were reported showing either no change or increased risk of viral infections (especially herpes virus) compared controls (206) (207) (208) . severe covid-19 cases have been reported in patients with multiple sclerosis on treatment with fingolimod that was stopped upon sars-cov-2 diagnosis; in all these cases patients fully recovered from infection (205, 209, 210 another molecule that has raised some interest to control the inflammatory response associated with sars-cov-2 infection is cd24fc, a recombinant fusion protein that comprises cd24 attached to the fc region of human igg1. cd24 is a glycosylated membrane protein expressed in hematopoietic cells (including immature b and t cells, granulocytes, macrophages and some epithelial cells) that plays a regulatory role on b and t cell homeostasis (211) . in humans, cd24 is able to suppress inflammation upon interaction with the prr siglec10 and several danger-associated molecular patterns (damps), helping to reduce the host immune response against proteins released by damaged cells. preclinical studies demonstrated that the chimeric molecule cd24fc mitigates the graft-versus-host disease, by decreasing the overall inflammatory response, and, in particular, the release of il1β, il6 and tnfα release (212) . these data provided the biological rationale for the clinical testing of cd24fc also for covid-19, and a randomized, double-blind, placebocontrolled, phase 3 study is currently recruiting severely ill infected patients [nct04317040]. a cell-based approach to modulate the damage deriving from inflammation and altered activation of dexamethasone is an old corticosteroid, i.e., a drug with broad anti-inflammatory and immunosuppressant activity that reduces cell-mediated immunity and various cytokine production. its clinical indications span from pain in the joints to asthma, irritable bowel disease/crohn disease, emesis, multiple sclerosis and various autoimmune diseases, as well as different types of cancer, to name just the most prevalent. it has also been used in the previous although the clinical manifestations of covid-19 are dominated by respiratory symptoms, the disease prognosis is largely influenced by the involvement of various organs, including the heart. cardiovascular complications (i.e., myocardial infarction, acute heart failure and cardiomyopathy, shock and cardiac arrest, dysrhythmias, venous thromboembolic events, acute myocarditis) are associated with a high mortality rate and occur in about 10% of hospitalized patients (227) . furthermore, patients with pre-existing cardiovascular diseases are predisposed to sars-cov-2-induced myocardial injury and infection is associated with a high mortality rate; in these patients, the risk of heart failure and myocardial damage increases to ≥35% (228) (229) (230) . the mechanisms involved in the cardiovascular injury of covid-19 include: i) direct damage upon virus entry through ace2 present in coronary endothelial cells, cardiomyocytes and cardiac fibroblasts; ii) increased oxygen consumption deriving from fever, enhanced adrenergic tone and tachycardia; iii) increased oxidative stress as a result of ros production; iv) massive cytokine release and a state of hyperinflammation that contribute to pneumonia/ards with consequent acute heart failure, as well as endotheliitis leading to disseminated intravascular coagulation, thrombosis and infarction; v) sars-cov-2-induced ace2 downregulation due to receptor shedding or internalization with consequent increase of angiotensin ii (228, 231) . in fact, normally, ace2 degrades angiotensin ii to produce angiotensin 1-7 that is endowed with vasodilating and anti-inflammatory effects. therefore, a reduction in ace2 function after viral infection may result in a dysfunctional renin-angiotensin system, associated with an increase of angiotensin ii, which would lead to vasoconstriction and inflammation. dysregulated immunothrombosis (i.e., clot formation triggered by the interaction of innate immune system components, like macrophages, neutrophils and the complement system, with platelets and coagulation factors, that provides a first line defense against infectious agents) with diffuse microvascular thrombi formation has been also described in covid-19 and involved in multi-organ damage (232, 233 in patients with severe covid-19, high rates of venous thromboembolism and disseminated intravascular coagulation, due to dysregulation of the coagulation and fibrinolytic systems are furthermore, heparin has anti-inflammatory properties by inhibiting il6, il8, tnfα release, c-reactive protein and adhesion of neutrophils to endothelial cells (241) (242) (243) . in a retrospective cohort study, the administration of lmwh to covid-19 patients besides producing anticoagulant effects also reduced il6 levels and increased lymphocyte counts suggesting a beneficial effect towards controlling the cytokine storm (244) . in addition, ufh and lmwh have been shown to inhibit the binding of the s protein of sars-cov-2 to its cellular receptor, ace2, in an in vitro cell system expressing ace2 and tmprss2. these results suggest another mechanism through which heparin may slow down or prevent disease progression in the early phases of covid-19. (245, 246) . fibrinolytic drugs, namely tissue-type plasminogen activator (tpa) as systemic intravenous treatment or as lung-targeted nebulizer form, have been proposed for covid-19 patients (247, 248) . several clinical trials are currently assessing different heparin regimens, other anticoagulants, systemic and local fibrinolytic approaches, and antiaggregants (e.g., rivaroxaban; defibrotide; clopidogrel, aspirin) (clinicaltrials.gov). the pde-5 inhibitor sildenafil, a vasodilator that is approved for treating erectile dysfunction and pulmonary arterial hypertension (249) , is also evaluated in a phase 3 trial for patients with mild to severe covid-19 [nct04304313]. in fact, sildenafil has a wide range of antiinflammatory, antioxidant, and vasodilatory actions resulting in cardioprotective effects and improved pulmonary circulation (250) (251) (252) . aviptadil is a synthetic form of vip that increases adenosine cyclase activity with consequent smooth muscle relaxation, approved in combination with phentolamine only in certain the vegf-a is considered the most potent inducer of vascular permeability. the potential involvement of vegf-a in covid-19 has been related to the excessive production of angiotensin ii, consequent to the sars-cov-2-mediated down-regulation of ace2. in fact, angiotensin ii is able to increase vegf-a expression which in turn may exacerbate inflammation stimulating the recruitment of inflammatory cells and the release of proinflammatory cytokines (255) (256) (257) . numerous studies have confirmed a key role of vegf-a as potential therapeutic target in ali and ards due do the increased vascular permeability and pulmonary edema (258) . furthermore, vegf-a has been involved in disruption of the blood-brain barrier and may contribute to brain inflammation in the course of sars-cov-2 infection (259 bleeding, delaying wound healing, thromboembolic events). all over the world, the scientific community is racing to evaluate a huge number of drugs or [46, 47] (see table 2 ) eidd-2801 inhibition of rdrp -nct04405739 nct04405570 a nct: clinicaltrials.gov identifier data from clinicaltrials.gov accessed on june, 2020. due to the rapidly evolving situation and the increasing number of clinical trials, the reported list of clinical trials does not mean to be exhaustive. b these agents might also have additional mechanisms contributing to the antiviral activity against sars-cov-2 rdrp; rna-dependent rna polymerase. human coronaviruses with emphasis on the covid-19 outbreak, virusdisease. 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vascular endothelial growth factor (vegf) as a vital target for brain inflammation during the covid-19 outbreak effectiveness of convalescent plasma therapy in severe covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma passive antibody therapy in covid-19 effect of convalescent plasma therapy on time to clinical improvement in patients with severe and lifethreatening covid-19: a randomized clinical trial challenges in the production of convalescent hyperimmune plasma in the age of covid-19 a human monoclonal antibody blocking sars-cov-2 infection we would like to acknowledge the support of the "fondazione airc" to the national civil protection to help tackle the covid-19 emergency in italy and its commitment to continue supporting cancer research during the challenging times of sars-cov-2 pandemic. g.graziani is principal investigator (pi) of the airc grant ig 2017 -id. 20353 project. orphan drug for the treatment of ards, ali and sarcoidosis nct04311697 nct04360096anti-vegf-a bevacizumab cancer treatment; age-related macular degeneration (off-label) nct04305106 nct04344782 nct04275414 a nct: clinicaltrials.gov identifier data from clinicaltrials.gov accessed on june, 2020. due to the rapidly evolving situation and the increasing number of clinical trials, the reported list of clinical trials does not mean to be exhaustive.ards: acute respiratory distress syndrome; ali: acute lung injury. key: cord-350992-l6l24pco authors: roldan, eugenia quiros; biasiotto, giorgio; magro, paola; zanella, isabella title: the possible mechanisms of action of 4-aminoquinolines (chloroquine/hydroxychloroquine) against sars-cov-2 infection (covid-19): a role for iron homeostasis? date: 2020-05-13 journal: pharmacol res doi: 10.1016/j.phrs.2020.104904 sha: doc_id: 350992 cord_uid: l6l24pco the anti-malarial drugs chloroquine (cq) and primarily the less toxic hydroxychloroquine (hcq) are currently used to treat autoimmune diseases for their immunomodulatory and anti-thrombotic properties. they have also been proposed for the treatment of several viral infections, due to their anti-viral effects in cell cultures and animal models, and, currently, for the treatment of coronavirus disease 2019 (covid-19), the pandemic severe acute respiratory syndrome caused by coronavirus 2 (sars-cov-2) infection that is spreading all over the world. although in some recent studies a clinical improvement in covid-19 patients has been observed, the clinical efficacy of cq and hcq in covid-19 has yet to be proven with randomized controlled studies, many of which are currently ongoing, also considering pharmacokinetics, optimal dosing regimen, therapeutic level and duration of treatment and taking into account patients with different severity degrees of disease. here we review what is currently known on the mechanisms of action of cq and hcq as anti-viral, anti-inflammatory and anti-thrombotic drugs and discuss the up-to-date experimental evidence on the potential mechanisms of action of cq/hcq in sars-cov2 infection and the current clinical knowledge on their efficacy in the treatment of covid-19 patients. given the role of iron in several human viral infections, we also propose a different insight into a number of cq and hcq pharmacological effects, suggesting a potential involvement of iron homeostasis in sars-cov-2 infection and covid-19 clinical course. chloroquine (cq) and its hydroxy-analogue hydroxychloroquine (hcq), both 4-aminoquinolines, have been extensively used in the treatment of malaria in the last century, but the continuous emergence of drugresistant strains of plasmodium falciparum has accelerated the development of new antimalarial drugs. nevertheless, these compounds, particularly the less toxic hcq, are currently widely used to treat autoimmune diseases like rheumatoid arthritis (ra), systemic lupus erythematosus (sle) and antiphospholipid syndrome (aps), due to their immunomodulatory and anti-thrombotic properties. cq and hcq have also been proposed for the treatment of viral infections, since they have been demonstrated to directly inhibit viral entry and spread in several in vitro and in vivo models. treatment with these drugs has then been proposed for several viruses in humans, including coronaviruses like sars-cov and sars-cov-2. nonetheless, a clear evidence of their utility in human viral infectious diseases still lacks [1] . translation from laboratory to clinic should be based on detailed analysis of results from randomized controlled studies and observational outcome registries focused on the efficacy, duration and toxicities of treatments with these drugs that could be useful to understand their real effectiveness. here we review the current knowledge on the mechanisms of action of cq and hcq as anti-viral, antiinflammatory and anti-thrombotic drugs and discuss the current experimental evidence on the potential mechanisms of action of cq/hcq on sars-cov2. we also propose a different insight into some of cq and hcq effects, suggesting a potential role of iron homeostasis in sars-cov-2 disease (covid-19), similarly to several other human viral infections [2] [3] [4] . finally, we briefly review and discuss the current knowledge on their efficacy in the treatment of patients with covid-19. we conducted a literature search using different database (pubmed, science direct and web of science) up to april 20 th 2020. the search strategy was to use different search terms alone and in any combination, such as "sars-cov-2 disease", "covid-19", "sars-cov-2", "coronavirus", "clinical trial", "treatment", "drug", "chloroquine", "hydroxychloroquine", "iron", "virus", "viral entry", "viral spread", "anti-viral activity", "infection", "inflammation", "immunity", "innate immunity", "cytokine", "il-6", "tnf-", "il-1", "adaptive immunity", "thrombosis", "in vitro". only english articles with available data were included. then, cq/hcq could have inhibitory effects on virus attachment and entry in the host cell, possibly resulting in blocking the viruses in endocytic vesicles. cq/hcq have also been shown to display anti-viral activity even when administered after viral infection. this effect has been observed in vitro also in sars-cov and sars-cov 2 infections [8, 9, 12] . further mechanisms could then be involved in antiviral drug action. through the alkalization of endosomes, cq/hcq might also act inhibiting or preventing endosomelysosome membrane fusion that leads to membrane viral receptor recycling, viral uncoating and viral genome release into the cytosol, as observed for sars-cov [18] . [7] . cleavage sites other than those recognized by cathepsin and tmprss2 have been identified in the s protein of sars-cov-2 that may be cleaved by furin-like proteases. furin is localized in the tgn, is highly expressed in the lung, its cleavage of s protein could be implicated in virus egress and spread [19] and might be inhibited by cq, as observed in chikungunya virus infection [20] . due to their basic properties and consequent disruption of cellular vesicle compartments, cq/hcq may also inhibit virion budding, occurring when encapsidated viral genomes bud into the ergic membranes in which viral envelope proteins are inserted, forming mature virions [21] . within infected airway epithelium, through their binding to toll-like receptors (tlrs), several respiratory viruses (among them some coronaviruses) activate the mitogen-activated protein kinase (mapk) pathways, particularly the p38 mapk. respiratory viruses harness mapk activation to their own advantage, exploiting the triggering of their downstream targets for trafficking their own proteins and for viral assembly and spread. these effects are particularly important in people suffering for underlying airway diseases, like asthma or chronic obstructive pulmonary disease (copd), who undergo a respiratory viral infection, since these pathways are already turned on and aberrantly activated [22] . cq has been shown to hinder viral infections through the inhibition of p38 mapk activation [23, 24] . this inhibitory action could then be of particular importance in covid-19 since people with copd are among the worst affected by sars-cov-2 infection. j o u r n a l p r e -p r o o f therefore, besides viral attachment and entry, also viral uncoating, genome release, protein maturation process and assembly of new virions for budding and spread may be inhibited by the basic drugs, resulting in reduced infectivity. little is currently known about the mechanism(s) of action of cq/hcq in the treatment of sars-cov-2 infection. the first published experimental evidence of the potential efficacy of cq/hcq in this disease comes from in vitro experiments. wang and colleagues [8] results suggested that cq could be more efficacious than hcq. the authors also confirmed that cq acts at both entry and post-entry stages and this double action was also observed for hcq. in order to shed light on potential mechanisms of action of the drugs, the authors studied the cellular localization of viral particles and found virions partly in early endosomes (ees) and more in late endosome-lysosomes (lels) in control conditions. when cells were treated with cq and hcq, more viral particles were observed in ees, suggesting that the drugs block endocytotic vesicle maturation at intermediate stages and probably stall the virus transport from ees to lels, a crucial step for the release of viral genome. the authors also observed that both cq and hcq treatments resulted in abnormally enlarged ees, but while hcq increased lel size and number, cq treatment induced no changes in number and size of lels but the vesicle structure was disrupted, suggesting partially distinct mechanisms of action of the two drugs. the anti-sars-cov-2 in vitro action of cq and hcq was confirmed in the same cellular model by [10] . these authors found ec 50 values of 23.90 and 5.47 for cq and 6.14 and 0.72 mm at 24 and 48h for cq and hcq respectively, that, in contrast with the previous report, was a better performance for hcq. they also confirmed that the drugs have anti-viral activity also when administered prior to viral infection, with ec 50 values of >100, and 18.01 for cq and 6.25 and 5.85 mm for hcq respectively at 24 and 48h. cq/hcq are also used as anti-inflammatory and immunomodulatory drugs in autoimmune diseases, like ra and sle. these properties derive from their multiple effects on the immune system cells and their modulation of crucial pro-inflammatory cytokines [25, 26] . see table 2 for a schematic list of cq/hcq biological activities as anti-inflammatory drugs. cq/hcq may impair the correct maturation and recognition of viral antigens by antigen-presenting cells (apcs) that require endosomal acidification for antigen processing. by phagocytosis and macropynocytosis, dendritic cells (dcs) capture pathogens, process and present their antigens to activate t cells. through their antigen-specific b cell receptor and cme and clathrin-independent endocytosis, b cells recognize specific antigens and present their peptides to specific t cells. through phagocytosis, cme, caveolin-mediated endocytosis and macropinocytosis, macrophages internalize pathogens, process their proteins and present antigens to t cells. t cells, through their antigen specific receptors, interact with apcs, recognizing antigenic peptides immobilized on their surface by major histocompatibility complex (mhc) class i (for cd8 + t cytotoxic cells) and mhc class ii (for cd4 + t helper cells) molecules. mhc class i molecules are loaded with those antigens mainly derived from proteasome degradation of cytosolic proteins (like viral proteins) in the er, but also from phagocytosed/endocytosed material, processed through endosomes and lysosomes. mhc class ii molecules bind antigens obtained by endosomal and lysosomal processing and, as mhc class i, require the low ph of these organelles to activate protease and other enzymes and form the complexed antigen [27, 28] . cq downregulates picalm affecting cme and possibly antigen-processing [15] . cq/hcq also alkalinize vesicles of the endosome-lysosome-autophagy pathway, possibly further interfering with antigen presentation by apcs, both acting on antigen degradation and mhc molecules processing. the inhibition of antigen presentation may reduce t cell activation and differentiation and in turn decrease the production of pro-inflammatory cytokines. tlrs are involved in innate immunity against bacteria and viruses. tlr7 and tlr9 are located in the er and move to endosomes and lysosomes where they recognize bacterial and viral nucleic acids. by this binding, their activation results in the production of pro-inflammatory cytokines and chemokine like interleukin-6 (il-6), interleukin-12 (il-12), type i interferons (ifn1s), interferon- (ifn-) and cytokines promoting the stimulation of th1 cells and disrupting the th1/th2 balance. the activation of tlr7 and tlr9 by their ligands requires the low ph of endosomes and lysosomes and an active autophagic pathway, then cq/hcq, through vesicle alkalinization, may inhibit this interaction and tlr signaling, in turn inhibiting the inflammatory response [31] . matrix metalloproteinases (mmps) are a family of zinc endopeptidases that regulate inflammation and tissue repair at several levels. mmps regulate pro-inflammatory and antiinflammatory cytokine and chemokine production by proteolytic processing and may in turn be induced by cytokines and chemokines. cq has been shown to downregulate mmp-9 expression through the inhibition of tlr9 signaling [32] . cq has also been demonstrated to inhibit phospholipase a2 and block the arachidonic acid cascade that leads to the production of prostaglandins and tromboxanes. in this context, cq may have both antiinflammatory and anti-thrombotic effects [33] . this is an important point, since sars-cov-2 can induce pulmonary microthrombi and coagulopathy, being characterized in its severe forms by d-dimer level elevation [34, 35] . interestingly, phospholipase a2 has recently been shown to be critically involved in coronavirus replication due to its role in the production of lipids required to form the membranous structures that are essential for virus replication and transcription [36] . then, inhibiting phospholipase a2, cq may be a triple arm against sars-cov-2, acting through the anti-inflammatory, anti-thrombotic and antiviral mechanisms. as stated above, respiratory viruses can activate the p38 mapk pathway in infected airway epithelium and harness the trafficking machinery of infected cells to assist viral assembly and spread. p38 mapk activation also leads to the release of cytokines like tumor necrosis factor- (tnf-α), interleukin-1 (il-1β), il-17 and il-6 that turn on the inflammatory response, but this reaction can be inhibited by the cq [22, 23] . as previously mentioned, covid-19 is characterized by the release of high levels of il-6, il-1 and tnf-that arekey modulators of inflammation [37, 38] . cq/hcq have been shown to inhibit their release by immune cells through several mechanisms like transcription and post-transcriptional regulation, p38 mapk signaling block as stated above or disruption of cellular iron metabolism among others [6, [39] [40] [41] [42] [43] [44] [45] [46] [47] . tnf- is then, the challenge for anti-il-6 therapy in covid-19 is to identify when il-6 is detrimental or beneficial that is when clinicians would act on this pathway [34] . a possible role of hcq in the prevention of thrombosis in hip replacement surgery has been proposed since the late 1970s [57-60]. in this pathology, a decrease of pulmonary embolism has been reported using hcq doses between 600 and 1600 mg per day [61, 62] . hcq is now commonly used as therapeutic in sle and j o u r n a l p r e -p r o o f aps, also due to its ability to decrease the incidence of thrombosis at the dose of 200 mg to 400 mg per day [63] [64] [65] [66] [67] [68] . some authors associated this anti-thrombotic effect to the decrease of the anti-phospholipid (apl) antibody titers [69] [70] [71] , while others to a more general and transversal contribute to immunomodulatory, anti-inflammatory, metabolic and anti-thrombotic effects [25, 72] . chinese cardiologists have reported diffuse micro-vascular thrombosis in various organs on autopsy examination in patients died for covid-19, particularly in the lung. taking into consideration this diffuse thrombosis, chinese physicians have recommended anti-coagulant therapy in these patients, but no pharmacological protocols have currently been published [73] . the anti-thrombotic effect of cq/hcq may derive from different mechanisms. see table 3 for a schematic list of cq/hcq biological activities as anti-thrombotic drugs. cq/hcq could act by interfering with platelet aggregation, decreasing the activation of collagen and alpha granule discharge [74, 75] . platelet activation induces degranulation with the release of adenine diphosphate (adp) and other molecules in order to amplify the platelet activation process. hcq has been shown to decrease adp effects on platelet activation and in vitro platelet aggregation induced by the antibiotic ristocetin [74, 76] . as mentioned above, cq/hcq could inhibit phospholipase a2 and decrease arachidonic acid, prostaglandin and tromboxane release [33, 77] . when administered within 48h before surgery, hcq induces the increase of fibrinogen causing a decrease of plasmatic and blood viscosity [78] . a possible role in rheological property decrease of red blood cells (rbcs) has been proposed, but this effect is not perfectly clarified [79] . neutrophil extracellular traps (nets) are released by activated neutrophils that unleash intracellular molecules such as granules, proteins, dna and histones in tissues or circulation to capture and kill pathogens during infections [80] . nets have been shown to increase thrombosis in autoimmune diseases and in inflammation [81, 82] . golonka and colleagues [83] have recently hypothesized that neutrophil abundance in covid-19 could be associated to the increase of nets. in a murine model of pancreatic adenocarcinoma, nets have been shown to stimulate the discharge of tissue factors and promote platelet activation and aggregation. in this model, cq/hcq showed, besides their proper anti-thrombotic characteristics, the ability to inhibit nets, in turn further decreasing hypercoagulability [84] . j o u r n a l p r e -p r o o f hcq has been shown to inhibit the binding of the apl antibody-2-glicoprotein i (gpi) complex to the phospholipid bilayer, decreasing the pro-thrombotic effect [85] . annexina5 (anxa5) is an anticoagulant protein with great affinity for negatively charged phospholipids. this protein forms a 2-dimensional crystal over membrane phospholipids and exerts its potent anticoagulant activity shielding the phospholipid bilayer from the interaction with coagulation enzymes [86] . in vitro experiments on human umbilical vein endothelial cells (huvec) have evidenced the protective effect of hcq, which acts restoring the anxa5 anticoagulant shield and therefore prevents the beginning of coagulation cascade [87, 88] . hcq is used in the treatment of aps. its ability to reduce endothelial dysfunction has been studied in animal models. vascular reactivity has been studied in mice injected for three weeks with apl antibodies directed against 2gpi to induce aps, with or without hcq co-treatment. hcq improved endotheliumdependent dilatation by the amelioration of no bioavailability and by the decrease of oxidative stress [89] . in another study, apl antibodies were administered in mice and in human aortic endotelial cells (haec), in the presence or absence of hcq, to evaluate endothelial function and endothelial nitric oxide synthetase (enos) modulation. in both models, thrombosis was evaluated by thrombin generating time (tgt) and tissue factor (tf) production. apl antibodies increased vascular cellular adhesion molecule 1 (vcam-1) expression and decreased endothelial relaxation induced by acetylcholine in haec cells. in mice, apl antibodies increased thrombus size and shortened the time needed to generate arterial occlusion. tgt and tf were increased in both models. hcq confirmed its anti-thrombotic activity decreasing clot formation, ameliorating tgt and improving endothelial relaxation. in addition, hcq ameliorated enos activation, increasing the p-enos/enos ratio with consequent improvement of nitric oxide (no) production [90] . cytokines like tnf-, il-1 and apl antibodies induce endosomal nadph oxidase (enox) that is implicated in the pro-inflammatory signal transduction. hcq inhibits these up-regulation in human monocytes, preventing the translocation of the catalytic subunit gp91phox in the endosome and so interfering with enox functions. further, hcq decreases the thrombotic effect induced by apl antibodies by inhibiting ndaph oxidase 2 (nox2), the nadph oxidase platelet isoform that is involved in platelet activation [91] . an additional indirect effect of hcq on thrombosis is its ability to improve blood lipid profile. studies in sle and ra patients treated with steroids have evidenced lower ldl cholesterol and triglyceride levels [92] . these data were confirmed in further sle cohorts treated with hcq in association with steroids: the treatment decreased total cholesterol and vldl level and increased hdl cholesterol [93, 94] . iron is an essential element for all organisms. this is due to its redox potential which makes it an essential cofactor for several proteins and enzymes involved in vital cellular functions like energy production, dna replication and transcription. even most viruses need iron, since they require the host metabolic apparatus to replicate their genome and produce mrnas for their translation in functional viral proteins [2] . therefore, while cellular iron repletion may boost viral replication and spread, iron deficiency may interfere with viral life cycle. during infections and inflammation, anemia is frequently observed and caused by pro-inflammatory cytokines. some of them directly affect iron homeostasis, like il-1, tnf-and il-6. the release of these cytokines, mainly il-6, results in the upregulation of the iron-regulatory hormone hepcidin (hamp), primarily produced by hepatocytes and released in the blood flow to regulate systemic iron homeostasis. systemic hamp blocks cellular iron export through ferroportin 1 (fpn1), resulting in reduced intestinal iron absorption, increased iron retention in hepatocytes and macrophages and ultimately anemia of infection/inflammation [4, 95] . several cells other than hepatocytes have been demonstrated to produce and release hamp that can act as autocrine and paracrine molecule, modulating local iron homeostasis [95, 96] . not only cells of the immune system like lymphocytes, monocytes and macrophages (including alveolar macrophages) but also airway epithelial cells have been demonstrated to produce hamp during infection and inflammation and potentially contribute to lung injury [97] [98] [99] . hamp is also a peptide involved in innate antimicrobial immunity and an acute phase protein [100] . further acute phase iron-related proteins like transferrin (tf), lactoferrin (lf), ferritin (ft), haptoglobin (hp) and hemopexin (hpx) are modulated by viral infections, further underlining the crucial role of iron in anti-viral host defense. the role of iron metabolism has been thoroughly investigated in several human viral infections. for extensive reviews on this topic and for details on systemic and cellular iron metabolism, please refer to [2] [3] [4] 101] . cq/hcq have been shown to modulate iron metabolism, impairing its homeostasis at different levels [43, 102] , and to decrease inflammatory cytokines like il-6, il-1 and tnf-. here we summarize the more striking evidences on cq/hcq action on cellular iron trafficking and their impact on cellular and systemic iron metabolism. we further propose alternative modes of action for these drugs, currently used in the treatment of covid-19, that might also work through the interference with local and/or systemic iron metabolism, restricting this essential element in infected cells and/or immune cells involved in virus clearance and/or acting on virus cell cycle (figure 1 ). in several experimental models, cq has been shown to restrict iron entry into the cells. in the eukaryotic model saccharomyces cerevisiae, cq interferes with iron uptake, acting as a competitive inhibitor of iron entry and inducing iron starvation. iron-deprived yeast, by means of knocking-out genes involved in iron uptake or by using iron chelators, shows increased sensitivity to cq [103] . in mammalian cells, cq has similar effects acting through the inhibition of the tf/transferrin receptor 1 (tfr1) complex endocytosis. the first evidence of cq inhibition of tf uptake by cells was obtained in cultured rat embryo fibroblasts [104] . tf is the main plasma iron carrier which, maintaining this cofactor in a redox inert state, distributes it to most cells of the human body. tf tightly binds two fe 3+ . tfr1, located on the plasma membrane of most types of cell, binds and internalizes tf into endocytic vesicles through cme [105] . as stated above, cq has been demonstrated to reduce picalm expression in macrophages of treated mice [15] . this ubiquitously expressed protein is involved in cme and its deficiency has been demonstrated to result in anemia and abnormal iron metabolism in mice [106, 107] and iron starvation, with increased surface tfr1 expression and decreased intracellular iron levels, in murine embryonic fibroblasts [108] . then, cq/hcq treatment may result in the inhibition of tf/tfr1 complex uptake and cellular iron starvation. the release of iron from tf after iron-loaded tf/tfr1 complex cme and its translocation into the cytosol is a fundamental step for iron cellular acquisition and further usage. the acidic milieau of endosomes weakens the binding of tf to fe 3+ , enabling its release within these vesicles [105] . cq/hcq, by virtue of their basic properties, raise ph of endocytic vesicles, possibly inhibiting iron removal from tf within endocytic vesicles. within endocytic vesicles, released fe 3+ is reduced to fe 2+ by the metalloreductase six-transmembrane epithelial antigen of the prostate 3 (steap3) and exported from the lysosomes to the cytosol through the divalent metal-ion transporter 1 (dmt1), mucolipin 1 (trpml1/mcoln1) and other transporters [105] . iron transport through dmt1 is ph dependent, being stimulated at low ph. dmt1 is indeed a h + /fe 2+ symporter that needs a proton electrochemical potential gradient as driving force to transport iron from endosomes into the cytoplasm [109] . trpml1/mcoln1 is a non-selective channel permeable to various cations such as ca 2+ , na + and k + that can also transport fe 2+ . it mainly localizes in late endosomes and lysosomes and the acidic environment of these vesicles activates this channel and the release of cations from the lumen into the cytosol [110] . then cq/hcq could also inhibit iron release from endosomes into the cytosol. the alkalizing properties of cq/hcq have been widely used to impair the endosome/lysosome fusion and inhibit autophagic flux [5] . ft is the main iron storage cellular protein that compartmentalizes iron in a nonreactive form within the cell, until use. iron release from ft mainly occurs through protein degradation by a selective lysosome-autophagy pathway named ferritinophagy [101, 111] that is inhibited by cq [112] . further, dmt1 and trpml1 have been implicated in the release of iron derived from ferritinophagy and entrapped in autophagic vesicles and, as stated above, both require an acidic environment for their channel functions. all the steps described above result in cellular iron starvation. this condition might possibly influence sars-cov-2 life cycle, although currently there is no experimental evidence in this new pandemic infection, but we suggest research in this direction. sars-cov-2 enter cells through ace2 receptor that is expressed broadly in the human body, mainly in the vascular endothelia, gastrointestinal system, heart, kidney, muscle, skin and bronchial and lung alveolar epithelial cells [113] . in all human cells, then also sars-cov-2 target cells, iron is a cofactor of several crucial proteins, involved in bioenergetics, cellular growth and a second important point raised by the iron starvation conditions induced by cq/hcq is their effect on immune cells, involved in the innate and adaptive immune responses against the virus. as all cells in the body, immune cells require iron for their proper functions and for their activation and proliferation. iron excess often results in impaired immune response to infections, as observed in patients suffering from hemochromatosis (hh) or thalassemia [116] . excessive or dysregulated immune response is of particular importance in the pathogenesis of covid-19 [117] and diseases caused by other coronaviruses [118] [119] [120] . direct infection of innate and adaptive immune cells has been described in some coronavirus infections [118, 119] , then iron starvation could also possibly inhibit these infections. resident macrophages can polarize under the stimuli of cytokines in classically activated pro-inflammatory macrophages (m1), induced by interferon- (ifn-) and tnf- or, under interleukin-4 (il-4) and 13 (il-13) stimuli, alternatively activated macrophages (m2), involved in pathogen clearance, tissue repair and inflammation reduction. while m2 macrophages have low iron levels, m1 macrophages are characterized by iron retention, secrete high levels of pro-inflammatory cytokines, produce high amounts of radicals to kill pathogens and produce hamp that acts in an autocrine manner to minimize iron exit. increased iron deposition in macrophages has been shown to induce m1 polarization and the persistence of a pro-inflammatory state due to an incomplete switch to m2 state [121] . iron retention in macrophages could then favor intracellular virus life cycle in the case of their infection and further promote the process of inflammation, while iron starvation could result in the opposite effects. further, iron excess in macrophages favors secondary infections with other j o u r n a l p r e -p r o o f microbes. chronic administration of cq has been shown to decrease iron content in a rat model. legssyer and colleagues [1232, 123] showed that cq decreased iron content in the liver of control, iron-loaded and iron-deficient rats, in the spleen of control and iron-loaded animals and, importantly, in alveolar macrophages of iron-loaded rats. they also interestingly observed that cq treatment of primary cultures of alveolar macrophages obtained from all three groups of rats and treated with lipopolysaccharide (lps) significatively reduced the oxidative response, measured by no 2 release, suggesting that cq might prevent infections, particularly those associated with diseases characterized by iron overload, through limiting iron availability not only in infected cells but also in macrophages, in turn reducing inflammation. however, heterozygous and homozygous mutations in the hemochromatosis gene (hfe) reduce the cq effect of iron removal in porphyria cutanea tarda [124] . also in murine models, cq has been shown to decrease macrophage activation syndrome (hemophagocytic syndrome) induced by pristane, by reducing macrophage infiltration, phagocytic functions, cytokine production and reducing ft, lactate dehydrogenase and triglyceride levels [125] . although immune cells need iron for their proliferation and activation, excess iron has also been reported to impair antigen processing and presentation by apcs, suppress cd4 + cells and alter cd4 + /cd8 + lymphocyte ratio, increase circulating immunoglobulin-producing b cells, reduce natural killer (nk) cell lysing efficiency and perturb complement activation [126] , while iron deficiency has an immunosuppressive effect on t cells [127] . then, cq/hcq treatment, through their action on iron homeostasis, could possibly have also a broad role in the modulation of the adaptive response to sars-cov-2. a third important point to highlight derives from the inhibitory action of cq/hcq on il-1, tnf- and il-6 release. first, tnf-α acts on iron homeostasis, reducing intestinal iron absorption and blocking iron recycling from macrophages, then inducing iron retention in these cells and their polarization to m1 proinflammatory phenotype [102] . conversely, cq inhibits tnf- acting also through the induction of cellular iron starvation [40] . then, the inhibition of the cytokine by cq/hcq, also through their effect on iron homeostasis, could alleviate inflammation, restoring a balanced systemic iron homeostasis and rescuing erythropoiesis. secondly, il-1, tnf- and il-6 as stated above, induce the systemic and local release of hamp. iron is essential for life, but excess iron is toxic, due to its redox potential that can hamper oxidative stress damaging crucial cellular components. iron availability is then tightly regulated both at the cellular and systemic levels [2-4, 95, 101] . the main systemic regulator is hamp. this hormone peptide is produced mainly in the liver and is regulated by circulating and tissue iron levels through the bone j o u r n a l p r e -p r o o f morphogenetic/small mother against decapentaplegic (bmp/smad) pathway in such a way that iron load induces while iron deficiency inhibits its expression, mainly acting on intestinal iron absorption and iron retention/release by macrophages and hepatocytes. hamp expression is also downregulated by hypoxia and erythropoietin (epo) through erythroferrone (erfe) to allow iron mobilization for erythropoiesis, while it is upregulated by inflammation. il-6 is the main signal that induce hamp during inflammation, through the jak/stat3 pathway in association with the bmp/smad pathway, but also il-1 and tnf- have a direct role on hamp regulation [95, 128, 129] . as stated above, cq/hcq not only interfere with cellular iron inducing its starvation in alveolar macrophages [122, 123] , then possibly resulting in a switch to m2 antiinflammatory state, but also inhibit il-6, il-1 and tnf- release, possibly reducing local hamp release by macrophages. this reduction can result in further decreased iron retention in these cells allowing direction towards inflammation resolution. further, cytokine decrease could result in systemic hamp decrease that, through increased intestinal iron absorption, may ameliorate anemia of infection. interestingly epo treatment has recently been found to attenuate covid-19 severe symptoms [130] while, through in silico modeling, sars-cov-2 has been found to possibly bind heme dissociating iron from porphyrin [131] . numerous studies correlated thrombocytosis induced by iron deficiency anemia (ida) with thrombotic events [132] [133] [134] [135] . a recent study has evidenced that ida patients manifesting thrombocytosis had 2-fold augmented risk of thrombosis when compared with ida patient with normal platelet count [136] . interestingly, covid-19 patients with severe pneumonia seem to have high platelet counts compared with patients with severe non-covid-19 pneumonia [137] and this increase is more evident among the nonsurvivor compared with survivor covid-19 patients [138] . to clarify the effect of iron deficiency on thrombotic propensity in animal models, thrombosis was induced in sprague-dawley rat fed with irondeficient diet or normal diet as control. iron deficiency induced thrombocytosis and platelet number resulted proportional with thrombus size. in addition, platelet adhesion and aggregation were impaired. taking into account data obtained in this model, the authors concluded that anemia of inflammation, caused by hamp-mediated iron sequestration in the liver, spleen and macrophages, as possibly occurs in covid-19 patients, may be considered a functional iron deficiency (id) and patients affected by this condition should be treated as patients with high risk of thrombosis [139] . in conclusion, the multiple action of cq and hcq as anti-viral, anti-inflammatory and anti-thrombotic drug may be also strictly linked to their effects on iron homeostasis, both at the local and systemic level. interestingly, another common drug, frequently used in the treatment of covid-19 patients with markedly j o u r n a l p r e -p r o o f elevated d-dimer levels is heparin. like cq and hcq, heparin is a versatile drug, as defined by jecko thachil [35] , due to its possible actions as anti-coagulant, anti-inflammatory and anti-viral drug. it is interesting to note that, like cq/hcq, heparin too has been demonstrated to modulate iron metabolism: this antithrombotic drug has indeed been demonstrated to inhibit hamp expression in human macrophages, to increase fpn1 plasma-membrane expression and promote iron export, resulting in cellular iron starvation [140] . all this evidence suggest a possible role of iron in sars-cov-2 infection that would be explored in future basic and clinical research, also considering it as a potential target for covid-19 therapy as proposed for other human infectious diseases [2] [3] [4] . molecular mechanism and theoretical mode of action of cq and hcq on multiple steps of the viral pathway and the demonstrated in vitro activity against covid-19 [8] [9] [10] have prompted their off-label use in clinical setting during this pandemic emergency. several clinical trials have been launched to evaluate the effectiveness of these drugs [141, 142] . until now, published clinical data on cq and hcq are limited and regard only small, poorly controlled or uncontrolled clinical studies. we found only four published reports of these trials in pubmed till april 20th, 2020. the first clinical study to evaluate the efficacy and safety of cq/hcq in patients with covid-19 has been reported by huang and colleagues [143] from china. the authors screened 22 patients tested positive for 12,5% of controls. treatment was more effective in patients with symptoms than in asymptomatic patients. the same researchers [145] further conducted an uncontrolled non-comparative observational study with 80 enrolled patients and the same treatment protocol for a maximum of 10 days. six of these patients were from the previous study. most patients (65, 81,3%) were discharged from the infectious disease unit (idu) with a mean length stay of 4.6 days, while 13 were still hospitalized, 1 died and 1 stopped treatment for the potential interaction with other drugs. on the total, 15% required oxygen therapy, 3 were transferred in icu and two of them improved and returned to idu, the third remained in icu. overall, for almost all enrolled patients the clinicians obtained a better clinical improvement with this treatment when compared with other treatments. further, viral load rapidly decreased, with 83% negative patients at day 7 and 93% at day 8 of treatment. only 2 patients had still detectable viral load at day 10. molina and colleagues [146] studied 11 covid-19 patients (10/11 with fever and nasal oxygen therapy, 8 with significant comorbidities) treated with hcq plus azithromycin, using the same dosing regimen reported by gautret and colleagues [144, 145] . one patient died and 2 were transferred to icu within 5 days after treatment initiation, one therapy was discontinued after 4 days for severe adverse event (prolongation of the qt interval), suggesting the poor clinical outcome of the combined treatment. in contrast with results obtained by [144, 145] , nasopharyngeal swabs were still positive for viral rna in 8/10 patients at days 5 to 6. researchers concluded by saying that, in their experience, they found no evidence of anti-viral activity and clinical benefit by using the combined therapy in severe covid-19. not only clinical efficacy but also optimal dosing regimen, therapeutic level, duration of treatment and pharmacokinetics in patients with different severity degrees of the disease are uncertain and currently there are no standard dosages or duration of treatment around the world for covid-19 patient treatment [148] [149] [150] [151] [152] . numerous studies are under way to evaluate their efficacy in treating and prevention of covid-19 and to establish benefit versus harm of cq/hqc treatment [153] . notwithstanding, in the emergency phase of covid-19 pandemic many old drugs have been off-label used for the treatment of the infection, based only on theoretical or in vitro efficacy and without enough clinical evidence based on randomized clinical trial. j o u r n a l p r e -p r o o f as we have described, cq/hcq are likely to have: (1) a direct antiviral action stopping viral infection in several steps; (2) a hypothetical ability to attenuate the progression of covid-19 to severe disease exploiting its inflammatory mechanisms, but also (3) through its potential anti-thrombotic effect. based on our review and because of their minimal toxicity profile and complex action, we suggest to use cq/hcq cq beyond 5-10 days of treatment in patients with covid-19 according to the hypothesis that their utility can extend also after ending sars-cov-2 high replication phase and considering also the possibility of a reactivation of the infection [154] . randomized controlled studies and observational outcome registries focused on efficacy, duration and toxicities of treatment with these drugs could be useful to understand their real effectiveness while more specific anti-covid-19 drugs are available. this study was partly supported by the university of brescia (fondi ex 60% to eugenia quiros-roldan, giorgio biasiotto and isabella zanella). the authors declare the following financial interests/personal relationships which may be considered as of chloroquine and covid-19 viral infection and iron metabolism hepcidin and the iron-infection axis iron and infection guidelines for the use and interpretation of assays for monitoring autophagy effects of chloroquine on viral infections: an old drug against today's diseases? coronaviruses: an overview of their replication and pathogenesis remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro 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miraculous therapeutic effects covid-19: attacks the 1-beta chain of hemoglobin and captures the porphyrin to inhibit human heme metabolism iron deficiency and thrombosis: literature review iron deficiency anemia as a risk factor for cerebrovascular events in early childhood: a case-control study association between ischemic stroke and irondeficiency anemia: a population-based study association between venous thromboembolism and iron-deficiency anemia: a population-based study characterization of the rate, predictors, and thrombotic complications of thrombocytosis in iron deficiency anemia difference of coagulation features between severe pneumonia induced by sars-cov2 and non-sars-cov2 anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy iron deficiency induced thrombocytosis increases thrombotic tendency in rats heparin inhibits intracellular mycobacterium tuberculosis bacterial replication by reducing iron levels in human macrophages treating covid-19 with chloroquine hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label nonrandomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection towards optimization of hydroxychloroquine dosing in intensive care unit covid-19 patients of the n-glycosylation of the cell surface viral receptor ace2  inhibition of the n-glycosylation of the viral spike (s) proteins  inhibition of the synthesis of cell membrane sialic acids  inhibition of picalm expression, cme and pathogen internalization  vesicle alkalinization and inhibition of endosomal/lysosomal antigen processing  vesicle alkalinization and inhibition of mhc processing and mhc-antigen we would like to thank andrea zanella for helpful language editing. isabella zanella wishes to dedicate this work to her father, antonio zanella, who recently died for covid-19.due to space constraints, we were unable to provide a comprehensive citation of all the relevant primary literature. we apologize to those whom we have even unintentionally omitted.j o u r n a l p r e -p r o o f key: cord-322913-sq9mq6f1 authors: ciabattini, annalisa; garagnani, paolo; santoro, francesco; rappuoli, rino; franceschi, claudio; medaglini, donata title: shelter from the cytokine storm: pitfalls and prospects in the development of sars-cov-2 vaccines for an elderly population date: 2020-11-06 journal: semin immunopathol doi: 10.1007/s00281-020-00821-0 sha: doc_id: 322913 cord_uid: sq9mq6f1 the sars-cov-2 pandemic urgently calls for the development of effective preventive tools. covid-19 hits greatly the elder and more fragile fraction of the population boosting the evergreen issue of the vaccination of older people. the development of a vaccine against sars-cov-2 tailored for the elderly population faces the challenge of the poor immune responsiveness of the older population due to immunosenescence, comorbidities, and pharmacological treatments. moreover, it is likely that the inflammaging phenotype associated with age could both influence vaccination efficacy and exacerbate the risk of covid-19-related “cytokine storm syndrome” with an overlap between the factors which impact vaccination effectiveness and those that boost virulence and worsen the prognosis of sars-cov-2 infection. the complex and still unclear immunopathological mechanisms of sars-cov-2 infection, together with the progressive age-related decline of immune responses, and the lack of clear correlates of protection, make the design of vaccination strategies for older people extremely challenging. in the ongoing effort in vaccine development, different sars-cov-2 vaccine candidates have been developed, tested in pre-clinical and clinical studies and are undergoing clinical testing, but only a small fraction of these are currently being tested in the older fraction of the population. recent advances in systems biology integrating clinical, immunologic, and omics data can help to identify stable and robust markers of vaccine response and move towards a better understanding of sars-cov-2 vaccine responses in the elderly. 0.86, while the m:f ratio of deaths is around 1.38, suggesting that, despite being more frequently infected, females are more capable of dealing with the infection [2, 3] . it is widely reported that most deaths occurred among patients with at least one underlying disease, such as hypertension [4] and diabetes mellitus [5] . a meta-analysis of seven clinical studies performed in china identified chronic obstructive pulmonary disease (copd), cardiovascular disease, and hypertension as risk factors for severe disease and intensity care unit (icu) admission [6] . analysis of risk factors associated with more than ten thousand deaths by covid-19 in the uk confirmed that age was linearly correlated with risk of death and that obesity, diabetes, severe asthma, respiratory disease, neurological disease (including stroke), recent (< 5 years) hematological malignancy, and recent (< 1 year) cancer diagnosis were all associated with higher death risk. as for hypertension, the hazard risk was higher only for the population < 70 years old, even if hypertension itself was strongly associated with other risk factors such as obesity and diabetes [7] . importantly, these epidemiological studies identify the categories of subjects who are at higher risk of developing severe sars-cov-2 infection and that should be prioritized in vaccine administration. the massive effort for the development of a vaccine against sars-cov-2 could be frustrated by the poor responsiveness to vaccination that characterizes a large proportion of the elderly population. in this rush against the time, we risk to pay a dear toll for the lack of knowledge in the response to vaccination of the elderly, a well-known issue, neglected notwithstanding its evident urgency and the annual reproposal through the seasonal influenza epidemic above all. interestingly, there is a consistent overlap between the factors hampering vaccination effectiveness in the elderly and those that boost the virulence and worsen the prognosis of sars-cov-2 infection. a common characteristic of the elderly people is the onset of a sterile low-grade increase of the basal inflammatory state named "inflammaging," which is considered a universal etiological agent of most of the age-related diseases [8] . it is likely that some specific components of the inflammaging phenotype could both influence vaccination efficacy and then increase the risk of the early massive production of inflammatory cytokines, termed the "cytokine storm syndrome." this is a condition reported in severe covid-19 cases during which the patient's immune system spins out of control and starts damaging healthy organs owing to the increased vascular permeability, vascular paralysis, and hypovolemic shock [9] . angiotensin-converting enzyme 2 (ace2) has been identified as the receptor for sars-cov-2, and it has been suggested that differential levels of ace2 in the cardiac and pulmonary tissues of younger versus older adults may be at least partially responsible for the spectrum of disease virulence observed among patients with covid-19 [10] . here, we analyze the different aspects that tackle sars-cov-2 vaccination in the elderly population, considering immunologic, genetic, and socio-economic factors that impact on the age-related changes of immune responses. a view of the current available vaccine platforms with a special focus on the clinical trials including older adults is reported. how the elderly condition can affect covid-19 disease progression and the response to vaccination immunosenescence for many reasons, it is difficult to clearly define what immunosenescence is: (i) immunosenescence is quite complex and involves cellular and molecular changes occurring lifelong (from newborns to centenarians) in both the innate and the adaptive immune systems; (ii) these changes can be at the same time detrimental and beneficial/adaptive [11] ; (iii) it is difficult to identify a unique common marker of immunosenescence, due to the overwhelming number of biological and non-biological factors that can impinge lifelong on the immune system of each individual; (iv) the changes occurring with age in the immune system are deeply correlated with the profound environmental, epidemiological, lifestyle, societal, medical, and public health changes, including vaccination policies and practices, that occurred in the last century. accordingly, immunosenescence is highly contextdependent [12] , different in different geographical and historical settings and in men and women, correlated to socioeconomic position, and sensitive to psychological stressors. indeed, both the adaptive and the innate immune systems have the capability of "remembering" all immunological stimuli a person has been exposed to lifelong. we have conceptualized this situation with the term immunobiography, which should help in understanding the enormous heterogeneity of the immune phenotype in old people. this is also the reason why there is a sort of imprinting in the immune responses favoring those towards antigens that have been experienced early in life [13] . the complex biological processes of aging are the result of alterations in gene regulation and protein expression, signaling pathways, and biological networks. complex changes, including pervasive epigenetic and metabolic modifications, affect most of the subsets of naïve, memory, regulatory effector t cells, and b cells [14] [15] [16] . despite the challenging complexity, a universally observed hallmark of immunosenescence is the decrease of naive t cells (particularly cd8+ t cells) in peripheral blood [17] consequent to thymic involution responsible for the early decline in the output of naïve t cells to the periphery and for the related shrinking of the t cell repertoire [18] [19] [20] . other important aging-related alterations are (i) the shift in the bone marrow maturation of hematopoietic cells towards myelocytic differentiation [21] , concomitant with a reduced lymphopoiesis, mainly due to changes in progenitor cells in the bone marrow [12, 22] ; (ii) the increased numbers of memory cells owing to large clonal expansion towards epitopes of persistent viral infections (cytomegalovirus [cmv] and epstein barr virus [ebv]) [23, 24] ; (iii) the compromised ability of cd4+ t cells to differentiate into functional subsets, resulting in a multitude of dysregulated responses, such as a reduced cognate help to b cells with consequent reduced humoral immunity, and the increased ratio of the proinflammatory th17 cells with respect to the immunosuppressive t regulatory cells, thus favoring a basal proinflammatory status [16, 25] ; (iv) accumulation of differentiated exhausted t cells, induced by a repeated pathogen encounter during chronological aging, and end-stage differentiated senescent t cells, characterized by a progressive reduction of telomere length leading to a state of proliferative arrest [26] . with aging, health conditions associated with immune senescence, comorbidities (particularly noncommunicable diseases such as heart disease, cancers, and metabolic and autoimmune diseases), and pharmacological treatments affect the immune responses to both vaccines and infectious diseases. overall, as a result of immunosenescence, the elderly population is more susceptible to infections, particularly to influenza, streptococcus pneumoniae rsv, and group b streptococcus but also to opportunistic, re-emergent chronic infections such as herpes zoster as well as antibiotic-resistant nosocomial pathogens. the reduced adaptive immune response, together with altered innate cell function, such as chemotaxis, phagocytosis, signaling pathways, and intracellular killing, prevents the appropriate control of the initial inflammatory response elicited upon viral infection. for rna virus, such as coronavirus, different pattern recognition receptors (prr) are triggered on the innate cells during the early phases of infection. these include the endosomic toll-like receptor 3 and 7 and the cytosolic rig-i/mda-5 molecules, which recognize viral rna [27] , and the cgas-sting pathway, which recognizes cytosolic dna [28] activated by cellular damage and mitochondrial dna release caused by viral infection [29] . the stimulation of these prr leads to the expression of type i ifn, a factor that limits viral replication through the stimulation of interferon-stimulated genes, and other inflammatory cytokines [30] . for middle east respiratory syndrome (mers)-cov, the timing of type i ifn production appears to dictate the outcome of infection in mouse models, and its administration within 1 day after infection was protective against lethal infection, while a delay in ifn production caused an inability to control viral replication, leading to cellular damage of airway epithelia and the lung parenchyma and an eventual lethal inflammatory cytokine storm [31] . the latter response often predominates in older individuals and in aged mouse models of sars-cov-1 infection [32, 33] . induction of innate immune responses is a crucial step in the pathophysiology of covid-19 disease (fig. 2) . on one hand, it triggers the anti-viral host defense mechanisms necessary for elimination of infection, but on the other hand, it may contribute to hyperinflammation and tissue damage during the later stages of the disease in a minority of patients [34] . this can be particularly relevant in the elderly population in which inflammaging, the state of chronic low-grade sterile inflammation [8] , characterized by high serum concentrations of c-reactive protein (crp), il-6, il-8, and tumor necrosis factor (tnf)-α, can be present. tissue damage in covid-19 is mainly mediated by an excess of immune response to the virus, which results in a cytokine storm, with activation of the il-6 signaling pathway. the pathophysiology of sars-cov-2 infection has strong similarities to other severe viral lung infections caused by sars-cov-1 and mers-cov. one of the first published studies on clinical features of covid patients hospitalized in wuhan showed that proinflammatory cytokines and chemokines, such as tnf-α, granulocyte-colony stimulating factor (g-csf), interferon g a m m a -i n d u c e d p r o t e i n -1 0 ( i p -1 0 ) , m o n o c y t e chemoattractant protein-1 (mcp-1), and macrophage inflammatory proteins 1-α (mip-1α), were significantly higher in patients admitted to the intensive care unit (icu) compared to those who were not in icu [35] . immune pathology in the form of vascular and cutaneous lesions has also been widely reported [36, 37] . the role of a dysregulated inflammatory response was proven in an animal model of sars-cov-1 infection using aged macaques. aged animals are more prone to develop severe disease and activate more readily the innate response, in particular the nf-kb pathway and proinflammatory cytokines such as il-8 and il-1β, while not inducing significantly ifn-β response. the innate immunity activation is not due to the viral load, which is comparable among young and aged macaques [38] . transcriptomic analysis performed in samples from subjects with severe covid-19 revealed the presence of low levels of type i and type iii interferon genes together with elevated levels of proinflammatory cytokines and chemokines, such as il-6, il1ra, ccl2, ccl8 cxcl2, cxcl8, cxcl9, and cxcl16 [39] . which type of cells elicits this cytokine storm and the virological mechanisms behind this inflammatory reaction are still unclear [40] . lung epithelial cells, alveolar macrophages, dendritic cells, and endothelial cells can effectively release the proinflammatory cytokines and chemokines, thus attracting monocytes, macrophages, and t cells to the site of infection [41] . the overproduction of proinflammatory cytokines in the lungs can damage the tissue infrastructure, recruit macrophages that infiltrate air spaces, and generate the respiratory failure from acute respiratory distress syndrome (ards), which is recognized as the leading cause of mortality. meanwhile, the direct attack on other organs by disseminated sars-cov-2, the immune pathogenesis caused by the systemic cytokine storm, and the microcirculation dysfunctions together may lead to multi-organ damage, even though whether sars-cov-2 can directly target organs other than the lung and how it can happen are aspects that need to be further investigated [40] (fig. 2) . together with the hyperinflammatory response, a significant lymphopenia, mainly related to cd4+ t and cd8+ t cells, which correlates with the severity of viral infection, was reported [42] [43] [44] . the causes of this adaptive immunity suppression are still unclear. pulmonary recruitment of immune cells from the blood and the infiltration of lymphocytes into the airways may explain the reduction in blood. the wellsystemic and local (lung) immune responses and their pathological role, following sars-cov-2 entry into the host are schematically represented. induction of innate immune responses is a crucial step in the pathophysiology of covid-19 disease, contributing to hyperinflammation and tissue damage during the later stages of the disease. infiltration of immune cells in the lungs causes overproduction of proinflammatory cytokines, which eventually damages the lung infrastructure, accumulation of macrophages in the air spaces and diffuse alveolar damage leading to acute respiratory distress syndrome (ards). furthermore, elevated levels of circulating proinflammatory cytokines can cause septic shock and multi-organ dysfunction. together with the hyperinflammatory response, overt disseminated intravascular coagulation has been reported and a significant lymphopenia, mainly related to cd4+ t and cd8+ t cells, has been observed, possibly due to pulmonary recruitment of lymphocytes from the blood. a possible immunopathological role can be mediated by non-neutralizing antibodies produced by b cells, which may enhance sars-cov-2 infection through antibody-dependent enhancement (ade), further exacerbating organ damage known age-related alteration of the immune function of t cell and b cells could lead to insufficient control of viral replication, thus increasing the macrophage infiltration and the lung injury (fig. 2) . finally, a possible immunopathological role can be mediated by non-neutralizing antibodies produced by b cells that may enhance sars-cov-2 infection through antibodydependent enhancement (ade), further exacerbating organ damage. it has recently been shown that sars-cov-1 and the mers-cov take advantage of non-or subneutralizing antibodies and enter cells via surface cd32a receptors, an fc receptor expressed on the surfaces of monocytes and alveolar macrophages. the antibody-cd32 interaction facilitates viral entry and infection, and activates intracellular signaling to upregulate proinflammatory cytokines [45] . the complex and still unclear immunopathological mechanisms of sars-cov-2 infection, together with the progressive age-related decline of innate and adaptive immune responses, and the lack of a clear correlate of protection, make the design of vaccination strategies for older people extremely challenging (fig. 3 ). an emerging class of instruments in the aging research is the development of markers capable of assessing the speed of the aging process. age is a major risk factor for a high number of diseases, and in general, it affects the fitness of each individual, including the capability of responding to vaccine administration and counteracting a severe infection [46] . however, it is also evident that the elderly population is extremely heterogeneous, so while chronological age is useful to identify macroscopic risk classes, it is poorly informative within age classes to get individual information. biological age is thus useful to evaluate clinical parameters and health risks on the basis of the individual aging pace, which tend to be more heterogeneous in the elderly population. several established biological age markers have been generated based on both classical anthropometric, clinical, and biochemical parameters as well as on innovative molecular characterizations such as dna methylation and the composition of the n-glycan shell of circulating proteins [47] . such biomarkers have shown in a number of studies that the aging pace is higher in the vast majority of the different elderly conditions, thus demonstrating that biological age assessment should be a critical information in a broad spectrum of clinical practices and in the development of strategies to tackle healthcare burden and emergencies. the detailed description of available biological age markers is out of the scope of the present manuscript, and an extensive overview is available in the review by jylhävä et al. [46] . to date, biological age has not been assessed in the sars-cov-2 clinical setting, but it is noteworthy that biological age has been associated with all the most important risk factors related to a poor prognosis of sars-cov-2 infection. the field of elderly vaccination could benefit from biological age information, but also in this case, the available data are rare. in a study from gensous et al. [48] , the whole genome methylation profile of pbmc was assessed in a group of volunteers of different ages who underwent influenza vaccination. the relationship between the vaccination response and the methylation profile was studied. while no difference in terms of biological age emerged in the study, an agedependent epigenetic remodeling emerged in elder non-responders. the study is limited owing to the very low number schematic interconnection between the main immune mechanisms elicited by the vaccination process, with the peculiarity of the elderly immune system-affected by both inflammaging and immunosenescence-and the still undefined correlates of protection from sars-cov-2 infection. the complex and still unclear immunopathological mechanisms of sars-cov-2 infection, together with the progressive agerelated decline of innate and adaptive immune responses, and the lack of a clear correlate of protection make the design of vaccination strategies for older people extremely challenging of analyzed subjects but confirmed that dna methylation is an informative instrument to be exploited in vaccination studies and strategies. immunobiography refers to the comprehensive immunological, clinical, socio-economic, and geographical history of each individual, and accounts for the large heterogeneity observed in the elderly regarding their health status, mirrored by their large individual variation in the responsiveness to vaccines. a major advantage of immunobiography is that it incorporates the most advanced conceptualization of immunosenescence which, according to the most recent literature [49] , has to be considered as a context-and population-dependent phenomenon. accordingly, in order to be properly interpreted, agerelated changes of immune parameters occurring in an elderly person necessitate a variety of other additional data regarding sex/gender, demographic cohort, population/country, individual immunological history, anthropometric parameters, socioeconomic status and education, cmv serostatus, morbidity and co-morbidity, among others. it is of critical importance taking in consideration the elderly vulnerability to direct the rational design of vaccines designed for this target population. gender is a critical issue in both vaccination of the elderly and in the sars-cov-2 pandemic. the pandemic epidemiological data show clearly that the risk of severe disease and mortality is sharply higher in men than in women. men's hospitalization exceeds women by about 50%, indicating a significantly higher susceptibility of men towards severe sars-cov-2 infection. available data show that men outnumber women 2 to 4 times in terms of icu admissions [50] [51] [52] . these numbers are concordant with the fatality rate that ranges between 1.2 and 1.4 men deaths for one women death. moreover, this unbalanced pattern is mirrored by the vaccine uptake, responses, and outcome in older-aged individuals. elderly women are indeed more responsive than men for several vaccine protocols recommended in older-aged individuals such as those against influenza, tetanus, pertussis, shingles, and pneumococcal infections [53] . on the other hand, an influenza vaccination study reported that aged men antibodies had higher affinity than those produced by women. moreover, men seem to respond better to pneumococcal vaccination in two independent studies [54, 55] . there is an impaired vaccination response in both old men and women with sex-specific weaknesses. the most striking data, however, is related to infection and all-cause mortality: indeed, in a number of reports, vaccine administration produces a sharper decrease of specific and all-cause mortality in vaccinated women compared to men, indicating that women have higher benefit from vaccination in the elderly [56] [57] [58] . these data indicate the need to consider sex-specific vaccination protocols for the elderly population [58, 59] and that the lack of such instruments could be critical in the sars-cov-2 pandemic since old men are both the most susceptible to severe sars-cov-2 infection and are those less likely protected by a possible sars-cov-2 vaccine [60] [61] [62] . another factor that could affect vaccine response is the intestinal microbiota that plays a crucial rule in the regulation of the immune system and is highly affected by age [63] [64] [65] [66] . microbial community composition indeed is influenced by age, environmental and socio-economic factors, diet, gender, chronic infections, immunosuppressive chemotherapy, antibiotic treatment, or probiotic use [64, [67] [68] [69] . the improvement in the nucleic acid sequencing obtained in the last 15 years hits massively the microbiological research and promotes the analysis of heterogeneous microbiological ecosystems such as those that reside in humans. the characterization of such ecological niches opens to the new conceptualization of humans as metaorganisms (organisms composed of different organisms) to stress the tight interdependencies between the host and the microbiological species residing in different anatomical districts. gut microbiota changes with age and that is likely an important contributor and modulator of the inflammaging phenotype [70, 71] . elderly people have less diverse gut microbiota and reduced beneficial microorganisms [72] . the general imbalance of gut microbiota, called "dysbiosis," is associated with both frailty, a geriatric syndrome leading to increased vulnerability for adverse health outcomes, and systemic inflammation. since a hyperinflammation status has been observed in most severe cases of sars-cov-2 infection, it is possible that gut dysbiosis may influence the clinical manifestation in covid-19 infection [73, 74] . interestingly, the gut microbiota has been shown to also affect pulmonary health through a bidirectional cross-talk between the gut microbiota and the lungs [75] . along this "gutlung axis," microbial products can reach the lung through blood and modulate pulmonary immune responses [76] , while inflammation processes occurring in the lung can impact on the gut microbiota [77] . some studies have demonstrated that respiratory infections are associated with a change in the composition of the gut microbiota [78] and the antibiotic treatment of mice for removing some gut bacteria has led to increased susceptibility to influenza virus infection in the lungs [79] . since one of the severe clinical manifestations of covid-19 is pneumonia and progression to acute respiratory distress syndrome (ards), especially in elderly and immunecompromised patients [80] , it can be speculated that sars-cov-2 infection can affect this gut-lung cross-talk which might influence the outcome of the clinical manifestation [81] . moreover, even though respiratory symptoms represent the principal clinical presentation of covid-19, clinical evidence suggests that the intestine may be another viral target organ. indeed, a high expression of ace2 has been observed in the brush border of intestinal enterocytes [82] and, using a human small intestinal organoid system, it has been demonstrated that sars-cov-2 readily replicates into the enterocytes, resulting in the production of large amounts of infective virus particles [83] . some reports show that sars-cov-2 rna can be detected in the stool of some patients of covid-19 [84, 85] , and patients often present gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain [86] . therefore, the characterization of the gut microbiota in patients with active sars-cov-2 intestinal infection could represent a striking aspect to investigate. these considerations on inflammaging, immunobiography, biological age, gender, and microbiota pertain to every vaccination strategy, but are particularly relevant for the development of vaccines against sars-cov-2 since it more seriously affects the elderly population and immunopathology is a crucial factor for the severity disease. need for the design of a sars-cov-2 vaccination strategies tailored for the elderly sars-cov-2 vaccines are urgently needed, and their design should take into consideration that the elderly population is the main target population for vaccination. while older adults are most likely to be severely affected by covid-19, they also may be less responsive to vaccination. efficacy of vaccination in the elderly is indeed strongly reduced compared to that of younger adults [87, 88] . sars-cov-2 vaccination strategies, tailored for the elderly, should take into consideration the delicate balance between immunosenescence/ inflammaging and the immunopathological aspects of the covid-19 disease (fig. 3) . vaccine adjuvants and vectors should be specifically designed for stimulating the elderly immune system without exacerbating the inflammatory status [87] . despite these considerations, the elderly are rarely included in vaccine clinical trials; in the last decades, the vast majority of randomized control trials did not include older adults and in particular frail older adults who are mostly at risk. we currently do not have full knowledge on the mechanisms of immunity to protect this population from sars-cov-2 [10] . the development of a sars-cov-2 vaccine is extremely challenging, since we are faced with a novel virus, just emerged in humans, and correlates of protection have not yet been fully identified, even though the induction of neutralizing antibodies is presumed to be a crucial target for an effective vaccination (fig. 3) . protection in older individuals against influenza virus appears to require higher neutralization titers than in younger individuals [89] , and this issue might need to be addressed for sars-cov-2. the knowledge obtained from the vaccine development efforts for mers and sars-cov-1 can be of high value for sars-cov-2, although no vaccines are licensed for these coronavirus strains [90] . memory cd4+ t cells, induced by infections with other coronavirus and capable of responding to sars-cov-2, have been detected in 20-50% of sars-cov-2 unexposed donors [91, 92] . the characterization of these cross-reactive t cells in the elderly and their impact on the immunogenicity of vaccine candidates should be taken into consideration in the ongoing covid-19 vaccination studies. sars-cov-2 vaccine candidates based on different vaccine platforms have been developed, and about 140 candidates have been tested in pre-clinical experiments, according to the who landscape documents of covid-19 candidate vaccines (https://www.who.int/publications/m/ item/draft-landscape-of-covid-19-candidate-vaccines) (fig. 4) . information on the specific sars-cov-2 molecules selected as vaccine antigens is limited, even though most candidates aim to elicit neutralizing antibodies against the spike (s) protein and its receptor-binding domain (rbd), as already performed with the sars and mers vaccines. a wide range of both innovative and traditional technology platforms has been deployed, including nucleic acid (dna and rna), recombinant viral vectors (replicating and non-replicating), recombinant protein combined with adjuvants, and live attenuated or inactivated virus [93] . some of these platforms were already tested in human studies for sars-cov-1 virus, such as inactivated virus, dna and soluble s proteins [94] [95] [96] , or for mers-cov [97] . the most advanced candidates for sars-cov-2 entered in human clinical testing with unprecedented rapidity employ nucleic acid (both mrna and dna), recombinant vaccine vectors (human or chimpanzee adenovirus vectors), subunit s protein combined or not with different adjuvants, and inactivated sars-cov-2 virus. other novel platforms based on the use of synthetic modified antigen presenting cells (apc) or cytotoxic t lymphocytes are also under study (fig. 4) . the platforms using mrna, nonreplicating viral vectors, and inactivated sars-cov-2 virus have already reached the clinical trial phase iii. some of the different platforms used may be tailored for specific population subtypes, such as the elderly, children, pregnant women, or immunocompromised patients [98] . in this regard, some of the ongoing clinical studies have specifically taken into consideration the older population, by including vaccination arms with people aged > 60 years. a schematic diagram of the ongoing phase i and ii clinical trials that have included older adults is reported in fig. 5 . enrolling older adult volunteers will help to better understand vaccination outcomes among the older population, who are most at risk of complications from covid-19. the ongoing clinical studies based on mrna technology (mrna-1273 from moderna n. nct04283461, and bnt162 from biontech se, n. nct04368728) aim to evaluate the safety, tolerability, immunogenicity, and potential efficacy of different sars-cov-2 rna vaccine candidates in the adult population, with a specific attention to older people (n.-n. releases. nih clinical trial of investigational vaccine for covid-19 begins. 2020. https://www.nih.gov/newsevents/news-releases/nih-clinical-trial-investigationalvaccine-covid-19-begins). the lipid nanoparticleencapsulated mrna-1273 vaccine, which encodes for the full-length s protein, is currently evaluated in a dose-ranging study in the adult population (18-55 years old), and in participants from 56 to 70 and > 71 years of age (fig. 5) . similarly, the large dose-finding study with the bnt162 biological component (7600 estimated participants) based on the administration of mrna coding for the full-length s protein, or for the two smaller receptor-binding domains, is going to test the immunogenicity in adults (18-55 years) and older adults (56-85 years) . an ongoing phase i/iia trial (n. nct04447781) is also aimed at evaluating the safety, tolerability, and immunological profile of the ino-4800 vaccine that, exploiting the dna technology, contains a plasmid encoding the full-length s glycoprotein. the ino-4800 vaccine is administered by intradermal injection followed by electroporation in healthy adults aged 19 to 64 years. another platform that is currently specifically tested in older people is based on the adenovirus type 5 vector that encodes the s protein from the sars-cov-2 strain (trials n. 2020-001228-32; pactr202006922165132; nct0439814; chictr2000031781 and nct04400838; fig. 5 ). different studies are ongoing, and one conducted in canada is a doseescalation designed study, from the younger adults (18 to < 55) to the older adults (65 to < 85). another huge phase 2/3 study (n. nct04400838) is aimed at determining the efficacy, safety, and immunogenicity of the candidate covid-19 vaccine based on the chimpanzee adenovirus vector (chadox1 ncov-19) in healthy uk volunteers, specifically divided in adults (18-55 years old), elderly (over the age of 56), and children (5-12 years old). the chadox1 platform has already been shown to be effective in the established rhesus macaque model of sars-cov-2 infection [99] . in this pre-clinical study, a single dose of chadox1 ncov-19 has protected six [100] . moreover, the chadox1 has been used to develop investigational vaccines against several pathogens, including the closely related coronavirus responsible for the mers [101] . adenovirus-based vectors are characterized by a broad range of tissue tropism that covers both respiratory and gastrointestinal epithelium, the two main sites that express the ace-2 receptor of sars-cov-2, even though a possible immunodominance mediated by vector genes rather than the transgenes should always be considered [102] . using the traditional recombinant protein technology to express the spike protein, a trial sponsored by clover biopharmaceuticals aus pty ltd. (n. nct04405908) is assessing the safety, reactogenicity, and immunogenicity of multiple doses of scb-2019 administered with as03 adjuvant, or with cpg 1018 plus alum adjuvants. data will be separately analyzed on adult (18 to 54 years of age) and elderly (55-75 years of age) healthy subjects enrolled in the study. in another study, the s protein has been administered with the advax adjuvant (n. nct04453852), a potent and safe immunopotentiator composed of delta inulin [103] . four trials are testing in the elderly population the inactivated sars-cov-2 virus (n. nct04456595; chictr2000031809; chictr2000032459), and one of these has been specifically performed only in people > 60 years (n nct04383574; fig. 5 ). numerous other vaccine developers have indicated plans to initiate human testing in 2020. despite the several vaccine candidates (fig. 4) , challenges including the need for optimizing antigen design and adjuvant formulation define the number of doses needed, induce the optimal immune response without exacerbating the inflammatory and antibody-dependent response involved in possible lung disease, and fully define correlates of protection and duration of immune responses have to be considered [104] . finally, a general consideration for the sars-cov-2 vaccine development regards safety issues that could arise with covid-19 vaccines developed under the strong pressure of the pandemic situation. animal studies on vaccines for sars-cov-1 and mers-cov report possible adverse effects mediated by vaccine-induced antibodies that have poor or no neutralizing activity [105] . safety and efficacy are two indissoluble properties of a vaccine to be administered to billions of people globally and need to be accurately evaluated for every sars-cov-2 candidate. the efforts in the development of covid-19 vaccines can benefit from the availability of most advanced tools and high-throughput technologies to decipher the effective immune responses in the older population and the correlates of protection. recent advances in systems biology integrating clinical, immunologic, and omics data can help to identify stable and robust markers of vaccine response and move towards a better understanding of sars-cov-2 vaccine responses in the elderly. machine/statistical learning applied to multi-omics data from clinical studies promises to revolutionize vaccine development by illuminating the mechanistic drivers of protective immunity. the high-performance data acquisition methods in molecular and cellular biology push the field of bioinformatics for the development and use application of the immunobiography approach could inform the stratification of elderly subjects and guide the implementation of vaccination strategies designed for specific elderly population clusters [87] . mathematical modeling allows the combination of different networks involved in biological aging such as epigenetic networks, cell-cell networks, and population genetics and can allow to generate hypothesis on response to treatment or vaccination [106] . recent progress in mathematical modeling can be utilized to generate biomarker models for prediction of disease and also response to vaccination taking into consideration biological age. currently, computational models have been applied to immunology data, for example, for the analysis of a high-dimensional dataset in vaccination studies [107, 108] , but these models are limited to particular aspects [109, 110] . there is the potential for these models to become more sophisticated and to predict how responses to pathogens and vaccines are affected by pre-disposing factors [111, 112] . the systems vaccinology approach has been applied to characterize the immune response to different vaccines providing the proof-of-concept evidence of the capacity of systems approaches to delineate "molecular signatures" predictive of vaccine responses [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] [130] [131] . this approach has also been applied to identify molecular signatures induced by immunization with the rvsv-zebov ebola vaccine, recently approved for human use. systems analysis has been conducted integrating clinical, immunologic, and omics data in clinical trials with different doses and in different continents (vianello et al. 2020 submitted, santoro et al. 2020 submitted). despite the great efforts made, unfortunately, many of the most useful clinical and multi-omics datasets are siloed in local databases to protect participant privacy and data confidentiality. creation of secure, faircompliant, federated learning databases in which predictive biological and mathematical models based on ai/ machine/statistical learning can be developed, refined, and tested on distributed datasets would have an enormous impact in supporting a rational vaccine development. sars-cov-2 vaccines are urgently needed, and their design should take into consideration that the elderly are the main target population for vaccination. the pandemic is stimulating the research on vaccine development, and this should be a tremendous opportunity to specifically include age and gender as critical factors for vaccination approaches and effectiveness. while older adults are most likely to be severely affected by covid-19, they also may be less responsive to vaccination. in the ongoing tremendous efforts for covid-19 vaccine development, only a limited number of clinical trials have included the older fraction of the population in the study design, and the platforms used are not specifically designed considering the peculiarity of the elderly immune system. indeed, vaccination strategies tailored for the sars-cov-2 infection in the elderly should take into consideration the delicate balance of immunosenescence and inflammaging with the immunopathological aspects of the sars-cov-2 infection, such as the cytokine storm reported in severe covid-19. therefore, the possible overlap between the factors hampering vaccination effectiveness in the elderly and those that boost the virulence and worsen the prognosis of sars-cov-2 infection should be carefully taken into consideration. thus, vaccine formulations, such as adjuvants and vectors, should be specifically designed for stimulating the elderly immune system without exacerbating the inflammatory status. the ongoing efforts in covid-19 vaccine development should fully exploit the availability of high-throughput technologies and recent advances in systems biology to decipher the effective immune responses in the older population and identify correlates of protection to guide towards sars-cov-2 vaccine strategies optimally designed to protect the older population. authors' contributions ac, pg, and dm, fs drafted the work; dm, rr, and cf revised it critically for important intellectual content; all the authors approved the version to be published. funding open access funding provided by università degli studi di siena within the crui-care agreement. this work was supported by commission of the european communities, horizon 2020 framework programme, grant number 730964 (transvac2), and russian ministry of science and education agreement no. 075-15-2020-808. conflict of interest the authors declare that they have no conflict of interest. consent for publication the authors are responsible for the correctness of the statements provided in the manuscript. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not 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responses but is attenuated by preexisting ad5 immunity a cell-based systems biology assessment of human blood to monitor immune responses after influenza vaccination publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-349262-gnqbyc6t authors: hemida, maged gomaa; ali, mohammed; alhammadi, mohammed; alnaeem, abdelmohsen title: the middle east respiratory syndrome coronavirus in the breath of some infected dromedary camels (camelus dromedarius) date: 2020-10-14 journal: epidemiol infect doi: 10.1017/s0950268820002459 sha: doc_id: 349262 cord_uid: gnqbyc6t dromedary camels remain the currently identified reservoir for the middle east respiratory syndrome coronavirus (mers-cov). the virus is released in the secretions of the infected camels, especially the nasal tract. the virus shedding curve through the nasal secretions was studied. although human transmission of the virus through the respiratory tract of close contact people with dromedary reported previously, the exact mechanism of transmission is still largely unknown. the main goal of this study was to check the possibility of mers-cov shedding in the exhaled air of the infected camels. to achieve this goal, we conducted a follow-up study in one of the dromedary camel herds, december 2018–april 2019. we tested nasal swabs, breath samples from animals within this herd by the real-time pcr. our results showed that some of the tested nasal swabs and breath were positive from 24 march 2019 until 7 april 2019. the phylogenetic analysis of the obtained s and n gene sequences revealed the detected viruses are clustering together with some human and camel samples from the eastern region, especially from al-hufuf city, as well as some samples from qatar and jordon. these results are clearly showing the possibility of shedding of the virus in the breath of the infected camels. this could explain, at least in part, the mechanism of transmission of mers-cov from animals to humans. this study is confirming the shedding of mers-cov in the exhaled air of the infected camels. further studies are needed for a better understanding of the mers-cov. it has been almost 8 years since the emergence of the middle east respiratory syndrome coronavirus (mers-cov) in 2012 in the arabian peninsula. the majority of human cases were reported in the arabian peninsula [1] [2] [3] . more recently, there are many reports about the potential risk of infection with mers-cov in africa [1] . the only known reservoir so far is the dromedary camels [4] . there are many reports about the animal-human transmission in the context of mers-cov [5, 6] . it is now well documented that mers-cov is released in the respiratory secretions detected in the nasal swabs by several research groups [5, [7] [8] [9] [10] [11] . although no report on the secretion of the virus in the milk, it is highly recommended to boil it before consumption to avoid any potential contamination of the raw milk during the process of milking and handling [12] [13] [14] . similar concept for eating the meats and raw organs, particularly the liver, it should be cooked well before consumption to avoid any potential hazards of infection as suggested by the world health organization (who) [12, 14] . the virus was also detected in the air around some positive camel herd. detection of the mers-cov-rnas of identical sequences from the owner and the herders of this particular herd was also reported [5, 6] . however, some other studies failed to detect the virus in the urine of some infected animals [15] . earlier studies succeeded in the detection of the mers-cov nucleic acids in the air circulated by an infected camel herd as well as in the people of close contact of this particular herd [5, 6] . meanwhile, several reports showed the possibility of detection of the mers-cov nucleic acids in the circulating air in some health care settings during the korean outbreak of mers-cov in 2015 [16, 17] . the main goal of the current study was to check the possibility of mers-cov shedding in the infected animal breath. we conducted this study according to the guidelines of the animal ethics protocols and the national committee of bio-ethics, king abdul-aziz city of science and technology, royal decree no. m/59 (http://www.kfsh.med.sa/kfsh_website/users uploadedfiles%5cncbe%20regulations%20english.pdf). the animal experiments were approved by the animal care committee of the deanship of scientific research, king faisal university, saudi arabia. we conducted molecular surveillance for the mers-cov in a dromedary camel herd during 2019. this herd consists of 52 animals held together in wire fenced yards. the male animals were kept in a separate individual partition while the female was kept together in multiple partitions. there is a shared open yard where all females may be allowed to be mixed together. the males only approach females during the mating season from november to march per each year. during the time from 10 march 2019 until 7 april 2019, we randomly selected nine animals out of the 52 (18%) from the camel herd to conduct our molecular surveillance. all animals were of variable ages ranging from 2 to 8 years old. the nine animals selected from different colour coat-based breeds, including magaheem, wodoah and sofr, were selected. these nine animals were mixed with the rest of the animals in the herd all the time. our molecular surveillance lasts from 10 march to 8 april 2019, with a biweekly sampling interval. we collected nasal swabs, breath samples from these nine animals, as previously described. a paired nasal and breath samples were collected per each animal at each time point, as described in detail below. nasal swabs were collected from at least 15% of the dromedary camel herd from november until march 2019. swabs were transferred into the tubes containing viral transport media including the dmem media containing 10% foetal bovine sera and antibiotic cocktail (penicillin and streptomycin). the processing of the collected swabs was done as previously described. briefly, the swabs were vortexed, then centrifuged at 5000 rpm for 10 min in a cooling centrifuge at 4°c. the supernatants were collected and stored at −80°c for further testing. the breath samples were collected in sterile tubes containing viral transport media as used for the collection of the nasal swabs. the breath collection technique was done in a simple way. briefly, the animals were secured in a specially designed camel crush. the animals were allowed to settle down for 5 min. with the help of an assistant, the nasal orifice is dilated, and the tube containing the media is slightly inserted inside (2-3 cm) the orifice without touching the skin or the mucous membranes of these animals during sampling. the collection tubes were held in a slope position facing the air stream coming from the animal during the exhalation. the tubes were kept in such position until the animal completes at least five cycles of breathing, including five exhalation streams. any container that touched the skin or the mucous membrane of the animals was discarded and not included in our analysis. the collection tubes were shaken immediately for 3 min and inverted upside down several times, then placed into ice tanks until transferred to the laboratory. the viral rnas were extracted from paired swabs and breath samples collected from dromedary camels using the qiagen viral rna (rneasy mini kit, qiagen, hilden, germany) extraction kit, according to the manufacturer's protocol. the concentration of the extracted viral rnas was measured by the nanodrop machine (thermo scientific nanodrop 2000, applied biosystems, 850 lincoln centre drive, foster city, ca 94404, usa). the eluted rnas were stored at −80°c for further testing. the rt-pcr targeting the upstream gene (up-e) of mers-cov was used for screening [13] . confirmation was done using the open reading frame (orf) 1a. five μl of the extracted rna was subjected to the real-time pcr testing using upe primers using lightmix molecular dx mers-cov-upe kits (roche, roche molecular systems, inc, pleasanton, ca 94588, usa) according to the manufacturer's protocol. all positive samples by the upe assay were confirmed by orf1a, as previously described [7] . positive samples were considered when there is an overlapping between the results from two targets. samples (nasal swabs and breath) that had ct values <39 were considered positive per each target. we used several sets of primers to amplify the partial mers-cov-s, mers-cov-n genes. the sequences of these primers are as follow: nf-5 ′ -cct tcg gta cag tgg agc ca-3 ′ and nr-5 ′ -gat ggg gtt gcc aaa cac aaa c-3 ′ , primers for the mers-cov-s gene sf-5 ′ -ccaattta-cgccaggatgat-3 ′ and sr-5 ′ -aatagaggcgg aaatagcac-3. we used the primers listed above to amplify the partial mers-cov-s and n genes using the qiagen one-step rt-pcr kit (cat no./id: 210210) to synthesis the cdna then doing the pcr in one reaction. the procedure of this one-step reaction was carried out as per the kit's instructions as well as previously described. we ran 5 μl per each amplified mers-n and s amplicons on a 1% agarose gel containing sybr® safe dna gel stain (invitrogen, thermo fisher scientific, waltham, ma, usa). the amplicons were visualised under ultraviolet light, then the gel pictures were photographed with the gel documentation system (bio-rad laboratories, inc., hercules, ca, usa). we purified the desired pcr products from the target bands by using the gel-based pcr extraction kits (qiagen, cat no/id: 28704), as per the instructions of the kits. we eluted the purified pcr products in 50 μl elution buffer as suggested by the kits, then stored at −20°c. we sequenced some positive samples from dromedary camels having low ct values (≤29) on the real-time pcr. the sequencing was done by the sanger method using the applied biosystems® we used the obtained sequences from dromedary camels to develop the phylogenetic tree based on the reported partial mser-cov-s and n genes sequences. the phylogenetic trees were built using the neighbour-joining method by the mega-7 software, as previously described. the scale bar represents the tree distance corresponding to 0.6 nucleotide substitution/nucleotides. a series of two by two tables, utilizing the fisher's exact test, were used to test the association between the results of the molecular surveillance, the sampling intervals and the sampling technique [18] . all the statistical data analyses were performed by spss version 21.0 (ibm corp, 2012). the values (<0.05) were considered significant (fig. 1) . our molecular testing for the selected animals at three time points starting 10 march 2019, until 7 april revealed that 1/9 (11%) of the tested nasal swabs were positive; all the nine animals were negative from the breath samples (table 1) . two weeks later, on 24 march, 6/9 (77%) nasal swabs were positive, and 5/9 (55%) breath samples were positive (table 1) . finally, on 7 april, 3/9 nasal swabs (33%) were positive, while 1/9 (11%) of the breath samples were positive ( table 1 ). the prevalence of positive nasal and breath samples was higher on the second sampling batch (24 march) compared to first (10 march) and third (7 april) batches. however, the difference was statistically significant (p < 0.05) only between the first vs. second sampling batches. similarly, consolidated results obtained from both nasal swaps and breath were significantly higher in the second batch vs. the first batch (p < 0.05). no significant statistical differences were observed between the prevalence of positive results obtained from nasal swabs and breath samples. the detection of the mers-cov-rnas in the breath of all three time intervals was statistically significant (p > 0.05) ( table 1) . the sequencing analysis of the partial mer-cov-s and n genes (figs 2 and 3 , respectively) from this positive mers-cov herd showing the detected viruses were closely related to the sequence from dromedary camels and humans from the arabian peninsula. the reported partial mers-cov-s gene clustered together with the other human sequences reported from al-hufuf city in the same regions in 2015 as well as sequences from qatar and jordon-2015 (fig. 2 ). the mers-cov continues to pose a significant risk for humans, especially some of those who may come in close contact with dromedary camels [12, 13] . despite the emergence of sars-cov-2 late 2019 [19] , mers-cov still has the high case fatality rates among the affected populations compared to sars-cov and sars-cov-2. the mers-cov case fatality rate started at 52% in 2012 then dropped down to 32% in 2019 [20] . the presence of mers-cov in the air of the proximity of patients and infected dromedary camels was previously documented independently by many research groups [6, 16, 17] . this result confirms that mers-cov may be transmitted through droplet infection. however, little is still known about the mechanism of transmission of mers-cov from the dromedary camels to humans. the roles of some camel secretions and excretions in the context of the mers-cov infection have been studied [5, 13, 15] . our results show that about 11% of the tested nasal swabs were positive during the first batch of the collection, while none of the breath samples was positive ( table 1 ). the reasons behind the inability to detect the viral rnas in the breath during the first round of sampling may be attributed to the dilution of the viral rna in the breath, and the rna may be present at the nondetectable limit by the real-time pcr. a high consistency was observed between the detection of the viral nucleic acids in both the nasal and breath samples in all three tested batches (p > 0.05), indicating similar opportunities to detect the virus nucleic acid in both methods (table 1 ). this may be attributed to several factors. first, during the early stage of the virus infection, the rna concentration in the breath may be at an undetectable level by the real-time pcr. second, the virus replication reached the peak of each animal; therefore, high virus concentration was achieved as previously reported [7, 8] . these results showed that mers-cov could be shed in the breath of infected animals for a while. however, the nasal swabs are still the sample of choice in the diagnosis of mers-cov among the infected dromedary camel population. spreading of the virus from animal to another in the same herd could potentially happen through the respiratory routes taking into consideration the very close contact between animals within the same herd. it may also occur through sharing the contaminated food and water from infected animals; however, all these aspects still need further investigations. detection of the virus in the air of positive camel's herd [5, 6] may suggest the virus is excreted in the breath of the infected animals in high concentration. however, this hypothesis was never tested before. detection of the foot and mouth diseases virus (fmdv) in the breath of some infected cattle was previously documented [21] . this study confirmed the potential spread of the fmdv between animals and among various herds in close proximity or within a distance. the aim of our study was to test the possibility of mers-cov shedding in the breath of the infected dromedary camels. our results are clearly showing the potential secretion of mers-cov in the breath of infected animals ( table 1) . the phylogenetic analysis based on the partial mers-cov-s and n genes in these infected animals revealed high identity to other mers-cov previously detected in the arabian peninsula (figs 2 and 3) [7, 8, 11] . a recent study showed that the sars-cov-2 could be transmitted through the droplet infection in the air [22] . although detection of the mers-cov-rnas in the breath does not conclude the viability of the detected virus particles. this may require virus isolation and the plaque assay to ensure the virus infectivity in the collected samples. these techniques should be done at a biosafety contaminant-3 laboratory. however, the overlapping between the viral detection in the nasal swabs and breath, as one of the gold standard techniques for the detection of the virus as well as the high degree of identity of the reported sequences in this study, may provide a piece of strong evidence about the potential shedding of the mers-cov in the breath of infected animals at various levels. further large-scale studies are highly recommended to document the curve of the mers-cov shedding in various body secretions and execrations as well as in the breath. this will lead to a better understanding of the dynamics of the virus spread among certain dromedary camel populations as well as in the environment. taken together all these evidence, we may conclude that mers-cov could be transmitted through the breath of infected animals. the virus spread from animal to animal and from animal to human come in their close contact (fig. 4) . however, large-scale controlled studies are still required to further enrich our understandings about the mechanism of mers-cov transmission between animals as well as from animals to humans. middle east respiratory syndrome coronavirus (mers-cov) neutralising antibodies in a high-risk human population comparative serological study for the prevalence of anti-mers coronavirus antibodies in high-and low-risk groups in qatar middle east respiratory syndrome coronavirus during pregnancy middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia evidence for camel-to-human transmission of mers coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient longitudinal study of middle east respiratory 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syndrome at an initial outbreak hospital in korea handbook of biological statistics a novel coronavirus from patients with pneumonia in china some one health based control strategies for the middle east respiratory syndrome coronavirus detection of foot-and-mouth disease virus in the breath of infected cattle using a hand-held device to collect aerosols airborne transmission of sars-cov-2: the world should face the reality acknowledgements. we wish to thank the king abdul-aziz city for science and technology (kacst), saudi arabia, for their generous funding through the mers-cov research grant programme (number 20-0004/24-1), which is a part of the targeted research program (trp). data availability statement. data are available upon request. key: cord-345356-gn1iwis0 authors: glebov, oleg o. title: understanding sars‐cov‐2 endocytosis for covid‐19 drug repurposing date: 2020-06-02 journal: febs j doi: 10.1111/febs.15369 sha: doc_id: 345356 cord_uid: gn1iwis0 the quest for the effective treatment against coronavirus disease 2019 pneumonia caused by the severe acute respiratory syndrome (sars)‐coronavirus 2(cov‐2) coronavirus is hampered by the lack of knowledge concerning the basic cell biology of the infection. given that most viruses use endocytosis to enter the host cell, mechanistic investigation of sars‐cov‐2 infection needs to consider the diversity of endocytic pathways available for sars‐cov‐2 entry in the human lung epithelium. taking advantage of the well‐established methodology of membrane trafficking studies, this research direction allows for the rapid characterisation of the key cell biological mechanism(s) responsible for sars‐cov‐2 infection. furthermore, 11 clinically approved generic drugs are identified as potential candidates for repurposing as blockers of several potential routes for sars‐cov‐2 endocytosis. more broadly, the paradigm of targeting a fundamental aspect of human cell biology to protect against infection may be advantageous in the context of future pandemic outbreaks. at the time of writing this piece, the pandemic of the atypical pneumonia coronavirus disease 2019 (covid-19) caused by a novel coronavirus severe acute respiratory syndrome (sars)-coronavirus 2 (cov-2) has spread globally, with more than 4 million cases and close to 300 000 deaths (https://www.worldometers.inf o/coronavirus/). this current epidemiological, economical and potentially political disaster in the making follows in the wake of earlier outbreaks of respiratory infections sars and middle east respiratory syndrome (mers), also caused by coronaviruses [1] . there is currently no known cure for covid-19, and available medical treatment is largely limited to alleviation of the respiratory dysfunction associated with the severe course of the disease. as a result of this, an enormous effort across biomedical sciences is mounting to develop therapeutic means of treating covid-19. one key area of this development is vaccination. as of may 11, there were five candidate vaccines in clinical evaluation, with as many as 71 in preclinical stage [2] . yet necessities of the research and development process dictate that none of the abovein the best possible scenariowill be clinically available earlier than in 18 months' time [3] . given the unprecedented scale of the current outbreak, there is immense pressure to find faster and cheaper therapeutic solutions to this rapidly worsening global health crisis. there is a broad agreement that perhaps the best way to produce an affordable treatment on a faster timescale is to repurpose existing drugs, and much interest is directed this way, especially focusing on drugs with previously demonstrated antiviral capabilities [4] . however, lack of the basic understanding of the cell biology of sars-cov-2 infection limits the efficacy of such an approach. although recent years have yielded some insight into the cell biology of the related human virus sars-cov, and the two viruses may bind to the same surface receptor protein angiotensin-converting enzyme 2 (ace2) [5] [6] [7] [8] , their genomes still differ by 20% [8] , and the associated epidemiological picture is very different indeed. furthermore, the extent of ace2 protein expression in the airways is subject to some controversy [9] [10] [11] [12] [13] , while preliminary evidence hints that other surface receptors may be involved in sars-cov-2 infection [14] . the resulting gap in knowledge forms a major obstacle on the road towards effective drug repurposing for treating covid-19. the critical step in any viral infection involves penetration of the viral particles into the cytosol; to this end, most viruses take advantage of the endocytic membrane trafficking of the host cell [15] . while the canonical endocytic pathways in mammalian cells involve budding of clathrin-coated vesicles through the action of a large gtpase dynamin, there are also multiple pathways of noncanonical endocytosis such as caveolae, flotillin-1-associated endocytosis, clathrin-independent carrier/glycosylphosphatidylinositol-anchored protein-enriched endosomal compartment endocytosis and macropinocytosis [16] , and viruses have been shown to use all of these [15] (fig. 1) . mechanistic details of these pathways may vary considerably between cell types, and diversity of endocytosis in airway epithelium is currently poorly understood. understanding endocytic viral entry in the respiratory tract may therefore offer a promising therapeutic strategy to treat viral infections. as far as coronaviruses are concerned, current evidence suggests that the mode of entry can vary between viruses and host cell types, and can include clathrin-dependent endocytosis [17] , caveolae [18] , and clathrin-and caveolae-independent mechanism involving lipid rafts [19, 20] . fusion at the cell surface, while proposed as a potential entry mechanism [5] , was not directly demonstrated. both sars-cov and sars-cov-2 appear to require endocytosis in cultured cell lines [6, [19] [20] [21] , and sars-cov may be endocytosed via more than one pathway depending on the cell line [19, 21] . however, there is little knowledge regarding the details of the epidemiologically relevant mode of viral entry, that is in the human lung. it is therefore imperative to consider sars-cov-2 endocytic trafficking in context of the correct host cell type. preliminary evidence suggests that infection with sars-cov-2 may begin in the upper respiratory tract, for example in the nasal epithelium, which expresses the highest levels of sars-cov-2 receptors [10] . the most significant morbidity and indeed mortality, however, appear to be associated with a further, severe stage of the disease, when infection spreads to the lower airways of the lung, resulting in respiratory failure and possibly multiorgan failure due to cytokine storm [22] . although the histopathology data are still scant, two case studies and preliminary findings from a primate disease model indicate that the lower airway sars-cov-2 infection may specifically affect the alveolar epithelial cells termed pneumocytes [23] [24] [25] . clearly, blockade of the sars-cov-2 entry at either of these stages is likely to yield considerable therapeutic benefits. to establish which endocytic pathways are likely to operate in nasal epithelial cells and pneumocytes, some clues can be gleaned from the local expression profile for the key endocytic proteins as evidenced by the human protein atlas (https://www.proteinatlas.org/). nasal epithelial cells abundantly express a wide variety of endocytic markers, suggesting the presence of multiple active pathways, in agreement with ultrastructural evidence [26] . conversely, pneumocytes show a more restricted expression pattern, with some key proteins associated with conventional endocytosis either low or missing. importantly, a large gtpase, dynamin, which is required for endocytosis through clathrin-coated vesicles and caveolae, is abundant in nasal epithelium but undetectable in pneumocytes. in contrast, there are medium-to-high expression levels of proteins involved in macropinocytosis, such as c-terminal binding protein (ctbp)1 & 2 and p21-activated kinase 1. therefore, macropinocytosis may be an important endocytic pathway in pneumocytes. the potentially distinct endocytic profile of upper and lower airways is reflected in the relevant infection mechanisms for known pathogens. upper respiratory epithelium can be infected by numerous airborne pathogens through multiple pathways of dynamin-dependent and dynamin-independent endocytosis [15] . conversely, pneumocyte infection is much less well understood; however, entry through macropinocytosis has been consistently demonstrated for airborne bacterial and viral pathogens such as mycobacterium tuberculosis [27], francisella tularensis [28], influenza a virus [29], adenovirus adenovirus serotype 35 [30] and haemophilus influenzae [31] , as well as for various nanoparticles [32, 33] . intriguingly, macropinocytosis in an alveolar epithelial cell line is upregulated by waterpipe smoke condensate, suggesting a possible mechanism underlying association of covid-19 morbidity with smoking [34] . taken together, the above evidence suggests that sars-cov-2 may employ distinct endocytic pathways for cell entry in the upper and lower respiratory tract (fig. 1) . what are these pathways? to address this question directly, we need to investigate the cell biology of sars-cov-2 endocytosis in detail. potential experimental model for investigating the key events of sars-cov-2 cell entry in vitro a suitable starting model for initial investigation of sars-cov-2 endocytosis can involve established immortalised cell lines derived from the respiratory tract epithelium (https://www.atcc.org/~/media/pdfs/ cancer%20and%20normal%20cell%20lines%20table s/lung%20cancer%20and%20normal%20cell%20line s.ashx). these lines provide several key methodological advantages: they are well-characterised, easy to maintain using standard cell culture protocols and retain the key characteristics of the primary cell type of origin. for emulation of the respiratory tract environment, the cell lines can be grown in an air-liquid interface culture as described before [35, 36] . immortalised cell cultures offer a simple and cost-effective platform for investigation of cell biology. there are, however, important caveats associated with immortalised cell cultures in vitro, which need addressing and further validation. one key consideration is expression profile, in which a cell line may be different from that in the original tissue. this can be especially relevant with regard to membrane trafficking, where discrepancy in expression of certain key proteins may affect the organisation of the whole network: for example, the lung cell line a549 can express multiple isoforms of dynamin [37] , which is not the case in pneumocytes in human lung tissue. as a result of this, any findings arising from the cell lines will need to be investigated further in a more expensive, but clinically relevant system of primary cells. cell preparations for both lower and upper [38, 39] . preliminary evidence shows that such systems can be efficiently infected with sars-cov-2 [40] . alternatively, cells can be directly obtained from human subjects, for example nasal epithelium, or alveolar epithelial cells can be isolated from surgically resected lung tissue material [41] . regardless of that, validation of findings in primary cells will be a key step in investigation. investigation of membrane trafficking of sars-cov-2 requires a probe that can adequately recapitulate the intracellular itinerary of the virus. using active, clinically isolated live virus samples would of course allow a closest approximation. however, a major drawback of this approach is a highly infectious nature of the virus, necessitating the use of a biosafety level 3 laboratory. an alternative approach would involve pseudoviruses, combining viral surface proteins responsible for cell receptor binding. the lack of sars-cov-2 genetic material renders them incapable of replication, allowing work in a biosafety level 2 laboratory. pseudoviruses have been successfully used before to investigate trafficking of sars-cov and mers-cov [5, 19] , and sars-cov-2 pseudovirus models have been recently published [6, 7] . for infection, the viral probe will be added to the cells for different lengths of time. in order to determine the endocytic pathway(s) involved in sars-cov-2 endocytosis, one can employ standard methods of multicolour fluorescence immunocytochemistry, light microscopy and colocalisation analysis. the proportion of the internalised virus colocalising with the classical markers of membrane trafficking compartments will indicate the intracellular itinerary of the virus [42] . for this approach, multiple well-characterised antibody markers for intracellular compartments, for example early endosomes, late endosomes and lysosomes are available. for a more detailed investigation of the endocytic route of the virus, viral infection can be combined with uptake of typical cargoes for different endocytic pathways. this approach would allow tracking of the virus in relation to other endocytic pathways and also to investigate the effect of viral infection on the general membrane trafficking network of the host cell. suitable cargo molecules include transferrin for clathrin-dependent endocytosis [43] , cholera toxin b subunit for caveolae [44] , and dextran for fluid-phase macropinocytosis and flotillin-dependent endocytosis [45] . what is the relative contribution of different endocytic pathways to sars-cov-2 infection? to address this question, loss-of-function analysis can be performed, involving knockdown of the key regulatory proteins for different endocytic pathways, for example dynamin 2, caveolin-1, flotillin-1 and ctbp [16, 44] (fig. 1) . the resulting findings can be further corroborated using available pharmacological blockers of endocytic pathways, including dynasore and pitstop for clathrin-dependent endocytosis, nystatin for caveolae, methyl-beta-cyclodextrin for dynamin-independent endocytosis and amiloride for macropinocytosis. however, care must be exercised in interpretation of these results due to the off-target effects of these agents, in which endocytic pathways other than the target pathway may be inhibited [46] [47] [48] . taken together, the combination of adequate cell models with the newly developed sars-cov-2 toolkit and established tools of membrane trafficking research is well-poised to deliver a key insight into the mechanisms underlying covid-19 infection. two broad approaches to developing drugs against sars-cov-2 involve targeting the biology of the host versus targeting the biology of the virus. one clear drawback associated with global perturbation of a general cell biological function is the potential for unwelcome side effects, whether in the same target tissue or elsewhere in the body. conversely, a key advantage of focusing on the cell biological mechanism is the relatively immutability of the host cell compared to virus, which may evolve drug resistance due to its high mutation rate [49, 50] . furthermore, considering that various viruses may use the same endocytic pathways of the host cell [15] , targeting viral entry at the point of endocytosis holds a more general promise for the development of broad-spectrum antiviral drugs [51] . once the endocytic route of sars-cov-2 in primary lung epithelial cells has been established, this insight can be capitalised upon for translational needs. specifically, a cell-based model of sars-cov-2 infection [40] will lend itself to establishment of a screening platform for drug development, aiming to identify compounds capable of blocking or hindering sars-cov-2 endocytic entry. there is already vivid interest in chloroquine and hydroxychloroquine, two related drugs that may block viral entry through endosomal acidification (fig. 1) , although evidence for their effectiveness is currently scant and safety hazards are a major concern [4, 52, 53] . large-scale screening of this sort will require considerable time and effort; in the meantime, a short-term screening of a limited number of candidate drugs is highly warranted. to this end, the range of candidates can be limited to the following 11 approved drugs ( table 1 , fig. 1 ). although most of these drugs are not currently prescribed against infection, they have shown ability to block various pathways of endocytosis in cell culture and may therefore have potential for blocking sars-cov-2 endocytosis. another addition to this list is a cholesterol chelating agent methyl-betacyclodextrin, used as a carrier to enhance bioavailability in drug formulations and generally recognised as safe by the food and drug administration (usa). importantly, 5 out of 11 drugs (chlorpromazine, nystatin, amiloride, vinblastine and itraconazole) are on the world health organization's list of essential medicines, ensuring their wide availability across the world. also, several of the above have been tested (or are being tested) for topical delivery into the respiratory tract (table 1) . these factors will be favourable for the rapid integration of the successful drugs into global clinical practice, subject to development of relevant clinical and epidemiological applications. despite frenzied efforts to find effective treatments for covid-19, very little is currently known about the fundamental cell biology of the sars-cov-2 infection. the pivotal event underpinning sars-cov-2 infection is likely endocytosis of viral particles; therefore, blockade of this process may provide for a major therapeutic breakthrough. the tools of membrane trafficking research can be readily applied to rapidly characterise the endocytic route of virus entry in a cell-based model of disease, providing a key insight into the disease mechanism (fig. 1) . crucially, the same model can be used as a screening platform for rapid repurposing of existing approved drugs as blockers of sars-cov-2 endocytic entry (table 1 ). in the longer term, insight into the poorly understood membrane trafficking mechanisms of the airway epithelium will aid development of novel drugs. the generalised strategy of targeting the membrane trafficking of the host cell biology to prevent viral infection holds great promise for future therapeutic applications, considering the existence of other zoonotic coronaviruses capable of binding to human cells [54] . more broadly, rapid repurposing of approved drugs in cell-based screening platforms is poised to play an instrumental role in future-proofing global health care against the interspecies transmission events. in the age of unparalleled spending on pharmaceutical research and development, humankind cannot afford to neglect one of its most valuable allies in the fight against the current and the future pandemicsthe basic foundations of human cell biology. covid-19: lessons from sars and mers who (2020) coronavirus disease coronavirus vaccine: when will it be ready? | world news | the guardian. available at discovering drugs to treat coronavirus disease 2019 (covid-19) structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a pneumonia outbreak associated with a new coronavirus of probable bat origin the protein expression profile of ace2 in human tissues sars-cov-2 entry genes are most highly expressed in nasal goblet and ciliated cells within human airways sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in 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inhibitors inhibit dynamin i guanosine triphosphatase (gtpase) suppression of dynamin gtpase activity by sertraline leads to inhibition of dynamin-dependent endocytosis herxheimer h (1949) antihistamines in bronchial asthma direct engagement of tlr9 ligand with t helper cells leads to cell proliferation & up-regulation of cytokines pulmonary moniliasis treated with nystatin aerosol amiloride inhalation therapy in cystic fibrosis: influence on ion content, hydration, and rheology of sputum identification of novel macropinocytosis inhibitors using a rational screen of food and drug administration-approved drugs vinblastine but not other microtubule inhibitors block transferrin endocytosis and iron uptake by reticulocytes inhaled itraconazole fast-tracked for allergic bronchopulmonary aspergillosis in asthma itraconazole inhalation receives ind approval for phase 2 clinical trial. available at pharmaceutical applications of cyclodextrins. iii. toxicological issues and safety evaluation 2020 the authors the authors declare no conflict of interest. key: cord-342383-ckswlo9o authors: pawlowski, c.; puranik, a.; bandi, h.; venkatakrishnan, a.; agarwal, v.; kennedy, r.; o'horo, j. c.; gores, g. j.; williams, a. w.; halamka, j.; badley, a. d.; soundararajan, v. title: exploratory analysis of immunization records highlights decreased sars-cov-2 rates in individuals with recent non-covid-19 vaccinations date: 2020-07-28 journal: nan doi: 10.1101/2020.07.27.20161976 sha: doc_id: 342383 cord_uid: ckswlo9o multiple clinical studies are ongoing to assess whether existing vaccines may afford protection against sars-cov-2 infection through trained immunity. in this exploratory study, we analyze immunization records from 137,037 individuals who received sars-cov-2 pcr tests. we find that polio, hemophilus influenzae type-b (hib), measles-mumps-rubella (mmr), varicella, pneumococcal conjugate (pcv13), geriatric flu, and hepatitis a / hepatitis b (hepa-hepb) vaccines administered in the past 1, 2, and 5 years are associated with decreased sars-cov-2 infection rates, even after adjusting for geographic sars-cov-2 incidence and testing rates, demographics, comorbidities, and number of other vaccinations. furthermore, age, race/ethnicity, and blood group stratified analyses reveal significantly lower sars-cov-2 rate among black individuals who have taken the pcv13 vaccine, with relative risk of 0.45 at the 5 year time horizon (n: 653, 95% ci: (0.32, 0.64), p-value: 6.9e-05). these findings suggest that additional pre-clinical and clinical studies are warranted to assess the protective effects of existing non-covid-19 vaccines and explore underlying immunologic mechanisms. we note that the findings in this study are preliminary and are subject to change as more data becomes available and as further analysis is conducted. since the genome for sars-cov-2 was released on january 11, 2020, scientists around the world have been racing to develop a vaccine 1 . however, vaccine development is a long and expensive process, which takes on average over 10 years under ordinary circumstances 2 . even for the previous epidemics of the past decade, including sars, zika, and ebola, vaccines were not available before the virus spread was largely contained 3 . conventionally vaccinations are intended to train the adaptive immune system by generating an antigen-specific immune response. however, studies are also demonstrating that certain vaccines lead to protection against other infections through trained immunity 4 . for instance, vaccination against smallpox showed protection against measles and whooping cough 5 . live vaccinia virus was successfully used against smallpox. due to the urgent need to reduce the spread of covid-19, scientists are turning to alternate methods to reduce the spread, such as repurposing existing vaccines. there are some hypotheses that the bacillus calmette-guérin (bcg) and live poliovirus vaccines may provide some protective effect against sars-cov-2 infection 6, 7 . there are several ongoing/recruiting clinical trials testing the protective effects of existing vaccines against sars-cov-2 infection, including: polio 8 , measles-mumps-rubella vaccine 9 , influenza vaccine 10 , and bcg vaccine 11, 12, 13, 14 . in this work, we conduct a systematic analysis to determine whether or not a set of existing non-covid-19 vaccines in the united states are associated with decreased rates of sars-cov-2 infection. in figure 1 , we provide an overview of the study design and statistical analyses. we consider data from 137,037 individuals from the mayo clinic electronic health record (ehr) database who received pcr tests for sars-cov-2 between february 15, 2020 and july 14, 2020 and have at least one icd diagnostic code recorded in the past five years (see methods). in table 1 , we show the clinical characteristics of the study population. in particular, 92,673 (67%) individuals have at least 1 vaccine in the past 5 years relative to the pcr testing date. in figure 2 , we present the sars-cov-2 infection rates for subsets of the study population with particular clinical covariates. we note that the rates of sars-cov-2 infection are higher in black, asian, and hispanic racial and ethnic subgroups compared to the overall study population. this is likely due to higher rates of covid-19 spread and/or decreased access to pcr testing. in addition, the rates of sars-cov-2 infection are lower in individuals with pre-existing conditions (e.g. hypertension, diabetes, obesity) possibly due to greater caution in avoiding exposure and/or higher pcr testing rates. given this study population, we assess the rates of sars-cov-2 infection among individuals who did and did not receive one of 18 vaccines in the past 1, 2, and 5 years relative to the date of pcr testing. in table 2 , we present the full names, common formulations, and counts for the 18 vaccines that we consider. first, we assess the overall association of vaccination status with the risk of sars-cov-2 infection (see methods). we use propensity score matching to construct unvaccinated control groups for each of the vaccinated populations at the 1 year, 2 year, and 5-year time horizons. the unvaccinated control groups are balanced in covariates including demographics, county-level incidence and testing rates for sars-cov-2, comorbidities, and number of other vaccines taken in the past 5 years. then, we compare the sars-cov-2 rates between each of the vaccinated cohorts and corresponding matched, unvaccinated control groups which have similar clinical characteristics. second, we repeat the analysis on a set of age, race, and blood type stratified subgroups of the study population. in particular, for each subgroup, we run propensity score matching and compute the difference in sars-cov-2 infection rate between the vaccinated and unvaccinated (matched) cohorts. finally, we run a series of sensitivity analyses to evaluate whether or not these results may be biased from unobserved confounders or other factors. the 1-year time horizon. in figure 3 , we present the vaccination coverage rates for each of these vaccines in the study population for all time horizons. overall, we observe that the polio and hib vaccinated cohorts generally have the lowest relative risks for sars-cov-2 infection across all time horizons. the relative risk of sars-cov-2 infection is 0.57 (n: 2,402, 95% ci: (0.42, 0.77), p-value: 0.003) for individuals who have taken the polio vaccine in the past 1 year, and 0.53 (n: 2,061, (95% ci: (0.37, 0.77), p-value: 3.2e-03) for individuals who have taken the hib vaccine in the past year. we note that these vaccines are almost exclusively administered to individuals under 18 years of age, as shown in figure 4 . other vaccines that are commonly administered to younger individuals with strong negative correlations with sars-cov-2 infection include mmr and varicella vaccines. the other vaccines which are consistently associated with lower sars-cov-2 rates include pcv13, geriatric flu, and hepa-hepb vaccines. at the 1 year time horizon, the relative risks of sars-cov-2 infection are 0.72 for pcv13 (n: 4,693, 95% ci: (0.56, 0.92), p-value: 0.03), 0.74 for geriatric flu (n: 12,085, 95% ci: (0.61, 0.89), p-value: 5.6e-03), and 0.80 for hepa-hepb (n: 5,858, 95% ci: (0.67, 0.97), p-value: 0.05). although the relative risks are less significant compared to polio and hib, these associations may be particularly interesting to explore further because these vaccines are commonly administered across a broader age range of the population (see figure 4 ). in order to identify vaccines which may be confounding factors for other vaccines that are linked to reduced rates of sars-cov-2 infection, we conduct a pairwise correlation analysis. for example, it is possible that the lower rates of sars-cov-2 infection that we observe for one vaccine are in fact caused by another vaccine which is highly correlated with the former. to measure the correlations we use cohen's kappa, which is a measure of correlation for categorical variables that ranges from -1 to +1. in particular, cohen's kappa = +1 indicates that the pair of vaccines are always administered together, cohen's kappa = 0 indicates that the pair of vaccines are independent of each other, and cohen's kappa = -1 indicates that the pair of vaccines are never administered together. in figure 5 , we present a heatmap of the pairwise correlations for each of the 18 vaccines administered in the 5 years prior to the pcr test date. sorted by cohen's kappa value, the top vaccine pairs with kappa ≥ 0.60 are: hib and rotavirus (0.83), hib and polio (0.80), mmr and varicella (0.74), polio and varicella (0.72), polio and rotavirus (0.71), mmr and polio (0.68). from this, we see that there is a cluster of vaccines which are commonly administered together, which includes: hib, polio, rotavirus, varicella, and mmr vaccines. the majority of individuals who receive this cluster of vaccines are children <18 years old (see figure 4 ). we note that in this cluster, the vaccines hib, polio, varicella, and mmr are all consistently associated with lower sars-cov-2 rates. this suggests that some of the lower rates of sars-cov-2 observed in these vaccinated cohorts may be confounded by the other vaccines in this group. in tables 7-9, we present the results of propensity score matching at the 1, 2, and 5-year time horizon, respectively, on study cohorts stratified by race. we observe that pcv13 vaccination is linked with significantly decreased sars-cov-2 rates in the black subpopulation. in particular, the relative risk of sars-cov-2 infection for black individuals who have been administered pcv13 is 0.24 at the 1 year time horizon (n: 197, 95% ci: (0.09, 0.71), p-value: 0.03); 0.33 at the 2 year time horizon (n: 239, 95% ci: (0.16, 0.74), p-value: 0.03); and 0.45 at the 5 year time horizon (n: 653, 95% ci (0.32, 0.64), p-value: 6.9e-5). furthermore, at the 5-year time horizon, the relative risk for the pcv13 vaccinated cohort of black individuals is significantly lower than the relative risk for the pcv13 vaccinated cohort overall (p-value: 0.03). in addition, we observe that polio, hib, and pcv13 vaccines are linked with decreased sars-cov-2 rates in the white subpopulation. however, since 119,979 (88%) of individuals in the study population are white, the relative risks for these vaccinated cohorts are close to the relative risks for the overall population (see tables 3-5) . matching within subgroups was done by age group (0-18, 19-49, 50-64, 65+) and blood group (a, b, ab, o) as well, but no significant within-subgroup associations between any vaccine and sars-cov-2 rates were found. this suggests that associations between vaccines and sars-cov-2 infection rates may not be strongly specific to particular age ranges/blood groups. in this retrospective study, we evaluate the correlations between vaccination and sars-cov-2 infection, taking into account a number of possible confounding variables, such as demographic variables and geographic covid-19 incidence rate (see methods). however, it is possible that the results from this study have been influenced by unobserved confounders. for example, we do not explicitly control for travel history, which was a significant risk factor for sars-cov-2 infection early on in the pandemic. in figure 6 , we present the results from the tipping point analysis on the statistically significant associations between vaccination and reduced rates of sars-cov-2 infection in the overall study population. for each time horizon, we show the relative prevalence and effect size that would be required for an unobserved confounder to overturn the conclusion for a given (vaccine, time horizon) pair. for reference, we show the effect size of the covariate (county-level covid-19 incidence rate ≥ median value) as a potential confounder, which has a large relative risk of 2.78. at the 1 year and 2 year time horizons, the associations of the polio vaccine to lower rates of sars-cov-2 infection are most robust to the impact of a potential unobserved confounder. in particular, an unobserved confounder with a large effect size of 2.78 would need to have an absolute difference in prevalence between vaccinated and unvaccinated cohorts of 17.8% (30.9%) in order to overturn the results for the 1 year (2 year) time horizon. on the other hand, at the 5 year time horizon, the association of pcv13 and lower rates of sars-cov-2 infection is most robust to the influence by unobserved confounders. an unobserved confounder with a large effect size of 2.78 would need to have an absolute difference in prevalence between vaccinated and unvaccinated cohorts of 19.1% in order to render the findings insignificant. ongoing clinical studies offer preliminary evidence that existing vaccines may reduce risk of sars-cov-2 infection. for example, interim results from the activate trial 12 indicate that the bcg vaccine reduces sars-cov-2 infection rates up to 53%. while specific vaccines such as bcg are being tested for cross-protective effects against sars-cov-2 infection based upon their prior potential for protection against other diseases 14 , to our knowledge, a systematic hypothesis-free analysis to identify potential vaccines that can have beneficial effects against sars-cov-2 infection is lacking. our retrospective study has analyzed 18 different vaccines and identified key vaccines that are correlated with lower rates of sars-cov-2 infection after controlling for confounding factors (see results). in particular, we find that individuals who have been recently vaccinated with one of polio, hib, mmr, varicella, pcv13, geriatric flu, or hepa-hepb vaccines have lower rates of sars-cov-2 infection. these vaccines are promising candidates for follow-up pre-clinical animal studies and clinical trials. we note that this list of vaccines is preliminary and may change as more data becomes available and as further analysis is conducted. for the rest of the 18 vaccines that we considered, the correlations with sars-cov-2 infection were either insignificant or varied across the time horizons of interest. in some cases, these vaccines may serve as negative controls in clinical trials testing the safety and efficacy of novel covid-19 vaccines. for example, a clinical trial evaluating the covid-19 vaccine candidate chadox1 uses meningococcal vaccine as a comparator arm 15 . preliminary results from this trial indicate that as expected, meningococcal vaccine does not induce antibody responses against sars-cov-2 spike protein. it may be interesting to evaluate the antibody responses for some of the vaccines that we have found to be significantly correlated with lower rates of sars-cov-2 infection, to explore if there is any underlying immunologic mechanism for the associations that we observe. because the bcg vaccine is rarely administered in the us, this vaccine did not meet the sample size threshold for inclusion in our analysis. from the limited data available, there were 51 individuals in the study population who had taken bcg vaccine in the past 5 years, and among these 0 individuals tested positive for sars-cov-2 infection (95% ci: (0.0%, 7.0%)). among the 198 individuals who had taken bcg vaccine at least once in their lifetime, there were 6 (3.0%) individuals who tested positive for sars-cov-2 infection (95% ci: (1.4%, 6.5%)). as a result, more data from additional medical centers would be required for us to assess the associations between bcg vaccine and sars-cov-2 infection. due to the observational nature of this study, there are potential biases which may have impacted the findings, including confounding, selection bias, and measurement bias. the motivation for using propensity score matching was to account for confounding. although we take into account some potential confounders through propensity score matching, there may still be residual confounding from unobserved factors (e.g. socioeconomic status, indications, contraindications, etc.) which may be different for each vaccine. for example, travel history is a risk factor for exposure to sars-cov-2 infection that we do not explicitly account for in this study. our motivation for the tipping point sensitivity analysis is to estimate the effect size and prevalence of an unobserved confounder which would be required to overturn the statistically significant findings (see figure 6 ). even among the variables that we consider, there is potential for bias if the cohorts are poorly matched on those covariates. in tables s1-s7, we present the propensity score matching results for a number of vaccines at the 1 year time horizon, in order to show the matching quality for each of these statistical comparisons. furthermore, we present plots showing the distribution of the age covariate in particular in figure s1 . we note that for some vaccines, differences in age between the vaccinated and unvaccinated (matched) cohorts may have influenced the results. in addition, it is possible that restricting the study population to sars-cov-2 pcr tested individuals may have introduced selection bias. for example, vaccinated individuals may engage in more health-seeking behaviors to reduce their potential covid-19 risk, and also have a higher likelihood of seeking out a pcr test. this type of bias is known as the "healthy user effect", which is suspected to have influenced the findings of recent covid-19 observational studies 16, 17 . we performed sensitivity analyses using breast cancer and colon cancer screening as negative controls which suggest that the propensity score matching analysis is in part effective in filtering out healthy user effect for the associations between vaccination status and sars-cov-2 risk. finally, measurement bias is a concern as vaccination records may be incomplete for some individuals in our cohort since they may have received the vaccines outside of the mayo clinic system. we plan to perform additional sensitivity analyses to further explore these potential sources of bias. as an initial exploratory analysis linking historical vaccination records to sars-cov-2 pcr testing results, more research is warranted in order to confirm the findings. we plan to update this analysis in coming months as more pcr testing data becomes available. also, we note that this study is based on data from one academic medical center in the united states, which restricts the analysis to vaccines administered in this geographic region. notably, we do not have sufficient immunization record data on the bcg vaccine, which has shown promise in early clinical trials. as a result, the findings from this study would be well complemented by similar studies from hospitals across the world. this is an observational study in a cohort of individuals who underwent polymerase chain reaction (pcr) testing for suspected sars-cov-2 infection at the mayo clinic and hospitals affiliated to the mayo health system. the full dataset includes 152,548 individuals who received pcr tests between february 15, 2020 and july 14, 2020. we restricted the study population to 137,037 individuals from this dataset who have at least one icd code recorded in the past 5 years. this exclusion criteria is applied in order to restrict the analysis to individuals with medical history data. within this pcr tested cohort, we define covidpos to be persons with at least one positive pcr test result for sars-cov-2 infection, which includes 5,679 individuals. similarly, we define covidneg to be persons with all negative pcr test results, which includes 131,358 individuals. for the study population of 137,037 individuals, we obtain a number of clinical covariates from the mayo clinic electronic health record (ehr) database, including: demographics (age, gender, race, ethnicity, county of residence), icd diagnostic billing codes from the past 5 years, and immunization records from the past 5 years (68 unique vaccines; we focus on the 18 taken by at least 1,000 individuals over the past 5 years). we use the elixhauser comorbidity index to map the icd codes from each individual from the past 5 years to a set of 30 medically relevant comorbidities 18 . in addition to the mayo clinic ehr database, we use the corona data scraper online database to obtain incidence rates of covid-19 at the county-level in the united states 18, 19 . by linking the county of residence data from the ehr with the incidence rates of covid-19 from corona data scraper, we are able to obtain county-level incidence rates of covid-19 for 136,313 individuals in the study population. we also obtain county-level testing data for 100,433 individuals in the study population from (i) minnesota state government records and (ii) public county-level testing data scraped from other state/county websites. in table 1 , we present the average values for each of the clinical covariates in the study population. given these clinical covariates, we conduct a series of statistical analyses to assess whether or not each of the 19 vaccines has an association with lower rates of sars-cov-2 infection at the 1 year, 2 years, and 5 year time horizons. for each vaccine and time horizon, the vaccinated cohort is defined as the set of individuals in the study population who received the vaccine within the past time horizon. for example, the "2-year polio vaccinated cohort" is the set of individuals who received the polio vaccine within the past two years. similarly, for each vaccine and time horizon, the unvaccinated cohort is defined as the set of individuals in the study population who did not receive the vaccine within the past time horizon. for example, the "5-year influenza unvaccinated cohort" is the set of individuals who did not receive the influenza vaccine within the past five years. in the following sections, we describe the statistical methods that we use to compare the rates of covid-19 between the vaccinated and unvaccinated cohorts for each of the (vaccine, time horizon) pairs. first, we describe the propensity score matching analysis to construct unvaccinated control groups that have similar clinical characteristics to the vaccinated cohorts. second, we describe the statistical tests that we use to determine which of the (vaccine, time horizon) pairs have the most significant association with lower rates of sars-cov-2 infection for the 1 year, 2 year, and 5 year time horizons, both overall and for particular demographic subgroups. third, we describe the covariate-level stratification analysis to identify vaccines which have the largest association with lower rates of sars-cov-2 infection for particular demographic subgroups. finally, we describe the sensitivity analyses that we use to evaluate the robustness of the statistical methods to potential biases from unobserved confounders or other factors that could impact the overall results from this observational study. before running the propensity score matching step, first we filtered to vaccinated cohorts with at least 1,000 persons. for the overall statistical analysis, there were 13, 15, and 18 vaccines which met this threshold for the 1 year, 2 year, and 5 year time horizons, respectively. for each vaccinated cohort with sufficient numbers of individuals, we applied 1:1 propensity score matching to construct a corresponding unvaccinated control group with similar clinical characteristics 20 . we refer to this as the "unvaccinated (matched)" cohort, which is a subset of the unvaccinated cohort. we considered the following clinical covariates in the propensity score matching step: • demographics (age, gender, race, ethnicity) • county-level covid-19 incidence rate: (number of positive sars-cov-2 pcr tests in county) / (total population of county) within +/-1 week of pcr testing date. • pregnancy: whether or not the individual had a pregnancy-related icd code recorded in the past 90 days relative to the pcr testing date. • number of other vaccines: count of the total number of unique vaccines (excluding the vaccine which is the treatment variable) taken by the individual in the past 5 years relative to the pcr testing date. for each of the vaccinated cohorts, we fit a logistic regression model to predict whether or not the individual was vaccinated, using these covariates as predictors. we trained the logistic regression model using the scikit-learn package in python 21 . then, we used the modelpredicted probability of an individual receiving the vaccine as the propensity score for the individual. matching was done without replacement using greedy nearest-neighbor matching within calipers. some subjects were dropped from the positive cohort in this procedure. the matching was performed with caliper width 0.2 * (pooled standard deviation of scores), as suggested in the literature 22 . after the propensity score matching step, we compare the covidpos rates for the vaccinated and unvaccinated (matched) cohorts. first, we compute the relative risk, which is equal to the covidpos rate for the vaccinated (matched) cohort divided by the covidpos rate for the unvaccinated (matched) cohort. we use a fisher exact test to compute the p-value for this association. we then apply the benjamini-hochberg (bh) adjustment 23 on the p-values over all vaccines for each time horizon to control the false discovery rate (at 0.05). we also compute and report 95% confidence intervals for the relative risks. we repeat the statistical analysis on subsets of the study population stratified by age, race/ethnicity, and blood type. for age, we consider the subgroups: 0 to 18 years, 19 to 49 years old, 50 to 64 years old, and ≥ 65 years old. for race/ethnicity, we consider the subgroups: white, black, asian, and hispanic. for blood type, we consider the subgroups: o, a, b, and ab. we note that age and race/ethnicity were recorded in the dataset for all subjects, but blood type information was only available for 41,828 subjects. for each vaccine, at the 1, 2, and 5 year time horizons, we use propensity score matching to construct unvaccinated control groups for each age bracket, race/ethnicity, and blood type subgroup. matching was done on the same covariates as in the overall analysis (apart from the race/ethnicity covariates for the race/ethnicity subgroups). we then compared the covidpos rates between the vaccinated and unvaccinated (matched) cohorts, and reported the relative risk, 95% confidence interval, and bh-corrected p-values. we performed two sets of sensitivity analyses, as described below. to assess the effectiveness of the propensity score matching procedure, we ran the statistical analysis using cancer screens as the exposure variable instead of vaccinations (i.e. negative control exposure). this set of experiments serves as a negative control because it is highly unlikely that cancer screenings are causally linked to risk of sars-cov-2 infection. in particular, we considered the following two cancer screens as negative controls: • colon cancer screen: whether or not the individual received a screening for colon cancer (within a specified time horizon relative to pcr testing date). • mammogram: whether or not the individual received a mammogram screening for breast cancer (within a specified time horizon relative to pcr testing date), in table 6 , we present the results from the negative control experiments. in the unmatched cohorts, we observe that persons who have had a mammogram in the past 1, 2, or 5 years have significantly lower rates of sars-cov-2 infection compared to persons who have not had mammograms during the same time period. for example, the sars-cov-2 infection rate is 2.5% among persons with mammograms in the past 5 years and 4.5% among persons without mammograms in the past 5 years (p-value: 1.9e-47). this significant difference in sars-cov-2 infection rate can be explained by confounding variables, because the unmatched cohorts have different underlying clinical characteristics. however, after propensity score matching, the sars-cov-2 infection rate is 2.8% among persons with mammograms in the past 5 years and 2.8% among persons without mammograms in the past 5 years (p-value: 1). we observe similar results for the colon cancer screening covariate. for example, the sars-cov-2 infection rate is 2.5% among persons with colon cancer screens in the past 5 years and 4.4% among persons without colon cancer screens in the past 5 years (p-value: 9.3e-44). after propensity score matching, the sars-cov-2 infection rate is 2.5% with and 2.4% without colon cancer screens in the past 5 years (p-value: 1). in total, 6 comparisons (2 controls, 3 time horizons each) were done. after applying fisher's method to combine p-values, we get a combined p-value of 0.22 (x 2 = 15, df=12) against the combined hypothesis that none of the controls have an association with sars-cov-2 after propensity score matching. we expect that the individuals who have recently taken cancer screens may have lower rates of sars-cov-2 infection due to the "healthy user effect" 17 . in particular, persons who have recently had mammograms or colonoscopies may engage in general health-seeking behaviors which decrease their risk of sars-cov-2 infection or generally decrease their risk of a positive pcr test result. the results from the negative control experiment demonstrates that the propensity score matching is able to correct for confounding variables which may contribute to spurious findings such as those caused by the healthy user effect. in order to evaluate how robust the associations between vaccinations and sars-cov-2 infection found in this study are to the effects of potential confounders, we conduct a "tipping point" analysis 24 . the purpose of this analysis is to find the point at which an unobserved confounder would "tip" the conclusion on each vaccine, making the results no longer statistically significant. here, there are two dimensions to consider: (1) the effect size (i.e. relative risk of sars-cov-2 infection) of the confounder, and (2) the relative prevalence of the confounder in the vaccinated vs. unvaccinated (matched) cohorts. for each vaccine, we compute the relative prevalence and effect size that would be required for an unobserved confounder to overturn the conclusion for a given (vaccine, time horizon) pair. we present the results from the tipping point analysis in figure 6 . this research was conducted under irb 20-003278, "study of covid-19 patient characteristics with augmented curation of electronic health records (ehr) to inform strategic and operational decisions". table 6 : summary of sars-cov-2 rates for individuals who did vs. did not receive negative control treatments before and after propensity score matching. sars-cov-2 positive rates, relative risks, and associated bh-adjusted fisher exact p-values for individuals who received or did not receive negative control treatments over the past 1 year, 2 years, and 5 years prior to pcr test. the negative control treatments considered are: (1) colon cancer screen and (2) mammogram. the bh adjustment is applied per time horizon, as in the main analysis. numbers are shown before and after propensity score matching. unmatched numbers are shown in red text. for each vaccine that is associated with lower sars-cov-2 rates in a particular time horizon, we plot the (prevalence, effect size) combinations of an unobserved confounder that would be required to overturn the results. the x-axis indicates the absolute difference in prevalence of the confounder between vaccinated and unvaccinated (matched) cohorts. for example, if the unobserved confounder is present in 25% of the vaccinated cohort and 5% of the unvaccinated cohort, then the absolute difference in prevalence would be 20%. the y-axis indicates the relative covidpos risk (effect size) of the unobserved confounder. for reference, we show the relative risk of (county-level covid-19 incidence rate ≥ median value) as a horizontal dotted line, which is equal to 2.78. each plot is annotated with the top 3 vaccines that are most robust to unobserved confounders, along with the intersection point between the vaccine curve and the reference line. for example, for the polio vaccine at the 1 year time horizon, an unobserved confounder with a relative risk of 2.78 which is prevalent in 17.8% of the vaccinated cohort and 0% of the unvaccinated cohort could explain the differences in sars-cov-2 infection rates that we observe in the data. the covid-19 vaccine development landscape risk in vaccine research and development quantified developing covid-19 vaccines at pandemic speed defining trained immunity and its role in health and disease trained immunity-based vaccines: a new paradigm for the development of broad-spectrum anti-infectious formulations considering bcg vaccination to reduce the impact of covid-19 can existing live vaccines prevent covid-19? opv as potential protection against covid-19 -full text view -clinicaltrials.gov measles vaccine in hcw -full text view -clinicaltrials influenza vaccination, acei and arb in the evolution of sars-covid19 infection -full text view -clinicaltrials bcg vaccination for healthcare workers in covid-19 pandemic -full text view -clinicaltrials bacillus calmette-guérin vaccination to prevent covid-19 -full text view -clinicaltrials bcg vaccination to protect healthcare workers against covid-19 -full text view -clinicaltrials bcg vaccine for health care workers as defense against covid 19 -full text view -clinicaltrials safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial collider bias undermines our understanding of covid-19 disease risk and severity healthy user and related biases in observational studies of preventive interventions: a primer for physicians comorbidity measures for use with administrative data an introduction to propensity score methods for reducing the effects of confounding in observational studies scikit-learn: machine learning in python optimal caliper widths for propensity-score matching when estimating differences in means and differences in proportions in observational studies controlling the false discovery rate: a practical and powerful approach to multiple testing assessing the sensitivity of regression results to unmeasured confounders in observational studies on jstor elixhauser -hypertension 1728 (36.8%) elixhauser -pulmonary 1013 (21.6%) 1390 (29.6%) 1013 (21.6%) elixhauser -pulmonary 1227 (20.9%) 1663 (28.4%) 1227 (20.9%) 28908 (22%) the authors thank murali aravamudan, patrick lenehan, and hilal maradit-kremers for their careful review and helpful feedback on this manuscript. key: cord-353777-t8q99tlq authors: jia, yong; shen, gangxu; zhang, yujuan; huang, keng-shiang; ho, hsing-ying; hor, wei-shio; yang, chih-hui; li, chengdao; wang, wei-lung title: analysis of the mutation dynamics of sars-cov-2 reveals the spread history and emergence of rbd mutant with lower ace2 binding affinity date: 2020-04-11 journal: biorxiv doi: 10.1101/2020.04.09.034942 sha: doc_id: 353777 cord_uid: t8q99tlq monitoring the mutation dynamics of sars-cov-2 is critical for the development of effective approaches to contain the pathogen. by analyzing 106 sars-cov-2 and 39 sars genome sequences, we provided direct genetic evidence that sars-cov-2 has a much lower mutation rate than sars. minimum evolution phylogeny analysis revealed the putative original status of sars-cov-2 and the early-stage spread history. the discrepant phylogenies for the spike protein and its receptor binding domain proved a previously reported structural rearrangement prior to the emergence of sars-cov-2. despite that we found the spike glycoprotein of sars-cov-2 is particularly more conserved, we identified a mutation that leads to weaker receptor binding capability, which concerns a sars-cov-2 sample collected on 27th january 2020 from india. this represents the first report of a significant sars-cov-2 mutant, and raises the alarm that the ongoing vaccine development may become futile in future epidemic if more mutations were identified. highlights based on the currently available genome sequence data, we proved that sars-cov-2 genome has a much lower mutation rate and genetic diversity than sars during the 2002-2003 outbreak. the spike (s) protein encoding gene of sars-cov-2 is found relatively more conserved than other protein-encoding genes, which is a good indication for the ongoing antiviral drug and vaccine development. minimum evolution phylogeny analysis revealed the putative original status of sars-cov-2 and the early-stage spread history. we confirmed a previously reported rearrangement in the s protein arrangement of sars-cov-2, and propose that this rearrangement should have occurred between human sars-cov and a bat sars-cov, at a time point much earlier before sars-cov-2 transmission to human. we provided first evidence that a mutated sars-cov-2 with reduced human ace2 receptor binding affinity have emerged in india based on a sample collected on 27th january 2020. monitoring the mutation dynamics of sars-cov-2 is critical for the development of effective approaches to contain the 20 pathogen. by analyzing 106 sars-cov-2 and 39 sars genome sequences, we provided direct genetic evidence that 21 sars-cov-2 has a much lower mutation rate than sars. minimum evolution phylogeny analysis revealed the putative 22 original status of sars-cov-2 and the early-stage spread history. the discrepant phylogenies for the spike protein and its 23 receptor binding domain proved a previously reported structural rearrangement prior to the emergence of sarsdespite that we found the spike glycoprotein of sars-cov-2 is particularly more conserved, we identified a mutation that 25 leads to weaker receptor binding capability, which concerns a sars-cov-2 sample collected on 27 th january 2020 from 26 india. this represents the first report of a significant sars-cov-2 mutant, and raises the alarm that the ongoing vaccine 27 development may become futile in future epidemic if more mutations were identified. 28 29 evolutionary rate assessment 95 the ratio of nonsynonymous mutations (dn) to synonymous mutations (ds) was calculated using codeml in the paml 96 (version 4.7) package (18). cds sequences for each protein encoding gene were filtered to remove redundant identical 97 sequences. then codon-based cds sequence alignment was performed using muscle program, and an individual nj 98 tree was generated using mega7.0 (17) with p-distance model. the obtained sequence alignment and phylogenetic tree 99 files were used as paml inputs for dn and ds calculations. 100 protein structural analyses 101 3d structure of the sars-cov-2 spike glycoprotein in complex with (pdb: 6vw1, 6vw1) has been determined recently 102 (5, 9) genetic diversity analyses identified a single amino acid mutation in rbd of the spike protein in sars-cov-2 112 as of 24 th march 2020, there are a total of 174 nucleotide sequences for sars-cov-2 in the ncbi database. by restricting 113 to the complete or near-complete genomes, 106 sequences from 12 countries were obtained and used for further analyses. 114 this encompasses 54 records from usa, 35 from china, and the rest from other countries: australia (1), brazil (2), finland 115 (1), india (2), italy (1), nepal (1), spain (3), south korea (1), and sweden (1). 116 based on the gene model of the reference sars-cov-2 genome (genebank: nc_045512.2), a total of 12 protein-encoding 117 open reading frames (orfs), plus 5utr and 3utr were annotated ( figure 1a) . overall, the gene sequences from 118 different samples are highly homologous, sharing > 99.1% identity, with the exception of 5utr (96.7%) and 3utr (98%) ( table 1) , which are relatively more divergent. sequence alignment showed that there is no mutation in orf6, orf7a, 120 and orf7b. the genetic diversity profile across the 106 genomes was displayed in figure 1a . a few nucleotide sites 121 within orf1a, orf1b, orf3a, and orf8 exhibiting high genetic diversity were identified ( figure 1a) . 122 the s protein is critical for virus infection and vaccine development. as shown in figure to assess how the mutation rate and genetic diversity of sars-cov-2, the ratio of nonsynonymous mutations (dn) and 138 synonymous mutations (ds), was calculated for each protein-encoding orf based on the 106 sars-cov-2 and 39 sars 139 genomes. for sars-cov-2, the highest dn was observed for orf8 (0.0111), followed by orf1a (0.0081), orf9 (0.0079), 140 and orf4 (0.0063) ( table 1) , indicating these genes may be more likely to accumulate nonsynonymous mutations. in 141 contrast, orf1b (0.0029), s gene (0.0040) encoding the spike protein, and orf5 (0.0023) are relatively more conserved 142 in terms of nonsynonymous mutation. noteworthy, orf6, orf7ab and orf10 are strictly conserved with no 143 nonsynonymous mutation. compared to sars-cov-2, sars displayed higher mutation rates for all of the orfs in the 144 virus genome (table 1) , suggesting an overall higher levels of genetic diversity and mutation rate. in particular, the dn and 145 ds values for the s gene in sars-cov is around 12 and 7 times higher than that for sars-cov-2. in contrast, the mutation 146 rate differences for orf1a and orf1b between sars-cov-2 and sars are relatively milder, varying from 1.5 times to 147 4.8 times only. in contrast to sars-cov-2, which has strictly conserved orf6, orf7a, and orf7b, sars displayed 148 mutation rates at different levels. notably, the ds for orf10 are comparable between the two genomes at 0.0326 and 0.0341, 149 respectively. 150 151 to trace the potential spread history of sars-cov-2 across the world, an unrooted minimum evolution (me) tree of the 153 106 genomes was developed based on whole-genome sequence alignment. the clustering pattern of the me phylogeny 154 the spike glycoprotein is critical for the virus infection. recent study suggested that the s protein in sars-cov-2 may 185 has underwent a structural rearrangement(13). to investigate this hypothesis, two separate phylogenies were developed 186 based on the full-s and rbd sequences, respectively. overall, the two phylogenies displayed similar clustering patterns, 187 separating into three major clades (figure 3) . sars-cov-2 was identified in the same major clade, and was clustered most 188 closely with two bat sars covs (highlighted in purple and green colors, figure 3 ) and the human sars-cov (orange 189 color, figure 3) . in both phylogenies, sars-cov-2 is most closely related to bat_cov_ratg13, suggesting sars2 may have originated from bat. however, the evolutionary positions of human sars-cov and bat-sl-covz45 were 191 swapped between the full-s and rbd-only phylogenies. in the full-s phylogeny, bat-sl-covz45 is relatively more similar 192 to human sars-cov-2, whilst human sars-cov is closer to sars-cov-2 than bat-sl-covz45. taken together, these results suggested that the rbd of sars-cov-2 is more likely originated from human sars-cov, whilst the rest part of 194 the s protein in sars-cov-2 may have originated from bat-sl-covz45, supporting the potential structural rearrangement 195 of s protein in sars-cov-2. bat_cov_ratg13 is similar to sars-cov-2, indicating the proposed structural 196 rearrangement may have occurred in bat first before its transmission to human. the rbd of virus s protein binds to a receptor in host cells, and is responsible for the first step of cov infection (3). thus, 204 amino acid mutation to rbd may have significant impact on receptor binding and vaccine development. the 3d structure 205 of the spike protein rbd of sars-cov-2 (pdb: 6vw1) has recently been determined in complex with human ace2 206 receptor (6). one of the12 amino acid mutations in the rbd of s protein (r408i) was identified among the 106 sars-207 cov-2 genomes. sequence alignment showed that 408r is strictly conserved in sars-cov-2, sars-cov and bat sars-208 like cov (figure 4a) . based on the determined cov2_rbd-ace2 complex structure, 408r is located at the interface 209 between rbd and ace2, but is positioned relatively far away from ace2, thus does not have direct interaction with ace2 210 ( figure 4b) . however, the determined rbd0-ace2 structure showed that 408r forms a hydrogen bond (3.3 å in length) 211 with the glycan attached to 90n from ace2 ( figure 4c ) (6). the hydrogen bond may have contributed to the exceptionally 212 higher ace2 binding affinity. in contrast, despite this arginine residue is also conserved in human sars-cov 213 (corresponding to 395r in pdb: 2ajf), it is positioned relatively distant (6.1 å) from the glycan bound to 90n from ace2 214 ( figure s1) . interestingly, the 408r-glycan hydrogen bond seem to be disrupted by the r408i mutation in one sars-cov-215 potential interventions for novel coronavirus in china: a systematic review timely development of vaccines against sars-cov-2. emerg microbes infec 9 structure of sars coronavirus spike receptor-binding 319 domain complexed with receptor evidence for a common evolutionary origin of coronavirus spike protein receptor-321 binding subunits cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis of receptor recognition by sars-cov-2 angiotensin receptor blockers as tentative sars-cov-2 therapeutics. drug 326 development research the spike protein of sars-cov -a target for vaccine and therapeutic 328 development structural basis for the recognition of sars-cov-2 by full-length human ace2 proof of principle for epitope-focused vaccine design influenza virosomes in vaccine 334 coronaviruses an rna 336 proofreading machine regulates replication fidelity and diversity genomic characterisation and epidemiology of 2019 novel coronavirus: 338 implications for virus origins and receptor binding evolution of the novel coronavirus from the ongoing wuhan outbreak and 340 modeling of its spike protein for risk of human transmission annotationsketch: a genome 343 annotation drawing library muscle: multiple sequence alignment with high accuracy and high throughput mega7: molecular evolutionary genetics analysis version 7.0 347 for bigger datasets phylogenetic analysis by maximum likelihood genomic variance of the 2019-ncov coronavirus on the origin and continuing evolution of sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin identifying sars-cov-2 related coronaviruses in malayan pangolins identifying sars-cov-2 related coronaviruses in malayan pangolins preliminary identification of potential vaccine targets 362 for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies contrast to the arginine residue, which is electrically charged and highly hydrophilic, the mutated isoleucine residue has a 217 highly hydrophobic side chain with no hydrogen-bond potential ( figure 4e ). to sum up, the r408i mutation identified 218 from the sars-cov-2 strain in india represents a sars-cov-2 mutant with potentially reduced ace2 binding affinity. 219 220 221 hydrophobic profile changes due to r408i mutation, with with red and white colours representing the highest hydrophobicity and the lowest 225 hydrophobicity respectively. all amino acid number according to the s protein of sars-cov-2 (nc_045512.2) and human ace2, respectively. based on the currently available genome sequence data, our results showed that the mutation rate of sars-cov-2 is much 229 lower than that for sars, which caused the 2002-2003 outbreak. our study is the first to provide a direct quantitative 230 comparison between sars-cov-2 and sars. a relatively stable genome of sars-cov-2 is a good indication for the 231 epidemic control, as less mutation raises the hope of the rapid development of validate vaccine and antiviral drugs. our 232 results are consistent with several recent genetic variance analyses on sars20) , which suggested the sars-233cov-2 genomes are highly homogeneous. molecular geneticists closely monitoring the virus development also suggested 234 that the mutation rate of sars-cov-2 maintains at a low level. whilst it is generally safe to say that sars-cov-2 tends 235 to mutate at a low rate, all current analyses are merely based on data collected at the early stage of this pandemic. as the 236 virus continues to spread rapidly around the world, and more genomic data is accumulated, the evolution and mutation 237 dynamics of sars-cov-2 still need to be monitored closely. 238 one critical aim of our study is to identify the original status of sars-cov-2 before its wide transmission across different 240 countries. due to the short time space of sample collection and a relatively low mutation rate for sars-cov-2, we believe 241 that a minimum evolution phylogeny may outperform other phylogenetic methods to achieve this aim. as expected, the 242 earliest few reported sars-cov-2 accessions collected from wuhan china were identified at the center of the phylogenetic 243 tree with the shortest branch. interestingly, a number of virus genomes from usa were found almost identical to these 244 putative original versions of virus from wuhan. however, according to public media, the outbreak of sars-cov-2 in usa 245 occurred relatively later than other countries. one possible explanation for this observation is that, the spread of sars-246cov-2 in usa might start much earlier than previously thought or reported. due to a dominant proportion of the samples 247 in this study were collected from china and usa, we observed a significantly higher level of genetic diversity from these 248 two countries. most sars-cov-2 accessions from the other countries can find their closely related sisters from either 249china or usa. this data bias, on the other hand, may give us an advantage to trace the spread history of sars-cov-2 in 250 different countries. this suggestion is reliable because all of the samples studies in this study were collected at the early 251 stage of the pandemic, which may avoid the potential data noise caused by recent published genomes of complex spread 252 background. one notable finding in our phylogenetic tree is that, the singleton sars-cov-2 accessions collected from 253 australia, brazil, south korea, italy and sweden were clustered together with two usa samples but without a chinese 254 version, suggesting that these infection cases may be somehow related. in addition, one of the three samples collected from 255 the cruise ship stranded in japan was found closely related to a sample collected from guangzhou, china, whilst the other 256 two were grouped with several cases from usa. noteworthy, out phylogeny seems to support the presence of two major 257 types of sars-cov-2 in the target samples, suggesting the potential existence of two spread sources. interestingly, this 258 speculation is corroborated by an independent clustering analyses using different phylogeny method (20). 259 260 until now, the origin of sars-cov-2, and how it has been transmitted to human remains largely a mystery. early genomic 261 data proved that human sars-cov-2 is an enveloped, positive-sense, and single-stranded rna virus in the subgenus 262sarbecovirus of the genus betacoronavirus (13, 14) . evolutionarily, sars-cov-2 is most closely related to bat sars-like 263cov (88% genome sequence identity) and human sars cov (79%), the latter of which has caused world pandemic in 264 2003 (13). based on the strong genome sequence identity between sars-cov-2 and bat sars-like covs, it was initially 265 speculated that sars-cov-2 may have originated from bat (14, 21). however, a more recent study proposed that pangolin 266 may be the most likely reservoir hosts due to the identification of closely related sars-covs from this species as well 267 (22). both of these two animals can harbor coronaviruses related to sars-cov-2. however, direct evidence of the 268 transmission of sars-cov-2 from either bat or pangolin to human is still missing. 269 270 prior to this study, several publications have suggested that sars-cov-2 may have originated from the genome 271 recombination of sars-like covs from different animal hosts, as evidenced by the discrepant clustering patterns for the 272 phylogenies using different genetic regions. lu (13) first observed that the rbd of s protein in sars-cov-2 is more 273 closely related to human sars-cov, whilst the other part of its genome is more similar to bat sars-cov. later peng (23) 274 identified a bat cov_ratg13 and several pangolin sars-covs that are consistently closer to sars-cov-2 than human 275 sars-cov in either full-s protein or rbd. by combining the data from these two studies, our study confirmed the 276 observations reported in both studies, and further determined that the s protein recombination actually happened between 277 human sars-cov and a bat sars-cov, much earlier before its transmission to human, with the newly identified bat 278 sars-cov-ratg13 as an intermediate. 279 another notable finding in this study corresponds to the identification of an amino acid mutation in the rbd of s protein 281 in sars-cov-2. mostly importantly, we showed that this amino acid mutation is very likely to cause a reduced binding 282 affinity to human ace2 receptor. the rbd of s protein binds to a receptor in host cells, and is responsible for the first 283 step of cov infection. the receptor binding affinity of rbd directly affects virus transmission rate. thus, it has been the 284 major target for antiviral vaccine and therapeutic development such as sars (8). despite the s protein gene seems to be 285 more conserved than the other protein-encoding genes in the sars-cov-2 genome, our study provide direct evidences 286 that a mutated version of sars-cov-2 s protein with varied transmission rate may have already emerged. based on the 287 close relationship of sars-cov-2 to sars, current vaccine and drug development for sars-cov-2 has also focused on 288 the s protein and its human binding receptor ace2 (7, 24). thus, the observation in this study raised the alarm that sars-289cov-2 mutation with varied epitope profile could arise at any time, which means current vaccine development against 290 sars-cov-2 is at great risk of becoming futile. because the receptor recognition mechanism seems to be highly conserved 291 between sars-cov-2 and sars-cov, which have been proved to share the common human cell receptor ace2. one 292 suggestion for the next step of therapeutic development is probably to focus on the identification of potential human ace2 293 receptor blocker, as suggested in a recent commentary (7). this approach will avoid the above key: cord-336775-d4hi9myk authors: kirtipal, nikhil; bharadwaj, shiv; kang, sang gu title: from sars to sars-cov-2, insights on structure, pathogenicity and immunity aspects of pandemic human coronaviruses date: 2020-08-13 journal: infection, genetics and evolution doi: 10.1016/j.meegid.2020.104502 sha: doc_id: 336775 cord_uid: d4hi9myk abstract human coronaviruses (hcov), periodically emerging across the world, are potential threat to humans such as severe acute respiratory syndrome coronavirus-2 (sars-cov-2) – diseases termed as covid-19. current sars-cov-2 outbreak have fueled ongoing efforts to exploit various viral target proteins for therapy, but strategies aimed at blocking the viral proteins as in drug and vaccine development have largely failed. in fact, evidence has now shown that coronaviruses undergoes repaid recombination to generate new strains of altered virulence; additionally, escaped the host antiviral defense system and target humoral immune system which further results in severe deterioration of the body such as by cytokine storm. this demands the understanding of phenotypic and genotypic classification, and pathogenesis of sars-cov-2 for the production of potential therapy. in lack of clear clinical evidences for the pathogenesis of covid-19, comparative analysis of previous pandemic hcovs associated immunological responses can provide insights into covid-19 pathogenesis. in this review, we summarize the possible origin and transmission mode of covs and the current understanding on the viral genome integrity of known pandemic virus against sars-cov-2. we also consider the host immune response and viral evasion based on available clinical evidences which would be helpful to remodel covid-19 pathogenesis; and hence, development of therapeutic against broad spectrum of coronaviruses. previously, the coronaviridae study group (csg) identified sars-cov and mers-cov strains into a new species under the new informal subgroup of betacov genus (van boheemen et al., 2012) . however, recent introduction of subgenus rank in virus taxonomy have established the two informal subgroups of sars-cov and mers-cov as subgenera sarbecovirus and merbecovirus (de groot et al., 2013; gorbalenya et al., 2004) , respectively. remarkably, newly emerged sars-cov-2 differs from previously reported zoonotic pandemic viruses, viz. sars-cov and mers-cov; and hence, taxonomic position of sars-cov-2 under subgenera sarbecovirus may be tentative to change based on further evidences (gorbalenya et al., 2020) ( fig. 1) . phylogenetic tree analysis of covs constructed based on s gene using molecular evolutionary genetics analysis 6 software under neighbor-joining method and 1000 bootstrap values (biswas et al., 2020) . before the emergence of sars-cov, construction of phylogenetic tree for covs was based on pol (polymerase) or nucleocapsid (n) gene as standard practice. initially, this method proposed sars-cov as a member of gammacov (rota et al., 2003) . however, further evidences on amino-terminal domain of sars-cov spike protein discovered that 19 out of 20 cysteine residues were structurally conserved with in betacov group while only 5 conserved residues were matched within alphacov and gammacov group (rota et al., 2003) . subsequently, whole genome-based phylogenetic analysis indicated sars-cov as member of betacov lineage b. likewise, mers-cov was also identified as novel betacov lineage c with high pathogenicity . remarkably, rna-dependent rna polymerase (rdrp) and spike (s) animal covs have been known since the late 1930s like transmissible gastroenteritis virus of swine (tgev), bovine coronavirus (bcov), feline infectious peritonitis virus (fipv), and infectious bronchitis virus (ibv) transmissible (saif, 2004) . recent studies associated hcovs evolution with expedited urbanization and poultry farming; these practices permitted frequent interchange of species and simplified crossing of species barrier and genomic recombination in these viruses (jones et al., 2013) . bats harbored a great diversity of covs; viral sampling conducted by the ecohealth alliance in china alone identified about 400 new strains of covs . besides, study of covs diversity harbored by bats in eastern thailand revealed forty-seven covs (wacharapluesadee et al., 2015) . moreover, bats were documented with unique immune systems that allowed them to cope with a variety of viruses by comparison to other terrestrial mammal (xie et al., 2018) . also, a high diversity of zoonotic alphacovs and betacovs were also detected in the circulating bats of western europe (ar gouilh et al., 2018) . generally, bats harbored rna viruses but can be infected by dna viruses (xie et al., 2018) . although, clear transmission of covs from bats to humans is not yet fully discovered but transmission by intermediary host to humans via direct contact has been widely suggested as one of the probable modes of transmission. the first sars-cov case was documented in november 2002 in foshan, china (ge et al., 2015) . it became an epidemic, affected 28 countries around the world with 8,096 cases and 774 deaths (ge et al., 2015) . during the sars epidemic, the first indication for the source of sars-cov viral strain was detected in masked palm civets (paguma larvata) and raccoon dog (nyctereutes procyonoides). later discovery of antibodies for virus in chinese ferret badgers (melogale moschata) in a live-animal market in shenzhen, china were suspected as source of human infection (drexler et al., 2014; guan et al., 2003; song et al., 2005) . however, later findings predicted these animals merely as incidental hosts in absence of concrete results to support the motion of sars-cov-like viruses in palm civets among the wild or in the breeding factors endorsing bats as animal reservoirs of mammalian viruses were also defined in terms of their densely packed colonies, longevity, close social interaction, and ability to fly (calisher et al., 2006; luis et al., 2013) . likewise, first human case of mers was reported in june 2012 in jeddah, saudi arabia (ge et al., 2015) . as of november 2019, 2,494 cases of mers have been reported in 27 countries, resulting in 858 fatalities (who, 2020) . initially, search for mers-cov reservoir was focused on bats; however, serological survey in dromedary camels (camelus dromedarius) from oman and the canary islands exhibited a significant prevalence of mers-cov-neutralizing antibodies (reusken et al., 2013) . additionally, dromedary camels were detected with mers-cov rna at a farm in qatar related to two human cases of mers; virus particles were detected from dromedary camels in qatar and saudi arabia (hemida et al., 2014; raj et al., 2014) . serological evidence supported that dromedary camels has been harboring mers-cov-like virus in eastern africa and northern africa, and middle east, dating back as far as 1983 (muller et al., 2014) . additionally, in saudi arabia, dromedary camels were detected with various viral genetic lineages (sabir et al., 2016) , including which crossed the species barrier and caused the outbreaks in humans (omrani et al., 2015) . collectively, these evidences strongly suggested the contribution of dromedary camels as considerable reservoir to mers-cov; the ancestral virus were predicted to cross the species barrier into dromedary camels from bats more than ≥30 years ago as supported by serological evidences (fig. 2) . recent sars-cov-2 outbreak highlights the hidden wild zoonotic reservoir of deadly viruses and possible threat of spillover zoonoses (malik et al., 2020) . in 2019, a food market that sold live in wuhan, china was linked to the outbreak of sars-cov-2. through genetic analyses, bats were again suggested as native host of sars-cov-2 as it shared 96% genome to two sars-like covs, viz. bat-sl-covzx45 and bat-sl-covzx2 from bats zhou et al., 2020b) . however, intermediate host and route which assisted viral transmission to humans is yet fully understood. initially, ji, et al., proposed snakes as an intermediate host of sars-cov-2 transmission from bats to humans which involved homologous recombination within the viral spike (s) protein (ji et al., 2020) . later, pangolins (manis spp.) were suggested as potential intermediate host for sars-cov-2 as it shared 99% genetic homology with covs in pangolins ( fig. 2) (yi et al., 2020) . however, 1% variance all over the two-genome sequence was considered as significant difference; and thus, conclusive and concrete evidences are still under j o u r n a l p r e -p r o o f journal pre-proof investigation (yi et al., 2020) . moreover, scientists also showed concerned about its validation genetics technique (codon usage bias) (callaway and cyranoski, 2020) . hence, diversity of covs in bat population needs further investigation in detail along with critical surveillance and monitoring of bats to prevent future outbreaks in animals and public. after infection in human, hcovs are transmitted among the human population by close personto-person contact. for example, close contact with infected person under in 3 feet distance has been concluded as major factor for cov transmission . the pandemic viruses, sars-cov, mers-cov and sars-cov-2, transmitted between humans mostly by nosocomial transmission; supported by evidences that health care workers and patients had higher frequency of infection against their relatives (de wit et al., 2016) . the collected data predicted that only 22-39% of sars and 13-21% of mers transmission occurred between family members. also, sars-cov transmission from infected patients to health care workers was very frequent (33-42%) and mers-cov transmission between patients was recorded as the most common course of infection (62-79% of cases) (chowell et al., 2015) . such a predominance of nosocomial transmission was predicted due to significant virus shedding that occurred following the onset of infection when most patients were already under medical care (cowling et al., 2015; peiris et al., 2003) . a study on hospital surfaces following treatment of mers-cov infected patients detected with viral rna for several days even after patients no longer detected for viral infection (bin et al., 2016) . moreover, many patients with sars or mers were infected through super spreaders (korea centers for disease control and prevention, 2015) (chowell et al., 2015; kucharski and althaus, 2015) . in addition, sars-cov was also suspected for transmission through air (airborne spread) or some other unkown transmission routes . (e), nucleocapsid (n) proteins, and hemagglutinin (ha), where s, m, and e proteins are embedded in viral envelop while n protein protects viral rna genome located as core of virus ( fig. 3 ) (zhou et al., 2020b) . s protein is characterized as heavily glycosylated protein and contained the receptor binding domain (rbd), which is the most variable structure in covs (zhou et al., 2020b) ; mediates viral entry into host cells (bosch et al., 2003) . the two different types of spike proteins have been discovered in covs: spike glycoprotein trimmer (s) that can be found in all covs, and hemagglutinin-esterase (he) that found in specific covs (belouzard et al., 2012; cui et al., 2019) . some covs, including sars-cov and sars-cov-2, contain polybasic cleavage site (rrar/s) at the junction of two subunits (s1 and s2) of s protein. this allowed digestion by host furin-like protease during viral replication (bosch et al., 2003) and contributed in determination of viral infectivity towards host range (nao et al., 2017) . although function of polybasic cleavage site in sars-cov-2 is not yet discovered; experimental studies with sars-cov and mers-cov have determined that insertion of a furin cleavage site (fcs) at s1-s2 junction enhanced the cell-cell fusion without affecting viral entry (follis et al., 2006) and efficient digestion enabled mers-cov like covs from bats to infect human cells (menachery et al., 2020) . furthermore, m protein exists in higher quantities against e protein in virion structure (nal et al., 2005) and critically required in orchestrating viral shape, assembly and in generation of mature viral envelopes (siu et al., 2008) . moreover, it also functions in intracellular virion formation without the requirement of s protein. in some occasions, such as in presence of tunicamycin, covs were reported to generate noninfectious virions without spike but contained m protein in envelop (de haan et al., 1998) . likewise, structural protein, e protein assisted in mature virions secretion from host cells, in addition to other functions like ion channel activity, inhibit the host cell stress response and implicate pathogenesis in host cell through virus (ruch and machamer, 2012) . n protein essentially assisted in genomic rna packing during viral particle assembly (chang et al., 2006; hurst et al., 2009) . whereas, he, which is a hemagglutinin similar to influenza virus hemagglutinin, conducts the acetyl-esterase activity (klausegger et al., 1999) . recently, role of he was also logged to catalyst viral invasion across host cell membrane and in pathogenesis (ashour et al., 2020) . hcovs, including sars-cov, mers-cov, and sars-cov-2, contained largest nonsegmented positive-sense single-stranded rna genome of size between 26.2 and 31.7 kb with varied g+c contents of 32% to 43% (mousavizadeh et al., 2020; yang et al., 2006) . the genome organization for a cov is 5′-leader-utr-replicase/transcriptase-spike (s)-envelope (e)membrane (m)-nucleocapsid (n)-3′utr-poly (a) tail (fig. 4) . the 5′utr (untranslated region) and 3′utr are involved in interand intramolecular interactions, essentially required in rna-rna interactions and for binding of viral and cellular proteins (yang and leibowitz, 2015) . the orf1a and orf1b occupied first two-thirds of rna genome and produced replicase/transcriptase polyprotein which undergoes autoproteolytic activity with the aid of papain-like proteinase (plpro) and 3c-like main protease (3clpro or m pro ) to form 16 nonstructural proteins (nsps) . plpro conducts cleavage at n-terminus of replicase polyprotein to produce nsp1, nsp2, and nsp3, required for viral replication (harcourt et al., 2004) . however, m pro is first protein which autoproteolytically separates polyprotein replicase 1ab (~790 kda, recognition sequence located at leu-gln↓ser,ala,gly; where ↓marks stand for cleavage site) to produced mature viral enzymes and further cleaved downstream nsps at 11 sites to release nsp4-nsp16 ( fig. 4) zhang et al., 2020) . these events indicated the direct role of m pro to mediate maturation of nsps, which are basically required for virus life cycle in host cells. hence, plpro and mpro have been suggested as potential targets in drug development against hcovs. the later reading frames on the genome encoded the four major structural proteins; s, e, m, and n, which are common proteins among all covs . all the viral structural and accessory proteins are decoded by subgenomic (sg) rnas of covs where accessory proteins are interspersed between orfs stands but their number and functional activities are restricted to cov strains . specifically, sars-cov genome translated 8 accessory gene (3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b), mers-cov encoded 5 accessory genes (3, 4a, 4b, 5, and 8b), and though exact number of functional proteins remains to be established in sars-cov-2 but based on previous study on sars-cov, sars-cov-2 has been predicted with translation of 4 structural proteins (yoshimoto, 2020 ) and at least 6 or 9 accessory proteins (3, 6, 7a, 7b, 8, 9b, 10b, 13, 14) (liu et al., 2014; wu et al., 2020c; zhou et al., 2020b) . also, orf1ab position in sars-cov-2 genome (251-21541 nt) was noticed with change at starting codon position by comparison to sars-cov j o u r n a l p r e -p r o o f (265-21486 nt) and mers-cov (279-21514 nt) . interestingly, conserved amino acid substitution at positions 439, 501, 493, 485 and 486 were also logged in rbd domain of sars-cov-2 s protein, which was considered as important in sars-cov . it is important to add that genomic material of hcovs is highly susceptible to frequent recombination process which have been directly associated with establishment of altered virulence in new hcov strains (hilgenfeld, 2014) . all the sars-like covs exhibits typical streadgy for replication and translation following infection in host cell. this section will briefly summarize sars-cov, mers-cov and sars-cov-2, their comparative lifecycle in host cell, which began on attachment of virus with host cell receptor and finished with release of newly generated progeny from infected cells. the binding of hcov to host-cell receptor is the initial step in viral infection which determined the severity of infection and pathogenesis. a better understanding of coupling of viral structural proteins with targeted receptor on host cells and other involved proteins, such as host protease, can help to predict the specific zoonotic covs infection in humans. hcovs infection begins with attachment of viral particle on host cell surface by densely glycosylated s protein, which is a trimeric class i fusion protein and consist of two major subunits; a receptor binding domain (s1; also known as rbd) and a second domain (s2) mediated viral fusion with host cell membrane. the fusion process of viral membrane with host cell membrane is triggered when s1 domain binds with host-cell receptor; for example, angiotensin-converting enzyme 2 (ace2) for sars-cov and sars-cov-2 (wu et al., 2020b, c) , also as an alternative receptor cd209l (a c-type lectin, also called l-sign) with low affinity for sars-cov (jeffers et al., 2004) , dipeptidyl peptidase-4 (dpp4, also known as cd26) for mers-cov . thus, cleavage of spike glycoprotein is performed by host cell proteases enables interaction of s2 domain for virus entry into the cell (de wit et al., 2016) . in the respiratory tract, ace2 receptor is widely distributed on the epithelial cells of trachea, bronchi, bronchial serous glands, and alveoli , as well as alveolar monocytes and macrophages ; sars-cov attacked these cells and mature virions are later released to infect other new target cells (zhang et al., 2004) . ace2 is also diffusely expressed on endothelial cells of arteries and veins, cerebral neurons and immune cells, tubular epithelial cells of kidneys, mucosal cells of intestines, and epithelial cells of renal tubules, presented as a variety of susceptible targets to sars-cov infection (gu and korteweg, 2007; guo et al., 2008) . whereas, mers-cov is known to target respiratory tract, liver small intestines, kidney, prostate and activated leukocytes (widagdo et al., 2016) . remarkably, unlike sars-cov in vitro studies, mers-cov was found to infect human dendritic cells and macrophages ; therefore, virus disrupted the immune system. besides, t (lymphocyte) cells are another potential target for mers-cov due to presence of high amounts of cd26 . hence, this cov was predicted to dysregulate antiviral t-cell responses because of stimulation of t-cell apoptosis yeung et al., 2016) . recent studies suggested that sars-cov-2 employs ace2 as main receptor as in sars-cov infection with higher affinity, suggesting the likelihood of same group of host-cells being targeted and infected (zhou et al., 2020b; zou et al., 2020) . after attachment to host-cell surface, virus entry into cell has been deciphered by two different paths based on availability of host cell protease to activate receptor-attached spike protein (fig. 5 ) (simmons et al., 2013) . in first path, covs invaded host cell as an endosome which is mediated by clathrin-dependent and-independent endocytosis (fig. 5) (kuba et al., 2010; wang et al., 2008a) . this phenomenon induced conformational changes in viral particle which subsequently fused viral envelope with endosomal wall (simmons et al., 2013) . alternatively, in second pathway, direct invasion of virus particles into host cell are mediated via proteolytic cleavage of receptor-attached spike protein by host's transmembrane serine protease 2 (tmprss2) or transmembrane serine protease 11d (tmprss11d) on the cell surface (heurich et al., 2014; zumla et al., 2016) . herein, s2 domain of spike protein accomplished direct membrane fusion between virus and plasma membrane as initially observed in sars-cov after virus and host cell membrane fusion event, virus released the nucleocapsid packed genomic rna into cellular cytoplasm under the influence of induced structural conformation changes . then, viral genome acted as a mrna and cell's ribosome translates two-thirds of this rna, crossponds to orf1a and orf1b into two large overlapping polyproteins (pp): pp1a and pp1ab. the larger polyprotein pp1ab translated from a -1 ribosomal frame shift triggered by slippery sequence (uuuaaac) and downstream rna pseudo knot at end of orf1a (masters, 2006) . this ribosomal frameshift enabled continuous translation of orf1a followed by orf1b . the encoded polyproteins possess the proteases; pl pro and m pro which assisted in generation of 16 nsps (nsp1-nsp16) from polyprotein pp1ab, including several replication proteins such as rdrp, rna helicase, and exoribonuclease (exon) . moreover, multifunction and enzyme activities for specific nsps in the previously reported hcov, i.e. sars-cov and mers-cov have been discovered over the past years and summarized elsewhere (masters, 2006; ziebuhr, 2005) . most of the newly translated nsps together with structural protein, i.e. n protein, formed the multi-protein replicase-transcriptase complex (rtc) which further conducted the viral genome replication and transcription . at the site of replicative organelles (knoops et al., 2008) , rtc complex contained rdrp as main replicase-transcriptase protein for the synthesis of negative-sense subgenomic (sg) rna strands from viral rna and transcription of negative-sense subgenomic rna molecules from corresponding positive-sense mrnas . the newly produced minus strands of genomic and sgrnas are subsequently employed as templates for production of positive sense strands (mrnas), j o u r n a l p r e -p r o o f specifically in generation of genomic rna (genome replication) and sg mrnas (transcription). these newly synthesized rna strands acted as genome for generated new viral progeny. also, several smaller mrnas are produced from viral last third of genome which tracks reading frames orf1a and orf1b, interpreted into viral four structural proteins (s, e, m, and n) and along with accessory proteins (orf3a to orf9b) become part of viral progeny . besides, other nsps in rtc complex also assist in viral replication and transcription process . for instance, nsp15 protein, a 3'-5' exoribonuclease, provides an extra fidelity to rtc complex via proofreading function in rna replication, which lacked in rdrp. likewise, nsp7 and nsp8 proteins generated a hexadecameric sliding clamp for rtc complex to catalyst the processivity of rdrp . this increased fidelity and processivity is essentially required in covs during rna synthesis because of their relatively bulky rna genome by comparison to other rna viruses (sexton et al., 2016) . the viral rna translation process occurred inside host cells' endoplasmic reticulum lead to formation of structural proteins, viz. s, e and m, which moved along the secretory pathway into golgi intermediate compartment. herein, n protein packed the newly produced rna genome and thereby shaped into a helical nucleocapsid. following, virion assembly is triggered by m protein via multiple protein-protein interactions which assisted in incorporation of nucleocapsid, envelope, and spike proteins into virus particles . later, viral progeny germinated into endoplasmic reticulum-golgi intermediate compartment (ergic) and released as secretory vesicles which fused with plasma membrane and secrete from host cell by exocytosis (fig. 6) lim et al., 2016) . generally, human population lacks immunity against covs and hence, susceptible to novel cov infections such as sars-cov-2. since, immune response against sars-cov-2 is not fully j o u r n a l p r e -p r o o f deciphered, recent studies suggested to refer the previous reports on other hcovs, especially sars-cov and mers-cov (fig.7) (yi et al., 2020) . thus, tlrs mainly mediated the detection of viral rna in mdcs (such as tlrs3 and some tlr7) and pdcs by tlr7 (and tlr8 in human pdcs) (schreibelt et al., 2010) . based on a specific prr, various-partially cross-talking-signaling pathways resulted in promoters transactivation of antiviral genes (o'neill et al., 2013) . for instance, a prototypical pamp relevant for covs is dsrna, a by-product from genome replication and transcription (weber et al., 2006; zielecki et al., 2013) . dsrna can also be detected by tlr3 in the endosome while sensed by rna helicases rig-i, mda5 and kinase pkr in the cytoplasm (rasmussen et al., 2009; yim and williams, 2014; yoneyama et al., 2016) . also, specific ssrnas are presented as pamps, either if they displayed particular features or in the wrong location; for example, tlr7 detects gu-rich ssrna in the endosome (heil et al., 2004) . thus, numerous types of prrs are regularly surveying the intracellular and extracellular space to sense virus infections in a sensitive and timely manner. the detection of viral infection triggers complex signaling cascades such as myd88 that induced manufacture of type i ifns and stimulation of transcription factor nuclear factor-κb (nf-κb). in turn, active nf-κb induced transcription of pro-inflammatory cytokines (fig.7) . moreover, type i ifns signal by ifnα/β receptor (ifnar) and following downstream signal activators and transducers of transcription (stat) proteins triggered the manufacture of antiviral proteins that are programmed by interferon-stimulated genes (isgs) like ifn-induced protein j o u r n a l p r e -p r o o f with tetratricopeptide repeats 1 (ifit1). altogether, this launches an antiviral immune response that restricts viral replication in infected and in neighbouring cells (de wit et al., 2016) . however, studies have showed that family of covs can significantly suppressed human immune responses against viral infection by evading immune detection machinery (kikkert, 2020) . for instance, papain-like protease (plpro) domain of nsp3 of sars-cov interrelated with ifn regulatory factor 3 (irf3) and inhibited its activation (devaraj et al., 2007; frieman et al., 2009) . besides, plpro was reported to cause deubiquitination (or inhibit ubiquitination) of tankbinding kinase 1 (tbk1), rig-i, and irf3 (clementz et al., 2010; devaraj et al., 2007; frieman et al., 2009; sun et al., 2012) . the several functions and mechanisms adopted to counteract ifn induction, and strategies to develop resistance against ifn by previously pandemic hcovs are summarized earlier (kindler et al., 2016) . remarkably, sars-cov and mers-cov were reported to induced production of double-membrane vesicles that lack prrs, replicated in these vesicles, and thereby escaped host detection system for viral dsrna (de wilde et al., 2013; knoops et al., 2008) . moreover, covs induced very little ifn production in most of cell types (kindler et al., 2016) . thus, high levels of dsrna, which are generated during viral replication cycle (weber et al., 2006; zielecki et al., 2013) , do not caused an adequate induction of ifn system. moreover, antigen presentation subsequently stimulated body's humoral and cellular immunity on viral invasion, which are mediated by virus-specific b and t cells. t lymphocytes, including cluster of differentiation 4 (cd4 + ) and cluster of differentiation 8 (cd8 + ) t cells, play a central role against viral infection, stimulated b cells to produce virus-specific antibodies while cd8 + t cells directly kills virus-infected cells. also, humoral immunity, including complements such as c3a, c5a and antibodies, are critical in fighting viral infection (fig. 7) (mathern and heeger, 2015; traggiai et al., 2004) . recent flow cytometric analysis of peripheral blood from sars-cov-2-infected patients showed substantially reduced cd4 + and cd8 + t cells number while their status was excessively activated as evident from high proportions of human leukocyte antigen -dr isotype (hla-dr) (cd4 3.47%) and cd38 (cd8 39.4%) double positive fractions . similarly, acute phase response in patients with sars-cov was associated with severe decrease of cd4 + t and cd8 + t cells. even if there is no antigen, cd4 + and cd8 + memory t cells are known to persist for four years as observed in sars-cov recovered individuals and were able to perform t cell proliferation, delayed-type hypersensitivity (dth) j o u r n a l p r e -p r o o f response and production of ifn-γ (fan et al., 2009) . also, six years after sars-cov infection, specific t-cell memory response to sars-cov s peptide library was identified in 14 of 23 recovered sars patients (tang et al., 2011) . the specific cd8 + t cells were documented to exhibit a similar effect on mers-cov clearance in mice (zhao et al., 2014) . these discoveries could be helpful to deduce a valuable information for the rational design of vaccines against sars-cov-2. for example, antibodies collected from a recovered patient were reported to neutralized mers-cov infection (niu et al., 2018) . moreover, t helper cells also produced proinflammatory cytokines to assist the defending cells. however, cov (sars-cov and mers-cov) were established to halt t cell function and induced their apoptosis mathern and heeger, 2015; yeung et al., 2016) . besides, sars-cov-2 assumed to attach with ace2 as in sars-cov infection, target and infect the same set of host cells zhou et al., 2020b; zou et al., 2020) . additionally, ace2-associated lung injury was documented in sars-cov infection kuba et al., 2005) ; s protein of sars-cov downregulates ace2 (glowacka et al., 2010; wang et al., 2008b) and induce detachment of catalytically active ace2 ectodomain (haga et al., 2008; jia et al., 2009) . this results in nonfunctional pulmonary ace2 receptor which has been advised to be linked with acute lung injury (imai et al., 2008; kuba et al., 2006) ; decrease activity of ace2 further triggered reninangiotensin system (ras) dysfunction and caused enhance inflammation and vascular permeability (gheblawi et al., 2020) . in animal studies with murine acute respiratory distress (ard) model showed association of reduced ace2 function with an increased lung edema, neutrophil accumulation, enhanced vascular permeability, and diminished lung function . also, in human airway epithelia, constitutive shedding of ace2 receptor by activity of a metalloprotease 17 (adam17), also called as tumour necrosis factor alpha (tnf-α) converting enzyme (tace), and disintegrin secrete functionally active soluble ace2 (sace2) (lambert et al., 2005) . hcovs infection and inflammatory cytokines like interleukin 1 beta (il-1β) and tnf-α were related with enhance ace2 shedding (haga et al., 2008; jia et al., 2009) . notably, in vitro studies presented close association of sars-cov s protein-induced ace2 shedding with tnf-α production (haga et al., 2008) . although, biological function of sace2 is yet unknown, however, recent studies have suggested direct involvement of sace2 in inflammatory responses against sars-cov, and possibly also connected with sars-cov-2 (fu et al., 2020) . additionally, apoptosis of type 2 alveolar cells caused release of specific j o u r n a l p r e -p r o o f inflammatory mediators which further instigate macrophages to secrete three essential immune substances; interleukin-1 (il-1), interleukin-6 (il-6), and tnf-α; also called as cytokines. these immune substances in the bloodstream produced infection symptoms linked to hcovs . recent in vitro cell experiments showed that delay release of cytokines and chemokines occurred in respiratory epithelial cells, dendritic cells (dcs), and macrophages at the early stage of sars-cov infection (choi and joo, 2020) . like sars, mers-cov also infected human airway epithelial cells, thp-1 cells (a monocyte cell line), human peripheral blood monocyte-derived macrophages and dcs, and induced late but elevated levels of proinflammatory cytokines and chemokines (tynell et al., 2016; zhou et al., 2014) . notably, sars-cov-2 infection is suspected to cause pyroptosis in lymphocytes and macrophages. it was observed that lymphocytopenia is often seen in severe sars-cov-2 patients and cytokine release syndrome (crs) induced by sars-cov-2 was also considered to be facilitate by leukocytes other than t cells . a vast majority of patients (82.1%) infected with sars-cov-2 have been logged for peripheral blood lymphopenia (guan et al., 2020) , supported the pulmonary infiltration of lymphocytes and/or cell damage via pyroptosis or apoptosis . previous pandemics triggered by mers-cov and sars-cov were also reported with massive production of chemokines and cytokines; sars-cov exhibited ccl2, ccl3, ccl5, cxcl8, cxcl9, cxcl10, etc.; and il-12, il-18, il-6, il-1beta, il-33, ifn-alpha, ifn-gamma, tnf-α and transforming growth factor (tgf)-beta. likewise, mers-cov was reported for elevated expression of cytokines ifn-α, il-6, and chemokine (cxcl-10, ccl-5, and cxcl-8) . hence, acute respiratory distress syndrome (ards) is caused by cytokine storm that triggers a destruction in host cells via immune system and subsequently results into multiple organs failure or death as stated in case of sars-cov-2 outbreak; similar observations were noted in case of sars-cov infection (kumar et al., 2020) . albeit, there is no direct data is available to corelate the participation of pro-inflammatory cytokines and chemokines in lung pathology through sars-cov and mers-cov, correlative observations and recorded from patients with severe disease suggested a role of hyper-inflammatory responses in hcov pathogenesis (fig.7) (channappanavar and perlman, 2017) . for instance, in mers-cov infection, plasmacytoid dendritic cells, but not mononuclear macrophages and dcs (scheuplein et al., 2015) , were induced to produce a large amount of ifns (choi and joo, 2020) . (yi et al., 2020) . despite of similar virus titers in respiratory tract, sars-cov-infected old nonhuman primates are more likely to develop immune dysregulation than infected young primates, leading to more severe disease manifestations (smits et al., 2010) . it seems that excessive inflammatory response rather than virus titer is more relevant to the death of the old nonhuman primates (smits et al., 2010) . similarly, in bagg albino/c (balb/c) mice infected with sars-cov, disease severity in old mice was related to early and disproportionately strong upregulation of ards-related inflammatory gene signals (rockx et al., 2009) . the rapid replication of sars-cov in balb/c mice induces delayed release of ifn-α/β, which was accompanied by influx of many pathogenic inflammatory mononuclear macrophages (channappanavar et al., 2016) . depleting inflammatory monocyte-macrophages or neutralizing inflammatory cytokine tnf protected the mice from fatal sars-cov infection. thus, inflammatory mediators contributes an vital role in pathogenesis of ards, which was estimated as an leading cause of death in patients infected with sars-cov or mers-cov lew et al., 2003) . it is now known that several proinflammatory cytokines consequences of cytokine storm contribute to the occurrence of ards (channappanavar and perlman, 2017; reghunathan et al., 2005) . these mechanisms also lead to activation of coagulation pathways. massive inflammation can impaired all the three control mechanisms like antithrombin iii, tissue factor pathway inhibitor, and protein c system (jose et al., 2014) ; with reduced anticoagulant concentrations due to reduced production and increasing consumption. this defective procoagulant-anticoagulant balance predisposes in the development of microthrombosis, disseminated intravascular coagulation, and multiorgan failure; as evident from severe sars-cov-2 pneumonia with raised d-dimer concentrations being a poor prognostic feature and disseminated intravascular coagulation common in nonsurvivors zhou et al., 2020a) . in this over all proposed mechanism, proteinase-activated receptor-1(par-1) acts as main thrombin receptor and mediates thrombininduced platelet aggregation, and interplay between coagulation, inflammatory, and fibrotic responses; all of these factors are an important aspects of pathophysiology of fibroproliferative lung disease (jose et al., 2014) , such as seen in sars-cov-2. on the other hand, the proposed events of cytokine storms in case of sars-cov-2 was suggested to increased inflammationrelated biomarkers, including c-reactive protein (crp), ferroprotein, erythrocyte sedimentation j o u r n a l p r e -p r o o f rate (esr) and il-6 huang et al., 2020) . in conclusion, based on previous hcovs infection, severity and pathogenesis of sarscov-2 was suggested to divided into three stages; stage-1, an asymptomatic incubation time with or without detectable virus; stage-2, nonsevere symptomatic period and detection of viral particles; stage-3, severe respiratory symptomatic stage and detection of high viral load . furthermore, based on previous studies of sars-cov, the inflammatory responses in sars-cov-2 infection can be classified into primary and secondary responses . initially, primary inflammatory responses produced subsequently viral infection, prior to arrival of neutralizing antibodies (nab). the activation of these factors is primarily driven by viralmediated ace2 downregulation and shedding, vigorous viral replication, and host antiviral defense system. secondary inflammatory responses are generated by the adaptive immunity and nab. moreover, virus-nab complex was also suggested to triggered fc receptor (fcr)-mediated inflammatory responses and acute lung injury that results in persistent viral replication and inflammatory responses from host macrophages (fig. 8) (fu et al., 2020) . it was observed that patients could survive in primary inflammatory responses but secondary inflammatory responses could be fatal. secondary inflammatory responses in which virus-nab complex formed was considered as targeted to develop potential therapeutic strategy along with anti-inflammatory drugs (fu et al., 2020) . accumulating data on hcovs exhibits the importance of understanding the zoonotic viral transmission and pathogenesis in humans. recent studies on virus have established the viral proteins as prime target in the drug designing and vaccine development; however, this review have highlighted the utmost importance of intermediate host for the viral transmission to human. future approaches, therefore, should considered the strains from intermediate hosts in the therapeutic development pipelines to better understand the severity of virulence via genome recombination process in covs. we also concluded major damaged caused by the failure of host j o u r n a l p r e -p r o o f immune system against hcov infection and further caused by insurgence of cytokines which lead to severe mutilation of the host body. synergistic and/or multi-target strategies that check viral growth and malfunctioned immune system of the host cells simultaneously might also be successful in battle against hcovs. nevertheless, synergistic approaches must take into account crossponding to the complementary target pathways. when developing novel therapeutic strategies to check the immunoregulatory cytokines such as tnfβ and il6, investigation should be considered on the viral strain and targeted organ specificity; for example, sars-cov-2 has more affinity to ace2 which are scattering on different organs like lung and kidney while mers-like cov can even infect t-cells. in addition, we should be cognizant of the feedback responses that produce with any treatment and consider the possibility to leveraged in a combinatorial fashion, taking into consideration the outcomes of preceding standard care and/or target immunoregulatory molecules will affect the efficacy and incidence of virulence. another utmost point to be consider is how to generalize the therapeutic decision under the different immunological responses to the viral infection; for instance, susceptibility of some persons and with asymptomatic symptoms or sever immune response to the infection. given the relatively high emergence of hcovs and potential threat to humans, future therapeutic strategies should involve deep immune profiling of the infected individuals and personalization of therapeutics when feasible to accelerate progress towards more effective treatment approaches. this goal is ever closer to becoming a reality as multiplexed imaging, immunophenotyping and mutational analysis tools are increasing the high-throughput process. although, many trials have failed before, but given the progress in our understanding on the interaction between host immune responses and viral escape plans, and the emerging sophisticated strategies we have reasons to be hopeful for the future successful treatment against hcovs. authors 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pneumonia in china coronavirus replication and reverse genetics human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection coronaviruses -drug discovery and therapeutic options middle east respiratory syndrome key: cord-333498-d25qfq0f authors: chitranshi, nitin; gupta, vivek k.; rajput, rashi; godinez, angela; pushpitha, kanishka; shen, ting; mirzaei, mehdi; you, yuyi; basavarajappa, devaraj; gupta, veer; graham, stuart l. title: evolving geographic diversity in sars-cov2 and in silico analysis of replicating enzyme 3cl(pro) targeting repurposed drug candidates date: 2020-07-09 journal: j transl med doi: 10.1186/s12967-020-02448-z sha: doc_id: 333498 cord_uid: d25qfq0f background: severe acute respiratory syndrome (sars) has been initiating pandemics since the beginning of the century. in december 2019, the world was hit again by a devastating sars episode that has so far infected almost four million individuals worldwide, with over 200,000 fatalities having already occurred by mid-april 2020, and the infection rate continues to grow exponentially. sars coronavirus 2 (sars-cov-2) is a single stranded rna pathogen which is characterised by a high mutation rate. it is vital to explore the mutagenic capability of the viral genome that enables sars-cov-2 to rapidly jump from one host immunity to another and adapt to the genetic pool of local populations. methods: for this study, we analysed 2301 complete viral sequences reported from sars-cov-2 infected patients. sars-cov-2 host genomes were collected from the global initiative on sharing all influenza data (gisaid) database containing 9 genomes from pangolin-cov origin and 3 genomes from bat-cov origin, wuhan sars-cov2 reference genome was collected from genebank database. the multiple sequence alignment tool, clustal omega was used for genomic sequence alignment. the viral replicating enzyme, 3-chymotrypsin-like cysteine protease (3cl(pro)) that plays a key role in its pathogenicity was used to assess its affinity with pharmacological inhibitors and repurposed drugs such as anti-viral flavones, biflavanoids, anti-malarial drugs and vitamin supplements. results: our results demonstrate that bat-cov shares > 96% similar identity, while pangolin-cov shares 85.98% identity with wuhan sars-cov-2 genome. this in-depth analysis has identified 12 novel recurrent mutations in south american and african viral genomes out of which 3 were unique in south america, 4 unique in africa and 5 were present in-patient isolates from both populations. using state of the art in silico approaches, this study further investigates the interaction of repurposed drugs with the sars-cov-2 3cl(pro) enzyme, which regulates viral replication machinery. conclusions: overall, this study provides insights into the evolving mutations, with implications to understand viral pathogenicity and possible new strategies for repurposing compounds to combat the ncovid-19 pandemic. in early january 2020, the world health organisation (who) reported cases of pneumonia of an unknown cause in wuhan city, hubei province of china, and by 30 january 2020, who escalated the warning to public health emergency of international concern. by 12 march 2020, the novel coronavirus (ncov) outbreak achieved a global pandemic status and was recognised as novel journal of translational medicine *correspondence: nitin.chitranshi@mq.edu.au; vivek.gupta@mq.edu.au 1 faculty of medicine, health and human sciences, macquarie university, f10a, 2 technology place, north ryde, nsw 2109, australia full list of author information is available at the end of the article page 2 of 15 chitranshi et al. j transl med (2020) 18:278 covid-19 disease (ncovid-19) [1] . the present coronavirus outbreak is associated with severe acute respiratory syndrome 2 (sars-cov-2), phylogeny and taxonomy designated [2] . worldometer reported the total sars-cov-2 infected cases on 31 may 2020 as 6,238,550 and deaths 374,374 worldwide (https ://www.world omete rs.info/coron aviru s/#count ries). the pathogen has been established to transmit from human to human contact and has quickly spread to more than 187 countries across the globe (https ://gisan ddata .maps.arcgi s.com/). coronaviruses are single and positive stranded rna viruses belonging to the genus coronavirus of the family coronaviridae that can cause acute and chronic respiratory and central nervous system illnesses in animals, including in humans [3, 4] . the infection can also cause mild episodes of follicular conjunctivitis in certain patients. in animal models, the infection has been shown to induce anterior uveitis, retinitis, and optic neuritis like symptoms [5] . recent study has shown formation of hyper-reflective lesions in the ganglion cell and inner plexiform layers of the retina particularly around the papillomacular bundles [6] . the disease has also been shown to affect sense of smell and taste bud sensitivity in patients [7] . all coronaviruses have a minimum of 3 basic viral proteins (i) an envelope protein (e), which is a highly hydrophobic protein involved in several aspects of the virus life cycle such as assembly and envelope formation [8] (ii) a spike protein (s), a glycoprotein involved in receptor recognition and membrane fusion [9] and (iii) a membrane protein (m), which plays a key role in virion assembly [10] (fig. 1) . the viral genome also encodes two open reading frames (orf), orfa and orfb that activate intracellular pathways and triggers the host innate immune response [11] . the polyprotein encoded by the virus are initially processed by two main viral proteases, which include a papain-like cysteine protease (pl pro ) and 26, 191) and nucleocapsid (n, nt 28,274-29,533) proteins in green. orf1a gene encodes papain-like protease and 3cl protease, orf1b gene encodes rna-dependent rna polymerase, helicase and endo ribo-nuclease, s, e, m and n gene encodes spike, membrane glycoprotein and nucleocapsid phosphoprotein respectively. three-dimensional crystal structure of 3cl-protese, endoribonuclease and sars-cov-2 spike protein receptor binding domain (rbd) engaged human angiotensin converting enzyme 2 (ace2) receptor were collected from protein data bank chymotrypsin-like cysteine protease, known as 3c-like protease (3cl pro ), into intermediate and mature nonstructural proteins [12] . the main proteinase 3cl pro , is one of the primary targets for development in an antiviral drug therapies, as it plays a critical role in the viral replication [13] . k11777, camostat and est, are cysteine protease inhibitors, which have been shown to inhibit sars-cov 3cl pro replication in cell culture conditions [14, 15] . recent release of the high-resolution crystal structure for the main proteinase 3cl pro (protein data bank, pdb id: 6y2g), describing an additional amide bond with the α-ketoamide inhibitor pyridone ring to enhance the half-life of the compound in plasma [16] is suggested to accelerate the targeted drug discovery efforts. two hiv-1 proteinase inhibitors, lopinavir and ritonavir, have been considered to target sars-cov [17] . interestingly, the substrate binding cleft is located between domains i and ii of both sars-cov 3cl pro and sars-cov-2 3cl pro enzymes [16, 18] . since the initial stages of the sars-cov-2 outbreak, laboratories and hospitals around the world have sequenced viral genome data with unprecedented speed, enabling real-time understanding of this novel disease process, which will hopefully contribute to the development of novel candidate drugs. the complete genomes of sars-cov-2 from all over the world have been deposited at the global initiative on sharing avian influenza data (gisaid) [19] database and more sequences continue to be deposited with the passage of time. development of a novel vaccine against sars-cov-2 so far remains elusive and requires a thorough understanding of molecular changes in viral genetics. this may be attained by freely accessing the gisaid database and processing the data to enhance our understanding of the fine biochemical and genetic differences that differentiate this virus from the previously known strains [20] . it is well known that viruses are non-living and that they require host cells to survive and to reproduce, with the sole aim to perpetuate themselves. when a virus jumps from animal to human, it is termed a zoonotic virus. this occurred during the sars outbreak of 2002, when a new coronavirus spread around the world and resulted in death of hundreds of people [21] . in 2012, another novel coronavirus outbreak, termed middle east respiratory syndrome (mers), caused over 400 fatalities and spread to over 20 different countries [22] . there are currently many circulating viruses, but why sars-cov-2 has achieved such a devastating pandemic status and whether this pandemic will subside remain unanswered. the purpose of this study is to characterise known viral variants that have spread across different countries, especially hot-spot regions, with a focus on recurrent mutations in south american and african geographical regions. we also focused on the sars-cov-2 main proteinase, 3cl pro which is highly conserved in most of the coronaviruses and has been suggested to be a potential drug target to fight against ncovid-19. repurposed drugs such as flavonoids and biflavanoids, known anti-malarial and anti-viral drugs and the inhibitory effects of vitamins could selectively inhibit this enzyme and can be used either alone or in combination with other disease management approaches to suppress the virulence of sars-cov-2. these bioinformatics, computational modelling and molecular docking approaches using repurposed drugs could be particularly useful in the current ncovid-19 outbreak. the global initiative on sharing avian influenza data (gisaid) is headquartered in munich, germany and is a public-private partnership project between german government and the non-profit organization founded by leading medical researchers in 2006. since december 2019, gisaid has become a repository storage database for ncovid-19 genome. the genome analysis was carried out for data deposited up to 31 may 2020 (https ://www. gisai d.org/). severe acute respiratory syndrome coronavirus 2 (sars-cov-2) wuhan genome was collected from ncbi, nc_045512.2. multiple sequence alignment (msa) of all nucleotide sequences were carried out in the embl-ebi clustal omega server to investigate sequence conservation [23, 24] . the newick format for the multiple align sequence was used to generate phylogeny [25] . the phylogenetic tree was constructed in the interactive tree of life (itol) online tool [26] . the itol server generate phylogeny trees in a circular (radial) and normal standard trees. the circular trees can be rooted and displayed in different arc sizes [27] [28] [29] . crystal structure of sars and sars-cov-2 3cl pro with bound inhibitors were collected from the protein data bank (pdb) [30] . pdb id: 3tnt, sars main protease was selected as reference to analyse the variants in sars-cov-2 3cl pro (pdb id: 6y2g). all the pdbs were visualised using ucsf chimera software [31] . multiple alignment, ribbon, surface and superimposition module in chimera software were used for analysis and image generation [24, 32] . the dataset comprises of flavones and biflavanoids, anti-viral, anti-malarial and vitamins as sars-cov-2 3cl pro inhibitors [16] . in total 17 repurposed drugs were collected from the pubchem database [33] . twodimensional (2d) structures were downloaded from the pubchem database in.sdf format. the inhibitor energies were minimized using the austin model-1 (am1) until the root mean square (rms) gradient value became smaller than 0.100 kcal/mol å and later re-optimization was done by mopac (molecular orbital package) method [34, 35] . later, all the inhibitors were converted to.pdb format in open babel software [36] and submitted to molecular docking studies. crystal structure of the sars-cov-2 3cl pro was retrieved from pdb (pdb id: 6y2g). the protein macromolecule (sars-cov-2 3cl pro ) optimization was carried out in ucsf chimera software [31, 37, 38] by adding polar hydrogen atoms, removing water molecules, implying amber parameters, followed by minimization with the mmtk method in 500 steps with a step size of 0.02 å. sars-cov-2 3cl pro contained chain a and b of 306 amino acids sequence length. chain a of pdb id: 6y2g containing alpha-ketoamide (o6k) inhibitor was used for identification of substrate binding site. the docking of sars-cov-2 3cl pro specific pharmacological inhibitors into the catalytic site was performed by the autodock 4.2 program [39] . the alpha-ketoamide (o6k) inhibitor was extracted from the sars-cov-2 3cl pro protein. the polar hydrogen atoms were added, the non-polar hydrogen atoms were merged, gasteiger charges were assigned and solvation parameters were added to the protease, sars-cov-2 3cl pro protein. the protonation state for all inhibitors and o6k were set to physiological ph and rotatable bonds of the ligands were set to be free. the autogrid program was also used to generate grid maps. cys145 residue in the sars-cov-2 3cl pro protein was selected with grid box dimensions of 40 × 40 × 40 å formed around the cys145 protease residue, which is present in the substrate binding site. protein rigid docking was performed using the empirical free energy function together with the lamarckian genetic algorithm (lga) [40] . lga default parameters were used in each docking procedure and 10 different poses were calculated. chimera and discovery studio (ds) visualizer2.5 [31] software were used for visualisation and calculation of protein-ligand interactions. a total of 9761 sars-cov-2 genomes were retrieved from the gisaid database (https ://www.gisai d.org) that contain 3 sequences from bat (betacoronavirus) and 9 sequences from malayan pangolin (manis javanica) (additional file 1: table s1 ). out of 9761 genome sequences, 2301 complete genome sequences of sars-cov-2 were selected randomly, aligned and compared with wuhan sars-cov-2 (nc_045512.2) reference genome. we have divided our dataset into 6 different geographic areas: europe (20.31%), north america (21.13%), asia (35.37%), oceania (20.86%), south america (16.63%) and africa (10.35%). the european group comprises of sars-cov-2 infected patient data from the following countries: austria, belgium, czech republic, denmark, estonia, finland, france, germany, greece, hungary, iceland, ireland, italy, latvia, lithuania, luxembourg, netherlands, poland, portugal,, slovakia, slovenia, spain, sweden, switzerland, and united kingdom. the north american group contains genomes from the united states and canada. the asian group comprises genomes obtained from patients located in china, indonesia, pakistan, philippines, taiwan, turkey, kuwait, georgia, south korea, japan, iran, india, thailand, hong kong, malaysia, singapore, and vietnam. the oceanian group comprises genomes from australia and new zealand. south america includes brazil, peru, chile, colombia, argentina, and ecuador ( fig. 2a-c) . sequences from bat-sars-cov and pangolin-sars-cov were aligned and compared to the wuhan sars-cov-2 (nc_045512.2) as a reference genome. to determine the evolutionary relationship among bat-cov, pangolin-cov and sars-cov-2, we estimated a phylogenetic tree based on the nucleotide sequences of the whole-genome sequence. bat-sars-cov and sars-cov-2 were grouped together and were observed to share > 96% similarity, whereas the pangolin-sars-cov was closest evolutionary ancestor (fig. 2d) . isolate of human wuhan sars-cov-2 (nc_045512.2) shared 85.98% identity with pangolin-sars-cov which suggests that pangolin may be associated with sars-cov-2 evolution or subsequent outbreak [41, 42] . recently, pachetti et al. [43] has reported eight novel recurrent mutations of sars-cov-2 that have been identified in positions 1397, 2891, 14,408, 17,746, same locus indicates the high susceptibility of these genetic regions to change as the virus evolves. for its actions, single-stranded sars-cov-2 rna viral genome encodes two protease polyproteins (i) papainlike cysteine-protease (pl pro ) and (ii) the chymotrypsinlike cysteine protease known as 3c-like protease (3cl pro ). 3cl pro , which is a main protease and therefore important in order to examine the incidence of any mutation in sars-cov-2 3cl pro . multiple sequence alignment of the sars-cov-2 genome collected from patients in six different geographical locations exhibited 100% similarity and no discernible variations in sequences obtained from diverse geographical regions, for this enzyme. sars and sars-cov-2 complete genomes were collected from ncbi, genbank database (nc_004718 and nc_045512). protease nucleotide sequences were extracted from sars (nc_004718) and were aligned with sars-cov-2 (nc_045512). clustal omega alignment of 918 sars nucleotides showed around 95% similarity with sars-cov-2 (additional file 2: table s2 ). higher amino acid sequence identity was also observed in sars-cov and sars-cov-2 main protease (3cl pro ) derived from wuhan and us patients. sars-cov and sars-cov-2 3cl pro showed highly conserved region in both the catalytic sites, his41 and cys145 [44] and substrate previously confirmed mutations at positions nt3036, nt8782, nt11083, nt14408, nt23403, nt28144 and nt28881 were also present in south american and african populations. we normalize the mutation frequency percentage by estimating the frequency of genomes carrying mutation and comparing it with the overall number of collected genomes per geographical area. the graph shows the cumulative mutation frequency of all given mutations present in south american and african regions. mutation localisation in viral genes are reported in the legend as well as the proteins (i.e. non-structural protein, nsp) presenting these mutations. b it is also evident that south american and african clusters show a differential pattern of novel mutations: mutation 1059 (black), 9477 (pink), 28,657 (green) and 28,878 (red) in south american, whereas mutation 1059 (black), 15,324 (orange), 28,878 (yellow) and 29,742 (magenta) are present with greater frequency in african patients (fig. 4a) , inferring that these proteases exhibit high similarities. furthermore, 12 variant positions (thr35val, ala46ser, ser65asn, leu86val, arg88lys, ser94ala, his-134phe, lys180asn, leu202val, ala267ser, thr285ala and ile286leu) were observed in sars-cov-2 3cl pro (fig. 4b, c) . the effects of mutations and potential resultant amino acids on sars-cov-2 3cl pro structure are expected to conserve the polarity and hydrophobicity, except when the resulting amino acid is leucine at 286 position. however, it is important to mention that these 12 variants are not present in catalytic and substrate binding regions which are involved in critical proteolytic activity of the sars-cov-2 protease molecule. the sars-cov-2 3cl pro receptor binding pocket was determined by superimposing sars and sars-cov-2 3cl pro with their respective inhibitors (fig. 4) . interestingly, needleman-wunsch alignment algorithm and blosum-62 matrix analysis revealed 94.44% sequence identity between sars (fig. 5a, grey) and sars-cov-2 3cl pro (fig. 5a, cyan) . cys-his catalytic dyad (cys145 and his41) comprises the active catalytic binding site in sars-cov-2 3cl pro (fig. 5a' , b) and indicated the strong possibility that intended pharmacological inhibitors of sars-cov-2 3cl pro may also suppress the activity of sars-cov-2 3cl pro viral enzymes. docking protocol for the autodock 4.2 program was optimized by extracting and re-docking the alpha-ketoamide inhibitor named o6k in the binding pocket of sars-cov-2 3cl pro . the lowest binding energy − 6.45 kcal/mol and 18.72 µm inhibitory constant (ki) was predicted for alpha-ketoamide inhibitor (shown in table 1 ). re-docking of o6k inhibitor occupied the similar docking pose in the sars-cov-2 3cl pro catalytic dyad active site as previously reported in the crystal structure (pdb id: 6y2g) (fig. 5c, d) . seven flavonoids and biflavonoid, three anti-malarial compounds, seven anti-viral drugs and three vitamin molecules were subjected to automated docking within the active site of sars-cov-2 3cl pro catalyticdyad. the superimposition of all docked flavones and biflavones (fig. 6a) , anti-malarial drugs (fig. 6b) , antiviral drugs (fig. 6c) and vitamins (fig. 6d) are shown in fig. 6 and various binding parameter have been tabulated in detail in table 2 . amentaflavone, a biflavonoid showed the highest binding energy (− 8.49 kcal/mol) implicating a strong affinity with sars-cov-2 3cl pro . this corresponded with previously reported enzyme inhibitory assays with amentaflavone that showed the highest ic 50 value at low concentrations of the molecule, 8.3 ± 1.2 µm [46] . however, bilobetin demonstrated the lowest ic 50 value at a higher concentration of 72.3 ± 4.5 µm in sars-cov enzyme activity assays [46] . in contrast, our docking studies revealed that bilobetin, predicted almost comparable binding energy with that of amentaflavone (− 8.29 kcal/mol) suggesting that mutation in sars-cov-2 3cl pro could potentially disrupt hydrogen bonding or induce some conformational change that could result in alterations in the binding site thus affecting inhibitor interactions with the enzyme active site residues. amentaflavone showed h-bond interactions with the catalytic dyad residues (cys145 and his41) as well as noteworthy interactions with the sars-cov-2 3cl pro residues thr26, ser46, ser144 and glu166 whereas his164, and gln189 amino acids contributed to the hydrophobic interactions for the sars-cov-2 3cl pro inhibitors (fig. 7a) . three antimalarial drugs were then selected to study their inhibitory actions on sars-cov-2 3cl pro . we found, artemisinin, a natural compound derived from chinese herb artemisia annua produces the highest docking score (− 6.40 kcal/mol) as compared to o6k, chloroquine (-4.95 kcal/mol) and hydroxychloroquine (− 5.77 kcal/mol) anti-malarial molecules. importantly, artemisinin has demonstrated broad anti-viral activity against human cytomegalovirus, herpes simplex virus type 1, epstein-barr virus, hepatitis b virus, hepatitis c virus, and bovine viral diarrhea virus [47] . artemisinin was shown to exhibit hydrogen bonding with his41, leu141, asn142, gly143, ser144 and glu166 sars-cov-2 3cl pro amino-acid residues (fig. 7b) . amongst the seven antiviral drugs, ritonavir showed the highest binding energy (-7.45 kcal/mol) and lowest inhibitory constant ki value (3.49 µm). ritonavir produced hydrogen bond interactions with thr26, his41 and cys145 sars-cov-2 amino acids (fig. 7c) . a combination of two hiv-1 protease inhibitors, lopinavir and ritonavir, were given to critically ill sars-cov 2 infected patients [48] . however, the combination therapy of lopinavir and ritonavir was also stopped early in 13 patients (total recruitment 99 patients) due to associated gastrointestinal adverse events [48] . the severity of antiviral therapy adverse events has led researchers to explore the potential of macro-, microand phytonutrients that can potentially promote an immune response and suppress viral induced effects. vitamins are known to modulate the host immune functions by providing anti-oxidants and anti-inflammatory activity [49, 50] . therefore, we selected vitamins, ascorbic acid (vitamin c), cholecalciferol (vitamin d) and alpha-tocopherol (vitamin e) to investigate their potential interactions with the enzyme sars-cov-2 3cl pro . our docking results interestingly, showed that vitamin d has the lowest binding energy and ki (− 7.75 kcal/mol and 2.08 µm respectively) as compared to vitamin c and vitamin e. amino acid residues thr24, thr26, his41 and cys145 of sars-cov-2 3cl pro showed hydrogen bond formation with vitamin d (fig. 7d) . amino acid thr is extensively involved in intracellular signalling changes through phosphorylation changes, and here we observed that cholecalciferol formed a strong hydrogen bond with thr residues and could potentially block the phosphorylation of thr residue in sars-cov-2 3cl pro enzyme. there is evidence that serious sars-cov-2 infected cases have reported severe vitamin d deficiency and thus therapeutic concentrations of this molecule could potentially be used clinically in sars-cov-2 cases [51, 52] . the novel coronavirus termed "ncovid-19" is now known as the third large-scale epidemic coronavirus introduced into the human population in the twenty-first century. at the time of writing, more than 3.67 million confirmed cases globally, with nearly 250,000 deaths had been reported by who. clinically, ncovid-19 is similar to sars regarding its presentation, however the sheer capacity and speed of which ncovid-19 has spread to global pandemic levels have left researchers asking what makes this outbreak so similar in presentation, yet so different in its virulence to previous coronaviruses. genome sequence analysis has looked to investigate similarities in the phylogeny of sars-cov-2, which like sars and mers, have now placed it in the betacoronavirus genus [53] . the known severe and often fatal pathogenicity of betacoronaviruses has been highlighted in these previous epidemics and has reported higher transmission and pathogenicity than the milder and lesser known a-covs, which are often compared to the common cold [54] . our study further compares the similarities between sars-cov and sars-cov-2 using clustal omega alignment to show that of 918 sars nucleotides, there was a similarity of approximately 95%. furthermore, we report high amino acid sequence identity in both sars-cov and sars-cov-2 main protease 3cl pro , which regulates coronavirus replication complexes [55] . such highly conserved regions in both catalytic sites and the substrate binding regions of the enzymes has also been validated previously in studies by huang et al. and muramatsu et al. [44, 45] . while this region provides an attractive target for anti-viral drug design, it also can begin to elucidate on viral origins and uncover its ease in transmission. based on more recent virus genome sequencing results and evolutionary analysis, the origins and transmission of ncovid-19 have uncovered bats as the natural host of the virus origins [42] . as such, studies earlier this year queried the unknown intermediate host between bats and humans, and recent studies have pointed this to pangolins [41, 42] . to determine the extent of the evolutionary relationship between bat-cov, pangolin-cov and sars-cov-2, we corroborate that based on the nucleotide sequences of the whole-genome sequence, bat-sars-cov and sars-cov-2 are grouped together and share > 96% similarity, with pangolin-sars-cov as the closest evolutionary ancestor [41, 42] . furthermore, we report that in isolates of human wuhan sars-cov-2 there is an 85.98% similarity in identity to pangolin-sars-cov, which suggests that pangolin may be associated with the evolution of subsequent outbreaks of covid-19. regarding ncovid-19 and its similarity in transmission to sars-cov, recent studies have also demonstrated that transmission occurs via the receptor angiotensinconverting enzyme 2 (ace2) [42] . this may indicate why sars-cov-2 has often led to severe and in many cases fatal respiratory tract infections, like its two sar-cov predecessors. since the sars-cov epidemic of 2002 was also known to use the ace2 receptor to infect humans [56] . bronchoalveolar lavage fluid taken from ncovid-19 patients have shown that ace2 is widely distributed in the lower respiratory tracts of humans [42] . furthermore, the virion s-glycoproteins expressed on the surface of coronaviruses adhere to ace2 receptors on human cells [57] . this location provides a target for uncovering the mechanistic insights into the severity of the disease and how this region has assisted in the zoonosis of sars-cov-2 specifically. additionally, mutations in the genomic structure of sars-cov-2 also might elucidate on the aggressiveness and pathogenicity of the viruses, which may in turn help to explain why some strains are evolutionarily much more virulent and contagious. angeletti et al. have described mutations in the endosome-associated-protein-like domain of the nsp2 and nsp3 proteins, the former possibly accounting for the high virulence and contagion, while the latter suggesting a mechanism that differentiates ncovid-19 from sars-cov [58] . our studies build on this knowledge and assist to begin to identify the sub-clinical causes for the virulence and unique pandemic pattern of this outbreak by identifying the evolving mutations from region to region. additionally, previous studies by pachetti et al. have reported novel recurrent mutations of the sars-cov-2, and our study corroborates these mutations in south america and africa regions [43] . drug discovery and vaccine development against sars-cov-2 infection require time and lengthy processes, however drug repurposing represents an alternative strategy in the current scenario. some of these antivirals are currently being used clinically in sars-cov-2 treatment, including lopinavir [59] , ritonavir [60] , remdesivir [61] , and oseltamivir [62] . however, in the clinical setting, lopinavir/ritonavir, a 3cl pro and rdrp inhibitors, showed no benefit in covid-19 adult patients [48] . the double point mutation in rdrp gene identified in our study can potentially lead to a drug-resistance event. moreover, other classes of drugs, such as chloroquine and hydroxychloroquine have shown antiviral properties by blocking viral entry into cells by inhibiting glycosylation of host receptors [63] . we observed no differences in the sars-cov-2 main proteinase, 3cl pro genome sequences, but important differences in sars-cov-2 3cl pro with sars-cov protein, underlining the extreme need for identification of inhibitors to target the viral life cycle. it is not known whether these mutations induce any alterations in the gene transcription or localisation of affected proteins which can be investigated in near future using biochemical and immunological approaches [64, 65] . various theories have been proposed regarding the origin of highly virulent sars-cov-2 particle. our analysis shows that bat-sars-cov shares > 90% similarity with the sars-cov-2, however it is possible that the bat coronavirus infected another "intermediate host", such as pangolin, which subsequently transmitted the virus to humans. pangolin isolates do share sequence identity with sars-cov-2 genomes and could be an intermediate host. we identified novel mutation hotspot regions from south american and african isolates of sars-cov-2 genome sequences. interestingly, double point mutations in rdrp at position 14,805 and 14,808 and triple point mutations in nucleocapsid protein at position 28,881, 28,882 and 28,883 were identified in both south american and african genomic sequences, suggesting the vulnerability of these genetic loci to undergo change. in addition, a novel mutation pattern specifically oriented towards nucleocapsid phosphoprotein in both south american and african sequences was noted while novel orf3a and rdrp specific variants were observed particularly from african genomic sequences. the potential effects of double and triple point mutations on translated proteins and the virulence of sars-cov-2 requires further investigations. sars-cov-2 main proteinase, 3cl pro genome was observed to be conserved across all collected genomic sequences. despite significant similarities in the sars-cov 3cl pro structure with sars-cov protein, sars-cov-2 3cl pro revealed certain key differences, which highlight the extreme need for identification of novel mechanism-based drugs to target the virus processing. repurposed drugs including natural flavonoids and bioflavonoids, antimalarial, antiviral and vitamins-based compounds have previously been shown to be beneficial in several viral infections and outbreaks. the novel data generated from this study enhances our knowledge of the fine molecular differences that differentiate sars-cov-2 virus sars-cov. it also highlights the emerging variations in the viral genome across different populations as the virus evolves to local genetic and environmental factors. these findings will likely play a key role in the development of mechanism-based and targeted therapeutic strategies to treat sars-cov-2 infection and reduce its virulence. supplementary information accompanies this paper at https ://doi. org/10.1186/s1296 7-020-02448 -z. additional file 1: table s1 . acknowledgement table containing information about authors, originating, and submitting laboratories of the sequences deposited to gisaid database. additional file 2: table s2 . sars-cov and sars-cov-2 sequence alignment of 3clpro shares around 95% similarity. note from the editors: world health organization 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middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega alpha-1-proteinase inhibitor is a heparin binding serpin: molecular interactions with the lys rich cluster of helix-f domain vcf2poptree: a client-side software to construct population phylogeny from genome-wide snps interactive tree of life (itol) v4: recent updates and new developments interactive tree of life (itol): an online tool for phylogenetic tree display and annotation interactive tree of life v2: online annotation and display of phylogenetic trees made easy interactive tree of life (itol) v3: an online tool for the display and annotation of phylogenetic and other trees the protein data bank ucsf chimera-a visualization system for exploratory research and analysis molecular determinants and interaction data of cyclic peptide inhibitor with the extracellular domain of trkb receptor pubchem 2019 update: improved access to chemical data exploring the molecular interactions of 7,8-dihydroxyflavone and its derivatives with trkb and vegfr2 proteins computational analysis unravels novel destructive single nucleotide polymorphisms in the non-synonymous region of human caveolin gene open babel: an open chemical toolbox molecular docking, dynamics, and pharmacology studies on bexarotene as an agonist of ligand-activated transcription factors, retinoid x receptors 18:278 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over ready to submit your research ? choose bmc and benefit from investigating the function of single nucleotide polymorphisms in the ctsb gene: a computational approach autodock4 and autodocktools4: automated docking with selective receptor flexibility new molecular scaffolds for the design of alzheimer's acetylcholinesterase inhibitors identified using ligand-and receptor-based virtual screening a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant 3c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism sars-cov 3cl protease cleaves its c-terminal autoprocessing site by novel subsite cooperativity biflavonoids from torreya nucifera displaying sars-cov 3cl(pro) inhibition the antiviral activities of artemisinin and artesunate a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 potential interventions for novel coronavirus in china: a systematic review induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths does vitamin d status impact mortality from sars-cov-2 infection? a novel coronavirus from patients with pneumonia in china sars and other coronaviruses as causes of pneumonia coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia structural insights into coronavirus entry covid-2019: the role of the nsp2 and nsp3 in its pathogenesis a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease-19 treatment option a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 arguments in favour of remdesivir for treating sars-cov-2 infections outbreak of covid-19 infection in children: fear and serenity covid-19: a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression phosphorylated grb14 is an endogenous inhibitor of retinal protein tyrosine phosphatase 1b, and light-dependent activation of src phosphorylates grb14 brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3beta (gsk3beta) signalling nextstrain: real-time tracking of pathogen evolution publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. nc and vkg designed and conducted the research. rr, ag, ts and kp helped in data collection and analysis. nc and vkg wrote the paper. vg, db, mm, yy page 14 of 15 chitranshi et al. j transl med (2020) 18:278 and slg supervised the data analysis and contributed to scientific discussion. all authors read and approved the final manuscript. no funding was used to conduct this research.availability of data and materials gisaid database (https ://www.gisai d.org/), sars-cov-2 isolate wuhan-hu-1, complete genome (https ://www.ncbi.nlm.nih.gov/nucco re/17981 74254 )ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. received: 8 may 2020 accepted: 6 july 2020 key: cord-345929-z7yfegr5 authors: thakur, suman s. title: proteomics and its application in pandemic diseases date: 2020-11-06 journal: j proteome res doi: 10.1021/acs.jproteome.0c00824 sha: doc_id: 345929 cord_uid: z7yfegr5 nan using in silico studies, barros et al. found that the antimalarial drug metaquine and anti-hiv antiretroviral saquinavir interact with four sars-cov-2 receptors, including nsp9 replicase, main protease (mpro), nsp15 endoribonuclease, and spike protein (s protein), interacting with human ace2; therefore, they may be repurposed for covid-19 treatment. the bioinformatics analysis of bcg antigens by glisic et al. suggested that four bacterial proteins, rv0934, rv3763, rv3875, and rv2997, have similar properties as the s1 protein of sars-cov-2; therefore, they might be effective against sars-cov-2. a molecular docking and dynamics simulation analysis suggested that noscapine binds with the main protease of sars-cov-2 and produces conformational changes (kumar et al.) . furthermore, maffucci and contini used an in silico approach to find drug candidates against the main proteinase and spike protein of sars-cov-2. this led to the finding that indinavir, polymyxin b, daptomycin, terlipressin, and thymopentin can be repurposed against the sars-cov-2 infection. interestingly, the studies by stamatakis et al. suggested that the antigenic peptides generated from the s1 spike glycoprotein of sars-cov-2 using aminopeptidases erap1, erap2, and irap might be helpful in selecting better epitopes for immunogenic studies and the design of a vaccine for covid-19. metabolites in sars-cov-2 patients were analyzed by nmr and mass spectrometry with the observations of an increase in the α-1-acid glycoprotein, an increased kynurenine/tryptophan ratio, low total and hdl apolipoprotein a1, low hdl triglycerides, high ldl and vldl triglycerides, elevated glutamine/glutamate, and fischer's ratios that are consistent with diabetes, coronary artery disease, and liver dysfunction risk (kimhofer et al.) . by using nmr spectroscopy, loo et al. reported that inactivation by heat causes the degradation of lipoproteins and changes in various metabolic information in sars-cov-2-infected plasma samples. saunders et al. described the coronavirus-specific web portal (https:// metatryp-coronavirus.whoi.edu/) in metatry v2.0 that can be used for coronavirus proteomics research. this web portal is helpful for finding peptide biomarkers and specific taxonomic groups. published: november 6, 2020 rosa-fernandes et al. reported a method to study the ocular surface proteome, especially for infants exposed to the zika virus during the gestation period without early clinical symptoms, and named it the cellular imprinting proteomic assay (cimpa). in the conjunctival epithelium of infants exposed to the zika virus, neutrophil, eosinophil infiltration, and degranulation were detected by this proteomic assay. furthermore, virus-like particles (vlps) have been applied in vaccine therapies. lavado-garcı́a et al. used quantitative proteomics to identify the changes in the secretome of vlps and the coproduction of extracellular vesicles (evs) under different conditions, including nontransfected and transfected with and without plasmid coding for hiv-1 gag polyprotein. interestingly, a computational method was used to find an allosteric site on the sars-cov-2 spike protein by di paola et al., as its detection would weaken the spike−ace2 interaction and thereby reduce the viral infection. another study by verkhivker suggests that the spike protein of sars-cov-2 may function as an allosteric regulatory engine fluctuating between dynamic functional states. hadi-alijanvand and rouhani reported the higher binding affinity of the closed state of ace2 for the s1 protein of sars-cov-2 compared with the open state of ace2 by using a computational approach. furthermore, nadeau et al. used a computational approach to study the sars-cov-2-interacting human proteins using the gonet algorithm. using a ferret model for the h1n1 2009 pandemic influenza a virus, chen et al. reported the host glycomic response and its age-dependent severity. they suggested that a high level of mannose may be related to the severity of the influenza a virus due to the overactive innate immune system. in the case of the ebola virus, banerjee and mitra proposed the tetrameric assembly model to the vp35 protein and suggested that the cterminal of vp35 interacts with human protein kinase r to stop its autophosphorylation. this special issue provides a platform to understand pandemic diseases. here several topics have been encouraged that connect proteomics and pandemic diseases, including proteomic technologies, biomarker discovery, pathogenesis, mechanistic details of proteins, protein−protein interactions, signaling pathways, post-translational modifications, computational proteomics, prevention and vaccination, drug repurposing, and therapeutic agents and their mode of action. it will be thrilling to find the bridge between proteomics and pandemic diseases, especially for covid-19. proteomics and informatics for understanding phases and identifying biomarkers in covid-19 disease computational immune proteomics approach to target covid-19 techniques and strategies for potential protein target discovery and active pharmaceutical molecule screening in a pandemic utility of proteomics in emerging and re-emerging infectious diseases caused by rna viruses sars-cov-2-encoded proteome and human genetics: from interaction-based to ribosomal biology impact on disease and risk processes molecular basis of sars-cov-2 infection and rational design of potential antiviral agents: modeling and simulation approaches targeting proteases for treating covid-19 a chain only as strong as its weakest link": an up-to-date literature review on the bidirectional interaction of pulmonary fibrosis and covid-19 rapid response to pandemic threats: immunogenic epitope detection of pandemic pathogens for diagnostics and vaccine development using peptide microarrays a novel strategy for the development of vaccines for sars-cov-2 (covid-19) and other viruses using ai and viral shell disorder the covid-19 pandemic from a human genetic perspective perspective on proteomics for virus detection in clinical samples mass-spectrometric detection of sars-cov-2 virus in scrapings of the epithelium of the nasopharynx of infected patients via nucleocapsid n serum proteomics in covid-19 patients: altered coagulation and complement status as a function of il-6 level quantitative in-vitro diagnostic nmr spectroscopy for lipoprotein and metabolite measurements in plasma and serum: recommendations for analytical artefact minimization with special reference to covid-19/sars-cov-2 samples evidence of structural protein damage and membrane lipid remodeling in red blood cells from covid-19 patients quantitative proteomic analysis of porcine intestinal epithelial cells infected with porcine deltacoronavirus using itraq-coupled lc-ms/ms age-dependent glycomic response to the 2009 pandemic h1n1 influenza virus and its association with disease severity cellular imprinting proteomics assay: a novel method for detection of neural and ocular disorders applied to congenital zika virus syndrome ebola virus vp35 protein: modeling of the tetrameric structure and an analysis of its interaction with human pkr shell disorder analysis suggests that pangolins offered a window for a silent spread of an attenuated sars-cov-2 precursor among humans computational identification of human biological processes and protein sequence motifs putatively targeted by sars-cov-2 proteins using protein−protein interaction networks interaction of drug candidates with various sars-cov-2 receptors: an in silico study to combat covid-19 the discovery of a putative allosteric site in the sars-cov-2 spike protein using an integrated structural/ dynamic approach molecular simulations and network modeling reveal an allosteric signaling in the sars-cov-2 studying the effects of ace2 mutations on the stability, dynamics, and dissociation process of sars-cov-2 s1/hace2 complexes maffucci, i.; contini, a. in silico drug repurposing for sars-cov-2 main proteinase and spike proteins biological rationale for the repurposing of bcg vaccine against sars-cov-2 deciphering the structural enigma of hla class-ii binding peptides for enhanced immunoinformatics-based prediction of vaccine epitopes repurposing of fda-approved toremifene to treat covid-19 by blocking the spike glycoprotein and nsp14 of sars-cov-2 molecular binding mechanism and pharmacology comparative analysis of noscapine for repurposing against sars-cov-2 protease nucleotide analogues as inhibitors of sars-cov-2 polymerase, a key drug target for covid-19 structural insights into the binding modes of viral rna-dependent rna polymerases using a function-site interaction fingerprint method for rna virus drug discovery integrative modeling of quantitative plasma lipoprotein, metabolic, and amino acid data reveals a multiorgan pathological signature of sars-cov-2 infection theoretical insights into the anti-sars-cov-2 activity of chloroquine and its analogs and in silico screening of main protease inhibitors metatryp v 2.0: metaproteomic least common ancestor analysis for taxonomic inference using specialized sequence assemblies−standalone software and web servers for marine microorganisms and coronaviruses key: cord-354394-zojhdnlu authors: wang, wei-kung; chen, shey-ying; liu, i-jung; chen, yee-chun; chen, hui-ling; yang, chao-fu; chen, pei-jer; yeh, shiou-hwei; kao, chuan-liang; huang, li-min; hsueh, po-ren; wang, jann-tay; sheng, wang-hwei; fang, chi-tai; hung, chien-ching; hsieh, szu-min; su, chan-ping; chiang, wen-chu; yang, jyh-yuan; lin, jih-hui; hsieh, szu-chia; hu, hsien-ping; chiang, yu-ping; wang, jin-town; yang, pan-chyr; chang, shan-chwen title: detection of sars-associated coronavirus in throat wash and saliva in early diagnosis date: 2004-07-17 journal: emerg infect dis doi: 10.3201/eid1007.031113 sha: doc_id: 354394 cord_uid: zojhdnlu the severe acute respiratory syndrome–associated coronavirus (sars-cov) is thought to be transmitted primarily through dispersal of droplets, but little is known about the load of sars-cov in oral droplets. we examined oral specimens, including throat wash and saliva, and found large amounts of sars-cov rna in both throat wash (9.58 x 10(2) to 5.93 x 10(6) copies/ml) and saliva (7.08 x 10(3) to 6.38 x 10(8) copies/ml) from all specimens of 17 consecutive probable sars case-patients, supporting the possibility of transmission through oral droplets. immunofluorescence study showed replication of sars-cov in the cells derived from throat wash, demonstrating the possibility of developing a convenient antigen detection assay. this finding, with the high detection rate a median of 4 days after disease onset and before the development of lung lesions in four patients, suggests that throat wash and saliva should be included in sample collection guidelines for sars diagnosis. s evere acute respiratory syndrome (sars) is an emerging infectious disease that spread rapidly from china to >30 countries, including canada, singapore, vietnam, and taiwan, in the first half of 2003 (1) (2) (3) (4) (5) . in the latest update from the world health organization, the number of probable sars cases is 8,096 (5) . the etiologic agent of sars has been identified as the novel sars-associated coronavirus (sars-cov) (6) (7) (8) (9) . the disease is highly contagious and has the potential to cause a very large epidemic in the absence of control measures (10) (11) . transmission appears to occur primarily through dispersal of droplets from the respiratory tract (12) , generated when the patient talks, coughs, or sneezes (4, 5, (10) (11) (12) (13) . although large amounts of sars-cov have been reported in sputum and nasal specimens, which may account for transmission during coughing and sneezing (7, 14) , little is known about the load of sars-cov in the oral cavity and how the virus is transmitted during talking. since sneezing and rhinorrhea are not common symptoms of sars, and cough with sputum is only seen in the later stage of infection (1) (2) (3) 13) , oral droplets generated during talking may represent an important route of transmission. we examined specimens derived from the oropharynx and oral cavity, including throat wash and saliva, from 17 patients with probable sars (15, 16) . using a quantitative real-time reverse transcription-polymerase chain reaction (rt-pcr) assay and fractionation experiment, we investigated the load of sars-cov in these samples and different components of the throat wash. from april 16, 2003 , through may 1, 2003, during a 2week period of the sars outbreak in taipei (16) , 17 adult patients, who were admitted to the emergency department of the national taiwan university hospital and met the clinical case definitions for probable sars (15) , were included in this study. physicians in the sars research group of national taiwan university hospital made the detection of sars-associated coronavirus in throat wash and saliva in early diagnosis diagnosis for each patient after thorough evaluation of their travel or contact history; symptoms; laboratory data including lymphopenia, thrombocytopenia, and elevated levels of lactate dehydrogenase or creatine kinase; and pneumonic patch in the chest x-ray. the first day of fever was defined as day 1 of illness. the serologic test of an indirect immunofluorescence assay performed on serum specimens collected 28 days after onset confirmed sars-cov infection in 13 of the 17 patients. the other four patients had at least two positive real-time rt-pcr results. therefore, all 17 cases with probable sars in this study were confirmed by laboratory tests (15) . with the patient's consent, saliva and throat wash (by gargling 10 ml normal saline) were collected in an airborne isolation room, according to the guidelines for aerosol-generating procedures (17) . all samples were transferred to the biosafety level 3 (bsl3) laboratory and stored at -80°c until use (18) . after thawing, 5 ml of the throat wash was centrifuged at 1,500 x g for 15 min to separate the supernatant from the mucous-cell pellet. four milliliters of the supernatant were collected as the throat wash supernatant. the remaining 1-ml portion that contained the mucous-cell pellet was treated with equal volume of n-acetyl-l-cysteine at room temperature for 25 min and centrifuged at 1,500 x g for 15 min to further separate the cell pellet from the supernatant, of which 1.12 ml was collected as the treated supernatant of throat wash. instead of extensively washing the potentially contagious cell pellet, we kept the remaining 0.88 ml as the cellular fraction of throat wash. equal amounts of the supernatant, treated supernatant, and cellular fractions were subjected to viral rna extraction. an aliquot of the saliva, to which an equal volume of 1 x phosphate-buffered saline (pbs) was added, was also subjected to viral rna extraction. viral rna was isolated from aliquots of saliva and different fractions of throat wash from the 17 probable sars patients and 12 healthy controls by using the qiaamp viral rna mini kit (qiagen, hilden, germany) in the bsl3 laboratory (18) . viral rna was also isolated from culture supernatants of the sars-cov isolate, tw1 (19) , human coronavirus 229e strain, and human enteric coronavirus dallas 1 strain (american type culture collection, manassas, va). the assay used forward and reverse primers and a fluorogenic probe of the sar1s_as taqman assay design (applied biosystems, foster city, ca). they matched to a region within a previously described region of the orf1b (6, 7) , which is also completely conserved by different isolates of sars-cov ( figure 1a ) (20, 21) . the sequences of the forward primer, reverse primers, and probe are 5′-cacaccgtttctacaggttagct-3′ (genome positions 15316 to 15338 of the urbani strain) (20) , 5′-gccacacatgaccatctcacttaat-3′ (positions 15380 to 15356) and 5′-acggttgcgcacactcggt-3′ (positions 15355 to 15339), respectively. a 200-bp product covering this region was generated by using the primers (f1 and r1), the superscript ii one-step rt-pcr system (invitrogen, san diego, ca), and the rna template derived from the sars-cov tw1 strain (19) . the sequences of the primers f1 and r1 are 5′-cagagccat-gcctaacatgc-3′ (genome positions 15239 to 15258) (20) and 5′-gcataagcagttgtagcatc-3′ (positions 15439 to 15420), respectively. rt-pcr conditions were 52°c for 40 min and 94°c for 2 min, followed by 35 cycles of 94°c for 1 min, 60°c for 1 min, and 68°c for 45 s. the (20) . (b) a schematic diagram of the construct, orf1b/pcrii-topo, and the protocol for generating the in vitro transcribed rna as the standard for the real-time rt-pcr assay is shown. the relationship between known input rna copies to the threshold cycle (ct) is shown at the bottom. product was subsequently cloned into the ta cloning vector (invitrogen, san diego, ca) to generate the construct, orf1b/pcrii-topo ( figure 1b ). the in vitro transcribed rna was purified and quantified to determine the copy number of rna as described previously (22) . an aliquot (5 µl) of rna isolated from the clinical sample and known amounts of the in vitro transcribed rna (5 to 50 million copies) were subjected to real-time rt-pcr by using the sar1s_as primers, probe, and the taqman onestep real-time rt-pcr master mix reagent kit (applied biosystems). the amplification conditions were 48°c for 30 min and 95°c for 10 min, followed by 45 cycles of 95°c for 15 s and 60°c for 1 min. the abi prism 7000 sequence detector was used to analyze the emitted fluorescence during amplification. a positive result is defined by the cycle number (ct value) required to reach the threshold as described previously (22) . precautions for pcr were followed to avoid contamination (23) . since 5 µl of 50 µl rna eluates that were derived from 560 µl throat wash supernatant, was used in each reaction, the number of sars-cov rna copies per reaction was divided by 56 µl (560 µl x 5 µl/50 µl) and multiplied by 1,000 to determine the rna copies per milliliter. the sensitivity of the assay is 5 copies rna per reaction, corresponding to 90 copies per milliliter throat wash. we used the following formula to calculate the copy numbers of sars-cov rna in different components including the supernatant (s), the mucus-associated (m), and the cell-associated (c) components in the 5-ml throat wash, which was the starting volume in our fractionation experiment. the numbers of rna copies in the s component equal the amount (copies/ml) in the supernatant times 5 (ml) (s = supernatant x 5 ml). since treatment of the mucus-cell pellet with n-acetyl-l-cysteine presumably released sars-cov from the mucus and increased the volume twofold (from 1 ml to 2 ml), the copy numbers in the m component equal the amount in the treated supernatant (copies/ml) times 2 ml minus that from the originally untreated supernatant (1 ml) (m = treated supernatant x 2 ml -supernatant x 1 ml). the copy numbers in the c component equal the amount in the cellular fraction (copies/ml) times the volume of the cell pellet (c = cellular fraction x volume of cell pellet [in ml]). taking patient id17 as an example, s = 4,790 copies (958 x 5), m = 8,402 copies (4,680 x 2 -958 x 1), and c = 330 copies (8,460 x 0.039). the amount of sars-cov rna in the cell-free component, which equals the amount in the s component plus that in the m component, is 13,192 copies, corresponding to 97.5% of the total sars-cov rna in 5 ml throat wash, and that in the cell-associated component is 330 copies, corresponding to 2.5% of the total (table 1) . aliquots of the cellular fraction of throat wash from patients and six healthy controls were fixed onto 12-well teflon-coated slides and subjected to a previously described immunofluorescence assay (19) . the first antibody was serum from a rabbit immunized with the recombinant nucleocapsid protein of the sars-cov (prepared by p.j. chen), and the secondary antibody was the fitcconjugated goat anti-rabbit immunoglobulin g (pierce biotechnology, rockford, il). regression analysis was performed to examine the correlation between the sampling day and the amount of sars-cov rna in throat wash or saliva and the correlation between the amount in throat wash and saliva (software spss base 10.0, spss inc., chicago, il). the demographic and clinical information of the 17 patients are summarized in table 2 . viral rna was extracted from saliva and supernatant of the throat wash and then subjected to a quantitative real-time rt-pcr assay by using the primers and probe within a highly conserved region of the orf1b ( figure 1a ) (20, 21) . known amounts of the in vitro transcribed rna covering this region were used as the standard for quantification. as shown in figure 1b , a linear curve was observed as the input rna increased from 5 to 50 million copies per reaction. positive signal was detected in the reactions containing rna template derived from the sars-cov taiwanese strain tw1 but not in those from 12 healthy controls and from two human coronaviruses (229e strain and human enteric coronavirus dallas 1 strain) and not in the reaction containing no rna (data not shown) (19, 22) . the results of the real-time rt-pcr assay on the throat wash and saliva are summarized in table 2 . the sampling day of these patients varied from day 2 to day 9 after onset of fever, with a median of day 4. sars-cov rna was readily detected in throat wash from all 17 patients. the amount of the sars-cov rna in the throat wash was 9.58 x 10 2 to 5.93 x 10 6 copies per ml (median 3.56 x 10 3 copies/ml). sars-cov rna was also detected in saliva from all 14 available specimens. the amount of sars-cov rna in the saliva was 7.08 x 10 3 to 6.38 x 10 8 copies per ml (median 9.92 x 10 4 copies/ml). the amount of sars-cov rna in the throat wash or saliva does not correlate with the sampling day (simple linear regression, coefficient of correlation r = 0.106 and 0.147, respectively), underscoring a more complex course of virus-host interaction. the amount of sars-cov rna in the saliva was greater than that in the throat wash for every patient from whom both type of specimens were available. a linear relationship existed between the amounts of sars-cov in the saliva and throat wash (simple linear regression, r = 0.848, p < 0.005), which suggests, but does not prove, that they could originate from a common source in the respiratory tract. to further investigate whether sars-cov is also present in the cellular component of the throat wash, we carried out the fractionation experiment and examined the amount of sars-cov rna in different components. as shown in table 1 , sars-cov rna was detected in the cell-associated component of the throat wash from all 16 specimens examined. the range of viral load was 34-4222,000 copies per 5 ml throat wash. while 0.1%-11.2% of sars-cov rna in the throat wash is present in the cell-associated component, a greater proportion of sars-cov rna, 88.8% to 99.9%, is present in the cell-free component. this finding suggests that sars-cov is released very efficiently. the possibility that virus is released from the cells during thawing is unlikely, since the fractionation experiment performed for aliquots of some samples without prior freezing and thawing showed a similar result. for example, in id7 the percentages of the cell-associated and cell-free components for an aliquot performed without freezing were 99.87% and 0.13%, respectively. these are similar to the results of 99.9% and 0.1% for another aliquot performed after freezing and thawing (table 1) . electron microscopic studies have shown sars-cov particles in the desquamated cells from bronchoalveolar lavage and lung tissues, both in the lower respiratory tract (6, 8, 24) . our detection of sars-cov rna in the cellassociated component of the throat wash suggested that sars-cov also replicates in the upper respiratory tract. to further explore this possibility, we prepared spot slides from the cellular fraction of the throat wash and examined them with an indirect immunofluorescence assay by using a polyclonal serum from a rabbit immunized with a recombinant nucleocapsid protein of sars-cov. when used in the epithelial cells prepared from two randomly chosen specimens (id11 and id17), the postimmune serum, but not the preimmune serum, reacted with epithelial cells with a speckle pattern (figure 2, c-d and f-g) . the classification of these cells as epithelial cells was supported by the size and morphologic features of them under light microscope ( figure 2h ). only background signal was seen in the cells prepared from a healthy control ( figure 2e ). these findings indicate that sars-cov can replicate in the epithelial cells of the upper respiratory tract, and such cells can be used in an antigen detection assay. we report large amounts of sars-cov rna in the throat wash and saliva from probable sars case-patients. this finding supports the possibility that sars-cov can be transmitted through oral droplets. most coronaviruses are known to replicate in the epithelial cells of the respiratory or enteric tract. after budding into the pre-golgi compartment, virus particles are released through an exocytosis-like process at the apical or basolateral surface, or both (25) . apical release is likely to facilitate the spread of virus in the respiratory or enteric tract, whereas basolateral release facilitates systemic spread. our findings that sars-cov can be detected in cells derived from throat wash by the immunofluorescence assay and that most of the sars-cov in throat wash is present in the cell-free component suggest that after its replication in the epithelial cells, sars-cov is released efficiently and accumulates in the oropharynx and oral cavity, which may contribute to its transmission through oral droplets. practicing droplet and contact precautions prevents nosocomial transmission of sars among healthcare workers (26) . however, a cluster of sars cases was reported among apparently protected healthcare workers during aerosol-generating procedures performed on sars patients (27) . this finding led to the controversial hypothesis of airborne transmission of sars, in which very small particles (<5 µm) are spread in the air (17, 27) . a substantial proportion, 88.8 % to 99.9%, of the sars-cov in the throat wash was present in cell-free form; this finding offers a mechanistic explanation of the possibility of airborne transmission of sars. three types of specimens from the upper respiratory tract, nasopharyngeal aspirates, nasopharyngeal swab, and oropharyngeal swab have been recommended to detect sars-cov (28, 29) . however, rt-pcr performed on nasopharyngeal aspirates from sars patients had positive rates of 32% at day 3, 50% at day 5, and 68% at day 14 (8, 14) . a recent study using nasopharyngeal aspirates reported a positive rate of 71% at a mean of 4.4 days (30) . in this study, we reported that sars-cov rna can be detected in both throat wash and saliva from all specimens examined at an average sampling day 4.8 (range day 2-9). furthermore, specimens of throat wash from four of our study participants who came to our emergency department, a designated sars screening site in taipei during the sars outbreak, were collected when radiographic evidence of pneumonia or respiratory distress syndrome had not been observed (table 2) . to our knowledge, this report is the first showing that sars-cov could be detected in probable sars patients before lung lesions developed. the high sars-cov detection rate in our study contrasts with those reported previously by using nasopharyngeal aspirates (8, 14, 30) . one possibility is that more sars-cov are present in the oropharynx and oral cavity than in the nasopharynx. in spite of the differences in the dilutional factors (for example, 10 ml of normal saline in the throat wash, 1.5-2 ml in the nasopharyngeal aspirates, and none in the saliva), the amounts of sars-cov rna in the throat wash and saliva in our study, 9.58 x 10 2 to 6.38 x 10 8 copies per ml, were in the same range as those previously reported for the nasopharyngeal specimens (10 3 -10 8 copies/ml) (7, 14) . a mutually nonexclusive possibility is that more respiratory secretions, mucus, and cells can be removed from the respiratory tract through throat wash than through nasopharyngeal aspiration, nasopharyngeal swabs, and oropharyngeal swabs. regardless of the reason, sars-cov was also detected in the throat wash of nine sars patients in a previous report (6) , which suggests the need to evaluate the benefit of collecting throat wash to diagnose sars. to our knowledge, this report is the first that demonstrates the possibility of devising a sars-cov antigen detection assay by using cells derived from throat wash. since a small number of patients were examined, future study with more patients and controls is required to develop a useful diagnostic test. technically, throat wash and saliva are easier to collect when compared with the collection of currently recommended respiratory specimens (28, 29) . in addition, they can be obtained without close contact between the patient and healthcare worker, and thus reduce the risk for infection of healthcare workers. another commonly obtained sample is sputum; however, it is rarely available at the early stage of infection, when virtually no cough or only dry cough is present (1-3,13) . these features, together with the high detection rate at early stage and before the development of lung lesions, suggest that throat wash and saliva are ideal specimens for early diagnosis of sars and should be included in guidelines for sample collection for sars diagnosis (28, 29) . further studies with longitudinally collected throat wash and saliva specimens from a larger number of sars patients would help determine the onset and duration of infectiveness, extent of infectiveness of some patients, such as superspreaders, and the response to antiviral agents. a cluster of cases of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada update: outbreak of severe acute respiratory syndrome-worldwide world health organization. summary of probable sars cases with onset of illness from 1 a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions transmission dynamics and control of the severe acute respiratory syndrome world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study world health organization. case definitions for surveillance of sars severe acute respiratory syndrome-taiwan interim domestic infection control precautions for aerosol-generating procedures on patients with severe acute respiratory syndrome (sars) interim laboratory biosafety guidelines for handling and processing specimens associated with severe acute respiratory syndrome (sars) microbiologic characterization, serologic responses, and clinical manifestations in severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time pcr assay avoiding false positive with pcr lung pathology of fatal severe acute respiratory syndrome coronaviridae: the virus and their replication effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) cluster of severe acute respiratory syndrome cases among protected health-care workers-toronto, canada sampling for severe acute respiratory syndrome (sars) diagnostic tests coronavirus-positive nasopharyngeal aspirate as predictor for severe acute respiratory syndrome mortality we are indebted to all the medical personnel at the national taiwan university hospital for taking care of sars patients during the outbreak in taipei and members of the sars research group of the national taiwan university college of medicine/national taiwan university hospital, including ding-shinn chen, yuan-teh lee, hong-nerng ho, chu-min teng, ming-fu chang, bor-liang chiang for discussion and coordination, tun-hou lee at the harvard school of public health for critical comments, and chao-min lee for assistance.applied biosystems taiwan inc. provided an abi prism 7000 sequence detector during the outbreak. this work was supported by the national science council (nsc92-2751-b-002-006-y), taiwan.dr. wei-kung wang is an associate professor of virology at the institute of microbiology, college of medicine, national taiwan university. his research interests include study of viral load, immune activation markers, and the pathogenesis of viral infectious diseases, such as dengue virus and sars-cov. key: cord-322957-clf8f90t authors: crespo, javier; andrade, raúl; parras, fernando alberca de las; balaguer, francesc; acosta, manuel barreiro-de; bujanda, luís; gutiérrez, ana; jorquera, francisco; iglesias-garcía, julio; sánchez-yagüe, andrés; calleja, josé luis title: resumption of activity in gastroenterology departments. recommendations by sepd, aeeh, geteccu and aeg date: 2020-04-28 journal: nan doi: 10.1016/j.gastre.2020.04.001 sha: doc_id: 322957 cord_uid: clf8f90t abstract the set of measures proposed by sepd, aeeh, geteccu and aeg are aimed to help departments in their resumption of usual activity. we have prepared a number of practical recommendations regarding patient management and the stepwise resumption of healthcare activity. these recommendations are based on the sparse, changing evidence available, and will be updated in the future according to daily needs and the availability of expendable materials to suit them; in each department they will be implemented depending upon the cumulative incidence of sars-cov-2 infection in each region, and the burden the pandemic has represented for each hospital. the general objectives of these recommendations include: • to protect our patients against the risks of infection with sars-cov-2 and to provide them with high-quality care. • to protect all healthcare professionals against the risks of infection with sars-cov-2. • to resume normal functioning of our departments in a setting of ongoing risk for infection with sars-cov-2. 3 each region, and the burden the pandemic has represented for each hospital. the general objectives of these recommendations include: • to protect our patients against the risks of infection with sars-cov-2 and to provide them with high-quality care. • to protect all healthcare professionals against the risks of infection with sars-cov-2. • to resume normal functioning of our departments in a setting of ongoing risk for infection with sars-cov-2. keywords: sars-cov2. gastroenterology departments. recommendations. resumption. restablecimiento de la actividad en los servicios de digestivo. recomendaciones de la sepd, aeeh, geteccu y aeg el artículo recoge el conjunto de medidas propuestas por la sepd, la aeeh, geteccu y la aeg que pretenden servir de ayuda a los servicios en su reincorporación a la actividad habitual. hemos confeccionado una serie de recomendaciones prácticas respecto al manejo y a la reintroducción progresiva de la actividad asistencial. estas recomendaciones están guiadas por la escasa y cambiante evidencia disponible y serán objeto de futuras actualizaciones, en base a las necesidades diarias y la disponibilidad del material fungible para adecuarse a las mismas; y se podrán implementar en cada servicio en función de la incidencia acumulada de sars-cov-2 en cada región y de la carga que la epidemia ha ocasionado en cada uno de los hospitales. los objetivos generales de estas recomendaciones son: • proteger a nuestros pacientes de los riesgos de la infección por sars-cov-2 y prestarles una atención de calidad. • recuperar el normal funcionamiento de nuestros servicios en un entorno de riesgo continuado de infección por sars-cov-2. palabras clave: sars-cov-2. servicios de digestivo. recomendaciones. restablecimiento. infection with the sars-cov-2 coronavirus and its potentially resulting disease, designated covid-19, is causing significant concern among the general population, and -needless to say-healthcare professionals and patients (1, 2) . in this regard, it has had a highly significant impact on our gastroenterology and hepatology departments, which have reduced both their hospitalization activity (by more than 50 %) and the number of diagnostic/therapeutic endoscopic procedures (by more than 50 %, unpublished data). besides affecting our activity, it also affected our work, with high numbers of gastroenterologists being moved to covid areas. finally, some -in fact many-of our colleagues have fallen ill as a consequence of caring for patients infected with sars-cov-2. let us not forget that some of the procedures we carry out on a daily basis are associated with a high risk for covid-19 transmission (3) (4) (5) . even if its incidence diminishes considerably, it will stay with us over the coming months, which should prompt us to take extreme precautions in a micro-environment with a high risk for coronavirus transmission as is the case with hospitals. times of crisis are usually accompanied by opportunities or else appropriate to reformulate activities and the way they are performed. in this crisis we had to respond to the exigencies of covid-19, but must also carry on providing essential care as defined within our specialty. because of this, this document also reflects on the opportunity to incorporate telemedicine into our usual practice in order to enhance page 5 of 47 j o u r n a l p r e -p r o o f 5 the care we provide to our chronic patients. since the present situation lacks consistency (different autonomous communities, hospitals, sars-cov-2 incidences, public/private centers, etc.), the right time to implement these recommendations may vary. be it as it may, we propose that the transition from the current state of alarm, which has brought activity in our departments to a virtually complete standstill, to a more normal situation be accomplished in three phases: activity resumption phase, stabilization phase, and normalization phase. the length of these phases is difficult to foretell in such dynamic, highly changing scenario, but will not foreseeably be shorter than 2-4 months. furthermore, when will the human and space resources redeployed to caring for covid-19 patients be recovered by our departments remains yet unknown. the set of measures proposed by sepd, aeeh, geteccu and aeg are aimed to help departments in their resumption of usual activity. we have prepared a number of practical recommendations regarding patient management and the stepwise resumption of healthcare activity. these recommendations are based on the sparse, changing evidence available, and will be updated in the future according to daily needs and the availability of expendable materials to suit them; in each department they will be implemented depending upon the cumulative incidence of sars-cov-2 infection in each region, and the burden the pandemic has represented for each hospital. the general objectives of these recommendations include:  to protect our patients against the risks of infection with sars-cov-2 and to provide them with high-quality care. 7 footwear (if possible, specific for hospital use; otherwise, with shoe covers). in addition to providing protection, these measures will prevent care providers from serving as vectors for transmission in and out of hospitals. c. all healthcare personnel with respiratory symptoms and/or fever and/or suspicion of recent contact with someone infected with sars-cov-2 must report it at the earliest possible time to the head of their department. under no circumstances whatsoever must they go to their workplace in case of suspicion. i. wherever possible, pharmacy departments shall facilitate drug dispensation for longer periods, even home delivery, as is now the case with some hospitals. resuming activity should lead us to pender over the structure of our traditional consultation schedules. the use of telematic tools (consultations over the phone, video calls, other) should be promoted both for patient care and work meetings, as it significantly decreases exposure for both patients and care providers (9) . objectives will be dependent on the current phase: in the first phase the primary goal is to reduce the risk for sars-cov-2 contagion among patients and professionals. in the second phase, and most particularly in the normalization phase, objectives will include: a) reducing non-value-added, on-site care (reporting normal results, further prescribing the same therapy, ordering supplementary examinations, etc.); b) facilitating care for patients unable to attend for work reasons; and c) reducing usual overcrowding in our clinics. some of the requirements telemedicine must meet are as follows (10, 11) :  telemedicine should be considered for all intents and purposes a medical act. this type of visit must be included in electronic records and appear in the agenda as an "off-site" visit. the electronic medical record may include screen captures of the prescriptions. having contact information available is important so that instructions and prescriptions may be mailed in writing and a follow-up strategy may be established.  ideally, telematic visits should be interspersed among in-person appointments in order to prolong intervals between the latter, thus reducing the number of people in waiting rooms.  appropriate coordination should be sought with primary care centers. we suggest appointing a department coordinator for each primary care center, who should currently favor telematic visits rather than referrals. a. differentiated circuits must be maintained for patients with and without covid-19. b. all admissions other than those strictly necessary should be avoided. c. in all patients admitted to hospital infection with sars-cov-2 must be ruled out regardless of symptoms. pcr is currently the most suitable technique but each hospital should follow their own previously approved protocol. d. since the risk of community transmission still lingers on, further rapid testing for sars-cov-2 at 10-14 days after admission is advisable to minimize the risk for in-hospital outbreaks. similarly, patients discharged after more than [10] [11] [12] [13] [14] days in hospital must be tested to prevent community outbreaks. e. for patients who remain hospitalized: -only one adequately equipped physician shall enter the room. stethoscopes and any other non-expendable materials coming in contact with patients will be subsequently cleaned with hydroalcoholic solution (or a disinfectant). -attempts should be made to monitor patients using telematic or telephony devices. -limit to a minimum all testing involving patient transportation within hospitals. -invasive procedures such as placement of nasogastric or bladder tubes should be avoided whenever possible, as well as ordering excessive lab tests. -foster early discharge and home hospitalization. f. the number of visits to inpatients must be minimized; this is particularly relevant for immunosuppressed patients. in no case should patients be accompanied by more than one person at a time. g. it is advisable that patients visiting day hospitals to receive intravenous medications be tested for temperature before entering the facility, and that infusion chairs be at least 2 meters away from each other. turns should be established if this is unfeasible. chairs and rooms should be adequately cleaned after infusion completion (12) . it is advisable that patients attend alone whenever possible. h. in case of day-hospital overcrowding efforts will be made to prescribe drugs with subcutaneous formulation. once the confinement phase is over, massive screening will likely be a most effective measure to gain insight into the population's immune status regarding sars-cov-2 infection. knowledge of this immune status will be particularly relevant in areas with high infection rates and, above all, among those who provide care for the rest of citizens. because of this we recommend: a. regular, universal screening of professionals. such screening will reveal immunization level, and will likely help establish the risk run by care providers, a key aspect for the management of a potential recurrence of the pandemic. b. universal screening of all patients who must undergo examinations involving sars-cov-2 infection transmission risks. c. systematic screening for sars-cov-2 among particularly vulnerable patients. ideally, such screening should be primarily offered to: i. patients on biologics or immunosuppressants: -inflammatory bowel disease. -autoimmune liver disease. -other. ii. patients with immunosuppression secondary to their underlying disease: -compensated and, most particularly, decompensated liver cirrhosis. iii. patients with liver cancer. iv. transplant recipients. two important considerations apply regarding the above screening:  obviously, a systematic screening of all these populations cannot be carried out simultaneously, hence we recommend setting up local screening plans.  screening is for the asymptomatic population; should a patient present with symptoms suggestive of infection with sars-cov-2, he or she should be diagnosed (pcr and/or serologic tests and/or chest x-rays and/or chest ct scan); in no case should the patient undergo an elective dianostic test. in our view, the best screening strategy should be established at any given time depending on the availability of rapid antibody tests and/or pcr and/or serological techniques such as elisa, as well as on endemic evolution.  interpretation of results obtained in asymptomatic patients without previous close contacts. -igg negative, igm negative. individual with no prior exposure to sars-cov-2. -igm positive, igg negative or positive. recent, potentially active infection. a pcr shall be made to rule out active infection.  activity shall be immediately resumed (first phase) for patients with urgent or preferential indications, and/or for undelayable therapeutic procedures.  the rate of activity resumption shall depend on local characteristics, both regarding pandemic incidence and healthcare personnel/ancillary staff availability.  transient elastography is only exceptionally an urgent procedure.  both the patient and physician must wear surgical masks during the examination.  activity shall be gradually resumed, preferentially starting in the second phase.  high-risk examinations beause of aerosol formation.  only exceptionally urgent.  resumption shall be held off until the third phase.  should an urgent indication arise in the first phase (highly unlikely), the course of action shall be the same as for gastroscopy, including screening for sars-cov-2 and using appropriate protective equipment.  high-risk examinations beause of aerosol formation.  only exceptionally urgent.  they may be substituted for by other testing modalities (fecal antigens, commercial tests).  these tests shall not be resumed until the third phase.  portal hemodynamics is a moderate-risk study regarding sars-cov-2 transmission (position and duration).  during the first phase of activity resumption in our departments a conservative attitude is advisable, prescribing this examination only in two situations: -liver biopsy in cases of severe acute liver failure where the test may play a key role. -urgent placement of tips for intractable bleeding secondary to portal hypertension.  in the second phase it seems reasonable to perform any necessary procedures to assess portal hypertension in patients with hepatocellular carcinoma potentially amenable to surgical resection.  finally, during the third phase (normalization) all indicated procedures will be carried out the same as before the crisis. the covid-19 pandemic represents an unprecedented challenge to our health system, and as regards specifically patients with inflammatory bowel disease (ibd) multiple concerns arise in connection with their management, as many are on treatment with immunity-impairing therapies. furthermore, ibd is a condition that evolves in flares alternating with remission periods, and may have potential complications that often require urgent, or at least preferential, care. a variable proportion of patients have digestive complaints such as nausea, vomiting, bowel habit changes, or abdominal pain (13) . these symptoms are common in patients with ibd, hence the importance of excluding covid in our patients. furthermore, the virus has been reported to be present in the stools of covid-19 patients, regardless of the presence of diarrhea, and to persist there even after respiratory symptoms are over or detection in the oropharynx is no longer feasible, its significance being uncertain concerning infectivity during endoscopic procedures or potential fecal-oral transmission (14) . the first question we posed ourselves from the start of the pandemic was whether patients with ibd are at increased risk of infection. patients with ibd do not seem to have a greater risk for infection with sars-cov-2 or for development of covid-19. according to data from bergamo in lombardy, a region especially affected of italy, no patient among their 522 cases of ibd was diagnosed with, or admitted to hospital for covid-19 (15) . a possible reason explaining this lower number of cases of covid-19 in ibd patients may be this population's adherence to protective measures. another common question is whether covid-19 may cause an ibd flare-up; current evidence does not seem to support that, albeit available data are scarce and caution is here advisable (16) . finally, the next question we posed ourselves was whether suffering from ibd may condition the course of covid-19. answering this is difficult since multiple factors may play a role: age, comorbidities, inflammatory activity, and trestments received, the available information being limited about these. there is an international registry called secure-ibd (17) that aims to collect the data of patients diagnosed with ibd where covid-19 has been confirmed (positive testing) (1). at the time of writing these recommendations a total of 457 patients have been recorded, 78 of them from spain. the overall rate of hospitalization has been 30 %, and those of icu admissions, ventilation requirement, and mortality have been 4 %, 4 %, y 3 %, respectively. therefore, it seems that the course of covid-19 in patients with ibd is not worse than in the general population, but we should bear in mind that our ibd patients are younger than the general population. in this respect the fact should be highlighted that patients with moderate-high activity required icu care/ventilation or died (pooled variable) in 17 % of cases, versus 5 % for patients with remission or low activity, with 27 % of subjects with an untoward outcome being on steroids. below we include a table with a theoretical stratification of the risk for poor outcomes based on recommendations by the british society of gastroenterology (bsg) (18), although, again, the dearth of data available about the therapies used for ibd should be borne in mind (table 1) . drug half-life must be taken into account, and so patients who discontinued immunosuppressants or biologics within the last 3 months remained exposed to their effects when it comes to risk categorization. recommendations concerning the treatment of ibd are based on those issued by the international organization for the study of ibd (ioibd), bsg, and american gastroenterology association (aga) (19) , differentiating between uninfected, sars-cov-2 infected, and covid-19 patients. general recommendations regarding the treatment of patients with ibd, which must remain in force during the gradual resumption of activities: a. patients must not discontinue medication or visits to the infusion center, or start self-medication, without consulting with their doctor first. b. medication must be available at home in case an isolation period is required. c. smoking should be stopped as it increases the risk and severity of covid-19. smoking augments gene expression of angiotensin converting enzyme 2 (ace2), the receptor for viral entry (20) . a. in case of suspect symptoms and a negative sars-cov-2 pcr, the potential for false negative results should be considered, as well as a repeat test. b. treatment must be maintained to prevent non-adherence-related relapse, which may represent a higher risk of infection because of steroid or hospitalization needs. e. when planning to initiate therapy with biologics or immunosuppressants, it is advisable that sars-cov-2 testing be included in the previous assessment routine (21) . f. steroids use should be minimized. if required, rapid tapering by 10 mg/week is advisable. a. if the patient is on steroids, reduce dose to below 20 mg or switch to budesonide should the clinical scenario allow. clostridium difficile, which requires specific treatment. ibd patients already underwent specific follow-up prior to the covid-19 crisis. most ibd units have clinics or free-access mechanisms in case of flare-ups to avoid visits to the emergency room (er) (23) . two further characteristics should be considered: nursing consultation (24) and telemedicine, long implemented in our ibd units pioneered in our country by geteccu platforms such as teccu (25) . in an initial phase it is recommended that all scheduled follow-up visits take place telematically. the duration of follow-up for patients in remission according to their medication should be met or at least deviated the least possible from the standards published and accepted by geteccu (26) . in the initial phase it is recommended that patients with severe disease requiring specific physical examinations attend. in the stabilization phase patient numbers will be adjusted according to the above indications. non-scheduled consultations. during the initial and stabilization phases, aiming to avoid physical visits to hospital, patients with urgent consultations should be advised to contact the ibd unit via the nursing clinic, telephone, or email. should the issue be serious or unsolvable through telematic means a visit will be scheduled according to the above indications. infection with sars-cov-2 must be ruled out if fever and diarrhea are present. (27) . a. it is recommended that patients with severe crohn's disease or ulcerative colitis flare-ups refractory to outpatient management be admitted to hospital. also patients with subocclusion and with septic complications. b. in case of high suspicion of covid-19 despite a negative test result on admission, it is advisable to place the patient in a pre-covid-19 area and then repeat testing given the potential for false negative results (28) . e. consider reducing and facilitating bureaucratic aspects using sponsor amendments given the care burden and activity reassignments of participating physicians. probably, from a healthcare perspective, managing endoscopy units is the most challenging activity of any gastroenterology department. patient presence is mandatory, work there involves risk for both patients and care providers (and society at large), and no published or accessible protocols deal with activity resumption. on the way back to normalization, we must bear in mind that the latter does not result from overcoming the pandemic but rather a reduction in infection rate allowing to decrease hospital overload, which may in turn permit a recovery of regulated activity. therefore, one must at all times recall that the risk of infection may persist both for patients and care workers. in general, social distancing and the use of adequate personal protective equipment must be maintained. it is crucial that consideration be given to the amount of hospital resources devoted to caring for patients with covid-19, and how their recovery for the care of non-covid patients is anticipated. when opening agenda windows the hospital's contingency plan to reclaim non-covid areas as covid areas should a new peak occur has to be taken into consideration. furthermore, the number of professionals available at the unit itself and their risk of infection must also be weighed up. a key point is the need for endoscopy units to have available all the materials necessary for a potential increase in activity (at least 3 ppes, for physician, nurse, and assistant, per procedure (4 if additional staff is required: anesthetist, nurse, etc.). without this minimum of materials (defined below in the present document) no endoscopic procedure should be performed. in preparation for activity resumption we should bear in mind a number of variables: a key point is the adjustment of the appointment schedule, which depends on the duration of endoscopic procedures (table 4 ). at present the times necessary to change clothes, clean instruments and room, disinfect, avoid waiting room overcrowding, etc., should be estimated. our proposal, considering transmisison risks and the need for cleaning and/or protective measures in case of examining a high-risk patient, is as follows (29): a. phase i: attempt to reach 50 % of usual activity at the endoscopy unit. b. phase ii and iii: attempt to reach 75 % of usual activity at the endoscopy unit. in order to define intervals between procedures when examining high-risk patients at least 45 minutes should be added to the established duration according to the eficad study (table 4) . a. appointments: patients will be called up on the day before endoscopy to fill out a risk checklist, which will be done again on the day of the procedure. the ideal scenario we must doubtless pursue is the running of a sars-cov-2 test in all patients before the treatment (see above) (30, 31) . b. access to the endoscopy unit: a surgical mask and gloves (or hydroalcoholic solution for hand washing) will be placed on the patient, and temperature will be measured. c. the patient shall attend at most with one companion, who will not enter the unit unless the patient requires specific help. social distancing is key.  risk by patient type. the risk of transmission by patient type may be seen in table 5 .  risk by procedure type. two kinds of procedure must be differentiated according to their potential to generate aerosols (32): i. aerosol-generating procedures, namely those involving upper endoscopy (ercp, gastroscopy, upper echoendoscopy, upper enteroscopy); these are deemed to be high-risk. when possible, sedation must be used for all upper examinations in order to reduce the risk for aerosol formation. ii. non-aerosol generating procedures, namely those involving lower endoscopy (colonoscopy, lower enteroscopy, lower echoendoscopy) or ostomy; these are deemed to be low-risk. a. some level of protection must be used during access to the endoscopy unit, including common areas (administrative area, corridor, living area, washing area, recovery room, etc.). a mask, body protection with scrubs and overcoat, and hospital-specific footwear. continuous use of gloves would not be required but regular hand washing would. b. once in the room the protective level will vary according to the risk allotted to each patient and procedure (32, 33, 34) : -perform the endoscopic study in a hospital-designated room (usually in the surgical area). -perform the endoscopic study within the endoscopy unit. in this case, it would be advisable to designate one specific room for these patients, with the procedures being undertaken at the end of the agenda in order to allow time and resources to clean the specific room. properties, which will minimize transmission risk for any type of virus. b. channel cleaning brushes must be single-use, and plastic connections to aspiration must be disposed of. c. endoscopes must travel to cleaning areas in a closed container (e.g., plastic bag); upon entering the disinfection room they must undergo immediate manual washing before entering the automated washing system. with telematic and phone-based care, during the present pandemic we have learned that patient acceptance is very high and their response to disease has changed. patients are now more reluctant to undergo procedures unless they are absolutely necessary. hence, the possibility and/or need emerges to clear the scheduled wait list. in this context patients could be contacted before their assigned appointment by a unit physician to assess their need for the scheduled procedure according to indication and clinical status. the possibility that the procedure could be delayed because of the pandemic should be laid out for their consideration. recommendations in case of patients with stable chronic liver disease (table 6) there is no evidence that patients with stable chronic liver disease of any origin will be more susceptible to infection with sars-cov-2, even though many of them have comorbidities such as hypertension and diabetes mellitus, which are associated with greater severity, particularly in patients with advanced fat deposition disease (37 immunocompromised patients might be more susceptible to sars-cov-2 infection, even though solid evidence is lacking in this respect. however, some data suggest that immune response is a key factor in pulmonary involvement, and so immunosuppression may even be protective (39, 40, 41) . in fact, post-transplant immunosuppression has not represented a risk factor for mortality during the sars or mers c coronavirus pandemic (40 in case the lt program was interrupted and resumption is under consideration, its restart will be subjected to the availability of an adequate number of icu/ccru beds and covid-19-free hospitalization areas. the course of the pandemic shall always be taken into account.  regarding donation, we shall preferentially use excellent donors with no risk factors whatsoever for covid-19.  in case of an offer, a coordinator will interview the potential recipient over the phone to assess the presence of covid-19.  performing an rt-pcr test on nasopharyngeal exudate samples from both donor and recipient is key to rule out covid-19; also a pulmonary assessment of the recipient should be undertaken (this will be according to each transplant group). in all cases a second recipient should be ready for the procedure.  when possible, acording to each center, the candidate recipient should undergo testing for measuring antibodies (igm and igg) in capillary blood, which will supplement the information obtained with the rt-pcr. this obviously extends and complicates logistics, but is indispensable.  confirmed covid-19 cases must be excluded as donors until at least 21 days after symptom disappearance and therapy completion. cases deemed cured are described in the ont document of april 13, 2020 (42) , as well as other technical issues whose review we deem advisable. the recommendations expressed in this document are aimed at helping departments in the resumption of their usual healthcare activity, which has been almost completely postponed in some of them. we are facing a changing reality that demands considerable plasticity of all of us; a relevant part of our way of working will change, and we must play a leading role in this process. for the above recommendations we have relied on pragmatism, although the scarce, changing evidence available will require future updates. the start of this journey towards a changing normality in every department will depend on the cumulative incidence of sars-cov-2 infection in each region, as well as on the burden the pandemic has inflicted on each hospital. afectación en aparato digestivo por covid 19 clinical characteristics of coronavirus disease 2019 in china protecting health care workers during the covid-19 coronavirus outbreaklessons from taiwan's sars response substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) science covid-19: protecting health-care workers. the lancet covid-19 and italy: what next? lancet telehealth seen as a key tool to fight covid-19 global telemedicine implementation and integration within health systems to fight the covid-19 pandemic: a call to action. jmir public health surveill clinical practice update on management of inflammatory bowel disease during the covid-19 pandemic: expert commentary evidence for gastrointestinal infection of sars-cov-2. gastroenterology prolonged presence of sars-cov-2 viral rna in faecal samples uneventful course in ibd patients during sars-cov-2 outbreak in northern italy 1st interview covid-19 ecco taskforce current data secure-ibd database inflammatory-bowel-diseases-during-the-covid-19-pandemic aga clinical practice update on management of inflammatory bowel disease during the covid-19 pandemic: expert commentary smoking upregulates angiotensin-converting enzyme-2 receptor: a potential adhesion site for novel coronavirus sars-cov-2 (covid-19) viral screening before initiation of biologics in patients with inflammatory bowel disease during the covid-19 outbreak a fatal case of covid-19 pneumonia occurring in a patient with severe acute ulcerative colitis. gut epub ahead of print british society of gastroenterology consensus guidelines on the management of inflammatory bowel disease in adults second n-ecco consensus statements on the european nursing roles in caring for patients with crohn's disease or ulcerative colitis telemedicine in inflammatory bowel disease: opportunity ahead delphi consensus statement: quality indicators for inflammatory bowel disease comprehensive care units switch to adalimumab in patients with crohn's disease controlled by maintenance infliximab: prospective randomised switch trial gastrointestinal symptoms of 95 cases with sars-cov-2 infection practice of endoscopy during covid-19 pandemic: position statements of the asian pacific society for digestive endoscopy (apsde-covid statements) preventing the spread of covid-19 in digestive endoscopy during the resuming period: meticulous execution of screening procedures suggestions for infection prevention and control in digestive endoscopy during current 2019-ncov pneumonia outbreak in wuhan gastrointestinal manifestations of sars-cov-2 infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis asociación española de gastroenterología. recommendations by the sepd and aeg, both in general and on the operation of gastrointestinal endoscopy and gastroenterology units, concerning the current sars-cov-2 pandemic (march, 18). rev espanola enfermedades dig organo of soc espanola patol dig esge and esgena position statement on gastrointestinal endoscopy and the covid-19 pandemic -european society of gastrointestinal endoscopy (esge) covid-19) outbreak: what the department of endoscopy should know reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: position statement of the european society of gastrointestinal endoscopy (esge) and european society of gastroenterology nurses and associates (esgena) -update liver injury in covid-19: management and challenges cancer patients in sars-cov-2 infection: a nationwide analysis in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china coronaviruses and immunosuppressed patients. the facts during the third epidemic aasld clinical insights for hepatology and liver transplant providers key: cord-351169-y91fdf66 authors: phillips, lia; pavisic, jovana; kaur, dominder; dorrello, n. valerio; broglie, larisa; hijiya, nobuko title: successful management of sars-cov-2 acute respiratory distress syndrome and newly diagnosed acute lymphoblastic leukemia date: 2020-09-14 journal: blood adv doi: 10.1182/bloodadvances.2020002745 sha: doc_id: 351169 cord_uid: y91fdf66 standard chemotherapy can still be used for new diagnosis of acute lymphoblastic leukemia in patients with sars-cov-2. corticosteroid can be given safely to patients with sars-cov-2 presenting with acute respiratory distress syndrome and all. although recommendations are emerging for the general management of oncology patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), 1,2 there is little experience in patients with newly diagnosed acute lymphoblastic leukemia (all). providers may have concern about initiating multiagent chemotherapy in patients with sars-cov-2, particularly corticosteroids, which are an essential part of induction regimens, but raise the theoretical possibility of delayed viral clearance. we describe our experience of successfully initiating therapy for an adolescent diagnosed with all, while managing severe sars-cov-2 infection marked by respiratory failure, systemic inflammation, and autoimmune hemolytic anemia (aiha). an 18-year-old adolescent male presented to our emergency room with pallor, having experienced cough and fever for 1 week. he tested positive for sars-cov-2 by polymerase chain reaction. he had multiple known exposures: his father had recently died of sars-cov-2 and his mother and siblings were symptomatic at home. initial laboratory evaluation was significant for the following counts: white blood cells (wbcs), 105 3 10 3 /ml with 95% blasts; hemoglobin (hgb), 3.7 g/dl; and platelets, 30 3 10 3 /ml. flow cytometry of peripheral blood confirmed the diagnosis of b-cell all. he had low-grade fever, normal oxygen saturation, and no respiratory distress at time of presentation, with the remainder of his laboratory results notable for hyperuricemia and mildly elevated lactate dehydrogenase. he received supportive care with hyperhydration and allopurinol, given his high risk for tumor lysis syndrome, and broad-spectrum antibiotics (piperacillin/tazobactam). shortly after admission, he developed persistent high fevers and intermittent hypoxia with sudden respiratory decompensation requiring mechanical ventilation and hypotension requiring vasopressor support on hospital day 3 (hd 3). a chest radiograph showed bibasilar opacities consistent with moderate acute respiratory distress syndrome (ards). 3 in the setting of worsening sars-cov-2 disease, his wbc count downtrended, with hydration but no specific antileukemic therapy, to 8 3 10 3 /ml; in addition, he developed transfusion-resistant anemia and thrombocytopenia and had a mild increase in bilirubin. he was found to have aiha with positive immunoglobulin g and c3 polyspecific direct antiglobulins, but a negative eluate and no alloantibodies. he also had rising ferritin and elevated interleukin 6 (il-6) and soluble il-2 receptor (sil2r) levels ( figure 1 ), which raised concern for a hemophagocytic lymphohistiocytosis (hlh) vs a sars-cov-2-related cytokine storm. due to the severity of his condition, antileukemic therapy was initiated with methylprednisolone alone on hd 5. his clinical condition steadily improved thereafter. he was extubated on hd 7, showed resolution of fevers by hd 8, and his inflammatory markers decreased ( figure 1 ). his response to transfusions improved, and hgb and platelet count stabilized. induction chemotherapy for all was then continued with vincristine on hd 11 and daunorubicin on hd 18. polyethylene glycol (peg)-asparaginase was held due to an elevated lipase level of 179 u/l (1.93 upper limit of normal), thought to be due to sars-cov-2 infection. he tolerated the chemotherapy well and was discharged home on hd 18. he received further chemotherapy (vincristine, daunorubicin) per the 4 peg-asparaginase was given 10 days after hospital discharge once his lipase level returned to normal. his end-of-induction bone marrow showed complete remission with minimal residual disease of 2.7% by flow cytometry. jak1 mutations (p.arg724cys, p.arg724his), jak2 mutations (p.arg938gln, p.arg867gln), loss of ikzf1, pax5, and cdkn2a/2b, and cytokine receptor-like factor 2 rearrangement were found, consistent with philadelphia chromosome-like all. he has subsequently been enrolled in a phase 2 study of ruxolitinib with chemotherapy (clinicaltrials.gov nct02723994). we collected the patient's medical records including clinical course, laboratory parameters, treatment record, and outcome. patients receiving myelosuppressive cancer therapy certainly have a theoretically increased risk for sars-cov-2 and more severe disease, although currently reported data paint a mixed picture. 5 reviews of pediatric oncology centers found a relatively low overall number of infected patients, with mild infection most common. 6, 7 whether treatments should be altered in pediatric cancer patients remains unclear, although emerging guidelines suggest postponing high-intensity treatments. 1, 2 in this case, intensive remission induction chemotherapy was initially delayed due to concern for potential worsening of sars-cov-2 disease by exacerbating the patient's already immunocompromised state in the setting of all. however, the patient developed progressively worsening symptoms, laboratory markers suggestive of cytokine storm, and rapid clinical deterioration to respiratory failure and shock. restoration of this patient's immune function was not expected without initiation of cytotoxic chemotherapy. he additionally showed evidence of immune dysregulation with persistent fevers, pancytopenia, and elevated serum ferritin, il-6, and sil2r. notably, as the patient's clinical condition deteriorated, his high initial wbc count decreased drastically with hyperhydration alone. this finding was concurrent with the development of transfusion-unresponsive aiha and thrombocytopenia. this constellation of findings could be attributed not only to sars-cov-2 infection but also to possible secondary hlh from untreated acute leukemia. 8, 9 additionally, secondary hlh may have contributed to sars-cov-2-induced lung inury. 10 reports from china at the onset of the sars-cov-2 pandemic reported that patients with more severe disease had higher ferritin, sil2r, il-6, il-8, il-10, and tumor necrosis factor a (tnf-a). 11 although lymphopenia is well described with sars-cov-2 infection, 12,13 this patient's case suggests that the hyperinflammatory response seen in severe sars-cov-2 infection may involve immune dysregulation leading to consumptive thrombocytopenia and aiha, which should be further explored among patients infected with sars-cov-2. aiha in this patient gave concern for breakdown of immunologic tolerance. 14 as there was evidence of both impaired and dysregulated immune function with evolving ards and shock, the decision was made to initiate corticosteroid therapy. significant systemic inflammation with elevated sil2r and il-6 levels, as seen in our patient, has led to the investigation of targeted cytokine blockade for patients with severe sars-cov-2 disease, with particular interest in targeting il6 with tocilizumab. [15] [16] [17] this was considered in our patient: however, in light of concurrent all and concern for possible secondary hlh, the decision was made to initiate therapy with corticosteroids and reserve tocilizumab as second-line therapy. the benefit to mortality with steroid use in patients with severe sars-cov-2 disease was unclear, with mixed outcomes in observational studies at the time of our patient's treatment. [18] [19] [20] [21] current recommendations for the management of severe sars-cov-2 infection do not support routine systemic corticosteroid use outside of a clinical trial, but do allow for clinical discretion with regard to moderate ards. 3, [22] [23] [24] [25] in this patient's case, however, corticosteroids were an integral part of his antileukemic therapy and management of aiha and hlh, and had anti-inflammatory potential; thus, benefits were determined to outweigh the risks. our patient's clinical condition and inflammatory marker elevation rapidly improved with steroid treatment, allowing him to continue standard all treatment. this suggests that in the setting of active sars-cov-2 infection in leukemia, systemic corticosteroids can be safely given without delay as a bridge to more myelosuppressive therapy. apart from supportive care and steroids, the patient did not receive any other sars-cov-2-directed therapy. he showed remarkable improvement upon initiation of steroids with resolution of fevers, decrease in inflammatory markers and d-dimer, stabilization of transfusion requirements, and rapid respiratory improvement with extubation possible within 3 days. given his rapid recovery, additional antileukemic therapy with a modified 4-drug combination 4 was given, first with addition of vincristine and subsequently daunorubicin and peg-asparaginase once the patient showed further clinical recovery from his sars-cov-2 infection. he tolerated the remainder of induction and achieved complete remission. we would thus recommend a similar stepwise approach in initiating systemic chemotherapy in patients with newly diagnosed all complicated by sars-cov-2 infection, utilizing steroids along with nonmyelosuppressive chemotherapy upfront, with addition of more toxic chemotherapy once the critical window of clinical deterioration from sars-cov-2 passes. early advice on managing children with cancer during the covid-19 pandemic and a call for sharing experiences caring for our cancer patients in the wake of covid-19 definition of acute respiratory distress syndrome replacing cyclophosphamide/cytarabine/mercaptopurine with cyclophosphamide/etoposide during consolidation/delayed intensification does not improve outcome for pediatric b-cell acute lymphoblastic leukemia: a report from the cog cancer patients in sars-cov-2 infection: a nationwide analysis in china lessons after the early management of the covid-19 outbreak in a pediatric transplant and hemato-oncology center embedded within a covid-19 dedicated hospital in lombardia, italy. estote parati flash survey on severe acute respiratory syndrome coronavirus-2 infections in paediatric patients on anticancer treatment a consensus review on malignancy-associated hemophagocytic lymphohistiocytosis in adults hemophagocytic lymphohistiocytosis in sars-cov-2 infection pulmonary involvement in patients with hemophagocytic lymphohistiocytosis dysregulation of immune response in patients with coronavirus 2019 (covid-19) in wuhan, china china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china covid-19 in critically ill patients in the seattle region -case series new insights in the pathogenesis of autoimmune hemolytic anemia the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression effective treatment of severe covid-19 patients with tocilizumab corticosteroids as adjunctive therapy in the treatment of influenza: an updated cochrane systematic review and meta-analysis national institutes of health. considerations for certain concomitant medications in patients with covid-19 risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury infectious diseases society of america guidelines on the treatment and management of patients with covid-19 interim clinical guidance for management of patients with confirmed coronavirus disease (covid-19 surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016 dexamethasone in hospitalized patients with covid-19 -preliminary report key: cord-335289-m4u96x2v authors: bhattacharya, manojit; sharma, ashish r.; patra, prasanta; ghosh, pratik; sharma, garima; patra, bidhan c.; lee, sang‐soo; chakraborty, chiranjib title: development of epitope‐based peptide vaccine against novel coronavirus 2019 (sars‐cov‐2): immunoinformatics approach date: 2020-03-05 journal: j med virol doi: 10.1002/jmv.25736 sha: doc_id: 335289 cord_uid: m4u96x2v recently, a novel coronavirus (sars‐cov‐2) emerged which is responsible for the recent outbreak in wuhan, china. genetically, it is closely related to sars‐cov and mers‐cov. the situation is getting worse and worse, therefore, there is an urgent need for designing a suitable peptide vaccine component against the sars‐cov‐2. here, we characterized spike glycoprotein to obtain immunogenic epitopes. next, we chose 13 major histocompatibility complex‐(mhc) i and 3 mhc‐ii epitopes, having antigenic properties. these epitopes are usually linked to specific linkers to build vaccine components and molecularly dock on toll‐like receptor‐5 to get binding affinity. therefore, to provide a fast immunogenic profile of these epitopes, we performed immunoinformatics analysis so that the rapid development of the vaccine might bring this disastrous situation to the end earlier. therefore, as the situation was getting worse and worse, the need for designing a suitable peptide vaccine component against the sars-cov-2 was growing. our work was to find suitable epitopes, which can generate enough immune response against the sars-cov-2 infection. using immunoinformatics, we could recognize and characterize potential b and t-cell epitopes for the generation of the the amino acid sequence of the targeted protein on sars-cov-2 was collected from the national centre for biotechnological information (ncbi) database. 11 the protein sequence is very crucial for identifying the potential epitopes of the targeted protein. in this subsection, we used the immune epitope database (iedb) to identify linear b-cell epitopes using the incorporated bepipred 2.0 prediction module. 12, 13 we provided the fasta sequence of the targeted protein as an input considering all default parameters. molecular docking is the most promising part of the modern drugdiscovery method. here, in this study, we adopted patchdock (beta we obtained a total of 34 sequential linear b-cell epitopes of varying lengths from the iedb server within spike glycoprotein of sars-cov-2. those b-cell epitopes were placed into table 1 based on their positional value, sequence, and length. in figure 1 the yellow-colored peaks represent the epitopic region, while the green-colored slopes, represent the nonepitopic region. we identified 29 mhc-i epitopes and 8 mhc-ii epitopes, which fall within the preidentified b-cell epitopic region. among them, tables 2 and 3 with encountering mhc alleles and antigenic scores. in this study, we linked the 13 mhc-i and 3 mhc-ii antigenic epitopes with (eaaak) 3 linker peptide to construct a vaccine component. this linker peptide was easily fused with the virus coat protein and increased stability as well as folding of the vaccine component. 22 the predicted structure of the vaccine component is shown in figure 2 . it has 90.0%, 7.1%, 1.6%, and 1.3% residues in most favored, additionally allowed, generously allowed and disallowed regions respectively within procheck as the validation server to generate ramachandran plot. using the prosa server, the "z" score was −3.82 and most of the residues had negative energy value as shown in all the observations of our present work depict the effectiveness of selected epitopes within the spike glycoprotein of sars-cov-2. these epitopes can be used to make an immunogenic multi-epitopic peptide vaccine against sars-cov-2. present immunoinformatic analysis pointed out 13 mhc-i and 3 mhc-ii epitopes within the spike glycoprotein of sars-cov-2. these epitopes are the ideal candidate to formulate a multi-epitopic peptide vaccine, not only because of being selected from the linear b-cell epitopic region but also because of their antigenic property was confirmed. moreover, the molecular docking of vaccine components with the tlr-5 proves the significance and effectiveness of these epitopes as an ideal vaccine candidate against sars-cov-2. however, these immunoinformatic analyses require several in vitro and in vivo validations before formulating the vaccine to resist covid-19. prasanta patra pratik ghosh garima sharma chiranjib chakraborty a novel coronavirus outbreak of global health concern a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan, china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating u r e 5 docking complex exhibiting the bonding interaction between vaccine component and toll-like receptor-5 | 13 person-to-person transmission: a study of a family cluster novel wuhan (2019-ncov) coronavirus another decade, another coronavirus the novel coronavirus originating in wuhan, china: challenges for global health governance a novel multi-epitope recombined protein for diagnosis of human brucellosis ct imaging features of 2019 novel coronavirus (2019-ncov) database resources of the national center for biotechnology information immune epitope database analysis resource bepipred-2.0: improving sequence-based b-cell epitope prediction using conformational epitopes propred1: prediction of promiscuous mhc class-i binding sites propred: prediction of hla-dr binding sites vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines improving protein fold recognition and template-based modeling by employing probabilistic-based matching between predicted onedimensional structural properties of query and corresponding native properties of templates prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins patchdock and symmdock: servers for rigid and symmetric docking pymol: an open-source molecular graphics tool. ccp4 newsletter on protein crystallography fusion protein linkers: property, design and functionality a homology model for clostridium difficile methionyl trna synthetase: active site analysis and docking interactions biocomputational analysis and in silico characterization of an angiogenic protein (rnase5) in zebrafish (danio rerio) in silico identification of catalytic residues in azobenzene reductase from bacillus subtilis and its docking studies with azo dyes detection of peptidebinding sites on protein surfaces: the first step toward the modeling and targeting of peptide-mediated interactions development of epitope-based peptide vaccine against novel coronavirus 2019 (sars-cov-2): immunoinformatics approach key: cord-342796-f7n8sxbu authors: stowe, j.; tessier, e.; zhao, h.; guy, r.; muller-pebody, b.; zambon, m.; andrews, n.; ramsay, m.; lopez bernal, j. title: interactions between sars-cov-2 and influenza and the impact of coinfection on disease severity: a test negative design date: 2020-09-18 journal: nan doi: 10.1101/2020.09.18.20189647 sha: doc_id: 342796 cord_uid: f7n8sxbu background: the potential impact of covid-19 alongside influenza on morbidity, mortality and health service capacity is a major concern as the northern hemisphere winter approaches. this study investigates the interaction between influenza and covid-19 during the latter part of the 2019-20 influenza season in england. methods: individuals tested for influenza and sars-cov-2 were extracted from national surveillance systems between 20/01/2020 and 25/04/2020. to estimate influenza infection on the risk of sars-cov-2 infection, univariable and multivariable analyses on the odds of sars-cov-2 in those who tested positive for influenza compared to those who tested negative for influenza. to assess whether a coinfection was associated with severe sars-cov-2 outcome, univariable and multivariable analyses on the odds of death adjusted for age, sex, ethnicity, comorbidity and coinfection status. findings: the risk of testing positive for sars-cov-2 was 68% lower among influenza positive cases, suggesting possible pathogenic competition between the two viruses. patients with a coinfection had a risk of death of 5.92 (95% ci, 3.21-10.91) times greater than among those with neither influenza nor sars-cov-2 suggesting possible synergistic effects in coinfected individuals. the odds of ventilator use or death and icu admission or death was greatest among coinfection patients showing strong evidence of an interaction effect compared to sars-cov-2/influenza acting independently. interpretation: cocirculation of these viruses could have a significant impact on morbidity, mortality and health service demand. testing for influenza alongside sars-cov-2 and maximising influenza vaccine uptake should be prioritised to mitigate these risks. it is likely that both sars-cov-2 and seasonal respiratory pathogens, most notably influenza, will be co-circulating as the northern hemisphere 2020/21 winter approaches. the potential impact of covid-19 alongside influenza on morbidity, mortality and health service capacity is a major concern, however, currently little is understood about the interaction between these two respiratory viruses 1,2 . there is existing evidence of pathogenic competition between respiratory viruses, including between influenza and seasonal coronaviruses [3] [4] [5] . this could be through immune-mediated interference resulting in some viruses to diminish during the peak of another virus, a phenomenon that has been recognised for many decades 3, 6, 7 . one study reported that influenza vaccination was associated with an increased risk of seasonal coronavirus 5 . to date there is some evidence of ectopic interaction between the sars-cov-2 protein and host proteins 8 , however there is no information on the pathogenic interaction between sars-cov-2 and influenza and the epidemiological impact of such interaction is unknown. if individuals are coinfected with both sars-cov-2 and influenza, this could lead to more severe disease outcomes. since the beginning of the sars-cov-2 pandemic, a number of case reports of sars-cov-2 and influenza coinfection with severe outcomes have been published 1, [9] [10] [11] [12] [13] . however, there is a propensity for case reports to highlight more severe cases and there has been no systematic analysis of disease outcomes in coinfected patients compared to non-coinfected controls. in the uk the 2019-2020 influenza season peaked early with activity declining significantly from january 2020 14 . the season saw lower activity with influenza a(h3n2) as the predominant strain 14 . the first sars-cov-2 infection occurred in late january 2020 arising from an imported case, and the distribution of sars-cov-2 rose with sustained community transmission from early march in the uk peaking on 7 april 2020 with 4,493 cases and on 21 april the total number of daily sars-cov-2 deaths peaked at 1,172 15 . as such there was only a limited period of overlap between influenza circulation and sars-cov-2 circulation. in this study, we explore the interaction between influenza and sars-cov-2 during the latter stages of the 2019-2020 influenza season in england. the aims of the study are two-fold; firstly, to assess whether infection with influenza is associated with a reduced risk of sars-cov-2 infection and secondly to assess whether coinfection with influenza is associated with a more severe sars-cov-2 outcome such as death, being admitted to hospital, admitted to icu or requiring ventilatory support. the sgss (second generation surveillance system) and datamart were used to obtain all influenza positive cases between 01/01/2020 and 02/06/2020 16, 17 . for the analyses data was restricted to the time period between 20/01/2020 up to 25/04/2020, when the first sars-cov-2 and influenza coinfection occurred and the last influenza sample was reported in datamart. individuals tested for influenza who had a negative result in datamart were also extracted. both groups were matched to sars-cov-2 test results (positive and negative) in sgss as of 02/06/2020. a coinfection was defined as positive for both influenza and sars-cov-2 within 7 days of each sample date. database coordinated by nhs digital that allows the tracing of information against personal demographics, and the date of death was extracted 18 . deaths from 6 days before to 28 days after the test result were included. test results were also linked to the secondary uses service (sus) dataset and the hospital episode statistics dataset, which contain information on all admitted patient care, outpatient and a&e attendances at nhs hospitals in england 19 20 . these datasets were used to identify patients in an intensive care unit (icu) and that required the use of a ventilator within 14 days before to 28 days after the earliest test sample date were as outcome variables as well as ethnicity 21 and comorbidities as covariates. comorbidities were identified using the international classification of diseases 10 th revision (icd-10) codes and grouped into the following categories: asplenia or dysfunction of the spleen, asthma, chronic heart disease, chronic kidney disease, chronic liver disease, chronic neurological disorders, chronic respiratory disease (excluding asthma), dementia including alzheimer's, diabetes, malignancies affecting the immune system, obesity, other neoplasms, rheumatological diseases, and transplantations and conditions affecting the immune system. for the comorbidities linkage, data was restricted to inpatient and outpatient hospital episodes in the last 5 years. effect of influenza infection on the risk of sars-cov-2 infection: the total number of positive and negative sars-cov-2 and influenza test results from weeks 1 to 17, 2020 were assessed. percent positivity was calculated for individuals with a sars-cov-2 and influenza coinfection and individuals with no influenza infection by dividing the number of individuals with sars-cov-2 positive results by the total number of individuals tested and multiplied by 100. additionally, the total number individuals with a sars-cov-2 and influenza coinfection were assessed by influenza type. to estimate the effect of recent influenza infection on the risk of sars-cov-2 infection, univariable and multivariable analyses on the odds of sars-cov-2 in those who tested positive for influenza compared to those who tested negative for influenza were conducted adjusting for age, sex, ethnicity, region, comorbidity and sample week. finally, to determine the influence of unmeasured confounding such as occupation, the analysis was stratified by age into children (under 19 years), working age adults and older adults (>65). severity and risk of death among individuals with a coinfection: the mortality rate among individuals with a sars-cov-2 and influenza coinfection and those with sars-cov-2 infection who tested negative for influenza was calculated by dividing the number of deaths by the total number of individuals tested by age group. to assess whether having a coinfection was associated with death, univariable and multivariable analyses on the odds of death adjusted for age, sex, ethnicity, comorbidity (0 or 1+) and coinfection status (flu negative/ sars-cov-2 negative; flu negative/ sars-cov-2 positive; flu positive/ sars-cov-2 negative; flu positive/ sars-cov-2 positive) was assessed. this analysis was repeated with a composite outcome of ventilator use or death use and a composite outcome of icu admission or death. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint a total of 19,256 individuals were tested for both influenza and sars-cov-2 between 20/01/2020 and 25/04/2020, when the last positive influenza test was detected in datamart. in total, 58 individuals had a sars-cov-2 and influenza coinfection, 992 had a positive influenza result and were negative for sars-cov-2, 4,443 had a positive sars-cov-2 result and were negative for influenza and the remaining 13,763 were negative for both sars-cov-2 and influenza during this period ( figure 1 ). of the 58 patients with a sars-cov-2 and influenza co-infection 32 (55.2%) cases were ages 70 years and older. of the 58 cases with a sars-cov-2 and influenza coinfection, 31 individuals had influenza type a (unsubtyped), 16 had influenza type b, 8 had influenza h1n1, one had influenza type a&b and two cases had unknown influenza type. week 12 had the highest reported sars-cov-2 and influenza coinfections (20 individuals, table 1 ). a total of 13,451 (70%) individuals linked to a hospital admission record in sus between 01/12/2020 and 24/08/2020 of which 12,253 individuals had an associated record in the 14 days before and up to 28 days after the earliest sars-cov-2 or influenza test date. a total 1,666 (6%) of individuals had an icu admission and 890 (5%) were ventilated (supplementary figure 1) . of the 19,256 cases, 2,469 (12.8%) died of which 25/58 (43.1%) of the sars-cov-2 and influenza coinfected cases died. sars-cov-2 positivity among influenza positive cases was generally lower than sars-cov-2 positivity among influenza test negatives ( table 1 ). the highest sars-cov-2 positivity rate for both influenza positive and negative cases was in week 14 (66.7% and 44.8%, respectively). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. overall 43.1% of cases with coinfection died compared to 26 .9% of those who tested positive only for sars-cov-2 (table 3) . age specific mortality rates were higher among older people with a sars-cov-2 and influenza coinfection (table 3) . for individuals with influenza only, the overall mortality rate was 48/992=4.8% and for those negative for both, the mortality rate was 1,203/13,763=8.7%. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint than those with only covid-19 where the odds of death was 2.61 time greater compared to no sars-cov-2 or influenza (table 4 ). for those only positive for influenza there was a slightly decreased mortality risk (or 0.64 (95% ci 0.47-0.89)). to formally test the interaction between influenza and sars-cov-2 the same model was fitted but with separate terms for influenza, sars-cov-2 and the interaction of influenza and sars-cov-2, this gave a significant interaction effect (p=<0.001) of an additional 3.60 odds of death (95% ci 1.83-7.11) compared to that expected if influenza and sars-cov-2 acted independently. when combining ventilator use or death into a composite variable, the odds was 6.43 times greater among individuals with coinfection (95% ci 3.61-11.47). the icu admission or death composite had an odds 6.33 times greater among individuals with coinfection (95% ci 3.57-11.23) ( table 4 ). a test for interaction for both the ventilator composite and icu composite gave a significant effect (p=<0.001) with additional 3.38 odds of coinfection (95% ci 1.81-6.34) and 3.39 odds of coinfection(95% ci 1.83-6.29), respectively compared to that expected if influenza and sars-cov-2 acted independently. we found that influenza infection was associated with a lower risk of sars-cov-2 infection, suggesting that there may be pathogenic competition between these two viruses. we also found strong evidence that coinfection with influenza and sars-cov-2 was associated with an increased risk of death or severe disease and that this appears to be beyond the additive effect of the two viruses acting independently. the risk of testing positive for sars-cov-2 was 68% lower among influenza positive cases. this is consistent with recent descriptive evidence from new york where <3% of those testing positive for . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint sars-cov-2 had coinfection with influenza whereas 13% of those testing negative for sars-cov-2 were influenza positive 22 . it is also consistent with existing evidence on the interaction between influenza and seasonal coronavirus and rhinovirus [3] [4] [5] 23 . there are biologically plausible mechanisms for such an effect, including stimulation of non-specific immune responses by the first infectious agent, such as the induction of a refractory state in bystander cells as a result of the antiviral effect of interferon induced as part of an innate immune response to an rna viral infection. our findings cannot distinguish between a reduced risk of sars-cov-2 among those first infected with influenza or vice versa. a recent study has suggests that sars-cov-2 has a lower growth rate than influenza and is suppressed if the infections start simultaneously, however, if an influenza infection were to occur after sars-cov-2 infection, a coinfection would be detected 24 . our findings would not support the relaxation of preventative measures against influenza, including vaccination, given the risk of morbidity and mortality from influenza 5,25,26 as well as our finding of adverse outcomes associated with influenza and sars-cov-2 coinfection. furthermore, results from brazil indicated a significantly lower odds of needing intensive care treatment, invasive respiratory support and death among patients with sars-cov-2 that received the inactivated trivalent influenza vaccine 27 . the international council on adult immunization highlights in their roadmap that influenza, pneumococcal and herpes zoster vaccines programmes are more urgent than ever before 28 . as a further potential implication on influenza vaccination, if there is a competitive effect between influenza and sars-cov-2, this effect may also be seen with live attenuated influenza vaccination (laiv) which if offered to children in england and could in turn have a role in outbreak management. further research on the pathology of influenza and sars-cov-2 coinfection such as the order of infection and the effect of timing of influenza infection on the risk of acquiring sars-cov-2 infection, as well as any effect of laiv is required. the results from this study indicate that the risk of death was nearly six times greater among individuals with a sars-cov-2 and influenza coinfection than those with neither influenza nor sars-cov-2 and that this effect is significantly higher than the risk associated with sars-cov-2 infection alone. similarly, the combined outcomes of ventilator use or death and icu admission or death gave similar results. these findings suggest a possible synergistic effect between sars-cov-2 and influenza once an individual is coinfected. the high mortality rate is consistent with case reports of severe outcomes in coinfected patients 12, 13, 29 . conversely, some case series have not seen increased severity with influenza and sars-cov-2 co-infection, where the outcomes have been similar to cases with sars-cov-2 only 30, 31 . synergistic effects have previously been reported between influenza and other respiratory viruses, for example by facilitating cell to cell spread 32 . these findings also emphasise the importance of influenza vaccination in at risk groups and early administration of antivirals where coinfection is identified or suspected. this also adds further weight to the need for effective vaccines against influenza, in particular among the elderly among whom vaccine effectiveness tends to be lower and among whom most coinfections were seen. this has been an area of development in recent years with the introduction of high dose and adjuvanted vaccines 33 . studies of other respiratory viral infections have not indicated adverse outcomes from coinfection, for example, a study assessing sars-cov and metapneumovirus in hong kong that showed that there was no significant difference in the outcomes, including deaths between those with a sars-cov and metapneumovirus coinfection versus sars-cov alone 34 . it is important to note, that these are case studies of hospitalised individuals and the comparisons do not adjust for potential confounders. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint to our knowledge, our study is the first epidemiological study that uses national level data on both positive and negative sars-cov-2 and influenza cases. by extracting all cases with a sars-cov-2 and influenza test result, and linking the data to hes we were able to assess the effects of sars-cov-2 and influenza co-infections compared to single infection and negative test results while controlling for variables such as ethnicity, comorbidities, sex and age which are known factors for sars-cov-2 morbidity [35] [36] [37] . furthermore, the test negative design controls for the propensity for more severe cases to be tested for other respiratory viruses. most of the sars-cov-2 tests were collected when the government policy was to test individuals on admission to hospital with lower respiratory tract infections and healthcare workers 38 . therefore, the majority of sars-cov-2 cases were individuals with moderate to severe symptoms and mild cases are likely to be missed. additionally, influenza test results collected from datamart are only collected from sentinel laboratories. however, the test negative controlled design means that none of the study arms were biased towards more severe outcomes as all were tested for both diseases. additionally, in our study the majority of cases with a sars-cov-2 coinfection had influenza subtype a. due to small numbers it was not possible to determine whether the risk of sars-cov-2 coinfection and severity of disease varied by influenza subtype. while in the 2019-2020 influenza season, the majority of influenza subtype a cases were h3n2, towards the end of the season there was a shift towards h1n1, which is consistent with our finding of more h1n1 cases among those coinfections that were subtyped 14 . the impact severity of influenza and sars-cov-2 coinfection by different subtypes should be further considered in the upcoming influenza season. furthermore, the influenza vaccination status of the patients was not available therefore we could not adjust for vaccination status of the patients in the model. while our findings provide evidence of pathogenic competition between influenza and sars-cov-2, a significant number of coinfections occur and they appear to be associated with higher mortality rates. further investigation is needed in order to understand the potential mechanisms for any synergistic interaction. cocirculation of these two viruses could have a significant impact on morbidity, mortality and health service demand. as the 2020-2021 northern hemisphere influenza season approaches, it is important that a high index of suspicion for coinfection is maintained. testing strategies should include influenza and other respiratory viruses as well as sars-cov-2 and measures should be adopted to prevent coinfection including maximising uptake of influenza vaccination, particularly in groups at higher risk of both diseases. authors jlb, et, js, na developed the study protocol. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint role of the funding source: the study was undertaken by authors at public health england as part of the routine functions of surveillance and control of communicable diseases. public health england, national infection service, immunisation and countermeasures division has provided vaccine manufacturers with post-marketing surveillance reports, which the marketing authorisation holders are required to submit to the uk licensing authority in compliance with their risk management strategy. a cost recovery charge is made for these reports. are co-infections with covid-19 and influenza low or underreported? an observational study examining current published literature including three new unpublished cases co-infections in people with covid-19: a systematic review and meta-analysis virus-virus interactions impact the population dynamics of influenza and the common cold increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine influenza vaccination and respiratory virus interference among department of defense personnel during the 2017-2018 influenza season viral interference and interferon in vitro growth profiles of respiratory syncytial virus in the presence of influenza virus covid-19, cilia, and smell co-infection with sars-cov-2 and influenza a virus high prevalence of sars-cov-2 and influenza a virus (h1n1) co-infection in dead patients in northeastern iran co-infection of influenza b virus and sars-cov-2: a case report from taiwan the epidemiology and clinical characteristics of co-infection of sars-cov-2 and influenza viruses in patients during covid-19 outbreak co-infection with covid-19 and influenza a virus in two died patients with acute respiratory syndrome surveillance of influenza and other respiratory viruses in the uk winter 2019 to 2020. 2020. 15. public health england. coronavirus (covid-19) in the uk a new laboratory-based surveillance system (respiratory datamart system) for influenza and other respiratory viruses in england: results and experience from laboratory reporting to public health england a guide for diagnostic laboratories data-tools-and-services/data-services/hospital-episode-statistics. 20. nhs digital. secondary uses service (sus) co-infection in sars-cov-2 infected patients: where are influenza virus and rhinovirus/enterovirus? interference between outbreaks of respiratory viruses sars-cov-2 coinfections: could influenza and the common cold be beneficial modeling the impact of mass influenza vaccination and public health interventions on covid-19 epidemics with limited detection capability influenza vaccination in the covid-19 era inactivated trivalent influenza vaccine is associated with lower mortality among covid-19 patients in brazil a global agenda for older adult immunization in the covid-19 era: a roadmap for action clinical characteristics of critically ill patients co-infected with sars-cov-2 and the influenza virus in wuhan, china. international journal of infectious diseases : ijid : official publication of the international society for coinfection with sars-cov-2 and influenza a virus a case series of patients coinfected with influenza and covid-19 enhanced growth of influenza a virus by coinfection with human parainfluenza virus type 2 end of season influenza vaccine effectiveness in adults and children in the united kingdom in 2017/18 co-circulation of human metapneumovirus and sars-associated coronavirus during a major nosocomial sars outbreak in hong kong asian and minority ethnic groups in england are at increased risk of death from covid-19: indirect standardisation of nhs mortality data intensive care national audit & research centre. icnarc report on covid-19 in critical care 08 preexisting comorbidities predicting severe covid-19 in older adults in the uk biobank community cohort key: cord-348899-vynk8q8c authors: jo, seri; kim, suwon; shin, dong hae; kim, mi-sun title: inhibition of sars-cov 3cl protease by flavonoids date: 2019-11-14 journal: j enzyme inhib med chem doi: 10.1080/14756366.2019.1690480 sha: doc_id: 348899 cord_uid: vynk8q8c there were severe panics caused by severe acute respiratory syndrome (sars) and middle-east respiratory syndrome-coronavirus. therefore, researches targeting these viruses have been required. coronaviruses (covs) have been rising targets of some flavonoids. the antiviral activity of some flavonoids against covs is presumed directly caused by inhibiting 3c-like protease (3clpro). here, we applied a flavonoid library to systematically probe inhibitory compounds against sars-cov 3clpro. herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of sars-cov 3clpro. the interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. an induced-fit docking analysis indicated that s1, s2 and s3′ sites are involved in binding with flavonoids. the comparison with previous studies showed that triton x-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. with the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors. coronaviruses (covs) are single-stranded rna viruses with large, enveloped and positive senses that can infect both animals and humans 1 . covs, along with artierivirdae and roniviridae, belong to the coronaviridae family in the order nidovirales. these covs can infect various hosts, including avian, swine and humans. human coronaviruses (hcovs) represent a major group of covs associated with various respiratory diseases from common cold to serious pneumonia and bronchiolitis 2 . today, hcovs are recognised as one of the fastest-evolving viruses derived from their characteristic high genomic nucleotide replacement rates and recombination 3 . severe acute respiratory syndrome (sars), the first confirmed atypical pneumonia in china's guangdong province, has spread to several countries. the most common symptoms of sars include coughing, high fever (>38 c), chills, convulsions, headaches, dizziness and progressive radiographic changes of the chest and lymphopenia 4 . the severity of the disease shows a death rate of about 3% to 6%, although this rate could rise up to 43% to 55% for senior citizens older than 60 years 5 . the primary epidemic of sars was eventually controlled, but a sars cov-like virus was detected in chinese bats 6, 7 . besides, a recent pandemic of middle east respiratory syndrome (mers) caused by a novel coronavirus mers-cov raises fear of possible recurrence of sars or related dangerous diseases 8, 9 . since there is no vaccine and effective therapy for these viral infections, developing anti-sars drugs against future outbreaks remains a formidable challenge. sars-and mers-covs genomes contain two open reading frames orf1a and orf1b translated to two respective viral polyproteins pp1a and pp1ab by host ribosomes. orf1a encodes two cysteine proteases, a papain-like protease (plpro) and a 3c-like protease (3clpro). while plpro cuts the first three cleavage sites of its polyprotein, 3clpro is responsible for cleavage of the remaining 11 locations resulting in release of a total of 16 nonstructural proteins (nsp) in both sars-and mers-covs. the homodimeric form of 3clpro is active in the presence of substrates. the crystal structures of both 3clpros showed that each monomer is composed of three structural domains: domains i and ii form a chymotrypsin-like architecture with a catalytic cysteine and are connected to a third c-terminal domain via a long loop 10 . in the proteolytic site, all 3clpros prefer glutamine at p1 position and leucine, basic residues, small hydrophobic residues at p2, p3 and p4 positions, respectively 11 . at p1 0 and p2 0 positions, small residues are required. however, p3 0 position shows no strong preference. since the autocleavage process is essential for viral propagation, 3clpro is a good drug target for anti-coronaviral infection. in this study, we employed a proteolytic method to probe sars-cov 3clpro inhibitory compounds. a synthetic peptide labelled with an edans-dabcyl fret (fluorescence resonance energy transfer) pair 12 was used to search sars-cov 3clpro inhibitory compounds against a flavonoid library. recent studies showed that flavonoids have antiviral activity in some viruses including sars-cov 13-17 . however, a molecular level study has not been reported for sars-cov. therefore, we performed the proteolytic assay with flavonoids followed by an induced-fit docking experiment with top hits. with the results, we tried to deduce a structural and functional relationship of flavonoids crucial to binding with sars-cov 3clpro. the information can be applied to develop synthetic compounds with better affinities. the coding sequence of sars-cov nsp5-pp1a/pp1ab 3cl (chymotrypsin-like) protease (3clpro) was obtained at the ncbi database (np_828863.1). the catalytic domain of 3clpro (m1$t196) was synthesised chemically by bioneer (daejeon, korea) and cloned into a bacteriophage t7-based expression vector, pbt7. the plasmid dna was transformed into e. coli bl21 (de3) for protein expression. e. coli bl21 (de3) cells were grown on luria-bertani (lb) agar plates containing 150 lg ml à1 ampicillin. several colonies were picked and grown in capped test tubes with 10 ml lb broth containing 150 lg ml à1 ampicillin. a cell stock composed of 0.85 ml culture and 0.15 ml glycerol was prepared and frozen at 193 k for use in a large culture. the frozen cell stock was grown in 5 ml lb medium and diluted into 1000 ml fresh lb medium. the culture was incubated at 310 k with shaking until an od 600 of 0.6-0.8 was reached. at this point, the expression of the sars-cov 3clpro was induced using isopropyl-b-d-1-thiogalactopyranoside (iptg) at a final concentration of 1 mm. the culture was further grown at 310 k for 3 h in a shaking incubator. cells were harvested by centrifugation at 7650g (6500 rev min à1 ) for 10 min in a high-speed refrigerated centrifuge at 277 k. the cultured cell paste was resuspended in 25 ml of a buffer consisting of 50 mm tris-hcl ph 8, 100 mm nacl, 10 mm imidazole, 1 mm phenylmethylsulfonyl fluoride (pmsf), 10 lg ml à1 dnase i. the cell suspension was disrupted using an ultrasonic cell disruptor (digital sonifier 450, branson, usa). cell debris was pelleted by centrifugation at 24,900g (15,000 rev min -1 ) for 30 min in a high-speed refrigerated ultra-centrifuge at 277 k. the protein was purified by cation chromatography using a 5 ml hi-trap sp column (ge healthcare, piscataway, new jersey, usa). the column was equilibrated with a buffer consisting of 50 mm mes ph 6.5 and the pooled fractions were loaded. the column was eluted using a linear nacl gradient to 1 m nacl and the protein was eluted at 0.23 m nacl. sds-page showed one band around 22 kda (21895.09 da), corresponding to the molecular weight of the catalytic domain of sars-cov 3clpro. the protein was concentrated to 16 mg ml à1 for the protease assay in a buffer consisting of 0.23 m nacl and 50 mm mes ph 6.5. the custom-synthesised fluorogenic substrate, dabcyl-ktsavlqsgfrkme-edans (anygen, gwangju, korea), was used as a substrate for the proteolytic assay using the sars-cov 3clpro 18 . this substrate contains the nsp4/nsp5 cleavage sequence, gvlq#sg 19 , and works as a generic peptide substrate for many coronavirus including the sars-cov 3clpro. the peptide was dissolved in distilled water and incubated with each protease. a spectramax i3x multi-mode microplate reader (molecular devices) was used to measure spectral-based fluorescence. the proteolytic activity was determined at 310 k by following the increase in fluorescence (k excitation ¼ 340 nm, k emission ¼ 490 nm, bandwidths ¼ 9, 15 nm, respectively) of edans upon peptide hydrolysis as a function of time. assays were conducted in black, 96-well plates (nunc) in 300 ll assay buffers containing protease and substrate as follow; for the sars-cov 3clpro assay, 4.05 ll of 0.074 mm protease containing 50 mm tris ph 6.5 was incubated with 7.5 ll of 0.1 mm substrate at 310 k for 2 h before measuring relative fluorescence unit (rfu). before the assay, the emission spectra of 64 flavonoids were surveyed after illuminating at 340 nm to avoid the overlapping with the emission spectrum of edans. every compound was suitable to be tested. the final concentration of the protease, peptide and chemical used at the assay was 1, 2.5 and 20 lm each. at first, the sars-cov 3clpro and chemical were mixed and pre-incubated at room temperature for 1 h. the reaction was initiated by the addition of the substrate and each well was incubated at 310 k for 16 h. after that, we measured the fluorescence of the mixture on the black 96-well plate using the endpoint mode of spectramax i3x where the excitation wavelength was fixed to 340 nm and the emission wavelength was set to 490 nm using 9, 15 nm bandwidth, respectively. all reactions were carried out in triplicate. among the first 64 flavonoids (supplementary table 1 ), three of them were picked up to further assay at a concentration range of 2-320 lm. the ic 50 value which is the value causing 50% inhibition of the catalytic activity of the sars-cov 3clpro was calculated by nonlinear regression analysis using graphpad prism 7.03 (graphpad software, san diego, ca, usa). fret protease assays with the sars-cov 3clpro in the presence of triton x-100 the proteolytic assay using the sars-cov 3clpro in the presence of triton x-100 has been performed to differentiate the artificial inhibitory activity of chemicals through non-specific binding with proteases by forming aggregate or complexation. the concentration used in this study was 0.01%. to confirm the feasibility of the assay method independently, the fluorescence spectra from the tryptophan of the sars-cov 3clpro with candidate inhibitors were investigated 20 . the fluorescence measurements were recorded with a spectramax i3x multi-mode microplate reader (molecular devices) at excitation and emission wavelengths of 290 nm and 300-500 nm, respectively. the optimal excitation and emission wavelengths were determined by softmax pro. one tryptophan of the sars-cov 3clpro showed a fluorescence emission with a peak at 350 nm after the excitation at the wavelength of 290 nm. in contrast, the flavonoids were almost non-fluorescent under the same experiment condition. each 40 lm chemical was incubated with 1 lm the sars-cov 3clpro for 1 h and the fluorescence intensity of the mixture was measured. all the docking and scoring calculation were performed using the schr€ odinger software suite (maestro, version 11.8.012). the compounds were extracted from the pubchem database in the sdf format and were combined in one file. the file was then imported into maestro and prepared for docking using ligprep. the atomic coordinates of the crystal structure of sars-cov 3clpro (4wy3) were retrieved from the protein data bank and prepared by removing all solvent and adding hydrogens and minimal minimisation in the presence of bound ligand using protein preparation wizard. ioniser was used to generate an ionised state of all compounds at the target ph 7 ± 2. this prepared low-energy conformers of the ligand were taken as the input for an inducedfit docking. the induced-fit docking protocol 21 was run from the graphical user interface accessible within maestro 11.8.012. receptor sampling and refinement were performed on residues within 5 å of each ligand for each of the ligand-protein complexes. with prime 22 , a side-chain sampling and prediction module, as well as the backbone of the target protein, were energy minimised. a total of induced-fit receptor conformations were generated for each of the ligands. re-docking was performed with the test ligands into their respective structures that are within 30 kcal/mol of their lowest energy structure. finally, the ligand poses were scored using a combination of prime and glidescore scoring functions 23 . the yield of cell harvested for purification of the catalytic domain of sars-cov 3clpro was 2.69 g per 1000 ml of e. coli culture. the amount of purified protein synthesised with no-tag was 16 mg. for storage and assay, the protein solution was concentrated to 16 mg ml à1 . the concentrated solution was diluted to 1 lm when the inhibitory assay was going on. a flavonoid library consisting of 10 different scaffolds was also built (figure 1 ). it contains five isoflavones, one isoflavane, 17 flavones, 11 flavonols, seven flavanols, seven flavanones, four flavanonol, one prenylflavonoid, nine chalcones and two unclassified flavonoids (supplementary table 1 ). we applied the library to assay sars-cov 3clpro. using 64 flavonoids, an inhibitory effect of each compound at 20 lm was tested. among them, herbacetin (3,4 0 ,5,7,8-pentahydroxyflavone), rhoifolin (apigenin-7-o-rhamnoglucoside) and pectolinarin (5,7-dihydroxy 4 0 ,6-dimethoxyflavone 7-rutinoside) were found to have prominent inhibitory activity ( figure 2 ). the binding affinity data were plotted as log inhibitor concentration versus percent fluorescence inhibition (figure 2 ). the three compounds showed the severely reduced fluorescent intensity and thus represented their sars-cov 3clpro inhibitory activity. the ic 50 values were calculated from the dose-dependent inhibitory curves of herbacetin, rhoifolin and pectolinarin. the measured values were 33.17, 27.45 and 37.78 lm, respectively. since flavonoids are known to be aggregated through complexity and thus non-specifically inhibit various proteases, the assay in the presence of triton x-100 was also performed 24 . before the examination, we tested the effects of triton x-100 on the catalytic activity of the sars-cov 3clpro. as shown in supplementary figure 1 , only a slight increase in catalyst activity was observed up to 0.1% triton x-100. therefore, the assay was performed at a concentration of 0.01% triton x-100 with no significant interference detected. to independently confirm the inhibitory activity of flavonoids, a general tryptophan-based assay method was employed. tryptophan was well known to emit its fluorescence. therefore, if tryptophan is positioned adequately in proteins, the change of fluorescence intensity can reflect the binding state of chemicals and be used to judge interaction between proteins and chemicals. the sars-cov 3clpro contains one tryptophan residue (trp31) at the catalytic domain. its backbone lines up at the entrance of the active site. therefore, its fluorescence change can reflect environmental variation of the active site. the catalytic domain of sars-cov 3clpro used in this study displays a fluorescence peak at 340 nm after the tryptophan excitation wavelength of 290 nm. we monitored the change of the fluorescence intensities depending on the presence or absence of all flavonoids. since each compound in the flavonoid library was almost non-fluorescent under the experiment condition, a change of fluorescence intensity reflects interactions between the catalytic domain and chemicals. intriguingly, the three inhibitory compounds obviously reduced the fluorescence intensity of the catalytic domain compared with others ( figure 3 ). the decreased emission intensity confirmed the complex formation between the catalytic domain and inhibitory compounds. in order to deduce the binding modes of the inhibitory flavonoids in the molecular level, an in-depth theoretical investigation through an induced-fit docking study was carried out. the interactions between sars-cov 3clpro and three inhibitory flavonoids were analysed to predict their binding affinities. top-ranked structures (according to the glide gscores) from the induced-fit docking results for herbacetin (-9.263), rhoifolin (-9.565) and pectolinarin (-8.054) were selected and hypothesised to be biological complexes. to find reasonable structural factors endowing the good affinity of herbacetin, the poses of kaempferol (-8.526) and morin (-8.930), two closest homologues, were also calculated and compared. the predicted complex structures and 2d schematic representations of them are illustrated in figure 4 . flavonoids are an important kind of natural products. in particular, they belong to a type of plant secondary metabolites with a polyphenolic structure widely found in fruits and vegetables. they have miscellaneous reciprocal biochemical and antioxidant effects associated with various diseases such as cancer, alzheimer's disease and atherosclerosis [25] [26] [27] . it is due to antioxidants, anti-inflammatory, anti-mutagens and anti-cancer-causing properties combined with the ability to control major cell enzyme functions 15 . intriguingly, some flavonoids also have antiviral activity 17 . specifically, apigenin, luteolin, quercetin, amentoflavone 28 , quercetin, daidzein, puerarin, epigallocatechin, epigallocatechin gallate, gallocatechin gallate 29 and kaempferol 30 were reported to inhibit the proteolytic activity of sars-cov 3clpro. therefore, the antiviral effect is presumed to be directly linked to suppress the activity of sars-cov 3clpro in some cases. however, the systematic analysis using various scaffolds of flavonoids was not reported. in order to find the best scaffold to inhibit the function of sars-cov 3clpro, an assay with various flavonoid derivatives classified in 10 scaffolds was built and performed. among them, the best compounds were one flavonol, herbacetin and two flavones, rhoifolin and pectolinarin. in the current assay method including 0.01% triton x-100, the three flavonoids showed the better inhibitory activity than the previously reported flavonoids as mentioned above [28] [29] [30] . the aggregating tendency of flavonoids frequently leads to obtain false bioassay results and can be avoided by adding 0.01% triton x-100 24 . it is worthwhile to note that amentoflavone, the most effective flavonoid inhibiting sars-cov 3clpro 28 , did not effectively function in the presence of 0.01% triton x-100 ( figure 5) . to elucidate the relationship between binding mode and binding affinity, a docking study for herbacetin homologues, kaempferol and morin, were also performed. the glide scores of three compounds were -9.263, -8.526 and -8.930, respectively. the tendency of the glide scores is matched with the binding affinity of the compounds. the comparison of the predicted binding modes of three complex structures showed critical factors governing their binding affinity. in common, they share the kaempferol motif. as shown in figure 4 , the phenyl moiety of kaempferol occupies the s1 site of sars-cov through a hydrogen bond with glu166. in contrast, the chromen-4-one scaffold of kaempferol locates in the s2 site. in herbacetin, total of four hydrogen bonds are formed 5 .011. s1 represents the polar s1 site of sars-cov 3clpro, s2 for the hydrophobic s2 site, and the s3 0 site with no strong tendency. the pink arrows represent hydrogen bond interaction. within a distance of 2.33 å in the s2 site. especially, the major binding force was driven by the presence of the additional 8hydroxyl group which plays a critical role in binding with glu166 and gln189. these bindings are predicted to confer a good glide score of herbacetin. in contrast, the binding modes of morin and kaemperol become different due to the absence of above two hydrogen bonds due the lack of the 8-hydroxyl group (figure 4) . the absence also induces the change nullifying the hydrogen bond formed by the 5-hydroxyl group of the chromen-4-one scaffold with asp187 observed in herbacetin. though there is a new hydrogen bond formed through the 3-hydroxyl group, it may not enough to overcome the loss of binding capacity induced by the 8-hydroxyl group. this survey shows the importance of the 8hydroxyl group at the strong binding affinity of herbacetin. the glide scores of rhoifolin and pectolinarin were -9.565 and -8.054, respectively. they belong to the flavone family. interestingly, their binding modes are different from those of the above three flavonols. rhoifolin and pectolinarin possess a-l-rhamnopyranosyl b-d-glucopyranoside and l-mannopyranosyl b-d-glucopyranoside, respectively. the additional bulky carbohydrate groups are attached to 7-position of the chromen-4-one scaffold. as a result, the hydrogen bond formed between the 7-hydroxyl group and the backbone of ile188 was abolished. in addition, the bulky groups require a large space to reside in. therefore, these carbohydrate groups occupy the s1 and s2 sites and the chromen-4-one moieties locate in the s2 and s3 0 sites, unlike the above three flavonols. the better affinity of rhoifolin may be due to orchestrated binding through s1, s2 and s3' sites. the assay and docking result indicates important conclusions. at first, flavonoids have a wide range of binding affinity to sars-cov clpro due to their hydrophobic aromatic rings and hydrophilic hydroxyl groups. second, the presence of carbohydrate groups influences severely to the binding affinity and mode of the chromen-4-one moiety. third, triton x-100 is critical to reduce false-positives and overestimates. based on the result, the strategy to cope with sars targeting sars-cov 3clpro can be newly designed. a further study is going on to make derivatives which lead to better inhibitory compounds based on this study. we have created a library of flavonoids to systematically investigate sars-cov 3clpro inhibitory compound by a fret method. using the library, a number of flavonoids with a wide range of inhibitory activity were detected. intriguingly, inhibitory activities of some flavonoids were turned out to be artificial when triton x-100 was treated. herbacetin, rhoifolin and pectolinarin were the best inhibitory compounds against sars-cov 3clpro in the flavonoid library. the binding of the flavonoids was independently demonstrated by a tryptophan-based fluorescence method. in order to predict the flavonoid scaffolds needed to interact with the catalytic site of sars-cov 3clpro, an induced-fit docking study was performed and analysed. the presence of additional 8hydroxyl group of herbacetin was expected to be critical to acquire its high binding affinity around the s1 and s2 sites. the occupation of the s1 and s2 sites by carbohydrate groups of rhoifolin and pectolinarin was anticipated to be another way of getting high affinity to sars-cov 3clpro in glycosylated flavonoids. this study suggests that the biochemical assay combined with the docking prediction may be useful to develop better inhibitory flavonoid derivatives from various flavonoid scaffolds. the molecular biology of coronaviruses a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes sars and mers: recent insights into emerging coronaviruses identification of a novel coronavirus in patients with severe acute respiratory syndrome epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong bats are natural reservoirs of sarslike coronaviruses sars-related perceptions in hong kong achilles' heel": current figure 5. the effect of triton x-100 on flavonoids. each of two bars represents the inhibitory activity of compounds w/wt 0.01% triton x-100. the first bar (shaded) represents the control. inhibitory compounds were used at 40 lm concentration. each bar is expressed as the mean ± standard error of the mean (n ¼ 3). rfu: relative fluorescence units. effective inhibitor targeting a 3c-like protease assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferasebased biosensors structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity profiling of substrate specificities of 3c-like proteases from group 1, 2a, 2b, and 3 coronaviruses screening of drugs by fret analysis identifies inhibitors of sars-cov 3cl protease activity of compounds from taxillus sutchuenensis as inhibitors of hcv ns3 serine protease flavonoids: biological activities and therapeutic potential the citrus flavanone naringenin impairs dengue virus replication in human cells inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, boesenbergia rotunda (l.), towards dengue-2 virus ns3 protease flavonoids: promising natural compounds against viral infections characterization of sars main protease and inhibitor assay using a fluorogenic substrate prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus spectroscopic investigation of interaction between mangiferin and bovine serum albumin novel procedure for modeling ligand/receptor induced fit effects a hierarchical approach to all-atom protein loop prediction extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes aggregating behavior of phenolic compounds-a source of false bioassay results? flavonoids and their antioxidant properties chemical studies of anthocyanins: a review epigallocatechin-3-gallate prevents lipopolysaccharide-induced elevation of b-amyloid generation and memory deficiency biflavonoids from torreya nucifera displaying sars-cov 3cl(pro) inhibition flavonoid-mediated inhibition of sars coronavirus 3c-like protease expressed in pichia pastoris kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus the authors declare no conflict of interest. key: cord-347767-aq9niccc authors: zhao, jie; yang, xiaodong; wang, chenghua; song, shuai; cao, kun; wei, taohua; ji, qiaoxue; zheng, wanqun; li, jiali; zhou, xue; liu, jie title: yidu-toxicity blocking lung decoction ameliorates inflammation in severe pneumonia of sars-cov-2 patients with yidu-toxicity blocking lung syndrome by eliminating il-6 and tnf-a date: 2020-06-19 journal: biomed pharmacother doi: 10.1016/j.biopha.2020.110436 sha: doc_id: 347767 cord_uid: aq9niccc the present study investigates the differences in inflammatory agents alterations, immune function, and leukocyte differential count evaluation in severe pneumonia of sars-cov-2 patients with yidu-toxicity blocking lung syndrome after the recommended chinese medicine prescription of yidu-toxicity blocking lung decoction. a total of 40 patients with yidu-toxicity blocking lung syndrome, diagnosed as severe pneumonia of sars-cov-2 following the latest chinese national recommendations for the diagnosis and treatment of pneumonia caused by sars-cov-2 (the 5th edition), were recruited. they were randomly divided into the pure western medicine therapy group (pwm) and integrated into chinese and western medicine therapy group (icw). the general strategies were given to both groups according to the national recommendations, and the icw group was given yidu-toxicity blocking lung decoction extraorally. a radioimmunoassay method was adopted to detect the content of il-6, il-8,il-2r,tnf-α, procalcitonin (pct) and high-sensitivity c-reactive protein (hs-crp) in sera. flow cytometry was used to determine the peripheral blood lymphocyte subsets (the levels of cd3+, cd4+, cd8+, and the ratios of cd4+/cd8+). the white blood cell counts (wbc#), neutrophils count(n#), and lymphocyte counts (l#) were measured using a fully automatic blood rheological instrument. the t test or rank sum test and spearman analysis were conducted to evaluate the differences. the results showed that il-6 (p = 0.013) and tnf-α (p = 0.035) levels in the pwm group were significantly higher than those in the icw group after treatment. infection related indicators such as wbc#, n#, l#, hs-crp showed no differences. the analysis showed that there was no statistical difference in the values of cd4 and cd8 between the two groups. by the end of day 29, all patients were discharged and the final cure rate for both group were 100%. taken together, we conclude that yidu-toxicity blocking lung decoction could relieve inflammation of sars-cov-2 patients with yidu-toxicity blocking lung syndrome by eliminating inflammatory agents. cm can serve as a complementary medication to western medicine, which should be highlighted in clinical settings. were recruited. they were randomly divided into the pure western medicine therapy group (pwm) and integrated into chinese and western medicine therapy group (icw). the general strategies were given to both groups according to the national recommendations, and the icw group was given yidu-toxicity blocking lung decoction extraorally. a radioimmunoassay method was adopted to detect the content of il-6, il-8,il-2r,tnf-α, procalcitonin (pct) and high-sensitivity c-reactive protein (hs-crp) in sera. flow cytometry was used to determine the peripheral blood lymphocyte subsets (the levels of cd3+, cd4+, cd8+, and the ratios of cd4+/cd8+). the white blood cell counts (wbc#), neutrophils count(n#), and lymphocyte counts (l#) were measured using a fully automatic blood rheological instrument. the t test or rank sum test and spearman analysis were conducted to evaluate the differences. the results j o u r n a l p r e -p r o o f showed that il-6 (p=0.013) and tnf-α (p=0.035) levels in the pwm group were significantly higher than those in the icw group after treatment. infection related indicators such as wbc#, n#, l#, hs-crp showed no differences. the analysis showed that there was no statistical difference in the values of cd4 and cd8 between the two groups. by the end of day 29, all patients were discharged and the final cure rate for both group were 100%. taken together, we conclude that yidu-toxicity blocking lung decoction could relieve inflammation of sars-cov-2 patients with yidu-toxicity blocking lung syndrome by eliminating inflammatory agents. cm can serve as a complementary medication to western medicine, which should be highlighted in clinical settings. in late 2019, wuhan in china became the focus of the world owing to an ongoing outbreak of pneumonia with a novel coronavirus named sars-cov-2 [1] [2] [3] .up to february 24, 2020, more than 77,779 cases in china have been confirmed. sars-cov-2 infections are mostly mild,but it can spread quickly.currently,the infection has affected 28 countries and 5 continents. middle-aged and elderly patients with underlying comorbidities are susceptible to respiratory failure and may have a poorer prognosis [4] .early identification, effective isolation measures, and appropriate treatment play an important role in improving its prognosis [1, 5] . unfortunately, no evidence-based western medicine treatment has been recommended for coronavirus infection, except for meticulous supportive treatments, until now [6]. recently, chinese medicine has not only for many common diseases had a significant effect, but also has played an important role in prevention and control of major diseases and emerging infectious diseases. unlike western medicine, which has j o u r n a l p r e -p r o o f comprehensive and logical theories, cm treatments are based on functional analysis of the patient's entire body and use the body's self-healing abilities along with drugs and other medications. one classical cm symptoms of sars-cov-2 named yidu-toxicity blocking lung syndrome are unflagging fever or alternating chills and fever, cough (little sputum or yellow sputum), abdominal distension or constipation, shortness of breath, dyspnea, red tongue, yellow greasy or burned furred tongue, rolling, and rapid pulse. from the cm dialectical point of view, the core pathogenic characteristics of this disease attributed to damp-toxin blocking lung. hence, aromatic chinese medicine remedies which can repel foulness and dispersing lung-qi are believed to be therapeutic principles. the recommendation of cm prescription of sars-cov-2 treatment in the fifth national recommendations are as follows: kuxingren, shengshigao, gualou, shengdahuang, shengmahuang, zhimahuang, tinglizi, taoren, caoguo, binglang and cangzhu. over time there has been little emphasis on studying the combination of levels of white blood cell, immune function, inflammatory agents, and cm treatments, and therefore, the objective of this study is to evaluate the role of cm in the treatment of sars-cov-2. the study was carried out with the approval of the ethics committee of the first affiliated hospital of anhui medical university. all participates were recruited by random selection and written informed consent was obtained prior to our study. 3. blood gas analysis: pao2 / fio2≤300mmhg (1mmhg=0.133kpa). patients with syndromes of unflagging fever or alternating chills and fever, cough (little sputum or yellow sputum), abdominal distension or constipation, shortness of breath, dyspnea, red tongue, yellow greasy or burned furred tongue, rolling, and rapid pulse. inclusion criteria were as follows: (1) patients with normal liver and kidney function, no concomitant metabolic, infectious or inflammatory respiratory disease; (2) patients were not treated with steroids and immunosuppressants; (3) patients with no extra intestinal malignancy or history of chemotherapy or radiotherapy; (4) patients meeting the diagnostic criteria of severe pneumonia and yidu-toxicity blocking lung syndrome. exclusion criteria were as follows: (1) patients who did not meet the above inclusion criteria; (2) patients with severe dysfunction of vital organ (heart, lung, kidney, and liver); (3) patients with psychiatric comorbidity or poor compliance. the general strategies were given to both groups according to the national recommendations for diagnosis and treatment of pneumonia caused by sars-cov-2 (the 5th edition), including bed rest and supportive treatments; ensuring sufficient calories and water intake; maintaining water electrolyte balance and homeostasis, and strengthening psychotherapy for elder children when necessary. the icw group received the 5th edition recommendation's cm prescription extraorally for two weeks. all crudes of the decoction drugs were purchased from guangdong e-fong pharmaceutical. drugs with license number of yue 20160214 are as follows (table 1 ). to characterize the effect of herbal medicine, immune function, and inflammatory agents, levels of white blood cells were detected for all patients according to the manufacturer's instructions at the beginning and at the end of the two weeks. flow cytometry was used to determine the peripheral blood lymphocyte subsets (the levels of cd3+, cd4+, cd8+, and the ratios of cd4+/cd8+). white blood cell counts (wbc#), neutrophils count (n#), and lymphocyte counts (l#) were measured using a fully automatic blood rheological instrument. an adopted radioimmunoassay method the graphpad prism 8.01 software (san diego, ca, usa) was used for statistical analysis. the t test or rank sum test was used to compare the difference between two individual groups, and one-way analysis of variance (anova) was used to compare the difference among multiple groups. all the data normality had been checked before applying the parametric test. data with normal distribution are presented as the mean ± sd while iqr (interquartile range) is used for non-normal distribution. a p value less than 0.05 was considered statistically significant.since all cases were cured, a cure rate curve is drawn according to the cure time of each patient. the baseline demographic characteristics including gender, age, exposure to epidemic area, signs and symptoms, onset of symptom to hospital admission and dyspnea (median (iqr), d) and respiratory rate (median (iqr)) of both groups are shown in table 2 . of these patients, 10(27.0%) cases were exposed to wuhan area, and j o u r n a l p r e -p r o o f onset of symptom to, median (iqr), d lymphocyte counts (on admission and discharge), and crp were compared between the two groups. procalcitonin (pct) was excluded because only one of the patients had a higher than normal procalcitonin. the analysis showed that there were no statistical differences in these indicators between the two groups of patients. the results for all the above indicators were listed in table 3 and table 4 and figure 1 . cd4 and cd8 in two different groups were also collected. the analysis showed that there was no statistical difference in the values of cd4 and cd8 between the two groups. the results were listed in table 3 and table 4 . we performed spearman analysis for cd4 and il6, while the results suggest that there was no significant relationship between the two indicators (p=0.772). because the value of il6 was not normal distribution, we try to calculate the lg (il-6) and lg (il-6+1) to convert il-6 value to normal distribution, and then did pearson analysis between the converted value and cd4, however the results still do not indicate that there was a significant correlation (p=0.864,p=0.935). in part a, the values of the data were shown with mean ± standard deviation, while in part b, the values of data were shown in quartiles (25, 75) . length of hospital stays for two group were collected and the mean hospital stay for pwm group was 17.08days(17.058 ± 4.98),while median survival time of the icw group was 13.00days(13.00(13.00,22.00)). no significant difference was shown between them(p=0.662). cure curve according to the cure time of patients in each group as showed in figure 3 . logrank test was used to analyze the two cure rate of the two group and there seemed to be no significant difference between them(p=0.878).by the end of day 29, all patients were discharged and the final cure rate for both group were 100%. no specific vaccines has been recommended for preventive control of this disease [7] .currently, the four principles "early identification", "early isolation", "early diagnosis", and "early treatment" were adopted [8] . antibacterial agents are ineffective. in addition, no clinical trial have assessed the efficacy and safety of antiviral agents used clinically such as lopinavir, arbidol, resochin and prezista [9] . methylprednisolone maxim.) constitute an herb pair known as mahuang-xingren according to the principles of mutual reinforcement and mutual assistance, which is used wildly to treat asthma and bronchitis. as a classical cm combination, mahuang-xingren is a core component of many herbal prescriptions and chinese patent drugs for asthma and cough, due to j o u r n a l p r e -p r o o f the synergistic efficacy of its components and its few associated side effects [11, 12] . shengshigao (gypsum fibrosum), another popular partner of mahuang in respiratory disease, is frequently working for the purpose of removing heat from the lung and controlling asthma [13] . the cm channel tropism theory, which identifies the area the drug acts on, and is the selective action of the drug on the different areas of the human body. compatible use of shengshigao is part of the theory of chinese materia medica [14] . . modern pharmacological studies proved that gualou exhibit an anti-inflammatory effect [15, 16] . semen persicae's beneficial effects on various pathological situations has been indicated and most commonly blood stasis, which can be expressed as anti-coagulate, antiinflammatory and antioxidant activity [17] [18] [19] . atractylodis rhizoma possesses an antiallergic and anti-inflammatory potential through suppressing various immune effector cells, thus can ameliorate symptoms of allergic diseases may potentially prevent the development of subsequent atopic disorder such as allergic asthma through the influence of the gut microbiota [20, 21] . inflammation is a natural defense mechanism of the body that involves migration of leucocytes to the damaged tissues to destroy the inflammatory trigger or challenge [22] . infectious and allergic triggers induce an initial phase, and uncontrolled inflammatory response to the virus in combination with viral virulence can lead to severe lung devastation [23] [24] [25] . acute inflammation is characterized by infiltration of neutrophilic cells followed by various cytokines [26] . therefore, it has been broadly suggested that disease severity depends on the number of cytokines released during a j o u r n a l p r e -p r o o f cytokine storm in response to infection. in symptomatic individuals, the major cytokine which undergoes the earliest increase is il-6 [22, [26] [27] [28] . il-6 is multifunctional cytokine in lungs, is produced from epithelial cells, interstitial fibroblasts, macrophages and other inflammatory cells in response to a variety of stimuli that include allergens, respiratory viruses, exercise, environmental particles, and inhaled toxic particles [23, 29, 30] . il-6 is an important modulator for the transition from acute phase to chronic phase of inflammation, participates actively in inflammatory and immunomodulatory mechanisms, plays both pro-inflammatory and anti-inflammatory roles in humans [31] . herein, it regulates the cd4+ t cell-mediated responses that include cytokine production (il-4, il-13, il-17 and il-21), sil-6r production, and suppressive activity on treg cells [24, 32] . these mediators or responses, in turn, contribute to damage lung through their effects on mucus production, matrix deposition, and proteases release from granulocytes among others [33, 34] . in this study, peripheral blood was prepared for radioimmunoassay method to detect the content of il-6, il-8, tnf-α, pct and hs-crp. compared with pmw, patients in icw group had lower levels of il-6 and tnfα after treatment. it is known to all that il-6 is the one that not only elicits acute phase reactions but also leads to the development of specific cellular and humoral immune responses via end-stage b-cell differentiation, immunoglobulin secretion and t-cell activation [35, 36] . the decreased il-6 may play a role in alleviating inflammatory response and protecting against development of severe illness. il-6 is a maturing agent for b lymphocytes which stimulates the synthesis and secretion of various immunoglobulins, and induces the proliferation of thymic and peripheral t-cells [37, 38] . these killer cells participate in causative mechanisms that are responsible for specific diseases associated with the inflammatory or immune system alternations. but in this article, we performed spearman analysis for the counts of peripheral cd4 and il6, the result suggested that there was no significant relationship between the two indicators(p=0.772). as the value of il6 was not normal j o u r n a l p r e -p r o o f distribution, we try to calculate the lg (il-6) and lg (il-6+1) to convert il-6 value to normal distribution, and then did pearson analysis between the converted value and cd4, however the results still do not indicate that there was a significant correlation(p=0.864,p=0.935).the results were different from the pathological findings of covid-19 patient, which showed that the counts of peripheral cd4 and cd8 t cells were substantially reduced, while their status was hyperactivated, as evidenced by the high proportions of hla-dr (cd4 3·47%) and cd38 (cd8 39·4%) double-positive fractions. moreover, there was an increased concentration of highly proinflammatory ccr4+ccr6+ th17 in cd4 t cells. additionally, cd8 t cells were found to harbor high concentrations of cytotoxicgranules [39] . meanwhile, il-6 also plays functional roles in a variety of other processes including macrophage differentiation, neural cell differentiation and proliferation [40] . in this study, the counts of white cells, immune function, and inflammatory agents' alterations in treating severe pneumonia with cm were observed. the analysis showed that there were no statistical differences in these indicators between the two groups. a possible explanation is that the amount of samples and lack of repeats in this study. in conclusion, our study suggests that the 5th edition recommendation's cm prescription, can be safely used in the treatment of severe pneumonia of sars-cov-2 with yidu-toxicity blocking lung syndrome. cm can serve as a complementary medication to western medicine, though not reversible at the late stage in most cases, organ failure, which may have great significance in the prognosis of the disease, can be ameliorated and the inflammatory medium could be eliminated with early and timely diagnosis and treatment, which should be 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cytokine release syndrome modeling cytokine release syndrome cytokine release syndrome associated with chimeric-antigen receptor tcell therapy: clinicopathological insights current concepts in the diagnosis and management of cytokine release syndrome 4-oxadiazole/chalcone hybrids: design, synthesis, and inhibition of leukemia cell growth and egfr, src, il-6 and stat3 activities inhibitory effects of compounds and extracts from ampelopsis brevipedunculata on il-6-induced stat3 activation inhibitory effects of suppressor of cytokine signaling 3 on inflammatory cytokine expression and migration and proliferation of il-6/ifn-gamma-induced vascular smooth muscle cells interleukin-6 signaling requires only few il-6 molecules: relation to physiological concentrations of extracellular il-6 toxicity, pharmacokinetics and metabolism of a novel inhibitor of il-6-induced stat3 activation il-6 in inflammation, immunity, and disease monocyte-derived il-1 and il-6 are differentially required for cytokinerelease syndrome and neurotoxicity due to car t cells pathological findings of covid-19 associated with acute respiratory distress syndrome rtvp-1 promotes mesenchymal transformation of glioma via a stat-3/il-6-dependent positive feedback loop the authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper. all authors declare no competing interests. this study was sponsored by the national natural science fund(81903994). key: cord-343317-97n1j0jj authors: duan, xiaohua; han, yuling; yang, liuliu; nilsson-payant, benjamin e.; wang, pengfei; zhang, tuo; xiang, jenny; xu, dong; wang, xing; uhl, skyler; huang, yaoxing; chen, huanhuan joyce; wang, hui; tenoever, benjamin; schwartz, robert e.; ho, david. d.; evans, todd; pan, fong cheng; chen, shuibing title: identification of drugs blocking sars-cov-2 infection using human pluripotent stem cell-derived colonic organoids date: 2020-05-02 journal: biorxiv doi: 10.1101/2020.05.02.073320 sha: doc_id: 343317 cord_uid: 97n1j0jj the current covid-19 pandemic is caused by sars-coronavirus 2 (sars-cov-2). there are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of sars-cov-2 biology. as a result, there is an urgent need to create new disease models to study sars-cov-2 biology and to screen for therapeutics using human disease-relevant tissues. covid-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. moreover, these symptoms are associated with worse covid-19 outcomes1. here, we report using human pluripotent stem cell-derived colonic organoids (hpsc-cos) to explore the permissiveness of colonic cell types to sars-cov-2 infection. single cell rna-seq and immunostaining showed that the putative viral entry receptor ace2 is expressed in multiple hesc-derived colonic cell types, but highly enriched in enterocytes. multiple cell types in the cos can be infected by a sars-cov-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. we used hpsc-derived cos in a high throughput platform to screen 1280 fda-approved drugs against viral infection. mycophenolic acid and quinacrine dihydrochloride were found to block the infection of sars-cov-2 pseudo-entry virus in cos both in vitro and in vivo, and confirmed to block infection of sars-cov-2 virus. this study established both in vitro and in vivo organoid models to investigate infection of sars-cov-2 disease-relevant human colonic cell types and identified drugs that blocks sars-cov-2 infection, suitable for rapid clinical testing. the current covid-19 pandemic is caused by sars-coronavirus 2 (sars-cov-2). there are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of sars-cov-2 biology. as a result, there is an urgent need to create new disease models to study sars-cov-2 biology and to screen for therapeutics using human disease-relevant tissues. covid-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. moreover, these symptoms are associated with worse covid-19 outcomes 1 . here, we report using human pluripotent stem cell-derived colonic organoids (hpsc-cos) to explore the permissiveness of colonic cell types to sars-cov-2 infection. single cell rna-seq and immunostaining showed that the putative viral entry receptor ace2 is expressed in multiple hesc-derived colonic cell types, but highly enriched in enterocytes. multiple cell types in the cos can be infected by a sars-cov-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. we used hpsc-derived cos in a high throughput platform to previously, we reported a chemically-defined protocol to derive cos from hpscs 2 , which we modified slightly based on published studies 3 . in brief, hues8 hescs were induced with chir99021 (chir) and activin a to generate definitive endoderm (de) (extended data fig. 1a) . after 4 days of culture with chir +fgf4 to induce hindgut endoderm (he), cells were treated with bmp2, epidermal growth factor (egf), and chir for 3 days to promote specification of colon progenitors (cps). starting on day 11, cps were treated with a colonic medium containing chir, ldn193189 (ldn), and egf. after embedding these organoids in matrigel, spheroids became pseudostratified and progressively cavitated into fully convoluted organoids (fig. 1a) . the organoids expressed cdx2, villin and satb2, confirming colonic identity (fig. 1b) . immunocytochemistry confirmed that cos contain cell types found in normal colon, including keratin 20 (krt20) + epithelial cells, mucin 2 (muc2) + goblet cells, eph receptor b2 (ephb2) + transit-amplifying (ta) cells, and chromogranin a (chga) + neuroendocrine (ne) cells (fig. 1c) . single cell rna-seq was used to examine global transcript profiles at single cell resolution (extended data fig. 1b) . consistent with the immunostaining results, most cells express cdx2 and vil1 (extended data fig. 1c) . five cell clusters were identified including krt20 + epithelial cells, muc2 + goblet cells, ephb2 + ta cells, chga + ne cells, and lgr5 + or bmi1 + stem cells (fig. 1d-e, extended data fig. 1d) . we examined the expression of two factors associated with sars-cov-2 cell entry, the putative receptor ace2 and the protease tmprss2 4 . both are expressed in all five cell clusters, but highly enriched in krt20 + enterocytes ( fig. 1f-g) . two-dimensional correlation confirmed the co-expression relationship for ace2 and krt20, as well as ace2 and tmprss2 (fig. 1h) . immunohistochemistry further validated the coexpression of krt20 and ace2 in hpsc-cos (fig. 1i) . to model infection of hpsc-cos with sars-cov-2, we used a vesicular stomatitis virus (vsv) based sars-cov-2 pseudo-entry virus, with the backbone provided by a vsv-g pseudo-typed δ g-luciferase virus and the sars-cov-2 spike protein incorporated into the surface of the viral particle (see methods for details) 5, 6 . cos were fragmentized and innoculated with the sars-cov-2 pseudo-entry virus. 24 or 48 hr post-infection (hpi), the cells were lysed and monitored for luciferase activity (extended data fig. 2a) . the organoids infected with sars-cov-2 pseudo-entry virus at moi=0.01 showed a strong signal at 24 hpi (fig. 2a) . single cell rna-seq was performed to examine the hpscderived cos at 24 hpi. the same five cell populations were identified in the cos postinfection ( fig. 2b and extended data fig. 2b-d) . compared to uninfected samples, the krt20 + enterocyte population decreased significantly (fig. 2c) . immunostaining confirmed increased cellular apoptosis, suggesting toxicity for these cells (extended data fig. 2e ). in addition, the ace2 + population was significantly depleted (fig. 2e) . the mrnas of sars-cov-2 pseudo-entry virus, including vsv-ns, vsv-n, and vsv-m, were detected in all five cell populations (fig. 2f) , but not in the uninfected cos (extended data fig. 2f) . immunostaining further validated the expression of luciferase in ace2 + , vil1 + , cdx2 + , krt20 + , and muc2 + cells (fig. 2g) . humanized mice carrying hpsc-cos in vivo provide a unique platform for modeling covid-19. in brief, hpsc-cos were transplanted under the kidney capsule of nodscid il2rg null mice. two weeks after transplantation, the organoid xenograft was removed and examined for cellular identities (fig. 2h) . consistent with in vitro culture, ace2 can be detected in hpsc-derived krt20 + enterocytes (fig. 2i) . sars-cov-2 pseudo-entry virus was inoculated locally. at 24 hpi, the xenografts were removed and analyzed by immuno-histochemistry. luciferase was detected in the xenografts inoculated with virus, but not in mock-infected controls (fig. 2j) . immunohistochemistry detected luciferase in ace2 + and villin + cells, suggesting these are permissive to sars-cov-2 pseudo-entry virus infection in vivo (fig. 2k) . next, we adapted hpsc-cos to a high throughput screening platform and probed the prestwick fda-approved drug library to identify drug candidates capable of blocking sars-cov-2 pseudo-virus infection. in brief, hpsc-cos were cultured in 384-well plates. after overnight incubation, organoids were treated with drugs from the library at 10 μ m. one hour post-exposure with drugs, the organoids were innoculated with the sars-cov-2 pseudo-entry virus. 24 hpi, the organoids were analyzed for luciferase activity (fig. 3a) . drugs that decreased the luciferase activity by at least 75% were chosen as primary hit drugs (fig. 3b) . eight drugs (extended data table 1 ) were identified as lead hits and further tested for their capacities to decrease the luciferase signal in a dose-dependent manner (extended data fig. 3) . these drugs could potentially function through blocking virus entry, by decreased cell survival, or even by directly inhibiting luciferase activity. to distinguish these possibilities, the lead hit drugs were tested in comparison to hpsc-cos infected with a control vsvg-luciferase reporter virus. four of the lead hit drugs showed specificity to sars-cov-2 pseudoentry virus, including mycophenolic acid (mpa, fig. 3c fig. 3h) . co-explanted humanized mice were treated with 50 mg/kg mpa by ip injection, followed by local inoculation of sars-cov-2 pseudo-entry virus. at 24 hpi, the mice were euthanized, and xenografts were analyzed by immunostaining. luc + cells in the xenografts of mpa-treated mice were significantly lower than those of vehicletreated mice (fig. 3i-k) . finally, hpsc-cos were infected with sars-cov-2 virus at moi=0.1 or 0.01. at 24 hpi, immunostaining detected the expression of sars-cov membrane protein in the infected hpsc-cos, which partially co-localized with cdx2 and krt20 (fig. 4a) . bulk rna sequencing confirmed viral transcripts in the sars-cov-2 infected hpsc-cos (moi=0 .1, fig. 4b) . the mock and infected hpsc-cos separated clearly into two distinct clusters in a pca plot (fig. 4c) . differential gene expression analysis showed striking induction of chemokine gene expression, including for il1a, cxcl8, cxcl6, cxcl11, and il1b, yet with no detectable levels of ifn-i or ifn-iii, which is consistent with recent reports [8] [9] [10] (fig. 4d) . ingenuity pathway analysis of the differential gene expression list highlighted the production of nitric oxide and reactive oxygen species, oxidative phosphorylation, as well as il-15 production (fig. 4e) . the hpsc-cos were pre-treated with mpa or qnhc and infected with a relatively high titer of sars-cov-2 virus (moi=0.1). immunostaining confirmed the decrease of sars-cov-2 + cells in mpa or qnhc-treated hpsc-cos (fig. 4f) . in summary, we report that hpsc-derived cos express ace2 and tmprs2s2 and are permissive to sars-cov-2 infection. there is currently a lack of physiologically relevant models for covid-19 disease that enable drug screens. previous studies were based on clinical data or transgenic animals, for example mice that express human ace2. however, such transgenic animals fail to fully recapitulate the cellular phenotype and host response of human cells 11, 12 . we adapted a hpsc-derived co platform for high throughput drug screening. using disease-relevant normal colonic human cells, we screened 1280 fda-approved compounds and identified mpa and qnhc, two drugs that can block the entry of sars-cov-2 into human cells. strikingly, in this assay, the efficacies of mpa and qnhc for blocking viral entry are more than 5 times higher than chloroquine, a drug recently authorized by the fda for emergency use to treat covid-19 patients. moreover, the mpa concentrations effective in blocking viral entry and replication are below that which is routinely used in clinical therapy 13 . mpa is a reversible, non-competitive inhibitor of inosine-5′-monophosphate dehydrogenase and is used widely and safely as an immunosuppressive drug (mycophenolate mofetil; cellcept) to prevent organ rejection after transplantation and for the treatment of autoimmune diseases 14 . mpa has been reported to block replication of human immunodeficiency virus 15 , dengue 16 , as well as middle east respiratory syndrome coronavirus (mers-cov) 17 . several studies on mers-cov suggest that mpa may noncompetitively inhibit the viral papain-like protease while also altering host interferon response 17, 18 . a recent study also predicts mpa would modulate the interaction between host protein inosine-5'-monophosphate dehydrogenase 2 (impdh2) and sars-cov-2 protein nonstructural protein 14 (nsp14) 19 . furthermore, a clinical study of mers-cov suggested that the patients treated with mycophenolate mofetil has 0% mortality rate, which is significantly lower than the overall mortality rate as 37% 20 . qnhc (acriquine ® , atabrine ® , atebrin ® , mepacrine ® ) is an fda-approved antimalarial drug used more recently as an anthelmintic, antiprotozoal, antineoplastic agent, and antirheumatic 21 . recent studies have shown that quinacrine protects mice against ebola virus infection in vivo 22 . both mpa and qnhc can be considered candidates for clinical trials of covid-19 therapy. recombinant indiana vsv (rvsv) expressing sars-cov-2 spikes were generated as previously described 23 . hek293t cells were grown to 80% confluency before transfection with pcmv3-sars-cov2-spike (kindly provided by dr. peihui wang, shandong university, china) using fugene 6 (promega). cells were cultured overnight at 37°c with 5% co2. the next day, medium was removed and vsv-g pseudotyped δ g-luciferase (g*δg-luciferase, kerafast) was used to infect the cells in dmem at an moi of 3 for 1 hr before washing the cells with 1x dpbs three times. dmem supplemented with 2% fetal bovine serum and 100 i.u./ml penicillin and 100 μ g/ml streptomycin was added to the infected cells and they were cultured overnight as described above. the next day, the supernatant was harvested and clarified by centrifugation at 300g for 10 min and aliquots stored at −80°c. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), isolate usa to assay pseudo-typed virus infection on colon organoids, cos were seeded in 24 well plates. pseudo-typed virus was added at moi=0.01 plus polybrene at a final concentration of 8 μ g/ml, and the plate centrifuged for 1 hr at 1200g. at 3 hpi, the infection medium was replaced with fresh medium. at 24 hpi, colon organoids were harvested for luciferase assays or immunostaining analysis. for chemical screening analysis, colon organoids were digested by tryple and seeded in 384 well plates at 1x10 4 cells per well. after chemical treatment, pseudo-typed virus was added at moi=0.01 and the plate centrifuged for 1 hr at 1200g. at 24 hpi, hpsc-cos were harvested for luciferase assays according to the luciferase assay system protocol (promega). cells were lysed in ripa buffer for protein analysis or fixed in 5% formaldehyde for 24 h for immunofluorescent staining, prior to safe removal from the bsl-3 facility. the colon organoids were released from matrigel using cell recovery solution (corning) on ice for 1 hr, followed by fixation in 4% paraformaldehyde for 4 hr at 4°c, washed twice with 1x pbs and allowed to sediment in 30% sucrose overnight. the organoids were then embedded in oct (tissuetek) and cryo-sectioned at 10 μ m thickness. for indirect immunofluorescence staining, sections were rehydrated in 1x pbs for 5 min, permeabilized with 0.2% triton in 1x pbs for 10 min, and blocked with blocking buffer containing 5% normal donkey serum in 1x pbs for 1 hr. the sections were then incubated with the corresponding primary antibodies diluted in blocking buffer at 4°c overnight. the following day, sections were washed three times with 1x pbs before incubating with fluorophore-conjugated secondary antibody for one hr at rt. the sections were washed three times with 1x pbs and mounted with prolong gold antifade mounting media with dapi (life technologies). images were acquired using an lsm880 laser scanning confocal microscope (zeiss) and processed with zen or imaris (bitplane) software. organoids and tissues were fixed in 4% pfa for 20 min at rt, blocked in mg 2+ /ca 2+ free pbs plus 5% horse serum and 0.3% triton-x for 1 hr at rt, and then incubated with primary antibody at 4°c overnight. the information for primary antibodies is provided in extended data table 2 . secondary antibodies included donkey anti-mouse, goat, rabbit or chicken antibodies conjugated with alexa-fluor-488, alexa-fluor-594 or alexa-fluor-647 fluorophores (1:500, life technologies). nuclei were counterstained by dapi. antibodies. antibody-mediated fluorescence was detected on a li-cor odyssey clx imaging system and analyzed using image studio software (li-cor). the colon organoids cultured in matrigel domes were dissociated into single cells using 0.25% trypsin (gibco) at 37°c for 10 min, and the trypsin was then neutralized with dmem f12 supplemented with 10% fbs. the dissociated organoids were pelleted and resuspended with l15 medium (gibco) supplemented with 10 mm hepes, and 10 ng/ml dnasei (sigma). the resuspended organoids were then placed through a 40 µm filter to obtain a single cell suspension, and stained with dapi followed by sorting of live we filtered cells with less than 300 or more than 8000 genes detected as well as cells with mitochondria gene content greater than 30%, and used the remaining cells (6175 cells for the uninfected sample and 2962 cells for the infected sample) for downstream analysis. we normalized the gene expression umi counts for each sample separately using a deconvolution strategy 24 implemented by the r scran package (v.1.14.1). in particular, we pre-clustered cells in each sample using the quickcluster function; we computed size factor per cell within each cluster and rescaled the size factors by normalization between clusters using the computesumfactors function; and we normalized the umi counts per cell by the size factors and took a logarithm transform using the normalize function. we further normalized the umi counts across samples using the multibatchnorm function in the r batchelor package (v1.2.1). we identified highly variable genes using the findvariablefeatures function in the r seurat (v3.1.0) 25 , and selected the top 3000 variable genes after excluding mitochondria genes, ribosomal genes and dissociation-related genes. the list of dissociation-related genes was originally built on mouse data 26 , we converted them to human ortholog genes using ensembl biomart. we aligned the two samples based on their mutual nearest neighbors (mnns) using the fastmnn function in the r batchelor package, this was done by performing a principal component analysis (pca) on the highly variable genes and then correcting the principal components (pcs) according to their mnns. we selected the corrected top 50 pcs for downstream visualization and clustering analysis. we ran the uniform manifold approximation and projection (umap) dimensional reduction using the runumap function in the r seurat 25 package with training epochs setting to 2000. we clustered cells into eight clusters by constructing a shared nearest neighbor graph and then grouping cells of similar transcriptome profiles using the findneighbors function and findclusters function (resolution set to 0.2) in the r seurat package. we identified marker genes for each cluster by performing differential expression analysis between cells inside and outside that cluster using the findmarkers function in the r seurat package. after reviewing the clusters, we merged four clusters that were likely from stem cell population into a single cluster (lgr5 + or bmi1 + stem cells) and kept the other four clusters (krt20 + epithelial cells, muc2 + goblet cells, ephb2 + ta cells, and chga + ne cells) for further analysis. we re-identified marker genes for the merged five clusters and selected the top 10 positive marker genes per cluster for heatmap plot using the doheatmap function in the r seurat package 25 . hpsc-cos were harvested by cell scraper, mixed with 20 µl matrigel (corning) and transplanted under the kidney capsule of 7-9 weeks old male nsg mice. two weeks post-transplantation, sars-cov-2 pseudo-entry virus was inoculated locally at 1x10 3 ffu. at 24 hpi, the mice were euthanized and used for immunohistochemistry analysis. to determine the mpa's activity in vivo, the mice were treated with 50 mg/kg mpa in (10%dmso/90% corn oil) by ip injection. two hours after drug administration, sars-cov-2 pseudo-entry virus was inoculated locally at 1x10 3 ffu. at 24 hpi, the mice were euthanized and used for immunohistochemistry analysis. all animal work was conducted in agreement with nih guidelines and approved by the wcm institutional animal care and use committee (iacuc) and the institutional biosafety committee (ibc). to perform the high throughput small molecule screen, hpsc-cos were dissociated using tryple for 20 min in a 37℃ waterbath and replated into 10% matrigel-coated 384well plates at 20,000 cells/40 µl medium/well. after 6 hr, cells were treated with compounds from an in-house library of ~1280 fda-approved drugs (prestwick) at 10 µm. dmso treatment was used as a negative control. one hour late, cells will be infected with sars-cov-2 pseudo virus (moi=0.01). after 24 hpi, hpsc-cos were harvested for luciferase assay following the luciferase assay system protocol (promega). the authors declare the following competing interests: r.e.s. is on the scientific advisory board of miromatrix inc. the other authors have no competing of interest. scrna-seq and rna-seq data is available from the geo repository database, accession number gse147975. expression level identity a. npm1 ciqbp ldhb mgst1 snhg8 ncl plin2 hist1h4c lcn15 gdf15 phlda2 igfbp3 lrrc75a s100a16 emp3 cd24 b4galt1 ceacam6 hnrnph1 anpep aldob rbp2 tm4sf2o selenop fabp6 tspan1 muc13 apoa1 kctd12 scgn tubaia or51e1 npw cyba2 tph1 chga mdk cdkn1c clca1 fcgbp muc2 guca2a selenom frzb st6galnac1 hes6 tff3 reg4 a. colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing differentiation of human pluripotent stem cells into colonic organoids via transient activation of bmp signaling sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines establishment and validation of a pseudovirus neutralization assay for sars-cov-2 chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice a pneumonia outbreak associated with a new coronavirus of probable bat origin characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov consensus report on therapeutic drug monitoring of mycophenolic acid in solid organ transplantation the inhibition of nucleic acid synthesis by mycophenolic acid effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo mycophenolic acid inhibits dengue virus infection by preventing replication of viral rna thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia repurposing quinacrine against ebola virus infection in vivo immunization-elicited broadly protective antibody reveals ebolavirus fusion loop as a site of vulnerability pooling across cells to normalize single-cell rna sequencing data with many zero counts comprehensive integration of single-cell data single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations moderated estimation of fold change and dispersion for rna-seq data with deseq2 key: cord-334976-53cd16w5 authors: jo, seri; kim, suwon; kim, dae yong; kim, mi-sun; shin, dong hae title: flavonoids with inhibitory activity against sars-cov-2 3clpro date: 2020-08-04 journal: journal of enzyme inhibition and medicinal chemistry doi: 10.1080/14756366.2020.1801672 sha: doc_id: 334976 cord_uid: 53cd16w5 coronavirus disease 2019 (covid-19) has been a pandemic disease of which the termination is not yet predictable. currently, researches to develop vaccines and treatments is going on globally to cope with this disastrous disease. main protease (3clpro) from severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is one of the good targets to find antiviral agents before vaccines are available. some flavonoids are known to inhibit 3clpro from sars-cov which causes sars. since their sequence identity is 96%, a similar approach was performed with a flavonoid library. baicalin, herbacetin, and pectolinarin have been discovered to block the proteolytic activity of sars-cov-2 3clpro. an in silico docking study showed that the binding modes of herbacetin and pectolinarin are similar to those obtained from the catalytic domain of sars-cov 3clpro. however, their binding affinities are different due to the usage of whole sars-cov-2 3clpro in this study. baicalin showed an effective inhibitory activity against sars-cov-2 3clpro and its docking mode is different from those of herbacetin and pectolinarin. this study suggests important scaffolds to design 3clpro inhibitors to develop antiviral agents or health-foods and dietary supplements to cope with sars-cov-2. before the nightmares of sars and mers has been forgotten, a novel coronavirus causing severe acute respiratory syndrome (sars-cov-2) triggered another pneumonia outbreak in wuhan, china. now it has posed significant threats to international health and the economy. coronaviruses (covs) are positive singlestranded rna virus that can infect both animals and humans 1 . human coronavirus (hcovs) represents a major group of covs associated with various respiratory diseases, from common colds to severe pneumonia and bronchiolitis 2 . today, hcov is recognised as one of the fastest-evolving viruses derived from their high element nucleotide replacement rates and recombination 3 . in particular, the virus, which is another characteristic of the past, is making some of the preceding experience obsolete. a virus genome analysis with a patient with sars-cov-2 infection revealed that its nucleotide identities with those of bat sarslike-covzxc21 and human sars-cov were $89% and $82%, respectively 4, 5 . like the sars and mers-cov genomes, sars-cov-2 includes two open reading frames orf1a and orf1b, each translated as two viral multi-protein pp1a and pp1ab by host ribosomes. orf1a encodes two cysteine proteases, a papain-like protease (plpro) and a 3 c-like protease (3clpro). plpro cleaves the first three sites at 181-182, 818-819, and 2763-2764 at the nterminus and 3clpro cuts at the remaining 11 sites at the c-terminus, and forming 15 non-structural proteins (nsp) 6, 7 . the crystal structure of 3clpro from sars-cov-2 has been deposited in the protein data bank (6lu7) and revealed the homodimeric form almost similar to that of sars-cov. they share common 294 same amino acids out of 306 residues and thus their sequence identity is 96%. except for two amino acids ser46 and val86, none is different in the active site pocket of sars-cov-2 3clpro. the putative cleavage sites of 16 nsp of sars-cov-2 predicted by bioinformatics contain qs and qa which are the same amino acid sequences found in 16 non-structural proteins of sars-cov. it coincides with the conserved active site together with 96% sequence identity of both 3clpros. therefore, in the proteolytic site, both 3clpros may prefer glutamine at p1 position. p2, p3, and p4 positions may require leucine, basic residues, small hydrophobic residues, respectively 8 . at p1 0 , p2 0 , and p3 0 positions, small residues are predicted for the two former sites and no strong preference for the last. in this study, we employed a proteolytic method to probe inhibitory compounds against sars-cov-2 3clpro. a synthetic peptide labelled with an edans-dabcyl fret (fluorescence resonance energy transfer) pair was used to probe sars-cov-2 3clpro inhibitory compounds. with the fret pair, an in-house flavonoid library was screened. for a molecular level study, we performed the proteolytic assay with flavonoids followed by an induced-fit docking experiment with top hits. with the results, we investigated a structural and functional relationship of flavonoids endowing binding affinities to sars-cov-2 3clpro. since the autocleavage process is indispensable with viral propagation, 3clpro is an attractive drug target for treating covid-19 patients. the information can be applied to develop synthetic medicine or healthfood compounds. the coding sequence of sars-cov-2 3 c-like proteinase (ncbi ref. seq. yp_009725301.1) was synthesised chemically by bioneer (daejeon, korea) and cloned into a bacteriophage t7-based expression vector. the plasmid dna was transformed into e. coli bl21 (de3) for protein expression. e. coli bl21 (de3) cells were grown on luria-bertani (lb) agar plates containing 150 lg ml à1 ampicillin. several colonies were picked and grown in capped test tubes with 10 ml lb broth containing 150 lg ml à1 ampicillin. a cell stock composed of 0.85 ml culture and 0.15 ml glycerol was prepared and frozen at 193 k for use in a large culture. the frozen cell stock was grown in 5 ml lb medium and diluted into 1000 ml fresh lb medium. the culture was incubated at 310 k with shaking until an od 600 of 0.6-0.8 was reached. at this point, the expression of sars-cov-2 3clpro was induced using isopropyl-b-d-1-thiogalactopyranoside (iptg) at a final concentration of 1 mm. the culture was further grown at 310 k for 3 h in a shaking incubator. cells were harvested by centrifugation at 7650 g (6500 rev min à1 ) for 10 min in a high-speed refrigerated centrifuge at 277 k. the cultured cell paste was resuspended in 25 ml of a buffer consisting of 50 mm tris-hcl ph 8.0, 100 mm nacl, 10 mm imidazole, 1 mm phenylmethylsulfonyl fluoride (pmsf), 10 lg ml à1 dnase i. the cell suspension was disrupted using an ultrasonic cell disruptor (digital sonifier 450, branson, usa). cell debris was pelleted by centrifugation at 24,900 g (15,000 rev min à1 ) for 30 min in a highspeed refrigerated ultra-centrifuge at 277 k. the protein was purified by affinity chromatography using a 5 ml hi-trap q his column (ge healthcare, piscataway, new jersey, usa). the column was equilibrated with a buffer consisting of 20 mm bis-tris ph 7.5. the column was eluted using a linear nacl gradient to 1.0 m nacl and the protein was eluted at 0.18 m nacl. the purified protein was buffer exchanged into 20 mm bis-tris ph 7.5 using vivaspin 20 mwco 10 kda (ge healthcare), a centrifugal device. sds-page showed one band around 34 kda (33796.64 da), corresponding to the molecular weight of sars-cov-2 3clpro. the custom-synthesised fluorogenic substrate, dabcyl-ktsavlqsgfrkme-edans (anygen, gwangju, korea), was used as a substrate for the proteolytic assay using the sars-cov-2 3clpro 9 . this substrate contains the nsp4/nsp5 cleavage sequence, gvlq#sg 10 , and works as a generic peptide substrate for many coronaviruses including the sars-cov-2 3clpro. the peptide was dissolved in distilled water and incubated with each protease. a spectramax i3x multi-mode microplate reader (molecular devices) was used to measure spectral-based fluorescence. the proteolytic activity was determined at 310 k by following the increase in fluorescence (k excitation ¼ 340 nm, k emission ¼ 490 nm, bandwidths ¼ 9, 15 nm, respectively) of edans upon peptide hydrolysis as a function of time. assays were conducted in black, 96-well plates (nunc) in 300 ll assay buffers containing protease and substrate as follow; for the sars-cov-2 3clpro assay, 2.04 ll of 0.294 mm protease containing 20 mm tris ph 7.5 was incubated with 7.5 ll of 0.1 mm substrate at 310 k for 2 h 30 min before measuring relative fluorescence units (rfu). before the assay, the emission spectra of antiviral agents and some of their adjuvants were surveyed after illuminating at 340 nm to avoid the overlapping with the emission spectrum of edans. every compound was suitable to be tested. the final concentration of the protease, peptide, and chemical used at the assay was 2 mm, 2.5 mm and 80 mm each. at first, sars-cov-2 3clpro and chemical were mixed and preincubated at room temperature for 1 h. the reaction was initiated by the addition of the substrate and each well was incubated at 310 k for 2 h 30 min. after that, we measured the fluorescence of the mixture on the black 96-well plate using the endpoint mode of spectramax i3x where the excitation wavelength was fixed to 340 nm and the emission wavelength was set to 490 nm using 9, 15 nm bandwidth, respectively. all reactions were carried out in triplicate. among the first seventy flavonoids (supplementary table 1 ), one of them was picked up to further assay at a concentration range of 4 mm $ 240 mm. the ic 50 value which is the value causing 50% inhibition of sars-cov-2 3clpro was calculated by nonlinear regression analysis using graphpad prism 7.03 (graphpad software, san diego, ca, usa). fret protease assays with sars-cov-2 3clpro in the presence of triton x-100 the proteolytic assay using sars-cov-2 3clpro in the presence of triton x-100 has been performed to differentiate the artificial inhibitory activity of chemicals through non-specific binding with proteases by forming aggregate or complexation. the concentration used in this study was 0.01%. to confirm the feasibility of the assay method independently, the fluorescence spectra from tryptophans of sars-cov-2 3clpro with candidate inhibitors were investigated 11 . the fluorescence measurements were recorded with a spectramax i3x multi-mode microplate reader (molecular devices) at excitation and emission wavelengths of 295 nm and 320-500 nm, respectively. the optimal excitation and emission wavelengths were determined by softmax pro. three tryptophans of sars-cov-2 3clpro showed a fluorescence emission with a peak at 340 nm after the excitation at the wavelength of 295 nm. in contrast, the flavonoids were almost non-fluorescent under the same experiment condition. each 80 mm chemical was incubated with 2 mm sars-cov-2 3clpro for 1 h and the fluorescence intensity of the mixture was measured. all the docking and scoring calculations were performed using the schr€ odinger software suite (maestro, version 11.8.012). the compounds were extracted from the pubchem database in the sdf format and were combined in one file. the file was then imported into maestro and prepared for docking using ligprep. the atomic coordinates of the crystal structure of sars-cov-2 3clpro (6lu7) were retrieved from the protein data bank and prepared by removing all solvent and adding hydrogens and minimal minimisation in the presence of bound ligand using protein preparation wizard. ioniser was used to generate an ionised state of all compounds at the target ph 7.0 ± 2.0. this prepared lowenergy conformers of the ligand were taken as the input for an induced-fit docking. the induced-fit docking protocol 12 was run from the graphical user interface accessible within maestro 11.8.012. receptor sampling and refinement were performed on residues within 5.0 å of each ligand for each of the ligand-protein complexes. with prime 13 , a side-chain sampling and prediction module, as well as the backbone of the target protein, were energy minimised. a total of induced-fit receptor conformations were generated for each of the ligands. re-docking was performed with the test ligands into their respective structures that are within 30.0 kcal/mol of their lowest energy structure. finally, the ligand poses were scored using a combination of prime and glidescore scoring functions 14 . the cell yield harvested for purification of sars-cov-2 3clpro was 2.9 g per 2000 ml of e. coli culture. the amount of purified protein synthesised was 29.85 mg. for storage and assay, the protein solution was concentrated to 99.5 mg ml à1 . the concentrate was diluted to 2 mm when the inhibitory assay was carried out. a flavonoid library consisting of 10 different scaffolds was also built (figure 1 ). it contains 5 isoflavones, 1 isoflavane, 18 flavones, 16 flavonols, 7 flavanols, 7 flavanones, 4 flavanonol, 1 prenylflavonoid, 9 chalcones, and 2 unclassified flavonoids (supplementary table 1 ). we applied the library to assay sars-cov-2 3clpro. using 70 flavonoids, an inhibitory effect of each compound at 80 mm was tested. five flavones; orientin (8-b-d-glucopyranosyl-3 0 ,4 0 ,5,7-tetrahydroxyflavone), baicalin (7-d-glucuronic acid-5,6-dihydroxyflavone), pectolinarin (5,7-dihydroxy 4 0 ,6-dimethoxyflavone 7rutinoside), homoplantaginin (6-methoxyapigenin 7-o-glucoside), rhoifolin (apigenin-7-o-rhamnoglucoside) and two flavonols; herbacetin (3,4 0 ,5,7,8-pentahydroxyflavone), rutin (3,3 0 ,4 0 ,5,7-pentahydroxyflavone-3-rutinoside) were found to have inhibitory activity. among them, baicalin, herbacetin, and pectolinarin were found to have prominent inhibitory activity. the binding affinity data were plotted as log inhibitor concentration versus percent fluorescence inhibition (figure 2 ). the compound showed the severely reduced fluorescent intensity and thus represented their sars-cov-2 3clpro inhibitory activity. the ic 50 values were calculated from the dose-dependent inhibitory curves of baicalin, herbacetin and pectolinarin. the measured values were 34.71 mm, 53.90 mm and 51.64 mm, respectively. since flavonoids are known to be aggregated through complexity and thus non-specifically inhibit various proteases, the assay in the presence of triton x-100 was also performed 15 . before the examination, we tested the effects of triton x-100 on sars-cov-2 3clpro. there was no difference in catalyst activity with or without 0.01% triton x-100. therefore, the assay was performed at a concentration of 0.01% triton x-100. to independently confirm the inhibitory activity of flavonoids, a general tryptophan based assay method was employed. tryptophan was well known to emit its fluorescence. thus, if the tryptophan is properly located in the protein, the change in fluorescence intensity can reflect the binding state of the chemicals and can be used to determine the interaction between the protein and the chemical. the sars-cov-2 3clpro contains three tryptophan residues. therefore, its fluorescence change can reflect the environmental variation of the protein. the sars-cov-2 3clpro used in this study displays a fluorescence peak at 340 nm after the tryptophan excitation wavelength of 295 nm. we monitored the change of the fluorescence intensities depending on the presence or absence of all flavonoids. since each compound in the flavonoid library was almost non-fluorescent under experimental conditions, the change in fluorescence intensity reflects the interaction between the protein and chemical. the decreased emission intensity confirmed the complex formation between the sars-cov-2 3clpro and the inhibitory compound ( figure 3 ). flavonoids constitute a large class of food constituents influencing a positive impact on health. many flavonoids occur as large molecules called tannins. these flavonoids form polymers through various carbon-carbon and ether linkages. flavonoids are important kinds of natural products widely found in fruits and vegetables. a wide range of beneficial roles with antioxidants, anti-inflammatory, anti-mutagens, and anti-cancer-causing properties have been reported against various diseases such as parkinson's disease, alzheimer's disease, and cancer [16] [17] [18] [19] [20] . intriguingly, some flavonoids possess antiviral activity and some of their direct proteolytic inhibitory activity against sars-cov 3clpro has been published 21 . the recent pandemic caused by sars-cov-2 is going on severely and thus patients with covid-19 are exponentially increasing. therefore, the development of vaccines and antiviral agents against sars-cov-2 is urgent. currently, some compounds designed for other rna viruses such as favipiravir, lopinavir, and tenofovir have been used in clinical trials in patients with covid-19 (http://clinicaltrials.gov). therefore, designing chemicals targeting directly against specific enzymes of sars-cov-2 is very important. as mentioned above 3clpro of sars-cov-2 is a good target to design inhibitory chemicals since some flavonoids inhibit the proteolytic activity of sars-cov 3clpro 21 . the 96% sequence identity between them together with their conserved active site suggests that similar flavonoids may work for sars-cov-2 3clpro. the full sequence of sars-cov-2 3clpro was purified and assayed against the in-house flavonoid library composed of various flavonoid derivatives with ten different scaffolds. the promising compounds include five flavones orientin, baicalin, pectolinarin, homoplantaginin and rhoifolin and two flavonols; herbacetin, rutin. among them, baicalin, herbacetin and pectolinarin revealed the prominent inhibitory activity against sars-cov-2 3clpro. the measured ic50 values were 34.71, 53.90 and 51.64 mm, respectively. an in silico docking study for baicalin, herbacetin and pectolinarin was performed to deduce their binding mode and binding affinity. the glide scores of three compounds were à8.776, à8.738 and à10.969, respectively. the binding modes of herbacetin and pectolinarin were similar to those obtained from the docking study of the catalytic domain of sars-cov 3clpro 21 . in the case of herbacetin, the phenyl moiety occupies the s1 site of sars-cov-2 with the aid of glu166 and the chromen-4-one scaffold locates in the s2 site with hydrogen bonds with his41 and gln189. the binding mode of pectolinarin showed that the l-mannopyranosyl b-d-glucopyranoside moiety occupies the s1 and s2 sites and the chromen-4-one moiety locates in the s2 and s3 0 site similar to the binding mode observed in sars-cov 3clpro. the binding mode of baicalin is described in figure 4 . due to the presence of the glucuronate moiety, its binding mode is more similar to that of pectolinarin than herbacetin. the important hydrogen bonds with glu166 are formed by the 6-hydroxyl group attached to the chromen-4-one moiety (2.778 ð) and the 5hydroxyl group attached to the glucuronate moiety (3.331 ð). as a result, more than half of the compound was docked in the s1 site. the two hydrogen bonds of the 3-hydroxyl and carboxylic acid groups from the glucuronate moiety formed with gly143 (2.962 ð) and asn142 (2.883 ð), respectively, stabilise the overall interaction. the p-p stacking between his41 and the phenyl moiety is first observed in baicalin. in the previous results of sars-cov 3clpro 21 , only the three effect flavonoids (herbacetin, pectolinarin, and rhoifolin) were mentioned. however, other four flavonoids (orientin, baicalin, homoplantaginin, and rutin) were also detected though their efficiency was low (data not shown). it is actually expected due to the 96% sequence identity of the two 3clpro. interestingly, the affinity of rhoifolin became weaken but the affinity of baicalin became stronger with sars-cov-2 3clpro. the efficiency of two flavonoids (herbacetin and pectolinarin) was still promising. considering almost the same active site composition of both 3clpros, the different binding affinity seemed to be a bit strange. however, this difference could be explained by the two factors. at first, the difference may be originated from the different constructs used in both cases. in this study, we used the whole sars-cov-2 3clpro but the catalytic domain of sars-cov 3clpro (amino acids met1-thr196) had been used in the previous study. in the crystal structures of both 3clpros, there is a dimerisation domain influencing proteolytic activity. that domain was not in the previous sars-cov 3clpro study. nevertheless, a long incubation result showed that the proteolytic function of the sars-cov 3clpro is still present without the dimerisation domain. second, there are some different residues comprising active site residues. ser46 and val86 in sars-cov-2 3clpro are replaced by ala46 and leu86 in sars-cov 3clpro, respectively. though their substitution seems to be minor in amino acid properties, the different residues may clearly contribute different affinities. the assay and docking result indicates important conclusions. at first, flavonoids have a wide range of binding affinity to sars-cov-2 3clpro as observed in sars-cov 3clpro 21 . second, the presence of the monocarbohydrate derivative, glucuronate, observed in baicalin severely influences to its mode compared with herbacetin and pectolinarin. the unique binding mode of baicalin can be applied to newly design inhibitory compounds against sars-cov-2 3clpro with the different scaffold. a further study is going on to make derivatives that lead to better inhibitory compounds and develop health-foods and dietary supplements to cope with sars-cov-2 based on this study. the molecular biology of coronaviruses a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes sars and mers: recent insights into emerging coronaviruses a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan profiling of substrate specificities of 3c-like proteases from group 1, 2a, 2b, and 3 coronaviruses characterization of sars main protease and inhibitor assay using a fluorogenic substrate prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus the enlarged view represents baicalin in the active site pocket with a semi-transparent view of the molecular surface of sars-cov-2 3clpro. (b) a 2d schematic representation of the interactions of baicalin with the side chains of sars-cov-2 3clpro spectroscopic investigation of interaction between mangiferin and bovine serum albumin novel procedure for modeling ligand/receptor induced fit effects a hierarchical approach to all-atom protein loop prediction extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes aggregating behavior of phenolic compounds-a source of false bioassay results? tissue distribution and neuroprotective effects of citrus flavonoid tangeretin in a rat model of parkinson's disease neuroprotective effects of a standardized flavonoid extract of safflower against neurotoxininduced cellular and animal models of parkinson's disease the flavonoid baicalein rescues synaptic plasticity and memory deficits in a mouse model of alzheimer's disease. behav flavonoid effects relevant to cancer the natural flavonoid fisetin inhibits cellular proliferation of hepatic, colorectal, and pancreatic cancer cells through modulation of multiple signaling pathways inhibition of sars-cov 3cl protease by flavonoids the authors declare no conflict of interest. key: cord-350855-gofzhff7 authors: hou, yixuan j.; okuda, kenichi; edwards, caitlin e.; martinez, david r.; asakura, takanori; dinnon, kenneth h.; kato, takafumi; lee, rhianna e.; yount, boyd l.; mascenik, teresa m.; chen, gang; olivier, kenneth n.; ghio, andrew; tse, longping v.; leist, sarah r.; gralinski, lisa e.; schäfer, alexandra; dang, hong; gilmore, rodney; nakano, satoko; sun, ling; fulcher, m. leslie; livraghi-butrico, alessandra; nicely, nathan i.; cameron, mark; cameron, cheryl; kelvin, david j.; de silva, aravinda; margolis, david m.; markmann, alena; bartelt, luther; zumwalt, ross; martinez, fernando j.; salvatore, steven p.; borczuk, alain; tata, purushothama r.; sontake, vishwaraj; kimple, adam; jaspers, ilona; o’neal, wanda k.; randell, scott h.; boucher, richard c.; baric, ralph s. title: sars-cov-2 reverse genetics reveals a variable infection gradient in the respiratory tract date: 2020-05-27 journal: cell doi: 10.1016/j.cell.2020.05.042 sha: doc_id: 350855 cord_uid: gofzhff7 summary the mode of acquisition and causes for the variable clinical spectrum of covid-19 remain unknown. we utilized a reverse genetics system to generate a gfp reporter virus to explore sars-cov-2 pathogenesis and a luciferase reporter virus to demonstrate sera collected from sars and covid-19 patients exhibited limited cross-cov neutralization. high-sensitivity rna in situ mapping revealed the highest ace2 expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of sars-cov-2 infection in proximal (high) vs distal (low) pulmonary epithelial cultures. covid-19 autopsied lung studies identified focal disease and, congruent with culture data, sars-cov-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. these findings highlight the nasal susceptibility to sars-cov-2 with likely subsequent aspiration-mediated virus seeding to the lung in sars-cov-2 pathogenesis. these reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis. we measured the relative infectivity of the sars-cov-2 gfp virus in primary 283 cells based on the average peak titers and observed that infectivity exhibited the same 284 pattern as the ace2 expression levels from the upper to lower respiratory tract ( figure 285 6bi-6biv). the icsars-cov-2-gfp virus replicated efficiently in hne and lae, with 286 peak viral titers significantly higher than the titers in sae, at2-like and at1-like cultures 287 ( figure 6bv ). although the viral peak titers were similar, the icsars-cov-2-gfp 288 infection in hne culture resulted in significantly higher titers than lae at 24h, 48h and 289 96h post-infection, suggesting more robust replication in the primary nasal cells ( figure 290 6bvi). collectively, these data indicate that virus infectivity/replication efficiency varies 291 markedly from proximal airway to alveolar respiratory regions. 292 whole mount immunohistochemistry of hne and lae cultures was utilized to 293 identify cell types infected by sars-cov-2 ( figure 6c , s4a). the ciliated cell was 294 routinely infected and extruded. in contrast, the other major cell type facing the airway 295 lumen, i.e., the muc5b+ club cell, was not infected, nor was the muc5ac+ metaplastic 296 goblet cell. we did note a cell type co-expressing the ciliated cell marker tubulin and 297 muc5b was rarely infected in hne, a finding consistent with infection of a 298 secretory/club cell transitioning to a ciliated cell phenotype. 299 there is debate whether at2 and/or at1 cells express sufficient ace2 to 300 mediate infection and whether at2, at1, or both cell types are infectable. previous 301 studies reported 2003 sars-cov infects at2 but not at1 pneumocytes (mossel et al., 302 14 standard at2/at1 cell cultures and a novel cell culture approach that well preserves 304 at2 and at1 cell populations over the infection/gfp expression interval were tested. 305 as shown in figure 6a and s4b, at2 cells appeared to be preferentially infected. second, to further characterize the infectivity of lae vs sae, replication rates of 317 three sars-cov-2 viruses in lae and sae cultures from the same donor were 318 compared. all three viruses replicated more slowly in sae than lae cells. the gfp 319 virus replicated modestly less effectively than the clinical isolate or wt virus in the two 320 regions ( figure 6e ). this observation differs from the equivalent replication noted in the 321 vero-e6 cells (figures 2a and 2b) , suggesting an intact orf7 gene contributes to 322 sars-cov-2 replication, and perhaps virulence, in human tissues. 323 third, the replication of sars-cov and sars-cov-2 in lae cells were 324 compared. sars-urbani wt and gfp viruses, in parallel with the three sars-cov-2 325 viruses, were administered to lae cultures from the same donor. gfp signals were 326 detected in lae cultures for both viruses, but the sars-cov-2-gfp exhibited delayed 327 and less intense signals than sars-cov-urbani-gfp ( figure s4d ). this phenotype is 328 consistent with the growth curve in which a lower titer of sars-cov-2 was recorded at 329 24h. 330 331 we utilized rna-ish/ihc to localize virus in four lungs from sars-cov-2-333 infected deceased subjects (table s1) were also infected. rna in situ and ihc co-localization of an at2 cell marker, spc 342 (sftpc) and at1 cell marker (ager) with sars-cov-2 indicated that at2 cells and 343 at1 cells (or at2 cells that had transitioned to at1 cells) were infected ( figure 7c we generated a sars-cov-2 reverse genetics system, characterized virus rna 362 transcription profiles, evaluated the impact of ectopically expressed proteases on virus 363 growth, and used reporter viruses to characterize virus tropisms, ex vivo replication, and 364 to develop a high-throughput neutralizing assay. these reagents were utilized to 365 explore aspects of early infectivity and disease pathogenesis relevant to sars-cov-2 366 respiratory infections. 367 our rnascope/cytospin technology extended the description of ace2 in 368 respiratory epithelia based on scrnaseq data (sungnak et al., 2020) . rna/cytospin 369 detected ~20% of upper respiratory cells expressing ace2 vs ~4% for scrnaseq 370 ( figure 4f ). most of the rna-ish-detected ace2-expressing cells were ciliated cells, 371 not normal muc5b+ secretory (club) cells or goblet cells. notably, the nose contained 372 the highest percentage of ace2-expressing ciliated cells in the proximal airways ( figure 373 4g). the higher nasal ace2 expression-level findings were confirmed by qpcr data 374 comparing nasal to bronchial airway epithelia. qpcr data also revealed that ace2 375 levels further waned in the more distal bronchiolar and alveolar regions. importantly, 376 these ace2 expression patterns were paralleled by high sars-cov-2 infectivity of 377 nasal epithelium with a gradient in infectivity characterized by a marked reduction in the 378 distal lung (bronchioles, alveoli) ( figures 6a and 6b) . 379 multiple aspects of the variability in sars-cov-2 infection of respiratory epithelia 380 were notable in these studies. first, significant donor variations in virus infectivity and 381 replication efficiency were observed. notably, the variability was less in the nose than 382 lower airways. the reason(s) for the differences in lower airway susceptibility are 383 important but remain unclear (cockrell et al., 2018) . we identified variations in ace2 384 receptor expression (figures 4a-d) but not numbers of ciliated cells as potential 385 variables ( figure 6d ). second, variation in infectivity of a single cell type, i.e., the 386 ciliated cell, was noted with only a fraction of ciliated cells having access to virus 387 infected at 72 h ( figure 6a ). third, the dominant secretory cell, i.e., the muc5b+ club 388 cell, was not infected in vitro or in vivo, despite detectable ace2 and tmprss2 389 expression ( figures 4g-4i ). collectively, these data suggest that measurements of 390 ace2/tmprss2 expression do not fully describe cell infectivity and that a description 391 of other variables that mediate susceptibility to infection, including the innate immune 392 system(s), is needed (menachery et al., 2014) . 393 the ace2 receptor gradient in the normal lung raised questions focused on the 394 initial sites of respiratory tract virus infection, the mechanisms that seed infection into 395 the deep lung, and the virus-host interaction networks that attenuate or augment intra-396 regional virus growth in the lung to produce severe disease, especially in vulnerable 397 patients experiencing chronic lung or inflammatory diseases (guan et al., 2020; leung 398 et al., 2020) . 399 we speculate that nasal surfaces may be the dominant initial site for sars-covin summary, our studies have quantitated differences in ace2 receptor 525 expression and sars-cov-2 infectivity in the nose (high) vs the peripheral lung (low). 526 these studies should provide valuable reference data for future animal models 527 development and expand the pool of tissues, e.g., nasal, for future study of disease 528 pathogenesis and therapy. while speculative, if the nasal cavity is the initial site 529 mediating seeding of the lung via aspiration, these studies argue for the widespread use 530 of masks to prevent aerosol, large droplet, and/or mechanical exposure to the nasal 531 passages. complementary therapeutic strategies that reduce viral titer in the nose early 532 24 in the disease, e.g., nasal lavages, topical antivirals, or immune modulation, may be 533 beneficial. finally, our studies provide key reagents and strategies to identify type 534 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1436 mathematical model describing the localization and spread of influenza a virus infection 1437 within the human respiratory tract influenza a viruses are 1440 transmitted via the air from the nasal respiratory epithelium of ferrets comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate 1445 model muc5b 1448 promoter polymorphism and development of acute respiratory distress syndrome type 2 and 1452 interferon inflammation strongly regulate sars-cov-2 related gene expression in the 1453 airway epithelium transmission potential of sars-cov-2 in viral shedding observed at the university of 1457 fiji: an open-source 1460 platform for biological-image analysis reverse 1463 genetics with a full-length infectious cdna of the middle east respiratory syndrome 1464 coronavirus structural basis of receptor recognition by sars-cov-2 severe acute respiratory syndrome coronavirus infection of human ciliated airway 1470 epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs a dynamic variation of pulmonary 1474 ace2 is required to modulate neutrophilic inflammation in response to pseudomonas 1475 aeruginosa lung infection in mice small molecule antipsychotic aripiprazole potentiates 1478 ozone-induced inflammation in airway epithelium sars-cov-2 entry 1481 factors are highly expressed in nasal epithelial cells together with innate immune genes high infectivity and 1484 pathogenicity of influenza a virus via aerosol and droplet transmission rapid reconstruction of sars-cov-1488 2 using a synthetic genomics platform characterization of mucins from cultured normal human tracheobronchial 1492 epithelial cells potent binding of 2019 novel coronavirus spike protein by a sars 1495 coronavirus-specific human monoclonal antibody structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural definition of a 1501 neutralization-sensitive epitope on the mers-cov s1-ntd proteolytic activation of the porcine epidemic 1505 diarrhea coronavirus spike fusion protein by trypsin in cell culture airborne transmission 1508 of severe acute respiratory syndrome coronavirus-2 to healthcare workers: a narrative 1509 review virological assessment 1512 of hospitalized patients with covid-2019 cryo-em structure of the 2019-ncov spike in the 1516 prefusion conformation genome composition and divergence of the novel coronavirus 1519 (2019-ncov) originating in china exposure to 1521 air pollution and covid-19 mortality in the united states characteristics of and important lessons from the 1524 coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 1525 cases from the chinese center for disease control and prevention an infectious cdna clone of 1529 sars-cov-2 imaging and clinical features of patients with 2019 novel 1532 coronavirus sars-cov-2 structural basis for the 1534 recognition of sars-cov-2 by full-length human ace2 junctional and allele-specific residues are critical for 1537 mers-cov neutralization by an exceptionally potent germline-like antibody strategy for systematic assembly of 1540 large rna and dna genomes: transmissible gastroenteritis virus model reverse genetics with a full-length 1544 infectious cdna of severe acute respiratory syndrome coronavirus structural basis for the neutralization of mers-cov by a human monoclonal 1548 antibody mers-27 a novel coronavirus from patients with pneumonia in china potent cross-reactive 1554 neutralization of sars coronavirus isolates by human monoclonal antibodies sars-cov-2 viral load in upper respiratory specimens of infected 1558 patients sars-cov-2 and sars-cov neutralization assays shows limited cross neutralization sars-cov-2 shows a gradient infectivity from the proximal to distal respiratory tract ciliated airway cells and at-2 cells are primary targets for sars-cov-2 infection present a reverse genetics system for sars-cov-2, which is then used to make reporter viruses to quantify the ability of patient sera and antibodies to neutralize infectious virus and to examine viral tropism along the human respiratory tract sequence, n gene, 3'utr, and a 25nt poly-a tail, was assembled under the control of a 1020 t7 promoter. two reporter viruses, one containing gfp and the other harboring, a gfp-1021 fused nluc gene, were generated by replacing the orf7 gene with the reporter genes. 1022 1023 seven genomic cdna fragments were digested with appropriate endonucleases, 1025 resolved on 0.8% agarose gels, excised and purified using a qiaquick gel extraction kit 1026 (qiagen). a full-length genomic cdna was obtained by ligating seven fragments in an 1027 equal molar ratio with t4 dna ligase (neb). we then purified the ligated cdna with 1028 chloroform and precipitated it in isopropanol. the full-length viral rna or sars-cov-2 1029 sgrna-n were synthesized using the t7 mmessage mmachine t7 transcription kit 1030 (thermo fisher) at 30℃ for 4h. the full-length sars-cov-2 transcript and sgrna-n 1031 were mixed and electroporated into 8×10 6 of vero e6 cells. the cells were cultured as 1032 usual in the medium for two to three days. the lumen, not in smg. (ciii-iv) h&e (iii) and dual-immunofluorescence staining using 1215 acetylated alpha tubulin (red) and anti-sars-cov-2 rabbit polyclonal antibody (green) 1216 (iv) from the trachea of a separate autopsy. related to figure 7b and s5di. (d) 1217regional distribution of sars-cov-2 rna from trachea to alveoli identified by rna-ish 1218 in one sars-2-cov autopsy lung (in i and ii, viral staining is red; in iii, viral staining is 1219 turquoise). rna-ish dual color images demonstrate sars-cov-2 rna and sftpc 1220 mrna (alveolar type 2 cell marker) localization in alveoli of a sars-cov-2 autopsy 1221 55 lung. sars-cov-2 (turquoise) was identified in a sftpc (red)-positive (iii, arrow) and a key: cord-346960-3empldlo authors: plebani, m.; padoan, a.; sciacovelli, l.; bonfante, f.; pagliari, m.; bozzato, d.; cosma, c.; bortolami, a.; negrini, d.; zuin, s. title: analytical and clinical performances of five immunoassays for the detection of sars-cov-2 antibodies in comparison with neutralization activity date: 2020-08-04 journal: nan doi: 10.1101/2020.08.01.20166546 sha: doc_id: 346960 cord_uid: 3empldlo background. reliable high-throughput serological assays for sars-cov-2 antibodies (abs) are urgently needed for the effective containment of the covid-19 pandemic, as it is of crucial importance to understand the strength and duration of immunity after infection, and to make informed decisions concerning the activation or discontinuation of physical distancing restrictions. methods. in 184 serum samples from 130 covid-19 patients and 54 sars-cov-2 negative subjects, the analytical and clinical performances of four commercially available chemiluminescent assays (abbott sars-cov-2 igg, roche elecsys anti-sars-cov-2, ortho sars-cov-2 total and igg) and one enzyme-linked immunosorbent assay (diesse enzy-well sars-cov-2 igg) were evaluated and compared with the neutralization activity achieved using the plaque reduction neutralization test (prnt). findings. precision results ranged from 0.9% to 11.8% for all assays. elecsys anti-sars-cov-2 demonstrated linearity of results at concentrations within the cut-off value. overall, sensitivity ranged from 78.5 to 87.8%, and specificity, from 97.6 to 100%. on limiting the analysis to samples collected 12 days after onset of symptoms, the sensitivity of all assays increased, the highest value (95.2%) being obtained with vitro anti-sars-cov-2 total and architect sars-cov-2 igg. the strongest prnt50 correlation with antibody levels was obtained with enzy-well sars-cov-2 igg (rho = 0.541, p < 0.001). interpretation. the results confirmed that all immunoassays had an excellent specificity, whereas sensitivity varied across immunoassays, depending strongly on the time interval between symptoms onset and sample collection. further studies should be conducted to achieve a stronger correlation between antibody measurement and prnt50 in order to obtain useful information for providing effective passive antibody therapy, and developing a vaccine against the sars-cov-2 virus. the continuing spread of coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has prompted concern worldwide, leading the world health organization (who) to declare covid-19 a pandemic on 11 march 2020 1 analytical pitfalls in both the pre-and analytical steps have been described 2 and negative molecular test results have been reported in the later stages of infection, thus being misleading from a clinical viewpoint. therefore, rrt-pcr precludes the identification of individuals who have been infected, but have had only minor, or no, symptoms and therefore have not sought medical attention. a wide range of immunoassays to detect sars-cov-2 antibodies (ab) have been developed to complement rrt-pcr, with different antigen targets and formats [3] [4] [5] . although not effective for making an early diagnosis, serological assays for sars-cov-2 play an important role in diagnosing covid-19 disease in individuals who present late, in understanding the virus epidemiology in the general population, and in identifying the disease prevalence in categories at higher risk of infection (e.g. healthcare workers). in addition, they should be used to ascertain the efficacy of containment measures both locally and globally, to screen convalescent sera for therapeutic and prophylactic purposes, and to improve knowledge of the immune response to the novel virus as the degree and duration of the response of specific antibodies is as yet poorly understood 6, 7 . like infections from other pathogens, sars-cov-2 infection elicits development of igm and igg specific ab which are the most available antibodies for assessing response, while little is known about iga response in the blood. the aim of this paper is to evaluate the performance characteristics and diagnostic specificity, sensitivity of four chemiluminescent assays (clia) and one enzyme-linked immunosorbent assay 122 hospitalized classified with moderate or severe disease, following who interim guidance 8 ) and 54 sars-cov-2 negative subjects (33 healthcare workers, 21 autoimmune patients, 8 pregnant women) were included in the study ( moreover, liaison sars-cov-2 s1/s2 igg (diasorin, sallugia-vc, italy), enzy-well sars-cov-2 iga and igm were evaluated for the correlation with the neutralization results. precision estimation was performed on clia assays using two human serum sample pools with different values, by means of quintuplicate measurements of same pool aliquots, performed for a total of four consecutive days. nested analysis of variance was used to estimate precision, following the clsi ep15-a3 protocol 9 the results for precision were compared to those claimed by the manufacturer when available, using the procedure recommended by ep15-a3. repeatability and within-laboratory precision were in accordance with the repeatability and intermediate precision conditions specified in the international vocabulary of metrology (vim, jcgm 100:2012) for precision estimation within a four-day period. sars-cov-2 igg. the pools were serially diluted with the corresponding low-level serum pools (0.174 s/co ratio for elecsys anti-sars-cov-2, 0.01 s/co for vitros anti-sars-cov-2 igg, 0.2 s/co for vitros anti-sars-cov-2 total, 0.04 s/co ratio for architect sars-cov-2 igg). all measurements were performed in triplicate. for a subgroup of 52 samples from sars-cov-2 positive subjects, the liaison sars-cov-2 s1/s2 igg (diasorin, sallugia, vc, italy) 5 for evaluation of precision, an in-house developed r (r foundation for statistical computing, vienna, austria) script for implementing the clsi ep15-a3 protocol was used for anova and for calculating the upper verification limit 9 module was used to estimate sensitivity, specificity, and positive and negative predictive values. results for precision of all the clia assays are reported in table 3 . the anova approach allowed us to estimate repeatability and intermediate precision separately. only the architect sars-cov-2 igg insert reported data on precision, claimed at levels of 0.04 and 3.53 s/co ratio. for this immunoassay, intermediate precision performances statistically deviated from the manufacturer's claims at both levels. all the immunoassays had acceptable analytical imprecision (cv%). linearity results for all the clia studies are summarized in figure 1 . all tested mixes of sample pools covered a wide range of values and included the manufacturers' cut-offs. all immunoassays, except for elecsys anti-sars-cov-2, deviated from linearity, the coefficients of the second-order polynomial fit attaining high statistical significance. sensitivities, specificities, and positive and negative likelihood ratios were estimated using the manufacturers' cut-offs, while receiver operating characteristic (roc) curves were used to evaluate overall performance(s). elecsys anti-sars-cov-2 immunoassay results were available for all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . 172/184 (93.4%) serum samples. table 4 summarizes estimated clinical performances for all clia and the elisa immunoassays considering the total time frame of 93 days and limiting the analyses to sera collected 12 days after the onset of symptoms. one hundred fifty-eight samples were included and evaluated in this restricted subgroup, while only 146 results were available for elecsys anti-sars-cov-2. table 4 shows data on positive and negative likelihood ratios, allowing an easy estimation of positive (ppv) and negative (npv) predictive values given disease prevalence. considering two different scenarios of disease prevalence settings: (a) 4%, as found in a veneto region (italy) survey (data not shown) 11 ; (b) 10%, as described in a survey conducted in geneva 12 , ppv and npv were then estimated, using vitros anti-sars-cov-2 total and architect sars since the results of serum samples and the corresponding immunoassays' cut-offs were used to derive either positive or negative test results, pairwise comparisons of all tests by cohen's kappa and overall agreements (in percentages) were calculated (table 5) . the signal-to-cut-off (s/co) ratios of the examined assays, including liaison sars-cov-2 s1/s2 (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . in this retrospective study, the analytical and clinical performances of four commercially available clia assays and one elisa assay (table 2 ) have been evaluated and compared with neutralization activity using the plaque reduction neutralization test (prnt). the neutralization activity was evaluated also with respect liaison sars-cov-2 s1/s2 igg and enzy-well sars-cov-2 iga and igm. before conducting the study, precision at two concentration levels and linearity were assessed for clia by using a standardized protocol according to the clsi ep15-a3 and clsi ep06-a (table 3 and fig. 1 ) 9, 10 the results obtained demonstrated that both repeatability and intermediate precisions were comparable with other immunoassays performances for the highest concentration levels 3, 5 , whilst for the lowest levels, less satisfactory results were obtained all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . to provide insight on neutralization activity compared with immunoassays results, prnt assay was performed on 52 samples from sars-cov-2 positive subjects. with the exception of all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . and an in-house developed igg elisa with recombinant rbd of the spike protein as coated antigen 21 . in addition, we found a highly significant correlation between prnt and igm elisa results, thus confirming the data reported by perera et al. 21 . another report, which compare igg or total antibodies measurement of three elisa, two clia and two lateral flow tests, in a total of 100 sars-cov-2 convalescent plasma donors, found a good correlation (rho > 0.700) between elisa (euroimmun igg and wantai total antibodies) and neutralization titer 22 . in asymptomatic/paucisymptomatics, prnt 50 titer values were lower than in moderate/severe sars-cov-2 patients. furthermore, a significant negative correlation was found between the prnt 50 titer and the time interval from symptom onset (figure 3 ). the present paper has limitations: first, neutralizing antibodies were tested in a limited number of samples the procedure being very complex; second, covid-19 positive patients were selected retrospectively on the basis of available leftover samples; therefore npv and ppv could be overestimated. finally, the relationship between igm antibodies and neutralizing activity should be studied further in a larger series of patients. in conclusion, although the performances of sars-cov-2 antibody immunoassays are of analytical and clinical value, they could be enhanced by considering the test purposes, emphasizing sensitivity in the screening and specificity in the second-line testing. in addition, a further search should be made for a better dynamic range and a stronger correlation with respect to antibody neutralization activity, in order to, above all, obtain information needed for effective passive antibody therapy and vaccine development against sars-cov-2 virus. we thank daniela rinaldi (medical laboratory scientists) for their valuable technical support. we acknowledge abbott laboratories, diesse diagnostica senese, diasorin, ortho clinical diagnostics, roche diagnostic for kindly supplying reagents without any influence in study design and data analysis. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted august 4, 2020. . timeline of who's response to covid-19 potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (covid-19) analytical performances of a chemiluminescence immunoassay for sars-cov-2 igm/igg and antibody kinetics iga-ab response to spike glycoprotein of sars-cov-2 in patients with covid-19: a longitudinal study diagnostic performances and thresholds: the key to harmonization in serological sars-cov-2 assays? antibody responses to sars-cov-2 in patients with covid-19 serology assays to manage covid-19 clinical management of covid-19, interim guidance user verification of precision and estimation of bias; approved guideline-third edition. clsi ep15-a3 interpreting diagnostic tests for sars-cov-2 clinical evaluation of serological igg antibody response on the abbott architect for established sars-cov-2 infection clinical performance of different sars-cov-2 igg antibody tests shortened title: sars-cov-2 igg antibody test performance key: cord-354597-xubsodnk authors: carvalho, alexandre; alqusairi, rana; adams, anna; paul, michelle; kothari, neelay; peters, stevany; debenedet, anthony t. title: sars-cov-2 gastrointestinal infection causing hemorrhagic colitis: implications for detection and transmission of covid-19 disease date: 2020-04-17 journal: am j gastroenterol doi: 10.14309/ajg.0000000000000667 sha: doc_id: 354597 cord_uid: xubsodnk nan the betacoronavirus, sars-cov-2, that is responsible for covid-19 disease and that was first described in wuhan, china, in late 2019, has swiftly made its way around our world, resulting in excess of 30,000 deaths to date (1) . efforts to recognize sars-cov-2 infection have focused on respiratory symptoms such as cough and shortness of breath (2, 3) . currently, the centers for disease control and prevention (cdc) criteria for identifying persons under investigation for sars-cov-2 infection in the united states comprise respiratory symptoms and/or fever only (4) . recent reports from china have described concomitant digestive symptoms, such as nausea, vomiting, diarrhea, and abdominal pain, in patients with confirmed sars-cov-2 pulmonary infection (5) (6) (7) (8) and the presence of sars-cov-2 rna in fecal samples (8, 9) . however, it remains unclear whether these digestive symptoms were causally related to sars-cov-2 gastrointestinal infection. because the main goals of the care in these cases were to treat the pulmonary disease and limit healthcare worker exposure, a comprehensive evaluation of the gastrointestinal system to implicate the virus and rule out alternative etiologies was not undertaken. we present a case of sars-cov-2 gastrointestinal infection causing acute hemorrhagic colitis and signaling covid-19 disease which endoscopy confirmed colonic injury and helped exclude other etiologies of disease. we believe that this observation has important implications for the detection and transmission of covid-19 disease. a 71-year-old woman with a history of hypertension, depression, and chronic back pain had returned to the united states in early march 2020 after a 10-day trip to egypt which included a 4-day cruise on the nile river. on her last day in egypt, she developed diffuse abdominal pain and nonbloody diarrhea. the next day, while traveling back to the united states, her diarrhea became bloody. over the next 4 days, she experienced nausea, vomiting, anorexia, diffuse abdominal pain and distention, and 10-20 bloody bowel movements daily. she presented to our emergency department 5 days after the onset of her symptoms. physical examination revealed a temperature of 36.4°c (97.6°f), blood pressure of 140/81 mm hg, pulse of 98 beats per minute, respiratory rate of 18 breaths per minute, and oxygen saturation of 99% on ambient air. lung auscultation was normal. abdominal examination demonstrated normal bowel sounds and diffuse tenderness to palpation, but no signs of peritonitis. red blood, mixed with loose stool, was present in her bedside commode. on further questioning, she denied fever, cough, shortness of breath, sore throat, or any other symptoms. she also denied a personal and family history of gastrointestinal disease and had undergone a normal screening colonoscopy 1 month earlier. she denied antibiotic, antidiarrheal, and nonsteroidal anti-inflammatory use, food allergies, lactose intolerance, alcohol abuse, smoking, and drug use. her medications did include lisinopril and desvenlafaxine, amlodipine, and morphine as needed for chronic back pain. she reported having been vaccinated against hepatitis a and b. laboratory evaluation was notable for an elevated white blood cell count of 24.4 k/ml, with 20.8 k/ml neutrophils and normal lymphocyte and eosinophil distributions, a normal hemoglobin, and slightly elevated creatinine at 1.31 mg/dl (baseline 0.90 mg/dl). ct scan of her abdomen and pelvis with intravenous contrast showed severe colonic inflammation that was most pronounced in the ascending, transverse, and descending colon but was also apparent in the sigmoid colon ( figure 1 ). there was also a small, right pleural effusion. given the presumptive diagnosis of traveler's diarrhea with dysentery, empiric ceftriaxone, azithromycin, and metronidazole were initiated intravenously. before administration of antimicrobials, a fecal sample was obtained and was negative for fecal leukocytes, stool culture (campylobacter, salmonella, shigella, shiga toxinproducing escherichia coli, and yersinia), ova and parasites, and clostridium difficile toxin (glutamate dehydrogenase antigen toxin screen). the next day, another fecal sample was negative for entamoeba histolytica antigen and giardia antigen. of note, later in the hospitalization (hospital day 7), fecal molecular testing (filmarray; biofire diagnostics, salt lake city, ut) was also negative for bacterial, viral, and parasitic pathogens. human immunodeficiency virus 1, 2 antibodies and legionella urine antigen were also negative. over the next 3 days, the patient's abdominal pain and bloody diarrhea persisted despite antimicrobial support. given a concern for inflammatory bowel disease, c-reactive protein on hospital day 3 was 11.6 mg/dl. in addition, on hospital day 3, the patient learned that someone in her travel group had been diagnosed with sars-cov-2 pulmonary infection. the patient was then immediately moved to a negative-pressure room, and sars-cov-2 precautions were instituted. on hospital day 4, 9 days after the onset of her digestive symptoms, the patient developed a cough; nasopharyngeal swabs were sent for comprehensive viral detection, including sars-cov-2 rna (quest diagnostics). given the patient's elevated c-reactive protein and persistent abdominal pain and bloody diarrhea, a flexible sigmoidoscopy was performed on hospital day 4 to evaluate for evidence of inflammatory bowel disease or ischemic colitis. endoscopic evaluation to 40 cm from the anal verge revealed patchy areas of focal erythema without ulceration in the descending colon, sigmoid colon, and rectum ( figure 2 ). histological examination of the colon and rectal biopsies by hematoxylin and eosin stain under light microscopy showed slight expansion of the lamina propria by edema with normal cellularity and intact crypts. no virocytes or protozoa were seen. there were no microscopic changes to indicate the presence of classic infectious colitis, ischemia, or inflammatory bowel disease. on the evening of hospital day 4, the patient's nasopharyngeal swab for comprehensive respiratory viral panel returned positive for rhinovirus and herpes simplex virus 1. her sars-cov-2 rna was also positive by reverse transcriptase polymerase chain reaction. over the next several days, the patient's abdominal pain and bloody diarrhea persisted and a sore throat developed. on hospital day 7, a sars-cov-2 reverse transcriptase polymerase chain reaction performed on a fecal sample using the swab and viral transport media from a sars-cov-2 nasopharyngeal testing kit was also positive. on hospital day 8, the patient's respiratory status worsened and her oxygen saturation declined to 91% on ambient air. she was given two 400 mg doses of hydroxychloroquine, followed by 200 mg twice daily. within the next 48 hours, she had improvement in her abdominal pain and bloody diarrhea. her respiratory symptoms did not evolve further, but she did require 5 l of oxygen via nasal cannula for several days. ct scan of the chest and ct angiogram of the abdomen and pelvis performed on hospital day 10 showed multifocal pneumonia consistent with pulmonary covid-19 disease and a resolution of colonic inflammation ( figure 3 ). there was no evidence of vascular compromise. over the next 12 days, the patient's respiratory status gradually improved and she was weaned off oxygen supplementation. her digestive symptoms also improved. the patient was discharged on hospital day 20 in good health, off all antimicrobials. unfortunately, at the time of the writing of this report, the patient has been readmitted with mental status changes that are currently being evaluated. there has been a growing appreciation of the importance of digestive symptoms (nausea, vomiting, anorexia, nonbloody diarrhea, and abdominal pain) in the spectrum of covid-19 disease. presumed gastrointestinal manifestations have been reported anywhere from 3 to 50% of patients with concomitant sars-cov-2 pulmonary infection (5-7,10). sars-cov-2 rna has been found in fecal samples from patients with covid-19 pulmonary disease, and initial case series have noted that 3%-10% of patients who are eventually found to have sars-cov-2 pulmonary infection initially presented with isolated digestive symptoms (5,7) . what has been more difficult to establish is whether sars-cov-2 infection is directly responsible for the digestive symptoms. because the focus of care in most hospitalized patients is the respiratory illness, and endoscopy-as a possible virusaerosolizing procedure-is used judiciously, diagnostic studies to implicate the virus in gastrointestinal pathology and to exclude other etiologies are generally not undertaken. because our patient presented with bloody diarrhea, which has not previously been described as a manifestation of covid-19, and our index of suspicion in early march 2020 was low, our patient did undergo a comprehensive evaluation. this strongly suggested that sars-cov-2 gastrointestinal infection was responsible for her acute hemorrhagic colitis. we demonstrated that sars-cov-2 rna was present in our patient's feces, and the endoscopic findings of coloproctopathy in her descending colon, sigmoid colon, and rectum confirmed colonic injury and pointed toward an infectious process. we were also able to eliminate, to the greatest extent possible, other potential etiologies of hemorrhagic colitis-such as alternative infections, inflammatory bowel disease, and ischemic colitis-through laboratory testing, radiological imaging, and colon and rectal biopsies. although fecal molecular testing was performed after the initiation of antimicrobials, it is well described that even in the treated patient, fecal molecular testing will remain positive for up to several weeks (11) . this, combined with the fact that our patient did not improve with standard antimicrobial therapy, makes a multi-infection scenario unlikely. interestingly, although our patient had endoscopic evidence of coloproctopathy and colonic thickening on ct, her sigmoid colon and rectal biopsies were histologically unremarkable. there is currently no commercially available assay in the united states to test tissue for the presence of sars-cov-2 rna, so we were not able to do this. however, such normal histologic findings are in line with the 2003 sars-cov experience wherein, under light microscopy, small intestinal and colonic specimens of patients with confirmed sars-cov gastrointestinal infection showed normal architecture, without evidence of villous atrophy, inflammatory infiltrates, or virocytes (12) . we also did not have access to electron microscopy; in the 2003 sars-cov experience, viral particles were seen by electron microscopy in the small intestinal and colonic epithelial cells (13) . similarly, there was also a disconnect between the degree of colonic inflammation seen on initial ct scan and the endoscopically observed coloproctopathy seen on flexible sigmoidoscopy. we suspect this is because the ct scan was performed 3 days before the flexible sigmoidoscopy and thus some healing was likely already taking place in at least the left colon. moreover, on ct scan, the colonic inflammation was most pronounced in the ascending and transverse colon, with the left colon not too far behind. our patient's continued bloody diarrhea after flexible sigmoidoscopy was likely from resolving mucosal damage in the ascending and transverse colon that was not observed on flexible sigmoidoscopy. it has been established that the target viral receptor for sars-cov-2 is angiotensinconverting enzyme 2 (ace2) (8, 13, 14) . this receptor is highly expressed on type ii alveolar cells, esophagus epithelial cells, and both small intestine and colonic cells, among other cell types (8, (15) (16) (17) . in addition, immunofluorescence analysis has shown that the ace2 receptor is abundantly expressed in gastric and rectal epithelia (8) . these data suggest that sars-cov-2 may gain entry into and potentially damage gastrointestinal host cells, causing the array of digestive symptoms that are currently being observed. our patient was taking lisinopril 40 mg daily as part of her regimen for hypertension. there have been some reports suggesting that patients treated with ace inhibitors and angiotensin receptor blockers may theoretically have increased numbers of ace2 receptors, making them more prone to infection with sars-cov-2 and perhaps higher risk for severe covid-19 disease (18) . it is certainly plausible that this applied to our patient. our patient did clinically improve with hydroxychloroquine administration, and there have been some reports suggesting a possible benefit (19, 20) . we are unsure whether this was truly a therapeutic effect or coincidental. more research is certainly needed regarding the clinical efficacy of hydroxychloroquine in the treatment of sars-cov-2 infection. from a transmission perspective, oral and respiratory droplets are well described as the major mode of transmission of sars-cov-2 viral particles. however, live sars-cov-2 virus has also been isolated from fecal samples and viral particles have been detected in the feces even after resolution of respiratory symptoms, suggesting the potential for fecal-oral transmission beyond the symptomatic period (8, 21) . when our patient was admitted, she did not meet the cdc guidelines at the time for persons under investigation for sars-cov-2 infection because she was afebrile, had no respiratory symptoms, and had not travelled to china, italy, iran, or south korea. we were unfortunately not aware of the washington post article that ran 3 days before her presentation, reporting a cluster of sars-cov-2 cases associated with nile river cruises (22) . awareness of the gastrointestinal manifestations of sars-cov-2 may have increased our index of suspicion and encouraged us to institute sars-cov-2 precautions on arrival, avoiding the exposure and subsequent quarantine of 72 healthcare workers, including many of us. to our knowledge, this is the first report of sars-cov-2 gastrointestinal infection causing hemorrhagic colitis in which colonic injury was demonstrated endoscopically and other etiologies were excluded. this case adds to the body of evidence implicating the gastrointestinal tract in the clinical expression and transmission of sars-cov-2 infection. on this basis, we believe it is important to institute sars-cov-2 precautions in patients who present with either respiratory or digestive symptoms. we also encourage the rapid development and deployment of fecal testing kits for sars-cov-2 rna and encourage institutions to use their nasopharyngeal kits for fecal testing in the interim. on march 29, 2020, new york city healthcare professionals made the recommendation that anyone presenting to new york city hospitals (even without respiratory or digestive symptoms) be considered sars-cov-2 positive and appropriate safeguards taken (23). we have not reached this level universally in our country yet. however, this emerging disease will continue to evolve, and so must we. the maxim "when you hear hoofbeats, think horses not zebras" works well, unless you are on a safari-or in the middle of a pandemic. guarantor of the article: anthony t. debenedet, md, msc. specific author contributions: a.c.: report concept, acquisition of the data, analysis and interpretation of the data, and drafting and finalizing the manuscript. r.a.: report concept, acquisition of the data, analysis and interpretation of the data, and drafting and finalizing the manuscript. a.a.: report concept, acquisition of the data, analysis and interpretation of the data, and drafting and finalizing the manuscript. m.p.: report concept, acquisition of the data, analysis and interpretation of the data, and drafting and finalizing the manuscript. n.k.: acquisition of data and intellectual manuscript revision. s.p.: acquisition of the data and intellectual manuscript revision. a.t.d.: report concept, acquisition of the data, analysis and interpretation of the data, and drafting and finalizing the manuscript. financial support: none to report. potential competing interests: none to report. world health organization director-general's opening remarks at covid-19 media briefing. world health organization a novel coronavirus from patients with pneumonia in china coronavirus disease testing (covid-19): symptoms & testing clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of covid-19 patients with digestive symptoms in hubei, china: a descriptive, cross-sectional, multicenter study evidence for gastrointestinal infection of sars-cov-2 covid-19 disease with positive fecal and negative pharyngeal and sputum viral tests clinical characteristics of 140 patients infected with sars-cov-2 in wuhan, china acg clinical practice guideline: diagnosis, treatment, and prevention of acute diarrheal infections in adults enteric involvement of severe acute respiratory syndrome-associated coronavirus infection angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection single-cell rna expression profiling of ace2, the putative receptor of wuhan the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid-19 efficacy of hydroxycholoroquine in patients with covid-19: results of a randomized clinical trial hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro detection of sars-cov-2 in different types of clinical specimens new coronavirus cluster, including 3 from md., linked to nile river cruise ship popular with tourists preparation in the big apple key: cord-345381-9cckppk2 authors: klimek, ludger; pfaar, oliver; worm, margitta; eiwegger, thomas; hagemann, jan; ollert, markus; untersmayr, eva; hoffmann-sommergruber, karin; vultaggio, alessandra; agache, ioana; bavbek, sevim; bossios, apostolos; casper, ingrid; chan, susan; chatzipetrou, alexia; vogelberg, christian; firinu, davide; kauppi, paula; kolios, antonios; kothari, akash; matucci, andrea; palomares, oscar; szépfalusi, zsolt; pohl, wolfgang; hötzenecker, wolfram; rosenkranz, alexander r.; bergmann, karl-christian; bieber, thomas; buhl, roland; buters, jeroen; darsow, ulf; keil, thomas; kleine-tebbe, jörg; lau, susanne; maurer, marcus; merk, hans; mösges, ralph; saloga, joachim; staubach, petra; jappe, uta; rabe, klaus f.; rabe, uta; vogelmeier, claus; biedermann, tilo; jung, kirsten; schlenter, wolfgang; ring, johannes; chaker, adam; wehrmann, wolfgang; becker, sven; freudelsperger, laura; mülleneisen, norbert; nemat, katja; czech, wolfgang; wrede, holger; brehler, randolf; fuchs, thomas; tomazic, peter-valentin; aberer, werner; fink-wagner, antje-henriette; horak, fritz; wöhrl, stefan; niederberger-leppin, verena; pali-schöll, isabella; pohl, wolfgang; roller-wirnsberger, regina; spranger, otto; valenta, rudolf; akdis, mübecell; matricardi, paolo m.; spertini, françois; khaltaev, nicolai; michel, jean-pierre; nicod, larent; schmid-grendelmeier, peter; idzko, marco; hamelmann, eckard; jakob, thilo; werfel, thomas; wagenmann, martin; taube, christian; jensen-jarolim, erika; korn, stephanie; hentges, francois; schwarze, jürgen; o´mahony, liam; knol, edward f.; del giacco, stefano; chivato pérez, tomás; bousquet, jean; bedbrook, anna; zuberbier, torsten; akdis, cezmi; jutel, marek title: use of biologicals in allergic and type-2 inflammatory diseases during the current covid-19 pandemic: position paper of ärzteverband deutscher allergologen (aeda)(a), deutsche gesellschaft für allergologie und klinische immunologie (dgaki)(b), gesellschaft für pädiatrische allergologie und umweltmedizin (gpa)(c), österreichische gesellschaft für allergologie und immunologie (ögai)(d), luxemburgische gesellschaft für allergologie und immunologie (lgai)(e), österreichische gesellschaft für pneumologie (ögp)(f) in co-operation with the german, austrian, and swiss aria groups(g), and the european academy of allergy and clinical immunology (eaaci)(h) date: 2020-09-07 journal: allergol select doi: 10.5414/alx02166e sha: doc_id: 345381 cord_uid: 9cckppk2 background: since the beginning of the covid-19 pandemic, the treatment of patients with allergic and atopy-associated diseases has faced major challenges. recommendations for “social distancing” and the fear of patients becoming infected during a visit to a medical facility have led to a drastic decrease in personal doctor-patient contacts. this affects both acute care and treatment of the chronically ill. the immune response after sars-cov-2 infection is so far only insufficiently understood and could be altered in a favorable or unfavorable way by therapy with monoclonal antibodies. there is currently no evidence for an increased risk of a severe covid-19 course in allergic patients. many patients are under ongoing therapy with biologicals that inhibit type 2 immune responses via various mechanisms. there is uncertainty about possible immunological interactions and potential risks of these biologicals in the case of an infection with sars-cov-2. materials and methods: a selective literature search was carried out in pubmed, livivo, and the internet to cover the past 10 years (may 2010 – april 2020). additionally, the current german-language publications were analyzed. based on these data, the present position paper provides recommendations for the biological treatment of patients with allergic and atopy-associated diseases during the covid-19 pandemic. results: in order to maintain in-office consultation services, a safe treatment environment must be created that is adapted to the pandemic situation. to date, there is a lack of reliable study data on the care for patients with complex respiratory, atopic, and allergic diseases in times of an imminent infection risk from sars-cov-2. type-2-dominant immune reactions, as they are frequently seen in allergic patients, could influence various phases of covid-19, e.g., by slowing down the immune reactions. theoretically, this could have an unfavorable effect in the early phase of a sars-cov-2 infection, but also a positive effect during a cytokine storm in the later phase of severe courses. however, since there is currently no evidence for this, all data from patients treated with a biological directed against type 2 immune reactions who develop covid-19 should be collected in registries, and their disease courses documented in order to be able to provide experience-based instructions in the future. conclusion: the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, chronic rhinosinusitis with nasal polyps, and spontaneous urticaria should be continued as usual in patients without suspected infection or proven sars-cov-2 infection. if available, it is recommended to prefer a formulation for self-application and to offer telemedical monitoring. treatment should aim at the best possible control of difficult-to-control allergic and atopic diseases using adequate rescue and add-on therapy and should avoid the need for systemic glucocorticosteroids. if sars-cov-2 infection is proven or reasonably suspected, the therapy should be determined by weighing the benefits and risks individually for the patient in question, and the patient should be involved in the decision-making. it should be kept in mind that the potential effects of biologicals on the immune response in covid-19 are currently not known. telemedical offers are particularly desirable for the acute consultation needs of suitable patients. course in allergic patients. many patients are under ongoing therapy with biologicals that inhibit type 2 immune responses via various mechanisms. there is uncertainty about possible immunological interactions and potential risks of these biologicals in the case of an infection with sars-cov-2. materials and methods: a selective literature search was carried out in pubmed, livivo, and the internet to cover the past 10 years (may 2010 -april 2020). additionally, the current german-language publications were analyzed. based on these data, the present position paper provides recommendations for the biological treatment of patients with allergic and atopy-associated diseases during the covid-19 pandemic. results: in order to maintain in-office consultation services, a safe treatment environment must be created that is adapted to the pandemic situation. to date, there is a lack of reliable study data on the care for patients with complex respiratory, atopic, and allergic diseases in times of an imminent infection risk from sars-cov-2. type-2-dominant immune reactions, as they are frequently seen in allergic patients, could influence various phases of covid-19, e.g., by slowing down the immune reactions. theoretically, this could have an unfavorable effect in the early phase of a sars-cov-2 infection, but also a positive effect during a cytokine storm in the later phase of severe courses. however, since there is currently no evidence for this, all data from patients treated with a biological directed against type 2 immune reactions who develop co-vid-19 should be collected in registries, and their disease courses documented in order to be able to provide experience-based instructions in the future. conclusion: the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, chronic rhinosinusitis with nasal polyps, and spontane-ous urticaria should be continued as usual in patients without suspected infection or proven sars-cov-2 infection. if available, it is recommended to prefer a formulation for self-application and to offer telemedical monitoring. treatment should aim at the best possible control of difficult-to-control allergic and atopic diseases using adequate rescue and add-on therapy and should avoid the need for systemic glucocorticosteroids. if sars-cov-2 infection is proven or reasonably suspected, the therapy should be determined by weighing the benefits and risks individually for the patient in question, and the patient should be involved in the decision-making. it should be kept in mind that the potential effects of biologicals on the immune response in covid-19 are currently not known. telemedical offers are particularly desirable for the acute consultation needs of suitable patients. the clinical symptoms of infection with the novel coronavirus (severe acute respiratory coronavirus 2; sars-cov-2) became known as the "coronavirus disease 2019 (covid-19)" on february 11, 2020 [1] . the international committee on taxonomy of viruses (ictv) called this novel human pathogenic virus sars-cov-2 [1] . the global spread of the sars-cov-2 pandemic and patients with severe covid-19 courses pose a major challenge to healthcare systems worldwide. the coronavirus that caused the severe acute respiratory syndrome (sars-cov) in 2002/2003 has approximately an 80% nucleotide sequence identity with sars-cov-2 [1] . sars-cov-2 is a betacoronavirus of the subgenus sarbecovirus, subfamily orthocoronavirinae, and the 7 th member of the coronaviridae family that can infect humans. it can be isolated from human samples obtained from respiratory secretions, nasal and pharyngeal swabs and isolated on cell cultures [1, 2, 3] . it is covered by a lipid membrane that can be disrupted by detergents and is different from the middle east respiratory syndrome-related coronavirus (mers-cov), from sars-cov, and from the coronaviruses responsible for the common cold (229e, oc43, nl63, and hku1) [1] . abbreviations. angiotensin-converting enzyme 2 the incubation period after an infection with sars-cov-2 can be of up to 14 days, during which the infected person can be asymptomatic but nevertheless transmit the virus. in a high number of patients, the infection leads to symptoms of the upper and lower airways, and, less frequently, also of other organ systems (nervous system, gastrointestinal tract, kidneys, blood vessels). in the worst case scenario, multi-organ failure and respiratory failure can result, as has been described for other coronavirus infections (sars-cov-1, mers-cov) [4, 5, 6] . in more severe cases, infection with sars-cov-2 can lead to pneumonia, severe acute respiratory syndrome, renal failure, and death [4, 7, 8, 9, 10] . higher age and comorbidities such as chronic airway diseases (particularly copd), diabetes mellitus, cardiovascular diseases, obesity, and immune deficiency of various origins have been described as risk factors of a severe course [4, 7, 8, 9] . the need for intensive care treatment and invasive ventilation is associated with high mortality. we will present clinical and immunological aspects that have to be considered with regard to the covid-19 pandemic in patients treated with biological therapy against ige and mediators of type 2 inflammation (table 1) . the characteristics of the immune response after infection with sars-cov-2 are still insufficiently understood. while various forms of the course of covid-19 and the infection with the virus have been described, it is still unclear which immunological background influences the course of the disease. this is also true for the role of the innate table 1 . recommendations on treatment with biologicals during the covid-19 pandemic in patients with asthma, atopic dermatitis, urticaria, or crswnp. recommendations for biological treatment in non-infected patients during the covid-19 pandemic or in patients who have recovered from covid-19 infection termination of biological treatment is not generally necessary; and biologicals should be continued as scheduled, particularly in severe cases, based on an individual risk-benefit analysis. in mild-to-moderate covid-19 courses, or when sars-cov-2 infection is suspected, biologicals can be continued in the indications discussed here if a patient-based risk-benefit analysis supports the decision and the patient consents after having been informed about the limited availability of data. prolongation of the injection interval can be considered (as indicated in the summary of product characteristics) to limit the necessary physician-patient contacts to a minimum. in severe covid-19 courses, prolongation of the injection interval (as indicated in the summary of product characteristics) or treatment interruption should be considered in the indications discussed here. the risk of the possible requirement of systemic glucocorticosteroids must be considered. biological treatment can be continued during the current covid-19 pandemic in asymptomatic patients with negative pcr tests, in patients without known exposure or contact with sars-cov-2-positive people, and in patients who have completed an adequate quarantine period. in a quarantine situation, telemedical support might be feasible, in particular with the aim of continuing the basic therapy with topical steroids, inhaled bronchodilators, antihistamines, etc. in accordance with the relevant guideline recommendations or with the aim to expand those therapies according to the patient's needs. biological therapy in patients without evidence of sars-cov-2 infection can be started for approved indications during the current covid-19 pandemic, self-administration of biologicals should generally be preferred; this is made easier if user-friendly pen systems for self-application are available. adequate patient training is required. practices and allergy centers must be prepared for the current covid-19 pandemic by following the recommendations of the who and of national and regional authorities. these recommendations should be continuously updated and adapted to new scientific findings and recommendations made by authorities. crswnp = chronic rhinosinusitis with nasal polyps. and adaptive immune system with regard to sars-cov-2 infection. while natural killer (nk) cells traditionally play an important role in the early phase of viral infections, cd8 + t helper cells come into action in the subsequent phase [11] . early antibody secretion and production in the mucosa-associated lymphatic tissue initially include antigenspecific igm, iga, and, later, igg antibodies, and are essential for immune response [12, 13, 14] . macrophages are activated and secrete inflammatory cytokines, with type 1 interferons (type 1 ifn) playing the most prominent role. in infections with other coronaviruses (e.g., sars-cov-1), type 1 ifn is responsible for the adequate initiation of the immune reaction, and patients with delayed or insufficient ifn production have a more severe disease course [6] . the activation of apoptosis or pyroptosis in epithelial cells serves as an antiviral defense, but excessive immune reactions can also contribute to local tissue damage through synergistic effects [15] . an excessive production of pro-inflammatory cytokines has already been observed in sars-cov-1, mers-cov, and recently also in sars-cov-2 infections, and has been described as a cytokine storm [4, 5] . natural igm, and probably also mannose-binding lectin (mbl), are believed to be the first line of defense against sars-cov-2 [16] . these antibodies and mbl recognize glycans and are abundant in children and young adults. however, they decrease dramatically with age and are over 50 times lower at the age of ≥ 60 than at the age of 20 -30 years [16] . as they are part of the innate immunity, they are the only antibodies able to recognize sars-cov-2 before the adaptive immune response is initiated [16] . if the virus enters the lungs early enough, it can replicate in an unhindered manner, as no or only little resistance exists. the resulting inflammation with a massive activation of local mediators (complement and coagulation cascades interleukin-6 (il-6), cytokine storm) can cause damages that lead to complications or, in some cases, even to death [16] . furthermore, the ability of sars-cov-2 viruses to penetrate the cell via receptors such as ace2 and tmprrss2 could explain the different severities of covid-19 in different patient groups [17] . extensive damage to the lungs leads to rapid clinical deterioration and usually to the need for intensive care treatment, which can be observed typically 7 -14 days after infection. the risk of kidney, liver, and/or other organ damage as well as of consumption coagulopathy is significantly increased. affected patients usually have highly increased interleukin (il)-1 β, il-6, il-8, and tnf-α levels ( figure 1 ) [18] . the therapeutic blockade of one or more of these cytokines has been discussed as a potential future therapy option for severely affected patients in whom il-6 can be massively increased [19] . il-6 plays a central role in the cytokine storm, and tocilizumab has already been used as a biological with anti-il-6 effects in covid-19 [20, 21] . approved indications for anti-il-6 or anti-il-6r antibodies (such as tocilizumab, sarilumab) currently include, for example, rheumatoid arthritis, juvenile rheumatoid arthritis, and castleman disease. the immune reactions of type 1 and type 3 described here are contained by other cytokines, such as il-10 and tgf-β, and type 2 inflammation could possibly counteract the cytokine storm. increased levels of eosinophilic granulocytes, as one of the key cells of type 2 inflammation, have been ascribed a protective effect in severe viral infections, although the mechanism of action has not yet been identified [22] . on the other hand, low blood eosinophil counts could simply reflect the severity of the infection. the interaction of sars-cov-2 with its receptor on the cells of the respiratory system, the membrane-bound angiotensin-converting enzyme 2 (ace2), which is responsible for the entry of the virus into the host cell, is far better investigated [17] . therefore, the reduced expression of ace2 in the airway epithelial cells of patients with allergic asthma is being discussed as a potentially protective factor against sars-cov-2 infection [23] . it can be assumed that only the interaction of the individual cytokine responses leads to an adequate and effective immune response in coronavirus infections. however, imbalances between type 1, type 2, and type 3 reactions might have a significant negative or positive impact on the course of the viral infection ( figure 1 ). figure 1 . sequence of immunological events in sars-cov-2 infection: if sars-cov-2 infects cells via the surface receptors ace2 and tmprss2, this leads to active replication and release of the virus, the cells decay through pyroptosis. damps are released that are recognized by neighboring epithelia, endothelia, and alveolar macrophages and trigger the release of pro-inflammatory cytokines such as il-6, ip-10, mip1α, mip1β, and mcp1. this attracts monocytes, macrophages, and t cells, which, when ifn-γ is added, activate another inflammatory, self-reinforcing cascade. in defective immune responses (left), this can lead to accumulation of immune cells and overproduction of pro-inflammatory cytokines, which then damage the lung and may lead to a cytokine storm with multi-organ failure. additionally, non-neutralizing antibodies produced by b cells may enhance the infection and lead to further organ damage. in a healthy immune response (right), the initial inflammation attracts virus-specific t cells to the infection site where they can eliminate the infected cells before the virus spreads further. neutralizing antibodies selectively block the virus, alveolar macrophages recognize and phagocytize affected cells and neutralized viruses. altogether, these processes clear the viral infection with minimal damage of respiratory tissue and lead to recovery. reproduced from tay et al. [65] . ace2 = angiotensin-converting enzyme 2; ade = antibodydependent enhancement; damp = damage-associated molecular pattern; g-csf = granulocyte colonystimulating factor; ifn = interferon; il = interleukin; mip1α = macrophage inflammatory protein 1α; sars-cov-2 = severe acute respiratory syndrome coronavirus 2; tnf = tumor necrosis factor. so far, there is insufficient evidence to indicate which risk factors cause a severe course of covid-19. a history of lung disease has been considered a potential risk factor for developing covid-19 and possibly also for a severe course. bronchial asthma, which is the most important allergic indication for biologicals targeting ige and type 2 inflammation, is possibly one of these diseases. however, since many patients with pulmonary disease also have other comorbidities, some of the suspected factors could turn out to be confounders once further studies are carried out. thus, it remains unclear whether patients with type-2-associated bronchial asthma without any other possible risk factors should be considered high risk for a severe covid-19 course. the currently available data rather contradict this. there is only limited data on covid-19 in connection with a type-2-associated disease, and its prognostic value is very limited. the currently available studies do not indicate an increased risk for patients with allergies, asthma, or other atopy-associated diseases ( table 2) [8, 24, 25, 26, 27, 28, 29] . for example, in wuhan and italy, the percentage of seriously ill or deceased covid-19 patients with known bronchial asthma was far below the prevalence of asthma in these places [18] . it also remains unclear why in many patients not only lymphopenia but also eosinopenia was detected at the time of admission [8] . neither decreased nor increased eosinophil levels have so far been clearly associated with certain clinical courses of sars-cov-2 infection. in recent years, several biologicals have been approved in europe that block ige antibodies or the interleukins il-4, il-5, and il-13, which are relevant in type 2 inflammation, or their receptors [30, 31, 32, 33, 34, 35, 36, 37, 38, 39] . omalizumab has been approved for the treatment of severe allergic bronchial asthma in adults and in children older than 6 years with proven sensitization against a perennial airborne allergen and reduced lung function. another indication is antihistamine-resistant chronic spontaneous urticaria in adults and in adolescents older than 12 years. mepolizumab, benralizumab, and reslizumab are report of 3 patients taking oral glucocorticosteroids because of breathing difficulties due to covid-19 and known asthma who were hospitalized 1 week later with acute respiratory insufficiency wang et al. [27] (wuhan, china) 2 of 69 studied patients had asthma zhang et al. [8] (wuhan, china) study of 140 patients of whom 2 had chronic urticaria, 1 had asthma, and 10 had unclear adverse drug reactions grasselli et al. [29] (lombardy, italy) study including 1,591, of whom 205 had a history of: bronchial asthma, anemia, inflammatory bowel disease, chronic respiratory insufficiency, endocrine disorders, chronic pancreatitis, diseases of the connective and supporting tissue, organ transplantation, epilepsy, neurological disease (reported as "other" in the study) dreher et al. [28] (aachen, germany) result: covid-19 patients with a history of respiratory disease develop ards more frequently (58 vs. 42%; 14 vs. 11 patients; of these, 4 vs. 2 patients with asthma; n = 50) ards = acute respiratory distress syndrome. il-5 or il-5 receptor blockers, available for adults and, partially, also for children. dupilumab has been approved for the treatment of: (i) atopic dermatitis in adolescents older than 12 years, (ii) severe type-2-dominant asthma in adults and (iii) chronic rhinosinusitis with nasal polyps (crswnp) in adults. table 2 shows the situation of approval in germany, austria, luxembourg, and switzerland. self-administration by the patient is listed separately as it significantly facilitates care for suitable patients during the sars-cov-2 pandemic. viral infections of the upper and lower airways have been associated with the development and exacerbation of allergic disease [40, 41, 42] . infection and the persistence of virus particles in the mucosa could inhibit the efficacy of the local innate immune system and promote type 2 inflammation. the blocking of type 2 inflammation by therapeutic antibodies against ige, il-5, or the il-5 or il-4/-13 receptors has so far not been suspected to increase the risk of viral infections. however, il-4 has a dual role in viral infections due to two different haplotypes in the il-4 gene. it can promote infections with the herpes virus and the norovirus [43] as well as with the ebola virus, which is related to the coronavirus [44] . on the other hand, il-4 can also inhibit viral infections by promoting innate immunity [45, 46, 47] . thus, more evidence from clinical observations is necessary to be able to provide clear recommendations with regard to covid-19. table 4 gives an overview of the frequency of viral infections occurring as adverse events in trials on these monoclonal antibodies. there have been reports on the lower incidence of viral infections under anti-ige treatment with omalizumab, since this therapy may increase the functionality and the production of ifn-α by plasmacytoid dendritic cells (pdc). this leads to an enhanced antiviral defense and to a reduction of virus-induced asthma exacerbations [40, 48] . also, for type 2 blockade with anti-il-5 antibodies (mepolizumab, reslizumab) or anti-il-5 receptor antibodies (benralizumab), the risk of respiratory viral infections in the active-agent study groups was equal to or lower than the risk in the placebo groups (table 4 ). it has not yet been clarified whether the blockade of type 2 inflammation or of ige influences the risk of developing covid-19 or its course. in the case of a cytokine storm, possible negative effects induced by blocking the type 2 immune response situation are conceivable; but these effects require further investigation. the first reports show that the disease course is not worse in covid-19 patients with eosinophilic diseases under biological therapy [24, 49] . however, further study results should be awaited, especially considering the fact that sars-cov-2 changes rapidly due to mutations [50] . meta-analyses by agache et al. [51, 52, 53] have shown a slightly increased rate (low to medium risk of association) of adverse events when anti-il-5/5r, anti-il-4/13r, and anti-ige are used in severe asthma, independently of covid-19. thus, there is no clear recommendation regarding the decision-making to continue or temporarily interrupt a biological therapy during an infection with sars-cov-2. treatment interruption could entail the risk of suboptimal control of the allergic disease or, in the case of exacerbations, the need for systemic glucocorticosteroids, for which an increased risk of a possibly more severe covid-19 course has been described [54] . recommendations for the management of allergic/atopyassociated diseases under anti-type-2 therapy during the covid-19 pandemic (table 1) to ensure an appropriate, high-quality, and accurate care for patients on anti-type-2 treatment with underlying atopic-eosinophilic or allergic disease, antibody therapy should be continued and remain unchanged during the ongoing pandemic when there is no evidence of sars-cov-2 infection. to cope with the current shortfalls in hospitals and the more difficult hygiene conditions, telephone or telemedical follow-up should be considered in suitable patients when technical and medical requirements allow for it. for this purpose, comprehensive patient training with regard to documentation of the disease activity and, where applicable, to self-administration of the medication is desirable. this is facilitated by the partial availability of user-friendly pen systems for self-application. in general, in countries with low infection numbers and a consequent relaxation of covid-19-associated restrictions, there is no contraindication for starting biological therapy in patients without evidence of a current sars-cov-2 infection. according to the current state of knowledge, biological therapy for the indications discussed here can be continued in mild to moderate cases of sars-cov2 infection/ covid-19 disease, if an individual consideration of risks and benefits supports this decision. the risks and benefits have to be assessed by a specialist, and it is recommended to in-form the patient about the fact that only limited data are available. in severe courses of covid-19, prolongation of the dosing interval or treatment interruption should be considered. when doing so, the risk of the potential requirement of treatment with systemic glucocorticoids should also be taken into account. in a quarantine situation, a telemedical approach might be feasible, in particular with the aim of continuing or expanding the basic therapy with topical steroids, inhaled bronchodilators, antihistamines, etc. in accordance with the relevant guideline recommendations [36, 37, 54, 55, 56, 57, 58] . if hospitalization due to the exacerbation of asthma-or type-2-associated diseases becomes necessary, current guidelines on diagnosis and treatment must be followed. sinus surgery for crswnp should, if possible, be delayed in patients with suspected or confirmed covid-19 disease. in the case of urgently indicated systemic therapy for severe atopic dermatitis, consideration should be given to therapy with either biologicals, classic immunosuppressants, or systemic glucocorticosteroids, although systemic glucocorticosteroids are not recommended due to their broad immunosuppressive effect (see above). for cyclosporin a (cya) as an approved therapeutic option for atopic dermatitis, in vitro studies have suggested antiviral effects [60] . t-celldirected immunosuppression performed after organ transplantation (cya, tacrolimus) is being discussed as a possible protective factor against serious clinical complications of sas-cov-2 infection [61] , as well as the use of cya in covid-19 [62, 63] . however, reliable clinical data have not yet been published. possible metabolic interactions between cya and lopinavir/ritonavir (cyp3 inhibitors) have to be taken into account. severe covid-19 courses have been reported in two patients with atopic dermatitis treated with dupilumab [64] . the currently available data suggest that the risk of developing a severe course of covid-19 is probably not increased in patients with allergies and atopy-associated diseases. however, there is a lack of study results including subgroup analyses on seriously ill atopy patients and their treatment. the effects of ige or type 2 inflammation blocking on sars-cov-2 infection have not yet been clarified. in cases of a mild to moderate covid-19 course, we advise to continue biological therapy for the indications mentioned here, if the patient-based assessment of the benefits and risks supports this approach and if the patient agrees after adequate information about the limited availability of data. in severe courses of covid-19, prolongation of the dosing interval or treatment interruption should be considered for the indications discussed here. this assessment should be patient-based and should consider the risk of the possible requirement of systemic glucocorticosteroids. in all other patients, in whom neither a suspected nor a proven sars-cov-2 infection is present, the use of biologicals for the treatment of bronchial asthma, atopic dermatitis, crswnp, and spontaneous urticaria can be continued unchanged or can be re-started in the current sars-cov-2 pandemic. the use of telemedicine for treatment support and patient education is recommended and can facilitate the continuation of biological administration by self-injection. s. korn received speaker and personal adviser's fees from astrazeneca, gsk, novartis, and sanofi outside the submitted work. u. darsow was a lecturer, principal investigator, and consultant for alk abello, bencard, and novartis pharma outside the submitted work. o. palomares received research grants and/ or personal fees from allergy therapeutics, amgen, astrazeneca, diater, glaxosmithkline, s.a., inmunotek s.l., novartis, sanofi-genzyme, and stallergenes outside the submitted work; he was a member of advisory boards of novartis and sanofi-genzyme. t. biedermann was a consultant to or received personal lecture fees or research grants from alk-abelló, celgene-bms, lilly deutschland gmbh, mylan, novartis, phadia-thermo fisher, sanofi-genzyme, regeneron. r. valenta received research grants from viravaxx, vienna, austria, and from hvd life sciences, vienna, austria, and acts as a consultant for viravaxx. r. buhl received personal lecture and/or consultant fees from astrazeneca, boehringer ingelheim, chiesi, cipla, novartis, roche, sanofi, and teva as well as research support for universitätsmedizin mainz from boehringer ingelheim, glaxosmithkline, novartis, and roche, outside the submitted work. r. brehler received personal fees for lectures and/or consultancy and/or clinical studies from alk, allergopharma, almirall, astra zeneca, bencard, gesellschaft zur förderung der dermatologischen forschung und fortbildung e.v., gesellschaft für information und organisation mbh, gsk, dr. pfleger, hal, leti, merck, novartis, oto-rhino-laryngologischer verein, pierre fabre, pohl boskamp, stallergenes, thermo-fischer, biotech tools, genentech, circassia. a. bossios reports personal consultant and/or lecture fees from novartis, astrazeneca, gsk, and teva outside the submitted work. j. bousquet reports personal fees from chiesi, cipla, hikma, menarini, mundipharma, mylan, novartis, sanofi-aventis, takeda, teva, uriach, kyomed-innov, and purina outside the submitted work. j. schwarze received personal fees from mylan, f2f events; support from industry for educational acivities of the scottish allergy and respiratory academy as well as the children and young people's allergy network scotland outside the submitted work; support from industry for eaaci; j. schwarze is eaaci secretary general 2019 -2021. j. hagemann received speaker fees from novartis pharma. m. wagenmann received personal consultant and/or speaker fees from alk-abelló, allergopharma, astrazeneca, bencard-allergie, genzyme, hal-allergie, infectopharm, leti pharma, meda pharma, novartis, sanofi aventis, stallergenes, teva. l. klimek reports grants and/or personal fees from allergopharma, meda/mylan, hal allergie, alk abelló, leti pharma, stallergenes, quintiles, sanofi, asit biotech, lofarma, allergy therapeut., astra-zeneca, gsk, inmunotk outside the submitted work; he is a member of the following organizations: aeda, dghno, deutsche akademie für allergologie und klinische immunologie, hno-bv gpa, eaaci. coronaviridae study group of the international committee on taxonomy of viruses. the species ssevere acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan 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guideline for the definition, classification, diagnosis and management of urticaria euforea consensus on biologics for crswnp with or without asthma dupilumab reduces local type 2 pro-inflammatory biomarkers in chronic rhinosinusitis with nasal polyposis immunological and hematological effects of il-5(rα)-targeted therapy: an overview randomized trial of omalizumab (anti-ige) for asthma in inner-city children viral infections in allergy and immunology: how allergic inflammation influences viral infections and illness bronchiolitis needs a revisit: distinguishing between virus entities and their treatments how helminths go viral: cellular signals during helminth infections can skew the immune response to favor viral spreading il-4/il-13 polarization of macrophages enhances ebola virus glycoprotein-dependent infection il-4 induced innate cd8+-t cells control persistent viral infection il-4 suppresses the expression and the replication of hepatitis b virus in the hepatocellular carcinoma cell line hep3b il-4 enhances interferon production by virus-infected human mast cells preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations no evidence of increased risk for covid-19 infection in patients treated with dupilumab for atopic dermatitis in a high-epidemic area -bergamo sars-cov-2 immunogenicity at the crossroads. allergy. 2020. epub ahead of print efficacy and safety of treatment with dupilumab for severe asthma: a systematic review of the eaaci guidelines-recommendations on the use of biologicals in severe asthma efficacy and safety of treatment with biologicals (benralizumab, dupilumab, mepolizumab, omalizumab and reslizumab) for severe eosinophilic asthma. a systematic review for the eaaci guidelines -recommendations on the use of biologicals in severe asthma stellungnahme zur anwendung von glukokortikosteroiden bei entzündlichen erkrankungen der oberen atemwege (u. a. allergische rhinitis/chronische 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of covid-19: immunity, inflammation and intervention considerations on biologicals for patients with allergic disease in times of the covid-19 pandemic: an eaaci statement praxis für kinderpenumologie/allergologie am kinderzentrum dresden (kid) praxis und klinik für dermatologie/allergologie am schwarzwald-baar klinikum germany 60 klinische abteilung für allgemeine hno 74 division of allergy and immunology 82 klinik für dermatologie, allergologie und venerologie medizinische hochschule hannover, 83 hno-klinik key: cord-351011-v4zmksio authors: golden, joseph w.; cline, curtis r.; zeng, xiankun; garrison, aura r.; carey, brian d.; mucker, eric m.; white, lauren e.; shamblin, joshua d.; brocato, rebecca l.; liu, jun; babka, april m.; rauch, hypaitia b.; smith, jeffrey m.; hollidge, bradley s.; fitzpatrick, collin; badger, catherine v.; hooper, jay w. title: human angiotensin-converting enzyme 2 transgenic mice infected with sars-cov-2 develop severe and fatal respiratory disease date: 2020-07-09 journal: biorxiv doi: 10.1101/2020.07.09.195230 sha: doc_id: 351011 cord_uid: v4zmksio the emergence of sars-cov-2 has created an international health crisis. small animal models mirroring sars-cov-2 human disease are essential for medical countermeasure (mcm) development. mice are refractory to sars-cov-2 infection due to low affinity binding to the murine angiotensin-converting enzyme 2 (ace2) protein. here we evaluated the pathogenesis of sars-cov-2 in male and female mice expressing the human ace2 gene under the control of the keratin 18 promotor. in contrast to non-transgenic mice, intranasal exposure of k18-hace2 animals to two different doses of sars-cov-2 resulted in acute disease including weight loss, lung injury, brain infection and lethality. vasculitis was the most prominent finding in the lungs of infected mice. transcriptomic analysis from lungs of infected animals revealed increases in transcripts involved in lung injury and inflammatory cytokines. in the lower dose challenge groups, there was a survival advantage in the female mice with 60% surviving infection whereas all male mice succumbed to disease. male mice that succumbed to disease had higher levels of inflammatory transcripts compared to female mice. this is the first highly lethal murine infection model for sars-cov-2. the k18-hace2 murine model will be valuable for the study of sars-cov-2 pathogenesis and the assessment of mcms. sars-cov-2 is a betacoronavirus and the causative agent of covid-19, a febrile 55 respiratory human disease that emerged in late 2019 in china and subsequently spread throughout the world (1, 2). covid-19 is primarily a respiratory disease with a wide spectrum of severity ranging from a mild cough, to the development of hypoxia, and in some cases resulting in a lifethreatening acute respiratory distress syndrome (ards) requiring mechanical ventilation (3, 4). the most severe cases are generally skewed towards the aged population (>50) and those with 60 underlying health conditions such as hypertension or cardiovascular disorders (5, 6) . sars-cov-2 human infections can also cause vasculature damage and coagulopathies, leading to infarction (7-9). acute disease often presents with elevated levels of inflammatory cytokines, including il-6, and these host molecules may play a role in the pathogenic process (10, 11) . additionally, ~30% of cases include signs of neurological disease such as headache, anosmia (loss of smell), ataxia, 65 meningitis, seizures and impaired consciousness (12) (13) (14) . to-date, sars-cov-2 has infected over eight million people world-wide and resulted in the death of more than 400,000. there is an urgent need for medical countermeasures to prevent this disease or limit disease severity in a post exposure setting. similar to sars-cov (15) , sars-cov-2 binds to target cells via an interaction between the 70 139 kda viral spike protein and the host angiotensin-converting enzyme 2 (ace2) protein (16) (17) (18) (19) ). an important factor in host tropism of the virus is this receptor interaction and reduced affinity between these two molecules greatly impacts host susceptibility to infection. both sars-cov and possibly to a greater extent sars-cov-2 bind to murine ace2 (mace) poorly compared human ace2 (hace2) (20) . accordingly, mice are inherently refractory to infection by ace2 utilizing 75 golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice human coronaviruses (21) (22) (23) . in these animals, infection by sars-cov (22) and sars-cov-2 (23) is rapidly controlled, although older mice are more permissive to lung replication, but nevertheless lung injury is limited and mortality is generally low. indeed, even infection in mice lacking adaptive immunity due to rag deficiencies (no t-cells or b-cells) are protected against severe sars-cov disease, whereas mice genetically devoid of stat-1, an important molecule 80 involved in innate immunity, are sensitive to infection by sars-cov(24-26). however, disease in stat-1 mice is protracted and not highly representative of human infections (25, 26) . in response to the need for small animal models to study sars-cov, several laboratories 32) . k18 limits expression to airway epithelial cells, colon and to a lesser extent kidney, liver, spleen and small intestine. a minor level 90 of hace2 expression was also detected in the brain. these mice are susceptible to sars-cov strain urbani and develop severe respiratory disease subsequent to intranasal exposure characterized by lung inflammation, serum cytokine and chemokine production as well as high lethality (30). as with several mouse strains transgenic for hace2, sars-cov infects the brains of k18-hace2 mice (30, 32) . cns localization is speculated to play a major role in host mortality 95 due to neuronal cell death, particular cell loss in cardiorespiratory control center (32) . some of these previous hace2 transgenic mice are being used for sars-cov-2 research and newer golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice systems have been developed using crsipr-cas9 (23, 33, 34). however, none were shown to produce a consistent lethal disease and in models where lung injury occurred, it was most pronounced in aged animals (23, 34). here, we evaluated susceptibility of k18-hace2 mice to 100 sars-cov-2. we found that mice developed severe disease that included respiratory distress with weight loss and mortality, as well as brain infection. sars-cov-2 produces severe and fatal infection of k18-hace2 mice. two groups of 9-week old k18-hace2 mice (n=14 per group) were intranasally infected with 2x10 4 or 2x10 3 pfu of 105 sars-cov-2. these groups equally divided by sex (n=7 per group/sex). on day 3, two mice per group were euthanized to assess disease severity. the remaining 5 mice per group were monitored up to 28 days. we also infected c57bl/6, balb/c and rag2 deficient mice with a challenge dose of 2x10 4 pfu. on day 4, all groups of infected k18-hace2 mice began to lose weight, which was more pronounced in the female mice compared to the male mice in either challenge dose group 110 ( fig. 1a & fig. s1a ). non-transgenic mice did not lose any weight and no animal succumbed to disease. starting on day 5, several k18-hace2 animals began to show signs of respiratory disease, included labored breathing and conjunctivitis. on day 5 through day 7 the majority of mice (15/20 mice) began to meet euthanasia criteria (n=13) or died (n=2). an additional animal in the low dose male group succumbed to disease on day 11, after a period of weight loss. the highest mean weight 115 loss was in the female groups (>20%), although male mice lost >12% weight (fig s1a,b) . of the k18-hace2 mice in the high dose challenge groups, all females and 80% of the male mice succumbed to disease. most female mice survived in the low dose group with 40% mortality, but all male mice succumbed to disease. the difference in survival between low dose male and female golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice mice was significant (log-rank; p=0.040), as was mortality between the high and low dose female 120 groups (log-rank; p=0.037). there was no statistical significance in survival between the other groups. lung homogenates taken on day 3 showed high levels of virus in k18-hace2, in contrast to c57bl/6 or rag2-deficient mice which had low levels of virus (fig. 1b,c) . the virus rna levels were nearly identical between all the infected k18-hace2 groups and remained high in most of the euthanized mice, with the exception of the mouse that died on day 11. that animal had 125 markedly lower levels of detectable genomic rna (fig. 1c) . compared to non-transgenic mice and uninfected k18-hace2 mice, infected k18-hace2 mice had comparatively higher serum levels of tnf-α, il-6 and il-10 as well as the monocyte chemoattractants mcp-1 (ccl2) and mcp-3 (ccl7) (fig. 1d) . levels of cytokines observed in mice that succumbed to disease were generally higher compared to those sampled on day 3. overall, these findings indicated that 130 sars-cov-2 causes a severe disease in k18-hace2 mice following intranasal exposure. lungs of k18-hace2 mice exposed to sars-cov-2 show signs of acute disease. lungs were collected from k18-hace2 on day 3 or at the time of euthanasia due to disease severity. in k18-hace2, viral rna was detected by in situ hybridization (ish) in all mice on day 3 and in most 135 mice succumbing to disease on days 5-7, but these levels diminished as disease progressed (fig 2a, fig s2, table s1 ). additionally, ish labeling was patchy (fig. s2) and most severe at the day 3 time point. ish labeling was present in both inflamed and normal appearing alveolar septa. positive ish labeling for sars-cov-2 was identified multifocally in alveolar septa in the lung, suggesting infection of pneumocytes and macrophages. this was confirmed by the detection of 140 golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice sars-cov-2 protein in e-cadherin positive cells (pneumocytes) and cd68 positive (alveolar and infiltrating macrophages) using immunofluorescence assay (ifa, fig. 2b ,c). in comparison with the normal lung architecture in uninfected control animals, infected mice necropsied on day 3, and those succumbing to disease on days 5-11, had varying levels of lung injury including area of lung consolidation characterized by inflammation/expansion of 145 alveolar septa with fibrin, edema and mononuclear leukocytes and infiltration of vessel walls by numerous mononuclear leukocytes (fig 3a, fig s4, and table s1 ). type ii pneumocyte hyperplasia was identified in less than half of infected animals. this lesion had a relatively patchy distribution except in the most severely affected animals where it is more abundant. in areas of septal inflammation, exudation of fibrin and edema into alveolar lumina from damaged septal 150 capillaries was observed in most animals. vasculitis was the most common finding and was present in ~95% percent of all mice (fig. 3a,b) . the lesion encompassed small and intermediate caliber vessels, and was often characterized by near circumferential infiltration of the vessel wall by numerous mononuclear inflammatory cells. this lesion also contained small amounts of fibrin and occasional necrotic debris, affecting all tunics and obscuring the vessel wall architecture. however, 155 the endothelial cells surrounding lung vasculature were largely free of viral rna (fig. 3b) . in one low dose male mouse that died on day 11, evidence of numerous fibrin thrombi were identified in the small and intermediate vessels, suggestive of a hypercoagulable state within the lung. this same animal had marginally detectable virus in the lung (fig. 1c) , and fibrin thrombi were not identified in other organs including liver and kidney. infected k18-hace2 mice had elevated 160 numbers of tunel positive cells, suggesting increased cell death (fig. 3c) . we also detected increased expression of ki-67, a marker for cellular proliferation, likely expressed by proliferating golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice pneumocytes and potentially by replicating alveolar macrophages (fig. 3d) . neutrophilia was detected by h&e staining, consistent with an increase in myeloperoxidase (mpo)-positive cells (neutrophils, basophil and eosinophil) detected by ifa (fig. s3a) . however, the mpo positive 165 cells were devoid of viral antigen (fig. s3a) . there was also an increase in the presence of cd68 positive macrophages (fig. s3b) and a pronounced increase in cd45 and cd3 positive cells in infected lungs, indicative of infiltrating leukocytes including t-cells, which is consistent with histologic findings (fig. s3c) . these data indicate that k18-hace2 mice develop a pronounced lung injury upon exposure to sars-cov-2. transcriptional profiles of host immunological and inflammatory genes in lung homogenates from sars-cov-2 infected k18-hace2 and c57bl/6 mice were examined by nanostring-based gene barcoding on day 3 (k18-hace2 and c57bl/6) or in k18-hace2 mice at the time euthanasia. 478 transcripts were increased in k18-hace2 mice and 430 decreased at a log2 fold cutoff of 1 and a p value <0.05. many of the increased transcripts in the k18-hace2 175 mice were inflammatory genes including il-6, interferon gamma and chemokines (ccl2, ccl5, ccl9 and cxcl10) (fig. 4a) . the type i interferon transcripts ifna1 and ifnb1 along with the cytokines il-9 and il-2 were decreased. consistent with the increased presence of cd68 macrophages, cd68 transcripts were also significantly increased in k18-hace2 mice. viral sensing pathways were elevated in infected mice with severe disease, indicated by high transcript was observed in the nasal turbinates in the majority of mice and rarely within the eyes of infected mice (fig. 5a, fig. s5 and table s1 ). viral rna in the eye was localized to the retina, suggesting 195 viral infection of neurons in the inner nuclear layer and ganglion cell layer (fig. s5) . despite infection, evidence of inflammation or other damage in the retina or elsewhere in the eye was not present. viral rna and spike protein were also detected to some degree in the nasal turbinate epithelium (predominantly olfactory epithelium) as early as day 3 post-infection (fig. 5b) , as indicated by co-staining with cytokeratin ( fig 5c) . pathology was minimal, predominantly 200 isolated to few areas of olfactory epithelium atrophy, with degeneration or erosion present in the epithelium lining the dorsal and lateral nasal meatuses (fig. 5d) . some cellular sloughing was also detected and these sloughed cells contained viral rna (fig. s5) . in mice succumbing to disease on days 5-7, virus was present in the olfactory bulb and most animals showed asymmetrical staining, with one bulb more positive than the other. viral rna was detected throughout the 205 olfactory bulb, including in the olfactory nerve layer (onl), glomerular layer (gl), external golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice plexiform layer (epl), and mitral cell layer (mcl) of olfactory bulbs in most of animals. viral protein co-localized with the neuronal marker neun, suggesting virus was present within neurons in the olfactory bulb (fig. s6) . virus was not detected in the olfactory bulb of animals taken on day 3, suggesting that virus trafficked to this region on day 4 or 5. these data indicated that sars-210 cov-2 infects cells within the nasal turbinates, eyes and olfactory system and that infection was observed in epithelial cells and neurons. brain infection was not observed in the majority of animals examined on day 3, but was prevalent in mice necropsied on days 5-11 (table 215 s1 ). evidence of sars-cov-2 was found throughout the brain including strong but variably diffuse signal in regions of the thalamus, hypothalamus, amygdala, cerebral cortex, medulla, pons and midbrain (fig. 6a, table s1 ). similar intense but less diffuse signal was present within the hippocampus. ish positive cells included neurons of thalamic nuclei. in contrast, cells within the vessel walls and perivascular spaces infiltrated by mononuclear inflammatory cells were negative 220 for viral genomic rna (fig. 6a) . histopathological changes were detected in the brains of several infected k18-hace2 mice euthanized on day 5-11, but not in most mice taken on day 3 (fig. 6b , table s1 and fig. s7 ). in the thalamus/hypothalamus, vasculitis was the most common lesion characterized by endothelial hypertrophy and increases in mononuclear leukocytes within the vessel wall and/or filling the perivascular space. small amounts of necrotic debris were also 225 identified. in the majority of these cases, the vascular lesion was characterized by the presence of increased numbers of microglia within the adjacent neuropil. occlusive fibrin thrombi were also detected within the thalamus in a few mice. meningitis was observed in a subset of animals and golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice was associated with infiltration of mononuclear leukocytes (majority lymphocytes) and is most prominent adjacent to vessels. in the mouse that died late on day 11 with massive pulmonary 230 clotting, the rostral cerebral cortex brain lesions included small to intermediate size vessel walls multifocally expanded by mononuclear inflammatory cells and perivascular hemorrhage extending into the adjacent neuropil (fig. 6b) . increased numbers of microglia were readily detected on h&e stained sections, expanding outward from the perivascular neuropil. an increase in numbers of microglia were found in the neuropil surrounding affected vessels. additionally, brains also 235 showed signs of neuroinflammation indicated by increased staining of iba-1 and gfap indicating microgliosis and astrocytosis, respectively (fig. 6c) . necrosis was identified in at least five animals, and was most prominent within the periventricular region of the hypothalamus as well as in the amygdala. the lesion was characterized by moderate numbers of shrunken, angular cells with hypereosinophilic cytoplasm, pyknotic nuclei and surrounded by a clear halo (fig. s7) . the 240 morphology and location of individual cells was suggestive of neuronal necrosis, but further investigation will be required to confirm this finding. viral spike protein was detected in neun positive cells, indicating viral infection of neurons (fig. 6d) . viral antigen was absent in gfap positive cells suggesting virus does not productively infect astrocytes. these data indicate that similar to sars-cov, sars-cov-2 also targets the brain of k18-hace2 mice, causing brain 245 injury. as indicated by duplex ish labeling of brain, neurons are positive for both hace2 transgene expression and viral genomic rna (fig. s8) . no animal showed outward signs of neurological deficient, such as hind limb paralysis, head tilting or tremors. 250 golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice other murine infection models for sars-cov-2 involving transgene expression of the human ace2 protein have been reported (23, 33, 34) . however in these models, sars-cov-2 only produces a transient weight loss with some lung injury, but the animals generally recover. additionally, several of these models required mice aged >30 weeks for the most severe disease to occur, diminishing the practicality of these systems given the urgent need for medical 255 countermeasures (mcms) (23, 34). one system tested sars-cov-2 infection in mice in which hace2 was expressed under the control of the hfh4 promoter (33) . infection in these mice was only ~40% lethal (2/5 mice) and lung injury (assessed by plethysmography) and weight loss were absent. lethality in this system was exclusive to animals where virus was detected in the brain. other recently reported sars-cov-2 murine models involved transduction of mouse lungs with 260 a replication-incompetent adenovirus virus or an adeno associated virus (aav) encoding the hace2 gene (35, 36) . transduced lung cells expressing hace2 supported sars-cov-2 replication and lung pathology ensued along with weight loss. however, disease was generally mild with no lethality. blockade of the type i interferon system using pharmacological intervention was needed to produce the most severe disease 26 . murine systems faithfully producing the major 265 elements of severe disease observed in humans will be more useful for identifying the most promising mcms. the k18-hace2 mice lost considerable weight >12% in males and >20% in females and lethality in the high dose exceeded 90%. acute lung injury was detected in all animals succumbing to disease, with vascular damage the most common lesion. similar to findings with sars-cov, sars-cov-2 infected the brains of k18-hace2 mice. brain infection resulted in 270 vasculitis and inflammation, with sars-cov-2 antigen detected in neurons. it is possible virus enters the brain via the olfactory bulb, as has been reported for sars-cov, although more studies golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice will be required as virus may also have entered the brain via inflamed vessels. whether mortality results directly from brain infection is not clear, but this has been speculated as the major cause of mortality in sars-cov infected mice (32) . infection in the brain was delayed by at least four 275 days, as it was an uncommon finding in day 3 animals. thus, early during infection lung appears to be the primary target. we have not yet evaluated the protective efficacy of mcms in this model, but it has been reported that antibodies protect against sars-cov, demonstrating the k18-hace2 system is useful for evaluating countermeasures against ace2-targeting human coronaviruses. importantly, in our study some animals at the lower dose survived infection despite significant infection of k18-hace2 mice by sars-cov-2 produces a disease similar to that observed in acute human cases, with development of an acute lung injury associated with edema, production 285 of inflammatory cytokines and the accumulation of mononuclear cells in the lung. impacted lungs had elevated levels of transcripts consistent with respiratory damage such as increased expression of himf, which is involved in activation of lung endothelial cells in response to lung inflammation (37) . we also found increased levels of sgpl1, a molecule associated with lung injury (38) and known to be increased in mechanically ventilated mice (39) . a prominent finding in infected k18-290 hace2 mice system was vasculitis. endotheliitis/vasculitis has been observed in human covid-19 patients and a role for the endothelium in acute disease is becoming more apparent (7, 8, 40) . direct viral infection in human endothelial cells has also been reported (7). curiously in the mice, virus was absent in these areas suggesting vasculitis was due to host inflammatory processes, but golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice further study will be required to determine if vascular damage is incurred by direct or indirect viral 295 effects. it has been speculated that during human acute disease, inflammatory cytokines (il-6 in particular) are major drivers of tissue damage and this is supported by data from humans showing il-6 receptor targeting with pharmacological antagonists (tocilizumab) can reduce morbidity (41). we found il-6 transcripts, as well chemokines, are elevated in mouse lungs. during human disease, pulmonary inflammation is associated with increases in lung granulocytes and an increase 300 in macrophages (4, 42, 43) . some have speculated that these macrophages play an important role in host injury (44) . it is still unclear if human macrophages are directly infected by sars-cov-2, though we found virus antigen in cd68 macrophages and others report infection in murine mac2-positive macrophages (23). further study will be required to determine the role macrophages play in sars-cov-2 lung injury and given the regents available, murine systems 305 may be highly suited for these studies. in addition to vascular issues, coagulopathies are a common findings in humans (9), with pulmonary embolism having been reported, along with clotting abnormities leading to loss of limbs (45) . at least some mice produced evidence of these clotting issues, with one mouse presenting with fibrin thrombi in the lungs. during human infections, males have been reported to have more severe outcomes despite 310 a similar infection rate (46). in our experiments, we observed a statistically significant difference in survival of female and male mice infected at the lower dose of virus, with 60% of females but no males surviving infection. despite surviving, the female mice challenged with the low dose still lost a significant amount of weight (>20%). transcriptomic profiling in mouse lungs indicated that female mice that succumbed to disease had modestly lower levels of il-6, cxcl-2 and il-1ra 315 suggesting a less intense inflammatory response. our work only involved a small number of golden jw, et al sars-cov-2 pathogenesis in k18-hace2 mice animals, and more work will be required to determine if the k18-hace2 system recapitulates this sex difference in disease severity. the neuroinvasive aspects of covid-19 are becoming more appreciated (12) . indeed, sars-cov-2 causes neurological sequela in at least a third of human cases including headache, infection of these cells may help explain the loss of smell associated with some covid-19 cases. however, the virus may also gain access via disruption of the blood brain barrier, as these were inflamed in most animals and neurovasculitis has been found in humans (48). overall, the k18were then heated in kit-provided antigen retrieval buffer and digested by kit-provided proteinase. 405 sections were exposed to ish target probe pairs and incubated at 40°c in a hybridization oven for 2 h. after rinsing, ish signal was amplified using kit-provided pre-amplifier and amplifier conjugated to alkaline phosphatase and incubated with a fast red substrate solution for 10 min at room temperature. sections were then stained with hematoxylin, air-dried, and coverslipped. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study targeting potential drivers of covid-19: neutrophil extracellular traps covid-19 and crosstalk with the hallmarks of aging the hallmarks of covid-19 disease pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 covid-19: the vasculature unleashed covid-19 and its implications for thrombosis and anticoagulation the pathogenesis and treatment of the `cytokine storm' in covid-19 the many faces of the anti-covid immune response neuromechanisms of sars-cov-2: a review early recovery following new onset anosmia during the covid-19 pandemic -an observational cohort study does sars-cov-2 invade the brain? translational lessons from animal models angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus structural basis of receptor recognition by sars-cov-2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven 520 cryo-em structure of the 2019-ncov spike in the prefusion conformation structure of the sars-cov-2 spike 525 receptor-binding domain bound to the ace2 receptor receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus animal models for sars and mers coronaviruses prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice sars-like wiv1-cov poised for human emergence severe acute respiratory 560 syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 a mouse-adapted sars-cov-2 model for the evaluation of covid-19 medical countermeasures a mouse model of sars-cov-2 infection and pathogenesis a sars-cov-2 infection model in mice demonstrates protection by neutralizing antibodies mouse model of sars-cov-2 reveals inflammatory role of type i interferon signaling hypoxiainduced mitogenic factor (himf/fizz1/relmalpha) increases lung inflammation and activates pulmonary microvascular endothelial cells via an il-4-dependent mechanism protection of lps-induced murine acute lung injury by sphingosine-1-phosphate lyase suppression sphingolipids in ventilator induced lung injury: role of sphingosine-1-phosphate lyase endothelial cell infection and endotheliitis in covid-19 effective treatment of severe covid-19 patients with tocilizumab macrophages: a trojan horse in covid-19? the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the 590 perspectives of clinical immunologists from china severe covid-19 and aging: are monocytes the key? geroscience acute pulmonary embolism associated with covid-19 pneumonia detected by pulmonary ct angiography gender differences in patients with covid-19: focus on severity and mortality 465 this project was funded by a grant awarded to j.w.g. and j.w.h. from the military infectious disease program. we thank the usamriid histology lab and comparative medicine division for their assistance. we also express gratitude to the jackson laboratory for providing early access to duplex in situ hybridization. duplex in situ hybridization was performed using the rnascope 2.5 hd duplex assay kit (advanced cell diagnostics) according to the manufacturer's instructions with minor modifications. in addition to sars-cov-2 genomic rna probe mentioned above (#854841, green), another probe with c2 channel (#848031-c2, red) specifically targeting human ace2 (nm_021804.3) was designed and synthesized by advanced cell diagnostics. ish signal was amplified using kit-provided pre-amplifiers and amplifiers conjugated to either alkaline phosphatase or horseradish peroxidase, and incubated sequentially with a fast red and green chromogenic substrate solution for 10 min at room temperature. sections were then stained with hematoxylin, air-dried, and coverslipped. key: cord-347734-0z2kin6r authors: armann, j. p.; unrath, m.; kirsten, c.; lueck, c.; dalpke, a.; berner, r. title: anti-sars-cov-2 igg antibodies in adolescent students and their teachers in saxony, germany (schoolcovidd19): very low seropraevalence and transmission rates date: 2020-07-17 journal: nan doi: 10.1101/2020.07.16.20155143 sha: doc_id: 347734 cord_uid: 0z2kin6r background: school closures are part of the sars-cov-2 pandemic control measures in many countries, based on the assumption that children play a similar role in transmitting sars-cov-2 as they do in transmitting influenza. we therefore performed a sars-cov-2 seropraevalence-study in students and teachers to assess their role in the sars-cov-2 transmission. methods: students grade 8-11 and their teachers in 13 secondary schools in eastern saxony, germany, were invited to participate in the schoolcovidd19 study. blood samples were collected between may 25th and june 30th, 2020. anti-sars-cov-2 igg were assed using chemiluminescence immunoassay technology and all samples with a positive or equivocal test result were re-tested with two additional serological tests. findings: 1538 students and 507 teachers participated in this study. the seropraevalence for sars-cov-2 was 0.6%. even in schools with reported covid-19 cases before the lockdown of march 13th no clusters could be identified. 23/24 participants with a household history of covid-91 were seronegative. by using a combination of three different immunoassays we could exclude 16 participants with a positive or equivocal results after initial testing. interpretation: students and teachers do not play a crucial role in driving the sars-cov-2 pandemic in a low prevalence setting. transmission in families occurs very infrequently, and the number of unreported cases is low in this age group, making school closures not appear appropriate as a strategy in this low prevalence settings. funding: this study was supported by a grant from the state of saxony methods: students grade 8-11 and their teachers in 13 secondary schools in eastern saxony, germany, were invited to participate in the schoolcovidd19 study. blood samples were collected between may 25th and june 30th, 2020. anti-sars-cov-2 igg were assed using chemiluminescence immunoassay technology and all samples with a positive or equivocal test result were re-tested with two additional serological tests. findings: 1538 students and 507 teachers participated in this study. the seropraevalence for sars-cov-2 was 0.6%. even in schools with reported covid-19 cases before the lockdown of march 13 th no clusters could be identified. 23/24 participants with a household history of covid-91 were seronegative. by using a combination of three different immunoassays we could exclude 16 participants with a positive or equivocal results after initial testing. interpretation: students and teachers do not play a crucial role in driving the sars-cov-2 pandemic in a low prevalence setting. transmission in families occurs very infrequently, and the number of unreported cases is low in this age group, making school closures not appear appropriate as a strategy in this low prevalence settings. funding: this study was supported by a grant from the state of saxony . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 17, 2020. . https://doi.org/10.1101/2020.07.16.20155143 doi: medrxiv preprint introduction: since the identification of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as the cause of covid-19 in december 2019 1 , the virus spread rapidly around the world, leading to the declaration of a pandemic by the world health organization on march 12 th 2020. by march 18 th 2020, 126 countries-including germany-had implemented school closures as part of their pandemic control measures, with the number of countries peaking at 194 on april 10 th 2020 and more than 90% of the world's student population being affected at this point 2 . these actions were mainly based on the assumption that children play a similar role in transmitting sars-cov-2 as they do in transmitting influenza during outbreaks, for which evidence exists that school closures reduce the peak of the outbreak 3 . however, there is reason to believe that children play a less significant role in sars-cov-2 transmission compared to influenza, making control measures focused on this age group less effective: most countries-including germany-report a much lower proportion of cases in children compared to their population size 4-6 . in addition a recent report from australia could only identify a very limited spread of covid-19 in primary schools, with no evidence of children infecting teachers 7 . however, currently available data is insufficient to rule out that children are as likely as adults to be infected by and to transmit sars-cov-2, but simply show little to no symptoms of the disease. we therefore aimed to quantify the proportion of adolescent schoolchildren and teachers in saxony, one of the eastern federal states of germany, that already have developed antibodies against sars-cov-2. in saxony, the infection rates were comparatively low with 139 laboratory-confirmed sars-cov-2 infections per 100,000 inhabitants as of 13 july 2020. after the reopening of the schools in saxony on may 18 th , 2020 students grade 8-11 and their teachers in 13 secondary schools in eastern saxony were invited to participate in the schoolcovidd19 study. after teachers, students, and their legal guardians provided informed consent, 5 ml of peripheral venous blood were collected from each individual during visits at each participating school between may 25 th and june 30 th , 2020. in addition, participants were asked to complete a questionnaire on age, household size, previously diagnosed sars-cov-2 infections in themselves or their household contacts, comorbidities and regular medication. in addition, students were asked about regular social contacts outside their household or classroom. the schoolcovidd19 study was approved by the ethics committee of the technische universität (tu) dresden (bo-ek-156042020) and has been assigned clinical trial number drks00022455. we assessed anti-sars-cov-2 igg antibodies in all samples using a commercially available chemiluminescence immunoassay (clia) technology for the quantitative determination of anti-s1 and anti-s2 specific igg antibodies to sars-cov-2 (diasorin liaison® sars-cov-2 s1/s2 igg assay). antibody levels > 15.0 au/ml were considered positive and levels between 12.0 and 15.0 au/ml were considered equivocal. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 17, 2020. . all samples with a positive or equivocal liaison® test result, as well as all samples from participants with a reported personal or household history of a sars-cov-2 infection, were re-tested with two additional serological tests: these were a chemiluminescent microparticle immunoassay (cmia) intended for the qualitative detection of igg antibodies to the nucleocapsid protein of sars-cov-2 (abbott diagnostics® architect sars-cov-2 igg ) (an index (s/c) of < 1.4 was considered negative whereas one >/= 1.4 was considered positive) and an elisa detecting igg against the s1 domain of the sars-cov-2 spike protein (euroimmun® anti-sars-cov-2 elisa) (a ratio < 0.8 was considered negative, 0.8-1.1 equivocal, > 1.1 positive) participants whose positive or equivocal liaison® test result could be confirmed by a positive test result in at least one additional serological test were considered having antibodies against sars-cov-2. analyses were performed using ibm spss 25.0 and microsoft excel 2010. results for continuous variables are presented as medians with interquartile ranges (iqr) and categorical variables as numbers with percentages, unless stated otherwise. the funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. a total of 1538 students and 507 teachers from 13 different schools participated in this study. demographic data is shown in table 1 . the number of participants ranged from 21 to 573 per individual school. twelve participants-11 students and one teacher-had detectable antibodies against sars-cov-2 in at least two different assays and were considered seropositive. in 7/13 schools, seropositive participants could be identified, with four seropositive participants in one school as the maximum. the seroprevalence ranged from 0 to 2. table 3 . the findings from this unique study in older students and their teachers indicate that the prevalence of igg antibodies against sars-cov-2 remains extremely low after the first wave of the corona pandemic in germany. while this finding is consistent with local surveillance data 8 that shows a prevalence of pcr-confirmed cases of 0,15%, it clearly indicates that schools did not develop into silent hotspots of sars-cov-2 transmission during this first wave of the pandemic. in fact, 5 of the 12 participants with antibodies against sars-cov-2 had a personal or household history of covid-19, yielding a ratio of unidentified to identified cases of 2.4, which is much smaller than that previously assumed by some authors 9 . we could not detect a single cluster of infections in the participating schools, even though at least three schools did have confirmed sars-cov-2 cases before the march 13 th lockdown in saxony. this is consistent with findings from the 2003 sars outbreak 10, 11 , and calls the effectiveness of transmission control measures focused mainly on the student population into question. this is especially relevant since there are clearly described adverse effects of school closures, as loss of education, loss of social contacts and social control, nutritional problems in children who rely on school meals, increases in harm to child welfare in vulnerable populations, as well as economic harm caused by loss to productivity due to parents being forced from work to childcare 12, 13 . additionally, even with school closures in place, social contacts continue as informal child care and non-school gatherings 14 , thereby reducing the potential benefit of school closures further. our data support this finding this finding since an overwhelming majority of not less than 80% of the participating students in our study reported to have regular social contacts outside their household or classroom. close contact with covid-19 patients-especially in the same household-has been shown to increase viral transmission 15 . however, in our study, only one out of 24 participants with a confirmed sars-cov-2 infection in the same household became indeed infected as measured by antibody production. this suggests that either the transmissibility of the virus is lower than previously assumed or that there are certain quarantine and separation measures than can effectively reduce the probability of viral transmission even in close contact situations. currently no gold standard serological testing strategy for sars-cov-2 exists. even though immunoassays yield better performance than rapid point-of-care tests 15 and the targeted sars-cov-2 s protein and nucleoprotein show a similarity of less than 30% to endemic betacoronaviruses 16 , false positive results are still a concern, especially in low-prevalence populations and when interpreting results on a personal rather than a population-based level. by using a combination of three different immunoassays and only regarding participants with at least two positive results as seropositive for sars-cov-2, we could exclude ten participants with a positive and six with an equivocal initial test by negative confirmatory testing. in our population a positive predictive value of 42.9% could be observed which was nearby an expected ppv of 45.3% for a prevalence of 0.59% population and the given test characteristics (sensitivity 97.6%, specificity 99.3%). by using this approach, we could reliably identify patients with confirmed seropositivity against sars-cov-2 in a low-prevalence population. as for now, students and teacher do not seem to play a crucial role in driving the sars-cov-2 pandemic in germany. transmission in families appears to occur very infrequently, and the number of unreported cases obviously is low in this age group. therefore, social distancing strategies such as the reduction of students of different classes mixing at school paired with symptom-based screening strategies, contact tracing and quarantine measures for identified cases are likely as effective as full school closures, with less adverse effects on the student population. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july 17, 2020. . https://doi.org/10.1101/2020.07.16.20155143 doi: medrxiv preprint a novel coronavirus from patients with pneumonia in china education: from disruption to recovery impact assessment of non-pharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study coronavirus disease 2019 in children -united states hospital admission in children and adolescents with covid-19 report: covid-19 in schools -the experience in nsw | ncirs sächsisches staatsministerium für soziales und gesellschaftlichen substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2) severe acute respiratory syndrome (sars) in children a probabilistic transmission dynamic model to assess indoor airborne infection risks school closure during novel influenza: a systematic review the effects of school closures on influenza outbreaks and pandemics: systematic review of simulation studies evidence compendium and advice on social distancing and other related measures for response to an influenza pandemic prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study. the lancet the role of antibody testing for sars-cov-2: is there one? for serological testing, a combination of different immunoassays seems to be effective to increase the number of true positive test results. all authors declare no conflict of interests key: cord-340516-9dfaqsv7 authors: moore, anne c.; dora, emery g.; peinovich, nadine; tucker, kiersten p.; lin, karen; cortese, mario; tucker, sean n. title: pre-clinical studies of a recombinant adenoviral mucosal vaccine to prevent sars-cov-2 infection date: 2020-09-06 journal: biorxiv doi: 10.1101/2020.09.04.283853 sha: doc_id: 340516 cord_uid: 9dfaqsv7 there is an urgent need to develop efficacious vaccines against sars-cov-2 that also address the issues of deployment, equitable access, and vaccine acceptance. ideally, the vaccine would prevent virus infection and transmission as well as preventing covid-19 disease. we previously developed an oral adenovirus-based vaccine technology that induces both mucosal and systemic immunity in humans. here we investigate the immunogenicity of a range of candidate adenovirusbased vaccines, expressing full or partial sequences of the spike and nucleocapsid proteins, in mice. we demonstrate that, compared to expression of the s1 domain or a stabilized spike antigen, the full length, wild-type spike antigen induces significantly higher neutralizing antibodies in the periphery and in the lungs, when the vaccine is administered mucosally. antigen-specific cd4+ and cd8+ t cells were induced by this leading vaccine candidate at low and high doses. this fulllength spike antigen plus nucleocapsid adenovirus construct has been prioritized for further clinical development. the emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of covid-19 disease, in 2019, has led to a global pandemic and significant morbidity, mortality and socio-economic disruption not seen in a century. coronavirus disease 2019 (covid-19) is a respiratory illness of variably severity; ranging from asymptomatic infection to mild infection, with fever and cough to severe pneumonia and acute respiratory distress 1 . current reports suggest that asymptomatic spread is substantial 2 , and sars-cov-2 infection induces a transient antibody response in most individuals 3 . therefore, development of successful interventions is an immediate requirement to protect the global population against infection and transmission of this virus and its associated clinical and societal consequences. mass immunization with efficacious vaccines has been highly successful to prevent the spread of many other infectious diseases and can also prevent disease in the vulnerable through the induction of herd immunity. significant effort and resources are being invested in urgently identifying efficacious sars-cov-2 vaccines. a number of different vaccine platforms have demonstrated pre-clinical immunogenicity and efficacy against pneumonia 4, 5 . several vaccines have demonstrated phase i or phase ii safety and immunogenicity [6] [7] [8] . however, at this time, no vaccine has demonstrated efficacy in the field. the most advanced sars-cov-2 vaccine candidates are all given by the intramuscular (im) route, with some requiring -80 °c storage. this is a major barrier for vaccine dissemination and deployment during a pandemic in which people are asked to practice social distancing and avoid congregation. the ultimate goal of any vaccine campaign is to protect against disease by providing enough herd immunity to inhibit viral spread, not to make a set number of doses of vaccine. an injected solution takes a long period of time to administer and distribute and requires costly logistics, which means dose availability does not immediately translate to immunity. further, systemic immunization can induce immunity in the periphery and lower respiratory tract. however, these vaccines cannot induce mucosal immunity in the upper respiratory tract. mucosal iga (with the polymeric structure and addition of the secretory component), creates more potent viral neutralization 9 , can block viral transmission 10, 11 , and in general, is more likely to create sterilizing immunity given that this is the first line of defense for a respiratory pathogen. mucosal vaccines can induce mucosal immune responses, antibodies and t cells at wet surfaces. we are developing oral vaccines for multiple indications, including influenza and noroviruses, delivered in a tablet form for people. our vaccine platform is a replication-defective adenovirus type-5 vectored vaccine that expresses antigen along with a novel toll-like receptor 3 agonist as an adjuvant. these vaccines have been well tolerated, and able to generate robust humoral and cellular immune responses to the expressed antigens [12] [13] [14] . protective efficacy in humans was demonstrated against a respiratory virus 90 days or more post vaccination, as shown in a well characterized experimental influenza infection model 15 . furthermore, the vaccine also has the advantage of room temperature stability and needle-free, ease of administration, providing several advantages over injected vaccine approaches with respect to vaccine deployment and access. here, we describe the pre-clinical development of a sars-cov-2 vaccine based on vaxart's oral adenovirus platform. the key approach was to develop several vaccine candidates in parallel, in order to create premanufacturing seeds while initial immunogenicity experiments were in progress. given that the vaccines were made during the pandemic, rapid decisions were required to keep the manufacturing and regulatory timelines from slipping. we assessed the relative immunogenicity of four candidate vaccines that expressed antigens based on the spike (s) and nucleocapsid (n) sars-cov-2 proteins. these proteins have been well characterized as antigens for related coronaviruses, such as sars-cov and mers (reviewed in yong, et al., 16 ) and, increasingly, for sars-cov-2 spike. the aim of our vaccine is to induce immunogenicity on three levels; firstly, to induce potent serum neutralizing antibodies to s, secondly to induce mucosal immune responses, and thirdly to induce t cell responses to both vaccine antigens. this three-fold approach aims to induce robust and broad immunity capable of protecting the individual from virus infection as well as disease, promote rapid dissemination of vaccine during a pandemic, and to protect the population from virus transmission through herd immunity. here, we report the induction of neutralizing antibody (nab), igg and iga antibody responses, and t cell responses in mice following immunization of rad vectors expressing one or more sars-cov-2 antigens. initially, three different rad vectors were constructed to express different sars-cov-2 antigens. these were a vector expressing the full-length s protein (rad-s), a vector expressing the s protein and the n protein (rad-s-n), and a vector expressing a fusion protein of the s1 domain with the n protein (rad-s1-n). the n protein of rad-s-n was expressed under control of the human beta actin promoter, which is much more potent in human cells than mouse cells. an additional construct where the expressed s protein was fixed in a prefusion conformation (rad-s(fixed)-n) was constructed at a later date as a control for exploring neutralizing antibody responses. these are described in figure 1 . expression of the various transgenes was confirmed following infection of 293 cells using flow cytometry and monoclonal antibodies to the s or n protein (supplemental figure 1) . the primary objective of the initial mouse immunogenicity studies was to determine which of the rad vectors induced significant antibody responses to s, and to obtain those results rapidly enough to provide a gmp seed in time for manufacturing. we and others 17 have observed that transgene expression by vaccine vectors orally administered to mice can be suppressed in their intestinal environment, so immunogenicity was assessed following intranasal (i.n.) immunization. animals were immunized i.n. and the antibody titers were measured over time by igg elisa. all three rad vectors induced nearly equivalent anti-s1 igg titers, at weeks 2 and 4 and the igg titer in all animals was significantly boosted by the second immunization (p < 0.05 mann whitney t-tests) ( fig. 2a) . however, the vector expressing full-length s (rad-s-n) induced higher neutralizing titers compared to the vector expressing only s1 (fig 2b) . this was measured by two different neutralizing assays, one based on sars-cov-2 infection of vero cells (cvnt) and one based on a surrogate neutralizing assay (svnt). furthermore, rad-s-n induced higher lung iga responses to s1 and unsurprisingly, to s2 (fig. 2c ) compared to rad-s1-n two weeks after the final immunization. notably, neutralizing titers in the lung were also significantly higher when rad-s-n was used compared to the s1-containing vaccine (rad-s1-n) (fig. 2d ). this demonstrated that the rad-s-n candidate induced greater functional responses (nab and iga) s1 s2 tm cmv s1 s2 furin furin compared to the vaccine containing the just the s1 domain. because the n protein is much more highly conserved than the s protein, and is a target of long term t cell responses induced by infection 18 , the vector rad-s-n was chosen for gmp manufacturing. c d three dose levels of rad-s-n were then tested to understand the dose responsiveness of this vaccine. the antibody responses to both s1 (fig. 3a) and s2 (fig. 3b) were measured. similar responses were seen at all three dose levels at all timepoints. responses to s1 and s2 were significantly increased at week 6 compared to earlier times, in all groups. the induction of s-specific t cells by rad-s-n at different doses was then assessed. induction of antigen-specific cd4+ and cd8+ t cells that produced effector cytokines such as ifn-g, tnf-a and il-2 was observed two weeks after 2 immunizations ( fig 4a) . notably, little il-4 was induced by this vaccine and only in cd4+ t cells; providing a level of assurance that the risk for vaccine dependent enhancement of disease was very low. furthermore, immunization with rad-s-n induced double and triple positive, multi-functional ifn-g, tnf-a and il-2 cd4+ t cells (fig. 4b) . a second dose response experiment was performed to focus on t cell responses to the s protein, 4 weeks after the final immunization (week 8 of the study). splenocytes were stimulated overnight with a peptide library to the s protein, divided in two separate peptide pools. t cell responses in the two pools were summed and plotted (fig. 4c ). animals administered the 1e7 iu and the 1e8 iu dose levels had significantly higher t cell responses compared to the untreated animals but produced a similar number of ifn-g secreting cells to each other, demonstrating a dose plateau at the 1e7 iu dose. notably, this t cell analysis was conducted 4 weeks after the second immunization, potentially after the peak of t cell responses. balb/c mice were immunized, in, on days 0 and 14 with 1 x 10 7 iu, 1 x 10 8 iu or 7.2 x 10 8 iu of rad co-expressing full length s and n (rad-s-n). the amount of igg specific for s1 (a) and s2 in serum diluted 1/4000, was evaluated using a mesoscale binding assay. points represent the mean and lines represent the standard deviation. a. c. rad-expressed wild-type s induces a superior neutralizing response compared to stabilized/pre-fusion s. an additional study was performed to compare rad-s-n to a vaccine candidate with the s-protein stabilized and with the transmembrane region removed (rad-s(fixed)-n). a stabilized version of the s protein has been proposed as a way to improve neutralizing antibody responses and produce less non-neutralizing antibodies. the s protein was stabilized through modifications as described by amanat et al., 19 . rad-s-n induced higher serum igg titers to s1 (fig. 5a ) at both timepoints tested, although these were not statistically significant at week 6 by mann-whitney (p = 0.067). however, rad-s-n induced significantly higher neutralizing antibody responses (fig. 5b ) than the stabilized version (p = 0.0152). these results suggest that a wild-type version of the s protein is superior for a rad based vaccine in mice. the endgame to the covid-19 pandemic requires the identification and manufacture of a safe and effective vaccine and a subsequent global immunization campaign. a number of vaccine candidates have accelerated to phase iii global efficacy testing and, if sufficiently successful in these trials, may form the first generation of an immunization campaign. however, all of these advanced candidates are s-based vaccines that are injected. such an approach will unlikely prevent virus transmission, but should prevent pneumonia and virus growth and damage in the lower respiratory tract and periphery, as evidenced in macaque challenge studies 4, 5 . one key constraint in a global covid-19 immunization campaign will be the cold chain distribution logistics and a bottleneck of requiring suitably trained health care workers (hcws) to inject the vaccine. current logistics costs, including cold chain and training, can double the cost of fully immunizing an individual in a low-middle income country (lmic) 20 . implementing a mass immunization campaign, requiring trained hcws for injection-based vaccines, will have a significant impact on healthcare resources in all countries. the need for cold chain, biohazardous sharps waste disposal and training will result in increased cost, inequitable vaccine access, delayed vaccine uptake and prolongation of this pandemic. these costs will be magnified if vaccines are unable to provide long-term protection (natural immunity to other beta-coronaviruses is short-lived 21 ), and annual injection-based campaigns are needed. vaxart's oral tablet vaccine platform provides a solution to these immunological as well as logistic, economic, access and acceptability problems. in this study we demonstrate, in an animal pre-clinical model, the immunogenicity of a sars-cov-2 vaccine using vaxart's vaccine platform; namely the induction of serum and mucosal neutralising antibodies and poly-functional t cells. mouse studies were designed to test immunogenicity of candidate vaccines rapidly in the spring of 2020, before moving onto manufacturing and clinical studies critical to addressing the pandemic. vaxart's oral tablet vaccine platform has previously proven to be able to create reliable mucosal (respiratory and intestinal), t cell, and antibody responses against several different pathogens in humans 12, 14, 22, 23 . we know from our prior human influenza virus challenge study that oral immunization was able to induce protective efficacy 90 days post immunization; on par with the commercial quadrivalent inactivated vaccine 15 . these features provide confidence that the adoption of the platform to covid-19 could translate to efficacy against this pathogenic coronavirus and could provide durable protection against virus infection. finally, a tablet vaccine campaign is much easier because qualified medical support is not needed to administer it. this ease of administration will result in increased vaccine access and potentially, acceptability, as has been evidenced by the success of easy-to-administer, oral polio vaccine, in the elimination of polio virus 24 . these features could be even more important during sars-cov-2 immunization campaigns compared to other vaccines, as substantially more resources may be required to ensure uptake of this vaccine, given the global levels of covid-19 denialism, mistrust and increased vaccine hesitancy 25, 26 . the tablet vaccine does not need refrigerators or freezers, does not require needles or vials, and can potentially be shipped via standard mail or by a delivery drone. these attributes significantly enhance deployment and distribution logistics, even permitting access to isolated regions with fewer technical resources. finally, from an immunological perspective, oral administration of this adenovirus is not compromised by pre-existing immunity to adenoviruses nor creates substantial anti-vector immunity 12, 13 ; issues that have been shown to cause significantly decreased vaccine potency in an rad5 based sars-cov2 vaccine 27 and can prevent durable increased immunity when the same adenovirus platform is re-administered by the im route 28 . the choice of antigen can be difficult during a novel pandemic, a time in which key decisions are needed quickly. the s protein is believed to be the major neutralizing antibody target for coronavirus vaccines, as the protein is responsible for receptor binding, membrane fusion, and tissue tropism. when comparing sars-cov-2 wu-1 to sars-cov, the s protein was found to have 76.2% identity 29 . both sars-cov and sars-cov-2 are believed to use the same receptor for cell entry: the angiotensin-converting enzyme 2 receptor (ace2), which is expressed on some human cell types 30 . thus, sars-cov-2 s protein is being used as the leading target antigen in vaccine development so far and is an ideal target given that it functions as the key mechanism for viral binding to target cells. however, the overall reliance on the s protein and an igg serum response in the vaccines could eventually lead to viral escape. for influenza, small changes in the hemagglutinin binding protein, including a single glycosylation site, can greatly affect the ability of injected vaccines to protect 31 . sars-cov-2 appears to be more stable than most rna viruses, but s protein mutations have already been observed without the selective pressure of a widely distributed vaccine. once vaccine pressure begins, escape mutations might emerge. we took two approaches to address this issue; firstly to include the more conserved n protein in the vaccine and secondly to induce broader immune responses, namely through mucosal iga. high expression levels of ace2 are present in type ii alveolar cells of the lungs, absorptive enterocytes of the ileum and colon, and possibly even in oral tissues such as the tongue 32 . transmission of the virus is believed to occur primarily through respiratory droplets and fomites between unprotected individuals in close contact 33 , although there is some evidence of transmission via the oral-fecal route as seen with both sars-cov and mers-cov viruses where coronaviruses can be secreted in fecal samples from infected humans 34 . there is also evidence that a subset of individuals exist that have gastrointestinal symptoms, rather than respiratory symptoms, are more likely to shed virus longer 35 . driving immune mucosal immune responses to s at both the respiratory and the intestinal tract may be able provide broader immunity and a greater ability to block transmission, than simply targeting one mucosal site alone. blocking transmission, rather than just disease, will be essential to reducing infection rates and eventually eradicating sars-cov-2. we have previously demonstrated that an oral, tableted rad-based vaccine can induce protection against respiratory infection and shedding following influenza virus challenge 15 as well as intestinal immunity to norovirus antigens in humans 12 . furthermore, mucosal iga is more likely to be able address any heterogeneity of the s proteins in circulating viruses than a monomeric igg response. miga has also been found to be more potent at cross reactivity than igg for other respiratory pathogens 36 . iga may also be a more neutralizing isotype than igg in covid-19 infection, and in fact neutralizing iga dominates the early immune response 37 . notably, we saw a higher ratio between neutralizing to non-neutralizing antibodies in our lung versus serum antibody results in our mouse study as well, which supports the concept that iga may have more potency compared to igg. polymeric iga, through multiple binding interactions to the antigen and to fc receptors can turn a weak single interaction into a higher overall affinity binding and activation signal, creating more cross-protection against heterologous viruses 38 . our second strategy to mitigate this potential vaccine-driven escape problem was to include the n protein in the vaccine construct. the n protein is highly conserved among b-coronaviruses, (greater than 90% identical) contains several immunodominant t cell epitopes, and long-term memory to n can be found in sars-cov recovered subjects as well as people with no known exposure to sars-cov or sars-cov-2 18, 39 . in an infection setting, t cell responses to the n protein seem to correlate to increased neutralizing antibody responses 40 . all of these reasons led us to add n to our vaccine approach. the protein was expressed in 293a cells (supp fig1) . however, as the human beta actin promoter is more active in human cells than mice, we did not explore immune responses in balb/c mice, but will examine them more carefully in future nhp and human studies. the optimum sequence and structure of the s protein to be included in a sars-cov-2 vaccine is a subject of debate. several labs have suggested that reducing the s protein to the key neutralizing domains within the receptor binding domain (rbd) would promote higher neutralizing antibody responses, and fewer non-neutralizing antibodies 41, 42 . we made a vaccine candidate composed of the s1 domain, which includes the rbd, in an attempt to promote this approach. although the s1based vaccine produced similar igg binding titers to s1, neutralizing antibody responses were significantly lower compared to the full-length s antigen. other gene-based vaccines have also shown the reductionist approach to s does not work so well, demonstrating that the dna vaccine expressing the full-length s-protein produced higher neutralizing antibodies than shorter s segments 5 . in agreement with these macaque studies, we observed that the sequence of the adenovirus encoded antigen had a significant impact on antibody function, here with respect to neutralization. while reducing the potential for exposing non-neutralizing antibody epitopes seems reasonable in theory, this might reduce the t cell help that allows for greater neutralizing antibodies to develop. indeed, of the spike protein t cell responses, which make up 54% of the responses to sars-cov-2, only 11% map to receptor binding domain 43 . stabilizing the s protein might be important for a protein vaccine, but not necessarily for a gene-based vaccine. the former is produced in vitro and it is produced to retain a homogenous, defined structure, ready for injection. in contrast, the latter, is expressed on the surface of a cell, in vivo, like natural infection, substantially in a prefusion form, and the additional stabilization may be unnecessary for b cells to create antibodies against the key neutralizing epitopes. we directly compared a stabilized version of s to the wild-type version. the wild-type version was significantly better at inducing neutralizing antibody responses. of interest, this was also observed in a dna vaccine study in nhps, where the stabilized version appeared to induce lower neutralizing antibody (nab) titers compared to the wild-type s 5 . a slightly different result was observed in studies of rad26 vectors by mercado, et al in nhps, where expressing a stabilized version of the s protein appeared to improve nab but lower t cell responses 44 . in summary, stabilization doesn't universally improve the immune responses in gene-based or vectorbased vaccines. multiple vaccine candidates are in, or are about to begin, clinical testing. due to known safety and immunogenicity for epidemic pathogens such as ebola virus, two leading candidate vaccines are based on recombinant adenovirus vectors; university of oxford's chadox1-ncov and janssen pharmaceutical's advac platforms [45] [46] [47] [48] . we saw stronger serum igg and nab titers in our study compared to a chadox1-ncov in balb/c mice 4 , however, this might reflect differences in assay components. a rad36 vaccine study was performed by hassan, et al., where doses of 1e10 vp were given by intranasal delivery 49 . the results were significant from the standpoint of blocking lung infection in a mouse sars-cov-2 challenge model. they reported titers of serum antibody titers of 1e4 above the background titers, similar to our results, despite using doses 2-to 3-log fold higher viral doses compared to our study. indeed, in our study, equivalently strong t cell and antibody responses were observed using 1e7 iu and 1e8 iu by the intranasal route. using these doses, we observed high percentages of cd8+ t cell responses (up to 14%) secreting ifn-g and tnf-a and strong cd4+ t cells after peptide restimulation. although we did not evaluate the trafficking properties of these antigen-specific t cells, we know that oral administration of this ad-based vaccine in humans induces high levels of mucosal homing lymphocytes 12, 15 . a proportion of the antigenspecific cd4+ and cd8+ t cells were polyfunctional in this mouse study. vaccine-induced t cells possessing multiple functions may provide more effective elimination of virus subsequent to infection and therefore could be involved in the prevention of disease. however, it is uncertain at this time what is the optimum t cell phenotype required for protection against disease. in summary, these studies in mice represent our first step in creating a vaccine candidate, demonstrating the immunogenicity of the construct at even low vaccine doses and the elucidation of the full-length spike protein as a leading candidate antigen to induce t cell responses and superior systemic and mucosal neutralizing antibody. future work will focus on the immune responses in humans. for this study, four recombinant adenoviral vaccine constructs were created based on the published dna sequence of sars-cov-2 publicly available as genbank accession no. mn908947.3. specifically, the published amino acid sequences of the sars-cov-2 spike protein (s protein) and the sars-cov-2 nucleocapsid protein (n protein) were used to synthesize nucleic acid sequences codon optimized for expression in homo sapiens cells (blue heron biotechnology, bothell, wa). these sequences were used to create recombinant plasmids containing transgenes cloned into the e1 region of adenovirus type 5 (rad5), as described by he, et al 50 , using the same vector backbone used in prior clinical trials for oral rad tablets 12, 15 . as shown in fig 1, c. rad-s1-n: rad5 vector using a fusion sequence combining the s1 region of sars-cov-2 s gene (including the native furin site between s1 and s2) with the full-length sars-cov-2 n gene. d. rad-s(fixed)-n: rad5 vector containing a stabilized s gene with the transmembrane region removed under the control of the cmv promoter and full-length sars-cov-2 n gene under control of the human beta-actin promoter. the s gene is stabilized through the following modifications: a) arginine residues at aa positions 682, 683, 685 were deleted to remove the native furin cleavage site b) two stabilizing mutations were introduced: k986p and v987p c) transmembrane region was removed following p1213 and replaced with bacteriophage t4 fibritin trimerization foldon domain sequence 51 (gyipeaprdgqayvrkdgewvllstfl) all vaccines were grown in the expi293f suspension cell-line (thermo fisher scientific), purified by cscl density centrifugation and provided in a liquid form for animal experiments. studies were approved for ethics by the animal care and use committees (iacuc). all of the procedures were carried out in accordance with local, state and federal guidelines and regulations. female 6-8 week old balb/c mice were purchased from jackson labs (bar harbor, me). because mice do not swallow pills, liquid formulations were instilled intranasally in 10 µl per nostril, 20 µl per mouse in order to test immunogenicity of the various constructs. serum was acquired by cheek puncture at various timepoints. elisas. specific antibody titers to proteins were measured similarly to methods described previously 52 . briefly, microtiter plates (maxisorp: nunc) were coated in 1 carbonate buffer (0.1 m at ph 9.6) with 1.0 ug/ml s1 protein (genscript). the plates were incubated overnight at 4°c in a humidified chamber and then blocked in pbs plus 0.05% tween 20 (pbst) plus 1% bsa solution for 1 h before washing. plasma samples were serially diluted in pbst. after a 2-h incubation, the plates were washed with pbst at least 5 times. antibodies were then added as a mixture of anti-mouse igg1-horseradish peroxidase (hrp) and anti-mouse igg2a-hrp (bethyl laboratories, montgomery, tx). each secondary antibody was used at a 1:5,000 dilution. the plates were washed at least 5 times after a 1-h incubation. antigen-specific mouse antibodies were detected with 3,3=,5,5=-tetramethylbenzidine (tmb) substrate (rockland, gilbertsville, pa) and h2so4 was used as a stop solution. the plates were read at 450 nm on a spectra max m2 microplate reader. average antibody titers were reported as the reciprocal dilution giving an absorbance value greater than the average background plus 2 standard deviations, unless otherwise stated. to measure responses to both s1 and s2 simultaneously, a multi-spot® 96-well, 2-spot plate (mesoscale devices; msd) was coated with sars cov-2 antigens. proteins were commercially acquired from a source (native antigen company) that produced them in mammalian cells (293 cells). these were biotinylated and adhered to their respective spots by their individual u-plex linkers. to measure igg antibodies, plates were blocked with msd blocker b for 1 hour with shaking, then washed three times prior to the addition of samples, diluted 1:4000. after incubation for 2 hours with shaking, the plates were washed three times. the plates were then incubated for 1 hour with the detection antibody at 1 μg/ml (msd sulfo-tag tm anti-mouse igg). after washing 3 times, the read buffer was added and the plates were read on the meso quickplex sq 120. neutralizing antibodies were routinely detected based on the sars-cov-2 surrogate virus neutralization test (svnt) kit (genscript). this elisa-based kit detects antibodies that hinder the interaction between the receptor binding domain (rbd) of the sars-cov-2 spike glycoprotein and the ace2 receptor on host cells, and is highly correlated to conventional virus neutralizing titers for sars-cov-2 infection of vero cells 53 . the advantage of this approach is that the assay can be done in a bsl-2 laboratory. sera from mice immunized with the candidate vaccines was diluted at 1:20, 1:100, 1:300, 1:500, 1:750 and 1:1000 using the provided sample dilution buffer. sera from non-immunized mice was diluted 1:20. lung samples were diluted 1:5, 1:20, and 1:100. positive and negative controls were prepared at a 1:9 volume ratio following the provided protocol. after dilution, sera or lung samples were individually incubated at a 1:1 ratio with hrp-rbd solution for 30 minutes at 37°c. following incubation, 100µl of the each hrp-rbd and sample or control mixture was added to the corresponding wells in the hace2-precoated capture plate and once again incubated at 37°c for 15 minutes. afterwards, wells were thoroughly washed and 100µl of the provided tmb (3,3=,5,5=tetramethyl-benzidine) solution was added to each well and left to incubate for 15 minutes at room temperature (20-25°c). lastly 50µl of stop solution was added to each well, and the plate was read on a spectra max m2 microplate reader at 450 nm. the absorbance of a given sample is inversely related on the titer of anti-sars-cov-2 rbd neutralizing antibody in a given sample. per test kit protocol, a cut-off of 20% inhibition when comparing the od of the sample versus the od of the negative control was determined to be positive for the presence of neutralizing antibodies. samples that were negative at the lowest dilution were set equal to ½ of the lowest dilution tested, either 10 for sera or 2.5 for lung samples. additional neutralizing antibodies responses were measured in some studies using a cvnt assay at visimederi under bsl3 conditions. the cvnt assay has a readout of cytopathic effect (cpe) to detect specific neutralizing antibodies against live sars-cov-2 in animal or human samples. the cvnt/cpe assay permits the virus to makes multiples cycles of infection and release from cells; its exponential grow in few days (usually 72 hours of incubation) causes the partial or complete cell monolayer detachment from the surface of the support, clearly identifiable as cpe. serum samples are heat inactivated for 30 min at 56°; two-fold dilutions, starting from 1:10 are performed then mixed with an equal volume of viral solution containing 100 tcid50 of sars-cov-2. the serum-virus mixture is incubated for 1 hour at 37° in humidified atmosphere with 5% co2. after incubation, 100 µl of the mixture at each dilution are added in duplicate to a cell plate containing a semiconfluent vero e6 monolayer. after 72 hours of incubation the plates are inspected by an inverted optical microscope. the highest serum dilution that protect more than 50% of cells from cpe is taken as the neutralization titer. two weeks after the final immunization (day 28 of the study), mice were sacrificed and bled via cardiac puncture. lungs were removed and snap frozen at -80 0 c. on thawing, lungs were weighed. lungs were homogenized in 150 µl dpbs using pellet pestles (sigma z359947). homogenates were centrifuged at 1300rpm for 3 minutes and supernatants were frozen. the total protein content in lung homogenate was evaluated using a bradford assay to ensure equivalent amounts of tissue in all samples before evaluation of iga content. antigen-specific iga titers in lungs were detected using a mouse iga elisa kit (mabtech) and pnpp substrate (mabtech). briefly, maxisorp plates (nunc) were coated with s1 or s2 (the native antigen company; 50 ng/well) in pbs for overnight adsorption at 4°c and then blocked in pbs plus 0.05% tween 20 (pbst) plus 0.1% bsa (pbs/t/b) solution for 1 h before washing. lung homogenates were serially diluted in pbs/t/b, starting at a 1:30 dilution. after 2 hours incubation and washing, bound iga was detected using mt39a-alp conjugated antibody (1:1000), according to the manufacturer's protocol. plates were read at 415nm. endpoint titers were taken as the x-axis intercept of the dilution curve at an absorbance value 3x standard deviations greater than the absorbance for naïve mouse serum. non-responding animals were set a titer of 15 or ½ the value of the lowest dilution tested. spleens were removed and placed in 5 ml hanks balanced salt solution (with 1m hepes and 5% fbs) before pushing through a sterile strainer with a 5ml syringe. after rbc lysis (ebiosolutions), resuspension, and counting, the cells were ready for analysis. cells were cultured at 5e5 cells/well with two peptide pools representing the full-length s protein at 1 µg/ml (genscript) overnight in order to stimulate the cells. the culture media consisted of rpmi media (lonza) with 0.01m hepes, 1x l-glutamine , 1x mem basic amino acids, 1x penstrep, 10% fbs, and 5.5e-5 mole/l beta-mercaptoethanol. antigen specific ifn-g elispots were measured using a mabtech kit. flow cytometric analysis was performed using an attune flow cytometer and flow jo version 10.7.1, after staining with the appropriate antibodies. for flow cytometry, 2e6 splenocytes per well were incubated for 18 hours at 37ºc with peptide pools representing full-length s at either 1 or 5 ug/ml, adding brefeldin a (thermofisher) for the last 4 hours of incubation. the antibodies used were apc-h7 conjugated cd4, fitc conjugated cd8, bv650 conjugated cd3, percp-cy5.5 conjugated ifn-y, bv421 conjugated il-2, pe-cy7 conjugated tnfa, apc conjugated il-4, alexa fluor conjugated cd44, and pe conjugated cd62l (bd biosciences). clinical features of patients infected with 2019 novel coronavirus in wuhan, china transmission and clinical characteristics of asymptomatic patients with sars-cov-2 infection clinical and immunological assessment of asymptomatic sars-cov-2 infections chadox1 ncov-19 vaccine prevents sars-cov-2 pneumonia in rhesus macaques dna vaccine protection against sars-cov-2 in rhesus macaques safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial immunogenicity and safety 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sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals a novel receptor-binding domain (rbd)-based mrna vaccine against sars-cov-2 development of a covid-19 vaccine based on the receptor binding domain displayed on virus-like particles selective and cross-reactive sars-cov-2 t cell epitopes in unexposed humans single-shot ad26 vaccine protects against sars-cov-2 in rhesus macaques safety and immunogenicity of a 2-dose heterologous vaccine regimen with ad26.zebov and mva-bn-filo ebola vaccines: 12-month data from a phase 1 randomized clinical trial in safety and immunogenicity of a 2-dose heterologous vaccination regimen with ad26.zebov and mva-bn-filo ebola vaccines: 12-month data from a phase 1 randomized clinical trial in uganda and tanzania clinical assessment of a novel recombinant simian adenovirus chadox1 as a vectored vaccine expressing conserved influenza a antigens a single intranasal dose of chimpanzee adenovirus-vectored vaccine confers sterilizing immunity against sars-cov-2 infection a simplified system for generating recombinant adenoviruses very fast folding and association of a trimerization domain from bacteriophage t4 fibritin systemic and mucosal antibody responses following retroductal gene transfer to the salivary gland a sars-cov-2 surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction the authors would like to thank alessandro torelli, laura palladino, emanuele montomoli and the team at vismederi for running neutralizing antibody titers (bsl3) and susan johnson for critically reviewing the manuscript. authorship acm, mc, and snt designed the experiments; acm and snt wrote the manuscript; np, kpt, and kl performed immunological assays; acm, np, kpt, kl, mc, and snt analyzed the data. egd and snt designed the vaccine vectors. egd, nd, kpt, klm mc and snt are current employees and/or own stock options in vaxart, the sponsor of the studies. egd and snt are named as inventors covering a sars-cov-2 (ncov19) vaccine. snt is named as an inventor on patent covering the vaccine platform. acm declares no competing interest. key: cord-347351-emdj66vj authors: kampf, günter; brüggemann, yannick; kaba, hani e.j.; steinmann, joerg; pfaender, stephanie; scheithauer, simone; steinmann, eike title: potential sources, modes of transmission and effectiveness of prevention measures against sars-cov-2 date: 2020-09-18 journal: j hosp infect doi: 10.1016/j.jhin.2020.09.022 sha: doc_id: 347351 cord_uid: emdj66vj during the current sars-cov-2 pandemic new studies are emerging daily providing novel information about sources, transmission risks and possible prevention measures. in this review, we aimed to comprehensively summarize the current evidence on possible sources for sars-cov-2, including evaluation of transmission risks and effectiveness of applied prevention measures. next to symptomatic patients, asymptomatic or pre-symptomatic carriers are a possible source with respiratory secretions as the most likely cause for viral transmission. air and inanimate surfaces may be sources; however, viral rna has been inconsistently detected. similarly, even though sars-cov-2 rna has been detected on or in personnel protective equipment, blood, urine, eyes, the gastrointestinal tract and pets, these sources are currently thought to play a negligible role for transmission. finally, various prevention measures such as hand washing, hand disinfection, face masks, gloves, surface disinfection or physical distancing for the healthcare setting and public are analysed for their expected protective effect. the end of the infectious period. in fact, real-time reverse transcriptase pcr results remained positive 6-8 days after the loss of transmissibility [9] . for sars coronavirus, viral rna was detectable in the respiratory secretions and stools of some patients after onset of illness for more than 1 month, but live virus could not be detected by culture after week 3 [10] . the inability to differentiate between infective and non-infective (dead or antibody-neutralised) viruses therefore remains a major limitation of nucleic acid detection methods. despite this limitation, given the difficulties in culturing infectious virus from clinical specimens during a pandemic, using viral rna load as a surrogate remains plausible for generating careful clinical hypotheses. the association between viral load and clinical outcome including severity of symptoms is still poorly characterized although the majority of studies reported an association between higher viral loads and more severe symptoms [11] [12] [13] [14] [15] [16] [17] [18] [19] . transmission dynamics of sars-cov-2 are heterogeneously [20] . numerous individual infection clusters in particular in asia with variable size have been reported [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . originating from a single travel-associated primary case from china, the first documented chain of multiple human-to-human transmissions of sars-cov-2 outside of asia allowed a detailed study of transmission events and identified several factors (e.g. cumulative face-toface contact, direct contact with secretions or body fluids of a patient, personal protective equipment) to classify contacts as low or high risk [32] . furthermore, factors such as immune suppression, increased disease severity and viral load, asymptomatic individuals, the practice of seeking care at multiple healthcare facilities, frequent inter-hospital transfer, large numbers of contacts and prolonged duration of exposure facilitate transmission [33] . household transmission is also common [34] . superspreading is regarded as a normal feature of disease spread and has also been described with sars-cov-2 [35, 36] . importantly, a recent study observed that transmission clusters occurred in many, predominantly indoor settings and most clusters involved fewer than 100 cases, with the exceptions being in healthcare (hospitals and elderly care), large religious gatherings, food processing plants, schools, shopping, and large co-habiting settings (worker dormitories, prisons and ships) [31] . given the predominately mild, non-specific symptoms, infectiousness before symptom onset the successful containment of covid-19 relies on stringent and urgent surveillance and infection-control measures. based on the definition of the who a confirmed case is a person with laboratory confirmation (detection of viral genomic material) of sars-cov-2, irrespective of clinical signs and symptoms [37] . asymptomatic coronavirus infections have been described before [38] and might together with pre-symptomatic spread form a potential source of covid-19 infections acquired in a social or nosocomial context [26, [39] [40] [41] [42] [43] [44] . in february 2020, a total of 44,672 confirmed cases were reported for china with a proportion of 1.2% of asymptomatic cases [45] . data from the first of april based on more rigorous testing of contact persons suggest in a small cohort of 166 new cases a proportion of 78% as asymptomatic cases [46] . irrespective of the frequency of asymptomatic carriers, they are considered to be important for the transmission of the disease [47] . various studies reported sars-cov-2 infections, originating from asymptomatic carriers during close contacts such as household contacts or residents of a long-term care skilled nursing facility [43, [48] [49] [50] [51] [52] . importantly, several studies have reported that viral rna loads in pre-symptomatic, asymptomatic and symptomatic patients do not differ significantly [53] [54] [55] . others have reported no transmission from 455 contacts (patients, family members, hospital staff) to asymptomatic carriers and concluded that the infectivity of some asymptomatic carriers may be weak [56] . as summarized in table i , the proportions of asymptomatic sars-cov-2 cases at the time point of testing have been determined for different cohorts of patients. in hospitalized patients it was described to range be between 5.0% and 27.8% [57] [58] [59] [60] . in a long-term care facility, it was quite high with 56.5% [61] . in family clusters it was found between 25% and 57.1% [62, 63] . in 171 children in china the proportion was 15.5% [64] . among japanese nationals evacuated from wuhan by chartered flights it was 30.8% in contrast to german nationals with 1.8% [65, 66] . on board of a cruise ship asymptomatic carriers were detected in 50.5% of the cases. the delay-adjusted asymptomatic proportion, however, was only 17.9% [67] . in iceland, a proportion of 3.6% of the general population (13080 out of 364000 inhabitants) were investigated. overall, 100 of them (0,8%) were positive with a proportion of 43% asymptomatic carriers. among inhabitants with a high risk for infection the proportion of asymptomatic cases was only 7% [68] . overall, asymptomatic sars-cov-2 infections seem to account for up to 56% of sars-cov-2 infections in selected cohorts, suggesting a significant factor for the rapid progression of the covid-19 pandemic [53, 69, 70] . for comparison, the prevalence of asymptomatic influenza virus carriage (total absence of symptoms) ranged from 5.2% to 35.5% and subclinical cases (illness that did not meet the criteria for acute respiratory or influenza-like illness) between 25.4% to 61.8% [71] . with mers a proportion of 9.5% of 1010 cases was asymptomatic [38] . follow-up examinations, however, indicate that the majority of initially tested asymptomatic cases (70.8% -100%) develop moderate but detectable clinical symptoms over time and therefore should be considered as pre-symptomatic. only in a small group of patients, no symptoms or radiological findings became apparent, but were described as potentially infectious for up to 29 d (table ii) [72] . of note, patients with negative pcr result prior to discharge may also become transient asymptomatic carriers again. one patient, for example, was retested positive for sars-cov-2 during the 2 weeks quarantine after discharge [73] . two healthcare workers (hcws) were also tested (throat swab) after discharge (covid-19) and were weakly positive in 2 of 7 samples and positive in 1 of 7 samples (case 1 sampled over 10 d), and weakly positive in 1 of 8 samples and positive in 1 of 8 samples (case 2 sampled over 8 d) [74] . however, these results have to be interpreted with caution as currently applied pcr methods can lead to fluctuating results in weakly positive samples due to detection limits of the assays. indeed, a single case was described with low viral rna loads or negative rt-qpcr results, despite a sars-cov-2 infection confirmed by the presence of anti-sars-cov-2 specific antibodies [75] . importantly, a systematic meta-analysis of different cohort studies observed that asymptomatic patients with covid-19 seems to correlate with young age and social activity [76, 77] . in particular, future studies aiming to understand the contribution of young aged patients such as children to asymptomatic transmission of sars-cov-2 should be prioritised [78] . in summary, the prevalence of asymptomatic sars-cov-2 infection and duration of pre-symptomatic infection are not well understood, as asymptomatic individuals are not routinely tested. studies on the immune response of asymptomatic carriers are lacking, which could contribute to a better characterization of the protective factors under natural conditions [79] . several sources have been described that could possibly be involved in sars-cov-2 transmission based on the detection of viral rna. these include the respiratory tract, air contamination, the gastrointestinal tract, eyes, inanimate surfaces, personnel protective equipment, pets, and rather less likely blood and the urinary tract. sars-cov-1 has been frequently associated with droplet-based transmission [80, 81] . likewise, person-to-person transmission has been assumed for sars-cov-2 very early [21] . importantly, a more efficient transmission of sars-cov-2 compared to sars-cov-1 has been suggested, due to active pharyngeal viral shedding while symptoms are still mild and typical of upper respiratory tract infection [7] . table iii summarizes the frequency and magnitude of sars-cov-2 viral rna loads in respiratory tract samples obtained from covid-19 patients. the viral rna load with sars-cov-2 can be as high as 11.1 log10 cpm (table iii) . it seems to be particularly high in the early and progressive stage of disease [16] or two days before and one day after symptom onset [82] . however, in some cases rna could still be found up to 51 days after the first positive test with negative results in-between [15, 83] . influenza a virus rna has even been released for up to 70 d with negative results in-between although infectious virus was only detected for 5 d after symptom onset [84] . age was also associated with high viral rna load [15] . most studies observed decreased viral rna loads over time [5, 7] . one study shows that sars-cov-2 was detected by culture in 19 out of 25 clinical samples (nasopharyngeal swab) from covid-19 patients [6] . the viral rna load detected in the asymptomatic patient was similar to that in the symptomatic patients, which suggests the transmission potential of asymptomatic or minimally symptomatic patients [5] . it is important to differentiate between detection of rna and the isolation of infectious virus in cell culture. pcr for rna of sars-cov-2 does not distinguish between infectious virus and non-infectious nucleic acid. thus, interpretation of duration of viral shedding and infection potential should be based on viable virus from cell culture and needs to be carefully evaluated when solely based on pcr results. j o u r n a l p r e -p r o o f a strict distinction between droplet versus airborne transmission routes for infections is not possible [85] . virus transmission via droplets and aerosols enables many viruses to spread efficiently between humans [86] . airborne transmission is defined as the transmission of infection by expelled particles of comparatively small in size and which can remain suspended in air for long periods of time [87] . the world health organization uses a particle diameter of 5 ߤm to delineate between airborne (≤5 ߤm) and droplet (>5 ߤm) transmission [88] . transmission of infectious diseases by the airborne route is dependent on the interplay of several factors, including particle size (i.e., particle diameter) and the extent of desiccation [87] . particle desiccation is a critical variable and depending on environmental factors as even large, moisture laden droplet particles desiccate rapidly [87] . for example, wells showed that particles begin desiccating immediately in a rapid fashion upon air expulsion: particles up to 50 ߤm can desiccate completely within approximately 0.5 seconds [89] . rapid desiccation is a concern since the smaller and lighter the infectious particle, the longer it will potentially remain airborne. hence, even when infectious agents are expelled from the respiratory tract in a matrix of mucus and other secretions, causing large, heavy particles, rapid desiccation can lengthen the time they remain airborne (the dried residuals of these large aerosols, termed droplet nuclei, are typically 0.5-12 ߤm in diameter) [87] . of further concern, very large aerosol particles may initially fall out of the air only to become airborne again once they have desiccated [87] . one of the challenges facing practitioners, particularly in an enclosed building, is that even large-sized droplets can remain suspended in air for long periods. the reason is that droplets settle out of air onto a surface at a velocity dictated by their mass [87] . if the upward velocity of the air in which they circulate exceeds this velocity, they remain airborne. hence, droplet aerosols up to 100 ߤm diameter have been shown to remain suspended in air for prolonged periods when the velocity of air moving throughout a room exceeds the terminal settling velocity of the particle [87] . respiratory virus shedding can occur via sneezing, coughing or talking. sneezing distributes approximately 40,000 particles (droplets or airborne microorganisms) per sneeze, coughing approximately 710 particles per cough, and talking approximately 36 particles per 100 words [87] . using highly sensitive laser light scattering observations a recent study describes that loud speech can emit thousands of oral fluid droplets per second [90] , indicating that normal speaking may also contribute to virus transmission in stagnant air. most of the 40,000 large droplet particles caused by a single sneeze will desiccate immediately into small, infectious droplet nuclei, with 80% of the particles being smaller than 100 ߤm [3] . the transmission of infectious diseases via airborne or droplet routes may further also depend on the frequency of the initiating activity. a single sneeze may produce more total infectious particles, while overall coughing may potentially be a more effective route of airborne transmission (e.g. during infection with coxsackievirus a) [91] . coronavirus-infected humans coughed on average 17 times over 30 min during exhaled breath collection [92] . given that dry cough is also a common symptom of covid-19 patients [93] , it may therefore contribute to potential airborne transmission of this pathogen. in this context, airborne transmission has been considered to be possible in a cluster of infections in a restaurant with air conditioning [94] . few studies are available to evaluate the role of air for transmission of sars-cov-2, most of them obtained in hospitals with covid-19 patients. from the data shown in table iv viral copies were only detected in large air volumes of 9000 l with a larger proportion on icu (35% detection rate) compared to general wards (12.5% detection rate). in smaller volumes such as 90 l, 1,200 l or 1.5 m 3 no virus was detected. even directly in front of a covid-19 patient it was not possible to detect the sars-cov-2 rna in the air [95] . the viral rna loads of the first confirmed case were 3.3×10 6 copies per ml in the pooled nasopharyngeal and throat swabs and 5.9×10 6 copies per ml in saliva on the day of air sampling [95] . the air samples of 1000 l were collected at a distance of 10 cm at the level of patient's chin while the patient performed 4 different manoeuvres (i.e., normal breathing, deep breathing, speaking "1, 2, 3" continuously, and coughing continuously) while putting on and putting off the surgical mask were all undetectable for sars-cov-2 rna [95] . nosocomial transmission of sars-cov-2 by an airborne route has been described to be very unlikely [96] . nonetheless, sars-cov-2 can remain infectious in air for 3 h measured in a goldberg drum with a decline of viral load from 3.5 log10 to 2.7 log10 per litre of air [97] . in a subset of 4 study participants with a symptomatic seasonal coronavirus infection but without any coughing during the 30 min exhaled breath collection no coronavirus rna was detected in respiratory droplets or aerosols [92] . other aspects influencing droplet or airborne transmission are temperature and humidity because they correlate with the spread of and deaths associated with covid-19 [98] [99] [100] . in china, the number of confirmed cases increased with higher temperature and higher humidity in most of the provinces [101, 102] . covid-19 lethality significantly worsened (4 times on average) with environmental temperatures between 4 °c and 12 °c and relative humidity between 60% and 80% [103] . biktasheva et al., however, described that the covid-19 mortality correlates with low air humidity, probably caused by a lower resistivity of dry or very dry mucous membranes [104] . huang et al. described that 60% of all covid-19 cases are found in places with an air temperature between 5 °c and 15 °c [105] . in brazil a 1 °c increase in temperature has been associated with a decrease in confirmed cases of 8% [106] . in wuhan and xiaogan temperature was the only meteorological parameter constantly but inversely correlated with covid-19 incidence [107] . at low temperature and low humidity, droplets tend to remain suspended in air [108] . high relative humidity will increase the droplet sizes due to the hygroscopic growth effect, which increases the deposition fractions on both humans and the ground [109] . overall, a seasonal pattern of covid-19 is very likely. sars-cov-2 aerosolized from infected patients and deposited on surfaces could remain infectious outdoors for considerable time during the winter in many temperate-zone cities, with continued risk for re-aerosolization and human infection [110] . conversely, sars-cov-2 should be inactivated in the environment relatively fast during summer in many populous cities of the world, indicating that sunlight should have a role in the occurrence, spread rate, and duration of coronavirus pandemics [110] . simulated sunlight has been described to rapidly inactive sars-cov-2 [111, 112] . indoor transmission of sars-cov-2 is much more likely compared to outdoor transmission [113] . in a closed seafood market, the risk of a costumer to acquire sars-cov-2 infection via the aerosol route after 1 h exposure in the market with one infected shopkeeper was about 2.23 × 10 − 5 . the risk rapidly decreased outside the market due to the dilution by ambient air j o u r n a l p r e -p r o o f and became below 10 − 6 at 5 m away from the exit [114] . outdoor, these virus particles are very strongly diluted by the open air [115] . some patients displayed diarrhoea at the beginning or during the course of infection suggesting that sars-cov-2 may also affect the gastrointestinal tract. viral rna was detected in a proportion between 9.1% and 100% in covid-19 patients with up to 8.1 log10 viral copies per g (table v) . one study including 46 patients with 16 of them reporting gastrointestinal manifestations (35%) reported diarrhea as the most common symptom (15%), followed by abdominal pain (11%), dyspepsia (11%), and nausea (2%) [116] . analysing two groups of overall 12 patients, none of the stool samples resulted in successful virus isolation in cell culture, irrespective of viral rna concentration [7, 117] . in contrast, one study described the successful isolation of virus by cell culture from 2 out of 3 patients [118] . of note, another study showed higher viral rna loads in fecal samples of mildly symptomatic or asymptomatic children compared to nasopharyngeal swabs [119] . these results indicate the possibility of fecal-oral transmission or fecal-respiratory transmission through aerosolized feces. furthermore, the presence of sars-cov-2 rna in bile juice was reported from one patient and speculated that rna in fecal specimens may partly originate from bile juice [120] . finally, a recent study suggested that detectable sars-cov-2 rna in the digestive tract could be a potential warning indicator of severe disease [121] , however further evidence will be needed. transmission of sars-cov-2 through the ocular surface was considered to be possible [122] . conjunctivitis has been reported in a patient in the middle phase of covid-19, the conjunctival swab specimens remained positive for sars-cov-2 on 14 and 17 days after onset and were negative on day 19 [123] . another study showed among 30 covid-19 patients that the virus was detected in tears and conjunctival secretions only in the one patient with conjunctivitis [124] . furthermore, in another group of 38 covid-19 patients two of them were identified with positive findings for sars-cov-2 in their conjunctival as well as nasopharyngeal specimens, a total of 12 patients had ocular manifestations consistent with conjunctivitis, including conjunctival hyperemia, chemosis, epiphora, or increased secretions [125] . in addition, no virus was detected on the conjunctiva in 5 other covid-19 patients [11] . one patient was described with persistent conjunctivitis with viral rna detection until day 27 after symptom onset and confirmation of infectious virus in the first rna-positive ocular sample [126] . even though the virus can be detected rarely in the conjunctival sac at very low levels [127, 128] , there is no evidence that it can replicate locally [129] . that is why the conjunctiva were considered not to be the preferred gateway into the respiratory tract [130] . a study analysed human post-mortem eyes for the expression of ace2 (the receptor for sars-cov-2) and tmprss2. in all samples the expression of ace2 and tmprss2 was detected in the conjunctiva, limbus, and cornea, with especially prominent staining in the superficial conjunctival and corneal epithelial surface [131] . in contrast, another study from germany found no relevant conjunctival expression of the ace2 receptor on mrna and protein levels [132] . in summary, the detailed pathophysiology of ocular transmission of sars-cov-2 remains not completely understood [133] and both the presence of viral particles in tears and conjunctiva, and the potential for conjunctival transmission remains controversial [134] . in conclusion, spread of covid-19 from ocular secretions cannot be ruled out but seems to be very unlikely. indirect transmission of covid-19 has been assumed to be possible via fomites although direct evidence is currently not available [135] . in hospitals some data were collected to describe the frequency of detection of sars-cov-2 rna on inanimate surfaces in the immediate patient surrounding. the detection rate was variable on icu surfaces (0% -75%), in isolation rooms (1.4 -100%) and on general wards (0% -61%). the mean virus concentration per swab were 4.4 -5.2 log10 on icus and 2.8 -4.0 log10 on general wards. a positive correlation between patient viral rna load and positivity rate of surface samples was described [136] . however, on cleaned and disinfected surfaces viral rna could mostly not be detected (table vi) . detection of viral rna on the floor is indicative for sedimentation of contaminated droplets. surfaces outside the covid-19 patient room were also investigated. on icu the virus was rarely detected as "weak positive" on the floor and on door knobs in 3 buffer rooms, 6 dressing rooms and a nurse station (6 of 84 samples; 7.1%) [137] . on the general ward the virus was rarely detected on the patient floor (23 samples; one "weak positive" result on the computer mouse or keyboard) and never detected on doorknobs and the floor in 3 buffer rooms and 5 dressing rooms (52 samples) [138] . viral rna could be detected even 28 d after discharge of covid-19 on surfaces of pagers and in drawers of the isolation wards. the relevance of finding, however, is not clear because it is not known if infectious virus was present at that time [139] . in a microbiology laboratory the detection rate on surfaces was 18.2%. in the domestic environment of sars-cov-2 carriers the detection rate on surfaces was overall low (0% -3.4%; table vi) . it has to be mentioned that in most studies only pcr was performed for rna. but detection of viral rna on surfaces does not provide any information about viral infectivity or viability [140] . new findings from a covid-19 cohort in gangelt, germany, and with cases in italy provide data on the detection of infectious sars-cov-2 on surfaces. although viral rna was detected in 3.4% of 119 surface samples in 21 households of confirmed covid-19-cases and on 7.7% of sampled surfaces around covid-19-cases in italy, infectious sars-cov-2 was not found in any sample [141, 142] . similar findings were described with sars-cov and influenza-virus. in canada, a total of 85 samples from inanimate surfaces were taken in a sars-hospital. viral sars-cov rna was present in 5.6% of samples, but none of the samples revealed infectious virus [143] . in thailand and taiwan rna of sars-cov was detected on 27.7% of 94 surface samples in a sars-hospital or in a sars-ward; in none of the samples infectious sars-cov was found [144] . similar data were reported from 90 households with proven h1n1 influenza virus infections in children. viral rna was detected on 17.8% of inanimate surfaces but virus could never be cultured [145] . in cell culture studies, sars-cov-2 has been described to remain infectious on stainless steel and plastic for 3 -4 d, on glass and banknote for 2 d, on wood for 1 d, all with a decrease of viral infectivity with time [97, 146] . in the close surrounding of covid-19 patients in hospitals sars-cov-2 rna is detected more frequently compared to surfaces outside the patient rooms but samples were so far consistently negative for infectious virus. if infectious sars-cov-2 may be detected in a relevant amount on various surfaces in the public when only a short exposure to potentially infected, may be even asymptomatic people exists, is currently unknown but very unlikely. surfaces in air planes or trains in coughing or sneezing distance for potentially infected long-distance travellers may theoretically have a higher risk for contamination. the rna of sars-cov-2 has so far mainly been found on ppe used by healthcare workers on icu (0% -50%), mainly on shoes and gloves. in other settings ppe was only very rarely contaminated with sars-cov-2 (table vii) . all studies performed pcr assays for sars-cov-2 rna detection. blood sars-cov-2 rna has occasionally been detected in blood of covid-19 patients, i.e. in 1 of 5 patients on days 7, 8, 9 and 12 after onset of disease [11] , in 5 of 23 covid-19 patients (21.7%) [15] , in 0 of 18 asymptomatic and symptomatic patients with sars-cov-2 infection [54] , or in 3 of 307 samples (1.0%) obtained from 205 covid-19 patients [147] . sars-cov-2 rna can very rarely (in 4 of 2430 samples) be detected in plasma during routine screening of blood donors considered to be healthy population [148] . detection of sars-cov-2 rna in blood is considered a strong indicator for further clinical severity [149] . so far, no cases of transmission due to transfusion of blood products have been reported for sars-cov, mers-cov, and sars-cov-2, and clinically ill patients are not considered as blood donors [54] . therefore, no immediate risk can be derived for the transfusion system [54] . based on the existing evidence, transmission of covid-19 by handling potentially contaminated blood products (laboratory technician) or by contact with blood e.g. from a wound to intact skin is very unlikely. sars-cov-2 rna has occasionally been detected in urine swabs from patients. in 9 patients with confirmed sars-cov-2 infections one of the patients was positive for viral rna in urine [150] . this observation is supported by observations among 12 sars-cov-2 positive children with 2 of them positive for viral rna in urine (17%) [119] . importantly, infectious virus could be detected from urine in one covd-19 patient [151] . however, other studies with a total of 47 patients [7, 15, 152] failed to detect sars-cov-2 rna in urine. these data indicate that urine might be a potential source of infection but further evidence is needed. there is evidence that the main entry receptor of sars-cov-2, ace2, is expressed in cells of the reproductive system [153, 154] . however, one study with 23 covid-19 patients in the acute (12 patients) and recovery phase (11 patients) failed to detect viral rna in semen [155] , indicating a low probability of sexual transmission through semen. sars-cov-2 rna has temporarily been detected in breast milk samples in one study in one of two infected mothers with approximately 10 5 viral copies per ml [156] . similarly, the presence of viral rna was reported in breast milk of an actively breastfeeding mildly symptomatic covid-19 patient raising the possibility of a potential transmission from breast milk [157] . so far, no evidence for transmission of the virus from pet animals to humans exists [158] . however, shi et al. reported that ferrets and cats were highly susceptible to sars-cov-2, while dogs had a low susceptibility and livestock including pigs, chickens, and ducks were not susceptible to the virus, under experimental conditions [159] . one of 22 cats (france) and two of 10 cats (wuhan) of covid-19 patients has been described to have a sars-cov-2 infection with mild respiratory and digestive symptoms whereas all 11 dogs (france) and 8 of 9 dogs (wuhan) were sars-cov-2 and serologically negative [160, 161] . interestingly, viral transmission between cats has been observed [159] . out of six naïve cats (three subadults and three juveniles), each exposed to a sars-cov-2 inoculated cat, transmission occurred in two cats (one cat of each age group). similar findings were reported by halfmann et al. [162] . this indicates that cats, being common companion animals, might theoretically transmit the virus to other animals and humans. however, there is so far no clear evidence that cat-to-human transmission of sars-cov-2 can occur. several practices are recommended with the aim to limit further transmission of sars-cov-2 in clinical practice but also public settings. these include hand washing, hand disinfection, wearing of face masks and gloves, disinfection of surfaces and physical distance. based on an integrated theoretical and statistical analysis of the influence of individual variation in infectiousness on disease emergence it has been suggested that individual-specific control measures outperform population-wide measures [35] . a hand soap solution (1:49) has been described to have some effect (≥ 3.6 log10 reduction of viral infectivity) against sars-cov-2 in 5 min [146] . for healthcare workers hand washing is only useful when hands are visibly soiled [163] . although sars-cov-2 has never been detected on hands of the public population yet, it seems reasonable to assume that the hand contamination by droplets from others may take place in the public with an unknown viral load. apart from avoiding hand-face-contacts in general, hand washing is first choice for the decontamination of hands, especially after returning home from public places with many close contacts to potentially infected people. ethanol and iso-propanol inactivate sars-cov-2 at concentration between 30% and 80% (both v/v) in 30 s [164] . both who-recommended hand rubs based on 75% iso-propanol or 80% ethanol (both v/v) also inactivate sars-cov-2 in only 30 s [164] . similar results were obtained with a propanol-based hand rub against sars-cov [165] . on clean hands use of an alcohol-based hand rub is first choice in healthcare for the decontamination of hands due to the better activity against nosocomial pathogens including bacteria and yeasts and a better dermal tolerance [163] . it may also be useful for covid-19 patients, e.g. before leaving the patients room for examinations. in this situation it is reasonable to recommend a hand disinfection in order to reduce potential transmission by direct hand contacts. the routine use of alcohol-based hand rubs for the general population should be discouraged, since there are currently no clear indications when to use them. it may be useful if a contamination of hands with sars-cov-2 is likely and a hand washing facility is not available. otherwise the widespread use of alcohol-based hand rubs may even enhance the shortage of the products in patient care which should be avoided by all means [166] . inadequate ppe including facemask at the beginning of the epidemic in china has resulted in infections and deaths among healthcare workers [167, 168] . unprotected patient care with long and close contacts was also later a major risk for healthcare workers to acquire covid-19 [169] . in covid-19 cases face masks can at least reduce the viral spread. in 17 individuals with a symptomatic seasonal coronavirus infection a surgical face mask was able to reduce the proportion of viral rna detection in droplets from 30% to 0% and in aerosols from 40% to 0% during 30 min exhaled breath collection suggesting a protective effect when worn by infected patients [92] . in another study 4 covid-19 patients coughed 5 times in front of a petri dish (20 cm distance) with a surgical mask, a cotton mask or without a mask. without a mask 2.6 log10 viral copies per ml were detected, with a surgical mask it was 2.4, and with a cotton mask 1.9 log10 viral copies per ml [170] . household transmission was more likely when the primary case and other household members did not wear a mask at home resulting in the possibility of unprotected transmission [171] . data on a protective effect of face masks when only worn by healthy subjects in an endemic covid-19 setting are not available. despite these results it was shown in south korea that none of 35 hcws with close contacts to a covid-19 patient developed symptoms or were pcr positive in the nasopharynx although they only wore a surgical mask for more than ten minutes during activities including aerosol-generating procedures such as intubation [172] . in addition, one study could show that a 4 days surgical mask partition between cages reduces the risk of noncontact transmission between artificially infected and naïve golden syrian hamsters [173] . importantly, a used face mask worn by a sars-cov-2 spreader will be contaminated. after only 5 coughs all surgical or cotton face masks worn by covid-19 patients were contaminated on the outer surface whereas samples from the inner surface were mostly negative [170] . chin et al. found that the virus can remain infectious or detectable for up to 7 days on the outer layer of a surgical mask, on the inner layer for 4 days [146] . although the results are only based on three independent triplicates, this finding should have implications for the re-use of face masks in a shortage situation [166] . wearing a face mask is recommended for healthcare workers in case of suspected or confirmed covid-19 patients [2, 174] although it was described in hong kong that 11 of 413 hcws had unprotected exposure to confirmed covid-19 cases, none of these were infected [95] . wearing a face mask may also be useful for hcws when mild respiratory symptoms occur because in the netherlands 4.1% of such healthcare workers were positive for sars-cov-2 [175] . even universal masking in hospitals by healthcare workers has been proposed although the expected effect was described as marginal [176] . suspected and confirmed covid-19 cases should wear a face mask to prevent the spread of infectious droplets [2] . so-called mass masking has been proposed as a considerable option [177, 178] . many countries have recommended or legally ordered the use of fabric masks or face coverings for the general public. the who, however, acknowledged that the widespread use of masks by healthy people in the community setting is not yet supported by high quality or direct scientific evidence and that there are potential benefits and harms to consider [179] . but in areas of known or suspected widespread community transmission and limited or no capacity to implement other containment measures, governments should encourage the general public to wear masks in specific situations and settings [179] . some recent studies suggest that general face mask usage by the healthy population in the community reduces the risk of transmission [180, 181] . but in order to evaluate only the effect of masks worn by healthy people in the community on the prevention of transmission in a country or region, some relevant variables with a proven impact on transmission should have been considered for the study period: the seasonal effect on the incidence (similar weather conditions), the main mode of transmission during the period of observation (mainly local clusters or mainly transmission in buildings or mainly transmission in the public), the total number of new cases in the observation period (mass masking in a region with 1 new case per day may have a different effect compared to a region with 10.000 new cases per day) and the extent of community lockdown (the less people are in the public the less likely a protective effect of general masks can be expected). in an endemic population scenario without restrictions regarding physical distance and close or long face-to-face contacts it may indeed be useful, especially for the part of the population which has a high risk for a severe covid-19 infection. it is, however, a controversial debate among the scientific community if any additional protective effect by mass masking is expectable if a minimum distance between people is assured (e.g. 2 m) and contacts are only of short duration. gloves can partially prevent the contamination of the hands with specific pathogens or all types of bioburden [182] . however, at the same time wearing gloves is associated in hospitals with a lower compliance in hand hygiene [183, 184] . use of gloves is recommended for hcws in specific patient care activities, e.g. when soiling of the hands is expected and when caring for covid-19 patients [2, 163] . if there is any protective effect by wearing gloves by the general population in the public is speculative. one aspect is that wearing gloves may result in more awareness too reduce face hand contacts. and yet it seems reasonable not be encourage the general population to routinely wear gloves in the public. even if a hand contact yields a transient contamination with sars-cov-2 on the hands it does not make a difference if the virus is found on the bare or gloved hand; the essential preventive measure in this case is to avoid hand-face contacts and to wash hands when returning from the public. the resident hand flora is even able to provide some colonisation resistance in contrast to the glove [185] . if wearing gloves by the general population has a similar effect on hand hygiene compliance as it has been described for healthcare workers wearing gloves in the public may even have the unwanted effect of less hand washing potentially increasing the risk of transmission via hands. some surface disinfectant agents have been described to inactivate sars-cov-2 in 30 s such as ethanol and iso-propanol (30% -80%, v/v) [164] . in 5 min household bleach (1:49 and 1:99) and 0.1% benzalkonium chloride were also very effective against sars-cov-2 [146] . limited data from surface samples in covid-19 settings support their efficacy [186, 187] . in healthcare settings routine cleaning and disinfection of surfaces with which the patient is in contact is recommended [2] . so far, no studies were reported to address if sars-cov-2 (viral rna or infectious virus) may be found on public inanimate surfaces. disinfection of surfaces in a household with chlorine-or ethanol-based products can reduce the risk of transmission when the primary case has diarrhoea [171] . the frequent use of household disinfectants also results in a remarkable increase of exposures reported to us poison centers, especially via ingestion in the age group between 0 and 5 years [188] . general disinfection of frequently touched surfaces in the public such as shopping carts or door handles is, however, unlikely to add any protective value because even in covid-19 wards inanimate surfaces were mainly contaminated in the permanent and immediate surrounding of symptomatic patients (detection of viral rna, not of infectious virus) and only rarely one room away [138] suggesting that the risk to find sars-cov-2 on frequently touched surfaces in the public is low. future research will hopefully clarify the role of public inanimate surfaces for the spread of sars-cov-2. close and long contacts are probably the main risk for transmission of sars-cov-2 from asymptomatic or symptomatic patients to healthy people as shown in clusters in families, a cruise ship, hospitals and nursing homes [189] . the mode of transmission is very likely by droplets during coughing, sneezing or talking. the risk of long and close contacts is supported with experimental data obtained with 8 syrian hamsters which were inoculated with 10 5 viral copies in 100 µl intranasally. 24 h later each hamster was transferred to a new cage with one naïve hamster as close contact. sars-cov-2 was detected in nasal secretions, trachea and lung after 4 days in all naïve contact hamsters [190] . physical distancing is another option to slow down the spread of sars-cov-2. early data from china suggests that quarantine, physical distancing, and isolation of infected populations can flatten the epidemic [191] . so far, there are no "real-life" data which provide conclusive evidence regarding effectiveness of physical distancing interventions. however, in a simulation model the likelihood of sars-cov-2 human-to-human transmission in a singaporean population was predicted [192] . they could demonstrate that the combined intervention, in which quarantine, school closure, and workplace distancing were implemented, was the most effective compared with the baseline scenario of no interventions, which reduced the estimated median number of covid-19 infections by 99.3% when r 0 was 1.5, by 93.0% when r 0 was 2.0, and by 78.2% when r 0 was 2.5 [192] . nevertheless, an evaluation of the effect of physical distancing alone is currently not possible. maintaining a physical distance of at least 1 m from other individuals is regarded as one of the most effective preventive measures by the who [193] . günter kampf has received personal fees from dr. schumacher gmbh, germany, for presentation and consultation. yannick brueggemann, hani e. j. kaba, joerg steinmann, stephanie pfaender, simone scheithauer and eike steinmann have no conflicts of interest. 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isolation of an asymptomatic carrier sars-cov-2 rna detection of hospital isolation wards hygiene monitoring during the coronavirus disease 2019 outbreak in a chinese hospital absence of contamination of personal protective equipment (ppe) by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) contamination of personal protective equipment by sars-cov-2 during routine care of patients with mild covid-19 j o u r n a l p r e -p r o o f [196] j o u r n a l p r e -p r o o f [198] *no absolute numbers reported; **first survey; ***second survey two weeks later.j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f samples significantly higher than for nasopharyngeal swab specimens cpm = copies per ml; cpg = copies per g; cps = copies per whole swab; pfu = plaque forming units. key: cord-341254-xnj6slby authors: li, hua; liu, zhe; he, yue; qi, yingjie; chen, jie; ma, yuanyuan; liu, fujia; lai, kaisheng; zhang, yong; jiang, liu; wang, xiangdong; ge, junbo title: a new and rapid approach for detecting covid‐19 based on s1 protein fragments date: 2020-06-05 journal: clin transl med doi: 10.1002/ctm2.90 sha: doc_id: 341254 cord_uid: xnj6slby the pandemic of novel coronavirus disease 2019 (covid‐19) seriously threatened the public health all over the world. a colloidal gold immunochromatography assay for igm/igg antibodies against the receptor‐binding domain of severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) s1 protein was established to assess its rapid diagnostic value. we first designed and manufactured all contents of the test cassette of sars‐cov‐2 rapid test kit: the colloidal gold‐labeled mouse‐antihuman lgm/lgg antibody, the recombinant sars‐cov‐2 antigen, the nitrocellulose membrane control line, and specimen diluents. furthermore, reverse transcription‐polymerase chain reaction (rt‐pcr) assay, colloidal gold immunochromatography assay, serological validation of cross reaction with other common viruses, and clinical validation were performed. the kit was finally evaluated by 75 serum/plasma samples of sars‐cov‐2 infection cases and 139 healthy samples as control, with the result of that the sensitivity, specificity, and accuracy for igm were 90.67%, 97.84%, and 95.33%, whereas for igg were 69.33%, 99.28%, and 88.79%, respectively; the combination of igm and igg could improve the value: 92.00%, 97.12%, and 95.33%, respectively. therefore, the rapid detection kit has high sensitivity and specificity, especially for igm&igg, showing a critical value in clinical application and epidemic control of covid‐19. in december 2019, some unexplained pneumonia cases started to be found in wuhan, hubei province, china. the pathogen was quickly clarified by china researchers as the positive-sense single-stranded rna coronavirus (severe acute respiratory syndrome coronavirus 2 [sars-cov-2]), belonging to the same family as of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). 1 and homology studies showed that sars-cov-2 had nearly 80% homology with sars-cov and 50% identity with mers-cov, whereas 96.3% identity with a bat's coronavirus. 2, 3 disease caused by the novel coronavirus was later named as coronavirus disease 2019 (covid-19) by the world health organization (who). covid-19 spread rapidly and has brought about a pandemic with more than 4.0 million laboratory confirmed cases until 11 may 2020 (https: //covid19.who.int). covid-19 diagnosis requires to be confirmed by sars-cov-2 nucleic acid detection via rt-pcr (reverse transcription-polymerase chain reaction) according to who covid-19 guideline. 4 however, nucleic acid detection of sars-cov-2 has obvious limitations in practice. 5 further researches indicated that the covid-19-infected patients would also produce specific antibodies by immune response, 6, 7 which was similar to those with sars-cov infection. based on it, the detection of igm/igg in blood became an optional approach to improve the diagnosis, especially for the covid-19 patient with negative nucleic acid test result. 8 for this reason, we designed and developed sars-cov-2 antibody test reagents. the kit can be performed in the site and took at most 15 minutes to obtain results with only one drop of blood sample, which is more convenient for large population screening and site inspection than nucleic acid test. 9 although a large number of antibody detection reagent kits were developed, evidence in terms of the clinical application value was still lacking. 10 in order to be more beneficial to improve the diagnosis timeliness and accuracy of covid-19, we supported following evidence to promote its clinical utility. we first designed and manufactured all contents of the test cassette of sars-cov-2 rapid test kit. 1. the contents of the rapid test kit for blood lgm/igg antibody were designed to include sample soleplate, reaction soleplate, test line (t), control line (c), suction filter paper, and plastic cassette ( figure 1a ). colloidal gold-labeled mouse-antihuman igm/igg antibody was on the reaction soleplate, the test line was on the nc membrane and covered by recombinant sars-cov-2 antigen, and the control line, used for quality control, was covered by goat-antimouse igm/igg antibody. 2. colloidal gold-labeled mouse-antihuman lgm/lgg antibody was manufactured by saiya hebei biotechnology co., ltd. to obtain the well-performance antibody, the antibody was selected for functional test including the positive and negative coincidence rates, minimum test threshold, and accelerated stability. first, the positive and negative coincidence rates were tested. the test line and control line were covered by 1 mg/ml recombinant sars-cov-2 antigen and 0.5 mg/ml goatantimouse lgm/lgg antibody, respectively. colloidal gold-labeled mouse-antihuman lgm/lgg antibody from four different batches were spread on the reaction soleplate for testing by positive reference (p1-p3) and negative reference (n1-n6). colloidal gold-labeled mouseantihuman lgm/lgg antibody evaluated the positive and negative coincidence rates, with samples m1, m3, and m4 showing good coincidence rate in both positive and negative references and selected for further testing (table s1) . second, minimum test threshold references with dilution of 1:2, 1:6, and 1:18 were used, and m1/m3 fulfilled the requirement of minimum threshold test (table s2 ). the test cassettes with m1 and m3 colloidal gold-labeled mouse-antihuman lgm/lgg antibody were further tested for the accelerated stability. the test cassettes were kept under dry and closed condition with 37 • c temperature for 21 days and tested by using threshold references (table s3) . finally, the result indicated that m3 fills the requirement. 3. the recombinant sars-cov-2 antigen was produced according to the procedure. 8 the recombinant protein was sent to beijing jorferin bio-technology co. ltd. for further process. to obtain the optimized antigen, strict functional selection steps including the positive and negative coincidence rated, minimum test threshold, and accelerated stability test were carried out. the expressed recombined sars-cov-2 protein was verified via protein electrophoresis ( figure 1b ), demonstrating the same molecular weight as that of predicted size around 30 kda in the reducing condition. and the reactogenicity of the recombinant antigen was further checked by elisa using biotin-labeled anti-sars-cov-2 antibodies. it was observed that the recombined sars-cov-2 antigen bound well with both anti-sars-cov-2 antibodies in varied concentration, indicating that the quality of the recombined sars-cov-2 antigen was viable ( figure 1c ). subsequently, to select the high-performance recombinant sars-cov-2 antigen, different batches of recombinant sars-cov-2 antigen were tested by using positive (p1-p3) and negative (n1-n5) references with a concentration of 1 mg/ml (table s4) . two samples (r1 and r2) out of three obtained 100% positive and negative coincidence rates and were brought to the minimum test threshold inspection with the threshold references in the dilution of 1:2, 1:6, and 1:18. ultimately, r1 sample obtained 100% positive coincidence rate with 1:2 and 1:6 dilution references and was selected as the final recombinant sars-cov-2 antigen (table s5 ). 4. the nitrocellulose membrane control line was covered by goat-antimouse lgm/lgg antibody, manufactured by beijing jorferin bio-technology co. ltd. colloidal gold-labeled mouse-antihuman lgm/lgg antibody covering the control line was also assessed at different concentrations from 5 to 0 mg/ml in a descending order with the same concentration (0.5 µm) of recombinant sars-cov-2 antigen. we found that as the concentration of the solution gradually decreased, the result turned from positive to negative and finally sample g2 demonstrated better performance than g1 (table s6 ). 5. specimen diluent contained 2.0% trehalose, 2.0% bovine serum albumin, 0.2% ethylenediaminetetraacetic acid disodium, 0.9% sodium chloride, 0.2% proclin 300, and 1.15% 0.5 m ph 7.2 tris buffer. furthermore, rt-pcr assay, 8 colloidal gold immunochromatography assay, 8 . it was observed that there was no cross reaction between sars-cov-2 and other common respiratory viruses ( figure 1e ). in the previous studies, igm was described as the earliest antibody produced after viral infection, whereas igg was produced in the recovery phase of viral infection and lasted for several months or years. 11 positive igm antibody usually indicated an acute phase of viral infection, whereas positive igg antibody suggested late or previous infection. due to only around 50% positive rate of sars-cov-2 nucleic acid test 8, 12 under various condition of sample collection and storage, viral infection regions, rna extraction methods, the quality of nucleic acid detection kit, and so on, 13 detection of igm/igg became a powerful approach for the early diagnosis of covid-19 and could help identify the patients with negative nucleic acid but with obvious clinical symptoms. 8, 14 in addition, detection of igm/igg can also provide the time course information of viral infection 12 5 we also reexamined 37 covid-19 convalescent patients and found that two patients' nucleic acid test was still positive with one of them igm + /igg + and the other igm − /igg − . in the other published studies, the positive nucleic acid test of covid-19 convalescent patients was also reported. 22, 23 therefore, antibody detection could assist to screen covid-19infected patients accurately, combined with nucleic acid detection. 13 in sum, the sars-cov-2 antibody rapid detection kits we demonstrated has high sensitivity and specificity. the reported data suggested that it could be a good prospect for wide application in individual serological qualitative monitoring and might play a valuable role in practical applications for the diagnosis and epidemic control of covid-19, with the development of the big database of epidemic investigation for sars-cov-2 igm/igg antibody ( figure 1f ). the authors declare no conflict of interest. hl, zl, yh, and yq collected, analyzed, and interpreted the data. hl, xw, and jg conceived and supervised the study. hl and fl wrote the manuscript. all authors read and approved the final manuscript. a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes the many faces of the anti-covid immune response sars-cov-2 lgm/lgg antibody detection confirms the infection after three negative nucleic acid detection developing antibody tests for sars-cov-2 diagnostic performance of covid-19 serology assays igm in microbial infections: taken for granted antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 scientific research progress of covid-19/ sars-cov-2 in the first five months longitudinal change of sars-cov2 antibodies in patients with covid-19 kinetics of sars-cov-2 specific igm and igg responses in covid-19 patients temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid-19 patients diagnosis and treatment guideline for novel coronavirus pneumonia. 0. 7th. beijing, china: national health commission of the prc recent advances in the detection of respiratory virus infection in humans sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup clinical evaluation of a rapid colloidal gold immunochromatography assay for sars positive rt-pcr test results in patients recovered from covid-19 application of the technology of serum specific antibody in detecting 2019 novel coronavirus we appreciate luming zhang of bestnovo (jiangsu) medical technology co., ltd. for his technical support. this work was supported by the national natural science foundation of china (81521001 and 81870182) and the national key basic research program (2016yfc1301204 and 2020yfc1316701). key: cord-346819-11fkgzaa authors: khan, mohd imran; khan, zainul a.; baig, mohammad hassan; ahmad, irfan; farouk, abd-elaziem; song, young goo; dong, jae-jun title: comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations and the effect of mutations on major target proteins: an in silico insight date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0238344 sha: doc_id: 346819 cord_uid: 11fkgzaa a novel severe acute respiratory syndrome-related coronavirus-2 (sars-cov-2) causing covid-19 pandemic in humans, recently emerged and has exported in more than 200 countries as a result of rapid spread. in this study, we have made an attempt to investigate the sars-cov-2 genome reported from 13 different countries, identification of mutations in major coronavirus proteins of these different sars-cov-2 genomes and compared with sars-cov. these thirteen complete genome sequences of sars-cov-2 showed high identity (>99%) to each other, while they shared 82% identity with sars-cov. here, we performed a very systematic mutational analysis of sars-cov-2 genomes from different geographical locations, which enabled us to identify numerous unique features of this viral genome. this includes several important country-specific unique mutations in the major proteins of sars-cov-2 namely, replicase polyprotein, spike glycoprotein, envelope protein and nucleocapsid protein. indian strain showed mutation in spike glycoprotein at r408i and in replicase polyprotein at i671t, p2144s and a2798v,. while the spike protein of spain & south korea carried f797c and s221w mutation, respectively. likewise, several important country specific mutations were analyzed. the effect of mutations of these major proteins were also investigated using various in silico approaches. main protease (mpro), the therapeutic target protein of sars with maximum reported inhibitors, was thoroughly investigated and the effect of mutation on the binding affinity and structural dynamics of mpro was studied. it was found that the r60c mutation in mpro affects the protein dynamics, thereby, affecting the binding of inhibitor within its active site. the implications of mutation on structural characteristics were determined. the information provided in this manuscript holds great potential in further scientific research towards the design of potential vaccine candidates/small molecular inhibitor against covid19. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in the last two decades, three coronaviruses viz. severe acute respiratory syndrome coronavirus (sars-cov) [1] , middle-east respiratory syndrome coronavirus (mers-cov) [2] and sars-cov-2 have crossed the species barrier to cause deadly pneumonia in humans. in 2002, sars-cov emerged in the guangdong province of china and spread to five continents, infecting 8,098 people with 774 deaths. in 2012, mers-cov emerged in the arabian peninsula, transmitted to 27 countries, infecting a total of~2,494 individuals and claiming 858 lives. the current outbreak of coronavirus disease caused by sars-cov-2, was first reported in december 2019 in wuhan, hubei province of china [3, 4] and spread across 200 countries, infecting over 2.5 million people and killed more than 1.5 lakh as of april, 23, 2020. sars-cov-2 was declared a pandemic by the world health organization on march 12, 2020. sars-cov-2 belongs to the family coronaviridae of genus betacoronavirus, having positive sense strand rna genome of 26-32 kb size. sars-cov-2 genome has six major open reading frames (orfs) viz. replication enzyme coding region (orf 1a and 1b), e gene (envelope protein), m gene (membrane protein), s gene (spike protein), and n gene (nucleocapsid protein) that are common to coronaviruses and a number of other accessory genes (orf 3a, 6, 7a, 7b and 8) (fig 1) [3] . the structural proteins: envelope protein, nucleocapsid protein, spike protein and membrane protein are essential for producing the structurally complete viral particle [5] [6] [7] [8] . entry of coronavirus into host cells is guided by spike glycoprotein. orf 1a and 1b encode replication enzyme consisting 16 non-structural proteins (nsp1-16) that are highly conserved among the coronaviruses. main protease (mpro, also known as 3clpro) is one of the important nsp encoded by orf 1a and 1b, play an essential role in the processing of polyproteins and control the replication of coronavirus [9, 10] . rna-dependent rna polymerase (rdrp) also known as nsp12, another important replicase catalyze the replication of rna using viral genomic rna template [11] . reports showed that mers-cov originated from bats, but the reservoir host fueling spillover to humans is unequivocally dromedary camels [12, 13] . both sars-cov and sars-cov-2 are originated from bats, which serve as reservoir host for these two viruses [3, 14, 15] . raccoon dogs and palm civets have been identified as intermediate hosts for zoonotic transmission of sars-cov between bats and humans [16] [17] [18] , however, the intermediate host of sars-cov-2 remains unknown. mutation rate is very high in rna viruses, up to a million times higher than their host, which enhance their virulence and evolvability (formation of new species) [19] . coronavirus replication is error prone as compared to other rna viruses and the estimated mutation rate is 4x10 -4 nucleotide substitutions/site/year [20] . the rate of sars-cov-2 mediated disease spread and the mortality varies from country to country. of several reasons affecting the rate of disease spread and mortality, mutations within the sars-cov-2 strains is also considered one of the major factors. this study was conducted to gather additional information on the sars-cov-2 sequences from different geographical locations infected with covid-19. the genome analysis of the sars-cov-2 strains from 13 different countries showed a large number of mutations within the major structural proteins. this is the first time we have comprehensively investigated these mutations and also discussed their potential roles in the pathogenicity, replication and entry of virus particle. this study provides a deeper insight into the emergence of these mutations within the major structural as well as nsp encoded by the sars-cov-2 genome from different countries. here, molecular dynamics and other in silico studies were also performed to investigate the effect of mutations on the dynamics of mpro. the findings of this study provide a clue for the futuristic development of potential vaccine candidate or therapeutic design against covid19. the genome sequence of orf1ab for sars with reference sequence id: nc_004718.3 and protein sequence with genbank id: aap41036. and visualization of sars and sars-cov-2 sequences from 13 different countries was performed using molecular evolutionary genetics analysis (mega) version 10.1.8. to delineate and analyze the mutation across different countries, an in-house script written in perl and python was used. mupro server was used to determine the effect of mutation on various sars-cov-2 proteins [21] . the crystal structure of mpro protein from sars-cov-2 in complex with boceprevir (pdb id: 7brp) was taken as a wildtype (wt). the structure of r60c was generated by inserting the point mutation and modelled using modeler 9v13 [22] . boceprevir was retrieved and redocked within the structure of mpro using ccdc gold. the rmsd for the crystal and redocked conformation of boceprevir were compared. further, boceprevir was subjected to dock within the active site of r60c mutant. the poses were visualized on pmv viewer [23] . the structure of boceprevir in complex with mpro (wt) and r60c mutant was subjected to energy minimization using gromacs-5 with the charmm27 all atom force field [24] [25] [26] . the models were solvated with a spc/e water model in a cubic periodic box with 1 nm distance from the edge of the complex atoms. the solvated system was neutralized by seven chloride ions. the system was, thereafter, minimized using steepest descent algorithm with convergence criteria of tolerance value 1000 kj mol −1 nm −2 . the complete simulation of minimized solvated proteins was performed under periodic boundary condition with time step of 2 fs. particle mesh ewald was used for long range electrostatic interactions with an interpolation order of 4 and a fourier spacing of 0.16. the first phase simulation was conducted under an nvt ensemble for 500 ps by keeping all bonds constrained using the lincs algorithm for temperature equilibration. the system was heated to 300 k using leap-frog integrator while pressure coupling was set off. a v-rescale thermostat was used to maintain constant temperature for each system, followed by pressure equilibration at 300 k using parrinello-rahman pressure coupling algorithm under an isothermal-isobaric ensemble for another 500 ps at 1.0 bar. isothermal compressibility of the solvent was set to 4.5e -5 bar −1 . further, simulation of 50000 ps production run was carried out at 300 k and 1 atm pressure for trajectory analysis. the final models obtained at the end of md were validated and taken for structural analysis. the complete nucleotide sequences of 13 sars-cov-2 reported from 13 different countries showed~82% sequence identity with sars-cov. also, all 13 sequences shared more than 99% sequence identity to each other. replicase polyprotein (orf 1ab) of 13 isolates, which are most conserved in all coronaviruses shared maximum identity (87%) with sars-cov (nc_0047180), which is less than the threshold value (90%) for demarcation of betacoronavirus species [27, 28] . phylogenetic analysis revealed that all 13 sars-cov-2 identified from different geographical locations clustered together in a single clad as compared to sars-cov (fig 2a and 2b ). further, we checked the mutation in all major proteins of 13 sars-cov-2 sequences and compared with sars-cov. orf 1a and 1b showed 11 changes among all 13 sars-cov-2. indian sars-cov-2 sequence showed three changes at 671 (isoleucine to threonine), 2144 (proline to serine) and 2798 (alanine to valine) compared to all other 12 isolates. here, we also noted two amino acid mutations (in orf1ab) in each sars-cov-2 sequences isolated from china (2708: asparagine to serine; 2908: phenylalanine to isoleucine), south korea (902: methionine to isoleucine; 6891: threonine to methionine) and sweden (818: glycine to serine; 4321: phenylalanine to leucine). brazil and vietnam isolate showed only one change at 3606 (leucine to phenylalanine) and 3323 (arginine to cystine), respectively (table 1) . 996 changes have been reported when orf 1a and 1b amino acid sequences of all 13 sars-cov-2 was compared with sars-cov (s1 file). the viral mpro controls the replication of coronavirus and is a key protein responsible for its life cycle [29] [30] [31] . mpro is an attractive drug discovery target. the analysis of mpro reveals that there was only one point mutation (r60c) in the vietnam strain of sars-cov-2 ( fig 3a) . rdrp, which is another important target for antiviral drugs functions by catalyzing the viral rna synthesis [32] . only one mutation (a406v) was observed in the rdrp of indian sars-cov-2 isolate (fig 3b) . spike proteins are the key surface glycoproteins and are well reported for their prominent role in interaction with host cell receptors [33, 34] . here, we analyzed the mutations in the spike protein of sars-cov-2 from different countries. it was found that this glycoprotein carried five different amino acid mutations at various positions within the investigated sars-cov-2 isolates. for instance, india, finland, australia, south korea and sweden sars-cov-2 isolates showed one amino acid change at 408 (arginine to isoleucine), 49 (histidine to tyrosine), 247 (serine to arginine), 221 (serine to tryptophan) and 797 (phenylalanine to cysteine), respectively ( table 2 and fig 3c) . the value of δδg show that the mutant r408i (0.49732107 kcal/mol) mutation was having stabilization effect on spike protein. it was found that the mutation on the receptor binding domain (rbd) of spike protein increases the stability. when these 13 sars-cov-2 isolates were compared to sars-cov sequence, 1338 changes have been reported (s1 file). the analysis of orf3a showed 3 mutations within different sars-cov-2 strains: w128l (south korea), l140v (japan), g251v (australia, south korea, brazil, italy, sweden) ( table 3) . one amino acid change occurred in each envelope protein of south korea sars-cov-2 isolate at 37 (leucine to histidine) and nucleocapsid protein of japan sars-cov-2 isolate at 344 (proline to serine) when compared among 13 sars-cov-2 isolates (tables 4 and 5 i i i i i i i i i i i i 818 t t m t t t t t t t t t t https://doi.org/10.1371/journal.pone.0238344.t001 when compared to sars-cov. mem glycoprotein did not show any amino acid change among 13 sars-cov-2 isolates, while 24 changes occurred when compared to sars-cov (s1 file). all the other point mutations occurring within the structural proteins of sars-cov-2 isolates from different countries were found to decrease protein stability (table 6 ). in the present study, we performed the md simulations for the boceprevir bound complexes of sars-cov-2 mpro and its r60c mutant to study the effect of mutation on the protein dynamics. the root mean square deviations of the backbone were calculated to analyze the trajectories of mpro from sars-cov-2 and its r60c mutant. in the complex form, a slight fluctuation in the comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations backbone rmsd was also noticed (fig 4a) . it was found that the rmsd of mutant was comparatively more stable than its wt. this variation in the average rmsd values suggests that this mutation was affecting the dynamic behavior of mpro. rmsf values of native as well as r60c mutant mpro were calculated to determine the impact of mutation on dynamic behavior of protein at residue level. rmsf plot clearly indicates the fluctuation in residues and showed the existence of higher degree of flexibility in r60c mutant mpro. it was found that the maximum amino acid fluctuation was in the region 50-76 and 127-222 ( fig 4d) . the binding studies also confirm that the residues falling within this region were very much involved in accommodating the inhibitor within the active site of mpro [29, 31] . throughout the md trajectory, the interaction energy of ligand in complex with the surrounding protein residues of wt and mutant mpro were calculated. the lennard-jones shortrange (lj-sr) and coulombic short-range (coul-sr) potential energies were calculated throughout the course of 50 ns of md simulation (fig 4e) . the average interaction energy for 50 ns are shown in table 7 . it was found that the binding of inhibitor within the active site of mpro (wt) was stronger as compared to the r60c mutant. the analysis of hydrogen bond network revealed that the r60c mutation also cause disturbance in the interactions with inhibitor as well as other surrounding active site residues of mpro. a large fluctuation was noticed in the hydrogen bond network of mpro and its r60c mutant (fig 5a) . it was found that r60c mutation results in the changes in local environment that cascade further to the short helix and loop of the catalytic active site of mpro. till date (april 23, 2020), 2.6 million cases of covid-19 have been reported worldwide. in this study, we analyzed 13 complete sequences of sars-cov-2 reported from 13 different countries and compared with sars-cov. phylogenetic analysis showed that sars-cov-2 sequences clustered together in a single clad irrespective of their geographic origin, whether from the same continent or neighboring countries. replicase polyprotein, which are most conserved among coronaviruses, shared 87% amino acid sequence similarity to sars-cov, less than the threshold value (90%) for demarcation of betacoronavirus species [27] . they belong to new virus species severe acute respiratory syndrome-related coronavirus of genus betacoronavirus [28] . among all known rna viruses, coronaviruses consist of the largest genome (26.4 to 31.7 kb) [36, 37] . the large genome size provides more plasticity in accommodating and modifying genes [36] [37] [38] . mutation frequency is very high in rna viruses, which enhances virulence and responsible for the formation of new species [19] . the high frequency of mutation within the viral genome at different geographical locations may be one of the reasons that sars-cov-2 is responsible for change in mortality rate and symptom of the disease [39] . the comparison of amino acid sequences of replicase polyprotein of 13 sars-cov-2 showed mutations in india, china, south korea, sweden, vietnam and brazil strains at different amino acid locations. earlier report showed the similar result i.e. a single mutation in replicase polyprotein at 3606 (l to f) [40] . we could identify few more single amino acid mutations at different positions in above mentioned sars-cov-2 strains ( table 1 ). the replicase polyprotein codes for nsp2 and nsp3 and it has been suggested in previous research that the mutation in nsp2 and nsp3 play a key role in infectious capability and are responsible for the differentiation mechanism of sars-cov-2 [39] . the rbd of spike protein is the region which specifically interact with ace2 leading to viral entry into the host cell [41] [42] [43] . the indian isolate of sars-cov-2 showed mutation within this region where at 408 position, arginine is replaced by isoleucine. for several years, the prediction of protein stability via theoretical or experimental approaches has been a profound area of research [44] . earlier findings suggest that a single point mutation at rbd is responsible for disrupting the antigenic structure, thereby, affecting the binding of rbd to ace2 [45, 46] . the mutation within this region of spike protein may affect the binding of rbd to its receptor, thus, affecting the viral entry within the host cells. further, in silico studies revealed that this point mutation within the rbd of spike glycoprotein was having stabilization effect on spike protein and found to increase the protein stability (δδg 0.49732107 kcal/mol). single amino acid mutation was observed in both mpro (r60c) of sars-cov-2 vietnam isolate and rdrp (a408v) of sars-cov-2 india isolate. the in silico findings revealed that the mutations in both strains decrease the stability of protein. the md simulation studies on mpro further confirmed that the point mutation on mpro affects the stability of proteins as well as the binding of inhibitor. our in silico study found that the catalytic active site of mpro is surrounded by amino acid residues of a loop (142-145, 175-200), short helix (40) (41) (42) (43) (46) (47) (48) (49) (50) , and beta sheet regions (25) (26) (27) (164) (165) (166) (167) . the r60c mutant lies at helix adjacent to the short helix (h2) that forms the catalytic channel (fig 5) . substitution of an amino acid with charged side chain to uncharged cysteine residue leads to loss of conserved ionic bond interaction and the effect cascades to other conserved ionic interactions. loss of conserved ionic interaction was observed between amide nitrogen of arginine and carboxylic oxygen atom of aspartic acid at position 48 of the catalytic channel. it was found that the short helix h2, that form the catalytic channel, have attained a more flexible loop like conformation in the mutant protein. conserved hydrogen bonded interactions that stabilizes the catalytic channel l1 loop between tyr54 oh� � �asp187 oδ1, tyr54 oh� � �asp187 o and leu50 o� � �arg188 ne were lost in the mutant enzyme, thereby, increasing the flexibility of structure forming the binding pocket ( fig 5b) . therefore, the local change of an ordered secondary structure to a more disordered loop like structure have increased the overall flexibility of the secondary structure elements forming the catalytic pocket, thereby, effecting the binding of ligand to residue in the catalytic channel. this is quite evident from the reduced lj-sd and coulombic-sr interaction energies between the enzyme and ligand in wild and mutant complexes ( fig 4e and table 7 ). the role of important active site residues, discussed in this study, has been reported before [47, 48] . the rmsf plot also reveals the key role of these residues in accommodating the inhibitor within the active site of mpro. envelop protein plays an important role in the assembly of viral genome and the formation of ion channels (ic), responsible for virus-host interaction, which is mainly associated with pathogenesis [5, 49] . we detected one amino acid mutation l37h in transmembrane domain (tmd) of envelop protein of sars-cov-2 south korea isolate. the tmd is hydrophobic in nature consisting mainly hydrophobic amino acids, while the mutation in tmd at 37 position changes hydrophobic to hydrophilic amino acid which changes the integrity of tmd. earlier report showed that mutations within the tmd domain of envelop protein completely disrupted ic activity [50] . this might be one of the reasons for slow spreading/low pathogenicity of sars-cov-2 in south korea. nucleocapsid protein of coronavirus is necessary for rna replication, transcription and genome packaging [51, 52] . a mutation p344s in nucleocapsid protein has been detected in sars-cov-2 japan strain. the p344s mutation on nucleocapsid was found to decrease the protein stability (δδg -1.2252261). this mutation is located in carboxy-terminal rna-binding domain (ctd) of nucleocapsid protein. earlier studies showed that ctd is responsible for oligomerization [53] . it was also revealed that among all the genomes studied in this study, the indian sars-cov-2 isolates were carrying maximum mutation. the indian isolates were carrying the r408i on the spike protein while a406v on the rdrp and several mutations on the replicase polyprotein of sars-cov-2. it is expected that these large number of mutations among the sars-cov-2 may affect the vaccine/inhibitor development against these isolates. to conclude, sars-cov-2 complete sequences from 13 countries were analyzed and compared with sars-cov. we identified country specific mutations in major proteins (replicase polyprotein, spike protein, envelop protein and nucleocapsid protein). further, molecular dynamics and other in silico studies revealed that mutations decrease the stability of protein and also hinders the binding of inhibitor. mutation r408i in spike protein of indian strain has significant influence on rbd domain of spike protein and this point mutation has a stabilization effect on the spike protein. the findings of the present study could help for the design of potential vaccine 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carboxyl terminal domain of the human coronavirus 229e nucleocapsid protein the authors are very grateful to prof. indranil dasgupta, university of delhi south campus, new delhi, india, for giving suggestions to improve the manuscript. key: cord-356166-fpno9zg5 authors: miyakawa, kei; jeremiah, sundararaj stanleyraj; ohtake, norihisa; matsunaga, satoko; yamaoka, yutaro; nishi, mayuko; morita, takeshi; saji, ryo; nishii, mototsugu; kimura, hirokazu; hasegawa, hideki; takeuchi, ichiro; ryo, akihide title: rapid quantitative screening assay for sars-cov-2 neutralizing antibodies using hibit-tagged virus-like particles date: 2020-09-15 journal: j mol cell biol doi: 10.1093/jmcb/mjaa047 sha: doc_id: 356166 cord_uid: fpno9zg5 nan due to the unavailability of any specific countermeasure, the constantly spreading covid-19 pandemic could only be partially and temporarily slowed down by implementing regional lockdowns that force people to stay at home and prevent their movement. with the progression of the pandemic, a considerable subset of the population would have acquired post-infection immunity and the tests that reveal the post-infection immune status of individuals are the need of the hour. a credible test, which accurately identifies the protected, can offer an immunity passport for the individual to be freed from the lockdown and resume routine activities without the fear of getting infected. at present, the semi-quantitative neutralization test (nt) and the quantitative plaque reduction neutralization test (prnt) identifying the presence of anti-sars-cov-2 neutralizing antibodies (nabs) are the only foolproof methods available for this purpose. however, practical feasibility of these highly specific tests is weighed down by drawbacks such as low throughput, long turnaround time (tat), and the need for a specialized laboratory setup with biosafety level 3 (bsl3) facilities to handle the live viruses used in these tests (cao et al., 2020) . to overcome these hurdles, pseudovirus-based nt (nie et al., 2020) , surrogate serodiagnostic tests (svnt) (tan et al., 2020) , and single-cell rna sequencing (cao et al., 2020) are being studied. however, a simple, convenient, rapid, and high-throughput test capable of directly detecting nabs with high specificity, which could act as an ideal alternative to the neutralization assay, is yet to be developed (ozcurumez et al., 2020) . virus-like particles (vlps) are self-assembling, non-replicating, nonpathogenic entities of similar size and conformation as that of infectious virions. vlps can be generated to possess the surface proteins of any kind of viruses on their membrane devoid of the viral nucleic acids. this genome-free feature of vlps overrides the need for bsl3 facilities while handling but has the drawback of difficulty in quantifying its cell entry and membrane fusion. hibit is an 11-amino acid peptide tag that can be attached to any protein-of-interest. lgbit, the counterpart of nanoluc luciferase is complementary to hibit and binds to the latter to produce a highly active luciferase enzyme. hibit-tagged proteins can be easily detected and quantified based on bioluminescence using the nano-glo assay system. hibit technology offers the advantages of high sensitivity, convenience of a single-reagent-addition step, and short tat of only a few minutes. in this report, we have developed a hibit-vlp-based neutralization test (hivnt) that can readily detect sars-cov-2 nabs ( figure 1a ). to establish hivnt, we inserted the hibit tag to the hiv-1 gag-pol protein, a minimal subunit to produce vlps. after testing several prototypes, we found that hibit tag insertion at the c-terminal region of the capsid gene in gag-pol performed best (supplementary figure s1a ). hibit tag can bind with high affinity to its complementary larger subunit lgbit and form luciferase (nanoluc) (sasaki et al., 2018) . as expected, since sars-cov-2 can infect veroe6/tmprss2 cells with high efficiency (matsuyama et al., 2020) , we next generated lgbit-expressing veroe6/tmprss2 cells. we noticed a robust increase in nanoluc activity when the lgbit-expressing we next tested whether our newly developed hivlp-sars2 system could detect nabs in the serum of covid-19 patients. the neutralization assay was carried out in accordance with the following steps. veroe6/tmprss2-lgbit cells were seeded on 96-well white polystyrene plates at a density of 10 4 cells/well one day prior to inoculation. decomplemented sera derived from convalescent covid-19 patients were mixed with hivlp-sars2 and the mixture was inoculated to veroe6/tmprss2-lgbit cells. three hours later, the cells were washed with pbs and measured for their nanoluc luminescence. we found that all the tested patients' sera could block the entry of hivlp-sars2 in varying degrees ( figure 1c ), suggesting the presence of nabs against sars-cov-2. two convalescent sera and one healthy donor serum were tested by hivnt and authentic sars-cov-2 nt only to show that all the samples gave similar results in both tests ( figure 1d ), suggesting that hivnt performance is in concordance with the nt using authentic sars-cov-2. since surrogate antibody detection is being widely studied as an alternative to nt, we wanted to check the correlation of nabs detected by hivnt with antibody titers detected by elisa. we matched the results of hivnt against the antibody titers detected by elisa in 74 covid-19-positive sera. nab levels were found to correlate with igg antibodies, but not igm antibodies, against both the nucleocapsid (np) and spike (sp) proteins ( figure 1e ). taken together, our novel hivnt could be useful for rapid detection of nabs in the sera of individuals recovered from covid-19. the hivnt assay principle is similar to conventional neutralization assays and is based on viral entry and membrane fusion with measurement of nanoluc luciferase activity to simplify the outcome. considering the drawbacks of nt and prnt, tests that may confer immunity passport to individuals are the need of the hour and different platforms are being exploited for this purpose, even though more detailed serological studies are needed. pseudovirus-based nts are the popular alternatives to detect nabs, as they overcome the need for bsl3 facility and have a high-throughput scale (muruato et al., 2020) . these tests employ pseudoviruses, which possess an envelope with sars-cov-2 spike proteins and also incorporate a reporter gene in their genome. the test depends on the expression of the reporter gene in the target cells infected with pseudovirus, which takes 24-48 h to be detected. in comparison, our hivnt does not involve gene expression and thus shortens the tat to only ~3 h. single-cell sequencing might have similar advantages (cao et al., 2020) but the need for specialized and expensive equipment hinders its practical application. patients is being studied for its potential to act as a surrogate marker to reflect the presence of sars-cov-2 nabs (tan et al., 2020) . our findings in a small sample size (n=74) reveal that igg could serve as a better surrogate marker than igm. however, the use of serodiagnostics for nab detection could have a few inherent drawbacks. although all covid-19 infections elicit a humoral immune response, the presence of antibodies does not reflect immunity. also, mild covid-19 infections elicit very low humoral response, which might not be detected by serological tests (ozcurumez et al., 2020) . in this setting, surrogate antibody detection tests can produce erroneous results. moreover, sars-cov-2 infects host cells through receptor binding domain (rbd) within s1 subunit in spike protein on the surface of viral particles that bind to host surface angiotensin converting enzyme-2 (ace2) receptor. elisa kits to detect anti-rbd antibodies use in vitro generated spike protein and its derivatives to detect nabs in serum. however, since spike proteins undergo glycosylation and oligomerization in vivo (xiao et al., 2004; walls et al., 2020) , the performance of in vitro generated spike proteins may vary depending on the 6 manufacturing method, as well as their clinical significance. also, nabs that target other regions of spike protein to suppress the function of fusion peptide of s2 subunit may exist, which could go undetected in s1 and rbd-based elisa detection (chi et al., 2020) . in this study, we established the hivnt, a simple, high-throughput assay system for the quantitative and rapid determination of sars-cov-2 nabs in the sera of individuals after recovery from symptomatic or subclinical covid-19. the hivnt system allows for bsl2-compliant testing and gives quantitative results at ultra-high-throughput. the luciferase-fragment complementation assay has been demonstrated to exhibit superior sensitivity with scope for further miniaturization into 384-or 1536-well plate format. based on our findings, we believe that the hivnt, by utilizing the nanoluc technology, can be instrumental in identifying individuals with protective immunity, carrying out epidemiological studies on population susceptibility, modeling virus propagation, and assessing convalescent plasma for therapy and vaccine evaluation. encouragingly, all these can be achieved on a high-throughput platform, with short tat, and in a lower biosafety setting. [ potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells a neutralizing human antibody binds to the n-terminal domain of the spike protein of sars-cov-2 enhanced isolation of sars-cov-2 by tmprss2-expressing cells a high-throughput neutralizing antibody assay for covid-19 diagnosis and vaccine evaluation establishment and validation of a pseudovirus neutralization assay for sars-cov-2 sars-cov-2 antibody testing-questions to be asked development of a rapid and quantitative method for the analysis of viral entry and release using a nanoluc luciferase complementation assay a sars-cov-2 surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction structure, function, and antigenicity of the sars-cov-2 spike glycoprotein oligomerization of the sars-cov s glycoprotein: dimerization of the n-terminus and trimerization of the ectodomain key: cord-355567-60sfv60p authors: azuma, kenichi; yanagi, u; kagi, naoki; kim, hoon; ogata, masayuki; hayashi, motoya title: environmental factors involved in sars-cov-2 transmission: effect and role of indoor environmental quality in the strategy for covid-19 infection control date: 2020-11-03 journal: environ health prev med doi: 10.1186/s12199-020-00904-2 sha: doc_id: 355567 cord_uid: 60sfv60p the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a new zoonotic agent that emerged in december 2019, causes coronavirus disease 2019 (covid-19). this infection can be spread by asymptomatic, presymptomatic, and symptomatic carriers. sars-cov-2 spreads primarily via respiratory droplets during close person-to-person contact in a closed space, especially a building. this article summarizes the environmental factors involved in sars-cov-2 transmission, including a strategy to prevent sars-cov-2 transmission in a building environment. sars-cov-2 can persist on surfaces of fomites for at least 3 days depending on the conditions. if sars-cov-2 is aerosolized intentionally, it is stable for at least several hours. sars-cov-2 is inactivated rapidly on surfaces with sunlight. close-contact aerosol transmission through smaller aerosolized particles is likely to be combined with respiratory droplets and contact transmission in a confined, crowded, and poorly ventilated indoor environment, as suggested by some cluster cases. although evidence of the effect of aerosol transmission is limited and uncertainty remains, adequate preventive measures to control indoor environmental quality are required, based on a precautionary approach, because covid-19 has caused serious global damages to public health, community, and the social economy. the expert panel for covid-19 in japan has focused on the “3 cs,” namely, “closed spaces with poor ventilation,” “crowded spaces with many people,” and “close contact.” in addition, the ministry of health, labour and welfare of japan has been recommending adequate ventilation in all closed spaces in accordance with the existing standards of the law for maintenance of sanitation in buildings as one of the initial political actions to prevent the spread of covid-19. however, specific standards for indoor environmental quality control have not been recommended and many scientific uncertainties remain regarding the infection dynamics and mode of sars-cov-2 transmission in closed indoor spaces. further research and evaluation are required regarding the effect and role of indoor environmental quality control, especially ventilation. in late december 2019, a cluster of severe pneumonia cases emerged in humans in wuhan, hubei province, china [1, 2] . the causative pathogen was identified as a novel coronavirus that was named the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [3, 4] . the disease rapidly spread internationally, raising global public health concerns, and was subsequently termed coronavirus disease [5, 6] . the most common clinical manifestations of patients with covid-19 are fever, cough, shortness of breath, and fatigue. some patients have also shown radiographic ground-glass lung changes and eventually died of acute respiratory distress syndrome (ards) [7, 8] . the world health organization (who) declared covid-19 as a global pandemic on march 11, 2020 [9] . sars-cov-2 is mainly transmitted human-to-human through close contact, respiratory droplets, fomites, and contaminated surfaces [10] [11] [12] [13] . the who adapted a 1m social distancing policy, based primarily on the assumption that the virus is transmitted through largely isolated droplets within this range [13] . however, the possibility of airborne transmission through airborne particles with diameters smaller than 5 μm has been suggested [14] . several factors are involved in the transmission of sars-cov-2 between individuals, including the environment in buildings and human behavior [15] [16] [17] [18] [19] [20] . similar to the transmission routes of other respiratory viruses, such as influenza or human coronavirus [21] [22] [23] , possible exposure pathways for covid-19 infection by the sars-cov-2 are finger contact with virus-contaminated surfaces (fomites) and subsequent finger contact with the facial membranes; inhalation of the virus carried in airborne particles (inhalable or respirable particles) exhaled from cough or vocalization; and droplet spray, the direct projection of the virus carried in particles exhaled from cough or vocalization onto the facial membranes. therefore, environmental factors in buildings, including temperature, humidity, stability on fomites, and ventilation and filtering systems, such as in public places, healthcare settings, restaurants, hotels, recreation facilities, or residential houses where people are close together, could have a significant influence on the infection. adequate control of these environmental factors and proper human behavior in accordance with these environmental conditions play a significant role in preventing the spread of covid-19 [24] . as most people spend more than 90% of their daily lives inside buildings, it is essential to understand the potential transmission dynamics of sars-cov-2 inside a building, the spatial dynamics, and the building operational factors that potentially promote and mitigate the transmission of sars-cov-2 and the spread of covid-19. this article aimed to review the effect of environmental factors in buildings, spatial dynamics, and building operational factors. in addition, a strategy to prevent sars-cov-2 transmission in building environments based on indoor environmental quality control recommended by the japanese ministries is also summarized. the characteristics of environmental air quality and environmental surfaces contaminated by the virus are important factors that determine the infectivity retention and extent and speed of the spread of the virus. the long-time persistence of these environmental factors influences the spread of covid-19 [25] . the sars-cov-2 genetic material, ribonucleic acid (rna), was detected in the rooms of both symptomatic and asymptomatic cruise ship passengers up to 17 days after the cabins were vacated [26] . the sars-cov-2 rna was detected on the surfaces of the floor around the toilet, in the bedroom, bed pillow, phone, table, television remote control, chair arm, toilet flush button, toilet seat, and other items in the cruise ship [27] . although the infectiousness of those materials is not known, the environment around the covid-19 cases was contaminated extensively with sars-cov-2 during the covid-19 outbreak on the cruise ship. the persistence of sars-cov-2 in aerosols in different environmental conditions has been reported. the results are summarized in table 1 . van doremalen et al. compared the survival rate and half-life of sars-cov-2 and sars-cov-1 within 3 h of aerosolization at a temperature of 21°c-23°c and a relative humidity (rh) of 65%. both viruses were detectable after 3 h of aerosolization and the median half-lives were 1.09 and 1.18 h for sars-cov-2 and sars-cov-1, respectively [28] . these results showed that the stability of sars-cov-2 was similar to that of sars-cov-1. smither et al. reported that sars-cov-2 is more stable at a medium rh of 40-60% (decay rate, 0.91%/min) compared with a higher rh of 68-88% (decay rate, 1.59%/min) in tissue culture media (tcm), whereas the converse was observed in artificial saliva, with a decay rate of 2.27% per minute at a medium rh and 0.40% per minute at a higher rh [29] . the results of the half-life obtained with tcm at a medium rh were similar to those described above [28] . although the infectious dose in humans is not known, sars-cov-2 may be able to remain viable and infectious in aerosols for hours (depending on the inoculum shed), if the virus is produced within smallparticle aerosols. sars-cov-2 is susceptible to heat treatment but can persist for at least 14 days at refrigerated temperatures of 4°c. with an increase in the incubation temperature, the time for virus inactivation was reduced dramatically to 2 days at 37°c and to 5 min at 70°c [30] . sars-cov-1 demonstrates tendencies similar to those of sars-cov-2 [31] . in simulated saliva on a stainless steel surface, sars-cov-2 exhibits negligible decay over 60 min in darkness but loses 90% of its infectivity every 6.8-12.8 min, depending on the intensity of simulated ultraviolet (uv) b radiation levels, when exposed to simulated sunlight representative of the summer solstice at 40°n latitude at sea level on a clear day [32] . these data indicate that sunlight may inactivate sars-cov-2 rapidly on surfaces, suggesting that persistence and subsequently exposure risk may vary significantly between indoor and outdoor environments. experimental studies using sars-cov-2 aerosols produced from artificial saliva found that simulated sunlight inactivates the virus rapidly [33] . in dark conditions, the half-life of aerosolized sars-cov-2 is approximately 86 min in simulated saliva. the high-intensity sunlightsimulated summer was found to show a 90% reduction in infectious concentration after 6 min. even with the low-intensity sunlight-simulated late winter or early fall, a 90% reduction was observed at 19 min. however, relative humidity (20, 37, 53, and 70%) had no significant effect on the survival of the aerosolized virus. these data indicate that sunlight is useful in mitigation strategies to minimize the potential for aerosol transmission. stability on surfaces of fomites sars-cov-2 can persist on plastic, stainless steel, and glass surfaces between 2 and 4 days at room temperature (table 2) [28, 30] . the persistence of sars-cov-2 on those surfaces was similar to that of sars-cov-1 [28, 30, 31] . the survivability of these viruses on metal surfaces differed according to the type of metal. both sars-cov-1 and sars-cov-2 survived for shorter periods on copper (8 and 4 h, respectively) than on stainless steel surfaces [28] . the antimicrobial properties of copper and copper alloy have been reported against various viruses [34, 35] . several mechanisms have been proposed regarding copper-induced cellular toxicity to the virus. reactive oxygen species generated by free copper ions cause the cells to commit metabolic suicide. copper ions can induce protein destabilization on the virus. copper ions have a direct effect on virus inactivation by causing aggregation of virus particles [35] . this might explain the short survival of sars-cov-1 and sars-cov-2 on copper surfaces compared with other metal surfaces. studies have reported that copper alloys (≥58% copper) reduced the surface microorganisms when incorporated into various hospital furnishings and fittings [36, 37] . the use of copper in combination with optimal infection-prevention strategies may further reduce the risk of patients and healthcare workers acquiring covid-19 infection in healthcare environments. sars-cov-2 showed variable persistence on different porous surfaces, such as paper, cardboard, wood, cloth, and mask. sars-cov-2 survived on the inner and outer layers of surgical facemasks for 4 and 7 days, respectively [30] . in other words, the infectious virus can be recovered from a surgical mask after 7 days (22°c, 65% rh). sars-cov-2 survived for 1 day on cardboard, wood, and cloth [28, 30] . it survived for 2 days on banknote paper [30] . however, the virus survived for only 30 min on paper and tissue paper, with complete decay after 3 h [30] . under the same conditions, sars-cov-2 survived for a longer time (1 day) than sars-cov-1, which survived for only 8 h [28] . interestingly, the stability of sars-cov-2 was enhanced when present with bovine serum albumin, which [29] is used commonly to represent sources of protein found in human sputum [38] . conversely, sars-cov-2 decayed more rapidly when either the humidity or the temperature was increased, but the droplet volume and surface type (stainless steel, plastic, or nitrile glove) did not impact the decay rate significantly. at room temperature (24°c), the virus half-life ranged from 6.3 to 18.6 h depending on the relative humidity but was reduced to 1.0-8.9 h when the temperature was increased to 35°c [39] . these findings suggest that a potential for fomite transmission may persist for hours to days in indoor environments and that the survivability of fomites is affected by temperature and relative humidity, as well as by the presence of protein found in human sputum. in summary, virus survival decreases with an increase in temperature. maintaining temperatures above 60°c for more than 60 min usually inactivate most viruses [40] . sars-cov-2 is also susceptible to heat treatment. persistence of sars-cov-2 on dry inanimate surfaces was range for 1-7 days. the persistence of influenza virus, rhinovirus, and norovirus was reported at 1-2 days, 2 h −7 days, and 8 h −7 days, respectively [41] . thus, sars-cov-2 can remain infectious on the surfaces compared with the influenza virus. however, the persistence of sars-cov-2 was significantly low on copper as compared with other surfaces such as plastics, stainless steel, glass, and fabrics. in addition, sunlight can rapidly inactivate sars-cov-2. these findings droplet and contact transmission have accounted for the main routes of infection related to covid-19. in addition, the who announced the possibility of aerosol infection in specific circumstances, such as endotracheal intubation, bronchoscopy, etc. [42] . on the other hand, some researchers have claimed a risk of short-and medium-range person-to-person distance in aerosol transmission, but evidently, airborne transmission (longrange distance) has not been acknowledged [43] . nicas et al. estimated that the relative contributions of four influenza virus exposure pathways, namely (1) virus-contaminated hand contact with facial mucous membranes, (2) inhalation of respirable cough particles, (3) inhalation of inspirable cough particles, and (4) the spray of cough droplets onto facial mucosa, account for 31, 17, 0.52, and 52% risk of influenza infection, respectively [22] . the transmission routes for these cough particles can be categorized as "droplet transmission," where droplets (>5 μm diameter, traveling <1 m) containing viable viruses make contact with the nose, mouth, eyes, or upper respiratory tract; and "airborne transmission" where droplet nuclei (<5 μm diameter, which can travel >1 m) are inhaled by susceptible individuals [44] . the who has stated that airborne transmission is different from droplet transmission as it refers to the presence of microbes within droplet nuclei, which are generally considered to be particles smaller than 5 μm in diameter, can remain in the air for long periods of time and can be transmitted to others over distances greater than 1 m [42] . from the view of this statement, the covid-19 virus can be transmitted by direct contact via infected people and indirect contact via surfaces in the immediate environment or with objects used by the infected person, not by airborne transmission through small airborne particles. recently, 36 researchers insisted on the potential risk of indoor airborne transmission of sars-cov-2 and the importance of sufficient and effective ventilation, particle filtration, and air sterilization as infection control measures inside buildings [43] . the who recognized the potential risks of the airborne spread of covid-19 [13] . however, a recent experimental study using transgenic mice indicated that sars-cov-2 could be experimentally transmitted among mice by close contact, through respiratory droplets, but is hardly transmitted through exposure to airborne particles [45] . a recent study also indicated that the role of airborne particles as carriers of the virus diffusion is not evident [46] . in discussing the airborne transmission of sars-cov-2, it is important to understand the characteristics of aerosol particles emitted by cough and speech in indoor environments. this section describes briefly the size distribution of droplets, the emission rate of droplets by cough and speech, and the resuspension of particles deposited on floor surfaces. sars-cov-2 has a diameter of 60-140 nm, with characteristic spikes ranging from 9 to 12 nm [47] . johnson et al. determined an aerosol droplet size distribution ranging from 700 nm to 1 mm, generated by breathing, speech, and voluntary coughing by using the expired droplet investigation system with an aerodynamic particle sizer (aps) and a droplet deposition analysis (dda) [48] . in the case of speech, three different droplet size distribution modes were identified, with median diameters at 1.6, 2.5, and 145 μm. in the case of voluntary coughing, the modes were located at 1.6, 1.7, and 123 μm. therefore, a wide range of droplet sizes is emitted by speaking and coughing. the key point in this study was that small droplets can be emitted not only by speaking and coughing but also through breathing. individuals infected by sars-cov-2 without symptoms can also transmit the infection to others. the emission by natural breathing without coughing or sneezing is also important to understand the transmission from infected individuals without symptoms. asadi et al. placed aps in a laminar flow hood to characterize the number and size distribution of particles emitted by individual human volunteers while performing various vocalizations and breathing activities [49] . the results showed that the rate of particle emission during normal human speech is correlated positively with the loudness (amplitude) of vocalization, ranging from approximately 1 to 50 particles per second (0.06 to 3 particles/cm 3 ) for low to high wavelengths, regardless of the language spoken (english, spanish, mandarin, or arabic). the results also indicated that the droplets could be emitted not only by speech but also by singing a song, cheering loudly for sports games, and so on. rahmani et al. reviewed the methods for the sampling and detection of coronaviruses, especially sars-cov-2. most of the samplers used, such as polytetrafluoroethylene filters, gelatin filters, and cyclones, showed a suitable performance to trap sars-cov and middle east respiratory syndrome (mers)-cov, followed by polymerase chain reaction (pcr) analysis [50] . some studies reported detecting sars-cov-2 from patients' rooms in hospitals, although it seems difficult to discriminate whether these were airborne or transmitted through respiratory droplets because sampling conditions (i.e., the patient's distance from the sampler, patient's activities, coughing and sneezing during sampling time, etc.) can affect the results. liu et al. investigated airborne sars-cov-2 by measuring viral rna in aerosols in two different hospitals in wuhan during the covid-19 outbreak in february and march 2020 [16] . the unique point in this study was to investigate the size distributions of airborne sars-cov-2 droplets. aerosol samples were collected using a miniature cascade impactor (sioutas impactor, skc) that could separate aerosols into five ranges (>2.5, 1.0-2.5, 0.50-1.0, and 0.25-0.50 μm on 25-mm filter substrates and 0-0.25 μm on 37-mm filters) and determined through the quantification of their genetic material (rna). sars-cov-2 aerosols were found to include mainly two size ranges, one in the submicrometer region (d p 0.25-1.0 μm) and the other in the supermicrometer region (d p > 2.5 μm), in isolation wards of ventilated patient rooms and medical staff areas. the authors hypothesized that the source of the submicrometer virus-laden aerosols may be resuspension from the surface of the protective apparel worn by medical staff while they are removing the equipment. the resuspension occurs largely because of the particle surface properties, human activities, environmental conditions, and so on. resuspension occurs mainly as a result of human activities, especially walking in indoor spaces. qian et al. reviewed particle resuspension owing to human walking in indoor environments [51] . from the results presented, resuspension is an important source compared with other indoor sources, such as cooking and stoves, and resuspension increases with particle size in the range of 0.7-10 μm. many researchers proposed resuspension terms, such as a resuspension rate coefficient (h −1 ), a resuspension fraction (-), an emission rate (mg/h), a resuspension factor (m −3 ), and a resuspension emission factor (mg/mg). rosati et al. experimentally investigated the characterization of the size distribution of resuspended particle matter from the carpet during walking events [52] . the resuspension emission factor by particle size was defined as the resuspension emission rate by particle load on the carpet by particle size. the emission factors were approximately 10 −4 to 10 −1 of particle diameter 1 to 10 μm. therefore, a small portion of particles deposited on the floor can be resuspended in indoor air. the resuspension is progressively more likely to occur by larger particles than by smaller ones. although there are a few studies on bioaerosols, and especially virus particles, bioaerosol resuspension is clearly not the same as infectious virus resuspension. it is important for airborne transmission to understand the droplet diameter emitted from patients. previous studies indicated that an aerosol droplet size distribution was ranged from 700 nm to 1 mm, generated not only by speaking and coughing but also through breathing. from the field measurements in the hospital, sars-cov-2 aerosols could be detected to include mainly two size ranges, 0.25-1.0 μm and larger than 2.5 μm, in isolation wards of ventilated patient rooms and medical staff areas. moreover, the resuspension is progressively more likely to occur by larger particles than by smaller ones, but bioaerosol resuspension cannot be clearly the same as infectious virus resuspension. the number of secondary infections of covid-19 varies widely, and most outbreaks of many secondary infections occur under common indoor environmental factors [53, 54] . given the lack of data to define environmental control measures against covid-19 based on quantitative analysis, it is important to understand the conditions common to outbreak cases and to take measures to minimize the indoor environmental factors that promote infection. in this section, we summarize the cases of outbreaks in which indoor environmental factors, including human behavior, are believed to have facilitated infection, as well as the environmental survey of sars-cov-2. on january 24, 2020, a covid-19 outbreak occurred in a restaurant in guangzhou, china, infecting ten people in three families. the minimum length of time during which the index-infected person was present with the secondary infected person was 46 min, and the possibility of contact infection was considered to be low based on in-store camera recordings. fifty percent (five of ten) of those at the same table as the infected person were found to be infected within the next 7 days. at the adjacent leeward table, 75% (three of four) were infected. two of the seven people at the windward table were infected. other diners who were located away from the airflow around the infected tables, as well as the staff, were not infected. lu et al. concluded that the airflow from the air-conditioners promoted droplet infection and recommended that distances between people are maintained and ventilation improved [55] . in this case, li et al. conducted a detailed investigation of the indoor environment by measuring the ventilation rate and conducting a numerical thermo-fluid analysis. as a result, they found that only the ventilation fan in the restroom was in operation. the ventilation fan on the wall of the restaurant was sealed and not in operation, and the ventilation rate ranged from 0.56 to 0.77 air changes per hour (ach) and 0.75 to 1.04 l/s per person. they suggested that poor ventilation may have been the primary cause of the spread of the infection by aerosol transmission [56] . ninety-four people tested positive for the virus on one floor of the call center, where 216 employees worked. [49, 58] . meat-processing plants have emerged as hotspots for sars-cov-2 around the world. an outbreak of sars-cov-2 at germany's largest meat-processing facility resulted in more than 1400 confirmed infections during tests conducted a month after the initial outbreak. guenther et al. conducted a causal investigation into the may 2020 outbreak. it was suggested that the infected person, who was the first example of a supplemental case, transmitted the virus to a colleague more than 8 m away over 3 consecutive working days. the low temperature in an environment with the low intake of outside air and air circulation through the airconditioning system in the hall, combined with the high physical workload of workers with heavy breathing, have been suggested as factors that enabled transmission over distances greater than 8 m by virus-containing aerosol particles. they stated that under these circumstances, distances of 1.5-3 m are not sufficient to prevent infection and that the wearing of masks, improved ventilation and airflow, and the installation of filtering devices are necessary to reduce the risk of infection [59] . these outbreaks strongly suggest that in a confined, crowded, and poorly ventilated environment where conversations, loud vocalizations, and heavy breathing take place, sars-cov-2 can spread through the air at a distance of 2 m or more and may result in a large number of secondary infections. the who defines droplets and droplet nuclei as respiratory aerosols more than 5 μm in diameter and the residue of dried respiratory aerosols up to 5 μm in diameter, produced by the evaporation of droplets coughed or sneezed into the atmosphere or aerosolized infective material, respectively [60] . based on field measurement results at wuhan hospitals during the covid-19 outbreak, liu et al. reported that the peak concentration of sars-cov-2 aerosols appears in two distinct size ranges: at the submicron scale with dominant aerodynamic diameters between 0.25 and 1.0 μm; and at the supermicron scale with diameters greater than 2.5 μm [16] . the main sources of sars-cov-2 aerosols are coughs and sneezes by infected persons. the capacity for droplets to travel long distances in airflow is determined largely by their size. most communicable respiratory infections are transmitted via large droplets over short distances or contact with contaminated surfaces. large droplets (diameter, >60 μm) tend to settle quickly from the air, and thus the risk of pathogen transmission is limited to individuals in close proximity to the saliva droplet source. small droplets (diameter, ≤60 μm) may be involved in short-range transmission (i.e., when the distance between individuals is less than 1 m) and are likely to evaporate into droplet nuclei (diameter, <10 μm) in favorable environments, making them candidates for long-distance aerosol transmission [61] . the terminal settling velocity of a particle increases rapidly with its size, as it is proportional to the square of particle diameter. the terminal settling velocity of a particle up to 10 μm in size is less than 0.3 cm/s (0.003 m/s) [62] , and because the particle diameter decreases as a result of the evaporation of the droplets during settling, droplets will remain suspended for a longer time indoors based on the relative indoor humidity [63] . therefore, aerosols up to 10 μm in diameter are carried easily over long distances (final inlet air) by the indoor airflow generated by air conditioning or ventilation equipment. field measurements show that the largest and average velocities in occupant space are 0.4 and 0.1 m/s, respectively [64, 65] . it is possible to control aerosols containing a virus with a proper indoor airflow plan. regarding the indoor airflow plan, there are different types of ventilation systems such as piston flow ventilation and mixing-type ventilation. when the infected individual's position can be fixed, piston flow ventilation or push-pull ventilation is applicable. however, as the positions of infected people cannot be pinpointed in general environments, mixing-type ventilation is more effective because it dilutes the infectious aerosols to decrease their concentration in the air. to describe the relationship between the air exchange rate and the probability of infection, the wells-riley model was used: this model is based on the assumption that the air in the room is well mixed, leading to a uniform concentration of bioaerosols throughout the space. to estimate the probability of infection in a room, the quantum generation rate must be determined. a quantum represents the minimum dose that can cause infection in the host [66] , and the quantum generation rate is the number of quanta produced per hour per infectious individual. table 3 provides the values of quantum generation rates reported for different infectious aerosols [77] . dai and zhao estimated the quantum generation rate of covid-19 by fitting the relationships between known rates and r0 (the basic reproduction number) of the infectious agents listed in table 3 [78] . the results indicate that the quantum generation rate for covid-19 ranges from 14 to 48 h −1 . figure 1 shows the schematics of the two typical heating, ventilation, and air-conditioning (hvac) systems used in japanese office buildings. system a is the centralized hvac system (cs) and system b is the individual hvac system (is). the ventilation rates of the cs are generally approximately 2 ach for outdoor air and 4 ach for return air. moreover, the cs normally uses an air filter with a collection efficiency approximately equivalent to a minimum efficiency reporting value (merv) of 12, indicating a removal efficiency of 90% of droplets from human respiration activities, most of which are less than 5-10 μm in diameter [79] [80] [81] . in this case, the equivalent change rate is approximately 5.6 ach (=2 ach + 4 ach × 90%). figure 2 shows the probability of infection plotted against the equivalent air change rate (hourly rate of room ventilation with clean air) based on eq. (1). the prediction conditions are shown as i = 1 person, p = 0.48 m 3 /(hour⋅person), t = 8 h, room floor area = 500 m 2 , ceiling height = 2.6 m. the higher the equivalent air change rate (the room ventilation rate with clean air/ room volume), the lower the probability of infection. furthermore, the cs shows a lower probability of infections than the is because of the larger amount of clean air. the use of highly efficient particle filtration in centralized hvac systems reduces the airborne load of infectious particles [82, 83] . the collection efficiency of air filters for suspended particles by particle size is given in table 4 . in japanese office buildings, medium-efficiency air filters (merv 11-13 in table 4 ) are typically used, whereas high-efficiency particulate air filters (hepa: 99.97% or higher particle collection efficiency for particles sized 0.3 μm at the rated airflow volume) are used for rooms such as hospital operating rooms that demand high air cleanliness. it is known that ultraviolet germicidal irradiation (uvgi) systems are used for air and surface disinfection. investigations of the bactericidal effect of sunlight in the late 19th century planted the seed of air disinfection by uv radiation. the first to nurture this seed was wells, who both discovered the spread of airborne infection by droplet nuclei and demonstrated the ability of uvgi to prevent such a spread. with modern concerns regarding multi-and extensive drug-resistant tuberculosis, bioterrorism, influenza pandemics, and severe acute respiratory syndrome, interest in uvgi continues to grow [84] . the centers for disease control and prevention (cdc) [85] and the american society of heating, refrigerating and air-conditioning engineers (ashrae) [86] recommend the application of uvgi to fight airborne diseases. microbes are uniquely vulnerable to light of wavelengths at or near 253.7 nm because the maximum absorption wavelength of a deoxyribonucleic acid (dna) molecule is 260 nm. the chemical compound pyrimidine in a dna base strongly absorbs uv light. after irradiation, the dna sequence where two pyrimidines link can form pyrimidine dimers. these dimers can change the dna double-helix structure and interfere with dna duplication, as well as lead to the destruction of the replication ability of cells and thus render the cells noninfectious [87, 88] . regarding the sterilization or inactive performance of uvc, the population st of microbes exposed to any biocidal factor is described by the characteristic logarithmic decay equation: where k = the standard decay-rate constant (cm 2 /mw⋅s), i = the intensity of uvgi (mw/cm 2 ), and t = the exposure time (s). the standard decay-rate constant of the influenza a virus has been reported as 0.001187 cm 2 /μw⋅s [89] [90] [91] . recently, upper-room uvgi and in-duct uvgi have been the two primary applications of uvgi air disinfection. in-duct uvgi is designed to disinfect air as it passes through the hvac system before it is recirculated or exhausted, and it has been also suggested to reduce nonspecific building-related illnesses [92, 93] . in summary, since small droplets (diameter, ≤60 μm) are likely to evaporate into droplet nuclei (diameter, <10 μm) in favorable environments, and the terminal settling velocity of a particle up to 10 μm in size is lower than 0.3 cm/s (0.003 m/s), it is possible to control aerosols containing a virus with a proper indoor airflow plan and sufficient ventilation rates. moreover, filtration is effective in the reduction of aerosol concentration. ventilation and filtration can contribute to the reduction of indoor infection probability. on the other hand, it is well-known that uvgi is an effective measure against microbes. the who, the cdc, and the ashrae recommend the application of uvgi to fight airborne diseases. the application in japan will be expected. it is important to decide the adequate measures against a new infectious agent by analyzing actual infection cases and to execute them as soon as possible. as of february 26, 2020, the ministry of health, labour and welfare (mhlw) covid-19 response team examined a total of 110 cases among 11 clusters and investigated who acquired infection from whom. all clusters were associated with close contact in indoor environments, including fitness gyms, a restaurant boat on a river, a club with live music, healthcare facilities, and a snow festival where there were eating spaces in tents with minimal ventilation rate [54] . therefore, the mhlw published a document titled "prevention of the covid-19 clusters" abbreviation: sars-cov severe acute respiratory syndrome coronavirus fig. 1 traditional japanese office building hvac systems: a a centralized hvac system; and b a centralized ventilation system with an individual air-conditioning system on march 1, 2020 [94] , showing the need for adequate ventilation in buildings because a ventilation standard for infection control has not been established in general buildings in japan and the characteristics of indoor spaces where the clusters occurred might include poor ventilation and crowding. therefore, the "3 cs," namely, "closed spaces with poor ventilation," "crowded spaces with many people," and "close contact," such as from intimate conversations, loud cheering, singing, or exercise within a short distance from other individuals, were proposed as important factors that result in a covid-19 cluster [95, 96] . the mhlw published "ventilation to improve "closed spaces with poor ventilation" in commercial facilities" on march 30, 2020, based on the analysis by the mhlw covid-19 response team concerning the actual ventilation state against the standards in the law for maintenance of sanitation in buildings, and recommended adequate ventilation rates in accordance with the standards of the law and the increase in the ventilation rates by adjusting the ventilation systems and opening the windows [97] . furthermore, this recommendation was added in the "measures to prevent the large-scale spread of covid-19 in workplaces" on march 31, 2020 [98] . on april 2, 2020, a document titled "maintenance of air-conditioning and ventilation systems in specific buildings" was published and adequate ventilation was requested again [99] . moreover, on april 3, 2020, the managers of commercial facilities were informed of the "methods of ventilation for improving "closed spaces with poor ventilation," and the guidelines of ventilation measures in both specific buildings and other buildings were provided [100] . in these documents, the required ventilation rate of approximately 30 m 3 /h per person was recommended [94] . when this ventilation rate was determined, the results of studies on tuberculosis [101] [102] [103] and measles [104] , which are infectious diseases known to be spread by airborne transmission, were considered [97] . the ventilation rates needed are indicated not per room but per person and if adequate ventilation is not secured in the room, the number of persons should be limited to avoid overcrowding, which may be a factor in clusters of infected individuals. the ventilation rates needed to prevent sars-cov-2 transmission have not been reported; thus, opening windows has also been recommended. however, it was difficult to recommend the specific methods because the conditions of the rooms (e.g., the number of windows, and the width and direction of the windows) are different. in the expert meeting on novel coronavirus disease control on may 4, a "new lifestyle" for the long-term fight against covid-19 was suggested, comprising three basic measures: social distancing, wearing face masks, and washing hands [105] . since the middle of april, researchers on building hygiene have collected recent scientific findings and discussed the ventilation measures towards summer [62] . during the rainy season and summer, opening the windows may create a poor indoor environment with insufficient air-conditioner cooling and dehumidification, resulting in increased risks of heatstroke, insomnia, and allergies owing to mold and mites. after discussions, the researchers produced a document titled "indoor environmental measures in summer against covid-19: the suggestion from the researchers on building hygiene" on may 20, 2020 and provided it to the expert meeting members on novel coronavirus disease control and headquarters of the mhlw [62] . from the present evidence, it is difficult to indicate specific standard values, such as ventilation rates. thus, ventilation and air-conditioning measures recommended under some conditions were shown, see table 5 . on may 26, 2020, the ministry of the environment and the mhlw served local governments a notice of "action for prevention of heatstroke in 2020," which requested that they take action to prevent heatstroke under the spread of covid-19 [106] . the headquarters of covid-19 prevention in the mhlw considered expert opinions, scientific literature, international standards, and laws and regulations in japan in the search for effective methods, and published a document titled "ventilation for improving "closed spaces with poor ventilation" with prevention of heatstroke" on june 17, 2020 [107] . in addition, it published the document titled "methods of ventilation for improving "closed spaces with poor ventilation" with prevention of heatstroke for owners using air-conditioning system without ventilation in commercial facilities" on june 24, 2020 [108] . the document shows points to remember while opening windows for ventilation, in using air cleaners, and so on. furthermore, in the "q&a for the public," information was provided on home air conditioners and ventilation during the hottest season. it was indicated that air conditioners should be used to prevent heatstroke, but that general air conditioners do not have a ventilatory function; thus, the whole air of a room should be ventilated twice or more per hour and the use of a 24-h mechanical ventilation system is effective. the society of heating, air-conditioning and sanitary engineering of japan started a special committee against covid-19 and published a document titled "is covid-19 spread by air-conditioning or ventilation?-experts' opinions" on june 16, 2020. in the document, the relationship between airconditioning or ventilation and the risk of infection, the possibility of the spread of covid-19 through air conditioners, the infection risk by inadequate ventilation and filters, and the prevention of heatstroke with infection control are discussed and the following remarks were obtained [109] : in japanese buildings, which are designed and managed in accordance with the law for maintenance of sanitation in buildings, considering the performance of ventilation and filters, the risk of the spread of covid-19 in a room through airconditioning systems is believed to be very low. however, in the case of buildings without the general-performance filters in air conditioners, commercial package-type air conditioners, or a fan coil unit (i.e., fcu), another ventilation method should be added. securing ventilation rates per person may prevent droplet infection through airflow. in hospitals and clinics where individuals can wear face masks all the time, the droplet from infected individuals can be prevented. when adequate ventilation rates are secured, it is necessary to use air conditioners to prevent heatstroke in summer. in hospitals and clinics where individuals are believed to be infected, the airflow from air conditioners should not be directed to any person. considering the fact that in a poorly ventilated room, an individual was infected when spending time with an infected person for less than an hour, not intermittent but continuous ventilation is desirable. in cases where natural ventilation is available, it is desirable to secure ventilation rates by opening the windows and so on if an adequate room temperature is secured. however, in the buildings designed and managed in accordance with the law for maintenance of sanitation in buildings, natural ventilation may cause a bad influence on air balance and may be beyond the standards of the law. thus, it is desirable to consult experts. an industry-classified guideline for the prevention of the spread of covid-19 was made based on the proposal by the experts' meeting on may 4, 2020. as of writing this article, approximately 100 guidelines were available on the website of the cabinet secretariat of the japanese government [110] . the items concerning building environments are cleaning, disinfection, ventilation, room density, etc., and guidelines are shown according to the characteristics of each industry. regarding ventilation, the specific rates or times are not indicated because of the various conditions of ventilation systems or windows. since pneumonia caused by sars-cov-2 was confirmed in december 2019, the mechanism of infection in an indoor environment has not been clarified. it is important to understand infection mechanism through various studies such as investigation of infection cluster spaces and experiments on the relationship between such environmental factors as temperature and humidity and the infectiousness of the virus. based on these studies, further adequate strategy for the infection risk management in an indoor environment is required. in environmental stabilities of sars-cov-2, this virus can persist on the fomite surfaces, such as plastic or metal, between 3 and 7 days in an indoor environment such as a building. sars-cov-2 in aerosols is also stable for at least several hours if the virus is produced within small-particle aerosols. sars-cov-2 is mainly transmitted between humans through close contact and respiratory droplets, including fomite transmission. however, close-contact aerosol transmission through smaller aerosolized particles is likely to be combined with respiratory droplet and contact transmission in a confined, crowded, and poorly ventilated indoor environment, as some cluster cases have suggested. therefore, adequate preventive measures to control indoor environmental quality are required. the risk of closecontact aerosol transmission can be reduced sufficiently by taking measures such as ensuring adequate ventilation in accordance with the number of individuals in a room and wearing masks to reduce the aerosols emitted into the indoor air. filtration and uvgi systems have also been developed. however, indoor environmental quality control measures such as ventilation alone cannot prevent exposure from an infected person at a short distance or through environmental surfaces; thus, adequate preventive measures against respiratory droplet and contact transmission, such as disinfection of fomites, hand hygiene, uncrowded spacing, and the wearing of masks, must be taken at appropriate times and places. in japan, epidemiological links to confirmed covid-19 cases showed that clusters of cases occurred in fitness gyms, a restaurant boat on a river, and a club with live music in february 2020. based on these findings, the expert panel for covid-19 focused on the "3 cs," namely, "closed spaces with poor ventilation," "crowded spaces with many people," and "close contact." in addition, the mhlw has recommended adequate ventilation in all closed spaces in accordance with the existing standard of the law for maintenance of sanitation in buildings. although specific ventilation standards for the control of sars-cov-2 transmission cannot be derived from the existing scientific evidence, covid-19 has caused serious global damage to public health, community, and the social economy. the united nations conference on environment and development adopted the rio declaration on environment and development in 1992 [111] . table 5 recommendations on the ventilation and air-conditioning measures to prevent covid-19 infection every indoor space • enough ventilation is necessary to prevent covid-19 infection. • opening windows is effective for ventilation and opening them wide and for a longer time is desirable. • in summer, air conditioners are necessary to prevent health risks such as heatstroke, etc. • general air conditioners do not function as ventilators, so mechanical ventilation and opening windows are necessary. • when opening windows, it is necessary to prevent harmful insects and animals from coming in. spaces in which air conditioning and ventilation systems are installed • it is necessary to check the systems and to confirm enough ventilation rates are secured. • it is necessary to limit the number of persons in a room or shorten the time they are inside secure enough ventilation rates per person. • it is necessary to investigate what the building is used for, how often it is used, what kind of airconditioning and ventilation system is used in the building when taking such measures as the better control of air-conditioning and ventilation systems, or the use of air cleaners, and the use of humidifiers in winter. principle 15 states: "in order to protect the environment, the precautionary approach shall be widely applied by states according to their capabilities. where there are threats of serious or irreversible damage, lack of full scientific certainty shall not be used as a reason for postponing cost-effective measures to prevent environmental degradation." this principle was also applied to the threats to human health in the public health domain [112] [113] [114] . based on principle 15, the implementation of practical precautionary measures that took into account technical feasibility, adequate control measures and strategies, and social, economic, and cultural conditions is essential to protect the public from sars-cov-2 transmission. however, the implementation of an approach based on the precautionary principle should start with a scientific evaluation, as complete as possible, and where possible, identifying at each stage the degree of scientific uncertainty [115] . therefore, further research and evaluation are required to evaluate the effect and role of indoor environmental quality control, especially ventilation. in this paper, we summarize the effect and role of environmental factors in buildings, spatial dynamics, building operational factors, and a strategy to prevent sars-cov-2 transmission in a building environment. most transmission has occurred in indoor environments in a closed space, especially inside a building. although ventilation based on the existing regulation has been recommended by the mhlw as one of the initial political actions to prevent the spread of this novel infectious disease, the specific standard has not been recommended. to protect public health from sars-cov-2 transmission, further research on infection dynamics and the mode of infection in sars-cov-2 transmission in closed spaces, investigations on indoor environments in the infected cluster spaces, experiments on the 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ventilation to improve "closed spaces with poor ventilation" in commercial facilities measures to prevent the large-scale spread of covid-19 in workplaces maintenance of air-conditioning and ventilation systems in specific buildings methods of ventilation for improving "closed spaces with poor ventilation hospital ventilation and risk for tuberculous infection in canadian health care workers. canadian collaborative group in nosocomial transmission of tb environmental factors relating to a mass outbreak of tuberculosis in a junior high school in association with tuberculous group infection in cram school: effect of ventilation on the infection risk measles outbreak in a pediatric practice: airborne transmission in an office setting emncdc. analysis of the response to the novel coronavirus disease (covid-19) and recommendations tokyo: ministry of health, labour and welfare ventilation for improving "closed spaces with poor ventilation" with prevention of heatstroke methods of ventilation for improving "closed spaces with poor ventilation" with prevention of heatstroke for owners using air-conditioning system without ventilation in commercial facilities tokyo: society of heating, air-conditioning and sanitary engineers of japan covid-19 information and resources. tokyo: cabinet secretariat united nations the precautionary principle also applies to public health actions the precautionary principle, public health, and public health nursing the precautionary principle: protecting public health, the environment and the future of our children. copenhagen: world health organization regional office for europe communication from the commission on the precautionary principle. brussels: commission of the european communities not applicable. the six authors are justifiably credited with authorship, according to the authorship criteria. in detail: ka-conception, design, acquisition, and interpretation of data, drafting of the manuscript, critical revision of the manuscript, final approval is given; uy and mh-conception, acquisition, and interpretation of data, drafting of the manuscript, critical revision of the manuscript, final approval given; nk, hk, and mo-acquisition and interpretation of data, drafting of the manuscript, critical revision of the manuscript, final approval given. no funder supported this work. ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-353524-3w970ycx authors: dömling, alexander; gao, li title: chemistry and biology of sars-cov-2 date: 2020-05-22 journal: chem doi: 10.1016/j.chempr.2020.04.023 sha: doc_id: 353524 cord_uid: 3w970ycx sars-cov-2 (previously 2019-ncov or wuhan coronavirus) caused an unprecedented fast-spreading worldwide pandemic. although currently with a rather low mortality rate, the virus spread rapidly over the world using the modern world’s traffic highways. the coronavirus (cov) family members were responsible for several deadly outbreaks and epidemics during the last decade. not only governments but also the scientific community reacted promptly to the outbreak, and information is shared quickly. for example, the genetic fingerprint was shared, and the 3d structure of key proteins was rapidly solved, which can be used for the discovery of potential treatments. an overview is given on the current knowledge of the spread, disease course, and molecular biology of sars-cov-2. we discuss potential treatment developments in the context of recent outbreaks, drug repurposing, and development timelines. sars-cov-2 (previously 2019-ncov or wuhan coronavirus) caused an unprecedented fast-spreading worldwide pandemic. although currently with a rather low mortality rate, the virus spread rapidly over the world using the modern world's traffic highways. the coronavirus (cov) family members were responsible for several deadly outbreaks and epidemics during the last decade. not only governments but also the scientific community reacted promptly to the outbreak, and information is shared quickly. for example, the genetic fingerprint was shared, and the 3d structure of key proteins was rapidly solved, which can be used for the discovery of potential treatments. an overview is given on the current knowledge of the spread, disease course, and molecular biology of sars-cov-2. we discuss potential treatment developments in the context of recent outbreaks, drug repurposing, and development timelines. in december 2019, an outbreak of pneumonia of an unknown cause was reported in wuhan, in hubei province, china. it was speculated that the first patient caught the infection from a seafood market that also traded wild animals. the causing agent was quickly identified as a novel coronavirus (cov) . the cov responsible for the outbreak is now called sars-cov-2. the respiratory illness caused by sars-cov-2 is called covid-19. the symptoms of the sars-cov-2 infection range from asymptomatic to mild to severe to death. 1 it soon became clear that person-to-person transmission was also occurring, as was the case with the previous human cov. in an unprecedented documented speed, the sars-cov-2 travels around the globe, and as of april 27 th led to >3.0 million infections and 200,000 fatalities. based on the previous experience with the sars-cov outbreak at the beginning of this century, very stringent measures were taken by the chinese government, and several multimillioninhabitant cities were isolated and put under quarantine in order to slow the pandemic spread. different hosts of the sars-cov-2 are proposed, including snails, bats, and pangolins. 2 covs are a large family of zoonotic viruses and their outbreaks are common to humans, although major outbreaks have been experienced in animals, especially in cattle. under the electron microscope, they exhibit formations that are reminiscent of the solar corona. the common cold is often caused by human covs. they are single-stranded enveloped positive rna viruses and stand out because of their rather large genome. as with viruses in general, the structure is rather simple. sars-cov-2 is generally less pathogenic than sars-cov, much less pathogenic than the middle east respiratory syndrome mers-cov, but more pathogenic than practically harmless hcov-oc43, hcov-hku1, hcov-229e, and hcov-nl63. the reported case-fatality rate of covid-19 is %3% and is thus rather low as compared with sars (30% , table 1 ). however, the transmission rate (tr) (number of newly infected people per infected person) of 2.5 to 3 is high and accounts for the danger of the current pandemic. for comparison, the tr of the yearly common cold is less than 1.4. challenges and opportunities: the sars-cov-2 pandemic has changed our world in just half a year: a large number of people have died from the virusinduced pneumonia covid-19, and the global economy is at an unprecedented low with unknown near-and long-term consequences. the coronavirus pandemic needs a two-pronged approach: a short-term solution to find a drug to treat the massive number of seriously ill patients, and long-term development of preventive and curative drugs for future corona outbreaks. de novo discovery of any therapeutic takes years to move from idea/ q5 pre-clinic to market release and is not a short-term solution for the current sars-cov-2 pandemic. drug repurposing is perhaps the only short-term solution, and vaccination is a middle-term solution. advice guidelines for diagnosis and treatment of sars-cov-2 infected pneumonia have been shared rapidly. 3 what are the issues and chances for a rapid approval of a new medicine to treat covid-19? in principle, there are several potential strategies to pharmacologically fight covid-19: vaccines, monoclonal antibodies, oligonucleotide-based therapies, peptides, interferon therapies, small-molecule drugs, or natural medicines (e.g., traditional chinese medicine [tcm]). the timelines for de novo development of a small-molecule drug are historically >6-7 years, and in the best case less than 2 years. vaccines can be developed much faster, but rapid development in the range of 1-2 years is very challenging. antibodies to support the body's immune system are also a strategy to combat viral diseases. again, the typical development timelines are several years. therefore, is there a hope for a q11 rapid drug to come to the market? a strategy that is promising in the current situation is drug repurposing. drug repurposing aims to discover novel indication areas for already approved drugs. 4 the overwhelming advantage of drug repurposing is the potential for much faster approval because of the already extensive knowledge of the drug's behavior in humans. an expert opinion on the potential for repurposing existing antiviral agents to treat covid-19, some of which are already clinically evaluated, was recently given. 5 here, we discuss molecular targets of the sars-cov-2, some of the known small molecules, and the potential for repurposing existing drugs. despite the rather large size of the rna virus genome of $30,000 bases, the sars-cov-2 genome encodes for few proteins (figure 1 ): the structural spike (s) protein, nucleocapsid (n) protein, membrane (m) protein, and the envelope (e) protein, which are needed to produce a structurally complete viral particle. additionally, the sars-cov-2 genome encodes 16-17 non-structural proteins (ns1 to ns17), such as 3-chymotrypsin-like protease (3clpro q13 ), papain-like protease (plpro), helicase, and rna-dependent rna polymerase (rdrp). both the virus-encoded proteases q14 are involved in the processing of the two viral polyproteins in a coordinated manner, and thus comprise important drug targets. the structure, function, and inhibition of cov 3clpro (also called mpro) has been recently comprehensively reviewed. 6 the 3clpro is a cysteine protease that cleaves and processes the viral polyproteins. sars-cov-2 and sars-cov share 96% sequence identity in their 3clpro. on the basis of the rapidly published virus sequence data, a homology model was created. moreover, an x-ray structure of the c3lpro covalently bound to a peptidomimetic acrylester (1) is now available ( figure 2 , pdb id 6lu7). 8 because of the high sequence similarity of different cov 3clpros, a lot of previously described inhibitors can be considered to be of great use in the current sars-cov-2. a majority of inhibitors of the 3clpro are covalent in nature, binding to the activesite cysteine (scheme 1). different electrophilic warheads are known, including a-halocarbonyl, acrylamides, sulfonyl chlorides, aldehydes (2), 9 isatines (3), 10 or a-ketoheteraromates (4). 6 many of the molecules are rather large and are based on extensive amide chemistry, mimicking part of the peptide substrate of the protease. moreover, their selectivity toward other potential targets in the human body has not been established. interestingly, some compounds binding to the active site of the 3clpro-although using a noncovalent mechanism-have been established . a high-throughput screening (hts) identified 3,3-dihydropyrazolidine (5), which displayed 1,3,5-triaryl substitution patterns, as sars-cov 3clpro inhibitors. 11 nitroanilides (6), derived from the drug niclosamide were also found to inhibit 3clpro. 12 a-aminoacylamides were identified by an hts, and a strong stereochemical effect was noted. the simple one-pot accessible scaffold by an ugi-four component condensation was the key to rapidly generate structure activity relationship (sar) for the putative p2-p1 and p1 subgroups. an optimized version ml188 (7) was designated as the probe status ( figure 3) . a p3 truncated version of 8 allowing for significant molecular weight (mw) reduction without diminishing potency was developed as a second probe ml300 (9) with potent enzyme inhibition and cellular activity. these compounds comprise rare examples of a noncovalent sars-cov 3clpro inhibitor of moderate mw with good enzyme and antiviral inhibitory activity. however, these molecules suffer from extensive metabolism and rapid clearance. nonetheless, they are a promising starting point for further drug development. even if such compounds cannot be rapidly developed to cope with the current situation, their development is highly warranted to be prepared for likely future cov outbreaks. it is noteworthy that different computational approaches, including machine learning, have been published to propose approved drugs potentially binding to 3clpro (drug repurposing). 13, 14 one such approach virtually screened commercial medicines in the drugbank database for binding into the active site of mpro. 15 ten different commercial medicines were proposed that might form hydrogen bonds to key residues within the binding pocket of sars-cov-2 3clpro, which might have higher mutation tolerance than lopinavir or ritonavir. flavonoids (10) (11) (12) (13) (14) are plant-derived natural products with diverse reported biological activities, and they have been shown to be also able to inhibit the 3clpro (figure 4) . 16, 17 the broad-spectrum and established use of plant-based medicines to combat infectious diseases in tcm is the basis of several currently ongoing clinical trials in china. one of the largest among them assesses shuanghuanglian, a chinese herbal medicine that contains extracts from the dried fruit lianqiao (forsythiae fructus), which is purported to have been used for treating infections for more than 2,000 years. several approved hiv protease inhibitors (15, 16, and 18) were previously repurposed for the treatment of sars (scheme 2). [18] [19] [20] they were hypothesized to inhibit the sars-cov 3clpro: hiv protease is a asp protease and differs considerably from the cys protease 3clpro, but it also shares some common elements, such as a tetrahedral transition state and receptor pockets to recognize the amino acid side chains of the substrates. given that sars-cov-2 and sars-cov share very high identical sequence in their 3clpro, these hiv protease inhibitors are currently again repurposed for the treatment of covid-19 (chinese clinical trial registry: chictr2000029539). [21] [22] [23] plpro the coronaviral plpro is another attractive antiviral drug target because it is essential for cov replication. the structure, function, and inhibition of the sars-cov plpro has been extensively reviewed. 24 although the primary function of plpro and 3clpro is to process the viral polyprotein in a coordinated manner, plpro has the additional function of stripping ubiquitin and isg15 from host-cell proteins to help cov to evade the host innate immune responses. therefore, it was recently argued that targeting plpro with antiviral drugs might have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that might lead to cell death in surrounding, uninfected cells. 24 different compounds forming a covalent bond to the active-site cys112 have been described, including epoxyketones, a-halo-ketones, alkynes, aldehydes, trifluoromethyl ketones, a,b-unsaturated ketone, activated esters, or vinyl sulfones. disulfiram (19) , an approved drug for the treatment of chronic alcohol dependence, has a great potential for drug repurposing because it has been shown to inhibit plpro of mers-cov and sars-cov. 25 the antimetabolites 6-mercaptopurine (20) and 6-thioguanine (21) are additional drugs inhibiting plpro. potent naphthyl methylamine q17 hits (22) which have been structurally characterized and have been subsequently optimized toward better metabolic stability were identified from an hts campaign, 27 . (23) 28 the naphthyl methylamines work through a noncovalent mechanism and show a rather drug-like appearance ( figure 5 ). the envelope-anchored s protein mediates cov entry into host cells by first binding to a host receptor and then fusing viral and host membranes. the sars-cov-2 s protein was solved by cryoelectron microscopy and just released; 29 knowing the å -resolution structure of the sars-cov-2 spike will allow for additional protein engineering efforts that could improve antigenicity and protein expression for vaccine development. moreover, the å -resolution detail will enable the design and screening of small molecules with fusion-inhibiting potential. it is the affinity between the viral receptor-binding domain (rbd) and the host receptor in the initial viral attachment step that primarily determines which host is susceptible to sars-cov infection. the sars-cov-2 entry through receptor binding was elucidated independently by several groups. 30, 31 on the basis of the rich knowledge about sars-cov and the sequence homology, it was suggested that sars-cov-2 uses angiotensin-converting enzyme 2 (ace2) as its receptor ( figure 6 ). it uses the sars-cov receptor ace2 and the cellular protease tmprss2 for entry into target cells. 30, 31 the interplay of the ace receptor in cardiovascular diseases (with the well-known drug class of ace inhibitors) and as the docking point for sars-cov-2 cellular infection is a current point of intense debate and research. 32 it was found that several critical residues in sars-cov-2 rbm (particularly gln493) provide favorable interactions with human ace2, consistent with sars-cov-2's capacity for human cell infection. on the basis of the extensive modeling of the virus-human receptor interactions, it was also predicted that a single n501t mutation might significantly enhance the binding affinity between sars-cov-2 rbd and human ace2 and thus potentially lead to much more virulent bodies. 30, 31 the emergency and evolution of novel mutations at the 501 position in sars-cov-2-infected and covid-19 patients have to be closely monitored. 33 mutations in the s protein are the ones causing the zoonosis, and as a result, not all of them will lead to their binding to ace2. therefore, drugs targeting the s protein-ace2 interaction might not apply to some of the future covs. on the other hand, the spike-shaped protein on the surface of the viruses causing sars, mers, and covid-19 provides a tantalizing target for antibodies or other compounds, which could prevent covs from invading human cells. 35 the virus' s protein seems to emerge as the consensus target antigen. the rdrp enzyme allows the viral genome to be transcribed into new rna copies by using the host cell's machinery. rdrp inhibitors are emerging as a new strategy to fight viral infections. 35 the chemistry and biology of rdrp have been extensively reviewed. 36 rna polymerase inhibitors are promising agents to fight covid-19. 5 rdrp proteins of sars-cov-2 and sars-cov share 96% sequence identity. favipiravir (24) is an approved rna polymerase inhibitor for the treatment of the influenza pandemic. it has been shown not only to be active in influenza but also active against other rna viruses (figure 7) . 34 favipiravir is a prodrug , which is metabolized in cells into the active purine nucleotide that mimics favipiravir-ribofuranosyl-5-triphosphate that inhibits the rna replication and thus the viral progression. 37 interestingly, it does not inhibit host dna and rna synthesis and inosine 5 0 -monophosphate dehydrogenase (impdh) activity. favipiravir reportedly demonstrated efficacy with minor side effects in an ongoing 70-patient clinical trial in shenzhen, guangdong province. 5 the drug's generic version received approval by the health authorities in china. 38 the broad-spectrum antiviral drug remdesivir together with chloroquine effectively inhibit the recently emerged novel cov (sars-cov-2) in vitro. 39, 40 remdesivir (25) reduced sars-cov-2 infection of monkey kidney cells with an ec 50 of 0.77 mm. the compound is in late-stage clinical development and has been recently described to inhibit multiple rna viruses on a cellular level, including ebola and sars. the presumed mode of action of the adenosine analog prodrug remdesivir is pre-mature rna synthesis termination by incorporation into nascent viral rna chains. 41 galidesivir (26) is another antiviral drug under clinical development with potential in covid-19 treatment (figure 7 ). it is an adenosine analog and is currently developed for ebola virus disease and marburg virus disease. it also shows broadspectrum antiviral activity against rna virus families including covs. 42 multiple other rdrp inhibitors are described in the literature, for example, broadspectrum antiviral 6 0 -bis fluorinated aristeromycin analog (27). 43 chloroquine (28) is an existing anti-malaria medicine also used to treat several other diseases. it blocked virus infection with an ec 50 of 1.13 mm. 39, 40 its mode of action is unclear. however, chloroquine inhibits endosomal acidification and thus could stop the release of viral dna into the cytoplasm. it is under assessment in more than 100 patients at over ten hospitals in beijing and guangdong province. plans for an additional study in hunan province are underway. the role of the other sars-cov-2 n proteins as drug-discovery targets is less clear. for the assembly of the replication/ q20 transcription complexes, there is a vast interaction network described between the non-structural proteins. similarly, viral particle assembly requires orchestrated interaction between n, s, m, and e proteins. all these interactions can be potential targets, but the structural information is currently minimal. resolution of the protein and complex structures will provide new unique drug targets. for example, the crystal structure of sars-cov-2 n protein rna-binding domain was just published and will give structural insight as a potential drug target. 44 it is rapidly detected by antibodies in serum, plasma, and peripheral blood, and might therefore serve to develop specific diagnostics. using methods of machine learning-enabled scientific literature analysis, the biotech company benevolentai q21 proposed the ap2-associated protein kinase 1 (aak1) as a host target to fight sars-cov-2. aak1 is the key enzyme of receptor-mediated endocytosis, which is the major mechanism of most viruses to enter their host cells. thus, they predict the approved (for rheumatoid arthritis) kinase inhibitor baricitinib 29 to reduce the ability of the virus to infect lung cells (figure 8 ). 45 the recent emergence of the wuhan cov (sars-cov-2) has put the world on alert. the rapid worldwide spread and the high human-to-human transmissibility, combined with the inability to contain the pandemic, is causing an increasing death toll and also considerable paralysis of the world economy (table 2 q22 ). the covid-19 could decrease and disappear or could be established worldwide in the human population and reoccur seasonally in future mutations through zoonosis from one of the animal reservoirs. however, it is very likely that in the upcoming years, we will see more outbreaks from cov and other viruses. the basic, translational, and public health research communities have to prepare for this much better. the outbreak has emphasized the urgent need for renewed efforts to develop broad-spectrum antiviral agents to combat covs. on the positive side, much new information of the virus biology and the spread was immediately shared, whereas, on the negative side, many past opportunities to develop antivirals against covs were not taken, despite a large number of promising approaches and compounds. 46 the past decade has shown that cov outbreaks are regularly reoccurring with more or less health effect on human and livestock. it remains to be hoped that the current pandemic will slow down and end as predicted in summer. furthermore, it turns out that containment measures are not effective to avoid more severe spread. it remains to be seen whether efficient and long-lasting immunity will develop in the infected population with regard to future outbreaks and whether pharmacological measures can be rapidly developed to be able to treat severely sick people. a.d. and l.g. wrote the manuscript. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges viral metagenomics revealed sendai virus and coronavirus infection of malayan pangolins (manis javanica) a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) drug repurposing: progress, challenges and recommendations therapeutic options for the 2019 novel coronavirus (2019-ncov) an overview of severe acute respiratory syndromecoronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small 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effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro insights from nanomedicine into chloroquine efficacy against covid-19 therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys galidesivir limits rift valley fever virus infection and disease in syrian golden hamsters design, synthesis, and anti-rna virus activity of 6 0 -fluorinated-aristeromycin analogues crystal structure of sars-cov-2 nucleocapsid protein rna binding domain reveals potential unique drug targeting sites baricitinib as potential treatment for 2019-ncov acute respiratory disease coronaviruses -drug discovery and therapeutic options key: cord-350959-bsbz3a1l authors: dovey, zachary; mohamed, nihal; gharib, yasmine; ratnani, parita; hammouda, nada; nair, sujit; chakravarty, dimple; stanislaw, sobotka; lantz, anna; wiklund, peter; kyprianou, natasha; tewari, ash title: impact of covid-19 on prostate cancer management: guidelines for urologists date: 2020-06-16 journal: nan doi: 10.1016/j.euros.2020.05.005 sha: doc_id: 350959 cord_uid: bsbz3a1l abstract context the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic has resulted in a global health emergency, the like of which has never been seen before. prostate cancer (pca) services across the globe have been on hold due to changing medical and surgical priorities. there is also epidemiological evidence that pca patients have increased incidence and mortality from sars-cov-2 infection due to gender differences, age, and higher propensity for risk factors (eg, respiratory disease, obesity, hypertension, and smoking status). objective to contribute to the emerging body of knowledge on the risks of sars-cov-2 infection to pca patients and, in the face of pca treatment delays, provide evidence-based recommendations for ongoing management of specific pca patient groups. evidence acquisition a literature search was performed using all sources (medline, embase, sciencedirect, cochrane libraries, and web of science) as well as the media to harness emerging data on the sars-cov-2 pandemic and its influence on pca. eligibility criteria were originality of data and relevance to pca management. the authors note that during these unprecedented times, retrospective data are constantly being updated from multiple sources globally. evidence synthesis a total of 72 articles and data sources were found initially. owing to repetition, lack of originality, or nonrelevance, six articles were rejected, leaving 23 retrospective studies, seven basic science research articles, 15 societal and journal guidelines, and 21 epidemiological data sources, from countries at different stages of sars-cov-2 pandemic. these were analyzed qualitatively to produce evidence-based guidelines for the management of pca patients at different stages of the patient journey, with strategies to reduce the risk of viral spread. conclusions pca patients may have an increased risk of sars-cov-2 infection as well as morbidity and mortality if infected. once appropriately triaged, and to reduce viral spread, pca patients can have surveillance by telemedicine, and institute lifestyle changes and social quarantining measures. if risk stratification suggests that treatment should be planned, androgen deprivation therapy can be started, or potentially surgery or radiation therapy is possible on a case-by-case basis. patient summary prostate cancer patients can be followed up remotely until the severe acute respiratory syndrome coronavirus 2 pandemic resolves, but higher-risk cases may have treatment expedited to limit any negative impact on prostate cancer outcomes. the subfamily coronavirinae, which contains positive-sense single-stranded rna, and genus betacoronavirus [5] [6] [7] . covs are pathogenic in humans and animals, causing disease syndromes of varying severity affecting mainly respiratory, but also the liver, gastrointestinal, and central nervous systems [7] . other notable covs were sars-cov, which caused a human epidemic of severe acute respiratory syndrome in 2003, with a patient mortality rate of 10% [8] , and mers-cov, which impacted the middle east in 2012, with a mortality rate of approximately 35% [9] . sars-cov-2 most likely arose from bats, based on genetic sequencing similarities [7] , with sars-cov-2 sharing 79.5% genetic sequence with sars-cov but 96.2% with a bat cov (batcov ratg13) [5] . as yet, the intermediate host from the bats to humans is not known [7] . structurally, the virion has an envelope spike (s) protein that drives entry of sars-cov-2 into target host cells by engaging the cellular receptor angiotensin-converting enzyme-2 (ace2) [10, 11] . this step is accompanied by activation of cellular tmprss2, a type-ii transmembrane serine protease [12] . the tmprss2 protein interaction is of major mechanistic significance in pca, as the androgenically regulated tmprrs2/erg fusion has been found in approximately 50% of pca patients. the incubation period for sars-cov-2 is around 5 d, varying from 1 to 14 d, and the main mode of transmission is human to human, via direct contact or aerosol spread, or through fomites [13, 14] . according to the cdc, infected patients have a low risk of being contagious until the onset of symptoms, most commonly in the 2nd week of illness, but as a precaution, it recommends limiting contact until 10 d after the illness has resolved [15] . examining the aerosol and surface stability, in comparison with sars-cov, sars-cov-2 remained stable in the aerosol drops for hours and on surfaces for days (depending on the amount of virus shed), the worst surfaces being stainless steel and plastic [14] . this means that the virus can spread at large-scale events, page 6 of 36 j o u r n a l p r e -p r o o f nosocomially exacerbating its pandemic potential [14] . the important epidemiological impact of the sars-cov-2 pandemic presents challenging questions to urologists and pca patients as to how long it will take for services hubei province following suit days after [7, 16] , and only recently did china announce plans to lift the lockdown on april 8, 2020 [17] . the presumption will be that once "lockdown" is lifted and social quarantining restrictions relaxed, hospitals will resume normal services. this timeline is in keeping with a statistical model recently developed by zhao and chen [16] , who predicted an end to the wuhan and hubei epidemic at the end of march 2020. based on this timeline, it seems unlikely that normal urological services will resume in affected countries until 3-6 mo following the onset of government restrictions. this means that pca patients awaiting biopsy or treatment may be in a triage system for up to 6 mo, with possibly starting androgen deprivation therapy (adt) to defer treatment until the pandemic is resolved. compelling epidemiological evidence suggests that pca patients have increased susceptibility to sars-cov-2 based on their male gender, age, and associated comorbidities. gender disparity has been reported for the sars-cov-2 pandemic, with some sources quoting twice as many men as women affected, although there is clear j o u r n a l p r e -p r o o f geographical variation (tables 1 and 2 ). in china, as of february 11, 2020, examination of 44 672 confirmed cases found that the male to female ratio was from 0.99 to 1 in wuhan, 1.04 to 1 in hubei, and 1.06 to 1 in the country [18] . another chinese study with a smaller cohort of 1099 confirmed cases has shown that 58% of those infected were men [19] . beyond asia, for the italian outbreak, livingston and bucher [20] also found that 58% of 22 512 confirmed cases were men (table 1 ). further evidence suggests that morbidity and mortality rates in confirmed cases are worse for men than for women [1] , with reports in chinese cohorts, albeit with small numbers, showing that 73% of patients are admitted to hospital [21] , 67% of patients are admitted to intensive treatment unit (itu), and 66% of mortalities in itu occur in male patients [22] (table 3) . similarly, another chinese cohort found that of 99 patients developing pneumonia, 67% were male [23] . in keeping with previous sars and mers outbreaks, older men with chronic morbidities and impaired immunity seem to be at maximum risk, once infection has taken hold [24] (summarized in tables 2 and 3 ). smoking rate, which is also associated with worse outcomes, may be a factor in male preponderance, and certainly in china, smoking is significantly more common in men than in women [23] . the potential role of ace2 in gender and ethnic disparity in sars-cov-2 infection has also been suggested, and although ace2 expression is similar in asians, caucasians, men, women, old, and young, it is much higher in asian smokers than in non-asian smokers [23] . the clinical syndrome of sars-cov-2 infection ranges from asymptomatic disease to pneumonia, multiorgan failure, and death. published retrospective studies, mainly from china, report symptoms in decreasing order of frequency page 8 of 36 j o u r n a l p r e -p r o o f as fever, cough, fatigue, arthralgia and myalgia, headache, hemoptysis, and diarrhea [19, 21] . in a chinese retrospective study, 78.7% of patients had mild disease, while 15.7% progressed to severe disease and 6.1% required itu admission [19] . there is also geographical variation, based on more virulent strains of sars-cov-2, with the italian epidemic showing higher rates of more severe disease [20] . ace2 expression (potential for sars-cov-2 infectivity) has been shown in 4% of proximal convoluted cells in the kidney and 2% of bladder urothelium [25] . this may cause acute kidney injury (aki) in 3-9% of cases [ 26, 27] . other urological abnormalities such as hematuria and proteinuria may be seen in up to 44% of admitted patients, as well as abnormal serum creatinine in 15.5% and blood urea in 14.1% [26, 27] . significantly, the development of an aki has been an independent risk factor for fatality [ 26, 27] . a clinical diagnosis may be made in a patient who has coryzal symptoms with fever, dyspnea, and signs of pneumonia [28] . chest x-ray may show interstitial consolidation, more common at the lung periphery, seen more clearly on a computed tomography (ct) scan [28] . laboratory diagnosis hinges on viral nucleic acid identification and viral isolation in respiratory tract specimens as well as in blood and feces [28] , primarily by reverse transcription polymerase chain reaction (rt-pcr) [7] , with detection rates improving with additional sars-cov-2 igg and igm serology [ 29] . the guidelines to urologists for the general approaches and clinical management of pca patients during the covid-19 pandemic are summarized in table 4 . smoking is associated with chronic obstructive pulmonary disease), which increases the risk and severity of sars-cov-2 infection. furthermore, smoking with vaping may independently increase sars-cov-2 infection risk and morbidity as well [30] . smokers have a 1.4 increased risk of sars-cov-2 infection and a 2.4 increased risk of icu admission, mechanical ventilation, and death [31] . a meta-analysis of 4202 smokers out of 22 549 pca patients revealed that the smoking status was associated with an increased risk of biochemical recurrence (bcr), progression to metastasis, and pcaspecific mortality [31] . as yet, there is no evidence on whether the combined effects of smoking and sars-cov-2 in pca patients will have a cumulative deleterious effect, but clearly a sensible recommendation on both counts is smoking cessation. retrospective studies from china revealed that patients with diabetes or hypertension were more likely to be admitted to itu [22] , and ace2 expression is increased in diabetics and also in patients treated with ace inhibitors and angiotensin receptor blockers (arbs) [32] . as shown in table 2 , pca patients with either diabetes or hypertension should seek advice from their physicians to optimize their treatment, especially if this includes ace inhibitors or arbs [32] , to reduce their risk of sars-cov-2 infection and morbidity. the human herpes family) showed a higher likelihood of a psa rise than controls, but the absolute change was small (only 5.9% with a rise of >0.50 ng/ml). they also found that patients with nonspecific systemic viral infections were more likely to have a psa rise than controls, but only >0.2 ng/ml [33] . in addition to the specific viral prostate infection, the effects of a viral immune response with systemic inflammation may affect local vascular permeability or prostate epithelium, resulting in psa elevation. thus, a systemic viral infection may produce a small rise in psa in pca patients in active surveillance or watchful waiting programs, but no such inference can be made on postradical prostatectomy patients. for those urologists who perform transrectal biopsies, there is evidence that sars-cov-2 may also be transmitted by fecal-oral route. a clinical study reported that over half of patients with sars-cov-2 infection had viral genome in the stool, and in just under a quarter, this viral shedding continued beyond when the virus was cleared from the respiratory tract, potentially as a result of ace2 expression within the gastrointestinal tract [34] . as discussed earlier, both renal proximal convoluted tubular cells and bladder urothelium express ace2 [25] ; however, reports of viral urinary shedding are conflicting. one study found urine samples to be negative for sars-cov-2 viral rna [35] , but another showed that 6.9% of infected patients had viral rna in their urine, which stayed positive until after their throat swabs were cleared [35] . if biopsy is the sars-cov-2 pandemic has caused widespread cancellations of pca surgeries, due to reduced anesthesiology cover, bed and resource availability, staffing levels, and policies aimed at reducing viral spread [3] . at the authors' institution, there is a triage system in place, where patients awaiting robotic-assisted radical prostatectomy (rarp) for national comprehensive cancer network (nccn) high-risk pca are assessed on a case-by-case basis, depending on wait time prior to suspension of surgery and individual disease parameters (summarized in table 5 ). a number of recent studies have examined the influence of delay between diagnosis of pca and surgical treatment on outcomes [36] [37] [38] [39] . a single-institution study of over 2600 patients undergoing radical prostatectomy with an average of 56 mo of follow-up found, despite a trend toward higher bcr rates after surgery, that this was significant for only nccn high-risk groups beyond 12 mo [36] . gupta et al [39] examined nearly 1250 patients with unfavorable intermediate-and high-risk pca, and found no difference in outcomes between groups waiting for <3 or 3-6 mo after diagnosis, with up to 10 yr of follow-up. loeb et al [37] studied delaying surgery for patients with gleason 6 pca, who under current guidelines will be in active surveillance programs, and park et al [38] evaluated open and minimally invasive radical prostatectomy performed before and after 4 wk following diagnosis of prostate biopsy, which is too short a time frame to influence the decision for expedited surgery in this context. optimization of operating room (or) times means that procedures should be performed by experienced prostatectomists with no training for page 12 of 36 j o u r n a l p r e -p r o o f residents or fellows during this period, and the number of trained or staff in the room should be kept to a bare minimum [40] . patients being considered for surgery should have a sars-cov-2-negative nasal swab pcr and potentially a ct thorax to rule out parenchymal lung infection, in case of a false negative nasal swab pcr. studies have shown that the pcr test is affected by technique and timing within disease course, with a false negative rate of 15-25%, and there are reports of negatively tested patients transmitting the disease [41] . at the authors' institution, nasal swab pcr is mandatory, but ct thorax is not; however, given the possibility of false negative results, full precautions should apply regardless. the primary risk of anesthesia relates to intubation as an aerosol-generating procedure, increasing the risk of acute respiratory virus infection in the staff [42] . for those involved, environmental and ppe control protocols are important, and intubation should be performed, if possible, under airborne isolation conditions and ors with negative pressure environments [43] . in patients for whom rarp is planned, or recommendations for minimally invasive surgery are available from a number of sources including erus, rcs, rcog, sages, american association of gynecologic laparoscopists (aagl), and aorn [44] . the putative risk is viral presence in the co2 plume, putting or staff at risk, based on prior evidence of hpv, hepatitis b, and hepatitis a smoke contamination [45] ; without evidence, this also applies to the sars-cov-2 virus. nevertheless, the prudent approach is a cautious one, with the procedural aim of limiting co2 and smoke release into the environment. this can be achieved by the lowest possible intra-abdominal pressure and closed systems with ultra-low penetrating air filters for plume evacuation to 0.01 m in size; sars-cov-2 size is 0.06-0.14 m) [43] . in addition, the ports should be closed when possible, instrument movement should be limited, intra-abdominal smoke should be kept to a minimum by suction, and smoke production should be reduced by avoiding the use of ultrasonic sealing and using low electrocautery power and bipolar vessel sealing, and sutures and clips whenever possible [40, 46, 47] . or personnel should also wear full protective equipment including masks with goggles or visors, and the console should be cleaned between cases [40, 46, 47] . radiation reduces white cell counts, but whether this increases the risk of sars-cov-2 for patients is not clear [48] . recently, a radiation oncology panel (from the usa and uk) published a systematic review with recommendations on how to manage pca patients safely during sars-cov-2 pandemic, applying a "rads framework" referring to remote visits, avoidance, deferment, and shortening of radiotherapy (rt) protocols [49] . for pca patients with nccn low-risk and favorable intermediate-risk disease, avoidance is recommended until the pandemic resolves (tables 4 and 5 ). for nccn unfavorable intermediate-risk, high-risk, and locally advanced disease, treatment can be deferred with the start of adt, depending on how the pandemic develops; however, if adt cannot be given, the risks of treatment for cure should be weighed against the risks of sars-cov-2 infection [49] . patients considered for adjuvant rt after surgery should have salvage rt instead, and the "rads framework" will also apply to patients referred for palliative rt (fig. 1 ). presently, there is no evidence that adt directly increases the risk of viral infection, so patients could be treated safely with luteinizing hormone-releasing hormone agonists, abiraterone, or enzalutamide [50] , unless convincing evidence emerges to the contrary. however, adt may exacerbate comorbidities such as obesity, diabetes, and hypertension, which may increase the risk of sars-cov-2 infection and complications [32, 51] . if present, patients should be advised regarding lifestyle changes and should be assessed by their internists on adt to optimize diabetic and hypertensive treatments (table 5) . sars-cov-2 requires tmprss2 as well as ace2 for cellular uptake, and it is known that upregulation of tmprss2 is caused by androgenic hormones in pca cells [12, 52] . given that sars-cov-2 has this novel mechanism of pathogenicity, further research is required to determine accurately the risk of sars-cov-2 infection in patients on adt. in the meantime, we must optimize comorbidities and monitor for adt complications, while adhering to government regulations to limit the risk of sars-cov-2 infection during progression to metastatic disease (fig. 1 ). advanced pca patients on chemotherapy treatments such as taxotere, carboplatin, or cabazitaxel may have an increased risk of sars-cov-2 infection. cancer patients undergoing chemotherapy, including taxanes, have been shown to have an increased risk of viral infections, specifically influenza, which may worsen their morbidity and mortality as a result of immunosuppression caused by the chemotherapy itself [53, 54] . although there are no data examining pca chemotherapy during the sars-cov-2 epidemic, a recent retrospective study by yu et al [55] reports on 1524 cancer patients treated at a center in wuhan during the chinese sars-cov-2 epidemic. less than 50% of the patient cohort was receiving active treatment, but all had regular hospital visits and besides the food and drug administration-approved autologous, active cellular immunotherapy sipuleucel, and checkpoint inhibitors ipilumab and nivolumab, which are used to treat metastatic castration-resistant prostate cancer [56] , most immunotherapies for different pca patient subpopulations with advanced disease will be given as a part of a clinical trial and will be suspended during the sars-cov-2 pandemic. telehealth and other technologies that facilitate remote patient interactions have previously been discussed as a means of providing healthcare in times of disaster [57] . the literature has focused on specific care for patients who are victims of the disaster (eg, those infected with sars-cov-2 during the viral pandemic), but there is much to be learnt for allied specialties in the current period of "lockdown." in the usa, over 50 large healthcare institutions have the digital telemedicine infrastructure in place, and from a urological perspective, the priority is triage of patients who are suitable compared with those who are not [58] . for pca clinic appointments, these can be screened and categorized as urgent or nonurgent (eg, a high suspicion of high-risk pca or uncomplicated active surveillance follow-up, respectively). nonurgent patients described as "well" can have telemedicine follow-up consultations as an alternative (table 4 ). any (table 4 ). the sars-cov-2 pandemic offers an opportunity to gain widespread experience of telehealth and associated technologies, and although all research in the usa is currently on hold, retrospective data will emerge, allowing the urological community to assess the success of these triage systems for both outpatient and inpatient care. one small silver lining to covid-19 is the opportunity to assess whether telemedicine has any real potential for providing pca clinical services over the long term. covid-19 as a pandemic has three phases: presurge, surge, and postsurge. table 4 deals with recommendations for the surge phase, but as hospitalization numbers gradually come down, the pandemic will enter the postsurge phase. however, given the predictions for more waves of the virus, the lines between the pre-and postsurge phases may become blurred. during these phases, stocks of ppe should be refilled, and departments should ensure that staff are fully cognizant of covid-19 guidelines. where possible, telemedicine infrastructure should be developed to allow remote followup, and all patients on treatment waiting lists should be meticulously stratified according to nccn risk guidelines and according to comorbidities and covid risk stratification [44] . during pandemic surge, nccn risk groups 5 and 6 (high and very high risk) may be offered surgery or alternatively adt according to their choice and their assessed risk of covid-19 infection due to comorbidities. after the surge, departments can work their way down through risk groups according to local capacity, preparing for subsequent pandemic waves as described above. similarly, patients with bcr or metastatic disease can be assessed according to table 4 and be referred for therapy as appropriate. these are extraordinary times, and there is much that is unknown. as just-in-time information is processed to improve understanding on how the pandemic should be managed at clinical, social, and economic levels, critical questions need to be asked so that lessons are learnt [3] . in affected countries, the aim has been to transfer pca services from predominantly hospitals to community-based systems, and surgeries have been delayed or cancelled. the questions are how long this will last and how long urological services can be put on hold. many countries have government-imposed targets for pca investigation and treatment from the point of referral, including italy and the uk [3] . at the best of times, urology departments find meeting these targets challenging, but clear guidance are now needed on how to manage delays in patients under diagnosis and treatment for pca, how long patients can wait to be seen, what red flags during initial assessment should expedite management, and how to deal with patients (and their families) who have suffered negative outcomes as a result of these delays. other direct effects of sars-cov-2 also need to be considered. service reductions and banning of group meetings necessarily have a deleterious effect on training and education. research efforts, both basic science and clinical, have generally been put on hold (barring local guidelines), and ramifications of interrupting projects and withholding grants will have long-term economic effects on institutional research programs and funding mechanisms, notwithstanding a negative impact on patient care. finally, although sars-cov-2 predictions point to that it will pass following containment, there is the potential for recurrence, and also emergence of mutant viruses with enhanced sensitivity. the question then arises whether formal guidelines be put in place for viral there is emerging evidence that patients with cancer have increased susceptibility to sars-cov-2 infection and potentially worse outcomes [84, 85] . general recommendations for pca patients waiting for treatment, under as, or after treatment should exercise ppe, social distancing, and quarantining according to local guidelines [83] . smoking cessation and optimization of comorbidities to reduce risk and severity of infection should be recommended to all [30] [31] [32] . lifestyle changes, physical exercise, mindfulness, as well as dietary changes and supplements, for example, pommi-t or zyflammend, as part of comprehensive lifestyle program are increasingly recommended to patients under as and after treatment, and should be emphasized to these patient groups during the pandemic, as well as to those awaiting diagnosis and treatment [86] . other specific recommendations with rationale for each patient group are given in the table. covid-19: the gendered impacts of the outbreak cancer patients in sars-cov-2 infection: a 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prostatectomy clinical characteristics of covid-19-infected cancer patients: a retrospective case study in three hospitals within wuhan, china interventions to mitigate early spread of sars-cov-2 in singapore: a modelling study building and implementing a lifestyle medicine program: from concept to clinical practice preventing lethal prostate cancer with diet, supplements, and rx: heart healthy continues to be prostate healthy and "first do no harm" part i do patients with cancer have a poorer prognosis of covid-19? an experience in impact of covid-19 on prostate cancer management: guidelines for urologists tewari prostate cancer (pca) patients may have an increased risk of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection and mortality. most can be followed up remotely until the pandemic resolves, but, once appropriately triaged, higher-risk cases can have treatment key: cord-348301-bk80pps9 authors: wahl, angela; gralinski, lisa; johnson, claire; yao, wenbo; kovarova, martina; dinnon, kenneth; liu, hongwei; madden, victoria; krzystek, halina; de, chandrav; white, kristen; schäfer, alexandra; zaman, tanzila; leist, sarah; grant, paul; gully, kendra; askin, frederic; browne, edward; jones, corbin; pickles, raymond; baric, ralph; garcia, j victor title: acute sars-cov-2 infection is highly cytopathic, elicits a robust innate immune response and is efficiently prevented by eidd-2801 date: 2020-09-24 journal: res sq doi: 10.21203/rs.3.rs-80404/v1 sha: doc_id: 348301 cord_uid: bk80pps9 all known recently emerged human coronaviruses likely originated in bats. here, we used a single experimental platform based on human lung-only mice (lom) to demonstrate efficient in vivo replication of all recently emerged human coronaviruses (sars-cov, mers-cov, sars-cov-2) and two highly relevant endogenous pre-pandemic sars-like bat coronaviruses. virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. our results indicate that bats harbor endogenous coronaviruses capable of direct transmission into humans. further detailed analysis of pandemic sars-cov-2 in vivo infection of lom human lung tissue showed predominant infection of human lung epithelial cells, including type ii pneumocytes present in alveoli and ciliated airway cells. acute sars-cov-2 infection was highly cytopathic and induced a robust and sustained type i interferon and inflammatory cytokine/chemokine response. finally, we evaluated a pre-exposure prophylaxis strategy for coronavirus infection. our results show that prophylactic administration of eidd-2801, an oral broad spectrum antiviral currently in phase ii clinical trials for the treatment of covid-19, dramatically prevented sars-cov-2 infection in vivo and thus has significant potential for the prevention and treatment of covid-19. the recently emerged human pandemic coronavirus severe acute respiratory syndrome coronavirus-2 (sars-cov-2), the causative agent of coronavirus disease (covid)-19, has spread to six continents resulting in substantial morbidity and mortality worldwide 1 . bats serve as a natural reservoir for coronaviruses and are the presumed source of sars-cov-2 and the highly pathogenic human coronaviruses sars-cov and middle east respiratory syndrome (mers)-cov 2 . transmission of coronaviruses from bats to other species is well-documented and adaptation in an intermediary host can facilitate their transmission to humans 2 . while it is possible that sars-cov-2 was transmitted to humans via an intermediate host such as pangolins, phylogenic analysis indicates that the sars-cov-2 lineage has circulated in bats for decades and evolved in bats into a virus capable of replicating in human cells 3 . thus, bats are a potential reservoir for coronaviruses with human pandemic potential that can be directly transmitted to humans. given the repeated and accelerating emergence of highly pathogenic coronaviruses, it has become increasingly important to monitor and characterize coronaviruses circulating in bats and to identify the viral determinants of human infection, disease, and global spread as well as to develop effective therapeutic interventions. animal models are useful in studying highly pathogenic human coronaviruses, the emergence potential of zoonotic coronaviruses, and to evaluate novel inhibitors for their ability to control coronavirus infection [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . however, human coronaviruses do not replicate in mice without either extensive adaptation of the virus or genetic modi cation of the host by genetic editing of the receptor or by introducing the individual human receptor genes for each virus [4] [5] [6] [7] [8] [9] [10] [11] 14, 15 . although existing rodent models have made several important contributions to our understanding of coronavirus infection and pathogenesis, none of these models possess the diverse set of primary human cells present in the human lung that can serve as targets for viral infection 16 . here, we show that human lung-only mice (lom), immune de cient mice implanted with authentic human lung tissue 17 , allow for the in vivo study of all recently emerged human coronaviruses (sars-cov, mers-cov, sars-cov-2) in a single platform that permits direct comparison of experimental outcomes. using lom, we also established that bats harbor novel coronaviruses capable of e cient replication in human lungs without prior adaptation. in addition, we performed an in-depth in vivo analysis of acute sars-cov-2 infection in the human lung. our results revealed robust sars-cov-2 replication, pathogenesis and sustained activation of the innate host immune response. finally, we used this platform for the in vivo evaluation of eidd-2801, an orally administered broad spectrum antiviral currently in phase ii clinical trials for covid-19 treatment, to prevent sars-cov-2 infection. our results show that eidd-2801 e ciently prevented sars-cov-2 infection in vivo strongly supporting its progression in clinical development for covid-19. lom are constructed by subcutaneous implantation of a small piece of human lung tissue into the back of immune de cient mice (fig. 1a) . previously, we demonstrated that the human lung tissue expands to form a highly vascularized palpable implant 17 (fig. 1a) . these lung implants contain human broblast, epithelial, endothelial, and mesenchymal cells that form highly relevant lung structures including cartilaginous and non-cartilaginous bronchial airways lined with ciliated and non-ciliated epithelium, alveolar sac structures, and extensive vasculature 17 (extended data fig. 1a,b) . we also showed that the human lung tissue of loms supports replication of a diverse set of emerging and clinically relevant human pathogens including zika virus, human cytomegalovirus, respiratory syncytial virus and mers-cov 17 . recently emerged human coronaviruses have used at least two different receptors to gain entry into host cells, human angiotensin converting enzyme-2 (ace2) and dipeptidyl peptidase 4 (dpp4). sars-cov and sars-cov-2 use human ace2 as a receptor while mers-cov uses dpp4 18-22 . these differences in receptor usage in uence viral tropism, pathogenesis and disease progression 23 . infection of lom with mers-cov resulted in robust virus replication and infection of human epithelial, endothelial and mesenchymal cells in vivo 17 . these ndings were consistent with the broad cellular distribution of its receptor, dpp4 24 . here, we rst evaluated the potential of lom to serve as a single platform to study all known recently emerged human coronaviruses and the potential of endogenous bat coronaviruses for human emergence. we rst con rmed that ace2, the receptor for sars-cov and sars-cov-2 in human cells, was present on the surface of human epithelial cells (cytokeratin 19+) in the human lung tissues of lom (extended data fig. 1c,d) . next, lom were inoculated with recently emerged coronaviruses sars-cov, mers-cov, or sars-cov-2 (extended data table 1 ). lom supported replication of all these viruses in vivo. speci cally, sars-cov and sars-cov-2 infection resulted in mean virus titers of 1.76x10 8 and 2.42x10 7 pfu/g respectively at 2 days post-infection (fig. 1b) . viral nucleoprotein antigen was abundantly observed in the human lung tissues of sars-cov and sars-cov-2 infected lom (extended data fig. 2a ). consistent with our previous results 17 , we also observed robust replication of mers-cov in the human lung tissues of lom with mean titers of 4.79x10 8 pfu/g in lom human lung tissues at 2 days postinfection (fig. 1b) and abundant viral antigen (extended data fig. 2a) . pre-pandemic bat coronaviruses wiv1-cov and shc014-cov have high sequence homology to sars-cov, use ace2 to infect human cells, and grow modestly in primary human airway cultures on liquid interface 4, 5 . lom were inoculated with wiv1-cov or shc014-cov and virus titers in human lung tissues measured 2 days post-infection (extended data table 1 ). wiv1-cov and shc014-cov e ciently replicated in the human lung tissue of lom with mean titers of 1.58x10 7 and 1.48x10 7 pfu/g, respectively (fig. 1b) and viral antigen was readily detected in human lung tissues (extended data fig. 2b ). collectively, these results demonstrate that lom serve as single platform where all newly emerged coronaviruses sars-cov, mers-cov, and sars-cov-2 replicated e ciently in human lung tissue. importantly, the fact that sars-like bat coronaviruses wiv1-cov and shc014-cov also replicated e ciently in the lom platform indicates that bats harbor coronaviruses that are potentially capable of direct transmission to humans, thus bypassing the need for further adaptation in an intermediary host. given the state of the current covid-19 pandemic and the urgent need to develop therapeutic and preventative approaches to control and prevent infection, we evaluated replication of sars-cov-2 in lom in detail. human lung tissues of lom were inoculated with sars-cov-2 and titers of replication competent virus determined 2, 6, and 14 days post-exposure (fig. 1c , extended data table 2 ). high titers of replication competent virus were noted at all time points although they were highest 2 days postinfection (fig. 1d) . the distribution of virus-infected cells was determined with rnascope (viral rna) and immuno uorescence microscopy (viral nucleoprotein). virus infection was widely distributed throughout the tissue with large numbers of cells positive for viral rna (fig. 1e ) and nucleoprotein (fig. 1f ). costaining with a human cytokeratin 19 antibody demonstrated that sars-cov-2 predominantly infects human epithelial cells in the lung (fig. 1g ). viral antigen was not detected in human cd34 expressing (endothelial) cells, and only a few human vimentin expressing (mesenchymal) cells were positive for viral nucleoprotein (fig. 1g) . to identify the epithelial cell types in the lung tissue that are susceptible to sars-cov-2 infection, we further identi ed infected cells by assessing co-localization of viral nucleoprotein with antibodies against acetylated alpha-tubulin iv (ciliated cells), cc10 (club cells), ht1-56 (alveolar type i [at1] pneumocytes), and pro-sp-c (alveolar type ii [at2] pneumocytes) (fig. 1h) . we were able to clearly identify virus antigen in cells which expressed pro-sp-c or acetylated alpha-tubulin iv; we did not detect virus antigen in ht1-56 or cc10 positive cells (fig. 1h) . these results demonstrate that sars-cov-2 has limited tropism in the lung with at2 pneumocytes and ciliated airway epithelial cells being the predominant lung cells infected by virus. to evaluate the cytopathogenic effects of sars-cov-2 during acute infection of human lung tissue in lom, we used a combination of histological analysis and electron microscopy. histopathologic analysis revealed several features of early diffuse alveolar damage that have been described in lung tissues of covid-19 patients including the accumulation of proteinaceous exudate and brin in alveolar spaces, desquamation of pneumocytes, multi-nucleated cell formation, and the appearance of brin thrombi in small vessels (fig. 2 ) 25-27 . proteinaceous exudate, including large globules of protein material, was observed in alveolar spaces, which overlapped with areas of virus accumulation (fig. 2a,b) . as early as 2 days post-infection, desquamation of pneumocytes was also noted; there were a large number of virally infected cells fully detached or detaching from the alveolar basement membrane into the alveolar space ( fig. 2c,d) . infected multi-nucleated cells were also observed (fig. 2c) . while the formation of hyaline membranes was not noted, brin was detected in alveolar spaces (fig. 2e) . importantly, we observed multiple occluded vessels containing brin thrombi as reported in the lungs of covid-19 patients ( fig. 2f ,g) [25] [26] [27] . electron microscopy demonstrated the normal architecture and integrity of uninfected at2 pneumocytes that were present in human lung tissue obtained from lom two days post-infection ( fig. 2h ). in contrast, at2 cells containing virus particles in the same sample had swollen mitochondria with loss of matrix and cristae as well as rough endoplasmic reticula with distended cisternae, protein accumulation, and virus particles (fig. 2i) . degenerative sars-cov-2 infected at2 cells detached from the alveolar basal membrane could also be observed in the alveolar luminal space (fig. 2j ). higher magni cation revealed subcellular accumulation of virus containing vesicles indicative of virus replication and egress. virions with electron dense nucleocapsids and distinctive crown-like spikes were observed (fig. 2i,j) . consistent with previous reports in human airway epithelial cell cultures and portmortem lung samples 26,28 , virions produced by human lung cells were pleomorphic in size (69 to 112 nm) and shape. despite the extensive damage in icted in the lung tissue by the virus, the endothelium in the majority of blood vessels was intact with tight junctions, numerous pinocytotic vesicles, and normal mitochondria and endoplasmic reticulum (fig. 2k ,l). virions were not detected within endothelial cells in agreement with a lack of infection as per our immuno uorescence analysis ( fig. 1g and fig. 2k ,l). however, pleomorphic virions were present in capillary lumen surrounded by brillar protein deposits and cell debris (fig. 2k,l) . together, these results demonstrate that acute sars-cov-2 infection of lom closely resembles infection of human lung in humans and is highly cytopathic resulting in signi cant injury to the fragile alveolar lung structures. to determine the effect of sars-cov-2 infection on human gene transcription, we performed rnasequencing analysis of human lung tissues collected from animals 2, 6 and 14 days post-infection. abundant viral transcripts were detected in infected lung tissue, ranging from 0.55% to 3.6% of the total reads at 2 days post-infection (extended data table 3 ). viral transcripts were still abundant but lower at 6 days and 14 days post-infection (extended data 3). sequencing data was consistent with previously identi ed canonical sars-cov-2 transcripts 29 and con rmed maintenance of the furin cleavage site throughout the course of infection in vivo. analysis of human gene transcripts revealed 1,504 differentially expressed cellular genes between naïve and infected human lung tissue at 2 days postexposure, the peak of infection (fig. 3a , supplementary tables 1 and 2) (adjusted p value <0.05 after correcting for multiple testing). of these, 1,043 genes were up-regulated and 461 genes were down regulated in the infected human lung tissue relative to non-infected lung tissue (fig. 3a , supplementary tables 1 and 2) . notably, numerous interferon-stimulated genes (isgs) and in ammatory cytokine genes, including pro-in ammatory cytokines genes il6, cxcl8 (il-8), cxcl10 (ip-10), tnf, and ccl5 (rantes) were potently induced in infected lung tissue (supplementary tables 1 and 2) . we also observed dramatic upregulation of ifnb1 expression (>1,000 fold) at 2 days post-exposure, suggesting that this cytokine plays a key role in the antiviral response to sars-cov-2 (supplementary tables 1 and 2 ). gene set enrichment analysis (gsea) showed over 840 gene pathways signi cantly upregulated (p<0.05) including response to type 1 interferon (p=0.0011), response to virus (p=0.0010), innate immune response (p=0.0010), cytokine mediated signaling (p=0.0010), cytokine production (p=0.0010), response to stress (p=0.0010), in ammatory response, (p=0.0010), nik nf-kb signaling (p=0.0011), acute in ammatory response (p=0.0035), regulation of cell death (p=0.0030), and coagulation pathways (p=0.0453) (fig. 3b ). complement activation, which contributes to sars-cov pathogenesis in mouse models 14 , was also increased (p=0.0470) (fig. 3b) . importantly, analysis of host gene expression at later time points demonstrated a sustained upregulation of antiviral and in ammatory genes that in some instances (e.g. isg15, ifitm1, tnf, cxcl9) persisted for up to 14 days post-infection (last time analyzed) (fig. 3c ,d, extended data table 4, supplementary tables 1 and 2 ). these results demonstrate that acute sars-cov-2 infection causes a potent and sustained upregulation of innate immune responses in virus-infected human lung tissue. currently, there is no vaccine to prevent sars-cov-2 infection or effective therapy to treat patients with covid-19. the ribonucleoside analog β-d-n 4 -hydroxycytidine (nhc) has been shown to broadly inhibit coronavirus infection in vitro in human airway epithelial cell cultures, with potent activity against sars-cov-2 as well as sars-cov, mers-cov, and bat sars-like and mers-like coronaviruses 7 . we therefore tested the ability of prophylactic eidd-2801 (also known as mk-4482), the oral pro-drug of nhc, to inhibit sars-cov-2 replication in vivo. for this purpose, lom were administered eidd-2801 starting 12 h prior to sars-cov-2 exposure and every 12 h thereafter (fig. 4a , extended data table 5 ). our results show that eidd-2801 had a dramatic effect on virus infection, signi cantly reducing the number of infectious particles in the human lung tissue of eidd-2801 treated animals in two independent experiments (fig. 4b ,c) by over 100,000 fold (fig. 4c,d) . furthermore, in contrast to eidd-2801 treated mice, abundant cell debris and nucleoprotein positive cells could be readily observed in the alveolar lumen of vehicle control treated mice consistent with the extensive pathogenic effects in icted on the lung by sars-cov-2 (fig.4e,f) . these results demonstrate that prophylactic administration eidd-2801 is highly effective at preventing sars-cov-2 infection and pathogenesis in vivo. the zoonotic transmission of the pathogenic sars-cov-2 resulted in a pandemic that has in icted signi cant morbidity and mortality as well as dire world-wide economic and social consequences. herein, we describe a unique model for the in vivo study of human coronavirus infection particularly well suited to model distal human lung virus infection. our results demonstrate replication of all known recently emerged human coronaviruses in lom. importantly, our results demonstrate that wiv1-cov and shc014-cov, two pre-pandemic endogenous bat viruses can replicate relatively e ciently in loms suggesting that coronaviruses circulating in bats have future pandemic potential without the need for further adaptation to the human host. we also show that interestingly, an analysis of post-mortem covid-19 patient lungs 12 did not reveal increased ifnb1 expression and it has been shown in vitro that its expression is blocked during sars-cov infection 33, 34 . these results suggest that in human lung tissue ifnb1 gene expression is induced during the acute phase of sars-cov-2 infection. we also observed increased expression of several human cytokine genes in lom infected with sars-cov-2. a substantial number of these cytokines were also increased in the serum of covid-19 patients and post-mortem lung tissue samples further establishing the similarities between lom and human infection with sars-cov-2 12, 35 . currently, there is no vaccine to prevent or therapeutics to treat covid-19. pre-exposure prophylaxis approaches to infectious diseases have proven to be highly e cacious and can contribute to reduce the risk of infection. the continued global spread of the virus and its associated morbidity and mortality are strong incentives to the development of prevention strategies for covid-19. in this regard, nhc was shown to have broad activity against human and bat coronavirus infection in vitro 7 . in addition, prophylactic and therapeutic administration of its oral pro-drug eidd-2801 reduced sars-cov and mers-cov replication and pathogenesis in mice 7 . here we show that prophylactic administration of eidd-2801 e ciently prevents sars-cov-2 infection in vivo highlighting its potential utility as an effective prophylactic agent against sars-cov-2 and other past and future zoonotic coronaviruses. there are some limitations of our study including the fact that lom do not possess the human nasal airway structures that are thought to be early sites of sars-cov-2 replication in humans 36 . since lom do not have an autologous human adaptive immune system they re ect the direct effect of viruses on their targets and bystander cells as well as their innate immune response to infection. collectively, our results indicate that lom re ect the pathogenic effects in icted by sars-cov-2 on the human lung and demonstrate their utility as a single in vivo platform to evaluate and compare the replication and pathogenesis of past, present, and future pre-emergent, epidemic, and pandemic coronaviruses accelerating the development and testing of pre-exposure prophylaxis agents such as eidd-2801. immunohistochemistry was performed as previously described 17 . brie y, xed (10% formalin) human lung tissues collected from coronavirus-infected lom were para n embedded and sectioned (5 um human lung tissues collected from mice were xed in 10% formalin and para n embedded. immuno uoresence staining of 5 um tissue sections was performed as previously described 17 . brie y, following depara nization and antigen retrieval (diva decloaker), tissue sections were incubated with a 10% normal donkey serum solution with 0.1% triton x-100 in 1x pbs to block non-speci c binding. tissue sections were then incubated overnight with primary antibodies at 4°c followed by incubation with uorescent conjugated secondary antibodies (supplementary table 7 ). primary antibodies were directed against sars nucleoprotein and human cytokeratin 19, cd34, vimentin, acetylated alpha-tubulin iv, cc10, ht1-56, and pro-sp-c (supplementary table 7 ). background auto uorescence was then quenched using a 0.1% sudan black b solution in 80% ethanol prior to staining with dapi. slides were mounted and then imaged using an olympus bx61 upright wide-eld microscope using volocity software (version 6.3) with a hamamatsu orca rc camera. appropriate negative controls without primary antibodies were also imaged using the same exposure time as matching stained sections. whole image contrast, brightness, and pseudocoloring were adjusted using imagej/fiji (version 2.0.0-rc-69/1.51w) and adobe photoshop tissue pieces were washed in deionized water, dehydrated in ethanol, and placed through two exchanges of propylene oxide before in ltration and embedment in polybed 812 epoxy resin (polysciences an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in pathogenesis of sars-cov-2 in transgenic mice expressing human angiotensin-converting enzyme 2 a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emergence evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic origin and evolution of pathogenic coronaviruses an interactive web-based dashboard to track covid-19 in real time precision mouse models with expanded tropism for human pathogens resident cellular components of the human lung: current knowledge and goals for research on cell phenotyping and function a mouse-adapted model of sars-cov-2 to test covid-19 countermeasures complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate imbalanced host response to sars-cov-2 drives development of covid-19 lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus the pathogenicity of sars-cov-2 in hace2 transgenic mice a mouse model for mers coronavirus-induced acute respiratory distress syndrome a novel coronavirus from patients with pneumonia in china pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer dysregulation of immune response in patients with covid-19 in wuhan sars-cov-2 reverse genetics reveals a variable infection gradient in the respiratory tract viruses were directly injected into the human lung tissue of loms and lung tissue collected either 2, 6, or 14 days post-exposure for virus titer determination and/or analysis by histology, electron microscopy, or rna-seq. lung tissue (advanced bioscience resources) subcutaneously into the upper and lower back of male and female 12-21 week old nod.cg-prkdc scid ll2rg tm1wjl /szj mice [nsg mice; the jackson laboratory] as previously described 17 . engraftment of lung tissue was assessed by palpation and by 8 weeks post-surgery animals were ready for experimentation. mice were housed and maintained by the division of comparative medicine at the university of north carolina-chapel hill shc014-cov, and wiv1-cov were derived from infectious virus clones and were prepared and titered on vero e6 (sars-cov, shc014-cov, and wiv1-cov) or vero ccl81 cells (mers-cov) (american type culture collection) as previously described 4,5,9,17 . a clinical isolate of sars-cov-2 (2019-ncov/usa-wa1/2020) was obtained from the reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus galloylglucoses of low molecular weight as mordant in electron microscopy. i. procedure, and evidence for mordanting effect star: ultrafast universal rna-seq aligner salmon provides fast and bias-aware quanti cation of transcript expression positive cells, brown) of human lung tissue of lom administered eidd-2801 (n=8 analyzed) or control vehicle (ctrl, n=8 analyzed) at 2 days post-exposure (scale bars acknowledgements: we thank current and past members of the garcia laboratory for technical assistance. the authors also thank technicians at the unc animal histopathology and laboratory animal medicine core and division of comparative medicine. we also thank the unc school of medicine bioinformatics and analytics research collaborative (barc) for providing technical support and k. author contributions: aw, cej, wy, mk, and cd constructed lom. aw contributed to experimental design, data interpretation, data presentation, and manuscript writing, coordinated the study, and the preparation of the manuscript. leg performed the virus inoculations, animal necropsies, virus tittering, processing of lung tissues for rna extraction, and contributed to the experimental design, planning, data analysis, data interpretation, and manuscript writing. cej performed immuno uorescence and h&e analyses, wy performed immunohistochemistry and h&e analyses, and mk performed the in-situ hybridization analysis of lom human lung tissues. khd, as, srl, and kg assisted with the in vivo experiments with coronavirus-infected loms. vjm in conjunction with kkw performed the electron microscopy analysis.fba assisted with the pathohistological analysis. hl, hmk, tz, pog, performed the rna-sequencing analysis. epb and cdj contributed to design of rna-sequencing experiments. rjp assisted with the immuno uorescence analysis and contributed to experimental design, data interpretation, data presentation, and manuscript writing. rsb conceived and designed experiments and contributed to data interpretation and manuscript writing. jvg conceived, designed and coordinated the study, conceived and designed experiments, and contributed to data interpretation, data presentation, and manuscript preparation.competing interests: the authors have no competing interests. supplementary information is available for this paper at correspondence: correspondence to j. victor garcia. further information on research design is available in the nature research reporting summary linked to this paper.data availability: gene-expression data are available at the gene expression omnibus (geo) repository (accession: gse155286). all other data is available in the manuscript or the supplementary materials. key: cord-337825-ujq9mxk7 authors: chen, bin; tian, er-kang; he, bin; tian, lejin; han, ruiying; wang, shuangwen; xiang, qianrong; zhang, shu; el arnaout, toufic; cheng, wei title: overview of lethal human coronaviruses date: 2020-06-10 journal: signal transduct target ther doi: 10.1038/s41392-020-0190-2 sha: doc_id: 337825 cord_uid: ujq9mxk7 coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. seven coronaviruses that infect humans have been identified: hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, sars-cov, mers-cov, and sars-cov-2. among them, sars-cov and mers-cov caused outbreaks in 2002 and 2012, respectively. sars-cov-2 (covid-19) is the most recently discovered. it has created a severe worldwide outbreak beginning in late 2019, leading to date to over 4 million cases globally. viruses are genetically simple, yet highly diverse. however, the recent outbreaks of sars-cov and mers-cov, and the ongoing outbreak of sars-cov-2, indicate that there remains a long way to go to identify and develop specific therapeutic treatments. only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. this paper mainly focuses on sars-cov, mers-cov, and sars-cov-2. here, recent studies are summarized and reviewed, with a focus on virus–host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment. coronaviruses are crown-like particles with spikes protruding from their surface. they are enveloped viruses with single-stranded, positive-sense rna (+ssrna) genomes of approximately 26-32 kb, which is currently the largest known genome size for an rna virus. 1 rna viruses are divided into five branches. 2, 3 certain groups in a particular branch might be related to and/or share features with viruses classified in another branch. in branch 1 are the leviviruses and eukaryotic relatives (e.g., mitoviruses, narnaviruses, ourmiaviruses); branch 2 represents several +rna virus families (of eukaryotes) (e.g., orders picornavirales and nidovirales, and the families caliciviridae, potyviridae, astroviridae, and solemoviridae) and several dsrna viruses (e.g., partitiviruses and picobirnaviruses); in branch 3 are certain +rna viruses such as the supergroups of alphaviruses and offlaviviruses, the nodaviruses, tombusviruses, and many small and novel groups; in branch 4 are dsrna viruses such as the cystoviruses, reoviruses, and totiviruses; branch 5 consists of −rna viruses. 2 coronaviruses were classified as most likely related to branch 2, and belong to the order nidovirales, and the family coronaviridae. in 2018, the international committee on taxonomy of viruses divided the coronaviridae into the orthocoronavirinae and letovirinae subfamilies. according to their host targets, coronaviruses can also be divided into animal and human coronaviruses. many diseases in domestic animals are related to animal coronaviruses, such as canine respiratory coronavirus, which causes respiratory disease in dogs. 4 the highly pathogenic human coronaviruses belong to the subfamily coronavirinae from the family coronaviridae. the viruses in this subfamily group into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the classic subgroup clusters are labeled 1a-1b for the alphacoronaviruses and 2a-2d for the betacoronaviruses. to date, including the recently discovered sars-cov-2, there are seven coronaviruses that infect humans. human coronavirus (hcov)-229e, hcov-nl63, hcov-oc43, or hcov-hku1 cause only the common cold, 5 whereas the severe acute respiratory syndrome coronavirus (sars-cov) or the middle east respiratory syndrome coronavirus (mers-cov) cause relatively high mortality and emerged in 2002 6 and 2012, 7 respectively. notably, sars-cov-2 is currently causing a worldwide epidemic. sars-cov and mers-cov belong to subgroups 2b and 2c of the betacoronaviruses, respectively, 1, 8 whereas sars-cov-2 is a new member of the betacoronaviruses distinct from sars-cov and mers-cov. 9 figure 1 shows the phylogenetic tree of rna viruses. the estimated mutation rates of coronaviruses (covs) are moderate to high compared to those of other ssrna viruses. 1 there are two gene loci that are sites of variation in sars-cov. one of these sites is located in the spike (s) protein gene. the second major site of variation is the accessory gene open reading frame orf8. in mers-cov, the major sites of variation are located in the s, orf4b, and orf3 genes. 2 the major differences in the sequence of the s gene of sars-cov-2 are three short insertions in the n-terminal domain and changes in four out of five of the key residues in the receptor-binding motif. 9 the organizations of the genome and gene expression are similar for all coronaviruses. orf1a/b, located at the 5′ end, encodes 16 nonstructural proteins (nsps) (named nsp1-nsp16); other orfs at the 3′ end encode structural proteins, including the s, envelope (e), membrane (m), and nucleocapsid (n) proteins. the mutations in sars-cov-2 have become a hot research topic. the surface proteins of sars-cov and batcov-ratg13, the nearest potential bat precursors of sars-cov-2, have 76 and 97% sequence identity, respectively, to that of sars-cov-2. 10 compared to that of sars-cov, the antigenic surface of sars-cov-2 is highly divergent compared to other covs. since its outbreak began at the end of 2019, sars-cov-2 has acquired mutations throughout its genome, and there are already hundreds of virus strains distributed worldwide. according to gisaid.org, as of may 8, there were 16,004 full genome sequences available, and the s clade of the full genome tree contains the largest group of viruses, indicating that mutations in orf8-l84s are most frequent. the temporal resolution assumes an average nucleotide substitution rate of 5 × 10 −4 substitutions per site per year. based on these mutations, researchers found that sars-cov-2 can be divided into two subtypes. they reported that the snps at locations 8782 and 28,144 show strong linkage; the "ct" haplotype was defined as the "l" type because t28,144 encodes leucine, while the "tc" haplotype was defined as the "s" type because c28,144 encodes serine, 11 which is in accordance with another study that divided sars-cov-2 into two types. 12 the "l" type seems to be more prevalent and aggressive, but the "s" type may be ancestral, as sites 8782 and 28,144 were identical to the orthologous sites in the most closely related viruses. 13 patients may be infected by either or both types. 11 from the gisaid update, compared with ratg13, there are differences in the receptor-binding interface, which may have favored host switching. data related to the sequence and mutations of sars-cov-2 are also available and continuously updated online through the china national center for bioinformation (cncb) (https://bigd.big.ac.cn/ncov). the history of sars-cov, mers-cov, and sars-cov-2 the first case of sars was discovered in foshan, china, in november 2002. 6 infections occurred through either direct or indirect contact with patients. by july 2003, sars-cov had spread to over 30 countries, 12 causing 8096 reported cases and 774 deaths. after that, no additional infections were detected, and the sars pandemic was declared over. sars-cov was first isolated from himalayan palm civets (hpcs) from a live-animal market in guangdong, china. 12, 14 other animals, such as raccoon dogs (nyctereutes procyonoides), along with human workers from the same market, also showed evidence of viral infection. 12 multiple studies have demonstrated that the reservoirs of several coronaviruses, including sars-cov-like and mers-cov-like viruses, were bats. 8, 15 although palm civets might have been intermediary hosts of sars-cov, 16 researchers often concluded there was no evidence that these animals were the ultimate sources of sars-cov, and the viruses cannot circulate directly in palm civets in the wild. 17 in 2003, guan and zheng's team investigated a live-animal retail market in guangdong, focusing on recently captured wild animals and human consumption. the animals sampled included seven wild and one domestic animal species. they collected nasal fig. 1 phylogenetic tree of coronaviruses based on full-length genome sequences. all complete genome sequences of coronavirus were downloaded from the ncbi reference sequence database, refseq. the tree was constructed using maximum likelihood estimation (mle) by mega x, with clustal omega as the multiple sequence alignment method, and 1000 bootstrap replicates. only bootstraps ≥50% values are shown. the seven known human-infecting coronaviruses are indicated with a red star and fecal samples with swabs and then used reverse transcriptionpolymerase chain reaction (rt-pcr) to test for viral nucleic acid from the n gene of the human sars-cov. the rt-pcr assay results showed that samples from four of six hpcs were positive; the other seven species (beaver, chinese ferret-badger, chinese hare, chinese muntjac, domestic cat, hog-badger, and raccoon dog) sampled were negative. 12 mers-cov was first found in 2012 in a lung sample from a 60year-old patient who died of respiratory failure in jeddah, saudi arabia. on 15 september 2012, a similar type of virus named human coronavirus was isolated from a patient with severe respiratory infection. cases have also been reported in other countries. 18 mers has a 35% mortality rate, and since it emerged in the human population in june 2012, it has caused substantial morbidity and mortality. 19, 20 from 2012 until january 15, 2020, the total number of laboratory-confirmed mers-cov infection cases reported globally to the world health organization (who) was 2506, with 862 associated deaths, covering 27 countries (www. who.int/csr/don/31-january-2020-mers-united-arab-emirates/en). cases of mers from other countries were linked to travel to the middle east. 20,21 mers-cov was identified from the saliva of a patient with acute pneumonia and renal failure in jeddah (ksa). mers-cov can be detected in respiratory tract secretions, as well as in feces, serum, and urine. [22] [23] [24] in addition, it has been isolated from environmental objects such as bedsheets, bedrails, intravenous fluid hangers, and x-ray devices. 25 in the case of known camel infection, mers-cov was transmitted from a camel to a human, which was confirmed by rna sequencing. 26 at the end of december 2019, the first clusters of patients with pneumonia caused by sars-cov-2 were reported in wuhan, china. 27 the basic reproduction number of sars-cov-2 is approximately 2.2, indicating that each patient would on average spread the infection to 2.2 people. 28 human-to-human transmission of sars-cov-2 occurred rapidly, and the atypical symptoms during the early stage may be a further disadvantage. 29 moreover, human infection might have begun months prior to the official announcement of the outbreak. 30 to prevent further spread, stricter screening and surveillance are needed at travel hubs. 31 early detection, diagnosis, and treatment are also effective measures to contain the epidemic. 32 nonetheless, the fatality rate of sars-cov-2 is lower than that of sars, 33 with an estimated mortality risk of 2%. 34 sars-cov-2 was first isolated from clinical specimens using human airway epithelial cells and the vero e6 and huh-7 cell lines. 27 approaches to assess virions include isolation from lower respiratory tract specimens 35 and artificially infected specific pathogen-free human airway epithelial cells. 36 fluorescence quantitative pcr with primers designed according to specific sequences and serological testing aimed at igg and igm can be used to detect the virus. 9 sars-cov, mers-cov, and sars-cov-2 pose major challenges to global health, causing infections in large parts of the world. sars-cov was found in 30 countries and mers-cov in 27 countries, while sars-cov-2 was found in 213 countries by 8th may, 2020. details on the pandemics caused by sars-cov-2 are shown in box 1, highlighting the status of the coronavirus-caused diseases worldwide. epidemiological analysis and symptoms of sars, mers, and coronavirus disease 2019 (covid19) the clinical manifestations of sars include hypoxia, cyanosis, high fever (above 38°c), accelerated breathing or respiratory distress syndrome, and shortness of breath. x-rays show changes to the lungs to varying degrees. the who case definition (2003) includes the following: (1) fever higher than 38°c or history of such in the past 2 days, (2) radiological evidence of new infiltrates consistent with pneumonia, (3) chills, cough, malaise, myalgia, or known history of exposure, and (4) positive test for sars-cov by one or more assays. 37 similar to sars-cov infection, mers-cov infection manifests as a severe lower respiratory tract infection with extrapulmonary involvement and high fatality rates. 19 symptomatic patients may present fever, chills, stiffness, myalgia, discomfort, cough, shortness of breath, and gastrointestinal symptoms of diarrhea, vomiting, and abdominal pain. pneumonia is common, and severe infection with acute respiratory failure, renal failure, and shock is particularly frequent among older patients. 20 previous studies have estimated that approximately 12.5-25% of mers-cov infections may be asymptomatic. 20, 38 it must be noted that immunocompromised people are at high risk of mers-cov infection. 21 for covid-19, fever, cough, myalgia, and fatigue are most commonly observed at illness onset; less commonly observed are sputum production, hemoptysis, and headache, among others. 29, 35 similar to sars and mers, some patients develop acute respiratory distress syndrome (ards). 39 around 81% of infected patients only develop mild symptoms, with mild pneumonia or no pneumonia. 14 and 5% are severe and critical status, respectively. 40 patients requiring icu care tend to be much older and are more likely to have underlying comorbidities, such as hypertension and diabetes. 29 additionally, symptoms such as pharyngeal pain and anorexia are reported at higher rates among those admitted to the icu than among non-icu patients. 29 histopathologically, symptoms such as diffuse alveolar damage (dad), loss of cilia, squamous metaplasia, denudation of bronchial epithelia, and giant-cell infiltrate often occur in the lungs of patients with sars. the number of macrophages increases prominently in the alveoli and the interstitium. 41 according to the morphological changes, observed, most authors have subclassified lung lesions in sars into two or three consecutive phases: an acute exudative inflammatory phase, a fibrous proliferative phase, and a final fibrotic stage, although there is considerable overlap in histological findings among these phases. 42 the main features of the first phase include extensive hyaline membrane formation, edema, alveolar hemorrhage, and fibrin exudation in alveolar spaces. the second phase includes widening of septae, pneumocyte hyperplasia, and organizing fibromyxoid and cellular exudates. 42, 43 the third phase includes septal and alveolar fibrosis. 42 several autopsies of sars patients have demonstrated secondary bronchopneumonia, and the pathogens identified mainly consist of staphylococcus aureus, mucor sp., aspergillus sp., pseudomonas aeruginosa, and cytomegalovirus. 43 sars-cov also greatly affects the immune system. for example, hemorrhagic necrosis is usually obvious in the lymph nodes and spleen. 44 as described above, sars-cov attacks immune cells, especially t cells and macrophages, with approximately 50% of lymphocytes and 30% of monocytes in the circulation becoming infected. 42, 45 under the influence of the virus, the patient's germinal centers disappear, and both t and b lymphocytes are depleted. in the spleen, the white pulp shrinks, hemorrhage and necrosis occur in the red pulp, and the periarterial lymphatic sheaths decrease. 42, 45, 46 with regard to the immune system, sars-cov can infect the lymphoid component of the intestine. for this reason, the patient's submucosal lymphoid tissue atrophies. 47 sars-cov can also infect epithelial cells in the mucosa of the small and large intestines, and the digestive tract may undergo mild diffuse inflammation and atrophy of the submucosal lymphoid tissues. 47 according to previous reports, symptoms in the liver include extensive hepatocyte mitosis, balloon degeneration of hepatocytes, mild to moderate lymphocytic infiltrates, fatty degeneration, and central lobular necrosis. 42 additionally, apoptosis of liver cells has been confirmed in some cases. 48 on autopsy, the kidneys of some sars patients showed focal bleeding and acute tubular necrosis of different degrees. one of the major complications of ards is acute kidney injury. 42, 49, 50 in many other cases, the features are nonspecific, including benign hypertensive nephrosclerosis and autolysis. 42 moreover, researchers have detected viral particles and genome sequences in the cytoplasm of neurons of the hypothalamus and cortex in the brain, 51 which suggests that the virus can cross the blood-brain barrier and cause degeneration and necrosis of neurons, edema, extensive glial cell hyperplasia, and cellular infiltration. 42 pneumocytes and epithelial syncytial cells are important targets of mers-cov. the lung tissue presents dad. severe peripheral vascular diseases, patchy cardiac fibrosis, and hepatic steatosis have also been found in other organs. 52 another symptom is bronchial submucosal gland necrosis. 52 renal biopsy may show acute tubulointerstitial nephritis and acute tubulosclerosis with protein casts. 22 in covid-19 patients, a bilateral (sometimes unilateral) distribution of patchy shadows and ground glass opacity in the lungs is typical, based on ct scans. 29 plasma concentrations of interleukins (ils) 2, 7, and 10, granulocyte-colony stimulating factor, interferon (ifn)-γ-inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and tumor necrosis factor α are increased in most dying patients. 39, 53 in addition, neutrophilia related to cytokine storms induced by virus invasion, coagulation activation related to a sustained inflammatory response, and acute kidney injury related to the effects of the virus, hypoxia, and shock, may be involved in mortality. 29 those who survive intensive care may experience long-term lung damage and fibrosis due to aberrant and excessive immune responses. 39 moreover, researchers found that as sars-cov-2 spread and changed across generations, clinical symptoms started to differentiate those infected previously from those infected more recently. the initial symptoms are becoming more insidious, which indicates that the virus may gradually evolve into an influenza-like virus and lie dormant for longer in asymptomatic patients. 54 in conclusion, in terms of outbreak times, sars-cov was the earliest, followed by mers-cov, then sars-cov-2. to date, mers-cov has the longest duration of infection, and sars-cov-2 appears to be the most infectious. sars-cov, mers-cov, and sars-cov-2 infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. the common transmission methods and symptoms of the three coronavirus infection are shown in fig. 2 . structural studies of sars-cov, mers-cov, and sars-cov-2 sars-cov is a coronavirus with a diameter of approximately 100 nm. coronaviruses are the largest +ssrna viruses and contain at least 14 orfs, 16 protein combines with viral rna to form a nucleocapsid, which is involved in the replication of sars-cov and is the most abundant protein in virus-infected cells. 56, 57 the m protein is a membrane glycoprotein that is involved in budding and envelope formation. the s protein of sars-cov is a type i transmembrane (tm) glycoprotein that is responsible for viral binding, fusion and entry into cells, as well as antibody induction. 16, 58 the s protein contains a signal peptide (sp: amino acids 1-12) and three domains called the extracellular domain (amino acids 13-1193), the tm domain (amino acids 1194-1215) and the intracellular region (amino acids 1216-1255). 16 the extracellular domain consists of two subunits: s1 and s2. the fragment located in the middle region of the s1 subunit (amino acids 318-510) is the angiotensin-converting enzyme 2 (ace2) receptor-binding region. the s2 subunit comprises a fusion peptide (fp) and two x 7-valent element repeats (hr1 and hr2) that are responsible for fusion between the virus and the target cell membrane. 58 thus, the s protein may be key to develop a vaccine to generate antibodies and block virus binding and fusion in the coronaviruses (fig. 3) . the 3′ end of the sars virus genome encodes 12 structural proteins and helper proteins, of which orf3a, orf6, orf7a, and orf7b have been proven to be viral structural proteins involved in the formation of viral particles. the 5′ end of the genome encodes 16 nonstructural proteins (nsps), which are important for virus assembly, and may enable the design of small molecule drugs and/or vaccines. mers-cov belongs to lineage c of the genus betacoronavirus (βcov) in the family coronaviridae, order nidovirales, and it is the first lineage c cov and the sixth cov known to cause human infection. mers-cov is an enveloped +ssrna virus similar to other covs, but the amino acid sequence homology between mers-cov and other known covs is less than 90%. 19 orf1a and orf1b located in the first two-thirds of the mers-cov genome are involved in virulence and encode nsps (nsp1-16). the remaining third of the genome encodes four structural proteins, the s, e, m, and n proteins, as well as five accessory proteins (orf3, orf4a, orf4b, orf5, and orf8b). [59] [60] [61] the flanking regions of the genome contain the 5′ and 3′ untranslated regions (utrs). 62 most mers-cov proteins are found in the cytoplasm. 63 to date, the ns4b protein and the orf3a-encoded protein are the only known coronavirus proteins to be detected in the nucleus. 64 nsp1 suppresses host gene expression in infected cells, and its rna cleavage-inducing function promotes virus assembly/budding. 65 nsp16 is a viral 2′-o-methyltransferase (2′-o-mtase) that encodes critical functions in immune modulation and infection, suggesting that nsp16 is a conserved universal candidate for rapid design of a live attenuated vaccine. 66 orf4a's protein can mask viral dsrna to play a role in immune evasion, including type 1 ifn and protein kinase r-mediated antiviral stress responses. 67 mers-cov nsp13 is a full-length coronavirus helicase that contains multiple domains, including an n-terminal cys/his rich domain with three zinc atoms, a beta-barrel domain and a c-terminal superfamily (sf) 1 helicase (sf1) core with two reca-like subdomains. this protein might interfere with the nonsensemediated mrna decay pathway to avoid degradation of viral rna. 68 the mers-cov s protein can recognize dipeptidyl peptidase 4 (dpp4) on the cell surface, promoting the fusion of the viral envelope and the cell membrane. the mers-cov s protein, a 1353-amino acid type i tm glycoprotein located on the viral envelope surface, is composed of s1 and s2 subunits. 69 the structure of the s protein consists of four parts: an sp located at the n terminus (amino acids 1-12), an extracellular domain (amino acids 13-1195), a tm domain (amino acids 1196-1215), and an intracellular domain (amino acids 1216-1255). 16 the s1 subunit contains two independent domains, an n-terminal domain (ntd, amino acids 18-353) 70 and a c-terminal domain (amino acids 367-588), both of which may function as receptor-binding domains (rbds); 71 these domains serve as critical targets for the development of vaccines and therapeutics 72, 73 and facilitate the involvement of the s1 subunit in cell receptor (dpp4) binding. the core structure of the mers-cov s protein rbd is a five-stranded antiparallel sheet with several short helices, in which three disulfide bonds stabilize the core by connecting c383 to c407, c425 to c478, and c437 to c585. 16, 73 the accessory subdomain of the c-domain is a hypervariable region for receptor recognition fig. 3 clustering of the spike protein of each coronavirus. fifty-four spike protein sequences filtered from each coronavirus coding sequences were clustered using the clans (cluster analysis of sequences) program on the website of mpi bioinformatics toolkit. each colored dot represents the spike protein sequence of each coronavirus. dots in the same color mean they are of the same genus, and each line shows the similarity of two sequences, with darker lines indicating higher similarity (lower e values). the coronaviridae family includes the following genera: alphacoronavirus (colored in green); betacoronavirus (red); gammacoronavirus (orange), and deltacoronavirus (blue). the indicated sars-cov, mers-cov, and sars-cov-2 belong to the genus of betacoronavirus and is called the receptor-binding motif (rbm). the accessory subdomains in sars-cov and mers-cov differ, resulting from divergent evolution and leading to the use of different receptors. 71 dpp4 consists of an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets 74 and the rbd. the s2 subunit of mers-cov contains an fp (residues 943-982), an hr1 domain (residues 984-1104), an hr2 domain (residues 1246-1295), a tm domain (residues 1296-1317), and an intracellular domain (residues 1318-1353). 69 the s2 subunit is responsible for fusion between the virus and the target cell membrane. after the s1 subunit binds to dpp4, the s2 subunit inserts its fp into the target cell membrane to change its conformation. its hr1 helix then forms a homotrimer with an exposed surface that has three highly conserved hydrophobic grooves, and binds with hr2 to form a six-helix bundle (6-hb) core structure, which facilitates fusion by bringing the viral and cell membranes into close proximity. 69 the genetic material of mers-cov then enters the host cell via the fusion pore. 69 sars-cov-2, which causes covid-19, is a spherical betacoronavirus with a diameter of 60-140 nm and unique spikes ranging from 9 to 12 nm. 27 its rna has the typical betacoronavirus organization, containing a 5′ utr, a gene encoding the replicase complex (orf1a/b), the s gene, e gene, m gene, and n gene, a 3′ utr, and several unidentified nonstructural orfs. 27, 75 the length of the sequences is 265 nt for the 5′ terminal region, 229 nt for the 3′ terminal region, 3822 nt for the s gene, 228 nt for the e gene, 669 nt for the m gene, and 1260 nt for the n gene. 75 the 21,291nt-long orf1a/b gene contains 16 predicted nsps. 75 similar to sars-cov, there is a predicted orf8 gene that is 366 nt in length and located between the m and n orf genes. 75 nucleotides 1, 1029, and 1652 may be the recombination breakpoints because based on the fragments from nt 1 to nt 1029 and nt 1652 to the end, sars-cov-2 seems to be related to bat-sl-covzc45 and bat-sl-covzxc21; however, when considering the region from nt 1030 to nt 1651, sars-cov-2 is mostly likely to be grouped with sars-cov and bat sars-like covs. more research is required to analyze the recombination of the genome as a whole. 75 with over 10% of its conserved replicase complex being different from those of other betacoronaviruses, sars-cov-2 solely occupies a newly created subclade within the sarbecovirus subgenus. 27, 75 bats may be its natural host, but owing to several arguments including the complicated environment in the wet markets in wuhan, this remains unclear, and further research is needed. 36, [75] [76] [77] the free energy of the spike protein of sars-cov-2 is much lower than that of sars-cov, indicating that sars-cov-2 is more stable than sars-cov. 78 the high similarity of the rbd sequences and of the spike protein structures between sars-cov-2 and sars-cov, 36, 75, 79 also the simulation of the spike protein of sars-cov-2 binding to ace2, 80 the receptor of sars-cov, and results shown that ace2 plays a vital role in sars-cov-2 entry into hela cells, 76 indicating that sars-cov-2 also uses ace2 for cell entry. structural modeling of the ace2-b0at1 complex (b0at1 is used to obtain stable ace2) suggests that the complex can bind two s proteins simultaneously, providing important clues to the molecular basis of coronavirus recognition and infection. 81 the rbd-ace2 binding free energy for sars-cov-2 is significantly lower than that for sars-cov, in accordance with the fact that sars-cov-2 is more infectious than sars-cov. 78 a recent study found that cd147, a kind of tm glycoprotein, facilitates cell entry of sars-cov-2 functionally, and its affinity constant with the s protein is 1.85 × 10 −7 m. 82 table 1 shows a comparison of the structures of sars-cov, mers-cov, and sars-cov-2. the specific function of the sars-cov-2 proteins, including s, e, m, and n proteins, need further study. in conclusion, sars-cov, mers-cov, and sars-cov-2 are similar in structure. their major proteins are structural proteins, including the s, e, m, and n proteins, accessory protein, and nsps (nsp1-16). all these viruses rely on the s protein to identify cell targets and fuse with membranes. their s proteins are composed of s1 and s2 subunits. the s1 subunit contains an ntd and a c-domain, in which there is an rbd. the accessory subdomain of the c-domain is a hypervariable region for receptor recognition and is called the rbm. the difference is that sars-cov recognizes ace2, sars-cov-2 recognizes ace2 and cd147, but mers recognizes dpp4, which results from the difference between the accessory subdomains of sars-cov, sars-cov-2, and mers-cov. the function of the accessory and nonstructural proteins of these three viruses is related to viral replication and assembly, hence linked to virulence. pathogenesis of sars-cov, mers-cov, and sars-cov-2 sars-cov and sars-cov-2 are nearly identical in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov-2 is ace2, plus cd147 for sars-cov-2, while that of mers-cov is dpp4. the human ace2 protein is a typical zinc metallopeptidase that comprises 805 amino acids and contains a single catalytic domain. it is a type i integral membrane glycoprotein that is oriented with an extracellular n terminus and a catalytic site that functions in metabolizing circulating peptides. 83 it is believed that there is a virus-binding hot spot on ace2 that is not pre-existent or preorganized but is induced to form upon virus binding. 84 the hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. the general structural features of the hot spot favor virus binding: it is located in a region on ace2 that is furthest from the membrane, relatively flat, and free of glycosylation and is thereby easily accessible to viruses. the hot spot is mainly an intrinsic property of ace2, but it is also a dynamic structure and receives structural contributions from both ace2 and the viruses, with the contributions of ace2 being more pronounced. dpp4 is a type ii transmembrane glycoprotein that consists of approximately 766 amino acids. dpp4 contains an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets. 74 the s protein rbd binds to the side surface of the dpp4 propeller and the amino acid residues in lancets 4 and 5, including k267, q286, t288, r317, r336, q344, a291, l294, and i295. 74 the pathogenic mechanism of sars-cov is believed to include two parts: the virus injures target cells directly, and subsequent immune system dysfunction leads to indirect injury. 42 sars-cov spreads via droplet and contact transmission and via the fecal-oral route. through droplet inhalation, the virus enters the respiratory tract and invades epithelial cells. infection and replication of the virus and local inflammatory changes contribute to lung tissue damage. subsequently, sars-cov infects circulating and resident immune cells. the key cells in this step consist mainly of t cells and macrophages. the viruses are then carried to other organs, including secondary lymphoid organs, by these circulating immune cells. 42 sars-cov resembles hiv because both viruses attack immune cells and cause immunodeficiency. 42 sars-cov causes lung injury mainly by inhibiting the function of ace2. the virus binds to ace2 via the spike protein, downregulating its function and contributing to lung injury. ace2 is an important component of the renin-angiotensin system (ras). in this system, angiotensinogen is first converted to angiotensini (angi) by renin, and then angiis converted to ang ii under the influence of ace2. ace2 downregulates the levels of angi and ang ii, which can bind to the ang ii type i (at1) receptor and cause certain types of lung injury, mainly pulmonary hypertension, pulmonary fibrosis, and acute lung injury. 85 ang ii can directly induce pulmonary artery smooth muscle cells to grow or proliferate rapidly through at1, thus causing pulmonary hypertension. 86 ang ii contributes to pulmonary fibrosis by promoting the expression of the profibrotic cytokine transforming growth factor-b1, causing fibroblasts to convert to myofibroblasts and accumulate collagen. 70 several strategies can be used by the virus to escape the innate immune response. normally, viral pathogen-associated molecular patterns (pamps) are detected by host pattern recognition receptors (prrs). pamps include viral dsrna and mrna, and prrs include retinoic acid-inducible gene i protein (rig-i) and melanoma differentiation-associated protein 5 (mda5). production of type i ifn is induced via the nuclear factor-κb (nf-κb) pathway. when ifn binds to the ifnα/β receptor, signal transducer and activator of transcription (stat) proteins are activated, which increases the production of other antiviral proteins and thus blocks sars-cov replication for further infection. 11 additionally, sars-cov could encode several proteins that hinder the signaling cascades downstream of prrs. the cell receptor of mers-cov is dpp4, which is widely expressed on epithelial cells in the prostate, alveoli, kidneys, liver, and small intestine and on activated leukocytes; thus, the range of mers-cov tissue tropism is broader than that of any other similar coronavirus. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of proinflammatory cytokines and chemokines (such as tnf-α, il-6, cxcl-10, ccl-2, ccl-3, ccl-5, and il-8). 87 compared to sars-cov infection, mers-cov infection can cause higher expression levels of il-12, ifn-γ, ip-10/cxcl-10, mcp-1/ccl-2, mip-1α/ccl-3, rantes/ccl-5, and il-8. 87 induction of immune cell-recruiting cytokines/chemokines might lead to the infiltration of a large number of immune cells into the lower respiratory tract, causing severe inflammation and tissue damage. 87 mers-cov can also infect t cells and lead to apoptosis, avoiding the host immune response and facilitating faster spread. 88 mers-cov has also evolved a mechanism to escape the host cell immune system. when host sensors initiate signaling pathways, transcription of type i ifn genes begins, and the type i ifn response, which is an essential component of antiviral innate immunity, is initiated. [89] [90] [91] ifn activates the transcription of numerous ifn-stimulated genes (isgs) and synthesizes 2′,5′oligoadenylate (2-5a), which causes rnase l activation. [91] [92] [93] activated rnase l can cleave both viral and host ssrna, leading to translation stagnation and apoptosis and limiting virus replication and transmission in vitro and in vivo. 92, 94 however, the mers-cov protein ns4b has phosphodiesterase activity and antagonizes rnase l via enzymatic degradation, leading to inference with ifn signaling. although ns4b is mainly expressed in the nucleus, the expression level of the ns4b protein in the cytoplasm is sufficient to prevent activation of rnase l. 88 in addition, the mers-cov m, orf4a, orf4b, and orf5 proteins are reported to be strong ifn antagonists, among which the orf4a, orf4b, and orf5 proteins inhibit both type i ifn induction 61, 95, 96 and nf-κb signaling pathways, which are similar to the type i ifn signaling pathway, 96 providing a possible mechanism for mers-cov evasion of innate immunity. 88, 96 for sars-cov-2, the first cluster of patients was believed to be associated with a seafood market. 27 however, due to the identification of patients without direct exposure to the market, transmission between humans was proven. 97 hospital-related transmission has also been detected via infected medical workers and hospitalized patients. main routes of transmission include respiratory droplets, close contact, and extended exposure to aerosol environments loaded with high concentrations of virions. in addition to transmission through the respiratory tract, exposure of unprotected eyes to sars-cov-2 might cause acute respiratory infection. 98 notably, the virus can be transmitted during the incubation period. 99 the tm protease serine type 2 (tmprss2) primes the s protein of sars-cov-2 for cell entry. 100 based on analysis of the ace2 rna expression profile in normal human lungs, sars-cov-2 mainly infects type ii alveolar (at2) cells (only 0.64% of human lung cells can express ace2, 83% of which are at2 cells), and they express many other genes that facilitate sars-cov-2 infection; 101 moreover, cd147 was also reported that it probably functionally facilitates cell entry of sars-cov-2. 82 high expression of ace2 has also been detected in cells of the digestive system, such as upper esophageal cells; accordingly, this system is a potential route of infection. 102 in addition to the lungs and intestines, other organs, such as the heart, esophagus, kidney, bladder, testis, ileum, and adipose tissue, also express ace2, and the expression level is higher than that in the lungs. 103, 104 certain tumor tissues have higher expression of ace2, making cancer patients more vulnerable than other people. 104 in conclusion, sars-cov and sars-cov-2 bind cells expressing ace2. they affect the ras system due to the affinity for ace2 (much stronger with sars-cov-2 than with sars-cov), leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. the specific pathogenic mechanisms of these three viruses are illustrated in fig. 4 . diagnostic methods in addition to clinical manifestations, definitive diagnosis and confirmation of a coronavirus infection requires specific testing. the criteria for the diagnosis of sars from the who include (1) fever higher than 38°c or history of such in the past 2 days, (2) radiological evidence of new infiltrates consistent with pneumonia, (3) chills, cough, malaise, myalgia, or known history of exposure, and (4) positive tests for by at least one assay. as for mers, the criteria for the diagnosis according to the who include (1) fever, cough, and hospitalization with suspicion of lower respiratory tract lesion, (2) history of contact with probable or confirmed cases, (3) history of travel or residence within the arabian peninsula, and (4) positive tests by a pcr-based detection method using respiratory samples, such as nasopharyngeal swabs, nasopharyngeal or tracheal aspirates, bronchoalveolar lavage (bal) and induced sputum, or by serological tests such as the rapid immunochromatographic assay using highly selective monoclonal antibodies at room temperature. the initial criteria for the diagnosis of sars included (1) a history of travel or residence in wuhan within 2 weeks before the onset of illness, (2) exposure to patients from wuhan with fever and respiratory symptoms within 2 weeks before the onset of illness or the existence of clusters of diseases, (3) fever accompanied by radiographic features of pneumonia, a normal or decreased total number of white blood cells, or a decreased lymphocyte count, and (4) fluorescence pcr testing positive for nucleic acids of sars-cov-2. 37, 59, [105] [106] [107] early diagnosis of mers is difficult, but several highly specific and sensitive molecular and serologic assays exist for diagnosis in both animals and humans. 108 among them, a rapid immunochromatographic assay can detect mers-cov antigens based on the detection of the mers-cov nucleocapsid protein in a short time frame; the relative sensitivity and specificity of this test are 93.9 and 100%, respectively. 105 nucleic acid tests, such as real-time pcr tests, were mostly used to detect virus in the early stage of the outbreak of sars-cov-2, although this approach requires a relatively long time and is not conducive to accelerating diagnosis or large-scale application. therefore, alternative, rapid diagnostic kits for sars-cov-2 may be used. for example, the colloidal gold detection kit uses serum, plasma, or whole blood samples to detect igm/igg antibodies for diagnosis on the 7th day of infection or the 3rd day after onset and requires only 15 min. the nucleic acid detection kit for six respiratory viruses, including sars-cov-2 and influenza a virus, uses thermostatic amplifier chips for diagnosis. by detecting different infections, it may help distinguish people with influenza from those with other viral infections. a newly developed fig. 4 pathogenesis of sars-cov, mers-cov, and sars-cov-2. sars-cov and sars-cov-2 play a pathogenic role by inhibiting ace2. under the influence of renin and ace, angiotensinogen is converted into ang ii. through the at1 receptor, ang ii acts as a lung injury-promoting factor, and in some cases, may cause vascular constriction, an inflammatory response, cell proliferation, fibrosis, and apoptosis of alveolar epithelial cells, resulting in diseases such as pulmonary hypertension, pulmonary fibrosis, and acute lung injury. ace2 converts ang ii into ang (1-7), and through the mas receptor, ang (1-7) play roles in vasodilation, antiproliferative activity, and antioxidant activity. sars-cov downregulates the activity of ace2 and causes an increase in the amount of ang ii and lung injury. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of pro-inflammatory cytokines and chemokines, leading to a large number of immune cells infiltrating a patient's lower respiratory tract, causing severe inflammation and tissue damage. mers-cov can infect t-cells from human lymphoid organs and causes the peripheral blood inducing apoptosis by intrinsic and extrinsic pathways, thus avoiding host immune response detection method, nanopore targeted sequencing, also has the potential for efficiently detecting viruses in a reasonable time. moreover, it could identify 40 other respiratory viruses and monitor changes in virulence and transmissibility caused by viral mutations. 109 therapeutic agents all patients should be treated in isolation. 53 based on previous studies, drugs may be developed to inhibit cell entry. several strategies have been investigated to identify specific antivirals targeting the s protein, including mers-rbd-targeted nabs that block viral attachment, peptide inhibitors targeting s2 to prevent the formation of the fusion core, and small molecules without defined mechanisms. 110 sdpp4 has been found to bind mers-cov rbd with high affinity, suggesting that sdpp4 could serve as a blocker for mers-cov attachment to and entry into cells. 111, 112 one study reported a monoclonal antibody, 7d10, that binds to the ntd to inhibit cellular entry of mers-cov. 113 experiments have shown that 7d10 not only inhibits the binding of mers-cov to cells but also has effects after viral-cell attachment. 7d10 inhibits virus invasion by blocking the binding of the s1 subunit to the receptor and interfering with the conformational transformation of the s2 subunit when the virus fuses with the cell membrane. 113 a peptide fragment spanning residues 736-761 of the s protein exerts neutralization activity by inhibiting membrane fusion and the entry of mers-cov, suggesting that it is possible to develop vaccines targeting this neutralizing epitope. 114 griffithsin, a lectin derived from red algae, binds to oligosaccharides on the surface of sars-cov s protein and may also prove effective against sars-cov-2. 34, 115 researchers found that antibodies raised against sars-cov, such as css-2, -3, -4, and -5, could cross-neutralize sars-cov-2 by reducing s-driven cell entry, although the efficiency was lower than that observed for sars-cov. 100 meplazumab, an anti-cd147 humanized antibody, binds with the cd147 in competition with the s protein, 116 thus significantly inhibiting virus from invading cells and reducing the time of negative conversion. 82, 106 chloroquine could effectively inhibit sars-cov-2 in vitro, and hydroxychloroquine was even more potent than chloroquine. 117, 118 hydroxychloroquine could significantly help reduce the virus load, and azithromycin could reinforce its effect. 119 arbidol could also contribute to condition improvement. 120 note that these studies were generally limited and further trials and development are necessary. protease inhibitors also have the potential for coronavirus therapeutics. protease inhibitors, including disulfiram, lopinavir, and ritonavir, have been reported to be effective against sars and mers, and disulfiram, a drug for alcohol dependence, was reported to inhibit the papain-like protease (plpro) of mers-cov and sars-cov in cell cultures. 34, 108 in vitro, sars-cov can be inhibited by both 6-mercaptopurine and 6-thioguanine targeting plpro. 121 when used together with ribavirin, the protease inhibitors lopinavir and ritonavir have improved the outcomes of patients with sars compared with those achieved with the use of ribavirin alone. 122 such inhibitors are also being tested in patients infected with sars-cov-2, but whether these inhibitors effectively suppress 3clpro and plpro of sars-cov-2 remains controversial. 34 drugs such as colistin, valrubicin, icatibant, bepotastine, epirubicin, epoprostenol, vapreotide, aprepitant, caspofungin, perphenazine, prulifloxacin, bictegravir, nelfinavir, and tegobuvir bind to the main protease of sars-cov-2, and thus, are potential candidates. 123 lianhua-qingwen formula, a kind of traditional chinese medicine, has been also recently described theoretically as a potential treatment candidate against the main virus protease. 124 the serine protease inhibitor camostat mesylate could partly block sars-cov-2 infection of lung cells by inhibiting tmprss2. 100 viral rna synthesis blockers also have potential. remdesivir has broad-spectrum activity against mers-cov and sars-cov in cell cultures and animal models and is currently in clinical development for the treatment of ebola virus disease. 34, 125 it was also reported positively based on a patient with sars-cov-2 infection in the us, 126 and another study showed that it was able to inhibit the virus. 117 ribavirin is often used to treat sars and mers, but it is uncertain whether it is potent enough against covid-19. 34, 108 regardless, when used alone, ribavirin has minimal activity against sars-cov and may cause strong side effects. it is often used together with corticosteroids. 14,127 a preclinical study of galidesivir (bcx4430), an adenosine analog, revealed antiviral activity against sars-cov and mers-covcov. 115 a study of favipiravir (t-705), a guanine analog, proved its activity against sars-cov-2, and randomized trials of favipiravir plus interferon-α (chictr2000029600), and favipiravir plus baloxavir marboxil (chictr2000029544) are in progress. 34 corticosteroids may be used to treat sars patients to suppress lung inflammation; although this therapy is not associated with mortality in patients with mers, it is associated with delayed clearance of mers-cov rna. 14,128 moreover, clinical evidence does not support the use of corticosteroids for covid-19 treatment. 129 regarding antibodies, plasma therapy using convalescent plasma from fully recovered patients is effective against these coronaviruses, including sars-cov-2. 108, 130 tocilizumab (antihuman il-6 receptor) may curb immunopathology and trials are ongoing now. 131 a study of novel monoclonal antibodies against the mers-cov spike protein suggests that mabs can be utilized for the identification of specific mutations of mers-cov. 132 wang et al. provided an all-hydrocarbon-stapled peptide that likely mimics the native conformation of the c-terminal short α-helical region of the mers-cov s protein, which can block the formation of the hexameric coiled-coil fusion complex to inhibit viral-cell membrane fusion. 133, 134 current treatments for sars mainly include ribavirin, ifn α, plasma therapy, host-directed therapies, and systemic corticosteroids. 14, 41 due to the lack of clinical trial data, adequate supportive care supplemented with different combinations of drugs remains the main treatment for patients. 14, 41 for mers, despite many studies in humans, there is no consensus on the best treatment; thus, randomized clinical trials are needed to assess potential treatment options. 134 several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn and novel therapies, including polyclonal antibodies and broad-spectrum antivirals. 108 antimicrobial peptides (amps) may be used as alternative therapeutic agents, 135 and many have been effective even against bacterial proteases agents. 136 antiviral drugs with effective activity in vitro include neutralizing monoclonal antibodies, antiviral peptides, mycophenolic acid, lopinavir, ifn, ribavirin, nitazoxamide, mycophenolate mofetil (mmf), alisporivir, silvestro, and corticosteroids. 19, 21, 59 although there is no agreement on the most ideal treatment option to cure covid-19, much research activities and pursued routes are underway. potential host-targeted agents the host-directed strategy is another approach for limiting viral replication. host proteases such as cathepsin b and cathepsin l can cleave the spike protein of sars-cov. drugs such as camostat inhibit host serine proteases and then interfere with the entry of sars-cov, but they may cause many significant side effects. 14, 135 pegylated ifn α-2a and -2b may be used to stimulate innate antiviral responses in patients, and chloroquine, an approved immune modulator, was reported to have inhibitory effects against sars-cov-2 under certain conditions. 34 researchers have also proposed that some commercially available drugs with suitable safety profiles, such as metformin, glitazones, fibrates, sartans, and atorvastin, as well as nutrient supplements and biologics, might reduce immunopathology, boost immune responses, and prevent or curb ards. in addition, ongoing cellular therapies using mesenchymal stromal cells from allogeneic donors have been shown to reduce nonproductive inflammation and affect tissue regeneration and are being evaluated in phase 1/2 trials in patients with ards; these therapies may be assayed in sars-cov-2-infected patients. 40, 137, 138 expansion of antiviral t cells as cellular drugs could aid in preparing t cell products for adjunct treatment of patients with severe infection. 40 overall, there are similarities and differences among the treatments for these three coronaviruses. notably, we summarize the potential drugs and vaccines in table 2 . vaccination could be used to prevent infection or to reduce disease severity. different kinds of vaccines, such as dna vaccines, recombinant proteins, subunit vaccines, and inactivated viruses, were described. 14 however, the highly sophisticated immune evasion mechanisms of viral pathogens and their high mutation rates make human vaccine development a major challenge. 136 the four nsps (3clpro, plpro, helicase, and rdrp), which are key enzymes in the viral life cycle, and the s protein, which is responsible for receptor binding during cell entry, are attractive targets for developing vaccines against coronaviruses. 115 among them, the s protein is most commonly targeted. 61 based on previous studies, it is believed that the s protein receptor-binding domain (s-rbd) located in the s1 subunit is an important target for the development of a sars vaccine, especially the key neutralizing region, cnd. indeed, cnd can induce a strong neutralizing antibody response and crossprotection against sars-cov mutants. one study showed that a recombinant fusion protein (designated rbd-fc) containing the 193-amino acid rbd and a human igg1 fc fragment can induce highly potent antibody responses in immunized rabbits. the antibodies inhibited sars-cov infection (serum dilution of 1:10,240), and they are believed to be safer than other types of vaccines. 139 additionally, with chloroplast transgenic technology, it is possible to combine the fusion gene s-rbd and the carrier molecule cholera toxin b subunit into the tobacco chloroplast genome to obtain a chloroplast transgenic tobacco plant that stably expresses the oral sars-cov subunit vaccine. 140 for mers, vaccines based on the viral s protein include full-length s or strimer protein-based vaccines such as a full-length s-based simian adenovirus vector vaccine (chadox1) and dna vaccine (gls-5300), 61 s1-based vaccines such as an ad vector encoding mers-cov s1 extracellular domain (ad5.mers-s1) and an rabv vector encoding an s1-elicited antibody, 141, 142 rbd-based vaccines, and vaccines based on other regions. it has been confirmed that the s proteins of sars-cov and sars-cov-2 are quite similar, but researchers recently found that the three monoclonal antibodies developed to bind to the s protein of sars-cov, s230, m396, and 80r do not cross-react with the s protein rbd of sars-cov-2, simian adenovirus vector vaccine (chadox1);an ad vector encoding mers-cov s1 extracellular domain (ad5.mers-s1); an rabv vector encoding s1 elicit antibody; rbd-based vaccines 61, 138, 139, 140 overview of lethal human coronaviruses chen et al. suggesting that tailored vaccines and antibodies against sars-cov-2 must be designed. 143 dna vaccines are also quite promising. specific igg antibodies against sars-cov can be promoted by the s gene dna vaccine. as mentioned above, the s protein of sars-cov plays an important role during the pathogenic process, and synthetic peptides induced by dna vaccine in escherichia coli elicit specific antibodies against the sars-cov s protein which might provide another approach for further developing sars-cov vaccines. 42, 144 to evaluate the safety and immunogenicity of a plasmid dna vaccine (gls-5300) that expresses the s protein of mers-cov, a phase i clinical trial on healthy volunteers was conducted in 2016, but the results were not reported. another phase i trial utilizing the viral vector, chimpanzee adenovirus, oxford university #1 (chadox1), containing the mers-cov s protein expression gene was started by oxford university in january 2018. 145 in addition, camel vaccines against mers-cov are a consideration. at present, at least two promising candidate camel vaccines are undergoing development, and field trial evaluation is in progress. 108, 146 one study found that the rbd fragment covering spike residues 377-588 is a key neutralizing receptor-binding fragment and an ideal candidate for mers vaccines. 147 another potential neutralizing epitope is a peptide fragment covering 736-761 residues of the s protein which blocks the membrane fusion and cellular entry of mers-cov 114 (fig. 5) . other approaches recombinant viruses may be employed to generate an immune response against the viruses. there are two kinds of recombinant viruses which are promising for designing a protective vaccine. the first is a defective or nonpathogenic vector capable of expressing viral proteins, and the second is a vector that can be assembled in a test tube to stimulate the assembly of virus-like particles. adenosine deaminase (ada), a kind of human enzyme, could interact with dpp4, therefore, ada could compete with mers-cov as a natural antagonist when the virus attempts to bind to cells. 148 in addition, anti-dpp4 can play a similar role, 149 however, it is not practical to use this method in vivo because dpp4 has important functions in the regulation of several different signaling pathways. 150 questions concerning the coronavirus-host interactions this topic has intrigued the community. understanding certain mechanisms about virus interactions will support drug research activities. for instance, it was found that sars-cov-2 has a stronger, more rapid spreading ability than many known viruses. is it possible that it invades host cells via additional novel routes, not limited to ace2? for example, cd147 is probably involved in virus invasion. 82 is there an s-like protein involved in invasion? the affinity of the human ace2 protein for the rbd of the new coronavirus is 10-20 times higher than it is for that of the sars virus, which likely explains why sars-cov-2 spreads more swiftly. there is a distinct insert present in the peptide fragment spanning of s1/s2 loop of the sars-cov-2 s protein, which is not shared the similarity with sars-cov or any sars-related viruses. does this peptide loop impact on the entry pathway type of sars-cov-2, compared to other known betacoronavirus lineage b? furthermore, the s protein is likely cleaved during virus assembly and delivery to the cell surface by golgi-resident proprotein convertases such as furin, 151 which is different from the behavior of its close relatives. furin is found in a variety of human tissues, including the lungs, liver, and small intestine. therefore, which are exactly the cell types that sars-cov-2 may attack? yet, in relation to the last two questions, it should be noted that studies are also necessary to investigate any possibility of receptor-independent virus entry. the sars-cov-2 genome encodes nonstructural proteins, structural proteins, and accessory proteins. 3clpro, plpro, helicase, and rdrp, which belong to the first type, and the s protein, which belongs to the second, have been recognized as promising targets for developing antiviral agents against sars-cov and mers-cov, which therefore may be applied to sars-cov-2. nonetheless, the rapid identification of effective interventions against sars-cov-2 is a major challenge. however, the subject of using currently known antiviral drugs has been frequently brought up by the community as a potential short-term strategy to combat sars-cov-2. drugs affecting such targets and their status for sars-cov-2 are listed below. the main candidates as inhibitors of 3clpro include other agents for sars-cov-2 fig. 5 the targets of the different drug candidates against the three coronaviruses. common targets against the three coronaviruses are mainly the s protein and the s1/s2 subunits, pl protein, rdrp, 3cl protein, and helicase. the figure shows drug candidates (in black) and vaccines (in red). among them, remdesivir has been trending in the news recently. it inhibits the rdrp, is in phase iii for sars-cov-2, and may have an effect on the three viruses. ribavirin in combination with a pegylated interferon may also have an effect against the three viruses. ritonavir and lopinavir, which inhibit the 3clpro and are in phase iii for sars-cov-2, have an effect on both sars-cov-2 and mers-cov. dna vaccines and vaccines based on the s protein or subunits of the s protein are in development lopinavir, ritonavir, darunavir, cobicistat, and asc09f (phase iii, in combination with oseltamivir, for sars-cov-2). 152 candidates as inhibitors of plpro include thiopurine analogs, compound 6 (preclinical), and remdesivir (phase iii for mars-cov). 121, 153 favipiravir, ribavirin (randomized trial for sars-cov-2), and remdesivir are among the candidate inhibitors of rdrp. 118 previously, compounds that may interfere with atpase and helicase activities were reported (before covid-19), such as bananins, 5-hydroxychromone derivatives, and triazole derivatives (preclinical). 34, 78 candidates that may suppress viral entry by targeting the s protein include peptide p9 and α-helical lipopeptides (preclinical), 154 and those targeting the s2 subunit of the s protein mainly include hr1p, hr1m, hr1l, hr2l, hr2p, and hr2l (preclinical). 155 due to the strong affinity between ace2 receptor and the rbd of sars-cov-2, another opportunity might be through the development of antibodies to block ace2. the second method is to use a large amount of soluble ace2 to directly block the spike protein. the third method is to find a drug that directly inhibits the spike membrane fusion process, such as the aids drug enfuvirtide. the fourth approach is to identify an agent that inhibits the activity of disintegrin and metalloproteinase 17 (adam17), which may also prevent viral release and proliferation in cells and protect ace2 and the lungs. 87 mrna vaccine technology sars-cov-2 vaccines are already under development. the mrna and dna vaccines are promising approaches to prevent cellular invasion by similar coronaviruses in the future. in january 2020, the coalition for epidemic preparedness innovations (cepi) announced three partnerships to develop a new coronavirus vaccine. among them, moderna and inovio presented mrna and dna vaccine technologies, respectively. the goal of the three teams is to have at least one candidate vaccine capable of preventing coronavirus infection in 16 weeks, to be tested in a phase i clinical trial. moderna's vaccine model is designed to use mrnas to safely pre-expose the immune system to a small amount of encoded proteins usually generated by the pathogen, so that the immune system becomes prepared to fight future pathogens and prevent infection. the candidate genes developed by moderna contain mrnas, and combining multiple mrnas into a single vaccine might be useful to rapidly induce responses, which is an approach that may be applied for future, emerging pandemic threats too. at the same time, because the mrna in the cell will be degraded over time, the mrna vaccine will not continue to produce antigen components for a long time, which can avoid the risk of continued stimulation of the immune system. generally, mrna vaccines are also described as simple to prepare and to yield remarkable results. additional advantages, as well as disadvantages, of nucleic acid-based vaccines were described in detail. furthermore, the basis of traditional vaccines, proteins or polysaccharides, cannot be straight forward applied at present against many pathogens. therefore, mrna vaccines would be suitable for achieving rapid, mass production of emergency vaccines in the event of a major epidemic. however, because rna is easily degraded, inducing cells to absorb mrna quickly is a challenge. in recent years, the method for delivery of mrna into cells has been greatly improved, and stemirna and moderna have adopted nanolipid (lpp or lnp (lipid nanoparticle)) drug-loading technology. however, this is still to be developed for mrnas. furthermore, ensuring safety and effectiveness is another challenge for viral mrna vaccines, and clinical validation will have to be carried out carefully. moderna is a worldwide leader in mrna therapy, with currently nine mrna-type vaccine candidates (e.g., mrna-1273 against sars-cov-2). the national institutes of allergy and infectious diseases (niaid) of the national institutes of health (nih) and the mrna vaccine giant moderna have teamed up to bring the new coronavirus vaccine possibly to phase ii within a few months, and phase iii late this year. simultaneously, in january 2020, the translational medicine platform of dongfang hospital affiliated with tongji university cooperated with stemirna (shanghai) biotechnology co., ltd. to rapidly promote the development of a new coronavirus mrna vaccine. in february 2020, the chinese scientific research team announced that animal testing of the newly developed coronavirus vaccine has begun. if animal testing goes well, this new vaccine will enter human clinical trials within a few months. also in february 2020, zhuhai livanda biotechnology co., ltd. announced that the first batch of new coronavirus mrna vaccine standard samples completed during the spring festival had been delivered to relevant national authorities on for animal testing and efficacy verification. the researchers detected the production of target antibodies in mouse serum at day 12 following immunization. the company claimed that this was also the first time worldwide that new coronavirus vaccines developed based on mrna technology have resulted in antibodies in animals. livanda is currently pushing ahead with the project through extensive cooperation with the academy of military medical sciences, the guangdong provincial institute of supervision, and the macau university of science and technology. the antigen used in its development is the same as that used by moderna. dna vaccine technology dna vaccine technology may have many advantages (e.g., safe, fast, less technical barriers), along with potential limitations too. 156 a phase i human clinical trial of the mers-cov vaccine has proven its safety and effectiveness. 157 the dna vaccine based on the sars-cov s protein developed by the team of barney graham and gary nabel of the us-nih vaccine research center (vrc) has achieved positive results in animal experiments and a clinical phase i trial. 158, 159 inovio pharmaceuticals has accumulated extensive safety and immunogenicity data for mers-cov vaccine studies. because of the high degree of similarity between mers-cov and sars-cov-2, lessons from such a vaccine development process may be beneficial for the development of a dna vaccine against sars-cov-2. since january 2020, inovio has been collaborating with several experts and companies (some in china), and has currently already begun phase i clinical trials of the dna vaccine ino-4800. china's cansino biologics might be the leader as it was announced it has moved to phase ii testing of a vaccine called ad5-ncov. the latter is currently often being reported as a dna vaccine, but to be precise, it uses viral vectors (adenovirus) to deliver dna related to sars-cov-2. part of the project is carried out currently with the chinese military medical research institute. the vaccine candidate is constructed using genetic engineering methods and defective human type 5 adenovirus as a vector that can express the sars-cov-2 s antigen, to stimulate the body to produce strong humoral or cellular immunity. sars-cov and mers-cov belong to subgroups 2b and 2c of the betacoronaviruses, respectively, and sars-cov-2 is a new member of betacoronaviruses, distinct from sars-cov and mers-cov. sars-cov and sars-cov-2 are similar in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov-2 is ace2, plus cd147 for sars-cov-2, while that of mers-cov is dpp4 (cd26). all of them evolved a mechanism to escape the host cell immune system. sars-cov and sars-cov-2 affect the ras system by suppressing ace2, leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. sars-cov, mers-cov, and sars-cov-2 infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. current treatments for sars mainly include ifn-α, antiviral treatments (e.g., ribavirin), plasma therapy, host-directed therapies, and systemic corticosteroids. for mers, several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn, and novel therapies, including polyclonal antibodies, broad-spectrum antivirals, and amps. for covid-19, candidates mainly include drugs targeting the s protein, nonstructural proteins (3clpro, plpro, helicase, and rdrp), and viral rna synthesis blockers such as remdesivir and ribavirin. vaccines such as dna vaccine, mrna vaccine, and recombinant vaccines are being rapidly developed. so far this century, human beings have experienced several epidemic outbreaks, and each outbreak had a negative impact at different levels, including health, economy, and even psychology and human behavior. in the future, more precautious measures should be available to guide 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to s1 protein that blocks receptor association potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies exceptionally potent neutralizationof middle east respiratory syndrome coronavirus by human monoclonalantibodies interaction between heptad repeat 1 and 2 regions inspike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors fusion mechanism of 2019-ncov andfusion inhibitors targeting hr1 domain in spike protein the authors gratefully acknowledge the financial support from the national natural science foundation of china (grant no. 31870836), and the 1.3.5 project for disciplines excellence, west china hospital, sichuan university (zyyc20005 to w.c.). competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-349643-jtx7ni9b authors: uyeki, timothy m.; erlandson, karl j.; korch, george; o’hara, michael; wathen, michael; hu-primmer, jean; hojvat, sally; stemmy, erik j.; donabedian, armen title: development of medical countermeasures to middle east respiratory syndrome coronavirus date: 2016-07-17 journal: emerg infect dis doi: 10.3201/eid2207.160022 sha: doc_id: 349643 cord_uid: jtx7ni9b preclinical development of and research on potential middle east respiratory syndrome coronavirus (mers-cov) medical countermeasures remain preliminary; advancements are needed before most countermeasures are ready to be tested in human clinical trials. research priorities include standardization of animal models and virus stocks for studying disease pathogenesis and efficacy of medical countermeasures; development of mers-cov diagnostics; improved access to nonhuman primates to support preclinical research; studies to better understand and control mers-cov disease, including vaccination studies in camels; and development of a standardized clinical trial protocol. partnering with clinical trial networks in affected countries to evaluate safety and efficacy of investigational therapeutics will strengthen efforts to identify successful medical countermeasures. f rom september 2012 through april 27, 2016, a total of 1,728 laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) infections, leading to 624 deaths (36% case-fatality proportion), had been reported to the world health organization (who) (1) . most infections (75%) have been identified in saudi arabia (2) . zoonotic transmission from exposure to mers-cov-infected arabian camels, known as dromedaries, or their raw milk and limited, nonsustained human-to-human transmission have been reported, including large outbreaks in healthcare facilities (3) (4) (5) . the recovery of infectious mers-cov in virus cultures of specimens from bed sheets, bedrails, intravenous fluid hangers, and radiograph equipment indicates the potential for fomite transmission of the virus in hospitals providing care for mers-cov patients (6) . however, sustained human-to-human transmission has not been documented, and some case-patients have no identified source of exposure to mers-cov. as of april 2016, a total of 26 countries had reported locally acquired or exported cases from the arabian peninsula, including 2 cases in the united states identified during may 2014 in healthcare personnel who became ill after working in saudi arabia (7, 8) . a traveler who visited saudi arabia, qatar, the united arab emirates, and bahrain and then returned to south korea infected with mers-cov in mid-2015 triggered 184 mers-cov cases, resulting in 38 deaths in multiple health facilities and 1 additional case in a person who traveled to china (9, 10) . human infections with mers-cov are expected to continue to occur on the arabian peninsula because of the prevalence of mers-cov in dromedaries and the cultural importance of these camels (i.e., for food, milk, and racing purposes) in the region. during the 2003 outbreak of severe acute respiratory syndrome (sars) in china, civet cats, the suspected reservoir of sars coronavirus (sars-cov), were culled aggressively; no outbreaks were identified after 2004. in contrast, culling of camels is culturally impractical in the middle east, and mers-cov zoonotic infections of humans have continued since 2012. the potential for emergence of mers-cov mutations that could facilitate sustained community transmission and global dissemination cannot be predicted. no vaccines against or specific treatments for human infection with sars-cov, mers-cov, or other coronaviruses have been approved. since 2013, efforts have focused on furthering development of animal models, vaccines, and therapies against mers-cov (11,12). in this report, we update the current state of development for mers-cov medical countermeasures, including regulatory challenges in the united states, and draw attention to areas in immediate need of increased infrastructure support for development of these countermeasures. persons at higher risk for more severe disease, including persons >65 years of age and those with chronic medical conditions. therapeutic drugs with specific activity against mers-cov (e.g., antiviral drugs, immunotherapeutic treatments) or that target the host immune response could be used for treatment of human illness caused by mers-cov infection or for pre-or postexposure prophylaxis. before human clinical trials of potential mers-cov medical countermeasures are started, proof-of-concept data must be obtained from in vivo studies of experimentally infected animals. such data may indicate a product's potential efficacy and provide a mechanism for selection of available medical countermeasure candidates. in addition, mers-cov vaccines could be developed for animals and used for vaccination of dromedaries on the arabian peninsula and in source countries for camel imports to the horn of africa to reduce mers-cov transmission among camels and possibly from camels to humans. preclinical development of mers-cov medical countermeasures has been hindered by several factors, including limited data on the natural history of mers-cov infection in humans; the lack of a small animal model that is naturally susceptible to mers-cov; and the inability to consistently replicate severe human disease in mers-cov-infected nonhuman primates (nhps). another factor is limited access to clinical samples and recent virus isolates; for example, a mers-cov strain isolated from a patient in 2012, rather than a more recently isolated strain, is currently used by most investigators worldwide. small animal and nhp models are useful for testing potential medical countermeasures for efficacy (table 1) . studies in mice, both dipeptidyl peptidase-4 (dpp4 or cluster differentiation 26) transduced and transgenic, and in rabbits, hamsters, and ferrets have been reviewed elsewhere (16, 20, 21) . these small animal models have been used for screening potential mers-cov medical countermeasures (13, 14, 22) . the major nhp models under development include rhesus macaques and common marmosets (17, 18, 23) . overall, common marmosets appear to be better suited than rhesus macaques for therapeutic studies designed to target severe disease because marmosets show slightly slower onset of illness and longer duration and severity of disease and their small size requires lower doses of therapeutic drugs. however, the marmoset model has not been standardized and is not consistent between laboratories (18, 24, 25) . furthermore, the size of marmosets substantially limits sequential blood sampling for virologic or pharmacokinetic testing. challenges to the development of nhp models include determination and standardization of the optimal mers-cov challenge dose and of the volume and route of exposure, as well as the limited availability of nhps, especially marmosets. large animal models in development include camels and camelids such as alpacas (19, 26, 27) . these models may be vital in understanding the virology and immunology of mers-cov infection in dromedaries, a natural host. in addition, serologic evidence of mers-cov infection in alpacas has been reported in qatar (28) . major gaps for all animal models include a lack of consensus and availability of the optimal animal model to replicate severe human illness from mers-cov infection; limited availability of currently or recently circulating mers-cov strains; the lack of understanding of clinically relevant symptoms that can be incorporated into clinical scores or used as a signal to begin treatment in animal models; and competition for funding, laboratory space, availability of animals, and expertise with other emerging or reemerging infectious diseases, such as ebola virus disease and zika virus disease. (29, 30) . the secretary of the us department of health and human services declared a potential public health emergency on may 29, 2013, regarding mers-cov infection that could have a high potential to affect national security or the health and security of us citizens living abroad. the us food and drug administration (fda) subsequently issued an emergency use authorization to the centers for diseases control and prevention (cdc) for an in vitro molecular diagnostic test to diagnose mers-cov infection in multiple types of clinical specimens from symptomatic patients. the use of this test was later expanded to include the ability to test asymptomatic contacts of a person infected with mers-cov who traveled from saudi arabia to the united states. the cdc made this test available to multiple us public health laboratories, the us department of defense, and who laboratories worldwide. although the test has been distributed extensively, it is limited in terms of the cdc's ability to scale up the supply of reagents to support a surge in mers-cov cases in the united states and in other countries where the test has been made available. therefore, an emergency use authorization was issued on july 17, 2015, for the commercially developed realstar mers-cov rt-pcr kit u.s. (altona diagnostics gmbh, hamburg, germany) for use in the in vitro qualitative detection of mers-cov rna in tracheal aspirate or tracheal secretion samples (31) . although this commercial assay is a first step in bridging the diagnostic test availability gap in case of a surge scenario, the current coverage, at least in the united states, is insufficient until alternative, fda-cleared commercial tests are available (table 2) . a worldwide gap exists in the lack of readily available, simple, rapid, and accurate diagnostic tests for use in outpatient and inpatient clinical settings where the ability of the facility to use currently available, higher complexity molecular tests is limited. the lack of commercial development of mers-cov assays may be partially related to the limited availability of clinical specimens and mers-cov isolates from infected patients. availability of serum specimens from rt-pcrconfirmed mers-cov patients who survived can help facilitate development of serologic tests. if paired acute and convalescent serum samples are available, serologic tests can be used to confirm mers-cov infection when viral shedding is not detectable, and for surveillance purposes such as measuring population exposures and immunity to mers-cov infection. no investigational therapeutic drugs have been evaluated for treatment of mers-cov patients in prospective randomized controlled clinical trials. potential therapeutic drugs for mers-cov patients include available approved drugs with nonspecific properties, such as immunomodulators, small-molecule drugs with broad antiviral activity, repurposed fda-approved small-molecule drugs that show activity against mers-cov in vitro (table 3 ) (34, 35) , and newly developed monoclonal or polyclonal antibody therapies with specific activity against mers-cov (table 4 ) (54). one promising approach has been to investigate libraries of drugs approved by the fda and the european medicines agency. considering development times and manufacturing requirements for new products, repurposing of existing drugs might potentially facilitate a rapid response to outbreaks of emerging viruses (see regulatory section for a discussion on repurposing). other early-stage work on mers-cov therapeutics includes studies focusing on the essential viral replication steps of fusion, proteolysis, and rna polymerization (table 3 ) (54) . immunotherapeutics under evaluation consist of convalescent plasma and monoclonal and polyclonal antibodies. most of the monoclonal antibodies in development have specific neutralizing activity against the mers-cov spike protein (55, 56) . platforms are being developed to rapidly discover monoclonal antibodies, either from fully human convalescent blood or from transgenic animals, which can be manufactured on a large scale and are likely to have a good safety profile. the most advanced immunotherapeutic for mers-cov uses a transchromosomal bovine production system to produce fully human polyclonal mers-cov antibodies; a phase i study of this product was recently implemented (57; https://clinicaltrials.gov/ct2/ show/nct02788188). preliminary results from immunoprophylaxis or treatment studies have shown efficacy of fully human monoclonal or polyclonal antibodies in mers-cov-infected mice and nhps ( profile and a defined set of preclinical toxicology studies, challenges to development of immunotherapeutics include ensuring the absence of antibody-dependent enhancement of disease and reducing the risk for generation of escape mutant viruses that would be resistant to treatment. development of mers-cov candidate vaccines was initiated by the national institute for allergy and infectious diseases at the national institutes of health, academic investigators, and several companies (table 5 ). most candidate vaccines are still being evaluated in animal models. they have generally targeted the spike protein of mers-cov and are recombinant virus, subunit, dna, or virus-like vector vaccines (60,63-67). one live-attenuated mers-cov candidate vaccine is in early development (66) . preliminary studies for several other mers-cov vaccine candidates have been initiated, and early results demonstrate immunogenicity; 2 have progressed to nhp challenge, and a phase 1 clinical study in adults of 3 different doses of a dna plasmid vaccine that expresses the mers-cov spike protein was started in january 2016 (61) . ongoing assessment of antigenic evolution of circulating mers-cov strains is essential for informing vaccine development (68) . a concern that must be addressed in the development of mers-cov vaccines is the potential for causing antibody-dependent enhancement of disease upon virus challenge, such as what was observed with a sars-cov candidate vaccine upon sars-cov challenge (69) . the lack of a precedent of coronavirus vaccines for humans poses another challenge for the evaluation of mers-cov vaccines for humans, although vaccines against other animal coronaviruses are safe and in use in animals. considering the cultural importance of dromedaries on the arabian peninsula for meat, milk, and racing, prevention of camel-to-camel mers-cov transmission and reduction of spread from dromedaries to humans by camel vaccination is being investigated by government, academic, and commercial investigators (table 6 ). young camels appear to be at high risk for mers-cov infection and could be a priority group for vaccination (73, 74) ; the loss of maternal mers-cov antibodies ≈5-6 months after birth suggests a short time window for vaccination (75) . a major challenge to this approach is that dromedaries can be reinfected with mers-cov; a study by farag et al. found no correlation between mers-cov rna levels and neutralizing antibodies in camels (76) , suggesting that antibodies may not be protective against infection. because older camels can be reinfected, a camel vaccination strategy may require multiple dosing and booster vaccination to increase effectivein the united states and overseas (19) . in addition, 3 doses of a dna vaccine containing the mers-cov spike protein induced humoral immunity in dromedaries (60) . in a recent study, a modified vaccinia virus ankara vaccine that expresses the mers-cov spike protein was administered intranasally and intramuscularly to dromedaries; when challenged intranasally with mers-cov, vaccinated dromedaries had fewer signs of respiratory infection and lower mers-cov titers in the upper respiratory tract compared with unvaccinated dromedaries (77) . alpacas (new world camelids) are being investigated as a suitable proxy for camels because of the lack of available dromedaries in the united states, the high cost of acquiring dromedaries, and the relatively smaller size of alpacas (26, 27) . regulatory considerations for mers-cov medical countermeasures in the united states are focused on a pathway to human clinical trials for drugs and vaccines through submission of investigational new drug applications. investigational new drug submissions must adhere to requirements set forth in the code of federal regulations, title 21, part 312 (21 cfr 312; http://www.ecfr.gov/cgibin/text-idx?tpl=/ecfrbrowse/title21/21cfr312_main_02. tpl). several guidance documents exist on the fda website related to virology, microbiology, pharmacology and toxicology, and clinical and medical considerations (78). the most appropriate approval pathway is likely to be product-specific and will require consideration of existing product data, proposed intended use and population for use, and validated endpoints for efficacy predictive of clinical benefit, if any. likewise, data needed for consideration of an emergency use authorization, including dose finding and dose ranging, duration, and safety, can be obtained through sources such as investigational new drug clinical trials. repurposing of drugs approved by the fda for other illnesses for a mers-cov indication can potentially be expedited or accelerated if 1) the mechanism of action for antiviral activity is defined, 2) there is no change to the approved final drug form and route of administration, 3) dosing does not exceed the currently approved dose and duration for the currently indicated population and adequate pharmacokinetics data support this dosing, and 4) the risk-benefit profile is acceptable for the intended population and indication. for example, the risk-benefit profile for an approved drug with an oncology indication may be unacceptable if the drug is repurposed for administration to a healthy population for mers-cov postexposure prophylaxis. however, data requirements to initiate human trials will depend on the characteristics of the drug product and its intended use against mers-cov. as such, sponsors should consider prioritizing drug development on the basis of the totality of scientific evidence and merit of the drug alone, not on whether the drug has been previously approved. in the absence of a standardized and accepted animal model that simulates human disease from mers-cov infection, it is unclear how the fda may be able to expedite licensure or approval when data are lacking. the best approach may be collection of preclinical safety data and implementation of adaptive human clinical trials. this approach was taken for medical countermeasures in response to the 2013-2016 ebola virus disease outbreak. for diagnostic devices, the current emergency use authorization pathway serves as a fast approach to make products available for emergency public health purposes. after an emergency has been terminated, premarket notifications for these products should be submitted to fda for a more thorough evaluation as 510(k)s (http://www.fda.gov/ e6 emerging infectious diseases • www.cdc.gov/eid • vol. 22, no. 7, july 2016 medicaldevices/productsandmedicalprocedures/deviceap provalsandclearances/510kclearances/default.htm). the overarching goal for clinical research of mers-cov patients is to optimize clinical management and to identify effective therapies to improve survival. although clinical data on some mers-cov patients have been published in case series (58, 79, 80) , there is a need for much more epidemiologic, clinical, virologic, and immunologic data to improve the limited understanding of the pathogenesis of mers-cov infection in humans. gaps include information on viral load and duration of viral shedding in blood, urine, respiratory, and other clinical specimens from infected persons; understanding of the innate and adaptive immune response to mers-cov infection; pathology data on the distribution of mers-cov in respiratory and extrapulmonary tissues in fatal cases; information from autopsies of persons who died of mers-cov; and an overall improved understanding of the pathogenesis of mers-cov in humans. only 1 study has investigated mers-cov infection in autopsy tissues of a patient who died from the disease (81) . collaborations are especially needed to pool and systematically collect serial clinical specimens from mers-cov patients for virologic, immunologic, and biomarker analyses to correlate with clinical illness, and to conduct long-term follow-up of survivors of severe disease (82) (83) (84) . detailed understanding of host factors and cofactors associated with disease severity from asymptomatic infection to fatal illness is needed. efforts to promote international sharing of clinical specimens and mers-cov isolates are needed to foster development of diagnostics, therapeutics, and vaccines. use of standardized clinical data collection instruments and common biologic sampling protocols for serial prospective data collection will facilitate data pooling from mers-cov cases and comparisons across clinical sites and countries. global collaborations among clinical networks are also needed to implement clinical trials, preferably randomized controlled clinical trials, of mers-cov investigational therapeutics (82) (83) (84) (85) . without an international agreement on protocols and systematic standardization of case reporting and data collection methods, haphazard or anecdotal reporting and analysis of disease course and outcome may continue. who and the international severe acute respiratory and emerging infection consortium are collaborating in adapting standardized protocols for controlled clinical trials for mers-cov (83) . prospective controlled clinical trials (ideally randomized clinical trials) of potential mers-cov therapies and vaccines in humans are needed urgently; however, there is uncertainty in estimating timelines for the development of potential mers-cov medical countermeasures because of the need to further characterize existing and new animal models, the unpredictability of demonstrating a favorable risk-benefit outcome during preclinical testing, and competition for resources with other emerging infectious diseases. in addition, the risk for antibody-dependent enhancement of disease may interrupt the timeline for conducting human clinical trials of mers cov vaccines and immunotherapeutics. researchers of all potential mers-cov medical countermeasures should have preclinical toxicology data available before initiating human clinical trials. although animal efficacy data are not technically required before implementing human clinical trials of potential countermeasures, such data are considered important for identifying the most promising medical countermeasure candidates, justifying risk in human volunteers, and informing the design of future clinical studies. timeframes for the production of specimen panels and repositories to aid commercial diagnostic development are also contingent on obtaining adequate funding and clinical samples. although preclinical development and research on potential mers-cov medical countermeasures has achieved appreciable progress to date, such development is preliminary, and substantive challenges must be overcome before most potential countermeasures are ready for human emerging infectious diseases • www.cdc.gov/eid • vol. 22, no. 7, july 2016 e7 results of substantial progress in establishing the infrastructure and platforms for preclinical and advanced clinical development of countermeasures can serve as a model to enable more timely response to other emerging infectious diseases of global public health concern in the future. world health organization. emergencies preparedness, response: middle east respiratory syndrome coronavirus saudi arabia world health organization. summary of current situation, literature update and risk assessment hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea first confirmed cases of middle east respiratory 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syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels animal models for sars and mers coronaviruses a comparative review of animal models of middle east respiratory syndrome coronavirus infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection animal models of middle east respiratory syndrome coronavirus infection treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a non-human primate model of common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas. emerg infect dis experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus. emerg infect dis mers-cov infection of alpaca in a region where mers-cov is endemic viral shedding and environmental cleaning in middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications regulatory and policy framework-emergency use authorization; middle east respiratory syndrome coronavirus (mers-vov) eus information assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections first international external quality assessment of molecular diagnostics for mers-cov screening of an fda-approved compound library identifies four small-molecule inhibitors of middle 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modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus camels emit dangerous mers virus, csu confirms mers surges again, but pandemic jitters ease immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice mers-cov in upper respiratory tract and lungs of 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international severe acute respiratory and emerging infection consortium. severe acute respiratory infection data tools open source clinical science for emerging infections clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance we thank john beigel, wendy carter, david cho, su-young choi, eric donaldson, heinz feldmann, barney graham, lisa hensley, cynthia kelley, peter miele, vincent munster, david spiro, kanta subbarao, and melissa willis for participating in foundational discussions and for help assessing potential therapeutic drugs and methods; vaccines under development or investigation; timelines for animal studies; scale-up of medical countermeasures; when human trials are to be started; regulatory issues for investigational new drug (ind) and emergency use authorization (eua) applications, and clinical studies; and safety issues and prioritization for clinical trials. we also thank dean erdman for insightful discussions on mers-cov diagnostic assay development, rick bright, tom dreier, and byron rippke for helpful commentary, and thuy phan for organizational assistance.dr. uyeki is the chief medical officer for the influenza division, national center for immunization and respiratory diseases, cdc. his research interests are in the epidemiology and clinical management of influenza and emerging infectious diseases. key: cord-338436-0z828org authors: tzou, philip l.; tao, kaiming; nouhin, janin; rhee, soo-yon; hu, benjamin d.; pai, shruti; parkin, neil; shafer, robert w. title: coronavirus antiviral research database (cov-rdb): an online database designed to facilitate comparisons between candidate anti-coronavirus compounds date: 2020-09-09 journal: viruses doi: 10.3390/v12091006 sha: doc_id: 338436 cord_uid: 0z828org background: to prioritize the development of antiviral compounds, it is necessary to compare their relative preclinical activity and clinical efficacy. methods: we reviewed in vitro, animal model, and clinical studies of candidate anti-coronavirus compounds and placed extracted data in an online relational database. results: as of august 2020, the coronavirus antiviral research database (cov-rdb; covdb.stanford.edu) contained over 2800 cell culture, entry assay, and biochemical experiments, 259 animal model studies, and 73 clinical studies from over 400 published papers. sars-cov-2, sars-cov, and mers-cov account for 85% of the data. approximately 75% of experiments involved compounds with known or likely mechanisms of action, including monoclonal antibodies and receptor binding inhibitors (21%), viral protease inhibitors (17%), miscellaneous host-acting inhibitors (10%), polymerase inhibitors (9%), interferons (7%), fusion inhibitors (5%), and host protease inhibitors (5%). of 975 compounds with known or likely mechanism, 135 (14%) are licensed in the u.s. for other indications, 197 (20%) are licensed outside the u.s. or are in human trials, and 595 (61%) are pre-clinical investigational compounds. conclusion: cov-rdb facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies prioritize the most promising compounds and repurposed drugs for further development. cov-rdb contains four main types of antiviral experimental data, six main lookup/explanation tables, and a registry of ongoing or planned clinical trials. the four main types of antiviral experimental data include (i) cell culture and entry assay experiments; (ii) biochemical experiments; (iii) animal model studies; and (iv) clinical studies. the six main lookup/explanation tables provide information on viruses, virus strains/isolates, tested compounds, compound targets, cell types, and animal models. cov-rdb data are stored in a postgresql relational database, but there is not necessarily a oneto-one relationship for the tables displayed on the web and their underlying database structure. indeed, several of the website tables contain information from more than one underlying database table. as of 14 august 2020, the cov-rdb contains data from more than 1800 virus cell culture experiments, 465 entry assay experiments, 519 biochemical experiments, 259 animal model experiments, and 71 clinical studies from more than 310 peer-reviewed publications and 90 preprints. research articles are identified through incremental daily searches of pubmed and biorxiv using the search term "coronavirus" and by citations identified through reading these papers. publications containing experimental data are imported into a staging zotero database and then annotated to extract the data described in the sections below. preprints that are subsequently published in a peer-reviewed journal are identfied using a computer script that parses datasets downloaded from the stephen b. thacker cdc (centers for disease control and prevention) library the cell culture experiments table contains 13 fields, including four fields present in each of the experiment tables: reference, compound, virus category, and virus isolate/strain. the nine fields unique to the cell culture experiments table include six that describe experimental conditions and the three experiment results are the half-maximal effective concentration (ec 50 ), percent inhibition, and the 50% cytotoxic concentration (cc 50 ). the ec 50 can only be determined using a series of compound dilutions. while the ec 50 is usually reported as µm, inhibitory activity for interferons is also often reported as international units (iu)/ml and inhibitory activity for monoclonal antibodies is often reported as ng/ml. the ec 50 is available for the vast majority of in vitro cell culture experiments. however, for a few experiments, the experimental setup involved a single compound concentration (rather than a dilution series). for these experiments, the percent virus inhibition with the single compound concentration is reported. there are two tables for entry assay experiments-one for pseudovirus entry assays and another for cell-cell fusion assays. the pseudovirus assay table contains the following six unique fields: (i) pseudovirus vector, (ii) pseudovirus number, (iii) target cell type, (iv) time to addition of drug, (v) indicator of virus replication, and (vi) ec 50 . in the pseudovirus experiments, the virus strain is a virus construct composed of a virus that does not require a biosafety level 3 (bsl-3) laboratory, such as vesicular stomatitis virus (vsv) or human immunodeficiency virus type 1 (hiv-1), into which the coronavirus spike (s) gene has been cloned. this construct also has a reporter gene such as luciferase or gfp. the cell-cell fusion assay table contains the following seven unique fields: (i) effector cell type, (ii) effector cell number, (iii) target cell type, (iv) target cell number, (v) time to addition of drug, (vi) indicator of virus replication, and (vii) ec 50 . the biochemical experiments table contains two unique fields: the biochemical target and the half maximal inhibitory concentration (ic 50 ). the biochemical target is usually one of the virus enzymes including rna-dependent rna polymerase (rdrp), main protease (also called 3c-like protease; 3cl pro ), papain-like protease (pl pro ), and helicase. however, cell-free assays that test inhibitors of the spike (s) protein binding to angiotensin converting enzyme 2 (ace2) are also included. the animal model experiments are characterized by comparisons between a group of animals receiving a treatment intervention either shortly before or after virus infection and a group of untreated virus-infected control animals. the animal model experiments table has two parts-experimental conditions and experimental results. the experimental conditions include the (i) animal model, (ii) size and route of virus challenge, (iii) treatment intervention, (iv) treatment dosage, (v) treatment timing in relation to the addition of virus, (vi) number of treated subjects, and (vii) number of control subjects. the experimental results, which often depend on the study, include endpoints such as mortality, weight loss, fever, respiratory rate, lung pathology, and virus load measurements. the reduction of endpoint severity is reported on an ordinal scale ranging from 0 to 3. there are more than 59 references containing more than 259 animal model experiments, nearly all involving sars-cov-2, sars-cov, or mers-cov. approximately 70% of the studies involve mice; 10% involve non-human primates (rhesus macaques, marmosets, and cynomolgus macaques); and 20% involve hamsters, ferrets, cats, dogs, or rabbits. the most commonly studied interventions have included monoclonal antibodies, fusion inhibitors, interferons, and the nucleoside analog polymerase inhibitors. the clinical studies are represented using several enumerated and free-text fields. the enumerated fields include the reference, virus category, and type of study (e.g., observational, randomized trial, randomized placebo-controlled trial). the free-text fields include descriptions of the interventions and regimen details, the study population and methods, and the study findings. cov-rdb does not randomized trial, randomized placebo-controlled trial). the free-text fields include descriptions of the interventions and regimen details, the study population and methods, and the study findings. cov-rdb does not provide an assessment of study quality such as validity and risk of bias as there are other research groups providing this type of assessment. antiviral data on coronaviruses other than sars-cov-2 provide insight into the robustness of an antiviral compound, in that, compounds that are active against multiple viral species will be more likely to inhibit future pandemic coronaviruses and will be less vulnerable to the development of drug-resistance mutations. indeed, for many drug targets, such as the virus rdrp and 3cl pro enzymes, and for host processes upon which coronaviruses depend, inhibitory compounds will likely have broad-spectrum activity. cov-rdb contains antiviral data for six categories of coronaviruses: sars-cov-2, sars-cov, mers-cov, endemic human coronaviruses, bat coronaviruses, and non-bat mammalian coronaviruses. sars-cov-2 (37%), sars-cov (31%), and mers-cov (17%) account for 85% of the data. however, the proportion of data associated with sars-cov-2 is rapidly increasing. figure 2 shows the distribution of study types for sars-cov-2, sars-cov, and mers-cov. sars-cov and sars-cov-2 belong to the same betacoronavirus 2b (sarbecovirus) clade, and their amino acids are approximately 97% identical in the rdrp and 3cl pro enzymes and 84% identical in the spike protein. in contrast, mers, a clade 2c betacoronavirus, is approximately 75% identical to sars-cov and sars-cov-2 in the rdrp gene, 60% identical in the 3cl pro gene, and 40% identical in the s gene. within each of these three viruses, there is little diversity, with median pairwise distances ranging between 0% and 0.2%. the four endemic human coronaviruses include two clade 2a betacoronaviruses and two alphacoronaviruses. bat coronaviruses are distributed widely among different clades [2, 3] . indeed, 4 of the 9 betacoronavirus clades and 7 of 11 coronavirus clades are found only in bats. the mammalian coronaviruses include murine hepatitis virus (mhv), which is a longstanding experimental model for coronavirus infection, and several other coronaviruses that have been studied because they are important livestock diseases [4] . although infectious bronchitis virus is an avian gammacoronavirus, we have included it in the non-bat mammalian coronavirus category. cov-rdb uses the terms isolate and strains to describe the different viruses used in antiviral studies, although "strains" is usually reserved for describing isolates that have distinct phenotypic properties [5] . where possible, isolates are named according to the recommendations from the international committee on taxonomy of viruses [6] , i.e., virus/host/location/isolate/date. most sars-cov-2 isolates are nearly identical to one another with the upper limit for the pairwise amino acid distance being about 0.1%, although this number varies depending upon the gene [7] . therefore, the biological significance of the isolate used for a particular study is not known. however, for some treatments such as monoclonal antibodies, changes in the sequence encoding the relevant epitope, specifically in the s protein receptor binding domain may prove to have biological and clinical significance [8, 9] . although sars-cov resulted from at least two zoonotic introductions from civet cats [4] , and although mers-cov resulted from multiple zoonotic introductions from dromedary camels, these viruses also demonstrate little genetic variability. several of the most commonly used isolates have been cloned, either as intact viruses (e.g., by plaque purification or limiting dilution) or by constructing a cdna copy representing a single sequence variant. modification of these clones, such as selection of a resistant variant in vitro [10] or introduction of a reporter gene like gfp or luciferase, presumably retains the characteristics of the original parental virus isolate or strain [11] . commonly used isolates that have been cloned and manipulated in the laboratory include mers-cov/human/amsterdam/emc/2012 [12] , sars-cov/human/hanoi/urbani/2003 [13] , sars-cov-2/human/usa/wa1/2020 [14] , and sars-cov-2/human/munich/929/2020 [15] . the cell lines table provides descriptions for the cell lines used in cell culture and entry assay experiments. it contains four fields: (i) the cell line's commonly used name, (ii) the source of the cell line, (iii) closely related cell lines, and (iv) a description of the cell line and one or more of the closely related cell lines. the most commonly used cell lines for sars-cov and sars-cov-2 include a variety of different vero cell clones [16] [17] [18] [19] [20] , huh7 [16, 21] , caco-2 [22] , calu-3 [23] , and 293t/ace2 cells [16] [17] [18] ( table 1) . while each of these cell lines expresses ace2, only calu-3 cells were originally derived from lung epithelial cells. the 293t cells are typically used for cell-cell fusion and pseudovirus entry experiments. several studies have also used human alveolar epithelial cells or a variety of different respiratory system or kidney organoids [24, 25] . the cell lines used for mers-cov are similar, with the main exception that 293t/dpp4 (dipeptidyl peptidase 4) cells are used instead of 293t/ace2 cells because dpp4 (aka cd26) is the mers-cov receptor [18] . vero cells support the replication of many viruses often producing a visual cytopathic effect [16] [17] [18] . they express ace2, the receptor for sars-cov and sars-cov2, and dpp4, the receptor for mers-cov. although vero cells are ifn-deficient, they express the ifn-α/β receptor and thus retain the ability to respond to exogenous ifn [19] . vero e6 cells engineered to express greater amounts of tmprss2 produce higher sars-cov-2 titers of sars-cov-2 [20] . drugs that target tmprss2 are often inactive in vero cells. human lung epithelial cell line calu-3 cells form differentiated pseudostratified columnar epithelia highly permissible to coronavirus infection. they are polarized with an apical domain facing the airway lumen and a basolateral domain facing internally. they produce a visual cytopathic effect. the 2b4 clone has high ace2 expression. they are often used for the preclinical development of respiratory drugs [23] . heterogeneous human epithelial colorectal adenocarcinoma caco-2 cells are considered to be more pharmacologically relevant than vero cells for some studies because of their human origin [22] . human hepatoma huh-7 cells express ace2 and tmprss2, yet do not support levels of replication as high as vero cells [16, 21] . the 293t cells are derived from the human embryonic kidney 293 cell line. 293t cells contain the sv40 large t-antigen, which facilitates replication of transfected plasmids containing the sv40 origin of replication. the 293t/ace2 cells are transfected to express ace2 and have been used for many sars-cov cell-cell fusion and pseudovirus entry inhibitor studies [16] [17] [18] . human airway epithelial cells differentiated human airway cells have occasionally been used to study antiviral agents, although they are more commonly used to study viral pathogenesis [24, 25] . tmprss2-transmembrane serine protease 2; ace2-angiotensin converting enzyme 2; ifn-interferon; dpp4-dipeptidyl peptidase 4; sv40-simian virus 40. the over 10 different animal models used in experiments described in the cov-rdb include three non-human primate models (rhesus macaques, cynomolgus macaque, and marmosets), multiple transgenic and non-transgenic mouse models, and several additional rodent models including hamsters and ferrets [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . the transgenic mice have been modified in multiple ways, including to express hdpp4 so that they can be infected with mers-cov, to knock out the interferon (ifn)-α/β receptor to compromise innate immunity [44] ; to knock out recombination activating gene 1 (rag1) to compromise adaptive immunity [45] ; to knock out carboxylesterase 1c, which causes poor plasma stability of remdesivir; and to express human rather than mouse ace2 [39] [40] [41] . table 2 describes the utility of the most common non-human primate and mammalian models for studies of the pandemic coronaviruses. pathological changes observed in the aged mouse model infected with sars-cov more closely resemble those observed in humans [29] . rag −/− mice lack t and b cells and lack adaptive immunity and experience prolonged coronavirus shedding [45] . ifnar −/− mice are vulnerable to greater coronavirus disease severity [44] . there are many hace2 transgenic mouse models. these mice are more likely to experience weight loss, detectable virus loads, and interstitial pneumonia following challenge with sars-cov-2 than those with the murine ace2 receptor [39] [40] [41] . rhesus macaque infection causes a self-limiting disease associated with virus replication. radiographic and pathologic examination of sars-cov-2-infected animals display evidence of pneumonia [26, 30, 32, 42] . cynomolgus macaque infection results in a productive infection in respiratory epithelial cells. symptoms are minimal but virus shedding can last up to 2 weeks. chest radiographs reveal unifocal or multifocal pneumonia. autopsy reveals variable amounts of foci of diffuse alveolar damage [31, 33] . rag-recombination activating gene; ifnar-interferon-α/β receptor; hace2-human angiotensin-converting enzyme 2. the target table has two main fields: name and description. the target classification organizes drugs, treatments, and compounds according to the virus or host process targeted by a compound including virus enzymes, virus entry into cells, host immunological responses, and other host processes. there are three virus enzyme inhibitor classes: rdrp, protease (including 3cl pro , pl pro ), and helicase inhibitors. there are four inhibitor classes targeting virus entry: convalescent plasma and polyclonal antibody preparations, monoclonal antibodies, miscellaneous other compounds that inhibit virus receptor binding, and fusion inhibitors. there are two classes that target host immunological responses: interferons and other potential immunostimulatory compounds. although there are potentially many mechanisms by which targeting a host processes may interfere with virus replication, we have divided these into two broad categories: host protease enzymes used by coronaviruses to cleave the spike protein, thus, priming it for fusion and other host proteins or pathways utilized by coronaviruses. the classification of host-acting compounds is likely to continue to evolve as mechanistic pathways become better defined. table 3 describes each of the targets and compound classes described above. figure 3 shows the distribution of experimental data types according to target. the database contains experiments involving approximately 1650 compounds. more than 1240 of these compounds appear in the online compounds tables, which contain the following fields: (i) name, (ii) synonyms including abbreviations, (iii) closely related compounds, (iv) drug availability, (v) drug class, (vi) target, and (vii) description. for interferons, the drug class is the type of interferon (α, β, γ, or λ). for monoclonal antibodies, additional data are stored and displayed, including the antibody source and information on sequence and structure availability. the closely related compounds are subjectively defined as those that we intend to be returned by a query even if that compound was not entered by the user. there are five broad categories of closely related compounds: (i) monoclonal antibodies described in the same publication, (ii) interferons belonging to the same type (i.e., α, β, γ, or λ), (iii) a series of compounds derived from the same lead compound, (iv) prodrugs such as those for gs-441524 (i.e., remdesivir) and β-nhydroxycytidine (i.e, eidd-28014), (v) drug combinations such as lopinavir and ritonavir-boosted lopinavir (lopinavir/r), and (vi) compounds presumed to act by a highly similar mechanism of action (e.g., hydroxychloroquine and chloroquine). the drug availability category indicates whether the compound has been licensed in the u.s. or another country or has been studied in humans. of 975 compounds with a known or likely mechanism of action, 135 (14%) are u.s. fda (u.s. food and drug administration) -approved drugs (for indications other than covid-19), 197 (20%) have been or are currently being evaluated in human clinical trials or are approved outside the u.s., and 595 (61%) are preclinical investigational compounds. the database contains experiments involving approximately 1650 compounds. more than 1240 of these compounds appear in the online compounds tables, which contain the following fields: (i) name, (ii) synonyms including abbreviations, (iii) closely related compounds, (iv) drug availability, (v) drug class, (vi) target, and (vii) description. for interferons, the drug class is the type of interferon (α, β, γ, or λ). for monoclonal antibodies, additional data are stored and displayed, including the antibody source and information on sequence and structure availability. the closely related compounds are subjectively defined as those that we intend to be returned by a query even if that compound was not entered by the user. there are five broad categories of closely related compounds: (i) monoclonal antibodies described in the same publication, (ii) interferons belonging to the same type (i.e., α, β, γ, or λ), (iii) a series of compounds derived from the same lead compound, (iv) prodrugs such as those for gs-441524 (i.e., remdesivir) and β-n-hydroxycytidine (i.e, eidd-28014), (v) drug combinations such as lopinavir and ritonavir-boosted lopinavir (lopinavir/r), and (vi) compounds presumed to act by a highly similar mechanism of action (e.g., hydroxychloroquine and chloroquine). the drug availability category indicates whether the compound has been licensed in the u.s. or another country or has been studied in humans. of 975 compounds with a known or likely mechanism of action, 135 (14%) are u.s. fda (u.s. food and drug administration) -approved drugs (for indications other than covid-19), 197 (20%) have been or are currently being evaluated in human clinical trials or are approved outside the u.s., and 595 (61%) are preclinical investigational compounds. table 3 . antiviral coronavirus therapy targets. inhibitors of the coronavirus rna-directed rna polymerase (rdrp) enzymes include nucleoside analogs that cause immediate chain termination, delayed chain termination, or viral mutagenesis. coronaviruses contain two protease enzymes: 3 chymotrypsin-like cysteine protease (3cl pro or main (m)-pro) and papain-like (pl pro ). coronavirus helicases catalyze the unwinding of duplex rna molecules into single strands. entry convalescent plasma and polyclonal sera. convalescent plasma is one of the most widely studied treatments for covid-19. polyclonal sera and immunoglobuline preparations have also entered clinical trials. recovery during sars-cov, mers-cov, and sars-cov-2 is usually associated with the development of neutralizing antibodies. most sars-cov-2 neutralizing mabs target the part of the receptor binding domain (rbd) that binds ace2 while most mers-cov neutralizing mabs target the part of the rbd that binds dpp4. many highly potent neutralizing mabs targeting each of the pandemic coronaviruses have shown protection in vitro and in animal models. structural studies have defined specific rbd epitopes recognized by individual mabs and identified amino acid residues that are critical for mab binding. other receptor binding inhibitors sars-cov and sars-cov-2 spike s1 binds to the cellular angiotensin converting enzyme 2 (ace2) receptor. mers-cov binds to dipeptidyl peptidase 4 (dpp4). a variety of compounds including non-antibody proteins, peptides, and small molecules have been shown to prevent the binding of the coronavirus spike protein to its cellular receptor. following receptor binding and spike s1/s2 cleavage and s2 priming, heptad region 1 (hr1), which is close to the fusion peptide sequence, and hr2, which is close to the virus membrane, collapse on to one another to bring virus and cell membranes together. nearly all fusion inhibitors are hr2-mimicking peptides less than 70 kda that bind hr1, thus preventing hr1−hr2 binding. interferons have been extensively studied for their ability to inhibit each of the pandemic coronaviruses in cell culture, animal models, and/or clinical studies [47] . sars-cov-2 may be more susceptible to interferons than sars-cov is [48] . there are several clinical trials using immunostimulatory cytokines and compounds purported to induce interferon. cleavage of coronavirus spike proteins is necessary for the virus to transition from receptor attachment to cell fusion. for sars-cov-2, there is a poly-basic furin cleavage site at the s1/s2 boundary and another cleavage site within s2 believed to be cleaved at the cell surface by host tmprss2 enzymes [49] . multiple intracellular processes essential to virus replication are vulnerable to pharmacologic inhibitors including endosomal acidification, membrane formation, various signaling pathways, nucleotide biosynthesis, and autophagy [50] . many compounds with uncertain mechanisms of action have been found to inhibit coronaviruses in vitro. several of these are also being studied in clinical trials. mabs-monoclonal antibodies. figure 4 displays ec 50 values for many of the directly acting antiviral compounds currently in clinical trials for the treatment of covid-19 including six polymerase inhibitors (remdesivir, eidd-2801, favipiravir, ribavirin, galidesivir, and sofosbuvir), three hiv-1 protease inhibitors (lopinavir, atazanavir, and darunavir), and three entry inhibitors (receptor binding monoclonal antibodies, soluble recombinant human ace2, and umifenovir). figure 5 displays ec 50 values for many of the repurposed compounds that target host processes required for virus replication including two host pis that target the transmembrane serine protease 2 (tmprss2) enzyme (camostat and nafamostat), three chloroquine analogs that interfere with endosomal acidification (chloroquine, hydroxychloroquine, and mefloquine), three other compounds believed to interfere with membrane trafficking (niclosamide, imatinib, and chlorpromazine), and four compounds acting by a variety of different cellular mechanisms (ivermectin, nitazoxanide, ciclesonide, and cyclosporin). trafficking (niclosamide, imatinib, and chlorpromazine), and four compounds acting by a variety of different cellular mechanisms (ivermectin, nitazoxanide, ciclesonide, and cyclosporin). figures 4 and 5 show that the potency of currently studied compounds extends over several orders of magnitude with monoclonal antibodies having ec50s in the high picomolar to low nanomolar range and some compounds displaying no activity at concentrations above 100 μm. however, there is also marked heterogeneity in the ec50 values for the same compound in different experiments. for several drugs, the heterogeneity can be explained by the type of cells used, inoculum size, drug timing, and culture duration. for example, the host tmprss2 inhibitors camostat and nafamostat are practically inactive against sars-cov-2 in vero cells but have ec50s consistently below 1 μm in caco-2 and calu-3 cells, because these cells require tmprss2 for virus replication whereas vero cells do not [22, [51] [52] [53] [54] [55] [56] . 5 show that the potency of currently studied compounds extends over several orders of magnitude with monoclonal antibodies having ec 50 s in the high picomolar to low nanomolar range and some compounds displaying no activity at concentrations above 100 µm. however, there is also marked heterogeneity in the ec 50 values for the same compound in different experiments. for several drugs, the heterogeneity can be explained by the type of cells used, inoculum size, drug timing, and culture duration. for example, the host tmprss2 inhibitors camostat and nafamostat are practically inactive against sars-cov-2 in vero cells but have ec 50 s consistently below 1 µm in caco-2 and calu-3 cells, because these cells require tmprss2 for virus replication whereas vero cells do not [22, [51] [52] [53] [54] [55] [56] . viruses 2020, 12, x for peer review 11 of 22 table 4 describes a set of the most promising compounds for the treatment of sars-cov-2 based on the following criteria: (i) act by a validated direct or indirect antiviral mechanism, (ii) display submicromolar activity in vitro and/or inhibitory activity in an animal model, and (iii) have a record of safety and favorable pharmacokinetics in human subjects. the majority of these compounds are being studied in clinical trials, although the numbers of these trials are far fewer than those for less promising compounds. table 4 . promising sars-cov-2 antiviral compounds. remdesivir is a delayed chain terminator monophosphate prodrug of a 1′cyano-substituted adenine c-nucleoside analog. it has high nanomolar inhibitory activity in vitro against sars-cov-2 particularly in cells other than vero cells [21, 22, [57] [58] [59] . it reduces viral replication and lung pathology in mice and rhesus macaques when administered shortly after infection [58, 60] . in a double-blind randomized clinical trial, its intravenous administration led to a significant reduction in time to recovery from 15 to 11 days (p < 0.001) and a non-statistically significant reduction in day 14 mortality of 11.9% vs. 7.1% (p = 0.06) [61] . based on this trial, the fda issued an emergency use authorization for remdesivir in patients with severe covid-19. ongoing trials are examining its safety and efficacy when administered subcutaneously or via inhalation. β-d-n4hydroxycytidine-5′isopropyl ester (eidd-2801) eidd-2801 is a nucleoside analog, which like remdesivir has high nanomolar inhibitory activity in vitro against sars-cov-2 [62] . it reduces sars-cov and mers-cov replication and lung pathology in a mouse model [62] . it is being evaluated in two phase ii clinical trials. table 4 describes a set of the most promising compounds for the treatment of sars-cov-2 based on the following criteria: (i) act by a validated direct or indirect antiviral mechanism, (ii) display sub-micromolar activity in vitro and/or inhibitory activity in an animal model, and (iii) have a record of safety and favorable pharmacokinetics in human subjects. the majority of these compounds are being studied in clinical trials, although the numbers of these trials are far fewer than those for less promising compounds. table 4 . promising sars-cov-2 antiviral compounds. remdesivir is a delayed chain terminator monophosphate prodrug of a 1 -cyano-substituted adenine c-nucleoside analog. it has high nanomolar inhibitory activity in vitro against sars-cov-2 particularly in cells other than vero cells [21, 22, [57] [58] [59] . it reduces viral replication and lung pathology in mice and rhesus macaques when administered shortly after infection [58, 60] . in a double-blind randomized clinical trial, its intravenous administration led to a significant reduction in time to recovery from 15 to 11 days (p < 0.001) and a non-statistically significant reduction in day 14 mortality of 11.9% vs. 7.1% (p = 0.06) [61] . based on this trial, the fda issued an emergency use authorization for remdesivir in patients with severe covid-19. ongoing trials are examining its safety and efficacy when administered subcutaneously or via inhalation. β-d-n4hydroxycytidine-5isopropyl ester (eidd-2801) eidd-2801 is a nucleoside analog, which like remdesivir has high nanomolar inhibitory activity in vitro against sars-cov-2 [62] . it reduces sars-cov and mers-cov replication and lung pathology in a mouse model [62] . it is being evaluated in two phase ii clinical trials. regn10933 and regn10987 are mabs with subnanomolar inhibitory activity that bind to non-overlapping ace2-competing sars-cov-2 spike receptor binding domain epitopes [8, 63] . this mab combination also reduces virus replication and lung pathology in syrian hamsters and rhesus macaques [64] . the combination is being evaluated in phase iii trials for preventing and treating sars-cov-2 infection. ly3819253 is a sars-cov-2 mab in phase iii trials for preventing and treating covid-19. as of august 2020, there are no associated published preclinical data. mabs (phase i/ii trials) azd7442, brii-196, js016, scta01, sti-1499, and ty027 are mabs in phase i/ii trials. as of august 2020, there are no associated published preclinical data linked to mabs with these names. many research groups that have published preclinical data on one or more mabs (or mab variants such as nanobodies) including their in vitro inhibitory activity, genetic sequence data, three-dimensional structural data, and/or animal model data [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] . interferons ifn-α, ifn-β, and ifn-λ ifn-α, ifn-β, and ifn-λ each inhibit sars-cov-2 by 90%-99% at low concentrations of about 100 international units (iu)/ml [48, [81] [82] [83] [84] . inhalational ifn-α and parenteral ifn-β were associated with modest reductions in disease severity and/or virus loads in two small open-label clinical trials [84, 85] . an inhaled formulation of ifn-β was reported in the news to significantly reduce the odds of developing severe disease or death in a blinded randomized control trial (sng016) of 220 patients that has not yet been published (https://www.synairgen.com/covid-19/). there are currently four planned or ongoing placebo-controlled trials of parenteral or inhaled ifn-β (~1800 patients) and of parenteral ifn-λ (~400 patients). camostat and nafamostat are tmprss2 inhibitors with nanomolar coronavirus inhibitory activity in biochemical and cell culture assays [51, [53] [54] [55] [56] [86] [87] [88] . both drugs are used in japan for the treatment of pancreatitis, while nafamostat is also used as an anticoagulant and for the treatment of disseminated intravascular coagulation. although nafamostat has approximately 10-fold greater inhibitory activity than camostat, it may be associated with greater toxicity. camostat is being studied in two blinded and two open-label randomized controlled studies totaling about 900 patients. nafamostat is being studied in three small randomized open-label studies totaling about 200 patients. apilimod was found to inhibit sars-cov-2 at two-digit nanomolar levels with high selectivity indexes in multiple drug screens [89] [90] [91] [92] . it inhibits the membrane protein pi (3, 5) p2 by inhibiting the enzyme pi-3p-5-kinase (pikfyve) thus interfering with endosomal trafficking of sars-cov-2 and additional viruses utilizing the same endosomal pathway [93, 94] . it has been studied in humans in multiple clinical trials and been found to be safe and well tolerated. it is being studied for the treatment of mild sars-cov-2 infections in one randomized placebo-controlled phase ii trial. ptc299 is an inhibitor of dihydroorotate dehydrogenase (dhodh), a rate limiting enzyme in the pyrimidine biosynthesis pathway [95] . dhodh inhibitors are therapeutic targets for autoimmune disases and viral infections [96, 97] . ptc299 has been found to be safe and have favorable pharmacokinetics in more than 300 human subjects and has low nanomolar sars-cov-2 inhibitory activity and a high selectivity index [98] . as both viral replication and cytokine overproduction depend on pyrmidine synthesis, dhodh inhibition may have a dual role in covid-19 treatment. dhodh inhibition is synergistic with viral polymerase inhibition [97] . there is one phase ii/iii trial of ptc299 for patients with severe but not critical covid-19. leflunomide, another repurposed dhodh inhibitor was found to reduce virus load in an open-label pilot study of 27 patients [99] . soluble recombinant human ace2 (rhace2) rhace2 protects mice for sars-cov-1 ards and has been studied as a treatment for ards in humans [100, 101] . it inhibits sars-cov-2 spike binding at nanomolar concentrations in a wide variety of cell lines [102, 103] . there are two ongoing phase ii trials of an intravenous commercial rhace2 preparation. notes: some compounds with sub-micromolar in vitro activity are not included in this table either because (i) they have not been used in humans, (ii) they have been identified only in high-throughput drug screens, or (iii) they have unfavorable pharmacokinetics. several compounds that inhibit sars-cov-2 less potently but are being studied as inhalational and/or intranasal therapies are also not shown. this last category includes (i) compounds that inhibit the interaction of sars-cov-2 spike and cell surface heparin sulfate proteoglycans such as lactoferrin and heparin; and (ii) ciclesonide, an inhaled corticosteroid that may interfere with membrane trafficking by binding directly to nsp-3 or nsp-4 or indirectly through a host protein. the clinical trials registry table is a regularly updated, annotated list of ongoing, planned, or completed clinical trials obtained from the clinicaltrials.gov, who ictrp, and chinese clinical trial websites. it contains trials of compounds with potential antiviral activity but not studies of non-antiviral interventions, such as those designed to optimize intensive-care management or reduce the inflammatory response associated with severe covid-19. the clinical trials registry classifies trials according to the compound target, the type of trial (e.g., observational or randomized controlled study), the status of the trial (pending, active, or completed), and the population studied. as of 6 august 2020, it contains more than 700 trials of which about 73% are listed on clinicaltrials.gov and 27% are listed only on the who international clinical trials platform. figure 6a displays the distribution of planned, ongoing, and published studies according to the compound targets of the drugs studied. figure 6b displays the same distribution for those drugs in three or more studies. it is notable that many of the most commonly studied compounds have either little or no activity against sars-cov-2, including several drugs used for non-coronavirus infections such as the hiv protease inhibitors lopinavir and darunavir and the influenza inhibitors favipiravir, oseltamivir, and umifenovir. the chloroquine analogs, chloroquine and hydroxychloroquine, have weak in vitro activity but have failed to show clinical efficacy in multiple clinical trials [104] [105] [106] [107] [108] [109] [110] . the search function allows users to specify one or more of the following options from four drop-down lists: (i) compound target, (ii) compound, (iii) virus category, and (iv) study type. if the user selects "any" for one of these and leaves the others in their default position, the search function returns the database's complete set of cell culture experiments, biochemical experiments, entry assay experiments, animal model studies, and published clinical studies. by selecting one or more of the above options, the search function restricts the data returned to those meeting the search criteria. by using the "copy to clipboard" link, users can import the results of any query into a spreadsheet where they can further sort and filter query results. the search function also provides a link to the trials in the clinical trials registry for selected compounds and compound targets. the compound drop-down list displays 60 of the most well recognized compounds. selecting a compound returns the data for that compound as well as for an additional 363 closely related compounds (as described in the compound table section 2.2.6). if the user selects the compound target from the dropdown menu, then the compound menu will list all compounds designed to inhibit the selected target. the compounds entry on the compounds page also links to all the data on that compound in the database. the compound drop-down list displays 60 of the most well recognized compounds. selecting a compound returns the data for that compound as well as for an additional 363 closely related compounds (as described in the compound table section 2.2.6). if the user selects the compound target from the dropdown menu, then the compound menu will list all compounds designed to inhibit the selected target. the compounds entry on the compounds page also links to all the data on that compound in the database. to prioritize licensed drugs and investigational compounds for the treatment of covid-19, it is necessary to compare their relative antiviral activities. compounds that are not active in vitro will almost certainly not be useful clinically. therefore, pre-clinical data are necessary to prioritize animal to prioritize licensed drugs and investigational compounds for the treatment of covid-19, it is necessary to compare their relative antiviral activities. compounds that are not active in vitro will almost certainly not be useful clinically. therefore, pre-clinical data are necessary to prioritize animal model and clinical studies. compounds that are active in vitro, however, may also not be clinically useful if their associated in vitro data do not reflect physiologic conditions or if standard dosing with these compounds does not result in sufficient inhibitory concentrations at sites of infection. the creation of the cov-rdb was primarily motivated by the observation that many of the drugs being evaluated in covid-19 clinical trials demonstrate little or no in vitro anti-coronavirus activity. for example, as recently as july 2020, four of the most commonly studied drugs-chloroquine analogs, azithromycin, lopinavir/r, and favipiravir-demonstrated little if any in vitro activity. the creation of the cov-rdb was secondarily motivated by the observation that results for the same compound often vary across different laboratories as a result of experimental design such as cell line, inoculum size, drug-addition timing, duration of culture, and method for measuring virus replication. given sufficient data, a database makes it possible to eventually identify the experimental features responsible for the heterogeneity in published results, thus improving the ability to compare the antiviral activity of different compounds. indeed, we have already noted in this manuscript several compound classes for which viral inhibition is influenced by the cells used for virus culture. the cov-rdb is also designed to be educational as it provides multiple lookup tables for the viruses, drugs, cell lines, and animal models used in reported experiments. these tables contain descriptions of viruses, virus isolate/strains, cell lines, animal models, and more than 300 licensed and investigational compounds. work is underway to also add comprehensive, yet detailed, summaries of sars-cov-2 monoclonal antibodies and of pharmacokinetic data for those drugs with well-documented in vitro inhibitory activity. there are several additional web resources devoted to coronavirus drug development including sites devoted to high-throughput drug screening [111] , the genetics of monoclonal antibodies [112] , and meta-analyses of published clinical trials [113, 114] . the national institutes of health (nih) recognizes the importance of such resources and has recently announced a notice of special interest: national institute of allergy and infectious diseases (niaid) priorities for biomedical knowledgebases and repositories (not-ai-20-044). the cov-rdb database, user interface, and underlying computer code represent a framework for organizing a vast amount of data and for facilitating data curation. however, the value of this resource depends upon ongoing manual data curation and annotation. in conclusion, the cov-rdb provides a uniquely integrated interdisciplinary synthesis of in vitro, animal model, and clinical studies of compounds with proven or possible anti-coronavirus activity. it helps researchers place their findings in the context of previously published data and it facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies to prioritize the most promising compounds and repurposed drugs for further development. the authors declare no conflict of 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hamsters a human neutralizing antibody targets the receptor binding site of sars-cov-2 human neutralizing antibodies elicited by sars-cov-2 infection cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody convergent antibody responses to sars-cov-2 in convalescent individuals isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model potent neutralizing antibodies from covid-19 patients define multiple targets of vulnerability potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells neutralization of sars-cov-2 by destruction of the prefusion spike neutralizing nanobodies bind sars-cov-2 spike rbd and block interaction with ace2 potently neutralizing and protective human antibodies against sars-cov-2 a cross-reactive human iga monoclonal antibody blocks sars-cov-2 spike-ace2 interaction a noncompeting pair of human neutralizing antibodies 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of rna-dependent rna polymerase inhibition by combination with modulators of pyrimidine metabolism the dhodh inhibitor ptc299 arrests sars-cov-2 replication and suppresses induction of inflammatory cytokines efficacy and safety of leflunomide for refractory covid-19: an open-label controlled study angiotensin-converting enzyme 2 protects from severe acute lung failure a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig effect of hydroxychloroquine in hospitalized patients with covid-19: preliminary results from a multi-centre, randomized hydroxychloroquine in nonhospitalized adults with early covid-19: a randomized trial hydroxychloroquine with or without azithromycin in mild-to-moderate covid-19 hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial a cluster-randomized trial of hydroxychloroquine as prevention of covid-19 transmission and disease hydroxychloroquine for early treatment of adults with mild covid-19: a randomized-controlled trial a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 an opendata portal to share covid-19 drug repurposing data in real time the coronavirus antibody database. biorxiv 2020 characteristics of registered studies for coronavirus disease 2019 (covid-19): a systematic review ongoing clinical trials for the management of the covid-19 pandemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-354824-7fdcu2f0 authors: wu, renyi; wang, lujing; kuo, hsiao-chen dina; shannar, ahmad; peter, rebecca; chou, pochung jordan; li, shanyi; hudlikar, rasika; liu, xia; liu, zhigang; poiani, george j.; amorosa, louis; brunetti, luigi; kong, ah-ng title: an update on current therapeutic drugs treating covid-19 date: 2020-05-11 journal: curr pharmacol rep doi: 10.1007/s40495-020-00216-7 sha: doc_id: 354824 cord_uid: 7fdcu2f0 the current pandemic of coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has presented unprecedented challenges to the healthcare systems in almost every country around the world. currently, there are no proven effective vaccines or therapeutic agents against the virus. current clinical management includes infection prevention and control measures and supportive care including supplemental oxygen and mechanical ventilatory support. evolving research and clinical data regarding the virologic sars-cov-2 suggest a potential list of repurposed drugs with appropriate pharmacological effects and therapeutic efficacies in treating covid-19 patients. in this review, we will update and summarize the most common and plausible drugs for the treatment of covid-19 patients. these drugs and therapeutic agents include antiviral agents (remdesivir, hydroxychloroquine, chloroquine, lopinavir, umifenovir, favipiravir, and oseltamivir), and supporting agents (ascorbic acid, azithromycin, corticosteroids, nitric oxide, il-6 antagonists), among others. we hope that this review will provide useful and most updated therapeutic drugs to prevent, control, and treat covid-19 patients until the approval of vaccines and specific drugs targeting sars-cov-2. coronavirus disease 2019 sars-cov-2 severe acute respiratory syndrome coronavirus 2 the horrific pandemic outbreak of covid-19 (coronavirus disease 2019) around the world caught the health care systems in every country by storm, most if not all were caught off guard without proper defense mechanisms to cope with and to control such a pandemic. covid-19, caused by a new and novel coronavirus (severe acute respiratory syndrome coronavirus 2, sars-cov-2), has recently been identified and characterized [1••] . coronaviruses are named for their crown-like spikes on their surface and there are four main sub-groupings of coronaviruses, known as alpha, beta, gamma, and delta [1, 2] . sars-cov-2 belongs to the beta sub-grouping, and is one of the seventh coronavirus to date infecting humans [1••] . some coronaviruses such as 229e alpha coronavirus [3] , oc43 beta coronavirus [4] , nl63 alpha coronavirus [5] , and hku1 beta coronavirus [6] were associated with mild clinical symptoms, whereas sars-cov beta coronavirus [7] , middle east respiratory syndrome coronavirus (mers-cov) beta coronavirus [8] , and sars-cov-2 caused severe diseases [2] . sars-cov-2 is a positive-sense single-stranded rna virus with 29,891 bases, 96% identical at the whole-genome level to a bat coronavirus, and shares 79.6% sequence identity to sars-cov [1••] . sars-cov-2 encodes spike s protein containing receptor binding domain (rbd) that binds to the human angiotensin-converting enzyme 2 (ace2), and promotes membrane fusion and uptakes of the virus into human cells such as the lung by endocytosis [1, [9] [10] [11] . upon entering the human cells, sars-cov-2, like other coronaviruses, will takeover or hijack the human cells' protein synthesis machinery to synthesize the viral proteins and assemble the proteins and subsequent viral replication [12•] . once inside the human body, viruses in general will trigger a series of good versus bad host responses including autophagy, apoptosis, stress response, and innate immunity [13] . fortunately, majority (more than 80%) of sars-cov-2infected individuals are asymptomatic or have mild symptoms, most likely due to the activation of the good response. these good responders would likely activate the body's innate immune system by activating the body's antiviral defense mechanisms including natural killer cells and antiviral t cells, and induction of interferon (ifn) [13] [14] [15] [16] . unfortunately, in about 20% of sars-cov-2-infected individuals including the immune compromised, elderly, patients with underlying health conditions such as cardiovascular and pulmonary problems, diabetics, hypertension, obesity, chronic obstructive pulmonary disease (or copd, such as emphysema), pulmonary fibrosis, asthma, and interstitial lung disease [17, 18] would encounter more severe disease characterized by significant respiratory symptoms leading to acute respiratory distress syndrome (ards) and even death. an important consideration to note is that ards occurs later in disease progression and is preceded by acute lung injury (ali) [19] . this distinction may inform treatment strategy in terms of drugs directed towards cytokine storm and thrombosis which is described in this manuscript. a study on sars-cov and mers-cov has found that these two coronaviruses appear to have evolved mechanisms to attenuate or delay ifn production, resulting in enhanced inflammatory host responses and severe lung injury [12, 13, [20] [21] [22] . this aberrant host immune response with the production of powerful inflammatory cytokines, known as "cytokine storm" found in sars-cov-and mers-covinfected patients, would correlate with disease severity and poor prognosis [13, 16, [20] [21] [22] [23] . severe covid-19 patients exhibit profound inflammatory response [24, 25] . transcriptomic rna-seq analysis of covid-19 patients has revealed that several immune pathways and pro-inflammatory cytokines cxcl, ccl2, cxcl2, ccl8, il33, and ccl3l1 in bronchoalveolar lavage fluid (balf) and tnfsf10, cxcl10, il10, timp1, c5, il18, areg, and nrg1 in peripheral blood mononuclear cells (pbmc) were induced by sars-cov-2 infection, suggesting a sustained inflammation and cytokine storm [26] . importantly, sars-cov-2 infection-induced excessive cytokine release correlates with lung tissue injury and covid-19 pathogenesis [26] . this estimated 20% of patients developing more severe disease with sars-cov-2 infection are most likely due to genetics, epigenetics, and or other factors, with dampened innate immune response to fight the virus coupled with enhanced viral load leading to cytokine storm, severe inflammatory/oxidative stress response, and severe lung injury secondary to ards. while there is clear understanding that the respiratory system is dramatically impacted in covid-19 patients, evidence suggests that other organ systems are also affected. emerging data show that sars-cov-2 may lead to damage to other organs including the heart and brain. nearly 20% of hospitalized patients with covid-19 have indication of cardiac damage [17] . furthermore, neurologic symptoms have been reported in patients and infection of sars-cov-2 has been found in the brainstem of both humans and experimental animals [18, 19] . currently, there is no vaccine and/or specific therapeutic drugs targeting the sars-cov-2. hence, it remains a major challenge to decide what potential therapeutic regimens to prevent and treat the severely sick covid-19 patients. effective vaccines are essential to combat against the extremely contagious sars-cov-2. at present, a lot of research efforts have been invested to develop vaccines around the world. until we have specific vaccines or therapeutic drugs targeting sars-cov-2, "repurposed" drugs that have been approved by the fda in the usa for other indications have been used to treat covid-19 patients. this review will summarize the most current pharmacotherapeutics prescribed in the treatment of severe cases of covid-19 patients. these include antiviral therapy, antibiotics, systemic corticosteroids and anti-inflammatory drugs (including anti-arthritis drugs), neuraminidase inhibitors, rna synthesis inhibitors, convalescent plasma, and traditional herbal medicines. in the absence of definitive and specific treatment regimens, strategies including early diagnosis, timely reporting, isolation, and supportive treatments are important line of actions against covid-19 infections. current social practices including timely release of epidemic information and maintenance of social orders and personal practices such as improving personal hygiene, wearing facial coverings or masks, adequate rest, and keeping rooms well ventilated remain some of first line of actions against covid-19 pandemic. at present, the treatments of patients with sars-cov-2 infection are mainly repurposing the available therapeutic drugs and based on symptomatic conditions. considering ards, followed by secondary infections, antibiotics, antiviral therapy, systemic corticosteroids, and anti-inflammatory drugs (including anti-arthritis drugs) are often used in the treatment regimens. in addition to antiviral interferers and antibiotics, neuraminidase inhibitors, rna synthesis inhibitors, convalescent plasma, and traditional herbal medicines have also been utilized in the treatment of covid-19 [27] . nevertheless, the efficacy of these treatment regimens remains to be verified by appropriately designed clinical trials. remdesivir is a potential drug for treatment of covid-19. it is a phosphoramidate prodrug of an adenosine c-nucleoside and a broad-spectrum antiviral agent synthesized and developed by gilead sciences in 2017 as a treatment for ebola virus infection [28] . remdesivir is metabolized into its active form, gs-441524, that obscures viral rna polymerase and evades proofreading by viral exonuclease, causing a decrease in viral rna production. the antiviral mechanism of remdesivir is a delayed chain cessation of nascent viral rna. animal experiments indicate that remdesivir can effectively reduce the viral load in lung tissue of mice infected with mers-cov, improve lung function, and alleviate pathological damage to lung tissue [29] . wang [31] . in order to evaluate the efficacy and safety of the drug in patients with covid-19, a randomized, placebo-controlled, double-blind, multicenter, phase iii clinical trial was launched on february 5, 2020 in china. patients in the experimental group received an initial dose of 200 mg of remdesivir and a subsequent dose of 100 mg for 9 consecutive days via intravenous infusion in addition to routine treatment. patients in the control group received same dose of placebo treatment. the trial is expected to conclude by the end of april 2020. the number of cases planned to be enrolled is 308 and 452, respectively [32, 33]. current recommendation for remdesivir includes a 10-day regimen of remdesivir treatment: 200 mg loading dose on day 1, followed by 100 mg once-daily maintenance doses for 9 days in both studies. this regimen of remdesivir therapy is similar to that of former randomized clinical trial against the ebola virus [32, 33]. in a summary of subjects receiving remdesivir via compassionate use in the usa, nearly 70% of patients had improvement in terms of oxygen requirements and many patients that were mechanically ventilated were extubated. this report did not include a control group; therefore, extrapolating these results is difficult. it is too early to conclude the direct antiviral effect of remdesivir on the enhanced clearing of viral loads in the respiratory tract, but it indeed suggests a promising therapeutic effect of remdesivir [34] . chloroquine and hydroxychloroquine are drugs with a long history of clinical use with similar chemical structures often used in the treatment of lupus erythematosus, rheumatoid arthritis, and malaria [35] . compared with chloroquine, hydroxychloroquine has a hydroxyl group, which makes it less toxic while maintaining similar activity. one mechanism of action of chloroquine and hydroxychloroquine is targeting lysosome which may be useful to control graft-versus-host disease in humans [36] . with the accumulation of chloroquine in lysosomes, the ph of lysosomes is significantly changed and the activity of proteases in lysosomes is directly affected, thus affecting the degradation of proteins and glycosaminoglycan [36, 37] . chloroquine can inhibit the entry of sars-cov-2 and prevent virus-cell fusion by interfering with glycosylation of ace2 receptor and its binding with spike protein, suggesting that chloroquine treatment might be more effective in the early stage of infection, before covid-19 reduces ace2 expression and activity [30, 38, 39] . hydroxychloroquine possesses anti-inflammatory effect on th17-related cytokines (il-6, il-17, and il-22) in healthy individuals, and systemic lupus erythematosus (sle) and rheumatoid arthritis (ra) patients [40] . there is some evidence that chloroquine and hydroxychloroquine can reduce cytokine storm. according to one analysis, the main cause of death of covid-19 patients is related to the triggering of the cytokine storm, which contributed to acute respiratory distress [41] . it has been reported that hydroxychloroquine is effective in inhibiting sars-cov-2 infection in vitro [1, 39, 42] . zinc inhibits sars-covand retrovirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture [43] . there is also evidence that zinc enhances chloroquine intracellular uptake [44] . as such, combining zinc with chloroquine or hydroxychloroquine is intriguing and is currently under investigation. overall, more clinical trials are underway to evaluate the safety and efficacy of hydroxychloroquine as a prophylactic and treatment for covid-19. the us fda has issued emergency authorization for the use of chloroquine and hydroxychloroquine for the treatment of covid-19. a recent study by tang et al. reported that hydroxychloroquine did not lead to higher negative conversion rates, but had reduced clinical symptoms through the anti-inflammatory properties and recovery of lymphopenia [45•] . it has also been reported that high doses of chloroquine (600 mg twice daily for 10 days or total dose of 12 g) may be associated with significant cardiac risks and should not be recommended for treating covid-19 [46• ]. there is still a lack of evidence regarding the safety and effectiveness of these agents in treating covid-19. in this regard, clinicians and patients should be made aware of the risk versus benefit profile of these medications [47] . lopinavir is a protease inhibitor with high specificity for hiv-1 protease. lopinavir is marketed and administered exclusively in combination with ritonavir. this combination was first marketed by abbott under the brand name kaletra in 2000 [48] . due to lopinavir's poor oral bioavailability and extensive biotransformation, it is co-formulated with ritonavir to enhance its exposure. ritonavir is a potent inhibitor of the enzymes that are responsible for lopinavir metabolism, and its co-administration "boosts" lopinavir exposure and improves antiviral activity [48] . lopinavir is a peptidomimetic molecule, containing a hydroxyethylene scaffold that mimics the peptide linkage typically targeted by the hiv-1 protease enzyme but which by itself cannot be cleaved, thus preventing the activity of the hiv-1 protease [49] . lopinavir-ritonavir was investigated in an open-label, individually randomized, controlled trial, where patients with covid-19 received either lopinavir-ritonavir 400 mg/ 100 mg, orally twice daily plus standard of care, or standard of care alone. no benefit was observed with lopinavirritonavir treatment beyond standard care. diarrhea, nausea, and asthenia were the most frequently reported adverse effects in patients receiving lopinavir-ritonavir-based regimen [50] . interestingly, in a report from korea, lopinavir-ritonavir administration significantly decreased coronavirus titers with no or little coronavirus titers were observed in the follow-up study. however, the analysis included a single patient in the initial phase of outbreak in korea [51] . umifenovir (branded as arbidol), a derivative of indole carboxylic acids, was first developed in 1988 in russia and has since been approved in russia and china for treating prophylaxis and infections associated with influenza a and b, and other arbovirus [52] . later on, umifenovir demonstrated in vitro antiviral efficacy in widely spreading virus strains such as the ebola virus, human herpesvirus 8 (hhv-8), hepatitis c virus (hcv), and tacaribe arenavirus [53] . its major mechanism of action is to block the virus-cell membrane fusion as well as virus-endosome fusion through incorporation into cell membranes and interference with the hydrogen bonding network of phospholipids [54] . in influenza virus, it has been shown to directly interact with virus particles to stabilize hemagglutinin (ha), reducing the likelihood of reaching the low ph threshold required for conformational transition into functional fusogenic ha [55] . blaising et al. reported the in vitro activity of umifenovir against sars-cov-1 and sars-cov-2 [56, 57] . a retrospective cohort study has reported that compared with lopinavir-ritonavir (lpv-rtv) only group, combination of umifenovir and lpv-rtv has shown increased negative conversion rate of sars-cov-2 and improved chest ct scan results [58] . however, another prospective study (chictr200030254) has shown that compared with favipiravir, umifenovir has inferior outcome in clinical recovery rate and relief of fever and cough [59] . there are two randomized and open-label trials ongoing in china, investigating the efficacy and safety of umifenovir against covid-19. the effect of umifenovir plus standard treatment versus lpv-rtv plus standard treatment will be evaluated in nct04252885, and the effect of umifenovir plus standard treatment versus standard treatment will be tested in nct04260594. favipiravir (branded as avigan) has been developed by fujifilm toyama chemical in 2014 in japan for the treatment of avian influenza or novel influenza resistant to neuraminid a s e i n h i b i t o r s . i t i s a g u a n i n e a n a l o g u e w i t h pyrazinecarboxamide structure, and its antiviral activity is decreased at the presence of purine nucleosides due to the competition [60] . the prodrug favipiravir first enters the infected cells through endocytosis and is then transformed into active f a v i p i r a v i r r i b o f u r a n o s y l p h o s p h a t e s t h r o u g h phosphoribosylation and phosphorylation [60, 61] . the antiviral activity is exhibited through selectively targeting conservative catalytic domain of rna-dependent rna polymerase (rdrp), interrupting the nucleotide incorporation process during viral rna replication [60] . the dysregulation in viral rna replication results in increased number and frequency of transition mutations including replacement of guanine (g) by adenine (a) and cytosine (c) by thymine (t) or c by uracil (u) which induces destructive mutagenesis in rna viruses [60] . favipiravir has been used in the treatment of infectious diseases caused by rna viruses such as influenza, ebola, and norovirus [62] . recent in vitro and human studies have repurposed favipiravir as an experimental agent against enveloped, positive-sense, single-strand rna virus sars-cov-2. an in vitro research has investigated seven potential anti-sars-cov-2 medicines including ribavirin, penciclovir, favipiravir, nafamostat, nitazoxanide, remdesivir, and chloroquine, showing that remdesivir and chloroquine have favorable selectivity index [30] . in addition, the study showed favipiravir has exerted efficacy in vero e6 cells infected with sars-cov-2 with half-maximal effective concentration (ec50) of 61.88 μm and half-cytotoxic concentration (cc50) at over 400 μm, implying the high concentration is needed for safe and effective treatment [30] . clinical trials testing favipiravir against covid-19 have been carried out vigorously in various countries including china and japan. a randomized control trial (chictr200030254) has shown that covid-19 patients treated with favipiravir have superior recovery rate (71.43%) than that treated with umifenovir (55.86%), and the duration of fever and cough relief time are significantly shorter in favipiravir group than in umifenovir group [59] . up to mid-april 2020, there are eight undergoing clinical trials in china and two in japan examining the anti-sars-cov-2 potential of favipiravir. these trials include non-randomized and randomized controlled trials evaluating t h e e f f i c a c y a n d s a f e t y o f f a v i p i r a v i r a l o n e (chictr2000030113, jprn-jrcts031190226, jprn-jrcts041190120) or in conjunction with interferon-α ( c h i c t r 2 0 0 0 0 2 9 6 0 0 ) , b a l o x a v i r m a r b o x i l (chictr2000029544, chictr2000029548), tocilizumab (chictr2000030894, nct04310228), or chloroquine phosphate (chictr2000030987, nct04319900). oseltamivir(branded as tamiflu) is a drug approved for treatment of influenza a and b. oseltamivir targets the neuraminidase distributed on the surface of the influenza virus to inhibit the spread of the influenza virus in the human body [63, 64] . a study in wuhan reported that no positive outcomes were observed after receiving antiviral treatment with oseltamivir [65] . several clinical trials are still evaluating the effectiveness of oseltamivir in treating sars-cov-2 infection. oseltamivir is also used in clinical trials in several combinations, such as with chloroquine and favipiravir [66] . in the absence of vaccine or specific antiviral drugs been proven against sars-cov-2, many adjunctive therapies are used as supportive care for covid-19 patients. the adjunctive therapies including azithromycin, ascorbic acid, corticosteroids, epoprostenol, sirolimus, tocilizumab, sarilumab, and anakinra are highlighted below. several of these therapies (i.e., tocilizumab and other interleukin-directed therapies) are administered in an effort to blunt the cytokine storm often seen in progressing disease. the optimal timing of administration is yet to be identified. conceptually, blocking cytokine production before it progresses to an exaggerated level would seem to be the most mechanistically idea. elevated serum concentration of il-6 is associated with worse outcome in covid-19 and blocking the activity of this pro-inflammatory mediator with directed therapies may be a key target [67] . other adjuncts are directed at viral replication, viral entry, or through some other alternative mechanisms. azithromycin is an antibiotic that can be used to fight many different types of infections caused by susceptible bacteria, such as respiratory infections, skin infections, and sexually transmitted diseases [68] . moreover, it has been proven to be active in vitro against zika and ebola viruses and to prevent severe respiratory tract infections when treated to patients suffering viral infection [69] [70] [71] . for the mechanism of action, azithromycin prevents bacteria from growing by interfering with their protein synthesis. it binds to the 50s subunit of the bacterial ribosome, thus inhibiting translation of mrna [72] . previously, azithromycin has been used as adjunctive therapy to provide antibacterial coverage and potential immunomodulatory and anti-inflammatory effects in the treatment of some viral respiratory tract infections (e.g., influenza) [73, 74] . currently, many trials are testing the effect of azithromycin conjunction with hydroxychloroquine on the course of disease in people with sars-cov-2. for example, pfizer has announced positive data for the use of its a z i t h r o m y c i n ( z i t h r o m a x ) d r u g , a l o n g w i t h hydroxychloroquine, in a covid-19 clinical trial that was performed in france. in brief, the clinical trial was conducted to assess hydroxychloroquine in 20 patients, 6 of which were co-administered with azithromycin. compared with 16 controls and 14 hydroxychloroquine alone group, the 6 patients treated with hydroxychloroquine + azithromycin presented with highest virologic cure rate following 6-day treatment [73] . three other clinical studies used azithromycin (500 mg on day 1, then 250 mg daily on days 2-5) co-treated with 10day regimen of hydroxychloroquine (600 mg daily) in an open-label non-randomized study in france (6 pts) [73] , open-label uncontrolled study in france (11 pts) [75] , and uncontrolled observational study in france (80 pts) [76] . specifically, gautret et al. reported a 100% viral clearance in nasopharyngeal swabs in their 6 patients after co-treated of hydroxychloroquine and azithromycin [73] . but the findings reported by molina et al. stand in contrast with those reported by gautret. molina et al. repeated the experiments, thought the rapid and full viral clearance was quite unexpected and found 8 of 11 patients had significant comorbidities [75] . .based on those results, data presented to date are insufficient to evaluate possible clinical benefits of azithromycin in patients with covid-19 [76] . furthermore, one must consider the additive cardiac toxicity of hydroxychloroquine and azithromycin. both agents are known to prolong the qt interval and may potentiate the risk for cardiac events in a population known to have cardiac-related comorbidities. vitamin c is an essential nutrient and plays significant roles within the human body. it can neutralize free radicals and assist to prevent or reverse cellular damage as a potent antioxidant agent. it is also involved in some biological processes, many of which are associated with immune health [77] . moreover, vitamin c appears to be effective as an antiviral agent, especially against influenza viruses [78] . many studies showed that vitamin c positively affects the development and maturation of t lymphocytes and nk (natural killer) cells involved in the immune response to viral agents. it also contributes to the inhibition of reactive oxygen species (ros) production and to the remodulation of the cytokine network typical of systemic inflammatory syndrome [79] . given this background, a phase ii clinical trial (nct04264533) is initiated in china to evaluate high-dose iv vitamin c in icu patients with severe covid-19-associated pneumonia [80] . some hospitals have reported giving infected patients 1500 mg of vitamin c as supportive treatment. high-dose iv vitamin c has been given in the treatment of 50 moderate to severe covid-19 patients in china [81] . the doses varied between 2 and 10 g per day, given over a period of 8--10-h iv infusion. the oxygenation index was improved in real time and all the patients eventually recovered and were discharged [81] . moreover, high-dose (1.5 mg/kg body weight) vitamin c has been used for several decades clinically and an nih panel also documented clearly that this dose regimen is safe and has no major side effects [81, 82] . as a potent anti-inflammatory and anti-fibrotic drug, low doses of methylprednisolone (depo-medrol or solu-medrol) have the potential to prevent an extended cytokine response and may accelerate resolution of pulmonary and systemic inflammation in pneumonia [83, 84] . recently, many medical researchers believe that corticosteroids, especially methylprednisolone, may improve dysregulated immune response caused by sepsis (possible complication of infection with covid-19) and increase blood pressure when it is low [85] . specifically, in a retrospective cohort study, 201 patients with confirmed covid-19 who developed ards were treated with methylprednisolone (1-2 mg/kg daily iv for 5-7 days) and the results showed that treatment with methylprednisolone may be beneficial for patients who develop ards in the reduction of the risk of death. briefly, of those patients with ards who received methylprednisolone treatment, 23 of 50 (46%) patients died, while those who did not receive methylprednisolone, 21 of 34 (61.8%) died [86] . in another study, 46 patients with severe covid-19 that progressed to acute respiratory failure, use of methylprednisolone was associated with improvement in clinical symptoms (i.e., fever, hypoxia) and a shortened disease course in patients who received the drug compared with those who did not [87] . moreover, according to expert consensus statement from chinese thoracic society, dosage regimen of methylprednisolone should be low to moderate (i.e., ≤ 0.5 to 1 mg/kg daily or equivalent) [88] and the most common regimens of methylprednisolone applied in china were typically 40-80 mg iv daily for a course of 3-6 days [89] . the appropriate dosage (low dose versus high dose), place in therapy (early versus late), and role for corticosteroids (cytokine storm or comorbidity management) require additional clarity. there is concern that the use of corticosteroids may have deleterious effects (i.e., inhibition of immune response and pathogen clearance) in patients with covid-19 [83] . one study reported no effect on mortality and decreased viral clearance with the use of corticosteroids [24] . furthermore, the infectious diseases society of american recommends against the routine use of corticosteroids in covid-19. however, they do recommend the use of corticosteroids in the setting of ards in the context of a clinical trial [90] . similarly, the surviving sepsis campaign recommends against corticosteroids in mechanically ventilated patients with acute lung injury in the absence of ards [91] . however, they provide a recommendation for the use of corticosteroids in patients with ards acknowledging the weak level of evidence. dexamethasone has demonstrated utility on ards by decreasing ventilator days and mortality on severe ards in patients without covid-19 [92] . whether the use of corticosteroids provides similar benefit in patients with covid-19 and ards remains to be seen. ultimately, the clinical utilization of corticosteroids still needs to be established and should be considered on a case by case basis. since patients with pre-existing pulmonary conditions are at higher risk of covid-19 and should be closely monitored and cared, pulmonary vasodilator agents have been used in some patients for hypoxemia refractory to conventional treatments, but no study has been performed specifically on covid-19 patients. the surviving sepsis campaign suggested a trial of inhaled pulmonary vasodilator method as rescue therapy in mechanically ventilated adults with covid-19, severe ards, and hypoxemia despite optimized ventilation and other rescue strategies. inhaled nitric oxide (ino) and inhaled epoprostenol (iepo, a naturally occurring prostaglandin) are two common pulmonary vasodilators that have been widely studied [93] [94] [95] . experience in patients with ards indicates that ino can substantially reduce mean pulmonary artery pressure and improve oxygenation in such patients. furthermore, in vitro evidence of direct antiviral activity against sars-cov was studied and the genetic similarity between sars-cov and sars-cov-2 suggests their potential effectiveness against sars-cov-2 [96] . for iepo, dosages up to 50 ng/kg per minute have been used [93, 94, 97, 98] . previous studies reported that to provide a clinically important increase in pao2 and reduction in pulmonary artery pressure, the most effective and safe dosage appears to be 20-30 ng/kg per minute in adults and 30 ng/kg per minute in pediatric patients [98] . for ino, therapy was given for ≥ 3 days (30 ppm on day 1, followed by 20 and 10 ppm on days 2 and 3, respectively, then weaned on day 4) in a pilot study on sars-cov [99] . additionally, clinical trials evaluating ino for treatment or prevention of covid-19 are planned or underway (nct04305457, nct04306393, nct04312243) [100, 101] . and on march 20, 2020, fda granted emergency expanded access allowing its ino delivery system (inopulse®) to be immediately used for the treatment of covid-19. finally, additional studies are needed to evaluate the potential role of iepo and ino in the treatment of covid-19 patients. sirolimus, also known as rapamycin, is an immunosuppressant that is used to prevent organ transplant rejection and to treat lymphangioleiomyomatosis (lam) by inhibiting mammalian target of rapamycin (mtor) kinase. it was originally isolated from the bacterium streptomyces hygroscopicus found on easter island (rapa nui) [102] and is commercially available as rapamune (pfizer). mtor, and more specifically a protein complex mtorc1 formed by mtor, plays a key role in viral replication. in an in vitro experiment, sirolimus has been shown to affect pi3k/akt/mtor pathway which inhibited mers-cov activity [103] . a new randomized double-blind placebo-controlled clinical trial (scope) by university of cincinnati is planned to be conducted between april and september 2020 to test the effect of sirolimus on progression of patients hospitalized with covid-19 to advanced respiratory support [104] . studies of patients hospitalized with influenza can further shed light on the antiviral effect of sirolimus. in a randomized clinical trial conducted on 38 patients with confirmed h1n1 pneumonia and on mechanical ventilator support, a group treated with corticosteroids and 2 mg/day of sirolimus for 14 days (n = 19) showed significantly better clinical outcomes compared with the group treated with corticosteroids only, including shorter median duration of ventilator used [105] . delayed oseltamivir plus sirolimus treatment in ph1n1-infected mouse model further suggested a significant association between the sirolimus treatment and improved outcomes [106] . additionally, a new trial by the chinese university of hong kong is planned to begin in august 2020 to investigate the effect of sirolimus and oseltamivir on normalization of respiratory status and changes in biomarkers (viral rna concentration, 10 cytokines/ chemokines and pro-inflammatory mediators) and several other clinical endpoints in influenza patients [107] . at least one in silico study identified sirolimus as one of the 16 potential candidates for treating covid-19 patients based on data from other human coronavirus infections using network-based drug repurposing model [108] . tocilizumab (branded as actemra) is a humanized mab developed by roche and chugai pharmaceutical for treating ra and systemic juvenile idiopathic arthritis patients. at the time of publishing this article, clinicaltrials.gov listed 20 planned studies that included tocilizumab treatment arm, all of them at the recruiting stage or earlier. a study published in april 2020 reported that 21 severe or critical covid-19 patients in china were treated with the compound, with 20 of them recovered at the time of publication and 1 on the way to recovery (but still in icu). encouraged by these results, a larger multicenter clinical trial was launched (chictr2000029765) and had about 500 patients treated with tocilizumab already enrolled [109, 110] . sarilumab, (branded as kefraza), a humanized mab, was developed by regeneron pharmaceuticals and sanofi for treatment of rheumatoid arthritis (ra). a phase 2/3 randomized double-blind placebo-controlled clinical trial was planned by regeneron pharmaceuticals and sanofi (and in partnership with northwell health's feinstein institutes for medical research) for march 2020 targeting to enroll 400 covid-19 patients, measuring percent change in c-protein (phase 2 only) and time to improvement on a 7-point scale (based on death and type of hospitalization) in patients with serum il-6 level above a threshold as primary endpoints. as of the time of this publication, the results of this study have not been made public [111] . anakinra (branded as kineret by swedish orphan biovitrum) is a modified human il-1 receptor antagonist (il-1ra) approved in 2001 in the usa and in 2002 in europe for use in ra patients. il-1 family of receptors triggers innate immune response and was associated with damaging inflammation [112] . out of 5 approved clinical trials involving anakinra treatment, 2 also have tocilizumab as a comparison: one multicenter open-label non-randomized trial in greece with estimated enrollment of 20 patients [113] , and another multicenter randomized open-label trial in belgium with estimated 342 patients has been enrolled to date [114] . angiotensin-converting enzyme 2 (ace2) receptor is regarded as an important target in the pathogenesis of covid-19. studies reveal that frequently observed comorbidities, including hypertension and diabetes in patients infected with sars-cov-2, are under medication with angiotensin-converting enzyme (ace) inhibitors or angiotensin receptor blocker (arb) [115] [116] [117] [118] that result in overexpression of ace2. it is speculated that sars-covand sars-cov-2 bind to human cells via interaction with ace2 receptors [118, 119] . the opposing physiological actions of ace and ace2 in the renin-angiotensin system are reviewed to determine the therapeutic efficacy of ace2 inhibitors or arbs [120, 121] . in hypertensive patients, chronic treatment with angiotensin ii type 1 receptor (at1r) antagonists like losartan, lisinopril, or olmesartan facilitates cardiac and renal ace2 overexpression according to some in vivo studies [120, 122] . in contrast, sar viral rna following entry into respiratory epithelial cells downregulates the activity of ace2, thereby increasing the levels of angiotensin 2. this may potentially cause severe lung damage [121, 123] . continued treatment with these drugs may be essential for the survival to attenuate the cardiac stress of advancing covid-19 infection and limit the vasoconstriction and profibrotic effects of angiotensin 2 in alveolar capillaries. some of the anti-inflammatory drugs such as ibuprofen, a nonsteroidal anti-inflammatory drug (nsaid), are activators of ace2 receptors, same as ace inhibitors or arbs. their usage can lead to increased risk of contracting covid-19 [118] . since fatal lung failure induced by sars-cov infections may be controlled by blocking renin-angiotensin pathway [123] , ibuprofen may not be harmful. however, there is no strong evidence, suggesting a link between intake of an nsaid and worsening symptoms due to infection caused by sars-cov-2. the fda considers ibuprofen and the likes as a potentially promising therapeutic agent against covid-19 [124] . studies have demonstrated that thiazolidinedione and its derivatives, which are type 2 diabetes mellitus drugs, show efficacious effect against pulmonary disease induced by respiratory syncytial virus (rsv) or h1n1 influenza infection [125, 126] . but their role as a therapeutic drug against coronavirus is not yet explored. interestingly, it is known that thiazolidinediones may have the potential to upregulate ace2 receptor, which is identified as a binding target for sars-cov-2 in host cells [118] . however, lack of clinical evidence makes it uncertain to determine its therapeutic efficacy against coronavirus infections. amici et al. have demonstrated that indomethacin, a wellknown nsaid and a potential cyclooxygenase (cox) inhibitor, exhibits antiviral activity against sars-cov and canine coronavirus (ccov). in vitro studies suggest that indomethacin exhibits dose-dependent response in canine a72 cell monolayers infected with ccov with an ic50 of 5 um after 24 h of exposure. also, remarkable inhibition against sars-cov-infected vero cells by more than 99% at concentrations that were non-toxic for uninfected cells is also observed. in addition, indomethacin significantly blocks viral rna synthesis in dogs infected with ccov following oral administration of the drug (1 mg/kg) [127] . this suggests probable efficacy of indomethacin against sars-cov-2 [127] . colchicine is an anti-inflammatory drug commonly used for gout management and a variety of other conditions sharing similar pathophysiology. its mechanisms of action are related to interfering with migration of neutrophils to sites of inflammation and blocking the inflammasome complex in both neutrophils and monocytes, thus reducing il-1beta activation [128] . colchicine also has inhibitory effects on macrophages via the inhibition of the nacht-lrrpyd-containing protein 3 (nalp3) inflammasome and pore formation activated by purinergic receptors p2x7 and p2x2. there may also be beneficial effects on endothelial function due to colchicine's antifibrotic activities. some patients with covid-19 present with myopathies and colchicine has been shown to reduce inflammation in the cardiac myocytes [129] . there are several ongoing studies investigating colchicine for cytokine storm (nct04326790, nct04322682, nct04322565). niclosamide, an anthelmintic drug, has been shown to be an effective sars-cov virus replication inhibitor at dose concentration of 1.56 um or higher in vero e6 cells without interfering with binding of corona virus onto the cells [130] . another study reveals the efficacy of niclosamide in inhibiting mers-cov replication in verob4 cells via reduction of skp2 regulated becn1 ubiquitination and enhancement of autophagic flux and its ic50 value is determined to be 0.3 um [131] . thus, the possibility of niclosamide to inhibit sars-cov-2 cannot be neglected. ivermectin, a potent anthelmintic drug, was first discovered to inhibit interaction between integrase (in) molecule of human immunodeficiency virus (hiv)-1 and its nuclear transport receptor importin α/β [132] . further studies exhibit its potential to prevent viral replication of a broad spectrum of viruses, including dengue virus, flavivirus, and influenza [133] [134] [135] .very recently, ivermectin has shown inhibition against sars-cov-2 up to 5000-fold at 48 h in vitro. inhibition of impα/β1-mediated nuclear import of viral proteins is suggested as the probable cause of its antiviral activity [136] . it will be interesting to know its inhibition effect against sars-cov-2 in vivo. both nitazoxanide and its metabolite and tizoxanide have shown inhibitory effects against mers-cov in llc-mk2 cells. besides, inhibition of other corona virus strains, including murine corona virus, mouse hepatitis virus strain a59 (mhv-a59), bovine corona virus strain l9 (bcov-l9), and human enteric corona virus 4408 (hecov-4408) by nitazoxanide is reported via suppression of viral n protein [137] . nitazoxanide is found to suppress pro-inflammatory cytokines in peripheral blood mononuclear cells (pbmcs) and il-6 in vivo. however, the relevance of this information is currently unknown [138] . this treatment option refers to transfusion of plasma loaded with antibodies from individuals after resolution from a specific pathogen. this technique has been used for decades [139] . transfusion can offer a short-term, immediate immunity for individuals. convalescent plasma can be used prophylactically and for already infected patients to attenuate clinical severity [140, 141] . mechanism of action is through binding of the transfused antibodies to the pathogen, resulting in cellular cytotoxicity, phagocytosis, or direct neutralization of the pathogen [142, 143] . previously, convalescent plasma was used for two coronaviruses, sars-cov and mers [144] . one large study in hong kong involving 80 patients with sars-cov supported early administration of antibodies for optimal clinical effect compared to later administration [145] . limited data from taiwan and south korea showed clinical benefits in severe cases of sars-cov and mers [146, 147] . reported dosage varied widely in terms of the amount of plasma transfused and antibody titer [148] . limited data on covid-19 patients from china illustrated clinical benefits [149, 150] . pilot study reported clinical improvement in terms of fever, cough, tightness of breath, and chest pain while no serious side effects were reported [150] . there has been considerable attention placed on the role of hypercoagulable state leading to micro-and macro-vascular thrombosis in covid-19. disseminated intravascular coagulation and elevated d-dimer level were identified as predictors of worse outcomes in a cohort study of patients with covid-19 [151] . patients receiving anticoagulants had a decreased mortality [152] . heparin has anti-inflammatory properties and may also inhibit viral attachment via conformational changes to the sars-cov-2 surface receptor (spike) s1 [153] . low molecular weight heparin in patients hospitalized with covid-19 was associated with lower serum il-6 concentrations, suggesting that there may be an added mechanism besides prevention/treatment of thrombosis [154] . based on available evidence, it is reasonable to administer venous thromboembolism prophylaxis with either a low molecular weight or unfractionated heparin in hospitalized patients. in patients with rapidly progressing respiratory deterioration or where clinical judgment suggests thrombosis, treatment doses of anticoagulants may be considered. historically, traditional herbal medicines have been used in the past to control and to treat epidemic outbreaks [155] including the past epidemic outbreaks, such as sars and h1n1 influenza [156, 157] . to date, china and south korea have issued traditional medicinal treatment guidelines on the prevention and treatment of covid-19 [158] . it is reported that greater than 85% of sars-cov-2-infected patients in china had received some forms of traditional chinese medicine (tcm) treatments [159] . similar to sars-cov, sars-cov-2 uses host receptor ace2 for the cellular entrance; it appears that some traditional medicines may have the capacity to target ace2 and these show some promises to prevent the infection of sars-cov-2 [160, 161] . due to the high similarity in epidemiologic, genomics, and pathogenesis between sars-cov-2 and sars-cov, some herbal medicinal products were used for the treatment of patients with infection of sars-cov-2 in china and korea [162, 163] . the top 10 most commonly used tcm herbal medicinal products in china to treat covid-19 patients include astragalus membranaceus, glycyrrhiza uralensis, saposhnikoviae divaricata, rhizoma atractylodis macrocephalae, lonicerae japonicae flos, fructus forsythia, atractylodis rhizoma, radix platycodonis, agastache rugosa, and cyrtomium fortunei j. sm [164] . in addition, some tcm herbal products, such as shen fu injection and re du ning injection, could manifest potential immunosuppressive effects and thereby decrease the level of tnf-α, il-1β, il-6, il-8, il-10, and other cytokines, resulting in inhibition of lung inflammation or acute lung injury [159, [165] [166] [167] . as discussed above, cytokine storm/inflammatory responses may contribute to the deaths of many covid-19 patients. thus, anti-inflammatory agents presumably could reduce the severity and mortality rate [24, 168, 169] . it is reported that qingfei paidu decoction could inhibit and alleviate excessive immune response and eliminate inflammation by regulating immune-related pathway and cytokine actionrelated pathway. the herbal formula qingfei paidu decoction was recommended by both chinese and korean guidelines. according to a recent publication, this herbal formula increases immunity and reduces inflammation by targeting the lung and spleen in covid-19 patients [170] . li et al. reported that lianhuaqingwen (lh), a tcm formula, significantly inhibited sars-cov-2 replication in vero e6 cells and markedly reduced pro-inflammatory cytokine (tnf-α, il-6, ccl-2/mcp-1, and cxcl-10/ip-10) production at the mrna levels [171] . sangju yin and yinqiao san are commonly used in clinical treatment to clear "lung heat," expel phlegm, relieve cough, regulate the patient's lungs, and restore normal lung function [172] . similarly, yinqiao san may have antibacterial and antiviral functions [172] . several clinical studies showed that tcm may bring new hope for the prevention and control of covid-19 [173] [174] [175] . in general, it appears that tcm products were commonly used in covid-19 patients with mild symptoms to severe symptoms and could prevent or block the diseases progression. although the precise molecular mechanisms currently are unknown, the potential role of anti-inflammatory/antioxidative stress, improving hypoxemia/hypoxia and antiviral activities, among others, could be some of the major drivers. further investigations in the future are needed to uncover the molecular mechanisms. conclusion the covid-19 pandemic represents the greatest global public health crisis in the past 100 years. hopefully vaccines and or specific therapeutic drugs targeting sars-cov-2 will be made available in the next few months or years. with the speed and volume of basic and clinical covid-19/ sars-cov-2 research to develop potential drugs and therapies for this disease, our hope will be on the horizon. particular interest, published recently, have been highlighted as: • of importance •• of major importance a pneumonia outbreak associated with a new coronavirus of probable bat origin the proximal origin of sars-cov-2 growth and intracellular development of a new respiratory virus the adaptation of two human coronavirus strains (oc38 and oc43) to growth in cell monolayers identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia sars-cov infection in a restaurant from palm civet isolation of a novel coronavirus from a man with pneumonia in saudi arabia bad news wrapped in protein: inside the coronavirus genome structural basis for the recognition of sars-cov-2 by full-length human ace2 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov emerging coronaviruses: genome structure, replication, and pathogenesis human coronavirus: host-pathogen interaction t cell-mediated immune response to respiratory coronaviruses viral innate immune evasion and the pathogenesis of emerging rna virus infections the host immune response in respiratory virus infection: balancing virus clearance and immunopathology groups at higher risk for severe illness coronavirus and covid-19: who is at higher risk? covid-19 does not lead to a "typical" acute respiratory distress syndrome middle east respiratory syndrome: emergence of a pathogenic human coronavirus sars-cov and ifn: too little, too late dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice sars and mers: recent insights into emerging coronaviruses clinical features of patients infected with 2019 novel coronavirus in wuhan, china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients drug treatment options for the 2019-new coronavirus (2019-ncov) discovery and synthesis of a phosphoramidate prodrug of a pyrrolo[2,1-f][triazin-4-amino] adenine c-nucleoside (gs-5734) for the treatment of ebola and emerging viruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel coronavirus in the united states compassionate use of remdesivir for patients with severe covid-19 antimalarial drugs in the treatment of rheumatological diseases the lysosomotropic amines, chloroquine and hydroxychloroquine: a potentially novel therapy for graftversus-host disease chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion new insights into the antiviral effects of chloroquine hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro hydroxychloroquine decreases th17-related cytokines in systemic lupus erythematosus and rheumatoid arthritis patients covid-19: immunopathology and its implications for therapy chloroquine and hydroxychloroquine as available weapons to f i g h t 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medicine treatment of covid-19 traditional chinese medicine for covid-19 treatment network pharmacology-based analysis of the role of traditional chinese herbal medicines in the treatment of covid-19 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations funding information this work was financially supported in part by institutional funds, by r01 ca200129, from the national cancer institute (nci), r01 at007065 and r01 at009152 from the national center for complementary, and integrative health (nccih) of the national institute of health (nih) awarded to dr. ah-ng tony kong. key: cord-350015-mg5wiihj authors: chen, yiyin; klein, sabra l.; garibaldi, brian t.; li, huifen; wu, cunjin; osevala, nicole m.; li, taisheng; margolick, joseph b.; pawelec, graham; leng, sean x. title: aging in covid-19: vulnerability, immunity and intervention date: 2020-10-31 journal: ageing res rev doi: 10.1016/j.arr.2020.101205 sha: doc_id: 350015 cord_uid: mg5wiihj the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) pandemic was first reported in wuhan, china in december 2019, moved across the globe at an unprecedented speed, and has caused a profound and yet still unfolding health and socioeconomic impacts. sars-cov-2, a β-coronavirus, is a highly contagious respiratory pathogen that causes a disease that has been termed the 2019 coronavirus disease (covid-19). clinical experience thus far indicates that covid-19 is highly heterogeneous, ranging from being asymptomatic and mild to severe and causing death. host factors including age, sex, and comorbid conditions are key determinants of disease severity and progression. aging itself is a prominent risk factor for severe disease and death from covid-19. we hypothesize that age-related decline and dysregulation of immune function, i.e., immunosenescence and inflammaging play a major role in contributing to heightened vulnerability to severe covid-19 outcomes in older adults. much remains to be learned about the immune responses to sars-cov-2 infection. we need to begin partitioning all immunological outcome data by age to better understand disease heterogeneity and aging. such knowledge is critical not only for understanding of covid-19 pathogenesis but also for covid-19 vaccine development. in december 2019, a cluster of novel infectious respiratory syndrome of unknown cause was observed in wuhan, china. thanks to the still fairly recent experience gained from the outbreak of severe acute respiratory syndrome (sars) in 2003, chinese scientists and clinicians worked together and quickly identified a novel coronavirus, sars coronavirus 2 (sars-cov-2) as the pathogen . a complete lock-down and other quarantine measures (e.g., staying at home, social distancing, wearing mask and gloves, hand washing, etc.) were implemented initially in wuhan city and then across much of china in the midst of its busy chinese new year holiday season. while the outbreak was brought under control in china quite promptly, the virus has quickly spread across the globe, causing significant morbidity and mortality initially in italy, then other european countries, united states (us), brazil and the rest of the world. on march 11, 2020, the world health organization (who) declared covid-19 a global pandemic. on march 13 th , the us government declared a national emergency. in the us, the new york city-new jersey area was the epicenter from march to may. by the end of june, covid-19 resurgence occurred in its sun belt states including arizona, texas, and florida, which became the new epicenter. on july 17, the us set a new record of over 77,255 new cases and the world saw over 260,000 new cases in a single day. as of cause pneumonia (cui et al., 2019; de wit et al., 2016) and, in severe cases, acute respiratory distress syndrome (ards). angiotensin-converting enzyme 2 (ace2) serves as the functional cellular receptor for both sars-cov and sars-cov-2 (bourgonje et al., 2020; hoffmann et al., 2020) . recent mechanistic and structural analyses indicate that the viral spike (s) protein of sars-cov-2 binds to ace2 in concert with s-protein priming by the host cell transmembrane serine protease tmprss2, mediating host cell entry of the virus mittal et al., 2020) . this binding of sars-cov-2 is shown to be at 10-to 20-fold higher affinity than that of sar-cov , accounting at least partially for the greater pathogenicity of sars-cov-2. in addition to mediating viral entry, binding of sars-cov-2 to ace2 and subsequent endocytosis dysregulate angiotensin system, leading to the loss of ace2-mediated health protection and adverse systemic effects (gheblawi et al., 2020) . moreover, these molecular events upregulate proteolytic cleavage mediated by a disintegrin and metalloproteinase 17 (adam17) of not only ace2 itself, which further dysregulates the angiotensin system, but also its primary substrate releasing tumor necrosis factor (tnf)-along with interleukin (il)-6 and other cytokine mediators, leading to cytokine storm in covid-19 described below [fig. 2, (gheblawi et al., 2020) ]. taken together, ace2 is the key human cellular receptor and plays a critical role in the pathogenesis of covid-19 through complex molecular mechanisms against which therapeutic agents can potentially be developed, such as recombinant human ace2 (rhace2) and inhibitors against tmprss2 and possibly adam17 (bourgonje et al., 2020; gheblawi et al., 2020; hoffmann et al., 2020) . influenza, another zoonotic virus which has a segmented negative-sense rna genome and is also a significant respiratory pathogen, is well known for its genetic instability, likely due to the lack of proofreading function of its rna polymerase complex (te velthuis and fodor, 2016) . point mutations j o u r n a l p r e -p r o o f or small deletions in individual rna segments cause relatively minor antigenic changes, termed antigenic drift, leading to new strains each winter season responsible for seasonal influenza. for this reason, annual updates of influenza vaccines are required. exchange or re-assortment of the viral genome rna segments between strains from humans and animals (e.g., birds or swine) can lead to major antigenic alterations, termed antigenic shift, so that the novel viruses can escape from existing herd immunity and cause influenza pandemics (kim et al., 2018; lyons and lauring, 2018) . coronaviruses, on the other hand, are not known for their major genomic re-assortment capacity. why the aforementioned three virulent novel coronaviruses have emerged in less than 20 years of this young century remains a mystery. among them, sars-cov-2 is the most contagious and sars-cov has not recurred since 2004, reasons for which also remain to be determined. specific mutations are being identified in the sars-cov-2 genome during this ongoing pandemic. for example, a mutation that changed the amino acid at position 614 of the spike protein from an aspartic acid to a glycine, known as g614, may increase infectivity of sars-cov-2 (korber et al., 2020; kupferschmidt, 2020) . unlike influenza and other common coronaviruses, it is now apparent that sars-cov-2 spread is not impeded by warm weather despite an early study suggesting distribution of covid-19 outbreaks along restricted latitudes, temperatures, and humidity (sajadi et al., 2020) ; increased international travel by air or cruise may also contribute to the spread of sars-cov-2 (gupta et al., 2020; tabari et al., 2020) . the mechanism for this unique and worrisome feature remains to be elucidated as well. addressing the challenges of these unknown aspects of sars-cov-2 biology is critically important, particularly in considering the current resurgence of new cases and the prospect of ongoing covid-19 for years to come. j o u r n a l p r e -p r o o f in their 60s, 8% in their 70s, to 14.8% in their 80s or older; the overall cfr is 2.3% . in comparison, the overall cfr was approximately 2.8% worldwide and 2.7% in the us as of october 19, 2020 (fig. 1) . the rapid growth of the number of older adults in the world's largest population, the so-called aging tsunami by some, coupled with the unique socioeconomic context, ongoing healthcare reform, and nascent development of geriatrics, creates significant challenges for china to fight againstcovid-19, particularly during the early days of the pandemic (fang et al., 2015; li et al., 2018; yip et al., 2019) . a more profound effect of aging is shown by covid-19 cfr data from italy, the first country affected by the pandemic after china. again, cfrs are from less than 0.4% or lower in patients aged in the 40s or younger, 1% among those in their 50s, 3.5% in their 60s, 12.8% in their 70s, to 20.2% in their 80s and above; the overall cfr is 7.2% (onder et al., 2020) . of note, the overall cfr is higher in italy than that in china (7.2% vs 2.3%, respectively). this is likely because italy not only has a higher cfr than china among adults over 70 years of age, but also has a higher proportion of older adults than china (22.8% vs 11.9%, respectively). the us: the first covid-19 outbreak reported in the us was at a long-term care facility in washington state (arons et al., 2020; mcmichael et al., 2020) . the first covid-19 death reported in new york city was an 82-year-old person in brooklyn. data from a large case series of 5700 covid-19 patients who were admitted to hospitals in new york city have shown a strikingly similar j o u r n a l p r e -p r o o f trend of age-related increases in covid-19 deaths. that is, deaths among patients in hospital at the study end point were 3.3% or lower in patients aged in the 40s or younger, 4.8% among those in their 50s, 6.4% in their 60s, 12.6% in their 70s, to 25.9% in their 80s and above (richardson et al., 2020) . as shown in fig. 4 , data reported by the us center for disease control and prevention (cdc) also demonstrate significantly higher rates of hospitalizations, icu admissions, and deaths secondary to covid-19 among older adults (> 65 years) than any younger age groups. [(cdc, 2020) , https://www.cdc.gov/mmwr/volumes/69/wr/mm6912e2.htm]. perhaps, the most striking evidence is the data on covid-19 cases and death in nursing homes across the us from a comprehensive analysis updated by the new york times on september 15 th [(new york times, 2020) , https://www.nytimes.com/interactive/2020/us/coronavirus-nursing-homes.html]. currently, there are up to 1.5 million nursing home residents in the us, less than 0.5% of its population. however, about 7% of confirmed covid-19 cases were among these vulnerable elderly individuals. moreover, they suffered 40% of covid deaths in the us. taken together, it is unmistakable that aging is an important risk factor for severe covid-19 disease and its adverse health outcomes including hospitalization, icu admission, and death. j o u r n a l p r e -p r o o f immunity is a cornerstone of host-pathogen interaction in any infectious disease. it involves three distinct but interrelated key aspects: vulnerability, immune response and protection, and potential immune pathology (fig. 5) . in most cases, immune response from prior exposure to the same pathogen or through vaccination with the same dominant antigen can provide at least partial immune protection (i.e., reduction of incidence of infection and/or its severity) via immune memory. the level of vulnerability also involves innate immunity independent of antigen-specific immune responses and other physiological protective mechanisms. if the immune response to the current infection is dysregulated, however, it may cause immune pathology and contribute to the pathogenesis of the disease. since sars-cov-2 is a novel coronavirus with no prior immune response, the entire population is susceptible with essentially no herd immunity. as discussed earlier, this was also the case in influenza pandemics, such as the pandemics in 1918, 1957, 1968, and more recently the swine flu pandemic in 2009 (petersen et al., 2020) . nonetheless, under certain circumstances, older adults may enjoy better protection than the young against strains that were circulating when they were young, by virtue of immunological memory and/or cross-reactivity. a considerable fraction of healthy individuals not infected with sars-cov-2 possess t cells reactive to sars-cov-2 antigens, perhaps because of cross-reactivity with other coronaviruses. it remains to be seen whether this provides any protection against covid-19 grifoni et al., 2020; mateus et al., 2020; sette and crotty, 2020) . in covid-19, a number of immunological studies were initially reported from clinical observations of covid-19 patients in wuhan, china. while more studies are being published almost every day, they often lack breadth and depth in interrogation of the immune system. we will first review some of the published data currently available, and then propose an immune hypothesis for age-related j o u r n a l p r e -p r o o f vulnerability to severe covid-19 and adverse health outcomes. instead of proposing age-related chronic inflammation (akbar and gilroy, 2020) or immunosenescence secondary to cytomegalovirus infection (kadambari et al., 2020; moss, 2020) individually as mechanisms underlying this vulnerability, this immune hypothesis integrates immunopathology secondary to cytokine storm and inflammaging and immunosenescence as complex immune mechanisms that could provide a basis for interventional strategies, such as anti-il-6 therapy and immunization with covid-19 vaccines ( fig. 5 ). we will also point out challenges in conducting comprehensive and in-depth immune studies and in interpretation of existing data and their implications for vaccine development. "cytokine storm" and immunopathology: severe covid-19 patients typically develop acute respiratory distress syndrome (ards) requiring intubation and ventilator support as well as significant involvement of other organ systems. for example, neurological manifestations, termed as "neuro-covid" by some, occur in over one-third of covid-19 patients (chiappelli, 2020; ferrarese et al., 2020; frontera et al., 2020; leonardi et al., 2020; wang et al., 2020b) . while sars-cov-2 has been shown for its neurotrophic and neuroinvasive properties (baig et al., 2020; puelles et al., 2020) , inflammatory cytokine release and immunopathology caused by sars-cov-2 infection may also play an important role in contributing to neuro-covid and other systemic manifestations (jose and manuel, 2020; wu and mcgoogan, 2020) . in fact, earlier clinical observations from wuhan, china indicated patients with covid-19 manifest an acute increase of serum levels of inflammatory mediators, such as il-6 and c-reactive protein (crp) . levels of other inflammatory mediators including interferon (ifn)- induced protein 10 (ip-10, or cxcl-10) and monocyte chemotactic protein-3 (mcp-3) are also acutely elevated in covid-19 patients, and such elevation is associated with disease severity and progression (lagunas-rangel and chavez-valencia, j o u r n a l p r e -p r o o f 2020; lin et al., 2020; yang et al., 2020) . cytokine storm, or cytokine release syndrome (moore and june, 2020) , has also been observed in sars and mers and is believed to play an important role in their development and progression (de wit et al., 2016) . although the sources and regulation of this cytokine storm remain to be elucidated, it is likely derived from dysregulated immune responses to these virulent coronaviruses, leading to immunopathology and severe disease. these observations have led to the ongoing therapeutic development targeting il-6, a cytokine mediator that is considered as a hallmark of inflammaging (franceschi et al., 2000; franceschi et al., 2017; maggio et al., 2006) . monoclonal antibodies (mab) targeting il-6 receptor (il-6r, tocilizumab and sarilumab) or il-6 itself (siltuximab) are now available. while in-depth discussion of differences in mechanisms regulating il-6 signaling between these mabs and implication in their efficacy and/or side effects is beyond the scope of this article, it is worthwhile noting that anti-il-6 and anti-il-6r mabs can all antagonize il-6 cis signaling (via canonical membrane-bound il-6r and gp130) and trans signaling (via soluble il-6r), but only anti-il-6r mabs can antagonize il-6 trans presentation, a newly described mode of il-6 signaling that involves membrane-bound il-6r of dendritic cells (dcs) to generate pathogenic th17 cells (heink et al., 2017; kang et al., 2019) . the immediate goal of such therapeutic intervention is to block the dysregulated cascade of immune activation and inflammation downstream to the cytokine storm to ameliorate severe covid-19 and its further progression. preliminary results available thus far did not show significant mortality benefit of sarilumab treatment (della-torre et al., 2020), further analyses of subgroup patients and studies of other mabs are needed. ultimately, effective vaccine and antivirals will be needed to prevent or ameliorate sars-cov-2 infection and its induced cytokine storm or immunopathology (tay et al., 2020) . ni et al., 2020; zhao et al., 2020) . one study also reported correlative igg, igm, and iga titers between serum and saliva (randad et al., 2020) . it is not currently known if asymptomatic infection induces detectable antibody responses or whether viral load determines antibody response. in addition, the titer threshold of anti-sars-cov-2 antibodies that correlates with clinical protection against covid-19 has yet to be determined. studies have shown higher anti-sars-cov-2 antibody titers in icu patients compared to mild patients and prolonged presence of neutralizing antibodies with viral shedding in covid-19 patients, raising questions about the protective efficiency of such antibody responses zhao et al., 2020) . moreover, a phenomenon called antibody-dependent enhancement (ade) was unexpectedly reported in covid-19 (arvin et al., 2020; wan et al., 2020) , further suggesting the complexity of the effects of such antibody responses. therapeutic use of convalescent plasma collected from recovered covid-19 patients is a promising passive immune therapy currently in clinical trials. observational findings suggest improved clinical outcomes in those who are transfused with covid-19 convalescent plasma (ccp), including radiological resolution, reduction in viral loads, and improved survival (duan et al., 2020; harvala et al., 2020; hegerova et al., 2020; joyner et al., 2020; zhang et al., 2020a) . while two randomized trials assessing ccp china and europe were terminated early and underpowered, they did not find clinically significant differences between the study arms ; gharbharan, et al). studies aimed at defining factors that impact the quality and titer of antibody, including sars-cov-2 neutralization reveal that older age, male sex, and hospitalization with severe covid-19 are all factors that contribute to greater antiviral antibody responses against sars-cov-2 . cell-mediated immune response: clinical observations have revealed significant lymphopenia and increased neutrophil counts in severe covid-19 disease and, therefore, lymphopenia and high neutrophil-lymphocyte ratio (nlr) are considered as useful predictors for covid-19 death whereas high lymphocyte counts predict better clinical outcomes (chen et al., 2020c; lagunas-rangel and chavez-valencia, 2020; qin et al., 2020; wang et al., 2020a; zhou et al., 2020a) . while neutrophil increases may reflect an acute inflammatory response related to the cytokine storm described above, lymphopenia indicates major impacts on cell-mediate immunity in the early stage of covid-19. lymphopenia consists of depletion of both cd4+ and cd8+ t cells . the reason for such depletion is not well understood at the present time. one may speculate that t cells are redistributed from the circulation to the site of the infection in the lungs. sánchez-cerrillo et al observed redistribution of activated monocytes and dendritic cells to the lungs in severe covid-19 patients (sanchez-cerrillo et al., 2020) . alternatively, as described in more detail below, viral proteins from sars-cov also shared by sars-cov-2 can suppress type 1 ifns leading to a poor cd8+ t cell response (fung et al., 2020; welsh et al., 2012) . whether the observed lymphopenia is a general phenomenon of acute viral infections versus unique or more profound in covid-19 remains to be determined. regardless, a retrospective study of 548 covid-19 patients showed that restored lymphocyte counts predicted recovery during hospitalization while lymphopenia persisted in j o u r n a l p r e -p r o o f non-survivors (chen et al., 2020c) . this further emphasizes the importance of cell-mediated immunity and its impact on clinical outcomes. t lymphocytes are a key cellular basis of adaptive immune protection and vaccination and play a critical role in assisting production of neutralizing antibodies and direct virus clearance. a number of studies have shown sars-cov-2-specific cd4+ and cd8+ t cell responses in covid-19 patients meckiff et al., 2020; neidleman et al., 2020; ni et al., 2020; weiskopf et al., 2020) . in the setting of sars, antigen-specific memory t cells to sars-cov have been shown to persist in convalescent sars patients at low frequency for up to six years and their responses to ex vivo stimulation with sars-cov persist up to 11 years (ng et al., 2016; oh et al., 2011) . the duration that sars-cov-2-specific memory t cells persist is yet to be established. not all t cell responses are beneficial. for example, kang et al observed hyperactivation of cytotoxic t cell responses as a determinant of covid-19 severity (kang et al., 2020) . zhang and colleagues suggest aberrant cd8 t cell activation as a potential mechanism for cardiac injury in severe covid-19 (zhang et al., 2020c) . th17 activation is also believed to play a role in contributing to cytokine storm in severe covid-19, suppression of which is proposed to be a mechanism underlying anti-il-6r therapy (lagunas-rangel and chavez-valencia, 2020) and other immunotherapies . comprehensive studies of cell-mediated immune responses are few and far between at the present time. peng et al observed broad and strong memory cd4+ and cd8+ t cells induced by sars-cov-2 in uk convalescent covid-19 patients, significantly more so in severe compared to mild covid-19 j o u r n a l p r e -p r o o f cases . a deep immune profiling of covid-19 patients conducted by matthew and colleagues, however, revealed great heterogeneity (mathew et al., 2020) . immune hypothesis for age-related vulnerability in older adults: given the disproportionate burden of severe covid-19 disease and death in older adults, it is important to understand mechanisms that underlie this age-related vulnerability. age-related immune system remodeling, or immunosenescence, is considered to be the major reason for increased susceptibility to infection, particularly respiratory infections such as influenza, as well as impaired immune responses to vaccination pawelec, 2018) . here we propose an immune hypothesis for covid-19 vulnerability of older adults. it involves age-related impairment of immune defense against sars-cov-2 infection, or immunosenescence, and increased risk for immunopathology (fig.5) . although age-related change in innate and adaptive immunity against sars-cov-2 infection are yet to be investigated in detail, its impairment and dysregulation in older adults can be inferred. for example, senescence-related impairment of type 1 ifn response is responsible for enhanced influenza viral replication in cell culture (kim et al., 2016) and older adults manifest impaired type 1 ifn response to influenza vaccination (thakar et al., 2015) . in addition, several sars-cov non-structural proteins that are shared by sars-cov-2 suppress type 1 ifn response and such suppression is shown to lead to poor cd8+ t cell response to viral infection (fung et al., 2020; manners et al., 2020; welsh et al., 2012) . therefore, age-associated reduction in type 1 ifn response coupled with direct viral suppression could serve as a critical innate immune mechanism that leads to poor cell mediated immunity and increased vulnerability of older adults against sars-cov-2 infection with therapeutic implication (sallard et al., 2020) . little data is currently available about the j o u r n a l p r e -p r o o f impact of aging on cd4+ and cd8+ t cell responses in covid-19. it is postulated that age-related decline of de novo t cell responsiveness and/or impact from comorbid conditions, particularly persistent viral infections such as chronic cytomegalovirus (cmv) infection could serve as potential causes of covid-19 vulnerability in older adults (kadambari et al., 2020; moss, 2020; nicoli et al., 2020) . available data on humoral immunity are fascinating and counter-intuitive. among covid-19 convalescent plasma donors, klein et al observed higher sars-cov-2-specific neutralizing and igg antibody titers in older donors compared with their young counterparts . in a study cited above, zhang et al also described the correlation between greater anti-sars-cov-2 igg titers and older age . the reason for these observations is unknown at the present time and deserves further investigation. substantial evidence supports the role of immunopathology in the pathogenesis of covid-19 overall as described above. based on their review of data on immune changes published during the early stage of this pandemic, lin and colleagues proposed a hypothesis for the role of immune and inflammatory factors in contributing to dysregulation of the coagulation system in the pathogenesis of covid-19 and suggested intravenous immune globulin (ivig) and low molecular weight heparin (lmwh) anticoagulant therapy . studies also suggested a link between complement activation and endothelial dysfunction, likely the key to microvascular thrombosis and multi-organ failure in severe covid-19 (mackman et al., 2020; magro et al., 2020; noris et al., 2020) . however, few studies are available with a focus on aging. inflammaging is well documented and can be derived from senescence-associated secretory phenotype (sasp), persistent viral infection such as cmv, and other potential sources franceschi et al., 2000; franceschi and campisi, 2014) . such an unbalanced pro-inflammatory environment could potentiate further inflammatory response j o u r n a l p r e -p r o o f upon sars-cov-2 infection, leading to the development of an exacerbated cytokine storm in older adults. it may also influence ace2 expression and facilitate viral entry (radzikowska et al., 2020) . further studies are urgently needed to address this important mechanism with a focus on aging. children are overwhelmingly spared from severe covid-19 disease except for the extremely rare occurrence of multisystem inflammatory syndrome in children (mis-c), also called kawasaki disease-like syndrome (lingappan et al., 2020; viner and whittaker, 2020) . insights into children's defense mechanisms against sars-cov-2 infection may shed light on age-related vulnerability in older adults from a different perspective. for example, chen et al observed significantly higher counts of total as well as both cd4+ and cd8+ t cells in pediatric covid-19 cases compared to their adult counterparts . on the other hand, children with covid-19 manifested lower levels of t cell activation than adult covid-19 patients (moratto et al., 2020) , suggesting better immune system control and regulation in response to sars-cov-2 infection in children. thymic function likely plays an important role in preserving t cells in covid-19 (rehman et al., 2020) . in fact, liu et al showed that thymosin 1 reversed lymphopenia, reversed exhausted t cells and reduced mortality of severe covid-19 in adults . in addition, children demonstrate strong innate immunity despite the fact that the immune system as a whole is yet to be fully developed. one possibility is "trained immunity" through scheduled immunization with many doses of pediatric vaccines (netea et al., 2020) . whether this can be applicable in older adults (i.e., potential protective effect from immunization with other unrelated vaccines such as influenza vaccine) remain to be investigated. challenges: despite the large number of research papers including preprints that are published almost every day, it remains a difficult task to obtain reliable knowledge of covid-19 that is accurate and precise. conflicting data and results that are not reproducible are not uncommon. here we discuss a few inherent difficulties that present themselves as great challenges for immune studies of covid-19, reminding readers of critically evaluating the literature and putting published results in appropriate context. this is especially true for assessing covid-19 information in social media, particularly in the current environment of rapid dissemination of misinformation and disinformation. first, sars-cov-2 infection is highly heterogeneous, from asymptomatic infection to mild, moderate, or severe covid-19. in addition, the infection can evolve through different stages and progress in either direction (improving and recovery versus worsening and death). most published immunological studies are cross-sectional and at one time point with a relatively small sample size. although immunological analysis itself can be cutting edge and in-depth, the results from these studies are only valid in the context of the study with limited generalizability. asymptomatic sars-cov-2 infection can be a challenge in selecting "healthy controls" as well. due to lack of universal covid-19 testing, it is difficult to know whether people are truly "healthy" (uninfected) or asymptomatic. secondly, complex impact of aging on the immune system and influences from comorbid conditions and concomitant use of medications may all contribute to the heterogeneity of the data on t-cell immune responses to sars-cov-2 (mathew et al., 2020) . finally, there is a high-sequence similarity in viral proteins (e.g., nuclear protein) between sars-cov-2 and other  coronaviruses (e.g. 229e strain) . as such, prior coronavirus infections may induce heterotypic cellular immunity against covid-19. this heterotypic cellular immune response may further complicate covid-19 immunological studies and interpretation of their results. therefore, it j o u r n a l p r e -p r o o f is important to take these challenges into consideration in interpretation of published data as well as in design and implementation of immunological and interventional studies including covid-19 vaccine development. effective and safe vaccination against sars-cov-2 is the best strategy to stop viral spread and control the pandemic. intense worldwide efforts for covid-19 vaccine development began after the genetic sequence of sars-cov-2 was published on january 11 th , and has since advanced at previously unimaginable speed with the first vaccine candidate entering human clinical trials on sequence became available and demonstrated promising preliminary results (jackson et al., 2020) . two phase 1/2 clinical trials of two different adenovirus-vectored covid-19 vaccines have reported robust antibody and t-cell responses to the vaccines with acceptable side effects in healthy adults (folegatti et al., 2020; zhu et al., 2020a) . however, the chadox1 ncov-19 vaccine trial was conducted among adults of 18-55 years of age with no enrollment of older adults (folegatti et al., 2020) . despite progress that has been made thus far, significant challenges lie ahead. for example, while focusing on the vaccine itself is important, attention must also be directed to the host. because of the lack of in-depth knowledge about the immune responses to sars-cov-2 infection, no specific immune parameter(s) of vaccine potency, or correlates of protection, are currently available. the phenomenon of ade discussed above (arvin et al., 2020) , also deserves careful consideration (lambert et al., 2020) . aging is a critical host factor to consider in the context of vaccination responses (pawelec and weng, 2020) . it may be necessary to develop different vaccines on specific vaccine platforms for older adults versus those for children and young adults. in the case of influenza vaccination, the standard dose of trivalent inactivated influenza vaccine (iiv3), the only influenza vaccine available for older adults for many years before the approval of high-dose and adjuvanted influenza vaccines, showed questionable effectiveness, if at all, for the elderly, particularly those who are frail and need vaccine protection the most (yao et al., 2011) . over a century after the 1918 spanish flu pandemic, the worst pandemic in human history, we are still in search of a universal influenza vaccine (paules et al., 2017) . the ongoing pandemic poses special challenges (diamond and pierson, 2020) . for vaccine trials, levels of pandemic activity as well as various quarantine measures and their implementation significantly impact study enrollment, protection of both research staff and participants, and other trial logistics. because of the low level of pandemic activity in china, covid-19 vaccine trials are not feasible at the present time, even for vaccines that were developed in china. when a safe and efficacious covid-19 vaccine becomes available, the demand of the ongoing pandemic can post huge challenges on large-scale production and distribution of the vaccine. addressing these and other challenges requires global corporation and coordination among governments, academia, and industry. with rapid progress in covid-19 vaccine development, it is promising and hopeful that there will be one or several safe and effective covid-19 vaccines in the near future. but the public should be fully educated about the alternative reality, that is, we may not have such a vaccine for a long time, if ever. to that end, authorities have begun to develop protective programs against covid-19 outbreaks at nursing homes, the setting with arguably highest risk for older adults as j o u r n a l p r e -p r o o f described above. for example, pennsylvania state has established a statewide regional response health collaborative program (rrhcp, https://www.media.pa.gov/pages/dhs_details.aspx?newsid=569) with nearly $23 million awarded to pennsylvania state university to support 244 nursing homes in the southcentral region of pennsylvania against covid-19 outbreaks and mitigation should covid-19 be present at these facilities. rrhcp provides a wide range of covid-19 related clinical and public health supportive services, such as testing performance and laboratory capacity, personal protective equipment (ppe) supplies, infection prevention training and advising, rapid response teams for sites with an active covid-19 outbreak, staffing support, alternate care settings, mental and behavioral health support for residents and staff, clinical support via telehealth and geriatrician site visits, contact tracing, and education through a statewide learning network [https://news.psu.edu/story/630235/2020/09/01/penn-state-health-receives-grant-mitigate-covid-1 9-care-facilities]. as to the public, every one of us should adhere to social distancing and other quarantine measures that are known to be effective to prevent virus spread. a historical lesson from the 1918 spanish flu pandemic is that compared to philadelphia, st. louis was able to minimize flu pandemic deaths and flatten the curve through its prompt and strictly enforced quarantine measures (fig. 6a) (hatchett et al., 2007) . the contemporary one is that europe flattened the curve during the summer, 2020, while the us suffers ongoing covid-19 resurgence (fig. 6b) j o u r n a l p r e -p r o o f fig. 2 . ace2 as the key human cellular receptor for sars-cov-2, its role and underlying molecular mechanisms in the pathogenesis of covid-19. in addition to viral entry, binding to ace2 of sars-cov-2 in concert with s-protein priming by tmprss2 and subsequent endocytosis result in dysregulation of the angiotensin system, leading to the loss of ace2-mediated systemic health protection. these molecular events also upregulate adam17-mediated proteolytic cleavage of not only ace2 itself, which further dysregulates the angiotensin system, but also its primary substrate releasing tnf- along with il-6 and other cytokine mediators, leading to cytokine storm. tmprss2: transmembrane serine protease 2; immune response and protection against sars-cov-2 and immunopathology. immune response includes humoral immunity (i.e., antibody response) and cell-mediated immunity (cmi) (right). while age-related immunosenescence is believed to weaken immune protection, vaccination enhances it. inflammaging and cytokine storm may lead to immunopathology (left). not all immune responses are protective as antibody-dependent enhancement (ade) in humoral immunity may promote sars-cov-2 infection while th17 response in cmi may contribute to cytokine storm. anti-il-6 therapy with monoclonal antibodies against either il-6 or il-6 receptor currently in clinical trials can block cytokine storm and its downstream event and/or suppress th17 response. age-related decrease of physiological reserve in respiratory and other organ systems may also contribute to vulnerability. together, they lead to disproportionately severe covid-19 and high mortality in older adults. all the authors have seen and approved the final version of the manuscript being submitted. all the authors warrant that the article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere. this study was supported in part by funding from national institutes of health (nih) (r01 ai108907 and r42ag054322) and funding from irma and paul milstein program for senior health, milstein medical asian american partnership (mmaap) foundation of usa (www.mmaapf.org) to sxl, nih r21 ag059742 to sxl and jbm, nih u54 ag062333 and department of defense for covid-19: w911qy-20-9-0012 to slk, and pennsylvania state regional response health collaborative program (rrhcp) funding to no. cjw is an irma and paul milstein program for senior health fellow supported by mmaap foundation. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. covid-19: zoonotic aspects aging immunity may exacerbate covid-19 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(sars-cov-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against sars-cov-2 spike protein and to evaluate the serological immunity in humans. since the accessibility of live virus microneutralisation (mn) assays with sars-cov-2 is limited and requires enhanced bio-containment, the approach based on “pseudotyping” can be considered a useful complement to other serological assays. after fully characterising lentiviral pseudotypes bearing the sars-cov-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an italian sero-epidemiological study. the results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and mn results. the overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context. in early december 2019, cases of severe pneumonia of unknown aetiology were reported by the china health authority. in january 2020, a novel coronavirus was identified as 2019-ncov (subsequently renamed as sars-cov-2). an initial site of infections was the huanan seafood market, where live animals are sold. progressively, human-to-human transmission occurred [1] , causing a disease named coronavirus disease 2019 . on 20th july 2020, the world health organization (who) estimated the global incidence of covid-19 as 14,348,858 cases and the number of the deaths as 603,691 [2] . hep g2 cells were cultured in rpmi 1640 (gibco) supplemented with 2 mm l-glutamine (lonza, milan, italy), 100 units/ml penicillin-streptomycin (lonza, milano, italy) and 10% foetal bovine serum (fbs) (euroclone, pero, italy). hek293 t/17, mdck and huh-7 cell lines were cultured in dulbecco's modified eagle's medium (dmem) high glucose (euroclone, pero, italy) supplemented with 2 mm l-glutamine (lonza, milan, italy), 100 units/ml penicillin-streptomycin (lonza, milan, italy) and 10% of fbs (fbs euroclone, pero, italy). vero e6 and caco2 cells were cultured in eagle's minimum essential medium (emem) (lonza, milan, italy) supplemented with 2 mm l-glutamine (lonza, milan, italy), 100 units/ml penicillin-streptomycin (lonza, milan, italy) and fbs (euroclone, pero, italy) to a final concentration of 10% for vero e6 and 20% for caco2. all the cell lines used were incubated at 37 • c, 5% co 2 in humidified atmosphere and were sub-cultured twice a week until passage 20. a total of 65 samples from an italian sero-epidemiological study, anonymously collected in compliance with italian ethics law, were provided by the laboratory of molecular epidemiology of the university of siena, italy. the human sera, derived from a sero-epidemiological study, had previously been tested in an elisa assay as pre-screening, and positive, borderline and negative elisa samples were tested in a micro-neutralisation assay, as previously described [24] . this panel of sera was subsequently tested with sars-cov-2 pseudotypes in order to compare the neutralisation profiles when they were tested against the live virus and the surrogate virus. as an internal positive control, a panel of samples collected from health care workers (confirmed positive for sars-cov-2 by reverse real-time pcr) were kindly provided by prof. valentina bollati, university of milan. in addition, three monoclonal antibodies (mabs) were included in the serological assay: human igg1 anti-s1 cr3022 (native antigen, oxford, uk), human igg1 anti-rbd (eenzyme, gaithersburg, md, usa) and human anti-igm sars-cov-2 spike s1 cr3022 (absolute antibody, 21 drydock avenue, 7th floor boston, ma 02210, usa (1:100 starting dilutions). the full-length s protein (genbank accession number: yp_009724390.1) was codon-optimised and synthesised (genscript, cina), and the s fragment was cloned into the expression vector as described previously [25] . the hiv gag-pol plasmid (p8.91) [26] , the firefly luciferase-expressing plasmid (pcsflw) [27] , the pcaggs-tmprss2 plasmid and the plasmid encoding for the spike's human ace2 receptor were kindly provided by dr. nigel temperton and have previously been described [28] [29] [30] . as a control, a vesicular stomatitis virus (vsv-g) plasmid was used (pcmv-vsv-g) (addgene plasmid 8454; http://n2t.net/addgene:8454) [31] . the day before transfection, confluent plates of hek 293t/17 cells were split 1:4 and seeded into 10 cm 2 plates in dmem 10% fbs. cells (at the 60% of confluence) were co-transfected with the s plasmid from sars-cov-2 (2 ug/ul), hiv gag-pol (1 ug/ul) and the pcsflw (1.5 ug/ul) using endofectin™ lenti transfection reagent (tebu bio-217ef002, via pretorio 4-c.p. 70. i-20013 magenta, milano) in accordance with the manufacturer's instructions. the following day, the supernatants were replaced with dmem without phenol red (thermo fisher scientific, 168 3rd ave, waltham, ma 02451, united states containing 10% fbs, and the plates were incubated for 48 h at 37 • c in an atmosphere of 5% co 2 . after 48 h, the supernatants of transfected cells were harvested and filtered by millex-ha 0.45 um filter. concurrently, hek 293t/17 cells were also transfected with vsv-g plasmid (1 µg/µl), and a no-spike control (∆ envelope) was generated by co-transfection with hiv gag-pol and pcsflw plasmids only. for sars-cov-2 titration, a further transfection is required in order to allow pseudotypes to enter target cells. the day before titration, hek 293t/17 cells were co-transfected with two plasmids encoding for the ace2 receptor gene and tmprss2, by means of endofectin™ lenti transfection reagent; after overnight incubation at 37 • c, the supernatant was replaced by dmem containing 10% fbs. the following day, supernatants were serially two-fold diluted in a fresh cell culture medium in 96-well, flat-bottomed culture plates, and 1 × 10 4 hek 293t/17 target cells were added to each well. as controls, vsv-g and ∆-envelope pseudotypes were also included. after 72 h, the luminescence of cell cultures (in relative luminescence units or rlus) was evaluated by luminometry (tecan infinite m1000 pro multi-detection plate reader) using the bright-glo assay system (bright-glo™ luciferase assay system, promega, viale piero e alberto pirelli, 6, 20126 milano mi). serial dilutions of sars-cov-2 pseudotype-containing media were tested by means of the lenti-x p24 rapid titre kit (cat. no. 632200) (takara, japan) in accordance with the manufacturer's instructions. in order to verify the incorporation of the s glycoprotein of sars-cov-2, the s protein expressed on the lentiviral pseudotypes was detected by western blot analysis. western blot analysis of spike protein was performed on the supernatant from sub-confluent hek 293t/17 cells co-transfected with hiv gag-pol plasmid, csflw plasmid and the s plasmid from sars-cov-2. western blot analysis was also performed on lv in a bsl3 facility. ∆-envelope (no-spike control) pseudotypes prepared with the same procedure were run as a negative control. sars-cov-2 pseudotypes, sars-cov-2 live virus and ∆-envelope pseudotypes were mixed with sds sample buffer. the mixture was heated for 10 min at 75 • c and electrophoresis (50 µg of protein/sample) was carried out in 4-12% bis-tris gels (life technologies, carlsbad, ca, usa). proteins were then blotted onto nitrocellulose membranes and incubated overnight with 500-fold diluted sera from convalescent sars-cov-2 patients. a goat anti-human igg (bethyl, 25043 fm 1097, montgomery, tx 77356, united states) was used as a secondary antibody (1:5000 dilution). the chemiluminescent signals from the nitrocellulose membranes were captured by a camera system (imagequant las 400 instrument). in this study, different cell lines have been tested in order to study their susceptibility to sars-cov-2 s protein driven entry, the role of the ace2 receptor and tmpssrs2 for s protein priming. one day before pseudotypes titration, mdck, vero e6, caco2, hep g2 and huh7 cells were transfected with the pcaggs-tmprss2 plasmid, while hek 293t/17 cells were co-transfected with ace2 and pcaggs-tmprss2 plasmids. after 24 h, the cells were removed by trypsinisation, counted and used for the subsequent titration. in parallel with sars-cov-2 pseudotypes, vsv-g and ∆-envelope pseudotypes were titrated as controls. plates were incubated for 48-72 h with the pseudotypes, at 37 • c in an atmosphere of 5% co2, and the efficiency of pseudotypes entry was characterised on the basis of luciferase activity (relative luminescence units or rlus). to measure the neutralisation activity of this panel of sera, neutralising antibody titres were defined as endpoint two-fold seral dilutions of test samples, and the 50% inhibitory concentration (ic 50 ) was determined as the serum dilution resulting in a 50% reduction of a single round of infection (reporter gene-mediated signal). values were expressed as a percentage in comparison with the signal from the cell-only control (equivalent to 100% neutralisation and/or no infection) and the signal from a pseudotype-only control (equivalent to 0% neutralisation or 100% infection). in brief, two-fold serial dilution of serum samples, starting from 1:10, was performed in a culture medium (dmem, 5% fbs, 1% pen-strep, 1% l-glutamine). the serum was mixed with sars-cov-2 pseudotypes in a 1:1 vol/vol ratio in a 96-well culture plate. the virus input used was 1 × 10 6 rlu/well (based on the previous titration). the serum-pseudotypes mixture was then incubated for 1 h at 37 • c in a humidified atmosphere with 5% co2. after 1 h, hek 293/ace2 transfected cell suspensions (1.5 × 10 4 cell/ml) were seeded into each well of flat-bottomed tissue culture plates. the plates were incubated at 37 • c for 48-72 h, and the neutralising antibodies were characterised on the basis of luciferase activity. the sars-cov-2 strain 2019-ncov/italy-inmi1-wild-type virus was purchased from the european virus archive goes global (evag, spallanzani institute, via portuense, 292, 00148-00153, rome) and propagated in vero e6 cells. the virus was titrated in a tissue culture infective dose 50% assay (tcid50) on 96-well culture plates with 1-log serial dilution. the plates were observed daily for a total of four days for the presence of cytopathic effect (cpe). the end-point titre was calculated according to the spearman-karber formula [32] . the mn assay was performed as previously reported by manenti et al. [24] . briefly, two-fold serial dilutions of serum samples were mixed with an equal volume of viral solution containing 100 tcid50 of sars-cov-2. the serum-virus mixture was incubated for 1 h at 37 • c in a humidified atmosphere with 5% co 2 , then passed to a vero e6 culture plate. the plates were incubated for four days at 37 • c in a humidified atmosphere with 5% co 2 . after the incubation time, each well of a 96-well plate was inspected by means of an inverted optical microscope to evaluate the percentage of cpe. pseudotype transduction titres were estimated by means of excel tm software; the pseudotype titres obtained at each point in a range of dilution points were expressed as rlu/ml, and the arithmetic mean was calculated. for the analyses of pseudotype-based neutralisation assays, titres were firstly normalised, and ic 50 values were calculated by a non-linear regression model (log (inhibitor) vs. normalised response-variable slope) analysis. titres were subsequently expressed as the range of dilution in which the ic 50 value lay. in order to evaluate cell infectivity, two-way analysis of variance (anova) with dunnett posttest was used to test for statistical significance (p > 0.05 (ns, not significant), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****)). for all statistical analyses, the graphpad prism version 8.4 package was used (graphpad software, graphpad, 2365 northside dr., suite 560, san diego, ca 92108, usa). the statistical analyses have been undertaken with the r software (version 3.6.2). different approaches drove our statistical analyses, as shown in figure 1 . all the titres underwent preliminary base-2 logarithmic transformation. in accordance with the classification approach, the titres greater than log2 (5) were labelled as "positive," and otherwise as "negative." with mnt taken as the reference ("true") results, the misclassifications were counted and displayed in an error matrix table. a linear regression model provided a measure of the strength of the relationship between pnt and mnt. as the dependent variable, we considered the log2 of pnt, and as the independent variable, the log2 of mnt. for the evaluation of the agreement between pnt and mnt, we used the intra-class correlation coefficient (icc). in addition, the bland-altman method (ba) enabled us to search for possible systematic difference (bias) between the pnt and mnt, as well as to identify the presence of outliers. the ba evaluation mainly consists in a scatter plot of the differences between the measurements vs. their means. the 95% limits of agreement (loas) were calculated around the mean of the differences. we set a maximum acceptable difference (mad) of 0.5 times the mnt, below which the observed pnt-mnt differences were considered as not having a significant biological effect. we interpreted the differences below the mad and within the 95% limits of agreement as interchangeable. by comparing the distributions of pnt and mnt, we got further insight into their similarity degree. thus, we applied the kolmogorov-smirnov test and measured the kullbac-leibler divergence (kld). the former considers the largest difference between the empirical distribution functions of the pnt and mnt tests, and the latter is an information theoretic-based value, which indicates how much information is lost when taking pnt as an approximation of mnt. the kld was calculated as the "true" reference the mnt distribution, and normalised as follows: where nkld is the normalised divergence, and pmnt and ppnt are the distributions of mnt and pnt, respectively. this normalisation restricts the divergence values in the range [−0.5, + 0.5], such that nkld = 0 if the pnt distribution perfectly reproduces the mnt distribution, while the extremes nkld = −0.5 or nkld = 0.5 are attained if kld tends towards infinite, (positive or negative infinite, respectively). lastly, we conducted a bootstrap test (100,000 resamples with replacement from the mnt and pnt data) on the kld statistic under the null hypothesis of nkld = 0. all the titres underwent preliminary base-2 logarithmic transformation. in accordance with the classification approach, the titres greater than log2 (5) were labelled as "positive," and otherwise as "negative." with mnt taken as the reference ("true") results, the misclassifications were counted and displayed in an error matrix table. a linear regression model provided a measure of the strength of the relationship between pnt and mnt. as the dependent variable, we considered the log2 of pnt, and as the independent variable, the log2 of mnt. for the evaluation of the agreement between pnt and mnt, we used the intra-class correlation coefficient (icc). in addition, the bland-altman method (ba) enabled us to search for possible systematic difference (bias) between the pnt and mnt, as well as to identify the presence of outliers. the ba evaluation mainly consists in a scatter plot of the differences between the measurements vs. their means. the 95% limits of agreement (loas) were calculated around the mean of the differences. we set a maximum acceptable difference (mad) of 0.5 times the mnt, below which the observed pnt-mnt differences were considered as not having a significant biological effect. we interpreted the differences below the mad and within the 95% limits of agreement as interchangeable. by comparing the distributions of pnt and mnt, we got further insight into their similarity degree. thus, we applied the kolmogorov-smirnov test and measured the kullbac-leibler divergence (kld). the former considers the largest difference between the empirical distribution functions of the pnt and mnt tests, and the latter is an information theoretic-based value, which indicates how much information is lost when taking pnt as an approximation of mnt. the kld was calculated as the "true" reference the mnt distribution, and normalised as follows: where nkld is the normalised divergence, and pmnt and ppnt are the distributions of mnt and pnt, respectively. this normalisation restricts the divergence values in the range [−0.5, + 0.5], such that nkld = 0 if the pnt distribution perfectly reproduces the mnt distribution, while the extremes nkld = −0.5 or nkld = 0.5 are attained if kld tends towards infinite, (positive or negative infinite, respectively). lastly, we conducted a bootstrap test (100,000 resamples with replacement from the mnt and pnt data) on the kld statistic under the null hypothesis of nkld = 0. sars-cov-2 s glycoprotein and gag-p24 in pseudotypes were characterised by immunoblot analysis and p24 elisa, respectively. to verify the expression of the spike protein in the sars-cov-2 pseudotypes, the spike was detected by western blot; sera from convalescent sars-cov-2 patients, which have been shown to have a high neutralising titre in microneutralisation with a live virus, were used as the primary antibody, and goat anti-human igg as the secondary antibody. sars cov-2 strain 2019-ncov/italy wild-type virus (lv), which was handled in a level 3 bio-containment facility (bsl 3), was used as positive control in order to evaluate the spike glycoprotein expression, while a ∆-envelope pseudotype, prepared with the same procedure, was used as a negative control. three different batches of pseudotypes were tested; specific bands were found for sars-cov-2 pseudotypes and for sars-cov-2 live virus, but not for the ∆-envelope control ( figure 2 ). sars-cov-2 s glycoprotein and gag-p24 in pseudotypes were characterised by immunoblot analysis and p24 elisa, respectively. to verify the expression of the spike protein in the sars-cov-2 pseudotypes, the spike was detected by western blot; sera from convalescent sars-cov-2 patients, which have been shown to have a high neutralising titre in microneutralisation with a live virus, were used as the primary antibody, and goat anti-human igg as the secondary antibody. sars cov-2 strain 2019-ncov/italy wild-type virus (lv), which was handled in a level 3 biocontainment facility (bsl 3), was used as positive control in order to evaluate the spike glycoprotein expression, while a δ-envelope pseudotype, prepared with the same procedure, was used as a negative control. three different batches of pseudotypes were tested; specific bands were found for sars-cov-2 pseudotypes and for sars-cov-2 live virus, but not for the δ-envelope control (figure 2 ). . the lv was used as a positive control and the δ-envelope (particles bearing no envelope protein), prepared with the same procedure, was used as a negative control. uncleaved s protein, about 180 kda; cleaved s protein, about 100 kda; dimeric-trimeric s protein, above 250 kda; nucleocapsid protein, about 50 kda. experiments were done twice and one is shown. regarding the pseudotypes, we observed three main bands: one just below 250 kda, and the remaining two bands at 180 kda and 100 kda, corresponding to the full-length and cleaved s protein, as shown in previous studies [33] . these two bands (180 kda and 100 kda) were barely detectable in the live virus, in which was observed one just above 250 kda, possibly reflecting the dimeric-trimeric s protein (detected in the pseudotypes below 250 kda), and the other band was around 50 kda, possibly corresponding to the nucleocapsid protein (np). the high glycosylation potential to which the spike is subjected during the infection differs from the spike expressed on the pseudotyped particles that do not undergo the same post-translational modifications [34] . this would explain the presence of a protein with a weight greater than 250 kda in the wild type virus, compared to the three isoforms with detectable molecular weight between 100 kda and below 250 kda related to the pseudotypes [33] . moreover, the quantitative data can slightly differ when a convalescence serum sample is used instead of an antibody (e.g., monoclonal antibody), specifically directed against a defined protein's portion. although, in this study, the evaluation of sars-cov-2 pseudotype titres is based on the reporter gene expression (rlus/ml), a number of eight different batches of sars-cov-2 lentiviral pseudotypes were also compared for the hiv-1 viral core protein p24 amount (directly correlating to the number of particles) by elisa (values reported as pg of p24 for ml). regarding the pseudotypes, we observed three main bands: one just below 250 kda, and the remaining two bands at 180 kda and 100 kda, corresponding to the full-length and cleaved s protein, as shown in previous studies [33] . these two bands (180 kda and 100 kda) were barely detectable in the live virus, in which was observed one just above 250 kda, possibly reflecting the dimeric-trimeric s protein (detected in the pseudotypes below 250 kda), and the other band was around 50 kda, possibly corresponding to the nucleocapsid protein (np). the high glycosylation potential to which the spike is subjected during the infection differs from the spike expressed on the pseudotyped particles that do not undergo the same post-translational modifications [34] . this would explain the presence of a protein with a weight greater than 250 kda in the wild type virus, compared to the three isoforms with detectable molecular weight between 100 kda and below 250 kda related to the pseudotypes [33] . moreover, the quantitative data can slightly differ when a convalescence serum sample is used instead of an antibody (e.g., monoclonal antibody), specifically directed against a defined protein's portion. although, in this study, the evaluation of sars-cov-2 pseudotype titres is based on the reporter gene expression (rlus/ml), a number of eight different batches of sars-cov-2 lentiviral pseudotypes were also compared for the hiv-1 viral core protein p24 amount (directly correlating to the number of particles) by elisa (values reported as pg of p24 for ml). the results showed that all batches tested consistently contained around 13-15 pg/ml of p24 gag capsid protein, corresponding approximately to a titre of 1.30e + 05 rlus/ml. similar vector infectivity was also identified for vsv-g pseudotyped vectors, around 15-16 pg/ml of p24 gag capsid protein corresponding to an approximate titre of 1.56e + 05 rlus/ml, while a value of 9-10 pg/ml, corresponding to a titre of 9.94e + 0 4, was obtained for ∆-envelope pseudotypes, as shown in figure 3 . the values obtained for the delta envelope are slightly higher for the p24 amount compared to the rlus. a possible explanation, as showed by geraerts [35] , is that p24 quantification by elisa will detect cores lacking envelope glycoproteins (non-functional) as well as cores belonging to transduction competent (functional) pseudotypes, and this technique usually overestimates the functional vector titre. in fact, it also been shown that omission of the envelope plasmid during the vector production resulted in p24 being comparable with those of a normal production although with a non-detectable functional titre. the results showed that all batches tested consistently contained around 13-15 pg/ml of p24 gag capsid protein, corresponding approximately to a titre of 1.30e + 05 rlus/ml. similar vector infectivity was also identified for vsv-g pseudotyped vectors, around 15-16 pg/ml of p24 gag capsid protein corresponding to an approximate titre of 1.56e + 05 rlus/ml, while a value of 9-10 pg/ml, corresponding to a titre of 9.94e + 0 4, was obtained for δ-envelope pseudotypes, as shown in figure 3 . the values obtained for the delta envelope are slightly higher for the p24 amount compared to the rlus. a possible explanation, as showed by geraerts [35] , is that p24 quantification by elisa will detect cores lacking envelope glycoproteins (non-functional) as well as cores belonging to transduction competent (functional) pseudotypes, and this technique usually overestimates the functional vector titre. in fact, it also been shown that omission of the envelope plasmid during the vector production resulted in p24 being comparable with those of a normal production although with a non-detectable functional titre. figure 3 . p24 quantification. vector particle concentration (pg p24 protein per ml) determined by p24 elisa and corresponding lentiviral vector infectivity (rlus/ml). values expressed as the means ± sd of independent measurements (n = 8). after the production of the sars-cov-2 pseudotypes, we asked which cell lines were susceptible to pseudotype-driven entry in order to have a panel of cell lines that can be used in downstream pseudotype-neutralisation assays. for this purpose, we used a panel of cell lines of human and animal origin. since sars-cov-2 live virus has been successfully isolated in vero (african green monkey kidney cell line), vero e6 and huh7 cell lines at high titres (as shown previously [24] ), these were chosen for the cell tropism study. the hep g2, mdck and caco cells were also included in this panel because a previous study evidenced ace2 receptor expression, except for hek 293t/17 [36] . however, hek 293/17 cells have also been included as control cell line due to their high transfectability, and they were firstly optimised using different ace2-expressing plasmid figure 3 . p24 quantification. vector particle concentration (pg p24 protein per ml) determined by p24 elisa and corresponding lentiviral vector infectivity (rlus/ml). values expressed as the means ± sd of independent measurements (n = 8). after the production of the sars-cov-2 pseudotypes, we asked which cell lines were susceptible to pseudotype-driven entry in order to have a panel of cell lines that can be used in downstream pseudotype-neutralisation assays. for this purpose, we used a panel of cell lines of human and animal origin. since sars-cov-2 live virus has been successfully isolated in vero (african green monkey kidney cell line), vero e6 and huh7 cell lines at high titres (as shown previously [24] ), these were chosen for the cell tropism study. the hep g2, mdck and caco cells were also included in this panel because a previous study evidenced ace2 receptor expression, except for hek 293t/17 [36] . however, hek 293/17 cells have also been included as control cell line due to their high transfectability, viruses 2020, 12, 1011 9 of 18 and they were firstly optimised using different ace2-expressing plasmid concentrations. based on these preliminary results, this panel of cell lines was tested against sars-cov-2, vsv-g and delta envelope pseudotypes. as also seen in previous studies [10, 20] , all the cell lines tested were highly susceptible to entry driven by vsv-g pseudotypes as demonstrated by titres of 10 7 -10 8 rlus/ml (figure 4) . all the cell lines tested were also susceptible to entry by sars-cov-2 pseudotypes, in particular, hek 293 ace2/tmprss2-transfected cells, as demonstrated by titres of 10 8 -10 9 rlus/ml. however, no statistical differences have been observed when sars-cov-2 pseudotypes have been tested against different cell lines. this comparable susceptibility can be due to the similar ace2 expression (except for mdck cell line as also showed by nie et al. [20] ). moreover, co-transfection with tmprss2 protease can potentially level the titres obtained with different cell lines except for hek 293/17. concentrations. based on these preliminary results, this panel of cell lines was tested against sars-cov-2, vsv-g and delta envelope pseudotypes. as also seen in previous studies [10, 20] , all the cell lines tested were highly susceptible to entry driven by vsv-g pseudotypes as demonstrated by titres of 10 7 -10 8 rlus/ml (figure 4) . all the cell lines tested were also susceptible to entry by sars-cov-2 pseudotypes, in particular, hek 293 ace2/tmprss2-transfected cells, as demonstrated by titres of 10 8 -10 9 rlus/ml. however, no statistical differences have been observed when sars-cov-2 pseudotypes have been tested against different cell lines. this comparable susceptibility can be due to the similar ace2 expression (except for mdck cell line as also showed by nie et al. [20] ). moreover, co-transfection with tmprss2 protease can potentially level the titres obtained with different cell lines except for hek 293/17. as expected, when cell lines were tested with δ-envelope control (particles without envelope proteins) the transduction activity dropped drastically (4-log), corresponding to 10 3 -10 4 rlus/ml (figure 4 ). as shown in table 1 , of 65 human serum samples tested, 24 proved positive for pnt, with titres ranging between 10-20 and > 1280, and 28 positives for mnt, with titres ranging between 10 and 1280 (table 2) . forty-one human samples were found negative for pnt and 37 negative for mnt. therefore, only titres obtained for 4 sera were found to be discordant. when mabs have been tested against pseudotypes and the live virus, no neutralisation activity was observed on mn, while an evident neutralisation profile was seen for pnt against human anti-igm [37] sars-cov-2 spike s1 (ic50 > 1280). as expected, when cell lines were tested with ∆-envelope control (particles without envelope proteins) the transduction activity dropped drastically (4-log), corresponding to 10 3 -10 4 rlus/ml ( figure 4 ). as shown in table 1 , of 65 human serum samples tested, 24 proved positive for pnt, with titres ranging between 10-20 and >1280, and 28 positives for mnt, with titres ranging between 10 and 1280 ( table 2) . forty-one human samples were found negative for pnt and 37 negative for mnt. therefore, only titres obtained for 4 sera were found to be discordant. table 2 . panel of human sera tested by live virus mn and pseudotype-based neutralisation assays. responses against sars-cov-2 are expressed as antibody titres for both assays (end-point dilution). titres (pnt) when mabs have been tested against pseudotypes and the live virus, no neutralisation activity was observed on mn, while an evident neutralisation profile was seen for pnt against human anti-igm [37] sars-cov-2 spike s1 (ic 50 > 1280). in addition, we defined the parameters of sensitivity, specificity and accuracy. from the error matrix (table 1) , we obtained the following results: accuracy = 93.8% (95% ci (85.0%-98.3%)), which was significantly higher (p < 0.0001) than the no-information rate (56.9%), sensitivity = 85.7% (95% ci (67.3%-96.0%)) and specificity = 100% (95% ci (90.5%-100%)). a simple linear regression was conducted to predict the log2 pnt based on the log2 mnt data ( figure 5 ). the linear regression was found to be significant, f (1, 63) = 344.6, p < 0.0001, with an r 2 = 0.84. the pnt increased 1.09 for each log2 of the mnt. viruses 2020, 12, x for peer review 11 of 18 47 5 <10 48 5 <10 49 5 <10 50 5 <10 51 5 <10 52 5 <10 53 5 <10 54 5 <10 55 5 <10 56 5 <10 57 5 <10 58 5 <10 59 5 <10 60 5 <10 61 5 <10 62 5 <10 63 5 <10 64 5 <10 65 5 <10 in addition, we defined the parameters of sensitivity, specificity and accuracy. from the error matrix (table 1) , we obtained the following results: accuracy = 93.8% (95% ci (85.0%-98.3%)), which was significantly higher (p < 0.0001) than the no-information rate (56.9%), sensitivity = 85.7% (95% ci (67.3%-96.0%)) and specificity = 100% (95% ci (90.5%-100%)). a simple linear regression was conducted to predict the log2 pnt based on the log2 mnt data ( figure 5 ). the linear regression was found to be significant, f (1, 63) = 344.6, p < 0.0001, with an r 2 = 0.84. the pnt increased 1.09 for each log2 of the mnt. to evaluate the agreement between pnt and mnt, we used the intra-class correlation coefficient (icc). the one-way random single score icc (table 3 ) calculated between the neutralisation titres pnt and mnt was 0.872 (95% ci (0.799-0.92)), p < 0.0001. this indicated excellent agreement [38] . to evaluate the agreement between pnt and mnt, we used the intra-class correlation coefficient (icc). the one-way random single score icc (table 3 ) calculated between the neutralisation titres pnt and mnt was 0.872 (95% ci (0.799-0.92)), p < 0.0001. this indicated excellent agreement [38] . table 3 . summary of the intra-class correlation analysis. the one-way random single score icc calculated between the neutralisation titres pnt and mnt was 0.872 (95% ci (0.799-0.92)), p < 0.0001. the bland-altman (ba) analysis ( figure 6 ) revealed the presence of a significant relationship between differences and means, which made applying a regression approach consistent [39] . calculated between the neutralisation titres pnt and mnt was 0.872 (95% ci (0.799-0.92)), p < 0.0001. the bland-altman (ba) analysis ( figure 6 ) revealed the presence of a significant relationship between differences and means, which made applying a regression approach consistent [39] . we found that most of the data-points were within the 95% limit of agreements (loas), while one outlier, corresponding to a means of titres equal to 6.56 (log2-units), was detected. these findings suggested that there was substantial agreement and interchangeability between pnt and mnt. in addition, the ba method enabled us to search for possible systematic difference (bias) between the pnt and mnt, and to identify the presence of outliers. the maximum acceptable differences (blue lines) embrace the loas (red lines), which makes the interpretation of the statistical results biologically plausible. we found a significant linear relationship between the differences and the means of the titres. moreover, except for one outlier, all the differences were both biologically and statistically acceptable. in other words, there was substantial agreement between pnt and mnt. we also concluded, on the basis of the kolmogorov-smirnov test, that there was not enough evidence to reject the null hypothesis of equal distributions of pnt and mnt, (ks test = 0.17, p = 0.31). the normalised kullback-leibler divergences (nkld) between the original pnt and mnt data was equal to −0.0098. the result of the bootstrap testing evidenced that the hypothesis of a zero nkld between pnt and mnt was consistent with the data, p = 0.44 (figure 7 ). the maximum acceptable differences (blue lines) embrace the loas (red lines), which makes the interpretation of the statistical results biologically plausible. we found a significant linear relationship between the differences and the means of the titres. moreover, except for one outlier, all the differences were both biologically and statistically acceptable. in other words, there was substantial agreement between pnt and mnt. viruses 2020, 12, x for peer review 13 of 18 since the observed nkld was lower (in absolute value) than the 5% lower-tail quantile, we concluded that the divergence was not significantly different from zero (p-value = 0.44). the recent emergence of the novel pathogenic sars-coronavirus-2 constitutes a global health emergency. as previously shown [16, 21] , infection with sars-cov-2 elicits antibodies that bind to the virus. although several studies are still ongoing regarding the virus and the complexity of the human immune responses, neutralising antibodies are known to be strongly correlated with protection [40] . currently, polyclonal antibodies from recovered sars-cov-2-infected patients have been used to treat the sars-cov-2 infection, but identification of sars-cov-2-specific neutralising mabs is still ongoing. once such antibodies are selected and produced, the subsequent steps will involve testing for neutralising and/or cross-neutralising activity [41] , which should simplify the analysis of functional humoral immune responses. the demand for serological testing for sars-cov-2 is high, as there is a need to better quantify the number of cases of covid-19, including asymptomatic carriers [42] and patients who have recovered. as we know, sars-cov-2 have a strong pathogenicity and working with the wild-type virus implies the need for level 3 bio-containment facility, according to the who guidelines. however, serological assays for the evaluation of neutralising activity against the sars-cov-2 currently require the use of isolated-live virus. indeed, high-throughput methods (such as elisa) that do not require the live virus are limited by the fact that they detect total antibody binding to sars-cov-2 or to some of its key constituent proteins [43, 44] . for this reason, we produced and characterised a lentiviral pseudotype system that expresses the s protein of sars-cov-2 with the same approach used by for other pathogenic coronaviruses including sars-cov and mers [29, 30] . this pseudotype system can be a useful tool because of its safety and versatility. its versatility lies in the fact that the virus can be pseudotyped with different envelope proteins [30, 45, 46] . moreover, this approach does not necessitate handling the live virus, and does not require high-level the nkld between the original mnt and pnt was −0.0098 (blue dotted line). the mnt and pnt data were re-sampled with replacement 100,000 times. the red dotted line shows the 5% lower-tail quantile (−0.0125) of the bootstrap distribution. since the observed nkld was lower (in absolute value) than the 5% lower-tail quantile, we concluded that the divergence was not significantly different from zero (p-value = 0.44). the recent emergence of the novel pathogenic sars-coronavirus-2 constitutes a global health emergency. as previously shown [16, 21] , infection with sars-cov-2 elicits antibodies that bind to the virus. although several studies are still ongoing regarding the virus and the complexity of the human immune responses, neutralising antibodies are known to be strongly correlated with protection [40] . currently, polyclonal antibodies from recovered sars-cov-2-infected patients have been used to treat the sars-cov-2 infection, but identification of sars-cov-2-specific neutralising mabs is still ongoing. once such antibodies are selected and produced, the subsequent steps will involve testing for neutralising and/or cross-neutralising activity [41] , which should simplify the analysis of functional humoral immune responses. the demand for serological testing for sars-cov-2 is high, as there is a need to better quantify the number of cases of covid-19, including asymptomatic carriers [42] and patients who have recovered. as we know, sars-cov-2 have a strong pathogenicity and working with the wild-type virus implies the need for level 3 bio-containment facility, according to the who guidelines. however, serological assays for the evaluation of neutralising activity against the sars-cov-2 currently require the use of isolated-live virus. indeed, high-throughput methods (such as elisa) that do not require the live virus are limited by the fact that they detect total antibody binding to sars-cov-2 or to some of its key constituent proteins [43, 44] . for this reason, we produced and characterised a lentiviral pseudotype system that expresses the s protein of sars-cov-2 with the same approach used by for other pathogenic coronaviruses including sars-cov and mers [29, 30] . this pseudotype system can be a useful tool because of its safety and versatility. its versatility lies in the fact that the virus can be pseudotyped with different envelope proteins [30, 45, 46] . moreover, this approach does not necessitate handling the live virus, and does not require high-level bio-containment facilities, as the pseudotype is devoid of virulent viral components and it is involved in a single round of replication [46] . the production of pseudotypes harbouring novel glycoproteins could permit elucidation of viral biological characteristics and a better understanding if they have potential to cause pandemics by the generation of mutants (via mutation of glycoprotein) without the risk of creating potentially dangerous viruses. it can also reflect key aspects of host cell entry and receptor binding specificity [10, 33] . however, the need of additional reagents seems a requirement due to cellular receptor specificity and tmprss2 priming for sars-cov-2 pseudotypes; thus, necessitating us to optimise the batch-to-batch variations in order to reduce the variability in terms of pseudotype titres and stability. since previous studies have evidenced that most amino acid residues for ace2 binding by sars-cov were also conserved in sars-cov-2 [47] , we have studied different cell susceptibility to sars-cov-2 pseudotype entry. understanding the entry mechanisms determined by the s glycoprotein and the susceptibility of different cells (based on specific cellular receptor expression) can also provide important information to study critical process in which the s spike protein of sars-cov-2 is involved. moreover, the use of multiple target cell lines is particularly valid since one of the advantages of the pseudotype-based assays is that they are deployable across different stakeholder laboratories who have access to different cell lines. it also represents a determinant for the development and optimisation of cell-based assays and for the screening of potential entry inhibitors. once the sars-cov-2 pseudotypes have been efficiently produced, we have also developed a lentiviral pseudotype-based assay that facilitates the accurate determination of neutralising antibody responses to sars-cov-2 that can be paired to the more intensive and laborious mn. although it is unclear as to how the display of s protein on heterologous virus impacts viral entry, antibody recognition and antibody neutralisation [48] , our results suggest that pseudotype-based neutralisation assays correlate well with the mn assays when testing human sera, and our findings show significant accuracy, sensitivity and specificity when both assays are compared. when employed in the screening of vaccinated human sera and other influenza serological studies [49] [50] [51] [52] , pseudotype-based neutralisation assays have been described to be more sensitive than classical mn. the sensitivity of pseudotypes can also detect particularly low antibody responses or facilitate the antibody's recognition that cannot always be determined by conventional assays, possibly due to lower density/quantity of glycoprotein expressed on the pseudotypes [53, 54] . this could explain the ability of human mab igm to recognise certain epitopes of s1 protein, as it has been seen for pseudotype-based neutralisation but not for live virus mn. although the model (pnt instead of mnt) returned a relatively high percentage of false-negatives, in large-scale serological testing, having a highly specific test is advantageous when a false-positive result has a great clinical impact. indeed, it guarantees against the risk of misclassification of the true-negative samples. undoubtedly, for a wider use of pseudotypes, it will be necessary to take into consideration additional aspects required for standardisation such as comparison between viral vectors/expression plasmids used, optimisation of particle titres and establishment of adequate positive, negative controls and reference standards (making it more difficult to evaluate the reproducibility of different serological assays). moreover, as in all the cell-based assays, cell input [55, 56] is an important aspect in standardisation: the use of an automatic cell counter, cell viability and requirement of different proteases should be taken into account and it could be important for the consistency of the assay during different analytical sessions. undoubtedly, a permanent cell line could be a valid alternative [18] (it would require only tmprss2 priming), and the generation of a cell line expressing proteases as a pseudotype producer could be investigated. our findings suggest that sars-cov-2 pseudotype-based assays and the live-virus microneutralisation correlate well when employed in testing antibody responses against the novel sars-cov-2 virus. undoubtedly, they would help to dissect out these diversities of immunological responses, and they can, additionally, have an important role in evaluating the neutralising 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coronaviruses sars-cov-2 infection among asymptomatic homebound subjects in serological assays for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) longitudinal evaluation and decline of antibody responses in sars-cov-2 infection the production and development of h7 influenza virus pseudotypes for the study of humoral responses against avian viruses current status on the development of pseudoviruses for enveloped viruses angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus neutralizing antibody and soluble ace2 inhibition of a replication-competent vsv-sars-cov-2 and a clinical isolate of sars-cov-2 pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to h5n1 influenza viruses multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies establishment of retroviral pseudotypes with influenza hemagglutinins from h1, h3, and h5 subtypes for sensitive and specific detection of neutralizing antibodies optimization and evaluation of an influenza a (h5) pseudotyped lentiviral particle-based serological assay investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison efficient generation of vesicular stomatitis virus (vsv)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies the united states pharmacopeial convention. design and development of biological assays; the united states pharmacopeial convention biological assay validation; the united states pharmacopeial convention this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we found that most of the data-points were within the 95% limit of agreements (loas), while one outlier, corresponding to a means of titres equal to 6.56 (log2-units), was detected. these findings suggested that there was substantial agreement and interchangeability between pnt and mnt.in addition, the ba method enabled us to search for possible systematic difference (bias) between the pnt and mnt, and to identify the presence of outliers.we also concluded, on the basis of the kolmogorov-smirnov test, that there was not enough evidence to reject the null hypothesis of equal distributions of pnt and mnt, (ks test = 0.17, p = 0.31). the normalised kullback-leibler divergences (nkld) between the original pnt and mnt data was equal to −0.0098. the result of the bootstrap testing evidenced that the hypothesis of a zero nkld between pnt and mnt was consistent with the data, p = 0.44 (figure 7) . developmental medicine, university of siena, italy, for providing us the human serum samples from the sero-epidemiological study on sars-cov-2. thanks to prof. valentina bollati, from the university of milan, for providing the sera sample from covid-19-positive healthcare workers. thanks to nigel temperton, and the pseudotypes unit, university of kent, for providing the panel of plasmids used for sars-cov-2 pseudotype production. we thank also: virginia cianchi and donata martinuzzi for their lab support in vismederi research. the authors declare no conflict of interest. key: cord-341453-9yrvjlpx authors: clay, candice c; donart, nathan; fomukong, ndingsa; knight, jennifer b; overheim, katie; tipper, jennifer; van westrienen, jesse; hahn, fletcher; harrod, kevin s title: severe acute respiratory syndrome-coronavirus infection in aged nonhuman primates is associated with modulated pulmonary and systemic immune responses date: 2014-03-19 journal: immun ageing doi: 10.1186/1742-4933-11-4 sha: doc_id: 341453 cord_uid: 9yrvjlpx background: many respiratory viruses disproportionately impact the elderly. likewise, advanced age correlated with more adverse disease outcomes following severe acute respiratory syndrome coronavirus (sars-cov) infection in humans. we used an aged african green monkey sars-cov infection model to better understand age-related mechanisms of increased susceptibility to viral respiratory infections. nonhuman primates are critical translational models for such research given their similarities to humans in immune-ageing as well as lung structure. results: significant ageand infection-dependent differences were observed in both systemic and mucosal immune compartments. peripheral lymphocytes, specifically cd8 t and b cells were significantly lower in aged monkeys preand postsars-cov infection, while neutrophil and monocyte numbers were not impacted by age or infection status. serum proinflammatory cytokines were similar in both age groups, whereas significantly lower levels of il-1beta, il-18, il-6, il-12 and il-15 were detected in the lungs of sars-cov-infected aged monkeys at either 5 or 10 days post infection. total lung leukocyte numbers and relative frequency of cd8 t cells, b cells, macrophages and dendritic cells were greatly reduced in the aged host during sars-cov infection, despite high levels of chemoattractants for many of these cells in the aged lung. dendritic cells and monocytes/macrophages showed age-dependent differences in activation and chemokine receptor profiles, while the cd8 t cell and b cell responses were significantly reduced in the aged host. in examination of viral titers, significantly higher levels of sars-cov were detected in the nasal swabs early, at day 1 post infection, in aged as compared to juvenile monkeys, but virus levels were only slightly higher in aged animals by day 3. although there was a trend of higher titers in respiratory tissues at day 5 post infection, this did not reach statistical significance and virus was cleared from all animals by day 10, regardless of age. conclusions: this study provides unique insight into how several parameters of the systemic and mucosal immune response to sars-cov infection are significantly modulated by age. these immune differences may contribute to deficient immune function and the observed trend of higher sars-cov replication in aged nonhuman primates. viral respiratory infections remain a predominant cause of morbidity and mortality in aged adults. the elderly have heightened susceptibility to infection, an increased risk of developing severe viral-induced pulmonary disease and have slower recovery rates [1] . several physiological parameters are thought to contribute to the poor outcomes of infectious disease in the elderly population, including the aging immune as well as respiratory system. almost all components of the immune system have been shown to undergo age-associated restructuring that greatly impacts immune function [2] [3] [4] [5] . the decline in immune function with age also results in reduced vaccine efficacy, further enhancing susceptibility to infection in the elderly [6] [7] [8] . age-associated alterations in the mucosal immune system are thought to occur at distinct times and in a distinct manner relative to systemic immunity [9] . data suggests that immunosenescence may occur earlier in the mucosa than the systemic immune system with a dramatic shift, with age, in the proportion of distinct t cell subsets and a decrease in total b lymphocytes [3, 10] . advanced age has also been associated with a reduction in antigen-specific iga, an important protective antibody predominantly localized to the mucosa [9] . in addition to the immunological remodeling as a function of age, there are also major alterations in respiratory physiology. the aging lung has been shown to undergo structural changes which include a loss in static recoil forces, a stiffening of the chest cavity and diminished alveolar surface area, ultimately resulting in reduced vital capacity [11] [12] [13] . in addition, respiratory muscle strength consistently declines with age making it more difficult for an elderly person to breath even when not suffering from a respiratory infection. the limitations of the aged immune and respiratory systems likely contributed to the increased mortality observed in elderly patients (>60 years old) with severe acute respiratory syndrome coronavirus (sars-cov). the sars-cov epidemic in 2002-2003 resulted in over 8000 human infections with an estimated 10% mortality rate [14] . advanced age and comorbidities were significantly associated with increased risk of sars-cov related death, due to acute respiratory distress syndrome [15] [16] [17] [18] . it is well appreciated that pulmonary damage in sars-cov infection is caused by direct viral effects as well as immunopathological factors [15] , however the pathogenic mechanisms in the vulnerable aged populations remain poorly defined. several aged animal models of sars-cov infection have been established to evaluate the response and elucidate mechanisms for increased sars-cov pathogenicity in the aged host. recombinant infectious clones and mouse passaged isolates of sars-cov show increased severity of disease and lethality in aged as compared to young mice [19] [20] [21] . interestingly, the aged and young hosts show similar levels of sars-cov replication in most experimental infections [22] . thus the increased acute lung injury in sars-covinfected aged animals is thought to be related to the over exuberant immune responses and not heightened viral-mediated damage. however, many aspects of the elderly immune response and how it may differ from the young adult are still unclear. furthermore, our study represents one of only two sars-cov infection studies in aged nonhuman primates as almost all aged sars-cov experiments to date have been conducted in mouse models with mouse-adapted viral strains. although murine models are often advantageous and informative, nonhuman primates may be better suited for studying the aging immune and respiratory systems. unlike mice, nonhuman primates show a high level of genetic homology to humans, are not inbred, have longer life spans and their lungs are more structurally similar to humans than other laboratory animals [23] . importantly, studies have shown that nonhuman primates undergo immune senescence similar to what has been described for humans [24, 25] . the aim of this study was to determine how the peripheral and mucosal immune responses to sars-cov infection compare in the aged and juvenile nonhuman primate host and to determine how this may impact viral replication levels. we report that sars-cov virus titers were significantly higher in the nasal cavity of aged monkeys at day 1 post infection but, by day 3, the difference in titers between age groups was negligible. although sars-cov virus levels were similar in aged and juvenile monkeys at later time points post infection there were significant age-dependent differences in systemic and mucosal immune responses to sars-cov. to evaluate the impact of advanced age on severity of sars-cov infection, aged and juvenile african green monkeys were inoculated intranasally with 10 7 plaqueforming units (pfu) sars-cov hku-39849 strain or mock-infected with sacrifice at 5 or 10 days post infection (d.p.i.). aged animals were roughly equivalent to 50 year old humans (n = 5 each time point post infection, n = 2 for mock-infected) [26, 27] while juvenile animals represented 6-12 year olds (n = 6 for all juvenile groups). some of the virology, pathology and immunology data for the juvenile animals have been previously reported [28] but are included here for age-related comparisons. in examination of clinical features, all animals showed a slight initial decrease in body weight with sars-cov inoculation that rebounded by 3 d.p.i. (figure 1a ; 2-way anova for age and d.p.i.). time post infection but not age was a significant source of variation for the fold change in body weight. body temperature however, fluctuated throughout the infection time course and was significantly affected by age but not infection ( figure 1b ; 2-way anova for age and d.p.i.). sars-cov titers were measured in nasal swabs and in several respiratory tissues by plaque assays. replicating virus was recovered from nasal swabs in 7 of the 10 aged animals at 1 d.p.i., in contrast to only one juvenile animal with detectable virus at this early time point ( figure 1c ; 2-way anova for age and d.p.i.). day 1 was the only time point in which sars-cov replication was significantly different; by 3 d.p.i., virus levels were only slightly higher in aged animals and titers remained similar in both age groups out to day 5. no significant age-dependent differences in sars-cov levels were observed in standardized collected respiratory tissues at 5 d.p.i. ( figure 1d ; 1-way anova for age). however, the levels of virus tended to be higher in all tissues from the aged as compared to the juvenile animals, except in the proximal portion of the right caudal lung lobe. no virus was detected in any sample collected from either age group at 10 d.p.i. to determine if advanced age correlated with increased severity of lung pathology, a comprehensive histological analysis of the respiratory tract following sars-cov infection was conducted in aged and juvenile animals. at 5 d.p.i. the sars-cov-induced histologic changes in the lung were higher in incidence in aged compared to juvenile monkeys (table 1) . these changes included perivascular cuffing with inflammatory cells, alveolitis and interstitial pneumonia. however, venous thrombosis was observed in 17-18% of the juvenile animals at both 5 and 10 d.p.i. whereas no venous thrombosis was detected in any aged animal at either time point. the severity of lung pathology was only slightly higher in aged as compared to juvenile animals at 5 d.p.i., and by 10 d.p.i., juvenile animals exhibited higher incidence and severity for most of the histologic changes. a marked increase in the incidence of interstitial pneumonia was observed in juvenile but not aged monkeys at 10 d.p.i. (83% in juvenile versus only 20% in aged). taken together, the histologic changes associated with sars-cov-induced interstitial pneumonia were slightly lower in incidence and severity in the aged compared to juvenile host. to determine if there were age-specific differences in immune activation following sars-cov infection, systemic inflammatory responses were compared in aged and juvenile animals. peripheral blood draws prior to and at days 1, 5 and 10 were used to monitor circulating immune cell and cytokine profiles. the total white blood cell counts for aged monkeys were lower than their juvenile counterparts at pre-and post sars-cov infection time points (figure 2a ; 2-way anova for age and d.p.i.). in examination of specific leukocyte populations, neutrophil and monocyte numbers significantly varied with sars-cov infection but were unaffected by age ( figure 2b -c; 2-way anova for age and d.p.i.). in contrast, age was a significant source of variation for lymphocyte numbers with reduced cd8 t cells and b cells in the peripheral blood of aged compared to juvenile animals ( figure 2d -f; 2-way anova for age and d.p.i.). the serum cytokine profile during sars-cov infection in aged and juvenile monkeys was defined using bead-based protein arrays focusing on inflammatory cytokines and chemokines associated with antiviral responses ( table 2 ). in comparing the aged and juvenile animals, the levels of il-12 and ifn-gamma were similar but had a trend of being higher in the aged monkeys whereas ccl2 was higher in juveniles, although not reaching statistical significance (2-way anova for age and d.p.i.). il-1 receptor agonist at baseline and ccl3 levels at day 10 were significantly elevated in the serum of aged animals over their juvenile counterparts (2-way anova for age and d.p.i. with bonferroni post-tests). as expected with an experiment using non-inbred animals the variation for serum cytokines was relatively high, particularly for the aged animals which is consistent with other previously published aged nonhuman primate studies [5, 25] . to determine how mucosal cytokines in sars-cov infection compared to systemic responses and how age may impact mucosal cytokine expression; the inflammatory protein profile was evaluated by bead-based arrays in standardized-collected lung tissue from the proximal portion of the right caudal lobe. the focus was on inflammatory cytokines thought to play a role in antiviral immunity. in contrast to the higher cytokine trend in the serum of aged animals, cytokines in the lung were often lower in aged as compared to the juvenile monkeys. proinflammatory cytokines il-1beta and il-6 were significantly lower in aged as compared to juvenile animals at 5 and 10 d.p.i. respectively ( figure 3a -b; unpaired student t-test). aged monkeys showed significantly lower levels of il-12 at day 5 and lower il-15 and il-18 at 10 d.p.i. as compared to juveniles (figures 3c-e; unpaired student t-test). there was also a trend of decreased il-21 levels in the lungs of aged versus juvenile monkeys although not reaching statistical significance (figures 3f). unlike the juvenile animals, interferongamma was below the level of detection in aged lung samples (data not shown). in general, cytokines tended to increase with sars-cov infection in juvenile severity is the average grade for the group (0 = normal, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked); incidence is the percentage of the group affected. monkeys whereas levels in the aged group showed no infection-induced increase. chemokines play a critical role in coordinating the migration of leukocytes into and out of the lung to trigger effective immune responses against viral pathogens [29, 30] . the chemokine milieu in the lungs following sars-cov infection was evaluated in aged and juvenile animals by targeted protein arrays. unlike data for proinflammatory cytokines in the lung, several of the chemokines showed increased expression with sars-cov infection and were detected at higher levels in the aged compared to the juvenile host. the t cell chemoattractant, cxcl11 was elevated in both age groups with sars-cov infection ( figure 4a ). however, cxcl11 levels in the aged lung did not reach the same magnitude as the juveniles and were significantly lower in comparison at 10 d.p.i. (unpaired student t-test). in contrast, ccl5, another activated t cell chemoattractant was significantly higher in aged compared to juvenile animals throughout the sars-cov infection period ( figure 4b ; unpaired student t-test). the b cell chemoattractant, cxcl13 was dramatically lower in aged as compared to juvenile animals, regardless of infection status whereas cxcl12 and ccl20, known dendritic cell (dc) chemoattractants, were much higher in mock and sars-cov infected aged animals ( figure 4d -f; unpaired student t-test). to determine if age impacted the kinetics and magnitude of mucosal inflammation in response to sars-cov, immune cell populations were quantified and characterized by flow cytometry in standardized collected lung tissue (proximal portion of the right caudal lobe) and lung draining lymph nodes. the number of total lung leukocytes per gram lung tissue was similar at 5 d. p.i. but was significantly lower in the aged as compared to juvenile animals at 10 d.p.i. (figure 5a ; unpaired student t-test). the frequency of distinct lung leukocyte populations, including macrophages (cd68 + hladr+), dendritic cells (dcs; cd68-hladr + cd11c+), cd8 t lymphocytes (cd3 + cd8+), and b cells (cd3-cd20+) differed in the two age groups during sars-cov infection ( figure 5b -e). gating strategies are shown in additional file 1: figures s1 and additional file 2: figure s2 and average values are summarized in additional file 3: table s1 . the frequency of lung macrophages was lower in aged as compared to juvenile animals in mock and sars-cov infection, reaching significance at 10 d.p.i. (figure 5b ; unpaired student t-test). the proportion of lung dcs was also significantly lower in aged animals at all infection time points examined ( figure 5c ; unpaired student t-test). although lung b cell frequencies were increased with infection in juvenile animals, no increase was observed in aged monkeys and, at most infection time points, significantly lower cd8 t cell and b cell frequencies were detected in aged monkeys ( figure 5d -e; unpaired student t-test). in contrast to the lung, no age-dependent differences in the leukocyte numbers or frequency of t and b cells were noted in the tracheobronchial lymph nodes ( figure 5f -h; unpaired student t-test). to further assess the age-specific differences in immunity to sars-cov infection, the activation status and chemokine receptor profile was assessed on monocytes/ macrophages, dcs and cd8 t cells. peripheral monocytes were defined by flow cytometry using cd14 and hladr expression and dcs as cd14-hladr + cd11c + (see peripheral blood monocyte and dc gating strategy in additional file 4: figure s3 ). expression of the activation marker cd86 showed significant age-dependent differences in the monocytes, lung macrophages and dc populations with significantly less expression in aged sars-cov-infected animals ( table 3 ; 2-way anova; additional file 2: figures s2 and additional file 4: figure s3 ). we also examined peripheral monocytes, dcs and cd8 t cells for ccl5 and ccl20 receptor expression as these chemokines were found at highest concentrations in the aged lung. for ccl5 receptors, aged monocytes showed reduced ccr3 but similar levels of ccr1 compared to their juvenile counterparts (table 3 , 2-way anova for age and d.p.i.). the frequency of cd8 t cells expressing ccr5 (another ccl5 receptor) was not significantly impacted by age. the receptor for ccl20, ccr6 was significantly down regulated with age and sars-cov infection on peripheral blood dcs (table 3 , 2-way anova for age and d.p.i.). cd8 t cell responses to sars-cov infection were evaluated in peripheral blood, lung and tracheobronchial lymph node by flow cytometric analysis (see gating strategy in additional file 1: figures s1 and additional file 2: figure s2 ). although no age-dependent differences were observed in the frequency of naã¯ve (cd45ra + ccr7+) cd8 t cells in peripheral blood, there were significantly lower levels of these cells in the lung and lymph node of aged animals during sars-cov infection ( figure 6a -c; unpaired student t-tests). a similar trend was observed with proliferating and cytotoxic granzyme b + cd8 t cells which were lower in the lungs and lymph nodes of aged compared to their juvenile counterparts at most infection time points (figure 6d -i; unpaired student ttest). however, granzyme b + t cell levels tended to be higher in the peripheral blood of aged compared to juvenile animals, reaching significance at 1 d.p.i. (figure 6g ; unpaired student t-test). to determine how the b cell response to sars-cov was impacted by age, anti-sars-cov antibodies were evaluated in the serum and lung. serum neutralization assays revealed increased antibodies in all juvenile animals with sars-cov infection as early as 5 d.p.i. (figure 7a ; 2-way anova for age and d.p.i.). in contrast, sars-cov neutralizing antibodies were not detected until 10 d.p.i. in aged monkeys, with antibody titers that were significantly lower as compared to juveniles. anti-sars-cov igg and iga antibodies in lung tissue homogenates were determined by elisa. lung anti-sars-cov igg was detected in 1 aged and 2 juvenile animals at 5 d.p.i. and although titers figure s2 and percent frequencies summarized in additional file 3: table s1. *p < 0.05, **p < 0.01 in unpaired student t-test comparing age groups. +p < 0.05 for comparing infection time points. increased in the juvenile lungs by day 10, no increase in anti-sars cov igg titers was observed in aged animals ( figure 7b , no significance by unpaired student t test). anti-sars-cov iga titers were significantly lower in aged as compared to juvenile animals at 10 d.p.i., with average titers that were up to 18-fold lower in the aged host ( figure 7c , unpaired student t test). the elderly (those over 65), are considered the fastestgrowing demographic in the united states and are expected to make up 19% of the population by 2030 [31] . it is well recognized that elderly individuals incur enhanced severity of respiratory infections and according to the center for disease control and prevention, an estimated 9 out of 10 flu-related deaths in the united states occur in people 65 and older. when sars-cov emerged in the human population in 2002, the elderly were also disproportionately affected, with individuals over 65 making up 50% of the total fatal cases [32, 33] . we have a poor understanding of the relationship between aging and the host response to respiratory virus infection. most of our understanding of the biological changes that occur with aging in humans has been limited to studies of peripheral blood, which may not reflect the immune dynamics of the respiratory tract. our study aimed at gaining insight into the kinetics and magnitude of the systemic and mucosal immune response to sars-cov infection in aged nonhuman primates, an important translational model for immune-aging research. the significantly higher levels of viral replication in the nasal secretions of aged monkeys as compared to the juvenile animals at 1 d.p.i. was unexpected as most aged sars-cov studies have detected no age-dependent differences in infection levels. higher sars-cov titers may have been missed in the previous aged nonhuman primate experiment as 2 d.p.i. was their earliest sampling time point, [22] and by day 3 in our study, aged and juvenile infection levels were similar. although we cannot be certain that we were measuring replicating virus and not virus delivered from the inoculum, the dramatic difference in the amount recovered suggests that the aged nasal epithelium may support higher levels of sars-cov, or that the nasal cavity drainage mechanisms are impaired in the aged host. despite the early ageassociated differences in viral shedding at mucosal sites, by 5 d.p.i., levels of sars-cov in respiratory tract tissues were only slightly higher in the aged monkeys. similar to previous reports, virus was not recovered from any animal at 10 d.p.i., suggesting that the kinetics of viral clearance may have been similar in both age groups. however, additional sampling between days 5 and 10 post infection would be necessary to confirm this observation. average frequency of chemokine receptor or activation marker positive cells of total perhipheral blood or lung leukocytes. monocytes (cd14 + hladr+), peripheral blood dendritic cells (dcs: cd14-hladr + cd11c+), cd8 t cells (cd3 + cd8+). lung macrophages are defined as cd68 + hladr + and lung dcs as cd68-hladr + cd11c+. gating strategies are show in additional file 1: figure s1 , additional file 2: figure s2 and additional file 4: figure s3 . statistical results of 2-way anovas for age and days post infection (d.p.i.) are indicated at the right with asterisks denoting degree of sigrificance: *p < 0.05, **p < 0.005 ****p < 0.0001. the aged monkeys in our study exhibited significantly decreased total white blood cell counts which is consistent with the inverse correlation of wbcs with age that has been observed at steady state in several rhesus and human studies [25, 34] . of the leukocyte populations in the blood, lymphocyte numbers were most dramatically affected by age, with flow cytometric results showing significantly reduced cd8 t cells and b cells in aged compared to juvenile monkeys. in contrast, peripheral cytokine responses showed only minimal age-related differences to sars-cov infection which may be related to the high variability, particularly in the aged group, which is consistent with previous reports in elderly rhesus monkeys [5, 25] . in regards to the mucosal inflammatory reactions, proinflammatory cytokines il-1beta, il-6, il-15, il-12 and il-18 were all significantly lower in the lungs of sars-cov-infected aged animals at either day 5 or 10 post infection. in particular, the reduced levels of il-1beta and il-18 are in line with recent reports of impaired nlrp3 inflammasome function in elderly mice during influenza infection [35] . the low cytokine response in aged monkeys may also be a reflection of the reduced total inflammatory cell numbers found in the lungs of aged as compared to juvenile animals as cells and cytokines were evaluated in adjacent lung regions. our data is not consistent with the exacerbated acute inflammatory responses shown to promote disease pathogenesis in aged sars-cov-infected mouse models [19, 21] . as we did not sample earlier than day 5 post infection in the lung, the increased inflammation in our aged monkeys may have been missed. our sampling schedule may have also precluded collections at optimal inflammatory response time points, as a biphasic pattern occurring in waves, at day 2 and 7 post infection with mouse-adapted sars-cov strains has been described in aged rodents [20, 36] . figure s2 . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 in unpaired student t-tests comparing age groups. in addition to the differences between our findings and those from aged mice, most of our results are also dissimilar to the previously reported aged nonhuman primate sars-cov study by smits et al., [22] as we did not observe stronger innate immune responses or more severe pathology in our aged monkeys. in comparing our results, there are several experimental design aspects to take into consideration, beyond differences in dose and route of sars-cov infection. first, cynomolgous macaques were used in the previous study whereas our sars-cov infection model utilizes african green monkeys. in addition, timing of lung sample collection differed between the two studies, days 2 and 4 were assessed in the cynomolgous macaques while days 5, and 10 post infection were evaluated in our study. furthermore, a standardized lung collection scheme was used in our immunology and pathology analysis, to give a more comprehensive perspective whereas their study assessed gene changes in specific regions of high sars-cov replication. despite the major differences in study design, there were several results that were comparable, including the similarity of sars-cov viral titers in the aged and juvenile lung at later infection time points (days 4 and 5). in addition, several chemokines, including ccl5, cxcl12 and ccl20 were significantly increased in the lungs of sars-cov-infected aged monkeys here, which is consistent with the finding of elevated chemokine signaling genes in the aged lungs of cynomolgous macaques during sars-cov infection by microarray [22] . immune senescence has been shown to impact innate as well as adaptive branches of the immune system, both contributing to the diminished immunity observed in the elderly [24, 37] . in examination of the kinetics of lung and lymph node cell expansion and/or trafficking following sars-cov infection, we found significantly reduced lung macrophages, dcs, cd8 t cells and b cells in the aged animals relative to their juvenile counterparts. not only were there fewer lung macrophages and dcs in the aged animals, but the frequency of costimulatory cd86+ cells were also significantly reduced. in contrast, the lymph node reaction showed no age-specific differences. interestingly, although lung leukocyte numbers were reduced in aged monkeys there were disproportionately high levels of the chemokines capable of recruiting these cells into the lung, ccl5, cxcl11, cxcl12 and ccl20. this prompted our examination of the expression of the corresponding ligandbinding receptors on peripheral blood leukocytes to determine if there were age-related differences in the migratory capacity of these cells into the lung. for ccl5 receptors, we found significantly reduced frequencies of ccr3 but not ccr1+ monocytes in aged animals and similar frequencies of ccr5+ cd8 t cells in both age groups. the frequency of aged dcs expressing the ccl20 receptor, ccr6 showed the most dramatic age-specific reduction which is reasonable, given that ccr6 is predominantly expressed by immature dcs and may reflect a reduction of these cells in our aged animals. the observed differences in lung chemokine and ligand receptor expression may reflect age-specific modulation of leukocyte trafficking into the lung. although we did not confirm these observations with mechanistic in vitro experiments, previous studies have shown that dcs acquired from older individuals display significantly impaired ability to migrate in response to chemokines [38] . we also examined age-specific differences in both t and b cell responses to sars-cov infection systemically and at mucosal sites. not surprisingly, the proportion of naã¯ve cd8 t cells in the lung and lymph node was significantly reduced in aged compared to juvenile animals in sars-cov infection. this corresponded to significantly lower levels of cd8 t cell proliferation at these sites in aged animals as well. interestingly, the frequency of cytotoxic enzyme positive (granzyme b) t cells was higher at 1 d.p.i. in the periphery of aged compared to juvenile animals. however, aged peripheral granzyme + t cell frequencies remained unchanged by sars-cov infection whereas juvenile cytotoxic t cells showed infectioninduced fluctuation. in contrast to peripheral blood, the lung and lymph node of aged animals had lower frequencies of granzyme + t cells compared to juvenile animals at 5 and 10 d.p.i.. the humoral response was also greatly reduced in aged sars-cov-infected monkeys with significantly reduced serum neutralizing antibodies and mucosal anti-sars-cov iga in the aged host. this limited antibody response may be related to the dramatically reduced lung and peripheral b lymphocyte populations observed in the aged monkeys which is consistent with reports in elderly humans [39] . despite the deficiencies observed in the aged adaptive responses, viral titers were only significantly higher in aged monkeys at day 1 post infection. this suggests that innate or other compensatory immune responses are sufficient for viral control in aged monkeys and serves to further support the minimal involvement of cytotoxic t cells and neutralizing antibodies in sars-cov clearance as demonstrated in mouse depletion models [36] . taken together, our data indicate that systemic and mucosal immunity to sars-cov infection differs in the aged as compared to the juvenile host. this knowledge will be important to consider in the design of effective intervention strategies for sars-cov and potentially other respiratory infections. our experimental results support future mechanistic studies to identify adjunctive strategies capable of overcoming the immune deficits of the aged airway mucosa, which may ultimately translate into novel approaches to enhance efficacy of vaccines against respiratory pathogens for the elderly population. in this study we found that viral titers in aged, as compared to juvenile monkeys were significantly higher early after infection, but levels became comparable in both age groups at later infection time points. we observed significant age and infection-dependent differences in both the systemic and mucosal immune compartments with more dramatic changes in cytokine levels and leukocyte frequencies in lung as compared to peripheral blood and tracheobronchial lymph nodes. in regards to the exacerbated sars-cov responses previously reported in aged nonhuman primates, we did not observe enhanced host immunity or pathology in our aged monkeys. instead, we detected less inflammatory cytokines and total lung leukocytes, as well as reduced adaptive responses with increased age. although our results are discrepant, both studies indicate that pulmonary immunity is intrinsically different in the aged as compared to the juvenile host, warranting further exploration given the important implications this has for vaccine and therapeutic design for the elderly. all procedures were conducted under protocols approved by the institutional animal care and use committee (iacuc) at lovelace respiratory research institute (lrri), all facilities were accredited by the association for assessment and accreditation of laboratory animal care international (aaalac), and guidelines for nonhuman primates described in the guide for the care and use of laboratory animals, national research council, were strictly adhered to. the animals used in this study were adult african green monkeys obtained from the barbados primate research centre, barbados, west indies through global research supply, llc (reno, nv), a class b usda licensed animal dealer. a permit to import the animals into new mexico was acquired through the fish and wildlife service by lrri. the aged african green monkeys were determined to be~10-20 yrs old while the remaining subjects were young adults (~2-6 years old). additionally, two female african green monkeys of 17 yrs and 18 yrs were acquired from the wake forest university primate center (medical center boulevard, winston-salem). as there is an estimated 3.2fold difference for relating age between humans and rhesus macaques [26, 27] , the aged african green monkeys in our study are expected to represent approximately, 50 year old humans while the juvenile african green monkeys correspond to 6-12 year olds. the rhesus-to-human age conversion was used given that the estimated age conversion is not as well established for african green monkeys and there is 95% genetic similarity [40] between the two old world monkey species belonging to the super family cercopithecoidea. randomization and group assignments for the aged monkey studies were performed with provantis integrated preclinical software (instem life science systems ltd.). all monkeys were quarantined for 6 weeks prior to the study, during which they were tested for tuberculosis. animals were given a subcutaneous microchip for identification and temperature measurements (iptt-300 implantable temperature transponder and a wrs-6007 handheld wireless reader system (bio medic data systems, inc, seaford, delaware)). during the infectious portion of the study, animals were individually housed indoors in stainless steel cages with wire mesh bottoms. temperature and humidity ranges were controlled along with 12 h light and dark cycles. animals were also given environmental enrichment including toys twice per day. tap water from the institutional watering system was available ad libitum and animals were fed twice a day harlan teklad certified 20% monkey diet (4 to 6 biscuits/kg body weight) with bananas or apples for enrichment 3 times per week. the sars-cov strain hku-39849 was provided by dr. leo poon (department of microbiology, university of hong kong, hong kong, china) and viral stocks were generated in vero e6 cells. experimental infection with 10 7 plaque-forming units (pfu) of sars-cov instilled intranasally was conducted as previously reported [28] . monkeys were examined by trained laboratory animal technicians twice per day (at least 6 h apart) on each day of the study for parameters including appetite, appearance, activity, stool, posture, neurological signs, respiration, ocular discharge and nasal discharge. onset of any abnormal clinical sign was documented and a score sheet completed each day beginning from the date the animal fell ill. euthanasia to alleviate suffering was conducted based on the clinical observation score sheet and consultation with the staff veterinarian. animals were sacrificed at 5 and 10 d.p.i. (n = 6 for juvenile and n = 5 for aged animals at each time point) by intravenous overdose of euthasolâ® (virbac ah, inc., fort worth, tx). virus-free cell culture medium was used to inoculate mock-infected controls (n = 6 for juveniles and n = 2 for aged). some of the immunology, virology and pathology data for the juvenile animals were previously reported [28] but are included in this manuscript for comparison with aged monkeys. virus titers were measured in plaque assays by applying serial dilutions of homogenized tissue supernatants onto vero e6 cell monolayers as previously described [28] . to measure sars-cov neutralizing antibodies in serum, samples were serially diluted and incubated with 2,000 pfu/ml sars-cov overnight before inoculation onto vero e6 cells. titers are expressed as the reciprocal of the highest dilution at which the cytopathic effect was completely inhibited. standardized collected lung tissue from the proximal portion of the right caudal lobe was processed into single-cell suspensions for flow cytometry. of note, the samples for flow cytometric analysis and cytokine protein evaluation were acquired from adjacent tissue regions. single cell suspensions were prepared as detailed in [28] . briefly, mechanical and enzymatic digestion with liberase (roche, pleasanton, ca) and dnase (sigma, st. louis, mo) solution was performed prior to overlaying samples on a percoll (sigma) gradient for 20 min at 500 ã� g with no brake. tracheobronchial lymph nodes were collected in a standardized manner in which the same region and size of tissue was collected from each animal. single-cell suspensions were made by mechanical disruption. peripheral blood mononuclear cells (pbmc) were isolated from heparinized blood with a ficol-hypaque (sigma) gradient as previously reported [41] . all flow cytometric analysis was conducted on previously frozen pbmc, lung or lymph node leukocytes. the following antibodies were used in 4-or 6-color staining panels: cd3 (clone sdp34-2); cd8 (clone sk1); cd11c (clone shcl-3); cd14 (clone m5e2); cd20 (clone l27); cd68 (clone kp1, santa cruz biotechnologies, dallas, tx); cd86 (clone 2331fun1); granzyme b (clone gb11, invitrogen, carlsbad ca); hla-dr (clone l243); ki67 (clone b56); ccr1 (clone 53504); ccr3 (clone 61828); ccr5 (clone 3a9); ccr6 (clone 11a9) and ccr7 (clone 150503, r&d systems, minneapolis, mn) all conjugated to fluorochromes fitc, pe, percp, percpcy5.5, apc, pecy7, alexa647 or apccy7 (bd biosciences, san jose, ca unless specified). for intracellular antigens, bd facs lyse and permeabilization solutions were used according to the manufacturer's instructions. cd4 was not assessed given the downregulation of this marker on african green monkey t lymphocytes [42] [43] [44] . antibody-stained samples were fixed for 16 hr in 1% paraformaldehyde and 3% fbs in phosphate buffered saline. sample data were acquired on a facs-calibur or facs-canto flow cytometer instrument (bd biosciences) and data files analyzed utilizing flowjo software 7.6.1 (treestar, medford, or). for all flow cytometric analysis, within one experiment, the gates applied to each sample were identical (see gating strategies in additional file 1: figure s1 , additional file 2: figure s2 and additional file 4: figure s3 ). when appropriate and possible, similar gates were used across the two different experiments (juvenile and aged samples). however, based on changes to flow cytometer settings and fluorescence minus one controls, some gates were adjusted between the two experiments. markers were chosen for characterization of specific leukocyte subsets in pbmc, lymph node, and lung based on previously published studies in humans or nonhuman primates [45] [46] [47] [48] [49] [50] [51] [52] [53] . lung tissue was acquired in a standardized collection scheme in which a section of the proximal right caudal lobe was divided into two portions, one for homogenization to assess cytokines and antibodies and virus while lung leukocytes were isolated from the other portion for flow cytometry (see previous section). for homogenization, a volume of rpmi media (gibco, life techologies, grand island, ny) equal to 10% of the lung tissue weight was added. sars-cov igg and iga antibodies were measured by elisa detailed in [28] . briefly, elisa plates were coated with purified recombinant sars-cov s protein in carbonatecoating buffer (s protein nr-686 obtained through nih biodefense and emerging infections research resources repository) and nonspecific binding blocked with power-block (biogenex, san ramon ca). starting at a 1:25 dilution, serially diluted lung tissue homogenate supernatants (clarified by centrifugation) were incubated on s-protein coated plates overnight at 37â°c. an anti-monkey igg or iga hrp-conjugated antibody (kpl inc., gaithersburg, md) was applied followed by substrate development with abts microwell peroxidase substrate system (kpl) and absorbance reading at 405 nm using a thermo electron corporation plate reader (thermo electron corporation, houston, tx). data was acquired with ascent software (ascent software, london, uk) and the elisa antibody titer recorded for each sample was the reciprocal of the highest dilution in which the optical density (o.d.) reading for s-protein bound wells was at least two-fold higher than that of the nonfat milk control. the o.d. of the highest titer chosen also had to fall within the linear range of the serial dilutions. cytokine and chemokine protein was measured in lung tissue homogenates and serum using both human and nonhuman primate multiplex bead-based array kits (millipore, billerica, ma). the assays were performed according to the manufacturer's instructions with an overnight incubation of the samples in antibodyimmobilized beads. data was collected on the bio-plex system (biorad, hercules, ca) and a weighted 5parameter logistic curve-fitting method used to calculate the concentration of individual analytes. all measurements were performed in duplicate and data reported as pg/ml. cd40l was undetectable in serum of either age group and il-5, tnf-alpha, il-17, il-2, ccl24 and ccl26 were below the level of detection in lung tissue homogenates. sampling of tissues for histopathology was performed as previously reported [28] in a standardized manner with random assessment. all organs were fixed in 4% paraformaldehyde solution prior to embedding in paraffin. 5 î¼m thick tissue sections were stained with hematoxylin and eosin for examination microscopically by a board certified veterinary pathologist. histologic lesions were graded for severity (0 = normal, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked) and distribution, (focal, multifocal, diffuse). infection and age differences were evaluated using a 2way anova with bonferroni post-tests or two-tailed student t-tests, where appropriate. all data for statistical evaluation was log transformed and analyzed with graphpad prism 5.0 software (graphpad, la jolla, ca). a p value of 0.05 or less was considered statistically significant. additional file 1: figure s1 . gating strategy for analysis of extracellular markers on b and t cells. the representative gating strategy for flow cytometric analysis of lung, lymph node and pbmc is illustrated based on pbmc from an aged monkey. first a lymphocyte gate based on forward and side scatter is applied with the percent frequency of total cells indicated in the plot (fsc/ssc). cd8 t cells were subsequently gated based on cd3 and cd8 expression (cd3 + cd8+). cd8 t cells were further evaluated for coexpression of naã¯ve cell markers ccr7 and cd45ra (ccr7 + cd45ra+) or ccr5 expression. b lymphocytes were gated based on cd20 expression and absence of cd3 (cd3-cd20+). the percent frequencies of total gated lymphocytes based on the fsc/ssc gate are indicated in each plot. additional file 2: figure s2 . gating strategy for analysis of intracellular markers. the gating strategy for flow cytometric analysis of macrophages, dendritic cells and cd8 t cells in which intracellular markers were used is shown using a representative lung sample from an aged animal. leukocytes were first gated on forward and side scatter with the percent frequency of total cells indicated in the plot (fsc/ssc). cells were subsequently gated on cd68 and hladr for macrophages and dendritic cells (dcs) (cd68/hladr). macrophages were defined as cd68 + hladr + and were evaluated for expression of cd86. cd68 negative hladr + cells were further gated on cd11c positive to define dcs (cd68-hladr + cd11c+) which were also evaluated for cd86 expression. cd8 t cells were defined as cd3 + cd8+ and were assessed for expression of intracellular antigens, ki67 and granzyme b. the percent frequencies of total gated leukocytes based on fsc/ssc are indicated in each plot. additional file 3: table s1 . relative frequency of leukocyte populations in the lung and lymph node during sars-cov infection. additional file 4: figure s3 . gating strategy for peripheral monocytes and dendritic cells. the gating strategy for monocytes and dcs in the peripheral blood is depicted using a representative aged pbmc sample. cells are first gated based on forward and side scatter with the percent frequency of total cells indicated in the plot. the subsequent gate is for cd14 and hladr expression in which monocytes are defined as cd14 + hladr+. monocytes are subsequently evaluated for cd86, ccr1 and hladr + cells are further gated on cd11c expression to define dcs (cd14-hladr + cd11c+). cd86 and ccr6 expression was then evaluated on dcs. the percent frequencies of total gated leukocytes based on fsc/ssc are indicated in each plot severe acute respiratory corona virus; d.p.i.: days post infection pfu: plaque-forming units; dc: dendritic cell inflamm-aging, cytokines and aging: state of the art, new hypotheses on the role of mitochondria and new perspectives from systems biology immunosenescence comes of age. symposium on aging research 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analysis of cd4 and cd8 t cell subsets in young and old people increase of ccr1 and ccr5 expression and enhanced functional response to mip-1 alpha during differentiation of human monocytes to macrophages differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis the cd14+ cd16+ blood monocytes: their role in infection and inflammation phenotype and function of myeloid dendritic cells derived from african green monkey blood monocytes submit your next manuscript to biomed central and take full advantage of: â�¢ convenient online submission â�¢ thorough peer review â�¢ no space constraints or color figure charges â�¢ immediate publication on acceptance â�¢ inclusion in pubmed, cas, scopus and google scholar â�¢ research which is freely available for redistribution we thank the absl3 animal care and pathology for assistance with these studies. we thank dr. fred cassels, niaid for helpful comments and suggestions regarding the experimental design. the authors declare no financial or non-financial competing interests in the work presented in this study.authors' contributions ccc data collection, analysis and interpretation of data, figure preparation, manuscript writing; nd data collection, analysis, manuscript editing; nf conception and design, analysis and interpretation of data; jbk data collection, analysis, manuscript editing; ko conception and design, study director, manuscript editing; jt data collection, data analysis, manuscript editing; jvw data collection, analysis, manuscript editing; fh board-certified pathologist, histopathology analysis, manuscript editing, ksh conception and design, interpretation of data, final approval of the manuscript. all authors read and approve the final manuscript. key: cord-353704-lfndq85x authors: ye, zi-wei; yuan, shuofeng; yuen, kit-san; fung, sin-yee; chan, chi-ping; jin, dong-yan title: zoonotic origins of human coronaviruses date: 2020-03-15 journal: int j biol sci doi: 10.7150/ijbs.45472 sha: doc_id: 353704 cord_uid: lfndq85x mutation and adaptation have driven the co-evolution of coronaviruses (covs) and their hosts, including human beings, for thousands of years. before 2003, two human covs (hcovs) were known to cause mild illness, such as common cold. the outbreaks of severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) have flipped the coin to reveal how devastating and life-threatening an hcov infection could be. the emergence of sars-cov-2 in central china at the end of 2019 has thrusted covs into the spotlight again and surprised us with its high transmissibility but reduced pathogenicity compared to its sister sars-cov. hcov infection is a zoonosis and understanding the zoonotic origins of hcovs would serve us well. most hcovs originated from bats where they are non-pathogenic. the intermediate reservoir hosts of some hcovs are also known. identifying the animal hosts has direct implications in the prevention of human diseases. investigating cov-host interactions in animals might also derive important insight on cov pathogenesis in humans. in this review, we present an overview of the existing knowledge about the seven hcovs, with a focus on the history of their discovery as well as their zoonotic origins and interspecies transmission. importantly, we compare and contrast the different hcovs from a perspective of virus evolution and genome recombination. the current cov disease 2019 (covid-19) epidemic is discussed in this context. in addition, the requirements for successful host switches and the implications of virus evolution on disease severity are also highlighted. coronaviruses (covs) belong to the family coronaviridae, which comprises a group of enveloped, positive-sensed, single-stranded rna viruses [1, 2] . these viruses harbouring the largest genome of 26 to 32 kilobases amongst rna viruses were termed "covs" because of their crown-like morphology under electron microscope [2, 3] . structurally, covs have non-segmented genomes that share a similar organization. approximately two thirds of the genome contain two large overlapping open reading frames (orf1a and orf1b), which are translated into the pp1a and pp1ab replicase polyproteins. the polyproteins are further processed to generate 16 non-structural proteins, designated nsp1~16. the remaining portion of the genome contains orfs for the structural proteins, including spike (s), envelope (e), membrane (m) and nucleoprotein (n). a number of lineage-specific accessory proteins are also encoded by different lineages of covs [2, 4] . based on the difference in protein sequences, covs are classified into four genera (alpha-cov, beta-cov, gamma-cov and delta-cov), among which the beta-cov genera contains most hcovs and is subdivided into four lineages (a, b, c and d) [2, 4, 5] . phylogenetic evidence has shown that bats and rodents serve as the gene source of most alpha-covs and beta-covs, while birds are the main reservoir of gamma-covs and delta-covs [2] . for thousands of years, covs have constantly crossed species barriers and some have emerged as important human pathogens [2, 4, [6] [7] [8] . to date, seven human covs (hcovs) are known. among them hcov-229e and hcov-nl63 are alpha-covs. the other five beta-covs include hcov-oc43, hcov-hku1, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus ivyspring international publisher (mers-cov) and sars-cov-2 [2, 5, 9] . hcov-229e, hcov-oc43, hcov-hku1 and hcov-nl63 usually cause mild symptoms, like common cold and/or diarrhea [10, 11] . in contrast, sars-cov, mers-cov and the newly-identified sars-cov-2 are highly pathogenic, causing severe lower respiratory tract infection in relatively more patients with a higher chance to develop acute respiratory distress syndrome (ards) and extrapulmonary manifestations. the first hcov-229e strain, b814, was isolated from the nasal discharge of patients with common cold in mid-1960s [12] . since then, more knowledge was accumulated through extensive studies on hcov-229e and hcov-oc43, both of which cause self-limiting symptoms [13] . indeed, the concept had been widely accepted that infection with hcovs is generally harmless until the outbreak of sars. the sars outbreak occurred in 2003 is one of the most devastating in current history, infecting over 8,000 people with a crude case fatality of approximately 10% [14, 15] . ten years later, the middle east respiratory syndrome (mers) outbreak resulted in a persistent epidemic in the arabian peninsula with sporadic spreading to the rest of the world [16] [17] [18] . the 2019 novel hcov (2019-ncov), which has subsequently been renamed sars-cov-2, is the causative agent of the ongoing epidemic of coronavirus disease 2019 (covid19) , which has claimed more than 3,120 lives and infected more than 91,000 people as of march 3, 2020 [19] . the alarm has been ringing and the world has to prepare for the coming pandemic of sars-cov-2. all seven hcovs have a zoonotic origin from bats, mice or domestic animals [2, 20] . multiple lines of evidence support an evolutionary origin of all hcovs from bats, where viruses are well adapted and non-pathogenic but show great genetic diversity. the covid-19 epidemic has presented enormous medical, scientific, social and moral challenges to china and the world. tracing the zoonotic origins of hcovs provides a framework to understand the natural history, driving force and restriction factors of species jumping. this might also guide or facilitate the search for the reservoir, intermediate and amplifying animal host(s) of sars-cov-2, with important implications in the prevention of future spillovers. in this review we present an overview of the zoonotic origins, interspecies transmission and pathogenesis of hcovs. particularly, we highlight and discuss the common theme that parental viruses of hcovs are typically non-pathogenic in their natural reservoir hosts but become pathogenic after interspecies transmission to a new host. we also review the trend of hcov evolution in which the increase in transmissibility often comes with the decrease in pathogenicity. the outcome of the ongoing sars-cov-2 outbreak is also discussed in this context. animal covs have been known since late 1930s. before the first isolation of hcov-229e strain b814 from the nasal discharge of patients who had contracted common cold, different covs had been isolated in various infected animals, including turkey, mouse, cow, pig, cat and dog [21] [22] [23] [24] [25] [26] . in the past decades, seven hcovs have been identified. a brief summary of the history of hcov discovery in chronological order (table 1) would be informative and instructive. the first hcov-229e strain was isolated from the respiratory tract of patients with upper respiratory tract infection in the year of 1966 [27] , and was subsequently adapted to grow in wi-38 lung cell lines [28] . patients infected with hcov-229e presented with common cold symptoms, including headache, sneezing, malaise and sore-throat, with fever and cough seen in 10~20% cases [29] . later in 1967, hcov-oc43 was isolated from organ culture and subsequent serial passage in brains of suckling mice [28] . the clinical features of hcov-oc43 infection appear to be similar to those caused by hcov-229e, which are symptomatically indistinguishable from infection with other respiratory tract pathogens such as influenza a viruses and rhinoviruses [28] . both hcov-229e and hcov-oc43 are distributed globally, and they tend to be predominantly transmitted during the season of winter in temperate climate [2] . generally, the incubation time of these two viruses is less than one week, followed by an approximately 2-week illness [28] . according to a human volunteer study, healthy individuals infected with hcov-229e developed mild common cold [30] . only a few immunocompromised patients exhibited severe lower respiratory tract infection. sars, also known as "atypical pneumonia", was the first well documented hcov-caused pandemic in human history and the etiological agent is sars-cov, the third hcov discovered [14, 15] . the first case of sars can be traced back to late 2002 in guangdong province of china. the sars epidemic resulted in 8,096 reported cases with 774 deaths, spreading across many countries and continents. apart from the super-spreaders, it was estimated that each case could give rise to approximately two secondary cases, with an incubation period of 4 to 7 days and the peak of viral load appearing on the 10 th day of illness [14, 15] . patients infected with sars-cov initially present with myalgia, headache, fever, malaise and chills, followed by dyspnea, cough and respiratory distress as late symptoms [14, 15] . lymphopenia, deranged liver function tests, and elevated creatine kinase are common laboratory abnormalities of sars [14, 15] . diffuse alveolar damage, epithelial cell proliferation and an increase of macrophages are also observed in sars patients [31] . approximately 20-30% of patients subsequently require intensive care and mechanical ventilation. in addition to lower respiratory tract, multiple organs including gastrointestinal tract, liver and kidney can also be infected in these severe cases, usually accompanied with a cytokine storm, which might be lethal particularly in immunocompromised patients. the virus was first isolated from the open lung biopsy of a relative of the index patient who travelled to hong kong from guangzhou [14, 15] . since then, tremendous efforts have been dedicated to hcov research. hcov-nl63 was isolated from a 7-month-old child from the netherlands during late 2004. it was initially found to be prevalent in young children, the elderly and immunocompromised patients with respiratory illnesses [32] . presentation of coryza, conjunctivitis, fever, and bronchiolitis is common in the disease caused by hcov-nl63 [33] . another independent study described the isolation of the same virus from a nasal specimen from an 8-month-old boy suffering from pneumonia in the netherlands [34] . although it was identified in netherlands, it is actually distributed globally [2] . it has been estimated that hcov-nl63 accounts for approximately 4.7% of common respiratory diseases, and its peak incidence occurs during early summer, spring and winter [2] . hcov-nl63 is associated with obstructive laryngitis, also known as croup [35] . in the same year, hcov-hku1 was isolated from a 71-year-old man who had been hospitalized with pneumonia and bronchiolitis in hong kong [36] . besides community-acquired pneumonia and bronchiolitis, hcov-hku1 was reported to be associated with acute asthmatic exacerbation [37] . similar to hcov-nl63, hcov-229e and hcov-oc43, hcov-hku1 was found worldwide, causing mild respiratory diseases [37] . all these four communityacquired hcovs have been well adapted to humans and are generally less likely to mutate to cause highly pathogenic diseases, though accidents did occur for unknown reasons as in the rare case of a more virulent subtype of hcov-nl63, which has recently been reported to cause severe lower respiratory tract infection in china [38] . generally, when these hcovs acquire the abilities to transmit efficiently and to maintain themselves continuously within humans, they also become less virulent or pathogenic. mers-cov was first isolated in 2012 from the lung of a 60-year-old patient who developed acute pneumonia and renal failure in saudi arabia [17, 18, 39] . whereas most of the laboratory-confirmed cases originate from the middle east, imported cases with occasional secondary spreads to close contacts have been reported in various european countries and tunisia. another secondary outbreak occurred in south korea in 2015 with 186 confirmed cases. clinical manifestations of mers resemble those of sars, characterized by progressive acute pneumonia [17, 18, 39] . unlike sars, many patients with mers also developed acute renal failure, which is thus far unique for mers among hcov-caused diseases [17, 18, 39] . more than 30% of patients present with gastrointestinal symptoms, such as diarrhea and vomiting [17, 18, 39] . as of february 14, 2020, over 2500 laboratory confirmed cases were reported with a high case fatality of 34.4%, making mers-cov one of the most devastating viruses known to humans. during middle to late december 2019, clusters of pneumonia patients retrospectively known to be associated with sars-cov-2 infection were detected in wuhan, hubei province, china [40] . world health organization declared the ongoing outbreak of lower respiratory tract infection caused by sars-cov-2 a public health emergency of international concern and also named the disease covid-19. as of march 3, 2020, 90,053 cases have been confirmed worldwide, with a crude case fatality of 3.4%. notably, the case fatality in hubei, china is 4.2%, whereas the one outside of it is 1.2%. sars-cov-2 causes severe respiratory infection like sars-cov and mers-cov, presented as fever, cough and dyspnea [40] . diarrhea is also seen in some patients [40] . pneumonia is one of the most severe symptoms and can progress rapidly to acute respiratory distress syndrome. although sars-cov and sars-cov-2 are very similar due to high nucleotide sequence homology of 82%, they cluster into different branches in the phylogenetic tree [5] . sars-cov-2 is apparently less pathogenic but more transmissible compared to sars-cov and mers-cov. asymptomatic subjects infected with sars-cov-2 have been reported and might contribute to its rapid spreading around the world. comparing and contrasting sars-cov-2 with the other six hcovs reveal similarities and differences of great interest. first, the incubation period and the duration of the course of hcov disease are very similar. in this regard, sars-cov-2 follows the general trend of the other six hcovs. second, the severity of symptoms of covid-19 lies between sars-cov and the four community-acquired hcovs (i.e. hcov-229e, hcov-oc43, hcov-hku1 and hcov-nl63). on one hand, sars-cov-2 infection exhibits features that are more commonly seen during infection with community-acquired hcovs, including the presentation of non-specific, mild or even no symptoms. on the other hand, a small subset of severe cases of covid-19 can also be seen as in the case of sars-cov infection, although the ratio is a bit lower. third, the transmission of sars-cov-2 also shows interesting patterns characteristic of both community-acquired hcovs and sars-cov. on one hand, the transmissibility of sars-cov-2 is at least as high as that of community-acquired hcovs. on the other hand, it remains to be verified whether the transmissibility of sars-cov-2 decreases after passages in humans as in the cases of sars-cov and mers-cov. finally, same as the other hcovs [41] , sars-cov-2 can be detected in fecal samples. whether fecal-oral transmission of sars-cov-2 plays an important role as in the case of sars-cov at least under some circumstance remains to be clarified by future studies. it is also of particularly great interest to see whether sars-cov-2 might exhibit seasonality as in the cases of community-acquired hcovs. nevertheless, the features of sars-cov-2 including its transmissibility, pathogenicity and sustainable spreading after passages in humans will be influential on the ultimate fate of the ongoing outbreak of covid-19. all four community-acquired hcovs causing mild symptoms have been well adapted to humans. from another perspective, it might also be true that humans have been well adapted to these four hcovs. in other words, both could be the survivors of ancient hcov pandemics. hcovs that cause severe diseases in humans and humans who developed severe hcov diseases have been eliminated. for this to happen, hcovs have to replicate in humans to sufficient extent to allow the accumulation of adaptive mutations that counteract host restriction factors. in this sense, the longer the sars-cov-2 outbreak persists and the more people that it infects, the greater chance that it will fully adapt to humans. if it adapts well, its transmission in humans would be difficult to stop by quarantine or other infection control measures. for many years, the four community-acquired covs circulate in human populations, triggering common cold in immunocompetent subjects. these viruses do not need an animal reservoir. in contrast, highly pathogenic sars-cov and mers-cov have not adapted to humans well and their transmission within humans cannot be sustained. they need to maintain and propagate in their zoonotic reservoirs and seek the chance to spillover to susceptible human targets, possibly via one or more intermediate and amplifying hosts. sars-cov-2 has features that are similar to both sars-cov/mers-cov and the four community-acquired hcovs. it is highly transmissible like community-acquired hcovs, at least for the time being. however, it is more pathogenic than community-acquired hcovs and less pathogenic than sars-cov or mers-cov. it remains to be seen whether it will adapt fully to humans and circulate within humans without a reservoir or intermediate animal host. before discussing the animal origins of hcovs, it will serve us well to discuss the definitions and characteristics of evolutionary, natural, reservoir, intermediate and amplifying hosts of hcovs. an animal serves as the evolutionary host of an hcov if it harbours a closely related ancestor sharing high homology at the level of nucleotide sequence. the ancestral virus is usually well adapted and nonpathogenic in this host. likewise, a reservoir host harbours hcov continuously and for long term. epidemiological data revealed retrospectively that the index case of sars had a contact history with game animals [42] . subsequent seroprevalence investigations indicated that animal traders had a higher prevalence of anti-sars-cov igg compared with that of the general population [42] . masked palm civets (paguma larvata) and a racoon dog in live animal markets were first identified to carry sars-cov-like viruses that are almost identical to sars-cov [43] . this was indirectly supported by the fact that no further sars was reported after killing all civets in the markets. however, it has been reported that masked palm civets from the wild or farms without exposure to the live animal markets were largely negative for sars-cov [44] , suggesting that masked palm civets might only serve as the intermediate amplifying host but not the natural reservoir of sars-cov. notably, since 80% of the different animals in the markets in guangzhou have anti-sars-cov antibodies [45] , the possibilities that multiple species of small mammals might also serve as intermediate amplifying hosts of sars-cov cannot be excluded. all of these appear to be dead-end hosts of sars-cov. subsequent search for the natural animal host of sars-cov unveiled a closely related bat cov, termed sars-related rhinolophus bat cov hku3 (sarsr-rh-batcov hku3), which exists in chinese horseshoe bats [46] . these bats are positive for anti-sars-cov antibodies and genome sequence of sarsr-rh-batcov hku3 [37, 47] . this and other bat covs share 88-92% nucleotide sequence homology with sars-cov. these studies have laid the foundation for the new concept that bats host emerging human pathogens. several sars-like covs (sl-covs) have also been identified from bats, but none except for one designated wiv1 can be isolated as live virus [7] . human angiotensin converting enzyme 2 (ace2) is known to be the receptor of sars-cov. wiv1 derived from fecal sample of bats was demonstrated to use bat, civet and human ace2 as receptor for cell entry [48] . intriguingly, sera of convalescent sars patients were capable of neutralizing wiv1 [48] . thus far, wiv1 represents the most closely related ancestor of sars-cov in bats [7, 48] , sharing 95% nucleotide sequence homology. albeit the high homology between these two viruses, it is generally believed that wiv1 is not the immediate parental virus of sars-cov and bats are not the immediate reservoir host of sars-cov. phylogenetic analysis clusters mers-cov to the same group as bat cov-hku4 and bat cov-hku5. bat cov-hku4 and mers-cov utilize the same host receptor, dipeptidyl peptidase 4 (dpp4), for virus entry [49] . rna-dependent rna polymerase sequences of mers-cov are phylogenetically closer to counterparts in bat beta-covs identified from europe and africa [50, 51] . up to now, no live mers-cov can be found in wild bats. mers-cov and its closest relative bat cov-hku25 share only 87% nucleotide sequence homology [52, 53] . thus, bats might not be the immediate reservoir host of mers-cov. on the other hand, studies in middle east have shown that dromedary camels are seropositive for mers-cov-specific neutralizing antibodies [54] , same as camels of middle east origin in multiple african countries [55] . live mers-cov identical to the virus found in humans was isolated from the nasal swabs of dromedary camels, further indicating that camels serve as the bona fide reservoir host of mers-cov [56] . it is also noteworthy that generally mild symptoms but massive virus shedding were observed in camels experimentally infected with mers-cov [57] . notably, infected camels shed viruses not only through respiratory route but also through fecal-oral route, which is also the main route for virus shedding from bats. however, questions still remain since many confirmed cases of mers have no contact history with camels prior to symptom onset [58] , plausibly ascribed to human-to-human transmission or unknown transmission routes involving unrecognized animal species that harbour mers-cov. these merit further investigations. sars-cov-2 shares 96.2% nucleotide homology with a bat cov ratg13 isolated from rhinolophus affinis bats [8] . as in the cases of sars-cov and mers-cov, the sequence divergence between sars-cov-2 and ratg13 is too great to assign parental relationship. that is to say, bats might not be the immediate reservoir host(s) of sars-cov-2 unless almost identical bat covs are found in future. presumably, the intermediate animal hosts of sars-cov-2 should be among the wildlife species sold and killed at the huanan seafood wholesale market, with which many of the initial cases of covid-19 were associated, indicative of a probable animal-to-human transmission event [40] . several recent studies based on metagenomic sequencing have suggested that a group of endangered small mammals known as pangolins (manis javanica) could also harbour ancestral beta-covs related to sars-cov-2 [59] . these novel pangolin cov genomes share 85-92% nucleotide sequence homology with sars-cov-2. however, they are equally closely related to ratg13 with about 90% identity at the level of nucleotide sequence. they cluster into two sub-lineages of sars-cov-2-like viruses in the phylogenetic tree, one of which share a more similar receptor binding domain (rbd) with sars-cov-2, with 97.4% amino acid sequence identity [59] . in stark contrast, the rbds of sars-cov-2 and ratg13 are more divergent, albeit a higher degree of sequence homology genome-wide. an earlier study on diseased pangolins also reported the detection of viral contigs from lung samples, which turn out to be similarly related to sars-cov-2 [60] . this study adopted different assembly methods and manual curation to generate a partial genome sequence comprising about 86.3% of the full-length viral genome [60] . we cannot exclude the possibility that pangolin is one of the intermediate animal hosts of sars-cov-2 [59] . however, currently there is no evidence in support of a direct pangolin origin of sars-cov-2 due to the sequence divergence between sars-cov-2 and pangolin sars-cov-2-related beta-covs. in addition, the distance between sars-cov-2 and ratg13 is even shorter than that between sars-cov-2 and pangolin sars-cov-2-related beta-covs. the evolutionary pathway of sars-cov-2 in bats, pangolins and other mammals remains to be established. whereas the highest sequence homology has been found in the rbds between sars-cov-2 and pangolin, sars-cov-2-related beta-covs, sars-cov-2 and ratg13 share the highest genome-wide sequence homology. it is highly speculative that the high degree of similarity between the rbds of pangolin sars-cov-2-related beta-covs and sars-cov-2 is driven by selectivitymediated convergent evolution. a counter-proposal is in favour of a recombination between a pangolin sars-cov-2-related beta-cov and ratg13 in the third wild animal species. as a driving force in evolution, recombination is widespread among beta-covs [61] . the jury is still out on the immediate zoonotic origin of sars-cov-2. besides the highly pathogenic hcovs, the zoonotic origin of hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1 have also been studied [9, [62] [63] [64] . phylogenetic evidence indicated that both hcov-nl63 and hcov-229e might have originated from bat covs [62] [63] [64] , while the parental viruses of hcov-oc43 and hcov-hku1 have been found in rodents [9] . it has been reported that a bat cov termed arcov.2 (appalachian ridge cov) detected in north american tricolored bat displayed close relationship with hcov-nl63 [62, 63] . on the other hand, hcov-229e was genetically related to another bat cov, termed hipposideros/ghanakwam/19/2008, which was detected in ghana [65] , while camelids have also been suspected as its intermediate host [66, 67] . for clarity, the current knowledge on animal origins of known hcovs is summarized in figure 1 and table 2 . phylogenetic analysis has provided evidence for interspecies transmission events of hcovs in the history. when hcov-oc43 crossed species to infect humans from domestic livestock around 1890, a pandemic of respiratory infection was recorded [68] . the interspecies transmission history of hcov-229e is less clear. bat alpha-covs closely related to hcov-229e have been found. between them there is an alpaca alpha-cov. several lines of evidence support the transmission of virus from bats to humans directly. first, humans but not alpacas might have contact with bats in a shared ecological niche. instead, humans have close contact with alpacas. second, hcov-229e-related bat alpha-covs are diverse and non-pathogenic in bats, whereas alpaca alpha-cov caused an outbreak of respiratory disease in infected animals [65] . finally, alpaca alpha-cov has not been found in feral animals. thus, the possibility cannot be excluded that alpacas obtain the hcov-229e-related alpha-cov from humans. in fact, bats are the direct source of human pathogenic viruses including rabies virus, ebola virus, nipah virus and hendra virus [69] . it is therefore not too surprising that bats might transmit hcov-229e to humans directly. alternatively, whereas bat alpha-covs serve as the gene pool of hcov-229e, alpacas and dromedary camels might serve as intermediate hosts that transmit viruses to humans, exactly as in the case of mers-cov [69] . mers-cov serves as an excellent example of interspecies transmission from bats to dromedary camels and from dromedary camels to humans. the evolutionary origin of mers-cov from bats is known at its initial identification and has also been strengthened by subsequent findings [49] [50] [51] . it is obvious that bats provide a rich pool of virus species for interspecies exchange of genetic fragments and interspecies transmission. longevity, densely packed colonies, close social interaction and strong ability to fly are all favourable conditions for bats to be an ideal 'virus spreader'. on the other hand, mers-cov has been introduced to dromedary camels for decades. it is well adapted to these camels that have turned from an intermediate host to a stable and natural reservoir host. mers-cov causes very mild disease and maintains a relatively low mutation rate in these animals. its sporadic transmission to humans is an accident and humans remain a dead-end host of mers-cov as its transmission cannot be sustained. in contrast to the role of camels in the transmission of mers-cov, the role of pangolins, if there is any, in the transmission of sars-cov-2 is different. particularly, pangolin beta-covs are highly pathogenic in pangolins. they might be a dead-end host for sars-cov-2-related beta-covs, similar to civets in the case of sars-cov. several possibilities for interspecies transmission of sars-cov-2 from animals to humans have to be ruled in or ruled out in future studies. first, bats could be the reservoir host of a sars-cov-2-related virus almost identical to sars-cov-2. humans might share the ecological niche with bats through butchering or coal mining. second, pangolins could be one of intermediate amplifying host to which a sars-cov-2-related virus had been newly introduced. humans contract the virus through butchering and consumption of game meat. it is possible that many mammals including domestic animals are susceptible to sars-cov-2. a survey of domestic and wild animals for antibodies is warranted. third, as mentioned above, recombination and adaptation of sars-cov-2 might have occurred in a third species that has contact with both bats and pangolins. the search for the animal origins of sars-cov-2 is still on. apart from different types of the animal hosts, three major factors on the viral side are also important in facilitating covs to cross species barriers [70] . first of all, their relatively high mutation rates in rna replication. in comparison to other single-stranded rna viruses, the estimated mutation rates of covs could be regarded as "moderate" to "high" with an average substitution rate being ~10 -4 substitution per year per site [2] , depending on the phase of cov adaptation to novel hosts. covs have a proof-reading exoribonuclease, deletion of which results in exceedingly high mutability and attenuation or even inviability. interestingly, the nucleotide analogue remdesivir is known to suppress cov replication through inhibition of this exoribonuclease and the rna-dependent rna polymerase [70] . remdesivir is one of most promising anti-sars-cov-2 agents to be tested in clinical trials. nevertheless, mutation rates of covs are about a million times higher than those of their hosts. in addition, mutation rate is often high when covs are not well adapted to the host [71] . compared to sars-cov with a high mutation rate [72] , the mutation rate of sars-cov-2 is apparently lower, suggestive of a higher level of adaptation to humans. presumably, it has already been adapted to another host close to humans. in addition to sars-cov-2, this also applies to mers-cov, which is well adapted to dromedary camels. theoretically, it is unlikely that genetic drift would render vaccines and antivirals against sars-cov-2 ineffective quickly. second, the large rna genome in covs exerts extra plasticity in genome modification for mutations and recombination, thereby increasing the probability for interspecies co-evolution, which is advantageous for the emergence of novel covs when the conditions become appropriate. this is supported by the copious unique open reading frames and protein functions encoded towards the 3â�² end of the genome. third, covs randomly and frequently switch templates during rna replication through a unique "copychoice" mechanism. in a host that serves as the mixing vessel, strand switching occurs frequently during cov rna transcription. highly homologous fulllength and subgenomic rnas could recombine to generate new covs. phylogenetic evidence of natural recombination has been found in both hcov-hku1 and hcov-oc43, as well as animal covs such as bat sl-cov and batcov-hku9 [73] . besides three viral factors stated above, viral interaction with host receptor is another key factor influential on interspecies transmission. herein, recombination of sars-cov is taken as a typical example, which also showed evidence of positive selection during interspecies transmission events [74] . based on the comparative analysis between isolates of human and civet sars-covs, sars-cov is thought to undergo rapid adaptation in different hosts, particularly with mutations at the rbd of the s protein [74] . generally, the rbd in the s protein of a cov interacts with the cellular receptor and is intensely selected by the host antibody response. in sars-cov, the rbd is in the 318 th to 510 th amino acids on the s1 fragment, which binds to the human ace2 as well as its coreceptors for viral entry [75] . the rbd of sars-cov is capable of recognizing the ace2 receptors of various animals, including bat, civet, mouse and raccoon dog, allowing interspecies transmission of the virus. in fact, only 6 amino acid residues were observed to be different from human and civet viral isolates in the rbd and 4 of them locate in the receptor-binding motif for interaction with the ace2 receptor. civet sars-cov has k479n and s487t mutations in its rbd, which might increase the affinity of the interaction of spike protein with human ace2 receptor. in other words, these two amino acid substitutions might be critical to viral adaption to humans [75] . it is noteworthy that sars-cov-2 shares the same cellular receptor with sars-cov [8] . a 30% difference between sars-cov-2 and sars-cov in the s1 unit of the s protein implicates that the binding affinity of its s protein with human ace2 might have altered [5] . indeed, a cryo-em study indicates a 10-to 20-fold higher affinity of this binding than that between human ace2 and sars-cov s protein [76] . it will also be of interest to determine whether any other coreceptor might be required for sars-cov-2 transmission. intriguingly, hcov-nl63 also binds to ace2 but with a different part of s [77] . there exist many other hcov receptors, such as aminopeptidase n for hcov-229e, and 9-o-acetylated sialic acid for hcov-oc43. they might also account for successful adaptation of these covs in humans after interspecies transmission from their animal hosts. in addition to cellular receptors, the outcome of interspecies transmission of hcovs is also governed by other host dependency and restriction factors. the divergence of these host proteins between humans and natural reservoir hosts of hcovs such as bats, dromedary camels and rodents might constitute a barrier to interspecies transmission. hcovs have to usurp host dependency factors and subvert host restriction factors for a successful interspecies transmission. in this regard, molecular determinants in this important area of virus-host interaction remain to be identified and characterized. an unbiased genome-wide screening of host dependency and restriction factors for sars-cov-2 using the state-ofthe-art technology of crispr might be fruitful. the diversity of bat covs provides ample opportunities for the emergence of novel hcovs. in this sense, bat covs serve as the gene pool of hcovs. in addition, rapid mutation and genetic recombination also drive hcov evolution and serve as two important steps in this process [2, 6] . for example, the acquisition or loss of novel proteincoding genes has the potential to drastically modify viral phenotypes. among sars-cov accessory proteins, orf8 has been thought to be important in adaptation to humans, as sars-cov-related bat viruses were isolated but found to encode divergent orf8 proteins [48] . a 29-nucleotide deletion characteristic of sars-covs has been found in strains isolated at the beginning of the human epidemic. this deletion splits orf8 into orf8a and orf8b and is thought to be an adaptive mutation that promotes the switch of hosts [78] . besides, sars-cov has a possible recombination history with lineages of alpha-and gamma-covs, where a large number of smaller recombinant regions were identified in the rnadependent rna polymerase [72] . recombination locations were also identified in the nsp9, most of nsp10, and parts of nsp14 [72] . likewise, it has been shown that the epidemic mers-cov experienced recombination events between different lineages, which occurred in dromedary camels in saudi arabia [79] . besides sars-cov and mers-cov, recombination events have also been observed in other hcovs, in which the hcovs recombine with other animal covs in their non-structural genes. it should also be cautioned that artificial selection can contribute to unintended changes in viral genomes, most likely resulting from relieving viruses from selection pressures exerted, such as by the host immune system. an example of these effects is the loss of a full-length orf4 in the hcov-229e prototype strain, owing to a two-nucleotide deletion [80] . while intact orf4 could be observed in bat and camel viruses related to hcov-229e, the alpaca alpha-cov displays a single nucleotide insertion, resulting in a frameshift [81] . last but not least, the evolution of novel hcovs is also driven by the selection pressure in their reservoir hosts. asymptomatic or only mild symptoms were detected when bats were infected with covs, indicating the mutual adaptation between covs and bats [82] . it appeared that bats are well adapted to covs anatomically and physiologically. for example, defects in the activation of proinflammatory response in bats efficiently reduce the pathology triggered by covs [83] . besides, the natural killer cell activity in bats is suppressed due to upregulation of inhibitory natural killer cell receptor nkg2/cd94 and low expression level of major histocompatibility complex class i molecules [84] . moreover, the high level of reactive oxygen species (ros) generated from high metabolic activity of bats could both suppress cov replication and affects proofreading by exoribonuclease [85] , thus providing the selection pressure for the generation of virus strains highly pathogenic when introduced into a new host. more pathogenic cov strains might also evolve by recombination, leading to the acquisition of novel proteins or protein features for host adaptation [82] . thus, it is not by chance that three novel hcovs have emerged in the past two decades. covs are non-pathogenic or cause mild symptoms in their reservoir hosts such as bats and camels. they replicate robustly without eliciting a strong host immune response. herein lie the secrets of why asymptomatic carriers are seen and what causes the severe cases in human infection. the severe symptoms are mainly due to the hyperactivation of immune response and the cytokine storm wherein the stronger the immune response, the more severe the lung damage. in contrast, in asymptomatic carriers, the immune response has been de-coupled from cov replication. the same strategy of delinking the immune response might have beneficial effects in anti-sars-cov-2 therapy. the interferon response is particularly strong in bats. thus, administration of type i interferon at least in the early phase of sars-cov-2 infection in humans should be beneficial. in addition, nlrp3 inflammasome activation in bats is defective [86] . by this reasoning, inhibition of nlrp3 inflammasome with mcc950 might be useful in the treatment of covid-19. the emergence of sars-cov-2 follows the general theme by which sars-cov and mers-cov arose. whereas a bat beta-cov sharing 95% nucleotide homology with sars-cov has been found, there also exists a bat-cov sharing 96% nucleotide homology with sars-cov-2. whereas civets and other animals in the markets have been found to harbour viruses identical to sars-cov, immediate intermediate hosts for sars-cov-2 have not been identified. pangolin beta-covs strikingly homologous to sars-cov-2 have been found, indicating that pangolins might serve as one of intermediate hosts or pangolin beta-covs could contribute gene fragments to the final version of sars-cov-2. although questions remain, there is no evidence that sars-cov-2 is man-made either deliberately or accidentally. covs have returned to the limelight due to the recent outbreak of sars-cov-2. the study of covs in bats and other animals has drastically changed our perception of the importance of zoonotic origins and animal reservoirs of hcovs in human transmission. pervasive evidence has shown that sars-cov, mers-cov and sars-cov-2 have a bat origin and are transmitted to humans via intermediate hosts. given that sars-cov infection originates from the contact between humans and civets in the markets, closing wet markets and killing civets therein could have effectively ended the sars epidemic. by the same reasoning, pangolins should be removed from wet markets to prevent zoonotic transmission, in view of the discovery of multiple lineages of pangolin beta-covs closely related to sars-cov-2. however, whether and how sars-cov-2 is transmitted to humans through pangolins and other mammals remain to be clarified in future investigations. on the other hand, mers-cov has existed in dromedary camels for a long time. these camels serve as an important tool for transportation as well as a main source of meat, milk, leather and wool products for the local people. they are widely distributed across the middle east and africa. it is therefore impossible to sacrifice all camels for the control of mers, as what was done in wild animal markets in china to prevent the spreading of sars-cov and sars-cov-2. to stop the recurrent outbreaks of mers, a comprehensive approach should be taken to develop effective vaccines against mers-cov for camels, in combination with other infection control measures. as we are not able to eliminate these viruses, new genotypes might emerge to cause outbreaks. a variety of zoonotic covs are circulating in the wild. particularly, bat covs with zoonotic potential are so diverse. there are plenty of opportunities that these zoonotic covs evolve and recombine, resulting in the emergence of new covs that are more transmissible and/or deadly in humans in future. the culture of eating wild animals in some places of china should be abandoned to reduce unnecessary contact between humans and animals. with the ordeals of sars, mers and covid-19, a better preparedness and response plan should be in place. in fact, many viruses have existed in the planet for a very long time. they stay in their own natural reservoirs until there is a chance for spillover. although bats have many features that favours the spreading of viruses, the chance for humans to be in contact with bats and other wildlife species can be minimized if people are educated to stay away from them. continuous surveillance in mammals is necessary for better understanding of the ecology of covs and their natural hosts, which will prove useful in preventing animal-to-human transmission and future outbreaks. to conclude, the most effective way to prevent viral zoonosis is for humans to stay away from the ecological niches of the natural reservoirs of the zoonotic viruses. several pieces in the puzzle of the zoonotic origin of sars-cov-2 are still missing. first, if bats transmit an ancestral virus of sars-cov-2 to pangolins, it will be of interest to see under what circumstances bats and pangolins could share the same ecological niche. second, if bats play a more direct role in human transmission, how humans get into contact with bats should be determined. third, if a third mammal acts as the true intermediate host, how it interacts with the different species including humans, bats and pangolins has to be clarified. finally, since many mammals including domestic animals might be susceptible to sars-cov-2, both surveillance and experimental infection should be conducted. should it be a bat, a pangolin or another mammal, it is expected that sars-cov-2 or its parental viruses that are almost identical will be identified in its natural 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species and recombination of mers-covs in saudi arabia coronavirus diversity, phylogeny and interspecies jumping why are rna virus mutation rates so damn high? moderate mutation rate in the sars coronavirus genome and its implications molecular evolution of human coronavirus genomes sars-cov and emergent coronaviruses: viral determinants of interspecies transmission recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission cryo-em structure of the 2019-ncov spike in the prefusion conformation human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry molecular evolution of the sars coronavirus during the course of the sars epidemic in china spread, circulation, and evolution of the middle east respiratory syndrome coronavirus human coronavirus 229e encodes a single orf4 protein between the spike and the envelope genes identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus 229e bats: important reservoir hosts of emerging viruses comparative analysis of bat genomes provides insight into the evolution of flight and immunity the egyptian rousette genome reveals unexpected features of jumping species-a mechanism for coronavirus persistence and survival dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host we thank pearl chan, hinson cheung, terence zwy and dyj wrote the manuscript with inputs from sy, ksy, syf and cpc. all authors read and approved the final version of the manuscript. the authors have declared that no competing interest exists. key: cord-353293-vjdwh19x authors: nan title: post-covid-19 global health strategies: the need for an interdisciplinary approach date: 2020-06-11 journal: aging clin exp res doi: 10.1007/s40520-020-01616-x sha: doc_id: 353293 cord_uid: vjdwh19x for survivors of severe covid-19 disease, having defeated the virus is just the beginning of an uncharted recovery path. what follows after the acute phase of sars-cov-2 infection depends on the extension and severity of viral attacks in different cell types and organs. despite the ridiculously large number of papers that have flooded scientific journals and preprint-hosting websites, a clear clinical picture of covid-19 aftermath is vague at best. without larger prospective observational studies that are only now being started, clinicians can retrieve information just from case reports and or small studies. this is the time to understand how covid-19 goes forward and what consequences survivors may expect to experience. to this aim, a multidisciplinary post-acute care service involving several specialists has been established at the fondazione policlinico universitario a. gemelli ircss (rome, italy). although covid-19 is an infectious disease primarily affecting the lung, its multi-organ involvement requires an interdisciplinary approach encompassing virtually all branches of internal medicine and geriatrics. in particular, during the post-acute phase, the geriatrician may serve as the case manager of a multidisciplinary team. the aim of this article is to describe the importance of the interdisciplinary approach––coordinated by geriatrician––to cope the potential post-acute care needs of recovered covid-19 patients. the coronavirus disease 2019 (covid19) is an infectious disease caused by sars-cov-2 that mainly affects the respiratory system, as interstitial pneumonia and acute respiratory distress syndrome (ards) [1] . infectious disease physicians, pneumologists, and intensive care physicians are the medical specialists primarily involved in the management of the acute phase of covid-19. however, as the number of confirmed covid-19 cases exceeds five million globally, the share of patients who have survived the disease is scaling up. clinicians and pathologists are now trying to better characterize the site(s), nature, and severity of damage caused by sars-cov-2. although the lungs are definitely the first target organ of sars-cov-2 infection, accumulating evidence indicates that the virus can spread to many different organs, including the heart, blood vessels, kidneys, gut, and brain [2] . for this reason, a multidisciplinary approach becomes crucial for the evaluation and the follow-up of patients with covid-19 disease (fig. 1) . although a substantial body of studies on short-term outcomes of covid-19 inpatients has already been produced, the literature is void of data on long-term outcomes of patients who survive the acute phase of the disease [3] . it may be assumed that the majority of survivors with a mildly symptomatic presentation (80%) will not be presenting longterm sequelae and will eventually fully recover. no midterm complications have been reported also for patients with moderately severe symptomatic presentation that required hospitalization but not mechanical ventilation. on the other hand, it may be expected that patients with severe symptomatic presentation requiring mechanical ventilation will be experiencing long-term complications and incomplete recovery (e.g., reduced exercise capacity). members of the gemelli against covid-19 post-acute care study group are listed in acknowledgement section. the follow-up of people who have recovered from covid-19 should be as comprehensive as possible in order to collect all the necessary information to better define the clinical and care needs. this comprehensive assessment should be linked to information on the acute phase of illness (signs and symptoms suffered during the hospital stay) and may be used to redefine the healthcare organizational model and plan what is necessary in the medium and long term. it therefore appears appropriate to propose a detailed model for the first assessment (minimum data set for the assessment of covid-19 patients), providing that subsequent stages can be customized based on the initial findings ( table 1) . the most important open question to be answered is as follows: "once recovered from covid-19 what happens to patients, and how has the virus impacted their body?" to answer this question, the fondazione policlinico universitario a. gemelli ircss (rome, italy) has set up a multidisciplinary healthcare service called "post-covid-19 day hospital." the specialist assessments offered to patients are outlined in the following sections. furthermore, the important role of geriatrician acting as a care manager of patients who suffered covid-19 disease is described. in fact, the geriatrician is the specialist who best can manage the multidimensional health problems, with a great aptitude and skill to cope multimorbid and complex patients. second, geriatricians are the doctors who best know the principles of teamwork in close collaboration with the other health care professionals and family. in particular, the geriatrician is able to manage the onset of the most important syndromes, such as sarcopenia, malnutrition, depression, and delirium. the study protocol was approved by the ethics committee of the università cattolica del sacro cuore (rome, italy). written informed consent has been obtained from the participants. due to possible contamination, a photograph was taken of the sheet and it was eliminated as a hazardous material. one of the most important problems to be addressed is the possibility of a sars-cov-2 reinfection. some reports suggest that covid-19 could relapse in patients who were considered to have recovered from the disease [4] . however, there are no definite data about possible sars-cov-2 reinfection. understanding the potential protective immunity to sars-cov-2 and its duration in time would represent a major scientific achievement both at the individual level and in a global health perspective. the role of the infectious disease physician is extremely important to evaluate the following: (a) the output and common findings in covid-19 hospitalized patients are respiratory failure, dry cough, dyspnoea, and ct scan lung abnormalities appearing as ground glass opacities and/or consolidations. during the acute phase, exercise tolerance cannot be assessed using standard tests like the 6-min walking test. moreover, some patients still need oxygen therapy or have respiratory symptoms at discharge. a respiratory follow-up is of pivotal importance to evaluate lung function, alveolar-arterial gas exchange, and exercise tolerance in recovered non-infective covid-19 patients [5] . nothing is known about long-term respiratory sequelae in covid-19 patients. the pneumological evaluation is also important to decide for whom and when to order a high-resolution lung ct scan to evaluate radiological resolution of pneumonia or the possible fibrotic evolution. sars-cov-2 infection affects the vasculature likely through immune-mediated reaction. occlusive phenomena of intravascular coagulation are expected to be more evident in smaller vascular districts [6] . in this scenario, ophthalmological assessment is particularly important to evaluate the degree of impairment of retinal vascularization in covid-19 survivors. indeed, the ophthalmologist will evaluate possible damages that covid-19 infection may have inflicted to the retina. it will be important to correlate the degree of retinal impairment with cerebrovascular and/or cognitive impairment. all patients should be offered complete ophthalmology evaluation including visual acuity assessment, anterior segment and ocular fundus photograph, 3d optical coherence tomography (oct), and oct angiography (octa). octa provides vascular analysis in vivo without dye injection. macula and optic nerve analyses are also performed to assess the degree of macula/optic nerve vascular impairment. the analysis of the fundus and the study of vascular detail with the octa technique may help evaluate the involvement of retinal layers from the most superficial to the deepest. all recovered covid-19 disease patients need to be investigated about nasal, hearing, and vestibular function. the ear-nose-throat (ent) evaluation consists of a complete physical inspection of the nose, throat, and ears, especially aimed at nasal cavities through anterior rhinoscopy [7] . five parameters are assessed: nasal mucosae, nasal septum, presence of polyps, degree of turbinate hypertrophy, and integrity of the olfactory cleft. each patient is asked to complete a visual analog scale (vas) for three symptoms: nasal obstruction, dysosmia, and dysgeusia. symptom severity during the acute phase and during the recovering period is collected and compared. scores range from 0 (total dysfunction) to 4 (normal function). the degree of olfactory and taste dysfunctions is also assessed by means of the chemosensory complaint score, a validated questionnaire, ranging from 0 to 16. all patients perform the identification test using the sniffin' sticks test [8] , which consists of 16 blue pens with black numbers. each pen is presented only once and an interval of at least 30 s is observed between each presentation to avoid olfactory desensitization. for each odorant pen, the patient is requested to choose from a list of four written proposals. the identification score corresponds to the number of correct responses. lastly, the refill taste strips are used to measure the taste ability. the test consists of four containers in the highest concentrations of sweet, sour, salty, and bitter. taste strips are applied by placing them on the tongue and asking the patient to close the mouth. to assess gustatory sensitivity of specific tongue areas, the mouth stays open and the strip is placed in contact only with this area until the patient can provide an answer. sars-cov-2 may infect nervous system and skeletal muscles [9] . neurologic features have been grouped into three categories: central nervous system (cns) manifestations (dizziness, headache, impaired consciousness, acute cerebrovascular disease, ataxia, and seizure), peripheral nervous system (pns) manifestations (taste impairment, smell impairment, vision impairment, and nerve pain), and skeletal muscular injury manifestations [10] . during the post-acute care assessment, neurologic signs and symptoms occurred during the acute phase of sars-cov-2 infection are retrospectively investigated to evaluate their persistence in the post-covid-19 phase. the follow-up of covid-19 patients also includes a specific neuropsychological assessment in order to evaluate cognitive functions (especially attention, memory, and language) and the interaction with psycho-behavioral aspects. patients recovered from covid-19 have passed through a dramatic experience, not only because of illness severity but also because of the peculiar conditions of their hospitalization. long-lasting fever, pain, difficulties of breathing, and exhaustion set up feelings of despair, hopelessness, and depression in most patients. in patients admitted to the intensive care unit, fear of dying reached a concrete and paroxysmal expression. in any case, during the hospitalization, they were forced to live isolated because of the biological risk. the isolation was generally long and intense. patients spent hours and days alone, meeting a few nurses or doctors for short time, which increased their sufferance and feelings of loneliness. some patient felt to be considered like plague and the fear of being contagious accompanied them even after hospital discharge. risk of dying, social isolation, illness severity, and sleep problems increase the risk of mental disorders such as anxiety, mood, and thought as well as acute and post-traumatic stress disorders. in addition, objective social isolation and subjective feelings of loneliness are associated with a higher risk of death, in general, and suicide, in particular. in light of the above considerations, mental health support is provided during the post-covid-19 phase to prevent the possible future development of severe psychiatric disorders [11] . sars-cov-2 impacts the cardiovascular system in multiple ways, although it is presently unclear whether the virus exacerbates pre-existing cardiovascular disease (cvd) or causes new cardiovascular abnormalities. many cardiac conditions have been described in covid-19 patients, including heart failure and cardiomyopathy [12] . heart failure is especially highly prevalent in hospitalized patients. cardiomyopathy was also reported and is thought to develop direct effects of the virus and/or toxic effects of the cytokines that are released during the infection. in many patients, a prothrombotic state develops during the acute phase, which may lead to pulmonary embolism, intracardiac thrombus, and exacerbation of coronary artery disease [13] . moreover, patients with cardiovascular risk factors, including male sex, diabetes, hypertension, and obesity, as well as pre-existing cvd are at highest risk of negative outcomes. echocardiography has an important role in managing critically ill patients, but the prolonged and close contact with patients makes it difficult to perform a detailed examination during the acute phase of disease. for these reasons, a complete trans-thoracic echocardiography is performed during the post-acute phase to explore the effects of sars-cov-2 infection on the heart. covid-19 is also recognized as a cause of severe vascular complications, mainly secondary to the inflammatory cytokine storm and rapidly progressing systemic inflammation [14] . these last conditions may lead to endothelial dysfunction, which in turn may promote the development of atherosclerosis, plaque instability, and myocardial infarction. furthermore, covid-19 patients show significant abnormalities in the coagulation pathway and are at increased risk of venous thromboembolic events. based on these elements, ultrasonographic evaluation is important to investigate the following: (a) the endothelial function, by evaluating the brachial artery reactivity, which is a well-established technique used in adults (endothelium-dependent vasodilation) [15] ; (b) the prevalence of current and/or previous deep vein thrombosis events; and (c) the whole atherosclerotic burden, using specific scores for carotid and lower limb districts as previously reported. nutrition is a major determinant of health. in covid-19, nutritional status is a crucial factor across all disease stages, especially in people at risk for negative outcomes, such as older adults and those with multimorbidity. it is widely acknowledged that malnutrition is both a cause and a consequence of immune dysfunction. in addition, in covid-19 patients, low levels of circulating markers of nutritional status (e.g., albumin, pre-albumin, and lymphocyte counts) are associated with worse outcomes [16] . prolonged intensive care unit stays are a well-established risk factor for malnutrition and lead to striking decline in muscle mass and strength and overall physical function. following sars-cov-2 infection, overactive inflammation may exacerbate catabolic processes and anorexia. these phenomena may, in turn, aggravate malnutrition and be responsible for poor recovery, loss of independence, disability, and reduced quality of life after icu discharge. not surprisingly, expert consensuses recommend accurate and timely nutritional assessment and interventions to improve clinical outcomes in people at risk of malnutrition, including persons with chronic/severe diseases across healthcare settings [17, 18] . recently, the european society for clinical nutrition and metabolism (espen) developed a practical guide for the nutritional management of patients with sars-cov-2 infection [19] . according to espen recommendations, nutritional screening, assessment and therapy should be considered as an integral part of the continuum of care for covid-19 patients. in this multidisciplinary post-acute care service, a comprehensive nutritional assessment is implemented according to the most recent guidelines. in particular, body composition changes (assessed through anthropometry and bioelectrical impedance analysis) as well as energy/protein intake is evaluated, and specific nutritional recommendations are provided to support the functional recovery of post-acute covid-19 patients. gastrointestinal involvement is common in covid-19, as reflected by a prevalence of anorexia, diarrhoea, vomiting, nausea, abdominal pain, and/or gastrointestinal bleeding as high as 50% (varying from 3 to 79% according to different reports), even in the absence of respiratory manifestations [20] . indeed, infectious sars-cov-2 has been detected in stool specimens, and ace2 receptors, which the virus uses to entry in host cells, are expressed in esophagus, stomach, small bowel, colon, liver, and pancreas. viral infections may cause post-infectious gastrointestinal disorders as well. moreover, drugs with potential gastrointestinal, pancreatic, and hepatobiliary side effects, such as antibiotics, antivirals, hydroxychloroquine, and biologics, are frequently used to treat patients affected by covid-19. the role of the gastroenterologist is to recognize covid-19 digestive manifestations, to make proper differential diagnosis, and to manage the infection-related complications and adverse drug events. sars-cov-2 infection can lead to an abnormal immunologic response causing severe respiratory failure and pneumonia, sustained by the so-called cytokine storm and thrombo-inflammation. indeed, several drugs used in severe covid-19 are immuno-modulators borrowed from the armamentarium of rheumatologic diseases. sars-cov-2 infection may also induce the development of autoimmune phenomena, as with other viral infections, and initial reports have described de novo development of autoantibodies in covid-19 patients, in particular, antiphospholipid antibodies, which may contribute to the thrombo-inflammation cascade [21] . in convalescent covid-19 patients, the role of the immuno-rheumatologist is therefore fundamental for the management of the autoinflammatory and autoimmune aspects of these patients. in particular, convalescent covid-19 patients deserve an immune-rheumatological evaluation aiming at (a) identifying possible risk factors requiring specific treatments (e.g., presence antiphospholipid antibodies); (b) re-assessing the eligibility for re-treatment of patients with severe pneumonia during hospitalization in a multidisciplinary manner or to integrate with further medications; and (c) managing patients exposed to immunological/immunosuppressant therapies during hospitalization for possible infection risk and adverse events. recent reports document that about 1% of children with covid-19 develop severe to critical disease. furthermore, several restrictive measures (parents' smart working and school closures) are heavily affecting the daily life of millions of children and their parents worldwide [22] . while the clinical impact of covid-19 in children is mild compared with adults, the real epidemiological burden of pediatric covid-19 is unknown, with several unanswered questions. do children play a role in this pandemic? do they play a role in the contagion chain? children are not infected, or most of them are asymptomatic, but do they spread the infection in family clusters? moreover, several patients with covid-19 are keen to understand whether children living in the same household got the infection or not. in order to provide a comprehensive support to families with known sars-cov-2 infection and to better understand the epidemiological impact of covid-19 in children, it is believed that it is important to evaluate with serological studies children whose parents have had a documented sars-cov-2 infection. the established post-acute service is a great opportunity to gain insights into the epidemiology of covid-19 disease among children. although no age group is safe from the sars-cov-2 infection, the burden is higher and most severe for persons aged 70 years and over, with documented mortality rates of more than 20% among octogenarians [23] . the presence of multiple pre-existing comorbidities is associated with more severe covid-19, possibly reflecting the presence of physical and/ or cognitive frailty. it is therefore clear that the population more susceptible to negative covid-19 outcomes involves older adults and/or with those with underlying medical conditions (such as cvd, diabetes mellitus, renal failure, and respiratory diseases), which requires more attention and care [1] . the complexity of patient follow-up, the different medical specialties involved, and the possibility that some patients may develop long-term sequelae call for a multi-domain organization to adequately match the needs of covid-19 survivors. in such a complex scenario, the involvement of internists and geriatricians is crucial for the adequate assessment and management of covid-19 patients. these individuals often experience a worsening of pre-existing medical conditions because of age and covid-19-associated physical and mental strain and are in need of doctors able to deal with multidimensional health problems. a case manager with great ability and competence in the management of multimorbid and complex patients is therefore needed for optimal post-acute care delivery [24] . in this regard, geriatricians are probably the doctors who best know the principles of teamwork [25] . in this post-covid-19 day hospital, internal medicine and geriatric specialists are integrated with infectious disease physicians, pneumologists, immuno-rheumatologists, and other specialists into the management of the sars-cov-2 infection. this organization allows developing tailored management strategies and the thorough investigation and description of the peculiar clinical consequence of covid-19 [26, 27] . the internal medicine and geriatric specialists have the clinical responsibility of the diagnostic schedule and therapeutic strategies. in particular, geriatricians act as case manager performing the initial assessment of the patients (telephone pre-screening, and clinical and functional assessment), managing health problems that arose from the specific evaluation, monitoring the provision of services, and providing additional diagnostic services when needed. in addition, together with the other specialities, they designed and implemented the individualized care plan and determined the services that each person was eligible for. in this respect, the geriatrician is responsible for the activation of specific rehabilitation and exercise program. finally, most patients do not need a specific rehabilitation program but a physical activity program; for this reason, a specific exercise protocol based on the sprintt project has been implemented [28, 29] . as a whole, the post-acute care service at the fondazione policlinico gemelli aims at expanding the knowledge of covid-19 and its impact on health status and care needs as well as at promoting healthcare strategies to treat and prevent the clinical consequence of sars-cov-2 infection across different organs and systems. the rapid spread of sars-cov-2 infection pandemic has led to the collection of an impressive amount of observational data addressing the acute phase of the disease. on the other hand, evidence on covid-19 clinical history following the acute phase is very limited and little is known about mid-and long-term outcomes. it is therefore of utmost importance that healthcare services are put in place to ensure a comprehensive follow-up of people discharged from hospital and the emergency department. patient follow-up will also offer the extraordinary opportunity to collect data in a standardized manner to better define the global impact of covid-19, identify specific clinical needs, and devise the organization of comprehensive and individualized care plans. the new challenge 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effects on frailty and mortality in old people with multimorbidity and high health care utilization the potential impact of multidimesional geriatric assessment in the social security system the prognostic significance of geriatric syndromes and resources the "sarcopenia and physical frailty in older people: multi-component treatment strategies" (sprintt) randomized controlled trial: design and methods physical activity and exercise as countermeasures to physical frailty and sarcopenia key: cord-351649-87g7g5au authors: haagmans, bart l.; osterhaus, albert d.m.e. title: sars date: 2009-01-30 journal: vaccines for biodefense and emerging and neglected diseases doi: 10.1016/b978-0-12-369408-9.00036-6 sha: doc_id: 351649 cord_uid: 87g7g5au abstract five years after the first severe acute respiratory syndrome (sars) outbreak, several candidate sars-coronavirus (cov) vaccines are at various stages of preclinical and clinical development. based on the observation that sarscov infection is efficiently controlled upon passive transfer of antibodies directed against the spike (s) protein of sars-cov, vaccines containing the s protein have been formulated. animals immunized with inactivated whole virus vaccines or live-recombinant vaccines expressing the sars-cov s protein (e.g., using rabies virus, vesicular stomatitis virus, bovine parainfluenza virus type 3, adenovirus, or attenuated vaccinia virus mva as a vector), as well as mice immunized with dna vaccines expressing the s protein gene all developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. although much effort has been focused on developing a sars vaccine, the commercial viability of such a vaccine for sars-cov will ultimately depend on whether the virus re-emerges in the near future. this vaccine should induce highly cross-reactive neutralizing antibodies to protect against newly emerging viruses related to sars-cov and protect both the gastrointestinal and respiratory tract in the absence of significant side effects. given the fact that in the previous outbreak mainly the elderly succumbed to the infection, special attention should be given to vaccines that are able to efficiently protect aged individuals. was successfully re-isolated from the nasal swabs and lung lesions of these animals, and a specific antibody response to the virus was shown in the infected animals, sars-cov proved to be the causative agent of this infectious disease kuiken et al., 2003 ) . sars-cov is a single-stranded, positive-sense rna virus, phylogentically related to coronaviruses from group 2 despite the fact that it does not encode a hemagglutinin-esterase protein ( snijder et al., 2003 ) . the genome is packaged together with the nucleocapsid protein, at least five membrane proteins (m, e, 3a, 7a, and 7b) and the spike (s) protein ( fig. 36.1 ) . the s1 region within the s protein, and more specifically a 193-amino acid fragment of the s protein (corresponding to residues 318-510), has been identified as the region that interacts with the cell receptor, angiotensin-converting enzyme 2 ( li et al., 2005a ) . the majority of neutralizing antibodies are directed against this region of the s protein. antibodies raised severe acute respiratory syndrome coronavirus (sars-cov) first emerged in the human population in november 2002. phylogenetic analysis of sars-cov isolates from animals indicated that this virus most probably originated from bats, was transmitted first to palm civets and subsequently to humans at the wet markets in southern china. subsequent outbreaks occurred early 2003 in hong kong, hanoi, toronto, and singapore, and could be directly traced back to one index patient who acquired the infection in guangdong and traveled to hong kong. a worldwide epidemic was halted through the efforts of the world health organization, which responded rapidly to this threat by issuing a global alert, rigorous local containment efforts, warning against unnecessary travel to affected areas, and by creating a network of international experts to combat this virus. in the end only 8096 people became ill, and 774 people died in this first sars epidemic. because sars-cov could re-emerge and cause another epidemic at any time, development of effective vaccines remains of vital importance. although several infectious agents, including chlamydia, influenza a subtype h5n1, and human metapneumovirus, were considered as a possible cause of sars, three groups independently reported the isolation of a previously unrecognized cov from clinical specimens of sars patients ( peiris et al., 2003 ; rota et al., 2003 ; drosten et al., 2003 ) . through electron microscopy, serology, and reversetranscription pcr with consensus-and randomprimers, and subsequent sequencing of the replicase gene, its identity could be revealed and consistently demonstrated in clinical specimens from patients with the disease but not in healthy controls. to conclusively establish a causal role for this cov, cynomolgous macaques were inoculated with a sars-cov isolate. because the disease in macaques caused by sars-cov infection was pathologically similar to that seen in human patients with sars, and since the virus should induce highly cross-reactive neutralizing antibodies to protect against newly emerging viruses related to sars-cov and protect both the gastrointestinal and respiratory tract in the absence of significant side effects. given the fact that in the previous outbreak mainly the elderly succumbed to the infection, special attention should be given to vaccines that are able to efficiently protect aged individuals. protein also inhibit sars-cov replication in vitro but their relevance in protection remains unclear. the genome also encodes two large poly-proteins with diverse enzymatic activities needed for efficient replication and several accessory proteins with unknown function (3b, 6, 8a, 8b, and 9b). at the end of 2004, 30 countries reported a total of 8096 probable cases of sars ( fig. 36.2 ) . pathogenic sars-covs do not circulate in the human population at the moment, but their re-emergence from animal reservoirs may likely occur in the future. because many of the early sars patients in guandong had epidemiological links to the live-animal market trade, different animal species were tested for the presence of sars-like viruses. soon after the outbreak, a sars-like coronavirus, which had more than 99% homology with human sars-cov, was detected by rt-pcr in the nasal and fecal swabs of palm civets ( paguma larvata ) and a raccoon dog ( nyctereutes procyonoides ) ( guan et al., 2003 ) . more recent studies indicate that bats may potentially act as natural reservoirs for sars-like covs ( li et al., 2005b ; lau et al., 2005 ) . however, sequence comparison of the s protein genes from bat sars-like cov and palm civet sars-like cov revealed only 64% genetic homology. subsequent studies by tang et al. (2006) have demonstrated that approximately 6% of bats sampled in china were positive for covs. interestingly, these covs are genetically diverse and many bat covs clustered with existing group 1 viruses, while others formed a separate lineage that included only viruses from bats (putative group 5). other sars-cov like viruses clustered in a putative group 4 consisting of two subgroups, one of bat covs and another of sars-covs from humans and other mammalian hosts. however, from these studies the direct progenitor of the sars-cov isolated from palm civets has not been identified. major genetic variations in the s protein gene of these viruses from civet cats, seemed essential for the transition from animal-to-human transmission epidemiology figure 36.2 reported suspected sars cases from november 1, 2002 to july 31, 2003 (data from the world health organization, http:// www.who.int/csr/sars/country/table2004_04_21/en/index.html ). countries with ͼ 5 suspected cases are indicated. patients. in fact, none of the sars-cov-infected children aged below 12 years in hong kong required intensive care or mechanical ventilation ( ng et al., 2004 ) . this is not totally explained by comorbid factors but similar age dependence in mortality is seen in patients with other (nonviral) causes of acute respiratory stress syndrome ( rubenfeld et al., 2005 ) . second, virus transmission is low in the first days of illness and peaks around day 10 after disease onset ( chu et al., 2004 ) . finally, several studies revealed that high viral load in the nasopharyngeal aspirate was found to be an independent predictor of mortality ( hung et al., 2004 ; chu et al., 2004 ) . therefore, vaccine strategies aimed at reducing the viral load may suffice to provide clinical benefit. the first efforts to treat sars patients were mainly based on the use of ribavirin and corticosteroids. ribavirin, which targets imp dehydrogenase, has been known a long time as a broad-spectrum antiviral agent. however, current data do not support the use of ribavirin for sars treatment; in vitro studies did not show significant antiviral activity ( cinatl et al., 2003 ) and ribavirin enhanced the infectivity of sars-cov in mice ( barnard et al., 2006 ) . on the other hand, a protective effect of interferon (ifn)-α has been observed in a preliminary study during the sars outbreak ( loutfy et al., 2003 ) . these results are in concordance with several studies that noted antiviral activity in vitro ( cinatl et al., 2003 ; hensley et al., 2004 ) and animal studies showing that pegylated ifn-α effectively reduced sars-cov replication and excretion, viral antigen expression by type 1 pneumocytes and the pulmonary damage in cynomolgous macaques that were infected experimentally with sars-cov ( haagmans et al., 2004 ) . however, despite an extensive literature reporting on sars treatments, it is not possible to determine whether treatments benefited patients during the sars outbreak. because of variation in treatment regimens-particularly the wide range in doses, duration of therapy, and route of administration of ribavirin and corticosteroids, no clear conclusion can be drawn regarding the efficacy of the drugs tested ( stockman et al., 2006 ) . in the event of a future outbreak of sars-cov or another novel agent, attempts should be made to develop treatment protocols, organize randomized trials and to collect and contribute information for a standardized minimum dataset that could facilitate analysis of treatment outcomes among different settings ( stockman et al., 2006 ) . to human-to-human transmission, which eventually caused the sars outbreak of 2002-2003. there is at present no evidence for the virus persisting in the human population. possible options for the re-emergence of sars include the escape of the virus from laboratories, which has already occurred on three occasions. the re-emergence of the virus from its animal reservoir remains possible, given that the virus is detectable in the feces and respiratory secretions of some animals. indeed, sars-cov re-emerged in four patients in guangdong in december 2003, although these sars-like covs caused milder clinical disease ( liang et al., 2004 ) . the us national institute of allergy and infectious diseases biodefense network classified sars-cov as a category c priority pathogen pointing out that sars-cov could be a potential biothreat agent. the clinical symptoms of sars-cov infection are those of lower respiratory tract disease and include fever, malaise, peripheral t cell lymphocytopenia, decreased platelet counts, prolonged coagulation profiles, and mildly elevated serum hepatic enzymes ( peiris et al., 2004 ; li et al., 2004 ) . chest radiography reveals infiltrates with subpleural consolidation or " ground glass " changes compatible with viral pneumonitis. around 20−30% of individuals with sars require management in intensive care units and the overall case:fatality rate reached approximately 10%. although the main clinical symptoms are those of severe respiratory illness, sars-cov actually also causes a gastrointestinal and urinary tract infection; sars-cov can be detected in the feces and urine of patients, and electron microscopic studies of biopsies of the upper and lower intestinal mucosae of patients with sars confirmed the presence of the virus in these tissues ( peiris et al., 2004 ) . fecal transmission proved to be important in at least one major community outbreak in hong kong (amoy gardens), in which over 300 patients were infected within a few days. three features of sars may be relevant for intervention strategies. first, progressive age dependence in mortality and disease severity is observed in sars the major sources of transmission in humans are droplets that deposit on the respiratory epithelium. unlike the situation in several other respiratory viral infections, viral load of sars-cov in the upper respiratory tract peaked around day 10 after disease onset ( peiris et al., 2004 ) . therefore, virus transmission may be less efficient in the first days of illness, a finding supported by epidemiological observations. realtime pcr assays detect sars-cov during the first week in specimens of the lower respiratory tract (e.g., bronchoalveolar lavage, sputum, endotracheal aspirates), nasopharyngeal aspirate, throat swabs, and/or serum ( chan et al., 2004 ) , whereas fecal samples may show very high viral loads toward the end of the first week and second week of illness. in typical cases, which were largely confined to adult and elderly individuals, sars presented with acute respiratory distress syndrome, characterized by the presence of diffuse alveolar damage and multiorgan dysfunction upon autopsy ( nicholls et al., 2003 ) . the pathological changes in lung alveoli most likely follow a common pathway characterized by an acute phase of proteinrich alveolar fluid influx into the alveolar lumina as a consequence of the injury to the alveolar wall. subsequently type-2 pneumocyte hyperplasia takes place to replace the loss of infected type-1 pneumocytes and to cover the denuded epithelial basement membrane, resulting in restoration of the normal alveolar architecture. severe alveolar injury may lead to fibrosis with loss of alveolar function in more protracted cases ( fig. 36 .3 ). it has been hypothesized that the pathological changes observed in the lungs are initiated by a disproportional innate immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such as cxcl10 (ip-10), ccl2 (mcp-1), il-6, il-8, il-12, il-1 β , and ifn-γ ( huang et al., 2005 ; wong et al., 2004 ) . these in vivo data have been confirmed in vitro, demonstrating that sars-cov infection induces a range of cytokines and chemokines in diverse cell types law et al., 2005 ) . although in vitro studies argued that production of type i ifns is inhibited or delayed by sars-cov, in sars-covinfected macaques, and also early during sars in humans, type i ifns can be readily demonstrated (de lang et al., 2007) . because prophylactic treatment of macaques with pegylated ifn-α reduces sars-cov replication in the lungs, regulation of the production of ifns may be important in controlling sars ( haagmans et al., 2004 ) . based on the observation that sars-cov infection is efficiently controlled upon passive transfer of antibodies directed against the s protein of sars-cov, a range of vaccines containing the s protein/gene has been developed. animals immunized with inactivated whole virus vaccines or live-recombinant vaccines expressing the sars-cov s protein (e.g., using rabies virus, vesicular stomatitis virus, bovine parainfluenza virus type 3, adenovirus, or attenuated vaccinia virus mva as a vector), as well as mice immunized with dna vaccines expressing the s protein gene all developed neutralizing antibodies to sars-cov and were protected against sars-cov challenge. table 36 .1 displays an overview of vaccines that have been tested for efficacy in animal models. inactivated sars vaccines have been reported to elicit high titers of s protein-specific neutralizing antibodies. few studies, however, have addressed whether inactivated whole sars-cov virions confer protection from virus challenge. mice that were immunized twice with a candidate sars-cov vaccine, produced through a two-step inactivation procedure involving sequential formaldehyde and u.v. inactivation developed high-antibody titers against the sars-cov s protein and high levels of neutralizing antibodies ( spruth et al., 2006 ) (see chapter 11). moreover, the vaccine conferred protective immunity as demonstrated by prevention of sars-cov replication in the respiratory tract of mice after intranasal challenge with sars-cov. protection of mice was correlated to the antibody titer against the sars-cov s protein and neutralizing antibody titer. similar results have been obtained using a beta-propiolactone inactivated sars-cov vaccine in mice . in addition, two chinese groups have demonstrated protective efficacy of inactivated sars vaccines in rhesus monkeys ( qin et al., 2006 ; zhou et al., 2005 ) . a soluble recombinant polypeptide containing the n-terminal segment of the s glycoprotein may suffice to induce neutralizing antibodies and protective immunity in mice ( bisht et al., 2005 ) . in addition, a trimeric recombinant s protein was able to elicit an efficacious protective immune response in hamsters ( kam et al., 2007 ) . one of the most promising vaccine candidates is based on the combination of recombinant s protein with the protollin adjuvant. in both young and aged mice, an intranasal protollin-formulated s protein seroconversion usually occurs in weeks 2 or 3 of illness and virus-neutralizing antibodies can be detected in convalescent human serum. in patients who had recovered from sars-neutralizing antibody titers peaked at month 4 but were undetectable in 16% of patients at month 36 ( cao et al., 2007 ) . neutralizing antibodies may be directed against different regions of the s protein (s1 and s2) as monoclonal antibodies against these epitopes exert potent neutralization of sars-cov in vitro ( sui et al., 2004 ; lip et al., 2006 ) . conversely, peptides which are located in these regions were able to induce neutralizing antibodies ( bisht et al., 2005 ; keng et al., 2005 ; zhang et al., 2004 ) . although experiments in diverse animal models have revealed that relatively low levels of neutralizing antibodies exert potent protection against lower respiratory tract infection, the neutralizing antibody titer necessary to achieve protection in humans exposed to sars-cov is not known. a concern in case of reemergence of sars is the possible absence of cross protection against these viruses. however, recent studies by he et al. (2006) have shown that the some neutralizing epitopes of sars-cov have been maintained during cross-species transmission, suggesting that receptor binding domain-based vaccines may induce broad protection against both human and animal sars-cov variants. in most sars autopsies, extensive necrosis of the spleen and atrophy of the white pulp with severe lymphocyte depletion have been observed. on the other hand, rapid phase in peripheral lymphocyte recovery usually coincided with improved clinical conditions of sars patients (peiris et al., 2004) . long-lived memory t cell responses against sars-cov nucleocapsid and s protein have been demonstrated in recovered sars patients, although their relevance in antiviral protection is not well understood peng et al., 2006 ; li et al., 2006 ) . however, despite potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients were not restored to normal levels . interestingly, mice that lack nk-t cells, or nk cells, or t and b cells all cleared the virus by day 9 after infection ( glass et al., 2004 ) . these data argue that cell-mediated immune responses are not essential to control virus clearance. vaccine elicited high levels of antigen-specific igg in serum and significant levels of antigen-specific lung iga ( hu et al., 2007 ) . in contrast, mice immunized intramuscularly with alum absorbed s protein did not develop detectable iga responses. following virus challenge of the aged mice, no virus was detected in the lungs of mice vaccinated intranasally, whereas intramuscularly immunized mice did not show significant control of virus replication compared to controls ( hu et al., 2007 ) . a dna vaccine encoding the s glycoprotein of the sars-cov induces t cell, neutralizing antibody responses, and protective immunity in a mouse model . these authors also demonstrated that antibody responses in mice vaccinated with an expression vector encoding a form of s protein that includes its transmembrane domain elicited neutralizing antibodies. viral replication was reduced by more than six orders of magnitude in the lungs of mice vaccinated with these s protein plasmid dna expression vectors, and protection was mediated by a humoral, but not a t-cell-dependent, immune mechanism. subsequent studies using a prime-boost combination of dna and whole killed sars-cov vaccines elicited higher antibody responses than dna or whole killed virus vaccines alone . apart from this study, several other groups have analyzed the immunogenicity of sars dna vaccines but none of these challenged the vaccinated animals with sars-cov. adenovirus-vector based vaccination strategies against sars-cov were employed early on after the sars outbreak to demonstrate that vaccinated rhesus macaques developed virus-neutralizing antibody responses against fragment s1 of the s protein and t cell responses against the nucleocapsid ( gao et al., 2003 ) . more recently, see et al. (2006) demonstrated that vaccination of c57b/l6 mice with adenovirus type 5-expressing s and nucleocapsid administered intranasally, but not intramuscularly, significantly limited sars-cov replication in the lungs. vaccines reduction in pulmonary sars-cov titer compared with control animals ( dinapoli et al., 2007 ) . finally, venezuelan equine encephalitis virus based vaccines have been tested extensively in young and aged mice. most importantly, different recombinant sars-cov bearing epidemic and zoonotic s protein variants were used to challenge the vaccinated mice. venezuelan equine encephalitis virus replicon particles expressing the 2003 epidemic urbani sars-cov strain s glycoprotein but not particles containing the nucleocapsid protein from the same strain provided complete short-and long-term protection against homologous strain challenge in young and senescent mice ( deming et al., 2006 ) . although the s protein encoding vaccine provided complete short-term protection against heterologous (strain gd03) challenge in young mice, only limited protection was seen in vaccinated senescent animals. interestingly, nucleocapsid-encoding vaccines not only failed to protect from homologous or heterologous challenge but also resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of sars-covchallenged mice ( deming et al., 2006 ) . a new generation of vaccines may be obtained from manipulating the full-length infectious cdna clone of sars-cov. one approach would be to delete the orfs 3a, 3b, 6, 7a, 7b, 8a, 8b, or 9b similar to other coronavirus mutants generated previously; some mouse hepatitis or feline infectious peritonitis deletion viruses replicate to the same extent as wild-type viruses in vitro but are severely attenuated in vivo making them potential vaccine candidates. however, sars-cov deletion mutants lacking orfs 3a, 3b, 6, 7a, or 7b, grew similar to that of the parental wild-type virus in the mouse model ( yount et al., 2005 ) . on the other hand, a recombinant sars-cov that lacks the e gene was attenuated both in vitro and in vivo . viable recombinant virus with the e gene deleted was recovered in vero cells with a titer around 10 6 pfu/ml but titers in the respiratory tract of hamsters were 100-1000-fold reduced compared to wild-type sars-cov replication, suggesting that this mutant is attenuated. multiplication of these viruses in packaging cell lines would provide the missing protein in trans and would make a promising sars-cov vaccine candidate that has the e gene deleted. live attenuated virus vaccines may revert to wildtype and recombine with other circulating human or the highly attenuated modified vaccinia virus ankara (mva) has been used to express the s glycoprotein of sars-cov in vaccination experiments using mouse, ferret, and rhesus monkey models ( bisht et al., 2004 ; chen et al., 2005 ; weingartl et al., 2004 ) . intranasal and intramuscular administration of mva encoding the sars-cov s protein led to the induction of a humoral immune response in balb/c mice, as well as reduced viral titers in the respiratory tract ( bisht et al., 2005 ) . recombinant bovine-human parainfluenza virus type 3 vector (bhpiv3) is being developed as a live attenuated, intranasal pediatric vaccine against human parainfluenza virus type 3. immunization of african green monkeys with a single dose of bhpiv3 expressing sars-cov s protein administered via the respiratory tract induced the production of sars-cov neutralizing antibodies . a recombinant bhpiv3 expressing sars-cov structural protein (s, m, and n) individually or in combination has been evaluated for immunogenicity and protective efficacy in hamsters . in the absence of s protein, expression of m, n, or e did not induce a detectable serum sars-cov-neutralizing antibody response and no protection against sars-cov challenge in the respiratory tract, whereas the vectors expressing the s protein induced neutralizing antibody responses and protection. recombinant rabies virus expressing the s protein of sars-cov induced a neutralizing antibody response in mice ( faber et al., 2005 ) . similarly, an attenuated vesicular stomatitis virus vector that encodes the sars-cov s protein may be used to induce neutralizing antibody responses ( kapadia et al., 2005 ) . mice vaccinated with recombinant vesicular stomatitis virus expressing s protein developed sars-cov-neutralizing antibody and were able to control a challenge with sars-cov performed at either 1 month or 4 months after a single vaccination. in addition, by passive antibody transfer experiments these authors demonstrated that the antibody response induced by the vaccine was sufficient to control sars-cov infection. the efficacy of these vectors was further demonstrated in studies using aged mice. in aged mice, vaccinated with recombinant vesicular stomatitis virus expressing s protein, antibody titers induced were sufficient to protect them against subsequent challenge with sars-cov ( vogel et al., 2007 ) . african green monkeys immunized via the respiratory tract with two doses of a recombinant newcastle disease virus encoding the s protein developed a relatively high titer of sars-cov neutralizing antibodies and upon challenge demonstrated a 1000-fold zoonotic coronaviruses. in order to prevent this, it has been proposed to delete an essential gene, located in a position distant from gene e, and the relocation of the deleted gene to the position previously occupied by gene e ( enjuanes et al., 2008 ) . a potential recombination leading to the rescue of gene e would lead to the loss of the essential gene. alternatively, the transcriptional regulatory sequences (trs) of a vaccine virus could be genetically manipulated to a sequence incompatible with the trs of any known circulating coronavirus as described by yount et al. (2006) . this virus could be further modified by building attenuating mutations on the genetic backbone of the recombination resistant trs rewired virus either for use as a safe high titer seed stock for making killed vaccines or as a live virus vaccine. one such attenuating mutation could be targeted to the nonstructural protein 1. recombinant mouse hepatitis virus (mhv) encoding a deletion in the nsp1-coding sequence grew normally in tissue culture but was severely attenuated in vivo ( züst et al., 2007 ) . low doses of nsp1 mutant mhv elicited potent cytotoxic t cell responses and protected mice against homologous and heterologous virus challenge. this attenuation strategy provides a new paradigm for the development of highly efficient coronavirus vaccines. although several types of vectored vaccines have been developed, several companies favored the classical approach using inactivated whole virus to develop a vaccine to be used for preclinical testing (see chapter 11). methods in place for the production of available vaccines could be easily used using wellestablished technologies. in the preclinical development stage, it is preferable that different animal species are used to evaluate the safety and efficacy of candidate vaccines. overall, a wide range of animal species, including rodents (mice and hamsters), carnivores (ferrets and cats), and nonhuman primates (cynomolgus and rhesus macaques, common marmosets, and african green monkeys) can be experimentally infected with sars-cov roberts et al., 2005b ; martina et al., 2003 ; kuiken et al., 2003 ; mcauliffe et al., 2004 ; haagmans and osterhaus, 2006 ) . most species show no clinical signs of disease, although the virus replicates efficiently in respiratory tissues. aged mice and ferrets on the other hand, show signs of clinical disease, albeit in the absence of the typical lung lesions seen in humans with sars ( roberts et al., 2005a ) . in contrast, inoculation of sars-cov in the respiratory tract of cynomolgus macaques causes infection of bronchial epithelial cells and type-1 pneumocytes 1-4 days postinfection, followed by extensive type-2 pneumocyte hyperplasia in the lungs at 4-6 days postinfection haagmans and osterhaus, 2006 ) . the lesions, consisting of multiple foci of acute diffuse alveolar damage and characterized by flooding of alveoli with protein-rich edema fluid mixed with variable numbers of neutrophils, are quite similar to those observed in humans in the acute stages of sars. remarkably, vaccine candidates tested in ferrets showed reduced efficacy. vaccination with mva encoding the s protein induced only moderate antibody responses and consequently did not protect against intranasal sars-cov infection and even resulted in an inflammatory response in the livers of the vaccinated ferrets (weingartl et al., 2004) . whether these aberrant responses resulted from immunopathological mechanisms, like antibody-dependent enhancement of infection, or represented recall responses to viral antigen in the liver is not clear at the moment but deserves further investigation. in addition, limited protection from sars-cov challenge was observed in ferrets vaccinated with inactivated whole virus ( darnell et al., 2007 ) . two out of four ferrets showed little or no neutralizing antibody even after the second immunization and none of the vaccinated ferrets were able to reduce virus excretion at day 2 after challenge but subsequently cleared the virus more rapidly compared to control animals. adenovirus-based vaccines tested in ferrets seemed more powerful as they protected the lower respiratory tract efficiently but had less effect on virus excretion in the upper respiratory tract ( kobinger et al., 2007 ) . five years after the first sars outbreak a range of candidate vaccines have been developed. early in 2006 some companies in china and the us initiated phase 1 trials. the first clinical trial has been initiated by a chinese company, sinovac biotech of beijing in collaboration with the chinese academy of medical sciences using an inactivated whole virus vaccine. to evaluate the safety and immunogenicity of this vaccine, 36 subjects received two doses of vaccine or placebo control. on day 42, all individuals showed seroconversion and peak titers of neutralizing antibodies were reached 2 weeks after the second vaccination followed clinical trials iii. virus vaccines with a type i coronavirus, feline infectious peritonitis virus (fipv), when subsequently exposed to fipv infection ( weiss and scott, 1981 ) . macrophages are able to take up feline coronavirus-antibody complexes more efficiently causing the virus to replicate to higher titers. interestingly, one study also demonstrated that antibodies against human sars-cov isolates enhance entry of pseudo-typed viruses expressing the civet cat sars-like cov s protein into cells, but not replication ( yang et al., 2005 ) . to date, there is no evidence for enhanced replication following sars-cov challenge in previously immunized animals. one other problem which may arise after vaccination with whole inactivated virus when absorbed with certain adjuvants such as alum, could relate to the induction of skewed th2 recall responses similar to what has been observed in children vaccinated with inactivated respiratory syncytial and measles virus vaccines. although much effort has been focused on developing a sars vaccine, the commercial viability of developing a vaccine for sars-cov will ultimately depend on whether the virus re-emerges in the near future. it is questionable whether possible future outbreaks will cause major outbreaks but vaccines, antivirals, or passive immunization would be relevant in the context of protecting high-risk individuals such as laboratory and health-care workers. • five years after the first sars outbreak, several candidate sars-cov vaccines are at various stages of preclinical and clinical development. • based on the observation that sars-cov infection is efficiently controlled upon passive transfer of antibodies directed against the s protein of sars-cov, vaccines encoding this protein have been developed. • animals immunized with diverse vaccines containing the s protein or s protein gene developed sars-cov-neutralizing antibodies and were protected against sars-cov challenge. • future challenges for the development of sars-cov vaccines are linked to the potential re-emergence of this virus. vaccines able to induce highly cross-reactive antibodies which efficiently protect the gastrointestinal and respiratory tract are needed. given the fact that in the previous outbreak mainly elderly humans succumbed to the infection, special attention should be given to protect specifically these individuals. by a significant decline 4 weeks later ( lin et al., 2007 ) . several other candidate sars vaccines are at various stages of preclinical and clinical development. in sars patients who recover, high levels of neutralizing antibody responses are observed, suggesting that antibody responses play a role in determining the ultimate disease outcome of sars-cov-infected patients . although attempts have been made to test the efficacy of serum preparations from seroconvalescent sars patients in the acute phase of sars, no conclusive evidence has been obtained regarding their efficacy. in mice, on the other hand, sars-cov infection is efficiently controlled upon passive transfer of convalescent immunoglobulines . the concept that antibodies protect against sars has been further explored through the generation of human monoclonal antibodies against sars-cov. prophylactic administration of a human monoclonal antibody reduced replication of sars-cov in the lungs of infected ferrets by 1000-fold, completely prevented the development of sars-cov-induced macroscopic lung pathology, and abolished shedding of virus in pharyngeal secretions ( ter meulen et al., 2004 ) . in subsequent studies, several other monoclonal antibodies were evaluated for their efficacy in mouse and hamster models ( sui et al., 2005 ; traggiai et al., 2004 ) . the importance of assessing immunogenicity of candidate sars-cov vaccines using virus neutralization assays is well acknowledged, but the variety of these tests in use is a significant problem since there is at this time no consensus on the most sensitive, specific, and reproducible assay system. to compare data from each of the candidate vaccines requires international standardization of the immunological assays and the availability of an antibody standard used for the evaluation of these vaccines. to test cross reactivity of antibodies generated by vaccination, murine leukemia virus was used to generate infectious particles containing different s proteins ( giroglou et al., 2004 ) . enhanced disease and mortality have been observed in kittens immunized against or infected enhancement of the infectivity of sars-cov in balb/c mice by imp dehydrogenase inhibitors, including 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rhesus monkeys prognostic factors for severe acute respiratory syndrome: a clinical analysis of 165 cases coronavirus nonstructural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines key: cord-351525-306syrrn authors: yang, yong-le; qin, pan; wang, bin; liu, yan; xu, guo-han; peng, lei; zhou, jiyong; zhu, shu jeffrey; huang, yao-wei title: broad cross-species infection of cultured cells by bat hku2-related swine acute diarrhea syndrome coronavirus and identification of its replication in murine dendritic cells in vivo highlight its potential for diverse interspecies transmission date: 2019-11-26 journal: j virol doi: 10.1128/jvi.01448-19 sha: doc_id: 351525 cord_uid: 306syrrn outbreaks of severe diarrhea in neonatal piglets in guangdong, china, in 2017 resulted in the isolation and discovery of a novel swine enteric alphacoronavirus (seacov) derived from the species rhinolophus bat coronavirus hku2 (y. pan, x. tian, p. qin, b. wang, et al., vet microbiol 211:15–21, 2017). seacov was later referred to as swine acute diarrhea syndrome cov (sads-cov) by another group (p. zhou, h. fan, t. lan, x.-l. yang, et al., nature 556:255–258, 2018). the present study was set up to investigate the potential species barriers of sads-cov in vitro and in vivo. we first demonstrated that sads-cov possesses a broad species tropism and is able to infect cell lines from diverse species, including bats, mice, rats, gerbils, hamsters, pigs, chickens, nonhuman primates, and humans. trypsin contributes to but is not essential for sads-cov propagation in vitro. furthermore, c57bl/6j mice were inoculated with the virus via oral or intraperitoneal routes. although the mice exhibited only subclinical infection, they supported viral replication and prolonged infection in the spleen. sads-cov nonstructural proteins and double-stranded rna were detected in splenocytes of the marginal zone on the edge of lymphatic follicles, indicating active replication of sads-cov in the mouse model. we identified that splenic dendritic cells (dcs) are the major targets of virus infection by immunofluorescence and flow cytometry approaches. finally, we demonstrated that sads-cov does not utilize known cov receptors for cellular entry. the ability of sads-cov to replicate in various cells lines from a broad range of species and the unexpected tropism for murine dcs provide important insights into the biology of this bat-origin cov, highlighting its possible ability to cross interspecies barriers. importance infections with bat-origin coronaviruses (covs) (severe acute respiratory syndrome cov [sars-cov] and middle east respiratory syndrome cov [mers-cov]) have caused severe illness in humans after “host jump” events. recently, a novel bat-hku2-like cov named swine acute diarrhea syndrome cov (sads-cov) has emerged in southern china, causing lethal diarrhea in newborn piglets. it is important to assess the species barriers of sads-cov infection since the animal hosts (other than pigs and bats) and zoonotic potential are still unknown. an in vitro susceptibility study revealed a broad species tropism of sads-cov, including various rodent and human cell lines. we established a mouse model of sads-cov infection, identifying its active replication in splenic dendritic cells, which suggests that sads-cov has the potential to infect rodents. these findings highlight the potential cross-species transmissibility of sads-cov, although further surveillance in other animal populations is needed to fully understand the ecology of this bat-hku2-origin cov. different animal species were tested for susceptibility to sads-cov treated with or without trypsin ( table 1) . as a brief summary of the results, 21 of the 24 cell lines showed significant susceptibility to sads-cov infection, defined by efficient viral replication, antigen expression, and the appearance of cytopathic effect (cpe). the three cell lines that were not infected by sads-cov were mdck, bfk, and raw 264.7. first, cpe was examined by inverted light microscopy at 48 hours postinfection (hpi), and scores are shown in table 1 . as the 293t, nih/3t3, cho, brl-3a, and nrk-52e cell lines were sensitive to trypsin, they could not be tested for sads-cov infection in mmt cells. apart from that, cpe was visible in vero, st, and brl-3a cell lines without trypsin, and prominent cpe appeared in or was enhanced with trypsin in marc-145, cos-7, bsc-40, vero, st, pk15, llc-pk1, and bhk-21 cell lines ( table 1) . as some cells did not display cpe after sads-cov infection, all cell lines were subsequently tested for viral m protein expression by immunofluorescence assay (ifa) fig. 1) , revealing the same range as seen by cpe in the different cell lines (data not shown). syncytium formation was prominent in huh-7, vero, and bhk-21 cells, whereas in mdck, bfk and raw 264.7 cells, the antigen expression was much less prominent than in the other cell lines (fig. 1a , c, and i). most cell lines tested showed evidence of productive infection, as indicated by expression of the m protein, while the inefficient antigen expression in marc-145, llc-pk1, and ipec-j2 cells suggested only a limited infection. next, viral load in the culture supernatants was detected over 5 days postinfection (dpi) by reverse transcriptase quantitative pcr (qrt-pcr) ( fig. 2a to c). a higher mean viral load was detected by qrt-pcr after trypsin treatment in hepg2, hela, marc-145, cos-7, bsc-40, vero, llc-pk1, ipec-j2, bhk-21, and df-1 cells. therefore, trypsin contributes to but is not essential for sads-cov propagation in these cell lines. there was no difference after trypsin treatment in the other cell lines, although huh-7 and tb-1 cells had high levels of sads-cov rna regardless of trypsin treatment. the progressive release of infectious sads-cov into the culture medium of six representative cell lines infected with sads-cov was determined by titration of superto determine the effect of trypsin on sads-cov infection, each cell line was infected in the following three conditions: "no trypsin" treatment, inoculated with sads-cov diluted in maintenance medium (mm) for 2 h and subsequently replaced with mm (a); "pretrypsin" treatment, inoculated with sads-cov diluted in mm containing 5 g/ml trypsin (mmt) for 2 h and subsequently replaced with mm (b); and "double-trypsin" treatment, inoculated with sads-cov in mmt and subsequently replaced with mmt (c). infection supernatants were collected at 12, 24, 36, 48, 72, and 120 hpi for viral load detection by a qrt-pcr assay targeting the viral n gene. data are expressed as the mean viral load (log 10 copies/l) ϯ standard deviation (sd), and all experiments were performed in triplicate. the 293t, cho, brl-3a, and nrk-52e cell lines did not survive in the presence of trypsin. (d) infectious titers (tcid 50 /ml) of sads-cov secreted from hela, vero, tb-1, bhk-21 pk-15, and mdck cells were determined on vero cells. natants in vero cells (fig. 2d ). unlike in mdck cells, sads-cov infection of hela, vero, tb-1, bhk-21, and pk-15 cells was productive, with hela cells showing the greatest susceptibility (fig. 2d) . wild-type c57bl/6j mice can be infected by sads-cov via oral and intraperitoneal routes. with the observation that sads-cov could infect diverse rodent cell lines (from mice, rats, and hamsters as well as gerbil primary kidney cells), we hypothesized that mice may be susceptible to sads-cov. to test this, we inoculated 6-to 8-week-old wild-type b6 mice with 5 ϫ 10 5 50% tissue culture infective dose (tcid 50 ) of sads-cov by the per oral (p.o.) or intraperitoneal (i.p.) route and monitored them for 28 days for clinical symptoms. the mice did not succumb to the infection nor did they develop diarrhea or experience weight loss during the incubation period (data not shown). to determine whether sads-cov infected the animals asymptomatically, tissue and fecal samples from inoculated mice were collected at 1, 3, 5, 7, 14, 21, and 28 dpi to determine viral growth kinetics and shedding. analysis of tissue samples by qrt-pcr suggested that sads-cov replicated modestly in the stomach early after i.p. or p.o. infection, declining and reaching undetectable levels at 7 or 14 dpi and thereafter (fig. 3a) . a very limited viral replication was observed in each region of the small intestine, with the ileum via i.p. infection showing continuous and decent detectable viral rna (fig. 3b ). in the large intestine, i.p. infection also resulted in viral rna loads slightly above the limit of detection at each time point in the ceca, whereas it led to higher viral rna levels at 1 to 3 dpi, and much lower viral rna at 21 to 28 dpi in the colon than that of the p.o. route (fig. 3c) . however, this replication in the large intestine did not translate into higher shedding, as hardly any viral genomes were detected, even at 1 dpi in the fecal samples collected from i.p.-infected mice (fig. 3f ). on the contrary, significantly more virus was detected in the feces of p.o.-infected mice at 1 and 3 dpi, indicating that i.p. inoculation does not lead to higher virus shedding. finally, sads-cov replicated more efficiently in the spleen following the i.p. route, with significantly higher viral rna loads at 21 dpi (fig. 3d ). more importantly, the virus was not cleared from this tissue by 28 dpi in the i.p.-infected group and by 14 dpi in the p.o.-infected group, suggestive of a sads-cov prolonged infection in the spleen independent of inoculation route. in contrast to the spleen, only very low levels of viral rna were detected in the local lymphoid tissue of mesenteric lymph nodes (mlns) at 1 to 3 dpi, and no virus was detectable at later time points (fig. 3d ). we also looked for virus in other extraintestinal sites, including the heart, lungs, liver, kidneys, and blood, but they were all negative or had extremely low levels (fig. 3e ). igg antibody levels after 7 days detected by sads-cov virion-based enzyme-linked immunosorbent assay (elisa) showed that the i.p. route could effectively elicit host immune responses (fig. 3g) . splenocytes support sads-cov replication. with the mouse infection model described above, our next step was to determine the cell tropism of sads-cov in vivo. thus, we performed immunohistochemistry (ihc) on sections of small and large intestine and spleen from mice infected i.p. with 5 ϫ 10 5 tcid 50 of sads-cov at 3 dpi. a monoclonal antibody against double-stranded rna (dsrna) was used to identify cells that supported active virus replication, as dsrna is an intermediate that only exists during intracellular viral replication. sads-cov dsrna signals were observed in the splenic white pulp in the marginal zone on the edge of lymphatic follicles and in the margins of the periarteriolar lymphocyte sheath (fig. 4a ). staining of tissue sections from mock-infected mice were used as a control (fig. 4b ). in addition to dsrna, we also used rabbit polyclonal antibodies (pabs) to detect the expression of the viral structural protein (m) or nonstructural protein (nsp3-ac). at 3 dpi, anti-m or anti-ac staining was observed in the white pulp around the lymphatic nodules (fig. 4c) , similar to the localization of dsrna staining (fig. 4a ). tissue sections from sads-cov or mockinfected mice probed with preimmune sera were negative, indicating the specificity of the sads-cov antibody. unfortunately, neither viral proteins (structural or nonstruc-tural) nor dsrna were detected in the intestine of infected mice, consistent with the detection of only very low levels of viral rna in these tissues by qrt-pcr (fig. 3) . next, sads-cov infection was quantified in the spleen using flow cytometry. we inoculated b6 wild-type mice with 5 ϫ 10 5 tcid 50 of virus either i.p. or p.o. and extracted the bulk immune cells from the spleen of infected animals at 3 dpi. the flow cytometry method was first validated in vero cells infected with sads-cov at a multiplicity of infection (moi) of 0.01, followed by staining with a pab against the n or ac protein at 24 hpi (fig. 4d ). as the anti-ac pab exhibited optimal intracellular staining for viral signals (fig. 4d) , it was used to determine the percentage of infected splenocytes. there were approximately 1.5-and 2.5-fold increases of total splenocytes positive for virus replication after p.o. and i.p. inoculation, respectively (fig. 4e , left; fig. 4f ), with a significant increase in the total number of ac-positive splenocytes in i.p.-infected mice compared with that of p.o. (fig. 4e , right). these data are consistent with the significantly lower viral loads in the spleen at 1 and 3 dpi in p.o.-inoculated mice (fig. 3d ), suggesting better virus dissemination and replication and escape from mucosal immune clearance. we then evaluated the growth characteristics of sads-cov in splenocytes by assessing antigen production and replication kinetics ex vitro. splenocytes were first extracted from naive mice, plated in 100-mm dishes, and infected with 1 ϫ 10 5 tcid 50 of sads-cov. we observed clusters of infected cells that appeared to have been engulfed by phagocytes (fig. 4g , middle), and the structural n protein was shown in the cytoplasm of infected cells by confocal microscopy (fig. 4g , middle and right). the percentage of infected cells was quantified by flow cytometry using anti-ac pab, revealing a nearly 2-fold increase in the splenocytes positive for viral signals (fig. 4h) , very similar to the percentage of infection observed in vivo. to further characterize the growth kinetic of sads-cov in primary splenocytes, cells were infected with 1 ϫ 10 5 tcid 50 of sads-cov, and culture supernatants were harvested at 0, 12, 24, 48, and 72 hpi. active viral replication was confirmed, with a 1.5-log time-dependent increase in genomic rna equivalents, plateauing from 24 to 72 hpi (fig. 4i ). these data suggest that although only ϳ2% of splenocytes were infected, and these cells supported a decent level of viral replication. together, these results indicate that sads-cov productively infects mouse splenocytes. splenic dcs support sads-cov replication. splenocytes were harvested from i.p.-infected mice at 3 dpi, and the extracted cells were costained with antibodies against sads-cov-ac and each of four cell surface markers (anti-cd19 for b cells, anti-cd4 for t cells, anti-cd11/c ϩ for dcs, and anti-f4/80 ϩ for macrophages) using flow cytometry (fig. 5a ). the percentage of infected cd11/c ϩ cells was significantly higher than the other cell subgroups, indicating that dcs are the major targets of sads-cov infection in the spleen. the phenotype was further confirmed by double-staining ifa with anti-dsrna, anti-m, or anti-ac antibody plus anti-cd11/c ϩ in splenic sections. as expected, dsrna staining overlapped with the cd11/c surface marker on the edges of lymphatic follicles (fig. 5b) , whereas no viral signals were seen in the mock-infected control (fig. 5c ). similar patterns of costaining were detected by m and ac antibodies (fig. 5d ). to gain insight into the relative quantity of dcs compared with other undefined target cells, cells positive for dsrna and cd11/c were counted in 10 to 15 different microscope fields of spleens from 3 infected mice (fig. 5e) , showing that 61.76% of sads-covinfected cells were dcs (fig. 5f ). to our knowledge, these results reveal the most extensive cell tropism among known covs, suggesting the functional receptor(s) for sads-cov is likely to be a very common molecule. in order to test this hypothesis, it was first necessary to find a cell line that was refractory to infection only at the internalization step. mdck cells, which showed undetectable virus production in early infection tests ( fig. 1 and 2) , were chosen as a potential candidate. there are four known types of functional cov protein receptors, including angiotensin converting enzyme 2 (ace2) for sars-cov (18), dipeptidyl peptidase 4 (dpp4) for mers-cov (19) , aminopeptidase n (apn) for tgev (20) and pdcov (21, 22) , and mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mceacam1a) for mouse hepatitis virus (mhv) (23) . to test whether one of these molecules serves as the sads-cov receptor, we attempted to inoculate nonsusceptible mdck cells overexpressing porcine apn, human dpp4, mouse ceacam1a, or human ace2 with sads-cov, but none of them allowed infection, as staining with anti-sads-cov-n pab was negative (fig. 6a) . meanwhile, the expression of each receptor in mdck cells was confirmed by ifa (fig. 6a) and western blot analysis (fig. 6b) using antibodies against the tags fused to the receptors. we confirmed that, as positive controls, lentiviruses pseudotyped with tgev, sars-cov, mers-cov, or mhv spike (i.e., pseudoviruses) efficiently entered mdck cells exogenously expressing the respective receptors (fig. 6c) . next, we demonstrated that mdck cells can confer sads-cov replication competency by transfection of a sads-cov/seacov infectious cdna clone established recently (24) , as simultaneous expression of nsp3-ac and n proteins were clearly detected by ifa (fig. 6d) . moreover, passaging of supernatants from psea-transfected mdck cells onto fresh vero cells resulted in progeny sads-cov infection, as evidenced by expression of the n protein (fig. 6e) , indicating that mdck cells can also support infectious sads-cov production without cell-to-cell spread. therefore, sads-cov apparently does not utilize any of the known cov receptors for cellular entry. the same conclusion was reached using hela cells overexpressing each of the four classical cov receptors followed by sads-cov inoculation by zhou et al. (14) ; however, the hela cell line itself was most susceptible to sads-cov infection in the present study (fig. 2d) . in order to assess the potential species barriers of sads-cov infection, a cell line susceptibility study was first conducted using 24 different cell lines. as sads-cov probably originated from a bat sadsr-cov (14) derived from hku2-cov identified in rhinolophus sinicus (chinese horseshoe bats) (12), we commenced testing viral susceptibility in two available bat cell lines, namely, bfk from myotis daubentonii (25) and tb-1 from tadarida brasiliensis. although bfk cells did not support sads-cov replication, it replicated efficiently in tb-1 cells (fig. 1a and fig. 2) , suggesting that other bat species in addition to horseshoe bats are likely susceptible to sads-cov infection. interestingly, sads-cov protein expression was detected in almost all of the rodent cells (hamster, gerbil, mouse, and rat) including bhk-21, which is not susceptible to other known human covs, such as sars-cov and mers-cov (26, 27) , as well as three swine enteric covs, namely, pedv, pdcov, and tgev (21) . given the fact that sads-cov infects both primary and passaged or primary cell lines originating from rodents, we hypothesized that rodents may be susceptible to sads-cov infection. to explore this possibility, we challenged wild-type b6 mice with sads-cov by two different inoculation routes. the challenged animals neither succumbed to infection nor manifested any signs of gastroenteritis. in fact, experimental infection of neonatal piglets with a higher dose of purified sads-cov in our laboratory resulted only in mild diarrheal signs or subclinical infection (11) . also, there was a lack of robust viral replication in the intestines during infection, and no tissue damage was detected throughout the intestines ( fig. 3b and c) , reflecting the suboptimal infection by sads-cov in immunocompetent wild-type mice. on the contrary, the virus had more efficient replication within the spleen, which was reflected by a continuous detection of viral genomic rna in the immune cells at all time points over a 28-day period (fig. 3d) . the phenotype was also consistent with the replication kinetics in extracted splenocytes in vitro, in which viral genomic rna peaked and plateaued at 72 hpi ( fig. 4g and i) . these data collectively led to speculation that sads-cov favors splenic cells over other tissues. the most logical explanation for these tissue-specific discrepancies in virus replication is (i) target cells are more concentrated in the spleen and more sporadic in the intestine or (ii) splenic immune cells have enhanced expression of the unknown receptor(s) over intestinal cells. the animals were more susceptible to i.p. infection, resulting in higher virus replication in the distal section of the small intestine, large intestine, and spleen, and perhaps a delayed clearance of viral infection in the cecum (fig. 3b to e) , suggesting the important role of mucosal immunity for controlling early infection in sads-cov in mice. it should be noted that mice (c57bl/6j mice in this study) may not be the optimal rodent species for sads-cov infection, as wild rats are more commonly seen in chinese pig farms. in addition, other transmission routes may be considered. recently, pdcov has been shown to possibly spread via the respiratory route in addition to fecal-oral transmission (28) . therefore, it will be interesting to try intranasal route for the inoculation in rats or the other rodent species to mimic sads-cov natural transmission in future studies. more interestingly, we identified dcs to be the precise cell population that supported sads-cov replication (fig. 5) . there have been a few reports of immune cell tropism for covs. macrophages are susceptible to mhv infection, representing the largest group of innate immune cells that infiltrate the central nervous system after infection with neurotropic mhv strains (29) . in addition, based on the fact that sars-cov spike-pseudotyped hiv-based vectors can efficiently transduce human dcs, kobinger (31) . these previous evidences support our present results, showing that sads-cov can efficiently replicate in dcs. furthermore, this study gives us a novel inspiration that rodents may potentially serve as susceptible hosts for sads-cov in addition to bats and pigs. of note, the species rhinolophus bat ␣-cov hku2, including sads-cov, possesses unique s genes closely related to the betacoronavirus (␤-cov), in a manner similar to some globally distributed rodent ␣-covs (11, 32, 33) , implying an unknown evolutionary connection between the bat ␣-cov hku2 and rodent ␣-covs. under the field conditions of china, direct contact between pigs and flying bats is a low probability; however, rodents (especially rats) are frequently visible in the swine industry, causing great nuisance due to feed loss. it is possible that as bats prey on insects near pig facilities, they leave feces containing hku2-like covs that contaminate pig feed, which is then eaten by pig and rodents that subsequently become carriers of sads-cov. rats and mice are increasingly implicated as external vectors for a wide range of different pig pathogens, such as lawsonia intracellularis (34) . rodents not only spread pathogens but also harm the practitioners of the swine industry, as they are thought to be the major source of leptospirosis in pigs and piggery workers (35) . future studies on identifying sads-covpositive samples in rodents near pig farms are warranted to test this hypothesis. in addition to rodents, we also measured the sads-cov susceptibility of cell lines from humans, monkeys, chickens, and dogs, revealing a remarkably broad spectrum of tropism (table 1 and fig. 1) . as for the ability of sads-cov to grow efficiently in human cell lines, we should not underestimate the risk that this bat-origin cov may "jump" from pigs to humans. it is noteworthy that camel workers with high rates of exposure to camel nasal and oral secretions had evidence of mers-cov infection (36) . considering that sars-cov and mers-cov originated from bats and spread from one species to another through intermediate hosts (civets and camels, respectively), sads-cov may pose a similar risk to human health through transmission from pigs or other susceptible hosts. the cell susceptibility study and testing of the overexpression of four known cov receptors in nonsusceptible mdck cells (fig. 6 ) demonstrated that sads-cov might use a new receptor molecule that is conserved in bats, pigs, rodents, chickens, monkeys, and humans, indicating a low barrier to cross-species transmission. this is in line with the unusual feature of broad species tropism of sads-cov. in summary, these results provide important insights into the ecology of this bat-origin cov, highlighting the possibility of its ability to jump interspecies barriers and the potential role of rodents as susceptible hosts in the field. identification of the unknown sads-cov cellular receptor and further surveillance of other animal populations are needed to fully understand the biology of sads-cov. the sads-cov isolate ch/gd-01/2017 at passage 10 was used in all experiments and cultured in vero cells (24) . the virus was passaged serially using the culture supernatant to infect fresh vero cells at a multiplicity of infection (moi) of 0.1, and viral titers were determined in vero cells by endpoint dilution as the 50% tissue culture infective dose (tcid 50 ). rabbit polyclonal antibodies (pab) against the membrane (m), nucleocapsid (n), and the nonstructural protein 3 (nsp3) acidic domain (ac) of sads-cov were generated in-house and validated in sads-cov-infected vero cells (24) . a mouse anti-sads-cov-n pab was also produced to allow double staining when mixed with the rabbit pab. a monoclonal antibody (mab) against dsrna (anti-dsrna mab j2; catalog number j2-1702, scicons, hungary) was used to specifically detect viral replication of sads-cov. cell lines and cell culture. twenty-four cell lines derived from tissues of different species were used (table 1) , including human (huh-7, hepg2/c3a, 293t, a549, and hela), monkey (marc-145, cos-7, bsc-40, and vero), swine (st, pk15, llc-pk1, and ipec-j2 [37] ), bat (bfk [25] and tb-1), canine (mdck), mouse (nih/3t3 and raw 264.7), hamster (bhk-21 and cho), rat (brl-3a and nrk-52e), and chicken (df-1) cell lines and a primary kidney cell line from mongolian gerbils (prepared in-house). the bfk cell line was a generous gift from changchun tu at the institute of military veterinary medicine, changchun, china. each cell line was cultured in dulbecco's modified eagle's medium (dmem; hyclone) supplemented with 10% (vol/vol) fetal bovine serum (fbs; biological industries), 100 u/ml penicillin, and 100 u/ml streptomycin under 37°c, 5% co 2 , and water-saturated humidity conditions. to determine viral susceptibility, each cell line was cultured at 70% confluence in 12-well plates with maintenance medium (mm) containing dmem, 0.3% tryptose phosphate broth (tpb), and 1% penicillin-streptomycin or mm with addition of 5 g/ml trypsin (mmt) (catalog number t7186-50tab; sigma, st. louis, mo, usa). after cells were washed with phosphate-buffered saline (pbs), they were inoculated with sads-cov diluted in mm or mmt at an moi of 0.01 for 2 h. nonattached viruses were removed by washing the cells three times with dmem, and cell monolayers were subsequently incubated in mm or mmt at 37°c for 5 days. to determine the effect of trypsin on viral entry, cell monolayers were infected by sads-cov in the following three conditions: (i) no trypsin treatment, infected with sads-cov diluted in mm, subsequently incubated in mm; (ii) pretrypsin treatment, inoculated with sads-cov diluted in mmt, subsequently incubated in mm; and (iii) double-trypsin treatment, inoculated with sads-cov in mmt, subsequently incubated in mmt. supernatants from cells were collected at 12, 24, 36, 48, 72, and 120 hours postinfection (hpi) for one-step quantitative rt-pcr analysis. cell cultures were examined for cytopathic effects (cpes) and immunofluorescence assay at 48 to 72 hpi. ifa for cell line susceptibility. different cells infected with sads-cov in 12-well plates were washed twice with pbs and fixed in 4% paraformaldehyde in pbs and then permeabilized with 0.1% triton x-100 in pbs. cells were then incubated with the rabbit anti-sads-cov-m pab at 1:5,000 dilution for 1 h at 37°c, washed with pbs, and stained with the alexa fluor 488-conjugated goat anti-rabbit secondary antibody (thermo fisher scientific, usa) at 1:1,000 dilution. after incubation for 1 h at 37°c, the cells were washed with pbs, stained with 4=,6-diamidino-2-phenylindole (dapi) at 1:1,000 dilution and visualized on a fluorescence microscope. one-step quantitative rt-pcr analysis targeting the n gene. the full-length sads-cov n gene was inserted into an appropriately digested pet-28a vector using two unique restriction sites, namely, ndei and xhoi, and then linearized with xhoi. the n gene was in vitro transcribed using the t7 high-efficiency transcription kit (transgen biotech co., ltd., beijing china). standard curves were generated using dilutions of a known quantity of the n gene rna to allow absolute quantitation of sads-cov rna copy numbers in samples. total rna was extracted from culture supernatants or tissue homogenates using trizol (thermofisher scientific, usa) following the manufacturer's instructions. sads-cov rna titer was determined by one-step qrt-pcr (toyobo co., ltd.) targeting the n gene with the primers 5=-ctaaaactagccccaca ggtc-3= and 5=-tgattgcgagaacgagactg-3= and the probe 6-carboxyfluorescein (fam)-gaaacccaa actgaggtgtagcagg-6-carboxytetramethylrhodamine (tamra). samples with a cycle threshold value of ͻ35 were considered positive based upon validation data using the rna standards. mouse infections, tissue harvest, and viral load determination. wild-type c57bl/6j mice (catalog number 000664; jackson laboratory) were purchased from the model animal research center of nanjing university and housed in animal facilities at the zhejiang university under specific-pathogen-free conditions. for sads-cov infections, 6-to 8-week-old female and male mice were inoculated with 5 ϫ 10 5 tcid 50 (equal to 6 ϫ 10 8 genome copies) of sads-cov, either per oral (p.o.) infection with 25-l inoculum (2 ϫ 10 7 tcid 50 /ml) or intraperitoneal (i.p.) infection with 200-l inoculum (2.5 ϫ 10 6 tcid 50 / ml). for viral load determination in specific tissues, mice were euthanized at 1, 3, 5, 7, 14, 21, and 28 days postinfection (dpi), and tissues were harvested, including stomach, duodenum, jejunum, ileum, cecum, colon, mesenteric lymph nodes, spleen, kidney, liver, heart, lung, blood, and feces. tissues were weighed and homogenized in medium (dmem contained 2% fbs) by bead beating using sterile zirconium oxide beads (catalog number zrob20; midsci). total rna was extracted from tissue homogenates and tested by quantitative rt-pcr analysis targeting the sads-cov n gene, as described above. blood samples were collected from the heart, and serum was separated for virus-specific antibody detection. enzyme-linked immunosorbent assay. sads-cov virus particles were purified from infected cell culture supernatants by sucrose density gradient centrifugation, and protein concentration was determined by the bicinchoninic acid (bca) protein assay kit (beyotime biotechnology, shanghai, china). the optimal dilution of antigen was determined by square titration. the igg antibodies contained in serum at a 1:100 dilution were detected in wells coated with purified sads-cov virus particles (6.25 ng/well) as antigen. histopathology, immunohistochemistry, and immunofluorescence assay for murine spleen. mice were infected i.p. with sads-cov, and at 3 dpi, spleens were harvested and fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. tissue sections were then deparaffinized and rehydrated in three changes of xylene, 15 min each, dehydrated in two changes of pure ethanol for 5 min, followed by rehydration in an ethanol gradient of 85% and 75% ethanol. after tissues were washed in distilled water, they were subjected to hematoxylin and eosin staining for histopathological examinations. for antigen retrieval, deparaffinized and rehydrated sections were immersed in sodium citrate antigen retrieval solution (ph 6.0) and maintained at a subboiling temperature for 8 min, were left at 98°c for 8 min, and then incubated again at subboiling temperature for 7 min. after the sections were allowed to cool to room temperature (rt) and were washed three times with pbs (ph 7.4), endogenous peroxidase was blocked by immersion in 3% hydrogen peroxide at rt for 30 min, and sections were again washed with pbs. tissue sections were blocked in 3% bovine serum albumin (bsa) at rt for 30 min and then incubated with a 1:500 dilution of each primary antibody (anti-dsrna mab, anti-sads-cov-m pab, or anti-sads-cov-ac pab) overnight at 4°c. after slides were washed three times with pbs (ph 7.4), they were stained with appropriate secondary antibodies labeled with horseradish peroxidase at rt for 50 min. freshly prepared diaminobenzidine chromogenic reagent was added and counterstained with hematoxylin and then dehydrated and visualized on a light microscope. spontaneous fluorescence quenching reagent (wuhan servicebio technology co., ltd., wuhan, china) was added to the tissue sections and incubated for 5 min after antigen retrieval. the sections were then washed in running water, followed with blocking and antibody staining as described above. in addition, the primary antibody was supplement with a cd11c antibody (wuhan servicebio technology) at a 1:200 dilution and then stained with appropriate secondary antibodies. finally, dapi was added, and sections were visualized on a fluorescence microscope; nuclei labeled with dapi appear blue, and positive cells are green by labeling with cd11/c or red by labeling with a virus-specific antibody. preparation of murine splenocytes and flow cytometry. mice infected with sads-cov were euthanized at 3 dpi, and spleens were removed and placed in 5 ml complete dmem. after the excised spleen was ground through a 100-m cell strainer using the plunger end of a 5-ml syringe, cells were washed with an excess of dmem and centrifuged at 200 ϫ g for 5 min. after cells were resuspended in 3 ml of red blood cell lysis buffer (solarbio life sciences, beijing, china) and incubated at rt for 10 min, 5 ml of dmem was added, and cells were passed through another strainer to remove clumps. after centrifugation at 200 ϫ g for 5 min, the supernatant was discarded, and cells were resuspended in 10 ml fresh dmem for cell counting and viability checks using trypan blue and a hemocytometer. for flow cytometry, cultured cells were resuspended in fc block buffer (containing anti-mouse cd16 fc block antibody at 1:500 dilution) and incubated on ice. cells in fc block buffer were added to 96-well plates at 1 ϫ 10 6 cells/well. after a 30-min incubation, cells were centrifuged at 200 ϫ g for 10 min, the supernatant was discarded, and pellets were resuspended in a 100-l cytofix/cytoperm solution (cytofix/cytoperm soln kit; bd biosciences, san jose, ca, usa) to fix cells. after incubation on ice for 20 min protected from light, cells were centrifuged at 800 ϫ g for 5 min at 4°c, supernatant was removed without disturbing cell pellets, and cells were washed twice in 150 l of 1ϫ perm/wash buffer. after the addition of 50-l virus-specific primary antibody (anti-dsrna mab, anti-sads-cov-n pab, or anti-sads-cov-ac pab) diluted in 1ϫ perm/wash buffer with 3% bsa and incubation at 4°c for 30 min, cells were centrifuged at 200 ϫ g for 10 min. cells were washed twice in 150 l of 1ϫ perm/wash buffer followed by staining with appropriate secondary antibodies conjugated to alexa fluor 488 (thermo fisher scientific, usa) at 4°c for 30 min. after pellets were centrifuged at 800 ϫ g for 5 min at 4°c and washed with 1ϫ perm/wash buffer, they were resuspended in 0.2 ml fluorescence-activated cell sorter (facs) buffer and analyzed by flow cytometry. to detect replication of sads-cov in mouse splenic cells in vitro, splenocytes were extracted from naive mice, plated in 100-or 35-mm dishes, and infected with sads-cov at an moi of 0.1. at 48 hpi, splenocytes were harvested and placed in a 15-ml tube, centrifuged at 200 ϫ g for 10 min at 4°c, and analyzed by flow cytometry as described above. infected mouse splenic cells in 35-mm dishes were detected by immunofluorescence assay with anti-sads-cov-n antibodies, and infection supernatants were collected at 0, 12, 24, 36, 48, and 72 hpi for one-step quantitative rt-pcr analysis. facs analysis of splenocytes with cell marker staining. mice infected with sads-cov were euthanized at 3 dpi, and splenocytes were prepared for flow cytometry by staining with the following appropriate antibodies: anti-sads-cov-ac following secondary antibodies conjugated to alexa fluor 647 (thermo fisher scientific, usa), anti-cd19-fitc (catalog number 4318813; ebioscience) for b cells, anti-cd4-pe (catalog number 4329629; ebioscience) for t cells, anti-cd11/c-pe-cy7 (catalog number 561022; bd bioscience) for dcs, and anti-f4/80-pe/cy5 (catalog number 123111; biolegend) for macrophages. stained cells were resuspended in 0.2 ml facs buffer and analyzed by flow cytometry. production and transduction of s protein-pseudotyped lentiviruses. pseudovirions with various cov spike proteins were produced as described previously (38) . briefly, each of the plasmids encoding tgev, sars-cov, mers-cov, and mouse hepatitis virus (mhv) s proteins were cotransfected into 293t cells with plenti-luc-green fluorescent protein (gfp) and pspax2 plasmids (kindly provided by zhaohui qian, chinese academy of medical sciences & peking union medical college) at a molar ratio of 1:1:1 by using polyethylenimine (pei). the cells were fed with fresh medium in the next 24 h, and the supernatant media containing pseudovirions were then collected and centrifuged at 800 ϫ g for 5 min to remove debris. to quantify s protein-mediated entry of pseudovirions, mdck cells were seeded at about 25% to 30% confluence in 24-well plates and transfected with either papn-flag, hdpp4-flag, mceacam1a-flag, hace2-gfp (kindly provided by zhaohui qian) (38) , or the control backbone vector by using lipofectamine 3000 (thermo fisher). the mdck cells overexpressing each receptor were inoculated with 500 l of 1:1 diluted corresponding pseudovirions at 24 h posttransfection. at 40 hpi, cells were lysed at room temperature with 110 l of medium with an equal volume of steady-glo (promega, madison, wi). the cell lysates were also used to confirm the expression of each receptor by using western blotting. transduction efficiency was monitored by quantitation of luciferase activity using a modulus ii microplate reader (turner biosystems, sunnyvale, ca). on the other hand, the mdck cells overexpressing each receptor were inoculated with sads-cov (moi, 1) at 24 h posttransfection. ifa was performed to test for sads-cov susceptibility using anti-n pab. the replication competency of sads-cov in mdck cells was further determined by a reverse genetics system. development of a dna-launched sads-cov (seacov) infectious cdna clone (named psea) and rescue of sads-cov by transfection of cultured cells with psea followed by passaging on vero cells have been described recently by our lab (24) . ethics statement. all animal experiments were performed in strict accordance with the experimental animal ethics committee of zhejiang university (approval number zju20170026). recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans feline coronavirus antibody titer in cerebrospinal fluid from cats with neurological signs origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states bat-to-human: spike features determining "host jump" of coronaviruses sars-cov, mers-cov, and beyond a novel coronavirus associated with severe acute respiratory syndrome ecology, evolution and classification of bat coronaviruses in the aftermath of sars middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease mers, sars and other coronaviruses as causes of pneumonia coronavirus spike protein and tropism changes discovery of a novel swine enteric alphacoronavirus (seacov) in southern china complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome a new bat-hku2-like coronavirus in swine fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin bat-origin coronaviruses expand their host range to pigs propagation of the virus of porcine epidemic diarrhea in cell culture proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins characterization of a novel bat-hku2-like swine enteric alphacoronavirus (seacov) infection in cultured cells and development of a seacov infectious clone characterization of a novel g3p[3] rotavirus isolated from a lesser horseshoe bat: a distant relative of feline/canine rotaviruses differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines coronavirus hku15 in respiratory tract of pigs and first discovery of coronavirus quasispecies in 5=-untranslated region protective role of toll-like receptor 3-induced type i interferon in murine coronavirus infection of macrophages human immunodeficiency viral vector pseudotyped with the spike envelope of severe acute respiratory syndrome coronavirus transduces human airway epithelial cells and dendritic cells ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign shared common ancestry of rodent alphacoronaviruses sampled globally discovery, diversity and evolution of novel coronaviruses sampled from rodents in china colonisation and shedding of lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms leptospirosis in piggery workers on trinidad high prevalence of mers-cov infection in camel workers in saudi arabia aminopeptidase-nindependent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins the professional editing service nb revisions was used for technical preparation of the text prior to submission. key: cord-349682-kpg0vley authors: ojha, probir kumar; kar, supratik; krishna, jillella gopala; roy, kunal; leszczynski, jerzy title: therapeutics for covid-19: from computation to practices—where we are, where we are heading to date: 2020-09-02 journal: mol divers doi: 10.1007/s11030-020-10134-x sha: doc_id: 349682 cord_uid: kpg0vley abstract: after the 1918 spanish flu pandemic caused by the h1n1 virus, the recent coronavirus disease 2019 (covid-19) brought us to the time of serious global health catastrophe. although no proven therapies are identified yet which can offer a definitive treatment of the covid-19, a series of antiviral, antibacterial, antiparasitic, immunosuppressant drugs have shown clinical benefits based on repurposing theory. however, these studies are made on small number of patients, and, in majority of the cases, have been carried out as nonrandomized trials. as society is running against the time to combat the covid-19, we present here a comprehensive review dealing with up-to-date information of therapeutics or drug regimens being utilized by physicians to treat covid-19 patients along with in-depth discussion of mechanism of action of these drugs and their targets. ongoing vaccine trials, monoclonal antibodies therapy and convalescent plasma treatment are also discussed. keeping in mind that computational approaches can offer a significant insight to repurposing based drug discovery, an exhaustive discussion of computational modeling studies is performed which can assist target-specific drug discovery. graphic abstract: [image: see text] a form of pulmonary disease was first reported in china from a city called wuhan in the hubei province on december 31, 2019 [1] . the deadly disease was later termed as covid-19 by the world health organization (who) on february 11, 2020. the identified causative novel coronavirus (2019-ncov) is termed as severe acute respiratory syndrome-related coronavirus sars-cov-2 as it shares around 79.6% of genome similarity with sars-cov which also previously emerged in china during 2002-2003 [2] . with the announcement of covid-19 as 'global pandemic' by who on march 11, 2020, sars-cov-2 has eventually affected 212 countries and territories around the world and 2 international conveyances. as of august 13, 2020, 20,881,635 cases have been confirmed with 748,503 deaths and 13,771,549 recovery cases, while among the active cases, 6,297,028 cases are in mild condition and 64,555 cases in a serious or critical condition [3] (fig. 1a) . the literature reported seven coronaviruses (covs) that are known to cause human disease where the strains 229e (α-cov), hku1 (β-cov), oc43 (β-cov) and nl63 (α-cov) caused mild infections of the upper respiratory tract in humans [4] . on the contrary, other two strains sars-cov (occurring in [2002] [2003] and mers-cov (middle east respiratory syndrome occurring in 2012) and the newly identified sars-cov-2 belonging to β-cov have caused serious health threat and fatality [5] . the present scenario and available pathophysiology specify that sars-cov-2 is highly transmittable and contagious than its progenitor affecting not only the respiratory system but also the gastrointestinal system, central nervous system, kidney, heart and liver leading to multiple organ failure [6] . the sars-cov-2 spike s glycoprotein has 72% identical sequence with human sars with a unique furin-like cleavage site, which is absent in other sars-like covs [7] . the cryo-em structural evidence has revealed that sars-cov-2 has 10-20 times higher binding affinity to the ace2 receptor than sars-cov which may lead to higher transmission and contagiousness [8] . therefore, blocking of the isolated viral s protein at its host receptor region and/or binding within the s protein-ace2 interface are the two most important strategies to design probable drugs for covid-19 ( fig. 1b) [9] . the sars-cov-2 virus replicates via multiple processes after entering into the host cell, and the proteins associated with these replication steps are the principal targets to treat the infected patients by blocking the viral replication. the replication-associated proteins are [10] : a. translation of genomic rna, b. proteolysis of the translated polyprotein with viral 3c-like proteinase, c. replication of genomic rna with the viral replication complex which comprises 3′-to-5′ exonuclease, rnadependent rna polymerase (rdrp), endornase and helicase, 2′-o-ribose methyltransferase, d. assembly of viral components. along with the above-mentioned targets, the most commonly employed drug targets and drug discovery strategies employed all over the world right now are illustrated in fig. 2 . presently, there is no specific treatment or approved drugs available to treat covid19 . in most of the active cases, physicians are relying on symptom-based treatment for mild cases and primarily oxygen therapy (if required, with ventilator support) for critically ill patients. a set of approved marketed drugs like hydroxychloroquine (hcq) [11, 12] , chloroquine (clq) [12] , combination of hcq and azithromycin [13] , remdesivir [14] , lopinavir [15] and ritonavir [15] are being evaluated for the infection treatment; their clinical trials are also going on in different pharmaceutical industries. a series of new vaccines are also under clinical trial such as mrna-1273 [16] , ad5-ncov [17] and chadox1 ncov19 [18] along with existing bacillus calmette-guerin (bcg) vaccine [19, 20] to check its efficiency in covid-19. due to severity and contagious nature of the sars-cov-2, researchers are exploring multiple in silico approaches and artificial intelligence [21] [22] [23] [24] [25] [26] with the aim of identifying target-specific and potent therapeutic agents to speed up the discovery process. the rcsb protein data bank (pdb) (www.rcsb.org) has already deposited around 110 protein crystal structures associated with sars-cov-2 and covid-19 to allow understanding important structural binding sites which can be explored in rational designing of small molecules. the current review discusses the most updated and probable drug candidates which are being experimentally used to treat patients in different parts of the world. also, their possible targets and pharmacological mechanisms of action which might not be clear in many cases and their pathophysiology along with the details about the status of convalescent plasma treatment and ongoing vaccine trials are discussed. we have compiled up-to-date in silico studies providing information related to computational tools, employed protein crystal structure used in the study followed by probable future drug candidates evolved from the repurposed virtual screening (vs) study employing docking, molecular dynamics (md) and homology modeling. therefore, the details related to sars-cov-2 transmission, protein structures, epidemiology, disease spectrum, diagnosis and testing are not discussed here at all as they have already been discussed in multiple literatures and separate reviews [1, 2, [4] [5] [6] [7] [27] [28] [29] . the present review is significantly different from the other recently published ones on a similar topic in that it covers and gives emphasis on the in silico modeling studies in at the time of writing this article, there are no clinically approved drugs or vaccine available for the treatment of covid-19 [30] . however, there are many drugs under trial for the treatment of covid-19 (chemical structures 1-19 in figs. 3, 4, 5) including angiotensin ii type i receptor (at1r) blockers, antiviral drugs, antimalarial drugs, interferon, il-6 inhibitors, corticosteroids, ascorbic acid, some antibacterial antibiotics, etc. an up-to-date list of drugs under trials against covid-19 with their targets, mechanisms of action, the developer companies or institutions, uses and recent status are tabulated in table 1 . several industries and research institute are also trying to develop miscellaneous drugs and/ or therapeutics agents, followed by investigation of the effectiveness of combination of drugs listed under box 1. among these tabulated drugs, most effective ones are discussed here. mechanisms of different categories of drugs used in covid-19 patients on various stages of sars-cov-2 life cycle are schematically depicted in fig. 6 . the entry of sars-cov-2 to the host cell can occur in two ways, i.e., either via plasma membrane fusion or via endosomes (endocytosis blockers: clq and hcq) (fig. 6 ). in both ways, spike proteins (s1, s2) of sars-cov-2 mediate attachment to the membrane of a host cell and engage angiotensin-converting enzyme 2 (ace2) as the entry receptor. inhibitors like convalescent plasma, monoclonal antibodies bind to the spike glycoprotein, thus preventing the viral entry. when virions are taken up into endosomes, the spike protein can be activated by the cellular serine protease tmprss2 in close proximity to the ace2 receptor, which initiates fusion of the viral membrane with the plasma membrane. camostat mesylate inhibits the tmprss2 receptor. the plasma membrane fusion entry is less likely to trigger host cell antiviral immunity and therefore more efficient for viral replication. after the viral rna is released into the host cell, polyproteins are translated. the coronavirus genomic rna encodes non-structural proteins that have a critical role in the synthesis of viral rna and structural proteins which are important for virion assembly. first, polyproteins are translated and cleaved by some of proteases like 3cl pro , pl pro , etc. (lopinavir, ritonavir and darunavir act as inhibitors of this step) to form rna replicase-transcriptase complex. the non-structural protein rdrp is responsible for replication of structural protein rna. (remdesivir, favilavir and ribavirin act as inhibitors of this enzyme). structural proteins s1, s2, envelope and membrane are translated by ribosomes that are bound to the endoplasmic reticulum (er) and presented on its surface as preparation of virion assembly. the nucleocapsids (n) remain in cytoplasm and are assembled from genomic rna. they fuse with the virion precursor which is then transported from the er through the golgi apparatus to the cell surface via small vesicles. the mature virions are then released from the infected cell through exocytosis, and then, they search another host cell (fig. 6 ). sars-cov-2 has transmembrane spikes (s protein). the spikes attached to the lipid membrane of the coronavirus recognize a host cell to attach and infect it with its viral rna. the attachment of the coronavirus s protein to angiotensin converting enzyme 2 (ace2) at its cellular binding site promotes the entry into human cells. the s protein contains two subunits such as an n-terminal s1 subunit which is responsible for receptor-virus binding and a c-terminal s2 subunit which is responsible for the fusion of virus with cell membrane [31, 32] . the s1 subunit has two domains such as a receptor-binding domain (rbd) and an n-terminal domain (ntd). at the time of infection, at first coronavirus binds to the human cell through interaction between the cell ace2 receptor and s1-rbd of coronavirus. as a result, conformational changes in the s2 subunit are triggered followed by virus-cell fusion and entry into the target cell. ace2 is a natural protein present in the lungs and the intestine (epithelial cells), and in the heart and the kidneys (endothelial cells). ace2 regulates the blood pressure by converting angiotensin molecules. it was found that the coronavirus which caused the sars outbreak in 2002 also binds to the same ace2 molecule, but in case of sars-cov-2, the binding affinity is 10 to 20 times more on human cells than the spike from the sars virus of 2002, which makes it a suitable target for covid-19. due to the high affinity of this virus to human cells, it is spread without any difficulty from one person to another than the earlier virus [33] . the entry of sars-cov-2 to the host cells via binding with ace2 enzyme leads to ace2 down-regulation. as a result, angiotensin ii is produced excessively by the correlated enzyme ace1, while a lower amount of ace2 is not capable of transforming it to the vasodilator heptapeptide angiotensin 1-7. thus, expression of higher ace2 resulting from frequently medicating covid-19 patients with at1r blockers may resist them against acute lung injury. this can be described by following two complementary mechanisms: (1) blocking the excessive angiotensin ii-mediated at1r activation caused by the viral infection, (2) upregulation of ace2 decreasing angiotensin ii production by ace and enhancing the production of the vasodilator angiotensin 1-7 [34] . the role of ace2 to enter the coronavirus into the host cell and mechanism of action of ace2 inhibitors to control the covid-19 are depicted in fig. 7 . table 1 drug candidates under trial against covid-19 with probable targets, mechanism of action, pathophysiology, application and current status [11, 12, 14, 30, 40, 43, 45, 53, 55, drug candidates under trial the sars-cov-2 uses the s protein to facilitate viral entry into the host cells. the pathogen s protein consists of two subunits s1 and s2, of which "s1" allows entry of pathogen and binding of s protein to ace2 (cellular receptor) (fig. 7 ). in addition, the entry requires s protein priming by cellular proteases, which is responsible for cleavage of s protein. after this, "s2" subunit employs fusion of viral and cellular membranes. sars-cov-2 engages ace2 as the entry receptor that can be blocked by ace2 inhibitors and arbidol, and it employs the cellular serine protease tmprss2 for s protein priming which is inhibited by camostat mesylate (fig. 7) . conclusively, sars-cov-2/ ace2 interface occurs in the molecular level, and the efficiency of ace2 usage is a key determinant of sars-cov-2 transmissibility. although it is widely accepted that coronavirus enters into the host cell through the ace2 receptor, due to limited number of studies, it is yet to establish how ace2, at1 and at2 receptors exert their activities in coronavirus-induced diseases [35, 36] . thus, ace2 inhibitors and at1 receptor antagonists [e.g., l-163491 as a partial antagonist of at1 receptor and partial agonists of at2 receptor; losartan, valsartan, irbesartan, candesartan cilexetil, telmisartan, and eprosartan (fda-approved at1 receptor blockers)] may be used as important drug candidates to control lung injury of covid-19 patients [34] . the binding of viral s protein with its receptor ace2 on host cells followed by viral endocytosis into the cells may also be a possible drug target. for example, the broad-spectrum antiviral drug arbidol recently entered the clinical trial for the treatment of sars-cov-2 which may act by inhibiting virus-host cell fusion, thus preventing the viral entry into host cells against influenza virus [37] [38] [39] . the serine protease tmprss2 produced by the host cells plays a key role for cell entry of coronaviruses by s protein priming to the receptor ace2 binding in human cells (fig. 7) . a recent study shows that camostat mesylate, a clinically approved inhibitor of tmprss2 (responsible for s protein priming), has been able to block sars-cov-2 infection of lung cells. thus, this drug may be a potential drug candidate for covid-19 [40] . remdesivir, a nucleoside analog and a monophosphoramidate prodrug of remdesivir-triphosphate (rdv-tp) developed by gilead sciences inc. (usa), was previously due to the incorporation of 3 additional nucleotides after rdv-tp, it does not cause instant chain termination because these three additional nucleotides may protect the inhibitor from excision by the viral 3′-5′ exoribonuclease activity (fig. 6) . recent reports showed that the ec 90 value of remdesivir against covid-19 in veroe6 cells was 1.76 µm, half-cytotoxic concentration (cc 50 ) was greater than 100 µm, and the selective index (si) was greater than 129.87, suggesting that its working concentrations are likely to be achieved in nonhuman primate (nhp) [14, 41] . this drug is also able to inhibit virus infection proficiently in human liver cancer huh-7 cells sensitive to covid-19. a recent case study revealed that treatment with remdesivir improved the clinical condition of the first patient infected by sars-cov-2 in the usa [42] . a recent in vitro data showed that remdesivir and chloroquine (cq) phosphate are capable of inhibiting sars-cov-2 infection [14] . remdesivir is currently being studied in phase-iii clinical trials against sars-cov-2 in wuhan, china, as on february 4, 2020, and in the usa. chloroquine (clq) and hydroxychloroquine (hcq) have received deep attention because of positive results from some small studies. an antimalarial drug, clq, has recently been reported to have potential in vitro activity against sars-cov-2. clq protects from viral infection by enhancing endosomal ph (making the environment unfavorable) which is required for virus-cell fusion. this drug may also block viral infection by inhibiting viral enzymes or processes like viral dna and rna polymerases, virus assembly, new virus particle transport, immunomodulation of cytokine release and virus release. clq also affects the glycosylation process of ace2 (as discussed earlier that to enter the host cell, the viral s protein binds with this receptor) [11, 14, 43, 44] . besides this mechanism, a recent report [12] showed that this drug also acts by inhibiting the sialic acid containing glycoprotein and gangliosides (act as primary attachment factors along the respiratory tract) mediated attachment to the s protein which is the first step for viral replication. in the ntd of the s protein of sars-cov-2, a ganglioside-binding site was recognized. the antimalarial drug clq was found to be a probable blocker of the s-ganglioside interaction. thus, this drug may be used to fight pathogenic coronaviruses especially sars-cov-2 which is responsible for covid-19. a detailed mechanism of action of clq and hcq against sars-cov-2 is illustrated in fig. 8 . a recent report showed that the ec 90 value of clq against the sars-cov-2 in veroe6 cells was 6.9 µm, which may be clinically achievable. although specific data are not available, this drug is able to inhibit the exacerbation of pneumonia patients with sars-cov-2 infection. figure 8 explains two possible mechanisms of clq and hcq against sars-cov-2. the mechanism 1 is that clq and its derivative hcq are weak bases, which can raise the ph of acidic intracellular organelles, such as endosomes/ lysosomes, essential for membrane fusion. on the other hand, mechanism-2 explains the entry of sars-cov-2 into the host cells also depends upon sialic acid (neu5ac) containing glycoproteins and gangliosides that act as the key binding factors along the respiratory tract. a ganglioside-binding site at the n-terminal domain (ntd) of the s glycoprotein of sars-cov-2 was recognized, and clq was found to be a possible blocker of the s-ganglioside interaction which occurs in the first step of the viral replication cycle (i.e., attachment to the surface of respiratory cells, intermediated by the s protein) [12] . the interaction was augmented by placing the negative charge of the carboxylate anion of neu5ac and one of the two positive charges of clq. sars-cov-2 especially interacted with 9-o-acetyl-n-acetylneuraminic acid (9-o-sia). in this case, the carboxylic acid group of the sialic acid interacted with the cationic group of the nitrogen-containing ring of clq. the formed complex of clq and 9-o-sia was further stabilized by oh-π and van der waals interactions. next, the complex developed from hcq was very close to that obtained from clq, although numerous conformational adjustments happened for the period of the simulations. interestingly, the -oh group of hcq reinforced the binding of clq to neu5ac via formation of a hydrogen bond. the formed complex of clq-oh and 9-o-sia will be stabilized again like clq to form a protective layer against fusion of the sars-cov-2. hcq is a hydroxy derivative of clq, which can block the viral infection by a similar mechanism as chloroquine; thus, this drug may also be a potential candidate against sars-cov-2. this drug is less toxic (~ 40%) than fig. 7 the role of ace2 receptor for the entry of coronavirus into the host cell, and mechanism of action of ace2 inhibitors and tmprss2 inhibitors to control covid-19 chloroquine in animals. it was found that there were seven clinical trials registered as on february 23, 2020, in the chinese clinical trial registry (http://www.chict r.org.cn), for using hcq to treat covid-19. the in vitro results suggested that this drug can efficiently inhibit sars-cov-2 infection. based on an in vitro report, it was suggested that hcq may be more potent than clq to treat covid-19 [11, 44] . a purine nucleoside presently labeled as avigan, developed by fujifilm toyama chemical of japan, has recently been approved for phase-iii clinical trial (march 31, 2020) for the covid-19 patients. this drug is approved for manufacturing and sale in japan for the treatment of influenza as an antiviral. in case of influenza virus, it selectively inhibits rna polymerase which is essential for viral replication when human cells are infected. it is believed that this drug may be effective for the treatment of covid-19 as sars-cov-2 uses same enzyme (rna polymerase) for replication and classification into the same type of single-stranded rna virus like influenza. thus, this drug acts by inhibiting the rdrp leading to inaccurate viral rna synthesis (fig. 6 ). this drug is recommended by the director of the china national center for biotechnology development under the ministry of science and technology to treat covid-19. italy has also approved the drug to treat covid-19 cases. due to the effectiveness of this drug against covid-19, it is being mass-produced as generic version in china [45, 46] . these drugs are approved hiv-1 protease inhibitors, used in combination with other anti-retroviral drugs to treat hiv-1 infection in both adults and pediatric patients who is older than 14 days. coronaviruses encode either two or three protease enzymes like papain-like proteases (pl pro ), a serinetype protease, the main protease, or m pro which cleave the polyproteins into non-structural polyproteins (nsps). these nsps are essential for viral rna synthesis. ritonavir and lopinavir act by inhibiting these protease enzymes. the mechanisms of action of these drugs suppressing coronavirus activity [47] are depicted in fig. 6 . a combination of these drugs is recommended in italy to treat covid-19 patients. several trials are going on worldwide using these drugs or in combination of other drugs. a collaborative research from china and the uk conducted a clinical trial to examine the effectiveness of a combination of these two drugs against covid-19 which was published in the new england journal of medicine (nejm) [15] . the output of their trial did not provide any significant benefit in the patients with covid-19. they suggest that "future trials in patients with severe illness may help to confirm or exclude the possibility of a treatment benefit." among those trials, two trials are investigating against pneumonia caused by covid-19. one trial is conducted in the tongji hospital, wuhan, china, using lopinavir-ritonavir against arbidol hydrochloride (influenza drugs) and oseltamivir (nct04255017). in south korea, a comparative study of lopinavir-ritonavir against hcq in patients with mild cases of covid-19 (nct04307693) was made. the two arms of the who solidarity trial are lopinavir-ritonavir alone and in combination with interferon-beta [48] . ivermectin is an fda-approved broad-spectrum antiparasitic drug, which recently showed in vitro antiviral activity against sars-cov-2. it acts by inhibiting the interaction between hiv-1 integrase protein (in) and the importin (imp) α/β1 heterodimer which is responsible for in nuclear import [49, 50] . therefore, (imp) α/β1 is unable to bind to the viral protein and preventing it from entering the nucleus, thus inhibiting hiv-1 replication [49, 50] . as a result, inhibition of the antiviral responses is reduced leading to a normal, more efficient antiviral response. the trial of potential monoclonal antibody-based therapy against covid-19 is going on by using the previous knowledge on the neutralizing monoclonal antibodies (nmab) against similar coronaviruses such as sars-cov and mers-cov. monoclonal antibodies targeting the vulnerable sites of trimeric spike (s) glycoproteins on the viral surface which are responsible for the entry to the host cell are increasingly being recognized as a promising class of drugs against covid-19. potential neutralizing monoclonal antibodies mainly targeting the receptor-interaction domain at s1 subunit ultimately disabled cell-receptor interactions [51] [52] [53] . the detailed mechanism of action of this class of drugs is depicted in fig. 9 . recently, several monoclonal antibodies, namely tocilizumab (atlizumab), mavrilimumab, lenzilumab, leronlimab, gimsilumab, sarilumab, siltuximab (sylvant), camrelizumab (airuika), eculizumab (soliris), etc., are being tried to investigate their potency against covid-19 disease, and these are tabulated in table 1 . the human monoclonal antibodies (hmabs) are developed by using several strategies against sars-cov-2 including preparation of hybridomas (using a transgenic mouse), phage display technologies and the immortalization of convalescent b cells. at present, the hmabs production has been carried out with two strains of transgenic mice, i.e., medarex humab-mouse and xeno-mouse [54] . the difference between these two strains of mice is that mouse l chain genes are still functional in the medarex humab-mouse; thus, these mice can produce chimeric mabs. the xeno-mouse from amgen has all the mouse l chain genes deleted, and b cells produce only human abs. one of these mice monoclonal antibodies [chimeric (medarex humab) (or) hybridomas (xeno-mouse)] was immunized to produce neutralizing antibodies and further evaluated. the developed neutralizing antibodies bind to a specific portion to the rbd (n-terminal (amino acids 12-261, 130-150), and c-terminal of the rbd (amino acids 548-567, 607-627)) to prevent fusion of sars-cov-2 with the target cells. due to emergency of covid-19 patients, recently, fda has recommended to healthcare providers and investigators the use of convalescent plasma collected from individuals who have recovered from covid-19 that may contain antibodies to sars-cov-2. however, till now, this therapy has not yet been shown to be effective and safe against this disease. thus, it is very essential to investigate the safety and effectiveness of covid-19 convalescent plasma in clinical trials. for this therapy, fda has recommended some guidelines as follows [55] : (1) the pathways for use of investigational covid-19 convalescent plasma; (2) patient eligibility; (3) collection of covid-19 convalescent plasma, including donor eligibility and donor qualifications; (4) labeling, and (5) record keeping. the pharmacological safety data including dose, drug-drug interactions and toxicities of selected potential drug candidates are provided in table 2 . due to the worldwide outbreak of the covid-19, the general public are keenly watching the progress of development of covid-19 vaccines [56] . dr. anthony s. fauci, director of national institute of allergy and infectious diseases (niaid), said that "finding a safe and effective vaccine to prevent infection with sars-cov-2 is an urgent public health priority." but development of a new vaccine against a new disease is not an easy task. various research institutes and industries are giving their full efforts to develop a vaccine against this pandemic disease with its earliest. thankfully, the progress is rapid due to various reasons such as: (1) sharing the efforts to sequence the genetic material of sars-cov-2 by china throughout the world, (2) coronaviruses were already on the radar of health science researchers, (3) the knowledge from sars and mers caused by corona viruses and (4) also learnings from the vaccines against sars and mers which were stopped or postponed when those outbreaks were controlled may still be used to defeat covid-19. the progress of most promising vaccines against the pandemic disease covid-19 being made by various industries and research institutes is tabulated in table 3 . several miscellaneous vaccines under investigation for covid-19 are listed under box 2. to combat the covid-19 pandemic, researchers must fight with the time to save as many lives as possible. to save the time and speed up the drug discovery process, in silico modeling provides one the best possible options. till now, in most of the cases, researchers are trying the computational repurposing theory of existing approved drugs (synthetic as well as from natural origin) for sars-cov-2 employing docking, homology modeling and molecular dynamics (md) studies to identify probable magic drugs for covid-19. huang et al. [21] computationally designed a short protein fragment or peptide which may block the coronaviruses' ability to enter human cells by binding to the viral protein which is one of the first kinds of peptide treatments routing for experimental efforts. smith and smith [22] analyzed 8000 small drug molecules and natural products (sweetlead library database) employing restrained temperature replica-exchange md simulations combining virtual screening through the ensemble docking to identify the effective drug for covid-19 which might stop the virus by two ways: (a) disrupting s protein and ace2 receptor interface stability; or (b) by troubling the capability of the s protein to recognize table 2 pharmacological safety data of selected potential drug candidates [11, 12, 14, 34, 38, 39, 43-45, 57-59, 64, 69, 70, 89] drug dose drug-drug interaction toxicity chloroquine phosphate (aralen) [11, 12, 14, 43, 89] this is a genetically engineered vaccine candidate with the replicationdefective adenovirus type 5 as the vector to express sars-cov-2 spike protein. preclinical animal studies of this vaccine candidate showed that it can persuade a strong immune response in animal models. preclinical animal safety studies also exhibited a good safety profile a phase-iii trial in saudi arabia is currently underway (fig. 10a) . ton et al. [23] identified 1000 noncovalent inhibitors for sars-cov-2 main protease (m pro ) using 1.36 billion compounds from the zinc15 library employing deep docking platform using glide sp module which utilizes qsar models trained on docking scores. the 4mds protein with 1.6 å resolution bound to a noncovalent inhibitor was used for the docking study, and among the identified 1000 noncovalent inhibitors, zinc000541677852 is the top hit drug candidate (fig. 10b) . α-ketoamide inhibitors are proposed as new drug candidates by designing, synthesis, followed by a docking study on the main protease (m pro , 3cl pro ) which has a crucial role in processing the polyproteins that are translated from the viral rna [24] . three different pdb structures were employed for the study; they are 6y2e, 6y2f and 6y2g. the authors modified previously designed inhibitor (1) (earlier used for beta-, alpha coronaviruses and 3c proteases of enteroviruses) by incorporating the p3-p2 amide bond into a pyridone ring to enhance the half-life of the newly designed (2) compound in plasma followed by replacing the hydrophobic cinnamoyl moiety with less hydrophobic boc group (fig. 11a) . although the inhibitory concentration decreased from 0.18 ± 0.02 µm (1) to 2.39 ± 0.63 µm (2), but molecule 2 reported three times higher plasma binding and 19 times better plasma solubility compared to molecule 1. further, to improve the antiviral activity against betacoronaviruses of clade b, the authors replaced the p2 cyclohexyl moiety of molecule 1 by the smaller cyclopropyl fragment to produce compound 3 which showed ic 50 value of 0.67 ± 0.18 μm for the purified recombinant sars-cov-2 m pro . zhou et al. [25] reported an integrative antiviral drug repurposing analysis employing pharmacology-based network medicine platform by a two-step process: (a) quantifying the relationship between the human coronaviruses oral bactrl-spike [91] symvivo corporation oral vaccine for covid-19 bactrl-spike-1 will be the firstin-human study of bactrl-spike, and the first-in-human use of orally delivered bactrl. it is a genetically modified, probiotic-based oral vaccine for covid-19. the study design is a phase 1, randomized, observer-blind, placebo-controlled trial in 84 healthy adults [63 receiving active vaccine in bacterial medium and 21 receiving placebo (bacterial medium only)] phase-i clinical trial (nct04334980) (hcov) and host interactome and (b) identifying the drug targets in the human protein-protein interaction network. as per phylogenetic analyses, sars-cov-2 has the highest nucleotide sequence identity (79.7%), followed by the envelope and nucleocapsid protein sequence identities of 96% and 89.6%, respectively, with sars-cov. employing the network proximity analyses between drug targets and hcov-associated proteins followed by gene set enrichment analysis (gsea), it was possible to identify 16 probable anti-hcov drugs (e.g., melatonin, mercaptopurine, and sirolimus, etc.). later, the drugs were validated through hcov-induced transcriptomics data in human cell lines and enrichment analyses of drug-gene signatures. the authors also reported three effective drug combinations among the probable hits identified through 'complimentary exposure' pattern (fig. 11b) . grifoni et al. [26] employed the immune epitope database and analysis resource (iedb) to characterize the sequence similarity between sars-cov and sars-cov-2 through homology modeling. the epitope prediction identified a priori potential b and t cell epitopes for sars-cov-2 which are the promising targets for immune recognition, followed by the discovery of diagnostics and future vaccines. within a short period of time, a huge number of in silico results were deposited in the preprint servers from all over the world and at the same time a few are already published. as in most of the cases, multiple in silico drug designing and virtual screening tools were employed for repurposing theory of approved existing drugs [94] , thus, major information is gathered in table 4 to avoid similar discussion several times . an interesting aspect is that most of the studies are based on repurposing theory of existing approved drug employing docking and md supported vs. majorly the authors relied on approved antiviral drugs along with drugbank database, zinc database, natural compounds' databases along with one of the most discussed molecules of recent time, hcq. thus, the basic ideas of implication of in silico tools and repurposing of approved drugs are same, but the only difference is selection of the target protein in different studies. without any doubt, the crystal structure of covid-19 main protease (m pro ) in complex with an inhibitor n3 (pdb: 6lu7) is the most accessed protein for the drug discovery. but based on the target type, the choice of proteins can be different. thus, to assist researchers, we have classified the target proteins into 14 types covering 110 pdb crystal id from pdb (https :// www.rcsb.org/) available until april 12, 2020, and enlisted in table 5 . multiple incidents illustrated the outbreak of corona virus in animals, especially in pets [120, 121] . animals from dogs, cats, tigers, lions to minks are already tested positive and showed mild to severe symptoms of corona virus, followed by death of few instances. although these events are scattered and not enough to study in the middle of human crisis, we cannot ignore the fact of human to animal transmission. if human to animal transmission is true, then there is a possibility of mutations, insertions and deletions in the genome sequence of this deadly virus in these animals in future with probabilities of zoonotic transfer of a stronger form of present sars-cov-2 virus from them to human in the near future. thus, these small incidents need to be checked very carefully to avoid any future transmission. the above-mentioned facts help to build an interspecies analysis by kar and leszczynski [122] for animals to human transmission or vice versa to aid in the drug discovery process of covid-19 in upcoming days. the in silico interspecies-quantitative structure-activity relationship (i-qsar) modeling correlates and then extrapolates the response (activity/toxicity) data from animal source to human which will be helpful for drug discovery. due to genome sequence similarity of sars-cov-2 with the pangolins and bat, experimental data of drug candidates to pangolins/bat along with structural and physicochemical properties of drugs can be correlated with human response endpoint which is the first step of modeling. in the next step, extrapolation of animal data to human data can lead to no human testing through developed i-qsar model. once the acceptable predictive i-qsar model is ready, there might be no need of future animal testing. as bat and pangolins are endangered species, thus future introspection can be performed in dogs and cats due to their better accessibility as well as considering them possible carriers of sars-cov-2 from the recent incidents. researchers from all over the world are trying to find the medicines which will help to stop the transmission of the virulent sars-cov-2 virus, mitigate the symptoms of the infected patients and help to lower the death toll throughout the world. unfortunately, till now there is no silver gunshot which can solve this pandemic covid-19. we have reported here a comprehensive and updated discussion on drug or drug candidates under investigation with their probable targets and mechanisms of action which might not be clear for many cases earlier, along with the effort of ongoing vaccine trials, monoclonal antibodies therapy and convalescent plasma treatment. we have presented here the ongoing computational efforts related to in silico tools to explore the probable drug candidates docking, md (sybyl-x 1.1) essential oils in garlic including organosulfur compounds (18) 6lu7 inhibitory effect of essential oils from garlic is established and found interactions with the amino acids of the human ace2 protein and the m pro of sars-cov-2 2 (allyl disulfide, allyl trisulfide) [110] against covid-19 along with the up-to-date target protein information. the information provided from in silico approaches may work as a magic bullet for the medicinal chemists to accelerate the drug discovery and development process. overall, this review provides a strong intellectual foundation to support progress of ongoing research related to thorough knowledge of drugs or investigational drugs, vaccines and in silico approaches which can be helpful for the development of new drug candidates for the treatment of covid-19. sars-cov-2 main protease (m pro /3cl pro ) 5r84, 5r83, 5r7y, 5r80, 5r82, 5r81, 5r8t, 5r7z, 5rea, 5rec, 5reb, 5ree, 5red, 5reg, 5ref, 5re9, 5re8, 5re5, 5re4, 5re7, 5re6, 5rfb, 5rfa, 5rfd, 5rfc, 5rff, 5rfe, 5rfh, 5rfg, 5rey, 5rex, 5rf9, 5rez, 5rf2, 5rep, 5rf1, 5res, 5rf4, 5rer, 5rf3, 5reu, 5rf6, 5ret, 5rf5, 5rew, 5rev, 5rf7, 5rei, 5reh, 5rek, 5rej, 5rem, 5rel, 5reo, 5rf0, 5ren, 5rfz, 5rfy, 5rfr, 5rfq, 5rft, 5rfs, 5rfv, 5rfu, 5rfx, 5rfw, 5rfj, 5rfi, 5rfl, 5rfk, 5rfn, 5rfm, 5rfp, 5rfo, 5rg0, 6y2e, 6y84, 6w63, 6yb7, 5rf8, 6y2g, 6y2f, 6lu7 adp ribose phosphatase of nsp3 from sars-cov-2 6vxs, 6w02. a new 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domestic animals to sars coronavirus-2 from animal to human-interspecies analysis provides novel way of ascertaining and fighting covid-19. the innovation acknowledgements pko thanks the ugc, new delhi, for finankey: cord-342756-rgm9ffpk authors: senger, mario roberto; evangelista, tereza cristina santos; dantas, rafael ferreira; santana, marcos vinicius da silva; gonçalves, luiz carlos saramago; de souza neto, lauro ribeiro; ferreira, sabrina baptista; silva-junior, floriano paes title: covid-19: molecular targets, drug repurposing and new avenues for drug discovery date: 2020-10-02 journal: mem inst oswaldo cruz doi: 10.1590/0074-02760200254 sha: doc_id: 342756 cord_uid: rgm9ffpk coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a highly contagious infection that may break the healthcare system of several countries. here, we aimed at presenting a critical view of ongoing drug repurposing efforts for covid-19 as well as discussing opportunities for development of new treatments based on current knowledge of the mechanism of infection and potential targets within. finally, we also discuss patent protection issues, cost effectiveness and scalability of synthetic routes for some of the most studied repurposing candidates since these are key aspects to meet global demand for covid-19 treatment. at the moment, the prevention aimed at reducing transmission in the community is the best alternative, indicating that enhanced public health interventions, including social distancing, use of masks and movement restrictions should be implemented to bring the covid-19 pandemic under control. aggressive isolation measures including travel restriction in china, have successfully led to a progressive reduction of covid-19 cases. (8) kissler and colleagues, simulated the relaxation of the protective measures using a post-pandemic mathematical model. (9) the authors demonstrated that social isolation will be necessary at the best scenario until 2021, and reaffirmed the need and importance of maintaining social isolation. they also suggested that in the absence of such restrictions, the pandemic could last until 2024. thus, in this context, the search for new medicines available to combat this disease is urgent. coronaviruses are members of the family coronaviridae that present crown-like spikes on their surface visualised by electron microscopy. the subfamily coronavirinae contains the four genera alpha, beta, gamma, and deltacoronavirus. coronaviruses infect birds (gamma and deltacoronaviruses) and several mammalian species (mainly alpha and betacoronaviruses), including humans. (10, 11) coronaviruses have been isolated from diverse species, including mammals like bats, rodents, bovines, swine, felines, pangolins, horses and others. (12, 13) human coronavirus was first identified in 1966 by tyrrell and bynoe. (14) nowadays the seven coronaviruses that can infect humans (hcovs) are classified in alpha coronavirus (229e, nl63) or beta coronavirus [oc43, hku1, middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov) and more recently the sars-cov-2]. 229e, nl63, oc43, and hku1 can cause upper and lower respiratory tract infection in adults and children. after the 2000s, two epidemic covs have arisen in humans: the sars-cov and the mers-cov which were reported in 2003 and 2012, respectively. (15, 16) in 2019, after the first report of novel pneumonia in wuhan, china, the seventh hcov was described. it also causes severe acute respiratory syndrome (therefore called sars-cov-2) and spreads very quickly worldwide . coronavirus are single-stranded positive-sense rna viruses and their genome size is approximately 30 kb, which encodes some important structural proteins. (17) the spike (s) glycoprotein is a well characterised protein that mediates coronavirus entry into host cells via fusion of the viral and cellular membranes through a pre to post fusion conformation transition. (18) the s protein s1-s2 subunits bind to cellular receptors that vary according to the coronavirus species: angiotensin-converting enzyme 2 (ace2) in sars-cov, sars-cov-2 and hcov-nl63; and dipeptidyl peptidase 4 (dpp4) and aminopeptidase n (apn) in mers or others alphacoronaviruses like tgev (porcine transmissible gastroenteritis coronavirus) and porcine respiratory coronavirus (prch). (18, 19, 20) other structural proteins are mandatory to assemble the complete viral particle like nucleocapsid protein (n), membrane protein (m) and the envelope protein (e). furthermore, they can be involved in other processes like morphogenesis, envelope formation, budding or pathogenesis. (17, 21, 22) by genomic sequencing analysis of other coronavirus strains and sars-cov-2, andersen and collaborators demonstrated that sars-cov-2 has mutations resulting in six different amino acids at the receptor-binding domain (rbd) that appears to be optimised for binding to the human receptor ace2. (23) they also showed that the gene encoding the spike protein has an insertion of 12 nucleotides giving it a polybasic (furin) cleavage site at the s1-s2. in this way, the high-affinity of the sars-cov-2 spike protein to the human ace2 is a consequence of natural selection on a human or human-like ace2. they suggest some possibilities to explain that: emergence in an animal host before zoonotic transfer; natural selection in humans following zoonotic transfer; or natural selection during the passage. (23) other researchers did a phylogenetic analysis of 160 genomes of sars-cov-2. (24) they showed 3 important variations in the composition of amino acids that allowed them to classify into different groups. group a has two subclusters that are distinguished by the synonymous mutation t29095c. while b is derived from a by two mutations t8782c and c28144t, type c differs from its parent type b by mutation of g26144t. the a and c types are found more often outside east asia, in europeans and americans. while the b type is the most common type in east asia. (24) in december 2019, covid-19 was initially reported as a new viral pneumonia, due to the clinical characteristics of the large number of cases that emerged in wuhan, china. (25, 26) sars-cov-2 typically causes respiratory sickness, the major clinical characteristics ob-served in infected patients are high fever, dry cough and dyspnea (shortness of breath or difficulty in breathing). minor symptoms include headache, diarrhea, nausea, vomiting, loss of smell and taste. (27) this clinical condition can progress to moderate or severe pneumonia. (28) in this case, first there is an accumulation of macrophages in alveoli, followed by release of cytokines and accumulation of fluids. neutrophils can also be recruited by the immune system leading to the destruction of type i and type ii alveolar epithelial cells causing a collapse of the alveoli function and consequently the acute respiratory distress syndrome (ards). in the severe condition, with an increase of the inflammation, the protein rich fluid from lungs enter in the bloodstream causing the systemic inflammatory syndrome (sirs). (29, 30) these complicating factors can lead to a multi-organ failure and septic shock, causing patient death. furthermore, some pre-existing conditions can enhance the risk to develop the severe form of the disease, including age over 60 years and a history of chronic diseases like chronic lung disease, asthma, heart diseases, immunosuppressed patients, cancer, diabetes or chronic kidney disease. (31, 32, 33, 34, 35) finally, the vast majority of people have mild symptoms or are asymptomatic, which is a big problem because they can also transmit the virus to the non-infected population. (36) drug repositioning, repurposing, reprofiling or retasking is the evaluation of existing drugs for new therapeutic purposes. (37) a candidate drug (investigational or approved) for repurposing efforts already has a known safety and toxicity profile, based on at least successful phase i or phase ii clinical trials. (38) considering the whole process, costs of bringing a repurposed drug to the market have been estimated to be ten times lower and the time is shortened by around a half, compared with a new drug. (38) even though the clinical phase iii and regulatory aspects remain similar for developing a new drug, drug repurposing possesses many advantages over developing a new drug from scratch: the reduced time and financial investment for development, the lower risk of failure and a consolidated pharmaceutical supply chain for production and distribution to the patients that effectively need treatment. (39) emerging or reemerging viruses pose major public health concerns globally. (40) for several pathogenic viruses, considerable efforts have focused on vaccine development and other therapies, like transfusion of convalescent plasma. (41, 42) however, during pandemics infected individuals need urgently to be treated on a large scale. a medicine armamentarium for the covid-19 outbreak is needed immediately and drug repurposing could be one of the best strategies to deal with this pandemic. (43, 44) computational and experimental approaches can be used, alone or combined, to achieve a more holistic point of view and increased chance of success in drug repurposing. in the following topic, we will review sars-cov-2 structure and mechanism of infection in order to discuss molecular targets from the virus or its human host that are being considered for drug repurposing and perhaps future development of new drugs. ongoing drug repurposing efforts will be described in more details later in this article, along with some clinical trials that have been carried out so far for covid-19 treatment. finally, as treatment availability is of utmost importance when dealing with a pandemic, we bring a discussion on patent protection and ease of large-scale production of some of the drugs that are more advanced in clinical studies. sars-cov-2: structure, mechanism of infection and drug targets sars-cov-2 structure -electron microscopy imaging of sars-cov-2 virions indicates that they have a spherical or pleomorphic shape, with diameters ranging from 60 to 140 nm, showing prominent spikes of 9-12 nm in their surfaces that resemble a solar corona, hence the name "coronavirus". (45, 46, 47, 48) sars-cov-2 is an enveloped virus with a single-stranded positive sense (5'-3') rna (+ssrna) (~ 30 kb) containing a 5'cap structure and a 3'-poly-a tail. (49, 50) its genomic rna (grna) has a variable number of open reading frames (orfs) that are predicted to encode 16 non-structural (nsp), 4 structural and several accessory proteins ( fig. 1 ). (26, 51, 52, 53, 54) orf1a and orf1b represent more than 2/3 of the whole length of grna, and encode two polyproteins: pp1a (440-500 kda) and pp1ab (740-810 kda). (53, 55) the polyprotein pp1a is translated from orf1a while pp1ab from orf1a/orf1b using a -1 ribosomal frameshift mechanism that occurs near the 3' end of orf1a which allows continued translation of orf1b. (53) together, pp1a and pp1ab originate all nsps (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) , such as m pro (nsp5) protease and rdrp (nsp12) rna polymerase, which form viral replicase/transcriptase complexes (rtcs), and are encapsulated in double-layered vesicles originated from the endoplasmic reticulum (er). (56, 57, 58) the orfs near the 3' end of the grna encode the structural and accessory proteins of sars-covs. (58) the first ones have a crucial role in the assembly of viral particles and virus invasion. (56, 58) the main structural proteins are named: spike (s), envelope (e), nucleocapsid (n) and membrane (m) proteins. most of them reside on the virion surface (s, e, m proteins) while n proteins are found in the core of the particle bound to grna. (59) s proteins are essential for virus attachment and entry into the host cells, tissue tropism and pathogenesis. (58, 60) e proteins exert several roles in virus infection, such as helping in virus assembly and release from infected cells, creating ion channels in cell membranes and suppressing host stress response. (58, 61, 62) n proteins interact with grna to form the ribonucleoprotein. (56, 62) m proteins have a role in virion assembly and in determining the shape of the envelope. they also bind to all other structural proteins promoting, for instance, the stabilisation of n protein-rna complexes. (56, 63) sars-cov-2 mechanism of infection -at present, the mechanisms that underlie sars-cov-2 infection have not been directly described. nonetheless, they seem to be similar to those proposed for other coronaviruses. (58) in one proposal, virus infection starts with the binding of its s proteins to host receptor ace2, a membrane protein largely expressed in the lung and small intestine cells (fig. 2 ). (44, 59, 64) after attachment, s protein is cleaved by host proteases initiating the fusion of virus and cell membranes that culminates in viral grna release into the cytoplasm. this event is proposed to occur through two distinct ways: via plasma membrane (early pathway) or via endosomes (late pathway). in the early pathway, s protein is cleaved by host plasma membrane proteases (e. g., tmprss2) while in the late pathway by endosomal proteases (e. g., cathepsin l). the route taken by the virus to enter the cell appears to be dependent on the availability of these proteases. (59, 64, 65) once in the cytoplasm, grna is readily translated into viral polyproteins (pp1a/pp1ab), which are cleaved into the individual nsps that compose the rtcs (fig. 2) . these complexes recognise transcriptional regulator sequences in grna and begin to transcribe a series of subgenomic rnas that encode structural and accessory proteins, otherwise the whole grna is replicated. (57, 58, 59) upon translation, s, e and m structural proteins are driven to the er-golgi intermediate compartment (ergic) where s proteins go through post-translational modifications (e. g., proteolysis, n-glycosylation). in parallel, a copy of the grna and n proteins bind in the cytoplasm to form the nucleocapsid and move into the ergic. in this compartment, nucleocapsid and the other (1) virus infection initiates with the binding of virus s proteins to the ace2 cellular receptors. after attachment, the virus may enter the cell through two distinct mechanisms: early and late pathways. (2) in the early pathway the genomic ssrna (grna) is liberated into the cytoplasm after the fusion between viral and cell cytoplasmic membranes, an event triggered by membrane proteases (e. g., tmprss2). (3) the grna is immediately translated into two polyproteins that undergo proteolytic cleavage giving rise to all nonstructural proteins (nsps). (4) the nsps form the replication-transcription complexes (rtcs) where the grna (blue ribbon) is replicated and the subgenomic rnas (red ribbon) are transcribed. (5) the subgenomic rnas are translated into viral structural and accessory proteins in the cytosol. (6) upon translation, e, m and s structural proteins are inserted into er and follow the secretory pathway to the er-golgi intermediate compartment (ergic). (7) meanwhile, a copy of the grna binds to n proteins in the cytoplasm forming the nucleocapsid, which is transported to the ergic. (8) virion assembly in the ergic. (9) the new virion travels through the cytoplasm inside a vesicle and leaves the cell by exocytosis. (10) alternatively, in the late pathway, the virus can undergo endocytosis to initiate the infection. (11) the virion membrane merges with the endosome membrane after s protein proteolysis by endosomal proteases (e. g., cathepsin l), allowing the grna to be released into the cytoplasm. from this point on, the cycle follows the same pathway described in 3-9 steps. the red arrows indicate the general replication pathway and the blue arrows indicate the grna movement through the cycle. some viral and host proteins have been explored as potential targets for drug repurposing. some of these drug candidates are shown with their site of action indicated in the cycle. (44, 59, 66, 67) viral proteins are assembled into a virion which travels through the cytoplasm inside a vesicle and leaves the cell by exocytosis. (48, 56, 59) candidate drug targets -in the search for a treatment for covid-19, several viral and host molecular proteins have been explored as potential drug targets. overall, they participate in key events of the virus infection cycle, such as cell entry and replication, as well in host metabolic pathways and immune response. in the following topics we will address in more details some of these targets. drugs under investigation for blocking the main steps of the sars-cov-2 virus replication cycle are indicated in fig. 2 . virus targets -during sars-cov-2 grna translation, two proteases, namely m pro and pl pro , act in concert to cleave and release from pp1a/pp1ab the 16 nsps that compose the rtc. (58) therefore, these proteases are essential for virus replication and represent useful targets for therapeutic intervention. recently, sars-cov-2 m pro and pl pro had their 3d structures published -pdb 6lu7, 2.16 å and pdb 6w9c, 2.70 å, respectivelywhich make them particularly useful for computational structure-based drug design methods. (68) 3-chymotrypsin-like protein (synonyms: coronavirus main protease, m pro , 3cl pro ) of sars-cov-2 is a 33.8 kda homodimeric protein (306 aa) that belongs to the cysteine protease class (possibly from c30 family and pa clan). (68, 69) this enzyme catalyses the hydrolysis of peptide bonds of polyproteins (possibly e.c. 3.4.22.69) at sites whose amino acid sequences generally follow the pattern leu-gln* (ser, ala, gly) (*marks the cleavage site). (70, 71) m pro is part of pp1a/pp1ab polyproteins (nsp5). (57) during polyproteins translation, m pro suffers autolyt-ic cleavage and is released from its polyprotein precursor, reaching a mature state that cleaves pp1a/pp1ab at no less than 11 sites downstream of the nsp4 coding region. (57, 68, 70, 72) each protomer of sars-cov-2 m pro is divided into three domains: chymotrypsin and picornavirus 3c protease-like i and ii domains, composed by antiparallel β-barrel structures, and domain iii which contains five α-helices arranged into an antiparallel globular cluster responsible for protease dimerisation (fig. 3a) . domains ii and iii are connected by a long loop, where lies a cleft that serves as a substrate binding site and where catalysis occurs using the cys 145 -his 41 dyad (fig. 3b ). (68, 70, 73) m pro has been widely explored in drug discovery campaigns using experimental and/or computational approaches. (68, 73, 74, 75, 76, 77) moreover, m pro has no human homologue, which reduces the chances of toxic effects of a given inhibitor. (68) potential sars-cov-2 m pro inhibitors include fda-approved antivirals, such as inhibitors of hiv-1 [e. g., lopinavir (1) /ritonavir (2) ] and hcv [e.g., boceprevir (3)] proteases, as well as antineoplastic [e.g. carmofur (4)] and antibacterial [e.g., doxycycline (5)] drugs. (73, 78, 79, 80, 81, 82) chemical structures for compounds 1-14 are shown in fig. 4 . nsp3 is the largest protein encoded by covs (~200 kda). in sars-covs it has 16 domains which include a papain-like proteolytic enzyme from the cysteine protease class (c16 family and ca clan for sars-cov). (57, 69, 83) pl pro is composed of a catalytic domain, an extended right-handed thumb-palm-finger structure with a cys-his-asp catalytic triad, and a ubiquitin-like domain (ubl) (fig. 5a ). the catalytic cys is located in the thumb subdomain while his and asp in the palm subdomain (fig. 5b ). pl pro catalyses the hydrolysis reaction of peptide bonds of pp1a/pp1ab at three sites (nsp1/nsp2, nsp2/nsp3 and nsp3/nsp4) that share the xlxgg* pattern (*represents the cleavage site). (57, 71, 83, 84) this activity is responsible for nsp3 release from polyproteins. pl pro also recognises and hydrolyses ubiquitin and isg15 from cellular proteins. (83, 84) the deubiquitination and deisgylation activities are proposed to modulate the post-translational modifications of signaling molecules that trigger innate immune response of the host. (85) these functions are pivotal for virus infection justifying the search for sars-covs pl pro inhibitors. (84, 86, 87, 88, 89, 90) putative inhibitors of sars-cov-2 pl pro include fda-approved drugs such as fostamatinib disodium (6) (a tyrosine kinase inhibitor used in the treatment of chronic immune thrombocytopenia) and natural products [e.g., platycodin d (7)]. (89, 91) rna-dependent rna polymerase (rdrp, nsp12) is an essential protein responsible for rna synthesis during viral rna transcription and replication cycles. (89) as described for sars-cov, the rna polymerase activity of sar-cov-2 rdrp (804 aa) also seems to require the binding of nsp7 and nsp8 cofactors to enhance rdrp binding and processivity. (92, 93) the overall rdrp structure has a "right hand" rdrp domain, composed by three subdomains (palm, fingers and thumb), and a nidovirus-unique n-terminal extension domain that forms a nidovirus rdrp-associated nucleotidyltransferase (niran) structure. these domains are connected by an interface domain. additionally, this protein also has a n-terminal β-hairpin (fig. 6a) . the active site of rdrp contains conserved polymerase motifs located in the palm subdomain and its configuration is similar to other rna polymerases. (94) rdrp substrates, rna template/primer and nucleotide triphosphate (ntps), access the catalytic centre through the template and ntp entry paths, respectively, while the product-template hybrid is released through the rna exit path. (92, 94) sars-cov-2 rdrp shares 96% identity in amino acid sequence with sars-cov protein. (93) moreover, the accessory proteins also have a high degree of amino acid sequence identity between these viruses: 98.1 % for nsp7 and 97.5 % for nsp8 -sequences obtained from pdb 7bv2 (2.5 å) and pdb 6nur (3.1 å) entries. (92, 95) therefore, it is reasonable to suggest that sars-cov rdrp inhibitors may also bind to the homologous enzyme from sars-cov-2, as demonstrated for remdesivir (8) (fig. 6b) , an adenosine triphosphate analog. (92, 93, 96) other potential inhibitors include clinically available drugs, such as favipiravir (9), a purine nucleic acid analog used in the treatment of influenza, and ribavirin (10), a synthetic guanosine nucleoside indicated for the treatment of hepatitis c virus (hcv) infection. (97, 98) the crucial role of rdrp and the lack of a host homolog turns this enzyme into a valuable target for anti-covs agents. (93) helicase (nsp13) is a multifunctional protein (predicted to have 596 aa in sars-cov-2) essential for sars-covs rna replication and proliferation. (74, 99) sars-cov helicase belongs to helicase superfamily 1 (sf1) and can unwind double-stranded dnas/rnas (helicase activity) in the 5'-3' direction, an energyconsuming process that is driven by the hydrolysis of nucleosides triphosphate (ntpase activity). (57, 100, 101) its structure contains five domains arranged in a triangular pyramid-shape: the triangular base is composed by two "reca-like" (1a and 2a) and 1b domains whereas the zinc binding domain (zbd), in the n-terminal, and the stalk domain are oriented towards the apex. the zbd and 1b domains are connected by the stalk domain ( fig. 7) . (101) the same domain arrangement is predicted to occur in sars-cov-2 enzyme. (74) sars-cov helicase has been explored as a potential target for drugs. (99, 100, 102) recently, some efforts have also been made to predict inhibitors for sars-cov-2 helicase. (74, 103, 104, 105) they include drugs used in the clinics to treat acquired immunodeficiency syndrome (aids), such as vapreotide (11) (somatostatin analog) and atazanavir (12) (hiv protease inhibitor), as well anti-hcv protease inhibitors [e.g., daclatasvir (13) ] and bismuth salts [e.g., bismuth potassium citrate (14) , a compound used in clinical treatment of gastrointestinal diseases]. (103, 104, 106) sars-covs replication also requires other enzymatic activities including (guanine-n7)-methyltransferase (n7-mtase), 2'-o-methyltransferase (2'-omtase) and exoribonuclease (exon). (57, 107, 108) the n7-mtase domain at the c-terminus of nsp14 is responsible for adding a methyl group in the n7 position of rna 5'-guanosine forming the 5'-cap structure of viral rnas. (108) additionally, nsp14 also has a catalytic domain in its nterminus with 3'-5' exon activity, which participates in rna proofreading mechanism (fig. 8 ). (57) 2'-omtase (nsp16) catalyses the methylation reaction at the ribose 2'-o position of the first and second nucleotide of the mrnas and it is one of the sars-cov-2 proteins whose 3d structure has already been resolved (pdb: 6w61, 2.0 å) (fig. 9 ). (109) both nsp14 and 16 use nsp10 as a cofactor to enhance their 3'-5' exon and 2'-omtase activities, respectively. (110) together, these proteins are responsible for inducing rna modifications that are essential for its stability and translation as well to avoid the activation of host immune response. (57, 107, 109) thus, nsp14 and 16 have been considered as potential targets for anti-sars agents that may affect their functions in a direct or indirect way (e.g., by blocking nsp 10 binding). (108,109,110,111,112 ,113,114,115) some compounds have been regarded as potential inhibitors of sars-cov-2 methyl transferases, such as adenine dinucleoside s-adenosylmethionine analogs [e.g., dinucleoside 13 (15) ], predicted for sars-cov n7-mtase activity, and some clinically available drugs [e.g., raltegravir (16) -a hiv integrase inhibitor], predicted for sars-cov-2 2'-omtase activity. (112, 115) chemical structures for compounds 15-29 are shown in fig. 10 . spike protein (s) is a viral type i transmembrane glycoprotein (~180-200 kda) responsible for covs interaction and invasion of host cells. (59, 64) this protein is synthesised as a monomer and suffers post-translational modifications in the er before becoming a trimeric glycosylated protein. (59, 116) its structure is divided into three main domains: an extracellular, a transmembrane and a short intracellular domain (fig. 11 ). (117) the former protrudes from the surface of the virus particle creating a crown-like halo and contains two functional subunits: s1 (bulbous shape), which binds to cellular receptors (e.g., ace2), and s2 (stalk shape) that promotes the fusion between cell and virus membranes. (59) in turn, s1 subunit has two domains: a n-terminal and a c-terminal domain. the latter serves as a rbd for sars-covs being responsible for ace2 recognition and binding. (59, 64, 117) apart from s1 and s2, sars-cov-2 s protein also has a ganglioside-binding subdomain at the tip of n-terminal domain, which may allow this protein to interact with gangliosides on cells' surface. in theory, this domain could facilitate virus attachment to the cell and facilitate the contact with ace2, being a potential site for drug interference. (118) fig. 9: ribbon representation and vdw surface of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 2'-o-methyltransferase 3d structure (pdb 6w61, 2.0 å, nsp16, pink) in complex with its nsp10 cofactor (light blue). the zinc and chloride ions are represented as gray and green spheres, respectively. image created using maestro, release 2020-1 (maestro, schrödinger, llc, new york, ny, 2020). covs s proteins require proteolytic priming or cleavage to become fusion competent. (59) this is achieved by host proteases (e.g., tmprss2, cathepsin l, furins) which cleave the s2 subunit of s protein at two main positions: s1/s2 interface and s2'. the latter is located immediately upstream of the fusion peptide (fp), the fusion functional element of s2. contrary to sars-cov, sars-cov-2 s2 subunit also has an additional cleavage site for furin proteases in the s1/s2 region, something that also occurs in mers-cov. (60) it is still unclear its role in sars-cov-2 infection, but it is speculated that the ubiquitous expression of furins may increase cell and tissue tropism of sars-cov-2 in comparison to sars-cov, as well altering its transmissibility and pathogenicity. (60, 117) taken together, host proteases represent potential targets for anti-sars-cov-2 drugs, and thus some of them will be discussed in the following topics. (60, 64, 119) covs e proteins are small integral membrane polypeptides (76 -109 aa) encoded by subgenomic rnas. in sars-cov, they are found in virions, but also in large amounts in the ergic where they participate in virus budding and trafficking. their structures are divided into three main domains: n-terminal, transmembrane (tmd) and c-terminal domain. tmd is able to form pentameric α-helical bundles creating ion conductive pores in membranes. (120, 121, 122) the ion channel (ic) activity of e protein has been proposed to alter ion homeostasis, as well induce inflammatory response, which may lead to pulmonary damage. (123, 124) thus, the use of inhibitors of ic activity may represent a possible therapeutic strategy for covs-related diseases, including covid-19. ( this can be exemplified by the fda-approved drugs gliclazide (17), a sulfonylurea administered to non-insulin-dependent diabetes mellitus patients, and memantine (18) , an n-methyl-d-aspartate receptor antagonist used in the management of alzheimer's disease, which have shown ic inhibitory activity in bacteria expressing sars-cov-2 e proteins. (126) host cell targets -a key step in sars-covs infection is the attachment of s protein to host angiotensinconverting enzyme 2 (ace2), a membrane-bound carboxypeptidase (family: m2, clan: ma). (25, 65, 69) this enzyme catalyses the hydrolysis of angiotensin i and angiotensin ii into angiotensin-(1-9) and angiotensin-(1-7) peptides, respectively. (127) ace2 is expressed in the upper respiratory system, type i and ii alveolar epithelial cells in the lungs, heart, endothelial cells, kidney tubular epithelium and other tissues. (128) its role as a functional receptor of sars-cov-2 s protein in host cells makes this protein a potential drug target to treat covid-19. there are several therapeutic strategies targeting ace2 which include: vaccines based on s protein, exogenous administration of a soluble form of ace2, and administration of small molecules [e.g., arbidol (19) , an anti-influenza drug] or antibodies (e.g., sti-1499, an anti-spike antibody) to block the interaction of ace2 with s protein. (66, 129, 130) additionally, compounds could also have an anti-sars-covs activity by disturbing ace2 glycosylation, as proposed for chloroquine (20) . (131) renin-angiotensin-aldosterone system (raas) inhibitors, such as ace inhibitors [ace-i, e.g., captopril (21) ] and angiotensin receptor blockers [arb, e.g., losartan (22) ], are commonly used in clinics to treat hypertension and cardiovascular/renal diseases. (132, 133) recently, some researchers have debated over the administration of these drugs to patients with known or suspected covid-19. (133, 134, 135, 136) the main reason for this discussion is that raas inhibitors may induce ace2 expression, which, in theory, could increase the severity of the infection. (133, 136) nonetheless, evidence suggests that these drugs may protect patients from lung injury by suppressing angiotensin ii signaling mediated by angiotensin receptor 1 (at1r). (135, 136, 137) further investigation is still required to determine whether ace-i and arb have a beneficial or deleterious role in covid-19 treatment. (134) in order to become fusion-competent, sar-covs s proteins must be cleaved by host proteases. in sars-cov-2, this process appears to be mediated primarily by tmprss2 in the plasma membrane. (65) tmprss2 (transmembrane protease, serine 2) is a transmembrane protein (predicted to have 492 aa) from the serine protease class (family: s1 clan: pa). (69, 138) it is highly expressed in the epithelial cells of the prostate, and relatively less in lungs, colon, liver, kidney, and pancreas. its physiological function is still unclear. (138) tmprss2 has a major role in sars-cov-2 cell entry and replication, and thus represents an interesting therapeutic target since its inhibitors could potentially block virus infection in its initial stages. (65, 138) potential tmprss2 blockers include some serine protease inhibitors [e.g., camostat mesylate (23)], commercially available compounds [e. g., zinc64606047 (24), 3-[[5-(3-fluorophenyl)pyrimidin-2-yl]amino~(n)-(4-methyl phenyl)benzamide]] and natural products [e.g., withanone (25) ]. (65, 139, 140, 141) in the absence of exogenous or membrane-bound proteases (e g. tmprss2) to promote sars-covs infection on cells' surface, the virus can also be internalised via endocytosis, also known as "late pathway". (59) this process comprises several factors that operate in a sequential and partially overlapping fashion. (142) endocytosis initiates with the recruitment of endocytic coat proteins (e. g., clathrins) from the cytosol to gather on the inner leaflet of the plasma membrane. then, protein-coated pits bud into vesicles originated from plasma membranes and fuse with each other or with pre-existing early endosomes. (142, 143) gradually, early endosomes mature to late endosomes which have an acid environment (approx. ph 5.5), activating endosomal cathepsin l cysteine proteases (family: c1, clan: ca). (59, 69, 143) these enzymes are responsible for triggering the fusion activity of s protein and subsequent insertion of viral sars-cov grna into the cytosol, especially in cells with lower expression of tmprss2. (64, 65) some key elements of endocytosis have been explored as potential targets for anti-sars-cov drugs, such as agents that increase endosomal ph (avoiding cathepsin l activation) [e.g., chloroquine (20) and hydroxychloroquine (26) ] and cathepsin l inhibitors [e.g., teicoplanin (27) , a glycopeptide antibiotic]. (49, 131, 144) cytokines are short-lived small size proteins (15-20 kda) that exert an important role in autocrine, paracrine, and endocrine signaling controlling the development and function of several immune and nonimmune cells. (145, 146) they are classified in families based on certain properties of their receptor complexes, such as their structure, specificity and composition. (145) one example is interleukin (il)-6 family which includes several cytokines, such as il-6, that use the common signaling receptor subunit glycoprotein 130 kda (gp130). il-6 signaling requires the binding of il-6 to il-6 receptor (il-6r) which consists of a soluble il-6 binding domain (cd126) and membrane-bound gp130. il-6 acts as the main inducer of fever, inflammation and of hepatic acute phase proteins. therefore, il-6/il-6r antagonists have a therapeutic effect in inflammatory diseases. (145, 146) their use in covid-19 treatment may be beneficial to avoid the "cytokines storm" syndrome that some patients with the severe form of the disease may develop. (147) this deleterious event is caused by an amplified immune response and cytokine release that can damage the organs, including the lungs. (66) tocilizumab and sarilumab are two examples of humanised monoclonal antibodies that act as il-6r antagonists which are currently under clinical trials for covid-19 treatment. (148) other targets -apart from sars-covs infection, some relevant molecular targets of other viral diseases and host metabolism have also been investigated in covid-19 drug discovery, such as viral neuraminidases and the dpp4 cell receptor. neuraminidases (na) or sialidases, are glycoside hydrolases (family: gh34) largely found attached to the envelope of influenza viruses. (149, 150) they catalyse the hydrolysis of glycosidic bonds between sialic acid and adjacent sugar residues (ec 3.2.1.18) in glycoproteins, glycolipids and oligosaccharides. (149) na is mainly responsible for cleaving sialic acid acids from cell receptors and on carbohydrate side chains of nascent virion, facilitating its release from infected cells. (150) its critical role in virus infection and proliferation has been exploited by na inhibitors [e.g., oseltamivir (28) ] which are administered in clinics to combat influenza infections. apparently, this therapeutic strategy was employed at the beginning of co-vid-19 outbreak during the peak of influenza season in china when the etiologic agent of this disease was yet unknown. so far, there is no evidence that na inhibitors may have a role in covid-19 management. (66) dipeptidyl peptidase-4 (ddp4), also known as adenosine deaminase complexing protein 2 or t-cell activation antigen cd26, is a type ii transmembrane glycoprotein of 766 aa largely expressed in many tissues (e.g. lungs and immune cells). (151, 152) ddp4 belongs to serine protease group (family: s9, clan: sc) and catalyses the hydrolysis of n-terminal dipeptide, xaa-yaa-|-zaa-, (preferentially when yaa is a proline, as long as zaa is neither proline or hydroxyproline), from a variety of peptide substrates (ec 3.4.14.5), such as chemokines, neuropeptides, vasoactive peptides and growth factors. (69, 71, 152) therefore, it participates in many physiologic and pathological processes, including glucose/insulin metabolism and immune/inflammatory response. (153) in addition, dpp4 acts as the functional cell receptor of mers-cov s protein, helping virus entry into the cell. theoretically, this role may also be played in sars-cov-2 infection, as predicted by computational analysis. (154) recently, there has been a discussion about the use of ddp4 inhibitors, such as sitagliptin (29) (anti-diabetic drug), in the treatment of covid-19. (152, 153, 155, 156) in principle, these inhibitors could be beneficial for type 2 diabetes patients (or even without diabetes) with covid-19 since it could potentially block virus cell entry/replication, as well suppress inflammatory response. (152, 155) however, there is not enough evidence yet to prove this hypothesis. (152, 153, 156) computational approaches -the sars-cov-2 pandemic is creating a fertile ground for computational approaches to identify viable therapeutic options in the short-term, with the number of published studies growing rapidly. (157, 158) classical target-and ligand-based strategies (e.g., molecular docking and similarity analysis), are being applied to fda-approved drugs and compounds in clinical trials to explore possible interactions with viral proteins. (75, 159, 160) artificial intelligence (ai) methods are also being extensively applied to large molecule databases to find existing drugs that could be repurposed based on previously reported antiviral activities. (103, 161, 162, 163) ai approaches can even explore hidden connections between drugs, human and viral targets to find novel bioactivities and even combinations of drugs. (52, 161, 164) these studies emphasize how important computational methods are in modern drug discovery to analyse the huge amount of biomedical data available. table i summarises a selection of studies that suggested potential drugs for repurposing. below we will describe the main computational methods and results. smith and smith performed a large virtual high-throughput screening to identify drugs, natural products and metabolites that could disrupt the interaction between sars-cov-2 s protein and human ace2 receptor. the authors used a supercomputer called summit to conduct the simulations, which included structural modeling of the s protein:ace2 complex, generation of an ensemble of conformations for the complex and molecular docking. (160) the ensemble generation was a crucial step in this work, allowing the authors to explore different conformations of the complex and identify drugs that could interact with binding sites not easily identified in static conformations. using the gromacs molecular dynamics simulation suite, six different clusters of conformations were generated by restrained temperature replica-exchange molecular dynamics simulation (t-remd) and submitted to docking with autodock vina using the sweet-lead compound collection. (170, 171, 172) the authors explored two strategies, consisting of disrupting the s protein: ace2 complex and preventing its formation by docking on the isolated proteins. from this analysis, four compounds [pemirolast (30) , nitrofurantoin (31), isoniazid pyruvate (32) and eriodictyol (33) ] displayed potential to disrupt the s protein: ace2 interaction, and three natural compounds [cepharanthine (34) , ergoloid (35) and hypericin (36) ] showed potential to block host recognition. (160) chemical structures for compounds 30-49 are shown in fig. 12 . ge and coworkers constructed a virus-related knowledge graph (or network) by combining seven networks with information about target-drug and protein-protein interactions, molecule similarity and sequence similarity of human and viral sources to predict drugs targeting sars-cov-2. (164) well known biological and interaction databases were employed, such as drugbank, chembl, bindingdb, and uniprot . (173, 174, 175, 176) the final knowledge graph was assembled by merging the nodes and edges of the seven networks, where each node represent drugs or targets, while the edges between them are the identified relations, such as similarity (i.e., molecular and sequence similarities) and drug-target or target-target interactions. (164) a graph convolutional network (gcn) was used to learn and extract the hidden information on the nodes and edges of the knowledge-graph, allowing the authors to access novel drug-target and target-target interactions and find molecules that could be repurposed to sars-cov-2. (164) gcn's are powerful neural networks that can access the rich information within the nodes of a graph and return insights about possible relations (e.g., potential drugs and targets interactions), representing a valuable tool in the modern drug discovery scenario, where massive biological data are available in databases such as drugbank and chembl. (177, 178) the final knowledge-graph was then mined to gather an initial list of drugs, which was further refined by extracting previous evidence of antiviral activity from pubmed using a deep learning method called biomedical entity relation extraction (bere) and manual inspection. (179) in a subsequent step, the authors elaborated a final list of drug candidates using transcriptome analysis of ten sars-cov patients. the poly-adp-ribose polymerase 1 (parp1) inhibitor, mefuparib hydrochloride (cvl218) (37) , currently in phase i clinical trials, was identified as a potential drug for repurposing. in vitro and in vivo validation showed promising inhibition and safety profiles. concretely, compared to arbidol (19) , one of the standard treatments for covid-19 in china, cvl218 showed higher antiviral efficacy and higher concentration in lungs of rats. (164) the authors also found that cvl218 significantly inhibited the production of il-6 induced by the cpg oligodeoxynucleotide 1826 (cpg-odn1826) in peripheral blood mononuclear cells (pmbc) indicating it might be an alternative to treat the inflammation caused by sars-cov-2. furthermore, cvl218 showed favorable pharmacokinetics and toxicity profiles in rats and monkey models. (164) this integration of artificial intelligence with wet-lab experiments highlights the importance of in silico methods to mine baricitinib (39) rheumatoid arthritis human ap2-associated protein kinase 1 (aak1) knowledge-graph a pilot trial indicated that baricitinib (39) improved clinical parameters (e.g., cough and dyspnea) in a small group of patients. c (161) toremifene (40) (41) and mercaptopurine (42) ] and angiotensin receptor blockers [irbesartan (43) ]. d (52) a: cepharanthin (34) has been shown to decrease the expression levels of s-protein in mrc-5 cells infected with human coronavirus oc43 (hcov-oc43). (165) flavonoids have been described as inhibitors of m pro and ligands of the s-protein of sars-cov, which could give insights on possible antiviral activities of eriodictyol (33), hypericin (36) and other flavonoids on sars-cov-2. (166, 167) b: a recent study showed that atazanavir (12) inhibited m pro in vitro with greater potency than lopinavir (1). (168) c: despite the small size (12 patients) and open label, non-randomised format, baricitinib (39) showed potential safety when used in combination with lopinavir (1) / ritonavir (2). (169) as large amounts of data from databases and accelerate the discovery of potential drugs for the covid-19 pandemic. beck et al. used a natural language processing (nlp)-based approach to estimate the binding affinity of 3,410 fda-approved drugs against potential targets of sars-cov-2, including 3cl pro , s protein, rdrp, helicase, endornase, 3'-to-5'-exonuclease and 2'-o-ribose methyltransferase. (103) the premise of their approach is analogous to understanding text in different languages, for instance by learning the semantic relationships of words to execute a task, such as predicting the most probable word in a text or the sentiment expressed by it. (180, 181, 182) their model, called molecular transformerdrug target interaction (mt-dti) was trained on 1 billion smiles strings and the fasta sequence of target proteins, which bypass the need for 3d structures (e.g., x-ray) of protein-target complexes. (183) the pre-training approach allows the model to be used for other related tasks without the need to train from scratch, which is especially important when not enough data is available, which is the case for sars-cov-2 inhibitors. in addition, this approach is able to transfer more general features learned by the model, making it extremely flexible to deal with related tasks. (184, 185) therefore, the task consisted of training the model to understand the chemical structure of small compounds and protein targets. the authors found that atazanavir (12), remdesivir (8) and efavirenz (38) are potential inhibitors of m pro , while atazanavir (12) also yielded nanomolar predicted binding affinity for rdrp, helicase, endornase, 3'-to-5'-exonuclease and 2'-o-ribose methyltransferase. (103) it is important to highlight that although these drugs were predicted as nanomolar inhibitors, experimental confirmation is essential to validate the computational analysis. researchers at the uk-based company benevolen-tai (https://www.benevolent.com/) analysed medical data from different sources to create a knowledge graph of biomedical information, showing possible drug-target interactions on its nodes and edges (similar to the previously mentioned approach adopted by ge et al.) to explore new strategies for sars-cov-2. (161, 164) among the drugs identified by mining the hidden information within the graph, there were 378 inhibitors of the p2associated protein kinase 1 (aak1), a regulator of viral endocytosis in at2 alveolar epithelial cells. (161) inhibitors of aak1 could then potentially block viral entry into alveolar cells. however, the authors argued that only one of these drugs, baricitinib (39), a janus kinase inhibitor, has an acceptable safety profile. in addition, the low therapeutic dosing of baricitinib (39) (2mg or 4mg daily) makes it a promising drug for repurposing. (161) baricitinib (39) will be further discussed below. zhou et al. developed a network-based approach to identify potential repurposable drugs based on the interactions between their human targets and coronavirusassociated proteins. (52) the premise is that protein targets that are associated with viral infections are part of a subnetwork of the human protein-protein interaction network. the targets of a repurposable drug should be in or close in proximity to this subnetwork. the authors identified many potential targets, such as poly [adp-ribose] polymerase 1 (parp-1), glycogen synthase kinase 3 (gsk-3), and also biological pathways that could be explored, such as nf-kappa b signaling. (52) the most interesting drugs selected by the authors are from a diverse set, including toremifene (40) (selective estrogen receptor modulator), sirolimus (41) (immunosuppressant), mercaptopurine (42) (antineoplastic) and irbesartan (43) (antihypertensive). an interesting finding was that the selected molecules and targets have reported links to viral infection and some have been used for treatment of coronavirus-related disease, such as emodin (44) in sars-cov, and mercaptopurine (42) and toremifene (40) in sars-cov and mers-cov. (52) in addition to identifying repurposable drugs, possible synergistic combinations between them were also suggested by the network, such as sirolimus (41) and dactinomycin (45) , and mercaptopurine (42) and melatonin (46) . (52) in vitro and in vivo studies -genome and replication kinetics of sars-cov, mers-cov and sars-cov-2 viruses are very similar, suggesting that these diseases could be treated by a single drug that modulates the activity of a common target or mechanism among them. (52, 186) so far, no clinically available antiviral drugs have been developed for any of these three viruses. (187) this fact demonstrates that we did not learn a suitable strategy for treatment from these two prior diseases, and we are not yet prepared to deal with this new coronavirus epidemic, regarding therapeutic options. (188) experimental target validation (e.g., by genetic and proteomic approaches), cellular in vitro and in vivo animal models of sars-cov-2 infection, are pivotal for the discovery of molecular pathways involved in pathogenesis, supporting the discovery of new drugs. (189, 190) sars-cov-2 genome encodes less than 30 proteins that can be explored for generating new avenues for further research. heterologous viral protein expression could be used in a myriad of structural and functional targetbased biochemical studies. (191) these assays do not need to be performed in biosafety level 3 laboratory, democratising the early research on searching for a new drug candidate for covid-19 treatment. (192) genome-wide expression studies, nucleotide sequences, protein structures and associated sequences are available online providing a foundation for preclinical drug discovery and development research against sars-cov-2 (available from: https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/). it is good to point out that many drugs show in vitro effect (e.g., target and/or cellular assays) that may not last in vivo, in animal models, including humans. an ideal animal model of covid-19 should reflect the clinical signs, viral replication and pathology reported in humans. (193) in vivo animal infection experiments with sars-cov-2 require infrastructure of a biosafety level 3 laboratory, restricting scientific research to well-equipped research groups. animal models are indispensable, because they could identify toxic hits or molecules that could enhance covid-19 pathogenicity. table ii lists some compounds that have the potential to be clinically tested and that will be discussed in more details below in this topic. remdesivir (8) , a nucleotide analog pro-drug developed by gilead sciences inc. to fight ebola, acts on viral replication by inhibiting rna polymerase. (96) this molecule was also active against sars and mers viruses. (200, 201, 202) wang et al. demonstrated reduced viral copy numbers in cell culture supernatant of vero e6 cells infected with ncov-2019betacov/wuhan/wiv04/20192, in the presence of varying concentrations of remdesivir (8) . (194) this compound blocked virus infection at lowmicromolar concentration (ec 50 = 0.77 μm) and showed a high selectivity index (> 100). the authors also disclosed that remdesivir (8) inhibited virus infection efficiently in human liver cancer huh-7 cells. chloroquine (cq; 20) and its less toxic derivative, hydroxychloroquine (hcq; 26) , are used to treat malaria and lupus/rheumatoid arthritis, respectively. both drugs have already been described as possible antivirals. (195, 203, 204) cq (20) and derivatives probably act by decreasing acidity in endosomes and lysosomes, intervening on glycosylation of cellular virus receptors and modulating host immunological activity. (118, 205) studies in cultures of vero e6 cells have suggested that hcq (26) can affect the virus sars-cov-2 in both entry and post entry stages at host cells. (194) this molecule presented an ec 50 (196) the authors also performed in vitro physiologically based pharmacokinetic models for both drugs, separately. interestingly, when comparing toxicity in five animal models, mcchesney et al. demonstrated that hcq (26) is two to three times less toxic than cq (20) itself. (207) it is also good to point out that chloroquine (20) has shown in vitro activity against many different viruses, but no significant beneficial effect on animal models. (208) the association ritonavir (2)/lopinavir (1) is used together with other antiretrovirals for the treatment of human immunodeficiency virus since the beginning of the century. (209) ritonavir (2) is a potent cyp3a inhibitor therefore inhibiting the metabolism of lopinavir (1), increasing its plasma levels. both are reported as peptidomimetic molecules that inhibit hiv-1 protease activity in a competitive manner. (210) the m pro plays a major role in homeostasis of viral polyproteins essential for viral function and replication, being considered a validated drug target. (211) this combination may be useful against (194) chloroquine (20) (194, 195, 196) ritonavir (2) + lopinavir (1) lopinavir (1) nd (197) nitazoxanide (47) broad-spectrum antiparasitic and antiviral still needs confirmation 2.1 μm 16.8 (194) ivermectin (48) broad-spectrum antiparasitic still needs confirmation 2 μm nd (198) teicoplanin (27) antibiotic for gram-positive bacteria still needs confirmation 1.7 μm nd (199) sars-cov-2 virus by acting on its main protease, an enzyme essential in processing polyproteins translated from viral rna. (212) choy et al. demonstrated that it inhibits sars-cov-2 replication in vero e6 cells with ec 50 value of 26.6 μm. (197) these results corroborate with the study of de wilde et al. that demonstrated antiviral in vitro effect of lopinavir (1), but not ritonavir (2), against sars-cov, mers-cov, and hcov-229e, with mean ec 50 varying from 6.6 to 17.1 μm. (213) other compounds were also tested against sars-cov-2 in vitro. nitazoxanide (47), a broad-spectrum antiparasitic and antiviral, inhibits a low-micromolar range with an ec 50 of 2.1 μm but has a selective index of only 16.8. (194) ivermectin (48), another broad spectrum antiparasitic agent also demonstrated in vitro anti-sars-cov-2 activity. its ec 50 was determined to be 2 μm when added to vero-hslam cells 2 h post-infection and measuring viral rna at 48h post-infection. (198) teicoplanin (27) , an antibiotic used against gram-positive bacterial infections, was found to be active in vitro against sars-cov-2 with an ec 50 value of 1.7 μm, but in vivo efficacy still needs to be determined. (199) a robust preclinical drug discovery pipeline comprising in vitro, and in vivo models of sars-cov-2 infection is particularly important to identify new antivirals for human covid-19 treatment. this pipeline can munition clinical studies with molecules that have a higher chance to become a drug, decreasing attrition rates. clinical studies -over 1452 clinical trials have been unveiled, until the publication of this manuscript, focusing on covid-19 treatment interventions (available from: https://clinicaltrials.gov/ct2/results?cond=covid-19). with a growing number of patients suffering from acute severe respiratory symptoms and hospital capacities reaching its limit, therapeutic options are urgently essential to avoid human mass deaths. drug repurposing approaches of approved (with safe and effectiveness proven) and unapproved molecules, that were promising in pre-clinical and early stages of clinical studies of sars and mers, are in vogue. with this premise, world health organization (who) organised a simplified dynamic platform comparing the effectiveness of treatment strategies around the globe. this clinical trial design, called solidarity, can shrink by 80% the time of clinical studies, compared with the "gold standard" double-blind, placebo-controlled trials. this strategy could overcome the uncertainty of multiple small trials that do not produce a solid base necessary to establish the relative success of arising probable treatments. on the other hand, this shorter time required reflects the compassionate characteristic of the studies, that cannot rule out placebo effects and patient severe adverse effects, including death. (214) currently, who is focusing on four most promising therapies: remdesivir (8); cq (20) or hcq (26) ; ritonavir (2)/lopinavir (1); ritonavir (2) /lopinavir (1) plus interferon-beta, an immune response modulator. (44) a selection of studies with these drugs, alone or in combination, are summarised in table iii. for other studies, the reader can refer to kupferschmidt and cohen, which summarised several clinical studies on drug repurposing for covid-19 reported so far. (44) it is worth noting that many clinical studies are beginning to be carried out in different parts of the world and their number is increasing rapidly day by day. remdesivir (8), a nucleoside analogue that acts by inhibiting rna synthesis, is already in clinical studies for treatment of covid-19. (96) this prodrug was already investigated in clinical trial testing for ebola virus with no effect but showed efficiency against different types of coronaviruses in cell culture and animal models. (200, 221) from the drugs in the solidarity trial, remdesivir (8) has the best potential to be used in clinics, having a low toxicity profile to humans. (44) this molecule was used compassionately in the first covid-19 patient diagnosed in the united states by intravenous treatment and improved patient clinical condition. (222) grein et al. reported a study in a cohort of patients hospitalised for severe covid-19 who were treated with compassionateuse remdesivir (8) , clinical improvement was observed in 68% of the patients (36 of 53). (223) there are two clinical trials at phase iii, being designed to evaluate the efficacy and safety of parenteral remdesivir (8) in mild/moderate (nct04252664) and severe (nct04257656) hospitalised adults with covid-19. (224) these trials are evaluating intravenous remdesivir (8) at a dose of 200 mg on the first day plus 100 mg once daily, for an additional 9 consecutive days. the randomised, double-blind, placebocontrolled, multicentre trials were recently suspended (nct04252664) or terminated (nct04257656) because no eligible patients for enrollment were found, due pandemic control in china. (215) the study nct04257656 showed that remdesivir (8) was not associated with statistically significant clinical benefits in severe forms of covid-19. however, the authors observed a numerical reduction in time to clinical improvement in those treated earlier, but still requires confirmation. another study, performed by beigel et al. (with the same dosage and treatment period than the previous works cited above) enrolled 1063 patients with lower respiratory tract infection, demonstrated that individuals that were treated with remdesivir (8) had a shorter time to recovery than placebo group (11 days compared with 15 days). (216) even with a small scientific ballast, due to limited information about safety and effectiveness of using remdesivir (8), u.s. food and drug administration (fda) approved its emergency use on severe covid-19. (225) the approval was based on review of the topline data on two studies that are recruiting patients, (nct04280705) and (nct04292899). other clinical studies are being conducted to address the effect of remdesivir (8) on co-vid-19 treatment (available from: https://clinicaltrials. gov/ct2/results?cond=covid-19&term=remdesivir&cn try=&state=&city=&dist=). other phase iii clinical trials evaluated for co-vid-19 treatment included cq (20)/hcq (26) . these studies were endorsed by promising in vitro data that suggested an impairment of viral replication by acting on endosome-mediated viral entry or later phases of viral replication. (194, 226) both molecules have a well-studied toxicity profile being used for more than half-century as antimalarials, but in some cases can induce heart side (8) was not associated with clinical benefits in severe forms. however, an observed numerical reduction in time to clinical improvement in patients treated earlier, but still requires confirmation (215) ; nct04257656 (terminated by epidemic control in china with no eligible patients) remdesivir (8) (216) ; nct04280705 chloroquine (20) multicentre > 100 patients 500 mg per day, for 10 days compilation of various clinical studies that are in course, authors conclude that the dosage used could be sufficient (217) phase iib double-blind, randomised 81 patients 600 mg twice daily for 10 days and 450 mg twice on the first day, once daily for more 4 days higher dosage of chloroquine (20) has toxic effects and increased lethality, with any clinical benefit (218) hydroxychloroquine (26) non-randomised, non-double-blind, non-placebo-controlled 36 patients 600 mg of hydroxychloroquine (26) daily for 10 days hydroxychloroquine (26) significantly reduces viral load despite small sample size (219) ritonavir ( effects. (227) these drugs have already been examined against at least five other viruses with good initial in vitro results, but without significant effects in animal models and humans. (205) clinical trials to measure the effectiveness of cq (20) at 500 mg of chloroquine diphosphate (corresponding to 300 mg of chloroquine (20) base) per day, for ten consecutive days on the treatment of covid-19 were performed. (217) based on results for a very limited number of patients (100) , it was concluded that cq (20) presented apparent efficacy and acceptable safety against cov-id-19 associated pneumonia. borba et al. performed a double-blind, randomised clinical trial designed to assess the safety of cq (20) in two dosages: 600 mg twice daily for 10 days with an initial dose of 450 mg twice daily on the first day, decreasing to once daily for four more days. (218) the study suggests that the higher dosage should not be recommended for critically ill patients with covid-19 because of its potential side effects. it is important to note that both doses studied were above the brazilian recommended dose. a controversial study performed by gautret et al. with a small sample size demonstrated that hcq (26) treatment daily at 600 mg significantly decreases viral load in covid-19 patients and its effect is reinforced by azithromycin (az; 49) combination. (219) until this moment there is clinical robustness to support az (49) therapy in covid-19. (228) mehra et al. performed a registry analysis of 96,032 hospitalised patients regarding the use of cq (20) or hcq (26, combined or not with a macrolide) for treatment of covid-19. (229) the observational study verified that with covid-19 requiring hospitalisation a regimen containing cq (20) or hcq (26) had no evidence of benefit, but instead was associated with an increase in the risk of ventricular arrhythmias and a greater hazard for in-hospital death with covid-19. these findings suggest that these drug regimens should not be used outside of clinical trials and urgent confirmation from randomised clinical trials is needed. after the publication, the paper was retracted, which influenced who to return the studies on cq (20) and hcq (26) . (230) recently, on 17 june 2020, who stopped the hcq (26) arm of the solidarity trials again based on the recovery trials (available from: https://www.recoverytrial.net/). in this study 1542 patients were randomised to hcq (26) and compared with 3132 patients randomised to usual care alone. there was no significant difference in any of the parameters evaluated: the primary endpoint of 28-day mortality, hazard ratio and hospital stay duration or other issues. other study reported that hcq (26) did not prevent covid-19 when used as postexposure prophylaxis within 4 days after moderate or high-risk exposure. (231) robust data from well-designed control randomised clinical trials with large number of patients is still expected for concluding on the efficacy of hydroxychloroquine (26) . ritonavir (2) and lopinavir (1) were developed to target hiv-1 protease and postulated to inhibit the 3-chymotrypsin-like protease of sars and mers, being associated with improved clinical outcomes. (232) the combination has beneficial effects in marmoset monkeys infected with the mers-cov virus. (233) (1)/(2) may have clinical efficacy against sars-cov-2, as seen in the response against sars-cov. (234) in vitro sensitivity and satisfactory response in a preliminary non-randomised clinical trial of sars-cov to (1)/(2) have already been reported, encouraging its testing in sars-cov-2. (235) a total of 199 patients confirmed severe sars-cov-2 infection were randomly designated to be given either (1)/(2) (400 mg/100 mg) twice a day for 14 consecutive days plus standard care or standard care alone. (220) this first trial was not promising, no benefit was observed on clinical improvement, mortality and viral loads on severe covid-19 patients. other clinical trials are being carried out and should decide whether these drugs are useful for covid-19 treatment or not. ritonavir (2)/lopinavir (1) plus interferon-beta, a cytokine involved in inflammatory modulation, is the last bet of the solidarity megatrial. sheahan et al. demonstrated that this combination improves pulmonary function but does not reduce virus replication or severe lung pathology in mice model of mers-cov. (236) this combination also showed promising results in mers-cov infection in a nonhuman primate model. (233) the study (nct04276688) administered ritonavir (2)/lopinavir (1) 400 mg + 100 mg/ml twice daily for 14 days and interferon beta-1b 0.25 mg subcutaneous, every alternate day for 14 days. another study (nct04343768) will perform a randomised trial to verify the effects of interferon beta 1a, compared to interferon beta 1b and the base therapeutic treatment in moderate to severe covid-19. currently, there is a lot of research around the world examining drugs that can be used for the treatment of covid-19. unfortunately, until this moment no therapeutic options are promptly effectively available. most of the studies have a small number of patients with variable dose and/or duration, fact that hinders a comparison. to successfully undertake the covid-19 pandemic with new medicines, synchronised clinical trials with randomised, double-blind, placebo-controlled are still needed. infection prevention by social distancing and supportive medical care, are the only strategies to deal with this disease until this moment. the goal of this session is to consider potential obstacles for effective adoption of a repurposed drug to widespread treatment of covid-19. both patent protection and scalability of manufacturing processes are key aspects to consider while choosing the best therapeutic option to adopt when dealing with a pandemic. a patent can be defined as a government license that confers the owner the exclusivity over a new invention or medication. with the patent, the inventor is able to exclude others from making and selling the invention for a determined period of time. when it comes to medication, the market exclusivity granted by the patent generates enormous economic profit for the patent holder, once the company will have the monopoly over the product for around 20 years. once the patent term finishes, generic companies can start producing and selling the drug, increasing the market competition over the product. a strategy adopted by some companies is to maximise the term of patent of successful products, extending market exclusivity, for instance, increasing economic rewards with the invention. in order to extend patent terms, the companies can apply for new formulations, new routes of administration, new uses of the drug among other strategies. (237, 238) this is very important when considering drug repurposing, once those strategies can make it harder to patent a new method of use for the drug, compromising the legal rights of the new repurposed indication. as a consequence, the expected profit associated with repurposing can be severely affected. (39) remdesivir (8) a good example of maximising the term of patent is the drug remdesivir (8) , which was developed by the american pharmaceutical company gilead sciences, inc. in 2011. nowadays, (8) has shown promising results as a suitable repurposed drug in the treatment of the covid-19 disease. indeed, there are many ongoing clinical trials with this drug in cov-id-19 patients. (239, 240) due to the good results concerning remdesivir (8) , in january 2020, the wuhan institute of virology applied for a patent on gilead's remdesivir (8) for the treatment of coronavirus, in an attempt to protect china's economic and medical interests. however, remdesivir (8) has already 133 patents related to the coronavirus filed by gilead science in 43 countries around the globe since 2011. in fact, the company has a robust patent portfolio that includes structures of remdesivir-related compounds (family 1), the manufacturing method of the drug (family 2), the use of remdesivir (8) in treating coronaviridae infection (family 3), among other patents that claims the use of (8) for the treatment of a series of viral infections (families 3 and 4) (fig. 13) . (241) practically, this means that if the (8) is proved to be efficient for the treatment of covid-19, gilead science is the only pharmaceutical company owing the market exclusivity of the drug, at least until 2031. in fact, the company has already started to produce larger amounts of the drug and they already made improvements to optimise the drug manufacturing timeline. now, the company can produce the drug in 6 months, half the time that they used to need to get the final product. in addition, the company has been donating remdesivir (8) to ongoing clinical trials, a gesture that will not only help the trial patients, but gilead itself. (242) baricitinib (39) -in 2009, a patent assigned to incyte corporation disclosed the preparation of several active compounds as jak inhibitors, including baricitinib (39). in the same year, lilly and incyte made an agreement allowing lilly co. to manufacture and commercialise the medicine worldwide, making it lilly co. the only supplier around the globe. (243) there are two patents that protect the drug, one concerning its synthetic pathway and another disclosing the use of (39) in the treatment of rheumatoid arthritis. both of them will expire in nine years, which could open opportunities for generic drug companies to produce baricitinib (39) . nowadays, (39) has been investigated as a drug that could be used in the treatment of covid 19 patients, since its anti-inflammatory activity could minimise inflammatory compli-cations in covid 19 patients. according to the platform clinicaltrials.gov, there are 14 ongoing clinical trials to evaluate the efficacy and safety of baricitinib (39) in the treatment of covid 19. (244) if the drug succeeds, there will be a need for larger and faster production and distribution of the medicine worldwide. there are two patents disclosing a synthetic method for the preparation of (39). the first one is from 2009 and owned by incyte co. and the latest is from 2016, by lilly co. (245, 246) the main difference between them is how the central pyrazole ring is installed in the molecule. in the patent from 2009, baricitinib (39) is obtained by a convergent synthesis. the synthetic pathway starts with the protection in position 7 of 4-chloro-7h-pyrrolo[2,3d]pyrimidine (50) using 2-(trimethylsilyl)ethoxymethyl chloride (51) , affording intermediate (52) . next step is a suzuki-miyaura reaction, coupling the fused ring system to a 4-pyrazoleboronic acid pinacol ester (53), giving key intermediate (54) . parallelly, a couple of steps starting from 2-(chloromethyl)oxirane (55) lead to 1-boc-3-azetidinone (58) that reacts with diethyl cyanomethylphosphonate (59), affording key intermediate (60) . next, the key intermediates (54) and (60) react in the presence of dbu, giving (61) . then, steps involving hydrolysis of the boc, sulfonation and pyrrolopyrimidine deprotection afford (39) with an overall yield of 21 % (fig. 14) . (246) the synthetic pathway described in the patent from 2016 has only six steps in a linear approach, as a consequence, the product is obtained in a higher overall yield when compared to the synthetic pathway discussed above. intermediate (67) is obtained from azetidine-3-ol (64) in a couple of steps, including a sulfonation, an oxidation and the installment of the cyanomethylene moiety (fig. 15 ). furthermore, it is not necessary to protect any position in this sequence. additionally, the oxidation step can be performed under flow conditions. then, intermediate (67) is reacted with ester (53), affording (68) . a suzuki-miyaura reaction involving 7-boc-4-chloro-7hpyrrolo [2,3-d] pyrimidine (69) is applied, allowing the formation of the bound between the azetidinylpyrazole group and the pyrrolo[2,3-d]pyrimidine system. last step is a hydrolysis of the boc, affording baricitinib (39) with an overall yield of 50 % (fig. 15 ). (245) off-patent drugs -fortunately, a number of drugs that have been tested as possible agents in the covid-19 treatment are off-patent, meaning that generic drug companies already manufacture and commercialise the medicine, making it easier for drug repurposing. (39) the off-patent drugs that have been tested in covid-19 clinical trials include cq (20) , hcq (26), ritonavir (2) and lopinavir (1) . (239, 240) a recent research article published by hill and collaborators estimated the minimum cost of production associated with these drugs, showing that all the treatments under evaluation in current clinical trials are cheap to manufacture. however, list prices can be over 100 times higher than the costs to produce the drug. (247) if any of those drugs become approved for the treatment of covid-19, its demand will increase dramatically. therefore, it is important to consider the challenges associated with scaling up the production to meet the demand. for instance, there are few regulatory approved production facilities, and drug manufacturers rely on low-cost suppliers of raw materials in india and china, which might be scarce during a pandemic. consequently, it would be difficult to ensure short term global availability of the treatment, since the production depends on different countries. (248) those conclusions are very helpful in showing the importance of optimising the way of manufacturing the candidate drugs presented above. for this reason, recent and optimised synthetic pathways found in patents and articles for some ritonavir (2) and lopinavir (1) are discussed below, as cq (20) and hcq (26) have now been abandoned. finally, we will briefly discuss the ease of manufacturing in large scale each of the drugs we had the synthesis reviewed here. ritonavir (2) -the first disclosure of ritonavir (2) is presented in a patent from 1994, assigned to abbott laboratories. even though the patent consists of a range of markush structures, among which is found (2), its synthesis is not presented. (249) the synthetic pathway to obtain (2) was disclosed for the first time in a second-generation patent in 1995, by the same company. (250, 251) some drawbacks related to the synthesis presented by abbott include the employment of expensive condensing agents, as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc) and poor reaction yields on the first steps of the synthetic pathway, making this strategy too expensive and not suitable for scale-up production. considering the above deficiencies, a recent chinese patent concerning the synthesis of ritonavir (2) has been released. the synthetic pathway described in the patent starts with a nucleophilic addition-elimination reaction between the starting material (2-isopropylthiazol-4-yl)-nitro-methylamine (71) and n-[(2,2,2-trichloroethoxy)carbonyl]-l-valine (72), generating intermediate (73) . the intermediate is mixed with p-toluenesulfonyl chloride and triethylamine to activate the acid function, followed by the addition of reagent (74) in a one-pot procedure. the condensation allows the formation of intermediate (75) , which is submitted to acidic conditions for a boc deprotection, followed by another nucleophilic additionelimination reaction with reagent (76), thus obtaining the final product ritonavir (2) (fig. 16) . (252) when comparing both patents, it is possible to highlight important advantages present in the latest one, including the use of cheap and easily available reagents, for instance, the use of ptoluenesulfonyl chloride as an amide condensing agent, the synthetic pathway has a high yield (79% overall), low cost and it is ease to scale-up the production. lopinavir (1) -the first synthetic methods related to the synthesis of lopinavir (1) are present in a patent from 1996 owned by abbott laboratories. (249) (1) has 4 chiral centres, and the synthetic strategies reported by abbott are similar, involving the synthesis of a key intermediate amino alcohol unit, that is then connected to the appropriated side chains. some drawbacks that can be pointed out in the synthesis include the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (edc) as a condensing agent and the weak base 1-hydroxybenzotriazole, both expensive reagents, making the synthesis not suitable from the viewpoint of cost and industrial ap-plication. in addition, one of the methods described by abbott includes the synthesis of an acid chloride as an intermediate, which is very unstable, and it can be easily decomposed by humidity, making the synthesis not suitable for industrial production. the most recent patent related to the synthesis of lopinavir (1) is dated 2018 and it is from the chinese company shanghai desano pharmaceuticals co. ltd. (253) the patent is described as technical, with the aim of improving the synthesis of (1) presented in the patent from 1996, by abbott laboratories. the patent describes the synthesis of lopinavir (1) by a one-pot procedure starting with the formation of an acid chloride from (2s)-(1tetrahydropyramid-2-one)-3-methylbutanoic acid (77) followed by addition of a weak base and the reactant (78) to the reaction system. the amine function of (78) allows a nucleophilic addition-elimination reaction to occur, affording (1) after treatment with nahco 3 (fig. 17 ). therefore, it is possible to synthesise lopinavir (1) in high yield (90%) in an optimised method, which involves a few reagents, mild conditions and is performed in a one-pot procedure. synthetic scalability and cost-effectiveness -the synthetic pathways described above show how much improvement has been done concerning the preparation of these drugs in a more cost-effective way. furthermore, when comparing the drugs discussed above, it is worth pointing out which one could be the easiest one to produce when analysing the factors that influence the final cost of a synthetic pathway, such as chiral centres, cost of starting materials, number of steps and overall yield. regarding chiral centres, ritonavir (2) and lopinavir (1) have four chiral centres each, and they are not sold as racemate mixtures. moreover, to synthesise those compounds the chiral centres do not come from natural molecules, but have to be synthetically installed, which makes the chiral starting materials more expensive and enantiomeric excess has to be accessed more carefully at the end of the synthetic pathway. as a consequence, it can take longer and more expensive to obtain the final product. on the other hand, baricitinib (39) does not have any chiral centres, making the synthetic pathway easier and cheaper to perform. concerning the number of steps and overall yield, baricitinib (39) has the highest number of steps and lower overall yield when compared to ritonavir (2) and lopinavir (1) . however, one of the steps in the synthesis of baricitinib (39) can be performed under flow conditions, which is very interesting from the industrial point of view. therefore, when taking into account the most recent synthetic pathways proposed for these drugs, the synthesis of baricitinib (39) is the most cost-effective of the three of them. in conclusion -sars-cov-2 infection is a lifethreatening disease with such a high transmission rate that can surpass even the most well-structured health systems in developed high-income countries. while vaccines are the ideal solution for preventing the spread of infectious diseases like covid-19 their development cycle have intrinsic challenges and safety checking steps that require several months or years to complete their development. a similar situation is found for developing new drugs for covid-19 (as with any other disease), because of the long process involving discovery, validation and safety evaluation of new chemical entities for use in human health. in this scenario, repositioning drugs already in clinical use for treatment of covid-19 is an important shortcut, as we have discussed. data are accumulating about the molecular pathology of covid-19 as well as structural information on the viral and host proteins involved in the infection mechanism. this knowledge is essential for allowing researchers to have new insights on approved or investigational drugs that can be repurposed to treat sars-cov-2 infection. as reviewed here, over 10 targets distributed amongst distinct steps of the sars-cov-2 replication cycle or host cell mediators are currently being investigated. here, we highlighted several up-to-date examples of potential repurposable drug candidates proposed from either computational approaches or experimental trials (or their combination) at different levels of validation and stages of development. noteworthy, ai methods hold a great promise in finding occult links between drugs, human and viral targets to find novel bioactivities and even combinations of drugs. remdesivir (8) , cq (20)/hcq (26) (alone or in association with other drugs) and the association lopinavir (1)/ritonavir (2) have been the focus of most clinical studies. so far, results have been mostly disappointing with these trials, stressing the importance of carefully performed studies in patients to attest that biological activities observed in vitro actually be translated into clinical efficacy and safety. at the moment, even with limited information about safety and effectiveness, remdesivir is the only drug approved by the fda for emergency use on severe covid-19. as discussed here, a major concern with drugs for effectively fighting the pandemic is the drug's industrial production cost and its impact on final treatment cost. gilead, remdesivir's owner company, has recently signed non-exclusive voluntary licensing agreements with five generic pharmaceutical 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potently blocks the cell entry of 2019-ncov broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection potential antivirals and antiviral strategies against sars coronavirus infections chloroquine and hydroxychloroquine in covid-19 targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro animal toxicity and pharmacokinetics of hydroxychloroquine sulfate of chloroquine and covid-19. antiviral res hiv protease inhibitors: a review of molecular selectivity and toxicity anti-hiv drugs: 25 compounds approved within 25 years after the discovery of hiv 3clpro inhibitors as a potential therapeutic option for covid-19: available evidence and ongoing clinical trials computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treating covid-19 -off-label drug use, compassionate use, and randomized clinical trials during pandemics remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for the treatment of covid-19 -preliminary report breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 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treatment of covid-19: a multinational registry analysis a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 coronaviruses -drug discovery and therapeutic options treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset molecular dynamic simulations analysis of ritronavir and lopinavir as sars-cov 3clpro inhibitors role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov patent protection strategies data exclusivity, and the development of new drugs. ssrn [internet coronavirus puts drug repurposing on the fast track research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases coronavirus patents: china files for remdesivir patent, but gilead sciences will still come out a winner fierce pharma gilead turbocharges production of covid-19 hopeful remdesivir lilly and incyte announce collaboration for development and commercialization of oral anti-inflammatory and autoimmune therapies search results: baricitinib | covid-19 processes and intermediates for the preparation of {1(ethylsulfonyl)-3 inventors; incyte corporation assignee 2009. azetidine and cyclobutane derivatives as jak inhibitors. united states patent us 2009/0233903 a1 minimum costs to manufacture new treatments for covid-19 dozens of coronavirus drugs are in development -what happens next? inventors; abbott laboratories assignee 1995. process for the preparations of a substituted 2,5-diamino-3-hydroxyhexane discovery of ritonavir, a potent inhibitor of hiv protease with high oral bioavailability and clinical efficacy zhou z, inventors; guizhou yongnuo feite bio pharmacy co ltd assignee 2019. preparation method of ritonavir. chinese patent cn 109369562a inventors; yancheng desano pharmaceutical co ltd assignee 2018. method used for preparing lopinavir using one-pot method. chinese patent cn 108218791a all authors contributed to writing, reviewing and editing the manuscript. mrs, tcse and rfd contributed equally to this manuscript; fps-jr performed the manuscript conceptualisation and final review. the authors declare no conflict of interest concerning this manuscript. key: cord-342569-ja96xfns authors: azer, samy a. title: covid-19: pathophysiology, diagnosis, complications and investigational therapeutics date: 2020-08-05 journal: new microbes new infect doi: 10.1016/j.nmni.2020.100738 sha: doc_id: 342569 cord_uid: ja96xfns abstract the novel coronavirus (covid-19) outbreak started early in december 2019 in the hubei province and its capital wuhan of the people’s republic of china and caused a global pandemic. the number of patients confined to this disease has exceeded nine million in more than 215 countries, and the number who died is over 480,600 (up to 25 june 2020). coronaviruses were identified in the 1960s and recently identified to cause the middle east respiratory syndrome (mers-cov) in 2012 and severe acute respiratory syndrome (sars) in 2003. the current severe acute respiratory syndrome coronavirus-2 (sars-cov-2) is the most recently identified. patients with covid-19 may be asymptomatic. typical symptoms including fever, dry cough, and shortness of breath. gastrointestinal symptoms such as nausea, vomiting, abdominal pain and diarrhea, have been reported—neurologically related symptoms, particularly anosmia, hyposmia, and dysgeusia, have also been reported. physical examination may reveal a fever in over 44% of patients (and could be documented in over 88% of patients after admission), increased respiratory rate, acute respiratory disease, and maybe decreased consciousness, agitation, and confusion. this article aims at presenting an up-to-date review on the pathogenesis, diagnosis and complications of covid-19 infection. currently, no therapeutics have been found to be effective. investigational therapeutics are briefly discussed. on 31 december 2019, the chinese authorities reported to the world health organisation (who) an emerging of a novice coronavirus, currently the virus is known as sars-cov-2 and the disease name is coronavirus-19 disease (covid19) , that has emerged in patients from wuhan city, hubel province [1] . this virus has a higher degree of lethality than other endemic viruses, and is more lethal to humans compared to earlier emerging outbreaks of single-stranded, positive-sense rna genomes and it is roughly 80% identical with other coronaviruses at a nucleotide level. the virus closely related (share 90% of nucleotide structure) to sars-cov-2 is ratg13-2013 which was identified in bats [2] . the complete genome of the severe acute respiratory syndrome coronavirus 2 isolated from wuhan hu-1 is available at (https://www.ncbi.nlm. nih.gov/nuccore/nc_045512). genetic epidemiology of hcv-19 and submitted data since december 2019 are available from gisaid database (https ://www.gisaid.org/). the sars-cov-2 is composed of at least 11 orfs (open reading frames) with the full length of 29,903 bp. four major structural protein-coding genes have been identified in the coronaviruses -spike protein (s), envelop protein (e), membrane protein (m), and nucleocapsid protein (n) [3] . the spike protein of sars-cov-2 utilizes angiotensin-converting enzyme (ace2) as its cell surface receptor and utilization influences the tropism of the virus. the covid-19 infects people of all ages. however, there are two main groups at a higher risk of developing severe disease including older people and people with underlying comorbidities such as diabetes mellitus, hypertension, cardiorespiratory disorders, chronic liver diseases, and renal failure. patients with cancer and those on immunosuppressive medication as well as pregnant women are also be at a higher risk of developing severe disease when infected. [4] . the transmission of infection is mainly person-to-person through respiratory droplets. fecal oral route is possible. the presence of the virus has been confirmed in sputum, pharyngeal swabs and feces [5] . vertical transmission of sars-cov-2 has been reported [6] and confirmed by positive nasopharyngeal swab for covid-19. the median incubation period of covid-19 is 5.2 days (most patients will develop symptoms in 11.5 to 15.5 days). therefore, it has been recommended to quarantine those exposed to infection (post-exposure) for 14 days. the sars-co-2 infection enters the host cells through the s spike protein by binding to ace2 for internalisation, and aided by tmprss2 protease. the high infectivity of the virus is related to mutations in the receptor binding domain and acquisition of a furan cleavage site in the s spike protein. the virus interaction with ace2 may down regulate the antiinflammatory function and heightened angiotensin ii effects in predisposed patients [7] . with the challenge we face with covid-19, there has been advocate for the use and the cessation of at1r blockers and ace inhibitors during the treatment of covid-19 in hypertensive j o u r n a l p r e -p r o o f patients. currently the recommendation of the council on hypertension of the european society of cardiology is that patients should continue their antihypertensive treatment with no changes because we do not have evidence supporting its cessation [8] . however, further research is needed to give more evidence to these questions. the invasion of the virus to the lung cells, myocytes and endothelial cells of the vascular system results in inflammatory changes including oedema, degeneration and necrotic changes. these changes are mainly related to pro-inflammatory cytokines including interleukins il-6, il-10, and tnf-oe, granulocyte colony stimulating factor, monocyte chemoattractant protein i, macrophage inflammatory protein 1 oe, and increased expression of programmed cell death marker-1 (pd-1) and t cell immunoglobulin and mucin domain 3 (tim-3) [9] . these changes contribute to lung injury pathogenesis, hypoxia-related myocyte injury, body immune response, and increased damage of myocardial cells, intestinal and cardiopulmonary changes. infection with sars-cov-2 has been also shown to cause hypoxaemia. these changes lead to the accumulation of oxygen free radicals, changes in intracellular ph, accumulation of lactic acid, electrolyte changes and further cellular damage. the respiratory system is the primary system affected in sars-cov-2, and multiple infiltrates of both lungs may be present. real-time reverse transcription polymerase (rt-qpcr) amplification of sars-cov-2 virus nucleic acid of nasopharyngeal swabs or sputum is needed to confirm the diagnosis. however, the test may be negative in early days of presentation. clinical picture, including shortness of breath, increased respiratory rate, decreased oxygen saturation, and raised c-reactive protein are non -specific. other tests such as igg and igm antibodies against sars-cov-2, cd4+ and cd8+ should be ordered. both j o u r n a l p r e -p r o o f cd4+ and cd8+ are substantially lowered in sara-cov-2. the pathology of the lungs shows microscopic bilateral diffuse alveolar damages, cellular fibromyxoid infiltrates, interstitial mononuclear inflammatory infiltrates with lymphocytes domination [10] . the cardiovascular system is usually involved in covid-19 infection. biomarkers such as elevated highly sensitive troponin-t, natriuretic peptides and il-6 are prognostic and their progressive rise are associated with poor outcomes. the inflammation of the vascular system results in these changes 1) diffuse microangiopathic thrombi, 2) inflammation of cardiac muscle (myocarditis), 3) cardiac arrhythmias, heart failure, acute coronary syndrome. these cardiovascular complications may cause death [11, 12] . the lymphocytopenia observed during the infection, potentially involves the cd4+ and some cd8+ t cells. these changes disturb the innate and acquired immune responses causing delayed viral clearance, and hyperstimulated macrophages and neutrophils. the notch signalling is known to be a major regulator of cardiovascular function and it is also implicated in several biological processes mediating viral infections. recently it was debated that targeting the notch signalling to prevent sars-cov-2 infection and interfering with the progression of covid-19associated heart and lungs disease pathogenesis [13] . the reported gastrointestinal manifestations of covid-19 include diarrhea, nausea, vomiting and abdominal pain. studies indicated that sars-cov-2 rna been isolated stool specimen and swabs taken from the anus/rectum [14] . the ace2 has been found expressed in the epithelial cells of the gastrointestinal tract suggesting that the virus entry through the ace2 receptors and its replication causing inflammatory changes and patient's symptoms. the sars-cov-2 also causes liver injury manifested by elevated serum alanine aminotransferase (alt) and aspartate aminotransferase (ast) [15] . mild elevation of serum bilirubin and j o u r n a l p r e -p r o o f gamma-glutamyl transpeptidase (ggt) have also been reported in some patients with covid-19 infection [16] . in most cases the liver injury was transient and mild. however, severe liver dysfunction/injury has been reported in patients with severe disease. high levels of alt of over 7500 u/l has been reported in a chinese study [17] . microscopically, microvesicular steatosis of the liver and mild lobular injury has been found in covid-19 infected patents [16] . it is not clear whether the observed sars-cov-2-associated liver injury is cause by direct viral injury or related to hepatoxic drugs, coexisting systemic inflammatory changes, sepsis, respiratory distress syndrome-induced hypoxia, and multiple organ failure [18] . there is clinical evidence that the sars-cov-19 has potential neuropathic properties. several neurologic related symptoms have been reported including headaches, dizziness, seizure, decreased level of consciousness, acute hemorrhagic necrotizing encephalopathy [19] , agitation, and confusion. in patients with type 2 diabetes mellitus who are infected with covid-19, it is important to remember that two receptor proteins ace-2 and dipeptidyl peptidase-4 (dpp-4) are test can detect igm, and igg antibodies against sars-cov-2 in the serum, plasma, and whole blood [23] . this test is a monoclonal antibody test against the sars-cov-2 nucleocapsid (n) protein. this protein is abnormally expressed in infected cells. monoclonal antibodies specifically directed against n protein, and by using enzyme-linked immunosorbent assay (elisa) it is possible to detect sars-cov-2. the test has a reported sensitivity of 84.1% and a specificity of 98.5%. no cross-reaction with human and animal coronaviruses in the assay were reported. there are no reports about applying this test yet on sars-cov-2 [24] . ultrasonography whole body point of care ultrasound has been used in patients with covid-19. ultrasound is considered an essential modality in the intensive care unit (icu) and the wards in these patients to guide the treatment in patients with cardiorespiratory failure. the current recommendations are to extend its use to multisystem and the whole-body sonographythoracic, cardiac, abdomen, and deep venous thrombosis [25] . earlier studies during the outbreak in china suggested that ct chest imaging together with clinical presentation, pneumonia patients with and without sars-cov-2 can be differentiated. the authors propose that radiological images and clinical features can form excellent diagnostic tools of covid-19 [26] . predictors of severe disease may include (i) high viral load, (ii) elevated neutrophil lymphocyte ratio (nlr), (iii) ct chest changes and extend of lesion, (iv) patient age, and (v) presence of comorbidities [27] . elevated age, and nlr are reported to be independent biomarkers for poor clinical outcomes [28] . the age and sex have been shown to affect the severity of complications of covid-19. the rates of hospitalization and death are less than 0.1% in children and increased to 10% or more in older patients. men are more likely to develop severe complications compared to women as a consequence of sars-cov-2 infection [29] . patients with cancer and solid organ • laryngeal oedema, and laryngitis in critically ill patients with covid-19. • necrotizing pneumonia as a result of superinfection caused by panton valentine leukocidin-secreting staphylococcus aureus infection. this super infection is usually fatal [30] . • cardiovascular complications including (i) acute pericarditis, (ii) left ventricular dysfunction, (iii) acute myocardial injury (associated with increased serum troponin), and (iv) new or worsening arrhythmias, (v) new or worsening heart failure. • acute respiratory failure. approximately 5% of covid-19 patients require to be admitted to intensive care unit because they develop severe disease complicated with acute respiratory distress syndrome [ards] [31] . • sepsis, septic shock and multiple organ failure. • patients with covid-19 infection are at a higher risk of death, particularly (i) males with severe disease, (ii) presence of heart injury and cardiac complications, (iii) hyperglycemia, and (iv) patients on high dose of corticosteroids [32] . • ventilation-associated pneumonia may occur in up to 30% of patients requiring intensive mechanical ventilation. • massive pulmonary embolism complicated with acute right-sided heart failure [33] . complications [34] . because of rapid spread of sars-cov-2, anti-hiv and anti-hcv medications have been tried in cases admitted to icu with severe pneumonia. table 1 summarized these drugs-possible mechanisms of action, side effects, precautions and recommendations. also, shows ongoing registered clinical trials. the covid-19 pandemic represents 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diagnosis of emerging human coronavirus infections -the state of the art sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome whole body point of care ultrasound for covid-19: a multisystem approach to a multi-system disease a diagnostic model for coronavirus disease 2019 (covid-19) based on radiological semantic and clinical features: a multi-center study epidemiological and initial clinical characteristics of patients with family aggregation of covid-19 the diagnostic and predictive role of nlr, d-nlr and plr in covid-19 patients a geroscience perspective on covid-19 mortality panton-valentine leukocidin-secreting staphylococcus aureus pneumonia complicating covid-19 german recommendations for critically ill patients with covid-19 empfehlungen zur intensivmedizinischen therapie von patienten mit covid-19 risk factors for severity and mortality in adult covid-19 inpatients in wuhan covid-19 complicated by acute pulmonary embolism and right-sided heart failure the centers for disease control and prevention (cdc). information for clinicians on investigational therapeutics for patients with covid-19. available at chloroquine and hydroxychloroquine in covid-19 of chloroquine and covid-19 use of hydroxychloroquine and chloroquine during the covid-19 pandemic: what every clinician should know a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 clinical pharmacology perspectives on the antiviral activity of azithromycin and use in covid-19 clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study compassionate use of remdesivir for patients with severe covid-19 remdesivir for severe acute respiratory syndrome coronavirus 2 causing covid-19: an evaluation of the evidence potential therapeutic agents against covid-19: what we know so far favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 an alternative approach to minimize the risk of coronavirus (covid-19) and similar infections tocilizumab treatment in covid-19: a single center experience bethesda (md): national library of medicine (us) bethesda (md): national institute of diabetes and digestive and kidney diseases deployment of convalescent plasma for the prevention and treatment of covid-19 research, king saud university, riyadh, saudi arabia. the author declares no financial or relationships that can be considered a conflict of interest.j o u r n a l p r e -p r o o f key: cord-351115-dy81dtnk authors: wang, chen; konecki, daniel m.; marciano, david c.; govindarajan, harikumar; williams, amanda m.; wastuwidyaningtyas, brigitta; bourquard, thomas; katsonis, panagiotis; lichtarge, olivier title: identification of evolutionarily stable sites across the sars-cov-2 proteome date: 2020-10-20 journal: res sq doi: 10.21203/rs.3.rs-95030/v1 sha: doc_id: 351115 cord_uid: dy81dtnk since the first recognized case of covid-19, more than 30 million people have been infected worldwide. despite global efforts in drug and vaccine development to fight the disease, there is currently no vaccine or drug cure for covid-19, though some drugs reduce severity and hasten recovery. here we interrogate the evolutionary history of the entire sars-cov-2 proteome to identify functional sites that can inform the search for treatments. combining this information with the mutations observed in the current covid-19 outbreak, we systematically and comprehensively define evolutionarily stable sites that are useful drug targets. several experimentally-validated effective drugs interact with these proposed target sites. in addition, the same evolutionary information can prioritize cross reactive antigens that are useful in directing multi-epitope vaccine strategies to illicit broadly neutralizing immune responses to the betacoronavirus family. although the results are focused on sars-cov-2, these approaches are based upon evolutionary principles and are agnostic to organism or infective agent. covid-19 is a worldwide a iction. since rst being reported in december 2019 in wuhan, hubei province, china, the world health organization (who) has tallied more than 950,000 covid-19 related deaths and over 30 million infections worldwide (as of september 19 th , 2020) (1) . although timely public health interventions can successfully curtail incidence, the threat of subsequent waves of infections remains widespread (1) (2) (3) . the novel betacoronavirus (sars-cov-2) that is causing the pandemic is closely related to other known human coronavirus pathogens sars-cov, mers-cov (4, 5) , hcov oc43, hku1 and is more distantly related to the human infectious alphacoronaviruses hcov 229e and hcov nl63 (6) . finding ways to control and prevent further infection are top priorities which include the targeted discovery of drugs that impair viral mechanisms (7-9) and antigenic epitopes through which vaccines raise immunity (10) (11) (12) . this study addresses both by utilizing evolutionary information from sars-cov-2 sequence and structural data to search for actionable functional sites for each protein in the sars-cov-2 genome. in a rst application, we note that the approval of new drugs under normal circumstances often takes more than 10 years (13, 14) . in order to hasten the response, many current clinical trials for covid-19 enlist antiviral agents that have targeted zika, sars-cov, ebola, and mers-cov in the past (13, 15) . in order to test more varieties of potential drugs, some studies screened thousands of clinical-stage or fdaapproved small molecules for antiviral activity, hoping to repurpose some of the top hits for covid-19 treatment (16) . however, the antiviral activity in these large-scale screens may, in part, be cell-line speci c (17) , and therefore of unclear clinical relevance. another approach to screen potential drugs for repurposing is to perform docking (18) of clinical-stage or fda-approved drugs to the sars-cov-2 proteome (19, 20) . however, selection of the correct binding sites on the target proteins is crucial and di cult as protein surface cavities far exceed actual ligand binding sites that modulate function (21) . here we systematically suggest potential drug target sites for most sars-cov-2 proteins based on evolutionary information. as these sites are chosen for their conserved functional roles, broad pancoronavirus/betacoronavirus relevance, and minimal variability across all known current sars-cov-2 variants, they should be prioritized in docking studies for drug repurposing. in a second application, we note that understanding the immune response to sars-cov-2 infection is critical for vaccine development (22) . most early sars-cov-2 immune epitope discovery studies rely heavily on bioinformatic prediction tools as well as sequence and epitope work already done in sars-cov and mers-cov. b-cell linear and discontinuous epitope prediction tools have been used by researchers to identify possible sars-cov-2 epitopes (23) (24) (25) . several more recent studies experimentally determined sars-cov-2 immune epitopes (11, 26, 27) . interestingly, several groups have reported signi cant t-cell reactivity against sars-cov-2 epitopes in individuals without virus exposure (22, 26, 28, 29) . mateus et al. suggested that this could be due to cross reactivity between sars-cov-2 and other common human coronaviruses, such as oc43, hku1, nl63 and 229e (28) . here we report an evolutionary metric, which can accurately separate cross-reactive epitopes from those that are not, and use this metric to suggest potential cross-reactive epitopes in sars-cov-2. prioritizing these crossreactive epitopes in vaccine development can potentially lead to broadly neutralizing immunity across the betacoronavirus family. here, we use the evolutionary trace (et) method, which predicts the importance of protein sequence positions, from most important (0.0) to least important (100.0). this relative ranking re ects the variation entropy of each sequence position within and across the branches of an associated phylogenetic tree, revealing evolutionary pressure points that correspond to functional and structural determinants, and the protein sites at which they often cluster (30) . past studies have shown that this method can predict binding and catalytic functional sites (31, 32) , guide protein engineering (33, 34) and predict function (35) . et rankings of residue importance can also be combined with amino acid substitution log odds to estimate the likely impact, or evolutionary action (ea), of coding variations on protein function (36) (37) (38) . here, this rst et and ea analysis of a full viral proteome identi es evolutionary important residues and functional sites in the sars-cov-2 proteome. evolutionary trace of sars-cov-2. in order to map functional determinants in sars-cov-2 proteins we applied the et approach. with the multiple sequence alignments ( figure s1a , dataset s1) and the corresponding phylogenetic trees ( figure s2 -s4) in hand for 24 of the 26 sars-cov-2 proteins (see si methods and materials), our protocol calculated the et ranking of importance for 99.5% of sars-cov-2 amino acid residue positions (dataset s2) generated from each of three protein databases (uniref90, uniref100, ncbi nr) and combined into a single average. to independently assess the quality of these ranks, rather than rely on the variety and breadth of sequences in the alignments as indicative of information content, we used a statistical measure that quanti es the distribution of et rankings in the 3d structure. residues with smaller et rankings tend to cluster together in active sites, protein-protein interaction sites or other functional sites (30, 31, (39) (40) (41) . such a clustering of top-ranked residues was particularly prominent in several sars-cov-2 proteins and complexes including the nsp5 main protease, the nsp7/nsp8/nsp12 rna-dependent rna polymerase complex and the nsp10/nsp16 rna cap methyltransferase complex and can be visualized as groups of warm colored residues in the protein structure ( figure 1 ). we evaluated the quality of et rankings using the selection cluster weighting (scw) z-score which measures how well highly ranked residues cluster relative to a randomized distribution of scores on the structure (see si materials and methods). for almost all proteins the scw z-score is 2 standard deviations above the randomized background, suggesting that the alignments are informative and that the resulting et rankings are meaningful ( figure s1 , dataset s3). for the proteins that do not reach signi cant z-scores there is a clear correlation to a lack of sequences in the alignments (e.g. nsp1, e, orf3, and orf7a), or, the structure belongs to a small domain within a larger protein (e.g. the macrodomain within nsp3 and the hr2 domain within the s protein). to probe these smaller domains within large proteins we further investigated the adp-ribose-phosphatase (adprp) subdomain and macro and papain-like protease (pl pro ) domains of nsp3. nsp3 was an intriguing case because top-ranked et residues cluster well in its pl pro domain but not in its macrodomain or in the adprp subdomain (dataset s3). in order to better resolve et rankings for nsp3, we generated new alignments, phylogenetic trees, and et residue rankings for the subsequences speci c to each nsp3 domain structure (see si materials and methods). in this focused analysis, the pl pro domain now yielded ~50% more sequences leading to a corresponding increase in the clustering of topranked residues ( figure s5 ). for the macrodomain and adprp subdomain, thousands of additional sequences spanning the three domains of life and distantly related viruses were included in the new data set which resulted in et rankings that rivaled the signi cance of clustering in the pl pro domain. the stark differences we nd in the phylogenetic trees of speci c nsp3 domains con rm previous observations of alternate domain con gurations in different coronavirus genera and even within clades of betacoronavirus (6) . the improvement in scw z-score corresponds to a cluster of highly ranked et residues within the ligand binding site of the macro domain and adprp subdomain ( figure s5d and e) which was missing in the analysis of the full nsp3 reference sequence. having better resolved et rankings in the nsp3 domains, we returned to the main data set to see how well et rankings captured functional sites in other proteins. phylogenetically conserved ligand binding sites. a catalog of sars-cov-2 ligand binding sites could serve as a timely resource for prioritizing therapeutic targets. previous studies have shown that evolutionary sequence information correlates well-enough with enzyme active sites so as to serve as 3dtemplates for functional signatures (35) and identify allosteric sites (42, 43) . here we used nsp12, nsp15 and nsp16 as examples to show how the evolutionary sequence information captured by et can successfully predict ligand binding sites for virus proteins. nsp12 is an rna dependent polymerase, nsp15 mediates the cleavage of both single-and double-stranded rna at uridine sites (44) and nsp16 is a m7gpppa-speci c, s-adenosylmethionine (sam)-dependent, 2'-o-mtase (45) . as shown in figure 2a -c, top ranked et residues cluster around the native ligands of nsp12 (rna) (46), nsp15 (gpu) (8) and nsp16 (m7gpppa and sam) (47) , indicating an accurate prediction of ligand binding sites for these proteins. several new functional sites are also predicted by et ( figure 2d and 2e). on the spike protein (s), one such et cluster partially overlaps the s2' protease cleavage site that is critical for membrane fusion and infectivity of the sars virus (48) . on the nucleoprotein (n), a cluster of highly ranked et residues lies adjacent to the putative rna binding site (49) and may contribute to formation of n protein-rna helical laments that are essential to packaging the rna genome. these results indicate et can provide alternative drug target sites with no currently available ligand-bound structures. in addition to being important to protein function, ideal drug target sites should also be rarely mutated in the current outbreak so as to avoid the potential emergence of drug resistance. thus, we focused on positions that do not have any mutations observed in the 52,061 high quality, full length sars-cov-2 sequences that were available as of september 14th, 2020. as more genomes and mutations within them are sequenced it may be necessary to lower the variant count stringency. in order to translate proteomewide et ranks and mutational pro les into potential drug target sites, we focused on clusters of mutationfree, surface-exposed residues that are highly ranked by et and fall within 5å of each other ( figure 3 , dataset s4). the resulting catalog of putative drug targets includes 116 sites at ~5 sites per structure with the largest structure (full-length model of spike, 6vsb_1_1_1) having the highest number of sites. for nsp12, nsp15 and nsp16, the predicted drug targets overlap the known ligand binding sites. in order to evaluate whether these et drug sites may correspond to druggable target sites, we examined their overlap with sites observed in ve sars-cov-2 protein-drug complex crystal structures. it is important to note that all 5 drugs showed an inhibitory effect in either cellular or biochemical assays. remdesivir has been shown to speed up the recovery of covid-19 patients in clinical trials (50) , while the α-ketoamide inhibitor 13b can suppress sars-cov-2 replication in cell lines (51) . vir251 and tipiracil were also shown to effectively inhibit the enzymatic activities of their targets (7, 8) . the remaining drug, sinefungin, is a pan-mtnase (nsp16) inhibitor that inhibits the growth of yeast cells ectopically expressing nsp16 from sars-cov (45) . the et drug sites were mapped onto the ve sars-cov-2 protein-drug complexes (7, 8, (51) (52) (53) and, as shown in figure 3 , all ve drugs reside in protein surface pockets that are within or very close to our predicted et drug sites. the et drug site for nsp5 is the least well recovered due to a single sars-cov-2 sequencing entry (strain mt745875) wherein several residues in the protease active site are mutated (g143s, s144e and c145i), including the catalytic cystine residue. s144e and c145i are both caused by two nucleotide substitutions in the codon, and only observed in this strain (sampled on 06/24/20). it is unclear whether this is a sequencing artifact or represents a genuine active site plasticity that compromises nsp5's active site as a stable drug target. it does however illustrate the importance of accurately detecting emerging sequence variations when choosing drug targets. overall, these results show that predicted et drug sites can recover experimentally tested drug binding pockets and suggest new sites that can be targeted in computational docking approaches. in addition, because these sites are conserved across multiple coronavirus genera, these predicted et drug sites are anticipated to be relevant for identifying inhibitors of sars-cov-2 as well as more distantly related coronaviruses. conserved linear epitopes. et drugs sites may prove valuable in guiding drug design, but these approaches are dependent upon having high resolution crystal structures and some structures are either not yet available (e.g. nsp2, nsp6, m, and several accessory proteins), do not cover a majority of the protein (nsp3 and nsp4) or are too low in resolution for accurate docking studies (nsp12, nsp14, ectodomain of s, n, orf3a and orf7a). however, et operates over linear protein sequences and thereby can identify phylogenetically important sequence fragments even in the absence of a 3d structure (54) . as in our approach to discover et drug sites, we combined et residue ranking information with sequencing data from sars-cov-2 isolates to arrive at linear peptides along the proteome that are evolutionarily important and also show little variation in the current outbreak ( figure s6 , dataset s5). in order to assess the value of these epitopes, we asked whether they could recapitulate et-derived drug sites. et-de ned linear peptides for nsp12 were mapped onto an available nsp12 structure and, as illustrated in figure 4a , the majority of the structural and linear peptides overlap with each other. linear et peptides and et drug sites overlap well for other sars-cov-2 proteins, which was quanti ed by jaccard similarity and fisher's exact test (dataset s6). these data suggest that linear et peptides contain functionally relevant information since they recapitulate et drug sites for proteins or domains without requiring 3d structural data. in the absence of a protein structure, these et peptides could be useful in designing inhibitory peptides (55, 56) . these peptides are also connected to a second main approach towards resolving the pandemic, by way of vaccine development. although vaccines for covid-19 may become available soon, ideally, effective protection against future outbreaks from related coronaviruses would require a broadly neutralizing effect wherein the immune system recognizes epitopes shared among coronavirus species. the prospect of raising a broadly neutralizing response is bolstered by a recent study wherein naïve patients, never exposed sars-cov-2, were found to possess a subset of t-cells that can cross-react to homologous epitopes shared by common cold coronaviruses and sars-cov-2 (28) . in this context, we note that et rankings re ect the degree of homology over the phylogenetic tree, so we reasoned that summing et scores over the length of an identi ed t-cell epitope may be able to estimate its potential for crossreactivity. as a rst step, we summed the et ranks for each of the 40 sars-cov-2 epitopes that had been shown to react with patient-derived t-cells so that they could be ranked by predicted cross-reactivity to 161 common cold coronavirus epitopes assayed by mateus et al. although summing et ranks could identify sars-cov-2 epitopes that are more likely to be cross-reactive ( figure s7 ), it did not account for the speci c amino acid differences in the potentially cross-reactive homolog. in other words, et ranks can predict whether or not a sars-cov-2 epitope will be cross-reactive in general, but they do not specify which epitope homologs will cross react. in order to improve resolution of our predictions to speci c epitope homologs, we next combined ea, a predictor of mutational impact, with the summed et rankings. ea calculates the predicted impact of amino acid variations on protein function aiding in the interpretation of coding variants (36) (37) (38) . summing the predicted impact of amino acid changes between a sars-cov-2 epitope and a homologous epitope in another virus (sumea) while adjusting for the sars-cov-2 epitope's overall evolutionary importance (sum(100-et ranking)) produced a metric that was able to separate cross-reactive epitopes from those that did not cross react ( figure 4b and s7, dataset s7). this metric, sumea/sum(100-et ranking), was then applied to 21 untested sars-cov-2 t-cell epitopes and their common cold homologs (28) . from a total of 92 homologs we identi ed 23 with potential to cross react to one of ve sars-cov-2 epitopes ( figure 4c , dataset s8). these 5 sars-cov-2 epitopes along with the 9 others experimentally shown to possess cross-reactivity could be used in a multi-epitope vaccination strategy that provides a broad neutralizing response to currently circulating coronaviruses, sars-cov-2 and, possibly, future outbreaks. moreover, the approach is not speci cally linked to any speci c virus, so it could be replicated in other families of pathogens. dissemination. to disseminate these results, a public website (http://cov.lichtargelab.org) makes these data and analyses fully accessible. the data include, for example, multiple sequence alignments, precalculated et ranks, and predicted epitopes (both linear and structural) for all sars-cov-2 proteins. in addition, an interactive structure viewer enables users to explore any one of the et-colored structures ( figure 1 ) and predicted et drug sites associated with those structures (dataset s4-5). the website will be updated as new sars-cov-2 isolates and protein structures become available. rapid progress has been made in response to the acute sars-cov-2 threat; from sequencing, to structural determination, and to drug and vaccine development (9, (57) (58) (59) (60) . here, by combining information from evolutionary history and the current outbreak of sars-cov-2 we systematically mapped potential therapeutic sites on all sars-cov-2 proteins. we make use of phylogenetics, sequence information and structure information to provide a functional map of sars-cov-2 proteins. the sites we determined are not only stable across coronavirus families but are also stable to mutations in the current pandemic, which make them ideal targets for pan coronavirus/betacoronavirus therapeutics. in so doing, we pinpoint functionally and structurally important sites in the sars-cov-2 proteome that reduce the search space for drug and vaccine development. in addition to focusing therapeutic studies, the data presented here will be important in identifying the mechanism of action for successful therapies, not only in the context of the current outbreak but across future coronavirus outbreaks. our ndings are available on the accompanying website, where results will be updated as more sars-cov-2 isolates are sequenced, and structures are completed. this should not only expand coverage of the sars-cov-2 proteome and re ne predicted therapeutic sites, but also provide a resource to monitor for variants that may signi cantly impact the virulence of sars-cov-2. there are limitations to this study. the quality of our results depends on the number and range of homologous sequences available. although most of the non-structural proteins yield et rankings that are likely informative (clustering z-score >=2 or >30 unique sequences between 25-98% identity), nsp1 and the accessory proteins do not reach signi cant z-scores or have many diverse sequences in their nal alignments. the inability to recover more sequence information could be due to a higher evolutionary rate in these proteins that limits our ability to recognize distantly related homologs with very little sequence identity. more likely, these peripheral genes have been more recently recruited through the frequent recombination events that occur in the coronavirus family (61) . such recruitment has occurred at the domain level in the nsp3 protein with its variable number of domains (10 to 16), some of which are unique to the betacoronavirus clade b containing sars-cov-1 and -2. therefore, it is unsurprising that the initial sequences returned and corresponding et rankings for full-length nsp3 are heavily in uenced by the less divergent pl pro domain that is present across coronavirus clades and families. domain-speci c analysis of nsp3 greatly improved both the number of sequences returned, phylogenetic coverage, and the resolution of et results. this suggests that future work should include domain speci c analyses for multidomain proteins. such domain speci c analyses are likely to provide et rankings that identify important functional sites for individual domains while full-length analysis can provide insight into how particular domains became recruited for speci c branches of the phylogenetic tree. several other groups have focused on experimentally screening clinical-stage or fda-approved small molecules with the hope of identifying and repurposing drugs for sars-cov-2 treatment. tens to hundreds of drug candidates are identi ed by these high-throughput assays. however, drug e cacy of top hits might be cell line speci c (17) and the mechanisms of drug action may be unclear or acting through modulation of the host cell rather than targeting the virus itself. in silico docking studies (19, 62) take a more targeted approach towards speci c sars-cov-2 sites that may complement the results of experimental screens. knowledge of the ligand binding site improves the chance of identifying drugs that inhibit protein function and although structural characterization of sars-cov-2 proteins is unprecedented, the structural information available is far from comprehensive. using the structures which have been solved, we identi ed clusters of surface residues that have low et rankings and a lack of mutations in the current outbreak as potential drug target sites. many of these et drug sites correspond to ligand bound active sites but others map to evolutionarily important sites that have yet to be fully characterized. et operates over the phylogenetic history of linear sequence space and can anticipate functional sites that may or may not be characterized in the future. these putative et drug targets can guide docking studies to additional sites not immediately apparent from currently available structural information. sites highlighted by et are evolutionarily conserved in the phylogenetic tree used in et calculation and this information can set expectations for how broadly a drug may inhibit different viral species. for instance, remdesivir targets the active site of rna-dependent rna polymerase (nsp12) in sars-cov-2 as well as homologs in sars, mers and the distantly related ebola rna virus (63, 64) . the nsp12 active site has a very strong et signal that is derived from one of deepest phylogenetic trees in our analysis and thereby would be expected to inhibit a wide swath of coronaviruses and related rna viruses. in contrast, the adp ribose phosphatase sub-domain of nsp3 has a phylogenetic tree that includes relatively few coronavirus sequences among a multitude of sequences that span three domains of life. drugs targeting this domain may inhibit coronavirus infectivity but could also have side effects if they inhibit host adp ribose phosphatases. however, adp ribose phosphatase inhibitors have been developed for cancer treatment and a wealth of information and expertise is available for this group of drugs (65) . as with the application of any new drug, particular care should be taken to ensure unwanted side effects do not overshadow any bene ts as a viral inhibitor. the linear epitopes we de ned here may also provide valuable information in drug development both for proteins with structure, and for those without, as amino acids connected linearly are guaranteed to be connected structurally. for protein regions that are exible or undergo large conformational changes during activation, structural proximity de ned in one conformation may not hold in other conformations. for example, the spike protein undergoes a large conformational change when mediating host-virus membrane fusion (66) . a structural epitope that is determined in the closed state might not be appropriate for the opened state. thus, linearly connected regions may identify cryptic binding sites that are revealed upon conformational change of the protein. linear epitopes are also a predominant mode of recognition of the adaptive immune system. studies have shown that some sars-cov-2 t-cell epitopes are capable of cross reacting with homologous peptides in other human coronaviruses (26, 28) . we performed evolutionary analysis on these crossreactive epitopes and developed a new metric that can distinguish cross reactive epitopes with a high accuracy that outperforms a simple percent identity metric. this sumea/sum(100-et ranking) metric was then used to suggest other potential sars-cov-2 cross-reactive t-cell epitopes. in general, cross-reactive epitopes have the potential of generating a pan-betacoronavirus immune response that can stimulate bcells to produce broadly neutralizing antibodies. although not directly addressed in this work, the sumea/sum(100-et ranking) metric may also be able to identify epitopes that stimulate cytotoxic t-cells through presentation on mhc-1 molecules. several groups are at the preclinical stage in multi-epitope vaccine development (milkeninstitute.org) but the speci c epitopes are not publicly available, and it is unknown whether or not they include any that are cross reactive. the ability to identify cross-reactive epitopes could inform a multi-epitope vaccine strategy that is speci cally designed to inoculate a susceptible population to a wide range of extant and undiscovered betacoronaviruses. this study was motivated by the current pandemic and uses evolutionary sequence information to guide the development of therapeutics for covid-19. although we are presently in the grip of covid-19, this pandemic was preceded by the sars and mers outbreaks and it should be anticipated that related coronaviruses will cause future outbreaks. and while this study is also focused upon sars-cov-2, it draws upon pieces of sequence information taken from the whole of the coronavirus family and thereby the ndings are extendable to other coronavirus species, including those that have not yet been encountered. indeed, the tools we present could be applied to any family of pathogen. putting a pandemic virus into the evolutionary context of related viruses can expose a path to managing a recovery and may offer therapeutics that cover future outbreaks. a brief description of the methods can be found here, for a more in-depth description of speci c methods please see the supplementary text. in order to map functional determinants in sars-cov-2 proteins we applied the evolutionary trace (et) approach (30, 31) . this method ranks each amino acid position from most to least important during evolution by tracking how they vary along the coronavirus phylogenetic tree. these rankings vary based on the precise choice of multiple sequence alignment (msa). in order to produce robust et rankings three separate alignments were generated for each protein in the sars-cov-2 wuhan-hu-1 reference genome (nc_045512.2) (57), by querying three protein databases (uniref90, uniref100, and ncbi nr) for sequences with identity between 25% and 98%, thus ltering out those that were either overly distant or redundant. only two proteins had too few matches for et, nsp11 and orf10, both of which have unknown function and have very short reference sequences (13 and 38 amino acids, respectively, figures1, dataset s1). the et scores for all other proteins for each alignment and for the average scores across alignments were evaluated with the previously presented selection cluster weighting (scw) zscore (30, (39) (40) (41) . the z-scores for each structure were then ranked 1-4 in order to determine if et scores from one database or the average of the three consistently outperforms the others. et scores from each of the three databases performed similarly well but the average et of the three provided better z-scores in most cases ( figure s1c ). et rankings were further investigated by comparing the highest scoring regions with known functional sites. therapeutic sites were predicted based on both the linear sequence as well as structural constraints. residues were nominated as members of potential therapeutic sites based on their et rankings, lack of variants as found in sars-cov-2 sequences retrieved from gisaid (67) and the china national center for bioinformation (68)(cncb), as well as surface accessibility, and structural proximity. structurally identi ed therapeutic sites were compared to drug binding sites for agents known to bind to sars-cov-2 proteins. to generalize this approach to proteins without structure, linear sites were predicted based on et rankings, current mutational pro le and linear connectivity. structural and linear predicted sites were compared to one another using jaccard similarity and fisher's exact test, to determine the usefulness of this method in the absence of a protein structure. several et metrics were also interrogated to determine their ability to highlight potential cross-reactive immunogenic epitopes (28) . the best metric, sumea/sum(100-et ranking), was used to predict cross-reactive t-cell epitopes which are good potential therapeutic sites. an interactive web-based dashboard to track covid-19 in real time the effect of human mobility and control measures on the covid-19 epidemic in china. science (80-. ) changes in contact patterns shape the dynamics of the covid-19 outbreak in china genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting 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sars coronavirus nsp14-nsp10 complex crystal structure of nsp15 endoribonuclease from sars cov-2 in the complex with uridine-5'-monophosphate rcsb pdb -6w4h: 1.80 angstrom resolution crystal structure of nsp16 -nsp10 complex from sars-cov developing a fully glycosylated full-length sars-cov-2 spike protein model in a viral membrane cryo-em structure of the sars-cov-2 3a ion channel in lipid nanodiscs structural model of the sars coronavirus e channel in lmpg micelles center for structural genomics of infectious diseases (csgid), rcsb pdb -6w37: structure of the sars-cov-2 orf7a encoded accessory protein crystal structure of rna binding domain of nucleocapsid phosphoprotein from sars coronavirus rcsb pdb -6zco: crystal structure of c-terminal dimerization domain of nucleocapsid phosphoprotein from sars-cov-2, crystal form ii the authors of this text have no con icts of interest to report. key: cord-356009-emn2w8if authors: roshandel, m. r.; nateqi, m.; lak, r.; aavani, p.; sari motlagh, r.; aghaei badr, t.; sfakianos, j.; kaplan, s. a.; shariat, s.; tewari, a. k. title: what specimen urologists should be most concerned about ? a systematic review and meta-analysis date: 2020-10-13 journal: nan doi: 10.1101/2020.10.08.20209544 sha: doc_id: 356009 cord_uid: emn2w8if objective:investigating the infectivity of body fluid can be useful for preventative measures in the community and ensuring safety in the operating rooms and on the laboratory practices. methods:we performed a literature search of clinical trials, cohorts, and case series using pubmed/medline, google scholar, and cochrane library, and downloadable database of cdc. we excluded case reports and searched all language articles for review and repeated until the final drafting. the search protocol was registered in the prospero database. results: thirty studies with urinary sampling for viral shedding were included. a total number of 1,271 patients were enrolled initially, among which 569 patients had undergone urinary testing. nine studies observed urinary viral shedding in urine from 41 patients. the total incidence of urinary sars-cov-2 shedding was 8%, compared to 21.3% and 39.5 % for blood and stool, respectively. the summarized risk ratio (rr) estimates for urine positive rates compared to the pharyngeal rate was 0.08. the pertaining rr urine compared to blood and stool positive rates were 0.20 and 0.33 respectively. conclusions: our review concludes that not only the sars-cov-2 can be excreted in the urine in eight ?percent of patients but also its incidence may have associations with the severity of the ?systemic disease, icu admission, and fatality rates. moreover, the findings in our review ?suggest that a larger population size may reveal more positive urinary cases possibly by ?minimizing biases. however, it is important to notice that it is the naso-pharyngeal specimens, ?stool, and serum that show more possibilities to became positive, respectively. urinary viral shedding can be important from the aspects of diagnosis, vertical and horizontal transmission of infection (1) . viral shedding has been proven for some other contagious viruses including the ebola virus, zika virus, and hepatitis-b virus (2, 3) . up to date, sars-cov-2 has spread to 213 countries and territories and infected over 28,000,000 patients around the globe with around 1,000,000 death toll (4) . before coronavirus disease-2019 (covid19) , the latest coronavirus outbreaks were the severe acute respiratory syndrome coronavirus-1 (sars-cov-1) and middle eastern respiratory syndrome coronavirus (mers-cov) outbreaks. for sars-cov-1, the urinary positivity rates were reported to be up to 42% (5) . sars-cov-2 structural features are similar to both sars-cov-1 and mers-cov which all belong to the family coronaviridae (6) . the presence of sars-cov-2 has been shown in urine (7) (8) (9) (10) (11) (12) . angiotensin-converting enzyme-ii (ace2) has been known as the cellular entry receptor of sars-cov-2 (13, 14) . the cells with ace-ii receptors such as epithelia of lung, kidney, and bladder may act as targets to sars-cov-2 (9, 15, 16) . although there are discrepancies in the reported results of the studies over the sars cov-2 urinary shedding, since the viral dynamics are yet to be fully determined, it's been recommended that the urethral or ureteral instrumentation and handling should be carried out cautiously (17) . determining whether the virus is detectable throughout the disease is critical to control transmission. considering the stability of sars-cov-2 for up to 72 hours (18), performing urological surgeries, or collecting infected urinary samples may put urologists and health care workers at risk (19) . the ebola virus epidemic (2014 to 2015) was an awakening alarm for the health care community regarding the lack of biosafety in the handling of samples containing suspected special pathogens (20) . this is true, particularly when responding to a not well-known pathogen, as the recommendations are often fluid (21) . learning about the infectivity also can alter preventative measures in the operating room and the settings needed for safety on laboratory practices (22) . furthermore, the probability of transmission by pets (23) (24) , leaves urine with a large potential to be a source of disease spread (25, 26) . although no data are available to confirm or exclude the possibility of such transmission, cdc advises restricting contact with pets and other animals while one has covid-19 (27) . by this review, we systematically investigated the findings on the urinary sars-cov-2 to points out the important methodological considerations needed to be considered in future studies. the protocol for this systematic review was registered with the international prospective register of systematic reviews (crd42020187294). the review follows the prisma (preferred reporting items for systematic reviews and meta-analysis) statement (28) . a systematic search of the literature was performed in pubmed/medline, google scholar, cochrane library, and covid-19 research articles downloadable database of cdc (centers for disease control and prevention). the comprehensive literature was performed in june 2020. no language restrictions were applied. articles published in 2019 and 2020 were included. searches were repeated until the final drafting of the manuscript, to capture emerging evidence from the ongoing studies. the searches included medical subject headings (mesh) and keywords for sars-cov-2, covid, corona, together with shedding, persistence, urine, urinary, specimen, viral load, or rna body fluids. searches were designed to be broad and comprehensive initially, using the following keywords and mesh terms: ("specimen" or "urine" or "urinary") and ("corona" or "coronavirus" or "covid" or "covid-19" or "covid-2019" or "sars" or "sars-cov" or "sars-cov-2"). study selection was based on predefined eligibility criteria within a cocopop (condition, context, population) and a pird (population, index test, reference test, diagnosis of interest) format (table 1 ) (29) . additional exclusion criteria were applied at the full-text stage. after finalizing the selection of studies on the urinary sars-cov-2, information on viral existence in stool and blood specimens were explored in the selected studies ( figure 1 ). all studies required a minimum of 2 patients were included. all potential studies were independently screened by two investigators. in the case disagreement, it resolved either through . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 13, 2020. . https://doi.org/10.1101/2020.10.08.20209544 doi: medrxiv preprint discussion or the involvement of a third researcher according to delphi consensus criteria. clinical trials, retrospective, prospective observational, case series, and cross-sectional studies were included as well as supplementary or non-peer-reviewed reports, correspondence, research letters, and preprints. review articles, case reports, or non-relevant articles were excluded from the pool. following reviewing and extraction of data, references of each manuscript were searched for relevant missing manuscripts. we created standardized forms for data extraction and the pilot tested the forms before the process of data extraction. we completed the data abstraction process using created forms to record study characteristics, clinical data, and laboratory data including study year and design, country of study origin, total initial population size, test type for disease diagnosis, test type for samples (urine/stool/rectal swab/blood), patients age (including mean and range), number of positive and total patients and/or (wherever applicable) number of positive and total specimens collected for each test category, disease severity, icu admission, and fatality rate. more details for study items are shown in table 2 . two investigators assessed the risk of bias for individual studies independently using jbi's (joanna briggs) critical appraisal tools for prevalence study and diagnostic test accuracy studies to assess the trustworthiness, and results of the studies (30-32). forest plots were used to assess risk ratio (rr) and summarized them to describe rr of viral shedding rates in the urine and control groups (i.e., nasopharynx, stool, blood). primary, secondary, and tertiary meta-analysis was conducted among all studies that reported urine and nasopharynx positive rates, urine, and stool positive rates, urine, and serum positive rates as an outcome, respectively. the heterogeneity across studies was evaluated using p-values, and q and i2 statistics. random effect and fixed effect meta-analysis were used when the heterogeneity was greater and lower than 50%, 6 respectively. p-values lower than 0.05 were considered statistically significant. all analyses were carried out using stata version 14. a total of 30 studies met the inclusion criteria and were included in the review (8-13, 17, 35-49) . the overall prevalence of urinary sars-cov-2 shedding was 8%. this was 21.3% and 39.5 % for blood and stool respectively. characteristics of the included studies, comparison among positive and negative studies are detailed in tables 2, 3, and figure 2 . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 13, 2020. . the confirmatory testing was positive pharyngeal swabs, except two studies that included four patients with initially negative pharyngeal but positive stool results (13, 40) . essentially, all studies included patients with a median age between 40s and 60s (ranged from 6 months to 92 years within the literature examined, the most commonly used assays for detection of sars-cov-2 in different samples such as urine, stool, blood, and pharyngeal swabs were rna extraction followed by semi-quantitative and quantitative real-time reverse transcriptase-polymerase chain reaction (real-time rt-pcr, or rrt-pcr). in some studies, serological and molecular methods such as enzyme-linked immunosorbent assay (elisa), partial and whole-genome sequencing were used for more verification. several specific primers pairs were used to amplification of gene regions including rdrp/helicase, spike, and nucleocapsid genes of sars-cov-2. such data were not available for all papers. severity, icu admission, fatality rate, and patient enrolling: . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 13, 2020. . https://doi.org/10.1101/2020.10.08.20209544 doi: medrxiv preprint 8 the covid-19 severity and icu admission rates are detailed in table 4 . • 20.1% of the total initial population were admitted into the icu, as reported in 13 studies. • 33.0% of the total initial population had severe respiratory disease because of sars-cov-2, as reported in 16 studies. • there was no information about the severity or icu admission in the rest of the studies. four studies that included the cases terminated by death reported a total fatality rate of 7.6% (11/144). the other 11 studies only enrolled survived cases. related information was not provided in 15 studies. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 13, 2020. . the results are shown in table 5 meta-analysis was performed across the urinary studies. the summarized rr of 30 retrospective studies that reported urine compared to nasopharynx positive rates, was 0.08 (95% confidence intervals (ci); 0.05-0.16). the pertaining rr urine compared to blood and stool positive rates were 0.20 (95% ci; 0.14-0.29) and 0.33 (95% ci; 0.15-0.72) respectively. the forest plots of the meta-analysis are shown in there was no significant heterogeneity across all studies that included in the all metaanalysis, therefore fixed-effect analyses have been used. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 13, 2020. . https://doi.org/10.1101/2020.10.08.20209544 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 13, 2020. . https://doi.org/10.1101/2020.10.08.20209544 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 13, 2020. . https://doi.org/10.1101/2020.10.08.20209544 doi: medrxiv preprint according to our three meta-analyses, stool and blood tests are associated with a significantly higher positive rate than urine (figures 1-3) . these results indicated that when the naso-/oro-pharyngeal sars-cov-2 test is negative, stool, and/or blood tests are more helpful for virus diagnosis than urine. nevertheless, different factors such as the nature of viruses, missing data, design flaws, or methodological limitations might have contributed to these findings. the importance of urinary viral infection should not be ignored in terms of protective measures. we reviewed some important factors that can influence the results. association between severity, fatality, icu admission rate, and sepsis, with urinary positivity: covid severity can be evidenced by the needs for mechanical ventilation, icu care, as well as higher fatality. viremia and sepsis are usually representative of more severe forms of diseases and a potential source for viral urinary shedding. in sars-cov-1, viremia was reported to be associated with disease severity (61) . similarly, severely ill patients in the setting of sars-cov-2 disease may have augmented and prolonged the existence of the . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 13, 2020. . https://doi.org/10.1101/2020.10.08.20209544 doi: medrxiv preprint virus in blood and other body fluids (10) . furthermore, urinary viral positivity was found in severe cases in a study (7) . sars-cov-2 viremia also may occur in patients with underlying comorbidities (62) . moreover, the duration of viral rna excretion in respiratory and stool specimens may be longer in the cases treated with glucocorticoid, an immunosuppressive medication (8, 10) . accordingly, we emphasize the importance of inclusion of severely ill patients and ones with an underlying disease in the shedding studies. in a study with a comparatively higher rate of urinary shedding (9.5%), a significantly higher proportion of the population had severe disease (40%) along with a case fatality of 6% (9) . in another study, the duration of the existence of the virus in the blood of icu patients was longer. the authors suggested its relationship with blood viral load and disease severity. viruses were not found in the urine samples. although their classification based on icu admission was informative, the study didn't have any information on whether the cases who ended in death -which might bear a higher urinary positive ratehadn't been excluded from the study (63) . similarly, some other studies with totally negative urinary results were limited to the populations with mild or moderate severity with null fatality (64, 65). according to figure 2, the incidence risk of viral shedding in stool or blood was similar for both groups of studies with and without positive results. while trying to justify why a study has positive urinary results but another study doesn't, the mentioned finding might not be in favor of the hypothesis that it could have arisen from systematic flaws in or in inclusion or exclusion criteria, sampling, or handling methods. nevertheless, a lack of concordance between the severity of disease and icu admission rates in two groups in table 2 indicates heterogeneity of study populations among the studies. as a result, since the difference in the severity of covid-19 may contribute to the urinary negative results, we suggest to consider it while planning the study. determining when the virus is detectable in the urine, is not simple. different phases of the disease may lead to considerable differences in viral loads and peak concentrations and can be another factor responsible for different urinary findings. toward the end of the period of the covid disease, the virus is shown to be only intermittently detectable in pharyngeal swabs (41) . since the pharyngeal samples have much higher positive rates, finding a virus in the other fluid types could be more challenging. as reported in sars-cov-1 studies, the urinary positivity rate was up to 42% at the end of the second week (5) with a peak occurring at weeks 3-4 and even shedding in the convalescent phase (66, 67) . similarly, for sars-cov-2, positive urine samples were detected at the latest available detection point (16 or 21 days after illness onset) (7) . another study with a urinary positivity rate of 6.9% for viral rna, revealed urine could stay positive after the throat swabs turned negative (8) . although regular serial sampling was performed for pharyngeal specimens in some of the studies in our review, that was not the case for urine. since repeat urinary testing is warranted especially in clinically suspected cases with an initially negative urinary result, we would emphasize the importance of systematic serial sample monitoring, throughout the disease phases, with an increased number of tested samples. improper exclusions, underpowered sample size, and bias in urinary results: failure to find urinary viruses in many studies may be explained by their undersized study population. this may contribute to the positivity of urinary results found in studies done in china which were larger in population. our review demonstrated that a considerable number of studies with a larger population were able to find positive urinary results (10, 34, 36, 53, 60, 68) . moreover, there was a considerable difference in number between the population initially enrolled and finally tested for urine. exclusions were not explained, as in some studies it was found to be more than half of the initial population. except for one study, all the mentioned studies were accompanied by no positive urinary results (9, 49, 53, 69) . . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 13, 2020. . https://doi.org/10.1101/2020.10.08.20209544 doi: medrxiv preprint 14 some studies with negative urinary results in this review had no stool positive findings (62, 65, 70) , even though stool has been proven to have a high possibility of viral shedding (48.1%) (71) . this co-negativity may also be explained by the errors in handling and laboratory technics. although real-time rt-pcr is considered as the gold standard for the diagnosis of covid-19 (72) , some factors such as the rna quality, operator variability, or processing methods can affect the test results (73) (74) (75) . also, this technique does not distinguish between rna residues and viable active viruses (76) . as sars-cov-2 shares a high nucleotide identity with sars-cov-1 (82%) (77), using nonspecific realtime rt-pcr (e.g. sybr green method) may cause false-positive results. some studies reviewed herein, lack information concerning the real-time rt-pcr type, primer, and probe sequences, candidate genes for virus detection, presence or absence of positive control, and the cycling parameters for pcr assay. real-time rt-pcr ct (cycle threshold) values may also differ because of specimen collection or handling. the presence of several enzymes such as protease, rnase, or bacteria and the absence of proteins that stabilize rna and virus in the urine may explain the quick degradation of viral rna (78, 79) . technical improvement in the sampling to prevent degradation of the urinary viral rna (such as immediate addition of lysis buffer to the fresh urine) may help to increase the diagnostic sensitivity and diminish false negative (78, 80) . further studies are needed to assess the efficacy of these methods in sars-cov-2. another source of false-positive urinary results can be passive contamination of urine samples with stool or other sources that can occur in severely ill or in the presence of diarrhea. the presence of different genotypes in urine or the comparatively higher rna concentrations in urine would indicate active replication in the urine rather than contamination and spillage. as can be noticed so far, this review encountered several limitations that resulted from a lack of highquality evidence. we just mentioned the most important topics that help in building researches with a deeper focus on the design and methodologic quality in the future and help assess the viral shedding in urine and other specimens more efficiently. based on review of the present literature, shedding of sars-cov-2 occurs in around 10 percent of population and it may have an association with the severity of systemic disease, need to admission in icu and fatality. investigating relationship between urine results and other factors is hardly possible without avoiding inappropriate exclusions. furthermore, our review suggests that a larger population size may reveal more positive urinary cases. moreover, using standardized laboratory quantitative control in realtime rt-pcr as well as repeat urinary testing would be warranted especially in patients with initially negative urinary results. we declare no competing interests. . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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quantitative detection and viral load analysis of sars-cov-2 in infected patients viral load of sars-cov-2 in clinical samples. the lancet infectious diseases gastrointestinal manifestations of sars-cov-2 infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19-rdrp/hel real-time reverse transcription-pcr assay validated in vitro and with clinical specimens the use of real-time reverse transcriptase pcr for the quantification of cytokine gene expression negative nasopharyngeal and oropharyngeal swabs do not rule out covid-19 clinical characteristics of imported cases of coronavirus disease 2019 (covid-19) in jiangsu province: a multicenter descriptive study use of propidium monoazide in reverse transcriptase pcr to distinguish between infectious and noninfectious enteric viruses in water samples severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection viral rna degradation makes urine a challenging specimen for detection of japanese encephalitis virus in patients with suspected cns infection ebola virus rna stability in human blood and urine in west africa's environmental conditions find the right sample: a study on the versatility of saliva and urine samples for the diagnosis of emerging viruses key: cord-352562-qfb478sf authors: yamamoto, lidia; dos santos, emilly henrique; pinto, lacyane silva; rocha, mussya cisotto; kanunfre, kelly aparecida; vallada, marcelo genofre; okay, thelma suely title: sars-cov-2 infections with emphasis on pediatric patients: a narrative review date: 2020-09-04 journal: revista do instituto de medicina tropical de sao paulo doi: 10.1590/s1678-9946202062065 sha: doc_id: 352562 cord_uid: qfb478sf this narrative review summarizes the main aspects underlying the new coronavirus sars-cov-2, its epidemiology, pathophysiology, pointing to differences of sars-cov-2 main receptors ace2, in terms of expression and the amount of soluble ace2 in the circulation of children, men and women, and also in those with risk factors such as the smokers and pregnant women or presenting with comorbidities (diabetes, obesity, hypertension and other cardiovascular diseases, renal and cns pre-existing diseases). clinical manifestations in adults and children were also described, emphasizing the particularities already seen in children, regarding signs, symptoms, viral excretion time and the involvement of all organs and systems. the covid-19 in the pediatric population was divided into two sections: one dedicated to previously healthy children and adolescents with covid-19, and the other to those who live with comorbidities and acquired covid-19. a few paragraphs were reserved to the recently described severe multisystemic inflammatory syndrome associated with covid-19 (mis-c) that shares certain characteristics with kawasaki disease. some studies on the infection in pregnant and postpartum women, as well as neonates were shown. this review has also covered the laboratory diagnosis of covid-19, passing through the imaging diagnosis made by the chest tomography revealing ground glass patching opacities, and results of non-specific exams such as the total blood with lymphopenia, the coagulation tests with increased prothrombin times, as well as marked increments of the d-dimer, troponin and proinflammatory cytokines. in the section devoted to the specific laboratory diagnosis of covid-19, the most used rt-pcr protocols were described and some studies on the serological diagnosis with iga, igm and igg detection were detailed, including the use of rapid immunochromatographic assays and discussing the ideal period after the onset of symptoms to perform each type of test. in the end, the management of pediatric patients with covid-19 based mainly on supportive measures has been briefly commented. in december 2019, an outbreak of pneumonia of unknown etiology was reported in the province of hubei, city of wuhan, china. in the following month, the etiologic agent was isolated and had its genome sequenced, revealing a new coronavirus, the sars-cov-2 or 2019-ncov, and the disease was called covid19 . the origin of the outbreak was attributed by the chinese government to a local seafood market. the incubation period of covid-19 ranges from 3-14 days, with a basic reproduction number (r0) varying from 1.4 to 6.49, depending on the country and the adopted control measures 5 . sars-cov-2 is transmitted by droplets released by sneezing and coughing. asymptomatic individuals might transmit the infection, as some reports described individuals in whom high viral loads were detected in respiratory secretions during the early stages of the infection, even before the onset of symptoms 5 . according to the severity of clinical manifestations, covid-19 can be classified as asymptomatic or symptomatic, the latter ranging from mild to severe and critical. mortality due to covid-19 was 1.4% in china and up to 15.2% in other countries 6 . covid-19 affects most frequently people over 60 years of age, especially smokers and those with comorbidities such as diabetes, hypertension, cardiovascular disease, obesity, chronic lung disease, kidney diseases 5 . what has drawn attention since the beginning of the pandemic is the low number of children and adolescents with severe and critical covid-19, and the large number of asymptomatic infections in this age group 7 . sars-cov and sars-cov-2 have high affinity with the angiotensin-converting enzyme 2 cellular transmembrane receptors (ace2), and this affinity is 10 to 20 times higher to sars-cov-2. the viral binding with ace2 receptors is the main mechanism of viral entry into the epithelial cells mainly of the lungs (type ii pneumocytes), intestines (enterocytes), kidneys and blood vessels. this entry takes place through the binding of the viral protein s (spike) receptor, more specifically of the receptor binding domain (rbd) region with the host cell transmembrane ace2 receptor. then, after the intronization of sars-cov-2 and the ace2 receptor, the s protein is cleaved by the transmembrane serine protease tmprss2 and the viral rna is released into the host cell, being ready to initiate the viral replication. the ace2 receptor, in turn, will undergo cleavage by tace (tumor necrosis factor alpha-converting enzyme), also known as adam 17, which is a metalloprotease that allows the shedding of the ace2 ectodomain, known as soluble or circulating ace2, into the extracellular space. soluble ace2 is enzymatically active and apparently able to bind with sars-cov-2, supporting the rationale for the administration of recombinant human ace2 to reduce the deleterious inflammatory effects of sars-cov-2 infections 8 . covid-19 symptomatology is nonspecific and includes fever, dry cough, dyspnea, headache, myalgia, fatigue, nausea, vomiting and abdominal pain 5 . in children, covid-19 symptoms do not differ from those of adults, although a higher proportion of asymptomatic and oligosymptomatic patients has been reported. in addition, there is a more prolonged viral excretion in respiratory secretions for up to 22 days after the onset of symptoms and in feces for more than 30 days 9 . cardiac complications associated with covid-19 have been frequently reported in adult patients and some of them can occur in severely ill children. the pathophysiology of cardiovascular complications involves the direct viral injury, hypoxemia, hemodynamic instability leading to hypoperfusion, enhanced systemic inflammation, local regulation of the expression of ace2 receptors, enhanced production of catecholamines and a higher incidence of medication toxicity. these signs and symptoms lead to chest pain due to acute cardiac injury (8-12%), heart failure (23-52%), arrhythmia (8.9-16.7%), acute myocarditis and ultimately shock 10 . sars-cov-2 can invade endothelial cells and enterocytes of the small intestine, being detected in fecal samples, esophagus, stomach, duodenal and rectal samples. patients present with anorexia (26.8%), diarrhea (12.5%), nausea and vomiting (10.2%), abdominal pain (9.2%) 11 . acute kidney injury due to sars-cov-2 infections occur in 0.5% of the patients overall, and in up to 23% of the severe cases that may progress to renal failure, sometimes requiring hemodialysis 12 . ace2 receptors are also present in the nervous system and in skeletal muscles, suggesting a mechanism for sars-cov-2-related neuromuscular injuries. dizziness (16.8%), headache (13.1%), skeletal muscle injury (10.7%) are more frequently seen, but covid-19 can also lead to impaired consciousness (7.5%), acute cerebrovascular disease (2.8%), ataxia (0.5%), seizures (0.5%), meningoencephalitis and the guillain-barré syndrome 13 . olfactory and gustatory disorders have been associated with viral infections and sars-cov-2 is not an exception. hyposmia (5.1-20.4%), anosmia (79.6%), dysgeusia (8.5%) and ageusia (1.7%) are the most common symptoms. a recent study has shown that ace2 is also highly expressed on the oral mucosa, granting the virus an easy access to the host 14 . this finding may explain, at least partially, the dysgeusia and ageusia reported in covid-19. on the contrary, a reduced expression and activity of ace2 receptors on the oral, nasal and respiratory epithelium of children has been reported and could explain the lower proportion of serious lung injuries of covid-19 in children 15 . although ace2 receptors have been detected in the retina, choroid and conjunctival epithelia, ocular involvement associated with sars-cov-2 infection, mainly acute conjunctivitis, has been reported in 31.6% of the patients 16 . skin lesions are unfrequently observed in covid-19, are highly polymorphic and have been reported in some pediatric patients presenting with chilblain-like skin lesions, urticarial eruptions, diffuse or disseminated erythema multiforme-like lesions, livedo racemosa, livedo reticularis, blue toe syndrome, retiform purpura, purpuric, non-purpuric or flexural exanthema, vesicles, acro-ischemic lesions 17 . another interesting feature of covid-19, observed so far in adult patients, is the higher proportion of deaths in men with respect to women. estrogens seem to participate in the upregulation of ace2 receptors activity leading to higher amounts of circulating ace2 in women. a recent report has shown that inhibitors of the 5 alpha reductases, a class of drugs commonly prescribed for prostatic disorders, dampen the androgen signaling and can reduce the expression of ace2 receptors and of the transmembrane serine protease tmprss2, theoretically leading to a decrement in sars-cov-2 binding with ace2 receptors 18 . long-term cigarette smokers and obese people have reduced circulating ace2 levels. in diabetics, the circulating ace2 down regulates the release of insulin by pancreatic beta cells worsening the glucose control. in pregnant women, the fluctuation of hormones, mainly of progesterone, may drastically upregulate the expression of ace2 receptors in the uterus and in other organs such as the kidneys, suggesting that pregnant women with covid-19 could be at higher risk to develop more severe infections 19 . in summary, it is possible that higher levels of ace2 in the circulation binding with sars-cov-2 could reduce the number of viruses able to bind with ace2 receptors. a trend toward this same kind of association has been proposed in children, who generally have higher levels of circulating ace2. in children from 6 months to 17 years of age, ace2 levels are 13-100 u/liter in comparison with 9-67 u/liter in adults, according to enzyme activity tests 20 . zimmermann and curtis 21 claimed that children have the same chance of contracting covid-19 than adults, but they develop more intestinal symptoms and less severe conditions. children with covid-19 generally have contact with an infected family member, while the exposure of adults usually occurs in hospitals. ong et al. 22 called attention to the group of children under one year of age, in whom the occurrence of severe cases of covid-19 is more frequent than in older children. lymphopenia, so common in adults (70%) occurs in only 3.5% of children, and the increment of inflammatory markers and cytokines are not so marked, except in the multisystem inflammatory syndrome associated with covid-19 (mis-c) cases. liguoro et al. 23 carried out a systematic review on children and adolescents with covid-19 (0-18 years), through the analysis of 65 articles published from february to may 2020, totaling 7,480 children, 52.1% of boys with a mean age of 7.6 years. most infections were mild (42.5%) or moderate (39.6%), only 2% needed icu and the mortality rate was 0.08%. however, the proportion of newborns with severe infections was 12%; 40% of them developed dyspnea, while in the other age groups the most frequent symptoms were fever (51.6%) and cough (47.3%). laboratory tests did not show significant changes; 73.9% of the patients underwent chest tomography and 67.3% of these exams revealed abnormalities. lu et al. 24 described 1,391 children in wuhan with severe acute respiratory syndrome. of these, 171 (12.3%) had covid-19, the mean age was 6.7 years, 60.8% were boys, 41.5% had fever, 48.5% cough, 64.9% pneumonia, three children needed icu (0.2%) and one child died (0.07%). a multicenter cohort study involving 82 healthcare institutions across 25 european countries included 582 children with pcr-confirmed sars-cov-2 infection, 62% of whom were inpatients and 8% admitted to the icu. the median age was 5 years, and 87% of children did not require respiratory support at any time, while 4% received mechanical ventilation for a median of seven days. the factors associated with icu admissions were being less than 30 days of life, male sex, preexisting comorbidities and lower respiratory tract symptoms on admission. four children, all of them older than 10 years, died 25 . jiang et al. 26 described 161 children with severe acute respiratory syndrome hospitalized in wuhan, from whom 239 respiratory viruses were identified with predominance of the respiratory syncytial virus in 76 children (31.80%) and influenza a in 72 (30.13%). sars-cov-2 was confirmed in only two cases by rt-pcr (0.84%), both children were coinfected by respiratory syncytial virus and human metapneumovirus or human metapneumovirus and metapneumovirus. the identification of coinfections could explain the severity of respiratory symptoms in these children. recent data reported a severe multisystem inflammatory syndrome in children (mis-c), adolescents and young adults with respiratory symptoms and positive rt-pcr to sars-cov-2, or even without respiratory symptoms, but with positive sars-cov-2 serology. clinical signs and symptoms are highly variable and include some characteristics of the kawasaki disease, with a tendency to thrombus formation and progression to shock. there is persistent fever, high levels of inflammatory markers such as c-reactive protein, troponin and cytokines. more than 50% of these patients present with cutaneous rash, abdominal pain, nausea, vomiting and diarrhea, and curiously, less than 50% of them have respiratory symptoms. some patients needed icu to receive cardiac (blood pressure maintenance) and respiratory support (pediatric intensive care society, 2020). whether this multisystem inflammatory syndrome (mis-c) associated with covid-19 will increase in incidence overtime, it is probably dependent on the host genetic susceptibility and/ or the pathogenicity of sars-cov-2 genetic variants 27,28 . feldstein et al. 27 conducted a surveillance on the multisystem inflammatory syndrome in children in the us and found 186 patients in 26 states. inclusion criteria were serious illness leading to hospitalization, age of less than 21 years, fever that lasted for at least 24 hours, laboratory evidence of inflammation, multisystem organ involvement, and evidence of infection caused by sars-cov-2. in this group, the median age was 8.3 years and 62% of patients were male. organ-system involvement included the gastrointestinal system (92%), cardiovascular (80%), hematological (76%), mucocutaneous (74%) and respiratory (70%). the median duration of hospitalization was seven days. due to the severity of illness, most children needed intensive care (80%), mechanical ventilation (20%) and vasoactive drugs support (48%). four children (2%) died. coronary-artery aneurysms were documented in 15 patients (8%), and kawasaki disease-like features were documented in 74 (40%). most patients (171 or 92%) had elevations in at least four biomarkers of inflammation. elizabeth dufort, the new york state and the centers for disease control and prevention formed the multisystem inflammatory syndrome in children investigation team (2020). they identified 191 cases in hospitalized patients younger than 21 years of age, reported by hospitals in the new york state with the diagnosis of kawasaki disease, toxic shock syndrome, myocarditis, and suspected multisystem inflammatory syndrome associated with covid-19 in children (mis-c). in this group of patients, 95 had laboratory-confirmed acute or recent sars-cov-2 infection and four patients met clinical and epidemiologic criteria for mis-c. thirty-one patients (31%) were between 0-5 years of old, 42 (42%) were 6-12 years; 26 (26%) were 13-20 years. fifty-three patients (54%) were male. all of them presented with subjective fever or chills; 97% had tachycardia, 80% had gastrointestinal symptoms, 60% had cutaneous rash, 56% had conjunctivitis and 27% had mucosal changes. increased levels of c-reactive protein, d-dimer and troponin were found in 100%, 91%, and 71% of the patients, respectively; 62% received vasopressor drugs, 53% had evidence of myocarditis, 80% were admitted to icus and two patients died 28 . another relevant issue is whether the severity and prognosis of covid-19 in children and adolescents with comorbidities are worse than in previously healthy counterparts. hrusak et al. 29 investigated 200 children undergoing cancer treatment and nine of them (4.5%) contracted covid-19, eight with mild symptoms and one asymptomatic, suggesting that the coexistence of cancer and covid-19 did not cause complications. minotti et al. 30 in a systematic review of 16 articles in which 116 adult patients and children with cancer, organ transplantation, primary or secondary immunodeficiencies were analyzed, concluded that immunosuppressed patients had more severe covid-19, but no worse prognosis, as there were no deaths among the studied patients. opposing the previous studies, shekerdemian et al. 31 analyzed 48 children and adolescents hospitalized due to covid-19 in icus in canada and the us. fifty-two percent were boys with a median age of 13 years, 83% with comorbidities, 76% had respiratory symptoms, 38% required ventilation mechanical, 23% presented with failure of two or more organs, 4% died and 31% remained hospitalized by the time the study was completed. the authors concluded that pediatric sars-cov-2 infections are actually less frequent in children than in adults and have a much better prognosis. however, the presence of comorbidities in the pediatric age group seems to act as a risk factor, as in adults, considering the number of symptomatic children with comorbidities who required mechanical ventilation, had multiple organs failure and died. regarding covid-19 during pregnancy and the possibility of vertical transmission, data in the literature are still scarce, but suggest that vertical transmission of sars-cov-2 may occur, although fetal malformations have not yet been described. nevertheless, there is evidence that indicates a higher frequency of hospitalizations in pregnant women, as in the context of influenza virus infections, and data around the world indicate a favorable prognosis of covid-19 in this group. on the contrary, a recent brazilian review based on the ministry of health reported data from february 26 to june 18, 2020, found 124 deaths of pregnant or postpartum women with covid19, corresponding to a high mortality rate of 12.7% 32 . lokken et al. 33 analyzed 46 pregnant women in the us with a mean age of 29 years, who contracted covid-19 predominantly in the second and third trimesters, two thirds presenting overweight or obesity. the infection was symptomatic in 93.5% of women with fever, cough, loss of smell and taste (30%) and dyspnea. the median duration of symptoms was 24 days. seven women (15%) needed hospitalization and one was admitted to the icu, with a favorable outcome. severe cases were associated with overweight and obesity, in addition to asthma, diabetes, hypertension and hypothyroidism. there was one induced preterm birth (33 weeks) due to the mother's respiratory failure and the neonate was uninfected. in addition, there was one stillbirth with placenta and fetal tissues testing negative for sars-cov-2 and cmv. in another us study, pierce-williams et al. 34 analyzed a cohort of 64 pregnant women hospitalized due to covid-19, 69% with severe infections and 31% in critical conditions. comorbidities were pre-existing lung (25%) and heart disease (17%), and a mean body mass index of 34 kg/m 2 . the mean gestational age was 30 weeks and hospitalizations occurred within seven days after the onset of symptoms and lasted for six days. there was one reversed cardiac arrest and no deaths; 88% of pregnant women had premature deliveries, 94% via cesarean sections. there were no stillbirths, neonatal deaths, or vertical transmission of sars-cov-2. zaigham and andersson 35 in a review of 18 articles on pregnant women and neonates published between december 8, 2019 and april 10, 2020, analyzed 108 pregnant women, mostly in the third trimester, with fever (68%), cough (34%), lymphopenia (58%) and high c-reactive protein (70%). most women (91%) had cesarean sections and three required icu; there were no maternal deaths, but one intrauterine death and one neonatal death were reported. it was not possible to exclude the vertical transmission of sars-cov-2 as fetal and neonatal samples, as well as placentas were not tested. in italy, ferrazzi et al. 36 studied 42 pregnant women with covid-19; 24 (57.1%) had vaginal delivery and 18 (42.9%) underwent elective cesarean sections. pneumonia was diagnosed in 19 pregnant women (54.2%), seven of them (36.8%) required respiratory support and four (21.1%) needed intensive care. two women breastfed their newborns without masks because maternal sars-cov-2 infection was only suspected after delivery, being confirmed in these two women. the two neonates have also tested positive some days after their mothers' diagnoses. in another case, the neonate tested positive a few days after a vaginal delivery, suggesting that this type of delivery may increase the risk of sars-cov-2 transmission. the only case in the literature suggestive of congenital sars-cov-2 infection so far, was that of a 29-year-old chinese primiparous woman, with 34 weeks and five days of gestational age, presenting with high fever, cough, progressing to respiratory distress. the chest ct showed a pattern suggestive of covid-19 and rt-pcr was positive for sars-cov-2 in nasopharyngeal secretions. at 35 weeks of gestation, she was hospitalized due to the unfavorable progression of respiratory symptoms, and other four rt-pcrs were performed and remained positive. three weeks after admission, she underwent serology for sars-cov-2, which was also positive (igm 279.72 au/ml and igg 107.89 au/ml). on that same occasion, the vaginal secretion was negative for sars-cov-2 by rt-pcr. due to the respiratory conditions, the patient, wearing a n95 mask, underwent a cesarean section and had no contact with the neonate, a girl weighing 3,120 g, with apgar score of 9 and 10 on the first and fifth minutes of life, without respiratory symptoms. at two hours of life, the neonate's serology was collected and revealed the presence of anti-sars-cov-2 igg (140.32 au/ml) and igm (45.83 au/ml). cytokines (il6 and il10) were also elevated, as well as the leukocyte count. the chest ct was normal and five rt-pcrs in the neonate's respiratory secretions performed from two hours of life until 160 days of age were negative. serology was repeated two weeks after the first collection, still revealing high levels of igg (69.94 au/ml) and detectable, although lower, igm antibody titers (11.75 au/ml). unfortunately, the amniotic fluid and the placenta were not tested for sars-cov-2, but the finding of igm antibodies in the neonate at two hours of life suggests that the infection was transmitted during pregnancy, since igm antibodies do not cross the placenta as do igg antibodies 37 . the chest tomography has been a key exam in elucidating the pulmonary involvement in sars-cov-2 infections due to the characteristic images of ground glass patching opacities found until the fourth day after the onset of symptoms, progressing to consolidations in large extensions of the lungs 38 . abnormalities in ancillary laboratory exams are also less frequent in children than in adults: blood count with leukopenia, lymphopenia (70% of adults vs. 3.5% in children), cd4 and cd8 lymphocyte depletion, thrombocytopenia (16-32%), changes in the neutrophil/ lymphocyte ratio in poorer prognosis cases. increased lactate dehydrogenase (dhl), aspartate aminotransferase, alanine aminotransferase, c-reactive protein (crp), ferritin, creatine kinase (ck) and d-dimer can also be present, as well as coagulation disorders, thrombotic events and the anti-phospholipid syndrome. significant increments of il2, il4, il6, il7, il10, tnf alpha, chemokine ligand 2 (ccl2), ccl3, ccl5, interferon gamma-induced protein 10 (ip10), characterizing the cytokine storm, can be found in more severe covid-19 cases 39 . the laboratory diagnosis of covid-19 are based on the detection of viral rna by real time amplifications (rt-pcr) 40 or the detection of antibodies (immunoglobulins) anti-sars-cov-2 from the classes igm, iga and igg, produced by the host's immune system. antibodies can be detected by enzyme immunoassays (elisa, eia) whose results are based on optical densities, reflecting the amount of antigen-antibody complexes, or through the detection of chemiluminescent signals, which are proportional to the amount of antigen-antibody complexes present in the sample [40] [41] [42] . after the exposure to the virus, the individual undergoes an incubation period of 1-2 weeks. viral detection by rt-pcr is possible from one week before the onset of symptoms, reaching a maximum positivity on the week of onset of symptoms. the positivity is still possible during the second week and it rapidly falls onwards 41 . the molecular assay, called rt-pcr (reverse transcriptase-polymerase chain reaction) is a test in which respiratory secretions undergo rna extraction, followed by a reverse transcription step, in which viral rna is converted into complementary dna (cdna) with the aid of an enzyme, the reverse transcriptase. then, there is the amplification step in which one or more sars-cov-2 targets (fragments of sars-cov-2 genes such as the envelope gene (e), nucleocapsid (n), spike (s), rna-dependent rna polymerase (rdrp ), several orfs, are amplified and these amplification products are identified and hybridized by fluorochrome-labeled probes, making amplification more specific, but at a higher cost. although it is possible to quantify the number of viral copies of the target genes, in most studies rt-pcr for sars-cov-2 has been used only qualitatively, generally releasing positive or negative results, which should be understood by clinicians as viruses detected or not detected in that specific sample 40 . hase et al. 43 reported a case of covid-19 with pneumonia and negative rt-pcr in an oropharyngeal swab. new oropharyngeal and induced sputum specimens were collected five days after hospitalization, being positive in sputum and negative in oropharynx, indicating that a negative rt-pcr in respiratory samples is not enough to rule out covid-19. in addition, three patients presenting with chest computed tomography suggestive of covid-19 but persistent negative rt-pcr from days 6 to 8 after the onset of symptoms, were very likely to be false-negatives of rt-pcr, suggesting a lack of sensitivity of rt-pcr in nasopharyngeal swabs depending on the time elapsed from the onset of symptoms 14 . even with sensitivity limitations, the detection of viral rna is considered the gold standard for the diagnosis of covid-19 in respiratory secretions, however, false negative results in the order of 25-30% have been reported. the detection of the virus depends on several aspect: presence of sufficient viruses in the sample, collection of respiratory samples during the "ideal" phase for viral detection, ideally within the first week after the onset of symptoms, adequate collection protocols, samples storage and processing procedures. inadequate procedures can limit the performance of molecular tests. rt-pcr sensitivity also varies according to the type of biological material. in a study of 1,070 samples from 250 patients with covid-19, the sensitivity of rt-pcr performed with orf1ab primers was: bronchoalveolar lavage (93%), sputum (72%), nasal swab (63%), oropharynx swab (32%), feces (29%), blood (1%) and there was no detection in urine samples. respiratory secretion swabs were collected 1-3 days after the onset of symptoms. although the best biological material would the bronchoalveolar lavage (93% sensitivity), in clinical practice this invasive procedure is not feasible and for this reason the analysis of nasopharyngeal and oropharyngeal (63%) and oropharynx (32%) secretions combined was adopted, expecting the analysis to reach around 70% sensitivity 44 . although there is no consensus, the most widely used rt-pcrs in the world to detect sars-cov-2 are those recommended by the centers for disease control (cdc) of usa 45 , the charité virology (berlin, germany) 46 , and the department of virology at the university of hong kong medical school and school of public health 47 . in the rt-pcr recommended by the cdc-usa, there is a simultaneous amplification of three distinct regions of the viral nucleocapsid (n1, n2 and n3) and of a control region from the rnase p, with hybridization of amplification products by fluorescent probes. the test is positive when it amplifies n1, n2 and n3 in addition to rnase p, and is negative when only rnase p is amplified. the test is invalid when none of the amplification products appears and is inconclusive (must be repeated after a new rna extraction) if the rnase p is positive together with n1 or n2 or n3 45 . in the diagnostic system described by corman et al. 46 in germany (charité virology), there is a first rt-pcr performed with primers from the envelope gene (e) that detects the subgenus sarbecovirus of the b-line of coronaviruses. these primers will recognize both, sars-cov and sars-cov-2, and amplification products are detected by a fluorescent probe. in the second rt-pcr, the target gene is rdrp, and in addition to the primer pair, there are two probes labeled with distinct fluorochromes, one recognizing sars-cov and sars-cov-2 and one specific to sars-cov-2. in the rt-pcr proposed by the hong kong university (hku), two reactions are carried out separately, the first one with orf1b primers (nsp14) and the second one targeting the n gene (viral nucleocapsid). the two amplification products are recognized and hybridized to fluorescent probes 47 . initial studies indicated that the rt-pcr performed with primers from the rdrp and e genes had high analytical sensitivity, being able to detect 3.6 -3.9 viral copies per reaction, while the amplifications performed with primers from the nucleocapsid gene detect 8.3 viral copies per reaction 48 . however, commercial rt-pcr kits for the detection of sars-cov-2 targeting the e, n and orf 1ab genes did not perform well when clinical samples were tested, reaching sensitivities ranging from 968 copies/ml to 7,744 copies/ml, making the occurrence of false negatives very likely 49 . serological assays, such as rapid immunochromatographic tests or immune assays with simple reading of optical densities or detection of fluorescent or chemiluminescent signals are faster, less expensive and easier to perform than rt-pcr [40] [41] [42] . anti-sars-cov-2 igm antibodies can be detected in covid-19 between 3-7 days after the onset of symptoms, on week 2 of illness, reaching peak levels on week 3 and beginning to decrease on weeks 4 and 5, but they can still be detected, depending on the method, on week 6. regarding the production of iga antibodies, some studies have reported their appearance at the same time as igm antibodies and others, along with igg antibodies. in any case, in covid-19, iga antibodies seem to present with higher levels than igm, with some studies indicating a more adequate diagnostic performance of the combined analysis of iga and igg antibodies in relation to igm and igg. as for igg antibodies, some studies have detected iga antibodies a few days after igm antibodies, while others, beginning 14 days after the onset of symptoms, remaining detectable for at least 6 weeks, but there is no information on long-term protection provided by these antibodies 40, 41 . lassaunière et al. 42 compared the efficiency of nine commercial tests, three elisa and six immunochromatographic kits. specificities were 100% for wantai sars-cov-2 total antibody elisa kit, 93% for euroimmun iga elisa and 96% for euroimmun igg elisa with sensitivities of 90%, 90% and 65%, respectively. among the six immunochromatographic kits (dynamiker biotechnology, ctk biotech, autobio diagnostics and artron laboratories) only acro biotech (80%) and hangzhou all test biotech (87%) showed specificity problems in detecting igm. then, sensitivities were analyzed in four periods: up to 6 days of infection, 7-13 days, 14-20 days and > 21 days. for elisa kits, sensitivities ranged from 40-86% in the earliest phase, 67-100% in the intermediate phase and 78-89% in the last two phases, with the chinese kit performing better than those from euroimmun. the performance of the immunochromatographic kits was worse than the elisas in the early phase, but it was comparable to elisa wantai total ab elisa and elisa euroimmun iga considering the four periods combined. huang et al. 50 evaluated an elisa assay for sars-cov-2 in plasma samples of confirmed cases of covid-19 or suspected cases with negative rt-pcr. in those with confirmed diagnosis, the average duration of igm and iga antibodies in the circulation was five days, and igg antibodies were detected from the 14 th day after the onset of symptoms. elisa-igm was more sensitive than rt-pcr in detecting cases from the 5 th day after the onset of symptoms. in the case of sars-cov-2, the main target of neutralizing antibodies is precisely the s protein, more specifically the rbd (receptor binding domain) region that will inhibit the viral recognition by the ace2 cell receptor. wu et al. 51 , followed-up the neutralizing antibody levels in 175 adult patients with mild or moderate covid-19 by means of a sensitive and reproducible neutralization assay. the mean age of the patients was 50 years, with 53% of women. the average hospital stay was 16 days, with the disease lasting 21 days. antibodies detection used a neutralization assay with a pseudovirus (psv), a technique widely used for the quantification of neutralizing antibodies in other viral infections, as the ones caused by influenza viruses, ebola virus, mers-cov and sars-cov. the quantification of neutralizing antibodies from recovered individuals was determined on the day of hospital discharge. low levels of neutralizing antibodies were detected until the 10 th day. from that day until the 150 th day of infection, concentrations of neutralizing antibodies have raised and remained stable afterwards, however, only 14% of the patients had high titers of neutralizing antibodies, in general older patients who had more prolonged infections, being only these the potential donors of plasma for patients with covid-19 in severe or critical conditions. the immunochromatographic tests with the best performance to date are the colloidal-gold-immunochromatographic assays (gica). xiang et al. 52 tested 126 serum samples from covid-19 patients, with 57.1% igm sensitivity, 100% specificity and 69% accuracy. for igg, they found 81.3% sensitivity, 100% specificity and 86.5% accuracy. the combined sensitivity (igm and igg) was 82.4%. pan et al. 53 also using gica, tested 134 samples from patients with covid-19 confirmed by rt-pcr, in addition to suspected cases with negative rt-pcr. the combined sensitivity (igm and igg) was 11.1% at the beginning of the infection (1-7 days after the onset of symptoms), 92.9% between 8-14 days and 96.8% after 15 days. surprisingly, the detection of igg in suspected cases with negative rt-pcr was 43.6%. knowledge on the treatment of sars-cov-2 is rapidly evolving, and it is essential that pediatricians are up to date, looking systematically for new guidelines and recommendations on the management of covid-19 in childhood 54 . there is no definitive evidence of any specific effective therapy for treating children with covid-19 so far. the outpatient management of children consists basically of symptomatic and supportive care, along with isolation measures. children at home should be closely monitored for signs of clinical deterioration: shortness of breathe or severe respiratory distress, chest pain, cyanosis and clinical signs associated with shock. clinical deterioration may occur suddenly and immediate reevaluation by a pediatrician is recommended 54, 55 . the length of home isolation is uncertain and depends on the duration of viral shedding which in turn is also variable. the who criteria (who 2020) for discharging patients from isolation without requiring a new specific test to sars-cov-2 are 10 days after the onset of symptoms for symptomatic patients, in addition to at least three additional days without symptoms. in the case of asymptomatic patients, at least 10 days after the last positive test to sars-cov-2 56 . children with severe clinical manifestations should be hospitalized. the basis for the management of covid-19 infections in hospitalized children is to provide supportive treatment 55, 56 . the team responsible for the care of the child must ensure an adequate respiratory support, including supplemental oxygen and noninvasive or invasive ventilatory support, as needed. children with severe illness and respiratory failure need special attention regarding the adequate fluid and electrolyte supplementation and they should be monitored for bacterial pneumonia and other secondary infections, with strict clinical control and through cultures and other microbiological tests. the empirical use of antibiotics may be initially necessary, but their maintenance should be based, whenever possible, on the identification of bacteria by culture methods 57 . there is a paucity of data on the use of antivirals for covid19 treatment in the pediatric population, and there is no indisputable evidence in the medical literature to support their use in children to date. antivirals may be considered only for children with severe or critical disease in the presence of a positive test to sars-cov-2 and on a case-by-case basis. as there are no definitive data establishing what are the risk factors to be considered in criteria for the progression of covid-19 to a serious or critical medical condition in children, a panel of experts extrapolated data from adult patients to establish possible risk factors in children that could be used to indicate the prescription of antivirals, such as the presence of immunological impairment, especially the ones revealing a severe immunocompromised status, young age, severe underlying cardiac and/or pulmonary disease, obesity, diabetes, among other conditions 57 . as there are no definitive studies on the use of antivirals for covid-19 in children, the panel of experts recommended that their use should be restricted only to severe cases, when potential benefits not yet perceived could outweigh possible risks and injuries, and whenever possible within a clinical trial. if an antiviral is to be used, remdesivir is, for the moment, the drug of choice 57 . for children presenting with the multisystem inflammatory syndrome (mis-c), there are currently no published guidelines from who, cdc or ecdc regarding the treatment of covid-19, or studies comparing the efficacy of various treatment options. a group of researchers from the university of california made some recommendations for the management of mis-c based on the extrapolation of data from other syndromes. children with clinical features meeting the criteria for kawasaki disease should be treated according to current recommendations, with high doses of intravenous immunoglobulins and aspirin. for children with severe disease or persistent inflammation, corticosteroids should be considered. in children with clinical manifestations consistent with a secondary hemophagocytic lymphohistiocytosis activation syndrome, the initial use of anakinra (recombinant and slightly modified version of the human interleukin 1 receptor antagonist protein) and intravenous immunoglobulins is suggested. the addition of pulsed corticosteroids and other immunosuppressive agents, such as cyclosporine and tacrolimus can be considered in severe cases 58 . although the advances made in the research of sars-cov-2 and covid-19 in such a short time are undeniable, at present we still do not have a consensual etiological treatment or a vaccine with proven efficacy. even so, it is important that pediatricians are kept up to date in relation to the particularities of covid-19 in pediatric patients, new findings on the virus itself, its characteristics and diagnosis and new therapeutic approaches. ly: made the final selection of the most interesting articles that would be part of the review based on the summary on molecular biology, serology, virology, epidemiology, and physiopathology. made the selection of the articles on maternal, fetal and neonatal infections; ehs: carried out a survey of the bibliography on the molecular diagnosis and summarized the selected articles; lsp: carried out the survey of the bibliography on the serological diagnosis and summarized the selected articles; mcr: carried out the survey of the bibliography on the virological and epidemiological aspects of sars-cov-2, sars-cov and mers and summarized the selected articles; kak: carried out the survey of the bibliography the physiopathology of sars-cov-2 infections, summarized the selected articles and organized all the references of the manuscript; mgv: revised the manuscript as a specialist in pediatric infectious diseases and wrote the management of patients' section; tso: idealized the review article, selected the manuscripts of children with and without comorbidities and covid19 and wrote the final manuscript. all the authors have read and approved the final version of the manuscript and declare that they have no conflict of interests whatsoever. world health organization. criteria for releasing covid-19 patients from isolation sars and mers: recent insights into emerging coronaviruses understanding evolution of sars-cov-2: a perspective from analysis of genetic diversity of rdrp gene emerging genetic diversity among clinical isolates of sars-cov-2: lessons for today the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak real estimates of mortality following covid-19 infection epidemiological and clinical characteristics of 26 asymptomatic sars-cov-2 carriers sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection in children and adolescents: a systematic review the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status gastrointestinal manifestations of sars-cov-2 infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis kidney involvement in covid-19 and rationale for extracorporeal therapies neurologic manifestations of hospitalized patients with coronavirus disease computed tomographic imaging of 3 patients with coronavirus disease 2019 pneumonia with negative virus real-time reversetranscription polymerase chain reaction test nasal gene expression of angiotensin-converting enzyme 2 in children and adults ocular manifestations of a hospitalised patient with confirmed 2019 novel coronavirus disease chilblain-like lesions in children following suspected covid-19 infection androgen regulates sars-cov-2 receptor levels and is associated with severe covid-19 symptoms in men physiological and pathological regulation of ace2, the sars-cov-2 receptor covid-19 infection and circulating ace2 levels: protective role in women and children coronavirus infections in children including covid-19: an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children coronavirus disease 2019 in critically ill children: a narrative review of the literature sars-cov-2 infection in children and newborns: a systematic review sars-cov-2 infection in children covid-19 in children and adolescents in europe: a multinational, multicentre cohort study coinfection of sars-cov-2 and multiple respiratory pathogens in children 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northern italy: a retrospective analysis possible vertical transmission of sars-cov-2 from an infected mother to her newborn time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia laboratory abnormalities in children with mild and severe coronavirus disease 2019 (covid-19): a pooled analysis and review sars-cov-2 and the covid-19 disease: a mini review on diagnostic methods serological approaches for covid-19: epidemiologic perspective on surveillance and control evaluation of nine commercial sars-cov-2 immunoassays a case of imported covid-19 diagnosed by pcr-positive lower respiratory specimen but with pcr-negative throat swabs detection of sars-cov-2 in different types of clinical specimens 2019-ncov) real-time rt-pcr diagnostic panel detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia diagnostic detection of wuhan coronavirus 2019 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with coronavirus: a rapid review to support covid-19 antimicrobial prescribing multi-system inflammatory syndrome in children (mis-c) following sars-cov-2 infection: review of clinical presentation, hypothetical pathogenesis, and proposed management key: cord-353484-q7d0ysbo authors: liu, xue; liu, chao; liu, gang; luo, wenxin; xia, ningshao title: covid-19: progress in diagnostics, therapy and vaccination date: 2020-06-19 journal: theranostics doi: 10.7150/thno.47987 sha: doc_id: 353484 cord_uid: q7d0ysbo coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has recently become a pandemic. as the sudden emergence and rapid spread of sars-cov-2 is endangering global health and the economy, the development of strategies to contain the virus's spread are urgently needed. at present, various diagnostic kits to test for sars-cov-2 are available for use to initiate appropriate treatment faster and to limit further spread of the virus. several drugs have demonstrated in vitro activity against sars-cov-2 or potential clinical benefits. in addition, institutions and companies worldwide are working tirelessly to develop treatments and vaccines against covid-19. however, no drug or vaccine has yet been specifically approved for covid-19. given the urgency of the outbreak, we focus here on recent advances in the diagnostics, treatment, and vaccine development for sars-cov-2 infection, helping to guide strategies to address the current covid-19 pandemic. the rapid spread of sars-cov-2, a novel coronavirus that emerged in december 2019, and the resulting disease, namely, coronavirus disease 2019 (covid19) , has posed a serious global public health emergency. whole-genome sequencing results show that the causative agent is a novel coronavirus that was initially named 2019-ncov by the world health organization (who) [1] [2] [3] . covid-19 was subsequently recommended as the disease name. as the genomic sequence of the new virus is closely related to that of severe acute respiratory syndrome (sars) coronavirus (sars-cov), the international committee on taxonomy of viruses (ictv) officially designated the virus sars-cov-2 [4] . patients infected by sars-cov-2 manifest a range of symptoms, such as dry cough, fever, headache and dyspnea, and are generally diagnosed with viral pneumonia [5, 6] , but asymptomatic infections have also been reported [7, 8] . coronaviruses, a genus in the coronaviridae family, are enveloped, positive-sense, single-stranded rna viruses with the largest known genomes (from 25 to 32 kb) among rna viruses [9, 10] . coronavirinae is subdivided into four genera: alpha-, beta-, gamma-, and delta coronaviruses [11] . phylogenetic analysis of coronavirus genomes has revealed that sars-cov-2 is a new member of the beta coronavirus genus, which includes sars-cov and middle east respiratory syndrome (mers) coronavirus (mers-cov), as well as other viruses that infect humans and diverse animal species. among the several coronaviruses that are pathogenic to humans, most are associated with mild clinical symptoms (e.g., hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1) [12, 13] , with three notable exceptions: sars-cov in november 2002 [14] [15] [16] , mers-cov in april 2012 [17] [18] [19] , and, more recently, sars-cov-2 causing covid-19. sars-cov provoked a large-scale epidemic beginning in china and involving 37 countries with approximately 8,000 cases and 774 deaths [20] ; mers-cov began in saudi arabia and caused a total of 2,519 mers cases with 866 associated deaths as of the end of january 2020 [21] . similar to sars-cov and mers-cov, the sars-cov-2 genome encodes nonstructural proteins (nsps, such as 3-chymotrypsin-like protease, papain-like protease, helicase, and rna-dependent rna polymerase), structural proteins and accessory proteins [22] . sars-cov-2 has four structural proteins: the spike (s) protein, envelope (e) protein, membrane (m) protein, and nucleocapsid (n) protein. among these proteins, the trimeric s protein is indispensable for virus-cell receptor interactions during viral entry [23, 24] . s protein comprises an n-terminal s1 subunit responsible for virus-receptor binding and a c-terminal s2 subunit responsible for virus-cell membrane fusion. s1 is further divided into an n-terminal domain (ntd) and a receptor-binding domain (rbd) [25, 26] . sars-cov-2 targets cells through the s protein, which binds to the human angiotensin-converting enzyme 2 (ace2) receptor and employs the cellular serine protease tmprss2 for s protein priming [27] [28] [29] [30] [31] . such binding triggers a cascade of events leading to fusion between the cellular and viral membranes for cell entry. the viral rna genome is released into the cytoplasm after membrane fusion (see the sars-cov-2 lifecycle in figure 1 ). polyproteins are subsequently synthesized to encode the viral replicase-transcriptase complex. the viral rna is then synthesized by rna-dependent rna polymerase. synthesis of structural proteins is followed by viral particle assembly and release [32, 33] . these viral lifecycle steps provide potential targets for vaccines and therapeutics to prevent and treat sars-cov-2 infection. promising targets include nsps, which are involved in transcription and replication of the virus, as the key enzymes in the viral lifecycle. additional targets include viral entry and immune regulation pathways. for example, the s protein plays key roles in the induction of t cell responses and neutralizing antibodies (nabs), as well as protective immunity, during infection with sars-cov-2. to elucidate the specific mechanisms of sars-cov-2 infection, the crystal structure of the sars-cov-2 spike rbd bound to the cell receptor ace2 at 2.45 å resolution was recently determined [34] . the overall binding mode of the sars-cov-2 rbd with ace2 is nearly identical to that of the sars-cov rbd, which also utilizes ace2 on the surface of host cells as a receptor. hela cells expressing ace2 are susceptible to sars-cov-2 infection, but cells without ace2 expression are not infected [1] . in vitro binding experiments also show that the sars-cov-2 rbd has affinity for ace2 in the low nm range, indicating that the rbd is the key functional component of the s1 subunit responsible for the binding of ace2 to sars-cov-2 [34] . more structural information at the atomic level would greatly enhance our understanding of the interaction between sars-cov-2 and host cells, providing precise targets for nabs, and assist us in urgently needed structure-based vaccine design in the ongoing fight against sars-cov-2. in addition, research on cell modeling tools might promote insight into our understanding of infection mechanisms and speed up the development of therapeutic agents and vaccines for covid-19 [35] . in this review, we aim to discuss the current status of diagnostics, therapeutics, and vaccines against covid-19. as rapid diagnostics and development of drugs and vaccines for the virus are important interventions to control the outbreak, a review article that encompasses the current research on this topic may provide help for guiding strategies to address the current covid-19 pandemic. rapid and accurate detection of covid-19 is essential to initiate the appropriate treatment rapidly, to limit further spread of the virus and to ultimately eliminate the virus from circulation. covid-19 patients present a wide range of clinical symptoms (e.g., cough, fever, and dyspnea) that are similar to influenza or other respiratory infections and thus cannot be used for accurate diagnosis [36, 37] . at present, accurate and rapid diagnosis has evolved as a tool for detecting this novel coronavirus ( table 1) . molecular-based approaches are the first-line methods to confirm suspected cases. nucleic acid testing is the main technique for laboratory diagnosis. as with other emerging viruses, the development of methods to detect antibodies and viral antigens began after identification of the viral genome. in fact, the genomic sequence of sars-cov-2 was released to public databases on january 10, 2020 (genbank accession no. mn908947), which facilitated the development of standardized laboratory pcr protocols for covid-19 in january of 2020 [38] . this was followed by the launch of a range of commercial sars-cov-2 pcr-based assays in the last 3 months [39] [40] [41] [42] [43] [44] [45] . following entry of the virus into the host cell, viral genomic rna is released and translated into viral polymerase proteins. in this process, subgenomic (−) rnas are synthesized and used as a template to form subgenomic (+) messenger rnas (mrnas). the nucleocapsid (n) structural protein and viral rna are replicated, transcribed, and synthesized in the cytoplasm, whereas other viral structural proteins, including the s protein, membrane (m) protein and envelope (e) protein, are transcribed and then translated in the endoplasmic reticulum (er). the resulting structural proteins are further assembled into the nucleocapsid and viral envelope at the er-golgi intermediate compartment (ergic) to form a mature virion, followed by release of the nascent virion from the host cell. antigen-detection low complexity; rapid; easy to perform best used to identify acute or early infection; more prone to false negatives [68, 69, [83] [84] [85] [86] reverse transcription quantitative pcr (rt-qpcr) is the most common and straightforward method for the detection of sars-cov-2 owing to its advantages as a specific, sensitive and simple quantitative assay, which greatly helps in the diagnosis of early infection. as of may 25, a total of 12 rt-qpcr tests to detect sars-cov-2 have been approved in china by the national medical products administration (nmpa) [46] . although rt-qpcr assays using respiratory samples provided sensitive and specific diagnostic tests at the initial phase of the outbreak, these rt-qpcr test kits have many limitations [47] . for example, this method is not efficient in rapidly screening a large number of individuals in places where thousands of people transit per hour. in addition, the performance of rt-qpcr depends on many factors, such as the sample type, stage of infection, skill of sample collection, and quality and consistency of the pcr assay being used [48, 49] . these problems lead to a noteworthy delay in early diagnosis and subsequent management and pose a serious challenge to providing timely life-support treatment and preventive quarantine. notably, another powerful and promising tool involves clustered regularly interspaced short palindromic repeats (crispr) technology, which is being quickly deployed in the molecular diagnostics landscape. on may 6, sherlock biosciences received emergency use authorization (eua) from the us food and drug administration for its sherlock™ crispr sars-cov-2 kit for detection of the virus that causes covid-19. based on the sherlock (specific high-sensitivity enzymatic reporter unlocking) method, the kit works by programming a crispr molecule to detect the presence of a specific genetic signature for sars-cov-2 [50] . if the signature is found, the crispr enzyme generates a fluorescent glow. furthermore, mammoth biosciences has developed a rapid (<40 min), easy-to-implement and accurate crispr-based assay for the detection of sars-cov-2 from respiratory swab rna extracts called the sars-cov-2 dna endonuclease-targeted crispr trans reporter (detectr) [51] . crispr-based diagnosis methods benefit from high sensitivity and specificity with efficiency and no requirement for elaborate instrumentation. as the assay uses similar specimen collection and nucleic acid extraction methods as the cdc and other rt-qpcr assays, it is subjected to the same potential limitations with regard to the availability of personal protective equipment, extraction kits and reagents. therefore, many clinicians have proposed that computed tomography (ct) scans should be a necessary auxiliary diagnostic method [52] [53] [54] [55] [56] . to identify and quarantine patients at an early date, patients clinically diagnosed by chest ct with typical ground-glass lung opacities were also counted as confirmed cases beginning in early february [57] [58] [59] [60] . however, ct scans also have some shortcomings, such as the hysteresis of abnormal ct imaging and indistinguishability from other viral pneumonia presentations. accordingly, a combination of chest ct scans and repeated rt-qpcr tests may be helpful for individuals with a high clinical suspicion of sars-cov-2 infection but who are negative by rt-qpcr screening. compared to the currently used nucleic acid detection and ct scans, other methods, such as virus antigen or serological antibody testing, are advantageous with fast turn-around times, high throughput and reduced workload for the detection of novel coronavirus infection. currently, immunological identification technologies (point-of-care testing (poct) of igm/igg and enzyme-linked immunosorbent assay (elisa)) kits for sars-cov-2 have become available, with detection rates higher than those of nucleic acid detection approaches. one type of rapid diagnostic test based on host antibody detection has become available more recently [61] [62] [63] [64] . antibodies are produced over days to weeks after infection with the virus. antibody testing is easy to administer and only requires minimal training. it may be helpful as a complement to nucleic acid testing for the diagnosis of suspected covid-19 cases with negative rt-qpcr results as well as for the identification of asymptomatic infections [65] . confirming suspected cases as early as possible with the benefit of antibody testing might reduce exposure to risk factors during repeated sampling and save valuable rt-qpcr tests. due to urgency and demand, many antibody test reagents are rapidly being developed and made available on the market to assist in the diagnosis of sars-cov-2 infection, including kits for testing total abs, igm antibody or igg antibody, by chemiluminescence, elisa or colloidal gold methods. on march 6, an anti-sars-cov-2 antibody test kit based on chemiluminescent particle immunoassay jointly developed by our team and beijing wantai biological pharmacy enterprise co., ltd. was approved for listing by the state drug administration for emergency approval and is the best performing commercial kit according to the latest who validation data [66] . the kit is based on the double-antigen sandwich principle that detects total antibodies (including igm, igg, iga and other antibody types). specific testing for sars-cov-2 has great significance for public health and clinical practice, and evidence from studies further demonstrate that rt-qpcr detection combined with serological testing enhances diagnosis sensitivity while maintaining high specificity [67] . another type of rapid diagnostic test (rdt) that detects the presence of viral antigens expressed by sars-cov-2 virus in a respiratory tract sample is of low complexity and may provide results typically within 30 minutes [68, 69] . the antigen(s) detected are expressed only when the virus is actively replicating; thus, such tests are best used to identify acute or early infection. monoclonal antibodies (mabs) against the nucleocapsid protein of sars-cov-2 have been developed and might constitute the basis of a future rapid antigen detection test. as of the time of this review, at least 5 antigen-detection rdts were under evaluation (https://www.finddx.org/covid-19/ sarscov2-eval-immuno/), though not all are ce marking approved or widely available. to date, there are no specific antiviral therapies or vaccines for covid-19 available for human use. given the urgency of treating these patients, rather than taking months to years to develop and test compounds from scratch, researchers are seeking to repurpose drugs that are already approved for other diseases and have acceptable safety profiles. small-molecule agents approved for other human diseases can exert their antiviral effect through multiple mechanisms, including blocking viral entry, inhibiting a virally encoded enzyme, targeting a host factor required for replication, or blocking virus particle formation [87] . notably, some antivirals and antimalarials have shown promising abilities to treat patients and reduce the danger of covid-19 ( figure 2 , table 2 ). remdesivir (gs-5734) is a nucleotide analog prodrug that shuts down viral replication by inhibiting a key viral enzyme, rna polymerase. a recent study reported that remdesivir potently blocks sars-cov-2 infection (ec50 = 0.77 μm) in vero e6 cells [88] , and the first patient with confirmed sars-cov-2 infection in the united states recovered after receiving intravenous remdesivir in january [89] . on april 10, a study reported clinical outcomes for compassionate use of remdesivir in a small cohort of patients with severe covid-19. specifically, clinical improvement was observed in 36 of 53 patients (68%) [90] . however, a trial (clinicaltrials.gov: nct04257656) found that compared with placebo, intravenous remdesivir did not provide significant clinical or antiviral effects in patients with serious covid-19. ongoing trials with larger sample sizes will provide information on the efficacy of remdesivir for covid-19 [91] . on may 1, the us food and drug administration granted emergency use authorization for remdesivir to treat covid-19 in adults and children hospitalized with severe disease. chloroquine (cq), a 9-aminoquinoline that is a widely used antimalarial and autoimmune disease drug, has recently been reported as a potential broad-spectrum antiviral drug [92] . cq inhibits virus infection by increasing the endosomal ph required for virus/cell fusion, and a recent study reported its activity against covid-19 (ec50=1.13 μm) in vero e6 cells [88, 93] . patients with covid-19 are being recruited in randomized trials (e.g., clinicaltrials.gov: nct04316377, nct04323527) to determine the safety and efficacy of cq. although a few clinical trials have suggested some beneficial effects of cq for covid-19, most of the reported data are preliminary [94, 95] . furthermore, it is likely that the risk of adverse reactions to cq will hamper the successful treatment of patients with covid-19. hydroxychloroquine (hcq), which exhibits an antiviral effect highly similar to that of chloroquine, might be a better therapeutic approach. studies have indicated that cq shows antagonism against sars-cov-2 in vitro. chen et al. partially confirmed the potential of hcq in patients with covid-19 (clinicaltrials.gov: chictr2000029559) [96] . although positive results from small studies have been reported for hydroxychloroquine, accumulating trial and observational evidence has prompted skepticism regarding whether there are any meaningful clinical benefits for patients with covid-19. indeed, a study from france did not support the use of hcq in patients hospitalized with covid-19 who require oxygen [97] . additionally, a randomized clinical trial (clinicaltrials.gov: chictr2000029868) from china shows that hospitalized patients with mild to moderate persistent covid-19 who received hcq did not eliminate the virus more quickly than those receiving standard care. moreover, adverse events were higher among hcq recipients than nonrecipients [98] . nonetheless, randomized clinical trials are needed to provide definitive causal evidence while offering an opportunity to continuously optimize the treatment plan. the lopinavir-ritonavir combination, which targets an enzyme that cleaves a long protein chain during the assembly of new viruses, can inhibit hiv protease [99, 100] . the combination has been evaluated in patients with sars and mers, though the results are ambiguous [101] [102] [103] [104] . clinical trials (e.g., clinicaltrials.gov: nct04255017, nct04307693) with covid-19 have been initiated to test the potency of the combination. recently, a randomized, controlled, open-label trial (clinicaltrials.gov: chictr2000029308) involving 199 patients with covid-19 was initiated, but no benefit beyond standard care was observed with lopinavir-ritonavir treatment. future trials including patients with severe illness may help to confirm or exclude the possibility of a treatment benefit. favipiravir (t-705), a purine nucleic acid analog approved for the treatment of influenza, can effectively inhibit the rna-dependent rna polymerase (rdrp) of rna viruses such as influenza, ebola and norovirus [105] . studies in vero e6 cells have suggested that favipiravir can cripple the sars-cov-2 virus (ec50 = 61.88 μm) [88] , and patients with covid-19 are being recruited in randomized trials to evaluate the efficacy of favipiravir plus other antivirals (e.g., clinicaltrials.gov: chictr2000029600, chictr2000029544). camostat mesylate is a potent serine protease inhibitor that has been used primarily to treat pancreatitis and some types of cancer [106] . sars-cov-2 uses ace2 for entry and the serine protease tmprss2 for s protein priming. utilizing research on sars-cov and the closely related sars-cov-2 cell entry mechanism, it has been demonstrated that camostat mesylate can block sars-cov-2 infection of lung cells [28] . patients with covid-19 are being recruited in randomized trials (clinicaltrials.gov: nct04374019, nct04355052) to determine whether this drug reduces sars-cov-2 viral load in early covid-19 disease. the antibody-mediated humoral response plays crucial roles in controlling viral infection. nabs, which can reduce viral infectivity by binding to the surface epitopes of viral particles and thereby blocking entry of the virus into host cells, are a promising approach to prevent and treat virus infection. passive antibody therapy, through transfusion of convalescent plasma from patients who have recovered from viral infections, can also be used as a treatment. importantly, this therapy offers the only short-term strategy of conferring immediate immunity to infected patients. the antibodies present in convalescent plasma can specifically bind to a given pathogen (e.g., an infectious virus), directly neutralizing its infectivity. other antibody-mediated pathways, such as antibody-dependent cellular cytotoxicity, complement activation, and/or phagocytosis, may also contribute to their therapeutic effect. there are numerous examples in which convalescent plasma therapy has been successfully applied to treat patients, as follows: sars; mers; h1n1 (pandemic 2009 influenza a); h5n1 (avian influenza a); several hemorrhagic fevers, such as ebola; and other viral infections [116] [117] [118] [119] [120] . as no specific therapeutic agents or vaccines are available for covid-19, this therapy is the only strategy that is immediately available for use to prevent and treat a novel, emerging infectious disease such as sars-cov-2 infection [121, 122] . shen et al reported findings from a preliminary study of 5 severely ill patients with covid-19 who were given plasma from recovered individuals [123] . in this case series, the clinical status of all patients improved by approximately 7 days after transfusion. in addition, the patients' specific nab titers increased following the transfusion, and respiratory samples became negative within 12 days. although the limited case size and study design preclude a definitive statement about the potential efficacy of this treatment, the results provide some evidence to support the possibility of evaluating this therapy in more rigorous investigations involving patients with covid-19. moreover, sophisticated technologies are devoted to identifying strongly neutralizing mabs that can be produced in large quantities. in coronaviruses, the viral component most frequently targeted by nabs is the surface-located envelope s protein [124] [125] [126] . to this end, several potently neutralizing human mabs against sars-cov-2 have been developed using various approaches, including single-cell culturing methods of memory b cells isolated from sars-cov-2 patients, fusing b cells with an appropriate partner to produce hybridomas, selection of antibodies from phage-display libraries of human antibody fragments, and hybridoma fusion of b cells from immunized transgenic mice encoding human variable immunoglobulin domains [127] [128] [129] [130] [131] . all of these antibodies were selected based on their in vitro neutralizing capacity, and most of them target the sars-cov-2 rbd. ju et al. isolated 206 mabs specific for the sars-cov-2 rbd derived from single b cells of eight sars-cov-2-infected individuals [130] . using bioinformatic and biologic characterization, several mabs with potent binding and neutralizing activity against pseudovirus encoding the s protein and live sars-cov-2 have been identified. in particular, the most potent antibodies, p2b-2f6 and p2c-1f11, out-competed ace2 with close to 100% efficiency, indicating that blocking the rbd and ace2 interaction is a useful surrogate for antibody neutralization. the results indicate that the humoral immune response is viral species specific and diverse and can produce potent nabs. wu et al. isolated 4 mabs specific to sars-cov-2 rbd from a convalescent patient [131] , and these antibodies effectively neutralized the sars-cov-2, with two of them exhibiting additive inhibition effects. moreover, a therapeutic study in a mouse model validated that these antibodies can reduce virus titers in infected lungs. these antibodies will serve as the most promising candidates for the development of prophylactic and therapeutic interventions against sars-cov-2. cd147-spike protein, a receptor on host cells, is a novel route for sars-cov-2 invasion [132] . meplazumab, an anti-cd147 antibody, effectively inhibited sars-cov-2 replication and virus-induced cytopathic effects in vero e6 cells; it also blocks cd147, the receptor of the proinflammatory factor cypa, thus attenuating inflammation [132, 133] . based on this evidence, the team designed an open-label, concurrent controlled trial (nct04275245) to assess the efficacy and safety of meplazumab in patients with covid-19 pneumonia. the data demonstrated that meplazumab efficiently improved the recovery of the patients, with a favorable safety profile. however, the results were limited by the absence of a parallel control and a small sample size. therefore, confirmation from a larger scale and randomized, double-blind trial is required to fully assess the efficacy of meplazumab in patients with covid-19 pneumonia. the s proteins of sars-cov-2 and sars-cov, which are structurally very similar, have an amino acid sequence identity of approximately 77% and commonly bind ace2 as a host receptor [134] [135] [136] . to reduce the time that is required to develop sars-cov-2-neutralizing antibodies de novo, researchers have assessed available sars-cov antibodies for cross-neutralization of sars-cov-2, given the high degree of sequence similarity. for example, a sars-cov rbd-specific human nab (cr3022) previously isolated from a convalescent sars patient is able to bind the sars-cov-2 rbd with high affinity (kd of 6.3 nm) and recognize an epitope on the rbd that does not overlap with the ace2-binding site [137] . however, cr3022 does not neutralize sars-cov-2 in vitro, and it is possible that this epitope can confer in vivo protection [138] . further studies will require suitable animal models, which have yet to be established. another anti-sars-cov antibody, 47d11, was isolated from transgenic h2l2 mice encoding the human immunoglobulin variable regions to generate antibodies that neutralize sars-cov-2 (and sars-cov). 47d11 binds a conserved epitope on the spike rbd, explaining its ability to cross-neutralize sars-cov and sars-cov-2, with diverse mechanisms of action that are independent of receptor binding inhibition [139] . this antibody offers potential for the prevention and treatment of covid-19. although antibodies are generally beneficial and protective, in rare cases, pathogen-specific antibodies could promote pathology, resulting in a phenomenon known as antibody-dependent enhancement (ade) [140, 141] . the ade phenomenon is documented for dengue virus and other viruses [142, 143] . however, the neutralizing antibodies can be engineered of fc region with molecular precision to avoid the potential ade. vaccination can be used to prevent infection or to reduce disease severity, viral shedding and thereby transmission, thus helping to control sars-cov-2 outbreaks. multiple strategies have been employed to generate sars-cov-2 vaccines, including dna-and rna-based vaccines, viral vector vaccines, inactivated virus vaccines, live-attenuated virus vaccines and recombinant protein vaccines (figure 3) . according to reports from the who, there are more than 100 vaccines against sars-cov-2 at various stages of development. specifically, ten candidate vaccines are already in clinical evaluation ( table 3) . mrna vaccines are an emerging new vaccine modality in which patients are treated with mrna oligonucleotides that encode either an antigen of interest or an antigen as well as its viral replication machinery [144] . the potential advantages of the mrna approach to prophylactic vaccines include the ability to mimic natural infection to stimulate a more potent immune response, combining multiple mrnas into a single vaccine, rapid discovery to respond to emerging pandemic threats and manufacturing agility derived from the platform nature of mrna vaccine design and production [145] . mrna vaccines have elicited potent immunity against infectious disease targets in animal models of infection with influenza virus, zika virus, rabies virus and others, especially in recent years, using lipid-encapsulated or naked forms of sequence-optimized mrna [146] [147] [148] [149] . after two doses, mrna-1273 elicited neutralizing antibody titer levels at or above the levels in convalescent sera in all initial participants. to continue progress on this potential vaccine during the ongoing global public health emergency, moderna intends to work with the fda and other government and nongovernment organizations to be ready for phase 2 and any subsequent trials, which are anticipated to include a large number of subjects and will seek to generate additional safety and immunogenicity data. furthermore, biontech and pfizer are jointly developing another mrna vaccine (bnt162), which has been approved for a phase 1/2 clinical trial to determine the optimal dose for further studies as well as to evaluate the safety and immunogenicity of the vaccine. the trial is the first clinical trial of a covid-19 vaccine candidate to start in germany and is part of a global development program. dna vaccines are appropriate for emerging infectious diseases because they allow for the rapid design of multiple candidates for novel antigens [150] . in addition, they are directly injected or otherwise inoculated into recipients. recently, various dna vaccine platforms have been developed to improve the efficacy of vaccines by using electroporation (ep) to deliver plasmids and adding adjuvants to enhance immune responses. inovio pharmaceuticals was the first to advance its vaccine (ino-4700) against mers-cov [151] , a related coronavirus, into evaluation in humans, and this company has developed a dna vaccine (ino-4800) to prevent infection by sars-cov-2. ino-4800 induces t cell activation by delivering dna plasmids that express the sars-cov-2 s protein. the effects based on measuring functional antibodies and antigen-specific t cell responses in multiple animal models are particularly encouraging [152] . inovio has also initiated an open-label trial (nct04336410) to evaluate the safety, tolerability and immunological profile of ino-4800 administered by intradermal (id) injection followed by ep using a cellectra® 2000 device in healthy adult volunteers. viral vector vaccines, which function as viral gene expression and delivery systems, rely on a host viral genome, and an unrelated viral genome lacking packaging elements is engineered to encode antigenic components of the virus of interest to elicit an immune response. because viral vector vaccines persist in the host as genetic material, they directly infect antigen-presenting cells and have strong immunogenicity, efficiently inducing b cell-and t cell-mediated immune responses. furthermore, viral vector vaccines can result in high-titer nabs. several different virus vectors have been developed as sars-cov-2 vaccines. cansinobio and the beijing institute of biotechnology (bib) have collaborated to develop an adenovirus type 5 vector-based novel coronavirus vaccine ("ad5-ncov") that encodes the full-length s protein of sars-cov-2. ad5-ncov protects against sars-cov-2 infection by relying on a recombinant replication-defective human adenovirus type-5 vector to induce the immune response. the vaccine candidate is built upon cansinobio's adenovirus-based viral vector vaccine technology platform, which has also been successfully applied to develop a globally innovative vaccine against ebola virus infection. the results from preclinical studies of ad5-ncov show that the vaccine candidate can induce a strong immune response in animal models, and preclinical animal safety studies demonstrate a good safety profile. this is currently the first novel coronavirus vaccine for covid-19 that has progressed to this stage in china. a first-in-human trial showed that the ad5-ncov vaccine is tolerable and immunogenic in healthy adults [153] , with specific humoral responses against sars-cov-2 peaking at day 28 postvaccination and rapid specific t-cell responses noted at day 14. there is potential for further investigation of the ad5-ncov vaccine for the control of the covid-19 outbreak. furthermore, university of oxford researchers have begun testing a new covid-19 vaccine called chadox1 ncov-19; it is derived from a virus (chadox1) that is an attenuated version of a common cold virus (adenovirus) causing infections in chimpanzees, but it has been genetically modified such that it is unable to proliferate in humans. the vaccine is based on an adenovirus vector (chadox1) and the sars-cov-2 s protein. the results showed that a single vaccination with chadox1 ncov-19 is effective for preventing lung damage upon high-dose challenge with sars-cov-2; however, all of the monkeys vaccinated with chadox1 ncov-19 became infected when challenged, and there was no difference in the amount of viral rna detected from nasal secretions in the vaccinated and unvaccinated monkeys [154] . as of may 13, more than 1,000 volunteers participated in the trial, which aims to assess whether healthy people can be protected from covid-19. it will also provide valuable information on the safety aspects of the vaccine and its ability to generate good immune responses against the virus. in addition to adenovirus, our team and the university of hong kong have used influenza virus vectors for the development of candidate sars-cov-2 vaccines that are in preclinical phases. inactivated virus vaccines use chemicals (such as formalin, β-propiolactone and diethylpyrocarbonate) or heat to render the viral genome noninfectious while maintaining the virion structure, thus preserving antigenicity but eliminating the potential to cause productive infection. thus, in theory, inactivated virus vaccines are easily prepared and antigenically similar to live viruses. such vaccines have been found to be effective and safe for the prevention of diseases caused by viruses such as poliovirus and influenza virus [155, 156] [157] . other vaccine approaches include liveattenuated vaccines that are produced by reducing or eliminating the virulence of a live virus, typically using chemical-driven or site-directed mutagenesis. thus, the virus is capable of productive infection, but the resulting disease is either diminished or eliminated. live-attenuated vaccines can elicit both innate and adaptive immune responses, and protection can be life-long. additionally, their production is inexpensive. codagenix and the serum institute of india are using viral deoptimization to synthesize "rationally designed", live-attenuated vaccines against the emergent coronavirus. recombinant-protein-based vaccines are advantageous over other types of vaccines in that they are safe and have few side effects, inducing the immune system without the introduction of infectious viruses. one example is the nvx-cov2373 vaccine developed by novavax, which is in the clinical stage [158] . it was created using recombinant nanoparticle technology to generate antigen derived from the s protein and contains matrix-m™ adjuvant to enhance the immune response and stimulate high levels of neutralizing antibodies. a trial of clover's s-trimer vaccine developed by clover biopharmaceuticals will be recruiting healthy adults in australia, and this vaccine will be widely available upon confirmation of its safety and efficacy. in addition, lv-smenp-dc and pathogen-specific aapc vaccines from shenzhen geno-immune medical institute are in phase 1 clinical trials to evaluate safety and immune reactivity [159,160]. despite tremendous efforts to prevent the spread of sars-cov-2 worldwide, the high mortality and person-to-person transmission pose a significant threat to global public health. diagnostics are important for dealing with virus outbreaks because they can enable healthcare workers to direct efforts to patients with covid-19. remarkably, various diagnostic kits to test for covid-19 are available. several drugs have demonstrated in vitro activity against the sars-cov-2 virus or potential clinical benefits in observational or small, nonrandomized studies. additionally, researchers are evaluating several different technologies, and more than 100 vaccines are under development against sars-cov-2 worldwide. specifically, ten candidate vaccines have already been administered to volunteers in safety trials; others have started testing in animals. however, as the development of vaccines for human use can take years, their development will likely come too late to impact the first wave of the pandemic. these challenges will remain in the long term. clearly, there is an urgent need for effective antiviral therapy and vaccines for this emerging global threat. there are a series of patient-associated and virologic factors that pose major clinical challenges to the development of anti-sars-cov-2 drugs. sars-cov-2 is a virus with diversity that becomes mutated, and novel sars-cov-2 mutants are likely to emerge repeatedly at unpredictable times [161, 162] . therefore, most drugs that specifically target the existing sars-cov-2 may not be effective against a mutant sars-cov-2. the lag time between mutant emergence and the development of new prophylactic treatments is of concern. thus, there is an urgent need for the development of new, broad-spectrum drugs that target conserved sites to prepare for future outbreaks involving mutants. recent studies have reported that lipopeptide ek1c4 shows exceptional promise to be developed as the first pan-cov fusion inhibitor-based antiviral therapeutic or prophylactic for treatment or prevention of infection by the currently circulating sars-cov-2 and mutants or emerging sars-related covs [163] [164] [165] . in addition, combining the advanced knowledge of bioinformatics and immunology to screen and confirm the conserved dominant epitopes of sars-cov-2, it will be helpful to develop therapeutic antibodies with broadspectrum neutralization activity and vaccines that can induce a broad-spectrum antiviral immune response. currently, there are a limited number of animal models available for infections caused by sars-cov-2. normally, antiviral therapy or vaccines enter into human trials after tests for safety and effectiveness in appropriate animal models. however, the virus does not grow in wild-type mice and induces only mild disease in transgenic animals expressing human ace2 [166, 167] . the limited availability of mouseadapted virus strains and ace2 transgenic mice remains a major obstacle to testing anti-sars-cov-2 drugs or vaccines. other potential animal models include ferrets and nonhuman primates (nhps), for which pathogenicity studies are ongoing. in addition, understanding how the immune system interacts with the pathogen and the vaccine itself is crucial for avoiding pitfalls, such as ade phenomenon, a process in which a virus leverages antibodies to promote infection. the viral load curve of covid-19 is similar to that of influenza, and the viral load is very high at the time of presentation [168, 169] . experience from the treatment of influenza patients, in this case of high-viral load patients, suggested that the combination of multiple antiviral drugs is more effective than single drug treatments [170] [171] [172] . indeed, the combination of appropriate antiviral drugs might rapidly suppress the high initial viral load, improve the clinical parameters, and reduce the risk to medical staff by controlling the spread of the virus. a multicenter randomized open-label phase 2 trial (chictr2000029308) showed that triple antiviral therapy with interferon beta-1b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavirritonavir alone in alleviating symptoms, suppressing shedding of sars-cov-2, and facilitating discharge of patients with mild to moderate covid-19 [168] . there are also many other studies investigating combination therapy; these smaller clinical studies examining virologic endpoints may be sufficient to identify which combinations can be used for large studies in risk groups or hospitalized patients. for combinations involving immunomodulatory interventions, the 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infection (covid-19) novavax initiates phase 1/2 clinical trial of covid-19 vaccine. revised 25 international expansion of a novel sars-cov-2 mutant attenuated sars-cov-2 variants with deletions at the s1/s2 junction inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein the pathogenicity of sars-cov-2 in hace2 transgenic mice pathogenesis of sars-cov-2 in transgenic mice expressing human angiotensin-converting enzyme 2. cell. in press triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial medical treatment of viral pneumonia including sars in immunocompetent adult investigating different mechanisms of action in combination therapy for influenza international severe acute r, emerging infection c. antiviral combinations for severe influenza efficacy of clarithromycin-naproxen-oseltamivir combination in the treatment of patients hospitalized for influenza a(h3n2) infection: an open-label randomized, controlled, phase iib/iii trial this work was supported by the xiamen science and technology major project the authors have declared that no competing interest exists. key: cord-346987-fbqqf00i authors: guo, yongwen; jing, yan; wang, yunshi; to, aileen; du, shufang; wang, liuzheng; bai, ding title: controls of sars-cov-2 transmission in orthodontic practice date: 2020-06-05 journal: am j orthod dentofacial orthop doi: 10.1016/j.ajodo.2020.05.006 sha: doc_id: 346987 cord_uid: fbqqf00i abstract the coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has attracted worldwide concerns because of its high person-to-person infectivity and lethality, and it was labeled as a pandemic as the rapid increase of confirmed cases in most areas around the world became evident. the sars-cov-2 is mainly transmitted through respiratory droplets and close contact. there are also evidences of transmission through aerosols and digestive tracts. since orthodontic treatment involves large population who need routine return-visits, it was significantly affected and suspended because of the covid-19 pandemic and the shutdown of the dental clinics and hospitals. although the spread of covid-19 has been effectively controlled in china and many areas have gradually resumed work and classes, orthodontic participants are still under high risks of sars-cov-2 infection. this is due to the asymptomatic carriers of sars-cov-2 or patients in the incubation period may cause the cross infection between orthodontic practitioners and patients. the close proximity between the practitioners and the patients, and the generation of droplets and aerosols that contain saliva and blood during treatment further increase the risks of transmission. in this review, we summarized the preventive strategies for controls of sars-cov-2 transmission to protect both staffs and patients during the orthodontic practice. since its emergence in december 2019, the coronavirus disease 2019 has spread rapidly and is now a global pandemic. the pathogen causing covid-19 was initially named 2019-novel coronavirus (2019-ncov), and then officially named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). a public health emergency of international concern over this disease has been announced by the world health organization (who) since 30th january 2020 1 . due to the particularity of the dental treatment procedures, the risk of sars-cov-2 transmission between dental practitioners and patients could be high 2 . thus, all the routine dental practices were suspended after the outbreak of covid-19 in many areas around the world, and only emergency services were provided. 3 as the covid-19 has been effectively controlled in china and some other areas, dental clinics and hospitals are gradually resuming regular services. however, the prevention and the control of sars-cov-2 transmission during dental practice are still serious and challenging. the most critical reason is that that asymptomatic patients and patients in their incubation period are also carriers of sars-cov-2 and have the ability to be infectious. 4 it is difficult to identify and quarantine these patients in time, which can result in the sars-cov-2 transmission in the population. what's more, the close contact between dental staffs and patients as well as the droplets and aerosols generated during treatment containing saliva and blood further increase the risk of sars-cov-2 transmission in dental practice 5 . in addition, due to the previous suspension, many orthodontists are currently under heavy workload to reschedule the accumulated return-visit patients whose treatments were significantly affected and postponed. a study from jordan found that although most jordanian dentists were aware of covid-19 symptoms, mode of transmission, infection control and measures in dental clinic, they had limited knowledge of the extra precautionary measures that are essential to protect the dental staffs and other patients from sars-cov-2 infection. 6 thus, the standard control measures in our previous daily orthodontic work may not be enough to prevent the transmission of sars-cov-2 and protect both practitioners and patients from the covid-19. effective control protocols during orthodontic practice are urgently needed. 7 the most common manifestations of the covid-19 infected patients are fever and dry cough. some have fatigue, diarrhea and other digestive tract symptoms. severe patients can rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation dysfunction and multiple organ failure. 8 most patients who underwent chest computed tomography (ct) showed bilateral pneumonia with ground-glass opacity and bilateral patchy shadows. 9 however, some patients who are sars-cov-2 carriers could be asymptomatic. 4 patients in the incubation period could also do not show the above typical symptoms. the incubation period of covid-19 has been estimated at 3 to 14 days with an average of 5 to 6 days, 10,11 but there is also evidence that it could be as long as 24 days. 12 studies have reported that majority of patients (50-80%) were considered asymptomatic at the early phase of infection but released large amounts of sars-cov-2, which were infectious and posed enormous challenges for controlling the spread of covid-19. 13, 14 the sars-cov-2 has been found the evidence of rapid person-to-person transmission and susceptible to different age groups in clinical epidemiology studies. 11 the virus is mainly transmitted by respiratory droplets form talking, coughing or sneezing, and direct or indirect contact with nasal, oral, and eye mucous. 15 it has been reported that sars-cov-2 was widely distributed on floors, computer mice, trash cans, sickbed handrails, and in air 4 meters away from patients in hospital wards. 16 another study also found that sars-cov-2 could remain viable and infectious on different types of environmental surfaces for a few hours or up to several days. 17 the contact with these contaminated surfaces would largely increase the risk of transmission. more importantly, studies have indicated that aerosol transmission of sars-cov-2 is plausible, since the virus in aerosols can stay viable and infectious for about 3 hours. [17] [18] [19] in addition, a growing number of clinical evidence reminds us that digestive system may also serve as an alternative route of infection as sars-cov-2 could be detected from the stool specimen of the confirmed patient. 20,21 sars-cov-2 could also be detected in the self-collected saliva of most infected patients even not in nasopharyngeal aspirate, suggesting the possibility of salivary gland infection and possible transmission. 22, 23 another study also suggested that ace2 (angiotensin-converting enzyme ii)-expressing cells in oral mucosa, especially in epithelial cells of tongue, might provide possible routes of entry for the sars-cov-2, which indicates that the oral cavity is a potentially high risk route for sars-cov-2 infectious susceptibility. 24 during the orthodontic practice, the patients' mouth and nose are in close proximity with orthodontists and assisting staffs for long periods. the communication, coughing, or sneezing during the practice can easily bring out the respiratory droplets. direct contact with saliva or blood of the infected patients during orthodontic procedures within the mouth include photographing, impression taking, oral scanning, bracket and attachment bonding or removal, archwire changing, anchorage screw implantation, and so on. indirect contact with contaminated dental settings and environmental surfaces will also increase risk for the virus transmission. the use of orthodontic tools and materials, including pliers, power chains, and adhesives which are usually not individually packaged or disposable, will increase the risk of cross infection. also, the use of high-speed handpieces and high-pressure 3-way syringes during bracket or attachment bonding and removal will generate a large number of saliva or blood-mixed droplets and aerosols which could remain suspended in the air for long periods before they settle on environmental surfaces or enter the respiratory tract. 5, 25 in addition, fecaloral routes may also be a potential transmission route during orthodontic procedures, especially the self-operation by patients, such as the placement and removal of the clear aligners, elastics and other removable appliances. thus, the major transmission routes of sars-cov-2 in orthodontic practice are respiratory droplets, direct or indirect contact, saliva or blood-mixed aerosols and the digestive tract. effective control strategies to prevent the transmission of sars-cov-2 through these routes are needed. 7 although vaccines are the most effective strategy for preventing infectious disease, research groups around the world are accelerating the development of covid-19 vaccines using various approaches, but there are still no vaccines available for covid-19 currently. 26, 27 therefore, it is critical to take infection prevention and strengthen the organization and management of the clinic or hospital, make systematic plans and procedures for the prevention and control of covid-19, and carry out training to all staffs including cleaners and security personnel to make sure everyone is aware. pay close attention to the changes of the pandemic situation and adjust the control strategies according to the management requirements of the national and local health administrative institutions. strictly perform the procedures of patient evaluation and pre-examination and triage before orthodontic practice to achieve early detection, early reporting and early isolation for covid-19 suspected or infected patients. manage only orthodontic emergencies in areas where covid-19 spreads, non-emergency orthodontic practices should be postponed. 32, 33 after the effective control of covid-19 in the area, first-visit or return-visit patients should be carefully scheduled and avoid the aggregation of patients and their companies. reinforce the hand hygiene, use personal protective equipment, and take disinfection measures for dental settings, tools and medical wastes to avoid iatrogenic infection. reduce the use of high-speed dental handpieces and high-pressure 3-way syringes during the practice, while increasing the use of saliva ejectors with high volume to minimize the environment pollution by droplets and aerosols. before entering the dental clinics or hospitals, each patient should be evaluated at the entrance whether they are suspected cases of covid-19. to date, the national health commission of the people's republic of china has released the 7th edition of the guideline for the diagnosis and treatment of covid-19. according to the guideline, first, the temperature of each patient should be taken using contact-free forehead thermometers. then, a questionnaire should be used to screen the suspected patients take different actions according to the triage of the patients as shown in the flow chart of figure 1 . notably, staffs responsible for temperature measurements and questionnaire should take protective measures as recommended in table 1 . what's more, a safety distance of at least 6 feet or 2 meters between people should be maintained while completing the questionnaire or in the waiting area in case that they spray droplets from their nose or mouth which may contain virus when someone coughs, sneezes, or speaks. 34, 35 in addition, patients are also suggested to wear face masks during the pre-examination and triage procedures as well as the waiting for treatment. 34 strict hand hygiene is a simple and effective way to cut off the spread of the virus. to reinforce the compliance of hand washing, a two-before and three-after hand hygiene guideline is proposed. specifically, the two-before is to wash hands before patient examination and before treatment procedures, the three-after is to wash hands after touching the patient, after touching the surroundings and equipment without disinfection and after touching the oral mucosa, blood, body fluid, and so on. also, orthodontic staffs should avoid touching their own eyes, mouth and nose. 7 hand hygiene should be also applied before eating as well as before and after using the bathroom to avoid the fecal-oral transmission. 21 since droplets, contact, and aerosols are the major transmission routes of sars-cov-2 during orthodontic practice, barrier-protection equipment is strongly recommended for all orthodontic staffs. the protective measures include wearing disposable working cap, disposable surgical mask, working clothes, protective goggles or face shield, disposable latex gloves or nitrile gloves, and disposable isolation clothing as well as waterproof boot covers. the protective levels and equipment for different applications are recommended in table 1 . notably, the orthodontist and the assistant working in close proximity to the patient are recommended the highest level of protection to wear the hooded medical isolation clothing that cover the body as much as possible, especially during the use of high-speed handpieces and high-pressure 3-way syringes. in addition, it is recommended to wear two layers of latex or nitrile gloves during long practice procedures. it is critical to pay special attention to the operation of the tools, especially sharp instruments. in case of inadvertent laceration, it would increase the risk of infection as the virus can enter the punctured skin directly and cause the infection. to avoid indirect contact with contaminated protective equipment, the order of wear and removal is critical during the epidemic period of the covid-19, it is recommended to manage only orthodontic emergencies to prevent further harm, such as brackets debonding, archwire/ligature wire deformation or shifting, oral mucosa irritations, and anchorage implants loosening. [37] [38] [39] nevertheless, orthodontists or assistants should evaluate the emergencies before allowing the patients to come to the office by requesting photos or videos from the patients. if the emergency could be managed at home by remote instructions to the patients over the phone or other communication tools, such as wechat and whatapps, it is unnecessary for them to come to the office. 33 first-visit and non-emergency return-visit patients are suggested to schedule after the covid-19 epidemic is effectively controlled in the area. the recommendations to prevent the transmission of sars-cov-2 during the main orthodontic procedures were summarized as below. preprocedural mouthrinse with antimicrobial agents is beneficial to reduce the salivary load of oral microbes. the commonly used chlorhexidine for mouthrinse has not been proven to be effective to disinfect sars-cov-2. however, 0.5%-1% povidone iodine solution is recommended as it has been reported to be able to disinfect sars-cov-1 and sars-cov-2. 40, 41 since sars-cov-2 is vulnerable to oxidation, mouthrinse containing oxidative agents such as 1% hydrogen peroxide is also recommended 7,41 . the patients should be instructed to swish the mouth rinse for 2-3min and spit gently into a disposable cup. saliva ejectors with low or high volume should be used to eject the gargle immediately to reduce the generation of droplets. photographic records of the facial and dental images could be taken in a separated unit. for patients with high throat sensitivity, the reflector should not be placed too deep in the mouth which may otherwise cause irritation, nausea, and vomiting leading to the generation of droplets. 42 photographers should strictly implement the principle of "one patient one use and one disinfection" for auxiliary equipment such as retractors and reflectors. although the intraoral x-ray examination is the most commonly used radiographic technique in dental practice; however, it can stimulate saliva secretion, gagging and coughing. 42 the direct contact to the saliva during examination would also increase the risk of infection transmission. therefore, extraoral radiographies, such as panoramic radiography and cone beam computed tomography (cbct) are more appropriate alternatives during the epidemic period of covid-19. 2 before taking the dental impression, patients should be informed of potential risks of nausea and gagging in advance. patients should also be instructed to inhale through the nose and exhale out the mouth. when taking the impression, the patient should wear a waterproof towel on the chest and hold a disposable cup in case of saliva splashing or nausea and vomiting caused by throat irritation from the alginate or silicone rubber materials. it is important to avoid contamination of the devices used in mixing the alginate material by putting it on a tray. after solidification and taking out of the impression, wash the saliva or blood on the surface gently with slow running water to prevent splashing and then disinfect the impressions. alginate and silicone rubber impressions should be disinfected by immersing in 1000mg/l chlorine containing disinfectants for 15-30 min before they are casted or sent to the factories. 43, 44 patients who need clear aligner orthodontic treatment and other customized appliances can obtain digital dental models through oral scanning. this may also help to reduce cross infection possibilities when compared to traditional methods of alginate or silicone rubber impressions during the storage and delivery. during the scanning, the staff should avoid inducing the pharyngeal reflex of the patient by gentle operation when scanning the molar regions. in order to avoid aerosol in the air caused by using 3-way syringe to dry the tooth surfaces during the scanning, cotton rolls could be alternatively used to wipe the patient's tooth surfaces and keep the mouth dry by using saliva ejectors. use the touch pad of the scanning bar to control and avoid the use of the touch screen which may not be effectively disinfected due to its special screen characteristics. after scanning, the handle of the scanner should be sprayed and wiped with 75% alcohol. the intraoral head of the scanner should be "one patient, one use and one disinfection". for the first-visit patients, after the initial examinations and records have been taken, it is recommended to make the next appointment for return-visit on a separate day to communicate with the patients about treatment alternatives. the purpose of this is to reduce the staying time that patients are in the clinic or hospital and to avoid the potential risk of cross infection caused by the aggregation of patients. during the treatment of the return-visit patients, especially the first-time bonding or the final removal of the appliances, the orthodontist and the assistant will be working in close proximity to the patient for an extended period. during bonding or removal of the brackets, trimming of the attachments, interproximal reduction, and so on, the staff should reduce the use of high-speed handpieces and high-pressure 3-way syringes. low-speed handpieces or the manual devices are alternatives that can be used to reduce the formation of droplets and aerosols. if high-speed handpieces are necessary to use, anti-retraction handpiece with specially designed anti-retractive valves or other anti-reflux designs are recommended. these alternative designs have a greater reduction in the backflow of oral microbes into the waterlines of the handpiece and dental unit as compared with the handpiece without anti-retraction function. 45 nevertheless, the use of high-speed handpieces and high-pressure 3-way syringes in the patient's mouth could easily splash the saliva or blood to form aerosols. therefore, in addition to the level iii personal protective measures (table 1) , the use of high-volume saliva ejectors and the four-handed or six-handed cooperation technique should be adopted to prevent cross infection as well as to improve working efficiency. what's more, a plasma air sterilizer should be turned on continuously for air disinfection, especially during the procedures related to the use of high-speed handpieces and high-pressure 3-way syringes, and the windows of the room should be open to allow natural ventilation. the principle of "one room one patient one disinfection" should also be followed. individually packaged archwires are recommended to use in the fixed orthodontic treatment to avoid cross infection. when the archwires need to be adjusted during the orthodontic treatment, such as the bending of three orders or the adding of the curve, spray and wipe the archwire with 75% alcohol after it is removed from the mouth. during bending, two layers of gloves are suggested to wear in case of glove tear or potential skin laceration from the archwires. removable appliances, including clear aligners, are directly in contact with the saliva and the oral mucosa making them potential transmission media of sars-cov-2. appliances should be washed and sprayed with 75% alcohol or 1000mg/l chlorine containing disinfectants before the adjustment. care should be taken to avoid pharyngeal reflex during wear and removal. after the completion of one patient, disinfection of dental settings and related surfaces should be done before treatment of the next patient. the saliva ejector tubes should be flushed with at least 150ml 1000mg/l chlorine containing disinfectant. the spittoon area should also be flushed and cleaned with 1000mg/l chlorine containing disinfectant. the handpieces and 3-way syringes should be run to discharge water for 30 seconds which will help to flush out patient materials that may have entered the waterlines. the dental chair should be sprayed with 75% alcohol and completely wiped and disinfected with 1000mg/l chlorine containing disinfectant. after disinfection, the possible touching areas of the dental setting by the orthodontist and the assistant during the treatment should be covered with new antifouling membrane. an interval of 3-5 min is recommended between two consecutive patients to allow for optimal disinfection. many aspects of orthodontic treatment require the cooperation of patients, such as the wearing and removal of the clear aligners, retainers, functional appliances, rubber band elastics and so on. orthodontic staffs should inform the patients to incorporate proper hand hygiene before and after self-operation as well as before and after meals and defecation. instruct the patients to use the six-step hand washing method to avoid the transmission of virus by contact and potential fecal-oral routes. in addition, patients who wear removable appliances should be instructed to keep the appliances stored in containers after removal rather than out on open surfaces to prevent the possible transmission of sars-cov-2. after the practice, the reusable orthodontic instruments, such as pliers, should be pretreated, cleaned, alcohol should also be used to spray and disinfect and then allowed to dry before preservation for future use. the medical waste generated by the treatment of patients should be stored in the specially made medical waste bags then transported and disposed in accordance with the management requirements for medical waste. since airborne infection is one of the major concerns in orthodontic practice, the disinfection and purification of the air is of great importance to prevent the spread of the covid-19 and other microbes. plasma air sterilizers can be left continuously running for air disinfection in an environment with human activity, especially in the working area and the waiting area. in addition, the ultraviolet light should be turned on after treatment or lunch break for environmental surface disinfection for 30-60 min, twice a day. if the ultraviolet light is not available, spray and wipe the surfaces, such as floor, desk and chair, with 1000mg/l chlorine containing disinfectant every 2-3 hours. also, the natural ventilation is a simple and effective way of air purification. under air circulation, the microbial colonies can be significantly reduced by 77.3% -79.3% within the first 30 minutes, and up to 96.4%-99.5% within 75 minutes. 46 it is recommended to follow up with patients digitally through photos or video calls using the phone or other communication tools, such as wechat and whatapps, to not only monitor the orthodontic progress, but also to minimize the repeated patient contact and ensure patient safety in case of orthodontic emergencies. 33, 47 it was reported that during the covid-19 epidemic period, 90% of the public dental hospitals in china provided dental consultations online, 3 which could also be helpful to relieve the patient anxiety caused by the suspension of the return-visit. although the covid-19 is currently under effective control in china and many areas are gradually resuming normal activities, the increase in population mobility and the rising cases worldwide also pose great challenges for the prevention and control of covid-19. since there may still be asymptomatic patients or patients in the incubation period after resumption of regular activities and a large number of orthodontic patients from a wide spread distribution in need of orthodontic return-visits, all procedures related to the orthodontic practice should be strictly performed with preventive measures to control the potential transmission of sars-cov-2. the control strategies include, but not limit to, pre-examination and triage of patients, hand hygiene, personal protective measures, mouthrinse, reducing the use of high-speed handpieces while increasing the use of high volume saliva ejectors during bracket or attachment bonding and removal, disinfection during archwire changing/bending and removable appliance adjustment, disinfection of dental settings between patients, instructions to patients, management of reusable items and medical wastes, air and environment disinfection, and digital patient follow-up. we must constantly bear in mind that the threat of infection is not visible which poses a challenge on the orthodontic practice thus effective control measures should be taken to prevent the transmission of sars-cov-2 and protect both practitioners and patients from the covid-19. introduced the covid-19 and sars-cov-2 charateristics and its impact on orthodontic practice summarized the potential transmission routes of sars-cov-2 during orthodontic practice. recommended the strategies for controls of sars-cov-2 transmission in orthodontic practice world health organization. statement on the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus coronavirus disease 2019 (covid-19): emerging and future challenges for dental and oral medicine health services provision of 48 public tertiary dental hospitals during the covid-19 epidemic in china transmission of 2019-ncov infection from an asymptomatic contact in germany droplets and aerosols in dental clinics and prevention and control measures of infection dentists' awareness, perception, and attitude regarding covid-19 and infection control: cross-sectional study among jordanian dentists transmission routes of 2019-ncov and controls in dental practice guideline for the diagnosis and treatment of novel coronavirus pneumonia clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of 2019 novel coronavirus infection in china novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures 2019-ncov transmission through the ocular surface must not be ignored aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 practical recommendations for critical care and anesthesiology teams caring for novel coronavirus (2019-ncov) patients covid-19 may transmit through aerosol first case of 2019 novel coronavirus in the united states covid-19: gastrointestinal manifestations and potential fecal-oral transmission consistent detection of 2019 novel coronavirus in saliva saliva: potential diagnostic value and transmission of 2019-ncov high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa airborne spread of infectious agents in the indoor environment current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019 (covid-19) developing covid-19 vaccines at pandemic speed guideline for the prevention and control of novel coronavirus pneumonia in medical institutes national health commission of the people's republic of china. guideline for the use of medical protective equipment in the prevention and control of novel coronavirus pneumonia suggestions on the prevention and control measures during the covid-19 epidemic period world health organization. country & technical guidance -coronavirus disease (covid-19) emergency management of prevention and control of novel coronavirus pneumonia in departments of stomatology management of orthodontic emergencies during 2019-ncov airborne transmission route of covid-19: why 2 meters/6 feet of inter-personal distance could not be enough personal protective equipment during the coronavirus disease (covid) 2019 pandemic -a narrative review handbook of covid-19 prevention and treatment a needle in a haystack: report of a retained archwire fragment in the pterygomandibular space ingestion of an orthodontic archwire resulting in a perforated bowel: a case report laryngeal impaction of an archwire segment after accidental ingestion during orthodontic adjustment inactivation of sars coronavirus by means of povidone-iodine, physical conditions, and chemical reagents persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents modern dental imaging: a review of the current technology and clinical applications in dental practice effect of rinsing alginate impressions using acidic electrolyzed water on dimensional change and deformation of stone models effects of chlorine-based and quaternary ammonium-based disinfectants on the wettability of a polyvinyl siloxane impression material risk of hepatitis b virus transmission via dental handpieces and evaluation of an anti-suction device for prevention of transmission factors influencing microbial colonies in the air of operating rooms urgent dental care for patients during the covid-19 pandemic key: cord-353826-owoec2ud authors: graham, simon p.; mclean, rebecca k.; spencer, alexandra j.; belij-rammerstorfer, sandra; wright, daniel; ulaszewska, marta; edwards, jane c.; hayes, jack w. p.; martini, veronica; thakur, nazia; conceicao, carina; dietrich, isabelle; shelton, holly; waters, ryan; ludi, anna; wilsden, ginette; browning, clare; bialy, dagmara; bhat, sushant; stevenson-leggett, phoebe; hollinghurst, philippa; gilbride, ciaran; pulido, david; moffat, katy; sharpe, hannah; allen, elizabeth; mioulet, valerie; chiu, chris; newman, joseph; asfor, amin s.; burman, alison; crossley, sylvia; huo, jiandong; owens, raymond j.; carroll, miles; hammond, john a.; tchilian, elma; bailey, dalan; charleston, bryan; gilbert, sarah c.; tuthill, tobias j.; lambe, teresa title: evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored covid-19 vaccine candidate chadox1 ncov-19 date: 2020-07-27 journal: npj vaccines doi: 10.1038/s41541-020-00221-3 sha: doc_id: 353826 cord_uid: owoec2ud clinical development of the covid-19 vaccine candidate chadox1 ncov-19, a replication-deficient simian adenoviral vector expressing the full-length sars-cov-2 spike (s) protein was initiated in april 2020 following non-human primate studies using a single immunisation. here, we compared the immunogenicity of one or two doses of chadox1 ncov-19 in both mice and pigs. whilst a single dose induced antigen-specific antibody and t cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in sars-cov-2 neutralising titres. as sars-cov-2 began to spread around the world at the beginning of 2020 several vaccine platform technologies were employed to generate candidate vaccines. several use replication-deficient adenoviral (ad) vector technology and express the sars-cov-2 spike (s) protein. the first phase i clinical study of an ad5-vectored vaccine has been reported 1 , chadox1 ncov-19 (azd1222) phase i trials (nct04324606) began in april 2020 with phase ii and iii trials (nct04400838) started soon thereafter, and an ad26-vectored vaccine is expected to enter phase i shortly. typically, only one dose of ad-vectored vaccines has been administered in early preclinical challenge studies or clinical studies against emerging or outbreak pathogens [2] [3] [4] [5] . rhesus macaques immunised with a single dose of chadox1 ncov-19 were protected against pneumonia but there was no impact on nasal virus titers after high dose challenge to both the upper and lower respiratory tract 6 . to increase antibody titres and longevity of immune responses, a booster vaccination may be administered. homologous prime-boost immunisation resulted in higher antibody titres including neutralising antibodies and a trend towards a lower clinical score in a mers-cov challenge study 7 . here, we set out to test the immunogenicity of either one or two doses of chadox1 ncov-19 in mice and pigs, to further inform clinical development. prime-only and prime-boost vaccination regimens in mice and pigs 'prime-boost' vaccinated inbred (balb/c) and outbred (cd1) mice (9-10 weeks of age) were immunised by intramuscular (i.m.) injection of 10 8 infectious units (iu) of chadox1 ncov-19 on 0 and 28 days post-vaccination (dpv), whereas, 'prime-only' mice received a single dose of chadox1 ncov-19 on day 28. spleens and serum were harvested from all mice on day 49 (3 weeks after boost or prime vaccination). analysis of sars-cov-2 s proteinspecific murine splenocyte responses by ifn-γ elispot assay showed no statistically significant difference between the primeonly and prime-boost vaccination regimens, in either strain of mouse (fig. 1a) . intracellular cytokine staining (ics) of splenocytes ( fig. 1b) showed, in both mouse strains, that the response was principally driven by cd8 + t cells. the predominant cytokine response of both cd8 + and cd4 + t cells was expression of ifn-γ and tnf-α, with negligible frequencies of il-4 + and il-10 + cells, consistent with previous data suggesting adenoviral vaccination does not induce a dominant th2 response 8, 9 . there were no signficant differences in cd4 + and cd8 + t cell cytokine responses between prime-only and prime-boost mice. prime-only and prime-boost pigs (8-10 weeks of age) were immunised by i.m. injection of 10 9 iu of chadox1 ncov-19 (the same dose as being used in human clinical trials) on 0 dpv and prime-boost pigs received a second immunisation on 28 dpv. blood samples were collected weekly until 42 dpv to analyse immune responses. ifn-γ elispot analysis of porcine peripheral blood mononuclear cells (pbmc) showed responses on 42 dpv (2 weeks after boost) that were significantly greater in the prime-boost pigs compared to prime-only animals (p < 0.05; fig. 1c ). the prime-boost 42 dpv responses were greater than responses observed in either group on 14 dpv, but interanimal variation meant this did not achieve statistical significance. ics analysis of porcine t cell reponses showed a dominance of th1-type cytokines (similar to the murine response) but with a higher frequency of s-specific cd4 + t cells compared to cd8 + t cells (fig. 1d) . however, cd4 + and cd8 + t cell cytokine responses did not differ significantly between vaccine groups or timepoints (14 vs. 42 dpv). sars-cov-2 s protein-specific antibody responses following chadox1 ncov-19 prime-only and prime-boost vaccination regimens in mice and pigs sars-cov-2 s protein-specific antibody titres in serum were determined by elisa using recombinant soluble trimeric s (fl-s) and receptor binding domain (rbd) proteins. a significant increase in fl-s binding antibody titres was observed in prime-boost balb/ c mice compared to their prime-only counterparts (p < 0.01), however, the difference between vaccine groups for cd1 mice was not significant (fig. 2a) . antibody responses were evaluated longitudinally in pig sera by fl-s and rbd elisa. compared to prevaccination sera, significant fl-s specific antibody titres were detected in both prime-only and prime-boost groups from 21 and 14 dpv, respectively (p < 0.01; fig. 2b ). fl-s antibody titres did not differ signifcantly between groups until after the boost, when titres in the prime-boost pigs became significantly greater with an average increase in titres of >1 log 10 (p < 0.0001). rbd-specific antibody titres showed a similar profile with significant titres in both groups from 14 dpv (p < 0.05) and a further significant increase in the prime-boost pigs from 35 dpv onwards which was greater than the prime-only pigs (p < 0.0001; fig. 2c ). sars-cov-2 neutralising antibody responses were assessed using a virus neutralisation test (vnt; fig. 2d ) and pseudovirus-based neutralisation test (pvnt; fig. 2e ). after the prime immunisation, sars-cov-2 neutralising antibody titres were detected by vnt in 14 and 28 dpv sera from 2/3 prime-boost and 2/3 prime-only pigs. two weeks after the boost (42 dpv), neutralising antibody titres were detected and had increased in all prime-boost pigs, which were significantly greater than the earlier timepoints and the titres measured in the prime-only group (p < 0.01). in agreement with this analysis, serum assayed for neutralising antibodies using the pvnt revealed that antibody titres in 42 dpv prime-boost pig sera were significantly greater than earlier timepoints and the primeonly group (p < 0.001). statistical analysis showed a highly significant correlation between pvnt and vnt titres (spearman's rank correlation r = 0.86; p < 0.0001). in this study, we utilised both a small and a large animal model to evaluate the immunogenicity of either one or two doses of a covid-19 vaccine candidate, chadox1 ncov-19 (now known as azd1222). small animal models have variable success in predicting vaccine efficacy in larger animals but are an important to analyse sars-cov-2 s-specific t cell responses, all mice were sacrificed on day 49 for isolation of splenocytes and pigs were blood sampled longitudinally to isolate pbmc. following stimulation with sars-cov-2 s-peptides, responses of murine splenocytes a and porcine pbmc c were assessed by ifn-γ elispot assays. using flow cytometry, cd4 + and cd8 + t cell responses were characterised by assessing expression of ifn-γ, tnf-α, il-2, il-4 and il-10 (mice; b) and ifn-γ, tnf-α, il-2 and il-4 (pigs; d). each data point represents an individual mouse/pig with bars denoting the median response per group/timepoint. data were analysed by anova and statistically significant differences between vaccine groups are indicated: *p < 0.05. stepping stone to facilitate prioritisation of vaccine targets. in contrast, larger animal models, such as the pig and non-human primates, have been shown to more accurately predict vaccine outcome in humans [10] [11] [12] . the mouse data generated in this study suggested that the immunogenicity profile was at the upper end of a dose response curve, which may have saturated the immune response and largely obscured our ability to determine differences between prime-only or prime-boost regimens. we have developed the pig as a model for generating and understanding immune responses to vaccination against human influenza [13] [14] [15] and nipah virus 16, 17 . the inherent heterogeneity of an outbred large animal model is more representative of immune responses in humans. extensive development of reagents to study immune responses in pigs in recent years has extended the usefulness and applicability of the pig as a model to study infectious disease. these data demonstrate the utility of the pig as a model for further evaluation of the immunogenicity of chadox1 ncov-19 and other covid-19 vaccines. we show here that t cell responses are higher in pigs that received a prime-boost vaccination when compared to prime only at day 42, whilst comparing responses 14 days after last immunisation demonstrates the prime-boost regimen trended toward a higher response. in addition, chadox1 ncov-19 immunisation induced robust th1-like cd4 + and cd8 + t cell responses in both pigs and mice. this has important implications for covid-19 vaccine development as virus-specific t cells are thought to play an important role in sars-cov-2 infection 18-22 . while no correlate of protection has been defined for covid-19, recent publications suggest that neutralising antibody titres may be correlated with protection in animal challenge models 23, 24 . a single dose of chadox1 ncov-19 induces antibody responses, but we demonstrate here that antibody responses are significantly enhanced after homologous boost in one mouse strain and to a greater extent in pigs. the sars-cov-2 neutralising antibody titres in pigs after a single immunisation appear broadly comparable to those in sera from humans following asymptomatic infection, whereas, titres in prime-boost pigs were more comparable to serum from recovered covid-19 patients 25 . however, it is likely that a combination of neutralising antibodies and antigen-specific t cells would act in synergy to prevent and control infection, as we have recently shown in the context of influenza vaccination 13, 26 . whilst human immunogenicity and clinical read-outs are a critically meaningful endpoint, studies in small animals and pigs will help prioritise candidates to be tested in humans. further clinical studies are needed to assess immunogenicity after primeboost vaccination and the impact on clinical efficacy and durability of the immune response. mouse and pig studies were performed in accordance with the uk animals inbred balb/c (n=5) and outbred cd1 (n=8) were immunised on day 0 and 28 with chadox1 ncov19 (prime-boost) or chadox1 ncov19 on day 28 (prime-only), whereas, pigs were immunised with chadox1 ncov-19 on days 0 and 28 (prime-boost), or only on day 0 (prime-only). to analyse sars-cov-2 s protein-specific antibodies in serum, all mice were sacrificed on day 49 and pigs were blood sampled weekly until day 42. antibody units or end-point titres (ept) were assessed by elisa using recombinant sars-cov-2 fl-s for both mice a and pigs b, and recombinant s protein rbd for pigs c. sars-cov-2 neutralising antibody titres in pig sera were determined by vnt, expressed as the reciprocal of the serum dilution that neutralised virus infectivity in 50% of the wells (nd 50 ; d), and pvnt, expressed as reciprocal serum dilution to inhibit pseudovirus entry by 50% (ic 50 ; e). each data point represents an individual mouse/pig sera with bars (a) denoting the median titre per group. data were analysed by anova and statistically significant differences between vaccine groups are indicated: **p < 0.01; ***p < 0.001; ****p < 0.0001. p9808b4f1 and university of oxford awerb, and pigs-project license pp1804248 and the pirbright institute awerb). the principles of the 3r's were applied for the duration of the study to ensure animal welfare was not unnecessarily compromised. vero e6 cells were grown in dmem containing sodium pyruvate and lglutamine (sigma-aldrich, poole, uk), 10% fbs (gibco, thermo fisher, loughborough, uk), 0.2% penicillin/streptomycin (10,000 u/ml; gibco) (maintenance media) at 37°c and 5% co 2 . sars-cov-2 isolate england-2 stocks were grown in vero e6 cells using a multiplicity of infection (moi) of 0.0001 for 3 days at 37°c in propagation media (maintenance media containing 2% fbs). sars-cov-2 stocks were titrated on vero e6 cells using mem (gibco), 2% fcs (labtech, heathfield, uk), 0.8% avicel (fmc biopolymer, girvan, uk) as overlay. plaque assays were fixed using formaldehyde (vwr, leighton buzzard, uk) and stained using 0.1% toluidine blue (sigma-aldrich). all work with live sars-cov-2 virus was performed in acdp hg3 laboratories by trained personnel. the propagation, purification and assessment of chadox1 ncov-19 titres were as described previously 7 . recombinant sars-cov-2 proteins and synthetic peptides a synthetic dna, encoding the spike (s) protein receptor binding domain (rbd; amino acids 330-532) of sars-cov-2 (genbank mn908947), codon optimised for expression in mammalian cells (idt technology) was inserted into the vector popinttgneo incorporating a c-terminal his6 tag. recombinant rbd was transiently expressed in expi293™ (thermo fisher scientific, uk) and protein purified from culture supernatants by immobilised metal affinity followed by a gel filtration in phosphatebuffered saline (pbs) ph 7.4 buffer. a soluble trimeric s (fl-s) protein construct encoding residues 1-1213 with two sets of mutations that stabilise the protein in a pre-fusion conformation (removal of a furin cleavage site and the introduction of two proline residues; k983p, v984p) was expressed as described 27 . the endogenous viral signal peptide was retained at the n-terminus (residues 1-14), a c-terminal t4-foldon domain incorporated to promote association of monomers into trimers to reflect the native transmembrane viral protein, and a c-terminal his6 tag included for nickel-based affinity purification. similar to recombinant rbd, fl-s was transiently expressed in expi293™ (thermo fisher scientific) and protein purified from culture supernatants by immobilised metal affinity followed by gel filtration in tris-buffered saline (tbs) ph 7.4 buffer. for analysis of t cell responses in pigs, overlapping 16mer peptides offset by 4 residues based on the predicted amino acid sequence of the entire s protein from sars-cov-2 wuhan-hu-1 isolate (ncbi reference sequence: nc_045512.2) were designed and synthesised (mimotopes, melbourne, australia) and reconstituted in sterile 40% acetonitrile (sigma-aldrich) at a concentration of 3 mg/ml. three pools of synthetic peptides representing residues 1-331 (pool 1), 332-748 (pool 2) and 749-1273 (pool 3) were prepared for use to stimulate t cells in ifn-γ elispot and intracellular cytokine staining (ics) assays. for analysis of t cell responses in mice, overlapping 15mer peptides offset by 11 residues were designed and synthesised (mimotopes) and reconstituted in sterile 100% dmso (sigma-aldrich) at a concentration of 100 mg/ml. two peptide pools spanning s1 region (pool 1: 1 to 77 and 317-321, pool 2: 78-167) and 2 peptide pools spanning s2 region (pool 3: 166 to 241, pool 4: 242 to 316) were used for stimulating splenocytes for ifn-γ elispot analysis, and single pools of s1 (pool 1 and pool 2) and s2 (pool 3 and pool 4) were used to stimulate splenocytes for ics. mice: inbred female balb/colahsd (balb/c) and outbred crl:cd1 (cd1) mice were purchase from commercial suppliers (envigo and charles river laboratories, respectively) and randomly allocated into 'prime-only' or 'prime-boost' vaccination groups (balb/c n = 5 and cd1 n = 8) upon arrival. at 9-10 weeks of age, prime-boost mice were immunised intramuscularly with 10 8 infectious units (iu) (6.02 × 10 9 virus particles; vp) chadox1 ncov-19 and boosted intramuscularly four weeks later with 1 × 10 8 iu chadox1 ncov-19. prime-only mice received a single dose of 10 8 iu chadox1 ncov-19 at the same time prime-boost mice were boosted. spleens and serum were harvested from all animals a further 3 weeks later. pigs: six 8-10-week-old, weaned, female, large white-landrace-hampshire cross-bred pigs from a commercial rearing unit were randomly allocated to two treatment groups (n = 3): 'prime-only' and 'prime-boost'. both groups were immunised on day 0 with 1 × 10 9 iu (5.12 × 10 10 vp) chadox1 ncov-19 in 1 ml pbs by intramuscular injection (brachiocephalic muscle). 'prime-boost' pigs received an identical booster immunisation on day 28. blood samples were taken from all pigs on a weekly basis at 0, 7, 14, 21, 28, 35 and 42 dpv by venepuncture of the external jugular vein: 8 ml/pig in bd sst vacutainer tubes (fisher scientific) for serum collection and 40 ml/pig in bd heparin vacutainer tubes (fisher scientific) for peripheral blood mononuclear cell (pbmc) isolation. mice. antibodies to sars-cov-2 fl-s protein were determined by performing a standardised elisa on serum collected 3-weeks after prime or prime-boost vaccination. maxisorp plates (nunc) were coated with 100 ng/well fl-s protein overnight at 4°c, prior to washing in pbs/tween (0.05% v/v) and blocking with blocker casein in pbs (thermo fisher scientific) for 1 h at room temperature (rt). standard positive serum (pool of mouse serum with high endpoint titre against fl-s protein), individual mouse serum samples, negative and an internal control (diluted in casein) were incubated for 2 h at rt. following washing, bound antibodies were detected by addition of alkaline phosphatase-conjugated goat anti-mouse igg (sigma-aldrich), diluted 1/5000 in casein, for 1 h at rt and detection of anti-mouse igg by the addition of pnpp substrate (sigma-aldrich). an arbitrary number of elisa units were assigned to the reference pool and od values of each dilution were fitted to a 4-parameter logistic curve using softmax pro software. elisa units were calculated for each sample using the od values of the sample and the parameters of the standard curve. pigs. serum was isolated by centrifugation of sst tubes at 1300 × g for 10 min at rt and stored at −80°c. sars-cov-2 rbd and fl-s specific antibodies in serum were assessed as detailed previously 27 with the exception of the following two steps. the conjugated secondary antibody was replaced with goat anti-porcine igg hrp (abcam, cambridge, uk) at 1/ 10,000 dilution in pbs with 0.1% tween 20 and 1% non-fat milk. in addition, after the last wash, a 100 µl of tmb (one component horse radish peroxidase microwell substrate, biofx, cambridge bioscience, cambridge, uk) was added to each well and the plates were incubated for 7 min at rt. a 100 µl of biofx 450 nm stop reagent (cambridge bioscience) was then added to stop the reaction and microplates were read at 450 nm. endpoint antibody titres (mean of duplicates) were calculated as follows: the log 10 od was plotted against the log 10 sample dilution and a regression analysis of the linear part of this curve allowed calculation of the endpoint titre with an od of twice the average od values of 0 dpv sera. the ability of pig sera to neutralise sars-cov-2 was evaluated using virus and pseudovirus neutralisation assays. for both assays, sera were first heatinactivated (hi) by incubation at 56°c for 2 h. virus neutralization test (vnt): starting at a 1 in 5 dilution, two-fold serial dilutions of sera were prepared in 96 well round-bottom plates using dmem containing 1% fbs and 1% antibiotic-antimycotic (gibco) (dilution media). 75 μl of diluted pig serum was mixed with 75 μl dilution media containing approximately 64 plaque-forming units (pfu) sars-cov-2 for 1 h at 37°c. vero e6 cells were seeded in 96-well flat-bottom plates at a density of 1 × 10 5 cells/ml in maintenance media one day prior to experimentation. culture supernatants were replaced by 100 µl of dmem containing 10% fcs and 1% antibiotic-antimycotic, before 100 µl of the virus-sera mixture was added to the vero e6 cells and incubated for six days at 37°c. cytopathic effect (cpe) was investigated by bright-field microscopy. cells were further fixed and stained as described above, and cpe scored. each individual pig serum dilution was tested in quadruplet on the same plate and no sera/sars-cov-2 virus and no sera/no virus controls were run concurrently on each plate in quadruplet. wells were scored for cytopathic effect and neutralisation titres expressed as the reciprocal of the serum dilution that completely blocked cpe in 50% of the wells (nd 50 ). researchers performing the vnts were blinded to the identity of the samples. pseudovirus neutralisation test (pvnt): lentiviral-based sars-cov-2 pseudoviruses were generated in hek293t cells incubated at 37°c, 5% co 2 . cells were seeded at a density of 7.5 × 10 5 in 6 well dishes, before being transfected with plasmids as follows: 500 ng of sars-cov-2 spike, 600 ng p8.91 (encoding for hiv-1 gag-pol), 600 ng csflw (lentivirus backbone expressing a firefly luciferase reporter gene), in opti-mem s.p. graham et al. (gibco) along with 10 µl pei (1 µg/ml) transfection reagent. a 'no glycoprotein' control was also set up using carrier dna (pcdna3.1) instead of the sars-cov-2 s expression plasmid. the following day, the transfection mix was replaced with 3 ml dmem with 10% fbs (dmem-10%) and incubated at 37°c. at both 48 and 72 h post transfection, supernatants containing pseudotyped sars-cov-2 (sars-cov-2 pps) were harvested, pooled and centrifuged at 1300 × g for 10 min at 4°c to remove cellular debris. target hek293t cells, previously transfected with 500 ng of a human ace2 expression plasmid (addgene, cambridge, ma, usa) were seeded at a density of 2 × 10 4 in 100 µl dmem-10% in a white flatbottomed 96-well plate one day prior to harvesting of sars-cov-2 pps. the following day, sars-cov-2 pps were titrated 10-fold on target cells, with the remainder stored at −80°c. for pvnts, pig sera were diluted 1:20 in serum-free media and 50 µl was added to a 96-well plate in quadruplicate and titrated 4-fold. a fixed titred volume of sars-cov-2 pps was added at a dilution equivalent to 10 6 signal luciferase units in 50 µl dmem-10% and incubated with sera for 1 h at 37°c, 5% co 2 . target cells expressing human ace2 were then added at a density of 2 × 10 4 in 100 µl and incubated at 37°c, 5% co 2 for 72 h. firefly luciferase activity was then measured with brightglo luciferase reagent and a glomax-multi + detection system (promega, southampton, uk). pseudovirus neutralization titres were expressed as the reciprocal of the serum dilution that inhibited luciferase expression by 50% (ic 50 ). mice. single cell suspension of mouse spleens were prepared by passing cells through 70 μm cell strainers and ack lysis (thermo fisher) prior to resuspension in complete media (αmem supplemented with 10% fcs, penstep, l-glut and 2-mercaptoethanol). for analysis of ifn-γ production by elispot assay, splenocytes were stimulated with s peptide pools at a final concentration of 2 μg/ml on ipvh-membrane plates (millipore) coated with 5 μg/ml anti-mouse ifn-γ (clone an18; mabtech). after 18-20 h of stimulation, ifn-γ spot forming cells (sfc) were detected by staining membranes with anti-mouse ifn-γ biotin mab (1 µg/ml; clone r46a2, mabtech) followed by streptavidin-alkaline phosphatase (1 µg/ml) and development with ap conjugate substrate kit (bio-rad). for analysis of intracellular cytokine production, cells were stimulated with 2 μg/ml s peptide pools, media or cell stimulation cocktail (containing pma-ionomycin, biolegend), together with 1 μg/ml golgiplug (bd biosciences) and 2 μl/ml cd107a-alexa647 for 6 h in a 96-well u-bottom plate, prior to placing at 4°c overnight. following surface staining with cd4-buv496, cd8-percp-cy5.5, cd62l-bv711, cd127-bv650, cd44-apc-cy7 and live/ dead aqua (thermo fisher), cells were fixed with 10% neutral buffered formalin (containing 4% paraformaldehyde) and stained intracellularly with tnf-α-af488, il-2-pe-cy7, il-4-bv605, il-10-pe and ifn-γ-e450 diluted in perm-wash buffer (bd biosciences). sample acquisition was performed on a fortessa (bd) and data analysed in flowjo v9 (treestar). an acquisition threshold was set at a minimum of 5000 events in the live cd3 + gate. antigen-specific t cells were identified by gating on live/dead negative, doublet negative (fsc-h vs. fsc-a), size (fsc-h vs. ssc), cd3 + , cd4 + or cd8 + cells and cytokine positive. the gating strategy is illustrated in supplementary fig. 1 . total sars-cov-2 s specific cytokine responses are presented after subtraction of the background response detected in the media stimulated control spleen sample of each mouse, prior to summing together the frequency of s1 and s2 specific cells. pigs. pbmcs were isolated from heparinised blood by density gradient centrifugation and cryopreserved in cold 10% dmso (sigma-aldrich) in hi fbs 17 . resuscitated pbmc were suspended in rpmi 1640 medium, glutamax supplement, hepes (gibco) supplemented with 10% hi fbs (new zealand origin, life science production, bedford, uk), 1% penicillin-streptomycin and 0.1% 2-mercaptoethanol (50 mm; gibco) (crpmi). to determine the frequency of sars-cov-2 s specific ifn-γ producing cells, an elispot assay was performed on pbmc from 0, 14, 28 and 42 dpv. multiscreen 96-well plates (mahas4510; millipore, fisher scientific) were pre-coated with 1 µg/ml anti-porcine ifn-γ mab (clone p2g10, bd biosciences) and incubated overnight at 4°c. after washing and blocking with crpmi, pbmcs were plated at 5 × 10 5 cells/well in crpmi in a volume of 50 µl/well. pbmcs were stimulated in triplicate wells with the sars-cov-2 s peptide pools at a final concentration of 1 µg/ml/peptide. crpmi alone was used in triplicate wells as a negative control. after 18 h incubation at 37°c with 5% co 2 , plates were developed as described previously 17 . the numbers of specific ifn-γ secreting cells were determined using an immunospot ® s6 analyzer (cellular technology, cleveland, usa). for each animal, the mean 'crpmi only' data was subtracted from the s peptide pool 1, 2 and 3 data which were then summed and expressed as the mediumcorrected number of antigen-specific ifn-γ secreting cells per 1 × 10 6 pbmc. to assess intracellular cytokine expression pbmc from 14 and 42 dpv were suspended in crpmi at a density of 2 × 10 7 cells/ml and added to 50 µl/well to 96-well round bottom plates. pbmcs were stimulated in triplicate wells with the sars-cov-2 s peptide pools (1 µg/ml/peptide). unstimulated cells in triplicate wells were used as a negative control. after 14 h incubation at 37°c, 5% co 2 , cytokine secretion was blocked by addition 1:1000 bd golgiplug (bd biosciences) and cells were further incubated for 6 h. pbmc were washed in pbs and surface labelled with zombie nir fixable viability stain (biolegend), cd4-percp-cy5.5 mab (clone 74-12-4, bd bioscience) and cd8β-fitc mab (clone ppt23, bio-rad antibodies). following fixation (fixation buffer, biolegend) and permeabilization (permeabilization wash buffer, biolegend), cells were stained with: ifn-γ-af647 mab (clone cc302, bio-rad antibodies, kidlington, uk), tnf-α-bv421 mab (clone mab11, biolegend), il-2 mab (clone a150d 3f1 2h2, invitrogen, thermo fisher scientific) and il-4 bv605 mab (clone mp4-25d2, biolegend) followed by staining with anti-mouse igg2a-pe-cy7 (clone rmg2a-62, biolegend). cells were analysed using a bd lsrfortessa flow cytometer and flowjo x software. the gating strategy is illustrated in supplementary fig. 2 . total sars-cov-2 s specific cytokine positive responses are presented after subtraction of the background response detected in the media stimulated control pbmc sample of each pig, prior to summing together the frequency of s-peptide pools 1-3 specific cells. graphpad prism 8.1.2 (graphpad software, san diego, usa) was used for graphical and statistical analysis of data sets. anova or a mixed-effects model were conducted to compare responses over time and between vaccine groups at different time points post-vaccination as detailed in the "results" section. antibody titre data were log transformed before analysis. neutralising antibody titre data generated by the vnt and pvnt assays were compared using spearman nonparametric correlation. p-values < 0.05 were considered statistically significant. safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial a single-dose chadox1-vectored vaccine provides complete protection against nipah bangladesh and malaysia in syrian golden hamsters safety and immunogenicity of a candidate middle east respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, openlabel, non-randomised, uncontrolled, phase 1 trial protective efficacy of a novel simian adenovirus vaccine against lethal mers-cov challenge in a transgenic human dpp4 mouse model a single dose of chadox1 mers provides broad protective immunity against a variety of mers-cov strains chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques a single dose of chadox1 mers provides protective immunity in rhesus macaques antigen encoded by vaccine vectors derived from human adenovirus serotype 5 is preferentially presented to cd8+ t lymphocytes by the cd8α+ dendritic cell subset immunization with an adenovirus-vectored tb vaccine containing ag85a-mtb32 effectively alleviates allergic asthma the pig: a model for human infectious diseases large animal models for vaccine development and testing the contribution of non-human primate models to the development of human vaccines comparison of heterosubtypic protection in ferrets and pigs induced by a single-cycle influenza vaccine immunogenicity and protective efficacy of seasonal human live attenuated cold-adapted influenza virus vaccine in pigs aerosol delivery of a candidate universal influenza vaccine reduces viral load in pigs challenged with pandemic h1n1 virus vaccine development for nipah virus infection in pigs bovine herpesvirus-4-vectored delivery of nipah virus glycoproteins enhances t cell immunogenicity in pigs targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients clinical and immunological features of severe and moderate coronavirus disease 2019 transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients presence of sars-cov-2 reactive t cells in covid-19 patients and healthy donors sars-cov-2 infection protects against rechallenge in rhesus macaques dna vaccine protection against sars-cov-2 in rhesus macaques antibody responses to sars-cov-2 at 8 weeks postinfection in asymptomatic patients vaccination with viral vectors expressing np, m1 and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice a serological assay to detect sars-cov-2 seroconversion in humans author contributions writing-review and editing, all authors further information on experimental design is available in the nature research reporting summary linked to this article. the data that support the findings of this study are available from the corresponding authors upon reasonable request. s.c.g. and t.l. are named on a patent application covering chadox1 ncov-19. the remaining authors declare no competing interests. the funders played no role in the conceptualisation, design, data collection, analysis, decision to publish, or preparation of the manuscript. supplementary information is available for this paper at https://doi.org/10.1038/ s41541-020-00221-3.correspondence and requests for materials should be addressed to s.p.g. or t.l. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-343302-g9vcchrh authors: agrawal, anurodh shankar; ying, tianlei; tao, xinrong; garron, tania; algaissi, abdullah; wang, yanping; wang, lili; peng, bi-hung; jiang, shibo; dimitrov, dimiter s.; tseng, chien-te k. title: passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection date: 2016-08-19 journal: sci rep doi: 10.1038/srep31629 sha: doc_id: 343302 cord_uid: g9vcchrh middle east respiratory syndrome coronavirus (mers-cov) has repeatedly caused outbreaks in the arabian peninsula. to date, no approved medical countermeasures (mcm) are available to combat mers-cov infections. several neutralizing human monoclonal antibodies (mabs), including m336, a germline-like human mab, have been chosen as promising mcm for mers-cov. however, their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections. we assessed the prophylactic and therapeutic efficacy of m336 by using well-characterized transgenic mice shown to be highly sensitive to mers-cov infection and disease. we found that mice treated with m336 prior to or post lethal mers-cov challenging were fully protected, compared to control mice which sufferered from profound weight loss and uniform death within days after infection. taken together, these results support further development of m336 and other human monoclonal antibodies as potential therapeutics for mers-cov infection. scientific reports | 6:31629 | doi: 10.1038/srep31629 and long-term threat to people, particularly those who interact closely with camels in the arabian peninsula. even though mers-cov presently has limited human-to-human transmission 2, 16 , the high mortality rate of this virus and limited information on the mechanism able to confer increased human-to-human transmission have raised concerns of a potential mers pandemic. indeed, the recent outbreaks in korea and the appearance of super-spreading events indicate that mers-cov has the ability to cause large outbreaks outside of the arabian peninsula [17] [18] [19] . currently, no approved vaccines or drugs are available to treat this viral infection. these facts highlight an urgent need to develop potent prophylactic and therapeutic agents to fight this lethal virus. similar to other coronaviruses, mers-cov uses the envelope spike (s) glycoprotein, a class i transmembrane protein, for interaction with its cellular receptor for binding, fusion and entry into the target cell 20 . the receptor binding domain (rbd) located in the s1 domain of the mers-cov spike is responsible for binding to the well-characterized cellular receptor identified as dpp4 (cd26) and is, therefore, critical for binding and entry of the virus [20] [21] [22] . therefore, neutralizing antibodies capable of blocking such interaction could be promising preventive and/or therapeutic candidates. recently, human monoclonal antibodies (mabs) capable of neutralizing mers-cov have been identified and characterized by several research groups [23] [24] [25] [26] [27] [28] . these antibodies have been isolated from naive human antibody libraries, from transgenic "humanized" mice, or from b cells of an infected individual, and they recognize different epitopes on mers-cov rbd. one of the most potent mabs, m336, is a germline-like antibody identified from a very large (~10 11 size) phage-displayed antibody library derived from b cells of healthy donors. this mab exhibits exceptionally potent neutralizing activity (ic 50 = 0.005 μ g/ml) in vitro 23 . moreover, because its epitope almost completely (~90%) overlaps with the receptor-binding site of dpp4 on mers-cov rbd, as is evident by its recently solved crystal structure 29 , the probability of generation of resistant mutants may be absent or very low. notably, although the functions of these mabs have been extensively characterized in vitro, their further clinical development has been hindered by the lack of an effective animal model of mers-cov infection. mers-cov cannot infect small laboratory animals (e.g., mice, hamsters and ferrets) as a consequence of species-specific differences in dpp4, while only causing mild-to-moderate symptoms in rhesus macaques. marmosets, which are more susceptible to mers-cov, developed a moderate-to-severe disease, but limited availability and high cost have hampered their use 30 . rabbits can be infected, but the infectious virus is challenging to detect 31, 32 . it was found that the expression of human dpp4 could overcome the lack of susceptibility in normal mice. with prior transduction of adenoviral human dpp4-expressing vectors, mice became susceptible to mers-cov infection without revealing any measurable clinical manifestations 33 . in contrast, transgenic (tg) mice with the human dpp4 gene integrated into the genome readily developed acute morbidity (weight loss), and uniform death occurred within a week 34, 35 , making it an ideal preclinical model for the development of vaccines and treatments against mers. some of the aforementioned human neutralizing monoclonal antibodies have been shown to protect engineered human dpp4-expressing mice and the naturally permissive rabbits, entirely based on their ability to inhibit mers-cov infection and/or alleviate histopathology of the lungs 27, 28, 36, 37 . to further verify the protective efficacy of these human monoclonal antibodies, particularly m336, against mers-cov infection, it is highly desirable to use the well-characterized human dpp4 tg mice known to result in acute disease (weight loss) and death 34, 35 . by using this highly permissive tg mouse model, we evaluated the prophylactic and therapeutic efficacy of m336 mab in vivo. we report in this study for the first time that treatment of tg mice with a single-dose of m336 antibody prior to or after challenging with 1,000 ld 50 of mers-cov protected mice from the lethality in a dose-dependent manner, thereby representing the first antibody tested for its protective efficacy against lethal mers-cov infection. prophylactic efficacy of mers-cov rbd-specific human monoclonal antibody, m336. we established a tg mouse model which is profoundly sensitive and susceptible to mers-cov infection, as determined by high viral titers in the lungs, as well as a high rate of morbidity and mortality 34, 38 . equipped with this small animal model of human mers-cov, we investigated the protective efficacy of mab m336. to accomplish this, each group (n = 6) of mice was treated via the intraperitoneal (i.p.) route with two different doses: 0.1 mg and 1 mg per mouse diluted in 100 μ l pbs, and challenged intranasally (i.n.) at 12 h post treatment with 10 4 tcid 50 (i.e., 1,000 ld 50 ) of mers-cov in a volume of 60 μ l 38 . challenged mice were monitored daily for clinical manifestations (weight loss) and mortality. as shown in fig. 1 , the group treated with 1 mg mab survived viral infection without showing any clinical symptoms. these mice initially showed either no weight loss or recovered from mild weight loss within three days (fig. 1a) . on the other hand, the group treated with 0.1 mg mab showed a gradual weight loss (15-20%) until day 13 just before starting to recover (fig. 1a ). all surviving mice (one died on day 13 in mice treated with 0.1 mg of m336) continued to recover and appeared well up to 21 dpi when the experiment was terminated (fig. 1b) . all mers-cov-challenged mice pretreated with a high dose (1 mg) of irrelevant mab m102.4 exhibited profound weight loss (> 15%) and succumbed to infection with 100% mortality by day 8 p.i. (fig. 1a,b ). therapeutic efficacy of mers-cov rbd-specific human monoclonal antibody, m336. to determine the therapeutic potential of this human monoclonal m336 antibody, groups of mice (n = 6 per group) were challenged (i.n.) with 10 4 tcid 50 of mers-cov (i.e., 1,000 ld 50 ) in a volume of 60 μ l and then treated (i.p.) 12 hours later with a single dose of either 1 mg or 0.1 mg of m336 or 1 mg of m102.4 antibody (control) in 100 μ l per mouse, followed by monitoring daily for wellbeing (weight loss and other clinical manifestations) and mortality of mice. we noted that whereas treatment with 1 mg of m336 antibodies was effective in the protection against the lethality caused by mers-cov infection, it failed to protect mice fully from the onset of clinical illness (weight loss). specifically, all of the challenged mice treated with 1 mg of m336 antibody suffered an attenuated (< 10%), and transient weight loss until day 9, and gradually recovered to day 21 when the experiment was terminated (fig. 2) . similarly, challenged mice treated with a low dose of 0.1 mg of m336 antibodies suffered from attenuated and transient weight loss until day 7 p.i. and gradually recovered. however, we noted a single death at day 9 in this low dose treatment group (fig. 2) . as expected, all mice treated with a single dose of 1 mg of control m102.4 antibody exhibited profound weight loss (> 15%) and succumbed to mers-cov infection with 100% mortality by day 8 p.i. (fig. 2) . taken together, these results indicate that this mers-cov rbd-specific human m336 antibody can be highly effective as prophylactic or therapeutic modalities in protecting highly permissive transgenic mice against mers-cov infection and disease. we also investigated the protective mechanism of m336 against mers-cov by determining the lung virus titers in challenged mice at day 2 after treatment. specifically, we sacrificed two mice (out of 6) in each group, as described above for figs 1 and 2 and their lung specimens were harvested for determining viral titers by using via vero e6 cell-based infectivity assay and quantitative pcr (q-pcr)-based assay targeting the upstream e gene of mers-cov. as shown in fig. 3a we were unable to recover infectious virus from any mouse treated with 1 mg of m336 antibody either before or after challenge with mers-cov. however, we were able to detect a barely detectable infectious virus, with the limit of detection (lod) of 2.3 log ticd 50 /g, from a single mouse receiving 0.1 mg of m336 prior to viral challenge. these results indicated that mab m336 most likely confers protection from lethal challenge by restricting viral replication within the lungs, thereby preventing viral infection in the brains and other organs. titers of viral rna copy number, as shown by qrt-pcr assays, were also compared among groups having different doses of mabs. lungs of infected mice were harvested on day 2 post-and pre-virus challenge group. all groups exhibited detectable viral rna. titers were significantly lower than those in the control group in all m336-treated groups. in the pretreatment group, mice treated with 1 mg of m336 showed a 2-log reduction in viral rna detection, while a ~1 log reduction in viral numbers was seen in mice treated within 0.1 mg m336 when compared to mice receiving control mab m102.4. in the post-treatment group, a smaller (~1 log) difference in viral rna copy number (compared to that in the pretreatment group) was observed between mice treated with 1 mg antibody compared with those receiving control antibody, while a more than 1 log reduction in viral rna number was seen in mice treated with 0.1 mg m336 when compared to mice receiving control mab (fig. 3b) . these data indicate that m336 confers significant protection to mice when administered pre-or post-viral challenge. taken together, these results suggest to us that the epitope targeted by this exceptionally potent rbd-specific m336 antibody has a great potential for further development as a potent preventive and therapeutic agent in the future. treatment with m336 attenuates lung pathology associated with mers-cov infection. the effect of m336 antibody treatment on the pulmonary pathology associated with mers-cov infection was evaluated by using formalin-fixed, paraffin embedded, and hematoxylin/eosin (h&e)-stained lung specimens harvested at day 2 p.i. pulmonary pathology was noted in all mice that were treated with different doses of m336 or control m102.4 antibodies either before or after viral infection. on a severity scale of 0 to 3 (none, mild, moderate, severe), h&e-stained samples from mice pretreated with 1 mg and 0.1 mg of m336 antibody were graded 0 and 1, respectively, for perivascular and intra-alveolar infiltration of mononuclear cells, including lymphocytes, macrophages/monocytes (fig. 4, middle panel) , whereas those obtained from mice that received post-infection lung specimens collected at day 2 after viral challenge were processed for assessing the viral titers by using both vero e6-based infectivity assay and qrt-pcr targeting upstream e gene of mers-cov, and expressed as log 10 tcid 50 /gram and log 10 tcid 50 equivalent (eq.)/gram, respectively. (a) prophylactic and therapeutic efficacy of human m336 antibody treatment in reducing the lung titers of infectious virus. (b) prophylactic and therapeutic efficacy of human m336 antibody in reducing the titers of viral rna. the data shown are representative of at least two independently conducted assays using the same samples. data is presented as mean ± standard error (se). * * * p < 0.001 as determined by using student's t test. scientific reports | 6:31629 | doi: 10.1038/srep31629 treatment with either dose of m336 were graded 1 (fig. 4, right panel) , compared to the grade 2 assigned to mice received control antibody treatment prior to infection (fig. 4, left panel) . mers-cov has attracted significant basic research and clinical studies since it was first discovered in early 2012. even though the transmissibility of mers-cov among humans remains low at present, as a mutation-prone rna virus, it could eventually evolve into a highly communicable and more virulent human pathogens. this emphasizes the urgent need for the development of an effective antiviral therapy which could restrict the spread of this deadly disease. in other viral infections, neutralizing antibodies have been shown to protect the host from disease progression and/or reduce the severity of clinical symptoms. passive immunotherapy for prophylaxis and treatment of infectious viral diseases has been widely used for many decades [39] [40] [41] [42] [43] . passive transfer of neutralizing antibodies is also a promising strategy for both prophylaxis and treatment against mers-cov infection. to this end, we and others have successfully demonstrated the protective efficacy of specific human neutralizing monoclonal antibodies in animal models of mers-cov infection 23, 24, 26, 28 . among a panel of mers-cov-specific mabs generated by using a vast phage display library 23 , we identified three mabs which specifically bind to the mers-cov rbd with very high affinity. among these three identified, we noted that mab m336 exhibited the highest potency in neutralizing live mers-cov. here, we further characterized this novel human mab in our tg mouse model of mers-cov infection and showed prophylactic and therapeutic protection of mice treated with m336 before and after a lethal challenge with the virus, respectively. thus, mab m336 is highly promising as a potent inhibitor for urgent prophylaxis in adjunctive treatment for patients infected with mers-cov. in our studies, we noted that passively transferred with 1 mg and 0.1 mg of m336 monoclonal antibodies to individual mice 12 h prior to challenge with 1,000 ld 50 of mers-cov resulted in 100% and 75% protection against lethality, respectively (fig. 1) , suggesting that using 0.1 mg m336/mouse as a prophylaxis is suboptimal to completely neutralize viral infection, thereby allowing residual viruses to replicate within lungs during the course of infection. these data demonstrate that m336 confers a dose-dependent reduction of mers-cov infection, corroborating lower viral rna levels and live virus isolation determined for these mice when compared to control mice. our study also confirmed the therapeutic efficacy of m336 in a dose-dependent manner. similar to the prophylactic studies, administration of a single-dose of m336 antibody at a concentration of either 1 or 0.1 mg per mouse at 12 h after mers-cov challenge provided 100% and 75% protection, respectively, against infection-induced lethality, accompanied by reduced viral loads (both infectious virus and viral rna) within the lungs. however, we also noted the recovery of bodyweight loss and the reduction of viral loads in mice treated with 1 mg of m336 at 12 hrs after infection were slower than those treated with 0.1 mg of m336, as shown in figs 2a and 3b, respectively. while there is no clear evidence showing an adverse impact on the overall wellbeing of mice imposed upon treatment with 1 mg of m336 antibody before mers-cov challenge (fig. 1) , it is difficult to completely rule out the existence of subtle "yet-to-be investigated" high-dose drug toxicity. we speculate that such a subtle high-dose drug toxicity in the phase of acute and dynamic mers-cov infection initiated at 12 hrs before treatment with 1 mg of m336 could exacerbate drug toxicity, resulting in reduction of appetite and antiviral capacity. however, such a negative impact imposed upon high-dose treatment of virally infected mice appeared to be transient and did not irreversibly alter the final outcome of infection, as judged by the mortality (fig. 2b) . additional studies, especially the pharmacokinetics and the dosing frequency of m336 are warranted in the future to optimize preventive and therapeutic strategies with this promising antibody. the transgenic mice that we used for evaluating the prophylactic and, especially, the therapeutic efficacy of this m336 antibody are extremely sensitive to mers-cov infection and disease, with ld 50 and id 50 of 4.5 and 0.4 tcid 50 of mers-cov, respectively (data not shown), titers which are lower than our original estimations 38 . such a striking ability of this m336 antibody, as a prophylactic or therapeutic agent, to significantly protect these transgenic mice against challenge with 1000 ld 50 of mers-cov is highly impressive. the rbd of the mers-cov, targeted by this m336 antibody, is highly conserved among various clinical isolates and the mutation rate of this rbd appears to be extremely low, compared to that of other rna viruses 23, 28 , thereby making the development of escape mutants to m336 unlikely. however, a combination treatment with multiple neutralizing mabs targeted at different epitopes or the mers-cov-specific hr2p fusion inhibitor targeting the hr1 domain of the s2 subunit of the mers-cov s protein 38,44 could be desirable. by immunizing mice with rbd of mers-cov s protein, li, y. et al. recently developed a humanized mab, named 4c2h, that exhibited strong neutralizing activity with nd 50 of ~0.71 and ~6.25 μ g/ml against the pseudotyped and live mers-cov, respectively 36 , which are about 100-fold less potent than m336 (nd 50 = 0.005 and 0.07 μ g/ml against the pseudotyped and live mers-cov, respectively) 23 . using ad5-hcd26-transduced mouse model 33 , they demonstrated that intravenous administration of a single dose of 4c2h one day before or after the mers-cov challenge resulted in reduction of viral titer by 2 log at 3 dpi. however, intraperitoneal administration of m336 to our hdpp4 tg mice lead to the reduction of viral titer as high as 4 log at 2 dpi. since mers-cov challenged ad5-hcd26-transduced mice showed no severe disease, the effect of 4c2h on the weight loss and mortality in these mice is unavailable. additionally, unlike hdpp4 transgenic mice that we used in this study with well-defined hdpp4 expression as well as 50% lethal dose (ld 50 ) and infectious dose (id 50 ), the intensities of hcd26 expression among the ad5-hcd26-transduecd mice are variable, ranging from undetectable to a high level 45 . although both 4c2h and m336 bind to the rbd of mers-cov s protein, some of the critical amino acid residues recognized by these two mabs are different 29, 36 . the epitope of m336 overlaps extensively with the dpp4-binding site, which is composed of mers-cov rbd residues n501-k502, s504, f506, d510, e513, w535-r542, w553, v555, s557 and s559 29 . the epitope of mab 4c2, the parental mouse mab of 4c2h, only overlaps with partial of dpp4-binding site, which is composed of five rbd residues, w535-e536 and d539-r542. most of other rbd amino acids recognized by 4c2, including y397-n398, k400, l495-k496, p525 and v527-s532, are not located on the dpp4-binding site, indicating that the neutralization efficacy of 4c2h is largely attributed to the steric hindrance created by its binding with mers-cov rbd 36 .these results suggest that combinational use of 4c2h and m336 may exhibit synergistic antiviral effect against both wild-type strains and escape mutants (if any) of mers-cov. taken together, these results suggest to us that the mers-cov rbd protein-specific m336 mab is an excellent candidate for passive immunotherapy to provide immediate and effective protection to individuals who may be exposed to mers-cov and to treat patients who have been exposed. testing in humans is needed for its potential use as a therapeutic for the treatment of mers-cov-infected patients. monoclonal antibody production. for expression of m336 igg1, the previously described m336 igg1 vector 23 was used to infect cho-k1 cells (atcc, manassas, va) with polyfect transfection reagent (qiagen, valencia, ca). after screening of 960 clones for antibody productivity by elisa and subsequent characterization, a stable cell line was generated and inoculated into a bioflo 410 bioreactor (new brunswick scientific, nj) for large-scale production of m336 igg1. purification was carried out by using a protein g column (ge healthcare), and endotoxin was removed by detoxi-gel endotoxin removing columns (thermo scientific) according to the manufacturer's instructions. transgenic mice expressing human dpp4 established by us were used throughout the study. animals were housed in on-site animal facilities at galveston national laboratory under a 12:12 light/dark cycle with room temperature and humidity kept between 21-25 °c and 31-47%, respectively, and with ad libitum access to food and water. all experiments were performed in accordance with the guide of nih and aaalac and were approved by the institutional animal care and use committee at the university of texas medical branch, as described previously 34 . briefly, groups of 6-8-weeks tg mice were challenged intranasally (in) with 10 4 tcid 50 /ml (~1,000 ld 50 ) of mers-cov-emc/2012, originally provided by heinz feldmann (nih, niaid rocky mountain laboratories, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, netherlands). the titers of individual virus stocks, stored at − 80 °c, were determined by using vero e6-based infectivity assays and expressed as 50% tissue culture infectious doses (tcid 50 )/ml. scientific reports | 6:31629 | doi: 10.1038/srep31629 viral infections and isolation. all of the animal studies involving infectious mers-cov were conducted within approved animal biosafety level 3 (absl-3) at the galveston national laboratory. experimental designs and strategies in different tg mouse groups involving intranasal challenge with live mers-cov were described in individual experiments in the results section. for live virus isolation, lung tissues were collected at day 2 post mers-cov challenge, weighed, and homogenized in phosphate-buffered saline (pbs) containing 10% fetal calf serum (fcs) by using tissuelyser (qiagen, retsch, haan, germany), as previously described 34 . the resulting suspensions of infected tissues were tittered in the standard vero e6 cell-based infectivity assays to quantify yields of infectious virus expressed as log 10 tcid 50 per gram (g) of tissue. rna extraction and viral titers determination by real-time q-pcr. lung tissue samples from each group of mice were transferred to individual vials having rna later solution (qiagen) and subsequently homogenized and subjected to total rna isolation, by using trizol reagent (life technologies), to assess mers-cov-specific genome targeting of virus-specific upstream e gene (upe) and endogenous control gene (mouse β -actin) by using a one-step rt-pcr kit (invitrogen), as previously described 34 . ct values for each sample were analyzed against ct values generated in our lab from the standard curve of mers-cov mrna copy number. relative mers-cov upe mrna expression value was calculated for each replicate and expressed as the equivalent of log10 tcid 50 per gram (g) of the tissue by the standard threshold cycle (∆∆ct) method. ct value analysis was done by using bio-rad cfx manager 3.0 software. is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sarslike pandemic? hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation 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neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germlinelike antibody infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits prophylaxis with a mers-cov-specific human monoclonal antibody protects rabbits from mers-cov infection rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease animal models of middle east respiratory syndrome coronavirus infection a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease the growth and potential of human antiviral monoclonal antibody therapeutics the spike protein of sars-cov-a target for vaccine and therapeutic development passive immunity in prevention and treatment of infectious diseases prophylaxis and therapy for chikungunya virus infection post-exposure treatment of ebola virus using passive immunotherapy: proposal for a new strategy structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor we thank dr. heinz feldmann, national institute of health at hamilton, montana, and dr. ron a. fouchier, erasmus medical center at rotterdam, the netherlands for the mers-cov. this research was supported in part by a national institutes of health grant, r21ai113206-01 (to c-t.k.t), and pilot grants from the center for biodefense and emerging infectious diseases and from the galveston national laboratory (grant number: 5uc7ai094660-05. project title: national biocontainment laboratories (nbls) operations support), university of texas medical branch, galveston, tx (to c-t.k.t.), intramural funding of nci (to dsd), and the national natural science foundation of china (#31570936 to sj, #31570936 to ty). t.g was supported in part by a t32 biodefense training program (5 t32 ai 60549-12) awarded to utmb by nih. histopathology. mice were necropsied, lung tissues were inflated and fixed in 10% neutral buffered formalin for 3 days before paraffin-embedded and processed for routine hematoxylin and eosin stain (h&e) for assessing the histopathology 46 . key: cord-346539-kxnrf5g5 authors: riggioni, carmen; comberiati, pasquale; giovannini, mattia; agache, ioana; akdis, mübeccel; alves‐correia, magna; antó, josep m.; arcolaci, alessandra; kursat azkur, ahmet; azkur, dilek; beken, burcin; boccabella, cristina; bousquet, jean; breiteneder, heimo; carvalho, daniela; de las vecillas, leticia; diamant, zuzana; eguiluz‐gracia, ibon; eiwegger, thomas; eyerich, stefanie; fokkens, wytske; gao, ya‐dong; hannachi, farah; johnston, sebastian l.; jutel, marek; karavelia, aspasia; klimek, ludger; moya, beatriz; nadeau, kari; o'hehir, robyn; o'mahony, liam; pfaar, oliver; sanak, marek; schwarze, jürgen; sokolowska, milena; torres, maría j.; van de veen, willem; van zelm, menno c.; wang, de yun; zhang, luo; jiménez‐saiz, rodrigo; akdis, cezmi a. title: a compendium answering 150 questions on covid‐19 and sars‐cov‐2 date: 2020-06-14 journal: allergy doi: 10.1111/all.14449 sha: doc_id: 346539 cord_uid: kxnrf5g5 in december 2019, china reported the first cases of the coronavirus disease 2019 (covid‐19). this disease, caused by the severe acute respiratory syndrome‐related coronavirus 2 (sars‐cov‐2), has developed into a pandemic. to date it has resulted in ~6.5 million confirmed cases and caused almost 400,000 related deaths worldwide. unequivocally, the covid‐19 pandemic is the gravest health and socio‐economic crisis of our time. in this context, numerous questions have emerged in demand of basic scientific information and evidence‐based medical advice on sars‐cov‐2 and covid‐19. although the majority of the patients show a very mild, self‐limiting viral respiratory disease, many clinical manifestations in severe patients are unique to covid‐19, such as severe lymphopenia and eosinopenia, extensive pneumonia, a “cytokine storm” leading to acute respiratory distress syndrome, endothelitis, thrombo‐embolic complications and multiorgan failure. the epidemiologic features of covid‐19 are distinctive and have changed throughout the pandemic. vaccine and drug development studies and clinical trials are rapidly growing at an unprecedented speed. however, basic and clinical research on covid‐19‐related topics should be based on more coordinated high‐quality studies. this paper answers pressing questions, formulated by young clinicians and scientists, on sars‐cov‐2, covid‐19 and allergy, focusing on the following topics: virology, immunology, diagnosis, management of patients with allergic disease and asthma, treatment, clinical trials, drug discovery, vaccine development and epidemiology. over 140 questions were answered by experts in the field providing a comprehensive and practical overview of covid‐19 and allergic disease. the first cases of the coronavirus disease 2019 (covid19) , caused by the novel severe acute respiratory syndrome-related coronavirus 2 (sars-cov-2), were reported in china in december 2019 1 and rapidly led to pandemic. currently, ~6.8 million confirmed cases of covid-19 and near 400,00 covid-19-related deaths have been reported globally. 2 these numbers, which are still rising, likely underestimate the cumulative incidence of covid-19 due to several factors; these include limitations of current diagnostic tests, the extent of population testing and reporting, and the type and timing of community mitigation strategies adopted by each country, among others. 3 covid-19 shows a complex clinical profile with many different presentations. like in many other viral infections, subclinical, mild, moderate, or severe cases (10-20% of patients require hospitalization and 2-4% intensive care unit, icu) presenting with or without pneumonia are observed. asymptomatic cases are common but, to date, there is a lack of epidemiological surveys that provide a clear percentage of asymptomatic cases. 4, 5 the covid-19 pandemic is the world's gravest public health crisis of the 21st century, and there is an urgent need for reliable and updated scientific and clinical information. covid-19 is a zoonosis to cd147 (known as basigin or extracellular matrix metalloproteinase inducer), which is expressed in human airway and kidney epithelium, as well as in innate cells and lymphocytes, 14 and to tmprss4, which is highly expressed in intestinal epithelial cells. 15 in addition, antibody-dependent enhancement of sars-cov-2 cell entry may also contribute to infection as reported for sars-cov. 16 sars-cov-2 may use receptors that have been reported for other coronaviruses, such as cd26, aminopeptidase n and glutamyl aminopeptidase for cell invasion. 13, 17, 18 among these, cd26 (encoded by dpp4) has emerged as a putative receptor for sars-cov-2 because structural analyses predict that the spike protein of sars-cov-2 binds to cd26. 19 this receptor has been shown to be expressed in the human epithelium and immune cells. 14 there is limited evidence about covid-19-associated polymorphisms. ace might be one of the candidate genes that influences pneumonia progression in sars. it is conceivable that the d allele influences the renin-angiotensin system via elevation of serum or local ace levels, which may damage the endothelium or epithelium of the lungs. 20 the variance in covid-19 prevalence and mortality cannot be explained by an ace insertion or deletion polymorphism alone, or one polymorphism of any single gene. however, polymorphisms in genes of toll-like receptors, inflammasome, intracellular molecular sensors, interferons (ifns) 21 and interleukins (ils) may contribute. structural proteins of sars-cov-2 virions, such as the spike glycoprotein, envelope, membrane and nucleocapsid, are the main immunogenic molecules (figure 1) . 22 ,23 sars-cov-2 adaptive responses develop mainly to the spike protein, and immunodominant t and b cell epitopes have been reported. 24 intracellularly, the viral rna replicase complex, and non-structural and translated proteins, activate innate immune pathways. this leads to an ifn type i response, nf-kb activation in epithelial cells, as well as activation of nlrp3 and other inflammasomes, in macrophages and dendritic cells. 23 this article is protected by copyright. all rights reserved the spike protein of sars-cov-2 has a receptor-binding domain that binds ace2 with higher affinity than sars-cov. 8 in addition, the sars-cov-2 spike protein harbors a polybasic furin cleavage site (prrar) with an insertion of 4 amino acid residues, which is distinct from that found in sars-cov and other sars-like viruses. this allows effective cleavage by furin and other proteases and determines viral infectivity and host range. 6 the severe lymphopenia observed in covid-19 25 is similar to that reported in hiv infection and acquired immune deficiency syndrome. the latter is characterized by cd4+ t cell lymphopenia, whereas covid-19 causes general lymphopenia. however, severe lymphopenia development in covid-19 happens in weeks, whereas hiv-induced lymphopenia takes years. 26 hiv and sars-cov-2 are both rna viruses and share some similarities in their replication pathways; hence certain rna replication drugs may work in both diseases (figure 1) . 27 there are 2 strains of sars-cov-2 that are clinically relevant. genome analysis of sars-cov-2 from human samples shows high rates of mutation and deletion in several viral genes, including the spike-glycoprotein gene. 28 covid-19 treatments, including future vaccination against sars-cov-2, may drive the genetic evolution of the virus affecting virulence and pathogenicity. for example, a report on a 382-nt deletion in orf8 (figure 1 ) of sars-cov2 isolated from patients in singapore implied that mutations may arise as a result of human adaptation and could be associated with attenuation. 29 nevertheless, the emergence of a sars-cov-3 is possible as long as there is close contact between humans and living animals that harbor coronaviruses. data from 96 covid-19 patients in china show sars-cov-2 detection in respiratory samples for a median of 18 days (13-29 days) . in this study, sputum and saliva were not analyzed separately. viral shedding was significantly longer in patients with severe disease, with a median of 21 days (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) days), compared to mild disease, 14 days (10-21 days). furthermore, glucocorticoids treatment this article is protected by copyright. all rights reserved longer than 10 days significantly extended the duration of sars-cov-2 shedding. 30 viral load differed significantly by sample type, with respiratory samples showing the highest, followed by stool samples, and serum samples showing the lowest (figure 2) . 30 another study of 78 patients with covid-19 (33 asymptomatic vs 42 symptomatic) has estimated that the duration of viral shedding from nasopharynx swabs was 8 days (3-12 days) for asymptomatic vs 19 days (16-24 days) for symptomatic patients. 31 the viral load range from 1.34 × 10 11 copies per ml to 7.52 × 10 5 in sputum of patients who died or survived, respectively. 32 tmprss2 and tmprss4 promote sars-cov-2 infection of ace-expressing human enterocytes 15 causing diarrhea in adults and children. 33,34 sars-cov-2 has been detected in stool samples by reverse transcription polymerase chain reaction (rt-pcr) (figure 2) . the median duration of the virus in stool samples (22 days, interquartile range 17-31 days) was significantly longer than in respiratory samples (18 days, 13-29 days) . 30 however, sars-cov-2 released into the intestinal lumen was inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of patients with covid-19. therefore, the intestine is a potential site of sars-cov-2 replication, which may contribute to local and systemic illness and overall disease progression but unlikely to contribute to the spreading of covid19. 15 section 2: immunology of covid-19 from previous sars studies, it is known that the median seroconversion time for detectable igg was 17 days after infection. 35 detectable levels of sars-specific igg and neutralizing antibodies persisted for up to 720 days. this suggests that there is antibody-mediated protection from sars-cov recurrent infection for up to 2 years. 36 there are inconsistent reports on the humoral response to sars-cov-2. one study with 285 covid-19 patients reported that sars-cov-2 virus-specific igg and igm peaked 17-19 days and 20-22 days after symptom onset, respectively. 37 on the other hand, another study of 26 hospitalized covid-19 patients showed that seroconversion could take up accepted article to 50 days. 38 these discrepancies may be related to the time of sars-cov-2 diagnosis or the clinical characteristics of each cohort and warrant additional studies. systemic iga responses may play a relevant role in the pathogenesis of covid-19. 39 mucosal iga likely exerts a protective role by preventing sars-cov-2 adherence to epithelial cells. circulatory iga may also contribute to sars-cov-2 neutralization. in addition, iga has the ability to either promote inflammation, through the formation of immune complexes, or to dampen it via fc-mediated inhibitory itam-signaling. 40, 41 a seroconversion study in covid-19 patients has found and association between disease severity and sars-cov-2-specific iga levels. these were significantly higher than sars-cov-2-specific-igm and -igg levels in critically ill covid-19 patients. 39 whether this association, previously unseen in sars-cov infection, 42 is due to a protective or detrimental role of iga in covid-19 remains to be elucidated. preliminary findings indicate that asymptomatic and mild cases of covid-19 can generate detectable levels of sars-cov-2-specific antibodies in serum. however, seroconversion is observed less frequently in asymptomatic compared to mild or severe cases, and many asymptomatic cases yield undetectable sars-cov-2-specific antibody responses. 37, [43] [44] [45] so far, no robust data are available on the qualitative differences in humoral responses between asymptomatic and symptomatic covid-19 patients. it is not clear which molecular mechanisms underlie the milder symptoms of covid-19 in children as compared to adults. children may mount a sars-cov-2 antibody response characterized by more efficient production of the so-called natural antibodies, which arise from activated igm+ memory b cells. 46 these cells, which are more prevalent in children than in adults, presumably produce broadly neutralizing antibodies early during the infection. this article is protected by copyright. all rights reserved b cell receptor-sequencing has been conducted in the blood of covid-19 patients. naive b cells exhibited little clonal expansion, whereas cd27+cd38+ memory b cells showed the highest expansion levels among diverse b cell subsets. covid-19 patients significantly expanded specific bcell receptor clones compared to those in the healthy controls. these findings suggest that b cells experience unique clonal variable, diversity, and joining gene segment rearrangements upon sars-cov-2 infection. 47 the lifespan and functionality of these b cells remain to be elucidated. the term "herd immunity" refers to the generation of population immunity that protects a region, or country, from infection. 48 the number of confirmed covid-19 cases has reached approximately 6.5 million. 2 the world population is estimated to be 7.8 billion. to ascertain the extent of herd immunity, it is pivotal to define the prevalence of sars-cov-2-exposed humans. it is thought that 67% is the minimum percentage of symptomatic or asymptomatic covid-19 population required for herd immunity. 48 that is to say that worldwide herd immunity may occur when ⁓5 billion humans have a protective immune response against sars-cov-2. to date, there are no reliable data, particularly on the number of asymptomatic individuals that show seroconversion, to determine the degree of herd immunity. 49 il-4 is pleiotropic and could theoretically cause negative effects on immune responses. however, based on phase ii and iii studies with dupilumab (an il-4rα-specific monoclonal antibody that blocks il-4 and il-13 signaling) in the context of atopic dermatitis, chronic rhinosinusitis with nasal polyps and asthma, no increased risk of infections to viral or bacterial pathogens have been documented. 50 furthermore, dupilumab had no impact on responses to non-live vaccines. 51 this article is protected by copyright. all rights reserved allergic airway disease patients appear to be underrepresented among covid-19 patients. 52-54 this could be partly attributed to the low ace2 expression detected in allergic patients, with or without concomitant asthma. 55 furthermore, allergen challenge, which induces t helper (th)-2 inflammation, 56 has been shown to reduce ace2 expression in a murine model of asthma, and ace2 expression was inversely associated with type 2 biomarkers (il-13, ige, exhaled nitric oxide fraction). 57 these results are in line with previous work showing that decreased ace expression in the airway epithelium of asthmatic subjects was associated with eosinophilic inflammation. 58 on the other hand, the analysis of nasal airway transcriptome data from 695 children identified that tmprss2 is highly upregulated by type 2 inflammation through the action of il-13. therefore, the reduced ace2 expression seen in asthmatic patients may be compensated by an increase in tmprss2 production. 59 eosinopenia has been reported in ⁓50-70% of severe covid-19 patients. a minority of covid-19 patients present with eosinophilic inflammation. 25,60 the th1/th2 cytokine balance may play a role, particularly as it pertains to il-5, which promotes eosinophilopoiesis and eosinophil survival and activation. eosinophilic inflammation suggests the dominance of type 2 inflammation, which may play a protective role against sars-cov-2. on the other hand, it may be the result of a hypersensitivity reaction to drugs used to treat covid-19. 61-63 anti-il-5 treatment, which induces eosinophil deficiency, results in a higher viral load in influenza and rhinovirus infection. this might be due to the ability of eosinophils to bind and inactivate the influenza a virus and respiratory syncytial virus (rsv). 64 a similar role seems possible in sars-cov-2 infection, where type-2 asthma patients potentially benefit from antiviral eosinophil responses. on the other hand, covid-19 post-mortems did not show lung eosinophilia 62 , which argues against its local protective role in sars-cov-2 infection, although it is important to control for glucocorticoid-driven eosinophil reduction in these studies. 61 this article is protected by copyright. all rights reserved eosinopenia is commonly reported in severe covid-19. 65, 66 the underlying mechanisms are largely unknown and most likely multifactorial. a number of possible explanations have been proposed: decreased eosinophilopoiesis; defective eosinophil egression from the bone marrow; and eosinophil apoptosis induced by type 1 ifn released during the acute infection. 61 also, increased eosinophil migration and retention within inflamed tissues has been described, 67 but disputed for the aforementioned reasons. 62 there is no evidence for an enhanced susceptibility of patients on anti-il-5/il-5r treatment to develop viral infections. observational studies in covid-19 patients reported elevated eosinophil counts with a favorable outcome, whereas eosinopenia was observed in more severe cases. 25, 68 neither was there proof of causation nor evidence for enhanced tissue presence in lungs of covid-19 patients. 69 there is neither evidence for a protective effect of these biologicals nor a negative effect regarding sars-cov-2 infection. importantly, maintaining proper asthma control is imperative and so is to follow up on severe asthmatics during the covid-19 pandemic, for example via telemedicine. 50 more than 1 billion people worldwide are infected with helminths, with those living in resource-poor tropical areas being disproportionately affected. helminth co-infection has been shown to influence the severity of viral infection in mice. for example, murid herpesvirus 4 respiratory infection, prior infection with schistosoma mansoni, reduced disease severity. 70 however, immune responses to pulmonary coronaviruses and murid herpesvirus 4 are different and therefore the impact of helminth co-infection is yet to be determined. this is particularly important as the pandemic is now spreading through the helminth-endemic regions of the word. 71 this article is protected by copyright. all rights reserved sars-cov-2 infects human t cells via cd147-binding. 72 t cells are severely affected by sars-cov-2, which reduces t cell counts nearly 2 times below the reference limit. this effect is more pronounced in critically ill covid-19 patients. 60, 73, 74 in addition to the reduction in t cell numbers, a recent study found that cd4+ and cd8+ t cells as well as natural killer cells displayed reduced antiviral cytokine production in covid-19 patients. a reduced cytotoxic potential was identified in covid-19 patients, particularly in those that required icu, and was associated with high il-6 serum levels. 75 circulating sars-cov-2−specific cd8+ and cd4+ t cells have been reported in ∼70% and 100% of covid-19 convalescent patients, respectively. 76 cd4+ t cell responses to the spike protein were robust and correlated with sars-cov-2-specific-igg and -iga titers. the m, spike and n proteins each accounted for 11-27% of the total cd4+ response, with additional responses commonly targeting nsp3, nsp4, orf3a and orf8, among others. for cd8+ t cells, spike and m proteins were recognized, with at least 8 sars-cov-2 orfs targeted. interestingly, sars-cov-2−reactive cd4+ t cells were detected in ∼40-60% of unexposed individuals, which indicate cross-reactive t cell recognition between circulating 'common cold' coronaviruses and sars-cov-2. 76 three sars-recovered individuals, 9-and 11-years post-infection, were analyzed for t-cell responses against 550 sars-cov peptides that may share homology with mers-cov. sarsspecific memory t cells persisted at 9 and 11 years post-sars infection in the absence of antigen exposure. 77 based on these data, it is likely that specific sars-cov-2 epitopes elicit a persistent t cell response, which may also confer protection against other 'common cold' coronaviruses. 76 however, long-term studies on the natural history of sars-cov-2 infection are pending. different mechanisms have been proposed for lymphopenia: 1) t cell exhaustion. the expression of programmed cell death-1 marker (also known as pd-1), which is associated with t-cell exhaustion, was higher in t cells from covid-19 patients than in healthy controls; the expression of pd-1 and tim-3 (another exhaustion marker) increased as covid-19 progressed. 78 2) activation of p53 signaling in lymphocytes, which suggests a role for apoptosis for in lymphopenia. 3 this article is protected by copyright. all rights reserved pyroptosis, which induces lymphopenia and may be proinflammatory. 79 cov-2, which may also cause a cytopathic effect on infected t cells. 5) other mechanisms of lymphopenia that remain to be studied are bone marrow suppression during cytokine storm syndrome (css; see below) and sequestration in the lungs during extensive bilateral pneumonia. 60 lymphopenia can be used as an early predictor of severity and clinical outcome. a significant reduction in lymphocyte counts was common in severe and critically ill covid-19 patients. a continuing or gradual decrease of lymphocyte counts was indicative of poor prognosis and usually required icu admission ( table 1) 60 . in agreement with this, a number of studies have identified lymphopenia as an independent risk factor for mortality in covid-19. 25,80 in covid-19 patients, decreases were observed in total lymphocytes, cd4+ and cd8+ t cells, b cells and natural killer cells. t cell and natural killer cell counts were below normal levels, while b cell counts were at the low end of the normal range. a reduction in specific subsets of lymphocytes, such as cd16+cd56+ natural killer cells and regulatory t cells, was reported in severe covid-19 patients. 60 css is associated with a wide variety of diseases, both infectious and noninfectious. it is a complex cascade of multicellular activation events that leads to an excessive or uncontrolled release of proinflammatory cytokines. css-associated inflammation begins at a local site and spreads throughout the body via the systemic circulation and can cause multi-organ failure and hyperferritinemia. 60,81 css encompasses the activation of large numbers of blood cells, including b cells, natural killer cells, macrophages, dendritic cells, neutrophils, monocytes, resident tissue cells and epithelial and endothelial cells. their activation cause a massive release of pro-inflammatory cytokines, which this article is protected by copyright. all rights reserved drives pathology. 82 the cells involved in css during covid-19 have not been fully determined yet. were the most important cell types releasing a large amount of proinflammatory cytokines. 60, 83 which cytokines are most elevated during css? multiple proinflammatory cytokines and inflammasome activation may contribute to css pathogenesis. 60 elevated serum ferritin, il-6, il-1β, ifn-γ , cxcl10 (known as ip-10) and ccl2 immunosuppression is a double-edged sword in viral infections. 86 this article is protected by copyright. all rights reserved primary immunodeficient patients are a high-risk group in the current pandemic, but to date it is unknown if a particular immunodeficiency poses a higher risk of severe disease. international primary immunodeficiency monitoring is being carried out and few cases have been documented. patients at higher risk are those with complications resulting from their primary immunodeficiency and strict follow-up must be done in those cases. a consensus has been established that baseline chronic treatment should be continued in those patients if they are asymptomatic or mildly symptomatic. furthermore, recommendations regarding primary immunodeficient patients adhere to individual national guidelines emphasizing social distancing and strict hygiene measures. systematic testing of primary immunodeficient patients is not advised, however recommendations may change as the pandemic evolves. 89 there are no longitudinal studies analyzing t regulatory cells in covid19 systemic dysregulation of metabolism, such as that seen in obesity and diabetes is a risk factor of sars-cov-1 and sars-cov-2 infection and of covid-19 severity 69 . these diseases lead to chronic systemic inflammation, upregulation of sars-cov-2 receptors in the lungs and the periphery, and they disturb the glucose and lipid metabolism of tissues and immune cells. 14, 91, 92 ards is an acute life-threatening inflammation of the lung due to infection, trauma, or inflammatory conditions. excessive inflammation leads to alveolar damage and increased permeability of endothelial and epithelial cells. this results in protein-rich fluid accumulation in the interstitium and the air space, which causes impaired gas exchange and hypoxemia. reactive oxygen species, leukocyte proteases, chemokines, and cytokines also contribute to lung injury. the barrier impairment of the lung microvascular barrier is central to the pathogenesis of ards. 93 in covid-19 patients, ards is more common in the elderly, those with multiple comorbidities, and those with continuing or gradually progressing neutrophilia and lymphopenia, and a higher level of creactive protein, lactate dehydrogenase, d-dimer and procalcitonin. 60, 88 there are at least 2 clinical phenotypes of ards: 1) near normal pulmonary compliance with isolated viral pneumonia; 2) decreased pulmonary compliance. 95, 96 what specific therapies can be suggested for ards? different treatments were suggested for ards. corticosteroid treatment is generally not recommended, although widely used in critically ill patients. convalescent plasma (cp) was administered to a small number of patients and was associated with virus clearance and clinical improvement ( table 2) . low tidal mechanical ventilation, positive end-expiratory pressure, prone positioning ventilation, and fluid management guidelines were associated with improved outcomes. extracorporeal membrane oxygenation could be used according to the inclusion and exclusion criteria of the eolia trial. other potential therapies such as mesenchymal stem cell therapy and cytokine inhibitors are still in trials and without definite results. 60,97 bcg is a live attenuated vaccine that was developed against tuberculosis at the beginning of the 20th century. bcg vaccination induces metabolic and epigenetic modifications by enhancing trained immunity (innate immunity to subsequent infections). 98 it was hypothesized that general bcg vaccination policies adopted by different countries might have impacted the transmission patterns and/or covid-19-associated morbidity and mortality. 99 the mechanisms underlying kawasaki disease -a generalized vasculitis, in young children, of unknown, potentially post-viral etiology-are poorly understood. the rare covid-19-associated inflammatory syndrome also features vasculitic changes, affects older children too and is often only associated with positive sars-cov-2 serology, but not viral shedding. its mechanisms need to be elucidated and may include post-infectious, antibody and immune-complex mediated pathology. in adults, there are occasional cases of covid-19-associated cutaneous vasculitis, possibly a localized manifestation of the disease that leads to severe generalized vasculitis in some children. [104] [105] [106] interestingly kawasaki-like disease was not reported in chinese cases and the first months of european cases. the season of the disease and environmental factors should be considered. the chinese epidemic was mainly from january to march whereas the usa epidemic started in mid-march and is still ongoing. initial results of acute phase reactants such as c-reactive protein, alanine transaminase, lactate dehydrogenase, d-dimer, procalcitonin, serum ferritin and il-6 on admission were used to evaluate the severity and predict the mortality. however, dynamic changes of these variables will be more precise in predicting the recovery or progression of covid-19. continuing or progressively increasing levels of c-reactive protein, procalcitonin, d-dimer and lactate dehydrogenase were shown to be associated with a high risk of death in severe covid-19 patients. 25,60,107 patients with acute respiratory illness (i.e., fever and at least one sign/symptom of respiratory disease such as cough or shortness of breath) and a history of contact with a confirmed or probable covid-19 case during the 14 days before symptom onset. patients with any acute respiratory illness in the context of a pandemic should have sars-cov-2 infection in their differential diagnosis. special attention should be given to patients with sudden onset of anosmia, loss of taste, gastrointestinal symptoms or skin lesions without respiratory symptoms who also have epidemiological links. 5,25,108 smell loss is now a well-established diagnostic symptom of covid-19 and can be present in otherwise asymptomatic patients, making it a useful tool in initial diagnosis. 109 this has resulted in anosmia to be included in the list of symptoms used in early screening tools for possible covid-19 in many international bodies. 109 rapidly progressive respiratory failure and sepsis, elevated serum proinflammatory cytokine levels, elevated acute phase reactants (e.g. c-reactive protein), cell-free-hemoglobin-leukopenia and markers of disseminated intravascular coagulation. 110 rt-pcr to generate cdna from sars-cov-2 rna extracted from respiratory samples, followed by quantitative pcr (figure 2 ). 111 common gene targets for sars-cov-2 include the envelope, nucleocapsid, spike, rna-dependent rna polymerase, and orf1 genes. it is recommended to include in the analysis, at least, 2 target genes. 112 nasopharyngeal and oropharyngeal (throat) swabs are the primary specimens for sars-cov-2 rt-pcr testing. lower respiratory tract specimens (i.e. sputum, endotracheal aspirate or bronchoalveolar lavage) may have higher viral loads and be more likely to yield positive tests ( figure 2 ). however, these locations carry a high risk of aerosolization and therefore should be reserved for severe patients with a negative test on an upper respiratory tract specimen and high suspicion for lower respiratory tract sars-cov-2 infection. 113, 114 serology is useful to determine prior exposure to sars-cov-2 within a given period of time (the length of time following infection that one remains positive is unknown) (figure 2) . detection of this article is protected by copyright. all rights reserved antibodies specific to the receptor binding domain of the spike protein indicates neutralization capacity, hence informing better about the development of protective immunity. 37, 111, 115 the antibody response occurs later than initiation of symptoms as well as of the detection of viral rna by rt-pcr in respiratory tract specimens, which usually peaks within the first week of symptom onset (figure 2) . although antibodies to sars-cov-2 have been detected as early as the first week after symptom onset, igm, iga and igg seroconversion commonly occurs between the 2 nd and 3 rd week of clinical illness onset. thereafter, igm starts to decline, reaching low levels by week 5 and almost disappears by week 7, while iga and igg persist beyond this period. 37, 39, 111, 116 the main approaches include nucleic acid amplification on respiratory samples using mobile devices (rt-pcr or isothermal nucleic acid amplification) and viral antigens or host antibodies (viral protein fragments) detection using immunoassays. 117 however, individual tests need validation in large populations before use and their sensitivity, specificity, positive and negative predictive values have to be accurately ascertained. otherwise, they may lead to covid-19 under or over diagnosis, thus undermining the public health efforts to control the disease. 118 a high rate of false negatives with antigen point-of-care assays may be due to the fact that the majority of patients produce antibodies against sars-cov-2 only after the second week after of infection ( figure 2 ). 119 furthermore, an effective antibody response is connected with several determinants, comprising severity of the disease, age and nutritional status of the patient, medications administered and concomitant infections. 118 nucleic acid amplification using rt-pcr directly targeting the virus is not affected by the above-mentioned limitations. 120 this article is protected by copyright. all rights reserved positive by the fifth and final test. 121 patients with an initial positive sars-cov-2 result had an increased risk of progressing to severe cases. altogether, these findings underscore how the timing of the immune response influences rt-pcr tests for sars-cov-2, and the importance of combining rt-pcr and seroconversion data for covid-19 diagnosis. the decision to discontinue home isolation/quarantine should be adapted to specific groups of patients based on factors such as symptom severity, healthcare systems´ capacity, laboratory diagnostic resources and local epidemic status. patients with suspected or confirmed symptomatic covid-19 can discontinue self-isolation/quarantine if all the following 4 conditions are met: a) resolution of fever (without the use of fever-reducing medications) for at least 3 days; b) clinical improvement in respiratory symptoms (e.g., cough, shortness of breath) for at least 3 days; c) at least 8 days have passed since the onset of symptoms for mild cases or at least 14 days for severe cases and immunocompromised patients; d) 2 negative rt-pcr tests from respiratory specimens taken 24 hours apart. if there is limited or no testing capacity, the combined symptom/test-based strategy should be reserved to hospitalized covid-19 cases and healthcare workers, whereas for mild or asymptomatic covid-19 cases (suspected or confirmed) the symptom-based strategy (condition a) and b) and c)) without lab testing is considered acceptable to end the self-isolation period. 122 pandemic strategies for risk minimization should be elaborated, harmonized and followed as such in allergy clinics, centers and practices. 123 in the eaaci/aria position paper by pfaar et al. 124 experts in the field have developed practical recommendations for optimizing allergic patients 'care whilst ensuring the safety of all health care professionals (figure 3) . general guidance from national health authorities should be strictly followed (i.e., world health organization, who; european centre for disease prevention). in-person consultations should be minimized to the lowest necessary level and triaged by telemedicine whenever possible (figure 4 ). 125 special attention should be paid to dataprotection in adherence to national data-security and -protection laws. non-delayable diagnostic and therapeutic measures should strictly follow reasonable preventive measures. several specific considerations regarding diagnostic and therapeutic measures are important in different allergic diseases ( figure 5) . moreover, socio-psychological aspects play a fundamental role in the care of allergic patients during the current pandemic and should be especially recognized and followed. stress caused by isolation and stigmatization due to allergic symptoms may amplify the development of allergic symptoms. 126 virtual doctor consultations have been regarded as an alternative to on-site clinical encounters and are increasing during the covid-19 pandemic. 124 initially, pre-visit telephonic communication is helpful to screen for patients with potential sars-cov-2 infection. 127 the epidemiological history should be investigated to determine if patients have fever or respiratory symptoms. in addition, previsit specific triage improves the efficiency of the patient's visit, thus reducing the length of stay in the hospital. to reduce face-to-face meetings, physicians can train some patients to self-treat at home based on the diagnosis obtained through a telephone consultation (figure 4) . a strict screening protocol is needed to identify sars-cov-2 infected patients (figure 4) . ideally, only sars-cov-2 negative patients (diagnosed via rt-pcr and/or rapid test) should come to the clinic. in places where systematic testing is unavailable, at least, normal temperature and negative epidemiological history should be mandatory to proceed to the outpatient departments. patients with a body temperature higher than 37.3ºc should have additional screening examinations, including routine blood tests, chest computed tomography scanning and even throat swabs for sars-cov-2 rt-pcr testing. 128 the indication and urgency of the tests for diagnosis should be considered. contraindications for skin, provocation and lung function tests can be explained beforehand to the patient, which helps to accepted article avoid unnecessary in-person consultations. 124 any test generating aerosol particles should be avoided because it is considered high risk (figure 4) . personal protective equipment (ppe) must be used when collecting biological samples. biological samples collected on-site from suspected or confirmed covid-19 patients (e.g. antibody assays, rna isolation, flow cytometry) should be processed following bsl-2 practices. during and after the covid-19 pandemic, the usage of bsl-2 facilities is mandatory for all newly arriving patient samples to prevent spreading the disease. research procedures involving sars-cov-2 isolation or culture should be conducted in a bsl-3 facility. 124,129 patients with common allergic diseases do not develop distinct symptoms or severe outcomes. allergic children show a mild course similar to non-allergic children 5 . in a recent study of 182 hospitalized children, 43 of them were reported with allergies. allergic rhinitis was the most prevalent allergic disease (83.7%), followed by drug allergy, atopic dermatitis, food allergy and asthma. in this study, allergic children showed a reduced increase in acute phase reactants, procalcitonin, d-dimer and aspartate aminotransferase levels compared to all patients. there were no deaths in allergic children in that study. 130 clinical history is very helpful to identify seasonality-and exposure-related symptoms driving the diagnosis of pollen-induced allergic rhinitis. an atopy test (in vivo or in vitro) reinforces the diagnosis. however, covid-19 can be superimposed on allergic rhinitis symptoms. 124 symptoms such as fever, fatigue and sudden loss of smell, are suggestive of covid-19 and should be closely monitored. pandemic? this article is protected by copyright. all rights reserved n95 facial masks have been proven useful in reducing allergen exposure by blocking pollen access to nose and mouth. on the other hand, surgical masks do not protect against inhalation of small airborne contaminants and are not designed to seal tightly against the user´s face, hence the contaminated air can pass through the gaps. 131 there are no conclusive data on the impact of allergic rhinitis on covid-19 susceptibility 132 . a recent study with 24 allergic rhinitis patients demonstrated a reduction of ace2 expression in nasal brush samples following an allergen challenge. 55 also, this study reported lower ace2-expression in the epithelium of asthmatic patients. on the other hand, tmprss2 is highly upregulated by type 2 inflammation through the action of il-13. 59 therefore, further studies are necessary to determine if allergic rhinitis patients have an altered risk of sars-cov-2 infection as compared to non-allergic individuals. although limited, the available evidence suggests that, compared to non-allergic individuals, allergic there is no scientific evidence that treatments for allergic rhinitis either increase susceptibility to sars-cov-2 infection or the severity of covid-19. therefore, allergen avoidance measures, nasal saline douches, and background controller therapies recommended by current guidelines for allergic rhinitis, such as nasal corticosteroids or second-generation h1-blockers, should be continued as prescribed, both in non-infected and covid-19 diagnosed patients. 124 the loss of smell in chronic rhinosinusitis is caused by type-2 inflammation of the olfactory epithelium. 135 in covid-19, the exact mechanism of potential olfactory neuropathy is still unclear. 136 however, a study found that sustentacular cells of the olfactory epithelium express ace2 and tmprss2, which enable sars-cov-2 entry and may subsequently impair the sense of smell. 137 a considerable percentage of covid-19 patients experience loss of smell as an early sign of the disease. 109 in many patients smell recovers in 1-2 weeks and there is no indication that intranasal corticosteroid treatment has a positive impact on the recovery. 138 on the other hand, there is no evidence suggesting that this treatment has a negative impact on symptomatology and/or accepted article development of covid-19. consequently, it is recommended to continue regular intranasal corticosteroid treatment for chronic rhinosinusitis (figure 5) . 124 an asthma exacerbation is difficult to differentiate from covid-19 ards or pneumonia by the patient, especially if it is triggered by rhinovirus, or other common respiratory viruses, because both conditions have dry cough and dyspnea. the british thoracic society advises patients with asthma experiencing fever, fatigue and loss of taste or smell to alert their physician as these are indicative of covid-19. 144 the distinction can be made by the physician based on the presence of wheeze, which is generally (but not always) absent in covid-19 pneumonia, as well as high-resolution chest tomography and viral diagnostic tests. 124 patients with controlled asthma are not at higher risk of severe infection than the general population. 53,54 ace2 expression was shown to be decreased in patients with allergic asthma 55 and in those receiving inhaled corticosteroids. 145 on the other hand, ace2 expression in asthmatic patients was increased in african-americans, in males and associated with diabetes, 55 and type 2 inflammation in children is associated with increased expression of tmprss2. 59 it is clear though that uncontrolled asthma is a risk factor of severe covid-19, thus all efforts should be focused on treating asthma by regular use of controller medication, including inhaled corticosteroids and biologicals. 53,146,147 there is no evidence available that patients on inhaled corticosteroids are at higher risk of covid-19 infection or of more severe symptoms than the general population. it is strongly advised by international scientific societies that patients continue with their routine control medication including inhaled corticosteroids during the pandemic (figure 5) . 132,148 this article is protected by copyright. all rights reserved recent evidence indicates that inhaled corticosteroid treatment reduces the expression of viral membrane receptors used to infect the human airways in a dose-dependent manner. 145 on the other hand, the immune suppression exerted by corticosteroids may impair anti-viral responses. 141 however, there are no clinical studies investigating the effect of inhaled corticosteroid on sars-cov-2 infection rates. spirometry is essential for the diagnosis of new asthma cases as stated by the global initiative for asthma guidelines. therefore, it should be conducted, but under special conditions (negative pressure chamber, etc.) and only in areas with low sars-cov-2 infection incidence. healthcare providers performing lung function testing need to wear maximum ppe (filtering face-piece particles 2 or 3 face mask, goggles, or disposable face shield covering the front and sides of the face, clean gloves, and clean isolation gowns), and the spirometer devices should be properly disinfected between patients (figure 3) . 149 an alternative, less precise, is monitoring morning and evening peak expiratory flow variability over a week. 150, 151 the global initiative for asthma guidelines state that routine spirometry should be avoided, especially in high-risk areas of covid-19 transmission. if spirometry needs to be performed, maximum ppe should be used (figures 3 and 4) . 148 the treatment of asthmatic patients can be monitored using personal devices measuring forced expiratory volume and peak expiratory flow. many of these devices are equipped with remote transmission functions and thus are amenable for the telemedicine management of patients. 152 pandemic? this article is protected by copyright. all rights reserved there is no evidence suggesting that the current approach to treat asthmatic patients during an exacerbation should change during the covid-19 pandemic. moreover, there is no proof that a short course of systemic corticosteroids impacts the evolution of covid-19. thus, oral corticosteroids should be given as usual for the treatment of an asthma exacerbation ( figure 5) . 144, 148 in the few cases in which patients are treated with long-term oral corticosteroids in addition to their high dose inhaled corticosteroids this should be continued in the lowest dose possible to prevent exacerbations. 148 the cause of the asthma exacerbation should be studied thoroughly to rule out potential exacerbations due to viral infections. 89 the preferred treatment is a pressurized metered-dose inhaler with a spacer. each patient should have an individual spacer, and this should not be shared at home. the use of nebulizers should be avoided when possible because they increase the risk of disseminating viral particles, which could affect other patients and healthcare personnel. 148 anti-ige treatment with omalizumab (or other biologics indicated for asthma) should be continued in non-infected patients. self-administration devices at home, whenever this option is available, are preferred, to minimize face-to-face contact in the clinic. in infected patients, omalizumab administration should be delayed until complete clinical recovery and viral clearance is achieved ( figure 5) . 50,153 endotype is associated with severe asthma in obese patients, are obese asthmatic patients more likely to develop severe covid-19? obesity, as part of the metabolic syndrome, increases the risk of severe covid-19. this is due to the pre-existent systemic low-grade inflammation and increased expression of sars-cov-2 entry receptors (ace2, tmprss2 and cd147). 154, 155 obese patients tend to have worse asthma control, increased hospitalizations and suboptimal response to standard controller therapy. thus, both difficult-to-control asthma and underlying metabolic syndrome are risk factors for severe covid-19. this article is protected by copyright. all rights reserved the il-6/th17 endotype encountered in late-onset obese asthma might be an additional risk factor. 156,157 the dermatological manifestations of covid-19 range from an un-specific macular erythematous rash, urticarial lesions, chickenpox-like vesicles and acro-ischemic lesions. 158, 159 they can result from local inflammation due to circulating immune complexes or from systemic manifestations leading to vasculitis and thrombosis. 160 these patients are also at increased risk of drug hypersensitivity lesions ( figure 6 ). 161 there is no evidence that patients with barrier defects such as atopic eczema have a higher risk for sars-cov-2 infection or skin complications during covid-19. however, patients with atopic dermatitis are often on systemic immunosuppressants and should be monitored closely. optimal topical treatment regime should also be encouraged in all patients. 162 hand hygiene procedures are pivotal to prevent self-infection and virus spreading. however, extensive water contact enhances dry skin, disturbs the commensal microbiota and leads to barrier disruption in healthy individuals. moreover, it exacerbates diseases with an intrinsic barrier defect such as atopic dermatitis. 163, 164 effective skin-care after hand hygiene is therefore essential to prevent barrier disruption and sensitization events. here, emollients containing hyaluronic acid, vitamin e, ceramide or urea are recommended. 165 dupilumab is approved for the treatment of moderate-to-severe atopic dermatitis. first data from italy on dupilumab-treated non-infected in high epidemic areas, and current evidence from dupilumab trials, suggest no negative effect of dupilumab regarding viral infections 166 with reports on a reduced number of herpes simplex superinfections and less bacterial superinfections. [167] [168] [169] accepted article this article is protected by copyright. all rights reserved the current eaaci statement on the usage of biologicals in the context of covid-19 advices no change of therapy in non-infected individuals and to withhold/delay the application of biologicals for a minimum of two weeks or the resolution of the disease in case of sars-cov-2 infection (figure 5) . 50 this is based on expert opinion in the light of missing data and may be adapted if more information becomes available. acro-ischemic lesions on toes and fingers have been identified in a subgroup of covid-19 patients. 25, 170 the data available are scarce and it is unclear if preventive or active anticoagulation should be initiated. however, acro-ischemic lesions could predate other sars-cov-2 symptoms in children and young adults. covid-19-induced skin lesions can be related to thrombovascular events (i.e. petechiae, acroischemia, dry gangrene) or to typical viral infections (i.e. erythematous rash, urticaria, maculopapular exanthema). 161 drug hypersensitivity has to be considered as a differential diagnosis, mainly in the second group, being a distinction difficult during the acute phase. diagnosis relies mostly on clinical observations. in that regard, an accurate chronology of the reaction and the drug exposure timeline is very informative 63 . laboratory and histopathological findings may also help. immunomodulatory drugs (including azithromycin), hydroxychloroquine/chloroquine and ifns, are the ones most frequently involved in hypersensitivity reactions. most reactions are non-immediate and further studies are required to clarify whether this increased frequency is caused by the drug immunogenicity or simply derives from a greater consumption as compared to other treatments. 161 this article is protected by copyright. all rights reserved drug provocation tests are not recommended because reactions can occur during the tests, including the generation and spreading of virus-containing aerosols. however, they may be considered after careful risk-benefit assessment in cases of urgent need, such as chemotherapy in cancer patients, perioperative drugs and radiocontrast media in subjects needing urgent procedures, and antibiotics if no effective alternative drug is available. 124 most ait products authorized for use in europe indicate that ait should be discontinued in case of infection; the same principle will apply to the covid-19 pandemic. patients on subcutaneous or sublingual ait, who are diagnosed with covid-19, those suspected of sars-cov-2 infection or symptomatic patients with a positive contact to sars-cov-2 individuals, ait should be interrupted until the patient has recovered. in patients not infected or who have recovered from the infection, ait could be continued ( table 3) . these recommendations are conditional and could change as clinical data evolve. 124 ,134 ait should continue in non-infected patients or those recovered from covid-19 ( figure 5 ). this is especially important in patients with life-threatening conditions such as venom allergy. it is possible to extend the intervals between vaccines during subcutaneous ait, as done for inhalant allergens, to minimize visits to the allergy clinic. if venom ait was stopped due to sars-cov-2 infection, it is unclear when it should be re-initiated because data from convalescent patients is scarce. 134 in patients diagnosed with covid-19 or cases with suspected sars-cov-2 infection, oral immunotherapy dosing should continue as indicated in the dosing plan and in coordination with the treating physician. oral immunotherapy can be continued in non-infected patients and those who have recovered from covid-19 ( figure 5 ). in areas with high level of sars-cov-2 community transmission, visits to the allergy clinic for oral immunotherapy up-dosing should be postponed. 124, 134 accepted article this article is protected by copyright. all rights reserved these patients are generally on symptomatic treatment. they need to look out for symptoms suggesting hypoxia or pneumonia, such as shortness of breath, deep shallow breathing, chest pains or persistent tachycardia. special attention needs to be given to those with risk factors for disease progression, such as patients older than 65 years, cardiac or pulmonary comorbidities and immunosuppression. 171, 172 prophylactic low molecular weight heparin, or heparin, has been recommended by the who in severe to critically ill covid-19 patients. 141 however, the international society on thrombosis and haemostasis recommended that all hospitalized covid-19 patients, not just those in icu, should receive prophylactic low molecular weight heparin in the absence of contraindications. 173 during the sars outbreak in 2003, corticosteroids did not change the course of the viral infection and delayed viral clearance. 174 on the other hand, a retrospective study on sars patients in hong kong suggested a better survival rate in patients treated with prednisolone for milder pneumonia or methylprednisolone in more severe cases. 175 recently, chinese experts stated that, in covid-19 patients, systemic corticosteroids should be considered on individual indications in a low-to-moderate dose and for no longer than a week. 176 the national institutes of health in their covid-19 treatment guidelines advises against the use of systemic corticosteroids in non-critically ill patients. 177 there are over 170 clinical trials on covid-19 treatment registered now in the international databases and very few have been completed. currently promoted pharmacological treatments are, at the most, based on anecdotic data collected in small numbers of covid-19 patients. these studies did not satisfy evidence-based medicine criteria, but caught general attention through news media, for example hydroxychloroquine (see below). tocilizumab is a humanized monoclonal antibody specific for il-6r, and it is approved for the treatment of rheumatoid arthritis. a positive response to tocilizumab points towards an imbalanced innate immune response in severe covid-19. luo et al. 178 reported that of the 15 patients treated with tocilizumab, 7 of them critically ill, 11 of the patients recovered within a week. prompt resolution of symptoms and encouraging results have also been reported in uncontrolled or retrospective trials. [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] these zoonotic beta-coronaviruses share structural and genomic similarities that are useful to patients? this article is protected by copyright. all rights reserved fang et al. 192 suggested that there is ace2 overexpression upon treatment with ace inhibitors, thiazolidinediones and ibuprofen. there were concerns pertaining to the use of nonsteroidal antiinflammatory drugs in covid-19 patients. the european medicines agency clarified that no scientific evidence established a link between ibuprofen, or other nonsteroidal anti-inflammatory drugs, and a risk to worsen covid19. 193 section 7: clinical trials and drug discovery in covid-19 adaptations for clinical trials during the pandemic must include all concerned parties such as there are drugs that interfere with ace2 and tmprss2, which are molecules used by the virus to enter the cell. 9, 195 for example, camostat mesylate is a clinically proven serine protease inhibitor with affinity for tmprss2. it has shown activity against sars-cov-2 in human lung calu-3 cells. 9 several drugs that target virus internalization are being investigated, including chloroquine phosphate and hydroxychloroquine, which have shown limited efficacy in humans and raised concerns due to side effects (see below). 196 drugs designed to inhibit the viral replication machinery may be effective against sars-cov-2. for example, remdesivir inhibits viral rna polymerases, which prevents sars-cov-2 replication (see below). it is uncertain whether lopinavir-boosted ritonavir and other antiretrovirals improve clinical outcomes or prophylaxis among patients at high risk of sars-cov-2 infection. 200 additional potential candidates include other broad-spectrum antiviral drugs such as arbidol and favipiravir and phytochemicals with anti-viral activity such as resveratrol ( figure. 1) . 197 in a cohort of severe covid-19 patients, compassionate-use of remdesivir showed clinical improvement in 68% of patients (36 out of 53). 201 of note, a double-blind, randomized, placebocontrolled trial of intravenous remdesivir was conducted in 1,063 adults hospitalized with covid-19 with evidence of lower respiratory tract involvement; remdesivir was superior to placebo in shortening the time to recovery in adults hospitalized with covid-19 and evidence of lower respiratory tract infection. 202 furthermore, in a study of 5 hiv-positive hospitalized patients with severe covid-19, three of them were given lopinavir-boosted ritonavir and 2 darunavir-boosted cobicistat for 14 days. four patients recovered and 1 remained hospitalized. 203 meplazumab is a cd147-specific humanized monoclonal antibody that has been shown to prevent sars-cov-2 infection of fibroblasts (veroe6 cells). 72 currently, there is insufficient evidence to draw any conclusions on the benefits of meplazumab for the therapy of covid-19 patients. in an observational chinese study, adults hospitalized with covid-19 pneumonia (n=17) who were treated with an intravenous infusion of meplazumab as an add-on therapy showed a higher recovery rate compared to controls (n=11). 198 however, these results should be interpreted with caution because they were generated in a non-randomized, non-stratified study, with a small sample size. large-scale studies are needed to assess the effectiveness and safety profile of meplazumab as a potential therapy for covid-19. cp therapy for covid-19 treatment has yielded promising results. for example, in a trial of 10 severe covid-19 patients, 205 cp therapy was well tolerated and improved the clinical outcomes. the viral load was undetectable after cp transfusion in 7 patients who had viremia. no severe adverse effects were observed. other clinical trials have shown the beneficial effect of cp therapy in covid-19 patients and ongoing clinical trials will provide additional data on its efficacy, safety and optimal timing for treatment ( table 2) . in this regard, it is unclear whether in patients with a high viral load, such as severely ill patients, cp therapy may drive tissue pathology through immune complexes or complement activation. baricitinib, fedratinib, and ruxolitinib are potent and selective jak-stat signaling inhibitors approved for indications such as rheumatoid arthritis and myelofibrosis. these drugs are powerful antiinflammatory medications that may reduce the systemic levels of cytokines associated with covid-19. 206 indeed, in a pilot study of 12 covid-19 patients, baricitinib limited the css and was beneficial for the patients. 207 the use of jak inhibitors has been associated with a higher risk of opportunistic viral infections, such as herpes zoster, which suggests that the reduced inflammation caused by jak inhibitors may limit, to some extent, anti-viral responses. 208 this article is protected by copyright. all rights reserved ivermectin (avermectin b1a and avermectin b1b) is an anti-parasitic drug that has shown broadspectrum anti-viral activity in vitro. in sars-cov-2-infected fibroblasts (vero-hslam cells), a single addition of ivermectin at 2 h post-infection reduced viral rna ~5000-fold at 48 hours. 209 however, plasma concentrations of total and unbound ivermectin did not reach the ic50 determined in vitro, even at a 10-times higher dose than approved by the food and drug administration (usa). 210 consequently, the likelihood of a successful clinical trial using ivermectin is low. in an observational study of 1,446 covid-19 patients, 811 received hydroxychloroquine treatment, which did not change the risk of intubation or death. 211 furthermore, in a brazilian randomized control study evaluating 2 different doses of chloroquine in covid-19 patients with severe respiratory symptoms, mortality was 2.5 times higher in the high-dose chloroquine arm. 212 moreover, pre-published results from us veterans health administration hospitals did not support any advantages of hydroxychloroquine administered alone or with azithromycin. 213 in addition, the results of a clinical study conducted in 821 individuals showed that hydroxychloroquine did not prevent illness compatible with covid-19 or confirmed infection when used as postexposure prophylaxis within 4 days after exposure. 214 however, because of the retraction of two main papers on hydroxychloroquine treatment for covid-19 patients, this area requires further attention by the european medicines agency and the food and drug administration. mesenchymal stem cells may exert antiviral mechanisms in the context of sars-cov-2 infection. the basal ifn-stimulated gene expression of mesenchymal stem cells is high, which enhances their responsiveness to ifn signaling, potentially inducing broad viral resistance. mesenchymal stem cell therapy may potentiate the low ifn-i and -iii levels and moderate ifn-stimulated gene response reported in sars-cov-2-infected ferrets and covid-19 patients. 215 it is is being used in some centers but its efficacy in covid-19 has not been proven. data available are mainly experimental with few records in humans and no reports on its efficacy in randomized clinical trials. 216 common anti-hypertensive drugs inhibit ace, but not ace2. importantly, ace2 opposes ace actions and lowers blood pressure by converting angiotensin-ii (a vasoconstrictor peptide) into its metabolites-angiotensin (1-7) (vasodilators). 217 other common related antihypertensive drugs are angiotensin-2 receptors blockers, which block at-1, a receptor for angiotensin-ii, through which it exerts its vasoconstrictor effect. however, at-1 is not known to be used by sars-cov-2 to infect cells. it was shown in animal models that ace inhibitors might increase ace2 expression, thus increasing susceptibility to infection. it has not been proven in humans but it raised the concerns during the covid-19 pandemic. 217 based on the data available to date, antihypertensive treatment with these medications should be continued. 218 at the moment, the animal model that resembles more closely human covid-19 is the rhesus macaque, whose ace2 receptor is identical to that in humans. this model recently showed that sars-cov-2 reinfection was hampered due to infection-acquired immunity and demonstrated the therapeutic effect of remdesivir in covid-19 prior use in human clinical trials. 219, 220 the murine ace2 receptor is different from humans, hence humanized murine models with recombinant human ace2 are necessary. 221 previous vaccine research for sars/mers facilitates rapid translation. 222 in the who vaccine single-domain antibodies have been investigated as potential therapeutics for influenza, rsv and hiv in addition to coronaviruses. sars-cov-2 mainly targets the respiratory tract, hence the development of vaccines directed to the respiratory epithelia and lung parenchyma using a nebulizer has been considered to maximize bioavailability and function. 229 although active research against respiratory viruses has focused on aerosolized plasmid dna vaccines, other forms of vaccine administration are currently further advanced in clinical trials. 222 veterinary medicine commonly uses aerosolized coronavirus vaccines for chicken farms. 230 a novel vaccine platform requires careful evaluation and should ideally include toxicological studies in valid animal models. early progress towards sars vaccines has facilitated a "running start" but standards of care and safety must be maintained. acceleration rather than omission of clinical trials is key. preliminary data from oxford university is anticipated by mid-2020. 222 of note, a doseescalation, single-center, open-label, non-randomized, phase 1 was conducted in 108 healthy individuals that received an ad5 vectored covid-19 vaccine. the vaccine was tolerable and this article is protected by copyright. all rights reserved immunogenic at 28 days post-vaccination. sars-cov-2-specific antibodies peaked at day 28 postvaccination and specific t-cell responses were detected from day 14 post-vaccination. 231 an important aspect is that covid-19-associated mortality is very high, almost unavoidable when the pandemic control fails. this is due to rapid community spread, high community virus, especially in the elderly and co-morbid, but also in younger non-comorbid persons, including healthcare workers, young adults and children. the covid-19 pandemic also seems to be characterized by a significant level of asymptomatic spread. [232] [233] [234] the iceberg of covid-19: are there asymptomatic cases below the surface? the the differences are almost entirely due to the timing and effectiveness of public health interventions. countries that failed to control did too little, too late, and allowed sars-cov-2 to rip through their population, with catastrophic outcomes. those that intervened early effectively stopped the disease transmission. 235 this article is protected by copyright. all rights reserved it is difficult to determine as it varies greatly from country to country, depending on how well countries control their epidemics with widespread testing, case isolation and vigorous contact tracing, testing and isolation if positive. in countries that do this well, the r0 can be very low indeed. in countries that fail to control the spread of the virus, the r0 is high but unknown as sars-cov-2 spreads untested and therefore undetected. it has been estimated to be ~2.2. 236 sars-cov-2 transmits more readily than either sars-cov or mers-cov. the r0 of sars-cov-2 is controversial but if left unchecked it is likely to be greater than 3-4. however, the r0 number cannot be precisely defined as no country has left it to spread completely unchecked. in any case, even when preventative measures are taken, the r0 of sars-cov-2 is higher than that of sars-cov (1.7-1.9) and mers-cov (<1). 237 there is a considerable frequency of very mild covid-19 patients as well as asymptomatic sars-cov-2-infected people. this makes transmission control more challenging than either sars-cov or mers-cov, where illness is frequently more severe. children are at low risk of severe covid-19 outcomes. 238, 239 most patients in pediatric age with sars-cov2 infection presented with no or mild clinical manifestations, including fever, fatigue and dry cough. they were typically managed with supportive treatments only and they had generally a favorable prognosis with a recovery within 2 weeks. [240] [241] [242] young children also frequently carry other respiratory viruses, which potentially limit sars-cov-2 infection, as reported for other viral infections. 243 differences between children and adults in the regulation of ace2 expression may also play a role. 46 ace2 mrna expression was high in type i and ii alveolar epithelial cells, in nasal and oral mucosa and nasopharynx, in smooth muscle cells and endothelium of vessels from the stomach, small intestine, colon, and in the kidney of human adults (mean age 52±22). 244 interestingly, a recent study demonstrated age-dependent ace2 gene expression in the nasal epithelium, which was lowest in younger children and increased with age. 245 in addition, cd147, cd26 and their molecular interaction proteins seem to be differently expressed in peripheral blood mononuclear cells and t cells in children in comparison with adults. 14 many children remain asymptomatic, even when they have radiologic pneumonia detected on screening. 238 given that children are effective transmitters of other respiratory viruses, 246 it is expected that they will be just as good at transmitting sars-cov-2. bats are likely the natural reservoir of sarsseverity (see questions below). data on ethnicity and covid-19 are scarce and further research on ethnicity and covid-19 outcomes is needed. 250 however, the data available show a disproportionate number of covid-19 deaths in black, asian and minority ethnic backgrounds. in fact, one third of uk icu admissions are reportedly from them. 251 in the usa, african americans had more covid-19 diagnoses and deaths, after adjusting for age, poverty, comorbidities, and epidemic duration. these disparities are also seen in the hispanic and asian communities. 252 pregnant women may be at a higher risk of poorer covid-19 outcomes because they have deficient ifn-α and ifn-λ responses to viral infections. 253 however, reported pregnancy outcomes in covid-19 are reassuring as they appear similar to non-pregnant adult females. 254 this article is protected by copyright. all rights reserved testing treatments is problematic because pregnant women are excluded from most trials. 255 it is known that azithromycin doubles innate ifn production from virus-infected lung cells. 256 it is safe for all trimesters of pregnancy 257 and has been shown effective in high-quality clinical trials of virusinduced lung disease. 258, 259 given that the human ace2 protein is encoded on the x chromosome, this may be relevant for malefemale differences in outcomes. particularly in males with rare ace2 coding variants as they will express those variants in all ace2-expressing cells compared to a mosaic pattern of expression in females. 260 males may also have differences in certain innate antiviral responses compared to female counterparts. 261 there is reasonably robust data of covid-19 deaths in hospitals because most people who die in hospital are tested. deaths outside hospitals are likely underestimated as people are dying in care homes where mortality approaches ~40%, 267 and may die without being tested and diagnosed. it is difficult to determine prevalence as testing practices vary so much from country to country. seroprevalence studies will help to collect these data. covid-19 was introduced rapidly to many industrialized countries as a result of air travel. 268 most of europe and the usa probably did not react in a timely and efficient manner, resulting in the rapid spread and subsequent high mortality rates. in light of the devastating situation in many european countries and the usa, less industrialized countries had a little more time to better prepare to control the pandemic. 269 an important factor for prevalence studies is the percentage of the population that has undergone a diagnostic test, which seems to be at lower levels in developing countries. this article is protected by copyright. all rights reserved respiratory viruses spread less readily in summer than in winter for reasons that are not well understood. dry air and higher temperatures are slowing down the spread of respiratory viruses. absence of school attendance, more time outdoors, greater household ventilation, warmer temperatures facilitating virus inactivation and higher vitamin d levels are all likely to play a part. although social distancing measures are implemented, the summer weather should play a role in hampering the spread of covid-19. however, based on the analogy of previous influenza pandemic, it is unlikely that summer, on its own, could stop transmission of sars-cov-2. [270] [271] [272] it largely depends on the sars-cov-2 seroprevalence developed in each country, which is still unknown. countries that have had widespread transmission may be hit by a second wave, but presumably with less severe consequences. countries that effectively controlled the pandemic are at a higher risk of second wave of covid-19 if those effective controls are relaxed due to the limited viral transmission and lack of active immunization. sars-cov-2 has spread worldwide in humans, causing mild or no disease in many cases. it will continue circulating similar to other human coronaviruses (229e, hku1, nl63, oc43), and it may well become an endemic, seasonal virus. 273 the main route of sars-cov-2 transmission is via respiratory droplets and aerosols. [274] [275] [276] avoidance of high virus loads, acquired through aerosol and droplet transmission, is paramount to prevent severe outcomes. consequently, social distancing, masks and hand sanitation are undoubtedly effective because they prevent the droplet and surface contact-associated initial high virus load and the increased risk of severe disease. [277] [278] [279] what is the evidence supporting social distancing and face mask to prevent sars-cov-2 infection? this article is protected by copyright. all rights reserved a systematic review and meta-analysis has found that transmission of viruses was lower with physical distancing of 1 m or more, compared with a distance of less than 1 (or 0.18) and protection was increased as distance was lengthened. in addition, face mask use could result in a large reduction in risk of infection (or 0.15), with stronger associations with n95 or similar respirators compared with disposable surgical masks or similar. eye protection also was associated with less infection (or 0.22). 280 therefore, the covid-19 pandemic can be controlled if social distancing is combined with widespread testing, case isolation, vigorous contact tracing and personal protection. indeed, severe and critical illness among chinese healthcare workers before january 10 th was 45%, a time when personal protection equipment and infectious control measures were likely not implemented. after february 1 st , when personal protection measures were in place, the percentage of severe and critically ill chinese healthcare workers dropped to 8.7%. 281 sars-cov-2 remained viable in aerosols for 3 h with a ~10-fold reduction in infectious titre. 282 sars-cov-2 was more stable on plastic and stainless steel than on copper and cardboard; viable virus was detected up to 3 days after application to plastic and 2 days to stainless steel, on each surface the virus titer was reduced nearly ~100-fold. 282 importantly, sunlight exposure inactivated 98% of infectious sars-cov-2 every 6.8 minutes in simulated saliva and every 14.3 minutes in culture media. this study suggests that persistence, and subsequently exposure risk, may vary significantly between indoor and outdoor environments. 272 therefore, it is convenient to minimize contact with surfaces touched by others (even before sars-cov-2 existed), particularly at indoor environments, for example when using public transportation. in 248 covi̇d-19 patients, the estimated median time from symptom onset to viral clearance in the nasal swabs was 11 days, while in asymptomatic cases it was 2 days. 283 in patients that recovered, the median duration of viral shedding was ⁓20 days, while in non-survivors it was detected until death. the longest duration of viral shedding in survivors was 37 days. 110 accepted article and found that a slow viral clearance is associated with an increased risk of high disease severity with a 1% mortality rate. 285 the individual variation in the transmission of an infection is described by a factor called "dispersion factor or k". the lower "k" value, the more transmission comes from a small proportion of individuals acting like superspreaders. superspreading clusters have been observed in past coronavirus outbreaks (sars/mers), where a small number of infected individuals was responsible for a large proportion of secondary transmissions, with an estimated "k" of about 0.16 for sars and 0.25 for mers. 286 it is unclear whether superspreading clusters have contributed to the covid-19 outbreak. a simulation of early outbreak trajectories estimated that "k" for covid-19 is higher than for sars and mers. 286 however, in a recent preprint study, the estimate of "k" for sars-cov-2 was around 0.1, suggesting that around 10% of infected patients may have been responsible for 80% of secondary transmissions. 287 individual variation in infectiousness is difficult to measure, as it is mostly empirical, but the identification of any sars-cov-2 superspreading will be of primary importance for pandemic control. the designation of covid-19-dedicated wards and personnel within hospitals is useful to limit nosocomial sars-cov-2 infections. 124 it also allows other non-covid-19 conditions to be treated using routine healthcare resources more safely and effectively. maintaining such separation requires intensive sars-cov-2 testing in view of the high asymptomatic infection rate. 288 community-based strategies are effective at controlling the transmission of sars-cov-2. australia, hong kong, japan, singapore, south korea, and new zealand have all controlled effectively. their cumulative covid-19 mortality is >100-fold less than that in belgium, france, italy, spain and the uk, countries which have had difficulties to adequately control the pandemic. 147, 289, 290 it is important to implement measures to contain the spread of the virus, such as developing models to predict sars-cov-2-related mortality. 291 closing live animal markets is likely to reduce the risk of future viral outbreaks although this is not a practical way to prevent viral outbreaks for multiple reasons including social and economic. lifestyle factors that may influence sars-cov-2 infection susceptibility and covid-19 severity include smoking, stress, diet and alcohol intake, among others. for example, smoking has been shown to increase the susceptibility to respiratory tract infections and its severity, 294 and it is a risk factor for severe covid-19. 295 moreover, alcohol consumption may impair anti-viral immunity; 296 in vitro studies with human monocytes have shown that both acute and prolonged alcohol exposures inhibit type i ifn induction upon toll-like receptor-8 and -4 stimulation 297 . dietary habits may also play a role as obese patients have been shown to have a higher risk of developing severe covid-19. 298 furthermore, there are bioactive food compounds with antiviral activity, such as resveratrol, 299 although the amount of them obtained through the diet is unlikely to play a relevant role in covidit is known that respiratory virus infection causes perturbations in the gut microbiota and that germfree mice are more susceptible to viral infections, which intimates a role for the microbiota in covid19 . 300 however, the impact of the commensal microbiota on sars-cov-2 infection susceptibility and covid-19 severity is unknown. 301 an essential step is identifying the bacterial species interacting with sars-cov-2. this is rather challenging given the large number of bacterial species in the lung and respiratory tract, 302 and especially in the gut. however, a number of lung metagenomic studies have reported an abundance of prevotella in the lung of sars-cov-2 infected patients 303 . while in accepted article silico analysis have revealed that prevotella proteins may promote viral infection 304 , prospective studies are necessary to ascertain if this is a consequence of the infection or a risk factor for it. it is well-established that epithelial barrier defects and/or damage favor the development of th2 immunity. 305, 306 increased hygiene, in general, as well as overexposure to epithelial barrier opening molecules, such as detergents, can promote the onset of allergic disease. 307 to date, there is no evidence linking the covid-19 protective measures (gloves, hand-sanitizers, etc.) with increased allergy prevalence. in this regard, multifactorial epidemiological studies are needed. these studies should consider the impact on allergic diseases of virus-specific type 1 responses and psychosocial and environmental changes caused by the pandemic and efforts to contain it. although there has been a significant change in pollution parameters, unfortunately this reduction in pollution is transient and consequently unlikely to be significant. the exposome-related allergy and asthma risk is multifactorial. it includes climate change, biodiversity, the microbiome and nutrition among others, which have not changed during the pandemic. 308 in addition, although pollution levels have dropped, climate change still occurs at an accelerated pace. lifestyle changes during the lockdown 309 , weight gain and increased exposure to indoor allergens and pollutants may even increase the incidence of allergic diseases in the long-run. with the rapid spread of covid-19 at a pandemic scale, we 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plasma for covid-19 patients in wuhan effect of convalescent plasma therapy on viral shedding and survival in covid-19 patients treatment of covid-19 patients with convalescent plasma the authors thank the european academy of allergy and clinical immunology this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-352230-8mazd3eu authors: beeraka, narasimha m.; sadhu, surya p.; madhunapantula, subbarao v.; rao pragada, rajeswara; svistunov, andrey a.; nikolenko, vladimir n.; mikhaleva, liudmila m.; aliev, gjumrakch title: strategies for targeting sars cov-2: small molecule inhibitors—the current status date: 2020-09-18 journal: front immunol doi: 10.3389/fimmu.2020.552925 sha: doc_id: 352230 cord_uid: 8mazd3eu severe acute respiratory syndrome-corona virus-2 (sars-cov-2) induced coronavirus disease 19 (covid-19) cases have been increasing at an alarming rate (7.4 million positive cases as on june 11 2020), causing high mortality (4,17,956 deaths as on june 11 2020) and economic loss (a 3.2% shrink in global economy in 2020) across 212 countries globally. the clinical manifestations of this disease are pneumonia, lung injury, inflammation, and severe acute respiratory syndrome (sars). currently, there is no vaccine or effective pharmacological agents available for the prevention/treatment of sars-cov2 infections. moreover, development of a suitable vaccine is a challenging task due to antibody-dependent enhancement (ade) and th-2 immunopathology, which aggravates infection with sars-cov-2. furthermore, the emerging sars-cov-2 strain exhibits several distinct genomic and structural patterns compared to other coronavirus strains, making the development of a suitable vaccine even more difficult. therefore, the identification of novel small molecule inhibitors (nsmis) that can interfere with viral entry or viral propagation is of special interest and is vital in managing already infected cases. sars-cov-2 infection is mediated by the binding of viral spike proteins (s-protein) to human cells through a 2-step process, which involves angiotensin converting enzyme-2 (ace2) and transmembrane serine protease (tmprss)-2. therefore, the development of novel inhibitors of ace2/tmprss2 is likely to be beneficial in combating sars-cov-2 infections. however, the usage of ace-2 inhibitors to block the sars-cov-2 viral entry requires additional studies as there are conflicting findings and severe health complications reported for these inhibitors in patients. hence, the current interest is shifted toward the development of nsmis, which includes natural antiviral phytochemicals and nrf-2 activators to manage a sars-cov-2 infection. it is imperative to investigate the efficacy of existing antiviral phytochemicals and nrf-2 activators to mitigate the sars-cov-2-mediated oxidative stress. therefore, in this review, we have reviewed structural features of sars-cov-2 with special emphasis on key molecular targets and their known modulators that can be considered for the development of nsmis. covid-19 is a devastating disease caused by a coronavirus related to the one that caused outbreaks of severe acute respiratory syndrome (sars) in the year 2002 (1, 2) . middle east respiratory syndrome (mers)-related coronavirus is an infamous member of this cohort. covid-19, which is caused by the sars-cov-2 infection, was detected in wuhan, china in december 2019. the world health organization (who) declared this infection a pandemic on march 11 2020 due to its severity and rapid spread across the globe. as of june 11 2020, sars-cov-2 had infected 7.4 million individuals, and caused 4,17,956 deaths across 212 countries worldwide ( table 1) . coronaviruses (cov) belongs to a family of single-stranded rna viruses (+rna) that can infect a variety of mammals such as bats and humans (3) . sars-cov-2 contains rna of 29,891nucleotide length, which codes for 9,860 amino acids (4) . the rna has a 5' cap and 3' poly-a tail and produces a poly-protein 1a/1ab (pp1a/pp1ab) in the host (4) . sars-cov-2 belongs to beta cov category and appears in a crown shape with a size of ∼60-140 nm (figure 1) . gene sequencing data revealed that sars-cov-2 has 89 and 82% sequence similarity with bat sars-like-cov-zxc21 and human sars-cov, respectively (4, 5) . the spike (s) proteincoding gene mutation in the nsp2 and nsp3 regions results in the replacement of glycine (g) with serine (s) at 723 position (g723s), and an isoleucine (i) replaced with proline (p) at 1010 amino acid position (i1010p). due to these mutations, the invading potential of sars-cov-2 has increased significantly toward host tissues. this virus can also be transmitted through the respiratory droplets from coughs and sneezes of infected individuals (4) . this mode of aerosol transmission is possible, especially, when protracted exposure occurs in closed areas (4) . the incubation time of the virus varies significantly from individual to individual. in general it takes about 6 days from the day of infection to the first appearance of symptoms. however, in a few cases the symptoms may appear only after 2 weeks (6) . members of coronaviridae are known to induce respiratory complications in humans (7, 8) . at first, sars-cov, mers-cov, and sars-cov-2 varieties were transmitted from animals to humans which triggered severe respiratory diseases (9) (10) (11) . however, subsequent transmission occurred among humans primarily due to physical contact. hence, conventional preventive measures such as physical isolation were implemented to avoid propagation of early infection across the human population (1, 12) . similar to the sars-cov, the pathological manifestations of sars-cov-2 could induce lung malfunction in humans as indicated by the severe acute respiratory syndrome and pneumonia (12) . recent studies reported that sars-cov-2 infection can induce mild, moderate, and severe illness in infected patients (4) . clinical manifestations of this infection include chronic pneumonia, sepsis, septic shock, fever, and dry cough (4) . a progressive respiratory failure during this infection may lead to sudden death (4) . mild illness resulting from a sars-cov-2 infection is characterized by the presence of malaise, headache, low fever and dyspnea. in the case of moderate illness from sars-cov-2, the complication is manifested by the presence of cough and mild pneumonia. severe illness from sars-cov-2 is associated with chronic pneumonia, cough, sars, hypoxia, and tachypnea (in children) followed by respiratory, and cardiovascular system failure (4). the autopsy and biopsy reports of sars-cov-2 patients revealed severe edema with pulmonary tissue exudates, focal reactive hyperplasia, damage to pneumocytes as well as alveolar macrophages, and patchy cellular infiltration (13) . coronavirus-induced lung damage has been demonstrated experimentally by several investigators in animal models (14) . for instance, the sialodacryoadenitis virus and parker's rcov were shown to induce damage to alveolar type-i cells through the expression of pro-inflammatory cytokines, and chemokines such as cinc-2, cinc-3, lix, mip-3α, and fractalkines (15) (16) (17) (18) (19) (20) (21) . for example, fractalkine promotes the infiltration of cytotoxic lymphocytes in the alveolar epithelium thereby inducing a severe inflammatory response (15, 22) . similarly, mip-3α confers the chemotaxis of immune cells via il-1β and tnf-α inflammatory mediators (17, (22) (23) (24) (25) . therefore, these animal models could be used to develop effective pharmacological agents against sars-cov-2 infections. studies from several laboratories have demonstrated that the entry of sars-cov-2 into human cells is facilitated by ace-2 (26) . ace-2 is a member of the renin-angiotensin system (ras), which plays a vital role in cardiovascular and renal homeostasis. ace-2 and tmprss2 facilitates the entry of the virus into host cells during sars-cov-2 infection (7). in addition, there are other proteases such as aminopeptidase n (apn) which plays a prominent role for the entry of hcov-nl63 and hcov-229e into host cells (27) (28) (29) (30) . apn is a membrane-bound glycoprotein that mediates the zincdependent protease activity during the entry and or replication of coronavirus strains into host cells (29, 31, 32) . hence, the ace-2 receptor's down-modulation may prevent sars-cov-2 viral entry/replication (33) . the s-protein of sars-cov and other coronavirus strains are different in their structural and functional domains (3). s-protein can bind to the n-terminus of ace-2 receptors on the outer surface of host cells including respiratory epithelium of the lungs (34) (35) (36) . identifying the key amino acid residues in s-protein of the sars-cov-2 strain may benefit virologists and medical scientists to develop better therapeutic agents. however, to date these details are not known, hence, there is an immediate requirement to identify the amino acids involved in binding s-proteins to ace-2 receptors on host cell surfaces. furthermore, investigations should also focus on establishing the structural similarities of s-protein motifs that are interacting with the ace-2 receptors of other coronavirus strains (37) (38) (39) (40) (41) . these investigations might help in deciphering molecular strategies to target receptor binding sites of ace-2 proteins with sars-cov-2 using novel therapeutics and vaccines to avoid membrane fusion process and viral entry (7) . the tmprss2 protease can foster the entry of the sars-cov-2 virus by activating the s-protein for virus-host cell membrane fusion, consequently enhancing viral replication in the host cells (7, (42) (43) (44) (45) (46) . tmprss2 plays a vital role in generating inflammatory cytokines and chemokines in lung epithelial cells by cleaving s-protein during coronavirus infections including sars-cov-2. hence, tmprss2 is another potential therapeutic target to consider for the novel drug development against sars-cov-2 (46) (47) (48) . prevention and treatment of sars-cov-2 infections are achieved at different levels (49) . the primary approach involves physical isolation to prevent the spread of virus from individual to individual; the second approach involves inhibiting the entry of virus into human cells and the third method includes treating the infected individuals to minimize inflammatory reactions and blocks cathepsin l required for sars-cov processing note: yet to be examined against sars-cov-2 infection blocks sars-cov interaction with ace-2 note: yet to be examined against sars-cov-2 infection [n-(9,10-dioxo-9,10-dihydroanthracen-2yl)benzamide] blocks sars-cov fusion to host cell membrane note: yet to be examined against sars-cov-2 infection (67) nct numbers were obtained from https://clinicaltrials.gov/. pulmonary damage. although physical isolation is the ideal way of limiting the spread, in reality this approach is difficult to execute, hence, many pharmacological companies are actively involved in developing small molecule inhibitors to prevent the entry of the virus into human hosts (7, 49) . in this regard several nsmis have been investigated to treat sars-cov; but, significant breakthroughs are yet to come for treating sars-cov-2 (48) ( table 2) . adedeji et al. (49) reported the discovery and characterization of novel inhibitors to block sars-cov replication via different mechanisms. one mechanism uses screening of small molecule inhibitors using "hiv-1 pseudotyped with sars-cov surface glycoprotein s (sars-s)" (49, 68) . "ssaa09e2" is a novel small molecule inhibitor, which blocks the interaction of cov sars-s with ace-2 receptors, thus blocking the viral entry (49) . another nsmi is "ssaa09e1" reported to be involved in blocking the cathepsin l, which is required for cov-sars-s processing to mediate viral entry into the host cell (49) . ssaa09e3 is another nsmi, which can block the fusion of viral membranes with host cell surfaces (49) (figure 2 ). since the pathological aspects and genomic similarity of sars-cov-2 virus with sars-cov, the above strategies of inhibition may figure 2 | molecular pathogenesis of sars-cov-2 in human lung cells. binding of s-protein of sars-cov-2 to the ace-2 receptors triggers the processing of ace-2 through adam-17/tnf-α-converting enzyme and induces the "ace-2 shedding" into the extracellular space and facilitates uptake of sars-cov-2 followed by the development of sars. alternatively, the entry of sars-cov-2 by membrane tmprss2 serine protease'/hat (human airway trypsin-like protease)-mediated cleavage of ace2 can facilitate sars-cov s-glycoprotein-mediated virus entry. even though, several nsmis targeting these processes were described and their mode of action against coronavirus were delineated, their efficacy against sars-cov-2 is yet to be tested. be considered for developing potent pharmacological agents to prevent sars-cov-2 infections (49) . however, the prospective research should address the efficacy of these inhibitors against sars-cov-2 infections. cytokine storm was predominantly reported during sars-cov-2 infection. targeting cytokine-mediated inflammatory responses induced by sars-cov-2 is another viable approach for mitigating the complications of viral infection. in this regard, chang et al. (35) , documented the inflammatory cascades mediated through intracellular signaling pathways conferred by the sars-cov in both lung epithelial cells and fibroblasts. authors of this study have reported that s-protein of sars-cov efficiently mediate the il-8 release in the infected lung cells by activating mapkinases, and activator protein-1 (ap-1) without intervention of nf-kb cascade (35) . this study suggested a promising lead for novel rational drug design through the identification of a "specific sequence motif of sprotein functional domain, " which is responsible for inducing il-8-mediated inflammatory response in lungs (35) . baricitinib is a pharmacological agent, which was reported to block the sars-cov-2 viral entry and inflammation through the inhibition of ap2-associated protein kinase 1 (aak1), cyclin g-associated kinase, and janus kinase-1 and 2 (69) . chloroquine (cq) and hydroxychloroquine (hcq) were reported to be effective in mitigating the coronaviral load (70, 71) . cq and hcq not only inhibit the entry of sars-cov-2 but also change the ph of acidic intracellular organelles such as endosomes and lysosomes thereby preventing membrane fusion reactions. however, many contradictions and queries prevail pertaining to the use of hcq for the treatment of covid-19. at the time of the submission of this review, results of many clinical trials are yet to be announced, hence, the efficacy of hcq for inhibiting sars-cov-2 infection is still a possibility. prospective studies should focus on testing the fda approved inhibitors of "abl-1 kinases, " "pi3k/akt/mtor" signaling, and "mapkinase" pathways against sars cov-2. since these pathways are involved in cell survival, inflammatory cytokines production, and proliferation of cells, targeted downregulation of these pathways is likely to mitigate the exacerbations induced by coronavirus. in this direction, many of these inhibitors are currently being tested against sars-cov-2 ( table 3 ) (72) (73) (74) . for instance, sorafenib, which inhibits raf, is being experimented in preclinical models and early clinical trials (72) . likewise, the efficacy of il-1 receptor antagonists and tnf-α receptor antagonists for blocking the rat coronavirus-mediated chemokine production was already proven effective in animal models (15, 75, 76) . further studies testing the safety and efficacy are warranted before considering these inhibitory agents for treating individuals infected with sars-cov-2 (76). however, the concept of "one drug to treat all" should be followed to combat several devastating viral infections (77, 78) . for instance, the ebola, marburg, and sars-cov-2 are undoubtedly devastating viral pathogens, which can induce high mortality as they transmit rapidly via air and body fluids (78) . outbreaks of these viruses occur sporadically and currently there are no clinically approved nsmis available to combat these viruses. a recent report by taylor et al. (79) demonstrated the efficacy of a synthetic adenosine analog, bcx4430 in blocking a broad spectrum of viral species viz., "coronaviruses, paramyxoviruses, and bunyaviruses" as these viruses could induce sars, measles, and mumps. bcx4430 could efficiently block both ebola and marburg viral titers in non-human primate models by targeting viral rna polymerase (78, 79) . hence, this molecule should be tested for further studies against sars-cov2 infections in humans. targeting the membrane protease involved in viral s-protein processing and the viral entry into host cells is another approach in mitigating sars-cov-2. the host cellular proteases viz., "trypsin, ", "miniplasmin, " "human airway trypsin like protease, " "tryptase clara, " and "tmprss2" could cleave the ha glycoprotein located in influenza a virus and thereby promote viral entry into lung cells (80) . the usage of serine protease inhibitors such as camostat and aprotinin significantly blocked the replication of influenza virus in epithelial cells of lungs and bronchioles (81) . in addition, these nsmis could block the release of inflammatory mediators such as cytokines, il-6 and tnf-α, during this infection (81) . tmprss2 is a key protein involved in the pathogenesis of several seasonal viral infections including influenza, h1n1, h3n2, and h7n9 (82) (83) (84) (85) . tmprss2 cleaves the s-protein of coronavirus to produce unlocked, fusion-catalyzing viral forms and binds to the host cell surface thereby enhancing rapid viral entry (43, 44, (86) (87) (88) (89) (90) . both sars-cov and mers-cov could rapidly enter into the host cells as tmprss2 can facilitate viral binding to the cell surface (42, 43, 45, 87, 91, 92) . tmprss2 also plays a vital role in the immuno-pathology of coronavirus infections including sars-cov-2 across lungs by inducing lung fibrosis (46) . hence, the emerging research should promote the development of nsmis to target these proteases thereby hindering the entry of sars-cov-2 into host cells. a proof-of-concept study by iwata-yoshikawa et al. (46) reported that sars-cov failed to replicate in the bronchioles and lungs of tmprss2 knockout mice. authors of this study reported elevated expression of tlr3-mrna expression in the lungs of "sars-cov-inoculated tmprss2-deficient mice" and showed enhanced tlr-3 mediated localization of dsrna into endosomes (46) . in this study, tmprss2 knockout has resulted in downregulation of inflammatory cytokines and chemokine expression, which are involved in the bronchiolitis obliterans organizing pneumonia (boop), sars, and pulmonary fibrosis in sars-cov infection (46, 93, 94) . the intricate sars-cov-2 pathogenesis is similar to that of sars-cov. studies have reported the efficacy of ifns to block sars-cov in cell line models but not against sars-cov-2. among ifnα/ -β/ and -γ, the ifn-β was reported to be the most potent blocker of sars-cov growth (3, (95) (96) (97) (98) . furthermore, ifn-β and-γ have a synergistic effect in blocking sars-cov viral replication (62, 63) . however, the effect of this combination against sars-cov-2 is not yet reported. therefore, future studies should focus on determining the efficacy of ifnα/ -β/ and -γ against sars-cov-2 infections. unlike small molecule inhibitors, sirnas are specific and can be designed to mitigate sars-cov associated structural proteins by targeting orf4 (99, 100), orf5 (101, 102), orf9a (50, 103, 104) , and orf7a (50, (105) (106) (107) . for example, sirnas sisc2 and sisc5 have shown success in cultured cells as well as in preclinical mouse models in inhibiting the sars infection without causing toxicity (108) . several other reports have also recently demonstrated the efficacy of sirnas to inhibit the expression of sars-cov genes coding for 3cl protease in cell line models (108) (109) (110) (111) (112) (113) (114) . the activity of sars-cov 3cl protease is essential for viral replication as this protein is involved in the processing of viral proteins (114) . selective optimization and screening of hexa-chlorophene analogs can be "active 3cl protease inhibitors" during a sars-cov infection (114) . hence, the pharmacological agents/sirnas targeting these pathways may likely produce effective clinical outcomes in sars-cov-2 infections. however, clinical studies should test the utility of these agents/sirna in reducing the burden of infections caused by sars-cov-2 (111). the genome of coronaviruses is reported to be significantly involved in coding both structural proteins, and non-structural proteins (nsp's) for the effective viral replication (115) . the nsp's (nsp8c and nsp7) are required for novice cov viral particle formation through viral orf 1ab polyprotein processing (115) . several nsmis were reported to target these non-structural proteins in coronavirus infections to treat sars (115) . for instance, grl0617, a bendioxolane derivative, could target papain-like proteinases like nsp3 (51, 52, 116, 117) , whereas 5choloropyridinyl indolecarboxylate targets nsp5 (53, (118) (119) (120) and a "combination of zinc derivatives with pyrithione" targets nsp12 (121, 122) ; ranitidine bismuth citrate targets nsp13 (123) (124) (125) (126) (127) . monoclonal antibodies; cr3014 (128), mab-201 (129), mdef-201 (130) , ampligen (131), polyiclc (61, 132) , stinging nettle lectin (131) , and tapi-2 (a tace-inhibitor) (59) are anticoronaviral agents tested in vivo models of sars. for instance, a study showed that amiodarone (a known anti-arrhythmic agent) effectively targets coronaviral spreading in in vitro models (55) . working in a similar fashion, 2878/10 humanized antibodies can neutralize coronaviruses thereby reduce the complications caused by viral infections (54) . however, the above nsmis should be tested against sars-cov-2 viral associated proteins and against the activity of nsp's to derive an effective therapeutic intervention. prospective research must focus on the development of novel "helicase inhibitors, viral attachment inhibitors, and activity of rhesus θ -defensin" that block sars-cov-2 infection using in vitro, in vivo, and clinical studies (115) . hence, the development of nsmis to target the synthesis of nsp's in sars-cov-2 may deliver cellular antiviral responses by blocking their replication in host cells (115, 133, 134) . repurposing existing drugs is another strategy widely under consideration to target key proteins involved in the sars-cov-2 infection. in this regard, the existing nsmis viz., antivirals (umefenovir, remdesivir, nitazoxanide, favipiravir, ritonavir, lopinavir, ifns), anticytokines, antimalaria drugs (chloroquine, hydroxychloroquine), and passive antibody therapies are currently being evaluated to improve clinical outcomes in sars-cov-2 infected patients (3, 47, 64, 135, 136) . however, these agents require additional experimental and clinical validations before being tested in sars-cov-2 infections. for example, hydroxychloroquine (anti-malarial drug) and the tocilizumab (immunosuppressive drug) are preferred currently to mitigate viral entry and cytokine production in the sars-cov-2 infection. these drugs are being tested in ongoing trails in china and italy (135, 137) . priming the spike (s)-protein of coronavirus by host cells using membrane proteases is a necessary process for viral entry and replication, which further determines zoonotic potential of coronaviruses (138) . a recent report by markus hoffmann et al. (7) investigated the protease dependence of sars-cov-2 for its entry into cells. for example, sars-cov-2 uses the tmprss2 protease for its priming (7) . inhibition of tmprss2 using camostat mesylate retarded the viral entry into caco-2 cells (7). camostat mesylate could be recommended as an nsmi for human clinical trials to combat the sars-cov-2 virus (7). this report delineated the ability of neutralizing antibody responses against s-protein to block the sars-cov-2 entry into host cells (139) . the serum antibody responses raised to combat the "sars-s protein/ace-2 interface" during the sars-cov-2 infection indicates that the vaccination strategy may be an effective therapeutic modality against the covid-19 infection (7). ace-2 catalytic efficacy is significantly higher than ace for angiotensin-ii (140) . several compounds, such as mln-4760, were screened according to structure-based/substrate-based studies through virtual screening for inhibiting ace-2 activity (140) (141) (142) (143) . ace-2 is predominantly expressed in lungs, brain, heart, blood vessels, and renal organs (144, 145) . ace-2 is essential for cardiovascular homeostasis, and cns homeostasis as ace-2 confer redox homeostasis by mitigating ang-ii-induced oxidative stress (146) . however, in covid-19, ace-2 acts as receptor on human respiratory epithelial cells for sars-cov-2 binding (7). a recent report by markus hoffmann et al. (7) provided evidence that the sars-cov-2 strain use its spike (s)-protein to bind to ace-2. authors of this paper have also demonstrated the efficiency of tmprss2 in sars-cov-2 viral strain priming in host cells (7) . therefore, targeting ace-2 could be a viable strategy to prevent the entry of sars-cov-2 into the human system. however, a recent report by guan et al. (147) cautioned that the administration of ace inhibitors significantly induced adverse clinical outcomes in covid-19 patients due to severe hypertension, coronary artery disease, and chronic renal failure; hence, further use of ace inhibitors to treat covid-19 infections was halted (147) (148) (149) . in another report diaz (149) hypothesized that covid-19 patients receiving i.v. infusions of aceis and arbs (at1-receptor blockers) are at a higher risk of attaining severe disease pathogenesis. hence, they supported the development of nsmis such as "tmprss2 inhibitors to treat sars-cov-2 infections (7)". the failure of disease management and lack of selective therapies could be due to the intricate covid-19 pathogenesis induced by the sars-cov-2 infection. hence, the early recognition of disease is essential for effective management of covid-19 (48) . although, several reports delineated the efficacy of certain nsmis viz., ribavirin, promazine, and imp dehydrogenase inhibitors to inhibit in vivo models of sars-cov replication, later, they were proven ineffective (60, (150) (151) (152) . a report by reghunathan et al. (153) showed that the immune response produced against sars-cov may be different from other viral infections as indicated by the lack of upregulation in mhc-i genes, cytokines, and ifns or complementmediated cytolysis in peripheral blood mononuclear cells (pbmcs). the failure in the development of a vaccine is due to antibody-dependent enhancement and th-2 immunopathology (154) (155) (156) . pegylated ifn-α inhibits viral replication of sars-cov and offers protection against type i pneumocytes in lungs (2) . a significant reason for the failure or lack of selective therapies against sars-cov-2-induced sars is the intricate immune system mediated pathophysiology (4) . other reports by law et al., also detailed similar mechanisms (157, 158) . sars-cov can evade host ifn-mediated viral growth inhibition by activating ifn-regulatory factor 3 (157) . furthermore, sars-cov could induce apoptosis in lymphocytes in vitro using "orf 7a, orf 3a, and orf 3b, e protein, and n protein" (159) (160) (161) (162) . for instance, the sars-cov can evade immunity as indicated by the decline in cd4 and cd8 t cells (163) . therefore, it is necessary to uncover the complement-based cytolysis in human patients in response to the sars-cov-2 strain as this virus executes unusual mechanisms to evade the human immune system consequently inducing pathogenesis and mortality. the prospective research should focus on this viral-mediated immune signaling with respect to sars-cov for developing effective nsmis. intravenous (iv) hyperimmune globulin therapy is one of the immunotherapies known to downmodulate pro-inflammatory cytokines and mitigate the severity of infection in covid-19 patients. iv infusion of immunoglobulins composed of a high dose of antibodies, which can bind to a number of inhibitory receptors viz., fc gamma receptor iib (fcγriib) (164, 165) and fcγriic (166) and confer anti-inflammatory responses against sars-cov-2 (completed clinical trials: hyperimmune plasma nct04321421). oxidative stress is significantly induced by several viral infections inside the lungs through the downregulation of redox regulator nuclear factor-erythroid 2 related factor 2 (nrf-2) (167). nrf-2 is a leucine-zipper transcription factor (167) expressed predominantly in nasal epithelium, epithelial cells of lungs, and alveolar macrophages (168, 169) . disruption of nrf-2 and keap1 interaction triggers the activation of the anti-oxidant defense mechanism (169) . for instance, nrf-2 activation offers protection against inflammation and lung injury induced by influenza viral infections and respiratory syncytial virus (rsv) through the anti-oxidant defense pathway (170) . several viral proteins in the host cells can foster optimum levels of ros-mediated oxidative stress to facilitate viral metabolism and the viral replication cycle without killing host cells (168, (171) (172) (173) (174) . recent seminal studies described the active role of viruses in inhibiting the nrf-2 pathway (175) (176) (177) . for instance, the positive regulation of nrf-2 in modulating the thiol redox system and oxidative stress for the survival of infected astrocytes was observed in moloney murine leukemia virus and hiv virus (178) . the hcv virus could induce the downregulation of nrf-2 dependent nqo1, gclc, and gpx and modulate oxidative stress (179, 180) . an rsv infection mediates proteasomal degradation, deacetylation, and sumoylation of nrf-2 consequently causing the downregulation of nqo1, cat, and sod1 gene expression (181) . hence, nrf-2 activators are potential anti-viral agents, which can be tested against the sars-cov-2 infection (167) . future research is highly imperative in unraveling the underlying activity of nrf-2 for emerging sars-cov-2 survival by analyzing nrf2 target genes nqo1, gclc, and gpx. in addition, the sars-cov-2 mediated expression of serine and cysteine proteases in different cell lines should be investigated in relation to nrf-2 activation, which is a beneficial strategy to combating sars-cov-2 pathogenesis. however, in the case of certain viral infections, it is imperative to develop nrf-2 inhibitors to protect the host cells (182) . for instance, the marburg virus (a causative agent for lethal hemorrhagic fever) can modulate oxidative stress by activating nrf-2 dependent signaling through the blockade of "vp-24 viral protein" binding to keap1 (183) . therefore, vp-24 dependent nrf2 activation can mediate the upregulation of genes ho (heme oxygenase)-1, nqo1, and gclm (183) . in the case of dengue virus, the viral particles could induce er stress and activate nrf-2 signaling, which then lead to tnf-α secretion (184) . in this scenario, it is crucial to uncover any underlying mechanisms of emerging sars-cov-2 survival through the modulation of oxidative stress via nrf-2 signaling in different cells of different organs including lungs (183) . the prospective lindera erythrocarpa makino (195) celastrol (quinone methide triterpene) inhibits tat-induced hiv-1 infection tripterygium wilfordii (197) bakuchiol (phenolic isoprenoid) psoralea corylifolia l. (199) rupestonic acid (sesquiterpene) artemisia rupestris l. (continued) frontiers in immunology | www.frontiersin.org research studies should focus on the development of nrf-2 modulators against sars-cov-2. natural products were proven to offer protection against virusinduced oxidative stress by modulating anti-oxidant defense pathways (185, 186) . for instance, the administration of egcg has mitigated viral replication of "influenza a/bangkok/1/79 infection" by activating nrf-2 to attenuate virus-induced oxidative stress, inflammation, and apoptosis in lung cells (186) . similarly, the cytoprotective and antioxidant efficacy of nrf-2 was reported against pr8 influenza-a viral infection in at-i and at-ii cells (186) . prospective research should focus on testing the efficacy of several natural products to block sars-cov-2 viral replication by ascertaining nrf-2 mediated antioxidant responses. studies have also shown the activation of host cellular transmembrane proteases (for example, serine proteases, cysteine proteases), which can further foster a prompt viral entry and viral replication in host cells by reducing nrf-2 expression (4). decline in proteolysis of the above proteases can actuate the propagation of several human viruses viz., influenza, hiv, nipah, ebola, and coronaviruses (sars-cov, mers-cov, sars-cov-2) (42, 90, (187) (188) (189) . in this scenario, similar to influenza-a virus (190) , it is highly important to unravel the influence of nrf-2 expression on tmprss2, and human airway trypsin-like protease during sars-cov-2-mediated inflammatory conditions and oxidative stress in lungs. the downregulation of the nrf-2 gene is correlated to serine protease activity and consequent influenza viral entry (185) . recent studies have demonstrated the efficacy of natural nrf-2 activators viz., egcg and sulforaphane (sfn) for blocking viral entry/viral replication as well as promoting antiviral mediators rig-i, ifn-β, and mxa (185) . in this context, it is essential to demonstrate the effects of nutritional interventions like sfn and egcg against sars-cov-2 induced oxidative stress by modulating nrf-2 signaling. evidence has demonstrated the use of naturally occurring nrf2 activators for mitigating viral infections/post-viral infection induced complications. for example, α-luminol is a natural nrf-2 activator, which confers the protection of astrocytes against the momul virus (191) . egcg enhances nuclear nrf-2 levels during tat-induced hiv-1 infection and offers protection against virus induced oxidative stress (192) . tanshinone ii a can induce upregulation of nrf-2 expression and mitigates ros production during tat-induced hiv-1 infection via modulating ampk/nampt/sirt1 signaling in host cells (193) . sfn enhances the phagocytic function of "hiv-infected alveolar macrophages in lungs" by activating nrf-2 signaling, which further induces downstream antioxidant cascades (194) . lucidone is effective for the nrf-2 mediated blockade of dengue virus by inducing heme oxygenase-1 (195) ; rographolide could induce nrf-2 induced antioxidant defenses against influenza a in lung cells (196) ; celastrol can mediate nrf-2 induced antioxidant defenses against hiv-1 tat-induced inflammation (197) . broccoli sprouts containing sfn acts as a nrf-2 activator to reduce influenza-induced infection in lung cells (198) . bakuchiol and rupestonic acid are phytoconstituents that confer nrf-2 activation thereby promoting nqo1 gene expression and ho-1-mediated interferon activity to enhance antioxidant response against influenza virus in lung cells (199, 200) . curcumin is another significant compound that can modulate nrf-2 signaling and enhance the generation of ifn-β to offer protection against the influenza virus (201) . curcumin can mitigate this viral infection by modulating tlr2/4, p38/jnk mapk, and nf-κb pathways (201) . however, studies are required to decipher the activity of these phytochemicals against sars-cov-2 ( table 4) . a recent report by drȃgoi (209) hypothesized that the potent natural nrf-2 activators viz., resveratrol, sfn, curcumin, and asea redox should be evaluated in different combinations with conventional drugs against sars-cov-2 infection in both in vitro and in vivo models and to further deduce a correlation between nrf-2 activity and sars-cov-2 viral entry/replication. sars-cov-2 is progressively inducing a high mortality rate across the globe due to the lack of selective therapeutic interventions or vaccination. recent reports by lu et al. (210) and xu et al. (148) delineated that the s-protein of sars-cov-2 and sars co-v exhibit similar 3-d pharmacophore in the receptor binding domain (rbd) of ace-2 of human cells. covid-19 patients are characterized by the severe viral pathogenesis due to extensive cytokine storm viz., tnf-α, il-1β, il-10, ifnγ, and mcp-1 in infected lung tissues (48) . a report by chen and du (211) hypothesized that the phyto-constituents such as "baicalin, scutellarin, hesperetin, nicotianamine, and glycyrrhizin" may deliver anti-sars-cov-2 effects. hesperetin glycoside abundant in citrus fruits, which can inhibit the sars-cov 3clpro (212) . the activity of this molecule must be examined against serine/cysteine proteases, which support sars-cov-2 viral entry/replication. traditional citrus flavonoids were frontiers in immunology | www.frontiersin.org reported to have a potential to act against sars-cov-2 as studied by molecular docking studies. molecular docking simulations, lc-ms studies described the efficacy of citrus flavonoids (ex. naringenin) in binding to ace-2, and mitigating inflammationinduced lung injury by the sars-cov-2 virus (213) . further studies should evaluate these compounds in preclinical models to determine the safety and efficacy against the sars-cov-2 infection. natural products such as di/tri-terpenoids, lignoids were proven to inhibit the viral replication of coronaviruses in vitro (214) ; griffithsin could block coronaviral entry by binding to the sars-cov spike glycoprotein (215) . tsl-1 can block coronaviral entry/replication; leaf extracts of toona sinesis roem effectively blocked sars-cov replication (57) . betulinic acid, savinin can act as competitive inhibitors against sars-cov 3cl protease to block viral entry (214) . the research gap must be filled to develop nutritional therapeutic interventions by investigating the efficacy of these phytochemicals against sars-cov-2 viral entry. seeds of psorelia corylifolia exhibit inhibitory effects against the sars-cov papain-like protease required for coronavirus entry/replication. the efficacy of these molecules should be examined against sars-cov-2 (216) ( table 5 ). the active site pockets of main proteases such as 6lu7 and 2 gtb in sars-cov-2 are reported to be involved in conferring viral entry/fusion; hence, these sites should be considered as the potential drug targets against sars-cov-2 (66) . a molecular docking study by khaerunnisa et al. (65) reported the predicted efficacy of bioactive compounds against above sars-cov-2 main protease (mpro) sites viz., "nelfinavir, lopinavir, kaempferol, quercetin, luteolin-7glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and allicin" ( table 5) . the life-threatening consequences of the covid-19 pandemic remain high due to lack of selective targeted therapies and vaccination strategies. this is primarily due to extreme genomic variability of rna viruses as well as variations in the host-cell invading mechanisms. hence, this review benefits virologists, medical scientists, and cell biologists to ascertain and develop nsmis, nrf-2 modulators, and clinically viable vaccines to combat this devastating sars-cov-2 strain. however, many more preclinical and clinical studies are required to uncover the therapeutic efficacy of potential phytochemicals, natural nrf2 modulators, and several nsmis against 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supplement" [ars]) should be clinically tested as adjuvants in all types of medium and severe cases of aggressive respiratory viral infections (including influenza a/b/c, sars, mers, covid-19, measles, avian influenza etc.) based on their extrapolated cytoprotective antioxidant effects (especially on vital organs), including the cytoprotection offered by ars on the cardiac muscle of dmd patients which can be extrapolated to the lungs -a very short medical communication genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding anti-sars coronavirus 3c-like protease effects of isatis indigotica root and plant-derived phenolic compounds citrus fruits are rich in flavonoids for immunoregulation and potential targeting ace2 specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia baicalin, a metabolite of baicalein with antiviral activity against dengue virus dual effect of glucuronidation of a pyrogallol-type phytophenol antioxidant: a comparison between scutellarein and scutellarin co-occurrence of nicotianamine and avenic acids in avena sativa and oryza sativa azetidine-2-carboxylic acid derivatives from seeds of fagus silvatica l. and a revised structure for nicotianamine betulin and betulinic acid: triterpenoids derivatives with a powerful biological potential the griffithsin dimer is required for high-potency inhibition of hiv-1: evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism research & consulting llc.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 beeraka, sadhu, madhunapantula, rao pragada, svistunov, nikolenko, mikhaleva and aliev. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms.